Antibody-drug conjugates and uses thereof

Abstract

To provide an effective antibody-drug conjugate comprising an anti-FRα antibody, with high specificity, low toxicity (improved therapeutic index) and differentiated mechanism of action of the payload.SOLUTION: Provided is an antibody-drug conjugate in which the antibody specifically binds to folate receptor alpha (FRα) and in which the drug is selected from topoisomerase I inhibitors, camptothecin analogs such as exatecan. Such an antibody-drug conjugate is especially useful for treating proliferative diseases including cancers such as ovarian, breast or lung cancer.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The antibody specifically binds to the folate receptor alpha (FRα) and the drug binds to a topoisomerase, preferably a topoisomerase. Inhibitors of ferrocene I, for example camptothecin analogues such as exatecan Disclosed below are antibody-drug conjugates that can be used in the treatment of cancer. The compounds are particularly useful in the treatment of proliferative disorders including cancers such as ovarian, breast or lung cancer. [Background technology]

[0002] Antibody-drug conjugates (hereafter referred to as "ADCs") represent a new class of therapeutic agents, particularly Such ADCs are primarily cancer treatments. ADCs comprise at least an antibody and a payload (e.g., a cytotoxic drug). Combining antibody target specificity with payload efficiency (e.g., cytotoxic activity of chemotherapeutic agents) Effective ADCs are designed to exhibit high specificity and low systemic toxicity. It is not.

[0003] Within the context of toxicity, the antibody used in the ADC must bind accurately and effectively to its antigen. This means that the appropriate target antigen is preferentially or exclusively expressed on the target cell. Taste.

[0004] When designing an ADC, the final active drug is covalently attached to a ligand targeting unit. On the other hand, active drug units by selective enzymatic mechanisms after cellular internalization or in the diseased tissue microenvironment. In this regard, several Several peptidase- and glycosidase-sensitive cleavable linker chemistry strategies have been developed. These cleavable linkers and their corresponding cleavage mechanisms are well known and have been described in several publications. (e.g., Bargh JG et al., Chem. Soc. R ev.,2019,48,4361,Toki et al.J.Org.Chem.2 002,67,6,1866-1872,Scott et al.Bioconjug (Late Chem. 2006, 17, 3, 831-840). The choice of site is a key design attribute of ADCs that influences the efficacy and tolerability of the conjugate. is.

[0005] Linker-type conjugates that have been used to conjugate cytotoxins or drugs to antibodies Examples include hydrazones, thioethers, esters, disulfides, and peptide-containing ligands. Linkers include, but are not limited to, those that are capable of transporting proteins to, for example, the lysosomal compartment. or proteases preferentially expressed in tumor tissues. are subject to cleavage by proteases such as cathepsins (e.g., cathepsins B, C, and D). Effective linkers are selected from those that are easy to release. In relation to toxicity, the linker itself may drive toxicity. The stability of the linker may affect the toxicity exerted by the payload, even if it is not visible to the naked eye. It is also clear that stable linkers can influence the targeting of the target molecule in a target-specific manner. While non-stable linkers can release the payload in an accurate manner, non-stable linkers can lead to inaccurate release of the payload. are more likely to be released (e.g., due to nonspecific cleavage) and to be nonspecifically systemically toxic. brings about sex.

[0006] The payloads used in the ADCs are highly potent, with in vitro inhibitory concentrations in the picomolar range. Common payloads are, for example, microtubule inhibitors. anti-inflammatory drugs (e.g., maytansine derivatives (DM1 / DM4), auristatins (MMAE / MM MAF), eribulin) and DNA alkylating agents (calicheamicin, pyrrolobenzodiazepines) azepines, indolinobenzodiazepines, or duocarmycins).

[0007] ADCs appear to be promising therapeutics, but some may be too toxic These compounds have various potential side effects, which may limit the therapeutic window of these compounds or prevent further clinical development. Most of the ADCs currently approved or under clinical investigation target the microtubules and DNA mentioned above. Therefore, they are resistant or resistant to microtubule and DNA targeting agents. To effectively treat tumors that become resistant to steroids, new drugs based on payloads with other mechanisms of action are being developed. There is a need for differentiated ADCs.

[0008] Therefore, an effective ADC that exhibits high specificity, maximum efficacy, and low toxicity must be formulated based on the specificity of its components. For a review of possible strategies, see e.g., Khong et al. orzul et al 2019(Molecular cancer resear ch,DOI:10.1158 / 1541-7786.MCR-19-0582) Please refer to International Publication No. 2019081455 and Conilh et al. (2021 , Pharmaceuticals, 14(3), 247) furthermore, especially topoisomers Based on the enzyme I inhibitor payload exatecan, hydrophilic monodisperse polysarcosine (PS AR) HER2-targeting antibody-drug conjugates using a drug-linker platform reports.

[0009] Cheng et al(2018,DOI:10 / 1158 / 1535-7163. MCT-17-1215) and WO 2017151979 are typically 3-4 Farletuzumab conjugated to the molecule eribulin (MORAb-202) was used. reported an ADC and its use in the treatment of tumors. The mechanism of action of erode is microtubule inhibition.

[0010] Moore et al(2018,Future Oncol.14(17)166 9-1678) is a drug that uses an average of 3-4 maytansine molecules as a payload for the treatment of ovarian cancer. mirvetuximab, a humanized anti-FRα antibody conjugated to a rhodopsin molecule; We report the results of a phase III trial using the ADC mirvetuximab soravtansine. The mechanism of action of the maytansine payload of this ADC is microtubule inhibition. Summary of the Invention

[0011] Therefore, it is effective, has high specificity, low toxicity (improved therapeutic index) and payload. Antibody-drug conjugates containing anti-FRα antibodies with distinct mechanisms of action have been developed. It is needed as.

[0012] As shown in the examples, the present disclosure is particularly directed to other compounds such as mirvetuximab soravtansine. Reference prior art ADCs targeting FRα-expressing antibodies with payloads and drug linkers It has superior in vivo efficacy and low toxicity in solid tumor cancer models when compared to The present invention provides anti-FRα antibody-drug conjugates based on topoisomerase I inhibitors.

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

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

[0015] In a preferred embodiment, the Ab is a mutant or chemically modified human IgG1 isotype. The variant or chemically modified constant region is an antibody having a wild-type The antibody has a IgG1 isotype constant region compared to a corresponding antibody with a human IgG1 isotype constant region. They do not confer ADCC activity or confer reduced ADCC activity.

[0016] In other specific embodiments, the Ab comprises a human IgG4 isotype constant region, or a variant or or a chemically modified IgG4 constant region, The constant regions are shown to be IgG4 isotype constant regions when compared to the corresponding antibodies with wild-type IgG4 isotype constant regions. In this case, the antibody is conferred no ADCC activity or reduced ADCC activity.

[0017] In a preferred embodiment, the Ab is: (a) HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 3 and a variable heavy chain polypeptide comprising an LCDR1 of SEQ ID NO: 4, an LCDR2 of SEQ ID NO: 5, and and a variable light chain polypeptide comprising an LCDR3 of SEQ ID NO: 6; or (b) a variable heavy chain polypeptide comprising a VH of SEQ ID NO: 7 and a variable heavy chain polypeptide comprising a VL of SEQ ID NO: 8 Light Chain Polypeptide The anti-FRα antibody comprises any one of the following:

[0018] In a specific embodiment, the Ab comprises a heavy chain of SEQ ID NO: 9 and a light chain of SEQ ID NO: 10. or consisting essentially of them.

[0019] In a specific embodiment, the Ab comprises a heavy chain of SEQ ID NO: 11 and a light chain of SEQ ID NO: 10. or consisting essentially of them.

[0020] In certain embodiments, D is an inhibitor of topoisomerase I, preferably camptothenate. and more preferably D is selected from the group consisting of exatecnic analogs of formula (II): It is the drug portion of

[0021] [ka]

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

[0023] [ka] During the ceremony, each R2 is independently selected from the group consisting of an electron-withdrawing group and a C1-C4 alkyl; n is 0, 1 or 2; T is a sugar cleavable unit or a polypeptide cleavable unit; When T is a sugar cleavable unit, Y is O, or when T is a polypeptide cleavable unit, if present, Y is NR3; R3 is H, C1~C 24 Alkyl, C2-C6 alkenyl; optionally substituted poly aryls with 6-10 ring atoms, C3-C8 cycloalkyls, 3-1 Heterocycloalkyl having 0 ring atoms, heteroaryl having 5 to 10 ring atoms and any combination thereof; The alkyl and alkenyl include -O-, -S-, -C(O)-, -NR''-, -C (O)NR''-, -NR''-C(O)-, -NR''-C(O)-NR'''-, - selected from NR″—C(O)—O—, —OC(O)NR″— and triazole optionally interrupted by one or more heteroatoms or chemical groups; R" and R"' are independently selected from H and C1-C6 alkyl, I), and pharmaceutically acceptable salts thereof.

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

[0025] [ka] During the ceremony, X1 is the connector unit, Z is an optional spacer, X2 is a connector unit, K is preferably selected from polysarcosine and polyethylene glycol; a hydrophobic masking entity of choice, R1 is H, C1~C 24 Alkyl, C2-C6 alkenyl; optionally substituted poly aryls with 6-10 ring atoms, C3-C8 cycloalkyls, 3-1 Heterocycloalkyl having 0 ring atoms, heteroaryl having 5 to 10 ring atoms and any combination thereof; The alkyl and alkenyl include -O-, -S-, -C(O)-, -NR''-, -C (O)NR''-, -NR''-C(O)-, -NR''-C(O)-NR'''-, - selected from NR″—C(O)—O—, —OC(O)NR″— and triazole optionally interrupted by one or more heteroatoms or chemical groups; R4 is selected from the group consisting of H, C1-C6 alkyl, and C2-C6 alkenyl; The alkyl and alkenyl are selected from -O-, -S-, -C(O)- and -NR''-. optionally interrupted by one or more heteroatoms or chemical groups selected from R5 is selected from the group consisting of H, C1-C6 alkyl, and C2-C6 alkenyl; The alkyl and alkenyl are selected from -O-, -S-, -C(O)- and -NR''-. The heteroatom may be optionally interrupted by one or more heteroatoms or chemical groups selected from the group consisting of aryl, ...

[0026] In more particular embodiments, X1 and X2 are one or more amino acids, one or more N-substituted Amino acids, optionally substituted polyethers, C1-C 12 Alkylene, 6 to 10 Arylene having ring atoms, C3-C8 cycloalkylene, having 5-10 ring atoms Heterocycloalkylene, heteroarylene with 5 to 10 ring atoms, C2 to C 10 alkenylene, and any combination thereof; The alkylene and alkenylene include -O-, -S-, -C(O)-, -NR''-, -C(O)NR''-, -NR''-C(O)-, -NR''-C(O)-NR'''- , —NR″—C(O)—O—, —OC(O)NR″—, and triazole. and optionally interrupted by one or more heteroatoms or chemical groups, The alkylene, arylene, cycloalkylene, heterocycloalkylene, heteroaromatic Arylene and alkenylene are substituted with halogen, oxo, -OH, -NO2, -CN, C1 C6 alkyl, C3-C6 cycloalkyl, heterocyclyl with 5-10 ring atoms aryl having 6 to 10 ring atoms; heteroaryl having 5 to 10 ring atoms; C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 haloalkoxy, -(CO )-R', -O-(CO)-R', -(CO)-O-R', -(CO)-NR''R'' one or more substituents selected from -NR''-(CO)-R', and -NR''R'''; optionally substituted with a substituent, R', R'' and R''' are independently selected from H and C1-C6 alkyl.

[0027] In more particular embodiments, Z is one or more amino acids, one or more N-substituted amino acids, Optionally substituted polyethers, C1-C 12 Alkylene, having 6 to 10 ring atoms arylene, C3-C8 cycloalkylene, heterocycloalkylene having 5-10 ring atoms, alkylene, heteroarylene having 5 to 10 ring atoms, C2 to C 10 Alkenyl and any combination thereof; The alkylene and alkenylene include -O-, -S-, -C(O)-, -NR''-, -C(O)NR''-, -NR''-C(O)-, -NR''-C(O)-NR'''- , —NR″—C(O)—O—, —OC(O)NR″—, and triazole. and optionally interrupted by one or more heteroatoms or chemical groups, The alkylene, arylene, cycloalkylene, heterocycloalkylene, heteroaromatic Arylene and alkenylene are substituted with halogen, oxo, -OH, -NO2, -CN, C1 C6 alkyl, C3-C6 cycloalkyl, heterocyclyl with 5-10 ring atoms aryl having 6 to 10 ring atoms; heteroaryl having 5 to 10 ring atoms; C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 haloalkoxy, -(CO )-R', -O-(CO)-R', -(CO)-O-R', -(CO)-NR''R'' one or more substituents selected from -NR''-(CO)-R', and -NR''R'''; optionally substituted with a substituent, R', R'' and R''' are independently selected from H and C1-C6 alkyl.

[0028] In certain embodiments, K is a polysarcosine, preferably a polysarcosine of formula (V): It is Shin,

[0029] [ka] In the formula, k is an integer of 2 to 50, preferably 4 to 30. R6 corresponds to OH or NH2.

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

[0031] In another particular embodiment, T is preferably Val-Cit, Val-Ala and Ph e-Lys.

[0032] In certain embodiments, L is covalently attached to one or more thiol residues of the antibody; Preferably, L corresponds to a linker of formula (VI).

[0033] [ka]

[0034] In certain embodiments, Abs comprise full-length antibodies or antibody fragments containing the antigen-binding portion.

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

[0036] [ka] where the Ab typically comprises leucine 234 and leucine 23 of the IgG1 Fc constant region. Anti-FRα antibodies, such as farletuzumab, or derivatives thereof, comprising an alanine substitution at position 5. It is an indifferent IgG1 variant, where p is 4 to 8.

[0037] In a preferred embodiment, the antibody drug conjugate corresponds to the following formula (VII):

[0038] [ka] where Ab is an anti-FRα antibody, such as farletuzumab, or its silent IgG. 1 variant, typically a leucine-binding domain variant of the IgG1 Fc constant region, also known as the LALA mutation Mutant variants of human IgG1 containing alanine substitutions at leucine 234 and leucine 235 and p is 8.

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

[0040] Another object of the present disclosure is to provide a method for treating a tumor, preferably a tumor, e.g., a solid tumor, for use as a medicament. more particularly from the group consisting of ovarian cancer, breast cancer, lung cancer, or mesothelioma for use in the treatment of The present invention relates to the above ADC selected from the group consisting of:

[0041] Another object of the present disclosure is to provide a method for treating tumors, such as solid tumors, more particularly ovarian cancer, breast cancer, lung cancer, or The present invention relates to the preparation of a medicament or pharmaceutical composition for treating a tumor selected from the group consisting of mesothelioma. The present invention relates to the use of the ADC in

[0042] In certain embodiments, the ADC is preferably used to treat ovarian cancer, triple-negative breast cancer, and It may be used to treat a cancer selected from the group consisting of non-small cell lung cancer.

[0043] The present disclosure further provides for the use of optionally other active ingredients, such as anti-cancer agents or immune checkpoint inhibitors. and combinations thereof with one or more pharmaceutically acceptable excipients, diluents, or carriers, including immunotherapeutic agents such as The present invention also relates to pharmaceutical compositions comprising the antibody-drug conjugates disclosed herein.

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

[0045] [ka] (d) conjugating the anti-FRα antibody to a compound of formula (VIII); thereby obtaining an ADC of the present disclosure; and Includes. [Brief explanation of the drawings]

[0046] [Figure 1] 1 depicts preclinical rodent efficacy, pharmacokinetic and tolerability data for the conjugate according to Example 4. [Figure 2] 1 shows flow cytometry assessment of extracellular expression of folate receptor alpha according to Example 5. [Figure 3] 1 shows in vitro cytotoxicity data of the compound exatecan mesylate against several folate receptor alpha positive cancer cell lines according to Example 6. [Figure 4] 10 depicts an in vitro ELISA binding experiment of the conjugate to recombinant human folate receptor alpha protein according to Example 7. [Figure 5] 1 depicts an in vitro surface plasmon resonance (SPR) binding experiment of the conjugate to recombinant human folate receptor alpha protein according to Example 8. [Figure 6] 10 shows the in vitro folate receptor alpha positive cancer cell binding affinity of the conjugates according to Example 9. [Figure 7] 1 shows the ex vivo human plasma stability of the conjugate according to Example 10. [Figure 8] 10 depicts an in vitro cytotoxicity assay of the conjugate against the folate receptor alpha negative breast cancer cell line BT-474 according to Example 11. [Figure 9] 10 depicts the in vivo efficacy evaluation of the conjugate in a folate receptor alpha-positive SW-620 xenograft cancer model according to Example 12. [Figure 10] 10 depicts in vivo efficacy evaluation of the conjugate in a second folate receptor alpha-positive SW-620 xenograft cancer model according to Example 12. [Figure 11]10 depicts the in vivo efficacy evaluation of the conjugate in a folate receptor alpha-positive OV-90 xenograft cancer model according to Example 12. [Figure 12] 10 depicts in vivo efficacy evaluation of the conjugate in a second folate receptor alpha-positive OV-90 xenograft cancer model according to Example 12. [Figure 13] 10 depicts the in vivo efficacy evaluation of the conjugate in a folate receptor alpha-positive KB xenograft cancer model according to Example 12. [Figure 14] 10 depicts the in vivo efficacy evaluation of the conjugate in a folate receptor alpha-positive PA-1 xenograft cancer model according to Example 12. [Figure 15] 10 depicts the in vivo efficacy evaluation of the conjugate in a third folate receptor alpha-positive OV-90 xenograft cancer model according to Example 12. [Figure 16] 10 depicts in vivo efficacy evaluation of conjugates in a folate receptor alpha-positive IGROV-1 xenograft cancer model and tumor re-implantation challenge according to Example 12. [Figure 17] 10 depicts the in vivo efficacy evaluation of the conjugate in a folate receptor alpha-negative BT-474 xenograft breast cancer model according to Example 12. [Figure 18] 10 shows in vivo SCID and CD-1 mouse tolerability evaluation of the conjugate at high doses according to Example 13. [Figure 19] 10 shows in vivo rat pharmacokinetic evaluation of total mAb, total ADC, and free exatecan subcomponents of the conjugate according to Example 14. [Figure 20] 10 shows in vivo mouse lung inflammatory evaluation of the conjugate according to Example 15. [Figure 21] 10 shows in vivo mouse lung inflammatory evaluation of the conjugate according to Example 15. [Figure 22] 10 depicts an in vivo cynomolgus monkey dose-ranging toxicity study according to Example 16. DETAILED DESCRIPTION OF THE INVENTION

[0047] definition In order that this disclosure may be more readily understood, certain terms are first defined. Further Definitions are described throughout the detailed description.

[0048] The term "FRα" or "folate receptor alpha" refers to SEQ ID NO: 1 unless otherwise specified. This sequence is the human folate receptor alpha as defined in SEQ ID NO:12. The FOLR1 gene (H) is also available in entry P15328 (FOLR1_HUMAN). The amino acid sequence of the folate receptor alpha encoded by omo sapiens Respond.

[0049] The term "antibody" as referred to herein includes whole antibodies and any antigen-binding fragments (i.e., A naturally occurring "antibody" contains a disulfide bond between the nucleotides of the antibody and the nucleotide sequence of the antibody. Contains at least two heavy (H) chains and two light (L) chains interconnected by bonds Each heavy chain is a glycoprotein. The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. Each light chain is composed 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 are The complementarity-determining regions (CDRs) are interspersed with more conserved regions called FRs. Each VH and VL can be further subdivided into three CDRs and four hypervariable regions called It consists of FR1, CDR1, FR2, CDR2, FR3, CDR3, The heavy and light chain variable regions are arranged in the order FR4 from the amino terminus to the carboxy terminus. The constant region of an antibody contains a binding domain that interacts with an antigen. The constant region of an antibody binds to various cells of the immune system ( effector cells) and the first component of the classical complement system (Clq), The "antigen-binding portion" (or simply "antigen-binding portion") of an antibody may mediate the binding of an immunoglobulin to a target or agent. The term "antigenic portion," as used herein, refers to a portion of an antigen (e.g., a portion of FRα). It refers to a full-length antibody or one or more fragments of an antibody that retains the ability to bind to an antigen. It has been shown that the function of "antigen binding" can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "portion" include Fab fragments, VL, VH, CL and Monovalent fragment consisting of the CH1 domain and the F(ab)2 fragment; disulfide bridge in the hinge region a bivalent fragment comprising two Fab fragments linked by a VH domain and a CH1 domain an Fd fragment consisting of the VL and VH domains of a single arm of an antibody; single-arm, domain-antibody having an IgG heavy chain modified in the hinge region, e.g., IgG4; Body fragments (Ward et al., 1989 Nature 341:544-546) or a UniBody comprising a Nanobody fragment comprising a VH domain; and an isolated phase complementarity-determining regions (CDRs); or any fusion protein containing such antigen-binding portions. Furthermore, the two domains of the Fv fragment, VL and VH, are encoded by separate genes. However, they are engineered using recombinant methods to pair VL and VH regions into monovalent molecules ( It can be produced as a single-chain protein forming a single-chain Fv (scFv). For example, the method of Bird et al. al., 1988 Science 242:423-426; and Huston et al. al.,1988 Proc.Natl.Acad.Sci.85:5879-588 3). Such single chain antibodies are also referred to within the term "antigen-binding portion" of an antibody. These antibody fragments can be produced using conventional techniques known to those skilled in the art. and the fragments are screened for utility in the same manner as are intact antibodies. do.

[0050] As used herein, an "isolated antibody" refers to an antibody that is isolated from other antibodies having different antigen specificities. (e.g., an isolated antibody that specifically binds to FRα is an antibody that is qualitatively free of FRα.) (The antibody is substantially free of antibodies that specifically bind to antigens other than FRα.) However, The isolated antibodies that specifically bind to FRα may interact with other antigens, such as FRα molecules from other species. Furthermore, isolated antibodies may have substantially different reactivities with other cellular material and / or chemicals. It may not be included in the calculation.

[0051] The phrases "antibody that recognizes an antigen" and "antibody that is specific to an antigen" are used herein to refer to "antibody The term "antibody that specifically binds to an antigen" is used interchangeably with the term "antibody that specifically binds to an antigen."

[0052] As used herein, the term "monoclonal antibody" or "monoclonal antibody composition" refers to a The term refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition is a preparation of antibody molecules of particular The antibodies show a single binding specificity and affinity for the epitope of

[0053] As used herein, "isotype" refers to the type of antibody provided by the heavy chain constant region genes. refers to the antibody class (e.g., IgM, IgE, IgG, e.g., IgG1 or IgG4) involved vinegar.

[0054] As used herein, "K" D The term "k off and k on The ratio of (i.e., k o ff / k on ) and expressed as a molar concentration (M) Figure. K D The K value is related to the antibody concentration (the amount of antibody needed for a particular experiment). D Lower values ​​(lower concentrations) result in higher affinity of the antibody. D The value is The K of a mAb can be determined using methods well established in the field. D Determine the value A preferred method for this is described in Harlow, et al., Antibodies:AL aboratory Manual,Cold Spring Harbor Labo ratory Press, Cold Spring Harbor, NY, 198 8),Coligan et al.,eds.,Current Protocols. in Immunology, Greene Publishing Assoc.a nd Wiley Interscience, NY, 1992, 1993, and M and Uller, Meth Enzymol 1983, these references being incorporated herein by reference. The entire contents of which are incorporated herein by reference. D The method for determining the By using resonance or biosensor systems such as Biacore® By using the system (affinity evaluation) Rich RL, Day YS, Morton T A,Myszka DG.High-resolution and high-thr oughput protocols for measuring drug / hum a serum albumin interaction using BIAC For more information about ORE® (Anal Biochem. 2001), See also).

[0055] As used herein, "k" assoc " or "k a " or "k on The term " While the term "antibody-antigen interaction" is intended to refer to the association rate of a given antibody-antigen interaction, as used herein, "k" dis " or "k d " or k off The term refers to a specific antibody-antigen interaction. It is intended to refer to the rate of dissociation.

[0056] As used herein, the term "affinity" refers to the affinity of an antibody to an antigen at a single antigenic site. Within each antigen site, the variable regions of the antibody "arms" interact with multiple The more interactions there are with the antigen, the greater the affinity. Become stronger.

[0057] As used herein, "specifically binds to an antigen," e.g., "specifically binds to FRα." An antibody or protein that "binds" to an epitope presented on an antigen such as the FRα of the present disclosure. It is intended to refer to an antibody that detectably binds, typically at a concentration of 200 nM or less. , 100 nM or less, 50 nM, 40 nM or less, or about 30 nM D Binds to human FRα Typically, K D is 10 -3 pM~20 0 nM, particularly 0.1 pM to 100 nM, particularly 0.1 pM to 50 nM, or 1 pM to 50 nM M, particularly 1 pM to 30 nM, 10 pM to 50 nM, 0.1 nM to 200 nM, or 0.1 Typically, the range is between 1 nM and 100 nM, or between 1 nM and 50 nM, in particular between 1 nM and 30 nM. The ADCs of the present disclosure are specific for FRα and have the K D It has.

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

[0059] As used herein, the term "ADCC" or "antibody-dependent cellular cytotoxicity" activity The term refers to cell depletion activity. ADCC activity can be measured using commercially available ADCC assays, e.g., Pro ADCC Reporter B marketed by mega under Ref# G7015 It can be measured by ioassay.

[0060] As used herein, the term "subject" includes any human or non-human animal. The term "non-human animals" refers to all vertebrates, including mammals and non-mammals, e.g., Examples include non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, and reptiles. The term "subject" also encompasses the term "patient."

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

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

[0063] Any formula shown herein also refers to a deuterium labeled compound or 14 C-labeled compounds It is intended to represent unlabeled and isotopically labeled forms of an entity.

[0064] The term "pharmaceutically acceptable salts" refers to salts that retain the biological effectiveness and properties of the compounds of the present disclosure. refers to salts that retain their original structure and are typically not biologically or otherwise undesirable. In this case, the compounds of the present disclosure may contain amino and / or carboxyl groups or groups similar thereto. Depending on the entity, acid salts and / or base salts can be formed. Acid addition salts can be formed with organic and / or inorganic acids. Addition salts can be formed with organic and / or inorganic bases. Such salts are well known to those skilled in the art. It is well known to

[0065] The term connector unit refers to the building block that connects different parts of a compound together, e.g. For example, a connector can connect Ab to a spacer or a spacer to an amide functional group -CO-NR1-. The connector can connect the components of the antibody-drug-conjugate, i.e., Ab, Spacer, hydrophobic masking entity, and / or bond for amide functionality -CO-NR1- A scaffold having a fusion site.

