Prodrugs of Topoisomerase I Inhibitors for ADC Conjugation and Methods of Use Thereof

Abstract

For example, protein-drug conjugates and compositions thereof useful for target-specific delivery of therapeutic moieties, such as camptothecin analogs and / or derivatives, are described herein. In certain embodiments, a specific and efficient method for producing protein-drug constructs (e.g., antibody-drug conjugates) utilizing a combination of transglutaminase and 1,3-cycloaddition technology is provided. Camptothecin analogs, antibody-drug conjugates, and compositions comprising glutaminyl-modified antibodies and camptothecin analog payloads are provided.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This patent application claims the benefit of U.S. Provisional Application No. 63 / 472,064, filed June 9, 2023, and U.S. Provisional Application No. 63 / 434,230, filed December 21, 2022, the disclosures of each of which are incorporated herein by reference in their entirety.

[0002] The present disclosure relates to protein-drug conjugates (e.g., antibody-drug conjugates), pharmaceutical compositions, and methods for treating diseases using the same. A specific and efficient method for producing protein-drug constructs utilizing a combination of transglutaminase and 1,3-cycloaddition technology is also provided. More specifically, the present disclosure relates to prodrugs of topoisomerase I inhibitors for ADC conjugation and methods for using the same.

[0003] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML file format, which is incorporated herein by reference in its entirety. The XML copy, created on December 21, 2023, is named 250298_000573_SL.xml and is 2,619,378 bytes in size. [Background technology]

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

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

[0006] Another approach uses passive drug carriers, such as polymers, liposomes, and polymeric micelles, to take advantage of the hyperpermeability of the vascular endothelium in tumor tissue. Macromolecular drugs and macromolecules accumulate in solid tumors due to enhanced permeability and retention mechanisms. However, barriers to using such targeted delivery include the rapid clearance of foreign particles from the blood and the technical hurdles of obtaining a highly standardized, pharmaceutically acceptable drug delivery system with the specificity and selectivity required to bind tumor cells.

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

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

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

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

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

[0012] In one aspect, the present disclosure provides a compound having formula (I):

[0013] [ka] or a pharmaceutically acceptable salt thereof, In the formula, R 1 , R 2 , R 3 , and R 4 are independently hydrogen, C 1-5alkyl or aryl, AA is a natural or unnatural amino acid; p is an integer from 1 to 6,

[0014] [ka] provides antibody-drug conjugates in which the present invention provides a point of attachment to the antibody or antigen-binding fragment thereof, either directly or via a linker.

[0015] In one embodiment, the compound of formula (I) is

[0016] [ka] Includes:

[0017] In one embodiment, the antibody or antigen-binding fragment thereof is a compound having a structure according to formula (II):

[0018] [ka] or a pharmaceutically acceptable salt thereof, wherein R 1 , R 2 , R 3 , and R 4 are independently hydrogen or C 1-5 is alkyl, A is a click chemistry adduct; W is NH, O, CO, CH, phenyl, or a combination of two or more thereof; AA is a natural or unnatural amino acid; m is an integer from 0 to 8, n is 0 or 1, p is an integer from 1 to 6,

[0019] [ka] indicates the point of attachment to the antibody or antigen-binding fragment thereof, either directly or via a linker.

[0020] In one embodiment, the click chemistry adduct is the product of a copper-free click chemistry reaction selected from (a) strain-promoted azide / dibenzocyclooctyne-amine (DBCO) click chemistry, (b) inverse electron demand Diels-Alder (IED-DA) tetrazine / trans-cyclooctene (TCO) click chemistry, (c) inverse electron demand Diels-Alder (IED-DA) tetrazine / norbornene click chemistry, (d) Diels-Alder maleimide / furan click chemistry, (e) Staudinger ligation, and (f) nitrile-oxide / norbornene cycloaddition click chemistry.

[0021] In one embodiment, the click chemistry adduct comprises a triazole or diazine.

[0022] In one embodiment, the click chemistry adduct is

[0023] [ka] and any positional isomer or entantiomer thereof, wherein R' is H or C 1-3 alkyl and Z is C or N.

[0024] In one embodiment, AA comprises a naturally occurring amino acid selected from glycine, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, threonine, lysine, asparagine, glutamine, aspartic acid, and glutamic acid.

[0025] In one embodiment, AA is an R-amino acid, an N-methyl amino acid,

[0026] [ka] The unnatural amino acid is selected from the group consisting of:

[0027] In one embodiment, the compound of formula (II) is

[0028] [ka]

[0029] [ka] Includes:

[0030] In one embodiment, the compound of formula (II) is

[0031] [ka] Includes:

[0032] In another aspect, the present disclosure provides an antibody-drug conjugate having a structure according to formula (III):

[0033] [ka] or a pharmaceutically acceptable salt thereof, wherein: Ab is an antibody or antigen-binding fragment thereof; R 1 , R 2 , R 3 , and R 4 are independently hydrogen or C 1-5 is alkyl, A is a click chemistry adduct; LL is a linker or bond connecting said Ab and said A; AA is a natural or unnatural amino acid; m is an integer from 0 to 8, n is 0 or 1, p is an integer from 1 to 6, q is an integer from 1 to 10.

[0034] In another aspect, the disclosure provides an antibody-drug conjugate having a structure according to formula (IVa or IVb):

[0035] [ka] or a pharmaceutically acceptable salt thereof, wherein: Ab is an antibody or antigen-binding fragment thereof; R is the side chain of any natural or unnatural amino acid; n is an integer of 1 to 5.

[0036] In another aspect, the disclosure provides an antibody-drug conjugate having a structure according to formula (IVc, IVd, IVe, IVf, IVg, IVh, IVi, IVj, or IVk):

[0037] [ka]

[0038] [ka] (SEQ ID NOs: 2115 and 2115, respectively),

[0039] [ka] (SEQ ID NOs: 2116 and 2116, respectively), or a pharmaceutically acceptable salt thereof, wherein: Ab is an antibody or antigen-binding fragment thereof; R is the side chain of any natural or unnatural amino acid; n is an integer of 1 to 5.

[0040] In one embodiment, the antibody or antigen-binding fragment thereof comprises Gln295 and / or Gln297, and the drug payload is conjugated to the antibody or antigen-binding fragment via the side chains of Gln295 and / or Gln297.

[0041] In one embodiment, the antibody or antigen-binding fragment thereof is selected from 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-FGFR2 antibody, an anti-FOLR1 antibody, an anti-HER2 / HER2 bispecific antibody, an anti-MET / MET bispecific antibody, or an antigen-binding fragment thereof.

[0042] In one embodiment, the antibody or antigen-binding fragment thereof is an anti-HER2 / HER2 bispecific antibody.

[0043] In one embodiment, the anti-HER2 / HER2 bispecific antibody a first antigen-binding domain (D1); and a second antigen-binding domain (D2), D1 specifically binds to the first epitope of human HER2; D2 specifically binds to a second epitope on human HER2.

[0044] In one embodiment, the antibody and linker-drug payload are site-specifically conjugated by using transglutaminase.

[0045] In one embodiment, the transglutaminase is a microbial transglutaminase.

[0046] In another aspect, the present disclosure provides a pharmaceutical composition comprising an antibody-drug conjugate according to any one of the above embodiments co-formulated with one or more pharmaceutically acceptable diluents, excipients, and / or additives.

[0047] In another aspect, the present disclosure provides a composition comprising a population of antibody-drug conjugates according to any one of the above embodiments, having a drug-to-antibody ratio (DAR) of from about 0.5 to about 30.0.

[0048] In one embodiment, the composition has a DAR of about 1.0 to about 2.5.

[0049] In one embodiment, the composition has a DAR of about 2.

[0050] In one embodiment, the composition has a DAR of about 3.0 to about 4.5.

[0051] In one embodiment, the composition has a DAR of about 4.

[0052] In one embodiment, the composition has a DAR of about 6.5 to about 8.5.

[0053] In one embodiment, the composition has a DAR of about 8.

[0054] In another aspect, the present disclosure provides a method for treating cancer in a subject in need thereof, comprising administering to the subject an antibody-drug conjugate according to any one of the above embodiments, or a pharmaceutical composition of the above embodiments.

[0055] In another aspect, the present disclosure provides a linker-payload compound having a formula selected from the group consisting of (D')-(N'):

[0056] [ka]

[0057] [ka]

[0058] [ka] or a pharmaceutically acceptable salt thereof, comprising: In the formula, R 1 , R 2 , R 3 , and R 4 are independently hydrogen or C 1-5 is alkyl, B,

[0059] [ka] is selected from the group consisting of W is NH, O, CO, CH, phenyl, or a combination of two or more thereof; R 5 , R 6 , R 7 , and R 8 is independently hydrogen, —NH, or the side chain of any natural or unnatural amino acid; The method comprises exposing a payload having an amino group to an activated intermediate having para-nitro-phenyl carbonate in the presence of a base and a coupling catalyst to obtain the linker-payload compounds (D')-(G'), wherein the coupling catalyst is 4-hydroxy-2-methylquinoline (MeHYQ).

