Camptothecin analogs conjugated to glutamine residues in proteins and uses thereof

Abstract

To provide: protein-drug conjugates (e.g., antibody-drug conjugates), pharmaceutical compositions, and methods of treating disease therewith; and specific and efficient methods for producing the protein-drug conjugates.SOLUTION: Described herein are, e.g., protein-drug conjugates and compositions thereof that are useful, for example, for target-specific delivery of therapeutic moieties, e.g., camptothecin analogs and / or derivatives. In certain embodiments, provided are specific and efficient methods for producing protein-drug constructs (e.g., antibody-drug conjugates) utilizing a combination of transglutaminase and 1,3-cycloaddition techniques. Camptothecin analogs, antibody-drug conjugates, and compositions which comprise glutaminyl-modified antibodies and camptothecin analog payloads are provided.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 051,172, filed July 13, 2020, and U.S. Provisional Patent Application No. 63 / 154,531, filed February 26, 2021, the contents of which are incorporated herein by reference in their entireties.

[0002] Field of the Disclosure The present disclosure relates to protein drug conjugates (e.g., antibody drug conjugates), pharmaceutical compositions, and methods for treating diseases using the same. Also provided are specific and efficient methods for producing protein drug conjugates using a combination of transglutaminase and 1,3-cycloaddition techniques. More specifically, the present disclosure relates to protein drug conjugates (e.g., antibody drug conjugates) containing camptothecin analogs and derivatives.

[0003] Sequence Listing An official copy of the Sequence Listing was submitted contemporaneously with the present specification as a paper copy of the ASCII Sequence Listing with filename 250298_000244_SL.txt, approximately 996 kilobytes in size, and created on July 12, 2021. The Sequence Listing contained in this paper copy of the ASCII document is a part of the present specification and is incorporated herein by reference in its entirety. [Background technology]

[0004] Proliferative diseases are characterized by uncontrolled growth and the spread of abnormal cells. Uncontrolled spread can lead to death. Abnormal proliferation, such as cancer, is caused by both external factors (e.g., tobacco, chemicals, radiation, infectious organisms) and internal factors (genetic mutations, immune system conditions, mutations resulting from metabolism). These causative factors may act together or sequentially to initiate or promote abnormal proliferation. Cancer is treated with surgery, radiation, chemotherapy, hormones, and immunotherapy. However, more effective antiproliferative drugs are needed.

[0005] An ideal antiproliferative therapy would enable the delivery of highly cytotoxic drugs that target tumor cells while leaving normal cells unaffected. Conventional chemotherapy treatments are limited by toxic side effects resulting from the drug's effect on non-cancerous cells. Various approaches to targeted drug delivery have been attempted, including the use of conjugates of tumor-targeting probes (e.g., antibodies or growth factors) with toxins, such as Pseudomonas or Diphtheria toxin, to block protein and cellular synthesis. However, side effects include immune system reactions due to the non-human components of the conjugates. Furthermore, the half-life of drug conjugates is limited due to excretion from the circulation via renal filtration, schematic degradation, uptake by the reticuloendothelial system (RES), and accumulation in non-target organs and tissues.

[0006] Other approaches use passive drug carriers, such as polymers, liposomes, and polymeric micelles, to take advantage of the increased permeability of the vascular endothelium in tumor tissue. Macromolecular drugs and macromolecules accumulate within solid tumors through enhanced permeability and retention mechanisms. However, obstacles to using such targeted delivery include the rapid clearance of foreign substances from the blood and technical hurdles in obtaining a drug delivery system that is highly standardized, pharmaceutically acceptable, and has the specificity and selectivity required for tumor cell binding.

[0007] Protein conjugates, such as antibody conjugates, utilize the selective binding of a binding agent to deliver a payload to a target within a tissue of interest, which can be a therapeutic moiety capable of taking action at the target.

[0008] There are several techniques available for conjugating linkers and payloads to antibodies.Many conjugates are prepared by non-selective covalent bonding to cysteine ​​or lysine residues in antibodies.This non-selective technique can result in heterogeneous mixtures of products with different conjugation sites and different numbers of conjugations per antibody.Therefore, the art requires methods and techniques that provide site-selective antibody conjugation.

[0009] There is a further need in the art for safe and effective anti-tumor targeting agents capable of binding to a variety of antigens that provide improved treatment of diseases such as cancer, for use in monotherapy and combination therapy. In certain embodiments, the present disclosure meets this need and provides other advantages as well.

[0010] The foregoing discussion is presented merely to provide a better understanding of the nature of the problem facing the art, but is not an admission of prior art, nor should the incorporation of any reference herein be construed as an admission that such reference constitutes "prior art" to the present application. Summary of the Invention

[0011] Various non-limiting aspects and embodiments of the disclosure are described below.

[0012] In one aspect, the present disclosure provides a compound of formula (A): BA-(Gln-NH-L1-B-(-L2-(-M-Dxd) m ) k ) n (A) wherein: BA is an antibody or antigen-binding fragment thereof; Gln is a glutamine residue, L1 is absent or is a first linker, B is a branching unit containing at least one addition of a group B′ and a group B″, and at least one of the groups B′ and B″ is selected from the group consisting of —N3 and

[0013] [ka] and the other of group B′ and group B″ is selected from

[0014] [ka] wherein Q is C or N; L2 is a second linker covalently attached to the branching unit B via at least one group B″; M is absent or has the structure:

[0015] [ka] wherein R, R', and R'' are independently at each occurrence hydrogen or C1-C4 alkyl, or R' and R'' together form a 5- or 6-membered ring; Dxd is the formula (P):

[0016] [ka] an antitumor drug having a structure according to k is an integer from 1 to 12, m is an integer from 1 to 30; n is an integer from 1 to 30. A compound is provided.

[0017] In another aspect, the present disclosure provides a compound of formula (I): BA-(Gln-NH-L1-B-(-L2-M-Dxd) k ) n (I) wherein: BA is an antibody or antigen-binding fragment thereof; Gln is a glutamine residue, L1 is absent or is a first linker, B is a branching unit containing at least one addition of group B', group B' being -N3,

[0018] [ka] wherein Q is C or N; L2 is a second linker covalently attached to the branching unit B via at least one group B″, wherein the groups B′ and B″ form at least one adduct; M is absent or has the structure:

[0019] [ka] wherein R, R', and R'' are independently at each occurrence hydrogen or C1-C4 alkyl, or R' and R'' together form a 5- or 6-membered ring; Dxd is the formula (P):

[0020] [ka] an antitumor drug having a structure according to k is an integer from 1 to 12, n is an integer from 1 to 30. A compound is provided.

[0021] In one embodiment, the BA is an antibody or an antigen-binding fragment thereof.

[0022] In one embodiment, the BA is an anti-HER2 antibody, an anti-HER2 / HER2 bispecific antibody, an anti-STEAP2 antibody, an anti-MET antibody, an anti-MET / MET bispecific antibody, an anti-EGFRVIII antibody, an anti-MUC16 antibody, an anti-PRLR antibody, an anti-PSMA antibody, an anti-FGFR2 antibody, an anti-FOLR1 antibody, or an antigen-binding fragment thereof.

[0023] In one embodiment, the BA is an anti-STEAP2 antibody comprising a HCVR / LCVR amino acid sequence pair specified in Table 1.

[0024] In one embodiment, the BA is an anti-HER2 / HER2 bispecific antibody. In one embodiment, the BA binds two distinct epitopes of the HER2 protein.

[0025] In one embodiment, the anti-HER2 / HER2 bispecific antibody a first antigen-binding domain (D1); and a second antigen-binding domain (D2); Including, D1 specifically binds the first epitope of human HER2; D2 specifically binds a second epitope on human HER2.

[0026] In one embodiment, D1 and D2 do not compete with each other for binding to human HER2.

[0027] In one embodiment, the BA is an anti-MET / MET bispecific antibody. In one embodiment, the BA binds two distinct epitopes of the MET protein.

[0028] In one embodiment, the anti-MET / MET bispecific antibody a first antigen-binding domain (D1); and a second antigen-binding domain (D2); Including, D1 specifically binds the first epitope of human MET, D2 specifically binds a second epitope on human MET.

[0029] In one embodiment, D1 and D2 do not compete with each other for binding to human MET.

[0030] In one embodiment, the BA is an anti-MET / MET bispecific antibody comprising a HCVR / LCVR amino acid sequence pair specified in Table 3.

[0031] In one embodiment, the BA is an anti-MET / MET bispecific antibody comprising a D1 antigen-binding domain and a D2 antigen-binding domain, wherein the D1 antigen-binding domain comprises the HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 2012 / 2092, or a set of heavy and light chain CDRs (HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) comprising SEQ ID NOs: 2014-2016-2018-2094-2096-2098, and the D2 antigen-binding domain comprises the HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 2036 / 2092, or a set of heavy and light chain CDRs (HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) comprising SEQ ID NOs: 2038-2040-2042-2094-2096-2098.

[0032] In one embodiment, the BA is the anti-MET / MET bispecific antibody H4H14639D, which comprises D1 from H4H13306P2 and D2 from H4H13312P2.

[0033] In one embodiment, the anti-STEAP2 comprises a HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 58, 74 / 58, 82 / 58, 90 / 58, 98 / 58, 106 / 114, 122 / 130, 138 / 146, 154 / 162, 170 / 178, 186 / 194, 202 / 210, 218 / 226, 234 / 242, 250 / 258, 266 / 274, 282 / 290, 298 / 306, 314 / 322, 330 / 338, 346 / 354, 362 / 370, and 378 / 386.

[0034] In one embodiment, the glutamine residue Gln is naturally present in the CH2 or CH3 domain of the BA.

[0035] In one embodiment, a glutamine residue, Gln, is introduced into the BA by modifying one or more amino acids.

[0036] In one embodiment, Gln is Q295 or N297Q.

[0037] In one embodiment, the BA targets a cancer selected from the group consisting of breast cancer, ovarian cancer, prostate cancer, lung cancer, liver cancer, or brain cancer.

[0038] In one embodiment, L1 is C 1-6 Alkyl, phenyl, -NH-, -C(O)-, -(CH2) u -NH-C(O)-, -(CH2) u -C(O)-NH-, -(CH2-CH2-O) v -, -(CH2) u -(O-CH2-CH2) v -C(O)-NH-, a peptide unit containing 2 to 4 amino acids, or a combination thereof, each of which may be optionally substituted by one or more of -S-, -S(O2)-, -C(O)-, -C(O2)-, and CO2H, and the subscripts u and v are independently integers from 1 to 8.

[0039] In one embodiment, L1 is

[0040] [ka] and combinations thereof, wherein R A is a group including alkyne, azide, tetrazine, trans-cyclooctene, maleimide, amine, ketone, aldehyde, carboxylic acid, ester, thiol, sulfonic acid, tosylate, halide, silane, cyano group, carbohydrate group, biotin group, and lipid residue, and the subscripts x, n, p, and q are independently integers from 0 to 12.

[0041] In one embodiment, B contains one appendage of group B'.

[0042] In one embodiment, -NH-L1-B' is

[0043] [ka] is selected from the group consisting of

[0044] [ka] is the amino point of attachment of BA to the glutamine residue.

[0045] In one embodiment, group B' is an azide (-N3) and the adduct of group B' comprises a triazole.

[0046] In one embodiment, B comprises two appendages of group B'.

[0047] In one embodiment, B contains three appendages of group B'.

[0048] In one embodiment, B comprises at least four appendages of group B'.

[0049] In one embodiment, B is

[0050] [ka] wherein (B') comprises the point of attachment of group B'.

[0051] In one embodiment, B is

[0052] [ka] is selected from the group consisting of:

[0053] In one embodiment, -NH-L1-B is

[0054] [ka] is selected from the group consisting of

[0055] [ka] is the amino point of attachment of BA to the glutamine residue.

[0056] In one embodiment, group B' is an azide (-N3) and the adduct of group B' comprises a triazole.

[0057] In one embodiment, the adduct of group B′ and group B″ is

[0058] [ka] wherein Q is C or N.

[0059] In one embodiment, M is absent.

[0060] In one embodiment, M is

[0061] [ka] wherein R, R', and R'' are hydrogen at each occurrence, i.e., M is

[0062] [ka] is.

[0063] In one embodiment, M is

[0064] [ka] wherein R is hydrogen and R′ and R″ together form a five-membered ring, i.e., M is

[0065] [ka] is.

[0066] In one embodiment, n is 2. In one embodiment, n is 4. In one embodiment, n is 8. In one embodiment, n is 12. In one embodiment, n is 16. In one embodiment, n is 24.

[0067] In one embodiment, L2 is represented by the formula (L2): B''-SP1-B2-(-SP2-AA-SP3) p (L2) wherein: B″ is a group capable of covalently bonding to group B′; SP1 is absent or is the first spacer unit, B2 is absent or a branching unit; SP2 is absent or a second spacer unit, AA is a peptide unit containing zero or two to four amino acids; SP3 is absent or a third spacer unit covalently linked to Dxd; p is an integer from 1 to 12.

[0068] In one embodiment, at least one group B'' is -N3,

[0069] [ka] and combinations thereof.

[0070] In one embodiment, SP1 is absent or

[0071] [ka] is selected from the group consisting of:

[0072] In one embodiment, B2 is absent or

[0073] [ka] is selected from the group consisting of:

[0074] In one embodiment, SP2 is absent or 1-6 Alkyl, -(CH2-CH2-O) v -, -NH-, -C(O)-, -NH-C(O)-, -NH-(CH2) u -, -NH-(CH2) u -C(O)-, -NH-(CH2-CH2-O) v -, -NH-(CH2-CH2-O) v -C(O)-, -NH-(CH2-CH2-O) v -(CH2) u -, -NH-(CH2-CH2-O) v -(CH2) u -C(O)-, -(CH2) u -NH-C(O)-, -NH-(CH2) u -NH-C(O)-, -NH-(CH2) u -C(O)-NH-, or combinations thereof, and the subscripts u and v are independently integers from 1 to 8.

[0075] In one embodiment, AA is a peptide unit comprising two to four amino acids selected from glycine, valine, phenylalanine, proline, glutamic acid, lysine, phenylalanine, and citrulline, and combinations thereof.

[0076] In one embodiment, AA is valine-citrulline, valine-alanine, or phenylalanine-lysine.

[0077] In one embodiment, AA is selected from the group consisting of glycine-glycine-glycine (GGG), glycine-glycine-glycine-glycine (GGGG (SEQ ID NO: 2113)), glycine-glycine-phenylalanine (GGF), glycine-glycine-phenylalanine-glycine (GGFG (SEQ ID NO: 2114)), L-glutamic acid-valine-citrulline (LEVC), and D-glutamic acid-valine-citrulline ( D EVC).

[0078] In one embodiment, SP3 is absent or

[0079] [ka] and combinations thereof, wherein R c is not present in each occurrence independently, or

[0080] [ka] is a group selected from

[0081] In one embodiment, M-Dxd is

[0082] [ka] wherein R is hydrogen or C1-C4 alkyl;

[0083] [ka] represents the point of attachment to L2.

[0084] In one embodiment, the compound is

[0085] [ka] It has the following structure.

[0086] In one embodiment, the compound is

[0087] [ka] It has the following structure.

[0088] In one embodiment, the compound is

[0089] [ka] It has the following structure.

[0090] In another aspect, the present disclosure provides a compound of formula (I): BA-(Gln-NH-L1-B-(-L2-M-Dxd) k ) n (I) wherein: BA is an antibody or antigen-binding fragment thereof; Gln is a glutamine residue, L1 is absent or is a first linker, B is

[0091] [ka] is a branched unit comprising M is absent or has the structure:

[0092] [ka] wherein R, R', and R'' are independently at each occurrence hydrogen or C1-C4 alkyl, or R' and R'' together form a 5- or 6-membered ring; Dxd is the formula (P):

[0093] [ka] an antitumor drug having a structure according to k is an integer from 1 to 12, n is an integer from 1 to 30. A compound is provided.

[0094] In another aspect, the present disclosure provides compounds of formula (L2-P), (L2'-P), or (L2''-P): B''-SP1-B2-(-SP2-AA-SP3-M-Dxd) p (L2-P), H2N-SP1-B2-(-SP2-AA-SP3-M-Dxd) p (L2'-P), Maleimide-N-SP1-B2-(-SP2-AA-SP3-M-Dxd) p (L2''-P) A compound according to the formula: B'' is -N3,

[0095] [ka] is selected from the group consisting of SP1 is either not present or

[0096] [ka] is a first spacer unit selected from the group consisting of: SP2 is not present or 1-6 Alkyl, -(CH2-CH2-O) v -, -NH-, -C(O)-, -NH-C(O)-, -NH-(CH2) u -, -NH-(CH2) u -C(O)-, -NH-(CH2-CH2-O) v , -NH-(CH2-CH2-O) v -C(O)-, -NH-(CH2-CH2-O) v -(CH2) u -, -NH-(CH2-CH2-O) v -(CH2) u-C(O)-, -(CH2) u -NH-C(O)-, -NH-(CH2) u -NH-C(O)-, -NH-(CH2) u a second spacer unit selected from the group consisting of: —C(O)—NH—, —C(O)—NH—, or combinations thereof; and the subscripts u and v are independently integers from 1 to 8; AA is a peptide unit containing zero or two to four amino acids; SP3 is either not present or

[0097] [ka] and a third spacer unit selected from the group consisting of: c is not present in each occurrence independently, or

[0098] [ka] is a group selected from M is absent or

[0099] [ka] wherein R, R', and R'' are independently at each occurrence hydrogen or C1-C4 alkyl, or R' and R'' together form a 5- or 6-membered ring; Dxd is the formula (P):

[0100] [ka] is an antitumor drug having the structure p is an integer from 1 to 12; A compound is provided.

[0101] In one embodiment, AA is a peptide unit comprising two to four amino acids selected from glycine, valine, phenylalanine, proline, glutamic acid, lysine, alanine, and citrulline, and combinations thereof.

[0102] In one embodiment, AA is valine-citrulline, valine-alanine, or phenylalanine-lysine.

[0103] In one embodiment, AA is selected from the group consisting of glycine-glycine-glycine (GGG), glycine-glycine-glycine-glycine (GGGG (SEQ ID NO: 2113)), glycine-glycine-phenylalanine (GGF), glycine-glycine-phenylalanine-glycine (GGFG (SEQ ID NO: 2114)), and glutamic acid-valine-citrulline (EVC).

[0104] In one embodiment, the compound is

[0105] [ka]

[0106] [ka] or a pharmaceutically acceptable salt thereof.

[0107] In one embodiment, the compound is

[0108] [ka] or a pharmaceutically acceptable salt thereof.

[0109] In one embodiment, the compound is

[0110] [ka]

[0111] [ka]

[0112] [ka]

[0113] [ka]

[0114] [ka]

[0115] [ka] and having a structure according to formula (L2-P), (L2'-P), or (L2''-P), selected from the group consisting of:

[0116] In another aspect, the present disclosure provides a compound of formula (II): Ab-(Gln-NH-L1-B-(SP1-B2-(-SP2-AA-SP3-M-Dxd) k ) p ) n (II) An antibody drug conjugate according to the formula: Ab is antibody, Gln is a glutamine residue, L1 is absent or is a first linker, B is a branching unit containing at least one addition of a group B' and a group B''; group B' is -N3,

[0117] [ka] and at least one group B″, B″-SP1-B2-(-SP2-AA-SP3-M-Dxd)p is a compound according to formula (L2-P) according to any of the previous embodiments, wherein the compound of formula (L2-P) is covalently attached to the antibody via the attachment of group B′ and group B″; k is an integer from 1 to 12; p is an integer from 1 to 30; and n is an integer from 1 to 30. Antibody drug conjugates are provided.

[0118] In one embodiment, the present disclosure provides an antibody drug conjugate comprising an antibody and a linker payload, or a pharmaceutically acceptable salt thereof, wherein the linker payload is

[0119] [ka] or a pharmaceutically acceptable salt thereof, During the ceremony,

[0120] [ka] represents the point of attachment to the antibody, either directly or through a second linker.

[0121] In another aspect, the disclosure provides a composition comprising a population of compounds according to any of the preceding embodiments, having a drug-to-antibody ratio (DAR) of from about 0.5 to about 30.0.

[0122] In one embodiment, the composition of the present disclosure has a DAR of about 1.0 to about 2.5. In one embodiment, the composition of the present disclosure has a DAR of about 2.

[0123] In one embodiment, the composition of the present disclosure has a DAR of about 3.0 to about 4.5. In one embodiment, the composition of the present disclosure has a DAR of about 4.

[0124] In one embodiment, the composition of the present disclosure has a DAR of about 6.5 to about 8.5. In one embodiment, the composition of the present disclosure has a DAR of about 8.

[0125] In one embodiment, the composition of the present disclosure has a DAR of about 10 to about 14. In one embodiment, the composition of the present disclosure has a DAR of about 12.

[0126] In one embodiment, the composition of the present disclosure has a DAR of about 14 to about 18. In one embodiment, the composition of the present disclosure has a DAR of about 16.

[0127] In one embodiment, the composition of the present disclosure has a DAR of about 22 to about 24.5. In one embodiment, the composition of the present disclosure has a DAR of about 24.

[0128] In another aspect, the present disclosure provides a compound of formula (PI):

[0129] [ka] or a pharmaceutically acceptable salt thereof, wherein R1, R2, R3, and R4 are independently hydrogen or C1-C4 alkyl, or R2 and R3 form a 5- or 6-membered ring. The present invention provides a compound or a pharmaceutically acceptable salt thereof.

[0130] In one embodiment, the compound is

[0131] [ka] or a pharmaceutically acceptable salt thereof.

[0132] An antibody drug conjugate, or a pharmaceutically acceptable salt thereof, comprising an antibody, a linker, and a payload, wherein the payload is

[0133] [ka] is.

[0134] An antibody drug conjugate, or a pharmaceutically acceptable salt thereof, comprising an antibody, a linker, and a payload, wherein the payload is

[0135] [ka] and

[0136] [ka] represents the point of attachment to the linker.

[0137] In one embodiment, the compound has the formula (P2):

[0138] [ka] or a pharmaceutically acceptable salt thereof, wherein R is hydrogen or C1-C4 alkyl.

[0139] In one embodiment, the compound is

[0140] [ka] or a pharmaceutically acceptable salt thereof.

[0141] In one aspect, the invention provides a pharmaceutical composition comprising a compound according to any of the preceding embodiments and a diluent, carrier, and / or excipient.

[0142] In one aspect, the invention provides a method of treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound according to any of the preceding embodiments, or a composition according to any of the preceding embodiments.

[0143] In one embodiment, the disease is cancer.

[0144] In one embodiment, the cancer is selected from the group consisting of breast cancer, ovarian cancer, prostate cancer, lung cancer, liver cancer, or brain cancer.

[0145] In one embodiment, the disease is HER2+ breast cancer.

[0146] In one aspect, the invention provides a method for selectively delivering a compound according to any one of the preceding embodiments to a cell.

[0147] In one aspect, the invention provides a method of selectively targeting an antigen on the cell surface with a compound according to any one of the preceding embodiments.

[0148] In one embodiment, the cell is a mammalian cell.

[0149] In one embodiment, the cell is a human cell.

[0150] In one embodiment, the cell is a cancer cell.

[0151] In one embodiment, the cancer cells are selected from the group consisting of breast cancer cells, ovarian cancer cells, prostate cancer cells, lung cancer cells, liver cancer cells, or brain cancer cells.

[0152] In one aspect, the present disclosure provides a compound of formula (A): BA-(Gln-NH-L1-B-(-L2-(-M-Dxd) m ) k ) n (A) A method for producing a compound having a structure according to the formula: BA is an antibody or antigen-binding fragment thereof; Gln is a glutamine residue, L1 is the first linker, B is a branching unit containing at least one addition of a group B' and a group B''; L2 is a second linker covalently attached to the branching unit B via at least one group B″; M is absent or

[0153] [ka] wherein R, R', and R'' are independently at each occurrence hydrogen or C1-C4 alkyl, or R' and R'' together form a 5- or 6-membered ring; Dxd is the formula (P):

[0154] [ka] an antitumor drug comprising a structure according to k and m are independently an integer of 1 to 12, and n is an integer of 1 to 30; The above method is (a) contacting a BA containing at least one glutamine residue Gln (BA-Gln-NH2) with a compound L1-B in the presence of a transglutaminase, wherein the branching unit B contains at least one group B'; (b) The product of step (a) is converted into compound L2-(-M-Dxd) m wherein the linker L2 comprises at least one group B″, and one of the groups B′ and B″ is —N3 and

[0155] [ka] and the other of group B′ and group B″ is selected from

[0156] [ka] wherein Q is C or N; (c) isolating the compound of formula (I) produced; The present invention provides a method comprising:

[0157] In one aspect, the present disclosure provides a compound of formula (A): BA-(Gln-NH-L1-B-(-L2-(-M-Dxd) m ) k ) n (A) 1. A method for producing a compound having a structure according to wherein BA is an antibody or antigen-binding fragment thereof, Gln is a glutamine residue, L1 is a first linker as described above, B is a branching unit comprising an appendage of at least one of group B' and group B'' as described above, L2 is a second linker as described above that is covalently bonded to branching unit B via at least one group B'' as described above, and M is absent or

[0158] [ka] wherein R, R′, and R″ are as described above, and Dxd is a group represented by the formula (P):

[0159] [ka] wherein k and m are independently integers of 1 to 12, and n is an integer of 1 to 30; The above method is (a) A compound L1-B in which the branching unit B contains at least one group B' is reacted with a compound L2-(-M-Dxd) m wherein the linker L2 comprises at least one group B″ capable of covalently bonding to group B′, and one of groups B′ and B″ is selected from the group consisting of —N3 and

[0160] [ka] and the other of group B′ and group B″ is selected from

[0161] [ka] wherein Q is C or N, and the contact forms L1-B-(-L2-(-M-Dxd) m ) k and (b) In the presence of transglutaminase, BA containing at least one glutamine residue Gln (BA-Gln-NH) is converted to L1-B-(L2-(-M-Dxd) m ) k contacting the product with (c) isolating the compound of formula (I) produced; The present invention provides a method comprising:

[0162] In one aspect, the present disclosure provides a compound of formula (I): BA-(Gln-NH-L1-B-(-L2-M-Dxd) k ) n (I) A method for producing a compound having a structure according to the formula: BA is an antibody or antigen-binding fragment thereof; Gln is a glutamine residue, L1 is the first linker, B is a branching unit containing at least one addition of a group B' and a group B''; L2 is a second linker covalently attached to the branching unit B via at least one group B″; M is absent or

[0163] [ka] wherein R, R', and R'' are independently at each occurrence hydrogen or C1-C4 alkyl, or R' and R'' together form a 5- or 6-membered ring; Dxd is the formula (P):

[0164] [ka] an antitumor drug comprising a structure according to k is an integer from 1 to 12, and n is an integer from 1 to 30. The above method is (a) contacting a BA containing at least one glutamine residue Gln (BA-Gln-NH2) with a compound L1-B in the presence of a transglutaminase, wherein the branching unit B contains at least one group B'; (b) contacting the product of step (a) with k or more equivalents of compound L2-M-Dxd, wherein linker L2 comprises at least one group B″ capable of covalently bonding to group B′, and one of groups B′ and B″ is selected from the group consisting of —N3 and

[0165] [ka] and the other of group B′ and group B″ is selected from

[0166] [ka] wherein Q is C or N; (c) isolating the compound of formula (I) produced; The present invention provides a method comprising:

[0167] In one aspect, the present disclosure provides a compound of formula (I): BA-(Gln-NH-L1-B-(-L2-M-Dxd) k ) n (I) 1. A method for producing a compound having a structure according to wherein BA is an antibody or antigen-binding fragment thereof, Gln is a glutamine residue, L1 is a first linker as described above, B is a branching unit comprising an appendage of at least one of group B' and group B'' as described above, L2 is a second linker as described above that is covalently bonded to branching unit B via at least one group B'' as described above, and M is absent or

[0168] [ka] wherein R, R′, and R″ are as described above, and Dxd is a group represented by the formula (P):

[0169] [ka] wherein k is an integer of 1 to 12 and n is an integer of 1 to 30; The above method is (a) contacting a compound L1-B, in which the branching unit B comprises at least one group B', with k or more equivalents of a compound L2-M-Dxd, in which the linker L2 comprises at least one group B'', to form L1-B-(-L2-M-Dxd) k is produced, and one of the groups B' and B'' is -N3 and

[0170] [ka] and the other of group B′ and group B″ is selected from

[0171] [ka] wherein Q is C or N; (b) In the presence of transglutaminase, a binder BA containing at least one glutamine residue Gln (BA-Gln-NH) is added to the L1-B-(L2(M-Dxd) k contacting the product with (c) isolating the compound of formula (I) produced; The present invention provides a method comprising:

[0172] In one aspect, the present disclosure provides a compound of formula (III): BA-(Gln-NH-L2'-P) n (III) 1. A method for producing a compound having a structure according to BA is an antibody or an antigen-binding fragment thereof, Gln is a glutamine residue, and L2'-P is H2N-SP1-B2-(SP2-AA-SP3-M-Dxd) as described above. p wherein n is an integer from 1 to 30, and SP1 is absent, or

[0173] [ka] and B2 is absent or a branching unit; SP2 is absent or a C 1-6 Alkyl, -(CH2-CH2-O) v -, -NH-, -C(O)-, -NH-C(O)-, -NH-(CH2) u -, -NH-(CH2) u -C(O)-, -NH-(CH2-CH2-O) v -, -NH-(CH2-CH2-O) v -C(O)-, -NH-(CH2-CH2-O) v -(CH2) u -, -NH-(CH2-CH2-O) v -(CH2) u -C(O)-, -(CH2) u -NH-C(O)-, -NH-(CH2) u -NH-C(O)-, -NH-(CH2) u a second spacer unit selected from the group consisting of -C(O)-NH-, -C(O)-NH-, or a combination thereof; the subscripts u and v are independently integers from 1 to 8; AA is absent or is a peptide unit containing 2 to 4 amino acids; SP3 is absent or is

[0174] [ka] and a third spacer unit selected from the group consisting of: c is not present in each occurrence independently, or

[0175] [ka] and M is absent, or

[0176] [ka] wherein R, R', and R'' are independently at each occurrence hydrogen or C1-C4 alkyl, or R' and R'' together form a 5- or 6-membered ring, and Dxd is a group represented by the formula (P):

[0177] [ka] wherein p is an integer of 1 to 30; The above method is (b) contacting BA containing at least one glutamine residue Gln (BA-Gln-NH2) with L2'-P in the presence of transglutaminase; (c) isolating the compound of formula (III) produced; The present invention provides a method comprising:

[0178] In one embodiment, the glutamine residue Gln is naturally present in the CH2 or CH3 domain of the BA.

[0179] In one embodiment, a glutamine residue, Gln, is introduced into the BA by modifying one or more amino acids.

[0180] In one embodiment, Gln is Q295 or N297Q.

[0181] In one embodiment, the transglutaminase is a microbial transglutaminase (MTG).

[0182] In one embodiment, M is absent or M-Dxd is

[0183] [ka] wherein R is hydrogen or C1-C4 alkyl;

[0184] [ka] represents the point of attachment to L2.

[0185] In one embodiment, the compound L2-Dxd is

[0186] [ka]

[0187] [ka] or a pharmaceutically acceptable salt thereof.

[0188] In one embodiment, the compound L2-Dxd is

[0189] [ka] or a pharmaceutically acceptable salt thereof.

[0190] In one embodiment, the compound L2-Dxd is

[0191] [ka]

[0192] [ka]

[0193] [ka]

[0194] [ka]

[0195] [ka]

[0196] [ka] or a pharmaceutically acceptable salt thereof.

[0197] These and other aspects of the present disclosure will become apparent to those skilled in the art upon reading the following detailed description of the disclosure, including the appended claims. [Brief explanation of the drawings]

[0198] The patent or patent application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0199] [Figure 1] 1 is a schematic demonstrating the two-step site-specific generation of Dxd-ADCs according to embodiments of the present disclosure. The first step is conjugation of one or more first linkers (L1-B') to glutamine residues on the antibody via a transglutaminase (e.g., MTG)-mediated conjugation reaction. The second step is conjugation of antibody L1-B to one or more linker 2-payloads (L2P). [Figure 2]2A and 2B are schematic diagrams demonstrating specific, non-limiting embodiments of the present disclosure. Figure 2A is a schematic diagram of two-step site-specific production of a Dxd-ADC with a glutamine residue at position 295 and a DAR of 2xnxm, according to an embodiment of the present disclosure. Figure 2B is a schematic diagram of two-step site-specific production of a Dxd-ADC with glutamine residues at positions 295 and 297 and a DAR of 4xnxm, according to an embodiment of the present disclosure. [Figure 3A] 1 is a schematic demonstrating the two-step site-specific generation of one specific embodiment of a Dxd-ADC according to the present disclosure. The first step is to conjugate a first linear linker 1 (L1-B') containing one azide moiety (-N3) to glutamine residues at positions 295 and 297 of the antibody via an MTG-mediated conjugation reaction to generate an antibody (Ab-(N3)4) to which four azide-containing linkers are attached. The second step is to attach Ab-(N3)4 to a specific Linker2-payload (L2P) via an azide-cycloalkyne 1,3 cycloaddition reaction to generate a Dxd-ADC with a DAR of 4. [Figure 3B] 3A depicts a schematic diagram of an ADC with a DAR of 2 or 4 and an exemplary aminoazide linker suitable for use in the embodiment of the present disclosure depicted in FIG. 3A. [Figure 4A] 1 is a schematic demonstrating the two-step site-specific generation of one specific embodiment of a Dxd-ADC according to the present disclosure. The first step is to conjugate a first branched linker 1 (L1-B') containing two azide moieties (-N3) to glutamine residues at positions 295 and 297 of the antibody via an MTG-mediated conjugation reaction, generating an antibody (Ab-(N3)8) to which eight azide-containing linkers are attached. The second step is to conjugate Ab-(N3)8 to a specific Linker2-payload (L2P) via an azide-cycloalkyne 1,3 cycloaddition reaction, generating a Dxd-ADC with a DAR of 8. [Figure 4B] 4A depicts a schematic diagram of an ADC and an exemplary branched alkyl azidoamine linker suitable for use in the embodiment of the present disclosure depicted in FIG. 4A. [Figure 5] FIG. 1 is a schematic diagram demonstrating three approaches to generate site-specific ADCs of DAR4-DAR24 on antibody Q295 / 297, according to embodiments of the present disclosure. [Figure 6] FIG. 1 is a schematic diagram demonstrating three approaches to producing site-specific ADCs bearing DAR2-DAR12 on antibody Q295, according to embodiments of the present disclosure. [Figure 7A] FIG. 1 is a schematic demonstrating a two-step approach I to generate an exemplary 8DAR branched linker payload ADC (Ab-AL1-LP39). [Figure 7B] FIG. 1 is a schematic demonstrating the two-step approach II to generate an exemplary 8DAR branched linker payload ADC (Ab-BL1-LP22). [Figure 7C] FIG. 1 is a schematic demonstrating one-step Approach III to generate 4 DAR ADCs with linear linker-P and 8 DAR ADCs with branched linker payloads. [Figure 8A] (SNU16, FGFR2-amplified gastic cancer) Figure 1 shows tumor volume versus days post-treatment for anti-FGFR2b Dxd ADCs (DAR8) of the present disclosure. These ADCs demonstrated significant anti-tumor efficacy against SNU-16 human gastric cancer xenografts. [Figure 8B] 1 shows tumor volume versus days post-treatment (SNU16 tumor-bearing mice) of anti-FGFR2b Dxd ADCs (DAR8) of the present disclosure. These ADCs demonstrated significant anti-tumor efficacy against SNU-16 human gastric cancer xenografts. [Figure 9A] (SNU16, FGFR2-amplified gastric cancer) Figure 1 shows tumor volume versus days post-treatment for anti-FGFR2b Dxd ADCs (DAR4) of the present disclosure. These ADCs demonstrated significant anti-tumor efficacy against SNU-16 human gastric cancer xenografts. [Figure 9B]1 shows tumor volume versus days post-treatment (SNU16 tumor-bearing mice) for anti-FGFR2b Dxd ADCs (DAR4) of the present disclosure. These ADCs demonstrated significant anti-tumor efficacy against SNU-16 human gastric cancer xenografts. [Figure 10] 1 is a diagram of a matrix illustrating the components of 272 exemplary MET×MET bispecific antibodies disclosed herein. Each numbered cell in the matrix identifies a unique bispecific antibody comprising a "D1" antigen-binding domain and a unique bispecific antibody comprising a "D2" antigen-binding domain, where the D1 antigen-binding domain comprises the immunoglobulin variable domain (HCVR / LCVR amino acid sequence pair) or CDR from the corresponding anti-MET antibody listed along the Y-axis, and the D2 antigen-binding domain comprises the immunoglobulin variable domain (HCVR / LCVR amino acid sequence pair) or CDR from the corresponding anti-MET antibody listed along the X-axis. DETAILED DESCRIPTION OF THE INVENTION

[0200] Detailed embodiments of the present disclosure are disclosed herein. However, it should be understood that the disclosed embodiments are merely illustrative of the disclosure, which may be embodied in various forms. In addition, the examples provided in connection with the various embodiments of the present disclosure are each illustrative and not intended to be limiting. Therefore, the specific structural and functional details disclosed herein should not be construed as limiting, but merely as a representative basis for teaching those skilled in the art how to utilize the present disclosure in various ways.

