Multispecific multi-drug antibody-drug conjugates

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 multi-drug antibody-drug conjugates.

Description

WSGR Docket No.: 58964-801.601 MULTISPECIFIC MULTI-DRUG ANTIBODY-DRUG CONJUGATES CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No.63 / 671,887, filed July 16, 2024, which is incorporated herein by reference in its entirety. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on July 11, 2025, is named 58964-801_601_SL.xml and is 245,123 bytes in size. TECHNICAL FIELD

[0003] 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 multi-drug antibody-drug conjugates (MMADC). BACKGROUND

[0004] 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 drugs, such as, for example, 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, such as, for example, 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, and inadequate selectivity, internalization and potency.

[0005] Multispecific antibodies are engineered proteins that can simultaneously bind to two or more different targets (e.g., different antigens or different epitopes of an antigen). TheWSGR Docket No.: 58964-801.601 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 incorporated herein by reference in their entirety. This technology is also referred to as the DEKK technology, and is used for producing Biclonics®antibodies.

[0006] 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

[0007] One of the objects of the present disclosure is to provide a new pharmaceutical agent for the treatment of human disease, including for the treatment of cancer. This object is met by the provision of a novel multispecific multi-drug antibody-drug conjugate (MMADC) format.

[0008] In certain embodiments, the present disclosure provides a multispecific multi-drug antibody-drug conjugate (MMADC) comprising: - a multispecific antibody that binds to two or more different targets; - a first linker connecting a first drug to the multispecific antibody at a first conjugation site; and - a second linker connecting a second drug to the multispecific antibody at a second conjugation site; wherein the first drug is different from the second drug.

[0009] In certain embodiments, the present disclosure provides a multispecific multi-drug antibody-drug conjugate (MMADC) comprising: a multispecific antibody; a first linker connecting a first drug to the multispecific antibody at a first conjugation site; and a second linker connecting a second drug to the multispecific antibody at a second conjugation site; wherein the first drug and the second drug are each independently a microtubule inhibitor, a topoisomerase inhibitor, a DNA- damaging agent, a DNA damage repair inhibitor, a chemotherapeutic agent, or an RNA polymerase inhibitor, wherein the first drug is different from the second drug.

[0010] In certain embodiments, the present disclosure provides a method for producing a multispecific multi-drug antibody-drug conjugate that binds to two or more different targets as described herein, and the method comprises conjugating two or more different drugs to the multispecific antibody.WSGR Docket No.: 58964-801.601

[0011] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising an effective amount of a multispecific multi-drug antibody-drug conjugate as described herein, and a pharmaceutically acceptable carrier.

[0012] In certain embodiments, the present disclosure provides a method of treating a disease, comprising administering an effective amount of a multispecific multi-drug antibody-drug conjugate as described herein, or the pharmaceutical composition described herein, to a subject in need thereof.

[0013] In certain embodiments, the present disclosure provides a method of treating cancer, comprising administering an effective amount of a multispecific multi-drug antibody-drug conjugate as described herein, or the pharmaceutical composition described herein, to a subject in need thereof.

[0014] In certain embodiments, the present disclosure provides a use of a multispecific multi-drug antibody-drug conjugate as described herein for the manufacture of a medicament for the treatment of a disease, in particular for the treatment of cancer.

[0015] In certain embodiments, the present disclosure provides a method of producing a multispecific multi-drug antibody-drug conjugate (MMADC), 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 first drug and a second drug to the antibody, wherein the first drug is different from the second drug.

[0016] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative instances of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different instances, and its several details are capable of modifications in various obvious aspects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive. INCOPORATION BY REFERENCE

[0017] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosureWSGR Docket No.: 58964-801.601 contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the present disclosure are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein) of which:

[0019] FIG.1A shows DAR distribution from 0 to 8 based on HIC data for EGFRxc-MET ADC.

[0020] FIG.1B shows DAR distribution from 0 to 8 based on HIC data for EGFRxLRG5 ADC.

[0021] FIG.2 shows degree of labeling (DOL) of Alexa Fluor 467 for dual labeled bispecific antibodies.

[0022] FIG.3A shows DOL of biotin for dual labeled bispecific antibodies.

[0023] FIG.3B shows a summary of dual DOL for dual labeled bispecific antibodies.

[0024] FIG.4 shows FACS staining results of dual labeled bispecific antibodies prepared from labeling reagents Biotin #1 and Alexa #2. IgG-PE is shown on the X-axis, Alexa FluorTM647 is shown on the Y-axis.

[0025] FIG.5 shows FACS staining results of dual labeled bispecific antibodies prepared from labeling reagents Biotin #1 and Alexa #4. IgG-PE is shown on the X-axis, Alexa FluorTM647 is shown on the Y-axis.

[0026] FIG.6 shows FACS staining results of dual labeled bispecific antibodies prepared from labeling reagents Biotin #3 and Alexa #2. IgG-PE is shown on the X-axis, Alexa FluorTM647 is shown on the Y-axis.

[0027] FIG.7 shows FACS staining results of dual labeled bispecific antibodies prepared from labeling reagents Biotin #3 and Alexa #4. IgG-PE is shown on the X-axis, Alexa FluorTM647 is shown on the Y-axis.

[0028] FIG.8 shows the elution profile of bispecific antibodies treated with DSMO only. The antibodies elute as a dimer (area A) without any detectable half antibodies (area B).

[0029] FIG.9 shows the elution profile of dual labeled bispecific antibodies. Dual labeled bispecific antibodies mainly elute as dimers ( area A) and show absorbance at 650 nm (red curve), without any detectable half antibodies (area B).

[0030] FIG.10 shows the ability of MSADC-1P and cetuximab at 10 µg / ml (upper graphs) and 1 µg / ml (lower graphs) to induce internalization as evaluated by an internalization assay using A549WSGR Docket No.: 58964-801.601 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. DETAILED DESCRIPTION

[0031] While various embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the present disclosure. It should be understood that various alternatives to the embodiments of the present disclosure described herein may be employed. Definitions

[0032] When a range of values is provided, it is to be understood that each intervening value between the upper and lower limit of that range, and any other stated or intervening value in that stated range is encompassed within the scope of the present disclosure. Where the stated range includes upper or lower limits, ranges excluding either of those included limits are also included in the present disclosure. When ranges are used herein for physical properties, such as molecular weight, or chemical properties, such as chemical formulas, all combinations and subcombinations of ranges and specific embodiments therein are intended to be included.

[0033] As used in the specification and appended claims, unless specified to the contrary, the following terms have the meaning indicated below.

[0034] As used herein, the term “C1-C6 alkyl” generally refers to a straight or branched hydrocarbon chain having from 1 to 6 carbon atoms, and the straight or branched hydrocarbon chain is attached to the rest of the molecule by a single bond. Likewise, an alkyl group comprising up to 3 carbon atoms is a C1-C3 alkyl group, and an alkyl group comprising up to 4 carbon atoms is a C1-C4 alkyl group. Examples of a C1-C6 alkyl group include, but are not limited to, methyl, ethyl, n- propyl, isopropyl, n-butyl, isobutyl, s-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3- dimethylbutyl, and 2-ethylbutyl. In some instances, a substituent of an alkyl group is specifically indicated. For example, “cyanoalkyl” refers to an alkyl group substituted with at least one cyano substituent.

[0035] The C1-C6alkyl group may be optionally substituted with a C1-C3alkoxy group. Examples include, but are not limited to, methoxyethyl, methoxypropyl, methoxyisopropyl, ethoxyethyl, ethoxypropyl, ethoxyisopropyl, propoxyethyl, propoxypropyl, and propoxyisopropyl.WSGR Docket No.: 58964-801.601

[0036] The C1-C6 alkyl group may be optionally substituted with a C3-C6 cycloalkyl group. Examples include, but are not limited to, 1-methylcyclopropyl, 1-methylcyclobutyl, and 1- methylcyclohexyl.

[0037] As used herein, the term “C1-C6 alkoxy” generally refers to a radical of the formula –OR wherein R is a C1-C6 alkyl group as defined. Likewise, an alkoxy group comprising up to 3 carbon atoms is a C1-C3 alkoxy group. Examples include, but are not limited to, methoxy, ethoxy, n- propoxy, isopropoxy, n-butoxy, isobutoxy, s-butoxy, tert-butoxy, n-pentoxy, isopentoxy, 2- methylbutoxy, neopentoxy, 1-ethylpropoxy, n-hexyloxy, isohexyloxy, 4-methylpentoxy, 3- methylpentoxy, 2-methylpentoxy, 1-methylpentoxy, 3.3-dimethylbutoxy, 2,2-dimethylbutoxy, 1,1- dimethylbutoxy, 1,2-dimethylbutoxy, 1.3-dimethylbutoxy, 2,3-dimethylbutoxy, and 2-ethylbutoxy.

[0038] The C1-C3 alkoxy group may be optionally substituted with a C1-C3 alkoxy group. Examples include, but are not limited to, methoxymethoxy, methoxyethoxy, methoxypropoxy, methoxyisopropoxy, ethoxymethoxy, ethoxyethoxy, ethoxypropoxy, ethoxyisopropoxy, propoxymethoxy, propoxyethoxy, propoxypropoxy, and propoxyisopropoxy.

[0039] As used herein, the term “C3-C6 cycloalkylamino” is, for example, azacyclobutyl, pyrrolidino, piperidino, or hexamethylenimino.

[0040] As used herein, the term “C3-C6 cycloalkyl” generally refers to a monocyclic non-aromatic radical having from 3 to 6 ring atoms, wherein each of the atoms forming the ring (i.e. skeletal atoms) is a carbon atom. Examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. The term “cycloalkenyl” as used herein generally refers to a group that comprises one or more unsaturated rings in which all ring members are carbon. Polycyclic cycloalkyls or carbocycles include, for example, adamantyl, norbornyl, decalinyl, bicyclo[3.3.0]octane, bicyclo[4.3.0]nonane, cis-decalin, trans-decalin, bicyclo[2.1.1]hexane, bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, bicyclo[3.2.2]nonane, and bicyclo[3.3.2]decane, and 7,7-dimethyl-bicyclo[2.2.1]heptanyl. Unless stated otherwise specifically in the specification, a cycloalkyl is optionally substituted with a C1-C3 alkoxy group or a C1-C3 alkyl group.

[0041] As used herein, the term “alkenyl” generally refers to straight or branched chain alkene groups, which comprise at least one unsaturated carbon-carbon double bond. Alkenyl groups include C2-8 alkenyl, C2-6 alkenyl and C2-4 alkenyl groups, which have from 2 to 8, 2 to 6, or 2 to 4 carbon atoms, respectively, including, for example, ethenyl, allyl or isopropenyl. The term “alkynyl” as used herein generally refers to straight or branched chain alkyne groups, which have one or more unsaturated carbon-carbon bonds, at least one of which is a triple bond. Alkynyl groups include C2-8 alkynyl, C2-6 alkynyl and C2-4 alkynyl groups, which have from 2 to 8, 2 to 6 or 2 to 4 carbon atoms, respectively.WSGR Docket No.: 58964-801.601

[0042] As used herein, the term “halogen” or “halide” generally refers to fluorine, chlorine, bromine, and iodine. The term “haloalkyl” as used herein generally refers to an alkyl group that is substituted with one or more independently chosen halogens (e.g., “C1-C6 haloalkyl” groups have from 1 to 6 carbon atoms and at least one halogen). Examples of haloalkyl groups include, but are not limited to, mono-, di- or tri-fluoromethyl; mono-, di- or tri-chloromethyl; mono-, di-, tri-, tetra- or penta-fluoroethyl; mono-, di-, tri-, tetra- or penta-chloroethyl; 2,2,2-trifluoroethyl; 1,2- difluoroethyl; 3-bromo-2-fluoropropyl; 1,2-dibromoethyl; and 1,2,2,2-tetrafluoro-l-trifluoromethyl- ethyl.

[0043] As used herein, the term “heteroalkyl” generally refers to an alkyl group in which one or more skeletal atoms of the alkyl are selected from an atom other than carbon, e.g., oxygen, nitrogen (e.g., -NH-, -N(alkyl)-), sulfur, or combinations thereof. In some instances, a heteroalkyl is attached to the rest of the molecule at a carbon atom of the heteroalkyl. In one aspect, a heteroalkyl is a C1-C6 heteroalkyl wherein the heteroalkyl is comprised of 1 to 6 carbon atoms and one or more atoms other than carbon, e.g., oxygen, nitrogen, sulfur, or combinations thereof. In some instances, a carbon atom or heteroatom is optionally oxidized (e.g., -C(O)OCH2-, -CH2OCH2-, - CH2S(O)2NHCH2-, -NHC(O)NHCH2-, -CH2NHC(O)CH2-). Further examples of such heteroalkyl are, for example, -CH2OCH3, -CH2CH2OCH3, -CH2CH2OCH2CH2OCH3, or -CH(CH3)OCH3. Unless stated otherwise specifically in the specification, a heteroalkyl is optionally substituted for example, with oxo, halogen, amino, nitrile, nitro, hydroxy, alkyl, alkenyl, alkynyl, haloalkyl, alkoxy, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, and the like. In certain embodiments, a heteroalkyl is optionally substituted with oxo, halogen, methyl, ethyl, -CN, -CF3, -OH, -OMe, -NH2, or -NO2. In certain embodiments, a heteroalkyl is optionally substituted with oxo, halogen, methyl, ethyl, -CN, - CF3, -OH, or -OMe.

[0044] As used herein, the term “heteroaryl” generally refers to a monocyclic aryl group that includes one or more ring heteroatoms selected from nitrogen, oxygen and sulfur. Examples include, but are not limited to, pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, pyridazinyl, triazinyl, oxadiazolyl, thiadiazolyl, and furazyl.

[0045] The terms “heterocyclic” or “heterocycle” or “heterocyclyl” or “cycloheteroalkyl” or “heterocycloalkyl” as used herein generally refer to a ring structure (monocycle or polycycle) containing 3-12 ring atoms (3-12 membered heterocycle), 3-8 ring atoms (3-8 membered heterocycle or 3-8 membered cycloheteroalkyl), 3-6 ring atoms (3-6 membered heterocycle or 3-6 membered cycloheteroalkyl), or 5-6 ring atoms (5-6 membered heterocycle or 5-6 membered cycloheteroalkyl), in which at least one ring atom is carbon, and at least one ring atom is aWSGR Docket No.: 58964-801.601 heteroatom selected from N, O, and S, or a heteroatom group selected from C(=O), S(=O), and S(=O)2. A heterocyclic group may be aromatic or non-aromatic. Piperidine and oxetane are non- limiting examples of non-aromatic heterocycles. Thiazole and pyridine are non-limiting examples of aromatic heterocycles. Other examples of heterocycle include: aziridinyl, azetidinyl, oxetanyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydrothienyl, piperidinyl, morpholinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, thiomorpholinyl, tetrahydropyranyl, 1,1-dioxothiomorpholinyl, butyrolactam, valerolactam, caprolactam, butyrolactone, valerolactone and caprolactone. Similarly, the term “cycloheteroalkenyl” refers to a monocycle or polycycle ring structure comprising carbon atom(s) and heteroatom(s) / heteroatom group(s), wherein the cycloheteroalkenyl comprises at least one C=C double bond, at least one ring atom that is carbon, and at least one ring atom that is a heteroatom selected from N, O, and S or a heteroatom group selected from C(=O), S(=O), and S(=O)2. Unless stated otherwise specifically in the specification, a heterocycle or heterocycloalkyl is optionally substituted, for example, with oxo, halogen, amino, nitrile, nitro, hydroxy, alkyl, alkenyl, alkynyl, haloalkyl, alkoxy, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, and the like. In certain embodiments, a heterocycloalkyl is optionally substituted with oxo, halogen, methyl, ethyl, -CN, -CF3, -OH, -OMe, -NH2, or -NO2. In certain embodiments, a heterocycloalkyl is optionally substituted with oxo, halogen, methyl, ethyl, -CN, - CF3, -OH, or -OMe.

[0046] As used herein, the term “aryl” generally refers to an all-carbon monocyclic or fused-ring polycyclic groups of 6 to 12 (C6-12 aryl) or 6 to 10 carbon atoms (C6-10 aryl) having a completely conjugated pi-electron system. Examples include, but are not limited to, phenyl, naphthalenyl, tetrahydronaphthyl, indanyl, biphenyl, and anthracenyl. The aryl group may be substituted or unsubstituted. Typical substituents include halo, trihalomethyl, alkyl, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, nitro, carbonyl, thiocarbonyl, C-carboxy, O-carboxy, O- carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, sulfinyl, sulfonyl, amino and -NRXRY, wherein RXand RYare independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl, carbonyl, acetyl, sulfonyl, trifluoromethanesulfonyl and, combined, a five- or six-membered heteroalicyclic ring. Illustrative substituted alkyl group include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, aminomethyl, aminoethyl, hydoxymethyl, methoxymethyl, 2-fluoroethyl, and 2-methoxyethyl, etc.

[0047] The term “heteroaryl” as used herein generally refers to an aromatic group in which at least one aromatic ring comprises at least one heteroatom selected from N, O and S. Heteroaryls include, for example, 5-12 membered heteroaryls, 5-10 membered heteroaryls, 5-7 membered monocyclic structures or 7-12 membered bicyclic structures. The number of heteroatoms in a heteroaryl can beWSGR Docket No.: 58964-801.601 1, 2, 3, 4, or more. Examples include, but are not limited to, thienyl, pyridyl, pyrimidinyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridine-2(1H)-keto, pyridine-4(1H)-keto, pyrrolyl, pyrazolyl, thiazolyl, 1,2 ,3-triazolyl, 1,2,4-triazolyl, 1,2,5-oxadiazolyl, imidazolyl, furanyl, tetrazolyl, isothiazolyl, oxazolyl, isoxazolyl, thiadiazolyl, oxadiazolyl, naphthyl ,benzothienyl, indolyl, benzimidazolyl, benzothiazolyl, benzofuranyl, quinolinyl, isoquinolinyl, and quinazolinyl. The heteroaryl group may be substituted or unsubstituted. Typical substituents include halo, trihalomethyl, alkyl, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, nitro, carbonyl, thiocarbonyl, C-carboxy, O- carboxy, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, sulfinyl, sulfonyl, amino and -NRXRY, with RXand RYas defined above.

[0048] As used herein, the term “amino” generally refers to primary amino group (−NH2),secondary amino group (−NH−), and tertiary amino group ( ).

[0049] As used herein, the term “alkylamino” generally refers to a secondary or tertiary amine that has the general structure -NH-R1or -N(R1)(R2), respectively, wherein R1and R2are selected independently from alkyl, cycloalkyl and (cycloalkyl)alkyl groups. Such alkylamino groups include, but are not limited to, mono- and di-(C1-6 alkyl)amino groups, in which each C1-6 alkyl may be the same or different. In this case, the definition of “alkyl” as used in the term “alkylamino” differs from the definition of “alkyl” used for all other alkyl-containing groups, in the inclusion of cycloalkyl and (cycloalkyl)alkyl groups.

[0050] The term “alkylthio” as used herein generally refers to an alkyl-substituted thio group, wherein the term alkyl is as defined above。

[0051] The terms “substituent” and “substituted,” as used herein, generally denote that a molecular moiety is covalently bonded to an atom within a molecule of interest. For example, a ring substituent may be a moiety such as a halogen, alkyl group, haloalkyl group or other group that is covalently bonded to an atom (preferably a carbon or nitrogen atom) that is a ring member. Substituents of aromatic groups are generally covalently bonded to a ring carbon atom. A straight chain substituent may be a moiety such as a halogen, alkyl group, haloalkyl group or other group that is covalently bonded to an atom (preferably a carbon or nitrogen atom) that is a member of a straight chain.

[0052] The term “cycloalkylamine” as used herein generally refers to either a ring structure with an amino group attached to a carbon atom in the ring or a ring structure with a nitrogen atom as member of the ring.

[0053] As used herein, the term “C1-C4 alkylcarbonyl” generally refers to a carbonyl radical that is substituted by a C1-C4 alkyl radical as defined above. Examples include, but are not limited to,WSGR Docket No.: 58964-801.601 methylcarbonyl, ethylcarbonyl, n-propylcarbonyl, isopropylcarbonyl, butylcarbonyl, and tert- butylcarbonyl.

[0054] As used herein, the term “C1-C3 alkylsulfonyl” generally refers to a sulfonyl radical that is substituted by a C1-C3 alkyl radical as defined above. Examples include, but are not limited to, methanesulfonyl, ethanesulfonyl, n-propanesulfonyl, and isopropanesulfonyl.

[0055] As used herein, the term “C1-C4 alkoxycarbonyl” generally refers to a carbonyl radical that is substituted by C1-C4 alkoxy radical, as defined above. Examples thereof include methoxycarbonyl, ethoxycarbonyl, n-propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, and tert-butoxycarbonyl.

[0056] The term “arylene”, as used herein, refers to a divalent aryl radical. Non-limiting examples of “arylene” include phenylene, pyridinylene, pyrimidinylene and thiophenylene. Substituents for arylene groups are selected from the group of acceptable substituents described herein.

[0057] The term “optionally substituted” as used herein generally refers to substituted or unsubstituted. Thus, when a substance, group or moiety is defined as optionally substituted, the substance, group or moiety can be a substituted group or an unsubstituted group. By way of example only, optionally substituted alkyl includes substituted alkyl and unsubstituted alkyl. Accordingly, the terms “substituted and unsubstituted” and “optionally substituted” may be used interchangeably.

[0058] The term “substituent(s)” generally refers to group(s) which can be used to replace another group on a molecule. Such substituent groups include, but are not limited to, halogen, C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, C1-C10 alkoxy, substituted C1-C10 alkoxy, C5-C12 aralkyl, C3-C12 cycloalkyl, C4-C12 cycloalkenyl, C2-C12 alkoxyalkyl, C5-C12 alkoxyaryl, C5-C12 aryloxyalkyl, C7-C12 oxyaryl, C1-C6 alkylsulfinyl, C1-C10 alkylsulfonyl, wherein m is from 1 to 8, aryl, substituted aryl (including but not limited to phenyl or substituted phenyl), haloalkyl (including but not limited to fluoroalkyl), heterocyclic radical, substituted heterocyclic radical, nitroalkyl, -SiR3, -NO2, -N3, - ONH2, -CN, -NRC(O)-(C1-C10 alkyl), -C(O)-(C1-C10 alkyl), C2-C10 alkthioalkyl, -C(O)O-(C1-C10 alkyl), -OH, -OR, -SR, -SO2, -S(O)R, -S(O)2R, -S(O)2NR2, -NRSO2R, , =NR, =N-OR, -OC(O)R, - C(O)R, -CO2R, -CONR2, -OC(O)NR2, -NRC(O)R, -NRC(O)NR2, -NR(O)2R, -NR-C(NR2)=NR, =S, -COOH, -NR2, carbonyl (-C(O)), -C(O)-(C1-C10 alkyl)-CF3, -C(O)-CF3, -C(O)NR2, -(C1-C10 aryl)-S- (C6-C10aryl), -C(O)-(C6-C10aryl), -(CH2)m-O-(CH2)m-O-(C1-C10alkyl), -(CH2)m-O-(C1-C10alkyl), - C(O)NR2, -C(S)NR2, -SO2NR2, -NRC(O)NR2, -NRC(S)NR2, a sugar; wherein each m is from 1 to 8; salts thereof, and the like. Each R group in the preceding list includes, but is not limited to, H, alkyl, substituted alkyl, aryl, substituted aryl, halogen or alkaryl. Where substituent groups are specified by their conventional chemical formulas, written from left to right, they equally encompass theWSGR Docket No.: 58964-801.601 chemically identical substituents that would result from writing the structure from right to left; for example, -CH2O- is equivalent to –OCH2-.

[0059] By way of example only, substituents for alkyl and heteroalkyl radicals (including those groups referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) include, but are not limited to: -OR, =O, =NR, =N-OR, -NR2, -SR, -halogen, -SiR3, -OC(O)R, -C(O)R, -CO2R, -CONR2, -OC(O)NR2, - NRC(O)R, -NRC(O)NR2, -NR(O)2R, -NR-C(NR2)=NR, -S(O)R, -S(O)2R, -S(O)2NR2, -NRSO2R, - CN, –NO2, -R, -N3, -ONH2, -CH(Ph)2, fluoro(C1-C4)alkoxy, fluoro(C1-C4)alkyl, a sugar, in a number ranging from zero to the total number of open valences on the alkyl or heteroalkyl group, and wherein each R group in the preceding list includes, but is not limited to, hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, including but not limited to, aryl substituted with 1-3 halogens, substituted or unsubstituted alkyl, alkoxy or thioalkoxy groups, or aralkyl groups. When two R groups are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 5-, 6-, or 7-membered ring. For example, -NR2 is meant to include, but not be limited to, 1-pyrrolidinyl and 4-morpholinyl.

[0060] By way of example, substituents for aryl and heteroaryl groups (including those groups referred to as arylene) include, but are not limited to, -OR, =O, =NR, =N-OR, -NR2, -SR, -halogen, - SiR3, -OC(O)R, -C(O)R, -CO2R, -CONR2, -OC(O)NR2, -NRC(O)R, -NRC(O)NR2, -NR(O)2R, -NR- C(NR2)=NR, -S(O)R, -S(O)2R, -S(O)2NR2, -NRSO2R, -CN, –NO2, -R, -N3, -ONH2, -CH(Ph)2, fluoro(C1-C4)alkoxy, fluoro(C1-C4)alkyl, a sugar, in a number ranging from zero to the total number of open valences on the aromatic ring system; and wherein each R group in the preceding list includes, but is not limited to, and is independently selected from hydrogen, alkyl, halogen, heteroalkyl, aryl and heteroaryl.

[0061] As used herein, the terms “functional group”, “active moiety”, “activating group”, “leaving group”, “reactive site”, “chemically reactive group” and “chemically reactive moiety”, generally refer to portions or units of a molecule at which chemical reactions occur. For example, a leaving group, can be replaced by another reactive group and lead to a molecular structure. Examples of a leaving group include, without limitation, halide or a sulfonate. Examples of sulfonates can include, without limitation, nonaflate, triflate, fluorosulfonate, tosylate, mesylate or besylate. In certain embodiments, for example and without limitation, the leaving group is bromide, mesylate or tosylate. The functional groups that can be converted into leaving groups, in accordance with the specification, are not particularly limited. In certain embodiments, for example the functional group can be a hydroxy group that can be converted into a leaving group as described above.WSGR Docket No.: 58964-801.601

[0062] The term “reactive compound” as used herein generally refers to a compound which under appropriate conditions is reactive toward another atom, molecule or compound.

[0063] The term “linkage” or “adduct moiety” as used herein generally refers to a bond or chemical moiety formed from a chemical reaction between the functional group of one group, such as a linker of the present disclosure, and another molecule. Such bonds may include, but are not limited to, covalent linkages and non-covalent bonds, while such chemical moieties may include, but are not limited to, esters, carbonates, imines, hydrazones, acetals, orthoesters, peptide linkages, oximes and oligonucleotide linkages. Hydrolytically stable linkages mean that the linkages are substantially stable in water and do not react with water at useful pH values, including but not limited to, under physiological conditions for an extended period of time, perhaps even indefinitely. Hydrolytically unstable or degradable linkages mean that the linkages are degradable in water or in aqueous solutions, including for example, blood. Enzymatically unstable or degradable linkages mean that the linkage can be degraded by one or more enzymes. Some hydrolytically degradable linkages include, but are not limited to, ester linkages, carbonate linkages; imine linkages resulted from reaction of an amine and an aldehyde; hydrazone linkages which are reaction product of a hydrazide and an aldehyde; acetal linkages that are the reaction product of an aldehyde and an alcohol; orthoester linkages that are the reaction product of a formate and an alcohol; and peptide linkages formed by an amine group, including but not limited to, at an end of a polymer such as polyethylene glycol (PEG), and a carboxyl group of a peptide.

[0064] As used herein, the term “linker” generally refers to a molecular group that connects, or is capable of connecting, a first group to at least one other group. In some cases, the linker uses covalent bonds to join the two other molecules. The term “cleavable linker” as used herein generally refers to a linker that can be degraded or otherwise severed to separate the two components connected by the cleavable linker. Cleavable linkers are generally cleaved by enzymes, typically peptidases, proteases, nucleases, lipases, and the like. Cleavable linkers may also be cleaved by environmental conditions, such as, for example, changes in temperature, pH, salt concentration, etc., when there is such a change in environment following transcytosis of the compound disclosed herein across a polarized epithelial membrane. Alternatively, linkers can be substantially resistant to cleavage (e.g., stable linker or non-cleavable linker).