[0060] In another aspect, the present disclosure provides a linker-payload compound having the formula (D-1):

[0061] [ka] A process for producing (D-1), or a pharmaceutically acceptable salt thereof, comprising: In the formula, R 1 , R 2 , R 3 , and R 4 are independently hydrogen or C 1-5 is alkyl, R 5 and R 6 is independently hydrogen, —NH, or the side chain of any natural or unnatural amino acid; The method comprises exposing a drug payload having an amino group to an activated intermediate having para-nitro-phenyl carbonate in the presence of a base and a coupling catalyst to obtain the linker-payload compound (D), wherein the coupling catalyst is 4-hydroxy-2-methylquinoline (MeHYQ).

[0062] In one embodiment, the activated intermediate having para-nitro-phenyl carbonate has a structure according to Formula II.

[0063] [ka]

[0064] The present disclosure also provides a linker-payload compound having the formula (D-1):

[0065] [ka] or a pharmaceutically acceptable salt thereof, comprising: In the formula, R 1 , R 2 , R 3 , and R 4 are independently hydrogen or C 1-5 is alkyl, R 5 and R 6 are independently hydrogen, —NH, or the side chain of any natural or unnatural amino acid; The process in question is: (a) A compound of formula (I-1) having the structure:

[0066] [ka] During the ceremony, X is,

[0067] [ka] providing a compound selected from the group consisting of: (b) reacting a compound of formula (I-1) with a compound of formula (PI),

[0068] [ka] During the ceremony, R is H or PG; by reacting with a compound of formula (PI) where PG is a suitable protecting group, and producing a compound of formula (D-1).

[0069] In one embodiment, the compound of formula (D-1) has the following structure:

[0070] [ka]

[0071] In one embodiment, step (b) of reacting a compound of Formula (I-1) with a compound of Formula (PI), wherein R is PG, further comprises reacting the compound of Formula (PI), wherein R is PG, with a deprotecting agent prior to said reacting with the compound of Formula (I-1).

[0072] In one embodiment, PG is selected from the group consisting of allyloxycarbonyl (Alloc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), and 9-fluorenylmethoxycarbonyl (Fmoc).

[0073] In one embodiment, the compound of formula (I-1) has the following structure:

[0074] [ka]

[0075] In one embodiment, the compound of formula (PI) has the structure:

[0076] [ka]

[0077] In one embodiment, the process for producing a linker-payload compound having formula (D-1) comprises the step of:

[0078] [ka] and, prior to step (a), forming a compound of formula (I-1) from the compound of formula (V).

[0079] In one embodiment, the step of forming a compound of formula (I-1) comprises reacting a compound of formula (V) with a compound of formula (VIa) or formula (VIb),

[0080] [ka] wherein X' is a halogen, to produce a compound of formula (I-1).

[0081] In one embodiment, the process comprises providing a compound of formula (VII) having the structure:

[0082] [ka] In the ceremony, P.G. 1 is a suitable protecting group; and forming a compound of formula (V) from the compound of formula (VII).

[0083] In one embodiment, PG 1 is selected from the group consisting of allyloxycarbonyl (Alloc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), and 9-fluorenylmethoxycarbonyl (Fmoc).

[0084] In one embodiment, the compound of formula (VII) has the structure:

[0085] [ka]

[0086] In one embodiment, the step of forming a compound of formula (V) comprises reacting a compound of formula (VII) with a compound of formula (VIII):

[0087] [ka] to produce a compound of formula (V).

[0088] In one embodiment, the process comprises reacting a compound of formula (IX) having the following structure:

[0089] [ka] and forming a compound of formula (VII) from the compound of formula (IX).

[0090] In one embodiment, the compound of formula (IX) has the structure:

[0091] [ka]

[0092] In one embodiment, the step of forming a compound of formula (VII) comprises reacting a compound of formula (IX) with a compound of formula (X):

[0093] [ka] to form a compound of formula (VII).

[0094] In one embodiment, the process comprises reacting a compound of formula (XI) having the following structure:

[0095] [ka] and forming a compound of formula (IX) from the compound of formula (XI).

[0096] In one embodiment, the compound of formula (XI) has the structure:

[0097] [ka]

[0098] In one embodiment, the step of forming a compound of formula (IX) comprises reacting a compound of formula (XI) with a compound of formula (XII):

[0099] [ka] to produce a compound of formula (IX).

[0100] In one embodiment, the process comprises reacting a compound of formula (XIII) having the following structure:

[0101] [ka] and and forming a compound of formula (VIII) from the compound of formula (XIII).

[0102] In one embodiment, the step of forming a compound of formula (VIII) comprises reacting a compound of formula (XIII) with a compound of formula (XII):

[0103] [ka] to form a compound of formula (VIII).

[0104] In one embodiment, the process comprises providing a compound of formula (XIV) having the structure:

[0105] [ka] During the ceremony, R a is a halogen, R b But C 1-6 providing a compound of formula (XIV), wherein XIV is alkyl; forming a compound of formula (XIII) from the compound of formula (XIV).

[0106] In one embodiment, R a is bromine.

[0107] In one embodiment, the compound of formula (XIV) has the structure:

[0108] [ka]

[0109] In one embodiment, the step of forming the compound of formula (XIII) comprises reacting a compound of formula (XIV) with a base to produce a compound of formula (XIII).

[0110] In one embodiment, the base is selected from the group consisting of sodium methoxide (NaOMe), potassium tert-butoxide (t-BuOK), sodium hydride (NaH), and lithium diisopropylamide (LDA).

[0111] In one embodiment, the process comprises the steps of providing a compound of formula (XV) having the structure:

[0112] [ka] forming a compound of formula (XIV) from the compound of formula (XV).

[0113] In one embodiment, the compound of formula (XV) has the structure:

[0114] [ka]

[0115] In one embodiment, the step of forming a compound of formula (XIV) comprises reacting a compound of formula (XV) with a compound of formula (XVI):

[0116] [ka] to produce a compound of formula (XIV).

[0117] In one embodiment, the process comprises reacting a compound of formula (XVII) having the structure:

[0118] [ka] and forming a compound of formula (XV) from the compound of formula (XVII).

[0119] In one embodiment, the step of forming the compound of formula (XV) comprises reacting a compound of formula (XVII) with a brominating agent to produce a compound of formula (XVII).

[0120] In one embodiment, the brominating agent is CHBr3.

[0121] In one embodiment, the process comprises reacting a compound of formula (XVIII) having the following structure:

[0122] [ka] and forming a compound of formula (PI) from the compound of formula (XVIII).

[0123] In one embodiment, the compound of formula (XVIII) has the structure:

[0124] [ka]

[0125] In one embodiment, the step of forming a compound of formula (PI) comprises reacting a compound of formula (XVIII) with a compound of formula (XIX):

[0126] [ka] to produce a compound of formula (PI).

[0127] In one embodiment, the process comprises reacting a compound of formula (XX) having the following structure:

[0128] [ka] and forming a compound of formula (XVIII) from the compound of formula (XX).

[0129] In one embodiment, the compound of formula (XX) has the structure:

[0130] [ka]

[0131] In one embodiment, the step of forming a compound of formula (XVIII) comprises reacting a compound of formula (XX) with a compound of formula (XXI):

[0132] [ka] to produce a compound of formula (XVIII).

[0133] In one embodiment, the process comprises reacting a compound of formula (XXII) having the structure:

[0134] [ka] and forming a compound of formula (XX) from the compound of formula (XXII).

[0135] In one embodiment, the compound of formula (XXII) has the structure:

[0136] [ka]

[0137] The present disclosure also provides a compound of formula (I-1):

[0138] [ka] or a pharmaceutically acceptable salt thereof, comprising: During the ceremony, X is,

[0139] [ka] is selected from the group consisting of R 1 , R 2 , R 3 , and R 4 are independently hydrogen or C 1-5 is alkyl, R 5 and R 6 are independently hydrogen, —NH, or the side chain of any natural or unnatural amino acid; The process is (a) providing a compound of formula (V) having the structure:

[0140] [ka] (b) forming a compound of formula (I-1) from a compound of formula (V).

[0141] In one embodiment, the compound of formula (I-1) has the following structure:

[0142] [ka]

[0143] In one embodiment, step (b) of forming a compound of Formula (I-1) comprises reacting a compound of Formula (V) with a compound of Formula (VIa) or Formula (VIb),

[0144] [ka] with a compound of formula (VIa) or (VIb), wherein X' is a halogen, The method includes producing a compound of formula (I-1).

[0145] In one embodiment, the process comprises providing a compound of formula (VII) having the structure:

[0146] [ka] In the ceremony, P.G. 1 is a suitable protecting group; forming a compound of formula (V) from the compound of formula (VII).

[0147] In one embodiment, the compound of formula (VII) has the structure:

[0148] [ka]

[0149] In one embodiment, the step of forming a compound of formula (V) comprises reacting a compound of formula (VII) with a compound of formula (VIII):

[0150] [ka] to produce a compound of formula (V).