[0201] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0202] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly indicates otherwise. Thus, for example, reference to "a method" includes one or more methods, and / or steps of the kind described herein and / or that will become apparent to those skilled in the art upon reading this disclosure.

[0203] The term "treat" or "treatment" of a condition, disorder, or disease includes (1) preventing, delaying, or reducing the likelihood of the onset and / or appearance of at least one clinical or subclinical symptom of the condition, disorder, or disease developing in a subject who may be afflicted with or prone to the condition, disorder, or disease, but who has not yet experienced or displayed clinical or subclinical symptoms of the condition, disorder, or disease; (2) inhibiting the condition, disorder, or disease, i.e., arresting, reducing, or delaying the progression of the disease or recurrence, or at least one clinical or subclinical symptom thereof; and / or (3) palliating the disease, i.e., causing regression of the condition, disorder, or disease, or at least one clinical or subclinical symptom thereof. The benefit to the subject to be treated is statistically significant or at least perceptible to the patient or physician. In some embodiments, treatment includes methods in which cells are ablated in a manner that indirectly affects the disease. In certain embodiments, treatment involves depleting immune cells as a hematopoietic conditioning regimen prior to treatment.

[0204] "Subject," "patient," "individual," or "animal," as used herein, refers to humans with a disease, veterinary animals (e.g., cats, dogs, cows, horses, sheep, pigs, etc.), and experimental animal models (e.g., mice, rats). In a preferred embodiment, the subject is a human.

[0205] As used herein, the term "effective" as applied to dose or amount refers to an amount of a compound or pharmaceutical composition that is sufficient to produce the desired activity upon administration to a subject in need thereof. It should be noted that when administered in combination with active ingredients, the effective amount of the combination may or may not include the amount of each ingredient that was effective when administered individually. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease being treated, the particular drug utilized, the mode of administration, etc.

[0206] The phrase "pharmaceutically acceptable salt" when used in reference to the compositions of the present disclosure refers to any salt suitable for administration to a patient. Suitable salts include, but are not limited to, those disclosed in "Pharmaceutical Salts" by Berge et al., J.Pharm.Sci., 1977, 66:1, which is incorporated herein by reference. Examples of salts include, but are not limited to, acid-derived salts, base-derived salts, organic salts, inorganic salts, amine salts, and alkali or alkaline earth metal salts, including, but not limited to, calcium salts, magnesium salts, potassium salts, sodium salts, hydrochloride, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, etc. In some examples, the payloads described herein (e.g., rifamycin analogs described herein) comprise a tertiary amine, and the nitrogen atom in the tertiary amine is the atom that connects the payload to the linker or linker-spacer. In such cases, the binding of the payload to the tertiary amine results in a quaternary amine in the linker-payload molecule. The positive charge on the quaternary amine can be balanced by a counterion (e.g., chloro, bromo, iodo, or any other suitably charged moiety, such as those described herein).

[0207] Ranges can be expressed herein as from "about" or "approximately" one particular value to "about" or "approximately" another particular value. When such a range is expressed, another embodiment includes from the one particular value to the other particular value.

[0208] "Comprising," "containing," or "including" means that at least the specified compounds, elements, particles, or method steps are present in a composition, article, or method, but does not exclude other compounds, materials, particles, or method steps, even if such other compounds, materials, particles, or method steps have the same function as the specified one.

[0209] The compounds of the present disclosure include those described throughout the present specification and are further exemplified by the classes, subclasses, and species disclosed herein. As used herein, the following definitions shall apply unless otherwise specified. For the purposes of this disclosure, chemical elements shall be identified according to the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Edition. In addition, the general principles of organic chemistry are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito: 1999, and March's Advanced Organic Chemistry, 5th Edition, Ed.: Smith, M.B. and March, J., John Wiley & Sons, New York: 2001, the entire contents of which are incorporated herein by reference.

[0210] As used herein, the term "alkyl" is given its ordinary meaning in the art and may include saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups. In certain embodiments, the backbone of a straight-chain or branched-chain alkyl group contains from about 1 to 20 carbon atoms (e.g., C1-C for a straight chain). 20 , C2-C for branched chains 20 ), and alternatively about 1 to 10 carbon atoms, or about 1 to 6 carbon atoms. In some embodiments, the ring structure of a cycloalkyl ring, where the ring is monocyclic or bicyclic, has about 3 to 10 carbon atoms, and alternatively about 5, 6, or 7 carbons. In some embodiments, an alkyl group can be a lower alkyl group, where the lower alkyl group contains 1 to 4 carbon atoms (e.g., C1-C4 for a straight chain lower alkyl).

[0211] As used herein, the term "alkenyl" refers to an alkyl group, as defined herein, having one or more double bonds.

[0212] As used herein, the term "alkynyl" refers to an alkyl group, as defined herein, having one or more triple bonds.

[0213] The term "heteroatom" means one or more of oxygen, sulfur, nitrogen, phosphorus, or silicon (including any oxidized form of nitrogen, sulfur, phosphorus, or silicon, the quaternized form of any basic nitrogen, or a substitutable nitrogen of a heterocycle).

[0214] The term "halogen" means F, Cl, Br, or I. The term "halide" refers to a halogen radical or substituent, i.e., -F, -Cl, -Br, or -I.

[0215] The term "adduct", e.g., "adduct of group B'" in the present disclosure, includes any moiety that comprises the product of an addition reaction, e.g., an addition reaction of group B', that is unrelated to the synthetic steps taken to produce any moiety that comprises the product of the addition reaction.

[0216] The term "covalent attachment" refers to the formation of a covalent bond, i.e., a chemical bond involving the sharing of one or more electron pairs between two atoms. Covalent bond formation can involve a variety of interactions, including, but not limited to, σ-bonds, π-bonds, metal-metal bonds, agostic interactions, bent bonds, and three-center, two-electron bonds. When a first group is said to be "capable of covalent attachment" to a second group, this means that the first group can form a covalent bond with the second group directly or indirectly, for example, through the use of a catalyst or under certain reaction conditions. Non-limiting examples of groups capable of covalent attachment to each other include, for example, amines and carboxylic acids (forming an amide bond), dienes and dienophiles (via a Diels-Alder reaction), and azides and alkynes (forming a triazole via a 1,3-cycloaddition reaction).

[0217] As used herein, compounds of the present disclosure may contain "optionally substituted" moieties. In general, the term "optionally," whether preceded or followed, means that one or more hydrogens of the specified moiety are replaced with a suitable substituent. Unless otherwise specified, an "optionally substituted" group may have a suitable substituent at each substitutable position of the group, and when more than one position in a given structure is substituted with more than one substituent selected from a specified group, the substituents may be the same or different at all positions. Combinations of substituents envisioned by the present disclosure are preferably those that result in the formation of stable or chemically feasible compounds. The term "stable," as used herein, refers to a compound that is not substantially modified when exposed to conditions that allow for production, detection, and in certain embodiments, recovery, purification, and use for one or more of the purposes disclosed herein.

[0218] Unless otherwise specified, structures depicted herein are also intended to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) forms of the structure, such as the R and S configurations for each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers. Thus, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present compounds are within the scope of the disclosure.

[0219] Unless otherwise stated, all tautomeric forms of the compounds of the present disclosure are within the scope of the present disclosure.

[0220] Additionally, unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms, for example, the replacement of a hydrogen with deuterium or tritium, or 11 C. 13 C, or 14 Compounds having the present structure except for the replacement of a carbon with a C-enriched carbon are within the scope of this disclosure.

[0221] It should also be understood that a reference to one or more method steps does not exclude the presence of additional method steps or intervening method steps between the explicitly identified steps. Similarly, it should also be understood that a reference to one or more components in a device or system does not exclude the presence of additional components or intervening components between the explicitly identified components.

[0222] Unless otherwise specified, all crystalline forms of the compounds of the present disclosure and their salts are also within the scope of the present disclosure.The compounds of the present disclosure may be isolated in various amorphous and crystalline forms, including, but not limited to, anhydrous, hydrated, unsolvated, or solvated forms.Examples of hydrates include hemihydrates, monohydrates, dihydrates, etc.In some embodiments, the compounds of the present disclosure are anhydrous and unsolvated."Anhydrous" means that the crystalline form of the compound does not substantially contain bound water in the crystal lattice structure, i.e., the compound does not form a crystalline hydrate.

[0223] As used herein, "crystalline form" is intended to refer to a particular lattice arrangement of a crystalline substance. Different crystalline forms of the same substance typically have different crystal lattices (e.g., unit cells) due to different physical properties characteristic of each crystalline form. In some instances, different lattice arrangements have different water or solvent contents. Different crystalline lattices can be identified by solid-state characterization methods such as X-ray powder diffraction (PXRD). Other characterization methods, such as differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), dynamic vapor sorption (DVS), and solid-state NMR, further aid in identifying crystalline forms, as well as determining stability and solvent / water content.

[0224] Crystalline forms of a substance include both solvated (e.g., hydrated) and unsolvated (e.g., anhydrous) forms. Hydrated forms are crystalline forms that contain water in the crystal lattice. Hydrated forms can be stoichiometric hydrates, where water is present in the lattice at a specific water / molecule ratio for hemihydrates, monohydrates, dihydrates, etc. Hydrated forms can also be non-stoichiometric, where the water content varies and is dependent on external conditions such as humidity.

[0225] In some embodiments, the compounds of the present disclosure are substantially isolated. "Substantially isolated" means that a particular compound is at least partially isolated from impurities. For example, in some embodiments, the compounds of the present disclosure contain less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 2.5%, less than about 1%, or less than about 0.5% of impurities. Impurities generally include anything that is not a substantially isolated compound, such as other crystalline forms or other substances.

[0226] Certain groups, moieties, substituents, and atoms are depicted with wavy lines. The wavy lines can cross or cap bonds. The wavy lines indicate the atom to which the group, moiety, substituent, or atom is attached. For example,

[0227] [ka] A phenyl group substituted with a propyl group, depicted as:

[0228] [ka] It has.

[0229] The term "HER2" or "human epidermal growth factor receptor 2" refers to a member of the human epidermal growth factor receptor family. The protein is also known as NEU, NGL, HER2, TKR1, CD340, HER-2, MLN 19, and HER-2 / neu. HER2 can refer to the amino acid sequence specified in NCBI accession number NP_004439.2. Amplification or overexpression of this oncogene has been shown to play a key role in the development and progression of certain aggressive forms of breast cancer. In recent years, the protein has become an important biomarker and target for treatment of approximately 30% of breast cancer patients. All references to proteins, polypeptides, and protein fragments herein are intended to refer to the human protein, polypeptide, or protein fragment, respectively, unless specifically identified as being derived from a non-human species. Thus, the term "HER2" refers to human HER2 unless specifically identified as being derived from a non-human species, e.g., "mouse HER2," "simian HER2," etc.

[0230] The phrase "antibody that binds HER2" or "anti-HER2 antibody" includes antibodies and antigen-binding fragments thereof that specifically recognize HER2.

[0231] The phrase "anti-HER2 / HER2" antibodies, e.g., "anti-HER2 / HER2 bispecific antibodies," includes antibodies and antigen-binding fragments thereof that specifically recognize two different HER2 epitopes. In some embodiments, bispecific antibodies and antigen-binding fragments thereof comprise an antigen-binding domain (D1) that specifically binds a first epitope of human HER2 and a second antigen-binding domain (D2) that specifically binds a second epitope of human HER2.

[0232] As used herein, the term "STEAP2" refers to six-transmembrane epithelial antigen of prostate 2. STEAP2 is an integral six-transmembrane protein that is highly expressed in prostate epithelial cells and is a cell surface marker for prostate cancer; for example, STEAP2 has been found to be expressed at significant levels in the LNCaP prostate cell line (Porkka et al., Lab Invest 2002, 82:1573-1582). STEAP2 (UniProtKB / Swiss-Prot:Q8NFT2.3) is a 490-amino acid protein encoded by the STEAP2 gene located in human chromosome region 7q21; see, for example, the amino acid sequence of human STEAP2 in Tables 1 and 2.

[0233] As used herein, "antibodies that bind STEAP2" or "anti-STEAP2 antibodies" include antibodies and antigen-binding fragments thereof that specifically recognize STEAP2.

[0234] The phrase "antibody that binds MET" or "anti-MET antibody" includes antibodies and antigen-binding fragments thereof that specifically recognize MET. The terms "MET," "c-MET," and the like, as used herein, refer to the human membrane spanning receptor tyrosine kinase.

[0235] The phrase "anti-MET / MET" antibodies, e.g., "anti-MET / MET bispecific antibodies," includes antibodies and antigen-binding fragments thereof that specifically recognize two different MET epitopes. In some embodiments, bispecific antibodies and antigen-binding fragments thereof comprise an antigen-binding domain (D1) that specifically binds a first epitope of human MET and a second antigen-binding domain (D2) that specifically binds a second epitope of human MET.

[0236] All amino acid anomalies used in this disclosure are those accepted by the United States Patent and Trademark Office and specified in 37 CFR § 1.822(B)(J).

[0237] The term "protein" refers to an amino acid polymer having more than about 20 amino acids covalently linked via amide bonds. As used herein, "protein" includes biotherapeutic proteins, recombinant proteins used in research and therapy, capture proteins and other Fc fusion proteins, chimeric proteins, antibodies, monoclonal antibodies, human antibodies, bispecific antibodies, antibody fragments, nanobodies, recombinant antibody chimeras, scFv fusion proteins, cytokines, chemokines, peptide hormones, and the like. Proteins can be produced using recombinant cell-based production systems, such as insect baculovirus systems, yeast systems (e.g., Pichia sp.), and mammalian systems (e.g., CHO cells, CHO derivatives such as CHO-K1 cells).

[0238] All references herein to proteins, polypeptides, and protein fragments are intended to refer to the respective human species protein, polypeptide, or protein fragment unless specifically identified as being from a non-human species. Thus, the term "STEAP2" refers to human STEAP2 unless specifically identified as being from a non-human species, e.g., "mouse STEAP2," "monkey STEAP2," etc.

[0239] The amino acid sequence of an antibody can be numbered using any known numbering scheme, including those described by Kabat et al. ("Kabat" numbering scheme), Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948 ("Chothia" numbering scheme), MacCallum et al., 1996, J. Mol. Biol., 262:732-745 ("Contact" numbering scheme), Lefranc et al., Dev. Comp. Immunol., 2003, 27:55-77 ("IMGT" numbering scheme), and Honegge and Pluckthun, J. Mol. Biol., 2001, 309:657-70 ("AHo" numbering scheme). Unless otherwise specified, the numbering scheme used herein is the Kabat numbering scheme. However, the choice of numbering scheme is not intended to suggest differences in sequences where none exist, and one of skill in the art can readily ascertain the location of sequences by examining the amino acid sequence of one or more antibodies. Unless otherwise specified, the "EU numbering scheme" is typically used when referring to residues of antibody heavy chain constant regions (e.g., as reported in Kabat et al., supra).

[0240] The term "glutaminyl-modified antibody" refers to an antibody having at least one covalent bond from a glutamine side chain to a primary amine compound of the present disclosure. In certain embodiments, the primary amine compound is linked via an amide linkage on the glutamine side chain. In certain embodiments, the glutamine is an endogenous glutamine. In other embodiments, the glutamine is an endogenous glutamine that has been made reactive by engineering the polypeptide (e.g., via amino acid deletion, insertion, substitution, or mutation on the polypeptide). In further embodiments, the glutamine is a polypeptide engineered with an acyl donor glutamine-containing tag (e.g., a glutamine-containing peptide tag, Q-tag, or TGase recognition tag).

[0241] The term "TGase recognition tag" refers to a sequence of amino acids containing an acceptor glutamine residue that, when incorporated (e.g., captured) into a polypeptide sequence, is recognized by a TGase under suitable conditions, resulting in TGase-mediated cross-linking through reaction of an amino acid side chain within the amino acid sequence with a reactive partner. A recognition tag can be a peptide sequence not normally present in a polypeptide that contains a TGase recognition tag. In some embodiments, a TGase recognition tag contains at least one Gln. In some embodiments, a TGase recognition tag comprises the amino acid sequence XXQX (SEQ ID NO: 1935), where X is any amino acid (e.g., the conventional amino acids Leu, Ala, Gly, Ser, Val, Phe, Tyr, His, Arg, Asn, Glu, Asp, Cys, Gln, Ile, Met, Pro, Thr, Lys, or Trp, or an unconventional amino acid). In some embodiments, the acyl donor glutamine-containing tag is LLQGG (SEQ ID NO: 1936), LLQG (SEQ ID NO: 1937), LSLSQG (SEQ ID NO: 1938), gGGLLQGG (SEQ ID NO: 1939), gLLQG (SEQ ID NO: 1940), LLQ, gSPLAQSHGG (SEQ ID NO: 1941), gLLQGGG (SEQ ID NO: 1942), gLLQGG (SEQ ID NO: 1943), gLLQ (SEQ ID NO: 1944), LLQ The amino acid sequence may be selected from the group consisting of LLQGA (SEQ ID NO: 1945), LLQGA (SEQ ID NO: 1946), LLQYQGA (SEQ ID NO: 1947), LLQGSG (SEQ ID NO: 1948), LLQYQG (SEQ ID NO: 1949), LLQLLQG (SEQ ID NO: 1950), SLLQG (SEQ ID NO: 1951), LLQLQ (SEQ ID NO: 1952), LLQLLQ (SEQ ID NO: 1953), and LLQGR (SEQ ID NO: 1954). See, for example, International Publication No. WO2012059882, the entire contents of which are incorporated herein.

[0242] The term "antibody," as used herein, refers to an antigen-binding molecule or molecular complex containing at least one complementarity-determining region (CDR) that specifically binds to or interacts with a particular antigen. The term "antibody" includes immunoglobulin molecules containing four polypeptide chains, two heavy (H) chains and two light (L) chains, interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain contains a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region contains three domains: CH1, CH2, and CH3. Each light chain contains a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region contains one domain (CL1). The VH and VL regions are further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), which can be interspersed with regions that are further conserved and called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments, the FRs of an antibody (or antigen-binding portion thereof) can be identical to human germline sequences or can be naturally or artificially modified. An amino acid consensus sequence can be determined based on parallel analysis of two or more CDRs.

[0243] The term "antibody," as used herein, also includes antigen-binding fragments of intact antibody molecules. The terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of antibodies can be obtained from intact antibody molecules using any suitable standard technique, such as, for example, proteolytic digestion or recombinant genetic engineering techniques, which involve the manipulation and expression of DNA-encoded antibody variable regions and, optionally, constant regions. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. The DNA can be chemically sequenced and manipulated, or sequenced and manipulated using molecular biology techniques, to place one or more variable and / or constant regions into the appropriate configuration, or to introduce codons, create cysteine ​​residues, modify, add, and / or delete amino acids.

[0244] Non-limiting examples of antigen-binding fragments include (i) Fab fragments, (ii) F(ab')2 fragments, (iii) Fd fragments, (iv) Fv fragments, (v) single-chain Fv (scFv) molecules, (vi) dAb fragments, and (vii) minimal recognition units consisting of amino acid residues that mimic a hypervariable region of an antibody (e.g., an isolated complementarity-determining region (CDR) such as a CDR3 peptide) or a restricted FR3-CDR3-FR4 peptide. Domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and other engineered molecules such as shark variable IgNAR domains are also encompassed by the expression "antigen-binding fragment" as used herein.

[0245] Antigen-binding fragments of antibodies typically contain at least one variable domain. The variable domain can be of any size or amino acid composition and generally contains at least one CDR adjacent to or in frame with one or more framework sequences. In antigen-binding fragments having a VH domain associated with a VL domain, the VH and VL domains can be positioned relative to each other in any suitable configuration. For example, the variable regions can be dimers, containing VH-VH, VH-VL, or VL-VL dimers.

[0246] Alternatively, an antigen-binding fragment of an antibody can contain a monomeric VH or VL domain.

[0247] In certain embodiments, an antigen-binding fragment of an antibody can contain at least one variable domain covalently linked to at least one constant region. Non-limiting exemplary configurations of variable and constant regions that may be found in an antigen-binding fragment of an antibody herein include: (i) VH-CH1, (ii) VH-CH2, (iii) VH-CH3, (iv) VH-CH1-CH2, (v) VH-CH1-CH2-CH3, (vi) VH-CH2-CH3, (vii) VH-Cl, (viii) VL-CH1, (ix) VL-CH2, (x) VL-CH3, (xi) VL-CH1-CH2, (xii) VL-CH1-CH2-CH3, (xiii) VL-CH2-CH3, and (xiv) VL-CL. In any configuration of variable and constant regions, including any of the exemplary configurations listed herein, the variable and constant regions can be directly linked to each other or can be linked by a complete or partial hinge or linker region, which can consist of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that provide a flexible or semi-flexible linkage between adjacent variable and / or constant regions in a single polypeptide molecule.

[0248] Furthermore, antigen-binding fragments of the antibodies herein can comprise homodimers or heterodimers (or other multimers) of any of the variable and constant domain arrangements listed herein that are covalently associated (e.g., by disulfide bonds) with each other and / or with one or more monomeric VH or VL domains.

[0249] Like intact antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically contain at least two different variable domains, each capable of specifically binding to a separate antigen or a different epitope on the same antigen. Multispecific antibody formats, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use in the context of the antigen-binding fragments of antibodies herein using routine techniques available in the art.

[0250] The antibodies herein can function via complement-dependent cytotoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC). "Complement-dependent cytotoxicity" (CDC) refers to the lysis of antigen-expressing cells by the antibodies herein in the presence of complement. "Antibody-dependent cellular cytotoxicity" (ADCC) refers to a cell-mediated reaction in which nonspecific cytotoxic cells expressing Fc receptors (FcRs), such as natural killer (NK) cells, neutrophils, and macrophages, recognize bound antibodies on target cells, resulting in lysis of the target cells. CDC and ADCC can be measured using assays well known and available in the art (see, e.g., U.S. Pat. Nos. 5,500,362 and 5,821,337, and Clynes et al. (1998) Proc. Natl. Acad. Sci. (USA) 95:652-656). The constant region of an antibody is important for the antibody's ability to fix complement and mediate cell-dependent cytotoxicity. Thus, the antibody isotype can be selected based on whether it is desirable for the antibody to mediate cytotoxicity.

[0251] In certain embodiments, an antibody of the present invention, e.g., an anti-HER2 antibody, an anti-HER2 / HER2 bispecific antibody, an anti-MET antibody, an anti-MET / MET bispecific antibody, or an anti-STEAP2 antibody, is a human antibody. The term "human antibody," as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies of the present invention may include, for example, in the CDRs and particularly the CDR3, amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody," as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of other mammalian species, such as a mouse, are grafted onto human framework sequences.

[0252] The antibody may, in some embodiments, be a recombinant human antibody. The term "recombinant human antibody," as used herein, is intended to include all human antibodies prepared, expressed, generated, or isolated by recombinant means, including antibodies expressed using recombinant expression vectors transfected into host cells (described in more detail below), antibodies isolated from recombinant combinatorial human antibody libraries (described in more detail below), antibodies isolated from animals (e.g., mice) transgenic for human immunoglobulin genes (see, e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies prepared, expressed, generated, or isolated by any other means involving splicing of human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when animals transgenic for human Ig sequences are used, in vivo somatic mutagenesis) so that the amino acid sequences of the VH and VL regions of the recombinant antibodies are derived from and related to human germline VH and VL sequences, but are sequences that may not normally be present within the human antibody germline repertoire in vivo.

[0253] Human antibodies can exist in two forms related to hinge heterogeneity. In one form, the immunoglobulin molecule contains a stable four-chain construct of approximately 150–160 kDa, in which the dimers are held together by interchain heavy chain disulfide bonds. In the other form, the dimers are not linked by interchain disulfide bonds, forming a molecule of approximately 75–80 kDa composed of covalently linked light and heavy chains (half antibodies). These forms have been extremely difficult to separate, even after affinity purification. The frequency of the second form among various intact IgG isotypes is due to structural differences associated with, but not limited to, the antibody hinge region isotype. A single amino acid substitution in the hinge region of a human IgG4 hinge can significantly reduce the occurrence of the second form (Angal et al., (1993) Molecular Immunology 30:105) to the level commonly observed using human IgG1 hinges. The present disclosure encompasses antibodies with one or more mutations in the hinge, CH2, or CH3 regions that may be desirable during production, for example, to improve the yield of the desired antibody form.

[0254] An antibody herein can be an isolated antibody or a purified antibody. An "isolated antibody" or "purified antibody," as used herein, refers to an antibody that has been identified, separated, and / or recovered from at least one component of its natural environment. For example, an antibody that has been separated or removed from at least one component of an organism, or from a tissue or cell in which it naturally occurs or is naturally produced, is an "isolated antibody" for purposes of this specification. For example, an antibody purified from at least one component of a reaction or series of reactions is a "purified antibody," or results from antibody purification. An isolated antibody also includes an antibody in situ within a recombinant cell. An isolated antibody is an antibody that has been subjected to at least one purification or isolation step. According to certain embodiments, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0255] The antibodies disclosed herein can contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences from which the antibodies are derived. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available, for example, from public antibody sequence databases. The present specification includes antibodies and antigen-binding fragments thereof derived from any of the amino acid sequences disclosed herein, in which one or more amino acids in one or more framework and / or CDR regions are mutated to the corresponding residue in the germline sequence from which the antibody is derived, to the corresponding residue in another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue (such sequence changes are collectively referred to herein as "germline mutations"). Starting with the heavy and light chain variable regions disclosed herein, one of skill in the art can readily produce numerous antibodies and antigen-binding fragments containing one or more germline mutations individually, or in combinations thereof. In some embodiments, all framework and / or CDR residues in the VH and / or VL domains are mutated back to the residues found in the original germline sequence from which the antibody is derived. In other embodiments, only specific residues are mutated back to the original germline sequence, e.g., only the mutated residues found in the first eight amino acids of FR1 or the last eight amino acids of FR4, or only the mutated residues found in CDR1, CDR2, or CDR3. In other embodiments, one or more of the framework and / or CDR residues are mutated to the corresponding residue in a different germline sequence (i.e., a different germline sequence from the germline sequence from which the antibody is originally derived).

[0256] Furthermore, the antibodies herein can contain any combination of two or more germline mutations in the framework and / or CDR regions, e.g., certain individual residues are mutated to the corresponding residues in a particular germline sequence, while other specific residues that differ from the original germline sequence are either maintained or mutated to the corresponding residues in a different germline sequence. Once obtained, antibodies and antigen-binding fragments containing one or more germline mutations can be easily tested for one or more desirable properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, or improved drug-antibody ratio (DAR) in antibody-drug conjugates. Such generally obtained antibodies and antigen-binding fragments are encompassed within the present specification.

[0257] The term "aglycosylated antibody" refers to an antibody that does not contain a glycosylation sequence that could interfere with transglutamination, e.g., an antibody that does not have a saccharide group at N297 on one or more heavy chains. In certain embodiments, the antibody heavy chain has an N297 mutation. In other words, the antibody is mutated so that it no longer has an asparagine residue at position 297 according to the EU numbering system disclosed by Kabat et al. In certain embodiments, the antibody heavy chain has an N297Q or N297D mutation. Such antibodies can be prepared by site-directed mutagenesis to remove or disable a glycosylation sequence or to insert a glutamine residue at a site separate from the interfering glycosylation site or other interfering structure. Such antibodies can also be isolated from natural or artificial sources. Aglycosylated antibodies also include antibodies containing T299 or S298P, other mutations, or combinations of mutations that result in the absence of glycosylation.

[0258] The term "deglycosylated antibody" refers to an antibody in which saccharide groups have been removed to facilitate transglutaminase-mediated conjugation. Saccharides include, but are not limited to, N-linked oligosaccharides. In some embodiments, deglycosylation is performed at residue N297. In some embodiments, removal of saccharide groups is accomplished enzymatically, including, but not limited to, via PNGase.

[0259] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule, known as the paratope. An antigen can have more than one epitope. Therefore, different antibodies can bind to different regions on the antigen and have different biological effects. Epitopes can be either conformational or linear. Conformational epitopes are generated by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear epitopes are epitopes generated by adjacent amino acid residues in a polypeptide chain. In some situations, epitopes can include saccharide, phosphoryl, or sulfonyl moieties on the antigen.

[0260] The term "conjugated protein" or "conjugated antibody," as used herein, refers to a protein or antibody covalently linked to one or more chemical moieties. The chemical moieties can include the amine compounds of the present disclosure. Linkers (L) and payloads (D) suitable for use in the present disclosure are described in detail herein. In certain embodiments, the conjugated antibody comprising a therapeutic moiety is an antibody-drug conjugate (ADC), also known as an antibody-payload conjugate or antibody-linker-payload conjugate.

[0261] The term "drug-to-antibody ratio" or (DAR) is the average number of therapeutic moieties, e.g., drugs, that are conjugated to a binding agent of the disclosure.

[0262] The term "linker-antibody ratio" or (LAR), represented as Case I below, in some embodiments, is the average number of reactive primary amine compounds conjugated to a binding agent of the present disclosure. Such binding agents, e.g., antibodies, can be conjugated with, for example, a suitable azide- or alkyne-containing primary amine compound. The resulting binding agent can be functionalized with the azide or alkyne and then reacted with the corresponding azide- or alkyne-containing therapeutic moiety via a 1,3-cycloaddition reaction.

[0263] The phrase "pharmaceutically acceptable amount" refers to an amount effective or sufficient to treat, reduce, alleviate, or modulate at least one effect or symptom of a health problem in a subject in need thereof. For example, a pharmaceutically acceptable amount of an antibody or antibody-drug conjugate is an amount effective for modulating a biological target using the antibody or antibody-drug conjugate provided herein. Suitable pharmaceutically acceptable amounts include, but are not limited to, from about 0.001% up to about 10%, and any amount therebetween, such as about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% of an antibody or antibody drug conjugate provided herein.

[0264] The phrase "reactive pH" refers to the pH of a reaction after all reaction components or reactants have been added.

[0265] The terms "substantial identity" or "substantially identical," when referring to a nucleic acid or fragment thereof, mean that, upon appropriate nucleotide insertions or deletions and optimal alignment with another nucleic acid (or its complementary strand), there is nucleotide sequence identity over at least about 95%, more preferably at least about 96%, 97%, 98%, or 99%, of the nucleic acid bases as measured by well-known sequence identity algorithms such as FASTA, BLAST, or GAP, discussed below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule can, in some instances, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0266] When applied to polypeptides, the terms "substantial similarity" or "substantially similar" mean that two peptide sequences, when best aligned using programs such as gAP or BESTFIT with default gap weights, share at least 95% sequence identity, more preferably at least 98% or 99% sequence identity. Preferably, non-identical residue positions differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced with another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functional properties of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or similarity may be adjusted upward to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24:307-331. Examples of groups of amino acids with side chains with similar chemical properties include: (1) glycine, alanine, valine, leucine, and isoleucine as aliphatic side chains, (2) serine and threonine as aliphatic hydroxyl side chains, (3) asparagine and glutamine as amide-containing side chains, (4) phenylalanine, tyrosine, and tryptophan as aromatic side chains, (5) lysine, arginine, and histidine as basic side chains, (6) aspartate and glutamate as acidic side chains, and (7) cysteine ​​and methionine as sulfur-containing side chains. In some embodiments, conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine.

[0267] Alternatively, a conservative substitution is one that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443-1445. A "moderately conservative" substitution is one that has a non-negative value in the PAM250 log-likelihood matrix.

[0268] Sequence similarity of polypeptides, also referred to as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, gCG software includes programs such as gap and Bestfit, which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms, or between wild-type proteins and their mutant proteins. See, for example, gCG Version 6.1. Polypeptide sequences can also be compared using FASTA, a program in gCG Version 6.1, using default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the best overlapping regions between the query and search sequences (Pearson (2000) supra). Another specific algorithm for comparing the sequences herein to a database containing a large number of sequences from various organisms is the computer program BLAST, specifically BLASTP or TBLASTN, using default parameters. See, for example, Altschul et al. (1990) J. Mol. Biol. 215:403-410, and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402.

[0269] Protein-drug conjugate compounds In accordance with the foregoing objects and others, the present disclosure provides protein drug conjugate compounds, e.g., antibody drug conjugate compounds, precursors and intermediates thereof, pharmaceutical compositions, and methods for treating certain diseases in subjects in need thereof. In accordance with the present disclosure, the protein drug conjugate compounds provided herein include glutaminyl-modified binding agents conjugated with a primary amine compound linked to a therapeutic moiety, e.g., a camptothecin analog moiety, as described herein.

[0270] In one aspect, the present disclosure provides compounds comprising a binding agent (e.g., an antibody or fragment thereof) according to the present disclosure, having one or more glutamine residues, wherein the one or more glutamine residues are conjugated to one or more camptothecin analogs, e.g., Dxd, via an optional first linker, a branching unit comprising at least one adduct, and an optional second linker. Illustrative, non-limiting examples include Formula (I) and Formula (II) described herein. In certain embodiments of protein drug conjugates according to the present disclosure, the binding agent is an antibody (e.g., a monoclonal antibody), and the term "antibody drug conjugate" or ADC is optionally used.

[0271] In one aspect, the present disclosure provides a compound of formula (C): BA-Gln-NH-L1-B-(-L2-(-M-Camp) m ) n (C) and providing a compound having a structure according to the invention: wherein BA is an antibody or antigen-binding fragment thereof, Gln is a glutamine residue, L1 is absent or is a first linker, B is a branching unit comprising at least one adduct of a group B′ and a group B″, group B′ is a first moiety, e.g., a first cycloaddition moiety, L2 is a second linker covalently attached to branching unit B via at least one group B″, B″ is a second moiety, e.g., a second cycloaddition moiety, wherein groups B′ and B″ form at least one adduct, and M is absent or has the structure:

[0272] [ka] wherein R, R', and R'' are independently at each occurrence hydrogen or C-C 20 is alkyl, or R′ and R″ together form a ring or rings; Camp is a camptothecin analog; and m and n are independently integers from 1 to 30.

[0273] In one aspect, the present disclosure provides a compound of formula (A): BA-(Gln-NH-L1-B-(-L2-(-M-Dxd) m ) k ) n (A) wherein: BA is an antibody or antigen-binding fragment thereof; Gln is a glutamine residue, L1 is absent or is a first linker, B is a branching unit containing at least one addition of a group B′ and a group B″, and at least one of the groups B′ and B″ is selected from the group consisting of —N3 and

[0274] [ka] and the other of group B′ and group B″ is selected from

[0275] [ka] wherein Q is C or N; L2 is a second linker covalently attached to the branching unit B via at least one group B″; M is absent or has the structure:

[0276] [ka] wherein R, R', and R'' are independently at each occurrence hydrogen or C1-C4 alkyl, or R' and R'' together form a 5- or 6-membered ring; Dxd is the formula (P):

[0277] [ka] an antitumor drug having a structure according to k is an integer from 1 to 12, m is an integer from 1 to 30; n is an integer from 1 to 30. A compound is provided.

[0278] In one aspect, the present disclosure provides a compound of formula (I): BA-(Gln-NH-L1-B-(-L2-M-Dxd) k ) n (I) A compound having a structure according to the formula: wherein BA is an antibody or antigen-binding fragment thereof, Gln is a glutamine residue, L1 is absent or is a first linker, B is a branching unit comprising at least one appendage of a group B', wherein the group B' is -N3,

[0279] [ka] wherein Q is C or N; L2 is a second linker covalently attached to the branching unit B via at least one group B″, and groups B′ and B″ form at least one adduct; and M is absent or has the structure:

[0280] [ka] wherein R, R', and R'' are independently at each occurrence hydrogen or C1-C4 alkyl, or R' and R'' together form a 5- or 6-membered ring, and Dxd is a moiety having the formula (P):

[0281] [ka] wherein k and n are independently integers of 1 to 30. A compound is provided.

[0282] Linker L1 In one embodiment, the linker L1 is absent.

[0283] In certain embodiments, the linker L1 is present and is covalently attached to the amine of a glutamine residue of the binding agent BA.

[0284] In certain embodiments, the linker L is alkyl (e.g., C 1-20 Alkyl, C 1-12 Alkyl, or C 1-6 alkyl), -NH-, -C(O)-, -(CH2) u -NH-C(O)-, -(CH2) u -C(O)-NH-, -(CH2-CH2-O) v -, -(CH2) u -(O-CH2-CH2) v -C(O)-NH-, a peptide unit containing 2 to 4 amino acids, or a combination thereof, each of which is optionally substituted by one or more of -S-, -S(O2)-, -C(O)-, -C(O2)-, or -CO2H, and the subscripts u and v are independently integers from 1 to 8.

[0285] In certain embodiments, the free (unconjugated) linker L1 comprises a primary amine for attachment to a glutamine residue via a transglutamation reaction.

[0286] In one embodiment, the linker L1 comprises one or more polyethylene glycol (PEG) units. In one embodiment, L1 comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 PEG units.

[0287] In one embodiment, the linker L1 comprises a disulfide (-SS-) bond.

[0288] In one embodiment, the linker L1 comprises a -S(O2)- moiety.

[0289] In one embodiment, one or more carbons on L1 are substituted with -CO2H.

