Antibody-drug conjugates and their use

Abstract

The present invention relates to an antibody-drug conjugate, wherein the antibody specifically binds to the folate receptor a and the drug is preferably selected from among cytotoxic drugs. Such antibody-drug conjugates are particularly useful in the treatment of proliferative diseases, including cancers such as ovarian cancer, breast cancer and non-small cell lung cancer.

Description

Technical Field

[0001] Antibody-drug conjugates are disclosed below, wherein the antibody specifically binds to folate receptor alpha (FRα) and the drug is preferably selected from inhibitors of topoisomerase I, such as camptothecin analogs such as exatecan. Such antibody-drug conjugates are particularly useful for the treatment of proliferative diseases including cancers such as ovarian cancer, breast cancer or lung cancer.

Background Art

[0002] Antibody-drug conjugates (hereinafter referred to as "ADCs") are a new class of therapeutic agents, particularly for cancer treatment. Such ADCs contain at least an antibody and a payload (e.g., a cytotoxic drug) covalently linked by a linker. Thus, ADCs are designed to combine the specificity of the antibody target and the efficiency of the payload (e.g., the cytotoxic activity of a chemotherapeutic agent). An effective ADC should exhibit high specificity and low systemic toxicity.

[0003] In the context of toxicity, the antibody used in an ADC needs to bind accurately and effectively to its antigen, which means that the appropriate target antigen is preferentially or exclusively expressed on the target cells.

[0004] When designing an ADC, it is necessary to covalently attach the final active drug to the ligand targeting unit while enabling the final release of the active drug unit by a selective enzymatic mechanism after cellular internalization or in the disease tissue microenvironment. In this regard, several peptidase- and glycosidase-sensitive cleavable linker chemical strategies (related to self-immolative chemistry) have been developed. These cleavable linkers and their corresponding cleavage mechanisms are well-known and described in several publications (e.g., Bargh JG et al., Chem. Soc. Rev., 2019, 48, 4361, Toki et al. J. Org. Chem. 2002, 67, 6, 1866 - 1872, Scott et al. Bioconjugate Chem. 2006, 17, 3, 831 - 840). The selection of this enzyme-sensitive cleavable entity is an important design attribute of the ADC that affects the efficacy and tolerability of the conjugate.

[0005] Examples of linker types that have been used to conjugate cytotoxins or drugs to antibodies include, but are not limited to, hydrazone, thioether, ester, disulfide, and peptide-containing linkers. The linker is selected, for example, from those that are susceptible to cleavage by the low pH within the lysosomal compartment or by proteases that are preferentially expressed in tumor tissue, such as cathepsins (e.g., cathepsin B, C, D, etc.). An effective linker should be able to ensure the accurate and timely release of the payload. In the context of toxicity, it is also clear that even if the linker itself does not appear to drive toxicity, the stability of the linker can affect the toxicity exerted by the payload. In fact, a stable linker can release the payload in a target-specific manner, while an unstable linker is more likely to undergo inaccurate release of the payload (e.g., due to non-specific cleavage), resulting in non-specific systemic toxicity.

[0006] The payloads used in ADCs are very potent and are often cytotoxic drugs with in vitro inhibitory concentrations in the picomolar range. Common payloads are, for example, microtubule inhibitors (e.g., maytansine derivatives (DM1 / DM4), auristatins (MMAE / MMAF), eribulin) and DNA alkylating agents (calicheamicin, pyrrolobenzodiazepine, indolinobenzodiazepine, or duocarmycin, etc.).

[0007] Although ADCs are considered promising therapeutic agents, some ADCs may be too toxic, limiting the therapeutic window of these compounds or preventing further clinical development. Furthermore, most of the currently approved or clinically studied ADCs are based on the above-mentioned microtubule and DNA targeting agents. Therefore, there is a need for new differentiated ADCs based on payloads with other mechanisms of action to effectively treat tumors that are resistant or become resistant to microtubule and DNA targeting agents.

[0008] Therefore, effective ADCs that exhibit high specificity, maximum efficiency, and low toxicity require an appropriate combination of each of their components. For a review of possible strategies, see, for example, Khongorzul et al 2019 (Molecular cancer research, DOI: 10.1158 / 1541-7786.MCR-19-0582). WO 2019081455 and Conilh et al (2021, Pharmaceuticals, 14(3), 247) further report HER2-targeted antibody-drug conjugates using a hydrophilic monodisperse polysarcosine (PSAR) drug-linker platform, particularly based on the topoisomerase I inhibitor payload exatecan.

[0009] Cheng et al (2018, DOI: 10 / 1158 / 1535-7163.MCT-17-1215) and International Publication No. 2017151979 reported an antibody-drug conjugate (ADC) using farletuzumab conjugated to typically 3 to 4 molecules of eribulin (MORAb-202) and its use in the treatment of tumors. The mechanism of action of the eribulin payload of this ADC is microtubule inhibition.

[0010] Moore et al (2018, Future Oncol. 14(17)1669-1678) reported the results of a Phase III trial using the ADC mirvetuximab soravtansine, a humanized anti-FRα antibody conjugated to an average of 3 to 4 maytansinoid molecules as a payload, for the treatment of ovarian cancer. The mechanism of action of the maytansinoid payload of this ADC is microtubule inhibition.

SUMMARY OF THE INVENTION

[0011] Therefore, there remains a need for antibody-drug conjugates that are effective, have high specificity, low toxicity (improved therapeutic index), and a mechanism of action different from that of other payloads.

[0012] As shown in the examples, the present disclosure provides an anti-FRα antibody-drug conjugate based on a topoisomerase I inhibitor that has excellent in vivo efficacy and low toxicity in solid tumor cancer models, particularly when compared to reference prior art ADCs that target FRα-expressing antibodies together with other payloads and drug linkers such as mirvetuximab soravtansine.

[0013] Accordingly, a first object of the present disclosure relates to an antibody-drug conjugate (ADC) of formula (I), Ab-[L-D]p (I), wherein, Ab is an anti-folate receptor alpha (FRα) antibody that specifically binds to SEQ ID NO: 12, L is a cleavable linker moiety that is preferably bound to the anti-folate receptor alpha (FRα) antibody via a thiol residue, D is a cytotoxic drug moiety bound to L, p is from 1 to 8, preferably from 6 to 8, more preferably p is 8.

[0014] In certain embodiments, Ab is an antibody comprising a human IgG1 isotype constant region.

[0015] In preferred embodiments, Ab is an antibody comprising a variant or chemically modified constant region of the human IgG1 isotype, and the variant or chemically modified constant region does not confer or confers reduced ADCC activity to the antibody when compared to the corresponding antibody having the wild-type human IgG1 isotype constant region.

[0016] In other certain embodiments, Ab is an antibody comprising a human IgG4 isotype constant region, or a variant or chemically modified IgG4 constant region, and the variant or chemically modified constant region does not confer or confers reduced ADCC activity to the antibody when compared to the corresponding antibody having the wild-type IgG4 isotype constant region.

[0017] In preferred embodiments, Ab is as follows: (a) a variable heavy chain polypeptide comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 3, and a variable light chain polypeptide comprising LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6; or (b) a variable heavy chain polypeptide comprising VH of SEQ ID NO: 7 and a variable light chain polypeptide comprising VL of SEQ ID NO: 8 and is an anti-FRα antibody comprising any of them.

[0018] In certain embodiments, the Ab comprises or consists essentially of a heavy chain of SEQ ID NO: 9 and a light chain of SEQ ID NO: 10.

[0019] In certain embodiments, the Ab comprises or consists essentially of the heavy chain of SEQ ID NO: 11 and the light chain of SEQ ID NO: 10.

[0020] In certain embodiments, D is an inhibitor of topoisomerase I, preferably selected from the group consisting of camptothecin analogs, and more preferably D is the drug moiety of exatecan of formula (II) below.

[0021]

Chemical formula

[0022] In certain embodiments, L is a cleavable linker moiety of the formula -A-W-, wherein A is an optional stretcher unit linked to the Ab and W is a cleavable moiety linked to D. In more particular embodiments, L is a lysosomal protease-sensitive linker and W comprises a cleavable peptide moiety selected from the group consisting of, for example, valine-citrulline (Val-Cit), alanine-alanine-asparagine (Ala-Asn), valine-alanine (Val-Ala), and phenylalanine-lysine (Phe-Lys). In another particular embodiment, L is a protease-sensitive cleavable linker and W preferably comprises a sugar-cleavable unit selected from a β-glucuronide or β-galactoside moiety. In another particular embodiment, L is a glutathione-sensitive linker and W comprises a disulfide moiety. In certain embodiments, W is of the following formula (III):

[0023]

Chemical formula

[0024] In a preferred embodiment, L corresponds to the linker -A-W- of formula (IV),

[0025]

Chemical formula

[0026] In a more specific embodiment, X1 and X2 are independently selected from the group consisting of one or more amino acids, one or more N-substituted amino acids, optionally substituted polyethers, C1-C 12 alkylene, arylene having 6-10 ring atoms, C3-C8 cycloalkylene, heterocycloalkylene having 5-10 ring atoms, heteroarylene having 5-10 ring atoms, C2-C 10 alkenylene, and any combination thereof, wherein the alkylene and alkenylene are optionally interrupted by one or more heteroatoms or chemical groups selected from -O-,-S-,-C(O)-,-NR''-,-C(O)NR''-,-NR''-C(O)-,-NR''-C(O)-NR'''-,-NR''-C(O)-O-,-O-C(O)NR''- and triazole; When the alkylene, arylene, cycloalkylene, heterocycloalkylene, heteroarylene, and alkenylene are optionally substituted with one or more substituents selected from halogen, oxo, -OH, -NO2, -CN, C1-C6 alkyl, C3-C6 cycloalkyl, heterocyclyl having 5-10 ring atoms, aryl having 6-10 ring atoms, heteroaryl having 5-10 ring atoms, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 haloalkoxy, -(CO)-R', -O-(CO)-R', -(CO)-O-R', -(CO)-NR''R''', -NR''-(CO)-R', and -NR''R''', R', R'', and R''' are independently selected from H and C1-C6 alkyl.

[0027] In a more specific embodiment, Z is one or more amino acids, one or more N-substituted amino acids, an optionally substituted polyether, C1-C 12 alkylene, arylene having 6-10 ring atoms, C3-C8 cycloalkylene, heterocycloalkylene having 5-10 ring atoms, heteroarylene having 5-10 ring atoms, C2-C 10 alkenylene, and is independently selected from the group consisting of any combination thereof, the alkylene and alkenylene are optionally interrupted by one or more heteroatoms or chemical groups selected from -O-, -S-, -C(O)-, -NR''-, -C(O)NR''-, -NR''-C(O)-, -NR''-C(O)-NR'''-,-NR''-C(O)-O-,-O-C(O)NR''-, and triazole, When the alkylene, arylene, cycloalkylene, heterocycloalkylene, heteroarylene, and alkenylene are optionally substituted with one or more substituents selected from halogen, oxo, -OH, -NO2, -CN, C1-C6 alkyl, C3-C6 cycloalkyl, heterocyclyl having 5-10 ring atoms, aryl having 6-10 ring atoms, heteroaryl having 5-10 ring atoms, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 haloalkoxy, -(CO)-R’, -O-(CO)-R’, -(CO)-O-R’, -(CO)-NR’’R’’’, -NR’’-(CO)-R’, and -NR’’R’’’, R’, R’’, and R’’’ are independently selected from H and C1-C6 alkyl.

[0028] In certain embodiments, K is polysarcosine, preferably polysarcosine of the following formula (V),

[0029]

Chemical formula

[0030] In certain embodiments, T is a sugar-cleavable unit that is glucuronide or galactoside.

[0031] In other certain embodiments, T is a dipeptide preferably selected from Val-Cit, Val-Ala, and Phe-Lys.

[0032] In certain embodiments, L is covalently bonded to one or more thiol residues of the antibody, and preferably, L corresponds to the linker of formula (VI).

[0033]

Chemical formula

[0034] In certain embodiments, Ab comprises a full-length antibody or an antibody fragment containing an antigen-binding portion.

[0035] In certain embodiments, the antibody-drug conjugate corresponds to the following formula (VII),

[0036]

Chemical formula

[0037] In preferred embodiments, the antibody-drug conjugate corresponds to the following formula (VII),

[0038]

Chemical formula

[0039] In a more preferred embodiment of the ADC of formula (I), Ab is (i) a variable heavy chain polypeptide comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 3, and (ii) a variable light chain polypeptide comprising LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6; an anti-folate receptor alpha antibody or an antigen-binding fragment thereof, L is a cleavable linker of the formula -A-W-, wherein A is any stretcher unit linked to Ab and W is a cleavable moiety linked to D. D is exatecan, p is from 1 to 8, for example from 4 to 8, preferably from 6 to 8, for example p is from 7 to 8.

[0040] Another object of the present disclosure relates to the above-mentioned ADC for use as a pharmaceutical, preferably for the treatment of tumors, such as solid tumors, more specifically selected from the group consisting of ovarian cancer, breast cancer, lung cancer, or mesothelioma.

[0041] Another object of the present disclosure relates to the use of the above-mentioned ADC in the preparation of a pharmaceutical or pharmaceutical composition for treating tumors, such as solid tumors, more specifically tumors selected from the group consisting of ovarian cancer, breast cancer, lung cancer, or mesothelioma.

[0042] In certain embodiments, the ADC can preferably be used for the treatment of cancers selected from the group consisting of ovarian cancer, triple-negative breast cancer, and non-small cell lung cancer.

[0043] The present disclosure further relates to a pharmaceutical composition comprising the antibody-drug conjugate disclosed herein in combination with one or more pharmaceutically acceptable excipients, diluents or carriers, optionally including other active ingredients, such as anti-cancer agents or immunotherapeutic agents such as immune checkpoint inhibitors.

[0044] The present disclosure also relates to a process for obtaining the ADC of the present disclosure, the method comprising (a) culturing a host cell under conditions suitable for the production of an anti-FRα antibody as defined herein, (b) isolating the anti-FRα antibody, (c) synthesizing exatecan conjugated to a linker L of formula (VIII),

[0045]

Chemical formula

Brief Description of the Drawings

[0046]

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Figure 22

Modes for Carrying Out the Invention

[0047] Definition To make the present disclosure more readily understandable, certain terms are first defined. Further definitions are set forth throughout the detailed description.

[0048] The term "FRα" or "folate receptor alpha" refers to the human folate receptor alpha as defined in SEQ ID NO: 12, unless otherwise specified. This sequence corresponds to the amino acid sequence of folate receptor alpha encoded by the FOLR1 gene (Homo sapiens), which is also available in the UniprotKB entry P15328 (FOLR1_HUMAN).

[0049] As used herein, the term "antibody" includes whole antibodies and any antigen-binding fragment (i.e., "antigen-binding portion") or single chain thereof. A naturally occurring "antibody" is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2 and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH region and the VL region can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, and are arranged from the amino terminus to the carboxy terminus in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain the binding domains that interact with the antigen. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq). The "antigen-binding portion" of an antibody (or simply the term "antigen portion" as used herein) refers to the full length or one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., a portion of FRα). It has been shown that the antigen-binding function of an antibody can be performed by fragments of the full-length antibody.Examples of binding fragments included within the term "antigen-binding portion" of an antibody include: Fab fragments, monovalent fragments consisting of the VL, VH, CL and CH1 domains; F(ab)2 fragments; divalent fragments comprising two Fab fragments linked by disulfide bridges in the hinge region; Fd fragments consisting of the VH domain and the CH1 domain; Fv fragments consisting of the VL and VH domains of a single arm of an antibody; a single arm having an IgG heavy chain modified in the hinge region, such as IgG4, a domain antibody fragment (Ward et al., 1989 Nature 341:544-546), or a UniBody consisting of a nanobody fragment consisting of the VH domain; and isolated complementarity determining regions (CDRs); or any fusion protein comprising such an antigen-binding portion. Furthermore, although the two domains of an Fv fragment, VL and VH, are encoded by separate genes, they can be linked by a synthetic linker that enables them to be made as a single-chain protein that pairs the VL and VH regions to form a monovalent molecule (known as a single-chain Fv (scFv)) using recombinant methods. See, for example, Bird et al., 1988 Science 242:423-426; and Huston et al., 1988 Proc. Natl. Acad. Sci. 85:5879-5883). Such single-chain antibodies are also intended to be included within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as intact antibodies.

[0050] As used herein, an "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds to FRα is substantially free of antibodies that specifically bind to other antigens other than FRα). However, an isolated antibody that specifically binds to FRα may have cross-reactivity to other antigens such as FRα molecules from other species. Furthermore, an isolated antibody may be substantially free of other cellular materials and / or chemical substances.

[0051] The terms "antibody that recognizes an antigen" and "antibody specific for an antigen" are used interchangeably in this specification with the term "antibody that specifically binds to an antigen".

[0052] As used herein, the term "monoclonal antibody" or "monoclonal antibody composition" refers to a preparation of antibody molecules of a single molecular composition. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope.

[0053] As used herein, "isotype" refers to the antibody class provided by the heavy chain constant region gene (e.g., IgM, IgE, IgG, such as IgG1 or IgG4).

[0054] As used herein, the term "K D " is intended to refer to the equilibrium dissociation constant obtained from the ratio of k off to k on (i.e., k off / k on ) and expressed as molar concentration (M). Since the K D value is related to the concentration of the antibody (the amount of antibody required for a particular experiment), the lower the K D value (lower concentration), the higher the resulting affinity of the antibody. The K D value of an antibody can be determined using methods well established in the art. Preferred methods for determining the K D value of an mAb can be found in Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988), Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, N.Y., 1992, 1993, and Muller, Meth Enzymol 1983, which are hereby incorporated by reference in their entirety. The K DThe method of determination may be by using surface plasmon resonance or by using a biosensor system such as Biacore® (see also detailed information regarding Affinity assessment Rich RL, Day YS, Morton TA, Myszka DG. High-resolution and high-throughput protocols for measuring drug / human serum albumin interactions using BIACORE®. Anal Biochem. 2001).

[0055] As used herein, the term "k assoc " or "k a " or "k on " is intended to refer to the association rate of a particular antibody-antigen interaction, while the term "k dis " or "k d " or k off is intended to refer to the dissociation rate of a particular antibody-antigen interaction.

[0056] As used herein, the term "affinity" refers to the strength of the interaction between an antibody and an antigen at a single antigenic site. Within each antigenic site, the variable regions of the antibody "arms" interact with the antigen via weak non-covalent forces at multiple sites, and the more interactions there are, the stronger the affinity.

[0057] As used herein, an antibody or protein that "specifically binds to an antigen", e.g., "specifically binds to FRα", is intended to refer to an antibody that detectably binds to an epitope presented on an antigen such as FRα of the present disclosure. It typically refers to an antibody or ADC that binds to human FRα with a K D of 200 nM or less, 100 nM or less, 50 nM, 40 nM or less, or about 30 nM. Typically, K D is 10 -3It is contained at pM of 200 nM, particularly 0.1 pM to 100 nM, particularly 0.1 pM to 50 nM, or 1 pM to 50 nM, particularly 1 pM to 30 nM, 10 pM to 50 nM, 0.1 nM to 200 nM, or 0.1 nM to 100 nM, or 1 nM to 50 nM, particularly 1 nM to 30 nM. Typically, the ADC of the present disclosure is specific for FRα and has K as defined above. D has.

[0058] As used herein, the term "host cell" refers to a prokaryotic or eukaryotic cell. Eukaryotic cells, such as mammalian host cells, yeast or filamentous fungi, are preferred, particularly mammalian cells, as they are more likely to assemble and secrete immunologically active antibodies that are properly folded than prokaryotic cells.

[0059] As used herein, the term "ADCC" or "antibody-dependent cell-mediated cytotoxicity" activity refers to cell depletion activity. ADCC activity can be measured by a commercially available ADCC assay, such as the ADCC Reporter Bioassay commercially available from Promega under Ref#G7015.

[0060] As used herein, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, and reptiles. The term "subject" also encompasses the term "patient".

[0061] As used herein, the term "drug" or D in formula (I) of the ADC also refers to the "payload", i.e., the moiety conjugated to the antibody (or fragment). The drug D should not be construed as being limited to classical chemotherapeutic agents. For example, D can include proteins, peptides or polypeptides having the desired biological activity. Preferably, it refers to a therapeutic moiety such as a cytotoxin. "Cytotoxin" or "cytotoxic agent" includes any agent that is harmful to cells (e.g., kills).

[0062] Unless otherwise specified, the present disclosure encompasses the compounds or drugs or cytotoxins described in this specification, as well as their tautomers, enantiomers, diastereomers, racemates or mixtures, and their hydrates, esters, solvates or pharmaceutically acceptable salts.

[0063] Any formula shown herein is also intended to represent both the unlabeled and isotopically labeled forms of the compounds, such as deuterium-labeled compounds or 14 13C-labeled compounds.

[0064] The term "pharmaceutically acceptable salt" refers to salts that retain the biological effectiveness and properties of the compounds of the present disclosure and are typically not biologically or otherwise undesirable. In many cases, the compounds of the present disclosure can form acidic salts and / or basic salts due to the presence of amino groups and / or carboxyl groups or similar groups. Pharmaceutically acceptable acid addition salts can be formed with organic acids and / or inorganic acids. Pharmaceutically acceptable base addition salts can be formed using organic bases and / or inorganic bases. Such salts are well known to those skilled in the art.

[0065] The term "connector unit" refers to a component that connects different parts of a compound to each other. For example, the connector can connect an Ab to a spacer or a spacer to an amide functional group -CO-NR1-. The connector is a scaffold having binding sites for the components of the antibody-drug conjugate, namely Ab, spacer, hydrophobic masking entity, and / or amide functional group -CO-NR1-.

[0066] One skilled in the art can select a suitable linker. A non-exhaustive list of linkers includes amino acids such as lysine, glutamic acid, aspartic acid, serine, tyrosine, cysteine, selenocysteine, glycine, homoalanine; amino alcohols; amino aldehydes; polyamines or any combination thereof. Advantageously, the linker units X1 and / or X2 are one or more natural or unnatural amino acids. In one embodiment, the linker units X1 and / or X2 are selected from glutamic acid, lysine, and glycine. The linker units X1 and X2 are one or more amino acids, one or more N-substituted amino acids, optionally substituted polyethers, C1-C 12 alkylene, arylene having 6-10 ring atoms, C3-C8 cycloalkylene, heterocycloalkylene having 5-10 ring atoms, heteroarylene having 5-10 ring atoms, C2-C 10 alkenylene, and can be independently selected from the group consisting of any combination thereof, and the alkylene and alkenylene are optionally interrupted by one or more heteroatoms or chemical groups selected from the group consisting of -O-, -S-, -C(O)-, -NR''-, -C(O)NR''-, -NR''-C(O)-, -NR''-C(O)-NR'''-,-NR''-C(O)-O-,-O-C(O)NR''- and triazole, and the alkylene, arylene, cycloalkylene, heterocycloalkylene, heteroarylene, and alkenylene are optionally substituted with one or more substituents selected from halogen, oxo, -OH, -NO2, -CN, C1-C6 alkyl, C3-C6 cycloalkyl, heterocyclyl having 5-10 ring atoms, aryl having 6-10 ring atoms, heteroaryl having 5-10 ring atoms, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 haloalkoxy, -(CO)-R', -O-(CO)-R', -(CO)-O-R', -(CO)-NR''R''', -NR''-(CO)-R', and -NR''R''', and R', R'' and R''' are independently selected from H and C1-C6 alkyl.

[0067] Examples of linker units include optionally substituted polyethers, amino acids, benzyl groups, amines, ketones,

[0068] [Chemical formula] and the like.

[0069] In particular, the linker unit can be divalent or trivalent. For example, when the hydrophobic masking entity K is present, X2 can be a trivalent linker unit.

[0070] The term "amino acid" refers to natural or non-natural amino acids. The CO portion of the -CONR1- or -CONR1'- group can be regarded as part of the X2 linker unit when X2 consists of one or more amino acids. Non-exhaustive listings of amino acids include lysine, glutamic acid, aspartic acid, serine, tyrosine, cysteine, selenocysteine, glycine, and homoalanine.

[0071] The spacer is a divalent arm that covalently binds to two components of the antibody-drug conjugate, such as two linker units.

[0072] Non-exhaustive lists of spacer units include alkylene, heteroalkylene (thus alkylene interrupted by at least one heteroatom selected from Si, N, O, and S); alkoxy; polyethers, such as polyalkylene glycols and typically polyethylene glycol; one or more natural or non-natural amino acids such as glycine, alanine, proline, valine, N-methylglycine, etc.; C3-C8 heterocycles; C3-C8 carbocycles; arylene, and any combination thereof. For example, the spacer is a divalent linear alkylene group, preferably (CH2)4.