[0151] In one embodiment, the process comprises reacting a compound of formula (IX) having the following structure:

[0152] [ka] and forming a compound of formula (VII) from the compound of formula (IX).

[0153] In one embodiment, the compound of formula (IX) has the structure:

[0154] [ka]

[0155] In one embodiment, the step of forming a compound of formula (VII) comprises reacting a compound of formula (IX) with a compound of formula (X):

[0156] [ka] to form a compound of formula (VII).

[0157] In one embodiment, the process comprises reacting a compound of formula (XI) having the following structure:

[0158] [ka] and forming a compound of formula (IX) from the compound of formula (XI).

[0159] In one embodiment, the compound of formula (XI) has the structure:

[0160] [ka]

[0161] In one embodiment, the step of forming a compound of formula (IX) comprises reacting a compound of formula (XI) with a compound of formula (XII):

[0162] [ka] to produce a compound of formula (IX).

[0163] The present disclosure also provides a compound of formula (XVIII):

[0164] [ka] or a pharmaceutically acceptable salt thereof, comprising: In the formula, R 1 , R 2 , R 3 , and R 4 are independently hydrogen or C 1-5 is alkyl, R 5 and R 6 are independently hydrogen, -NH, or the side chain of any natural or unnatural amino acid. (a) providing a compound of formula (XX) having the structure:

[0165] [ka] (b) forming a compound of formula (XVIII) from a compound of formula (XX).

[0166] In one embodiment, the compound of formula (XVIII) has the structure:

[0167] [ka]

[0168] In one embodiment, the compound of formula (XX) has the structure:

[0169] [ka]

[0170] In one embodiment, the step of forming a compound of formula (XVIII) comprises reacting a compound of formula (XX) with a compound of formula (XXI):

[0171] [ka] to produce a compound of formula (XVIII).

[0172] In one embodiment, the process comprises reacting a compound of formula (XXII) having the structure:

[0173] [ka] and forming a compound of formula (XX) from the compound of formula (XXII).

[0174] In one embodiment, the compound of formula (XXII) has the structure:

[0175] [ka]

[0176] The present disclosure also provides a compound of formula (D-1):

[0177] [ka] or a pharmaceutically acceptable salt thereof, comprising: In the formula, R 1 , R 2 , R 3 , and R 4 are independently hydrogen or C 1-5 is alkyl, R 5 and R 6 are independently hydrogen, -NH, or the side chain of any natural or unnatural amino acid. (a) A compound of formula (I-1) having the structure:

[0178] [ka] During the ceremony, X is,

[0179] [ka] providing a compound selected from the group consisting of: (b) reacting a compound of formula (I-1) with a compound of formula (PI),

[0180] [ka] During the ceremony, R is H or PG; by reacting with a compound of formula (PI) where PG is a suitable protecting group, and producing a compound of formula (D-1).

[0181] In one embodiment, the compound of formula (D-1) has the following structure:

[0182] [ka]

[0183] In one embodiment, the compound of formula (I-1) has the following structure:

[0184] [ka]

[0185] In one embodiment, step (b) of reacting a compound of Formula (I-1) with a compound of Formula (PI), wherein R is PG, further comprises reacting the compound of Formula (PI), wherein R is PG, with a deprotecting agent prior to said reacting with the compound of Formula (I-1).

[0186] In one embodiment, PG is selected from the group consisting of allyloxycarbonyl (Alloc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), and 9-fluorenylmethoxycarbonyl (Fmoc).

[0187] In one embodiment, the compound of formula (PI) has the structure:

[0188] [ka]

[0189] In one embodiment, the process comprises reacting a compound of formula (XVIII) having the following structure:

[0190] [ka] and forming a compound of formula (PI) from the compound of formula (XVIII).

[0191] In one embodiment, the compound of formula (XVIII) has the structure:

[0192] [ka]

[0193] In one embodiment, the step of forming a compound of formula (PI) comprises reacting a compound of formula (XVIII) with a compound of formula (XIX):

[0194] [ka] to produce a compound of formula (PI).

[0195] The present disclosure also provides a compound of formula (D-1):

[0196] [ka] or a pharmaceutically acceptable salt thereof, comprising: In the formula, R 1 , R 2 , R 3 , and R 4 are independently hydrogen or C 1-5 is alkyl, R 5 and R 6 are independently hydrogen, —NH, or the side chain of any natural or unnatural amino acid; The process is (a) providing a compound of formula (XXIII);

[0197] [ka] (b) reacting a compound of formula (XXIII) with a compound having the following structure in the presence of an activating agent and a base:

[0198] [ka] to produce a compound of formula (D-1).

[0199] In one embodiment, the compound of formula (D-1) has the following structure:

[0200] [ka]

[0201] In one aspect, the present disclosure provides a compound of formula (I-1):

[0202] [ka] or a pharmaceutically acceptable salt thereof, During the ceremony, X is,

[0203] [ka] is selected from the group consisting of R 1 , R 2 , R 3 , and R 4 are independently hydrogen or C 1-5 is alkyl, R 5 and R 6 are independently hydrogen, —NH 2 , or the side chain of any natural or unnatural amino acid, or a pharmaceutically acceptable salt thereof.

[0204] In one embodiment, the compound of formula (I-1) has the following structure:

[0205] [ka]

[0206] In one aspect, the present disclosure provides a compound of formula (XVIII):

[0207] [ka] or a pharmaceutically acceptable salt thereof, In the formula, R 1 , R 2 , R 3 , and R 4 are independently hydrogen or C 1-5 is alkyl, R 5 and R 6 are independently hydrogen, —NH 2 , or the side chain of any natural or unnatural amino acid, or a pharmaceutically acceptable salt thereof.

[0208] In one embodiment, the compound of formula (XVIII) has the structure:

[0209] [ka]

[0210] In another aspect, the present disclosure provides a linker-payload compound of formula (D):

[0211] [ka] or a pharmaceutically acceptable salt thereof, In the formula, R 1 , R 2 , R 3 , and R 4 are independently hydrogen or C 1-5 is alkyl, R 5 and R 6 is independently hydrogen, —NH 2 , or the side chain of any natural or unnatural amino acid, or a pharmaceutically acceptable salt thereof.

[0212] In another aspect, the present disclosure provides a linker-payload compound having a formula selected from the group consisting of (D')-(N'):

[0213] [ka]

[0214] [ka]

[0215] [ka] or a pharmaceutically acceptable salt thereof, In the formula, R 1 , R 2 , R 3 , and R 4 are independently hydrogen or C 1-5 is alkyl, B,

[0216] [ka] is selected from the group consisting of W is NH, O, CO, CH, phenyl, or a combination of two or more thereof; R 5 , R 6 , R 7 , and R 8 are independently hydrogen, —NH, or the side chain of any natural or unnatural amino acid, or a pharmaceutically acceptable salt thereof; The method comprises exposing a payload having an amino group to an activated intermediate having para-nitro-phenyl carbonate in the presence of a base and a coupling catalyst to obtain the linker-payload compounds (D')-(G'), wherein the coupling catalyst is 4-hydroxy-2-methylquinoline (MeHYQ).

[0217] In one embodiment, the structure is selected from the group consisting of:

[0218] [Table 1-1]

[0219] [Table 1-2]

[0220] [Table 1-3]

[0221] [Table 1-4]

[0222] In one embodiment, the structure is selected from the group consisting of:

[0223] [Table 2]

[0224] These and other aspects of the present disclosure will become apparent to those skilled in the art after reading the following detailed description of the present disclosure, including the appended claims. [Brief explanation of the drawings]

[0225] [Figure 1] 1 is a schematic diagram showing the two-step site-specific generation of Dxd-ADCs according to embodiments of the present disclosure. The first step is the conjugation of one or more first linkers (L1-B') to glutamine residues on the antibody via a transglutaminase (e.g., MTG)-mediated conjugation reaction. The second step is the conjugation of antibody-L1-B to one or more linker-2-payloads (L2P). [Figure 2A] 1 is a schematic diagram illustrating certain non-limiting embodiments of the present disclosure.

[0023] FIG. 1 is a schematic diagram of the two-step site-specific generation of a Dxd-ADC with a glutamine residue at position 295 with a DAR of 2×n×m, according to an embodiment of the present disclosure. [Figure 2B]1 is a schematic diagram illustrating certain non-limiting embodiments of the present disclosure.

