Antibody-drug conjugates and uses thereof

Abstract

The present invention relates to antibody-drug conjugates wherein the antibody specifically binds to folate receptor alpha, and wherein the drug is preferably selected from cytotoxic drugs. Such antibody-drug conjugates are particularly useful for 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 wherein the drug is preferably selected from inhibitors of topoisomerase I, such as camptothecin analogs such as exotecan. Such antibody-drug conjugates are particularly useful for treating 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, especially cancer therapeutic agents. Such ADCs contain at least an antibody and a payload (e.g., a cytotoxic drug), both of which are covalently bonded via a linker. Therefore, ADCs are designed to combine the specificity of the antibody target with the efficiency of the payload (e.g., the cytotoxic activity of a chemotherapeutic agent). Effective ADCs should show high specificity and low systemic toxicity.

[0003] In the context of toxicity, the antibodies used in ADCs need to bind accurately and efficiently to their antigens, implying that the appropriate target antigen is preferentially or exclusively expressed on target cells.

[0004] When designing ADC, it is necessary to covalently attach the final active drug to the ligand targeting unit, while allowing the final release of the active drug unit by a selective enzymatic mechanism after cell internalization or in the microenvironment of diseased tissue. In this regard, several peptidase and glycosidase sensitive cleavable linker chemical strategies (related to self-destructive chemistry (self-immolativechemistries)) have been developed. These cleavable linkers and their corresponding cleavage mechanisms are well known and have been described in multiple 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 a key design attribute of ADC, affecting the efficacy and tolerability of the conjugate.

[0005] The example of the joint type that has been used for cytotoxin or drug is conjugated with antibody includes but is not limited to hydrazone, thioether, ester, disulfide and peptide-containing joint.Joint is for example selected from those joints that are susceptible to the cutting of low pH in lysosomal compartment or susceptible to the cutting of protease, and the protease is for example preferentially expressed in tumor tissue, such as cathepsin (such as cathepsin B, C, D).Effective joint can ensure accurate and timely payload release.Under the background of toxicity, it seems that the stability of joint can also affect the toxicity played by payload, even if joint itself does not seem to drive toxicity.In fact, stable joint can release payload in a target-specific manner, and unstable joint is more likely to experience inaccurate payload release (such as due to nonspecific cutting), causing nonspecific systemic toxicity.

[0006] The payloads used in ADCs are highly potent, 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 (e.g., calicheamicin, pyrrolobenzodiazepines, indolebenzodiazepines, or duocarmycins).

[0007] Although ADCs appear to be promising therapeutic agents, some ADCs may be too toxic, which limits the therapeutic window of these compounds or hinders further clinical development. In addition, most ADCs currently approved or under clinical investigation are based on microtubule and DNA targeting agents as cited above. As such, new differentiated ADCs based on payloads with other mechanisms of action are needed in order to effectively treat tumors that are resistant or become resistant to microtubule and DNA targeting agents.

[0008] Effective ADCs showing high specificity, maximum efficiency and low toxicity therefore require appropriate combinations 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). WO2019081455 and Conilh et al. (2021, Pharmaceuticals, 14 (3), 247) further reported HER2-targeted antibody-drug conjugates based in particular on the topoisomerase I inhibitor payload exitecan and using a hydrophilic monodisperse polysarcosine (PSAR) drug-linker platform.

[0009] Cheng et al. (2018, DOI: 10 / 1158 / 1535-7163.MCT-17-1215) and WO2017151979 reported ADCs with fatuinumab conjugated to typically 3 to 4 eribulin (MORAb-202) molecules and their use in treating 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 study using the ADC somituximab for the treatment of ovarian cancer, which contained the humanized anti-FRα antibody metuximab conjugated to an average of 3 to 4 maytansine molecules as a payload. The mechanism of action of the maytansine payload of this ADC is microtubule inhibition.

[0011] Therefore, there remains a need for antibody-drug conjugates comprising anti-FRα antibodies that are potent, have high specificity, low toxicity (improved therapeutic index), and differentiated mechanisms of action for the payload.

[0012] As shown in the Examples, the present disclosure provides anti-FRα antibody-drug conjugates based on topoisomerase I inhibitors with excellent in vivo efficacy and low toxicity in solid tumor cancer models, particularly when compared to reference prior art ADCs targeting antibodies expressing FRα with other payloads and drug-linkers, such as somituximab. SUMMARY OF THE INVENTION

[0014] Accordingly, a first object of the present disclosure relates to an antibody-drug conjugate (ADC) of formula (I):

[0015] Ab-[LD] p (I)

[0016] in:

[0017] Ab is an anti-folate receptor α (FRα) antibody that specifically binds to SEQ ID NO: 12,

[0018] L is a cleavable linker moiety that is bonded to the anti-folate receptor alpha (FRα) antibody, preferably via a thiol residue,

[0019] D is a cytotoxic drug moiety bonded to L,

[0020] p is 1 to 8, preferably 6 to 8, and more preferably p is 8.

[0021] In a specific embodiment, the Ab is an antibody comprising a constant region of the human IgG1 isotype.

[0022] In a preferred embodiment, the Ab is an antibody comprising a mutant or chemically modified constant region of the human IgG1 isotype, wherein the mutant or chemically modified constant region does not confer ADCC activity or confers reduced ADCC activity to the antibody when compared to a corresponding antibody having a wild-type human IgG1 isotype constant region.

[0023] In other specific embodiments, the Ab is an antibody comprising a human IgG4 isotype constant region, or a mutant or chemically modified IgG4 constant region, wherein the mutant or chemically modified constant region does not confer ADCC activity or confers reduced ADCC activity to the antibody when compared to a corresponding antibody having a wild-type IgG4 isotype constant region.

[0024] In a preferred embodiment, the Ab is an anti-FRα antibody comprising any of:

[0025] (a) a variable heavy chain polypeptide comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, HCDR3 of SEQ ID NO: 3, and a variable light chain polypeptide comprising LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6; or,

[0026] (b) 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.

[0027] In a specific embodiment, the Ab comprises or consists essentially of the heavy chain of SEQ ID NO: 9 and the light chain of SEQ ID NO: 10;

[0028] In a specific embodiment, the Ab comprises or consists essentially of the heavy chain of SEQ ID NO:11 and the light chain of SEQ ID NO:10.

[0029] In a specific embodiment, D is an inhibitor of topoisomerase I, preferably selected from camptothecin analogs, and more preferably D is the drug moiety of exitecan of the following formula (II):

[0030]

[0031] In a specific embodiment, L is a cleavable linker moiety of the formula -AW-, wherein A is an optional stretcher unit linked to Ab, and W is a cleavable moiety linked to D. In a more specific embodiment, L is a lysosomal protease-sensitive linker, and W comprises a cleavable peptide moiety, for example, selected from valine-citrulline (Val-Cit), alanine-alanine-asparagine (Ala-Ala-Asn), valine-alanine (Val-Ala) and phenylalanine-lysine (Phe-Lys). In another specific embodiment, L is a protease-sensitive cleavable linker, and W comprises a sugar cleavable unit preferably selected from a β-glucuronide or β-galactoside moiety. In another specific embodiment, L is a glutathione-sensitive linker, and W comprises a disulfide moiety. In a specific embodiment, W has the following formula (III)

[0032]

[0033] in

[0034] Each R2 is independently selected from an electron withdrawing group and a C1-C4 alkyl group;

[0035] n is 0, 1, or 2;

[0036] T is a sugar cleavable unit or a polypeptide cleavable unit;

[0037] When T is a Sugar Cleavable Unit, Y is O, or when T is a Polypeptide Cleavable Unit, Y is NR3; and

[0038] R3 is selected from H, C1-C 24 alkyl, C2-C6 alkenyl; optionally substituted polyether, aryl having 6 to 10 ring atoms, C3-C8 cycloalkyl, heterocycloalkyl having 3 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms, and any combination thereof,

[0039] The alkyl and alkenyl groups 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-, -OC(O)NR"-, and triazole,

[0040] R" and R"' are independently selected from H and C1-C6 alkyl,

[0041] and pharmaceutically acceptable salts thereof

[0042] In a preferred embodiment, L corresponds to a linker -AW- of formula (IV)

[0043]

[0044] in

[0045] X1 is the connector unit;

[0046] Z is an optional spacer;

[0047] X2 is the connector unit;

[0048] K is an optional hydrophobic masking entity, preferably selected from polysarcosine and polyethylene glycol;

[0049] R1 is selected from H, C1-C 24 alkyl, C2-C6 alkenyl; optionally substituted polyether, aryl having 6 to 10 ring atoms, C3-C8 cycloalkyl, heterocycloalkyl having 3 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms, and any combination thereof,

[0050] The alkyl and alkenyl groups 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-, -OC(O)NR"-, and triazole;

[0051] R4 is selected from H, C1-C6 alkyl and C2-C6 alkenyl, wherein the alkyl and alkenyl are optionally interrupted by one or more heteroatoms or chemical groups selected from: -O-, -S-, -C(O)- and -NR"-;

[0052] R5 is selected from H, C1-C6 alkyl and C2-C6 alkenyl, wherein the alkyl and alkenyl are optionally interrupted by one or more heteroatoms or chemical groups selected from: -O-, -S-, -C(O)- and -NR"-.

[0053] In a more specific embodiment, X1 and X2 are independently selected from 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,

[0054] The alkylene and alkenylene groups 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-, -OC(O)NR"- and triazole,

[0055] and the alkylene, arylene, cycloalkylene, heterocycloalkylene, heteroarylene and alkenylene are optionally substituted with one or more substituents selected from the group consisting of 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)-OR′, -(CO)-NR″R″′, -NR″-(CO)-R′ and -NR″R″′;

[0056] R', R'" and R'" are independently selected from H and C1-C6 alkyl.

[0057] In a more specific embodiment, Z is independently selected from 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,

[0058] The alkylene and alkenylene groups 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-, -OC(O)NR"- and triazole,

[0059] and the alkylene, arylene, cycloalkylene, heterocycloalkylene, heteroarylene and alkenylene are optionally substituted with one or more substituents selected from the group consisting of 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)-OR′, -(CO)-NR″R″′, -NR″-(CO)-R′ and -NR″R″′;

[0060] R', R'" and R'" are independently selected from H and C1-C6 alkyl.

[0061] In a specific embodiment, K is polysarcosine, preferably of the following formula (V)

[0062]

[0063] wherein k is an integer between 2 and 50, preferably between 4 and 30,

[0064] And R6 corresponds to OH or NH2.

[0065] In specific embodiments, T is a sugar cleavable unit which is a glucuronide or a galactoside.

[0066] In other specific embodiments, T is a dipeptide preferably selected from Val-Cit, Val-Ala and Phe-Lys.

[0067] In a specific embodiment, L is covalently bonded to one or more thiol residues of the antibody, preferably, L corresponds to a linker of formula (V1):

[0068]

[0069] In specific embodiments, Ab comprises a full-length antibody or an antibody fragment containing an antigen-binding portion.

[0070] In a specific embodiment, the antibody-drug conjugate corresponds to the following formula (VII):

[0071]

[0072] Wherein the Ab is an anti-FRα antibody, such as facilitation mab, or a silent IgG1 variant thereof, typically comprising alanine substitutions in leucine 234 and leucine 235 of the IgG1 Fc constant region, and p is 4 to 8.

[0073] In a preferred embodiment, the antibody-drug conjugate corresponds to the following formula (VII):

[0074]

[0075] wherein Ab is an anti-FRα antibody, such as facilitation mab, or a silent IgG1 variant thereof, typically a mutant variant of human IgG1 comprising alanine substitutions in leucine 234 and leucine 235 in the IgG1 Fc constant region, also commonly referred to as a LALA mutation, and p is 8.

[0076] In a more preferred embodiment of the ADC of formula (I),

[0077] Ab is an anti-folate receptor alpha antibody or an antigen-binding fragment thereof, which comprises

[0078] (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

[0079] (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;

[0080] L is a cleavable linker of the formula -AW-, wherein A is an optional stretcher unit linked to Ab, and W is a cleavable moiety linked to D

[0081] D is ixotecan;

[0082] p is 1 to 8, for example between 4 and 8, preferably between 6 and 8, and for example p is between 7 and 8.

[0083] Another object of the present disclosure relates to the above ADC for use as a medicament, preferably for the treatment of a tumor, such as a solid tumor, more particularly selected from ovarian cancer, breast cancer, lung cancer or mesothelioma.

[0084] Another object of the present disclosure relates to the use of the above-mentioned ADC in the preparation of a medicament or pharmaceutical composition for treating a tumor, such as a solid tumor, more specifically selected from ovarian cancer, breast cancer, lung cancer or mesothelioma.

[0085] In a specific embodiment, the ADC may be preferably used to treat a cancer selected from ovarian cancer, triple-negative breast cancer, and non-small cell lung cancer.

[0086] The present disclosure further relates to pharmaceutical compositions comprising an antibody-drug conjugate as disclosed herein, optionally comprising other active ingredients, such as anti-cancer drugs or immunotherapeutic drugs such as immune checkpoint inhibitors, in combination with one or more pharmaceutically acceptable excipients, diluents or carriers.

[0087] The present disclosure also relates to a method for obtaining the ADC of the present disclosure, wherein the method comprises:

[0088] (a) culturing a host cell under conditions suitable for producing an anti-FRα antibody as defined herein,

[0089] (b) isolating the anti-FRα antibody,

[0090] (c) Synthesis of Exetecan bonded to a linker L of formula (VIII):

[0091]

[0092] (d) conjugating the anti-FRα antibody to a compound of formula (VIII);

[0093] Thus, the ADC of the present disclosure is obtained. BRIEF DESCRIPTION OF THE DRAWINGS

[0094] Figure 1 Preclinical rodent efficacy, pharmacokinetic and tolerability data of the conjugate according to Example 4 are presented.

[0095] Figure 2 FIG. 5 shows the flow cytometric evaluation of the extracellular expression of folate receptor α according to Example 5.

[0096] Figure 3 The in vitro cytotoxicity data of the compound exotecan mesylate according to Example 6 against several folate receptor α-positive cancer cell lines are shown.

[0097] Figure 4 The figure shows the in vitro ELISA binding experiment of the conjugate against recombinant human folate receptor α protein according to Example 7.

[0098] Figure 5 The figure shows the in vitro surface plasmon resonance (SPR) binding experiment of the conjugate against recombinant human folate receptor α protein according to Example 8.

[0099] Figure 6 The in vitro folate receptor α-positive cancer cell binding affinity of the conjugate according to Example 9 is shown.

[0100] Figure 7 The ex vivo human plasma stability of the conjugate according to Example 10 is shown.

[0101] Figure 8 The in vitro cytotoxicity assay of the conjugate according to Example 11 against the folate receptor α negative breast cancer cell line BT-474 is shown.

[0102] Fig. 9 Shows the in vivo efficacy evaluation of the conjugate according to Example 12 in the folate receptor alpha positive SW-620 xenograft cancer model.

[0103] Fig.10 Shown is the in vivo efficacy evaluation of the conjugate according to Example 12 in a second folate receptor alpha positive SW-620 xenograft cancer model.

[0104] Fig.11 Shows the in vivo efficacy evaluation of the conjugate according to Example 12 in the folate receptor alpha positive OV-90 xenograft cancer model.

[0105] Fig.12 Shown is the in vivo efficacy evaluation of the conjugate according to Example 12 in a second folate receptor alpha positive OV-90 xenograft cancer model.

[0106] Fig.13 Figure 1 shows the in vivo efficacy evaluation of the conjugate according to Example 12 in the folate receptor α-positive KB xenograft cancer model.

[0107] Fig.14 Shown is the in vivo efficacy evaluation of the conjugate according to Example 12 in the folate receptor alpha positive PA-1 xenograft cancer model.

[0108] Fig.15 Shows the in vivo efficacy evaluation of the conjugate according to Example 12 in a third folate receptor alpha positive OV-90 xenograft cancer model.

[0109] Fig.16 In vivo efficacy evaluation of the conjugate in the folate receptor alpha positive IGROV-1 xenograft cancer model and tumor re-implantation challenge according to Example 12 are shown.

[0110] Fig.17 Shown is the in vivo efficacy evaluation of the conjugate according to Example 12 in the folate receptor alpha negative BT-474 xenograft breast cancer model.

[0111] Fig.18 Figure 1 shows the in vivo SCID and CD-1 mouse tolerability evaluation of the conjugate at high doses according to Example 13.

[0112] Fig.19 Figure 14 shows the in vivo rat pharmacokinetic evaluation of total mAb, total ADC and free exetitecan subfractions of the conjugate according to Example 14.

[0113] Fig. 20 and 21 Shows in vivo mouse lung inflammation evaluation of the conjugate according to Example 15.

[0114] Fig. 22 Represents an in vivo cynomolgus monkey dose-ranging toxicology study according to Example 16.

[0115] Details

[0116] definition

[0117] In order that the present disclosure may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

[0118] Unless otherwise indicated, the term "FRα" or "folate receptor alpha" refers to human folate receptor alpha as defined in SEQ ID NO: 12. This sequence corresponds to the amino acid sequence of folate receptor alpha as encoded by the FOLR1 gene (Homo sapiens), which is also available in UniprotKB under entry P15328 (FOLR1_HUMAN).

[0119] As mentioned herein, the term "antibody" includes whole antibodies and any antigen binding fragment (i.e., "antigen binding portion") or a 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 consists 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 and VL regions can be further subdivided into hypervariable regions called complementary determining regions (CDRs), interspersed with more conservative regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody can mediate the binding of an immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. As used herein, the term "antigen-binding portion" (or simply "antigen portion") of an antibody refers to the full length or one or more fragments of an antibody that retains 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 a fragment of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include Fab fragments, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; F(ab)2 fragments; a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CH1 domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a UniBody consisting of a single arm with a modified IgG heavy chain, such as IgG4, at the hinge region, a domain antibody fragment (Ward et al., 1989 Nature 341:544-546) or a nanobody fragment consisting of a VH domain; and isolated complementarity determining regions (CDRs); or any fusion protein comprising such an antigen-binding portion. In addition, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be connected by synthetic linkers using recombinant methods, which allow them to be prepared as single-chain proteins in which the VL and VH regions are paired to form monovalent molecules (called single-chain Fv (scFv); see, e.g., Bird et al., 1988 Science 242: 423-426; and Huston et al., 1988 Proc. Natl. Acad. Sci. 85: 5879-5883). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody.These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0120] As used herein, "isolated antibodies" refer to antibodies that are substantially free of other antibodies with different antigenic specificities (e.g., an isolated antibody that specifically binds to FRα is substantially free of antibodies that specifically bind to antigens other than FRα). However, an isolated antibody that specifically binds to FRα may have cross-reactivity with other antigens, such as FRα molecules from other species. In addition, an isolated antibody may be substantially free of other cellular materials and / or chemicals.

[0121] The phrases "an antibody that recognizes an antigen" and "an antibody specific for an antigen" are used interchangeably herein with the term "an antibody that specifically binds to an antigen."

[0122] As used herein, the term "monoclonal antibody" or "monoclonal antibody composition" refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

[0123] As used herein, "isotype" refers to the antibody class provided by the heavy chain constant region genes (eg, IgM, IgE, IgG such as IgGl or IgG4).

[0124] As used herein, the term "K D "Expected" refers to the equilibrium dissociation constant, which is obtained from k off / k on The ratio (i.e. k off / k on ) and is expressed as a molar concentration (M). K D The value is related to the antibody concentration (the amount of antibody required for a particular experiment), and thus K D The lower the value (the lower the concentration), the higher the affinity of the antibody. D The K value can be determined using methods well established in the art. DPreferred methods for determining K values ​​can be found in Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988), Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, NY, 1992, 1993, and Muller, Meth Enzymol 1983, all of which are incorporated herein by reference. D The method is to use surface plasmon resonance, or by using a biosensor system such as (For details on affinity evaluation, see also Rich RL, Day YS, Morton TA, Myszka DG. High-resolution and high-throughput protocols forming drug / human serum albumin interactions using Anal Biochem. 2001).

[0125] 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, and as used herein, the term "k dis ″ or ″k d ″ or k off Expected refers to the off-rate of a particular antibody-antigen interaction.

[0126] As used herein, the term "affinity" refers to the strength of interaction between an antibody and an antigen at a single antigenic site. Within each antigenic site, the variable region of the antibody "arm" interacts with the antigen at numerous sites through weak non-covalent forces; the more interactions, the stronger the affinity.

[0127] As used herein, an antibody or protein that "specifically binds to an antigen", such as "specifically binds to FRα", is intended to refer to an antibody that detectably binds to an antigen, such as an epitope present on FRα in the present disclosure. It is generally intended to refer to an antibody or ADC that binds to human FRα, whose K D is 200 nM or less, 100 nM or less, 50 nM, 40 nM or less, or about 30 nM. Typically, K DBetween 10 -3 The ADCs of the present disclosure are typically between 1 pM and 200 nM, particularly between 0.1 pM and 100 nM, particularly between 0.1 pM and 50 nM, or between 1 pM and 50 nM, particularly between 1 pM and 30 nM, between 10 pM and 50 nM, between 0.1 nM and 200 nM, or between 0.1 nM and 100 nM, or between 1 nM and 50 nM, particularly between 1 nM and 30 nM. Typically, the ADCs of the present disclosure are specific for FRα and have a K as defined above. D .

[0128] As used herein, the term "host cell" refers to a prokaryotic cell or a eukaryotic cell. Eukaryotic cells, such as mammalian host cells, yeast, or filamentous fungi are preferred, and particularly mammalian cells, because they are more likely than prokaryotic cells to assemble and secrete correctly folded and immunologically active antibodies.

[0129] As used herein, the term "ADCC" or "antibody-dependent cellular cytotoxicity" activity refers to cell depleting activity. ADCC activity can be measured by commercially available ADCC assays, for example, as commercialized by Promega under Ref#G7015, the ADCCReporter Bioassay.

[0130] 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, reptiles. The term "subject" also encompasses the term "patient".

[0131] As used herein, the term "drug" or D in formula (I) of ADC also refers to a "payload", i.e., a portion conjugated to an antibody (or fragment). Drug D should not be construed as being limited to classical chemotherapeutic agents. For example, D may encompass a protein, peptide, or polypeptide having a 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 (e.g., kills) cells.

[0132] Unless otherwise specified, the present disclosure encompasses the compounds or drugs or cytotoxins as described herein and their tautomers, enantiomers, diastereomers, racemates or mixtures thereof, and their hydrates, esters, solvates or pharmaceutically acceptable salts.

[0133] Any formula given herein is also intended to represent unlabeled forms of the compounds as well as isotopically labeled forms, such as deuterium-labeled compounds or 14 C-labeled compounds.

[0134] The term "pharmaceutically acceptable salt" refers to salts that retain the biological effectiveness and properties of the compounds of the present disclosure and are generally not biologically or otherwise undesirable. In many cases, the compounds of the present disclosure are capable of forming acid and / or base salts through the presence of amino and / or carboxyl groups or groups similar thereto. Pharmaceutically acceptable acid addition salts can be formed from organic and / or inorganic acids. Pharmaceutically acceptable base addition salts can be formed from organic and / or inorganic bases. Such salts are well known to those skilled in the art.

[0135] The term connector unit refers to a component that connects different parts of a compound together, for example, a connector can connect an Ab to a spacer, or a spacer to an amide functional group -CO-NR1-. A connector is a scaffold that carries attachment sites for the components of an antibody-drug conjugate (i.e., Ab, spacer, hydrophobic masking entity, and / or amide functional group -CO-NR1-).

[0136] Based on their knowledge, the skilled person will be able to select an appropriate connector. A non-exhaustive list of connectors 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, connector units X1 and / or X2 are one or more natural or unnatural amino acids. In one embodiment, connector units X1 and / or X2 are selected from glutamic acid, lysine and glycine. Connector units X1 and X2 may be independently selected from 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, 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-, -OC(O)NR"- and triazole,

[0137] And the alkylene, arylene, cycloalkylene, heterocycloalkylene, heteroarylene and alkenylene are optionally substituted by one or more substituents selected from the following: 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)-OR′, -(CO)-NR″R″′, -NR″-(CO)-R′ and -NR″R″′; R′, R″ and R″′ are independently selected from H and C1-C6 alkyl.

