Camptothecin analogs conjugated to glutamine residues in proteins and their use

Abstract

Described herein are, for example, protein-drug conjugates useful for target-specific delivery of therapeutic moieties, such as camptothecin analogs and / or derivatives, and compositions thereof. In certain embodiments, specific and efficient methods are provided 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 comprising glutaminyl-modified antibodies and camptothecin analog payloads are provided.

Description

[Technical Field]

[0001] Cross-references to related applications This application claims priority under 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 whole.

[0002] Areas of this disclosure This disclosure relates to protein-drug conjugates (e.g., antibody-drug conjugates), pharmaceutical compositions, and methods for treating diseases using them. It also provides a specific and efficient method for producing protein-drug conjugates using a combination of transglutaminase and 1,3-addition cyclization techniques. More specifically, this disclosure relates to protein-drug conjugates (e.g., antibody-drug conjugates) comprising camptothecin analogs and derivatives.

[0003] Sequence List An official copy of the sequence listing was submitted simultaneously with this specification as a paper copy of the sequence listing in ASCII format, filenamed 250298_000244_SL.txt, size approximately 996 kilobytes, and was created on July 12, 2021. The sequence listing contained in this paper copy of the ASCII document is part of this specification and is incorporated herein by reference to the entire document. [Background technology]

[0004] Proliferative disorders are characterized by uncontrolled growth and the proliferation of abnormal cells. If the proliferation is uncontrolled, it can be fatal. Abnormal proliferation, such as cancer, can result from both external factors (e.g., tobacco, chemicals, radiation, infectious organisms) and internal factors (genetic mutations, immune system status, metabolic mutations). These causative factors may act collectively or sequentially to initiate or promote abnormal proliferation. Cancer is treated with surgery, radiation, chemotherapy, hormones, and immunotherapy. However, more effective growth inhibitors are needed.

[0005] An ideal proliferative therapy would allow for the delivery of highly cytotoxic drugs targeting tumor cells, leaving normal cells unaffected. Conventional chemotherapy is limited due to toxic side effects resulting from the drug's effects on non-cancerous cells. Various approaches to targeted drug delivery have been attempted, including the use of toxins such as Pseudomonas or diphtheria toxin to conjugate tumor-targeting probes (antibodies, growth factors, etc.) to inhibit protein and cell synthesis. However, side effects include immune system reactions caused by the non-human components of the conjugate. Furthermore, the half-life of drug conjugates is limited due to excretion from circulation through renal filtration, schematic degradation, uptake by the reticuloendothelial system (RES), and accumulation in non-target organs and tissues.

[0006] Other approaches utilize passive drug carriers such as polymers, liposomes, and macromolecular micelles to take advantage of 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 bloodstream and technical challenges in obtaining a highly normalized, pharmaceutically acceptable drug delivery system with the specificity and selectivity required for binding to tumor cells.

[0007] Protein conjugates, such as antibody conjugates, utilize the selective binding of a conjugate to deliver the payload to the target within the target tissue. The payload can be a therapeutic portion capable of acting at the target.

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

[0009] In the art, there is a need for safe and effective antitumor targeting agents capable of binding to various antigens, which can be used in monotherapy and combination therapy to improve the treatment of diseases such as cancer. In some embodiments, this disclosure satisfies this need and also provides other advantages.

[0010] The aforementioned discussion is presented solely to help you better understand the nature of the challenges facing the technical field in question, but even if any reference in this specification is recognized as prior art, it should not be interpreted as allowing such reference to constitute “prior art” to this application. [Overview of the project]

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

[0012] In one embodiment, this disclosure relates to formula (A): BA-(Gln-NH-L1-B-(-L2-(-M-Dxd) m ) k ) n (A) A compound having the structure of the formula, in which, BA is an antibody or its antigen-binding fragment. Gln is a glutamine residue, L1 is either non-existent or the first linker. B is a branched unit comprising at least one adduct of base B' and base B'', where at least one of base B' and base B'' is -N3 and

[0013] [ka] Selected from, the other of base B' and base B'' is,

[0014] [ka] Selected from, where Q is C or N, L2 is a second linker covalently bonded to branch unit B via at least one group B'', M is either non-existent or has no structure:

[0015] [ka] The portion having the following characteristics, where R, R', and R'' are independently hydrogen or C1-C4 alkyl when they appear, or R' and R'' combine to form a 5-membered ring or a 6-membered ring. Dxd is given by equation (P):

[0016] [ka] It is an antitumor drug having a structure such as k is an integer between 1 and 12. m is an integer between 1 and 30. n is an integer between 1 and 30. Provides compounds.

[0017] In another aspect, this disclosure is defined as formula (I): BA-(Gln-NH-L1-B-(-L2-M-Dxd) k ) n (I) A compound having the structure of the formula, in which, BA is an antibody or its antigen-binding fragment. Gln is a glutamine residue, L1 is either non-existent or the first linker. B is a branched unit comprising at least one adduct of base B', where base B' is -N3,

[0018] [ka] Selected from, where Q is C or N, L2 is a second linker covalently bonded to branch unit B via at least one group B'', where groups B' and B'' form at least one adduct. M is either non-existent or has no structure:

[0019] [ka] The portion having the following characteristics, where R, R', and R'' are independently hydrogen or C1-C4 alkyl when they appear, or R' and R'' combine to form a 5-membered ring or a 6-membered ring. Dxd is given by equation (P):

[0020] [ka] It is an antitumor drug having a structure such as k is an integer between 1 and 12. n is an integer between 1 and 30. Provides compounds.

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

[0022] In one embodiment, 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, BA is an anti-STEAP2 antibody containing the HCVR / LCVR amino acid sequence pair specified in Table 1.

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

[0025] In one embodiment, the anti-HER2 / HER2 bispecific antibody is The first antigen-binding domain (D1), The second antigen-binding domain (D2) and Includes, D1 specifically binds to the first epitope of human HER2. D2 specifically binds to the second epitope of human HER2.

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

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

[0028] In one embodiment, the anti-MET / MET bispecific antibody is The first antigen-binding domain (D1), The second antigen-binding domain (D2) and Includes, D1 specifically binds to the first epitope of human MET, D2 specifically binds to the second epitope of human MET.

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

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

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

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

[0033] In one embodiment, anti-STEAP2 includes 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.

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

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

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

[0037] In one embodiment, 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 comprising 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 each independently an integer of 1 to 8.

[0039] In one embodiment, L1 is

[0040] [Chemical Formula] as well as combinations thereof, wherein R A is a group comprising an alkyne, azide, tetrazine, trans-cyclooctene, maleimide, amine, ketone, aldehyde, carboxylic acid, ester, thiol, sulfonic acid, tosylate, halide, silane, cyano group, carbohydrate group, biotin group, or lipid residue, and the subscripts x, n, p, and q are each independently an integer of 0 to 12.

[0041] In one embodiment, B includes one adduct of base B'.

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

[0043] [ka] Selected from the group consisting of,

[0044] [ka] This is the amino site of the binding of BA to the glutamine residue.

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

[0046] In one embodiment, B includes two adducts of base B'.

[0047] In one embodiment, B includes three adducts of base B'.

[0048] In one embodiment, B includes at least four adducts of base B'.

[0049] In one embodiment, B is

[0050] [ka] The formula includes a group selected from the group consisting of the following, where (B') includes the bonding site of the adduct of group B'.

[0051] In one embodiment, B is

[0052] [ka] It is selected from the group consisting of the following.

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

[0054] [ka] Selected from the group consisting of,

[0055] [ka] This is the amino site of the binding of BA to the glutamine residue.

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

[0057] In one embodiment, the adducts of base B' and base B'' are

[0058] [ka] The structure is selected from the group consisting of the following, where Q is C or N.

[0059] In one embodiment, M does not exist.

[0060] In one embodiment, M is

[0061] [ka] In the formula, R, R', and R'' are hydrogen atoms in their respective appearances, i.e., M is

[0062] [ka] That is the case.

[0063] In one embodiment, M is

[0064] [ka] In this formula, R is hydrogen, and R' and R'' combine to form a 5-membered ring, that is, M is

[0065] [ka] That is the case.

[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 given by formula (L2): B''-SP1-B2-(-SP2-AA-SP3) p (L2) It has a structure such that, in the formula, B'' is a group that can covalently bond to group B', SP1 is either non-existent or is the first spacer unit. B2 either does not exist or is a branch unit. SP2 is either non-existent or a second spacer unit. AA is either absent or a peptide unit containing 2-4 amino acids. SP3 is a third spacer unit that is either absent or covalently bonded to Dxd. p is an integer between 1 and 12.

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

[0069] [ka] The group is selected from the group consisting of these combinations.

[0070] In one embodiment, SP1 is absent or

[0071] [ka] It is selected from the group consisting of the following.

[0072] In one embodiment, B2 is absent or

[0073] [ka] It is selected from the group consisting of the following.

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

[0075] 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, as well as combinations thereof.

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

[0077] In one embodiment, AA is 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 Selected from the group consisting of EVC).

[0078] In one embodiment, SP3 is either absent or

[0079] [ka] A group consisting of these and their combinations is selected, in the formula R c They either do not exist independently at the time of each occurrence, or

[0080] [ka] It is a base selected from among them.

[0081] In one embodiment, M-Dxd is,

[0082] [ka] The structure is selected from the group consisting of the following, where R is hydrogen or C1-C4 alkyl,

[0083] [ka] This represents the connection point to L2.

[0084] In one embodiment, the compound is

[0085] [ka] It has the structure of [the object].

[0086] In one embodiment, the compound is

[0087] [ka] It has the structure of [the object].

[0088] In one embodiment, the compound is

[0089] [ka] It has the structure of [the object].

[0090] In another aspect, this disclosure is defined as formula (I): BA-(Gln-NH-L1-B-(-L2-M-Dxd) k ) n (I) A compound having the structure of the formula, in which, BA is an antibody or its antigen-binding fragment. Gln is a glutamine residue, L1 is either non-existent or the first linker. B is

[0091] [ka] It is a branching unit that includes, M is either non-existent or has no structure:

[0092] [ka] The portion having the following characteristics, where R, R', and R'' are independently hydrogen or C1-C4 alkyl when they appear, or R' and R'' combine to form a 5-membered ring or a 6-membered ring. Dxd is given by equation (P):

[0093] [ka] It is an antitumor drug having a structure such as k is an integer between 1 and 12. n is an integer between 1 and 30. Provides compounds.

[0094] In another aspect, this disclosure relates to formulas (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 by which, in the formula, B'' is -N3,

[0095] [ka] Selected from the group consisting of, SP1 does not exist, or

[0096] [ka] A first spacer unit selected from the group consisting of, SP2 does not exist, 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 A second spacer unit selected from the group consisting of -C(O)-NH- or combinations thereof, where the subscripts u and v are independently integers from 1 to 8. AA is either absent or a peptide unit containing 2-4 amino acids. SP3 does not exist, or

[0097] [ka] A third spacer unit selected from the group consisting of, where R c They either do not exist independently at the time of each occurrence, or

[0098] [ka] It is a base selected from, M does not exist, or

[0099] [ka] In the formula, R, R', and R'' are independently hydrogen or a C1-C4 alkyl group when they appear, or R' and R'' combine to form a 5-membered ring or a 6-membered ring. Dxd is given by equation (P):

[0100] [ka] It is an antitumor drug having the structure, p is an integer between 1 and 12. Provides compounds.

[0101] In one embodiment, AA is a peptide unit comprising 2 to 4 amino acids selected from glycine, valine, phenylalanine, proline, glutamic acid, lysine, alanine, and citrulline, as well as 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] It has a structure selected from the group consisting of, or a pharmaceutically acceptable salt thereof.

[0107] In one embodiment, the compound is

[0108] [ka] It has a structure selected from the group consisting of, 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] It has a structure according to formula (L2-P), (L2'-P), or (L2''-P), selected from the group consisting of the following.

[0116] In another aspect, this disclosure relates to formula (II): Ab-(Gln-NH-L1-B-(SP1-B2-(-SP2-AA-SP3-M-Dxd) k ) p ) n (II) An antibody-drug conjugate by which, Ab is an antibody, Gln is a glutamine residue, L1 is either non-existent or the first linker. B is a branched unit comprising at least one adduct of base B' and base B'', where base B' is -N3,

[0117] [ka] and selected from at least one base B'', B''-SP1-B2-(-SP2-AA-SP3-M-Dxd)p is a compound of formula (L2-P) according to any of the embodiments described above, wherein the compound of formula (L2-P) is covalently bound to the antibody via adducts of groups B' and B'', where 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. We provide antibody-drug conjugates.

[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] The structure of, or a pharmaceutically acceptable salt thereof, During the ceremony,

[0120] [ka] This represents a binding site to the antibody, either directly or via a second linker.

[0121] In another embodiment, the present disclosure provides a composition comprising a group of compounds according to any of the above embodiments having a drug-antibody ratio (DAR) of about 0.5 to about 30.0.

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

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

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

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

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

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

[0128] In another aspect, this disclosure relates to formula (PI):

[0129] [ka] A compound having the structure thereof or a pharmaceutically acceptable salt thereof, In the formula, R1, R2, R3, and R4 are independently hydrogen or C1-C4 alkyl, or R2 and R3 form a 5-membered ring or a 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 comprising an antibody, a linker, and a payload, or a pharmaceutically acceptable salt thereof, wherein the payload is

[0133] [ka] That is the case.

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

[0135] [ka] And,

[0136] [ka] This represents the linking point to the linker.

[0137] In one embodiment, the compound is given by formula (P2):

[0138] [ka] The structure is as follows, or a pharmaceutically acceptable salt thereof, where R is hydrogen or a C1-C4 alkyl group.

[0139] In one embodiment, the compound is

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

[0141] In one embodiment, the present invention provides a pharmaceutical composition comprising a compound according to any of the embodiments described above, and a diluent, a carrier, and / or an excipient.

[0142] In one embodiment, the present invention provides a method for treating a disease of a subject requiring treatment, comprising the step of administering a compound according to any of the embodiments described above, or a composition according to any of the embodiments described above, to the subject in a therapeutically effective amount.

[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 embodiment, the present invention provides a method for selectively delivering a compound according to any one of the embodiments described above to a cell.

[0147] In one embodiment, the present invention provides a method for selectively targeting an antigen on the cell surface using a compound according to any one of the embodiments described above.

[0148] In one embodiment, the cells are mammalian cells.

[0149] In one embodiment, the cells are human cells.

[0150] In one embodiment, the cells are cancer cells.

[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 embodiment, this disclosure relates to formula (A): BA-(Gln-NH-L1-B-(-L2-(-M-Dxd) m ) k ) n (A) A method for producing a compound having the structure of, in the formula, BA is an antibody or its antigen-binding fragment. Gln is a glutamine residue, L1 is the first linker, B is a branched unit comprising at least one adduct from base B' and base B'', L2 is a second linker covalently bonded to branch unit B via at least one group B'', M does not exist, or

[0153] [ka] In the formula, R, R', and R'' are independently hydrogen or a C1-C4 alkyl group when they appear, or R' and R'' combine to form a 5-membered ring or a 6-membered ring. Dxd is given by equation (P):

[0154] [ka] It is an antitumor drug that contains a structure, k and m are independent integers between 1 and 12, and n is an integer between 1 and 30. The above method, (a) A step of contacting compound L1-B with BA (BA-Gln-NH2) containing at least one glutamine residue Gln in the presence of transglutaminase, wherein branched unit B contains at least one group B', (b) The product of step (a) is compound L2-(-M-Dxd) m A step of bringing into contact with k or more equivalents of, wherein the linker L2 includes at least one group B'', and one of groups B' and B'' is -N3 and

[0155] [ka] Selected from, the other of base B' and base B'' is,

[0156] [ka] Selected from, where Q is C or N, process and, (c) A step to isolate the compound of formula (I) produced and We provide a method that includes [this].

[0157] In one embodiment, this disclosure relates to formula (A): BA-(Gln-NH-L1-B-(-L2-(-M-Dxd) m ) k ) n (A) A method for producing a compound having the structure of, In the formula, BA is an antibody or its antigen-binding fragment, Gln is a glutamine residue, L1 is the first linker described above, B is a branched unit comprising an adduct of at least one of the groups B' and B'' described above, L2 is the second linker described above, covalently bonded to branched unit B via at least one of the groups B'' described above, and M is absent or

[0158] [ka] In the formula, R, R', and R'' are as described above, and Dxd is given by formula (P):

[0159] [ka] An antitumor drug containing the structure, where k and m are independent integers between 1 and 12, and n is an integer between 1 and 30. The above method, (a) Compound L1-B, in which branched unit B contains at least one group B', is given compound L2-(-M-Dxd) m A step of bringing into contact with k or more equivalents of, wherein the linker L2 includes at least one group B'' that can covalently bond with group B', and one of groups B' and B'' is -N3 and

[0160] [ka] Selected from, the other of base B' and base B'' is,

[0161] [ka] Selected from, where Q is C or N, and by contact L1-B-(-L2-(-M-Dxd) m ) k The process by which it is produced, (b) In the presence of transglutaminase, BA (BA-Gln-NH2) containing at least one glutamine residue Gln is converted to L1-B-(L2-(-M-Dxd) in step (a) m ) k The process of bringing the product into contact with the product, (c) A step to isolate the compound of formula (I) produced and We provide a method that includes [this].

[0162] In one embodiment, this disclosure relates to formula (I): BA-(Gln-NH-L1-B-(-L2-M-Dxd) k ) n (I) A method for producing a compound having the structure of, in the formula, BA is an antibody or its antigen-binding fragment. Gln is a glutamine residue, L1 is the first linker, B is a branched unit comprising at least one adduct from base B' and base B'', L2 is a second linker covalently bonded to branch unit B via at least one group B'', M does not exist, or

[0163] [ka] In the formula, R, R', and R'' are independently hydrogen or a C1-C4 alkyl group when they appear, or R' and R'' combine to form a 5-membered ring or a 6-membered ring. Dxd is given by equation (P):

[0164] [ka] It is an antitumor drug that contains a structure, k is an integer between 1 and 12, and n is an integer between 1 and 30. The above method, (a) A step of contacting compound L1-B with BA (BA-Gln-NH2) containing at least one glutamine residue Gln in the presence of transglutaminase, wherein branched unit B contains at least one group B', (b) A step of contacting the product of step (a) with k or more equivalents of compound L2-M-Dxd, wherein linker L2 includes at least one group B'' that can be covalently bonded to group B', and one of groups B' and B'' is -N3 and

[0165] [ka] Selected from, the other of base B' and base B'' is,

[0166] [ka] Selected from, where Q is C or N, process and, (c) A step to isolate the compound of formula (I) produced and We provide a method that includes [this].

[0167] In one embodiment, this disclosure relates to formula (I): BA-(Gln-NH-L1-B-(-L2-M-Dxd) k ) n (I) A method for producing a compound having the structure of, In the formula, BA is an antibody or its antigen-binding fragment, Gln is a glutamine residue, L1 is the first linker described above, B is a branched unit comprising an adduct of at least one of the groups B' and B'' described above, L2 is the second linker described above, covalently bonded to branched unit B via at least one of the groups B'' described above, and M is absent or

[0168] [ka] In the formula, R, R', and R'' are as described above, and Dxd is given by formula (P):

[0169] [ka] An antitumor drug containing the structure, where k is an integer from 1 to 12 and n is an integer from 1 to 30. The above method, (a) A step of contacting a compound L1-B in which branch unit B contains at least one group B' with k or more equivalents of compound L2-M-Dxd, wherein linker L2 contains at least one group B'', and contact results in L1-B-(-L2-M-Dxd) k A is produced, and one of the groups B' and B'' becomes -N3 and

[0170] [ka] Selected from, the other of base B' and base B'' is,

[0171] [ka] Selected from, where Q is C or N, process and, (b) In the presence of transglutaminase, the binder BA (BA-Gln-NH2) containing at least one glutamine residue Gln is used in step (a) L1-B-(L2(M-Dxd) k The process of bringing the product into contact with the product, (c) A step to isolate the compound of formula (I) produced and We provide a method that includes [this].

[0172] In one embodiment, this disclosure relates to formula (III): BA-(Gln-NH-L2'-P)) n (III) A method for producing a compound having the structure of, BA is an antibody or its antigen-binding fragment, Gln is a glutamine residue, and L2'-P is the aforementioned H2N-SP1-B2-(SP2-AA-SP3-M-Dxd) p And n is an integer from 1 to 30, and SP1 does not exist or

[0173] [ka] A first spacer unit selected from the group consisting of B2, where B2 is absent or is a branching unit, and SP2 is absent 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 A second spacer unit selected from the group consisting of -C(O)-NH- or combinations thereof, where the subscripts u and v are independently integers from 1 to 8, AA is a peptide unit that is absent or contains 2 to 4 amino acids, and SP3 is absent or

[0174] [ka] A third spacer unit selected from the group consisting of, where R c They either do not exist independently at the time of each occurrence, or

[0175] [ka] It is a base selected from, where M is either absent or

[0176] [ka] In the formula, R, R', and R'' are independently hydrogen or a C1-C4 alkyl group when they appear, or R' and R'' combine to form a 5-membered ring or a 6-membered ring, and Dxd is given by formula (P):

[0177] [ka] An antitumor drug having the structure, where p is an integer from 1 to 30. The above method, (b) A step of contacting BA (BA-Gln-NH2) containing at least one glutamine residue Gln with L2'-P in the presence of transglutaminase, (c) A step to isolate the compound of formula (III) produced and We provide a method that includes [this].

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

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

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

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

[0182] In one embodiment, M does not exist, or M-Dxd is

[0183] [ka] The structure is selected from the group consisting of the following, where R is hydrogen or C1-C4 alkyl,

[0184] [ka] This represents the connection point to L2.

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

[0186] [ka]

[0187] [ka] It has a structure selected from the group consisting of, or a pharmaceutically acceptable salt thereof.

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

[0189] [ka] It has a structure selected from the group consisting of, or a pharmaceutically acceptable salt thereof.

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

[0191] [ka]

[0192] [ka]

[0193] [ka]

[0194] [ka]

[0195] [ka]

[0196] [ka] It has a structure selected from the group consisting of, or a pharmaceutically acceptable salt thereof.

[0197] These and other aspects of the present disclosure will be apparent to those skilled in the art by reading the detailed description of the present disclosure below, including the appended claims. [Brief explanation of the drawing]

[0198] This patent or patent application file shall include at least one drawing prepared in color. A copy of this patent or patent application publication accompanied by the color drawing shall be provided by the authorities after the request and payment of the required fees.