[0023] FIG. 1 is a schematic diagram of the two-step site-specific generation of Dxd-ADC with glutamine residues at positions 295 and 297 with a DAR of 4×n×m, according to an embodiment of the present disclosure. [Figure 3A] 1 is a schematic diagram illustrating the two-step site-specific generation of one specific embodiment of a Dxd-ADC according to the present disclosure. The first step is to conjugate a linear first 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, generating an antibody with a four azide-containing linker attached thereto (Ab-(N3)4). The second step is to attach Ab-(N3)4 to a specific linker 2-payload (L2P) via an azide-cycloalkyne 1,3 cycloaddition reaction, generating a Dxd-ADC with a DAR of 4. [Figure 3B] 3A shows a schematic diagram of an ADC with a DAR of 2 or 4 and an exemplary aminoazide linker suitable for use in the embodiments of the present disclosure shown in FIG. 3A. [Figure 4A-1] 1 is a schematic diagram illustrating the two-step site-specific generation of one specific embodiment of a Dxd-ADC according to the present disclosure. The first step is to conjugate a branched first linker 1 (L1-B') containing two azide moieties (-N3) to glutamine residues at positions 295 and 297 of the antibody via an MTG-mediated conjugation reaction, generating an antibody having an eight azide-containing linker attached thereto (Ab-(N3)8). The second step is to attach Ab-(N3)8 to a specific linker 2-payload (L2P) via an azide-cycloalkyne 1,3 cycloaddition reaction, generating a Dxd-ADC with a DAR of 8. [Figure 4A-2]1 is a schematic diagram illustrating the two-step site-specific generation of one specific embodiment of a Dxd-ADC according to the present disclosure. The first step is to conjugate a branched first linker 1 (L1-B') containing two azide moieties (-N3) to glutamine residues at positions 295 and 297 of the antibody via an MTG-mediated conjugation reaction, generating an antibody having an eight azide-containing linker attached thereto (Ab-(N3)8). The second step is to attach Ab-(N3)8 to a specific linker 2-payload (L2P) via an azide-cycloalkyne 1,3 cycloaddition reaction, generating a Dxd-ADC with a DAR of 8. [Figure 4B] FIG. 4B shows a schematic diagram of an ADC and an exemplary branched alkyl azidoamine linker suitable for use in embodiments of the present disclosure shown in FIG. 4A. [Figure 5] Schematic diagram of two-step antibody-drug conjugation according to an embodiment of the present disclosure. Step 1: Site-specific conjugation of a handle-functionalized amine with an antibody to generate drug conjugates containing 2, 4, or 8 handles per antibody. Where AL = unbranched handle-functionalized amine, BL = branched handle-functionalized amine. Step 2: Click reaction between the handle-functionalized antibody and a linker-payload (LP) to generate a site-specific ADC. [Figure 6] 1 shows an exemplary conjugation procedure according to the present disclosure. [Figure 7A] Three approaches to preparing antibody-drug conjugates according to the present disclosure are shown. In approaches 1 and 2, the handle can be bivalent or multivalent. The amine handle can be conjugated to an antibody via transglutaminase-mediated conjugation to generate an Ab-Handle, and another moiety within the handle of the Ab-Handle can be clicked with a linker-payload to generate an ADC. If the handle has a diene, the linker-payload will have a dienophile, or vice versa. [Figure 7B]For approach 3, the linker-payload may be conjugated directly to the antibody; LL containing an amine moiety can be conjugated to the antibody via transglutaminase-mediated conjugation; LL containing a moiety reactive with cysteine-SH can be conjugated to the antibody-cystine via Michael addition. [Figure 8] 10 is a graph showing linker-ProDXd LP1 in mouse whole blood (SEQ ID NO: 2121). [Figure 9] 1 shows a schematic process for the preparation of liver S9 and liver microsomes from hepatocytes. DETAILED DESCRIPTION OF THE INVENTION

[0226] Detailed embodiments of the present disclosure are disclosed herein; however, it should be understood that the disclosed embodiments are merely exemplary of the present disclosure, which may be embodied in various forms. Furthermore, each of the examples shown in connection with various embodiments of the present disclosure is intended to be illustrative and not limiting. Therefore, the specific structural and functional details disclosed herein should not be construed as limiting, but merely as a representative basis for teaching those skilled in the art how to variously employ the present disclosure.

[0227] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0228] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a method" includes one or more methods, and / or steps, of the type described herein and / or that will become apparent to those skilled in the art upon reading this disclosure.

[0229] The terms "treating" or "treatment" of a state, disorder, or condition include (1) preventing, delaying, or reducing the likelihood of the onset and / or appearance of at least one clinical or subclinical symptom of a state, disorder, or condition developing in a subject who may be affected by or prone to the state, disorder, or condition, but who has not yet experienced or exhibited clinical or subclinical symptoms of the state, disorder, or condition; or (2) inhibiting the state, disorder, or condition, i.e., preventing, reducing, or delaying the onset or recurrence of the disease, or at least one clinical or subclinical symptom thereof; or (3) palliating the disease, i.e., causing regression of the state, disorder, or condition, or at least one clinical or subclinical symptom thereof. The benefit to the subject being treated is either statistically significant or at least perceptible to the patient or physician. In some embodiments, the treatment comprises ablation of cells in such a way that the disease is indirectly affected, hi certain embodiments, the treatment comprises depleting immune cells as a hematopoietic modulating regimen prior to treatment.

[0230] As used herein, "subject" or "patient" or "individual" or "animal" refers to humans, veterinary animals (e.g., cats, dogs, cows, horses, sheep, pigs, etc.), and experimental animal models of disease (e.g., mice, rats). In a preferred embodiment, the subject is a human.

[0231] As used herein, the term "effective" as applied to dose or amount refers to the amount of a compound or pharmaceutical composition that is sufficient to produce the desired activity when administered to a subject in need of the desired activity. It should be noted that when a combination of active ingredients is administered, the effective amount of the combination may or may not include the amount of each ingredient that was effective when administered individually. The exact amount required will vary from subject to subject, depending on the species, age, and general health of the subject, the severity of the condition being treated, the specific drug or drugs used, the mode of administration, etc.

[0232] When used in connection with the compositions of the present disclosure, the phrase "pharmaceutically acceptable salt" refers to any salt suitable for administration to a patient. Suitable salts include, but are not limited to, those disclosed in Berge et al., "Pharmaceutical Salts," J. Pharm. Sci., 1977, 66:1, which is incorporated herein by reference. Examples of salts include, but are not limited to, calcium salts, magnesium salts, potassium salts, sodium salts, hydrochloric acid, 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, and include, but are not limited to, acid-derived, base-derived, organic, inorganic, amine, and alkali or alkaline earth metal salts. In some examples, the payloads described herein (e.g., rifamycin analogs described herein) comprise a tertiary amine, and the nitrogen atom in the tertiary amine is the atom through which the payload is attached to the linker or linker-spacer. In such cases, the attachment of the payload to the tertiary amine results in a quaternary amine in the linker-payload molecule. The positive charge on the quaternary amine can be balanced by a counterion (e.g., chloro, bromo, iodo, or any other suitable charge moiety, such as those described herein).

[0233] Ranges may be expressed herein as from "about" or "approximately" one particular value, and / or to "about" or "approximately" another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value.

[0234] "Comprising" or "containing" or "including" means that at least the named compound, element, particle, or method step is present in the composition or article or method, but does not exclude the presence of other compounds, materials, particles, or method steps, even if other such compounds, materials, particles, or method steps have the same function as the named one.

[0235] The compounds of the present disclosure include those generally described herein and are further exemplified by the classes, subclasses, and species disclosed herein. As used herein, the following definitions shall apply unless otherwise indicated. For the purposes of this disclosure, chemical elements are identified according to the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed. Additionally, 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, M.B. and March, J., John Wiley & Sons, New York: 2001, the entire contents of which are incorporated herein by reference.

[0236] As used herein, the term "alkyl" is given its ordinary meaning in the art and can include saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups. In certain embodiments, a straight-chain or branched-chain alkyl has about 1 to 20 carbon atoms in its backbone (e.g., C1-C6 for straight chain). 20 , C2-C for branched chain 20 ), alternatively having about 1-10 carbon atoms, or about 1-6 carbon atoms. In some embodiments, cycloalkyl rings have from about 3-10 carbon atoms in their ring structure when such rings are monocyclic or bicyclic, and alternatively have about 5, 6, or 7 carbons in the ring structure. In some embodiments, alkyl groups can be lower alkyl groups, which lower alkyl groups contain from 1-4 carbon atoms (e.g., C1-C4 for straight chain lower alkyls).

[0237] As used herein, the term "alkenyl" refers to an alkyl group, as defined herein, having one or more double bonds.

[0238] As used herein, the term "alkynyl" refers to an alkyl group, as defined herein, having one or more triple bonds.

[0239] The term "aryl," used alone or as part of a larger moiety such as "aralkyl," "aralkoxy," or "aryloxyalkyl," refers to a monocyclic or bicyclic ring system having a total of 5 to 14 ring members, wherein at least one ring of the system is aromatic and each ring of the system contains 3 to 7 ring members. The term "aryl" may be used interchangeably with the term "aryl ring." In certain embodiments of the present invention, "aryl" refers to an aromatic ring system, including, but not limited to, phenyl, biphenyl, naphthyl, anthracyl, and the like, which may bear one or more substituents. Also included within the scope of the term "aryl," as used herein, are groups in which an aromatic ring is fused to one or more non-aromatic rings, such as indanyl, phthalimidyl, naphthimidyl, phenanthridinyl, or tetrahydronaphthyl.