[0073] For example, the spacer is -C1-C 10 alkylene-, -C1-C 10heteroalkylene-, -C3-C8 carbocyclo-, -O-(C1-C8 alkyl)-, -arylene-, -C1-C 10 alkylene-arylene-, -arylene-C1-C 10 alkylene-, -C1-C 10 alkylene-(C3-C8 carbocyclo)-, -(C3-C8 carbocyclo)-C1-C 10 alkylene-, -C3-C8 heterocyclo-, -C1-C 10 alkylene-(C3-C8 heterocyclo)-, -(C3-C8 heterocyclo)-C1-C 10 alkylene-, -C1-C 10 alkylene-C(=O)-, -C1-C 10 heteroalkylene-C(=O)-, -C3-C8 carbocyclo-C(=O)-, -O-(C1-C8 alkyl)-C(=O)-, -arylene-C(=O)-, -C1-C 10 alkylene-arylene-C(=O)-, -arylene-C1-C 10 alkylene-C(=O)-, -C1-C 10 alkylene-(C3-C8 carbocyclo)-C(=O)-, -(C3-C8 carbocyclo)-C1-C 10 alkylene-C(=O)-, -C3-C8 heterocyclo-C(=O)-, -C1-C 10 alkylene-(C3-C8 heterocyclo)-C(=O)-, -(C3-C8 heterocyclo)-C1-C 10 alkylene-C(=O)-, -C1-C 10 alkylene-NH-, -C1-C 10 heteroalkylene-NH-, -C3-C8 carbocyclo-NH-, -O-(C1-C8 alkyl)-NH-, -arylene-NH-, -C1-C 10 alkylene-arylene-NH-, -arylene-C1-C 10 alkylene-NH-, -C1-C 10 alkylene-(C3-C8 carbocyclo)-NH-, -(C3-C8 carbocyclo)-C1-C 10 alkylene-NH-, -C3-C8 heterocyclo-NH-, -C1-C 10Alkylene-(C3-C8 heterocycle)-NH-, -(C3-C8 heterocycle)-C1-C 10 Alkylene-NH-, -C1-C 10 Alkylene-S-, -C1-C 10 Heteroalkylene-S-, -C3-C8 carbocycle-S-, -O-(C1-C8 alkyl)-)-S-, -Arylene-S-, -C1-C 10 Alkylene-arylene-S-, -Arylene-C1-C 10 Alkylene-S-, -C1-C 10 Alkylene-(C3-C8 carbocycle)-S-, -(C3-C8 carbocycle)-C1-C 10 Alkylene-S-, -C3-C8 heterocycle-S-, -C1-C 10 Alkylene-(C3-C8 heterocycle)-S-, -(C3-C8 heterocycle)-C1-C 10 Alkylene-S-, -C1-C 10 Alkylene-O-C(=O)-, -C3-C8 carbocycle-O-C(=O)-, -O-(C1-C8 alkyl)-O-C(=O)-, -Arylene-O-C(=O)-, -C1-C 10 Alkylene-arylene-O-C(=O)-, -Arylene-C1-C 10 Alkylene-O-C(=O)-, -C1-C 10 Alkylene-(C3-C8 carbocycle)-O-C(=O)-, -(C3-C8 carbocycle)-C1-C 10 Alkylene-O-C(=O)-, -C3-C8 heterocycle-O-C(=O)-, -C1-C 10 Alkylene-(C3-C8 heterocycle)-O-C(=O)-, and -(C3-C8 heterocycle)-C1-C 10 It may be selected from the group consisting of alkylene-O-C(=O)-.

[0074] Any of the above groups is -X, -R', -O - , -OR', =O, -SR', -S - , -NR'2, -NR'3 +, =NR’, -CX3, -CN, -OCN, -SCN, -N=C=O, -NCS, -NO, -NO2, =N2, -N3, -NR’C(=O)R’, -C(=O)R’, -C(=O)NR’2, -SO3 - , -SO3H, -S(=O)2R’, -OS(=O)2OR’, -S(=O)2NR’, -S(=O)R’, -OP(=O)(OR’)2, -P(=O)(OR’)2, -PO3 - , -PO3H2, -C(=O)X, -C(=S)R’, -CO2R’, -CO2, -C(=S)OR’, C(=O)SR’, C(=S)SR’, C(=O)NR’2, C(=S)NR’2, and one or more of the substituents selected from C(=NR’)NR’2, wherein each X is independently halogen: -F, -Cl, -Br, or -I, and each R’ is independently -H, -C1-C 20 alkyl, -C6-C 10 aryl, or -C3-C 10 heterocycle.

[0075] As used herein, the term “alkyl” refers to a monovalent saturated hydrocarbon chain (having a straight or branched chain). For example, alkyl refers to C1-C 20 alkyl. Preferably, alkyl is “lower alkyl”, i.e., an alkyl group having 1, 2, 3, 4, 5, or 6 carbons (a straight or branched C1-C6 alkyl group). For example, this includes methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, etc.

[0076] For example, alkylene, used alone or as part of an alkylene glycol, refers to a divalent saturated, straight or branched alkyl group as defined herein.

[0077] Alkenyl and alkynyl refer to a hydrocarbon group having 2-20 carbon atoms, preferably 2-12, more preferably 2-6, particularly 2-4, of at least partially unsaturated straight or branched chains. An alkenyl group contains at least one C=C double bond, and an alkynyl group contains at least one

[0078]

Chem.

[0079] As used herein, the term "C3-C8 cycloalkyl" or "carbocyclic ring" refers to a saturated or unsaturated cyclic group having 3 to 8, preferably 3 to 6 carbon atoms. The cycloalkyl can have a single ring or multiple rings fused to each other. The cycloalkyl can also include spiro rings. Suitable cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

[0080] As used herein, the term "C3-C8 cycloalkylene" or "carbocyclo" refers to a divalent cycloalkyl as defined herein.

[0081] As used herein, the term "halogen" refers to a fluoro (-F), chloro (-Cl), bromo (-Br), or iodo (-I) group.

[0082] As used herein, the term "C1-C6 haloalkyl" refers to a C1-C6 alkyl as defined herein substituted by one or more halogen groups as defined herein. Suitable C1-C6 haloalkyl groups include trifluoromethyl and dichloromethyl.

[0083] As used herein, the term "heteroalkyl" refers to a straight or branched hydrocarbon chain consisting of 1 to 12 carbon atoms, preferably 1 to 10, more preferably 1 to 6 carbon atoms, and 1 to 3 heteroatoms selected from the group consisting of O, N, Si, and S, wherein the nitrogen and sulfur atoms may optionally be oxidized (e.g., sulfoxide or sulfone), and the nitrogen heteroatom may optionally be quaternized. The heteroatoms O, N, and S may be located at any internal position of the heteroalkyl group or at the position where the alkyl group is attached to the remainder of the molecule.

[0084] Heteroalkylene refers to a divalent heteroalkyl as defined above. In the case of a heteroalkylene group, the heteroatoms may also occupy either or both of the chain termini.

[0085] As used herein, the term "C1-C6 alkoxy" refers to an -O-alkyl group, wherein the alkyl group is C1-C6 alkyl as defined herein. Suitable C1-C6 alkoxy groups include methoxy, ethoxy, propoxy.

[0086] As used herein, the term "C1-C6 haloalkoxy" refers to a C1-C6 alkoxy group as defined herein substituted by one or more halogen groups as defined herein. Suitable haloalkoxy includes trifluoromethoxy.

[0087] As used herein, the term "aryl having 6 to 10 ring atoms" refers to a polyunsaturated aromatic hydrocarbyl group having a single ring or a plurality of aromatic rings fused together, containing 6 to 10 ring atoms, wherein at least one ring is aromatic. The aromatic ring may optionally contain 1 to 2 additional rings (cycloalkyl, heterocyclyl or heteroaryl as defined herein) fused thereto. Suitable aryl groups include phenyl, naphthyl and phenyl rings fused to heterocyclyls such as benzopyranyl, benzodioxolyl, benzodioxanyl.

[0088] Arylene refers to the divalent aryl group defined above.

[0089] As used herein, the term "heteroaryl having 5 to 10 ring atoms" refers to a polyvalent unsaturated aromatic ring system having a single ring or a plurality of aromatic rings that are fused together or covalently bonded and contain 5 to 10 atoms, at least one of the rings being aromatic and at least one of the ring atoms being a heteroatom selected from N, O, and S. The nitrogen and sulfur heteroatoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized. Such rings may be fused to an aryl, cycloalkyl, or heterocyclyl ring. Non-limiting examples of such heteroaryl include furanyl, thiophenyl, pyrrolyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, oxadiazolyl, thiadiazolyl, tetrazolyl, oxatriazolyl, thiatriazolyl, pyridinyl, pyrimidyl, pyrazinyl, pyridazinyl, oxazinyl, dioxinyl, thiazinyl, triazinyl, indolyl, isoindolyl, benzofuranyl, isobenzofuranyl, benzothiophenyl, isobenzothiophenyl, indazolyl, benzimidazolyl, benzoxazolyl, purinyl, benzothiadiazolyl, quinolinyl, isoquinolinyl, cinnolinyl, quinazolinyl, and quinolinyl.

[0090] As used herein, the term "heterocyclyl having 3 to 10 ring atoms", "heterocycloalkyl having 3 to 10 ring atoms", or "heterocyclyl" refers to a saturated or unsaturated cyclic group having 3 to 10 ring atoms, preferably 3 to 8 ring atoms, at least one of the ring atoms being a heteroatom selected from N, O, and S. The nitrogen and sulfur heteroatoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized. The heterocycle can include fused or bridged rings as well as spiro rings. Examples of heterocycles include, but are not limited to, tetrahydropyridyl, piperidinyl, morpholinyl, tetrahydrofuranyl, tetrahydrothienyl, piperazinyl, 1-azepanyl, imidazolinyl, 1,4-dioxanyl, etc.

[0091] As used herein, the terms "heterocycle" or "heterocycloalkylene" refer to a divalent heterocyclic ring as defined herein.

[0092] Furthermore, the terms alkyl, alkenyl, alkynyl, aryl, alkylene, arylene, heteroalkyl, heteroalkylene, C3-C8 carbocycle, C3-C8 carbocycle, C3-C8 heterocycle, C3-C8 heterocycle, polyether refer to -X, -R', -O - , -OR', =O, -SR', -S - , -NR'2, -NR'3, =NR', -CX3, -CN, -OCN, -SCN, -N=C=O, -NCS, -NO, -NO2, =N2, -N3, -NRC(=O)R', -C(=O)R', -C(=O)NR'2, -SO3 - , -SO3H, -S(=O)2R', -OS(=O)2OR', -S(=O)2NR', -S(=O)R', -OP(=O)(OR')2, -P(=O)(OR')2, -PO3 - , -PO3H2, -C(=O)R', -C(=O)X, -C(=S)R', -CO2R', -CO2, -C(=S)OR', C(=O)SR', C(=S)SR', C(=O)NR'2, C(=S)NR'2, and C(=NR')NR'2, and refers to a group optionally substituted with one or more of the substituents selected from, each X is independently halogen: -F, -Cl, -Br, or -I, and each R' is independently -H, -C1-C 20 alkyl, -C6-C 10 aryl, or -C3-C 10 heterocycle.

[0093] As used herein, the term "polyether" refers to a polymer containing an ether bond. The number of ether moieties in the polyether can be between 2 and 100, preferably between 2 and 25, particularly between 2 and 10. Examples of polyethers include polyethylene glycol.

[0094] An electron-withdrawing group refers to an atom or group that draws electron density towards itself from an adjacent atom, usually by resonance or inductive effects. Electron-withdrawing groups include halogens, haloalkyls (-CF3, etc.), -CN, -SO3H, -NO2, and -C(O)R groups (where R = H, OH, or alkoxy). Advantageously, the electron-withdrawing group is -NO2. In one embodiment, the electron-withdrawing group is in the ortho position relative to the Y-T substituent of the phenyl ring.

[0095] As used herein, the term "protecting group" refers to a chemical substituent that can be selectively removed by an easily accessible reagent that does not attack the regenerable functional group or other functional groups in a molecule. Suitable protecting groups are known in the art and are continuously being developed. Suitable protecting groups can be found, for example, in Wutz et al. ("Greene’s Protective Groups in Organic Synthesis, Fourth Edition," Wiley-Interscience, 2007). In certain embodiments, protecting groups for protecting amino groups as described by Wutz et al. (pages 696 - 927) are used. Representative examples of amino protecting groups include, but are not limited to, t-butyloxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl (Fmoc), acetyl (Ac), carboxybenzyl group (Cbz), benzyl group (Bn), allyl, trifluoroacetyl, allyloxycarbonyl (Alloc) group, and 2,2,2-trichloroethoxycarbonyl (Troc).

[0096] The hydrophobic masking entity refers to a group capable of reducing the apparent hydrophobicity of a compound. The hydrophobic masking entity can be selected from polysarcosine and polyethylene glycol. The number of ethylene glycol or sarcosine moieties can vary widely. For example, the number of ethylene glycol or sarcosine moieties in the hydrophobic masking entity can be between 2 and 500, preferably between 5 and 100, particularly between 5 and 25. In one embodiment, the hydrophobic masking entity is polysarcosine containing 6 to 24 sarcosine moieties, preferably 10 to 12 sarcosine moieties.

[0097] As used herein, the term "linked" refers to a linkage. This linkage is also represented by the dash "-" in formula (I). The linkage can be a covalent bond or a non-covalent interaction such as an electrostatic force. Preferably, the bond is a covalent bond. As used herein, the "wavy line" on the formula represents the binding site between each part (Ab, L, Z, X, and D) of the ADC of the present disclosure.

[0098] As used herein, the terms "treat", "treating", or "treatment" refer to (1) inhibiting a disease, e.g., inhibiting a disease, condition, or disorder in an individual experiencing or showing the pathology or overall symptoms of the disease, condition, or disorder (i.e., preventing further development of the pathology and / or overall symptoms); (2) ameliorating a disease, e.g., ameliorating a disease, condition, or disorder in an individual experiencing or showing the pathology or overall symptoms of the disease, condition, or disorder (i.e., reversing the pathology and / or overall symptoms), e.g., reducing the severity of the disease, or alleviating or mitigating one or more symptoms of the disease; and refer to one or more of the above. In particular, with respect to the treatment of tumors, the term "treatment" can refer to inhibiting tumor growth or reducing tumor size.

[0099] As used herein, a "therapeutically effective amount" or "effective amount" of an ADC is an amount sufficient to effect the specific purpose for which it is described, e.g., an amount sufficient to bring about a therapeutic effect such as inhibition or reduction of tumor growth rate or tumor volume, reduction of cancer symptoms, or some indication of therapeutic efficacy after administration. In the case of cancer, a therapeutically effective amount of an ADC can reduce the number of cancer cells, reduce tumor size, inhibit (e.g., slow or stop) tumor metastasis, inhibit (e.g., slow or stop) tumor growth, and / or alleviate one or more symptoms.

[0100] As used herein, the percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity = number of identical positions / total number of positions x 100), taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences. Comparison of sequences and determination of the percent identity between two sequences can be accomplished using a mathematical algorithm as described below.

[0101] The percent identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (NEEDLEMAN, and Wunsch).

[0102] The percent identity between two nucleotide or amino acid sequences can also be determined, for example, using EMBOSS Needle (pairwise alignment; available at www.ebi.ac.uk). For example, EMBOSS Needle can be used with a BLOSUM62 matrix, a "gap open penalty" of 10, a "gap extend penalty" of 0.5, a false "end gap penalty", an "end gap open penalty" of 10 and an "end gap extend penalty" of 0.5. Generally, the "percent identity" is a function of dividing the number of matching positions by the number of positions compared and multiplying by 100. For example, after alignment, if 6 out of 10 sequence positions are identical between two compared sequences, the identity is 60%. The % identity is typically determined over the entire length of the query sequence being analyzed. Two molecules having the same primary amino acid sequence or nucleic acid sequence are identical regardless of chemical and / or biological modifications.

[0103] Antibody Ab for use in the preparation of the ADCs of the present disclosure The antibody Ab for use in making the ADCs of the present disclosure is an anti-folate receptor alpha (FRα) antibody that specifically binds to SEQ ID NO: 12.

[0104] Preferably, such antibodies include the following antibodies that are isolated and structurally characterized by their variable heavy and light chain amino acid sequences as set forth in Table 1 below and human constant isotypes.

[0105]

Table 1

[0106] IgG1 LALA corresponds to a mutant IgG1 Fc region containing amino acid substitutions from leucine to alanine at residues 234 and 235, also disclosed in J. Virol 2001 Dec;75(24):12161-8 (by Hezareh et al).

[0107] The full-length light and heavy chains of mAb1 and the corresponding coding sequences are shown in Table 2 below.

[0108] [Table 2]

[0109] Examples of the amino acid sequences of VH CDR1 (also called HCDR1), VH CDR2 (also called HCDR2), VH CDR3 (also called HCDR1), VL CDR1 (also called LCDR1), VL CDR2 (also called LCDR2), and VL CDR3 (also called HCDR3) of the mAb1 antibody are shown in Table 3.

[0110] In Table 3, the CDR regions of some antibodies of the present disclosure are depicted using the Kabat system. For ease of reading, the CDR regions are hereinafter referred to as HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively.

[0111] [Table 3]

[0112] The following Tables 4 and 5 provide amino acid and nucleotide sequences that are useful for comparison with the antibody of the ADC.

[0113] [Table 4] * aa is the amino acid sequence nt is the nucleotide sequence

[0114] [Table 5-1]

[0115] [Table 5-2]

[0116]

Table 5-3

[0117] In one embodiment, Ab is (i) a heavy chain variable region comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 3; and (ii) a light chain variable region comprising LCDR1 of SEQ ID NO: 4; LCDR2 of SEQ ID NO: 5 or 8; and LCDR3 of SEQ ID NO: 6; an isolated recombinant antibody having said antibody specifically binds to folate receptor alpha of SEQ ID NO: 12.

[0118] In certain embodiments, Ab is (a) a variable heavy chain polypeptide comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 3, and a variable light chain polypeptide comprising LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5 and LCDR3 of SEQ ID NO: 6; or (b) a variable heavy chain polypeptide comprising VH of SEQ ID NO: 7 and a variable light chain polypeptide VL of SEQ ID NO: 8 a recombinant antibody comprising any of

[0119] In certain embodiments, Ab is a recombinant antibody comprising or consisting essentially of a heavy chain of SEQ ID NO: 9 and a light chain of SEQ ID NO: 10.

[0120] In certain embodiments, Ab is a recombinant antibody comprising or consisting essentially of a heavy chain of SEQ ID NO: 11 and a light chain of SEQ ID NO: 10.

[0121] In certain embodiments, Ab is a recombinant antibody comprising or consisting essentially of a heavy chain of SEQ ID NO: 16 and a light chain of SEQ ID NO: 17.

[0122] In certain embodiments, Ab is an anti-FRα antibody having one or more of the following characteristics.

[0123] (i) When measured by surface plasmon resonance such as a Biacore® assay, Ab has a K D of 100 nM or less, preferably 50 nM or less, and binds to folate receptor alpha (FRα). D (ii) Ab is an internalizing antibody and, in particular, internalizes in FRα-expressing tumor cells. (iii) Ab enables conjugation of 4 to 8 payloads per antibody, particularly without issues of stability or aggregation.

[0124] In certain embodiments that can be combined with the previous embodiments, Ab is an internalizing antibody fragment of a recombinant antibody as defined above.

[0125] As used herein with respect to an antibody, "internalizing" refers to an antibody that, upon specific binding to a cell, can be taken up into an internal compartment (i.e., "internalized"), preferably an intracellular degradation compartment, through the outer lipid bilayer membrane of the cell.

[0126] Antibody fragments include, but are not limited to, Fab, Fab’, Fab’-SH, F(ab’)2, Fv, UniBody and scFv fragments, diabodies, single domains or nanobodies and other fragments. The term “diabody” refers to a small antibody fragment having two antigen-binding sites, which fragment comprises a heavy-chain variable domain (VH) linked to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains on another chain, creating two antigen-binding sites. A single-domain antibody is an antibody fragment that comprises all or part of the heavy-chain variable domain of an antibody or all or part of the light-chain variable domain of an antibody. In certain embodiments, the single-domain antibody is a human single-domain antibody (see Domantis, Inc., Waltham, MA; for example, U.S. Patent No. 6,248,516). Antibody fragments can be made by a variety of techniques including, but not limited to, proteolytic digestion of intact antibodies, as well as production by recombinant host cells as described herein.

[0127] In certain embodiments, Ab is a humanized antibody. Typically, non-human antibodies are humanized to reduce their immunogenicity in humans while having at least the same (or improved) affinity as the parental non-human antibody. In preferred embodiments, the antibodies of the present disclosure are humanized antibodies. Generally, a humanized antibody comprises one or more variable domains in which the CDR (or a portion thereof) is derived from a non-human antibody, such as a mouse anti-FRα internalizing antibody, and the framework region (or a portion thereof) is derived from a human antibody sequence. A humanized antibody may also optionally include at least a portion of a human constant region. In some embodiments, some framework residues in the humanized antibody are replaced with the corresponding residues from a non-human antibody (e.g., the anti-FRα mouse antibody from which the CDR residues are derived) to, for example, restore or improve the specificity or affinity of the antibody. In some particular embodiments, some CDR residues in the humanized antibody are also replaced to, for example, restore or improve the specificity or affinity of the antibody. Humanized antibodies and methods for their production are reviewed, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described, for example, in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Natl Acad. Sci. USA 86:10029-10033 (1989); U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing specificity determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "resurfacing"); Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing a "guided selection" approach to FR shuffling).

[0128] Preferably, Ab is a humanized or human silent antibody, preferably a humanized silent IgG1 antibody.

[0129] As used herein, the term "silent" antibody refers to an antibody that does not exhibit or exhibits low ADCC activity when measured in an ADCC activity assay.

[0130] In one embodiment, the term "no or low ADCC activity" means that the silent antibody exhibits at least less than 10%, e.g., less than 50%, of the ADCC activity observed for the corresponding antibody having the wild-type corresponding IgG isotype.

[0131] The silenced effector function can be obtained by mutations in the Fc constant region of the antibody and is described in the art: Strohl 2009 (AA&N297A) Baudino 2008, D265A (Baudino et al., J. Immunol. 181 (2008): 6664-69, Strohl, CO Biotechnology 20 (2009): 685-91), or also outlined in Saunders 2019 (Front. Immunol., 07 June 2019, doi:10.3389 / fimmu.2019.01296). Examples of silent IgG1 antibodies include the so-called LALA mutations comprising L234A and L235A mutations in the IgG1 Fc amino acid sequence, or those comprising Ser228Pro paired with Leu235Glu. Pro331Ser may also be used, optionally in combination with Ledu234Glu and Leu235Phe (LALA-PG), to generate a silent IgG1 antibody. Another example of a silent IgG1 antibody comprises the N297A mutation that results in an aglycosylated or non-glycosylated antibody. An example of a silent IgG4 antibody comprises Ser228Pro.

[0132] In certain embodiments, Ab is farletuzumab, or another anti-FRα antibody disclosed in WO 2005080431 or WO 2017151979.

[0133] In other specific embodiments, Ab is mirvetuximab or a silent LALA variant of mirvetuximab.

[0134] In preferred embodiments, Ab is a silent LALA variant of farletuzumab, or another anti-FRα antibody disclosed in WO 2005 / 080431 or WO 2017 / 151979.

[0135] Antibodies having a variant amino acid sequence can be obtained by testing the mutated antibody encoded for retained function (i.e., the functions described above) using the functional assays described herein following mutagenesis of the encoding nucleic acid molecule (e.g., site-directed or PCR-mediated mutagenesis).

[0136] Antibody having conservative modifications In certain embodiments, Ab has a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 sequences, and one or all of these CDR sequences have a specific amino acid sequence based on the mAb1 (silent LALA variant version of farletuzumab) antibody described herein, or a functional variant of the antibody having a similar CDR sequence that differs from the CDR sequences of farletuzumab by 1, 2, or 3 amino acid conservative modifications, and the antibody or protein retains the desired functional properties of the mAb1 antibody, particularly when used as an ADC.

[0137] In certain embodiments, Ab has a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 sequences, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 sequences, and one or more of these CDR sequences have a specific amino acid sequence based on the mirvetuximab (or silent LALA variant version of mirvetuximab) antibody described herein, or a functional variant of said antibody having a similar CDR sequence that differs from the CDR sequence of farletuzumab by 1, 2, or 3 amino acid conservative modifications, and the antibody or protein retains the desired functional properties of the mAb1 antibody, particularly when used as an ADC.

[0138] Desired functional properties of the anti-FRα antibody include, but are not limited to, the following. (i) The anti-FRα antibody binds to folate receptor α (FRα) with an EC50 of less than 5 nM, e.g., about 0.5 nM, as determined by an Elisa assay (described in the Examples). (ii) The anti-FRα antibody has a K D of 100 nM or less, preferably 50 nM or less, when measured by surface plasmon resonance such as a Biacore® assay, D and binds to folate receptor receptor α (FRα) (e.g., as determined using the SPR Biacore affinity assay described in the Examples). (iii) The anti-FRα is an internalizing antibody, particularly internalizing in FRα-expressing tumor cells. (iv) The anti-FRα enables conjugation of 4 to 8 payloads per antibody, particularly without stability issues or aggregation, and the drug-antibody ratio is determined by RPLC-MS as described, for example, in the Examples. (v) An ADC comprising such a variant anti-FRα antibody provides in vitro efficacy in an FRα-negative cell line (such as the BT-474 breast cancer cell line) similar to or lower than that of the corresponding control ADC comprising mAb1 as an anti-FRα antibody, using, for example, an in vitro efficacy assay in an FRα-negative cell line used in the Examples, and / or (vi) An ADC having such a variant anti-FRα antibody provides in vivo efficacy similar to or higher than that of the corresponding control ADC having mAb1 as the anti-FRa antibody in a tumor-bearing mouse xenograft model, for example using one of the xenograft models used in the examples.