[0199] [Figure 1] This is a schematic diagram demonstrating the two-step site-specific generation of Dxd-ADC according to embodiments of the present disclosure. The first step is to conjugate one or more first linkers (L1-B') and glutamine residues on an antibody via a transglutaminase (e.g., MTG)-mediated conjugation reaction. The second step is to conjugate one or more linker 2-payloads (L2P) with antibody L1-B. [Figure 2]These are schematic diagrams illustrating specific and non-limiting embodiments of the present disclosure. Figure 2A is a schematic diagram of a two-step site-specific synthesis of a Dxd-ADC having a glutamine residue at position 295 and a 2×n×m DAR according to an embodiment of the present disclosure. Figure 2B is a schematic diagram of a two-step site-specific synthesis of a Dxd-ADC having glutamine residues at positions 295 and 297 and a 4×n×m DAR according to an embodiment of the present disclosure. [Figure 3A] This is a schematic diagram demonstrating the two-step site-specific generation of a specific embodiment of the Dxd-ADC according to the present disclosure. The first step is to generate an antibody (Ab-(N3)4) by conjugating 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 which four azide-containing linkers are attached. The second step is to conjugate Ab-(N3)4 to a specific Linker 2-payload (L2P) via an azide-cycloalkyne 1,3 cycloaddition reaction to generate a Dxd-ADC having four DARs. [Figure 3B] Figure 3A shows schematic diagrams of ADCs having 2 or 4 DARs and exemplary aminoazid linkers suitable for use in the embodiments of this disclosure. [Figure 4A] This is a schematic diagram demonstrating the two-step site-specific generation of a specific embodiment of the Dxd-ADC according to the present disclosure. The first step is to generate an antibody (Ab-(N3)8) by conjugating 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, to which eight azide-containing linkers are attached. The second step is to conjugate Ab-(N3)8 to a specific Linker 2-payload (L2P) via an azide-cycloalkyne 1,3 cycloaddition reaction to generate a Dxd-ADC having eight DARs. [Figure 4B] Figure 4A shows schematic diagrams of ADCs and exemplary branched alkylazidoamine linkers suitable for use in the embodiments of this disclosure. [Figure 5] This diagram illustrates three methods for producing site-specific ADCs (ADCs) of DAR4-DAR24 on antibody Q295 / 297 according to embodiments of the present disclosure. [Figure 6] This diagram illustrates three methods for producing site-specific ADCs having DAR2-DAR12 on antibody Q295 according to embodiments of the present disclosure. [Figure 7A] This is a schematic diagram demonstrating a two-step method I for fabricating an exemplary 8DAR branched linker payload ADC (Ab-AL1-LP39). [Figure 7B] This is a schematic diagram demonstrating a two-step method II for fabricating an exemplary 8DAR branch linker payload ADC (Ab-BL1-LP22). [Figure 7C] This is a schematic diagram demonstrating a one-step method III for fabricating a 4DAR ADC with a linear linker-P and an 8DAR ADC with a branched linker payload. [Figure 8A] This figure shows tumor volume as a function of days after treatment with the anti-FGFR2b Dxd ADC (DAR8) of this disclosure (SNU16, FGFR2-amplified gastic cancer). These ADCs demonstrated significant antitumor efficacy against SNU-16 human gastric cancer xenografts. [Figure 8B] This figure shows tumor volume as a function of days after treatment with the anti-FGFR2b Dxd ADC (DAR8) of this disclosure (in mice with SNU16 tumors). These ADCs demonstrated significant antitumor efficacy against SNU-16 human gastric cancer xenografts. [Figure 9A] (SNU16, FGFR2-amplifying gastric cancer) This figure shows tumor volume as a function of the number of days after treatment with the anti-FGFR2b Dxd ADC (DAR4) of this disclosure. These ADCs demonstrated significant antitumor efficacy against SNU-16 human gastric cancer xenografts. [Figure 9B]This figure shows tumor volume as a function of days after treatment with the anti-FGFR2b Dxd ADC (DAR4) of this disclosure (in mice with SNU16 tumors). These ADCs demonstrated significant antitumor efficacy against SNU-16 human gastric cancer xenografts. [Figure 10] This 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 containing a “D1” antigen-binding domain and a unique bispecific antibody containing 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. [Modes for carrying out 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 various ways in which the disclosure can be embodied. In addition, the examples provided in relation to the various embodiments of the present disclosure are illustrative and not intended to be limiting. Therefore, the details of the specific structures and functions disclosed herein should not be construed as limiting, but merely as representative grounds to teach those skilled in the art how to make diverse uses of the present disclosure.

[0201] definition Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which this disclosure pertains.

[0202] In this specification and the accompanying claims, the singular forms “a,” “an,” and “the” also include the plural form unless the context makes it clear. Thus, for example, “a method” includes one or more methods and / or processes of the kind described herein and / or which would be obvious to those skilled in the art by reading this disclosure.

[0203] The terms “treat” or “treatment” of a condition, disorder, or disease include (1) preventing, delaying, or reducing the onset and / or likelihood of the appearance of at least one clinical or subclinical symptom of a progressing condition, disorder, or disease in a subject who is currently suffering from or prone to suffering from the condition, disorder, or disease but has not yet experienced or presented any clinical or subclinical symptoms of the condition, disorder, or disease; (2) inhibiting the condition, disorder, or disease, i.e., preventing, reducing, or delaying the progression of the disease or its recurrence or at least one clinical or subclinical symptom; and / or (3) alleviating the disease, i.e., causing the regression of at least one clinical or subclinical symptom of the condition, disorder, or disease. The benefit received by the subject to be treated must be statistically significant or at least perceptible to the patient or physician. In some embodiments, treatment includes methods of excising cells in a manner that indirectly affects the disease. In one embodiment, the procedure includes removing immune cells as part of a hematopoietic conditioning regimen prior to treatment.

[0204] When used herein, “subject,” “patient,” “individual,” or “animal” refers to a human being with a disease, a veterinary animal (e.g., a cat, dog, cow, horse, sheep, pig, etc.), and an experimental animal model (e.g., a mouse, rat). In preferred embodiments, the subject is a human being.

[0205] As used herein, the term “effective” as applied to dose or quantity means the amount of compound or pharmaceutical composition sufficient to produce the desired activity when administered to a subject requiring administration. Note that when administered in combination with an active ingredient, the effective dose of this combination may or may not include the amount of each component that would have been effective when administered individually. The exact amount required will vary among subjects depending on the species, age, and general condition of the subject, the severity of the disease being treated, the specific drug used, and the form of administration.

[0206] The phrase “pharmaceutically acceptable salt” means, when used in reference to the compositions of this disclosure, 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, salts derived from acids, salts derived from bases, organic salts, inorganic salts, amine salts, and alkali or alkaline earth metal salts, as well as calcium salts, magnesium salts, potassium salts, sodium salts, hydrochloride salts, 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, and the like. In some examples, the payload described herein (e.g., the rifamycin analog described herein) contains a tertiary amine, and the nitrogen atom in the tertiary amine is the atom that bonds the payload to the linker or linker spacer. In such cases, the bonding 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, iodine, or any other suitably charged moiety described herein).

[0207] A range may be expressed herein as from one specific number "about" or "approximately" to another specific number "about" or "approximately". In other embodiments, such a range includes from one specific number to another specific number.

[0208] "Comprising," "containing," or "including" means that at least one of the specified compound, element, particle, or method steps is present in the composition, article, or method, but does not exclude any other compound, material, particle, or method steps, even if those steps have the same function as the specified one.

[0209] The compounds disclosed herein include those described throughout this specification, and are further illustrated by the classes, subclasses, and species disclosed herein. When used herein, the following definitions shall apply unless otherwise specified. For the purposes of this disclosure, chemical elements are identified in accordance with "Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed." 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 Ed., Ed.: Smith, MB and March, J., John Wiley & Sons, New York: 2001, and their entire contents are incorporated herein by reference.

[0210] As used herein, the term "alkyl" is given in its ordinary sense in the art and may include saturated aliphatic groups, including linear alkyl groups, branched alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups. In some embodiments, the backbone of a linear or branched alkyl group consists of about 1 to 20 carbon atoms (for example, C1-C in a linear group). 20 In branched chains, C2-C 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 carbon atoms. In some embodiments, the alkyl group may be a lower alkyl group, in which case the lower alkyl group contains 1 to 4 carbon atoms (e.g., C1-C4 in a linear lower alkyl group).

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

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

[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, any quaternized form of basic nitrogen, or a heterocyclic, replaceable nitrogen).

[0214] The term "halogen" refers to 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,” for example, “adduct of group B'” in this disclosure, encompasses any part containing the product of an addition reaction, such as an addition reaction of group B', which is unrelated to the synthetic steps taken to produce any part containing the product of an addition reaction.

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

[0217] When used herein, the compounds of this disclosure may contain “optionally substituted” moieties. Generally, the term “optionally” means that one or more hydrogens of a designated moiety, whether preceding or following, are substituted with a preferred substituent. Unless otherwise specified, an “optionally substituted” group may have preferred substituents at each of its substituted positions, and when more than one position in a given structure is substituted with more than one substituent selected from a particular group, the substituents may be the same or different at all positions. The substituent combinations envisioned herein are preferably those that result in the formation of stable or chemically feasible compounds. The term “stable” when used herein means a compound that, when exposed to conditions that enable production, detection, and in some embodiments, recovery, purification, and use for one or more purposes disclosed herein, is substantially unmodified.

[0218] Unless otherwise specified, the structures described herein are also intended to include all isomers (e.g., enantiomers, diastereomers, and geometric (or stereostructural)) of said structures, such as the R and S configurations for each chiral center, the (Z) and (E) double bond isomers, and the (Z) and (E) conformational isomers. Thus, in addition to single stereochemical isomers, enantiomers, diastereomers, and geometric (or stereostructural) mixtures of the compound are within the scope of this disclosure.

[0219] Unless otherwise specified, all tautomer forms of the compounds disclosed herein are within the scope of this disclosure.

[0220] In addition, unless otherwise specified, the structures described herein are also intended to contain different compounds only in the presence of one or more isotopically enriched atoms, for example, by substitution of hydrogen with deuterium or tritium, or 11 C, 13 C, or 14 Compounds having this structure, excluding carbon substitution by C-enriched carbon, are within the scope of this disclosure.

[0221] It should be understood that reference to one or more process steps in a method does not preclude the existence of additional process steps or process steps intervening between clearly identified process steps. Similarly, reference to one or more components in a device or system does not preclude the existence of additional components or components intervening between clearly identified components.

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

[0223] As used herein, “crystal morphology” is intended to refer to a specific lattice arrangement of a crystalline material. Different crystal morphologies of the same material have different crystal lattices (e.g., unit cells), which typically result from the various physical properties that characterize each crystal morphology. In some cases, different lattice arrangements have different water or solvent content. Different crystal 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 adsorption (DVS), and solid-state NMR further assist in identifying crystal morphologies and determining stability and solvent / water content.

[0224] The crystalline forms of a substance include both solvated forms (e.g., hydrated forms) and unsolvated forms (e.g., anhydrous forms). A hydrated form is a crystalline form in which water is present in the crystal lattice. A hydrated form can be a stoichiometric hydrate, in which case water is present in the lattice at a specific water / molecular ratio relative to hemihydrates, monohydrates, dihydrates, etc. A hydrated form can also be non-stoichiometric, in which case the water content varies and depends on external conditions such as humidity.

[0225] In some embodiments, the compounds of the 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 Disclosure contain impurities in amounts of 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%. Impurities generally include all substances that are not substantially isolated compounds, such as other crystalline forms or other materials.

[0226] Certain groups, subgroups, substituents, and atoms are drawn with wavy lines. Wavy lines can intersect or cap bonds. Wavy lines indicate atoms that bond a group, subgroup, substituent, or atom. For example,

[0227] [ka] The phenyl group substituted with a propyl group, as depicted, has the following structure:

[0228] [ka] It holds.

[0229] The expression "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 crucial role in the development and progression of certain invasive species of breast cancer. In recent years, the protein has become an important biomarker and target for the treatment of approximately 30% of breast cancer patients. All references to proteins, polypeptides, and protein fragments herein are intended to refer to human proteins, polypeptides, or protein fragments, respectively, unless explicitly identified as being of non-human origin. Thus, the expression "HER2" means human HER2 unless specifically identified as being of non-human origin, such as "mouse HER2" or "monkey HER2."

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

[0231] An “anti-HER2 / HER2” antibody, for example, the phrase “anti-HER2 / HER2 bispecific antibody,” includes an antibody that specifically recognizes two distinct HER2 epitopes and its antigen-binding fragment. In some embodiments, the bispecific antibody and its antigen-binding fragment include an antigen-binding domain (D1) that specifically binds to a first epitope of human HER2 and a second antigen-binding domain (D2) that specifically binds to a second epitope of human HER2.

[0232] In this specification, the term "STEAP2" refers to the six-transmembrane epithelial antigen of prostate 2. STEAP2 is a unified 6-multilateral transmembrane protein that is highly expressed in prostate epithelial cells and is a cell surface marker for prostate cancer. For example, STEAP2 has been shown to be expressed at significant levels on 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 the human chromosome region 7q21. For example, please refer to the amino acid sequences of human STEAP2 listed in Tables 1 and 2.

[0233] When used herein, "STEAP2-binding antibody" or "anti-STEAP2 antibody" includes an antibody that specifically recognizes STEAP2 and its antigen-binding fragment.

[0234] The phrases “MET-binding antibody” or “anti-MET antibody” include antibodies that specifically recognize MET and their antigen-binding fragments. “MET,” “c-MET,” and other similar terms, as used herein, refer to human membrane spanning receptor tyrosine kinase.

[0235] An “anti-MET / MET” antibody, for example, the phrase “anti-MET / MET bispecific antibody,” includes an antibody that specifically recognizes two distinct MET epitopes and its antigen-binding fragment. In some embodiments, the bispecific antibody and its antigen-binding fragment include an antigen-binding domain (D1) that specifically binds to a first epitope of human MET and a second antigen-binding domain (D2) that specifically binds to a second epitope of human MET.

[0236] All amino acid abnormalities used in this disclosure are accepted by the United States Patent and Trademark Office and are specified in 37 CFR § 1 .822(B)(J).

[0237] The term "protein" refers to an amino acid polymer having approximately 20 or more amino acids covalently linked via amide bonds. As used herein, "protein" includes biopharmaceutical 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, and peptide hormones. Proteins can be produced using recombinant cell production systems, such as insect baculoviruses, yeasts (e.g., Pichia sp.), and mammals (e.g., CHO cells, CHO derivatives such as CHO-K1 cells).

[0238] All references to proteins, polypeptides, and protein fragments herein are intended to refer to human proteins, polypeptides, or protein fragments, respectively, unless explicitly identified as being of non-human origin. Therefore, the term "STEAP2" means human STEAP2 unless specifically identified as being of non-human origin, such as "mouse STEAP2" or "monkey STEAP2."

[0239] The amino acid sequences of antibodies may 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 Hongge 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 selection of a numbering scheme is not intended to suggest a difference in sequences where no difference exists, and those skilled in the art can easily determine the position of the sequence by examining the amino acid sequences of one or more antibodies. Unless otherwise specified, the "EU numbering scheme" is usually used when referring to residues in the constant region of the antibody heavy chain (e.g., those reported by Kabat et al. above).

[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 this disclosure. In certain embodiments, the primary amine compound is linked via an amide linkage on the glutamine side chain. In some embodiments, glutamine is endogenous glutamine. In other embodiments, glutamine is endogenous glutamine made reactive by polypeptide manipulation (e.g., via amino acid deletion, insertion, substitution, or mutation on the polypeptide). In further embodiments, glutamine is a polypeptide manipulated 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 an amino acid sequence containing an acceptor glutamine residue that, when incorporated into a polypeptide sequence (e.g., captured), is recognized by TGase under favorable conditions, causing TGase crosslinking via a reaction between the amino acid side chains within the amino acid sequence and a reaction partner. The recognition tag may be a peptide sequence not normally present in the polypeptide containing the TGase recognition tag. In some embodiments, the TGase recognition tag contains at least one Gln. In some embodiments, the TGase recognition tag comprises the amino acid sequence XXQX (SEQ ID NO: 1935), where X is any amino acid (e.g., 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 a non-conventional amino acid). In some embodiments, the acyl-doner glutamine-containing tags are 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 sequences include those 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). For example, see International Publication No. WO2012059882, the entirety of which is incorporated herein.

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

[0243] The term “antibody,” as used herein, also includes the antigen-binding fragment of a complete antibody molecule. Terms such as “antigen-binding portion” and “antigen-binding fragment” of an antibody, as used herein, include any spontaneously occurring, enzymatically available, 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 a complete antibody molecule using any suitable standard technique, such as proteolytic digestion or recombinant genetic engineering techniques, which are involved in the manipulation and expression of DNA-coding antibody variable regions and optionally invariant regions. Such DNA is publicly known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. DNA can be chemically sequenced and manipulated, or sequenced and manipulated using molecular biological techniques, to position one or more variable and / or constant regions into appropriate configurations, or to introduce codons, create cysteine ​​residues, modify, add, and / or delete amino acids.

[0244] Non-restrictive 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 mimicking the hypervariable region of an antibody (e.g., an isolated complementarity-determining region (CDR) such as the CDR3 peptide) or a restricted FR3-CDR3-FR4 peptide. Domain-specific antibodies, single-domain antibodies, domain deletion antibodies, chimeric antibodies, CDR graft antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and other manipulated molecules such as shark variable IgNAR domains are also included in the expression “antigen-binding fragment” as used herein.

[0245] Antibody antigen-binding fragments 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 within a frame having 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 preferred configuration. For example, the variable region can be a dimer, containing VH-VH, VH-VL, or VL-VL dimers.

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

[0247] In some embodiments, the antigen-binding fragment of an antibody may contain at least one variable domain covalently bound to at least one constant region. Non-limiting and exemplary arrangements of variable and constant regions that can be found within the 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 may be directly linked to each other or linked by a complete or partial hinge region or linker region. A hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that result in a mobile or semi-mobile link between adjacent variable and / or constant regions within a single polypeptide molecule.

[0248] Furthermore, the antigen-binding fragments of the antibodies herein may include homodimers or heterodimers (or other polymers) of any of the variable domain configurations and constant domain configurations listed herein, covalently bound to each other and / or to one or more monomeric VH or VL domains (e.g., by disulfide bonds).

[0249] Similar to complete antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). A multispecific antigen-binding fragment of an antibody typically comprises at least two distinct variable domains, each of which is capable of specifically binding to a different antigen or a different epitope on the same antigen. Forms of multispecific antibodies, including the exemplary bispecific antibody forms disclosed herein, can be adapted for use in terms of antigen-binding fragments of antibodies herein using conventional techniques available in the art.

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

[0251] In some embodiments, the antibodies described herein, such as anti-HER2 antibodies, anti-HER2 / HER2 bispecific antibodies, anti-MET antibodies, anti-MET / MET bispecific antibodies, or anti-STEAP2 antibodies, are human antibodies. 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 described herein may include, for example, amino acid residues in the CDR and certain CDR3 that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro, or somatic mutations 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 mice, are graphed onto the human framework sequence.

[0252] In some embodiments, the antibodies may be recombinant human antibodies. The term “recombinant human antibody” is intended, as used herein, to include all human antibodies prepared, expressed, produced, or isolated by recombinant means, such as antibodies expressed using recombinant expression vectors transfected into host cells (detailed below), antibodies isolated from recombinant combination human antibody libraries (detailed below), antibodies isolated from animals transgenic to human immunoglobulin genes (e.g., mice) (see, for example, Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies prepared, expressed, produced, or isolated by any other means involving splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in one embodiment, such recombinant human antibody is subjected to in vitro mutagenesis (or, in vivo somatic mutagenesis when using an animal transgenic to the human Ig sequence), thereby obtaining amino acid sequences in the VH and VL regions of the recombinant antibody that are derived from and related to human germline VH and VL sequences, but which may not normally be present in the in vivo repertoire of human antibody germline sequences.

[0253] Human antibodies can exist in two forms related to hinge heterogeneity. In one form, the immunoglobulin molecule contains four stable, approximately 150-160 kDa chain constructs 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, and a molecule of approximately 75-80 kDa is formed, consisting of covalently bonded light and heavy chains (half-antibodies). These forms have been extremely difficult to separate even after affinity purification. The frequency of the appearance of the second form in various intact IgG isotypes is not limited but is attributed to structural differences related to the hinge region isotype of the antibody. A single amino acid substitution in the hinge region of the human IgG4 hinge can significantly reduce the appearance of the second form (Angal et al., (1993) Molecular Immunology 30:105) to the extent normally observed using the human IgG1 hinge. This specification includes antibodies having one or more mutations in, for example, the hinge region, CH2 region, or CH3 region, which may be desirable during production, in order to improve the yield of a desired antibody morphology.

[0254] The antibodies described herein may be isolated antibodies or purified antibodies. “Isolated antibody” or “purified antibody,” as used herein, means an antibody identified, separated, and / or recovered from at least one component of its natural environment. For example, an antibody separated or removed from at least one component of a living organism, or from a tissue or cell in which antibodies naturally exist or are naturally produced, is an “isolated antibody” for the purposes of this specification. For example, an antibody purified from at least one component of one reaction or a series of reactions is a “purified antibody” or a result of antibody purification. Isolated antibodies also include antibodies in situ within recombinant cells. An isolated antibody is an antibody that has undergone at least one purification or isolation step. According to one embodiment, an isolated antibody may substantially contain no other cellular material and / or chemicals.

[0255] The antibodies disclosed herein may contain one or more amino acid substitutions, insertions, and / or deletions in the framework of the heavy and light chain variable domains and / or the CDR region compared to the corresponding germline sequence from which the antibody is obtained. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein with germline sequences available, for example, from public antibody sequence databases. This specification includes antibodies obtained from any of the amino acid sequences disclosed herein and their antigen-binding fragments, in which one or more amino acids in the framework and / or CDR region are mutated to corresponding residues in the germline sequence from which the antibody is obtained, to corresponding residues in another human germline sequence, or to conserved amino acid substitutions of the corresponding germline residues (such sequence changes are collectively referred to herein as “germline mutations”). Those skilled in the art can readily produce a number of antibodies and antigen-binding fragments, including one or more individual germline mutations or combinations thereof, starting from the heavy and light chain variable regions disclosed herein. In one embodiment, all framework residues and / or CDR residues within the VH and / or VL domains are mutated back to residues found in the original germline sequence from which the antibody is obtained. In another embodiment, only specific residues, for example, 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, are mutated back to the original germline sequence. In yet another embodiment, one or more framework residues and / or CDR residues are mutated to corresponding residues in a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antibody is originally obtained).

[0256] Furthermore, the antibodies of this specification may contain any combination of two or more germline mutations within the framework region and / or CDR region, for example, certain individual residues may be mutated to corresponding residues in a particular germline sequence, while other specific residues that differ from the original germline sequence are maintained or mutated to corresponding residues in a different germline sequence. Antibodies and antigen-binding fragments containing one or more germline mutations, once obtained, can be readily tested for one or more desirable properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or reactive bioproperties (as applicable), decreased immunogenicity, or improved drug-to-antibody ratio (DAR) in the antibody-drug conjugate. Antibodies and antigen-binding fragments commonly obtained in this manner are incorporated herein.

[0257] The term "aglycosylated antibody" refers to an antibody that does not contain a glycosylated sequence that can interfere with the transglutamination reaction, for example, an antibody that lacks 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, when the antibody mutates, 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 the glycosylated sequence, or by site-directed mutagenesis to insert a glutamine residue at a site other than the interfering glycosylated 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 from which a saccharide group has been removed to facilitate transglutaminase-mediated conjugation. Examples of saccharides include, but are not limited to, N-linked oligosaccharides. In some embodiments, deglycosylation occurs at residue N297. In some embodiments, the removal of the saccharide group is achieved using enzymes, but is not limited to, PNGase.

[0259] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site within the variable region of an antibody molecule, also known as a paratope. A single antigen can have more than one epitope. Therefore, different antibodies can bind to different regions on antigens and exhibit diverse biological effects. Epitopes can be either structural or linear. Structural epitopes are produced by spatially parallel amino acids from various segments of a linear polypeptide chain. Linear epitopes are produced by adjacent amino acid residues in a polypeptide chain. In some situations, epitopes can contain saccharide, phosphoryl, or sulfonyl groups on the antigen.

[0260] The terms “conjugated protein” or “conjugated antibody,” as used herein, refer to a protein or antibody covalently linked to one or more chemical moieties. The chemical moieties may include the amine compounds of this disclosure. Linkers (L) and payloads (D) suitable for use in this disclosure are described in detail herein. In certain embodiments, a conjugated antibody containing 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-antibody ratio" or (DAR) refers to the average number of therapeutic portions, such as drugs, conjugated to the binder of this disclosure.