[0240] The term "heteroatom" means one or more of oxygen, sulfur, nitrogen, phosphorus, or silicon (including any oxidized form of nitrogen, sulfur, phosphorus, or silicon; the quaternized form of any basic nitrogen; or a substitutable nitrogen of a heterocycle).

[0241] The term "halogen" means F, Cl, Br, or I, and the term "halide" refers to a halogen radical or substituent, i.e., -F, -Cl, -Br, or -I.

[0242] The term "click chemistry" refers to a class of biocompatible small molecule reactions commonly used in bioconjugation, allowing selected substrates to be linked with specific biomolecules. Click chemistry is a method for generating products that follow examples from nature, rather than a single specific reaction, and also generates substances by linking small modular units. Click chemistry is not limited to biological contexts, and the concept of the "click" reaction can be used in chemical proteomics, pharmacology, and various biomimetic applications. Specific non-limiting examples of click chemistry reactions include: (a) Strain-promoted azide / dibenzocyclooctyne-amine (DBCO) click chemistry; (b) inverse electron demand Diels-Alder (IED-DA) tetrazine / trans -cyclooctene (TCO) click chemistry; (c) Inverse electron demand Diels-Alder (IED-DA) tetrazine / norbornene click chemistry, (d) Diels-Alder maleimide / furan click chemistry, (e) Staudinger ligation, and (f) Nitrile-oxide / norbornene cycloaddition click chemistry.

[0243] The term "adduct," e.g., "adduct of group B" or "click chemistry adduct," in the present disclosure, encompasses any moiety that comprises the product of an addition reaction, e.g., a group B addition reaction or a click chemistry addition reaction, regardless of the synthetic steps taken to produce the moiety.

[0244] The term "covalent bond" refers to the formation of a covalent bond, i.e., a chemical bond involving the sharing of one or more electron pairs between two atoms. Covalent bonds can include different interactions, including, but not limited to, σ-bonds, π-bonds, metal-metal bonds, agostic interactions, bent bonds, and three-center, two-electron bonds. When a first group is said to be "capable of covalent bonding" to a second group, this means that the first group can form a covalent bond with the second group, directly or indirectly, for example, through the use of a catalyst or under certain reaction conditions. Non-limiting examples of groups that can be covalently bonded to each other include, for example, amines and carboxylic acids (forming amide bonds), dienes and dienophiles (via Diels-Alder reactions), and azides and alkynes (forming triazoles via 1,3-cycloaddition reactions).

[0245] As described herein, compounds of the present disclosure may contain "optionally substituted" moieties. In general, the term "substituted," whether preceded by the term "optionally," means that one or more hydrogen atoms of the specified moiety are replaced with a suitable substituent. Unless otherwise indicated, an "optionally substituted" group may have a suitable substituent at each substitutable position of the group, and if more than one position in any given structure can be substituted with more than one substituent selected from a specified group, the substituents may be either the same or different at all positions. Combinations of substituents envisioned by the present disclosure preferably result in the formation of stable or chemically feasible compounds. The term "stable," as used herein, refers to compounds that are substantially unchanged when subjected to conditions that allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein.

[0246] Unless otherwise stated, structures depicted herein are also meant to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) forms of the structure, such as the R and S configurations of each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers. Accordingly, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present compounds are within the scope of the disclosure.

[0247] Unless otherwise stated, all tautomeric forms of the compounds of the present disclosure are within the scope of the present disclosure.

[0248] Additionally, unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms, for example, the replacement of a hydrogen by deuterium or tritium, or 11 C- or 13 C- or 14Compounds having the present structures except for the replacement of a carbon with a C-enriched carbon are within the scope of this disclosure.

[0249] It should also be understood that the reference to one or more method steps does not exclude the presence of additional or intervening method steps between those explicitly identified steps. Similarly, it should also be understood that the reference to one or more components in a device or system does not exclude the presence of additional or intervening components between those explicitly identified components.

[0250] Unless otherwise specified, all crystalline forms of the compounds of the present disclosure and their salts are also within the scope of the present disclosure.The compounds of the present disclosure can be isolated in various amorphous and crystalline forms, including but not limited to anhydrous, hydrated, non-solvated, or solvated forms.Exemplary hydrates include hemihydrate, monohydrate, dihydrate, etc.In some embodiments, the compounds of the present disclosure are anhydrous and non-solvated."Anhydrous" means that the crystalline form of the compound is essentially free of bound water in the crystal lattice structure, i.e., the compound does not form crystalline hydrate.

[0251] As used herein, "crystalline form" refers to a specific lattice arrangement of a crystalline substance. Different crystalline forms of the same substance typically have different crystal lattices (e.g., unit cells) due to the different physical properties characteristic of each crystalline form. In some instances, different lattice arrangements have different water or solvent contents. Different crystalline lattices can be identified by solid-state characterization methods such as X-ray powder diffraction (PXRD). Other characterization methods, such as differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), dynamic vapor sorption (DVS), and solid-state NMR, further help identify crystalline forms and determine stability and solvent / water content.

[0252] Crystalline forms of a substance include both solvated (e.g., hydrated) and non-solvated (e.g., anhydrous) forms. Hydrated forms are crystalline forms that contain water in the crystal lattice. Hydrated forms can be stoichiometric hydrates, where water is present in the lattice at a specific water / molecule ratio, such as hemihydrate, monohydrate, or dihydrate. Hydrated forms can also be non-stoichiometric, where the water content is variable and dependent on external conditions such as humidity.

[0253] In some embodiments, the compounds of the present disclosure are substantially isolated. "Substantially isolated" means that a particular compound is at least partially isolated from impurities. For example, in some embodiments, the compounds of the present disclosure contain less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 2.5%, less than about 1%, or less than about 0.5% impurities. Impurities generally include those that are not substantially isolated compounds, including, for example, other crystalline forms and other substances.

[0254] Certain groups, moieties, substituents, and atoms are indicated with a wavy line. The wavy line may cross or cap a junction(s). The wavy line indicates the atom to which the group, moiety, substituent, or atom is attached. For example,

[0255] [ka] The phenyl group substituted with a propyl group, shown as

[0256] [ka] It has.

[0257] The term "HER2" or "human epidermal growth factor receptor 2" refers to a member of the human epidermal growth factor receptor family. This protein is also known as NEU, NGL, HER2, TKR1, CD340, HER-2, MLN19, and HER-2 / neu. HER2 may refer to the amino acid sequence set forth in NCBI accession number NP_004439.2. Amplification or overexpression of this oncogene has been shown to play a key role in the development and progression of certain aggressive types of breast cancer. In recent years, this protein has become an important biomarker and therapeutic target for approximately 30% of breast cancer patients. All references to proteins, polypeptides, and protein fragments herein are intended to refer to the human form of the respective protein, polypeptide, or protein fragment unless specifically identified as being derived from a non-human species. Thus, the term "HER2" refers to human HER2 unless otherwise specified as being derived from a non-human species, e.g., "mouse HER2," "simian HER2," etc.

[0258] The phrase "antibody that binds to HER2" or "anti-HER2 antibody" includes antibodies and antigen-binding fragments thereof that specifically recognize HER2.

[0259] The phrase "anti-HER2 / HER2" antibodies, e.g., "anti-HER2 / HER2 bispecific antibodies," include antibodies and antigen-binding fragments thereof that specifically recognize two different HER2 epitopes. In some embodiments, bispecific antibodies and antigen-binding fragments thereof comprise 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.

[0260] As used herein, the term "STEAP2" refers to the six-transmembrane epithelial antigen of prostate 2. STEAP2 is an endogenous six-transmembrane protein that is highly expressed in prostate epithelial cells and is a cell surface marker for prostate cancer; for example, STEAP2 has been found to be expressed at significant levels in 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 that is encoded by the STEAP2 gene located in human chromosome region 7q21. For example, see the amino acid sequence of human STEAP2 in Tables 5 and 6.

[0261] As used herein, "antibodies that bind to STEAP2" or "anti-STEAP2 antibodies" include antibodies and antigen-binding fragments thereof that specifically recognize STEAP2.

[0262] The phrase "antibody that binds to MET" or "anti-MET antibody" includes antibodies and antigen-binding fragments thereof that specifically recognize MET. As used herein, the terms "MET," "c-Met," and the like refer to the human transmembrane receptor tyrosine kinase.

[0263] The phrase "anti-MET / MET" antibodies, e.g., "anti-MET / MET bispecific antibodies," includes antibodies and antigen-binding fragments thereof that specifically recognize two different MET epitopes. In some embodiments, bispecific antibodies and antigen-binding fragments thereof comprise 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.

[0264] All amino acid abbreviations used in this disclosure are those accepted by the United States Patent and Trademark Office as set forth in 37 C.FR §1.822(B)(J).