[0065] In certain embodiments, the linker connects two or more groups comprising, or consisting of, at least one moiety, wherein each at least one moiety is independently selected from the group consisting of a bond, unsubstituted alkylene, substituted alkylene, –(alkylene–O)nn–, optionally substituted arylene, -O-, -C(O)-, -C(S)-, -N(Rw)-, -S(O)0-2-, methine (-CH)-, an amino acid, a peptide, and a disulfide (-S-S-) moiety; and combinations thereof; wherein: each nn is independentlyWSGR Docket No.: 58964-801.601 an integer from 1 to 100; and each Rw is independently H, C1-C8 alkyl or a bond. Unless expressly indicated otherwise, no orientation of the linker is implied by the direction in which the formula of the linker group is written. By way of example, the formula –C(O)CH2CH2– represents both – C(O)CH2CH2– and –CH2CH2C(O)–. In another example, the formula –C(O)CH2CH2– represents both *–C(O)CH2CH2– and –C(O)CH2CH2–*, wherein * denotes a point of connection, for example, connection to a drug. In certain embodiments, when a selected moiety occurs two or more times in the same linker, the two or more occurrences are not adjacent. In certain embodiments, a linker is not a bond.

[0066] In certain embodiments, a linker connects a first group and a second group. In some other embodiments, the linker connects a first group, a second group, and a third group. In a non-limiting example, a linker that connects three groups is C(H) (i.e., methine) or N. In certain embodiments, a linker connects four groups: a first group, a second group, a third group, and a fourth group.

[0067] In certain embodiments, a linker connects at least a first group and a second group, wherein the first group is a drug, and the second group is a biologically active polypeptide or protein, such as an antibody, including, but not limited to, a bispecific or trispecific antibody. In certain embodiments, the biologically active polypeptide or protein contains at least one engineered amino acid to facilitate conjugation. In certain embodiments, the linker connects the drug to an engineered amino acid of the biologically active polypeptide or protein. Thus, the antibody connected to a drug via a linker can be an antibody-drug conjugate (ADC), such as a MMADC of the present disclosure.

[0068] In certain embodiments, a linker connects at least a first group and a second group, wherein the first group is a drug, and the second group is a reactive moiety. In certain embodiments, the second group is a reactive moiety that is capable of reacting with a biologically active polypeptide or protein. In certain embodiments, the biologically active polypeptide or protein contains at least one engineered amino acid to facilitate conjugation. Thus, in certain embodiments, the reactive moiety is capable of reacting with an engineered amino acid of the biologically active polypeptide or protein. In certain embodiments, the biologically active polypeptide or protein is an antibody.

[0069] In certain embodiments, a first linker is connected to a second linker, and the combined linkers (i.e., a composite linker) connects at least a first group and a second group. A composite linker of the present disclosure can contain 2, 3, 4, 5, 6, 7, 8, 9, 10 or more linker groups. In a non- limiting example, a first, second and third linker group are joined together to provide a composite linker that can connect a first group (e.g., a drug) to at least one other group, such as a reactive moiety and / or a biologically active polypeptide or protein (e.g., an antibody). In certain embodiments, the biologically active polypeptide or protein (e.g., antibody) contains an engineered amino acid to facilitate conjugation.WSGR Docket No.: 58964-801.601

[0070] In certain embodiments, a linker is linear. In certain embodiments, a linker is branched.

[0071] In certain embodiments, the disclosure concerns polymers such as a bifunctional polymer. A “bifunctional polymer”, also referred to as a “bifunctional linker”, refers to a polymer comprising two functional groups that are capable of reacting specifically with other moieties to form covalent or non-covalent linkages. Many procedures and linker molecules for attachment of various compounds to peptides are known. A “multi-functional polymer” also referred to as a “multi- functional linker”, refers to a polymer comprising two or more functional groups that are capable of reacting with other moieties.

[0072] As used herein, the term “amino acid” generally refers to naturally occurring and non- natural or unnatural amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally encoded amino acids are the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine) and pyrolysine and selenocysteine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, by way of example only, an α-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and a functional R group. Such analogs may have modified R groups (by way of example, norleucine) or may have modified peptide backbones while still retaining the same basic chemical structure as a naturally occurring amino acid. Non-limiting examples of amino acid analogs include homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Amino acids may be referred to herein by either their name, their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Additionally, nucleotides, may be referred to by their commonly accepted single-letter codes.

[0073] As used herein, the term “amino or carboxy terminus modification group” generally refers to any molecule that can be attached to a terminal amine group or terminal carboxy group, respectively. By way of example, such terminal amine groups or terminal carboxy groups may be at the end of polymeric molecules, wherein such polymeric molecules include, but are not limited to, polypeptides, polynucleotides, and polysaccharides. Terminus modification groups include but are not limited to, various water-soluble polymers, peptides or proteins. By way of example only, terminus modification groups include polyethylene glycol or serum albumin. Terminus modification groups may be used to modify therapeutic characteristics of the polymeric molecule, including but not limited to increasing the serum half-life of peptides, polypeptides or proteins.WSGR Docket No.: 58964-801.601

[0074] As used herein, the term “conjugate” generally refers to a compound disclosed herein linked to a biologically active polypeptide or protein, such as a multispecific antibody, via a linker. In some cases, the linker can be a cleavable linker. In some cases, the linker can be a non-cleavable linker.

[0075] The term “drug,” as used herein, refers to any substance used in the prevention, diagnosis, alleviation, treatment, or cure of a disease or condition such as cancer, including but not limited to oral, colorectal, gastric, esophageal, hepatocellular, non-small-cell-lung (NSCL), small-cell lung (SCL), ovarian, breast including triple-negative breast, prostate, pancreatic, head and neck, squamous, renal, bladder, cervical, endometrial, thyroid, and glioblastoma cancer.

[0076] The term “drug-to-antibody ratio” (“DAR”) as used herein refers to the average (mean) number of drugs that are conjugated to an antibody in an antibody-drug conjugate (ADC) or multispecific multi-drug antibody-drug conjugate (MMADC) composition. The DAR value indicates the amount of “payload” (e.g., drug or drug-linker) that is loaded onto an antibody and can be delivered to a target (e.g., cell or diseased tissue). DAR can be determined by methods known to a person of ordinary skill in the art, for example, LC-MS (e.g., see Tang, Y. et al., Real-Time Analysis on Drug-Antibody Ratio of Antibody-Drug Conjugates for Synthesis, Process Optimization and Quality Control, Sci Rep 7, 7763 (2017). doi: 10.1038 / s41598-017-08151-2; and Chen, Y. Drug- to-antibody ratio (DAR) by UV / Vis spectroscopy, Methods Mol. Biol., 2013;1045:267-73. doi: 10.1007 / 978-1-62703-541-5_16). In a non-limiting example, an ADC or MMADC can have a population distribution of 25% of drug-loaded antibody, wherein the drug load is two (2) drugs per antibody; 20% of drug-loaded antibody, wherein the drug load is three (3) drugs per antibody; and 55% of drug-loaded antibody, wherein the drug load is four (4) drugs per antibody; thus, in this example, DAR is [(0.25 x 2) + (0.20 x 3) + (0.55 x 4)] = 3.30.

[0077] 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.

[0078] 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 canWSGR Docket No.: 58964-801.601 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 tumor size, decrease 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.

[0079] 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.

[0080] The term “agent” refers to a therapeutically active substance, in the present case a multispecific binding moiety of the present disclosure, or a pharmaceutical composition of the present disclosure.

[0081] The term “pharmaceutically acceptable” as used herein generally refers to a form of the compound that is safe for administration to a subject. For example, a free base, a salt form, a solvate, a hydrate, a prodrug or derivative form of a compound described herein, which has been approved for mammalian use, via oral ingestion or any other route of administration, by a governing authority or regulatory agency, such as the Food and Drug Administration (FDA) of the United States, is pharmaceutically acceptable.

[0082] As used herein, "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded.

[0083] 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. Multispecific Multi-Drug Antibody-Drug Conjugate (MMADC)

[0084] One of the objects of the present disclosure is to provide a new pharmaceutical agent for the treatment of human disease, including for the treatment of cancer. This object is met by the provision of a novel multispecific multi-drug antibody-drug conjugate (MMADC) format, and pharmaceutical compositions comprising this format.

[0085] Disclosed herein is a new ADC format: a multispecific multi-drug antibody-drug conjugate (MMADC) comprising: - a multispecific antibody that binds to two or more different targets; - a first linker connecting a first drug to the multispecific antibody at a first conjugation site; andWSGR Docket No.: 58964-801.601 - a second linker connecting a second drug to the multispecific antibody at a second conjugation site; wherein the first drug is different from the second drug.

[0086] The four components of the MMADC are the antibody, the linker, the drug (or payload), and the conjugation site, each of which are described below.

[0087] In some embodiments, a multispecific multi-drug antibody-drug conjugate (MMADC) is administered to a subject in need thereof, wherein the MMADC comprises a multispecific antibody; a first linker connecting a first drug to the multispecific antibody at a first conjugation site; and a second linker connecting a second drug to the multispecific antibody at a second conjugation site; wherein the first drug is different from the second drug. The first and second drugs can independently be any drugs described herein.

[0088] In some embodiments, the multispecific multi-drug antibody-drug conjugate (MMADC) comprising: a multispecific antibody; a first linker connecting a first drug to the multispecific antibody at a first conjugation site; and a second linker connecting a second drug to the multispecific antibody at a second conjugation site; wherein the first drug and the second drug are each independently a microtubule inhibitor, a topoisomerase inhibitor, a DNA-damaging agent, a DNA damage repair inhibitor, a chemotherapeutic agent, or an RNA polymerase inhibitor, wherein the first drug is different from the second drug. Antibodies and Antibodies Sequences

[0089] Multispecific antibodies suitable for use in an MMADC of the present disclosure include any multispecific antibody that allows for conjugation of at least two different drugs. In certain embodiments, the multispecific antibody comprises at least two or three, in particular two or three, different binding domains and an Fc region. The advantage of an Fc region is that can provide conjugation sites for linkage with drugs. In certain embodiments, the multispecific antibody comprises at least two or three, in particular two or three, different binding domains that comprise at least one light chain constant region. In certain embodiments, each binding domain comprises a light chain constant region. The advantage of a light chain constant region is that it can provide conjugation sites for linkage with drugs.

[0090] Multispecific antibodies, or multispecific antibody formats, suitable for use in an MMADC of the present disclosure are for instance described in WO 2013 / 157953 and WO 2013 / 157954.

[0091] In certain embodiments, the multispecific antibody is a bispecific antibody or a trispecific antibody. In certain embodiments, the multispecific antibody is a trispecific antibody. In certain embodiments, the trispecific antibody binds to three different targets. In certain embodiments, the multispecific antibody is a bispecific antibody. In certain embodiments, the bispecific antibody bindsWSGR Docket No.: 58964-801.601 to two different targets. In certain embodiments, the targets are independently EGFR, c-MET, LGR5, HER2, and HER3. In certain embodiments, the targets are independently EGFR, c-MET, and LGR5. In certain embodiments, the multispecific antibody is a multispecific IgG1 antibody.

[0092] In certain embodiments, the multispecific antibody comprises a first heavy chain, a second heavy chain, a first light chain, and a second light chain.

[0093] In certain embodiments, the present disclosure provides a multispecific multi-drug 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.

[0094] In certain embodiments, the present disclosure provides a multispecific multi-drug 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. Such substitution is made with respect to the wildtype sequence of the 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).

[0095] 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 multispecific antibody comprises a first and a second CH3 domain, wherein the first CH3 domain comprises amino acid substitution T366K and L351K and the second CH3 domain comprises amino acid substitution L351D and L368E.

[0096] In certain embodiments, the first CH3 domain or the CH3 domain of the first heavy chain comprises amino acid substitutions T366K and L351K.WSGR Docket No.: 58964-801.601

[0097] In certain embodiments, the second CH3 domain or the CH3 domain of the second heavy chain comprises amino acid substitution L351D and Y349E.

[0098] In certain embodiments, the second CH3 domain or the CH3 domain of the second heavy chain comprises amino acid substitution L351D and Y349D.

[0099] In certain embodiments, the second CH3 domain or the CH3 domain of the second heavy chain comprises amino acid substitution L351D and L368E.

[0100] 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.

[0101] 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.

[0102] 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.

[0103] In certain embodiments, the first CH3 domain or the 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.

[0104] In certain embodiments, the first heavy chain has SEQ ID NO: 257 the second heavy chain has SEQ ID NO: 258, and the first light chain and the second light chain have SEQ ID NO: 259. In certain embodiments, the first heavy chain has SEQ ID NO: 260, the second heavy chain has SEQ ID NO: 261, and the first light chain and the second light chain have SEQ ID NO: 259. In some embodiments, the MMADC is selected from Table 3. In some embodiments, the MMADC is selected from Table 4.

[0105] In certain embodiments, the present disclosure provides a multispecific multi-drug 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.

[0106] In certain embodiments, the present disclosure provides a multispecific multi-drug 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, andWSGR Docket No.: 58964-801.601 the CH3 domain of the second heavy chain comprises a negatively charged amino acid residue at positions 368 according to the EU numbering system.

[0107] 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.

[0108] Further multispecific multimer formats suitable for use in an MMADC of the present disclosure are for instance described in WO 2019 / 190327.

[0109] In certain embodiments, the antibody of the MMADC 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.

[0110] In certain embodiments, the multispecific antibody is capable of binding two or more targets or epitopes selected from the group consisting of cytokines, cell surface proteins, enzymes and receptors. In certain embodiments, the multispecific antibody is capable of modulating a biological function of one or more targets. In certain embodiments, the multispecific antibody is capable of neutralizing one or more targets.

[0111] 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.

[0112] 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 moreWSGR Docket No.: 58964-801.601 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.

[0113] 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. 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 variant 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.

[0114] In certain embodiments, an antibody of an antibody-drug conjugate antibody 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.

[0115] 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. 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 CH1WSGR Docket No.: 58964-801.601 region comprising a variation of an amino acid selected from D148, Y149, V154, N159, Al72, 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.

[0116] 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.

[0117] A “Fab” typically means a binding domain comprising a heavy chain variable region, a light chain variable region, a CH1 and a CL region.

[0118] An “Fc region” typically comprises a hinge, CH2, and CH3 region. A suitable hinge, CH2, and CH3 region include any hinge, CH2, and CH3 of any IgG isotype and combinations thereof, and include, but are not limited to, those as described herein. The Fc region mediates effector functions of an antibody, such as complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cell phagocytosis (ADCP). Depending on the therapeutic antibody or Fc fusion protein application, it may be desired to either reduce or increase the effector function.

[0119] In certain embodiments, a MMADC of the present disclosure has Fc effector function. In certain embodiments, a MMADC of the present disclosure has enhanced Fc effector function. In certain embodiments, a MMADC of the present disclosure exhibits antibody-dependent cell- mediated cytotoxicity (ADCC). A binding moiety, such as an antibody, can be engineered to enhance the ADCC activity (for review, see Kubota T et al. Cancer Sci.2009;100(9):1566-72). For instance, ADCC activity of an antibody can be improved when the antibody itself has a low ADCC activity, by slightly modifying the constant region of the antibody (Junttila TT. et al. Cancer Res. 2010;70(11):4481-9). Changes are sometimes also made to improve storage or production or toWSGR Docket No.: 58964-801.601 remove C-terminal lysines (Kubota T et al. Cancer Sci.2009;100(9):1566-72). Another way to improve ADCC activity of an antibody is by enzymatically interfering with the glycosylation pathway resulting in a reduced fucose (von Horsten HH. et al. Glycobiology.2010;20(12):1607-18). Alternatively, or additionally, multiple other strategies can be used to achieve ADCC enhancement, for instance including glycoengineering (Kyowa Hakko / Biowa, GlycArt (Roche) and Eureka Therapeutics) and mutagenesis, all of which seek to improve Fc binding to low-affinity activating FcγRIIIa, and / or to reduce binding to the low affinity inhibitory FcγRIIb. In certain embodiments, a MMADC of the present disclosure exhibits enhanced antibody-dependent cell-mediated cytotoxicity (ADCC). In certain embodiments, a MMADC of the present disclosure is afucosylated.

[0120] In certain embodiments, the Fc region of the MMADC has reduced immune cell effector function, in particular reduced ADCC and / or ADCP activity. In certain embodiments, a MMADC of the present disclosure has reduced Fc-receptor interaction or reduced C1q binding. In certain embodiments, a MMADC of the present disclosure exhibits reduced ADCC and / or ADCP. A binding moiety, such as an antibody, can be engineered to reduce the ADCC and / or ADCP activity (Liu R, et. al. Fc-Engineering for Modulated Effector Functions-Improving Antibodies for Cancer Treatment. Antibodies (Basel).2020 Nov 17;9(4):64). For instance, ADCC and / or ADCP activity of an antibody can be reduced by modifying the CH2 and / or lower hinge region of an IgG antibody, such that the interaction of the antibody to a Fc-gamma receptor is reduced.

[0121] 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 c-MET. 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.

[0122] 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.

[0123] 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, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, or 217. 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 notWSGR Docket No.: 58964-801.601 comprise any amino acid variations. In certain embodiments, the amino acid variation is a conservative amino acid substitution.

[0124] 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), Gly (G); (3) aliphatic: Ala (A), Val (V), Leu (L), Ile (I), Gly (G), Met (M), Pro (P); (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), Phe (F), Trp (W), Tyr (Y); (2) neutral hydrophilic: Ser (S), Thr (T), Asn (N), Gln (Q); (3) acidic hydrophilic: Asp (D), Glu (E); (4) basic hydrophilic: 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).

[0125] 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.

[0126] 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 heavyWSGR Docket No.: 58964-801.601 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).

[0127] 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; 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; 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: 30, SEQ ID NO: 31, and SEQ ID NO: 32, 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: 34, SEQ ID NO: 35, and SEQ ID NO: 36, 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: 38, SEQ ID NO: 39, and SEQ ID NO: 40, 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: 42, SEQ ID NO: 43, and SEQ ID NO: 44, respectively;WSGR Docket No.: 58964-801.601 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: 46, SEQ ID NO: 47, and SEQ ID NO: 48, respectively; j) 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; k) 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; l) 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; m) 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; or n) 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: 218, SEQ ID NO: 219, and SEQ ID NO: 220, respectively.

[0128] 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.

[0129] In certain embodiments, the variable domain of the antibody drug conjugate that binds to c- MET 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: 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169, 173, 177, 181, 185, 189, 193, 197, 201, 205, 209, or 213. 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.

[0130] In certain embodiments, the variable domain of the antibody drug conjugate that binds to c- MET comprises a heavy chain variable region comprising:WSGR Docket No.: 58964-801.601 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: 66, SEQ ID NO: 67, and SEQ ID NO: 68, 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: 70, SEQ ID NO: 71, and SEQ ID NO: 72, 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: 74, SEQ ID NO: 75, and SEQ ID NO: 76, 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: 78, SEQ ID NO: 79, and SEQ ID NO: 80, 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: 82, SEQ ID NO: 83, and SEQ ID NO: 84, 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: 86, SEQ ID NO: 87, and SEQ ID NO: 88, 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: 90, SEQ ID NO: 91, and SEQ ID NO: 92, 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: 94, SEQ ID NO: 95, and SEQ ID NO: 96, respectively; 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: 98, SEQ ID NO: 99, and SEQ ID NO: 100, respectively; j) 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: 102, SEQ ID NO: 103, and SEQ ID NO: 104, respectively; k) 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: 106, SEQ ID NO: 107, and SEQ ID NO: 108, respectively;WSGR Docket No.: 58964-801.601 l) 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: 110, SEQ ID NO: 111, and SEQ ID NO: 112, respectively; m) 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: 114, SEQ ID NO: 115, and SEQ ID NO: 116, respectively; n) 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: 118, SEQ ID NO: 119, and SEQ ID NO: 120, respectively; o) 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: 122, SEQ ID NO: 123, and SEQ ID NO: 124, respectively; p) 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: 126, SEQ ID NO: 127, and SEQ ID NO: 128, respectively; q) 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: 130, SEQ ID NO: 131, and SEQ ID NO: 132, respectively; r) 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: 134, SEQ ID NO: 135, and SEQ ID NO: 136, respectively; s) 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: 138, SEQ ID NO: 139, and SEQ ID NO: 140, respectively; t) 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: 142, SEQ ID NO: 143, and SEQ ID NO: 144, respectively; u) 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: 146, SEQ ID NO: 147,and SEQ ID NO: 148, respectively; v) 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: 150, SEQ ID NO: 151, and SEQ ID NO: 152, respectively;WSGR Docket No.: 58964-801.601 w) 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: 154, SEQ ID NO: 155, and SEQ ID NO: 156, respectively; x) 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: 158, SEQ ID NO: 159, and SEQ ID NO: 160, respectively; y) 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: 162, SEQ ID NO: 163, and SEQ ID NO: 164, respectively; z) 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: 166, SEQ ID NO: 167, and SEQ ID NO: 168, respectively; aa) 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: 170, SEQ ID NO: 171, and SEQ ID NO: 172, respectively; bb) 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: 174, SEQ ID NO: 175, and SEQ ID NO: 176, respectively; cc) 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: 178, SEQ ID NO: 179, and SEQ ID NO: 180, respectively; dd) 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: 182, SEQ ID NO: 183, and SEQ ID NO: 184, respectively; ee) 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: 186, SEQ ID NO: 187, and SEQ ID NO: 188, respectively; ff) 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: 190, SEQ ID NO: 191, and SEQ ID NO: 192, respectively; gg) 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: 194, SEQ ID NO: 195, and SEQ ID NO: 196, respectively;WSGR Docket No.: 58964-801.601 hh) 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: 198, SEQ ID NO: 199, and SEQ ID NO: 200, respectively; ii) 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: 202, SEQ ID NO: 203, and SEQ ID NO: 204, respectively; jj) 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: 206, SEQ ID NO: 207, and SEQ ID NO: 208, respectively; kk) 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: 210, SEQ ID NO: 211, and SEQ ID NO: 212, respectively; or ll) 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: 214, SEQ ID NO: 215, and SEQ ID NO: 216, respectively.

[0131] 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.

[0132] 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: 221, 225, 229, 233, 237, 241, 245, 249, or 253. 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.

[0133] 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: 222, SEQ ID NO: 223, and SEQ ID NO: 224, respectively;WSGR Docket No.: 58964-801.601 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: 226, SEQ ID NO: 227, and SEQ ID NO: 228, 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: 230, SEQ ID NO: 231, and SEQ ID NO: 232, 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: 234, SEQ ID NO: 235, and SEQ ID NO: 236, 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: 238, SEQ ID NO: 239, and SEQ ID NO: 240, 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: 242, SEQ ID NO: 243, and SEQ ID NO: 244, 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: 246, SEQ ID NO: 247, and SEQ ID NO: 248, 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: 250, SEQ ID NO: 251, and SEQ ID NO: 252, 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: 254, SEQ ID NO: 255, and SEQ ID NO: 256, respectively.

[0134] 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.

[0135] In certain embodiments, the variable domain of the antibody-drug conjugate that binds to EGFR and / or c-MET 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-conservativeWSGR Docket No.: 58964-801.601 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 c-MET 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, AAS, 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.

[0136] 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: 62, HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 63, and HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 64; - a variable domain that binds to c-MET comprising a heavy chain variable region comprising HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 154, HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 155, and HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 156; and wherein the variable domain of both the variable domain that binds to EGFR and the variable domain that binds to c-MET 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 AAS, and LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 4.

[0137] 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: 30, HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 31, and HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 32; - 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: 246, HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 247, and HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 248; andWSGR Docket No.: 58964-801.601 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 AAS, and LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 4.

[0138] 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, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, or 217, or having at least 80%, at least 85%, at least 90%, or at least 95%, sequence identity thereto.

[0139] The term “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.

[0140] 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).

[0141] 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). 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.WSGR Docket No.: 58964-801.601

[0142] 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.

[0143] In certain embodiments, the variable domain of the antibody-drug conjugate that binds to c- MET comprises a heavy chain variable region comprising any one of the amino acid sequences as set forth in SEQ ID NO: 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169, 173, 177, 181, 185, 189, 193, 197, 201, 205, 209, or 213, or having at least 80%, at least 85%, at least 90%, or at least 95%, sequence identity thereto.

[0144] 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: 221, 225, 229, 233, 237, 241, 245, 249, or 253, or having at least 80%, at least 85%, at least 90%, or at least 95%, sequence identity thereto.

[0145] In certain embodiments, the variable domain of the antibody-drug conjugate that binds to EGFR and / or c-MET 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.

[0146] 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: 61, or having at least 80%, at least 85%, at least 90%, or at least 95%, sequence identity thereto; - a variable domain that binds to c-MET comprising a heavy chain variable region comprising the amino acid sequence as set forth in SEQ ID NO:153, 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 c-MET 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.

[0147] 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: 29, or having at least 80%, at least 85%, at least 90%, or at least 95%, sequence identity thereto;WSGR Docket No.: 58964-801.601 - 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: 245, 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.

[0148] 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.

[0149] 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: 61; - a variable domain that binds to c-MET comprising a heavy chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 153; and wherein the variable region of both the variable domain that binds to EGFR and the variable domain that binds to c- MET comprise a light chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 1.

[0150] 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: 29; - 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: 245; 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.WSGR Docket No.: 58964-801.601

[0151] 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 c-MET or EGFR and / or LGR5.

[0152] 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 c-MET, or 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 c-MET, or EGFR or 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.

[0153] 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. Linkers

[0154] Linkers known and available in the industry are capable of use in the present disclosure of MMADC. 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,WSGR Docket No.: 58964-801.601 particularly useful for their relative stability in systemic circulation and the ability to efficiently release the drug in tumor.

[0155] In certain embodiments, the second linker is the same as the first linker, and the second conjugation site is the same as the first conjugation site. In this configuration, a single linker (the first linker) connects with two different drugs (the first drug and the second drug) at different sites on the single linker, and connects with the multispecific antibody at a single conjugation site (the first conjugation site). In this configuration, the link is multivalent and comprises three or more chemically reactive groups to connect with the first drug, the second drug, and the multispecific antibody, respectively.

[0156] In certain embodiments, the first linker is different from the second linker, and the first conjugation site is different from the second conjugation site. In this configuration, the first linker and the second linker are two distinct chemical entities, and connect with the multispecific antibody at different conjugation sites (the first conjugation site and the second conjugation site), respectively. For example, lysine can be the first conjugation site while cysteine can be the second conjugation site. One linker can selectively react with the lysine in the presence of the cysteine. Another linker can selectively react with the cysteine in the presence of the lysine. By connecting with different drugs to different linkers, different drugs can be connected with the antibody comprising cysteine and lysine in a site-specific fashion. Additionally, different conjugation sites can be engineered into the heavy chains, each is bio-orthogonal to the other, thereby allowing site-specific introduction of different drugs to the multispecific antibody. For example, different non-natural amino acids can be engineered into different heavy chains. In certain embodiments, each of the first linker and the second linker may independently 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-WSGR Docket No.: 58964-801.601 (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 carboxyl groups 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 a wide variety of active carboxyl groups (e.g., esters) where the 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 a-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.

[0157] In certain embodiments, the first linker or the second linker is attached to the multispecific antibody via a lysine residue on the antibody. In certain embodiments, the first linker or the second 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-WSGR Docket No.: 58964-801.601 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).

[0158] In certain embodiments, the first linker or the second 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 interspersed 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.

[0159] 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-dibenzocyclooctyneWSGR Docket No.: 58964-801.601 (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.

[0160] In certain embodiments, the first linker or the second 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 multispecific antibody or an antigen-binding fragment thereof. In certain embodiments, the non- cleavable linker is covalently attached to the first drug or the second drug, respectively. 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.

[0161] In certain embodiments, the first linker and the second linker is independently connected with the multispecific antibody via a reaction using a reactive group of:, ,WSGR Docket No.: 58964-801.601LG is halide, triflate, fluorosulfonate, tosylate, mesylate, or besylate; RAis 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; RBis 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, R7and R8is 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 R9is 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-C4alkyl), –OH, –O(C1-C4alkyl), –O(C1-C4haloalkyl), –O(C2-C4alkylene)-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

[0162] In certain embodiments, the reactive group is:WSGR Docket No.: 58964-801.601

[0163] In certain embodiments, the first linker comprises a first reactive group to connect with the antibody and the second linker comprises a second reactive group to connect with the antibody. In certain embodiments, the first reactive group and the second reactive group are the same. In certain embodiments, the first reactive group and the second reactive group are different.

[0164] In certain embodiments, the first reactive group can be orthogonal to the second reactive group in terms of reactivity towards the antibody. For example, the second reactive group can be masked such that the masked second reactive group does not react with an antibody comprising an additional reactive group tailored for the first reactive group to react with. Alternatively, different and orthogonal conjugation reactions (e.g., maleimide conjugation, amide formation, click chemistry, displacement reaction) can be relied upon to conjugate the drug-linker combinations with the antibody. For example, an orthogonal pair of reactive groups can be (1) a click chemistry reactive group (e.g., azide or alkyne, including a strained cyclooctyne); and (2) a thiol group (to react with maleimide conjugation). Another orthogonal pair of reactive groups can be (1) a click chemistry reactive group (e.g., azide or alkyne, including a strained cyclooctyne); and (2) a reactive group (e.g., an activated acyl group) to react with a lysine. With the aid of the orthogonal pair of reactive group, the two different drug-linker combinations can selectively and sequentially react with the antibody and form covalent bonds, respectively, in a controlled manner. For example, an antibody comprising a lysine and a cysteine residues can selectively react with a maleimide reagent followed by reacting with an activated acyl group with the amine on lysine. An antibody comprising an azide moiety and a cysteine residue can selectively react with a maleimide reagent and a strained cyclooctyne.