[0138] Examples of connector units include optionally substituted polyethers, amino acids, benzyl groups, amines, ketones,

[0139] In particular, the connector unit may be divalent or trivalent. For example, when a hydrophobic masking entity K is present, X2 may be a trivalent connector unit.

[0140] The term "amino acid" refers to natural or unnatural amino acids. When X2 consists of one or more amino acids, the CO portion of the -CONR1- or -CONR1'- group can be considered as part of the X2 connector unit. A non-exhaustive list of amino acids includes lysine, glutamic acid, aspartic acid, serine, tyrosine, cysteine, selenocysteine, glycine and homoalanine.

[0141] A spacer is a bivalent arm that covalently binds two components of the antibody-drug-conjugate, eg, two connector units.

[0142] A non-exhaustive list of spacer units includes: alkylene, heteroalkylene (i.e., 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 unnatural amino acids, such as glycine, alanine, proline, valine, N-methylglycine; C3-C8 heterocycle; C3-C8 carbocycle; arylene, and any combination thereof. For example, the spacer is a divalent linear alkylene group, preferably (CH2)4.

[0143] For example, the spacer may be selected from -C1-C 10 Alkylene-, -C1-C 10 heteroalkylene-, -C3-C8 carbocycle-, -O-(C1C8 alkyl)-, -arylene-, -C1-C 10 Alkylene-arylene-, -arylene-C1-C 10 Alkylene-, -C1-C 10Alkylene-(C3-C8 carbocyclic ring)-, -(C3-C8 carbocyclic ring)-C1-C 10 Alkylene-, -C3-C8 heterocyclic-, -C1-C 10 Alkylene-(C3-C8 heterocycle)-, -(C3-C8 heterocycle)-C1-C 10 Alkylene-, -C1-C 10 Alkylene-C(=O)-, -C1-C 10 heteroalkylene-C(=O)-, -C3-C8 carbocyclic-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 carbocyclic ring)-C(=O)-, -(C3-C8 carbocyclic ring)-C1-C 10 Alkylene-C(=O)-, -C3-C8 heterocyclic-C(=O)-, -C1-C 10 Alkylene-(C3-C8 heterocycle)-C(=O)-, -(C3-C8 heterocycle)-C1-C 10 Alkylene-C(=O)-, -C1-C 10 Alkylene-NH-, -C1-C 10 Heteroalkylene-NH-, -C3-C8 carbocyclic-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 carbocyclic ring)-NH-, -(C3-C8 carbocyclic ring)-C1-C 10 Alkylene-NH-, -C3-C8 heterocyclic-NH-, -C1-C 10 Alkylene-(C3-C8 heterocycle)-NH-, -(C3-C8 heterocycle)-C1-C 10 Alkylene-NH-, -C1-C 10 Alkylene-S-, -C1-C 10 heteroalkylene-S-, -C3-C8 carbocyclic-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 carbocyclic ring)-S-, -(C3-C8 carbocyclic ring)-C1-C 10Alkylene-S-, -C3-C8 heterocyclic-S-, -C1-C 10 Alkylene-(C3-C8 heterocycle)-S-, -(C3-C8 heterocycle)-C1-C 10 Alkylene-S-, -C1-C 10 Alkylene-OC(=O)-, -C3-C8 carbocycle-OC(=O)-, -O-(C1-C8 alkyl)-OC(=O)-, -arylene-OC(=O)-, -C1-C 10 Alkylene-arylene-OC(=O)-, -arylene-C1-C 10 Alkylene-OC(=O)-, -C1-C 10 Alkylene-(C3-C8 carbocyclic ring)-OC(=O)-, -(C3-C8 carbocyclic ring)-C1-C 10 Alkylene-OC(=O)-, -C3-C8 heterocyclic-OC(=O)-, -C1-C 10 Alkylene-(C3-C8 heterocycle)-OC(=O)- and -(C3-C8 heterocycle)-C1-C 10 Alkylene-OC(═O)—.

[0144] Any of the above mentioned groups is optionally substituted by one or more substituents selected from the following: -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 C(=NR′)NR′2, wherein each X is independently a halogen: -F, -CI, -Br or -I; and each R′ is independently -H, -C 1- C 20 Alkyl, -C6-C 10 Aryl or -C3-C 10 Heterocyclic ring.

[0145] As used herein, the term "alkyl" refers to a monovalent saturated hydrocarbon chain (having a straight chain or a branched chain). For example, an alkyl group refers to a C1-C20 Preferably, the alkyl group is a "lower alkyl group", i.e., an alkyl group having 1, 2, 3, 4, 5 or 6 carbon atoms (straight or branched C1-C 20 alkyl group). This includes, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, and the like.

[0146] Alkylene used alone or as part of an alkylene glycol, for example, refers to a divalent saturated straight chain or branched alkyl group as defined herein.

[0147] Alkenyl and alkynyl refer to at least partially unsaturated, straight-chain or branched hydrocarbon groups having 2 to 20, preferably 2 to 12, more preferably 2 to 6 and in particular 2 to 4 carbon atoms. Alkenyl groups contain at least one C=C double bond; alkynyl groups contain at least one C≡C triple bond.

[0148] As used herein, the term "C3-C8 cycloalkyl" or "carbocycle" refers to a saturated or unsaturated cyclic group having 3 to 8, preferably 3 to 6 carbon atoms. The cycloalkyl group may have a single ring or multiple rings fused together. The cycloalkyl group may also include a spirocycle. Suitable cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

[0149] As used herein, the term "C3-C8 cycloalkylene" or "carbocycle" refers to a divalent cycloalkyl group as defined herein.

[0150] As used herein, the term "halogen" refers to a fluoro (-F), chloro (-Cl), bromo (-Br), or iodo (-I) group.

[0151] As used herein, the term "C1-C6 haloalkyl" refers to a C1-C6 alkyl group as defined herein, substituted by one or more halogen groups as defined herein. Suitable C1-C6 haloalkyl groups include trifluoromethyl and dichloromethyl.

[0152] 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 one to three heteroatoms selected from O, N, Si and S, and wherein the nitrogen and sulfur atoms may be optionally oxidized (e.g., sulfoxide or sulfone), and the nitrogen heteroatom may be optionally quaternized. The heteroatoms O, N and S may be placed at any interior position of the heteroalkyl group or at the position where the alkyl group is attached to the remainder of the molecule.

[0153] Heteroalkylene refers to a divalent heteroalkyl group as defined above. For heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini.

[0154] As used herein, the term "C1-C6 alkoxy" refers to an -O-alkyl group, wherein the alkyl group is a C1-C6 alkyl group as defined herein. Suitable C1-C6 alkoxy groups include methoxy, ethoxy, propoxy.

[0155] 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 groups include trifluoromethoxy.

[0156] As used herein, the term "aryl having 6 to 10 ring atoms" refers to a polyunsaturated, aromatic hydrocarbon group having a single ring or multiple aromatic rings fused together containing 6 to 10 ring atoms, wherein at least one ring is aromatic. The aromatic ring may optionally include one to two additional rings (cycloalkyl, heterocyclyl or heteroaryl as defined herein) fused thereto. Suitable aryl groups include benzene rings, naphthalene rings and benzene rings fused to heterocyclyls, such as benzopyranyl, benzodioxolyl, benzodioxanyl and the like.

[0157] Arylene refers to a divalent aromatic group as defined above.

[0158] As used herein, the term "heteroaryl having 5 to 10 ring atoms" refers to a polyunsaturated aromatic ring system containing 5 to 10 atoms, having a single ring or multiple aromatic rings fused together or covalently linked, wherein at least one ring is aromatic and at least one ring atom is 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 ring, a cycloalkyl ring, or a heterocyclyl ring. Non-limiting examples of such heteroaryl groups include furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, oxadiazolyl, thiadiazolyl, tetrazolyl, oxatriazolyl, thiatriazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, oxazinyl, dioxinyl, thiazinyl, triazinyl, indolyl, isoindolyl, benzofuranyl, isobenzofuranyl, benzothienyl, isobenzothienyl, indazolyl, benzimidazolyl, benzoxazolyl, purinyl, benzothiadiazolyl, quinolyl, isoquinolyl, cinnolinyl, quinazolinyl, and quinoxalinyl.

[0159] 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, wherein at least one ring atom is 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 may include fused or bridged rings and spirocycles. Examples of heterocycles include, but are not limited to, tetrahydropyridyl, piperidinyl, morpholinyl, tetrahydrofuranyl, tetrahydrothienyl, piperazinyl, 1-azepanyl, imidazolinyl, 1,4-dioxanyl and the like.

[0160] As used herein, the term "heterocycle" or "heterocycloalkylene" refers to a divalent heterocycle as defined herein.

[0161] In addition, 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 optionally substituted groups having one or more substituents selected from the group consisting of -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, wherein each X is independently a halogen: -F, -CI, -Br or -I; and each R′ is independently -H, -C 1- C 20 Alkyl, -C6-C 10 Aryl or -C3-C 10 Heterocyclic ring.

[0162] As used herein, the term "polyether" refers to a polymer containing ether linkages. The number of ether moieties in the polyether may be between 2 and 100, preferably between 2 and 25, especially between 2 and 10. Examples of polyethers include polyethylene glycol.

[0163] Electron withdrawing groups refer to atoms or groups that pull electron density from adjacent atoms toward themselves, usually through resonance or inductive effects. Electron withdrawing groups include halogens, haloalkyls (such as -CF3), -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 YT substituent of the benzene ring.

[0164] As used herein, the term "protecting group" refers to a chemical substituent that can be selectively removed by readily available reagents, which do not attack the regenerated functional groups or other functional groups in the molecule. Suitable protecting groups are known in the art and will continue to be 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, a protecting group for protecting an amino group as described by Wutz et al. (page 696-927). Representative examples of amino protecting groups include, but are not limited to, tert-butyloxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl (Fmoc), acetyl (Ac), carboxybenzyl group (Cbz), benzyl group (Bn), allyl, trifluoroacetyl, allyloxycarbonyl (Alloc) group and 2,2,2-trichloroethoxycarbonyl (Troc).

[0165] Hydrophobic masking entities refer to groups that can reduce 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 over a wide range. 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 a polysarcosine comprising 6 to 24 sarcosine moieties, preferably comprising 10 to 12 sarcosine moieties.

[0166] As used herein, the term "bonded" refers to bonding. The bonding is also represented by the dash "-" in formula (I). The bonding can be a covalent bond, or a non-covalent interaction, such as by electrostatic forces. Preferably, the bond is a covalent bond. As used herein, the "wavy line" on the formula represents the attachment site between each part (Ab, L, Z, X and D) of the ADC of the present disclosure.

[0167] As used herein, the terms "treat," "treating," or "treatment" refer to one or more of: (1) inhibiting a disease; e.g., inhibiting a disease, condition, or disorder in an individual who is experiencing or exhibiting pathology or symptomology of the disease, condition, or disorder (i.e., preventing further development of the pathology and / or symptomology); and (2) ameliorating a disease; e.g., ameliorating a disease, condition, or disorder in an individual who is experiencing or exhibiting pathology or symptomology of the disease, condition, or disorder (i.e., reversing the pathology and / or symptomology), such as reducing the severity of the disease or reducing or alleviating one or more symptoms of the disease. In particular, with respect to the treatment of a tumor, the term "treat" may refer to the inhibition of tumor growth or the reduction of tumor size.

[0168] As used herein, a "therapeutically effective amount" or "effective amount" of an ADC is an amount sufficient to perform a particular stated purpose, such as to produce a therapeutic effect after administration, such as inhibiting or reducing tumor growth rate or tumor volume, alleviating cancer symptoms, or some indicator of therapeutic efficacy. 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 down or stop) tumor metastasis, inhibit (e.g., slow down or stop) tumor growth, and / or alleviate one or more symptoms.

[0169] As used herein, the percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., the total number of % identity=number of identical positions / positions x 100), taking into account the number of spaces and the length of each space, which spaces need to be introduced for the best comparison of the two sequences. A mathematical algorithm can be used to complete the comparison of the sequence and the determination of the percent identity between the two sequences, as described below.

[0170] The percent identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (NEEDLEMAN AND WUNSCHI).

[0171] Percent identity between two nucleotide sequences or amino acid sequences can also be determined using, for example, algorithms such as EMBOSS Needle (paired comparisons; Available at www.ebi.ac.uk). For example, EMBOSS Needle can be used together with BLOSUM62 matrix, "gap opening penalty" 10, "gap extension penalty" 0.5, false "end gap penalty", "end gap opening penalty" 10 and "end gap extension penalty" 0.5. Generally speaking, "percent identity" is a function that the number of matching positions is divided by the number of comparison positions and multiplied by 100. For example, if 6 of the 10 sequence positions after the comparison are identical between two comparison sequences, then the identity is 60%. % identity is determined conventionally on the entire length of the query sequence analyzed. Two molecules with identical primary amino acid sequence or nucleotide sequence are identical, regardless of any chemical and / or biological modification.

[0172] Antibodies Ab used to prepare ADCs of the present disclosure

[0173] The antibody Ab used to prepare the ADC of the present disclosure is an anti-folate receptor alpha (FRα) antibody, which specifically binds to SEQ ID NO:12.

[0174] Preferably, such antibodies include the following antibodies isolated and structurally characterized by their variable heavy and light chain amino acid sequences and human constant isotypes as described in Table 1 below:

[0175]

[0176] Table 1: Variable heavy and light chain amino acid sequences

[0177] IgG1 LALA corresponds to a mutant IgG1 Fc region comprising amino acid substitutions from leucine to alanine at residues 234 and 235, which is also disclosed by Hezareh et al. J. Virol 2001 Dec;75(24):12161-8.

[0178] The full length light and heavy chains of mAb1 and the corresponding coding sequences are shown in Table 2 below.

[0179]

[0180] Table 2 : Full-length heavy and light chain amino acid and DNA coding sequences

[0181] Examples of 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), VL CDR3 (also called HCDR3) of the mAb1 antibody are shown in Table 3.

[0182] In Table 3, the CDR regions of some antibodies of the present disclosure are delineated using the Kabat system. For ease of reading, the CDR regions are hereinafter referred to as HCDR1, HCDR2, HCDR3, L, CDR1, L, CDR2, L, CDR3, respectively.

[0183]

[0184] Table 3 : CDR regions of mAb 1 according to Kabat (also disclosed in WO2017151979)

[0185] Tables 4 and 5 below provide useful amino acid sequences and nucleotide sequences of antibodies relative to ADCs.

[0186] Table 4: Brief description of amino acid sequences and nucleotide sequences useful for practicing the present invention

[0187]

[0188] *aa is the amino acid sequence

[0189] nt is the nucleotide sequence

[0190] Table 5: Useful amino acid sequences and nucleotide sequences for practicing the present invention

[0191]

[0192]

[0193]

[0194]

[0195] In one embodiment, Ab is an isolated recombinant antibody having:

[0196] (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,

[0197] (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;

[0198] The antibody specifically binds to the folate receptor α of SEQ ID NO:12.

[0199] In a specific embodiment, Ab is a recombinant antibody comprising any of:

[0200] (a) a variable heavy chain polypeptide comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, HCDR3 of SEQ ID NO: 3, and a variable light chain polypeptide comprising LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6; or,

[0201] (b) 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.

[0202] In a specific embodiment, Ab is a recombinant antibody comprising or consisting essentially of the heavy chain of SEQ ID NO:9 and the light chain of SEQ ID NO:10.

[0203] In a specific embodiment, Ab is a recombinant antibody comprising or consisting essentially of the heavy chain of SEQ ID NO:11 and the light chain of SEQ ID NO:10.

[0204] In a specific embodiment, Ab is a recombinant antibody comprising or consisting essentially of the heavy chain of SEQ ID NO:16 and the light chain of SEQ ID NO:17.

[0205] In a specific embodiment, the Ab is an anti-FRα antibody having one or more of the following properties:

[0206] (i) by surface plasmon resonance, e.g. The assay measures the K of 100 nM or lower. D , preferably a K of 50 nM or lower D Binds to folate receptor α (FRα);

[0207] (ii) it is an internalizing antibody, in particular, it internalizes in tumor cells expressing FRα;

[0208] (iii) It allows the conjugation of 4 to 8 payloads / antibody, in particular without stability issues or aggregation.

[0209] In certain embodiments that may be combined with the preceding embodiments, Ab is an internalizing antibody fragment of a recombinant antibody as defined above.

[0210] As used herein, "internalization" with reference to an antibody refers to an antibody that, after specifically binding to a cell, is able to cross the lipid bilayer outer membrane of the cell into an internal compartment (ie, "internalized"), preferably into a degradation compartment in the cell.

[0211] 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 "diabodies" refers to small antibody fragments with two antigen binding sites, the fragments comprising a heavy chain variable domain (VH) connected 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 two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and produce two antigen binding sites. Single domain antibodies are antibody fragments that contain all or part of the heavy chain variable domain of an antibody or all or part of the light chain variable domain. In certain embodiments, single domain antibodies are human single domain antibodies (Domantis, Inc., Waltham, MA; see, e.g., U.S. Pat. No. 6,248,516 B1). Antibody fragments can be prepared by various techniques, including but not limited to proteolytic digestion of intact antibodies and production by recombinant host cells as described herein.

[0212] In certain embodiments, Ab is a humanized antibody. Typically, non-human antibodies are humanized to reduce immunogenicity to humans while having at least the same affinity (or higher affinity) as the parent non-human antibody. In a preferred embodiment, the antibody of the present disclosure is a humanized antibody. Generally, a humanized antibody comprises one or more variable domains, wherein CDR (or its various parts) are derived from non-human antibodies, such as mouse anti-FRα internalizing antibodies, and framework regions (or its various parts) are derived from human antibody sequences. Humanized antibodies optionally also include at least a portion of a human constant region. In some embodiments, some framework residues in a humanized antibody are replaced by corresponding residues from a non-human antibody (e.g., CDR residues are derived from anti-FRα mouse antibodies), for example, to restore or improve antibody specificity or affinity. In some specific embodiments, some CDR residues in a humanized antibody are also replaced, for example, to restore or improve antibody specificity or affinity. Humanized antibodies and methods for their preparation are reviewed, e.g., in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and are further described, e.g., 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 "surface remodeling"); Dall'Acqua et al., Methods 36:25-34 (2005) (describing specificity determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "surface remodeling"); Dall'Acqua et al., Methods 36:25-34 (2005) (describing specificity determining region (SDR) grafting); 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 the "guided selection" method of FR shuffling).

[0213] Preferably, the Ab is a humanized or human silent antibody, preferably a humanized silent IgG1 antibody.

[0214] As used herein, the term "silent" antibody refers to an antibody that shows no ADCC activity or shows low ADCC activity as measured in an ADCC activity assay.

[0215] In one embodiment, the term "no or low ADCC activity" means that the silent antibody exhibits an ADCC activity at least less than 10%, such as less than 50% of the ADCC activity observed with a corresponding antibody having a wild-type corresponding IgG isotype.

[0216] Silent effector functions can be obtained by mutations in the Fc constant part of antibodies and have been 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 in Saunders 2019 (Front. Immunol., 07 June 2019, doi: 10.3389 / fimmu.2019.01296 ) are reviewed in. Examples of silent IgG1 antibodies include the so-called LALA mutation, which comprises L234A and L235A mutations in the IgG1 Fc amino acid sequence, or Ser228Pro paired with Leu235Glu. Pro331Ser, optionally in combination with Leu234Glu and Leu235Phe (LALA-PG), can also be used to generate silent IgG1 antibodies. Another example of a silent IgG1 antibody comprises the N297A mutation, which results in an aglycosylated or non-glycosylated antibody. An example of a silent IgG4 antibody comprises Ser228Pro.

[0217] In specific embodiments, Ab is fatutumomab, or other anti-FRα antibodies as disclosed in WO2005080431 or WO2017151979.

[0218] In other specific embodiments, the Ab is metuximab or a silent LALA mutant form of metuximab.

[0219] In a preferred embodiment, the Ab is a silent LALA mutant form of fatutumomab, or other anti-FRα antibodies as disclosed in WO2005080431 or WO2017151979.

[0220] Antibodies with mutant amino acid sequences can be obtained by mutagenesis (e.g., site-directed mutagenesis or PCR-mediated mutagenesis) of the encoding nucleic acid molecule, followed by testing the encoded altered antibody for retained function (i.e., the function set forth above) using the functional assays described herein.

[0221] Antibodies with conservative modifications

[0222] In certain embodiments, the Ab has a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 sequences, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 sequences, wherein one or all of these CDR sequences have a specified amino acid sequence based on the mAb 1 (silent LALA mutant form of facilitation) antibody described herein, or a functional variant of the antibody having an analogous CDR sequence that differs from the CDR sequence of facilitation by 1, 2, or 3 conservative modifications of amino acids, and wherein the antibody or protein retains the desired functional properties of the mAb1 antibody, particularly when used as an ADC.

[0223] In certain embodiments, the Ab has a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 sequences, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 sequences, wherein one or more of these CDR sequences has a specified amino acid sequence based on the mituximab (or a silent LALA mutant form of mituximab) antibody described herein, or a functional variant of the antibody having a similar CDR sequence that differs from the CDR sequence of facilitation by 1, 2, or 3 conservative amino acid modifications, and wherein the antibody or protein retains the desired functional properties of the mAb1 antibody, particularly when used as an ADC.

[0224] Desirable functional properties of anti-FRα antibodies include, but are not limited to, the following:

[0225] (i) It binds to folate receptor alpha (FRα) with an EC50 of less than 5 nM, such as about 0.5 nM, as determined by Elisa assay (as described in the Examples).

[0226] (ii) such as by surface plasmon resonance e.g. assay; for example as determined using the SPR Biacore affinity assay described in the Examples, it binds to the folate receptor alpha (FRα) with a KD of 100 nM or less, preferably a KD of 50 nM or less,

[0227] (iii) it is an internalizing antibody, in particular, it internalizes in tumor cells expressing FRα;

[0228] (iv) it allows the conjugation of 4 to 8 payloads / antibody, in particular without stability issues or aggregation, wherein the drug-antibody ratio is determined, for example, by RPLC-MS as described in the Examples;

[0229] (v) an ADC having such variant anti-FRα antibody provides similar or lower in vitro efficacy in a FRα-negative cell line (e.g., BT-474 breast cancer cell line) than a corresponding control ADC having mAb1 as the anti-FRα antibody, e.g., using an in vitro efficacy assay in a FRα-negative cell line as used in the Examples; and / or,

[0230] (vi) ADCs with such variant anti-FRα antibodies provided similar or higher in vivo efficacy than the corresponding control ADC with mAbl as the anti-FRa antibody in a tumor-bearing mouse xenograft model, for example using one of the xenograft models as used in the Examples.

[0231] As used herein, the term "conservative sequence modification" is intended to refer to amino acid substitutions in which amino acid residues are replaced by amino acid residues with similar side chains. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, one or more amino acid residues in the CDR region of an antibody of the present disclosure can be replaced by other amino acid residues from the same side chain family, and the altered antibody can be tested for the retained function using functional assays described herein.

[0232] Modifications can be introduced into the antibodies of the disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.

[0233] In a specific embodiment, the Ab is an anti-FRα antibody comprising 6 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 in SEQ ID NOs: 7 and 8, respectively, wherein the anti-FRα antibody has the following properties:

[0234] (i) It binds to folate receptor alpha (FRα) with an EC50 of less than 5 nM, such as about 0.5 nM, as determined by Elisa assay (as described in the Examples).

[0235] (ii) such as by surface plasmon resonance e.g. The assay measures the K of 100 nM or lower. D , preferably a K of 50 nM or lower D Binds to folate receptor a (FRa);

[0236] (iii) it is an internalizing antibody, in particular, it internalizes in tumor cells expressing FRα;

[0237] (iv) it allows the conjugation of 4 to 8 payloads / antibody, in particular without stability issues or aggregation, wherein the drug-antibody ratio is determined, for example, by RPLC-MS as described in the Examples;

[0238] (v) an ADC having such variant anti-FRα antibody provides similar or lower in vitro efficacy in a FRα-negative cell line (e.g., BT-474 breast cancer cell line) than a corresponding control ADC having mAb1 as the anti-FRα antibody, e.g., using an in vitro efficacy assay in a FRα-negative cell line as used in the Examples; and / or,

[0239] (vi) ADCs with such variant anti-FRα antibodies provided similar or higher in vivo efficacy than the corresponding control ADC with mAbl as the anti-FRa antibody in a tumor-bearing mouse xenograft model, for example using one of the xenograft models as used in the Examples.