[0139] As used herein, the term "conservative sequence modification" is intended to refer to amino acid substitutions in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within the CDR regions of the antibodies of the present disclosure can be replaced with other amino acid residues from the same side chain family, and the altered antibodies can be tested for retained function using the functional assays described herein.

[0140] The modifications can be introduced into the antibodies of the present disclosure by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis.

[0141] In certain embodiments, the Ab is an anti-FRα antibody comprising six CDRs that are 100% identical to the corresponding CDRs of SEQ ID NOs: 1-6, and framework amino acid regions that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the corresponding framework amino acid regions specified by SEQ ID NOs: 7 and 8, respectively, and the anti-FRα antibody has the following characteristics. (i) The anti-FRα antibody binds to folate receptor α (FRα) as determined by an ELISA assay (described in the Examples) with an EC50 of less than 5 nM, for example, about 0.5 nM. (ii) The anti-FRα has a KD of 100 nM or less, preferably 50 nM or less, when measured by surface plasmon resonance such as a Biacore® assay and binds to folate receptor receptor α (FR). D with a KD of preferably 50 nM or less D and binds to folate receptor receptor α (FR). (iii) The anti-FRα is an internalizing antibody, particularly internalizing in FRα-expressing tumor cells. (iv) The anti-FRα enables conjugation of 4 to 8 payloads per antibody, particularly without stability issues or aggregation. The drug-antibody ratio is determined by RPLC-MS as described in the Examples, for example. (v) An ADC comprising such a variant anti-FRα antibody provides in vitro efficacy in FRα-negative cell lines (such as the BT-474 breast cancer cell line) similar to or lower than the corresponding control ADC comprising mAb1 as the anti-FRα antibody, using, for example, the in vitro efficacy assay in the FRα-negative cell lines used in the Examples. (vi) An ADC having such a variant anti-FRα antibody provides in vivo efficacy in a tumor-bearing mouse xenograft model similar to or higher than the corresponding control ADC having mAb1 as the anti-FRa antibody, using, for example, one of the xenograft models used in the Examples.

[0142] In certain embodiments, the Ab is an anti-FRα antibody comprising six CDRs that are 100% identical to the corresponding CDRs of mirvetuximab and a framework amino acid region that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding framework amino acid region of mirvetuximab, and the anti-FRα antibody has the following characteristics. (i) The anti-FRα binds to folate receptor α (FRα) as determined by an ELISA assay (described in the Examples) with an EC50 of less than 5 nM, for example, about 0.5 nM. (ii) The anti-FRα binds to folate receptor alpha (FRα) with a K of 100 nM or less, preferably 50 nM or less, when measured by surface plasmon resonance such as a Biacore® assay. D wherein the "K" is preferably 50 nM or less. D (iii) The anti-FRα is an internalizing antibody, particularly internalizing in FRα-expressing tumor cells. (iv) The anti-FRα enables conjugation of 4 to 8 payloads per antibody, particularly without stability issues or aggregation, and the drug-antibody ratio is determined by RPLC-MS as described, for example, in the Examples. (v) An ADC comprising such a variant anti-FRα antibody provides in vitro efficacy in an FRα-negative cell line (such as the BT-474 breast cancer cell line) that is the same as or lower than that of a corresponding control ADC comprising mAb1 as the anti-FRα antibody, using, for example, an in vitro efficacy assay in the FRα-negative cell line used in the Examples. And / or (vi) An ADC having such a variant anti-FRα antibody provides in vivo efficacy in a tumor-bearing mouse xenograft model that is the same as or higher than that of a corresponding control ADC having mAb1 as the anti-FRα antibody, using, for example, one of the xenograft models used in the Examples.

[0143] Framework or Fc engineering For use in the ADCs of the present disclosure, the Abs can also include engineered versions of the antibodies disclosed in the previous section, including, for example, those in which framework residues within the VH and / or VL have been modified to improve antibody properties. Typically, such framework modifications are made to reduce the immunogenicity of the antibody. For example, one approach is to "backmutate" one or more framework residues to their corresponding germline sequences. More specifically, an antibody that has undergone somatic mutations may contain framework residues that differ from the germline sequences from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequence to the germline sequences from which the antibody is derived. To return the framework region sequences to their germline configurations, somatic mutations can be "backmutated" to the germline sequences, for example, by site-directed mutagenesis or PCR-mediated mutagenesis. Such "backmutated" antibodies are also intended to be encompassed by the present invention.

[0144] Another type of framework modification involves mutating one or more residues within the framework region, or even within one or more CDR regions, to remove T cell epitopes and thereby reduce the potential immunogenicity of the antibody.

[0145] In addition to, or instead of, modifications made within the framework or CDR regions, the antibody can typically be engineered to include modifications within the Fc region to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antibody-dependent cell cytotoxicity.

[0146] Furthermore, the Abs can be antibodies that can be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or modified to alter their glycosylation, where again one or more functional properties of the antibody are altered. Each of these embodiments will be described in more detail below.

[0147] As used herein, the terms "isotype constant region" or "Fc region" are used interchangeably to define the C-terminal region of an immunoglobulin heavy chain that includes the native sequence Fc region and variant Fc regions. The human IgG heavy chain Fc region is generally defined as including the amino acid residues from position C226 or P230 to the carboxyl terminus of the IgG antibody. The numbering of the residues in the Fc region is that of the Kabat EU index. The C-terminal lysine (residue K447) of the Fc region can be removed, for example, during antibody production or purification. Thus, the antibody compositions of the present disclosure can include an antibody population in which all K447 residues have been removed, an antibody population in which the K447 residues have not been removed, and an antibody population having a mixture of antibodies with and without the K447 residue.

[0148] In one particular embodiment, the hinge region of CH1 is modified such that the number of cysteine residues in the hinge region is changed, e.g., increased or decreased. This approach is further described in U.S. Patent No. 5,677,425 to Bodmer et al. The number of cysteine residues in the hinge region of CH1 is changed, for example, to facilitate assembly of the light and heavy chains, or to increase or decrease the stability of the antibody.

[0149] In other embodiments, the Fc region is modified to decrease the ability of the antibody to mediate antibody-dependent cell cytotoxicity (ADCC) and / or to decrease the affinity of the antibody for Fcγ receptors by modifying one or more amino acids. Such antibodies with reduced effector function, particularly reduced ADCC, include silent antibodies.

[0150] In certain embodiments, the Fc domain of the IgG1 isotype is used. In some specific embodiments, a variant of the IgG1 Fc fragment is used, such as a silent IgG1 Fc that reduces or eliminates the ability of an antibody-drug conjugate (ADC) to mediate antibody-dependent cell cytotoxicity (ADCC) and / or bind to Fcγ receptors. A preferred example of an IgG1 isotype silent variant is an IgG1 in which leucine is replaced by alanine at amino acid positions 234 and 235, as described in J. Virol 2001 Dec;75(24):12161-8 (by Hezareh et al). Another example is an IgG1 isotype silent variant having the LALA-PG triple mutation, in which, in addition to the LALA mutation, the proline at position 329 is replaced by glycine.

[0151] In certain embodiments, the Fc domain is a silent Fc variant that prevents glycosylation at position 297 of the Fc domain. For example, the Fc domain includes an asparagine amino acid substitution at position 297. Examples of such amino acid substitutions are the substitution of N297 with glycine or alanine.

[0152] In yet another embodiment, the glycosylation of the antibody is modified. For example, a non-glycosylated antibody can be produced (i.e., the antibody lacks glycosylation). Glycosylation can be altered, for example, to increase the affinity of the antibody for an antigen. Such carbohydrate modifications can be achieved, for example, by modifying one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in the elimination of glycosylation sites in one or more variable region frameworks, thereby eliminating glycosylation at those sites. Such non-glycosylation can increase the affinity of the antibody for an antigen. Such approaches are described in more detail in U.S. Pat. Nos. 5,714,350 and 6,350,861 by Co et al.

[0153] Another modification of the antibodies of the present disclosure contemplated by the present disclosure is pegylation or heshylation or related techniques. The antibody can be pegylated, for example, to extend the biological (e.g., serum) half-life of the antibody. To pegylate an antibody, the antibody or a fragment thereof is typically reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions such that one or more PEG groups become attached to the antibody or antibody fragment. Pegylation can be carried out by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or a similar reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any form of PEG that has been used to derivatize other proteins, such as mono(1-C 10 ) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody to be pegylated is a non-glycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to the antibodies of the present disclosure. See, for example, European Patent No. 0154316 by Nishimura et al. and European Patent No. 0401384 by Ishikawa et al.

[0154] Another modification of the antibodies contemplated by the present disclosure is a conjugate or protein fusion of at least the antigen-binding region of the antibodies of the present disclosure with a serum protein, such as human serum albumin or a fragment thereof, to increase the half-life of the resulting molecule. Such an approach is described, for example, in European Patent No. 0322094 by Ballance et al.

[0155] Preparation of antibodies for use in the preparation of the ADCs of the present disclosure The antibodies of the present disclosure can be obtained using conventional techniques known to those skilled in the art. For further information regarding the nucleic acids encoding the antibodies of the present disclosure and the generation of transfectomas that produce these antibodies, those skilled in the art can also refer to International Application No. International Publication No. 2005080431.

[0156] For example, to express an antibody or an antibody fragment thereof, DNA encoding a partial or full-length light chain and heavy chain can be obtained by standard molecular biology or biochemical techniques (e.g., DNA chemical synthesis, PCR amplification, or cDNA cloning using a hybridoma that expresses the desired antibody), and the DNA can be inserted into an expression vector such that the gene is operably linked to transcriptional and translational control sequences. In this context, the term "operably linked" is intended to mean that the antibody gene is ligated to the vector such that the transcriptional and translational control sequences within the vector perform their intended function of regulating the transcription and translation of the antibody gene. The expression vector and expression control sequences are selected to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors, or more typically, both genes are inserted into the same expression vector. The antibody gene is inserted into the expression vector by standard methods (e.g., ligation of antibody gene fragments and complementary restriction sites on the vector, or blunt-end ligation if no restriction sites are present). The light chain and heavy chain variable regions of the antibodies described herein are used to insert them into an expression vector that already encodes the heavy chain constant region and light chain constant region of the desired isotype such that the VH segment is operably linked to the CH segment within the vector and the VL segment is operably linked to the CL segment within the vector, thereby producing a full-length antibody gene of any antibody isotype. Additionally or alternatively, the recombinant expression vector can encode a signal peptide that promotes the secretion of the antibody chain from the host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is in-frame and linked to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

[0157] In addition to the antibody chain gene, the recombinant expression vectors disclosed herein have regulatory sequences that control the expression of the antibody chain gene in a host cell. The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain gene. Those skilled in the art will appreciate that the design of the expression vector, including the selection of regulatory sequences, may depend on factors such as the choice of host cell to be transformed and the level of expression of the desired protein. Regulatory sequences for mammalian host cell expression include viral elements that direct high-level protein expression in mammalian cells, such as the cytomegalovirus (CMV), simian virus 40 (SV40), adenovirus (e.g., adenovirus major late promoter (AdMLP)), and promoters and / or enhancers derived from polyomavirus. Alternatively, non-viral regulatory sequences such as the ubiquitin promoter or the P-globin promoter may be used. Additionally, regulatory elements are composed of sequences from different sources such as the SRa promoter system, which includes sequences from the SV40 early promoter and sequences from the long terminal repeat of human T-cell leukemia virus type 1.

[0158] In addition to the antibody chain gene and regulatory sequences, the recombinant expression vectors of the present disclosure may have additional sequences such as sequences that regulate the replication of the vector in a host cell (e.g., origin of replication) and selectable marker genes. The selectable marker gene facilitates the selection of host cells into which the vector has been introduced (see, e.g., all of U.S. Pat. Nos. 4,399,216, 4,634,665, and 5,179,017 by Axel et al.). For example, typically, the selectable marker gene confers resistance to drugs such as G418, hygromycin, or methotrexate on the host cells into which the vector has been introduced. Selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr host cells with methotrexate selection / amplification) and the neo gene (for G418 selection).

[0159] For the expression of the light and heavy chains, expression vector(s) encoding the heavy and light chains are transfected into host cells by standard techniques. The various forms of the term "transfection" are intended to encompass the diverse techniques commonly used for the introduction of exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. It is theoretically possible to express the antibodies of the present disclosure in either prokaryotic host cells or eukaryotic host cells. The expression of antibodies in eukaryotic cells, such as mammalian host cells, yeast, or filamentous fungi, is discussed because such eukaryotic cells, particularly mammalian cells, are more likely than prokaryotic cells to assemble and secrete properly folded immunologically active antibodies.

[0160] The nucleotide sequences encoding the heavy and light chains of the preferred antibodies for use in the preparation of the ADCs of the present disclosure are set forth in Tables 3 and 4 (see particularly SEQ ID NOs: 13-15).

[0161] Mammalian host cells for expressing the recombinant antibodies of the present disclosure include Chinese hamster ovary (CHO cells) including dhfr-CHO cells (described in Urlaub and Chasin, 1980) used with a DHFR selectable marker (described in Kaufman and Sharp, 1982), CHOK1 dhfr+ cell line, NSO myeloma cells, COS cells, and SP2 cells. When a recombinant expression vector encoding the antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period sufficient for the expression of the antibody in the host cell and, optionally, the secretion of the antibody into the culture medium in which the host cell is grown. The antibodies can be recovered and purified from the culture medium after their secretion, for example, using standard protein purification methods.

[0162] Linker L As used herein, "linker" or "linker moiety" refers to any chemical moiety capable of covalently linking a compound such as a drug moiety to another moiety such as an antibody moiety.

[0163] The ADC of the present disclosure includes a cleavable linker moiety L that binds to Ab on one side and to drug D on the other side, and the anti-FRα antibody is disclosed in the previous section.

[0164] In certain embodiments, the linker L is covalently attached to one or more thiol residues of the antibody Ab, such as natural or engineered cysteine residues in the antibody sequence.

[0165] As used herein, the term "cleavable" refers to a linker that can be cleaved under certain environmental conditions (such as redox potential or pH) or in response to the intracellular environment, for example, under certain lysosomal enzymes after internalization of the ADC within the cell.

[0166] In certain embodiments, L is a cleavable linker moiety of the formula -A-W- (wherein A is any stretcher unit linked to Ab and W is a cleavable moiety linked to D).

[0167] In certain embodiments, the stretcher unit A of any element can be selected from the group consisting of one or more amino acids, one or more N-substituted amino acids, optionally substituted polyethers, C1-C 12 alkylene, arylene having 6 to 10 ring atoms, C3-C8 cycloalkylene, heterocycloalkylene having 5 to 10 ring atoms, heteroarylene having 5 to 10 ring atoms, C2-C 10 alkenylene, and any combination thereof, and the alkylene and alkenylene are optionally interrupted by one or more heteroatoms or chemical groups selected from -O-, -S-, -C(O)-, -NR''-, -C(O)NR''-, -NR''-C(O)-, -NR''-C(O)-NR'''-,-NR''-C(O)-O-,-O-C(O)NR''- and triazole. When the alkylene, arylene, cycloalkylene, heterocycloalkylene, heteroarylene, and alkenylene are optionally substituted with one or more substituents selected from halogen, oxo, -OH, -NO2, -CN, C1-C6 alkyl, C3-C6 cycloalkyl, heterocyclyl having 5-10 ring atoms, aryl having 6-10 ring atoms, heteroaryl having 5-10 ring atoms, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 haloalkoxy, -(CO)-R', -O-(CO)-R', -(CO)-O-R', -(CO)-NR''R''', -NR''-(CO)-R', and -NR''R''', R', R'', and R''' are independently selected from H and C1-C6 alkyl,

[0168] Examples of cleavable linkers that can be used in the ADCs of the present disclosure include acid-sensitive or acid-labile linkers, such as acid-labile hydrazone linkers, lysosomal protease-sensitive linkers, β-glucuronide linkers, or glutathione-sensitive disulfide linkers.

[0169] In certain embodiments where L is a lysosomal protease-sensitive linker, W can include a cleavable peptide moiety selected from the group consisting of valine-citrulline (Val-Cit), alanine-alanine-asparagine (Ala-Ala-Asn), valine-alanine (Val-Ala), and phenylalanine-lysine (Phe-Lys). Preferably, W includes a valine-alanine peptide moiety.

[0170] In other certain embodiments where L is a protease-sensitive linker, W preferably includes a sugar-cleavable unit selected from a β-glucuronide or β-galactoside moiety.

[0171] In other certain embodiments where L is a glutathione-sensitive linker, W includes a disulfide moiety.

[0172] In a preferred embodiment, L is a cleavable linker moiety of the formula -A-W-, wherein W is of the following formula (III):

[0173] [Chemical Formula] wherein each R2 is independently selected from the group consisting of an electron-withdrawing group and C1-C4 alkyl, n is 0, 1 or 2, T is a sugar-cleavable unit or a polypeptide-cleavable unit, when T is a sugar-cleavable unit, Y is O, or when T is a polypeptide-cleavable unit, Y is NR3, R3 is H, C1-C 24 alkyl, C2-C6 alkenyl; optionally substituted polyether, aryl having 6-10 ring atoms, C3-C8 cycloalkyl, heterocycloalkyl having 3-10 ring atoms, heteroaryl having 5-10 ring atoms, and any combination thereof, selected from the group consisting of; the alkyl and alkenyl are optionally interrupted by one or more heteroatoms or chemical groups selected from -O-, -S-, -C(O)-, -NR''-, -C(O)NR''-, -NR''-C(O)-, -NR''-C(O)-NR'''-,-NR''-C(O)-O-, -O-C(O)NR''- and triazole, R'' and R''' are independently selected from H and C1-C6 alkyl, formula (III) and its pharmaceutically acceptable salts.

[0174] In certain embodiments, T is a sugar-cleavable unit that is glucuronide or galactoside.

[0175] In other certain embodiments, T is a dipeptide preferably selected from Val-Cit, Val-Ala and Phe-Lys.

[0176] In a more specific embodiment, L corresponds to the linker - A - W - of formula (IV),

[0177]

Chemical formula

[0178] In a preferred embodiment of the linker of formula (IV), X1 and X2 are one or more amino acids, one or more N - substituted amino acids, optionally substituted polyether, C1 - C12 alkylene, arylene having 6 to 10 ring atoms, C3-C8 cycloalkylene, heterocycloalkylene having 5 to 10 ring atoms, heteroarylene having 5 to 10 ring atoms, C2-C 10 alkenylene, and are independently selected from the group consisting of any combination thereof, the alkylene and alkenylene are optionally interrupted by one or more heteroatoms or chemical groups selected from -O-, -S-, -C(O)-, -NR''-, -C(O)NR''-, -NR''-C(O)-, -NR''-C(O)-NR'''-,-NR''-C(O)-O-,-O-C(O)NR''- and triazole, the alkylene, arylene, cycloalkylene, heterocycloalkylene, heteroarylene, and alkenylene are halogen, oxo, -OH, -NO2, -CN, C1-C6 alkyl, C3-C6 cycloalkyl, heterocyclyl having 5 to 10 ring atoms, aryl having 6 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 haloalkoxy, -(CO)-R', -O-(CO)-R', -(CO)-O-R', -(CO)-NR''R''', -NR''-(CO)-R', and -NR''R''' optionally substituted with one or more substituents selected from, R', R'' and R''' are independently selected from H and C1-C6 alkyl.

[0179] In another preferred embodiment of the linker of formula (IV), Z is one or more amino acids, one or more N-substituted amino acids, optionally substituted polyethers, C1-C 12 alkylene, arylene having 6 to 10 ring atoms, C3-C8 cycloalkylene, heterocycloalkylene having 5 to 10 ring atoms, heteroarylene having 5 to 10 ring atoms, C2-C 10 alkenylene, and are independently selected from the group consisting of any combination thereof, When the alkylene and alkenylene are optionally interrupted by one or more heteroatoms or chemical groups selected from -O-, -S-, -C(O)-, -NR''-, -C(O)NR''-, -NR''-C(O)-, -NR''-C(O)-NR'''-,-NR''-C(O)-O-,-O-C(O)NR''-, and triazole, the alkylene, arylene, cycloalkylene, heterocycloalkylene, heteroarylene, and alkenylene are optionally substituted with one or more substituents selected from halogen, oxo, -OH, -NO2, -CN, C1-C6 alkyl, C3-C6 cycloalkyl, heterocyclyl having 5-10 ring atoms, aryl having 6-10 ring atoms, heteroaryl having 5-10 ring atoms, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 haloalkoxy, -(CO)-R', -O-(CO)-R', -(CO)-O-R', -(CO)-NR''R''', -NR''-(CO)-R', and -NR''R''', R', R'', and R''' are independently selected from H and C1-C6 alkyl.

[0180] In another embodiment of the linker L of formula (IV) or (V), K is preferably polysarcosine of the following formula (V),

Chemical formula

[0181] In another embodiment of the linker L of formula (IV) or (V), K is preferably a polyethylene glycol moiety (PEG) containing ethylene glycol moieties between 2 and 50.

[0182] In a preferred embodiment, L corresponds to the linker of formula (VI).

Chemical formula

[0183] Preferably, the linker L is covalently bound to one or more thiol residues of the antibody Ab, for example, eight thiol residues of the antibody Ab.

[0184] Payload In one embodiment, D of the present disclosure is a payload linked to X on the side of the carbonyl functional group of X. In one embodiment, the linkage between X and D occurs between the carbonyl functional group of X and the amino group of D.

[0185] The payload D is an important component of the ADC design. The payload can be a therapeutic agent or a drug. The term "drug" particularly refers to an agent that can regulate biological processes and / or has biological activity.

[0186] In certain embodiments, the payload D is a cytotoxic drug that is activated after release from the internalized ADC in the cytoplasm of tumor cells. Ideally, it should be able to destroy tumor cells without affecting non-tumor cells (when linked to the antibody). The payload should also ideally have high stability in systemic circulation and lysosomes. Preferably, it should have an IC50 value of sub-nanomolar concentration in vitro for cancer cell lines and sufficient solubility in an aqueous environment.

[0187] In certain embodiments, the cytotoxic drug is selected from auristatins (including monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF)), maytansinoids (such as maytansine), calicheamicin, duocarmycin, and DNA damaging agents such as anthracyclines (e.g., daunorubicin, doxorubicin, dihydroxyanthracenedione), or topoisomerase I inhibitors such as camptothecin or their analogs.

[0188] In certain embodiments, D is selected from the group consisting of a taxon, cytochalasin B, an auristatin (including monomethyl auristatin E), gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracinedione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, a glucocorticoid, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.

[0189] In certain embodiments, D is selected from the group consisting of antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), ablative agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamine platinum(II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mitramycin and anthramycin (AMC)) and antimicrotubule agents (e.g., vincristine and vinblastine).

[0190] Preferably, D is an inhibitor of topoisomerase I and includes, but is not limited to, camptothecin analogs, indolocarbazole analogs, phenanthridine analogs, fluoroquinolone analogs, quinoxaline, evodiamine, acridine, naphthyridine, deoxynybomycin analogs, angustine analogs, stilbenethiazole analogs, pyrroloquinazolinquinoline alkaloids or indenoisoquinoline analogs (for reviews, see Selas et al., A patent review of topoisomerase I inhibitors (2016 - present), 2021, Expert Opinion on Therapeutic Patents, 31(6), 473 - 508), and is preferably selected from the group consisting of camptothecin and its analogs, including, but not limited to, irinotecan, topotecan, camptothecin, SN - 38, exatecan, DXd, silatecan, cositecan, lutetcan, gimatecan, belotecan, rubitecan (for reviews, see Sriram et al., Camptothecin and its analogues: a review on their chemotherapeutic potential, 2005, Natural Product Research, 14(9), 393 - 412).

[0191] In a preferred embodiment, D is the drug moiety of exatecan of formula (II) below.

[0192]

Chemical Structure

[0193] The ADCs of the present disclosure In one embodiment, the present disclosure provides an ADC in which an anti - FRα antibody is linked to a drug (particularly exatecan). The linker L is as defined above.

[0194] Preferably, such an ADC can selectively deliver an effective dose of a drug, such as an inhibitor of topoisomerase I, preferably exatecan, to tumor cells expressing FRα.

[0195] In one embodiment, the present disclosure provides an ADC of formula (I), Ab-[L-D]p (I), wherein -Ab is an anti-folate receptor alpha (FRα) antibody that specifically binds to SEQ ID NO: 12, -L is a cleavable linker moiety attached to the antibody via a thiol residue, -D is a cytotoxic drug moiety attached to L, such as an inhibitor of topoisomerase I, -p is from 1 to 8, preferably from 6 to 8, more preferably p is 8.

[0196] Ab, L, and D are as defined in the previous section.

[0197] The term "p", also referred to as the drug-to-antibody ratio or "DAR", corresponds to the number of drug moieties per antibody moiety in the ADC of formula (I), or the number of -L-D moieties per antibody Ab.

[0198] The drug-antibody ratio generally has an exact value for a single ADC, but that value is typically an average value when used to describe a composition containing many ADCs due to some degree of heterogeneity associated with the conjugation process. The average loading of a sample of ADC is also referred to herein as the drug-antibody ratio or "DAR". In some embodiments, DAR(p) is from about 1 to about 8 (i.e., 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, and 8), preferably from about 4 to about 8, more preferably from about 6 to about 8, even more preferably about 8.

[0199] Thus, in a composition comprising multiple copies of the ADC of formula (I), "p" refers to the average number of -L-D moieties per antibody Ab. In the case of an average p number close to 8, the ADC is considered to be "DAR8".