[0265] The term "protein" refers to any amino acid polymer having more than about 20 amino acids covalently linked via amide bonds. As used herein, "protein" includes biotherapeutic proteins, recombinant proteins used in research or therapy, trap proteins and other Fc fusion proteins, chimeric proteins, antibodies, monoclonal antibodies, human antibodies, bispecific antibodies, antibody fragments, nanobodies, recombinant antibody chimeras, scFv fusion proteins, cytokines, chemokines, peptide hormones, and the like. Proteins can be produced using recombinant cell-based production systems such as insect baculovirus systems, yeast systems (e.g., Pichia species), and mammalian systems (e.g., CHO cells and CHO derivatives such as CHO-K1 cells).

[0266] The terms "natural amino acid" and "natural amino acid side chain" refer to any naturally occurring amino acid and its side chain, respectively. These include the 20 L-amino acids that occur naturally in the human body.

[0267] The terms "unnatural (also spelled unnatural and unnatural) amino acid" and "unnatural amino acid side chain" refer to amino acids and their side chains, respectively, that do not naturally occur in a target organism, e.g., humans. Such unnatural amino acids can be produced synthetically or can occur naturally in a different environment, e.g., a different organism. Non-limiting examples of unnatural amino acids can include D-amino acids, homo-amino acids, β-homo-amino acids, N-methyl amino acids, α-methyl amino acids, and amino acids present in microbial peptides, such as, for example, citrulline (Cit), hydroxyproline (Hyp), norleucine (Nle), 3-nitrotyrosine, nitroarginine, ornithine (Orn), naphthylalanine (Nal), Abu, DAB, methionine sulfoxide, or methionine sulfone.

[0268] All references herein to proteins, polypeptides, and protein fragments are intended to refer to the human form of the respective protein, polypeptide, or protein fragment unless specifically identified as being from a non-human species. Thus, the term "STEAP2" refers to human STEAP2 unless it is specified as being from a non-human species, e.g., "mouse STEAP2," "monkey STEAP2," etc.

[0269] The amino acid sequence of an antibody 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 Honegge and Pluckthun, J. Mol. Biol., 2001, 309:657-70 ("AHo" numbering scheme). Unless otherwise specified, the numbering scheme used herein is the Kabat numbering scheme. However, the choice of numbering scheme is not intended to imply sequence differences where they do not exist, and one of skill in the art can readily ascertain sequence positions by examining the amino acid sequences of one or more antibodies. Unless otherwise specified, the "EU numbering scheme" is generally used when referring to residues within antibody heavy chain constant regions (e.g., as reported in Kabat et al., supra).

[0270] The term "glutaminyl-modified antibody" refers to an antibody of the present disclosure having at least one covalent bond from a glutamine side chain to a primary amine compound. In certain embodiments, the primary amine compound is linked via an amide bond on the glutamine side chain. In certain embodiments, the glutamine is endogenous glutamine. In other embodiments, the glutamine is endogenous glutamine that has been made reactive by polypeptide engineering (e.g., via amino acid deletion, insertion, substitution, or mutation of the polypeptide). In additional embodiments, the glutamine is a polypeptide engineered with an acyl donor glutamine-containing tag (e.g., a glutamine-containing peptide tag, Q tag, or TGase recognition tag).

[0271] The term "TGase recognition tag" refers to a sequence of amino acids containing an acceptor glutamine residue, which, when incorporated (e.g., added) into a polypeptide sequence under suitable conditions, is recognized by a TGase and crosslinked by the TGase through a reaction between an amino acid side chain within the sequence of amino acids and a reactive partner. The recognition tag can be a peptide sequence that does not naturally occur in a polypeptide that contains 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, where X is any amino acid (e.g., the conventional amino acids Leu, Ala, Gly, Ser, Val, Phe, Tyr, His, Arg, Asn, Glu, Asp, Cys, Gln, Ile, Met, Pro, Thr, Lys, or Trp, or a non-conventional amino acid). In some embodiments, the acyl donor glutamine-containing tag is LLQGG (SEQ ID NO: 1936), LLQG (SEQ ID NO: 1937), LSLSQG (SEQ ID NO: 1938), gGGLLQGG (SEQ ID NO: 1939), gLLQG (SEQ ID NO: 1940), LLQ, gSPLAQSHGG (SEQ ID NO: 1941), gLLQGGG (SEQ ID NO: 1942), gLLQGG (SEQ ID NO: 1943), gLLQ (SEQ ID NO: 1944), LLQ The amino acid sequence may be selected from the group consisting of LLQGA (SEQ ID NO: 1945), LLQGA (SEQ ID NO: 1946), LLQYQGA (SEQ ID NO: 1947), LLQGSG (SEQ ID NO: 1948), LLQYQG (SEQ ID NO: 1949), LLQLLQG (SEQ ID NO: 1950), SLLQG (SEQ ID NO: 1951), LLQLQ (SEQ ID NO: 1952), LLQLLQ (SEQ ID NO: 1953), and LLQGR (SEQ ID NO: 1954). See, e.g., WO2012059882, the entire contents of which are incorporated herein.

[0272] As used herein, the term "antibody" refers to any antigen-binding molecule or molecular complex containing at least one complementarity-determining region (CDR) that specifically binds to or interacts with a particular antigen. The term "antibody" includes immunoglobulin molecules, which include four polypeptide chains, two heavy (H) chains and two light (L) chains, interconnected by disulfide bonds, and multimers thereof (e.g., IgM). Each heavy chain contains a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region contains three domains: CH1, CH2, and CH3. Each light chain contains a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region contains one domain (CL1). The VH and VL regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In different embodiments, the FRs of an antibody (or antigen-binding portion thereof) can be identical to human germline sequences or can be naturally or artificially modified. An amino acid consensus sequence can be defined based on a parallel analysis of two or more CDRs.

[0273] As used herein, the term "antibody" also includes antigen-binding fragments of intact antibody molecules. As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of antibodies can be derived from intact antibody molecules using any suitable standard technique, such as, for example, proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable domains and, optionally, constant domains. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. DNA can be sequenced and manipulated chemically or by using molecular biology techniques, for example, to place one or more variable and / or constant domains in a suitable configuration, or to introduce codons, create cysteine ​​residues, modify, add, or delete amino acids, etc.

[0274] Non-limiting examples of antigen-binding fragments include (i) Fab fragments, (ii) F(ab')2 fragments, (iii) Fd fragments, (iv) Fv fragments, (v) single-chain Fv (scFv) molecules, (vi) dAb fragments, and (vii) minimal recognition units consisting of amino acid residues mimicking a hypervariable region of an antibody (e.g., an isolated complementarity-determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the term "antigen-binding fragment" as used herein.

[0275] Antigen-binding fragments of antibodies typically contain at least one variable domain. The variable domain can be of any size or amino acid composition and generally contains at least one CDR adjacent to or in-frame with one or more framework sequences. In antigen-binding fragments having a VH domain associated with a VL domain, the VH domain and the VL domain can be positioned relative to each other in any suitable configuration. For example, the variable region can be a dimer and contain a VH-VH, VH-VL, or VL-VL dimer. Alternatively, the antigen-binding fragment of an antibody can contain a monomeric VH or VL domain.

[0276] In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found in an antigen-binding fragment of an antibody herein include: (i) VH-CH1, (ii) VH-CH2, (iii) VH-CH3, (iv) VH-CH1-CH2, (V) VH-CH1-CH2-CH3, (vi) VH-CH2-CH3, (vii) VH-CL, (viii) VL-CH1, (ix) VL-CH2, (x) VL-CH3, (xi) VL-CH1-CH2, (xii) VL-CH1-CH2-CH3, (xiii) VL-CH2-CH3, and (xiv) VL-CL. In any configuration of variable and constant domains, including any of the exemplary configurations listed herein, the variable and constant domains may be either directly linked to one another or may be linked by a complete or partial hinge or linker region, which may consist of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that provide a flexible or semi-flexible connection between adjacent variable and / or constant domains in a single polypeptide molecule.

[0277] Furthermore, antigen-binding fragments of the antibodies herein can comprise homodimers or heterodimers (or other multimers) of any of the variable and constant domain configurations enumerated herein in non-covalent association with each other and / or with one or more monomeric VH or VL domains (e.g., via disulfide bond(s)).

[0278] Like intact antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically contain at least two different variable domains, each capable of specifically binding to a separate antigen or a different epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use in the context of antigen-binding fragments of antibodies herein using routine techniques available in the art.

[0279] The antibodies herein may function via complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC). "Complement-dependent cytotoxicity" (CDC) refers to the lysis of antigen-expressing cells by the antibodies herein in the presence of complement. "Antibody-dependent cell-mediated 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 bound antibodies on target cells, thereby resulting in lysis of the target cells. CDC and ADCC are well known in the art and can be measured using available assays. (See, e.g., U.S. Patent Nos. 5,500,362 and 5,821,337, and Clynes et al. (1998) Proc. Natl. Acad. Sci. (USA) 95:652-656.) The constant region of an antibody is important for the antibody's ability to fix complement and mediate cell-dependent cytotoxicity. Thus, the isotype of the antibody can be selected based on whether it is desirable for the antibody to mediate cytotoxicity.