[0290] In one embodiment, the linker L1 comprises a peptide unit containing 2 to 4 amino acids, a peptide unit containing 2 amino acids, a peptide unit containing 3 amino acids, or a peptide unit containing 4 amino acids.

[0291] In one embodiment, the linker L1 comprises a peptide unit containing two amino acids selected from glycine, valine, phenylalanine, proline, glutamic acid, and citrulline, and combinations thereof. In a particular embodiment, the linker L1 comprises a valine-citrulline unit.

[0292] In one embodiment, the linker L1 is

[0293] [ka] and combinations thereof, wherein R A is a group including alkyne, azide, tetrazine, trans-cyclooctene, maleimide, amine, ketone, aldehyde, carboxylic acid, ester, thiol, sulfonic acid, tosylate, halide, silane, cyano group, carbohydrate group, biotin group, and lipid residue, and the subscripts x, n, p, and q are independently integers from 0 to 12.

[0294] Branch Unit B The branching unit B comprises at least one adduct of group B'. In some embodiments, B comprises one adduct of group B'. In some embodiments, B comprises two adducts of group B'. In some embodiments, B comprises three adducts of group B'.

[0295] In some embodiments, B comprises at least four adducts of group B'. In some embodiments, B comprises four adducts of group B'. In some embodiments, B comprises five adducts of group B'. In some embodiments, B comprises six adducts of group B'.

[0296] In general, adducts of group B' according to the present disclosure encompass moieties that comprise the product of the addition reaction of group B', regardless of the synthetic steps taken to produce the moiety that comprises the product of the addition reaction of group B'.

[0297] In some embodiments, the adduct of group B' is a product of a substituted maleimide and, for example, a thiol, or a substituted trans-cyclooctene, e.g.

[0298] [ka] And, where n is an integer from 0 to 12. Substituted trans-cyclooctenes and, for example, tetrazines may be used.

[0299] In some embodiments, the adduct of group B' may be the product of a 1,3-cycloaddition reaction between an azide moiety and an alkyne moiety. Without being bound by theory, the azide-alkyne cycloaddition is a 1,3-dipolar cycloaddition between an azide and a terminal or internal alkyne to give a 1,2,3-triazole.

[0300] More specifically, -N3,

[0301] [ka] wherein Q is C or N, the adduct is -N3,

[0302] [ka] wherein Q is C or N, and the group B″ is complementary to the group B′ to form the 1,3-cycloaddition adduct.

[0303] As a non-limiting example, group B' can be an azide (-N3) and group B'' can be an alkyne-containing group, e.g.,

[0304] [ka] There are cases where...

[0305] As another non-limiting example, group B' can be an alkyne-containing moiety, e.g.,

[0306] [ka] and the group B'' may be an azide.

[0307] In one embodiment, the adduct of group B' and group B" comprises a triazole moiety. In one particular embodiment, the adduct of group B' and group B" comprises

[0308] [ka] wherein Q is C or N.

[0309] As noted above, in one embodiment, B includes one appendage of group B'.

[0310] In certain embodiments, L1-B is

[0311] [ka] is selected from the group consisting of During the ceremony,

[0312] [ka] is the amino point of attachment of BA to the glutamine residue, and (B') is the attachment of group B'.

[0313] In one embodiment, group B' is an azide (-N3) and the adduct of group B' comprises a triazole.

[0314] According to one embodiment of the present disclosure, the linker L1-B can be an azidoamine linker (AL), which comprises an amine group that directly binds to the antibody, a PEG-containing substructure, and an azide functional group B' (n=1).

[0315] Non-limiting exemplary building block structures of azidoamine linkers are shown in Figure 3B. Specific structures that have been synthesized as examples are provided below.

[0316] In one embodiment, B comprises at least two adducts of group B'. In certain embodiments, B is

[0317] [ka] where (B') comprises the point of attachment of the group B'.

[0318] In certain embodiments, B is

[0319] [ka] is selected from the group consisting of:

[0320] In certain embodiments, L1-B is

[0321] [ka] is selected from the group consisting of

[0322] [ka] is the amino point of attachment of BA to the glutamine residue.

[0323] According to another embodiment of the present disclosure, the linker L1-B may be a branched alkyl azidoamine linker (BL) comprising an amine group that directly bonds to the BA (e.g., an antibody), a branched alkyl PEG-containing substructure, and 2-6 azide functional groups B' (n=2-6).

[0324] Exemplary, non-limiting, building block structures of branched alkyl azidoamine linkers are listed in Figure 4B. Specific structures that have been synthesized as examples are provided below.

[0325] Linker L2 In certain embodiments of the present disclosure, L2 is represented by the formula (L2): B''-SP1-B2-(-SP2-AA-SP3) p (L2) wherein: B″ is a group capable of covalently bonding to group B′; SP1 is absent or is the first spacer unit, B2 is absent or a branching unit; SP2 is absent or a second spacer unit, AA is a peptide unit containing zero or two to four amino acids; SP3 is absent or a third spacer unit, p is an integer from 1 to 12.

[0326] In certain embodiments of the present disclosure, L2 is represented by formula (L2'): H2N-SP1-B2-(-SP2-AA-SP3) p (L2') wherein: SP1 is absent or is the first spacer unit, B2 is absent or a branching unit; SP2 is absent or a second spacer unit, AA is a peptide unit containing zero or two to four amino acids; SP3 is absent or a third spacer unit, p is an integer from 1 to 12.

[0327] In certain embodiments of the present disclosure, L2 is represented by the formula (L2″): Maleimide-N-SP1-B2-(-SP2-AA-SP3) p (L2'') wherein: SP1 is absent or is the first spacer unit, B2 is absent or a branching unit; SP2 is absent or a second spacer unit, AA is a peptide unit containing zero or two to four amino acids; SP3 is absent or a third spacer unit, p is an integer from 1 to 12.

[0328] In some embodiments, the linker L2 comprises a group B'' that can be covalently bonded to group B' described above.

[0329] In certain embodiments, the group B″ is —N3,

[0330] [ka] wherein Q is C or N.

[0331] As a non-limiting example, group B″ can be an alkyne-containing moiety, such as

[0332] [ka] There are cases where...

[0333] As another non-limiting example, group B'' can be an azide.

[0334] In one embodiment, the adduct of group B' and group B" comprises a triazole moiety. In one particular embodiment, the adduct of group B' and group B" comprises

[0335] [ka] or a positional isomer thereof, wherein Q is C or N.

[0336] In one embodiment, the first spacer SP1 is absent.

[0337] In another embodiment, SP1 is

[0338] [ka] is selected from the group consisting of:

[0339] In one embodiment, branching unit B2 is absent.

[0340] In one embodiment, the branching unit B2 of the present disclosure has one of the structures B1 to B5 described below.

[0341] [ka]

[0342] In one embodiment, the second spacer SP2 is absent.

[0343] In another embodiment, SP2 is alkyl (e.g., C 1-20 Alkyl, C 1-12Alkyl, C 1-10 Alkyl, C 1-8 Alkyl, or C 1-6 alkyl), -(CH2-CH2-O) v -, -NH-, -C(O)-, -NH-C(O)-, -NH-(CH2) u -, -NH-(CH2) u -C(O)-, -NH-(CH2-CH2-O) v -, -NH-(CH2-CH2-O) v -C(O), -NH-(CH2-CH2-O) v -(CH2) u -, -NH-(CH2-CH2-O) v -(CH2) u -C(O)-, -(CH2) u -NH-C(O)-, -NH-(CH2) u -NH-C(O)-, -NH-(CH2) u -C(O)-NH-, or combinations thereof, and the subscripts u and v are independently integers from 1 to 8.

[0344] In one embodiment, AA is a peptide unit comprising 2 to 4 amino acids selected from glycine, valine, phenylalanine, proline, glutamic acid, lysine, phenylalanine, and citrulline, and combinations thereof.

[0345] In one embodiment, AA is a peptide unit comprising 2 amino acids. In one embodiment, AA is a peptide unit comprising 3 amino acids. In one embodiment, AA is a peptide unit comprising 4 amino acids.

[0346] In one particular embodiment, AA is valine-citrulline, valine-alanine, or phenylalanine-lysine.

[0347] In another specific embodiment, AA is selected from the group consisting of glycine-glycine-glycine (GGG), glycine-glycine-glycine-glycine (GGGG (SEQ ID NO: 2113)), glycine-glycine-phenylalanine (GGF), glycine-glycine-phenylalanine-glycine (GGFG (SEQ ID NO: 2114)), and glutamic acid-valine-citrulline (EVC).

[0348] In one embodiment, the third spacer SP3 is absent.

[0349] In another embodiment, SP3 is

[0350] [ka] and combinations thereof, wherein R c is not present in each occurrence independently, or

[0351] [ka] is a group selected from

[0352] In one embodiment, the spacer SP3 is covalently linked to a camptothecin analog, eg, Dxd or M-Dxd.

[0353] In one embodiment, the linker L2 comprises about 1 to about 12, about 1 to about 10, about 1 to about 8, about 1 to about 6, about 1 to about 4, or about 1 to about 2 (SP2-AA-SP3) moieties, and the linker payload L2-Dxd comprises about 1 to about 12, about 1 to about 10, about 1 to about 8, about 1 to about 6, about 1 to about 4, or about 1 to about 2 Dxd payload molecules.

[0354] Part M In some embodiments, the moiety M is absent.

[0355] In certain embodiments, M is present and

[0356] [ka] wherein R, R', and R'' are independently at each occurrence hydrogen or alkyl, or R' and R'' together form a ring, for example a 3- to 8-membered ring.

[0357] In certain embodiments, M is present and

[0358] [ka] wherein R, R', and R'' are independently at each occurrence hydrogen or C1-C4 alkyl, or R' and R'' together form a 5- or 6-membered ring.

[0359] In one embodiment, R is hydrogen.

[0360] In one embodiment, R' is hydrogen. In one embodiment, R' is C1-C4 alkyl.

[0361] In one embodiment, R" is hydrogen. In one embodiment, R" is C1-C4 alkyl.

[0362] In one embodiment, R' and R" are taken together to form a five-membered ring. In one embodiment, R' and R" are taken together to be -(CH2)3-.

[0363] In one embodiment, R' and R" are taken together to form a six-membered ring. In one embodiment, R' and R" are taken together to be -(CH2)4-.

[0364] In one embodiment, R, R', and R'' are hydrogen at each occurrence, i.e., M is

[0365] [ka] is.

[0366] In another embodiment, R is hydrogen, R′ and R″ together form a five-membered ring, e.g., R′ and R″ together are —(CH 2 ) 3 —, and M is

[0367] [ka] is.

[0368] payload In certain embodiments, the payloads of the present disclosure are camptothecin analogs and / or derivatives.

[0369] [ka]

[0370] Camptothecin (CPT), mentioned above, is a topoisomerase poison. It was discovered in 1966 by M.E. Wall and M.C. Wani during a systematic screening of natural products for potential anticancer drugs. Camptothecin was isolated from the bark and stem of Camptotheca (Happy Tree), an endemic Chinese tree used in traditional Chinese medicine as a cancer treatment. Camptothecin exhibited significant anticancer activity in preliminary clinical trials. However, due to its poor solubility, synthetic and pharmaceutical chemists have developed numerous successful compounds of camptothecin with various derivatives to enhance the chemical's benefits. Four camptothecin analogs, topotecan, irinotecan, belotecan, and deruxtecan (DXD), have been approved and are currently used in cancer chemotherapy.

[0371] Trastuzumab deruxtecan (T-Dxd) is an antibody-drug conjugate containing the human epidermal growth factor receptor 2 (HER2)-directed antibody trastuzumab and the topoisomerase I inhibitor conjugate deruxtecan (Dxd, a derivative of exatecan). It was approved for use in the United States in December 2019. Exatecan, shown below, is a camptothecin analog.

[0372] [ka]

[0373] In one embodiment, the payload of the present disclosure is deruxtecan (Dxd).

[0374] In one embodiment, the payload of the present disclosure has the structure PI:

[0375] [ka] or a pharmaceutically acceptable salt thereof, wherein R1, R2, R3, and R4 are independently hydrogen or alkyl, e.g., C1-C 12 alkyl, C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl, or R2 and R3 together form a 5- or 6-membered ring.

[0376] In one embodiment, R1 is hydrogen.

[0377] In one embodiment, R2 is hydrogen. In one embodiment, R2 is C1-C4 alkyl.

[0378] In one embodiment, R3 is hydrogen. In one embodiment, R3 is C1-C4 alkyl.

[0379] In one embodiment, R4 is hydrogen. In one embodiment, R4 is C1-C4 alkyl.

[0380] In one embodiment, R, R, R, and R are hydrogen at each occurrence. In one embodiment, the compounds of the present disclosure are

[0381] [ka] or a pharmaceutically acceptable salt thereof

[0382] In one embodiment, R2 and R3 together form a five-membered ring. In one embodiment, R2 and R3 together are -(CH2)3-.

[0383] In one embodiment, R2 and R3 together form a 6-membered ring. In one embodiment, R2 and R3 together are -(CH2)4-.

[0384] In one embodiment, R1 is hydrogen and R2 and R3 together form a five-membered ring.

[0385] In one embodiment, the compound of the present disclosure has formula (P-II):

[0386] [ka] or a pharmaceutically acceptable salt thereof, wherein R is hydrogen or alkyl, e.g., C1-C 12 alkyl, C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl.

[0387] It should be understood by those skilled in the art that compound P-II, as depicted above, is also intended to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (i.e., conformational)) forms of the structure. For example, R and S configurations at each asymmetric center are within the scope of this disclosure. By way of example, the two isomers depicted below are within the scope of this disclosure:

[0388] [ka]

[0389] In one embodiment, the compound of the present disclosure is

[0390] [ka] or a pharmaceutically acceptable salt thereof.

[0391] In one embodiment, a payload according to the present disclosure is conjugated to form a protein drug conjugate (e.g., an antibody drug conjugate). In one embodiment, the payload is covalently attached to a moiety M. In one embodiment, the payload is M-Dxd. In one embodiment, M-Dxd is

[0392] [ka] wherein R is hydrogen or C1-C4 alkyl;

[0393] [ka] represents the point of attachment to L2.

[0394] The present disclosure also relates to pharmaceutical compositions comprising a therapeutically effective amount of the compound described above, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0395] In one embodiment, a compound according to the present disclosure has the following structure:

[0396] [ka] where BA is a binding agent (e.g., an antibody or antigen-binding fragment thereof).

[0397] In one embodiment, a compound according to the present disclosure has the following structure:

[0398] [ka] where BA is a binding agent (e.g., an antibody or antigen-binding fragment thereof).

[0399] In one embodiment, a compound according to the present disclosure has the following structure:

[0400] [ka] where BA is a binding agent (e.g., an antibody or antigen-binding fragment thereof).

[0401] In one embodiment, a compound according to the present disclosure has the following structure:

[0402] [ka] where BA is a binding agent (e.g., an antibody or antigen-binding fragment thereof).

[0403] The present disclosure also relates to pharmaceutical compositions comprising a therapeutically effective amount of the compound described above, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0404] Linker Payload (L2P) In another aspect, the present disclosure provides a compound of formula (L2-P): B''-SP1-B2-(-SP2-AA-SP3-M-Dxd) p (L2-P) A compound according to the formula: B'' is -N3,

[0405] [ka] is selected from the group consisting of SP1 is either not present or

[0406] [ka] is a first spacer unit selected from the group consisting of: B2 is absent or a branching unit; SP2 is not present or 1-6 Alkyl, -(CH2-CH2-O) v -, -NH-, -C(O)-, -NH-C(O)-, -NH-(CH2) u -, -NH-(CH2) u -C(O)-, -NH-(CH2-CH2-O) v -, -NH-(CH2-CH2-O) v -C(O)-, -NH-(CH2-CH2-O) v -(CH2) u -, -NH-(CH2-CH2-O) v -(CH2) u -C(O)-, -(CH2) u -NH-C(O)-, -NH-(CH2) u -NH-C(O)-, -NH-(CH2) u a second spacer unit selected from the group consisting of: —C(O)—NH—, —C(O)—NH—, or combinations thereof; and the subscripts u and v are independently integers from 1 to 8; AA is a peptide unit containing zero or two to four amino acids; SP3 is either not present or

[0407] [ka] and a third spacer unit selected from the group consisting of: c is not present in each occurrence independently, or

[0408] [ka] is a group selected from M is absent or

[0409] [ka] wherein R, R', and R'' are independently at each occurrence hydrogen or C1-C4 alkyl, or R' and R'' together form a 5- or 6-membered ring; Dxd is the formula (P):

[0410] [ka] is an antitumor drug having the structure p is an integer from 1 to 12; A compound is provided.

[0411] In another aspect, the present disclosure provides a compound of formula (L2'-P): H2N-SP1-B2-(-SP2-AA-SP3-M-Dxd)p(L2'-P) A compound according to the formula: B'' is -N3,

[0412] [ka] is selected from the group consisting of SP1 is either not present or

[0413] [ka] is a first spacer unit selected from the group consisting of: B2 is absent or a branching unit; SP2 is not present or 1-6 Alkyl, -(CH2-CH2-O) v -, -NH-, -C(O)-, -NH-C(O)-, -NH-(CH2) u -, -NH-(CH2) u -C(O)-, -NH-(CH2-CH2-O) v -, -NH-(CH2-CH2-O) v-C(O)-, -NH-(CH2-CH2-O) v -(CH2) u -, -NH-(CH2-CH2-O) v -(CH2) u -C(O)-, -(CH2) u -NH-C(O)-, -NH-(CH2) u -NH-C(O)-, -NH-(CH2) u a second spacer unit selected from the group consisting of: —C(O)—NH—, —C(O)—NH—, or combinations thereof; and the subscripts u and v are independently integers from 1 to 8; AA is a peptide unit containing zero or two to four amino acids; SP3 is either not present or

[0414] [ka] and a third spacer unit selected from the group consisting of: c is not present in each occurrence independently, or

[0415] [ka] is a group selected from M is absent or

[0416] [ka] wherein R, R', and R'' are independently at each occurrence hydrogen or C1-C4 alkyl, or R' and R'' together form a 5- or 6-membered ring; Dxd is the formula (P):

[0417] [ka] is an antitumor drug having the structure p is an integer from 1 to 12; A compound is provided.

[0418] In another aspect, the present disclosure provides a compound of formula (L2″-P): Maleimide-N-SP1-B2-(-SP2-AA-SP3-M-Dxd)p(L2''-P) A compound according to the formula: B'' is -N3,

[0419] [ka] is selected from the group consisting of SP1 is either not present or

[0420] [ka] is a first spacer unit selected from the group consisting of: B2 is absent or a branching unit; SP2 is not present or 1-6 Alkyl, -(CH2-CH2-O) v -, -NH-, -C(O)-, -NH-C(O)-, -NH-(CH2) u -, -NH-(CH2) u -C(O)-, -NH-(CH2-CH2-O) v -, -NH-(CH2-CH2-O) v -C(O)-, -NH-(CH2-CH2-O) v -(CH2) u -, -NH-(CH2-CH2-O) v -(CH2) u -C(O)-, -(CH2) u -NH-C(O)-, -NH-(CH2) u -NH-C(O)-, -NH-(CH2) u a second spacer unit selected from the group consisting of: —C(O)—NH—, —C(O)—NH—, or combinations thereof; and the subscripts u and v are independently integers from 1 to 8; AA is a peptide unit containing zero or two to four amino acids; SP3 is either not present or

[0421] [ka] and a third spacer unit selected from the group consisting of: c is not present in each occurrence independently, or

[0422] [ka] is a group selected from M is absent or

[0423] [ka] wherein R, R', and R'' are independently at each occurrence hydrogen or C1-C4 alkyl, or R' and R'' together form a 5- or 6-membered ring; Dxd is the formula (P):

[0424] [ka] is an antitumor drug having the structure p is an integer from 1 to 12; A compound is provided.

[0425] In one embodiment, the linker payload L2-P, L2'-P, L2''-P according to the present disclosure is

[0426] [ka]

[0427] [ka]

[0428] [ka] or a pharmaceutically acceptable salt thereof.

[0429] Branched Linker 2-Payload (BL2P)

[0430] In another aspect, the present disclosure provides L2-P comprising one or more branching units. Exemplary branching units B1-B5 according to the present disclosure are depicted below:

[0431] [ka]

[0432] The structure of an exemplary branched linker 2-payload (BL2P) according to the present disclosure is provided below.

[0433] [ka]

[0434] [ka]

[0435] [ka]

[0436] [ka]

[0437] [ka]

[0438] In one embodiment, the compound (i.e., the linker payload) according to the present disclosure comprises:

[0439] [ka] or a pharmaceutically acceptable salt thereof.

[0440] In one embodiment, the compound (i.e., the linker payload) according to the present disclosure comprises:

[0441] [ka] or a pharmaceutically acceptable salt thereof.

[0442] In another aspect, the present disclosure provides a compound of formula (II): Ab-(Gln-NH-L1-B-(SP1-B2-(-SP2-AA-SP3-M-Dxd) k ) p ) n (II) and providing an antibody drug conjugate according to the formula: wherein Ab is an antibody, Gln is a glutamine residue, L1 is absent or is a first linker as described above, B is a branching unit as described above containing at least one addition of a group B' and a group B''; and group B' is -N3,

[0443] [ka] and at least one group B″, selected from B″-SP1-B2-(-SP2-AA-SP3-M-Dxd) p is a compound according to formula (L2-P) described above, wherein the compound of formula (L2-P) is covalently attached to the antibody via the attachment of group B' and group B''; k is an integer from 1 to 12; and p and n are independently integers from 1 to 30.

[0444] In one embodiment, an antibody drug conjugate according to the present disclosure comprises an antibody and a linker payload, wherein the linker payload is

[0445] [ka] or a pharmaceutically acceptable salt thereof, wherein:

[0446] [ka] represents the point of attachment to a binding agent (e.g., an antibody) either directly or via a second linker.

[0447] Binder In one embodiment, the efficacy of embodiments of the protein drug conjugates described herein depends on the selectivity with which the binding agent binds to its binding partner. In one embodiment of the present disclosure, the binding agent is a molecule that can bind to a given binding partner with some selectivity. In one embodiment, the binding agent is in a mammal where the interaction may occur for therapeutic applications. In an alternative embodiment, the binding agent is in vitro where the interaction may occur for diagnostic applications. In some aspects, the binding agent is capable of binding to a cell or cell population.

[0448] Suitable binding agents of the present disclosure include proteins that bind to a binding partner, where the binding agent comprises one or more glutamine residues. Suitable binding agents include, but are not limited to, antibodies, lymphokines, hormones, growth factors, viral receptors, interleukins, or any other cell- or peptide-binding molecule or substance.

[0449] In one embodiment, the binding agent is an antibody. In some embodiments, the antibody is selected from a monoclonal antibody, a polyclonal antibody, an antibody fragment (e.g., Fab, Fab', and F(ab)2, a minibody, a diabody, a triabody, etc.). The antibodies herein can be humanized using the methods described in U.S. Pat. No. 6,596,541 and U.S. Patent Application Publication No. 2012 / 0096572, each of which is incorporated by reference in its entirety. In some embodiments of the protein drug conjugate compounds of the present disclosure, the BA is a humanized monoclonal antibody. For example, the BA can be a monoclonal antibody that binds HER2, MET, or STEAP2. In some embodiments of the protein drug conjugate compounds of the present disclosure, the BA is a bispecific antibody, e.g., an anti-HER2 / HER2 bispecific antibody or an anti-MET / MET bispecific antibody.

[0450] In the present disclosure, the antibody can be any antibody deemed suitable by one skilled in the art. In some embodiments, the antibody comprises at least one glutamine residue in at least one polypeptide chain sequence. In certain embodiments, the antibody comprises one or more gln295 residues. In certain embodiments, the antibody comprises two heavy chain polypeptides, each having one gln295 residue. In further embodiments, the antibody comprises one or more glutamine residues at a site other than heavy chain 295. Such antibodies can be isolated from natural sources or engineered to contain one or more glutamine residues. Techniques for engineering glutamine residues into antibody polypeptide chains are within the purview of those skilled in the art. In certain embodiments, the antibody is aglycosylated.

[0451] The antibody can be in any form known to those of skill in the art. In some embodiments, the antibody comprises a light chain. In some embodiments, the light chain is a kappa light chain. In some embodiments, the light chain is a lambda light chain.

[0452] In some embodiments, the antibody comprises a heavy chain. In some aspects, the heavy chain is IgA. In some aspects, the heavy chain is IgD. In some aspects, the heavy chain is IgE. In some aspects, the heavy chain is IgG. In some aspects, the heavy chain is IgM. In some aspects, the heavy chain is IgG1. In some aspects, the heavy chain is IgG2. In some aspects, the heavy chain is IgG3. In some aspects, the heavy chain is IgG4. In some aspects, the heavy chain is IgA1. In some aspects, the heavy chain is IgA2.

[0453] In some embodiments, the antibody is an antibody fragment. In some aspects, the antibody fragment is an Fv fragment. In some aspects, the antibody fragment is a Fab fragment. In some aspects, the antibody fragment is a F(ab')2 fragment. In some aspects, the antibody fragment is a Fab' fragment. In some aspects, the antibody fragment is an scFv (sFv) fragment. In some aspects, the antibody fragment is an scFv-Fc fragment.

[0454] In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a polyclonal antibody.

[0455] In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a human antibody.

[0456] The antibody can have binding specificity for any antigen deemed appropriate by one of skill in the art. In certain embodiments, the antigen is a transmembrane molecule (e.g., a receptor) or a growth factor. Exemplary antigens include molecules such as renin, growth hormones, including human growth hormone and bovine growth hormone, growth hormone-releasing factor, parathyroid hormone, thyroid-stimulating hormone, lipoproteins; α1-antitrypsin, insulin A chain, insulin B chain, proinsulin, follicle-stimulating hormone, calcitonin, luteinizing hormone, glucagon, clotting factors such as vmc factor, factor IX, tissue factor (TF), and von Willebrand factor; anticoagulants such as protein C; atrial natriuretic factor; and pulmonary surfactant. plasminogen activators such as urinary tract plasminogen activator (t-PA), bombesin, thrombin, hematopoietic growth factors, tumor necrosis factors-α and -β, enkephalinase, RANTES (normally expressed and secreted and regulated upon T cell activation), human macrophage inflammatory protein (MIP-I-α), serum albumins such as human serum albumin; Muellerian inhibitor, relaxin A chain, relaxin B chain, prorelaxin, mouse gonadotropins related peptides, microbial proteins such as beta-lactamase, DNase, 19E, cytotoxic T lymphocyte-associated antigens (CTLA) such as CTLA-4, inhibin, activin, vascular endothelial growth factor (VEGF), hormone or growth factor receptors, protein A or D, rheumatoid factor, neurotrophic factors such as bone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT4, NT-5, or NT-6), or nerve growth factors such as NGF-β, platelet-derived neurotrophic factors, fibroblast growth factors such as PDGF, aFGF and bFGF, fibroblast growth factor receptor 2 (FGFR2), epidermal growth factor (EGF), transforming growth factors (TGFs) such as TGF-α and TGF-β, including TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5, insulin-like growth factors-1 and -2 (IGF-1 and IGF-2), des(I-3)-IGF-l (brain IGF-l), insulin-like growth factor binding proteins, EpCAM, gD3, FLT3, PSMA,PSCA, MUC1, MUC16, STEAP, STEAP2, CEA, TENB2, EphA receptor, EphB receptor, folate receptor, FOLRI, mesothelin, cripto, alphabeta6, integrin, VEGF, VEGFR, EGFR, transferrin receptor, lRTAI, lRTA2, lRTA3, lRTA4, lRTA5, CD proteins such as CD2, CD3, CD4, CD5, CD6, CD8, CDII, CD14, CD19, CD20, CD21, CD22, CD25, CD26, CD28, CD30, CD33, CD3 6, CD37, CD38, CD40, CD44, CD52, CD55, CD56, CD59, CD70, CD79, CD80, CD81, CD103, CD105, CD134, CD137, CD138, CD152, or antibodies that bind to one or more tumor-associated antigens or cell surface receptors, erythropoietin, osteoinductive factors, immunotoxins, bone morphogenetic proteins (BMPs), interferon-α, -β, -γ, as disclosed in U.S. Patent Application Publication No. 2008 / 0171040 or 2008 / 0305044 and incorporated by reference in their entireties. Interferons such as M-CSF, gM-CSF, g-CSF, interleukins (ILs) such as IL-1 to IL-10, superoxide dismutase, T cell receptors, surface membrane proteins, dissociation promoting factors, viral antigens such as parts of the HIV envelope, transport proteins, homing receptors, addressins, regulatory proteins, integrins such as CD11a, CD11b, CD11c, CD118, ICAM, VLA-4, and VCAM, tumor-associated antigens such as AFP, ALK, B7H4, and BAGE proteins. , β-catenin, brc-abl, BRCA1, BORIS, CA9 (carbonic anhydrase IX), caspase-8, CD20, CD40, CD123, CDK4, CEA, CLEC12A, c-kit, cMET, CTLA4, cyclin-B1, CYP1B1, EGFR, EGF-VIII, endoglin, Epcam, EphA2, ErbB2 / HER2, ErbB3 / HER3, ErbB4 / HER4, ETV6-AML, Fra-1, FOLR1, gAGE proteins (e.g., gAGE-1, -2), gD2, gD3, globoH, glyptocan-3,gM3, gp100, HER2, HLA / B-raf, HLA / EBNA1, HLA / k-ras, HLA / MAGE-A3, hTERT, IGF1R, LGR5, LMP2, MAGE proteins (e.g., MAGE-1, -2, -3, -4, -6, -12), MART-1, Sotelin, mL-IAP, Muc1, Muc16(CA-125), MET, MUM1, NA17, NGEP, NY-BR1, NY-BR62, NY-BR85, NY-ESO1, OX40, p15, p53, PAP, PAX3, PAX5, PCTA-1, PDGFR-α, PD Examples of polypeptides include, but are not limited to, GFR-β, PDGF-A, PDGF-B, PDGF-C, PDGF-D, PLAC1, PRLR, PRAME, PSCA, PSGR, PSMA (FOLH1), RAGE protein, Ras, RGS5, Rho, SART-1, SART-3, STEAP1, STEAP2, STn, survivin, TAG-72, TGF-β, TMPRSS2, Tn, TNFRSF17, TRP-1, TRP-2, tyrosinase, and uroplakin-3, as well as fragments of any of the polypeptides listed herein.

[0457] Exemplary antigens also include, but are not limited to, BCMA, SLAMF7, B7H4, gPNMB, UPK3A, and LGR5. Exemplary antigens also include, but are not limited to, MUC16, PSMA, STEAP2, and HER2.

[0458] In some embodiments, antigens also include, but are not limited to, blood targets such as CD22, CD30, CD33, CD79a, CD79b.

[0459] Some embodiments herein are target-specific for therapeutic or diagnostic applications. In one embodiment, the binding agent is tailored to interact with and bind to antigens defined as tumor antigens, including antigens specific to a single antigen and antigens shared, overexpressed, or modified on specific tumor types. Examples include α-actinin-4 with lung cancer, ARTC1 with melanoma, BCR-ABL fusion protein with chronic myeloid leukemia, B-RAF, CLPP, or Cdc27 with melanoma, CASP-8 with squamous cell carcinoma, and hsp70-2 with renal cell carcinoma, as well as shared tumor-specific antigens such as BAGE-1, gAGE, gnTV, KK-LC-1, MAGE-A2, NA88-A, and TRP2-INT2. In some embodiments, the antigen is PRLR or HER2. In some embodiments, the antibody binds STEAP2, MUC16, EGFR, EGF VIII, FGR2, or PRLR.

[0460] In some embodiments, the antigen comprises HER2. In some embodiments, the antigen comprises STEAP2. In some embodiments, the antigen comprises MET. In some embodiments, the antigen comprises EGFRVIII. In some embodiments, the antigen comprises MUC16. In some embodiments, the antigen comprises PRLR. In some embodiments, the antigen comprises PSMA. In some embodiments, the antigen comprises FGFR2.

[0461] In some embodiments, the BA is an anti-HER2 antibody, an anti-STEAP2 antibody, an anti-MET antibody, an anti-EGFRVIII antibody, an anti-MUC16 antibody, an anti-PRLR antibody, an anti-PSMA antibody, or an anti-FGFR2 antibody, an anti-HER2 / HER2 bispecific antibody, an anti-MET / MET bispecific antibody, or an anti-FOLR1 antibody, or an antigen-binding fragment thereof.

[0462] In some embodiments, the BA targets a cancer selected from the group consisting of breast cancer, ovarian cancer, prostate cancer, lung cancer, liver cancer, or brain cancer.

[0463] Anti-HER2 antibodies suitable for protein-drug conjugates In some embodiments, the antibody is an anti-HER2 antibody. In some embodiments, the antibody is trastuzumab, pertuzumab (2C4), or margetuximab (MGAH22). In some embodiments, the antibody is trastuzumab. According to certain embodiments, a protein drug conjugate, e.g., an ADC, according to the present disclosure comprises an anti-HER2 antibody. In some embodiments, the anti-HER2 antibody may include those described in International Publication No. WO2019 / 212965A1.

[0464] In some embodiments, the antibody is an anti-HER2 / HER2 bispecific antibody, which comprises a first antigen-binding domain (D1) that specifically binds a first epitope of human HER2 and a second antigen-binding domain (D2) that specifically binds a second epitope of human HER2.

[0465] In one embodiment, the D1 and D2 domains of an anti-HER2 / HER2 bispecific antibody do not compete with each other. Lack of competition between D1 and D2 for binding to HER2 means that the respective monospecific antigen-binding proteins from which D1 and D2 are derived do not compete with each other for binding to human HER2. Exemplary antigen-binding protein competition assays are known in the art.

[0466] In certain embodiments, D1 and D2 bind to different (eg, non-overlapping or partially overlapping) epitopes on HER2.

[0467] In one non-limiting embodiment, the present disclosure provides: a first antigen-binding domain (D1); and a second antigen-binding domain (D2); and a protein drug conjugate comprising a dual-potential antigen-binding molecule comprising: D1 specifically binds the first epitope of human HER2; D2 specifically binds a second epitope on human HER2.

[0468] An anti-HER2 / HER2 bispecific antibody may be constructed using the antigen-binding domains of two separate monospecific anti-HER2 antibodies. For example, a population of monoclonal monospecific anti-HER2 antibodies may be produced using standard methods known in the art. The individual antibodies thus produced may be tested pairwise in cross-competition with HER2 protein. If two different anti-HER2 antibodies are capable of binding to HER2 simultaneously (i.e., do not compete with each other), the antigen-binding domain of a first anti-HER2 antibody and the antigen-binding domain of a second, non-competitive anti-HER2 antibody may be engineered into a single anti-HER2 / HER2 bispecific antibody according to the present disclosure.

[0469] According to the present disclosure, a bispecific antigen-binding molecule can be a single multifunctional polypeptide or a multimer of two or more polypeptides that are covalently or non-covalently associated with each other. As will be apparent from the present disclosure, an antigen-binding construct capable of simultaneously binding two distinct, non-identical epitopes on the HER2 molecule is considered a bispecific antigen-binding molecule. Any of the antigen-binding molecules described herein, or variants thereof, can be constructed using standard molecular biology techniques (e.g., recombinant DNA and protein expression techniques) known to those skilled in the art.

[0470] In another aspect, the present disclosure provides a pharmaceutical composition comprising a recombinant human antibody or fragment thereof that specifically binds HER2 and a pharmaceutically acceptable carrier. In one non-limiting embodiment, the antibody may bind two distinct epitopes on the HER2 protein, i.e., the antibody is a HER2 / HER2 bispecific antibody. In a related aspect, the present disclosure features a composition that is a combination of an anti-HER2 / HER2 antibody and a second therapeutic agent. In one embodiment, the second therapeutic agent is any agent that is advantageously combined with an anti-HER2 / HER2 antibody. Additional combination therapies and co-formulations involving the anti-HER2 / HER2 bispecific antibodies of the present disclosure are disclosed elsewhere herein.

[0471] In another aspect, the present disclosure provides a therapeutic method for targeting / killing HER2-expressing tumor cells using an anti-HER2 / HER2 bispecific antibody of the present disclosure, the therapeutic method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising an anti-HER2 / HER2 antibody of the present disclosure. Optionally, the anti-HER2 / HER2 antibody (or antigen-binding fragment thereof) can be used to treat breast cancer or may be modified to enhance cytotoxicity by methods including, but not limited to, modified Fc domains that increase ADCC (see, e.g., Shield et al. (2002) JBC 277:26733), radioimmunotherapy, antibody-drug conjugates, or other methods that improve the efficiency of tumor resection.

[0472] The present disclosure also includes the use of an anti-HER2 antibody of the present disclosure in the manufacture of a medicament for the treatment of a disease or disorder (e.g., cancer) associated with or caused by cells expressing HER2. In one aspect, the present disclosure relates to a compound comprising an anti-HER2 antibody or antigen-binding fragment, or a HER2 / HER2 bispecific antibody disclosed herein, for use in medicine. In one aspect, the present disclosure relates to a compound comprising an antibody-drug conjugate (ADC) disclosed herein, for use in medicine.

[0473] In yet another aspect, the present disclosure provides bispecific anti-HER2 / HER2 antibodies for diagnostic applications, for example, as imaging reagents.

