Antibody drug conjugates targeting c-Met and methods of use

Abstract

Antibody-drug conjugates (ADCs) are provided that include antibody constructs that specifically bind to c-Met conjugated via a linker to one or more drugs, e.g., compound (1), which is an auristatin analog. Also described are anti-cMet antibody constructs that have been engineered to contain one or more cysteine ​​insertion mutations, with each inserted cysteine ​​residue providing a conjugation "handle" that allows for conjugation of a drug-linker to provide an ADC. Additionally, multivalent drug-linkers comprising multiple auristatin analogs suitable for use in the ADCs described herein are described. TIFF2025531990000170.tif42165

Description

[Technical Field]

[0001] Field The present disclosure relates to the field of immunotherapy, and in particular to antibody-drug conjugates comprising anti-cMet antibodies and auristatin analogs, and their use in therapy. [Background technology]

[0002] background c-Met (also known as hepatocyte growth factor receptor (HGFR)) is a receptor tyrosine kinase encoded by the MET proto-oncogene. c-Met is frequently overexpressed in cancer, and activation of c-Met signaling is associated with drug resistance, as well as the processes of carcinogenesis, invasion, and metastasis (Gherardi et al., 2012, Nat Rev Cancer, 12:89-103). Therefore, disruption of MET signaling is considered a promising approach for cancer therapy. Various therapeutic strategies targeting c-Met are currently being explored, including MET kinase inhibitors, HGF inhibitors, and antibodies and antibody-drug conjugates (ADCs) that bind to c-Met.

[0003] Several ADCs targeting c-Met are in development, including telisotuzumab vedotin (formerly ABBV-399) (AbbVie Inc.), BYON3521 (Byondis BV), RC108 (RemeGen Co., Ltd.), and REGN5093-M114 (Regeneron Pharmaceuticals). Of these, telisotuzumab vedotin, which comprises the anti-cMet antibody telisotuzumab conjugated to monomethyl auristatin E (MMAE) (see U.S. Pat. No. 8,545,839), is the most advanced and is currently in Phase III clinical trials. In a phase II trial, telisotuzumab vedotin was reported to exhibit antitumor activity in cMet-positive non-small cell lung cancer (NSCLC), with 65% of patients reporting grade >3 treatment-emergent adverse events (Camidge, et al., 2021, Clin Cancer Res, 27(21):5781-5792 (Non-Patent Document 2)).

[0004] This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present disclosure. No admission is necessarily intended, nor should it be construed, that any of the preceding information constitutes prior art against the claimed invention. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] U.S. Patent No. 8,545,839 [Non-patent literature]

[0006] [Non-Patent Document 1] Gherardi et al,2012,Nat Rev Cancer,12:89-103 [Non-patent document 2] Camidge,et al.,2021,Clin Cancer Res,27(21):5781-5792 Summary of the Invention

[0007] overview Described herein are antibody-drug conjugates (ADCs) that target c-Met and methods of using these ADCs in therapy. One aspect of the disclosure is a compound represented by Formula I: A-(L-(D) n ) p (I) In another aspect, the invention relates to an antibody-drug conjugate having the formula: A is an antibody construct comprising an antigen-binding domain and an immunoglobulin (Ig) hinge region, wherein the antigen-binding domain specifically binds to c-Met and comprises heavy chain CDR sequences (HCDR1, HCDR2, and HCDR3) of a VH domain sequence set forth in SEQ ID NO: 1 and light chain CDR sequences (LCDR1, LCDR2, and LCDR3) of a VL domain sequence set forth in SEQ ID NO: 2, and the Ig hinge region comprises an upper hinge sequence having the amino acid sequence of a native IgG1, IgG2, or IgG4 upper hinge sequence; L is a cleavable linker; D is, TIFF2025531990000002.tif37165, where * is the point of attachment to L, n is 1 to 4, p is 1 to 8.

[0008] In certain embodiments, the antibody-drug conjugate has the structure: TIFF2025531990000003.tif59165 (wherein A is an antibody construct that specifically binds to c-Met and p is 6), or TIFF2025531990000004.tif79165 (in the structure, A is an antibody construct that specifically binds to c-Met, and p is 2) It has.

[0009] Another aspect of the present disclosure is a method for manufacturing a semiconductor device comprising: an antigen-binding domain comprising a VL domain and a VH domain, and optionally a CH1 domain and a CL domain, which specifically binds to c-Met; an Fc region comprising a CH2 domain having two CH2 domain sequences and a CH3 domain having two CH3 domain sequences; An antibody construct comprising: the antigen-binding domain comprises heavy chain CDR sequences (HCDR1, HCDR2, and HCDR3) of the VH domain sequence set forth in SEQ ID NO: 1, and light chain CDR sequences (LCDR1, LCDR2, and LCDR3) of the VL domain sequence set forth in SEQ ID NO: 2; the antibody construct comprising: (a) a cysteine ​​residue inserted between positions 40 and 41 in the VL domain; (b) a cysteine ​​residue inserted between positions 126 and 127 in the CL domain; (c) a cysteine ​​residue inserted between positions 9 and 10 in the VH domain; (d) a cysteine ​​residue inserted between positions 237 and 238 in the CH2 domain sequence; and (e) A cysteine ​​residue inserted between positions 299 and 300 in the CH2 domain sequence. wherein the numbering of amino acids in the VL domain, CL domain, and VH domain is Kabat numbering, and the numbering of amino acids in the CH2 domain is EU numbering.

[0010] Another aspect of the present disclosure relates to the use of an antibody construct comprising one or more cysteine ​​insertion mutations described herein for the preparation of an antibody drug conjugate.

[0011] Another aspect of the present disclosure relates to an antibody-drug conjugate comprising an antibody construct having one or more cysteine ​​insertion mutations described herein conjugated via a linker to a cytotoxin.

[0012] In certain embodiments, the antibody-drug conjugate, comprising an antibody construct with one or more cysteine ​​insertion mutations conjugated to a cytotoxin via a linker, has the formula I: A-(L-(D) n ) p (I) wherein A is an antibody construct; L is a cleavable linker; D is, TIFF2025531990000005.tif37165, where: * is the point of attachment to L, n is 1 to 4, p is 1 to 8; Each L is conjugated to the sulfhydryl group of an inserted cysteine ​​residue.

[0013] Another aspect of the present disclosure is a compound having the structure: TIFF2025531990000006.tif79165, wherein p is 2 and A is an antibody construct an antigen-binding domain comprising a VL domain and a VH domain, and optionally a CH1 domain and a CL domain, wherein the antigen-binding domain specifically binds to c-Met; an Fc region comprising a CH2 domain having two CH2 domain sequences and a CH3 domain having two CH3 domain sequences; the antigen-binding domain comprises heavy chain CDR sequences (HCDR1, HCDR2, and HCDR3) of the VH domain sequence set forth in SEQ ID NO: 1, and light chain CDR sequences (LCDR1, LCDR2, and LCDR3) of the VL domain sequence set forth in SEQ ID NO: 2; the antibody construct comprises a cysteine ​​residue inserted between positions 299 and 300 in each CH2 domain sequence; The antibody construct is a bivalent antibody comprising two antigen-binding domains, each of which specifically binds to c-Met.

[0014] Another aspect of the present disclosure pertains to a polynucleotide or set of polynucleotides that encodes an antibody construct comprising one or more cysteine ​​insertion mutations described herein.

[0015] Another aspect of the present disclosure pertains to a vector or set of vectors comprising a polynucleotide or set of polynucleotides encoding an antibody construct comprising one or more cysteine ​​insertion mutations described herein.

[0016] Another aspect of the present disclosure relates to a host cell comprising a vector or set of vectors comprising a polynucleotide or set of polynucleotides encoding an antibody construct comprising one or more cysteine ​​insertion mutations described herein.

[0017] Another aspect of the present disclosure relates to a pharmaceutical composition comprising an antibody-drug conjugate described herein and a pharmaceutically acceptable carrier or diluent.

[0018] Another aspect of the present disclosure relates to a method of treating cancer in a subject, the method comprising administering to the subject an effective amount of an antibody-drug conjugate described herein.

[0019] Another aspect of the present disclosure pertains to an antibody-drug conjugate described herein for use in therapy, e.g., the treatment of cancer in a subject in need thereof.

[0020] Another aspect of the present disclosure relates to the use of an antibody-drug conjugate described herein in the manufacture of a medicament for the treatment of cancer.

[0021] Another aspect of the present disclosure is a method for manufacturing a semiconductor device comprising: TIFF2025531990000007.tif241165TIFF2025531990000008.tif173165. [Brief explanation of the drawings]

[0022] [Figure 1] An alignment of the hinge sequences of human IgG1 (SEQ ID NO: 24), IgG2 (SEQ ID NO: 26), and IgG4 (SEQ ID NO: 28) is presented. The upper, middle, and lower hinge regions are marked. [Figure 2A] SDS-PAGE results (M = molecular weight marker) for a representative anti-cMet cysteine ​​insertion variant v29001 under non-reducing (NR) and reducing (R) conditions are presented. [Figure 2B] A UPLC-SEC chromatogram for a representative anti-cMet cysteine ​​insertion variant v29001 is presented. [Figure 2C] A zoomed-in view of the peak at 7.827 min shown in Figure 2B is presented. [Figure 3-1] Exemplary reaction routes are presented for the preparation of trivalent drug-linkers, Drug-Linker 007 and Drug-Linker 008. [Figure 3-2] See description of Figure 3-1. [Figure 4] Figure 1 shows the effects of cMet pathway activation on the proliferation of H596 lung cancer cells treated with anti-cMet antibodies (v17429, v17606, and v17427) and ADCs (v17427-drug linker 001 and v17427-MCvcPABC-MMAE). Data are shown as the mean (±SEM) of three independent replicate experiments. [Figure 5] Figure 1 shows the results of assessing activation of the cMet pathway by measuring AKT phosphorylation by ELISA in (A) H596 lung cancer cells and (B) H441 lung cancer cells treated with anti-cMet antibodies (v17429, v17606, and v17427) and ADCs (v17427-drug linker 001 and v17427-MCvcPABC-MMAE). Data are shown as the mean (±SEM) of three independent replicate experiments. [Figure 6]1 presents the results of assessing the effect of cMet pathway activation by proliferation of H596 lung cancer cells treated with anti-cMet antibodies (v17429 and v17427) and ADCs containing various anti-cMet cysteine ​​insertion variants conjugated at DAR2 to drug-linker 001. Data are shown as the mean (±SEM) of four independent replicate experiments. [Figure 7A] 1 presents the results of assessing activation of the cMet pathway by measuring AKT phosphorylation by ELISA for H441 lung cancer cells treated with ADCs containing anti-cMet antibodies (v17429, v17606, and v17427), cysteine-conjugated v17427-drug-linker 001 at DAR 4, lysine-conjugated v17427-drug-linker 002 at DAR 2, and an anti-cMet antibody site-specifically conjugated to drug-linker 001 at DAR 1. Data are shown as the mean (±SEM) of two independent replicate experiments. [Figure 7B] 1 presents the results of assessing activation of the cMet pathway by measuring AKT phosphorylation by ELISA for H441 lung cancer cells treated with ADCs comprising anti-cMet antibodies (v17429, v17606, and v17427), cysteine-conjugated v17427-drug-linker 001 at DAR 4, lysine-conjugated v17427-drug-linker 002 at DAR 2, and anti-cMet antibodies site-specifically conjugated to drug-linker 001 at DAR 2. Data are shown as the mean (±SEM) of two independent replicate experiments. [Figure 7C] 1 presents the results of assessing activation of the cMet pathway by measuring AKT phosphorylation by ELISA for H441 lung cancer cells treated with ADCs containing anti-cMet antibodies (v17429, v17606, and v17427), cysteine-conjugated v17427-drug-linker 001 at DAR 4, lysine-conjugated v17427-drug-linker 002 at DAR 2, and anti-cMet antibodies site-specifically conjugated to drug-linker 001 at DAR 3. Data are shown as the mean (±SEM) of two independent replicate experiments. [Figure 8A] 1 presents the results of an in vivo evaluation of the antitumor activity of ADCs comprising an anti-cMet antibody conjugated with DAR4 to drug-linker 001 or MCvcPABC-MMAE in a cMet-high HCC827 lung cancer model. [Figure 8B] 1 presents the results of an in vivo evaluation of the antitumor activity of ADCs comprising anti-cMet antibodies conjugated with DAR4 to drug-linker 001 or MCvcPABC-MMAE in a cMet-high EBC1 lung cancer model. [Figure 8C] 1 presents the results of an in vivo evaluation of the antitumor activity of ADCs comprising an anti-cMet antibody conjugated with DAR4 to drug-linker 001 or MCvcPABC-MMAE in a cMet-high H1975 lung cancer model. [Figure 8D] 1 presents the results of an in vivo evaluation of the antitumor activity of ADCs comprising anti-cMet antibodies conjugated with DAR4 to drug-linker 001 or MCvcPABC-MMAE in a cMet-medium / high HT29 colon cancer model. [Figure 8E] 1 presents the results of an in vivo evaluation of the antitumor activity of ADCs comprising an anti-cMet antibody conjugated with DAR4 to drug-linker 001 or MCvcPABC-MMAE in a cMet-low H292 lung cancer model. [Figure 8F] 1 presents the results of an in vivo evaluation of the antitumor activity of ADCs comprising anti-cMet antibodies conjugated with DAR4 to drug-linker 001 or MCvcPABC-MMAE in a cMet-low SW48 colon cancer model. [Figure 9] 1 presents the results of in vivo evaluation of the antitumor activity of ADCs comprising an anti-cMet antibody conjugated to drug-linker 001 at DAR 1, 2, 3, or 4, or conjugated to drug-linker 002 at DAR 2, at (A) toxin match doses of 24, 12, 8, and 6 mg / kg, and (B) toxin match doses of 4, 2, 1.3, and 1 mg / kg in a cMet-high H1975 lung cancer model. [Figure 10]1 presents the results of in vivo evaluation of the antitumor activity of ADCs comprising an anti-cMet antibody conjugated to drug-linker 001 at DAR 1, 2, 3, or 4, or conjugated to drug-linker 002 at DAR 2, at (A) toxin match doses of 12, 6, 4, and 3 mg / kg, and (B) toxin match doses of 6, 3, 2, and 1.5 mg / kg in a cMet-medium / high HT29 colon cancer model. [Figure 11A] 1 presents the results of an in vivo evaluation of the anti-tumor activity of ADCs comprising anti-cMet antibodies conjugated to Drug-Linker 002, Drug-Linker 003, Drug-Linker 004, or MCvcPABC-MMAE at various DARs and doses (as indicated) in a cMet-high H1975 lung cancer model. [Figure 11B] 1 presents the results of an in vivo evaluation of the antitumor activity of ADCs comprising anti-cMet antibodies conjugated to Drug-Linker 002, Drug-Linker 003, Drug-Linker 004, or MCvcPABC-MMAE at various DARs and doses (as indicated) in a cMet-medium / high HT29 colon cancer model. [Figure 11C] 1 presents the results of an in vivo evaluation of the anti-tumor activity of ADCs comprising anti-cMet antibodies conjugated to Drug-Linker 002, Drug-Linker 003, Drug-Linker 004, or MCvcPABC-MMAE at various DARs and doses (as indicated) in a cMet-low H292 lung cancer model. [Figure 11D] 1 presents the results of an in vivo evaluation of the antitumor activity of ADCs comprising anti-cMet antibodies conjugated to Drug-Linker 002, Drug-Linker 003, Drug-Linker 004, or MCvcPABC-MMAE at various DARs and doses (as indicated) in a cMet-medium / high Hs746t gastric cancer model. [Figure 11E]1 presents the results of an in vivo evaluation of the antitumor activity of ADCs comprising anti-cMet antibodies conjugated to Drug-Linker 002, Drug-Linker 003, Drug-Linker 004, or MCvcPABC-MMAE at various DARs and doses (as indicated) in a cMet HCT116 colon cancer model. [Figure 12] 1 presents the results of in vivo evaluation of the antitumor activity at the indicated doses for ADCs comprising an anti-cMet antibody conjugated to Drug-Linker 003 at DAR 4, Drug-Linker 004 at DAR 6, or MCvcPABC-MMAE at DAR 3 in various PDX models. *v29001-Drug-Linker 004 DAR 6 model was not tested. [Figure 13] Pharmacokinetics in Tg32 mice: (A) serum total IgG concentration over time; and (B) serum total ADC concentration over time are presented for ADCs comprising anti-cMet antibodies conjugated to Drug-Linker 002 at DAR 4 or DAR 6, Drug-Linker 003 at DAR 4, or Drug-Linker 004 at DAR 6, and the corresponding free antibodies. [Figure 14] 1 presents results from an evaluation of the in vivo stability of ADCs comprising an anti-cMet antibody conjugated to Drug-Linker 002 at a DAR of 4 or 6, Drug-Linker 003 at a DAR of 4, or Drug-Linker 004 at a DAR of 6, as assessed by (A) % DAR remaining (Drug-Linker 002 ADC), and (B) % thiosuccinimide ring-opening (RO) and % DAR remaining (Drug-Linker 003 ADC and Drug-Linker 004 ADC). DETAILED DESCRIPTION OF THE INVENTION

[0023] Detailed Description The present disclosure relates to antibody-drug conjugates (ADCs) comprising an antibody construct that specifically binds c-Met (an "anti-cMet antibody construct") conjugated via a linker to a drug, such as a cytotoxin. In an ADC, the anti-cMet antibody construct may be conjugated to one drug molecule, or it may be conjugated to two or more drug molecules.

[0024] Certain embodiments of the present disclosure relate to ADCs comprising an anti-cMet antibody construct conjugated via a linker to the auristatin analog Compound 1. TIFF2025531990000009.tif42165

[0025] In such embodiments, the anti-cMet antibody construct may be conjugated to one of the auristatin analogs, or it may be conjugated to two or more of the auristatin analogs.

[0026] The present disclosure also relates to anti-cMet antibody constructs engineered to contain one or more cysteine ​​insertion mutations. Each inserted cysteine ​​residue provides a conjugation "handle" that allows for drug-linker conjugation to provide an ADC. Certain embodiments of the present disclosure relate to ADCs comprising anti-cMet antibody constructs engineered to contain one or more cysteine ​​insertion mutations and conjugated to one or more drug molecules via the inserted cysteine(s).

[0027] The present disclosure further relates to multivalent drug-linkers comprising multiple auristatin analogs suitable for use in the ADCs described herein. Certain embodiments of the present disclosure relate to ADCs comprising an anti-cMet antibody construct conjugated to a multivalent drug-linker comprising multiple auristatin analogs.

[0028] The ADCs of the present disclosure can be used as therapeutic agents, for example, for the treatment of cancer.

[0029] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0030] As used herein, the term "about" refers to approximately a + / - 10% variation from a given value. It is to be understood that such a variation is always included in any given value provided herein, whether or not it is specifically referred to.

[0031] The use of the words "a" or "an," when used herein in conjunction with the term "comprising," can mean "one," but is also consistent with the meanings of "one or more," "at least one," and "one or more than one."

[0032] As used herein, the terms "comprising," "having," "including," and "containing," and grammatical variations thereof, are inclusive, i.e., open-ended, and do not exclude additional, unrecited elements and / or method steps. When used herein in connection with a composition, use, or method, the term "consisting essentially of" means that additional elements and / or method steps may be present, but that these additions do not materially affect the manner in which the recited composition, method, or use functions. When used herein in connection with a composition, use, or method, the term "consisting of" excludes the presence of additional elements and / or method steps. A composition, use, or method described herein as including certain elements and / or steps may also consist essentially of those elements and / or steps in certain embodiments, and may consist of those elements and / or steps in other embodiments, whether or not those embodiments are specifically referred to.

[0033] "Complementarity-determining regions" or "CDRs" are amino acid sequences that contribute to antigen-binding specificity and affinity. "Framework" regions (FRs) may help maintain the proper conformation of the CDRs to promote binding between the antigen-binding region and the antigen. From the N-terminus to the C-terminus, both the light chain variable region (VL) and heavy chain variable region (VH) of an antibody typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The three heavy chain CDRs are referred to herein as HCDR1, HCDR2, and HCDR3, and the three light chain CDRs are referred to herein as LCDR1, LCDR2, and LCDR3. CDRs provide the majority of contact residues for antibody binding to an antigen or epitope. In many cases, three heavy chain CDRs and three light chain CDRs are required for antigen binding. However, in some cases, even a single variable domain can confer antigen-binding specificity. Furthermore, as is known in the art, in some cases antigen binding may also occur through a combination of one or more CDRs, e.g., HCDR3s, selected from the VH and / or VL domains.

[0034] Several different definitions of CDR sequences are commonly used, including those described by Kabat et al. (1983, Sequences of Proteins of Immunological Interest, NIH Publication No. 369-847, Bethesda, MD), Chothia et al. (1987, J Mol Biol, 196:901-917), and IMGT, AbM (University of Bath), and Contact (MacCallum, et al., 1996, J Mol Biol, 262(5):732-745). By way of example, the definitions of CDRs according to Kabat, Chothia, IMGT, AbM, and Contact are provided in Table 1 below. Thus, as will be readily apparent to one skilled in the art, the exact numbering and arrangement of CDRs may vary based on the numbering system used. However, it should be understood that disclosure of a VH herein includes disclosure of the associated (unique) heavy chain CDRs (HCDRs), as defined by any of the known numbering systems. Similarly, disclosure of a VL herein includes disclosure of the associated (unique) light chain CDRs (LCDRs), as defined by any of the known numbering systems.

[0035] [Table 1]

[0036] As used herein, the terms "subject" and "patient" refer to an animal, and in some embodiments, a mammal, that is the object of treatment, observation, or experiment. The animal may be a human, a non-human primate, a companion animal (e.g., dog, cat, etc.), a livestock animal (e.g., cow, sheep, pig, horse, etc.), or a laboratory animal (e.g., rat, mouse, guinea pig, non-human primate, etc.). In certain embodiments, the subject is a human.

[0037] It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method, use, or composition disclosed herein, and vice versa.

[0038] Particular features, structures, and / or characteristics described in connection with an embodiment disclosed herein may be combined in any suitable manner with features, structures, and / or characteristics described in connection with other embodiments disclosed herein to provide one or more additional embodiments.

[0039] It should also be understood that the affirmative recitation of a feature in one embodiment serves as a basis for the exclusion of that feature in an alternative embodiment. For example, where a list of alternatives is presented for a given embodiment or claim, it should be understood that one or more alternatives may be deleted from the list, and the shortened list may form an alternative embodiment, whether or not such alternative embodiment is specifically mentioned.

[0040] Antibody-drug conjugates Certain embodiments of the present disclosure are directed to compounds of formula I: TIFF2025531990000011.tif13128, wherein: A is an antibody construct that specifically binds to c-Met; L is a linker, D is the structure: TIFF2025531990000012.tif37165, in which: * is the point of attachment to L, n is 1 to 4, p is 1 to 8.

[0041] The linker L can be monovalent (attaching a single D, where n=1), or it can be multivalent (attaching multiple D, where n=2, 3, or 4).

[0042] In Formula I above, the parameters n and p define the number of auristatin analog molecules D conjugated to the antibody construct A. More specifically, the product of n x p defines the drug-to-antibody ratio, or "DAR," for the ADC. One of skill in the art will appreciate that a given DAR can be achieved by various combinations of n and p. For example, an ADC with a DAR of 4 may comprise an antibody construct conjugated to four drug-linkers, each containing a single D (i.e., n = 1 and p = 4), or an antibody construct conjugated to two drug-linkers, each containing two Ds (i.e., n = 2 and p = 2). Similarly, in another example, an ADC with a DAR of 6 may comprise an antibody construct conjugated to six drug-linkers, each drug-linker comprising a single D (i.e., n=1 and p=6), or an antibody construct conjugated to three drug-linkers, each drug-linker comprising two Ds (i.e., n=2 and p=3), or an antibody construct conjugated to two drug-linkers, each drug-linker comprising three Ds (i.e., n=3 and p=2).

[0043] Those skilled in the art will also understand that within an ADC preparation comprising multiple ADCs, although each anti-cMet antibody construct A is conjugated to an integer number of auristatin analogs D, the DAR determined for the ADC preparation may reflect a statistical average of the individual DARs for the multiple ADCs comprised by the preparation, resulting in a non-integer result. Thus, ADC preparations with both integer and non-integer DARs are intended to be encompassed by Formula I.

[0044] In some embodiments, the ADC of Formula I has a DAR of about 1 to about 6. In some embodiments, the ADC of Formula I has a DAR of about 2 to about 6. In some embodiments, the ADC of Formula I has a DAR of about 4 to about 6.

[0045] In some embodiments, in the ADC of Formula I, n is 1 and p is 4, and a preparation of the ADC has a DAR of about 4. In some embodiments, in the ADC of Formula I, n is 2 and p is 2, and a preparation of the ADC has a DAR of about 4. In some embodiments, in the ADC of Formula I, n is 3 and p is 2, and a preparation of the ADC has a DAR of about 6. In some embodiments, in the ADC of Formula I, n is 1 and p is 6, and a preparation of the ADC has a DAR of about 6.

[0046] Anti-cMet antibody construct The ADCs of the present disclosure include anti-cMet antibody constructs. In this context, the term "antibody construct" refers to a polypeptide or set of polypeptides comprising one or more antigen-binding domains, each of which specifically binds to an epitope or antigen. When an antibody construct comprises two or more antigen-binding domains, each of the antigen-binding domains may bind to the same epitope or antigen (i.e., the antibody construct is monospecific), or they may bind to different epitopes or antigens (i.e., the antibody construct is bispecific or multispecific). According to the present disclosure, an anti-cMet antibody construct comprises at least one antigen-binding domain that specifically binds to c-Met. In certain embodiments, the anti-cMet antibody construct may further comprise a scaffold, and at least one of the one or more antigen-binding domains may be fused or covalently attached to the scaffold, optionally via a linker.

[0047] In certain embodiments, an anti-cMet antibody construct comprises two antigen-binding domains, each of which specifically binds to c-Met. In some embodiments, an anti-cMet antibody construct comprises two antigen-binding domains (each of which specifically binds to c-Met) and a scaffold. In some embodiments, an anti-cMet antibody construct may comprise three or four antigen-binding domains and a scaffold. In these formats, at least the first antigen-binding domain is operably linked to the scaffold, and the remaining antigen-binding domain(s) may each independently be operably linked to the scaffold, or to the first antigen-binding domain, or to another antigen-binding domain if more than two antigen-binding domains are present.

[0048] In certain embodiments, the anti-cMet antibody construct may be in an immunoglobulin (Ig)-based antibody format. In certain embodiments, the anti-cMet antibody construct may be based on an IgG class immunoglobulin, e.g., an IgG1, IgG2, IgG3, or IgG4 immunoglobulin. In some embodiments, the anti-cMet antibody construct may be based on an IgG1 immunoglobulin. In the context of the present disclosure, when an anti-cMet antibody construct is based on a specified immunoglobulin isotype, it means that the anti-cMet antibody construct comprises all or a portion of the constant region of the specified immunoglobulin isotype. For example, an anti-cMet antibody construct based on a given Ig isotype may comprise at least one antigen-binding domain operably linked to an Ig scaffold, the scaffold comprising an Fc region from the given isotype and, optionally, an Ig hinge region from the same or a different isotype. It should be understood that the anti-cMet antibody construct may also comprise isotype and / or subclass hybrids in some embodiments. It is also understood that the Fc region and / or hinge region may optionally be modified to impart one or more desirable functional properties as known in the art.

[0049] In some embodiments, the anti-cMet antibody construct may be derived from two or more immunoglobulins from different species, e.g., the anti-cMet antibody construct may be a chimeric antibody or a humanized antibody. The terms "chimeric antibody" and "humanized antibody" both generally refer to antibodies that combine immunoglobulin regions or domains from two or more species.

[0050] A "chimeric antibody" typically comprises at least one variable domain from a non-human antibody, such as a rabbit or rodent (e.g., murine) antibody, and at least one constant domain from a human antibody. The human constant domain of a chimeric antibody need not be of the same isotype as the non-human constant domain it replaces. Chimeric antibodies are discussed, for example, in Morrison et al., 1984, Proc. Natl. Acad. Sci. USA, 81:6851-55, and U.S. Pat. No. 4,816,567.

[0051] A "humanized antibody" is a type of chimeric antibody that contains minimal sequence derived from a non-human antibody. Generally, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region or CDR of the recipient are replaced by residues from a hypervariable region or CDR of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity and affinity for the target antigen. This technique for producing humanized antibodies is often referred to as "CDR grafting."

[0052] In some cases, additional modifications are made to further refine antibody performance. For example, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues, or humanized antibodies may comprise residues that are not found in either the recipient antibody or the donor antibody. Generally, the variable domains in a humanized antibody will contain all or substantially all of the hypervariable regions or CDRs from the non-human immunoglobulin and all or substantially all of the FRs from the human immunoglobulin sequence. Humanized antibodies are further described in, for example, Jones, et al., 1986, Nature, 321:522-525; Riechmann, et al., 1988, Nature, 332:323-329; and Presta, 1992, Curr. Op. Struct. Biol., 2:593-596.

[0053] Several approaches for selecting the most appropriate human framework for grafting nonhuman CDRs are known in the art. Early approaches used a limited subset of well-characterized human antibodies, regardless of their sequence identity with the nonhuman antibody providing the CDRs (the "fixed framework" approach). More recent approaches use variable regions with high amino acid sequence identity with the variable regions of the nonhuman antibody providing the CDRs (the "homology matching" or "best-fit" approach). An alternative approach is to select fragments of framework sequences within each light or heavy chain variable region from several different human antibodies. CDR grafting can, in some cases, partially or completely lose the affinity of the grafted molecule for its target antigen. In such cases, affinity can be restored by backmutating some of the human-derived residues to the corresponding nonhuman-derived residues. Methods for preparing humanized antibodies by these approaches are well known in the art (see, for example, Tsurushita & Vasquez, 2004, Humanization of Monoclonal Antibodies, Molecular Biology of B Cells, 533-545, Elsevier Science (USA); Jones et al., 1986, Nature, 321:522-525; Riechmann et al., 1988, Nature, 332:323-329; Presta et al., 1997, Cancer Res, 57(20):4593-4599).

[0054] Alternatively or in addition to these traditional approaches, more recent techniques may be used to further reduce the immunogenicity of CDR-grafted humanized antibodies. For example, a framework based on a human germline sequence or consensus sequence may be used as the acceptor human framework, rather than a human framework with somatic mutation(s). Another technique aimed at reducing the potential immunogenicity of non-human CDRs is to graft only specificity-determining residues (SDRs). In this approach, only the minimum CDR residues ("SDRs") required for antigen-binding activity are grafted onto a human germline framework. This method can help improve the "humanness" of humanized antibodies (i.e., similarity to human germline sequences), thus reducing the risk of immunogenicity of the variable regions. These techniques are described in various publications (see, e.g., Almagro & Fransson, 2008, Front Biosci, 13:1619-1633; Tan, et al., 2002, J Immunol, 169:1119-1125; Hwang, et al., 2005, Methods, 36:35-42; Pelat, et al., 2008, J Mol Biol, 384:1400-1407; Tamura, et al., 2000, J Immunol, 164:1432-1441; Gonzales, et al., 2004, Mol Immunol, 1:863-872; and Kashmiri, et al., 2005, Methods, 36:25-34).

[0055] In certain embodiments, an anti-cMet antibody construct comprises an antigen-binding domain that specifically binds to c-Met and an immunoglobulin (Ig) hinge region. In some embodiments, an antibody construct comprises two antigen-binding domains, each of which specifically binds to c-Met, and an Ig hinge region. In some embodiments, an anti-cMet antibody construct comprises at least one antigen-binding domain that specifically binds to c-Met, an Ig hinge region, and a scaffold.

[0056] In certain embodiments, an anti-cMet antibody construct comprises an antigen-binding domain that specifically binds c-Met, an Ig hinge region, and a scaffold that is an Fc region, hi some embodiments, the antibody construct comprises two antigen-binding domains, each of which specifically binds c-Met, an Ig hinge region, and an Fc region.

[0057] In certain embodiments, the anti-cMet antibody construct is an antibody or an antigen-binding antibody fragment. In some embodiments, the anti-cMet antibody construct is a bivalent antibody. In some embodiments, the anti-cMet antibody is a monospecific or bispecific antibody. In some embodiments, the anti-cMet antibody is a monospecific antibody. In some embodiments, the anti-cMet antibody is a bivalent monospecific antibody.

[0058] antigen-binding domain The anti-cMet antibody constructs of the present disclosure comprise at least one antigen-binding domain that specifically binds to c-Met. "Specifically binds" to c-Met means that the antibody construct binds to c-Met and does not exhibit significant binding to non-c-Met proteins. In certain embodiments, at least one antigen-binding domain that specifically binds to c-Met is capable of binding to human c-Met. In some embodiments, at least one antigen-binding domain that specifically binds to c-Met is capable of binding to human c-Met and cynomolgus c-Met. In some embodiments, at least one antigen-binding domain that specifically binds to c-Met is capable of binding to human c-Met and cynomolgus c-Met and does not exhibit significant binding to c-Met from other species.

