Antibody drug conjugates targeting C-MET and methods of use

Abstract

An antibody-drug conjugate (ADC) comprising an antibody construct that specifically binds c-Met conjugated via a linker to one or more drugs such as, for example, Auristatin analogue Compound (1). Also described are anti-cMet antibody constructs that have been engineered to include one or more cysteine insertion mutations wherein each inserted cysteine residue provides a "handle" of conjugation, thereby allowing drug-linker conjugation to provide an ADC. Further described are multivalent drug-linkers comprising a plurality of auristatin analogs suitable for use in the ADCs described herein. # imgabs0 #

Description

Technical Field

[0001] The present disclosure relates to the field of immunotherapeutics, and in particular to antibody-drug conjugates comprising an anti-cMet antibody and an auristatin analog and their use in therapy. Background Art

[0002] c-Met (also known as hepatocyte growth factor receptor (HGFR)) is a receptor tyrosine kinase encoded by the MET proto-oncogene. c-Met is often overexpressed in cancer, and activation of c-Met signaling has been associated with drug resistance and carcinogenic, invasive, and metastatic processes (Gherardi et al., 2012, Nat Rev Cancer, 12: 89-103). Therefore, disruption of MET signaling is considered a promising cancer therapy. Various therapeutic strategies targeting c-Met are 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 under development, including telisotuzumab vedotin (formerly known as ABBV-399) (AbbVie Inc.), BYON3521 (Byondis BV), RC108 (RemeGen Co., Ltd), and REGN5093-M114 (Re generon Pharmaceuticals). Among them, telisotuzumab vedotin, which comprises the anti-cMet antibody telisotuzumab vedotin (see U.S. Patent No. 8,545,839) conjugated to monomethyl auristatin E (MM AE), is the most advanced and is currently in Phase III clinical trials. In Phase II trials, telisotuzumab vedotin reportedly exhibited antitumor activity in cMet-positive non-small cell lung cancer (NSC LC), with 65% of patients reporting > Grade 3 treatment-emergent adverse events (Camidge et al., 2021, Clin Cancer Res, 27(21):5781-5792).

[0004] This background information is provided for the purpose of making known information believed by the applicant to be potentially relevant to the present disclosure. It is not necessarily intended to, nor should it be construed as, an admission that any of the foregoing information constitutes prior art to the claimed invention. Summary of the invention

[0005] Described herein are antibody-drug conjugates (ADCs) targeting c-Met and methods of using these ADCs in therapy. One aspect of the disclosure relates to an antibody-drug conjugate having Formula I:

[0006] A-(L-(D) n ) p (I)

[0007] in:

[0008] 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 a heavy chain CDR sequence (HCDR1, HCDR2, and HCDR3) of the VH domain sequence shown in SEQ ID NO: 1 and a light chain CDR sequence (LCDR1, LCDR2, and LCDR3) of the VL domain sequence shown in SEQ ID NO: 2, and the Ig hinge region comprises an upper hinge sequence having an amino acid sequence of a native IgG1, IgG2, or IgG4 upper hinge sequence;

[0009] L is a cleavable linker;

[0010] D is:

[0011]

[0012] Where * is the connection point with L,

[0013] n is between 1 and 4, and

[0014] p is between 1 and 8.

[0015] In certain embodiments, the antibody-drug conjugate has the following structure:

[0016] ADC 002

[0017]

[0018] wherein A is an antibody construct that specifically binds to c-Met, and p is 6, or

[0019] ADC 004

[0020]

[0021] wherein A is the antibody construct that specifically binds to c-Met, and p is 2.

[0022] Another aspect of the present disclosure relates to an antibody construct comprising:

[0023] 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;

[0024] An Fc region comprising a CH2 domain having two CH2 domain sequences and a CH3 domain having two CH3 domain sequences,

[0025] wherein the antigen binding domain comprises the heavy chain CDR sequence (HCDR1, HCDR2 and HCDR3) of the VH domain sequence shown in SEQ ID NO: 1, and the light chain CDR sequence (LCDR1, LCDR2 and LCDR3) of the VL domain sequence shown in SEQ ID NO: 2, and

[0026] Wherein the antibody construct comprises one or more cysteine ​​insertion mutations independently selected from:

[0027] (a) a cysteine ​​residue inserted between positions 40 and 41 in said VL domain;

[0028] (b) a cysteine ​​residue inserted between positions 126 and 127 in the CL domain;

[0029] (c) a cysteine ​​residue inserted between positions 9 and 10 in said VH domain;

[0030] (d) a cysteine ​​residue inserted between positions 237 and 238 in the CH2 domain sequence, and

[0031] (e) a cysteine ​​residue inserted between positions 299 and 300 in the CH2 domain sequence,

[0032] The numbering of amino acids in the VL, CL and VH domains is the Kabat numbering, and the numbering of amino acids in the CH2 domain is the EU numbering.

[0033] Another aspect of the present disclosure relates to the use of an antibody construct comprising one or more cysteine ​​insertion mutations as described herein for the preparation of an antibody drug conjugate.

[0034] Another aspect of the disclosure relates to an antibody-drug conjugate comprising an antibody construct having one or more cysteine ​​insertion mutations as described herein conjugated to a cytotoxin via a linker.

[0035] In certain embodiments, an antibody-drug conjugate comprising an antibody construct having one or more cysteine ​​insertion mutations conjugated to a cytotoxin via a linker has Formula I:

[0036] A-(L-(D) n ) p (I)

[0037] in:

[0038] A is the antibody construct;

[0039] L is a cleavable linker;

[0040] D is:

[0041]

[0042] Where * is the connection point with L,

[0043] n is between 1 and 4, and

[0044] p is between 1 and 8, and

[0045] wherein each L is conjugated to the sulfhydryl group of an inserted cysteine ​​residue.

[0046] Another aspect of the present disclosure relates to an antibody-drug conjugate having the following structure:

[0047] ADC 004

[0048]

[0049] wherein p is 2, and A is an antibody construct comprising:

[0050] 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;

[0051] An Fc region comprising a CH2 domain having two CH2 domain sequences and a CH3 domain having two CH3 domain sequences,

[0052] wherein the antigen binding domain comprises the heavy chain CDR sequence (HCDR1, HCDR2 and HCDR3) of the VH domain sequence shown in SEQ ID NO: 1, and the light chain CDR sequence (LCDR1, LCDR2 and LCDR3) of the VL domain sequence shown in SEQ ID NO: 2, and

[0053] wherein the antibody construct comprises a cysteine ​​residue inserted between positions 299 and 300 in each CH2 domain sequence, and

[0054] The antibody construct is a bivalent antibody comprising two antigen binding domains, each of which specifically binds to c-Met.

[0055] Another aspect of the present disclosure relates to a polynucleotide or set of polynucleotides encoding an antibody construct as described herein comprising one or more cysteine ​​insertion mutations.

[0056] Another aspect of the present disclosure relates to a vector or set of vectors comprising a polynucleotide or set of polynucleotides encoding an antibody construct as described herein comprising one or more cysteine ​​insertion mutations.

[0057] 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 as described herein comprising one or more cysteine ​​insertion mutations.

[0058] Another aspect of the present disclosure relates to a pharmaceutical composition comprising an antibody-drug conjugate as described herein and a pharmaceutically acceptable carrier or diluent.

[0059] Another aspect of the present disclosure relates to a method of treating cancer in a subject, comprising administering to the subject an effective amount of an antibody-drug conjugate as described herein.

[0060] Another aspect of the present disclosure relates to an antibody-drug conjugate as described herein for use in therapy, e.g., in the treatment of cancer in a subject in need thereof.

[0061] Another aspect of the present disclosure relates to the use of an antibody-drug conjugate as described herein in the manufacture of a medicament for treating cancer.

[0062] Another aspect of the present disclosure relates to a multivalent drug-linker selected from the group consisting of:

[0063] Drug-Connector 003

[0064]

[0065] Drug-Connector 004

[0066]

[0067] Drug-Connector 005

[0068]

[0069] Drug-Connector 006

[0070]

[0071] Drug-Connector 007

[0072]

[0073] Drug-Connector 008

[0074] BRIEF DESCRIPTION OF THE DRAWINGS

[0075] 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 indicated.

[0076] Figure 2 presents (A) SDS-PAGE results of representative anti-cMet cysteine ​​insertion variant v29001 under non-reducing (NR) and reducing (R) conditions (M = molecular weight marker), (B) UPLC-SEC chromatogram of representative anti-cMet cysteine ​​insertion variant v29001, and (C) an enlarged view of the peak at 7.827 min shown in the upper figure (B).

[0077] Figure 3 Exemplary reaction pathways for preparing trivalent drug-linkers Drug-Linker 007 and Drug-Linker 008 are presented.

[0078] Figure 4 Results of evaluation of cMet pathway agonism by 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) are presented. Data are presented as the mean (± SEM) of 3 independent experimental replicates.

[0079] Figure 5 presents the results of the evaluation of cMet pathway agonism 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) by measuring AKT phosphorylation by ELISA. Data are presented as the mean (± SEM) of 3 independent experimental replicates.

[0080] Figure 6 Results are presented for the evaluation of cMet pathway agonism by proliferation of H596 lung cancer cells treated with anti-cMet antibodies (v17429 and v17427) and ADCs comprising various anti-cMet cysteine ​​insertion variants conjugated to the drug-linker 001 with DAR 2. Data are presented as the mean (± SEM) of 4 independent experimental replicates.

[0081] Figure 7 presents the results of evaluating cMet pathway agonism of H441 lung cancer cells treated with anti-cMet antibodies (v17429, v17606, and v17427), v17427-Drug-Linker 001 conjugated to the cysteine ​​of DAR 4, v17427-Drug-Linker 002 conjugated to the lysine of DAR 2, and ADCs comprising anti-cMet antibodies site-specifically conjugated to Drug-Linker 001 at (A) DAR 1, (B) DAR 2, and (C) DAR 3 by measuring AKT phosphorylation by ELISA. Data are presented as the mean (± SEM) of two independent experimental replicates.

[0082] Figure 8 presents the results of in vivo evaluation of the anti-tumor activity of ADCs comprising anti-cMet antibodies conjugated to drug-linker 001 or MCvcPABC-MMAE with DAR 4 in the (A) cMet high HCC827 lung cancer model, (B) cMet high EBC1 lung cancer model, (C) cMet high H1975 lung cancer model, (D) cMet medium / high HT29 colorectal cancer model, (E) cMet low H292 lung cancer model, and (F) cMet low SW48 colorectal cancer model.

[0083] Figure 9 presents the results of in vivo evaluation of anti-tumor activity of ADCs comprising an anti-cMet antibody conjugated to drug-linker 001 with DAR1, 2, 3 or 4 or to drug-linker 002 with DAR2 in the cMet high H1975 lung cancer model at (A) toxin-matched doses of 24 mg / kg, 12 mg / kg, 8 mg / kg and 6 mg / kg, and (B) toxin-matched doses of 4 mg / kg, 2 mg / kg, 1.3 mg / kg and 1 mg / kg.

[0084] Figure 10 presents the results of in vivo evaluation of anti-tumor activity of ADCs comprising an anti-cMet antibody conjugated to Drug-Linker 001 with DAR1, 2, 3 or 4 or to Drug-Linker 002 with DAR2 in the cMet-medium / high HT29 colorectal cancer model at (A) toxin-matched doses of 12 mg / kg, 6 mg / kg, 4 mg / kg and 3 mg / kg, and (B) toxin-matched doses of 6 mg / kg, 3 mg / kg, 2 mg / kg and 1.5 mg / kg.

[0085] Figure 11 presents the results of in vivo evaluation of the anti-tumor activity of anti-cMet antibodies comprising drug-linker 002, drug-linker 003, drug-linker 004 or MCvcPABC-MMAE conjugated with various DARs at various doses (as indicated) in the (A) cMet high H1975 lung cancer model, (B) cMet medium / high HT29 colorectal cancer model, (C) cMet low H292 lung cancer model, (D) cMet medium / high Hs746t gastric cancer model, and (E) cMet medium HCT116 colorectal cancer model.

[0086] Fig.12 Results of in vivo evaluation of anti-tumor activity of ADCs comprising anti-cMet antibodies conjugated to drug-linker 003 with DAR4, drug-linker 004 with DAR6, or MCvcPABC-MMAE with DAR3 in various PDX models at the indicated doses are presented. *Models with v29001-Drug-Linker 004DAR6 were not tested.

[0087] Figure 13 presents the results of the evaluation of the pharmacokinetics of ADCs comprising anti-cMet antibodies conjugated to drug-linker 002 with DAR4 or DAR 6, to drug-linker 003 with DAR4, or to drug-linker 004 with DAR6, and the corresponding free antibodies in Tg32 mice, (A) total IgG concentration in serum over time, and (B) total ADC concentration in serum over time.

[0088] Figure 14 presents the results of the assessment of the in vivo stability of ADCs comprising an anti-cMet antibody conjugated to Drug-Linker 002 with DAR4 or DAR 6, to Drug-Linker 003 with DAR4, or to Drug-Linker 004 with DAR6, 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

[0089] The present disclosure relates to antibody-drug conjugates (ADCs) comprising an antibody construct that specifically binds c-Met ("anti-cMet antibody construct") conjugated to a drug (such as a cytotoxin) via a linker. In the ADCs shown, the anti-cMet antibody construct can be conjugated to one drug molecule, or it can be conjugated to more than one drug molecule.

[0090] Certain embodiments of the present disclosure relate to an ADC comprising an anti-cMet antibody construct conjugated to an auristatin analog Compound 1 via a linker.

[0091]

[0092] In such embodiments, the anti-cMet antibody construct may be conjugated to one of the auristatin analogs or may be conjugated to more than one of the auristatin analogs.

[0093] The present disclosure also relates to anti-cMet antibody constructs that have been engineered to contain one or more cysteine ​​insertion mutations. Each inserted cysteine ​​residue provides a conjugation "handle" that allows conjugation of a drug-linker to provide an ADC. Certain embodiments of the present disclosure relate to ADCs comprising anti-cMet antibody constructs that have been engineered to contain one or more cysteine ​​insertion mutations, wherein the antibody construct is conjugated to one or more drug molecules via the one or more inserted cysteines.

[0094] The present disclosure also relates to multivalent drug-linkers comprising a plurality of 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 a plurality of auristatin analogs.

[0095] The ADCs of the present disclosure can be used as therapeutic agents, for example, for treating cancer.

[0096] definition

[0097] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0098] As used herein, the term "about" refers to a variation of approximately + / - 10% from a given value. It should be understood that such variations are always included in any given value provided herein, whether or not specifically mentioned.

[0099] When used in conjunction with the term "comprising" in this document, the use of the word "a / kind" may mean "one / kind", but it also has the meaning of "one / kind or more / kinds", "at least one / kind" and "one / kind or more than one / kind".

[0100] As used herein, the terms "comprising," "having," "including," and "containing," and grammatical variations thereof, are inclusive or open-ended, and do not exclude additional, unlisted elements and / or method steps. When used in conjunction with compositions, uses, or methods herein, the term "consisting essentially of means that additional elements and / or method steps may be present, but these additions do not substantially affect the manner in which the enumerated compositions, methods, or uses function. The term "consisting of" does not include the presence of additional elements and / or method steps when used in combination with compositions, uses, or methods herein. Compositions, uses, or methods described herein as comprising certain elements and / or steps may also consist essentially of those elements and / or steps in certain embodiments, and consist of those elements and / or steps in other embodiments, whether or not these embodiments are specifically mentioned.

[0101] "Complementarity determining region" or "CDR" is an amino acid sequence that contributes to antigen binding specificity and affinity. "Framework" region (FR) can help maintain the correct conformation of CDR to promote the binding between antigen binding region and antigen. From N-terminus to C-terminus, the light chain variable region (VL) and heavy chain variable region (VH) of an antibody generally include 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. CDR provides most of the contact residues for the binding of an antibody to an antigen or epitope. Typically, three heavy chain CDRs and three light chain CDRs are required to bind antigen. However, in some cases, even a single variable domain can also confer binding specificity to an antigen. In addition, as known in the art, in some cases, antigen binding can also occur through a combination of one or more CDRs (e.g., HCDR3) selected from VH and / or VL domains.

[0102] Many 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). For example, the definitions of CDRs according to Kabat, Chothia, IMGT, AbM and Contact are provided in Table 1 below. Therefore, it will be apparent to one skilled in the art that the exact numbering and placement of CDRs may differ based on the numbering system employed. However, it should be understood that the disclosure of VH herein includes disclosure of the associated (intrinsic) heavy chain CDRs (HCDRs) as defined by any known numbering system. Similarly, disclosure of a VL herein includes disclosure of the associated (intrinsic) light chain CDRs (LCDRs) as defined by any known numbering system.

[0103] Table 1: Common CDR definitions 1

[0104]

[0105]

[0106] 1 Either the Kabat or Chothia numbering system may be used for all defined HCDR2, HCDR3 and light chain CDRs except for Contact which uses Chothia numbering.

[0107] 2 Kabat numbering is used. The positions in the Kabat numbering scheme that delineate the ends of the Chothia and IMGT CDR-H1 loops vary depending on the length of the loop, as Kabat places insertions outside of those CDR definitions at positions 35A and 35B. However, the IMGT and Chothia CDR-H1 loops can be clearly defined using Chothia numbering. CDR-H1 definitions using Chothia numbering: Kabat H31-H35, Chothia H26-H32, AbM H26-H35, IMGT H26-H33, Contact H30-H35.

[0108] As used herein, the terms "subject" and "patient" refer to an animal, in some embodiments a mammal, that is the subject of treatment, observation or experiment. The animal can be a human, a non-human primate, a companion animal (e.g., a dog, a cat, etc.), a farm animal (e.g., a cow, a sheep, a pig, a horse, etc.), or a laboratory animal (e.g., a rat, a mouse, a guinea pig, a non-human primate, etc.). In certain embodiments, the subject is a human.

[0109] It is contemplated that any embodiment discussed herein can be implemented by any method, use, or composition disclosed herein, and vice versa.

[0110] Certain features, structures, and / or characteristics described in connection with one embodiment disclosed herein may be combined with features, structures, and / or characteristics described in connection with another embodiment disclosed herein in any suitable manner to provide one or more further embodiments.

[0111] It should also be understood that the positive recitation of a feature in one embodiment is a basis for excluding that feature in another embodiment. For example, where a list of options is presented for a given embodiment or claim, it should be understood that one or more options may be deleted from the list and the shortened list may form an alternative embodiment, regardless of whether such an alternative embodiment is specifically mentioned.

[0112] Antibody-drug conjugates

[0113] Certain embodiments of the present disclosure relate to antibody-drug conjugates (ADCs) having Formula I:

[0114] A-(L-(D) n ) p

[0115] I

[0116] in,

[0117] A is an antibody construct that specifically binds to c-Met;

[0118] L is the connector;

[0119] D is an auristatin analog having the following structure:

[0120]

[0121] Where * is the connection point with L;

[0122] n is between 1 and 4, and

[0123] p is between 1 and 8.

[0124] The linker L can be monovalent (bonded to a single D, where n=1) or multivalent (bonded to multiple Ds, where n=2, 3, or 4).

[0125] 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 nxp defines the drug to antibody ratio, or "DAR," of the ADC. One skilled 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 of which comprises a single D (i.e., n=1 and p=4), or an antibody construct conjugated to two drug linkers, each of which comprises 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 of which comprises a single D (i.e., n=1 and p=6), or an antibody construct conjugated to three drug-linkers, each of which comprises two Ds (i.e., n=2 and p=3), or an antibody construct conjugated to two drug-linkers, each of which comprises three Ds (i.e., n=3 and p=2).

[0126] Those skilled in the art will also understand that in an ADC preparation comprising a plurality of ADCs, each anti-cMet antibody construct A is conjugated to an integer number of auristatin analogs D, however, a DAR determined for an ADC preparation may give a non-integer result reflecting the statistical average of the individual DARs of the plurality of ADCs comprised by the preparation. Thus, ADC preparations having both integer and non-integer DARs are intended to be encompassed by Formula I.

[0127] In some embodiments, the ADC of Formula I has a DAR between about 1 and about 6. In some embodiments, the ADC of Formula I has a DAR between about 2 and about 6. In some embodiments, the ADC of Formula I has a DAR between about 4 and about 6.

[0128] 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.

[0129] Anti-cMet antibody constructs

[0130] The ADC of the present disclosure comprises an anti-cMet antibody construct. In this context, the term "antibody construct" refers to a polypeptide or polypeptide group comprising one or more antigen binding domains, wherein each of the one or more antigen binding domains specifically binds to an epitope or antigen. In the case where the antibody construct comprises two or more antigen binding domains, each antigen binding domain can bind to the same epitope or antigen (ie, the antibody construct is monospecific) or they can bind to different epitopes or antigens (ie, the antibody construct is bispecific or multispecific). According to the present disclosure, the 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 also comprise a scaffold, and at least one of the one or more antigen binding domains may be optionally fused or covalently attached to the scaffold via a linker.

[0131] In certain embodiments, the anti-cMet antibody construct comprises two antigen binding domains, each of which specifically binds to c-Met. In some embodiments, the anti-cMet antibody construct comprises two antigen binding domains, each of which specifically binds to c-Met; and a scaffold. In some embodiments, the 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 domains can each be independently operably linked to the scaffold or the first antigen binding domain, or when there are more than two antigen binding domains, operably linked to another antigen binding domain.

[0132] In certain embodiments, the anti-cMet antibody construct may be an antibody format based on an immunoglobulin (Ig). In certain embodiments, the anti-cMet antibody construct may be based on an IgG class immunoglobulin, such as 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 the anti-cMet antibody construct is based on a specified immunoglobulin isotype, it means that the anti-cMet antibody construct comprises all or part 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, wherein the scaffold comprises an Fc region from a given isotype and optionally an Ig hinge region from the same or different isotypes. It should be understood that in some embodiments, the anti-cMet antibody construct may also comprise a hybrid of an isotype and / or subclass. It should also be understood that the Fc region and / or hinge region may be optionally modified to confer one or more desired functional properties known in the art.

[0133] In some embodiments, the anti-cMet antibody construct can be derived from two or more immunoglobulins from different species, for example, the anti-cMet antibody construct can be a chimeric antibody or a humanized antibody. The terms "chimeric antibody" and "humanized antibody" generally refer to antibodies that combine immunoglobulin regions or domains from more than one species.

[0134] "Chimeric antibodies" typically comprise at least one variable domain from a non-human antibody, such as a rabbit or rodent (e.g., mouse) antibody, and at least one constant domain from a human antibody. The human constant domain of a chimeric antibody need not have 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.

[0135] "Humanized antibodies" are a class of chimeric antibodies containing minimal sequences derived from non-human antibodies. Typically, humanized antibodies are human immunoglobulins (recipient antibodies) in which residues from the hypervariable regions or CDRs of the recipient are replaced with residues from hypervariable regions or CDRs of non-human species (donor antibodies) having the desired specificity and affinity for the target antigen, such as mice, rats, rabbits, or non-human primates. This technique for creating humanized antibodies is often referred to as "CDR transplantation."

[0136] In some cases, additional modifications are made to further improve antibody performance. For example, the framework region (FR) residues of human immunoglobulins are replaced by corresponding non-human residues, or humanized antibodies may include residues not found in recipient antibodies or donor antibodies. In general, the variable domains in humanized antibodies will include all or nearly all hypervariable regions or CDRs from non-human immunoglobulins and all or nearly all FRs from human immunoglobulin sequences. Humanized antibodies are described in more detail 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.

[0137] Many methods are known in the art for selecting the most appropriate human framework for transplanting non-human CDR therein. Early methods use a limited subset of fully characterized human antibodies, independent of the sequence identity of the non-human antibodies providing CDR ("fixed framework" method). The most recent method has adopted a variable region with high amino acid sequence identity to the variable region of the non-human antibodies providing CDR ("homologous matching" or "best fit" method). An alternative method is to select a fragment of framework sequence from each light chain or heavy chain variable region from several different human antibodies. In some cases, CDR transplantation may cause the affinity of the transplanted molecule to its target antigen to be partially or completely lost. In such cases, affinity can be restored by backmutating some of the residues in some human sources to corresponding non-human residues. Methods for preparing humanized antibodies by these methods are well known in the art (see, e.g., 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).

[0138] Alternatively, or in addition to these traditional methods, newer technology can be used to further reduce the immunogenicity of the humanized antibody of CDR transplantation.For example, a framework based on human germline sequence or consensus sequence can be used as a receptor human framework rather than a human framework with somatic mutations.Another technology intended to reduce the potential immunogenicity of non-human CDR is to transplant specificity determining residues (SDR) only.In this method, only the minimum CDR residues (" SDR ") required for antigen binding activity are transplanted into the human germline framework.This method improves the "humanity" (i.e., similarity to human germline sequence) of humanized antibodies, and therefore can contribute to reducing the immunogenicity risk of variable regions. These techniques have been described in various publications (see, e.g., Almagro and 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).

[0139] In certain embodiments, the anti-cMet antibody construct comprises an antigen binding domain that specifically binds to c-Met and an immunoglobulin (Ig) hinge region. In some embodiments, the antibody construct comprises two antigen binding domains, each of which specifically binds to c-Met; and an Ig hinge region. In some embodiments, the anti-cMet antibody construct comprises at least one antigen binding domain that specifically binds to c-Met, an Ig hinge region, and a scaffold.

[0140] In certain embodiments, the anti-cMet antibody construct comprises an antigen binding domain that specifically binds to c-Met, an Ig hinge region, and a scaffold that is an Fc region. In some embodiments, the antibody construct comprises two antigen binding domains, each antigen binding domain specifically binds to c-Met; an Ig hinge region, and an Fc region.

[0141] In certain embodiments, the anti-cMet antibody construct is an antibody or 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 antibody or a bispecific antibody. In some embodiments, the anti-cMet antibody is a monospecific antibody. In some embodiments, the anti-cMet antibody construct is a bivalent, monospecific antibody.

[0142] Antigen binding domain

[0143] The anti-cMet antibody constructs of the present disclosure comprise at least one antigen binding domain that specifically binds to cMet. By "specifically binding" c-Met is meant 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 monkey 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 monkey c-Met, and does not exhibit significant binding to c-Met from other species.

[0144] The at least one antigen binding domain can be an immunoglobulin-based antigen binding domain, such as an antigen binding antibody fragment. Examples of antigen binding antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, single-chain Fab (scFab), single-chain Fv (scFv) and single domain antibodies (sdAb).

[0145] A "Fab fragment" contains the constant domain (CL) of the light chain and the first constant domain (CH1) of the heavy chain, as well as the variable domains of the light and heavy chains (VL and VH, respectively). A Fab' fragment differs from a Fab fragment in that it has several amino acid residues added to the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the hinge region of the antibody. A Fab fragment can also be a single-chain Fab molecule, i.e., a Fab molecule in which a Fab light chain and a Fab heavy chain are connected by a peptide linker to form a single peptide chain. For example, the C-terminus of a Fab light chain can be connected to the N-terminus of a Fab heavy chain in a single-chain Fab molecule.