[0066] A person skilled in the art will be able to select an appropriate connector. The stock contains amino acids such as lysine, glutamic acid, aspartic acid, serine, and tyrosine. , cysteine, selenocysteine, glycine, homoalanine; amino alcohol; amino aldehyde; polyamine or any combination thereof. Advantageously, the connector unit X1 and / or X2 are one or more natural or unnatural amino acids. The necta units X1 and / or X2 are selected from glutamic acid, lysine and glycine. Connector units X1 and X2 may be one or more amino acids, one or more N-substituted amino acids, Polyethers substituted by C1-C 12 Alkylene, having 6 to 10 ring atoms Arylene, C3-C8 cycloalkylene, heterocycloalkylene having 5-10 ring atoms alkylene, heteroarylene with 5 to 10 ring atoms, C2 to C 10 Alkenylene, and any combination thereof, wherein the alkylene and alkenylene is -O-, -S-, -C(O)-, -NR''-, or -C(O)NR'. '-, -NR''-C(O)-, -NR''-C(O)-NR'''-, -NR''-C (O)—O—, —OC(O)NR″—, and triazole. optionally interrupted by one or more heteroatoms or chemical groups, The alkylene, arylene, cycloalkylene, heterocycloalkylene, heteroaromatic Arylene and alkenylene are substituted with halogen, oxo, -OH, -NO2, -CN, C1 C6 alkyl, C3-C6 cycloalkyl, heterocyclyl with 5-10 ring atoms aryl having 6 to 10 ring atoms; heteroaryl having 5 to 10 ring atoms; C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 haloalkoxy, -(CO )-R', -O-(CO)-R', -(CO)-O-R', -(CO)-NR''R'' one or more substituents selected from -NR''-(CO)-R', and -NR''R'''; R', R'' and R''' are selected from H and C1-C6 alkyl. are selected independently.

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

[0068] [ka] Examples include:

[0069] In particular, the connector unit may be bivalent or trivalent. For example, if a hydrophobic masking entity K is present, If present, X2 can be a trivalent connector unit.

[0070] The term "amino acid" refers to a natural or unnatural amino acid. The CO moiety of the ONR1'-group is the X2 connector only when X2 consists of one or more amino acids. A non-exhaustive list of amino acids includes lysine, glutamic acid, , aspartic acid, serine, tyrosine, cysteine, selenocysteine, glycine and phosphatase Contains moaranine.

[0071] The spacer may be used to separate two components of an antibody-drug-conjugate, e.g., two connector units. It is a covalently linked bivalent arm.

[0072] A non-exhaustive list of spacer units includes alkylene, heteroalkylene (and thus Ar interrupted by at least one heteroatom selected from Si, N, O and S alkylene); alkoxy; polyethers, such as polyalkylene glycols and typically Polyethylene glycol; glycine, alanine, proline, valine, N-methylglycine one or more natural or unnatural amino acids such as; C3-C8 heterocyclo; C3-C8 carboxyl For example, the spacer may be a divalent group, such as chloro; arylene, or any combination thereof. It is a straight chain alkylene group, preferably (CH2)4.

[0073] For example, the spacer is -C1 to C 10 Alkylene-, -C1~C 10 Heteroalkylene -, -C3-C8 carbocyclo-, -O-(C1C8 alkyl)-, -arylene-, -C1~C 10 Alkylene-arylene-, -arylene-C1-C 10 alkylene-, -C1~C 10 Alkylene-(C3-C8 carbocyclo)-, -(C3-C8 carbocyclo) b)-C1~C 10 Alkylene-, -C3-C8 heterocyclo-, -C1-C 10 Archi Ren-(C3-C8 heterocyclo)-, -(C3-C8 heterocyclo)-C1-C 10 a Alkylene-, -C1~C 10 Alkylene -C(=O)-, -C1 to C10 Heteroalkyl Cyclo-C(=O)-, -C3-C8 carbocyclo-C(=O)-, -O-(C1-C8 alkane Arylene-C(=O)-, -C1~C 10 Alkylene-Aryl Arylene-C(=O)-, -arylene-C1~C 10 Alkylene-C(=O)-, -C1 ~C 10 Alkylene-(C3-C8 carbocyclo)-C(=O)-, -(C3-C8 carbocyclo) Boshikuro)-C1~C 10 Alkylene-C(=O)-, -C3-C8 heterocyclo-C( =O)-, -C1~C 10 Alkylene-(C3-C8 heterocyclo)-C(=O)-, - (C3-C8 heterocyclo)-C1-C 10 Alkylene -C(=O)-, -C1 to C 10 Alkylene-NH-, -C1~C 10 Heteroalkylene-NH-, -C3~C8 carboxy chloro-NH-, -O-(C1-C8 alkyl)-NH-, -arylene-NH-, -C1 ~C 10 Alkylene-arylene-NH-, -arylene-C1-C 10 Alkylene-N H-, -C1~C 10 Alkylene-(C3-C8 carbocyclo)-NH-, -(C3-C 8Carbocyclo)-C1~C 10 Alkylene-NH-, -C3~C8 heterocyclo-NH -, -C1~C 10 Alkylene-(C3-C8 heterocyclo)-NH-, -(C3-C8 Heterocyclo)-C1~C 10 Alkylene-NH-, -C1~C 10 alkylene-S-, -C1~C 10 Heteroalkylene-S-, -C3-C8 carbocyclo-S-, -O-(C 1-C8 alkyl)-)-S-, -arylene-S-, -C1-C 10 Alkylene-Aryl Arylene-S-, -Arylene-C1~C 10 Alkylene-S-, -C1~C 10 Alkire -(C3-C8 carbocyclo)-S-, -(C3-C8 carbocyclo)-C1-C 10 Alkylene-S-, -C3-C8 heterocyclo-S-, -C1-C 10 Alkylene-(C (3~C8 heterocyclo)-S-, -(C3~C8 heterocyclo)-C1~C 10 Alkire N-S-, -C1~C 10 Alkylene-OC(=O)-, -C3~C8 carbocyclo- OC(=O)-, -O-(C1-C8 alkyl)-OC(=O)-, -arylene- OC(=O)-, -C1~C 10 Alkylene-arylene-OC(=O)-, -Arylene Len-C1~C 10 Alkylene -OC(=O)-, -C1 to C 10 Alkylene-(C -(C3-C8 carbocyclo)-OC(=O)-, -(C3-C8 carbocyclo)-C1-C 10 Alkylene-OC(=O)-, -C3-C8 heterocyclo-OC(=O)-, - C1~C 10 Alkylene-(C3-C8 heterocyclo)-OC(=O)-, and -(C 3-C8 heterocyclo)-C1-C 10 alkylene -OC(=O)- can be selected.

[0074] Any of the above groups may be -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 optionally substituted with one or more substituents selected from, each X independently represents halogen: -F, -CI, -Br, or -I, and each R' is independently -H, -CI, - C 20 Alkyl, -C6~C 10 Aryl or -C3~C 10 It is a heterocycle.

[0075] As used herein, the term "alkyl" refers to a univalent saturated hydrocarbon chain (straight chain For example, alkyl refers to C1-C 20 Refers to alkyl. Alternatively, alkyl may be a "lower alkyl," i.e., alkyl having 1, 2, 3, 4, 5, or 6 carbon atoms. The alkyl group is a straight or branched C1 to C6 alkyl group. , methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, sec-butyl butyl, tert-butyl, n-pentyl, n-hexyl, and the like.

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

[0077] Alkenyl and alkynyl are alkyl groups having 2 to 20 carbon atoms, preferably 2 to 12 carbon atoms, more preferably 1 to 20 carbon atoms. Preferably 2 to 6, especially 2 to 4, at least partially unsaturated linear or branched hydrocarbons An alkenyl group contains at least one C=C double bond, and an alkynyl group is , at least one

[0078] [ka] Contains a triple bond.

[0079] 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. A cycloalkyl can have a single ring or multiple rings fused together. The alkyl group may also include a spiro ring. Suitable cycloalkyl groups include cycloalkyl groups such as cycloalkyl groups, ... Examples include propyl, cyclobutyl, cyclopentyl and cyclohexyl.

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

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

[0082] As used herein, the term "C1-C6 haloalkyl" means any of the groups defined herein. C1-C6 alkyl esters as defined herein, substituted with one or more halogen groups as defined herein. Suitable C1-C6 haloalkyl groups include trifluoromethyl and difluoromethyl. Examples include chloromethyl.

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

[0084] Heteroalkylene refers to a divalent heteroalkyl as defined above. In cases where heteroatoms are present, they can also occupy either or both of the chain termini.

[0085] As used herein, the term "C1-C6 alkoxy" refers to an -O-alkyl group. wherein the alkyl group is C1-C6 alkyl as defined herein. 6Alkoxy groups include methoxy, ethoxy, and propoxy.

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

[0087] As used herein, the term "aryl having 6 to 10 ring atoms" means Polyunsaturated aromatic rings containing 6 to 10 ring atoms, either a single ring or multiple aromatic rings fused together The term "aromatic hydrocarbyl group" refers to an aromatic hydrocarbyl group in which at least one ring is aromatic. 1 to 2 additional rings (cycloalkyl, heterocyclyl or heterocyclic as defined herein) joined together Suitable aryl groups include benzopyranyl, benzyl, Phenyl, naphthyl, etc. fused to heterocyclyl such as benzodioxolyl, benzodioxanyl, etc. Examples include aryl and phenyl rings.

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

[0089] As used herein, the term "heteroaryl having 5 to 10 ring atoms" The term refers to a single ring or multiple aromatic rings fused or covalently bonded together containing 5 to 10 atoms. wherein at least one ring is aromatic and at least one One ring atom is a heteroatom selected from N, O, and S. The nitrogen and sulfur heteroatoms are It may be optionally oxidized and the nitrogen heteroatom may be optionally quaternized. It may be fused to an aryl, cycloalkyl or heterocyclyl ring. Non-limiting examples of aryl include furanyl, thiophenyl, pyrrolyl, pyrazolyl, Imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl Zolyl, oxadiazolyl, thiadiazolyl, tetrazolyl, oxatriazolyl, thia Triazolyl, pyridinyl, pyrimidyl, pyrazinyl, pyridazinyl, oxazinyl, di Oxynil, thiazinyl, triazinyl, indolyl, isoindolyl, benzofuranyl , isobenzofuranyl, benzothiophenyl, isobenzothiophenyl, indazolyl, Benzimidazolyl, benzoxazolyl, purinyl, benzothiadiazolyl, quinolinyl Examples include quinolinyl, isoquinolinyl, cinnolinyl, quinazolinyl and quinalinyl.

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

[0091] As used herein, the term "heterocyclo" or "heterocycloalkylene" The term refers to a divalent heterocycle as defined herein.

[0092] Further, alkyl, alkenyl, alkynyl, aryl, alkylene, arylene, heterocyclic alkyl, heteroalkylene, C3-C8 carbocyclic, C3-C8 carbocyclic, C3-C8 heterocyclic The terms ring, C3-C8 heterocycle, and polyether are used to refer to -X, -R', -O - ,-OR', =O, -SR', -S - , -NR'2, -NR'3, =NR', -CX3, -CN, -O CN, -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 - , -PO3H 2, -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( and C(=NR')NR'2. refers to an optionally substituted group, wherein each X is independently a halogen: -F, -CI, -Br, or is -I, and each R' is independently -H, -C 20 Alkyl, -C6~C 10 a Reel or -C3~C 10 It is a heterocycle.

[0093] As used herein, the term "polyether" refers to a polyether containing ether linkages. The number of ether moieties in the polyether is between 2 and 100, preferably between 2 and 2 The molecular weight of the polyether may be between 5 and 5, particularly between 2 and 10. Examples of polyethers include polyethylene glycols, Contains rules.

[0094] Electron-withdrawing groups attract electrons from neighboring atoms toward themselves, usually by resonance or inductive effects. refers to an atom or group that withdraws electron density. Electron-withdrawing groups include halogens, haloalkyls (- CF3, etc.), -CN, -SO3H, -NO2, and -C(O)R groups (where R = H, OH, or Advantageously, the electron withdrawing group is -NO2. In one embodiment, In the example, the electron-withdrawing group is ortho to the YT substituent on the phenyl ring.

[0095] As used herein, the term "protecting group" refers to the protection of a regenerating functional group or other functional group in a molecule. Chemical substitution that can be selectively removed by readily available reagents that do not attack the functional group Suitable protecting groups are known in the art and continue to be developed. The groups are described, for example, in Wutz et al. ("Greene's Protective Groups in Organic Synthesis,Fourth Editi on,” Wiley-Interscience, 2007). In embodiments, the method described by Wutz et al. (pp. 696-927) A protecting group is used to protect the amino group. Representative examples of amino protecting groups include t-butyl. 9-Fluorenylmethoxycarbonyl (Boc), 9-Fluorenylmethoxycarbonyl (Fmoc), Cetyl (Ac), carboxybenzyl (Cbz), benzyl (Bn), allyl, tri Fluoroacetyl, allyloxycarbonyl (Alloc) group and 2,2,2-trichloro These include, but are not limited to, thiathoxycarbonyl (Troc).

[0096] Hydrophobic masking entities refer to groups that can reduce the apparent hydrophobicity of a compound The hydrophobic masking entity may be selected from polysarcosine and polyethylene glycol. The number of ethylene glycol or sarcosine moieties can vary widely. For example, the number of ethylene glycol or sarcosine moieties in the hydrophobic masking entity is 2 to 50. 0, preferably between 5 and 100, in particular between 5 and 25. In one embodiment, The hydrophobic masking entity contains 6 to 24 sarcosine moieties, preferably 10 to 1 It is a polysarcosine containing two sarcosine moieties.

[0097] As used herein, the term "coupled" refers to a linkage. It is also represented by a dash "-" in formula (I). The bond may be a covalent bond, an electrostatic force, or the like. The bond may be a non-covalent interaction due to the interaction of the two or more molecules. Preferably, the bond is a covalent bond. When used, the "wavy lines" in the formulas represent the individual moieties (Ab, L, Z, X, and D) of the ADCs of the present disclosure. Represents the binding site between

[0098] As used herein, the terms "treat," "treating," or "treatment" (1) inhibiting a disease, e.g., preventing the symptoms or symptomology of a disease, condition, or disorder from being experienced; inhibiting a disease, condition, or disorder in an individual who has or is exhibiting the disease, condition, or disorder (i.e., (2) preventing further development of pathology and / or symptomatology; (3) ameliorating the disease, e.g. , a disease, condition in an individual experiencing or exhibiting the pathology or symptomology of the disease, condition, or disorder or ameliorating the disorder (i.e., reversing the pathology and / or symptomatology), e.g. For example, reducing the severity of a disease or alleviating or alleviating one or more symptoms of a disease. In particular, with respect to the treatment of tumors, the term "treatment" refers to one or more of the following: It may refer to an inhibition of growth or a reduction in size of a tumor.

[0099] As used herein, a "therapeutically effective amount" or "effective amount" of an ADC is defined as an amount sufficient to carry out its intended purpose, e.g., to produce a therapeutic effect, e.g., tumor growth, after administration. inhibition or reduction in tumor growth rate or tumor volume, reduction in cancer symptoms, or any indication of therapeutic efficacy. In the case of cancer, a therapeutically effective amount of an ADC reduces the number of cancer cells and reducing tumor size, inhibiting (e.g., slowing or stopping) tumor metastasis, inhibiting tumor growth ( For example, the blood flow may be slowed or stopped) and / or one or more symptoms may be alleviated.

[0100] As used herein, the percent identity between two sequences refers to the best fit between the two sequences. Consider the number of gaps that need to be introduced for the alignment and the length of each gap. Also, it is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions (number of sequences / total number of positions × 100). Sequence comparison and determination of percent identity between two sequences The determination can be accomplished using a mathematical algorithm, as described below.

[0101] The percent identity between two amino acid sequences is calculated using the Needleman and Wuns method can be determined using the ch algorithm (Needleman and Wunsch) can.

[0102] The percent identity between two nucleotide or amino acid sequences can also be determined using the EMBO S.S. Needle (pairwise alignment; available at www.ebi.ac.uk For example, the EMBOSS Needle can be determined using BLOSUM62 matrix, a "Gap Open Penalty" of 10, a "Gap Extension" of 0.5 "End Gap Penalty", "False End Gap Penalty", 10 "End Gap Open" It can be used with an "End Gap Extend Penalty" of 0.5 and an "End Gap Extend Penalty" of 0.5. Generally, "percent identity" is calculated by dividing the number of matching positions by the number of positions compared, to give a 10 For example, after alignment, 6 out of 10 sequence positions are 2 If the two compared sequences are identical, the identity is 60%. The percent identity is typically expressed as is determined over the entire length of the query sequence being analyzed. Two molecules that have a nucleic acid sequence are identical regardless of chemical and / or biological modifications.

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

[0104] Preferably, such antibodies include those isolated and listed in Table 1 below. Structurally characterized by variable heavy and light chain amino acid sequences and human constant isotypes The following antibodies are included:

[0105] [Table 1]

[0106] IgG1 LALA J. Virol 2001 Dec;75(24):1216 Residues 234 and 235, also disclosed in 1-8 (Hezareh et al.) Corresponding to a mutant IgG1 Fc region containing a leucine-to-alanine amino acid substitution in do.

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

[0108] [Table 2]

[0109] VH CDR1 (also called HCDR1), VH CDR2 (HCDR2) of mAb1 antibody R2), VH CDR3 (also called HCDR1), VL CDR1 (L CDR1), VL CDR2 (also called LCDR2), VL CDR3 Examples of amino acid sequences of (also called HCDR3) are shown in Table 3.

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

[0111] [Table 3]

[0112] Tables 4 and 5 below show useful amino acid and nucleotide sequences compared to antibodies in ADCs. to provide.

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

[0114] [Table 5-1]

[0115] [Table 5-2]

[0116] [Table 5-3]

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

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

[0119] In a specific embodiment, the Ab comprises a heavy chain of SEQ ID NO: 9 and a light chain of SEQ ID NO: 10, or and a recombinant antibody consisting essentially of them.

[0120] In a specific embodiment, the Ab comprises a heavy chain of SEQ ID NO: 11 and a light chain of SEQ ID NO: 10. is a recombinant antibody consisting essentially of them.

[0121] In a specific embodiment, the Ab comprises a heavy chain of SEQ ID NO: 16 and a light chain of SEQ ID NO: 17 or is a recombinant antibody consisting essentially of them.

[0122] In certain embodiments, the Ab is an anti-FRα antibody that has one or more of the following properties: do.

[0123] (i) Abs are identified by surface plasmon resonance, such as a Biacore® assay. K below 100 nM when measured D and preferably a K of 50 nM or less D Folate receptor receptor binds to receptor alpha (FRα), (ii) the Ab is an internalizing antibody, and is internalized specifically in FRα-expressing tumor cells; (iii) Abs have 4-8 payloads per antibody without particular stability issues or aggregation. This allows for the conjugation of

[0124] In certain embodiments which may be combined with the preceding embodiments, Ab is a group defined above. It is an internalizing antibody fragment of a recombinant antibody.

[0125] As used herein with respect to antibodies, "internalizes" refers to the ability of an antibody to bind to a cell and then internalize the cell upon specific binding to the cell. through the outer lipid bilayer membrane of the cell to an internal compartment (i.e., "internalization"), preferably within the cell. It refers to an antibody that can be incorporated into the solution compartment.

[0126] Antibody fragments include Fab, Fab', Fab'-SH, F(ab')2, Fv, and UniB These include fibroblast and scFv fragments, diabodies, single domain or nanobodies and other fragments. The term "diabody" refers to a molecule that contains two antigen-binding sites. This refers to small antibody fragments having a VH-VL domain, which are light chains in the same polypeptide chain (VH-VL). It contains a heavy chain variable domain (VH) linked to a single chain variable domain (VL). By using a linker that is too short to allow pairing between the domains, The domains are forced to pair with the complementary domains of another chain, creating two antigen-binding sites. Single domain antibodies are antibodies that contain all or part of the heavy chain variable domain or the light chain variable domain of an antibody. In certain embodiments, antibody fragments containing all or part of a single domain are The antibody is a human single domain antibody (Domantis, Inc., Waltham , MA; see, e.g., U.S. Pat. No. 6,248,516). Antibody fragments include, but are not limited to: , proteolytic digestion of intact antibodies, and recombinant host cells as described herein. They can be produced by a variety of techniques, including production.

[0127] In certain embodiments, the Ab is a humanized antibody. Typically, a non-human antibody is a humanized antibody. It has at least the same affinity (or better affinity) as a human antibody, while retaining its immunity to humans. In a preferred embodiment, the antibodies of the present disclosure are humanized antibodies. Generally, a humanized antibody is one in which the CDRs (or portions thereof) are derived from a non-human antibody, e.g., a mouse antibody. Derived from an anti-FRα internalizing antibody, the framework region (or a part thereof) is derived from a human antibody sequence. Humanized antibodies also optionally contain one or more variable domains derived from human constant regions. In some embodiments, some of the framework regions in the humanized antibody are The amino acid residues may be modified, for example, by the addition of amino acid residues to a non-human antibody (e.g., a non-human antibody) to restore or improve antibody specificity or affinity. For example, CDR residues are substituted with the corresponding residues from the anti-FRα mouse antibody from which they are derived. In some particular embodiments, some CDR residues in a humanized antibody may also be humanized, e.g., The substitutions are made to restore or improve specificity or affinity. Humanized antibodies and methods for making them are also described. See, e.g., Almagro and Fransson, Front. Biosci. 13:1 619-1633 (2008), and further see, for example, Riechmann et al.,Nature 332:323-329(1988);Queen et al. al.,Proc.Natl Acad.Sci.USA 86:10029-100 33 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, and No. 6,982,321 and No. 7,087,409;Kashmiri et al. .,Methods 36:25-34(2005)(Specificity Determining Region (SDR) graph Padlan, Mol. Immunol. 28:489-498 (199 1) (referring to "resurfacing"); Dall'Acqua et al., Metho ds 36:43-60(2005) (referring to "FR shuffling"); and Osbo Urnet al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (FR Sha This is further described in (describes a "guided selection" approach to buffering).

[0128] Preferably, the Ab is a humanized or human silenced antibody, preferably a humanized silenced Ig. It is a G1 antibody.

[0129] As used herein, the term "silent" antibody refers to an antibody that is capable of inhibiting ADCC activity in an ADCC activity assay. This refers to antibodies that exhibit no or low ADCC activity when measured by

[0130] In one embodiment, the term "no or low ADCC activity" refers to a silenced antibody that: The ADCC activity observed with the corresponding antibody with the wild-type IgG isotype was reduced. In both cases, the ADCC activity is less than 10%, for example, less than 50%.

[0131] Silenced effector functions are mediated by mutations in the Fc constant portion of antibodies. and in the art: Strohl 2009 (AA&N297A) Baud ino 2008, D265A (Baudino et al., J. Immunol. 181(2008):6664-69,Strohl,CO Biotechnolog y 20(2009):685-91) or Saunders 2 019(Front.Immunol.,07 June 2019,doi:10.3 389 / fimmu.2019.01296). Silent IgG1 An example antibody contains the L234A and L235A mutations in the IgG1 Fc amino acid sequence. The so-called LALA mutation, or Ser228Pro paired with Leu235Glu, is involved. Pro331Ser can also be used to generate silent IgG1 antibodies using Ledu23. 4Glu and Leu235Phe (LALA-PG), optionally in combination Another example of a silent IgG1 antibody is an aglycosylated or non-glycosylated antibody. An example of a silent IgG4 antibody contains the N297A mutation, resulting in an antibody with a Ser228P Includes ro.

[0132] In certain embodiments, the Ab is farletuzumab, or 1 or other anti-FRα antibodies disclosed in WO 2017151979. do.

[0133] In other specific embodiments, the Ab is a silencing agent of mirvetuximab or mirebutuximab. It is a TRALALA mutant.

[0134] In a preferred embodiment, the Ab is a silent LALA mutant of farletuzumab, or The compounds disclosed in International Publication No. 2005080431 or International Publication No. 2017151979 Other anti-FRα antibodies that have been reported include:

[0135] Antibodies having variant amino acid sequences can be produced by mutagenesis (e.g., site-directed or PCR-mediated mutagenesis) followed by the functional assays described herein were used to confirm the presence of retained by testing the encoded altered antibodies for their desired function (i.e., the functions described above). You can get this.

[0136] Antibodies with conservative modifications In certain embodiments, the Ab comprises a duplicated fragment comprising HCDR1, HCDR2, and HCDR3 sequences. and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 sequences. One or all of these CDR sequences are present in mAb1 (farletuzumab) described herein. a silent LALA mutant version of the antibody) based on a specific amino acid sequence, or or a group that differs from the CDR sequence of farletuzumab by three conservative amino acid modifications. and functional variants of the antibody having similar CDR sequences, and the antibody or protein is When used as an ADC, it retains the desired functional properties of the mAb1 antibody.

[0137] In certain embodiments, the Ab comprises a duplicated fragment comprising HCDR1, HCDR2, and HCDR3 sequences. and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 sequences. One or more of these CDR sequences may be used in the mirebutuximab (or mirbetuximab) described herein. a silent LALA mutant version of ximab) antibody based on a specific amino acid sequence, or The CDR sequences of farletuzumab are modified by one, two, or three conservative amino acid modifications. and functional variants of the antibody having different but similar CDR sequences, The quality retains the desirable functional properties of the mAb1 antibody, particularly when used as an ADC.