[0280] In certain embodiments, the antibodies described herein, e.g., anti-HER2 antibodies, or anti-HER2 / HER2 bispecific antibodies, or anti-MET antibodies, or anti-MET / MET bispecific antibodies, or anti-STEAP2 antibodies, are human antibodies. As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies herein may include, for example, amino acid residues in the CDRs, particularly CDR3, that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random or site-specific mutagenesis or in vivo somatic mutation). However, the term "human antibody," as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0281] The antibody may, in some embodiments, be a recombinant human antibody. As used herein, the term "recombinant human antibody" is intended to include all human antibodies prepared, expressed, generated, or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described below), antibodies isolated from a recombinant combinatorial human antibody library (described below), antibodies isolated from an animal (e.g., a mouse) transgenic for human immunoglobulin genes (see, e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies prepared, expressed, generated, or isolated by any other means, including splicing of human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when animals transgenic for human Ig sequences are used, in vivo somatic mutagenesis), and therefore the amino acid sequences of the VH and VL regions of the recombinant antibodies, while derived from and related to human germline VH and VL sequences, are sequences that may not naturally occur in the human antibody germline repertoire in vivo.

[0282] Human antibodies can exist in two forms related to hinge heterogeneity. In one form, the immunoglobulin molecule contains a stable four-chain construct of approximately 150–160 kDa, in which dimers are held together by interchain heavy chain disulfide bonds. In the second form, the dimers are not linked by interchain disulfide bonds, forming approximately 75–80 kDa molecules consisting of covalently linked light and heavy chains (half antibodies). These forms are extremely difficult to separate, even after affinity purification. The frequency of the second form in various intact IgG isotypes depends on, but is not limited to, structural differences associated with the antibody hinge region isotype. A single amino acid substitution in the hinge region of a human IgG4 hinge can significantly reduce the occurrence of the second form to levels typically observed using a human IgG1 hinge (Angal et al. (1993) Molecular Immunology 30:105). The present specification encompasses antibodies with one or more mutations in the hinge, CH2 region, or CH3 region, which may be desirable, for example, to improve the yield of a desired antibody form in production.

[0283] An antibody herein may be an isolated or purified antibody. An "isolated antibody" or "purified antibody," as used herein, refers to an antibody that has been identified and separated and / or recovered from at least one component of its natural environment. For example, an antibody that has been separated or removed from at least one component of an organism, or from a tissue or cell in which it naturally occurs or is naturally produced, is an "isolated antibody" for purposes of this specification. For example, an antibody purified from at least one component of a reaction or reaction sequence is a "purified antibody," or is the result of purifying an antibody. An isolated antibody also includes an antibody in situ within a recombinant cell. An isolated antibody is an antibody that has undergone at least one purification or isolation step. According to certain embodiments, an isolated or purified antibody may be substantially free of other cellular material and / or chemicals.

[0284] The antibodies disclosed herein can contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences from which the antibody was derived. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein to germline sequences available, for example, from public antibody sequence databases. The present disclosure includes antibodies and antigen-binding fragments thereof derived from any of the amino acid sequences disclosed herein, in which one or more amino acids in one or more framework and / or CDR regions are mutated to the corresponding residue(s) in the germline sequence from which the antibody was derived, or to the corresponding residue(s) in another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are collectively referred to herein as "germline mutations"). Starting from the heavy and light chain variable region sequences disclosed herein, one skilled in the art can readily produce numerous antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof. In certain embodiments, all framework and / or CDR residues in the VH and / or VL domains are mutated back to the residue found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., a mutated residue is found within the first 8 amino acids of FR1, or a mutated residue is found within the last 8 amino acids of FR4, or a mutated residue is found only in CDR1, CDR2, or CDR3. In other embodiments, one or more of the framework and / or CDR residue(s) is mutated to the corresponding residue(s) in a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antibody was originally derived).

[0285] Furthermore, the antibodies herein can contain any combination of two or more germline mutations within the framework and / or CDR regions, e.g., certain individual residues are mutated to the corresponding residue in a particular germline sequence, while certain other residues that differ from the original germline sequence are maintained or mutated to the corresponding residue in a different germline sequence. Once obtained, antibodies and antigen-binding fragments thereof containing one or more germline mutations can be readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonist or agonist biological properties (as the case may be), reduced immunogenicity, or improved drug-to-antibody ratio (DAR) for antibody-drug conjugates. Antibodies and antigen-binding fragments obtained in this general manner are encompassed herein.

[0286] The term "aglycosylated antibody" refers to an antibody that does not contain a glycosylation sequence that could interfere with transglutamination, e.g., an antibody that does not have a sugar group at N297 on one or more heavy chains. In certain embodiments, the antibody heavy chain has an N297 mutation. In other words, the antibody is mutated so that it no longer has an asparagine residue at position 297 according to the EU numbering system disclosed by Kabat et al. In certain embodiments, the antibody heavy chain has an N297Q or N297D mutation. Such antibodies can be prepared by site-directed mutagenesis to remove or disable glycosylation sequences or by site-directed mutagenesis to insert a glutamine residue at a site away from any interfering glycosylation sites or any other interfering structures. Such antibodies can also be isolated from natural or artificial sources. Aglycosylated antibodies also include antibodies containing T299 or S298P or other mutations, or combinations of mutations that result in the absence of glycosylation.

[0287] The term "deglycosylated antibody" refers to an antibody in which sugar groups have been removed to facilitate transglutaminase-mediated conjugation. Saccharides include, but are not limited to, N-linked oligosaccharides. In some embodiments, deglycosylation is performed at residue N297. In some embodiments, removal of sugar groups is achieved enzymatically, including, but not limited to, via PNGase.

[0288] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule, known as the paratope. A single antigen can have more than one epitope. Thus, different antibodies may bind to different regions on the antigen and have different biological effects. Epitopes can be either conformational or linear. Conformational epitopes are produced by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear epitopes are produced by adjacent amino acid residues in a polypeptide chain. In certain circumstances, epitopes may include carbohydrate, phosphoryl, or sulfonyl moieties on the antigen.

[0289] As used herein, the term "conjugated protein" or "conjugated antibody" refers to a protein or antibody covalently attached to one or more chemical moieties. The chemical moieties may include the amine compounds of the present disclosure. Linkers (LL) and payloads (P) suitable for use in the present disclosure are described in detail herein. In certain embodiments, a conjugated antibody comprising a therapeutic moiety is an antibody-drug conjugate (ADC), also referred to as an antibody-payload conjugate or an antibody-linker-payload conjugate.

[0290] The term "drug-to-antibody ratio" or (DAR) is the average number of therapeutic moieties, e.g., drugs, conjugated to a binding agent of the disclosure.

[0291] The term "linker-antibody ratio" or (LAR), also referred to in some embodiments as a lowercase l, is the average number of reactive primary amine compounds conjugated to a binding agent of the present disclosure. Such binding agents, e.g., antibodies, can be conjugated, for example, with a suitable azide- or alkyne-containing primary amine compound. The resulting binding agent can be functionalized with the azide or alkyne and then reacted with the corresponding azide- or alkyne-containing therapeutic moiety via a 1,3-cycloaddition reaction.

[0292] The phrase "pharmaceutically acceptable amount" refers to an amount effective or sufficient to treat, reduce, alleviate, or modulate the effects or symptoms of at least one health problem in a subject in need thereof. For example, a pharmaceutically acceptable amount of an antibody or antibody-drug conjugate is an amount effective to modulate a biological target using an antibody or antibody-drug conjugate provided herein. Suitable pharmaceutically acceptable amounts include, but are not limited to, about 0.001% up to about 10% of an antibody or antibody-drug conjugate provided herein, and any amount therebetween, 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%.

[0293] The phrase "reaction pH" refers to the pH of the reaction after all reaction components or reactants have been added.

[0294] The terms "substantial identity" or "substantially identical," when referring to a nucleic acid or fragment thereof, indicate that when optimally aligned with another nucleic acid (or its complementary strand) using appropriate nucleotide insertions or deletions, there is at least about 95%, more preferably about 96%, 97%, 98%, or 99% nucleotide sequence identity of the nucleotide bases as measured by any well-known algorithm for sequence identity, such as FASTA, BLAST, or gap, as discussed below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule can, in certain cases, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0295] When applied to polypeptides, the term "substantial similarity" or "substantially similar" means that two peptide sequences share at least 95% sequence identity, and even more preferably at least 98% or 99% sequence identity, when optimally aligned, such as by the programs gAP or BESTFIT, using predetermined gap weights. Preferably, non-identical residue positions differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functional properties of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upward to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24:307-331. Examples of groups of amino acids having side chains with similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine, (2) aliphatic-hydroxyl side chains: serine and threonine, (3) amide-containing side chains: asparagine and glutamine, (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan, (5) basic side chains: lysine, arginine, and histidine, (6) acidic side chains: aspartic acid and glutamic acid, and (7) sulfur-containing side chains: cysteine ​​and methionine. In some embodiments, conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine.