[0059] The at least one antigen-binding domain may be an immunoglobulin-based antigen-binding domain, such as an antigen-binding antibody fragment, including, but not limited to, a Fab fragment, a Fab' fragment, a single-chain Fab (scFab), a single-chain Fv (scFv), and a single-domain antibody (sdAb).

[0060] A "Fab fragment" contains the light and heavy chain variable domains (VL and VH, respectively), as well as the light chain constant domain (CL) and the first heavy chain constant domain (CH1). Fab' fragments differ from Fab fragments by the addition of a few amino acid residues at the C-terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab fragments may also be single-chain Fab molecules, i.e., Fab molecules in which the Fab light chain and Fab heavy chain are connected by a peptide linker to form a single peptide chain. For example, in a single-chain Fab molecule, the C-terminus of the Fab light chain may be connected to the N-terminus of the Fab heavy chain.

[0061] An "scFv" comprises an antibody heavy chain variable domain (VH) and light chain variable domain (VL) in a single polypeptide chain. An scFv may optionally further comprise a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding. For example, an scFv may comprise a VL connected from the C-terminus of the VL to the N-terminus of the VH by a polypeptide linker. Alternatively, an scFv may comprise a VH connected via the C-terminus of the VH to the N-terminus of the VL by a polypeptide linker (see review in Plückthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994)).

[0062] The "sdAb" format refers to a single immunoglobulin domain. sdAbs may, for example, be of camelid origin. Camelid antibodies lack light chains and their antigen-binding site consists of a single domain, termed "VHH". sdAbs comprise three CDR / hypervariable loops that form the antigen-binding site: CDR1, CDR2, and CDR3. sdAbs are fairly stable and easy to express, for example as fusions with the Fc of an antibody (see, for example, Harmsen & De Haard, 2007, Appl. Microbiol Biotechnol., 77(1):13-22).

[0063] In embodiments in which the anti-cMet antibody construct comprises two or more antigen-binding domains, each additional antigen-binding domain may independently be an immunoglobulin-based antigen-binding domain, such as an antigen-binding antibody fragment, or a non-immunoglobulin-based antigen-binding domain, such as a non-immunoglobulin-based antibody mimetic, or other polypeptide or small molecule capable of specifically binding to its target, e.g., a natural or engineered ligand. Non-immunoglobulin-based antibody mimetic formats include, for example, anticalins, finomers, affimers, alphabodies, DARPins, and avimers. The additional antigen-binding domains may bind to the same epitope within c-Met, different epitopes within c-Met, or different antigens.

[0064] In certain embodiments, at least one antigen-binding domain that specifically binds c-Met comprised by an anti-cMet antibody construct comprises heavy chain CDR sequences (HCDR1, HCDR2, and HCDR3) of the VH domain sequence set forth in SEQ ID NO: 1 and light chain CDR sequences (LCDR1, LCDR2, and LCDR3) of the VL domain sequence set forth in SEQ ID NO: 2 (see Table 2).

[0065] In certain embodiments, at least one antigen-binding domain that specifically binds to c-Met comprised by an anti-cMet antibody construct comprises an HCDR1 sequence selected from the sequences set forth in SEQ ID NOs: 3, 9, 14, 16, and 22, an HCDR2 sequence selected from the sequences set forth in SEQ ID NOs: 4, 10, 15, 17, and 23, and an HCDR3 sequence selected from the sequences set forth in SEQ ID NOs: 5, 11, and 18 (see Table 2). In certain embodiments, at least one antigen-binding domain that specifically binds to c-Met comprised by an anti-cMet antibody construct comprises an LCDR1 sequence selected from the sequences set forth in SEQ ID NOs: 6, 12, and 19, an LCDR2 sequence selected from the sequences set forth in SEQ ID NOs: 7, 13, and 20, and an LCDR3 sequence selected from the sequences set forth in SEQ ID NOs: 8 and 21 (see Table 2).

[0066] In certain embodiments, at least one antigen-binding domain that specifically binds to c-Met comprised by the anti-cMet antibody construct comprises an HCDR1 sequence selected from the sequences set forth in SEQ ID NOs: 3, 9, 14, 16, and 22; an HCDR2 sequence selected from the sequences set forth in SEQ ID NOs: 4, 10, 15, 17, and 23; an HCDR3 sequence selected from the sequences set forth in SEQ ID NOs: 5, 11, and 18; an LCDR1 sequence selected from the sequences set forth in SEQ ID NOs: 6, 12, and 19; an LCDR2 sequence selected from the sequences set forth in SEQ ID NOs: 7, 13, and 20; and an LCDR3 sequence selected from the sequences set forth in SEQ ID NOs: 8 and 21 (see Table 2).

[0067] In certain embodiments, at least one antigen-binding domain that specifically binds to c-Met comprised by the anti-cMet antibody construct comprises heavy chain CDR (HCDR1, HCDR2, and HCDR3) sequences set forth in SEQ ID NOs: 3, 4, and 5, respectively, and light chain CDR (LCDR1, LCDR2, and LCDR3) sequences set forth in SEQ ID NOs: 6, 7, and 8, respectively (see Table 2).

[0068] [Table 2] TIFF2025531990000014.tif35165

[0069] In certain embodiments, an anti-cMet antibody construct comprises two antigen-binding domains, each of which specifically binds to c-Met. In some embodiments, each of the two antigen-binding domains comprised by the anti-cMet antibody construct that specifically bind to c-Met comprises a heavy chain CDR sequence (HCDR1, HCDR2, and HCDR3) of the VH domain sequence set forth in SEQ ID NO: 1 and a light chain CDR sequence (LCDR1, LCDR2, and LCDR3) of the VL domain sequence set forth in SEQ ID NO: 2 (see Table 2).

[0070] In certain embodiments, an anti-cMet antibody construct comprises two antigen-binding domains, each of which comprises an HCDR1 sequence selected from the sequences set forth in SEQ ID NOs: 3, 9, 14, 16, and 22, an HCDR2 sequence selected from the sequences set forth in SEQ ID NOs: 4, 10, 15, 17, and 23, and an HCDR3 sequence selected from the sequences set forth in SEQ ID NOs: 5, 11, and 18 (see Table 2). In certain embodiments, an anti-cMet antibody construct comprises two antigen-binding domains, each of which comprises an LCDR1 sequence selected from the sequences set forth in SEQ ID NOs: 6, 12, and 19, an LCDR2 sequence selected from the sequences set forth in SEQ ID NOs: 7, 13, and 20, and an LCDR3 sequence selected from the sequences set forth in SEQ ID NOs: 8 and 21 (see Table 2).

[0071] In certain embodiments, the anti-cMet antibody construct comprises two antigen-binding domains, each of which comprises an HCDR1 sequence selected from the sequences set forth in SEQ ID NOs: 3, 9, 14, 16, and 22, an HCDR2 sequence selected from the sequences set forth in SEQ ID NOs: 4, 10, 15, 17, and 23, an HCDR3 sequence selected from the sequences set forth in SEQ ID NOs: 5, 11, and 18, an LCDR1 sequence selected from the sequences set forth in SEQ ID NOs: 6, 12, and 19, an LCDR2 sequence selected from the sequences set forth in SEQ ID NOs: 7, 13, and 20, and an LCDR3 sequence selected from the sequences set forth in SEQ ID NOs: 8 and 21 (see Table 2).

[0072] In certain embodiments, the anti-cMet antibody construct comprises two antigen-binding domains, each of which comprises the heavy chain CDR (HCDR1, HCDR2, and HCDR3) sequences set forth in SEQ ID NOs: 3, 4, and 5, respectively, and the light chain CDR (LCDR1, LCDR2, and LCDR3) sequences set forth in SEQ ID NOs: 6, 7, and 8, respectively (see Table 2).

[0073] Hinge Area In certain embodiments, the anti-cMet antibody construct comprises an immunoglobulin (Ig) hinge region. The Ig hinge region may be based on the hinge region sequence of native human IgG1, IgG2, or IgG4, or it may be a modified version of the hinge region sequence of native human IgG1, IgG2, or IgG4.

[0074] As known in the art, the hinge region of an immunoglobulin is a flexible, hydrophilic region connecting the CH1 and CH2 domains and is generally defined as extending from positions 216 to 238 of IgG1 (Burton, 1985, Molec. Immunol., 22:161-206). Hinge regions of other IgG isotypes may be aligned with the IgG1 sequence by aligning the first and last cysteine ​​residues that form the inter-heavy chain disulfide bond, as shown in Figure 1. The Ig hinge region can be considered to include three subparts: the upper hinge, the middle hinge, and the lower hinge (see Figure 1) (Burton, 1985 (ibid.); see also Deveuve, et al., 2019, Med Sci (Paris), 35(12):1098-1105). The sequences of the native whole and upper hinge regions of human IgG1, IgG2, and IgG4 are provided in Table 3.

[0075] [Table 3]

[0076] In certain embodiments, an anti-cMet antibody construct comprises an Ig hinge region comprising an upper hinge sequence having the amino acid sequence of a native IgG1, IgG2, or IgG4 upper hinge sequence. In some embodiments, an anti-cMet antibody construct comprises an Ig hinge region comprising an upper hinge sequence having the amino acid sequence of a native IgG1 upper hinge sequence. In some embodiments, an anti-cMet antibody construct comprises an Ig hinge region comprising an upper hinge sequence having the amino acid sequence of a native IgG1 upper hinge sequence. In some embodiments, an anti-cMet antibody construct comprises an Ig hinge region comprising the amino acid sequence of SEQ ID NO: 25.

[0077] In certain embodiments, an anti-cMet antibody construct comprises an Ig hinge region comprising at least a portion of a native IgG1, IgG2, or IgG4 hinge sequence, e.g., the upper hinge sequence or the upper and middle (or "core") hinge sequences. In some embodiments, an anti-cMet antibody construct comprises an Ig hinge region comprising at least a portion of a native IgG1 hinge sequence. In some embodiments, an anti-cMet antibody construct comprises an Ig hinge region comprising at least a portion of a native IgG1 hinge sequence, this portion having the amino acid sequence: EPKSCDKTHTCPPCP (SEQ ID NO: 35).

[0078] In certain embodiments, an anti-cMet antibody construct comprises an Ig hinge region comprising the amino acid sequence of the hinge region of a native IgG1, IgG2, or IgG4. In some embodiments, an anti-cMet antibody construct comprises an Ig hinge region comprising the amino acid sequence set forth in any one of SEQ ID NOs: 24, 26, or 28. In some embodiments, an anti-cMet antibody construct comprises an Ig hinge region comprising the amino acid sequence of the hinge region of a native IgG1. In some embodiments, an anti-cMet antibody construct comprises an Ig hinge region comprising the amino acid sequence set forth in SEQ ID NO: 24.

[0079] In certain embodiments, an anti-cMet antibody construct comprises an Ig hinge region that is a modified version of the hinge region sequence of native human IgG1, IgG2, or IgG4. In some embodiments, an anti-cMet antibody construct comprises an Ig hinge region that is a modified version of the hinge region sequence of native human IgG1. For example, in some embodiments, an anti-cMet antibody construct may comprise one of the modified hinge sequences described in U.S. Patent No. 8,545,839 or U.S. Patent No. 8,741,290. In some embodiments, an anti-cMet antibody construct may comprise the modified hinge sequence: EPKSCDCHCPPCP (SEQ ID NO: 36).

[0080] scaffold In certain embodiments, at least one of the one or more antigen-binding domains comprised by the anti-cMet antibody constructs of the present disclosure is operably linked to a scaffold. As used herein, the term "operably linked" means that the described components are in a relationship permitting them to function in their intended manner. Examples of suitable scaffolds are further detailed below and include, but are not limited to, immunoglobulin Fc regions, albumin, albumin analogs and derivatives, heterodimerizing peptides (such as leucine zippers, heterodimer-forming "zipper" peptides derived from Jun and Fos, IgG CH1 and CL domains, or barnase barstar toxin), cytokines, chemokines, or growth factors. Other examples include antibodies based on DOCK-AND-LOCK™ (DNL™) technology developed by IBC Pharmaceuticals, Inc. and Immunomedics, Inc. (see, e.g., Chang, et al., 2007, Clin. Cancer Res., 13:5586s-5591s).

[0081] The scaffold can be a peptide, polypeptide, polymer, nanoparticle, or other chemical substance.When the scaffold is a polypeptide, the antigen-binding domain can be linked to either the N-terminus or C-terminus of the polypeptide scaffold.Also contemplated in certain embodiments are anti-cMet antibody constructs comprising polypeptide scaffolds in which one or more antigen-binding domains are linked to regions other than the N-terminus or C-terminus, for example, via amino acid side chains with or without a linker.

[0082] The antigen-binding domain(s) of the anti-cMet antibody construct may be linked to the scaffold by genetic fusion or chemical conjugation. In certain embodiments, when the scaffold is a peptide or polypeptide, the antigen-binding domain(s) are linked to the scaffold by genetic fusion. In some embodiments, when the scaffold is a polymer or nanoparticle, the antigen-binding domain(s) may be linked to the scaffold by chemical conjugation.

[0083] In certain embodiments, the anti-cMet antibody construct may comprise a protein scaffold. The use of protein scaffolds in combination with antigen-binding moieties has been described (see, e.g., Muller et al., 2007, J. Biol. Chem., 282:12650-12660; McDonaugh et al., 2012, Mol. Cancer Ther., 11:582-593; Vallera et al., 2005, Clin. Cancer Res., 11:3879-3888; Song et al., 2006, Biotech. Appl. Biochem., 45:147-154; and U.S. Patent Application Publication No. k009 / 0285816). Appl.

[0084] In certain embodiments, anti-cMet antibody constructs may comprise protein scaffolds based on immunoglobulin Fc regions, albumin, or albumin analogs or derivatives. For example, fusing antigen-binding moieties, such as scFvs, diabodies, or single-chain diabodies, to albumin has been shown to improve the serum half-life of the antigen-binding moieties (Muller et al., ibid.). The antigen-binding moieties may be fused at the N-terminus and / or C-terminus of albumin, optionally via a linker. Derivatives of albumin in the form of heteromultimers have been described, comprising two transporter polypeptides obtained by segmenting the albumin protein such that the transporter polypeptides self-assemble to form quasi-native albumin (see International Patent Applications WO 2012 / 116453 and WO 2014 / 012082). As a result of albumin segmentation, the heteromultimer comprises four termini and can therefore be fused to up to four different antigen-binding moieties, optionally via linkers.

[0085] In some embodiments, the anti-cMet antibody construct may comprise a protein scaffold based on an immunoglobulin Fc region, for example, an IgG Fc region.

[0086] Fc area As used herein, the term "Fc region," "Fc," or "Fc domain" refers to the C-terminal region of an immunoglobulin heavy chain comprising at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. Unless otherwise specified herein, the numbering of amino acid residues in an Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0087] In certain embodiments, the anti-cMet antibody constructs of the present disclosure may comprise a scaffold that is based on the immunoglobulin (Ig) Fc region. The Fc region may be dimeric, composed of two Fc polypeptides, or alternatively, the Fc region may be composed of a single polypeptide.

[0088] An "Fc polypeptide" in the context of a dimeric Fc refers to one of the two polypeptides that form the dimeric Fc domain, i.e., a polypeptide comprising one or more C-terminal constant regions of an immunoglobulin heavy chain capable of stable self-association. When referring to a dimeric Fc region, the terms "first Fc polypeptide" and "second Fc polypeptide" may be used interchangeably, provided that the Fc region comprises one first Fc polypeptide and one second Fc polypeptide.

[0089] An Fc region may comprise a CH3 domain, or it may comprise both a CH3 and a CH2 domain. For example, in certain embodiments, the Fc polypeptides of a dimeric IgG Fc region may comprise an IgG CH2 domain sequence and an IgG CH3 domain sequence. In such embodiments, the CH3 domain comprises two CH3 domain sequences, one from each of the two Fc polypeptides of the dimeric Fc region, and the CH2 domain comprises two CH2 domain sequences, one from each of the two Fc polypeptides of the dimeric Fc region.

[0090] In some embodiments, the anti-cMet antibody construct may comprise a scaffold that is based on an IgG Fc region and includes an IgG CH2 domain and an IgG CH3 domain. In some embodiments, the anti-cMet antibody construct may comprise a scaffold that is based on a human IgG Fc region. In some embodiments, the anti-cMet antibody construct may comprise a scaffold that is based on an IgG1 Fc region. In some embodiments, the anti-cMet antibody construct may comprise a scaffold that is based on a human IgG1 Fc region.

[0091] In certain embodiments, an anti-cMet antibody construct may comprise an IgG Fc region-based scaffold that is a homodimeric Fc region comprising a first Fc polypeptide and a second Fc polypeptide, each comprising a CH3 domain sequence and optionally a CH2 domain sequence, wherein the first and second Fc polypeptides are the same. In certain embodiments, an anti-cMet antibody construct may comprise an IgG Fc region-based scaffold that is a heterodimeric Fc region comprising a first Fc polypeptide and a second Fc polypeptide, each comprising a CH3 domain sequence and optionally a CH2 domain sequence, wherein the first and second Fc polypeptides are different. In some embodiments, an anti-cMet antibody construct may comprise an Fc region-based scaffold that comprises two CH3 domain sequences, at least one of which comprises one or more amino acid modifications. In some embodiments, an anti-cMet antibody construct may comprise an Fc region-based scaffold that comprises two CH3 domain sequences and two CH2 domain sequences, at least one of the CH2 domain sequences comprises one or more amino acid modifications.

[0092] In some embodiments, an anti-cMet antibody construct may comprise a heterodimeric Fc region comprising a modified CH3 domain, the modified CH3 domain being an asymmetrically modified CH3 domain comprising one or more asymmetric amino acid modifications. As used herein, "asymmetric amino acid modification" refers to a modification, such as a substitution or insertion, in which an amino acid at a particular position on a first CH3 or CH2 domain sequence differs from the amino acid at the same position on a second CH3 or CH2 domain sequence. These asymmetric amino acid modifications may be the result of modification of only one of the two amino acids at the same amino acid position on each sequence, or different modifications of both amino acids at the same position on each of the first and second CH3 or CH2 domain sequences. Each of the first and second CH3 or CH2 domain sequences of the heterodimeric Fc may comprise one or more asymmetric amino acid modifications.

[0093] In some embodiments, the anti-cMet antibody construct may comprise a heterodimeric Fc comprising a modified CH3 domain, the modified CH3 domain comprising one or more amino acid modifications that promote heterodimeric Fc formation over homodimeric Fc formation, in some embodiments, one or more of the amino acid modifications that promote heterodimeric Fc formation are asymmetric amino acid modifications.

[0094] Amino acid modifications that can be made to the CH3 domain of an Fc to promote heterodimeric Fc formation are known in the art and include, for example, those described in WO 96 / 027011 ("knobs into holes"), Gunasekaran et al., 2010, J Biol Chem, 285, 19637-46 ("electrostatic steering"), Davis et al., 2010, Prot Eng Des Sel, 23(4):195-202 (strand exchange engineered domain (SEED) technology), and Labrijn et al., 2013, Proc Natl Acad Sci USA, 110(13):5145-50 (Fab arm exchange). Other examples include approaches that combine positive and negative design strategies to produce stable asymmetrically modified Fc regions, such as those described in International Publication Nos. WO2012 / 058768 and WO2013 / 063702. In certain embodiments, an anti-cMet antibody construct may comprise a scaffold based on a modified Fc region as described in International Publication Nos. WO2012 / 058768 or WO2013 / 063702.

[0095] Table 4 provides the amino acid sequence of the human IgG1 Fc sequence (SEQ ID NO: 30), which corresponds to amino acids 231 to 447 of the full-length human IgG1 heavy chain. The CH3 sequence includes amino acids 341 to 447 of the full-length human IgG1 heavy chain. Table 4 also lists amino acid modifications in the CH3 domain that promote the formation of heterodimeric Fc, as described in International Patent Applications WO2012 / 058768 and WO2013 / 063702.

[0096] In certain embodiments, an anti-cMet antibody construct may comprise a heterodimeric Fc scaffold with a modified CH3 domain comprising any one of variant 1, variant 2, variant 3, variant 4, or variant 5 modifications as shown in Table 4.

[0097] In certain embodiments, the anti-cMet antibody construct may comprise a heterodimeric Fc scaffold having a modified CH3 domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein: a) the first Fc polypeptide comprises the amino acid modifications L351Y, F405A, and Y407V and the second Fc polypeptide comprises the amino acid modifications T366L, K392M, and T394W, or b) the first Fc polypeptide comprises the amino acid modifications L351Y, F405A, and Y407V and the second Fc polypeptide comprises the amino acid modifications T366L, K392L, and T394W; or c) the first Fc polypeptide comprises the amino acid modifications T350V, L351Y, F405A, and Y407V and the second Fc polypeptide comprises the amino acid modifications T350V, T366L, K392M, and T394W; or d) the first Fc polypeptide comprises the amino acid modifications T350V, L351Y, F405A, and Y407V and the second Fc polypeptide comprises the amino acid modifications T350V, T366L, K392L, and T394W; or e) the first Fc polypeptide comprises the amino acid modifications T350V, L351Y, S400E, F405A, and Y407V, and the second Fc polypeptide comprises the amino acid modifications T350V, T366L, N390R, K392M, and T394W.

[0098] [Table 4]

[0099] In some embodiments, an anti-cMet antibody construct may comprise an Fc region-based scaffold comprising two CH3 domain sequences and two CH2 domain sequences, wherein at least one of the CH2 domain sequences comprises one or more amino acid modifications. Modifications in the CH2 domain can affect binding of Fc receptors (FcRs) to Fc, such as receptors of the FcγRI, FcγRII, and FcγRIII subclasses. In some embodiments, an anti-cMet antibody construct comprises an IgG Fc-based scaffold having a modified CH2 domain, wherein the modification of the CH2 domain results in altered binding to one or more of the FcγRI, FcγRII, and FcγRIII receptors.

[0100] Several amino acid modifications to the CH2 domain that selectively alter the affinity of Fc for different Fcγ receptors are known in the art (e.g., Lu, et al., 2011, J Immunol Methods, 365(1-2):132-41; Stavenhagen, et al. 2007, Cancer Res 67(18):8882-90; Nordstrom, et al., 2011, Breast Cancer Res, 13(6):R123; Stewart, et al., 2011, Protein Eng Des Sel., 24(9):671-8; Shields, et al., 2001, J Biol Chem, 276(9):6591-604; Lazar, et al., 2006, Proc Natl Acad Sci USA, 103(11):4005-10; Chu, et al., 2008, Mol Immunol, 45(15):3926-33, International Publication No. WO2021 / 232162, and Therapeutic Antibody Engineering (Strohl & Strohl, Woodhead Publishing series in Biomedicine No 11, ISBN 1 907568 37 9, October 2012, page 283).

[0101] Amino acid modifications that result in increased FcγR binding and decreased FcγR binding may each be useful in certain indications. For example, increasing the binding affinity of Fc to FcγRIIIa (an activating receptor) can result in increased antibody-dependent cell-mediated cytotoxicity (ADCC), which in turn leads to increased lysis of target cells. Similarly, decreased binding to FcγRIIb (an inhibitory receptor) may be beneficial in some situations. In certain indications, reducing or eliminating ADCC and complement-mediated cytotoxicity (CDC) may be desirable. In such cases, modified CH2 domains containing amino acid modifications that result in increased binding to FcγRIIb or that reduce or eliminate binding of the Fc region to all Fcγ receptors ("knockout" variants) may be useful.

[0102] Various publications describe strategies that have been used to engineer antibodies to produce "knockout" variants (see, for example, Strohl, 2009, Curr Opin Biotech 20:685-691, and Strohl & Strohl, "Antibody Fc engineering for optimal antibody performance" In Therapeutic Antibody Engineering, Cambridge: Woodhead Publishing, 2012, pp 225-249; U.S. Patent Publication No. 2011 / 0212087; International Publication No. WO2006 / 105338; U.S. Patent Publication No. 2012 / 0225058; U.S. Patent Publication No. 2012 / 0251531; and Strop et al., 2012, J. Mol. Biol., 420:204-219). Other examples of mutations that can be introduced into the hinge or CH2 domain to produce "knockout" variants include the amino acid modifications L234A / L235A, and L234A / L235A / D265S.

[0103] In certain embodiments, the anti-cMet antibody constructs described herein may comprise a scaffold based on an IgG Fc in which the native glycosylation has been modified. As is known in the art, glycosylation of the Fc can be modified to increase or decrease effector function. For example, mutation of the conserved asparagine residue at position 297 to alanine, glutamine, lysine, or histidine (i.e., N297A, Q, K, or H) results in a deglycosylated Fc lacking all effector function (Bolt et al., 1993, Eur. J. Immunol., 23:403-411; Tao & Morrison, 1989, J. Immunol., 143:2595-2601).

[0104] Conversely, removal of fucose from the oligosaccharide attached to heavy chain N297 has been shown to enhance ADCC due to improved binding to FcγRIIIa (see, e.g., Shields et al., 2002, J. Biol. Chem., 277:26733-26740, and Niwa et al., 2005, J. Immunol. Methods, 306:151-160). Such low-fucose antibodies can be produced, for example, in knockout Chinese hamster ovary (CHO) cells lacking fucosyltransferase (FUT8) (Yamane-Ohnuki et al., 2004, Biotechnol. Bioeng., 87:614-622), in the variant CHO cell line Lec13 with reduced ability to attach fucose to the carbohydrate attached to N297 (International Publication No. WO 03 / 035835), or in other cells that produce defucosylated antibodies (see, e.g., Li et al., 2006, Nat Biotechnol, 24:210-215; Shields et al., 2002 (ibid.); and Shinkawa et al., 2003, J. Biol. Chem., 278:3466-3473). Additionally, International Publication No. WO 2009 / 135181 describes the addition of a fucose analog to the culture medium during antibody production to inhibit the incorporation of fucose into the carbohydrate on the antibody. Other methods for producing antibodies with little or no fucose at the Fc glycosylation site (N297) are known in the art, such as GlymaX® technology (ProBioGen AG) (see von Horsten et al., 2010, Glycobiology, 20(12):1607-1618 and U.S. Pat. No. 8,409,572).

[0105] Other glycosylation variants include those with bisected oligosaccharides, for example, variants in which biantennary oligosaccharides attached to the Fc region of an antibody are bisected by N-acetylglucosamine (GlcNAc). Such glycosylation variants may have reduced fucosylation and / or improved ADCC function (see, e.g., International Publication No. WO2003 / 011878, U.S. Patent No. 6,602,684, and U.S. Patent Application Publication No. US2005 / 0123546). Useful glycosylation variants also include those with at least one galactose residue in the oligosaccharide attached to the Fc region, which may have improved CDC function (see, e.g., International Publication Nos. WO1997 / 030087, WO1998 / 58964, and WO1999 / 22764).

[0106] Anti-cMet antibody constructs containing cysteine ​​mutations Certain embodiments of the present disclosure relate to anti-cMet antibody constructs as described above, further comprising one or more non-native cysteine ​​residues that provide a "conjugation handle" that allows for conjugation of a drug-linker as described herein.

[0107] Modification of an antibody to include a non-native cysteine ​​residue may be achieved by substitution of a native residue with a cysteine ​​residue (see, e.g., U.S. Pat. Nos. 7,521,541, 8,455,622, and 9,000,130) or by insertion of a cysteine ​​residue between two native residues in the antibody sequence (see, e.g., U.S. Pat. No. 10,744,206).

[0108] The anti-cMet antibody construct may comprise one or more cysteine ​​substitution mutations, one or more cysteine ​​insertion mutations, or a combination thereof. In some embodiments, the anti-cMet antibody construct may comprise one to four cysteine ​​substitution mutations, cysteine ​​insertion mutations, or a combination thereof. In some embodiments, the anti-cMet antibody construct may comprise one or more cysteine ​​substitution mutations, for example, one to four cysteine ​​substitution mutations. In some embodiments, the anti-cMet antibody construct may comprise one or more cysteine ​​insertion mutations, for example, one to four cysteine ​​insertion mutations.

[0109] In certain embodiments, the anti-cMet antibody construct comprises at least one antigen binding domain comprising a VL domain and a VH domain, and optionally a CH1 domain and a CL domain, and an Fc region comprising a CH2 domain and a CH3 domain. In some such embodiments, the anti-cMet antibody construct may comprise one or more cysteine ​​insertion mutations, each of which: (a) a cysteine ​​residue inserted between positions 39 and 40 in the VL domain; (b) a cysteine ​​residue inserted between positions 40 and 41 in the VL domain; (c) a cysteine ​​residue inserted between positions 126 and 127 in the CL domain; (d) a cysteine ​​residue inserted between positions 148 and 149 in the CL domain; (e) a cysteine ​​residue inserted between positions 149 and 150 in the CL domain; (f) a cysteine ​​residue inserted between positions 9 and 10 in the VH domain; (g) a cysteine ​​residue inserted between positions 169 and 170 in the CH1 domain; (h) a cysteine ​​residue inserted between positions 237 and 238 in the CH2 domain; (i) a cysteine ​​residue inserted between positions 295 and 296 in the CH2 domain; and (j) A cysteine ​​residue inserted between positions 299 and 300 in the CH2 domain are independently selected from

[0110] The numbering of amino acids in the VL, CL, and VH domains used herein when describing cysteine ​​insertion mutations is Kabat numbering, and the numbering of amino acids in the CH2 domain is EU numbering.

[0111] In some embodiments, the anti-cMet antibody construct may comprise one or more cysteine ​​insertion mutations, each of which is (i) a cysteine ​​residue inserted between positions 40 and 41 in the VL domain; (ii) a cysteine ​​residue inserted between positions 126 and 127 in the CL domain; (iii) a cysteine ​​residue inserted between positions 9 and 10 in the VH domain; (iv) a cysteine ​​residue inserted between positions 237 and 238 in the CH2 domain; and (v) a cysteine ​​residue inserted between positions 299 and 300 in the CH2 domain are independently selected from

[0112] In some embodiments, the anti-cMet antibody construct may be monovalent and comprise one antigen-binding domain, comprising a VL domain and a VH domain, and optionally a CH1 domain and a CL domain. In some embodiments, the monovalent anti-cMet antibody construct may comprise a cysteine ​​residue inserted between positions 39 and 40 in the VL domain, a cysteine ​​residue inserted between positions 40 and 41 in the VL domain, a cysteine ​​residue inserted between positions 126 and 127 in the CL domain, a cysteine ​​residue inserted between positions 148 and 149 in the CL domain, a cysteine ​​residue inserted between positions 149 and 150 in the CL domain, a cysteine ​​residue inserted between positions 9 and 10 in the VH domain, and / or a cysteine ​​residue inserted between positions 169 and 170 in the CH1 domain.

[0113] In some embodiments, the anti-cMet antibody construct may be bivalent and comprise two antigen-binding domains, each comprising a VL domain and a VH domain, and optionally a CH1 domain and a CL domain. Both antigen-binding domains may bind to the same antigen, or they may each bind to a different antigen. A bivalent anti-cMet antibody construct may comprise one or more cysteine ​​insertion mutations in one antigen-binding domain, or it may comprise one or more cysteine ​​insertion mutations in each antigen-binding domain. When the anti-cMet antibody construct comprises one or more cysteine ​​insertion mutations in each antigen-binding domain, each antigen-binding domain may comprise the same cysteine ​​insertion mutation(s), or they may comprise different cysteine ​​insertion mutation(s).

[0114] In some embodiments, a bivalent anti-cMet antibody construct may comprise a cysteine ​​residue inserted between positions 39 and 40 in one VL domain. In some embodiments, a bivalent anti-cMet antibody construct may comprise a cysteine ​​residue inserted between positions 39 and 40 in each VL domain. In some embodiments, a bivalent anti-cMet antibody construct may comprise a cysteine ​​residue inserted between positions 40 and 41 in one VL domain. In some embodiments, a bivalent anti-cMet antibody construct may comprise a cysteine ​​residue inserted between positions 40 and 41 in each VL domain. In some embodiments, a bivalent anti-cMet antibody construct may comprise a cysteine ​​residue inserted between positions 126 and 127 in one CL domain. In some embodiments, a bivalent anti-cMet antibody construct may comprise a cysteine ​​residue inserted between positions 126 and 127 in each CL domain. In some embodiments, a bivalent anti-cMet antibody construct may comprise a cysteine ​​residue inserted between positions 148 and 149 in one of the CL domains. In some embodiments, a bivalent anti-cMet antibody construct may comprise a cysteine ​​residue inserted between positions 148 and 149 in each of the CL domains. In some embodiments, a bivalent anti-cMet antibody construct may comprise a cysteine ​​residue inserted between positions 149 and 150 in one of the CL domains. In some embodiments, a bivalent anti-cMet antibody construct may comprise a cysteine ​​residue inserted between positions 149 and 150 in each of the CL domains. In some embodiments, a bivalent anti-cMet antibody construct may comprise a cysteine ​​residue inserted between positions 9 and 10 in one of the VH domains. In some embodiments, a bivalent anti-cMet antibody construct may comprise a cysteine ​​residue inserted between positions 9 and 10 in each of the VH domains. In some embodiments, a bivalent anti-cMet antibody construct may comprise a cysteine ​​residue inserted in one CH1 domain between positions 169 and 170. In some embodiments, a bivalent anti-cMet antibody construct may comprise a cysteine ​​residue inserted in each CH1 domain between positions 169 and 170.