[0165] In certain embodiments, each of the first linker and the second linker independently comprises *–(L2)p–(L1)s–; wherein: * denotes a connection leading to the first drug or the second drug; each L1and L2is independently a bond, –O–, –S–, –NH–, –NRD–, –C(=O)–, –C(=O)O–, –WSGR Docket No.: 58964-801.601 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; RDis independently –CR3R4R5; each R3, R4, and R5is 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 R9and / 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 R9is 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, p and s is independently an integer of 1-12.

[0166] In certain embodiments, each of the first linker and the second linker further independently comprises a spacer connected to either end of *–(L2)p–(L1)s–, 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,WSGR Docket No.: 58964-801.601 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.

[0167] In certain embodiments, each of the spacer is independently , 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

[0168] In certain embodiments, each of the spacer is independently, wherein: each Y1 and Y2 is independently a bond, O, S, or NRE; REis 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 REtogether 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.

[0169] In certain embodiments, the linker is a branched linker to connect to two different drugs and the antibody. In certain embodiments, the branched linker comprises, wherein denotes a connection leading to the drug, whereindenotes a connection leading to the antibody, wherein each of e, d and f is independently 0, 1, 2, 3, 4, 5, 6, 7 or 8, with the proviso that at least one of e, d, and f is not 0.WSGR Docket No.: 58964-801.601 Drugs and Linker-Drug Combinations

[0170] In certain embodiments, the multispecific multi-drug antibody-drug conjugate (MMADC) of the present disclosure comprises two different drugs, each of which is coupled to the antibody of the MMADC via a linker. The moiety of a linker-connected drug is herein referred to as linker-drug combination.

[0171] In certain embodiments, the drug is independently a tubulin inhibitor, a deoxynucleic acid (DNA) damaging agent, a chemotherapeutic agent, a protein degrader, or an immune stimulant.

[0172] In certain embodiments, the drugs are selected from a microtubule inhibitor, a topoisomerase inhibitor, a DNA-damaging agent, a DNA damage repair inhibitor, a chemotherapeutic agent, or an RNA polymerase inhibitor. In certain embodiments, the first drug and the second drug are each independently a microtubule inhibitor, a topoisomerase inhibitor, a DNA- damaging agent, a DNA damage repair inhibitor, a chemotherapeutic agent, or an RNA polymerase inhibitor, wherein the first drug is different from the second drug. In certain embodiments, the first drug and the second drug are each independently a microtubule inhibitor, a topoisomerase inhibitor, a DNA-damaging agent, a DNA damage repair inhibitor, or an RNA polymerase inhibitor, wherein the first drug is different from the second drug. In certain embodiments, the first drug and the second drug are each independently a microtubule inhibitor, a topoisomerase inhibitor, a DNA-damaging agent, or an RNA polymerase inhibitor, wherein the first drug is different from the second drug.

[0173] In certain embodiments, when the first drug is a microtubule inhibitor, the second drug is a topoisomerase inhibitor, a DNA-damaging agent, a DNA damage repair inhibitor, a chemotherapeutic agent, or an RNA polymerase inhibitor; or when the first drug is a topoisomerase inhibitor, the second drug is a microtubule inhibitor, a DNA-damaging agent, a DNA damage repair inhibitor, a chemotherapeutic agent, or an RNA polymerase; or when the first drug is a DNA- damaging agent, the second drug is a microtubule inhibitor, a topoisomerase inhibitor, a DNA damage repair inhibitor, a chemotherapeutic agent, or an RNA polymerase inhibitor; or when the first drug is a DNA damage repair inhibitor, the second drug is a microtubule inhibitor, a topoisomerase inhibitor, a DNA-damaging agent, a chemotherapeutic agent, or an RNA polymerase inhibitor; or the first drug is a chemotherapeutic agent, the second drug is a microtubule inhibitor, a topoisomerase inhibitor, a DNA-damaging agent, a DNA damage repair inhibitor, or an RNA polymerase inhibitor; or the first drug is an RNA polymerase inhibitor, the second drug is a microtubule inhibitor, a topoisomerase inhibitor, a DNA-damaging agent, a DNA damage repair inhibitor, or a chemotherapeutic agent.

[0174] In certain embodiments, when the first drug is a microtubule inhibitor, the second drug is a different microtubule inhibitor; or when the first drug is a topoisomerase inhibitor, the second drugWSGR Docket No.: 58964-801.601 is a different topoisomerase inhibitor; or when the first drug is a DNA-damaging agent, the second drug is a different DNA-damaging agent; or when the first drug is a DNA damage repair inhibitor, the second drug is a different DNA damage repair inhibitor; or when the first drug is a chemotherapeutic agent, the second drug is a different chemotherapeutic agent; or when the first drug is an RNA polymerase inhibitor, the second drug is a different RNA polymerase inhibitor.

[0175] In certain embodiments, the first drug is a microtubule inhibitor and the second drug is a topoisomerase inhibitor. In certain embodiments, the first drug is a microtubule inhibitor and the second drug is a DNA-damaging agent. In certain embodiments, the first drug is a microtubule inhibitor and the second drug is a DNA damage repair inhibitor. In certain embodiments, the first drug is a microtubule inhibitor and the second drug is a chemotherapeutic agent. In certain embodiments, the first drug is a microtubule inhibitor and the second drug is an RNA polymerase inhibitor. In certain embodiments, the first drug is a topoisomerase inhibitor and the second drug is a DNA-damaging agent. In certain embodiments, the first drug is a topoisomerase inhibitor and the second drug is a DNA damage repair inhibitor. In certain embodiments, the first drug is a topoisomerase inhibitor and the second drug is a chemotherapeutic agent. In certain embodiments, the first drug is a topoisomerase inhibitor and the second drug is an RNA polymerase inhibitor.

[0176] In certain embodiments, the first drug is a microtubule inhibitor and the second drug is a different microtubule inhibitor. In certain embodiments, the first drug is a topoisomerase I inhibitor and the second drug is a topoisomerase II inhibitor. In certain embodiments, the first drug is DNA- damaging agent and the second drug is a different DNA-damaging agent. In certain embodiments, the first drug is a DNA damage repair inhibitor and the second drug is a different DNA damage repair inhibitor. In some embodiments, the first drug is a chemotherapeutic agent and the second drug is a different chemotherapeutic agent. In certain embodiments, the first drug is an RNA polymerase inhibitor, and the second drug is a different RNA polymerase inhibitor.

[0177] In certain embodiments, the drug is a tubulin inhibitor that is an auristatin or derivative thereof, a tubulysin or derivative thereof, or a maytansine or derivative thereof.

[0178] In certain embodiments, the drug 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 DNA damaging agent is duocarmycins or pyrrolobenzodiazepines. In certain embodiments, the DNA damaging agent is duocarmycins. In certain embodiments, the DNA damaging agent is pyrrolobenzodiazepines.

[0179] In certain embodiments, the drug is a DNA damage repair response inhibitor. In certain embodiments, the DNA damage repair response inhibitor is a PARP inhibitor, an ATM inhibitor, an ATR inhibitor, an NHEJ inhibitor, or a MRN inhibitor. In certain embodiments, the DNA damageWSGR Docket No.: 58964-801.601 repair response inhibitor is LY294002, KU55922, CP466722, AZD6738, PFM39, MPF01, PFM03, INO-1001, E7016, CEP-9722, tuvusertib, M9466, lartesertib, peposertib, olaparib, talazoparib, veliparib, rucaparib, or niraparib. In certain embodiments, the DNA damage repair response inhibitor is tuvusertib, M9466, lartesertib, peposertib, olaparib, talazoparib, veliparib, rucaparib, or niraparib. In certain embodiments, the DNA damage repair response inhibitor is olaparib, talazoparib, veliparib, rucaparib, or niraparib.

[0180] In certain embodiments, the drug is monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), tubulysin A, maytansine, ansamitocin P3, mertansine (DM1), ravtansine (DM4), calicheamicin, duocarmycin, topotecan, exatecan, DXd, irinotecan, cisplatin, oxaliplatin, paclitaxel, teniposide, SN38, hexylresorcinal, camptothecin, MM398, etoposide, novobiocin, doxorubicin, nemorubicin, daunorubicin, idarubicin, epipodophyllotoxin, toposide, teniposide, mitoxanthrone, pyrrolobenzodiazepine (PDB), TAS-103, 7-MAD_MDCPT, SN38, SG2199, amanitins, 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 pharmaceutically acceptable salt or derivative thereof. In certain embodiments, the first drug and the second drug are each independently monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), mertansine (DM1), ravtansine (DM4), Dxd, exatecan, doxorubicin, TAS-103, irinotecan, cisplatin, oxaliplatin, paclitaxel, 7-MAD-MDCPT, camptothecin, SN38, SG3199, or Amanitins, or a pharmaceutically acceptable salt or derivative thereof. In certain embodiments, the first drug and the second drug are each independently MMAE, MMAF, DM1, Dxd, doxorubicin, TAS-103, exatecan, cisplatin, or irinotecan.

[0181] In certain embodiments, the first drug and second drug are: MMAE and MMAF; MMAE and DM1; MMAE and Dxd; MMAE and doxorubicin; MMAE and TAS-103; MMAF and DM1; MMAF and Dxd; MMAF and doxorubicin; MMAF and TAS-103; DM1 and Dxd; DM1 and doxorubicin; DM1 and TAS-103; Dxd and doxorubicin; Dxd and exatecan; or cisplatin and irinotecan.

[0182] In certain embodiments, the drug is a chemotherapeutic agent which is an anticancer drug, in particular an anticancer drug approved by a regulatory authority.

[0183] In certain embodiments, the drug is a chemotherapeutic agent which is independently Abraxane, Actinomycin, Alitretinoin, All-trans retinoic acid, Altretamine, Azacitidine, Azathioprine, Belotecan, Bendamustine, Bexarotene, Bleomycin, Bortezomib, Busulfan, Cabazitaxel, Camptothecin, Carboplatin, Carboquone, Carmustine, Capecitabine, Cisplatin, Chlorambucil, Chlormethine, Chlorozotocin, Cyclophosphamide, Cytarabine, Dacarbazine, Daunorubicin, Docetaxel, Doxifluridine, Doxorubicin, Epirubicin, Epothilone, Erlotinib, Etoposide, Exatecan,WSGR Docket No.: 58964-801.601 Fluorouracil, Fotemustine, Gefitinib, Gemcitabine, Gimatecan, Hydroxyurea, Idarubicin, Ifosfamide, Imatinib, Irinotecan, Ixabepilone, Larotaxel, Lomustine, Melphalan, Melphalan flufenamide, Mercaptopurine, Methotrexate, Mitobronitol, Mitomycin C, Mitoxantrone, Nimustine, Nitrosoureas, Oxaliplatin, Paclitaxel, Pemetrexed, Pipobroman, Ranimustine, Romidepsin, Semustine, Streptozotocin, Tafluposide, Taxotere, Temozolomide, Tesetaxel, Teniposide, Thiotepa, Tioguanine, Topotecan, Treosulfan, Tretinoin, Triaziquone, Triethylenemelamine, Valrubicin, Vemurafenib, Vinblastine, Vincristine, Vindesine, Vinorelbine, Vismodegib, or Vorinostat, or a derivative thereof.

[0184] 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.

[0185] 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.

[0186] 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.

[0187] MMAF is also 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.

[0188] 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 beWSGR Docket No.: 58964-801.601 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).

[0189] Exatecan (or DXd, or DX-8951f) is a synthetic derivative 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.

[0190] 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).

[0191] 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’-acetyl- phenoxy)butanoic acid moiety, which linker-drug combination is referred to as ozogamicin.

[0192] 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 an 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.WSGR Docket No.: 58964-801.601

[0193] The drug of an antibody-drug conjugate of the present disclosure can be conjugated to a multispecific antibody by any suitable linker. In certain embodiments, the multispecific multi-drug antibody-drug conjugate comprises a linker-drug combination selected from the group consisting of vedotin, mafodotin, deruxtecan, DM1, tesirine, govitecan, ozogamicin, saratolacan, and soravtansine.

[0194] 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.

[0195] 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.

[0196] In certain embodiments, the drug reacts with a linker via a reactive group on the drug. In certain embodiments, the reactive group on the drug is –OH, –SH, –NH2, –NRH (R is C1-C6 alkyl or C3-C6 cycloalkyl), a carbonyl, –PO3H2, or –CO2H. In certain embodiments, the first drug comprises a first reactive group and the second drug comprises a second reactive group. In certain embodiments, the first reactive group and the second reactive group are the same. In certain embodiments, the first reactive group and the second reactive group are different.

[0197] In certain embodiments, the first linker and the second linker is independently connected to the multispecific antibody via a reaction using the reactive group selected from:

[0198] In certain embodiments, the branched linker comprises the following structure:WSGR Docket No.: 58964-801.601 wherein denotes a connection leading to the drug, wherein denotes a connection leading to the antibody, wherein each of e, d and f is independently 0, 1, 2, 3, 4, 5, 6, 7 or 8, wherein at least one of e, d, and f is not 0.

[0199] In certain embodiments, the first linker and second linker independently comprises maleimidocaproyl-valine-citrulline-paminobenzyloxycarbonyl (MC-VCP) or glutamic acid-valine- citrulline (GluValCit). In certain embodiments, the first linker and the second linker further independently comprises a first spacer and a second spacer. In certain embodiments, the first spacer and second spacer independently comprises a polyethylene glycol (PEG).

[0200] In certain embodiments, the drugs can have different drug-to antibody (DAR) ratios. In certain embodiments, the first drug has a first drug-to-antibody (DAR) ratio of about 1.5 to about 8. In certain embodiments, the first drug has a first DAR ratio of about 2 to about 6. In certain embodiments, the first drug has first DAR ratio of about 2, about 4, about 6 or about 8. In certain embodiments, the second drug has a second drug-to-antibody (DAR) ratio of about 0.7 to about 9.1. In certain embodiments, the second DAR ratio is about 1 to about 9. In certain embodiments, the second DAR ratio is about 2 to about 8. In certain embodiments, the second DAR ratio is about 2 to about 6. In certain embodiments, the first drug has the second DAR ratio of about 2, about 4, about 6 or about 8. Conjugation Sites

[0201] In certain embodiments, the conjugation site on the antibody is an amino acid. In certain embodiments, the first conjugate site and the second conjugation site is independently a reactive amino acid of the multispecific antibody. In certain embodiments, the conjugation site on the antibody is a side chain on an amino acid. In certain embodiments, the reactive amino acid has the following functional group: –SH, –SeH, –NH2, –CO2H, or –C(=O)–, or a derivative thereof.

[0202] 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.

[0203] In certain embodiments, there are multiple conjugation sites on a multispecific antibody for a particular linker-payload, i.e., "a first conjugation site" means there can be multiple conjugations sites of the same amino acid or the same side chain on an amino acid. In this way, multiple linker- payload can be connect with the same multispecific antibody.

[0204] 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 naturallyWSGR Docket No.: 58964-801.601 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.

[0205] 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.

[0206] 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.

[0207] 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.

[0208] 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.WSGR Docket No.: 58964-801.601

[0209] In certain embodiments, the conjugation sites on the antibody belong to two bioorthogonal reactive groups, each of which can react with a linker-drug combination selectively in the presence of the other conjugation site. For example, the biorthogonal reactive groups can be (i) p- acetylphenylalanine and lysine; (ii) p-acetylphenylalanine and cysteine, (iii) p-acetylphenylalanine and azide; or (iv) p-acetylphenylalanine and alkyne. Synthesis of antibody-drug conjugate

[0210] 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 a multispecific multi-drug 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 a multispecific multi-drug 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 method for producing a multispecific multi-drug antibody-drug conjugate of the present disclosure is based on maleimide conjugation or NHS conjugation.

[0211] Further suitable methods include, but are not limited to, the methods as described herein.

[0212] Payloads can be conjugated to engineered cysteine (Cys) / selenocysteine (sec) residues on an antibody, for instance, by using the THIOMAB / SELENOMAB technologies. Drug-linker compounds that can be used in this method are for instance PNU-159682-Gly3-iodoacetamide and MMAF-nc-MSODA. The method can be performed using sequential dual conjugation, wherein Sec residues are initially reduced and conjugated with PNU-159682, followed by conjugation with MMAF-nc-MSODA after reducing and partially oxidizing the antibody. The method can also be performed with other linkers and / or drugs.WSGR Docket No.: 58964-801.601

[0213] Payloads can be conjugated to an antibody via site-specific enzymatic conjugation using engineered glycan sites, for instance by chemoenzymatic conjugation technology which combines targeted glycosite-specific conjugation with traditional random conjugation. A first payload can be attached to glycans and a second payload can be attached to lysine residues on an antibody. In order to achieve N-glycosylation remodeling, an egg-yolk sialylglycopeptide (SGP) or its modified versions with azido or alkyne tags can be used. Dual drug ADCs can be generated by treatment of azido-tagged antibodies with specific linkers for lysine conjugation and click chemistry reactions. Similarly, glycoengineering technology can be utilized, employing for instance GnT-I and GnT-II (UDP N-Acetyl glucosamine transferases) to attach tri-mannosyl to antibodies.

[0214] Site- specific enzymatic transglutaminase-mediated conjugations can be utilized to improve ADC homogeneity by adjusting the DARs. For example, microbial transglutaminase can be utilized for site-specific linker installation to generate highly uniform ADCs containing a first and a second drug with various defined DARs. Further examples of site-specific enzymatic-mediated conjugation methods include a method for site-specific conjugation utilizing aerobic formylglycine-generating enzymes (FGE). This approach allows for controlled conjugation of two distinct payloads to an antibody, facilitated by for instance a Dibenzocyclooctyne (DBCO) linker.

[0215] Dual payload ADCs can also be generated by combining two single payload ADCs with different linker types. For example, a solid-phase, site-specific conjugation method can be used employing two orthogonal conjugation methodologies: enzymatic transglutaminase- and chemical maleimide conjugation to engineered cysteines. In this method, for instance, an auristatin-based protease cleavable linker-payload (mcValCit-PABC_Aur-0101) and a maleimide-functionalized non- cleavable linked auristatin (mc_Aur-0131) can be used as first and second linker-drug moieties.

[0216] Branched chemical linkers containing two orthogonally masked cysteine residues can be used for preparing homogeneous ADCs with multiple payloads. This approach involves sequential unmasking of cysteine residues on the multiplexing drug carrier using orthogonal protection, allowing for site-specific conjugation of each drug into the native, non-engineered, antibody cysteine residues. For instance, the commonly used auristatin drug-linkers mc-MMAF (1), mc-vc- MMAF (2), and mc-vc-MMAE (3) can be used in this method. Furthermore, branched linkers can be used for attaching multiple payload molecules by microbial transglutaminase-mediated conjugation. A flexible heterotrifunctional engineered branched linker can be used to facilitate the site-specific attachment of two payload molecules onto a single antibody through orthogonal strain-promoted azide–dibenzocyclooctyne compound (DBCO) cycloaddition. These designed branched linkers bear azide and methyltetrazine groups as orthogonal clickable features. Also, a tetrapeptide linker can be used to conjugate two different payloads to achieve synergistic effects. Further linkers that can beWSGR Docket No.: 58964-801.601 used include for instance a cleavable linker, such as Val-Cit for, for instance, MMAE conjugation a non-cleavable linker, such as Succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC)) for, for instance, DM1 conjugation.

[0217] ADCs combining two distinct payloads can be generated by covalent attachment of two different drugs through two distinct conjugation methods: a thiol-maleimide reaction and a Cu(I)- catalyzed alkyne-azide cycloaddition (CuAAC), and by utilizing suitable linkers, such as, maleimide-Val-Cit-PAB for, for instance conjugation of α-amanitin, and azide-PEG4-Val-Cit-PAB for, for instance conjugation of MMAE.

[0218] The Synthemer platform can be used to attach different payloads to a synthetic scaffold in a controlled manner, followed by chemically linking the loaded scaffold to an antibody, thus, ensuring that the payloads are consistently integrated in a predetermined ratio into the scaffold and then into the ADC.

[0219] In certain embodiments, the present disclosure provides a method for producing a multispecific multi-drug antibody-drug conjugate, the method comprising: - providing a multispecific antibody; and - coupling a first drug and a second drug the antibody, wherein the first drug is different from the second drug.

[0220] In certain embodiments, the present disclosure provides a method for producing a multispecific multi-drug antibody-drug conjugate, the method comprising: - providing a multispecific antibody; and - coupling a first drug to the antibody by means of a first conjugation method; and - coupling a second drug to the antibody by means of a second conjugation method.

[0221] In certain embodiments, the present disclosure provides a method for producing a multispecific multi-drug antibody-drug conjugate, the method comprising: - providing a multispecific 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; - coupling a first drug and a second drug the antibody, wherein the first drug is different from the second drug.

[0222] In certain embodiments, the present disclosure provides a method for producing a multispecific multi-drug antibody-drug conjugate, the method comprising:WSGR Docket No.: 58964-801.601 - providing a multispecific 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; - coupling a first drug to the antibody by means of a first conjugation method; and - coupling a second drug to the antibody by means of a second conjugation method.

[0223] In certain embodiments, the present disclosure provides a method for producing a multispecific multi-drug antibody-drug conjugate, the method comprising: - providing a multispecific antibody comprising 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; and - coupling a first drug and a second drug the antibody, wherein the first drug is different from the second drug.

[0224] In certain embodiments, the present disclosure provides a method for producing a multispecific multi-drug antibody-drug conjugate, the method comprising: - providing a multispecific antibody comprising a first CH3 domain and 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; - coupling a first drug to the antibody by means of a first conjugation method; and - coupling a second drug to the antibody by means of a second conjugation method.

[0225] In certain embodiments, the multispecific antibody is a multispecific antibody as described herein.

[0226] In certain embodiments, the method comprises a step of modifying the multispecific antibody to provide for one or more conjugations sites. Said one or more conjugations sites may be in addition to any conjugation sites already present in the antibody. Modifying the multispecific antibody can be done by any method known in the art, and as described herein. Examples of modifications include, but are not limited to, modification of the amino acid sequence of the antibody, introduction of non-natural amino acids at selected positions on the multispecific antibody, selective protection and deprotection of amino acids on or close to the surface of the multispecific antibody, and modification of the glycosylation of one or more amino acid residues of the antibody.WSGR Docket No.: 58964-801.601

[0227] In certain embodiments, the first conjugation method and the second conjugation method are the same. In certain embodiments, the first conjugation method and the second conjugation method are different.

[0228] In certain embodiments, the first conjugation method and the second conjugation method are performed simultaneously, i.e. in the same reaction mixture. In certain embodiments, the first conjugation method and the second conjugation method are performed consecutively, for instance in two separate steps, wherein the first conjugation method is performed prior to the second conjugation method or the second conjugation method is performed prior to the first conjugation method.

[0229] Attachment of the antibody to the drug via a non-cleavable linker can be performed in various ways. For example, one method is to prepare the antibody and the drug separately. Then prepare a linker reagent which has two or more differently activated / activatable groups at its end. Each of the two differently activated / activatable groups can selectively react with the antibody and the drug, respectively, thereby completing the synthesis of the antibody-drug conjugate. For example, one activated / activatable group can be BrCH2C(=O)-, which can react with thiol groups of the antibody, in particular the surface thiol groups, to attach the linker to the antibody. Another activated / activatable group can be an activated carbonyl group such as acyl chloride or mixed anhydride or simply carboxylic acid, which can react with an amino group of the drug. A covalent bond formation reaction can be used to attach the linker to the antibody or the drug.

[0230] Another method is to incorporate the linker group or a fragment thereof during the synthesis of the drug. After the synthesis of the drug is complete, modification of the incorporated linker group or the fragment thereof can lead to the addition / unmask of an activated / activatable group at the free end of the linker. The newly revealed activated / activatable group of the linker can react with a surface reactive group (e.g., a thiol group) of the antibody.Pharmaceutical compositions and methods of use

[0231] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising an effective amount of a MMADC as described herein, and optionally a pharmaceutically acceptable carrier. In certain embodiments, the antibody of the MMADC is a multispecific IgG1 antibody. In certain embodiments, the antibody of the MMADC is a bispecific IgG1 antibody.

[0232] In certain embodiments, the present disclosure provides a MMADC as described herein, and a pharmaceutical composition as described herein, for use in therapy. In certain embodiments, the antibody of the MMADC is a multispecific IgG1 antibody. In certain embodiments, the antibody of the MMADC is a bispecific IgG1 antibody.WSGR Docket No.: 58964-801.601

[0233] In certain embodiments, the present disclosure provides a MMADC as described herein, or the pharmaceutical composition as described herein, for use in the treatment of cancer. In certain embodiments, the antibody of the MMADC is a multispecific IgG1 antibody. In certain embodiments, the antibody of the MMADC is a bispecific IgG1 antibody.

[0234] In certain embodiments, the present disclosure provides a method for treating a disease, comprising administering an effective amount of a MMADC as described herein, or a pharmaceutical composition as described herein, to an individual in need thereof. In certain embodiments, the antibody of the MMADC is a multispecific IgG1 antibody. In certain embodiments, the antibody of the MMADC is a bispecific IgG1 antibody.

[0235] In certain embodiments, the present disclosure provides a method for treating cancer, comprising administering an effective amount of a MMADC as described herein, or a pharmaceutical composition as described herein, to an individual in need thereof. In certain embodiments, the antibody of the MMADC is a multispecific IgG1 antibody. In certain embodiments, the antibody of the MMADC is a bispecific IgG1 antibody.

[0236] 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.

[0237] 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.

[0238] An effective amount can be administered to a subject in one or more administrations.

[0239] 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.

[0240] 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.

[0241] All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.

[0242] 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.WSGR Docket No.: 58964-801.601 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. Amino acids in the constant regions are indicated according to the EU numbering system.

[0243] 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 a MMADC 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.

[0244] 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.WSGR Docket No.: 58964-801.601 Numbered Embodiments

[0245] The following embodiments recite non-limiting permutations of combinations of features disclosed herein. Other permutations of combinations of features are also contemplated. In particular, each of these numbered embodiments is contemplated as depending from or relating to every previous or subsequent numbered embodiment, independent of their order as listed.

[0246] Embodiment 1. A multispecific multi-drug antibody-drug conjugate (MMADC) comprising: - a multispecific antibody that binds to two or more different targets; - a first linker connecting a first drug to the multispecific antibody at a first conjugation site; and - a second linker connecting a second drug to the multispecific antibody at a second conjugation site; wherein the first drug is different from the second drug.

[0247] Embodiment 2. The MMADC according to embodiment 1, wherein each of the first drug and the second drug is independently a tubulin inhibitor, a deoxynucleic acid (DNA) damaging agent, a chemotherapeutic agent, a protein degrader, or an immune stimulant.

[0248] Embodiment 3. The MMADC according to embodiment 2, wherein the tubulin inhibitor is an auristatin or derivative thereof, a tubulysin or derivative thereof, or a maytansine or derivative thereof.

[0249] Embodiment 4. The MMADC according to embodiment 2, wherein the DNA damaging agent 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.

[0250] Embodiment 5. The MMADC according to embodiment 2, wherein the chemotherapeutic agent is an anticancer drug.

[0251] Embodiment 6. The MMADC according to embodiment 1, wherein the first drug and the second drug is independently 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.

[0252] Embodiment 7. The MMADC according to embodiment 2, wherein the chemotherapeutic agent is independently Abraxane, Actinomycin, Alitretinoin, All-trans retinoic acid, Altretamine,WSGR Docket No.: 58964-801.601 Azacitidine, Azathioprine, Belotecan, Bendamustine, Bexarotene, Bleomycin, Bortezomib, Busulfan, Cabazitaxel, Camptothecin, Carboplatin, Carboquone, Carmustine, Capecitabine, Cisplatin, Chlorambucil, Chlormethine, Chlorozotocin, Cyclophosphamide, Cytarabine, Dacarbazine, Daunorubicin, Docetaxel, Doxifluridine, Doxorubicin, Epirubicin, Epothilone, Erlotinib, Etoposide, Exatecan, Fluorouracil, Fotemustine, Gefitinib, Gemcitabine, Gimatecan, Hydroxyurea, Idarubicin, Ifosfamide, Imatinib, Irinotecan, Ixabepilone, Larotaxel, Lomustine, Melphalan, Melphalan flufenamide, Mercaptopurine, Methotrexate, Mitobronitol, Mitomycin C, Mitoxantrone, Nimustine, Nitrosoureas, Oxaliplatin, Paclitaxel, Pemetrexed, Pipobroman, Ranimustine, Romidepsin, Semustine, Streptozotocin, Tafluposide, Taxotere, Temozolomide, Tesetaxel, Teniposide, Thiotepa, Tioguanine, Topotecan, Treosulfan, Tretinoin, Triaziquone, Triethylenemelamine, Valrubicin, Vemurafenib, Vinblastine, Vincristine, Vindesine, Vinorelbine, Vismodegib, or Vorinostat, or a derivative thereof.