[0138] Desirable functional properties of an anti-FRα antibody include, but are not limited to: (i) The anti-FRα antibody exhibits an ELISA assay with an EC50 of less than 5 nM, for example, about 0.5 nM. binds to folate receptor alpha (FRα) as determined by SEQ ID NO: 1 (described in the Examples); (ii) The anti-FRα antibody is capable of detecting surface plasmon resonance in a Biacore® assay or the like. K below 100 nM as measured by D and preferably a K of 50 nM or less D Leaves Binds to acid receptor receptor alpha (FRα) (e.g., SPR Biacor described in the Examples) e) as determined using affinity assays. (iii) Anti-FRα is an internalizing antibody, and is internalized especially in FRα-expressing tumor cells. Ru, (iv) Anti-FRα can be used with 4-8 payloads per antibody without any particular stability issues or aggregation. The drug-antibody ratio can be, for example, as described in the Examples. As determined by RPLC-MS, (v) ADCs comprising such variant anti-FRα antibodies are, for example, those used in the Examples. Using an in vitro efficacy assay in FRα-negative cell lines, BT-474 breast cancer cell line), and a corresponding control containing mAb1 as the anti-FRα antibody. provide in vitro efficacy similar to or lower than ADCs, and / or (vi) ADCs having such variant anti-FRα antibodies are useful, for example, as used in the Examples. Using one of the xenograft models available, we demonstrated the efficacy of anti-cancer drugs in tumor-bearing mouse xenograft models. Similar or higher in vivo efficacy than the corresponding control ADC with mAb1 as the FRa antibody Provides sex.

[0139] As used herein, the term "conservative sequence modifications" refers to modifications in which the amino acid residues are similar. It is intended to refer to an amino acid substitution in which an amino acid residue having a side chain is substituted. Families of amino acid residues having similar side chains have been defined in the art. The family includes basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, beta-branched side chains (e.g., threonine, valine, isoleucine) and and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine) Thus, the present disclosure also includes amino acids having one or more amino acids within the CDR regions of the antibodies. The amino acid residue can be replaced with another amino acid residue from the same side chain family, resulting in a change The resulting antibodies are tested for retained function using the functional assays described herein. It is possible.

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

[0141] In certain embodiments, the Ab is 100% identical to the corresponding CDRs of SEQ ID NOs: 1-6. The six CDRs and corresponding framework amino acids are identified in SEQ ID NOs: 7 and 8, respectively. amino acid region and at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 9 Anti-FRα antibodies containing framework amino acid regions that are 7%, 98%, or 99% identical. The anti-FRα antibody has the following properties: (i) The anti-FRα antibody exhibits an ELISA assay with an EC50 of less than 5 nM, for example, about 0.5 nM. binds to folate receptor alpha (FRα) as determined by SEQ ID NO: 1 (described in the Examples); (ii) Anti-FRα is detected by surface plasmon resonance assay such as Biacore® assay. Therefore, when measured, K values ​​of 100 nM or less D and preferably a K of 50 nM or less D Folic acid intake binds to receptor α (FR), (iii) Anti-FRα is an internalizing antibody, and is internalized especially in FRα-expressing tumor cells. Ru, (iv) Anti-FRα can be used with 4-8 payloads per antibody without any particular stability issues or aggregation. The drug-antibody ratio can be, for example, as described in the Examples. As determined by RPLC-MS, (v) ADCs comprising such variant anti-FRα antibodies are, for example, those used in the Examples. Using an in vitro efficacy assay in FRα-negative cell lines, BT-474 breast cancer cell line), and a corresponding control containing mAb1 as the anti-FRα antibody. provide in vitro efficacy similar to or lower than ADCs, and / or (vi) ADCs having such variant anti-FRα antibodies are useful, for example, as used in the Examples. Using one of the xenograft models available, we demonstrated the efficacy of anti-cancer drugs in tumor-bearing mouse xenograft models. Similar or higher in vivo efficacy than the corresponding control ADC with mAb1 as the FRa antibody Provides sex.

[0142] In certain embodiments, the Ab is 100% identical to the corresponding CDR of mirebutuximab. The six CDRs are identical to the corresponding framework amino acid regions of mirvetuximab at least once a year. Also 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and an anti-FRα antibody comprising a framework amino acid region identical to that of the anti-FRα antibody. has the following properties: (i) Anti-FRα has an EC50 of less than 5 nM, e.g., about 0.5 nM, in an ELISA assay. binds to folate receptor alpha (FRα) as determined by (described in the Examples); (ii) Anti-FRα is detected by surface plasmon resonance assay such as Biacore® assay. Therefore, when measured, K values ​​of 100 nM or less D and preferably a K of 50 nM or less D Folic acid intake binds to receptor α (FR), (iii) Anti-FRα is an internalizing antibody, and is internalized especially in FRα-expressing tumor cells. Ru, (iv) Anti-FRα can be used with 4-8 payloads per antibody without any particular stability issues or aggregation. The drug-antibody ratio can be, for example, as described in the Examples. As determined by RPLC-MS, (v) ADCs comprising such variant anti-FRα antibodies are, for example, those used in the Examples. Using an in vitro efficacy assay in FRα-negative cell lines, BT-474 breast cancer cell line), and a corresponding control containing mAb1 as the anti-FRα antibody. provide in vitro efficacy similar to or lower than ADCs, and / or (vi) ADCs having such variant anti-FRα antibodies are useful, for example, as used in the Examples. Using one of the xenograft models available, we demonstrated the efficacy of anti-cancer drugs in tumor-bearing mouse xenograft models. Similar or higher in vivo efficacy than the corresponding control ADC with mAb1 as the FRα antibody Provides sex.

[0143] Framework or Fc manipulation For use in the ADCs of the present disclosure, Abs can also be used in the manipulation of antibodies disclosed in the previous section. These may include engineered versions, e.g., within the VH and / or VL to improve antibody properties. Typically, such framework residues are modified. Key modifications are made to reduce the immunogenicity of antibodies. For example, one approach is to , "backmutating" one or more framework residues to the corresponding germline sequence ) More specifically, antibodies that have undergone somatic mutations are not related to the germline from which they are derived. The sequence may contain framework residues that differ from the antibody framework. The sequence can be identified by comparing it to the germline sequence from which the antibody is derived. To restore framework region sequences to their germline configuration, somatic mutations can be performed at, e.g., sites It can be "backmutated" to the germline sequence by directed mutagenesis or PCR-mediated mutagenesis. Such "backmutated" antibodies are also intended to be encompassed by the present invention.

[0144] Another type of framework modification removes T cell epitopes, thereby modifying the antibody. To reduce potential immunogenicity, one or more C This involves mutating one or more residues within the DR region.

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

[0146] Additionally, Abs can be chemically modified (e.g., by attaching one or more chemical moieties to the antibody). or antibodies that can be modified to alter their glycosylation. , again altering one or more functional properties of the antibody. Each of these embodiments is described below. This will be explained in more detail.

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

[0148] In one particular embodiment, the hinge region of CH1 is The number is modified to change, e.g., increase or decrease. Further described in U.S. Patent No. 5,677,425 to Bodmer et al. The number of cysteine ​​residues in the hinge region of CH1, for example, facilitates assembly of the light and heavy chains. The alterations may be made to facilitate the synthesis or to increase or decrease the stability of the antibody.

[0149] In other embodiments, the Fc region comprises an Fc region of an antibody that mediates antibody-dependent cellular cytotoxicity (ADCC). Fcγ by modifying one or more amino acids to reduce its potency Modifications that reduce the affinity of the antibody for the receptor. Reduced effector function. In particular, such antibodies with reduced ADCC include silent antibodies.

[0150] In certain embodiments, an Fc domain of the IgG1 isotype is used. In certain embodiments, mutant variants of IgG1 Fc fragments, e.g., antibody-dependent cell proliferation, Reduced ability of ADC to mediate ADCC and / or bind to Fcγ receptors Alternatively, a silent IgG1 Fc that excludes the IgG1 isotype may be used. Preferred examples of the mutants are described in J. Virol 2001 Dec;75(24):12161 As described in Hezareh et al., leucine is an amino acid IgG1 in which positions 234 and 235 are replaced by alanines. An example is an IgG1 isotype silent mutant with a LALA-PG triple mutation. In addition to the LALA mutation, the proline at position 329 is replaced by a glycine.

[0151] In certain embodiments, the Fc domain comprises a glycosylated nucleotide at position 297 of the Fc domain. For example, the Fc domain contains an amino acid sequence at position 297, which is a silent Fc variant that prevents cleavage. Examples of such amino acid substitutions include amino acid substitutions of glycine or alanine. The substitution of N297 by methylamino acid.

[0152] In yet another embodiment, the glycosylation of the antibody is modified. For example, a non-glycosylated antibody The antibody can be made to be glycosylated (i.e., the antibody lacks glycosylation). For example, alterations can be made to increase the affinity of the antibody for the antigen. Such carbohydrate modifications can be made, for example, by altering one or more sites of glycosylation within the antibody sequence. For example, glycosylation of one or more variable region frameworks can be achieved. one or more amino acids that result in the elimination of a glycosylation site, thereby eliminating glycosylation at that site Such aglycosylation can increase the affinity of the antibody for the antigen. Such an approach is described in U.S. Patent No. 5,714,350 by Co et al. and 6,350,861.

[0153] Another modification of the antibodies herein contemplated by this disclosure is pegylation or hexylation, or Related Art: Antibodies are, for example, immunosuppressants that increase the biological (e.g., serum) half-life of antibodies. To pegylate an antibody, the antibody or fragment thereof may be pegylated with one or more under conditions such that the above PEG group becomes attached to the antibody or antibody fragment, typically Polyethylene glycol (PEG), such as reactive ester or aldehyde derivatives of PEG PEGylation is performed by reacting a reactive PEG molecule (or an analogous reactive water-soluble polymer) with As used herein, the term "aromatic" refers to a carboxylic acid or a carboxylic acid derivative that is a carboxylic acid derivative of a carboxylic acid. In this case, the term "polyethylene glycol" refers to mono(1-C 10 )Alkoxy-young aryloxy-polyethylene glycol or polyethylene glycol-maleimide, etc. This includes any form of PEG that has been used to derivatize other proteins. In certain embodiments, the antibody to be pegylated is an aglycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to the antibodies of the disclosure. For example, Nishimura et al., European Patent No. 0154316 See European Patent No. 0401384 by Ishikawa et al. .

[0154] Another modification of the antibodies contemplated by the present disclosure is to increase the half-life of the resulting molecule. and a serum protein, such as human serum albumin, Such approaches include conjugates or protein fusions with the ribozyme or fragments thereof. is described, for example, in European Patent No. 0322094 to Ballance et al. are.

[0155] Generation of antibodies for use in generating ADCs of the present disclosure The antibodies of the present disclosure can be obtained using conventional techniques known to those skilled in the art. Further details regarding the generation of nucleic acids encoding these antibodies and transfectomas producing these antibodies are available. For further information, the skilled artisan is referred to International Application No. WO 2005080431. It is also possible to do so.

[0156] For example, to express an antibody or antibody fragment thereof, partial or full-length light and heavy chains can be synthesized using The DNA encoding the desired antibody can be isolated using standard molecular biology or biochemistry techniques (e.g., Chemical DNA synthesis, PCR amplification, or cDNA cloning using expressing hybridomas ), wherein the gene is operably linked to transcriptional and translational control sequences. The DNA can be inserted into an expression vector so that it can be used to express the gene. The term "operably linked" refers to the transcription and translation control sequences in a vector that allow transcription of the antibody gene. The antibody genes are inserted into the vector so that they perform their intended functions of regulating transcription and translation. The term "ligated" is intended to mean "ligated." Expression vectors and expression control sequences The sequence is selected to be compatible with the expression host cell used. The chain genes can be inserted into separate vectors, or more typically, both genes are inserted into the same vector. The antibody gene is inserted into the same expression vector as the antibody gene fragment. Ligation of complementary restriction sites on the cleavage site and the vector, or if no restriction sites are present, The vector is inserted into the expression vector by blunt end ligation (if desired). The light and heavy chain variable regions of an antibody containing the VH segment are used to express the antibody. the VL segment is operably linked to the CL segment in the vector The heavy and light chain constant regions of the desired isotype are all operably linked. The entire antibody of any antibody isotype can be expressed by inserting it into an expression vector already encoding it. Additionally or alternatively, recombinant expression vectors can be used to generate long antibody genes. A signal peptide can be encoded that facilitates secretion of the antibody chain from the host cell. The chain genes are constructed such that the signal peptide is attached in-frame to the amino terminus of the antibody chain gene. The signal peptide can be cloned into a vector as follows: a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein) It may also be a peptide.

[0157] In addition to the antibody chain genes, the recombinant expression vectors disclosed herein can also The term "regulatory sequence" refers to a regulatory sequence that controls the expression of the antibody chain genes. Promoters, enhancers and other expression control elements (e.g., The expression vector containing the selection of regulatory sequences is intended to include a sequence encoding the desired sequence (e.g., a polyadenylation signal). The design of the target depends on factors such as the choice of host cell to be transformed and the level of expression of the desired protein. It will be understood by those skilled in the art that the regulatory sequences for mammalian host cell expression may depend on the Cytomegalovirus (CMV), Simian virus 40 (SV40), adenovirus genes (e.g., adenovirus major late promoter (AdMLP)), and polyoma-derived High levels of proteins in mammalian cells, such as promoters and / or enhancers It contains viral elements that direct expression. Alternatively, it may contain a ubiquitin promoter or a P-globin promoter. Non-viral regulatory sequences may also be used, such as the SV40 promoter. Contains sequences from the early promoter and long terminal repeat of human T-cell leukemia virus type 1. The sequences were constructed from different sources, including the SRa promoter system.

[0158] In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the disclosure also contain the antibody chain genes and regulatory sequences required for expression in a host cell. Addition of sequences that regulate replication of the vector (e.g., replication origin) and selectable marker genes, etc. The selectable marker gene allows for the selection of host cells into which the vector has been introduced. (See, e.g., U.S. Patent No. 4,399,216, all by Axel et al.) (See, for example, US Pat. Nos. 4,634,665 and 5,179,017). Typically, the selectable marker gene is selected from the group consisting of G418, G418- ... Conferring resistance to drugs such as hygromycin or methotrexate. The mosquito gene contains the dihydrofolate reductase (DHFR) gene (methotrexate selection / amplification (for use in dhfr host cells with dhfr) and neo genes (for G418 selection) It can be enjoyed.

[0159] For expression of the light and heavy chains, expression vector(s) encoding the heavy and light chains are targeted. The host cell is transfected by a conventional technique called "transfection." Various forms of the term generally refer to the introduction of exogenous DNA into prokaryotic or eukaryotic host cells. A wide variety of techniques are used to DEAE-dextran transfection, etc. It is understood that the antibodies can be expressed in either prokaryotic or eukaryotic host cells. It is theoretically possible to produce antibodies in eukaryotic cells, such as mammalian host cells, yeast, or filamentous fungi. Expression of the nucleotides in the nucleotide sequence ... This is argued to be due to the fact that they have a higher potential than prokaryotic cells to assemble and secrete specific antibodies.

[0160] Nucleic acids encoding the heavy and light chains of preferred antibodies for use in making the ADCs of the disclosure The peptide sequences are set out in Tables 3 and 4 (see in particular SEQ ID NOS: 13-15).

[0161] Mammalian host cells for expressing the recombinant antibodies of the disclosure may contain a DHFR selectable marker. (described in Kaufman and Sharp, 1982) CHO cells (described in Urlaub and Chasin, 1980), CHOK1 d Chinese ham containing hfr+ cell lines, NSO myeloma cells, COS cells and SP2 cells These include star ovary (CHO) cells. When introduced into a mammalian host cell, the antibody is conjugated to the host cell, resulting in expression of the antibody and optionally and culturing the host cells for a period of time sufficient for secretion of the antibody into the culture medium in which the host cells are grown. Antibodies can be produced, for example, by culturing the antibodies using standard protein purification methods. After their secretion, they can be recovered and purified from the culture medium.

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

[0163] The ADCs of the present disclosure comprise a cleavable antibody conjugated to an Ab on one side and a drug D on the other side. The anti-FRα antibodies comprising the linker moiety L are disclosed in the previous section.

[0164] In certain embodiments, the linker L is linked to one or more thiol residues of an antibody Ab, e.g., an antibody It is covalently linked to a natural or artificial cysteine ​​residue in the sequence.

[0165] As used herein, the term "cleavable" refers to a molecule that can be cleaved under certain environmental conditions (e.g., oxidation-reduction). in response to the intracellular environment, e.g., potential or pH), or the intracellular environment, e.g., after internalization of the ADC within the cell, It refers to a linker that can be cleaved under lysosomal enzymes.

[0166] In certain embodiments, L has the formula -AW-, where A is any string linked to Ab. and W is a cleavable moiety linked to D. - part.

[0167] In certain embodiments, the optional stretcher unit A comprises one or more amino acids, one or more N-substituted amino acids above, optionally substituted polyethers, C1-C 12 alkylene, Arylene having 6 to 10 ring atoms, C3 to C8 cycloalkylene, 5 to 10 ring atoms heterocycloalkylene having 5 to 10 ring atoms; heteroarylene having 5 to 10 ring atoms; C2~C 10 alkenylene, and any combination thereof; The alkylene and alkenylene include -O-, -S-, -C(O)-, -NR''-, -C(O)NR''-, -NR''-C(O)-, -NR''-C(O)-NR'''- , —NR″—C(O)—O—, —OC(O)NR″—, and triazole. and optionally interrupted by one or more heteroatoms or chemical groups, The alkylene, arylene, cycloalkylene, heterocycloalkylene, heteroaromatic Arylene and alkenylene are substituted with halogen, oxo, -OH, -NO2, -CN, C1 C6 alkyl, C3-C6 cycloalkyl, heterocyclyl with 5-10 ring atoms aryl having 6 to 10 ring atoms; heteroaryl having 5 to 10 ring atoms; C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 haloalkoxy, -(CO )-R', -O-(CO)-R', -(CO)-O-R', -(CO)-NR''R'' one or more substituents selected from -NR''-(CO)-R', and -NR''R'''; optionally substituted with a substituent, R', R'', and R''' are independently selected from H and C1-C6 alkyl;

[0168] Examples of cleavable linkers that can be used in the ADCs of the disclosure include acid-sensitive or acid-labile linkers. Qualitative linkers, e.g., acid-labile hydrazone linkers, lysosomal protease-sensitive linkers, Examples of linkers include a β-glucuronide linker, a glutathione-sensitive disulfide linker, and a glutathione-sensitive disulfide linker. can be done.

[0169] In certain embodiments where L is a lysosomal protease-sensitive linker, W is valine. -Citrulline (Val-Cit), Alanine-Alanine-Asparagine (Ala-Ala) -Asn), valine-alanine (Val-Ala) and phenylalanine-lysine (Ph e-Lys). Preferably, W comprises a valine-alanine peptide moiety.

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

[0171] In other particular embodiments, where L is a glutathione-sensitive linker, W is a disulfide moiety. Includes minutes.

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

[0173] [ka] During the ceremony, each R2 is independently selected from the group consisting of an electron-withdrawing group and a C1-C4 alkyl; n is 0, 1 or 2; T is a sugar cleavable unit or a polypeptide cleavable unit; When T is a sugar cleavable unit, Y is O, or when T is a polypeptide cleavable unit, In some cases, Y is NR3; R3 is H, C1~C 24 Alkyl, C2-C6 alkenyl; optionally substituted poly aryls with 6-10 ring atoms, C3-C8 cycloalkyls, 3-1 Heterocycloalkyl having 0 ring atoms, heteroaryl having 5 to 10 ring atoms and any combination thereof; The alkyl and alkenyl include -O-, -S-, -C(O)-, -NR''-, -C (O)NR''-, -NR''-C(O)-, -NR''-C(O)-NR'''-, - selected from NR″—C(O)—O—, —OC(O)NR″— and triazole optionally interrupted by one or more heteroatoms or chemical groups; R" and R"' are independently selected from H and C1-C6 alkyl, I) and pharmaceutically acceptable salts thereof.

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

[0175] In another particular embodiment, T is preferably Val-Cit, Val-Ala and Ph e-Lys.

[0176] In a more particular embodiment, L corresponds to the linker -AW- of formula (IV):

[0177] [ka] During the ceremony, X1 is the connector unit, Z is an optional spacer, X2 is a connector unit, K is preferably selected from polysarcosine and polyethylene glycol; a hydrophobic masking entity of choice, R1 is H, C1~C 24 Alkyl, C2-C6 alkenyl; optionally substituted poly aryls with 6-10 ring atoms, C3-C8 cycloalkyls, 3-1 Heterocycloalkyl having 0 ring atoms, heteroaryl having 5 to 10 ring atoms and any combination thereof; The alkyl and alkenyl include -O-, -S-, -C(O)-, -NR''-, -C (O)NR''-, -NR''-C(O)-, -NR''-C(O)-NR'''-, - selected from NR″—C(O)—O—, —OC(O)NR″— and triazole optionally interrupted by one or more heteroatoms or chemical groups; R4 is selected from the group consisting of H, C1-C6 alkyl, and C2-C6 alkenyl; The alkyl and alkenyl are selected from -O-, -S-, -C(O)- and -NR''-. optionally interrupted by one or more heteroatoms or chemical groups selected from R5 is selected from the group consisting of H, C1-C6 alkyl, and C2-C6 alkenyl; The alkyl and alkenyl are selected from -O-, -S-, -C(O)- and -NR''-. The heteroatom may be optionally interrupted by one or more heteroatoms or chemical groups selected from the group consisting of aryl, ...

[0178] In a preferred embodiment of the linker of formula (IV), X1 and X2 are each independently one or more amino acids. , one or more N-substituted amino acids, optionally substituted polyethers, C1-C 12 Al alkylene, arylene having 6 to 10 ring atoms, C3 to C8 cycloalkylene, 5 to 1 Heterocycloalkylene having 0 ring atoms, heteroaryl having 5 to 10 ring atoms Len, C2~C 10 Independently from the group consisting of alkenylene, and any combination thereof Selected, The alkylene and alkenylene include -O-, -S-, -C(O)-, -NR''-, -C(O)NR''-, -NR''-C(O)-, -NR''-C(O)-NR'''- , —NR″—C(O)—O—, —OC(O)NR″—, and triazole. and optionally interrupted by one or more heteroatoms or chemical groups, The alkylene, arylene, cycloalkylene, heterocycloalkylene, heteroaromatic Arylene and alkenylene are substituted with halogen, oxo, -OH, -NO2, -CN, C1 C6 alkyl, C3-C6 cycloalkyl, heterocyclyl with 5-10 ring atoms aryl having 6 to 10 ring atoms; heteroaryl having 5 to 10 ring atoms; C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 haloalkoxy, -(CO )-R', -O-(CO)-R', -(CO)-O-R', -(CO)-NR''R'' one or more substituents selected from -NR''-(CO)-R', and -NR''R'''; optionally substituted with a substituent, R', R'' and R''' are independently selected from H and C1-C6 alkyl.

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

[0180] In another embodiment of the linker L of formula (IV) or (V), K is preferably of the following formula: V) polysarcosine, [ka] In the formula, k is an integer of 2 to 50, preferably 4 to 30. R6 corresponds to OH or NH2.

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

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

[0183] Preferably, the linker L is linked to one or more thiol residues of the antibody Ab, e.g., It is covalently bound to eight thiol residues in

[0184] payload In one embodiment, D of the present disclosure is a payload linked to X at the carbonyl functional group of X. In one embodiment, the link between X and D is the carbonyl functionality of X and the amino functionality of D. It occurs between the base.

[0185] The payload D is a key component of ADC design. The payload D is a therapeutic agent or drug. The term "drug" refers in particular to a substance capable of modulating a biological process and refers to an agent that has biological activity and / or

[0186] In certain embodiments, payload D is released from an internalized ADC within the cytoplasm of a tumor cell. It is a cytotoxic drug that is activated after administration. It is ideally (when linked to an antibody) It must be able to destroy tumor cells without affecting non-tumor cells. Erode should also ideally have high stability in the systemic circulation and lysosomes. It preferably has a sub-nanomolar IC5 0 value and have sufficient solubility in an aqueous environment.

[0187] In certain embodiments, the cytotoxic agent is an auristatin (monomethyl auristatin E (MMAE), monomethylauristatin F (MMAF), maytansinoids (e.g., maytansine), calicheamicin, duocarmycin, and anthracyclines Phosphorus (e.g., daunorubicin, doxorubicin, dihydroxyanthracindione) or topoisomerase I inhibitors such as camptothecin or their analogs. The compound is selected from the group consisting of:

[0188] In certain embodiments, D is selected from the group consisting of taxon, cytochalasin B, auristatin (monomethicone), auristatin E), gramicidin D, ethidium bromide, emetine, mitomycin cin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin Rubicin, Daunorubicin, Dihydroxyanthracindione, Mitoxantrone, Mito ramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, Procaine, tetracaine, lidocaine, propranolol, and puromycin, and analogs or homologs thereof.

[0189] In certain embodiments, D is an antimetabolite (e.g., methotrexate, 6-mercapto Purine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), ointments (e.g., mechlorethamine, thioepacloraxumbucil, Myfalan, Lumustine (BSNU) and lomustine (CCNU), cyclosulfonamide, busulfan , dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodi amineplatinum(II) (DDP) cisplatin), anthracyclines (e.g., Daunol bicin (formerly daunomycin and doxorubicin), antibiotics (e.g., dactino Mycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin Antimitotic agents (e.g., vincristine and vinblastine) and antimitotic agents (e.g., vincristine and vinblastine) is selected from the group consisting of:

[0190] Preferably, D is an inhibitor of topoisomerase I, such as a camptothecin analogue, Carbazole analogues, phenanthridine analogues, fluoroquinolone analogues, quinoxalines , evodiamine, acridine, naphthyridine, deoxynivomicin analogues, angusti quinazolinoquinoline alkaloids or quinazolinoquinoline alkaloids Ndenoisoquinoline analogues (for review, see Selas et al., Apate nt review of topoisomerase I inhibitors( 2016-present),2021,Expert Opinion on The (See, e.g., J. Applied Physics, 31(6), 473-508) The compounds include, but are not limited to, preferably selected from the group consisting of camptothecin and analogs thereof. Selected from irinotecan, topotecan, camptothecin, SN-38, exatecan, DXd, ciratecan, cositecan, lutotecan, gimatecan, belotecan, rubitecan ( For a review, see Sriram et al., Camptothecin and its s analogues:a review on their chemothera peutic potential,2005,Natural Product Re (see search,14(9),393-412) do not have.

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

[0192] [ka]

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

[0194] Preferably, such an ADC delivers an effective dose of a drug, e.g., For example, it can selectively deliver an inhibitor of topoisomerase I, preferably exatecan. do.

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

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

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

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

[0199] Thus, in a composition comprising multiple copies of an ADC of formula (I), "p" represents the number of copies of antibody A This refers to the average number of -LD moieties per b. For an average p number close to 8, the ADC is "DAR8 "It is thought that this is the case.