[0474] Anti-STEAP2 antibodies suitable for protein-drug conjugates In some embodiments, the antibody is an anti-six-transmembrane epithelial antigen of prostate 2 (STEAP2) antibody, i.e., an anti-STEAP2 antibody. STEAP2 is a metalloreductase that acts as a shuttle between the Golgi complex and the plasma membrane, reducing iron and copper and facilitating their entry into cells. STEAP2 is primarily localized in epithelial cells of the prostate. STEAP2 is also expressed in normal heart, brain, pancreas, ovary, skeletal muscle, mammary gland, testis, uterus, kidney, lung, trachea, colon, and liver. STEAP2 is overexpressed in cancerous tumors, including prostate, bladder, cervix, lung, colon, kidney, breast, pancreas, stomach, uterine, and ovarian tumors (Gomes, IM et al., 2012, Mol. Cancer Res. 10:573-587; Challita-Eid-PM et al., 2003, International Publication No. WO03 / 087306; Emtage, PCR, 2005, International Publication No. WO2005 / 079490).

[0475] In one embodiment, suitable anti-STEAP antibodies are those disclosed in U.S. Patent Application No. 2018 / 0104357. Exemplary anti-STEAP2 antibodies according to the present disclosure are listed in Tables 1 and 2 herein. Table 1 specifies the amino acid sequence identifiers for the heavy chain variable region (HCVR) and light chain variable region (LCVR) of exemplary anti-STEAP2 antibodies, as well as the heavy chain complementarity determining regions (HCDR1, HCDR2, HCDR3) and light chain complementarity determining regions (LCDR1, LCDR2, LCDR3). Table 2 specifies the sequence identifiers for nucleic acid molecules encoding the HCVR, LCVR, HCDR1, HCDR2 HCDR3, LCDR1, LCDR2, and LCDR3 of exemplary anti-STEAP2 antibodies.

[0476] The present disclosure provides antibodies or antigen-binding fragments thereof comprising an HCVR comprising an amino acid sequence selected from any of the HCVR amino acid sequences listed in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0477] The present disclosure also provides an antibody or antigen-binding fragment thereof comprising an LCVR comprising an amino acid sequence selected from any of the LCVR amino acid sequences listed in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0478] The present disclosure also provides antibodies or antigen-binding fragments thereof comprising an HCVR and LCVR amino acid sequence pair (HCVR / LCVR) comprising any of the HCVR amino acid sequences listed in Table 1 paired with any of the LCVR amino acid sequences listed in Table 1. According to certain embodiments, the present disclosure provides antibodies or antigen-binding fragments thereof comprising the HCVR / LCVR amino acid sequence included in any of the exemplary anti-STEAP2 antibodies listed in Table 1. In certain embodiments, the HCVR / LCVR amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 250 / 258 (e.g., H2M11162N).

[0479] The present disclosure also provides an antibody or antigen-binding fragment thereof comprising a heavy chain CDR1 (HCDR1) comprising an amino acid sequence selected from any of the HCDR1 amino acid sequences listed in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0480] The present disclosure also provides antibodies or antigen-binding fragments thereof comprising a heavy chain CDR2 (HCDR2) comprising an amino acid sequence selected from any of the HCDR2 amino acid sequences listed in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0481] The present disclosure also provides antibodies or antigen-binding fragments thereof comprising a heavy chain CDR3 (HCDR3) comprising an amino acid sequence selected from any of the HCDR3 amino acid sequences listed in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0482] The present disclosure also provides an antibody or antigen-binding fragment thereof comprising an LCDR1 comprising an amino acid sequence selected from any of the light chain CDR1 (LCDR1) amino acid sequences listed in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0483] The present disclosure also provides antibodies or antigen-binding fragments thereof comprising an LCDR2 comprising an amino acid sequence selected from any of the light chain CDR2 (LCDR2) amino acid sequences listed in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0484] The present disclosure also provides antibodies or antigen-binding fragments thereof comprising an LCDR3 comprising an amino acid sequence selected from any of the light chain CDR3 (LCDR3) amino acid sequences listed in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0485] The present disclosure also provides an antibody or antigen-binding fragment thereof comprising an HCDR3 and LCDR3 amino acid sequence pair (HCDR3 / LCDR3) comprising any of the HCDR3 amino acid sequences listed in Table 1 paired with any of the LCDR3 amino acid sequences listed in Table 1. According to certain embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof comprising an HCDR3 / LCDR3 amino acid sequence included in any of the exemplary anti-STEAP2 antibodies listed in Table 1. In certain embodiments, the HCDR3 / LCDR3 amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 256 / 264 (e.g., H2M11162N).

[0486] The present disclosure also provides antibodies or antigen-binding fragments thereof comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained in any of the exemplary anti-STEAP2 antibodies listed in Table 1. In certain embodiments, the set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences is selected from the group consisting of SEQ ID NOs: 252-254-256-260-262-264 (e.g., H2M11162N).

[0487] In related embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained in the HCVR / LCVR amino acid sequence defined by any of the exemplary anti-STEAP2 antibodies listed in Table 1. For example, the present disclosure includes an antibody or antigen-binding fragment thereof comprising the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence contained in the HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 250 / 258 (e.g., H2M11162N). Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within specific HCVR and / or LCVR amino acid sequences disclosed herein. Exemplary conventions that can be used to identify CDR boundaries include, for example, the Kabat definition, the Chothia definition, and the AbM definition. Generally speaking, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, e.g., Kabat, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989). Public databases are also available for identifying CDR sequences within antibodies.

[0488] The present disclosure also provides nucleic acid molecules encoding anti-STEAP2 antibodies or portions thereof. For example, the present disclosure provides nucleic acid molecules encoding any of the HCVR amino acid sequences listed in Table 1, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0489] The present disclosure also provides nucleic acid molecules encoding any of the LCVR amino acid sequences listed in Table 1, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0490] The present disclosure also provides nucleic acid molecules encoding any of the HCDR1 amino acid sequences listed in Table 1, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR1 nucleic acid sequences listed in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0491] The present disclosure also provides nucleic acid molecules encoding any of the HCDR2 amino acid sequences listed in Table 1, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR2 nucleic acid sequences listed in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0492] The present disclosure also provides nucleic acid molecules encoding any of the HCDR3 amino acid sequences listed in Table 1, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR3 nucleic acid sequences listed in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0493] The present disclosure also provides nucleic acid molecules encoding any of the LCDR1 amino acid sequences listed in Table 1, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR1 nucleic acid sequences listed in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0494] The present disclosure also provides nucleic acid molecules encoding any of the LCDR2 amino acid sequences listed in Table 1, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR2 nucleic acid sequences listed in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0495] The present disclosure also provides nucleic acid molecules encoding any of the LCDR3 amino acid sequences listed in Table 1, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR3 nucleic acid sequences listed in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0496] The present disclosure also provides nucleic acid molecules encoding HCVRs, which comprise a set of three CDRs (i.e., HCDR1-HCDR2-HCDR3), and the set of HCDR1-HCDR2-HCDR3 amino acid sequences is defined by any of the exemplary anti-STEAP2 antibodies listed in Table 1.

[0497] The present disclosure also provides nucleic acid molecules encoding LCVRs, which comprise a set of three CDRs (i.e., LCDR1-LCDR2-LCDR3), and the set of LCDR1-LCDR2-LCDR3 amino acid sequences is defined by any of the exemplary anti-STEAP2 antibodies listed in Table 1.

[0498] The disclosure also provides nucleic acid molecules encoding both an HCVR and an LCVR, wherein the HCVR comprises any of the HCVR amino acid sequences listed in Table 1, and the LCVR comprises any of the LCVR amino acid sequences listed in Table 1. In certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR amino acid sequences listed in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto, and a polynucleotide sequence selected from any of the LCVR amino acid sequences listed in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments according to this aspect of the disclosure, the nucleic acid molecule encodes an HCVR and an LCVR, wherein the HCVR and LCVR are both obtained from the same anti-STEAP2 antibody listed in Table 1.

[0499] The present disclosure also provides recombinant expression vectors capable of expressing polypeptides comprising heavy or light chain variable regions of anti-STEAP2 antibodies. For example, the present disclosure includes recombinant expression vectors comprising any of the nucleic acid molecules described above, i.e., nucleic acid molecules encoding any of the HCVR, LCVR, and / or CDR sequences listed in Table 1. Also included within the scope of the present disclosure are host cells into which such vectors have been introduced, as well as methods for producing antibodies or portions thereof by culturing the host cells under conditions permissive for the production of antibodies or antibody fragments, and recovering the antibodies and antibody fragments so produced.

[0500] The present disclosure includes anti-STEAP2 antibodies with modified glycosylation patterns. In some embodiments, modifications to remove unnecessary glycosylation sites or antibodies lacking fucose moieties present in the oligosaccharide chains may be useful, for example, to increase complement-dependent cell cytotoxicity (ADCC) function (see Shield et al. (2002) JBC 277:26733). In other applications, galactosylation modifications can be made to modify complement-dependent cytotoxicity (CDC).

[0501] In another aspect, the disclosure provides a pharmaceutical composition comprising a recombinant human antibody or fragment thereof that specifically binds STEAP2 and a pharmaceutically acceptable carrier. In a related aspect, the disclosure features a composition that is a combination of an anti-STEAP2 antibody and a second therapeutic agent. In one embodiment, the second therapeutic agent is any agent that is advantageously combined with an anti-STEAP2 antibody. Additional combination therapies and co-formulations that include the anti-STEAP2 antibodies of the disclosure are disclosed elsewhere herein.

[0502] In another aspect, the present disclosure provides a therapeutic method for targeting / killing STEAP2-expressing tumor cells using an anti-STEAP2 antibody of the present disclosure, the therapeutic method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising an anti-STEAP2 antibody of the present disclosure. Optionally, the anti-STEAP2 antibody (or antigen-binding fragment thereof) can be used to treat prostate cancer or may be modified to enhance cytotoxicity by methods including, but not limited to, modified Fc domains that increase ADCC (see, e.g., Shield et al. (2002) JBC 277:26733), radioimmunotherapy, antibody-drug conjugates, or other methods that improve the efficiency of tumor resection.

[0503] The present disclosure also includes the use of an anti-STEAP2 antibody of the present disclosure in the manufacture of a medicament for the treatment of a disease or disorder (e.g., cancer) associated with or caused by cells expressing STEAP2. In one aspect, the present disclosure relates to a compound comprising an anti-STEAP2 antibody or antigen-binding fragment, or a STEAP2xCD3 bispecific antibody disclosed herein, for use in medicine. In one aspect, the present disclosure relates to a compound comprising an antibody-drug conjugate (ADC) disclosed herein, for use in medicine.

[0504] In yet another aspect, the present disclosure provides bispecific anti-STEAP2 antibodies for diagnostic use, for example, as imaging reagents.

[0505] In yet another aspect, the present disclosure provides a therapeutic method for stimulating T cell activation using an anti-CD3 antibody or antigen-binding portion of an antibody of the present disclosure, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising the antibody.

[0506] In another aspect, the present disclosure provides an isolated antibody or antigen-binding fragment thereof that binds C4-2 cells expressing STEAP2 with an EC50 of less than 50 nM as measured by FACS analysis. In another aspect, the present disclosure provides an isolated antibody or antigen-binding fragment thereof that binds C4-2 cells expressing STEAP2 and is internalized by the cells.

[0507] The present disclosure further provides antibodies or antigen-binding fragments that compete with a reference antibody comprising a pair of HCVR / LCVR amino acid sequences specified in Table 1 for binding to human STEAP2. In another aspect, the disclosure provides an antibody or antigen-binding fragment that competes for binding to human STEAP2 with a reference antibody comprising a pair of HCVR / LCVR amino acid sequences selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 58, 74 / 58, 82 / 58, 90 / 58, 98 / 58, 106 / 114, 122 / 130, 138 / 146, 154 / 162, 170 / 178, 186 / 194, 202 / 210, 218 / 226, 234 / 242, 250 / 258, 266 / 274, 282 / 290, 298 / 306, 314 / 322, 330 / 338, 346 / 354, 362 / 370, and 378 / 386.

[0508] The present disclosure provides antibodies or antigen-binding fragments thereof that bind to the same epitope on human STEAP2 as a reference antibody comprising a HCVR / LCVR amino acid sequence pair specified in Table 1. In another embodiment, the antibody or antigen-binding fragment binds to the same epitope on human STEAP2 as a reference antibody comprising a HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 58, 74 / 58, 82 / 58, 90 / 58, 98 / 58, 106 / 114, 122 / 130, 138 / 146, 154 / 162, 170 / 178, 186 / 194, 202 / 210, 218 / 226, 234 / 242, 250 / 258, 266 / 274, 282 / 290, 298 / 306, 314 / 322, 330 / 338, 346 / 354, 362 / 370, and 378 / 386.

[0509] The present disclosure further provides an isolated antibody or antigen-binding fragment thereof that binds human STEAP2, wherein the antibody or antigen-binding fragment comprises a complementarity-determining region (CDR) of a heavy chain variable region (HCVR) having an amino acid sequence set forth in Table 1, and a CDR of a light chain variable region (LCVR) having an amino acid sequence set forth in Table 1. In another embodiment, the isolated antibody or antigen-binding fragment comprises the heavy chain CDRs and light chain CDRs of a pair of HCVR / LCVR amino acid sequences selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 58, 74 / 58, 82 / 58, 90 / 58, 98 / 58, 106 / 114, 122 / 130, 138 / 146, 154 / 162, 170 / 178, 186 / 194, 202 / 210, 218 / 226, 234 / 242, 250 / 258, 266 / 274, 282 / 290, 298 / 306, 314 / 322, 330 / 338, 346 / 354, 362 / 370, and 378 / 386.In another embodiment, the isolated antibodies or antigen-binding fragments are selected from the group consisting of SEQ ID NOs: 4-6-8-12-14-16, 20-22-24-28-30-32, 36-38-40-44-46-48, 52-54-56-60-62-64, 68-70-72-60-62-64, 76-78-80-60-62-64, 84-86-88-60-62-64, 92-94-96-6 0-62-64, 100-102-104-60-62-64, 108-110-112-116-118-120, 124-126-128-132-134-136, 140-142-144-148-150-152, 156-158-160-164-166-168, 172-174-176-180-182-184, 188-190-192-196-198-200, 204-206-208-212-214-216, 220-222-224-228-230-232, 236-238-240-244-246-248, 252-254-256-260-262-264, 268-270-272-276-278-280, 284-286-288-292-294-296, 300-302-304-308-310-312, 316-3 and 380-382-384-388-390-392.

[0510] In another aspect, the disclosure provides an isolated antibody or antigen-binding fragment thereof that binds human STEAP2, wherein the antibody or antigen-binding fragment is selected from the group consisting of (a) SEQ ID NOs: 2, 18, 34, 50, 66, 74, 82, 90, 98, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, 298, 314, 330, 346, 362, and and 378; and (b) a heavy chain variable region (HCVR) selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, 290, 306, 322, 338, 354, 370, and 386. In a further embodiment, the isolated antibody or antigen-binding fragment of claim 10 comprises a pair of HCVR / LCVR amino acid sequences selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 58, 74 / 58, 82 / 58, 90 / 58, 98 / 58, 106 / 114, 122 / 130, 138 / 146, 154 / 162, 170 / 178, 186 / 194, 202 / 210, 218 / 226, 234 / 242, 250 / 258, 266 / 274, 282 / 290, 298 / 306, 314 / 322, 330 / 338, 346 / 354, 362 / 370, and 378 / 386.

[0511] According to another aspect, the present disclosure provides an antibody-drug conjugate comprising the above-described anti-STEAP2 antibody or antigen-binding fragment thereof and a therapeutic agent (e.g., an anti-tumor agent, such as a camptothecin analog, e.g., Dxd). In some embodiments, the antibody or antigen-binding fragment and the anti-tumor agent are covalently linked via a linker as discussed above. In various embodiments, the anti-STEAP2 antibody or antigen-binding fragment can be any of the anti-STEAP2 antibodies or fragments described herein.

[0512] Heavy and light chain variable region amino acid and nucleic acid sequences of anti-STEAP2 antibodies

[0513] Table 1 lists the amino acid sequence identifiers for the heavy and light chain variable regions and CDRs of selected anti-STEAP2 antibodies according to the present disclosure. The corresponding nucleic acid sequence identifiers are listed in Table 2.

[0514] [Table 1]

[0515] [Table 2]

[0516] Anti-MET antibodies suitable for protein-drug conjugates

[0517] In some embodiments, the antibody is an anti-MET antibody. According to certain embodiments, a protein drug conjugate, e.g., an ADC, according to the present disclosure comprises an anti-MET antibody. In some embodiments, the anti-MET antibody may include those described in U.S. Patent Application Publication No. 2018 / 0134794.

[0518] In some embodiments, the antibody is an anti-MET / MET bispecific antibody, which comprises a first antigen-binding domain (D1) that specifically binds a first epitope of human MET and a second antigen-binding domain (D2) that specifically binds a second epitope of human MET. In some embodiments, the anti-MET / MET bispecific antibody may include those described in U.S. Patent Application No. 2018 / 0134794.

[0519] In certain embodiments, the D1 and D2 domains of an anti-MET / MET bispecific antibody do not compete with each other. Lack of competition between D1 and D2 for binding to MET means that the respective monospecific antigen-binding proteins from which D1 and D2 are derived do not compete with each other for binding to human MET. Exemplary antigen-binding protein competition assays are known in the art.

[0520] In certain embodiments, D1 and D2 bind to different (eg, non-overlapping or partially overlapping) epitopes on MET.

[0521] In one non-limiting embodiment, the present disclosure provides: a first antigen-binding domain (D1); and a second antigen-binding domain (D2); and a protein drug conjugate comprising a dual-potential antigen-binding molecule comprising: D1 specifically binds the first epitope of human MET, D2 specifically binds a second epitope on human MET.

[0522] Anti-MET / MET bispecific antibodies may be constructed using the antigen-binding domains of two separate, monospecific anti-MET antibodies. For example, a population of monoclonal, monospecific anti-MET antibodies may be produced using standard methods known in the art. The individual antibodies thus produced may be tested pairwise in cross-competition with the MET protein. If two different anti-MET antibodies are capable of binding to MET simultaneously (i.e., do not compete with each other), the antigen-binding domain of a first anti-MET antibody and the antigen-binding domain of a second, non-competing anti-MET antibody can be engineered into a single anti-MET / MET bispecific antibody according to the present disclosure.

[0523] According to the present disclosure, a bispecific antigen-binding molecule can be a single multifunctional polypeptide or a multimer of two or more polypeptides that are covalently or noncovalently associated with each other. As will be apparent from the present disclosure, an antigen-binding construct capable of simultaneously binding two distinct, non-identical epitopes on the MET molecule is considered a bispecific antigen-binding molecule. Any of the antigen-binding molecules described herein, or variants thereof, may be constructed using standard molecular biology techniques (e.g., recombinant DNA and protein expression techniques) as known to those skilled in the art.

[0524] A bispecific antigen-binding molecule comprises a first antigen-binding domain (D1) that specifically binds a first epitope of human MET and a second antigen-binding domain (D2) that specifically binds a second epitope of human MET, and may be referred to herein as a "MET / MET bispecific antibody," "METxMET bispecific antibody," "MET / MET," "METxMET," or other related terminology. In some embodiments, the first epitope of human MET comprises amino acids 192-204 of SEQ ID NO: 2109. In some embodiments, the second epitope of human MET comprises amino acids 305-315 and 421-455 of SEQ ID NO: 2109. In some embodiments, the first epitope of human MET comprises amino acids 192-204 of SEQ ID NO: 2109, and the second epitope of human MET comprises amino acids 305-315 and 421-455 of SEQ ID NO: 2109.

[0525] Exemplary antigen-binding domains (D1 and D2) that can be included in the METxMET bispecific antigen-binding molecules provided herein include antigen-binding domains derived from any of the anti-MET antibodies disclosed herein. For example, the present disclosure includes METxMET bispecific antigen-binding molecules comprising a D1 or D2 antigen-binding domain that comprises an HCVR comprising an amino acid sequence selected from any of the HCVR amino acid sequences listed in Table 3, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0526] Also provided herein are METxMET bispecific antigen-binding molecules comprising a D1 or D2 antigen-binding domain comprising an LCVR comprising an amino acid sequence selected from any of the LCVR amino acid sequences listed in Table 3, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0527] Provided herein are METxMET bispecific antigen-binding molecules comprising a D1 or D2 bispecific antigen-binding molecule comprising a pair of HCVR and LCVR amino acid sequences (HCVR / LCVR) comprising any of the HCVR amino acid sequences listed in Table 3 paired with any of the LCVR amino acid sequences listed in Table 3. According to certain embodiments, the present invention provides METxMET bispecific antigen-binding molecules comprising a D1 or D2 antigen-binding domain comprising a pair of HCVR / LCVR amino acid sequences included in any of the exemplary anti-MET antibodies listed in Table 3.

[0528] Also provided herein are METxMET bispecific antigen-binding molecules comprising a D1 or D2 antigen-binding domain comprising an HCDR1 that comprises an amino acid sequence selected from any of the heavy chain CDR1 (HCDR1) amino acid sequences listed in Table 3, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0529] Also provided are METxMET bispecific antigen-binding molecules comprising a D1 or D2 antigen-binding domain comprising an HCDR2 that comprises an amino acid sequence selected from any of the heavy chain CDR2 (HCDR2) amino acid sequences listed in Table 3, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0530] Also provided are METxMET bispecific antigen-binding molecules comprising a D1 or D2 antigen-binding domain comprising an HCDR3 that comprises an amino acid sequence selected from any of the heavy chain CDR3 (HCDR3) amino acid sequences listed in Table 3, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0531] Also provided are METxMET bispecific antigen-binding molecules comprising a D1 or D2 antigen-binding domain comprising an LCDR1 comprising an amino acid sequence selected from any of the light chain CDR1 (LCDR1) amino acid sequences listed in Table 3, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0532] Also provided are METxMET bispecific antigen-binding molecules comprising a D1 or D2 antigen-binding domain comprising an LCDR2 comprising an amino acid sequence selected from any of the light chain CDR2 (LCDR2) amino acid sequences listed in Table 3, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0533] Also provided are METxMET bispecific antigen-binding molecules comprising a D1 or D2 antigen-binding domain comprising an LCDR3 comprising an amino acid sequence selected from any of the light chain CDR3 (LCDR3) amino acid sequences listed in Table 3, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0534] Provided herein are MET×MET bispecific antigen-binding molecules comprising a D1 or D2 bispecific antigen-binding molecule comprising a pair of HCDR3 and LCDR3 amino acid sequences (HCDR3 / LCDR3), including any of the HCDR3 amino acid sequences listed in Table 3 paired with any of the LCDR3 amino acid sequences listed in Table 3. According to certain embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof comprising an HCDR3 / LCDR3 amino acid sequence included in any of the exemplary anti-MET antibodies listed in Table 3.

[0535] Additionally, METxMET bispecific antigen-binding molecules are provided that comprise a D1 or D2 antigen-binding domain that contains a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained in any of the exemplary anti-MET antibodies listed in Table 3.

[0536] In related embodiments, the present disclosure provides METxMET bispecific antigen-binding molecules comprising a D1 or D2 antigen-binding domain that includes a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained in the HCVR / LCVR amino acid sequence defined by any of the exemplary anti-MET antibodies listed in Table 3.

[0537] The METxMET bispecific antigen-binding molecules provided herein can comprise a D1 antigen-binding domain obtained from any of the anti-MET antibodies in Table 3 and a D2 antigen-binding domain obtained from any of the other anti-MET antibodies in Table 3. Non-limiting examples of METxMET bispecific antibodies of the present disclosure are depicted in Figure 10. Figure 10 is a diagram of a matrix illustrating the components of 272 exemplary METxMET bispecific antibodies. Each numbered cell in the matrix (numbered 1-272) identifies a unique bispecific antibody comprising a "D1" antigen-binding domain and a unique bispecific antibody comprising a "D2" antigen-binding domain, where the D1 antigen-binding domain comprises an immunoglobulin variable domain (HCVR / LCVR amino acid sequence pair) or CDR from the corresponding anti-MET antibody listed along the Y-axis, and the D2 antigen-binding domain comprises an immunoglobulin variable domain (HCVR / LCVR amino acid sequence pair) or CDR from the corresponding anti-MET antibody listed along the X-axis. Thus, for example, the METxMET bispecific antigen-binding molecule "Number 10" shown in the matrix comprises a D1 antigen-binding domain comprising the HCVR / LCVR pair or six-CDR set of the exemplary anti-MET antibody H4H13290P2 and a D2 antigen-binding domain comprising the HCVR / LCVR pair or six-CDR set of the exemplary anti-MET antibody H4H13321P2.

[0538] As a non-limiting, illustrative example, the present disclosure includes a MET×MET bispecific antigen-binding molecule comprising a D1 antigen-binding domain and a D2 antigen-binding domain, wherein the D1 antigen-binding domain comprises the HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 2012 / 2092, or a set of heavy and light chain CDRs (HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) comprising SEQ ID NOs: 2014-2016-2018-2094-2096-2098, and the D2 antigen-binding domain comprises the HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 2036 / 2092, or a set of heavy and light chain CDRs (HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) comprising SEQ ID NOs: 2038-2040-2042-2094-2096-2098. An exemplary MET×MET bispecific antibody having these sequence characteristics is designated H4H14639D, also referred to as bispecific antibody number 2076, and comprises D1 derived from H4H13306P2 and D2 derived from H4H13312P2.

[0539] Amino acid and nucleic acid sequences of the heavy and light chain variable regions of anti-MET and MET / MET antibodies

[0540] Table 3 lists the amino acid sequence identifiers for the heavy and light chain variable regions and CDRs of selected anti-MET antibodies disclosed herein (as noted above, all of the anti-MET antibodies of this disclosure have the same light chain variable region and therefore also the same light chain CDR sequences). The corresponding nucleic acid sequence identifiers are listed in Table 4.

[0541] [Table 3]

[0542] [Table 4]

[0543] Antibodies are typically referred to herein by a nomenclature consisting of an Fc prefix (e.g., "H4H"), followed by a numerical identifier (e.g., "13290," "13291," "13295," etc.), and finally a "P2" suffix, as shown in Tables 3 and 4. Thus, according to this nomenclature, antibodies may be referred to herein as, for example, "H4H13290P2," "H4H13291P2," "H4H13295P2," etc. The prefixes in antibody names used herein indicate the particular Fc region isotype of the antibody. Specifically, an "H4H" antibody has a human IgG4 Fc (all variable regions are fully human, as indicated by the initial "H" in the antibody name). As one of skill in the art will recognize, an antibody having a particular Fc isotype can be converted to an antibody having a different Fc isotype (e.g., an antibody having a murine IgG4 Fc can be converted to an antibody having a human IgG1, etc.), but in each case the variable domains (including the CDRs) indicated by the numerical identifiers in Tables 3 and 4 will remain the same, and the binding characteristics are expected to be the same or substantially similar regardless of the nature of the Fc domain.

[0544] Antibody conjugation Techniques and linkers for conjugating to residues of an antibody or antigen-binding fragment are known in the art. Exemplary amino acid linkages that can be used in the context of this embodiment include, for example, lysine (see, e.g., U.S. Pat. No. 5,208,020; U.S. Patent Application No. 2010 / 0129314; Hollander et al., Bioconjugate Chem., 2008, 19:358-361; International Publication No. WO2005 / 089808; U.S. Pat. No. 5,714,586; U.S. Patent Application No. 2013 / 0101546; and U.S. Patent Application No. 2012 / 0585592), cysteine ​​(see, e.g., U.S. Patent Application No. 2007 / 0258987; U.S. Patent Application No. WO2013 / 0585592), thiazolinone ... No. 55993, International Publication No. WO2013 / 055990, International Publication No. WO2013 / 053873, International Publication No. WO2013 / 053872, International Publication No. WO2011 / 130598, U.S. Patent Application No. 2013 / 0101546, and U.S. Patent No. 7,750,116), selenocysteine ​​(e.g., , WO 2008 / 122039, and Hofer et al., Proc. Natl. Acad. Sci., USA, 2008, 105:12451-12456), formylglycine (e.g., Carrico et al., Nat. Chem. Biol., 2007, 3:321-322, Agarwal et al., Proc. Natl. Acad. Sci., USA, 2013, 110:46-51, and Rabuka et al., Nat. Protocols, 2012, 10:1052-1067), unnatural amino acids (e.g., WO 2013 / 068874, and WO 2012 / 166559), and acidic amino acids (e.g., WO 2012 / 05982). Lysine conjugation can also proceed via NHS (N-hydroxysuccinimide). Linkers can also be conjugated to cysteine ​​residues, including those of cleaved interchain disulfide bonds, by forming a carbon bridge between the thiols (see, e.g., U.S. Pat. Nos. 9,951,141 and 9,950,076).Linkers can also be conjugated to antigen-binding proteins via attachment to carbohydrates (see, e.g., U.S. Patent No. 2008 / 0305497, WO 2014 / 065661, and Ryan et al., Food & Agriculture Immunol., 2001, 13:127-130) and disulfide linkers (see, e.g., WO 2013 / 085925, WO 2010 / 010324, WO 2011 / 018611, and Shaunak et al., Nat. Chem. Biol., 2006, 2:312-313). Site-specific conjugation techniques can also be used to target conjugation to specific residues of an antibody or antigen-binding protein (see, e.g., Schumacher et al., J Clin Immunol (2016) 36(Suppl 1):100). In specific embodiments discussed in more detail below, site-specific conjugation techniques include transglutaminase-mediated glutamine conjugation (see, e.g., Schibli, Angew Chemie Inter Ed. 2010, 49, 9995).

[0545] Payloads of the present disclosure that are linked through lysines and / or cysteines, for example, via maleimide or amide conjugation, are included within the scope of the present disclosure.

[0546] In some embodiments, the protein drug conjugations of the present disclosure are produced by a two-step process, where step 1 is the conjugation of a lysine-based linker, e.g., an NHS-ester linker, and step 2 is a payload conjugation reaction (e.g., a 1,3-cycloaddition reaction).

[0547] In some embodiments, the protein drug conjugations of the present disclosure are produced by a two-step process, where step 1 is the conjugation of a cysteine-based linker, e.g., a maleimide linker, and step 2 is a payload conjugation reaction (e.g., a 1,3-cycloaddition reaction).

[0548] In some embodiments, the protein drug conjugations of the present disclosure are produced by a two-step process, where step 1 is a transglutaminase-mediated site-specific conjugation and step 2 is a payload conjugation reaction (e.g., a 1,3-cycloaddition reaction).

[0549] Step 1: Transglutaminase-mediated site-specific conjugation In some embodiments, proteins (e.g., antibodies) can be modified according to known methods to provide glutaminyl-modified proteins. Techniques for conjugating antibodies and primary amine compounds are known in the art. Site-specific conjugation techniques are utilized herein to target conjugation to glutamine using transglutaminase-mediated glutamine conjugation (see, for example, Schibli, Angew Chemie Inter Ed. 2010, 49, 9995).

[0550] A primary amine-containing compound (e.g., linker L1) of the present disclosure can be conjugated to one or more glutamine residues of a binder (e.g., a protein, e.g., an antibody) via chemoenzymatic conjugation using transglutaminase (see, e.g., Protein Conjugate Chem. 2014, 25, 569-578 by Dennler et al. and International Publication No. WO 2017 / 147542). For example, in the presence of transglutaminase, one or more glutamine residues of an antibody can be linked to a primary amine linker compound. Briefly, in some embodiments, a binder having a glutamine residue (e.g., gln295, i.e., Q295 residue) is treated with the above-described primary amine-containing linker L1 in the presence of the enzyme transglutaminase. In some embodiments, the binder is not aglycosylated. In some embodiments, the binder is deglycosylated.

[0551] In some embodiments, a binding agent (e.g., a protein, e.g., an antibody) comprises at least one glutamine residue in at least one polypeptide chain sequence. In some embodiments, a binding agent comprises two heavy chain polypeptides, each having a gln295 residue. In further embodiments, a binding agent comprises one or more glutamine residues at a site separate from 295 on the heavy chain.

[0552] In some embodiments, binding agents, such as antibodies, can be prepared by site-directed mutagenesis to insert a glutamine residue at a single site without impairing the function or binding of the antibody. For example, the present specification includes antibodies with the Asn297Gln (N297Q) mutation described herein. In some embodiments, antibodies with the gln295 residue and / or N297Q mutation contain one or more naturally occurring additional glutamine residues in their variable regions, which are accessible to transglutaminase and therefore allow conjugation to a linker or linker payload. An exemplary naturally occurring glutamine residue can be found, for example, at Q55 in the light chain. In such cases, binding agents, such as antibodies, conjugated via transglutaminase may have higher than expected LAR values ​​(e.g., LARs greater than 4). All such antibodies can be isolated from natural or artificial sources.

[0553] In certain embodiments of the present disclosure, the linker to antibody ratio, or LAR, is 1, 2, 3, 4, 5, 6, 7, or 8 linker L1 molecules per antibody. In some embodiments, the LAR is 1 to 8. In some embodiments, the LAR is 1 to 6. In some embodiments, the LAR is 2 to 4. In some cases, the LAR is 2 to 3. In some cases, the LAR is 0.5 to 3.5. In some embodiments, the LAR is about 1, about 1.5, about 2, about 2.5, about 3, or about 3.5. In some embodiments, the LAR is 2. In some embodiments, the LAR is 4.

[0554] Step 2: Payload conjugation reaction In some embodiments, the linker L1 of the present disclosure comprises a branching unit B comprising at least one reactive group B' that allows for further reaction after transglutamination. In these embodiments, the glutaminyl-modified protein (e.g., an antibody) is capable of further reaction with a reactive payload compound or a reactive linker payload compound (e.g., L2-P disclosed herein) to form a protein-payload conjugate. More specifically, the reactive linker payload compound L2-P can comprise a reactive group B" that can react with the reactive group B' of the linker L1. In some embodiments, the reactive group B' of the present disclosure comprises a moiety capable of undergoing a 1,3-cycloaddition reaction. In some embodiments, the reactive group B' is an azide. In some embodiments, the reactive group B" comprises an alkyne (e.g., a terminal alkyne or an internal alkyne under strain). In some embodiments of the present disclosure, the reactive group B' is compatible with the conjugation agent and transglutamination reaction conditions.

[0555] In some embodiments of the present disclosure, the linker L1 molecule comprises a branching unit B comprising one reactive group B'. In some embodiments of the present disclosure, the linker L1 molecule comprises a branching unit B comprising more than one reactive group B'.

[0556] In certain embodiments, the reactive linker payload L2-P comprises one payload molecule (n=1). In certain other embodiments, the reactive linker payload L2-P comprises two or more payload molecules (n≧2). In certain embodiments, the reactive linker payload L2-P comprises 1 to 12 payload molecules, 1 to 10 payload molecules, 1 to 8 payload molecules, 1 to 6 payload molecules, 1 to 4 payload molecules, or 1 to 2 payload molecules.

[0557] In one embodiment, the reactive linker payload L2-P contains one payload molecule. When such an L2-P reacts with BA-L1-B, the DAR is approximately equal to the LAR of BA-L1-B. For example, if an L2-P containing one payload molecule reacts with BA-L1-B having a LAR of 4 (e.g., via Q295 and N297Q transglutamination), the resulting protein-drug conjugate will have a DAR of 4.

[0558] In one embodiment, the reactive linker payload L2-P contains two payload molecules. When such an L2-P reacts with BA-L1-B, the DAR is approximately twice the LAR of BA-L1B. For example, if an L2-P containing two payload molecules reacts with BA-L1-B, which has a LAR of 4 (e.g., via Q295 and N297Q transglutamination), the resulting protein-drug conjugate will have a DAR of 8.

[0559] For example, if L2-P containing three payload molecules reacts with BA-L1-B having a LAR of 8 (e.g., via Q295 and N297Q transglutamination of a branched L1-B unit containing two B groups), the resulting protein drug conjugate will have a DAR of 24.

[0560] In certain embodiments of the present disclosure, the drug-to-antibody ratio, or DAR (e.g., abbreviated with a lowercase n), is about 1 to about 30, about 1 to about 24, about 1 to about 20, about 1 to about 16, about 1 to about 12, about 1 to about 10, about 1 to about 8, or about 1, 2, 3, 4, 5, 6, or 8 payload molecules per antibody. In some embodiments, the DAR is 1 to 30. In some embodiments, the DAR is 1 to 24. In some embodiments, the DAR is 1 to 16. In some embodiments, the DAR is 1 to 8. In some embodiments, the DAR is 1 to 6. In some embodiments, the DAR is 2 to 4. In some cases, the DAR is 2 to 3. In some cases, the DAR is 0.5 to 3.5. In some cases, the DAR is 10 to 14. In some cases, the DAR is 14 to 18. In some cases, the DAR is 20 to 24.5. In some embodiments, the DAR is about 1, about 1.5, about 2, about 2.5, about 3, or about 3.5. In some embodiments, the DAR is 2. In some embodiments, the DAR is 4. In some embodiments, the DAR is 8. In some embodiments, the DAR is 12. In some embodiments, the DAR is 16. In some embodiments, the DAR is 24.