[0253] Embodiment 8. The MMADC according to embodiment 1, wherein the first drug is independently 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, and wherein the second drug is independently Abraxane, Actinomycin, Alitretinoin, All- trans retinoic acid, Altretamine, Azacitidine, Azathioprine, Belotecan, Bendamustine, Bexarotene, Bleomycin, Bortezomib, Busulfan, Cabazitaxel, Camptothecin, Carboplatin, Carboquone, Carmustine, Capecitabine, Cisplatin, Chlorambucil, Chlormethine, Chlorozotocin, Cyclophosphamide, Cytarabine, Dacarbazine, Daunorubicin, Docetaxel, Doxifluridine, Doxorubicin, Epirubicin, Epothilone, Erlotinib, Etoposide, Exatecan, Fluorouracil, Fotemustine, Gefitinib, Gemcitabine, Gimatecan, Hydroxyurea, Idarubicin, Ifosfamide, Imatinib, Irinotecan, Ixabepilone, Larotaxel, Lomustine, Melphalan, Melphalan flufenamide, Mercaptopurine, Methotrexate, Mitobronitol, Mitomycin C, Mitoxantrone, Nimustine, Nitrosoureas, Oxaliplatin, Paclitaxel, Pemetrexed, Pipobroman, Ranimustine, Romidepsin, Semustine, Streptozotocin, Tafluposide, Taxotere, Temozolomide, Tesetaxel, Teniposide, Thiotepa, Tioguanine, Topotecan, Treosulfan, Tretinoin, Triaziquone, Triethylenemelamine, Valrubicin, Vemurafenib, Vinblastine, Vincristine, Vindesine, Vinorelbine, Vismodegib, or Vorinostat, or a derivative thereof.WSGR Docket No.: 58964-801.601

[0254] Embodiment 9. The MMADC according to any one of embodiments 1-8, wherein each of the first linker and the second linker independently comprises *–(L2)p–(L1)s–; wherein: * denotes a connection leading to the first drug or the second drug; each L1and L2is independently a bond, –O–, –S–, –NH–, –NRD–, –C(=O)–, –C(=O)O–, –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; RDis independently –CR3R4R5; each R3, R4, and R5is 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 R9and / 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 R9is 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-C4alkylene)-C(O)O-(C1-C4alkyl), –O(C2-C4alkenyl), –O(C1-C4alkylene)-(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;WSGR Docket No.: 58964-801.601 each of n, p and s is independently an integer of 1-12.

[0255] Embodiment 10. The MMADC according to embodiment 9, wherein each of the first linker and the second linker further independently comprises a spacer connected to either end of *–(L2)p– (L1)s–, 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.

[0256] Embodiment 11. The MMADC according to embodiment 10, wherein each of the spacer is independently , 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.

[0257] Embodiment 12. The MMADC according to embodiment 10, wherein each of the spacer is independently , wherein: each Y1 and Y2 is independently a bond, O, S, or NRE; REis independently H, deuterium, C1-C6 alkyl, C3-C6 cycloalkyl, C1-C6 alkyl; C6-C12 aryl, 5- 12 membered heteroaryl, C3-C12cycloalkyl or 3-12 membered heteroalicyclic, or REtogether 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.

[0258] Embodiment 13. The MMADC according to any one of embodiments 1-12, wherein the second linker is the same as the first linker, and wherein the second conjugation site is the same as the first conjugation site.

[0259] Embodiment 14. The MMADC according to any one of embodiments 1-12, wherein the first linker is different from the second linker, and wherein the first conjugation site is different from the second conjugation site.WSGR Docket No.: 58964-801.601

[0260] Embodiment 15. The MMADC according to any one of embodiments 1-14, wherein each of the first linker and the second linker is independently connected with the multispecific antibody via a reaction using a reactive group of:LG is halide, triflate, fluorosulfonate, tosylate, mesylate, or besylate; RAis 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; RBis 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, R7and R8is 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 R9is 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-C4WSGR Docket No.: 58964-801.601 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.

[0261] Embodiment 16. The MMADC according to embodiment 15, wherein the reactive group is:

[0262] Embodiment 17. The MMADC according to any one of embodiments 1-16, wherein each of the first conjugation site and the second conjugation site is independently a reactive amino acid of the multispecific antibody.

[0263] Embodiment 18. The MMADC according to embodiment 17, wherein the reactive amino acid reacts with the first linker or the second linker via a moiety of –SH, –SeH, –NH2, –CO2H, or – C(=O)–, or a derivative thereof.

[0264] Embodiment 19. The MMADC according to embodiment 17 or embodiment 18, wherein the reactive amino acid is a natural amino acid.

[0265] Embodiment 20. The MMADC according to embodiment 17 or embodiment 18, wherein the naked multispecific antibody does not require modification for conjugation.

[0266] Embodiment 21. The MMADC according to embodiment 17 or embodiment 18, wherein the naked multispecific antibody is modified for conjugation.

[0267] Embodiment 22. The MMADC according to embodiment 17 or embodiment 18, wherein the reactive amino acid is an unnatural amino acid.WSGR Docket No.: 58964-801.601

[0268] Embodiment 23. The MMADC according to any one of embodiments 1-22, wherein the multispecific antibody is a bispecific antibody or a trispecific antibody.

[0269] Embodiment 24. The MMADC according to any one of embodiments 1-23, wherein the two or more different targets are two different epitopes of the same antigen.

[0270] Embodiment 25. The MMADC according to embodiment 24, wherein the two or more different targets are epitopes of two different antigens.

[0271] Embodiment 26, The MMADC according to any one of embodiments 1-25, wherein the multispecific antibody is an IgG1 antibody.

[0272] Embodiment 27. The MMADC according to any one of embodiments 1-25, wherein the multispecific antibody comprises two binding domains comprising a common light chain.

[0273] Embodiment 28. The MMADC according to any one of embodiments 1-27, wherein the multispecific antibody comprises three binding domains comprising a common light chain.

[0274] Embodiment 29. The MMADC according to any one of embodiments 1-28, wherein the multispecific antibody comprises a first and a second CH3 domain, wherein the first CH3 domain comprises amino acid substitution T366K and L351K and the second CH3 domain comprises amino acid substitution L351D and L368E.

[0275] Embodiment 30. The MMADC according to any one of embodiment 1-29, wherein the first drug is a first cytotoxic agent and the second drug is a second cytotoxic agent.

[0276] Embodiment 31. The MMADC according to any one of embodiments 1-29, wherein the first drug is a first chemotherapeutic agent and the second drug is a second chemotherapeutic agent.

[0277] Embodiment 32. The MMADC according to embodiment 30 or embodiment 31, wherein the first cytotoxic agent and the second cytotoxic agent, or the first chemotherapeutic agent and the second chemotherapeutic agent, interfere with one cellular pathways.

[0278] Embodiment 33. The MMADC according to embodiment 30 or embodiment 31, wherein the first cytotoxic agent and the second cytotoxic agent, or the first chemotherapeutic agent and the second chemotherapeutic agent, interfere with two different cellular pathways.

[0279] Embodiment 34. The MMADC according to embodiment 30, wherein the first cytotoxic agent and second cytotoxic agent are from the same class of cytotoxic agents.

[0280] Embodiment 35. The MMADC according to embodiment 30, wherein the first cytotoxic agent and second cytotoxic agent are from different classes of cytotoxic agents.

[0281] Embodiment 36. The MMADC according to embodiment 30, wherein: (i) the first cytotoxic agent is a tubulin inhibitor, and the second cytotoxic agent is a DNA damaging agent, a protein degrader, or an immune stimulant; orWSGR Docket No.: 58964-801.601 (ii) the first cytotoxic agent is a DNA damaging agent, and the second cytotoxic agent is a tubulin inhibitor, a protein degrader, or an immune stimulant; or (iii) the first cytotoxic agent is a protein degrader, and the second cytotoxic agent is a tubulin inhibitor, a DNA damaging agent, or an immune stimulant; or (iv) the first cytotoxic agent is an immune stimulant, and the second cytotoxic agent is a tubulin inhibitor, a DNA damaging agent, or a protein degrader.

[0282] Embodiment 37. The MMADC according to embodiment 30, wherein: (i) the first cytotoxic agent is a tubulin inhibitor, and the second cytotoxic agent is another tubulin inhibitor; or (ii) the first cytotoxic agent is a DNA damaging agent, and the second cytotoxic agent is another DNA damaging agent; or (iii) the first cytotoxic agent is a protein degrader, and the second cytotoxic agent is another protein degrader; or (iv) the first cytotoxic agent is an immune stimulant, and the second cytotoxic agent is another immune stimulant.

[0283] Embodiment 38. The MMADC according to embodiment 31, wherein: (i) the first chemotherapeutic agent is a compound selected from the group consisting of: Abraxane, Actinomycin, Alitretinoin, All-trans retinoic acid, Altretamine, Azacitidine, Azathioprine, Belotecan, Bendamustine, Bexarotene, Bleomycin, Bortezomib, Busulfan, Cabazitaxel, Camptothecin, Carboplatin, Carboquone, Carmustine, Capecitabine, Cisplatin, Chlorambucil, Chlormethine, Chlorozotocin, Cyclophosphamide, Cytarabine, Dacarbazine, Daunorubicin, Docetaxel, Doxifluridine, Doxorubicin, Epirubicin, Epothilone, Erlotinib, Etoposide, Exatecan, Fluorouracil, Fotemustine, Gefitinib, Gemcitabine, Gimatecan, Hydroxyurea, Idarubicin, Ifosfamide, Imatinib, Irinotecan, Ixabepilone, Larotaxel, Lomustine, Melphalan, Melphalan flufenamide, Mercaptopurine, Methotrexate, Mitobronitol, Mitomycin C, Mitoxantrone, Nimustine, Nitrosoureas, Oxaliplatin, Paclitaxel, Pemetrexed, Pipobroman, Ranimustine, Romidepsin, Semustine, Streptozotocin, Tafluposide, Taxotere, Temozolomide, Tesetaxel, Teniposide, Thiotepa, Tioguanine, Topotecan, Treosulfan, Tretinoin, Triaziquone, Triethylenemelamine, Valrubicin, Vemurafenib, Vinblastine, Vincristine, Vindesine, Vinorelbine, Vismodegib, or Vorinostat, or a derivative thereof; and (ii) the second chemotherapeutic agent is another compound selected from the group consisting of: Abraxane, Actinomycin, Alitretinoin, All-trans retinoic acid, Altretamine, Azacitidine, Azathioprine, Belotecan, Bendamustine, Bexarotene, Bleomycin, Bortezomib, Busulfan, Cabazitaxel, Camptothecin, Carboplatin, Carboquone, Carmustine, Capecitabine, Cisplatin,WSGR Docket No.: 58964-801.601 Chlorambucil, Chlormethine, Chlorozotocin, Cyclophosphamide, Cytarabine, Dacarbazine, Daunorubicin, Docetaxel, Doxifluridine, Doxorubicin, Epirubicin, Epothilone, Erlotinib, Etoposide, Exatecan, Fluorouracil, Fotemustine, Gefitinib, Gemcitabine, Gimatecan, Hydroxyurea, Idarubicin, Ifosfamide, Imatinib, Irinotecan, Ixabepilone, Larotaxel, Lomustine, Melphalan, Melphalan flufenamide, Mercaptopurine, Methotrexate, Mitobronitol, Mitomycin C, Mitoxantrone, Nimustine, Nitrosoureas, Oxaliplatin, Paclitaxel, Pemetrexed, Pipobroman, Ranimustine, Romidepsin, Semustine, Streptozotocin, Tafluposide, Taxotere, Temozolomide, Tesetaxel, Teniposide, Thiotepa, Tioguanine, Topotecan, Treosulfan, Tretinoin, Triaziquone, Triethylenemelamine, Valrubicin, Vemurafenib, Vinblastine, Vincristine, Vindesine, Vinorelbine, Vismodegib, Vorinostat, or a derivative thereof.

[0284] Embodiment 39. The MMADC according to embodiment 31, wherein: (i) the first chemotherapeutic agent is a compound selected from the group consisting of: alkylating agent, antimetabolite, antimicrotubular agent, plant alkaloid, and antitumor antibiotics; and (ii) the second chemotherapeutic agent is another compound selected from the group consisting of: alkylating agent, antimetabolite, antimicrotubular agent, plant alkaloid, and antitumor antibiotics.

[0285] Embodiment 40. The MMADC according to embodiment 31, wherein: (i) the first chemotherapeutic agent is a compound selected from the group consisting of: bendamustine, cyclophosphamide, ifosfamide, carmustine, lomustine, carboplatin, cisplatin, oxaliplatin, dacarbazine, procarbazine, temozolamide, busulfan, thiotepa, azacitidine, decitabine, cytarabine, gemcitabine, methotrexate, pemetrexed, cladribine, clofarabine, nelarabine, fluorouracil (5-FU), capecitabine, doxorubicin, daunorubicin, idarubicin, mitoxantrone, irinotecan, topotecan, paclitaxel, docetaxel, cabazitaxel, vinblastine, vincristine, vinorelbine, actinomycin D, bleomycin, daunomycin, hydroxyurea, tretinoin, and bortezomib; and (ii) the second chemotherapeutic agent is another compound selected from the group consisting of: bendamustine, cyclophosphamide, ifosfamide, carmustine, lomustine, carboplatin, cisplatin, oxaliplatin, dacarbazine, procarbazine, temozolamide, busulfan, thiotepa, azacitidine, decitabine, cytarabine, gemcitabine, methotrexate, pemetrexed, cladribine, clofarabine, nelarabine, fluorouracil (5-FU), capecitabine, doxorubicin, daunorubicin, idarubicin, mitoxantrone, irinotecan, topotecan, paclitaxel, docetaxel, cabazitaxel, vinblastine, vincristine, vinorelbine, actinomycin D, bleomycin, daunomycin, hydroxyurea, tretinoin, and bortezomib.WSGR Docket No.: 58964-801.601

[0286] Embodiment 41. The MMADC according to any one of embodiments 1 to 40, wherein a first drug-to-antibody ratio (DAR) for the first drug in the MMADC is from 1.52 to 7.95, and wherein a second DAR for the second drug in the MMADC is from 0.79 to 9.08.

[0287] Embodiment 42. The MMADC according to embodiment 41, wherein the first DAR and the second DAR are: (1) about 3.29 : about 2.51; (2) about 2.60 : about 5.00; (3) about 3.79 : about 4.91; (4) about 4.13 : about 9.08; (5) about 2.88 : about 2.52; (6) about 2.41 : about 3.02; (7) about 3.40 : about 4.31; (8) about 3.84 : about 7.92; (9) about 2.32 : about 5.64; (10) about 2.34 : about 6.41; (11) about 1.77 : about 4.44; (12) about 1.52 : about 6.59; (13) about 7.95 : about 3.58; (14) about 7.44 : about 4.57; (15) about 5.25 : about 1.26; or (16) about 4.46 : about 0.79.

[0288] Embodiment 43. A method for producing a multispecific multi-drug antibody-drug conjugate that binds to two or more different targets according to any one of embodiments 1-42, the method comprising conjugating two or more different drugs to the multispecific antibody.

[0289] Embodiment 44. The method according to embodiment 43, wherein two different drugs are conjugated to the multispecific antibody by random conjugation.

[0290] Embodiment 45. The method according to embodiment 43, wherein two different drugs are conjugated to the multispecific antibody by site-specific conjugation, optionally by two different site-specific conjugation methods.

[0291] Embodiment 46. The method according to any one of embodiments 43-45, wherein a first of two different drugs is conjugated to the multispecific antibody by random conjugation and a second of the two different drugs is conjugated to the multispecific antibody by site-specific conjugation.WSGR Docket No.: 58964-801.601

[0292] Embodiment 47. A pharmaceutical composition comprising an effective amount of a multispecific multi-drug antibody-drug conjugate according to any one of embodiments 1-42, and a pharmaceutically acceptable carrier.

[0293] Embodiment 48. The multispecific multi-drug antibody-drug conjugate according to embodiment 1-42, or the pharmaceutical composition according to embodiment 47, for use in therapy.

[0294] Embodiment 49. The multispecific multi-drug antibody-drug conjugate according to embodiment 1-42, or the pharmaceutical composition according to embodiment 47, for use in the treatment of cancer.

[0295] Embodiment 50. A method of treating a disease, comprising administering an effective amount of a multispecific multi-drug antibody-drug conjugate according to embodiment 1-42, or the pharmaceutical composition according to embodiment 47, to a subject in need thereof.

[0296] Embodiment 51. A method of treating cancer, comprising administering an effective amount of a multispecific multi-drug antibody-drug conjugate according to embodiment 1-42, or the pharmaceutical composition according to embodiment 47, to a subject in need thereof.

[0297] Embodiment 52. Use of a multispecific multi-drug antibody-drug conjugate according to embodiment 1-42 for the manufacture of a medicament for the treatment of a disease, in particular for the treatment of cancer.

[0298] Embodiment 53. The use according to embodiment 52, wherein the cancer is a heterogenous and / or a resistant cancer.

[0299] Embodiment 54. A method of producing a multispecific multi-drug antibody-drug conjugate (MMADC), 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 first drug and a second drug to the antibody, wherein the first drug is different from the second drug.

[0300] Embodiment 55. The method according to embodiment 54, wherein each of the first drug and the second drug is independently a tubulin inhibitor, a deoxynucleic acid (DNA) damaging agent, a chemotherapeutic agent, a protein degrader, or an immune stimulant.WSGR Docket No.: 58964-801.601

[0301] Embodiment 56. The method according to embodiment 55, wherein the tubulin inhibitor is an auristatin or derivative thereof, a tubulysin or derivative thereof, or a maytansine or derivative thereof.

[0302] Embodiment 57. The method according to embodiment 55, wherein the DNA damaging agent 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.

[0303] Embodiment 58. The method according to any one of embodiments 54-57, wherein - a first linker connects the first drug to the antibody at a first conjugation site; and - a second linker connects the second drug to the antibody at a second conjugation site.

[0304] Embodiment 59. The method according to embodiment 58, wherein each of the first conjugation site and the second conjugation site is independently a reactive amino acid of the antibody.

[0305] Embodiment 60. The method according to embodiment 57, wherein the reactive amino acid reacts with the first linker or the second linker via a moiety of –SH, –SeH, –NH2, –CO2H, or – C(=O)–, or a derivative thereof.

[0306] Embodiment 61. The method according to embodiment 59, wherein the reactive amino acid is a natural amino acid.

[0307] Embodiment 62. The method according to embodiment 59, wherein the reactive amino acid is an unnatural amino acid.

[0308] Embodiment 63. The method of any one of embodiments 54-62, wherein the second linker is the same as the first linker, and wherein the second conjugation site is the same as the first conjugation site.

[0309] Embodiment 64. The method according to embodiment 63, further comprising: - prior to the coupling, attaching the first drug and the second drug to the first linker, thereby creating a drug-linker conjugate comprising the first drug and the second drug; and - coupling the drug-linker conjugate to the first conjugation site.

[0310] Embodiment 65. The method according to embodiment 64, further comprising: - during the attaching, reacting a first reactive group on the first linker with the first drug; and - after attaching the first drug to the first linker , reacting a second reactive group on the first linker with the second drug.

[0311] Embodiment 66. The method according to embodiment 65, wherein the first drug reacts with the first reactive group faster than with the second reactive group.WSGR Docket No.: 58964-801.601

[0312] Embodiment 67. The method according to embodiment 65 or embodiment 66, wherein the second drug reacts with the second reactive group faster than with the first reactive group.

[0313] Embodiment 68. The method according to any one of embodiments 54-62, wherein the first linker is different from the second linker, and wherein the first conjugation site is different from the second conjugation site.

[0314] Embodiment 69. The method according to embodiment 68, further comprising: - prior to the coupling, attaching the first drug to the first linker, thereby creating a first drug-linker conjugate comprising the first drug; - prior to the coupling, attaching the second drug to the second linker, thereby creating a second drug-linker conjugate comprising the second drug;. - coupling the first drug-linker conjugate to the first conjugation site; and - coupling the second drug-linker conjugate to the second conjugation site.

[0315] Embodiment 70. The method of embodiment 69, wherein the first drug-linker conjugate reacts faster with the first conjugation site than with the second conjugation site.

[0316] Embodiment 71. The method of embodiment 69 or embodiment 70, wherein the second drug-linker conjugate reacts faster with the second conjugation site than with the first conjugation site.

[0317] Embodiment 72. A polypeptide composition comprising a polypeptide; first linker connecting a first drug to the polypeptide at a first conjugation site, and a second linker connecting a second drug to the polypeptide; wherein the first drug and second drug are each independently a microtubule inhibitor, a topoisomerase inhibitor, a DNA-damaging agent, or an RNA polymerase inhibitor, wherein the first drug is different from the second drug.

[0318] Embodiment 73. The polypeptide of embodiment 72, wherein the polypeptide is an antibody.

[0319] Embodiment 74. The polypeptide of embodiment 73, wherein the antibody is a multispecific antibody.

[0320] Embodiment 75. A multispecific multi-drug antibody-drug conjugate (MMADC) comprising: a multispecific antibody; a first linker connecting a first drug to the multispecific antibody at a first conjugation site; and a second linker connecting a second drug to the multispecific antibody at a second conjugation site; wherein the first drug and the second drug are each independently a microtubule inhibitor, a topoisomerase inhibitor, a DNA-damaging agent, a DNA damage repair inhibitor, a chemotherapeutic agent, or an RNA polymerase inhibitor, wherein the first drug is different from the second drug.WSGR Docket No.: 58964-801.601

[0321] Embodiment 76. The MMADC of embodiment 75, wherein when: (a) the first drug is a microtubule inhibitor, the second drug is a topoisomerase inhibitor, a DNA-damaging agent, a DNA damage repair inhibitor, a chemotherapeutic agent, or an RNA polymerase inhibitor; or (b) the first drug is a topoisomerase inhibitor, the second drug is a microtubule inhibitor, a DNA-damaging agent, a DNA damage repair inhibitor, a chemotherapeutic agent, or an RNA polymerase; or (c) the first drug is a DNA-damaging agent, the second drug is a microtubule inhibitor, a topoisomerase inhibitor, a DNA damage repair inhibitor, a chemotherapeutic agent, or an RNA polymerase inhibitor; or (d) the first drug is a DNA damage repair inhibitor, the second drug is a microtubule inhibitor, a topoisomerase inhibitor, a DNA-damaging agent, a chemotherapeutic agent, or an RNA polymerase inhibitor; or (e) the first drug is a chemotherapeutic agent, the second drug is a microtubule inhibitor, a topoisomerase inhibitor, a DNA-damaging agent, a DNA damage repair inhibitor, or an RNA polymerase inhibitor; or (f) the first drug is an RNA polymerase inhibitor, the second drug is a microtubule inhibitor, a topoisomerase inhibitor, a DNA-damaging agent, a DNA damage repair inhibitor, or a chemotherapeutic agent.

[0322] Embodiment 77.The MMADC of embodiment 76, wherein the first drug is a microtubule inhibitor and the second drug is a topoisomerase inhibitor.

[0323] Embodiment 78. The MMADC of embodiment 75, wherein when: (a) the first drug is a microtubule inhibitor, the second drug is a different microtubule inhibitor; or (b) the first drug is a topoisomerase inhibitor, the second drug is a different topoisomerase inhibitor; or (c) the first drug is a DNA-damaging agent, the second drug is a different DNA-damaging agent; or (d) the first drug is a DNA damage repair inhibitor, the second drug is a different DNA damage repair inhibitor; or (e) the first drug is a chemotherapeutic agent, the second drug is a different chemotherapeutic agent; or (f) the first drug is an RNA polymerase inhibitor, the second drug is a different RNA polymerase inhibitor.

[0324] Embodiment 79. The MMADC of embodiment 78, wherein the first drug is a microtubule inhibitor and the second drug is a different microtubule inhibitor.

[0325] Embodiment 80. The MMADC of embodiment 78, wherein the first drug is a topoisomerase I inhibitor and the second drug is a topoisomerase II inhibitor.

[0326] Embodiment 81. The MMADC of embodiment 78, wherein the first drug is a chemotherapeutic agent and the second drug is a different chemotherapeutic agent.

[0327] Embodiment 82. The MMADC of any one of embodiments 75, 76, or 78, wherein the DNA-damaging agent is duocarmycins or pyrrolobenzodiazepines (PDBs).WSGR Docket No.: 58964-801.601

[0328] Embodiment 83. The MMADC of any one of embodiments 75,76, or 78, wherein the DNA damage response inhibitor (DDRi) is tuvusertib, M9466, lartesertib, peposertib, olaparib, talazoparib, veliparib, rucaparib, or niraparib.

[0329] Embodiment 84. The MMADC of any one of embodiments 75 to 83, wherein the first drug and the second drug is independently monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), tubulysin A, maytansine, ansamitocin P3, mertansine (DM1), ravtansine (DM4), calicheamicin, duocarmycin, topotecan, exatecan, DXd, irinotecan, cisplatin, oxaliplatin, paclitaxel, teniposide, SN38, hexylresorcinal, camptothecin, MM398, etoposide, novobiocin, doxorubicin, nemorubicin, daunorubicin, idarubicin, epipodophyllotoxin, toposide, teniposide, mitoxanthrone, pyrrolobenzodiazepine (PDB), TAS-103, 7-MAD_MDCPT, SN38, SG2199, amanitins, 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 pharmaceutically acceptable salt or derivative thereof.

[0330] Embodiment 85. The MMADC of embodiment 84, wherein the first drug and the second drug are each independently monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), mertansine (DM1), ravtansine (DM4), Dxd, exatecan, doxorubicin, TAS-103, irinotecan, cisplatin, oxaliplatin, paclitaxel, 7-MAD-MDCPT, camptothecin, SN38, SG3199, or Amanitins, or a pharmaceutically acceptable salt or derivative thereof.

[0331] Embodiment 86. The MMADC of embodiment 85, wherein the first drug and the second drug are each independently MMAE, MMAF, DM1, Dxd, doxorubicin, TAS-103, exatecan, cisplatin, or irinotecan.

[0332] Embodiment 87. The MMADC of any one of embodiments 75 to 86, wherein the first drug and the second drug are: (a) MMAE and MMAF; (b) MMAE and DM1; (c) MMAE and Dxd; (d) MMAE and doxorubicin; (e) MMAE and TAS-103; (f) MMAF and DM1; (g) MMAF and Dxd; (h) MMAF and doxorubicin; (i) MMAF and TAS-103;(j) DM1 and Dxd; (k) DM1 and doxorubicin; (l) DM1 and TAS-103; (m) Dxd and doxorubicin; (n) Dxd and exatecan; or (o) Cisplatin and irinotecan.

[0333] Embodiment 88. The MMADC of any one of embodiments 75 to 87, wherein the first drug has first drug-to-antibody (DAR) ratio of about 1.5 to about 8.

[0334] Embodiment 89. The MMADC of any one of embodiments 75 to 88, wherein the second drug has a second drug-to-antibody ratio (DAR) of about 0.7 to about 9.1.

[0335] Embodiment 90. The MMADC of any one of embodiments 75 to 89, wherein the first linker and the second linker is independently connected to the multispecific antibody via a reaction using the reactive group selected from:WSGR Docket No.: 58964-801.601

[0336] Embodiment 91. The MMADC of any one of embodiments 75 to 89, wherein the first linker and second linker is independently a branched linker.

[0337] Embodiment 92. The MMADC of embodiment 91, wherein the branched linker comprises the following structure:wherein denotes a connection leading to the drug, wherein denotes a connection leading to the antibody, wherein each of e, d and f is independently 0, 1, 2, 3, 4, 5, 6, 7 or 8, wherein at least one of e, d, and f is not 0.

[0338] Embodiment 93. The MMADC of any one of embodiments 75 to 92, wherein the first linker and second linker independently comprises maleimidocaproyl-valine-citrulline- paminobenzyloxycarbonyl (MC-VCP) or glutamic acid-valine-citrulline (GluValCit).

[0339] Embodiment 94. The MMADC of any one of embodiments 75 to 93, wherein the first linker and the second linker further independently comprises a first spacer and a second spacer.

[0340] Embodiment 95. The MMADC of embodiment 94, wherein the first spacer and second spacer independently comprises a polyethylene glycol (PEG).

[0341] Embodiment 96. The MMADC of any one of embodiments 75 to 95, wherein the first conjugate site and the second conjugation site is independently a reactive amino acid of the multispecific antibody.

[0342] Embodiment 97. The MMADC of embodiment 96, wherein the reactive amino acid has the following functional group: –SH, –SeH, –NH2, –CO2H, or –C(=O)–, or a derivative thereof.WSGR Docket No.: 58964-801.601

[0343] Embodiment 98. The MMADC of any one of embodiments 75 to 97, wherein the multispecific antibody is a bispecific antibody or a trispecific antibody.