[0200] Methods for measuring the drug-antibody ratio are disclosed in the Examples or described, for example, in Conilh et al (Pharmaceuticals 2021, 14(3), 247).

[0201] Drug loading onto the antibody via the linker L can be limited by the number of attachment sites on the antibody moiety. In some embodiments, the linker portion (L) of the ADC binds to the antibody moiety via a chemically active group on one or more amino acid residues on the antibody moiety. For example, the linker can bind to the antibody moiety via a free amino acid, imino, hydroxyl, thiol, or carboxyl group (e.g., the N-terminus or C-terminus, the ε-amino group of one or more lysine residues, the free carboxyl group of one or more glutamic acid residues or aspartic acid residues, or the sulfhydryl group of one or more cysteine residues). The site to which the linker binds can be a native residue in the amino acid sequence of the antibody moiety or can be introduced into the antibody moiety, for example, by DNA recombination techniques (e.g., by introducing cysteine or non-native amino acid residues into the amino acid sequence) or by protein biochemistry (e.g., by reduction, pH adjustment, or hydrolysis).

[0202] If the binding site is an interchain cysteine thiol group, the antibody can have only one or a few cysteine thiol groups to which the linker can bind. In fact, most reactive cysteine thiols generally exist as interchain disulfide bridges. Excessive binding of the linker-toxin to the antibody can destabilize the antibody by reducing the cysteine residues available to form interchain disulfide bridges. Thus, the optimal drug-antibody ratio should increase the efficacy of the ADC (by increasing the number of bound drug moieties per antibody) without destabilizing the antibody moiety and without reducing the pharmacokinetic properties.

[0203] In certain embodiments, -L-D is attached to the interchain reactive thiol residues of the antibody. A typical antibody with a full-length heavy chain and light chain contains 8 available interchain reactive thiol residues. Thus, in certain embodiments, the ADC is a DAR8 ADC in which 8 -L-D moieties are covalently attached to the 8 interchain reactive thiol residues of the antibody Ab, and most preferably, -L- corresponds to formula (VI).

[0204] In certain embodiments, the ADC of the present disclosure corresponds to the following formula (VII),

[0205]

Chemical formula

[0206] In a more preferred embodiment, the ADC of the present disclosure corresponds to the following formula (VII),

[0207]

Chemical formula

[0208] In certain embodiments, the ADC of the present disclosure corresponds to formula (I), wherein (i) Ab is · a variable heavy chain polypeptide comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 3 and · a variable light chain polypeptide comprising LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6; and is an anti-folate receptor alpha antibody or an antigen-binding fragment thereof, (ii) L is a cleavable linker of the formula -A-W-, wherein A is any stretcher unit linked to Ab, and W is a cleavable moiety linked to D, (iii) D is an inhibitor of topoisomerase I, for example, exatecan, and (iv) p is from 1 to 8, preferably 8.

[0209] In certain embodiments, the ADCs of the present disclosure correspond to formula (I), wherein (i) Ab is an antibody comprising the heavy chain of SEQ ID NO: 9 and the light chain of SEQ ID NO: 10, (ii) L is a cleavable linker of the formula -A-W-, wherein A is any stretcher unit linked to Ab and W is a cleavable moiety linked to D, (iii) D is an inhibitor of topoisomerase I, for example, exatecan, and (iv) p is from 1 to 8, preferably from 6 to 8, for example about 8.

[0210] In certain embodiments, the ADCs of the present disclosure are ADCs of formula (I), wherein (i) Ab is an antibody comprising the heavy chain of SEQ ID NO: 11 and the light chain of SEQ ID NO: 10, (ii) L is a cleavable linker of the formula -A-W-, wherein A is any stretcher unit linked to Ab and W is a cleavable moiety linked to D, (iii) D is an inhibitor of topoisomerase I, for example, exatecan, and (iv) p is from 1 to 8, preferably from 6 to 8, for example about 8.

[0211] In a preferred embodiment, the ADCs of the present disclosure are ADCs of formula (VII) wherein Ab is an antibody comprising the heavy chain of SEQ ID NO: 9 and the light chain of SEQ ID NO: 10.

[0212] In another particular embodiment, the ADCs of the present disclosure are ADCs of formula (VII) wherein Ab is an antibody comprising the heavy chain of SEQ ID NO: 11 and the light chain of SEQ ID NO: 10.

[0213] Pharmaceutical composition - formulation In another aspect, the present disclosure provides a composition, e.g., a pharmaceutical composition, containing one or a combination of the ADCs disclosed herein (e.g., mAb1 conjugated to linker L of formula (VI), which itself is conjugated to a drug such as exatecan) formulated with a pharmaceutically acceptable carrier.

[0214] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The pharmaceutical formulations of the present disclosure may further include one or more pharmaceutically acceptable excipients selected from stabilizers, surfactants, buffers, antibacterial preservatives, protective agents, antioxidants, chelating agents, and bulking agents.

[0215] As used herein, "solvent" is any pharmaceutically acceptable (i.e., safe and non-toxic for administration to humans or other mammals) and useful component for the preparation of liquid formulations such as aqueous formulations. Exemplary solvents include water such as sterile water for injection (WFI) or bacteriostatic water for injection (BWFI), pH buffers (e.g., phosphate buffered saline), sterile saline, Ringer's solution or dextrose solution, and combinations thereof. Preferably, the solvent is sterile water or bacteriostatic water for injection (BWFI) for the disclosure.

[0216] As used herein, a stabilizer is a compound that increases protein stability, particularly against unfolding and aggregation. Preferably, the stabilizer is approved by the authorities as a suitable additive or excipient in pharmaceutical formulations.

[0217] The stabilizer may be a saccharide. "Saccharide" herein includes a general composition (CH2O) including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, etc. nand its derivatives. Examples of saccharides herein include glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerin, dextran, erythritol, glycerol, arabitol, xylitol, sorbitol, mannitol, melibiose, melezitose, raffinose, mannotriose, stachyose, maltose, lactulose, maltulose, glucitol, maltitol, lactitol, isomaltulose, etc.

[0218] The concentration of the stabilizer in the pharmaceutical preparation of the present disclosure is included between 1 and 500 mM.

[0219] As used herein, "surfactant" refers to a surface-active agent. Surfactants are generally added to protein formulations to reduce the exposure of hydrophobic regions and thus reduce protein-protein interactions and interface-induced aggregation, which are also prevented by competition for adsorption sites.

[0220] Examples of surfactants in this specification include polysorbates (e.g., polysorbate 20 and polysorbate 80); poloxamers (e.g., poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium lauryl sulfate; sodium octyl glucoside; lauryl-, myristyl-, linoleyl- or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- or stearyl-sarcosine; linoleyl-, myristyl- or cetyl-betaine; lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl- or isostearamidopropyl-betaine (e.g., lauroamidopropyl); myristamidopropyl-, palmidopropyl- or isostearamidopropyl-dimethylamine; sodium methyl cocoyl- or disodium methyl oleoyl taurate; polyethylene glycol, polypropylene glycol, and copolymers of ethylene and propylene glycol (e.g., Pluronics, PF68, etc.). Other examples of pharmaceutically acceptable surfactants include polyoxyethylene-sorbitan fatty acid esters (Tween), polyethylene-polypropylene glycol, polyoxyethylene-stearate, polyoxyethylene alkyl ethers, such as polyoxyethylene monolauryl ether, alkylphenyl polyoxyethylene ether (Triton-X), polyoxyethylene-polyoxypropylene copolymers (poloxamers, pluronics) and sodium dodecyl sulfate (SDS). The most suitable polyoxyethylene sorbitan-fatty acid esters are polysorbate 20 (sold under the trademark of Tween 20(trademark)) and polysorbate 80 (sold under the trademark of Tween 80(trademark)).

[0221] The most suitable polyethylene - polypropylene copolymer is the one sold under the name Pluronic® F68 or Poloxamer 188™. The most suitable polyoxyethylene alkyl ether is the one sold under the Brij® trademark. The most suitable alkylphenol - polyoxyethylene ether is sold under the trade name Triton - X.

[0222] The concentration of the surfactant in the pharmaceutical formulations of the present disclosure can be included between 0.01 - 0.1% (w / v).

[0223] As used herein, the term "buffer" refers to an agent that causes the solution containing it to withstand changes in pH due to the action of its acid / base conjugate components. Examples of buffers that control the pH within this range include acetate, succinate, gluconate, histidine, citrate, glycylglycine, and other organic acid buffers.

[0224] A "preservative" is a compound that can be added to the formulations herein to reduce contamination and / or the action of bacteria, fungi, or other infectious agents. The addition of a preservative can, for example, facilitate the manufacture of multi - use (multiple - dose) formulations. Examples of possible preservatives include octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides with long - chain alkyl groups), and benzethonium chloride. Other types of preservatives include phenol, aromatic alcohols such as butyl and benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3 - pentanol, and w - cresol.

[0225] As generally used herein, a "protective agent" is a substance that, when combined with a protein, significantly reduces the chemical and / or physical instability of the protein during lyophilization and / or subsequent refrigerated storage. Exemplary protective agents include sugars and their corresponding sugar alcohols, such as sucrose, lactose, trehalose, dextran, erythritol, arabinitol, xylitol, sorbitol, and mannitol; amino acids, such as arginine or histidine; lyotropic salts, such as magnesium sulfate; polyols, such as propylene glycol, glycerol, poly(ethylene glycol), or poly(propylene glycol); and combinations thereof. Further examples of protective agents include gelatin, dextrin, modified starch, and carboxymethyl cellulose.

[0226] The protective agent can be added to the pre-lyophilized formulation in a "cryoprotective amount". This means that after lyophilization of the protein in the presence of a cryoprotective amount of the protective agent, the protein essentially retains its physical and chemical stability and integrity.

[0227] As generally used herein, an "antioxidant" is a pharmaceutically acceptable excipient that is commonly used to limit oxidation reactions and maintain the stability and safety of proteins. Examples of antioxidants are ascorbic acid, sodium metabisulfite, histamine, methionine, ascorbic acid, glutathione, vitamin E, polyethyleneimine.

[0228] The antioxidant concentration in the pharmaceutical formulations of the present disclosure can be included between 5 and 25 mM.

[0229] A "chelating agent" is a pharmaceutically acceptable excipient that is commonly used to maintain the stability of proteins. Examples of chelating agents include disodium edetate, diethylenetriaminepentaacetic acid, citric acid, hexaphosphate, thioglycolic acid, zinc.

[0230] As generally used herein, an "extender" is a pharmaceutically acceptable excipient generally used to add mass to a lyophilized mixture and contribute to the physical structure of the lyophilized cake (e.g., facilitating the production of an essentially uniform lyophilized cake that maintains an open pore structure). Exemplary extenders include mannitol, glycine, lactose, modified starch, polyethylene glycol), and sorbitol.

[0231] The form, route of administration, dosage, and regimen of the pharmaceutical composition naturally depend on the condition being treated, the severity of the disease, and the age, weight, and gender of the patient.

[0232] The pharmaceutical compositions of the present disclosure can be formulated for topical, oral, parenteral, intraperitoneal, intranasal, intravenous, intramuscular, subcutaneous, or intraocular administration, etc., preferably for intraperitoneal or intravenous administration.

[0233] Preferably, the pharmaceutical composition contains a pharmaceutically acceptable vehicle for an injectable formulation. These can be, in particular, isotonic sterile physiological saline (sodium monophosphate or diphosphate, sodium chloride, potassium, calcium, or magnesium, etc., or a mixture of such salts), or, optionally, a dry composition, in particular a lyophilized composition, that allows the constitution of an injectable solution when sterile water or physiological saline is added.

[0234] The dosage used for administration can be adapted as a function of various parameters, in particular as a function of the mode of administration used, the associated pathological condition, or the desired treatment period.

[0235] To prepare the pharmaceutical composition, an effective amount of the ADC can be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

[0236] Pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions; preparations containing sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders or lyophilized products for the immediate preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy injectability exists. It must be stable under the conditions of manufacture and storage and must be protected against the contaminating action of microorganisms such as bacteria and fungi.

[0237] Solutions of the active compound as the free base or a pharmaceutically acceptable salt can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. These preparations usually contain a preservative to prevent the growth of microorganisms under normal storage and use conditions.

[0238] The ADCs of the present disclosure can be formulated into compositions in the neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed at the free amino groups of proteins) and are formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. Salts formed at the free carboxyl groups can also be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, etc.

[0239] Sterile injectable solutions are prepared by incorporating the required amount of the active compound, optionally with various other ingredients enumerated above, into a suitable solvent and then filtering sterilizing. Generally, dispersions are prepared by incorporating various sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and lyophilization techniques by which powders of the active ingredient + any additional desired ingredients are obtained from previously sterile filtered solutions.

[0240] The preparation of more or higher concentration solutions for direct injection is also contemplated, and the use of DMSO as a solvent is expected to result in extremely rapid penetration and deliver a high concentration of the active agent to small tumor areas.

[0241] Upon formulation, the solution is administered in a therapeutically effective amount in a manner compatible with the dosage formulation. The formulation can be easily administered in various dosage forms such as the types of injection solutions described above, but drug release capsules and the like can also be used.

[0242] In the case of parenteral administration in an aqueous solution, for example, the solution should be appropriately buffered as needed, and the liquid diluent should first be made isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, sterile aqueous media that can be used are known to those skilled in the art in light of the present disclosure. For example, one dosage can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous infusion fluid or injected into the proposed injection site (see, for example, "Remington’s Pharmaceutical Sciences" 15th Edition, pages 1035 - 1038 and 1570 - 1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. In any case, the person responsible for administration will determine the appropriate dosage for each individual subject.

[0243] The ADCs of the present disclosure can be formulated in a therapeutic mixture to contain from about 0.0001 to 1.0 milligrams, or from about 0.001 to 0.1 milligrams, or from about 0.1 to 1.0 milligrams, or even from 1.0 to about 10 milligrams per dose. Multiple administrations can also be made.

[0244] The pharmaceutical formulation containing the ADC of the present disclosure can be an "immediately available" injection formulation or a lyophilized formulation.

[0245] In certain embodiments, pharmaceutical formulations comprising the ADCs of the present disclosure may be provided in pre-filled syringes.

[0246] Use and method of the ADCs of the present disclosure The ADCs of the present disclosure have therapeutic utility. For example, these molecules can be administered to a subject, e.g., in vivo, to treat or prevent various disorders.

[0247] It is contemplated herein to use the ADCs of the present disclosure as pharmaceuticals, particularly for the treatment of cancer in a subject in need thereof, particularly cancer having tumor cells that express FRα, more particularly cancer having solid tumors, more particularly cancer selected from the group consisting of ovarian cancer, breast cancer, lung cancer, or mesothelioma.

[0248] The term “cancer” typically refers to or describes a physiological state in a mammal characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinomas, lymphomas, blastomas (including medulloblastoma and retinoblastoma), sarcomas (including liposarcoma and synovial sarcoma), neuroendocrine tumors (including carcinoid tumors, gastrinomas, and pancreatic islet cell carcinomas), mesotheliomas, schwannomas (including acoustic neuromas), meningiomas, adenocarcinomas, melanomas, and leukemias or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinomas (e.g., epidermoid squamous cell carcinomas), small cell lung cancer, non-small cell lung cancer, lung cancer including squamous cell carcinoma of the lung, peritoneal cancer, hepatocellular carcinoma, gastric cancer (gastric cancer or stomach cancer including gastrointestinal cancers), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer or renal carcinoma, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, testicular cancer, esophageal cancer, biliary tract tumors, and head and neck cancers.

[0249] The ADCs of the present disclosure are particularly useful for the treatment of cancer selected from the group consisting of ovarian cancer, triple negative breast cancer, and non-small cell lung cancer.

[0250] Accordingly, the present disclosure relates to a method for treating cancer, particularly cancer selected from the group consisting of one of the cancers listed above, more preferably ovarian cancer, triple-negative breast cancer, and non-small cell lung cancer, the method comprising administering a therapeutically effective amount of the ADC of formula (I) disclosed herein.

[0251] The ADC for use as disclosed above may be administered as the sole active ingredient, for example, in combination with or concomitantly with other drugs for the treatment or prevention of the above-mentioned diseases, such as antiviral drugs, anti-inflammatory drugs or cytotoxic drugs, antiproliferative drugs, chemotherapeutic drugs or antitumor drugs, for example as an adjuvant.

[0252] For example, the ADC for use as disclosed above may be used in combination with AZT, IFN-α, anti-CD20 mAb, anti-CD25 mAb, anti-PD1 mAb, anti-PDL-1 mAb, anti-CTLA4 mAb, chemotherapeutic agents.

[0253] Examples of such anti-PD1 or anti-PDL1 antibodies include, but are not limited to, nivolumab, pembrolizumab, avelumab, durvalumab, semiprilumab, or atezolizumab.

[0254] Suitable anti-tumor agents include, but are not limited to, alkylating agents (e.g., cyclophosphamide, mechlorethamine, chlorambucil, melphalan, nitrosourea, temozolomide), anthracyclines (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin), taxanes (paclitaxel, docetaxel, etc.), epothilones, inhibitors of topoisomerase I (irinotecan or topotecan, etc.), inhibitors of topoisomerase II (etoposide, teniposide or telfluposide, etc.), nucleotide analogs and precursor analogs (azacitidine, azathioprine, capecitabine, cytarabine, fluorouracil, gemcitabine, hydroxyurea, mercaptopurine, methotrexate or thioguanine, etc.), peptide antibiotics (bleomycin, colistin, gramicidin, etc.), platinum-based anti-neoplastic agents (carboplatin, cisplatin and oxaliplatin, etc.), retinoids (tretinoin, alitretinoin, bexarotene, etc.), vinca alkaloids and derivatives (vinblastine, vincristine, vindesine, vinorelbine), anti-VEGF agents (bevacizumab, ranibizumab, sunitinib, sorafenib, pazopanib, targeted therapies, e.g., kinase inhibitors (e.g., ibrutinib, idelalisib, erlotinib, gefitinib, imatinib, vemurafenib, vismodegib), proteasome inhibitors (e.g., bortezomib, carfilzomib), histone deacetylase inhibitors (vorinostat or romidepsin, etc.) may be mentioned.

[0255] As described above, in still further aspects, the present disclosure provides a method comprising co-administration, e.g., simultaneous or sequential administration, of a therapeutically effective amount of the ADC of the present disclosure and at least one second prodrug, wherein the second prodrug is an antiviral agent, an anti-inflammatory agent or a cytotoxic agent, an anti-proliferative agent, a chemotherapeutic agent or another anti-tumor agent, e.g., as shown above.

[0256] Kits comprising the compositions (e.g., including ADCs) and instructions disclosed herein are also within the scope of the present disclosure. The kits can further include at least one additional reagent, or one or more additional antibodies or proteins (e.g., antibodies having complementary activity that binds to an epitope on a target antigen different from the first antibody). The kits typically include a label indicating the purpose of use of the contents of the kit. The term label includes any written or recorded material supplied on or with the kit or associated with the kit. The kits can further include a tool for diagnosing whether a patient belongs to a group that would respond to ADC treatment, such as having an FRα-expressing tumor, as defined above.

[0257] Process for preparing the ADCs of the present disclosure The antibodies of the present disclosure can be conjugated to at least one drug by a linker L by any technique known in the art. Such techniques are described, for example, in Greg T. Hermanson, Bioconjugate Techniques, 3rd Edition, 2013, Academic Press (eBook ISBN: 9780123822406). For further information regarding methods for conjugating therapeutic agents to antibodies, see Lyon et al., "Chapter six - Conjugation of Anticancer Drugs Through Endogenous Monoclonal Antibody Cysteine Residues", 2012, Methods in Enzymology, 502, 123 - 138 (doi:10.1016 / B978 - 0 - 12 - 416039 - 2.00006 - 9); Chapter 2 - 7 of "Antibody - Drug Conjugates: Fundamentals, Drug Development, and Clinical Outcomes to Target Cancer", 3 November 2016, eBook ISBN: 9781119060727, doi:10.1002 / 9781119060727 and Panowksi S et al. 2014 Jan 1;6(1):34 - 45.

[0258] In one embodiment, the process for obtaining the ADC of formula (I) comprises the following steps: - culturing a host cell under conditions suitable for the expression of a nucleic acid encoding the antibody Ab defined in the previous section, - isolating the antibody, - synthesizing exatecan conjugated to the linker L of formula (VIII),

[0259]

Chemical formula

[0260] Antibodies can be obtained as described above. Antibodies contain four accessible interchain disulfide bonds that can be used as potential conjugation sites. The four interchain disulfide bonds can be reduced, for example, by tris(2-carboxyethyl)phosphine (TCEP) or dithiothreitol (DTT), resulting in eight thiol groups available for conjugation.

[0261] The moiety-L-D of formula (VIII) can be prepared according to procedures known in the field of organic synthesis (chemical reactions, extractions, evaporations, precipitations, chromatography, filtrations, triturations, crystallizations, etc.) and analytical procedures known to those skilled in the art of analytical chemistry. Details of such reactions and techniques can be found in many treatises including Richard Larock, Comprehensive Organic Transformations, A Guide to Functional Group Preparations, 2nd Ed (2010), and the multi-volume serie edited (by Michael B. Smith et al.) Compendium of Organic Synthetic Methods (1974 et seq.). Starting materials and reagents may be obtained from commercial sources or prepared using literature methods.

[0262] Exatecan compounds can be synthesized according to known procedures (see U.S. Patent No. 5,834,476; International Publication No. 2019 / 044946 or Sugimori et al., 1998, J. Med. Chem., 41(13), 2308-2318 doi:10.1021 / jm970765q). Exatecan can also be purchased from reputable suppliers (e.g., MedChemExpress catalog number HY-13631A or Carbosynth catalog number FE72401).

[0263] The final ADC of formula (I) is obtained following known procedures after conjugation of the -L-D component to the native or engineered cysteine thiol residues of the antibody. See, for example, Lyon et al., "Chapter six-Conjugation of Anticancer Drugs Through Endogenous Monoclonal Antibody Cysteine Residues", 2012, Methods in Enzymology, 502, 123-138 (doi:10.1016 / B978-0-12-416039-2.00006-9) or SJ Walsh et al., Site-selective modification strategies in antibody-drug conjugates, Chem. Soc. Rev., 2021, 50, 1305-1353, doi:10.1039 / D0CS00310G. Typically, the antibody component is reduced with a reducing agent such as tris(2-carboxyethyl)phosphine (TCEP) or dithiothreitol (DTT), the -L-D component is added, reacted covalently with the cysteine thiol residues of the antibody, and the final ADC compound is purified and buffer exchanged.

[0264] The invention, which is fully described, is further illustrated by the following embodiments and examples, which are illustrative only and are not intended to be further limiting.

[0265] Specific embodiments 1. An antibody-drug conjugate of formula (I), Ab-[L-D]p (I), wherein -Ab is an anti-folate receptor alpha (FRα) antibody that specifically binds to SEQ ID NO: 12, -L is a cleavable linker moiety preferably attached to the antibody via a thiol residue, -D is a cytotoxic drug moiety attached to L, -p is from 1 to 8, preferably from 6 to 8, more preferably p is 8, An antibody-drug conjugate of formula (I). 2. D is an inhibitor of topoisomerase I, preferably selected from the group consisting of camptothecin analogs, and more preferably D is the drug moiety of exatecan of formula (II) as described in Embodiment 1, the antibody-drug conjugate.

[0266]

Chemical formula

[0267]

Chemical formula

[0268]

Chemical formula

[0269]

Chemical formula

[0270]

Chemical formula

[0271]

Chemical formula

[0272]

Chemical formula

Examples

[0273] Functional assay Human FRα binding affinity by ELISA The sandwich ELISA assay is performed using a 96-well high-binding ELISA plate (Corning Inc., New York, NY, USA, catalog number 3590). The plate is coated with 100 μL / well of recombinant human FRα protein (Sino Biological, catalog number 11241-H08H) in 2 μg / mL PBS (pH 7.4) and incubated overnight at 4°C. After washing twice with PBS-T (PBS + 0.05% Tween-20), the plate is blocked with 200 μL / well of incubation buffer (PBS-T + 0.1% BSA) for 1 hour at room temperature. The plate is washed four times with PBS-T, and 100 μL of a three-fold dilution series of the test compound (antibody or antibody-drug conjugate) is added. The plate is then incubated for 2 hours at room temperature in the dark. After five washes with PBS-T, the plate is incubated with 100 μL / well of goat anti-human IgG (H+L) HRP-conjugated antibody (Jackson Immunoresearch, catalog number 109-035-088), which is pre-diluted 1:250,000 with incubation buffer, for 1 hour at room temperature. After washing five times with PBS-T, TMB substrate solution (Thermo-Fisher, catalog number N301) is added. The peroxidase activity is stopped with 0.18 M H2SO4, and the absorbance is read at 450 nm (reference wavelength 650 nm) using a Thermo Scientific MultiSkan EX microplate reader. Sigmod fitting is performed using GraphPad Prism 9 software.