[0200] Methods for determining the drug-antibody ratio are disclosed in the Examples or can be found, for example, in Con ilh et al(Pharmaceuticals 2021,14(3),247 ) is described in

[0201] Drug loading onto an antibody via a linker L is limited by the number of attachment sites on the antibody moiety. In some embodiments, the linker moiety (L) of the ADC comprises one or more The linker is attached to the antibody moiety via a chemically active group on an amino acid residue of , free amino acids, imino, hydroxyl, thiol or carboxyl groups (e.g., N-terminus or C-terminus, the ε-amino group of one or more lysine residues, one or more glutamic acid residues or aspartic acid residues the free carboxyl group of a paragic acid residue or the sulfhydryls of one or more cysteine ​​residues The site at which the linker is attached can be the amino group of the antibody moiety. It may be a naturally occurring residue in the amino acid sequence or may be introduced, for example, by recombinant DNA techniques (e.g., cysteine or by introducing unnatural amino acid residues into the amino acid sequence) or protein biochemistry can be introduced into the antibody portion by (eg, by reduction, pH adjustment, or hydrolysis).

[0202] If the attachment site is an interchain cysteine ​​thiol group, the antibody will have a cysteine ​​residue to which a linker can be attached. In fact, most reactive cysteine ​​groups are The ol is generally present as an interchain disulfide bridge. The binding occurs by reducing cysteine ​​residues available to form interchain disulfide bridges. Therefore, the optimal drug-antibody ratio is determined by determining the ratio of the antibody moiety to the drug-antibody moiety. without increasing the size and reducing the pharmacokinetic properties (the number of bound drug moieties per antibody) This should increase the potency of the ADC (by increasing the number of

[0203] In certain embodiments, -LD is attached to an interchain reactive thiol residue of the antibody. A typical antibody with long heavy and light chains has eight available interchain reactive thiol residues. Thus, in certain embodiments, the ADC comprises eight -LD moieties on the eighth amino acid of the antibody Ab. and most preferably a DAR8 ADC in which the α- and β-actin groups are covalently linked to two interchain reactive thiol residues. -L- corresponds to formula (VI).

[0204] In certain embodiments, the ADCs of the disclosure correspond to formula (VII):

[0205] [ka] where Ab is anti-FRα as defined in the previous section, and p is 4 to 8, preferably It is 6 to 8.

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

[0207] [ka] where Ab is anti-FRα as defined in the previous section.

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

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

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

[0211] In a preferred embodiment, the ADC of the present disclosure comprises an Ab having a heavy chain of SEQ ID NO: 9 and a heavy chain of SEQ ID NO: 10. and a light chain.

[0212] In another specific embodiment, the ADC of the disclosure comprises an Ab having a heavy chain of SEQ ID NO: 11 and a heavy chain of SEQ ID NO: 1 and a light chain of formula (VII).

[0213] Pharmaceutical Compositions and Formulations In another aspect, the present disclosure provides a method for treating a rheumatoid arthritis, comprising administering to a subject therapies described herein, comprising administering to a subject therapies ... and The ADC shown (e.g., mAb1 linked to a linker L of formula (VI), which itself is an exogenous a composition, e.g., a pharmaceutical composition, containing one or a combination of to provide.

[0214] As used herein, a "pharmaceutically acceptable carrier" refers to a physiologically compatible Any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and The pharmaceutical formulations of the present disclosure may also contain stabilizers, surfactants, buffers, antibacterial agents, and the like. one or more pharmaceutically acceptable salts selected from preservatives, protectants, antioxidants, chelating agents, and bulking agents; The composition may further comprise an excipient.

[0215] As used herein, a "solvent" refers to any solvent for the preparation of a liquid formulation, such as an aqueous formulation. Pharmaceutically acceptable (i.e., safe and non-toxic for administration to humans or other mammals) Exemplary solvents include sterile water for injection (WFI) or bacteriostatic injection (BFI). Water for injection (BWFI), pH buffer solutions (e.g., phosphate-buffered saline), sterile saline These include saline, Ringer's solution, or dextrose solution, and combinations thereof. The solvent is sterile water for injection or bacteriostatic water for injection (BWFI).

[0216] As used herein, a stabilizer is a substance that specifically inhibits unfolding and aggregation. Preferably, the stabilizer is a compound that increases protein stability in a pharmaceutical formulation. It is recognized by authorities as an additive or excipient.

[0217] The stabilizer may be a sugar. As used herein, "sugar" refers to a monosaccharide, disaccharide, trisaccharide, or the like. , polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, etc. (CHO) n and Examples of sugars herein include glucose, sucrose, trehalose, and derivatives thereof. Lactose, fructose, maltose, dextran, glycerin, dextrose erythritol, glycerol, arabitol, sorbitol, mannitol Toll, melibiose, melezitose, raffinose, mannotriose, stachyose , maltose, lactulose, maltulose, glucitol, maltitol, lactitol These include maltol, isomaltulose, etc.

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

[0219] As used herein, "surfactant" refers to a surface-active agent. Generally, it reduces the exposure of hydrophobic regions and therefore also prevents competition for adsorption sites. Proteins are intercalated to reduce inhibited protein-protein interactions and interface-induced aggregation. It is added to protein preparations.

[0220] Examples of surfactants herein include polysorbates (e.g., polysorbate 20 and polysorbate 80); poloxamers (e.g., poloxamer 188); Trito Triton; sodium dodecyl sulfate (SDS); lauryl Sodium sulfate; Sodium octylglycerol; Lauryl, myristyl, linoleyl lauryl-, myristyl-, linoleyl- or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- or stearyl-sulfobetaine Tearyl sarcosine; Linoleyl, myristyl or cetyl betaine; Lauroamine Dopropyl, cocamidopropyl, linoleamidopropyl, myristamidopropyl -, palmidopropyl- or isostearamidopropyl-betaine (e.g., lauroamidopropyl myristamidopropyl-, palmidopropyl- or isostearamidopropyl Pyridyl-dimethylamine; sodium methyl cocoyl or disodium methyl oleyl urates; polyethyl glycols, polypropyl glycols, and ethylene and propylene glycols copolymers with polyethylene glycol (e.g., Pluronics, PF68, etc.) Other examples of pharmaceutically acceptable surfactants include polyoxyethylene-sorbitan fatty acids. Fatty acid ester (Tween), polyethylene-polypropylene glycol, polyoxyethylene ethylene stearate, polyoxyethylene alkyl ethers, e.g., polyoxyethylene Monolauryl ether, alkylphenyl polyoxyethylene ether (Triton -X), polyoxyethylene-polyoxypropylene copolymers (poloxamer, Pluroxamer, The most suitable polyoxyethylene glycol monoacrylates include PEG-100, ... Tween 20™ is a sorbitan-based fatty acid ester. (sold under the trademark Tween 80™) and Polysorbate 80 (sold under the trademark Tween 80™) (It is on sale).

[0221] The most suitable polyethylene-polypropylene copolymer is Pluronic® ) F68 or Poloxamer 188 (trademark). The most suitable polyoxyethylene alkyl ethers are those sold under the Brij™ trademark. The most suitable alkylphenol-polyoxyethylene ether is T It is sold under the trade name riton-X.

[0222] The concentration of the surfactant in the pharmaceutical formulation of the present disclosure is comprised between 0.01 and 0.1% (w / v). It is possible.

[0223] As used herein, the term "buffer" refers to a solution containing it that This refers to a drug that can withstand changes in pH due to the action of the conjugate component. Examples of buffers that control this range include acetate, succinate, gluconate, Examples include stigmine, citrate, glycylglycine and other organic acid buffers.

[0224] An "antiseptic" is a substance that is used to reduce contamination and / or the action of bacteria, fungi, or other infectious agents. Preservatives are compounds that can be added to the formulations herein to prevent the formation of plaque. May facilitate the production of multi-use (multi-dose) formulations. Examples of potential preservatives include octadecanoate, Decyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride ammonium chloride (alkylbenzyldimethyammonium chloride with long alkyl chains) Other types of preservatives include fluoride, benzophenone, benzotriazole ... Aromatic alcohols such as phenol, butyl and benzyl alcohol, methyl or propyl alcohol Alkylparabens such as lavendone, catechol, resorcinol, cyclohexanol, 3- pentanol, and w-cresol.

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

[0226] A protectant may be added to the pre-lyophilized formulation in a "cryoprotectant amount." This allows the freeze-drying of the protectant. After lyophilization of the protein in the presence of a lyophilizing agent, the protein is stabilized to its physical and chemical stability. This means essentially preserving the integrity and validity of the product.

[0227] As generally used herein, "antioxidants" are compounds that limit oxidation reactions and maintain protein stability. It is a commonly used pharmaceutically acceptable excipient to maintain efficacy and safety. Examples of antioxidants include ascorbic acid, sodium metabisulfite, histamine, methionine, Ascorbic acid, glutathione, vitamin E, and polyethyleneimine.

[0228] The concentration of antioxidants in the pharmaceutical formulations of the present disclosure may be comprised between 5 and 25 mM.

[0229] "Chelating agents" are pharmaceutical agents commonly used to maintain protein stability. Examples of chelating agents include edetate disodium, diethylenetriamine, Contains amine pentaacetic acid, citric acid, hexaphosphate, thioglycolic acid, and zinc.

[0230] As generally used herein, a "bulking agent" is an agent that adds mass to the lyophilization mixture and acts as a bulking agent during lyophilization. Contributes to the physical structure of the cake (e.g., an essentially uniform freeze-dried cake that maintains an open pore structure) It is a pharmaceutically acceptable excipient commonly used to facilitate the manufacture of pharmaceutical preparations. Exemplary bulking agents include mannitol, glycine, lactose, modified starch, polyethylene glycol, and the like. ethylene glycol), and sorbitol.

[0231] The form of the pharmaceutical composition, the route of administration, the dosage and the regimen will depend on the condition to be treated, the severity of the disease. , which naturally depends on the age, weight and sex of the patient.

[0232] The pharmaceutical compositions of the present disclosure may be administered topically, orally, parenterally, intraperitoneally, intranasally, intravenously, intramuscularly, subcutaneously, or intravenously. Alternatively, the compound may be formulated for intraocular administration, preferably intraperitoneally or intravenously.

[0233] Preferably, the pharmaceutical composition comprises a pharmaceutically acceptable vehicle for an injectable formulation. These include, in particular, isotonic sterile saline (monosodium phosphate or disodium phosphate) sodium chloride, potassium, calcium or magnesium, or the like, or A mixture of 100% ethanol and 100% saline, or sterile water or saline, may be added to form an injectable solution. The composition may be a dry composition, particularly a freeze-dried composition, which allows for the synthesis of the composition.

[0234] The dose used for administration will vary as a function of various parameters, in particular the mode of administration used, It can be adapted as a function of the pathology involved or the desired duration of treatment.

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

[0236] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; sesame oil, peanut oil or aqueous propane. Preparations containing propylene glycol; and sterile for the extemporaneous preparation of sterile injectable solutions or dispersions This includes powders or lyophilized forms. In all cases, the form must be sterile and the container It must be fluid to the extent that easy injectability exists. It must be stable under ambient conditions and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. It must be.

[0237] A solution of the active compound as a free base or a pharmacologically acceptable salt is prepared by adding hydroxypropyl The dispersion can be prepared in water suitably mixed with a surfactant such as cellulose. , glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. These preparations, under normal conditions of storage and use, do not inhibit the growth of microorganisms. Contains preservatives to prevent

[0238] The ADCs of the disclosure can be formulated into compositions in either a neutral or salt form. Acceptable salts include acid addition salts (formed with the free amino groups of the protein) and inorganic salts. Acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. Salts formed with the free carboxyl groups can be easily reacted with inorganic bases, such as sodium hydroxide. , potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, and Derived from organic bases such as isopropylamine, trimethylamine, histidine, and procaine It is also possible to do so.

[0239] Sterile injectable solutions may be prepared by combining the active compound in the required amount with various other ingredients as enumerated above, as required. Both are prepared by incorporating them in a suitable solvent followed by filter sterilization. The solutions may contain various sterilized active ingredients in a basic dispersion medium and the required amount from those enumerated above. The preparation of sterile injectable solutions is carried out by incorporating the compound into a sterile vehicle containing other ingredients. For sterile powders for the preparation of pharmaceuticals, the preferred method of preparation is to mix a powder of the active ingredient plus any additional desired ingredients. from its previously sterile filtered solution.

[0240] The preparation of more or more concentrated solutions for direct injection is also contemplated, and DMS as the solvent is also preferred. The use of O results in extremely rapid penetration, delivering high concentrations of active agent to small tumor areas. It is expected that this will happen.

[0241] Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The agent is easily administered in a variety of dosage forms, including the types of injectable solutions mentioned above, but also drug-release capsules and the like. It can be used.

[0242] For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary. The liquid diluent should first be made isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dose is dissolved in 1 ml of isotonic NaCl solution and then added to 1000 ml of skin It can be added to the infusion solution or injected at the proposed infusion site (e.g., "R emington's Pharmaceutical Sciences" 15th See Edition, pages 1035-1038 and 1570-1580 Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

[0243] The ADCs of the disclosure may be administered in an amount of about 0.0001 to 1.0 milligrams per dose, or about 0.001 up to 0.1 milligrams, or about 0.1 to 1.0 milligrams, or even 1.0 to about 10 milligrams The therapeutic mixture may be formulated to contain grams of the compound. Multiple doses may also be administered.

[0244] Pharmaceutical formulations comprising the ADCs of the disclosure may be "ready-to-use" injectable or lyophilized formulations. could be.

[0245] In certain embodiments, pharmaceutical formulations comprising the ADCs of the disclosure are supplied in pre-filled syringes. This may be done.

[0246] Uses and Methods of the ADCs of the Disclosure The ADCs of the present disclosure have therapeutic utility. For example, these molecules can be used to treat a variety of disorders. The compounds can be administered to a subject, for example in vivo, for treatment or prevention.

[0247] The ADCs of the present disclosure can be used as pharmaceuticals, particularly for the treatment of cancers, particularly those expressing FRα, in subjects in need thereof. Cancers having tumor cells that are tumour-forming, more particularly cancers having solid tumors, more particularly ovarian cancer, breast cancer It is used as a pharmaceutical for the treatment of cancer selected from the group consisting of cancer, lung cancer, and mesothelioma. Use thereof is contemplated herein.

[0248] The term "cancer" refers to a cancer that occurs in mammals and is typically characterized by unregulated cell growth. Cancer refers to or describes a physiological condition in which the body is in a state of stasis. Examples of cancer include carcinoma, lymphoma, blastoma (myeloma), and blastoma and retinoblastoma), sarcomas (including liposarcoma and synovial cell sarcoma), neuroendocrine Tumors (including carcinoid tumors, gastrinomas, and islet cell carcinomas), mesothelioma, Schwann cell carcinoma Cysts (including acoustic neuromas), meningiomas, adenocarcinomas, melanomas, and leukemia or lymphoid malignancies More specific examples of such cancers include, but are not limited to, squamous cell carcinoma. Cancer (e.g., epithelial squamous cell carcinoma), small cell lung cancer, non-small cell lung cancer, lung cancer, and squamous cell carcinoma of the lung Lung cancer, including cancer of the peritoneum, hepatocellular carcinoma, gastric cancer, including gastrointestinal cancer (stomach cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, Bladder cancer, liver cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, Kidney cancer or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, testicular cancer, esophageal cancer , tumors of the biliary tract, and head and neck cancer.

[0249] The ADCs of the present disclosure are directed to the treatment of cancers from the group consisting of ovarian cancer, triple-negative breast cancer, and non-small cell lung cancer. The present invention is particularly useful in the treatment of cancers selected from the following:

[0250] Thus, the present disclosure provides a method for treating cancer, particularly one of the cancers listed above, more preferably ovarian cancer, avian ... and non-small cell lung cancer. administering a therapeutically effective amount of an ADC of Formula (I) disclosed herein. Regarding.

[0251] An ADC for use as disclosed above may contain, as the only active ingredient, a compound such as those described above. Other drugs for the treatment or prevention of disease, such as antivirals, anti-inflammatory drugs or cytotoxic drugs in association with, for example, an anti-inflammatory, anti-proliferative, chemotherapeutic or anti-tumor agent, or may be administered in combination.

[0252] For example, ADCs for use as disclosed above may include AZT, IFN-α, anti-CD40 20 mAb, anti-CD25 mAb, anti-PD1 mAb, anti-PDL-1 mAb, anti-CT LA4 mAb may be used in combination with a chemotherapeutic agent.

[0253] Examples of such anti-PD1 or anti-PDL1 antibodies include nivolumab, pembrolizumab, and avastin. These include velumab, durvalumab, cemiplimab, or atezolizumab. Not limited.

[0254] Suitable anti-tumor agents include, but are not limited to, alkylating agents (e.g., cyclophosphamide, mechlorethamine, chlorambucil, melphalan, nitrosoureas, temozolomide), Anthracyclines (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin) bicin, mitoxantrone, valrubicin), taxanes (paclitaxel, docetaxel epothilones, topoisomerase I inhibitors (irinotecan or topotecan, etc.), Inhibitors of ribosomal enzyme II (etoposide, teniposide, or tafluposide, etc.), nucleoside tide analogs and precursor analogs (azacytidine, azathioprine, capecitabine, cytarabine) flurouracil, gemcitabine, hydroxyurea, mercaptopurine, methotrexate peptide antibiotics (bleomycin, colistin, gramicin, etc.), platinum-based antineoplastic agents (carboplatin, cisplatin, and oxaliplatin); retinoids (tretinoin, alitretinoin, bexarotene, etc.), vinca albicans kaloids and derivatives (vinblastine, vincristine, vindesine, vinorelbine), Anti-VEGF agents (bevacizumab, ranibizumab, sunitinib, sorafenib, pazopanib, Targeted therapy, such as kinase inhibitors (e.g., ibrutinib, idelalisib, erlotinib) , gefitinib, imatinib, vemurafenib, vismodegib), proteasome inhibitors (e.g., bortezomib, carfilzomib), histone deacetylase inhibitors (borino stat or romidepsin, etc.

[0255] As noted above, the present disclosure provides, in still further aspects, a therapeutically effective amount of an ADC of the present disclosure; and The method includes co-administration, e.g., simultaneous or sequential administration, with at least one second drug substance. The second drug substance is an antiviral agent, an anti-inflammatory or cytotoxic agent, an antiproliferative agent, a chemotherapeutic agent, or a combination thereof. or other anti-tumor agents, such as those listed above.

[0256] Kits comprising compositions disclosed herein (e.g., comprising ADCs) and instructions for use. Also within the scope of this disclosure are kits containing at least one additional reagent, or one or more additional a second antibody or protein (e.g., a second antibody or protein that binds to a different epitope on the target antigen than the first antibody) The kit may further comprise an antibody having a complementary activity to the antibody. The term label includes any label on or in a kit that indicates the intended use of the contents of the kit. This includes any written or recorded material supplied with or accompanying the kit. The patient is a patient who responds to ADC treatment, particularly with FRα-expressing tumors, as defined above. The method may further include a tool for diagnosing whether a patient belongs to a group that would be susceptible to the disease.

[0257] Processes for Making the ADCs of the Disclosure The antibodies of the present disclosure may be linked by at least one linker, L, by any technique known in the art. Such techniques can be used, for example, to conjugate Gr eg T.Hermanson,Bioconjugate Techniques,3 rd Edition,2013,Academic Press(eBook ISB Therapeutic agents conjugated to antibodies are described in (N:9780123822406). For more information on methods for achieving this, see Lyon et al., "Chapt er six-Conjugation of Anticancer Drugs T through Endogenous Monoclonal Antibody Cy steine ​​residues”,2012,Methods in Enzymol ogy,502,123-138(doi:10.1016 / B978-0-12-41 6039-2.00006-9);Chapter 2-7 of “Antibody- Drug Conjugates:Fundamentals,Drug Develo pment,and Clinical Outcomes to Target Ca ncer”,3 November 2016,eBook ISBN:9781119 060727,doi:10.1002 / 9781119060727 and Pan See owksi S et al.2014 Jan 1;6(1):34-45 sea ​​bream.

[0258] In one embodiment, the process for obtaining an ADC of formula (I) comprises the steps of: - a host under conditions suitable for expression of a nucleic acid encoding an antibody (Ab) as defined in the previous section Culturing the cells; - isolating the antibody, - synthesis of exatecan linked to a linker L of formula (VIII),

[0259] [ka] - conjugating said antibody to a compound of formula (VIII), thereby Obtaining an ADC.

[0260] The antibody can be obtained as described above. It contains four accessible interchain disulfide bonds that can be used as linkage sites. The four interchain disulfide bonds are formed by, for example, tris(2-carboxyethyl)phosphine ( It can be reduced by TCEP or dithiothreitol (DTT), resulting in This results in eight thiol groups available for conjugation.

[0261] The moiety -LD of formula (VIII) can be prepared by procedures known in the art of organic synthesis (chemical reactions, extraction, evaporation, etc.). precipitation, chromatography, filtration, trituration, crystallization, etc.) and analytical chemistry. These reactions and techniques can be prepared according to known analytical procedures. hard Larock,Comprehensive Organic Transf ormations,A Guide to Functional Group Pr eparations, 2nd Ed(2010), and the multi-volume me series edited (Michael B. Smith et al.) Comp endium of Organic Synthetic Methods(1974 The starting materials and reagents can be found in many publications, including: They may be obtained from commercial sources or prepared using literature methods.

[0262] The exatecan compound can be synthesized according to known procedures (U.S. Pat. No. 5,833,383). 4476; WO 2019044946 or Sugimori et al., 1998, J.Med.Chem.,41(13),2308-2318 doi:10 Exatecan is available from reputable sources (e.g., see .1021 / jm970765q). , MedChemExpress Catalog Number HY-13631A or Carbosyn It can also be purchased from the National Instruments catalog number FE72401.

[0263] The final ADC of formula (I) can be prepared by converting the natural or engineered cysteine ​​residues of the antibody according to known procedures. It is obtained after conjugation of the -LD moiety to a thiol residue. et al., “Chapter six-Conjugation of Antic ancer Drugs Through Endogenous Monoclonal l Antibody Cysteine ​​Residues”,2012,Metho ds in Enzymology,502,123-138(doi:10.1016 / B978-0-12-416039-2.00006-9) or SJ Walsh e t al.,Site-selective modification strategy gies in antibody-drug conjugates,Chem.So c.Rev.,2021,50,1305-1353,doi:10.1039 / D0C See S00310G. Typically, the antibody component is tris(2-carboxyethyl) It is reduced with a reducing agent such as trichloroethylene phosphine (TCEP) or dithiothreitol (DTT), The -LD component is added and reacts covalently with the cysteine ​​thiol residues of the antibody to give the final A The DC compound is purified and buffer exchanged.

[0264] The invention having been fully described will be further illustrated by the following embodiments and examples. These are examples only and are not intended to be further limiting.