[0344] Embodiment 99. The MMADC of embodiment 98, wherein the multispecific antibody is a bispecific antibody.

[0345] Embodiment 100. The MMADC of embodiment 99, wherein the bispecific antibody binds to two different targets, wherein the targets are independently EGFR, c-MET, LGR5, HER2, and HER3.

[0346] Embodiment 101. The MMADC of embodiment 100, wherein the targets are independently EGFR, c-MET, and LGR5.

[0347] Embodiment 102. The MMADC of any one of embodiments 75 to 100, wherein the multispecific antibody is a multispecific IgG1 antibody.

[0348] Embodiment 103. The MMADC of any one of embodiments 75 to 102, wherein the multispecific antibody comprises a first heavy chain, a second heavy chain, a first light chain, and a second light chain.

[0349] Embodiment 104. The MMADC of any one of embodiments 75 to 103, wherein the multispecific antibody comprises a first and a second CH3 domain, wherein the first CH3 domain comprises amino acid substitution T366K and L351K and the second CH3 domain comprises amino acid substitution L351D and L368E.

[0350] Embodiment 105. The MMADC of embodiment 104, wherein the first heavy chain has SEQ ID NO: 257, the second heavy chain has SEQ ID NO: 258, and the first light chain and the second light chain have SEQ ID NO: 259.

[0351] Embodiment 106. The MMADC of embodiment 104, wherein the first heavy chain has SEQ ID NO: 260, the second heavy chain has SEQ ID NO: 261, and the first light chain and the second light chain have SEQ ID NO: 259.

[0352] Embodiment 107. The MMADC of any one of embodiments 75 to 104, wherein the MMADC is selected from Table 3.

[0353] Embodiment 108. The MMADC of any one of embodiments 75 to 104, wherein the MMADC is selected from Table 4.

[0354] Embodiment 109. The MMADC of any one of embodiments 75 to 108, for use in the treatment of cancer.

[0355] Embodiment 110. The MMADC of embodiment 109, wherein the cancer is resistant to either the first drug or second drug.WSGR Docket No.: 58964-801.601

[0356] Embodiment 111. A pharmaceutical composition comprising a therapeutically effective amount of a multispecific multi-drug antibody-drug conjugate (MMADC) according to any one of embodiments 75 to 110, and a pharmaceutically acceptable carrier.

[0357] Embodiment 112. A method of treating a disease, the method comprising administering a therapeutically effective amount of the multispecific multi-drug antibody-drug conjugate (MMADC) of any one of embodiments 75 to 110, or the pharmaceutical composition according to embodiment 111, to a subject in need thereof

[0358] Embodiment 113. A method of treating a disease, the method comprising administering a therapeutically effective amount of a multispecific multi-drug antibody-drug conjugate (MMADC) to a subject in need thereof, wherein the MMADC comprises a multispecific antibody; a first linker connecting a first drug to the multispecific antibody at a first conjugation site; and a second linker connecting a second drug to the multispecific antibody at a second conjugation site; wherein the first drug is different from the second drug.

[0359] Embodiment 114.The method of embodiment 113, wherein the first drug and second drug is each independently a microtubule inhibitor, a topoisomerase inhibitor, a DNA-damaging agent, a DNA damage repair inhibitor, an RNA polymerase inhibitor, a chemotherapeutic agent, a protein degrader, or an immune stimulant.

[0360] Embodiment 115. The method of embodiment 113 or 114, wherein when: (a) the first drug is a microtubule inhibitor, the second drug is a topoisomerase inhibitor, a DNA-damaging agent, a DNA damage repair inhibitor, a chemotherapeutic agent, or an RNA polymerase inhibitor; or (b) the first drug is a topoisomerase inhibitor, the second drug is a microtubule inhibitor, a DNA-damaging agent, a DNA damage repair inhibitor, a chemotherapeutic agent, or an RNA polymerase; or (c) the first drug is a DNA-damaging agent, the second drug is a microtubule inhibitor, a topoisomerase inhibitor, a DNA damage repair inhibitor, a chemotherapeutic agent, or an RNA polymerase inhibitor; or (d) the first drug is a DNA damage repair inhibitor, the second drug is a microtubule inhibitor, a topoisomerase inhibitor, a DNA-damaging agent, a chemotherapeutic agent, or an RNA polymerase inhibitor; or (e) the first drug is a chemotherapeutic agent, the second drug is a microtubule inhibitor, a topoisomerase inhibitor, a DNA-damaging agent, a DNA damage repair inhibitor, or an RNA polymerase inhibitor; or (f) the first drug is an RNA polymerase inhibitor, the second drug is a microtubule inhibitor, a topoisomerase inhibitor, a DNA-damaging agent, a DNA damage repair inhibitor, or a chemotherapeutic agent.

[0361] Embodiment 116. The method of embodiment 115, wherein the first drug is a microtubule inhibitor and the second drug is a topoisomerase inhibitor.WSGR Docket No.: 58964-801.601

[0362] Embodiment 117. The method of embodiment 113 or 114, wherein when: (a) the first drug is a microtubule inhibitor, the second drug is a different microtubule inhibitor; or (b) the first drug is a topoisomerase inhibitor, the second drug is a different topoisomerase inhibitor; or (c) the first drug is a DNA-damaging agent, the second drug is a different DNA-damaging agent; or (d) the first drug is a DNA damage repair inhibitor, the second drug is a different DNA damage repair inhibitor; or (e) the first drug is a chemotherapeutic agent, the second drug is a different chemotherapeutic agent; or (f) the first drug is an RNA polymerase inhibitor, the second drug is a different RNA polymerase inhibitor.

[0363] Embodiment 118. The method of embodiment 117, wherein the first drug is a microtubule inhibitor and the second drug is a different microtubule inhibitor.

[0364] Embodiment 119. The method of embodiment 117, wherein the first drug is a topoisomerase I inhibitor and the second drug is a topoisomerase II inhibitor.

[0365] Embodiment 120. The method of embodiment 113, wherein the first drug and second drug is independently monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), tubulysin A, maytansine, ansamitocin P3, mertansine (DM1), ravtansine (DM4), calicheamicin, duocarmycin, topotecan, exatecan, DXd, irinotecan, cisplatin, oxaliplatin, paclitaxel, teniposide, SN38, hexylresorcinal, camptothecin, MM398, etoposide, novobiocin, doxorubicin, nemorubicin, daunorubicin, idarubicin, epipodophyllotoxin, toposide, teniposide, mitoxanthrone, pyrrolobenzodiazepine (PDB), TAS-103, 7-MAD_MDCPT, SN38, SG2199, amanitins, 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 pharmaceutically acceptable salt or derivative thereof.

[0366] Embodiment 121. The method of embodiment 120, wherein the first drug and the second drug are each independently monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), mertansine (DM1), ravtansine (DM4), Dxd, exatecan, doxorubicin, TAS-103, irinotecan, 7-MAD- MDCPT, camptothecin, SN38, SG3199, or Amanitins, or a pharmaceutically acceptable salt or derivative thereof.

[0367] Embodiment 122. The method of embodiment 121, wherein the first drug and the second drug are each independently MMAE, MMAF, DM1, Dxd, doxorubicin, TAS-103, exatecan, cisplatin, or irinotecan.

[0368] Embodiment 123. The method of any one of embodiments 113 to 122, wherein the first drug and the second drug are: (a) MMAE and MMAF; (b) MMAE and DM1; (c) MMAE and Dxd; (d) MMAE and doxorubicin; (e) MMAE and TAS-103; (f) MMAF and DM1; (g) MMAF and Dxd; (h) MMAF and doxorubicin; (i) MMAF and TAS-103;(j) DM1 and Dxd; (k) DM1 and doxorubicin;WSGR Docket No.: 58964-801.601 (l) DM1 and TAS-103; (m) Dxd and doxorubicin; (n) Dxd and exatecan; or (o) Cisplatin and irinotecan.

[0369] Embodiment 124. The method of any one of embodiments 113 to 123, wherein the first linker and the second linker is independently connected to the multispecific antibody via a reaction using the reactive group selected from:

[0370] Embodiment 125. The method of any one of embodiments 113 to 123, wherein the first linker and the second linker is independently a branched linker.

[0371] Embodiment 126. The method of embodiment 125, wherein the branched linker comprises the following structure:wherein denotes a connection leading to the drug, wherein denotes a connection leading to the antibody, wherein each of e, d and f is independently 0, 1, 2, 3, 4, 5, 6, 7 or 8, wherein at least one of e, d, and f is not 0.

[0372] Embodiment 127. The method of any one of embodiments 113 to 126, wherein the first linker and second linker independently comprises maleimidocaproyl-valine-citrulline- paminobenzyloxycarbonyl (MC-VCP) or glutamic acid-valine-citrulline (GluValCit).

[0373] Embodiment 128. The method of any one of embodiments 113 to 127, wherein the first linker and the second linker further independently comprises a first spacer and a second spacer.

[0374] Embodiment 129. The method of embodiment 128, wherein the first spacer and second spacer independently comprises a polyethylene glycol (PEG).WSGR Docket No.: 58964-801.601

[0375] Embodiment 130. The method of any one embodiments 113 to 129, wherein the first conjugate site and the second conjugation site is independently a reactive amino acid of the multispecific antibody.

[0376] Embodiment 131. The method of embodiment 130, wherein the reactive amino acid has the following functional group: –SH, –SeH, –NH2, –CO2H, or –C(=O)–, or a derivative thereof.

[0377] Embodiment 132. The method of any one of embodiments 113 to 131, wherein the multispecific antibody is a bispecific antibody or a trispecific antibody.

[0378] Embodiment 133. The method of embodiment 132, wherein the multispecific antibody is a bispecific antibody or a trispecific antibody.

[0379] Embodiment 134. The method of embodiment 133, wherein the multispecific antibody is a bispecific antibody.

[0380] Embodiment 135. The method of embodiment 134, wherein the bispecific antibody binds to two different targets, wherein the targets are independently EGFR, c-MET, LGR5, HER2, and HER3.

[0381] Embodiment 136. The method of any one of embodiments 113 to 135, wherein the multispecific antibody is a multispecific IgG1 antibody.

[0382] Embodiment 137. The method of any one of embodiments 113 to 136, wherein the multispecific antibody comprises a first heavy chain, a second heavy chain, a first light chain, and a second light chain.

[0383] Embodiment 138. The method of embodiment 137, wherein the first heavy chain and the second heavy chain have different amino acid sequences.

[0384] Embodiment 139. The method of any one of embodiments 113 to 138, wherein the multispecific antibody comprises a first and a second CH3 domain, wherein the first CH3 domain comprises amino acid substitution T366K and L351K and the second CH3 domain comprises amino acid substitution L351D and L368E.

[0385] Embodiment 140. The method of embodiment 139, wherein the first heavy chain has SEQ ID NO: 257, the second heavy chain has SEQ ID NO: 258, and the first light chain and the second light chain has SEQ ID NO: 259.

[0386] Embodiment 141. The method of embodiment 139, wherein the first heavy chain has SEQ ID NO: 260, the second heavy chain has SEQ ID NO: 261, and the first light chain and the second light chain has SEQ ID NO: 259.

[0387] Embodiment 142. The method of any one of embodiments 113 to 139, wherein the MMADC is selected from Table 3.WSGR Docket No.: 58964-801.601

[0388] Embodiment 143. The method of any one of embodiments 113 to 139, wherein the MMADC is selected from Table 4.

[0389] Embodiment 144. The method of any one of embodiments 112 to 143, wherein the disease is cancer.

[0390] Embodiment 145. The method of embodiment 144, wherein the cancer is a heterogenous cancer and / or a resistant cancer.

[0391] Embdoiment 146. The method of embodiment 144 or 145, wherein the cancer is oral cancer, colorectal cancer, gastric cancer, esophageal cancer, hepatocellular cancer, non-small-cell lung cancer (NSCLC), small-cell lung cancer (SCLC), ovarian cancer, breast cancer, prostate cancer, pancreatic cancer, head and neck cancer, squamous cancer, renal cancer, bladder cancer, cervical cancer, endometrial cancer, thyroid cancer, or glioblastoma cancer.

[0392] Embodiment 147. The method of embodiments 114 to 146, wherein the cancer is resistant to treatment with MMAE, MMAF, DM1, Dxd, cisplatin, or irinotecan, or a combination thereof.

[0393] Embodiment 148. Use of the MMADC of any one of embodiments 75 to 111, for the manufacture of a medicament for the treatment of a disease.

[0394] Embodiment 149. The use according to embodiment 148, wherein the disease is cancer.

[0395] Embodiment 150. The use according to embodiment 149, wherein the cancer is a heterogenous and / or a resistant cancer. Examples Example 1 – Cell Lines

[0396] The following cell lines are used to characterize the multispecific multi-drug antibody-drug conjugates (MMADCs). HCC827 (ATCC; CRL-2868) is an epithelial cell line that was isolated from the lung of a White, 39-year-old female patient with adenocarcinoma. NCI-H596 [H596] (ATCC; HTB-178) is an epithelial-like cell line that was isolated from the lung of a 73-year-old, white, male with lung adenosquamous carcinoma. A549 cells (ATCC; CCL-185) were isolated from the lung tissue of a white, 58-year-old male with lung cancer. NCI-H1792 [H1792] (ATCC; CRL-5895) is a cell line exhibiting epithelial morphology that was isolated from the lungs of a 50-year-old, white male patient with stage 4 adenocarcinoma. NCI-H1975 [H-1975, H1975] (ATCC; CRL-5908) is a cell line exhibiting epithelial morphology that was isolated in 1988 from the lungs of a nonsmoking female with non-small cell lung cancer.WSGR Docket No.: 58964-801.601 NCI-H292 (ATCC; CRL-1848) is a cell line exhibiting epithelial morphology that was isolated from the lungs of a female with mucoepidermoid pulmonary carcinoma. A-427 (ATCC; HTB-53) is a cell line exhibiting epithelial morphology that was isolated from the lungs of a 52-year-old, white male patient with carcinoma. NCI-H358 [H-358, H358] (ATCC; CRL-5807) is an epithelial-like cell that was isolated from the bronchiole of a male patient with bronchioalveolar carcinoma. NCI-H23 [H23] (ATCC; CRL-5800) is an epithelial-like cell that was isolated from the lung of a black, 51-year-old, male patient with adenocarcinoma. NCI-H1573 [H1573] (ATCC; CRL-5877) is a cell line isolated from the lungs of a 35-year- old, white female patient with stage 4 adenocarcinoma. A-431 (ATCC; CRL-1555) is a cell line exhibiting epithelial morphology that was isolated from the epidermis of an 85-year-old female patient with epidermoid carcinoma. HCT 116 cell line (ATCC; CCL-247) was isolated from the colon of an adult male with colon cancer. BxPC-3 (ATCC; CRL-1687) is a cell line exhibiting epithelial morphology that was isolated from the pancreas tissue of a 61-year-old, female patient with adenocarcinoma. HT-29 (HTB-38) is a cell line with epithelial morphology that was isolated in 1964 from a primary tumor obtained from a 44-year-old, white, female patient with colorectal adenocarcinoma. Hs 746T (ATCC; HTB-135) is a cell line with epithelial morphology that was isolated from the stomach of a 74-year-old male patient with gastric carcinoma. SNU-5 (ATCC; CRL-5973) is a cell line with epithelial morphology that was isolated from the stomach of a 33-year-old female patient with gastric carcinoma. 786-O (ATCC; CRL-1932) is a cell line with epithelial morphology that was isolated from the kidney of a 58-year-old male patient with renal cell adenocarcinoma. AsPC-1 (ATCC; CRL-1682) is cell line derived from nude mouse xenografts initiated with cells from the ascites of a 62-year-old, white, female patient with cancer of the pancreas. A-498 (ATCC; HTB-44) is a cell line with epithelial morphology that was isolated from kidney tissue obtained from a 52-year-old, female, kidney cancer patient. EBC-1 (JCRB CellBank; JCRB0820) is a human squamous cell carcinoma cell line. MKN-45 (AcceGen; ABC-TC0687) is a human gastric cancer cell line derived from poorly differentiated gastric adenocarcinoma. NCI-H441 [H441] cells (ATCC; HTB-174) were isolated in 1982 from the pericardial fluid of a male patient with papillary adenocarcinoma of the lung.WSGR Docket No.: 58964-801.601 Example 2 –Conjugation of bispecific antibodies with cytotoxic payload

[0397] Two bispecific antibodies were used in this study: a bispecific antibody targeting EGFR and c-MET and a bispecific antibody targeting EGFR and LGR5. 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 “DE” substitutions refer the amino acid substitutions L351D and L368E. The “KK” substitutions refer to the amino acid substitutions T366K and L315K.

[0398] 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.

[0399] Both bispecific antibodies were coupled with monomethyl auristatin E (MMAE) through a maleimidocaproyl-valine-citrulline-paminobenzyloxycarbonyl (MC-VCP) 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 MMAE through a MC-VCP linker, referred to herein as trastuzumab- MC-VCP-MMAE or “trastuzumab ADC”, was generated as a control.

[0400] 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. FractionsWSGR Docket No.: 58964-801.601 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.

[0401] 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 ^m pore size, PVDF membrane filter.

[0402] 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. EGFRxc-MET MC-VCP MMAE and EGFRxLGR5 MC-VCP- MMAE conjugation resulted in a DAR distribution of between DAR0-DAR8, as determined by HIC and shown in FIG.1A and FIG.1B, respectively. Specifically, FIG.1A shows 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. FIG.1B shows the DAR distribution of EGFRxLRG5 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. Table 1. Conjugation of MMAE to bispecific antibodies and trastuzumab. LMW = low molecular weight. 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 ADCWSGR Docket No.: 58964-801.601 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

[0403] 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), 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.

[0404] The EGFRxc-MET ADC and EGFRxLGR5 ADC were shown to be stable, having thermal stabilities of above 55°C and with more than 70% of the payload still conjugated to the antibodies in human serum for up to 4 days (data not shown). Target binding was unaffected by the conjugation and both ADCs show internalization on, and killing of, dual target expressing cells (data not shown). Example 3 – Dual conjugation of bispecific antibodies

[0405] In this study, a bispecific IgG1 antibody comprising a variable region that binds tetanus toxoid (TT) and a variable region that binds CD137 in DEKK format was labeled with biotin (label 1) and Alexa FluorTM647 (label 2) to determine conjugation of two different labels by using conjugation chemistry that is commonly used for generating antibody-drug conjugates (ADCs). Amino acid sequences of the CH1, hinge, CH2 and CH3 of the TT binding arm are provided as SEQ ID NOs: 7, 6, 9, and 12, respectively; amino acid sequences of the CH1, hinge, CH2 and CH3 of the CD137 binding arm are provided as SEQ ID NOs: 7, 6, 9, and 11, respectively. Conjugation chemistry that was used includes 1) maleimide conjugation, wherein maleimide reacts with thiol groups created by reduction of cysteine bridges present in the antibody; and 2) NHS conjugation, wherein activated esters (NHS esters) react with primary amino groups present in the antibody. Labeling of antibodies

[0406] Labeling was carried out in 96-well format using plates that do not react with DMSO. The antibody concentration during labeling is ~ 6 µM (~ 1 mg / mL). The TCEP concentration for reduction was ~7x molar excess (~42 ^M). Concentrations of individual labels during the labelingWSGR Docket No.: 58964-801.601 reaction were 2.5x, 12.5x and 25x molar excess (15 ^M, 75 ^M, 150 ^M concentration) for labels #1-3 (Table 2). For label #4 (Table 2), 5x and 25x molar excess were used (30 ^M and 150 ^M).

[0407] Reagents and materials were equilibrated at room temperature. The labels are light sensitive and were kept in the dark. The reagents that were used to label the antibody are listed in Table 2, and were all obtained from Thermo Fisher Scientific Inc. Table 2. Reagents used to label the bispecific antibody. Name Reagent Cat# MW Amount mL DMSO for 7.5 mM (g / mol) per vial stock (mg) Biotin #1 EZ-Link A39261 525.62 2 0.507 Maleimide-PEG2- Biotin Alexa #2 Alexa Fluor™ A20347 1250 1 0.107 647 C2 Maleimide Biotin #3 EZ-Link NHS 20217 341.38 100 39.1 Biotin Alexa #4 Alexa Fluor™ A37573 1250 3x 0.1 3x 0.0107 647 NHS Ester

[0408] A 1:100 dilution of the 0.5 M TCEP stock in MQ was prepared and 1 ^L of this dilution was added per well of plate I.120 ^L of antibody was added per well and mixed with the TCEP by gently pipetting up and down. The plate was covered and incubated for 1 hour at 37^C in a titramax shaker without shaking. After incubation, the plate was cooled down to room temperature (~20^C), briefly centrifuged to collect the samples at the bottom of the wells, and directly proceeded to incubation with the labels.

[0409] Prior to labeling, labels were freshly reconstituted in anhydrous DMSO as listed in Table 2 and diluted in DMSO to arrive at concentrations of 7.5 mM, 3.7 mM, and 0.75 mM for labels #1-3, and 1.5 mM and 7.5 mM for label #4 (FIG.2.2.5 ^L of DMSO or biotin and Alexa FluorTM647 were added per well of plate II, with a final volume per well of 5 ^L.115 ^L of the antibody solution from plate I was added to the respective wells of plate II and gently mixed by pipetting up and down. The plate was covered and incubated at room temperature for 2 hours protected from light. Samples were collected by brief spinning down and 100 ^L was used for buffer exchange into PBS pH 7.4. The plate was covered and kept at 4^C protected from light.

[0410] Absorbance was measured at 280 nm to determine protein concentration and at 650 nm to measure the amount of Alexa FluorTM647 labeling with a Lunatic UV / Vis spectrophotometer.WSGR Docket No.: 58964-801.601 Protein concentration and degree of labeling (DOL) for Alexa FluorTM647 were calculated with an online tool, selecting IgG as target and Alexa FluorTM647 as label.

[0411] Results indicate that the antibody concentration was not affected by conjugation to the labels. FIG.2 shows that in all dual labeled bispecific antibody samples, on average each antibody is labeled with Alexa FluorTM647. Dual labeled bispecific antibodies contain an average of between 1.52-7.95 Alexa FluorTM647 molecules when using NHS conjugation, depending on the concentration of label used. At a stock concentration of 1.5 mM Alexa FluorTM647 NHS Ester, bispecific antibodies contain an average of between 1.52-2.67 Alexa FluorTM647 molecules. At a stock concentration of 1.5 mM Alexa FluorTM647 NHS Ester and 3.7 mM EZ-Link NHS biotin, bispecific antibodies contain an average of 1.77 Alexa FluorTM647 molecules. The lowest concentration Alexa FluorTM647 (stock concentration 0.75 mM) did not result in detectable levels of labeled antibody (data not shown). HABA assay

[0412] A 2-(4-hydroxy-phenylazo)-benzoic acid (HABA) assay was performed to confirm labeling with biotin and to quantify the average level of biotinylation of antibodies. Dilutions of D-biotin (200 µg / ml, Sigma, #47867) for the standard curve were prepared in duplicate in a round-bottom 96- wells plate according to the scheme below: Conc. D-Biotin Preparation 16 µg / ml 16 µl of 200 µg / ml D-Biotin + 184 µl PBS 12 µg / ml 12 µl of 200 µg / ml D-Biotin + 188 µl PBS 8 µg / ml 8 µl of 200 µg / ml D-Biotin + 192 µl PBS 6 µg / ml 6 µl of 200 µg / ml D-Biotin + 194 µl PBS 4 µg / ml 4 µl of 200 µg / ml D-Biotin + 196 µl PBS 2 µg / ml 2 µl of 200 µg / ml D-Biotin + 198 µl PBS 1 µg / ml 1 µl of 200 µg / ml D-Biotin + 199 µl PBS 0 µg / ml 200 µl PBS

[0413] IgG samples used in the assay were undiluted. The HABA / Avidin solution was prepared by reconstituting the lyophilized powder of one unit HABA / Avidin reagent (Sigma, #H2153) with 10 ml MQ water.90 µl of the HABA / Avidin solution was added per well to a half area 96-wells plate. 25 µl of the standard curve D-Biotin solutions or 25 µl sample were added to the 90 µl HABA / Avidin solution and mixed. Absorbance was measured at 500 nm by using EPOCH2 (BioTek). The Δ (delta) A500 was calculated as follows: ΔA500 = A500 of (90 µl HABA / Avidin +WSGR Docket No.: 58964-801.601 25 µl PBS) – A500 of biotin-sample (90 µl HABA / Avidin + 25 µl sample or D-Biotin dilution). A standard curve was made to determine the trendline and the equation of the trendline as follows: y- axis: ΔA500 vs. x-axis concentration D-biotin µg / ml. The concentration biotin (µg / ml) present in the samples was calculated using the equation of the standard curve: Y = aX + b, X = (Y-b) / a, where Y = ΔA500, and X = concentration biotin (µg / ml). The concentration biotin was calculated in µmol / ml as follows: concentration biotin (µg / ml) / 244.3 (MW of D-Biotin = 244.3). The concentration of protein was calculated in µmol / ml as follows: concentration IgG antibody (µg / ml) / 150,000 (MW of IgG antibody = 150,000). The ratio mol biotin / mol IgG antibody was calculated as follows: concentration biotin (µmol / ml) / concentration IgG antibody (µmol / ml).

[0414] Results indicate that the antibody concentration was not affected by conjugation to the labels. FIG.3A shows that in all dual labeled bispecific antibody samples, on average each antibody is labeled with biotin. Dual labeled bispecific antibodies contain an average of between 0.79-9.55 biotin molecules, depending on the concentration of label used. At a stock concentration of 3.7 mM EZ-Link NHS biotin, bispecific antibodies contain an average of between 1.26-5.38 biotin molecules. At a stock concentration of 1.5 mM Alexa FluorTM647 NHS Ester and 3.7 mM EZ-Link NHS biotin, bispecific antibodies contain an average of 4.44 biotin molecules. The lowest concentration biotin (stock concentration 0.75 mM) did not result in detectable levels of labeled antibody (data not shown). The data for the bispecific antibodies labeled with the highest concentrations (7.5 mM) biotin and Alexa FluorTM647 using NHS conjugation suggests that using the same conjugation method for both labels leads to competition of labeling. FIG.3B shows the summary of dual degrees of labeling (DOL) for dual labeled bispecific antibody prepared by treating the bispecific antibody with different concentrations of the two individual labels. The results show that the relative amount of the two labels on the same bispecific antibody can be tuned by varying the concentrations of the labeling reagents, using different reactive group on the labeling reagents, and varying the ratio of antibody vs. labeling reagents. Such tunability can help delivering the correct total amount and desired ratio of the two labels to the targeted binding site of the bispecific antibody. FACS assay

[0415] A FACS assay was performed to confirm that bispecific antibodies contain both the biotin and the Alexa FluorTM647 labels. In short, biotinylated bispecific antibodies were captured with streptavidin beads and these beads were analyzed for the presence of (A) IgG and (B) Alexa 647 dye in iQue. As controls, unlabeled bispecific antibodies and bispecific antibodies that are only labeled with either biotin or Alexa FluorTM647 were taken along. Coupling of bispecific antibodies to the streptavidin beadsWSGR Docket No.: 58964-801.601

[0416] Streptavidin beads were washed by diluting these in PBSB to 54 µg / ml and centrifuged for 4 minutes at 3200 rpm. Supernatant was discarded and the bead pellet was resuspended in PBSB. Wash was repeated twice and the pellet was resuspended in the desired volume.

[0417] 12.5 µl of the prepared beads was added per well of a 96-wells FACS plate.

[0418] Bispecific antibodies were prepared by diluting these to 1-4 ug / mL in PBSB.

[0419] According to the manufacturer, the capacity of beads is 10 ug biotinylated IgG per mg beads. Using 0.675 ug beads per well and incubation with 37.5-150 ng (biotinylated) IgG results in applying an excess of IgG sample per bead and potential enrichment for the biotinylated IgG portion in a given sample. Empty beads only stained with PBSB and empty beads stained with a Goat anti- human IgG PE conjugate (Invitrogen catalogue number H10104) were included as controls.

[0420] 37.5 µl per well of the diluted IgG was added to the beads already in the FACS plate. The plate was sealed, kept in the dark, and incubated for 1 hour at 30-37°C in a titramax shaker while gently swirling. FACS staining

[0421] The plate was briefly spun to collect all material at the bottom of the well.100 µl PBSB was added and centrifuged for 4 minutes at 3200 rpm. Supernatant was removed by inverting the plate over a plastic container and afterwards stamping the plate on a stack of paper towels, leaving the beads at the bottom of the plate. Plate was washed twice using 100 µl PBSB. The bead pellet was resuspended after the last wash in 50 µl PBSB containing the secondary antibody Goat anti-human IgG PE conjugate (Invitrogen catalogue number H10104), detecting IgG and diluted 1:100, and incubated for 15 minutes on ice.100 µl PBSB was added and centrifuged for 4 minutes at 3200 rpm. Plate was washed twice using 100 µl PBSB. The bead pellet was resuspended after the last wash in 75 µl PBSB.