[0240] In a specific embodiment, Ab is an anti-FRα antibody comprising 6 CDRs that are 100% identical to the corresponding CDRs of metuximab, 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 of metuximab, wherein the anti-FRα antibody has the following properties:

[0241] (i) It binds to folate receptor alpha (FRα) with an EC50 of less than 5 nM, such as about 0.5 nM, as determined by Elisa assay (as described in the Examples).

[0242] (ii) such as by surface plasmon resonance e.g. The assay measures the K of 100 nM or lower. D , preferably a K of 50 nM or lower D Binds to folate receptor a (FRa);

[0243] (iii) it is an internalizing antibody, in particular, it internalizes in tumor cells expressing FRα;

[0244] (iv) it allows the conjugation of 4 to 8 payloads / antibody, in particular without stability issues or aggregation, wherein the drug-antibody ratio is determined, for example, by RPLC-MS as described in the Examples;

[0245] (v) an ADC having such variant anti-FRα antibody provides similar or lower in vitro efficacy in a FRα-negative cell line (e.g., BT-474 breast cancer cell line) than a corresponding control ADC having mAb1 as the anti-FRα antibody, e.g., using an in vitro efficacy assay in a FRα-negative cell line as used in the Examples; and / or,

[0246] (vi) ADCs with such variant anti-FRα antibodies provided similar or higher in vivo efficacy than the corresponding control ADC with mAbl as the anti-FRα antibody in a tumor-bearing mouse xenograft model, for example using one of the xenograft models as used in the Examples.

[0247] Frame or Fc modification

[0248] For use in the ADC of the present disclosure, Ab may also include a modified form of the antibody disclosed in the previous section, and includes those in which the framework residues in VH and / or VL have been modified, for example, to improve the properties of the antibody. Typically, such framework modifications are performed to reduce the immunogenicity of the antibody. For example, one method is to "revert" one or more framework residues to the corresponding germline sequence. More specifically, an antibody that has undergone somatic mutations may contain framework residues different from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequence with the germline sequence from which the antibody is derived. In order to restore the framework region sequence to its germline configuration, the somatic mutation can be "reverted" to the germline sequence by, for example, site-directed mutagenesis or PCR-mediated mutagenesis. Such "reverted" antibodies are also expected to be covered by the present invention.

[0249] 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, thereby reducing the potential immunogenicity of the antibody.

[0250] In addition to or in place of modifications made within the framework or CDR regions, antibodies can be engineered to include modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity.

[0251] In addition, Ab is an antibody that can be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or modified to alter its glycosylation, again to alter one or more functional properties of the antibody. Each of these embodiments is described in further detail below.

[0252] 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, including native sequence Fc regions and variant Fc regions. The human IgG heavy chain Fc region is generally defined as comprising amino acid residues from position C226 or P230 of an IgG antibody to the carboxyl terminus. The residue numbering in the Fc region is the residue numbering of the EU index of Kabat. The C-terminal lysine (residue K447) in the Fc region can be removed, for example, during the production or purification of the antibody. Accordingly, the antibody compositions of the present disclosure can include antibody populations in which all K447 residues are removed, antibody populations in which the K447 residue is not removed, and antibody populations of a mixture of antibodies with and without the K447 residue.

[0253] In a specific embodiment, the hinge region of CH1 is modified so that the number of cysteine ​​residues in the hinge region is changed, e.g., increased or decreased. This method is further described in U.S. Pat. No. 5,677,425 by Bodmer et al. The number of cysteine ​​residues in the hinge region of CH1 is changed, e.g., to facilitate assembly of light and heavy chains or to increase or decrease the stability of the antibody.

[0254] In other embodiments, the Fc region is modified by modifying one or more amino acids to reduce the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or reduce the affinity of the antibody for Fcγ receptors. Such antibodies with reduced effector function and particularly reduced ADCC include silent antibodies.

[0255] In certain embodiments, an Fc domain of the IgG1 isotype is used. In some specific embodiments, a mutant variant of the IgG1 Fc fragment is used, such as a silent IgG1 Fc, which reduces or eliminates the ability of the ADC to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or bind to Fcγ receptors. A preferred example of an IgG1 isotype silent mutant is an IgG1 in which the leucine at amino acid positions 234 and 235 is replaced by alanine (the so-called LALA mutation), as described by Hezareh et al. in J. Virol 2001 December; 75(24): 12161-8. Another example is an IgG1 isotype silent mutant with a LALA-PG triple mutation, in which, in addition to the LALA mutation, the proline at position 329 is also replaced by glycine.

[0256] In certain embodiments, the Fc domain is a silent Fc mutant that prevents glycosylation at position 297 of the Fc domain. For example, the Fc domain contains an amino acid substitution of asparagine at position 297. An example of such an amino acid substitution is replacement of N297 with glycine or alanine.

[0257] In another embodiment, the glycosylation of the antibody is modified. For example, an aglycosylated antibody (i.e., the antibody lacks glycosylation) can be prepared. Glycosylation can be changed, for example, to increase the affinity of the antibody for an antigen. Such carbohydrate modifications can be achieved by, for example, changing one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made, which lead to the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at the site. Such aglycosylation can increase the affinity of the antibody for an antigen. Such methods are described in further detail in U.S. Patent Nos. 5,714,350 and 6,350,861 by Co et al.

[0258] Another modification of the antibodies herein contemplated by the present disclosure is pegylation or hesylation or related techniques. Antibodies can be pegylated, for example, to increase the biological (e.g., serum) half-life of the antibody. To pegylate an antibody, the antibody or its fragment is typically reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or antibody fragment. Pegylation can be performed with a reactive PEG molecule (or a similar reactive water-soluble polymer) by an acylation reaction or an alkylation reaction. 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(C1-C 10 ) alkoxy or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody to be pegylated is an aglycosylated antibody. Methods for pegylation of proteins are known in the art and can be applied to the antibodies of the present disclosure. See, for example, EP 0 154 316 by Nishimura et al. and EP 0 401 384 by Ishikawa et al.

[0259] Another modification of the antibodies contemplated by the present disclosure is the conjugation or protein fusion of at least the antigen binding region of the antibodies of the present disclosure with serum proteins such as human serum albumin or fragments thereof to increase the half-life of the resulting molecule. Such methods are described, for example, in Ballance et al. EP 0 322 094.

[0260] Generation of Antibodies for Preparation of ADCs of the Disclosure

[0261] The antibodies of the present disclosure can be obtained using conventional techniques known to those skilled in the art. For more information on nucleic acids encoding the antibodies of the present disclosure and the generation of transfectomas producing these antibodies, those skilled in the art can also refer to International Application No. WO2005080431.

[0262] For example, in order to express antibody or its antibody fragment, DNA encoding part or full-length light chain and heavy chain can be obtained by standard molecular biology or biochemical techniques (for example, DNA chemical synthesis, PCR amplification or cDNA cloning using hybridomas expressing antibodies of interest), and DNA can be inserted into expression vectors so that genes are operably connected to transcription and translation control sequences. In this context, the term "operably connected" is expected to mean that antibody genes are connected to vectors so that transcription and translation control sequences in the vectors play the expected functions of transcription and translation of the antibody genes. Expression vectors and expression control sequences are selected to be compatible with the expression host cells used. Antibody light chain genes and antibody heavy chain genes can be inserted into separate vectors, or more generally, two genes are inserted into the same expression vector. Antibody genes are inserted into expression vectors by standard methods (for example, connection of complementary restriction sites on antibody gene fragments and vectors, or if there is no restriction site, then flat ends are connected). The light chain and heavy chain variable regions of the antibodies described herein can be used to produce full-length antibody genes of any antibody isotype by inserting them into an expression vector encoding the heavy chain constant region and the light chain constant region of the desired isotype, so that the VH segment is operably connected to the CH segment in the vector, and the VL segment is operably connected to the CL segment in the vector. In addition or alternatively, the recombinant expression vector can encode a signal peptide that promotes secretion of the antibody chain from a host cell. The antibody chain gene can be cloned into a vector so that the signal peptide is connected to the amino terminus of the antibody chain gene in a frame. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin).

[0263] In addition to the antibody chain genes, the recombinant expression vectors disclosed herein also carry regulatory sequences that control the expression of the antibody chain genes in host cells. 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 genes. It will be appreciated by those skilled in the art that the design of the expression vector, including the selection of regulatory sequences, may depend on such factors as the selection of the host cell to be transformed, the expression level of the desired protein, and the like. Regulatory sequences for mammalian host cell expression include viral elements that direct high-level protein expression in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV), simian virus 40 (SV40), adenovirus (e.g., adenovirus major late promoter (AdMLP)) and polyoma virus. Alternatively, non-viral regulatory sequences such as ubiquitin promoters or P-globin promoters may be used. Further, regulatory elements composed of sequences from different sources, such as the SRa promoter system, contain sequences from the SV40 early promoter and the long terminal repeats of human T-cell leukemia virus type 1.

[0264] In addition to antibody chain genes and regulatory sequences, the recombinant expression vector of the present disclosure can also carry other sequences, such as sequences (e.g., replication origins) and selectable marker genes that regulate the replication of the vector in the host cell. The selectable marker gene promotes the selection of the host cell into which the vector has been introduced (see, for example, U.S. Patent Nos. 4,399,216, 4,634,665 and 5,179,017 by Axel et al.). For example, the selectable marker gene generally gives the host cell into which the vector has been introduced resistance to drugs such as G418, hygromycin or methotrexate. The selectable marker gene includes dihydrofolate reductase (DHFR) gene (used in dhfr-host cells together with methotrexate selection / amplification) and neo gene (selected for G418).

[0265] In order to express light chain and heavy chain, the expression vector encoding heavy chain and light chain is transfected into the host cell by standard techniques. The various forms of the term "transfection" are expected to cover the widely varied techniques commonly used for introducing exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, etc. It is theoretically possible to express the antibody of the present disclosure in prokaryotic 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, and particularly mammalian cells, are more likely to assemble and secrete correctly folded and immunocompetent antibodies than prokaryotic cells.

[0266] Nucleotide sequences encoding the heavy and light chains of preferred antibodies for preparing ADCs of the present disclosure have been described in Tables 3 and 4 (see in particular SEQ ID NOs 13-15).

[0267] 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 (as described in Kaufman and Sharp, 1982), CHO K1 dhfr+ cell lines, NSO myeloma cells, COS cells, and SP2 cells. When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period of time sufficient to express the antibody in the host cell and, optionally, secrete the antibody into the culture medium in which the host cell is grown. The antibody can be recovered and purified, for example, from the culture medium after its secretion using standard protein purification methods.

[0268] Connector L

[0269] 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.

[0270] The ADC of the present disclosure contains a cleavable linker moiety L bonded to the Ab (such as the anti-FRα antibody disclosed in the previous section) on one side and to the drug D on the other side.

[0271] In a specific embodiment, linker L is covalently bonded to one or more thiol residues of antibody Ab, such as natural or artificial cysteine ​​residues from the antibody sequence.

[0272] As used herein, the term "cleavable" refers to a linker that can be cleaved under specific environmental conditions (eg, redox potential or pH) or by specific lysosomal enzymes in response to the intracellular environment (eg, after the ADC is internalized in the cell).

[0273] In specific embodiments, L is a cleavable linker moiety of the formula -AW-, wherein A is an optional Stretcher linked to Ab, and W is a cleavable moiety linked to D.

[0274] In a specific embodiment, the optional stretcher unit A can be selected from one or more amino acids, one or more N-substituted amino acids, optionally substituted polyethers, C1-C 12alkylene, 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,

[0275] The alkylene and alkenylene groups 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-, -OC(O)NR"- and triazole,

[0276] and the alkylene, arylene, cycloalkylene, heterocycloalkylene, heteroarylene and alkenylene are optionally substituted with one or more substituents selected from the group consisting of 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)-OR′, -(CO)-NR″R″′, -NR″-(CO)-R′ and -NR″R″′;

[0277] R', R" and R'" are independently selected from H and C1-C6 alkyl.

[0278] 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, b-glucuronide linkers, or glutathione-sensitive disulfide linkers.

[0279] In a specific embodiment where L is a lysosomal protease-sensitive linker, W comprises a cleavable peptide portion which may be selected from valine-citrulline (Val-Cit), alanine-alanine-asparagine (Ala-Ala-Asn), valine-alanine (Val-Ala) and phenylalanine-lysine (Phe-Lys). Preferably, W comprises a valine-alanine peptide portion.

[0280] In other specific embodiments wherein L is a protease-sensitive linker, w comprises a sugar cleavable unit preferably selected from a b-glucuronide or b-galactoside moiety.

[0281] In other specific embodiments wherein L is a glutathione-sensitive linker, w comprises a disulfide bond moiety.

[0282] In a preferred embodiment, L is a cleavable linker moiety of the formula -AW-, wherein w has the following formula (III)

[0283]

[0284] in

[0285] Each R2 is independently selected from an electron withdrawing group and a C1-C4 alkyl group;

[0286] n is 0, 1, or 2;

[0287] T is a sugar cleavable unit or a polypeptide cleavable unit;

[0288] When T is a Sugar Cleavable Unit, Y is O, or when T is a Polypeptide Cleavable Unit, Y is NR3; and

[0289] R3 is selected from H, C1-C 24 alkyl, C2-C6 alkenyl; optionally substituted polyether, aryl having 6 to 10 ring atoms, C3-C8 cycloalkyl, heterocycloalkyl having 3 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms, and any combination thereof,

[0290] The alkyl and alkenyl groups 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-, -OC(O)NR"- and triazole,

[0291] R" and R"' are independently selected from H and C1-C6 alkyl,

[0292] and pharmaceutically acceptable salts thereof

[0293] In specific embodiments, T is a sugar cleavable unit which is a glucuronide or a galactoside.

[0294] In other specific embodiments, T is a dipeptide preferably selected from Val-Cit, Val-Ala and Phe-Lys.

[0295] In a more specific embodiment, L corresponds to a linker -AW- of formula (IV)

[0296]

[0297] in

[0298] X1 is the connector unit;

[0299] Z is an optional spacer;

[0300] X2 is the connector unit;

[0301] K is an optional hydrophobic masking entity, preferably selected from polysarcosine and polyethylene glycol;

[0302] R1 is selected from H, C1-C 24 alkyl, C2-C6 alkenyl; optionally substituted polyether, aryl having 6 to 10 ring atoms, C3-C8 cycloalkyl, heterocycloalkyl having 3 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms, and any combination thereof,

[0303] The alkyl and alkenyl groups 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-, -OC(O)NR"-, and triazole;

[0304] R4 is selected from H, C1-C6 alkyl and C2-C6 alkenyl, wherein the alkyl and alkenyl are optionally interrupted by one or more heteroatoms or chemical groups selected from: -O-, -S-, -C(O)- and -NR"-;

[0305] R5 is selected from H, C1-C6 alkyl and C2-C6 alkenyl, wherein the alkyl and alkenyl are optionally interrupted by one or more heteroatoms or chemical groups selected from: -O-, -S-, -C(O)- and -NR"-.

[0306] In a preferred embodiment of the linker of formula (IV), X1 and X2 are independently selected from 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,

[0307] The alkylene and alkenylene groups 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-, -OC(O)NR"- and triazole,

[0308] and the alkylene, arylene, cycloalkylene, heterocycloalkylene, heteroarylene and alkenylene are optionally substituted with one or more substituents selected from the group consisting of 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)-OR′, -(CO)-NR″R″′, -NR″-(CO)-R′ and -NR″R″′;

[0309] R', R" and R'" are independently selected from H and C1-C6 alkyl.

[0310] In other preferred embodiments of the linker of formula (IV), Z is independently selected from 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,

[0311] The alkylene and alkenylene groups 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-, -OC(O)NR"- and triazole,

[0312] and the alkylene, arylene, cycloalkylene, heterocycloalkylene, heteroarylene and alkenylene are optionally substituted with one or more substituents selected from the group consisting of 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)-OR′, -(CO)-NR″R″′, -NR″-(CO)-R′ and -NR″R″′;

[0313] R', R" and R'" are independently selected from H and C1-C6 alkyl.

[0314] In other embodiments of the linker L of formula (IV) or (V), K is polysarcosine, preferably of the following formula (V)

[0315]

[0316] wherein k is an integer between 2 and 50, preferably between 4 and 30,

[0317] And R6 corresponds to OH or NH2.

[0318] In other embodiments of the linker L of formula (IV) or (V), K is a polyethylene glycol moiety (PEG) preferably comprising from 2 to 50 ethylene glycol moieties.

[0319] In a preferred embodiment, L corresponds to a linker of formula (VI):

[0320]

[0321] Preferably, the linker L is covalently bonded to one or more thiol residues of antibody Ab, for example eight thiol residues of antibody Ab.

[0322] Payload

[0323] In one embodiment, D of the present disclosure is a payload attached to X on one side of the carbonyl functional group of X. In one embodiment, the bonding between X and D occurs between the carbonyl functional group of X and the amino group of D.

[0324] Payload D is an important component of ADC design. Payload can be a therapeutic agent or a drug. The term "drug" refers in particular to an agent that can modulate a biological process and / or has biological activity.

[0325] In a specific embodiment, the payload D is a cytotoxic drug that is activated after release from the internalized ADC in the cytoplasm of tumor cells. It should ideally be able to destroy tumor cells without affecting non-tumor cells (when connected to the antibody). The payload should also ideally have high stability in the systemic circulation and lysosomes. It should preferably have an in vitro sub-nanomolar IC50 value for cancer cell lines and sufficient solubility in an aqueous environment.

[0326] In a specific embodiment, the cytotoxic drug is selected from a microtubule disrupting agent or an antimitotic agent such as auristatins (including monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF)), maytansines (e.g., maytansine), DNA damaging agents such as calicheamicin, duocarmycins and anthracyclines (e.g., daunorubicin, doxorubicin, dihyrdroxyanthracindione), or an inhibitor of topoisomerase I such as camptothecin or its analogs.

[0327] In a specific embodiment, D is selected from the group consisting of: taxon, cytochalasin B, auristatins (including monomethyl auristatins), gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthraquinone, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol and puromycin and analogs or homologs thereof.

[0328] In a specific embodiment, D is selected from antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, dacarbazine), ablative agents (e.g., mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-diaminedichloroplatinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin)), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin and anthramycin (AMC)), and antimitotic agents (e.g., vincristine and vinblastine).

[0329] Preferably, D is an inhibitor of topoisomerase I, including but not limited to camptothecin analogs, indolecarbazole analogs, phenanthridine analogs, fluoroquinolone analogs, quinoxaline, evodiamine, acridine, naphthyridine, deoxynipomycin analogs, augustine analogs, stilbenethiazole analogs, pyrroloquinazolinoquinoline alkaloids or indenoisoquinoline analogs (for review, see: Selas et al., A patent review of topoisomerase I inhibitors (2016-present), 2021, Expert Opinion on Therapeutic Patents, 31(6), 473-508); preferably selected from camptothecin and its analogs, including but not limited to irinotecan, topotecan, camptothecin, SN-38, exitecan, DXd, silatecan, cositecan, lortotecan, gimatidecan, belotecan, rubitecan (for review, see: Sriram et al., Camptothecin and its analogues: a review on their chemotherapeutic potential, 2005, Natural Product Research, 14(9), 393-412).

[0330] In a preferred embodiment, D is the drug moiety of exitecan of formula (II)

[0331]

[0332] ADC of the present disclosure

[0333] In one embodiment, the present disclosure provides an ADC, wherein the anti-FRα antibody is linked to a drug, in particular exotecan. The linker L is as defined above.

[0334] Preferably, such ADCs can selectively deliver an effective dose of a drug, such as an inhibitor of topoisomerase I, preferably exotecan, to tumor cells expressing FRα.

[0335] In one embodiment, the present disclosure provides an ADC of formula (I)

[0336] Ab-[LD]p (I),

[0337] in:

[0338] -Ab is an anti-folate receptor alpha (FRα) antibody that specifically binds to SEQ ID NO: 12,

[0339] -L is a cleavable linker moiety bonded to the antibody via a thiol residue,

[0340] -D is a cytotoxic drug moiety bonded to L, such as an inhibitor of topoisomerase I,

[0341] - p is 1 to 8, preferably 6 to 8, and more preferably p is 8.

[0342] Ab, L and D have been defined in the previous section.

[0343] The term "p", also referred to as the drug-antibody ratio or "DAR", corresponds to the number of drug moieties per antibody moiety in the ADC of Formula (I) or the number of -LD moieties per antibody Ab.

[0344] Although the drug-antibody ratio has an exact value for a single ADC in principle, it is understood that when used to describe a composition containing many ADCs, the value is often an average value due to a certain degree of heterogeneity generally associated with the conjugation step. The average loading of a sample of ADCs is also referred to herein as the drug-antibody ratio or "DAR". In some embodiments, DAR (p) is between about 1 and 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 between about 4 and about 8, more preferably between about 6 and about 8, and even more preferably about 8.

[0345] Thus, in a composition comprising multiple copies of the ADC of formula (I), "p" refers to the average number of -LD moieties per antibody Ab. For an average p number close to 8, the ADC will be referred to as "DAR8".

[0346] Methods for measuring the drug-to-antibody ratio are disclosed in the Examples or described, for example, in Conilh et al. (Pharmaceuticals 2021, 14(3), 247).

[0347] The drug loaded to the antibody via the linker L may be limited by the number of attachment sites on the antibody portion. In some embodiments, the linker portion (L) of the ADC is attached to the antibody portion via a chemically active group on one or more amino acid residues on the antibody portion. For example, the linker can be attached to the antibody portion (e.g., N- or C-terminus, the epsilon amino group of one or more lysine residues, the free carboxyl group of one or more glutamic acid or aspartic acid residues, or the sulfhydryl group of one or more cysteine ​​residues) via a free amino acid, an imino group, a hydroxyl group, a thiol group, or a carboxyl group. The site to which the linker is attached can be a natural residue in the amino acid sequence of the antibody portion, or it can be introduced into the antibody portion, for example, by DNA recombinant technology (e.g., by introducing cysteine ​​or non-natural amino acid residues into the amino acid sequence) or by protein biochemistry (e.g., by reduction, pH adjustment, or hydrolysis).

[0348] When the attachment site is an interchain cysteine ​​thiol group, the antibody may have only one or a few cysteine ​​thiol groups through which the linker can be attached. In fact, most reactive cysteine ​​thiols generally exist as interchain disulfide bridges. Excessive attachment of linker-toxin to the antibody may destabilize the antibody by reducing the cysteine ​​residues available for forming interchain disulfide bridges. Therefore, the optimal drug-antibody ratio should increase the efficacy of the ADC (by increasing the number of drug moieties attached per antibody) without destabilizing the antibody moiety and reducing pharmacokinetic properties.

[0349] In a specific embodiment, -LD is bonded to an interchain reactive thiol residue of an antibody. A typical antibody having full-length heavy and light chains contains 8 available interchain reactive thiol residues. Thus, in a specific embodiment, the ADC is a DAR8 ADC, wherein eight -LD moieties are covalently bonded to 8 interchain reactive thiol residues of an antibody Ab, and most preferably, wherein -L- corresponds to formula (VI).

[0350] In a specific embodiment, the ADC of the present disclosure corresponds to the following formula (VII):

[0351]

[0352] wherein Ab is anti-FRα as defined in the previous paragraph and p is 4-8, preferably 6-8.

[0353] In a more preferred embodiment, the ADC of the present disclosure corresponds to the following formula (VII):

[0354]

[0355] wherein Ab is anti-FRα as defined in the previous section.