[0265] Specific Embodiments 1. An antibody-drug conjugate of formula (I): Ab-[LD]p (I), During the ceremony, -Ab is an anti-folate receptor alpha (FRα) antibody that specifically binds to SEQ ID NO: 12 can be, -L is a cleavable linker moiety attached to the antibody, preferably via a thiol residue minutes, -D is a cytotoxic drug moiety attached to L; -p is 1 to 8, preferably 6 to 8, more preferably p is 8; An antibody-drug conjugate of formula (I): 2. D is an inhibitor of topoisomerase I, preferably consisting of a camptothecin analogue. More preferably, D is the drug moiety of exatecan of formula (II): 2. The antibody-drug conjugate of embodiment 1,

[0266] [ka] 3. L is a cleavable linker moiety of formula -AW-, where A is linked to Ab. and W is a cleavable moiety linked to D. 1. The antibody-drug conjugate according to any one of claims 1 to 2. 4. L is a lysosomal protease-sensitive linker and W is, for example, valine-citrol. Phosphorus (Val-Cit), Alanine-Alanine-Asparagine (Ala-Ala-Asn ), valine-alanine (Val-Ala) and phenylalanine-lysine (Phe-Lys) s) wherein the antibody of embodiment 3 comprises a cleavable peptide moiety selected from the group consisting of: -Drug conjugates. 5. L is a protease-sensitive cleavable linker and W is preferably β-glucuronide 4. The method of claim 3, wherein the sugar cleavable unit is selected from a β-galactoside or β-galactoside moiety. Antibody-drug conjugates. 6. The embodiment wherein L is a glutathione-sensitive linker and W comprises a disulfide moiety 3. The antibody-drug conjugate according to claim 3. 7. W is of formula (III):

[0267] [ka] During the ceremony, each R2 is independently selected from the group consisting of an electron-withdrawing group and a C1-C4 alkyl; n is 0, 1 or 2; T is a sugar cleavable unit or a polypeptide cleavable unit; When T is a sugar cleavable unit, Y is O, or when T is a polypeptide cleavable unit, In some cases, Y is NR3; R3 is H, C1~C 24 Alkyl, C2-C6 alkenyl; optionally substituted poly aryls with 6-10 ring atoms, C3-C8 cycloalkyls, 3-1 Heterocycloalkyl having 0 ring atoms, heteroaryl having 5 to 10 ring atoms and any combination thereof; The alkyl and alkenyl include -O-, -S-, -C(O)-, -NR''-, -C (O)NR''-, -NR''-C(O)-, -NR''-C(O)-NR'''-, - selected from NR″—C(O)—O—, —OC(O)NR″— and triazole optionally interrupted by one or more heteroatoms or chemical groups; R" and R"' are independently selected from H and C1-C6 alkyl, I) and pharmaceutically acceptable salts thereof. to. 8. L corresponds to the linker -AW- of formula (IV),

[0268] [ka] During the ceremony, X1 is the connector unit, Z is an optional spacer, X2 is a connector unit, K is preferably selected from polysarcosine and polyethylene glycol; a hydrophobic masking entity of choice, R1 is H, C1~C 24 Alkyl, C2-C6 alkenyl; optionally substituted poly aryls with 6-10 ring atoms, C3-C8 cycloalkyls, 3-1 Heterocycloalkyl having 0 ring atoms, heteroaryl having 5 to 10 ring atoms and any combination thereof; The alkyl and alkenyl include -O-, -S-, -C(O)-, -NR''-, -C (O)NR''-, -NR''-C(O)-, -NR''-C(O)-NR'''-, - selected from NR″—C(O)—O—, —OC(O)NR″— and triazole optionally interrupted by one or more heteroatoms or chemical groups; R4 is selected from the group consisting of H, C1-C6 alkyl, and C2-C6 alkenyl; The alkyl and alkenyl are selected from -O-, -S-, -C(O)- and -NR''-. optionally interrupted by one or more heteroatoms or chemical groups selected from R5 is selected from the group consisting of H, C1-C6 alkyl, and C2-C6 alkenyl; The alkyl and alkenyl are selected from -O-, -S-, -C(O)- and -NR''-. optionally interrupted by one or more heteroatoms or chemical groups selected from 8. The antibody-drug conjugate of embodiment 7. 9. X1 and X2 are one or more amino acids, one or more N-substituted amino acids, optionally Substituted polyethers, C1-C 12 Alkylene, aryl with 6 to 10 ring atoms C3-C8 cycloalkylene, heterocycloalkylene having 5-10 ring atoms heteroarylenes having 5 to 10 ring atoms, C2 to C 10 Alkenylene and its Independently selected from the group consisting of any combination of these: The alkylene and alkenylene include -O-, -S-, -C(O)-, -NR''-, -C(O)NR''-, -NR''-C(O)-, -NR''-C(O)-NR'''- , —NR″—C(O)—O—, —OC(O)NR″—, and triazole. and optionally interrupted by one or more heteroatoms or chemical groups, The alkylene, arylene, cycloalkylene, heterocycloalkylene, heteroaromatic Arylene and alkenylene are substituted with halogen, oxo, -OH, -NO2, -CN, C1 C6 alkyl, C3-C6 cycloalkyl, heterocyclyl with 5-10 ring atoms aryl having 6 to 10 ring atoms; heteroaryl having 5 to 10 ring atoms; C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 haloalkoxy, -(CO )-R', -O-(CO)-R', -(CO)-O-R', -(CO)-NR''R'' one or more substituents selected from -NR''-(CO)-R', and -NR''R'''; optionally substituted with a substituent, R', R'' and R''' are independently selected from H and C1-C6 alkyl; 9. The antibody-drug conjugate of embodiment 8. 10. Z is one or more amino acids, one or more N-substituted amino acids, optionally substituted polyethers, C1-C12 alkylenes, arylenes with 6-10 ring atoms, C 3-C8 cycloalkylene, heterocycloalkylene having 5-10 ring atoms, 5- Heteroarylene with 10 ring atoms, C2-C 10 Alkenylene and any of the above are independently selected from the group consisting of any combination of The alkylene and alkenylene include -O-, -S-, -C(O)-, -NR''-, -C(O)NR''-, -NR''-C(O)-, -NR''-C(O)-NR'''- , —NR″—C(O)—O—, —OC(O)NR″—, and triazole. and optionally interrupted by one or more heteroatoms or chemical groups, The alkylene, arylene, cycloalkylene, heterocycloalkylene, heteroaromatic Arylene and alkenylene are substituted with halogen, oxo, -OH, -NO2, -CN, C1 C6 alkyl, C3-C6 cycloalkyl, heterocyclyl with 5-10 ring atoms aryl having 6 to 10 ring atoms; heteroaryl having 5 to 10 ring atoms; C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 haloalkoxy, -(CO )-R', -O-(CO)-R', -(CO)-O-R', -(CO)-NR''R'' one or more substituents selected from -NR''-(CO)-R', and -NR''R'''; optionally substituted with a substituent, R', R'' and R''' are independently selected from H and C1-C6 alkyl; An antibody-drug conjugate according to any one of embodiments 8 to 9. 11. K is preferably polysarcosine of formula (V) below,

[0269] [ka] In the formula, k is an integer of 2 to 50, preferably 4 to 30. R6 corresponds to OH or NH2; An antibody-drug conjugate according to any one of embodiments 8 to 10. 12. Embodiments 7 to 12, wherein T is a sugar cleavable unit that is a glucuronide or a galactoside. 12. The antibody-drug conjugate according to any one of 11. 13. T is preferably selected from Val-Cit, Val-Ala and Phe-Lys 12. The antibody-drug conjugate of any one of embodiments 7 to 11, wherein the dipeptide is to. 14. L is covalently attached to one or more thiol residues of the antibody; Preferably, L corresponds to a linker of formula (VI): An antibody-drug conjugate according to any one of embodiments 1 to 3.

[0270] [ka] 15. Embodiment 1, wherein the antibody comprises a full-length antibody or an antibody fragment containing the antigen-binding portion. 15. The antibody-drug conjugate according to any one of claims 1 to 14. 16. Corresponding to formula (VII) below:

[0271] [ka] where the Ab typically comprises leucine 234 and leucine 23 of the IgG1 Fc constant region. Anti-FRα antibodies, such as farletuzumab, or derivatives thereof, comprising an alanine substitution at position 5. an isoform of an IgG1 variant, and p is 4 to 8, preferably 8; 16. The antibody-drug conjugate according to any one of embodiments 1 to 15. 17. -Ab is a human IgG1 isotype constant region, or a mutant or chemically modified The antibody comprises a mutant or chemically modified constant region, The antibody when compared to a corresponding antibody having a native human IgG1 isotype constant region conferring no or reduced ADCC activity to -Ab is a human IgG4 isotype constant region, or a mutant or chemically modified The antibody comprises a mutant or chemically modified constant region, The antibody has A when compared to a corresponding antibody with a native IgG4 isotype constant region. conferring no or reduced ADCC activity; 17. The antibody-drug conjugate according to any one of embodiments 1 to 16. 18.Ab is (i) HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, HCDR3 of SEQ ID NO: 3 and a variable heavy chain polypeptide comprising an LCDR1 of SEQ ID NO: 4, an LCDR2 of SEQ ID NO: 5, and a variable light chain polypeptide comprising an LCDR3 of SEQ ID NO: 6; or (ii) a variable heavy chain polypeptide comprising a VH of SEQ ID NO: 7 and a VL of SEQ ID NO: 8 Variable Light Chain Polypeptide 18. The antibody according to any one of embodiments 1 to 17, which is an anti-FRα antibody comprising any one of -Drug conjugates. 19.Ab is (i) a heavy chain of SEQ ID NO: 9 and a light chain of SEQ ID NO: 10; or (ii) a heavy chain of SEQ ID NO: 11 and a light chain of SEQ ID NO: 10 Any one of embodiments 1 to 18, comprising or consisting essentially of any of 1. The antibody-drug conjugate described herein. 20. (i) Ab is HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, HCDR3 of SEQ ID NO: 3 and a variable heavy chain polypeptide comprising LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5 and LCDR3 of SEQ ID NO: 6 a variable light chain polypeptide comprising: an anti-folate receptor alpha antibody or antigen-binding fragment thereof, comprising: (ii) L is a cleavable linker of formula -AW-, where A is linked to Ab and W is a cleavable moiety linked to D; (iii) D is an inhibitor of topoisomerase I, e.g., exatecan, and (iv) p is 1 to 8, preferably 8; An antibody-drug conjugate according to any one of embodiments 1 to 19. 21.Ab is below (i) an antibody or antigen-binding fragment comprising a heavy chain of SEQ ID NO: 9 and a light chain of SEQ ID NO: 10; or (ii) the antibody or antigen-binding fragment comprises a heavy chain of SEQ ID NO: 11 and a light chain of SEQ ID NO: 10; nothing, 21. The antibody-drug conjugate of embodiment 20, wherein 22. For use as a medicament, preferably in a subject in need of treatment of a tumor tumors, for example solid tumors, more particularly selected from the group consisting of ovarian cancer, breast cancer, lung cancer or mesothelioma The antibody according to any one of embodiments 1 to 21 for use in the treatment of selected tumors. Drug conjugates. 23. Selected from the group consisting of ovarian cancer, triple-negative breast cancer, and non-small cell lung cancer any of embodiments 1 to 21 for use in treating cancer in a subject in need thereof. The antibody-drug conjugate according to any one of the above. 24. The antibody-drug conjugate according to any one of embodiments 1 to 21, In combination with one or more pharmaceutically acceptable excipients, diluents or carriers, including other active ingredients. together with a pharmaceutical composition. 25. A method for obtaining the antibody-drug conjugate according to any one of claims 1 to 23. A process comprising: - under conditions suitable for the production of an anti-FRα antibody as defined in any one of embodiments 1 to 21. Culturing host cells in isolating the anti-FRα antibody; and - the synthesis of exatecan linked to a linker L of formula (VIII),

[0272] [ka] conjugating the anti-FRα antibody to a compound of formula (VIII); thereby obtaining an ADC as defined in any one of embodiments 1 to 21; To obtain the antibody-drug conjugate according to any one of claims 1 to 23, process. [Example]

[0273] Functional assays Human FRα binding affinity by ELISA Sandwich ELISA assays were performed in 96-well high-binding ELISA plates (Corn ing Inc., New York, NY, USA, Catalog No. 3590) 100 μL / well of recombinant human FR-1 in PBS (pH 7.4) at 2 μg / mL a protein (Sino Biological, Cat. No. 11241-H08H) Coat the plate with PBS-T (PBS-T) and incubate overnight at 4°C. After washing the plate twice with PBS (S+0.05% Tween-20), 200 μL / well of The cells were then blocked with incubation buffer (PBS-T + 0.1% BSA) for 1 hour at room temperature. Wash the plate four times with PBS-T and add 100 μL of 3-fold serially diluted test compounds. The antibody or antibody-drug conjugate is then added to the plate. The plate is then left in the dark to warm to room temperature. After washing five times with PBS-T, the plate was washed with 100 μL / well goat anti-human IgG (H+L) HRP-conjugated antibody (Jackson Immunoresearch, Catalog No. 109-035-088) at room temperature for 1 hour. Incubate and pre-dilute 1:250,000 in incubation buffer. After washing five times with ST, TMB substrate solution (Thermo-Fisher, catalog no. N301) was added. Peroxidase activity was stopped with 0.18M H2SO4, and Th ermo Scientific MultiSkan EX microplate reader Read absorbance at 450 nm (reference wavelength 650 nm) using GraphPad P Sigmoid fixation was performed using rism 9 software.

[0274] SPR Biacore affinity Surface plasmon resonance (SPR) experiments are performed on a Biacore T200 instrument at 25°C. The antibody or ADC to be tested is subjected to Human Antibody Capture Ki. CM5 series S pre-functionalized with t (Cytiva, Cat. No. BR100839) Captured at low density (300-500RU) on a sensor chip. The functionalized surface / flow cell without the band is used as a reference. Kinetics and affinity To measure the FRα, recombinant human FRα (Sino Biological, Cat. No. 11241-H08H) Analyte samples were run using a single cycle kinetic strategy. A constant 70°C of buffer solution (HBS-EP+, Cytiva, Cat. No. BR100669) At a flow rate of 1 μL / mL, five concentrations (0.5, 1, 2, 4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 The dissociation phase was performed by injecting running buffer for 900 seconds. Between duplicates, the surface / flow cell was regenerated with 3M MgCl2 to separate the analyte and the Both ligands are removed and a fresh ligand capture is performed using identical conditions. , after subtraction of the reference surface and zero concentration signal of the analyte, Biacore Evaluation The data is processed and fitted to a 1:1 binding model to determine the binding rate constant k a (on speed) and k d (off rate), and KD (also called "affinity") Determine the equilibrium dissociation constant (EQ) and report the mean and standard deviation of replicate experiments.

[0275] Cell-binding affinity by flow cytometry Antibody or ADC binding to extracellular human FRα expressed on cancer cell lines was analyzed by flow cytometry. The antibody or ADC to be tested is evaluated using LYNX Rapid APC. Use the Antibody Conjugation Kit according to the manufacturer's protocol. and conjugated to the APC fluorophore (Bio-Rad, catalog number LNK 032APC). 500,000 cells (in a flow cytometry plastic tube) 10 μL of the APC-labeled antibody or ADC to be tested (suspended in 100 μL of PBS) Add 5 μL of 100 μg / mL solution. Incubate the cells in the dark for 20 minutes and then centrifuge. Wash three times with PBS and resuspend in 200 μL of PBS for analysis. Measurements were performed using BD FACSDiva software (BD Biosciences). The data was collected using a BD Fortessa flow cytometer controlled by a Analyze using FlowJo software (BD Bioscience).

[0276] Stability assay in human plasma ADC samples (>6 mg / mL solution in PBS) were purified by centrifugation in screw-cap centrifuge tubes. Dilute with sterile human plasma (GeneTex, Cat. No. GTX73265) at 200 Obtain a final ADC concentration of 37 μg / mL (less than 5% v / v residual PBS volume). Incubate at °C and aliquot at 5 min, 6 hr, 1 day, 2 days, 3 days, and 7 days. (Aliquots were kept frozen at -80°C until analysis). ADC was Human folate receptor alpha recombinant protein (Sino Biologicals, Catalog Dynabeads™ M pre-coated with ELISA (Product ID: 11241-H08H) -280 Streptavidin (Thermo Scientific) magnetic beads were used The beads are isolated from plasma by immunocapture. Briefly, 600 μL of a commercially available bead solution is added to the HO tube. Wash twice with BS-EP buffer (Cytiva, Cat. No. BR100188) and 1. Resuspend in 2 mL of HBS-EP buffer. Add 65 μL of biotinylated recombinant FRα solution (tan). The beads were then added to HBS-E1000 (48 μg of protein), and the solution was stirred at room temperature for 2 hours. Wash three times with P buffer and resuspend in 1.2 mL of HBS-EP buffer. For the preparation, add 100 μL of the previous bead solution to 100 μL of HBS-EP in a microcentrifuge tube. Add 10 μL of the ADC solution in plasma (theoretical ADC amount 2 μg) and add the solution to the chamber. After incubation, the bead-ADC complex was diluted with HBS-EP buffer. Wash the cells twice with PBS, resuspend them in 200 μL of HBS-EP buffer, and mix them for 3 minutes with gentle agitation. Incubate at 7°C overnight with 2 μL / 1000 U of PNGase F (New England Biol abs, Cat. No. P0705L) is added to deglycosylate the The beads were washed twice with HBS-EP buffer, twice with distilled water, and once with 10% acetonitrile in water ( Wash the beads once with 50 μL of 300 mL of 0.1% (v / v) formic acid. Ink with 0% aqueous acetonitrile (v / v) at room temperature for 30 min under gentle stirring. The eluted sample containing the deglycosylated ADC is then purified using a Bruker I Thermo UltiMat with mpact II™ Q-ToF mass spectrometer e Denaturing reversed-phase chromatography-mass spectrometry using a 3000 UHPLC system Mobile phase A is water + 0.1% formic acid, and mobile phase B is acetonitrile + 0.1 % formic acid. The column was Agilent PEEK PLRP-S 1000Å 2.1 × 100 mm, 5 μm (80°C). The linear gradient was 20% B to 50% B in 25 minutes. The flow rate is 0.4 mL / min. UV detection is monitored at 280 nm. Q-ToF mass spectrometer The analyzer was designed for the m / z range of 500–5000 (ESI + ) used in Bruker Co The MaxEnt algorithm included in the mpass® software is used to For stability data analysis, the selected light chain (LC) Deconvolution of the raw spectra within the heavy chain (HC) elution time window is performed. Loss or modification of the product-linker is identified according to the corresponding mass shift from the starting ADC material. The relative ratio of ADCs with different DARs can be used to assess the strength of a particular ADC subspecies relative to the total ADC species. The final DAR value is calculated by dividing the intensity from Xu et al.,An Calculated as described in al. Biochem., 2011, 412(1), 56-66 will be done.

[0277] In vitro efficacy in FRa-negative cell lines (BT-474 cell lines) To assess the nonspecific (off-target) cytotoxicity of ADCs, in vitro cell injury The toxicity assay is carried out in the BT-474 (FRa negative) cancer cell line. Culture at an appropriate density (1,000–10,000 cells / well in 100 µL of appropriate culture medium) depending on the ) into a 96-well plate and incubate at 37°C for 24 hours. Serial dilutions (50 μL) of the dissolved test compound were added and the incubation was continued at 37°C. MTT (5 mg / mL, 20 μL, Sigma-Aldrich) was added for 144 hours. The wells were then incubated at 37°C for 1-2 hours. The well contents are carefully removed and dissolved homogeneously with acidified isopropanol. MultiskanTM Sky microplate reader (Ther Measured at a wavelength of 570 nm (reference wavelength 690 nm) using a Fluorescence Intensity Analyzer (Fluorescence Intensity Analyzer, ... IC50 concentration values ​​compared to untreated control cells were calculated using inhibitory dose-response curve fitting ( This was determined using GraphPad Prism 9).

[0278] In vivo efficacy in cancer xenograft models Obtain 4-5 week-old female CB-17 severe combined immunodeficient (SCID) mice and sterilize them for 7 days before the start of the study. PBS (OV-90, SW-620, KB, BT-474 cell lines) or P 50% BD Matrigel (Corning®) in BS (PA-1, Cells (mouse) resuspended in IGROV-1, OVCAR-3, and NCI-H2110 cell lines The mice were subcutaneously inoculated with 5-10 x 106 cells per tumor. Once they reached 150 mm3, mice were randomized (typically 7 mice per group) Treated with a single (unless otherwise stated) intravenous injection of PBS (negative control), or Antibody-drug conjugates were examined. Tumor volume was measured using a caliper device (length x width). Measurements were taken every 3 to 5 days using the following formula: V = 4 / 3 × π × R3 (where R represents the radius). If the tumor volume exceeds 1500 mm3 or if the tumor becomes ulcerated, , and sacrifice the mice.

[0279] Drug-to-antibody ratio (DAR) by reversed-phase liquid chromatography-mass spectrometry (RPLC-MS) )evaluation: Denaturing RPLC-QToF analysis was used to determine the drug-to-antibody ratio (DAR) of the conjugates. Briefly, the ADC was dissolved in water / acetonitrile + 0.1% formic acid (0.4 mL / min) A mobile phase gradient of 1000Å was used on an Agilent PLRP-S column (1000Å x 2.1 × 150 mm column). Elution was performed at 8 μm (80 °C) and the m / z range was 500–3500 (ESI + ) scanning Br Detection is performed using a Berker Impact II™ Q-ToF mass spectrometer. The MaxEnt algorithm included in the UKer Compass® software The data is deconvolved using a

[0280] Example 1: Synthesis of Exatecan-Based Chemical Drug-Linker Materials and general organic synthesis methods All solvents and reagents were obtained from reliable commercial sources (Sigma-Aldrich) unless otherwise stated. Aldrich, Fluorochem, TCI Chemicals, Acros O rganics, Alfa Aesar, Enamine, Thermo Fisher , Carbosynth, WuXi AppTec, Iris Biotech) Anhydrous solvents were purchased from Sigma-Aldrich. Fmoc-amino acids, 2-chlorotrityl, Wang and Rink Amidopoly Styrene 1% DVB 100-200 mesh resin (first Fmoc-sarcosine amino Pre-filled with acid) is manufactured by Christof Senn Laboratories and Sigma-Aldrich. Exatecan mesylate was purchased from MedChem Purchased from Express.

[0281] On-resin synthesis was performed using a 20 μm polyethylene frit (Sigma-Aldrich). The Titramax 101 platform was used in an empty SPE plastic tube containing A foam shaker (Heidolph) was used for agitation. The chemical reaction was carried out at room temperature under an inert argon atmosphere.

[0282] Liquid-crystal nuclear magnetic resonance spectra were obtained from Bruker using residual solvent peaks for calibration. Recorded on a Fourier 300HD or Bruker AVANCE III HD400 spectrometer Mass spectrometry was performed at the University Claude Bernard Lyon 1 UMR5246 CNRS Institute Centre Commun d e Spectrometrie de Masse (CCSM).

[0283] Macherey-Nagel Chromabond® Flash Cart Ridges (40–63 μm) were used to measure the size of the Teledyne Isco CombiFla Normal phase flash chromatography was performed on a sh® Rf200 device. B on ledyne Isco Combiflash® Rf200 device iotage® Sfar C18 Duo 100Å 30μm cartridge or Interchim PuriFlash RP-AQ (30 μm) cartridges were used. or reversed-phase chromatography using an Agilent 1100 preparative binary HPLC system. I did the math.

[0284] Pre-coated 40-63 microplates for monitoring chemical reactions and compound characterization, respectively μm silica gel (Macherey-Nagel), HPLC-UV (Agilent 1100 system) or UHPLC-UV / MS (Bruker Impact II ( Thermo UltiMate 3000 UH with a Q-ToF mass spectrometer Agi equipped with a PLC system or a Bruker MicroToF-QII mass spectrometer Analyzed by thin layer chromatography using a Lent 1260 HPLC system did.

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

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

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

[0288] HPLC Method 4: Thermo UltiMate 3000 UHPLC System+ Bruker Impact II™ Q-ToF Mass Spectrometer. Mobile phase A is water +0. The mobile phase A was acetonitrile + 0.1% formic acid, and the mobile phase B was acetonitrile + 0.1% formic acid. The column was Agil The PLRP-S was 1000Å, 2.1×150mm, and 8μm (80℃). The linear gradient was from 10% B to 50% B in 25 min. The flow rate was 0.4 mL / min. V detection was monitored at 280 nm. The Q-ToF mass spectrometer was operated in the m / z range of 500–35 00(ESI + ) used with Bruker Compass® software Data were deconvoluted using the MaxEnt algorithm included in the .

[0289] HPLC Method 5 (Preparative Method): Teledyne Isco Co with DAD detection mbiFlash® Rf200 binary MPLC system. Mobile phase A was water + 0 The mobile phase B was acetonitrile. , Biotage® Sfar C18 Duo 100Å 30μm (30g) The linear gradient was from 10% B to 50% B in 35 min, followed by 50% B for 5 min. The flow rate was 25 mL / min.

[0290] HPLC Method 6 (Preparative Method): Dual Loop Autoinjector, DAD Detection and Fractionation Agilent 1100 preparative binary HPLC system equipped with a cyclohexane collector. Mobile phase A The mobile phase A was water + 0.1% TFA, and the mobile phase B was acetonitrile. rs SunFire C18 OBD Prep Column, 100Å, 5μm, The dimensions were 19 mm x 250 mm (room temperature). The linear gradient was from 10% B to 60% B in 40 minutes. followed by a 5-minute hold at 60% B. The flow rate was 25 mL / min.

[0291] 1.1) Monodisperse polysarcosine intermediate 1.1.1) General method On-resin synthesis of monodisperse polysarcosines using Rink amide and 2-chlorotrityl resins Using an iterative submonomer synthesis procedure (described in WO 2019081455) or commercially available Fmoc-Sar-Sar-OH dipeptoid building blocks for Wang resin. Classic Fmoc cleavage with a cleavage block (Cat. No. 2313534-20-0) The on-resin dimerization step (n=2) was achieved according to the SPPS methodology. All synthetic yields are based on the initial Fmoc- Reported based on sarcosine loading. All reactions were performed at room temperature unless otherwise stated. The first Fmoc-sarcosine residue (Christof Senn Laboratories) was preloaded onto Ri nk amide, 2-chlorotrityl or Wang polystyrene 1% DVB 100-20 0 mesh resin was used (typical initial loading of 0.6-1 mmol / g). ratories) were used (typical initial loading of 0.6-1 mmol / g).

[0292] 1.1.2) Polysarcosine elongation Preloaded Rink amide with Fmoc-sarcosine, 2-chlorotrityl or Wan The resin was treated with 20% piperidine in DMF (1 mL per 100 mg of resin) at room temperature for 15 min. The resin was then washed with DMF (4 times) and DCM (4 times). Fmoc-Sar-Sar-OH (3 equivalents), HATU (2.9 equivalents) and DIPEA ( A solution of 6 equivalents of HCl in DMF (1 mL per 100 mg of resin) was added. The reaction vessel was left for 2 hours. After stirring, the resin was washed with DMF (4 times) and DCM (4 times). The mixture was treated twice with piperidine (1 mL per 100 mg of resin) for 15 minutes at room temperature. The resin was washed with DMF (4 times) and DCM (4 times).

[0293] For synthesis on Rink amide or 2-chlorotrityl resin, WO 20190 A classical submonomer synthesis procedure was used, as described in JP 81455. The desired length can be achieved by alternating the monoacetylation and amine substitution steps. The polysarcosine oligomer with n = 3 was elongated up to 1000 kJ / mol. The bromoacetylation step was carried out as follows: 10 equivalents of bromoacetic acid and 13 equivalents of diisopropylcarbodiimide (resin 100 The mixture was stirred for 30 minutes, drained, and Washed with DMF (4 times). For the amine substitution step, 40% (wt) methylamine in water was added. Add 1.5 mL of ethanol (1.5 mL per 100 mg of resin), shake the vessel for 30 minutes, drain, and Washed with MF (4 times) and DCM (4 times).

[0294] For synthesis on Wang resin, the classical Fmoc / SPPS procedure was used. n=3 The elongation of polysarcosine oligomers was carried out using Fmoc-Sar-Sar-OH dipeptoid biphenyls. Iterative coupling of the bonding block (Cat. No. 2313534-20-0) The resin was loaded with Fmoc-Sar-Sar-OH (3 equivalents), HATU (2.9 equivalents), and A solution of 100 mg of resin (1 mL) and DIPEA (6 equiv.) in DMF (1 mL per 100 mg of resin) was added. The reaction vessel was agitated for 90 min and the resin was washed extensively with DMF (4 times) and DCM (4 times). The resin was then washed with 20% piperidine in DMF (per 100 mg of resin) at room temperature for 15 minutes. The resin was treated twice with 1 mL of HCl (1 mL per 100 ml). The resin was washed with DMF (4 times) and DCM (4 times). The coupling / Fmoc deprotection cycle was repeated until the desired polysarcosine length was obtained. If necessary, the final coupling was repeated to obtain the final polysarcosine of even length. To achieve this, commercially available Fmoc-S instead of the Fmoc-Sar-Sar-OH dipeptoid unit was used. This is done using ar-OH amino acids.

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

[0296] 1.1.3.1) 2-Azidoethan-1-amine Side-Functionalized Polysarcosine Rink or 2-chlorotrityl resin was treated with 10 equivalents of bromoacetic acid and 13 equivalents of Add 2 mL of diisopropylcarbodiimide (2 mL per 100 mg of resin). Stir for 30 minutes, drain, and wash with DMF (4 times). A 3 molar solution in DMF (1 mL per 100 mg of resin) was added and the vessel was shaken for 45 minutes. The mixture was drained and washed with DMF (4 times) and DCM (4 times). Gly-OH coupling (5 equiv. Fmoc-Gly-OH, 4.9 equiv. HATU, 1 DMF (1 mL per 100 mg of resin) containing 0 equivalents of DIPEA and 20% piperidine Fmoc deprotection with 1 mL of methylpropanol in DMF (1 mL per 100 mg of resin) at room temperature for 15 min This was done twice. The resin was washed with DMF (4 times) and DCM (4 times).