[0422] Acquisition and analysis was performed on the iQue flow cytometer.

[0423] FIGs.4-7 show that in all dual labeled bispecific antibody samples, except one, bispecific antibodies are present that contain both labels. Only in the bispecific antibody sample with the lowest concentrations (stock concentration 0.75 mM) biotin and Alexa FluorTM647, it was not possible to detect beads comprising antibodies with both labels. HP-SEC

[0424] HP-SEC was performed to confirm the presence of IgG in the bispecific antibody samples that were only incubated with DMSO and selected bispecific antibody samples that contain detectable amounts of dual-labeled bispecific antibodies.

[0425] The concentration of IgG samples were in range of 0.2-2 mg / ml (in PBS, Gibco, #10010- 015 or 10010-031).10 ug material was injected. The samples were transferred into HPLC glass vialsWSGR Docket No.: 58964-801.601 (Waters, cat# 186000234) or Agilent 96-wells plate (Agilent, cat# 5043-9314). The HP-SEC run was performed on the HPLC-system (Agilent 1260 series) and the samples were acquired at 280 nm and 650 nm to identify Alexa FluorTMlabeling on the eluting protein material. Samples were spun down (at 4^C, >20 minutes at >3000 xg) and supernatant was visually inspected for aggregates before injecting the material. The HP-SEC sequence run started with injection of Milli-Q water, the IgG buffer (PBS), the system QC sample (Gel Filtration Standard (GFS), BioRad, #151-1901), followed by the IgG samples. The sequence ended with a second injection of the system QC sample (GFS). The maximum injection volume was 100 µl. Data is analyzed via the Data Analysis software(Agilent BioTek).

[0426] FIG.8 shows the elution profile of bispecific antibodies only treated with DSMO, where the antibodies elute as a dimer without any detectable half antibodies. FIG.9 shows that bispecific antibodies labeled with Alexa FluorTM647 (stock concentration 3.7 mM) and biotin (stock concentration 1.5 mM) using NHS conjugation mainly elute as dimers (95%).

[0427] The dual labeled bispecific antibodies are shown to be stable.

[0428] In conclusion, these data demonstrate that bispecific antibodies conjugated to two different moieties can successfully be generated. Example 4 – Bispecific antibodies and antibody drug conjugates

[0429] In the Examples described herein, bispecific antibodies targeting EGFR and cMET and targeting EGFR and LGR5 are chosen as examples of the antibody component of the MMADCs of the present disclosure. As an example of a bispecific antibody targeting EGFR and c-MET, a bispecific antibody comprising a heavy chain comprising the amino acid sequence as set forth in SEQ ID NO: 257, a heavy chain comprising the amino acid sequence as set forth in SEQ ID NO: 258, and two light chains comprising the amino acid sequence as set forth in SEQ ID NO: 259, is chosen (BsAb1). As an example of a bispecific antibody targeting EGFR and LGR5, a bispecific antibody comprising a heavy chain comprising the amino acid sequence as set forth in SEQ ID NO: 260, a heavy chain comprising the amino acid sequence as set forth in SEQ ID NO: 261, and two light chains comprising the amino acid sequence as set forth in SEQ ID NO: 259, is chosen (BsAb2). Both BsAb1 and BsAb2 are conjugated to several combinations of two different payloads, and referred to herein as MMADC-1 and MMADC-2, respectively. The MMADCs and their corresponding multispecific single drug antibody drug conjugates (MSADCs) for use in the Examples are listed in Tables 3-6. BsAb1 and / or BsAb2 may be engineered to facilitate conjugation as appropriate.WSGR Docket No.: 58964-801.601 Table 3. MMADCs with BsAb1. ADC First payload Second payload 1stHC: SEQ ID NO: 257 2ndHC: SEQ ID NO: 258 Both LC: SEQ ID NO: 259 MMADC-1A MMAE MMAF MMADC-1B MMAE DM1 MMADC-1C MMAE Dxd MMADC-1D MMAE Doxorubicin MMADC-1E MMAE TAS-103 MMADC-1F MMAF DM1 MMADC-1G MMAF Dxd MMADC-1H MMAF Doxorubicin MMADC-1I MMAF TAS-103 MMADC-1J DM1 Dxd MMADC-1K DM1 Doxorubicin MMADC-1L DM1 TAS-103 MMADC-1M Dxd Doxorubicin MMADC-1N Dxd Exatecan MMADC-1O Cisplatin Irinotecan Table 4. MMADCs with BsAb2. ADC First payload Second payload 1stHC: SEQ ID NO: 260 2ndHC: SEQ ID NO: 261 Both LC: SEQ ID NO: 259 MMADC-2A MMAE MMAF MMADC-2B MMAE DM1 MMADC-2C MMAE Dxd MMADC-2D MMAE Doxorubicin MMADC-2E MMAE TAS-103 MMADC-2F MMAF DM1 MMADC-2G MMAF Dxd MMADC-2H MMAF Doxorubicin MMADC-2I MMAF TAS-103 MMADC-2J DM1 Dxd MMADC-2K DM1 Doxorubicin MMADC-2L DM1 TAS-103 MMADC-2M Dxd DoxorubicinWSGR Docket No.: 58964-801.601 MMADC-2N Dxd Exatecan MMADC-2O Cisplatin Irinotecan Table 5. MSACDs with BsAB1. ADC First payload Second payload 1stHC: SEQ ID NO: 257 2ndHC: SEQ ID NO: 258 Both LC: SEQ ID NO: 259 MSADC-1P MMAE None MSADC-1Q MMAF None MSADC-1R DM1 None MSADC-1S Dxd None MSADC-1T Cisplatin None Table 6. MSADCs with BsAb2. ADC First payload Second payload 1stHC: SEQ ID NO: 260 2ndHC: SEQ ID NO: 261 Both LC: SEQ ID NO: 259 MSADC-2P MMAE None MSADC-2Q MMAF None MSADC-2R DM1 None MSADC-2S Dxd None MSADC-2T Cisplatin None Example 5 - Dual conjugation of a bispecific antibody binding EGFR and c-MET

[0430] Any suitable combination of conjugation methods can be used to conjugate the two different payloads to the bispecific antibody. In this particular Example, microbial transglutaminase (MTG) is used to site-specifically conjugate a branched ADC linker construct to a bsAb, as reported by Yamazaki et al., 2021. Synthesis of branched ADC linkers

[0431] To enable site-specific conjugation of two different payload molecules, a branched azido- methyltetrazine di-arm linker is designed and synthesized as described in Yamazaki et al., 2021. Materials for chemical synthesis are purchased from commercial suppliers (for example, Acros Organics, AnaSpec, Broadpharm, Chem-Impex International, ThermoFisher Scientific, Levena Biopharma, Millipore Sigma, and TCI America), unless stated otherwise. The branched linker is an azido-methyltetrazine di-arm linker characterized by a methyltetrazine group and an azide group asWSGR Docket No.: 58964-801.601 orthogonal clickable handles. The methyltetrazine group reacts with a methyltetrazine-trans- cyclooctene (TCO) payload module and the azide group reacts with an azide-dibenzocyclooctyne (DBCO) payload module, as explained in the following sections. Payload modules are designed with click pair DBCO and TCO, for the installation of two different payload molecules as listed in Tables 3 and 4, a polyethylene glycol (PEG) spacer, a glutamic acid-valine-citrulline (GluValCit) cleavable linker, and a p aminobenzyloxycarbonyl (PABC) group. MTG-mediated conjugation of branched ADC linkers

[0432] Microbial transglutaminase (MTG)-mediated transpeptidation allows conjugation of the bi- functional branched linker onto the side chain of glutamine 295 (Q295), affording a highly homogeneous antibody–linker conjugate (Anami and Tsuchikama, 2019; Dennler et al., 2014). A bsAb with a N297A variation (714 μL in PBS, 12.6 mg / mL, 9.0 mg antibody) is incubated with the azido-methyltetrazine di-arm linker (24 μL of 100 mM stock in dimethyl sulfoxide (DMSO), 40 equivalent) and Activa TI® transglutaminase preparation (180 μL of 40% solution in PBS, Ajinomoto, purchased from Modernist Pantry) at room temperature for 16–20 hours. The reaction is monitored using an Agilent G1946D LC / electrospray ionization (ESI)–MS system equipped with a MabPac RP column (3 × 50 mm, 4 μm, Thermo Scientific). Elution conditions are as follows: mobile phase A = water (0.1% formic acid); mobile phase B = acetonitrile (0.1% formic acid); gradient over 6.8 min from A : B = 75 : 25 to 1 : 99; flow rate = 0.5 mL / min. The conjugated bsAb is purified by SEC (Superdex 200 increase 10 / 300 GL, GE Healthcare, solvent: PBS, flow rate = 0.6 mL / min), and yield is determined by BCA assay. Double click reaction for payload installation

[0433] The first payload module designed as TCO click pair (2.5 equivalent per tetrazine group) is added to a solution of the mAb–azido-methyltetrazine di-arm linker conjugated bsAb (4.0 mg / mL) in PBS, and the mixture is incubated at room temperature for 2 hours. The reaction is monitored using an Agilent G1946D LC / ESI-MS system equipped with a MabPac RP column. The second payload module designed as DBCO click pair (1.5 equivalent per azide group) is added to the mixture and incubated at room temperature for additional 2 hours. The crude products are then purified by SEC to yield a dual-drug ADC (% yield is determined by BCA assay). Dual conjugation and average DAR values are determined based on liquid chromatography (LC)-mass spectrometry (MS) analysis. The resulting multidrug multispecific antibody drug conjugates (MMADCs) have a DAR of 4 [2+2]. This technology also allows for the generation of MMADCs with a DAR of 6 (4+2 or 2+4).WSGR Docket No.: 58964-801.601

[0434] In case it is desired to preserve the original antibody sequence, a similar conjugation strategy can be employed using different linkers, such as for instance described in Wehrmuller at al., 2024. Example 6 - Dual conjugation of a bispecific antibody using Fab Arm Exchange (FAE) Transfection, expression and purification of IgG half antibodies for use in the generation of ADCs using Fab Arm Exchange (FAE)

[0435] Expi293F™ cells (ThermoFisher Scientific) cultured in 100 mL Expi293F™ expression medium (ThermoFisher Scientific, cat.# A14351010), are transiently transfected with various expression vectors coding for heavy chains (HC) and light chains LC, see Table 7), using ExpiFectamine™ 293 Transfection Kit (ThermoFisher Scientific, cat.# A14635) and OptiMEM I Reduced serum medium (Gibco, cat.# 31985062). Table 7. Expression vectors (#1-4) are used for transfections and production of HC and LC molecules. Expression vectors contain a DNA construct that encodes a CH3 domain with the indicated modifications, a heavy chain (HC) and a light chain (LC). # Half antibody CH3 modification HC sequence LC sequence as expressed 1 HAb1-KK KK SEQ ID NO: 262 SEQ ID NO: 266 2 HAb2-KK KK SEQ ID NO: 263 SEQ ID NO: 267 3 HAb3-DE DE SEQ ID NO: 264 SEQ ID NO: 268 4 HAb4-DE DE SEQ ID NO: 265 SEQ ID NO: 259

[0436] Briefly, a DNA-Opti-MEM mixture and an Expifectamine-Opti-MEM mixture is prepared by diluting, respectively, 50 µg plasmid DNA (0.1 mL from DNA stock with a concentration of 0.5 mg / mL) with 3 mL Opti-MEM™ I Medium or 0.16 mL ExpiFectamine™ 293 Reagent with 2.8 mL Opti-MEM by swirl or inversion, followed by incubation for 5 minutes before the DNA-Opti-MEM mixture is added to the Expifectamine-Opti-MEM mixture and inverting the tube 4-5 times and incubation at room temperature for 15 minutes. Next, the entire volume of the ExpiFectamine™ 293 / plasmid DNA complexes (about 6.06 mL) are added dropwise to said cells whilst gently swirling the flask during addition followed by incubation (37°C incubator with ≥80% relative humidity and 8% CO2 on an orbital shaker at 155 rpm).

[0437] 18–22 hours post transfection, Enhancer 1 and Enhancer 2 of the ExpiFectamine™ 293 Transfection Kit are added to the transfectionflask while gently swirling. Day 6 post transfection, medium is collected and cells are spun down at 500 g for 10 minutes at RT, supernatant is collected and transferred into a new 50 ml tube and cells are spun down at 3000 g for 20 minutes.WSGR Docket No.: 58964-801.601 Supernatants are filtered using a bottle top 0.45 µm filter and IgG concentrations are measured using the ForteBIO Octet-QK system, which is based on Bio-Layer Interferometry (BLI). This enables real-time quantitation and kinetic characterization of biomolecular interactions. Supernatants are used for ÄKTA purification.

[0438] Volumes of culture supernatants that contained between 9-12 mg IgG are purified using ÄKTA pure system (Cytiva, EN490, serial number: 2031829) using protein A columns (GE Healthcare / cat# 11-0034-95) according to manufacturer’s instructions and eluted in 0.1 M citrate buffer, pH 3.0 and immediately neutralized in an equal volume of 1.0 M Tris-HCL pH 8.0 or directly rebuffered to PBS using a desalting column (Cytiva #17-1408-01).

[0439] The concentration of the half antibodies is determined with Lunatic system (Unchained Labs) in a 96-wells format, using 2 µl of sample based on absorbance at 280nm using the generic E1% of 14.5 for all products according to the manufacturer’s instructions. IgG concentrations are adjusted to 1.2 mg / mL in PBS pH 7. Conjugation of the antibodies

[0440] Labeling of the DE and KK half antibodies from Table 7 with one or more of the labels in Table 2 is carried out as described in Example 3. Alternatively, labeling of deglycosylated (N297A- substituted) DE and KK half antibodies with one or more labels is carried out at specific sites, such as described in Example 5. Fab Arm Exchange

[0441] Briefly, labeled DE half antibodies are mixed with labeled KK half antibodies in a 1:1 molar ratio and incubated at 31°C at pH 7.4, in the presence of 75 mM β-Mercapto-ethylamine hydrochloride (β-Mercapto-ethylamine hydrochloride)), without shaking. After 5 hours, samples are buffer exchanged to PBS pH 7.4 (Bex) at room temperature using Zeba plates. For reoxidation, samples are kept at 4°C for at least 1 night.

[0442] The FAE reaction is performed in 96-well format to produce the IgG molecules listed in Table 8. Table 8. Mixtures of labeled IgG antibodies for use in FAE. FAE # Half antibody Expression CH3 Half antibody Expression vector CH3 as expressed vector as expressed 1 HAb3-DE MG14471C1708 DE HAb1-KK MG9312C3130 KK 2 HAb4-DE MG6744C1708 DE HAb2-KK MG14256C3130 KKWSGR Docket No.: 58964-801.601

[0443] Fresh MEA stock solution (750mM) is prepared by dissolving 852 mg Cysteamine hydrochloride (Sigma cat# 30078) in 5-6 mL PBS (pH 7.4). pH is adjusted to pH 7.3 - 7.5 by adding NaOH (5M) to the solution at room temperature and the solution is filled with PBS pH 7.4 up till the final volume of 10 mL to achieve 750mM stock solution. The solution is filtered through 0.2 µm filter before use.

[0444] Briefly, IgG samples listed in Table 8 are prepared in a deep well plate (plate 1, 175 μL final volume adjusted by adding PBS pH 7.4) using IgG at a concentration of 1.1 mg / mL. In 12 wells of another deep well plate (plate 2), 11 μL of reducing agent 750 mM MEA is pipetted.100 μL of each prepared sample of plate 1 is gently mixed with the MEA in plate 2, which is covered with an aluminum seal and kept at 31°C for 5 hours without shaking. Material remaining in plate 1 (75 μL) is used as non-reacted controls i.e. not exposed to MEA or Bex and stored at 4°C in the dark until further use. After incubation, 100 µL of each sample is used for buffer exchange (bex) with the Zeba™ Spin Desalting plates, 96-well (Thermo Fisher Scientific, cat.# 89807). Buffer exchange:

[0445] Samples are buffer exchanged against PBS pH 7.4 (1x, Gibco cat. # 10010-015) using Zeba™ Spin Desalting kit (Thermo Fisher Scientific, cat. # 89807) to remove the reduction agent and allow reformation of disulfide bonds.

[0446] Briefly, ZebaTMspin desalting plates are equilibrated to room-temperature and assembled on top of the wash plate. The plate assembly is centrifuged to remove storage solution and the wash plate is blotted dry on paper towel. Three washing steps are performed by addition of washing buffer (1x, Gibco cat. # 10010-015), after which plates are centrifuged, flow through is discarded and the ZebaTMplate dry blotted on towel paper.

[0447] Next, ZebaTMdesalting plates are stacked on top of the collection plate and IgG samples are loaded into the wells and centrifuged. The flow through containing IgG is transferred into another ZebaTMplate and centrifuged. To achieve a more thorough buffer exchange, the flow through is transferred again into another ZebaTMplate and centrifuged. All centrifugations are performed at 1000x g for 2 minutes. The third flow-through that contains the sample which is passed three times through the ZebaTMplate, is retained for IgG concentration measurements. Buffer-exchanged samples are kept in the collector plate covered and kept at 4°C overnight (without further pipetting, shaking or mixing) to allow material to fully re-oxidize. IgG molecules as obtained are used in SDS- PAGE using Labchip under reducing and non-reducing conditions and as appropriate using CIEX analysis and HP-SEC. Confirmation of dual labeling is performed using a HABA and FACS assay as described in Example 3 or by LC-MS analysis as described in Example 5.WSGR Docket No.: 58964-801.601 Example 7 – Generation of payload resistant cell lines Generation of A549 (MDR1+) resistant cell line by lentivirus transduction

[0448] The coding sequence of the human MDR1 (ABCB1) gene is cloned into a custom-made LentifectTMlentiviral vector (GeneCopoeiaTM) and the final construct is confirmed by DNA sequencing. A549 cells (ATCC; cat. no. CCL-185) are seeded in a 24-well plate (50,000 cells per well in 0.5 mL culture medium) and incubated overnight at 37 °C under 5% CO2. Subsequently, cells are transduced with lentiviral particles at a multiplicity of infection of 5, according to the manufacturer’s instructions. After 24 hours, the lentiviral particles are removed and transduced cells stably expressing the MDR1 efflux pump are selected using a medium containing the antibiotic as informed by the vector used. MDR1 expression is confirmed using flow cytometry. Generation of A549 (MDR1+) resistant cell line by stable transfection

[0449] The coding sequence of the human MDR1 (ABCB1) gene is cloned into pEF1 / myc-His A vector (Invitrogen, cat. no. V921-20). A549 cells (ATCC; cat. no. CCL-185) are seeded in culture medium and transfected with this vector using LipofectamineTM2000 (Invitrogen, cat. no.11668- 019), according to the manufacturer’s instructions. Subsequently, cells are incubated at 37°C, 5% CO2. MDR1 expression is confirmed using flow cytometry, and, if MDR1 is detectable (>8% transfected cells), cells are selected using the antibiotic as informed by the vector used. The optimal dose of antibiotic is determined in a prior experiment treating the A549 WT cells with a 2-fold serial dilution of antibiotic to establish the dilution that kills >80% of cells. For single cell colonies selection, a 2-fold serial dilution is made starting from 20,000 cells / mL and cells are treated with the optimal dose of antibiotic. Cells are regularly inspected for growth and, after 10-14 days, single cell colonies should form. Single cell colonies are monitored until they cover ~25% of the well surface. Single cell colonies are then sub-cultured, daily inspected for growth, and screened for MDR1 overexpression using flow cytometry. The single cell clones showing the best growth (~75% confluence) and MDR1 expression are selected. Generation of A549 MMAE-resistant cell line

[0450] A549 MMAE-resistant cell lines are established by continuous treatment with MSADC-1P or MSADC-2P. A549 wildtype (WT) cells (ATCC; cat.no. CCL-185) are exposed to 0.23 nM MSADC-1P or 0.29 nM MSADC-2P for 48 hours. Media is removed and cells are washed with PBS. Fresh media without MSADC is added to allow cells to recover until cell proliferation is restored. Subsequently, cells are washed with PBS and fresh media is added containing a higher concentration of MSADC-1P (0.28 nM) or MSADC-2P (0.35 nM). Exposed cells are followed visually along time to inspect for cell death. The MSADC-1P and MSADC-2P concentrations are increased with 20% at each cycle until clones with robust resistance remain, which are then selectedWSGR Docket No.: 58964-801.601 and grown individually. Resistant cell lines are confirmed to be resistant to MMAE and not to the antibody component. Alternatively, A549 MMAE resistant cell lines are similarly established by continuous exposure to MMAE as free payload instead of to MSADC-1P or MSADC-2P. Generation of A549 MMAF-resistant cell line

[0451] A549-resistant cell lines are established by continuous treatment with MSADC-1Q or MSADC-2Q. A549 wildtype (WT) cells (ATCC; cat.no. CCL-185) are exposed to 0.22 nM MSADC-1Q or 0.15 nM MSADC-2Q for 48 hours. Media is removed and cells are washed with PBS. Fresh media without MSADC is added to allow cells to recover until cell proliferation is restored. Subsequently, cells are washed with PBS and fresh media is added containing a higher concentration of MSADC-1Q (0.26 nM) or MSADC-2Q (0.18 nM). Exposed cells are followed visually along time to inspect for cell death. The MSADC-1Q and MSADC-2Q concentrations are increased with 20% at each cycle until clones with robust resistance remain, which are then selected and grown individually. Resistant cell lines are confirmed to be resistant to MMAF and not to the antibody component.

[0452] Alternatively, A549 MMAF resistant cell lines are similarly established by continuous exposure to MMAF as free payload instead of to MSADC-1Q or MSADC-2Q. Generation of A549 DM1-resistant cell line

[0453] A549 DM1-resistant cell lines are established by continuous treatment with MSADC-1R or MSADC-2R. A549 wildtype (WT) cells (ATCC; cat.no. CCL-185) are exposed to 0.40 nM MSADC-1R or 0.10 nM MSADC-2R for 48 hours. Media is removed and cells are washed with PBS. Fresh media without ADC is added to allow cells to recover until cell proliferation is restored. Subsequently, cells are washed with PBS and fresh media is added containing a higher concentration of MSADC-1Q (0.48 nM) or MSADC-2Q (0.12 nM). Exposed cells are followed visually along time to inspect for cell death. The MSADC-1R and MSADC-2R concentrations are increased with 20% at each cycle until clones with robust resistance remain, which are then selected and are grown individually. Resistant cell lines are confirmed to be resistant to DM1 and not to the antibody component.

[0454] Alternatively, A549 DM1 resistant cell lines are similarly established by continuous exposure to DM1 as free payload instead of to MSADC-1R or MSADC-2R. Generation of A549 Dxd-resistant cell line

[0455] A549 Dxd-resistant cell lines are established by continuous treatment with MSADC-1S or MSADC-2S. A549 wildtype (WT) cells (ATCC; cat.no. CCL-185) are exposed to a concentration and for a period previously established with A549 WT cells and MSADC that result in measurableWSGR Docket No.: 58964-801.601 IC50 values. Cells are exposed to the IC20 concentration until proliferation is restored. Exposed cells are followed visually along time to inspect for cell death. The MSADC-1S and MSADC-2S concentrations are increased with 20% at each cycle until clones with robust resistance remain, which are then selected and are grown individually. Resistant cell lines are confirmed to be resistant to Dxd and not to the antibody component.

[0456] Alternatively, A549 Dxd resistant cell lines are similarly established by continuous exposure to Dxd as free payload instead of to MSADC-1S or MSADC-2S. Generation of A549 cisplatin-resistant cell line

[0457] A549 cisplatin-resistant cell lines are established by continuous treatment with MSADC-1T or MSADC-2T. A549 wildtype (WT) cells (ATCC; cat.no. CCL-185) are exposed to a concentration and for a period previously established with A549 WT cells and MSADC that result in measurable IC50 values. Cells are exposed to the IC20 concentration until proliferation is restored. Exposed cells are followed visually along time to inspect for cell death. The MSADC-1T and MSADC-2T concentrations are increased with 20% at each cycle until clones with robust resistance remain, which are then selected and are grown individually. Resistant cell lines are confirmed to be resistant to cisplatin and not to the antibody component.

[0458] Alternatively, A549 cisplatin resistant cell lines are similarly established by continuous exposure to cisplatin as free payload instead of to MSADC-1T or MSADC-2T. Example 8 – Binding of MMADCs to resistant cell lines

[0459] This experiment is to show that the ADCs bind to the resistant cell lines and display similar binding activity as the naked BsAbs, confirming that the conjugation does not affect binding of the bispecific antibody to its target antigens.

[0460] Binding of BsAb1, BsAb2, MSADC-1P-T, MSADC-2P-T, MMADC-1A-O, and MMADC- 2A-O to A549 resistant cell lines, is assessed by FACS. A549 resistant cells as described in Example 7 are recovered from liquid nitrogen storage and prepared at 1 x 106cells / ml in flow cytometry buffer (PBS (Thermo Fisher Scientific, 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 ADCs are prepared at 10 µg / ml starting concentration, and subjected to a 10-point, 3-fold serial dilution. Cells are re-suspended with either titrated ADC, naked BsAb, or assay control antibody in duplicate (buffer; control wells) and incubated for 60 minutes at 4°C. After washing with buffer, the cells are 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 are washed in buffer and fixed with 10% BD CellFix (BD Biosciences,WSGR Docket No.: 58964-801.601 Oxford, UK). Fluorescence is measured using an Attune NxT Acoustic Focusing Cytometer (ThermoFisher Scientific, Loughborough, UK), and data is analyzed using FlowJo v10 (FlowJo, Ashland, OR). Data is fitted using a 4-parameter logistic (4PL) regression in GraphPad Prism 10 (GraphPad Software, La Jolla, CA). Example 9 – Internalization of MMADCs

[0461] This experiment is to generate data supporting that MMADC-1A-O exhibit internalization activity on A549 resistant cell lines.

[0462] The ability of MSADC-1P 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. Cetuximab (Selleck Chemicals A2000) was used as reference control antibody. To determine ADC internalization capacity in target cells, cells were monitored live for 24 hours, using Incucyte®S3 live cell analysis system. 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 IncuCyte®Fabfluor-pH Red antibody labeling reagent (Sartorius cat. No.4722), 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 IncuCyte®Fabfluor-pH reagent at 3:1 IncuCyte®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. For analysis, 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.

[0463] The results shown in FIG.10 show that MSADC-1P induced internalization in A549 WT cells and A549 c-MET-KO cells. Also, MSADC-1P showed relative higher internalization than cetuximab in A549 WT cells and A549 c-MET-KO cells. It can be reasonably expected that MMADC-1A-O similarly internalize in A549 WT cells. Example 10 – In vitro potency of MMADCs

[0464] The in vitro potency of the MMADCs is measured in a panel of different cancer cell lines, selected from for instance the cell lines described in Example 1.

[0465] Cells are plated at suitable densities in their recommended culture media, supplemented with 10% fetal bovine serum as appropriate. Serial dilutions of the ADC and controls are prepared using culture medium. The ADC and controls are added to the cells and incubated for 72 hours atWSGR Docket No.: 58964-801.601 37^C in a humidified incubator. The metabolic activity is determined using CellTiter-Glo Luminescent Viability Assay from Promega according to manufacturer’s instructions.

[0466] This experiment is to show that the MMADCs are capable of killing cancer cells having different expression levels of EGFR, or EGFR and cMET, and does so with a relative higher potency as compared to a benchmark compound. Experiment 1

[0467] This experiment is to show that the MMADCs comprising BsAb1 induce concentration- dependent cell death of NCI-H1975 cells, at least comparable to AZ9592, and that the MMADCs comprising BsAb2 induce concentration-dependent cell death of A549 WT cells, at least comparable to their respective MSADCs.

[0468] The in vitro potency of MMADC-1A-O at DAR 4 (2+2) or DAR 6 (2+4 or 4+2) is measured on NCI-H1975 cells and compared with AZ9592. The in vitro potency of MMADC-2A-O at DAR 4 (2+2) or DAR 6 (2+4 or 4+2) is measured on A549 WT cells and compared with their respective MSADCs. Suitable assay controls are taken along.

[0469] NCI-H1975 WT (ATCC; cat. no. CRL-5908) cells are seeded into 96-well plates at a density of 2 x 103cells per well in complete medium (50 µL). Plates are then incubated for 24 hours at 37 °C / 5% CO2. After 24 hours, samples are prepared in complete medium at previously validated final assay concentrations and added to the cells in duplicate (25 µL).The ADCs are added at the same total antibody concentration. Assay plates are analyzed after 120 hours and viability assessment is undertaken utilizing the CellTiter-Glo® luminescence assay. Briefly, assay plates are equilibrated at room temperature for 20 minutes before addition of CellTiter-Glo® reagent (100 µL per well, Sartorius, #4633). The plates are 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 is recorded using a SpectraMax i3x plate reader (Molecular Devices, Wokingham, UK). Data is then analyzed on GraphPad Prism version 10 (GraphPad Software, La Jolla, CA) using a four-parameter non-linear regression model. Viability of treated cultures is compared to viability of control, untreated cells (100% viable) and expressed as percentage. The percentage viability is plotted against the drug concentration in nM. Example 11 – Potency of MMADCs in killing A549 resistant cells

[0470] This experiment is to show that MMADCs have a higher potency in killing A549 cells resistant against one of the MMADC payloads than their corresponding MSADCs.