[0356] In a specific embodiment, the ADC of the present disclosure corresponds to formula (I), wherein

[0357] (i) Ab is an anti-folate receptor α antibody or an antigen-binding fragment thereof, comprising

[0358] a variable heavy chain polypeptide comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, HCDR3 of SEQ ID NO: 3, and

[0359] 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;

[0360] (ii) L is a cleavable linker of the formula -AW-, wherein A is an optional stretcher linked to Ab, and W is a cleavable moiety linked to D

[0361] (iii) D is an inhibitor of topoisomerase I, such as exotecan;

[0362] (iv) p is 1 to 8, preferably 8.

[0363] In a specific embodiment, the ADC of the present disclosure corresponds to formula (I), wherein

[0364] (i) Ab is an antibody comprising the heavy chain of SEQ ID NO: 9 and the light chain of SEQ ID NO: 10;

[0365] (ii) L is a cleavable linker of the formula -AW-, wherein A is an optional stretcher linked to Ab, and W is a cleavable moiety linked to D

[0366] (iii) D is an inhibitor of topoisomerase I, such as exotecan;

[0367] (iv) p is 1 to 8, preferably between 6 and 8, such as about 8.

[0368] In a specific embodiment, the ADC of the present disclosure is an ADC of formula (I), wherein

[0369] (i) Ab is an antibody comprising the heavy chain of SEQ ID NO: 11 and the light chain of SEQ ID NO: 10;

[0370] (ii) L is a cleavable linker of the formula -AW-, wherein A is an optional stretcher linked to Ab, and W is a cleavable moiety linked to D

[0371] (iii) D is an inhibitor of topoisomerase I, such as exotecan;

[0372] (iv) p is 1 to 8, preferably between 6 and 8, such as about 8.

[0373] In a preferred embodiment, the ADC of the present disclosure is an ADC of formula (VII), wherein Ab is an antibody comprising a heavy chain of SEQ ID NO:9 and a light chain of SEQ ID NO:10.

[0374] In another specific embodiment, the ADC of the present disclosure is an ADC of formula (VII), wherein Ab is an antibody comprising a heavy chain of SEQ ID NO:11 and a light chain of SEQ ID NO:10.

[0375] Pharmaceutical compositions - preparations

[0376] In another aspect, the disclosure provides a composition, e.g., a pharmaceutical composition, containing one or a combination of the ADCs disclosed herein (e.g., mAb1 conjugated to a linker L of formula (VI), itself conjugated to a drug such as exotecan) formulated together with a pharmaceutically acceptable carrier.

[0377] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. that are physiologically compatible. The pharmaceutical formulations of the present disclosure may further comprise one or more pharmaceutically acceptable excipients selected from the group consisting of stabilizers, surfactants, buffers, antimicrobial preservatives, protectants, antioxidants, chelating agents, and fillers.

[0378] As used herein, "solvent" is any pharmaceutically acceptable (i.e., safe and non-toxic for administration to a human or another mammal) and useful ingredient for preparing a liquid formulation, such as an aqueous formulation. Exemplary solvents include water, such as sterile water for injection (WFI) or bacteriostatic water for injection (BWFI), pH buffered solutions (e.g., phosphate-buffered saline), sterile saline solutions, Ringer's solution, or dextrose solution, and combinations thereof. Preferably, the solvent is sterile water or bacteriostatic water for injection (BWFI) for use in the present disclosure.

[0379] As used herein, a stabilizer is a compound that increases protein stability, especially with respect to protein unfolding and aggregation. Preferably, the stabilizer is approved by the authorities as a suitable additive or excipient in pharmaceutical formulations.

[0380] The stabilizer can be a sugar. In this context, "sugar" comprises the general composition (CH2O) nand derivatives thereof, including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, etc. Examples of sugars herein include glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerol, dextran, erythritol, glycerol, arabitol, sylitol, sorbitol, mannitol, melibiose, melezitose, raffinose, mannotriose, stachyose, maltose, lactulose, maltulose, glucitol, maltitol, lactitol, isomaltulose, etc.

[0381] The concentration of the stabilizer in the pharmaceutical formulations of the present disclosure is between 1 and 500 mM.

[0382] As used herein, "surfactant" refers to a surface active agent. Surfactants are generally added to protein formulations to reduce exposure of hydrophobic regions and thus reduce protein-protein interactions and interface-induced aggregation, which is also prevented by competition for adsorption sites.

[0383] Examples of surfactants herein 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 stearoyl sulfobetaine; lauryl, myristyl, linoleyl, or stearoyl sarcosine; linoleyl, myristyl, or cetyl betaine; Base; lauramide propyl, cocamidopropyl, linoleamide propyl, myristamide propyl, palmitamide propyl or isostearamide propyl betaine (such as lauramide propyl); myristamide propyl, palmitamide propyl or isostearamide propyl dimethylamine; sodium cocoyl methyl taurate or disodium oleyl methyl taurate; polyethylene glycol, polypropylene glycol and copolymers of ethylene glycol and propylene glycol (such as Pluronics, PF68, etc.). Other examples of pharmaceutically acceptable surfactants include polyoxyethylene-sorbitan fatty acid esters (Tween), polyethylene glycol-polypropylene glycol, polyoxyethylene-stearate, polyoxyethylene alkyl ethers such as polyoxyethylene monolauryl ether, alkylphenol polyoxyethylene ether (Triton-X), polyoxyethylene-polyoxypropylene copolymers (poloxamers, Pluronics) and sodium lauryl sulfate (SDS). The most suitable polyoxyethylene sorbitan fatty acid ester is polysorbate 20 (available under the trade name Tween 20 TM ) and polysorbate 80 (sold under the trademark Tween 80 TM sell).

[0384] The most suitable polyethylene-polypropylene copolymers are known under the names F68 or Poloxamer 188 TMThe most suitable polyoxyethylene alkyl ethers are sold under the trademark Brij TM Those polyoxyethylene alkyl ethers sold as such. The most suitable alkylphenol-polyoxyethylene ether is sold under the trade name Triton-X.

[0385] The concentration of surfactant in the pharmaceutical formulations of the present disclosure may be between 0.01 and 0.1% (w / v).

[0386] As used herein, the term "buffer" refers to an agent that allows a solution containing it to resist pH changes through the action of its acid / base conjugate components. Examples of buffers that control pH within this range include acetate, succinate, gluconate, histidine, citrate, glycylglycine, and other organic acid buffers.

[0387] "Preservative" is a compound that can be added to the preparation herein to reduce contamination and / or its effect by bacteria, fungi or another infectious agent. For example, the addition of preservative can promote the production of multiple-use (multiple dose) preparations. Examples of potential preservatives include octadecyl dimethyl benzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride (wherein the alkyl group is a mixture of long-chain alkyl benzyl dimethyl ammonium chloride) and benzethonium chloride. Other types of preservatives include aromatic alcohols, such as phenol, butanol and benzyl alcohol, alkyl parabens such as methyl paraben or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol and w-cresol.

[0388] As generally used herein, a "protectant" is a substance that, when combined with a protein, significantly reduces the chemical and / or physical instability of the protein upon lyophilization and / or subsequent frozen storage. Exemplary protectants include sugars and their corresponding sugar alcohols, such as sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol, and mannitol; amino acids, such as arginine or histidine; lyotropic salts, such as magnesium sulfate; polyols, such as propylene glycol, glycerol, poly(ethylene glycol) or poly(propylene glycol); and combinations thereof. Additional examples of protectants include gelatin, dextrin, modified starch, and carboxymethyl cellulose.

[0389] The protective agent may be added to the pre-lyophilized formulation in a "lyophilization-protective amount." This means that after lyophilization of the protein in the presence of the lyophilization-protective amount of the protective agent, the protein substantially maintains its physical and chemical stability and integrity.

[0390] As generally used herein, "antioxidants" are pharmaceutically acceptable excipients generally used to limit oxidation reactions and maintain the stability and safety of proteins. Examples of antioxidants are ascorbic acid, sodium pyrosulfite, histamine, methionine, ascorbic acid, glutathione, vitamin E, polyethyleneimine.

[0391] The concentration of antioxidants in the pharmaceutical formulations of the present disclosure may be between 5 and 25 mM.

[0392] "Chelating agents" are pharmaceutically acceptable excipients generally used to maintain the stability of proteins. Examples of chelating agents include disodium edetate, diethylenetriaminepentaacetic acid, citric acid, hexaphosphate, thioglycolic acid, zinc.

[0393] As generally used herein, "bulking agents" are pharmaceutically acceptable excipients that are generally used to increase the mass of the lyophilized mixture and contribute to the physical structure of the lyophilized cake (e.g., to promote the production of a substantially uniform lyophilized cake that maintains an open-pore structure). Exemplary bulking agents include mannitol, glycine, lactose, modified starches, polyethylene glycols) and sorbitol.

[0394] The form of the pharmaceutical composition, the route of administration, the dosage and the regimen will naturally depend on the condition to be treated, the severity of the disease, the age, weight and sex of the patient.

[0395] The pharmaceutical compositions of the present disclosure may be formulated for topical, oral, parenteral, intraperitoneal, intranasal, intravenous, intramuscular, subcutaneous, or intraocular administration, etc., preferably intraperitoneal or intravenous.

[0396] Preferably, the pharmaceutical composition contains a vehicle which is pharmaceutically acceptable for an injectable formulation. These may be in particular isotonic, sterile saline solutions (mono- or disodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, etc. or mixtures of such salts), or dry, in particular lyophilized, compositions which, after addition of sterile water or physiological saline, as the case may be, allow the constitution of injectable solutions.

[0397] The dosage for administration can be adjusted according to various parameters and in particular according to the mode of administration used, the pathology concerned or, alternatively, the desired duration of treatment.

[0398] To prepare a pharmaceutical composition, an effective amount of ADC can be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

[0399] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders or lyophilizates for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

[0400] Solutions of the active compound as free alkali or pharmacologically acceptable salts can be prepared in water by suitable mixing with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof and oils. Under normal storage and use conditions, these preparations contain preservatives to prevent the growth of microorganisms.

[0401] The ADC of the present disclosure can be formulated into compositions in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed by free amino groups of proteins) and are formed by inorganic acids such as hydrochloric acid or phosphoric acid or such organic acids as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. Salts formed by free carboxyl groups can also be derived from inorganic bases, such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or ferric hydroxide, and such organic bases as isopropylamine, trimethylamine, histidine, procaine, etc.

[0402] Sterile injectable solutions are prepared by mixing the active compound in the desired amount with the various other ingredients listed above in an appropriate solvent, as required, followed by filtration sterilization. Generally, dispersions are prepared by mixing the various sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and the required other ingredients from the above list. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred preparation methods are vacuum drying and freeze drying techniques, which obtain a powder of the active ingredient plus any additional required ingredients from a previously sterile filtered solution thereof.

[0403] The preparation of more concentrated or highly concentrated solutions for direct injection is also contemplated, with the use of DMSO as a solvent envisioned to result in extremely rapid penetration, delivering high concentrations of active agent to small tumor areas.

[0404] After formulation, the solution is administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulation is readily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like may also be employed.

[0405] For parenteral administration in aqueous solution, for example, if necessary, the solution should be properly buffered, and the liquid diluent is first made isotonic with enough saline or glucose. These specific aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this regard, according to the present disclosure, the sterile aqueous medium that can be adopted will be known to those skilled in the art. For example, a dose can be dissolved in 1ml isotonic NaCl solution, and either added to 1000ml subcutaneous injection or injected at the infusion site of the proposal (see, for example, "Remington's Pharmaceutical Sciences" the 15th edition, pages 1035-1038 and 1570-1580). Depending on the condition of the subject to be treated, some changes in the dosage will inevitably occur. In any case, the individual responsible for administration will determine the appropriate dosage for each subject.

[0406] The ADCs of the present disclosure may be formulated within a therapeutic mixture to contain about 0.0001 to 1.0 mg, or about 0.001 to 0.1 mg, or about 0.1 to 1.0 or even 1.0 to about 10 mg per dose. Multiple doses may also be administered.

[0407] The pharmaceutical formulations comprising the ADCs of the present disclosure may be "ready-to-use" injectable formulations or lyophilized formulations.

[0408] In a specific embodiment, the pharmaceutical formulation comprising the ADC of the present disclosure may be supplied in a pre-filled syringe.

[0409] Uses and methods of ADC disclosed herein

[0410] The ADCs of the present disclosure have therapeutic utility. For example, these molecules can be administered in a subject, such as in vivo, to treat or prevent various conditions.

[0411] Contemplated herein are the use of the ADCs of the present disclosure as medicaments, particularly for treating cancer in a subject in need thereof, particularly cancer having tumor cells expressing FRα, more particularly cancer having solid tumors, and more particularly selected from ovarian cancer, breast cancer, lung cancer or mesothelioma.

[0412] The term "cancer" refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma (including medulloblastoma and retinoblastoma), sarcoma (including liposarcoma and synovial cell sarcoma), neuroendocrine tumors (including carcinoid tumors, gastrinomas, and islet cell carcinomas), mesothelioma, schwannoma (including acoustic neuroma), meningioma, adenocarcinoma, melanoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous carcinoma), peritoneal cancer, hepatocellular carcinoma, stomach or gastric cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, testicular cancer, esophageal cancer, biliary tract tumors, and head and neck cancer.

[0413] The ADCs of the present disclosure may be particularly useful for treating a cancer selected from ovarian cancer, triple-negative breast cancer, and non-small cell lung cancer.

[0414] Accordingly, the present disclosure relates to a method of treating cancer, in particular one of the cancers listed above, and more preferably a cancer selected from ovarian cancer, triple-negative breast cancer and non-small cell lung cancer, comprising administering a therapeutically effective amount of an ADC of formula (I) as disclosed herein.

[0415] The ADC for use as disclosed above can be administered as the sole active ingredient, or in conjunction with other drugs, such as antiviral, anti-inflammatory or cytotoxic agents, antiproliferative agents, chemotherapeutic or anti-tumor agents, e.g. for the treatment or prevention of the diseases mentioned above, for example as an adjuvant or in combination.

[0416] For example, the ADC for use as disclosed above can 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.

[0417] Examples of such anti-PD1 or anti-PDL1 antibodies include, but are not limited to, nivolumab, pembrolizumab, avelumab, durvalumab, cemiplimab, or atezolizumab.

[0418] Suitable anti-tumor agents may include, but are not limited to, alkylating agents (e.g., cyclophosphamide, mechlorethamine, chlorambucil, melphalan, nitrosoureas, temozolomide), anthracyclines (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin), taxanes (e.g., paclitaxel, docetaxel), epothilones, inhibitors of topoisomerase I (e.g., irinotecan or topotecan), inhibitors of topoisomerase II (e.g., etoposide, teniposide or taflutoposide), nucleotide analogs and precursor analogs (e.g., azacitidine, azathioprine, capecitabine, cytarabine, fluorouracil, gemcitabine, hydroxyurea, mercaptopurine, methotrexate or thioguanine), peptide antibiotics (e.g., bovine serum albumin, beta-thiocyanate, cytosine, thioguan ...

[00136] In some embodiments, the present invention relates to anticancer drugs, such as levofloxacin, colistin, gramicidin, platinum-based antineoplastic drugs (e.g., carboplatin, cisplatin, and oxaliplatin), retinoids (e.g., tretinoin, alitretinoin, bexarotene), vinca alkaloids and derivatives (e.g., vinblastine, vincristine, vindesine, vinorelbine), anti-VEGF agents (e.g., bevacizumab, ranibizumab, sunitinib, sorafenib, pazopanib, targeted therapies such as kinase inhibitors (e.g., ibrutinib, idelalisib, erlotinib, gefitinib, imatinib, vemurafenib, vismodegib), proteasome inhibitors (e.g., bortezomib, carfilzomib), histone deacetylase inhibitors (e.g., vorinostat or romidepsin).

[0419] In accordance with the foregoing, the present disclosure provides in a still further aspect a method comprising co-administration, e.g. concomitantly or sequentially, of a therapeutically effective amount of an ADC of the present disclosure and at least one second drug substance, the second drug substance being an antiviral, anti-inflammatory or cytotoxic agent, antiproliferative agent, chemotherapeutic or other anti-tumor agent, e.g. as indicated above.

[0420] Also within the scope of the present disclosure is a kit consisting of a composition disclosed herein (e.g., comprising an ADC) and instructions for use. The kit may further contain at least one additional reagent, or one or more additional antibodies or proteins (e.g., an antibody with complementary activity that binds to an epitope on the target antigen that is different from the first antibody). The kit typically includes a label indicating the intended use of the contents of the kit. The term label includes any written or recorded material on the kit or supplied with the kit, or otherwise accompanying the kit. The kit may further include a tool for diagnosing whether the patient belongs to a population (as defined above) that responds to ADC treatment, particularly a population with FRα-expressing tumors.

[0421] Methods of preparing the ADC of the present disclosure

[0422] The antibodies of the present disclosure can be conjugated to at least one drug via 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 more information with respect to 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); Chapters 2-7 of "Antibody-Drug Conjugates: Fundamentals, Drug Development, and Clinical Outcomes to Target Cancer", November 3, 2016, eBook ISBN: 9781119060727, doi: 10.1002 / 9781119060727 and Panowksi S et al. 2014 Jan 1; 6(1): 34-45.

[0423] In one embodiment, the method for obtaining the ADC of formula (I) comprises the following steps:

[0424] - culturing the host cell under conditions suitable for the expression of a nucleic acid encoding an antibody Ab as defined in the previous paragraph,

[0425] - Isolation of antibodies,

[0426] -Synthesis of Exetecan bonded to a linker L of formula (VIII):

[0427]

[0428] - conjugating the antibody with a compound of formula (VIII) to obtain an ADC of formula (I).

[0429] Antibodies can be obtained as described above. Antibodies contain four accessible interchain disulfide bonds, which can be used as potential conjugation sites. The four interchain disulfide bonds can be reduced, for example, by tris(2-carboxyethyl)phosphine (TCEP) or dithiothreitol (DTT), which results in eight thiol groups that can be used for conjugation.

[0430] Part-LD in formula (VIII) can follow procedures known in the field of organic synthesis (chemical reaction, extraction, evaporation, precipitation, chromatography, filtration, grinding, crystallization, etc.), and analytical procedures known to those of ordinary skill in analytical chemistry to prepare. The details of such reactions and techniques can be found in many papers, including Richard Larock, Comprehensive Organic Transformations, A Guide to Functional Group Preparations, Second Edition (2010), and the multi-volume series Compendium of Organic Synthetic Methods (1974 and later) edited by Michael B. Smith and others. Raw materials and reagents can be obtained from commercial sources, or can be prepared using literature methods.

[0431] Exotecan compounds can be synthesized following known procedures (see US5834476; WO2019044946 or Sugimori et al., 1998, J. Med. Chem., 41(13), 2308-2318 doi: 10.1021 / jm970765q). Exotecan can also be purchased from reputable sources (e.g., MedChemExpress catalog #HY-13631A or Carbosynth catalog #FE72401).

[0432] Following known procedures, the final ADC of formula (I) is obtained after the -LD component is conjugated to the natural or modified cysteine ​​thiol residue 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 / DOCS00310G. Typically, the antibody component is reduced with a reducing agent such as tris(2-carboxyethyl)phosphine (TCEP) or dithiothreitol (DTT); the -LD component is added and allowed to covalently react with the cysteine ​​thiol residues of the antibody; and the final ADC compound is purified and buffer exchanged.

[0433] The invention having been fully described will now be further illustrated by the following embodiments and examples which are illustrative only and are not meant to be further limiting. Specific implementation plan

[0434] 1. An antibody-drug conjugate having the molecular formula (I):

[0435] Ab-[LD]p (I),

[0436] in:

[0437] -Ab is an anti-folate receptor alpha (FRα) antibody that specifically binds to SEQ ID NO: 12,

[0438] -L is a cleavable linker moiety bonded to the antibody preferably via a thiol residue,

[0439] -D is the cytotoxic drug moiety bonded to L,

[0440] - p is 1 to 8, preferably 6 to 8, and more preferably p is 8.

[0441] 2. The antibody-drug conjugate according to embodiment 1, wherein D is an inhibitor of topoisomerase I, preferably selected from camptothecin analogs, and more preferably D is the drug moiety of exitecan of the following formula (II):

[0442]

[0443] 3. The antibody-drug conjugate according to any one of embodiments 1-2, wherein L is a cleavable linker moiety of the formula -AW-, wherein A is an optional stretcher unit linked to Ab, and W is a cleavable moiety linked to D.

[0444] 4. An antibody-drug conjugate according to embodiment 3, wherein L is a lysosomal protease-sensitive linker and W comprises a cleavable peptide portion, e.g., selected from valine-citrulline (Val-Cit), alanine-alanine-asparagine (Ala-Ala-Asn), valine-alanine (Val-Ala) and phenylalanine-lysine (Phe-Lys).

[0445] 5. The antibody-drug conjugate according to embodiment 3, wherein L is a protease-sensitive cleavable linker and W comprises a sugar cleavable unit preferably selected from a β-glucuronide or β-galactoside moiety.

[0446] 6. The antibody-drug conjugate according to embodiment 3, wherein L is a glutathione-sensitive linker and W comprises a disulfide moiety.

[0447] 7. The antibody-drug conjugate according to embodiment 3, wherein W has the following formula (III)

[0448]

[0449] in

[0450] Each R2 is independently selected from an electron withdrawing group and a C1-C4 alkyl group;

[0451] n is 0, 1, or 2;

[0452] T is a sugar cleavable unit or a polypeptide cleavable unit;

[0453] When T is a Sugar Cleavable Unit, Y is O, or when T is a Polypeptide Cleavable Unit, Y is NR3; and

[0454] R3 is selected from H, C1-C 24 alkyl, C2-C6 alkenyl; optionally substituted polyether, aryl having 6 to 10 ring atoms, C3-C8 cycloalkyl, heterocycloalkyl having 3 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms, and any combination thereof,

[0455] The alkyl and alkenyl groups 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-, -OC(O)NR"-, and triazole,

[0456] R" and R"' are independently selected from H and C1-C6 alkyl,

[0457] and pharmaceutically acceptable salts thereof.

[0458] 8. The antibody-drug conjugate according to embodiment 7, wherein L corresponds to the linker -AW- of formula (IV)

[0459]

[0460] in

[0461] X1 is the connector unit;

[0462] Z is an optional spacer;

[0463] X2 is the connector unit;

[0464] K is an optional hydrophobic masking entity, preferably selected from polysarcosine and polyethylene glycol;

[0465] R1 is selected from H, C1-C 24 alkyl, C2-C6 alkenyl; optionally substituted polyether, aryl having 6 to 10 ring atoms, C3-C8 cycloalkyl, heterocycloalkyl having 3 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms, and any combination thereof,

[0466] The alkyl and alkenyl groups 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-, -OC(O)NR"-, and triazole;

[0467] R4 is selected from H, C1-C6 alkyl and C2-C6 alkenyl, wherein the alkyl and alkenyl groups are optionally interrupted by one or more heteroatoms or chemical groups selected from: -O-, -S-, -C(O)- and -NR"-; and

[0468] R5 is selected from H, C1-C6 alkyl and C2-C6 alkenyl, wherein the alkyl and alkenyl are optionally interrupted by one or more heteroatoms or chemical groups selected from: -O-, -S-, -C(O)- and -NR"-.

[0469] 9. The antibody-drug conjugate according to embodiment 8, wherein X1 and X2 are independently selected from 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,

[0470] The alkylene and alkenylene groups 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-, -OC(O)NR"- and triazole,

[0471] and the alkylene, arylene, cycloalkylene, heterocycloalkylene, heteroarylene and alkenylene are optionally substituted with one or more substituents selected from the group consisting of 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)-OR′, -(CO)-NR″R″′, -NR″-(CO)-R′ and -NR″R″′;

[0472] R', R" and R'" are independently selected from H and C1-C6 alkyl.

[0473] 10. The antibody-drug conjugate according to any one of embodiments 8-9, wherein Z is independently selected from 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,

[0474] The alkylene and alkenylene groups 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-, -OC(O)NR"- and triazole,

[0475] and the alkylene, arylene, cycloalkylene, heterocycloalkylene, heteroarylene and alkenylene are optionally substituted with one or more substituents selected from the group consisting of 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)-OR′, -(CO)-NR″R″′, -NR″-(CO)-R′ and -NR″R″′;

[0476] R', R" and R'" are independently selected from H and C1-C6 alkyl.