[0561] In one aspect, the present disclosure provides a compound of formula (A): BA-(Gln-NH-L1-B-(-L2-(-M-Dxd) m ) k ) n (A) A method for producing a compound having a structure according to the formula: BA is an antibody or antigen-binding fragment thereof; Gln is a glutamine residue, L1 is the first linker, B is a branching unit containing at least one addition of a group B' and a group B''; L2 is a second linker covalently attached to the branching unit B via at least one group B″; M is absent or

[0562] [ka] wherein R, R', and R'' are independently at each occurrence hydrogen or C1-C4 alkyl, or R' and R'' together form a 5- or 6-membered ring; Dxd is the formula (P):

[0563] [ka] an antitumor drug comprising a structure according to k and m are independently an integer of 1 to 12, and n is an integer of 1 to 30; The above method is (a) contacting a BA containing at least one glutamine residue Gln (BA-Gln-NH2) with a compound L1-B in the presence of a transglutaminase, wherein the branching unit B contains at least one group B'; (b) The product of step (a) is converted into compound L2-(-M-Dxd) m wherein the linker L2 comprises at least one group B″, and one of the groups B′ and B″ is —N3 and

[0564] [ka] and the other of group B′ and group B″ is selected from

[0565] [ka] wherein Q is C or N; (c) isolating the compound of formula (I) produced; The present invention provides a method comprising:

[0566] In one aspect, the present disclosure provides a compound of formula (A): BA-(Gln-NH-L1-B-(-L2-(-M-Dxd) m )k ) n (A) 1. A method for producing a compound having a structure according to wherein BA is an antibody or antigen-binding fragment thereof, Gln is a glutamine residue, L1 is a first linker as described above, B is a branching unit comprising an appendage of at least one of group B' and group B'' as described above, L2 is a second linker as described above that is covalently bonded to branching unit B via at least one group B'' as described above, and M is absent or

[0567] [ka] wherein R, R′, and R″ are as described above, and Dxd is a group represented by the formula (P):

[0568] [ka] wherein k and m are independently integers of 1 to 12, and n is an integer of 1 to 30; The above method is (a) A compound L1-B in which the branching unit B contains at least one group B' is reacted with a compound L2-(-M-Dxd) m wherein the linker L2 comprises at least one group B″ capable of covalently bonding to group B′, and one of groups B′ and B″ is selected from the group consisting of —N3 and

[0569] [ka] and the other of group B′ and group B″ is selected from

[0570] [ka] wherein Q is C or N, and the contact forms L1-B-(-L2-(-M-Dxd) m ) kand (b) In the presence of transglutaminase, BA containing at least one glutamine residue Gln (BA-Gln-NH) is converted to L1-B-(L2-(-M-Dxd) m ) k contacting the product with (c) isolating the compound of formula (I) produced; The present invention provides a method comprising:

[0571] In one aspect, the present disclosure provides a compound of formula (I): BA-(Gln-NH-L1-B-(-L2-M-Dxd) k ) n (I) A method for producing a compound having a structure according to the formula: BA is an antibody or antigen-binding fragment thereof; Gln is a glutamine residue, L1 is the first linker, B is a branching unit containing at least one addition of a group B' and a group B''; L2 is a second linker covalently attached to the branching unit B via at least one group B″; M is absent or

[0572] [ka] wherein R, R', and R'' are independently at each occurrence hydrogen or C1-C4 alkyl, or R' and R'' together form a 5- or 6-membered ring; Dxd is the formula (P):

[0573] [ka] an antitumor drug comprising a structure according to k is an integer from 1 to 12, and n is an integer from 1 to 30. The above method is (a) contacting a BA containing at least one glutamine residue Gln (BA-Gln-NH2) with a compound L1-B in the presence of a transglutaminase, wherein the branching unit B contains at least one group B'; (b) contacting the product of step (a) with k or more equivalents of compound L2-M-Dxd, wherein linker L2 comprises at least one group B″ capable of covalently bonding to group B′, and one of groups B′ and B″ is selected from the group consisting of —N3 and

[0574] [ka] and the other of group B′ and group B″ is selected from

[0575] [ka] wherein Q is C or N; (c) isolating the compound of formula (I) produced; The present invention provides a method comprising:

[0576] In one aspect, the present disclosure provides a compound of formula (I): BA-(Gln-NH-L1-B-(-L2-M-Dxd) k ) n (I) 1. A method for producing a compound having a structure according to wherein BA is an antibody or antigen-binding fragment thereof, Gln is a glutamine residue, L1 is a first linker as described above, B is a branching unit comprising an appendage of at least one of group B' and group B'' as described above, L2 is a second linker as described above that is covalently bonded to branching unit B via at least one group B'' as described above, and M is absent or

[0577] [ka] wherein R, R′, and R″ are as described above, and Dxd is a group represented by the formula (P):

[0578] [ka] wherein k is an integer of 1 to 12 and n is an integer of 1 to 30; The above method is (a) contacting a compound L1-B, in which the branching unit B comprises at least one group B', with k or more equivalents of a compound L2-M-Dxd, in which the linker L2 comprises at least one group B'', to form L1-B-(-L2-M-Dxd) k is produced, and one of the groups B' and B'' is -N3 and

[0579] [ka] and the other of group B′ and group B″ is selected from

[0580] [ka] wherein Q is C or N; (b) In the presence of transglutaminase, a binder BA containing at least one glutamine residue Gln (BA-Gln-NH) is added to the L1-B-(L2(M-Dxd) k contacting the product with (c) isolating the compound of formula (I) produced; The present invention provides a method comprising:

[0581] In one aspect, the present disclosure provides a compound of formula (III): BA-(Gln-NH-L2'-P) n (III) A method for producing a compound having a structure according to the formula: BA is an antibody or an antigen-binding fragment thereof, Gln is a glutamine residue, and L2'-P is H2N-SP1-B2-(SP2-AA-SP3-M-Dxd) as described above. p and n is an integer from 1 to 30. SP1 is either not present or

[0582] [ka] is a first spacer unit selected from the group consisting of: B2 is absent or a branching unit; SP2 is not present or 1-6 Alkyl, -(CH2-CH2-O) v -, -NH-, -C(O)-, -NH-C(O)-, -NH-(CH2) u -, -NH-(CH2) u -C(O)-, -NH-(CH2-CH2-O) v -, -NH-(CH2-CH2-O) v -C(O)-, -NH-(CH2-CH2-O) v -(CH2) u -, -NH-(CH2-CH2-O) v -(CH2) u -C(O)-, -(CH2) u -NH-C(O)-, -NH-(CH2) u -NH-C(O)-, -NH-(CH2) u a second spacer unit selected from the group consisting of: —C(O)—NH—, —C(O)—NH—, or combinations thereof; and the subscripts u and v are independently integers from 1 to 8; AA is a peptide unit containing zero or two to four amino acids; SP3 is either not present or

[0583] [ka] and a third spacer unit selected from the group consisting of: cis not present in each occurrence independently, or

[0584] [ka] is a group selected from M is absent or

[0585] [ka] wherein R, R', and R'' are independently at each occurrence hydrogen or C1-C4 alkyl, or R' and R'' together form a 5- or 6-membered ring; Dxd is the formula (P):

[0586] [ka] is an antitumor drug having the structure p is an integer from 1 to 30, The above method is (b) contacting BA containing at least one glutamine residue Gln (BA-Gln-NH2) with L2'-P in the presence of transglutaminase; (c) isolating the compound of formula (III) produced; The present invention provides a method comprising:

[0587] In one aspect, the present disclosure provides a compound of formula (I): BA-(Gln-NH-L1-B-(-L2-M-Dxd) k ) n (I) 1. A method for producing a compound having a structure according to wherein BA is an antibody or antigen-binding fragment thereof, Gln is a glutamine residue, L1 is a first linker as described above, B is a branching unit comprising at least one group B' as described above, L2 is a second linker as described above that is covalently bonded to branching unit B via at least one group B'' as described above, and groups B' and B'' form at least one adduct as described above, and M is absent or

[0588] [ka] wherein R, R′, and R″ are as described above, and Dxd is a group represented by the formula (P):

[0589] [ka] wherein k is an integer of 1 to 12 and n is an integer of 1 to 30; The above method is (a) contacting a binder BA containing at least one glutamine residue Gln (BA-Gln-NH2) with a compound L1-B in the presence of transglutaminase, wherein the branching unit B is -N3,

[0590] [ka] wherein the contacting produces BA-Gln-NH-L1-B; (b) contacting the product of step (a) with k or more equivalents of compound L2-M-Dxd, wherein linker L2 comprises at least one group B″ capable of covalently bonding to group B′; (c) isolating the compound of formula (I) produced; The present invention provides a method comprising:

[0591] In another aspect, the present disclosure provides a compound of formula (I): BA-(Gln-NH-L1-B-(-L2-M-Dxd)k ) n (I) 1. A method for producing a compound having a structure according to wherein BA is an antibody or antigen-binding fragment thereof, Gln is a glutamine residue, L1 is a first linker as described above, B is a branching unit comprising at least one group B' as described above, L2 is a second linker as described above that is covalently bonded to branching unit B via at least one group B'' as described above, and groups B' and B'' form at least one adduct as described above, and M is absent or

[0592] [ka] wherein R, R′, and R″ are as described above, and Dxd is a group represented by the formula (P):

[0593] [ka] wherein k is an integer of 1 to 12 and n is an integer of 1 to 30; The above method is (a) Branching unit B is -N3,

[0594] [ka] and contacting a compound L1-B containing at least one group B' selected from the group k and (b) In the presence of transglutaminase, a binder BA containing at least one glutamine residue Gln (BA-Gln-NH) is added to the L1-B-(L2(M-Dxd) k contacting the product with (c) isolating the compound of formula (I) produced; The present invention provides a method comprising:

[0595] In one embodiment, the glutamine residue Gln is naturally present in the CH2 or CH3 domain of the BA. In another embodiment, the glutamine residue Gln is introduced into the BA by modifying one or more amino acids. In one embodiment, the Gln is Q295 or N297Q.

[0596] In one embodiment, the transglutaminase is a microbial transglutaminase (MTG). In one embodiment, the transglutaminase is a bacterial transglutaminase (BTG).

[0597] In one embodiment, M is absent. In another embodiment, M-Dxd is

[0598] [ka] wherein R is hydrogen or C1-C4 alkyl;

[0599] [ka] represents the point of attachment to L2.

[0600] In one embodiment, the compound L2-Dxd is

[0601] [ka]

[0602] [ka]

[0603] [ka]

[0604] [ka] or a pharmaceutically acceptable salt thereof.

[0605] In one embodiment, compound L2-Dxd includes an optional branching unit B2. In such an embodiment, compound L2-Dxd comprises two or more Dxd units.

[0606] In one embodiment, the branching unit B2 is

[0607] [ka] has a structure selected from the group consisting of:

[0608] In one embodiment, the compound L2-Dxd is

[0609] [ka]

[0610] [ka]

[0611] [ka]

[0612] [ka]

[0613] [ka]

[0614] [ka] or a pharmaceutically acceptable salt thereof.

[0615] Therapeutic Formulation and Administration The present disclosure provides pharmaceutical compositions comprising the protein drug conjugates of the present disclosure.

[0616] In one aspect, the present disclosure provides a composition comprising a population of protein drug conjugates of the present disclosure having a drug-to-antibody ratio (DAR) of about 0.5 to about 30.0.

[0617] In one embodiment, the composition has a DAR of about 1.0 to about 2.5.

[0618] In one embodiment, the composition has a DAR of about 2.

[0619] In one embodiment, the composition has a DAR of about 3.0 to about 4.5.

[0620] In one embodiment, the composition has a DAR of about 4.

[0621] In one embodiment, the composition has a DAR of about 6.5 to about 8.5.

[0622] In one embodiment, the composition has a DAR of about 8.

[0623] In one embodiment, the composition has a DAR of about 10 to about 14.

[0624] In one embodiment, the composition has a DAR of about 12.

[0625] In one embodiment, the composition has a DAR of about 14 to about 18.

[0626] In one embodiment, the composition has a DAR of about 16.

[0627] In one embodiment, the composition has a DAR of about 20 to about 24.5.

[0628] In one embodiment, the composition has a DAR of about 24.

[0629] The compositions of the present disclosure are formulated with suitable carriers, excipients, and other agents to improve transport, delivery, tolerability, etc. Many suitable formulations can be found in "Remington's Pharmaceutical Sciences," Mack Publishing Company, Easton, PA, a formulary known to all pharmacists. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (e.g., LIPOFECTIN™, Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsion carbowax (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also "Compendium of Excipients for Parenteral Formulations" by Powell et al., PDA (1998) J Pharm Sci Technol 52:238-311.

[0630] The dose of a protein drug conjugate administered to a patient may vary depending on the patient's age and size, the target disease or condition, the route of administration, and other factors. Suitable doses are typically calculated based on body weight and body surface area. When using the protein drug conjugate of the present disclosure for therapeutic purposes in adult patients, it may be convenient to administer the protein drug conjugate of the present disclosure intravenously at a single dose of about 0.01 to about 20 mg / kg, more preferably about 0.02 to 0.07 mg / kg, about 0.03 to about 5 mg / kg, or about 0.05 to about 3 mg / kg. The frequency and duration of treatment can be adjusted depending on the severity of the disease. Effective dosages and schedules for administering a protein drug conjugate may be determined empirically; for example, patient progress is monitored by periodic evaluation, and the dosage adjusted accordingly. Furthermore, interspecies scaling of dosages can be performed using methods known in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

[0631] Various delivery systems are known and can be used to administer the pharmaceutical compositions of the present disclosure, such as those encapsulated in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, or receptor-mediated endocytosis (see, e.g., Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions may be administered by any convenient route, such as injection or bolus injection, or absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal, intestinal mucosa, etc.), and may be administered together with other bioactive agents. Administration can be systemic or local.

[0632] The pharmaceutical compositions of the present disclosure can be delivered subcutaneously or intravenously using a standard needle and syringe. Additionally, for subcutaneous delivery, pen delivery devices are easily adapted to deliver the pharmaceutical compositions of the present disclosure. Such pen delivery devices can be reusable or disposable. Reusable pen delivery devices generally utilize a replaceable cartridge containing the pharmaceutical composition. Once all of the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be quickly discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. Disposable pen delivery devices do not have a replaceable cartridge. Instead, disposable pen delivery devices are pre-filled with the pharmaceutical composition held in a reservoir within the device. Once the pharmaceutical composition is released and the reservoir is emptied, the entire device is discarded.

[0633] Numerous reusable pen delivery devices and autoinjector delivery devices are adapted for subcutaneous delivery of the pharmaceutical compositions of the present disclosure, including, but not limited to, the AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), the DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), the HUMALOG MIX 75 / 25™ pen, the HUMALOG™ pen, the HUMALIN 70 / 30™ pen (Eli Lilly and Co., Indianapolis, IN), the NOVOPEN™ I, II, and III (Novo Nordisk, Copenhagen, Denmark), the NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), the BD™ pen (Becton Dickinson, Franklin Lakes, NJ), the OPTIPEN™, the OPTIPEN PRO™, the OPTIPEN™, and the OPTIPEN™ pen, to name a few. Examples of disposable pen delivery devices applicable to subcutaneous delivery of the pharmaceutical composition of the present disclosure include, but are not limited to, SOLOSTAR pen (Sanofi-Aventis), FLEXPEN pen (Novo Nordisk), KWIKPEN pen (Eli Lilly), SURECLICK pen (Amgen, Thousand Oaks, CA), PENLET pen (Haselmeier, Stuttgart, Germany), EPIPEN pen (Dey, LP), HUMIRA pen (Abbott Labs, Abbott Park, IL), to name a few.

[0634] In certain circumstances, pharmaceutical compositions can be delivered in controlled-release systems. In one embodiment, a pump may be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Press, Boca Raton, Florida). In yet another embodiment, a controlled-release system can be placed near the target of the composition, so that only systemic administration is necessary (see, e.g., Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled-release systems are discussed in the review by Langer, 1990, Science 249:1527-1533.

[0635] Injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal, and intramuscular injections or infusions. These injectable preparations can be prepared by known methods. For example, injectable preparations can be prepared by dissolving, suspending, or emulsifying the antibody or its salt in a sterile aqueous or oily medium conventionally used for injections. Aqueous media for injection include, for example, saline, isotonic solutions containing glucose or other additives, and the like. These may be used in combination with suitable solubilizers such as alcohols (e.g., ethanol), polyhydric alcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants (e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)). Sesame oil and soybean oil are used as oily media, and may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. The injections prepared in this manner are preferably filled into appropriate ampoules.

[0636] Conveniently, the above-mentioned oral or parenteral pharmaceutical compositions are prepared into dosage forms in unit doses suitable for the dosage of the active ingredient. Examples of such dosage forms in unit doses include tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the antibody contained therein is usually about 5 to about 500 mg per dosage form in a unit dose. In particular, the antibody is preferably contained in an amount of about 5 to about 100 mg in injection forms, and about 10 to about 250 mg in other dosage forms.

[0637] Therapeutic Applications of Protein Drug Conjugates, Linker Payloads, and Payloads In another aspect, the protein drug conjugates, eg, ADCs, disclosed herein are useful for the treatment, prevention, and / or amelioration of, among other diseases, disorders, or conditions in need of treatment.

[0638] In one embodiment, the present invention provides a method of treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound according to the present disclosure (e.g., antibody drug conjugate, linker payload, and / or payload), or a composition comprising any compound according to the present disclosure.

[0639] In one embodiment, the protein drug conjugates, e.g., ADCs, disclosed herein are useful for the treatment of cancer. In one embodiment, the protein drug conjugates, e.g., ADCs, disclosed herein are useful for the treatment of a cancer selected from the group consisting of breast cancer, ovarian cancer, prostate cancer, lung cancer, liver cancer, or brain cancer. In one embodiment, the protein drug conjugates, e.g., ADCs, disclosed herein are useful for the treatment of HER2+ breast cancer. In one embodiment, the protein drug conjugates, e.g., ADCs, disclosed herein are useful for the treatment of prostate cancer.

[0640] In one aspect, the present disclosure provides a method for selectively delivering a compound to a cell. In one embodiment, the method for selectively delivering a compound to a cell comprises linking the compound to a targeted antibody. In one embodiment, the compound is a payload as described above. In one embodiment, the cell is a mammalian cell. In one embodiment, the cell is a human cell. In one embodiment, the cell is a cancer cell. In one embodiment, the cancer cell is selected from the group consisting of a breast cancer cell, an ovarian cancer cell, a prostate cancer cell, a lung cancer cell, a liver cancer cell, or a brain cancer cell.

[0641] In one embodiment, the present disclosure provides a compound of the structure PI:

[0642] [ka] or a pharmaceutically acceptable salt thereof, wherein R1, R2, R3, and R4 are independently hydrogen or alkyl, e.g., C1-C 12 alkyl, C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl, or R2 and R3 together form a 5- or 6-membered ring.

[0643] In one aspect, the present disclosure provides a method for selectively targeting an antigen on a cell surface with a compound. In one embodiment, the method for selectively targeting an antigen on a cell surface with a compound comprises linking the compound to a targeted antibody. In one embodiment, the compound is a payload as described above. In one embodiment, the cell is a mammalian cell. In one embodiment, the cell is a human cell. In one embodiment, the cell is a cancer cell. In one embodiment, the cancer cell is selected from the group consisting of a breast cancer cell, an ovarian cancer cell, a prostate cancer cell, a lung cancer cell, a liver cancer cell, or a brain cancer cell.

[0644] In one embodiment, the present disclosure provides a compound of the structure PI:

[0645] [ka] or a pharmaceutically acceptable salt thereof, wherein R1, R2, R3, and R4 are independently hydrogen or alkyl, e.g., C1-C 12 alkyl, C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl, or R2 and R3 together form a 5- or 6-membered ring.

[0646] Anti-HER2 antibody-drug conjugate In certain embodiments, the protein drug conjugates, e.g., ADCs, disclosed herein are useful for treating, preventing, and / or ameliorating, inter alia, any disease or disorder associated with or mediated by HER2 expression or activity, or treatable by binding HER2 without competition with modified LDL, and / or by promoting internalization of the HER2 receptor and / or reducing the number of cell surface receptors.

[0647] The protein drug conjugates of the present disclosure (and therapeutic compositions comprising the same) are particularly useful for treating any disease or disorder in which stimulating, activating, and / or targeting an immune response is beneficial. Specifically, anti-HER2 protein drug conjugates, including both monospecific anti-HER2 antibodies and bispecific anti-HER2 / HER2 antibodies of the present disclosure, can be useful for treating, preventing, and / or ameliorating any disease or disorder associated with or mediated by HER2 expression or activity or the proliferation of HER2+ cells. The mechanism of action for achieving the therapeutic methods of the present disclosure involves killing HER2-expressing cells in the presence of effector cells, for example, by CDC, apoptosis, ADCC, phagocytosis, or a combination of two or more of these mechanisms. HER2-expressing cells that can be inhibited or killed using the protein drug conjugates of the present disclosure include, for example, breast tumor cells.

[0648] In one embodiment, the protein drug conjugates (and therapeutic compositions and dosage forms comprising same) of the present disclosure comprise: a first antigen-binding domain (D1); and a second antigen-binding domain (D2); and a bispecific antigen-binding molecule comprising: D1 specifically binds the first epitope of human HER2; D2 specifically binds a second epitope on human HER2.

[0649] In one embodiment of the above, D1 and D2 do not compete with each other for binding to human HER2.

[0650] The protein drug conjugates of the present disclosure can be used to treat primary and / or metastatic tumors occurring in, for example, the prostate, bladder, cervix, lung, colon, kidney, breast, pancreas, stomach, uterus, and / or ovary. In certain embodiments, the protein drug conjugates of the present disclosure are used to treat one or more of prostate cancer, bladder cancer, cervical cancer, lung cancer, colon cancer, kidney cancer, breast cancer, pancreatic cancer, stomach cancer, uterine cancer, and ovarian cancer. According to certain embodiments of the present disclosure, anti-HER2 antibodies or anti-HER2 / HER2 bispecific antibodies are useful for treating patients with breast cancer cells that are IHC2+ or higher. According to other related embodiments of the present disclosure, methods are provided that include administering an anti-HER2 antibody or anti-HER2 / HER2 antibody disclosed herein to a patient with breast cancer cells that are IHC2+ or higher. Analytical / diagnostic methods known in the art, such as tumor scanning, can be used to determine whether a patient has a castration-resistant tumor.

[0651] In certain embodiments, the present disclosure also includes methods of treating residual cancer in a subject. The term "residual cancer" refers to the presence or persistence of one or more cancer cells in a subject after treatment with an anti-cancer therapy.

[0652] The protein drug conjugates of the present disclosure (and therapeutic compositions comprising them) are particularly useful for treating any disease or disorder in which stimulating, activating, and / or targeting the immune response is beneficial. Specifically, protein drug conjugates comprising anti-HER2 antibodies or anti-HER2 / HER2 antibodies of the present disclosure can be useful for treating, preventing, and / or ameliorating any disease or disorder associated with or mediated by HER2 expression or activity, or the proliferation of HER2+ cells. The mechanism of action for achieving the therapeutic methods of the present disclosure involves killing HER2-expressing cells in the presence of effector cells, for example, by CDC, apoptosis, ADCC, phagocytosis, or a combination of two or more of these mechanisms. HER2-expressing cells that can be inhibited or killed using the protein drug conjugates of the present disclosure include, for example, breast tumor cells.

[0653] According to certain aspects, the present disclosure provides methods of treating a disease or disorder associated with HER2 expression (e.g., breast cancer), the method comprising administering to a subject one or more of the anti-HER2 protein drug conjugates or anti-HER2 / HER2 bispecific protein drug conjugates described elsewhere herein after determining that the subject has breast cancer (e.g., IHC2+ breast cancer). For example, the present disclosure includes methods of treating breast cancer, the method comprising administering to a patient an anti-HER2 antibody or antigen-binding molecule, or a protein drug conjugate comprising an anti-HER2 / HER2 bispecific antibody or antigen-binding molecule, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 4 months, 6 months, 8 months, 1 year or more after the subject has received hormone therapy (e.g., anti-androgen therapy).

[0654] In certain embodiments, the present disclosure also includes the use of an anti-HER2 antibody of the present disclosure in the manufacture of a medicament for the treatment of a disease or disorder (e.g., cancer) associated with or caused by cells expressing HER2. In one aspect, the present disclosure relates to a protein-drug conjugate comprising an anti-HER2 antibody or antigen-binding fragment, or a HER2 / HER2 bispecific antibody or antigen-binding fragment, disclosed herein, for use in medicine. In one aspect, the present disclosure relates to a compound comprising an antibody-drug conjugate (ADC) disclosed herein, for use in medicine.

[0655] Anti-STEAP2 antibody-drug conjugate In certain embodiments, the protein drug conjugates, e.g., ADCs, disclosed herein are useful for treating, preventing, and / or ameliorating, inter alia, any disease or disorder associated with or mediated by the expression or activity of STEAP2, or treatable by binding STEAP2 without competition with modified LDL, and / or by promoting internalization of the STEAP2 receptor and / or reducing the number of cell surface receptors.

[0656] The protein drug conjugates of the present disclosure (and therapeutic compositions comprising them) are particularly useful for treating any disease or disorder in which stimulating, activating, and / or targeting the immune response is beneficial. Specifically, the HER2 or HER2HER2-STEAP2 protein drug conjugates of the present disclosure can be useful for treating, preventing, and / or ameliorating any disease or disorder associated with or mediated by HER2 expression or activity, or the proliferation of HER2+ cells. The mechanism of action for achieving the therapeutic methods of the present disclosure involves killing cells expressing STEAP2 in the presence of effector cells, for example, by CDC, apoptosis, ADCC, phagocytosis, or a combination of two or more of these mechanisms. Cells expressing STEAP2 that can be inhibited or killed using the protein drug conjugates of the present disclosure include, for example, prostate tumor cells.

[0657] The protein drug conjugates of the present disclosure can be used to treat primary and / or metastatic tumors occurring, for example, in the prostate, bladder, cervix, lung, colon, kidney, breast, pancreas, stomach, uterus, and / or ovary. In certain embodiments, the protein drug conjugates of the present disclosure are used to treat one or more of prostate cancer, bladder cancer, cervical cancer, lung cancer, colon cancer, kidney cancer, breast cancer, pancreatic cancer, stomach cancer, uterine cancer, and ovarian cancer. Analytical / diagnostic methods known in the art, such as tumor scanning, can be used to determine whether a patient has a tumor that is castration-resistant.

[0658] In certain embodiments, the present disclosure also includes methods of treating residual cancer in a subject. The term "residual cancer" refers to the presence or persistence of one or more cancer cells in a subject after treatment with an anti-cancer therapy.

[0659] According to certain aspects, the present disclosure provides a method of treating a disease or disorder associated with STEAP2 expression (e.g., prostate cancer), comprising administering to a subject one or more of the anti-STEAP2 protein-drug conjugates described elsewhere herein after the subject has been determined to have prostate cancer. For example, the present disclosure includes a method of treating prostate cancer, comprising administering to a patient a protein-drug conjugate comprising an anti-STEAP antibody or antigen-binding molecule 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 4 months, 6 months, 8 months, 1 year or more after the subject has received hormone therapy (e.g., anti-androgen therapy).

[0660] In certain embodiments, the present disclosure also includes the use of an anti-STEAP2 antibody of the present disclosure in the manufacture of a medicament for the treatment of a disease or disorder (e.g., cancer) associated with or caused by cells that express STEAP2. In one aspect, the present disclosure relates to a protein-drug conjugate comprising an anti-STEAP2 antibody or antigen-binding fragment disclosed herein for use in medicine. In one aspect, the present disclosure relates to a compound comprising an antibody-drug conjugate (ADC) disclosed herein for use in medicine.

[0661] Anti-MET antibody-drug conjugate In certain embodiments, the protein drug conjugates, e.g., ADCs, disclosed herein are useful for treating, preventing, and / or ameliorating, inter alia, any disease or disorder associated with or mediated by MET expression or activity, or treatable by binding MET without competition with modified LDL, and / or by promoting internalization of the MET receptor and / or reducing the number of cell surface receptors.

[0662] The protein drug conjugates of the present disclosure (and therapeutic compositions comprising the same) are particularly useful for treating any disease or disorder in which stimulating, activating, and / or targeting the immune response is beneficial. Specifically, the anti-MET or anti-MET / MET protein drug conjugates of the present disclosure may be useful for treating, preventing, and / or ameliorating any disease or disorder associated with or mediated by MET expression or activity, or the proliferation of MET+ cells. The mechanism of action by which the therapeutic methods of the present disclosure are achieved involves killing MET-expressing cells in the presence of effector cells, e.g., by CDC, apoptosis, ADCC, phagocytosis, or a combination of two or more of these mechanisms. Cells expressing MET that can be inhibited or killed using the protein drug conjugates of the present disclosure include, for example, lung tumor cells.

[0663] The protein drug conjugates of the present disclosure can be used to treat primary and / or metastatic tumors occurring, for example, in the prostate, bladder, cervix, lung, colon, kidney, breast, pancreas, stomach, uterus, and / or ovary. In certain embodiments, the protein drug conjugates of the present disclosure are used to treat one or more of prostate cancer, bladder cancer, cervical cancer, lung cancer, colon cancer, kidney cancer, breast cancer, pancreatic cancer, stomach cancer, uterine cancer, and ovarian cancer. Analytical / diagnostic methods known in the art, such as tumor scanning, can be used to determine whether a patient has a tumor that is castration-resistant.

[0664] In certain embodiments, the present disclosure also includes methods of treating residual cancer in a subject. The term "residual cancer" refers to the presence or persistence of one or more cancer cells in a subject after treatment with an anti-cancer therapy.

[0665] According to certain aspects, the present disclosure provides methods of treating a disease or disorder associated with MET expression (e.g., lung cancer), comprising administering to a subject one or more of the anti-MET or anti-MET / MET protein drug conjugates described elsewhere herein after the subject has been determined to have lung cancer. For example, the present disclosure includes methods of treating lung cancer, comprising administering to a patient a protein drug conjugate comprising an anti-MET or anti-MET / MET bispecific antibody or antigen-binding molecule 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 4 months, 6 months, 8 months, 1 year or more after the subject has received hormone therapy (e.g., anti-androgen therapy).

[0666] For example, the anti-MET antibody drug conjugates and METxMET bispecific antibody drug conjugates of the present disclosure are useful for treating tumors that express (or overexpress) MET. For example, the anti-MET antibody drug conjugates and METxMET bispecific antibody drug conjugates may be used to treat primary and / or metastatic tumors arising in the brain and meninges, oropharynx, lungs and bronchial tree, gastrointestinal tract, male and female reproductive organs, muscle, bone, skin and appendages, connective tissue, spleen, immune system, blood forming cells and bone marrow, liver and urinary tract, specialized sensory organs such as the eye. In certain embodiments, anti-MET antibody drug conjugates and METxMET bispecific antibody drug conjugates are used to treat one or more of acute myeloid leukemia, adult T-cell leukemia, astrocytoma, bladder cancer, breast cancer, cervical cancer, intrahepatic cholangiocarcinoma, chronic myeloid leukemia, colorectal cancer, endometrial cancer, esophageal cancer, gastric cancer (e.g., gastric cancer with MET amplification), glioblastoma, head and neck cancer (e.g., head and neck squamous cell carcinoma [HNSCC]), Kaposi's sarcoma, kidney cancer, leiomyosarcoma, liver cancer, lung cancer (e.g., non-small cell lung cancer [NSCLC]), lymphoma, malignant glioma, malignant mesothelioma, melanoma, mesothelioma, MFH / fibrosarcoma, multiple myeloma, nasopharyngeal carcinoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, rhabdomyosarcoma, small cell lung cancer, synovial sarcoma, thyroid cancer, and Wilms' tumor.

[0667] In certain embodiments, the present disclosure also includes the use of an anti-MET antibody drug conjugate or a METxMET bispecific antibody drug conjugate of the present disclosure in the manufacture of a medicament for the treatment of a disease or disorder associated with or caused by cells expressing MET (e.g., cancer). In one aspect, the present disclosure relates to a protein drug conjugate, comprising an anti-MET antibody drug conjugate or a METxMET bispecific antibody drug conjugate disclosed herein, for use in medicine. In one aspect, the present disclosure relates to a compound comprising an antibody drug conjugate (ADC) disclosed herein, for use in medicine.

[0668] Combination Therapies and Formulations The present disclosure provides methods comprising administering a pharmaceutical composition comprising any of the exemplary protein drug conjugates (e.g., antibody drug conjugates), linker payloads, and payloads described herein in combination with one or more additional therapeutic agents. Exemplary additional therapeutic agents that may be combined with or administered in combination with the protein drug conjugates (e.g., antibody drug conjugates), linker payloads, and payloads of the present disclosure include, for example, HER2 antagonists (e.g., anti-HER2 antibodies [e.g., trastuzumab] or small molecule inhibitors of HER2 or anti-HER2 antibody drug conjugates, or anti-HER2 / HER2 bispecific antibodies or anti-HER2 / HER2 bispecific antibody drug conjugates), EGFR antagonists (e.g., anti-EGFR antibodies [e.g., cetuximab or panitumumab] or small molecule inhibitors of EGFR [e.g., gefitinib or erlotinib]), HER2 / ErbB2, ErbB3, ErbB4, etc. antagonists of another EGFR family member (e.g., anti-ErbB2, anti-ErbB3, or anti-ErbB4 antibody, or small molecule inhibitor of ErbB2, ErbB3, or ErbB4 activity), antagonists of EGFRvIII (e.g., an antibody that specifically binds EGFRvIII), cMET antagonists (e.g., anti-cMET antibody), IGF1R antagonists (e.g., anti-IGF1R antibody), B-raf inhibitors (e.g., vemurafenib, sorafenib, gDC-0879, PLX-4720), PDGFR-α inhibitors (e.g., anti-PDGFR-α antibody), PDGFR-β inhibitors (e.g., anti-PDGFR-β antibody), VEGF antagonists (e.g., VEGF-Trap, see, e.g., U.S. Pat. No. 7,087,087,411 (also referred to herein as "fusion proteins that inhibit VEGF")), anti-VEGF antibodies (e.g., bevacizumab), small molecule kinase inhibitors of VEGF receptors (e.g., sunitinib, sorafenib, or pazopanib), DLL4 antagonists (e.g., anti-DLL4 antibodies disclosed in U.S. Patent No. 2009 / 0142354), Ang2 antagonists (e.g., anti-A antibodies disclosed in U.S. Patent No. 2011 / 0027286, such as H1H685P), and the like. ng2 antibody), FOLH1 (PSMA) antagonist, PRLR antagonist (e.g., anti-PRLR antibody), STEAP1 or STEAP2 antagonist (e.g., anti-STEAP1 antibody or anti-STEAP2 antibody), TMPRSS2 antagonist (e.g., anti-TMPRSS2 antibody), MSLN antagonist (e.g., anti-MSLN antibody), CA9 antagonist (e.g., anti-CA9 antibody), uroplakin antagonist (e.g., anti-uroplakin antibody), etc.

[0669] Other agents that may be beneficially administered in combination with the protein drug conjugates (e.g., antibody drug conjugates), linker payloads, and payloads of the present disclosure include cytokine inhibitors, including small molecule cytokine inhibitors and antibodies, that bind to cytokines such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-11, IL-12, IL-13, IL-17, IL-18, or their respective receptors. Pharmaceutical compositions of the present disclosure (e.g., pharmaceutical compositions comprising an anti-HER2, anti-HER2 / HER2 bispecific, anti-MET, anti-MET / MET bispecific, or anti-STEAP2 protein drug conjugate (e.g., an antibody drug conjugate disclosed herein)) also include other anti-cancer drugs such as "ICE": ifosfamide (e.g., Ifex®), carboplatin (e.g., Paraplatin®), etoposide (e.g., Etopophos®, Toposar®, VePesid®, VP-16); "DHAP": dexamethasone (e.g., Decadron®), trademark), cytarabine (e.g., Cytosar-U®, cytosin arabinoside, ara-C), cisplatin (e.g., Platinol®-AQ); and "ESHAP": etoposide (e.g., Etopophos®, Toposar®, VePesid®, VP-16), methylprednisolone (e.g., Medrol®), high-dose cytarabine, cisplatin (e.g., Platinol®-AQ).

[0670] The present disclosure also includes therapeutic combinations comprising any of the protein drug conjugates (e.g., antibody drug conjugates), linker payloads, and payloads mentioned herein and one or more of HER2, VEGF, Ang2, DLL4, EGFR, ErbB2, ErbB3, ErbB4, EGFRvIII, cMet, IGF1R, B-raf, PDGFR-α, PDGFR-β, FOLH1 (PSMA), PRLR, STEAP1, STEAP2, TMPRSS2, MSLN, CA9, uroplakin, or any of the foregoing cytokines, wherein the inhibitor is an aptamer, antisense molecule, ribozyme, siRNA, peptibody, nanobody, or antibody fragment (e.g., Fab fragment; F(ab')2 fragment; Fd fragment; Fv fragment; scFv; dAb fragment; or other engineered molecule such as a diabody, triabody, tetrabody, minibody, minimal recognition unit, etc.). The antigen-binding molecules of the present disclosure may also be administered in combination with and / or co-administered with antivirals, antibiotics, analgesics, corticosteroids, and / or NSAIDs. The antigen-binding molecules of the present disclosure may also be administered as part of a treatment regimen that further includes radiation therapy and / or conventional chemotherapy.