[0274] SPR Biacore affinity Surface plasmon resonance (SPR) experiments are performed on a Biacore T200 instrument at 25 °C. The antibody or ADC to be tested is captured at low density (300 - 500 RU) on a CM5 series S sensor chip pre-functionalized with the Human Antibody Capture kit (Cytiva, catalog number BR100839). A similarly treated functionalized surface / flow cell without ligand is used as a reference. To measure the kinetic rate and affinity, recombinant human FRα (Sino Biological, catalog number 11241-H08H) analyte samples are injected in duplicate at five consecutive concentrations (0.5, 1, 2, 4, 8 nM) with a 300-second contact pulse at a constant flow rate of 70 μL / mL of running buffer (HBS-EP+, Cytiva, catalog number BR100669) using a single-cycle kinetic strategy. The dissociation phase is measured by injecting the running buffer for 900 seconds. Between duplicates, the surface / flow cell is regenerated with 3 M MgCl2 to remove both analyte and ligand and fresh ligand capture is performed using the same conditions. Data analysis is performed using Biacore Evaluation software after subtracting the reference surface and zero-concentration signal of the analyte. The data is processed and fitted to a 1:1 binding model to determine the association rate constant k a (on-rate) and k d (off-rate), as well as K D (equilibrium dissociation constant, also called "affinity"). The mean and standard deviation of replicate experiments are reported.

[0275] Cell binding affinity by flow cytometry The antibody or ADC binding to extracellular human FRα expressed on cancer cell lines is evaluated by flow cytometry. The antibody or ADC to be tested is conjugated to an APC fluorophore using the LYNX Rapid APC Antibody Conjugation Kit according to the manufacturer's protocol (Bio-Rad, catalog number LNK032APC). To 500,000 cells (suspended in 100 μL of PBS in a flow cytometry plastic tube), 5 μL of a 10 μg / mL solution of the APC-labeled antibody or ADC to be tested is added. The cells are incubated in the dark for 20 minutes, washed 3 times with PBS by centrifugation, and resuspended in 200 μL of PBS for analysis. Flow cytometry is performed using a BD Fortessa flow cytometer controlled by BD FACSDiva software (BD Biosciences), and the data is analyzed using FlowJo software (BD Bioscience).

[0276] Stability assay in human plasma The ADC sample (solution > 6 mg / mL in PBS) is diluted with pure, sterile human plasma (GeneTex, catalog number GTX73265) in screw-cap centrifuge tubes to obtain a final ADC concentration of 200 μg / mL (residual PBS volume < 5% v / v). The sample is incubated at 37 °C and aliquots are taken at 5 minutes, 6 hours, 1 day, 2 days, 3 days, and 7 days (aliquots were kept frozen at -80 °C until analysis). The ADC is isolated from plasma by immunocapture using Dynabeads™ M-280 streptavidin (Thermo Scientific) magnetic beads pre-coated with biotinylated human folate receptor alpha recombinant protein (Sino Biologicals, catalog number 11241-H08H). Briefly, 600 μL of a commercially available bead solution is washed twice with HBS-EP buffer (Cytiva, catalog number BR100188) and resuspended in 1.2 mL of HBS-EP buffer. 65 μL of the biotinylated recombinant FRα solution (protein amount 48 μg) is added and the solution is stirred at room temperature for 2 hours. The beads are then washed three times with HBS-EP buffer and resuspended in 1.2 mL of HBS-EP buffer. For a single immunocapture, 100 μL of the previous bead solution is added onto 100 μL of HBS-EP in a microcentrifuge tube. 10 μL of the ADC solution in plasma (theoretical ADC amount 2 μg) is added and the solution is stirred at room temperature for 2 hours. After incubation, the bead-ADC complex is washed twice with HBS-EP buffer, resuspended in 200 μL of HBS-EP buffer, and deglycosylated by adding 2 μL / 1000 U of PNGase F (New England Biolabs, catalog number P0705L) and stirring gently at 37 °C overnight. The beads are then washed twice with HBS-EP buffer, twice with distilled water, and once with 10% acetonitrile (v / v) in water. The beads are incubated for 30 minutes at room temperature with gentle stirring with 50 μL of a 30% aqueous acetonitrile solution (v / v) containing 0.1% (v / v) formic acid.Next, the elution sample containing the deglycosylated ADC is analyzed by denaturing reverse-phase chromatography-mass spectrometry using a Thermo UltiMate 3000 UHPLC system equipped with a Bruker Impact II™ Q-ToF mass spectrometer. Mobile phase A is water + 0.1% formic acid, and mobile phase B is acetonitrile + 0.1% formic acid. The column is an Agilent PEEK PLRP-S 1000Å 2.1×100mm 5μm (80 °C). The linear gradient is 20% B to 50% B in 25 minutes. The flow rate is 0.4 mL / min. UV detection is monitored at 280 nm. The Q-ToF mass spectrometer is used in the m / z range 500 - 5000 (ESI. + ) The data is deconvoluted using the MaxEnt algorithm included in the Bruker Compass® software. For stability data analysis, deconvolution of the raw spectra within the selected light chain (LC) and heavy chain (HC) elution time windows is performed. Loss or modification of the drug-linker is identified according to the corresponding mass shift from the starting ADC material. The relative ratio of ADCs with different DARs is calculated by dividing the intensity of a particular ADC subspecies by the intensity from the total ADC species. The final DAR value is calculated as described in Xu et al., Anal. Biochem., 2011, 412(1), 56 - 66.

[0277] In vitro efficacy in FRa-negative cell line (BT-474 cell line) To evaluate the non-specific (off-target) cytotoxicity of the ADC, an in vitro cytotoxicity assay is performed in the BT-474 (FRα negative) cancer cell line. Cells are seeded in a 96-well plate at an appropriate density (1000 - 10000 cells / well in 100 μL of appropriate culture medium) according to the cell line and incubated at 37 °C for 24 hours. Serial dilutions (50 μL) of the test compound pre-dissolved in the culture medium are added, and incubation is carried out at 37 °C for 144 hours. MTT (5 mg / mL, 20 μL, Sigma-Aldrich) is added to the wells, and incubation is continued at 37 °C for 1 - 2 hours. Then, the culture medium is carefully removed, and the well contents are uniformly dissolved with acidified isopropanol. Absorbance values are measured using a MultiskanTM Sky microplate reader (Thermo Scientific) at a wavelength of 570 nm (reference wavelength 690 nm). The IC50 concentration value compared to untreated control cells is determined using inhibition dose-response curve fitting (GraphPad Prism 9).

[0278] In vivo efficacy in cancer xenograft model Four- to five-week-old female CB-17 severe combined immunodeficiency (SCID) mice are obtained and isolated for 7 days before the start of the study. Cells resuspended in PBS (OV-90, SW-620, KB, BT-474 cell lines) or 50% BD Matrigel (Corning®) in PBS (PA-1, IGROV-1, OVCAR-3, NCI-H2110 cell lines) (5 - 10 × 106 cells per mouse) are inoculated subcutaneously into the mice. When the average tumor volume reaches approximately 120 - 150 mm3, the mice are randomized (typically 7 mice per group) and treated by single (unless otherwise specified) intravenous injection of PBS (negative control) or the antibody-drug conjugate is examined. Tumor volume is measured every 3 - 5 days using a caliper device (length × width) and calculated using the following formula V = 4 / 3 × π × R3, where R represents the radius. If the tumor volume exceeds 1500 mm3 or the tumor ulcerates, the mice are sacrificed.

[0279] Drug-antibody ratio (DAR) evaluation by reverse-phase liquid chromatography - mass spectrometry (RPLC-MS): Use modified RPLC-QToF analysis to evaluate the drug-antibody ratio (DAR) of the conjugate. Briefly, the ADC is eluted on an Agilent PLRP-S 1000 Å 2.1×150 mm 8 μm column (80 °C) using a mobile phase gradient of water / acetonitrile + 0.1% formic acid (0.4 mL / min) and detected using a Bruker Impact II™ Q-ToF mass spectrometer that scans the 500-3500 m / z range (ESI + ). Deconvolute the data using the MaxEnt algorithm included in Bruker Compass® software.

[0280] Example 1: Synthesis of exatecan-based chemical drug-linker Materials and general organic synthesis methods All solvents and reagents were obtained from reliable commercial sources (Sigma-Aldrich, Fluorochem, TCI Chemicals, Acros Organics, Alfa Aesar, Enamine, Thermo Fisher, Carbosynth, WuXi AppTec, Iris Biotech) and used without further purification, unless otherwise specified. Anhydrous solvents were purchased from Sigma-Aldrich. Fmoc-amino acids, 2-chlorotrityl, Wang and Rink amide polystyrene 1% DVB 100-200 mesh resin (pre-loaded with the first Fmoc-sarcosine amino acid) were purchased from Christof Senn Laboratories and Sigma-Aldrich. Exatecan mesylate was purchased from MedChemExpress.

[0281] Synthesis on resin was carried out in empty SPE plastic tubes equipped with 20 μm polyethylene frits (Sigma-Aldrich). A Titramax 101 platform shaker (Heidolph) was used for stirring. Unless otherwise specified, all chemical reactions were carried out at room temperature under an inert argon atmosphere.

[0282] Liquid nuclear magnetic resonance spectra were recorded on a Bruker Fourier 300HD or Bruker AVANCE III HD400 spectrometer using the residual solvent peak for calibration. Mass spectrometry was performed by the Centre Commun de Spectrometrie de Masse (CCSM) of the UMR5246 CNRS Institute of University Claude Bernard Lyon 1.

[0283] Normal-phase flash chromatography was carried out on a Teledyne Isco CombiFlash® Rf200 device using Macherey-Nagel Chromabond® flash cartridges (40 - 63 μm). Reverse-phase chromatography was carried out on a Teledyne Isco Combiflash® Rf200 device using a Biotage® Sfar C18 Duo 100Å 30μm cartridge or an Interchim PuriFlash RP-AQ (30μm) cartridge, or using an Agilent 1100 preparative binary HPLC system.

[0284] Chemical reactions and compound characterizations were monitored and analyzed by thin-layer chromatography using pre-coated 40 - 63μm silica gel (Macherey-Nagel), HPLC-UV (Agilent 1100 system), or UHPLC-UV / MS (Thermo UltiMate 3000 UHPLC system equipped with a Bruker Impact II™ Q-ToF mass spectrometer or Agilent 1260 HPLC system equipped with a Bruker MicrOTOF-QII mass spectrometer).

[0285] HPLC method 1: Agilent 1100 HPLC system equipped with DAD detection. Mobile phase A was water + 0.1% TFA, and mobile phase B was acetonitrile. The column was Agilent Zorbax SB-Aq 4.6×150 mm 5 μm (room temperature). The linear gradient was from 0% B to 50% B in 30 minutes, followed by holding at 50% B for 5 minutes. The flow rate was 1.0 mL / min.

[0286] HPLC method 2: Agilent 1100 HPLC system equipped with DAD detection. Mobile phase A was water + 0.1% TFA, and mobile phase B was acetonitrile. The column was Agilent Poroshell 120 EC-C18 3.0×50 mm 2.7 μm (room temperature). The linear gradient was from 5% B to 80% B in 9 minutes, followed by holding at 80% B for 1 minute. The flow rate was 0.8 mL / min.

[0287] HPLC method 3: Agilent 1100 HPLC system equipped with DAD detection. Mobile phase A was water + 0.1% TFA, and mobile phase B was acetonitrile. The column was Agilent Poroshell 120 EC-C18 3.0×50 mm 2.7 μm (room temperature). The linear gradient was from 5% B to 80% B in 20 minutes, followed by holding at 80% B for 2 minutes. The flow rate was 0.8 mL / min.

[0288] HPLC method 4: Thermo UltiMate 3000 UHPLC system + Bruker Impact II (trademark) Q-ToF mass spectrometer. Mobile phase A was water + 0.1% formic acid, and mobile phase B was acetonitrile + 0.1% formic acid. The column was Agilent PLRP-S 1000 Å 2.1×150 mm 8 μm (80 °C). The linear gradient was from 10% B to 50% B in 25 minutes. The flow rate was 0.4 mL / min. UV detection was monitored at 280 nm. The Q-ToF mass spectrometer was used in the m / z range 500 - 3500 (ESI + )). The data was deconvoluted using the MaxEnt algorithm included in Bruker Compass (registered trademark) software.

[0289] HPLC method 5 (fractionation method): Teledyne Isco CombiFlash (registered trademark) Rf200 binary MPLC system equipped with DAD detection. Mobile phase A was water + 0.1% TFA, and mobile phase B was acetonitrile. The reusable cartridge was Biotage (registered trademark) Sfar C18 Duo 100Å 30μm (30 g). The linear gradient was from 10% B to 50% B in 35 minutes, followed by holding at 50% B for 5 minutes. The flow rate was 25 mL / min.

[0290] HPLC method 6 (fractionation method): Agilent 1100 preparative binary HPLC system equipped with a dual-loop autoinjector, DAD detection, and fraction collector. Mobile phase A was water + 0.1% TFA, and mobile phase B was acetonitrile. The column was a Waters SunFire C18 OBD Prep Column, 100Å, 5μm, 19mm × 250mm (room temperature). The linear gradient was from 10% B to 60% B in 40 minutes, followed by holding at 60% B for 5 minutes. The flow rate was 25 mL / min.

[0291] 1.1) Monodisperse polysarcosine intermediate 1.1.1) General method The on-resin synthesis of monodisperse polysarcosine was achieved using the submonomer synthesis iteration procedure (described in WO 2019 / 081455) for Rink amide and 2-chlorotrityl resins or according to the classical Fmoc / SPPS methodology using a commercially available Fmoc-Sar-Sar-OH dipeptoid building block (catalog number 2313534-20-0) for Wang resin. The on-resin dimerization step (n = 2) was avoided for diketopiperazine formation. All synthetic yields are reported based on the initial Fmoc-sarcosine loading indicated by the manufacturer. Unless otherwise specified, all reactions were carried out at room temperature. Pre-packed Rink amide, 2-chlorotrityl or Wang polystyrene 1% DVB 100-200 mesh resin with the first Fmoc-sarcosine residue (Christof Senn Laboratories) was used (typical initial loading of 0.6 - 1 mmol / g). Laboratories) was used (typical initial loading of 0.6 - 1 mmol / g).

[0292] 1.1.2) Extension of polysarcosine Rink amide, 2-chlorotrityl or Wang resin pre-packed with Fmoc-sarcosine was treated twice with 20% piperidine in DMF (1 mL per 100 mg of resin) at room temperature for 15 minutes. The resin was then washed with DMF (4 times) and DCM (4 times). To the resin was added a solution of Fmoc-Sar-Sar-OH (3 equiv), HATU (2.9 equiv) and DIPEA (6 equiv) in DMF (1 mL per 100 mg of resin). The reaction vessel was stirred for 2 hours and the resin was washed with DMF (4 times) and DCM (4 times). The resin was treated twice with 20% piperidine in DMF (1 mL per 100 mg of resin) at room temperature for 15 minutes. The resin was then washed with DMF (4 times) and DCM (4 times).

[0293] For synthesis on rink amide or 2-chlorotrityl resin, classical submonomer synthesis procedures were used as described in WO 2019 / 081455. By alternately performing the bromoacetylation step and the amine substitution step, the poly(sarcosine) oligomer with n = 3 was extended until the desired length was obtained. The bromoacetylation step was carried out by adding 10 equivalents of bromoacetic acid and 13 equivalents of diisopropylcarbodiimide (2 mL per 100 mg of resin) in DMF. The mixture was stirred for 30 minutes, drained, and washed with DMF (4 times). For the amine substitution step, 40% (wt) methylamine in aqueous solution (1.5 mL per 100 mg of resin) was added, the vessel was shaken for 30 minutes, drained, and washed with DMF (4 times) and DCM (4 times).

[0294] For synthesis on Wang resin, classical Fmoc / SPPS procedures were used. The extension of the poly(sarcosine) oligomer with n = 3 was carried out by iterative coupling of the Fmoc-Sar-Sar-OH dipeptoid building block (catalog number 2313534-20-0). To the resin was added a solution of Fmoc-Sar-Sar-OH (3 equivalents), HATU (2.9 equivalents), and DIPEA (6 equivalents) in DMF (1 mL per 100 mg of resin). The reaction vessel was stirred for 90 minutes, and the resin was extensively washed with DMF (4 times) and DCM (4 times). The resin was then treated twice with 20% piperidine in DMF (1 mL per 100 mg of resin) at room temperature for 15 minutes. The resin was washed with DMF (4 times) and DCM (4 times). This coupling / Fmoc deprotection cycle was repeated until the desired poly(sarcosine) length was obtained. Optionally, the final coupling was carried out using a commercially available Fmoc-Sar-OH amino acid instead of the Fmoc-Sar-Sar-OH dipeptoid unit to obtain an even-length final poly(sarcosine).

[0295] 1.1.3) Side functionalization of polysarcosine on the final resin, optional capping, cleavage and purification of the resin Once the desired poly(sarcosine) monodisperse oligomer length on the resin is reached, orthogonal chemical functionalization is carried out. Optionally, this is followed by final capping with Fmoc-amino acids (e.g., Fmoc-Gly-OH, Fmoc-β-Ala-OH, Fmoc-amino-3,6-dioxaoctanoic acid, Fmoc-9-amino-4,7-dioxanonanoic acid). The Fmoc protecting group that caps the N-terminus of the final compound can be removed before or after resin cleavage, depending on the orthogonal functionalization chemistry used (see below).

[0296] 1.1.3.1) 2-Azidoethane-1-amine side-functionalized polysarcosine To Rink or 2-chlorotrityl resin, add 10 equivalents of bromoacetic acid and 13 equivalents of diisopropylcarbodiimide (2 mL per 100 mg of resin) in DMF. Stir the mixture for 30 minutes, drain, and wash with DMF (4 times). Add a 3 molar solution of 2-azidoethan-1-amine in DMF (1 mL per 100 mg of resin), shake the vessel for 45 minutes, drain, and wash with DMF (4 times) and DCM (4 times). Then perform Fmoc-Gly-OH coupling for 1 hour (DMF containing 5 equivalents of Fmoc-Gly-OH, 4.9 equivalents of HATU, 10 equivalents of DIPEA, 1 mL per 100 mg of resin) and Fmoc deprotection with DMF containing 20% piperidine (1 mL per 100 mg of resin) twice at room temperature for 15 minutes each. Wash the resin with DMF (4 times) and DCM (4 times).

[0297] The final poly(sarcosine) compound was cleaved from the resin (100% TFA twice for 30 minutes for Rink resin and Wang resin, 20% TFA twice for 15 minutes for 2-chlorotrityl resin). The resin was filtered and the volatiles were removed under reduced pressure to obtain the crude product, which was purified on an Interchim® RP-AQ (30 μm) cartridge. Mobile phase A was water + 0.1% TFA and mobile phase B was acetonitrile.

[0298] 1.1.3.2) Glutamic acid side-functionalized polysarcosine To the Rink, Wang or 2-chlorotrityl resin, a solution of DMF (1 mL per 100 mg of resin) containing Fmoc-Glu(OAll)-OH (3 equivalents), HATU (2.9 equivalents) and DIPEA (6 equivalents) was added. The reaction vessel was stirred for 90 minutes and the resin was extensively washed with DMF (4 times) and DCM (4 times). Then, the resin was treated twice with 20% piperidine in DMF (1 mL per 100 mg of resin) for 15 minutes at room temperature. The resin was washed with DMF (4 times) and DCM (4 times). Thereafter, Fmoc-amino-3,6-dioxaoctanoic acid (3 equivalents), HATU (2.9 equivalents), DIPEA (6 equivalents) in DMF (1 mL per 100 mg of resin) were coupled for 1 hour. The resin was washed with DMF (4 times) and DCM (4 times). The alloc protecting group was removed by treatment twice for 30 minutes with a DCM solution containing 0.25 equivalent of Pd(PPh3)4 and 20 equivalents of phenylsilane (gently stirred under an argon stream). Then, the resin was washed with DMF (5 times) and DCM (5 times). Optionally, the N-hydroxysuccinimide (NHS) ester was introduced into the carboxylic acid side chain of the final polysarcosine compound by treatment for 90 minutes with a DMF solution containing 50 equivalents of DIC and 60 equivalents of N-hydroxysuccinimide (1.5 mL per 100 mg of resin). Then, the resin was washed with DMF (4 times) and DCM (4 times).

[0299] The final polysarcosine compound was cleaved from the resin (100% TFA twice for 30 minutes for Rink resin and Wang resin, 20% TFA in DCM twice for 15 minutes for 2-chlorotrityl resin). The resin was filtered and the volatiles were removed under reduced pressure to obtain the crude product, which was purified on an Interchim® RP-AQ (30 μm) cartridge. Mobile phase A was water + 0.1% TFA and mobile phase B was acetonitrile.

[0300] 1.1.4) Final polysarcosine intermediate The obtained compounds are shown in Table 6 below.

[0301]

Table 6

[0302] 1.2) Synthesis of intermediate compounds 1.2.1) Synthesis of compound INT1

[0303] [Chemistry] Compound INT1 was synthesized according to the procedure described in International Patent Application Publication No. WO 2019 / 081455. This compound is an equimolar mixture of diastereoisomers (stereocenters indicated by asterisks).

[0304] 1.2.2) Synthesis of compounds INT2, INT2-S and INT2-R

[0305] [Chemistry]

[0306] 1.2.2.1) Synthesis of tert-butyl (2-hydroxy-2-(4-hydroxy-3-nitrophenyl)ethyl)carbamate (±)-Octopamine hydrochloride (1690 mg / 11 mmol) was weighed in a round-bottom flask and suspended in 4 mL of distilled water. The flask was cooled to 0 °C and 4 mL of pre-cooled 65% nitric acid solution was slowly added. The reaction mixture was held at 0 °C for 20 minutes and evaluated by HPLC, which showed complete mononitration of the starting material. The contents of the flask were transferred to a pre-cooled 250 mL Erlenmeyer flask and slowly neutralized at 0 °C with saturated NaHCO3 solution (about 50 mL) until a pH value of 8 - 9 was reached. Then, 30 mL of dioxane was added, followed by Boc2O (7202 mg / 13.2 mmol). The reaction mixture was then allowed to reach room temperature and stirred overnight. The reaction mixture was then diluted with EtOAc and washed three times with saturated citric acid solution and once with saturated NaCl solution. The organic phase was dried over MgSO4, filtered, and evaporated under vacuum to give the crude product, which was purified by silica gel chromatography (petroleum ether / EtOAc, gradient 70:30 to 20:80) to give the title compound (1320 mg / 40%) as a thick yellow - brown oil. 1H NMR (300 MHz, DMSO-d6) δ 10.79 (s, 1H), 7.79 (d, J = 2.1 Hz, 1H), 7.47 (dd, J = 8.6, 2.1 Hz, 1H), 7.08 (d, J = 8.6 Hz, 1H), 6.74 (t, J = 5.9 Hz, 1H), 4.56 (t, J = 6.3 Hz, 1H), 3.07 (td, J = 6.1, 1.6 Hz, 2H), 1.31 (s, 9H). MS m / z (ESI+): Calc [M+H]+ = 299.1; Exp [M+H]+ = 299.1. HPLC method 2, retention time = 5.5 minutes.

[0307] 1.2.2.2) Synthesis of (2S,3R,4S,5S,6S)-2-(4-(2-((tert-butoxycarbonyl)amino)-1-hydroxyethyl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate. During the round-bottom flash, Ag2CO3 (1500 mg / 5.4 mmol) and 1,1,4,7,10,10-hexamethyltriethylenetetramine (251 mg / 1.1 mmol) were dissolved in 4 mL of anhydrous acetonitrile and stirred at room temperature for 2 hours. The previous compound tert-butyl (2-hydroxy-2-(4-hydroxy-3-nitrophenyl)ethyl)carbamate (292 mg / 0.98 mmol) and 1-bromo-2,3,4-tri-O-acetyl-α-D-glucuronide methyl ester (583 mg / 1.46 mmol) were added at 0 °C, and the solution mixture was stirred at room temperature for 4 hours. Then, the reaction mixture was filtered through Celite, diluted with EtOAc, and washed three times with saturated citric acid solution and once with saturated NaCl solution. The organic phase was dried over MgSO4, filtered, and evaporated under vacuum to obtain the crude product, which was purified by silica gel chromatography (petroleum ether / EtOAc, gradient 70:30 to 30:70) to give the title compound (244 mg / 48%) as a yellow foam. 1H NMR (300 MHz, DMSO-d6) δ 7.76 (dd, J = 3.3, 2.1 Hz, 1H), 7.60 (t, J = 7.6 Hz, 1H), 7.36 (dd, J = 8.7, 2.6 Hz, 1H), 6.77 (s, 1H), 5.71 (d, J = 7.8 Hz, 1H), 5.61 (s, 1H), 5.46 (td, J = 9.5, 1.1 Hz, 1H), 5.21 - 5.02 (m, 3H), 4.75 (dd, J = 9.9, 1.4 Hz, 1H), 4.62 (s, 1H), 3.65 (s, 3H), 3.17 (s, 2H), 3.10 (t, J = 6.1 Hz, 2H), 2.81 - 2.59 (m, 6H), 2.05 - 1.96 (m, 9H), 1.30 (d, J = 1.7 Hz, 9H). MS m / z (ESI+): Calc [M+Na]+ = 637.2; Exp [M+Na]+ = 637.2. HPLC method 2, retention time = 6.75 minutes.