[0477] 11. The antibody-drug conjugate according to any one of embodiments 8-10, wherein K is polysarcosine, preferably of the following formula (V)

[0478]

[0479] wherein k is an integer between 2 and 50, preferably between 4 and 30,

[0480] And R6 corresponds to OH or NH2.

[0481] 12. The antibody-drug conjugate according to any one of embodiments 7-11, wherein T is a sugar cleavable unit which is a glucuronide or a galactoside.

[0482] 13. The antibody-drug conjugate according to any one of embodiments 7-11, wherein T is a dipeptide, preferably selected from Val-Cit, Val-Ala and Phe-Lys.

[0483] 14. The antibody-drug conjugate according to any one of embodiments 1-3, wherein L is covalently bonded to one or more thiol residues of the antibody,

[0484] Preferably, said L corresponds to a linker of formula (VI):

[0485]

[0486] 15. The antibody-drug conjugate according to any one of embodiments 1-14, wherein the antibody comprises a full-length antibody or an antibody fragment containing an antigen binding portion.

[0487] 16. The antibody-drug conjugate according to any one of embodiments 1-15, which corresponds to the following formula (VII):

[0488]

[0489] Wherein Ab is an anti-FRα antibody, such as fatutumomab, or a silent IgG1 variant thereof typically comprises alanine substitutions in Leucine 234 and Leucine 235 of the IgG1 Fc constant region, and p is 4 to 8, preferably 8.

[0490] 17. The antibody-drug conjugate according to any one of embodiments 1-16, wherein:

[0491] - Ab is an antibody comprising a human IgG1 isotype constant region or a mutant or chemically modified constant region, wherein the mutant or chemically modified constant region does not confer ADCC activity or confers reduced ADCC activity to the antibody when compared to a corresponding antibody having a wild-type human IgG1 isotype constant region; or,

[0492] -Ab is an antibody comprising a human IgG4 isotype constant region or a mutant or chemically modified constant region, wherein the mutant or chemically modified constant region confers no ADCC activity or reduced ADCC activity to the antibody when compared to a corresponding antibody having a wild-type IgG4 isotype constant region.

[0493] 18. The antibody-drug conjugate according to any one of embodiments 1-17, wherein Ab is an anti-FRα antibody comprising any of:

[0494] (i) a variable heavy chain polypeptide comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, HCDR3 of SEQ ID NO: 3, and a variable light chain polypeptide comprising LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6; or,

[0495] (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.

[0496] 19. The antibody-drug conjugate according to any one of embodiments 1-18, wherein Ab comprises or consists essentially of:

[0497] (i) the heavy chain of SEQ ID NO: 9 and the light chain of SEQ ID NO: 10; or,

[0498] (ii) the heavy chain of SEQ ID NO:11 and the light chain of SEQ ID NO:10.

[0499] 20. The antibody-drug conjugate according to any one of embodiments 1-19, wherein

[0500] (i) Ab is an anti-folate receptor α antibody or an antigen-binding fragment thereof, comprising

[0501] a variable heavy chain polypeptide comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, HCDR3 of SEQ ID NO: 3, and

[0502] 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;

[0503] (ii) L is a cleavable linker of the formula -AW-, wherein A is an optional stretcher linked to Ab, and W is a cleavable moiety linked to D

[0504] (iii) D is a topoisomerase I inhibitor, such as exotecan; and

[0505] (iv) p is 1 to 8, preferably 8.

[0506] 21. The antibody-drug conjugate according to embodiment 20, wherein Ab is

[0507] (i) an antibody or antigen-binding fragment comprising the heavy chain of SEQ ID NO: 9 and the light chain of SEQ ID NO: 10, or

[0508] (ii) an antibody or antigen-binding fragment comprising the heavy chain of SEQ ID NO:11 and the light chain of SEQ ID NO:10.

[0509] 22. The antibody-drug conjugate according to any one of embodiments 1-21 for use as a medicament, preferably for treating a tumor, such as a solid tumor, more particularly selected from ovarian cancer, breast cancer, lung cancer or mesothelioma, in a subject in need thereof.

[0510] 23. The antibody-drug conjugate according to any one of embodiments 1-21, for use in treating a cancer selected from ovarian cancer, triple-negative breast cancer, and non-small cell lung cancer in a subject in need thereof.

[0511] 24. A pharmaceutical composition comprising the antibody-drug conjugate according to any one of embodiments 1-21 in combination with one or more pharmaceutically acceptable excipients, diluents or carriers, optionally comprising other active ingredients.

[0512] 25. A method for obtaining an antibody-drug conjugate according to any one of claims 1 to 23, wherein the method comprises:

[0513] - culturing the host cell under conditions suitable for producing an anti-FRα antibody as defined in any one of embodiments 1-21,

[0514] - isolating said anti-FRα antibody,

[0515] -Synthesis of Exetecan bonded to a linker L of formula (VIII):

[0516]

[0517] - conjugating the anti-FRα antibody to a compound of formula (VIII),

[0518] Thereby an ADC as defined in any one of embodiments 1-21 is obtained.

[0519] Example

[0520] Functional assays

[0521] Human FRα binding affinity by ELISA

[0522] Sandwich ELISA assays were performed using 96-well high binding ELISA plates (Corning Inc., New York, NY, USA, catalog #3590). The plates were coated with 100 μL / well of recombinant human FRa protein (SinoBiological catalog #11241-H08H) at 2 μg / mL in PBS (pH 7.4) and incubated overnight at 4°C. After 2 washes with PBS-T (PBS + 0.05% Tween-20), the plates were blocked with 200 μL / well incubation buffer (PBS-T + 0.1% BSA) at room temperature for 1 hour. The plates were washed 4 times with PBS-T, and 100 μL of a three-fold dilution series of test compounds (antibodies or antibody-drug conjugates) were added. The plates were then incubated in the dark at room temperature for 2 hours. After 5 washes with PBS-T, the plate was incubated at room temperature for 1 hour with 100 μL / well of goat anti-human IgG (H+L) HRP conjugated antibody (Jackson Immunoresearch catalog #109-035-088), which was previously diluted 1:250 000 in incubation buffer. After 5 washes with PBS-T, TMB substrate solution (Thermo-Fisher catalog #N301) was added. Peroxidase activity was stopped with 0.18M H2SO4, and absorbance was read at 450nm (reference wavelength 650nm) using a Thermo Scientific MultiSkan EX microplate reader. S-shaped fitting was performed using GraphPad Prism 9 software.

[0523] SPR Biacore Affinity

[0524] Surface plasmon resonance (SPR) experiments were performed on a Biacore T200 instrument at 25°C. Test antibodies or ADCs were captured at low density (300-500RU) on a CM5 series S sensor chip that was functionalized with a Human Antibody Capture kit (Cytiva catalog #BR100839) in advance. A functionalized surface / flow cell with similar treatment but omitting the ligand was used as a reference. To measure kinetic rates and affinities, a single-cycle kinetic strategy was used with a constant 70 μL / mL flow rate of running buffer (HBS-EP+, Cytiva catalog #BR100669), followed by a 300-second contact pulse, and a series of 5 concentrations (0.5, 1, 2, 4, 8 nM) of recombinant human FRα (SinoBiological catalog #11241-H08H) analyte samples were injected in duplicate. The dissociation phase was measured by injecting running buffer for 900 seconds. Between replicates, the surface / flow cell was regenerated with 3M MgCl2 to remove both analyte and ligand, and fresh ligand capture was performed using the same conditions. Data analysis was performed using BiacoreEvaluation software after subtracting the reference surface and analyte zero concentration signals. The data were processed and fitted to a 1:1 binding model to determine the binding kinetic rate constants, k a (binding rate) and k d (dissociation rate) and K D (Equilibrium dissociation constant, also called "affinity"). Means and standard deviations of replicates are reported.

[0525] Cell binding affinity by flow cytometry

[0526] The combination of antibody or ADC with the extracellular human FRα expressed on cancer cell line was evaluated by flow cytometry. According to the manufacturer's scheme (Bio-Rad, catalog #LNK032APC), LYNX Rapid APC Antibody Conjugation Kit was used to conjugate the antibody or ADC tested with APC fluorophore. 5 μL 10 μg / mL of antibody or ADC tested APC labeled was added to 500,000 cells (suspended in 100 μL PBS in flow cytometry plastic tubes). Cells were incubated in the dark for 20 minutes, washed 3 times with PBS by centrifugation, and resuspended in 200 μL PBS for analysis. Flow cytometry was performed using the BD Fortessa flow cytometer controlled by BD FACS Diva software (BD Biosciences), and data were analyzed using FlowJo software (BD Bioscience).

[0527] Stability assay in human plasma

[0528] ADC samples (>6 mg / mL in PBS) were diluted with pure sterile human plasma (GeneTex catalog # GTX73265) in a screw-capped centrifuge tube to produce a final ADC concentration of 200 μg / mL (residual PBS volume less than 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). Dynabeads were used. TMM-280Streptavidin (ThermoScientific) magnetic beads, ADC was separated from plasma by immunocapture, and the magnetic beads were previously coated with biotinylated human folate receptor alpha recombinant protein (Sino Biologicals catalog #11241-H08H). In brief, 600 μL of commercial bead solution was washed twice with HBS-EP buffer (Cytiva, catalog #BR100188) and resuspended in 1.2 mL HBS-EP buffer. 65 μL (48 μg protein amount) of biotinylated recombinant FRα solution was added, and the solution was stirred at room temperature for 2 hours. The beads were then washed 3 times with HBS-EP buffer and resuspended in 1.2 mL HBS-EP buffer. For an immunocapture, 100 μL of the previous bead solution was added to 100 μL HBS-EP in a microcentrifuge tube. 10 μL of ADC solution in plasma (theoretical 2 μg ADC amount) 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 with 200 μL HBS-EP buffer, and deglycosylated by adding 2 μL / 1000U PNGase F (NewEngland Biolabs catalog # P0705L) at 37°C with gentle agitation overnight. The beads were then washed twice with HBS-EP buffer, twice with distilled water, and once with 10% acetonitrile in water (v / v). The beads were incubated with 50 μL of 30% acetonitrile in water (v / v) containing 0.1% (v / v) formic acid at room temperature for 30 minutes with gentle agitation. The beads were then lysed using a Bruker Impact II TM The eluted samples containing deglycosylated ADC were analyzed by denaturing reverse phase chromatography-mass spectrometry using a Thermo UltiMate 3000 UHPLC system with a 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 2.1x100mm 5μm (80°C). Linear gradient 20% B to 50% B in 25 minutes. Flow rate 0.4mL / min. UV detection monitored at 280nm. Q-ToF mass spectrometer in m / z range 500-5000 (ESI + ) are used. Use Bruker The MaxEnt algorithm included in the software deconvolutes the data. For stability data analysis, deconvolution of the original spectrum within the selected light chain (LC) and heavy chain (HC) elution time window is implemented. Drug-linker loss or modification is identified based on 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 specific 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.

[0529] In vitro efficacy in FRa-negative cell line (BT-474 cell line)

[0530] In order to evaluate the non-specific (off-target) cytotoxicity of ADC, in vitro cytotoxicity assay was achieved in BT-474 (FRa negative) cancer cell line. Cells were plated in 96-well plates (1000 to 10 000 cells / well in 100 μL appropriate culture medium) at an appropriate density depending on the cell line, and incubated at 37 ° C for 24 hours. Serial dilutions of the test compound previously dissolved in the culture medium (50 μL) 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 continued for 1 to 2 hours at 37 ° C. The culture medium was then carefully removed, and the well contents were evenly dissolved with acidified isopropanol. Absorbance values ​​were measured on a MultiskanTMSky microplate reader (Thermo Scientific) using a wavelength of 570 nm (with a reference wavelength of 690 nm). Inhibitory dose-response curve fitting (GraphPad Prism 9) was used to determine IC50 concentration values ​​compared to untreated control cells.

[0531] In vivo efficacy in cancer xenograft models

[0532] Female CB-17 severe combined immunodeficient (SCID) mice were obtained and isolated for 7 days at the beginning of the study. The mice were suspended in PBS (OV-90, SW-620, KB, BT-474 cell lines) or 50% BD Matrigel Cells in PBS solution (PA-1, IGROV-1, OVCAR-3, NCI-H2110 cell lines) were inoculated subcutaneously. When the average tumor volume reached about 120-150mm3, mice were randomized (usually 7 mice / group) and treated by a single (unless otherwise stated) intravenous injection of PBS (negative control) or the antibody-drug conjugate investigated. Tumor volume was measured (length x width) using a caliper device every 3-5 days and calculated using the following formula V=4 / 3×π×R3, where R represents radius. When the tumor volume exceeded 1500mm3 or in the case of tumor ulceration, mice were killed.

[0533] Drug-Antibody Ratio (DAR) Evaluation by Reverse Phase Liquid Chromatography-Mass Spectrometry (RPLC-MS):

[0534] Denaturing RPLC-QToF analysis was used to evaluate the drug-antibody ratio (DAR) of the conjugates. Briefly, ADC was analyzed using a mobile phase gradient of water / acetonitrile + 0.1% formic acid (0.4 mL / min) on an Agilent PLRP-S Elution was performed on a 2.1x150mm 8μm (80°C) and using a Bruker Impact II TM The Q-ToF mass spectrometer scanned the range of 500-3500 m / z (ESI + ) was used for detection. The MaxEnt algorithm included in the software deconvolutes the data.

[0535] Example 1: Synthesis of chemical drug-linker based on Exitecan

[0536] Materials and general organic synthesis methods

[0537] All solvents and reagents were obtained from reputable 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 stated. 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. Exotecan mesylate was purchased from MedChemExpress.

[0538] On-resin synthesis was performed in empty SPE plastic tubes equipped with 20 μm polyethylene frits (Sigma-Aldrich). A Titramax 101 platform shaker (Heidolph) was used for agitation. Unless otherwise stated, all chemical reactions were performed at room temperature under an inert argon atmosphere.

[0539] Liquid NMR spectra were recorded on a Bruker Fourier 300HD or Bruker AVANCE III HD400 spectrometer using the residual solvent peak for calibration.Mass spectrometry analyses have been performed by the Centre Commun de Spectrométrie de Masse (CCSM) at the UMR5246 CNRS Institute at University Claude Bernard Lyon 1.

[0540] Normal Phase Flash Chromatography at Teledyne Isco Rf200 apparatus, using the Macherey-Nagel Flash cartridges (40-63 μm). Reverse phase chromatography using Teledyne Isco On the Rf200 device C18 Duo 30 μm column or Interchim PuriFlash RP-AQ (30 μm) column; or Agilent 1100 preparative binary HPLC system.

[0541] Chemical reactions and compound characterization were monitored and analyzed by thin layer chromatography (Macherey-Nagel) using pre-coated 40-63 μm silica gel, HPLC-UV (Agilent 1100 system), or UHPLC-UV / MS (equipped with Bruker Impact II TM Thermo UltiMate 3000 UHPLC system equipped with a Q-ToF mass spectrometer or Agilent 1260 HPLC system equipped with a Bruker MicroTOF-QII mass spectrometer).

[0542] 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 an Agilent Zorbax SB-Aq 4.6x150mm 5μm (room temperature). The linear gradient was 0% B to 50% B in 30 minutes, followed by a 5 minute hold at 50% B. The flow rate was 1.0 mL / min.

[0543] 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 120EC-C18 3.0x50mm 2.7μm (room temperature). The linear gradient was 5% B to 80% B in 9 minutes, followed by a 1 minute hold at 80% B. The flow rate was 0.8 mL / min.

[0544] 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 120EC-C18 3.0x50mm 2.7μm (room temperature). The linear gradient was 5% B to 80% B in 20 minutes, followed by a 2 minute hold at 80% B. The flow rate was 0.8 mL / min.

[0545] HPLC method 4: Thermo UltiMate 3000 UHPLC system + Bruker Impact II TM Q-ToF mass spectrometer. Mobile phase A was water + 0.1% formic acid, and mobile phase B was acetonitrile + 0.1% formic acid. Column was Agilent PLRP-S 2.1x150mm 8μm (80°C). Linear gradient 10% B to 50% B in 25 minutes. Flow rate 0.4mL / min. UV detection monitored at 280nm. Q-ToF mass spectrometer in the m / z range 500-3500 (ESI + ) are used. Use Bruker The MaxEnt algorithm included in the software deconvolutes the data.

[0546] HPLC Method 5 (Preparative): Teledyne Isco with DAD detection Rf200 binary MPLC system. Mobile phase A was water + 0.1% TFA, and mobile phase B was acetonitrile. The reusable chromatography column was C18 Duo 30 μm (30 g). Linear gradient was 10% B to 50% B in 35 min, followed by a 5 min hold at 50% B. Flow rate was 25 mL / min.

[0547] HPLC method 6 (preparative method): Agilent 1100 preparative binary HPLC system equipped with dual loop autosampler, DAD detection and fraction collector. Mobile phase A was water + 0.1% TFA, and mobile phase B was acetonitrile. Column was Waters SunFire C18 OBD Prep Column, 5 μm, 19 mm x 250 mm (room temperature). Linear gradient 10% B to 60% B in 40 min, followed by a 5 min hold at 60% B. Flow rate 25 mL / min.

[0548] 1.1) Monodisperse polysarcosine intermediate

[0549] 1.1.1) General Methods

[0550] The on-resin synthesis of monodisperse polysarcosine was achieved using a submonomer synthesis iterative procedure (as described in WO2019081455) for Rink amide and 2-chlorotrityl resins, or using a commercial Fmoc-Sar-Sar-OH class II peptide building block (CAS#2313534-20-0) for Wang resin following the classical Fmoc / SPPS method. Due to the formation of diketopiperazine, the on-resin dimerization stage (n=2) is avoided. All synthesis yields are reported based on the initial Fmoc-sarcosine loading indicated by the manufacturer. Unless otherwise stated, all reactions were performed at room temperature. Rink amide, 2-chlorotrityl or Wang polystyrene 1% DVB 100-200 mesh resin (Christ of Senn Laboratories) pre-loaded with the first Fmoc-sarcosine residue was used (typically with an initial loading of 0.6-1 mmol / g).

[0551] 1.1.2) Extension of polysarcosine

[0552] Rink amide, 2-chlorotrityl or Wang resin pre-loaded with Fmoc-sarcosine was treated with 20% piperidine in DMF solution (1 mL per 100 mg resin) at room temperature for 15 minutes twice. The resin was then washed with DMF (4 times) and DCM (4 times). A DMF solution of Fmoc-Sar-Sar-OH (3 equivalents), HATU (2.9 equivalents) and DIPEA (6 equivalents) was added to the resin (1 mL per 100 mg 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 with 20% piperidine in DMF solution (1 mL per 100 mg resin) at room temperature for 15 minutes twice. The resin was then washed with DMF (4 times) and DCM (4 times).

[0553] For the synthesis on Rink amide or 2-chlorotrityl resin, the classical submonomer synthesis procedure is then used, as described in WO2019081455. The extension of n=3 polysarcosine oligomers is performed by alternating bromoacetylation and amine displacement steps until the desired length is obtained. The bromoacetylation step is performed by adding 10 equivalents of bromoacetic acid and 13 equivalents of diisopropylcarbodiimide in DMF (2 mL per 100 mg of resin). The mixture is stirred for 30 minutes, drained and washed with DMF (4 times). For the amine displacement step, 40% (wt) aqueous methylamine solution (1.5 mL per 100 mg of resin) is added, and the container is shaken for 30 minutes, drained and washed with DMF (4 times) and DCM (4 times).

[0554] For the synthesis on Wang resin, the classic Fmoc / SPPS procedure was used. The extension of n=3 polysarcosine oligomers was performed by iterative coupling of Fmoc-Sar-Sar-OH class II peptide building blocks (CAS#2313534-20-0). A DMF solution of Fmoc-Sar-Sar-OH (3 equiv.), HATU (2.9 equiv.) and DIPEA (6 equiv.) was added to the resin (1 mL per 100 mg resin). The reaction vessel was agitated for 90 minutes, and the resin was washed thoroughly with DMF (4 times) and DCM (4 times). The resin was then treated with 20% piperidine in DMF (1 mL per 100 mg resin) for 15 minutes twice at room temperature. The resin was washed with DMF (4 times) and DCM (4 times). This coupling / Fmoc deprotection cycle was repeated until the desired polysarcosine length was obtained. If necessary, the last coupling was performed using commercial Fmoc-Sar-OH amino acids instead of Fmoc-Sar-Sar-OH dipeptide units in order to obtain a uniform length of the final polysarcosine.

[0555] 1.1.3) Final resin functionalization, optional end-capping, resin cleavage and purification of polysarcosine

[0556] When the desired length of the polysarcosine monodisperse oligomer on the resin is reached, an orthogonal chemical functionalization is performed. It is optionally followed by a final end-capping with an Fmoc-amino acid (e.g., Fmoc-Gly-OH, Fmoc-β-Ala-OH, Fmoc-amino-3,6-dioxa-octanoic acid, Fmoc-9-amino-4,7-dioxa-nonanoic acid). The Fmoc protecting group at the N-terminus of the end-capped final compound can be removed before or after resin cleavage, depending on the orthogonal functionalization chemistry used (see below).

[0557] 1.1.3.1) Polysarcosine functionalized with 2-azidoethyl-1-amine

[0558] To Rink or 2-chlorotrityl resin, 10 equivalents of bromoacetic acid and 13 equivalents of diisopropylcarbodiimide in DMF (2 mL per 100 mg resin) were added. The mixture was agitated for 30 minutes, drained and washed with DMF (4 times). 3 moles of 2-azidoethyl-1-amine in DMF (1 mL per 100 mg resin) were added, and the container was vibrated for 45 minutes, drained and washed with DMF (4 times) and DCM (4 times). It was followed by 1 hour of Fmoc-Gly-OH coupling (5 equivalents of Fmoc-Gly-OH, 4.9 equivalents of HATU, 10 equivalents of DIPEA in DMF (1 mL per 100 mg resin), and Fmoc deprotection with 20% piperidine in DMF (1 mL per 100 mg resin) at room temperature for 15 minutes twice. The resin was washed with DMF (4 times) and DCM (4 times).

[0559] The final polysarcosine compound was cleaved from the resin (100% TFA 2x 30 min for Rink and Wang resins, 20% TFA in DCM 2x 15 min for 2-chlorotrityl resin). The resin was filtered and the volatiles removed under reduced pressure to provide The crude product was purified on a RP-AQ (30 μm) column. Mobile phase A was water + 0.1% TFA and mobile phase B was acetonitrile.

[0560] 1.1.3.2) Polysarcosine functionalized with glutamic acid side

[0561] To Rink, Wang or 2-chlorotrityl resin was added a DMF solution of Fmoc-Glu(OAll)-OH (3 eq.), HATU (2.9 eq.), and DIPEA (6 eq.) (1 mL per 100 mg resin). The reaction vessel was agitated for 90 minutes, and the resin was washed thoroughly with DMF (4 times) and DCM (4 times). The resin was then treated with 20% piperidine in DMF (1 mL per 100 mg resin) twice for 15 minutes at room temperature. The resin was washed with DMF (4 times) and DCM (4 times). It was followed by a 1 hour coupling with a DMF solution of Fmoc-amino-3,6-dioxanoic acid (3 eq.), HATU (2.9 eq.), DIPEA (6 eq.) (1 mL per 100 mg resin). The resin was washed with DMF (4 times), DCM (4 times). The Alloc protecting group is removed by treating with a DCM solution containing 0.25 equivalents of Pd(PPh3)4 and 20 equivalents of phenylsilane (gently stirred under an argon stream) for 2 times for 30 minutes. The resin is then washed with DMF (5 times) and DCM (5 times). Optionally, N-hydroxysuccinimide (NHS) ester is introduced into the carboxylic acid side chain of the final polysarcosine compound by treating with a DMF solution containing 50 equivalents of DIC and 60 equivalents of N-hydroxysuccinimide (1.5 mL per 100 mg of resin) for 90 minutes. The resin is then washed with DMF (4 times) and DCM (4 times).