[0471] A549 WT and A549 resistant cells as described in Example 7 are seeded into 96-well plates at a density of 2 x 103cells per well in 50 µL of complete medium (Ham’s F12K (Kaighn’s) medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U / mL penicillin, 100 µg / mLWSGR Docket No.: 58964-801.601 streptomycin). Plates are incubated for 0-24 hours at 37 °C / 5% CO2. After 24 hours, 8-point 1:2.5 serial dilution of test samples: MMADC or MSADC, are prepared in complete medium and added to the cells in duplicate (25 µL). Cells are incubated with samples without reagent replacement for a duration previously established with A549 WT cells and MSADC that result in measurable IC50 values.

[0472] Viability endpoint assessment is performed with the CellTiter-Glo® luminescence assay. Assay plates are equilibrated to room temperature for 20 minutes before addition of CellTiter-Glo® reagent (100 µL per well). The plates are 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 is recorded using a SpectraMax i3x plate reader. Viability of treated cultures is compared to viability of control, untreated cells (100% viable) and expressed as percentage. The percentage viability is plotted against the drug concentration in nM and the software is used to calculate the IC₅₀ values for all test samples from the generated dose response curves. Example 12 - In vivo efficacy Anti-tumor activity in in vivo xenograft models

[0473] This experiment is to show that the MMADCs are well tolerated and not obviously toxic to the mice, as well as are effective in reducing tumor volume or tumor growth.

[0474] The MMADCs are tested for their effect on tumor growth in vivo in a NCI-H1975 xenograft mouse model. About 5 × 105, or about 5 × 106, NCI-H1975 cells are injected subcutaneously in B- NDG mice (Biocytogen Pharmaceuticals (Beijing) Co., Ltd., Cat#: B-CM-002). When the tumors in the mice reach a volume of about 300 mm3, or 400 mm3, the mice are randomly placed into different groups based on the volume of the tumor. The mice are then injected with phosphate buffer saline (PBS), the dual payload EGFRxcMET bispecific ADC, or controls by intravenous (i.v.) administration once a week for two weeks.

[0475] The lengths of the long axis and the short axis of the tumor are measured and the volume of the tumor is calculated as 0.5 × (long axis) × (short axis)2. The tumor growth inhibition (TGI) is calculated using the following formula: TGI (%) = [1- (Ti-T0) / (Vi-V0)] × 100, wherein Ti is the average tumor volume in the treatment group on day I; T0 is the average tumor volume in the treatment group on day zero; Vi is the average tumor volume in the control group on day I; and V0 is the average tumor volume in the control group on day zero. T-test is performed for statistical analysis. The body weight of the mice is measured twice a week.

[0476] The experiment is also performed in an NCI-H292 xenograft mouse model, using about 5 × 106, or about 1 × 107, NCI-H292 cells. When the tumors in the mice reach a volume of about 300WSGR Docket No.: 58964-801.601 mm3, or 200 mm3, the mice are randomly placed into different groups based on the volume of the tumor.

[0477] The experiment is also performed in a SNU-5 xenograft mouse model, using about 1 × 107SNU-5 cells. When the tumors in the mice reach a volume of about 200 mm3, the mice are randomly placed into different groups based on the volume of the tumor. Experiment 1

[0478] This experiment is to show that the dual payload MMADC-1C and MMADC-2C have an increased tumor inhibitory effect compared to the vehicle and the single payload MSADC-1P, MSADC-1S, MSADC-2P, and MSADC-2S in this mouse model.

[0479] The dual payload MMADC-1C and MMADC-2C are tested for their effect on tumor growth in vivo in a human non-small cell lung cancer A549 xenograft mouse model using MMAE or Dxd resistant A549 cells. BALB / nude mice (6-8 weeks old; Jiangsu GemPharmatech Co.,Ltd) are injected with 1 x107A549 cells subcutaneously into the right flank of each mouse. Tumors are measured using a caliper according to the formula volume = 0.5 × (long axis) × (short axis)2. When the tumors reach ~200-250 mm3volume, mice are randomized. In order to determine the maximum tolerated dose (MTD), the highest dose that does not induce >20% weight loss and / or signs of distress, a dose range of MMADC-1C or MMADC-2C (0.1-15 mg / kg) is applied to mice via tail vein, as a single dose for two weeks. Mice are monitored for potential side effects, including lack of energy and loss of appetite. Taking side effects, including weight loss, into consideration, the MTD is selected.

[0480] For the in vivo efficacy experiment, mice are randomized in 5 groups for each resistance model: the MMADC-1C group, the MSADC-1P group, the MMADC-2C group, the MSADC-2P group, and the control group for the MMAE resistance model, and the MMADC-1C group, the MSADC-1S group, the MMADC-2C group, the MSADC-2S group, and the control group for the Dxd resistance model. The sample size per group is determined according to recommendations and the power calculations provided by Crown Bioscience. Mice are injected with a single dose of either MMADC-1C, MSADC-1P, MMADC-2C, or MSADC-2P at the determined MTD dose and a 50% lower dose, or phosphate buffer saline (PBS). Weight of the mice and tumor size is monitored once every three days, and study is terminated after 3 weeks or when reaching a humane endpoint (tumor volume exceeding 2000 m3). The tumor growth inhibition (TGI) is calculated using the following formula: TGI (%) = [1- (Ti-T0) / (Vi-V0)] × 100, wherein Ti is the average tumor volume in the treatment group on day 1; T0 is the average tumor volume in the treatment group on day 0; Vi is the average tumor volume in the control group on day 1; and V0 is the average tumor volume in theWSGR Docket No.: 58964-801.601 control group on day 0. For statistical analysis, Bartlett's test is used for comparison among more than 2 groups.

[0481] Alternatively, several in vitro, in vivo and ex vivo resistance cancer models are commercially available which can be used to demonstrate the ability of the MMADCs to overcome resistance to a specific prior line of therapy, including to therapy with topoisomerase inhibitors, taxanes, and platinum-based compounds. Example 13 – MMADCs MMADCs comprising BsAb1 or BsAb2 conjugated to a topoisomerase inhibitor as the first drug and a platinum-based chemotherapeutic agent as the second drug are generated. Combinations of the topoisomerase inhibitor and platinum-based therapeutic agent are listed in Table 9. Table 9. Combinations of topoisomerase inhibitors and platinum-based chemotherapeutic agents for use in MMADCs Topoisomerase inhibitor Platinum-based chemotherapeutic agent Exatecan K2PtCl4 Cisplatin (cis-DDP) cis-aq Oxaliplatin (O-LH) DACHPt Methylininodiacetato-1,2-daminocyclohexane platinum(II) (MIDP) Pt(DACH)Glu) Carboplatin Nedaplatin Dicycloplatin Triplatin tetranitrate Phenanthriplatin Picoplatin Satraplatin C8Pt(IV) Dxd K2PtCl4Cisplatin (cis-DDP) cis-aq Oxaliplatin (O-LH) DACHPt Methylininodiacetato-1,2-daminocyclohexane platinum(II) (MIDP) Pt(DACH)Glu) CarboplatinWSGR Docket No.: 58964-801.601 Nedaplatin Dicycloplatin Triplatin tetranitrate Phenanthriplatin Picoplatin Satraplatin C8Pt(IV) Irinotecan K2PtCl4 Cisplatin (cis-DDP) cis-aq Oxaliplatin (O-LH) DACHPt Methylininodiacetato-1,2-daminocyclohexane platinum(II) (MIDP) Pt(DACH)Glu) Carboplatin Nedaplatin Dicycloplatin Triplatin tetranitrate Phenanthriplatin Picoplatin Satraplatin C8Pt(IV) Topotecan K2PtCl4Cisplatin (cis-DDP) cis-aq Oxaliplatin (O-LH) DACHPt Methylininodiacetato-1,2-daminocyclohexane platinum(II) (MIDP) Pt(DACH)Glu) Carboplatin Nedaplatin Dicycloplatin Triplatin tetranitrate Phenanthriplatin Picoplatin Satraplatin C8Pt(IV) Belotecan K2PtCl4WSGR Docket No.: 58964-801.601 Cisplatin (cis-DDP) cis-aq Oxaliplatin (O-LH) DACHPt Methylininodiacetato-1,2-daminocyclohexane platinum(II) (MIDP) Pt(DACH)Glu) Carboplatin Nedaplatin Dicycloplatin Triplatin tetranitrate Phenanthriplatin Picoplatin Satraplatin C8Pt(IV)

[0482] While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the disclosure. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.WSGR Docket No.: 58964-801.601 SEQUENCES SEQ ID NO: 1 – Light chain variable region DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIK SEQ ID NO: 2 – Light chain CDR1 according to IMGT QSISSY Light chain CDR2 according to IMGT AAS SEQ ID NO: 4 – Light chain CDR3 according to IMGT QQSYSTPPT SEQ ID NO: 5 – CL region RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 6 – hinge region EPKSCDKTHTCPPCP SEQ ID NO: 7 - CH1 region ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV SEQ ID NO: 8 – CH2 region APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK SEQ ID NO: 9 – CH2-DM region APELGRGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK SEQ ID NO: 10 – CH3 regionWSGR Docket No.: 58964-801.601 GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 11 – CH3-DE region GQPREPQVYTDPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 12 – CH3-KK region GQPREPQVYTKPPSREEMTKNQVSLKCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 13 – Heavy chain variable region QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNT NYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDSYWHWWLGAFDYWGQG TLVTVSS 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 DSYWHWWLGAFDY SEQ ID NO: 17 – Heavy chain variable region QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNT NYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCAKDRHWHWWLDAFDYWGQG TLVTVSS SEQ ID NO: 18 – Heavy chain CDR1 according to Kabat SYGIS SEQ ID NO: 19 – Heavy chain CDR2 according to KabatWSGR Docket No.: 58964-801.601 WISAYNGNTNYAQKLQG SEQ ID NO: 20 – Heavy chain CDR3 according to Kabat DRHWHWWLDAFDY SEQ ID NO: 21 – Heavy chain variable region QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNT NYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARGYLDHWWLGAFDYWGQG TLVTVSS SEQ ID NO: 22 – Heavy chain CDR1 according to Kabat SYGIS SEQ ID NO: 23 – Heavy chain CDR2 according to Kabat WISAYNGNTNYAQKLQG SEQ ID NO: 24 – Heavy chain CDR3 according to Kabat GYLDHWWLGAFDY SEQ ID NO: 25 – Heavy chain variable region EVQLVESGPEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGTINPSGGST YYAQKFQGRVTLTRDTSTSTVYMVLSSLRSEDTAVYYCARDRNWGWDFDYWGQGTLVT VSS SEQ ID NO: 26 – Heavy chain CDR1 according to Kabat SYYMH SEQ ID NO: 27 – Heavy chain CDR2 according to Kabat TINPSGGSTYYAQKFQG SEQ ID NO: 28 – Heavy chain CDR3 according to Kabat DRNWGWDFDY SEQ ID NO: 29 – Heavy chain variable regionWSGR Docket No.: 58964-801.601 QVQLVQSGSELKKPGASVKISCKASGYDFTNYAMNWVRQAPGHGLEWMGWINANTGDP TYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDSAVYYCTRERFLEWLHFDYWGQGTLVTV SS SEQ ID NO: 30 – Heavy chain CDR1 according to Kabat NYAMN SEQ ID NO: 31 – Heavy chain CDR2 according to Kabat WINANTGDPTYAQGFTG SEQ ID NO: 32 – Heavy chain CDR3 according to Kabat ERFLEWLHFDY SEQ ID NO: 33 – Heavy chain variable region QVQLVQSGAEVKKPGASVKVSCKVSGYTLTELSMHWVRQAPGKGLEWMGGFDPEYGK TFFAQNFQGRVTMTEDTSADTAYMELSSLRSEDTAVYYCATEGYYETTTYYYNLFDSWG QGTLVTVSS SEQ ID NO: 34 – Heavy chain CDR1 according to Kabat ELSMH SEQ ID NO: 35 – Heavy chain CDR2 according to Kabat GFDPEYGKTFFAQNFQG SEQ ID NO: 36 – Heavy chain CDR3 according to Kabat EGYYETTTYYYNLFDS SEQ ID NO: 37 – Heavy chain variable region QVQLQESGPGLVKPSETLSLTCTVSGDSISSSSYYWGWIRQSPGKGLEWIGSIYYGGITYYN PSLKSRVTISADTSKNQFSLKLSSVTAADTAVYYCASGDLFITGTLDYWGQGTLVTVSS SEQ ID NO: 38 – Heavy chain CDR1 according to Kabat SSSYYWGWSGR Docket No.: 58964-801.601 SEQ ID NO: 39 – Heavy chain CDR2 according to Kabat SIYYGGITYYNPSLKS SEQ ID NO: 40 – Heavy chain CDR3 according to Kabat GDLFITGTLDY SEQ ID NO: 41 – Heavy chain variable region QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYSGNT NYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARGYLDHWWLGAFDYWGQG TLVTVSS SEQ ID NO: 42 – Heavy chain CDR1 according to Kabat SYGIS SEQ ID NO: 43 – Heavy chain CDR2 according to Kabat WISAYSGNTNYAQKLQG SEQ ID NO: 44 – Heavy chain CDR3 according to Kabat GYLDHWWLGAFDY SEQ ID NO: 45 – Heavy chain variable region QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNANT NYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARGYLDHWWLGAFDYWGQG TLVTVSS SEQ ID NO: 46 – Heavy chain CDR1 according to Kabat SYGIS SEQ ID NO: 47 – Heavy chain CDR2 according to Kabat WISAYNANTNYAQKLQG SEQ ID NO: 48 – Heavy chain CDR3 according to Kabat GYLDHWWLGAFDYWSGR Docket No.: 58964-801.601 SEQ ID NO: 49 – Heavy chain variable region QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYSGNT NYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDSYWHWWLGAFDYWGQG TLVTVSS SEQ ID NO: 50 – Heavy chain CDR1 according to Kabat SYGIS SEQ ID NO: 51 – Heavy chain CDR2 according to Kabat WISAYSGNTNYAQKLQG SEQ ID NO: 52 – Heavy chain CDR3 according to Kabat DSYWHWWLGAFDY SEQ ID NO: 53 – Heavy chain variable region QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNANT NYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDSYWHWWLGAFDYWGQG TLVTVSS SEQ ID NO: 54 – Heavy chain CDR1 according to Kabat SYGIS SEQ ID NO:55 – Heavy chain CDR2 according to Kabat WISAYNANTNYAQKLQG SEQ ID NO: 56 – Heavy chain CDR3 according to Kabat DSYWHWWLGAFDY SEQ ID NO: 57 – Heavy chain variable region QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYSGNT NYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCAKDRHWHWWLDAFDYWGQG TLVTVSS SEQ ID NO: 58 – Heavy chain CDR1 according to KabatWSGR Docket No.: 58964-801.601 SYGIS SEQ ID NO: 59 – Heavy chain CDR2 according to Kabat WISAYSGNTNYAQKLQG SEQ ID NO: 60 – Heavy chain CDR3 according to Kabat DRHWHWWLDAFDY SEQ ID NO: 61 – Heavy chain variable region QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNANT NYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCAKDRHWHWWLDAFDYWGQG TLVTVSS SEQ ID NO: 62 – Heavy chain CDR1 according to Kabat SYGIS SEQ ID NO: 63 – Heavy chain CDR2 according to Kabat WISAYNANTNYAQKLQG SEQ ID NO: 64 – Heavy chain CDR3 according to Kabat DRHWHWWLDAFDY SEQ ID NO: 65 – Heavy chain variable region EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGKSHYSWDAFDYWGQGTLVT VSS SEQ ID NO: 66 – Heavy chain CDR1 according to Kabat SYAMS SEQ ID NO: 67 – Heavy chain CDR2 according to Kabat AISGSGGSTYYADSVKG SEQ ID NO: 68 – Heavy chain CDR3 according to KabatWSGR Docket No.: 58964-801.601 GKSHYSWDAFDY SEQ ID NO: 69 – Heavy chain variable region QVQLQESGPGLVKPSETLSLTCTVSGGSMSSYYWSWIRQPPGKGLEWIGYIFNSGSTTYKP SFESRVSMSVDTSNNQFSLRLTSVTAADTAVYYCARGTYYYGSGSFSTRVFDAFDVWGQG TMVTVSS SEQ ID NO: 70 – Heavy chain CDR1 according to Kabat SYYWS SEQ ID NO: 71 – Heavy chain CDR2 according to Kabat YIFNSGSTTYKPSFES SEQ ID NO: 72 – Heavy chain CDR3 according to Kabat GTYYYGSGSFSTRVFDAFDV SEQ ID NO: 73 – Heavy chain variable region QVQLVQSGAEVKKPGESLKISCKGSGNSFTNYWIGWVRQVPGKGLEWMGIIYPGDSDTR YSPSFQGQVTISADKSLSTAYLQWSSLKASDTAMYYCARQSRRYSGYASYFDYWGQGTLV TVSS SEQ ID NO: 74 – Heavy chain CDR1 according to Kabat NYWIG SEQ ID NO: 75 – Heavy chain CDR2 according to Kabat IIYPGDSDTRYSPSFQG SEQ ID NO: 76 – Heavy chain CDR3 according to Kabat QSRRYSGYASYFDY SEQ ID NO: 77 – Heavy chain variable region EVQLVQSGAEVKKPGESLKISCKGPEYSFTNYWIGWVRQMPGKGLEWMGIIYPGDSDTT YSPSFQGQVTISADKSISTAYLQWNSLKASDTAMYYCARQRRAYSGYNWYFDLWGRGTL VTVSSWSGR Docket No.: 58964-801.601 SEQ ID NO: 78 – Heavy chain CDR1 according to Kabat NYWIG SEQ ID NO: 79 – Heavy chain CDR2 according to Kabat IIYPGDSDTTYSPSFQG SEQ ID NO: 80 – Heavy chain CDR3 according to Kabat QRRAYSGYNWYFDL SEQ ID NO: 81 – Heavy chain variable region QVQLQESGPGLVKPSETLSLTCNVSGGSISNFYWSWIRQPPGKGLEWIGHIYYSGSTNYNP SLKSRVTISIDTSKNQFSLNLSSVTAADTAVYYCARRNDFWSGYLFDYWGQGTLVTVSS SEQ ID NO: 82 – Heavy chain CDR1 according to Kabat NFYWS SEQ ID NO: 83 – Heavy chain CDR2 according to Kabat HIYYSGSTNYNPSLKS SEQ ID NO: 84 – Heavy chain CDR3 according to Kabat RNDFWSGYLFDY SEQ ID NO: 85 – Heavy chain variable region QMQLVQSGAEVKKPGSSVKVSCKASGGTFSIYAISWVRQAPGQGLEWMGGIIPISDTPNY AQKFQGRVTITADNSTNTAYMELSSLRSEDTAVYYCATKTTVGYYYYYMDVWGKGTTVT VSS SEQ ID NO: 86 – Heavy chain CDR1 according to Kabat IYAIS SEQ ID NO: 87 – Heavy chain CDR2 according to Kabat GIIPISDTPNYAQKFQGWSGR Docket No.: 58964-801.601 SEQ ID NO: 88 – Heavy chain CDR3 according to Kabat KTTVGYYYYYMDV SEQ ID NO: 89 – Heavy chain variable region EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSSAINWVRQAPGQGLEWMGGIVPIFGTLD DAQKFQGRVKFTADKSTSTAYMELSSLRSEDTAVYYCARGPELGYYYYYMDIWGKGTTV TVSS SEQ ID NO: 90 – Heavy chain CDR1 according to Kabat SSAIN SEQ ID NO: 91– Heavy chain CDR2 according to Kabat GIVPIFGTLDDAQKFQG SEQ ID NO: 92 – Heavy chain CDR3 according to Kabat GPELGYYYYYMDI SEQ ID NO: 93 – Heavy chain variable region EVQLVESGGGLVKPGGSLRLSCAASGFTFSKAWMNWVRQAPGKGLEWVGRIKSKTDGG TTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTASSMITFGGVIVSWFDPW GQGTLVTVSS SEQ ID NO: 94 – Heavy chain CDR1 according to Kabat KAWMN SEQ ID NO: 95 – Heavy chain CDR2 according to Kabat RIKSKTDGGTTDYAAPVKG SEQ ID NO: 96 – Heavy chain CDR3 according to Kabat ASSMITFGGVIVSWFDP SEQ ID NO: 97 – Heavy chain variable regionWSGR Docket No.: 58964-801.601 EVQLVQSGSELKKPGSSVKVSCKASGGTFSTYAISWVRQAPGQGLEWMGGIIPIFGTTDY AQKFQDRVKITADKSTSTAYMELSSLRSEDTAVYYCARKLEPTGYYYYYMDVWGKGTTV TVSS SEQ ID NO: 98 – Heavy chain CDR1 according to Kabat TYAIS SEQ ID NO: 99 – Heavy chain CDR2 according to Kabat GIIPIFGTTDYAQKFQD SEQ ID NO: 100 – Heavy chain CDR3 according to Kabat KLEPTGYYYYYMDV SEQ ID NO: 101 – Heavy chain variable region EVQLVQSGAEVKKPGESLKISCKGSGNSFNNYWIGWVRQMPGKGLEWMGIIYPGDSDTR YSPSFQGQVIISADKSISTAYLQWSSLKASDTAMYFCVRRVNRYSGYATYFDLWGRGTLVT VSS SEQ ID NO: 102 – Heavy chain CDR1 according to Kabat NYWIG SEQ ID NO: 103 – Heavy chain CDR2 according to Kabat IIYPGDSDTRYSPSFQG SEQ ID NO: 104 – Heavy chain CDR3 according to Kabat RVNRYSGYATYFDL SEQ ID NO: 105 – Heavy chain variable region QVQLVQSGSELKKPGASVKVSCKASGYTFTSYSMNWVRQAPGQGLEWMGWINTYTGDP TYAQGFTGRYVFSLDTSVNTAYLQISSLKAEDTAVYYCARETYYYDRGGYPFDPWGQGTL VTVSS SEQ ID NO: 106 – Heavy chain CDR1 according to Kabat SYSMNWSGR Docket No.: 58964-801.601 SEQ ID NO: 107 – Heavy chain CDR2 according to Kabat WINTYTGDPTYAQGFTG SEQ ID NO: 108 – Heavy chain CDR3 according to Kabat ETYYYDRGGYPFDP SEQ ID NO: 109 – Heavy chain variable region EVQLVESGGGLVQPGGSLRLSCAASGFIFSGYDMHWVRQATGKGLEWVSGIDTTGDTYY PDSVKGRFTISREDDKNSLFLQMNSLRAGDTAVYYCARKTSRYSGYHYYMDVWGKGTT VTVSS SEQ ID NO: 110 – Heavy chain CDR1 according to Kabat GYDMH SEQ ID NO: 111 – Heavy chain CDR2 according to Kabat GIDTTGDTYYPDSVKG SEQ ID NO: 112 – Heavy chain CDR3 according to Kabat KTSRYSGYHYYMDV SEQ ID NO: 113 – Heavy chain variable region EVQLVESGGGLVQPGGSLRLSCAASGFTFSGYDMHWVRQATGKGLEWVSAIGSTGDTYY PGSVKGRFTISRENAKNSLYLQMNSLRAGDTAVYYCARRTSRYSGYHYYLDVWGSGTTV TVSS SEQ ID NO: 114 – Heavy chain CDR1 according to Kabat GYDMH SEQ ID NO: 115 – Heavy chain CDR2 according to Kabat AIGSTGDTYYPGSVKG SEQ ID NO: 116 – Heavy chain CDR3 according to Kabat RTSRYSGYHYYLDVWSGR Docket No.: 58964-801.601 SEQ ID NO: 117 – Heavy chain variable region QLQLQESGPGLVKPSETLSLTCTVSGGSITSSTYYWGWIRQPPGKGLDWIGTIYYSGNTYY NPSLRSRVNISVDTSKNQFSLKLSSVTAADTAVYYCARLLYDLFDLWGRGTLVTVSS SEQ ID NO: 118 – Heavy chain CDR1 according to Kabat SSTYYWG SEQ ID NO: 119 – Heavy chain CDR2 according to Kabat TIYYSGNTYYNPSLRS SEQ ID NO: 120 – Heavy chain CDR3 according to Kabat LLYDLFDL SEQ ID NO: 121 – Heavy chain variable region QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSHAVSWVRQAPGQGLEWMGGIIPIFGTES NAEKFRGRVTITADKSTNTAYMELISLISEDTAVYYCARSIDMATITDAFDIWGHGTMVTV SS SEQ ID NO: 122 – Heavy chain CDR1 according to Kabat SHAVS SEQ ID NO: 123 – Heavy chain CDR2 according to Kabat GIIPIFGTESNAEKFRG SEQ ID NO: 124 – Heavy chain CDR3 according to Kabat SIDMATITDAFDI SEQ ID NO: 125 – Heavy chain variable region QLQLQESGPGLVKPSETLSLTCTVSGGSIGSSSYYWGWIRQPPGKGLEWIGSIYDSGNTYY NPSLKSRVTISVDTSKIQFSLKLSSVTAADTAVYYCARAHYDILTGWGQGTLVTVSS SEQ ID NO: 126 – Heavy chain CDR1 according to Kabat SSSYYWGWSGR Docket No.: 58964-801.601 SEQ ID NO: 127 – Heavy chain CDR2 according to Kabat SIYDSGNTYYNPSLKS SEQ ID NO: 128 – Heavy chain CDR3 according to Kabat AHYDILTG SEQ ID NO: 129 – Heavy chain variable region QVQLVQSGAEVKKPGSSVKVSCKGSGDTFNSYAISWVRQAPGQGLEWMGGIVPIFGTAD YAQRFQDRVTITADKSTSTAYMDLSSLRSEDTAVYYCARGTTGNPYYFYYYMDVWGNGT TVTVSS SEQ ID NO: 130 – Heavy chain CDR1 according to Kabat SYAIS SEQ ID NO: 131 – Heavy chain CDR2 according to Kabat GIVPIFGTADYAQRFQD SEQ ID NO: 132 – Heavy chain CDR3 according to Kabat GTTGNPYYFYYYMDV SEQ ID NO: 133 – Heavy chain variable region QVQLVQSGSELKKPGASVKVSCKASGYTFTDYAMNWVRQAPGQGLEWMGWINTYTGN PTYVQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARETYYYDSSGYPFDPWGQGTL VTVSS SEQ ID NO: 134 – Heavy chain CDR1 according to Kabat DYAMN SEQ ID NO: 135 – Heavy chain CDR2 according to Kabat WINTYTGNPTYVQGFTG SEQ ID NO: 136 – Heavy chain CDR3 according to Kabat ETYYYDSSGYPFDPWSGR Docket No.: 58964-801.601 SEQ ID NO: 137 – Heavy chain variable region QVELVQSGSELKKPGASVKVSCKASGYTFTTYSMNWVRQAPGQGLEWMGWINTNTGTP TYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARETYYYDSSGYPFDPWGQGTLV TVSS SEQ ID NO: 138 – Heavy chain CDR1 according to Kabat TYSMN SEQ ID NO: 139 – Heavy chain CDR2 according to Kabat WINTNTGTPTYAQGFTG SEQ ID NO: 140 – Heavy chain CDR3 according to Kabat ETYYYDSSGYPFDP SEQ ID NO: 141 – Heavy chain variable region QVQLVQSGSELKKPGASVKVSCKASGYTFTTYAMNWVRQAPGQGLEWMGWINTNTGN PTYAQGFTGRFVFSLDTSDSTAFLQISSLKAEDTAVYYCARETYYYDSSGYPFDPWGQGTL VTVSS SEQ ID NO: 142 – Heavy chain CDR1 according to Kabat TYAMN SEQ ID NO: 143 – Heavy chain CDR2 according to Kabat WINTNTGNPTYAQGFTG SEQ ID NO: 144 – Heavy chain CDR3 according to Kabat ETYYYDSSGYPFDP SEQ ID NO: 145 – Heavy chain variable region QVQLMQSGSELKKPGASVKVSCKASGYTFTTYSMNWVRQAPGQGLEWMGWINTNTGN PTYAQDFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARETYYYDSSGYPFDPWGQGTL VTVSSWSGR Docket No.: 58964-801.601 SEQ ID NO: 146 – Heavy chain CDR1 according to Kabat TYSMN SEQ ID NO: 147 – Heavy chain CDR2 according to Kabat WINTNTGNPTYAQDFTG SEQ ID NO: 148 – Heavy chain CDR3 according to Kabat ETYYYDSSGYPFDP SEQ ID NO: 149 – Heavy chain variable region QVQLVQSGSELKKPGASVKVSCKASGYTFTTYSLNWVRQAPGQGLEWMGWINTYTGNP TYAQGFTGRFVFFLDTSVSTAYLQISSLKAEDTAVYYCARETYYYDSSGYPFDPWGQGTLV TVSS SEQ ID NO: 150 – Heavy chain CDR1 according to Kabat TYSLN SEQ ID NO: 151 – Heavy chain CDR2 according to Kabat WINTYTGNPTYAQGFTG SEQ ID NO: 152 – Heavy chain CDR3 according to Kabat ETYYYDSSGYPFDP SEQ ID NO: 153 – Heavy chain variable region QVQLVQSGSELKKPGASVKVSCKASGYTFTTYSMNWVRQAPGQGLEWMGWINTYTGD PTYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARETYFYDRGGYPFDPWGQGT LVTVSS SEQ ID NO: 154 – Heavy chain CDR1 according to Kabat TYSMN SEQ ID NO: 155 – Heavy chain CDR2 according to Kabat WINTYTGDPTYAQGFTG SEQ ID NO: 156 – Heavy chain CDR3 according to KabatWSGR Docket No.: 58964-801.601 ETYFYDRGGYPFDP SEQ ID NO: 157 – Heavy chain variable region QVQVVQSGSEVKKPGASVKVSCKASGYTFTTYSMNWVRQAPGQGLEWMGWINTYTGD PTYVQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARETYFYDRGGYPFDPWGQGT LVTVSS SEQ ID NO: 158 – Heavy chain CDR1 according to Kabat TYSMN SEQ ID NO: 159 – Heavy chain CDR2 according to Kabat WINTYTGDPTYVQGFTG SEQ ID NO: 160 – Heavy chain CDR3 according to Kabat ETYFYDRGGYPFDP SEQ ID NO: 161 – Heavy chain variable region QVQLVQSGSELEKPGASVKVSCKASGYTFISYAMNWVRQAPGQGLEWMGWINTYTGNP TYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARETYYYDSGGYPFDPWGQGTL VTVSS SEQ ID NO: 162 – Heavy chain CDR1 according to Kabat SYAMN SEQ ID NO: 163 – Heavy chain CDR2 according to Kabat WINTYTGNPTYAQGFTG SEQ ID NO: 164 – Heavy chain CDR3 according to Kabat ETYYYDSGGYPFDP SEQ ID NO: 165 – Heavy chain variable region QVQLVQSGSELKKPGASVKVSCKASGYTFTTYSMNWVRQAPGQGLEWMGWINTNTGT PTYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARETYYYGSSGYPFAPWGQGTL VTVSSWSGR Docket No.: 58964-801.601 SEQ ID NO: 166 – Heavy chain CDR1 according to Kabat TYSMN SEQ ID NO: 167 – Heavy chain CDR2 according to Kabat WINTNTGTPTYAQGFTG SEQ ID NO: 168 – Heavy chain CDR3 according to Kabat ETYYYGSSGYPFAP SEQ ID NO: 169 – Heavy chain variable region QVQLVQSGSELKKPGASVKVSCKASGYTFTTYSMNWVRQAPGQGLEWMGWINTYTGN PTYAQGFTGRFVFSLDTSVSTAYLQISSLTTEDTAVYYCARETYYYESSGYPFDPWGQGTL VTVSS SEQ ID NO: 170 – Heavy chain CDR1 according to Kabat TYSMN SEQ ID NO: 171 – Heavy chain CDR2 according to Kabat WINTYTGNPTYAQGFTG SEQ ID NO: 172 – Heavy chain CDR3 according to Kabat ETYYYESSGYPFDP SEQ ID NO: 173 – Heavy chain variable region QVQLVQSGSELKKPGASVKVSCKASGYTFTSFGMSWVRQAPGQGLEWMGWINTNTGNP TYAQGFTGRFVFSLDTSVSTAYLQINSLKAEDTAVYYCARESYYYDRNDYPFDPWGQGTL VTVSS SEQ ID NO: 174 – Heavy chain CDR1 according to Kabat SFGMS SEQ ID NO: 175 – Heavy chain CDR2 according to Kabat WINTNTGNPTYAQGFTGWSGR Docket No.: 58964-801.601 SEQ ID NO: 176 – Heavy chain CDR3 according to Kabat ESYYYDRNDYPFDP SEQ ID NO: 177 – Heavy chain variable region QVQLVQSGSELEKPGASVKVSCKASGYTFTTYSMNWVRQAPGQGLEWMGWINTYTGSP TYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAIYYCARETFYFDSGGYPFDPWGQGTLV TVSS SEQ ID NO: 178 – Heavy chain CDR1 according to Kabat TYSMN SEQ ID NO: 179 – Heavy chain CDR2 according to Kabat WINTYTGSPTYAQGFTG SEQ ID NO: 180 – Heavy chain CDR3 according to Kabat ETFYFDSGGYPFDP SEQ ID NO: 181 – Heavy chain variable region QVQLVQSGSELKKPGASVKVSCKASGYTFTDYAMNWVRQVPGQGLEWMGWINTYTGN PTYVQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARETYYYDSSGFPFDPWGQGTL VTVSS SEQ ID NO: 182 – Heavy chain CDR1 according to Kabat DYAMN SEQ ID NO: 183 – Heavy chain CDR2 according to Kabat WINTYTGNPTYVQGFTG SEQ ID NO: 184 – Heavy chain CDR3 according to Kabat ETYYYDSSGFPFDP SEQ ID NO: 185 – Heavy chain variable regionWSGR Docket No.: 58964-801.601 QVQLVQSGSELKKPGASVKVSCKASGYTFTTYSMNWVRQAPGQGLEWMGWINTNTGN PTYAQGFTGRFVFSLDTSVSTAYLQISSLNTEDTAVYYCARETYYYDVGGYPFDPWGQGT LVTVSS SEQ ID NO: 186 – Heavy chain CDR1 according to Kabat TYSMN SEQ ID NO: 187 – Heavy chain CDR2 according to Kabat WINTNTGNPTYAQGFTG SEQ ID NO: 188 – Heavy chain CDR3 according to Kabat ETYYYDVGGYPFDP SEQ ID NO: 189 – Heavy chain variable region QVQLVQSGSELEKPGASVKVSCKASGYTFTTYSMNWVRQAPGQGLEWMGWINTYTGSP TYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARETYYFDSGDYPFDPWGQGTLV TVSS SEQ ID NO: 190 – Heavy chain CDR1 according to Kabat TYSMN SEQ ID NO: 191 – Heavy chain CDR2 according to Kabat WINTYTGSPTYAQGFTG SEQ ID NO: 192 – Heavy chain CDR3 according to Kabat ETYYFDSGDYPFDP SEQ ID NO: 193 – Heavy chain variable region QVQLVQSGSELKKPGASVKVSCKASGYTFTNYAMNWVRQAPGQGLEWMGWINTNTGN PTYAQGFTGRFVFPLDTSVSTTYLQISSLKAEDTAVYYCARETYYYQSSGYLFDPWGQGT LVTVSS SEQ ID NO: 194 – Heavy chain CDR1 according to Kabat NYAMNWSGR Docket No.: 58964-801.601 SEQ ID NO: 195 – Heavy chain CDR2 according to Kabat WINTNTGNPTYAQGFTG SEQ ID NO: 196 – Heavy chain CDR3 according to Kabat ETYYYQSSGYLFDP SEQ ID NO: 197 – Heavy chain variable region QVQLVQSGSELKKPGASVKVSCKASGYTFTSYAMNWVRQAPGQGLEWMGWINTNTGN PTYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARETYYYDRGGYPFDPWGQGT LVTVSS SEQ ID NO: 198 – Heavy chain CDR1 according to Kabat SYAMN SEQ ID NO: 199 – Heavy chain CDR2 according to Kabat WINTNTGNPTYAQGFTG SEQ ID NO: 200 – Heavy chain CDR3 according to Kabat ETYYYDRGGYPFDP SEQ ID NO: 201 – Heavy chain variable region QVQLVQSGSELEKPGASVKVSCKASGYTFTTYSMNWVRQAPGQGLEWMGWINTYTGSP TYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARETYYFDSGGYPFDPWGQGTL VTVSS SEQ ID NO: 202 – Heavy chain CDR1 according to Kabat TYSMN SEQ ID NO: 203 – Heavy chain CDR2 according to Kabat WINTYTGSPTYAQGFTG SEQ ID NO: 204 – Heavy chain CDR3 according to Kabat ETYYFDSGGYPFDPWSGR Docket No.: 58964-801.601 SEQ ID NO: 205 – Heavy chain variable region QVQLVQSGSELKKPGASVKVSCKASGYTFTSYAVNWVRQAPGQGLEWMGWINTYTGNP TYAQGFTGRFVFSSDTSVNTAYLQISSLKAEDTAVYYCARETYFYDSSGYPFDPWGQGTLV TVSS SEQ ID NO: 206 – Heavy chain CDR1 according to Kabat SYAVN SEQ ID NO: 207 – Heavy chain CDR2 according to Kabat WINTYTGNPTYAQGFTG SEQ ID NO: 208 – Heavy chain CDR3 according to Kabat ETYFYDSSGYPFDP SEQ ID NO: 209 – Heavy chain variable region QVQLVQSGSELKKPGASVKVSCKASGYTFTDYAMNWVRQAPGQGLEWMGWINTYTGN PTYVQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARETYYYDSSAYPFDPWGQGTL VTVSS SEQ ID NO: 210 – Heavy chain CDR1 according to Kabat DYAMN SEQ ID NO: 211 – Heavy chain CDR2 according to Kabat WINTYTGNPTYVQGFTG SEQ ID NO: 212 – Heavy chain CDR3 according to Kabat ETYYYDSSAYPFDP SEQ ID NO: 213 – Heavy chain variable region QVQLVQSGSELKKPGASVKVSCKASGYTFTTYAINWVRQAPGQGLEWMGWINTNTGNP TYAQGFTGRFVFSLDTSVSTAHLQISSLKAEDTAVYYCARETYYYATSGYPFDPWGQGAL VTVSSWSGR Docket No.: 58964-801.601 SEQ ID NO: 214 – Heavy chain CDR1 according to Kabat TYAIN SEQ ID NO: 215 – Heavy chain CDR2 according to Kabat WINTNTGNPTYAQGFTG SEQ ID NO: 216 – Heavy chain CDR3 according to Kabat ETYYYATSGYPFDP SEQ ID NO: 217 – Heavy chain variable region QVQLVQSGSELKKPGASVKVSCKTSGYTFTDYAMTWVRQAPGQGLEWMGWITTNTGDP TYAPGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARVYHWIRGFEFWGQGTLVTVSS SEQ ID NO: 218 – Heavy chain CDR1 according to Kabat DYAMT SEQ ID NO: 219 – Heavy chain CDR2 according to Kabat WITTNTGDPTYAP...