[0115] In certain embodiments, an anti-cMet antibody construct may comprise an Fc region. In some embodiments, an anti-cMet antibody construct may comprise a dimeric Fc region composed of two Fc polypeptides as described above, wherein the CH3 domain of the Fc region comprises two CH3 domain sequences, one from each of the two Fc polypeptides of the dimeric Fc region, and the CH2 domain of the Fc region comprises two CH2 domain sequences, one from each of the two Fc polypeptides of the dimeric Fc region. In some embodiments, an anti-cMet antibody construct may comprise an Fc region composed of a single Fc polypeptide as described above, wherein the CH3 domain of the Fc region comprises two CH3 domain sequences, and the CH2 domain of the Fc region comprises two CH2 domain sequences, and both CH3 domain sequences and both CH2 domain sequences are comprised by the single Fc polypeptide.

[0116] In some embodiments, an anti-cMet antibody construct may comprise an Fc region and a cysteine ​​residue inserted between positions 237 and 238 in one of the CH2 domain sequences. In some embodiments, an anti-cMet antibody construct may comprise an Fc region and a cysteine ​​residue inserted between positions 237 and 238 in each of the CH2 domain sequences. In some embodiments, an anti-cMet antibody construct may comprise an Fc region and a cysteine ​​residue inserted between positions 295 and 296 in one of the CH2 domain sequences. In some embodiments, an anti-cMet antibody construct may comprise an Fc region and a cysteine ​​residue inserted between positions 295 and 296 in each of the CH2 domain sequences. In some embodiments, an anti-cMet antibody construct may comprise an Fc region and a cysteine ​​residue inserted between positions 299 and 300 in one of the CH2 domain sequences. In some embodiments, the anti-cMet antibody construct may comprise an Fc region and a cysteine ​​residue inserted between positions 299 and 300 in each CH2 domain sequence.

[0117] In certain embodiments, the anti-cMet antibody construct specifically binds c-Met and comprises an antigen binding domain comprising a VL domain and a VH domain, and optionally a CH1 domain and a CL domain, and an Fc region comprising a CH2 domain having two CH2 domain sequences and a CH3 domain having two CH3 domain sequences, wherein the antibody construct comprises: (a) a cysteine ​​residue inserted between positions 40 and 41 in the VL domain; (b) a cysteine ​​residue inserted between positions 126 and 127 in the CL domain; (c) a cysteine ​​residue inserted between positions 9 and 10 in the VH domain; (d) a cysteine ​​residue inserted between positions 237 and 238 in the CH2 domain sequence; and (e) A cysteine ​​residue inserted between positions 299 and 300 in the CH2 domain sequence. and one or more cysteine ​​insertion mutations independently selected from:

[0118] Various combinations of the above-described cysteine ​​insertion mutations are contemplated and may be selected based on whether the antibody construct is monovalent, bivalent, or multivalent and the nature of the antigen-binding domain (e.g., whether the antigen-binding domain is a Fab or scFv). In certain embodiments, an anti-cMet antibody construct may comprise a combination of cysteine ​​insertions. In some embodiments, an anti-cMet antibody construct may comprise a combination of cysteine ​​insertions, where the combination is: (a) cysteine ​​residues inserted between positions 299 and 300 and between positions 237 and 238 in the CH2 domain; or (b) a cysteine ​​residue inserted between positions 299 and 300 in the CH2 domain and between positions 9 and 10 in the VH domain; or (c) cysteine ​​residues inserted between positions 299 and 300 in the CH2 domain and between positions 40 and 41 in the VL domain; or (d) a cysteine ​​residue inserted between positions 237 and 238 in the CH2 domain and between positions 9 and 10 in the VH domain; or (e) Cysteine ​​residues inserted between positions 9 and 10 in the VH domain and between positions 40 and 41 in the VL domain Includes:

[0119] In some embodiments, an anti-cMet antibody construct comprises at least one antigen-binding domain comprising a VL domain, a VH domain, and optionally a CH1 domain and a CL domain, and an Fc region, wherein the anti-cMet antibody construct comprises (i) a cysteine ​​insertion between positions 299 and 300 in one CH2 domain sequence, and (ii) a cysteine ​​residue inserted between positions 299 and 300 and between positions 237 and 238 in the other CH2 domain sequence. In some embodiments, an anti-cMet antibody construct comprises at least one antigen-binding domain comprising a VL domain, a VH domain, and optionally a CH1 domain and a CL domain, and an Fc region, wherein the anti-cMet antibody construct comprises (i) a cysteine ​​insertion between positions 299 and 300 in each CH2 domain sequence, and (ii) a cysteine ​​residue inserted between positions 9 and 10 in one VH domain.

[0120] In some embodiments, an anti-cMet antibody construct comprises two antigen-binding domains, each comprising a VL domain, a VH domain, and optionally a CH1 domain and a CL domain, and an Fc region, wherein the anti-cMet antibody construct comprises (i) a cysteine ​​insertion between positions 299 and 300 in one CH2 domain sequence, and (ii) a cysteine ​​residue inserted between positions 299 and 300 and between positions 237 and 238 in the other CH2 domain sequence. In some embodiments, an anti-cMet antibody construct comprises two antigen-binding domains, each comprising a VL domain, a VH domain, and optionally a CH1 domain and a CL domain, and an Fc region, wherein the anti-cMet antibody construct comprises (i) a cysteine ​​insertion between positions 299 and 300 in each CH2 domain sequence, and (ii) a cysteine ​​residue inserted between positions 9 and 10 in one VH domain.

[0121] In some embodiments, an anti-cMet antibody construct comprises two antigen-binding domains, a VL domain, a VH domain, and optionally a CH1 domain and a CL domain, respectively, and an Fc region, wherein the anti-cMet antibody construct comprises a cysteine ​​residue inserted between positions 40 and 41 in each VL domain, and either (i) a cysteine ​​residue inserted between positions 299 and 300 in one of the CH2 domain sequences, or (ii) a cysteine ​​residue inserted between positions 9 and 10 in one of the VH domains. In some embodiments, an anti-cMet antibody construct comprises two antigen-binding domains, a VL domain, a VH domain, and optionally a CH1 domain and a CL domain, respectively, and an Fc region, wherein the anti-cMet antibody construct comprises a cysteine ​​residue inserted between positions 9 and 10 in each VH domain, and a cysteine ​​insertion between positions 237 and 238 in one of the CH2 domain sequences.

[0122] In certain embodiments, the anti-cMet antibody construct comprises an Fc region comprising at least one VH domain, at least one VL domain, and two CH2 domain sequences, and the following combination of cysteine ​​insertion mutations: (a) cysteine ​​residues inserted between positions 299 and 300 and between positions 237 and 238 in one or both CH2 domain sequences; or (b) a cysteine ​​residue inserted between positions 299 and 300 in one or both of the CH2 domain sequences and a cysteine ​​residue inserted between positions 9 and 10 in the VH domain; or (c) a cysteine ​​residue inserted between positions 299 and 300 in one or both of the CH2 domain sequences and a cysteine ​​residue inserted between positions 40 and 41 in the VL domain; or (d) a cysteine ​​residue inserted between positions 237 and 238 in one or both of the CH2 domain sequences and a cysteine ​​residue inserted between positions 9 and 10 in the VH domain; or (e) a cysteine ​​residue inserted between positions 9 and 10 in the VH domain and between positions 40 and 41 in the VL domain; Contains one of the following:

[0123] In certain embodiments, the anti-cMet antibody construct comprises an Fc region comprising one or two VH domains, one or two VL domains, and two CH2 domain sequences; (i) a cysteine ​​residue inserted between positions 299 and 300 in one of the CH2 domain sequences; or (ii) a cysteine ​​residue inserted between positions 299 and 300 in each CH2 domain sequence; or (iii) a cysteine ​​residue inserted between positions 237 and 238 in one of the CH2 domain sequences; or (iv) a cysteine ​​residue inserted between positions 237 and 238 in each CH2 domain sequence; or (v) a cysteine ​​residue inserted between positions 9 and 10 in one VH domain; or (vi) a cysteine ​​residue inserted between positions 9 and 10 in each VH domain; or (vii) a cysteine ​​residue inserted between positions 40 and 41 in each VL domain; or (viii) a cysteine ​​residue inserted between positions 126 and 127 in each CL domain; or (ix) a cysteine ​​insertion between positions 299 and 300 in the first CH2 domain sequence, a cysteine ​​residue inserted between positions 299 and 300 in the second CH2 domain sequence, and a cysteine ​​residue inserted between positions 237 and 238 in the second CH2 domain sequence; or (x) a cysteine ​​residue inserted between positions 9 and 10 in one VH domain and a cysteine ​​inserted between positions 299 and 300 in each CH2 domain sequence; or (xi) a cysteine ​​residue inserted between positions 40 and 41 in each VL domain and a cysteine ​​residue inserted between positions 299 and 300 in one CH2 domain sequence; or (xii) a cysteine ​​residue inserted between positions 40 and 41 in each VL domain and a cysteine ​​residue inserted between positions 9 and 10 in one VH domain; or (xiii) a cysteine ​​residue inserted between positions 9 and 10 in each VH domain and a cysteine ​​inserted between positions 237 and 238 in one CH2 domain sequence Further includes:

[0124] In certain embodiments, an anti-cMet antibody construct comprises a VH domain comprising the amino acid sequence set forth in SEQ ID NO: 59. In certain embodiments, an anti-cMet antibody construct comprises two VH domains, each comprising the amino acid sequence set forth in SEQ ID NO: 59.

[0125] In certain embodiments, an anti-cMet antibody construct comprises a VL domain comprising the amino acid sequence set forth in SEQ ID NO: 56. In certain embodiments, an anti-cMet antibody construct comprises two VL domains, each comprising the amino acid sequence set forth in SEQ ID NO: 56.

[0126] In certain embodiments, an anti-cMet antibody construct comprises a first heavy chain and a second heavy chain, wherein the first heavy chain and the second heavy chain each comprise a CH2 domain, and one of the CH2 domains comprises an amino acid sequence selected from the sequences set forth in SEQ ID NOs: 76, 77, and 78. In certain embodiments, an anti-cMet antibody construct comprises a first heavy chain and a second heavy chain, wherein the first heavy chain and the second heavy chain each comprise a CH2 domain, and both of the CH2 domains comprise amino acid sequences independently selected from the sequences set forth in SEQ ID NOs: 76, 77, and 78.

[0127] In certain embodiments, an anti-cMet antibody construct comprises a first light chain and a second light chain, wherein the first light chain and the second light chain each comprise a CL domain, and one of the CL domains comprises the amino acid sequence set forth in SEQ ID NO: 79. In certain embodiments, an anti-cMet antibody construct comprises a first light chain and a second light chain, wherein the first light chain and the second light chain each comprise a CL domain, and both of the CL domains comprise the amino acid sequence set forth in SEQ ID NO: 79.

[0128] Auristatin Analogue Drug-Linker In certain embodiments, the ADCs of the disclosure comprise an anti-cMet antibody construct as described above conjugated via a linker to the auristatin analog Compound 1. Thus, certain embodiments comprise an ADC of Formula II: TIFF2025531990000017.tif13128, wherein: L is a linker, D is the structure: TIFF2025531990000018.tif37165, where * is the point of attachment to L, n is 1 to 4.

[0129] The linker L comprised by the drug-linker of Formula II and the ADC of Formula I functions to link one or more auristatin analogs to the anti-cMet antibody construct and may be monovalent or multivalent. A monovalent linker L functions to link a single auristatin analog to a single site on the anti-cMet antibody construct, while a multivalent (or polyvalent) linker L functions to link two or more auristatin analogs to a single site on the anti-cMet antibody construct. A linker linking one auristatin analog to more than one site on the anti-cMet antibody construct may also be considered multivalent in some embodiments.

[0130] In certain embodiments, linker L is linked to anti-cMet antibody construct A via a functional group capable of reacting with targeting group(s) on the antibody construct and to auristatin analog(s) via a functional group capable of reacting with targeting amino groups on the auristatin analog. Suitable functional groups are known in the art and include, for example, those described in Bioconjugate Techniques (G.T. Hermanson, 2013, Academic Press). Groups on anti-cMet antibody constructs that can serve as targeting groups for linker attachment include, but are not limited to, thiol, hydroxyl, carboxyl, amine, aldehyde, and ketone groups.

[0131] Non-limiting examples of functional groups that can react with thiols include maleimides, haloacetamides, haloacetyls, activated esters (such as succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters, and tetrafluorophenyl esters), anhydrides, acid chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates. Also useful in this context are "self-stabilizing" maleimides, such as those described in Lyon et al., 2014, Nat. Biotechnol., 32:1059-1062.

[0132] Non-limiting examples of functional groups for reaction with free amines include activated esters (such as N-hydroxysuccinamide (NHS) esters and sulfo-NHS esters), imidoesters (such as Traut's reagent), tetrafluorophenyl (TFP) esters, sulfodichlorophenyl esters, isothiocyanates, aldehydes, and acid anhydrides (such as diethylenetriaminepentaacetic anhydride (DTPA)). Other examples include the conversion of carboxylic acids to activated esters using succinimido-1,1,3,3-tetramethyluronium tetrafluoroborate (TSTU) or benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), which can then be reacted with amines.

[0133] Non-limiting examples of functional groups that can react with electrophilic groups such as aldehyde or ketone carbonyl groups include hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide.

[0134] In certain embodiments, the linker L may comprise a functional group that allows for cross-linking of two interchain cysteines on the anti-cMet antibody construct, such as a ThioBridge™ linker (Badescu et al., 2014, Bioconjug. Chem. 25:1124-1136), a dithiomaleimide (DTM) linker (Behrens et al., 2015, Mol. Pharm. 12:3986-3998), a dithioaryl (TCEP) pyridazinedione-based linker (Lee et al., 2016, Chem. Sci., 7:799-802), or a dibromopyridazinedione-based linker (Maruani et al., 2015, Nat. Commun., 6:6645).

[0135] Alternatively, the anti-cMet antibody construct may be modified to contain a non-natural reactive group, such as an azide, that allows conjugation to a linker via a complementary reactive group on the linker. For example, conjugation of the linker to the targeting moiety may utilize a click chemistry reaction, such as the azide-alkyne cycloaddition (AAC) reaction, which has been successfully used in the development of antibody-drug conjugates (see, e.g., Chio & Bane, 2020, Methods Mol. Biol., 2078:83-97). The AAC reaction may be a copper-catalyzed AAC (CuAAC) reaction, which involves coupling an azide with a linear alkyne, or a strain-promoted AAC (SPAAC) reaction, which involves coupling an azide with a cyclooctyne.

[0136] The linker L may be a cleavable or non-cleavable linker. A cleavable linker is one that is susceptible to cleavage under certain conditions, such as intracellular conditions (such as endosomes or lysosomes) or in the vicinity of target cells (such as the tumor microenvironment). Examples include protease-, acid-, or reduction-sensitive linkers. In contrast, non-cleavable linkers rely on antibody degradation in cells, which typically results in release of the amino acid-linker-drug moiety.

[0137] Examples of cleavable linkers include linkers containing an amino acid sequence that is a cleavage recognition sequence for a protease. Many such cleavage recognition sequences are known in the art. For conjugates that are not intended to be internalized by cells, an amino acid sequence that is recognized and cleaved by a protease present in the extracellular matrix near target cells, such as cancer cells, may be used. Examples of extracellular tumor-associated proteases include plasmin, matrix metalloproteinase (MMP), elastase, and kallikrein-related peptidase. For conjugates that are intended to be internalized by cells, the linker L may contain an amino acid sequence that is recognized and cleaved by an endosomal or lysosomal protease. Examples of such proteases include cathepsin B, C, D, H, L, and S, and legumain.

[0138] The cleavage recognition sequence may be, for example, a dipeptide, tripeptide, or tetrapeptide. Non-limiting examples of dipeptide recognition sequences that may be included in a cleavable linker include, but are not limited to, Ala-(D)Asp, Ala-Lys, Ala-Phe, Asn-Lys, Asn-(D)Lys, Asp-Val, His-Val, Ile-Cit, Ile-Pro, Ile-Val, Leu-Cit, Me3Lys-Pro, Met-Lys, Met-(D)Lys, NorVal-(D)Asp, Phe-Arg, Phe-Cit, Phe-Lys, PhenylGly-(D)Lys, Pro-(D)Lys, Trp-Cit, Val-Ala, Val-(D)Asp, Val-Cit, Val-Gly, Val-Gln, and Val-Lys. Examples of tripeptide and tetrapeptide cleavage sequences include, but are not limited to, Ala-Ala-Asn, Ala-Val-Cit, (D)Ala-Phe-Lys, Asp-Val-Ala, Asp-Val-Cit, Gly-Cit-Val, Lys-Val-Ala, Lys-Val-Cit, Met-Cit-Val, (D)Phe-Phe-Lys, Asn-Pro-Val, Ala-Leu-Ala-Leu, Gly-Phe-Leu-Gly, Gly-Gly-Phe-Gly, and Gly-Phe-Gly-Gly.

[0139] Additional examples of cleavable linkers include disulfide-containing linkers such as N-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB) and N-succinimidyl-4-(2-pyridyldithio)-2-sulfobutanoate (sulfo-SPDB). Disulfide-containing linkers may optionally contain additional groups adjacent to the disulfide bond to provide steric hindrance (e.g., incorporation of geminal dimethyl groups) to improve the extracellular stability of the linker. Other cleavable linkers include linkers that are hydrolyzable at a specific pH or within a pH range, such as hydrazone linkers. Linkers containing a combination of these functional groups may also be useful; for example, linkers containing both hydrazones and disulfides are known in the art.

[0140] A further example of a cleavable linker is a linker containing a β-glucuronide that is cleavable by β-glucuronidase, an enzyme present in lysosomes and tumor stroma (see, e.g., De Graaf et al., 2002, Curr. Pharm. Des. 8:1391-1403, and International Patent Publication No. WO 2007 / 011968). The β-glucuronide can also function to improve the hydrophilicity of the linker L.

[0141] Another example of a linker that is cleaved intracellularly to improve hydrophilicity is a linker that contains a pyrophosphate diester moiety (see, e.g., Kern et al., 2016, J Am Chem Soc., 138:2430-1445).

[0142] In certain embodiments, the linker L comprised by the ADC of Formula I and the drug-linker of Formula II is a cleavable linker. In some embodiments, the linker L comprises a cleavage recognition sequence. In some embodiments, the linker L may comprise an amino acid sequence that is recognized and cleaved by a lysosomal protease.

[0143] The cleavable linker may optionally further comprise one or more additional functional groups, such as a self-immolative or self-leaving group, a stretcher, or a hydrophilic moiety.

[0144] Self-immolative and self-leaving groups utilized in linkers include, for example, p-aminobenzyl (PAB) and p-aminobenzyloxycarbonyl (PABC) groups, and methylated ethylenediamine (MED). Other examples of self-immolative groups include aromatic compounds electronically similar to PAB or PABC groups, such as heterocyclic derivatives, for example, but not limited to, 2-aminoimidazole-5-methanol derivatives as described in U.S. Patent No. 7,375,078. Other examples include groups that undergo cyclization upon hydrolysis of the amide bond, such as substituted and unsubstituted 4-aminobutyric acid amide (Rodrigues et al., 1995, Chemistry Biology 2:223-227) and 2-aminophenylpropionic acid amide (Amsberry, et al., 1990, J. Org. Chem. 55:5867-5877). Self-immolative / self-leaving groups are typically attached to amino or hydroxyl groups on the payload drug. Self-immolative / self-leaving groups, alone or in combination, are often included in peptide-based linkers, but can also be included in other types of linkers.

[0145] Stretchers utilized in linkers for drug conjugates include, for example, alkylene groups and stretchers based on fatty acids, diacids, amines, or diamines, such as diglycolate, malonate, caproate, and caproamide. Other stretchers include, for example, glycine-based stretchers and polyethylene glycol (PEG) or monomethoxypolyethylene glycol (mPEG) stretchers.

[0146] PEG and mPEG stretchers can also function as hydrophilic moieties within the linker. For example, PEG or mPEG may be included in the linker either "in-line" or as a pendant group to increase the hydrophilicity of the linker (see, e.g., U.S. Patent Application Publication No. US2016 / 0310612). Various PEG-containing linkers are also commercially available from companies such as Quanta BioDesign, Ltd. (Plain City, OH). Other hydrophilic groups that can optionally be incorporated into the linker L include, for example, β-glucuronide, sulfonate group, carboxylate group, and pyrophosphate diester.

[0147] The selection of an appropriate linker for a given ADC can be readily made by one skilled in the art, taking into account relevant factors such as the attachment site to the antibody construct, any structural constraints of the payload drug, and the hydrophobicity of the payload drug (see, for example, the review in Nolting, Chapter 5, Antibody-Drug Conjugates: Methods in Molecular Biology, 2013, Ducry (Ed.), Springer).

[0148] In certain embodiments, the drug-linker of Formula II and the ADC of Formula I may comprise a cleavable linker. In some embodiments, the drug-linker of Formula II and the ADC of Formula I may comprise a peptide-containing linker. In some embodiments, the drug-linker of Formula II and the ADC of Formula I may comprise a protease-cleavable linker.

[0149] In certain embodiments, in the Drug-Linker of Formula II, n is 1 and the Drug-Linker is of Formula III: TIFF2025531990000019.tif27165, wherein Z is a functional group capable of reacting with a targeting group on the anti-cMet antibody construct A; Str is the stretcher, AA1 and AA2 are each independently an amino acid, wherein AA1-[AA2] m forms a protease cleavage site, X is a self-immolative group; s is 0 or 1, m is 1, 2, or 3; o is 0, 1, or 2; D has the structure shown in Formula II.

[0150] When incorporated into the ADCs of the disclosure, the drug-linker of Formula III has the formula IV: TIFF2025531990000020.tif27165, wherein Z' is a linking group that connects the linker to the targeting group on the anti-cMet antibody construct A; Str, AA1, AA2, X, s, m, o, and D are as defined for formula III; # indicates the point of attachment to anti-cMet antibody construct A.

[0151] In some embodiments, s is 1 in Formulas III and IV.

[0152] In some embodiments, in formulas III and IV, o is 0 (ie, X is absent).

[0153] In some embodiments, in Formula III, Z is a functional group capable of reacting with a thiol or amino group on the anti-cMet antibody construct A.

[0154] In some embodiments, in Formula III: Z is TIFF2025531990000021.tif37165, where * is the point of attachment to the rest of the linker.

[0155] In some embodiments, in Formula IV: Z' is a carbonyl group (-C(O)-) or TIFF2025531990000022.tif32165, where # is the point of attachment to anti-cMet antibody construct A; * is the point of attachment to the rest of the linker.

[0156] In some embodiments, in Formula III and IV, Str is Selected from TIFF2025531990000023.tif53165, where: each R is independently H or C1-C6 alkyl; each p is independently an integer from 2 to 10; each q is independently an integer from 1 to 10; $ is the point of attachment to Z or Z', * is the point of attachment to the rest of the linker.

[0157] In some embodiments, in Formula III and IV, Str is TIFF2025531990000024.tif37165, where p, q, $ and * is as defined above.

[0158] In some embodiments, in Formula III and IV, Str is TIFF2025531990000025.tif37165, where $ and * is as defined above, p is an integer of 2 to 6, and q is an integer of 2 to 8.

[0159] In some embodiments, in Formula III and IV, AA1-[AA2] mare Val-Lys, Ala-Lys, Phe-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Arg, Ala-Phe, Val-Ala, Met- Lys, Asn-Lys, Ile-Pro, Ile-Val, Asp-Val, His-Val, Met-(D)Lys, Asn-(D)Lys, Val-(D)Asp, NorVal-(D)Asp, Ala -(D)Asp, Me3Lys-Pro, PhenylGly-(D)Lys, Met-(D)Lys, Asn-(D)Lys, Pro-(D)Lys, Met-(D)Lys, Met-Cit-Val, Gly- selected from Cit-Val, (D)Phe-Phe-Lys, (D)Ala-Phe-Lys, Gly-Phe-Leu-Gly, Ala-Leu-Ala-Leu, and Gly-Gly-Phe-Gly.

[0160] In some embodiments, in Formula III and IV, m is 1 (i.e., AA1-[AA2] m is a dipeptide).

[0161] In some embodiments, in Formula III and IV, AA1-[AA2] m is a dipeptide selected from Val-Lys, Ala-Lys, Phe-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit, and Trp-Cit.

[0162] In some embodiments, in Formula III: Z is TIFF2025531990000026.tif37165, where * is the point of attachment to the rest of the linker, Str is TIFF2025531990000027.tif22165, where: $ is the point of attachment to Z, * is the point of attachment to the remainder of the linker, p is an integer from 2 to 6, and q is an integer from 2 to 8; m is 1, and AA1-[AA2] mis a dipeptide selected from Val-Lys, Ala-Lys, Phe-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit, and Trp-Cit; s is 1, o is 0.

[0163] In some embodiments, in Formula IV: Z' is a carbonyl group (-C(O)-) or TIFF2025531990000028.tif32165, where # is the point of attachment to anti-cMet antibody construct A; * is the point of attachment to the rest of the linker, Str is TIFF2025531990000029.tif22165, where $ is the point of attachment to Z', * is the point of attachment to the remainder of the linker, p is an integer from 2 to 6, and q is an integer from 2 to 8; m is 1, and AA1-[AA2] m is a dipeptide selected from Val-Lys, Ala-Lys, Phe-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit, and Trp-Cit; s is 1, o is 0.

[0164] In certain embodiments, in the Drug-Linker of Formula II, n is 1 and the Drug-Linker is represented by Formula V: TIFF2025531990000030.tif27165, wherein Z is a functional group capable of reacting with a targeting group on the anti-cMet antibody construct A; Str is the stretcher, AA1 and AA2 are each independently an amino acid, wherein AA1-[AA2] m forms a protease cleavage site, m is 1, 2, or 3; D has the structure shown in Formula II.

[0165] When incorporated into the ADCs of the disclosure, the drug-linker of Formula V has the formula VI: TIFF2025531990000031.tif27165, wherein Z' is a linking group that connects the linker to the targeting group on the anti-cMet antibody construct A; Str, AA1, AA2, X, m, and D are as defined for formula V; # indicates the point of attachment to anti-cMet antibody construct A.

[0166] In some embodiments, in formula V, Z is a functional group capable of reacting with a thiol or amino group on the anti-cMet antibody construct A.

[0167] In some embodiments, in Formula V: Z is TIFF2025531990000032.tif37165, where * is the point of attachment to the rest of the linker.

[0168] In some embodiments, in Formula VI: Z' is a carbonyl group (-C(O)-) or TIFF2025531990000033.tif32165, where # is the point of attachment to anti-cMet antibody construct A; * is the point of attachment to the rest of the linker.

[0169] In some embodiments, in Formulas V and VI, Str is Selected from TIFF2025531990000034.tif53165, where: each R is independently H or C1-C6 alkyl; each p is independently an integer from 2 to 10; each q is independently an integer from 1 to 10; $ is the point of attachment to Z or Z', *is the point of attachment to the rest of the linker.

[0170] In some embodiments, in Formulas V and VI, Str is TIFF2025531990000035.tif37165, where p, q, $ and * is as defined above.

[0171] In some embodiments, in Formulas V and VI, Str is TIFF2025531990000036.tif37165, where $ and * is as defined above, p is an integer of 2 to 6, and q is an integer of 2 to 8.

[0172] In some embodiments, in Formulas V and VI, AA1-[AA2] m are Val-Lys, Ala-Lys, Phe-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Arg, Ala-Phe, Val-Ala, Met- Lys, Asn-Lys, Ile-Pro, Ile-Val, Asp-Val, His-Val, Met-(D)Lys, Asn-(D)Lys, Val-(D)Asp, NorVal-(D)Asp, Ala -(D)Asp, Me3Lys-Pro, PhenylGly-(D)Lys, Met-(D)Lys, Asn-(D)Lys, Pro-(D)Lys, Met-(D)Lys, Met-Cit-Val, Gly- selected from Cit-Val, (D)Phe-Phe-Lys, (D)Ala-Phe-Lys, Gly-Phe-Leu-Gly, Ala-Leu-Ala-Leu, and Gly-Gly-Phe-Gly.

[0173] In some embodiments, in Formulas V and VI, m is 1 (i.e., AA1-[AA2] m is a dipeptide).

[0174] In some embodiments, in Formulas V and VI, AA1-[AA2]m is a dipeptide selected from Val-Lys, Ala-Lys, Phe-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit, and Trp-Cit.

[0175] In some embodiments, in Formula V: Z is TIFF2025531990000037.tif37165, where * is the point of attachment to the rest of the linker, Str is TIFF2025531990000038.tif22165, where $ is the point of attachment to Z, * is the point of attachment to the remainder of the linker, p is an integer from 2 to 6, and q is an integer from 2 to 8; m is 1, and AA1-[AA2] m is a dipeptide selected from Val-Lys, Ala-Lys, Phe-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit, and Trp-Cit.

[0176] In some embodiments, in Formula VI: Z' is a carbonyl group (-C(O)-) or TIFF2025531990000039.tif32165, where # is the point of attachment to the anti-cMet antibody construct; * is the point of attachment to the rest of the linker, Str is TIFF2025531990000040.tif22165, where $ is the point of attachment to Z', * is the point of attachment to the remainder of the linker, p is an integer from 2 to 6, and q is an integer from 2 to 8; m is 1, and AA1-[AA2] m is a dipeptide selected from Val-Lys, Ala-Lys, Phe-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit, and Trp-Cit.

[0177] In certain embodiments, in the Drug-Linker of Formula II, n is 1 and the Drug-Linker is of Formula VII or Formula VIII: TIFF2025531990000041.tif99165, wherein Z is a functional group capable of reacting with a targeting group on the anti-cMet antibody construct A; D has the structure shown in Formula II.

[0178] When incorporated into the ADCs of the disclosure, the drug-linkers of Formula VII and Formula VIII are represented by Formula IX and Formula X, respectively: TIFF2025531990000042.tif87128, wherein Z' is a linking group that connects the linker to the targeting group on the anti-cMet antibody construct A; # is the point of attachment to anti-cMet antibody construct A, D has the structure shown in Formula II.

[0179] In certain embodiments, in the Drug-Linker of Formula II, n is >1 and the Drug-Linker is of Formula XI: TIFF2025531990000043.tif32165, wherein Z is a functional group capable of reacting with a targeting group on the anti-cMet antibody construct A; Str 1 and Str 2 are each independently a stretcher, BU is a branching unit, AA1 and AA2 are each independently an amino acid, wherein AA1-[AA2] m forms a protease cleavage site, X is a self-immolative group; s and s' are each independently 0 or 1; m is 1, 2, or 3; o is 0, 1, or 2; t is 2 or 3, D has the structure shown in Formula II.

[0180] When incorporated into the ADCs of the disclosure, the drug-linker of Formula XI has the structure of Formula XII: TIFF2025531990000044.tif32165, wherein Z' is a linking group that connects the linker to the targeting group on the anti-cMet antibody construct A; Str 1 , Str 2 , BU, AA1, AA2, X, s, s', m, o, t, and D are as defined for formula XI; # indicates the point of attachment to anti-cMet antibody construct A.

[0181] In Formulas XI and XII, BU is a multifunctional (trifunctional or tetrafunctional) group that allows multiple components of the drug linker to be linked together. Examples of multifunctional groups that can be used as branching units (BU) include, but are not limited to, tris, amino acids with side chain functional groups (such as glutamic acid, aspartic acid, tyrosine, lysine, cysteine, serine, or threonine), trisubstituted aromatic compounds (such as 5-aminoisophthalic acid), Behera's amine (di-tert-butyl 4-amino-4-(3-(tert-butoxy)-3-oxopropyl)heptanedioate), and various dendron cores (see, for example, Newkome & Shreiner, 2010, Chem. Reviews, 110(10):6338-6442).

[0182] In some embodiments, in Formulas XI and XII, BU is an amino acid or beheramine.In some embodiments, in Formulas XI and XII, BU is glutamic acid or beheramine.

[0183] In some embodiments, in Formulas XI and XII, s is 1. In some embodiments, in Formulas XI and XII, s' is 1. In some embodiments, in Formulas XI and XII, s and s' are each 1.

[0184] In some embodiments, in formulas XI and XII, o is 0 (ie, X is absent).

[0185] In some embodiments, in formula XI, Z is a functional group capable of reacting with a thiol or amino group on the anti-cMet antibody construct A.

[0186] In some embodiments, in Formula XI: Z is TIFF2025531990000045.tif37165, where * is the point of attachment to the rest of the linker.

[0187] In some embodiments, in Formula XI: Z is TIFF2025531990000046.tif32165 where: * is the point of attachment to the rest of the linker.

[0188] In some embodiments, in Formula XII: Z' is a carbonyl group (-C(O)-) or TIFF2025531990000047.tif32165, where # is the point of attachment to anti-cMet antibody construct A; * is the point of attachment to the rest of the linker.