[0562] The final polysarcosine compound was cleaved from the resin (100% TFA 2x 30 min for Rink and Wang resins, 20% TFA in DCM 2x 15 min for 2-chlorotrityl resin). The resin was filtered and the volatiles removed under reduced pressure to provide The crude product was purified on a RP-AQ (30 μm) column. Mobile phase A was water + 0.1% TFA and mobile phase B was acetonitrile.

[0563] 1.1.4) Final polysarcosine intermediate

[0564] The obtained compounds are listed in Table 6 below.

[0565]

[0566]

[0567] 1.2) Synthesis of intermediate compounds

[0568] 1.2.1) Synthesis of compound INT1

[0569]

[0570] Compound INT1 was synthesized following the procedure described in patent application WO2019081455. The compound is an equimolar mixture of diastereomers (stereoisomerogenic centers are indicated by asterisks).

[0571] 1.2.2) Synthesis of compounds INT2, INT2-S and INT2-R

[0572]

[0573] 1.2.2.1) Synthesis of tert-butyl (2-hydroxy-2-(4-hydroxy-3-nitrophenyl)ethyl) carbamate

[0574] (±)-octopamine hydrochloride (1690mg / 11mmol) is weighed into a round-bottomed flask and suspended in 4mL distilled water. The flask is cooled at 0°C and 4mL pre-cooled 65% nitric acid solution is slowly added. The reaction is kept at 0°C for 20 minutes, showing complete mononitration of the raw material as evaluated by HPLC. The contents of the flask are transferred to a 250mL pre-cooled conical flask and slowly neutralized at 0°C with saturated NaHCO solution (about 50mL) until a pH value of 8-9 is reached. Then 30mL dioxane is added, followed by Boc O (7202mg / 13.2mmol). The reaction is then allowed to reach room temperature and stirred overnight. The reactant is then diluted with EtOAc, and washed 3 times with saturated citric acid solution, and washed 1 time with saturated NaCl solution. The organic phase was dried over MgSO4, filtered and evaporated under vacuum to provide a crude product which was purified by chromatography on silica gel (petroleum ether / EtOAc, gradient from 70:30 to 20:80) to give the title compound (1320 mg / 40%) as a thick yellow to 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 min.

[0575] 1.2.2.2 Synthesis of (2S, 3R, 4S, 5S, 6S)-2-(4-(2-((tert-butyloxycarbonyl)amino)-1-hydroxyethyl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate

[0576] In a round bottom flask, AgCO (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. The reaction was then filtered on celite, diluted with EtOAc, and washed 3 times with saturated citric acid solution and 1 time with saturated NaCl solution. The organic phase was dried over MgSO4, filtered and evaporated under vacuum to provide a crude product, which was purified by chromatography on silica gel (petroleum ether / EtOAc, gradient from 70:30 to 30:70) to provide the title compound as a yellow foam (244 mg / 48%). 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 min.

[0577] 1.2.2.3 Synthesis of (2S,3R,4S,5S,6S)-2-(4-(2-((tert-butyloxycarbonyl)amino)-1-(((4-nitrophenoxy)carbonyl)oxy)ethyl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate

[0578] 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 anhydrous 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 on a 0.45 μm PTFE filter and purified by chromatography on silica gel (petroleum ether / EtOAc, gradient from 85:15 to 30:70) to provide the title compound as a yellow foam (380 mg / 90%). NMR (300MHz, DMSO-d6) δ8.39-8.26 (m, 2H), 7.93 (d, J=2.2Hz, 1H), 7.75 (d, J=8.8Hz, 1H) , 7.55 (dd, J=9.2, 1.2Hz, 2H), 7.46 (d, J=8.8Hz, 1H), 7.20 (d, J=4.8Hz, 1H), 5.77 (dd, J= 5.7 (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 min.

[0579] 1.2.2.4) Synthesis of compound INT2

[0580] 381 mg (0.49 mmol) of the previous compound (2S,3R,4S,5S,6S)-2-(4-(2-((tert-butyloxycarbonyl)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 exitecan mesylate and 51 mg (0.38 mmol) of HOBt were dissolved in 5 mL of anhydrous DMF / pyridine 85:15 (v / v) mixture. 53.5 mg (0.51 mmol) of DIPEA was added. The reaction was stirred at 40° C. for 2 hours and the volatiles were evaporated under reduced pressure. The crude residue was purified by chromatography on silica gel (DCM / MeOH gradient from 99:1 to 95:5) to provide 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 / light green solid. ESI+[M+Na]+ = 1098.3. HPLC Method 3 Retention time = 14.7 and 14.9 minutes (mixture of diastereomers).

[0581] 355mg (0.33mmol) of this intermediate compound was dissolved in 8mL MeOH / THF 75:25 at 0°C. LiOH monohydrate (138mg / 3.3mmol) was dissolved in water (1mL) 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 (258mg / 4.3mmol) and concentrated under reduced pressure. The resulting crude product was redissolved with TFA / DCM (30:70v / v) solution at 0°C and stirred at room temperature for 20 minutes. The volatiles were evaporated under reduced pressure, the crude residue was dissolved in water / ACN (1:1v / v) solution, and purified using HPLC preparation method 5 to provide 172mg (62%) of compound INT2 as a yellow solid. ESI+[M+H]+=836.2. HPLC method 3 retention time=7.3 and 7.8 minutes (diastereoisomer mixture).

[0582]

[0583] 1.2.2.5.1) Chiral separation of a racemic mixture of tert-butyl (2-hydroxy-2-(4-hydroxy-3-nitrophenyl)ethyl)carbamate

[0584] At Teledyne Isco On the Rf200 system, use Chiral separation of racemic tert-butyl (2-hydroxy-2-(4-hydroxy-3-nitrophenyl)ethyl) carbamate (synthesized as previously described) was performed on an IC MPLC column 30x100 mm, 20 μm (Daicel catalog #83M73). 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 of the two enantiomers after separation was over 80%.

[0585] The retention time of tert-butyl (S)-(2-hydroxy-2-(4-hydroxy-3-nitrophenyl)ethyl)carbamate was 15 minutes, and the retention time of tert-butyl (R)-(2-hydroxy-2-(4-hydroxy-3-nitrophenyl)ethyl)carbamate was 25 minutes.

[0586] To determine the absolute configuration, the phenolic position of the two enantiomers was esterified with 1.2 molar equivalents of 4-nitrobenzoyl chloride and 2 molar equivalents of triethylamine in anhydrous THF. The compound was purified by chromatography on silica gel (petroleum ether / EtOAc, gradient from 90:10 to 10:90) to provide 4-(2-((tert-butoxycarbonyl)amino)-1-hydroxyethyl)-2-nitrophenyl 4-nitrobenzoate. 1H NMR (300MHz, DMSO-d6) δ8.51 (d, J=9.1Hz, 2H), 8.44 (d, J=9.1Hz, 2H), 8.19 (d, J=1.9Hz, 1H), 7.88 (d, J=10.2Hz, 1H), 7.7 2 (d, J=8.4Hz, 1H), 6.93 (t, J=5.9Hz, 1H), 5.84 (d, J=4.7Hz, 1H), 4.86-4.76 (m, 1H), 3.24 (t, J=6.1Hz, 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 allowed to slowly evaporate for 3 weeks to induce the formation of crystals) was confirmed by X-ray crystallography. Bulk crystals were fixed to nylon loops in perfluoroether oil. Data were collected using Xcalibur, Atlas, Gemini superdiffractometers equipped with Oxford Cryosystems cryogenic devices operating at T = 150.00 (5) K. Data were measured using ω scanning using Cu Kα radiation. The structure was resolved using the ShelXT solution program using a dual approach and by using Olex2 (OV Dolomanov et al., Olex2: A complete structure solution, refinement and analysis program, J. Appl. Cryst., 2009, 42, 339-341) as a graphical interface. The model was refined using full-matrix least squares minimization on F2 with ShelXL 2018 / 3 (Sheldrick, GM, Crystal structure refinement with ShelXL, ActaCryst., 2015, C71, 3-8).

[0587] 1.2.2.5.2) Synthesis of stereopure INT2-S and INT2-R compounds

[0588] Stereopure compounds INT2-S and INT2-R were synthesized as described in the previous section 1.2.2 without any significant changes in reaction conditions, reactivity or overall yield.

[0589] Final purification using HPLC preparation method 5 provided 33 mg of compound INT2-S as a yellow solid. ESI+[M+H]+ = 836.2. HPLC method 3 retention time = 7.3 minutes.

[0590] Final purification using HPLC preparation method 5 provided 21 mg of compound INT2-R as a yellow solid. ESI+[M+H]+ = 836.2. HPLC method 3 retention time = 7.8 minutes.

[0591] 1.2.3) Synthesis of compound INT3

[0592]

[0593] 1.2.3.1) Synthesis of Ac-Val-Ala-OH (acetyl-L-valyl-L-alanine)

[0594] To a solution of L-alanine benzyl ester hydrochloride (542 mg / 2.5 mmol) in 30 mL 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 sequentially added. The mixture was then cooled to 0° C. and EDC-HCl (530 mg / 2.75 mmol) was added. The resulting mixture was stirred overnight at 0° C. The reaction was diluted with 20 mL 2M HCl and the layers were separated. The organic phase was washed twice with 2M HCl, twice with saturated NaHCO solution, and once with saturated NaCl solution. The organic phase was dried over MgSO, filtered and evaporated under vacuum to provide 714 mg (89%) of acetyl-L-valyl-L-alanine benzyl ester as a white solid intermediate.

[0595] This intermediate is dissolved in 10mL EtOAc / MeOH 1: 1 (v / v) and transferred into a stainless steel hydrogenation reactor. After the first argon purge, a catalytic amount of 5%wt Pd / C is added. The reactor is then purged twice with H2 and kept overnight at room temperature at 10 bar of H2 pressure. After filtering the reaction with a 0.45 μm PTFE filter and removing the solvent under vacuum, a quantitative pure acetyl-L-valyl-L-Alanine as a white solid is obtained. 1H NMR (300MHz, DMSO-d6) δ12.45 (s, 1H), 8.23 ​​(d, J = 6.9Hz, 1H), 7.85 (d, J = 9.0Hz, 1H), 4.25-4.11 (m, 2 H), 1.94 (dt, J=13.6, 6.8Hz, 1H), 1.85 (s, 3H), 1.26 (d, J=7.3Hz, 3H), 0.85 (dd, J=12.1, 6.8Hz, 6H).

[0596] 1.2.3.2 Synthesis of (2S)-2-acetylamino-N-((2S)-1-((4-(1-hydroxybut-3-yn-1-yl)phenyl)amino)-1-oxoprop-2-yl)-3-methylbutanamide

[0597] In a round-bottom flask, 420 mg (2.60 mmol) of 1-(4-aminophenyl)but-3-yn-1-ol (synthesized following 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. The volatiles were then evaporated under reduced pressure, and the crude residue was dry loaded and purified by chromatography on silica gel (DCM / MeOH gradient from 99: 1 to 85: 15) to provide 809 mg (83%) of the title compound as a white solid. 1H NMR (300MHz, DMSO-d6) δ9.84 (s, 1H), 8.18 (d, J = 7.0Hz, 1H), 7.90 (d, J = 8.6Hz, 1H), 7.5 3(d, J=8.6Hz, 2H), 7.28 (d, J=8.6Hz, 2H), 5.44 (d, J=4.4Hz, 1H), 4.62 (q, J=6.2Hz, 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 minutes.

[0598] 1.2.3.3) Synthesis of 1-(4-((S)-2-((S)-2-acetylamino-3-methylbutyrylamino)propionylamino)phenyl)but-3-yn-1-yl(4-nitrophenyl)carbonate

[0599] 94 mg (0.25 mmol) of (2S)-2-acetylamino-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 were added, and the reaction mixture was stirred at room temperature overnight. The volatiles were removed under reduced pressure, and the crude residue was purified by chromatography on silica gel (DCM / MeOH gradient from 99:1 to 90:10) to provide 118 mg (87%) of the title compound as a yellow solid. 1H NMR (300MHz, DMSO-d6) δ9.99 (s, 1H), 8.35-8.26 (m, 2H), 8.22 (d, J=7.0Hz, 1H), 7.89 (d , J=8.6Hz, 1H), 7.63 (d, J=8.7Hz, 2H), 7.58-7.48 (m, 2H), 7.43 (d, J=8.7Hz, 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 minutes.

[0600] 1.2.3.4) Synthesis of compound INT3

[0601] 43 mg (0.081 mmol) of the previous compound 1-(4-((S)-2-((S)-2-acetylamino-3-methylbutyrylamino)propionylamino)phenyl)but-3-yn-1-yl(4-nitrophenyl)carbonate, 55.5 mg (0.11) exotecan mesylate and 11.0 mg (0.08 mmol) of HOBt were dissolved in 2 mL of anhydrous DMF / pyridine 85:15 (v / v) mixture. 11.5 mg (0.09 mmol) of DIPEA was added. The reaction was stirred at 40° C. for 3 hours. After removing the volatiles under reduced pressure, the reaction mixture was purified by chromatography on silica gel (DCM / MeOH gradient from 99:1 to 95:5) to provide 42 mg (62%) of compound INT3 as a yellow / light green solid. ESI+[M+H]+=835.3. HPLC Method 2 Retention time = 6.50 and 6.60 minutes (mixture of diastereomers).

[0602] 1.2.4) Synthesis of compounds INT4, INT4-S and INT4-R

[0603]

[0604] 1.2.4.1) Synthesis of tert-butyl (2-(4-aminophenyl)-2-hydroxyethyl) carbamate

[0605] A MeOH solution containing 911 mg (3.23 mmol) of commercially available tert-butyl (2-hydroxy-2-(4-nitrophenyl)ethyl) carbamate (CAS# 939757-25-2) was transferred to a stainless steel hydrogenation reactor. After the first 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 under 10 bar H2 pressure for 5 hours while the reaction was kept stirring. After filtering the reaction with a 0.45 μm PTFE filter and MeOH removal under vacuum, 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 minutes.

[0606] 1.2.4.2) Synthesis of tert-butyl (2-(4-((S)-2-((S)-2-acetylamino-3-methylbutyrylamino)propionylamino)phenyl)-2-hydroxyethyl)carbamate

[0607] 150 mg (0.60 mmol) of the previous compound tert-butyl (2-(4-aminophenyl)-2-hydroxyethyl) carbamate, 222 mg (0.71 mmol) Fmoc-Ala-OH and 81 mg (0.62 mmol) DIPEA were dissolved in 5 mL anhydrous DMF. 181 mg (0.71 mmol) HATU was added and the reaction was stirred at room temperature overnight. The volatiles were then removed under reduced pressure and the crude residue was purified by chromatography on silica gel (DCM / MeOH gradient from 100:0 to 90:10) to quantitatively provide the first intermediate tert-butyl (2-(4-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)phenyl)-2-hydroxyethyl)carbamate, which directly participated in the Fmoc deprotection. HPLC Method 2 Retention time = 7.7 minutes.

[0608] Tert-butyl (2-(4-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)phenyl)-2-hydroxyethyl)carbamate was dissolved in 5 mL DMF / piperidine 9:1 (v / v) and stirred at room temperature for 15 minutes. The volatiles were then removed under reduced pressure and the crude residue was purified by chromatography on silica gel (DCM / MeOH gradient from 99:1 to 80:20) to provide 130 mg (68% in two steps) of the second intermediate tert-butyl (2-(4-((S)-2-aminopropionamido)phenyl)-2-hydroxyethyl)carbamate as a white foamy solid. HPLC Method 2 Retention time = 3.75 minutes.

[0609] 130 mg (0.40 mmol) of tert-butyl (2-(4-((S)-2-aminopropionylamino)phenyl)-2-hydroxyethyl)carbamate and 124 mg (0.48 mmol) of commercially available Ac-Val-OSu (CAS#56186-37-9) were dissolved in 3 mL of anhydrous DMF, and the reaction mixture was stirred at room temperature overnight. The volatiles were then removed under reduced pressure and the crude residue was triturated with 3 mL of DCM to provide 95 mg (51%) of the title compound as a white solid. 1H NMR (300MHz, DMSO-d6) δ9.82 (s, 1H), 8.16 (d, J = 7.1Hz, 1H), 7.88 (d, J = 8.6Hz, 1H) , 7.53 (d, J = 8.4Hz, 2H), 7.22 (d, J = 8.5Hz, 2H), 6.73-6.58 (m, 1H), 5.27 (d, J = 4.4Hz , 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 minutes.

[0610] 1.2.4.3) Synthesis of tert-butyl (2-(4-((S)-2-((S)-2-acetylamino-3-methylbutyrylamino)propionylamino)phenyl)-2-(((4-nitrophenoxy)carbonyl)oxy)ethyl)carbamate

[0611] 286 mg (0.62 mmol) of tert-butyl (2-(4-((S)-2-((S)-2-acetylamino-3-methylbutyrylamino) propionylamino) 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 were added, and the reaction mixture was stirred at room temperature for 3 hours. The volatiles were removed under reduced pressure, and the crude residue was purified by chromatography on silica gel (DCM / MeOH gradient from 99:1 to 90:10) to provide 336 mg (87%) of the title compound as a yellow solid. 1H NMR (300MHz, DMSO-d6) δ9.99 (s, 1H), 8.36-8.26 (m, 2H), 8.22 (d, J=6.9Hz, 1H), 7.89 (d, J=8.6H z, 1H), 7.63 (d, J = 8.6Hz, 2H), 7.56-7.46 (m, 2H), 7.34 (d, J = 8.6Hz, 2H), 7.22 (t, J = 5.6Hz, 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 minutes.

[0612] 1.2.4.4) Synthesis of compound INT4

[0613] 231 mg (0.37 mmol) of the previous compound tert-butyl (2-(4-((S)-2-((S)-2-acetylamino-3-methylbutyrylamino)propionylamino)phenyl)-2-(((4-nitrophenoxy)carbonyl)oxy)ethyl)carbamate, 150 mg (0.28) exitecan mesylate and 38 mg (0.28 mmol) of HOBt were dissolved in 5 mL of anhydrous DMF / pyridine 85:15 (v / v) mixture. 40 mg (0.31 mmol) of DIPEA was added. The reaction was stirred at 40° C. for 3 hours and the volatiles were evaporated under reduced pressure. The crude residue was purified by chromatography on silica gel (DCM / MeOH gradient from 99:1 to 90:10). 220 mg (85%) of the intermediate compound 1-(4-((S)-2-((S)-2-acetylamino-3-methylbutyrylamino)propionylamino)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 were obtained as a brown-yellow solid. ESI+[M+H]+=926.4. HPLC method 2 retention time=6.7 and 6.8 minutes (mixture of diastereomers).

[0614] The obtained solid was redissolved with TFA / DCM (30:70 v / v) solution at 0°C and stirred at room temperature for 20 minutes. The volatiles were evaporated under reduced pressure, the crude residue was dissolved in water / ACN (1:1 v / v) solution, and purified using HPLC preparation method 5 to provide 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 minutes (diastereoisomer mixture).

[0615]

[0616] 1.2.4.5.1) Chiral separation of a racemic mixture of tert-butyl (2-(4-aminophenyl)-2-hydroxyethyl) carbamate

[0617] At Teledyne Isco On the Rf200 system, use IC MPLC column 30x100mm, 20μm (Daicel catalog #83M73) was used to perform the chiral separation of racemic tert-butyl (2-(4-aminophenyl)-2-hydroxyethyl) carbamate. The mobile phase was DCM + 0.2% (v / v) EtOH (isocratic gradient). The flow rate was 12mL / min. The sample solvent was DCM + 0.2% (v / v) EtOH. The mass recovery of the two enantiomers after separation was over 75%.

[0618] 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 (previously dissolved in a 1:1 mixture of heptane / ethanol and allowed to slowly evaporate for 1 week to induce the formation of crystals) was confirmed by X-ray crystallography. The bulk crystals were fixed to a nylon loop in perfluoroether oil. Data were collected using an Xcalibur, Atlas, Gemini superdiffractometer equipped with an Oxford Cryosystems cryogenic device operating at T=150.00(10)K. The data were measured using Cu Kα radiation using ω scanning. The structure was solved using a dual approach and by using Olex2 (OV Dolomanov et al., Olex2: A complete structure solution, refinement and analysis program, J. Appl. Cryst., 2009, 42, 339-341) as a graphical interface using the ShelXT solution program. The model was refined using ShelXL 2018 / 3 (Sheldrick, GM, Crystal structure refinement with ShelXL, Acta Cryst., 2015, C71, 3-8) using full matrix least squares minimization on F2.

[0619] 1.2.4.5.2) Synthesis of stereopure INT4-S and INT4-R compounds

[0620] Stereopure compounds INT4-S and INT4-R were synthesized as described in the previous section 1.2.4 without any significant changes in reaction conditions, reactivity or overall yield.

[0621] Final purification using HPLC preparation method 5 provided 54 mg of compound INT4-S as a yellow solid. ESI+[M+H]+ = 826.4. HPLC method 3 retention time = 8.45 minutes.

[0622] Final purification using HPLC preparative method 5 provided 46 mg of compound INT4-R as a yellow solid. ESI+[M+H]+ = 826.4. HPLC method 3 retention time = 8.90 min.

[0623] 1.3) Synthesis of drug-linker

[0624] 1.3.1) Synthesis of Glucuronide-Based Drug-Linkers

[0625] 1.3.1.1) Synthesis of compound LNK1

[0626]

[0627] 44.5 mg (0.049 mmol) of compound PSR1 (azide-polysarcosine intermediate) and 27.5 (0.033 mmol) of compound INT1 (alkyne-payload) were dissolved in a 1:1 (v / v) solution of 100 mM PBS (pH=7.5) and DMSO to reach a concentration of 0.060 M of compound INT1. Freshly prepared CuSO4 pentahydrate and sodium ascorbate solution (approximately 250 mg / mL) were then added sequentially to the reaction vial to reach 0.08 molar equivalents of Cu and 1 molar equivalent of sodium ascorbate (based on the molar equivalent of compound INT 1 in the reaction mixture). The reaction was purged with argon and stirred at 40°C. The reaction was monitored by HPLC and was completed in less than 2 hours. The reaction mixture was then stirred in water / ACN The mixture was diluted with 0.1% TFA solution in 1:1 (v / v) and purified using HPLC preparation method 5 to provide 44 mg (77% based on the starting compound INT1) of the intermediate compound (2S,3S,4S,5R,6S)-6-(4-(2-(1-(35-amino-3-glycyl-6,9,12,15,18,21,24,27,30,33-decamethyl-5,8,11,14,17,20,23,26,29,32,35-undecaoxo-3,6,9,12,1 =5,5-dihydro-1H-pyran-2-carboxylic acid. ESI+[M+H]+=1755.7. HPLC Method 3 Retention time=7.51 min and 7.82 min (mixture of diastereomers).

[0628] 26.0 mg (0.015 mmol) of this compound and 4.11 mg (0.016 mmol) of maleimidoacetic acid N-hydroxysuccinimide ester were dissolved in anhydrous DMF (0.1 M concentration of maleimide compound). 2.25 mg (0.022 mmol) of triethylamine were added and the reaction was stirred for 2 hours until complete conversion of the reaction was observed by HPLC. The reaction mixture was then diluted with a 1% TFA solution in water / ACN 1:1 (v / v) and purified using HPLC preparation method 6 to provide 16.0 mg (57%) of compound LNK1 as a yellow solid. ESI+[M+H]+=1892.7. HPLC method 3 retention time=7.95 minutes and 8.20 minutes (equimolar diastereoisomer mixture).