[0308] 1.2.2.3) Synthesis of (2S,3R,4S,5S,6S)-2-(4-(2-((tert-butoxycarbonyl)amino)-1-(((4-nitrophenoxy)carbonyl)oxy)ethyl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate The previous compound (2S,3R,4S,5S,6S)-2-(4-(2-((tert-butoxycarbonyl)amino)-1-hydroxyethyl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (334 mg / 0.54 mmol) and 4-nitrophenyl chloroformate (219 mg / 1.09 mmol) were dissolved in 6 mL of dry DCM at 0 °C. Anhydrous pyridine (112 mg / 1.41 mmol) was added and the mixture was stirred at room temperature for 30 minutes. The reaction was filtered through a 0.45 μm PTFE filter and purified by silica gel chromatography (petroleum ether / EtOAc, gradient 85:15 to 30:70) to give the title compound (380 mg / 90%) as a yellow foam. 1H NMR (300 MHz, DMSO-d6) δ 8.39 - 8.26 (m, 2H), 7.93 (d, J = 2.2 Hz, 1H), 7.75 (d, J = 8.8 Hz, 1H), 7.55 (dd, J = 9.2, 1.2 Hz, 2H), 7.46 (d, J = 8.8 Hz, 1H), 7.20 (d, J = 4.8 Hz, 1H), 5.77 (dd, J = 7.7, 3.7 Hz, 1H), 5.47 (t, J = 9.5 Hz, 1H), 5.11 (q, J = 9.6 Hz, 2H), 4.77 (d, J = 9.9 Hz, 1H), 3.73 - 3.59 (m, 3H), 3.59 - 3.37 (m, 2H), 2.05 - 1.96 (m, 9H), 1.48 - 1.35 (m, 1H), 1.32 (s, 9H). MS m / z (ESI+): Calc [M+Na]+ = 802.15; Exp [M+Na]+ = 802.15. HPLC method 2, retention time = 8.5 minutes.

[0309] 1.2.2.4) Synthesis of Compound INT2 381 mg (0.49 mmol) of the previous compound (2S,3R,4S,5S,6S)-2-(4-(2-((tert-butoxycarbonyl)amino)-1-(((4-nitrophenoxy)carbonyl)oxy)ethyl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate, 200 mg (0.38 mmol) of exatecan mesylate, and 51 mg (0.38 mmol) of HOBt were dissolved in 5 mL of an 85:15 (v / v) mixture of anhydrous DMF / pyridine. 53.5 mg (0.51 mmol) of DIPEA was added. The reaction mixture was stirred at 40 °C for 2 h and the volatiles were evaporated under reduced pressure. The crude residue was purified by silica gel chromatography (gradient of DCM / MeOH from 99:1 to 95:5) to give 360 mg (87%) of the intermediate compound (2S,3R,4S,5S,6S)-2-(4-(2-((tert-butoxycarbonyl)amino)-1-((((1R,9R)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)ethyl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate as a yellow / greenish solid. ESI+ [M+Na]+ = 1098.3. HPLC method 3, retention times = 14.7 and 14.9 min (mixture of diastereoisomers).

[0310] 355 mg (0.33 mmol) of this intermediate compound was dissolved in 8 mL of MeOH / THF 75:25 at 0 °C. LiOH monohydrate (138 mg / 3.3 mmol) was dissolved in water (1 mL) and added to the reaction vessel. After stirring at 0 °C for 30 minutes (reaction, followed by HPLC), the mixture was neutralized with acetic acid (258 mg / 4.3 mmol) and concentrated under reduced pressure. The resulting crude product was redissolved at 0 °C using a TFA / DCM (30:70 v / v) solution and stirred at room temperature for 20 minutes. The volatile substances were evaporated under reduced pressure, and the crude residue was dissolved in a water / ACN (1:1 v / v) solution and purified using HPLC preparative method 5 to obtain 172 mg (62%) of compound INT2 as a yellow solid. ESI+[M+H]+ = 836.2. HPLC method 3, retention times = 7.3 and 7.8 minutes (mixture of diastereoisomers).

[0311] 1.2.2.5) Synthesis of Stereochemically Pure Compounds INT2-S and INT2-R

[0312]

Chem.

[0313] 1.2.2.5.1) Chiral Resolution of the Racemic Mixture of tert-Butyl (2-Hydroxy-2-(4-Hydroxy-3-Nitrophenyl)Ethyl)Carbamate Chiral separation of racemic tert-butyl (2-hydroxy-2-(4-hydroxy-3-nitrophenyl)ethyl)carbamate (synthesized as described above) was performed using a Chiralflash® IC MPLC column 30x100 mm, 20 μm (Daicel catalog number 83M73) on a Teledyne Isco CombiFlash® Rf200 system. The mobile phase was DCM + 0.2% (v / v) EtOH (isocratic gradient). The flow rate was 12 mL / min. The sample solvent was DCM + 0.2% (v / v) EtOH. The mass recovery rates of the two enantiomers after separation were over 80%.

[0314] The retention time of tert-butyl (S)-(2-hydroxy-2-(4-hydroxy-3-nitrophenyl)ethyl)carbamate was 15 minutes, while the retention time of tert-butyl (R)-(2-hydroxy-2-(4-hydroxy-3-nitrophenyl)ethyl)carbamate was 25 minutes.

[0315] To determine the absolute configuration, the phenolic positions of both enantiomers were esterified with 1.2 molar equivalents of 4-nitrobenzoyl chloride and 2 molar equivalents of triethylamine in anhydrous THF. The compounds were purified by chromatography on silica gel (petroleum ether / EtOAc, gradient 90:10 to 10:90) to afford 4-(2-((tert-butoxycarbonyl)amino)-1-hydroxyethyl)-2-nitrophenyl 4-nitrobenzoate. 1H NMR (300 MHz, DMSO-d6) δ 8.51 (d, J = 9.1 Hz, 2H), 8.44 (d, J = 9.1 Hz, 2H), 8.19 (d, J = 1.9 Hz, 1H), 7.88 (d, J = 10.2 Hz, 1H), 7.72 (d, J = 8.4 Hz, 1H), 6.93 (t, J = 5.9 Hz, 1H), 5.84 (d, J = 4.7 Hz, 1H), 4.86 - 4.76 (m, 1H), 3.24 (t, J = 6.1 Hz, 2H), 1.38 (s, 9H). ESI+ [M+Na]+ = 470.1. The absolute configuration of the enantiomers (previously dissolved in a 1:1 mixture of heptane / dichloromethane and slowly evaporated over 3 weeks to induce crystal formation) was confirmed by X-ray crystallography. Block-shaped crystals were mounted on a nylon loop in perfluorinated ether oil. The data were collected using an Xcalibur, Atlas, Gemini diffractometer equipped with an Oxford Cryosystems cryostat operating at T = 150.00(5) K. The data were measured using ω scans with CuKα radiation. The structure was solved using the ShelXT solution program with the dual-space algorithm and refined using ShelXL 2018 / 3 (Sheldrick, G.M., Crystal structure refinement with ShelXL, Acta Cryst., 2015, C71, 3 - 8) using full-matrix least-squares minimization of F2.

[0316] 1.2.2.5.2) Synthesis of Stereochemically Pure INT2-S and INT2-R Compounds Stereochemically pure compounds INT2-S and INT2-R were synthesized as described in the previous section 1.2.2 without any appreciable change in reaction conditions, reactivity, or overall yield.

[0317] By final purification using HPLC preparative method 5, 33 mg of compound INT2-S was obtained as a yellow solid. ESI+ [M+H]+ = 836.2. HPLC method 3, retention time = 7.3 minutes.

[0318] By final purification using HPLC preparative method 5, 21 mg of compound INT2-R was obtained as a yellow solid. ESI+ [M+H]+ = 836.2. HPLC method 3, retention time = 7.8 minutes.

[0319] 1.2.3) Synthesis of Compound INT3

[0320]

Chemical formula

[0321] 1.2.3.1) Synthesis of Ac-Val-Ala-OH (Acetyl-L-Valyl-L-Alanine) To a solution of L-alanine benzyl ester hydrochloride (542 mg / 2.5 mmol) in 30 mL of DCM, triethylamine (254 mg / 2.5 mmol), distilled water (30 mL), N-α-acetyl-L-valine (400 mg / 2.5 mmol), and HOBt (339 mg / 2.5 mmol) were added sequentially. The mixture was then cooled to 0 °C and EDC-HCl (530 mg / 2.75 mmol) was added. The resulting mixture was stirred at 0 °C overnight. The reaction was diluted with 20 mL of 2 M HCl and the layers were separated. The organic phase was washed twice with 2 M HCl, twice with saturated NaHCO3 solution, and once with saturated NaCl solution. The organic phase was dried over MgSO4, filtered, and evaporated under vacuum to give 714 mg (89%) of benzyl acetyl-L-valyl-L-alaninate as a white solid intermediate.

[0322] This intermediate was solubilized in 10 mL of EtOAc / MeOH 1:1 (v / v) and transferred to a stainless steel hydrogenation reactor. After an initial argon purge, a catalytic amount of 5%wt Pd / C was added. The reactor was then purged twice with H2 and maintained at room temperature overnight under a H2 pressure of 10 bar. The reaction mixture was filtered through a 0.45 μm PTFE filter and the solvent was removed under vacuum to afford a quantitative amount of pure acetyl-L-valyl-L-alanine as a white solid. 1H NMR (300 MHz, DMSO-d6) δ 12.45 (s, 1H), 8.23 (d, J = 6.9 Hz, 1H), 7.85 (d, J = 9.0 Hz, 1H), 4.25 - 4.11 (m, 2H), 1.94 (dt, J = 13.6, 6.8 Hz, 1H), 1.85 (s, 3H), 1.26 (d, J = 7.3 Hz, 3H), 0.85 (dd, J = 12.1, 6.8 Hz, 6H).

[0323] 1.2.3.2) Synthesis of (2S)-2-Acetamido-N-((2S)-1-((4-(1-Hydroxybut-3-yn-1-yl)phenyl)amino)-1-oxopropan-2-yl)-3-Methylbutanamide During the round-bottom flash, 420 mg (2.60 mmol) of 1-(4-aminophenyl)but-3-yn-1-ol (synthesized according to the procedure described in Sharma A. et al., Chem 2018, 4(10), 2370-2383) and 600 mg (2.60 mmol) of the previous compound Ac-Val-Ala-OH were suspended in 20 mL of anhydrous THF. Then, 676 mg (2.74 mmol) of 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) previously dissolved in 5 mL of anhydrous DMF was added to the flask, and the turbid reaction mixture was stirred at room temperature overnight. Then, the volatile substances were evaporated under reduced pressure, the crude residue was dry loaded, and purified by silica gel chromatography (DCM / MeOH gradient from 99:1 to 85:15) to obtain 809 mg (83%) of the title compound as a white solid. 1H NMR (300 MHz, DMSO-d6) δ 9.84 (s, 1H), 8.18 (d, J = 7.0 Hz, 1H), 7.90 (d, J = 8.6 Hz, 1H), 7.53 (d, J = 8.6 Hz, 2H), 7.28 (d, J = 8.6 Hz, 2H), 5.44 (d, J = 4.4 Hz, 1H), 4.62 (q, J = 6.2 Hz, 1H), 4.39 (p, J = 7.6, 7.2 Hz, 1H), 4.17 (dd, J = 8.5, 6.8 Hz, 1H), 2.70 (t, J = 2.6 Hz, 1H), 1.96 (dt, J = 13.2, 6.6 Hz, 1H), 1.88 (s, 3H), 1.30 (d, J = 7.1 Hz, 3H), 0.86 (dd, J = 10.9, 6.8 Hz, 6H). ESI+[M+H]+ = 374.2. HPLC method 2, retention time = 3.95 min.

[0324] 1.2.3.3) Synthesis of 1-(4-((S)-2-((S)-2-Acetamido-3-Methylbutanamide)Propanamide)phenyl)but-3-yn-1-yl(4-Nitrophenyl)Carbonate 94 mg (0.25 mmol) of (2S)-2-acetamido-N-((2S)-1-((4-(1-hydroxybut-3-yn-1-yl)phenyl)amino)-1-oxopropan-2-yl)-3-methylbutanamide and 153 mg (0.50 mmol) of bis(4-nitrophenyl) carbonate were dissolved in 2 mL of anhydrous DMF. 98 mg (0.76 mmol) of DIPEA was added and the reaction mixture was stirred at room temperature overnight. Volatiles were removed under reduced pressure and the crude residue was purified by silica gel chromatography (gradient of 99:1 to 90:10 DCM / MeOH) to give 118 mg (87%) of the title compound as a yellow solid. 1H NMR (300 MHz, DMSO-d6) δ 9.99 (s, 1H), 8.35 - 8.26 (m, 2H), 8.22 (d, J = 7.0 Hz, 1H), 7.89 (d, J = 8.6 Hz, 1H), 7.63 (d, J = 8.7 Hz, 2H), 7.58 - 7.48 (m, 2H), 7.43 (d, J = 8.7 Hz, 2H), 5.74 (d, J = 7.5 Hz, 1H), 4.39 (p, J = 7.2 Hz, 1H), 4.17 (dd, J = 8.5, 6.9 Hz, 1H), 3.01 - 2.84 (m, 3H), 1.99 - 1.91 (m, 1H), 1.88 (s, 3H), 1.31 (d, J = 7.1 Hz, 3H), 0.86 (dd, J = 11.2, 6.8 Hz, 6H). ESI+ [M+H]+ = 539.1. HPLC method 2, retention time = 6.48 min.

[0325] 1.2.3.4) Synthesis of Compound INT3 43 mg (0.081 mmol) of the previous compound 1-(4-((S)-2-((S)-2-acetamido-3-methylbutanamido)propanamido)phenyl)but-3-yn-1-yl (4-nitrophenyl) carbonate, 55.5 mg (0.11) of exatecan mesylate and 11.0 mg (0.08 mmol) of HOBt were dissolved in a 2 mL mixture of anhydrous DMF / pyridine 85:15 (v / v). 11.5 mg (0.09 mmol) of DIPEA was added. The reaction was stirred at 40 °C for 3 h. After removal of the volatiles under reduced pressure, the reaction mixture was purified by silica gel chromatography (DCM / MeOH gradient from 99:1 to 95:5), and 42 mg (62%) of compound INT3 was obtained as a yellow / greenish solid. ESI+[M+H]+ = 835.3. HPLC method 2, retention time = 6.50 and 6.60 min (mixture of diastereoisomers).

[0326] 1.2.4) Synthesis of Compounds INT4, INT4-S and INT4-R

[0327]

Chemical formula

[0328] 1.2.4.1) Synthesis of tert-Butyl (2-(4-Aminophenyl)-2-Hydroxyethyl)Carbamate A MeOH solution containing 911 mg (3.23 mmol) of commercially available tert-butyl (2-hydroxy-2-(4-nitrophenyl)ethyl)carbamate (CAS No. 939757-25-2) was transferred to a stainless steel hydrogenation reactor. After an initial argon purge, a catalytic amount of 5% wt Pd / C was added. The reactor was then purged twice with H2 and the reaction was maintained at room temperature for 5 h under a H2 pressure of 10 bar with stirring. The reaction was filtered through a 0.45 μm PTFE filter and MeOH was removed under vacuum, and 749 mg (92%) of the title compound was obtained as a white solid. ESI+[M+H]+ = 253.2. HPLC method 2, retention time = 2.73 min.

[0329] 1.2.4.2) Synthesis of tert-Butyl (2-(4-((S)-2-((S)-2-Acetamido-3-Methylbutanamide)Propanamide)phenyl)-2-Hydroxyethyl)Carbamate 150 mg (0.60 mmol) of the previous compound tert-butyl (2-(4-aminophenyl)-2-hydroxyethyl)carbamate, 222 mg (0.71 mmol) of Fmoc-Ala-OH and 81 mg (0.62 mmol) of DIPEA were dissolved in 5 mL of anhydrous DMF. 181 mg (0.71 mmol) of HATU was added and the reaction was stirred overnight at room temperature. The volatiles were then removed under reduced pressure and the crude residue was purified by silica gel chromatography (DCM / MeOH gradient from 100:0 to 90:10) to give quantitatively the first intermediate tert-butyl (2-(4-(((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)phenyl)-2-hydroxyethyl)carbamate which is directly involved in the Fmoc deprotection. HPLC method 2 retention time = 7.7 min.

[0330] tert-Butyl (2-(4-(((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)phenyl)-2-hydroxyethyl)carbamate was dissolved in 5 mL of DMF / piperidine 9:1 (v / v) and stirred at room temperature for 15 min. The volatiles were then removed under reduced pressure and the crude residue was purified by silica gel chromatography (DCM / MeOH gradient from 99:1 to 80:20) to give 130 mg (68% over 2 steps) of the second intermediate tert-butyl (2-(4-(((S)-2-aminopropanamido)phenyl)-2-hydroxyethyl)carbamate as a white foamy solid. HPLC method 2, retention time = 3.75 min.

[0331] 130 mg (0.40 mmol) of tert-butyl (2-(4-((S)-2-aminopropanamido)phenyl)-2-hydroxyethyl)carbamate and 124 mg (0.48 mmol) of commercially available Ac-Val-OSu (CAS number 56186-37-9) were dissolved in 3 mL of anhydrous DMF, and the reaction mixture was stirred overnight at room temperature. Subsequently, the volatile substances were removed under reduced pressure, and the crude residue was triturated with 3 mL of DCM to afford 95 mg (51%) of the title compound as a white solid. 1H NMR (300 MHz, DMSO-d6) δ 9.82 (s, 1H), 8.16 (d, J = 7.1 Hz, 1H), 7.88 (d, J = 8.6 Hz, 1H), 7.53 (d, J = 8.4 Hz, 2H), 7.22 (d, J = 8.5 Hz, 2H), 6.73 - 6.58 (m, 1H), 5.27 (d, J = 4.4 Hz, 1H), 4.58 - 4.47 (m, 1H), 4.40 (q, J = 7.1 Hz, 1H), 4.17 (dd, J = 8.4, 6.8 Hz, 1H), 3.15 - 2.90 (m, 2H), 1.88 (s, 4H), 1.35 (s, 9H), 1.30 (d, J = 7.1 Hz, 3H), 0.92 - 0.73 (m, 6H). ESI+ [M+H]+ = 487.3. HPLC method 2, retention time = 4.50 min.

[0332] 1.2.4.3) Synthesis of tert-Butyl (2-(4-((S)-2-((S)-2-Acetamido-3-Methylbutanamide)Propanamide)phenyl)-2-(((4-Nitrophenoxy)Carbonyl)Oxy)Ethyl)Carbamate 286 mg (0.62 mmol) of tert-butyl (2-(4-((S)-2-((S)-2-acetamido-3-methylbutanamido)propanamido)phenyl)-2-hydroxyethyl)carbamate and 375 mg (1.23 mmol) of bis(4-nitrophenyl) carbonate were dissolved in 3 mL of anhydrous DMF. 318 mg (2.46 mmol) of DIPEA was added and the reaction mixture was stirred at room temperature for 3 h. Volatiles were removed under reduced pressure and the crude residue was purified by silica gel chromatography (gradient of DCM / MeOH from 99:1 to 90:10) to afford 336 mg (87%) of the title compound as a yellow solid. 1H NMR (300 MHz, DMSO-d6) δ 9.99 (s, 1H), 8.36 - 8.26 (m, 2H), 8.22 (d, J = 6.9 Hz, 1H), 7.89 (d, J = 8.6 Hz, 1H), 7.63 (d, J = 8.6 Hz, 2H), 7.56 - 7.46 (m, 2H), 7.34 (d, J = 8.6 Hz, 2H), 7.22 (t, J = 5.6 Hz, 1H), 5.68 (t, J = 6.0 Hz, 1H), 4.38 (p, J = 7.1 Hz, 1H), 4.17 (dd, J = 8.5, 6.9 Hz, 1H), 3.49 - 3.34 (m, 2H), 2.01 - 1.90 (m, 1H), 1.87 (s, 3H), 1.37 (s, 9H), 1.30 (d, J = 7.1 Hz, 3H), 0.86 (dd, J = 11.1, 6.8 Hz, 6H). ESI+ [M+Na]+ = 652.2. HPLC HPLC method 2, retention time = 7.13 min.

[0333] 1.2.4.4) Synthesis of Compound INT4 231 mg (0.37 mmol) of the previous compound tert-butyl (2-(4-((S)-2-((S)-2-acetamido-3-methylbutanamido)propanamido)phenyl)-2-(((4-nitrophenoxy)carbonyl)oxy)ethyl)carbamate, 150 mg (0.28) of exatecan mesylate and 38 mg (0.28 mmol) of HOBt were dissolved in a 85:15 (v / v) mixture of 5 mL of anhydrous DMF / pyridine. 40 mg (0.31 mmol) of DIPEA was added. The reaction was stirred at 40 °C for 3 h and the volatiles were evaporated under reduced pressure. The crude residue was purified by silica gel chromatography (DCM / MeOH gradient from 99:1 to 90:10). 220 mg (85%) of the intermediate compound 1-(4-((S)-2-((S)-2-acetamido-3-methylbutanamido)propanamido)phenyl)-2-((tert-butoxycarbonyl)amino)ethyl ((1R,9R)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate was obtained as a brown-yellow solid. ESI+ [M+H]+ = 926.4. HPLC method 2, retention time = 6.7 and 6.8 min (mixture of diastereoisomers).

[0334] The solid obtained was redissolved at 0 °C using a TFA / DCM (30:70 v / v) solution and stirred at room temperature for 20 min. The volatiles were evaporated under reduced pressure and the crude residue was dissolved in a water / ACN (1:1 v / v) solution and purified using preparative HPLC method 5 to give 171.4 mg (73%) of compound INT4 as a yellow solid. ESI+ [M+H]+ = 826.4. HPLC method 3, retention time = 8.3 and 8.75 min (mixture of diastereoisomers).

[0335] 1.2.4.5) Synthesis of Stereochemically Pure Compounds INT4-S and INT4-R

[0336]

Chemical Structure

[0337] 1.2.4.5.1) Chiral Resolution of the Racemic Mixture of tert-Butyl (2-(4-Aminophenyl)-2-Hydroxyethyl)Carbamate Using a Teledyne Isco CombiFlash (registered trademark) Rf200 system with a Chiralflash (registered trademark) IC MPLC column 30 x 100 mm, 20 μm (Daicel catalog number 83M73), chiral separation of racemic tert-butyl (2-(4-aminophenyl)-2-hydroxyethyl) carbamate was performed. The mobile phase was DCM + 0.2% (v / v) EtOH (isocratic gradient). The flow rate was 12 mL / min. The sample solvent was DCM + 0.2% (v / v) EtOH. The mass recovery rate of the two enantiomers after separation was over 75%.

[0338] The retention time of tert-butyl (S)-(2-(4-aminophenyl)-2-hydroxyethyl) carbamate was 21 minutes, while the retention time of tert-butyl (R)-(2-(4-aminophenyl)-2-hydroxyethyl) carbamate was 29 minutes. The absolute configuration of the enantiomers (dissolved in a 1:1 mixture of heptane / ethanol in advance and slowly evaporated for one week to induce crystal formation) was confirmed by X-ray crystallography. Block-shaped crystals were placed on a nylon loop in perfluorinated ether oil. The data was collected using an Xcalibur, Atlas, Gemini diffractometer equipped with an Oxford Cryosystems cryogenic device operating at T = 150.00(10) K. The data was measured using a ω scan with CuKα radiation. The structure was analyzed by using the ShelXT solution program with the dual method and Olex2 (O.V. Dolomanov et al., Olex2: A complete structure solution, refinement and analysis program, J. Appl. Cryst., 2009, 42, 339-341). The model was refined with ShelXL 2018 / 3 (Sheldrick, G.M., Crystal structure finement with ShelXL, Acta Cryst., 2015, C71, 3-8) using full-matrix least-squares minimization of F2.

[0339] 1.2.4.5.2) Synthesis of Stereochemically Pure INT4-S and INT4-R Compounds Without any appreciable change in reaction conditions, reactivity, or overall yield, stereochemically pure compounds INT4-S and INT4-R were synthesized as described in the previous section 1.2.4.

[0340] By final purification using HPLC preparative method 5, 54 mg of compound INT4-S was obtained as a yellow solid. ESI+[M+H]+ = 826.4. HPLC method 3, retention time = 8.45 min.

[0341] By final purification using HPLC preparative method 5, 46 mg of compound INT4-R was obtained as a yellow solid. ESI+[M+H]+ = 826.4. HPLC method 3, retention time = 8.90 min.

[0342] 1.3) Synthesis of Drug-Linker 1.3.1) Synthesis of Glucuronide-Based Drug-Linker 1.3.1.1) Synthesis of Compound LNK1

[0343]

Chem.

[0344] 26.0 mg (0.015 mmol) of this compound and 4.11 mg (0.016 mmol) of N-hydroxysuccinimide ester of maleimidoacetic acid were dissolved in anhydrous DMF (maleimide compound at a concentration of 0.1 M). 2.25 mg (0.022 mmol) of triethylamine was added and the reaction mixture was stirred for 2 hours until complete conversion of the reactants was observed by HPLC. The reaction mixture was then diluted with 1% aqueous TFA / ACN 1:1 (v / v) and purified using HPLC preparative method 6 to obtain 16.0 mg (57%) of compound LNK1 as a yellow solid. ESI+[M+H]+ = 1892.7. HPLC method 3, retention times = 7.95 minutes and 8.20 minutes (equimolar mixture of diastereoisomers).

[0345] 1.3.1.2) Synthesis of Compound LNK2.