[0671] The additional therapeutically active ingredient may be administered immediately before, simultaneously with, or immediately after administration of the antigen-binding molecule of the present disclosure (for purposes of the present disclosure, such administration regimens are considered to be administration of the antigen-binding molecule "in combination" with the additional therapeutically active ingredient).

[0672] The present disclosure includes pharmaceutical compositions in which a protein drug conjugate (e.g., an antibody drug conjugate), linker payload, and / or payload of the present disclosure is co-administered with one or more additional therapeutically active ingredients as described elsewhere herein.

[0673] Dosing regimen According to certain embodiments of the present disclosure, multiple doses of a protein drug conjugate (e.g., an anti-HER2, anti-HER2 / HER2 bispecific, anti-MET, anti-MET / MET bispecific, or anti-STEAP2 antibody drug conjugate), linker payload, and / or payload may be administered to a subject over a predetermined period of time. The method according to this aspect of the present disclosure comprises sequentially administering to a subject multiple doses of a protein drug conjugate (e.g., an anti-HER2, anti-HER2 / HER2 bispecific, anti-MET, anti-MET / MET bispecific, or anti-STEAP2 antibody drug conjugate), linker payload, and / or payload of the present disclosure. As used herein, "sequential administration" means that each dose of a protein drug conjugate (e.g., an anti-HER2, anti-HER2 / HER2 bispecific, anti-MET, anti-MET / MET bispecific, or anti-STEAP2 antibody drug conjugate), linker payload, and / or payload is administered to a subject at different time points, e.g., on different days, separated by predetermined intervals (e.g., hours, days, weeks, months). The present disclosure includes methods comprising sequentially administering to a patient a single initial dose of a protein drug conjugate (e.g., an anti-HER2, anti-HER2 / HER2 bispecific, anti-MET, anti-MET / MET bispecific, or anti-STEAP2 antibody drug conjugate), linker payload, and / or payload, followed by one or more second doses of the protein drug conjugate (e.g., an anti-HER2, anti-HER2 / HER2 bispecific, anti-MET, anti-MET / MET bispecific, or anti-STEAP2 antibody drug conjugate), linker payload, and / or payload, and optionally a third dose or doses of the protein drug conjugate (e.g., an anti-HER2, anti-HER2 / HER2 bispecific, anti-MET, anti-MET / MET bispecific, or anti-STEAP2 antibody drug conjugate), linker payload, and / or payload.

[0674] The terms "first dose," "second dose," and "third dose" refer to a series of administrations of a protein drug conjugate (e.g., an anti-HER2, anti-HER2 / HER2 bispecific, anti-MET, anti-MET / MET bispecific, or anti-STEAP2 antibody drug conjugate), linker payload, and / or payload of the present disclosure. Thus, a "first dose" is a dose administered at the beginning of a treatment regimen (also referred to as a "baseline dose"), a "second dose" is a dose administered after the first dose, and a "third dose" is a dose administered after the second dose. The first, second, and third doses all contain the same amount of protein drug conjugate (e.g., an anti-HER2, anti-HER2 / HER2 bispecific, anti-MET, anti-MET / MET bispecific, or anti-STEAP2 antibody drug conjugate), linker payload, and / or payload, although the amounts may differ from one another, generally in terms of administration frequency. However, in certain embodiments, the amounts of protein drug conjugate (e.g., anti-HER2, anti-HER2 / HER2 bispecific, anti-MET, anti-MET / MET bispecific, or anti-STEAP2 antibody drug conjugate), linker payload, and / or payload included in the first dose, second dose, and / or third dose are varied relative to one another (e.g., adjusted upward or downward as appropriate) during the treatment period. In certain embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered as "loading doses" at the beginning of the treatment regimen, followed by subsequent doses administered less frequently (e.g., "maintenance doses").

[0675] In an exemplary embodiment of the present disclosure, the second and / or third doses are administered 1 to 26 weeks (e.g., 1, 1 1 / 2, 2, 2 1 / 2, 3, 3 1 / 2, 4, 4 1 / 2, 5, 5 1 / 2, 6, 6 1 / 2, 7, 7 1 / 2, 8, 8 1 / 2, 9, 9 1 / 2, 10, 10 1 / 2, 11, 111 / 2, 12, 12 1 / 2, 13, 13 1 / 2, 14, 14 1 / 2, 15, 15 1 / 2, 16, 16 1 / 2, 17, 17 1 / 2, 18, 18 1 / 2, 19, 19 1 / 2, 20, 20 1 / 2, 21, 21 1 / 2, 22, 22 1 / 2, 23, 23 1 / 2, 24, 24 1 / 2, 25, 25 1 / 2, 26, 26 1 / 2 weeks or more). The phrase "immediately preceding dose" as used herein means a dose of protein drug conjugate (e.g., anti-HER2, anti-HER2 / HER2 bispecific, anti-MET, anti-MET / MET bispecific, or anti-STEAP2 antibody drug conjugate), linker payload, and / or payload administered to a patient prior to administration of the next dose in a multiple dose series, with no intervening doses.

[0676] Methods according to this aspect of the disclosure can include administering to the patient any number of second and / or third doses of protein drug conjugate (e.g., anti-HER2, anti-HER2 / HER2 bispecific, anti-MET, anti-MET / MET bispecific, or anti-STEAP2 antibody drug conjugate), linker payload, and / or payload. For example, in certain embodiments, the second dose is administered to the patient only once. In other embodiments, the second dose is administered to the patient two or more times (e.g., 2, 3, 4, 5, 6, 7, or 8 or more times). Similarly, in certain embodiments, the third dose is administered to the patient only once. In other embodiments, the third dose is administered to the patient two or more times (e.g., 2, 3, 4, 5, 6, 7, or 8 or more times).

[0677] In embodiments involving multiple second doses, each second dose may be administered with the same frequency as the other second doses. For example, each second dose may be administered to the patient 1-2 weeks after the immediately preceding dose. Similarly, in embodiments involving multiple third doses, each third dose may be administered with the same frequency as the other third doses. For example, each third dose may be administered to the patient 2-4 weeks after the immediately preceding dose. Alternatively, the frequency with which the second and / or third doses are administered to the patient may vary over the duration of the treatment regimen. The administration frequency may also be adjusted during treatment by the physician after clinical examination according to the patient's individual needs. [Example]

[0678] The following examples illustrate specific aspects of the present invention, but are not to be construed as limiting, but merely as providing a concrete understanding and practice of the embodiments and various aspects thereof.

[0679] [Table 5-1]

[0680] [Table 5-2]

[0681] [Table 5-3]

[0682] Most of the starting materials are commercially available from Sigma-Aldrich®, J&K®, ChemExpress®, etc. The following compounds were synthesized according to the corresponding references.

[0683] [Table 6]

[0684] general law Example 1: Camptothecin Derivatives (Payload)

[0685] [Table 7]

[0686] Payload P1, exatecan methylate, was commercially available from MCE. Payloads P2 and P3 were synthesized as described in International Publication No. WO2015155998, which is incorporated herein by reference, and payload P4, a camptothecin derivative, was synthesized as described in Scheme 1A2 and by the synthetic steps outlined in Examples 1A-1E.

[0687] [ka]

[0688] Example 1A: Synthesis of 9H-fluoren-9-ylmethyl N-[2-(2-hydroxypyrrolidin-1-yl)-2-oxoethyl]carbamate (P4-2)

[0689] [ka]

[0690] To a mixture of Fmoc-Gly-Pro-OH P4-1 (0.10 g, 0.26 mmol) in dry DMF (1 mL) was added lead acetate (0.14 g, 0.31 mmol). The resulting mixture was stirred at room temperature for 30 minutes, and the reaction progress was monitored by LCMS. The resulting mixture was filtered through Celite, and the filtrate was diluted with ethyl acetate, washed with water and brine, dried over anhydrous sodium sulfate, and concentrated in vacuo. The residue was purified by silica gel column chromatography (0–10% ethyl acetate in petroleum ether) to give compound P4-2 (50 mg, 53% yield) as a white solid, but the acetate intermediate was not obtained. ESI m / z: 389 (M+23). + .1 H NMR(400MHz,DMSO)δ7.90(d,J=7.4Hz,2H),7.73(d,J=7.5Hz,2H),7.47-7.37(m,3H),7.33(t,J=7.3Hz,2H),5.86(br s,1H),5.48(d,J=4.0Hz,0.25H),5.39(d,J=4.0Hz,0.75H),4.33-4.18(m,3H),3.96(d,J=6.0H z,1.5H),3.75(d,J=6.0Hz,0.5H),3.59-3.33(m,1H),3.22-3.11(m,1H),2.00-1.59(m,4H)ppm.

[0691] Example 1B: Synthesis of benzyl 2-{[1-(2-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}acetyl)pyrrolidin-2-yl]oxy}acetate (P4-3)

[0692] [ka]

[0693] To a solution of compound P4-2 (0.30 g, 0.82 mmol) in DCM (25 mL) was added chlorotrimethylsilane (TMSCl) (0.27 g, 2.5 mmol). The reaction mixture was stirred at room temperature for 3 h, and the reaction progress was monitored by LCMS. The resulting mixture was concentrated in vacuo, and the residue was diluted with DCM (25 mL). Benzyl glycolate (0.27 g, 1.6 mmol) and DIPEA (0.21 g, 1.6 mmol) were added to the solution, and the reaction mixture was stirred at room temperature for 1 h. The completion of the reaction was monitored by LCMS. The resulting mixture was concentrated in vacuo, and the residue was purified by reverse-phase flash chromatography (0-100% acetonitrile in aqueous ammonium bicarbonate (0.05%)) to give compound P4-3 (0.11 g with a purity of >99%, 25% yield, and 50 mg with a purity of 75%) as a white solid. ESI m / z: 537.3 (M+Na) + . 1 H NMR (400 MHz, DMSO d6)δ7.91-7.89(m,2H),7.74-7.69(m,2H),7.63-7.48(m,1H),7.42-7.25(m,9H),5.51-5.09(m,2H), 4.35-4.21(m,5H),4.00-3.77(m,2H),3.52-3.38(m,2H),3.30-3.18(m,1H),2.19-1.64(m,4H)ppm.

[0694] Example 1C: Synthesis of 2-{[1-(2-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}acetyl)pyrrolidin-2-yl]oxy}acetic acid (P4-4)

[0695] [ka]

[0696] To a solution of compound P4-3 (89 mg, 0.17 mmol) in methanol (3 mL) and THF (7 mL), wet palladium on carbon (10% Pd, 20 mg) was added under nitrogen protection. The mixture was degassed and stirred at room temperature under hydrogen balloon pressure for 2 hours, and the reaction completion was monitored by LCMS. The reaction mixture was filtered through Celite, and the filtrate was concentrated in vacuo. The residue was purified by reverse-phase flash chromatography (0-100% acetonitrile in aqueous ammonium bicarbonate (0.05%)) to give compound P4-4 (36 mg, 49% yield) as a white solid. ESI m / z: 447.1 (M+Na). + .

[0697] Example 1D: 9H-Fluoren-9-ylmethyl N-{2-[2-({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24]tetracosa-1,6(11),12,14,16,18,20(24)-heptan-23-yl]carbamoyl}methoxy)pyrrolidin-1-yl]-2-oxoethyl}carbamate (P4-5)

[0698] [ka]

[0699] To a mixture of compound P4-4 (63 mg, 0.15 mmol) and exatecan methylate (66 mg, 0.12 mmol) in DMF (2 mL), HATU (61 mg, 0.16 mmol) and DIPEA (46 mg, 0.36 mmol) were added. The mixture was stirred at room temperature for 2 h, and the reaction completion was monitored by LCMS. The reaction mixture was directly purified by reverse-phase flash chromatography (0–100% acetonitrile in 10 mM aqueous ammonium bicarbonate) to give compound P4-5 (45 mg, 44% yield) as a yellow solid. ESI m / z: 842.3 (M+H). + .

[0700] Example 1E: 2-{[1-(2-aminoacetyl)pyrrolidin-2-yl]oxy}-N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]tetracosa-1,6(11),12,14,16,18,20(24)-heptan-23-yl]acetamide (P4)

[0701] [ka]

[0702] To a solution of compound P4-5 (45 mg, 54 μmol) in DCM (4 mL) was added diethylamine (20 mg, 0.27 mmol), and the mixture was stirred at room temperature overnight. The completion of the reaction was monitored by LCMS. The reaction mixture was concentrated in vacuo, and the residue was purified by silica gel flash chromatography (0-10% methanol in DCM) to give compound P4 (9.5 mg, 28% yield) as a colorless oil. ESI m / z: 620.3 (M+H). + Table 6 below provides cytotoxicity and ADME (absorption, distribution, metabolism, excretion) data for payloads P1-P3 according to the present disclosure.

[0703] [Table 8]

[0704] In the table, "-v" indicates that verapamil is not included, and "+v" indicates that verapamil is included. Verapamil is known as an inhibitor of P-glycoprotein and may function to prevent P-glycoprotein-mediated efflux.

[0705] Example 2: Linker 2 - Payload

[0706] Example 2A: Linear Linker 2-Payload (LL2P)

[0707] Table 7 below provides the structures of exemplary Linear Linker 2-Payload (LL2P) molecules according to the present disclosure.

[0708] [Table 9-1]

[0709] [Table 9-2]

[0710] [Table 9-3]

[0711] Table 8 below provides the chemical properties of exemplary Linear Linker 2-Payload (LL2P) compounds according to the present disclosure.

[0712] [Table 10-1]

[0713] [Table 10-2]

[0714] * A mixture of lactone and ring-opened product. MW and m / z values ​​are those of the corresponding lactone.

[0715] Example 2B: Branched Linker 2-Payload (BL2P)

[0716] Table 9 below provides structures of exemplary branching units B1-B5 according to the present disclosure.

[0717] [Table 11]

[0718] Table 10 below provides exemplary branched linker 2-payload (BL2P) structures according to the present disclosure.

[0719] [Table 12-1]

[0720] [Table 12-2]

[0721] [Table 12-3]

[0722] [Table 12-4]

[0723] [Table 12-5]

[0724] Table 11 below provides the chemical properties of exemplary Branched Linker 2-Payload (BL2P) molecules according to the present disclosure.

[0725] [Table 13-1]

[0726] [Table 13-2]

[0727] Example 3: Synthesis of vcPAB-carbamate linker payload

[0728] Linker payloads LP1 and LP2 were synthesized as described in Scheme 22 and in Examples 3A-3C (LP1) and 2D (LP2) below. Starting materials L1-1 (CAS2226472-26-8) and L2-3 (CAS2226472-28-0) were synthesized according to International Publication No. WO2018089373A2, which is incorporated by reference in its entirety.

[0729] [ka]

[0730] Example 3A: N-[(1S)-1-{[(1S)-4-(carbamoylamino)-1-{[4-(hydroxymethyl)phenyl]carbamoyl}butyl]carbamoyl}-2-methylpropyl]-1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadecan-15-amide (L1-2)

[0731] [ka]

[0732] To a solution of compound L1-1 (0.17 g, 0.33 mmol) in DMF (10 mL), DIPEA (0.13 g, 1.0 mmol) and vcPAB (0.13 g, 0.34 mmol) were added sequentially, and the reaction mixture was stirred at room temperature for 1 h. The completion of the reaction was monitored by LCMS. The resulting mixture was directly purified by reverse-phase flash chromatography (0–80% acetonitrile in water) to give compound L1-2 (0.18 g, 70% yield) as a colorless oil. ESI m / z: 791.3 (M+H). + . 1 H NMR (400 MHz, DMSO d6)δ9.91(s,1H),8.11(d,J=8.4Hz,1H),7.89(d,J=8.8Hz,1H),7.61(t,J=5.6Hz,1H),7. 55(d,J=8.4Hz,2H),7.23(d,J=8.4Hz,2H),5.98(t,J=5.6Hz,1H),5.42(s,2H),5.10(br s,1H),),4.43(s,2H),4.39-4.37(m,1H),4.30-4.21(m,2H),3.87(d,J=14.8Hz,1H),3.7 5(d,J=14.8Hz,1H),3.62-3.58(m,2H),3.50-3.46(m,12H),3.43(t,J=6.0Hz,2H),3.27- 3.22(m,2H),3.06-2.92(m,2H),2.41-2.32(m,2H),2.26-2.05(m,3H),1.99-1.66(m,6H) ,1.62-1.55(m,3H),1.44-1.35(m,3H),0.89(d,J=6.8Hz,3H),0.83(d,J=6.8Hz,3H)ppm.

[0733] Example 3B: {4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadecan-15-amido}-3-methylbutanamido]pentanamido]phenyl}methyl 4-nitrophenyl carbonate (L1-3)

[0734] [ka]

[0735] A suspension of compound L1-2 (80 mg, 0.10 mmol), DMAP (12 mg, 0.10 mmol), and DIPEA (26 mg, 0.20 mmol) in dry DMF (5 mL) was stirred at room temperature for 10 minutes, and then bis(4-nitrophenyl)carbonate (61 mg, 0.20 mmol) was added. The reaction mixture was stirred at room temperature for 2 hours. The completion of the reaction was monitored by LCMS. The resulting mixture was directly purified by reverse-phase flash chromatography (0-80% acetonitrile in water) to give compound L1-3 (53 mg, 55% yield) as a white solid. ESI m / z: 956.3 (M+H). + .

[0736] Example 3C: {4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadecan-15-amido}-3-methylbutanamido]pentanamido]phenyl}methyl N-({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]tetracosa-1,6(11),12,14,16,18,20(24)-heptan-23-yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamate (LP1)

[0737] [ka]

[0738] To a yellow solution of compound L1-3 (16 mg, 17 μmol) and P3 (12 mg, 17 μmol) in dry DMF (2 mL), DIPEA (6.5 mg, 51 μmol) was added, and the resulting clear reaction solution was stirred at room temperature for 2 h. Completion of the reaction was monitored by LCMS. The resulting mixture was directly purified by reverse-phase flash chromatography (0.01% aqueous TFA, 0-60% acetonitrile) to afford the linker payload LP1 (15 mg as the TFA salt, 63% yield) as a white solid. ESI m / z: 698.8 (M / 2+H). + . 1 H NMR (400 MHz, DMSO d6 )δ9.99(s,1H),8.80(t,J=6.8Hz,1H),8.50(d,J=9.2Hz,1H),8.12(d,J=7.2Hz,1H),7.87(d,J=8.8Hz,1H),7 .79(d,J=10.8Hz,1H),7.62-7.58(m,3H),7.42(t,J=6.0Hz,1H),7.31(s,1H),7.28(d,J=8.4Hz,2H),6.53(br s,1H),5.98(t,J=5.2Hz,1H),5.63-5.57(m,1H),5.46-5.37(m,3H),5.21(s,2H),4.93(s,2H),4.63(d,J=6.4Hz,2H),4 .41-4.35(m,1H),4.29-4.21(m,2H),4.02(s,2H),3.87(d,J=14.4Hz,1H),3.75(d,J=14.8Hz,1H),3.63-3.58(m,4H),3 0.50-3.48 (m, 12H), 3.46-3.41 (m, 2H), 3.27-3.24 (m, 2H), 3.23-3.12 (m, 2H), 3.07-2.91 (m, 2H), 2.47-2.45 (m, 0.5H), 2.41-2.33 (m, 4.5H), 2.25-2.04 (m, 5H), 1.99-1.69 (m, 9H), 1.63-1.54 (m, 3H), 1.44-1.33 (m, 3H), 0.88-0.82 (m, 9H) ppm. (TFA protons were not observed.) 19 F NMR (376 MHz, DMSO d6 ) δ-74(TFA),-111(Ar-F)ppm.

[0739] Example 3D: {4-[(2S)-2-[(2S)-2-[1-(4-{2-azatricyclo[10.4.0.0 4 , 9 ]hexadeca-1(12),4(9),5,7,13,15-hexaen-10-yn-2-yl}-4-oxobutanamido)-3,6,9,12-tetraoxapentadecan-15-amido]-3-methylbutanamido]-5-(carbamoylamino)pentanamido]phenyl}methyl N-({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]tetracosa-1,6(11),12,14,16,18,20(24)-heptan-23-yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamate (LP2)

[0740] [ka]

[0741] Following the procedure for the preparation of LP1, but replacing L1-3 with L2-3, the linker payload LP2 (12 mg, 46% yield) was obtained as a mixture of the lactone product (LP2, shown above) and the ring-opened product (LP2-RO, shown below) as a white solid after purification by reverse-phase flash chromatography (0–100% methanol in 10 mM aqueous ammonium bicarbonate).

[0742] [ka]

[0743] Lactone LP2: HPLC purity: 67%, retention time: 7.41 min, ESI m / z: 507.3 (M / 3+H) +, 760.5(M / 2+H) + ; Ring-opening product LP2-RO: HPLC purity: 33%, retention time: 6.61 min, ESI m / z: 513.3 (M / 3+H) + , 769.5(M / 2+H) + .

[0744] A mixture of lactone product and ring-opened product 1 H NMR (400 MHz, DMSO d6 )δ9.99(s,1H),8.80(t,J=6.4Hz,1H),8.50(d,J=8.8Hz,1H),8.12(d,J=7.2Hz,1H),7.87(d, J=8.4Hz,1H),7.80-7.75(m,2H),7.69-7.67(m,1H),7.63-7.58(m,3H),7.51-7.46(m,3H),7 .45-7.33(m,3H),7.32-7.26(m,4H),6.53(s,1H),5.98(t,J=6.0Hz,1H),5.63-5.57(m,1H), 5.42(s,4H),5.21(s,2H),5.03(d,J=14.0Hz,1H),4.93(s,2H),4.63(d,J=6.8Hz,2H),4.41- 4.35(m,1H),4.25-4.21(m,1H),4.02(s,2H),3.62-3.57(m,5H),3.48-3.45(m,12H),3.31-3 .28(m,2H),3.23-3.14(m,2H),3.11-3.07(m,2H),3.05-2.98(m,1H),2.96-2.91(m,1H),2.6 0-2.55(m,1H),2.46-2.44(m,1H),2.39(s,3H),2.35-2.33(m,1H),2.26-2.15(m,3H),2.03- 1.94(m,2H),1.88-1.67(m,4H),1.63-1.57(m,1H),1.46-1.33(m,2H),0.88-0.81(m,9H)ppm. 19 F NMR (376 MHz, DMSO d6 )δ-111 ppm.

[0745] Linker payloads LP16 and LP17 were synthesized as described in Scheme 3 and in Examples 3E-3F (LP16) and 3G (LP17) below.

[0746] [ka]

[0747] Example 3E: {4-[(2S)-2-[(2S)-2-amino-3-methylbutanamido]-5-(carbamoylamino)pentanamido]phenyl}methyl N-({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]tetracosa-1,6(11),12,14,16,18,20(24)-heptan-23-yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamate (LP16-1)

[0748] [ka]

[0749] To a solution of payload P3 (0.11 g, 0.15 mmol) in DMF (2 mL), DIPEA (39 mg, 0.30 mmol) and Fmoc-vcPAB-PNP (CAS: 863971-53-3, 77 mg, 0.10 mmol) were added, and the reaction mixture was stirred at room temperature for 1 h until clear. P was completely consumed by LCMS. The resulting solution was separated by reverse-phase flash chromatography (0-70% acetonitrile in aqueous TFA (0.01%)) to obtain Fmoc-LP16-1 (98 mg, ESI m / z: 494 (M DXD +H) + , 714.2(MM DXD +H) +was obtained as a pale yellow solid, which was dissolved in dry DMF (4.5 mL). Diethylamine (0.5 mL) was slowly added to this solution, and the reaction mixture was stirred at room temperature for 2 h until the Fmoc group was completely removed by LCMS. The volatiles were removed in vacuo, and the residue was purified by reverse-phase flash chromatography (0-40% acetonitrile in aqueous TFA (0.01%)) to give LP16-1 (TFA salt, 45 mg, 41% yield from P3). ESI m / z: 493.1 (M / 2+H). + . 1 H NMR (400 MHz, DMSO d6 )δ10.20(s,1H),8.80(t,J=6.8Hz,1H),8.68(d,J=7.6Hz,1H),8.50(d,J=8.8Hz, 1H),8.09-8.01(m,3H),7.79(d,J=10.8Hz,1H),7.58(d,J=8.4Hz,2H),7.41(t,J =6.0Hz,1H),7.31(s,1H),7.29(d,J=8.4Hz,2H),6.53(s,1H),6.05(t,J=5.6Hz, 1H),5.63-5.57(m,1H),5.54-5.45(m,2H),5.42-5.41(m,2H),5.21(s,2H),4.93 (s,2H),4.63(d,J=6.4Hz,2H),4.55-4.50(m,1H),4.02(s,2H),3.69-3.61(m,3H ),3.24-3.12(m,1H),3.08-2.94(m,2H),2.40(s,3H),2.23-2.18(m,2H),2.14-2 0.03 (m, 1H), 1.90-1.81 (m, 2H), 1.78-1.68 (m, 1H), 1.64-1.53 ​​(m, 1H), 1.47-1.36 (m, 2H), 0.96 (d, J = 3.2 Hz, 3H), 0.94 (d, J = 3.2 Hz, 3H), 0.87 (t, J = 7.6 Hz, 3H) ppm. (TFA protons were not identified.) 19 F NMR (376 MHz, DMSO d6 )δat -111,-73 ppm.

[0750] Example 3F: {4-[(2S)-2-[(2S)-2-(1-amino-3,6,9,12-tetraoxapentadecan-15-amido)-3-methylbutanamido]-5-(carbamoylamino)pentanamido]phenyl}methyl N-({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]tetracosa-1,6(11),12,14,16,18,20(24)-heptan-23-yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamate (LP16)

[0751] [ka]

[0752] To a solution of LP16-1 (16 mg, 15 μmol) in anhydrous DMF (1 mL), DIPEA (4 mg, 29 μmol) was added until the pH reached between 8.0 and 9.0. Then, a solution of compound If (CAS 1314378-14-7, 9 mg, 15 μmol) in anhydrous DMF (1 mL) was added to the reaction mixture. The mixture was stirred at room temperature for 1 h until the starting material was completely consumed by LCMS. The resulting solution was separated by reverse-phase flash chromatography (0.01% aqueous TFA, 0-100% acetonitrile) to give LP16f (18 mg, ESI m / z: 728.3 (M / 2+H). +) was obtained as a white solid, which was dissolved in anhydrous DMF (1.9 mL). Diethylamine (0.1 mL) was added to this solution, and the yellow reaction mixture was stirred at room temperature for 30 min until the Fmoc group was completely removed by LCMS. The resulting solution was concentrated in vacuo, and the residue was purified by preparative HPLC (10-95% acetonitrile in aqueous formic acid (0.01%)) to give the linker payload LP16 (6 mg, 34% yield) as a pale yellow solid. ESI m / z: 616.9 (M / 2+H). + . 1 H NMR (400 MHz, DMSO d6 )δ10.03(s,1H),8.80(d,J=6.8Hz,1H),8.51(d,J=9.2Hz,1H),8.41(s,1H),8.15(d, J=6.8Hz,1H),7.90(d,J=8.8Hz,1H),7.79(d,J=6.8Hz,1H),7.60(d,J=8.0Hz,2H),7 .42(t,J=6.4Hz,1H),7.32(s,1H),7.27(d,J=8.0Hz,2H),6.56-6.50(m,1H),6.05-6 .01(m,1H),5.63-5.58(m,1H),5.43(s,4H),5.21(s,2H),4.93(s,2H),4.63(d,J=6.8 Hz,2H),4.42-4.35(m,1H),4.23(t,J=7.6Hz,1H),4.02(s,2H),3.63-3.58(m,4H),3 .53-3.49(m,12H),3.46-3.43(m,2H),3.18-3.15(m,2H),3.06-2.93(m,2H),2.79-2 .76(m,2H),2.42-2.36(m,5H),2.24-2.12(m,2H),2.03-1.93(m,1H),1.89-1.81(m, 2H), 1.74-1.65 (m, 1H), 1.65-1.54 (m, 1H), 1.48-1.31 (m, 3H), 0.89-0.82 (m, 9H) ppm. 19 F NMR (400 MHz, DMSO) d6 )δ-111 ppm.

[0753] Example 3G: {4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-{1-[3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido]-3,6,9,12-tetraoxapentadecan-15-amido}-3-methylbutanamido]pentanamido]phenyl}methyl N-({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]tetracosa-1,6(11),12,14,16,18,20(24)-heptan-23-yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamate (LP17)

[0754] [ka]

[0755] To a yellow solution of LP16-1 (30 mg, 30 μmol) in anhydrous DMF (1.5 mL) were added DIPEA (8 mg, 62 μmol) and Ia (CAS: 756525-99-2, commercially available, 16 mg, 30 μmol). The reaction mixture was stirred at room temperature for 1 h until the starting material was completely consumed by LCMS. The resulting mixture was directly purified by preparative HPLC (0.05% aqueous TFA, 0-100% acetonitrile) to give LP17 (TFA salt, 15 mg, 38% yield) as a pale yellow solid. ESI m / z: 692.4 (M / 2+H). 1 H NMR (400 MHz, DMSO d6)δ9.99(s,1H),8.79(t,J=6.8Hz,1H),8.50(d,J=8.4Hz,1H),8.11(d,J=8.0Hz,1H),8.02(t,J=6.0Hz,1H),7.87(d,J=8.8Hz,1H) ,7.79(d,J=10.8Hz,1H),7.59(d,J=8.4Hz,2H),7.41(t,J=6.0Hz,1H),7.32(s,1H),7.28(d,J=8.4Hz,2H),7.00(s,2H),6.52(br s,1H),5.98(t,J=6.0Hz,1H),5.63-5.57(m,1H),5.43-5.41(m,4H),5.21(s,2H),4.93(s,2H),4.63(d,J=6 .8Hz,2H),4.43-4.34(m,1H),4.25-4.21(m,1H),4.02(s,2H),3.61-3.57(m,6H),3.49-3.48(m,12H),3.21 -3.12 (m, 4H), 3.05-2.91 (m, 4H), 2.49-2.39 (m, 5H), 2.33 (t, J = 7.2 Hz, 2H), 2.22-2.14 (m, 2H), 2.02-1.94 (m, 1H), 1.89-1.80 (m, 2H), 1.75-1.65 (m, 1H), 1.65-1.54 (m, 1H), 1.49-1.32 (m, 2H), 0.87-0.82 (m, 9H) ppm. (The protons of TFA were not revealed.) 19 F NMR (376 MHz, DMSO d6 )δ-111,-73 ppm.

[0756] Linker payloads LP1, LP2, LP13, LP14, LP15, LP19, LP20, LP21, and LP22 were synthesized as described in Scheme 4 and in Examples 3H-3AR below.

[0757] [ka]

[0758] Example 3H: Methyl (4R)-4-amino-4-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-{[4-(hydroxymethyl)phenyl]carbamoyl}butyl]carbamoyl}-2-methylpropyl]carbamoyl}butanoate ( D EvcPAB)

[0759] [ka]

[0760] To a solution of vcPAB (0.25 g, 0.95 mmol) in DMF (3 mL), DIPEA (0.37 g, 2.9 mmol) and HATU (0.25 g, 0.95 mmol) were added, and the mixture was stirred at room temperature for 10 min before adding Boc-DGlu(OMe)-OH (0.40 g, 1.1 mmol). The reaction mixture was stirred at room temperature for 3 h and monitored by LCMS. The resulting mixture was purified by reverse-phase flash chromatography (0-70% acetonitrile in water) to give Boc-DEvcPAB (0.35 g, ESI m / z: 623.4 (M+H)). + ) was obtained as a white solid, which was dissolved in DCM (5 mL). TFA (1.5 mL) was added to the solution, and the reaction mixture was stirred at room temperature for 3 h until the Boc was completely removed, which was monitored by LCMS. The volatiles were removed in vacuo, and the residue was purified by reverse-phase flash chromatography (10-40% acetonitrile in water) to give D EvcPAB (0.27 g, 48% yield) was obtained as a white solid. ESI m / z: 523.4 (M+H) + .

[0761] Example 3I: Methyl (4S)-4-amino-4-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-{[4-(hydroxymethyl)phenyl]carbamoyl}butyl]carbamoyl}-2-methylpropyl]carbamoyl}butanoate ( L EvcPAB)

[0762] [ka]

[0763] Except for using Boc-LGlu(OMe)-OH instead of Boc-DGlu(OMe)-OH D Follow the same procedure as for EvcPAB and prepare the intermediate L EvcPAB (0.18 g, 49% yield) was obtained as a pale yellow solid. ESI m / z: 523.3 (M+H)+, 545.3 (M+Na). + .

[0764] Example 3J: General Procedure for LP#-2 and Synthesis of LP13-2, LP16-2, LP1-2, and LP2-2

[0765] To a solution of LP13-1, LP16-1, LP1-1, or LP2-1 (1.0 equiv.) in DMF (0.25 mM) was added DIPEA (3.3 equiv.) and HATU (1.0 equiv.). The mixture was stirred at room temperature for 10 min, followed by the addition of vcPAB or EvcPAB (1.1–1.5 equiv.). The reaction mixture was stirred at room temperature for 3 h and monitored by LCMS. The resulting mixture was directly purified by reverse-phase flash chromatography (0–100% acetonitrile in water) to afford linker LP#-2 (LP13-2, LP16-2, LP1-2, or LP2-2) as a white solid.

[0766] Example 3K: (9H-Fluoren-9-yl)methyl N-{2-[2-(2-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-{[4-(hydroxymethyl)phenyl]carbamoyl}butyl]carbamoyl}-2-methylpropyl]carbamoyl}ethoxy)ethoxy]ethyl}carbamate (LP13-2)

[0767] [ka]

[0768] Following the general procedure using vcPAB (0.85 g, 2.2 mmol) and LP13-1 (0.60 g, 1.5 mmol), compound LP13-2 (1.0 g, 87% yield) was obtained as a white solid. ESI m / z: 761.3 (M+H) + ,783.3(M+Na) + .

[0769] Example 3L: Methyl (4R)-4-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-{[4-(hydroxymethyl)phenyl]carbamoyl}butyl]carbamoyl}-2-methylpropyl]carbamoyl}-4-(3-{2-[2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)ethoxy]ethoxy}propanamido)butanoate (LP14-2)

[0770] [ka]

[0771] Following the general procedure starting with LP14-1 (0.19 g, 0.37 mmol) and DEvcPAB (0.14 g, 0.34 mmol), linker LP14-2 (0.20 g, 66% yield) was obtained as a white solid after purification by reverse-phase flash chromatography (0-70% acetonitrile in water). ESI m / z: 904.4 (M+H). + .

[0772] Example 3M: Methyl (4S)-4-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-{[4-(hydroxymethyl)phenyl]carbamoyl}butyl]carbamoyl}-2-methylpropyl]carbamoyl}-4-(3-{2-[2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)ethoxy]ethoxy}propanamido)butanoate (LP15-2)

[0773] [ka]

[0774] LP13-1 (0.45 g, 1.1 mmol) and L Following the general procedure starting with EvcPAB (0.65 g, 1.2 mmol), linker LP15-2 (0.70 g, 69% yield) was obtained as a white solid after purification by reverse-phase flash chromatography (0-100% acetonitrile in water). ESI m / z: 904.5 (M+H). + . 1 H NMR (400 MHz, DMSO d6 )δ9.94(s,1H),8.15-8.07(m,2H),7.91-7.87(m,2H),7.78(d,J=8.8Hz,1H),7.69(d,J=7.2Hz,2H),7.55-7.52(m,2H),7.41(t,J=7.6Hz,2H),7 .35-7.30(m,3H),7.23(d,J=8.4Hz,2H),6.00-5.93(m,1H),5.42(bs,1H) ),4.42(s,2H),4.39-4.34(m,2H),4.31-4.27(m,2H),4.24-4.16(m,2H) ,3.60-3.56(m,5H),3.47(s,3H),3.36-3.34(m,2H),3.14-3.10(m,2H) ,3.04-3.00(m,1H),2.96-2.91(m,1H),2.44-2.38(m,2H),2.35-2.29(m ,4H),2.00-1.95(m,1H),1.92-1.86(m,1H),1.77-1.66(m,2H),1.61-1. 55(m,1H), 1.46-1.34(m,2H), 1.25-1.18(m,1H), 0.87-0.80(m,6H)ppm.

[0775] Example 3N: N-[(1S)-1-{[(1S)-4-(carbamoylamino)-1-{[4-(hydroxymethyl)phenyl]carbamoyl}butyl]carbamoyl}-2-methylpropyl]-1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadecan-15-amide (LP1-2)

[0776] [ka]

[0777] Following the general procedure starting with LP1-1 and vcPAB (3.3 g, 8.0 mmol), linker LP1-2 (4.3 g, 68% yield) was obtained as a white solid after purification by reverse-phase flash chromatography (0–100% acetonitrile in water). ESI m / z: 791.5 (M+H). + .

[0778] Example 3O: Methyl (4R)-4-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-{[4-(hydroxymethyl)phenyl]carbamoyl}butyl]carbamoyl}-2-methylpropyl]carbamoyl}-4-[1-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-3,6,9,12-tetraoxapentadecan-15-amido]butanoate (LP19-2)

[0779] [ka]

[0780] Following the general procedure starting with DEvcPAB (0.17 g, 0.34 mmol) and LP16-1 (0.17 g, 0.34 mmol), linker LP19-2 (0.18 g, 53% yield) was obtained as a pale yellow solid. ESI m / z: 993.5 (M+H). + .