[0146] "scFv" comprises the heavy chain variable domain (VH) and light chain variable domain (VL) of an antibody in a single polypeptide chain. ScFv may optionally further comprise a polypeptide linker between the VH and VL domains so that the scFv can form a structure required for antigen binding. For example, scFv may include a VL connected to the N-terminus of VH from the C-terminus via a polypeptide linker. Alternatively, scFv may comprise a VH connected to the N-terminus of VL via its C-terminus via a polypeptide linker (see the review in Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)).

[0147] The "sdAb" format refers to a single immunoglobulin domain. sdAb can be, for example, of camel origin. Camel antibodies lack light chains, and their antigen binding sites consist of a single domain called "VHH". sdAb contains three CDR / hypervariable loops CDR1, CDR2, and CDR3 that form the antigen binding site. sdAb is quite stable and easy to express, for example, expressed as a fusion with the Fc of an antibody (see, e.g., Harmsen and De Haard, 2007, Appl. Microbiol Biotechnol., 77 (1): 13-22).

[0148] In those 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 polypeptides or small molecules capable of specifically binding to its target (e.g., a natural or engineered ligand). Non-immunoglobulin-based antibody mimetic forms include, for example, anticalins, fynomers, affimers, alphabodies, DARPins, and avimers. The additional antigen binding domains may bind to the same epitope within c-Met, may bind to different epitopes within c-Met, or may bind to different antigens.

[0149] In certain embodiments, the anti-cMet antibody construct comprises at least one antigen binding domain that specifically binds to c-Met comprising the heavy chain CDR sequence (HCDR1, HCDR2, and HCDR3) of the VH domain sequence shown in SEQ ID NO: 1, and the light chain CDR sequence (LCDR1, LCDR2, and LCDR3) of the VL domain sequence shown in SEQ ID NO: 2 (see Table 2).

[0150] In certain embodiments, the anti-cMet antibody construct comprises at least one antigen binding domain that specifically binds to c-Met comprising a HCDR1 sequence selected from the sequences shown in SEQ ID NOs: 3, 9, 14, 16, and 22; a HCDR2 sequence selected from the sequences shown in SEQ ID NOs: 4, 10, 15, 17, and 23; and a HCDR3 sequence selected from the sequences shown in SEQ ID NOs: 5, 11, and 18 (see Table 2). In certain embodiments, the anti-cMet antibody construct comprises at least one antigen binding domain that specifically binds to c-Met comprising a LCDR1 sequence selected from the sequences shown in SEQ ID NOs: 6, 12, and 19; a LCDR2 sequence selected from the sequences shown in SEQ ID NOs: 7, 13, and 20; and a LCDR3 sequence selected from the sequences shown in SEQ ID NOs: 8 and 21 (see Table 2).

[0151] In certain embodiments, the anti-cMet antibody construct comprises at least one antigen binding domain that specifically binds to c-Met comprising a HCDR1 sequence selected from the sequences shown in SEQ ID NOs: 3, 9, 14, 16 and 22; a HCDR2 sequence selected from the sequences shown in SEQ ID NOs: 4, 10, 15, 17 and 23; a HCDR3 sequence selected from the sequences shown in SEQ ID NOs: 5, 11 and 18; a LCDR1 sequence selected from the sequences shown in SEQ ID NOs: 6, 12 and 19; a LCDR2 sequence selected from the sequences shown in SEQ ID NOs: 7, 13 and 20; and a LCDR3 sequence selected from the sequences shown in SEQ ID NOs: 8 and 21 (see Table 2).

[0152] In certain embodiments, the anti-cMet construct comprises at least one antigen binding domain that specifically binds to c-Met comprising heavy chain CDRs (HCDR1, HCDR2, and HCDR3) as shown in SEQ ID NOs: 3, 4, and 5, respectively, and light chain CDRs (LCDR1, LCDR2, and LCDR3) as shown in SEQ ID NOs: 6, 7, and 8, respectively (see Table 2).

[0153] Table 2: VH, VL and CDR sequences

[0154]

[0155]

[0156] In certain embodiments, the 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 that specifically bind to c-Met that the anti-cMet antibody construct comprises comprises a heavy chain CDR sequence (HCDR1, HCDR2, and HCDR3) of the VH domain sequence shown in SEQ ID NO: 1, and a light chain CDR sequence (LCDR1, LCDR2, and LCDR3) of the VL domain sequence shown in SEQ ID NO: 2 (see Table 2).

[0157] In certain embodiments, the anti-cMet antibody construct comprises two antigen binding domains, each antigen binding domain comprising a HCDR1 sequence selected from the sequence shown in SEQ ID NO: 3, 9, 14, 16 and 22; a HCDR2 sequence selected from the sequence shown in SEQ ID NO: 4, 10, 15, 17 and 23; and a HCDR3 sequence selected from the sequence shown in SEQ ID NO: 5, 11 and 18 (see Table 2). In certain embodiments, the anti-cMet antibody construct comprises two antigen binding domains, each antigen binding domain comprising a LCDR1 sequence selected from the sequence shown in SEQ ID NO: 6, 12 and 19; a LCDR2 sequence selected from the sequence shown in SEQ ID NO: 7, 13 and 20; and a LCDR3 sequence selected from the sequence shown in SEQ ID NO: 8 and 21 (see Table 2).

[0158] In certain embodiments, the anti-cMet antibody construct comprises two antigen binding domains, each antigen binding domain comprising a HCDR1 sequence selected from the sequences shown in SEQ ID NOs: 3, 9, 14, 16 and 22; a HCDR2 sequence selected from the sequences shown in SEQ ID NOs: 4, 10, 15, 17 and 23; a HCDR3 sequence selected from the sequences shown in SEQ ID NOs: 5, 11 and 18; a LCDR1 sequence selected from the sequences shown in SEQ ID NOs: 6, 12 and 19; a LCDR2 sequence selected from the sequences shown in SEQ ID NOs: 7, 13 and 20; and a LCDR3 sequence selected from the sequences shown in SEQ ID NOs: 8 and 21 (see Table 2).

[0159] In certain embodiments, the anti-cMet antibody construct comprises two antigen binding domains, each antigen binding domain comprising the heavy chain CDR (HCDR1, HCDR2, and HCDR3) sequences shown in SEQ ID NOs: 3, 4, and 5, respectively, and the light chain CDR (LCDR1, LCDR2, and LCDR3) sequences shown in SEQ ID NOs: 6, 7, and 8, respectively (see Table 2).

[0160] Hinge area

[0161] In certain embodiments, the anti-cMet antibody construct comprises an immunoglobulin (Ig) hinge region. The Ig hinge region can be based on a native human IgG1, IgG2 or IgG4 hinge region sequence, or it can be a modified form of a native human IgG1, IgG2 or IgG4 hinge region sequence.

[0162] As is 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 position 216 to 238 in IgG1 (Burton, 1985, Molec. Immunol., 22: 161-206). Hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by aligning the first and last cysteine ​​residues that form inter-heavy chain disulfide bonds, such as Figure 1 The Ig hinge region can be considered to consist of three sub-parts: the upper hinge, the middle hinge, and the lower hinge (see Figure 1 ) (Burton, 1985, supra; see also Deveuve et al., 2019, Med Sci (Paris), 35(12): 1098-1105). Table 3 provides the sequences of the native complete hinge region and upper hinge region of human IgG1, IgG2 and IgG4.

[0163] Table 3: Hinge region sequences

[0164]

[0165] In certain embodiments, the anti-cMet antibody construct comprises an Ig hinge region comprising an upper hinge sequence having an amino acid sequence of a natural IgG1, IgG2 or IgG4 upper hinge sequence. In some embodiments, the anti-cMet antibody construct comprises an Ig hinge region comprising an amino acid sequence as shown in any one of SEQ ID NO: 25, 27 or 29. In some embodiments, the anti-cMet antibody construct comprises an Ig hinge region comprising an upper hinge sequence having an amino acid sequence of a natural IgG1 upper hinge sequence. In some embodiments, the anti-cMet antibody construct comprises an Ig hinge region comprising an amino acid sequence as shown in SEQ ID NO: 25.

[0166] In certain embodiments, the anti-cMet antibody construct comprises an Ig hinge region comprising at least a portion of a native IgG1, IgG2, or IgG4 hinge sequence, such as an upper hinge sequence or an upper hinge sequence and a middle (or "core") hinge sequence. In some embodiments, the anti-cMet antibody construct comprises an Ig hinge region comprising at least a portion of a native IgG1 hinge sequence. In some embodiments, the anti-cMet antibody construct comprises an Ig hinge region comprising at least a portion of a native IgG1 hinge sequence, wherein the portion has the amino acid sequence: EPKSCDKTHTCPPCP (SEQ ID NO: 35).

[0167] In certain embodiments, the anti-cMet antibody construct comprises an Ig hinge region comprising the amino acid sequence of a native IgG1, IgG2, or IgG4 hinge region. In some embodiments, the anti-cMet antibody construct comprises an Ig hinge region comprising an amino acid sequence as shown in any one of SEQ ID NO: 24, 26, or 28. In some embodiments, the anti-cMet antibody construct comprises an Ig hinge region comprising the amino acid sequence of a native IgG1 hinge region. In some embodiments, the anti-cMet antibody construct comprises an Ig hinge region comprising the amino acid sequence as shown in SEQ ID NO: 24.

[0168] In certain embodiments, the anti-cMet antibody construct comprises an Ig hinge region that is a modified form of a native human IgG1, IgG2, or IgG4 hinge region sequence. In some embodiments, the anti-cMet antibody construct comprises an Ig hinge region that is a modified form of a native human IgG1 hinge region sequence. For example, in some embodiments, the anti-cMet antibody construct may comprise one of the modified hinge sequences described in U.S. Pat. No. 8,545,839 or U.S. Pat. No. 8,741,290. In some embodiments, the anti-cMet antibody construct may comprise a modified hinge sequence: EPKSCDCHCPPCP (SEQ ID NO: 36).

[0169] Bracket

[0170] In certain embodiments, at least one of the one or more antigen binding domains contained in 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 that allows them to function in their intended manner. Examples of suitable scaffolds are described in more detail below and include, but are not limited to, immunoglobulin Fc regions, albumin, albumin analogs and derivatives, heterodimerizing peptides (such as leucine zippers, "zipper" peptides that form heterodimers derived from Jun and Fos, IgG CH1 and CL domains, or barnase-barstar toxins), cytokines, chemokines, or growth factors. Other examples include DOCK-AND-LOCK based on the DOCK-AND-LOCK developed by IBC Pharmaceuticals, Inc. and Immunomedics, Inc. TM (DNL TM ) technology (see, e.g., Chang et al., 2007, Clin. Cancer Res., 13:5586s-5591s).

[0171] The scaffold can be a peptide, polypeptide, polymer, nanoparticle or other chemical entity. When the scaffold is a polypeptide, the antigen binding domain can be connected to the N-terminus or C-terminus of the polypeptide scaffold. Anti-cMet antibody constructs comprising polypeptide scaffolds are also contemplated in certain embodiments, wherein one or more of the antigen binding domains are connected to regions other than the N-terminus or C-terminus, for example, via the side chains of amino acids with or without a linker.

[0172] One or more antigen binding domains of the anti-cMet antibody construct can be attached to the scaffold by genetic fusion or chemical conjugation. In certain embodiments, when the scaffold is a peptide or polypeptide, the one or more antigen binding domains are attached to the scaffold by genetic fusion. In some embodiments, where the scaffold is a polymer or nanoparticle, the antigen binding domain can be attached to the scaffold by chemical conjugation.

[0173] 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., Müller 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. 2009 / 0285816).

[0174] In certain embodiments, the anti-cMet antibody construct may comprise a protein scaffold based on an immunoglobulin Fc region, albumin, or an albumin analog or derivative. For example, it has been demonstrated that fusing an antigen binding moiety such as a scFv, a diabody, or a single chain diabody to albumin can improve the serum half-life of the antigen binding moiety (Müller et al., supra). The antigen binding moiety may optionally be fused to the N-terminus and / or C-terminus of albumin via a linker. A derivative of albumin in the form of a heteromultimer has been described, comprising two transporter polypeptides obtained by albumin segmentation, such that the transporter polypeptides self-assemble to form a natural albumin-like protein (see International Patent Application Publication Nos. WO 2012 / 116453 and WO 2014 / 012082). Due to the segmentation of albumin, the heteromultimer comprises four termini and may therefore optionally be fused to up to four different antigen binding moieties via a linker.

[0175] In some embodiments, the anti-cMet antibody construct may comprise a protein scaffold based on an immunoglobulin Fc region (eg, an IgG Fc region).

[0176] Fc region

[0177] As used herein, the term "Fc region," "Fc" or "Fc domain" refers to the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. Unless otherwise indicated 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).

[0178] In certain embodiments, the anti-cMet antibody constructs of the present disclosure may comprise a scaffold based on an immunoglobulin (Ig) Fc region. The Fc region may be dimeric and consist of two Fc polypeptides, or alternatively, the Fc region may consist of a single polypeptide.

[0179] In the case of a dimeric Fc, "Fc polypeptide" refers to one of the two polypeptides forming 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" can be used interchangeably, provided that the Fc region comprises one first Fc polypeptide and one second Fc polypeptide.

[0180] The 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 polypeptide 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.

[0181] In some embodiments, the anti-cMet antibody construct may comprise a scaffold based on an IgG Fc region and comprise an IgG CH2 domain and an IgG CH3 domain. In some embodiments, the anti-cMet antibody construct may comprise a scaffold based on a human IgG Fc region. In some embodiments, the anti-cMet antibody construct may comprise a scaffold based on an IgG1 Fc region. In some embodiments, the anti-cMet antibody construct may comprise a scaffold based on a human IgG1 Fc region.

[0182] In certain embodiments, the anti-cMet antibody construct may include a scaffold based on an IgG Fc region, the IgG Fc region being 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, and wherein the first and second Fc polypeptides are the same. In certain embodiments, the anti-cMet antibody construct may include a scaffold based on an IgG Fc region, the IgG Fc region being 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, and wherein the first and second Fc polypeptides are different. In some embodiments, the anti-cMet antibody construct may include a scaffold based on an Fc region, the Fc region comprising two CH3 domain sequences, wherein at least one CH3 domain sequence comprises one or more amino acid modifications. In some embodiments, the anti-cMet antibody construct may include a scaffold based on an Fc region, the Fc region comprising two CH3 domain sequences and two CH2 domain sequences, wherein at least one CH2 domain sequence comprises one or more amino acid modifications.

[0183] In some embodiments, the anti-cMet antibody construct may comprise a heterodimeric Fc region comprising a modified CH3 domain, wherein the modified CH3 domain is an asymmetrically modified CH3 domain containing 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 the amino acid at a specific position on the first CH3 domain sequence or CH2 domain sequence is different from the amino acid at the same position on the second CH3 domain sequence 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 corresponding amino acid position on each sequence, or different modifications of the two amino acids at the same corresponding 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.

[0184] In some embodiments, the anti-cMet antibody construct may comprise a heterodimeric Fc containing a modified CH3 domain, wherein the modified CH3 domain comprises one or more amino acid modifications that promote the formation of heterodimeric Fc rather than homodimeric Fc. In some embodiments, one or more of the amino acid modifications that promote the formation of heterodimeric Fc are asymmetric amino acid modifications.

[0185] Amino acid modifications that can be made to the CH3 domain of Fc to promote the formation of heterodimeric Fc are known in the art and include, for example, International Publication No. WO 96 / 027011 ("knob-mortise"), Gunasekaran et al., 2010, J Biol Chem, 285, 19637-46 ("electrostatic manipulation"), Davis et al., 2010, Prot Eng Des Sel, 23(4): 195-202 (chain 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 methods of combining positive and negative design strategies to generate stable asymmetric modified Fc regions, such as those described in International Publication Nos. WO 2012 / 058768 and WO 2013 / 063702. In certain embodiments, the anti-cMet antibody construct may comprise a modified Fc region based scaffold as described in International Publication Nos. WO 2012 / 058768 or WO 2013 / 063702.

[0186] Table 4 provides the amino acid sequence of the human IgG1 Fc sequence (SEQ ID NO: 30), corresponding to amino acids 231 to 447 of the full-length human IgG1 heavy chain. The CH3 sequence comprises amino acids 341-447 of the full-length human IgG1 heavy chain. Table 4 also shows CH3 domain amino acid modifications that promote heterodimeric Fc formation, as described in International Patent Application Publication Nos. WO 2012 / 058768 and WO2013 / 063702.

[0187] In certain embodiments, the anti-cMet antibody construct may comprise a heterodimeric Fc scaffold having a modified CH3 domain comprising modifications of any of variant 1, variant 2, variant 3, variant 4, or variant 5, as shown in Table 4.

[0188] 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

[0189] a) the first Fc polypeptide comprises amino acid modifications L351Y, F405A and Y407V, and the second Fc polypeptide comprises amino acid modifications T366L, K392M and T394W, or

[0190] b) the first Fc polypeptide comprises amino acid modifications L351Y, F405A and Y407V, and the second Fc polypeptide comprises amino acid modifications T366L, K392L and T394W, or

[0191] c) the first Fc polypeptide comprises amino acid modifications T350V, L351Y, F405A and Y407V, and the second Fc polypeptide comprises amino acid modifications T350V, T366L, K392M and T394W, or

[0192] d) the first Fc polypeptide comprises amino acid modifications T350V, L351Y, F405A and Y407V, and the second Fc polypeptide comprises amino acid modifications T350V, T366L, K392L and T394W, or

[0193] e) said first Fc polypeptide comprises amino acid modifications T350V, L351Y, S400E, F405A and Y407V, and said second Fc polypeptide comprises amino acid modifications T350V, T366L, N390R, K392M and T394W.

[0194] Table 4: Human IgG1 Fc sequences that promote heterodimer formation 1 and CH3 domain amino acid modifications

[0195]

[0196]

[0197] 1 Sequence from position 231 to 447 (EU numbering)

[0198] In some embodiments, the anti-cMet antibody construct may comprise a scaffold based on an Fc region comprising two CH3 domain sequences and two CH2 domain sequences, at least one of the CH2 sequences comprising one or more amino acid modifications. Modifications in the CH2 domain may affect the binding of an Fc receptor (FcR) to Fc, such as receptors of the FcγRI, FcγRII, and FcγRIII subclasses. In some embodiments, the anti-cMet antibody construct comprises a scaffold based on an IgG Fc with 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.

[0199] A variety of amino acid modifications to the CH2 domain are known in the art that selectively alter the affinity of Fc for different Fcγ receptors (see, 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 Biol Chem, 23(12):154-16; Immunol, 45(15):3926-33; International Publication No. WO2021 / 232162, and Therapeutic Antibody Engineering (Strohl and Strohl, Woodhead Publishing series in Biomedicine No. 11, ISBN 1 907568 37 9, October 2012, p. 283).

[0200] Amino acid modifications that result in increased FcγR binding and amino acid modifications that result in reduced FcγR binding are each 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 results in increased lysis of target cells. Reducing binding to FcγRIIb (an inhibitory receptor) may also be beneficial in some cases. In certain indications, it may be necessary to reduce or eliminate ADCC and complement-mediated cytotoxicity (CDC). In such cases, a modified CH2 domain ("knockout" variant) comprising an amino acid modification that results in increased binding to FcγRIIb or an amino acid modification that reduces or eliminates binding of the Fc region to all Fcγ receptors may be useful.

[0201] Various publications describe strategies that have been used to engineer antibodies to generate “knockout” variants (see, e.g., Strohl, 2009, Curr Opin Biotech 20:685-691, and Strohl et al., “Antibody Fc engineering for optimal antibody performance” Therapeutic Antibody Engineering, Cambridge: Woodhead Publishing, 2012, pp. 225-249; U.S. Patent Publication No. 2011 / 0212087, International Publication No. WO 2006 / 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 generate "knockout" variants include amino acid modifications L234A / L235A and L234A / L235A / D265S.

[0202] 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, the glycosylation of 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 the production of an aglycosylated Fc lacking all effector functions (Bolt et al., 1993, Eur. J. Immunol., 23: 403-411; Tao & Morrison, 1989, J. Immunol., 143: 2595-2601).

[0203] In contrast, removal of fucose from heavy chain N297-linked oligosaccharides has been shown to enhance ADCC based on 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 that lack fucosyltransferase (FUT8) (Yamane-Ohnuki et al., 2004, Biotechnol. Bioeng., 87:614-622); in a variant CHO cell line Lec 13 with reduced ability to attach fucose to N297-linked carbohydrates (International Publication No. WO 03 / 035835), or other cells that produce afucosylated 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). In addition, International Publication No. WO 2009 / 135181 describes adding a fucose analog to the culture medium during antibody production to inhibit fucose incorporation into carbohydrates on the antibody. Other methods for producing antibodies containing little or no fucose on the Fc glycosylation site (N297) are well known in the art. For example, Technology (ProBioGen AG) (see von Horsten et al., 2010, Glycobiology, 20(12): 1607-1618 and US Pat. No. 8,409,572).

[0204] Other glycosylation variants include those with bisected oligosaccharide variants, for example, wherein the biantennary oligosaccharide connected to the Fc region of the antibody is bisected by N-acetylglucosamine (GlcNAc) variants. Such glycosylation variants can have reduced fucosylation and / or improved ADCC function (see, for example, International Publication No. WO 2003 / 011878, U.S. Patent No. 6,602,684 and U.S. Patent Application Publication No. US2005 / 0123546). Useful glycosylation variants also include those variants having at least one galactose residue in the oligosaccharide connected to the Fc region, which can have improved CDC function (see, for example, International Publication No. WO 1997 / 030087, WO 1998 / 58964 and WO 1999 / 22764).

[0205] Anti-cMet antibody constructs containing cysteine ​​mutations

[0206] Certain embodiments of the present disclosure relate to an anti-cMet antibody construct as described above, further comprising one or more non-native cysteine ​​residues. The one or more non-native cysteine ​​residues provide a "conjugation handle" allowing conjugation of a drug-linker as described herein.

[0207] The antibody can be modified to contain a non-native cysteine ​​residue by substituting a native cysteine ​​residue for the native residue (see, e.g., U.S. Pat. Nos. 7,521,541; 8,455,622 and 9,000,130) or by inserting a cysteine ​​residue between two native residues in the antibody sequence (see, e.g., U.S. Pat. No. 10,744,206).

[0208] 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 between one and 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, such as between one and four cysteine ​​substitution mutations. In some embodiments, the anti-cMet antibody construct may comprise one or more cysteine ​​insertion mutations, such as between one and four cysteine ​​insertion mutations.

[0209] 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 cysteine ​​insertion mutation being independently selected from:

[0210] (a) a cysteine ​​residue inserted between positions 39 and 40 in the VL domain;

[0211] (b) a cysteine ​​residue inserted between positions 40 and 41 in said VL domain;

[0212] (c) a cysteine ​​residue inserted between positions 126 and 127 in said CL domain;

[0213] (d) a cysteine ​​residue inserted between positions 148 and 149 in the CL domain;

[0214] (e) a cysteine ​​residue inserted between positions 149 and 150 in said CL domain;

[0215] (f) a cysteine ​​residue inserted between positions 9 and 10 in said VH domain;

[0216] (g) a cysteine ​​residue inserted between positions 169 and 170 in the CH1 domain;

[0217] (h) a cysteine ​​residue inserted between positions 237 and 238 in the CH2 domain;

[0218] (i) a cysteine ​​residue inserted between positions 295 and 296 in the CH2 domain, and

[0219] (j) Cysteine ​​residue inserted between positions 299 and 300 in the CH2 domain.

[0220] The numbering of amino acids in the VL, CL and VH domains used herein when describing cysteine ​​insertion mutations is the Kabat numbering, while the numbering of amino acids in the CH2 domain is the EU numbering.

[0221] In some embodiments, the anti-cMet antibody construct may comprise one or more cysteine ​​insertion mutations, each cysteine ​​insertion mutation being independently selected from:

[0222] (i) a cysteine ​​residue inserted between positions 40 and 41 in said VL domain;

[0223] (ii) a cysteine ​​residue inserted between positions 126 and 127 in the CL domain;

[0224] (iii) a cysteine ​​residue inserted between positions 9 and 10 in said VH domain;

[0225] (iv) a cysteine ​​residue inserted between positions 237 and 238 in the CH2 domain; and

[0226] (v) A cysteine ​​residue inserted between positions 299 and 300 in the CH2 domain.

[0227] In some embodiments, the anti-cMet antibody construct can be monovalent and comprises 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 can 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.

[0228] In some embodiments, the anti-cMet antibody construct can be bivalent and comprise two antigen binding domains, each antigen binding domain comprising a VL domain and a VH domain, and optionally a CH1 domain and a CL domain. The antigen binding domains can all bind to the same antigen, or they can each bind to a different antigen. The bivalent anti-cMet antibody construct can comprise one or more cysteine ​​insertion mutations in one antigen binding domain, or it can 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 can comprise the same cysteine ​​insertion mutation, or they can comprise different cysteine ​​insertion mutations.

[0229] In some embodiments, the bivalent anti-cMet antibody construct may comprise a cysteine ​​residue inserted between positions 39 and 40 in one VL domain. In some embodiments, the bivalent anti-cMet antibody construct may comprise a cysteine ​​residue inserted between positions 39 and 40 in each VL domain. In some embodiments, the bivalent anti-cMet antibody construct may comprise a cysteine ​​residue inserted between positions 40 and 41 in one VL domain. In some embodiments, the bivalent anti-cMet antibody construct may comprise a cysteine ​​residue inserted between positions 40 and 41 in each VL domain. In some embodiments, the bivalent anti-cMet antibody construct may comprise a cysteine ​​residue inserted between positions 126 and 127 in one CL domain. In some embodiments, the bivalent anti-cMet antibody construct may comprise a cysteine ​​residue inserted between positions 126 and 127 in each CL domain. In some embodiments, the bivalent anti-cMet antibody construct may comprise a cysteine ​​residue inserted between positions 148 and 149 in one CL domain. In some embodiments, the bivalent anti-cMet antibody construct may comprise a cysteine ​​residue inserted between positions 148 and 149 in each CL domain. In some embodiments, the bivalent anti-cMet antibody construct may comprise a cysteine ​​residue inserted between positions 149 and 150 in one CL domain. In some embodiments, the bivalent anti-cMet antibody construct may comprise a cysteine ​​residue inserted between positions 149 and 150 in each CL domain. In some embodiments, the bivalent anti-cMet antibody construct may comprise a cysteine ​​residue inserted between positions 9 and 10 in one VH domain. In some embodiments, the bivalent anti-cMet antibody construct may comprise a cysteine ​​residue inserted between positions 9 and 10 in each VH domain. In some embodiments, the bivalent anti-cMet antibody construct may comprise a cysteine ​​residue inserted between positions 169 and 170 in one CH1 domain. In some embodiments, the bivalent anti-cMet antibody construct may comprise a cysteine ​​residue inserted between positions 169 and 170 in each CH1 domain.