[0346]

Chemical Structure

[0347] 254 mg (0.14 mmol) of this compound and 39.1 mg (0.15 mmol) of N-hydroxysuccinimide ester of maleimidoacetic acid were dissolved in anhydrous DMF (maleimide compound at 0.1 M concentration). 22.6 mg (0.22 mmol) of triethylamine was added and the reactants were stirred for 1 hour until the overall conversion of the reaction as observed by HPLC. The reaction mixture was then diluted with 1% aqueous TFA / ACN 1:1 (v / v) and purified using HPLC preparative method 6 to obtain 161 mg (58%) of the final compound LNK2 as a yellow solid. HRMS m / z (ESI+): Calc [M+2H]2+ = 985.8900; Exp [M+2H]2+ = 985.8896; Error = 0.4 ppm. HPLC method 2, retention time = 7.9 min and 8.1 min (equimolar mixture of diastereoisomers).

[0348] 1.3.1.3) Synthesis of Compounds LNK2-S and LNK2-R

[0349]

Chemical formula

[0350] 53.4 mg of compound LNK2-S was obtained as a yellow solid. ESI+ [M+2H]2+ = 985.9. HPLC method 3, retention time = 7.8 min.

[0351] 44.0 mg of compound LNK2-R was obtained as a yellow solid. ESI+ [M+2H]2+ = 985.9. HPLC method 3, retention time = 8.1 min.

[0352] 1.3.2) Synthesis of Dipeptide-Based Drug-Linker 1.3.2.1) Synthesis of Compound LNK3

[0353]

Chemical formula

[0354] 14.0 mg (36% over 2 steps) of compound LNK3 was obtained as a yellow solid. ESI+ [M+H]+ = 1883.8. HPLC method 3, retention time = 8.82 min and 9.07 min (equimolar mixture of diastereoisomers).

[0355] 1.3.2.2) Synthesis of Compound LNK4

[0356]

Chemical Structure

[0357] 34.9 mg (46% over 2 steps) of compound LNK4 was obtained as a yellow solid. HRMS m / z (ESI+): Calc [M+2H]2+ = 981.4394; Exp [M+2H]2+ = 981.4398; Error = -0.4 ppm. HPLC method 3, retention time = 8.81 min and 8.94 min (equimolar mixture of diastereoisomers).

[0358] 1.3.2.3) Synthesis of Compounds LNK4-S and LNK4-R

[0359]

Chemical Structure

[0360] 18.1 mg of compound LNK4-S was obtained as a yellow solid. ESI+[M+2H]2+ = 981.4. HPLC method 3, retention time = 8.78 min.

[0361] 16.3 mg of compound LNK4-R was obtained as a yellow solid. ESI+[M+2H]2+ = 981.4. HPLC method 3, retention time = 8.96 min.

[0362] Example 2: Preparation and Characterization of Antibody-Drug Conjugates 2.1) Antibody Production The amino acid sequences of the monoclonal antibodies were obtained from the literature. The amino acid sequences of the light chain (SEQ ID NO: 10) and heavy chain (SEQ ID NO: 11) of farletuzumab were obtained from the International Nonproprietary Names for Pharmaceutical Substances (INN) List 62 published in World Health Organization (WHO) Drug Information Vol 23, No. 3, 2009 and International Publication No. WO 2017 / 151979. Farletuzumab-LALA containing the Fc silencing mutations L234A and L235A ("LALA") on the heavy chain of farletuzumab (Wines et al. The Journal of Immunology May 15, 2000, 164(10)5313-5318) was also produced (heavy chain "LALA" SEQ ID NO: 9). The amino acid sequences of the light chain (SEQ ID NO: 17) and heavy chain (SEQ ID NO: 16) of mirvetuximab were obtained from International Patent Application Publication No. WO 2011 / 106528. The human IgG1k non-binding isotype control was Catalog No. HG1K (proprietary amino acid sequence) from Sino Biologicals. Trastuzumab (Herceptin® 150 mg) was purchased from Roche. Enhertu® (trastuzumab deruxtecan) was purchased from Daiichi Sankyo / AstraZeneca.

[0363] Monoclonal antibodies were produced by transient transfection of CHO K1 cells using techniques recognized in the art (contracted to Evitria AG, Switzerland). cDNA was cloned into Evitria's vector system using conventional (non-PCR based) cloning techniques. pDNA was prepared under low endotoxin conditions based on anion exchange chromatography. DNA concentration was determined by measuring absorption at a wavelength of 260 nm. The accuracy of the sequence was verified by Sanger sequencing (up to two sequencing reactions per plasmid). Suspension-adapted CHO K1 cells (originally received from ATCC and adapted to serum-free growth in suspension culture at Evitria) were used for antibody production. Seeds were grown in an animal component and serum-free medium owned by Evitria. Cells were transfected with Evitria's proprietary transfection reagent. The supernatant was recovered by centrifugation and subsequent filtration (0.2 μm filter). Antibodies were purified using MabSelect SuRe protein A purification resin (Cytiva) and SEC fractionation resin. The purity of the antibody material was confirmed by SDS-PAGE and size exclusion chromatography and was greater than 95%. Endotoxin content was measured using the Charles River Endosafe PTS system.

[0364] 2.2) Preparation of Cysteine-Conjugated Antibody-Drug Conjugates A solution of the antibody (10 mg / mL in PBS 7.4 + 1 mM EDTA) was treated with the required amount (2.2 molar equivalents for the final ~DAR4 ADC or 14 molar equivalents for the final DAR8 ADC) of tris(2-carboxyethyl)phosphine (TCEP) at 37 °C for 1 to 2 hours. The reduced antibody was buffer-exchanged with 100 mM potassium phosphate pH 7.4 + 1 mM EDTA by three dilutions / centrifugations using an Amicon 30K centrifugal filter device (Millipore). For the final ~DAR4 ADC, 6 molar equivalents of the drug-linker (from a 12 mM DMSO stock solution) were added to the antibody. For the final DAR8 ADC, 10 - 12 molar equivalents of the drug-linker were added. The solution was incubated at room temperature for 30 minutes. The final conjugate was buffer-exchanged / purified either with PBS pH7.4 buffer or histidine sucrose buffer (20 mM histidine buffer pH 6.0, 4% (w / v) sucrose + 75 mM NaCl) by four dilutions / centrifugations using an Amicon 30K centrifugal filter device and sterile filtered (0.20 μm PES filter).

[0365] The final protein concentration was evaluated spectrophotometrically at 280 nm using a Nanodrop One device (Thermo Fisher Scientific).

[0366] 2.3) Characterization of Cysteine-Conjugated Antibody-Drug Conjugates The resulting conjugate was characterized as follows: Drug-antibody ratio (DAR) evaluation by reverse-phase liquid chromatography-mass spectrometry (RPLC-MS): Denaturing RPLC-QToF analysis was performed using HPLC method 4 described above in Example 1. Briefly, the conjugate was eluted on an Agilent PLRP-S 1000 Å 2.1×150 mm 8 μm (80 °C) using a mobile phase gradient of water / acetonitrile + 0.1% formic acid (0.4 mL / min), and detected using a Bruker Impact IITM Q-ToF mass spectrometer scanning the 500-3500 m / z range (ESI+). Data was deconvoluted using the MaxEnt algorithm included in Bruker Compass® software.

[0367] Reverse-phase liquid chromatography (RPLC-UV): Denaturing RPLC-UV analysis was also performed on an Agilent 1100 HPLC-DAD system using a slightly modified version of HPLC method 4 above. The mobile phase modifier 0.1% formic acid was replaced with 0.1% TFA, and detection was performed using only DAD UV absorbance (no mass spectrometry detector).

[0368] Size-exclusion chromatography (SEC): SEC was performed on an Agilent 1100 HPLC system with a column dead volume of less than 15 μL (equipped with a short section of peak tube with an inner diameter of 0.12 mm and a microvolume UV flow cell). The column was an Agilent AdvanceBio SEC 300 Å 4.6×150 mm 2.7 μm (maintained at 30 °C). The mobile phase was 100 mM sodium phosphate and 200 mM sodium chloride (pH 6.8). 10% acetonitrile (v / v) was added to the mobile phase to minimize secondary hydrophobic interactions with the stationary phase and prevent bacterial growth. The flow rate was 0.35 mL / min. UV detection was monitored at 280 nm or any other relevant wavelength.

[0369] 2.4) Preparation of Lysine-Conjugated Antibody-Drug Conjugates A solution of antibody milbemycin mab or farletuzumab (10 mg / mL in 100 mM KH2PO4 pH 8.0) was treated with a 12 mM DMSO solution of sulfo-SPDB-DM4 CAS number 1626359-59-8 (MedChemExpress) to a final concentration of 7.5 molar equivalents of sulfo-SPDB-DM4. The solution was incubated at room temperature for 3 hours and filtered using a 0.20 μm PES filter. The final conjugate was buffer exchanged / purified by 5 dilutions / centrifugations using an Amicon 30K centrifugal filter device with either PBS pH 7.4 buffer or histidine sucrose buffer (20 mM histidine buffer pH 6.0, 4% (w / v) sucrose + 75 mM NaCl) and sterile filtered (0.20 μm PES filter).

[0370] The final protein concentration was spectrophotometrically evaluated at 280 nm using a Nanodrop One device (Thermo Fisher Scientific).

[0371] 2.5) Characterization of Lysine-Conjugated Antibody-Drug Conjugates The resulting conjugate was characterized as follows: Evaluation of Average Drug-Antibody Ratio (DAR) by Native SEC-MS: Prior to analysis, the ADC was deglycosylated by adding 50 U of IgGZERO® (Genovis) enzyme to 50 μg of the ADC. The ADC was separated on an Agilent AdvanceBio SEC 200A 1.9 μm 2.1×150 mm PEEK column (catalog number PL1980-3201PK) maintained at 30 °C. The column was equilibrated in 50 mM ammonium acetate + 10% (v / v) HPLC grade isopropanol. During the run, the flow rate was maintained at 0.075 mL / min and the ADC typically eluted between 3.5 and 4.5 minutes. After elution of the mAb or ADC, the flow and buffer composition were maintained. The column eluate was directed to a Bruker Impact II™ Q-ToF mass spectrometer in the 300 - 8000 m / z range (ESI +) was scanned. The capillary voltage of the raw material was set at 4500 V. The drying gas of the raw material was set at 8.0 L / min, and the spray gas was set at 25 Psi. The drying temperature of the source was set at 200 °C. The ADC mass spectrum was deconvoluted using the MaxEnt algorithm included in Bruker Compass (registered trademark) software, and the DAR was calculated using the ion intensity peak height of each ADC subspecies.

[0372] Size Exclusion Chromatography (SEC): SEC was performed on an Agilent 1100 HPLC system with a column dead volume of less than 15 μL (equipped with a short section of peak tube with an inner diameter of 0.12 mm and a microvolume UV flow cell). The column was an Agilent AdvanceBio SEC 300 Å 4.6×150 mm 2.7 μm (maintained at 30 °C). The mobile phase was 100 mM sodium phosphate and 200 mM sodium chloride (pH 6.8). 10% isopropanol (v / v) was added to the mobile phase to minimize secondary hydrophobic interactions with the stationary phase and prevent bacterial growth. The flow rate was 0.35 mL / min. UV detection was monitored at 280 nm or any other relevant wavelength.

[0373] 2.6) Summary of Synthesized Antibody-Drug Conjugates

[0374]

Table 7-1

[0375]

Table 7-2

[0376] Example 3: Performance of General Cell Culture and Animal Experiments The human cancer cell lines used in this project were purchased from either the American Type Culture Collection (ATCC), The Leibniz Institute DSMZ German Collection of Microorganisms and Cell Cultures GmbH (DSMZ), or the European Collection of Authenticated Cell Cultures (ECACC). For use in experiments, the cells were cultured according to the instructions from the original ATCC / DSMZ / ECACC suppliers regarding cell culture medium and supplements, cryopreservation, and subculture procedures, following the implementation of good established cell culture. The cells were incubated at 37 °C in a 5% CO2 atmosphere for within 2 months.

[0377] All animal procedures were carried out in accordance with the European Union Directive 86 / 609 / EEC. The experiments were conducted in an animal breeding facility certified by the French Ministry of Agriculture under individual permits. This study was approved by the local Animal Ethics Committee (CECCAPP).

[0378] Example 4: Comparison of Rodent Therapeutic Index for Glucuronide or Dipeptide Drug-Linker Design The mouse therapeutic indices of the drug-linker constructs LNK1 (glucuronide-exatecan) and LNK3 (dipeptide Val-Ala-exatecan) were evaluated to compare the mouse therapeutic indices of the two enzyme cleavage modalities glucuronide and dipeptide Val-Ala. Using the model human HER2 that targets the trastuzumab monoclonal antibody as the targeting moiety, the T-GLC-EXA ADC and the T-VA-EXA ADC were formulated. These ADCs were compared regarding their in vivo efficacy and tolerance (therapeutic index) in a HER2+ gastric cancer model. Since trastuzumab does not cross-react in mice, this assay provides useful information regarding the apparent toxicity of the drug-linker component of the ADC (target-mediated toxicity that may occur is excluded).

[0379] In vivo efficacy evaluation: NCI-N87 gastric cancer cells were transplanted subcutaneously into female SCID mice (4 weeks old). The ADC was intravenously administered once at a sub-curative dose of 1 mg / kg when the tumors had grown to approximately 150 mm 3 (6 animals per group assigned to minimize the difference in initial tumor volume between groups). Tumor volume was measured every 3 - 5 days using a caliper device and calculated using the formula (L×W 2 ) / 2. Mice were sacrificed when the tumor volume exceeded 1000 mm 3 .

[0380] Pharmacokinetic profile evaluation: PK profile in rats (total antibody-drug conjugate concentration based on the mAb component over time) after a single intravenous dose of 3 mg / kg of the conjugate. The ADC was injected at 3 mg / kg into female Sprague-Dawley rats (4 - 6 weeks old, Charles River) via the tail vein (3 animals per group, randomly assigned). Blood was collected into citrate tubes via retro-orbital bleeding at various time points, processed into plasma, and stored at -80 °C until analysis. The ADC concentration based on the antibody component was evaluated using a human IgG ELISA kit (Stemcell™ Technologies) according to the manufacturer's protocol. A standard curve of the corresponding monoclonal antibody was used for quantification. PK parameters (clearance, half-life, and AUC) were calculated by two-compartment analysis using Microsoft® Excel® software incorporating a PK function (add-in developed by Usansky et al., Department of Pharmacokinetics and Drug Metabolism, Allergan, Irvine, USA).

[0381] In vivo tolerability evaluation: Female SCID mice (n = 3) were treated with a single intraperitoneal high dose of 200 mg / kg of the ADC compound. Weight loss or obvious signs of toxicity in the mice were observed over 12 days. In this experiment, Enhertu® (trastuzumab deruxtecan) was used as a positive control.

[0382] The results of this study are shown in Figure 1. T-GLC-EXA and T-VA-EXA showed similar in vivo efficacy (Figure 1A) and similar rat PK profiles (Figure 1B), but showed a significant difference in mouse tolerance at high doses (Figure 1C). It was concluded that the dipeptide Val-Ala cleavable modality provided a better efficacy / tolerance profile, at least for use with the exatecan payload. Therefore, this entity was favorable for other ADC constructs.

[0383] Example 5: Flow Cytometry Evaluation of the Extracellular Binding Site of Folic Acid Receptor Alpha (FRa) For FRa cell surface quantification, cells were incubated with a PE anti-FOLR1 antibody (BioLegend, catalog number 908304) for 20 minutes at room temperature. The eBioscience™ Fixable Viability Dye eFluor™ 780 kit (Thermo Fisher Scientific, catalog number 65-0865-18) was used according to the manufacturer's instructions to evaluate cell viability. Analyses were performed using a BD Fortessa flow cytometer controlled by BD FACSDiva software (BD Biosciences), and data were analyzed using FlowJo software (BD Bioscience).

[0384] The results are shown in Figure 2. The cell lines of this study program showed different levels of extracellular expression of FRa. BT-474 breast cancer cells did not express extracellular FRa and were thus considered negative control cell lines in the context of this study program.

[0385] Example 6: In Vitro Cytotoxicity Assay of Exatecan Mesylate Against FRa+ Cancer Cell Lines The in vitro cytotoxicity of the compound exatecan mesylate was evaluated in several FRα-positive cancer cell lines. Cells were seeded into 96-well plates at an appropriate density (1000 - 10000 cells / well in 100 μL of appropriate culture medium) according to the cell line and incubated at 37 °C for 24 hours. Serial dilutions (50 μL) of the test compound pre-dissolved in the culture medium were added, and incubation was carried out at 37 °C for 144 hours. MTT (5 mg / mL, 20 μL, Sigma-Aldrich) was added to the wells, and incubation was continued at 37 °C for 1 - 2 hours. Then, the culture medium was carefully removed, and the well contents were uniformly dissolved with acidic isopropanol. Absorbance values were measured using a MultiskanTM Sky microplate reader (Thermo Scientific) at a wavelength of 570 nm (reference wavelength 690 nm). The IC50 concentration values compared to untreated control cells were determined using inhibition dose-response curve fitting (GraphPad Prism 9).

[0386] The results are shown in Figure 3. Exatecan mesylate showed in vitro potencies with IC50s in the sub-nM to low nM range, prompting the inventors to investigate the use of this compound as an ADC payload for FRα-expressing malignancies.

[0387] Example 7: Recombinant FRα Binding Affinity by ELISA The sandwich ELISA assay was performed using a 96-well high-binding ELISA plate (Corning Inc., New York, NY, USA, catalog number 3590). The plate was coated with 2 μg / mL of recombinant human FRα protein (Sino Biological, catalog number 11241-H08H); recombinant cynomolgus FRα protein (Sino Biological catalog number 90950-C08H); recombinant rat FRα protein (Sino Biological, catalog number 81073-R08H) or recombinant mouse FRα protein (Sino Biological, catalog number 50573-M08H) in PBS (pH 7.4) at 100 μL / well and incubated overnight at 4°C. After washing twice with PBS-T (PBS + 0.05% Tween-20), the plate was blocked with 200 μL / well of incubation buffer (PBS-T + 0.1% BSA) for 1 hour at room temperature. The plate was washed four times with PBS-T and 100 μL of a 3-fold dilution series of the test compound (antibody or antibody-drug conjugate) was added. The plate was then incubated for 2 hours at room temperature in the dark. After five washes with PBS-T, the plate was incubated with 100 μL / well of goat anti-human IgG (H+L) HRP-conjugated antibody (Jackson Immunoresearch, catalog number 109-035-088) diluted 1:250000 in incubation buffer in advance for 1 hour at room temperature. After washing five times with PBS-T, TMB substrate solution (Thermo-Fisher, catalog number N301) was added. The peroxidase activity was stopped with 0.18 M H2SO4 and the absorbance was read at 450 nm (reference wavelength 650 nm) using a Thermo Scientific MultiSkan EX microplate reader. Sigmodial fitting was performed using GraphPad Prism 9 software.

[0388] The results are shown in Figure 4. Figure 4A: All antibodies and ADCs had an approximately EC 50showed a similar recombinant human FRα binding affinity of = 0.1 nM. Loss of binding was not observed between the native antibody and each ADC construct. Loss of binding was not observed between trastuzumab and trastuzumab-LALA and their respective ADC constructs. No significant loss of binding was observed between mirvetuximab and the M-SORAV ADC construct. For the negative control ADC NEG-VA-EXA, human FRα binding was not detected. Figure 4B: F-LALA-VA-EXA bound equally to recombinant FRa proteins of human and cynomolgus monkeys and did not bind to recombinant FRa proteins of rats and mice. Thus, F-LALA-VA-EXA is cynomolgus monkey cross-reactive rather than rodent cross-reactive.

[0389] Example 8: Human FRα Binding Affinity by SPR Surface plasmon resonance (SPR) experiments were performed at 25 °C on a Biacore T200 instrument. The antibodies or ADCs tested were captured at low density (300 - 500 RU) on a CM5 series S sensor chip pre-functionalized with a Human Antibody Capture kit (Cytiva, catalog number BR100839). A similarly treated functionalized surface / flow cell without ligand was used as a reference. To measure the kinetic rate and affinity, recombinant human FR (Sino Biological, catalog number 11241-H08H) analyte samples were injected in duplicate at five concentrations (0.5, 1, 2, 4, 8 nM) in series by a 300-second contact pulse at a constant flow rate of 70 μL / mL of running buffer (HBS-EP+, Cytiva, catalog number BR100669) using a single-cycle kinetic strategy. The dissociation phase was measured by injecting running buffer for 900 seconds. Between duplicates, the surface / flow cell was regenerated with 3 M MgCl2 to remove both analyte and ligand and fresh ligand capture was performed using the same conditions. Data analysis was performed using Biacore Evaluation software after subtracting the reference surface and zero concentration signal of the analyte. The data were processed and fitted to a 1:1 binding model to obtain the association rate constant k a (on-rate) and kd (off-rate), and K D (equilibrium dissociation constant, also referred to as "affinity") were determined. The mean and standard deviation of the repeated experiments are reported.

[0390] The results are shown in Figure 5. Loss of recombinant human FRα binding was not observed between the native antibody and each ADC construct. Loss of binding was not observed between trastuzumab and trastuzumab-LALA and each ADC construct. Surprisingly, about a 10-fold decrease in K D value (affinity constant) was observed for trastuzumab and trastuzumab-LALA-based constructs compared to mirvetuximab and mirvetuximab-based constructs. This was the result of an increase in the k d value (dissociation rate constant) for trastuzumab and trastuzumab-LALA-based constructs (considering that the association rate constant k a was similar). No human FRα binding events were detected for the negative control IgG1 (Sino Biologicals, catalog number HG1K).

[0391] Example 9: Cell Binding Affinity by Flow Cytometry The antibody or ADC binding to extracellular human FRα expressed on cancer cell lines was evaluated by flow cytometry. The tested antibody or ADC was conjugated to an APC fluorophore using the LYNX Rapid APC Antibody Conjugation Kit according to the manufacturer's protocol (Bio-Rad, catalog number LNK032APC). 5 μL of a 10 μg / mL solution of the tested APC-labeled antibody or ADC was added to 500,000 cells (suspended in 100 μL of PBS in a flow cytometry plastic tube). The cells were incubated in the dark for 20 minutes, washed three times with PBS by centrifugation, and resuspended in 200 μL of PBS for analysis. Flow cytometry was performed using a BD Fortessa flow cytometer controlled by BD FACSDiva software (BD Biosciences), and the data was analyzed using FlowJo software (BD Bioscience).

[0392] The results are shown in Figure 6. Equivalent human FRα cell binding was observed between farletuzumab and farletuzumab-LALA and their respective ADC constructs. The binding affinities of native mirvetuximab and native farletuzumab appeared to be the same. Surprisingly, a significant loss of binding was observed for the ADC derivative of mirvetuximab (M-SORAV) compared to its parental antibody, mirvetuximab. This could be explained by either (1) the heterogeneous stochastic conjugation of the sulfo-SPDB-DM4 drug-linker, which can react with lysine amino acids that are part of the antibody variable region, and thus a decrease in binding affinity; (2) the lower APC labeling efficiency of M-SORAV when the LYNX Rapid APC conjugation kit reacts with lysine amino acids such as sulfo-SPDB-DM4, or (3) a combination of (1) and (2). No binding was observed in the FRα-negative control cell line BT-474 for all compounds tested.

[0393] Example 10: Ex Vivo ADC Human Plasma Stability F-VA-EXA and F-LALA-VA-EXA ADC samples (solutions > 6 mg / mL in PBS) were diluted with pure sterile human plasma (GeneTex, catalog number GTX73265) in screw-cap centrifuge tubes to obtain a final ADC concentration of 200 μg / mL (residual PBS volume < 5% v / v). Samples were incubated at 37 °C and aliquots were taken at 5 minutes, 6 hours, 1 day, 2 days, 3 days and 7 days (aliquots were kept frozen at -80 °C until analysis). ADCs were isolated from plasma by immunocapture using Dynabeads™ M-280 streptavidin (Thermo Scientific) magnetic beads pre-coated with biotinylated human folate receptor alpha recombinant protein (Sino Biologicals, catalog number 11241-H08H). Briefly, 600 μL of commercially available bead solution was washed twice with HBS-EP buffer (Cytiva, catalog number BR100188) and resuspended in 1.2 mL of HBS-EP buffer. 65 μL of biotinylated recombinant FRa solution (protein amount 48 μg) was added and the solution was stirred at room temperature for 2 hours. The beads were then washed three times with HBS-EP buffer and resuspended in 1.2 mL of HBS-EP buffer. For one immunocapture, 100 μL of the previous bead solution was added onto 100 μL of HBS-EP in a microcentrifuge tube. 10 μL of ADC solution in plasma (theoretical ADC amount 2 μg) was added and the solution was stirred at room temperature for 2 hours. After incubation, the bead-ADC complex was washed twice with HBS-EP buffer, resuspended in 200 μL of HBS-EP buffer and deglycosylated by adding 2 μL / 1000 U of PNGase F (New England Biolabs, catalog number P0705L) and incubating overnight at 37 °C with gentle stirring. The beads were then washed twice with HBS-EP buffer, twice with distilled water and once with 10% acetonitrile (v / v) in water. The beads were incubated for 30 minutes at room temperature with gentle stirring with 50 μL of 30% aqueous acetonitrile (v / v) containing 0.1% (v / v) formic acid.Next, the elution sample containing the deglycosylated ADC was analyzed by modified reverse-phase chromatography-mass spectrometry using a Thermo UltiMate 3000 UHPLC system equipped with a Bruker Impact II™ Q-ToF mass spectrometer. Mobile phase A was water + 0.1% formic acid, and mobile phase B was acetonitrile + 0.1% formic acid. The column was an Agilent PEEK PLRP-S 1000 Å 2.1×100 mm 5 μm (80 °C). The linear gradient was from 20% B to 50% B in 25 minutes. The flow rate was 0.4 mL / min. UV detection was monitored at 280 nm. The Q-ToF mass spectrometer was used in the m / z range 500 - 5000 (ESI. + )). Data were deconvoluted using the MaxEnt algorithm included in Bruker Compass® software. For stability data analysis, deconvolution of the raw spectra within the selected light chain (LC) and heavy chain (HC) elution time windows was performed. Loss or modification of the drug-linker was identified according to the corresponding mass shift from the starting ADC material. The relative ratios of ADCs with different DARs were calculated by dividing the intensity of a specific ADC subspecies by the intensity from the total ADC species. The final DAR value was calculated as described in Xu et al., Anal. Biochem., 2011, 412(1), 56 - 66.