[0629] 1.3.1.2) Synthesis of compound LNK2

[0630]

[0631] 409 mg (0.31 mmol) of compound PSR3 (NHS activated polysarcosine intermediate) and 172 mg (0.21 mmol) of compound INT2 (NH2 payload) were dissolved in anhydrous DMF in a vial (0.080 M concentration of compound INT2). 83.5 mg (0.83 mmol) of triethylamine was added and the reaction was stirred at room temperature for 30 minutes. After complete conversion of the reaction as observed by HPLC, piperidine was added directly to the reaction vial to achieve an 8% (v / v) solution of piperidine in DMF. The reaction was then stirred at room temperature for 5-10 minutes until complete deprotection of Fmoc was observed by HPLC. The reaction was washed with water / ACN A 10% TFA solution in a 1:1 (v / v) ratio was slowly neutralized and purified using HPLC preparation method 5 to provide 254 mg (67% yield based on the starting compound INT2) of the intermediate compound (2S, 3S, 4S, 5R, 6S)-6-(4-((9S)-40-amino-9-(2-(2-(2-aminoethoxy)ethoxy)acetylamino)-1-(((1S, 9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H- =1,2H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl))amino)-11,14,17,20,23,26,29,32,35,38-decamethyl-1,6,10,13,16,19,22,25,28,31,34,37,40-tridecaoxo-2-oxa-5,11,14,17,20,23,26,29,32,35,38-undecaazatetracontan-3-yl)-2-nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid. ESI+[M+H]+=1819.7. HPLC Method 2 Retention time=4.60 minutes and 4.69 minutes (equimolar mixture of diastereomers).

[0632] 254 mg (0.14 mmol) of this compound and 39.1 mg (0.15 mmol) of maleimidoacetic acid N-hydroxysuccinimide ester were dissolved in anhydrous DMF (0.1 M concentration of maleimide compound). 22.6 mg (0.22 mmol) of triethylamine were added and the reaction was stirred for 1 hour until complete conversion of the reaction as observed by HPLC. The reaction mixture was then diluted with a 1% TFA solution in water / ACN 1:1 (v / v) and purified using HPLC preparation method 6 to provide 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 minutes and 8.1 minutes (equimolar diastereoisomer mixture).

[0633] 1.3.1.3) Synthesis of compounds LNK2-S and LNK2-R

[0634]

[0635] Compounds LNK-2-S and LNK2-R were synthesized as described above, using the same procedure as for compound LNK2. The starting materials were compounds INT2-S and INT2-R (NH2 payload) and compound PSR3 (NHS-activated polysarcosine intermediate), respectively.

[0636] 53.4 mg of compound LNK2-S were obtained as a yellow solid. ESI+[M+2H]2+=985.9. HPLC method 3 retention time=7.8 minutes.

[0637] 44.0 mg of compound LNK2-R were obtained as a yellow solid. ESI+[M+2H]2+=985.9. HPLC method 3 retention time=8.1 min.

[0638] 1.3.2) Synthesis of dipeptide-based drug-linkers

[0639] 1.3.2.1) Synthesis of compound LNK3

[0640]

[0641] Final compound LNK3 was synthesized as described above, using the same procedure as for compound LNK 1. The starting materials were compound PSR2 (azide-polysarcosine intermediate) and compound INT3 (alkyne-payload).

[0642] 14.0 mg (36% in two steps) of compound LNK3 were 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 diastereomers).

[0643] 1.3.2.2) Synthesis of compound LNK4

[0644]

[0645] Compound LNK4 was synthesized as described above, using the same procedure as for compound LNK2. The starting materials were compound PSR4 (NHS-activated polysarcosine intermediate) and compound INT4 (NH2 payload).

[0646] 34.9 mg (46% in two steps) of compound LNK4 were 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 diastereomers).

[0647] 1.3.2.3) Synthesis of compounds LNK4-S and LNK4-R

[0648]

[0649] Compounds LNK4-S and LNK4-R were synthesized as described above, using the same procedure as for compound LNK2. The starting materials were compounds INT4-S and INT4-R (NH2 payload) and compound PSR4 (NHS-activated polysarcosine intermediate), respectively.

[0650] 18.1 mg of compound LNK4-S were obtained as a yellow solid. ESI+[M+2H]2+=981.4. HPLC method 3 retention time=8.78 minutes.

[0651] 16.3 mg of compound LNK4-R were obtained as a yellow solid. ESI+[M+2H]2+=981.4. HPLC method 3 retention time=8.96 minutes.

[0652] Example 2: Preparation and characterization of antibody-drug conjugates

[0653] 2.1) Antibody production

[0654] The amino acid sequence of monoclonal antibody was obtained from the literature. The light chain (SEQ ID.10) and heavy chain (SEQ ID.11) sequences of fatugumab were obtained from the International Nonproprietary Names for Pharmaceutical Substances (INN) list 62 and WO2017151979 published in the 23rd volume, the 3rd issue of the World Health Organization (WHO) drug information in 2009. fatugumab LALA (heavy chain "LALA" SEQ ID.9 sequence) including Fc silent mutations L234A and L235A ("LALA") on the heavy chain (Wines et al. The Journal of Immunology May 15, 2000, 164 (10) 5313-5318) of fatugumab was also produced. The sequences of the light chain (SEQ ID. 17) and heavy chain (SEQ ID. 16) of trastuzumab were obtained from patent application WO2011106528. The human IgG1k non-binding isotype control was from catalog #HG1K (proprietary amino acid sequence) of Sino Biologicals. 150 mg) was purchased from Roche. (trastuzumab) was purchased from Daiichi Sankyo / AstraZeneca.

[0655] Monoclonal antibodies are produced by transient transfection of CHO K1 cells using field-recognized techniques (outsourced to Evitria AG, Switzerland). Using conventional (non-PCR-based) cloning techniques, cDNA is cloned into Evitria's vector system. Based on anion exchange chromatography, pDNA is prepared under low endotoxin conditions. DNA concentration is determined by measuring the absorbance at a wavelength of 260nm. The correctness of the sequence is 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) are used for antibody production. Seeds are grown in Evitria's proprietary animal component-free and serum-free culture medium. Cells are transfected with Evitria's proprietary transfection reagent. Supernatant is harvested by centrifugation and subsequent filtration (0.2 μm filter). Antibodies are purified using MabSelect SuRe protein A purification resin (Cytiva) and SEC preparative resin. The purity of the antibody material is confirmed by SDS-PAGE and size exclusion chromatography and exceeds 95%. Endotoxin levels were measured using the Charles River Endosafe PTS system.

[0656] 2.2) Preparation of Cysteine-Conjugated Antibody-Drug Conjugates

[0657] The antibody solution (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 1-2 hours. The reduced antibody was buffer exchanged with potassium phosphate 100 mM pH 7.4+1 mM EDTA by three rounds of dilution / centrifugation using an Amicon 30K centrifugal filter device (Millipore). For the final ~DAR4 ADC, 6 molar equivalents of drug-linker (from a 12 mM DMSO stock solution) were added to the antibody. For the final DAR8 ADC, 10-12 molar equivalents of drug-linker were added. The solution was incubated at room temperature for 30 minutes. The final conjugate was buffer exchanged / purified with PBS pH 7.4 buffer or histidine sucrose buffer (20 mM histidine buffer pH 6.0, 4% (w / v) sucrose + 75 mM NaCl) by four rounds of dilution / centrifugation using Amicon 30K centrifugal filter devices and sterile filtered (0.20 μm PES filter).

[0658] Final protein concentration was estimated spectrophotometrically at 280 nm using a Nanodrop One apparatus (Thermo Fisher Scientific).

[0659] 2.3) Characterization of Cysteine-Conjugated Antibody-Drug Conjugates

[0660] The resulting conjugate was characterized as follows:

[0661] Drug-Antibody Ratio (DAR) Evaluation by Reverse Phase Liquid Chromatography-Mass Spectrometry (RPLC-MS):

[0662] Denaturing RPLC-QToF analysis was performed using HPLC Method 4 described above in Example 1. Briefly, a mobile phase gradient of water / acetonitrile + 0.1% formic acid (0.4 mL / min) was used on an Agilent PLRP-S The conjugate was eluted on a 2.1x150mm 8μm (80°C) and detected using a Bruker Impact IITM Q-ToF mass spectrometer scanning the 500-3500 m / z range (ESI+). The MaxEnt algorithm included in the software deconvolutes the data.

[0663] Reverse Phase Liquid Chromatography (RPLC-UV):

[0664] Denaturing RPLC-UV analysis was also performed on an Agilent 1100 HPLC-DAD system using a slightly modified version of the above HPLC method 4. The mobile phase modifier 0.1% formic acid was replaced by 0.1% TFA and detection was performed using DAD UV absorbance only (no mass spectrometer detector).

[0665] Size Exclusion Chromatography (SEC):

[0666] SEC was performed on an Agilent 1100 HPLC system with an extra-column volume of less than 15 μL (equipped with a short section of 0.12 mm id peek tubing and a micro-UV flow cell). The column was Agilent AdvanceBioSEC 4.6x150mm 2.7μm (maintained at 30°C). The mobile phase was 100mM sodium phosphate and 200mM 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 to prevent bacterial growth. The flow rate was 0.35mL / min. UV detection was monitored at 280nm or any other relevant wavelength.

[0667] 2.4) Preparation of lysine-conjugated antibody-drug conjugates

[0668] A solution of antibody Mituximab or Fatuinumab (10 mg / mL in 100 mM KH2PO4 pH 8.0) was treated with a 12 mM DMSO solution of Sulfo-SPDB-DM4 CAS#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 with PBS pH 7.4 buffer or histidine sucrose buffer (20 mM histidine buffer pH 6.0, 4% (w / v) sucrose + 75 mM NaCl) by five rounds of dilution / centrifugation using an Amicon 30K centrifugal filter device and sterile filtered (0.20 μm PES filter).

[0669] Final protein concentration was estimated spectrophotometrically at 280 nm using a Nanodrop One apparatus (Thermo Fisher Scientific).

[0670] 2.5) Characterization of lysine-conjugated antibody-drug conjugates

[0671] The resulting conjugate was characterized as follows:

[0672] Evaluation of average drug-antibody ratio (DAR) by native SEC-MS:

[0673] Before analysis, ADC was prepared by adding 50 U Deglycosylation was performed using the (Genovis) enzyme. ADCs were separated on an Agilent AdvanceBio SEC 200A 1.9 μm 2.1x150 mm PEEK column (Catalog # PL1980-3201PK) maintained at 30°C. The column was equilibrated in 50 mM ammonium acetate + 10% (v / v) HPLC grade isopropanol. The flow rate was maintained at 0.075 mL / min during the run, and the ADCs typically eluted between 3.5 and 4.5 minutes. The flow rate and buffer composition were maintained after elution of the mAb or ADC. The column eluate was introduced to scan the 300-8000 m / z range (ESI + )'s Bruker Impact II TM The source capillary voltage was set to 4500 V. The source drying gas and nebulizing gas were set to 8.0 L / min and 25 Psi, respectively. The source drying temperature was set to 200 °C. The MaxEnt algorithm included in the software deconvoluted the ADC mass spectra, and the DAR was calculated using the ion intensity peak height of each ADC subspecies.

[0674] Size Exclusion Chromatography (SEC):

[0675] SEC was performed on an Agilent 1100 HPLC system with an extra-column volume of less than 15 μL (equipped with a short section of 0.12 mm id peek tubing and a micro-UV flow cell). The column was Agilent AdvanceBioSEC 4.6x150mm 2.7μm (maintained at 30°C). The mobile phase was 100mM sodium phosphate and 200mM 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 to prevent bacterial growth. The flow rate was 0.35mL / min. UV detection was monitored at 280nm or any other relevant wavelength.

[0676]

[0677]

[0678]

[0679]

[0680]

[0681] Example 3: General cell culture practice and animal experiments

[0682] The human cancer cell lines used in this project are purchased from 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 the experiment, follow the instructions from the original ATCC / DSMZ / ECACC supplier about cell culture medium and supplements, cryopreservation and subculture procedures, and cells follow good and established cell culture practices for cultivation. Cells are incubated at 37°C for no more than 2 months under 5% CO2 atmosphere.

[0683] All animal procedures were performed in accordance with EU Directive 86 / 609 / EEC. Experiments were performed under personal permission and in an animal care facility approved by the French Ministry of Agriculture. The study was approved by the local animal ethics committee (CECCAPP).

[0684] Example 4: Comparison of the Therapeutic Index of Glucuronide or Dipeptide Drug-Linker Designs in Rodents

[0685] The mouse therapeutic index of drug-linker constructs LNK1 (glucuronide-exitecan) and LNK3 (dipeptide Val-Ala-exitecan) was evaluated to compare the mouse therapeutic index of two enzyme-cleavable patterns glucuronide and dipeptide Val-Ala. Using model human HER2-targeted trastuzumab monoclonal antibody as the targeting part, T-GLC-EXA ADC and T-VA-EXA ADC were formulated. These ADCs were compared according to the in vivo efficacy in the HER2+ gastric cancer model and according to tolerability (therapeutic index). Since trastuzumab does not cross-react in mice, this assay provides useful information about the apparent toxicity of the drug-linker component of the ADC (excluding possible target-mediated toxicity).

[0686] Evaluation of in vivo efficacy: NCI-N87 gastric cancer cells were subcutaneously implanted into female SCID mice (4 weeks old). When the tumor had grown to approximately 150 mm 3ADC was administered once intravenously at a subcurative dose of 1 mg / kg (6 animals per group, allocated to minimize differences in initial tumor volume between groups). Tumor volume was measured every 3-5 days by caliper device and calculated using the formula (L x W 2 ) / 2. When the tumor volume exceeds 1000mm 3 When , the mice were killed.

[0687] Evaluation of pharmacokinetic profile: PK profile in rats (total concentration of antibody-drug conjugate based on mAb component over time) following a single intravenous 3 mg / kg dose of the conjugate. ADC was injected into female Sprague-Dawley rats (4-6 weeks old, Charles River) at 3 mg / kg via the tail vein (3 animals per group, randomly assigned). Blood was drawn into citrate tubes via retro-orbital bleeding at various time points, processed into plasma and stored at -80°C until analysis. Human IgG ELISA kit (Stemcell) was used according to the manufacturer's protocol. TM Technologies) to evaluate the concentration of ADC based on antibody components. 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 the ANALYZER® software (plug-in developed by Usansky et al., Department of Pharmacokinetics and Drug Metabolism, Allergan, Irvine, USA).

[0688] Evaluation of in vivo tolerability: Female SCID mice (n=3) were treated with a single intraperitoneal high dose of 200 mg / kg ADC compound. Mice were observed over the course of 12 days for weight loss or overt signs of toxicity. (de-trastuzumab) served as a positive control.

[0689] The results of this study are shown in Figure 1 T-GLC-EXA and T-VA-EXA showed similar in vivo efficacy ( Figure 1 A) and similar rat PK profiles ( Figure 1 B), but showed significant differences in mouse tolerance at high doses ( Figure 1 C). It was concluded that at least for use with the exotecan payload, the dipeptide Val-Ala cleavable pattern provides a better efficacy / tolerability profile. As such, this entity is preferred for other ADC constructs.

[0690] Example 5: Flow cytometric evaluation of the extracellular binding site of folate receptor alpha (FRa)

[0691] For FRa cell surface quantification, cells were incubated with PE anti-FOLR1 antibody (BioLegend catalog #908304) for 20 minutes at room temperature. TM Fixable Viability DyeeFluor TM Cell viability was assessed using the BIOMED® 780 kit (Thermo Fisher Scientific catalog #65-0865-18). Analysis was performed using a BD Fortessa flow cytometer controlled by BD FACSDiva software (BD Biosciences), and data were analyzed using FlowJo software (BD Bioscience).

[0692] Results are shown in Figure 2 The cell lines in this research project exhibited different levels of extracellular expression of FRa. BT-474 breast cancer cells do not express extracellular FRa and are therefore considered a negative control cell line in the context of this research project.

[0693] Example 6: In vitro cytotoxicity assay of exitecan mesylate against FRa+ cancer cell lines

[0694] The in vitro cytotoxicity of compound exitecan mesylate is evaluated on several FRa positive cancer cell lines.Cells were plated in 96-well plates (1000 to 10000 cells / well in 100 μL appropriate culture medium) at an appropriate density depending on the cell line, and incubated at 37°C for 24 hours.Serial dilutions of the test compound previously dissolved in the culture medium (50 μL) 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 continued for 1 to 2 hours at 37°C.The culture medium was then carefully removed, and the well contents were uniformly dissolved with acidified isopropanol.Absorbance values ​​were measured on a MultiskanTM Sky microplate reader (Thermo Scientific) using a wavelength of 570 nm (with a reference wavelength of 690 nm).IC50 concentration values ​​compared to untreated control cells were determined using inhibitory dose response curve fitting (GraphPad Prism 9).

[0695] Results are shown in Figure 3 Exotecan mesylate showed in vitro potency of sub-nM to low nM IC50, prompting us to investigate the use of this compound as an ADC payload for FRa-expressing malignancies.

[0696] Example 7: Recombinant FRα Binding Affinity by ELISA

[0697] Sandwich ELISA assays were performed using 96-well high-binding ELISA plates (Corning Inc., New York, NY, USA, catalog #3590). Plates were coated with 100 μL / well of recombinant human FRa protein (SinoBiological catalog #11241-H08H) at 2 μg / mL in PBS (pH 7.4); recombinant cynomolgus monkey FRa protein (SinoBiological catalog #90950-C08H); recombinant rat FRa protein (Sino Biological catalog #81073-R08H) or recombinant mouse FRa protein (SinoBiological catalog #50573-M08H) and incubated overnight at 4°C. After 2 washes with PBS-T (PBS + 0.05% Tween-20), the plates were blocked with 200 μL / well of incubation buffer (PBS-T + 0.1% BSA) at room temperature for 1 hour. The plate was washed 4 times with PBS-T, and 100 μL of a three-fold dilution series of the test compound (antibody or antibody-drug conjugate) was added. The plate was then incubated for 2 hours in the dark at room temperature. After 5 washes with PBS-T, the plate was incubated for 1 hour at room temperature with 100 μL / well of goat anti-human IgG (H+L) HRP conjugated antibody (Jackson Immunoresearch catalog #109-035-088), which was previously diluted 1:250 000 in incubation buffer. After 5 washes with PBS-T, TMB substrate solution (Thermo-Fisher catalog #N301) was added. Peroxidase activity was stopped with 0.18M H2SO4, and absorbance was read at 450nm (reference wavelength 650nm) using a Thermo Scientibic MultiSkan EX microplate reader. S-shaped fitting was performed using GraphPad Prism 9 software.

[0698] Results are shown in Figure 4 middle. Figure 4 A: All antibodies and ADCs show approximately EC 50=0.1 nM. No binding loss was observed between native antibodies and respective ADC constructs. No binding loss was observed between facilitation and facilitation LALA and their respective ADC constructs. No significant binding loss was observed between metuximab and M-SORAV ADC constructs. No human FRα binding was detected for the negative control ADC NEG-VA-EXA. Figure 4 B: F-LALA-VA-EXA binds equally to human and cynomolgus monkey recombinant FRa proteins, but does not bind to rat and mouse recombinant FRa proteins. As such, F-LALA-VA-EXA is not rodent cross-reactive, but cynomolgus monkey cross-reactive.

[0699] Example 8: Human FRα Binding Affinity by SPR

[0700] Surface plasmon resonance (SPR) experiments were performed on a Biacore T200 instrument at 25°C. Test antibodies or ADCs were captured at low density (300-500RU) on a CM5 series S sensor chip that was functionalized with a Human Antibody Capture kit (Cytiva catalog #BR100839) in advance. A functionalized surface / flow cell with similar treatment but omitting the ligand was used as a reference. To measure kinetic rates and affinities, a single-cycle kinetic strategy and a running buffer (HBS-EP+, Cytiva catalog #BR100669) at a constant flow rate of 70 μL / mL were used, with a contact pulse of 300 seconds, and recombinant human FRa (Sino Biological catalog #11241-H08H) analyte samples were injected in duplicate at a series of 5 concentrations (0.5, 1, 2, 4, 8 nM). The dissociation phase was measured by injecting the running buffer for 900 seconds. Between replicates, the surface / flow cell was regenerated with 3M MgCl2 to remove both analyte and ligand, and fresh ligand capture was performed using the same conditions. Data analysis was performed using BiacoreEvaluation software after subtracting the reference surface and analyte zero concentration signals. The data were processed and fitted to a 1:1 binding model to determine the binding kinetic rate constants, k a (binding rate) and k d (dissociation rate) and K D (Equilibrium dissociation constant, also called "affinity"). Means and standard deviations of replicates are reported.

[0701] Results are shown in Figure 5No loss of binding of recombinant human FRa was observed between native antibody and respective ADC constructs. No loss of binding was observed between facilitation and facilitation LALA and respective ADC constructs. Surprisingly, a ∼10-fold reduction in K was observed for facilitation and facilitation LALA-based constructs compared to metuximab and metuximab-based constructs. D This is the k for the fatuinumab and fatuinumab LALA based constructs compared to metuximab and metuximab based constructs. d The consequence of increasing the value (dissociation rate constant) (taking into account the association rate constant k a For the negative control IgG1 (SinoBiologicals catalog #HG1K), no human FRα binding events were detected.

[0702] Example 9: Cell Binding Affinity by Flow Cytometry

[0703] The combination of antibody or ADC with extracellular human FRa expressed on cancer cell line was evaluated by flow cytometry. According to the manufacturer's protocol (Bio-Rad, catalog #LNK032APC), LYNX Rapid APC Antibody Conjugation Kit was used to conjugate the antibody or ADC tested with APC fluorophore. 5 μL 10 μg / mL of antibody or ADC tested APC labeled was added to 500,000 cells (suspended in 100 μL PBS in flow cytometry plastic tubes). Cells were incubated in the dark for 20 minutes, washed 3 times with PBS by centrifugation, and resuspended in 200 μL PBS for analysis. Flow cytometry was performed using a BD Fortessa flow cytometer controlled by BD FACS Diva software (BD Biosciences), and data were analyzed using FlowJo software (BD Bioscience).

[0704] Results are shown in Figure 6Comparable human FRα cell binding was observed between facilitation and facilitation LALA to the respective ADC constructs. Native metoximab and native metoximab binding affinities appear to be identical. Surprisingly, a significant loss of binding was observed for the metoximab ADC derivative (M-SORAV) compared to its parent antibody metoximab. This may be explained by either: (1) heterogeneous random conjugation of sulfo-SPDB-DM4 drug-linker, which can react with lysine amino acids that are part of the antibody variable region, thereby reducing binding affinity; (2) lower APC labeling efficiency of M-SORAV because 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 on the FRα negative control cell line BT-474 for all compounds tested.

[0705] Example 10: Ex vivo ADC human plasma stability

[0706] F-VA-EXA and F-LALA-VA-EXA ADC samples (>6 mg / mL in PBS) were diluted with pure sterile human plasma (GeneTex catalog # GTX73265) in screw-capped centrifuge tubes to produce a final ADC concentration of 200 μg / mL (residual PBS volume less than 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). Dynabeads were used. TMM-280Streptavidin (Thermo Scientibic) magnetic beads, ADC was separated from plasma by immune capture, and the magnetic beads were previously coated with biotinylated human folate receptor alpha recombinant protein (Sino Biologicals catalog #11241-H08H). In brief, 600 μL of commercial bead solution was washed twice with HBS-EP buffer (Cytiva, catalog #BR100188) and resuspended in 1.2 mL HBS-EP buffer. 65 μL (48 μg protein amount) of biotinylated recombinant FRa solution was added, and the solution was stirred at room temperature for 2 hours. The beads were then washed 3 times with HBS-EP buffer and resuspended in 1.2 mL HBS-EP buffer. For an immune capture, 100 μL of previous bead solution was added to 100 μL HBS-EP in a microcentrifuge tube. 10 μL of ADC solution in plasma (theoretical 2 μg ADC amount) 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 with 200 μL HBS-EP buffer, and deglycosylated by adding 2 μL / 1000U PNGase F (New England Biolabs catalog # P0705L) with gentle agitation overnight at 37°C. The beads were then washed twice with HBS-EP buffer, twice with distilled water, and once with 10% acetonitrile in water (v / v). The beads were incubated with 50 μL of 30% acetonitrile in water (v / v) containing 0.1% (v / v) formic acid at room temperature for 30 minutes with gentle agitation. The beads were then lysed using a Bruker Impact II TM The eluted samples containing deglycosylated ADC were analyzed by denaturing reverse phase chromatography-mass spectrometry on a Thermo UltiMate 3000 UHPLC system with a 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 PEEK PLRP-S 2.1x100mm 5μm (80°C). Linear gradient 20% B to 50% B in 25 minutes. Flow rate 0.4mL / min. UV detection monitored at 280nm. Q-ToF mass spectrometer in m / z range 500-5000 (ESI + ) are used. Use Bruker The MaxEnt algorithm included in the software deconvolutes the data. For stability data analysis, deconvolution of the original spectrum within the selected light chain (LC) and heavy chain (HC) elution time window is implemented. Drug-linker loss or modification is identified based on 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 specific 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.