[0230] In some embodiments, the anti-cMet antibody construct may comprise an Fc region. In some embodiments, the anti-cMet antibody construct may comprise a dimeric Fc region consisting of two Fc polypeptides as described above, wherein the CH3 domain of the Fc region comprises two CH3 domain sequences, i.e., one each of the two Fc polypeptides from the dimeric Fc region, and the CH2 domain of the Fc region comprises two CH2 domain sequences, i.e., one each of the two Fc polypeptides from the dimeric Fc region. In some embodiments, the anti-cMet antibody construct may comprise an Fc region consisting 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, wherein both the two CH3 domain sequences and the two CH2 domain sequences are comprised by the single Fc polypeptide.

[0231] In some embodiments, the anti-cMet antibody construct may comprise an Fc region and a cysteine ​​residue inserted between positions 237 and 238 in one CH2 domain sequence. In some embodiments, the anti-cMet antibody construct may comprise an Fc region and a cysteine ​​residue inserted between positions 237 and 238 in each CH2 domain sequence. In some embodiments, the anti-cMet antibody construct may comprise an Fc region and a cysteine ​​residue inserted between positions 295 and 296 in one CH2 domain sequence. In some embodiments, the anti-cMet antibody construct may comprise an Fc region and a cysteine ​​residue inserted between positions 295 and 296 in each CH2 domain sequence. In some embodiments, the anti-cMet antibody construct may comprise an Fc region and a cysteine ​​residue inserted between positions 299 and 300 in one CH2 domain sequence. 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.

[0232] In certain embodiments, the anti-cMet antibody construct comprises an antigen binding domain that specifically binds to c-Met and comprises 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 one or more cysteine ​​insertion mutations independently selected from the following:

[0233] (a) a cysteine ​​residue inserted between positions 40 and 41 in said VL domain;

[0234] (b) a cysteine ​​residue inserted between positions 126 and 127 in the CL domain;

[0235] (c) a cysteine ​​residue inserted between positions 9 and 10 in said VH domain;

[0236] (d) a cysteine ​​residue inserted between positions 237 and 238 in the CH2 domain sequence, and

[0237] (e) Cysteine ​​residue inserted between positions 299 and 300 in the CH2 domain sequence.

[0238] Various combinations of the above cysteine ​​insertion mutations are contemplated and may be selected depending 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, the anti-cMet antibody construct may comprise a combination of cysteine ​​insertions. In some embodiments, the anti-cMet antibody construct may comprise a combination of cysteine ​​insertions, wherein the combination comprises:

[0239] (a) cysteine ​​residues inserted between positions 299 and 300 and between positions 237 and 238 in the CH2 domain, or

[0240] (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

[0241] (c) a cysteine ​​residue inserted between positions 299 and 300 in the CH2 domain and between positions 40 and 41 in the VL domain, or

[0242] (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

[0243] (e) cysteine ​​residues inserted between positions 9 and 10 in the VH domain and between positions 40 and 41 in the VL domain.

[0244] In some embodiments, the 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 a cysteine ​​residue inserted between positions 237 and 238 in another CH2 domain sequence. In some embodiments, the 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.

[0245] In some embodiments, the anti-cMet antibody construct comprises two antigen binding domains, each 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 a cysteine ​​residue inserted between positions 237 and 238 in another CH2 domain sequence. In some embodiments, the anti-cMet antibody construct comprises two antigen binding domains, each 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.

[0246] In some embodiments, the anti-cMet antibody construct comprises two antigen binding domains, each 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 a cysteine ​​residue inserted between positions 40 and 41 in each VL domain, and (i) a cysteine ​​residue inserted between positions 299 and 300 in a CH2 domain sequence, or (ii) a cysteine ​​residue inserted between positions 9 and 10 in a VH domain. In some embodiments, the anti-cMet antibody construct comprises two antigen binding domains, each 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 a cysteine ​​residue inserted between positions 9 and 10 in each VH domain and a cysteine ​​insertion between positions 237 and 238 in a CH2 domain sequence.

[0247] In certain embodiments, the anti-cMet antibody construct comprises at least one VH domain, at least one VL domain, and an Fc region comprising two CH2 domain sequences and one of the following cysteine ​​insertion mutation combinations:

[0248] (a) a cysteine ​​residue inserted between positions 299 and 300 and between positions 237 and 238 in one or both CH2 domain sequences, or

[0249] (b) a cysteine ​​residue inserted between positions 299 and 300 in one or both CH2 domain sequences and a cysteine ​​residue inserted between positions 9 and 10 in the VH domain, or

[0250] (c) a cysteine ​​residue inserted between positions 299 and 300 in one or both CH2 domain sequences and a cysteine ​​residue inserted between positions 40 and 41 in the VL domain, or

[0251] (d) a cysteine ​​residue inserted between positions 237 and 238 in one or both CH2 domain sequences and a cysteine ​​residue inserted between positions 9 and 10 in the VH domain, or

[0252] (e) A cysteine ​​residue inserted between positions 9 and 10 in the VH domain, and a cysteine ​​residue inserted between positions 40 and 41 in the VL domain.

[0253] In certain embodiments, the anti-cMet antibody construct comprises one or two VH domains, one or two VL domains, and an Fc region comprising two CH2 domain sequences, and further comprises:

[0254] (i) a cysteine ​​residue inserted between positions 299 and 300 in a CH2 domain sequence, or

[0255] (ii) a cysteine ​​residue inserted between positions 299 and 300 in each CH2 domain sequence, or

[0256] (iii) a cysteine ​​residue inserted between positions 237 and 238 in a CH2 domain sequence, or

[0257] (iv) a cysteine ​​residue inserted between positions 237 and 238 in each CH2 domain sequence, or

[0258] (v) a cysteine ​​residue inserted between positions 9 and 10 in a VH domain, or

[0259] (vi) a cysteine ​​residue inserted between positions 9 and 10 in each VH domain, or

[0260] (vii) a cysteine ​​residue inserted between positions 40 and 41 in each VL domain, or

[0261] (viii) a cysteine ​​residue inserted between positions 126 and 127 in each CL domain, or

[0262] (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

[0263] (x) a cysteine ​​residue inserted between positions 9 and 10 in one VH domain and a cysteine ​​insertion between positions 299 and 300 in each CH2 domain sequence, or

[0264] (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

[0265] (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

[0266] (xiii) a cysteine ​​residue inserted between positions 9 and 10 in each VH domain, and a cysteine ​​insertion between positions 237 and 238 in one CH2 domain sequence.

[0267] In certain embodiments, the anti-cMet antibody construct comprises a VH domain comprising the amino acid sequence as shown in SEQ ID NO: 59. In certain embodiments, the anti-cMet antibody construct comprises two VH domains, each VH domain comprising the amino acid sequence as shown in SEQ ID NO: 59.

[0268] In certain embodiments, the anti-cMet antibody construct comprises a VL domain comprising the amino acid sequence as shown in SEQ ID NO: 56. In certain embodiments, the anti-cMet antibody construct comprises two VL domains, each VL domain comprising the amino acid sequence as shown in SEQ ID NO: 56.

[0269] 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, wherein one of the CH2 domains comprises an amino acid sequence selected from the group consisting of the sequences shown 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, wherein both CH2 domains comprise an amino acid sequence selected from the group consisting of the sequences shown in SEQ ID NOs: 76, 77, and 78.

[0270] In certain embodiments, the 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, wherein one of the CL domains comprises the amino acid sequence as shown in SEQ ID NO. 79. In certain embodiments, the 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, wherein both CL domains comprise the amino acid sequence as shown in SEQ ID NO: 79.

[0271] Auristatin Analog Drug-Linker

[0272] In certain embodiments, the ADC of the present disclosure comprises an anti-cMet antibody construct as described above conjugated to an auristatin analog, Compound 1, via a linker. Certain embodiments therefore relate to a drug-linker (auristatin analog-linker) having Formula II:

[0273] L-(D) n

[0274] II

[0275] in

[0276] L is the connector;

[0277] D has the following structure:

[0278]

[0279] where * is the connection point to L, and

[0280] n is between 1 and 4.

[0281] The linker L is composed of a drug-linker of Formula II and an ADC of Formula I, and functions to link one or more auristatin analogs to the anti-cMet antibody construct, and can 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 more than one auristatin analog to a single site on the anti-cMet antibody construct. In some embodiments, a linker that links one auristatin analog to more than one site on the anti-cMet antibody construct can also be considered multivalent.

[0282] In certain embodiments, the linker L is linked to the anti-cMet antibody construct A via a functional group capable of reacting with one or more target groups on the antibody construct, and is linked to the auristatin analog via a functional group capable of reacting with a target amino group on the auristatin analog. Suitable functional groups are known in the art and include, for example, those described in Bioconjugate Techniques (GTHermanson, 2013, Academic Press). Groups on the anti-cMet antibody construct that can be used as target groups for linker attachment include, but are not limited to, thiol, hydroxyl, carboxyl, amine, aldehyde, and ketone groups.

[0283] Non-limiting examples of functional groups capable of reacting with thiols include maleimide, haloacetamide, haloacetyl, activated esters (such as succinimidyl esters, 4-nitrophenyl esters, pentafluorophenyl esters and tetrafluorophenyl esters), anhydrides, acyl chlorides, sulfonyl chlorides, isocyanates and isothiocyanates. In this case, "self-stabilizing" maleimides as described in Lyon et al., 2014, Nat. Biotechnol., 32: 1059-1062 can also be used.

[0284] Non-limiting examples of functional groups for reacting with free amines include activated esters such as N-hydroxysuccinamide (NHS) esters, sulfo-NHS esters, imidoesters such as Traut's reagent, tetrafluorophenyl (TFP) esters, sulfodichlorophenyl esters, isothiocyanates, aldehydes, and anhydrides such as diethylenetriaminepentaacetic anhydride (DTPA). Other examples include the use of succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate (TSTU) or benzotriazol-1-yl-oxytripyrrolephosphonium hexafluorophosphate (PyBOP) to convert carboxylic acids into activated esters, which can then react with amines.

[0285] Non-limiting examples of functional groups capable of reacting with an electrophilic group such as an aldehyde or ketone carbonyl group include hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide.

[0286] In certain embodiments, the linker L may comprise a functional group that allows for bridging of two interchain cysteines on the anti-cMet antibody construct, such as ThioBridge TM linker (Badescu et al., 2014, Bioconjug. Chem. 25: 1124-1136), dithiomaleimide (DTM) linker (Behrens et al., 2015, Mol. Pharm. 12: 3986-3998), dithioaryl (TCEP) pyridazinedione-based linker (Lee et al., 2016, Chem. Sci., 7: 799-802) or dibromopyridazinedione-based linker (Maruani et al., 2015, Nat. Commun., 6: 6645).

[0287] Alternatively, the anti-cMet antibody construct may be modified to include a non-natural reactive group, such as an azide, which allows conjugation to the linker through a complementary reactive group on the linker. For example, the conjugation of the linker to the targeting moiety can utilize a click chemistry reaction (see, e.g., Chio and Bane, 2020, Methods Mol. Biol., 2078: 83-97), such as an azide-alkyne cycloaddition (AAC) reaction, which has been successfully used to develop antibody-drug conjugates. The AAC reaction can be a copper-catalyzed AAC (CuAAC) reaction, which involves the coupling of an azide to a linear alkyne, or a strain-promoted AAC (SPAAC) reaction, which involves the coupling of an azide to a cyclooctyne.

[0288] The linker L can be a cleavable or non-cleavable linker. A cleavable linker is a linker that is easily cleaved under specific conditions, such as intracellular conditions (such as in endosomes or lysosomes) or near target cells (such as in a tumor microenvironment). Examples include protease-sensitive, acid-sensitive or reduction-sensitive linkers. In contrast, non-cleavable linkers rely on the degradation of the antibody in the cell, which generally results in the release of the amino acid-linker-drug moiety.

[0289] The example of cleavable linker includes, for example, a linker comprising an amino acid sequence as a cleavage recognition sequence of a protease. Many such cleavage recognition sequences are known in the art. For conjugates that are not intended to be internalized by cells, for example, amino acid sequences recognized and cleaved by proteases present in the extracellular matrix near target cells such as cancer cells can be used. Examples of extracellular tumor-associated proteases include, for example, plasmin, matrix metalloproteinases (MMPs), elastases, and kallikrein-related peptidases. For conjugates that are intended to be internalized by cells, linker L may include an amino acid sequence recognized and cleaved by endosomal or lysosomal proteases. Examples of such proteases include, for example, cathepsins B, C, D, H, L, and S, and legumin.

[0290] The cleavage recognition sequence can be, for example, a dipeptide, a tripeptide or a tetrapeptide. Non-limiting examples of dipeptide recognition sequences that can be included in the 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, phenyl Gly-(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.

[0291] Additional examples of cleavable linkers include disulfide-containing linkers, such as N-succinimidyl-4-(2-pyridyldithio)butyrate (SPDB) and N-succinimidyl-4-(2-pyridyldithio)-2-sulfobutyrate (sulfo-SPDB). Linkers containing disulfide bonds may optionally include additional groups to provide steric hindrance near the disulfide bond to improve the extracellular stability of the linker, for example, including a geminal dimethyl group. Other cleavable linkers include linkers that are hydrolyzable at a specific pH or pH range, such as hydrazone linkers. Linkers containing combinations of these functionalities may also be useful, for example, linkers containing both hydrazone and disulfide bonds are known in the art.

[0292] Another example of a cleavable linker is a linker comprising β-glucuronide, which can be cleaved 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). β-glucuronide can also be used to improve the hydrophilicity of the linker L.

[0293] Another example of a linker that is cleaved internally within the cell and improves hydrophilicity is a linker comprising a pyrophosphate diester moiety (see, e.g., Kern et al., 2016, J Am Chem Soc., 138:2430-1445).

[0294] 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.

[0295] The cleavable linker may optionally further comprise one or more additional functional groups, such as self-immolative and self-degradable groups, extenders, or hydrophilic moieties.

[0296] Self-decomposition and self-eliminating groups that can be used for joints include, for example, p-aminobenzyl (PAB) and p-aminobenzyloxycarbonyl (PABC) groups, and methylated ethylenediamine (MED). Other examples of self-decomposition groups include, but are not limited to, aromatic compounds similar to PAB or PABC group electronics, such as heterocyclic derivatives, such as 2-aminoimidazole-5-methanol derivatives described in U.S. Patent No. 7,375,078. Other examples include groups that are cyclized when amide bonds are hydrolyzed, 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-decomposition / self-eliminating groups are generally connected to amino or hydroxyl groups on payload drugs. Self-decomposition / self-eliminating groups alone or in combination are generally included in peptide-based joints, but may also be included in other types of joints.

[0297] Extenders that can be used in the linker of the drug conjugate include, for example, alkylene groups and extenders based on aliphatic acids, diacids, amines or diamines, such as diglycolates, malonates, caproates and caproamides. Other extenders include, for example, glycine-based extenders and polyethylene glycol (PEG) or monomethoxy polyethylene glycol (mPEG) extenders.

[0298] PEG and mPEG extenders can also be used as hydrophilic moieties in joints. For example, PEG or mPEG can be "directly inserted" or included in the joint as a side group to increase the hydrophilicity of the joint (see, for example, U.S. Patent Application Publication No. US2016 / 0310612). Various PEG-containing joints can also be commercially available from companies such as Quanta BioDesign, Ltd (Plain City, OH). Other hydrophilic groups that can be optionally incorporated into joint L include, for example, β-glucuronide, sulfonic acid group, carboxylic acid group, and pyrophosphate diester.

[0299] A skilled artisan with knowledge in the art can readily select an appropriate linker for a given ADC by considering relevant factors such as the site of attachment to the antibody construct, any structural constraints of the payload drug, and the hydrophobicity of the payload drug (see, e.g., review in Nolting, Chapter 5, Antibody-Drug Conjugates: Methods in Molecular Biology, 2013, Ducry (ed.), Springer).

[0300] 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.

[0301] In certain embodiments, in the drug-linker of Formula II, n is 1, and the drug-linker has Formula III:

[0302]

[0303] in:

[0304] Z is a functional group capable of reacting with a target group on the anti-cMet antibody construct A;

[0305] Str is an extender;

[0306] AA1 and AA2 are each independently an amino acid, wherein AA1-[AA2] m Formation of protease cleavage sites;

[0307] X is a self-decomposing group;

[0308] s is 0 or 1;

[0309] m is 1, 2, or 3;

[0310] o is 0, 1, or 2; and

[0311] D has the structure shown in Formula II.

[0312] When incorporated into the ADCs of the present disclosure, the drug-linker of Formula III has Formula IV:

[0313]

[0314] in:

[0315] Z' is a linker group that connects the linker to the target group on the anti-cMet antibody construct A;

[0316] Str, AA1, AA2, X, s, m, o and D are as defined for Formula III, and

[0317] # is the connection point with the anti-cMet antibody construct A.

[0318] In some embodiments, in Formulas III and IV, s is 1.

[0319] In some embodiments, in Formulas III and IV, o is 0 (ie, X is absent).

[0320] 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.

[0321] In some embodiments, in Formula III:

[0322] Z is Where * is the point of connection to the rest of the joint.

[0323] In some embodiments, in Formula IV:

[0324] Z' is a carbonyl group (-C(O)-) or Where # is the point of attachment to anti-cMet antibody construct A and * is the point of attachment to the rest of the linker.

[0325] In some embodiments, in Formula III and IV, Str is selected from:

[0326]

[0327] in:

[0328] Each R is independently H or C1-C6 alkyl;

[0329] Each p is independently an integer between 2 and 10;

[0330] Each q is independently an integer between 1 and 10;

[0331] $ is a point of connection with Z or Z', and

[0332] * is the connection point to the rest of the connector.

[0333] In some embodiments, in Formulas III and IV, Str is:

[0334]

[0335] Among them, p, q, $ and * As defined above.

[0336] In some embodiments, in Formulas III and IV, Str is:

[0337]

[0338] in $ and * As defined above, p is an integer between 2 and 6, and q is an integer between 2 and 8.

[0339] In some embodiments, in Formula III and IV, AA1-[AA2] m Selected from Val-Lys, Ala-Lys, Phe-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Arg, Ala-Phe, Val-Ala, M et-Lys, Asn-Lys, Ile-Pro, Ile-Val, Asp-Val, His-Val, Met-(D)Lys, Asn-(D)Lys, Val-(D)Asp, NorVal-(D)As p, Ala-(D)Asp, Me3Lys-Pro, PhenylGly-(D)Lys, Met-(D)Lys, Asn-(D)Lys, Pro-(D)Lys, Met-(D)Lys, Met-Cit-Va l, Gly-Cit-Val, (D)Phe-Phe-Lys, (D)Ala-Phe-Lys, Gly-Phe-Leu-Gly, Ala-Leu-Ala-Leu and Gly-Gly-Phe-Gly.

[0340] In some embodiments, in Formula III and IV, m is 1 (i.e., AA1-[AA2] m is a dipeptide).

[0341] In some embodiments, in Formula III and IV, AA1-[AA2] m It is a dipeptide selected from Val-Lys, Ala-Lys, Phe-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit and Trp-Cit.

[0342] In some embodiments, in Formula III:

[0343] Z is Where * is the point of connection to the rest of the joint;

[0344] Str is in $ is the connection point with Z, * is the point of attachment to the rest of the linker, p is an integer between 2 and 6, and q is an integer between 2 and 8;

[0345] m is 1 and AA1-[AA2] m is a dipeptide selected from the group consisting of Val-Lys, Ala-Lys, Phe-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit and Trp-Cit;

[0346] s is 1, and

[0347] o is 0.

[0348] In some embodiments, in Formula IV:

[0349] Z' is a carbonyl group (-C(O)-) or Wherein # is the point of attachment to anti-cMet antibody construct A, and * is the point of attachment to the rest of the linker;

[0350] Str is in $ is the connection point with Z', * is the point of attachment to the rest of the linker, p is an integer between 2 and 6, and q is an integer between 2 and 8;

[0351] m is 1 and AA1-[AA2] m is a dipeptide selected from the group consisting of Val-Lys, Ala-Lys, Phe-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit and Trp-Cit;

[0352] s is 1, and

[0353] o is 0.

[0354] In certain embodiments, in the drug-linker of Formula II, n is 1, and the drug-linker has Formula V:

[0355]

[0356] in:

[0357] Z is a functional group capable of reacting with a target group on the anti-cMet antibody construct A;

[0358] Str is an extender;

[0359] AA1 and AA2 are each independently an amino acid, wherein AA1-[AA2] m Formation of protease cleavage sites;

[0360] m is 1, 2, or 3, and

[0361] D has the structure shown in Formula II.

[0362] When incorporated into an ADC of the present disclosure, the drug-linker of Formula V has Formula VI:

[0363]

[0364] in:

[0365] Z' is a linker group that connects the linker to the target group on the anti-cMet antibody construct A;

[0366] Str, AA1, AA2, X, m and D are as defined for Formula V, and

[0367] # is the connection point with the anti-cMet antibody construct A.

[0368] 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.

[0369] In some embodiments, in Formula V:

[0370] Z is Where * is the point of connection to the rest of the joint.

[0371] In some embodiments, in Formula VI:

[0372] Z' is a carbonyl group (-C(O)-) or wherein # is the point of attachment to the anti-cMet antibody construct A, and * is the point of attachment to the rest of the linker.

[0373] In some embodiments, in Formulas V and VI, Str is selected from:

[0374]

[0375] in:

[0376] Each R is independently H or C1-C6 alkyl;

[0377] Each p is independently an integer between 2 and 10;

[0378] Each q is independently an integer between 1 and 10;

[0379] $ is a point of connection with Z or Z', and

[0380] * is the connection point to the rest of the connector.

[0381] In some embodiments, in Formulas V and VI, Str is:

[0382]

[0383] Among them, p, q, $ and * As defined above.

[0384] In some embodiments, in Formulas V and VI, Str is:

[0385]

[0386] in $ and * As defined above, p is an integer between 2 and 6, and q is an integer between 2 and 8.

[0387] In some embodiments, in Formulas V and VI, AA1-[AA2] m Selected from Val-Lys, Ala-Lys, Phe-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Arg, Ala-Phe, Val-Ala, M et-Lys, Asn-Lys, Ile-Pro, Ile-Val, Asp-Val, His-Val, Met-(D)Lys, Asn-(D)Lys, Val-(D)Asp, NorVal-(D)As p, Ala-(D)Asp, Me3Lys-Pro, PhenylGly-(D)Lys, Met-(D)Lys, Asn-(D)Lys, Pro-(D)Lys, Met-(D)Lys, Met-Cit-Va l, Gly-Cit-Val, (D)Phe-Phe-Lys, (D)Ala-Phe-Lys, Gly-Phe-Leu-Gly, Ala-Leu-Ala-Leu and Gly-Gly-Phe-Gly.

[0388] In some embodiments, in Formulas V and VI, m is 1 (i.e., AA1-[AA2] m is a dipeptide).

[0389] In some embodiments, in Formulas V and VI, AA1-[AA2] m It is a dipeptide selected from Val-Lys, Ala-Lys, Phe-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit and Trp-Cit.

[0390] In some embodiments, in Formula V:

[0391] Z is Where * is the point of connection to the rest of the joint;

[0392] Str is in $ is the connection point with Z, * is the point of attachment to the rest of the linker, p is an integer between 2 and 6, and q is an integer between 2 and 8, and

[0393] m is 1 and AA1-[AA2] m It is a dipeptide selected from Val-Lys, Ala-Lys, Phe-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit and Trp-Cit.

[0394] In some embodiments, in Formula VI:

[0395] Z' is a carbonyl group (-C(O)-) or Where # is the point of attachment to the anti-cMet antibody construct and * is the point of attachment to the rest of the linker.

[0396] Str is in $ is the connection point with Z', * is the point of attachment to the rest of the linker, p is an integer between 2 and 6, and q is an integer between 2 and 8, and

[0397] m is 1 and AA1-[AA2] m It is a dipeptide selected from Val-Lys, Ala-Lys, Phe-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit and Trp-Cit.

[0398] In certain embodiments, in the drug-linker of Formula II, n is 1, and the drug-linker has Formula VII or Formula VIII:

[0399]

[0400]

[0401] in:

[0402] Z is a functional group capable of reacting with a target group on the anti-cMet antibody construct A, and

[0403] D has the structure shown in Formula II.

[0404] When incorporated into the ADCs of the present disclosure, the drug-linkers of Formula VII and Formula VIII have Formula IX and Formula X, respectively:

[0405]

[0406] in:

[0407] Z' is a linker group that connects the linker to the target group on the anti-cMet antibody construct A;

[0408] # is the connection point with the anti-cMet antibody construct A, and

[0409] D has the structure shown in Formula II.

[0410] In certain embodiments, in the drug-linker of Formula II, n>1, and the drug-linker has Formula XI:

[0411]

[0412] in:

[0413] Z is a functional group capable of reacting with a target group on the anti-cMet antibody construct A;

[0414] Str 1 and Str 2 each independently an extender;

[0415] BU is a branch unit;

[0416] AA1 and AA2 are each independently an amino acid, wherein AA1-[AA2] m Formation of protease cleavage sites;

[0417] X is a self-decomposing group;

[0418] s and s' are each independently 0 or 1;

[0419] m is 1, 2, or 3;

[0420] o is 0, 1, or 2;

[0421] t is 2 or 3, and

[0422] D has the structure shown in Formula II.

[0423] When incorporated into an ADC of the present disclosure, the drug-linker of Formula XI has Formula XII:

[0424]

[0425] in:

[0426] Z' is a linker group that connects the linker to the target group on the anti-cMet antibody construct A;

[0427] Str 1 , Str 2 , BU, AA1, AA2, X, s, s', m, o, t and D are as defined for Formula XI, and

[0428] # is the connection point with the anti-cMet antibody construct A.

[0429] In formula 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 functional side groups (such as glutamic acid, aspartic acid, tyrosine, lysine, cysteine, serine or threonine), trisubstituted aromatic compounds (such as 5-aminoisophthalic acid), Behera amine (di-tert-butyl 4-amino-4-(3-(tert-butoxy)-3-oxopropyl) pimelate) and various dendritic cores (see, e.g., Newkome and Shreiner, 2010, Chem. Reviews, 110(10): 6338-6442).

[0430] In some embodiments, in Formula XI and XII, BU is an amino acid or Behera amine. In some embodiments, in Formula XI and XII, BU is glutamic acid or Behera amine.

[0431] In some embodiments, in Formula XI and XII, s is 1. In some embodiments, in Formula XI and XII, s′ is 1. In some embodiments, in Formula XI and XII, s and s′ are each 1.

[0432] In some embodiments, in Formulae XI and XII, o is 0 (ie, X is absent).

[0433] 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.

[0434] In some embodiments, in Formula XI:

[0435] Z is Where * is the point of connection to the rest of the joint.

[0436] In some embodiments, in Formula XI:

[0437] Z is Where * is the point of connection to the rest of the joint.

[0438] In some embodiments, in Formula XII:

[0439] Z' is a carbonyl group (-C(O)-) or Where # is the point of attachment to anti-cMet antibody construct A and * is the point of attachment to the rest of the linker.