[0262] The term "linker-antibody ratio" or (LAR) is expressed as Example I below and, in some embodiments, is the average number of reactive primary amine compounds conjugated to the binder of the present disclosure. Such a binder, e.g., an antibody, can be conjugated with primary amine compounds, e.g., suitable azides or alkynes. The resulting binder can be functionalized with an azide or alkyne and then reacted with a therapeutic moiety containing the corresponding azide or alkyne via a 1,3-addition cycloaddition reaction.

[0263] The phrase "pharmaceutically acceptable amount" means an amount that is effective or sufficient to treat, reduce, alleviate, or modulate the action or symptoms of at least one health problem of the target being treated. For example, a pharmaceutically acceptable amount of an antibody or antibody-drug conjugate is an amount that is effective in modulating a biological target using the antibody or antibody-drug conjugate provided herein. Suitable pharmaceutically acceptable amounts include, but are not limited to, about 0.001% to a maximum of about 10% of the antibody or antibody-drug conjugate provided herein, any amount in between, for example, 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%.

[0264] The term "reactive pH" refers to the pH of the reactants after all the reactants or reactants have been added.

[0265] The terms "substantial identity" or "substantially identical," when referring to a nucleic acid or fragment thereof, mean that, given appropriate nucleotide insertions or deletions and optimal alignment with another nucleic acid (or its complementary strand), at least about 95%, more preferably at least about 96%, 97%, 98%, or 99% of the nucleic acid bases are identical, as measured by well-known sequence identity algorithms such as FASTA, BLAST, or GAP, which are discussed below. A nucleic acid molecule having substantial identity with a reference nucleic acid molecule may, in some cases, 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 identical” mean that, when two peptide sequences are best aligned using default gap weights, such as by programmed gAP or BESTFIT, they share at least 95% sequence identity, and more preferably at least 98% or 99% sequence identity. Preferably, the non-identical residue positions differ by conservative amino acid substitutions. A “conservative amino acid substitution” is an amino acid substitution in which one amino acid residue is replaced by another amino acid residue that has 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 the protein. If two or more amino acid sequences differ from each other by conservative substitutions, the degree of sequence identity or similarity can be adjusted upward to compensate for the conservative nature of the substitutions. Means for making this adjustment are well known to those skilled in the art. See, for example, “Pearson (1994) Methods Mol. Biol. 24:307-331”. Examples of amino acids having 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, the conserved amino acid substituents are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine.

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

[0268] Polypeptide sequence similarity, also known as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using similarity measurements assigned to various substitutions, deletions, and other modifications, including conserved amino acid substitutions. For example, gCG software includes programs such as gap and Bestfit, which, when used with default parameters, can determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from various 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, with default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides the best overlap region alignment and sequence identity ratio between the query sequence and the search sequence (Pearson (2000) above). Another specific algorithm for comparing the sequences described herein with databases containing numerous sequences from various organisms is the computer program BLAST, specifically BLASTP or TBLASTN, using default parameters. For example, see 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 According to the aforementioned objectives and the like, the present disclosure provides protein drug conjugate compounds, such as antibody drug conjugate compounds, precursors and intermediates thereof, pharmaceutical compositions, and methods of treating specific diseases in a subject in need of treatment. According to the present disclosure, the protein drug conjugate compounds provided herein comprise a glutaminyl-modified binding agent that is conjugated with a primary amine compound linked to a therapeutic moiety described herein, such as a camptothecin analog moiety.

[0270] In one aspect, the present disclosure provides a compound comprising a binding agent (e.g., an antibody or a fragment thereof) according to the present disclosure, which has one or more glutamine residues, wherein the one or more glutamine residues are conjugated to one or more camptothecin analogs, such as 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 the 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 Formula (C): BA-Gln-NH-L1-B-(-L2-(-M-Camp) m ) n (C) provides a compound having a structure according to: wherein BA is an antibody or an 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 group B' and group B'', group B' is a first component, e.g., a first cycloaddition component, L2 is a second linker covalently bonded to branching unit B via at least one group B'', B'' is a second component, e.g., a second cycloaddition component, group B' and group B'' form at least one adduct, M is absent or has the structure:

[0272] [ka] This is a part having a C1-C, where R, R', and R'' are independently hydrogen or C1-C when they appear. 20 It is alkyl, or R' and R'' form a ring or ring, Camp is a camptothecin analog, and m and n are independent integers from 1 to 30.

[0273] In one embodiment, this disclosure relates to formula (A): BA-(Gln-NH-L1-B-(-L2-(-M-Dxd) m ) k ) n (A) A compound having the structure of the formula, in which, BA is an antibody or its antigen-binding fragment. Gln is a glutamine residue, L1 is either non-existent or the first linker. B is a branched unit comprising at least one adduct of base B' and base B'', where at least one of base B' and base B'' is -N3 and

[0274] [ka] Selected from, the other of base B' and base B'' is,

[0275] [ka] Selected from, where Q is C or N, L2 is a second linker covalently bonded to branch unit B via at least one group B'', M is either non-existent or has no structure:

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

[0277]

Chemical Formula

[0278] In one aspect, the present disclosure relates to a compound of formula (I): BA-(Gln-NH-L1-B-(-L2-M-Dxd) k ) n (I) A compound having a structure represented by, wherein BA is an antibody or an 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 group B', and group B' is -N3,

[0279]

Chemical Formula

[0280]

Chemical Formula

[0281] [ka] An antitumor drug having the structure, where k and n are independent integers from 1 to 30. Provides compounds.

[0282] Linker L1 In one embodiment, linker L1 does not exist.

[0283] In one embodiment, linker L1 is present and is covalently bonded to the amine of the glutamine residue of binder BA.

[0284] In one embodiment, linker L1 is an 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 It contains -C(O)-NH-, a peptide unit containing 2 to 4 amino acids, or a combination thereof, each of which is optionally substituted with 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 one embodiment, the free (unconjugated) linker L1 contains a primary amine for binding to a glutamine residue via a transglutamation reaction.

[0286] In one embodiment, 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 includes a disulfide (-SS-) bond.

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

[0289] In one embodiment, one or more carbon atoms on L1 are replaced with -CO2H.

[0290] In one embodiment, linker L1 includes 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, 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, linker L1 comprises a valine-citrulline unit.

[0292] In one embodiment, the linker L1 is

[0293] [ka] Furthermore, selected from the group consisting of combinations thereof, in the formula, R A The groups include alkynes, azides, tetrazines, trans-cyclooctene, maleimides, amines, ketones, aldehydes, carboxylic acids, esters, thiols, sulfonic acids, tosylates, halides, silanes, cyano groups, carbohydrate groups, biotin groups, and lipid residues, and the subscripts x, n, p, and q are independently integers from 0 to 12.

[0294] Branching Unit B Branching unit B includes at least one adduct of base B'. In one embodiment, B includes one adduct of base B'. In one embodiment, B includes two adducts of base B'. In one embodiment, B includes three adducts of base B'.

[0295] In one embodiment, B includes at least four adducts of base B'. In another embodiment, B includes four adducts of base B'. In yet another embodiment, B includes five adducts of base B'. In yet another embodiment, B includes six adducts of base B'.

[0296] In general, the adducts of group B' according to this disclosure include a portion containing the product of the addition reaction of group B', independently of the synthetic steps taken to produce the portion containing 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, for example.

[0298] [ka] And, In the formula, n is an integer between 0 and 12, and may be a substituted trans-cyclooctene, or, for example, tetrazine.

[0299] In some embodiments, the adduct of group B' may be the product of a 1,3-cycloaddition reaction between the azide and alkyne moieties. While not limited to theory, azide-alkyne cycloaddition is a 1,3-dipolar cycloaddition between an azide and a terminal or internal alkyne to obtain 1,2,3-triazole.

[0300] More specifically, -N3,

[0301] [ka] An adduct of base B' selected from, where Q is C or N, is -N3.

[0302] [ka] Adducts of 1,3-cycloadductal groups B' and B'' selected from the formula, which may include adducts where Q is C or N, and where group B'' is complementary to group B' in order to form a 1,3-cycloadductal adduct.

[0303] As a non-restrictive example, group B' may be an azide (-N3), and group B'' may be an alkyne-containing group, for example,

[0304] [ka] There are cases where this is the case.

[0305] As another non-limiting example, group B' is an alkyne-containing moiety, for example,

[0306] [ka] In some cases, base B'' may be azide.

[0307] In one embodiment, the adducts of groups B' and B'' include a triazole moiety. In a particular embodiment, the adducts of groups B' and B'' include

[0308] [ka] The structure is selected from the group consisting of the following, where Q is C or N.

[0309] As described above, in one embodiment, B includes one adduct of base B'.

[0310] In a particular embodiment, L1-B is

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

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

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

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

[0315] Figure 3B shows the basic component structure of a non-restrictive and exemplary azidoamine linker. A specific structure synthesized as an example is provided below.

[0316] In one embodiment, B includes at least two adducts of base B'. In a particular embodiment, B is

[0317] [ka] The formula includes, where (B') includes the bond point of the adduct of base B'.

[0318] In a particular embodiment, B is

[0319] [ka] It is selected from the group consisting of the following.

[0320] In a particular embodiment, L1-B is

[0321] [ka] Selected from the group consisting of,

[0322] [ka] This is the amino site of the binding of BA to the glutamine residue.

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

[0324] The basic component structures of exemplary and non-limiting branched alkyl azidoamine linkers are listed in Figure 4B. Specific structures synthesized as examples are provided below.

[0325] Linker L2 In one embodiment of this disclosure, L2 is given by formula (L2): B''-SP1-B2-(-SP2-AA-SP3) p (L2) It has a structure such that, in the formula, B'' is a group that can covalently bond to group B', SP1 is either non-existent or is the first spacer unit. B2 either does not exist or is a branch unit. SP2 is either non-existent or a second spacer unit. AA is either absent or a peptide unit containing 2-4 amino acids. SP3 is either non-existent or a third spacer unit. p is an integer between 1 and 12.

[0326] In one embodiment of this disclosure, L2 is given by formula (L2'): H2N-SP1-B2-(-SP2-AA-SP3) p (L2') It has a structure such that, in the formula, SP1 is either non-existent or is the first spacer unit. B2 either does not exist or is a branch unit. SP2 is either non-existent or a second spacer unit. AA is either absent or a peptide unit containing 2-4 amino acids. SP3 is either non-existent or a third spacer unit. p is an integer between 1 and 12.

[0327] In one embodiment of this disclosure, L2 is given by formula (L2''): Maleimide-N-SP1-B2-(-SP2-AA-SP3) p (L2'') It has a structure such that, in the formula, SP1 is either non-existent or is the first spacer unit. B2 either does not exist or is a branch unit. SP2 is either non-existent or a second spacer unit. AA is either absent or a peptide unit containing 2-4 amino acids. SP3 is either non-existent or a third spacer unit. p is an integer between 1 and 12.

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

[0329] In one embodiment, base B'' is -N3,

[0330] [ka] Selected from the given options, where Q is either C or N.

[0331] As a non-limiting example, group B'' is an alkyne-containing moiety, for example,

[0332] [ka] There are cases where this is the case.

[0333] As another non-restrictive example, base B'' may be azid.

[0334] In one embodiment, the adducts of groups B' and B'' include a triazole moiety. In a particular embodiment, the adducts of groups B' and B'' include

[0335] [ka] The structure is selected from the group consisting of the following, or has a positional isomer thereof, where Q is C or N.

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

[0337] In another embodiment, SP1 is

[0338] [ka] It is selected from the group consisting of the following.

[0339] In one embodiment, the branching unit B2 does not exist.

[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 an 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 The subscripts u and v are selected from the group consisting of -C(O)-NH- or combinations thereof, and 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, as well as combinations thereof.

[0345] In one embodiment, AA is a peptide unit containing two amino acids. In one embodiment, AA is a peptide unit containing three amino acids. In one embodiment, AA is a peptide unit containing four amino acids.

[0346] In a 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] A group consisting of these and their combinations is selected, in the formula R c They either do not exist independently at the time of each occurrence, or

[0351] [ka] It is a base selected from among them.

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

[0353] In one embodiment, 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) portions, and 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 one embodiment, part M does not exist.

[0355] In one embodiment, M exists,

[0356] [ka] The structure is such that, in the formula, R, R', and R'' are independently hydrogen or alkyl when they appear, or R' and R'' combine to form a ring, for example, a 3-membered to 8-membered ring.

[0357] In one embodiment, M exists,

[0358] [ka] The structure is such that, in the formula, R, R', and R'' are independently hydrogen or C1-C4 alkyl when they appear, or R' and R'' combine to form a 5-membered ring or a 6-membered ring.

[0359] In one embodiment, R is hydrogen.

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

[0361] In one embodiment, R'' is hydrogen. In another embodiment, R'' is a C1-C4 alkyl group.

[0362] In one embodiment, R' and R'' combine to form a five-membered ring. In another embodiment, R' and R'' combine to form -(CH2)3-.

[0363] In one embodiment, R' and R'' combine to form a 6-membered ring. In another embodiment, R' and R'' combine to form -(CH2)4-.

[0364] In one embodiment, R, R', and R'' are hydrogen in their respective appearances, i.e., M is

[0365] [ka] That is the case.

[0366] In another embodiment, R is hydrogen, R' and R'' combine to form a five-membered ring, for example, R' and R'' combine to form -(CH2)3-, and M is

[0367] [ka] That is the case.

[0368] payload In one embodiment, the payload of the Disclosure is a camptothecin analog and / or derivative.

[0369] [ka]

[0370] Camptothecin (CPT), mentioned above, is a topoisomerase toxin. It was discovered in 1966 by Mewall and McWani during a systematic screening of natural products for anticancer drugs. Camptothecin was isolated from the bark and trunk of Camptotheca (Happy Tree), a tree native to China that has been used in traditional Chinese medicine for cancer treatment. Camptothecin showed remarkable anticancer activity in preliminary clinical trials. However, due to its low solubility, synthetic and medicinal chemists have developed numerous compounds of camptothecin with various derivatives that yield favorable results in order to increase the chemical benefits. Four camptothecin analogs—topotecan, irinotecan, berotecan, and deruxtecan (Dxd)—are approved and are used in cancer chemotherapy today.

[0371] Trastuzumab deruxtecan (T-Dxd) is an antibody-drug conjugate containing trastuzumab, a human epidermal growth factor receptor 2 (HER2)-directed antibody, and deruxtecan (Dxd, a derivative of exatecan), a topoisomerase I inhibitor. 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 Disclosure is deruxtecan (Dxd).

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

[0375] [ka] A compound having, or a pharmaceutically acceptable salt thereof, wherein R1, R2, R3, and R4 are independently hydrogen or alkyl, for example, C1-C 12 It is alkyl, C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl, or R2 and R3 combine to form a 5-membered ring or a 6-membered ring.

[0376] In one embodiment, R1 is hydrogen.

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

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

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

[0380] In one embodiment, R1, R2, R3, and R4 are hydrogen when they appear. 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 combine to form a five-membered ring. In another embodiment, R2 and R3 combine to form -(CH2)3-.

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

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

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

[0386] [ka] It has a structure or a pharmaceutically acceptable salt thereof, where R is hydrogen or alkyl, for example C1-C 12 It is alkyl, C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl.

[0387] Those skilled in the art will understand that compound P-II described above is also intended to include all structural isomers (e.g., enantiomers, diastereomers, and geometric (i.e., stereostructural) forms). For example, the R and S configurations at each chiral center are within the scope of this disclosure. As an example, the two isomers described 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, the payload according to the Disclosure is conjugated to form a protein-drug conjugate (e.g., an antibody-drug conjugate). In one embodiment, the payload is covalently bonded to a portion M. In one embodiment, the payload is M-Dxd. In one embodiment, M-Dxd is

[0392] [ka] The structure is selected from the group consisting of the following, where R is hydrogen or C1-C4 alkyl,

[0393] [ka] This represents the connection point to L2.

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

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

[0396] [ka] The formula comprises a binder (e.g., an antibody or its antigen-binding fragment).

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

[0398] [ka] The formula comprises a binder (e.g., an antibody or its antigen-binding fragment).

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

[0400] [ka] The formula comprises a binder (e.g., an antibody or its antigen-binding fragment).

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

[0402] [ka] The formula comprises a binder (e.g., an antibody or its antigen-binding fragment).

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

[0404] Linker Payload (L2P) In another aspect, this disclosure relates to formula (L2-P): B''-SP1-B2-(-SP2-AA-SP3-M-Dxd) p (L2-P) A compound by which, in the formula, B'' is -N3,

[0405] [ka] Selected from the group consisting of, SP1 does not exist, or

[0406] [ka] A first spacer unit selected from the group consisting of, B2 either does not exist or is a branch unit. SP2 does not exist, 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 A second spacer unit selected from the group consisting of -C(O)-NH- or combinations thereof, where the subscripts u and v are independently integers from 1 to 8. AA is either absent or a peptide unit containing 2-4 amino acids. SP3 does not exist, or

[0407] [ka] A third spacer unit selected from the group consisting of, where R c They either do not exist independently at the time of each occurrence, or

[0408] [ka] It is a base selected from, M does not exist, or

[0409] [ka] In the formula, R, R', and R'' are independently hydrogen or a C1-C4 alkyl group when they appear, or R' and R'' combine to form a 5-membered ring or a 6-membered ring. Dxd is given by equation (P):

[0410] [ka] It is an antitumor drug having the structure, p is an integer between 1 and 12. Provides compounds.

[0411] In another aspect, this disclosure relates to formula (L2'-P): H2N-SP1-B2-(-SP2-AA-SP3-M-Dxd)p(L2'-P) A compound by which, in the formula, B'' is -N3,

[0412] [ka] Selected from the group consisting of, SP1 does not exist, or

[0413] [ka] A first spacer unit selected from the group consisting of, B2 either does not exist or is a branch unit. SP2 does not exist, 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 A second spacer unit selected from the group consisting of -C(O)-NH- or combinations thereof, where the subscripts u and v are independently integers from 1 to 8. AA is either absent or a peptide unit containing 2-4 amino acids. SP3 does not exist, or

[0414] [ka] A third spacer unit selected from the group consisting of, where R c They either do not exist independently at the time of each occurrence, or

[0415] [ka] It is a base selected from, M does not exist, or

[0416] [ka] In the formula, R, R', and R'' are independently hydrogen or a C1-C4 alkyl group when they appear, or R' and R'' combine to form a 5-membered ring or a 6-membered ring. Dxd is given by equation (P):

[0417] [ka] It is an antitumor drug having the structure, p is an integer between 1 and 12. Provides compounds.

[0418] In another aspect, this disclosure relates to formula (L2''-P): maleimide-N-SP1-B2-(-SP2-AA-SP3-M-Dxd)p(L2''-P) A compound by which, in the formula, B'' is -N3,

[0419] [ka] Selected from the group consisting of, SP1 does not exist, or

[0420] [ka] A first spacer unit selected from the group consisting of, B2 either does not exist or is a branch unit. SP2 does not exist, 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 A second spacer unit selected from the group consisting of -C(O)-NH- or combinations thereof, where the subscripts u and v are independently integers from 1 to 8. AA is either absent or a peptide unit containing 2-4 amino acids. SP3 does not exist, or

[0421] [ka] A third spacer unit selected from the group consisting of, where R c They either do not exist independently at the time of each occurrence, or

[0422] [ka] It is a base selected from, M does not exist, or

[0423] [ka] In the formula, R, R', and R'' are independently hydrogen or a C1-C4 alkyl group when they appear, or R' and R'' combine to form a 5-membered ring or a 6-membered ring. Dxd is given by equation (P):

[0424] [ka] It is an antitumor drug having the structure, p is an integer between 1 and 12. Provides compounds.

[0425] In one embodiment, the linker payloads L2-P, L2'-P, and L2''-P according to this disclosure are:

[0426] [ka]

[0427] [ka]

[0428] [ka] It has a structure selected from the group consisting of, or a pharmaceutically acceptable salt thereof.

[0429] Branch Linker 2-Payload (BL2P)

[0430] In another embodiment, the Disclosure provides L2-P comprising one or more branching units. Exemplary branching units B1-B5 according to the Disclosure are shown below.

[0431] [ka]

[0432] An exemplary branch linker 2-payload (BL2P) structure according to this disclosure is provided below.

[0433] [ka]

[0434] [ka]

[0435] [ka]

[0436] [ka]

[0437] [ka]

[0438] In one embodiment, the compound according to this disclosure (i.e., the linker payload) is

[0439] [ka] It has the structure of, or a pharmaceutically acceptable salt thereof.

[0440] In one embodiment, the compound according to this disclosure (i.e., the linker payload) is

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

[0442] In another aspect, this disclosure relates to formula (II): Ab-(Gln-NH-L1-B-(SP1-B2-(-SP2-AA-SP3-M-Dxd) k ) p ) n (II) We provide antibody-drug conjugates. In the formula, Ab is an antibody, Gln is a glutamine residue, L1 is either absent or the first linker described above, B is the branched unit described above comprising at least one adduct of group B' and group B'', and group B' is -N3,

[0443] [ka] and selected from at least one base B'', B''-SP1-B2-(-SP2-AA-SP3-M-Dxd) p This is a compound according to the formula (L2-P) described above, in which the compound of formula (L2-P) is covalently bound to the antibody via adducts of groups B' and B'', where k is an integer from 1 to 12, and p and n are independently integers from 1 to 30.

[0444] In one embodiment, the antibody-drug conjugate according to this disclosure comprises an antibody and a linker payload, the linker payload being

[0445] [ka] The structure of, or a pharmaceutically acceptable salt thereof, comprising,

[0446] [ka] This represents a binding site to a binder (e.g., an antibody), either directly or via a second linker.

[0447] Binder In one embodiment, the effectiveness of the protein-drug conjugate embodiments described herein depends on the selectivity of the conjugate to bind to its binding partner. In one embodiment of this disclosure, the conjugate is a molecule capable of binding to a given binding partner with some selectivity. In one embodiment, the conjugate is in a mammal where the interaction may occur for therapeutic purposes. In an alternative embodiment, the conjugate is in vitro where the interaction may occur for diagnostic purposes. In some embodiments, the conjugate is capable of binding to cells or cell populations.

[0448] A preferred binder of this disclosure is a protein that binds to a binding partner, wherein the binder comprises one or more glutamine residues. Examples of preferred binders include, but are not limited to, antibodies, lymphokines, hormones, growth factors, viral receptors, interleukins, or any other cell-binding or peptide-binding molecule or substance.

[0449] In one embodiment, the conjugate is an antibody. In some embodiments, the antibody is selected from monoclonal antibodies, polyclonal antibodies, and antibody fragments (Fab, Fab', and F(ab)2, minibody, diabody, tribody, etc.). The antibodies herein can be humanized using the methods described in U.S. Patent No. 6,596,541 and U.S. Patent Application Publication No. 2012 / 0096572, which are invoked by reference to the whole, respectively. In some embodiments of the protein-drug conjugate compounds of the present disclosure, BA is a humanized monoclonal antibody. For example, BA may be a monoclonal antibody that conjugates HER2, MET, or STEAP2. In some embodiments of the protein-drug conjugate compounds of the present disclosure, BA is a bispecific antibody, for example, an anti-HER2 / HER2 bispecific antibody or an anti-MET / MET bispecific antibody.

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

[0451] The antibody may be in any form known to those skilled in the art. In some embodiments, the antibody includes a light chain. In some embodiments, the light chain is a κ light chain. In some embodiments, the light chain is a λ light chain.