[0189] In some embodiments, in Formula XII: Z' is TIFF2025531990000048.tif32165, where # is the point of attachment to anti-cMet antibody construct A; * is the point of attachment to the rest of the linker.

[0190] In some embodiments, in Formulas XI and XII, Str 1 teeth, Selected from TIFF2025531990000049.tif53165, where: each R is independently H or C1-C6 alkyl; each p is independently an integer from 2 to 10; each q is independently an integer from 1 to 10; $ is the point of attachment to Z or Z', * is the point of attachment to the rest of the linker.

[0191] In some embodiments, in Formulas XI and XII, Str 2 teeth, Selected from TIFF2025531990000050.tif68165, where: each R is independently H or C1-C6 alkyl; each p is independently an integer from 2 to 10; each q is independently an integer from 1 to 10; $ is the attachment point to BU, * is the point of attachment to the rest of the linker.

[0192] In some embodiments, in Formulas XI and XII, Str 1 teeth, TIFF2025531990000051.tif37165, where p, q, $ and * Str 1 As defined above with respect to

[0193] In some embodiments, in Formulas XI and XII, Str 2 teeth, TIFF2025531990000052.tif53165, where p, q, $ and * Str 2 As defined above with respect to

[0194] In some embodiments, in Formulas XI and XII, Str 1 teeth, TIFF2025531990000053.tif37165, where $ and * Str 1 where p is an integer from 2 to 6 and q is an integer from 2 to 8.

[0195] In some embodiments, in Formulas XI and XII, Str 2 teeth, TIFF2025531990000054.tif46165, where $ and * Str 2 where p is an integer from 2 to 6 and q is an integer from 2 to 8.

[0196] In some embodiments, in Formulas XI and XII, AA1-[AA2] m are Val-Lys, Ala-Lys, Phe-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Arg, Ala-Phe, Val-Ala, Met- Lys, Asn-Lys, Ile-Pro, Ile-Val, Asp-Val, His-Val, Met-(D)Lys, Asn-(D)Lys, Val-(D)Asp, NorVal-(D)Asp, Ala -(D)Asp, Me3Lys-Pro, PhenylGly-(D)Lys, Met-(D)Lys, Asn-(D)Lys, Pro-(D)Lys, Met-(D)Lys, Met-Cit-Val, Gly- selected from Cit-Val, (D)Phe-Phe-Lys, (D)Ala-Phe-Lys, Gly-Phe-Leu-Gly, Ala-Leu-Ala-Leu, and Gly-Gly-Phe-Gly.

[0197] In some embodiments, in Formulas XI and XII, m is 1 (i.e., AA1-[AA2] m is a dipeptide).

[0198] In some embodiments, in Formulas XI and XII, AA1-[AA2] mis a dipeptide selected from Val-Lys, Ala-Lys, Phe-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit, and Trp-Cit.

[0199] In some embodiments, in Formula XI: Z is TIFF2025531990000055.tif37165, where * is the point of attachment to the rest of the linker, BU is an amino acid (e.g., glutamic acid) or beheramine; Str 1 teeth, TIFF2025531990000056.tif22165, where $ is the point of attachment to Z, * is the point of attachment to the remainder of the linker, p is an integer from 2 to 6, and q is an integer from 2 to 8; Str 2 teeth, TIFF2025531990000057.tif31165, where $ is the attachment point to BU, * is the point of attachment to the remainder of the linker, p is an integer from 2 to 6, and q is an integer from 2 to 8; m is 1, and AA1-[AA2] m is a dipeptide selected from Val-Lys, Ala-Lys, Phe-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit, and Trp-Cit; s is 1, s' is 1, o is 0.

[0200] In some embodiments, in Formula XII: Z' is a carbonyl group (-C(O)-) or TIFF2025531990000058.tif32165, where # is the point of attachment to anti-cMet antibody construct A; * is the point of attachment to the rest of the linker, BU is an amino acid (e.g., glutamic acid) or beheramine; Str 1 teeth, TIFF2025531990000059.tif22165, where $ is the point of attachment to Z', * is the point of attachment to the remainder of the linker, p is an integer from 2 to 6, and q is an integer from 2 to 8; Str 2 teeth, TIFF2025531990000060.tif32165, where $ is the attachment point to BU, * is the point of attachment to the remainder of the linker, p is an integer from 2 to 6, and q is an integer from 2 to 8; m is 1, and AA1-[AA2] m is a dipeptide selected from Val-Lys, Ala-Lys, Phe-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit, and Trp-Cit; s is 1, s' is 1, o is 0.

[0201] In certain embodiments, the drug-linker of formula II has formula XIII: TIFF2025531990000061.tif32165, wherein Z is a functional group capable of reacting with a targeting group on the anti-cMet antibody construct A; Str 1 and Str 2 are each independently a stretcher, BU is a branching unit, AA1 and AA2 are each independently an amino acid, wherein AA1-[AA2] m forms a protease cleavage site, s and s' are each independently 0 or 1; m is 1, 2, or 3; t is 1, 2, or 3; x is 0 or 1, where when x is 0, t is 1, and when x is 1, t is 2 or 3; D has the structure shown in Formula II.

[0202] When incorporated into the ADCs of the disclosure, the drug-linker of Formula XIII can be represented by Formula XIV: TIFF2025531990000062.tif32165, wherein Z' is a linking group that connects the linker to the targeting group on the anti-cMet antibody construct A; Str 1 , Str 2 , BU, AA1, AA2, s, s', m, t, x, and D are as defined for formula XIII; # indicates the point of attachment to anti-cMet antibody construct A.

[0203] In some embodiments, in Formulas XIII and XIV, x is 1 and BU is an amino acid or beheramine. In some embodiments, in Formulas XIII and XIV, x is 1 and BU is glutamic acid or beheramine.

[0204] In some embodiments, in Formulas XIII and XIV, s is 1. In some embodiments, in Formulas XI and XII, x is 1 and s' is 1. In some embodiments, in Formulas XI and XII, x is 1 and s and s' are each 1.

[0205] In some embodiments, in Formula XIII, Z is a functional group capable of reacting with a thiol or amino group on the anti-cMet antibody construct A.

[0206] In some embodiments, in Formula XIII: Z is TIFF2025531990000063.tif37165, where * is the point of attachment to the rest of the linker.

[0207] In some embodiments, in Formula XIV: Z' is a carbonyl group (-C(O)-) or TIFF2025531990000064.tif32165, where # is the point of attachment to anti-cMet antibody construct A; * is the point of attachment to the rest of the linker.

[0208] In some embodiments, in Formulas XIII and XIV, Str 1 teeth, Selected from TIFF2025531990000065.tif53165, where: each R is independently H or C1-C6 alkyl; each p is independently an integer from 2 to 10; each q is independently an integer from 1 to 10; $ is the point of attachment to Z or Z', * is the point of attachment to the rest of the linker.

[0209] In some embodiments, in Formulas XIII and XIV, Str 2 teeth, Selected from TIFF2025531990000066.tif68165, where: each R is independently H or C1-C6 alkyl; each p is independently an integer from 2 to 10; each q is independently an integer from 1 to 10; $ is the attachment point to BU, * is the point of attachment to the rest of the linker.

[0210] In some embodiments, in Formulas XIII and XIV, Str 1 teeth, TIFF2025531990000067.tif37165, where p, q, $ and * Str 1As defined above with respect to

[0211] In some embodiments, in Formulas XIII and XIV, Str 2 teeth, TIFF2025531990000068.tif53165, where p, q, $ and * Str 2 As defined above with respect to

[0212] In some embodiments, in Formulas XIII and XIV, Str 1 teeth, TIFF2025531990000069.tif37165, where $ and * Str 1 where p is an integer from 2 to 6 and q is an integer from 2 to 8.

[0213] In some embodiments, in Formulas XIII and XIV, Str 2 teeth, TIFF2025531990000070.tif45165, where $ and * Str 2 where p is an integer from 2 to 6 and q is an integer from 2 to 8.

[0214] In some embodiments, in Formulas XIII and XIV, AA1-[AA2] mare Val-Lys, Ala-Lys, Phe-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Arg, Ala-Phe, Val-Ala, Met- Lys, Asn-Lys, Ile-Pro, Ile-Val, Asp-Val, His-Val, Met-(D)Lys, Asn-(D)Lys, Val-(D)Asp, NorVal-(D)Asp, Ala -(D)Asp, Me3Lys-Pro, PhenylGly-(D)Lys, Met-(D)Lys, Asn-(D)Lys, Pro-(D)Lys, Met-(D)Lys, Met-Cit-Val, Gly- selected from Cit-Val, (D)Phe-Phe-Lys, (D)Ala-Phe-Lys, Gly-Phe-Leu-Gly, Ala-Leu-Ala-Leu, and Gly-Gly-Phe-Gly.

[0215] In some embodiments, in Formulas XIII and XIV, m is 1 (i.e., AA1-[AA2] m is a dipeptide).

[0216] In some embodiments, in Formulas XIII and XIV, AA1-[AA2] m is a dipeptide selected from Val-Lys, Ala-Lys, Phe-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit, and Trp-Cit.

[0217] Non-limiting examples of drug-linkers of Formula II are shown in Table 5, and non-limiting examples of ADCs comprising these drug-linkers are shown in Table 6. In certain embodiments, an ADC of Formula I comprises a drug-linker selected from the drug-linkers shown in Table 5. In certain embodiments, a drug-linker of Formula II is selected from the drug-linkers shown in Table 5. In certain embodiments, an ADC of Formula I is selected from the ADCs shown in Table 6, wherein A is an anti-cMet antibody construct and p is an integer from 1 to 8. In some embodiments, an ADC of Formula I is selected from the ADCs shown in Table 6, wherein A is an anti-cMet antibody construct and p is an integer from 2 to 6. In some embodiments, an ADC of Formula I is ADC001 or ADC002 shown in Table 6, wherein A is an anti-cMet antibody construct and p is an integer from 2 to 6. In some embodiments, the ADC of Formula I is selected from ADC003, ADC004, ADC005, and ADC006 shown in Table 6, wherein A is an anti-cMet antibody construct and p is an integer from 2 to 4. In some embodiments, the ADC of Formula I is ADC003 or ADC005 shown in Table 6, wherein A is an anti-cMet antibody construct and p is 2 or 3. In some embodiments, the ADC of Formula I is ADC004 or ADC006 shown in Table 6, wherein A is an anti-cMet antibody construct and p is 2.

[0218] [Table 5] TIFF2025531990000072.tif231150TIFF2025531990000073.tif231156TIFF2025531990000074.tif231101

[0219] [Table 6] TIFF2025531990000076.tif231106TIFF2025531990000077.tif231149TIFF2025531990000078.tif231103

[0220] preparation Anti-cMet antibody construct The anti-cMet antibody constructs described herein may be produced using standard recombinant methods known in the art (see, e.g., U.S. Pat. No. 4,816,567 and “Antibodies: A Laboratory Manual,” 2nd Edition, Ed. Greenfield, Cold Spring Harbor Laboratory Press, New York, 2014).

[0221] Typically, for recombinant production of an antibody construct, a polynucleotide or set of polynucleotides encoding the anti-cMet antibody construct is generated and inserted into one or more vectors for further cloning and / or expression in a host cell. The polynucleotide(s) encoding the anti-cMet antibody construct may be produced by standard methods known in the art (see, e.g., Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1994 & update, and "Antibodies: A Laboratory Manual," 2nd Edition, Ed. Greenfield, Cold Spring Harbor Laboratory Press, New York, 2014). As will be understood by those skilled in the art, the number of polynucleotides required for expression of an anti-cMet antibody construct will depend on the format of the construct, including whether the antibody construct includes a scaffold. For example, if the anti-cMet antibody construct is in a full-size antibody format with a homodimeric Fc, three polynucleotides, each encoding one polypeptide chain, will be required. If multiple polynucleotides are required, they may be incorporated into one vector or into two or more vectors.

[0222] Generally, for expression, a polynucleotide or set of polynucleotides is incorporated into an expression vector(s) along with one or more control elements, such as transcriptional elements required for efficient transcription of the polynucleotide. Examples of such control elements include, but are not limited to, promoters, enhancers, terminators, and polyadenylation signals. Those skilled in the art will understand that the selection of control elements will depend on the host cell selected for expression of the antibody construct and that such control elements may be derived from a variety of sources, including bacterial, fungal, viral, mammalian, or insect genes. The expression vector may optionally further comprise a heterologous nucleic acid sequence that facilitates expression or purification of the expressed protein. Examples include, but are not limited to, signal peptides and affinity tags such as metal affinity tags, histidine tags, avidin / streptavidin coding sequences, glutathione-S-transferase (GST) coding sequences, and biotin coding sequences. The expression vector may be an extrachromosomal or integrating vector.

[0223] Suitable host cells for cloning or expressing the anti-cMet antibody constructs include a variety of prokaryotic or eukaryotic cells known in the art. Prokaryotic host cells include, for example, E. coli, A. salmonicida, and B. subtilis cells. Eukaryotic host cells include, for example, mammalian cells, plant cells, insect cells, and yeast cells (such as Saccharomyces or Pichia cells). The selected host cells containing the expression vector(s) encoding the anti-cMet antibody construct may be cultured using conventional methods.

[0224] In certain embodiments, anti-cMet antibody constructs may be produced in eukaryotic cells. In some embodiments, anti-cMet antibody constructs may be produced in mammalian cells. Mammalian cell lines adapted to grow in suspension may be particularly useful for expressing antibody constructs. Examples include SV40 (COS-7) transformed monkey kidney CV1 cell line, human embryonic kidney (HEK) cell line 293 or 293 cells (see, e.g., Graham et al., 1977, J. Gen Virol., 36:59), baby hamster kidney cells (BHK), mouse Sertoli TM4 cells (see, e.g., Mather, 1980, Biol Reprod, 23:243-251), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma (HeLa) cells, canine kidney cells (MDCK), buffalo rat liver cells (BRL3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary carcinoma cells (MMT060562), TRI cells (see, e.g., Mather et al., 1982, Annals of Pharmacology, 2000, 12:101-102), and the like. Sci, 383:44-68), MRC5 cells, FS4 cells, Chinese hamster ovary (CHO) cells (including DHFR-CHO cells (Urlaub et al., 1980, Proc Natl Acad Sci USA, 77:4216)), and myeloma cell lines (such as Y0, NS0, and Sp2 / 0). Various examples of mammalian host cell lines suitable for the production of antibody constructs are reviewed in Yazaki & Wu, Methods in Molecular Biology, Vol. 248, pp. 255-268 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003).

[0225] In certain embodiments, the host cell can be a transient or stable higher eukaryotic cell line, such as a mammalian cell line. In some embodiments, the host cell can be a mammalian HEK293T, CHO, HeLa, NS0, or COS cell line, or a cell line derived from any one of these cell lines. In some embodiments, the host cell can be a stable cell line that allows mature glycosylation of the antibody construct.

[0226] Certain embodiments of the present disclosure relate to an isolated polynucleotide or set of polynucleotides that encode the anti-cMet antibody constructs described herein. A polynucleotide in this context can encode all or part of an anti-cMet antibody construct.

[0227] The terms "polynucleotide," "nucleic acid," and "nucleic acid molecule" are used interchangeably herein and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Non-limiting examples of polynucleotides include genes, gene fragments, messenger RNA (mRNA), cDNA, recombinant polynucleotides, plasmids, vectors, isolated DNA, isolated RNA, nucleic acid probes, and primers.

[0228] A polynucleotide "encoding" a given polypeptide is one that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A transcription termination sequence may be located 3' to the coding sequence.

[0229] Certain embodiments of the present disclosure relate to vectors (such as expression vectors) that contain one or more polynucleotides encoding the anti-cMet antibody constructs described herein. The polynucleotide(s) may be contained by a single vector or by two or more vectors. In some embodiments, the polynucleotides are contained by a multicistronic vector.

[0230] Certain embodiments of the present disclosure relate to host cells comprising a polynucleotide(s) encoding an anti-cMet antibody construct described herein, or one or more vectors comprising said polynucleotide(s). In some embodiments, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell, a human embryonic kidney (HEK) cell, or a lymphoid cell (e.g., a Y0, NS0, Sp20 cell).

[0231] Typically, anti-cMet antibody constructs are purified after expression. Proteins may be isolated or purified in a variety of ways known to those skilled in the art (see, e.g., Protein Purification: Principles and Practice, 3rd Ed., Scopes, Springer-Verlag, NY, 1994). Standard purification methods include one or more chromatographic techniques, including ion exchange chromatography, hydrophobic interaction chromatography, affinity chromatography, sizing (or gel) filtration chromatography, and reverse-phase chromatography, performed at atmospheric or elevated pressure using systems such as FPLC and HPLC. Additional purification methods include electrophoretic, immunological, precipitation, dialysis, and chromatofocusing techniques. Ultrafiltration and diafiltration techniques in conjunction with protein concentration may also be useful. As is well known in the art, various natural proteins bind to antibodies, and these proteins may be used to purify certain antibody constructs. For example, bacterial proteins A and G bind to the Fc region. Similarly, bacterial protein L binds to the Fab region of some antibodies. Purification may also be enabled by specific fusion partners. For example, the antibody may be attached to a glutathione resin if a GST fusion is used, or to a Ni tag if a His tag is used. +2 Purification may be performed using affinity chromatography or, if a flag tag is used, using an immobilized anti-flag antibody. The degree of purification required will vary depending on the intended use of the anti-cMet antibody construct. In some cases, no purification may be necessary.

[0232] In certain embodiments, the anti-cMet antibody construct is substantially pure. The term "substantially pure" (or "substantially purified"), when used with reference to the anti-cMet antibody constructs described herein, means that the antibody construct is substantially or essentially free from components that normally accompany or interact with the protein as found in its naturally occurring environment, such as in natural cells or, in the case of recombinantly produced constructs, in host cells. In certain embodiments, a substantially pure anti-cMet antibody construct is a protein preparation having less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5% (by dry weight) of contaminating protein.

[0233] Certain embodiments of the present disclosure relate to methods for producing an anti-cMet antibody construct, the method comprising culturing host cells into which one or more polynucleotides encoding the anti-cMet antibody construct or one or more expression vectors encoding the anti-cMet antibody construct have been introduced under conditions suitable for expression of the anti-cMet antibody construct, and optionally recovering the anti-cMet antibody construct from the host cells (or from the culture medium of the host cells). In some embodiments, the method further comprises subjecting the anti-cMet antibody construct to one or more purification steps.

[0234] Post-translational modifications In certain embodiments, the anti-cMet antibody constructs described herein may comprise one or more post-translational modifications. Such post-translational modifications may occur in vivo or may be performed in vitro after isolation of the anti-cMet antibody construct from a host cell.

[0235] Post-translational modifications include various modifications known in the art, such as glycosylation, acetylation, phosphorylation, amidation, deamidation, derivatization with known protecting / blocking groups, formylation, oxidation, reduction, proteolytic cleavage with cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4, etc., or specific chemical cleavage (see, e.g., Proteins - Structure and Molecular Properties, 2nd Ed., TECreighton, W.H. Freeman and Company, New York, 1993; Post-Translational Covalent Modification of Proteins, B.C. Johnson, Ed., Academic Press, New York, pp. 1-12, 1983; Seifter et al., 1990, Meth. Enzymol., 182:626-646; and Rattan et al., 1992, Ann. NY Acad. Sci., 663:48-62). In embodiments in which the anti-cMet antibody construct contains one or more post-translational modifications, the construct may contain the same type of modification at one or several sites, or it may contain different modifications at different sites.

[0236] Additional examples of post-translational modifications include, but are not limited to, the addition or removal of N- or O-linked carbohydrate chains, chemical modification of N- or O-linked carbohydrate chains, N- or C-terminal processing, attachment of chemical moieties to the amino acid backbone, and the addition or deletion of N-terminal methionine residues resulting from expression in prokaryotic host cells. Post-translational modifications can also include modification with a detectable label, such as an enzyme label, a fluorescent label, a luminescent label, an isotopic label, or an affinity label, to enable detection and isolation of the protein. Examples of suitable enzyme labels include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, and acetylcholinesterase. Examples of suitable prosthetic group complexes include, but are not limited to, streptavidin / biotin and avidin / biotin. Examples of suitable fluorescent materials include, but are not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, and phycoerythrin. Examples of luminescent materials include luminol and bioluminescent materials such as luciferase, luciferin, and aequorin. Examples of suitable radioactive materials include iodine, carbon, sulfur, tritium, indium, technetium, thallium, gallium, palladium, molybdenum, xenon, and fluorine.

[0237] Further examples of post-translational modifications include acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, gamma-carboxylation, GPI anchor formation, hydroxylation, iodination, methylation, myristylation, pegylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA-mediated addition of amino acids to proteins such as arginylation, and ubiquitination.

[0238] ADC ADCs of Formula I comprising anti-cMet antibody constructs may be prepared by standard methods known in the art (see, e.g., Bioconjugate Techniques (G.T. Hermanson, 2013, Academic Press)). Exemplary methods are provided herein. A variety of linkers and linker components are commercially available or may be prepared using standard synthetic organic chemistry techniques (see, e.g., March's Advanced Organic Chemistry (Smith & March, 2006, Sixth Ed., Wiley); Toki et al., (2002) J. Org. Chem. 67:1866-1872; Frisch et al., (1997) Bioconj. Chem. 7:180-186; Bioconjugate Techniques (G.T. Hermanson, 2013, Academic Press)). Additionally, various antibody-drug conjugation services are commercially available from companies such as Lonza Inc. (Allendale, NJ), Abzena PLC (Cambridge, UK), ADC Biotechnology (St. Asaph, UK), Baxter BioPharma Solutions (Baxter Healthcare Corporation, Deerfield, IL), and Piramal Pharma Solutions (Grangemouth, UK), and may be used to prepare ADCs.

[0239] Typically, preparation of the ADC involves first preparing a drug-linker DL (e.g., a drug-linker of Formula II) comprising one or more auristatin analogs and a linker L, and then conjugating the drug-linker DL to a suitable group on the anti-cMet antibody construct A. However, ligation of linker L to the anti-cMet antibody construct A, followed by ligation of the anti-cMet antibody construct-linker AL to one or more auristatin analogs D, remains an alternative approach that may be used in some embodiments.

[0240] Suitable groups on the anti-cMet antibody construct A for attachment of the linker L include sulfhydryl groups (e.g., on the side chain of a cysteine ​​residue), amino groups (e.g., on the side chain of a lysine residue), carboxylic acid groups (e.g., on the side chain of an aspartic acid or glutamic acid residue), and carbohydrate groups.

[0241] In certain embodiments, one or more naturally occurring cysteine ​​residues on the anti-cMet antibody construct A may be used to attach to the linker L via a sulfhydryl group of the cysteine. In certain embodiments, one or more naturally occurring lysine residues on the anti-cMet antibody construct A may be used to attach to the linker L via an amino group of the lysine.

[0242] Alternatively, one or more lysine residues on the anti-cMet antibody construct A may be chemically modified to introduce one or more sulfhydryl groups. Reagents that may be used to modify lysine residues include, but are not limited to, N-succinimidyl S-acetylthioacetate (SATA), N-succinimidyl-3-(2-pyridyldithio)propionate ("SPDP"), and 2-iminothiolane hydrochloride (Traut's reagent). Alternatively, one or more carbohydrate groups on the anti-cMet antibody construct A may be chemically modified to include one or more sulfhydryl groups.

[0243] Carbohydrate groups on the anti-cMet antibody construct A may also be oxidized to provide aldehyde (-CHO) groups (see, e.g., Laguzza et al., 1989, J. Med. Chem. 32(3):548-55), which can then be reacted with a linker L, for example, via a hydrazine or hydroxylamine group on the linker L.

[0244] Anti-cMet antibody construct A may also be modified to include additional cysteine ​​residues as described above or, for example, in U.S. Pat. Nos. 7,521,541, 8,455,622, and 9,000,130. Alternatively, anti-cMet antibodies may be modified to include one or more unnatural amino acids that provide a reactive handle, such as selenomethionine, p-acetylphenylalanine, formylglycine, or p-azidomethyl-L-phenylalanine, to allow for site-specific conjugation (see, e.g., Hofer et al., 2009, Biochemistry, 48:12047-12057; Axup et al., 2012, PNAS, 109:16101-16106; Wu et al., 2009, PNAS, 106:3000-3005; Zimmerman et al., 2014, Bioconj. Chem., 25:351-361). Anti-cMet antibody construct A may also be modified to include a non-naturally reactive group, such as an azide, that allows for conjugation to a linker via a complementary reactive group on the linker, e.g., by click chemistry (see, e.g., Chio & Bane, 2020, Methods Mol. Biol., 2078:83-97). A further option is the use of GlycoConnect™ technology (Synaffix BV, Nijmegen, Netherlands), which involves enzymatic remodeling of antibody glycans to enable attachment of a linker via metal-free click chemistry (see, e.g., European Patent No. EP 2911699).

[0245] Other protocols for modifying proteins for the attachment or association of linkers L are known in the art (see, e.g., Coligan et al., Current Protocols in Protein Science, vol. 2, John Wiley & Sons (2002)).

[0246] Alternatively, ADCs may be prepared using the enzyme transglutaminase, particularly bacterial transglutaminase (BTG) from Streptomyces mobaraensis (see, e.g., Jeger et al., 2010, Angew. Chem. Int. Ed., 49:9995-9997). BTG forms an amide bond between the side chain carboxamide of glutamine (typically an amine acceptor on an antibody) and an alkyleneamino group (typically an amine donor on a drug-linker), which may be, for example, the ε-amino group of lysine or a 5-amino-n-pentyl group. Antibodies may also be modified to include a glutamine-containing peptide, or "tag," that allows the antibody to be conjugated to a drug-linker using BTG conjugation (see, e.g., U.S. Patent Application Publication No. US2013 / 0230543 and International (PCT) Publication No. WO2016 / 144608).

[0247] A similar conjugation approach utilizes the enzyme sortase A. In this approach, an antibody is typically modified to contain a sortase A recognition motif (LPXTG, where X is any naturally occurring amino acid), and the drug-linker is designed to contain an oligoglycine motif (typically GGG) to enable sortase A-mediated transpeptidation (see, e.g., Beerli, et al., 2015, PLos One, 10:e0131177; Chen et al., 2016, Nature: Scientific Reports, 6:31899).

[0248] Once conjugation is complete, the average number of auristatin analog molecules conjugated to the anti-cMet antibody construct A (i.e., the "drug-to-antibody ratio" or DAR) may be determined by standard techniques such as UV / VIS spectroscopy, ELISA-based techniques, chromatographic techniques such as hydrophobic interaction chromatography (HIC), UV-MALDI mass spectrometry (MS), or MALDI-TOF MS. Additionally, optionally, the distribution of drug-conjugated forms (e.g., the fraction of anti-cMet antibody construct A containing zero, one, two, three, etc. conjugated auristatin analog molecules) may be analyzed, for example, by MS (with or without an accompanying chromatographic separation step), hydrophobic interaction chromatography, reverse-phase HPLC, or isoelectric focusing gel electrophoresis (IEF) (see, e.g., Wakankar et al., 2011, mAbs, 3:161-172).

[0249] Certain embodiments of the present disclosure relate to methods for preparing an ADC of Formula I, the method comprising conjugating a drug-linker of Formula II to an anti-cMet antibody construct. In some embodiments, the method comprises conjugating the drug-linker to a cysteine ​​residue on the anti-cMet antibody construct. In some embodiments, the anti-cMet antibody construct comprises one or more cysteine ​​insertion mutations, and the method comprises conjugating the drug-linker to the inserted cysteine ​​residue. In some embodiments, the method comprises conjugating the drug-linker to a lysine residue on the anti-cMet antibody construct.

[0250] Pharmaceutical Compositions For therapeutic use, the ADCs of the present disclosure are typically formulated as pharmaceutical compositions. Thus, certain embodiments of the present disclosure relate to pharmaceutical compositions comprising an ADC described herein and a pharmaceutically acceptable carrier, diluent, or excipient. Such pharmaceutical compositions may be prepared by known procedures using well-known and readily available ingredients.

[0251] Pharmaceutical compositions may be formulated for administration to a subject, for example, orally (including, for example, buccal or sublingually), topically, parenterally, rectally, or vaginally, or by inhalation or spray. As used herein, the term "parenteral" includes subcutaneous injection, as well as intradermal, intraarticular, intravenous, intramuscular, intravascular, intrasternal, and intrathecal injection or infusion. Pharmaceutical compositions will typically be formulated in a format suitable for the selected route of administration to a subject, for example, as a syrup, elixir, tablet, troche, lozenge, hard capsule, soft capsule, pill, suppository, oily or aqueous suspension, dispersible powder or granule, emulsion, injectable solution, or liquid. Pharmaceutical compositions may be provided as unit-dosage formulations.

[0252] In certain embodiments, a pharmaceutical composition comprising an ADC is formulated for parenteral administration, e.g., as a lyophilized preparation or an aqueous solution. Such a pharmaceutical composition may be provided, for example, in a unit dosage injectable form.

[0253] Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed. Examples of such carriers include buffers such as phosphate, citric acid, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl alcohol, benzyl alcohol, alkylparabens (such as methyl or propylparaben), catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol; low molecular weight (less than about 10 residues) polypeptides; serum albumin or These include, but are not limited to, proteins such as gelatin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates such as glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes such as Zn-protein complexes, and non-ionic surfactants such as polyethylene glycol (PEG).

[0254] In certain embodiments, compositions containing the ADC may be in the form of a sterile injectable aqueous or oily solution or suspension. Such suspensions may be formulated using suitable dispersing or wetting agents and / or suspending agents known in the art. Sterile injectable solutions or suspensions may contain the ADC in a non-toxic, parenterally acceptable diluent or carrier. Acceptable diluents and carriers that may be used include, for example, 1,3-butanediol, water, Ringer's solution, or isotonic sodium chloride solution. Additionally, sterile, fixed oils may be used as carriers. For this purpose, various bland, fixed oils may be used, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectable solutions. Auxiliaries such as local anesthetics, preservatives, and / or buffers may also be included in the injectable solution or suspension.

[0255] In certain embodiments, a composition comprising an ADC may be formulated for intravenous administration to humans. Typically, a composition for intravenous administration is a solution in sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizing agent and / or a local anesthetic such as lignocaine to ease pain at the injection site. Generally, the ingredients are supplied either separately or mixed together in a unit dosage form, e.g., as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent. When the composition is to be administered by inhalation, it can be dispensed from an inhalation bottle containing pharmaceutical-grade sterile water or saline. When the composition is administered by injection, an ampoule containing, for example, sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0256] Other pharmaceutical compositions and methods for preparing pharmaceutical compositions are known in the art and are described, for example, in "Remington: The Science and Practice of Pharmacy" (formerly "Remington's Pharmaceutical Sciences"), Gennaro, A., Lippincott, Williams & Wilkins, Philadelphia, PA (2000).

[0257] How to use Certain embodiments of the present disclosure relate to therapeutic uses of the ADCs described herein. Some embodiments relate to the use of the ADCs as therapeutic agents, e.g., as anti-cancer agents.

[0258] In certain embodiments, the ADCs described herein may be used in the treatment of cancer. Certain embodiments relate to methods of inhibiting the growth of cancer or tumor cells, inhibiting the proliferation of cancer or tumor cells, or treating cancer in a subject, the methods comprising administering an ADC of Formula I.

[0259] Certain embodiments of the present disclosure relate to methods of inhibiting the growth of cancer or tumor cells, the method comprising contacting the cells in vitro or in vivo with an ADC of Formula I. Some embodiments relate to methods of killing cancer or tumor cells, the method comprising contacting the cells in vitro or in vivo with an ADC of Formula I. Certain embodiments relate to the use of an ADC of Formula I in a method of inhibiting tumor growth in a subject.

[0260] Some embodiments relate to methods of treating a subject with cancer by administering to the subject an ADC of Formula I. In this context, treating the subject may result in one or more of: a reduction in tumor size; a slowing or prevention of tumor size increase; an increase in disease-free survival between the disappearance or elimination of a tumor and its reappearance; prevention of subsequent tumor development (e.g., metastasis); an increase in the time to progression; a reduction in one or more adverse symptoms associated with the tumor; and / or an increase in overall survival of the subject with cancer.

[0261] Exemplary cancers that may be treated with the ADCs described herein in certain embodiments include carcinomas (including adenocarcinomas and squamous cell carcinomas), melanomas, and sarcomas. Carcinomas and sarcomas are also often referred to as "solid tumors." Examples of commonly occurring solid tumors that may be treated with the ADCs described herein in certain embodiments include, but are not limited to, brain cancer, breast cancer, cervical cancer, colon cancer, head and neck cancer, kidney cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, uterine cancer, non-small cell lung cancer (NSCLC), and colorectal cancer. Various forms of lymphoma can also result in the formation of solid tumors and, therefore, may also be considered solid tumors in certain circumstances. Typically, the cancer to be treated is a cMet-expressing cancer.

[0262] Certain embodiments relate to methods of inhibiting the growth of cMet-positive tumor cells, the method comprising contacting the cells with an ADC of Formula I. The cells may be in vitro or in vivo. In certain embodiments, the ADC may be used in methods of treating a cMet-positive or cMet-overexpressing cancer or tumor in a subject.