[0440] In some embodiments, in Formula XII:

[0441] Z is Where # is the point of attachment to anti-cMet antibody construct A and * is the point of attachment to the rest of the linker.

[0442] In some embodiments, in Formulas XI and XII, Str 1 Selected from:

[0443]

[0444]

[0445] in:

[0446] Each R is independently H or C1-C6 alkyl;

[0447] Each p is independently an integer between 2 and 10;

[0448] Each q is independently an integer between 1 and 10;

[0449] $ is a point of connection with Z or Z', and

[0450] * is the connection point to the rest of the connector.

[0451] In some embodiments, in Formulas XI and XII, Str 2 Selected from:

[0452]

[0453] in:

[0454] Each R is independently H or C1-C6 alkyl;

[0455] Each p is independently an integer between 2 and 10;

[0456] Each q is independently an integer between 1 and 10;

[0457] $ is the connection point to the BU, and

[0458] * is the connection point to the rest of the connector.

[0459] In some embodiments, in Formulas XI and XII, Str 1 yes:

[0460]

[0461] Among them, p, q, $ and * As above for Str 1 defined.

[0462] In some embodiments, in Formulas XI and XII, Str 2 yes:

[0463]

[0464] Among them, p, q, $ and * As above for Str 2 defined.

[0465] In some embodiments, in Formulas XI and XII, Str 1 yes:

[0466]

[0467] in $ and * As above for Str 1 As defined herein, p is an integer between 2 and 6, and q is an integer between 2 and 8.

[0468] In some embodiments, in Formulas XI and XII, Str 2 yes:

[0469]

[0470]

[0471] in $ and * As above for Str 2 As defined herein, p is an integer between 2 and 6, and q is an integer between 2 and 8.

[0472] In some embodiments, in Formulas XI and XII, AA1-[AA2] mSelected from Val-Lys, Ala-Lys, Phe-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Arg, Ala-Phe, Val-Ala, M et-Lys, Asn-Lys, Ile-Pro, Ile-Val, Asp-Val, His-Val, Met-(D)Lys, Asn-(D)Lys, Val-(D)Asp, NorVal-(D)As p, Ala-(D)Asp, Me3Lys-Pro, PhenylGly-(D)Lys, Met-(D)Lys, Asn-(D)Lys, Pro-(D)Lys, Met-(D)Lys, Met-Cit-Va l, Gly-Cit-Val, (D)Phe-Phe-Lys, (D)Ala-Phe-Lys, Gly-Phe-Leu-Gly, Ala-Leu-Ala-Leu and Gly-Gly-Phe-Gly.

[0473] In some embodiments, in Formulas XI and XII, m is 1 (i.e., AA1-[AA2] m is a dipeptide).

[0474] In some embodiments, in Formulas XI and XII, AA1-[AA2] m It is a dipeptide selected from Val-Lys, Ala-Lys, Phe-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit and Trp-Cit.

[0475] In some embodiments, in Formula XI:

[0476] Z is Where * is the point of connection to the rest of the joint;

[0477] BU is an amino acid (e.g. glutamic acid) or Behera amine,

[0478] Str 1 yes in $ is the connection point with Z, * is the point of attachment to the rest of the linker, p is an integer between 2 and 6, and q is an integer between 2 and 8;

[0479] Str 2 yes

[0480] in $It is the connection point with BU. * is the point of attachment to the rest of the linker, p is an integer between 2 and 6, and q is an integer between 2 and 8;

[0481] m is 1 and AA1-[AA2] m is a dipeptide selected from the group consisting of Val-Lys, Ala-Lys, Phe-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit and Trp-Cit;

[0482] s is 1;

[0483] s' is 1, and

[0484] o is 0.

[0485] In some embodiments, in Formula XII:

[0486] Z' is a carbonyl group (-C(O)-) or Wherein # is the point of attachment to anti-cMet antibody construct A, and * is the point of attachment to the rest of the linker;

[0487] BU is an amino acid (e.g. glutamic acid) or Behera amine,

[0488] Str 1 yes in $ is the connection point with Z', * is the point of attachment to the rest of the linker, p is an integer between 2 and 6, and q is an integer between 2 and 8;

[0489] Str 2 yes

[0490] in $ It is the connection point with BU. * is the point of attachment to the rest of the linker, p is an integer between 2 and 6, and q is an integer between 2 and 8;

[0491] m is 1 and AA1-[AA2] m is a dipeptide selected from the group consisting of Val-Lys, Ala-Lys, Phe-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit and Trp-Cit;

[0492] s is 1;

[0493] s' is 1, and

[0494] o is 0.

[0495] In certain embodiments, the drug-linker of Formula II comprises a drug-linker of Formula XIII:

[0496]

[0497] in:

[0498] Z is a functional group capable of reacting with a target group on the anti-cMet antibody construct A;

[0499] Str 1 and Str 2 each independently an extender;

[0500] BU is a branch unit;

[0501] AA1 and AA2 are each independently an amino acid, wherein AA1-[AA2] m Formation of protease cleavage sites;

[0502] s and s' are each independently 0 or 1;

[0503] m is 1, 2, or 3;

[0504] t is 1, 2, or 3;

[0505] x is 0 or 1, wherein when x is 0, t is 1, and when x is 1, t is 2 or 3, and

[0506] D has the structure shown in Formula II.

[0507] When incorporated into the ADCs of the present disclosure, the drug-linker of Formula XIII has Formula XIV:

[0508]

[0509] in:

[0510] Z' is a linker group that connects the linker to the target group on the anti-cMet antibody construct A;

[0511] Str 1 , Str 2 , BU, AA1, AA2, s, s', m, t, x and D are as defined for Formula XIII, and

[0512] # is the connection point with the anti-cMet antibody construct A.

[0513] In some embodiments, in Formulas XIII and XIV, x is 1 and BU is an amino acid or Behera amine. In some embodiments, in Formulas XIII and XIV, x is 1 and BU is glutamic acid or Behera amine.

[0514] 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.

[0515] 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.

[0516] In some embodiments, in Formula XIII:

[0517] Z is Where * is the point of connection to the rest of the joint.

[0518] In some embodiments, in Formula XIV:

[0519] Z' is a carbonyl group (-C(O)-) or Where # is the point of attachment to anti-cMet antibody construct A and * is the point of attachment to the rest of the linker.

[0520] In some embodiments, in Formulas XIII and XIV, Str 1 Selected from:

[0521]

[0522] in:

[0523] Each R is independently H or C1-C6 alkyl;

[0524] Each p is independently an integer between 2 and 10;

[0525] Each q is independently an integer between 1 and 10;

[0526] $ is a point of connection with Z or Z', and

[0527] * is the connection point to the rest of the connector.

[0528] In some embodiments, in Formulas XIII and XIV, Str 2 Selected from:

[0529]

[0530] in:

[0531] Each R is independently H or C1-C6 alkyl;

[0532] Each p is independently an integer between 2 and 10;

[0533] Each q is independently an integer between 1 and 10;

[0534] $ is the connection point to the BU, and

[0535] * is the connection point to the rest of the connector.

[0536] In some embodiments, in Formulas XIII and XIV, Str 1 yes:

[0537]

[0538] Among them, p, q, $ and * As above for Str 1 defined.

[0539] In some embodiments, in Formulas XIII and XIV, Str 2 yes:

[0540]

[0541]

[0542] Among them, p, q, $ and * As above for Str 2 defined.

[0543] In some embodiments, in Formulas XIII and XIV, Str 1 yes:

[0544]

[0545] in $ and * As above for Str 1 As defined herein, p is an integer between 2 and 6, and q is an integer between 2 and 8.

[0546] In some embodiments, in Formulas XIII and XIV, Str 2 yes:

[0547]

[0548] in $ and* As above for Str 2 As defined herein, p is an integer between 2 and 6, and q is an integer between 2 and 8.

[0549] In some embodiments, in Formulas XIII and XIV, AA1-[AA2] m Selected from Val-Lys, Ala-Lys, Phe-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Arg, Ala-Phe, Val-Ala, M et-Lys, Asn-Lys, Ile-Pro, Ile-Val, Asp-Val, His-Val, Met-(D)Lys, Asn-(D)Lys, Val-(D)Asp, NorVal-(D)As p, Ala-(D)Asp, Me3Lys-Pro, PhenylGly-(D)Lys, Met-(D)Lys, Asn-(D)Lys, Pro-(D)Lys, Met-(D)Lys, Met-Cit-Va l, Gly-Cit-Val, (D)Phe-Phe-Lys, (D)Ala-Phe-Lys, Gly-Phe-Leu-Gly, Ala-Leu-Ala-Leu and Gly-Gly-Phe-Gly.

[0550] In some embodiments, in Formulas XIII and XIV, m is 1 (i.e., AA1-[AA2] m is a dipeptide).

[0551] In some embodiments, in Formulas XIII and XIV, AA1-[AA2] m It is a dipeptide selected from Val-Lys, Ala-Lys, Phe-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit and Trp-Cit.

[0552] 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, the ADC of Formula I comprises a drug-linker selected from the drug-linkers shown in Table 5. In certain embodiments, the drug-linker of Formula II is selected from the drug-linkers shown in Table 5. In certain embodiments, the 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 between 1 and 8. In some embodiments, the 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 between 2 and 6. In some embodiments, the ADC of Formula I is ADC 001 or ADC 002 shown in Table 6, wherein A is an anti-cMet antibody construct and p is an integer between 2 and 6. In some embodiments, the ADC of Formula I is selected from ADC 003, ADC 004, ADC 005, and ADC 006 shown in Table 6, wherein A is an anti-cMet antibody construct and p is an integer between 2 and 4. In some embodiments, the ADC of Formula I is ADC 003 or ADC 005 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 ADC 004 or ADC 006 shown in Table 6, wherein A is an anti-cMet antibody construct and p is 2.

[0553]

[0554]

[0555]

[0556]

[0557]

[0558]

[0559]

[0560] preparation

[0561] Anti-cMet antibody constructs

[0562] The anti-cMet antibody constructs described herein can 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, Greenfield, ed., Cold Spring Harbor Laboratory Press, New York, 2014).

[0563] Typically, to recombinantly produce an antibody construct, a polynucleotide or set of polynucleotides encoding an anti-cMet antibody construct is generated and inserted into one or more vectors for further cloning and / or expression in a host cell. Polynucleotides encoding anti-cMet antibody constructs can 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 and updates, and "Antibodies: A Laboratory Manual," 2nd edition, Greenfield ed., Cold Spring Harbor Laboratory Press, New York, 2014). As will be appreciated by those skilled in the art, the number of polynucleotides required to express an anti-cMet antibody construct will depend on the format of the construct, including whether the antibody construct contains a scaffold. For example, when the anti-cMet antibody construct is in the form of a full-size antibody with a homodimeric Fc, three polynucleotides will be required, each encoding one polypeptide chain. When multiple polynucleotides are required, they can be incorporated into one vector or more than one vector.

[0564] Typically, for expression, a polynucleotide or a group of polynucleotides is incorporated into one or more expression vectors together with one or more regulatory elements, such as transcription elements, which are required for the effective transcription of the polynucleotides. Examples of such regulatory elements include, but are not limited to, promoters, enhancers, terminators, and polyadenylation signals. It will be appreciated by those skilled in the art that the selection of regulatory elements depends on the host cell selected for expressing the antibody construct, and such regulatory elements may be derived from a variety of sources, including bacteria, fungi, viruses, mammals, or insect genes. The expression vector may optionally further contain a heterologous nucleic acid sequence, which 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 vector or an integration vector.

[0565] Suitable host cells for cloning or expressing anti-cMet antibody constructs include various prokaryotic or eukaryotic cells known in the art. Prokaryotic host cells include, for example, Escherichia coli (E. coli), Aeromonas salmonicida (A. salmonicida) and Bacillus subtilis (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 cell containing the expression vector encoding the anti-cMet antibody construct can be cultured using conventional methods.

[0566] In certain embodiments, the anti-cMet antibody constructs can be produced in eukaryotic cells. In some embodiments, the anti-cMet antibody constructs can be produced in mammalian cells. Mammalian cell lines adapted to grow in suspension are particularly useful for expressing antibody constructs. Examples include, but are not limited to, monkey kidney CV1 line transformed by SV40 (COS-7), human embryonic kidney (HEK) 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 (BRL 3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor cells (MMT 060562), TRI cells (see, e.g., Mather et al., 1982, Annals NY Acad Sci, 383:44-68), MRC 5 cells, FS4 cells, Chinese hamster ovary (CHO) cells (including DHFR - CHO cells, see 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 producing antibody constructs are reviewed in Yazaki and Wu, Methods in Molecular Biology, Vol. 248, pp. 255-268 (BKCLo ed., Humana Press, Totowa, NJ, 2003).

[0567] 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 of these cell lines. In some embodiments, the host cell can be a stable cell line that allows mature glycosylation of the antibody construct.

[0568] Certain embodiments of the present disclosure relate to an isolated polynucleotide or set of polynucleotides encoding an anti-cMet antibody construct described herein. A polynucleotide in this context may encode all or part of an anti-cMet antibody construct.

[0569] The terms "polynucleotide", "nucleic acid" and "nucleotide molecule" are used interchangeably herein and refer to a polymeric form of nucleotides (deoxyribonucleotides or ribonucleotides or their analogs) of any length. 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.

[0570] A polynucleotide that "encodes" a given polypeptide is one that is transcribed (in the case of DNA) or translated (in the case of mRNA) into the 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' (carboxyl) terminus. A transcription termination sequence may be located 3' to the coding sequence.

[0571] Certain embodiments of the present disclosure relate to vectors (such as expression vectors) comprising one or more polynucleotides encoding the anti-cMet antibody constructs described herein. The one or more polynucleotides may be contained in a single vector, or contained in more than one vector. In some embodiments, the polynucleotides are contained in a multicistronic vector.

[0572] Certain embodiments of the present disclosure relate to a host cell comprising a polynucleotide encoding an anti-cMet antibody construct described herein or one or more vectors comprising the polynucleotide. In some embodiments, the host cell is eukaryotic, 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).

[0573] Typically, the anti-cMet antibody construct is purified after expression. Proteins can 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 Edition, Scopes, Springer-Verlag, NY, 1994). Standard purification methods include one or more chromatographic techniques, including ion exchange, hydrophobic interaction, affinity, size exclusion or gel filtration, and reverse phase chromatography, performed at atmospheric pressure or under high pressure using systems such as FPLC and HPLC. Additional purification methods include electrophoresis, immunostaining, precipitation, dialysis, and chromatofocusing techniques. Ultrafiltration and diafiltration techniques combined with protein concentration may also be useful. As is well known in the art, a variety of natural proteins bind to antibodies, and these proteins can be used to purify certain antibody constructs. For example, bacterial proteins A and G bind to the Fc region. Similarly, the bacterial protein L binds to the Fab region of some antibodies. Purification can also be performed by specific fusion partners. For example, if a GST fusion is used, glutathione resin can be used to purify the antibody, and if a His tag is used, Ni can be used. +2 The antibody is purified by affinity chromatography, or if a Flag tag is used, an immobilized anti-flag antibody may be used to purify the antibody. The degree of purification necessary will vary depending on the intended use of the anti-cMet antibody construct. In some cases, no purification may be necessary.

[0574] In certain embodiments, the anti-cMet antibody construct is substantially pure. The term "substantially pure" (or "substantially purified"), when used in reference to the anti-cMet antibody constructs described herein, means that the antibody construct is substantially or essentially free of components that normally accompany or interact with the protein as found in its naturally occurring environment (such as a native cell, or in the case of a recombinantly produced construct, a host cell). 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.

[0575] Certain embodiments of the present disclosure relate to methods for preparing an anti-cMet antibody construct, the method comprising culturing a host cell 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 expressing the anti-cMet antibody construct, and optionally recovering the anti-cMet antibody construct from the host cell (or from the host cell culture medium). In some embodiments, the method further comprises subjecting the anti-cMet antibody construct to one or more purification steps.

[0576] Post-translational modification

[0577] 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 isolating the anti-cMet antibody construct from a host cell.

[0578] Post-translational modifications include various modifications known in the art, such as glycosylation, acetylation, phosphorylation, amidation, deamidation, derivatization by known protecting / blocking groups, formylation, oxidation, reduction, proteolytic cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4, etc., or specific chemical cleavage (see, e.g., Proteins-Structure and Molecular Properties, 2nd Edition, TECreighton, WH Freeman and Company, New York, 1993; Post-Translational Covalent Modification of Proteins, BC 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 those embodiments in which the anti-cMet antibody construct comprises one or more post-translational modifications, the construct may comprise the same type of modification at one or a few sites, or it may comprise different modifications at different sites.

[0579] Additional examples of post-translational modifications include, but are not limited to, addition or removal of N-linked or O-linked carbohydrate chains, chemical modification of N-linked or O-linked carbohydrate chains, treatment of the N-terminus or C-terminus, attachment of chemical moieties to the amino acid backbone, and addition or deletion of N-terminal methionine residues produced by prokaryotic host cell expression. Post-translational modifications may also include modification with a detectable label (such as an enzyme label, a fluorescent label, a luminescent label, an isotope label, or an affinity label) to allow detection and separation of proteins. Examples of suitable enzyme labels include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-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, dichlorotriazineamine 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.

[0580] Additional examples of post-translational modifications include acetylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or a nucleotide derivative, covalent attachment of a lipid or a 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.

[0581] ADC

[0582] ADCs of Formula I comprising anti-cMet antibody constructs can be prepared by standard methods known in the art (see, e.g., Bioconjugate Techniques (GT Hermanson, 2013, Academic Press)). Exemplary methods are provided herein. Various linkers and linker components are commercially available or can be prepared using standard synthetic organic chemistry techniques (see, e.g., March's Advanced Organic Chemistry (Smith and March, 2006, 6th edition, Wiley); Toki et al., (2002) J. Org. Chem. 67: 1866-1872; Frisch et al., (1997) Bioconj. Chem. 7: 180-186; Bioconjugate Techniques (GT Hermanson, 2013, Academic Press)). In addition, 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 can be used to prepare ADCs.

[0583] Typically, the preparation of the ADC includes 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 an appropriate group on the anti-cMet antibody construct A. However, the connection of the linker L to the anti-cMet antibody construct A and the subsequent connection of the anti-cMet antibody construct-linker AL to one or more auristatin analogs D remains an alternative method that can be used in some embodiments.

[0584] Suitable groups on the anti-cMet antibody construct T for attachment to 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 chains of aspartic acid or glutamic acid residues), and carbohydrate groups.

[0585] In certain embodiments, one or more naturally occurring cysteine ​​residues on the anti-cMet antibody construct A can be used to bond to the linker L via the thiol group of cysteine. In certain embodiments, one or more naturally occurring lysine residues on the anti-cMet antibody construct A can be used to bond to the linker L via the amino group of lysine.

[0586] 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 can 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.

[0587] Carbohydrate groups on anti-cMet antibody construct A can 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 react with linker L, e.g., via a hydrazine or hydroxylamine group on linker L.

[0588] 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, the anti-cMet antibody 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 (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), to allow site-specific conjugation. Alternatively, the anti-cMet antibody construct A can also be modified to include a non-native reactive group, such as an azide, which allows conjugation to the linker via a complementary reactive group on the linker, for example by click chemistry (see, e.g., Chio and Bane, 2020, Methods Mol. Biol., 2078: 83-97). Another option is to use GlycoConnect TMTechnology (Synaffix BV, Nijmegen, Netherlands) involves enzymatic remodeling of antibody glycans to allow attachment of linkers via metal-free click chemistry (see, e.g., European Patent No. EP 2 911 699).

[0589] Other protocols for modifying proteins to attach or associate a linker L are known in the art (see, e.g., Coligan et al., Current Protocols in Protein Science, Vol. 2, John Wiley & Sons (2002)).

[0590] Alternatively, ADCs can 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 (amine acceptor, usually on antibodies) and an alkylene amino group (amine donor, usually on drug-linker), which can be, for example, the ε-amino group of lysine or a 5-amino-n-pentyl group. Antibodies can also be modified to include a peptide or "tag" containing glutamine, which 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. WO 2016 / 144608).

[0591] A similar conjugation approach utilizes the enzyme transpeptidase A. In this approach, the antibody is typically modified to include a transpeptidase A recognition motif (LPXTG, where X is any natural amino acid), and the drug-linker is designed to include an oligoglycine motif (usually GGG) to allow transpeptidase A-mediated transpeptidation (see, e.g., Beerli et al., 2015, PLos One, 10:e0131177; Chen et al., 2016, Nature: Scientific Reports, 6:31899).

[0592] Once conjugation is complete, the average number of auristatin analog molecules conjugated to the anti-cMet antibody construct A (i.e., the "drug-antibody ratio" or DAR) can 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. In addition, the distribution of drug-linked forms (e.g., the fraction of anti-cMet antibody construct A containing zero, one, two, three, etc. conjugated auristatin analog molecules) can optionally 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).

[0593] Certain embodiments of the present disclosure relate to methods for preparing an ADC of formula I, 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.

[0594] Pharmaceutical composition

[0595] For therapeutic use, the ADC of the present disclosure is generally formulated as a pharmaceutical composition. Therefore, certain embodiments of the present disclosure relate to a pharmaceutical composition comprising an ADC as described herein and a pharmaceutically acceptable carrier, diluent or excipient. Such pharmaceutical compositions can be prepared by known procedures using well-known and readily available ingredients.

[0596] The pharmaceutical composition can be formulated for administration by, for example, oral (including, for example, buccal or sublingual), topical, parenteral, rectal or vaginal routes, or by inhalation or spraying to a subject. The term "parenteral" as used herein includes subcutaneous injection, as well as intradermal, intraarticular, intravenous, intramuscular, intravascular, intrasternal, intrathecal injection or infusion. The pharmaceutical composition will generally be formulated into the form of a selected route of administration suitable for use to a subject, such as a syrup, elixir, tablet, lozenge, pastille, hard or soft capsule, pill, suppository, oily or aqueous suspension, dispersible powder or granules, emulsion, injection or solution. The pharmaceutical composition can be provided as a unit dose formulation.

[0597] In certain embodiments, the pharmaceutical composition comprising the ADC is formulated for parenteral administration, for example, in the form of a lyophilized formulation or an aqueous solution. Such pharmaceutical compositions can be provided, for example, in a unit dose injectable form.

[0598] Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed. Examples of such carriers include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl alcohol, benzyl alcohol, alkyl parabens (such as methyl paraben or propyl paraben), catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol; low molecular weight (less than about 10 residues) polypeptides; Proteins such as serum albumin or 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 ions; metal complexes such as Zn-protein complexes; and non-ionic surfactants such as polyethylene glycol (PEG).

[0599] In certain embodiments, the composition comprising ADC can be in the form of a sterile injectable aqueous or oily solution or suspension. Such suspensions can be prepared using suitable dispersants or wetting agents and / or suspending agents known in the art. Sterile injectable solutions or suspensions can contain ADC in non-toxic parenteral acceptable diluents or carriers. Acceptable diluents and carriers that can be used include, for example, 1,3-butanediol, water, Ringer's solution, or isotonic sodium chloride solution. In addition, sterile fixed oils can be used as carriers. To this end, various mild fixed oils can be used, including synthetic monoglycerides or diglycerides. In addition, fatty acids such as oleic acid can be used to prepare injections. Adjuvants, such as local anesthetics, preservatives, and / or buffers can also be included in injectable solutions or suspensions.

[0600] In certain embodiments, the composition comprising ADC can be formulated for intravenous administration to humans. Typically, the composition for intravenous administration is a solution in a sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizing agent and / or a local anesthetic, such as lidocaine, to relieve pain at the injection site. Typically, the components are provided separately or mixed together in unit dosage form, for example, as a dried lyophilized powder or anhydrous concentrate in a sealed container (such as an ampoule or a sachet) indicating the amount of the active agent. When the composition needs to be administered by infusion, it can be distributed with an infusion bottle containing sterile pharmaceutical grade water or saline. When the composition is administered by injection, for example, an ampoule of sterile water for injection or saline can be provided so that the components can be mixed before administration.

[0601] Other pharmaceutical compositions and methods of preparing pharmaceutical compositions are known in the art and are described, for example, in "Remington: The Science and Practice of Pharmacy" (formerly "Remingtons Pharmaceutical Sciences"); Gennaro, A., Lippincott, Williams & Wilkins, Philadelphia, PA (2000).

[0602] How to use

[0603] Certain embodiments of the present disclosure relate to therapeutic uses of the ADCs described herein. Some embodiments relate to the use of ADCs as therapeutic agents, for example, as anticancer agents.

[0604] In certain embodiments, the ADC described herein can be used to treat cancer. Certain embodiments relate to methods of inhibiting cancer cell or tumor cell growth; inhibiting cancer cell or tumor cell proliferation in a subject, or treating cancer, comprising administering an ADC of Formula I.

[0605] Certain embodiments of the present disclosure relate to methods of inhibiting proliferation of cancer or tumor cells, 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, 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.

[0606] Some embodiments relate to methods of treating a subject with cancer by administering to the subject an ADC of Formula I. In this case, treating the subject may result in one or more of the following: a reduction in tumor size, a slowing or prevention of an increase in tumor size, a prolongation of the disease-free survival time between the disappearance or removal of a tumor and its reappearance, prevention of subsequent occurrences of a tumor (e.g., metastasis), a prolongation of the time to progression, a reduction in one or more adverse symptoms associated with a tumor, and / or a prolongation of the overall survival time of the subject with cancer.

[0607] Examples of cancers that can 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 common solid tumors that can 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 lymphomas can also lead to the formation of solid tumors and therefore can also be considered solid tumors in certain cases. Typically, the cancer to be treated is a cancer that expresses cMet.

[0608] Certain embodiments relate to methods of inhibiting the growth of cMet-positive tumor cells, comprising contacting the cells with an ADC of Formula I. The cells may be in vitro or in vivo. In certain embodiments, the ADC can be used in methods of treating a cMet-positive or cMet-overexpressing cancer or tumor in a subject.

[0609] Cancers that overexpress c-Met are typically solid tumors. Examples include, but are not limited to, ovarian cancer, lung cancer, breast cancer, gastric cancer, colorectal cancer, head and neck cancer, kidney cancer, and pancreatic cancer. In certain embodiments, the ADC of Formula I can be used in a method of treating a subject with cMet-positive or cMet-overexpressing ovarian cancer, lung cancer, breast cancer, gastric cancer, colorectal cancer, head and neck cancer, kidney cancer, or pancreatic cancer.

[0610] Drug kit

[0611] Certain embodiments relate to pharmaceutical kits comprising the ADC of Formula I.

[0612] The kit will typically include a container for containing the ADC and a label and / or package insert on or accompanying the container. The label or package insert contains instructions typically included in the commercial packaging of the therapeutic product, providing information about the indications, usage, dosage, administration, contraindications and / or warnings for using such therapeutic products. The label or package insert may also include a notice in the form of a governmental agency regulation for the manufacture, use or sale of regulated drugs or biological products, which reflects the approval of the use or sale of the manufacturing agency for human or animal administration. In some embodiments, the container may have a sterile inlet. For example, the container may be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic needle.