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

[0453] In some embodiments, the antibody is an antibody fragment. In some embodiments, the antibody fragment is an Fv fragment. In some embodiments, the antibody fragment is a Fab fragment. In some embodiments, the antibody fragment is an F(ab')2 fragment. In some embodiments, the antibody fragment is a Fab' fragment. In some embodiments, the antibody fragment is an scFv(sFv) fragment. In some embodiments, 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] An antibody may have binding specificity to any antigen deemed suitable by those skilled in the art. In some 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, follicular-stimulating hormone, calcitonin, luteinizing hormone, glucagon, vmc factor, factor IX, tissue factor (TF), and coagulation factors such as von Willebrand factor, anticoagulant factors such as protein C, atrial natriuretic factor, and pulmonary surfactant. Plasminogen activators such as uterine urokinase, human urine, or human tissue plasminogen activator (t-PA), bombesin, thrombin, hematopoietic growth factor, tumor necrosis factor-α and -β, enkephalinase, RANTES (regulated in the activation of normally expressed and secreted T cells), human macrophage inflammatory protein (MLP-I-α), serum albumin such as human serum albumin; Muellerian inhibitors, relaxin A chain, relaxin B chain, prorelaxin, mouse gonadotropins Related peptides, microbial proteins such as β-lactamase, DNase, 19E, cytotoxic T lymphocyte-associated antigens (CTLAs) such as CTLA-4, inhibin, activin, vascular endothelial growth factor (VEGF), hormone or growth factor receptors, protein A or D, rheumatoid factor, bone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT4, NT-5, or NT-6), or neurotrophic factors such as nerve growth factors like NGF-β, platelet-derived compounds Growth factors (PDGF), fibroblast growth factors such as aFGF and bFGF, fibroblast growth factor receptor 2 (FGFR2), epidermal growth factor (EGF), transforming growth factors (TGF) such as TGF-α, TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5, insulin-like growth factor-1 and -2 (IGF-1 and IGF-2), des(I-3)-IGF-1 (brain IGF-1), insulin-like growth factor binding protein, EpCAM, gD3, FLT3, PSMA,PSCA, MUC1, MUC16, STEAP, STEAP2, CEA, TENB2, EphA receptor, EphB receptor, folate receptor, FOLRI, mesothelin, cripto, alphavbeta6, integrin, VEGF, VEGFR, EGFR, transferrin receptor, lRTAI, lRTA2, lRTA3, lRTA4, lRTA5, CD proteins, e.g., CD2, CD3, CD4, CD5, CD6, CD8, CDII, CDI4, CDI9, 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, CDI52, or antibodies that bind to one or more tumor-associated antigens or cell surface receptors, erythropoietin, bone-inducing factors, immunotoxins, bone morphogenetic proteins (BMPs), interferon-α, -β, -γ, disclosed and incorporated by reference in U.S. Patent Application Publication No. 2008 / 0171040 or 2008 / 0305044. Interferons, colony-stimulating factors (CSFs) 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, adresins, regulatory proteins, integrins such as CDlla, CDllb, CDllc, CDI8, ICAM, VLA-4, 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, EGFRVIII, endoglin, Epcam, EphA2, ErbB2 / HER2, ErbB3 / HER3, ErbB4 / HER4, ETV6-AML, Fra-1, FOLR1, gAGE protein (e.g., gAGE-1, -2), gD2, gD3, globoH, glypcan-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 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 any fragment of the polypeptides listed herein.

[0457] Exemplary antigens 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, the antigen may be a blood target, such as CD22, CD30, CD33, CD79a, or CD79b, but is not limited to these.

[0459] Some embodiments described herein are targets specific to therapeutic or diagnostic applications. In one embodiment, the conjugate is prepared to interact with and bind to an antigen designated as a tumor antigen, which may be an antigen specific to one type of tumor, or an antigen that is shared, overexpressed, or modified on a particular type of tumor. Examples include α-actinin-4 for lung cancer, ARTC1 for melanoma, BCR-ABL fusion protein for chronic myeloid leukemia, B-RAF, CLPP, or Cdc27 for melanoma, CASP-8 for squamous cell carcinoma, and hsp70-2 for renal cell carcinoma, as well as the following shared tumor-specific antigens, e.g., BAGE-1, gAGE, gnTV, KK-LC-1, MAGE-A2, NA88-A, TRP2-INT2. In some embodiments, the antigen is PRLR or HER2. In some embodiments, the antibody conjugates STEAP2, MUC16, EGFR, EGFRVIII, FGR2, or PRLR.

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

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

[0462] In some embodiments, the BA targets 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 one embodiment, the protein-drug conjugate, e.g., ADC, according to this 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 comprising a first antigen-binding domain (D1) that specifically binds to a first epitope of human HER2 and a second antigen-binding domain (D2) that specifically binds to 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. The non-competition between D1 and D2 for binding to HER2 means that the respective monospecific antigen-binding proteins obtained from D1 and D2 do not compete with each other for binding to human HER2. Exemplary antigen-binding protein competition assays are known in the art.

[0466] In one embodiment, D1 and D2 bind to various (e.g., non-overlapping or partially overlapping) epitopes on HER2.

[0467] In one non-limiting embodiment, this disclosure includes: The first antigen-binding domain (D1), The second antigen-binding domain (D2) and We provide a protein-drug conjugate comprising a bipolar antigen-binding molecule containing the following: D1 specifically binds to the first epitope of human HER2. D2 specifically binds to the second epitope of human HER2.

[0468] Anti-HER2 / HER2 bispecific antibodies may be constructed using the antigen-binding domains of two distinct 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 in pairs in cross-competition with the HER2 protein. If two different anti-HER2 antibodies can bind to HER2 simultaneously (i.e., do not compete with each other), the antigen-binding domain of the first anti-HER2 antibody and the antigen-binding domain of the second non-competitive anti-HER2 antibody can be manipulated to form a single anti-HER2 / HER2 bispecific antibody according to this disclosure.

[0469] According to this disclosure, a bispecific antigen-binding molecule may be a single multifunctional polypeptide or a polymer of two or more polypeptides that are covalently or noncovalently related to one another. As will become apparent from this disclosure, an antigen-binding construct having the ability to simultaneously bind two distinct, non-identical epitopes in the HER2 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 biotechnologies known to those skilled in the art (e.g., recombinant DNA and protein expression techniques).

[0470] In another embodiment, the Disclosure provides a pharmaceutical composition comprising a recombinant human antibody or a fragment thereof that specifically binds HER2 to a pharmaceutically acceptable carrier. In one non-limiting embodiment, the antibody may bind to two distinct epitopes on the HER2 protein, i.e., the antibody is a HER2 / HER2 bispecific antibody. In a relevant embodiment, the Disclosure features a composition which 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 conveniently combined with the anti-HER2 / HER2 antibody. Further combination therapies and co-formulations involving the anti-HER2 / HER2 bispecific antibody of the Disclosure are disclosed elsewhere in this Specification.

[0471] In another aspect, the Disclosure provides a therapeutic method for targeting / killing HER2-expressing tumor cells using the anti-HER2 / HER2 bispecific antibody of the Disclosure, comprising the step of administering a therapeutically effective dose of a pharmaceutical composition comprising the anti-HER2 / HER2 antibody of the Disclosure to a subject requiring administration. Optionally, the anti-HER2 / HER2 antibody (or its antigen-binding fragment) may be used for the treatment of breast cancer, or may be modified to increase cytotoxicity by means of a modified Fc domain that increases ADCC (see, for example, Shield et al. (2002) JBC 277:26733), radioimmunotherapy, antibody-drug conjugates, or other methods that increase the efficiency of tumor resection.

[0472] The disclosure also includes the use of the anti-HER2 antibodies of the disclosure in the manufacture of agents for the treatment of diseases or disorders (e.g., cancer) associated with or resulting from cells expressing HER2. In one embodiment, the disclosure relates to compounds comprising the anti-HER2 antibodies or antigen-binding fragments, or HER2 / HER2 bispecific antibodies, disclosed herein, for pharmaceutical use. In one embodiment, the disclosure relates to compounds comprising antibody-drug conjugates (ADCs) disclosed herein, for pharmaceutical use.

[0473] In another embodiment, the disclosure provides a bispecific anti-HER2 / HER2 antibody for diagnostic use, such as an imaging reagent.

[0474] Anti-STEAP2 antibody suitable for protein-drug conjugates In some embodiments, the antibody is anti-six-transmembrane epithelial antigen of prostate 2 (STEAP2), i.e., an anti-STEAP2 antibody. STEAP2 is a metalloreductase that acts as a shuttle between the Golgi complex and the cell membrane, reducing iron and copper and facilitating their translocation into cells. STEAP2 is primarily localized to prostate epithelial cells. STEAP2 is also expressed in normal heart, brain, pancreas, ovaries, skeletal muscle, mammary glands, testes, uterus, kidneys, lungs, trachea, colon, and liver. STEAP2 is overexpressed in cancerous tumors, including those of the prostate, bladder, neck, lung, colon, kidney, breast, pancreas, stomach, uterus, 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, a suitable anti-STEAP antibody is disclosed in U.S. Patent Application No. 2018 / 0104357. Exemplary anti-STEAP2 antibodies according to this disclosure are listed in Tables 1 and 2 herein. Table 1 specifies the amino acid sequence identifiers of the heavy chain variable region (HCVR) and light chain variable region (LCVR) of the 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 of the nucleic acid molecules encoding the HCVR, LCVR, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the exemplary anti-STEAP2 antibodies.

[0476] This disclosure provides an antibody or an antigen-binding fragment thereof comprising an HCVR containing 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 disclosure also provides an antibody or an antigen-binding fragment thereof comprising an LCVR containing 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 Disclosure also provides antibodies or antigen-binding fragments comprising HCVR / LCVR amino acid sequence pairs (HCVR / LCVR), in which any of the HCVR amino acid sequences listed in Table 1 is paired with any of the LCVR amino acid sequences listed in Table 1. According to one embodiment, the Disclosure provides antibodies or antigen-binding fragments comprising HCVR / LCVR amino acid sequences contained in any of the exemplary anti-STEAP2 antibodies listed in Table 1. In one embodiment, the HCVR / LCVR amino acid sequence pairs are selected from the group consisting of SEQ ID NOs: 250 / 258 (e.g., H2M11162N).

[0479] The disclosure also provides antibodies or antigen-binding fragments comprising an HCDR1 containing an amino acid sequence selected from any of the heavy chain CDR1 (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 disclosure also provides antibodies or antigen-binding fragments comprising an HCDR2 having an amino acid sequence selected from any of the heavy chain CDR2 (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 disclosure also provides antibodies or antigen-binding fragments comprising an HCDR3 containing an amino acid sequence selected from any of the heavy chain CDR3 (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 disclosure also provides antibodies or antigen-binding fragments 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 disclosure also provides antibodies or antigen-binding fragments 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 disclosure also provides antibodies or antigen-binding fragments 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 Disclosure also provides an antibody or an antigen-binding fragment thereof comprising an HCDR3-LCDR3 amino acid sequence pair (HCDR3 / LCDR3), in which any of the HCDR3 amino acid sequences listed in Table 1 is paired with any of the LCDR3 amino acid sequences listed in Table 1. According to one embodiment, the Disclosure provides an antibody or an antigen-binding fragment thereof comprising an HCDR3 / LCDR3 amino acid sequence contained in any of the exemplary anti-STEAP2 antibodies listed in Table 1. In one embodiment, the HCDR3 / LCDR3 amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 256 / 264 (e.g., H2M11162N).

[0486] The disclosure also provides an antibody or its antigen-binding fragment 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 one embodiment, 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 relevant embodiments, the Disclosure provides an antibody or its antigen-binding fragment comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained in an HCVR / LCVR amino acid sequence defined by one of the exemplary anti-STEAP2 antibodies listed in Table 1. For example, the Disclosure includes an antibody or its antigen-binding fragment comprising the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence contained in a pair of HCVR / LCVR amino acid sequences 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 conventional methods 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 methods. For example, see Kabat's "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 to identify CDR sequences within antibodies.

[0488] The disclosure also provides nucleic acid molecules encoding an anti-STEAP2 antibody or a portion thereof. For example, the disclosure provides nucleic acid molecules encoding any of the HCVR amino acid sequences listed in Table 1, and in some 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 disclosure also provides nucleic acid molecules encoding any of the LCVR amino acid sequences listed in Table 1, in some embodiments the nucleic acid molecule comprising 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 disclosure also provides nucleic acid molecules encoding any of the HCDR1 amino acid sequences listed in Table 1, in some embodiments the nucleic acid molecule comprising 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 disclosure also provides nucleic acid molecules encoding any of the HCDR2 amino acid sequences listed in Table 1, in some embodiments the nucleic acid molecule comprising 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 disclosure also provides nucleic acid molecules encoding any of the HCDR3 amino acid sequences listed in Table 1, in some embodiments the nucleic acid molecule comprising 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 disclosure also provides nucleic acid molecules encoding any of the LCDR1 amino acid sequences listed in Table 1, in some embodiments the nucleic acid molecule comprising 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 disclosure also provides nucleic acid molecules encoding any of the LCDR2 amino acid sequences listed in Table 1, in some embodiments the nucleic acid molecule comprising 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 disclosure also provides nucleic acid molecules encoding any of the LCDR3 amino acid sequences listed in Table 1, in some embodiments the nucleic acid molecule comprising 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] This disclosure also provides nucleic acid molecules encoding HCVR, which comprises a set of three CDRs (i.e., HCDR1-HCDR2-HCDR3), and the set of HCDR1-HCDR2-HCDR3 amino acid sequences is determined by one of the exemplary anti-STEAP2 antibodies listed in Table 1.

[0497] This disclosure also provides nucleic acid molecules encoding LCVR, which comprises a set of three CDRs (i.e., LCDR1-LCDR2-LCDR3), and the set of LCDR1-LCDR2-LCDR3 amino acid sequences is determined by one of the exemplary anti-STEAP2 antibodies listed in Table 1.

[0498] The Disclosure also provides nucleic acid molecules encoding both HCVR and LCVR, where HCVR comprises one of the HCVR amino acid sequences listed in Table 1, and LCVR comprises one of the LCVR amino acid sequences listed in Table 1. In some 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 some embodiments of this aspect of the Disclosure, the nucleic acid molecule encodes both HCVR and LCVR, and both HCVR and LCVR are obtained from the same anti-STEAP2 antibody listed in Table 1.

[0499] The disclosure also provides recombinant expression vectors capable of expressing polypeptides containing heavy chain or light chain variable regions of anti-STEAP2 antibodies. For example, the 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. Within the scope of the disclosure are further host cells into which such vectors have been introduced, as well as methods for producing antibodies or portions thereof by culturing these host cells under conditions that allow for the production of antibodies or antibody fragments, and recovering the antibodies and antibody fragments thus produced.

[0500] This disclosure includes anti-STEAP2 antibodies having modified glycosylation patterns. In some embodiments, modifications that remove unwanted glycosylation sites or antibodies lacking fucose moieties present in the oligosaccharide chain 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 embodiment, the Disclosure provides a pharmaceutical composition comprising a recombinant human antibody or a fragment thereof that specifically conjugates STEAP2 to a pharmaceutically acceptable carrier. In a related embodiment, the Disclosure features a composition which 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 conveniently combined with the anti-STEAP2 antibody. Further combination therapies and co-formulations comprising anti-STEAP2 of the Disclosure are disclosed elsewhere in this Specification.

[0502] In another aspect, the Disclosure provides a therapeutic method for targeting / killing tumor cells expressing STEAP2 using the anti-STEAP2 of the Disclosure, comprising the step of administering a therapeutically effective dose of a pharmaceutical composition comprising the anti-STEAP2 antibody of the Disclosure to a subject requiring administration. Optionally, the anti-STEAP2 antibody (or its antigen-binding fragment) may be used for the treatment of prostate cancer, or may be modified to increase cytotoxicity by means of a modified Fc domain that increases ADCC (see, for example, Shield et al. (2002) JBC 277:26733), radioimmunotherapy, antibody-drug conjugates, or other methods that increase the efficiency of tumor resection.

[0503] The disclosure also includes the use of the anti-STEAP2 antibody of the disclosure in the manufacture of agents for the treatment of diseases or disorders (e.g., cancer) associated with or resulting from cells expressing STEAP2. In one embodiment, the disclosure relates to a compound comprising the anti-STEAP2 antibody or antigen-binding fragment disclosed herein, or a STEAP2xCD3 bispecific antibody, for pharmaceutical use. In one embodiment, the disclosure relates to a compound comprising the antibody-drug conjugate (ADC) disclosed herein, for pharmaceutical use.

[0504] In another embodiment, the disclosure provides a bispecific anti-STEAP2 antibody for diagnostic applications, such as imaging reagents.

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

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

[0507] This disclosure further provides antibody or antigen-binding fragments that compete with reference antibodies containing the HCVR / LCVR amino acid sequence pairs specified in Table 1 for binding to human STEAP2. In another aspect, the disclosure provides an antibody or antigen-binding fragment that competes 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 for binding to human STEAP2.

[0508] This disclosure provides an antibody or its antigen-binding fragment that binds to the same epitope as a reference antibody containing the HCVR / LCVR amino acid sequence pair specified in Table 1 on human STEAP2. In another embodiment, the antibody or antigen-binding fragment binds to the same epitope on human STEAP2 as a reference antibody containing 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.

[0509] This disclosure further provides isolated antibodies or antigen-binding fragments thereof that bind to human STEAP2, wherein the antibody or antigen-binding fragment comprises a complementation-determining region (CDR) of a heavy chain variable region (HCVR) having the amino acid sequence described in Table 1, and a CDR of a light chain variable region (LCVR) having the amino acid sequence described in Table 1. In another embodiment, the isolated antibody or antigen-binding fragment comprises a heavy chain CDR and a light chain CDR 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 antibody or antigen-binding fragments are, respectively, sequence numbers 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 92-94-96-6 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 It includes HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains selected from the group consisting of 18-320-324-326-328, 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.

[0510] In another aspect, the Disclosure provides an isolated antibody or antigen-binding fragment thereof that binds to human STEAP2, the antibody or antigen-binding fragment being (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 (b) A heavy chain variable region (HCVR) that selects an amino acid sequence selected from the group consisting of 378, and a light chain variable region (LCVR) that selects an amino acid sequence 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 according to 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] In another embodiment, the present disclosure provides an antibody-drug conjugate comprising the aforementioned anti-STEAP2 antibody or its antigen-binding fragment and a therapeutic agent (e.g., an antitumor agent, e.g., a camptothecin analog, e.g., Dxd). In some embodiments, the antibody or antigen-binding fragment and the antitumor agent are covalently linked via a linker as discussed above. In various embodiments, the anti-STEAP2 antibody or antigen-binding fragment may be any of the anti-STEAP2 antibodies or fragments described herein.

[0512] Amino acids in the heavy and light chain variable regions, 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 the CDRs for the anti-STEAP2 antibodies selected in this 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 one embodiment, the protein-drug conjugate, e.g., ADC, according to this disclosure comprises an anti-MET antibody. In some embodiments, the anti-MET antibody may include one described in U.S. Patent Application 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 to a first epitope of human MET and a second antigen-binding domain (D2) that specifically binds to a second epitope of human MET. In some embodiments, the anti-MET / MET bispecific antibody may include the one described in U.S. Patent Application No. 2018 / 0134794.

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

[0520] In one embodiment, D1 and D2 are coupled to various (e.g., non-overlapping or partially overlapping) epitopes on MET.

[0521] In one non-limiting embodiment, this disclosure includes: The first antigen-binding domain (D1), The second antigen-binding domain (D2) and We provide a protein-drug conjugate comprising a bipolar antigen-binding molecule containing the following: D1 specifically binds to the first epitope of human MET, D2 specifically binds to the second epitope of human MET.

[0522] Anti-MET / MET bispecific antibodies may be constructed using the antigen-binding domains of two distinct 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 in pairs in cross-competition with the MET protein. If two different anti-MET antibodies can bind to MET simultaneously (i.e., do not compete with each other), the antigen-binding domain of the first anti-MET antibody and the antigen-binding domain of the second non-competitive anti-MET antibody can be manipulated to form a single anti-MET / MET bispecific antibody according to this disclosure.

[0523] According to this disclosure, a bispecific antigen-binding molecule may be a single multifunctional polypeptide or a polymer of two or more polypeptides that are covalently or noncovalently related to one another. As will become apparent from this disclosure, an antigen-binding construct having the ability to simultaneously bind two distinct, non-identical epitopes in a 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 biotechnologies known to those skilled in the art (e.g., recombinant DNA and protein expression techniques).

[0524] A bispecific antigen-binding molecule comprises a first antigen-binding domain (D1) that specifically binds to a first epitope of human MET and a second antigen-binding domain (D2) that specifically binds to a second epitope of human MET, and may be referred to herein as “MET / MET bispecific antibody,” “MET×MET bispecific antibody,” “MET / MET,” “MET×MET,” or other relevant technical terms. 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 may be included in the MET×MET bispecific antigen-binding molecules provided herein include antigen-binding domains derived from any of the anti-MET antibodies disclosed herein. For example, the disclosure includes a MET×MET bispecific antigen-binding molecule comprising a D1 or D2 antigen-binding domain containing an HCVR, which is 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] This specification also provides MET×MET bispecific antigen-binding molecules comprising a D1 or D2 antigen-binding domain containing an LCVR, which is 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] This specification provides a MET×MET bispecific antigen-binding molecule comprising a D1 or D2 bispecific antigen-binding molecule containing a pair of HCVR and LCVR amino acid sequences (HCVR / LCVR), in which one of the HCVR amino acid sequences listed in Table 3 is paired with one of the LCVR amino acid sequences listed in Table 3. According to one embodiment, the present invention provides a MET×MET bispecific antigen-binding molecule comprising a D1 or D2 antigen-binding domain containing a pair of HCVR / LCVR amino acid sequences contained in one of the exemplary anti-MET antibodies listed in Table 3.

[0528] This specification also provides MET×MET bispecific antigen-binding molecules comprising a D1 or D2 antigen-binding domain containing an HCDR1 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] Furthermore, MET×MET bispecific antigen-binding molecules are also provided, comprising a D1 or D2 antigen-binding domain containing 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] Furthermore, MET×MET bispecific antigen-binding molecules are also provided, comprising a D1 or D2 antigen-binding domain containing an HCDR3 containing 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] Furthermore, MET×MET bispecific antigen-binding molecules are also provided, comprising a D1 or D2 antigen-binding domain containing an LCDR1 containing 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] Furthermore, MET×MET bispecific antigen-binding molecules are also provided, comprising a D1 or D2 antigen-binding domain containing an LCDR2, which is 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] Furthermore, MET×MET bispecific antigen-binding molecules are also provided, comprising a D1 or D2 antigen-binding domain containing 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] This specification provides a MET×MET bispecific antigen-binding molecule comprising a D1 or D2 bispecific antigen-binding molecule containing a pair of HCDR3 and LCDR3 amino acid sequences (HCDR3 / LCDR3), in which one of the HCDR3 amino acid sequences listed in Table 3 is paired with one of the LCDR3 amino acid sequences listed in Table 3. According to one embodiment, the present disclosure provides an antibody or an antigen-binding fragment thereof comprising an HCDR3 / LCDR3 amino acid sequence contained in any of the exemplary anti-MET antibodies listed in Table 3.

[0535] Furthermore, MET×MET bispecific antigen-binding molecules are also provided, comprising a D1 or D2 antigen-binding domain containing a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) found in any of the exemplary anti-MET antibodies listed in Table 3.