[0263] Cancers that overexpress c-Met are typically solid tumors. Examples include, but are not limited to, ovarian cancer, lung cancer, breast cancer, gastric cancer, colon cancer, head and neck cancer, renal cancer, and pancreatic cancer. In certain embodiments, an ADC of Formula I may be used in methods of treating a subject with cMet-positive or cMet-overexpressing ovarian cancer, lung cancer, breast cancer, gastric cancer, colon cancer, head and neck cancer, renal cancer, or pancreatic cancer.

[0264] Medicine Kit Certain embodiments relate to pharmaceutical kits comprising an ADC of Formula I.

[0265] The kit typically includes a container holding the ADC and a label and / or package insert on or associated with the container. The label or package insert includes instructions customarily included in commercial packaging of therapeutic products, providing information about the indications, usage, dosage, administration, contraindications, and / or warnings regarding the use of such therapeutic product. The label or package insert may further include a notice in a form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceutical or biological products, such notice reflecting approval by that agency for manufacture, use, or sale for human or animal administration. In some embodiments, the container may have a sterile access port. For example, the container may be an intravenous solution bag or a vial having a stopper that can be pierced by a hypodermic injection needle.

[0266] In addition to the container holding the ADC, the kit may optionally include one or more additional containers containing other components of the kit, for example, a pharmaceutically acceptable buffer (such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, or dextrose solution), other buffers, or diluents.

[0267] Suitable containers include, for example, bottles, vials, syringes, intravenous solution bags, etc. The containers may be formed from a variety of materials, such as glass or plastic. Where appropriate, one or more components of the kit may be lyophilized or provided in a dried form, such as a powder or granules, and the kit may additionally include a suitable solvent for the reconstitution of the lyophilized or dried component(s).

[0268] Kits may further include other materials desirable from a commercial or user standpoint, including filters, needles, and syringes.

[0269] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. [Example]

[0270] Example 1: Anti-cMET antibody preparation 1.1 Antibody generation A cMet-targeting antibody, variant v32634, was prepared as described below. This antibody is based on telisotuzumab (ABT-700) (see Wang et al., 2016, BMC Cancer, 16:105), but lacks any modifications to the hinge region. The full-length, VH, VL, and CDR sequences of v32634 are provided in the Sequence Listing. Additionally, VH, VL, and CDR sequences are provided in Table 2 above. In addition to variant v32634, antibody variants containing various cysteine ​​insertion mutations, as described below, and a control variant, v17606, containing the hinge sequence modification contained by telisotuzumab, were also prepared. An additional control antibody, variant v17429, which is a bivalent version of the anti-cMet antibody MetMab (Onartuzumab), was also prepared. These variants are summarized in Table 1.3.

[0271] Variants were produced in a full-size antibody (FSA) format containing either two identical full-length heavy chains resulting in a homodimeric Fc region (HomoFc), or heterodimeric full-length heavy chains containing complementary mutations in the CH3 region to drive heterodimeric heavy chain pairing, resulting in a heterodimeric Fc region (HetFc).

[0272] Two identical full-length heavy chains encompassed by the HomoFc region bind to IGHG1 * The full-length heavy chains of the heterodimer (HetFc-A and HetFc-B) contained the human CH1-hinge-CH2-CH3 domain sequence of IGHG1 (SEQ ID NO: 31; see Table 1.1). * It contains the human CH1-hinge-CH2-CH3 domain sequence of 01, with the following mutations in the Fc region:

[0273] HetFc-A:T350V_L351Y_F405A_Y407V

[0274] HetFc-B:T350V_T366L_K392L_T394W

[0275] The sequences of HetFc-A (SEQ ID NO: 33) and HetFc-B (SEQ ID NO: 34) are provided in Table 1.1. * The human kappa CL sequence of 01 (SEQ ID NO: 32; see Table 1.1) was used in all constructs.

[0276] [Table 1.1]

[0277] The wild-type (WT) hinge sequence contained by variant v32634 and the cysteine ​​insertion variant, and the modified hinge sequence contained by variant v17606 and telisotuzumab are provided in Table 1.2.

[0278] [Table 1.2]

[0279] [Table 1.3] TIFF2025531990000082.tif220136

[0280] 1.2 Antibody production The antibody variants listed in Table 1.3 were produced using a heavy chain expression vector containing a heavy chain vector insert containing the signal peptide MRPTWAWWLFLVLLLALWAPARG (SEQ ID NO: 37) (Barash et al., 2002, Biochem and Biophys Res. Comm., 294:835-842) ligated into a pTT5 vector and a heavy chain clone terminating at residue G446 (EU numbering) of the CH3 domain, and a light chain expression vector containing a light chain vector insert containing the same signal peptide ligated into a pTT5 vector. The resulting heavy and light chain expression vectors were sequenced to confirm the correct reading frame and sequence of the encoding DNA. A representative example of a protocol for producing antibodies is as follows.

[0281] Heavy and light chains were expressed in 200 ml cultures of CHO-3E7 cells. Briefly, 1.7-2 x 10 6 CHO-3E7 cells at a density of 100 cells / ml with >95% viability were cultured in FreeStyle™ F17 medium (Thermo Fisher Scientific, Waltham, MA) supplemented with 4 mM glutamine (GE Life Sciences, Marlborough, MA) and 0.1% Pluronic® F-68 (Gibco / Thermo Fisher Scientific, Waltham, MA) at 37°C. A total volume of 200 ml of CHO-3E7 cells plus 1× antibiotic / antimycotic (GE Life Sciences, Marlborough, MA) was transfected with a total of 200 μg of DNA (100 μg antibody DNA and 100 μg GFP / AKT / stuffer DNA) using PEI-MAX® (Polyscience, Inc., Philadelphia, PA) at a DNA:PEI ratio of 1:4 (w / w). 24 hours after addition of the DNA-PEI mixture, 0.5 mM valproic acid (final concentration) + 1% (w / v) tryptone (final concentration) was added to the cells, which were then transferred to 32°C and incubated for a further 6 days before harvesting.

[0282] Protein-A purification was performed in batch mode or using a 1 mL HiTrap™ MabSelect™ SuRe™ column (Cytiva, Marlborough, MA). In batch mode, clarified supernatant samples were incubated in batch with mAb Select SuRe™ resin (GE Healthcare, Chicago, IL) that had been cleaned in place (CIP) with NaOH and equilibrated with Dulbecco's PBS (DPBS). The resin was poured into the CIPed column, which was then washed with DPBS. In both purification modes, protein was eluted with 100 mM sodium citrate buffer (pH 3.0). The pH of the eluted fractions was adjusted by adding 10% (v / v) 1 M HEPES (pH approximately 10.6-10.7) to a final pH of 6-7. Samples were buffer exchanged into DPBS. Protein was quantified based on absorbance at 280 nm (A280nm). Based on purity, variants were further purified by Protein-A purification followed by preparative SEC chromatography on a Superdex™ 200 Increase 10 / 30 column (GE Healthcare, Chicago, IL) in DPBS mobile phase.

[0283] Following purification, sample purity was assessed by electrophoresis under non-reducing and reducing conditions using the High Throughput Protein Express assay and a Caliper LabChip® GXII or GXII Touch HT (Perkin Elmer, Waltham, MA). The procedure was performed according to the HT Protein Express LabChip® User Guide Version 2, with the following modifications: Either 2 μl or 5 μl of antibody sample (concentration range 5–2000 ng / μl) was added to separate wells in a 96-well plate (BioRad, Hercules, CA) along with 7 μl of HT Protein Express Sample Buffer (Perkin Elmer, catalog no. 760328). The antibody samples were then denatured at 70°C for 15 minutes. The LabChip® instrument was run using the HT Protein Express Chip (Perkin Elmer, Waltham, MA) and the Ab-200 assay settings. In some cases, sample purity was also monitored by SDS-PAGE under reducing and non-reducing conditions.

[0284] Yields for the variants (after Protein-A purification) range from mg to gram and are summarized in Table 1.4.

[0285] 1.3 Assessment of purity by analytical size exclusion chromatography (SEC) The purity of the variants was determined by UPLC-SEC. For analytical SEC runs, an Agilent Advance Bio SEC column (300 Å, 2.7 μm, 7.8 × 150 mm) (Agilent Technologies, Inc., Santa Clara, CA; serial number 6377910-24) was loaded with 5 column volumes of buffer A (150 mM NaCl) at room temperature. xThe column was equilibrated with HCl (pH 6.95). Typically, 20-30 μg of sample at a concentration of 2-3 mg / mL was loaded onto the column and pumped isocratically at 1 mL / min for 7 minutes. Absorbance at 280 nm was reported. For each sample, the chromatogram was integrated to obtain full baseline-to-baseline integration of each peak, while providing reasonable separation between partially resolved peaks. Based on the SEC profile of the control IgG1 antibody, trastuzumab, the peak corresponding to the major IgG component (approximate retention time 3.3 min) was reported as monomeric. Any peaks occurring before 3.3 min were designated HMWS, and any peaks occurring after 3.3 min, excluding the solvent peak (>5.2 min), were designated LMWS.

[0286] The purity for each of the cysteine ​​insertion variants is summarized in Table 1.4.

[0287] [Table 1.4]

[0288] SDS-PAGE results for a representative cysteine ​​insertion variant v29001 under non-reducing (NR) and reducing (R) conditions, corresponding to the full-size antibody and intact heavy and light chains, are shown in Figure 2A, and the UPLC-SEC chromatogram for variant v29001 is shown in Figure 2B. Based on the UPLC-SEC chromatogram, the sample purity for this variant was approximately 99%, reflecting high species homogeneity.

[0289] Example 2: Characterization of cysteine ​​insertion variants The molecular weights of the cysteine ​​insertion variants described in Example 1 were assessed by liquid chromatography-mass spectrometry (LC-MS). Differential scanning calorimetry (DSC) was used to assess the thermal stability of two of the variants.

[0290] 2.1 Liquid Chromatography-Mass Spectrometry (LC-MS) The exact mass of the purified variants was determined by LC-MS. The variants were diluted to 1 mg / mL in PBS (pH 7.4) and then deglycosylated. For deglycosylation, typically 1 μg of EndoS was used per 10 μg of antibody, and the reaction mixture was incubated at room temperature for 1 hour. In some cases, the samples were also reduced by adding 1 μL of 500 mM tris(2-carboxyethyl)phosphine (TCEP) to each 10 μL sample, followed by incubation at 70°C for 1 hour. Finally, the samples were analyzed on an LC-MS quadrupole time-of-flight (QTOF) system (Agilent 1290 HPLC coupled to an Agilent 6545 QTOF; Agilent Technologies, Inc., Santa Clara, CA) with an injection volume of 1 μL each. The detailed procedure is described below. Column: PLRP-S 1000Å, 8µM, 50×2.1mm (Agilent Technologies, Inc., Santa Clara, CA) Mobile phase C: 0.1% formic acid, 0.025% trifluoroacetic acid, and 10% isopropyl alcohol in H2O Mobile phase D: 0.1% formic acid and 10% isopropyl alcohol in acetonitrile Detection: Signal A (280 nm, bandwidth 4.0), Signal B (220 nm, bandwidth 4.0) ●Slope: TIFF2025531990000084.tif53165 ● Post-run time: 2 minutes

[0291] The measured deglycosylated masses of the full-size variants were in agreement with the theoretical masses (see Table 2.1).

[0292] [Table 2.1]

[0293] 2.2 Differential scanning calorimetry (DSC) The melting temperature (Tm) difference between two of the cysteine ​​insertion variants, v28983 and v29001, and the corresponding parent antibody, v17427, was determined by DSC as follows: 400 μL of purified sample at a concentration of 0.2 mg / mL or 0.4 mg / mL in PBS was used for DSC analysis on a MicroCal VP-Capillary DSC (GE Healthcare, Chicago, IL). At the beginning of each DSC run, five buffer blank injections were performed to stabilize the baseline, and a buffer injection was provided before each sample injection for reference. Each sample was scanned from 20°C to 100°C at a rate of 60°C / hour with low feedback, an 8-second filter, a 5-minute pre-Tstat, and 70 psi nitrogen pressure. The resulting thermograms were referenced and analyzed using Origin 7 software (OriginLab Corporation, Northampton, MA). DSC measurements are summarized in Table 2.2.

[0294] [Table 2.2]

[0295] Example 3: Preparation of Drug-Linker The following abbreviations are used in this example: ACN = acetonitrile, DCM = dichloromethane, DMF = dimethylformamide, DMSO = dimethyl sulfoxide, LC / MS = liquid chromatography / mass spectrometry, LC-MSD = liquid chromatography-mass selective detector, SEC = size exclusion chromatography, HIC = hydrophobic interaction chromatography, RP-UPLC = reversed-phase ultra high performance liquid chromatography, HPLC = high performance liquid chromatography, MT = maleimidotriethyleneglycolate, TCEP = tris(2-carboxyethyl)phosphine, TFA = trifluoroacetic acid, VC = valine-citrulline, UHPLC = ultra high performance liquid chromatograph.

[0296] The following general method was used.

[0297] Flash chromatography: Crude reaction products were purified on a Biotage® Isolera™ automated flash system (Biotage, Charlotte, NC) using a Biotage® Snap Ultra column (10, 25, 50, or 100 g) (Biotage, Charlotte, NC) eluting with a linear gradient of ethyl acetate / hexane or methanol / dichloromethane. Alternatively, reverse-phase flash purification was performed using a Biotage® Snap Ultra C18 column (12, 30, 60, or 120 g) eluting with a linear gradient of CH3CN + 0.1% TFA / HO + 0.1% TFA. Purified compounds were isolated either by removal of organic solvents on a rotary evaporator or by lyophilization from acetonitrile / water mixtures.

[0298] Preparative HPLC: Reverse-phase HPLC of crude compounds was performed on an Agilent 1260 Infinity II preparative LC-MSD system (Agilent Technologies, Inc., Santa Clara, CA) using a Kinetex® 5-μm EVO C18 100 Å (250 × 21.2 mm) column (Phenomenex, Torrance, CA) eluting with a linear gradient of CH3CN + 0.1% TFA / HO + 0.1% TFA. Purified compounds were isolated by lyophilization of acetonitrile / water mixtures.

[0299] NMR: 1 H NMR spectra were collected on either a Bruker AVANCE III 300 spectrometer (300 MHz) or a Bruker AVANCE III 400 spectrometer (400 MHz) (Bruker Corporation, Billerica, MA). Chemical shifts are in parts per million (ppm).

[0300] 2,2-Dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azaheptadecan-17-oic acid: TIFF2025531990000087.tif18165 was obtained from ChemPep Inc. (Wellington, FL).

[0301] 3.1 (S)—N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((4-aminophenyl)sulfonamido)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamide)-N,3-dimethylbutanamide (Compound 1) TIFF2025531990000088.tif27165 Prepared as described in International Publication No. WO2016 / 041082.

[0302] 3.2 (S)-2-((S)-2-amino-3-methylbutanamido)-N-(4-(N-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)sulfamoyl)phenyl)-5-ureidopentanamide (Compound 15) TIFF2025531990000089.tif43165 Prepared as described in International Publication No. WO2019 / 173911.

[0303] 3.3 2,3,5,6-Tetrafluorophenyl 3-(2-(2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethoxy)ethoxy)propanoate (Compound 2) TIFF2025531990000090.tif27165 Flask 1: In a dry 250 mL RB flask, 3-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}propanoic acid (10.0 g, 45.2 mmol, 1 equiv.) and maleic anhydride (4.43 g, 45.2 mmol, 1 equiv.) were dissolved in anhydrous DMF (15 mL) and then stirred overnight at room temperature under nitrogen. The next day, the reaction was determined to be complete by LC / MS. To the reaction was added 2,3,5-collidine (21.9 g, 24.1 mL, 181 mmol, 4 equiv.), and the mixture was cooled to 0 °C. Flask 2: In a separate flask, 2,3,4,5-tetrafluorophenol (30.0 g, 181 mmol, 4 equiv.) was dissolved in 45 mL of anhydrous DMF and cooled to 0° C., at which point trifluoroacetic anhydride (38.0 g, 25.4 mL, 1.49 g / mL, 181 mmol, 4 equiv.) was added dropwise over 2 minutes. The resulting solution was stirred at 0° C. for 10 minutes, after which 2,3,5-collidine (21.9 g, 24.1 mL, 0.91 g / mL, 181 mmol, 4 equiv.) was added over 3 minutes. The final mixture was allowed to stir at 0° C. for 15 minutes and then added to the solution in Flask 1 over 3 minutes, after which the final mixture was allowed to warm to room temperature and stirred for 48 hours. A small amount of intermediate remained as determined by LC / MS, and the reaction was stirred for an additional 72 hours, at which point the conversion was determined to be fully complete by LC / MS. The reaction was acidified with 1 M HCl (75 mL) and extracted with EtO (3 × 100 mL). The combined organic layers were then washed with 5% LiCl (60 mL) and brine (30 mL), then dried over MgSO and concentrated in vacuo. The crude product was purified via reverse-phase flash chromatography with a gradient of 10-50% ACN / HO + 0.1% TFA. Fractions containing the product were pooled for lyophilization, and the title compound was recovered as a pale orange oil (14.5 g, 32.4 mmol, 71.4%).

[0304] LC / MS: C 19 H 19 Calculated m / z for F4NO7 = 449.11, Found [M+H] + = 450.2 m / z. 1H NMR (300 MHz, MeOD) δ 7.42 (tt, J = 10.5, 7.2 Hz, 1H), 6.81 (s, 2H), 3.87 (t, J = 6.0 Hz, 2H), 3.71 - 3.55 (m, 12H), 2.98 (t, J = 6.0 Hz, 2H).

[0305] 3.4 Di-tert-butyl 4-(3-(tert-butoxy)-3-oxopropyl)-4-(3-(2-(2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethoxy)ethoxy)propanamido)heptanedioate (Compound 3) TIFF2025531990000091.tif481651,7-Di-tert-butyl 4-amino-4-[3-(tert-butoxy)-3-oxopropyl]heptanedioate (2.89 g, 6.95 mmol, 1.1 equiv.) and compound 2 (2.84 g, 6.32 mmol, 1 equiv.) were dissolved in 10 mL of anhydrous DMF along with 1-hydroxybenzotriazole monohydrate (0.968 g, 6.32 mmol, 1 equiv.). N-Ethyldiisopropylamine (1.63 g, 2.21 mL, 0.74 g / mL, 12.6 mmol, 2 equiv.) was added over 1 min at room temperature with rapid stirring. After 1 h, the reaction was determined to be complete by LC / MS and was diluted with 1 M HCl (5 mL) and ACN (3 mL) and then purified via reverse-phase chromatography with a gradient of 10-100% ACN / HO + 0.1% TFA. The products were pooled and concentrated in vacuo, then brine (5 mL) was added and the aqueous layer was extracted with EtO (3 × 50 mL). The organics were pooled and dried with brine (5 mL) and MgSO, then filtered and evaporated in vacuo to recover the title compound as an oily off-white solid (4.18 g, 6.00 mmol, 94.9%).

[0306] LC / MS: C 35 H 58 N2O 12 Calculated m / z value for = 698.40, Found value [M+H] + = 699.6 m / z. 1H NMR (400 MHz, CDCl3) δ 6.72 (s, 2H), 6.13 (s, 1H), 3.78 - 3.69 (m, 4H), 3.67 - 3.63 (m, 2H), 3.63 - 3.61 (m, 8H), 2.40 (t, J = 5.8 Hz, 2H), 2.27 - 2.15 (m, 6H), 2.03 - 1.92 (m, 6H), 1.44 (s, 27H).

[0307] 3.5 4-(2-carboxyethyl)-4-(3-(2-(2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethoxy)ethoxy)propanamido)heptanedioic acid (compound 4) TIFF2025531990000092.tif34165Compound 3 (4.18 g, 6.00 mmol, 1 equiv) was dissolved in 33% TFA / DCM (15 mL) in a 500 mL round-bottom flask. After stirring at room temperature for 18 hours, the reaction was determined to be complete by LC / MS. The reaction was concentrated to dryness in vacuo, and the residue was co-evaporated with ACN (3 x 10 mL). The residue was taken up in 10 mL of 2:1 HO / ACN and lyophilized. The title compound was recovered as an oil, which was approximately 25% overweight and assumed to contain residual TFA and HO (4.0 g, assuming a quantitative yield of 6.00 mmol, 100%).

[0308] LC / MS: C 23 H 34 N2O 12 Calculated m / z for = 530.21, Found [M+H] + = 531.4 m / z. 1 H NMR (300 MHz, MeOD) δ 6.84 (s, 2H), 3.74 - 3.68 (m, 4H), 3.68 - 3.63 (m, 4H), 3.63 - 3.57 (m, 8H), 2.46 - 2.39 (m, 2H), 2.39 - 2.25 (m, 4H), 2.10 - 1.97 (m, 6H).

[0309] 3.6 Bis(2,3,5,6-tetrafluorophenyl) 4-(3-(2-(2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethoxy)ethoxy)propanamido)-4-(3-oxo-3-(2,3,5,6-tetrafluorophenoxy)propyl)heptanedioate (Compound 5) Compound 4 (3.34 g, 6.30 mmol, 1 equiv.) was dissolved in ACN (50 mL) along with 2,3,5,6-tetrafluorophenol (4.18 g, 25.2 mmol, 4 equiv.) in a 50 mL round-bottom flask and stirred at 0 °C. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (4.83 g, 25.2 mmol, 4 equiv.) was added. After 30 min, LC / MS indicated approximately 60% conversion, with the major impurity being a partially esterified intermediate. Additional 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (500 mg) was added and stirred for 1 h, at which point conversion was nearly complete. The reaction was concentrated in vacuo to a volume of approximately 8 mL, then diluted with 1 M HCl (5 mL) and HO (5 mL) and purified via reverse-phase chromatography with a gradient of 10-75% ACN / HO + 0.1% TFA. Product fractions were pooled, concentrated in vacuo, and then extracted with EtO (2 x 40 mL). The organics were pooled and washed with brine (30 mL), then dried over MgSO and filtered to recover the title compound as a clear, colorless oil (2.60 g, 2.67 mmol, 42.3% yield).

[0310] LC / MS: C 41 H 34 F 12 N2O 12 Calculated m / z value for = 974.2, Detected value [M+H] + = 975.4 m / z. 1H NMR (300 MHz, CDCl3) δ 7.03 (tt, J = 9.8, 7.0 Hz, 3H), 6.71 (s, 2H), 6.62 (s, 1H), 3.82 - 3.76 (m, 2H), 3.76 - 3.69 (m, 2H), 3.69 - 3.59 (m, 10H), 2.81 (dd, J = 9.0, 6.8 Hz, 6H), 2.54 (t, J = 5.5 Hz, 2H), 2.37 - 2.26 (m, 6H).

[0311] 3.7 Bis(2,3,5,6-tetrafluorophenyl)(tert-butoxycarbonyl)-L-glutamic acid (compound 6) To a 250 mL round-bottom flask containing glutamic acid (1.80 g, 7.28 mmol, 1 equiv.), ACN (30 mL) and 2,3,5,6-tetrafluorophenol (2.54 g, 15.3 mmol, 2.1 equiv.) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (2.93 g, 15.3 mmol, 2.1 equiv.) were added. The reaction was stirred at room temperature for 18 h, at which point it was determined to be complete by LC / MS. The reaction mixture was concentrated in vacuo to a crude oil and then redissolved in EtOAc (50 mL) and 1 M HCl (20 mL). The organic layer was washed with 1 M HCl (2 × 20 mL), saturated NaHCO (20 mL), and 1 × brine (20 mL), then dried over MgSO, filtered, and evaporated in vacuo to give the title compound as a white solid (3.27 g, 6.02 mmol, 82.7% yield).

[0312] LC / MS: C 22 H 17 Calculated m / z for F8NO6 = 543.09, Found [M+Na] + = 556.4 m / z. 1H NMR (400 MHz, CDCl3) δ 7.13 - 6.97 (m, 2H), 5.17 (s, 1H), 4.83 (s, 1H), 2.94 (q, J = 7.2 Hz, 2H), 2.63 - 2.50 (m, 1H), 2.39 - 2.24 (m, 1H), 1.50 (s, 9H).

[0313] 3.8 (S)-15-((tert-butoxycarbonyl)amino)-14,18-dioxo-4,7,10,22,25,28-hexaoxa-13,19-diazahentriacontanedioic acid (compound 7) To a flask containing compound 6 (1.00 g, 1.84 mmol, 1 equiv.) and 3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)propanoic acid (0.855 g, 3.87 mmol, 2.1 equiv.), ACN (15 mL) and saturated NaHCO solution (1.14 M, 9.59 mL, 11.0 mmol, 6 equiv.) were added. The reaction was stirred at room temperature for 18 h, at which point it was determined to be complete by LC / MS. Assuming a quantitative yield, the reaction was used without purification.

[0314] LC / MS: C 28 H 51 N3O 14 Calculated m / z value for = 653.34, Found value [M+H] + = 654.7 m / z.

[0315] 3.9 Bis(2,3,5,6-tetrafluorophenyl)(S)-15-((tert-butoxycarbonyl)amino)-14,18-dioxo-4,7,10,22,25,28-hexaoxa-13,19-diazahentriacontanedioate (Compound 8) To a reaction solution containing compound 7 (1.20 g, 1.84 mmol, 1 equiv.), 1 M NaHPO (5 mL) and 1 M HCl (5 mL) were added to lower the pH to approximately 5. 2,3,5,6-Tetrafluorophenol (0.641 g, 3.87 mmol, 2.1 equiv.) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.06 g, 5.52 mmol, 3.0 equiv.) were added, and the reaction was stirred at room temperature for 18 h, at which point LC / MS showed the presence of an intermediate species as well as the product. Additional 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (300 mg) was added to drive the reaction to completion. The reaction mixture was concentrated in vacuo to remove most of the ACN and then purified via reverse-phase chromatography with a gradient of 10-100% ACN / HO + 0.1% TFA. Product fractions were pooled and concentrated in vacuo to remove ACN, then extracted with EtO (50 mL) and 3×DCM (50 mL). The organics were pooled and dried over MgSO, then filtered and evaporated in vacuo to recover the title compound as a clear oil (1.05 g, 1.11 mmol, 60.1%).

[0316] LC / MS: C 40 H 51 F8N3O 14 Calculated value m / z = 949.32, detected value [M+H] + = 950.8 m / z. 1 H NMR (400 MHz, CDCl3) δ 7.12 (s, 1H), 7.07 - 6.97 (m, 2H), 6.76 (s, 1H), 5.66 (d, J = 7.6 Hz, 1H), 4.12 (d, J = 7.2 Hz, 1H), 3.89 (t, J = 6.2 Hz, 4H), 3.71 - 3.61 (m, 16H), 3.61 - 3.57 (m, 2H), 3.54 - 3.36 (m, 3H), 2.96 (td, J = 6.2, 1.8 Hz, 4H), 2.40 - 2.19 (m, 2H), 2.11 - 1.92 (m, 2H), 1.43 (s, 9H).

[0317] 3.10 tert-Butyl((6S,9S,25S,43S,46S)-1,51-diamino-6,46-bis((4-(N-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidine-2- (yl)-3-methoxy-2-methylpropanoyl)sulfamoyl)phenyl)carbamoyl)-9,43-diisopropyl-1,8,11,24,28,41,44,51-octaoxo-14,17,20,32,35,38-hexaoxa-2,7,10,23,29,42,45,50-octaazahenpentacontan-25-yl)carbamate (Compound 9) TIFF2025531990000097.tif49165

[0318] Compound 8 (570 mg, 0.6 mmol, 1 equiv.) was dissolved in 2 mL of DMF, and compound 15 (1.66 g, 80 w / w%, 1.32 mmol, 2.2 equiv.) was added as a DMF solution (3 mL). The resulting solution was stirred at room temperature, and N-ethyldiisopropylamine (0.310 g, 0.419 mL, 0.741 g / mL, 2.40 mmol, 4 equiv.) was added. The reaction was allowed to stir at room temperature for 42 hours, at which point LC / MS indicated the reaction was complete. The reaction was diluted with 1 M NaH2PO4 (3 mL) and 1 M HCl (1 mL) and purified via reverse-phase chromatography with a gradient of 10-55% ACN / HO + 0.1% TFA. Product fractions were pooled and evaporated in vacuo to recover the title compound as a white solid (1.00 g, 0.379 mmol, 63.2%).

[0319] LC / MS: C 124 H 215 N 23 O 34 Calculated m / z for S2 = 2635.53, Found [M+3H] 3+ = 879.8 m / z. 1H NMR (400 MHz, MeOD) δ 7.90 (d, J = 8.9 Hz, 3H), 7.77 (d, J = 20.8 Hz, 5H), 4.70 (t, J = 8.7 Hz, 1H), 4.60 - 4.52 (m, 3H), 4.28 (dd, J = 17.8, 7.2 Hz, 2H), 4.11 (d, J = 18.5 Hz, 3H), 3.77 (s, 3H), 3.66 - 3.57 (m, 15H), 3.54 (d, J = 5.5 Hz, 2H), 3.33 (dt, J = 3.3, 1.7 Hz, 61H), 3.22 - 3.11 (m, 5H), 2.66 - 2.47 (m, 8H), 2.41 (d, J = 5.4 Hz, 8H), 2.32 (d, J = 7.9 Hz, 2H), 2.21 - 1.98 (m, 4H), 1.94 (d, J = 19.9 Hz, 2H), 1.68 - 1.53 (m, 2H), 1.45 (s, 11H), 1.21 - 1.09 (m, 6H), 1.03 (dq, J = 17.9, 6.7 Hz, 37H), 0.93 - 0.83 (m, 10H).

[0320] 3.11 (S)-2-Amino-N1,N5-bis((6S,9S)-1-amino-6-((4-(N-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)sulfamoyl)phenyl)carbamoyl)-9-isopropyl-1,8,11-trioxo-14,17,20-trioxa-2,7,10-triazadocosan-22-yl)pentanediamide (Compound 10) TIFF2025531990000098.tif51165 Compound 9 (850 mg, 0.322 mmol, 1 equiv.) was dissolved in 10% TFA / DCM (10 mL) and stirred at room temperature. The reaction was complete within 2 hours as determined by LC / MS. The reaction was evaporated under vacuum to an oily residue, co-evaporated with ACN (10 mL), and then redissolved in 10 mL of 2:1 HO / ACN and purified via reverse-phase chromatography using a gradient of 10-50% ACN / HO+0.1%. Product fractions were pooled and lyophilized to recover the title compound as a white solid (0.845 g, 0.294 mmol, 91.1%).

[0321] LC / MS: C 119 H 207 N 23 O 32 Calculated m / z for S2 = 2535.48 Found [M+3H] 3+ = 846.6 m / z. 1H NMR (400 MHz, MeOD) δ 8.03 - 7.92 (m, 7H), 7.92 (s, 2H), 4.72 (t, J = 8.4 Hz, 1H), 4.56 (dd, J = 9.3, 4.8 Hz, 3H), 4.30 - 4.20 (m, 2H), 4.16 - 4.02 (m, 3H), 3.98 (d, J = 14.4 Hz, 1H), 3.92 - 3.82 (m, 3H), 3.77 (qd, J = 6.5, 3.2 Hz, 9H), 3.68 - 3.45 (m, 36H), 3.46 - 3.37 (m, 7H), 3.37 - 3.28 (m, 31H), 3.29 - 3.19 (m, 1H), 2.95 (d, J = 15.1 Hz, 16H), 2.59 (dd, J = 8.1, 3.8 Hz, 8H), 2.51 (d, J = 7.7 Hz, 2H), 2.43 (dq, J = 9.1, 6.7 Hz, 5H), 2.13 (dt, J = 11.9, 6.7 Hz, 7H), 2.05 - 1.87 (m, 1H), 1.80 (ddd, J = 20.2, 14.6, 9.2 Hz, 2H), 1.71 - 1.54 (m, 4H), 1.49 - 1.36 (m, 1H), 1.18 - 0.96 (m, 42H), 0.88 (q, J = 7.1 Hz, 9H).

[0322] 3.12 (S)-N 1 、N 5-bis((6S,9S)-1-amino-6-((4-(N-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)sulfa (2-(2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethoxy)ethoxy)propanamido)pentanediamide (Drug-Linker 003) TIFF2025531990000099.tif42165

[0323] In a 50 mL round-bottom flask, compound 10 (840 mg, 0.292 mmol, 1 equiv.) and 2,3,5,6-tetrafluorophenyl 3-(2-{2-[2-(2,5-dioxopyrrol-1-yl)ethoxy]ethoxy}ethoxy)propanoate (0.144 g, 0.321 mmol, 1.1 equiv.) were dissolved in DMF (5 mL). N-Ethyldiisopropylamine (0.189 g, 0.255 mL, 0.74 g / mL, 1.46 mmol, 5 equiv.) was added and stirred at room temperature. After 1 h, LC / MS showed the reaction was complete. The reaction was diluted with 1 M HCl (5 mL) and purified via reverse-phase chromatography using a gradient of 10–60% ACN / HO + 0.1% TFA. Product fractions were pooled and lyophilized to recover the title compound as a white solid (0.660 g, 0.217 mmol, 74.2%).