[0707] Results are shown in Figure 7 In. For both 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 study (no premature exotecan release or metabolism), except for the +18Da (+H2O) mass shift from the self-stabilizing hydrolysis of the maleimide group (which was completed after 48-72 hours ADC incubation time). The only observed instability was caused by the reverse Michael maleimide deconjugation of the entire drug-linker on the heavy chain of the ADC (deconjugation was not observed on the light chain).

[0708] Example 11: In vitro cytotoxicity experiment on FRa-negative cell lines

[0709] In order to evaluate the non-specific (off-target) cytotoxicity of ADC, in vitro cytotoxicity assay was achieved in BT-474 (FRa negative) cancer cell line. Cells were plated in 96-well plates (1000 to 10 000 cells / well in 100 μL appropriate culture medium) at an appropriate density depending on the cell line, and incubated at 37 ° C for 24 hours. Serial dilutions of the test compound previously dissolved in the culture medium (50 μL) 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 continued for 1 to 2 hours at 37 ° C. The culture medium was then carefully removed, and the well contents were evenly dissolved with acidified isopropanol. Absorbance values ​​were measured on a MultiskanTMSky microplate reader (Thermo Scientific) using a wavelength of 570 nm (with a reference wavelength of 690 nm). Inhibitory dose-response curve fitting (GraphPad Prism 9) was used to determine IC50 concentration values ​​compared to untreated control cells.

[0710] Results are shown in Figure 8Soravtansine (sulfo-SPDB-DM4)-based ADCs showed higher levels of off-target (non-FRa-mediated) cell killing efficacy compared to VA-EXA (LNK4-S drug-linker)-based ADCs. This has been observed for both metuximab- and facilitation-based ADCs. The VA-EXA drug-linker component produces less off-target toxicity compared to the soravtansine drug-linker, which is a good prerequisite for increasing the tolerability of ADCs in clinical settings.

[0711] Table 8:

[0712]

[0713] Example 12: In vivo efficacy xenograft model

[0714] Female CB-17 severe combined immunodeficient (SCID) mice aged four to five weeks were obtained from Janvier labs (Le Genest-Saint-Isle, France) and isolated for 7 days before the start of the study. Mice were cultured with 10% PBS (OV-90, SW-620, KB, BT-474 cell lines) or 50% BD Matrigel Cells in PBS solution (PA-1, IGROV-1, OVCAR-3, NCI-H2110 cell lines) were inoculated subcutaneously. When the average tumor volume reached about 120-150mm3, mice were randomized (usually 7 mice / group) and treated by a single (unless otherwise stated) intravenous injection of PBS (negative control) or the antibody-drug conjugate investigated. Tumor volume was measured (length x width) using a caliper device every 3-5 days and calculated using the following formula V=4 / 3×π×R3, where R represents radius. When the tumor volume exceeded 1500mm3 or in the case of tumor ulceration, mice were killed.

[0715] In the IGROV-1 tumor model, a tumor re-implantation challenge was performed on day 94 of the study. Mice treated with F-LALA-VA-EXA (12 mg / kg IV once) were re-implanted with IGROV-1 cells (on the other flank of the animal) using the same procedure used at the beginning of the study. A control group of 5 new SCID animals were also re-implanted following the exact same procedure.

[0716] exist Fig. 9Tumor xenograft experiments in the SW-620 cancer model are presented in. F-VA-EXA and M-SORAV conjugates were injected once IV at 5, 10, or 15 mg / kg. F-VA-EXA was highly active at doses of 5 mg / kg and above, while the comparator M-SORAV was inactive even when administered at 15 mg / kg.

[0717] exist Fig.10 Tumor xenograft experiments in the SW-620 cancer model are presented in . F-VA-EXA and F-LALA-VA-EXA were injected once IV at 1, 3, and 6 mg / kg. The non-targeted isotype-matched negative control ADC NEG-VA-EXA was injected once IV 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 tested dose of 6 mg / kg, confirming that the anti-tumor activity of the conjugate is target selective.

[0718] exist Fig.11 Tumor xenograft experiments in the OV-90 cancer model are presented in. F-VA-EXA, F-LALA-VA-EXA, and M-SORAV conjugates were injected once with 30 mg / kg IV. All conjugates were highly active, with a 100% remission rate in all groups. However, significant toxicity was observed with the M-SORAV conjugate: 6 of 6 mice showed a disheveled appearance (dull and tangled hair), 3 of 6 mice showed signs of collapse, and 2 of 6 mice suffered from diarrhea. No toxicity was observed for F-VA-EXA and F-LALA-VA-EXA conjugates. These data suggest a better preclinical therapeutic window for F-VA-EXA and F-LALA-VA-EXA constructs compared to M-SORAV.

[0719] exist Fig.12 In presents the tumor xenograft experiment in OV-90 cancer model.F-VA-EXA, F-LALA-VA-EXA and M-SORAV conjugates were injected once with sub-curative doses of 5 and 10 mg / kg IV.At 5 mg / kg, when compared with M-SORAV, F-VA-EXA and F-LALA-VA-EXA showed similar efficacy and improved efficacy.At 10 mg / kg, F-VA-EXA and M-SORAV showed similar efficacy (although more heterogeneity was observed in the M-SORAV group).At 10 mg / kg, the performance of F-LALA-VA-EXA was better than both F-VA-EXA and M-SORAV.

[0720] exist Fig.13 Tumor xenograft experiments in the KB cancer model are presented in. F-VA-EXA, F-LALA-VA-EXA, and M-SORAV conjugates were injected once IV at 3, 6, and 12 mg / kg. The non-targeted isotype-matched negative control ADC NEG-VA-EXA was injected once IV at 12 mg / kg. F-VA-EXA, F-LALA-VA-EXA, and M-SORAV were equally active at doses of 3 mg / kg and above. The non-targeted conjugate NEG-VA-EXA was inactive at the highest tested dose of 12 mg / kg, confirming that the anti-tumor activity of the conjugate was target selective.

[0721] exist Fig.14 Tumor xenograft experiments in the PA-1 cancer model are presented in . The F-LALA-VA-EXA and M-SORAV conjugates were injected once IV at subcurative doses of 3, 6, and 12 mg / kg. The non-targeted isotype-matched negative control ADC NEG-VA-EXA was injected once IV at 12 mg / kg. At 3 mg / kg, F-LALA-VA-EXA performed better than M-SORAV. At 6 mg, F-LALA-VA-EXA performed slightly better than M-SORAV. At 12 mg, M-SORAV performed better than F-LALA-VA-EXA. Overall, both conjugates showed similar levels of efficacy. At the highest tested 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.

[0722] exist Fig.15 Tumor xenograft experiments in the OV-90 cancer model are presented in. F-LALA-VA-EXA and M-SORAV conjugates were injected once IV at 3, 6, and 12 mg / kg doses. At 3, 6, and 12 mg / kg, F-LALA-VA-EXA outperformed M-SORAV.

[0723] exist Fig.16In the present invention, the tumor xenograft experiment in the IGROV-1 cancer model is presented.F-LALA-VA-EXA and M-SORAV conjugates are injected once with 3, 6 and 12 mg / kg doses IV.Negative control ADC NEG-VA-EXA, which is matched with non-targeted isotypes, is injected once with 12 mg / kg IV.At 3, 6 and 12 mg / kg, F-LALA-VA-EXA and M-SORAV show similar efficacy levels, accompanied by strong tumor regression.At the highest tested dose of 12 mg / kg, non-targeted conjugate NEG-VA-EXA shows a certain efficacy level, although not as obvious as other FRa targeting ADCs in the study.The tumor regrowth re-challenge performed after the 94th day of the study confirmed that F-LALA-VA-EXA can induce protective immune memory response, because after the re-implantation of the group treated once with F-LALA-VA-EXA at ​​12 mg / kg IV, no tumor growth was observed (the positive control group with new untreated SCID mice showed tumor growth after implantation).

[0724] exist Fig.17 Tumor xenograft experiments in the folate receptor alpha negative (FRa neg) BT-474 breast cancer model are presented in. F-LALA-VA-EXA and M-SORAV conjugates were injected once IV at doses of 5 and 10 mg / kg. (HER2-targeted trastuzumab) was used as a positive control and was injected once IV at a dose of 10 mg / kg. No efficacy was observed for F-LALA-VA-EXA and M-SORAV at both doses, confirming that the efficacy of these conjugates is FRa selective. As expected and as previously observed (Conilh et al., 2021, Pharmaceuticals, 14(3), 247, doi: 10.3390 / ph14030247), It was effective in this HER2-positive BT-474 cancer model.

[0725] Table 9:

[0726]

[0727] Example 13: Mouse tolerance experiment

[0728] To evaluate the preclinical therapeutic index in mice, mouse tolerance experiments were performed using F-VA-EXA, F-LALA-VA-EXA and M-SORAV conjugates. Female SCID mice (n=5 mice / 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). Mice were carefully monitored over the course of 21 days for weight loss or overt signs of toxicity.

[0729] The same experiment was also performed in female CD-1 mice treated once with F-LALA-VA-EXA 0, 50, 100, 150 or 200 mg / kg IV.

[0730] The results are shown below and Fig.18 (for SCID mouse experiments). F-VA-EXA and F-LALA-VA-EXA were well tolerated with no obvious signs of toxicity at a dose of 200 mg / kg. In contrast, all mice treated with M-SORAV at a dose of 100 mg / kg showed obvious signs of toxicity (untidy appearance with unkempt hair, diarrhea, collapse, closed eyes, sluggish behavior) and were found dead or had to be euthanized on days 2-3. These results, together with the xenograft efficacy data mentioned above, show the excellent preclinical therapeutic index of the F-VA-EXA and F-LALA-VA-EXA constructs compared to the comparator M-SORAV.

[0731] In female CD-1 mice, MBK-103 did not cause any dose-dependent adverse systemic or local effects at every dose level investigated (up to 200 mg / kg IV once). No weight loss or behavioral changes were observed. The only notable observation was a slight decrease in thymus and spleen weights in all groups compared to the control group during final necropsy.

[0732] Table 10:

[0733]

[0734] Example 14: Sprague-Dawley rat pharmacokinetic study

[0735] F-VA-EXA and F-LALA-VA-EXA conjugates were injected into female Sprague-Dawley rats (4-6 weeks old - Charles River) at 5 mg / kg via the tail vein (6 animals per group, randomly assigned). Blood was drawn 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 into plasma; and stored at -80°C until analysis.

[0736] Total mAb concentrations were 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 concentrations were evaluated by ELISA using rabbit polyclonal anti-exitecan antibody (ref#6294 / 00000920, custom made at 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 using ADC was used for quantification.

[0737] Pharmacokinetic parameters (clearance, half-life, Vss and AUC) were calculated by two-compartment analysis using Microsoft Excel software incorporating PK functions (a plug-in developed by Usansky et al., Department of Pharmacokinetics and Drug Metabolism, Allergan, Irvine, CA, USA).

[0738] The free isotecan concentration was evaluated using an LC / MS-MS method using an Agilent 1100 HPLC system and a Sciex API 4000 MS / MS system. Rat plasma samples were protein precipitated using an organic solution consisting of 80:20 (v / v) acetonitrile / methanol + 1% formic acid + d5-isotecan (20 ng / mL) as an internal standard. A Phenomenex HPLC system maintained at 45°C was used in gradient elution mode. Samples were analyzed on a C8 2.1x30mm 2.6μm 100A column (Phenomenex catalog #00D-4497-AN). 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 scans were used in positive ion mode. A calibration curve was plotted using peak area ratios analyte / deuterated internal standard versus nominal analyte concentration using linear least squares regression with 1 / x weighting. The calibration curve range was 0.2 (LLOQ) to 500 ng / mL.

[0739] Results are shown in Fig.19 F-VA-EXA and F-LALA-VA-EXA showed a biphasic PK profile in the circulation (a rapid distribution phase followed by a slower elimination phase), with similar distribution volumes, slow clearance rates, and half-lives in the range of 13-15 days. The total mAb and total ADC curves were similar in intensity, slope, and shape, suggesting good ADC stability (no major payload deconjugation over time). Free exitecan payload (at a concentration of 1 / 10 of the parent ADC component) was detected only during the 0-48 hour period of the experiment. 6 ), suggesting good ADC stability and no premature deconjugation of exitecan.

[0740] Example 15: Pulmonary inflammation and toxicity assessment in mice

[0741] Bleomycin has been widely used in rodents to simulate pulmonary fibrosis, for studying the mechanisms involved in fibrosis and for evaluating potential therapies. To evaluate ADC pulmonary toxicity, follow the known procedures described by Walter and Kleeberger, Mouse Models of Bleomycin-Induced Pulmonary Fibrosis, 2008, Curr. Protoc. Pharmacol. 40: 5.46.1-5.46.17, induced bleomycin-induced pulmonary fibrosis in mice. On days 0, 4 and 8, female C57 / BL6 mice (Janvier Labs, Le Genest-Saint-Isle, France) were lightly anesthetized with isoflurane and intranasally administered with 20 μL 1 mg / kg bleomycin PBS solution (Bleomycine Bellon, 15 mg for injection) or PBS (negative control mice). Non-bleomycin conditioned mice were included as negative control groups. On day 11, mice (n=6 / group) were treated with the tested conjugate (10 mg / kg i.p.) and monitored for overt signs of toxicity and weight loss for the duration of the experiment. (10 mg / kg i.p.) for the positive control group, the conjugate It is known to induce interstitial lung disease and pneumonia in preclinical and in a few patients. All mice were sacrificed on the 25th day, bronchoalveolar supernatant was obtained (by bronchoalveolar lavage), and lung organs were harvested, 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 and embedded in paraffin for histological staining. Slides were stained with anti-CD45 antibodies (Abcam catalog # ab10558, 1: 500) to quantify the level of leukocyte infiltration in the lungs. Secondary biotinylated goat anti-rabbit antibodies (Vector Laboratories catalog # BA-1000, 1: 300) and avidin-HRP (Vector Laboratories catalog # A-2004) were used for detection. Histological experiments and imaging were outsourced to Centre d′ImagerieQuantitative Lyon-Est (CiQLE) platform (Lyon, France). Using Fiji software (developed and maintained by the Laboratory for Optical and Computational Instrumentation of University of Wisconsin-Madison, USA), random parts of IHC images (magnification 30%) were processed using local functionalization to count CD45 positive cells (1 slide / lung) in slides, and to evaluate the level of leukocyte infiltration. The monitoring of lymphocyte (CD3 / CD4), eosinophil (CCR3 / SiglecF), neutrophil (Ly6g / Ly6C) and macrophage (F4 / 80) total counts in the bronchoalveolar supernatant obtained from non-bleomycin conditioned mice was completed by flow cytometry. The level of inflammatory cytokines in the bronchoalveolar supernatant obtained from non-bleomycin conditioned mice was quantitatively determined by ELISA. Follow the manufacturer's instructions for using Bio-Plex Pro TM Mouse Cytokine Th2 Panel (Bio-Rad catalog #L60000UKVT) was used to quantify IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13. Mouse TGF-beta 1 DuoSet and Mouse IL-17 DuoSet ELISA kits (R&D systems catalog #DY1679 and DY421) were used to quantify TGF-β and IL-17, respectively, following the manufacturer's instructions.

[0742] Statistical significance was assessed using one-way ANOVA (Tukey method) using GraphPad Prism 9 software. p values ​​are indicated as * (p < 0.033), ** (p < 0.002), *** (p < 0.0002) and **** (p < 0.0001), and "ns" represents not significant (p > 0.123).

[0743] IHC CD45 quantification results are shown in Fig. 20 When compared to untreated (***) and bleomycin-conditioned (****) negative controls, the positive control ADC causes a significant increase in leukocyte inflammatory infiltration. When compared with an untreated group, F-VA-EXA ADC does not cause a significant increase in leukocyte inflammatory infiltration, and when compared with a bleomycin conditioned (****) control group, causes a significant increase. When compared with both untreated and bleomycin conditioned negative control groups, F-LALA-VA-EXA does not cause a significant increase in leukocyte inflammatory infiltration. Based on these preclinical results, it can be assumed that F-VA-EXA and especially F-LALA-VA-EXA (Fc silent variants) rarely induce or do not induce interstitial lung disease or pneumonia in clinical settings.

[0744] The levels of inflammatory cytokines in bronchoalveolar supernatant are shown in Fig.21 A. Compared with the untreated group, the positive control The ADCs caused a significant increase in IL-13, IL-17, and TGF-β cytokine levels. The F-VA-EXA and F-LALA-VA-EXA ADCs did not cause a significant increase in cytokine levels when compared to the untreated group.

[0745] The total counts of lymphocytes, eosinophils, neutrophils, and macrophages in the bronchoalveolar supernatant are shown in Fig.21 B. Compared with the untreated control group, the positive control Caused a significant increase in lymphocyte, macrophage, neutrophil, and eosinophil counts. F-VA-EXA and F-LALA-VA-EXA showed a significant increase in lymphocyte counts compared to the untreated control group, but did not show a statistically significant increase in macrophages, neutrophils, and eosinophils compared to the untreated control group.

[0746] Table 11:

[0747]

[0748] Example 16: Dose Range Finding Toxicology Study in Cynomolgus Monkeys

[0749] A dose-ranging toxicology study in nonhuman primates was conducted using purpose-bred native female cynomolgus macaques (Macaca fascicularis) of Vietnamese origin. France) was implemented. The research protocol was approved by the Institutional Animal Care and Use Committee of the testing facility. F-LALA-VA-EXA ADC was intravenously administered (5mL / kg / h, within a 30-minute injection period) at a dosage level of 30, 40, 50 or 60 mg / kg at three-week intervals (3 doses in total). Two female monkeys / groups (8 animals in total) were used in the study. After the final sacrifice 5 days after the last administration, macroscopic autopsy, organ weight measurement and histopathological examination were performed. The parameters evaluated during the study included mortality, clinical signs (including estimates of food consumption), body weight, ophthalmological examination, body temperature, hematology, coagulation, clinical chemistry, urinalysis, organ weight and a wide list of tissues for macroscopic and microscopic examination. The ADC formulation buffer was 20mM histidine pH 6.0, 4% (w / v) sucrose and 75mM NaCl. The vehicle was 0.9% saline.

[0750] Results are shown in Fig. 22 In. F-LALA-VA-EXA ADC is well tolerated, with an estimated maximum non-serious toxic dose (HNSTD) of 50mg / kg three times. Due to acute renal failure in one of the two animals (most likely target-related, since folate receptor α is endogenously expressed in the kidney), administration at 60mg / kg is not tolerated. The preclinical therapeutic window of F-LALA-VA-EXA seems to be favorable, because the cynomolgus monkey HNSTD is much higher than the effective therapeutic dose in rodent cancer models (after allometric scaling of the dose).

Claims

1. An antibody-drug conjugate of formula (I): Ab-[LD]p (I), in: -Ab is an anti-folate receptor alpha (FRα) antibody that specifically binds to SEQ ID NO: 12, -L is a cleavable linker moiety bonded to said antibody, preferably via a thiol residue, -D is the cytotoxic drug moiety bonded to L, - p is 1 to 8, preferably 6 to 8, and more preferably p is 8.

2. The antibody-drug conjugate according to claim 1, wherein D is an inhibitor of topoisomerase I, preferably selected from camptothecin analogs, and more preferably D is the drug moiety of exitecan of the following formula (II):

3. The antibody-drug conjugate of any one of claims 1-2, wherein L is a cleavable linker moiety of the formula -AW-, wherein A is an optional stretcher unit linked to Ab, and W is a cleavable moiety linked to D.

4. The antibody-drug conjugate according to claim 3, wherein W has the following formula (III) in Each R 2 Independently selected from electron withdrawing groups and C1-C4 alkyl groups; n is 0, 1, or 2; T is a sugar cleavable unit or a polypeptide cleavable unit; When T is a carbohydrate cleavable unit, Y is O, or when T is a polypeptide cleavable unit, Y is NR 3 ;and R3 is selected from H, C1-C24 alkyl, C2-C6 alkenyl; optionally substituted polyether, aryl having 6 to 10 ring atoms, C3-C8 cycloalkyl, heterocycloalkyl having 3 to 10 ring atoms, heteroaryl having 5 to 10 ring atoms, and any combination thereof, The alkyl and alkenyl groups 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-, -OC(O)NR"-, and triazole, R" and R"' are independently selected from H and C1-C6 alkyl, and pharmaceutically acceptable salts thereof.

5. The antibody-drug conjugate according to claim 4, wherein L corresponds to the linker -AW- of formula (IV) in X 1 is a connector unit; Z is an optional spacer; X 2 is a connector unit; K is an optional hydrophobic masking entity, preferably selected from polysarcosine and polyethylene glycol; R 1 is selected from H, C 1-C 24 alkyl, C 2 -C 6 an optionally substituted polyether, an aryl group having 6 to 10 ring atoms, a C3-C8 cycloalkyl group, a heterocycloalkyl group having 3 to 10 ring atoms, a heteroaryl group having 5 to 10 ring atoms, and any combination thereof, The alkyl and alkenyl groups 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-, -OC(O)NR"-, and triazole; R 4 is selected from H, C1-C6 alkyl and C2-C6 alkenyl, wherein the alkyl and alkenyl are optionally interrupted by one or more heteroatoms or chemical groups selected from: -O-, -S-, -C(O)- and -NR"-; and R 5 Selected from H, C 1 -C 6 Alkyl and C 2 -C 6 The alkyl and alkenyl groups are optionally interrupted by one or more heteroatoms or chemical groups selected from the group consisting of -O-, -S-, -C(O)- and -NR"-.

6. The antibody-drug conjugate according to claim 5, wherein K is polysarcosine, preferably the following formula (V): wherein k is an integer between 2 and 50, preferably between 4 and 30, And R 6 Corresponding to OH or NH 2 .

7. The antibody-drug conjugate according to any one of claims 5-6, wherein T is a sugar cleavable unit which is a glucuronide or a galactoside.

8. The antibody-drug conjugate according to any one of claims 5 to 7, wherein T is a dipeptide, preferably selected from Val-Cit, Val-Ala and Phe-Lys.

9. The antibody-drug conjugate according to any one of claims 1 to 3, wherein L corresponds to a linker of formula (VI): And L is covalently bonded to one or more thiol residues of the antibody.

10. The antibody-drug conjugate according to any one of claims 1 to 9, which corresponds to the following formula (VII): Wherein the Ab is an anti-FRα antibody, such as facilitation mab, or a silent IgG1 variant thereof, typically comprising alanine substitutions in leucine 234 and leucine 235 of the IgG1 Fc constant region, and p is 4 to 8.

11. The antibody-drug conjugate according to any one of claims 1 to 10, wherein Ab is an anti-FRα antibody comprising any of: (i) a variable heavy chain polypeptide comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, HCDR3 of SEQ ID NO: 3, and a variable light chain polypeptide comprising LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6; or, (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.

12. The antibody-drug conjugate according to any one of claims 1 to 11, wherein (i) Ab is an anti-folate receptor α antibody or an antigen-binding fragment thereof, comprising a variable heavy chain polypeptide comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, HCDR3 of SEQ ID NO: 3, and a variable light chain polypeptide comprising LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6; (ii) L is a cleavable linker of the formula -AW-, wherein A is an optional stretcher linked to Ab, and W is a cleavable moiety linked to D (iii) D is a topoisomerase I inhibitor, such as exotecan; and (iv) p is 1 to 8, preferably 4 to 8, more preferably about 8.

13. The antibody-drug conjugate according to any one of claims 1 to 12 for use as a medicament, preferably for treating a tumor, such as a solid tumor, more particularly selected from ovarian cancer, breast cancer, lung cancer or mesothelioma, in a subject in need thereof.

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

15. A pharmaceutical composition comprising the antibody-drug conjugate according to any one of claims 1 to 12 in combination with one or more pharmaceutically acceptable excipients, diluents or carriers, optionally comprising other active ingredients.

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