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

[0537] The MET×MET bispecific antigen-binding molecules provided herein may include 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 MET×MET bispecific antibodies in this disclosure are shown in Figure 10. Figure 10 is a diagram of a matrix illustrating the components of 272 exemplary MET×MET bispecific antibodies. The numbered cells in the matrix (numbers 1 to 272) each identify an intrinsic bispecific antibody containing a "D1" antigen-binding domain and an intrinsic bispecific antibody containing a "D2" antigen-binding domain, where the D1 antigen-binding domain includes 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 includes an immunoglobulin variable domain (HCVR / LCVR amino acid sequence pair) or CDR from the corresponding anti-MET antibody listed along the X axis. Therefore, for example, the MET×MET bispecific antigen-binding molecule "number 10" shown in the matrix includes a D1 antigen-binding domain containing an HCVR / LCVR pair or 6-CDR set of exemplary anti-MET antibody H4H13290P2, and a D2 antigen-binding domain containing an HCVR / LCVR pair or 6-CDR set of exemplary anti-MET antibody H4H13321P2.

[0538] As a non-limiting and illustrative example, the 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 a pair of HCVR / LCVR amino acid sequences of SEQ ID NO: 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 a pair of HCVR / LCVR amino acid sequences of SEQ ID NO: 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 possessing these sequence features was designed as H4H14639D, also known as bispecific antibody number 2076, and contains D1 derived from H4H13306P2 and D2 derived from H4H13312P2.

[0539] Amino acid sequences 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 the CDRs for the selected anti-MET antibodies disclosed herein (as stated above, all anti-MET antibodies in this disclosure have the same light chain variable region and therefore the same light chain CDR sequence). 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 the suffix "P2", as shown in Tables 3 and 4. Therefore, according to this nomenclature, antibodies may be referred to herein as, for example, "H4H13290P2", "H4H13291P2", "H4H13295P2", etc. The prefix in antibody names used herein indicates a specific Fc region isotype of the antibody. Specifically, an "H4H" antibody has human IgG4 Fc (as indicated by the initial "H" in the antibody name, the variable region is entirely human). As those skilled in the art will recognize, antibodies with a particular Fc isotype can be converted to antibodies with a different Fc isotype (for example, an antibody with mouse IgG4 Fc can be converted to an antibody with human IgG1, etc.), but in either case, the variable domain (including the CDR) indicated by the numerical identifiers shown in Tables 3 and 4 remains the same, and the binding properties are expected to be identical or substantially similar regardless of the properties of the Fc domain.

[0544] Antibody conjugation Techniques and linkers for conjugating residues of antibody or antigen-binding fragments are known in the art. Exemplary amino acid conjugates usable in this embodiment include, for example, lysine (see, e.g., U.S. Patent No. 5,208,020, U.S. Patent Application No. 2010 / 0129314, Bioconjugate Chem., 2008, 19:358-361 by Holander et al., International Publication No. WO2005 / 089808, U.S. Patent 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, International Publication No. WO2013 / 0 See Patent 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.) See International Publication No. 2008 / 122039 and Proc. Natl. Acad. Sci., USA, 2008, 105:12451-12456 by Hofer et al., formylglycine (e.g., Nat. Chem. Biol., 2007, 3:321-322 by Carrico et al., Proc. Natl. Acad. Sci., USA, 2013, 110:46-51 by Agarwal et al., and Nat. Protocols, 2012, 10:1052-1067 by Rabuka et al.), unnatural amino acids (e.g., see International Publication No. WO2013 / 068874 and International Publication No. WO2012 / 166559), and acidic amino acids (e.g., see International Publication No. WO2012 / 05982). Lysine conjugation can further proceed via NHS (N-hydroxysuccinimide). The linker can further conjugate to a cysteine ​​residue containing the cleaved interchain disulfide bond by forming a carbon bridge between thiols (see, for example, U.S. Patent Nos. 9,951,141 and 9,950,076).The linker can further be conjugated to antigen-binding proteins via binding to carbohydrates (see, e.g., U.S. Patent No. 2008 / 0305497, International Publication No. WO2014 / 065661, and Food & Agriculture Immunol., 2001, 13:127-130 by Ryan et al.) and disulfide linkers (see, e.g., International Publication No. WO2013 / 085925, International Publication No. WO2010 / 010324, International Publication No. WO2011 / 018611, and Nat. Chem. Biol., 2006, 2:312-313 by Shaunak et al.). Site-directed conjugation techniques can also be used to target the conjugation of specific residues of antibodies or antigen-binding proteins (see, e.g., Schumacher et al., J Clin Immunol (2016) 36(Suppl 1):100). In specific embodiments discussed in more detail below, a site-directed conjugation technique is transglutaminase-mediated glutamine conjugation (see, e.g., Schibli, Angew Chemie Inter Ed. 2010, 49, 9995).

[0545] Payloads of the Disclosure, which are linked by lysine and / or cysteine, for example, via maleimide or amide conjugations, are included within the scope of the 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, such as an NHS-ester linker, and step 2 is a payload conjugation reaction (e.g., a 1,3-addition 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, such as a maleimide linker, and step 2 is a payload conjugation reaction (e.g., a 1,3-addition 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-directed conjugation, and step 2 is a payload conjugation reaction (e.g., a 1,3-addition cycloaddition reaction).

[0549] Step 1: Transglutaminase-mediated site-specific conjugation In some embodiments, proteins (e.g., antibodies) may 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-directed conjugation techniques are utilized herein to target conjugation to glutamine using transglutaminase-mediated glutamine conjugation (see, for example, Angew Chemie Inter Ed. 2010, 49, 9995 by Schibli).

[0550] Compounds containing primary amines of this disclosure (e.g., linker L1) can be conjugated to one or more glutamine residues of a binder (e.g., a protein, e.g., an antibody) via chemoenzyme conjugation using transglutaminase (see, for example, Dennler et al., Protein Conjugate Chem. 2014, 25, 569-578, and International Publication WO2017 / 147542). For example, in the presence of transglutaminase, one or more glutamine residues of an antibody can be conjugated 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 primary amine-containing linker L1 described above in the presence of the enzyme transglutaminase. In some embodiments, the binder is aglycosylated. In some embodiments, the binder is deglycosylated.

[0551] In one embodiment, the binder (e.g., a protein, e.g., an antibody) comprises at least one glutamine residue in at least one polypeptide chain sequence. In another embodiment, the binder comprises two heavy chain polypeptides, each having one gln295 residue. In yet another embodiment, the binder comprises one or more glutamine residues at a site other than heavy chain 295.

[0552] In some embodiments, conjugates such as antibodies can be prepared by site-directed mutagenesis, which involves inserting a glutamine residue at a single site without impairing the function or binding of the antibody. For example, this specification includes an antibody having the Asn297Gln(N297Q) mutation described herein. In some embodiments, antibodies having the gln295 residue and / or the N297Q mutation contain one or more naturally occurring additional glutamine residues in their variable region, where access to transglutaminase is possible, thus enabling 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, the conjugate, such as an antibody, conjugated via transglutaminase may have a higher-than-expected LAR value (e.g., LAR greater than 4). Any such antibody can be isolated from natural or synthetic sources.

[0553] In some embodiments of this disclosure, the linker-to-antibody ratio, i.e., 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 Disclosure includes a branched unit B containing at least one reactive group B' that allows for further reactions after transglutamation. In these embodiments, the glutaminyl-modified protein (e.g., an antibody) allows for further reactions 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 may contain a reactive group B'' that can react with the reactive group B' of linker L1. In some embodiments, the reactive group B' of the Disclosure includes a moiety capable of undergoing a 1,3-addition cycloaddition reaction. In some embodiments, the reactive group B' is an azide. In some embodiments, the reactive group B'' includes an alkyne (e.g., a terminal alkyne, or a strained alkyne internal alkyne). In some embodiments of the Disclosure, the reactive group B' is compatible with a binder and transglutamation reaction conditions.

[0555] In some embodiments of the present disclosure, the linker L1 molecule includes a branched unit B containing one reactive group B'. In some embodiments of the present disclosure, the linker L1 molecule includes a branched unit B containing one or more reactive groups B'.

[0556] In one embodiment, the reactive linker payload L2-P contains one payload molecule (n=1). In other specific embodiments, the reactive linker payload L2-P contains two or more payload molecules (n≧2). In one embodiment, the reactive linker payload L2-P contains 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 L2-P reacts with BA-L1-B, the DAR becomes approximately equivalent to the LAR of BA-L1-B. For example, if L2-P containing one payload molecule reacts with BA-L1-B having 4 LARs (e.g., via Q295 and N297Q transglutamation), the resulting protein-drug conjugate will have 4 DARs.

[0558] In one embodiment, the reactive linker payload L2-P contains two payload molecules. When such L2-P reacts with BA-L1-B, the DAR becomes approximately twice the LAR of BA-L1B. For example, if L2-P containing two payload molecules reacts with BA-L1-B having an LAR of 4 (e.g., via Q295 and N297Q transglutamation), 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 8 LARs (e.g., via Q295 and N297Q transglutamation of a branched L1-B unit containing two B groups), the resulting protein-drug conjugate will have 24 DARs.

[0560] In some embodiments of this disclosure, the drug-to-antibody ratio, i.e., DAR (for example, abbreviated with 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, DAR is about 1, about 1.5, about 2, about 2.5, about 3, or about 3.5. In some embodiments, DAR is 2. In some embodiments, DAR is 4. In some embodiments, DAR is 8. In some embodiments, DAR is 12. In some embodiments, DAR is 16. In some embodiments, DAR is 24.

[0561] In one embodiment, this disclosure relates to formula (A): BA-(Gln-NH-L1-B-(-L2-(-M-Dxd) m ) k ) n (A) A method for producing a compound having the structure of, in the formula, BA is an antibody or its antigen-binding fragment. Gln is a glutamine residue, L1 is the first linker, B is a branched unit comprising at least one adduct from base B' and base B'', L2 is a second linker covalently bonded to branch unit B via at least one group B'', M does not exist, or

[0562] [ka] In the formula, R, R', and R'' are independently hydrogen or a C1-C4 alkyl group when they appear, or R' and R'' combine to form a 5-membered ring or a 6-membered ring. Dxd is given by equation (P):

[0563] [ka] It is an antitumor drug that contains a structure, k and m are independent integers between 1 and 12, and n is an integer between 1 and 30. The above method, (a) A step of contacting BA (BA-Gln-NH2) containing at least one glutamine residue Gln with compound L1-B in the presence of transglutaminase, wherein branched unit B contains at least one group B', (b) The product of step (a) is compound L2-(-M-Dxd) m A step of bringing into contact with k or more equivalents of, wherein the linker L2 includes at least one group B'', and one of groups B' and B'' is -N3 and

[0564] [ka] Selected from, the other of base B' and base B'' is,

[0565] [ka] Selected from, where Q is C or N, process and, (c) A step to isolate the compound of formula (I) produced and This provides a method that includes this.

[0566] In one embodiment, this disclosure relates to formula (A): BA-(Gln-NH-L1-B-(-L2-(-M-Dxd) m )k ) n (A) A method for producing a compound having the structure of, In the formula, BA is an antibody or its antigen-binding fragment, Gln is a glutamine residue, L1 is the first linker described above, B is a branched unit comprising an adduct of at least one of the groups B' and B'' described above, L2 is the second linker described above, covalently bonded to branched unit B via at least one of the groups B'' described above, and M is absent or

[0567] [ka] In the formula, R, R', and R'' are as described above, and Dxd is given by formula (P):

[0568] [ka] An antitumor drug containing the structure, where k and m are independent integers between 1 and 12, and n is an integer between 1 and 30. The above method, (a) Compound L1-B, in which branched unit B contains at least one group B', is given compound L2-(-M-Dxd) m A step of bringing into contact with k or more equivalents of, wherein the linker L2 includes at least one group B'' that can covalently bond with group B', and one of groups B' and B'' is -N3 and

[0569] [ka] Selected from, the other of base B' and base B'' is,

[0570] [ka] Selected from, where Q is C or N, and by contact L1-B-(-L2-(-M-Dxd) m ) kThe process by which it is produced, (b) In the presence of transglutaminase, BA (BA-Gln-NH2) containing at least one glutamine residue Gln is converted to L1-B-(L2-(-M-Dxd) in step (a) m ) k The process of bringing the product into contact with the product, (c) A step to isolate the compound of formula (I) produced and This provides a method that includes this.

[0571] In one embodiment, this disclosure relates to formula (I): BA-(Gln-NH-L1-B-(-L2-M-Dxd) k ) n (I) A method for producing a compound having the structure of, in the formula, BA is an antibody or its antigen-binding fragment. Gln is a glutamine residue, L1 is the first linker, B is a branched unit comprising at least one adduct from base B' and base B'', L2 is a second linker covalently bonded to branch unit B via at least one group B'', M does not exist, or

[0572] [ka] In the formula, R, R', and R'' are independently hydrogen or a C1-C4 alkyl group when they appear, or R' and R'' combine to form a 5-membered ring or a 6-membered ring. Dxd is given by equation (P):

[0573] [ka] It is an antitumor drug that contains a structure, k is an integer between 1 and 12, and n is an integer between 1 and 30. The above method, (a) A step of contacting BA (BA-Gln-NH2) containing at least one glutamine residue Gln with compound L1-B in the presence of transglutaminase, wherein branched unit B contains at least one group B', (b) A step of contacting the product of step (a) with k or more equivalents of compound L2-M-Dxd, wherein linker L2 includes at least one group B'' that can be covalently bonded to group B', and one of groups B' and B'' is -N3 and

[0574] [ka] Selected from, the other of base B' and base B'' is,

[0575] [ka] Selected from, where Q is C or N, process and, (c) A step to isolate the compound of formula (I) produced and This provides a method that includes this.

[0576] In one embodiment, this disclosure relates to formula (I): BA-(Gln-NH-L1-B-(-L2-M-Dxd) k ) n (I) A method for producing a compound having the structure of, In the formula, BA is an antibody or its antigen-binding fragment, Gln is a glutamine residue, L1 is the first linker described above, B is a branched unit comprising an adduct of at least one of the groups B' and B'' described above, L2 is the second linker described above, covalently bonded to branched unit B via at least one of the groups B'' described above, and M is absent or

[0577] [ka] In the formula, R, R', and R'' are as described above, and Dxd is given by formula (P):

[0578] [ka] An antitumor drug containing the structure, where k is an integer from 1 to 12 and n is an integer from 1 to 30. The above method, (a) A step of contacting a compound L1-B in which branch unit B contains at least one group B' with k or more equivalents of compound L2-M-Dxd, wherein linker L2 contains at least one group B'', and contact results in L1-B-(-L2-M-Dxd) k A is produced, and one of the groups B' and B'' becomes -N3 and

[0579] [ka] Selected from, the other of base B' and base B'' is,

[0580] [ka] Selected from, where Q is C or N, process and, (b) In the presence of transglutaminase, the binder BA (BA-Gln-NH2) containing at least one glutamine residue Gln is used in step (a) L1-B-(L2(M-Dxd) k The process of bringing the product into contact with the product, (c) A step to isolate the compound of formula (I) produced and This provides a method that includes this.

[0581] In one embodiment, this disclosure relates to formula (III): BA-(Gln-NH-L2'-P)) n (III) A method for producing a compound having the structure of, in the formula, BA is an antibody or its antigen-binding fragment, Gln is a glutamine residue, and L2'-P is the aforementioned H2N-SP1-B2-(SP2-AA-SP3-M-Dxd) p And n is an integer from 1 to 30. SP1 does not exist, or

[0582] [ka] A first spacer unit selected from the group consisting of, B2 either does not exist or is a branch unit. SP2 does not exist, 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 A second spacer unit selected from the group consisting of -C(O)-NH- or combinations thereof, where the subscripts u and v are independently integers from 1 to 8. AA is either absent or a peptide unit containing 2-4 amino acids. SP3 does not exist, or

[0583] [ka] A third spacer unit selected from the group consisting of, where R cThey either do not exist independently at the time of each occurrence, or

[0584] [ka] It is a base selected from, M does not exist, or

[0585] [ka] In the formula, R, R', and R'' are independently hydrogen or a C1-C4 alkyl group when they appear, or R' and R'' combine to form a 5-membered ring or a 6-membered ring. Dxd is given by equation (P):

[0586] [ka] It is an antitumor drug having the structure, p is an integer between 1 and 30. The above method, (b) A step of contacting BA (BA-Gln-NH2) containing at least one glutamine residue Gln with L2'-P in the presence of transglutaminase, (c) A step to isolate the compound of formula (III) produced and This provides a method that includes this.

[0587] In one embodiment, this disclosure relates to formula (I): BA-(Gln-NH-L1-B-(-L2-M-Dxd) k ) n (I) A method for producing a compound having the structure of, In the formula, BA is an antibody or its antigen-binding fragment, Gln is a glutamine residue, L1 is the first linker described above, B is a branched unit containing at least one group B' described above, L2 is the second linker described above, covalently bonded to branched unit B via at least one group B'' described above, groups B' and B'' form at least one adduct described above, and M is absent or

[0588] [ka] In the formula, R, R', and R'' are as described above, and Dxd is given by formula (P):

[0589] [ka] An antitumor drug containing the structure, where k is an integer from 1 to 12 and n is an integer from 1 to 30. The above method, (a) A step of contacting a binder BA containing at least one glutamine residue Gln(BA-Gln-NH2) with compound L1-B in the presence of transglutaminase, wherein branched unit B is -N3,

[0590] [ka] A step comprising: a compound containing at least one group B' selected from, wherein contact produces BA-Gln-NH-L1-B; (b) A step of contacting the product of step (a) with k or more equivalents of compound L2-M-Dxd, wherein linker L2 includes at least one group B'' that can covalently bond with group B', (c) A step to isolate the compound of formula (I) produced and This provides a method that includes this.

[0591] In another aspect, this disclosure relates to formula (I): BA-(Gln-NH-L1-B-(-L2-M-Dxd)k ) n (I) A method for producing a compound having the structure of, In the formula, BA is an antibody or its antigen-binding fragment, Gln is a glutamine residue, L1 is the first linker described above, B is a branched unit containing at least one group B' described above, L2 is the second linker described above, covalently bonded to branched unit B via at least one group B'' described above, groups B' and B'' form at least one adduct described above, and M is absent or

[0592] [ka] In the formula, R, R', and R'' are as described above, and Dxd is given by formula (P):

[0593] [ka] An antitumor drug containing the structure, where k is an integer from 1 to 12 and n is an integer from 1 to 30. The above method, (a) Branch unit B is -N3,

[0594] [ka] A compound L1-B containing at least one group B' selected from is brought into contact with k or more equivalents of a compound L2-M-Dxd containing at least one group B'' that can covalently bond with group B' by linker L2, thereby forming L1-B-(L2-M-Dxd) k The process of producing and (b) In the presence of transglutaminase, the binder BA (BA-Gln-NH2) containing at least one glutamine residue Gln is used in step (a) L1-B-(L2(M-Dxd) k The process of bringing the product into contact with the product, (c) A step to isolate the compound of formula (I) produced and This provides a method that includes this.

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

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

[0597] In one embodiment, M does not exist. In another embodiment, M-Dxd is

[0598] [ka] The structure is selected from the group consisting of the following, where R is hydrogen or C1-C4 alkyl,

[0599] [ka] This represents the connection point to L2.

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

[0601] [ka]

[0602] [ka]

[0603] [ka]

[0604] [ka] It has a structure selected from the group consisting of, or a pharmaceutically acceptable salt thereof.

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

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

[0607] [ka] It has a structure selected from the group consisting of the following.

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

[0609] [ka]

[0610] [ka]

[0611] [ka]

[0612] [ka]

[0613] [ka]

[0614] [ka] It has a structure selected from the group consisting of, or a pharmaceutically acceptable salt thereof.

[0615] Therapeutic formulations and administration This disclosure provides pharmaceutical compositions comprising the protein-drug conjugate of this disclosure.

[0616] In one embodiment, the Disclosure provides a composition comprising a group of protein-drug conjugates of the Disclosure having a drug-antibody ratio (DAR) of about 0.5 to about 30.0.

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

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

[0619] In one embodiment, the DAR of the composition is approximately 3.0 to approximately 4.5.

[0620] In one embodiment, the DAR of the composition is approximately 4.

[0621] In one embodiment, the DAR of the composition is approximately 6.5 to approximately 8.5.

[0622] In one embodiment, the DAR of the composition is approximately 8.

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

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

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

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

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

[0628] In one embodiment, the DAR of the composition is approximately 24.

[0629] The compositions of this disclosure are formulated with suitable carriers, excipients, and other agents to improve transport, delivery, and tolerability. Numerous suitable formulations can be found in "Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA," a formulary known to all pharmacists. Examples of these formulations include powders, pastes, ointments, jellies, waxes, oils, lipids, vesicle-containing (cationic or anionic) lipids (e.g., LIPOFECTIN®, Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, carbowax emulsions (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 protein-drug conjugate administered to a patient may vary depending on the patient's age and size, the target disease, condition, and route of administration. A suitable dose is typically calculated based on body weight or body surface area. When the protein-drug conjugates of this disclosure are used for therapeutic purposes in adult patients, it may be convenient to administer them intravenously as a single dose of approximately 0.01 to 20 mg / kg, more preferably 0.02 to 0.07, 0.03 to 5, or 0.05 to 3 mg / kg. The frequency and duration of treatment can be adjusted according to the severity of the disease. Effective doses and schedules for administering protein-drug conjugates may be determined empirically; for example, patient progression may be monitored through periodic assessments and doses adjusted as needed. Furthermore, interspecies scaling of doses can be performed using methods known in the art (e.g., Mordenti et al., Pharmaceut. Res. 8:1351, 1991).

[0631] Various delivery systems are known and can be used to administer the pharmaceutical compositions of this disclosure, such as liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, or receptor-dependent endocytosis (see, for example, Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of delivery 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 by absorption via the epithelium or mucocutaneous lining (e.g., oral mucosa, rectal mucosa, intestinal mucosa), and may be administered together with other bioactive agents. Administration may be systemic or topical.

[0632] The pharmaceutical compositions of this disclosure can be delivered subcutaneously or intravenously using standard needles and syringes. In addition, for subcutaneous delivery, pen delivery devices are readily applicable to the delivery of the pharmaceutical compositions of this disclosure. Such pen delivery devices can be reusable or disposable. Reusable pen delivery devices generally utilize replaceable cartridges 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 promptly 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 replaceable cartridges. Instead, disposable pen delivery devices are pre-filled with the pharmaceutical composition held in a reservoir within the device. Once the pharmaceutical composition has been released and the reservoir is empty, the entire device is discarded.

[0633] Numerous reusable pen delivery devices and auto-injector delivery devices are applicable to the subcutaneous delivery of the pharmaceutical compositions of this disclosure. Examples include, but are not limited to, AUTOPEN® (Owen Mumford, Inc., Woodstock, UK), DISETRONIC® pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75 / 25® pen, HUMALOG® pen, HUMALIN 70 / 30® pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN® I, II, and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR® (Novo Nordisk, Copenhagen, Denmark), BD® pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN®, OPTIPEN PRO®, and OPTIPEN®. Examples of disposable pen delivery devices applicable to the subcutaneous delivery of the pharmaceutical compositions of this disclosure include, but are not limited to, STARLET® and OPTICLIK® (Sanofi-Aventis, Frankfurt, Germany).