[0297] The final polysarcosine compound was cleaved from the resin (for Rink and Wang resins). For 2-chlorotrityl, 100% TFA was used twice for 30 minutes, and for 2-chlorotrityl, 20% TFA was used twice for 1 minute. The resin was filtered and the volatiles were removed under reduced pressure to give the crude product, which was then purified by Intermediate Ion Exchange. Purification was performed using a chim® RP-AQ (30 μm) cartridge. Mobile phase A was water + 0.1% TFA and mobile phase B was acetonitrile.

[0298] 1.1.3.2) Glutamic Acid Side-Functionalized Polysarcosine Fmoc-Glu(OAll)- was added to Rink, Wang or 2-chlorotrityl resin. OH (3 equiv.), HATU (2.9 equiv.) and DIPEA (6 equiv.) in DMF (resin A solution of 1 mL of DMF (100 mg per 1 mL of DMF) was added. The reaction vessel was stirred for 90 minutes and the resin was diluted with DMF. The resin was then washed extensively with DMSO (4 times) and DCM (4 times). The resin was treated twice with 20% piperidine in F (1 mL per 100 mg of resin). The resin was washed with DMF (1 mL per 100 mg of resin) and DCM (4 times). Fmoc-amino-3,6-dioxaoctanoic acid (3 equivalents), HATU (2.9 equivalents) The resin was then coupled with DIPEA (6 equiv.) for 1 hour. The resin was then washed with DMF (4 times), DCM (4 The mixture was washed with 0.25 equivalents of Pd(PPh3)4 and 20 equivalents of phenylsilane (a The reaction was carried out by two 30-minute treatments with a DCM solution containing 100 mg of methylcellulose (with gentle stirring under a stream of argon). The alloc protecting group was then removed by rinsing the resin with DMF (5 times) and DCM (5 times). Optionally, N-hydroxysuccinimide (NHS) ester was added in an amount of 50 equivalents. of DIC and 60 equivalents of N-hydroxysuccinimide (1.5 mg per 100 mg of resin). The final polysarcosine compound was prepared by treatment with a DMF solution containing 1,2-dichloro-2,4-dichloro-1,4 ... The resin was then washed with DMF (4 times) and DCM (4 times). .

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

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

[0301] [Table 6]

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

[0303] [ka] Compound INT1 was prepared according to the procedure described in patent application WO 2019081455. This compound was synthesized according to the following procedure. (stereocenter indicated by the

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

[0305] [ka]

[0306] 1.2.2.1) tert-butyl(2-hydroxy-2-(4-hydroxy-3-dimethylamino)methyl) Synthesis of cyclophenylethyl carbamate (±)-Octopamine hydrochloride (1690 mg / 11 mmol) was weighed into a round-bottom flask. The flask was cooled to 0°C and suspended in 4 mL of pre-cooled 65% nitric acid solution. 4 mL was added slowly. The reaction was kept at 0°C for 20 minutes and evaluated by HPLC. The contents of the flask were added to a pre-cooled 2 Transfer to a 50 mL Erlenmeyer flask and add saturated NaHCO3 solution (approximately 10 mL) until a pH value of 8–9 is reached. The mixture was slowly neutralized with 50 mL of dioxane at 0°C. Then, 30 mL of dioxane was added, followed by B C0C (7202 mg / 13.2 mmol) was added. The reaction was then allowed to reach room temperature. The reaction was then diluted with EtOAc and washed three times with saturated citric acid solution, The organic phase was dried over MgSO4, filtered and concentrated under vacuum. Evaporation gave the crude product, which was purified by silica gel chromatography (petroleum ether / EtOA c, gradient 70:30 to 20:80 to give the title compound (1320 mg / 4 0%) as a thick yellow-brown oil. 1H NMR (300 MHz, DMSO-d6 )δ10.79(s,1H),7.79(d,J=2.1Hz,1H),7.47(dd ,J=8.6,2.1Hz,1H),7.08(d,J=8.6Hz,1H),6.74 (t,J=5.9Hz,1H),4.56(t,J=6.3Hz,1H),3.07(t d,J=6.1,1.6Hz,2H),1.31(s,9H). MS m / z(ESI+ ): Calc[M+H]+=299.1; Exp[M+H]+=299.1. HPLC method Method 2, retention time = 5.5 minutes.

[0307] 1.2.2.2)(2S,3R,4S,5S,6S)-2-(4-(2-((tert -butoxycarbonyl)amino)-1-hydroxyethyl)-2-nitrophenoxy)- 6-(Methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriazol- Synthesis of acetate. In a round-bottom flash chamber, Ag2CO3 (1500 mg / 5.4 mmol) and 1,1,4, 7,10,10-Hexamethyltriethylenetetramine (251mg / 1.1mmol) The compound was dissolved in 4 mL of anhydrous acetonitrile and stirred at room temperature for 2 hours. -butyl(2-hydroxy-2-(4-hydroxy-3-nitrophenyl)ethyl)chlor Bamate (292 mg / 0.98 mmol) and 1-bromo-2,3,4-tri-O-a Cetyl-α-D-glucuronide methyl ester (583 mg / 1.46 mmol) at 0°C The reaction mixture was stirred at room temperature for 4 hours. The reaction mixture was then filtered through Celite and The organic phase was diluted with tOAc and washed three times with saturated citric acid solution and once with saturated NaCl solution. The extract was dried over MgSO4, filtered and evaporated in vacuo to give the crude product, which was then passed through silica gel. by column chromatography (petroleum ether / EtOAc, gradient 70:30 to 30:70). The title compound (244 mg / 48%) was obtained as a yellow foam. 300MHz,DMSO-d6)δ7.76(dd,J=3.3,2.1Hz,1H), 7.60(t,J=7.6Hz,1H),7.36(dd,J=8.7,2.6Hz,1 H),6.77(s,1H),5.71(d,J=7.8Hz,1H),5.61(s, 1H),5.46(td,J=9.5,1.1Hz,1H),5.21-5.02(m, 3H),4.75(dd,J=9.9,1.4Hz,1H),4.62(s,1H),3 .65(s,3H),3.17(s,2H),3.10(t,J=6.1Hz,2H), 2.81-2.59(m,6H),2.05-1.96(m,9H),1.30(d,J =1.7Hz,9H). MS m / z(ESI+):Calc[M+Na]+=637. 2;Exp[M+Na]+=637.2. HPLC method 2, retention time = 6.75 minutes.

[0308] 1.2.2.3)(2S,3R,4S,5S,6S)-2-(4-(2-((tert -butoxycarbonyl)amino)-1-(((4-nitrophenoxy)carbonyl)oxy (Ci)ethyl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2 Synthesis of H-pyran-3,4,5-triyl triacetate The previous compound (2S,3R,4S,5S,6S)-2-(4-(2-((tert-butyl) (1-hydroxyethyl)-2-nitrophenoxy)-6-(2-hydroxycarbonyl)amino) (Methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate 4-nitrophenyl chloroformate (334 mg / 0.54 mmol) and 4-nitrophenyl chloroformate (21 9 mg / 1.09 mmol) was dissolved in 6 mL of dry DCM at 0° C. Anhydrous pyridine (1 12 mg / 1.41 mmol) was added and the mixture was stirred at room temperature for 30 minutes. Filtration through a 0.45 μm PTFE filter and silica gel chromatography (petroleum ether / Purification with EtOAc, gradient 85:15 to 30:70 gave the title compound (380m g / 90%) as a yellow foam. 1H NMR (300 MHz, 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),7.4 6(d,J=8.8Hz,1H),7.20(d,J=4.8Hz,1H),5.77( dd,J=7.7,3.7Hz,1H),5.47(t,J=9.5Hz,1H),5. 11(q,J=9.6Hz,2H),4.77(d,J=9.9Hz,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(ES I+):Calc[M+Na]+=802.15;Exp[M+Na]+=802.15 . HPLC method 2, retention time = 8.5 minutes.

[0309] 1.2.2.4) Synthesis of Compound INT2 381 mg (0.49 mmol) of the previous compound (2S,3R,4S,5S,6S)-2 -(4-(2-((tert-butoxycarbonyl)amino)-1-(((4-nitrophenyl) (phenoxy)carbonyl)oxy)ethyl)-2-nitrophenoxy)-6-(methoxy ... (carbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate, 200 mg (0.38 mmol) of exatecan mesylate and 51 mg (0.38 mmol) of HOBt was dissolved in 5 mL of an 85:15 (v / v) mixture of anhydrous DMF / pyridine. 3.5 mg (0.51 mmol) of DIPEA was added. The reaction was stirred at 40° C. for 2 hours. The crude residue was purified by silica gel chromatography (99:1 Purification by elution with a 95:5 to 95:5 DCM / MeOH gradient gave 360 ​​mg (87%) of Intermediate compound (2S,3R,4S,5S,6S)-2-(4-(2-((tert-but 9-(((1R,9R)-9-ethyl-5-fluoro-9-hydroxycarbonyl)amino)-1-( ... -Hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexyl 1H,12H-benzo[de]pyrano[3',4':6,7]indolizino [1,2-b]quinolin-1-yl)carbamoyl)oxy)ethyl)-2-nitrophen (oxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-trimethylsilyl Iodotriacetate was obtained as a yellow / greenish solid. ESI+[M+Na]+=1 098.3. HPLC Method 3, retention times = 14.7 and 14.9 min (diastereoisomers) mixture).

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

[0311] 1.2.2.5) Synthesis of stereopure compounds INT2-S and INT2-R

[0312] [ka]

[0313] 1.2.2.5.1) tert-butyl(2-hydroxy-2-(4-hydroxy-3 Chiral Separation of Racemic Mixture of (-Nitrophenyl)ethyl)carbamate Teledyne Isco CombiFlash® Rf200 System Chiralflash® IC MPLC column 30x100mm, 20μ m (Daicel catalog number 83M73) to obtain racemic tert-butyl (2- Hydroxy-2-(4-hydroxy-3-nitrophenyl)ethyl)carbamate (as described above) The mobile phase was DCM + 0.2% (v / v) EtOH ( The flow rate was 12 mL / min. The sample solvent was DCM+0.2% ( The mass recovery of the two enantiomers after separation was greater than 80%. It was.

[0314] tert-Butyl (S)-(2-hydroxy-2-(4-hydroxy-3-nitrophenyl) The retention time of tert-butyl ( R)-(2-hydroxy-2-(4-hydroxy-3-nitrophenyl)ethyl)carba The mat retention time was 25 min.

[0315] To determine the absolute configuration, the phenolic position of both enantiomers was analyzed by HCl in anhydrous THF. Esterified with 1.2 molar equivalents of 4-nitrobenzoyl chloride and 2 molar equivalents of triethylamine. The compound was purified by chromatography on silica gel (petroleum ether / EtOAc, Purification by a gradient of 90:10 to 10:90 gave 4-(2-((tert-butoxy) Carbonyl)amino)-1-hydroxyethyl)-2-nitrophenyl 4-nitrobenzo Art. 1H NMR (300MHz, DMSO-d6) δ 8.51 (d, J = 9 .1Hz,2H),8.44(d,J=9.1Hz,2H),8.19(d,J=1.9 Hz,1H),7.88(d,J=10.2Hz,1H),7.72(d,J=8.4H z,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. Absolute configuration of enantiomers (Previously dissolved in a 1:1 mixture of heptane / dichloromethane and allowed to slowly evaporate for 3 weeks. The formation of block-shaped crystals was confirmed by X-ray crystallography. The sample was mounted on a nylon loop in fluoroether oil. Data were obtained at T = 150.00(5) K. Xcalibur, equipped with Oxford Cryosystems cryogenic equipment operating at The data were collected using an Atlas Gemini superdiffractometer. The measurements were performed using the ω scan. The ShelXT solution program and O lex2(OVDolomanov et al.,Olex2:A complete te structure solution, refinement and ana lysis program,J.Appl.Cryst.,2009,42,339- The structure was analyzed using the full matrix least squares (F2) method. Using minimization, we used ShelXL 2018 / 3 (Sheldrick, GM, Cr ystal structure finement with ShelXL,Act a Cryst.,2015,C71,3-8) and improved.

[0316] 1.2.2.5.2) Synthesis of stereopure INT2-S and INT2-R compounds The previous section was repeated without any appreciable change in reaction conditions, reactivity, or overall yield. As described in Section 1.2.2, stereo-pure compounds INT2-S and INT2-R was synthesized.

[0317] Final purification using HPLC preparative method 5 afforded 33 mg of compound INT2-S as a yellow solid. ESI+ [M+H]+ = 836.2. HPLC Method 3, retention time = 7.3 Minutes.

[0318] Final purification using HPLC preparative method 5 afforded 21 mg of compound INT2-R as a yellow solid. ESI+ [M+H]+ = 836.2. HPLC Method 3, retention time = 7.8 Minutes.

[0319] 1.2.3) Synthesis of compound INT3

[0320] [ka]

[0321] 1.2.3.1) Ac-Val-Ala-OH (acetyl-L-valyl-L-alanine) ) synthesis 30 mL of L-alanine benzyl ester hydrochloride (542 mg / 2.5 mmol) A solution of triethylamine (254 mg / 2.5 mmol) in CM and distilled water (30 mL) , N-α-acetyl-L-valine (400 mg / 2.5 mmol) and HOBt (339 mg / 2.5 mmol) was added sequentially. The mixture was then cooled to 0° C., and EDC-HC l (530 mg / 2.75 mmol) was added. The resulting mixture was stirred overnight at 0°C. The reaction was diluted with 20 mL of 2M HCl and the layers were separated. The organic phase was washed with 2M HCl for 2 min. The organic phase was washed twice with saturated NaHCO3 solution and once with saturated NaCl solution. 4, filtered, and evaporated under vacuum to give 714 mg (89%) of benzyl acetylacetonate. L-valyl-L-alaninate was obtained as a white solid intermediate.

[0322] This intermediate was solubilized in 10 mL of 1:1 EtOAc / MeOH (v / v) and added to the stencil. After the initial argon purge, a catalytic amount of 5% wt Pd / C was added. The reactor was then purged twice with H2 and stirred at room temperature under 10 bar H2 pressure. The reaction mixture was filtered through a 0.45 μm PTFE filter and the solvent was removed under vacuum. After the reaction, a quantitative amount of pure acetyl-L-valyl-L-alanine was obtained as a white solid. 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,2H),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).

[0323] 1.2.3.2)(2S)-2-Acetamido-N-((2S)-1-((4-(1- Hydroxybut-3-yn-1-yl)phenyl)amino)-1-oxopropane-2- Synthesis of (( ... In a round-bottom flash container, 420 mg (2.60 mmol) of 1-(4-aminophenyl)butanol was added. Ter-3-yn-1-ol (Sharma A. et al., Chem 2018, 4 (10), 2370-2383) and 600 mg (2.60 mmol) of the previous compound Ac-Val-Ala-OH was dissolved in 20 mL of anhydrous THCM. F. Then, 676 mg (2.74 mmol) of HCl (previously dissolved in 5 mL of anhydrous DMF) was added to the suspension. l) 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) The resulting cloudy reaction mixture was stirred at room temperature overnight. The volatiles were then removed under reduced pressure. The crude residue was dried and purified by silica gel chromatography (99:1 to 85: Purification by HCl (15 ml DCM / MeOH gradient) gave 809 mg (83%) of the title compound. Obtained as a white solid. 1H NMR (300MHz, DMSO-d6) δ 9.84 (s, 1H),8.18(d,J=7.0Hz,1H),7.90(d,J=8.6Hz,1H ),7.53(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=7.6,7.2Hz,1H),4.17(dd,J=8.5,6.8Hz ,1H),2.70(t,J=2.6Hz,1H),1.96(dt,J=13.2,6 .6Hz,1H),1.88(s,3H),1.30(d,J=7.1Hz,3H),0 .86(dd,J=10.9,6.8Hz,6H). ESI+[M+H]+=374.2 . HPLC method 2, retention time = 3.95 minutes.

[0324] 1.2.3.3) 1-(4-((S)-2-((S)-2-acetamido-3-methyl Butanamido)propanamido)phenyl)but-3-yn-1-yl(4-nitrophenyl) Synthesis of (nyl)carbonates 94 mg (0.25 mmol) of (2S)-2-acetamido-N-((2S)-1- ((4-(1-hydroxybut-3-yn-1-yl)phenyl)amino)-1-oxo propan-2-yl)-3-methylbutanamide and 153 mg (0.50 mmol) Bis(4-nitrophenyl) carbonate was dissolved in 2 mL of anhydrous DMF. 0.76 mmol) of DIPEA was added and the reaction mixture was stirred at room temperature overnight. The solids were removed under reduced pressure and the crude residue was purified by silica gel chromatography (99:1 to 90:10 Purification by HCl / MeOH gradient (DCM / MeOH) afforded 118 mg (87%) of the title compound as a yellow solid 1H NMR (300MHz, DMSO-d6) δ 9.99 (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. 5Hz,1H),4.39(p,J=7.2Hz,1H),4.17(dd,J=8.5 ,6.9Hz,1H),3.01-2.84(m,3H),1.99-1.91(m,1 H),1.88(s,3H),1.31(d,J=7.1Hz,3H),0.86(dd ,J=11.2,6.8Hz,6H). ESI+[M+H]+=539.1. HPLC method Method 2, retention time = 6.48 minutes.

[0325] 1.2.3.4) Synthesis of compound INT3 43 mg (0.081 mmol) of the above compound 1-(4-((S)-2-((S)-2 -acetamido-3-methylbutanamido)propanamido)phenyl)but-3-yne -1-yl(4-nitrophenyl) carbonate, 55.5 mg (0.11) mesylate Exatecan and 11.0 mg (0.08 mmol) of HOBt were dissolved in 2 mL of anhydrous DMF. The resulting solution was dissolved in a 85:15 (v / v) mixture of 11.5 mg (0.09 mmol) of HCl / pyridine. ) of DIPEA was added. The reaction was stirred at 40°C for 3 hours. The volatiles were removed under reduced pressure. After evaporation, the reaction mixture was purified by silica gel chromatography (99:1 to 9 5:5 DCM / MeOH gradient), 42 mg (62%) of compound INT3 was obtained in a yellow / green Obtained as a colorless solid. ESI+ [M+H]+ = 835.3. HPLC Method 2, retention Time = 6.50 and 6.60 min (diastereoisomeric mixture).

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

[0327] [ka]

[0328] 1.2.4.1) tert-butyl(2-(4-aminophenyl)-2-hydroxyethyl) Synthesis of ethyl)carbamates 911 mg (3.23 mmol) of commercially available tert-butyl(2-hydroxy-2-( Contains 4-nitrophenyl)ethyl)carbamate (CAS number 939757-25-2) The MeOH solution containing the catalyst was transferred to a stainless steel hydrogenation reactor. After the initial argon purge, A quantity of 5% wt Pd / C was added. The reactor was then purged twice with H2 and the reaction was stirred. The reaction was kept under 10 bar H pressure with stirring at room temperature for 5 hours. After filtration through a PTFE filter and removal of the MeOH under vacuum, 749 mg (92%) of the product was obtained. The title compound was obtained as a white solid. ESI+ [M+H]+ = 253.2. HPLC Method 2. Retention time = 2.73 minutes.

[0329] 1.2.4.2) tert-Butyl (2-(4-((S)-2-((S)-2-acetonitrile) Amido-3-methylbutanamido)propanamido)phenyl)-2-hydroxyethyl ) Synthesis of carbamates 150 mg (0.60 mmol) of the above compound tert-butyl (2-(4-aminopropyl) (phenyl)-2-hydroxyethyl)carbamate, 222 mg (0.71 mmol) of F Dissolve moc-Ala-OH and 81 mg (0.62 mmol) of DIPEA in 5 mL of anhydrous D 181 mg (0.71 mmol) of HATU was added and the reaction was stirred at room temperature. Stir overnight. The volatiles are then removed under reduced pressure and the crude residue is chromatographed on silica gel. Purification by chromatography (DCM / MeOH gradient from 100:0 to 90:10) afforded F The first intermediate directly involved in the moc deprotection, tert-butyl (2-(4-((S)-2- ((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamide )phenyl)-2-hydroxyethyl)carbamate was obtained quantitatively. Duration = 7.7 minutes.

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

[0331] 130 mg (0.40 mmol) of tert-butyl (2-(4-((S)-2-amino)methyl) (2-hydroxyethyl) carbamate and 124 mg ( 0.48 mmol) of commercially available Ac-Val-OSu (CAS number 56186-37-9) was dissolved in 3 mL of anhydrous DMF and the reaction mixture was stirred at room temperature overnight. The solvent was removed under reduced pressure and the crude residue was triturated with 3 mL of DCM to give 95 mg (51%) of the title compound. The product was obtained as a white solid. 1H NMR (300MHz, DMSO-d6) δ 9.82( 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),4.5 8-4.47(m,1H),4.40(q,J=7.1Hz,1H),4.17(dd, J=8.4,6.8Hz,1H),3.15-2.90(m,2H),1.88(s,4 H),1.35(s,9H),1.30(d,J=7.1Hz,3H),0.92-0. 73(m,6H). ESI+[M+H]+=487.3. HPLC method 2, retention time = 4 .50 minutes.

[0332] 1.2.4.3) tert-Butyl (2-(4-((S)-2-((S)-2-acetonitrile) Amido-3-methylbutanamido)propanamido)phenyl)-2-(((4-nitro Synthesis of phenoxy)carbonyl)oxy)ethyl)carbamate 286 mg (0.62 mmol) of tert-butyl (2-(4-((S)-2-(( S)-2-acetamido-3-methylbutanamido)propanamido)phenyl)-2- hydroxyethyl)carbamate and 375 mg (1.23 mmol) of bis(4-nitro 318 mg (2.46 mmHg) of methyltriphenyl carbonate was dissolved in 3 mL of anhydrous DMF. 100 ml of DIPEA was added and the reaction mixture was stirred at room temperature for 3 hours. Volatiles were removed under reduced pressure. The crude residue was purified by silica gel chromatography (99:1 to 90:10 DCM / M Purification by hexane (HCl) (pH 7.0) gave 336 mg (87%) of the title compound as a yellow solid. 1H NMR (300MHz, DMSO-d6) δ 9.99 (s, 1H), 8.36 -8.26(m,2H),8.22(d,J=6.9Hz,1H),7.89(d,J= 8.6Hz,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,1 H),5.68(t,J=6.0Hz,1H),4.38(p,J=7.1Hz,1H) ,4.17(dd,J=8.5,6.9Hz,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=7.1Hz,3H),0.86(dd,J=11.1,6.8Hz,6 H). ESI+[M+Na]+=652.2. HPLC HPLC method 2, retention time = 7 .13 minutes.

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

[0334] The resulting solid was redissolved in TFA / DCM (30:70 v / v) solution at 0°C. The mixture was stirred at room temperature for 20 minutes. The volatiles were evaporated under reduced pressure and the crude residue was diluted with water / ACN (1:1 v / v) solution and purified using HPLC preparative method 5 to give 171.4 mg (7 3%) of compound INT4 was obtained as a yellow solid. ESI+ [M+H]+ = 826.4. PLC method 3, retention times = 8.3 and 8.75 min (diastereoisomeric mixture).

[0335] 1.2.4.5) Synthesis of stereopure compounds INT4-S and INT4-R

[0336] [ka]

[0337] 1.2.4.5.1) tert-butyl(2-(4-aminophenyl)-2-hydroxybenzoate Chiral separation of racemic mixtures of diethyl carbamates. Teledyne Isco CombiFlash® Rf200 System Chiralflash® IC MPLC column 30x100mm, 20μ m (Daicel catalog number 83M73) to obtain racemic tert-butyl (2- Chiral separation of (4-aminophenyl)-2-hydroxyethyl)carbamate was carried out. The mobile phase was DCM + 0.2% (v / v) EtOH (isocratic gradient). The flow rate was 12 mL. The sample solvent was DCM + 0.2% (v / v) EtOH. The mass recoveries of the two enantiomers were greater than 75%.

[0338] tert-Butyl (S)-(2-(4-aminophenyl)-2-hydroxyethyl)carbonyl The retention time of tert-butyl (R)-(2-(4-amino)-2-methyl-2-propanol was 21 min. The retention time of enantiomer (phenyl-2-hydroxyethyl)carbamate was 29 minutes. The absolute configuration of the mer (previously dissolved in a 1:1 mixture of heptane / ethanol and slowly stirred for 1 week) The formation of crystals was confirmed by X-ray crystallography. was mounted on a nylon loop in perfluoroether oil. Data were obtained at T = 150.0 Xca equipped with Oxford Cryosystems cryostat operating at 0(10)K The data were collected using the libur, Atlas, and Gemini ultradiffractometers. Measurements were taken using ω scans with α radiation. The ShelXT solution probe was used with the dual method. Gram and Olex2 (OV Dolomanov et al., Olex2:A complete structure solution, refinement a nd analysis program,J.Appl.Cryst.,2009,4 The structure was solved using the 2D model (2,339-341). Using matrix least squares minimization, we use ShelXL 2018 / 3 (Sheldrick, G .M.,Crystal structure finement with Shell XL, Acta Cryst., 2015, C71, 3-8).

[0339] 1.2.4.5.2) Synthesis of stereopure INT4-S and INT4-R compounds The previous section was repeated without any appreciable change in reaction conditions, reactivity, or overall yield. As described in Section 1.2.4, stereo-pure compounds INT4-S and INT4-R was synthesized.

[0340] Final purification using HPLC preparative method 5 yielded 54 mg of compound INT4-S as a yellow solid. ESI+ [M+H]+ = 826.4. HPLC Method 3, retention time = 8.4 5 minutes.

[0341] Final purification using HPLC preparative method 5 afforded 46 mg of compound INT4-R as a yellow solid. ESI+ [M+H]+ = 826.4. HPLC Method 3, retention time = 8.9 0 minutes.