[0324] LC / MS:C 132 H 224 N 24 O 38 Calculated m / z for S2 = 2818.58, Detected value [M+3H] 3+ = 941.0 m / z. 1H NMR (300 MHz, MeOD) δ 7.99 - 7.82 (m, 8H), 6.85 (s, 2H), 4.77 - 4.70 (m, 1H), 4.62 - 4.49 (m, 3H), 4.30 - 4.20 (m, 2H), 4.17 - 4.03 (m, 2H), 3.86 (d, J = 7.3 Hz, 2H), 3.83 - 3.66 (m, 9H), 3.66 - 3.46 (m, 40H), 3.43 - 3.23 (m, 48H), 3.16 (s, 6H), 2.95 (d, J = 10.9 Hz, 12H), 2.59 (d, J = 6.8 Hz, 6H), 2.53 (s, 1H), 2.45 (d, J = 5.8 Hz, 4H), 2.22 (d, J = 9.1 Hz, 5H), 2.01 (s, 4H), 1.60 (d, J = 6.9 Hz, 2H), 1.52 - 1.37 (m, 2H), 1.18 - 0.96 (m, 53H), 0.89 (q, J = 6.8 Hz, 6H).

[0325] 3.13 2,3,5,6-Tetrafluorophenyl 2,2-dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azaheptadecan-17-oic acid ester (compound 11) In a 50 mL round-bottom flask, 2,2-dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azaheptadecan-17-oic acid (1.412 g, 4.394 mmol, 1 equiv.), 2,3,5,6-tetrafluorophenol (0.803 g, 4.83 mmol, 1.1 equiv.), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.926 g, 4.83 mmol, 1.1 equiv.) were dissolved in ACN (15 mL). The reaction was stirred at room temperature for 18 h, at which point LC / MS indicated the reaction was complete. The crude product was recovered by evaporation of the solvent and redissolved in EtO (100 mL) and HO (20 mL). The organic layer was washed with saturated NaHCO3 (3 x 20 mL), 1 M HCl (2 x 20 mL) and brine (20 mL), then dried over MgSO4, filtered and the filtrate evaporated to dryness in vacuo to recover the title compound as a clear / colorless oil (2.06 g, 4.39 mmol, 99%).

[0326] LC / MS: C 20 H 27 Calculated m / z for F4NO7 = 469.17 Found [M+H-Boc] + = 370.2 m / z. 1 H NMR (300 MHz, CDCl3) δ 7.03 (tt, J = 9.9, 7.1 Hz, 1H), 5.05 (d, J = 15.3 Hz, 1H), 3.92 (t, J = 6.2 Hz, 2H), 3.77 - 3.62 (m, 8H), 3.60 - 3.51 (m, 2H), 3.34 (t, J = 5.2 Hz, 2H), 2.99 (t, J = 6.3 Hz, 2H), 1.47 (s, 9H).

[0327] 3.14 tert-Butyl ((6S,9S)-1-amino-6-((4-(N-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)sulfamoyl)phenyl)carbamoyl)-9-isopropyl-1,8,11-trioxo-14,17,20-trioxa-2,7,10-triazadocosan-22-yl)carbamate (compound 12) Compound 15 (3.00 g, 80% w / w, 2.38 mmol, 1.1 equiv.) and compound 11 (1.02 g, 2.16 mmol, 1 equiv.) were dissolved in DMF (10 mL) in a round-bottom flask. N-Ethyldiisopropylamine (0.838 g, 1.13 mL, 0.74 g / mL, 6.49 mmol, 3 equiv.) was added and stirred at room temperature. The reaction was complete within 30 min as determined by LC / MS. The reaction was concentrated to approximately 5 mL on a rotary evaporator, diluted with a premixed solution of HO (7 mL) and TFA (1 mL), and then purified via reverse-phase chromatography using a gradient of 10–45% ACN / HO + 0.1% TFA. Product fractions were pooled and lyophilized to recover the title compound as a white solid (1.80 g, 1.26 mmol, 84.6%).

[0328] LC / MS: C 62 H 109 N 11 O 17 Calculated m / z for S = 1311.77 Found [M+2H] +2 = 657.2 m / z. 1H NMR (300 MHz, MeOD) δ 8.02 - 7.91 (m, 2H), 7.90 - 7.81 (m, 1H), 4.73 (t, J = 8.4 Hz, 1H), 4.56 (dt, J = 8.7, 4.2 Hz, 1H), 4.23 (ddd, J = 8.6, 5.8, 2.6 Hz, 1H), 4.16 - 4.01 (m, 1H), 3.94 - 3.82 (m, 1H), 3.82 - 3.69 (m, 3H), 3.62 (d, J = 2.3 Hz, 7H), 3.57 - 3.47 (m, 2H), 3.38 (s, 1H), 3.35 - 3.27 (m, 1H), 3.23 (t, J = 5.6 Hz, 2H), 3.16 (s, 2H), 2.99 - 2.90 (m, 6H), 2.65 - 2.55 (m, 2H), 2.53 - 2.33 (m, 1H), 2.19 - 2.08 (m, 1H), 2.05 (s, 1H), 2.02 - 1.84 (m, 1H), 1.84 - 1.71 (m, 1H), 1.68 - 1.55 (m, 1H), 1.45 (s, 9H), 1.18 - 0.94 (m, 22H), 0.89 (q, J = 6.9 Hz, 3H)

[0329] 3.15 (S)-2-((S)-1-amino-14-isopropyl-12-oxo-3,6,9-trioxa-13-azapentadecan-15-amido)-N-(4-(N-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)sulfamoyl)phenyl)-5-ureidopentanamide (Compound 13) Compound 12 (1.80 g, 1.26 mmol, 1 equiv.) was dissolved in 10% TFA / DCM (15 mL) in a 50 mL round-bottom flask and stirred at room temperature for 2 h, at which point LC / MS indicated the reaction was complete. The reaction was evaporated to dryness in vacuo, redissolved in 2:1 HO / ACN solution (8 mL), and lyophilized to recover the title compound as an oily white solid (1.81 g, 1.25 mmol, 99%).

[0330] LC / MS: C 57 H 101 N 11 O 15 Calculated m / z for S = 1211.72 Found [M+2H-Boc] +2 = 607.2 m / z. 1 H NMR (300 MHz, MeOD) δ 8.02 - 7.93 (m, 2H), 7.93 - 7.81 (m, 2H), 4.73 (d, J = 8.7 Hz, 1H), 4.60 - 4.46 (m, 1H), 4.24 (d, J = 6.9 Hz, 1H), 4.15 - 4.03 (m, 1H), 3.87 (d, J = 5.7 Hz, 1H), 3.77 (dd, J = 6.1, 2.8 Hz, 1H), 3.72 (t, J = 5.2 Hz, 2H), 3.69 - 3.62 (m, 7H), 3.35 - 3.29 (m, 11H), 3.19 - 3.10 (m, 4H), 3.03 - 2.90 (m, 6H), 2.59 (t, J = 6.2 Hz, 2H), 2.56 - 2.49 (m, 1H), 2.49 - 2.32 (m, 3H), 2.12 (dt, J = 13.4, 6.7 Hz, 1H), 2.05 (s, 1H), 1.98 (d, J = 4.8 Hz, 1H), 1.90 (t, J = 6.8 Hz, 1H), 1.82 - 1.71 (m, 1H), 1.65 - 1.54 (m, 1H), 1.52 - 1.38 (m, 1H), 1.20 - 0.96 (m, 13H), 0.89 (q, J = 7.0 Hz, 3H).

[0331] 3.16 4-((6S,9S)-1-amino-6-((4-(N-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)sulfamoyl)phenyl)carbamoyl)-9-isopropyl-1,8,11,24-tetraoxo-14,17,20-trioxa-2,7,10,23-tetraazahexacosan-26-yl)-N 1 , N 7 -bis((6S,9S)-1-amino-6-((4-(N-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)sulfa (2-(2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethoxy)ethoxy)propanamide)heptanediamide (Drug-Linker 004) TIFF2025531990000103.tif68165

[0332] Compound 13 (3.104 g, 2.154 mmol, 4.2 equiv) was dissolved in anhydrous DMF (8 mL). Compound 5 (500 mg, 0.513 mmol, 1 equiv) was dissolved separately in DMF (1 mL). The two solutions were combined and rinsed with DMF (3 mL). N-ethyldiisopropylamine (0.796 g, 1.07 mL, 0.74 g / mL, 6.16 mmol, 12 equiv) was added and stirred at room temperature. The reaction was complete within 3 h as determined by LC / MS. The reaction was neutralized with TFA (1 mL), diluted with 1 M HCl (3 mL) and HO (12 mL), and then purified via reverse-phase chromatography using a gradient of 10-50% ACN / HO + 0.1% TFA. The product fraction was isolated and lyophilized to recover the title compound as a white solid (1.07 g, 0.513 mmol, 46.6%).

[0333] LC / MS: C 194 H 331 N 35 O 54 Calculated m / z for S3 = 4112.34, Found [M+4H] +4 = 1029.8 m / z. 1H NMR (300 MHz, MeOD) δ 8.03 - 7.81 (m, 9H), 6.85 (s, 2H), 4.74 (d, J = 8.8 Hz, 2H), 4.61 - 4.51 (m, 3H), 4.24 (t, J = 6.4 Hz, 3H), 4.12 - 4.06 (m, 3H), 3.86 (d, J = 7.6 Hz, 3H), 3.82 - 3.67 (m, 7H), 3.67 - 3.57 (m, 31H), 3.54 (t, J = 5.5 Hz, 5H), 3.38 (s, 5H), 3.36 (s, 3H), 3.35 - 3.32 (m, 39H), 3.31 (d, J = 3.8 Hz, 9H), 3.16 (d, J = 7.8 Hz, 6H), 2.95 (d, J = 10.8 Hz, 18H), 2.56 (d, J = 15.7 Hz, 6H), 2.49 - 2.41 (m, 3H), 2.26 - 2.17 (m, 3H), 2.06 - 1.97 (m, 5H), 1.64 - 1.56 (m, 2H), 1.48 - 1.42 (m, 2H), 1.17 - 0.96 (m, 49H), 0.89 (q, J = 6.8 Hz, 8H).

[0334] 3.17 Atomic acid (2,3,5,6-Turonic acid) (Compound 14) TIFF2025531990000104.tif321652,3,5,6-Tetrafluorophenol (37.0 g, 222 mmol, 2.1 equiv.) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (44.7 g, 233 mmol, 2.2 equiv.) were dissolved in ACN (200 mL) at 0 °C. Adipic acid (15.5 g, 106 mmol, 1 equiv.) was added in small portions. The reaction became clear and colorless. The reaction was stirred at room temperature for 18 h, at which point LC / MS indicated the reaction was complete. The solvent was evaporated in vacuo, and the residue was redissolved in EtO (200 mL) and 1 M HCl (50 mL). The organic layer was washed with 1 M HCl (2 × 50 mL) and brine (2 × 50 mL). Due to the formation of significant emulsion, the separatory funnel was allowed to stand for 1 hour between shaking. The organic layer was dried over MgSO4, filtered, and the solvent evaporated under vacuum. A white solid was collected, dissolved in hot DCM (30 mL), filtered, and then allowed to stand in a fume hood exposed to air for 54 hours, at which point crystal formation became evident. The solution was allowed to stand at -20 °C for an additional 18 hours, then the crystals were collected by filtration and rinsed with cold DCM (50 mL). 32.1 g of crystals were recovered from the first crop. The crystallization protocol was repeated on the mother liquor, and an additional 5.4 g of crystals was recovered. The two crops of crystals were combined to give the title compound as a crystalline solid (37.5 g, 84.8 mmol, 79.9%).

[0335] LC / MS: C 18 H 10 m / z calculated for F8O4 = 442.05, m / z not detected. 1 H NMR (300 MHz, DMSO) δ 7.95 (tt, J = 10.9, 7.4 Hz, 2H), 2.95 - 2.78 (m, 4H), 1.87 - 1.55 (m, 4H).

[0336] 3.18 2,3,5,6-tetrafluorophenyl 6-(((S)-1-(((S)-1-((4-(N-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)sulfamoyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-6-oxohexanoate (Drug-Linker 002) In a 50 mL round-bottom flask, compound 15 (2.35 g, 1.90 mmol, 1 equiv.) was dissolved in DMF (2 mL). Separately, compound 14 (5.00 g, 11.4 mmol, 6 equiv.) was dissolved in DMF (10 mL) and then added to the first solution. The reaction was stirred at room temperature, and N,N-diisopropylethylamine (0.736 g, 0.992 mL, 5.70 mmol, 3 equiv.) was added. The reaction was complete within 30 min as indicated by LCMS. TFA (1 mL) was added, and the reaction was concentrated in vacuo to a volume of 12 mL. The concentrate was purified via reverse-phase chromatography using a gradient of 10-50% ACN / HO + 0.1% TFA. Product fractions were pooled and lyophilized to recover the title compound as a white solid powder (2.17 g, 1.55 mmol, 81.5%).

[0337] LC / MS: C 60 H 92 F4N 10 O 14 Calculated m / z for S = 1284.56, Found [M+H] + = 1285.8 m / z. 1H NMR (300 MHz, MeOD) δ 8.02 - 7.93 (m, 2H), 7.89 (d, J = 9.0 Hz, 1H), 7.83 (d, J = 9.0 Hz, 1H), 7.43 (tt, J = 10.6, 7.3 Hz, 1H), 4.74 (d, J = 8.8 Hz, 1H), 4.56 (dt, J = 9.4, 4.8 Hz, 1H), 4.20 (dd, J = 9.7, 7.5 Hz, 1H), 4.10 (s, 1H), 3.96 - 3.82 (m, 1H), 3.72 (t, J = 4.7 Hz, 1H), 3.51 (d, J = 9.3 Hz, 1H), 3.38 (s, 1H), 3.36 - 3.32 (m, 6H), 3.30 (d, J = 5.4 Hz, 3H), 3.17 (s, 2H), 3.12 (d, J = 6.9 Hz, 1H), 2.95 (d, J = 10.2 Hz, 6H), 2.83 - 2.74 (m, 1H), 2.63 - 2.48 (m, 2H), 2.46 (d, J = 6.9 Hz, 1H), 2.44 - 2.34 (m, 3H), 2.21 - 2.01 (m, 1H), 1.89 (dd, J = 14.3, 7.7 Hz, 1H), 1.80 (q, J = 3.5 Hz, 1H), 1.60 (d, J = 7.4 Hz, 2H), 1.18 - 0.95 (m, 19H), 0.89 (q, J = 6.9 Hz, 3H).

[0338] 3.19 2,5-dioxopyrrolidin-1-yl(6S,9S,27S)-1-amino-27-(((6S,9S)-1-amino-6-((4-(N-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)sulfamoyl)phenyl)carbamoyl)-9-isopropyl-1,8,11-trioxo-14,17,20-trioxa-2,7,10-triazadocosane -22-yl)carbamoyl)-6-((4-(N-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)sulfamoyl)phenyl)carbamoyl)-9-isopropyl-1,8,11,24,29-pentaoxo-14,17,20-trioxa-2,7,10,23,28-pentaazatetratriacontan-34-oic acid ester (Drug-Linker 005) In a 50 mL round-bottom flask, compound 10 (104.0 mg, 0.0361 mmol, 1 equiv.) was dissolved in 2 mL of DMF. Bis(2,5-dioxopyrrolidin-1-yl) adipate (98.4 mg, 0.289 mmol, 8.0 equiv.) and N-ethyldiisopropylamine (23.3 mg, 0.0314 mL, 0.74 g / mL, 0.181 mmol, 5 equiv.) were added and stirred at room temperature. The reaction was determined to be complete after 30 min by LC / MS. The reaction mixture was acidified with 0.5 mL of 1 M HCl, diluted with 4 mL of HO, and then purified via reverse-phase chromatography using a gradient of 10-50% ACN / HO + 0.1% TFA. The product-containing fractions were lyophilized to recover the title compound as a white solid (93.6 mg, 0.0313 mmol, 86.7%).

[0339] LC / MS: C129 H 218 N 24 O 37 Calculated m / z value for S2 = 2760.54, detected value [M+2H] +2 = 1382.0 m / z. 1 H NMR (400 MHz, MeOD) δ 8.01 - 7.94 (m, 3H), 7.91 (d, J = 8.8 Hz, 1H), 7.86 (d, J = 8.8 Hz, 1H), 4.74 (d, J = 8.6 Hz, 1H), 4.60 - 4.51 (m, 2H), 4.43 - 4.33 (m, 1H), 4.24 (t, J = 7.3 Hz, 1H), 4.11 (s, 1H), 3.93 - 3.82 (m, 1H), 3.81 - 3.68 (m, 4H), 3.61 (s, 10H), 3.56 - 3.52 (m, 2H), 3.52 - 3.49 (m, 0H), 3.38 (s, 2H), 3.19 - 3.11 (m, 3H), 2.97 (s, 3H), 2.93 (s, 5H), 2.85 (t, J = 1.7 Hz, 3H), 2.67 (s, 2H), 2.63 - 2.55 (m, 3H), 2.52 (d, J = 8.0 Hz, 1H), 2.46 (t, J = 6.6 Hz, 1H), 2.35 - 2.27 (m, 1H), 2.16 - 2.00 (m, 2H), 1.99 - 1.87 (m, 1H), 1.80 - 1.68 (m, 4H), 1.68 - 1.57 (m, 1H), 1.49 - 1.37 (m, 1H), 1.17 - 1.​​​3.20 2,3,5,6-tetrafluorophenyl(6S,9S,27S)-1-amino-27-(((6S,9S)-1-amino-6-((4-(N-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)sulfamoyl)phenyl)carbamoyl)-9-isopropyl-1,8,11-trioxo-14,17,20-trioxa-2,7,10-triazadocosane -22-yl)carbamoyl)-6-((4-(N-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)sulfamoyl)phenyl)carbamoyl)-9-isopropyl-1,8,11,24,29-pentaoxo-14,17,20-trioxa-2,7,10,23,28-pentaazatetratriacontan-34-oic acid ester (Drug-Linker 006) TIFF2025531990000107.tif42165 Drug-Linker 005 (17 mg, 0.00568 mmol, 1 equiv.) was dissolved in a 2:1 mixture of diH2O:ACN (3 mL). The pH was raised to approximately 11 with saturated NaHCO3 (0.2 mL), and the reaction was allowed to stir at room temperature for 18 h. Hydrolysis of the NHS ester was determined to be complete by LC / MS. The reaction was acidified to pH 5 with 1 M NaH2PO4 (1 mL), and 2,3,5,6-tetrafluorophenol (4.72 mg, 0.0284 mmol, 5 equiv.) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (5.45 mg, 0.0284 mmol, 5 equiv.) were added. After 2 h, the reaction was determined to be complete by LC / MS. The reaction mixture was purified via reverse-phase chromatography with a gradient of 10-50% ACN / HO+0.1% TFA. The product-containing fractions were pooled and lyophilized to recover the title compound as a white solid (3.7 mg, 0.00122 mmol, 21.4%).

[0341] LC / MS: C 131 H 215 F4N 23 O 35 Calculated m / z for S2 = 2811.52, Found [M+2H] +2 = 1407.0 m / z.

[0342] Alternatively, Drug-Linker 006 may be prepared via the same methods as Drug-Linker 005.

[0343] 3.21 (S)—N-(4-(N-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)sulfamoyl)phenyl)-2-((S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-14-isopropyl-12-oxo-3,6,9-trioxa-13-azapentadecan-15-amido)-5-ureidopentanamide (Drug-Linker 001) TIFF2025531990000108.tif37165 Prepared as described in International Publication No. WO2019 / 173911.

[0344] Example 4: Preparation of additional multivalent drug-linkers Drug-Linker 007 and Drug-Linker 008 Alternatively, the trivalent Drug-Linker structure exemplified in Drug-Linker 004 (Example 3.16) can be adapted for conjugation to lysine residues by utilizing 2,3,4,5-tetrafluorophenol or N-hydroxysuccinimide activated esters similar to those utilized in Drug-Linker 002 (Example 3.18) and Drug-Linker 006 (Example 3.20), and Drug-Linker 005 (Example 3.19), respectively. Such Drug-Linkers (Drug-Linker 007 and Drug-Linker 008) may be prepared by reacting adipic anhydride with 1,7-di-tert-butyl 4-amino-4-[3-(tert-butoxy)-3-oxopropyl]heptanedioate under amide bond-forming conditions similar to those used to generate Compound 3, followed by further evolution into the final Drug-Linker. See Scheme 1 (Figure 3).

[0345] Example 5: Conjugation and characterization of anti-cMET antibody-drug conjugates The antibody-drug conjugates (ADCs) shown in Table 5.1 were prepared as described below.

[0346] 5.1 Preparation of ADCs by stochastic lysine conjugation An exemplary protocol for the preparation of ADCs via stochastic lysine conjugation is provided below.

[0347] A solution (2.3 mL) of variant v17427 (25 mg) in PBS (pH 7.4) was reacted with 4–22 molar equivalents of drug-linker 002 (10–20 mM dissolved in DMSO) at 5 mg / mL in PBS (pH 7.4) in the presence of 5–10% (v / v, final) DMSO. The reaction mixture was mixed by pipetting and then centrifuged at 400 × g for 3 min. The resulting solution was incubated at room temperature for 16–20 h before purification.

[0348] 5.2 Preparation of ADCs by Stochastic Cysteine ​​Conjugation An exemplary protocol for the preparation of ADCs by stochastic cysteine ​​conjugation is provided below.

[0349] A solution of variant v17427 in PBS (pH 7.4) was reduced (5-10 mg / mL final concentration) by the addition of 5 mM diethylenetriaminepentaacetic acid (DTPA) (11.4 mL in PBS, pH adjusted to 7.4) and 10 mM aqueous tris(2-carboxyethyl)phosphine (TCEP) (591 μL, 2.2 equiv.). The reduction reaction proceeded for 1-3 hours at 37°C. The reduced protein was then reacted with an excess of Drug-Linker 001 (6-10 equiv., 10-20 mM DMSO stock solution) on ice for 1-2 hours. The conjugation reaction was quenched by the addition of an excess of N-acetyl-L-cysteine ​​solution (6-10 equiv.) from a 10 mM stock solution in water. The quenched reaction was incubated on ice for 30 minutes before purification.

[0350] 5.3 Preparation of ADCs by site-specific conjugation to cysteine ​​inserts An exemplary protocol for the preparation of ADCs by site-specific conjugation to an inserted cysteine ​​residue is provided below. This protocol, or a similar protocol, has been applied to the site-specific conjugation of Drug-Linker 001, Drug-Linker 003, and Drug-Linker 004.

[0351] A solution (202 mL) of variant v29001 (2 g) in PBS (pH 7.4) was reduced by the addition of 10 mM DTPA (24 mL in PBS, pH adjusted to 7.4) and 25 mM aqueous TCEP (13.7 mL, 25 equivalents). After 3-4 hours at 37°C, the reduced antibody was diluted to approximately 250 mL with PBS and filtered through a Pellicon® XL ultrafiltration module (Ultracel 30 kDa 0.005 m) using approximately 3 dialysis volumes of PBS (pH 7.4). 2 The antibody was purified using a 500-kJ / mL (MilliporeSigma, Burlington, MA; PXC030C50) HPLC system. The purified antibody was then reoxidized with 25 molar equivalents of dehydroascorbic acid (DHAA) (50 mM stock solution in DMSO) for 16-20 hours at 2-8°C. To the reoxidized antibody (1.6 g, 220 mL) was added 5.9 mL of Drug-Linker 004 (5.5 molar equivalents) from a 10 mM stock solution in DMSO. The conjugation reaction was allowed to proceed for 3-4 hours at room temperature with mixing. The conjugation reaction was quenched by adding an excess of N-acetyl-L-cysteine ​​solution (5.6 mL, 5.25 equivalents) from a 10 mM stock solution (10% in DMSO). The quenched reaction was incubated at room temperature with mixing for 30 minutes, then incubated at 2-8°C for 16-20 hours before purification.

[0352] 5.4 Purification and Characterization of ADCs ADCs prepared at scales greater than 50 mg were purified using a Pellicon® XL ultrafiltration module (MilliporeSigma, Burlington, MA). Briefly, the crude ADC solution was diluted or concentrated to approximately 5-10 mg / mL with 10 mM NaOAc (pH 5.5) and purified using 8-15 diavolumes of 10 mM NaOAc (pH 4.5) on a Pellicon® XL ultrafiltration module (Ultracel 30 kDa 0.005 m 2 The purified ADC was then sterile filtered (0.2 μm).

[0353] ADCs prepared on a scale of less than 50 mg were purified by Zeba™ Spin desalting column, 40 MWCO (ThermoFisher Scientific, Waltham, MA) pre-equilibrated with 10 mM NaOAc (pH 4.5) according to the manufacturer's instructions.

[0354] The control variant v17606 conjugated to the drug-linker MCvcPABC-MMAE was purified using column chromatography as described in International Publication No. WO 2017 / 201204. Briefly, the crude ADC sample was applied to a HiTrap™ Butyl HP column (Cytiva Life Sciences, Marlborough, MA) using an ammonium sulfate / sodium phosphate buffer and eluted with sodium phosphate buffer containing 20% ​​isopropyl alcohol. ADC species with average drug-to-antibody ratios (DARs) of 2 and 4 were enriched and combined at a 1:1 ratio to yield an ADC with an average DAR of approximately 3. The purified ADC was formulated in 10 mM histidine (pH 6.0) buffer and sterile filtered.

[0355] Following purification, the concentrations of the ADCs were determined by BCA assay with reference to standard curves generated using the respective unconjugated parent antibodies. Alternatively, concentrations were estimated by measuring absorbance at 280 nm using the calculated extinction coefficients of the antibody sequences. The ADCs were also characterized by hydrophobic interaction chromatography (HIC) and size exclusion chromatography (SEC), as described below.

[0356] 5.4.1 Hydrophobic Interaction Chromatography The ADCs were analyzed by HIC to estimate the drug-to-antibody ratio (DAR). Chromatography was performed using a TSKgel® Butyl-NPR column (2.5 μm, 4.6 × 35 mm; TOSOH Bioscience GmbH, Griesheim, Germany) with a gradient of 95 / 5% MPA / MPB to 5 / 95% MPA / MPB (MPA = 1.5 M (NH4)2SO4, 25 mM NaCl) over a 12-minute period at a flow rate of 0.5 mL / min. x PO4 (pH 7) and MPB = 75% 25 mM Na x Analysis was performed on an Agilent Infinity II 1290 HPLC (Agilent Technologies, Santa Clara, CA) using a gradient of 25% HCl (pH 7, 25% isopropanol). Detection was by absorbance at 280 nm.

[0357] 5.4.2 Size Exclusion Chromatography The degree of aggregation of the ADC (approximately 15-150 μg, 5 μL injection volume) was assessed by SEC on an Agilent Infinity II 1260 HPLC (Agilent Technologies, Santa Clara, CA) using an AdvanceBio SEC column (300 Å, 2.7 μm, 7.8 × 150 mm) (Agilent, Santa Clara, California) and a mobile phase consisting of 150 mM phosphate (pH 6.95) and a flow rate of 1 mL / min. Detection was by absorbance at 280 nm.

[0358] Table 5.1 summarizes the properties evaluated for each of the ADCs.

[0359] [Table 5.1]

[0360] Example 6: In vitro cytotoxicity of stochastic cysteine-conjugated anti-cMET antibody-drug conjugate (DAR4) The cell growth inhibitory (cytotoxic) potency of anti-cMet ADCs containing variant v17427 (HetFc, unmodified hinge) conjugated to drug-linker 001 or MCvcPABC-MMAE by stochastic cysteine ​​conjugation at an average DAR of 4 was determined in a panel of four cMet-expressing cell lines as described below: EBC-1 (lung squamous cell carcinoma), H292 (lung cancer), BT-20 (breast cancer), and SW48 (colon cancer).

[0361] Briefly, cells were seeded at densities of 1,000–1,500 cells / well in 384-well plates and treated with increasing concentrations of test substances formulated in complete cell growth medium. Treated cells were incubated for 4–5 days under standard culture conditions (37°C / 5% CO2). After incubation, CellTiter-Glo® Reagent (Promega Corporation, Madison, WI; Catalog No. G7570) was spiked into each well, and luminescence corresponding to the ATP present in each well was measured using a Synergy™ H1 microplate (BioTek Instruments, Winooski, VT). Percent cytotoxicity values ​​were calculated using ATP-measured RLU values ​​(relative light units) based on blank wells (no test substance, vehicle only) and plotted against test substance concentration using GraphPad Prism 8 software (GraphPad Software, San Diego, CA).

[0362] result The results are summarized in Table 5.1. The DAR-matched v17427-MCvcPABC-MMAE DAR4 ADC exhibited lower in vitro cytotoxicity compared to the v17427-drug-linker 001 ADC in the high- and medium-cMet-expressing cell lines EBC-1, HT-29, and BT-20. The difference in cytotoxicity was less evident in the highest cMet-expressing cell line tested, EBC-1. Neither ADC exhibited significant cytotoxicity in the low-cMet-expressing cell line W48. The free payload from Drug-Linker 001 (Compound 1) exhibited the expected cytotoxicity in all tumor cell lines, although it was inferior to the v17427-drug-linker 001 ADC.

[0363] [Table 6.1]

[0364] Example 7: Activation of the cMET pathway by a stochastic cysteine-conjugated anti-cMET antibody-drug conjugate (DAR4) The cMet pathway activation activity of ADCs containing anti-cMet antibody variant v17427 (HetFc, unmodified hinge) and variant v17427 stochastically cysteine-conjugated to drug-linker 001 or MCvcPABC-MMAE at DAR4 was assessed by ELISA to measure cell proliferation and AKT phosphorylation as downstream indicators of cMET activation.

[0365] Cell proliferation: Briefly, H596 lung cancer cells were serum-starved overnight by replacing the complete growth medium with serum-free RPMI-1640 (Thermo Fisher Scientific Inc., Waltham, MA; Catalog No. A1049101) at 37°C and 5% CO2. Cells were then detached with cell dissociation buffer (Thermo Fisher Scientific Inc., Catalog No. 13151014), resuspended in RPMI-1640 supplemented with 1% FBS (v / v) (Thermo Fisher Scientific Inc., Catalog No. 12483-020), and seeded at a density of 1,000 cells / well into 384-well tissue culture plates. Cells were treated with increasing concentrations of test substances prepared in RPMI-1640 + 1% FBS (v / v) and incubated for 6 days at 37°C and 5% CO2. After incubation, CellTiter-Glo® reagent (Promega Corporation, Madison, WI; catalog number G7570) was spiked into all wells, and luminescence corresponding to the ATP present in each well was measured using a Synergy™ H1 microplate (BioTek Instruments, Winooski, VT). Percent viability values, based on untreated cells (no test substance added), were calculated using the ATP-measured RLU values ​​(relative light units) and plotted against the test substance concentration using GraphPad Prism 8 software (GraphPad Software, San Diego, CA).

[0366] Phospho-AKT ELISA: Briefly, H441 or H596 lung cancer cells were detached with cell dissociation buffer and seeded at a density of 50,000 cells / well in 24-well tissue culture plates containing RPMI-1640 + 10% FBS and incubated overnight (37°C, 5% CO2). Cells were then serum-starved overnight by replacing the complete growth medium with serum-free RPMI-1640 (37°C, 5% CO2). The culture medium was removed, and the cells were then treated with test substances prepared at 100 nM in serum-free RPMI-1640 and incubated for the appropriate time points (37°C, 5% CO2). Cell lysates were generated by adding cell lysis buffer (Cell Signaling Technology, Danvers, MA; Cat. No. 9803S) + 1 mM PMSF (Cell Signaling Technology, Cat. No. 8553S). Lysate protein concentrations were assessed by BCA protein assay (Thermo Fisher Scientific Inc., catalog numbers 23223 and 23224). Phosphorylated AKT levels in 10 μg of lysate were assessed using the PathScan® Phosphorylated Akt1 (Ser473) Sandwich ELISA Kit (Cell Signaling Technology, catalog number 7160C) according to the manufacturer's instructions. Absorbance at 450 nm was measured using a Synergy™ H1 microplate (BioTek Instruments, Winooski, VT). AKT fold phosphorylation was calculated by subtracting the background A450 nm signal from all wells using a signal blank well and then normalizing to the A450 nm signal of untreated cells at each corresponding time point. Fold changes were then plotted for each time point using GraphPad Prism 8 software (GraphPad Software, San Diego, CA).

[0367] result The results are shown in Figure 4 and Figures 5A and B. As can be seen from Figure 4, bivalent MetMab (v17429) induced a dose-dependent increase in H596 proliferation compared to untreated controls, while hinge-modified telisotuzumab (v17606), as expected, showed minimal effects. The unconjugated variant v17427 (HetFc, unmodified hinge) showed a small but consistent increase in H596 proliferation after 6 days. Variant v17427 stochastically conjugated to drug-linker 001 or MCvcPABC-MMAE at DAR4 showed a significant dose-dependent increase in proliferation compared to untreated controls.