[0781] Example 3P: Methyl (4R)-4-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-{[4-(hydroxymethyl)phenyl]carbamoyl}butyl]carbamoyl}-2-methylpropyl]carbamoyl}-4-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadecan-15-amido}butanoate (LP20-2)

[0782] [ka]

[0783] D Following the general procedure starting with EvcPAB (0.28 g, 0.54 mmol) and LP1-1 (0.25 g, 0.48 mmol), compound LP20-2 (0.20 g, 44% yield) was obtained as a pale yellow solid. ESI m / z: 934.5 (M+H). + .

[0784] Example 3Q: Methyl (4S)-4-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-{[4-(hydroxymethyl)phenyl]carbamoyl}butyl]carbamoyl}-2-methylpropyl]carbamoyl}-4-[1-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-3,6,9,12-tetraoxapentadecan-15-amido]butanoate (LP21-2)

[0785] [ka]

[0786] L Following the general procedure starting with EvcPAB (0.20 g, 0.38 mmol) and LP16-1 (0.19 g, 0.39 mmol), compound LP21-2 (0.20 g, 53% yield) was obtained as a white solid. ESI m / z: 992.5 (M+H) + . 1 H NMR (400 MHz, DMSO d6)δ9.94(s,1H),8.13(d,J=7.2Hz,1H),8.08(d,J=7.6Hz,1H),7.89(d,J=7.2Hz,2H),7.77(d,J=8.4Hz,1H),7.70(d,J=7 .6Hz,2H),7.54(d,J=8.4Hz,2H),7.44-7.39(m,2H),7.35-7.31(m,3H),7.23(d,J=8.4Hz,2H),5.98(brs,1H),5.41(br s,1H),4.43(s,2H),4.39-4.33(m,2H),4.31-4.29(m,2H),4.23-4.17(m,2H),3. 63-3.57(m,6H),3.50-3.46(m,12H),3.41(t,J=6.0Hz,2H),3.16-3.10(m,2H),3. 10-3.00 (m, 1H), 2.99-2.89 (m, 1H), 2.40-2.30 (m, 4H), 2.02-1.87 (m, 2H), 1.78-1.55 (m, 3H), 1.48-1.32 (m, 2H), 0.86 (d, J = 6.8 Hz, 3H), 0.83 (d, J = 6.8 Hz, 3H) ppm. (The protons of benzyl alcohol were not revealed.)

[0787] Example 3R: Methyl (4S)-4-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-{[4-(hydroxymethyl)phenyl]carbamoyl}butyl]carbamoyl}-2-methylpropyl]carbamoyl}-4-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadecan-15-amido}butanoate (LP22-2)

[0788] [ka]

[0789] L Following the general procedure starting with EvcPAB (0.10 g, 0.19 mmol) and LP1-1 (81 mg, 0.19 mmol), compound LP22-2 (0.11 g, 63% yield) was obtained as a white solid. ESI m / z: 934.5 (M+H). + .

[0790] Example 3S: General Procedure for LP#-3

[0791] To a solution of LP#-2 (1.0 equiv.) in DMF (0.15 mM) was added DMAP (1.0 equiv.), DIPEA (3.0 equiv.), and bis(4-nitrophenyl)carbonate (3.0 equiv.). The reaction mixture was stirred at room temperature for 1 h and monitored by LCMS. The resulting mixture was purified by reverse-phase flash chromatography (0–60% acetonitrile in water) to give LP#-3 as a pale yellow oil.

[0792] Example 3T: {4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-(3-{2-[2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)ethoxy]ethoxy}propanamido)-3-methylbutanamido]pentanamido]phenyl}methyl 4-nitrophenyl carbonate (LP13-3)

[0793] [ka]

[0794] Following the general procedure starting from LP13-2 (0.50 g, 0.66 mmol), linker LP13-3 (0.40 g, 68% yield) was obtained as a pale yellow oil that solidified in air. ESI m / z: 926.5 (M+H) + ,948.4(M+Na) + .

[0795] Example 3U: Methyl (4R)-4-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-{[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]carbamoyl}butyl]carbamoyl}-2-methylpropyl]carbamoyl}-4-(3-{2-[2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)ethoxy]ethoxy}propanamido)butanoate (LP14-3)

[0796] [ka]

[0797] Following the general procedure starting from LP14-2 (0.20 g, 0.22 mmol), linker LP14-3 (0.18 g, 77% yield) was obtained as a white solid. ESI m / z: 1069.2 (M+H). + .

[0798] Example 3V: Methyl (4S)-4-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-{[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]carbamoyl}butyl]carbamoyl}-2-methylpropyl]carbamoyl}-4-(3-{2-[2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)ethoxy]ethoxy}propanamido)butanoate (LP15-3)

[0799] [ka]

[0800] Following the general procedure starting from LP15-2 (0.70 g, 0.77 mmol), linker LP15-3 (0.50 g, 61% yield) was obtained as a pale yellow oil. ESI m / z: 1069.5 (M+H). + .

[0801] Example 3W: {4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadecan-15-amido}-3-methylbutanamido]pentanamido]phenyl}methyl 4-nitrophenyl carbonate (LP1-3)

[0802] [ka]

[0803] Following the general procedure starting from LP1-2 (2.3 g, 2.9 mmol), linker LP1-3 (1.9 g, 69% yield) was obtained as a pale yellow oil. ESI m / z: 978.5 (M+Na). + .

[0804] Example 3X Methyl (4R)-4-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-{[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]carbamoyl}butyl]carbamoyl}-2-methylpropyl]carbamoyl}-4-[1-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-3,6,9,12-tetraoxapentadecan-15-amido]butanoate (LP19-3)

[0805] [ka]

[0806] Following the general procedure starting from LP19-2 (0.18 g, 0.18 mmol), linker LP19-3 (0.18 g, 86% yield) was obtained as a pale yellow oil. ESI m / z: 1157.5 (M+H) + .

[0807] Example 3Y: Methyl (4R)-4-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-{[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]carbamoyl}butyl]carbamoyl}-2-methylpropyl]carbamoyl}-4-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadecan-15-amido}butanoate (LP20-3)

[0808] [ka]

[0809] Following the general procedure starting from LP20-2 (0.20 g, 0.21 mmol), linker LP20-3 (0.20 g, 85% yield) was obtained as a yellow solid. ESI m / z: 1099.6 (M+H). + .

[0810] Example 3Z: Methyl (4S)-4-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-{[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]carbamoyl}butyl]carbamoyl}-2-methylpropyl]carbamoyl}-4-[1-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-3,6,9,12-tetraoxapentadecan-15-amido]butanoate (LP21-3)

[0811] [ka]

[0812] Following the general procedure starting from LP21-2 (0.20 g, 0.20 mmol), linker LP21-3 (0.19 g, 81% yield) was obtained as a pale yellow oil. ESI m / z: 1157.4 (M+H) + . 1 H NMR (400 MHz, DMSO d6)δ10.11(s,1H),8.32(d,J=8.8Hz,2H),8.17(d,J=7.2Hz,1H),8.09(d,J=8.0Hz,1H),7.89(d,J=7.6Hz,2H),7.77(d,J=8.4 Hz,1H),7.69(d,J=7.2Hz,2H),7.65(d,J=8.4Hz,2H),7.57(d,J=9.2Hz,2H),7.44-7.40(m,4H),7.35-7.31(m,3H),5.99(br s,1H),5.42(br s,1H),5.25(s,2H),4.42-4.36(m,2H),4.29(d,J=6.8Hz,2H),4.23-4.17(m,2H), 3.61-3.57(m,6H),3.50-3.46(m,12H),3.41(t,J=6.0Hz,2H),3.16-3.11(m,2H), 3.12-3.00(m,1H),3.00-2.89(m,1H),2.40-2.30(m,4H),2.01-1.88(m,2H),1.80 -1.56(m,3H),1.50-1.33(m,2H),0.87(d,J=6.8Hz,3H),0.83(d,J=6.8Hz,3H)ppm.

[0813] Example 3AA: Methyl (4S)-4-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-{[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]carbamoyl}butyl]carbamoyl}-2-methylpropyl]carbamoyl}-4-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadecan-15-amido}butanoate (LP22-3)

[0814] [ka]

[0815] Following the general procedure starting from LP08b-2 (0.10 g, 0.11 mmol), linker LP08b-3 (71 mg, 60% yield) was obtained as a white solid. ESI m / z: 550.5 (M / 2+H). + .

[0816] Example 3AB: General Procedure for LP1, LP2, and LP#-4

[0817] To a solution of LP#-3 (1.0–1.2 equiv.) in DMF (0.15 mM), HOBt (0.5 equiv.), DIPEA (3.0 equiv.), and payload P (1.0 equiv.) were added. The reaction mixture was stirred at room temperature for 2 h and monitored by LCMS. The resulting mixture was directly purified by reverse-phase flash chromatography to give LP1, LP2, or LP#-4 as a white solid.

[0818] Example 3AC: {4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadecan-15-amido}-3-methylbutanamido]pentanamido]phenyl}methyl N-({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]tetracosa-1,6(11),12,14,16,18,20(24)-heptan-23-yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamate (LP1)

[0819] [ka]

[0820] Following the general procedure starting with payload P3 (0.85 g, 1.2 mmol) and LP1-3 (1.2 g, 1.2 mmol), the linker payload LP1 (1.1 g, 62% yield, formate salt) was obtained as a white solid after purification by preparative HPLC (5-60% acetonitrile in aqueous formic acid (0.1%)). ESI m / z: 699.0 (M / 2+H). + . 1 H NMR (400 MHz, DMSO d6 )δ10.00(s,1H),8.80(t,J=6.4Hz,1H),8.51(d,J=8.8Hz,1H),8.13(d,J=7. 6Hz,1H),7.90(d,J=8.8Hz,1H),7.79(d,J=10.8Hz,1H),7.65-7.50(m,3H), 7.43(t,J=6.0Hz,1H),7.31(s,1H),7.27(d,J=8.8Hz,2H),6.54(s,1H),5.9 8(t,J=5.2Hz,1H),5.63-5.57(m,1H),5.41(s,4H),5.21(s,2H),4.92(s,2H) ),4.62(d,J=6.4Hz,2H),4.43-4.33(m,1H),4.31-4.17(m,2H),4.01(s,2H) ,3.86(d,J=14.4Hz,1H),3.75(d,J=14.8Hz,1H),3.67-3.46(m,15H),3.44- 3.39(m,2H),3.27-3.10(m,4H),3.06-2.90(m,2H),2.47-2.32(m,5H),2.26 -1.64(m,14H),1.63-1.52(m,3H),1.47-1.32(m,3H),0.90-0.80(m,9H)ppm. 19 F NMR (376 MHz, DMSO d6 )δ-111 ppm.

[0821] Example 3AD: {4-[(2S)-2-[(2S)-2-[1-(4-{2-azatricyclo[10.4.0.0 4 , 9]hexadeca-1(12),4(9),5,7,13,15-hexan-10-yn-2-yl}-4-oxobutanamido)-3,6,9,12-tetraoxapentadecan-15-amido]-3-methylbutanamido]-5-(carbamoylamino)pentanamido]phenyl}methyl N-({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]tetracosa-1,6(11),12,14,16,18,20(24)-heptan-23-yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamate (LP2)

[0822] [ka]

[0823] Following the general procedure, starting with payload P3 (10 mg, 17 μmol) and LP2-3 (CAS 2226472-28-0, synthesized according to International Publication No. WO2018089373, 18 mg, 17 μmol), the linker payload LP2 (12 mg, 46% yield) was obtained as a white solid after purification by preparative HPLC (5-95% acetonitrile in 10 mM aqueous ammonium bicarbonate). ESI m / z: 513.4 (M / 3+H). + , Rt=6.63 min in HPLC (E-open ring form, 34%); 507.4 (M / 3+H), 760.5 (M / 2+H), Rt=7.45 min in HPLC (lactone form, 64%). 1 H NMR (400 MHz, DMSO d6)δ9.99(s,1H),8.80(t,J=6.4Hz,1H),8.50(d,J=8.8Hz,1H),8.12(d,J=7.2Hz,1H),7.87(d, J=8.4Hz,1H),7.80-7.75(m.2H),7.69-7.67(m,1H),7.63-7.58(m,3H),7.51-7.46(m,3H),7 .45-7.33(m,3H),7.32-7.26(m,4H),6.53(s,1H),5.98(t,J=6.0Hz,1H),5.63-5.57(m,1H), 5.42(s,4H),5.21(s,2H),5.03(d,J=14.0Hz,1H),4.93(s,2H),4.63(d,J=6.8Hz,2H),4.41- 4.35(m,1H),4.25-4.21(m,1H),4.02(s,2H),3.62-3.57(m,5H),3.48-3.45(m,12H),3.31-3 .28(m,2H),3.23-3.14(m,2H),3.11-3.07(m,2H),3.05-2.98(m,1H),2.96-2.91(m,1H),2.6 0-2.55(m,1H),2.46-2.44(m,1H),2.39(s,3H),2.35-2.33(m,1H),2.26-2.15(m,3H),2.03- 1.94(m,2H),1.88-1.67(m,4H),1.63-1.57(m,1H),1.46-1.33(m,2H),0.88-0.81(m,9H)ppm. 19F NMR(376MHz,DMSO d6 )δ-111 ppm.

[0824] Example 3AE: (9H-Fluoren-9-yl)methyl N-{2-[2-(2-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-{[4-({[({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24]tetracosa-1,6(11),12,14,16,18,20(24)-heptan-23-yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamoyl]oxy}methyl)phenyl]carbamoyl}butyl]carbamoyl}-2-methylpropyl]carbamoyl}ethoxy)ethoxy]ethyl}carbamate (LP13-4)

[0825] [ka]

[0826] Following the general procedure starting from LP13-3 (0.10 g, 0.11 mmol) and payload P3 (77 mg, 0.11 mmol), compound LP13-4 (0.12 g, 78% yield) was obtained as a pale yellow oil after purification by reverse-phase flash chromatography (0–100% acetonitrile in water for 10 min, then 100% acetonitrile for 5 min). ESI m / z: 684.0 (M / 2+H)+.

[0827] Example 3AF: Methyl (4R)-4-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-{[4-({[({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 ,11.020,24]tetracosa-1,6(11),12,14,16,18,20(24)-heptan-23-yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamoyl]oxy}methyl)phenyl]carbamoyl}butyl]carbamoyl}-2-methylpropyl]carbamoyl}-4-(3-{2-[2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)ethoxy]ethoxy}propanamido)butanoate (LP14-4)

[0828] [ka]

[0829] Following the general procedure starting with LP14-3 (0.12 g, 0.11 mmol) and payload P3 (64 mg, 0.11 mmol), compound LP14-4 (0.13 g, 78% yield) was obtained as a white solid after purification by reverse-phase flash chromatography (0–60% acetonitrile in aqueous ammonium bicarbonate (10 mM)). ESI m / z: 755.7 (M / 2+H). + .

[0830] Example 3AG: Methyl (4S)-4-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-{[4-({[({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .04,13.0 6 , 11 .0 20 , 24 ]tetracosa-1,6(11),12,14,16,18,20(24)-heptan-23-yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamoyl]oxy}methyl)phenyl]carbamoyl}butyl]carbamoyl}-2-methylpropyl]carbamoyl}-4-(3-{2-[2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)ethoxy]ethoxy}propanamido)butanoate (LP15-4)

[0831] [ka]

[0832] Following the general procedure starting from LP15-3 (0.50 g, 0.47 mmol) and P3 (0.22 g, 0.38 mmol), compound LP15-4 (0.40 g, 56% yield) was obtained as a white solid. ESI m / z: 755.5 (M / 2+H). + .

[0833] Example 3AH: Methyl (4R)-4-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-{[4-({[({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]tetracosa-1,6(11),12,14,16,18,20(24)-heptan-23-yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamoyl]oxy}methyl)phenyl]carbamoyl}butyl]carbamoyl}-2-methylpropyl]carbamoyl}-4-[1-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-3,6,9,12-tetraoxapentadecan-15-amido]butanoate (LP19-4)

[0834] [ka]

[0835] Following the general procedure starting from LP19-3 (80 mg, 69 μmol) and payload P3 (40 mg, 69 μmol), compound LP19-4 (70 mg, 64% yield) was obtained as a white solid. ESI m / z: 799.5 (M / 2+H). + .

[0836] Example 3AI: Methyl (4R)-4-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-{[4-({[({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .06 , 11 .0 20 , 24 ]tetracosa-1,6(11),12,14,16,18,20(24)-heptan-23-yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamoyl]oxy}methyl)phenyl]carbamoyl}butyl]carbamoyl}-2-methylpropyl]carbamoyl}-4-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadecan-15-amido}butanoate (LP20-4)

[0837] [ka]

[0838] Following the general procedure starting from LP20-3 (95 mg, 86 μmol) and payload P3 (58 mg, 0.10 mmol), compound LP20-4 (60 mg, 45% yield) was obtained as a pale yellow solid. ESI m / z: 770.6 (M / 2+H). + .

[0839] Example 3AJ: Methyl (4S)-4-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-{[4-({[({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24]tetracosa-1,6(11),12,14,16,18,20(24)-heptan-23-yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamoyl]oxy}methyl)phenyl]carbamoyl}butyl]carbamoyl}-2-methylpropyl]carbamoyl}-4-[1-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-3,6,9,12-tetraoxapentadecan-15-amido]butanoate (LP21-4)

[0840] [ka]

[0841] Following the general procedure starting from LP21-3 (58 mg, 50 μmol) and payload P3 (35 mg, 50 μmol), compound LP21-4 (51 mg, 64% yield) was obtained as a white solid. ESI m / z: 799.8 (M / 2+H). + . 1 H NMR (400 MHz, DMSO d6)δ10.04(s,1H),8.80(t,J=6.4Hz,1H),8.51(d,J=8.4Hz,1H),8.16(d,J=7.6Hz,1H),8.09(d,J=7.6Hz,1H),7.88(d,J=7.6Hz,2H),7.7 8(d,J=10.8Hz,2H),7.69(d,J=7.2Hz,2H),7.58(d,J=8.8Hz,2H),7.44-7.39(m,3H),7.34-7.31(m,4H),7.27(d,J=8.4Hz,2H),6.53(br s,1H),5.99(br s,1H),5.62-5.57(m.1H),5.42(s,2H),5.46-5.37(m.1H),5.20(s,2H),4.93(s,2H),4.63(d,J=5.6Hz,2H),4 .41-4.33(m,2H),4.30-4.28(m,2H),4.22-4.16(m,2H),4.02(s,2H),3.63-3.57(m,8H),3.50-3.46(m,12H), 3.40(t,J=6.0Hz,2H),3.26-3.18(m,1H),3.15-3.10(m,3H),3.07-2.99(m,1H),2.99-2.90(m,1H),2.42-2.3 0(m,7H),2.21-2.14(m,2H),1.97-1.82(m,4H),1.77-1.59(m,3H),1.54-1.32(m,2H),0.88-0.82(m,9H)ppm.

[0842] Example 3AK: Methyl (4S)-4-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-{[4-({[({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20,24]tetracosa-1,6(11),12,14,16,18,20(24)-heptan-23-yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamoyl]oxy}methyl)phenyl]carbamoyl}butyl]carbamoyl}-2-methylpropyl]carbamoyl}-4-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadecan-15-amido}butanoate (LP22-4)

[0843] [ka]

[0844] Following the general procedure starting from LP22-3 (80 mg, 73 μmol) and payload P3 (43 mg, 73 μmol), compound LP22-4 (60 mg, 60% yield) was obtained as a white solid after purification by reverse-phase flash chromatography (0-60% acetonitrile in aqueous ammonium bicarbonate (10 mM)). ESI m / z: 770.5 (M / 2+H). + .

[0845] Example 3AL: {4-[(2S)-2-[(2S)-2-{3-[2-(2-aminoethoxy)ethoxy]propanamido}-3-methylbutanamido]-5-(carbamoylamino)pentanamido]phenyl}methyl N-({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]tetracosa-1,6(11),12,14,16,18,20(24)-heptan-23-yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamate (LP13)

[0846] [ka]

[0847] To a solution of LP13-4 (0.12 g, 84 μmol) in anhydrous DMF (1.8 mL) was added diethylamine (0.2 mL), and the mixture was stirred at room temperature for 1 h until the Fmoc group was completely removed by LCMS. The resulting solution was directly separated by reverse-phase flash chromatography (10 min in aqueous TFA (0.01%), 0–100% acetonitrile) to give LP13 (50 mg, 52% yield) as a pale yellow solid. ESI m / z: 573.0 (M / 2+H). + .

[0848] Example 3AM: (4R)-4-{3-[2-(2-aminoethoxy)ethoxy]propanamide}-4-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-{[4-({[({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]tetracosa-1,6(11),12,14,16,18,20(24)-heptan-23-yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamoyl]oxy}methyl)phenyl]carbamoyl}butyl]carbamoyl}-2-methylpropyl]carbamoyl}butanoic acid (LP14)

[0849] [ka]

[0850] To a solution of compound LP14-4 (0.10 g, 66 μmol) in DMF (3 mL) was added piperidine (56 mg, 0.66 mmol), and the mixture was stirred at room temperature for 2 h until Fmoc cleavage, as monitored by LCMS. Aqueous lithium hydroxide (0.2 mM, 1 mL) and THF (3 mL) were added to the reaction mixture, and the mixture was stirred at room temperature for an additional 1 h until the methyl ester was completely hydrolyzed by LCMS. After filtration, the resulting mixture was acidified to pH 5.0 with PBS buffer (pH 3.0) and then concentrated under vacuum. The residue was purified by preparative HPLC (10–95% acetonitrile in aqueous TFA (0.01%)) to afford the linker payload LP14 (40 mg, 47% yield) as a white solid. ESI m / z: 637.5 (M / 2+H). + .

[0851] Example 3AN: (4S)-4-{3-[2-(2-aminoethoxy)ethoxy]propanamide}-4-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-{[4-({[({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .020, 24 ]tetracosa-1,6(11),12,14,16,18,20(24)-heptan-23-yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamoyl]oxy}methyl)phenyl]carbamoyl}butyl]carbamoyl}-2-methylpropyl]carbamoyl}butanoic acid (LP15)

[0852] [ka]

[0853] Following the same procedure as for LP14, except replacing LP14-4 with LP15-4, the linker payload LP15 (50 mg, 14% yield) was obtained as a white solid after purification by preparative HPLC (10-95% acetonitrile in aqueous TFA (0.05%)). ESI m / z: 637.4 (M / 2+H). + .

[0854] Example 3AO: (4R)-4-(1-amino-3,6,9,12-tetraoxapentadecan-15-amido)-4-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-{[4-({[({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]tetracosa-1,6(11),12,14,16,18,20(24)-heptan-23-yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamoyl]oxy}methyl)phenyl]carbamoyl}butyl]carbamoyl}-2-methylpropyl]carbamoyl}butanoic acid (LP19)

[0855] [ka]

[0856] Following the same procedure as for LP14, except replacing LP14-4 with LP19-4 (60 mg, 38 μmol), the linker payload LP19 (12 mg, 24% yield) was obtained as a white solid after purification by preparative HPLC (10-95% acetonitrile in aqueous TFA (0.05%)). ESI m / z: 681.5 (M / 2+H). + . 1 H NMR (400 MHz, DMSO d6)δ9.80(s,1H),8.81(t,J=6.4Hz,1H),8.52(d,J=8.8Hz,1H),8.19-8.13(m,2H),8.08(d,J=8.4Hz,1H),7. 81-7.75(m,3H),7.61(d,J=6.4Hz,2H),7.43(t,J=6.0Hz,1H),7.31(s,1H),7.27(d,J=8.4Hz,2H),6.55(br s,1H),6.04-5.99(m,1H),5.64-5.57(m,1H),5.43-5.40(m,2H),5.20(s,2H),4.93(s,2H),4.63(d,J=6.4Hz,2H),4.40-4. 32(m,2H),4.23-4.19(m,1H),4.02(s,2H),3.63-3.60(m,4H),3.59-3.57(m,4H),3.56-3.54(m,3H),3.51-3.49(m,5H),3. 01-2.95(m,4H), 2.44-2.41(m,1H), 2.39(s,3H), 2.31-2.28(m,1H), 2.26-2.21(m,3H), 2.20-2.15(m,2H), 2.09-2.01(m,2H), 1.90-1.82(m,4H), 1.78-1.72(m,2H), 1.66-1.61(m,1H), 1.50-1.41(m,2H), 1.40-1.35(m,1H), 0.90-0.82(m,11H) ppm. (COOH protons were not identified.) 19 F NMR (376 MHz, DMSO d6 )δ-73.70,-111.28 ppm.

[0857] Example 3AP: (4R)-4-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-{[4-({[({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24]tetracosa-1,6(11),12,14,16,18,20(24)-heptan-23-yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamoyl]oxy}methyl)phenyl]carbamoyl}butyl]carbamoyl}-2-methylpropyl]carbamoyl}-4-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadecan-15-amido}butanoic acid (LP20)

[0858] [ka]

[0859] To a solution of compound LP20-4 (60 mg, 39 μmol) in water (1 mL) and THF (3 mL) was added aqueous lithium hydroxide (0.12 M, 1 mL). The reaction mixture was stirred at room temperature for 2 h until the methyl ester was completely hydrolyzed, as monitored by LCMS. The mixture was acidified with PBS buffer (pH 4.0) to pH 6.0 and then concentrated in vacuo. The residue was purified by preparative HPLC (10–95% acetonitrile in aqueous TFA (0.05%)) to give LP20 (15 mg, 25% yield) as a white solid. ESI m / z: 763.5 (M / 2+H). + . 1 H NMR (400 MHz, DMSO d6)δ9.78(s,1H),8.80(t,J=6.8Hz,1H),8.51(t,J=8.8Hz,1H),8.18-8.12(m,2H),8.07(d,J=6.0Hz,1H),7.78(d ,J=11.2Hz,1H),7.63-7.59(m,3H),7.43(t,J=6.0Hz,1H),7.32-7.22(m,4H),7.11-6.96(m,1H),6.61-6.45(br s,1H),6.02-5.96(m,1H),5.63-5.57(m,1H),5.44-5.38(m,3H),5.20(s,2H),4.92(s,2H),4.63 (d,J=6.0Hz,2H),4.40-4.33(m,2H),4.29-4.25(m,1H),4.24-4.18(m,1H),4.01(s,2H),3.89-3 .84(m,1H),3.78-3.73(m,1H),3.63-3.60(m,2H),3.57-3.55(m,1H),3.51-3.47(m,8H),3.27-3 .21(m,4H),3.17-3.12(m,1H),3.03-2.99(m,1H),2.97-2.93(m,1H),2.44-2.41(m,1H),2.39(br s, 3H), 2.28-2.14 (m, 7H), 2.08-1.99 (m, 2H), 1.94-1.81 (m, 6H), 1.80-1.71 (m, 5H), 1.63-1.54 (m, 3H), 1.40-1.33 (m, 4H), 0.90-0.81 (m, 11H) ppm. (The protons of the acid and TFA were not revealed.) 19 F NMR (400 MHz, DMSO) d6 )δ-73.86,-111.30 ppm.

[0860] Example 3AQ: (4S)-4-(1-amino-3,6,9,12-tetraoxapentadecan-15-amido)-4-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-{[4-({[({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6, 11 .0 20 , 24 ]tetracosa-1,6(11),12,14,16,18,20(24)-heptan-23-yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamoyl]oxy}methyl)phenyl]carbamoyl}butyl]carbamoyl}-2-methylpropyl]carbamoyl}butanoic acid (LP21)

[0861] [ka]

[0862] Following the same procedure as for LP14, except replacing LP14-4 with LP21-4 (45 mg, 28 μmol), the linker payload LP21 (10 mg, 26% yield) was obtained as a white solid after purification by preparative HPLC (10-95% acetonitrile in aqueous TFA (0.05%)). ESI m / z: 681.4 (M / 2+H). + . 1 H NMR (400 MHz, DMSO d6)δ10.05(s,1H),8.81(t,J=7.2Hz,1H),8.52(d,J=8.4Hz,1H),8.19(d,J=7.6H z,1H),8.09(d,J=8.0Hz,1H),7.81-7.68(m,5H),7.58(d,J=8.4Hz,2H),7.43(t ,J=6.8Hz,1H),7.32(s,1H),7.27(d,J=8.4Hz,2H),6.54(brs,1H),6.03-5.97( m,1H),5.63-5.57(m,1H),5.48-5.42(m,3H),5.21(s,2H),4.92(s,2H),4.63(d ,J=6.4Hz,2H),4.42-4.31(m,2H),4.22-4.17(m,1H),4.02(s,2H),3.62-3.55 (m,8H),3.51-3.48(m,12H),3.20-3.12(m,2H),3.05-2.89(m,5H),2.40(s,3H) , 2.26-2.21 (m, 2H), 2.20-2.12 (m, 2H), 2.02-1.95 (m, 1H), 1.90-1.80 (m, 3H), 1.74-1.63 (m, 2H), 1.59-1.54 (m, 1H), 1.49-1.31 (m, 2H), 0.88-0.82 (m, 9H) ppm. (COOH protons were not identified.) 19 F NMR (376 MHz, DMSO d6 )δ-73,-111 ppm.

[0863] Example 3AR: (4S)-4-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-{[4-({[({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24]tetracosa-1,6(11),12,14,16,18,20(24)-heptan-23-yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamoyl]oxy}methyl)phenyl]carbamoyl}butyl]carbamoyl}-2-methylpropyl]carbamoyl}-4-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadecan-15-amido}butanoic acid (LP22)

[0864] [ka]

[0865] Following the same procedure as for LP20, except replacing LP20-4 with LP22-4 (60 mg, 39 μmol), the linker payload LP22 (15 mg, 26% yield) was obtained as a white solid after purification by preparative HPLC (10-95% acetonitrile in aqueous TFA (0.05%)). ESI m / z: 763.5 (M / 2+H). + . 1 H NMR (400 MHz, DMSO d6)δ9.78(br s,1H),8.80(t,J=6.8Hz,1H),8.51(t,J=8.8Hz,1H),8.18-8.12(m,2H),8.07(d,J=6.0Hz,1H),7.78(d,J=1 1.2Hz,1H),7.63-7.59(m,3H),7.43(t,J=6.0Hz,1H),7.32-7.22(m,4H),7.11-6.96(m,1H),6.61-6.45(br s,1H),6.02-5.96(m,1H),5.63-5.57(m,1H),5.43-5.40(m,2H),5.20( s,2H),4.92(s,2H),4.63(d,J=6.0Hz,2H),4.40-4.33(m,2H),4.29-4. 25(m,1H),4.23-4.18(m,1H),4.01(s,2H),3.89-3.84(m,1H),3.78-3. 73(m,1H),3.63-3.60(m,2H),3.57-3.55(m,1H),3.51-3.47(m,8H),3. 46-3.45 (m, 2H), 3.27-3.21 (m, 4H), 3.17-3.13 (m, 1H), 3.03-2.99 (m, 1H), 2.97-2.93 (m, 1H), 2.46-2.41 (m, 1H), 2.39 (s, 3H), 2.36-2.31 (m, 1H), 2.27-2.16 (m, 7H), 2.09-2.00 (m, 1H), 2.00-1.81 (m, 7H), 1.80-1.68 (m, 4H), 1.62-1.54 (m, 3H), 1.40-1.35 (m, 3H), 0.90-0.81 (m, 11H) ppm. (COOH protons were not identified.) 19 F NMR (376 MHz, DMSO d6 )δ-73.86,-111.30 ppm.

[0866] Example 4: Synthesis of Peptide Linker Payload

[0867] Linker payloads LP3, LP4, LP7 / LP7', and LP9 were synthesized as described in Scheme 5 and in Examples 4A-4F below.

[0868] The starting materials L3-2 (CAS 1353016-71-3) and L4-2 (CAS 1425803-45-7) were commercially obtained from Accela.

[0869] [ka]

[0870] Example 4A: 2-(2-{2-[2-(cyclooct-2-yn-1-yloxy)acetamido]acetamido}acetamido)acetic acid (L3-3)

[0871] [ka]

[0872] To a suspension of peptide L3-1 (Gly-Gly-Gly-OH, 0.34 g, 1.8 mmol) in DMF (13 mL), a solution of L3-2 (0.50 g, 1.8 mmol) in THF (6 mL) and DIPEA (0.69 g, 5.4 mmol) was added, and the cloudy mixture was stirred at room temperature for 20 h. The mixture was filtered, the clear filtrate was concentrated in vacuo, and the residue was purified by reverse-phase flash chromatography (0–20% acetonitrile in water) to give compound L3-3 (0.13 g, 21% yield) as a white solid. ESI m / z: 354.2 (M+H). + . 1 H NMR (400 MHz, DMSO d6 )δ12.6(s,1H),8.20(t,J=5.6Hz,1H),8.15(t,J=6.0Hz,1H),7.82(t,J=5.6Hz,1H),4.35-4.31(m,1H),3.94(d,J=14.8Hz, ppm

[0873] Example 4B: 2-[2-(2-{2-[2-(cyclooct-2-yn-1-yloxy)acetamido]acetamido}acetamido)acetamido]-N-[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]tetracosa-1,6(11),12,14,16,18,20(24)-heptan-23-yl]carbamoyl}methoxy)methyl]acetamide (LP3)

[0874] [ka]

[0875] To a yellow solution of compound L3-3 (9.0 mg, 25 μmol) in dry DMF (14 mL) was added DIPEA (9.0 mg, 70 μmol) and HATU (10 mg, 26 μmol). The mixture was stirred at room temperature for 30 min, followed by the addition of payload P3 (15 mg, 22 μmol). The reaction mixture was stirred at room temperature for 2 h until the majority of the starting material was consumed by LCMS. The resulting mixture was directly purified by preparative HPLC (0-100% acetonitrile in aqueous TFA (0.01%)) to afford linker payload LP3 (8.0 mg, 36% yield, TFA salt) as a pale yellow solid. ESI m / z: 915.5 (M+H). + . 1 H NMR (400 MHz, DMSO d6)δ8.68(t,J=6.6Hz,1H),8.51(d,J=8.8Hz,1H),8.25-8.12(m,3H),7.86-7.75(m,2H),7.31( s,1H),6.53(s,1H),5.59(s,1H),5.43(s,2H),5.20(s,2H),4.63(d,J=6.5Hz,2H),4.31(m,1H) ),4.01(s,2H),3.92(d,J=14.9Hz,1H),3.75(m,9H),3.18(s,2H),2.40(s,3H),2.26-2.02(m ,5H),1.96-1.70(m,6H),1.63-1.53(m,2H),1.39(d,J=8.7Hz,1H),0.87(t,J=7.3Hz,3H)ppm. 19 F NMR (376 MHz, DMSO d6 ) δ-74(TFA),-111(Ar-F)ppm.

[0876] Example 4C: (2S)-2-{2-[2-(4-{2-azatricyclo[10.4.0.0 4 , 9 ]Hexadeca-1(12),4(9),5,7,13,15-hexane-10-yn-2-yl}-4-oxobutanamido)acetamido]acetamido}-3-phenylpropanoic acid (L4-3)

[0877] [ka]

[0878] To a solution of compound L4-2 (0.28 g, 0.69 mmol) and peptide L4-1 (Gly-Gly-Phe-OH, 0.19 g, 0.69 mmol) in DMF (10 mL), DIPEA (0.37 mL, 2.1 mmol) was added, and the reaction mixture was stirred at room temperature for 1 h. Completion of the reaction was monitored by LCMS. The resulting mixture was directly purified by reverse-phase flash chromatography (0–100% acetonitrile in 10 mM aqueous ammonium bicarbonate) to give compound L4-3 (0.31 g, 78% yield) as a white solid. ESI m / z: 567.0 (M+H). + .

[0879] Example 4D: 4-{2-azatricyclo[10.4.0.0 4 , 9 ]hexadeca-1(12),4(9),5,7,13,15-hexan-10-yn-2-yl}-N-{[({[(1S)-1-[({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]tetracosa-1,6(11),12,14,16,18,20(24)-heptan-23-yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamoyl]-2-phenylethyl]carbamoyl}methyl)carbamoyl]methyl}-4-oxobutanamide (LP4)

[0880] [ka]

[0881] Following the procedure for the preparation of LP3, except for replacing L3-3 with L4-3, the ring-opened lactone LP4-RO (14 mg, 56% yield) with and without the linker payload, LP4, was obtained as a white solid after purification by preparative HPLC (0–100% acetonitrile in aqueous ammonium bicarbonate (10 mM)).