Claims

WSGR Docket No.: 58964-801.601 CLAIMS 1. A multispecific multi-drug antibody-drug conjugate (MMADC) comprising: a multispecific antibody; a first linker connecting a first drug to the multispecific antibody at a first conjugation site; and a second linker connecting a second drug to the multispecific antibody at a second conjugation site; wherein the first drug and the second drug are each independently a microtubule inhibitor, a topoisomerase inhibitor, a DNA-damaging agent, a DNA damage repair inhibitor, a chemotherapeutic agent, or an RNA polymerase inhibitor, wherein the first drug is different from the second drug.

2. The MMADC of claim 1, wherein when: (a) the first drug is a microtubule inhibitor, the second drug is a topoisomerase inhibitor, a DNA-damaging agent, a DNA damage repair inhibitor, a chemotherapeutic agent, or an RNA polymerase inhibitor; or (b) the first drug is a topoisomerase inhibitor, the second drug is a microtubule inhibitor, a DNA-damaging agent, a DNA damage repair inhibitor, a chemotherapeutic agent, or an RNA polymerase; or (c) the first drug is a DNA-damaging agent, the second drug is a microtubule inhibitor, a topoisomerase inhibitor, a DNA damage repair inhibitor, a chemotherapeutic agent, or an RNA polymerase inhibitor; or (d) the first drug is a DNA damage repair inhibitor, the second drug is a microtubule inhibitor, a topoisomerase inhibitor, a DNA-damaging agent, a chemotherapeutic agent, or an RNA polymerase inhibitor; or (e) the first drug is a chemotherapeutic agent, the second drug is a microtubule inhibitor, a topoisomerase inhibitor, a DNA-damaging agent, a DNA damage repair inhibitor, or an RNA polymerase inhibitor; or (f) the first drug is an RNA polymerase inhibitor, the second drug is a microtubule inhibitor, a topoisomerase inhibitor, a DNA-damaging agent, a DNA damage repair inhibitor, or a chemotherapeutic agent.

3. The MMADC of claim 2, wherein the first drug is a microtubule inhibitor and the second drug is a topoisomerase inhibitor.

4. The MMADC of claim 1, wherein when: (a) the first drug is a microtubule inhibitor, the second drug is a different microtubule inhibitor; or (b) the first drug is a topoisomerase inhibitor, the second drug is a different topoisomerase inhibitor; orWSGR Docket No.: 58964-801.601 (c) the first drug is a DNA-damaging agent, the second drug is a different DNA- damaging agent; or (d) the first drug is a DNA damage repair inhibitor, the second drug is a different DNA damage repair inhibitor; or (e) the first drug is a chemotherapeutic agent, the second drug is a different chemotherapeutic agent; or (f) the first drug is an RNA polymerase inhibitor, the second drug is a different RNA polymerase inhibitor.

5. The MMADC of claim 4, wherein the first drug is a microtubule inhibitor and the second drug is a different microtubule inhibitor.

6. The MMADC of claim 4, wherein the first drug is a topoisomerase I inhibitor and the second drug is a topoisomerase II inhibitor.

7. The MMADC of claim 4, wherein the first drug is a chemotherapeutic agent and the second drug is a different chemotherapeutic agent.

8. The MMADC of any one of claims 1, 2 or 4, wherein the DNA-damaging agent is duocarmycins or pyrrolobenzodiazepines (PDBs).

9. The MMADC of any one of claims 1, 2, or 4, wherein the DNA damage response inhibitor (DDRi) is tuvusertib, M9466, lartesertib, peposertib, olaparib, talazoparib, veliparib, rucaparib, or niraparib.

10. The MMADC of any one of claims 1 to 9, wherein the first drug and the second drug is independently monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), tubulysin A, maytansine, ansamitocin P3, mertansine (DM1), ravtansine (DM4), calicheamicin, duocarmycin, topotecan, exatecan, DXd, irinotecan, cisplatin, oxaliplatin, paclitaxel, teniposide, SN38, hexylresorcinal, camptothecin, MM398, etoposide, novobiocin, doxorubicin, nemorubicin, daunorubicin, idarubicin, epipodophyllotoxin, toposide, teniposide, mitoxanthrone, pyrrolobenzodiazepine (PDB), TAS-103, 7-MAD_MDCPT, SN38, SG2199, amanitins, 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 pharmaceutically acceptable salt or derivative thereof.

11. The MMADC of any claim 10, wherein the first drug and the second drug are each independently monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), mertansine (DM1), ravtansine (DM4), Dxd, exatecan, doxorubicin, TAS-103, irinotecan, cisplatin, oxaliplatin, paclitaxel, 7-MAD-MDCPT, camptothecin, SN38, SG3199, or Amanitins, or a pharmaceutically acceptable salt or derivative thereof.WSGR Docket No.: 58964-801.601 12. The MMADC of claim 11, wherein the first drug and the second drug are each independently MMAE, MMAF, DM1, Dxd, doxorubicin, TAS-103, exatecan, cisplatin, or irinotecan.

13. The MMADC of any one of claims 1 to 12, wherein the first drug and the second drug are: (a) MMAE and MMAF; (b) MMAE and DM1; (c) MMAE and Dxd; (d) MMAE and doxorubicin; (e) MMAE and TAS-103; (f) MMAF and DM1; (g) MMAF and Dxd; (h) MMAF and doxorubicin; (i) MMAF and TAS-103; (j) DM1 and Dxd; (k) DM1 and doxorubicin; (l) DM1 and TAS-103; (m) Dxd and doxorubicin; (n) Dxd and exatecan; or (o) Cisplatin and irinotecan.

14. The MMADC of any one of claims 1 to 13, wherein the first drug has first drug-to- antibody (DAR) ratio of about 1.5 to about 8.

15. The MMADC of any one of claims 1 to 14, wherein the second drug has a second drug-to-antibody ratio (DAR) of about 0.7 to about 9.

1.

16. The MMADC of any one of claims 1 to 15, wherein the first linker and the second linker is independently connected to the multispecific antibody via a reaction using the reactive group selected from:WSGR Docket No.: 58964-801.60117. The MMADC of any one of claims 1 to 15, wherein the first linker and second linker is independently a branched linker.

18. The MMADC of claim 17, wherein the branched linker comprises the following structure:wherein denotes a connection leading to the drug, whereindenotes a connection leading to the antibody, wherein each of e, d and f is independently 0, 1, 2, 3, 4, 5, 6, 7 or 8, wherein at least one of e, d, and f is not 0.

19. The MMADC of any one of claims 1 to 18, wherein the first linker and second linker independently comprises maleimidocaproyl-valine-citrulline-paminobenzyloxycarbonyl (MC-VCP) or glutamic acid-valine-citrulline (GluValCit).

20. The MMADC of any one of claims 1 to 19, wherein the first linker and the second linker further independently comprises a first spacer and a second spacer.

21. The MMADC of claim 20, wherein the first spacer and second spacer independently comprises a polyethylene glycol (PEG).

22. The MMADC of any one of claims 1 to 21, wherein the first conjugate site and the second conjugation site is independently a reactive amino acid of the multispecific antibody.

23. The MMADC of claim 22, wherein the reactive amino acid has the following functional group: –SH, –SeH, –NH2, –CO2H, or –C(=O)–, or a derivative thereof.

24. The MMADC of any one of claims 1 to 23, wherein the multispecific antibody is a bispecific antibody or a trispecific antibody.

25. The MMADC of claim 24, wherein the multispecific antibody is a bispecific antibody.WSGR Docket No.: 58964-801.601 26. The MMADC of claim 25, wherein the bispecific antibody binds to two different targets, wherein the targets are independently EGFR, c-MET, LGR5, HER2, and HER3.

27. The MMADC of claim 26, wherein the targets are independently EGFR, c-MET, and LGR5.

28. The MMADC of any one of claims 1 to 27, wherein the multispecific antibody is a multispecific IgG1 antibody.

29. The MMADC of any one of claims 1 to 28, wherein the multispecific antibody comprises a first heavy chain, a second heavy chain, a first light chain, and a second light chain.

30. The MMADC of any one of claims 1 to 29, wherein the multispecific antibody comprises a first and a second CH3 domain, wherein the first CH3 domain comprises amino acid substitution T366K and L351K and the second CH3 domain comprises amino acid substitution L351D and L368E.

31. The MMADC of claim 30, wherein the first heavy chain has SEQ ID NO: 257, the second heavy chain has SEQ ID NO: 258, and the first light chain and the second light chain have SEQ ID NO:

259.

32. The MMADC of claim 30, wherein the first heavy chain has SEQ ID NO: 260, the second heavy chain has SEQ ID NO: 261, and the first light chain and the second light chain have SEQ ID NO:

259.

33. The MMADC of any one of claims 1 to 30, wherein the MMADC is selected from Table 3.

34. The MMADC of any one of claims 1 to 30, wherein the MMADC is selected from Table 4.

35. The MMADC of any one of claims 1 to 34, for use in the treatment of cancer.

36. The MMADC of claim 35, wherein the cancer is resistant to either the first drug or second drug.

37. A pharmaceutical composition comprising a therapeutically effective amount of a multispecific multi-drug antibody-drug conjugate (MMADC) according to any one of claims 1 to 36, and a pharmaceutically acceptable carrier.

38. A method of treating a disease, the method comprising administering a therapeutically effective amount of the multispecific multi-drug antibody-drug conjugate (MMADC) of any one of claims 1 to 36, or the pharmaceutical composition according to claim 37, to a subject in need thereof.

39. A method of treating a disease, the method comprising administering a therapeutically effective amount of a multispecific multi-drug antibody-drug conjugate (MMADC)WSGR Docket No.: 58964-801.601 to a subject in need thereof, wherein the MMADC comprises a multispecific antibody; a first linker connecting a first drug to the multispecific antibody at a first conjugation site; and a second linker connecting a second drug to the multispecific antibody at a second conjugation site; wherein the first drug is different from the second drug.

40. The method of claim 39, wherein the first drug and second drug is each independently a microtubule inhibitor, a topoisomerase inhibitor, a DNA-damaging agent, a DNA damage repair inhibitor, an RNA polymerase inhibitor, a chemotherapeutic agent, a protein degrader, or an immune stimulant.

41. The method of claim 39 or 40, wherein when: (a) the first drug is a microtubule inhibitor, the second drug is a topoisomerase inhibitor, a DNA-damaging agent, a DNA damage repair inhibitor, a chemotherapeutic agent, or an RNA polymerase inhibitor; or (b) the first drug is a topoisomerase inhibitor, the second drug is a microtubule inhibitor, a DNA-damaging agent, a DNA damage repair inhibitor, a chemotherapeutic agent, or an RNA polymerase; or (c) the first drug is a DNA-damaging agent, the second drug is a microtubule inhibitor, a topoisomerase inhibitor, a DNA damage repair inhibitor, a chemotherapeutic agent, or an RNA polymerase inhibitor; or (d) the first drug is a DNA damage repair inhibitor, the second drug is a microtubule inhibitor, a topoisomerase inhibitor, a DNA-damaging agent, a chemotherapeutic agent, or an RNA polymerase inhibitor; or (e) the first drug is a chemotherapeutic agent, the second drug is a microtubule inhibitor, a topoisomerase inhibitor, a DNA-damaging agent, a DNA damage repair inhibitor, or an RNA polymerase inhibitor; or (f) the first drug is an RNA polymerase inhibitor, the second drug is a microtubule inhibitor, a topoisomerase inhibitor, a DNA-damaging agent, a DNA damage repair inhibitor, or a chemotherapeutic agent.

42. The method of claim 41, wherein the first drug is a microtubule inhibitor and the second drug is a topoisomerase inhibitor.

43. The method of claim 39 or 40, wherein when: (a) the first drug is a microtubule inhibitor, the second drug is a different microtubule inhibitor; or (b) the first drug is a topoisomerase inhibitor, the second drug is a different topoisomerase inhibitor; orWSGR Docket No.: 58964-801.601 (c) the first drug is a DNA-damaging agent, the second drug is a different DNA- damaging agent; or (d) the first drug is a DNA damage repair inhibitor, the second drug is a different DNA damage repair inhibitor; or (e) the first drug is a chemotherapeutic agent, the second drug is a different chemotherapeutic agent; or (f) the first drug is an RNA polymerase inhibitor, the second drug is a different RNA polymerase inhibitor.

44. The method of claim 43, wherein the first drug is a microtubule inhibitor and the second drug is a different microtubule inhibitor.

45. The method of claim 43, wherein the first drug is a topoisomerase I inhibitor and the second drug is a topoisomerase II inhibitor.

46. The method of claim 39, wherein the first drug and second drug is independently monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), tubulysin A, maytansine, ansamitocin P3, mertansine (DM1), ravtansine (DM4), calicheamicin, duocarmycin, topotecan, exatecan, DXd, irinotecan, cisplatin, oxaliplatin, paclitaxel, teniposide, SN38, hexylresorcinal, camptothecin, MM398, etoposide, novobiocin, doxorubicin, nemorubicin, daunorubicin, idarubicin, epipodophyllotoxin, toposide, teniposide, mitoxanthrone, pyrrolobenzodiazepine (PDB), TAS-103, 7-MAD_MDCPT, SN38, SG2199, amanitins, 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 pharmaceutically acceptable salt or derivative thereof.

47. The method of claim 46, wherein the first drug and the second drug are each independently monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), mertansine (DM1), ravtansine (DM4), Dxd, exatecan, doxorubicin, TAS-103, irinotecan, 7-MAD-MDCPT, camptothecin, SN38, SG3199, or Amanitins, or a pharmaceutically acceptable salt or derivative thereof.

48. The method of claim 47, wherein the first drug and the second drug are each independently MMAE, MMAF, DM1, Dxd, doxorubicin, TAS-103, exatecan, cisplatin, or irinotecan.

49. The method of any one of claims 39 to 48, wherein the first drug and second drug are: (a) MMAE and MMAF; (b) MMAE and DM1;WSGR Docket No.: 58964-801.601 (c) MMAE and Dxd; (d) MMAE and doxorubicin; (e) MMAE and TAS-103; (f) MMAF and DM1; (g) MMAF and Dxd; (h) MMAF and doxorubicin; (i) MMAF and TAS-103; (j) DM1 and Dxd; (k) DM1 and doxorubicin; (l) DM1 and TAS-103; (m) Dxd and doxorubicin; (n) Dxd and exatecan; or (o) Cisplatin and irinotecan.

50. The method of any one of claims 39 to 49, wherein the first linker and the second linker is independently connected to the multispecific antibody via a reaction using the reactive group selected from:

51. The method of any one of claims 39 to 50, wherein the first linker and the second linker is independently a branched linker.

52. The method of claim 51, wherein the branched linker comprises the following structure:WSGR Docket No.: 58964-801.601 wherein denotes a connection leading to the drug, wherein denotes a connection leading to the antibody, wherein each of e, d and f is independently 0, 1, 2, 3, 4, 5, 6, 7 or 8, wherein at least one of e, d, and f is not 0.

53. The method of any one of claims 39 to 52, wherein the first linker and second linker independently comprises maleimidocaproyl-valine-citrulline-paminobenzyloxycarbonyl (MC-VCP) or glutamic acid-valine-citrulline (GluValCit).

54. The method of any one of claims 39 to 53, wherein the first linker and the second linker further independently comprises a first spacer and a second spacer.

55. The method of claim 54, wherein the first spacer and second spacer independently comprises a polyethylene glycol (PEG).

56. The method of any one of claims 39 to 55, wherein the first conjugate site and the second conjugation site is independently a reactive amino acid of the multispecific antibody.

57. The method of claim 56, wherein the reactive amino acid has the following functional group: –SH, –SeH, –NH2, –CO2H, or –C(=O)–, or a derivative thereof.

58. The method of any one of claims 39 to 57, wherein the multispecific antibody is a bispecific antibody or a trispecific antibody.

59. The method of claim 58, wherein the multispecific antibody is a bispecific antibody or a trispecific antibody.

60. The method of claim 59, wherein the multispecific antibody is a bispecific antibody.

61. The method of claim 60, wherein the bispecific antibody binds to two different targets, wherein the targets are independently EGFR, c-MET, LGR5, HER2, and HER3.

62. The method of any one of claims 39 to 61, wherein the multispecific antibody is a multispecific IgG1 antibody.

63. The method of any one of claims 39 to 62, wherein the multispecific antibody comprises a first heavy chain, a second heavy chain, a first light chain, and a second light chain.

64. The method of claim 63, wherein the first heavy chain and the second heavy chain have different amino acid sequences.

65. The method of any one of claims 39 to 64, wherein the multispecific antibody comprises a first and a second CH3 domain, wherein the first CH3 domain comprises amino acid substitution T366K and L351K and the second CH3 domain comprises amino acid substitution L351D and L368E.

66. The method of claim 65, wherein the first heavy chain has SEQ ID NO: 257, the second heavy chain has SEQ ID NO: 258, and the first light chain and the second light chain has SEQ ID NO: 259.WSGR Docket No.: 58964-801.601 67. The method of claim 65, wherein the first heavy chain has SEQ ID NO: 260, the second heavy chain has SEQ ID NO: 261, and the first light chain and the second light chain has SEQ ID NO:

259.

68. The method of any one of claims 39 to 65, wherein the MMADC is selected from Table 3.

69. The method of any one of claims 39 to 65, wherein the MMADC is selected from Table 4.

70. The method of any one of claims 38 to 69, wherein the disease is cancer.

71. The method of claim 70, wherein the cancer is a heterogenous cancer and / or a resistant cancer.

72. The method of claim 70 or 71, wherein the cancer is oral cancer, colorectal cancer, gastric cancer, esophageal cancer, hepatocellular cancer, non-small-cell lung cancer (NSCLC), small-cell lung cancer (SCLC), ovarian cancer, breast cancer, prostate cancer, pancreatic cancer, head and neck cancer, squamous cancer, renal cancer, bladder cancer, cervical cancer, endometrial cancer, thyroid cancer, or glioblastoma cancer.

73. The method of any one of claims 70 to 72, wherein the cancer is resistant to treatment with MMAE, MMAF, DM1, Dxd, cisplatin, or irinotecan, or a combination thereof.

74. Use of the MMADC of any one of claims 1 to 37, for the manufacture of a medicament for the treatment of a disease.

75. The use according to claim 74, wherein the disease is cancer.

76. The use according to claim 75, wherein the cancer is a heterogenous and / or a resistant cancer.

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