[0634] In certain circumstances, pharmaceutical compositions can be delivered in controlled-release systems. In one embodiment, a pump may be used (see Langer; Sefton, 1987, CRC Crit.Ref. Biomed.Eng. 14:201 above). In another embodiment, polymer materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida). In yet another embodiment, the controlled-release system can be positioned near the target of the composition so that only systemic administration is required (see, for example, 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 such as intravenous, subcutaneous, intradermal, and intramuscular injection or infusion. These injectable preparations may be prepared by known methods. For example, injectable preparations may be prepared by dissolving, suspending, or emulsifying the antibodies or salts thereof in a sterile aqueous or oily medium conventionally used for injection. Examples of aqueous media for injection include physiological saline, isotonic solutions containing glucose or other adjuvants, which may be used in combination with appropriate solubilizers such as alcohol (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)). Examples of oily media include sesame oil and soybean oil, which may be used in combination with solubilizers such as benzyl benzoate or benzyl alcohol. The injectable preparations thus prepared are preferably filled into appropriate ampoules.

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

[0637] Protein-drug conjugates, linker payloads, and therapeutic applications of payloads In another embodiment, protein-drug conjugates disclosed herein, such as ADCs, are particularly useful for the treatment, prevention, and / or relief of diseases, disorders, or illnesses requiring treatment.

[0638] In one embodiment, the present invention provides a method for treating a disease of a subject requiring treatment, comprising the step of administering to the subject a therapeutically effective dose of a compound according to the present disclosure (e.g., an antibody-drug conjugate, a linker payload, and / or a payload), or a composition comprising any of the compounds according to the present disclosure.

[0639] In one embodiment, the protein-drug conjugates disclosed herein, such as ADCs, are useful for treating cancer. In one embodiment, the protein-drug conjugates disclosed herein, such as ADCs, are useful for treating cancers 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 disclosed herein, such as ADCs, are useful for treating HER2+ breast cancer. In one embodiment, the protein-drug conjugates disclosed herein, such as ADCs, are useful for treating prostate cancer.

[0640] In one embodiment, the 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 includes the step of linking the compound to a targeted antibody. In one embodiment, the compound is the payload 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 breast cancer cells, ovarian cancer cells, prostate cancer cells, lung cancer cells, liver cancer cells, or brain cancer cells.

[0641] In one embodiment, the present disclosure relates to structure PI:

[0642] [ka] The present invention provides a method for selectively delivering a compound having or a pharmaceutically acceptable salt thereof to cells, wherein R1, R2, R3, and R4 are independently hydrogen or alkyl, for example, C1-C 12 It is alkyl, C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl, or R2 and R3 combine to form a 5-membered ring or a 6-membered ring.

[0643] In one embodiment, the 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 includes the step of linking the compound with a targeted antibody. In one embodiment, the compound is the payload 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 breast cancer cells, ovarian cancer cells, prostate cancer cells, lung cancer cells, liver cancer cells, or brain cancer cells.

[0644] In one embodiment, the present disclosure relates to structure PI:

[0645] [ka] This provides a method for selectively targeting antigens on the cell surface using a compound having or a pharmaceutically acceptable salt thereof, wherein R1, R2, R3, and R4 are independently hydrogen or alkyl, for example, C1-C 12 It is alkyl, C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl, or R2 and R3 combine to form a 5-membered ring or a 6-membered ring.

[0646] Anti-HER2 antibody drug conjugate In some embodiments, the protein-drug conjugates disclosed herein, such as ADCs, are useful for treating, preventing, and / or alleviating any disease or disorder that is related to or mediated by HER2 expression or activity, or that can be treated by binding HER2 without competing with modified LDL, and / or by promoting the internalization of HER2 receptors and / or by reducing the number of cell surface receptors.

[0647] The protein-drug conjugates (and therapeutic compositions comprising them) of this disclosure are particularly useful for treating any disease or disorder in which stimulation, activation, and / or targeting of an immune response is beneficial. Specifically, anti-HER2 protein-drug conjugates comprising both monospecific anti-HER2 antibodies and bispecific anti-HER2 / HER2 antibodies of this disclosure can be useful for treating, preventing, and / or alleviating 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 this 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. Examples of HER2-expressing cells that can be inhibited or killed using the protein-drug conjugates of this disclosure include breast tumor cells.

[0648] In one embodiment, the protein-drug conjugate (and therapeutic compositions and dosage forms containing the same) of the present disclosure are The first antigen-binding domain (D1), The second antigen-binding domain (D2) and It comprises a bispecific antigen-binding molecule containing, D1 specifically binds to the first epitope of human HER2. D2 specifically binds to the second epitope of human HER2.

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

[0650] The protein-drug conjugates of this 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 ovaries. In one embodiment, the protein-drug conjugates of this disclosure are used to treat one or more of the following cancers: 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 one embodiment of this disclosure, an anti-HER2 antibody or an anti-HER2 / HER2 bispecific antibody is useful for treating patients with breast cancer cells that are IHC2+ or higher. According to another relevant embodiment of this disclosure, a method is provided that includes the step of administering an anti-HER2 antibody or an 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 some embodiments, the disclosure also includes a method for treating residual cancer in a subject. The term “residual cancer” means that one or more cancer cells are present or persist in the subject after treatment with anti-cancer therapy.

[0652] The protein-drug conjugates (and therapeutic compositions comprising them) of this disclosure are particularly useful for treating any disease or disorder in which stimulation, activation, and / or targeting of an immune response is beneficial. Specifically, the protein-drug conjugates comprising anti-HER2 antibodies or anti-HER2 / HER2 antibodies of this disclosure can be useful for treating, preventing, and / or alleviating 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 this 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. Examples of HER2-expressing cells that can be inhibited or killed using the protein-drug conjugates of this disclosure include breast tumor cells.

[0653] In one aspect, the Disclosure provides a method for treating a disease or disorder associated with HER2 expression (e.g., breast cancer), comprising the step of administering one or more anti-HER2 protein-drug conjugates or anti-HER2 / HER2 bispecific protein-drug conjugates, as separately described herein, to a subject after determining that the subject has breast cancer (e.g., IHC2+ breast cancer). For example, the Disclosure includes a method for treating breast cancer, comprising the step of administering to a patient one, two, three, four, five, six, one, two, three, four weeks, two months, six, eight months, or more than one year after the subject has received hormone therapy (e.g., anti-androgen therapy), a protein-drug conjugate containing an anti-HER2 antibody or antigen-binding molecule, or an anti-HER2 / HER2 bispecific antibody or antigen-binding molecule.

[0654] In some embodiments, the Disclosure also includes the use of an anti-HER2 antibody of the Disclosure in the manufacture of a drug for the treatment of a disease or disorder (e.g., cancer) associated with or resulting from cells expressing HER2. In one embodiment, the Disclosure relates to a protein-drug conjugate comprising an anti-HER2 antibody or antigen-binding fragment disclosed herein, or a HER2 / HER2 bispecific antibody or antigen-binding fragment, for use in pharmaceuticals. In one embodiment, the Disclosure relates to a compound comprising an antibody-drug conjugate (ADC) disclosed herein for use in pharmaceuticals.

[0655] Anti-STEAP2 antibody drug conjugate In some embodiments, the protein-drug conjugates disclosed herein, such as ADCs, are useful for treating, preventing, and / or alleviating any disease or disorder that is related to or mediated by the expression or activity of STEAP2, or that can be treated by binding STEAP2 without competing with modified LDL, and / or by promoting the internalization of STEAP2 receptors and / or by reducing the number of cell surface receptors.

[0656] The protein-drug conjugates (and therapeutic compositions comprising them) of this disclosure are particularly useful for treating any disease or disorder in which stimulation, activation, and / or targeting of an immune response is beneficial. Specifically, the HER2 or HER2-HER2-STEAP2 protein-drug conjugates of this disclosure can be useful for treating, preventing, and / or alleviating 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 this 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. Examples of STEAP2-expressing cells that can be inhibited or killed using the protein-drug conjugates of this disclosure include prostate tumor cells.

[0657] The protein-drug conjugates of this disclosure can be used, for example, to treat primary and / or metastatic tumors occurring in the prostate, bladder, cervix, lung, colon, kidney, breast, pancreas, stomach, uterus, and / or ovaries. In some embodiments, the protein-drug conjugates of this disclosure are used to treat one or more of the following cancers: 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 resistant to castration.

[0658] In some embodiments, the disclosure also includes a method for treating residual cancer in a subject. The term “residual cancer” means that one or more cancer cells are present or persist in the subject after treatment with anti-cancer therapy.

[0659] In one embodiment, the Disclosure provides a method for treating a disease or disorder associated with STEAP2 expression (e.g., prostate cancer), comprising the step of administering one or more anti-STEAP2 protein-drug conjugates, as separately described herein, to a subject after the subject has been determined to have prostate cancer. For example, the Disclosure provides a method for treating prostate cancer, comprising the step of administering a protein-drug conjugate containing an anti-STEAP antibody or antigen-binding molecule to a patient at 1, 2, 3, 4, 5, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 4 months, 6 months, 8 months, or more than 1 year after the subject has received hormone therapy (e.g., anti-androgen therapy).

[0660] In some embodiments, the Disclosure also includes the use of the anti-STEAP2 antibody of the Disclosure in the manufacture of a drug for the treatment of a disease or disorder (e.g., cancer) associated with or resulting from cells expressing STEAP2. In one embodiment, the Disclosure relates to a protein-drug conjugate comprising the anti-STEAP2 antibody or antigen-binding fragment disclosed herein for pharmacopoeial use. In one embodiment, the Disclosure relates to a compound comprising an antibody-drug conjugate (ADC) disclosed herein for pharmacopoeial use.

[0661] Anti-MET antibody drug conjugate In some embodiments, the protein-drug conjugates disclosed herein, such as ADCs, are useful for treating, preventing, and / or alleviating any disease or disorder that is related to or mediated by the expression or activity of MET, or that can be treated by binding MET without competing with modified LDL, and / or by promoting the internalization of MET receptors and / or by reducing the number of cell surface receptors.

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

[0663] The protein-drug conjugates of this disclosure can be used, for example, to treat primary and / or metastatic tumors occurring in the prostate, bladder, cervix, lung, colon, kidney, breast, pancreas, stomach, uterus, and / or ovaries. In some embodiments, the protein-drug conjugates of this disclosure are used to treat one or more of the following cancers: 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 resistant to castration.

[0664] In some embodiments, the disclosure also includes a method for treating residual cancer in a subject. The term “residual cancer” means that one or more cancer cells are present or persist in the subject after treatment with anti-cancer therapy.

[0665] In one aspect, the Disclosure provides a method for treating a disease or disorder associated with MET expression (e.g., lung cancer), comprising the step of administering one or more anti-MET or anti-MET / MET protein-drug conjugates, as separately described herein, to a subject after the subject has been determined to have lung cancer. For example, the Disclosure includes a method for treating lung cancer, comprising the step of administering to a patient a protein-drug conjugate containing an anti-MET or anti-MET / MET bispecific antibody or antigen-binding molecule at 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, or more than 1 year after the subject has received hormone therapy (e.g., anti-androgen therapy).

[0666] For example, the anti-MET antibody drug conjugates and MET×MET bispecific antibody drug conjugates of this disclosure are useful for treating tumors that express (or overexpress) MET. For example, anti-MET antibody drug conjugates and MET×MET bispecific antibody drug conjugates may be used to treat primary and / or metastatic tumors occurring in the brain and meninges, oral pharynx, lungs and bronchial trees, gastrointestinal tract, male and female reproductive organs, muscles, bones, skin and appendages, connective tissue, spleen, immune system, blood forming cells and bone marrow, liver and urinary tract, and specific sensory organs such as the eyes. In some embodiments, anti-MET antibody drug conjugates and MET×MET bispecific antibody drug conjugates are used to treat one or more of the following: 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 cancer, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, rhabdomyosarcoma, small cell lung cancer, synovial sarcoma, thyroid cancer, and Wilms' tumor.

[0667] In some embodiments, the Disclosure also includes the use of the anti-MET antibody-drug conjugate or MET×MET bispecific antibody-drug conjugate of the Disclosure in the manufacture of a drug for the treatment of a disease or disorder (e.g., cancer) associated with or resulting from cells expressing MET. In one embodiment, the Disclosure relates to a protein-drug conjugate, including the anti-MET antibody-drug conjugate or MET×MET bispecific antibody-drug conjugate disclosed herein, for use in pharmaceuticals. In one embodiment, the Disclosure relates to a compound, including an antibody-drug conjugate (ADC) disclosed herein, for use in pharmaceuticals.

[0668] Combination therapy and formulations This disclosure provides a method comprising the step of 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. Examples of exemplary additional therapeutic agents that can be combined with or administered in combination with the protein-drug conjugates (e.g., antibody-drug conjugates), linker payloads, and payloads of this 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., antibodies against EGFR [e.g., cetuximab or panitumumab] or small molecule inhibitors of EGFR [e.g., gefitinib or erlotinib]), HER2 / ErbB2, ErbB3, ErbB4, etc. Antagonists of other EGFR family members (e.g., anti-ErbB2, anti-ErbB3, or anti-ErbB4 antibodies, or small molecule inhibitors of ErbB2, ErbB3, or ErbB4 activity), EGFRvIII antagonists (e.g., antibodies that specifically bind EGFRvIII), cMET antagonists (e.g., anti-cMET antibodies), IGF1R antagonists (e.g., anti-IGF1R antibodies), B-raf inhibitors (e.g., vemurafenib, sorafenib, gDC-0879, PLX-4720), PDGFR-α inhibitors (e.g., anti-PDGFR-α antibodies), PDGFR-β inhibitors (e.g., anti-PDGFR-β antibodies), VEGF antagonists (e.g., VEGF-Trap, e.g., U.S. 7,087,See U.S. Patent No. 411 (also referred to herein as “VEGF-inhibiting fusion proteins”), anti-VEGF antibodies (e.g., bevacizumab), small molecule kinase inhibitors of the VEGF receptor (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-A2 antibodies disclosed in U.S. Patent No. 2011 / 0027286, such as H1H685P) Examples include ng2 antibodies, FOLH1 (PSMA) antagonists, PRLR antagonists (e.g., anti-PRLR antibodies), STEAP1 or STEAP2 antagonists (e.g., anti-STEAP1 or anti-STEAP2 antibodies), TMPRSS2 antagonists (e.g., anti-TMPRSS2 antibodies), MSLN antagonists (e.g., anti-MSLN antibodies), CA9 antagonists (e.g., anti-CA9 antibodies), and uroplakin antagonists (e.g., anti-uroplakin antibodies).

[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 this 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, and IL-18, or their respective receptors. The pharmaceutical compositions of this disclosure (e.g., pharmaceutical compositions comprising anti-HER2, anti-HER2 / HER2 bispecific, anti-MET, anti-MET / MET bispecific, or anti-STEAP2 protein drug conjugates (e.g., antibody drug conjugates disclosed herein)) also include: "ICE": ifosfamide (e.g., Ifex®), carboplatin (e.g., Paraplatin®), etoposide (e.g., Etopophos®, Toposar®, VePesid®, VP-16); "DHAP": dexamethasone (e.g., Decadron®) It may be administered as part of a treatment regimen that includes one or more therapeutic combinations selected from "ESHAP": etoposide (e.g., Etopophos®, Toposar®, VePesid®, VP-16), methylprednisolone (e.g., Medrol®), high-dose cytarabine, 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] This disclosure also includes a protein-drug conjugate (e.g., an antibody-drug conjugate), a linker payload, and a therapeutic combination of any of the payloads with one or more of the following cytokines: 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 aforementioned cytokines, wherein the inhibitor is an aptamer, antisense molecule, ribozyme, siRNA, peptide body, nanobody, or antibody fragment (e.g., Fab fragment; F(ab')2 fragment; Fd fragment; Fv fragment; scFv; dAb fragment; or other manipulated molecules such as diabody, triabody, tetrabody, minibody, minimal recognition unit). The antigen-binding molecules of this disclosure may be administered in combination with and / or concurrently with antiviral agents, antibiotics, analgesics, corticosteroids, and / or NSAIDs. The antigen-binding molecules of this disclosure may also be administered as part of a treatment regimen that further includes radiotherapy and / or conventional chemotherapy.

[0671] Additional therapeutically active components may be administered immediately before, concurrently with, or immediately after the administration of the antigen-binding molecule of this disclosure (for the purposes of this disclosure, such administration regimens are considered to be administrations of antigen-binding molecules "combined" with additional therapeutically active components).

[0672] This disclosure includes pharmaceutical compositions in which the protein-drug conjugate (e.g., antibody-drug conjugate), linker payload, and / or payload are administered concurrently with one or more additional therapeutically active components as separately described herein.

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

[0674] The terms “initial dose,” “second dose,” and “third dose” refer to a series of administrations of the 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 as disclosed herein. Therefore, the “initial dose” is the dose administered at the start of the treatment regimen (also referred to as the “baseline dose”), the “second dose” is the dose administered after the initial dose, and the “third dose” is the dose administered after the second dose. The initial, second, and third doses all contain equal amounts of the 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, although these amounts may differ from one another in terms of administration frequency. However, in some embodiments, the 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 amounts contained in the initial dose, second dose, and / or third dose vary from one another during the treatment period (e.g., adjusted upward or downward as appropriate). In some embodiments, two or more doses (e.g., two, three, four, or five) are administered as a “loading dose” at the start of the treatment regimen, followed by subsequent doses administered at a lower frequency (e.g., “maintenance doses”).

[0675] In one exemplary embodiment of this disclosure, the second and / or third doses are each administered 1 to 26 weeks after the previous dose (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 It is administered over a period of two weeks or more. The phrase “immediate dose” as used herein means the dose of the 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 that is administered to the patient before the next dose in a series of multiple doses without any intervening doses.

[0676] Methods according to this aspect of the Disclosure may include the step of administering a protein-drug conjugate (e.g., anti-HER2, anti-HER2 / HER2 bispecific, anti-MET, anti-MET / MET bispecific, or anti-STEAP2 antibody-drug conjugate), a linker payload, and / or a second and / or third dose of the payload to a patient in any number of doses. For example, in one embodiment, the second dose is administered to the patient only once. In another embodiment, the second dose is administered to the patient two or more times (e.g., two, three, four, five, six, seven, or eight or more times). Similarly, in one embodiment, the third dose is administered to the patient only once. In another embodiment, the third dose is administered to the patient two or more times (e.g., two, three, four, five, six, seven, or eight or more times).

[0677] In embodiments involving multiple second doses, each second dose may be administered at the same frequency as the other second doses. For example, each second dose may be administered to the patient 1-2 weeks after the preceding dose. Similarly, in embodiments involving multiple third doses, each third dose may be administered at the same frequency as the other third doses. For example, each third dose may be administered to the patient 2-4 weeks after the preceding dose. Alternatively, the frequency with which 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 by the physician during the treatment period according to the individual patient's needs after clinical examinations. [Examples]

[0678] The following embodiments illustrate specific aspects of this specification. These embodiments are merely intended to provide a concrete understanding and implementation of embodiments and their various forms, and should not be construed as limiting.

[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 derivative (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, incorporated herein by reference, and payload P4, a camptothecin derivative, was synthesized as described in Scheme 1A2 and by the synthesis steps outlined in Examples 1A to 1E.

[0687] [ka]

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

[0689] [ka]

[0690] Lead acetate (0.14 g, 0.31 mmol) was added to a mixture of Fmoc-Gly-Pro-OH P4-1 (0.10 g, 0.26 mmol) in 1 mL of dry DMF. The resulting mixture was stirred at room temperature for 30 minutes, and the reaction progress was monitored by LC-MS. The resulting mixture was filtered through Celite, the filtrate was diluted with ethyl acetate, washed with water and brine, dried over anhydrous sodium sulfate, and concentrated under vacuum. The residue was purified by silica gel column chromatography (in petroleum ether with 0-10% ethyl acetate) to obtain compound P4-2 (50 mg, 53% yield) as a white solid, but no acetate intermediate was 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]aminoacetyl)pyrrolidine-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), chlorotrimethylsilane (TMSCl) (0.27 g, 2.5 mmol) was added. The reaction mixture was stirred at room temperature for 3 hours, and the progress of the reaction was monitored by LC-MS. The resulting mixture was concentrated under vacuum, 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 hour, and the completion of the reaction was monitored by LC-MS. The resulting mixture was concentrated under vacuum, and the residue was purified by reverse-phase flash chromatography (0-100% acetonitrile in 0.05% aqueous ammonium bicarbonate solution) to obtain compound P4-3 (0.11 g with a purity of over 99%, yielding 25%, and 50 mg with a purity of 75%) as a white solid. ESI m / z: 537.3 (M+Na) + . 1 1H NMR (400MHz, 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]aminoacetyl)pyrrolidine-2-yl]oxy}acetic acid (P4-4)

[0695] [ka]

[0696] Compound P4-3 (89 mg, 0.17 mmol) was dissolved in methanol (3 mL) and THF (7 mL), to which wet palladium carbon (10% Pd, 20 mg) was added under nitrogen protection. The mixture was degassed and stirred under hydrogen balloon pressure at room temperature for 2 hours, and the completion of the reaction was monitored by LC-MS. The reaction mixture was filtered through Celite, and the filtrate was concentrated under vacuum. The residue was purified by reverse-phase flash chromatography (0-100% acetonitrile in 0.05% aqueous ammonium bicarbonate solution) to obtain 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)pyrrolidine-1-yl]-2-oxoethyl}carbamate (P4-5)

[0698] [ka]

[0699] Compound P4-4 (63 mg, 0.15 mmol) and exatecan methylate (66 mg, 0.12 mmol) were mixed in DMF (2 mL). HATU (61 mg, 0.16 mmol) and DIPEA (46 mg, 0.36 mmol) were added, and the mixture was stirred at room temperature for 2 hours. The completion of the reaction was monitored by LC-MS. The reaction mixture was directly purified by reverse-phase flash chromatography (0-100% acetonitrile in 10 mM aqueous ammonium bicarbonate) to obtain 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)pyrrolidine-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] Diethylamine (20 mg, 0.27 mmol) was added to a solution of compound P4-5 (45 mg, 54 μmol) in DCM (4 mL), and the mixture was stirred overnight at room temperature. The completion of the reaction was monitored by LC-MS. The reaction mixture was concentrated under vacuum, and the residue was purified by silica gel flash chromatography (0-10% methanol in DCM) to obtain 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, and excretion) data for payloads P1-P3 according to this 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 a P-glycoprotein inhibitor and may have the function of preventing P-glycoprotein-mediated efflux.

[0705] Example 2: Linker 2-Payload

[0706] Example 2A: Linear linker 2-payload (LL2P)

[0707] Table 7 below provides an exemplary linear linker 2-payload (LL2P) structure according to this disclosure.

[0708] [Table 9-1]

[0709] [Table 9-2]

[0710] [Table 9-3]

[0711] Table 8 below provides the chemical properties of an exemplary linear linker 2-payload (LL2P) according to this disclosure.

[0712] [Table 10-1]

[0713] [Table 10-2]

[0714] * A mixture of lactone and ring-opening product. The MW and m / z values ​​are data for the corresponding lactone.

[0715] Example 2B: Branched linker 2-payload (BL2P)

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

[0717] [Table 11]

[0718] Table 10 below provides an exemplary branch linker 2-payload (BL2P) structure according to this 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 an exemplary branched linker 2-payload (BL2P) according to this 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 (CAS 2226472-26-8) and L2-3 (CAS 2226472-28-0) were synthesized in accordance with International Publication No. WO2018089373A2, which is incorporated by reference in whole.