[0613] In addition to the container holding the ADC, the kit may optionally contain 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.

[0614] Suitable containers include, for example, bottles, vials, syringes, and intravenous solution bags, etc. The container may be made of various materials such as glass or plastic. Where appropriate, one or more components of the kit may be lyophilized or provided in a dry form (such as a powder or granules), and the kit may additionally contain a suitable solvent for reconstituting the lyophilized or dried component.

[0615] The kit may also include other materials desirable from a commercial or user standpoint, such as filters, needles, and syringes.

[0616] The following examples are offered for illustrative purposes and are not intended to limit the scope of the present invention in any way.

[0617] Example

[0618] Example 1: Preparation of anti-cMET antibodies

[0619] 1.1 Antibody Production

[0620] The antibody targeting cMet, variant v32634, was prepared as described below. The antibody is based on terituzumab (ABT-700) (see Wang et al., 2016, BMC Cancer, 16: 105), but lacks any modification to the hinge region. The full length, VH, VL and CDR sequences of v32634 are provided in the sequence table. In addition, the VH, VL and CDR sequences are provided in Table 2 above. In addition to variant v32634, antibody variants comprising various cysteine ​​insertion mutations as described below and control variant v17606 comprising hinge sequence modifications included in terituzumab were prepared. Another control antibody variant v17429 was also prepared, which is a bivalent form of the anti-cMet antibody MetMab (onartuzumab). The variants are summarized in Table 1.3.

[0621] The variants are produced in a full-size antibody (FSA) format containing two identical full-length heavy chains, generating a homodimeric Fc region (HomoFc); or a heterodimeric full-length heavy chain comprising complementary mutations in the CH3 region to drive heterodimeric heavy chain pairing, generating a heterodimeric Fc region (HetFc).

[0622] The two identical full-length heavy chains contained in the HomoFc region contain the human CH1-hinge-CH2-CH3 domain sequence of IGHG1*01 (SEQ ID NO: 31; see Table 1.1). The heterodimeric full-length heavy chains (HetFc-A and HetFc-B) contained in the HetFc region contain the human CH1-hinge-CH2-CH3 domain sequence of IGHG1*01, and have the following mutations in the Fc region:

[0623] HetFc-A:T350V_L351Y_F405A_Y407V

[0624] HetFc-B:T350V_T366L_K392L_T394W

[0625] The sequences of HetFc-A (SEQ ID NO: 33) and HetFc-B (SEQ ID NO: 34) are provided in Table 1.1. For all constructs, the human kappa CL sequence of IGKC*01 (SEQ ID NO: 32; see Table 1.1) was used.

[0626] Table 1.1: Antibody sequences

[0627]

[0628]

[0629] Table 1.2 provides the wild-type (WT) hinge sequences comprised by variant v32634 and the cysteine ​​insertion variants and the modified hinge sequences comprised by variant v17606 and terituzumab.

[0630] Table 1.2: Wild-type and modified hinge sequences

[0631] sequence SEQ ID NO WT Hinge EPKSCDKTHTCPPCP 35 Modified hinges EPKSCDCHCPPCP 36

[0632]

[0633]

[0634] 1.2 Antibody Production

[0635] The antibody variants listed in Table 1.3 were produced using a heavy chain expression vector and a light chain expression vector, wherein the heavy chain expression vector comprises a heavy chain vector insert, wherein the heavy chain vector insert comprises a signal peptide MRPTWAWWLFLVLLLALWAPARG (SEQ ID NO: 37) (Barash et al., 2002, Biochem and Biophys Res. Comm., 294: 835–842) and a heavy chain clone terminating at residue G446 (EU numbering) of the CH3 domain, and the heavy chain clone is ligated into a pTT5 vector; wherein the light chain expression vector comprises a light chain vector insert, wherein the light chain vector insert comprises the same signal peptide, and the light chain vector insert is 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:

[0636] The heavy and light chains were expressed in 200 ml CHO-3E7 cell cultures. Briefly, the cells were grown at 37°C in a 4% CO medium supplemented with 4 mM glutamine (GE Life Sciences, Marlborough, MA) and 0.1% FreeStyle for F-68 (Gibco / ThermoFisher Scientific, Waltham, MA) TM CHO-3E7 cells were cultured in F17 medium (Thermo Fisher Scientific, Waltham, MA) at a density of 1.7-2 x 10 6 cells / ml, viability>95%. Use (Polyscience, Inc., Philadelphia, PA), a total of 200ug DNA (100ug antibody DNA and 100ug GFP / AKT / filler DNA) was used to transfect CHO-3E7 cells in a total volume of 200ml at a DNA:PEI ratio of 1:4 (w / w) + 1x antibiotic / antimycotic (GE Life Sciences, Marlborough, MA). Twenty-four hours after adding the DNA-PEI mixture, 0.5mM 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 another 6 days before harvesting.

[0637] In batch mode or using 1mL HiTrap TM MabSelect TM SuRe TM Protein A purification was performed using a Cytiva 500 column (Cytiva, Marlborough, MA). In batch mode, the clarified supernatant samples were mixed with mAb Select SuRe TM The resin (GE Healthcare, Chicago, IL) was incubated in batches, cleaned in place (CIP'd) with NaOH and equilibrated in Dulbecco's PBS (DPBS). The resin was poured into a CIP'd column and the column was washed with DPBS. The protein was eluted with 100 mM sodium citrate buffer (pH 3.0) in both purification modes. The eluted fractions were pH adjusted by adding 10% (v / v) 1 M HEPES (pH approximately 10.6-10.7) to give a final pH of 6-7. The sample buffer was exchanged into DPBS. The protein was quantified based on the absorbance at 280 nm (A280 nm). After protein A purification, the variants were purified on a Superdex TM It was further purified by preparative SEC chromatography on a 200 μL Increase 10 / 30 column (GE Healthcare, Chicago, IL) in DPBS mobile phase.

[0638] After purification, high-throughput protein expression assays and Caliper The purity of the samples was assessed by electrophoresis under non-reducing and reducing conditions using a GXII or GXII Touch HT (Perkin Elmer, Waltham, MA). The procedure was followed according to the User Guide, Version 2, with the following modifications. 2 μl or 5 μl of antibody sample (concentration range 5-2000 ng / μl) was added to individual wells in a 96-well plate (BioRad, Hercules, CA) along with 7 μl of HT protein expression sample buffer (Perkin Elmer, catalog number 760328). The antibody samples were then denatured at 70°C for 15 min. The instrument was operated using the HT Protein Express Chip (Perkin Elmer, Waltham, MA) and the Ab-200 assay setup. In some cases, the purity of the samples was also monitored by SDS-PAGE under reducing and non-reducing conditions.

[0639] The yields of the variants (after Protein A purification) ranged from mg to grams and are summarized in Table 1.4.

[0640] 1.3 Purity assessment by analytical size exclusion chromatography (SEC)

[0641] The purity of the variants was determined by UPLC-SEC. For analytical SEC runs, 5 column volumes of buffer A (150 mM Na x PO4, pH 6.95) to balance the Agilent Advance Bio SEC column ( 2.7 μm, 7.8×150 mm) (Agilent Technologies, Inc., Santa Clara, CA; Serial No. 6377910-24). Typically, 20-30 ug of a sample with a concentration of 2-3 mg / mL is loaded onto the column, run at 1 mL / min for 7 min in a constant mode, and then report the absorbance at 280 nm. For each sample, the chromatogram is integrated to provide a complete baseline-baseline integration of each peak, and the separation position between the partially separated peaks is reasonable. Based on the SEC spectrum of the control IgG1 antibody trastuzumab, the peak corresponding to the main component of IgG (approximately retention time 3.3 min) is reported as a monomer. Any peak appearing before 3.3 min is designated as HMWS, and any peak appearing after 3.3 min is designated as LMWS, excluding solvent peaks (over 5.2 min).

[0642] The purity of each cysteine ​​insertion variant is summarized in Table 1.4.

[0643] Table 1.4: Summary of Generation of Representative Cysteine ​​Insertion Variants

[0644]

[0645] Figure 2A The SDS-PAGE results of the representative cysteine ​​insertion variant v29001 under non-reducing (NR) and reducing (R) conditions are shown, corresponding to the full-size antibody and intact heavy and light chains, Figure 2B A UPLC-SEC chromatogram of variant v29001 is shown. Based on the UPLC-SEC chromatogram, the sample purity of this variant was about 99%, reflecting high species homogeneity.

[0646] Example 2: Characterization of Cysteine ​​Insertion Variants

[0647] The molecular weight of the cysteine ​​insertion variants described in Example 1 was estimated by liquid chromatography-mass spectrometry (LC-MS). The thermal stability of both variants was evaluated using differential scanning calorimetry (DSC).

[0648] 2.1 Liquid chromatography-mass spectrometry (LC-MS)

[0649] The exact mass of the purified variants was measured by LC-MS. The variants were diluted to 1 mg / mL in PBS (pH 7.4) and then deglycosylated. For deglycosylation, 1 ug EndoS was usually used for every 10 ug of antibody, and the reaction mixture was incubated at room temperature for one hour. In some cases, the samples were also reduced by adding 1 uL of 500 mM tris(2-carboxyethyl)phosphine (TCEP) to each 10 uL sample, and then incubated at 70 ° C for one hour. Finally, the samples were run on an LC-MS quadrupole time-of-flight (QTOF) system (Agilent 1290HPLC coupled to Agilent 6545QTOF; Agilent Technologies, Inc., Santa Clara, CA), with 1 uL injected each time. The detailed procedure is described below.

[0650] ·column: 8uM, 50x2.1mm (Agilent Technologies, Inc., SantaClara, CA)

[0651] Mobile phase C: 0.1% formic acid, 0.025% trifluoroacetic acid and 10% isopropanol in H2O

[0652] Mobile phase D: 0.1% formic acid and 10% isopropanol in acetonitrile

[0653] Detection: Signal A (280nm, 4.0 bandwidth), Signal B (220nm, 4.0 bandwidth)

[0654] ·gradient:

[0655]

[0656] Post-run time: 2 minutes

[0657] The measured deglycosylated masses of the full-size variants were consistent with the theoretical masses (see Table 2.1).

[0658] Table 2.1: Mass verification of representative cysteine ​​insertion variants by LC-MS

[0659] Variant number Expected mass (Da) Observed mass (Da) Quality difference (Da) v28983 146269 146272 3 v29001 146269 146273 4 v35527 147019 147027 8 v33967 146166 146174 8 v33968 146166 146170 4 v33970 146371 146376 5 v33971 146371 146378 7 v33979 146371 146381 10

[0660] 2.2 Differential Scanning Calorimetry (DSC)

[0661] The difference in melting temperature (Tm) between the two cysteine ​​insertion variants v28983 and v29001 and the corresponding parent antibody v17427 was determined by DSC as follows: 400 μL of purified samples at a concentration of 0.2 mg / mL or 0.4 mg / mL in PBS were used for DSC analysis using MicroCal VP-Capillary DSC (GE Healthcare, Chicago, IL). At the beginning of each DSC run, 5 buffer blank injections were performed to stabilize the baseline, and buffer injections were arranged 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, 8 second filter, 5min preTstat and 70psi nitrogen pressure. The resulting thermogram was referenced and analyzed using Origin 7 software (OriginLab Corporation, Northampton, MA). The DSC measurement results are summarized in Table 2.2.

[0662] Table 2.2: Comparison of thermal stability of cysteine ​​insertion variants and parent antibody

[0663]

[0664] Example 3: Preparation of drug-linker

[0665] 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-performance liquid chromatography; HPLC = high performance liquid chromatography; MT = maleimidotriethylene glycol ester; TCEP = tris(2-carboxyethyl)phosphine; TFA = trifluoroacetic acid; VC = valine-citrulline; UHPLC = ultra-performance liquid chromatography.

[0666] The following general approach was adopted:

[0667] Flash chromatography : The crude reaction product was used Snap Ultra columns (10 g, 25 g, 50 g or 100 g) (Biotage, Charlotte, NC) were used for purification. Isolera TM The elution was performed on an automated rapid system (Biotage, Charlotte, NC) using a linear gradient of ethyl acetate / hexane or methanol / dichloromethane. Alternatively, the elution was performed using Reverse phase flash purification was performed on a SnapUltra C18 column (12 g, 30 g, 60 g or 120 g) using a linear gradient of CH3CN + 0.1% TFA / H2O + 0.1% TFA. The purified compound was isolated by removing the organic solvent by rotary evaporation or lyophilizing the acetonitrile / water mixture.

[0668] Preparative HPLC: Reverse phase HPLC of crude compound was performed using 5-μm EVO C18 The HPLC-MS / MS analysis was performed on an Agilent 1260 Infinity II preparative LC / MSD system (Agilent Technologies, Inc., Santa Clara, CA) using a linear gradient of CH3CN+0.1% TFA / H2O+0.1% TFA. The purified compound was isolated by lyophilization of acetonitrile / water mixtures.

[0669] NMR: 1 H NMR spectra were collected using a Bruker AVANCE III 300 spectrometer (300 MHz) or a Bruker AVANCE III 400 spectrometer (400 MHz) (Bruker Corporation, Billerica, MA). Chemical shifts are reported in parts per million (ppm).

[0670] 2,2-Dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azaheptadecan-17-oic acid:

[0671]

[0672] Obtained from ChemPep Inc. (Wellington, FL).

[0673] 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-oxoheptane-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamide (Compound 1)

[0674]

[0675] Prepared as described in International Publication No. WO 2016 / 041082.

[0676] 3.2(S)-2-((S)-2-amino-3-methylbutyramido)-N-(4-(N-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutyramido)-N,3-dimethylbutyramido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)sulfamoyl)phenyl)-5-ureidopentanamide (Compound 15)

[0677]

[0678] Prepared as described in International Publication No. WO 2019 / 173911.

[0679] 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)

[0680]

[0681] Flask 1: 3-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}propanoic acid (10.0 g, 45.2 mmol, 1 eq) and maleic anhydride (4.43 g, 45.2 mmol, 1 eq) were dissolved in anhydrous DMF (15 mL) in a dry 250 mL RB flask and stirred overnight at room temperature under nitrogen. The next morning, the reaction was determined to be complete by LC / MS. 2,3,5-Trimethylpyridine (21.9 g, 24.1 mL, 181 mmol, 4 eq) was added to the reaction 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, then trifluoroacetic anhydride (38.0 g, 25.4 mL, 1.49 g / mL, 181 mmol, 4 equiv) was added dropwise over 2 min. The resulting solution was stirred at 0 °C for 10 min, then 2,3,5-trimethylpyridine (21.9 g, 24.1 mL, 0.91 g / mL, 181 mmol, 4 equiv) was added over 3 min. The final mixture was stirred at 0 °C for 15 min, then added to the solution in flask 1 over 3 min, then the final mixture was warmed to room temperature and stirred for 48 h. A small amount of intermediate still remained as determined by LC / MS, and the reaction was stirred for an additional 72 h, then the conversion was determined to be complete by LC / MS. The reaction was acidified with 1M HCl (75 mL) and extracted with Et2O (3×100 mL). The combined organic layers were then washed with 5% LiCl (60 mL) and brine (30 mL), then dried over MgSO4 and concentrated in vacuo. The crude product was purified by reverse phase flash chromatography over a 10-50% ACN / H2O+0.1% TFA gradient. The fractions containing the product were pooled for lyophilization and the title compound (14.5 g, 32.4 mmol, 71.4%) was recovered as a light orange oil.

[0682] LC / MS:for C 19 H 19 The calculated value of F4NO7 is m / z = 449.11, and the detected [M+H] + =450.2m / z. 1 H NMR (300MHz, MeOD) δ7.42(tt,J=10.5,7.2Hz,1H),6.81(s,2H),3.87(t,J=6.0Hz,2H),3.71–3.55(m,12H),2.98(t,J=6.0Hz,2H).

[0683] 3.4 4-(3-(tert-butoxy)-3-oxopropyl)-4-(3-(2-(2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethoxy)ethoxy)propionamido)di-tert-butyl pimelate (Compound 3)

[0684]

[0685] 1,7-Di-tert-butyl 4-amino-4-[3-(tert-butoxy)-3-oxopropyl]heptanedioate (2.89 g, 6.95 mmol, 1.1 eq) and compound 2 (2.84 g, 6.32 mmol, 1 eq) were dissolved in 10 mL of anhydrous DMF along with 1-hydroxybenzotriazole monohydrate (0.968 g, 6.32 mmol, 1 eq). N-ethyldiisopropylamine (1.63 g, 2.21 mL, 0.74 g / mL, 12.6 mmol, 2 eq) was added over 1 min at room temperature with rapid stirring. After 1 h, the reaction was determined to be complete by LC / MS, diluted with 1 M HCl (5 mL) and ACN (3 mL), and then purified by reverse phase chromatography on a 10-100% ACN / H2O + 0.1% TFA gradient. The products were pooled and concentrated in vacuo, then brine (5 mL) was added and the aqueous layer was extracted with Et2O (3 x 50 mL). The organics were pooled and dried over brine (5 mL) and MgSO4, then filtered and evaporated in vacuo to recover the title compound (4.18 g, 6.00 mmol, 94.9%) as an oily off-white solid.

[0686] LC / MS:C 35 H 58 N2O 12 Calculated value of m / z = 698.40, detected [M+H] + =699.6m / z. 1 H NMR (400MHz, CDCl3) δ6.72(s,2H),6.13(s,1H),3.78–3.69(m,4H),3.67–3.63(m,2H),3.6 3–3.61(m,8H),2.40(t,J=5.8Hz,2H),2.27–2.15(m,6H),2.03–1.92(m,6H),1.44(s,27H).

[0687] 3.5 4-(2-carboxyethyl)-4-(3-(2-(2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethoxy)ethoxy)propionamido)pimelic acid (Compound 4)

[0688]

[0689] Compound 3 (4.18 g, 6.00 mmol, 1 equivalent) was dissolved in 33% TFA / DCM (15 mL) in a 500 mL round-bottom flask. Stirred at room temperature for 18 h, when the reaction was determined to be complete by LC / MS. The reactant was concentrated to dryness in vacuo and the residue was co-evaporated with ACN (3 x 10 mL). The residue was dissolved in 10 mL 2:1 H2O / ACN and lyophilized. The title compound was recovered as an oil, which was about 25% overweight, assuming that it contained residual TFA and H2O. (4.0 g, assuming quantitative yield 6.00 mmol, 100%).

[0690] LC / MS:C 23 H 34 N2O 12 Calculated value of m / z = 530.21, detected [M+H] + =531.4m / z. 1 H NMR(300MHz,MeOD)δ6.84(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).

[0691] 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)propionamido)-4-(3-oxo-3-(2,3,5,6-tetrafluorophenoxy)propyl)pimelate (Compound 5)

[0692]

[0693] Compound 4 (3.34 g, 6.30 mmol, 1 eq) was dissolved in ACN (50 mL) along with 2,3,5,6-tetrafluorophenol (4.18 g, 25.2 mmol, 4 eq) and stirred at 0 °C in a 250 mL round bottom flask. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (4.83 g, 25.2 mmol, 4 eq) was added. After 30 min, LC / MS indicated a conversion of about 60%, 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 hour, at which point the conversion was almost complete. The reaction was concentrated in vacuo to a volume of about 8 mL, then diluted with 1 M HCl (5 mL) and H2O (5 mL), and purified via reverse phase chromatography over a 10-75% ACN / H2O+0.1% TFA gradient. Product fractions were pooled and concentrated in vacuo, then extracted with Et2O (2 x 40 mL). The organics were pooled and washed with brine (30 mL), then dried over MgSO4 and filtered to recover the title compound (2.60 g, 2.67 mmol, 42.3% yield) as a clear colorless oil.

[0694] LC / MS:C 41 H 34 F 12 N2O 12 Calculated value of m / z = 974.2, detected [M+H] + =975.4m / z. 1 H NMR(300MHz, CDCl3)δ7.03(tt,J=9.8,7.0Hz,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=9.0,6.8Hz,6H),2.54(t,J=5.5Hz,2H),2.37–2.26(m,6H).

[0695] 3.7 Bis(2,3,5,6-tetrafluorophenyl)(tert-butoxycarbonyl)-L-glutamic acid (Compound 6)

[0696]

[0697] To a 250 mL round bottom flask containing glutamic acid (1.80 g, 7.28 mmol, 1 eq), ACN (30 mL), 2,3,5,6-tetrafluorophenol (2.54 g, 15.3 mmol, 2.1 eq) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (2.93 g, 15.3 mmol, 2.1 eq) was added. The reaction was stirred at room temperature for 18 h, at which time the reaction was determined to be complete by LC / MS. The reaction mixture was concentrated in vacuo to a crude oil, which was then redissolved in EtOAc (50 mL) and 1 M HCl (20 mL). The organic layer was washed with 1 M HCl (2 x 20 mL), saturated NaHCO3 (20 mL), and 1 x brine (20 mL), then dried over MgSO4, filtered, and evaporated in vacuo to afford the title compound as a white solid (3.27 g, 6.02 mmol, 82.7% yield).

[0698] LC / MS:C 22 H 17 The calculated value of F8NO6 is m / z = 543.09, and the detected [M+Na] + =556.4m / z. 1 H NMR (400MHz, CDCl3) δ7.13–6.97(m,2H),5.17(s,1H),4.83(s,1H),2.94(q,J=7.2Hz,2H),2.63–2.50(m,1H),2.39–2.24(m,1H),1.50(s,9H).

[0699] 3.8(S)-15-((tert-Butoxyhydroxy)amino)-14,18-dioxo-4,7,10,22,25,28-hexaoxa-13,19-diazaheteronecanedioic acid (Compound 7)

[0700]

[0701] To a flask containing compound 6 (1.00 g, 1.84 mmol, 1 eq) and 3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)propanoic acid (0.855 g, 3.87 mmol, 2.1 eq) was added ACN (15 mL) and saturated NaHCO3 solution (1.14 M, 9.59 mL, 11.0 mmol, 6 eq). The reaction was stirred at room temperature for 18 h, at which time the reaction was determined to be complete by LC / MS. Assuming quantitative yield, the reaction was used without purification.

[0702] LC / MS: calculated value for C28H51N3O14 m / z = 653.34, detected [M+H] + =654.7m / z.

[0703] 3.9(S)-15-((tert-butoxycarbonyl)amino)-14,18-dioxo-4,7,10,22,25,28-hexaoxa-13,19-triacontanedioic acid bis(2,3,5,6-tetrafluorophenyl) ester (Compound 8)

[0704]

[0705] 1M NaH2PO4 (5 mL) and 1M HCl (5 mL) were added to the reaction solution containing compound 7 (1.20 g, 1.84 mmol, 1 eq) to lower the pH to about 5. 2,3,5,6-tetrafluorophenol (0.641 g, 3.87 mmol, 2.1 eq) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.06 g, 5.52 mmol, 3.0 eq) were added and the reaction was stirred at room temperature for 18 h, at which time LC / MS indicated the presence of intermediate material and product. Additional 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (300 mg) was added to complete the reaction. The reaction mixture was concentrated in vacuo to remove most of the ACN and then purified by reverse phase chromatography over a 10-100% ACN / H2O+0.1% TFA gradient. Product fractions were pooled and concentrated in vacuo to remove ACN, then extracted with Et2O (50 mL) and 3xDCM (50 mL). The organics were pooled and dried over MgSO4, then filtered and evaporated in vacuo to recover the title compound (1.05 g, 1.11 mmol, 60.1%) as a clear oil.

[0706] LC / MS: calculated value for C40H51F8N3O14 m / z = 949.32, detected [M+H] + =950.8m / z. 1 H NMR (400MHz, CDCl3) δ7.12(s,1H),7.07–6.97(m,2H),6.76(s,1H),5.66(d,J=7.6Hz,1H),4.12(d,J=7.2Hz,1H),3.89(t,J=6.2Hz,4H),3 .71–3.61(m,16H),3.61–3.57(m,2H),3.54–3.36(m,3H),2.96(td,J=6.2,1.8Hz,4H),2.40–2.19(m,2H),2.11–1.92(m,2H),1.43(s,9H).

[0707] 3.10((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-methylbutyramido)-N,3-dimethylbutyramido)-3-methoxy-5-methylheptanoyl)pyrrolidine -2-yl)-3-methoxy-2-methylpropionyl)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-octaazapentadecane-25-yl)carbamic acid tert-butyl ester (Compound 9)

[0708]

[0709] Compound 8 (570 mg, 0.6 mmol, 1 eq) was dissolved in 2 mL of DMF, and compound 15 (1.66 g, 80 w / w%, 1.32 mmol, 2.2 eq) 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 eq) was added. The reactants were stirred at room temperature for 42 h, at which point LC / MS indicated that the reaction was complete. The reactants were diluted with 1 M NaH2PO4 (3 mL) and 1 M HCl (1 mL) and purified by reverse phase chromatography through a 10-55% ACN / H2O+0.1% TFA gradient. The product fractions were collected and evaporated in vacuo to recover the title compound (1.00 g, 0.379 mmol, 63.2%) as a white solid.

[0710] LC / MS:C 124 H 215 N 23 O 34 The calculated value of S2 is m / z = 2635.53, and the detected value is [M+3H] 3+ =879.8m / z. 1H NMR (400MHz, MeOD) δ7.90(d,J=8.9Hz,3H),7.77(d,J=20.8Hz,5H),4.70(t,J=8.7Hz,1H),4.60–4.52(m,3H),4.28(dd,J =17.8,7.2Hz,2H),4.11(d,J=18.5Hz,3H),3.77(s,3H),3.66–3.57(m,15H),3.54(d,J=5.5Hz,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=5.4Hz,8H),2.32(d,J=7.9Hz,2H),2.21–1.98(m,4H),1.94(d ,J=19.9Hz,2H),1.68–1.53(m,2H),1.45(s,11H),1.21–1.09(m,6H),1.03(dq,J=17.9,6.7Hz,37H),0.93–0.83(m,10H).

[0711] 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-methylbutyramido)-N,3-dimethylbutyramido)-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)glutaramide (Compound 10)

[0712]

[0713] Compound 9 (850 mg, 0.322 mmol, 1 equivalent) was dissolved in 10% TFA / DCM (10 mL) and stirred at room temperature. The reaction was completed within 2 h as determined by LC / MS. The reactant was evaporated in vacuo to an oily residue, co-evaporated with ACN (10 mL), then redissolved in 10 mL 2:1 H2O / ACN, and purified by reverse phase chromatography with a 10-50% ACN / H2O+0.1% gradient. The product fractions were pooled and lyophilized to recover the title compound (0.845 g, 0.294 mmol, 91.1%) as a white solid.