[0368] Figures 5A and 5B show that bivalent MetMab (v17429) induced strong AKT phosphorylation in both H596 and H441 cells, which was maintained for up to 60 minutes after treatment. In contrast, hinge-modified telisotuzumab (v17606) showed minimal effects on AKT phosphorylation in either cell line. Unconjugated variant v17427 induced a small, transient increase in AKT phosphorylation in both cell lines. Stochastic ADCs generated from variant v17427 appeared to exhibit increased phosphorylation compared to the unconjugated parent antibody.

[0369] Example 8: In vitro cytotoxicity of lysine-conjugated anti-cMET antibody-drug conjugates (DAR2, 4, and 6) The cell growth inhibitory potency of anti-cMet ADCs containing variant v17427 (HetFc, unmodified hinge) conjugated to drug-linker 002 by stochastic lysine conjugation at DARs ranging from DAR2 to DAR6 was determined in a panel of seven cMet-expressing cell lines. Variant v17427 conjugated to the MCvcPABC-MMAE drug linker at DAR4 and the anti-RSV antibody palivizumab (v22277) conjugated to drug-linker 002 or MCvcPABC-MMAE at DAR4 were used as controls. The cell lines were SNU-5 (gastric cancer), EBC-1 (squamous cell lung carcinoma), HCC827 (lung cancer), H1975 (non-small cell lung cancer), HCT-116 (colon cancer), H292 (lung cancer), and BT-20 (breast cancer).

[0370] Cytotoxicity was determined as described in Example 6 by incubating treated cells (1,000 cells / well) under standard culture conditions (37° C. / 5% CO 2 ) for 4 days.

[0371] result The results are summarized in Table 8.1. Stochastic v17427-drug linker 002 ADCs with DARs ranging from 1.9 to 6.2 produced variable cytotoxic activity across a panel of seven cMet-expressing cell lines, with higher DAR conjugates generally exhibiting lower EC50 values ​​across all cell lines tested. In the high-expressing cell lines SNU-5 and EBC-1, differences between ADCs with DARs between 1.9 and 6.2 were minimal, with EC50s for the lowest and highest DAR conjugates ranging from 0.043 to 0.021 nM and 0.012 to 0.004 nM, respectively. In the medium- to low-expressing cell lines HCC827, H1975, HCT-116, H292, and BT-20, differences between ADCs with DARs between 1.9 and 6.2 were more pronounced. Stochastic palivizumab-drug-linker 002 and palivizumab-MCvcPABC-MMAE conjugates, as expected, did not exhibit cytotoxicity in cMET-expressing cell lines.

[0372] [Table 8.1]

[0373] Example 9: In vitro cytotoxicity of anti-cMET antibody-drug conjugates with HomoFc or HetFc scaffolds The cell growth inhibitory potency of ADCs containing anti-cMet antibodies (v32634 and v17427, respectively) with either a HomoFc or HetFc backbone conjugated to Drug-Linker 002 via stochastic lysine conjugation at approximately DAR 4 and DAR 6 was determined in a panel of four cMet-expressing tumor cell lines and one cMet-negative cell line. The anti-RSV antibody palivizumab (v22277), conjugated to Drug-Linker 002 at DAR 3.7, and free payload (Compound 1) were used as controls. Cytotoxicity was determined as described in Example 8. The cell lines were SNU-5 (gastric cancer), EBC-1 (lung squamous cell carcinoma), H1975 (non-small cell lung cancer), H292 (lung cancer), and cMet-negative T-47D (breast cancer).

[0374] result The results are summarized in Table 9.1. DAR-matched ADCs containing anti-cMet antibodies with HetFc or HomoFc backbones conjugated to drug-linker 002 produced comparable cytotoxicity in cMet-expressing cell lines. None of the ADCs showed activity in the cMet-negative cell line T-47D, as expected. In the high-expressing cell lines SNU-5 and EBC-1, the difference in potency was discrete between the conjugates with DARs of 3.9-4.0 and 5.8. In the intermediate-expressing cell lines H1975 and H292, the difference in potency between the conjugates with DARs of 3.9-4.0 and 5.8 was more pronounced. The conjugates with DARs of 5.8 produced 2.0- to 3.4-fold higher EC50s than the conjugates with DARs of 4 in the H1975 and H292 cell lines, respectively.

[0375] [Table 9.1]

[0376] Example 10: Cellular equilibrium binding of lysine-conjugated anti-cMET antibody-drug conjugates The on-cell binding capabilities of ADCs containing anti-cMet antibodies with either HomoFc or HetFc backbones (v32634 and v17427, respectively), conjugated to Drug-Linker 002 by stochastic lysine conjugation at approximately DAR4 and DAR6, were assessed for cMet binding by flow cytometry using SNU-5 and H292 endogenously cMet-expressing cell lines as described below. The unconjugated anti-RSV antibody palivizumab (v22277) was used as a control.

[0377] Briefly, cells were seeded at 50,000 cells / well in V-bottom 96-well plates and treated with antibody for 24 hours at 4°C to prevent internalization. Following incubation, cells were washed and stained with anti-human IgG Fc AF647 conjugate (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA; catalog number 109-605-098) for 30 minutes at 4°C. Following incubation and washing, fluorescence was detected by flow cytometry on a BD LSRFortessa™ cell analyzer (BD Biosciences, Franklin Lake, NJ), collecting a minimum of 1,000 events per well. AF647 / APC-A GeoMean (fluorescence signal geometric mean, proportional to anti-human AF647 binding) in the live cell population was plotted using GraphPad Prism version 8 (GraphPad Software, San Diego, CA).

[0378] result The results are summarized in Table 10.1. Both unconjugated variants, v32634 and v17427 (HomoFc and HetFc regions, respectively), yielded comparable apparent Kd and Bmax values ​​in both SNU-5 and H292 cell lines (high and moderate endogenous cMet expression, respectively). In SNU-5 and H292 cell lines, unconjugated HomoFc variant v32634 yielded Kd values ​​of 0.59 and 0.02 nM, respectively. Similarly, in SNU-5 and H292 cell lines, unconjugated HetFc variant v17427 yielded Kd values ​​of 0.33 and 0.03 nM, respectively. The binding affinities of the unconjugated HomoFc and HetFc variants were comparable to their lysine-conjugated ADC counterparts (DAR4 and DAR6) in both cMet-expressing cell lines. The unconjugated palivizumab antibody v22277, as expected, showed no binding to the cMet-expressing cell lines.

[0379] [Table 10.1]

[0380] Example 11: In vitro cytotoxicity of site-specific cysteine-conjugated anti-cMET antibody-drug conjugates The cell growth inhibitory potency of site-specific cysteine-conjugated ADCs containing various cysteine-inserted antibody variants conjugated to drug-linker 001 at approximately DARs 1, 2, or 3 was evaluated in a panel of four cMet-expressing tumor cell lines: SNU-5 (gastric cancer), EBC-1 (lung squamous cell carcinoma), H292 (lung cancer), and H1975 (non-small cell lung cancer), as well as one cMet-negative cell line: T-47D (breast cancer). An ADC containing the anti-RSV antibody palivizumab (v22277) conjugated to drug-linker 001 at approximately DAR 4 was used as a non-targeting control. The actual DARs are shown in Table 11.1. ADCs with "DAR 1" had DARs ranging from 0.85 to 0.89, and ADCs with "DAR 3" had DARs ranging from 2.54 to 2.84. Cytotoxicity was determined as described in Example 7.

[0381] result The results are summarized in Table 11.1. The cytotoxicity observed in cMet-expressing cell lines for site-specific ADCs with average DARs of 1, 2, and 3 was drug-load dependent. In general, the three site-specific ADCs with average DARs of 1 exhibited lower cytotoxicity than the two ADCs with average DARs of 2 and the five ADCs with average DARs of 3. The stochastic ADC with average DARs of 4 exhibited higher cytotoxicity than the site-specific ADCs with DARs of 3, 2, and 1 in all cell lines tested, resulting in lower EC50 values ​​and higher % maximum cytotoxicity. Two of the three DAR1 site-specific ADCs produced comparable cytotoxicity; these ADCs contained variants v33967 and v33968 (see Table 1.3). The third DAR1 ADC, containing variant v33969, produced higher cytotoxicity than the other DAR1 ADCs in HT-29 and H441 cell lines. Both DAR2 site-specific ADCs produced comparable cytotoxicity in the cell lines tested. Finally, the five DAR3 ADCs also produced comparable cytotoxicity. As expected, the palivizumab stochastic DAR4 ADC showed no cytotoxicity in cMet-expressing cell lines.

[0382] [Table 11.1]

[0383] Example 12: In vitro cytotoxicity of site-specific anti-cMET antibody-drug conjugates containing multivalent linkers The cell growth inhibitory ability of ADCs containing variant v29001 with two cysteine ​​insertions conjugated to multivalent drug-linker 003 or multivalent drug-linker 004 was evaluated in a panel of cMet-expressing tumor cell lines, along with ADCs containing the parent variant v17427 conjugated to monovalent drug-linker 002 at DAR4 or DAR6. The cell lines used were SNU-5 (gastric cancer), EBC-1 (lung squamous cell carcinoma), H292 (lung cancer), H1975 (non-small cell lung cancer), and T-47D (breast cancer, cMet negative). An ADC containing the anti-RSV antibody palivizumab (v22277) conjugated to drug-linker 002 was used as a non-targeting control. Free payload compound 1 was also included as a control. Cytotoxicity was determined as described in Example 8.

[0384] result The results are summarized in Table 12.1. In all cMet-expressing cell lines tested, ADCs with an average DAR of 6 exhibited greater cytotoxicity than ADCs with an average DAR of 4, regardless of whether they used monovalent or multivalent drug linkers.

[0385] In the highly cMet-expressing cell lines SNU-5 and EBC-1, monovalent drug-linker ADCs exhibited comparable cytotoxicity to DAR-matched multivalent drug-linker ADCs. In SNU-5 and EBC-1 cells, the monovalent drug-linker DAR4 ADCs produced EC50 values ​​of 38.5 pM and 9.6 pM, respectively. In the same cell lines, the multivalent drug-linker DAR4 ADCs produced EC50 values ​​of 33.1 pM and 8.5 pM, respectively. Similarly, in SNU-5 cells, the monovalent and multivalent drug-linker DAR6 ADCs produced EC50 values ​​of 13.6 pM and 18.7 pM, respectively. In EBC-1 cells, the monovalent and multivalent drug-linker DAR6 ADCs produced EC50 values ​​of 6.2 pM and 4.1 pM, respectively.

[0386] In the moderately expressing cMet cell lines H1975 and H292, monovalent drug-linker ADCs exhibited comparable cytotoxicity to DAR-matched multivalent drug-linker ADCs. In H1975 and H292 cells, the monovalent drug-linker DAR4 ADCs produced EC50 values ​​of 83.6 pM and 118.0 pM, respectively. In the same cell lines, the multivalent drug-linker DAR4 ADCs produced EC50 values ​​of 48.2 pM and 93.2 pM, respectively. Similarly, in H1975 cells, the monovalent and multivalent drug-linker DAR6 ADCs produced EC50 values ​​of 43.7 pM and 27.7 pM, respectively. In H292 cells, the monovalent and multivalent drug-linker DAR6 ADCs produced EC50 values ​​of 118.0 pM and 93.2 pM, respectively. In the cMet-negative T-47D cell line, none of the monovalent or multivalent drug-linker DAR4 and DAR6 ADCs exhibited cytotoxicity, as expected. The palivizumab control ADC, as expected, did not exhibit cytotoxicity in any of the cell lines tested.

[0387] [Table 12.1]

[0388] Example 13: Cellular equilibrium binding of site-specific anti-cMET antibody-drug conjugates The on-cell binding capabilities of the following unconjugated antibodies and ADCs were assessed by flow cytometry in SNU-5 and H292 endogenously cMet-expressing cell lines: unconjugated variant v17427 (HetFc, unmodified hinge) and cysteine-inserted variant v29001 (two cysteine ​​insertions), a lysine-conjugated ADC comprising variant v17427 and drug-linker 002 at DAR 4 or DAR 6, and a cysteine-conjugated ADC comprising the cysteine-inserted variant v29001 conjugated to drug-linker 001, drug-linker 003, or drug-linker 004 at DAR 2, 4, or 6, respectively. Unconjugated palivizumab v22277 was used as a non-targeting control. Cell binding was determined as described in Example 10.

[0389] result The results are summarized in Table 13.1. All unconjugated anti-cMet antibodies and ADCs exhibited comparable apparent Kd and Bmax values ​​in both SNU-5 and H292 cell lines (high and moderate endogenous cMet expression, respectively). Unconjugated antibodies v17427 and v29001 yielded Kd values ​​of 0.328 nM and 0.428 nM, respectively, in the cMet-high expressing cell line NU-5. Similarly, variant v17427 DAR4 and DAR6 ADCs yielded Kd values ​​of 0.428 nM and 0.435 nM, respectively, in SNU-5 cells. Variant v29001 ADC yielded Kd values ​​ranging from 0.584 nM to 1.131 nM in SNU-5 cells.

[0390] In the cMet-expressing cell line H292, unconjugated antibodies v17427 and v29001 yielded Kd values ​​of 0.026 nM and 0.024 nM, respectively. Similarly, variant v17427 DAR4 and DAR6 ADCs yielded Kd values ​​of 0.040 nM and 0.047 nM, respectively, in H292 cells. Variant v29001 ADC yielded Kd values ​​in the range of 0.028 nM and 0.064 nM in H292 cells. The palivizumab negative control, as expected, showed no binding to any of the cMet-expressing cell lines.

[0391] [Table 13.1]

[0392] Example 14: Internalization of anti-cMET antibody-drug conjugates The receptor-mediated internalization capabilities of the unconjugated variant v17427 (HetFc, unmodified hinge) and v17427-drug-linker002 DAR6.7 ADCs were evaluated in two cMet-expressing cell lines, IGROV-1 and OVCAR-3, using high-content imaging as described below. The unconjugated anti-RSV antibody palivizumab (v22277) was used as a negative control.

[0393] Briefly, antibodies were fluorescently labeled by conjugation to anti-human IgG Fc Fab fragment AF488 conjugate (Jackson Immuno Research Labs, West Grove, PA; catalog number 109-547-008) at a 1:1 molar ratio in PBS (pH 7.4) (Thermo Fisher Scientific, Waltham, MA; catalog number 10010-023) for 24 hours at 4°C. Cells were seeded at 5,000 cells / well in 384-well plates and incubated overnight under standard culture conditions (37°C / 5% CO2) to allow binding. The next day, conjugated antibodies were added to the cells at various concentrations (70-0.3 nM) and incubated for 5 hours under standard culture conditions to allow internalization. Following incubation, cells were fixed at room temperature for 30 minutes using 20 μL / well of FluoroFix™ buffer (BioLegend, San Diego, CA; catalog number 422101). Nuclear stain Hoechst 33342 (Thermo Fisher Scientific, Waltham, MA; catalog number 62249) was added to the wells at 10 μM, and the assay plate was incubated at 37°C / 5% CO2 for 1 hour. Following incubation with the nuclear stain, fluorescent images were acquired using a Cytation 5 cell imaging multimode reader (BioTek Instruments, Winooski, VT) and analyzed using Gen 5 software (BioTek Instruments, Winooski, VT) to determine the average object fluorescence per well. Mean object fluorescence (GFP channel) values ​​were plotted using GraphPad Prism version 9 (GraphPad Software, San Diego, CA).

[0394] result The results are summarized in Table 14.1. Unconjugated variant v17427 exhibited receptor-mediated internalization comparable to the variant v17427-drug-linker 002 DAR6.7 ADC in EBC-1 and HT-29 (high- and moderate-cMet-expressing cell lines, respectively), suggesting that drug-linker conjugation does not affect the antibody's binding and internalization capabilities. Both the unconjugated antibody and ADC exhibited dose-dependent internalization between 70 nM and 0.3 nM treatments in both cMet-expressing cell lines. The palivizumab control (v22277), as expected, did not exhibit any internalization in EBC-1 or HT-29 cells.

[0395] [Table 14.1]

[0396] Example 15: Activation of the cMET pathway by site-specific cysteine- or lysine-conjugated anti-cMET antibody-drug conjugates The cMet pathway activation activity of ADCs containing various cysteine ​​insertion variants conjugated to Drug-Linker 001 at DAR 2 was assessed by cell proliferation. The cMet pathway activation activity was also assessed by ELISA to measure AKT phosphorylation as a downstream indicator of cMet activation for ADCs containing cysteine ​​insertion variants conjugated to Drug-Linker 001 at DAR 1, 2, or 3, as well as for the cysteine ​​conjugate v17427-Drug-Linker 001 at DAR 4 and the lysine conjugate v17427-Drug-Linker 002 at DAR 2, according to the protocol described in Example 7.

[0397] result The results of the cell proliferation assessment are shown in Figure 6. The bivalent MetMab (v17429) demonstrated a strong dose-dependent increase in proliferation of H596 cells compared to untreated controls, whereas variant v17427 (HetFc, unmodified hinge) demonstrated only a modest increase in proliferation. Site-specific DAR2 ADCs containing variants v22761, v22765, v28983, v28989, or v29001 did not demonstrate a significant increase in proliferation of H596 cells.

[0398] The results of AKT phosphorylation assessment are shown in Figures 7A-C. Bivalent MetMab (v17429) potently induced phosphorylation in H441 cells, whereas hinge-modified telisotuzumab (v17606) had minimal effects. Consistent with the results described in Example 7, variant v17427 (HetFc, unmodified hinge) induced modest, transient AKT phosphorylation, which was further increased by stochastic cysteine ​​conjugation of Drug-Linker 001. In contrast, the phosphorylation profiles for ADCs with site-specific cysteine ​​conjugation of Drug-Linker 001 at various DARs were not different from those of variant v17427. These observations suggest that reduction of hinge cysteines during stochastic conjugation may increase cMET activation, which can be bypassed by site-specific conjugation to inserted cysteine ​​residues. An ADC with stochastic DAR2 lysine conjugation of drug-linker 002 to variant v17427 also resulted in minimal changes in AKT phosphorylation compared to the unconjugated parent antibody (Figure 7B), further supporting the potential impact of stochastic cysteine ​​conjugation on cMET activation.

[0399] Example 16: In vivo activity of stochastic cysteine-conjugated anti-cMET antibody-drug conjugate (DAR4) The in vivo antitumor activity of ADCs containing variant v17427 (HetFc, unmodified hinge) stochastically cysteine-conjugated to drug-linker 001 or MCvcPABC-MMAE at DAR4 was evaluated in several cell line-derived xenograft (CDX) models expressing various cMet levels, as described below. The cell lines were: cMet-high HCC827 lung cancer, cMet-high EBC1 lung cancer, cMet-high H1975 lung cancer, cMet-medium / high HT29 colon cancer, cMet-low H292 lung cancer, and cMet-low SW48 colon cancer.

[0400] For the cMet-high HCC827 lung cancer model, 5 x 10 6 Cells were implanted into BALB / c nude mice in 0.1 ml of 1:1 PBS:Matrigel, resulting in tumor volumes of approximately 125 mm 3 Once the mice reached 100 mg / mL, they were assigned to groups (n=5 per group) and treated as specified in Table 16.1 on day 0 for a 60-day study duration. For the cMet-high EBC1 lung cancer model, 3 x 10 6 The cells were implanted into female BALB / c nude mice, and tumor volumes reached approximately 135 mm 3 Once the mice reached 100 mg / mL, they were assigned to groups (n = 6 per group) and treated as specified in Table 16.1 on day 0 for a 60-day study duration. For the cMet-high H1975 lung cancer model, 5 x 10 6 BALB / c-Foxn1 cells nu The tumor volume was approximately 155 mm 3 Once the mice reached 100 mg / mL, they were assigned to groups (n=5 per group) and treated as specified in Table 16.1 on day 0 for a 56-day study duration. For the cMet medium / high HT29 colon model, 3 x 10 6 The cells were transplanted into BALB / c nude mice, and tumor volumes were approximately 150 mm 3 Once the mice reached 100 mg / mL, they were assigned to groups (n=5 per group) and treated as specified in Table 16.1 on day 0 for a 60-day study duration. For the cMet-low H292 lung cancer model, 5 x 10 6The cells were transplanted into SCID / beige mice, and tumor volumes were approximately 160 mm 3 Once the mice reached 1×10 β-glucan in 0.1 ml of PBS, they were assigned to groups (n=5 per group) and treated as specified in Table 16.1 on day 0, for a 46-day study duration. For the cMet-low SW48 colon cancer model, 1×10 β-glucan in 0.1 ml of PBS was administered. 7 The cells were transplanted into BALB / c nude mice, and tumor volumes were approximately 120 mm 3 Once the NIH score reached 0, mice were assigned to groups (n=5 per group) and treated as specified in Table 16.1 on Day 0, for a study duration of 42 days.

[0401] For all models, tumor volume and body weight were measured twice weekly. Tumor volume plots represent the mean and standard error of the mean. Average data were plotted only if 80% or more of the mice remained in the study at that time point. For statistical analysis, a linear mixed-effects model was fitted to the log-transformed tumor volumes, followed by an F-test and post-hoc pairwise comparisons for the null hypothesis of equal mean growth rates.

[0402] [Table 16.1]

[0403] result The results are shown in Figures 8A-F and summarized below.

[0404] In the cMet-high HCC827 lung cancer model (Figure 8A), variant v17427 conjugated to Drug-Linker 001 or MCvcPABC-MMAE demonstrated potent inhibition of tumor growth at both the 3 and 10 mg / kg dose levels compared to vehicle control (p<0.0001). Tumor growth inhibition tended to be more sustained following administration of v17427-MCvcPABC-MMAE than following administration of v17427-Drug-Linker 001.

[0405] In the cMet-high EBC1 lung cancer model (FIG. 8B), v17427-drug-linker001 and v17427-MCvcPABC-MMAE demonstrated potent inhibition of tumor growth at both the 5 and 10 mg / kg dose levels compared to vehicle controls (p<0.005).

[0406] In the cMet-high H1975 lung cancer model (Figure 8C), v17427-drug-linker001 and v17427-MCvcPABC-MMAE demonstrated tumor growth inhibition at the 2, 4, and 8 mg / kg dose levels compared to vehicle control (p<0.01), with a dose-response trend for both ADCs. v17427-drug-linker001 tended to demonstrate greater tumor growth inhibition than v17427-MCvcPABC-MMAE, with this comparison being significant at the 2 mg / kg dose level (p<0.01).

[0407] In the cMet-high HT29 colon cancer model (Figure 8D), v17427-drug-linker001 and v17427-MCvcPABC-MMAE demonstrated potent tumor growth inhibition at both the 3 and 10 mg / kg dose levels compared to vehicle control (p<0.0001). Tumor growth inhibition tended to be more sustained after administration of v17427-drug-linker001 than after administration of v17427-MCvcPABC-MMAE, and regrowth rates were lower after administration of v17427-drug-linker001 than after administration of v17427-MCvcPABC-MMAE when each was dosed at 10 mg / kg (p<0.005).

[0408] In the cMet-low H292 lung cancer model (Figure 8E), v17427-drug-linker001 and v17427-MCvcPABC-MMAE demonstrated modest inhibition of tumor growth at both the 3 and 10 mg / kg dose levels compared to vehicle control (p<0.05). No significant dose response was observed between the 3 and 10 mg / kg dose levels or between the response of v17427-drug-linker001 and v17427-MCvcPABC-MMAE.

[0409] In the cMet-low SW48 colon cancer model (Figure 8F), neither v17427-drug-linker001 nor v17427-MCvcPABC-MMAE demonstrated potent inhibition of tumor growth at the administered dose levels, and there was no significant difference in the activity of v17427-drug-linker001 and v17427-MCvcPABC-MMAE.

[0410] Collectively, these data demonstrate that the v17427-drug-linker001 and v17427-MCvcPABC-MMAE ADCs are active in vivo in cMet-expressing models. A trend was observed between the magnitude of response and cMet expression. v17427-drug-linker001 and v17427-MCvcPABC-MMAE exhibited largely comparable activity, with superior activity of v17427-drug-linker001 observed in the HT29 and H1975 models.

[0411] Example 17: In vivo activity of anti-cMET antibody-drug conjugates (DAR1, 2, 3, and 4) The in vivo antitumor activity of cMet-targeting ADCs containing stochastic lysine-conjugated drug-linker 002 at DAR 2 and site-specific cysteine-conjugated drug-linker 001 at DARs 1, 2, 3, and 4 was evaluated in H1975 (cMet-high) lung cancer and HT29 (cMet-medium / high) colon cancer CDX models. The ADCs and their dosing are listed in Table 17.1. Doses were selected for comparison at antibody-matched and toxin-matched dose levels. Studies using these models were performed according to the methods described in Example 16, except that the H1975 model used n = 8 mice per group and had a study duration of 41 days, and the HT29 model used n = 8 mice per group and had a study duration of 32 days. Mean data were plotted only if 80% or more of the mice remained in the study at that time.

[0412] [Table 17.1]

[0413] result Results for the cMet-high H1975 CDX lung cancer model are shown in Figures 9A and 9B. For the H1975 model, the DAR1 Drug-Linker 001, DAR2 Drug-Linker 001 and Drug-Linker 002, DAR3 Drug-Linker 001, and DAR4 Drug-Linker 001 ADCs all significantly inhibited tumor growth compared to vehicle at toxin-matched antibody doses of 24, 12, 8, and 6 mg / kg, respectively (Figure 9A). The DAR2 Drug-Linker 001 and Drug-Linker 002, DAR3 Drug-Linker 001, and DAR4 Drug-Linker 001 ADCs also all significantly inhibited tumor growth compared to vehicle at toxin-matched antibody doses of 2, 1.3, and 1 mg / kg, respectively (Figure 9B). The DAR2, DAR3, and DAR4 Drug-Linker 001 site-specific ADCs, and the v17427-Drug-Linker 001 and v17427-Drug-Linker 002 ADCs showed comparable activity at the toxin-matched dose, whereas the DAR1 Drug-Linker 001 ADC appeared to be less active at the toxin-matched dose.

[0414] Results for the cMet medium / high HT29 colon cancer CDX model are shown in Figures 10A and 10B. In the HT29 model, the DAR2 Drug-Linker 001 and Drug-Linker 002, DAR3 Drug-Linker 001, and DAR4 Drug-Linker 001 ADCs all significantly inhibited tumor growth compared to vehicle at toxin-matched antibody doses of 6, 4, and 3 mg / kg, respectively (p<0.05, mixed-effects model for tumor growth rate) (Figure 10A). The DAR1 Drug-Linker 001 ADC did not inhibit tumor growth at the toxin-matched dose of 12 mg / kg. All of the DAR2 Drug-Linker 001 and Drug-Linker 002, DAR3 Drug-Linker 001, and DAR4 Drug-Linker 001 ADCs, except for v28983-Drug-Linker 001, also significantly inhibited tumor growth compared to vehicle at toxin-matched antibody doses of 3, 2, and 1.5 mg / kg, respectively (Figure 10B). In comparison, v17427-MCvcPABC-MMAE DAR4 did not significantly inhibit tumor growth at the 1.5 mg / kg dose tested. When doses were matched by toxin, there was a trend toward a positive correlation between DAR and antitumor activity. The activity of the site-specific cysteine ​​conjugate v29001-Drug-Linker 001 DAR2 ADC was comparable to that of the stochastic lysine conjugate v17427-Drug-Linker 002 DAR2.

[0415] Collectively, these data demonstrate the activity of stochastic and site-specific anti-cMet ADCs in an in vivo cMet expression model, as well as a trend toward lower toxin-match activity for low-DAR ADCs.

[0416] Example 18: In vivo activity of anti-cMET antibody-drug conjugates containing multivalent drug linkers (CDX model) The in vivo antitumor activity of ADCs containing anti-cMet antibodies stochastically conjugated to drug-linker 002 via lysine or stochastically conjugated to MCvcPABC-MMAE via cysteine, and ADCs containing anti-cMet antibodies conjugated to multivalent drug-linker 003 or drug-linker 004 via site-specific cysteine ​​insertion, at DARs of 4 or 6, respectively, was evaluated in several CDX models expressing various cMet levels. The ADCs were administered as specified in Table 18.1 to allow comparison at toxin-matched dose levels.

[0417] Studies using the H1975, HT29, and H292 models were performed according to the methods described in Example 16, with the following differences: the H1975 model used n = 12 mice per group with a study duration of 28 days, and the HT29 model used n = 12 mice per group with a study duration of 25 days. For the cMet medium / high Hs746t gastric cancer model, 5 x 10 mice were cultured in 0.1 ml of 1:1 PBS:Matrigel. 6 The cells were transplanted into BALB / c nude mice, and tumor volumes were approximately 150 mm 3 Once the mice reached 100 mg / mL, they were assigned to groups (n=7 per group) and treated as specified in Table 18.1 for the 28-day study duration. For the HCT116 colon cancer model in cMet, 5 x 10 5 The cells were transplanted into BALB / c mice, and tumor volumes were approximately 150 mm 3 Once the mean age reached 18.2, mice were assigned to groups (n = 7 per group) and treated as specified in Table 18.1 for the 27-day study duration. Mean data were plotted only if 80% or more of the mice remained on study at that time.

[0418] [Table 18.1]

[0419] result The results are shown in Figures 11A-E and summarized below.

[0420] In the cMet-high H1975 lung cancer model, tumor growth inhibitory activity was evaluated at 0.4 mg / kg and 0.8 mg / kg for the DAR4 ADC and at 0.27 mg / kg and 0.53 mg / kg for the DAR6 ADC. These relatively low doses were used to allow comparison at toxin-matched doses. All ADCs produced statistically significant tumor growth inhibition compared to vehicle controls (p<0.03) except for v17427-MCvcPABC-MMAE DAR4 at 0.8 mg / kg and lysine-conjugated v17427-drug-linker 002 DAR6 at 0.27 mg / kg (Figure 11A). The drug-linker 002, 003, and 004 ADCs all demonstrated statistically greater tumor growth inhibition than v17427-MCvcPABC-MMAE at toxin-matched dose levels. At the 0.8 and 0.53 mg / kg dose levels of the DAR4 and DAR6 ADCs, respectively, there was no differentiation in activity among any of the drug-linker 002, 003, and 004 ADCs. At the lower 0.4 and 0.27 mg / kg dose levels of the DAR4 and DAR6 ADCs, respectively, the site-specific v29001-drug-linker 004 DAR6 ADC demonstrated superior activity to the stochastic lysine conjugate v17427-drug-linker 002 DAR4 and DAR6 ADCs. The site-specific v29001-drug-linker 003 DAR4 ADC demonstrated greater activity compared to the stochastic lysine v17427-drug-linker 002 DAR4 ADC. Overall, these data demonstrate the superior activity of ADCs containing drug-linkers conjugated via an inserted cysteine ​​over ADCs containing drug-linkers conjugated by stochastic lysine conjugation, and the superior activity of all Compound 1-containing ADCs over the comparator MMAE ADC.

[0421] In a cMet-medium / high HT29 colon cancer model, tumor growth inhibitory activity was evaluated at 0.8 and 0.53 mg / kg for the DAR4 and DAR6 ADCs, respectively. These relatively low doses were used to allow comparison at toxin-matched dose levels. The site-specific ADCs v29001-Drug-Linker004 DAR6 and v29001-Drug-Linker003 DAR4, as well as the stochastic lysine conjugate v17427-Drug-Linker002 DAR4, all resulted in statistically significant tumor growth inhibition compared with the vehicle control and the comparator ADC v17427-MCvcPABC-MMAE DAR4 (Figure 11B). The stochastic lysine conjugate v17427-Drug-Linker002 DAR6 was not differentiated from the vehicle control or the comparator ADC v17427-MCvcPABC-MMAE DAR4. Overall, these data demonstrate the activity advantage of ADCs containing drug-linkers conjugated via an inserted cysteine ​​over ADCs containing drug-linkers conjugated by stochastic lysine conjugation and over the comparator MCvcPABC-MMAE ADC.

[0422] In the cMet-low H292 lung cancer model, both the site-specific v29001-drug-linker004 DAR6 and the stochastic lysine-conjugated v32634-drug-linker002 DAR6 ADCs demonstrated modest inhibition of tumor volume compared to vehicle controls and greater activity compared to v17427-MCvcPABC-MMAE DAR4 (Figure 11C).

[0423] In the cMet-medium / high Hs746t gastric cancer model, all ADCs demonstrated tumor growth inhibition compared to vehicle controls (Figure 11D). In this model, the stochastic lysine-conjugated ADC, v32634-drug-linker002 DAR4, demonstrated the greatest activity, outperforming the site-specific v35527 DAR4 and DAR6 ADCs. v32634-drug-linker002 DAR4 also tended to outperform v17606-MCvcPABC-MMAE DAR3.

[0424] In the cMet HCT116 colon cancer model, all tested ADCs demonstrated modest inhibition of tumor growth compared to vehicle controls (Figure 11E). The site-specific DAR4 and DAR6 ADCs and the stochastic DAR4 ADC were not statistically distinct from each other, but all demonstrated statistically superior activity to v17606-MCvcPABC-MMAE DAR3 (p<0.01).