[0729] [ka]

[0730] Example 3A: N-[(1S)-1-{[(1S)-4-(carbamoylamino)-1-{[4-(hydroxymethyl)phenyl]carbamoyl}butyl]carbamoyl}-2-methylpropyl]-1-[2-(cycloocta-2-in-1-yloxy)acetamide]-3,6,9,12-tetraoxapentadecane-15-amide(L1-2)

[0731] [ka]

[0732] Compound L1-1 (0.17 g, 0.33 mmol) was dissolved in DMF (10 mL) and DIPEA (0.13 g, 1.0 mmol) and vcPAB (0.13 g, 0.34 mmol) were added sequentially. The reaction mixture was stirred at room temperature for 1 hour. The completion of the reaction was monitored by LC-MS. The resulting mixture was directly purified by reverse-phase flash chromatography (in water, 0-80% acetonitrile) to obtain compound L1-2 (0.18 g, 70% yield) as a colorless oil. ESI m / z: 791.3 (M+H) + . 1 1H NMR (400MHz, 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-(cycloocta-2-in-1-yloxy)acetamide]-3,6,9,12-tetraoxapentadecane-15-amide}-3-methylbutanamide]pentanamide]phenyl}methyl 4-nitrophenyl carbonate (L1-3)

[0734] [ka]

[0735] A suspension of compounds 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, after which 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 LC-MS. The resulting mixture was directly purified by reverse-phase flash chromatography (in water, 0-80% acetonitrile) to obtain 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-(cycloocta-2-in-1-yloxy)acetamide]-3,6,9,12-tetraoxapentadecane-15-amide}-3-methylbutanamide]pentanamide]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}methyl]carbamoyl}methyl)carbamate (LP1)

[0737] [ka]

[0738] Compounds L1-3 (16 mg, 17 μmol) and P3 (12 mg, 17 μmol) were dissolved in a 2 mL yellow solution of dry DMF. DIPEA (6.5 mg, 51 μmol) was added, and the clear reaction solution was stirred at room temperature for 2 hours. The completion of the reaction was monitored by LC-MS. The resulting mixture was directly purified by reverse-phase flash chromatography (0-60% acetonitrile in 0.01% aqueous TFA solution) to obtain linker payload LP1 (15 mg as TFA salt, 63% yield) as a white solid. ESI m / z: 698.8 (M / 2+H) + . 1 1H NMR (400MHz, 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. (No TFA protons were 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-in-2-yl}-4-oxobutanamide)-3,6,9,12-tetraoxapentadecane-15-amide]-3-methylbutanamide]-5-(carbamoylamino)pentanamide]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}methyl]carbamoyl}methyl)carbamate (LP2)

[0740] [ka]

[0741] By replacing L1-3 with L2-3 and following the procedure for preparing LP1, the linker payload LP2 (12 mg, 46% yield) was purified by reverse-phase flash chromatography (0-100% methanol in 10 mM aqueous ammonium bicarbonate solution) to obtain a mixture of the lactone product (LP2, shown in the figure above) and the ring-opening product (LP2-RO, shown in the figure below) as a white solid.

[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-opening product 1 1H NMR (400MHz, 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-methylbutanamide]-5-(carbamoylamino)pentanamide]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 2 mL solution of payload P3 (0.11 g, 0.15 mmol) in DMF, DIPEA (39 mg, 0.30 mmol) and Fmoc-vcPAB-PNP (CAS: 863971-53-3, 77 mg, 0.10 mmol) were added. The reaction mixture was stirred at room temperature for 1 hour until clear, and P was completely consumed by LC-MS. The resulting solution was separated by reverse-phase flash chromatography (0-70% acetonitrile in 0.01% TFA aqueous solution), revealing Fmoc-LP16-1 (98 mg, ESI m / z: 494 (M DXD +H) + , 714.2(MM DXD +H) +The compound was obtained as a pale yellow solid and 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 hours until Fmoc was completely removed by LC-MS. Volatile substances were removed under vacuum, and the residue was purified by reverse-phase flash chromatography (0-40% acetonitrile in 0.01% aqueous TFA solution) to obtain LP16-1 (TFA salt, 45 mg, 41% yield from P3). ESI m / z: 493.1 (M / 2+H) + . 1 1H NMR (400MHz, 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=3.2Hz, 3H), 0.94 (d, J=3.2Hz, 3H), 0.87 (t, J=7.6Hz, 3H) ppm. (The protons of TFA were not determined.) 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-tetraoxapentadecane-15-amide)-3-methylbutanamide]-5-(carbamoylamino)pentanamide]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}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 was between 8.0 and 9.0. Then, a solution of compound If (CAS1314378-14-7, 9 mg, 15 μmol) in anhydrous DMF (1 mL) was added to the reaction solution. The mixture was stirred at room temperature for 1 hour until the starting materials were completely consumed by LC-MS. The resulting solution was separated by reverse-phase flash chromatography (in an aqueous TFA solution (0.01%) with 0-100% acetonitrile), revealing LP16f (18 mg, ESI m / z: 728.3 (M / 2+H)). +The reaction was obtained as a white solid and dissolved in anhydrous DMF (1.9 mL). Diethylamine (0.1 mL) was added to this solution, and the reaction solution, which turned yellow, was stirred at room temperature for 30 minutes until Fmoc was completely removed by LC-MS. The resulting solution was concentrated under vacuum, and the residue was purified by preparative HPLC (in 0.01% aqueous formic acid solution with 10-95% acetonitrile) to obtain linker payload LP16 (6 mg, 34% yield) as a pale yellow solid. ESI m / z: 616.9 (M / 2+H) + . 1 1H NMR (400MHz, 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-pyrrole-1-yl)propanamide]-3,6,9,12-tetraoxapentadecane-15-amide}-3-methylbutanamide]pentanamide]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), DIPEA (8 mg, 62 μmol) and Ia (CAS: 756525-99-2, commercially available, 16 mg, 30 μmol) were added, and the reaction mixture was stirred at room temperature for 1 hour until the starting material was completely consumed by LC-MS. The resulting mixture was directly purified by preparative HPLC (in 0-100% acetonitrile in 0.05% aqueous TFA solution) to obtain LP17 (TFA salt, 15 mg, 38% yield) as a pale yellow solid. ESI m / z: 692.4 (M / 2+H). 1 1H NMR (400MHz, 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=7.2Hz, 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 determined.) 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 Examples 3H to 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 minutes before Boc-DGlu(OMe)-OH (0.40 g, 1.1 mmol) was added. The reaction mixture was stirred at room temperature for 3 hours and monitored by LC-MS. The resulting mixture was purified by reverse-phase flash chromatography (in water, 0-70% acetonitrile) to obtain Boc-DEvcPAB (0.35 g, ESI m / z: 623.4 (M+H)). + The substance was obtained as a white solid and 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 hours until Boc was completely removed, and monitored by LC-MS. Volatile substances were removed under vacuum, and the residue was purified by reverse-phase flash chromatography (in water, 10-40% acetonitrile). 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] Aside from using Boc-LGlu(OMe)-OH instead of Boc-DGlu(OMe)-OH, D Following the same procedure as with EvcPAB, 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 equivalent) in DMF (0.25 mM), DIPEA (3.3 equivalents) and HATU (1.0 equivalent) were added. The mixture was stirred at room temperature for 10 minutes, and then vcPAB or EvcPAB (1.1-1.5 equivalents) was added. The reaction mixture was stirred at room temperature for 3 hours and monitored by LC-MS. The resulting mixture was directly purified by reverse-phase flash chromatography (in water, 0-100% acetonitrile) to obtain 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] Compound LP13-2 (1.0 g, 87% yield) was obtained as a white solid following a general procedure using vcPAB (0.85 g, 2.2 mmol) and LP13-1 (0.60 g, 1.5 mmol). 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}propanamide)butanoate (LP14-2)

[0770] [ka]

[0771] Following a general procedure starting with LP14-1 (0.19 g, 0.37 mmol) and DEvcPAB (0.14 g, 0.34 mmol), the linker LP14-2 (0.20 g, 66% yield) was obtained as a white solid after purification by reverse-phase flash chromatography (in water, 0-70% acetonitrile). 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}propanamide)butanoate (LP15-2)

[0773] [ka]

[0774] LP13-1 (0.45g, 1.1mmol) and L Following a standard procedure starting with EvcPAB (0.65 g, 1.2 mmol), the linker LP15-2 (0.70 g, 69% yield) was obtained as a white solid after purification by reverse-phase flash chromatography (in water, 0-100% acetonitrile). ESI m / z: 904.5 (M+H) + . 1 1H NMR (400MHz, 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-(cycloocta-2-in-1-yloxy)acetamide]-3,6,9,12-tetraoxapentadecane-15-amide(LP1-2)

[0776] [ka]

[0777] Following a general procedure starting with LP1-1 and vcPAB (3.3 g, 8.0 mmol), the linker LP1-2 (4.3 g, 68% yield) was obtained as a white solid after purification by reverse-phase flash chromatography (in water, 0-100% acetonitrile). 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-tetraoxapentadecane-15-amide]butanoate (LP19-2)

[0779] [ka]

[0780] Following a 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-(cycloocta-2-in-1-yloxy)acetamide]-3,6,9,12-tetraoxapentadecane-15-amide}butanoate (LP20-2)

[0782] [ka]

[0783] D Compound LP20-2 (0.20 g, 44% yield) was obtained as a pale yellow solid following a general procedure starting with EvcPAB (0.28 g, 0.54 mmol) and LP1-1 (0.25 g, 0.48 mmol). 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-tetraoxapentadecane-15-amide]butanoate (LP21-2)

[0785] [ka]

[0786] L Compound LP21-2 (0.20 g, 53% yield) was obtained as a white solid following a general procedure starting with EvcPAB (0.20 g, 0.38 mmol) and LP16-1 (0.19 g, 0.39 mmol). ESI m / z: 992.5 (M+H) + . 1 1H NMR (400MHz, 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=6.8Hz,3H), 0.83 (d,J=6.8Hz,3H) ppm. (The protons of benzyl alcohol were not identified.)

[0787] Example 3R: Methyl(4S)-4-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-{[4-(hydroxymethyl)phenyl]carbamoyl}butyl]carbamoyl}-2-methylpropyl]carbamoyl}-4-{1-[2-(cycloocta-2-in-1-yloxy)acetamide]-3,6,9,12-tetraoxapentadecane-15-amide}butanoate (LP22-2)

[0788] [ka]

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

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

[0791] To a solution of LP#-2 (1.0 equivalent) in DMF (0.15 mM), DMAP (1.0 equivalent), DIPEA (3.0 equivalent), and bis(4-nitrophenyl) carbonate (3.0 equivalent) were added. The reaction mixture was stirred at room temperature for 1 hour and monitored by LC-MS. The resulting mixture was purified by reverse-phase flash chromatography (in water, 0-60% acetonitrile) to obtain 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}propanamide)-3-methylbutanamide]pentanamide]phenyl}methyl 4-nitrophenyl carbonate (LP13-3)

[0793] [ka]

[0794] Following a general procedure starting with LP13-2 (0.50 g, 0.66 mmol), linker LP13-3 (0.40 g, 68% yield) was obtained as a pale yellow oil and solidified in the atmosphere. 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}propanamide)butanoate (LP14-3)

[0796] [ka]

[0797] Following a general procedure starting with 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}propanamide)butanoate (LP15-3)

[0799] [ka]

[0800] Following the general procedure starting with 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-(cycloocta-2-in-1-yloxy)acetamide]-3,6,9,12-tetraoxapentadecane-15-amide}-3-methylbutanamide]pentanamide]phenyl}methyl 4-nitrophenyl carbonate (LP1-3)

[0802] [ka]

[0803] Following the general procedure starting with LP1-2 (2.3g, 2.9 mmol), linker LP1-3 (1.9g, 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-tetraoxapentadecane-15-amide]butanoate (LP19-3)

[0805] [ka]

[0806] Following the general procedure starting with 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-(cycloocta-2-in-1-yloxy)acetamide]-3,6,9,12-tetraoxapentadecane-15-amide}butanoate (LP20-3)

[0808] [ka]

[0809] Following the general procedure starting with 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-tetraoxapentadecane-15-amide]butanoate (LP21-3)

[0811] [ka]

[0812] Following the general procedure starting with 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 1H NMR (400MHz, 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-(cycloocta-2-in-1-yloxy)acetamide]-3,6,9,12-tetraoxapentadecane-15-amide}butanoate (LP22-3)

[0814] [ka]

[0815] Following a general procedure starting with 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 equivalents) in DMF (0.15 mM), HOBt (0.5 equivalents), DIPEA (3.0 equivalents), and Payload P (1.0 equivalent) were added. The reaction mixture was stirred at room temperature for 2 hours and monitored by LC-MS. The resulting mixture was directly purified by reverse-phase flash chromatography to obtain LP1, LP2, or LP#-4 as a white solid.

[0818] Example 3AC: {4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-{1-[2-(cycloocta-2-in-1-yloxy)acetamide]-3,6,9,12-tetraoxapentadecane-15-amide}-3-methylbutanamide]pentanamide]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}methyl]carbamoyl}methyl)carbamate (LP1)

[0819] [ka]

[0820] Following a general procedure starting with payload P3 (0.85 g, 1.2 mmol) and LP1-3 (1.2 g, 1.2 mmol), preparative HPLC (5-60% acetonitrile in 0.1% formic acid aqueous solution) was performed to obtain linker payload LP1 (1.1 g, 62% yield, formate) as a white solid. ESI m / z: 699.0 (M / 2 + H) + . 1 1H NMR (400MHz, 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-Hexane-10-in-2-yl}-4-oxobutanamide)-3,6,9,12-tetraoxapentadecane-15-amide]-3-methylbutanamide]-5-(carbamoylamino)pentanamide]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}methyl]carbamoyl}methyl)carbamate (LP2)

[0822] [ka]

[0823] Following a 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), preparative HPLC (5-95% acetonitrile in 10 mM aqueous ammonium bicarbonate) was performed to obtain linker payload LP2 (12 mg, 46% yield) as a white solid. ESI m / z: 513.4 (M / 3+H) + Rt = 6.63 min in HPLC (E-ring-opened form, 34%); 507.4 (M / 3 + H), 760.5 (M / 2 + H), Rt = 7.45 min in HPLC (lactone form, 64%). 1 1H NMR (400MHz, 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 a general procedure starting with LP13-3 (0.10 g, 0.11 mmol) and payload P3 (77 mg, 0.11 mmol), the compound LP13-4 (0.12 g, 78% yield) was obtained as a pale yellow oil after purification by reverse-phase flash chromatography (10 minutes in water with 0-100% acetonitrile, followed by 5 minutes in 100% acetonitrile). 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}Propanamide)Butanoate (LP14-4)

[0828] [ka]

[0829] Following a general procedure starting with LP14-3 (0.12 g, 0.11 mmol) and payload P3 (64 mg, 0.11 mmol), the 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 10 mM aqueous ammonium bicarbonate). 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}propanamide)butanoate (LP15-4)

[0831] [ka]

[0832] Compound LP15-4 (0.40 g, 56% yield) was obtained as a white solid following a general procedure starting with LP15-3 (0.50 g, 0.47 mmol) and P3 (0.22 g, 0.38 mmol). 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-tetraoxapentadecane-15-amide]butanoate (LP19-4)

[0834] [ka]

[0835] Compound LP19-4 (70 mg, 64% yield) was obtained as a white solid following a general procedure starting with LP19-3 (80 mg, 69 μmol) and payload P3 (40 mg, 69 μmol). 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-(cycloocta-2-in-1-yloxy)acetamide]-3,6,9,12-tetraoxapentadecane-15-amide}butanoate (LP20-4)

[0837] [ka]

[0838] Compound LP20-4 (60 mg, 45% yield) was obtained as a pale yellow solid following a general procedure starting with LP20-3 (95 mg, 86 μmol) and payload P3 (58 mg, 0.10 mmol). 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-tetraoxapentadecane-15-amide]butanoate (LP21-4)

[0840] [ka]

[0841] Compound LP21-4 (51 mg, 64% yield) was obtained as a white solid following a general procedure starting with LP21-3 (58 mg, 50 μmol) and payload P3 (35 mg, 50 μmol). ESI m / z: 799.8 (M / 2 + H) + . 1 1H NMR (400MHz, 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-(cycloocta-2-in-1-yloxy)acetamide]-3,6,9,12-tetraoxapentadecane-15-amide}butanoate (LP22-4)

[0843] [ka]

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

[0845] Example 3AL: {4-[(2S)-2-[(2S)-2-{3-[2-(2-aminoethoxy)ethoxy]propanamide}-3-methylbutanamide]-5-(carbamoylamino)pentanamide]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] Diethylamine (0.2 mL) was added to a solution of LP13-4 (0.12 g, 84 μmol) in anhydrous DMF (1.8 mL), and the mixture was stirred at room temperature for 1 hour until Fmoc was completely removed by LC-MS. The resulting solution was directly separated by reverse-phase flash chromatography (0-100% acetonitrile in 0.01% TFA aqueous solution for 10 minutes) to obtain 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), piperidine (56 mg, 0.66 mmol) was added, and the mixture was stirred at room temperature for 2 hours until Fmoc was removed, and monitored by LC-MS. Aqueous lithium hydroxide solution (0.2 mM, 1 mL) and THF (3 mL) were added to the reaction mixture, and the mixture was stirred at room temperature for a further 1 hour until the methyl ester was completely hydrolyzed by LC-MS. After filtration, the resulting mixture was acidified with PBS buffer (pH 3.0) to pH 5.0, and then concentrated under vacuum. The residue was purified by preparative HPLC (in aqueous TFA solution (0.01%) with 10-95% acetonitrile) to obtain 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] Except for replacing LP14-4 with LP15-4, the procedure was the same as for LP14. After purification by preparative HPLC (10-95% acetonitrile in 0.05% TFA aqueous solution), linker payload LP15 (50 mg, 14% yield) was obtained as a white solid. ESI m / z: 637.4 (M / 2+H) + .

[0854] Example 3AO:(4R)-4-(1-amino-3,6,9,12-tetraoxapentadecane-15-amide)-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] Except for replacing LP14-4 with LP19-4 (60 mg, 38 μmol), the procedure was the same as for LP14. After purification by preparative HPLC (10-95% acetonitrile in 0.05% TFA aqueous solution), linker payload LP19 (12 mg, 24% yield) was obtained as a white solid. ESI m / z: 681.5 (M / 2 + H) + . 1 1H NMR (400MHz, 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. 0.1-2.95 (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. (The protons of COOH 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-(cycloocta-2-in-1-yloxy)acetamide]-3,6,9,12-tetraoxapentadecane-15-amide}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), an aqueous lithium hydroxide solution (0.12 M, 1 mL) was added. The reaction mixture was stirred at room temperature for 2 hours until the methyl ester was completely hydrolyzed, and monitored by LC-MS. The mixture was acidified with PBS buffer (pH 4.0) until the pH reached 6.0, and then concentrated under vacuum. The residue was purified by preparative HPLC (in an aqueous TFA solution (0.05%) with 10-95% acetonitrile) to obtain LP20 (15 mg, 25% yield) as a white solid. ESI m / z: 763.5 (M / 2 + H) + . 1 1H NMR (400MHz, 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 identified.) 19 F NMR (400 MHz, DMSO d6 )δ-73.86,-111.30 ppm.

[0860] Example 3AQ: (4S)-4-(1-amino-3,6,9,12-tetraoxapentadecane-15-amide)-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] Except for replacing LP14-4 with LP21-4 (45 mg, 28 μmol), the procedure was the same as for LP14. After purification by preparative HPLC (10-95% acetonitrile in 0.05% TFA aqueous solution), linker payload LP21 (10 mg, 26% yield) was obtained as a white solid. ESI m / z: 681.4 (M / 2+H) + . 1 1H NMR (400MHz, 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. (The protons of COOH 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-(cycloocta-2-in-1-yloxy)acetamide]-3,6,9,12-tetraoxapentadecane-15-amide}butanoic acid (LP22)

[0864] [ka]

[0865] Except for replacing LP20-4 with LP22-4 (60 mg, 39 μmol), the procedure was the same as for LP20. After purification by preparative HPLC (10-95% acetonitrile in 0.05% TFA aqueous solution), linker payload LP22 (15 mg, 26% yield) was obtained as a white solid. ESI m / z: 763.5 (M / 2 + H) + . 1 1H NMR (400MHz, 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. (The protons of COOH 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 Examples 4A to 4F below.

[0868] Starting materials L3-2 (CAS1353016-71-3) and L4-2 (CAS1425803-45-7) were commercially available from Accela.

[0869] [ka]

[0870] Example 4A: 2-(2-{2-[2-(cycloocta-2-in-1-yloxy)acetamide]acetamide}acetamide)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 turbid mixture was stirred at room temperature for 20 hours. The mixture was filtered, and the clear filtrate was concentrated under vacuum. The residue was purified by reverse-phase flash chromatography (in water, 0-20% acetonitrile) to obtain compound L3-3 (0.13 g, 21% yield) as a white solid. ESI m / z: 354.2 (M+H) + . 1 1H NMR (400MHz, 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-(cycloocta-2-in-1-yloxy)acetamide]acetamide}acetamide)acetamide]-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), DIPEA (9.0 mg, 70 μmol) and HATU (10 mg, 26 μmol) were added. The mixture was stirred at room temperature for 30 minutes, and then payload P3 (15 mg, 22 μmol) was added. The reaction mixture was stirred at room temperature for 2 hours until most of the starting material was consumed by LC-MS. The resulting mixture was directly purified by preparative HPLC (0-100% acetonitrile in 0.01% aqueous TFA solution) to obtain linker payload LP3 (8.0 mg, 36% yield, TFA salt) as a pale yellow solid. ESI m / z: 915.5 (M+H) + . 1 1H NMR (400MHz, 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-in-2-yl}-4-oxobutanamide)acetamide]Acetamide}-3-phenylpropanoic acid (L4-3)

[0877] [ka]

[0878] Compound L4-2 (0.28 g, 0.69 mmol) and peptide L4-1 (Gly-Gly-Phe-OH, 0.19 g, 0.69 mmol) were dissolved in DMF (10 mL), to which DIPEA (0.37 mL, 2.1 mmol) was added. The reaction mixture was stirred at room temperature for 1 hour. The completion of the reaction was monitored by LC-MS. The resulting mixture was directly purified by reverse-phase flash chromatography (0-100% acetonitrile in 10 mM aqueous ammonium bicarbonate) to obtain 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-Hexane-10-in-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] Except for replacing L3-3 with L4-3, the procedure for preparing LP3 was followed. After purification by preparative HPLC (0-100% acetonitrile in 10 mM aqueous ammonium bicarbonate solution), a linker payload containing the ring-opened lactone LP4-RO (14 mg, 56% yield) and a linker payload LP4 without it were obtained as white solids.

[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 1H NMR (400MHz, 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-(cycloocta-2-in-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)pyrrolidine-1-yl]-2-oxoethyl}carbamoyl)methyl]carbamoyl}methyl)carbamoyl]methyl}acetamide (diastereomer 1, LP7 and diastereomer 2, LP7')

[0884] [ka]

[0885] Except for replacing P3 with P4, the procedure for LP3 was followed, and after purification by preparative HPLC (0-100% acetonitrile in 8 mM aqueous solution of ammonium bicarbonate containing ammonia (0.05% v)), diastereomers LP7 (with and without ring-opening lactone product, 3.0 mg, 7% yield) and LP7' (with and without ring-opening 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 1H NMR (400MHz, 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 1H NMR (400MHz, 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-(cycloocta-2-in-1-yloxy)acetamide]acetamide}acetamide)acetamide]-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, and then payload P (15 mg, 22 μmol) was added. The reaction mixture was stirred at room temperature for 2 hours until most of the starting material was consumed by LC-MS. The resulting mixture was directly purified by preparative HPLC (in 0-100% acetonitrile in 0.01% aqueous TFA solution) to obtain linker payload LP9 (8.0 mg, 36% yield, TFA salt) as a pale yellow solid. ESI m / z: 915.5 (M+H) + . 1 1H NMR (400MHz, 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] The linker payload LP8 was synthesized as described in Scheme 6 and 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-Hexane-10-in-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)pyrrolidine-1-yl]-2-oxoethyl}-3,6,9,12-tetraoxapentadecane-15-amide (LP8)

[0896] [ka]

[0897] Compound L2-1 (6.5 mg, 10 μmol) and DIPEA (3.9 mg, 30 μmol) were added to a solution of payload P4 (6.2 mg, 10 μmol) in DMF (1.0 mL), and the reaction mixture was stirred at room temperature for 2 hours. The completion of the reaction was monitored by LC-MS. The resulting mixture was directly purified by preparative HPLC (5-95% acetonitrile in 10 mM aqueous ammonium bicarbonate solution) to obtain linker payload LP8 (containing lactone ring-opening 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 1H NMR (400MHz, 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 in schemes 7A to 7B and examples 6A to 6N below.