[0394] The results are shown in Figure 7. For both the F-VA-EXA and F-LALA-VA-EXA ADCs, >90% drug-linker human plasma stability was observed. No drug-linker mass shift was observed during this test (no early exatecan release or metabolism), except for a +18 Da (+H2O) mass shift from the self-stabilizing hydrolysis of the maleimide group, which was completed after 48 - 72 hours of ADC incubation time. The only instability observed was caused by retro-Michael maleimide deconjugates of the total drug-linker on the heavy chain of the ADC (no deconjugation was observed on the light chain).

[0395] Example 11: In Vitro Cytotoxicity Experiment Against FRa-Negative Cell Lines To evaluate the non-specific (off-target) cytotoxicity of the ADC, an in vitro cytotoxicity assay was performed in the BT-474 (FRa-negative) cancer cell line. Cells were seeded in 96-well plates at an appropriate density (1000 - 10000 cells / well in 100 μL of appropriate culture medium) according to the cell line and incubated at 37 °C for 24 hours. Serial dilutions (50 μL) of the test compound pre-dissolved in the culture medium were added, and incubation was carried out at 37 °C for 144 hours. MTT (5 mg / mL, 20 μL, Sigma-Aldrich) was added to the wells, and incubation was continued at 37 °C for 1 - 2 hours. Then, the culture medium was carefully removed, and the well contents were uniformly dissolved with acidic isopropanol. Absorbance values were measured using a Multiskan™ Sky microplate reader (Thermo Scientific) at a wavelength of 570 nm (reference wavelength 690 nm). The IC50 concentration values were determined using inhibition dose-response curve fitting (GraphPad Prism 9) compared to untreated control cells.

[0396] The results are shown in Figure 8. The soravtansine (sulfo-SPDB-DM4)-based ADC showed a higher level of off-target (non-FRa-mediated) cell killing efficacy compared to the VA-EXA (LNK4-S drug-linker)-based ADC. This has been observed in both the milatuzumab-based ADC and the farletuzumab-based ADC. The VA-EXA drug-linker component has less off-target toxicity compared to the soravtansine drug-linker, which is a good prerequisite for enhancing the tolerability of ADCs in a clinical setting.

[0397] [Table 8]

[0398] Example 12: In Vivo Efficacy Xenograft Model Female CB-17 severe combined immunodeficiency (SCID) mice, 4 - 5 weeks old, were obtained from Janvier labs (Le Genest-Saint-Isle, France) and isolated for 7 days before the start of the experiment. Cells resuspended in 50% BD Matrigel (Corning®) in PBS (OV-90, SW-620, KB, BT-474 cell lines) or PBS (PA-1, IGROV-1, OVCAR-3, NCI-H2110 cell lines) (5 - 10×10 6 cells per mouse) were inoculated subcutaneously into the mice. When the average tumor volume reached approximately 120 - 150 mm 3 , the mice were randomized (typically 7 mice per group) and treated by single intravenous injection of PBS (negative control) (unless otherwise specified), or the antibody-drug conjugate was examined. Tumor volume was measured every 3 - 5 days using a caliper device (length × width) and calculated using the following formula V = 4 / 3×π×R 3 (where R represents the radius). When the tumor volume exceeded 1500 mm3 or the tumor ulcerated, the mice were sacrificed.

[0399] In the IGROV-1 tumor model, a tumor re-transplantation challenge was performed on day 94 of the study. Mice treated with F-LALA-VA-EXA (12 mg / kg single intravenous) were re-transplanted with IGROV-1 cells (on the other flank of the animal) using the same procedure as used at the start of the study. A control group of 5 new SCID animals was also re-transplanted following exactly the same procedure.

[0400] Figure 9 shows the tumor xenograft experiment in the SW-620 cancer model. F-VA-EXA and M-SORAV conjugates were injected IV at 5, 10 or 15 mg / kg. F-VA-EXA was very active at doses of 5 mg / kg and above, while the comparator M-SORAV was inactive even when administered at 15 mg / kg.

[0401] Figure 10 shows the tumor xenograft experiments in the SW-620 cancer model. F-VA-EXA and F-LALA-VA-EXA were intravenously injected once at 1, 3, and 6 mg / kg. The non-targeted isotype-matched negative control ADC NEG-VA-EXA was IV injected at 6 mg / kg. F-VA-EXA and F-LALA-VA-EXA were equally highly active at doses of 1 mg / kg and above. The non-targeted conjugate NEG-VA-EXA was inactive at the highest test dose of 6 mg / kg, demonstrating that the antitumor activity of the conjugate is target-selective.

[0402] Figure 11 shows the tumor xenograft experiments in the OV-90 cancer model. F-VA-EXA, F-LALA-VA-EXA, and M-SORAV conjugate were intravenously injected as a single dose at 30 mg / kg. All conjugates were highly active, with a 100% remission rate in all groups. However, significant toxicity was observed with the M-SORAV conjugate: 6 out of 6 mice showed a disheveled appearance (a dull, non-glossy hair coat), 3 out of 6 mice showed signs of collapse, and 2 out of 6 mice had diarrhea. No toxicity was observed with the F-VA-EXA and F-LALA-VA-EXA conjugates. These data suggest that the preclinical therapeutic profile of the F-VA-EXA and F-LALA-VA-EXA constructs is better compared to M-SORAV.

[0403] Figure 12 shows the tumor xenograft experiments in the OV-90 cancer model. F-VA-EXA, F-LALA-VA-EXA, and M-SORAV conjugate were IV injected once at sub-curative 5 and 10 mg / kg doses. At 5 mg / kg, F-VA-EXA and F-LALA-VA-EXA showed similar and improved efficacy compared to M-SORAV. At 10 mg / kg, F-VA-EXA and M-SORAV showed similar efficacy (although more heterogeneity was observed within the M-SORAV group). At 10 mg / kg, F-LALA-VA-EXA was superior to both F-VA-EXA and M-SORAV.

[0404] Figure 13 shows the tumor xenograft experiment in the KB cancer model. The F-VA-EXA, F-LALA-VA-EXA, and M-SORAV conjugates were injected intravenously once at 3, 6, and 12 mg / kg. The non-targeted isotype-matched negative control ADC NEG-VA-EXA was injected intravenously once at 12 mg / kg. F-VA-EXA, F-LALA-VA-EXA, and M-SORAV were equally active at doses of 3 mg / kg or higher. The non-targeted conjugate NEG-VA-EXA was inactive at the highest test dose of 12 mg / kg, demonstrating that the antitumor activity of the conjugate is target-selective.

[0405] Figure 14 shows the tumor xenograft experiment in the PA-1 cancer model. The F-LALA-VA-EXA and M-SORAV conjugates were injected intravenously once at the sub-curative 3, 6, and 12 mg / kg doses. The non-targeted isotype-matched negative control ADC NEG-VA-EXA was injected intravenously once at 12 mg / kg. At 3 mg / kg, F-LALA-VA-EXA exceeded M-SORAV. At 6 mg of F-LALA-VA-EXA, it slightly exceeded M-SORAV. At 12 mg of M-SORAV, it was superior to F-LALA-VA-EXA. Overall, both conjugates showed similar levels of efficacy. At the highest test dose of 12 mg / kg, the non-targeted conjugate NEG-VA-EXA showed a slightly reduced tumor growth rate compared to the untreated control group.

[0406] Figure 15 shows the tumor xenograft experiment in the OV-90 cancer model. The F-LALA-VA-EXA and M-SORAV conjugates were injected intravenously once at 3, 6, and 12 mg / kg doses. At 3, 6, and 12 mg / kg, F-LALA-VA-EXA exceeded M-SORAV.

[0407] Figure 16 shows the tumor xenograft experiment in the IGROV-1 cancer model. The F-LALA-VA-EXA and M-SORAV conjugates were injected IV once at doses of 3, 6, and 12 mg / kg. The non-targeted isotype-matched negative control ADC NEG-VA-EXA was injected IV once at 12 mg / kg. At 3, 6, and 12 mg / kg, F-LALA-VA-EXA and M-SORAV showed similar levels of efficacy and strong tumor regression. At the highest test dose of 12 mg / kg, the non-targeted conjugate NEG-VA-EXA showed some level of efficacy, although it was not as prominent as the other FRα-targeted ADCs in the study. Tumor regrowth rechallenge performed 94 days after the study confirmed that F-LALA-VA-EXA could induce a protective immune memory response, as no tumor growth was observed after re-transplantation in the group treated with F-LALA-VA-EXA at a single 12 mg / kg IV (the positive control group using new untreated SCID mice showed tumor growth after transplantation).

[0408] Figure 17 shows the tumor xenograft experiment in the folate receptor alpha negative (FRα neg) BT-474 breast cancer model. The F-LALA-VA-EXA and M-SORAV conjugates were injected IV once at doses of 5 and 10 mg / kg. Enhertu® (HER2-targeted trastuzumab deruxtecan) was used as a positive control and injected IV once at a dose of 10 mg / kg. No efficacy was observed for F-LALA-VA-EXA and M-SORAV at either dose, confirming that the efficacy of these conjugates is FRα-selective. As expected and as previously observed (Conilh et al., 2021, Pharmaceuticals, 14(3), 247, doi:10.3390 / ph14030247), Enhertu® was effective in this HER2-positive BT-474 cancer model.

[0409]

Table 9

[0410] Example 13: Tolerance Experiment in Mice To evaluate the preclinical therapeutic index of the mouse, a tolerance experiment of the mouse was conducted using F-VA-EXA, F-LALA-VA-EXA and M-SORAV conjugates. Female SCID mice (n = 5 mice per group) were treated with a single intraperitoneal dose of F-VA-EXA (200 mg / kg), F-LALA-VA-EXA (200 mg / kg) or M-SORAV (100 mg / kg). The mice were carefully monitored for weight loss or obvious signs of toxicity over a period of 21 days.

[0411] The same experiment was also conducted in female CD-1 mice treated once with 0, 50, 100, 150 or 200 mg / kg IV of F-LALA-VA-EXA.

[0412] The results (for the SCID mouse experiment) are shown below and in Figure 18. F-VA-EXA and F-LALA-VA-EXA showed no obvious signs of toxicity at a dose of 200 mg / kg and were well tolerated. In contrast, all mice treated with a dose of 100 m / kg of M-SORAV showed significant signs of toxicity (a disheveled appearance with ruffled fur, diarrhea, prostration, closed eyes, suppressed behavior), and death was found on the 2nd to 3rd day or euthanasia was necessary. These results, together with the above xenograft efficacy data, indicate an excellent preclinical therapeutic index of the F-VA-EXA and F-LALA-VA-EXA constructs compared to the comparative example M-SORAV.

[0413] In female CD-1 mice, MBK-103 did not cause dose-dependent harmful systemic or local effects at all dose levels investigated (up to 200 mg / kg, IV, once). No weight loss or change in behavior was observed. The only notable observation was a slight decrease in the weights of the thymus and spleen in all groups compared to the control group during the final autopsy.

[0414]

Table 10

[0415] Example 14: Pharmacokinetics Study in Sprague-Dawley Rats F-VA-EXA and F-LALA-VA-EXA conjugates were injected at 5 mg / kg via the tail vein into female Sprague-Dawley rats (4 - 6 weeks old - Charles River), six animals per group assigned randomly. Blood was collected into citrate tubes via retro-orbital bleeding at 5 minutes, 4 hours, 1 day, 2 days, 4 days, 7 days, 14 days and 21 days, processed to plasma and stored at -80 °C until analysis.

[0416] Total mAb concentration was evaluated by ELISA using goat polyclonal anti-human IgG (H+L) primary antibody (Jackson Immunoresearch) as capture reagent and mouse polyclonal anti-human IgG (H+L) HRP conjugate (Jackson Immunoresearch) as secondary detection antibody. Total ADC concentration was evaluated by ELISA using rabbit polyclonal anti-exatecan antibody (custom order from ref#6294 / 00000920, Biotem, Apprieu, France) as capture reagent and mouse polyclonal anti-human IgG (H+L) HRP conjugate (Jackson Immunoresearch) as secondary detection antibody. A standard curve of ADC was used for quantification.

[0417] Pharmacokinetic parameters (clearance, half-life, Vss and AUC) were calculated by two-compartment analysis using Microsoft Excel software incorporating a PK function (add-in developed by Usansky et al., Department of Pharmacokinetics and Drug Metabolism, Allergan, Irvine, CA, USA).

[0418] The free exatecan concentration was evaluated using LC / MS-MS method with an Agilent 1100 HPLC system and a Sciex API 4000 MS / MS system. Rat plasma samples were protein precipitated using an organic solution composed of 80:20 (v / v) acetonitrile / methanol + 1% formic acid + d5-exatecan (20 ng / mL) as the internal standard. The samples were analyzed using gradient elution mode on a Phenomenex Kinetex® C8 2.1×30 mm 2.6 μm 100A column (Phenomenex, catalog number 00D-4497-AN) maintained at 45 °C. Mobile phase A was water + 0.15% formic acid, and mobile phase B was acetonitrile / isopropanol 80:20 (v / v) + 0.15% formic acid. The flow rate was 0.8 mL / min. For detection, MRM scanning was used in the positive ion mode. A calibration curve was plotted using linear least squares regression with 1 / x weighting and the peak area ratio analyte / deuterated internal standard against the nominal analyte concentration. The calibration curve was in the range of 0.2 (LLOQ) to 500 ng / mL.

[0419] The results are shown in Figure 19. F-VA-EXA and F-LALA-VA-EXA showed a biphasic PK profile in the circulation with similar volume of distribution, slow clearance rate, and half-life in the range of 13 - 15 days (fast distribution phase followed by slow elimination phase). The total mAb curve and the total ADC curve were similar in intensity, gradient, and shape, suggesting good ADC stability (no major payload deconjugation over time). The free exatecan payload was detected only during the 0 - 48-hour period of the experiment (10 6 times less concentrated than the parental ADC component), suggesting good ADC stability and no early deconjugation of exatecan.

[0420] Example 15: Evaluation of Lung Inflammation and Toxicity in Mice Bleomycin is widely used in rodents to model pulmonary fibrosis for the study of mechanisms involved in fibrogenesis and the evaluation of potential therapies. To evaluate ADC pulmonary toxicity, bleomycin-induced pulmonary fibrosis was induced in mice by the following known procedure as described by Walter and Kleeberger, Mouse Models of Bleomycin-Induced Pulmonary Fibrosis, 2008, Curr. Protoc. Pharmacol. 40:5.46.1-5.46.17. On days 0, 4, and 8, female C57 / BL6 mice (Janvier Labs, Le Genest-Saint-Isle, France) were lightly anesthetized with isoflurane, and 20 μL of a 1 mg / kg bleomycin PBS solution (bleomycin Veron, injectable 15 mg) or PBS (negative control mice) was administered intranasally. Non-bleomycin-acclimated mice were included as the negative control group. On day 11, the mice (n = 6 per group) were treated with the conjugate being tested (10 mg / kg i.p.), and monitored for obvious signs of toxicity and weight loss during the experimental period. A positive control group treated with the clinically approved conjugate Enhertu® (10 mg / kg i.p.) was included, which is known to induce interstitial lung disease and pneumonitis preclinically and in a small subset of patients. On day 25, all mice were sacrificed, bronchoalveolar lavage supernatant was obtained (by bronchoalveolar lavage), the lung organs were recovered, weighed, and fixed in 0.1% (w / v) formaldehyde in PBS. After fixation, the lungs were rinsed with 0.02% (w / v) sodium azide in PBS solution and embedded in paraffin for histological staining. Slides were stained with an anti-CD45 antibody (Abcam, catalog number ab10558, 1:500) to quantify the level of leukocyte infiltration in the lungs. A secondary biotinylated goat anti-rabbit antibody (Vector Laboratories, catalog number BA-1000, 1:300) and avidin-HRP (Vector Laboratories, catalog number A-2004) were used for detection.Histological experiments and imaging were outsourced at the Centre d’Imagerie Quantitative Lyon-Est (CiQLE) platform (Lyon, France). Random sections (30% zoom) of IHC images were processed using native Fiji software (developed and maintained by the Laboratory for Optical and Computational Instrumentation, University of Wisconsin-Madison, US) to count CD45-positive cells within the slides (1 slide per lung) and evaluate the level of leukocyte infiltration. The total numbers of lymphocytes (CD3 / CD4), eosinophils (CCR3 / SiglecF), neutrophils (Ly6g / Ly6C), and macrophages (F4 / 80) in bronchoalveolar lavage supernatants obtained from non-bleomycin-acclimated mice were monitored by flow cytometry. The inflammatory cytokine levels in bronchoalveolar lavage supernatants obtained from non-bleomycin-acclimated mice were quantified by ELISA. IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13 were quantified using the Bio-Plex Pro™ Mouse Cytokine Th2 Panel (Bio-Rad, catalog number L60000UKVT) according to the manufacturer's instructions. TGF-β and IL-17 were quantified using the Mouse TGF-β1 DuoSet and Mouse IL-17 DuoSet ELISA kits (R&D systems, catalog numbers DY1679 and DY421), respectively, according to the manufacturer's instructions.

[0421] Statistical significance was evaluated using one-way ANOVA (Tukey's method) with GraphPad Prism 9 software. p-values are * (p < 0.033), ** (p < 0.002), *** (p < 0.0002), and **** (p < 0.0001) as represented, and “ns” represents non-significant (p > 0.123).

[0422] The quantitative results of IHC CD45 are shown in Figure 20. The positive control Enhertu (registered trademark) ADC caused a significant increase in leukocyte inflammatory infiltration when compared to both the untreated ( *** ) and bleomycin-acclimated ( **** ) negative control groups. F-VA-EXA ADC did not cause a significant increase in leukocyte inflammatory infiltration when compared to the untreated group, but caused a significant increase when compared to the bleomycin-acclimated ( *** ) control group. F-LALA-VA-EXA did not cause a significant increase in leukocyte inflammatory infiltration when compared to both the untreated and bleomycin-acclimated negative control groups. Based on these preclinical results, it can be hypothesized that F-VA-EXA, especially F-LALA-VA-EXA (Fc silent variant), will rarely or not at all induce interstitial lung disease or pneumonitis in the clinical setting.

[0423] The inflammatory cytokine levels in bronchoalveolar lavage supernatant are shown in Figure 21A. The positive control Enhertu (registered trademark) ADC caused a significant increase in IL-13, IL-17, and TGF-β cytokine levels when compared to the untreated group. F-VA-EXA and F-LALA-VA-EXA ADCs did not cause a significant increase in cytokine levels when compared to the untreated group.

[0424] The total numbers of lymphocytes, eosinophils, neutrophils, and macrophages in bronchoalveolar lavage supernatant are shown in Figure 21B. The positive control Enhertu (registered trademark) caused a significant increase in the numbers of lymphocytes, macrophages, neutrophils, and eosinophils when compared to the untreated control group. F-VA-EXA and F-LALA-VA-EXA showed a significant increase in lymphocyte numbers when compared to the untreated control group, but did not show a statistically significant increase in macrophages, neutrophils, and eosinophils when compared to the untreated control group.

[0425]

Table 11

[0426] Example 16: Dose Range Determination Toxicity Test in Cynomolgus Monkeys A non-human primate dose range finding toxicity study was conducted using purpose-bred naive female cynomolgus monkeys (Macaca fascicularis) of Vietnamese origin. The study was performed at Cynbiose SAS (Marcy-l’Etoile, France). The study protocol was approved by the animal experimentation committee of the study facility. F-LALA-VA-EXA ADC was administered intravenously at dose levels of 30, 40, 50 or 60 mg / kg at 3-week intervals (total of 3 times) (5 mL / kg / h over a 30-minute injection period). Two female monkeys / group were used in the study (total of 8 animals). At necropsy, 5 days after the last dose, gross necropsy, organ weight measurements and histopathological examinations were performed. Parameters evaluated during the study included mortality, clinical signs (including estimation of food consumption), body weight, ophthalmic examination, body temperature, haematology, coagulation, clinical chemistry, urinalysis, organ weights, and gross and microscopic examination of an extensive list of tissues. The ADC formulation buffer was 20 mM histidine pH 6.0, 4% (w / v) sucrose and 75 mM NaCl. The vehicle was 0.9% saline.

[0427] The results are shown in Figure 22. F-LALA-VA-EXA ADC was well tolerated and the estimated highest non-severe toxic dose (HNSTD) was 50 mg / kg, 3 times. Administration at 60 mg / kg was not tolerated in one of the two animals due to acute renal failure (presumably target-related as folate receptor alpha is endogenously expressed in the kidney). The preclinical therapeutic window of F-LALA-VA-EXA appears favourable as the cynomolgus monkey HNSTD is well above the effective therapeutic dose in rodent cancer models (after dose allometric scaling). The present invention includes the following aspects. <1> An antibody-drug conjugate of formula (I), Ab-[L-D]p (I), wherein, - Ab is an anti-folate receptor alpha (FRα) antibody that specifically binds to SEQ ID NO: 12, - L is a cleavable linker moiety preferably bound to the antibody via a thiol residue, - D is a cytotoxic drug moiety bound to L, - p is from 1 to 8, preferably from 6 to 8, more preferably p is 8, The antibody-drug conjugate of formula (I). <2> The antibody-drug conjugate according to <1>, wherein D is an inhibitor of topoisomerase I, preferably selected from the group consisting of camptothecin analogs, and more preferably D is the drug moiety of exatecan of the following formula (II).

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Claims

1. An antibody-drug conjugate of formula (I): Ab-[L-D]p (I), wherein: - Ab is an anti-folate receptor alpha (FRα) antibody that specifically binds to folate receptor alpha of SEQ ID NO: 12; - L is a cleavable linker moiety represented by the following formula (VI), and L is covalently bound to one or more thiol residues of the antibody; 【Chemical Formula 5】 - D is a drug moiety of exatecan represented by the following formula (II) that is bound to L; 【Chemical Formula 1】 - p is from 1 to 8; The antibody-drug conjugate of formula (I).

2. The antibody-drug conjugate according to claim 1, wherein p is from 6 to 8.

3. The antibody-drug conjugate according to claim 1 or 2, wherein p is 8.

4. The antibody-drug conjugate is an antibody-drug conjugate of the following formula (VII): 【Chemical Formula 6】 wherein Ab is an anti-FRα antibody, typically an IgG1 Fc constant region containing alanine substitutions at leucine 234 and leucine 235, or a silent IgG1 variant thereof, and p is from 4 to 8; The antibody-drug conjugate according to claim 1.

5. The antibody-drug conjugate according to claim 4, wherein Ab is farletuzumab or a silent IgG1 variant thereof.

6. The antibody-drug conjugate according to any one of claims 1 to 5, wherein Ab is any of the following: (i) A variable heavy chain polypeptide comprising the HCDR1 of SEQ ID NO: 1, the HCDR2 of SEQ ID NO: 2, and the HCDR3 of SEQ ID NO: 3, and a variable light chain polypeptide comprising the LCDR1 of SEQ ID NO: 4, the LCDR2 of SEQ ID NO: 5, and the LCDR3 of SEQ ID NO: 6; or (ii) A variable heavy chain polypeptide comprising the VH of SEQ ID NO: 7 and a variable light chain polypeptide comprising the VL of SEQ ID NO: 8 and is an anti-FRα antibody containing any of them.

7. An antibody-drug conjugate of the following formula (VII): wherein: 【Chemical Formula 6】 Ab is A variable heavy chain polypeptide comprising the VH of SEQ ID NO: 7 and a variable light chain polypeptide comprising the VL of SEQ ID NO: 8 and is an anti-FRα antibody; The IgG1 Fc constant region contains alanine substitutions at leucine 234 and leucine 235; p is 8; The antibody-drug conjugate of formula (VII).

8. The antibody-drug conjugate according to any one of claims 1 to 7 for use as a medicament in a subject in need thereof.

9. The antibody-drug conjugate according to any one of claims 1 to 8 for use in the treatment of tumors in a subject in need thereof.

10. The antibody-drug conjugate according to claim 9, wherein the tumor is a solid tumor.

11. The antibody-drug conjugate according to claim 10, wherein the solid tumor is selected from the group consisting of ovarian cancer, breast cancer, lung cancer, or mesothelioma.

12. The antibody-drug conjugate according to any one of claims 1 to 7 for use in the treatment of cancer in a subject in need of treatment for cancer selected from the group consisting of ovarian cancer, triple-negative breast cancer, and non-small cell lung cancer.

13. A pharmaceutical composition comprising the antibody-drug conjugate according to any one of claims 1 to 7 in combination with one or more pharmaceutically acceptable excipients, diluents or carriers, optionally comprising other active ingredients.

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