[0714] LC / MS:C 119 H 207 N23 O 32 The calculated value of S2 is m / z = 2535.48, and the detected value is [M+3H] 3+ =846.6m / z. 1 H NMR (400MHz, MeOD) δ8.03–7.92(m,7H),7.92(s,2H),4.72(t,J=8.4Hz,1H),4.56(dd,J=9.3,4.8Hz,3H),4.30–4.20(m,2H),4.16–4.02(m,3H) ,3.98(d,J=14.4Hz,1H),3.92–3.82(m,3H),3.77(qd,J=6.5,3.2Hz,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=15.1Hz,16H),2.59(dd,J=8.1,3.8Hz,8H),2.51(d,J=7.7Hz,2H),2.43(dq,J=9.1,6.7Hz,5H),2.13(dt,J=11.9,6.7Hz, 7H),2.05–1.87(m,1H),1.80(ddd,J=20.2,14.6,9.2Hz,2H),1.71–1.5 4(m,4H),1.49–1.36(m,1H),1.18–0.96(m,42H),0.88(q,J=7.1Hz,9H).

[0715] 3.12(S)-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-methylbutyryl)-N,3-dimethylbutyryl)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy- (2-methylpropionyl)sulfamoyl)phenyl)carbamoyl)-9-isopropyl-1,8,11-trioxo-14,17,20-trioxa-2,7,10-triazadocosan-22-yl)-2-(3-(2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethoxy)propionylamino)glutaramide (Drug-Linker 003)

[0716]

[0717] Compound 10 (840 mg, 0.292 mmol, 1 eq) 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 eq) were dissolved in DMF (5 mL) in a 50 mL round bottom flask. N-ethyldiisopropylamine (0.189 g, 0.255 mL, 0.74 g / mL, 1.46 mmol, 5 eq) was added and stirred at room temperature. After 1 hour, LC / MS indicated that the reaction was complete. The reactant was diluted with 1M HCl (5 mL) and purified by reverse phase chromatography with a 10-60% ACN / HO+0.1% TFA gradient. The product fractions were pooled and lyophilized to recover the title compound (0.660 g, 0.217 mmol, 74.2%) as a white solid.

[0718] LC / MS: calculated value for C132H224N24O38S2 m / z = 2818.58, detected [M+3H] 3+ =941.0m / z. 1 HNMR(300MHz,MeOD)δ7.99–7.82(m,8H),6.85(s,2H),4.77–4.70(m,1H),4.62–4.49(m,3H),4.30–4.20(m,2 H),4.17–4.03(m,2H),3.86(d,J=7.3Hz,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=10.9Hz,12H),2.59(d,J=6.8Hz,6H),2.53(s,1H),2.45(d,J=5.8Hz,4H),2.22(d,J=9. 1Hz, 5H), 2.01 (s, 4H), 1.60 (d, J = 6.9Hz, 2H), 1.52–1.37 (m, 2H), 1.18–0.96 (m, 53H), 0.89 (q, J = 6.8Hz, 6H).

[0719] 3.13 2,2-Dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azaheptadecan-17-oic acid 2,3,5,6-tetrafluorophenyl ester (Compound 11)

[0720]

[0721] 2,2-Dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azaheptadecan-17-acid (1.412 g, 4.394 mmol, 1 eq.), 2,3,5,6-tetrafluorophenol (0.803 g, 4.83 mmol, 1.1 eq.) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.926 g, 4.83 mmol, 1.1 eq.) were dissolved in ACN (15 mL) in a 250 mL round bottom flask. The reaction was stirred at room temperature for 18 h, at which point LC / MS indicated the reaction was complete. The solvent was evaporated to recover the crude product and redissolved in Et2O (100 mL) and H2O (20 mL). The organic layer was washed with saturated NaHCO3 (3 x 20 mL), 1M 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 (2.06 g, 4.39 mmol, 99%) as a clear / colorless oil.

[0722] LC / MS:C 20 H 27 The calculated value of F4NO7 is m / z = 469.17, and the detected [M+H-Boc] + =370.2m / z. 1 H NMR(300MHz, CDCl3)δ7.03(tt,J=9.9,7.1Hz,1H),5.05(d,J=15.3Hz,1H),3.92(t,J=6.2Hz,2H), 3.77–3.62(m,8H),3.60–3.51(m,2H),3.34(t,J=5.2Hz,2H),2.99(t,J=6.3Hz,2H),1.47(s,9H).

[0723] 3.14((6S,9S)-1-amino-6-((4-(N-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutyramido)-N,3-dimethylbutyramido)-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)carbamic acid tert-butyl ester (Compound 12)

[0724]

[0725] Compound 15 (3.00 g, 80 w / w%, 2.38 mmol, 1.1 equivalents) and compound 11 (1.02 g, 2.16 mmol, 1 equivalent) 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 equivalents) was added and stirred at room temperature. As determined by LC / MS, the reaction was completed within 30 min. The reactant was concentrated to about 5 mL on a rotary evaporator, diluted with a premixed solution of H2O (7 mL) and TFA (1 mL), and then purified by reverse phase chromatography through a 10-45% ACN / H2O+0.1% TFA gradient. The product fractions were pooled and lyophilized to recover the title compound (1.80 g, 1.26 mmol, 84.6%) as a white solid.

[0726] LC / MS:C 62 H 109 N 11 O 17 Calculated value for S: m / z = 1311.77, detected [M+2H] +2 =657.2m / z. 1 H NMR (300MHz, MeOD) δ8.02–7.91(m,2H),7.90–7.81(m,1H),4.73(t,J=8.4Hz,1H),4.56(dt,J=8.7,4.2Hz,1H),4.23(ddd,J=8.6, 5.8,2.6Hz,1H),4.16–4.01(m,1H),3.94–3.82(m,1H),3.82–3.69(m,3H),3.62(d,J=2.3Hz,7H),3.57–3.47(m,2H),3.38(s,1H), 3.35–3.27(m,1H),3.23(t,J=5.6Hz,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=6.9Hz,3H).

[0727] 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-methylbutyramido)-N,3-dimethylbutyramido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)sulfamoyl)phenyl)-5-ureidopentanamide (Compound 13)

[0728]

[0729] Compound 12 (1.80 g, 1.26 mmol, 1 eq) was dissolved in 10% TFA / DCM (15 mL) in a 250 mL round bottom flask and stirred at room temperature for 2 h, at which time LC / MS indicated the reaction was complete. The reactants were evaporated to dryness in vacuo, redissolved in 2:1 H2O / ACN solution (8 mL) and lyophilized to recover the title compound (1.81 g, 1.25 mmol, 99%) as an oily white solid.

[0730] LC / MS:C 57 H 101 N 11 O 15 Calculated value of S m / z = 1211.72, detected [M+2H-Boc] +2 =607.2m / z. 1HNMR(300MHz,MeOD)δ8.02–7.93(m,2H),7.93–7.81(m,2H),4.73(d,J=8.7Hz,1H),4.60–4.46(m,1H),4.24(d,J=6.9Hz,1H),4.15–4.03( m,1H),3.87(d,J=5.7Hz,1H),3.77(dd,J=6.1,2.8Hz,1H),3.72(t,J=5.2Hz,2H),3.69–3.62(m,7H),3.35–3.29(m,11H),3.19–3.10(m,4 H),3.03–2.90(m,6H),2.59(t,J=6.2Hz,2H),2.56–2.49(m,1H),2.49–2.32(m,3H),2.12(dt,J=13.4,6.7Hz,1H),2.05(s,1H),1.98(d,J =4.8Hz,1H),1.90(t,J=6.8Hz,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=7.0Hz,3H).

[0731] 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-methylbutyramido)-N,3-dimethylbutyramido)-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-tetraazahexacosane-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-methylbutyramido)-N,3-dimethylbutyramido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl (2-(2-(2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethoxy)propionamido)pimelamide (Drug-Linker 004)

[0732]

[0733] Compound 13 (3.104 g, 2.154 mmol, 4.2 equivalents) was dissolved in anhydrous DMF (8 mL). Compound 5 (500 mg, 0.513 mmol, 1 equivalent) 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 equivalents) was added and stirred at room temperature. As determined by LC / MS, the reaction was completed within 3 h. The reactant was neutralized with TFA (1 mL), diluted with 1M HCl (3 mL) and H2O (12 mL), and then purified by reverse phase chromatography through a 10-50% ACN / H2O+0.1% TFA gradient. The product fractions were separated and lyophilized to recover the title compound (1.07 g, 0.513 mmol, 46.6%) as a white solid.

[0734] LC / MS:C 194 H 331 N 35 O 54 Calculated value of S3 m / z = 4112.34, detected [M+4H] +4 =1029.8m / z. 1HNMR(300MHz,MeOD)δ8.03–7.81(m,9H),6.85(s,2H),4.74(d,J=8.8Hz,2H),4.61–4.51(m,3H),4.24(t,J=6.4Hz,3H),4.12–4 .06(m,3H),3.86(d,J=7.6Hz,3H),3.82–3.67(m,7H),3.67–3.57(m,31H),3.54(t,J=5.5Hz,5H),3.38(s,5H),3.36(s,3H),3. 35–3.32(m,39H),3.31(d,J=3.8Hz,9H),3.16(d,J=7.8Hz,6H),2.95(d,J=10.8Hz,18H),2.56(d,J=15.7Hz,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=6.8Hz,8H).

[0735] 3.17 Bis(2,3,5,6-tetrafluorophenyl) adipate (Compound 14)

[0736]

[0737] 2,3,5,6-Tetrafluorophenol (37.0 g, 222 mmol, 2.1 eq) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (44.7 g, 233 mmol, 2.2 eq) were dissolved in ACN (200 mL) at 0°C. Adipic acid (15.5 g, 106 mmol, 1 eq) was added in portions. The reaction became clear and colorless. The reaction was stirred at room temperature for 18 h, at which time LC / MS indicated that the reaction was complete. The solvent was evaporated in vacuo and the residue was redissolved in Et2O (200 mL) and 1M HCl (50 mL). The organic layer was washed with 1M HCl (2 x 50 mL) and mL brine (2 x 50 mL). Due to the presence of a large amount of emulsification, the separatory funnel was left to stand for 1 h between shaking. The organic layer was dried over MgSO4, filtered and the solvent was evaporated in vacuo. Reclaim the white solid, dissolve it in hot DCM (30mL) and filter, then expose it to the atmosphere in a fume hood and let it stand for 54h, at which point crystal formation is obvious. Allow the solution to stand for 18h at -20°C, then filter and reclaim the crystals and rinse with cold DCM (50mL). 32.1g of crystals were recovered from the first batch of product. The mother liquor was repeated with the crystallization scheme to recover another 5.4g of crystals. Two batches of crystals were combined to obtain the title compound (37.5g, 84.8mmol, 79.9%) as a crystalline solid.

[0738] LC / MS:C 18 H 10 Calculated for F8O4 m / z = 442.05, no m / z detected. 1 HNMR (300MHz, DMSO) δ7.95 (tt, J = 10.9, 7.4Hz, 2H), 2.95–2.78 (m, 4H), 1.87–1.55 (m, 4H).

[0739] 3.18 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-oxohexanoic acid 2,3,5,6-tetrafluorophenyl ester (Drug-Linker 002)

[0740]

[0741] Compound 15 (2.35 g, 1.90 mmol, 1 equivalent) was dissolved in DMF (2 mL) in a 250 mL round-bottom flask. Separately, compound 14 (5.00 g, 11.4 mmol, 6 equivalents) was dissolved in DMF (10 mL) and then added to the first solution. The reactant was stirred at room temperature, and N, N-diisopropylethylamine (0.736 g, 0.992 mL, 5.70 mmol, 3 equivalents) was added. As indicated by LCMS, the reaction was completed within 30 min. TFA (1 mL) was added and the reactant was concentrated in vacuo to a volume of 12 mL. The concentrate was purified by reverse phase chromatography through a 10-50% ACN / H2O+0.1% TFA gradient. The product fractions were collected and lyophilized to recover the title compound (2.17 g, 1.55 mmol, 81.5%) as a white solid powder.

[0742] LC / MS:C 60 H 92 F4N 10 O 14 Calculated value for S: m / z = 1284.56, detected [M+H] + =1285.8m / z. 1 H NMR (300MHz, MeOD) δ8.02–7.93(m,2H),7.89(d,J=9.0Hz,1H),7.83(d,J=9. 0Hz,1H),7.43(tt,J=10.6,7.3Hz,1H),4.74(d,J=8.8Hz,1H),4.56(dt,J=9 .4,4.8Hz,1H),4.20(dd,J=9.7,7.5Hz,1H),4.10(s,1H),3.96–3.82(m,1H) ,3.72(t,J=4.7Hz,1H),3.51(d,J=9.3Hz,1H),3.38(s,1H),3.36–3.32(m,6H ),3.30(d,J=5.4Hz,3H),3.17(s,2H),3.12(d,J=6.9Hz,1H),2.95(d,J=10. 2Hz,6H),2.83–2.74(m,1H),2.63–2.48(m,2H),2.46(d,J=6.9Hz,1H),2.44 –2.34(m,3H),2.21–2.01(m,1H),1.89(dd,J=14.3,7.7Hz,1H),1.80(q,J=3 .5Hz,1H),1.60(d,J=7.4Hz,2H),1.18–0.95(m,19H),0.89(q,J=6.9Hz,3H).

[0743] 3.19 (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-methylbutyramido)-N,3-dimethylbutyramido)-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)amino ((4-(N-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutyramido)-N,3-dimethylbutyramido)-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-pentaazatetratriacontanoic acid 2,5-dioxopyrrolidin-1-yl ester (Drug-Linker 005)

[0744]

[0745] Compound 10 (104.0 mg, 0.0361 mmol, 1 equivalent) was dissolved in 2 mL DMF in a 50 mL round-bottom flask. Bis (2,5-dioxopyrrolidine-1-yl) adipate (98.4 mg, 0.289 mmol, 8.0 equivalents) and N-ethyldiisopropylamine (23.3 mg, 0.0314 mL, 0.74 g / mL, 0.181 mmol, 5 equivalents) were added and stirred at room temperature. After 30 min, the reaction was determined to be complete by LC / MS. The reaction mixture was acidified with 0.5 mL 1 M HCl and diluted with 4 mL H2O, then purified by reverse phase chromatography through 10-50% ACN / H2O+0.1% TFA gradient. The fraction containing the product was lyophilized to recover the title compound (93.6 mg, 0.0313 mmol, 86.7%) as a white solid.

[0746] LC / MS:C 129 H 218 N 24 O 37 Calculated value of S2 m / z = 2760.54, detected [M+2H] +2 =1382.0m / z. 1HNMR(400MHz,MeOD)δ8.01–7.94(m,3H),7.91(d,J=8.8Hz,1H),7.86(d,J=8.8Hz,1H),4.74(d,J=8.6Hz,1H),4.60–4.51(m,2H),4.43–4.33(m,1H),4.24(t,J=7.3Hz,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=1.7Hz,3H),2.67(s,2H),2.63–2.55(m,3H),2.52(d,J=8.0Hz,1H),2.46(t,J=6.6Hz,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),1.17–1.09(m,7H),1.09–1.06(m,2H),1.06–0.98(m,13H),0.89(q,J=7.4Hz,4H)。

[0747] 3.20(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-methylbutyramido)-N,3-dimethylbutyramido)-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)amino 6-((4-(N-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutyramido)-N,3-dimethylbutyramido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)sulfamoyl)phenyl)carbamoyl)-9-isopropyl-1,8,11,24,29-anooxo-14,17,20-trioxa-2,7,10,23,28-pentaazatetratriacontano-34-oic acid 2,3,5,6-tetrafluorophenyl ester (Drug-Linker 006)

[0748]

[0749] Drug-joint 005 (17 mg, 0.00568 mmol, 1 equivalent) was dissolved in 2: 1 diH2O:ACN mixture (3 mL). The pH was raised to about 11 with saturated NaHCO3 (0.2 mL), and the reaction was stirred at room temperature for 18 hours. The hydrolysis of NHS ester was determined to be complete by LC / MS. The reaction was acidified to pH 5 with 1M NaH2PO4 (1 mL), followed by the addition of 2,3,5,6-tetrafluorophenol (4.72 mg, 0.0284 mmol, 5 equivalents) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (5.45 mg, 0.0284 mmol, 5 equivalents). After 2 hours, the reaction was determined to be complete by LC / MS. The reaction mixture was purified by reverse phase chromatography through a 10-50% ACN / H2O+0.1% TFA gradient. Product containing fractions were pooled and lyophilized to recover the title compound (3.7 mg, 0.00122 mmol, 21.4%) as a white solid.

[0750] LC / MS:C 131 H 215 F4N 23 O 35 Calculated value of S2 m / z = 2811.52, detected [M+2H] +2=1407.0m / z.

[0751] Alternatively, drug-linker 006 can be prepared by the same method as drug-linker 005.

[0752] 3.21 (S)-N-(4-(N-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutyramido)-N,3-dimethylbutyramido)-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-amino)-5-ureidopentanamide (Drug-Linker 001)

[0753]

[0754] Prepared as described in International Publication No. WO 2019 / 173911.

[0755] Example 4: Preparation of Additional Multivalent Drug-Linkers

[0756] Drug-Linker 007 and Drug-Linker 008

[0757]

[0758] Drug-Connector 007

[0759]

[0760] Drug-Connector 008

[0761] Alternatively, the trivalent drug-linker structure exemplified in Drug-Linker 004 (Example 3.16) can be adjusted to conjugate to a lysine residue by utilizing 2,3,4,5-tetrafluorophenol or N-hydroxysuccinimide activated esters similar to those used 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) can be prepared by the reaction of adipic anhydride with 4-amino-4-[3-(tert-butoxy)-3-oxopropyl]pimelic acid 1,7-di-tert-butyl ester under amide bond formation conditions similar to those used to generate Compound 3, followed by further elaboration into the final drug-linker. See Scheme 1 ( Figure 3 ).

[0762] Example 5: Conjugation and characterization of anti-cMET antibody-drug conjugates

[0763] The antibody-drug conjugates (ADCs) shown in Table 5.1 were prepared as follows.

[0764] 5.1 Preparation of ADCs by random lysine conjugation

[0765] An exemplary scheme for preparing ADCs by random lysine conjugation is provided below.

[0766] 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 in DMSO) in 5-10% (v / v, final) DMSO (5 mg / mL) in PBS (pH 7.4). The reaction mixture was mixed by pipetting and then centrifuged at 400 x g for 3 minutes. The resulting solution was incubated at room temperature for 16-20 hours and then purified.

[0767] 5.2 Preparation of ADCs by random cysteine ​​conjugation

[0768] An exemplary scheme for preparing ADCs by random cysteine ​​conjugation is provided below.

[0769] By adding 5mM diethylenetriamine pentaacetic acid (DTPA) (11.4mL PBS solution, pH adjusted to 7.4) and 10mM tris (2-carboxyethyl) phosphine (TCEP) aqueous solution (591uL, 2.2 equivalents) reduction variant v17427 solution in PBS (pH 7.4), the final concentration is 5-10mg / mL. The reduction reaction is carried out at 37°C for 1-3 hours. Then the reduced protein is reacted on ice for 1-2 hours with excess drug-linker 001 (6-10 equivalents, 10-20mM DMSO stock solution). Add excess N-acetyl-L-cysteine ​​solution (6-10 equivalents) from 10mM stock aqueous solution to quench the conjugation reaction. Before purification, the quenched reactant is incubated on ice for 30 minutes.

[0770] 5.3 Preparation of ADCs by site-specific conjugation to cysteine ​​insertions

[0771] An exemplary scheme for preparing ADCs by site-specific conjugation of inserted cysteine ​​residues is provided below. This scheme or similar schemes have been applied to the site-specific conjugation of drug-linker 001, drug-linker 003, and drug-linker 004.

[0772] A solution of variant v29001 (2 g) in PBS (202 mL) (pH 7.4) was reduced by adding 10 mM DTPA (24 mL PBS solution, pH adjusted to 7.4) and 25 mM TCEP aqueous solution (13.7 mL, 25 equivalents). After 3-4 hours at 37°C, the reduced antibody was diluted to approximately 250 mL with PBS and washed with XL ultrafiltration module (Ultracel 30kDa0.005m 2 ; MilliporeSigma, Burlington, MA; PXC030C50) was purified with approximately 3 diavolumes of PBS (pH 7.4). The purified antibody was then reoxidized with 25 molar equivalents of dehydroascorbic acid (DHAA) (50 mM DMSO stock) at 2-8°C for 16-20 hours. 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 DMSO stock. The conjugation reaction was allowed to proceed for 3-4 hours at room temperature with mixing. Excess N-acetyl-L-cysteine ​​solution (5.6 mL, 5.25 equivalents) from a 10 mM stock solution (10% DMSO in water) was added to quench the conjugation reaction. The quenched reaction was incubated with mixing at room temperature for 30 minutes and then incubated at 2-8°C for 16-20 hours before purification.

[0773] 5.4 Purification and Characterization of ADC

[0774] use ADCs prepared at a scale greater than 50 mg were purified using a XL ultrafiltration module (MilliporeSigma, Burlington, MA). Briefly, crude ADC solutions were diluted or concentrated to approximately 5-10 mg / mL with 10 mM NaOAc (pH 5.5) and purified using XL ultrafiltration module (Ultracel 30kDa 0.005m 2 ; MilliporeSigma, Burlington, MA; PXC030C50) was purified with 8-15 diafiltration volumes of 10 mM NaOAc (pH 4.5). The purified ADC was then sterile filtered (0.2 um).

[0775] Follow the manufacturer's instructions by Zeba TM ADC prepared at a scale of less than 50 mg was purified using a Spin Desalting column 40 MWCO, (ThermoFisher Scientific, Waltham, MA) pre-equilibrated with 10 mM NaOAc, pH 4.5.

[0776] 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 using ammonium sulfate / sodium phosphate buffer. TM The ADCs were purified by centrifugation on a butyl HP column (Cytiva Life Sciences, Marlborough, MA) and eluted with sodium phosphate buffer containing 20% ​​isopropanol. ADC species with average drug to antibody ratios (DAR) of 2 and 4 were enriched and combined at a 1:1 ratio to obtain ADCs with an average DAR of approximately 3. The purified ADCs were formulated in 10 mM histidine (pH 6.0) buffer and sterile filtered.

[0777] After purification, the concentration of ADC is determined by BCA assay with reference to the standard curve generated using the corresponding unconjugated parent antibody. Alternatively, the concentration is estimated by measuring the absorption at 280 nm using the calculated extinction coefficient of the antibody sequence. ADC is also characterized by hydrophobic interaction chromatography (HIC) and size exclusion chromatography (SEC) as described below.

[0778] 5.4.1 Hydrophobic interaction chromatography

[0779] ADCs were analyzed by HIC to estimate the drug to antibody ratio (DAR). The chromatographic analysis was performed on a Butyl-NPR column (2.5 μm, 4.6×35 mm; TOSOH Bioscience GmbH, Griesheim, Germany) with a gradient from 95 / 5% MPA / MPB to 5 / 95% MPA / MPB in 12 min at a flow rate of 0.5 mL / min (MPA = 1.5 M (NH 4 ) 2 SO 4 , 25 mM Na x PO4, pH 7 and MPB = 75% 25 mM Na x PO4, pH 7, 25% isopropanol). Detection was by absorbance at 280 nm.

[0780] 5.4.2 Size Exclusion Chromatography

[0781] On Agilent Infinity II 1260HPLC (Agilent Technologies, Santa Clara, CA), using AdvanceBio SEC column (300 angstroms, 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, the aggregation degree of ADC (about 15-150 ug, 5 uL injection volume) was evaluated by SEC. Detection was performed by absorbance at 280 nm.

[0782] Table 5.1 summarizes the characteristics evaluated for each ADC.

[0783] Table 5.1: Characteristics of ADC

[0784]

[0785]

[0786] 1 SS = site specific

[0787] Example 6: In vitro cytotoxicity of random cysteine-conjugated anti-cMET antibody-drug conjugate (DAR 4)

[0788] As described below, the cell growth inhibition (cytotoxicity) ability of anti-cMet ADCs comprising variant v17427 (HetFc, unmodified hinge) conjugated to drug-linker 001 or MCvcPABC-MMAE by random cysteine ​​conjugation with an average DAR of 4 was determined in a panel of four cMet-expressing cell lines. The cell lines were EBC-1 (lung squamous cell carcinoma), H292 (lung cancer), BT-20 (breast cancer), and SW48 (colorectal cancer).

[0789] Briefly, cells were seeded at a density of 1,000-1,500 cells / well in 384-well plates and treated with titrations of test articles generated in complete cell growth medium. Treated cells were incubated for 4-5 days under standard culture conditions (37°C / 5% CO2). Reagent (Promega Corporation, Madison, WI; Catalog No. G7570) was added to each well and the cells were analyzed using Synergy TMH1 microplates (BioTek Instruments, Winooski, VT) measure the luminescence corresponding to the ATP present in each well. Based on blank wells (no test article added, only blank medium), ATP was used to measure the RLU value (relative light unit) to calculate the cytotoxicity value % and the test article concentration was plotted using GraphPad Prism 8 software (GraphPad Software, San Diego, CA).

[0790] result

[0791] The results are summarized in Table 5.1. The DAR-matched v17427-MCvcPABC-MMAE DAR 4 ADC exhibited lower in vitro cytotoxicity than the v17427-Drug-Linker 001 ADC in high and medium cMet expressing cells EBC-1, HT-29, and BT-20. The difference in cytotoxicity was less pronounced in EBC-1, the cell line with the highest cMet expression level tested. Neither ADC exhibited significant cytotoxicity in the low cMet expressing cell line SW48. As expected, the free payload from Drug-Linker 001 (Compound 1) showed cytotoxicity in all tumor cell lines, but not as well as the v17427-Drug-Linker 001 ADC.

[0792] Table 6.1: Summary of in vitro cytotoxicity - EC50

[0793]

[0794] Example 7: Agonistic effect of random cysteine-conjugated anti-cMET antibody-drug conjugate (DAR 4) on the cMET pathway

[0795] The cMet pathway agonism of anti-cMet antibody variant v17427 (HetFc, unmodified hinge) and ADCs comprising variant v17427 conjugated to drug-linker 001 or MCvcPABC-MMAE at DAR 4 random cysteine ​​was evaluated by cell proliferation and ELISA to measure AKT phosphorylation as a downstream indicator of cMET activation.

[0796] Cell proliferation:Briefly, H596 lung cancer cells were serum starved overnight (37°C, 5% CO2) by replacing complete growth medium with serum-free RPMI-1640 (Thermo Fisher Scientific Inc., Waltham, MA; catalog number A1049101). Cells were then detached with cell dissociation buffer (Thermo Fisher Scientific Inc., catalog number 13151014), resuspended in RPMI-1640 (Thermo Fisher Scientific Inc., catalog number 12483-020) containing 1% FBS (v / v), and seeded in 384-well tissue culture plates at a density of 1,000 cells / well. Cells were treated with test article titrations prepared in RPMI-1640 + 1% FBS (v / v) and incubated for 6 days (37°C, 5% CO2). After incubation, CellTiter- Reagent (Promega Corporation, Madison, WI; Catalog No. G7570) was added to all wells and the cells were analyzed using Synergy TM H1 microplates (BioTek Instruments, Winooski, VT) measure the luminescence corresponding to the ATP present in each well. Based on untreated cells (no test article added), ATP was used to measure the RLU value (relative light unit) to calculate the activity value % and GraphPad Prism 8 software (GraphPad Software, San Diego, CA) was used to plot the test article concentration.