[0425] Collectively, these data demonstrate that ADCs containing compound 1 as the payload (i.e., drug-linkers 002, 003, and 004), conjugated at DAR4 or DAR6 by either stochastic lysine conjugation or site-specific cysteine ​​conjugation, are active against a cMet-expressing in vivo xenograft model, with activity primarily superior to that of ADCs containing MMAE as the payload (i.e., drug-linker MCvcPABC-MMAE).

[0426] Example 19: In vivo activity of anti-cMET antibody-drug conjugates containing multivalent drug linkers (PDX model) The in vivo antitumor activity of the lysine-conjugated DAR4 ADC (v32634-Drug-Linker002) and the site-specific cysteine-conjugated DAR6 ADC (v35527-Drug-Linker004 or v29001-Drug-Linker004) was compared to v17606-MCvcPABC-MMAE DAR3 (either the HIC-purified v36198 ADC or the non-HIC-purified v19875 ADC—see Table 5.1) in a panel of lung cancer PDX models expressing various cMet levels. The site-specific DAR6 ADC was dosed at 2 mg / kg to toxin-match the 3 mg / kg dose of the lysine-conjugated DAR4 ADC. Variants v32634, v35527, and v29001 all contain an unmodified hinge, while variant v17606 contains a modified hinge (see Table 1.3). Variant v17606-MCvcPABC-MMAE is equivalent to telisotuzumab vedotin (ABBV399).

[0427] Tumor fragments derived from stock mice were transplanted into mice (NudeFoxn1nu or BALB / c nude) until the tumor volume reached approximately 150–300 mm. 3 Patients were assigned to treatment groups when they reached a median age of 18.5 years. The ADC was administered by single intravenous injection as specified in Table 19.1. Study duration ranged from 25 to 42 days. Tumors from untreated mice were excised, formalin-fixed, paraffin-embedded, and subjected to immunohistochemical (IHC) analysis for relative cMet levels using antibody clone SP44 (Abcam) according to standard IHC methods.

[0428] [Table 19.1] TIFF2025531990000123.tif47165

[0429] result Tumor growth rate inhibition values ​​are shown in Figure 12. The v32634-drug-linker 002 DAR4 ADC demonstrated similar or superior activity to the v17606-MCvcPABC-MMAE DAR3 ADC in 12 of 16 models. The site-specific v29001-drug-linker 004 DAR6 ADC demonstrated superior activity to both the v17606-MCvcPABC-MMAE DAR3 ADC and the v32634-drug-linker 002 DAR4 ADC in 6 of 8 models evaluated, and non-inferior activity in the remaining 2 models.

[0430] Taken together, these data indicate that ADCs comprising site-specifically conjugated Compound 1 at DAR 6 are primarily more active than ADCs comprising lysine-conjugated Compound 1 when administered at toxin-matched doses. Overall, ADCs comprising lysine-conjugated Compound 1 at DAR 4 and ADCs comprising site-specifically conjugated Compound 1 at DAR 6 have superior activity to the MCvcPABC-MMAE ADC.

[0431] Example 20: Pharmacokinetics of anti-cMET antibody-drug conjugates in Tg32 mice The pharmacokinetics of anti-cMet ADCs and corresponding free antibodies were evaluated in humanized FcRn Tg32 mice as described below. The humanized FcRn Tg32 mouse model was chosen for this study because it is a good predictor of drug pharmacokinetics in humans. The ADCs and antibodies evaluated were v29001 (HetFc, unmodified hinge, two cysteine ​​insertions), v17427 (HetFc, unmodified hinge), v29001-drug-linker003 DAR4, v29001-drug-linker004 DAR6, v17427-drug-linker002 DAR4, and v17427-drug-linker002 DAR6.

[0432] All test articles were administered intravenously to hFcRn Tg32 mice (The Jackson Laboratory, Sacramento, CA; stock number 014565) at 5 mg / kg. For each test article, blood was collected from n=4 animals by retro-orbital or terminal bleeding at 1, 4, and 8 hours and 1, 3, 7, 10, 14, and 21 days post-dose. Blood was processed to serum and stored frozen at -80°C in 96-well storage plates prior to pharmacokinetic analysis.

[0433] Total IgG and total ADC concentrations of test articles in mouse serum were measured by sandwich ELISA using an anti-human IgG1 Fc capture antibody (Jackson ImmunoResearch Labs, West Grove, PA; catalog number 709-005-098) or a rabbit antitoxin capture antibody and an HRP-conjugated anti-IgG1 Fab detection antibody (Jackson ImmunoResearch Labs; catalog number 109-035-097). Absorbance at 450 nm was measured using a Synergy™ H1 Hybrid Multimode Plate Reader (BioTek Instruments, Winooski, VT). Sample data were analyzed using SoftMax® Pro 7.1 (Molecular Devices, San Jose, CA). Pharmacokinetic parameters were calculated from noncompartmental analysis using Phoenix WinNonlin™ software (Certara, Princeton, NJ).

[0434] result The results are shown in Figures 13A and B and Table 20.1. All mAbs and ADCs exhibited typical antibody-like prolonged exposure. Variant v17427 and the cysteine-insertion variant v29001 showed minimal differences in their PK parameters. The ADCs utilizing multivalent drug linkers, v29001-drug-linker003 DAR4 and v29001-drug-linker004 DAR6, exhibited PK comparable to their parent antibody, variant v29001.

[0435] [Table 20.1]

[0436] Example 21: In vivo stability of anti-cMET antibody-drug conjugates The in vivo stability of the four ADCs described in Example 20 (v17427-drug-linker002 DAR4, v17427-drug-linker002 DAR6, v29001-drug-linker003 DAR4, and v29001-drug-linker004 DAR6) in Tg32 mice was assessed using immunoprecipitation / mass spectrometry as described below. Serum samples collected from Tg32 mice at various time points in the circulation (1 hour to 10 days) were used, as described in Example 20. For all groups, serum samples from mice from each time point (1 hour to 21 days post-dose) were tested.

[0437] Briefly, biotinylated anti-human IgG F(ab')2 antibodies were bound to streptavidin-coated magnetic beads (11 μg antibody per sample) for 30 minutes at room temperature. Following binding, the beads were incubated with the test samples at room temperature for 1.5 hours to allow for immunocapture. The samples were washed with PBS (pH 7.4) using a DynaMag™-2 magnet (ThermoFisher Scientific Corporation, Waltham, MA). The immunocaptured samples were reduced for 1 hour at room temperature using dithiothreitol (DTT) in PBS (pH 7.4) (v29001-Drug-Linker003 and v29001-Drug-Linker004 only). The v17427-Drug-Linker002 ADC was not reduced. After further washing with PBS (pH 7.4), the samples were eluted by incubation with pH 3.0 buffer (distilled water containing 20% ​​acetonitrile and 1% formic acid) for 1 hour at room temperature. The isolated ADC samples were then analyzed by mass spectrometry to quantify the DAR or drug loading, or kept frozen at -80°C until further analysis.

[0438] For LC-MS analysis, samples were injected onto an Agilent™ PLRP-S 1000Å 8 μM 50 × 2.1 mm column using an Agilent™ 1290 Infinity™ II LC system coupled to an Agilent™ 6545 quadrupole time-of-flight (Q-TOF) analyzer at a column temperature of 70°C and a flow rate of 0.3 ml / min. The mobile phase consisted of A: LC-MS grade water containing 0.1 v / v% formic acid, 0.025 v / v trifluoroacetic acid, and 10 v / v% isopropyl alcohol, and B: acetonitrile containing 0.1 v / v% formic acid and 10 v / v% isopropyl alcohol. The column was pre-equilibrated with 20% mobile phase B before sample injection. A gradient of 20 to 40% mobile phase B was then applied over 20 minutes, followed by a gradient of 40 to 90% mobile phase B over 2 minutes, with a 2.5-minute column wash at 99% mobile phase B. Between runs, the column was re-equilibrated to 10% mobile phase B for 2 minutes. Electrospray ionization (ESI) was performed in positive mode on a Dual AJS™ ESI source (Agilent Technologies, Santa Clara, CA) using a capillary voltage of 5000 V, a nozzle voltage of 2000 V, a fragmentor voltage of 170 V, a skimmer voltage of 65 V, an Octople RFPeak voltage of 750 V, a gas temperature of 300 °C, a gas flow rate of 13 L / min, a nebulizer voltage of 45 psig, and a sheath gas temperature of 400 °C. Data were acquired at a scan rate of 1 spectrum / second using an m / z range of 500 to 7000.

[0439] Peak integration, MS deconvolution, and mass assignment were performed in Protein Metrics Byos™ v4.0 (Protein Metrics Inc., Cupertino, CA) using a deconvolution window of 50,000-170,000 Da (for v17427-drug-linker002 ADC) or 20,000-60,000 Da (for v29001-drug-linker003 and v29001-drug-linker004) and an m / z range of 600-6,000 (v17427-drug-linker002 ADC) or 850-4,000 (v29001-drug-linker003 and v29001-drug-linker004). For the reduction analysis of v29001-drug-linker003 and v29001-drug-linker004, the reference mass was defined as the average mass of each heavy chain of the parent antibody v29001, with one 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-[alpha-L-fucopyranose-(1-6)] stub resulting from EndoS activity on the N-glycan and the formation of pyroglutamic acid if a glutamine residue was present at the N-terminus of the protein sequence. For the intact analysis of the v17427-drug-linker002 ADC, the reference mass was defined as the average mass of the parent antibody v17427, containing two 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-[alpha-L-fucopyranose-(1-6)] stubs, 16 disulfide bonds, and the formation of pyroglutamic acid(s) if glutamine residue(s) were present at the N-terminus of the protein sequence. Drug loading was assessed based on a mass shift equal to the x linker-drug mass relative to the reference mass, and the weighted average DAR was calculated using the deconvoluted MS peak intensity. For the predicted drug-linker conjugated to each variant, see Example 3. The thiosuccinimide ring-opening was defined as the deconvoluted MS peak with a mass shift of 18 Da relative to the deconvoluted MS peak for drug loading. A mass tolerance of ±10 Da was applied to the mass assignment.The extent of drug loading and % thiosuccinimide ring opening at each time point were graphed using GraphPad Prism software (GraphPad Software, San Diego, CA).

[0440] result The results are summarized in Figure 14 and Table 21.1. All four ADCs showed >84% DAR remaining after 21 days. v29001-drug-linker003 and v29001-drug-linker004 showed thiosuccinimide ring opening. For the v17427-drug-linker002 ADC, minimal drug-linker degradation was observed at 21 days (observed as a loss of approximately 582 Da from the drug-loaded species). For v29001-drug-linker003 and v29001-drug-linker004, no linker-drug degradation was observed after 21 days.

[0441] [Table 21.1]

[0442] The disclosures of all patents, patent applications, publications, and database entries mentioned in this specification are specifically incorporated herein by reference in their entirety to the same extent as if each individual patent, patent application, publication, and database entry was specifically and individually indicated to be incorporated by reference.

[0443] Modifications of the specific embodiments described herein that will be obvious to those skilled in the art are intended to be included within the scope of the following claims.

[0444] Array Table [Table A]

[0445] [Table B] TIFF2025531990000128.tif240165TIFF2025531990000129.tif236165TIFF2025531990000130.tif242165TIFF2025531990000131.tif239165TIFF2025531990000132.tif230165TIFF2025531990000133.tif226165TIFF2025531990000134.tif237165TIFF2025531990000135.tif236165TIFF2025531990000136.tif237165TIFF2025531990000137.tif241165TIFF2025531990000138.tif236165TIFF2025531990000139.tif239165TIFF2025531990000140.tif240165TIFF2025531990000141.tif240165TIFF2025531990000142.tif238165TIFF2025531990000143.tif237165TIFF2025531990000144.tif189165

[0446]

Table C

Claims

1. Formula I: A-(L-(D) ｎ ) ｐ (I) An antibody-drug conjugate having the formula: A is an antibody construct comprising an antigen-binding domain and an immunoglobulin (Ig) hinge region, wherein the antigen-binding domain specifically binds to c-Met and comprises heavy chain CDR sequences (HCDR1, HCDR2, and HCDR3) of a VH domain sequence set forth in SEQ ID NO: 1 and light chain CDR sequences (LCDR1, LCDR2, and LCDR3) of a VL domain sequence set forth in SEQ ID NO: 2, and the Ig hinge region comprises an upper hinge sequence having the amino acid sequence of a native IgG1, IgG2, or IgG4 upper hinge sequence; L is a cleavable linker; D is, where: ＊ is the point of attachment to L, n is 1 to 4; p is 1 to 8; The antibody-drug conjugate.

2. 2. The antibody-drug conjugate of claim 1, wherein the antigen-binding domain comprises an HCDR1 sequence selected from the sequences set forth in SEQ ID NOs: 3, 9, 14, 16, and 22, an HCDR2 sequence selected from the sequences set forth in SEQ ID NOs: 4, 10, 15, 17, and 23, an HCDR3 sequence selected from the sequences set forth in SEQ ID NOs: 5, 11, and 18, an LCDR1 sequence selected from the sequences set forth in SEQ ID NOs: 6, 12, and 19, an LCDR2 sequence selected from the sequences set forth in SEQ ID NOs: 7, 13, and 20, and an LCDR3 sequence selected from the sequences set forth in SEQ ID NOs: 8 and 21.

3. The antibody-drug conjugate of claim 1 or 2, wherein the antibody construct comprises a VH domain sequence having the amino acid sequence set forth in SEQ ID NO: 1 and a VL domain sequence having the amino acid sequence set forth in SEQ ID NO:

2.

4. The antibody-drug conjugate of any one of claims 1 to 3, wherein the Ig hinge region comprises an upper hinge sequence having the amino acid sequence of the upper hinge sequence of native IgG1.

5. The antibody-drug conjugate of any one of claims 1 to 3, wherein the Ig hinge region comprises an upper hinge sequence having the amino acid sequence set forth in SEQ ID NO:

25.

6. The antibody-drug conjugate of any one of claims 1 to 5, wherein the antibody construct further comprises a scaffold based on an immunoglobulin Fc region.

7. The antibody-drug conjugate of claim 6, wherein the immunoglobulin Fc region is an IgG1 Fc region.

8. 8. The antibody-drug conjugate of claim 6 or 7, wherein the Fc region is a heterodimeric Fc comprising a modified CH3 domain comprising one or more amino acid modifications, wherein the one or more amino acid modifications promote the formation of the heterodimeric Fc over the formation of a homodimeric Fc.

9. the heterodimeric Fc comprising a first Fc polypeptide and a second Fc polypeptide, (a) the first Fc polypeptide comprises the amino acid modifications L351Y, F405A, and Y407V, and the second Fc polypeptide comprises the amino acid modifications T366L, K392M, and T394W, or (b) the first Fc polypeptide comprises the amino acid modifications L351Y, F405A, and Y407V, and the second Fc polypeptide comprises the amino acid modifications T366L, K392L, and T394W; or (c) the first Fc polypeptide comprises the amino acid modifications T350V, L351Y, F405A, and Y407V, and the second Fc polypeptide comprises the amino acid modifications T350V, T366L, K392M, and T394W; or (d) said first Fc polypeptide comprises the amino acid modifications T350V, L351Y, F405A, and Y407V, and said second Fc polypeptide comprises the amino acid modifications T350V, T366L, K392L, and T394W; or (e) the first Fc polypeptide comprises the amino acid modifications T350V, L351Y, S400E, F405A, and Y407V, and the second Fc polypeptide comprises the amino acid modifications T350V, T366L, N390R, K392M, and T394W; The antibody-drug conjugate of claim 8.

10. The antibody-drug conjugate of any one of claims 1 to 9, wherein the antibody construct is a bivalent antibody comprising two antigen-binding domains, both of which specifically bind to c-Met.

11. The antibody-drug conjugate of any one of claims 1 to 10, wherein L is a protease-cleavable linker.

12. The antibody-drug conjugate of any one of claims 1 to 11, wherein each L is conjugated to a sulfhydryl group of a cysteine ​​residue of the antibody construct.

13. The antibody-drug conjugate of claim 12, wherein each cysteine ​​residue is a naturally occurring cysteine ​​residue.

14. The antibody-drug conjugate of claim 13, wherein p is 2 or 4.

15. The antibody-drug conjugate of claim 12, wherein each cysteine ​​residue is a non-naturally occurring cysteine ​​residue.

16. The antibody-drug conjugate of claim 15, wherein each non-native cysteine ​​residue is a cysteine ​​insertion mutation or a cysteine ​​substitution mutation.

17. each non-native cysteine ​​residue is (a) a cysteine ​​residue inserted between positions 40 and 41 in the VL domain; (b) a cysteine ​​residue inserted between positions 126 and 127 in the CL domain; (c) a cysteine ​​residue inserted between positions 9 and 10 in the VH domain; (d) a cysteine ​​residue inserted between positions 237 and 238 in the CH2 domain; and (e) a cysteine ​​residue inserted between positions 299 and 300 in the CH2 domain; and a cysteine ​​insertion mutation independently selected from The numbering of amino acids in the VL domain, the CL domain, and the VH domain is Kabat numbering, and the numbering of amino acids in the CH2 domain is EU numbering. The antibody-drug conjugate of claim 15.

18. The antibody-drug conjugate of any one of claims 15 to 17, wherein p is 1, 2, 3, or 4.

19. The antibody-drug conjugate of any one of claims 1 to 11, wherein each L is conjugated to an amino group of a lysine residue of the antibody construct.

20. 20. The antibody-drug conjugate of claim 19, wherein p is 2, 4, or 6.

21. L-D has the following structure: (a) Formula IV (In the formula, Z' is a linking group that connects the linker to a targeting group on the antibody construct A; Str is a stretcher, A.A. １ and A.A. ２ are each independently an amino acid, １ - [AA ２ ] ｍ forms a protease cleavage site, X is a self-immolative group; s is 0 or 1; m is 1, 2, or 3; o is 0, 1, or 2; # is the point of attachment to said antibody construct A), or (b) Formula XII (In the formula, Z' is a linking group that connects the linker to a targeting group on the antibody construct A; Str １ and Str ２ are each independently a stretcher, BU is a branching unit, A.A. １ and A.A. ２ are each independently an amino acid, １ - [AA ２ ] ｍ forms a protease cleavage site, X is a self-immolative group; s and s' are each independently 0 or 1; m is 1, 2, or 3; o is 0, 1, or 2; t is 2 or 3; # is the point of attachment to said antibody construct A), The antibody-drug conjugate of any one of claims 1 to 20, wherein the conjugate has one of the following structures:

22. L-D has structure IV, wherein: Z' is a carbonyl group (-C(O)-) or where # is the point of attachment to anti-cMet antibody construct A; ＊ is the point of attachment to the remainder of the linker.

23. L-D has structure IV, wherein: s is 1, Str is, where: ＄ is the point of attachment to Z′, ＊ is the point of attachment to the remainder of the linker, p is an integer from 2 to 6, and q is an integer from 2 to 8; The antibody-drug conjugate of claim 21 or 22.

24. L-D has structure IV, wherein: m is 1 and AA １ - [AA ２ ] ｍ is a dipeptide selected from Val-Lys, Ala-Lys, Phe-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit, and Trp-Cit; o is 0; The antibody-drug conjugate according to any one of claims 21 to 23.

25. LD has the structure XII, wherein: Z' is a carbonyl group (-C(O)-) or where # is the point of attachment to anti-cMet antibody construct A; ＊ is the point of attachment to the remainder of the linker.

26. LD has the structure XII, wherein: s is 1, s' is 1, Str １ but, where: ＄ is the point of attachment to Z′, ＊ is the point of attachment to the remainder of the linker, p is an integer from 2 to 6, and q is an integer from 2 to 8; Str ２ but, where: ＄ is the attachment point to BU, ＊ is the point of attachment to the remainder of the linker, p is an integer from 2 to 6, and q is an integer from 2 to 8; The antibody-drug conjugate of claim 21 or 25.

27. LD has the structure XII, wherein: m is 1 and AA １ - [AA ２ ] ｍ is a dipeptide selected from Val-Lys, Ala-Lys, Phe-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit, and Trp-Cit; o is 0; The antibody-drug conjugate of any one of claims 21, 25, and 26.

28. LD has the structure XII, wherein: The antibody-drug conjugate of any one of claims 21 and 25 to 27, wherein BU is an amino acid or Behera's amine.

29. The following structure: The antibody-drug conjugate of claim 1, wherein the antibody-drug conjugate has one of the following structures:

30. structure:

2. The antibody-drug conjugate of claim 1, having the structure: wherein A is the antibody construct that specifically binds to c-Met and p is 6.

31. structure:

2. The antibody-drug conjugate of claim 1, having the structure:

32. an antigen-binding domain comprising a VL domain and a VH domain, and optionally a CH1 domain and a CL domain, which specifically binds to c-Met; an Fc region comprising a CH2 domain having two CH2 domain sequences and a CH3 domain having two CH3 domain sequences; An antibody construct comprising: the antigen-binding domain comprises heavy chain CDR sequences (HCDR1, HCDR2, and HCDR3) of the VH domain sequence set forth in SEQ ID NO: 1, and light chain CDR sequences (LCDR1, LCDR2, and LCDR3) of the VL domain sequence set forth in SEQ ID NO: 2; the antibody construct comprising: (a) a cysteine ​​residue inserted between positions 40 and 41 in the VL domain; (b) a cysteine ​​residue inserted between positions 126 and 127 in the CL domain; (c) a cysteine ​​residue inserted between positions 9 and 10 in the VH domain; (d) a cysteine ​​residue inserted between positions 237 and 238 in the CH2 domain sequence; and (e) a cysteine ​​residue inserted between positions 299 and 300 in the CH2 domain sequence; wherein the numbering of amino acids in the VL domain, the CL domain, and the VH domain is Kabat numbering, and the numbering of amino acids in the CH2 domain is EU numbering. The antibody construct.

33. 33. The antibody construct of claim 32, further comprising an immunoglobulin (Ig) hinge region, wherein the Ig hinge region comprises an upper hinge sequence having the amino acid sequence of a native IgG1, IgG2, or IgG4 upper hinge sequence.

34. 34. The antibody construct of claim 33, wherein the Ig hinge region comprises an upper hinge sequence having the amino acid sequence of the upper hinge sequence of a native IgG1.

35. 34. The antibody construct of claim 33, wherein the Ig hinge region comprises an upper hinge sequence having the amino acid sequence set forth in SEQ ID NO:

25.

36. 36. The antibody construct of any one of claims 32 to 35, wherein the antigen binding domain comprises an HCDR1 sequence selected from the sequences set forth in SEQ ID NOs: 3, 9, 14, 16, and 22, an HCDR2 sequence selected from the sequences set forth in SEQ ID NOs: 4, 10, 15, 17, and 23, an HCDR3 sequence selected from the sequences set forth in SEQ ID NOs: 5, 11, and 18, an LCDR1 sequence selected from the sequences set forth in SEQ ID NOs: 6, 12, and 19, an LCDR2 sequence selected from the sequences set forth in SEQ ID NOs: 7, 13, and 20, and an LCDR3 sequence selected from the sequences set forth in SEQ ID NOs: 8 and 21.

37. The antibody construct of any one of claims 32 to 36, wherein the Fc region is an IgG1 Fc region.

38. 38. The antibody construct of any one of claims 32 to 37, wherein the Fc region is a heterodimeric Fc comprising a modified CH3 domain comprising one or more amino acid modifications, wherein the one or more amino acid modifications favor formation of the heterodimeric Fc over formation of a homodimeric Fc.

39. the heterodimeric Fc comprising a first Fc polypeptide and a second Fc polypeptide, (a) the first Fc polypeptide comprises the amino acid modifications L351Y, F405A, and Y407V, and the second Fc polypeptide comprises the amino acid modifications T366L, K392M, and T394W, or (b) the first Fc polypeptide comprises the amino acid modifications L351Y, F405A, and Y407V, and the second Fc polypeptide comprises the amino acid modifications T366L, K392L, and T394W; or (c) the first Fc polypeptide comprises the amino acid modifications T350V, L351Y, F405A, and Y407V, and the second Fc polypeptide comprises the amino acid modifications T350V, T366L, K392M, and T394W; or (d) said first Fc polypeptide comprises the amino acid modifications T350V, L351Y, F405A, and Y407V, and said second Fc polypeptide comprises the amino acid modifications T350V, T366L, K392L, and T394W; or (e) the first Fc polypeptide comprises the amino acid modifications T350V, L351Y, S400E, F405A, and Y407V, and the second Fc polypeptide comprises the amino acid modifications T350V, T366L, N390R, K392M, and T394W; 39. The antibody construct of claim 38.

40. 40. The antibody construct of any one of claims 32 to 39, wherein the antibody construct is a bivalent antibody comprising two antigen-binding domains, both antigen-binding domains specifically binding to c-Met.

41. The antibody construct comprises a combination of cysteine ​​insertions, the combination comprising: (a) cysteine ​​residues inserted between positions 299 and 300 and between positions 237 and 238 in one or both CH2 domain sequences; or (b) a cysteine ​​residue inserted between positions 299 and 300 in one or both of the CH2 domain sequences and a cysteine ​​residue inserted between positions 9 and 10 in the VH domain; or (c) a cysteine ​​residue inserted between positions 299 and 300 in one or both of the CH2 domain sequences and a cysteine ​​residue inserted between positions 40 and 41 in the VL domain; or (d) a cysteine ​​residue inserted between positions 237 and 238 in one or both of the CH2 domain sequences and a cysteine ​​residue inserted between positions 9 and 10 in the VH domain; or (e) a cysteine ​​residue inserted between positions 9 and 10 in the VH domain and between positions 40 and 41 in the VL domain; 41. The antibody construct of any one of claims 32 to 40, comprising:

42. (i) a cysteine ​​residue inserted between positions 299 and 300 in one of the CH2 domain sequences; (ii) a cysteine ​​residue inserted between positions 299 and 300 in each CH2 domain sequence; (iii) a cysteine ​​residue inserted between positions 237 and 238 in one of the CH2 domain sequences; (iv) a cysteine ​​residue inserted between positions 237 and 238 in each CH2 domain sequence; (v) a cysteine ​​residue inserted between positions 9 and 10 in one VH domain; (vi) a cysteine ​​residue inserted between positions 9 and 10 in each VH domain; (vii) a cysteine ​​residue inserted between positions 40 and 41 in each VL domain; (viii) a cysteine ​​residue inserted between positions 126 and 127 in each CL domain; (ix) a cysteine ​​insertion between positions 299 and 300 in the first CH2 domain sequence, a cysteine ​​residue inserted between positions 299 and 300 in the second CH2 domain sequence, and a cysteine ​​residue inserted between positions 237 and 238 in the second CH2 domain sequence; (x) a cysteine ​​residue inserted between positions 9 and 10 in one VH domain and a cysteine ​​inserted between positions 299 and 300 in each CH2 domain sequence; (xi) a cysteine ​​residue inserted between positions 40 and 41 in each VL domain and a cysteine ​​residue inserted between positions 299 and 300 in one CH2 domain sequence; (xii) a cysteine ​​residue inserted between positions 40 and 41 in each VL domain and a cysteine ​​residue inserted between positions 9 and 10 in one VH domain; or (xiii) a cysteine ​​residue inserted between positions 9 and 10 in each VH domain and a cysteine ​​inserted between positions 237 and 238 in one CH2 domain sequence.

41. The antibody construct of claim 40, comprising:

43. 41. The antibody construct of claim 40, comprising a cysteine ​​residue inserted between positions 299 and 300 in each CH2 domain sequence.

44. 41. The antibody construct of any one of claims 32 to 40, comprising a VH domain sequence having the amino acid sequence set out in SEQ ID NO: 1 or SEQ ID NO: 59, and a VL domain sequence having the amino acid sequence set out in SEQ ID NO: 2 or SEQ ID NO:

56.

45. 45. The antibody construct of any one of claims 32-40 and 44, wherein the anti-cMet antibody construct comprises a first heavy chain and a second heavy chain, wherein the first heavy chain and the second heavy chain each comprise a CH2 domain, and wherein one or both of the CH2 domains comprise an amino acid sequence selected from the sequences set forth in SEQ ID NOs: 76, 77, and 78.

46. 46. ​​The antibody construct of any one of claims 32-40, 44, and 45, wherein the anti-cMet antibody construct comprises a first light chain and a second light chain, each of the first light chain and second light chain comprising a CL domain, wherein one or both of the CL domains comprises the amino acid sequence set forth in SEQ ID NO:

79.

47. Use of an antibody construct according to any one of claims 32 to 46 for preparing an antibody drug conjugate.

48. An antibody-drug conjugate comprising the antibody construct of any one of claims 32 to 46 conjugated to a cytotoxin via a linker.

49. Formula I: A-(L-(D) ｎ ) ｐ (I) wherein A is an antibody construct according to any one of claims 30 to 44, L is a cleavable linker; D is, where: ＊ is the point of attachment to L, n is 1 to 4; p is 1 to 8; each L is conjugated to the sulfhydryl group of an inserted cysteine ​​residue; The antibody-drug conjugate of claim 48.

50. 50. The antibody-drug conjugate of claim 49, wherein L is a protease-cleavable linker.

51. L-D has the following structure: (a) Formula IV (In the formula, Z' is a linking group that connects the linker to a targeting group on the antibody construct A; Str is a stretcher, A.A. １ and A.A. ２ are each independently an amino acid, １ - [AA ２ ] ｍ forms a protease cleavage site, X is a self-immolative group; s is 0 or 1; m is 1, 2, or 3; o is 0, 1, or 2; # is the point of attachment to said antibody construct A), or (b) Formula XII (In the formula, Z' is a linking group that connects the linker to a targeting group on the antibody construct A; Str １ and Str ２ are each independently a stretcher, BU is a branching unit, A.A. １ and A.A. ２ are each independently an amino acid, １ - [AA ２ ] ｍ forms a protease cleavage site, X is a self-immolative group; s and s' are each independently 0 or 1; m is 1, 2, or 3; o is 0, 1, or 2; t is 2 or 3; # is the point of attachment to said antibody construct A), 51. The antibody-drug conjugate of claim 49 or 50, having one of the following:

52. L-D has structure IV, wherein: Z' is a carbonyl group (-C(O)-) or where # is the point of attachment to said antibody construct A, ＊ is the point of attachment to the rest of the linker; The antibody-drug conjugate of claim 51.

53. L-D has structure IV, wherein: s is 1, Str is, where: ＄ is the point of attachment to Z′, ＊ is the point of attachment to the remainder of the linker, p is an integer from 2 to 6, and q is an integer from 2 to 8; The antibody-drug conjugate of claim 51 or 52.

54. L-D has structure IV, wherein: m is 1 and AA １ - [AA ２ ] ｍ is a dipeptide selected from Val-Lys, Ala-Lys, Phe-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit, and Trp-Cit; o is 0; The antibody-drug conjugate according to any one of claims 51 to 53.

55. LD has the structure XII, wherein: Z' is a carbonyl group (-C(O)-) or where # is the point of attachment to said antibody construct A, ＊ is the point of attachment to the rest of the linker; The antibody-drug conjugate of claim 51.

56. LD has the structure XII, wherein: s is 1, s' is 1, Str １ but, where: ＄ is the point of attachment to Z′, ＊ is the point of attachment to the remainder of the linker, p is an integer from 2 to 6, and q is an integer from 2 to 8; Str ２ but, where: ＄ is the attachment point to BU, ＊ is the point of attachment to the remainder of the linker, p is an integer from 2 to 6, and q is an integer from 2 to 8; The antibody-drug conjugate of claim 51 or 55.

57. LD has the structure XII, wherein: m is 1 and AA １ - [AA ２ ] ｍ is a dipeptide selected from Val-Lys, Ala-Lys, Phe-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit, and Trp-Cit; o is 0; 57. The antibody-drug conjugate of any one of claims 51, 55 and 56.

58. 58. The antibody-drug conjugate of any one of claims 51 and 55-57, wherein LD has the structure XII, wherein BU is an amino acid or beheramine.

59. The antibody-drug conjugate of any one of claims 49 to 58, wherein p is 1, 2, 3, or 4.

60. The following structure:

50. The antibody-drug conjugate of claim 49, having one of the following:

61. structure: wherein A is the antibody construct of claim 43 and p is 2.

62. A pharmaceutical composition comprising the antibody-drug conjugate of any one of claims 1 to 31 and 48 to 61 and a pharmaceutically acceptable carrier or diluent.

63. 62. A method of treating cancer in a subject, comprising administering to the subject an effective amount of the antibody-drug conjugate of any one of claims 1 to 31 and 48 to 61.

64. An antibody-drug conjugate according to any one of claims 1 to 31 and 48 to 61 for use in therapy.

65. 65. The antibody-drug conjugate for use according to claim 64, wherein said therapy comprises treating cancer in a subject in need thereof.

66. 62. Use of the antibody-drug conjugate of any one of claims 1 to 31 and 48 to 61 in the manufacture of a medicament for the treatment of cancer.

67. A polynucleotide or set of polynucleotides encoding the antibody construct of any one of claims 32 to 46.

68. 68. A vector or set of vectors comprising the polynucleotide or set of polynucleotides of claim 67.

69. 69. A host cell comprising the vector or set of vectors of claim 68.

70. A polyvalent drug-linker selected from the following:

Images