[0882] Lactone: HPLC purity: 75%, retention time: 7.93 minutes, ESI m / z: 1128.3 (M+H) + ,564.8(M / 2+H) + ;Ring-opening product: HPLC purity: 20%, retention time: 6.94 minutes, ESI m / z: 1169.4 (M+Na) + ,1146.3(M+H) + . 1 H NMR (400 MHz, DMSO d6)δ8.62(s,1H),8.50(d,J=8.8Hz,1H),8.28(s,1H),8.18-7.91(m,3H),7.78(d,J=11.5Hz,1H),7.70-7.58(m,2H),7.50 -7.38(m,3H),7.29(m,3H),7.24-7.11(m,5H),6.51(s,1H),5.58(s,1H),5.41(s,1H),5.19(s,1H),4.99(d,J=13.8Hz, 1H),4.62(d,J=6.1Hz,2H),4.46(s,1H),4.01(s,2H),3.70(m,3H),3.56(m,3H),3.22-3.06(m,2H),2.99(m,2H),2.75( m,1H),2.67(m,1H),2.38(m,3H),2.33(m,4H),2.17(m,2H),2.07(m,2H),1.95-1.70(m,2H),0.86(t,J=7.2Hz,3H)ppm.

[0883] Example 4E: 2-(cyclooct-2-yn-1-yloxy)-N-{[({[({2-[2-({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]tetracosa-1,6(11),12,14,16,18,20(24)-heptan-23-yl]carbamoyl}methoxy)pyrrolidin-1-yl]-2-oxoethyl}carbamoyl)methyl]carbamoyl}methyl)carbamoyl]methyl}acetamide (diastereomer 1, LP7 and diastereomer 2, LP7')

[0884] [ka]

[0885] Following the procedure for LP3, except replacing P3 with P4, after purification by preparative HPLC (0–100% acetonitrile in aqueous ammonium bicarbonate (8 mM) containing ammonia (0.05% v)), diastereomers LP7 (with and without the ring-opened lactone product, 3.0 mg, 7% yield) and LP7′ (with and without the ring-opened lactone product, 4.0 mg, 9.3% yield) were obtained separately as white solids.

[0886] LP7: Lactone: HPLC purity: 11%, retention time: 6.87 minutes, ESI m / z: 955.3 (M+H) + , Ring-opening product: HPLC purity: 89%, retention time: 5.91 min, ESI m / z: 996.5 (M+Na) + 1 H NMR (400 MHz, DMSO d6 )δ8.71-8.57(m,1H),8.30-8.13(m,2H),7.99-7.70(m,3H),7.32-6.67(m,2H),5.63-5.48(m,1H),5.43-5.01(m,5H),4 .36-4.27(m,1H),4.21-3.68(m,10H),2.40-2.24(m,4H),2.14-1.33(m,22H),1.14-0.97(m,2H),0.89-0.84(m,3H)ppm. 19 F NMR (376 MHz, DMSO d6 )δ-111,-112 ppm.

[0887] LP7': Lactone: HPLC purity: 21%, retention time: 6.98 min, ESI m / z: 955.3 (M+H) + , Ring-opening product: HPLC purity: 79%, retention time: 6.02 min, ESI m / z: 996.5 (M+Na) + 1 H NMR (400 MHz, DMSO d6)δ8.60-8.55(m,1H),8.31-7.69(m,5H),7.32-7.21(m,1H),6.66-6.53(m,1H),5.64-5.56(m,1H),5.43-5.09(m,5H),4.32-4.30(m,1H),4 .16-4.02(m,2H),3.98-3.89(m,2H),3.82-3.55(m,6H),2.40-2.31(m,4H),2.22-1.36(m,22H),1.15-0.98(m,2H),0.89-0.84(m,3H)ppm. 19 F NMR (376 MHz, DMSO d6 )δ-111,-112 ppm.

[0888] Example 4F: 2-[2-(2-{2-[2-(cyclooct-2-yn-1-yloxy)acetamido]acetamido}acetamido)acetamido]-N-[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]tetracosa-1,6(11),12,14,16,18,20(24)-heptan-23-yl]carbamoyl}methoxy)methyl]acetamide (LP9)

[0889] [ka]

[0890] To a yellow solution of compound L3-3 (Example 4A) (9.0 mg, 25 μmol) in dry DMF (14 mL), DIPEA (9.0 mg, 70 μmol) and HATU (10 mg, 26 μmol) were added. The mixture was stirred at room temperature for 30 minutes, followed by the addition of payload P (15 mg, 22 μmol). The reaction mixture was stirred at room temperature for 2 hours until most of the starting material was consumed by LCMS. The resulting mixture was directly purified by preparative HPLC (0-100% acetonitrile in aqueous TFA (0.01%)) to afford linker payload LP9 (8.0 mg, 36% yield, TFA salt) as a pale yellow solid. ESI m / z: 915.5 (M+H). + . 1 H NMR (400 MHz, DMSO d6 )δ8.68(t,J=6.6Hz,1H),8.51(d,J=8.8Hz,1H),8.25-8.12(m,3H),7.86-7.75(m,2H),7.31( s,1H),6.53(s,1H),5.59(s,1H),5.43(s,2H),5.20(s,2H),4.63(d,J=6.5Hz,2H),4.31(m,1H) ),4.01(s,2H),3.92(d,J=14.9Hz,1H),3.75(m,9H),3.18(s,2H),2.40(s,3H),2.26-2.02(m ,5H),1.96-1.70(m,6H),1.63-1.53(m,2H),1.39(d,J=8.7Hz,1H),0.87(t,J=7.3Hz,3H)ppm. 19 F NMR (376 MHz, DMSO d6 ) δ-74(TFA),-111(Ar-F)ppm.

[0891] Example 5: Synthesis of Acid-Sensitive Linker Payload

[0892] Linker payload LP8 was synthesized as shown in Scheme 6 and described below.

[0893] The starting material L2-1 (CAS1427004-19-0) was commercially available from Accela.

[0894] [ka]

[0895] 1-(4-{2-azatricyclo[10.4.0.0 4 , 9 ]hexadeca-1(12),4(9),5,7,13,15-hexan-10-yn-2-yl}-4-oxobutanamide)-N-{2-[2-({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]tetracosa-1,6(11),12,14,16,18,20(24)-heptan-23-yl]carbamoyl}methoxy)pyrrolidin-1-yl]-2-oxoethyl}-3,6,9,12-tetraoxapentadecan-15-amide (LP8)

[0896] [ka]

[0897] To a solution of payload P4 (6.2 mg, 10 μmol) in DMF (1.0 mL), compound L2-1 (6.5 mg, 10 μmol) and DIPEA (3.9 mg, 30 μmol) were added, and the reaction mixture was stirred at room temperature for 2 h. Completion of the reaction was monitored by LCMS. The resulting mixture was directly purified by preparative HPLC (5–95% acetonitrile in 10 mM aqueous ammonium bicarbonate) to afford linker payload LP8 (containing the lactone ring-opened product, 3.0 mg, 26% yield) as a yellow solid.

[0898] Lactone: HPLC purity: 80%, retention time: 8.12 minutes, ESI m / z: 577.6 (M / 2+H) +;Ring-opening product: HPLC purity: 20%, retention time: 6.91 min, ESI m / z: 586.7 (M / 2+H) + .

[0899] 1 H NMR (400 MHz, DMSO d6 )δ7.74-7.67(m,2H),7.63-7.58(m,1H),7.56-7.52(m,1H),7.44-7.36(m,3H),7.32-7.20(m,4H),6.60-6.44(m,1H),5.56-5.4 9(m,1H),5.34(s,2H),5.28-5.16(m,1H),5.11-5.06(m,1H),4.98-4.92(m,1H),4.10-3.91(m,2H),3.75-3.74(m,1H),3.55-3.5 0(m,3H),3.40-3.37(m,13H),3.22-3.16(m,5H),3.02-2.99(m,3H),2.54-2.48(m,2H),2.31(d,J=2.8Hz,2H),2.22-2.11(m,4H) ),1.96-1.88(m,4H),1.82-1.72(m,2H),1.67-1.64(m,2H),1.01(t,J=7.2Hz,2H),0.92(t,J=7.2Hz,2H),0.82-0.76(m,3H)ppm. 19 F NMR (376 MHz, DMSO d6 )δ-111 ppm.

[0900] Example 6: Synthesis of glucose linker payload

[0901] Linker payloads LP5 and LP6 were synthesized as described below in Schemes 7A-7B and Examples 6A-6N.

[0902] [ka]

[0903] [ka]

[0904] Example 6A: N-(2-aminoethyl)-2-(cyclooct-2-yn-1-yloxy)acetamide (L5-1)

[0905] [ka]

[0906] To a solution of ethylenediamine (0.71 g, 12 mmol) in DMF (2.0 mL), DIPEA (0.30 g, 2.4 mmol) and a solution of compound L3-2 (0.33 g, 1.2 mmol) in DMF (3.0 mL) were slowly added, and the mixture was stirred at room temperature for 30 min. The completion of the reaction was monitored by LCMS. The resulting mixture was purified by reverse-phase flash chromatography (0-100% acetonitrile in aqueous ammonium bicarbonate (0.8 mM)) to give compound L5-1 (0.18 g, 68% yield) as a colorless oil. ESI m / z: 225.2 (M+H). + . 1 H NMR (400 MHz, DMSO d6 )δ7.74-7.63(m,1H),4.28(t,J=5.8Hz,1H),3.88-3.73(m,2H),3.11-3.00(m,4H),2.58(t,J =6.4Hz,2H),2.27-2.06(m,3H),1.94-1.71(m,4H),1.66-1.54(m,2H),1.45-1.33(m,1H)ppm.

[0907] Example 6B: Methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-({2-[2-(cyclooct-2-yn-1-yloxy)acetamido]ethyl}carbamoyl)-4-(hydroxymethyl)phenoxy]oxane-2-carboxylate (L5-3)

[0908] [ka]

[0909] To a mixture of compound L5-2 (synthesized according to International Publication No. WO2018182341A1, the entirety of which is incorporated herein by reference) (0.11 g, 0.23 mmol) and HATU (96 mg, 0.25 mmol) in dry DMF (4 mL) was added compound L5-1 (51 mg, 0.23 mmol) and DIPEA (89 mg, 0.69 mmol). The reaction mixture was stirred at room temperature for 2 h until L5-2 was completely consumed, as monitored by LCMS. The resulting mixture was directly purified by reverse-phase flash chromatography (0-100% acetonitrile in aqueous TFA (0.01%)) to give compound L5-3 (0.14 g, 90% yield) as a white solid. ESI m / z: 691.4 (M+H). + . 1 H NMR(400MHz,CDCl3)δ8.06-8.04(m,1H),7.64-7.59(m,1H),7.50-7.47(m,1 H),7.22-7.18(m,1H),7.01-6.98(m,1H),5.44-5.28(m,5H),4.68(s,2H),4 .30-4.21(m,2H),4.10-4.06(m,1H),3.93-3.88(m,1H),3.75(s,3H),3.67- 3.48(m,2H),2.21-2.07(m,15H),1.93-1.79(m,3H),1.70-1.38(m,3H)ppm.

[0910] Example 6C: Methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-({2-[2-(cyclooct-2-yn-1-yloxy)acetamido]ethyl}carbamoyl)-4-{[(4-nitrophenoxycarbonyl)oxy]methyl}phenoxy]oxane-2-carboxylate (L5-4)

[0911] [ka]

[0912] To a solution of compound L5-3 (0.14 g, 0.20 mmol) in DMF (2.0 mL) was added bis(4-nitrophenyl)carbonate (55 mg, 0.18 mmol) and DIPEA (26 mg, 0.20 mmol) under nitrogen at 0 °C. The reaction mixture was stirred at 0 °C for 30 min and then at room temperature for 3 h. The reaction mixture was diluted with ethanol (10 mL) and extracted with ethyl acetate (3 x 20 mL). The combined organic solution was washed with brine (10 mL), dried over anhydrous sodium sulfate, and concentrated in vacuo. The residue was purified by flash chromatography (40-60% ethyl acetate in petroleum ether) to give compound L5-4 (85 mg, 49% yield) as a colorless oil. ESI m / z: 856.0 (M+H). + .

[0913] Example 6D: Methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-({2-[2-(cyclooct-2-yn-1-yloxy)acetamido]ethyl}carbamoyl)-4-({[({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]tetracosa-1,6(11),12,14,16,18,20(24)-heptan-23-yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamoyl]oxy}methyl)phenoxy]oxane-2-carboxylate (L5-5)

[0914] [ka]

[0915] To a solution of compound L5-4 (17 mg, 20 μmol) in DMF (1.0 mL) were added P3 (12 mg, 20 μmol), HOBt (2.7 mg, 20 μmol), and DIPEA (5.1 mg, 40 μmol). The reaction mixture was stirred at room temperature for 16 h and monitored by LCMS. The resulting mixture was directly purified by reverse-phase flash chromatography (0-100% acetonitrile in aqueous TFA (0.01%)) to give compound L5-5 (13 mg, 20% yield) as a yellow solid. ESI m / z: 649.0 (M / 2+H). + .

[0916] Example 6E: (2S,3S,4S,5R,6S)-6-[2-({2-[2-(cyclooct-2-yn-1-yloxy)acetamido]ethyl}carbamoyl)-4-({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]tetracosa-1,6(11),12,14,16,18,20(24)-heptan-23-yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamoyl]oxy}methyl)phenoxy]-3,4,5-trihydroxyoxane-2-carboxylic acid (LP5)

[0917] [ka]

[0918] To a mixture of compound L5-5 (13 mg, 10 μmol) in methanol (2 mL) was added aqueous lithium hydroxide (0.1 M, 2 mL), and the mixture was stirred at room temperature for 1 h. The reaction completion was monitored by LCMS. After quenching with aqueous HCl (1 N) to pH 4, the resulting mixture was purified by reverse-phase flash chromatography (5–95% acetonitrile in aqueous TFA (0.01%)) to afford the linker payload LP5 (5 mg, 43% yield) as a white solid. ESI m / z: 1156.3 (M+H). + .

[0919] Example 6F: [(2R,3R,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-({2-[2-(cyclooct-2-yn-1-yloxy)acetamido]ethyl}carbamoyl)-4-(hydroxymethyl)phenoxy]oxan-2-yl]methyl acetate (L6-3)

[0920] [ka]

[0921] Following the procedure for making L5-3, except replacing L5-2 with L6-2, compound L6-3 (0.10 g, 80% yield) was obtained as a white solid. ESI m / z: 705.3 (M+H). + .

[0922] Example 6G: [(2R,3R,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-({2-[2-(cyclooct-2-yn-1-yloxy)acetamido]ethyl}carbamoyl)-4-{[(4-nitrophenoxycarbonyl)oxy]methyl}phenoxy]oxan-2-yl]methyl acetate (L6-4)

[0923] [ka]

[0924] Following the procedure for making L5-4, except replacing L5-3 with L6-3, compound L6-4 (62 mg, 50% yield) was obtained as a white solid. ESI m / z: 870.3 (M+H). + .

[0925] Example 6H: [(2R,3R,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-({2-[2-(cyclooct-2-yn-1-yloxy)acetamido]ethyl}carbamoyl)-4-({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]tetracosa-1,6(11),12,14,16,18,20(24)-heptan-23-yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamoyl]oxy}methyl)phenoxy]oxan-2-yl]methyl acetate (L6-5)

[0926] [ka]

[0927] Following the procedure for making L5-5, except replacing L5-4 with L6-4, compound L6-5 (30 mg, 66% yield) was obtained as a white solid. ESI m / z: 655.7 (M / 2+H). + .

[0928] Example 6I: [3-({2-[2-(cyclooct-2-yn-1-yloxy)acetamido]ethyl}carbamoyl)-4-{[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}phenyl]methyl N-({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]tetracosa-1,6(11),12,14,16,18,20(24)-heptan-23-yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamate (LP6)

[0929] [ka]

[0930] Following the procedure for making LP5, except replacing L5-5 with L6-5, the linker payload LP6 (9 mg, 34% yield) was obtained as a white solid. ESI m / z: 1142.3 (M+H). + .

[0931] Example 6J: Benzyl 2-({2-[2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)acetamido]acetamido}methoxy)acetate (GP-2)

[0932] [ka]

[0933] To a solution of compound GP-1 (CAS: 1599440-07-9, synthesized according to International Publication No. WO2014057687, 0.20 g, 0.42 mmol) in DMF (5 mL) was added diethylamine (0.15 g, 2.1 mmol). The reaction mixture was stirred at room temperature overnight and monitored by LCMS. The resulting mixture was directly separated by reverse-phase flash chromatography (0-100% acetonitrile in 0.05% aqueous ammonium bicarbonate) to give a white solid (0.1 g, ESI m / z: 253.1). This was added to a mixture of Fmoc-glycine (0.14 g, 0.48 mmol) and HATU (0.23 g, 0.59 mmol) in DMF (5 mL), followed by the addition of DIPEA (0.15 g, 0.59 mmol). The reaction mixture was stirred at room temperature for 4 h and monitored by LCMS. The resulting mixture was directly purified by preparative HPLC (10-95% acetonitrile in aqueous TFA (0.05%)) to give compound GP-2 (0.14 g, 64% yield) as a white solid. ESI m / z: 554.3 (M+Na). + .

[0934] Example 6K: 2-({2-[2-({[(9H-Fluoren-9-yl)methoxy]carbonyl}amino)acetamido]acetamido}methoxy)acetic acid (GP-3)

[0935] [ka]

[0936] Palladium on carbon (0.10 g) was added to a solution of compound GP-2 (0.10 g, 0.19 mmol) in ethyl acetate (10 mL) under nitrogen protection. The reaction mixture was stirred at room temperature under a hydrogen balloon for 4 hours and monitored by LCMS. The resulting mixture was filtered through Celite, and the filtrate was concentrated in vacuo to give compound GP-3 (56 mg, 65% yield) as a white solid. ESI m / z (weak): 464.0 (M+Na). + .

[0937] Example 6L: 2-amino-N-({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]tetracosa-1,6(11),12,14,16,18,20(24)-heptan-23-yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)acetamide (Gly-P3)

[0938] [ka]

[0939] To a solution of compound GP-3 (41 mg, 93 μmol) in dry DMF (5 mL), HATU (39 mg, 0.10 mmol), exatecan (mesylate, 41 mg, 93 μmol), and DIPEA (36 mg, 0.28 mmol) were added sequentially. The reaction mixture was stirred at room temperature for 2 h and monitored by LCMS. The resulting mixture was separated by reverse-phase flash chromatography (0.01% aqueous TFA, 0-100% acetonitrile) to give Fmoc-Gly-P3 (50 mg, 63% yield, ESI m / z: 859.0) as a white solid, which was dissolved in DMF (5 mL). Diethylamine (20 mg, 0.27 mmol) was added to the solution, and the mixture was stirred at room temperature overnight. The resulting mixture was separated by reverse-phase flash chromatography (0.01% aqueous TFA, 0-100% acetonitrile) to give Gly-P3 (TFA salt, 40 mg, 57% yield from exatecan) as a white solid. ESI m / z: 637.3 (M+H). + .

[0940] Example 6M: Methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-({2-[2-(cyclooct-2-yn-1-yloxy)acetamido]ethyl}carbamoyl)-4-{[({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]tetracosa-1,6(11),12,14,16,18,20(24)-heptan-23-yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamoyl]methyl}carbamoyl)oxy]methyl}phenoxy]oxane-2-carboxylate (LP12-5)

[0941] [ka]

[0942] Following the same procedure as for LP10-5, except replacing P3 with Gly-P3, compound LP12-5 (23 mg, 39% yield) was obtained as a yellow solid. ESI m / z: 860.5 (MM DXD +H) + ,677.4(M / 2+H) + .

[0943] Example 6N: (2S,3S,4S,5R,6S)-6-[2-({2-[2-(cyclooct-2-yn-1-yloxy)acetamido]ethyl}carbamoyl)-4-{[({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .020 , 24 ]tetracosa-1,6(11),12,14,16,18,20(24)-heptan-23-yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamoyl]methyl}carbamoyl)oxy]methyl}phenoxy]-3,4,5-trihydroxyoxane-2-carboxylic acid (LP12)

[0944] [ka]

[0945] Following the same procedure as for LP10, except replacing LP10-5 with LP12-5, the linker payload LP12 (3 mg, 27% yield) was obtained as a white solid. ESI m / z: 607.4 (M / 2+H). + . 1 H NMR (400 MHz, DMSO d6 )δ8.72(t,J=6.5Hz,1H),8.62-8.49(m,2H),8.19(t,J=5.1Hz,1H),7.98-7.90(m,1H),7.82-7.71(m,2H),7.53(t,J=6.0Hz,1H),7.4 7-7.41(m,1H),7.37-7.28(m,2H),6.56(s,1H),5.69-5.55...

Claims

1. Formula (A): BA-(NH-L1-B-(-L2-(-M-Dxd) ｍ ) ｋ ) ｎ (A) wherein: L1 is absent or is a first linker linked to the BA via the side chain of a glutamine residue (Gln); B is a branching unit containing at least one addition of a group B′ and a group B″, one of which is —N ３ and 【Chemistry 1】 and the other of group B′ and group B″ is selected from: 【Chemistry 2】 wherein Q is CH or N; L2 is a second linker covalently attached to the branching unit B via at least one group B″; M has the structure: 【Transformation 3】 wherein R, R′, and R″ are independently at each occurrence hydrogen or C １ -C ４ alkyl, or R′ and R″ together form a 5- or 6-membered ring; Dxd is represented by the formula (P): 【Chemistry 4】 is an antitumor drug having a structure according to k is a number from about 1 to about 12; m is a number from about 1 to about 30; n is a number from about 1 to about 30; BA is an anti-STEAP2 antibody or antigen-binding fragment thereof, or an anti-MET bispecific antibody or antigen-binding fragment thereof comprising a first antigen-binding domain (D1) and a second antigen-binding domain (D2); D1 specifically binds a first epitope of human MET; D2 specifically binds a second epitope of human MET; compound.

2. the BA is an anti-MET bispecific antibody or an antigen-binding fragment thereof; D1 comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 2012, and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 2092; D2 comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 2036, and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 2092; The compound of claim 1.

3. The compound of claim 1 , wherein the BA is an anti-STEAP2 antibody or an antigen-binding fragment thereof.

4. 4. The compound of claim 3, wherein the anti-STEAP2 antibody or antigen-binding fragment thereof comprises the heavy chain CDRs and light chain CDRs of an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 58, 74 / 58, 82 / 58, 90 / 58, 98 / 58, 106 / 114, 122 / 130, 138 / 146, 154 / 162, 170 / 178, 186 / 194, 202 / 210, 218 / 226, 234 / 242, 250 / 258, 266 / 274, 282 / 290, 298 / 306, 314 / 322, 330 / 338, 346 / 354, 362 / 370, and 378 / 386.

5. said L1 is absent, B is a branching unit containing at least one addition of a group B′ and a group B″, one of which is —N ３ and 【Transformation 5】 and the other of group B′ and group B″ is selected from: 【Transformation 6】 wherein Q is CH or N; L2 is a second linker comprising a self-immolative moiety and / or an enzyme-labile moiety; The M has the structure: 【Transformation 7】 a portion having The Dxd is represented by the formula (P): 【Transformation 8】 is an antitumor drug having a structure according to n is a number from about 1 to about 10; The compound of claim 1.

6. The compound of claim 1 , wherein the glutamine residue Gln is naturally present in the CH2 or CH3 domain of the BA.

7. The compound of claim 6, wherein the Gln is Q295 and / or N297Q.

8. A compound having the structure: 【Chemistry 9】 The compound according to claim 5, wherein n is an integer from 1 to 8.

9. 9. A pharmaceutical composition comprising a compound according to any one of claims 1 to 8 and a pharmaceutically acceptable carrier.

10. A compound having the structure: 【Chemistry 10】 n is an integer from 1 to 8, BA is an anti-MET bispecific antibody or antigen-binding fragment thereof comprising a first antigen-binding domain (D1) and a second antigen-binding domain (D2); D1 specifically binds a first epitope of human MET; D2 specifically binds a second epitope of human MET; A pharmaceutical composition comprising the compound.

11. The first antigen-binding domain (D1) of the bispecific antibody or antigen-binding fragment thereof comprises: HCDR1 comprising the amino acid sequence of SEQ ID NO: 2014; HCDR2 comprising the amino acid sequence of SEQ ID NO: 2016; HCDR3 comprising the amino acid sequence of SEQ ID NO: 2018; LCDR1 comprising the amino acid sequence of SEQ ID NO: 2094; LCDR2 comprising the amino acid sequence of SEQ ID NO: 2096, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 2098; Including, The second antigen-binding domain (D2) of the bispecific antibody or antigen-binding fragment thereof comprises: HCDR1 comprising the amino acid sequence of SEQ ID NO: 2038; HCDR2 comprising the amino acid sequence of SEQ ID NO: 2040; HCDR3 comprising the amino acid sequence of SEQ ID NO: 2042; LCDR1 comprising the amino acid sequence of SEQ ID NO: 2094; LCDR2 comprising the amino acid sequence of SEQ ID NO: 2096, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 2098; 11. The pharmaceutical composition of claim 10, comprising:

12. Use of a compound of any one of claims 1 to 8, or a pharmaceutical composition of any one of claims 9 to 11, in the manufacture of a medicament for treating cancer in a subject afflicted with a tumor associated with or mediated by MET expression or activity, or the proliferation of MET+ cells.

13. 13. The use according to claim 12, wherein the cancer is selected from the group consisting of lung cancer, prostate cancer, bladder cancer, cervical cancer, colon cancer, kidney cancer, breast cancer, pancreatic cancer, stomach cancer, uterine cancer and ovarian cancer.

14. 14. The use according to claim 13, wherein the cancer is non-small cell lung cancer.

15. The compound has the following structure: 【Chemistry 11】 n is a number from about 1 to about 8; BA is a bispecific antibody or antigen-binding fragment thereof comprising a first antigen-binding domain (D1) and a second antigen-binding domain (D2); D1 specifically binds a first epitope of human MET; D2 specifically binds a second epitope of human MET; 13. The use according to claim 12.

16. The first antigen-binding domain (D1) of the bispecific antibody or antigen-binding fragment thereof comprises: HCDR1 comprising the amino acid sequence of SEQ ID NO: 2014; HCDR2 comprising the amino acid sequence of SEQ ID NO: 2016; HCDR3 comprising the amino acid sequence of SEQ ID NO: 2018; LCDR1 comprising the amino acid sequence of SEQ ID NO: 2094; LCDR2 comprising the amino acid sequence of SEQ ID NO: 2096, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 2098; Including, The second antigen-binding domain (D2) of the bispecific antibody or antigen-binding fragment thereof comprises: HCDR1 comprising the amino acid sequence of SEQ ID NO: 2038; HCDR2 comprising the amino acid sequence of SEQ ID NO: 2040; HCDR3 comprising the amino acid sequence of SEQ ID NO: 2042; LCDR1 comprising the amino acid sequence of SEQ ID NO: 2094; LCDR2 comprising the amino acid sequence of SEQ ID NO: 2096, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 2098; 16. The use according to claim 15, comprising:

17. A compound having the structure: 【Chemistry 12】 n is an integer from 1 to 8, BA is an anti-STEAP2 antibody or an antigen-binding fragment thereof, A pharmaceutical composition comprising:

18. The anti-STEAP2 antibodies or antigen-binding fragments thereof are represented by SEQ ID NOs: 4-6-8-12-14-16, 20-22-24-28-30-32, 36-38-40-44-46-48, 52-54-56-60-62-64, 68-70-72-60-62-64, 76-78-80-60-62-64, 84-86-88-60-62-64, 92-94-96-60-62-64, 100-102-104-60-62-64, 108-110-112-116-118-120, 124-126-128-132-134-136, 140-142-144-148-150-152, 156-158-160-164-166-168, 172-174-176-180-182-184, 188-190-192-196-198-200, 204-206-208-2 12-214-216, 220-222-224-228-230-232, 236-238-240-244-246-248, 252-254-256-260-262-264, 268-270-272-276-278-280, 284-286-288-292-294-296, 300-302-304-308-310-312, 316-318-320-324-326-328 18. The pharmaceutical composition of claim 17, comprising an HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domain comprising an amino acid sequence selected from the group consisting of: 332-334-336-340-342-344, 348-350-352-356-358-360, 364-366-368-372-374-376, and 380-382-384-388-390-392.

19. 20. Use of a pharmaceutical composition according to any one of claims 17 to 18 in the manufacture of a medicament for treating a disease or disorder associated with or mediated by STEAP2 expression or activity or proliferation of STEAP2+ cells.

20. 20. The use of claim 19, wherein the disease or disorder is prostate cancer.

21. 1. A method for preparing an antibody drug conjugate having the formula: 【Chemistry 13】 n is an integer from 1 to 8, The method comprises: a) 【Chemistry 14】 A compound having the structure 【Chemistry 15】 contacting the compound having the structure 【Chemistry 16】 obtaining an intermediate compound having the structure b) exposing the intermediate to a BA in the presence of a microbial transglutaminase to obtain the antibody drug conjugate; wherein the BA is an anti-STEAP2 antibody or an antigen-binding fragment thereof, or an anti-MET bispecific antibody or an antigen-binding fragment thereof comprising a first antigen-binding domain (D1) and a second antigen-binding domain (D2); D1 specifically binds a first epitope of human MET; D2 specifically binds a second epitope of human MET; method.

22. the BA is an anti-MET bispecific antibody or an antigen-binding fragment thereof; D1 comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 2012, and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 2092; D2 comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 2036, and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 2092; 22. The method of claim 21.

23. the BA is an anti-STEAP2 antibody or an antigen-binding fragment thereof; 22. The method of claim 21, wherein the anti-STEAP2 antibody or antigen-binding fragment thereof comprises the heavy chain CDRs and light chain CDRs of an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 58, 74 / 58, 82 / 58, 90 / 58, 98 / 58, 106 / 114, 122 / 130, 138 / 146, 154 / 162, 170 / 178, 186 / 194, 202 / 210, 218 / 226, 234 / 242, 250 / 258, 266 / 274, 282 / 290, 298 / 306, 314 / 322, 330 / 338, 346 / 354, 362 / 370, and 378 / 386.

24. 1. A method for preparing an antibody drug conjugate having the formula: 【Chemistry 17】 n is an integer from 1 to 8, The method comprises: a) in the presence of microbial transglutaminase; [Chemistry 18] exposing a compound having the structure 【Chemistry 19】 obtaining an intermediate antibody having a handle of the structure: b) coupling an intermediate antibody having said handle to 【Chemistry 20】 and obtaining the antibody-drug conjugate, the BA is an anti-STEAP2 antibody or an antigen-binding fragment thereof, or an anti-MET bispecific antibody or an antigen-binding fragment thereof, comprising a first antigen-binding domain (D1) and a second antigen-binding domain (D2); D1 specifically binds a first epitope of human MET; D2 specifically binds a second epitope of human MET; method.

25. the BA is an anti-MET bispecific antibody or an antigen-binding fragment thereof; D1 comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 2012, and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 2092; D2 comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 2036, and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 2092; 25. The method of claim 24.

26. the BA is an anti-STEAP2 antibody or an antigen-binding fragment thereof; The anti-STEAP2 antibody or antigen-binding fragment thereof comprises the heavy chain CDRs and light chain CDRs of an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 58, 74 / 58, 82 / 58, 90 / 58, 98 / 58, 106 / 114, 122 / 130, 138 / 146, 154 / 162, 170 / 178, 186 / 194, 202 / 210, 218 / 226, 234 / 242, 250 / 258, 266 / 274, 282 / 290, 298 / 306, 314 / 322, 330 / 338, 346 / 354, 362 / 370, and 378 / 386; 25. The method of claim 24.

27. A compound having a structure according to the formula: 【Chemistry 21】 n is an integer from 1 to 8, BA is a bispecific antibody or antigen-binding fragment thereof comprising a first antigen-binding domain (D1) and a second antigen-binding domain (D2); D1 specifically binds a first epitope of human MET; D2 specifically binds a second epitope of human MET; D1 comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 2012, and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 2092; D2 comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 2036, and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 2092; compound.

28. D1 comprises an HCVR comprising the amino acid sequence of SEQ ID NO: 2012 or an amino acid sequence at least 95% identical thereto; and LCVR comprising the amino acid sequence of SEQ ID NO: 2092 or an amino acid sequence at least 95% identical thereto; 28. The compound of claim 27.

29. D1 comprises an HCVR comprising the amino acid sequence of SEQ ID NO: 2012, and comprising an LCVR comprising the amino acid sequence of SEQ ID NO: 2092; 29. The compound of claim 28.

30. D2 comprises an HCVR comprising the amino acid sequence of SEQ ID NO: 2036 or an amino acid sequence at least 95% identical thereto; and LCVR comprising the amino acid sequence of SEQ ID NO: 2092 or an amino acid sequence at least 95% identical thereto; 28. The compound of claim 27.

31. D2 comprises an HCVR comprising the amino acid sequence of SEQ ID NO: 2036, and comprising an LCVR comprising the amino acid sequence of SEQ ID NO: 2092; 31. The compound of claim 30.

32. 32. A pharmaceutical composition comprising a compound of any one of claims 27 to 31 and a pharmaceutically acceptable carrier.

33. Use of a compound of any one of claims 27 to 31 or a pharmaceutical composition of claim 32 in the manufacture of a medicament for treating cancer in a subject afflicted with a tumor associated with or mediated by MET expression or activity, or the proliferation of MET+ cells.

34. 34. The use of claim 33, wherein the cancer is selected from the group consisting of lung cancer, prostate cancer, bladder cancer, cervical cancer, colon cancer, kidney cancer, breast cancer, pancreatic cancer, stomach cancer, uterine cancer and ovarian cancer.

35. 35. The use of claim 34, wherein the cancer is non-small cell lung cancer.

36. A compound having the structure: 【Chemistry 22】 n ranges from about 1 to about 8; BA is an anti-STEAP2 antibody or an antigen-binding fragment thereof, or an anti-MET bispecific antibody or an antigen-binding fragment thereof comprising a first antigen-binding domain (D1) and a second antigen-binding domain (D2); compound.

37. the BA is an anti-MET bispecific antibody or an antigen-binding fragment thereof; D1 specifically binds a first epitope of human MET; D1 is HCDR1 comprising the amino acid sequence of SEQ ID NO: 2014; HCDR2 comprising the amino acid sequence of SEQ ID NO: 2016; HCDR3 comprising the amino acid sequence of SEQ ID NO: 2018; LCDR1 comprising the amino acid sequence of SEQ ID NO: 2094; LCDR2 comprising the amino acid sequence of SEQ ID NO: 2096, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 2098; Including, D2 specifically binds a second epitope of human MET; D2 comprises the second antigen-binding domain (D2) of the bispecific antibody or antigen-binding fragment thereof, and HCDR1 comprises the amino acid sequence of SEQ ID NO: 2038; HCDR2 comprising the amino acid sequence of SEQ ID NO: 2040; HCDR3 comprising the amino acid sequence of SEQ ID NO: 2042; LCDR1 comprising the amino acid sequence of SEQ ID NO: 2094; LCDR2 comprising the amino acid sequence of SEQ ID NO: 2096, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 2098; 37. The compound of claim 36, comprising:

38. D1 comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 2012, and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 2092; D2 comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 2036, and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 2092; 38. The compound of claim 37.

39. 40. Use of a compound according to any one of claims 36 to 38 in the manufacture of a medicament for treating cancer in a subject afflicted with a tumor associated with or mediated by MET expression or activity, or the proliferation of MET+ cells.

40. 40. The use of claim 39, wherein the cancer is selected from the group consisting of lung cancer, prostate cancer, bladder cancer, cervical cancer, colon cancer, kidney cancer, breast cancer, pancreatic cancer, stomach cancer, uterine cancer and ovarian cancer.

41. 41. The use of claim 40, wherein the cancer is non-small cell lung cancer.

42. the BA is an anti-STEAP2 antibody or an antigen-binding fragment thereof; The anti-STEAP2 antibodies or antigen-binding fragments thereof are represented by SEQ ID NOs: 4-6-8-12-14-16, 20-22-24-28-30-32, 36-38-40-44-46-48, 52-54-56-60-62-64, 68-70-72-60-62-64, 76-78-80-60-62-64, 84-86-88-60-62-64, and 92-94-96-60-62-64, respectively. , 100-102-104-60-62-64, 108-110-112-116-118-120, 124-126-128-132-134-136, 140-142-144-148-150-152, 156-158-160-164-166-168, 172-174-176-180-182-184, 188-190-192-196-198-200, 204-206-208- 212-214-216, 220-222-224-228-230-232, 236-238-240-244-246-248, 252-254-256-260-262-264, 268-270-272-276-278-280, 284-286-288-292-294-296, 300-302-304-308-310-312, 316-318-320-324-326-32 37. The compound of claim 36, comprising an HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domain comprising an amino acid sequence selected from the group consisting of: 376-378-379-380-381-382-383-384-385-386-387-388-390-392; 378-379-383-384-385-386-387-388-390-392;

43. 43. The compound of claim 42, wherein the anti-STEAP2 antibody or antigen-binding fragment thereof comprises the heavy chain CDRs and light chain CDRs of an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 58, 74 / 58, 82 / 58, 90 / 58, 98 / 58, 106 / 114, 122 / 130, 138 / 146, 154 / 162, 170 / 178, 186 / 194, 202 / 210, 218 / 226, 234 / 242, 250 / 258, 266 / 274, 282 / 290, 298 / 306, 314 / 322, 330 / 338, 346 / 354, 362 / 370, and 378 / 386.

44. 44. Use of a compound according to any one of claims 42 to 43 in the manufacture of a medicament for treating a disease or disorder associated with or mediated by STEAP2 expression or activity, or proliferation of STEAP2+ cells.

45. 45. The use of claim 44, wherein the disease or disorder is prostate cancer.

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