[0797] Phospho-AKT ELISA:In brief, H441 or H596 lung cancer cells were isolated with cell dissociation buffer, seeded in 24-well tissue culture plates containing RPMI-1640+10% FBS at a density of 50,000 cells / well, and incubated overnight (37°C, 5% CO2). Then, the cells were serum-starved overnight (37°C, 5% CO2) by replacing the complete growth medium with serum-free RPMI-1640. The culture medium was removed, and the cells were then treated with 100nM test articles prepared in serum-free RPMI-1640 and incubated at appropriate time points (37°C, 5% CO2). Cell lysates were generated by adding cell lysis buffer (CellSignaling Technology, Danvers, MA; Catalog No. 9803S)+1mM PMSF (Cell Signaling Technology, Catalog No. 8553S). The protein concentration of the lysate was assessed by BCA protein assay (Thermo Fisher Scientific Inc., catalog number 23223 and 23224). Phospho-Akt1 (Ser473) sandwich ELISA kit (Cell Signaling Technology, catalog number 7160C) was used to assess the level of phosphorylated AKT in 10 μg of lysate. TM The absorbance at 450 nm was measured using an H1 microplate (BioTek Instruments, Winooski, VT). The background A450 nm signal was subtracted from all wells using a signal blank well and then normalized to the A450 nm signal of untreated cells at each corresponding time point to calculate the AKT phosphorylation fold. The fold change at each time point was then plotted using GraphPad Prism 8 software (GraphPad Software, San Diego, CA).

[0798] result

[0799] The results are as follows Figure 4 and Figure 5A and 5B As shown. Figure 4As can be seen, the bivalent MetMab (v17429) induced a dose-dependent increase in H596 proliferation compared to the untreated control, while the hinge-modified Terituzumab (v17606) showed minimal effect, as expected. The unconjugated variant v17427 (HetFc, unmodified hinge) showed a slight but sustained increase in H596 proliferation after 6 days. Variants v17427 randomly conjugated to drug-linker 001 or MCvcPABC-MMAE with DAR 4 showed a clear dose-dependent increase in proliferation compared to the untreated control.

[0800] Figure 5A and 5B The bivalent MetMab (v17429) was shown to induce strong AKT phosphorylation in both H596 and H441 cells, and this effect could be sustained for up to 60 minutes after treatment. In contrast, the hinge-modified terituzumab (v17606) had minimal effect on AKT phosphorylation in both cell lines. The unconjugated variant v17427 induced a slight temporary increase in AKT phosphorylation in both cell lines. Random ADCs generated by variant v17427 appeared to exhibit an increase in phosphorylation compared to the unconjugated parent antibody.

[0801] Example 8: In vitro cytotoxicity of lysine-conjugated anti-cMET antibody-drug conjugates (DAR 2, 4 and 6)

[0802] The cell growth inhibition ability of an anti-cMet ADC comprising variant v17427 (HetFc, unmodified hinge) conjugated to drug-linker 002 with DARs DAR 2 to DAR 6 via random lysine conjugation was determined in a panel of seven cMet-expressing cell lines. Variant v17427 conjugated to MCvcPABC-MMAE drug linker with DAR 4, and anti-RSV antibody palivizumab (v22277) conjugated to drug-linker 002 or MCvcPABC-MMAE with DAR 4 were used as controls. The cell lines were SNU-5 (gastric cancer), EBC-1 (lung squamous cell carcinoma), HCC827 (lung cancer), H1975 (non-small cell lung cancer), HCT-116 (colorectal cancer), H292 (lung cancer), and BT-20 (breast cancer).

[0803] Cytotoxicity was determined as described in Example 6, where treated cells (1,000 cells / well) were incubated under standard culture conditions (37° C. / 5% CO 2 ) for 4 days.

[0804] result

[0805] The results are summarized in Table 8.1. Randomized v17427-drug linker 002 ADCs with DARs ranging from DAR 1.9 to DAR 6.2 produced a range of cytotoxic activities in the panel of seven cMet-expressing cell lines, with higher DAR conjugates generally showing lower EC50 values ​​in all tested cell lines. In the high-expressing cell lines SNU-5 and EBC-1, the differences between ADCs with DAR 1.9 to DAR 6.2 were small, with EC50s ranging from 0.043 to 0.021 nM and 0.012 to 0.004 nM for the lowest and highest DAR conjugates, respectively. In the medium-to-low-expressing cell lines HCC827, H1975, HCT-116, H292, and BT-20, the differences between ADCs with DAR 1.9 to DAR 6.2 were more pronounced. Randomized palivizumab-drug-linker 002 and palivizumab-MCvcPABC-MMAE conjugates did not show expected cytotoxicity in cMET-expressing cell lines.

[0806] Table 8.1: Summary of in vitro cytotoxicity - EC50

[0807]

[0808] Example 9: In vitro cytotoxicity of anti-cMET antibody-drug conjugates with HomoFc or HetFc scaffolds

[0809] The cell growth inhibitory ability of ADCs containing anti-cMet antibodies (v32634 and v17427, respectively) with HomoFc or HetFc backbones conjugated to drug-linker 002 at approximately DAR 4 and DAR 6 by random lysine conjugation was evaluated in a panel of four cMet-expressing tumor cell lines and one cMet-negative cell line. 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).

[0810] result

[0811] 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. As expected, all ADCs showed no activity in the cMet-negative cell line T-47D. In the high-expressing cell lines SNU-5 and EBC-1, the potency difference between DAR3.9-4.0 and DAR 5.8 conjugates was discrete. In the medium-expressing cell lines H1975 and H292, the potency difference between DAR 3.9-4.0 and DAR 5.8 conjugates was more obvious. In the H1975 and H292 cell lines, the EC50 of the DAR5.8 conjugate was 2.0 to 3.4 times that of the DAR 4 conjugate, respectively.

[0812] Table 9.1: Summary of in vitro cytotoxicity - EC50

[0813]

[0814] Example 10: Cellular equilibrium binding of lysine-conjugated anti-cMET antibody-drug conjugates

[0815] As described below, the cell binding ability of ADCs containing anti-cMet antibodies with HomoFc or HetFc backbones (v32634 and v17427, respectively) conjugated to drug-linker 002 by random lysine conjugation at approximately DAR 4 and DAR 6 was evaluated for cMet binding by flow cytometry using SNU-5 and H292 endogenous cMet-expressing cell lines. Unconjugated anti-RSV antibody palivizumab (v22277) was used as a control.

[0816] Briefly, cells were seeded at 50,000 cells / well in V-bottom 96-well plates and treated with antibodies for 24 hours at 4°C to prevent internalization. After 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 min at 4°C. After incubation and washing, cells were stained with BD LSRFortessa TM Fluorescence was detected by flow cytometry on a cell analyzer (BD Biosciences, Franklin Lake, NJ), with a minimum of 1,000 events collected per well. The 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).

[0817] result

[0818] The results are summarized in Table 10.1. Both unconjugated variants, v32634 and v17427 (HomoFc and HetFc regions, respectively), produced comparable apparent Kd and Bmax values ​​in both SNU-5 and H292 cell lines (high and medium endogenous cMet expression, respectively). In the SNU-5 and H292 cell lines, the unconjugated HomoFc variant v32634 produced Kd values ​​of 0.59 and 0.02 nM, respectively. Similarly, in the SNU-5 and H292 cell lines, the unconjugated HetFc variant v17427 produced Kd values ​​of 0.33 and 0.03 nM, respectively. In both cMet-expressing cell lines, the binding affinities of the unconjugated HomoFc and HetFc variants were comparable to the lysine-conjugated ADC counterparts (DAR 4 and DAR 6). As expected, unconjugated palivizumab antibody v22277 showed no binding to cMet-expressing cell lines.

[0819] Table 10.1: Cell Binding - Kd and Bmax Summary

[0820]

[0821] Example 11: In vitro cytotoxicity of site-specific cysteine-conjugated anti-cMET antibody-drug conjugates

[0822] The cell growth inhibition ability of site-specific cysteine-conjugated ADCs containing various cysteine ​​insertion antibody variants conjugated to drug-linker 001 at approximately DAR 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) and 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. The "DAR 1" ADC ranged from DAR 0.85–0.89 and the "DAR 3" ADC ranged from DAR 2.54–2.84. Cytotoxicity was determined as described in Example 7.

[0823] result

[0824] The results are summarized in Table 11.1. The cytotoxicity of site-specific ADCs with average DARs of 1, 2, and 3 observed in cMet-expressing cell lines depends on the drug load. Overall, three site-specific ADCs with an average of DAR 1 showed lower cytotoxicity than two ADCs with an average of DAR 2 and five ADCs with an average of DAR 3. In all cell lines tested, random ADCs with an average of DAR 4 showed higher cytotoxicity than DAR 3, 2, and 1 site-specific ADCs, producing lower EC50 values ​​and higher maximum cytotoxicity%. Two of the three DAR 1 site-specific ADCs produced comparable cytotoxicity, including variants v33967 and v33968 (see Table 1.3). The third DAR 1 ADC includes variant v33969, which produced higher cytotoxicity than other DAR 1 ADCs in HT-29 and H441 cell lines. Both DAR 2 site-specific ADCs produced comparable cytotoxicity in the cell lines tested. Finally, five DAR 3 ADCs also produced comparable cytotoxicity. As expected, the palivizumab randomized DAR 4 ADC showed no cytotoxicity in cMet-expressing cell lines.

[0825] Table 11.1: Summary of in vitro cytotoxicity - EC50

[0826]

[0827] *Incomplete curve

[0828] Example 12: In vitro cytotoxicity of site-specific anti-cMET antibody-drug conjugates comprising a multivalent linker

[0829] The cell growth inhibition ability of ADCs containing variant v29001 with two cysteine ​​insertions conjugated to multivalent drug-linker 003 or multivalent drug-linker 004 and ADCs containing parental variant v17427 conjugated to monovalent drug-linker 002 with DAR 4 or DAR 6 was evaluated in a panel of tumor cell lines expressing cMet. 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.

[0830] result

[0831] The results are summarized in Table 12.1. In all cMet-expressing cell lines tested, the average DAR 6 ADC exhibited higher cytotoxicity than the average DAR 4 ADC, regardless of whether monovalent or multivalent drug linkers were used.

[0832] In the high-expressing cMet cell lines SNU-5 and EBC-1, the monovalent drug-linker ADCs showed comparable cytotoxicity to the DAR-matched multivalent drug-linker ADCs. In SNU-5 and EBC-1 cells, the monovalent drug-linker DAR 4 ADC produced EC50 values ​​of 38.5 pM and 9.6 pM, respectively. In the same cell lines, the multivalent drug-linker DAR 4 ADC produced EC50 values ​​of 33.1 pM and 8.5 pM, respectively. Similarly, in SNU-5 cells, the monovalent and multivalent drug-linker DAR 6 ADCs produced EC50 values ​​of 13.6 pM and 18.7 pM, respectively. In EBC-1 cells, the monovalent and multivalent drug-linker DAR 6 ADCs produced EC50 values ​​of 6.2 pM and 4.1 pM, respectively.

[0833] In the medium-expressing cMet cell lines H1975 and H292, the monovalent drug-linker ADC showed comparable cytotoxicity to the DAR-matched multivalent drug-linker ADC. In H1975 and H292 cells, the monovalent drug-linker DAR 4ADC produced EC50 values ​​of 83.6pM and 118.0pM, respectively. In the same cell line, the multivalent drug-linker DAR 4ADC produced EC50 values ​​of 48.2pM and 93.2pM, respectively. Similarly, in H1975 cells, the monovalent and multivalent drug-linker DAR 6ADC produced EC50 values ​​of 43.7pM and 27.7pM, respectively. In H292 cells, the monovalent and multivalent drug-linker DAR 6ADC produced EC50 values ​​of 118.0pM and 93.2pM, respectively. In the cMet-negative T-47D cell line, neither the monovalent nor the multivalent drug-linker DAR 4 and DAR 6 ADCs showed cytotoxicity as expected. As expected, the palivizumab control ADC showed no cytotoxicity in any of the cell lines tested.

[0834] Table 12.1: Summary of in vitro cytotoxicity - EC50

[0835]

[0836] Example 13: Cellular equilibrium binding of site-specific anti-cMET antibody-drug conjugates

[0837] The following unconjugated antibodies and ADCs were evaluated for their on-cell binding ability by flow cytometry on SNU-5 and H292 endogenous cMet-expressing cell lines: unconjugated variant v17427 (HetFc, unmodified hinge); and cysteine ​​insertion variant v29001 (two cysteine ​​insertions); lysine-conjugated ADCs comprising variant v17427 with DAR 4 or DAR 6 and drug-linker 002; and cysteine-conjugated ADCs comprising cysteine ​​insertion variant insertion v29001 conjugated to drug-linker 001, drug-linker 003, or drug-linker 004 with DAR 2, 4, or 6, respectively. Unconjugated palivizumab v22277 was used as a non-targeting control. Cellular binding was determined as described in Example 10.

[0838] result

[0839] The results are summarized in Table 13.1. All unconjugated anti-cMet antibodies and ADCs exhibited comparable apparent Kd and Bmax values ​​in SNU-5 and H292 cell lines (high and medium endogenous cMet expression, respectively). Unconjugated antibodies v17427 and v29001 produced Kd values ​​of 0.328 nM and 0.428 nM, respectively, in the high cMet expressing cell line SNU-5. Similarly, variant v17427 DAR 4 and DAR 6 ADCs produced Kd values ​​of 0.428 nM and 0.435 nM, respectively, in SNU-5 cells. Variant v29001 ADCs produced Kd values ​​ranging between 0.584 nM and 1.131 nM in SNU-5 cells.

[0840] In the medium cMet expressing cell line H292, the unconjugated antibodies v17427 and v29001 produced Kd values ​​of 0.026 nM and 0.024 nM, respectively. Similarly, the variant v17427 DAR 4 and DAR 6 ADCs produced Kd values ​​of 0.040 nM and 0.047 nM, respectively, in H292 cells. The variant v29001 ADC produced Kd values ​​ranging between 0.028 nM and 0.064 nM in H292 cells. As expected, the palivizumab negative control did not exhibit cellular binding to any of the cMet expressing cell lines.

[0841] Table 13.1: Cell Binding - Kd and Bmax Summary

[0842]

[0843] Example 14: Internalization of anti-cMET antibody-drug conjugates

[0844] The receptor-mediated internalization capacity of the unconjugated variant v17427 (HetFc, unmodified hinge) and v17427-drug-linker 002DAR 6.7 ADC was assessed in two cMet-expressing cell lines, IGROV-1 and OVCAR-3, using high-content imaging as described below. Unconjugated anti-RSV antibody palivizumab (v22277) was used as a negative control.

[0845] Briefly, antibodies were fluorescently labeled by coupling with anti-human IgG Fc Fab fragment AF488 conjugate (Jackson ImmunoResearch Labs, West Grove, PA; catalog number 109-547-008) at a 1:1 molar ratio in PBS (pH 7.4) (ThermoFisher Scientific, Waltham, MA; catalog number 10010-023) at 4°C for 24 hours. Cells were seeded in 384-well plates at 5,000 cells / well and incubated overnight under standard culture conditions (37°C / 5% CO2) to allow attachment. The next day, the conjugated antibodies were added to the cells at various concentrations (70 to 0.3 nM) and incubated under standard culture conditions for 5 hours to allow internalization. After incubation, the cells were stained with FluoroFix TM Buffer (BioLegend, San Diego, CA; Catalog No. 422101), 20uL / well fixed cells at room temperature for 30min. Nuclear stain Hoechst 33342 (Thermo Fisher Scientific, Waltham, MA; Catalog No. 62249) was added to the wells at 10uM, and the assay plate was incubated at 37°C / 5% CO2 for 1 hour. After incubation with nuclear stain, fluorescent images were captured 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 of each well. Average object fluorescence (GFP channel) values ​​were plotted using GraphPad Prism version 9 (GraphPad Software, San Die go, CA).

[0846] result

[0847] The results are summarized in Table 14.1. The unconjugated variant v17427 showed comparable receptor-mediated internalization to the ADC variant v17427-drug-linker 002DAR 6.7 in EBC-1 and HT-29 (high and medium cMet expressing cell lines, respectively), indicating that the conjugation of the drug-linker does not affect the binding and internalization ability of the antibody. In both cMet expressing cell lines, both the unconjugated antibody and the ADC showed dose-dependent internalization at 70nM to 0.3nM treatment. As expected, the palivizumab control (v22277) did not show any internalization in EBC-1 and HT-29 cells.

[0848] Table 14.1: Receptor-mediated internalization

[0849]

[0850]

[0851] Example 15: cMET pathway agonism of site-specific cysteine-conjugated or lysine-conjugated anti-cMET antibody-drug conjugates

[0852] cMet pathway agonism was assessed by cell proliferation for ADCs comprising various cysteine ​​insertion variants conjugated to Drug-Linker 001 with DAR 2. cMet pathway agonism was also assessed by ELISA to measure AKT phosphorylation as a downstream indicator of cMet activation, following the protocol described in Example 7 for ADCs comprising cysteine ​​insertion variants conjugated to Drug-Linker 001 with DAR 1, 2, or 3, as well as v17427-Drug-Linker 001 conjugated to cysteine ​​with DAR 4 and v17427-Drug-Linker 002 conjugated to lysine.

[0853] result

[0854] The results of cell proliferation assessment were Figure 6 As shown. Compared with the untreated control, the bivalent MetMab (v17429) showed a strong dose-dependent increase in H596 cell proliferation, while the variant v17427 (HetFc, unmodified hinge) showed a slight increase in proliferation. Site-specific DAR 2 ADCs containing variants v22761, v22765, v28983, v28989 or v29001 did not show a significant increase in H596 cell proliferation.

[0855] The results of AKT phosphorylation assessment were as follows Fig. 7A-C. The bivalent MetMab (v17429) strongly induced phosphorylation in H441 cells, while minimal effects were observed with hinge-modified terituzumab (v17606). Consistent with the results described in Example 7, variant v17427 (HetFc, unmodified hinge) induced slight transient AKT phosphorylation, which was further increased with random cysteine ​​conjugation of drug-linker 001. In contrast, the phosphorylation profiles of ADCs with site-specific cysteine ​​conjugation of drug-linker 001 with different DARs did not differ from those of variant v17427. These observations suggest that the reduction of hinge cysteines during random conjugation may increase cMET agonism, which can be avoided by site-specific conjugation with inserted cysteine ​​residues. ADCs with random DAR 2 lysine conjugation of drug-linker 002 with variant v17427 also resulted in minimal changes in AKT phosphorylation compared to the unconjugated parent antibody ( Figure 7B ), which further supports the potential impact of random cysteine ​​conjugation on cMET agonism.

[0856] Example 16: In vitro activity of random cysteine-conjugated anti-cMET antibody-drug conjugate (DAR 4)

[0857] As described below, the in vivo antitumor activity of ADCs containing variant v17427 (HetFc, unmodified hinge) conjugated to drug-linker 001 or MCvcPABC-MMAE with random cysteine ​​at DAR 4 was evaluated in multiple cell line-derived xenograft (CDX) models expressing a range of cMet levels. The cell lines were as follows: cMet high HCC827 lung cancer, cMet high EBC1 lung cancer, cMet high H1975 lung cancer, cMet medium / high HT29 colorectal cancer, cMet low H292 lung cancer, and cMet low SW48 colorectal cancer.

[0858] For the cMet high HCC827 lung cancer model, 5x10 6 Cells were implanted into BALB / c nude mice in 0.1 ml of 1:1 PBS:Matrigel, and when the tumor volume reached approximately 125 mm 3 At 4 hr, mice were assigned to groups (n=5 per group) and treated as specified in Table 16.1 on day 0, with a study duration of 60 days. For the cMet high EBC1 lung cancer model, 3x10 6 The cells were implanted into female BALB / c nude mice, and when the tumor volume reached approximately 135 mm 3At 4 hr, mice were assigned to groups (n=6 per group) and treated on day 0 as specified in Table 16.1, with a study duration of 60 days. For the cMet high H1975 lung cancer model, 5x10 6 cells were implanted into BALB / c-Foxn1 nu In mice, when the tumor volume reached approximately 155 mm 3 At 4 hr, mice were assigned to groups (n=5 per group) and treated on day 0 as specified in Table 16.1, with a study duration of 56 days. For the cMet medium / high HT29 colorectal cancer model, 3x10 6 The cells were implanted into BALB / c nude mice, and when the tumor volume reached approximately 150 mm 3 At 4 hr, mice were assigned to groups (n=5 per group) and treated on day 0 as specified in Table 16.1, with a study duration of 60 days. For the cMet low H292 lung cancer model, 5x10 6 cells were implanted into SCID / beige mice, and when the tumor volume reached approximately 160 mm 3 At 4 pm, mice were assigned to groups (n=5 per group) and treated on day 0 as specified in Table 16.1, with a study duration of 46 days. For the cMet low SW48 colorectal cancer model, 1x10 7 cells were implanted into BALB / c nude mice, and when the tumor volume reached approximately 120 mm 3 Mice were assigned to groups (n=5 per group) and treated on day 0 as specified in Table 16.1, with a study duration of 42 days.

[0859] For all models, tumor volume and body weight were measured twice a week. Tumor volume graphs represent mean values ​​and standard errors of the mean. Average data were plotted only if greater than or equal to 80% of the mice continued on study at that time point. For statistical analysis, linear mixed effects models were fitted to log-transformed tumor volumes, followed by F-tests for the null hypothesis of equal mean growth rates and post hoc pairwise comparisons.

[0860] Table 16.1: Test articles and doses

[0861]

[0862] result

[0863] The results are shown in Fig. 8A -F and are summarized as follows.

[0864] In the cMet high HCC827 lung cancer model ( Fig. 8A ), compared with the vehicle control, the variant v17427 conjugated to drug-linker 001 or MCvcPABC-MMAE showed strong inhibition of tumor growth at both 3 and 10 mg / kg dose levels (p<0.0001). Compared with v17427-drug-linker 001, the trend of tumor growth inhibition after v17427-MCvcPABC-MMAE administration was more persistent.

[0865] In the cMet high EBC1 lung cancer model ( Figure 8B ), v17427-Drug-Linker 001 and v17427-MCvcPABC-MMAE showed strong inhibition of tumor growth at both 5 and 10 mg / kg dose levels compared to vehicle control (p<0.005).

[0866] In the cMet high H1975 lung cancer model ( Figure 8C ), v17427-drug-linker 001 and v17427-MCvcPABC-MMAE showed inhibition of tumor growth at 2, 4 and 8 mg / kg dose levels compared to vehicle control (p<0.01), and both ADCs showed a dose-response trend. v17427-drug-linker 001 tended to show stronger tumor growth inhibition than v17427-MCvcPABC-MMAE, and this comparison was significant at the 2 mg / kg dose level (p<0.01).

[0867] In the cMet medium / high HT29 colorectal cancer model ( Fig.8D ), compared with the vehicle control, v17427-drug-linker 001 and v17427-MCvcPABC-MMAE showed strong inhibition of tumor growth at 3 and 10 mg / kg dose levels (p<0.0001). Compared with v17427-MCvcPABC-MMAE, the trend of tumor growth inhibition after v17427-drug-linker 001 administration was more persistent, and when each was administered at 10 mg / kg, the re-growth rate after v17427-drug-linker 001 administration was slower than that after v17427-MCvcPABC-MMAE administration (p<0.005).

[0868] In the cMet low H292 lung cancer model ( Fig. 8E), v17427-Drug-Linker 001 and v17427-MCvcPABC-MMAE both showed moderate inhibition of tumor growth at 3 and 10 mg / kg dose levels compared to vehicle control (p<0.05). No significant dose response was observed between 3 and 10 mg / kg dose levels or between v17427-Drug-Linker 001 and v17427-MCvcPABC-MMAE responses.

[0869] In the cMet low SW48 colorectal cancer model ( Fig.8F ), neither v17427-Drug-Linker001 nor v17427-MCvcPABC-MMAE showed strong inhibition of tumor growth at the administered dose level, and there was no significant difference in the activity of v17427-Drug-Linker001 and v17427-MCvcPABC-MMAE.

[0870] In summary, these data demonstrate that v17427-Drug-Linker001 and v17427-MCvcPABC-MMAE ADCs are active against in vivo cMet expression models. A trend was shown between the magnitude of response and cMet expression. v17427-Drug-Linker001 and v17427-MCvcPABC-MMAE exhibited roughly comparable activity, with superior activity observed for v17427-Drug-Linker001 in the HT29 and H1975 models.

[0871] Example 17: In vivo activity of anti-cMET antibody-drug conjugates (DAR 1, 2, 3 and 4)

[0872] The in vivo antitumor activity of cMet-targeted ADCs containing drug-linker 001 conjugated to site-specific cysteines at DARs 1, 2, 3, and 4, drug-linker 002 conjugated to random lysines at DAR 2, was evaluated in the H1975 (cMet high) lung cancer and HT29 (cMet medium / high) colorectal cancer CDX models. Table 17.1 lists the ADCs and their doses. The doses were selected to allow for comparison at antibody-matched and toxin-matched dose levels. Studies using these models were performed as described in Example 16, except that the H1975 model used n=8 mice per group and the study duration was 41 days, while the HT29 model used n=8 mice per group and the study duration was 32 days. Average data were plotted only if greater than or equal to 80% of the mice remained on study at that time point.

[0873] Table 17.1: Test articles and doses

[0874]

[0875]

[0876] result

[0877] Results of the cMet high H1975 CDX lung cancer model Fig. 9A and 9B For the H1975 model, all DAR 1 Drug-Linker 001, DAR 2 Drug-Linker 001 and Drug-Linker 002, DAR 3 Drug-Linker 001 and DAR 4 Drug-Linker 001 ADCs significantly inhibited tumor growth compared to vehicle at toxin-matched antibody doses of 24, 12, 8 and 6 mg / kg, respectively ( Fig. 9A All DAR 2 Drug-Linker 001 and Drug-Linker 002, DAR 3 Drug-Linker 001 and DAR 4 Drug-Linker 001 ADCs also significantly inhibited tumor growth compared to vehicle at toxin-matched antibody doses of 2, 1.3 and 1 mg / kg, respectively ( Fig. 9B ). While DAR 2, DAR 3, and DAR 4 drug-linker 001 site-specific ADCs, as well as v17427-drug-linker 001 and v17427-drug-linker 002 ADCs, showed comparable activity at toxin-matched doses, the activity of DAR 1 drug-linker 001 ADC appeared to be lower at toxin-matched doses.