[0902] [ka]

[0903] [ka]

[0904] Example 6A: N-(2-aminoethyl)-2-(cycloocta-2-in-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 minutes. The completion of the reaction was monitored by LC-MS. The resulting mixture was purified by reverse-phase flash chromatography (0-100% acetonitrile in 0.8 mM aqueous ammonium bicarbonate) to obtain compound L5-1 (0.18 g, 68% yield) as a colorless oil. ESI m / z: 225.2 (M+H) + . 1 1H NMR (400MHz, 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-(cycloocta-2-in-1-yloxy)acetamidoethyl}carbamoyl)-4-(hydroxymethyl)phenoxy]oxane-2-carboxylate(L5-3)

[0908] [ka]

[0909] Compound L5-2 (synthesized according to International Publication No. WO2018182341A1, incorporated herein by reference in its entirety) (0.11 g, 0.23 mmol) and HATU (96 mg, 0.25 mmol) were mixed in dry DMF (4 mL). Compound L5-1 (51 mg, 0.23 mmol) and DIPEA (89 mg, 0.69 mmol) were added, and the reaction mixture was stirred at room temperature for 2 hours until L5-2 was completely consumed, and monitored by LC-MS. The resulting mixture was directly purified by reverse-phase flash chromatography (0-100% acetonitrile in 0.01% aqueous TFA solution) to obtain 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-(cycloocta-2-in-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), bis(4-nitrophenyl) carbonate (55 mg, 0.18 mmol) and DIPEA (26 mg, 0.20 mmol) were added under nitrogen at 0°C. The reaction mixture was stirred at 0°C for 30 minutes, then stirred at room temperature for 3 hours. The reaction mixture was diluted with (10 mL) and extracted with ethyl acetate (20 mL three times). The combined organic solution was washed with brine (10 mL), dried over anhydrous sodium sulfate, and concentrated under vacuum. The residue was purified by flash chromatography (in petroleum ether with 40-60% ethyl acetate) to obtain 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-(cycloocta-2-in-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-carboxylate (L5-5)

[0914] [ka]

[0915] Compound L5-4 (17 mg, 20 μmol) was dissolved in DMF (1.0 mL) and P3 (12 mg, 20 μmol), HOBt (2.7 mg, 20 μmol), and DIPEA (5.1 mg, 40 μmol) were added. The reaction mixture was stirred at room temperature for 16 hours and monitored by LC-MS. The resulting mixture was directly purified by reverse-phase flash chromatography (0-100% acetonitrile in 0.01% TFA aqueous solution) to obtain 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-(cycloocta-2-in-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] A mixture of compound L5-5 (13 mg, 10 μmol) and methanol (2 mL) was mixed with lithium hydroxide aqueous solution (0.1 M, 2 mL), and the mixture was stirred at room temperature for 1 hour. The completion of the reaction was monitored by LC-MS. 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 solution (0.01%)) to obtain 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-(cycloocta-2-in-1-yloxy)acetamido]ethyl}carbamoyl)-4-(hydroxymethyl)phenoxy]oxan-2-yl]methylacetate (L6-3)

[0920] [ka]

[0921] Compound L6-3 (0.10 g, 80% yield) was obtained as a white solid by following the preparation procedure for L5-3, except that L5-2 was replaced with L6-2. ESI m / z: 705.3 (M+H) + .

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

[0923] [ka]

[0924] Compound L6-4 (62 mg, 50% yield) was obtained as a white solid by following the preparation procedure for L5-4, except that L5-3 was replaced with L6-3. ESI m / z: 870.3 (M+H) + .

[0925] Example 6H:[(2R,3R,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-({2-[2-(cycloocta-2-in-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]methylacetate (L6-5)

[0926] [ka]

[0927] Compound L6-5 (30 mg, 66% yield) was obtained as a white solid by following the preparation procedure for L5-5, except that L5-4 was replaced with L6-4. ESI m / z: 655.7 (M / 2 + H) + .

[0928] Example 6I: [3-({2-[2-(cycloocta-2-in-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}methyl]carbamoyl}methyl)carbamate (LP6)

[0929] [ka]

[0930] Except for replacing L5-5 with L6-5, the procedure for preparing LP5 was followed, and 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)acetamide]acetamide}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), diethylamine (0.15 g, 2.1 mmol) was added. The reaction mixture was stirred overnight at room temperature and monitored by LC-MS. The resulting mixture was directly separated by reverse-phase flash chromatography (0.05% aqueous ammonium bicarbonate solution with 0-100% ace...

Claims

1. Formula (A) shown below BA-(NH-L1-B-(-L2-(-M-Dxd) ｍ ) ｋ ) ｎ (A) A compound having the structure of the formula, in which, BA is an antibody or its antigen-binding fragment. L1 is a first linker linked to BA via the side chain of a glutamine residue of BA, B is a branched unit comprising at least one adduct from base B' and base B'', where one of base B' and base B'' is -N ３ and 【Chemistry 1】 Selected from, the other of base B' and base B'' is, 【Chemistry 2】 Selected from, where Q is C or N, L2 is a second linker covalently bonded to branch unit B via at least one group B'', M does not exist, or has the following structure 【Transformation 3】 This is the part having, where R, R', and R'' independently represent hydrogen or C when they appear. １ -C ４ It is alkyl, or R' and R'' combine to form a five-membered or six-membered ring. Dxd is given by the following formula (P) 【Chemistry 4】 It is an antitumor drug having a structure such as k is an integer between 1 and 12. m is an integer between 1 and 30. n is in the range of approximately 1 to approximately 30. compound.

2. Formula (I) shown below BA-(NH-L1-B-(-L2-M-Dxd) ｋ ) ｎ (I) A compound having the structure of the formula, in which, BA is an antibody or its antigen-binding fragment. L1 is a first linker linked to BA via the side chain of a glutamine residue of BA, B is a branching unit comprising at least one adduct of group B', and group B' is -N ３ , 【Transformation 5】 Selected from, where Q is C or N, L2 is a second linker covalently bonded to branch unit B via at least one group B'', Base B'' is -N ３ , 【Transformation 6】 Selected from, Base B' and base B'' form the at least one adduct, M does not exist, or has the following structure 【Transformation 7】 This is the part having, where R, R', and R'' independently represent hydrogen or C when they appear. １ -C ４ It is alkyl, or R' and R'' combine to form a five-membered or six-membered ring. Dxd is given by the following formula (P) 【Transformation 8】 It is an antitumor drug having a structure such as k is an integer between 1 and 12. n is in the range of approximately 1 to approximately 30. compound.

3. The compound according to claim 1 or 2, wherein 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, an anti-FFFR2 antibody, an anti-FOLR1 antibody, an anti-HER2 / HER2 bispecific antibody, an anti-MET / MET bispecific antibody, or an antigen-binding fragment thereof.

4. The compound according to any one of claims 1 to 3, wherein the glutamine residue is naturally present in the CH2 or CH3 domain of the BA, or the glutamine residue is introduced into the BA by modifying one or more amino acids.

5. The compound according to any one of claims 1 to 3, wherein the glutamine residue is Q295 or N297Q.

6. The compound according to any one of claims 1 to 5, wherein the BA targets a cancer selected from the group consisting of breast cancer, ovarian cancer, prostate cancer, lung cancer, liver cancer, or brain cancer.

7. L1 is C １ - ６ Alkylene, phenylene, -NH-, -C(O)-, -(CH ２ ) ｕ -NH-C(O)-,-(CH ２ ) ｕ -C(O)-NH-, -(CH ２ -CH ２ -O) ｖ -, - (CH ２ ) ｕ - (O-CH ２ -CH ２ ) ｖ -C(O)-NH-, a peptide unit containing 2 to 4 amino acids, or a combination thereof, each of which is -S-, -S(O ２ )-, -C(O)-, -C(O ２ ) - and CO ２ The compound according to any one of claims 1 to 6, wherein one or more H may be optionally substituted, and the subscripts u and v are independently integers from 1 to 8.

8. L1 is 【Chemistry 9】 Furthermore, selected from the group consisting of combinations thereof, in the formula, R Ａ The compound according to claim 1, wherein is a group comprising an alkyne, azide, tetrazine, trans-cyclooctene, maleimide, amine, ketone, aldehyde, carboxylic acid, ester, thiol, sulfonic acid, tosylate, halide, silane, cyano group, carbohydrate group, biotin group, or lipid residue, and the subscripts x, n, p, and q are independently integers from 0 to 12.

9. The compound according to any one of claims 1 to 8, wherein B comprises one adduct of group B'.

10. -NH-L1-B' is 【Chemistry 10】 Selected from the group consisting of, 【Chemistry 11】 The compound according to claim 9, wherein is the amino site of the binding of BA to the glutamine residue.

11. The compound according to any one of claims 1 to 8, wherein B comprises two adducts of group B', or B comprises three adducts of group B', or B comprises at least four adducts of group B'.

12. B, 【Chemistry 12】 A compound according to any one of claims 1 to 8, comprising a group selected from the group consisting of, wherein (B') comprises a bonding site of an adduct of the group B' and the group B''.

13. B, 【Chemistry 13】 A compound according to claim 12, selected from the group consisting of the following.

14. -NH-L1-B is 【Chemistry 14】 Selected from the group consisting of, 【Chemistry 15】 The compound according to claim 1, wherein is the amino site of the binding of BA to the glutamine residue.

15. The aforementioned group B' is azide (-N ３ The compound according to claim 10 or 14, wherein the adducts of group B' and group B'' contain triazole.

16. The appendages of base B' and base B'' are, 【Chemistry 16】 A compound according to any one of claims 1 to 15, having a structure selected from the group consisting of, wherein Q is C or N in the formula.

17. A compound according to any one of claims 1 to 16, wherein M is absent.

18. M is 【Chemistry 17】 In the formula, R, R', and R'' are hydrogen in their respective appearances, i.e., M is [Chemistry 18] is, or, M is 【Chemistry 19】 In this formula, R is hydrogen, and R' and R'' combine to form a five-membered ring, that is, M is 【Chemistry 20】 The compound according to any one of claims 1 to 16.

19. The compound according to any one of claims 1 to 18, wherein n is in the range of about 1 to about 16.

20. L2 is given by the following equation (L2): B''-SP1-B2-(-SP2-AA-SP3) ｐ (L2) It has a structure such that, in the formula, B'' is a group that can covalently bond to the aforementioned group B', SP1 is either non-existent or is the first spacer unit. B2 either does not exist or is a branching unit. SP2 is either non-existent or a second spacer unit. AA is either absent or a peptide unit containing 2 to 4 amino acids. SP3 is either absent or a third spacer unit covalently coupled to Dxd. p is an integer between 1 and 12. The compound according to any one of claims 1 to 19.

21. The at least one base B'' is -N ３ , 【Chemistry 21】 The compound according to claim 20, selected from the group consisting of and combinations thereof.

22. SP1 does not exist, or 【Chemistry 22】 , C １ - ６ Alkylene, -(CH ２ -CH ２ -O) ｖ -, -NH-, -C(O)-, -NH-C(O)-, -NH-(CH ２ ) ｕ -, -NH-(CH ２ ) ｕ -C(O)-, -NH-(CH ２ -CH ２ -O) ｖ -, -NH-(CH ２ -CH ２ -O) ｖ -C(O)-, -NH-(CH ２ -CH ２ -O) ｖ - (CH ２ ) ｕ -, -NH-(CH ２ -CH ２ -O) ｖ - (CH ２ ) ｕ -C(O)-, -(CH ２ ) ｕ -NH-C(O)-, -NH-(CH ２ ) ｕ -NH-C(O)-, -NH-(CH ２ ) ｕ The compound according to any one of claims 20 or 21, selected from the group consisting of -C(O)-NH- or combinations thereof, wherein the subscripts u and v are independently integers from 1 to 8.

23. B2 does not exist, or 【Chemistry 23】 A compound according to any one of claims 20 to 22, selected from the group consisting of the following.

24. SP2 is absent, or C １ - ６ alkylene, -(CH ２ -CH ２ -O) ｖ -, -NH-, -C(O)-, -NH-C(O)-, -NH-(CH ２ ) ｕ -, -NH-(CH ２ ) ｕ -C(O)-, -NH-(CH ２ -CH ２ -O) ｖ -, -NH-(CH ２ -CH ２ -O) ｖ -C(O)-, -NH-(CH ２ -CH ２ -O) ｖ -(CH ２ ) ｕ -, -NH-(CH ２ -CH ２ -O) ｖ -(CH ２ ) ｕ -C(O)-, -(CH ２ ) ｕ -NH-C(O)-, -NH-(CH ２ ) ｕ -NH-C(O)-, -NH-(CH ２ ) ｕ -C(O)-NH-, or a combination thereof, wherein the subscripts u and v are each independently an integer from 1 to 8, the compound according to any one of claims 20 to 22.

25. The compound according to any one of claims 20 to 23, wherein AA is a peptide unit comprising 2 to 4 amino acids selected from glycine, valine, phenylalanine, proline, glutamic acid, lysine, alanine, and citrulline, and combinations thereof.

26. AA is valine-citrulline, valine-alanine, phenylalanine-lysine, 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 ( Ｌ EVC), and D-glutamic acid-valine-citrulline ( Ｄ EVC), the compound according to claim 25.

27. SP3 does not exist, or 【Chemistry 24】 A group consisting of combinations thereof is selected, and in the formula, R ｃ They either do not exist independently at the time of each occurrence, or 【Chemistry 25】 A compound according to any one of claims 20 to 26, wherein the group is selected from the following.

28. M-DXD is 【Chemistry 26】 The structure is selected from the group consisting of the following, where R is hydrogen or C １ -C ４ It is alkyl, 【Chemistry 27】 The compound according to any one of claims 1 to 27, wherein is a bond site to L2.

29. The following: 【Chemistry 28】 or 【Chemistry 29】 or 【Transformation 30】 A compound according to claim 1 or 2, having the structure described above.

30. Formula (I) shown below BA-(NH-L1-B-(-L2-M-Dxd) ｋ ) ｎ (I) A compound having the structure of the formula, in which, BA is an antibody or its antigen-binding fragment. L1 is a first linker linked to BA via the side chain of a glutamine residue of BA, B is, 【Chemistry 31】 It is a branching unit that includes, where Q is C or N, L2 is a second linker covalently bonded to branch unit B, M is either non-existent or has no structure: 【Chemistry 32】 This is the part having, where R, R', and R'' independently represent hydrogen or C when they appear. １ -C ４ It is alkyl, or R' and R'' combine to form a five-membered or six-membered ring. Dxd is given by the following formula (P) 【Transformation 33】 It is an antitumor drug having a structure such as k is an integer between 1 and 12. n is in the range of approximately 1 to approximately 30. compound.

31. The following formula (L2-P) B''-SP1-B2-(-SP2-AA-SP3-M-Dxd) ｐ (L2P) A compound by which, in the formula, B'' is -N ３ , 【Transformation 34】 Selected from the group consisting of, SP1 does not exist, or 【Chemistry 35】 A first spacer unit selected from the group consisting of, B2 either does not exist or is a branching unit. SP2 does not exist, or C １ - ６ Alkylene, -(CH ２ -CH ２ -O) ｖ -, -NH-, -C(O)-, -NH-C(O)-, -NH-(CH ２ ) ｕ -, -NH-(CH ２ ) ｕ -C(O)-, -NH-(CH ２ -CH ２ -O) ｖ -, -NH-(CH ２ -CH ２ -O) ｖ -C(O)-, -NH-(CH ２ -CH ２ -O) ｖ - (CH ２ ) ｕ -, -NH-(CH ２ -CH ２ -O) ｖ - (CH ２ ) ｕ -C(O)-, -(CH ２ ) ｕ -NH-C(O)-, -NH-(CH ２ ) ｕ -NH-C(O)-, -NH-(CH ２ ) ｕ A second spacer unit selected from the group consisting of -C(O)-NH- or combinations thereof, where the subscripts u and v are independently integers from 1 to 8. AA is either absent or a peptide unit containing 2 to 4 amino acids. SP3 does not exist, or 【Transformation 36】 A third spacer unit selected from the group consisting of, where R ｃ They either do not exist independently at the time of each occurrence, or 【Chemistry 37】 It is a base selected from, M does not exist, or 【Transformation 38】 In the formula, R, R', and R'' are independently either hydrogen or C when they appear. １ -C ４ It is alkyl, or R' and R'' combine to form a five-membered or six-membered ring. Dxd is given by the following formula (P) 【Chemistry 39】 It is an antitumor drug having a structure such as p is an integer between 1 and 12. compound.

32. The following formula (L2'-P) or (L2''-P) H ２ N-SP1-B2-(-SP2-AA-SP3-M-Dxd) ｐ (L2'-P) Maleimide-N-SP1-B2-(-SP2-AA-SP3-M-Dxd) ｐ (L2''-P) A compound by which, in the formula, SP1 does not exist, or 【Chemistry 40】 A first spacer unit selected from the group consisting of, B2 either does not exist or is a branching unit. SP2 does not exist, or C １ - ６ Alkylene, -(CH ２ -CH ２ -O) ｖ -, -NH-, -C(O)-, -NH-C(O)-, -NH-(CH ２ ) ｕ -, -NH-(CH ２ ) ｕ -C(O)-, -NH-(CH ２ -CH ２ -O) ｖ -, -NH-(CH ２ -CH ２ -O) ｖ -C(O)-, -NH-(CH ２ -CH ２ -O) ｖ - (CH ２ ) ｕ -, -NH-(CH ２ -CH ２ -O) ｖ - (CH ２ ) ｕ -C(O)-, -(CH ２ ) ｕ -NH-C(O)-, -NH-(CH ２ ) ｕ -NH-C(O)-, -NH-(CH ２ ) ｕ A second spacer unit selected from the group consisting of -C(O)-NH- or combinations thereof, where the subscripts u and v are independently integers from 1 to 8. AA is a peptide unit comprising 2 to 4 amino acids selected from valine, phenylalanine, proline, glutamic acid, lysine, alanine, and citrulline, as well as combinations thereof. SP3 does not exist, or 【Chemistry 41】 A third spacer unit selected from the group consisting of, where R ｃ They either do not exist independently at the time of each occurrence, or 【Chemistry 42】 It is a base selected from, M does not exist, or 【Chemistry 43】 In the formula, R, R', and R'' are independently either hydrogen or C when they appear. １ -C ４ It is alkyl, or R' and R'' combine to form a five-membered or six-membered ring. Dxd is given by the following formula (P) 【Chemistry 44】 It is an antitumor drug having a structure such as p is an integer between 1 and 12. compound.

33. The compound according to claim 31, wherein AA is a peptide unit comprising 2 to 4 amino acids selected from glycine, valine, phenylalanine, proline, glutamic acid, lysine, alanine, and citrulline, and combinations thereof.

34. The compound according to claim 33, wherein AA is selected from the group consisting of valine-citrulline, valine-alanine, phenylalanine-lysine, glycine-glycine-glycine (GGG), glycine-glycine-glycine-glycine (GGGG (SEQ ID NO: 2113)), glycine-glycine-phenylalanine (GGF), glycine-glycine-phenylalanine-glycine (GGGFG (SEQ ID NO: 2114)), and glutamic acid-valine-citrulline (EVC). 【Request Item 35】 【Chemistry 45-1】 【Chemistry 45-2】 The compound according to claim 31, having a structure by formula (L2-P) selected from the group consisting of, or a pharmaceutically acceptable salt thereof. 【Request Item 36】 【Chemistry 46】 A compound selected from the group consisting of the following, or a pharmaceutically acceptable salt thereof. 【Request Item 37】 【Chemistry 47-1】 【Chemistry 47-2】 【Chemistry 47-3】 【Chemistry 47-4】 【Chemistry 47-5】 【Chemistry 47-6】 The compound according to claim 31 or claim 32, having a structure according to formula (L2-P), (L2'-P), or (L2''-P) selected from the group consisting of the above.

38. Formula (II) shown below BA-(NH-L1-B-(SP1-B2-(-SP2-AA-SP3-M-Dxd) ｋ ) ｐ ) ｎ (I) An antibody-drug conjugate by which, BA is an antibody, L1 is a first linker linked to BA via the side chain of a glutamine residue of BA, B is a branched unit comprising at least one adduct from base B' and base B'', wherein base B' is -N ３ , 【Chemistry 48】 Selected from, B''-SP1-B2-(-SP2-AA-SP3-M-Dxd) ｐ The compound is a compound of formula (L2-P) according to any one of claims 31, 33 to 35, wherein the compound of formula (L2-P) is covalently bound to the antibody via the adducts of the groups B' and B'', where k is an integer from 1 to 12, p is an integer from 1 to 30, and n is in the range of about 1 to about 30. Antibody-drug conjugate.

39. An antibody-drug conjugate comprising an antibody and a linker payload, wherein the linker payload is The following are shown: 【Chemistry 49】 The structure of, or a pharmaceutically acceptable salt thereof, During the ceremony, [Transformation 50] an antibody-drug conjugate representing a binding site to the antibody, either directly or via a second linker, through the side chain of a glutamine residue of the antibody.

40. A composition comprising a group of compounds according to any one of claims 1 to 30, having a drug-antibody ratio (DAR) of approximately 0.5 to approximately 30.

41. The composition according to claim 40, having a DAR of approximately 1 to approximately 16.

42. The composition according to claim 41, having a DAR of approximately 2 to approximately 8.

43. The following formula (P - I) 【Chemistry 46】 A compound having the structure of, or a pharmaceutically acceptable salt thereof, wherein R １ C １ -C ４ It is alkyl, R ２ , R ３ , and R ４ These are independently hydrogen or C １ -C ４ Alkyl, or R ２ and R ３ This refers to a compound or a pharmaceutically acceptable salt thereof that forms a five-membered or six-membered ring.

44. The following formula (P2) 【Chemistry 47】 A compound having the structure of , or a pharmaceutically acceptable salt thereof, wherein R is hydrogen or C １ -C ４ A compound that is alkyl, or a pharmaceutically acceptable salt thereof.

45. A pharmaceutical composition comprising the compound according to claim 43 or claim 44, and one or more of a diluent, a carrier, and / or an excipient.

46. A compound according to any one of claims 1 to 36, 43, or 44, an antibody-drug conjugate according to claim 38 or claim 39, a composition according to any one of claims 40 to 42, or a pharmaceutical composition according to claim 45, for treating a target disease requiring treatment.

47. The compound, antibody-drug conjugate, composition, or pharmaceutical composition according to claim 46, wherein the disease is cancer.

48. The compound, antibody-drug conjugate, composition, or pharmaceutical composition according to claim 47, wherein the cancer is selected from the group consisting of breast cancer, ovarian cancer, prostate cancer, lung cancer, liver cancer, or brain cancer.

49. The compound according to claim 43 or claim 44 for selective delivery to cells.

50. A compound according to claim 43 or claim 44 for selectively targeting an antigen on the cell surface.

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