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

[0343] [ka] 44.5 mg (0.049 mmol) of compound PSR1 (azido-polysarcosine intermediate 27.5 (0.033 mmol) of compound INT1 (alkyne payload) , to reach a 0.060 M concentration of compound INT1, add 100 mM PBS (pH = 7. 5) and DMSO in a 1:1 (v / v) solution. Then, freshly prepared CuSO 4 pentahydrate and sodium ascorbate solution (approximately 250 mg / mL) in 0.08 M equivalent of Cu and 1 molar equivalent of sodium ascorbate (compound INT1 in the reaction mixture). The reaction was then flushed with argon. The mixture was heated and stirred at 40° C. The reaction was monitored by HPLC and was complete in less than 2 hours. The reaction mixture was diluted with 0.1% aqueous TFA / ACN 1:1 (v / v) and analyzed by HPLC. Purification using preparative method 5 yielded 44 mg (77% based on starting compound INT1) of 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- 3,6,9,12,15,18,21,24,27,30,33-Undecazapentato riacontyl)-1H-1,2,3-triazol-4-yl)-1-((((1R,9 R)-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)-3,4,5-trihydroxytetrahydrogen rho-2H-pyran-2-carboxylic acid was obtained as a yellow solid. ESI+ [M+H]+ = 17 55.7. HPLC Method 3, retention times = 7.51 min and 7.82 min (diastereoisomers) mixture).

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

[0345] 1.3.1.2) Synthesis of compound LNK2.

[0346] [ka] 409 mg (0.31 mmol) of compound PSR3 (NHS-activated polysarcosine intermediate) 172 mg (0.21 mmol) of compound INT2 (NH2-payload) Compound INT2 was dissolved in anhydrous DMF in a small vial (0.080M concentration). 83. 5 mg (0.83 mmol) of triethylamine was added and the reaction was stirred at room temperature for 30 min. After the total conversion of the reaction as monitored by HPLC, 8% (v / v) piperazine in DMF was added. To achieve a methylated solution, piperidine is added directly to the reaction vial. Stir the reaction at room temperature for 5-10 minutes until complete Fmoc deprotection is observed. The reaction mixture was slowly neutralized with 10% TFA aqueous solution / ACN 1:1 (v / v) and analyzed by HPLC. Purification using Preparative Method 5 gave 254 mg (67% yield based on starting compound INT2). The intermediate compound (2S,3S,4S,5R,6S)-6-(4-((9S)-40-amino) No-9-(2-(2-(2-aminoethoxy)ethoxy)acetamido)-1-(((1 S,9S)-9-Ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-di Oxo-2,3,9,10,13,15-hexahydro-1H,12H-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-Tridecao oxo-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 was obtained as a yellow solid. I+[M+H]+=1819.7. HPLC method 2, retention time = 4.60 min and 4.69 min (equimolar mixture of diastereoisomers).

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

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

[0349] [ka] Compounds LNK-2-S and LNK2-R were synthesized using the same procedure as used for compound LNK2. The starting materials were compounds INT2-S and IN2-S, respectively, synthesized as described above. T2-R (NH2-payload) and compound PSR3 (NHS-activated polysarcosine (internal body).

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

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

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

[0353] [ka] Using the same procedure used for compound LNK1, prepare the final compound LNK3 as described above. The starting materials were compound PSR2 (azido-polysarcosine intermediate) and compound INT3 (alkyne-payload).

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

[0355] 1.3.2.2) Synthesis of Compound LNK4

[0356] [ka] Compound LNK4 was synthesized as described above using the same procedure used for compound LNK2. The starting materials were compound PSR4 (NHS-activated polysarcosine intermediate) and compound The substance was INT4 (NH2-payload).

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

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

[0359] [ka] Compounds LNK4-S and LNK4-R were synthesized using the same procedure as that used for compound LNK2. The starting materials were compounds INT4-S and INT4-S, respectively. -R (NH2-payload) and compound PSR4 (NHS-activated polysarcosine intermediate) ) was.

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

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

[0362] Example 2: Preparation and characterization of antibody-drug conjugates 2.1) Antibody production The amino acid sequence of the monoclonal antibody was obtained from the literature. Farletuzumab light chain (SEQ ID NO: 10) and heavy chain (SEQ ID NO: 11) sequences were obtained from the World Health Organization tion(WHO)Drug Information Vol 23,No.3,20 International Nonproprietary N ames for Pharmaceutical Substances(INN)L ist 62 and WO 2017151979. Heavy chain (Wines et al., The Journal of Immunology May 15, 2000, 164(10)5313-5318) on Fc silencing Farletuzumab-LALA, which contains the mutations L234A and L235A (“LALA”), is also Also produced was the mirvetuximab light chain (SEQ ID NO: 1) (heavy chain "LALA" sequence SEQ ID NO: 9). 7) and heavy chain (SEQ ID NO: 16) sequences are taken from patent application WO 2011106528 Human IgG1k non-binding isotype control was obtained from Sino Biologicals. Catalog number HG1K (proprietary amino acid sequence). Trastuzumab (Herce Enhertu® 150 mg was purchased from Roche. Daiichi Sankyo / Astra Purchased from Zeneca.

[0363] Transient transfection of CHO K1 cells using art-recognized techniques Monoclonal antibodies were produced by PCR (commissioned to Evitria AG, Switzerland). cDNA was cloned into Evita using conventional (non-PCR-based) cloning techniques. The pDNA was cloned into the ria vector system. The DNA concentration was determined by measuring the absorbance at 260 nm. The accuracy of the sequence was determined by Sanger sequencing (plasmid equivalent). The results were validated in suspension-adapted CHO K1 cells (originally (received from ATCC and adapted for serum-free growth in suspension culture in Evitria) Seeds were grown in Evitria's proprietary animal component and serum-free medium. The cells were transfected with Evitria's proprietary transfection reagent. The supernatant was collected by centrifugation and subsequent filtration (0.2 μm filter). The purified product was purified using MabSelect SuRe Protein A purification resin (Cytiva) and SEC analysis. The purity of the antibody material was confirmed by SDS-PAGE and size exclusion chromatography. Endotoxin content was determined by fluoroscopy and was greater than 95%. Measurements were performed using the River Endosafe PTS system.

[0364] 2.2) Preparation of Cysteine-Conjugated Antibody-Drug Conjugates Add the antibody (10 mg / mL in PBS 7.4 + 1 mM EDTA) solution to the required volume (maximum 2.2 molar equivalents for the final DAR4 ADC or 1 for the final DAR8 ADC 4 molar equivalents) of tris(2-carboxyethyl)phosphine (TCEP) at 37 °C. The reduced antibody was then filtered through an Amicon 30K centrifugal filter device (Mi Potassium phosphate 100 mM was obtained by three dilutions / centrifugations using a Lithium Ion Transfection (Lithium Ion Transfection) The buffer was exchanged into pH 7.4 + 1 mM EDTA. 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 mixture was incubated at room temperature for 30 minutes. The final conjugate was purified by Amicon 3 PBS pH 7 by four dilutions / centrifugation using 0K centrifugal filter devices. 4 buffer or histidine sucrose buffer (20 mM histidine buffer pH 6.0, 4% Buffer exchange / purification with either (w / v) sucrose + 75 mM NaCl) and sterile filter The solution was filtered (0.20 μm PES filter).

[0365] Final protein concentrations were determined using a Nanodrop One device (Thermo Fish The assay was evaluated spectrophotometrically at 280 nm using a Fluorescent Scientific.

[0366] 2.3) Characterization of Cysteine-Conjugated Antibody-Drug Conjugates The resulting conjugate was characterized as follows: Drug-to-antibody ratio (DAR) by reversed-phase liquid chromatography-mass spectrometry (RPLC-MS) )evaluation: Modified RPLC-QToF analysis was performed using HPLC method 4 described above in Example 1. For cleaning, a mobile phase gradient of water / acetonitrile + 0.1% formic acid (0.4 mL / min) was used. Agilent PLRP-S 1000Å 2.1×150mm 8μm (80℃) Elute the conjugate and scan the 500-3500 m / z range (ESI+). Detection was performed using an er Impact II™ Q-ToF mass spectrometer. r Using the MaxEnt algorithm included in Compass® software The data were deconvoluted using

[0367] Reversed Phase Liquid Chromatography (RPLC-UV): The denaturing RPLC-UV analysis also uses a slightly modified version of the HPLC method described above. The analysis was performed on an Agilent 1100 HPLC-DAD system using mobile phase modification. Replace the reagent 0.1% formic acid with 0.1% TFA and measure only the DAD UV absorbance (mass spectrometry detector) Detection was performed using ELISA kits (none).

[0368] Size Exclusion Chromatography (SEC): SEC was performed on an Agilent 1100 HPLC column with an extra-column volume of less than 15 μL. The system (short section of peak tube with an inner diameter of 0.12 mm and microvolume UV filter) The column was an Agilent AdvanceBioS EC 300Å 4.6×150mm 2.7μm (maintained at 30℃). , 100 mM sodium phosphate and 200 mM sodium chloride (pH 6.8). 10% acetonitrile to minimize secondary hydrophobic interactions with the stationary phase and prevent bacterial growth Tolyl (v / v) was added to the mobile phase. The flow rate was 0.35 mL / min. UV detection was performed for 2 min. Monitoring was at 80 nm or any other relevant wavelength.

[0369] 2.4) Preparation of Lysine-Conjugated Antibody-Drug Conjugates Antibody mirvetuximab or farletuzumab (100 mM KH2PO4 pH 8.0 A solution of sulfo-SPDB-DM4 (CAS No. 1626359- Treat with a 12 mM DMSO solution of 59-8 (MedChemExpress) and 7. The final concentration was 5 molar equivalents of sulfo-SPDB-DM4. The solution was incubated at room temperature for 3 hours. The final conjugate was diluted with 1000 ribonucleotides and filtered through a 0.20 μm PES filter. , by five dilutions / centrifugation using Amicon 30K centrifugal filter devices , PBS pH 7.4 buffer or histidine sucrose buffer (20 mM histidine buffer Buffer exchange was performed using either HCl (pH 6.0, 4% (w / v) sucrose + 75 mM NaCl) or HCl (pH 6.0, 4% (w / v) sucrose + 75 mM NaCl). The resulting solution was then purified and sterile filtered (0.20 μm PES filter).

[0370] Final protein concentrations were determined using a Nanodrop One device (Thermo Fish The assay was evaluated spectrophotometrically at 280 nm using a Fluorescent Scientific.

[0371] 2.5) Characterization of Lysine-Conjugated Antibody-Drug Conjugates The resulting conjugate was characterized as follows: Average Drug-to-Antibody Ratio (DAR) Assessment by Native SEC-MS: Prior to analysis, 50 U of IgGZERO® (Geno) was administered per 50 μg of ADC. The ADC was deglycosylated by adding the enzyme vis. Agilent AdvanceBio SEC 200A 1.9 μm 2 on a 1 x 150 mm PEEK column (catalog number PL1980-3201PK) The column was filled with 50 mM ammonium acetate + 10% (v / v) HPLC grade The flow rate was maintained at 0.075 mL / min during the run, and the ADC The mAb or ADC was typically eluted between 3.5 and 4.5 minutes. After elution of the mAb or ADC, the flow and relaxation time were The buffer composition was maintained. The column eluate was then purified using a Bruker Impact II™ Q- The ToF mass spectrometer was used to measure the m / z range of 300–8000 (ESI + ) was scanned. The capillary voltage was set to 4500 V. The raw material drying gas was 8.0 L / min, and the nebulizing gas was 2 The pressure was set to 5 Psi. The source drying temperature was set to 200°C. ADC mass spectra were obtained. MaxEnt algorithm included in Bruker Compass® software The DAR was calculated using the ion intensity peaks of each ADC subspecies. The calculation was performed using the arc height.

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

[0373] 2.6) Overview of synthesized antibody-drug conjugates

[0374] [Table 7-1]

[0375] [Table 7-2] LC-0d: light chain; LC-1d: light chain with one drug-linker; HC-0d: heavy HC-1d: heavy chain with one drug-linker; HC-2d: two drug-linkers HC-3d: heavy chain with three drug-linkers. Major glycoforms have been reported for HC.

[0376] Example 3: General cell culture practices and animal studies The human cancer cell lines used in this project are from the American Type Culture Collection. ure Collection(ATCC), The Leibniz Institu te DSMZ German Collection of Microorgani sms and Cell Cultures GmbH (DSMZ) or Europe a Collection of Authenticated Cell Cults For experimental use, well-established Cell culture media and supplements, cryopreservation, and subculture procedures are described according to established cell culture practices. Cells were cultured according to the instructions from the original ATCC / DSMZ / ECACC supplier. The cells were incubated at 37°C in a 5% CO2 atmosphere for up to 2 months.

[0377] All animal procedures were performed in accordance with European Union Directive 86 / 609 / EEC. The study was carried out in an animal facility certified by the French Ministry of Agriculture under the permission of the , was approved by the local animal ethics committee (CECCAPP).

[0378] Example 4: Rodent Therapeutic Index Comparison of Glucuronide or Dipeptide Drug-Linker Designs Drug-linker constructs LNK1 (glucuronide-exatecan) and LNK3 (dipeptide-exatecan) We evaluated the therapeutic index of exatecan (Val-Ala-exatecan) in mice and compared its efficacy against two enzyme cleavage modalities. The therapeutic index of the dipeptide Val-Ala was compared in mice. Using a model human HER2 targeting trastuzumab monoclonal antibody as a moiety T-GLC-EXA ADC and T-VA-EXA ADC were formulated using these The ADC was evaluated for in vivo efficacy and tolerability (therapeutic index) in a HER2+ gastric cancer model. Because trastuzumab does not cross-react in mice, this assay was provides useful information regarding the apparent toxicity of the drug-linker component of the DC (potential target (internal-mediated toxicity is excluded).

[0379] In vivo efficacy evaluation: NCI-N87 gastric cancer cells were subcutaneously injected into female SCID mice (4 weeks old). ADC was implanted into the tumors approximately 150 mm 3 (To minimize the difference in initial tumor volume between groups) When the animals reached adulthood (6 animals per group, assigned to each group), a sub-curative dose of 1 mg / kg was obtained. Tumor volumes were measured every 3 to 5 days using a caliper device. Formula (L×W 2 ) / 2. 3 When it exceeds The animals were slaughtered.

[0380] Evaluation of the pharmacokinetic profile: rats after a single intravenous 3 mg / kg dose of the conjugate PK profile in a study (total antibody-drug conjugate based on mAb component over time) The ADC was administered to female rats via the tail vein (3 animals per group, randomly assigned). Sprague-Dawley rats (4-6 weeks old, Charles River) were given 3 At various time points, blood was collected via retroorbital bleeding into citrate tubes. The ADC concentration based on the antibody component was determined using human plasma. An IgG ELISA kit (Stemcell™ Technologies) was manufactured. The assay was performed according to the manufacturer's protocol. was used for quantification. PK parameters (clearance, half-life and AUC) were calculated using the PK function ( Usansky et al.,Department of Pharmacokin etics and Drug Metabolism,Allergan,Irvin e, an add-in developed by USAA) ) Calculated by two-compartment analysis using Excel® software did.

[0381] Assessment of in vivo tolerability: Female SCID mice (n=3) were treated with 200 mg / kg of the ADC. Mice were treated with a single high intraperitoneal dose of the compound. Weight loss or obvious signs of toxicity were not observed until 12 days after administration. In this experiment, Enhertu® ( trastuzumab deruxtecan) was used.

[0382] The results of this study are shown in Figure 1. T-GLC-EXA and T-VA-EXA exhibited similar effects. showed similar in vivo efficacy (Figure 1A) and rat PK profile (Figure 1B), but There was a significant difference in the tolerability of the mice depending on the dose (Figure 1C). For use in It was concluded that this entity provided a tolerable profile. It was good for the building.

[0383] Example 5: Flow cytometric evaluation of folate receptor alpha (FRa) extracellular binding sites For FRa cell surface quantification, cells were incubated with PE anti-FOLR1 antibody (BioLegend, The mixture was incubated with eBiosci (Cat. No. 908304) for 20 minutes at room temperature. ence(TM)Fixable Viability Dye eFluor(TM) 780 Kit (Thermo Fisher Scientific, Cat. No. 65 Cell viability was assessed using BD Fluorescence Immunosorbent Assay (BD Fluorescence Immunosorbent Assay -0865-18) according to the manufacturer's instructions. B was controlled by FACSDiva software (BD Biosciences). Analysis was performed using a Fortessa flow cytometer and FlowJo software Data were analyzed using software (BD Bioscience).

[0384] The results are shown in Figure 2. The cell lines in this research program express different levels of extracellular FRa. BT-474 breast cancer cells did not express extracellular FRa, so this study It was considered a negative control cell line in the context of Gram.

[0385] Example 6: In vitro cytotoxicity of exatecan mesylate against FRa+ cancer cell lines Assay The in vitro cytotoxicity of the compound exatecan mesylate was investigated in several FRa-positive cancer cells. Cells were cultured at an appropriate density (100 μL in 100 μL of appropriate culture medium) depending on the cell line. Cells were seeded at 0.000–10,000 cells / well in a 96-well plate and incubated at 37°C for 24 hours. Serial dilutions (50 μL) of test compounds pre-dissolved in culture medium were added. The mixture was incubated at 37°C for 144 hours. MTT (5 mg / mL, 20 μL (Sigma-Aldrich) was added to the wells and incubated at 37°C for 1-2 min. The incubation period was continued for 2 hours. The culture medium was then carefully removed and the well contents were reconstituted with acidified isopropanol. The absorbance values ​​were measured using a Multiskan™ Sky microp A latent reader (Thermo Scientific) was used at a wavelength of 570 nm ( The IC50 concentration values ​​were measured using a reference wavelength of 690 nm compared to untreated control cells. Inhibition was determined using dose-response curve fitting (GraphPad Prism 9). Ta.

[0386] The results are shown in Figure 3. Exatecan mesylate exhibited an IC50 of sub-nM to low nM. The compound demonstrated in vitro efficacy, allowing us to demonstrate its utility as an ADC payload for FRa-expressing malignancies. The agency urged the government to investigate the use of these compounds.

[0387] Example 7: Recombinant FRα binding affinity by ELISA Sandwich ELISA assays were performed in 96-well high-binding ELISA plates (Corn ing Inc., New York, NY, USA, Catalog No. 3590) 100 μL / well of recombinant human FRa protein (Sino Biolog ical, Catalog No. 11241-H08H); recombinant cynomolgus monkey FRa protein (Sino Biological Catalog No. 90950-C08H); Recombinant Rat FRα protein (Sino Biological, catalog number 81073-R08 H) or recombinant mouse FRα protein (Sino Biological, catalog no. Plate at 2 μg / mL in PBS (pH 7.4) using the antibody (No. 50573-M08H). The plates were coated with PBS-T (PBS + 0.05%) and incubated overnight at 4°C. After washing twice with Tween-20, the plate was filled with 200 μL / well of incubation buffer. The plate was blocked with blocking buffer (PBS-T + 0.1% BSA) for 1 hour at room temperature. The tray was washed four times with PBS-T, and 100 μL of a 3-fold dilution series of the test compound (antibody or The plate was then incubated in the dark at room temperature for 2 hours. After washing five times with PBS-T, the plates were bated with 100 μL / well of goat anti- Human IgG (H+L) HRP-conjugated antibody (Jackson Immunore Incubate with 1 mL of 10 ... The cells were pre-diluted 1:250,000 in incubation buffer. Washed five times with PBS-T. Afterwards, TMB substrate solution (Thermo-Fisher, Cat. No. N301) was added. Peroxidase activity was stopped with 0.18 M H2SO4 and the solution was then lysed at 100°C for 1 hour at 4°C. 450 nm using an Intelific MultiSkan EX microplate reader The absorbance was read at a reference wavelength of 650 nm. Sigmoid fixation was performed using software.

[0388] The results are shown in Figure 4. Figure 4A: All antibodies and ADCs showed approximately EC 50 =0.1n The recombinant human FRα binding affinities of M were similar to those of the native antibody and each ADC structure. No loss of binding was observed between the constructs. No significant differences were observed between letuzumab-LALA and their respective ADC constructs. No significant loss of binding was observed between rubetuximab and the M-SORAV ADC construct. For the negative control ADC NEG-VA-EXA, human FRα binding was not detected. Figure 4B: F-LALA-VA-EXA was found to be a recombinant FR-1 gene of human and cynomolgus monkeys. It binds equally to the recombinant FRa protein and does not bind to the recombinant rat or mouse FRa protein. Therefore, F-LALA-VA-EXA is not rodent cross-reactive but rather shows cytotoxicity. It is cross-reactive with rhesus monkeys.

[0389] Example 8: Human FRα binding affinity by SPR Surface plasmon resonance (SPR) experiments were performed on a Biacore T200 instrument at 25°C. The tested antibodies or ADCs were subjected to Human Antibody Capture Ki. CM5 series S pre-functionalized with t (Cytiva, Cat. No. BR100839) The captured samples were then placed on a sensor chip at low density (300-500 RU). The functionalized surface / flow cell without the band was used as a reference. Kinetics and affinity To measure the FR, recombinant human FR (Sino Biological, Cat. No. 1 1241-H08H) Analyte samples were run using a single cycle kinetic strategy. A constant 70 μL of buffer (HBS-EP+, Cytiva, Cat. No. BR100669) Five concentrations (0.5, 1, 2, 4) were serially administered at a flow rate of 1 L / mL with a 300-second contact pulse. The dissociation phase was performed by injecting the running buffer for 900 seconds. Between duplicates, the surface / flow cell was regenerated with 3M MgCl2 to remove the analyte. Both ligands were removed and a fresh ligand capture was performed using identical conditions. The analysis was performed using Biacore Evaluation after subtraction of the reference level and zero concentration signal of the analyte. The data were processed and fitted to a 1:1 binding model. , the binding rate constant k a (on speed) and k d (off rate), and K D (also called "affinity") The equilibrium dissociation constant (Eq. 1.0) was determined. The mean and standard deviation of replicate experiments are reported.

[0390] The results are shown in Figure 5. The loss of recombinant human FRa binding was observed with the native antibody and each No loss of binding was observed between farletuzumab and the ADC construct. No significant differences were observed between Zumab-LALA and the respective ADC constructs. , farletuzumab compared to mirvetuximab and mirvetuximab-based constructs and about 10-fold reduced K for farletuzumab-LALA-based constructs D Value (Affinity A constant (constant) was observed. This was due to the In comparison, the k of farletuzumab and farletuzumab-LALA based constructs d value( This resulted in an increase in the association rate constant k a (Considering that the Regarding the negative control IgG1 (Sino Biologicals, Cat. No. HG1K), No human FRα binding events were detected.

[0391] Example 9: Cell Binding Affinity by Flow Cytometry Antibody or ADC binding to extracellular human FRa expressed on cancer cell lines was analyzed using flow cytometry. The antibodies or ADCs tested were evaluated by LYNX Rapid APC Use the Antibody Conjugation Kit according to the manufacturer's protocol. The fluorophore was conjugated to an APC fluorophore using a fluorophore kit (Bio-Rad, Cat. No. LN K032APC). 500,000 cells (in a flow cytometry plastic tube) 10 μL of the APC-labeled antibody or ADC to be tested was added to the 100 μL of PBS suspension. 5 μL of 100 μg / mL solution was added. The cells were incubated in the dark for 20 minutes and then centrifuged. The cells were washed three times with PBS and resuspended in 200 μL of PBS for analysis. BD F controlled by SDiva software (BD Biosciences) Flow cytometry was performed using an Ortessa flow cytometer and FlowJo Data were analyzed using software (BD Bioscience).

[0392] The results are shown in Figure 6. Equivalent human FRα cell binding was observed between the ADC constructs. The binding affinity of the antibody to native farletuzumab appeared to be identical. Compared with the parent antibody mirvetuximab, the ADC derivative of mirvetuximab (M-SORA A significant loss of binding was observed for V), which is due to (1) the presence of a portion of the antibody variable region. Heterogeneity of the sulfo-SPDB-DM4 drug-linker, which can react with lysine amino acids (2) LYNX Rapid conjugation, thus reducing binding affinity; d APC conjugation kits are available for lysine amino acids such as sulfo-SPDB-DM4. Lower APC labeling efficiency of M-SORAV when reacting with acid, or (3)(1) and (2) For all compounds tested, F No binding was observed in the Ra negative control cell line BT-474.

[0393] Example 10: Ex vivo ADC human plasma stability F-VA-EXA and F-LALA-VA-EXA ADC samples (>6 mg / in PBS) mL solution) in a screw-cap centrifuge tube containing pure sterile human plasma (GeneTex, Catalog number GTX73265) to a final ADC concentration of 200 μg / mL (5% The sample was incubated at 37°C and aliquots were Time points were collected at 5 min, 6 h, 1 day, 2 days, 3 days, and 7 days (aliquots were kept at -80°C until analysis). The ADC was prepared by mixing the recombinant human folate receptor alpha protein with the recombinant human folate receptor alpha protein. Pre-treated with cellulose (Sino Biologicals, Cat. No. 11241-H08H). Coated Dynabeads™ M-280 streptavidin (The isolated from plasma by immunocapture using rmo Scientific magnetic beads Briefly, 600 μL of commercial bead solution was dissolved in HBS-EP buffer (Cytiva, Wash twice with PBS (catalog number BR100188) and resuspend in 1.2 mL of HBS-EP buffer. 65 μL of biotinylated recombinant FRa solution (protein amount: 48 μg) was added, and the solution The beads were then stirred at room temperature for 2 hours. Then, the beads were washed three times with HBS-EP buffer and diluted with 1.2 mL of Resuspended in HBS-EP buffer. For one immunocapture, 100 μL of the previous bead solution was used. The solution was added onto 100 μL of HBS-EP in a microcentrifuge tube. 1 μL (theoretical ADC amount 2 μg) was added, and the solution was stirred at room temperature for 2 hours. After the reaction, the bead-ADC complex was washed twice with HBS-EP buffer and then resuspended in 200 μL of HBS Resuspend in -EP buffer and incubate at 2 μL / 1000 U of PN overnight at 37 °C with gentle agitation. Gase F (New England Biolabs, catalog number P0705L) The beads were then deglycosylated by adding 2 ml of HBS-EP buffer. The beads were washed once with distilled water, twice with distilled water, and once with 10% acetonitrile in water (v / v). 50 μL of 30% aqueous acetonitrile (v / v) containing 0.1% (v / v) formic acid The mixture was incubated with HCl for 30 minutes at room temperature with gentle agitation. The elution samples containing the methylated ADC were analyzed using a Bruker Impact II™ Q-ToF A Thermo UltiMate 3000 UHPLC system equipped with a mass spectrometer was used. The analysis was carried out by denaturing reversed phase chromatography-mass spectrometry using mobile phase A: water + 0.1% ethanol. % formic acid, and mobile phase B was acetonitrile + 0.1% formic acid. The column was Agile nt PEEK PLRP-S 1000Å 2.1×100mm 5μm (80℃) The linear gradient was from 20% B to 50% B in 25 min. The flow rate was 0.4 mL / min. UV detection was monitored at 280 nm. The Q-ToF mass spectrometer measured the m / z range of 5 00~5000(ESI + ) used in Bruker Compass® software. The data was deconvolved using the MaxEnt algorithm included in the software. For stability data analysis, selected light chain (LC) and heavy chain (HC) elution times were Deconvolution of the raw spectra within the window was performed. Drug-linker loss or modification were identified according to the corresponding mass shift from the starting ADC material. The relative ratio of ADCs was calculated by dividing the intensity of a specific ADC subspecies by the intensity from the total ADC species. The final DAR values ​​were calculated according to Xu et al., Anal. Biochem., 201 1,412(1),56-66.