[0878] Fig. 10A and 10B Results from the cMet medium / high HT29 CDX colorectal cancer model are shown. In the HT29 model, all DAR 2 Drug-Linker 001 and Drug-Linker 002, DAR 3 Drug-Linker 001, and DAR 4 Drug-Linker 001 ADCs 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) ( Fig. 10A ). DAR 1 Drug-Linker 001 ADC did not inhibit tumor growth at a toxin-matched dose of 12 mg / kg. All DAR 2 Drug-Linker 001 and Drug-Linker 002, DAR 3 Drug-Linker 001 and DAR 4 Drug-Linker 001 ADCs (except 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 ( Fig. 10B). In contrast, v17427-MCvcPABC-MMAE DAR 4 did not significantly inhibit tumor growth at the 1.5 mg / kg dose tested. When doses were matched to toxins, there was a trend of positive correlation between DAR and antitumor activity. The activity of the site-specific cysteine-conjugated v29001-Drug-Linker001 DAR 2 ADC was comparable to that of the random lysine-conjugated v17427-Drug-Linker002 DAR 2.

[0879] Together, these data demonstrate activity of random and site-specific anti-cMet ADCs in an in vivo cMet expression model, as well as a trend toward lower toxin-matched activity for low-DAR ADCs.

[0880] Example 18: In vivo activity of anti-cMET antibody-drug conjugates containing multivalent drug linkers (CDX model)

[0881] The in vivo anti-tumor activity of ADCs comprising anti-cMet antibodies randomly conjugated to drug-linker 002 via lysine or to MCvcPABC-MMAE via cysteine, and ADCs comprising anti-cMet antibodies conjugated to multivalent drug-linker 003 or drug-linker 004 at DAR 4 or 6, respectively, via site-specific cysteine ​​insertion, was evaluated in various CDX models expressing a range of cMet levels. ADCs were administered as specified in Table 18.1 for comparison at toxin-matched dose levels.

[0882] Studies using the H1975, HT29, and H292 models were performed as described in Example 16, with the following differences: the H1975 model used had n=12 mice per group and the study duration was 28 days, while the HT29 model used had n=12 mice per group and the study duration was 25 days. For the cMet medium / high Hs746t gastric cancer model, 5 x 10 6 The cells were implanted into BALB / c nude mice, and when the tumor volume reached approximately 150 mm 3 At 4 pm, mice were assigned to groups (n=7 per group) and treated as specified in Table 18.1 for a study duration of 28 days. For the cMet medium HCT116 colorectal cancer model, 5 x 10 5 The cells were implanted into BALB / c mice, and when the tumor volume reached approximately 150 mm 3 At 18.0, mice were assigned to groups (n=7 per group) and treated as specified in Table 18.1 for a study duration of 27 days. Average data were plotted only if greater than or equal to 80% of the mice continued on study at that time point.

[0883] Table 18.1: Test articles and doses

[0884]

[0885] result

[0886] The results are shown in Fig.11A -E and summarized as follows.

[0887] In the cMet high H1975 lung cancer model, the tumor growth inhibitory activity of DAR 4 ADC was evaluated at 0.4mg / kg and 0.8mg / kg, and the tumor growth inhibitory activity of DAR 6 ADC was evaluated at 0.27mg / kg and 0.53mg / kg. These relatively low doses are to allow comparison at toxin-matched doses. All ADCs resulted in statistically significant inhibition of tumor growth compared to vehicle control (p<0.03), except for v17427-MCvcPABC-MMAE DAR 4 at 0.8mg / kg and lysine-conjugated v17427-drug-linker 002 DAR 6 at 0.27mg / kg ( Fig.11A ). All drug-linker 002, 003 and 004 ADCs showed statistically higher tumor growth inhibition than v17427-MCvcPABC-MMAE at toxin-matched dose levels. At dose levels of 0.8 and 0.53 mg / kg for DAR 4 and DAR 6 ADCs, respectively, there was no difference in activity between any two of the drug-linker 002, 003 and 004 ADCs. At lower dose levels of DAR 4 and DAR 6 ADCs (0.4 and 0.27 mg / kg, respectively), the site-specific v29001-drug-linker 004 DAR 6 ADCs showed superior activity to random lysine-conjugated v17427-drug-linker 002 DAR 4 and DAR 6 ADCs. Compared with random lysine v17427-drug-linker 002 DAR 4 ADCs, the site-specific v29001-drug-linker 003 DAR 4 ADCs showed higher activity. Overall, these data demonstrate that ADCs containing drug-linkers conjugated via inserted cysteines are more active than ADCs containing drug-linkers conjugated via random lysine conjugation, and that all ADCs containing Compound 1 are more active than the comparator MMAE ADC.

[0888] In the cMet medium / high HT29 colorectal cancer model, the tumor growth inhibitory activity of DAR 4 and DAR 6 ADCs was evaluated at 0.8 and 0.53 mg / kg, respectively. These relatively low doses were intended to allow comparisons at toxin-matched dose levels. Site-specific ADCs v29001-drug-linker 004 DAR 6 and v29001-drug-linker 003 DAR 4, as well as random lysine-conjugated v17427-drug-linker 002 DAR 4, all resulted in statistically significant inhibition of tumor growth compared to vehicle control and comparator ADC v17427-MCvcPABC-MMAE DAR 4 ( Fig. 11B ). Random lysine conjugated v17427-Drug-Linker 002 DAR 6 was not different from the vehicle control or the comparator ADC v17427-MCvcPABC-MMAE DAR 4. Overall, these data indicate that ADCs comprising drug-linkers conjugated via inserted cysteine ​​are superior in activity to ADCs comprising drug-linkers conjugated via random lysine conjugation and the comparator MCvcPABC-MMAE ADC.

[0889] In the cMet low H292 lung cancer model, both the site-specific v29001-Drug-Linker 004DAR 6 and the random lysine-conjugated v32634-Drug-Linker 002DAR 6 ADCs showed moderate tumor volume inhibition compared to vehicle control and showed higher activity compared to v17427-MCvcPABC-MMAE DAR 4 ( Fig. 11C ).

[0890] In the cMet medium / high Hs746t gastric cancer model, all ADCs showed inhibition of tumor growth compared to vehicle control ( Fig.11D In this model, the random lysine conjugated ADC v32634-Drug-Linker002DAR 4 showed the greatest activity and outperformed the site-specific v35527 DAR 4 and DAR6 ADCs. v32634-Drug-Linker002DAR4 tended to outperform v17606-MCvcPABC-MMAE DAR 3.

[0891] In the cMet-mediated HCT116 colorectal cancer model, all tested ADCs demonstrated moderate tumor growth inhibition compared to vehicle control ( Fig.11E The site-specific DAR 4 and DAR 6 ADCs and the random DAR 4 ADC were not statistically different, but all showed statistically superior activity to v17606-MCvcPABC-MMAE DAR 3 (p<0.01).

[0892] In summary, these data demonstrate that ADCs containing compound 1 as a payload (i.e., drug-linker 002, 003, and 004) conjugated with DAR4 or DAR 6 by random lysine conjugation or site-specific cysteine ​​conjugation are active against an in vivo xenograft model expressing cMet and are significantly more active than ADCs containing MMAE as a payload (i.e., drug-linker MCvcPABC-MMAE).

[0893] Example 19: In vivo activity of anti-cMET antibody-drug conjugates containing multivalent drug linkers (PDX model)

[0894] In vivo anti-tumor activity of lysine-conjugated DAR 4 ADC (v32634-Drug-Linker 002) and site-specific cysteine-conjugated DAR 6 ADC (v35527-Drug-Linker 004 or v29001-Drug-Linker 004) was compared to v17606-MCvcPABC-MMAE DAR 3 (HIC-purified v36198 ADC or non-HIC-purified v19875 ADC - see Table 5.1) in a panel of lung cancer PDX models expressing a range of cMet levels. The site-specific DAR 6 ADC was dosed at 2 mg / kg toxin-match the lysine-conjugated DAR 4 ADC dose of 3 mg / kg. Variants v32634, v35527, and v29001 all contained an unmodified hinge, while variant v17606 contained a modified hinge (see Table 1.3). The variant v17606-MCvcPABC-MMAE is equivalent to Vidin-Terituzumab (ABBV399).

[0895] Mice (NudeFoxn1nu or BALB / c nude mice) were implanted with tumor fragments from stock mice and were grown when tumors reached approximately 150 to 300 mm 3 Mice were assigned to treatment groups at 1:10 p.m. ADC was administered by a single IV injection as indicated in Table 19.1. The duration of the study was 25 to 42 days. Tumors from untreated mice were resected, formalin-fixed, paraffin-embedded, and analyzed by immunohistochemistry (IHC) for relative cMet levels using antibody clone SP44 (Abcam) according to standard IHC methods.

[0896] Table 19.1: Test articles and doses

[0897]

[0898]

[0899] 1 v19875 ADC (HIC purified)2 v36198 ADC (non-HIC purified)

[0900] result

[0901] Tumor growth rate inhibition value Fig.12 As shown. v32634-Drug-Linker 002DAR 4ADC showed similar or superior activity to v17606-MCvcPABC-MMAE DAR 3ADC in 12 of 16 models. In 6 of 8 evaluated models, the site-specific v29001-Drug-Linker 004DAR 6ADC showed superior activity to both v17606-MCvcPABC-MMAE DAR 3ADC and v32634-Drug-Linker 002DAR 4ADC, and non-inferior activity in the remaining two models.

[0902] In summary, these data demonstrate that ADCs comprising Compound 1 site-specifically conjugated at DAR 6 are significantly more active than ADCs comprising Compound 1 conjugated to lysine when administered at toxin-matched doses. Overall, ADCs comprising Compound 1 conjugated to lysine at DAR 4 and ADCs comprising Compound 1 site-specifically conjugated at DAR 6 have superior activity to the MCvcPABC-MMAE ADC.

[0903] Example 20: Pharmacokinetics of anti-cMET antibody-drug conjugates in Tg32 mice

[0904] The pharmacokinetics of anti-cMet ADCs and corresponding free antibodies were evaluated in humanized FcRn Tg32 mice as described below. Humanized FcRn Tg32 mouse models were selected for this study because they are good predictors of drug pharmacokinetics in humans. The ADCs and antibodies evaluated were: v29001 (HetFc, unmodified hinge, two cysteine ​​insertions), v17427 (HetFc, unmodified hinge), v29001-Drug-Linker003DAR 4, v29001-Drug-Linker004DAR6, v17427-Drug-Linker002DAR 4, and v17427-Drug-Linker002DAR 6.

[0905] All test articles were administered to hFcRn Tg32 mice (The Jackson Laboratory, Sacramento, CA; stock number 014565) at 5 mg / kg by intravenous injection. For each test article, blood was collected from n=4 animals by retro-orbital or peripheral bleeding at 1, 4 and 8 hours and 1, 3, 7, 10, 14 and 21 days after dosing. The blood was processed into serum and stored frozen at -80°C in 96-well storage plates before pharmacokinetic analysis.

[0906] Total concentrated IgG and total ADC concentrations of the test articles in mouse serum were measured by sandwich ELISA using anti-human IgG1 Fc capture antibody (Jackson ImmunoResearch Labs, West Grove, PA; catalog number 709-005-098) or rabbit antitoxin capture antibody and HRP-conjugated anti-IgG1 Fab detection antibody (Jackson ImmunoResearch Labs; catalog number 109-035-097). TM The absorbance at 450 nm was measured using a BioTek H1 Hybrid Multimode Microplate Reader (BioTek Instruments, Winooski, VT). Pro 7.1 (Molecular Devices, San Jose, CA) was used to analyze the sample data. Phoenix WinNonlin TM Pharmacokinetic parameters were calculated by noncompartmental analysis using Certara software (Certara, Princeton, NJ).

[0907] result

[0908] The results are as follows Fig.13A and 13B As shown in Table 20.1. All mAbs and ADCs showed typical antibody-like extended exposure. Variant v17427 and cysteine ​​insertion variant v29001 showed little difference in PK parameters. ADCs v29001-drug-linker003DAR 4 and v29001-drug-linker004DAR 6 using multivalent drug linkers showed comparable PK to their parent antibody variant v29001.

[0909] Table 20.1: Test Articles and PK Values

[0910]

[0911] Example 21: In vivo stability of anti-cMET antibody-drug conjugates

[0912] The in vivo stability of the four ADCs described in Example 20 (v17427-Drug-Linker002DAR 4, v17427-Drug-Linker002DAR 6, v29001-Drug-Linker003DAR 4, and v29001-Drug-Linker004DAR 6) in Tg32 mice was evaluated using immunoprecipitation / mass spectrometry as described below. Serum samples taken from Tg32 mice as described in Example 20 at different time points in the circulation (1 hour to 10 days) were used. For all groups, serum samples from mice at each time point (1 hour to 21 days after dosing) were tested.

[0913] Briefly, biotinylated anti-human IgG F(ab')2 antibodies were coupled to magnetic beads coated with streptavidin (11 ug antibody per sample) for 30 min at room temperature. After coupling, the beads were incubated with the test samples at room temperature for 1.5 hours to allow for immunocapture. TM -2 magnet (ThermoFisher Scientific Corporation, Waltham, MA) with PBS (pH 7.4) to wash the samples. Dithiothreitol (DTT) (pH 7.4) in PBS was used to reduce the immunocapture samples (v29001-drug-linker 003 and v29001-drug-linker 004 only) for 1 hour at room temperature. The v17427-drug-linker 002 ADC was not reduced. After additional 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 separated ADC samples were then analyzed by mass spectrometry to quantify DAR or drug loading, or kept frozen at -80°C until further analysis.

[0914] For LC-MS analysis, use the same TM Agilent 6545 Quadrupole Time-of-Flight (Q-TOF) TM 1290 Infinity TM II LC system injects the sample into the Agilent TM 8uM 50x2.1mm column with a column temperature of 70°C and a flow rate of 0.3ml / min. The mobile phase consisted of: A: LC-MS grade water containing 0.1% v / v formic acid, 0.025v / v trifluoroacetic acid and 10% v / v isopropanol, and B: acetonitrile containing 0.1% v / v formic acid and 10% v / v isopropanol. Prior to sample injection, the column was pre-equilibrated in 20% mobile phase B. Then, a 20min, 20% to 40% mobile phase B gradient was applied, followed by a 2min, 40% to 90% mobile phase B gradient, and a 2.5min column wash at 99% mobile phase B. The column was re-equilibrated to 10% mobile phase B for 2min between runs. Electrospray ionization (ESI) was performed on a Dual AJS TM ESI source (Agilent Technologies, Santa Clara, CA) was used in positive mode with capillary voltage 5000 V, nozzle voltage 2000 V, fragmentor voltage 170 V, skimmer 65 V, octopole RFPeak 750 V, gas temperature 300° C., gas flow 13 L / min, nebulizer 45 psig, and shielding gas temperature 400° C. Data were acquired at a scan rate of 1 spectrum / sec with an m / z range from 500 to 7000.

[0915] Peak integration, MS deconvolution, and mass assignment were performed in Protein Metrics Biosystems 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). TMv4.0 (Protein Metrics Inc., Cupertino, CA) with an m / z range of 600-6000 (v17427-Drug-Linker002 ADC) or 850-4000 (v29001-Drug-Linker003 and v29001-Drug-Linker004). For simplified 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, which has one 2-acetylamino-2-deoxy-β-D-glucopyranosyl-(1-4)-[α-L-fucopyranosyl-(1-6)] residue derived from the activity of EndoS on N-glycans and pyroglutamate formation if a glutamine residue is present at the N-terminus in the protein sequence. For the complete analysis of v17427-drug-linker 002 ADC, the reference mass was defined as the average mass of the parent antibody v17427, which has two 2-acetylamino-2-deoxy-β-D-glucopyranose-(1-4)-[α-L-fucopyranose-(1-6)] residues, 16 disulfide bonds, and pyroglutamate formation if a glutamine residue is present at the N-terminus in the protein sequence. Drug loading was assessed based on a mass shift equal to the mass of x linkers-drugs relative to the reference mass, and a weighted average DAR was calculated using the deconvoluted MS peak intensity. For the expected drug-linker attached to each variant, see Example 3. Thiosuccinimide ring opening was defined as a deconvoluted MS peak with a mass shift of 18Da relative to the deconvoluted MS peak of drug loading. The mass tolerance for mass assignment was ±10Da. The extent of drug loading and maleimide ring opening % at each time point was plotted using GraphPad Prism software (GraphPad Software, San Diego, CA).

[0916] result

[0917] 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 v17427-Drug-Linker002 ADC, minimal drug-linker decomposition was observed on day 21 (a loss of approximately 582 Da of drug carrier was observed). After 21 days, no linker drug decomposition was observed for v29001-Drug-Linker003 and v29001-Drug-Linker004.

[0918] Table 21.1: DAR Remaining and Sulfosuccinimide Ring Opening %*

[0919]

[0920] *Changes within 21 days

[0921] The disclosures of all patents, patent applications, publications, and database entries mentioned in this specification are hereby expressly incorporated by reference in their entirety to the same extent as if each such individual patent, patent application, publication, and database entry was specifically and individually indicated to be incorporated by reference.

[0922] Modifications of the specific embodiments described herein which are apparent to those skilled in the art are intended to be within the scope of the following claims.

[0923] SEQUENCE LISTING TABLE A: CLONE NUMBER OF VARIANTS

[0924]

[0925] Table B: Cloned sequences

[0926]

[0927]

[0928]

[0929]

[0930]

[0931]

[0932]

[0933]

[0934]

[0935]

[0936]

[0937]

[0938]

[0939]

[0940]

[0941]

[0942]

[0943]

[0944]

[0945]

[0946]

[0947]

[0948]

[0949]

[0950] Table C: Additional sequences of cysteine ​​insertion variants

[0951]

[0952]

Claims

1. An antibody-drug conjugate having formula I: A-(L-(D) n ) p (I) in: 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 a heavy chain CDR sequence (HCDR1, HCDR2, and HCDR3) of the VH domain sequence shown in SEQ ID NO: 1 and a light chain CDR sequence (LCDR1, LCDR2, and LCDR3) of the VL domain sequence shown in SEQ ID NO: 2, and the Ig hinge region comprises an upper hinge sequence having an amino acid sequence of a native IgG1, IgG2, or IgG4 upper hinge sequence; L is a cleavable linker; D is: Where * is the connection point with L, n is between 1 and 4, and p is between 1 and 8.

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

3. The antibody-drug conjugate according to claim 1 or 2, wherein the antibody construct comprises a VH domain sequence having an amino acid sequence as shown in SEQ ID NO: 1 and a VL domain sequence having an amino acid sequence as shown in SEQ ID NO:

2. 4 . The antibody-drug conjugate according to any one of claims 1 to 3 , wherein the Ig hinge region comprises an upper hinge sequence having an amino acid sequence of a native IgG1 upper hinge sequence.

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

25.

6. The antibody-drug conjugate according to any one of claims 1 to 5, wherein the antibody construct further comprises a scaffold, wherein the scaffold is based on an immunoglobulin Fc region. The antibody-drug conjugate of claim 6 , wherein the immunoglobulin Fc region is an IgG1 Fc region.

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

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

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

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

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

13. The antibody-drug conjugate of claim 12, wherein each cysteine ​​residue is a native cysteine ​​residue. 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-natural 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. The antibody-drug conjugate of claim 15, wherein each non-native cysteine ​​residue is a cysteine ​​insertion mutation independently selected from: (a) a cysteine ​​residue inserted between positions 40 and 41 in said 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 said VH domain; (d) a cysteine ​​residue inserted between positions 237 and 238 in said CH2 domain; and (e) a cysteine ​​residue inserted between positions 299 and 300 in said CH2 domain, The numbering of amino acids in the VL, CL and VH domains is the Kabat numbering, and the numbering of amino acids in the CH2 domain is the EU numbering.

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

21. The antibody-drug conjugate according to any one of claims 1 to 20, wherein LD has one of the following structures: a) Formula IV in: Z' is a linking group that connects the linker to the target group on the antibody construct A; Str is an extender; AA1 and AA2 are each independently an amino acid, wherein AA1-[AA2] m Formation of protease cleavage sites; X is a self-decomposing group; s is 0 or 1; m is 1, 2, or 3; o is 0, 1, or 2; and # is the connection point with the antibody construct A, or b) Formula XII in: Z' is a linking group that connects the linker to the target group on the antibody construct A; Str 1 and Str 2 each independently an extender; BU is a branch unit; AA1 and AA2 are each independently an amino acid, wherein AA1-[AA2] m Formation of protease cleavage sites; X is a self-decomposing 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, and # is the connection point with the antibody construct A.

22. The antibody-drug conjugate of claim 21, wherein LD has structure IV, and wherein: Z' is a carbonyl group (-C(O)-) or wherein # is the point of attachment to the anti-cMet antibody construct A, and * is the point of attachment to the rest of the linker.

23. The antibody-drug conjugate of claim 21 or 22, wherein LD has structure IV, and wherein: s is 1, and Str is in $ is the connection point with Z', * is the point of attachment to the rest of the linker, p is an integer between 2 and 6, and q is an integer between 2 and 8.

24. The antibody-drug conjugate of any one of claims 21 to 23, wherein LD has structure IV, and wherein: m is 1 and AA1-[AA2] m is a dipeptide selected from the group consisting of Val-Lys, Ala-Lys, Phe-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit and Trp-Cit, and o is 0.

25. The antibody-drug conjugate of claim 21, wherein LD has structure XII, and wherein: Z' is a carbonyl group (-C(O)-) or wherein # is the point of attachment to the anti-cMet antibody construct A, and * is the point of attachment to the rest of the linker.

26. The antibody-drug conjugate of claim 21 or 25, wherein LD has structure XII, and wherein: s is 1; s' is 1; Str 1 yes in $ is the connection point with Z', * is the point of attachment to the rest of the linker, p is an integer between 2 and 6, and q is an integer between 2 and 8, and Str 2 yes in $ It is the connection point with BU. * is the point of attachment to the rest of the linker, p is an integer between 2 and 6, and q is an integer between 2 and 8.

27. The antibody-drug conjugate of any one of claims 21, 25 and 26, wherein LD has structure XII, and wherein: m is 1 and AA1-[AA2] m is a dipeptide selected from the group consisting of Val-Lys, Ala-Lys, Phe-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit and Trp-Cit, and o is 0.

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

29. The antibody-drug conjugate according to claim 1, having one of the following structures: ADC 001 ADC 002 ADC 003 ADC 004 ADC 005 or ADC 006 30. The antibody-drug conjugate of claim 1, having the following structure: ADC 002 wherein A is the antibody construct that specifically binds to c-Met, and p is 6.

31. The antibody-drug conjugate according to claim 1, having the following structure: ADC 004 wherein A is the antibody construct that specifically binds to c-Met, and p is 2.

32. An antibody construct comprising: 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, wherein the antigen binding domain comprises the heavy chain CDR sequence (HCDR1, HCDR2 and HCDR3) of the VH domain sequence shown in SEQ ID NO: 1, and the light chain CDR sequence (LCDR1, LCDR2 and LCDR3) of the VL domain sequence shown in SEQ ID NO: 2, and Wherein the antibody construct comprises one or more cysteine ​​insertion mutations independently selected from: (a) a cysteine ​​residue inserted between positions 40 and 41 in said 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 said 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, The numbering of amino acids in the VL, CL and VH domains is the Kabat numbering, and the numbering of amino acids in the CH2 domain is the EU numbering.

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 an amino acid sequence of a native IgG1, IgG2 or IgG4 upper hinge sequence.

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

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

25.

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

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

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 that promote the formation of the heterodimeric Fc rather than the formation of a homodimeric Fc.

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

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

41. The antibody construct according to any one of claims 32 to 40, wherein the antibody construct comprises a combination of cysteine ​​insertions comprising: (a) a cysteine ​​residue 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 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 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 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 a cysteine ​​residue inserted between positions 40 and 41 in the VL domain.

42. The antibody construct of claim 40, wherein the antibody construct comprises: (i) a cysteine ​​residue inserted between positions 299 and 300 in a CH2 domain sequence; (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 a CH2 domain sequence; (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 ​​insertion 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 ​​insertion between positions 237 and 238 in one CH2 domain sequence.

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

44. The antibody construct of any one of claims 32 to 40, wherein the antibody construct comprises a VH domain sequence having an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 59 and a VL domain sequence having an amino acid sequence as shown in SEQ ID NO: 2 or SEQ ID NO:

56.

45. The antibody construct of any one of claims 32 to 40 and 44, wherein the anti-cMet antibody construct comprises a first heavy chain and a second heavy chain, each of the first heavy chain and the second heavy chain comprising a CH2 domain, and wherein one or both of the CH2 domains comprise an amino acid sequence selected from the sequence shown in SEQ ID NO: 76, 77 and 78.

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

79.

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

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

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

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

51. The antibody-drug conjugate of claim 49 or 50, wherein LD has one of the following structures: a) Formula IV in: Z' is a linking group that connects the linker to the target group on the antibody construct A; Str is an extender; AA1 and AA2 are each independently an amino acid, wherein AA1-[AA2] m Formation of protease cleavage sites; X is a self-decomposing group; s is 0 or 1; m is 1, 2, or 3; o is 0, 1, or 2; and # is the connection point with the antibody construct A, or b) Formula XII in: Z' is a linking group that connects the linker to the target group on the antibody construct A; Str 1 and Str 2 each independently an extender; BU is a branch unit; AA1 and AA2 are each independently an amino acid, wherein AA1-[AA2] m Formation of protease cleavage sites; X is a self-decomposing 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, and # is the connection point with the antibody construct A.

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

53. The antibody-drug conjugate of claim 51 or 52, wherein LD has structure IV, and wherein: s is 1, and Str is in $ is the connection point with Z', * is the point of attachment to the rest of the linker, p is an integer between 2 and 6, and q is an integer between 2 and 8.

54. The antibody-drug conjugate of any one of claims 51 to 53, wherein LD has structure IV, and wherein: m is 1 and AA1-[AA2] m is a dipeptide selected from the group consisting of Val-Lys, Ala-Lys, Phe-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit and Trp-Cit, and o is 0.

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

56. The antibody-drug conjugate of claim 51 or 55, wherein LD has structure XII, and wherein: s is 1; s' is 1; Str 1 yes in $ is the connection point with Z', * is the point of attachment to the rest of the linker, p is an integer between 2 and 6, and q is an integer between 2 and 8, and Str 2 yes in $ It is the connection point with BU. * is the point of attachment to the rest of the linker, p is an integer between 2 and 6, and q is an integer between 2 and 8.

57. The antibody-drug conjugate of any one of claims 51, 55 and 56, wherein LD has structure XII, and wherein: m is 1 and AA1-[AA2] m is a dipeptide selected from the group consisting of Val-Lys, Ala-Lys, Phe-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit and Trp-Cit, and o is 0.

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

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

60. The antibody-drug conjugate of claim 49, having one of the following structures: ADC 001 ADC 003 ADC 004 61. The antibody-drug conjugate of claim 49, having the following structure: ADC 004 wherein A is an antibody construct according to claim 43 and p is 2.

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

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

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

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

68. A vector or a vector set comprising the polynucleotide or the polynucleotide set according to claim 67.

69. A host cell comprising the vector or vector set according to claim 68.

70. A multivalent drug-linker selected from the group consisting of: Drug-Connector 003 Drug-Connector 004 Drug-Connector 005 Drug-Connector 006 Drug-Connector 007 and drugs-connector 008

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