[0394] The results are shown in Figure 7. For both, a human plasma stability of greater than 90% of the drug-linker was observed. The mass shift of +18 Da (+H2O) from the self-stabilizing hydrolysis of α-H2O (which occurs between 48 and 72 h) The drug-linker was not used during this study, except for the ADC incubation time between No mass shift was observed (no early exatecan release or metabolism). The only instability observed was the retro-Michael maleimide deprotection of the entire drug-linker on the heavy chain of the ADC. Deconjugation was caused by the conjugate (no deconjugation was observed on the light chain). Ta).

[0395] Example 11: In vitro cytotoxicity experiments against FRa-negative cell lines To assess the nonspecific (off-target) cytotoxicity of ADCs, in vitro cell injury The toxicity assay was performed in the BT-474 (FRa negative) cancer cell line. Culture at an appropriate density (1,000–10,000 cells / well in 100 µL of appropriate culture medium) depending on the ) into a 96-well plate and incubated at 37°C for 24 hours. Serial dilutions (50 μL) of the dissolved test compound were added and the incubation was continued at 37°C. MTT (5 mg / mL, 20 μL, Sigma-Aldrich) was added for 144 hours. was added to the wells and incubation continued for 1-2 hours at 37°C. The medium was carefully removed and the well contents were dissolved homogeneously with acidified isopropanol. Values ​​were measured using a Multiskan™ Sky microplate reader (Th ermo Scientific) at a wavelength of 570 nm (reference wavelength 690 nm). IC50 concentration values ​​compared to untreated control cells were determined by fitting an inhibitory dose-response curve. The results were determined using GraphPad Prism 9.

[0396] The results are shown in Figure 8. Soravtansine (Sulfo-SPDB-DM4)-based ADC is higher compared to VA-EXA (LNK4-S drug-linker)-based ADCs showed off-target (non-FRa-mediated) cell killing efficacy at the level of Mirbetsukishi. This has been observed in both mab-based and farletuzumab-based ADCs. -EXA drug-linker moieties have been shown to be favorable for enhancing the tolerability of ADCs in the clinical setting Prerequisite: less off-target toxicity compared to soravtansine drug-linker .

[0397] [Table 8]

[0398] Example 12: In vivo efficacy xenograft model Four to five-week-old female CB-17 severe combined immunodeficient (SCID) mice were cultured at Janvier I abs (Le Genest-Saint-Isle, France) and tested The cells were isolated for 7 days before the start of the experiment. PBS (OV-90, SW-620, KB, BT-474 cells) strain) or PBS (PA-1, IGROV-1, OVCAR-3, NCI-H2110 cells Cells were resuspended in 50% BD Matrigel (Corning®) in 100% PBS. cells (5–10 × 10 per mouse) 6 The average tumor volume was approximately 120~150mm 3 Once the mice reached the target age, they were randomized (typically 7 mice per group). mice), or PBS (negative control) by a single intravenous injection (unless otherwise stated). or antibody-drug conjugates were examined. Tumor volume was measured using a caliper device (length × width) every 3 to 5 days and calculate the following formula: V = 4 / 3 × π × R 3 (where R is the radius The tumor volume was calculated using the following formula: 1500 mm3 or ulceration. If so, the mice were sacrificed.

[0399] In the IGROV-1 tumor model, a tumor re-implantation challenge was performed on day 94 of the study. - Mice treated with LALA-VA-EXA (12 mg / kg intravenously, single dose) were started. The animals were reimplanted with IGROV-1 cells using the same procedure as used previously (other flanks of the animals). A control group of five new SCID animals was also reimplanted following the exact same procedure.

[0400] Figure 9 shows tumor xenograft experiments in the SW-620 cancer model. and M-SORAV conjugate were injected IV at 5, 10, or 15 mg / kg. -VA-EXA was highly active at doses of 5 mg / kg or higher, whereas the comparator M-SORA V was inactive even at 15 mg / kg.

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

[0402] Figure 11 shows tumor xenograft experiments in the OV-90 cancer model. F-LALA-VA-EXA and M-SORAV conjugates were administered intravenously at 30 mg / kg. All conjugates were highly active, with 100% success in all groups. However, significant toxicity was observed with the M-SORAV conjugate. Six out of six mice showed a disheveled appearance (dull, matte hair coat), Three of the six mice showed signs of collapse and two of the six mice had diarrhea. No toxicity was observed with the F-VA-EXA and F-LALA-VA-EXA conjugates. These data show that F-VA-EXA and F- This suggests a better preclinical therapeutic window for the LALA-VA-EXA construct.

[0403] Figure 12 shows tumor xenograft experiments in the OV-90 cancer model. , F-LALA-VA-EXA and M-SORAV conjugates were administered at sub-curative doses of 5 and 1. At 5 mg / kg, F-VA-EXA and F- LALA-VA-EXA showed similar efficacy and improved response compared to M-SORAV. At 10 mg / kg, F-VA-EXA and M-SORAV showed similar efficacy. Efficacy was demonstrated (although more heterogeneity was observed within the M-SORAV group). At 0 mg / kg, F-LALA-VA-EXA outperformed F-VA-EXA and M-SORAV was better than both.

[0404] Figure 13 shows tumor xenograft experiments in the KB cancer model. LALA-VA-EXA and M-SORAV conjugates were administered at 3, 6, and 12 mg / kg The non-targeting isotype-matched negative control ADC NEG-VA-E was administered intravenously once at 100 mg / kg. XA was administered by a single IV injection at 12 mg / kg. F-VA-EXA, F-LALA-VA-E XA and M-SORAV were equally active at doses of 3 mg / kg or higher. Conjugate NEG-VA-EXA was inactive at the highest tested dose of 12 mg / kg. The antitumor activity of the conjugate was demonstrated to be target selective.

[0405] Figure 14 shows tumor xenograft experiments in the PA-1 cancer model. -EXA and M-SORAV conjugate at sub-curative doses of 3, 6 and 12 mg / kg The non-targeting isotype-matched negative control ADC NEG-VA-EX was administered intravenously once. A was administered as a single IV injection at 12 mg / kg. At 3 mg / kg, F-LALA-VA-EX A outperformed M-SORAV. 6 mg of F-LALA-VA-EXA outperformed M-SORAV. The 12 mg M-SORAV slightly exceeded the F-LALA-VA-EXA Overall, both conjugates demonstrated 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.

[0406] Figure 15 shows tumor xenograft experiments in the OV-90 cancer model. A-EXA and M-SORAV conjugates were administered once at doses of 3, 6, and 12 mg / kg. At 3, 6, and 12 mg / kg, F-LALA-VA-EXA was administered IV. It surpassed ORAV.

[0407] Figure 16 shows tumor xenograft experiments in the IGROV-1 cancer model. -VA-EXA and M-SORAV conjugate at doses of 3, 6, and 12 mg / kg A single IV injection. Non-targeting isotype-matched negative control ADC NEG-VA-EXA was administered IV once at 12 mg / kg. A-EXA and M-SORAV demonstrated similar levels of efficacy and strong tumor regression At the highest tested dose of 12 mg / kg, the non-targeted conjugate NEG-VA-EXA It showed some level of efficacy, although less pronounced than other FRa-targeting ADCs in the study Tumor regrowth rechallenge performed 94 days after the study demonstrated that F-LALA-VA-E It was confirmed that XA can induce a protective immune memory response, which was observed in a single 12 mg / kg dose. g Tumor growth was observed after reimplantation in the group treated with IV F-LALA-VA-EXA. (The positive control group using new untreated SCID mice showed no tumor growth after transplantation.) showed proliferation).

[0408] Figure 17 shows the results of the folate receptor alpha-negative (FRa neg) BT-474 breast cancer model. Tumor xenograft experiments showing F-LALA-VA-EXA and M-SORAV conjugates Gates were administered IV once at doses of 5 and 10 mg / kg. HER2-targeted trastuzumab deruxtecan was used as a positive control, at 10 mg / kg The dose was administered intravenously once. No efficacy was observed with FRα, confirming that the efficacy of these conjugates is FRa selective. As expected and as previously observed (Conilh et al., 2014), 21,Pharmaceuticals,14(3),247,doi:10.3390 / ph14030247), Enhertu® is a HER2-positive BT-4 It was effective in 74 cancer models.

[0409] [Table 9]

[0410] Example 13: Mouse Tolerance Study To assess the preclinical therapeutic index in mice, F-VA-EXA, F-LALA-VA- A mouse tolerance study was conducted using EXA and M-SORAV conjugates. ID mice (n = 5 mice per group) were treated with F-VA-EXA (200 mg / kg), F-LALA-VA-EXA (200 mg / kg) or M-SORAV (100 mg / kg) Mice were treated with a single intraperitoneal dose of 100 mg / kg of 1000 mg ... Patients were closely monitored for any signs of illness.

[0411] The same experiment was performed with 0, 50, 100, 150, or 200 mg of F-LALA-VA-EXA. A single dose of 100 mg / kg IV was also administered to female CD-1 mice.

[0412] The results (for SCID mouse experiments) are shown below and in Figure 18. F-VA-EXA and F-LALA-VA-EXA was effective at a dose of 200 mg / kg without any obvious signs of toxicity. In contrast, treatment with M-SORAV at a dose of 100 mg / kg resulted in a significant improvement in survival. All mice showed significant signs of toxicity (disheveled appearance with shaggy fur, diarrhea, prostration, closed eyes). If the animals showed signs of dysentery (inhibited behavior) and died within 2-3 days, they had to be euthanized. These results, along with the xenograft efficacy data described above, were consistent with those of the comparative M-SO Superior prevalence of F-VA-EXA and F-LALA-VA-EXA constructs compared to RAV The clinical therapeutic index is shown.

[0413] In female CD-1 mice, MBK-103 inhibited the growth of the ovarian tumors at all dose levels investigated (up to 200 mg / mL). g / kg IV, 1 dose) did not cause dose-related adverse systemic or local effects. No weight loss or behavioral changes were observed. The only notable observation was that the There was a slight decrease in thymus and spleen weights in all groups compared to the control group.

[0414] [Table 10]

[0415] Example 14: Sprague-Dawley rat pharmacokinetic study F-VA-EXA and F-LALA-VA-EXA conjugates were administered via the tail vein (randomized). Female Sprague-Dawley rats were administered via a 10-day immunization program (6 animals per group assigned to each group). The rats (4-6 weeks old - Charles River) were injected with 5 mg / kg of IgG. Quenched via retro-orbital bleeding at 5 min, 4 hr, 1 day, 2 days, 4 days, 7 days, 14 days, and 21 days The blood was collected in acid tubes, processed to plasma, and stored at −80°C until analysis.

[0416] Goat polyclonal anti-human IgG (H+L) primary antibody (Jackson) was used as the capture reagent. Immunoresearch) and mouse polyclonal anti-human as secondary detection antibody IgG(H+L) HRP conjugate (Jackson Immunoresearch h) Total mAb concentration was assessed by ELISA. Total ADC concentration was determined by the capture assay. Drug: Rabbit polyclonal anti-exatecan antibody (ref#6294 / 0000092 0, custom-made by Biotem, Apprieu, France) and mouse as secondary detection antibody Polyclonal anti-human IgG (H+L) HRP conjugate (Jackson Immunol. The ADC was evaluated by ELISA using the immunoresearch (immunoresearch). was used for quantification.

[0417] Pharmacokinetic parameters (clearance, half-life, Vss and AUC) were calculated using the PK function (Us ansky et al.,Department of Pharmacokinet ics and Drug Metabolism,Allergan,Irvine, Microsoft Excel with an add-in developed by CA, USA The data were calculated by two-compartment analysis using the software.

[0418] An LC / MS-MS method was used, using an Agilent 1100 HPLC system and Sc Free exatecan concentrations were assessed using the iex API 4000 MS / MS system. 80:20 (v / v) acetonitrile / methanol + 1% formic acid + d5-exate Rat plasma samples using an organic solution composed of ethanol (20 ng / mL) as an internal standard The samples were protein precipitated in a Phenomenex Kinet™ tube maintained at 45°C. ex® C8 2.1 x 30 mm 2.6 μm 100A column (Phenom Analyzed using gradient elution mode on a 1000-kJ / kg ion exchanger (Berkeley, Cat. No. 00D-4497-AN). Mobile phase A was water + 0.15% formic acid, and mobile phase B was acetonitrile / isopropanol. The mixture was 80:20 (v / v) + 0.15% formic acid. The flow rate was 0.8 mL / min. For the detection, MRM scans were used in positive ion mode. Linear optimization with 1 / x weighting was used. Using least squares regression, peak area ratios of analyte / deuterated internal standard versus nominal analyte concentrations were used. The calibration curve was plotted in the range of 0.2 (LLOQ) to 500 ng / mL. there were.

[0419] The results are shown in Figure 19. F-VA-EXA and F-LALA-VA-EXA were 13 to 1 Biphasic circulation with similar volume of distribution, slow clearance rate and half-life in the range of 5 days The PK profile was characterized by a rapid distribution phase followed by a slow elimination phase. The DC curves were similar in intensity, slope, and shape, indicating good ADC stability (mainly the time course). The free exatecan payload was measured in the experimental detected only during the 0-48 h period (10 times higher than the parent ADC component) 6 (2x less concentrated) , suggesting good ADC stability and no premature deconjugation of exatecan.

[0420] Example 15: Pulmonary inflammation and toxicity assessment in mice Bleomycin has been used to study the mechanisms involved in fibrosis and to evaluate potential therapeutics. It is widely used in rodents to model pulmonary fibrosis. To evaluate the s of Bleomycin-Induced Pulmonary Fibrosi s,2008,Curr.Protoc.Pharmacol.40:5.46.1-5 Bleomycin was administered in mice following known procedures such as those described by [46.17]. On days 0, 4, and 8, female C57 / BL6 mice (Janv) were treated with IFN-γ-α-glucan (FcG) and IFN-γ-α-glucan (FcG). Isoir Labs, Le Genest-Saint-Isle, France Lightly anesthetize the animal with flurane and administer 20 μL of 1 mg / kg bleomycin in PBS (bleomycin). Synberon (15 mg for injection) or PBS (negative control mice) were administered intranasally. Omycin-acclimated mice were included as a negative control group. On day 11, mice (n = 6 / group) were treated with the tested conjugates (10 mg / kg ip) and showed no signs of toxicity for the duration of the experiment. Patients were monitored for overt signs and weight loss. A positive control group treated with nhertu® (10 mg / kg ip) was included. However, this induces interstitial lung disease and pneumonitis preclinically and in a small proportion of patients. On day 25, all mice were sacrificed and bronchoalveolar supernatant was obtained (tracheal After bronchial lavage, lung organs were harvested, weighed, and resuspended in 0.1% (w / v) folate in PBS. After fixation, the lungs were fixed in 0.02% (w / v) sodium azide in PBS. The slides were then rinsed in sodium and embedded in paraffin for histological staining. The white blood cells in the lungs were stained with IgG (Abcam, Cat. No. ab10558, 1:500). The level of hemocyte infiltration was quantified using a secondary biotinylated goat anti-rabbit antibody (Vector Labs). oratories, Cat. No. BA-1000, 1:300) and avidin-HRP (Vector Laboratories, Cat. No. A-2004) was used for detection. Histological experiments and imaging were performed at the Centre d'Imagerie Qua ntitative Lyon-Est(CiQLE)platform(Lyon,F rance) and Fiji Software (University of W Laboratory for Optic in isconsin-Madison,US Developed by al and Computational Instrumentation Random sections (30%) of IHC images were imaged using native features (preserving the original image). (zoom) and count CD45-positive cells in the slides (one slide per lung). ), and the level of leukocyte infiltration was assessed. Lymphocytes (CD3 / CD4), eosinophils (CCR3 / SiglecF), and neutrophils in the cell supernatant Monitoring of total numbers of macrophages (F4 / 80) and lysine monophosphate (Ly6g / Ly6C) was performed. The results were analyzed by cytometry. The levels of inflammatory cytokines in the supernatant were quantified by ELISA. IL-5, IL-6, IL-9, IL-10, IL-13, Bio-Plex Pro Mouse Cytokine Th2 Panel (Bio-Rad, Cat. No. L60000 TGF-β and IL-17 were quantified using the UKVT according to the manufacturer's instructions. Mouse TGF-β1 DuoSet and Mouse IL-17 DuoSet EL, respectively ISA kit (R&D systems, catalog numbers DY1679 and DY421) Quantification was performed using the manufacturer's instructions.

[0421] Statistical significance was determined using one-way A regression analysis using GraphPad Prism 9 software. Evaluation was performed using NOVA (Tukey's method). p-values ​​are * (p<0.033), ** (p <0.002), *** (p<0.0002), and **** (p<0.0001) The results are expressed as "n / s" and "ns" indicates non-significant (p>0.123).

[0422] The quantitative results of IHC CD45 are shown in Figure 20. Positive control Enhertu® A DCs were untreated ( *** ) and bleomycin-conditioned ( **** ) compared with both negative control groups When administered, F-VA-EXA ADC caused a significant increase in leukocyte inflammatory infiltration. Compared with the untreated group, it did not cause a significant increase in leukocyte inflammatory infiltration and suppressed bleomycin. Habituation ( *** ) caused a significant increase when compared to the control group. A-EXA significantly reduced the number of white blood cells when compared to both untreated and bleomycin-conditioned negative controls. Based on these preclinical results, F-VA did not cause a significant increase in inflammatory infiltrates. -EXA, especially F-LALA-VA-EXA (Fc silent variant), has been shown to be a potential target in clinical situations. It can be assumed that it induces little or no interstitial lung disease or pneumonitis in .

[0423] The inflammatory cytokine levels in the bronchoalveolar supernatant are shown in Figure 21A. The tu® ADC significantly reduced IL-13, IL-17, and TGF-β levels compared to the untreated group. F-VA-EXA and F-LALA caused a significant increase in the levels of β cytokines. -VA-EXA ADC demonstrated a significant increase in cytokine levels compared to the untreated group did not cause any

[0424] The total numbers of lymphocytes, eosinophils, neutrophils, and macrophages in the bronchoalveolar supernatant are shown in Figure 21B. The positive control, Enhertu®, increased lymphocytes, myocytes, and lymphocytes compared to the untreated control group. F-VA-EXA and VA-EXA caused a significant increase in the number of macrophages, neutrophils, and eosinophils. F-LALA-VA-EXA showed a significant increase in lymphocyte counts compared to the untreated control group. However, there was a statistically significant increase in macrophages, neutrophils, and eosinophils compared to the untreated control group. No addition was shown.

[0425] [Table 11]

[0426] Example 16: Dose-ranging toxicity study in cynomolgus monkeys A non-human primate dose-ranging toxicity study was conducted in purpose-reared wild-caught female cynomolgus monkeys (Macaca fascicularis) of Vietnamese origin. The test was performed using a mouse (Macaca fascicularis). e SAS (Marcy-l'Etoile, France). Study protocol The study was approved by the Animal Care and Use Committee of the study facility. C at dose levels of 30, 40, 50 or 60 mg / kg, at 3-week intervals (total of 3 doses) The drug was administered intravenously (5 mL / kg / h over a 30-minute infusion period). Two female monkeys / Groups were used in the study (total of 8 animals). After terminal sacrifice 5 days after the last administration, gross necropsy was performed. Organ weights and histopathological examinations were performed. Parameters evaluated during the study included mortality, Mortality, clinical signs (including estimates of food consumption), body weight, ophthalmology, temperature, hematology, coagulation, clinical Chemistry, urinalysis, organ weights, and gross and microscopic examination of an extensive list of tissues were included. The ADC formulation buffer was 20 mM histidine pH 6.0, 4% (w / v) sucrose, and and 75 mM NaCl. The vehicle was 0.9% saline.

[0427] The results are shown in Figure 22. F-LALA-VA-EXA ADC was well tolerated and The highest known non-severe toxic dose (HNSTD) was 50 mg / kg, given three times. Administration at 0.05 mg / kg / day was not tolerated due to acute renal failure in one of the two animals. (This is likely target-related, since the folate receptor alpha is endogenously expressed in the kidney.) - The preclinical therapeutic window for LALA-VA-EXA is based on the cynomolgus monkey HNSTD rodent cancer model Because the dose far exceeds the effective therapeutic dose in after aging), appears to be advantageous.

Claims

1. An antibody-drug conjugate of formula (I), Ab-[LD]p(I), During the ceremony, Ab is an anti-folate receptor alpha (FRα) antibody that specifically binds to folate receptor alpha, as indicated by SEQ ID NO:

12. L is a cleavable linker portion bound to the anti-folate receptor alpha (FRα) antibody, D is a topoisomerase I inhibitor bound to L, p is between 1 and 8. Antibody-drug conjugate.

2. The antibody-drug conjugate according to claim 1, wherein L is a hydrophobic masking entity containing polysarcosine.

3. The antibody-drug conjugate according to claim 1, wherein the topoisomerase I inhibitor is selected from the group consisting of exatecan, camptothecin, irinotecan, topotecan, SN-38, DXd, silatecan, cocitecan, rutothecan, gimatecan, berotecan, and rubitecan.

4. The antibody-drug conjugate according to claim 1, wherein the topoisomerase I inhibitor is an indenoisoquinoline analog or an indocarbazole analog.

5. The antibody-drug conjugate according to claim 1, wherein the topoisomerase I inhibitor is exatecan.

6. L is given by the following formula: 【Chemistry 2】 Includes a severable portion W represented by, During the ceremony, Each R ２ These are independently selected from the group consisting of electron-withdrawing groups and C1-C6 alkyl groups. n is 0, 1, or 2. T is a sugar-cleavable unit or a polypeptide-cleavable unit. The antibody-drug conjugate according to claim 1, wherein if T is a sugar-cleavable unit, Y is O, or if T is a polypeptide-cleavable unit, Y is NH.

7. L is given by the following formula (IV): 【Transformation 3】 It is represented as, L is covalently bonded to one or more thiol residues of Ab via X1, During the ceremony, X １ This is per connector, Z is an arbitrary spacer, X ２ This is per connector, K is a hydrophobic masking entity containing polysarcosine, T is a polypeptide cleavable unit. The antibody-drug conjugate according to claim 1.

8. X1 is given by the following formula: 【Chemistry 101】 The antibody-drug conjugate according to claim 7, comprising a structure represented by [formula].

9. The antibody-drug conjugate according to claim 7, wherein X2 comprises an amino acid.

10. The antibody-drug conjugate according to claim 7, wherein X2 comprises an amino acid selected from the group consisting of glutamic acid, aspartic acid, lysine, serine, tyrosine, cysteine, selenocysteine, glycine, and homoalanine.

11. The antibody-drug conjugate according to claim 7, wherein K comprises a polysarcosine containing 2 to 500 sarcosine moieties.

12. The antibody-drug conjugate according to claim 7, wherein K comprises a polysarcosine containing 5 to 25 sarcosine moieties.

13. The antibody-drug conjugate according to claim 7, wherein T is selected from the group consisting of valine-citrulline (Val-Cit), alanine-alanine-asparagine (Ala-Ala-Asn), valine-alanine (Val-Ala), and phenylalanine-lysine (Phe-Lys).

14. The antibody-drug conjugate according to claim 7, wherein p is 4 to 8.

15. The antibody-drug conjugate according to claim 1, wherein Ab is an anti-FRα antibody comprising a variable heavy chain polypeptide comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 3, and a variable light chain polypeptide comprising LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO:

6.

16. The antibody-drug conjugate according to claim 15, wherein Ab is an anti-FRα antibody comprising the heavy chain of SEQ ID NO: 9 and the light chain of SEQ ID NO:

10.

17. The antibody-drug conjugate according to claim 7, wherein Ab is an anti-FRα antibody comprising a variable heavy chain polypeptide comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 3, and a variable light chain polypeptide comprising LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO:

6.

18. The antibody-drug conjugate according to claim 17, wherein Ab is an anti-FRα antibody comprising the heavy chain of SEQ ID NO: 9 and the light chain of SEQ ID NO:

10.

19. The antibody-drug conjugate according to claim 1, wherein Ab is an anti-FRα antibody comprising the heavy chain of SEQ ID NO: 16 and the light chain of SEQ ID NO:

17.

20. The antibody-drug conjugate according to claim 7, wherein Ab is an anti-FRα antibody comprising the heavy chain of SEQ ID NO: 16 and the light chain of SEQ ID NO:

17.

21. A pharmaceutical composition comprising an antibody-drug conjugate according to any one of claims 1 to 20 in combination with one or more pharmaceutically acceptable excipients, diluents, or carriers.

22. A pharmaceutical composition according to claim 21 for use in a method of treating cancer in a subject requiring cancer treatment, A pharmaceutical composition comprising the method of administering the pharmaceutical composition described in claim 21.

23. The pharmaceutical composition according to claim 22, for use in the treatment of cancer in a subject requiring treatment for cancer, wherein the cancer is selected from the group consisting of ovarian cancer, triple-negative breast cancer, non-small cell lung cancer, and mesothelioma.

24. Use of an antibody-drug conjugate according to any one of claims 1 to 20 in the manufacture of a pharmaceutical composition for use in treating cancer in a subject requiring cancer treatment.

25. The use according to claim 24, wherein the cancer is selected from the group consisting of ovarian cancer, triple-negative breast cancer, non-small cell lung cancer, and mesothelioma.

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