[0296] Alternatively, a conservative replacement is any change that has a positive value in the PAM250 log-likelihood matrix as disclosed in Gonnet et al. (1992) Science 256:1443-1445, incorporated herein by reference. A "moderately conservative" replacement is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0297] Sequence similarity for polypeptides, also referred to as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using similarity measures assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, gCG software includes programs such as gap and Bestfit, which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms, or between a wild-type protein and its mutant protein. See, for example, gCG version 6.1. Polypeptide sequences can also be compared using FASTA, a program in gCG version 6.1, using default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of best overlap between the query and search sequences (Pearson (2000) supra). Another specific algorithm for comparing the sequences herein to a database containing a large number of sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402.

[0298] Protein-drug conjugate compounds In accordance with the above and other objectives, the present disclosure provides protein-drug conjugate compounds, e.g., antibody-drug conjugate compounds, as well as precursors and intermediates thereof, pharmaceutical compositions, and methods for treating specific diseases in subjects in need of such treatment. According to the present disclosure, the protein-drug conjugate compounds provided herein comprise a glutaminyl-modified binding agent conjugated to a therapeutic moiety, e.g., a primary amine compound linked to a camptothecin analog moiety, as described herein. Also provided is a specific and efficient method for producing protein-drug conjugates, e.g., antibody-drug conjugates, utilizing a combination of transglutaminase and 1,3-cycloaddition technology. According to the present disclosure, the protein-drug conjugate compounds provided herein comprise a prodrug of a topoisomerase I inhibitor, e.g., a prodrug of Dxd.

[0299] In one aspect, the present disclosure provides a compound having formula (I):

[0300] [ka] or a pharmaceutically acceptable salt thereof, In the formula, R 1 , R 2 , R 3 , and R 4 are independently hydrogen, C 1-5 alkyl or aryl, AA is a natural or unnatural amino acid; p is an integer from 1 to 6,

[0301] [ka] provides an antibody or antigen-binding fragment thereof, wherein:

[0302] In one embodiment, the compound of formula (I) is directly conjugated to an antibody or antigen-binding fragment thereof.

[0303] In another embodiment, the compound of formula (I) is conjugated to the antibody or antigen-binding fragment thereof via a bivalent linker.

[0304] In one embodiment, p is 1. In another embodiment, p is 2, i.e., [AA]2 is a peptide dimer of two amino acids. In another embodiment, p is 3. In another embodiment, p is 4. In another embodiment, p is 5. In another embodiment, p is 6. In any embodiment where p is greater than 1, the amino acids can be the same or different from one another. In one embodiment, p is 2 and the two amino acids are different from one another.

[0305] In one embodiment, p is 1 and the amino acid is a natural amino acid. In one embodiment, p is 1 and the natural amino acid is selected from glycine, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, threonine, lysine, asparagine, glutamine, aspartic acid, and glutamic acid. In one embodiment, p is 1 and the natural amino acid is selected from glycine, phenylalanine, threonine, lysine, glutamine, and glutamic acid.

[0306] In one embodiment, p is 1 and the amino acid is an unnatural amino acid. In one embodiment, p is 1 and the unnatural amino acid is an R-amino acid, an N-methyl amino acid,

[0307] [ka] is selected from the group consisting of:

[0308] In another embodiment, p is 2, i.e., [AA]2 is a peptide dimer of two amino acids. In one embodiment, p is 2 and both amino acids are glycine.

[0309] In one embodiment, R 1 is H.

[0310] In one embodiment, R 2 is H. In one embodiment, R 3 is H. In one embodiment, R 2 and R 3 are both H.

[0311] In one embodiment, R 4 is H. In another embodiment, R 4 is C 1-5 In one particular embodiment, R 4 is a C1 alkyl (methyl).

[0312] In one embodiment, the compound of formula (I) is referred to as a payload.

[0313] In one embodiment, the compound of formula (I) is

[0314] [ka] and is conjugated to the antibody or antigen-binding fragment via the amino group.

[0315] In one embodiment, the antibody or antigen-binding fragment thereof is a compound having a structure according to formula (II):

[0316] [ka] or a pharmaceutically acceptable salt thereof, wherein R 1 , R 2 , R 3 , and R 4 are independently hydrogen or C 1-5 is alkyl, A is a click chemistry adduct; W is NH, O, CO, CH, phenyl, or a combination of two or more thereof; AA is a natural or unnatural amino acid; m is an integer from 0 to 8, n is 0 or 1, p is an integer from 1 to 6,

[0317] [ka] indicates the point of attachment to the antibody or antigen-binding fragment thereof, either directly or via a linker.

[0318] In one embodiment, the click chemistry adduct is (a) Strain-promoted azide / dibenzocyclooctyne-amine (DBCO) click chemistry; (b) inverse electron demand Diels-Alder (IED-DA) tetrazine / trans -cyclooctene (TCO) click chemistry; (c) Inverse electron demand Diels-Alder (IED-DA) tetrazine / norbornene click chemistry, (d) Diels-Alder maleimide / furan click chemistry, (e) Staudinger ligation, and (f) The product of a copper-free click chemistry reaction selected from nitrile-oxide / norbornene cycloaddition click chemistry.

[0319] In one non-limiting embodiment, the click chemistry adduct is the product of a strain-promoted azide / dibenzocyclooctyne-amine (DBCO) click chemistry reaction. In another embodiment, the click chemistry adduct is the product of an inverse electron demand Diels-Alder (IED-DA) tetrazine / trans-cyclooctene (TCO) click chemistry reaction.

[0320] In one embodiment, the click chemistry adduct comprises a triazole. In another embodiment, the click chemistry adduct comprises a diazine.

[0321] In one embodiment, the click chemistry adduct is

[0322] [ka] and any positional isomer or entantiomer thereof, wherein R' is H or C 1-3 alkyl and Z is C or N.

[0323] In one embodiment, the click chemistry adduct is

[0324] [ka] is.

[0325] In one embodiment, R 1 is H.

[0326] In one embodiment, R 2 is H. In one embodiment, R 3 is H. In one embodiment, R 2 and R 3 are both H.

[0327] In one embodiment, R 4 is H. In another embodiment, R 4 is C 1-5 In one particular embodiment, R 4 is a C1 alkyl (methyl).

[0328] In one embodiment, W is O. In one embodiment, W is NH. In one embodiment, W is CO. In one embodiment, W is CH. In one embodiment, W is phenyl. In one embodiment, W is OCH. In one embodiment, W is -OCH-CO-NH-. In one embodiment, W is -O-CO-NH-. In one embodiment, W is

[0329] [ka] is.

[0330] In one embodiment, m is 0. In another embodiment, m is 1. In another embodiment, m is 2. In another embodiment, m is 3. In another embodiment, m is 4. In another embodiment, m is 5. In another embodiment, m is 6. In another embodiment, m is 7. In another embodiment, m is 8.

[0331] In one particular embodiment, m is 4.

[0332] In one embodiment, n is 0. In another embodiment, n is 1.

[0333] In one embodiment, p is 1. In another embodiment, p is 2, i.e., [AA]2 is a peptide dimer of two amino acids. In another embodiment, p is 3. In another embodiment, p is 4. In another embodiment, p is 5. In another embodiment, p is 6. In any embodiment where p is greater than 1, the amino acids can be the same or different from one another. In one embodiment, p is 2 and the two amino acids are different from one another.

[0334] In one embodiment, p is 1 and the amino acid is a natural amino acid. In one embodiment, p is 1 and the natural amino acid is selected from glycine, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, threonine, lysine, asparagine, glutamine, aspartic acid, and glutamic acid. In one embodiment, p is 1 and the natural amino acid is selected from glycine, phenylalanine, threonine, lysine, glutamine, and glutamic acid.

[0335] In one embodiment, p is 1 and the amino acid is an unnatural amino acid. In one embodiment, p is 1 and the unnatural amino acid is an R-amino acid, an N-methyl amino acid,

[0336] [ka] is selected from the group consisting of:

[0337] In another embodiment, p is 2, i.e., [AA]2 is a peptide dimer of two amino acids. In one embodiment, p is 2 and both amino acids are glycine.

[0338] In one embodiment, the compound of formula (II) is

[0339] [ka]

[0340] [ka] The present invention includes compounds having a structure selected from the group consisting of:

[0341] In one embodiment, the compound of formula (II) is

[0342] [ka] Includes:

[0343] In one aspect, provided herein is an antibody-drug conjugate having a structure according to formula (III):

[0344] [ka] or a pharmaceutically acceptable salt thereof, wherein: Ab is an antibody or antigen-binding fragment thereof; R 1 , R 2 , R 3 , and R 4 are independently hydrogen or C 1-5 is alkyl, A is a click chemistry adduct; W is NH, O, CO, CH, phenyl, or a combination of two or more thereof; LL is a linker or bond connecting said Ab and said A; AA is a natural or unnatural amino acid; m is an integer from 0 to 8, n is 0 or 1, p is an integer from 1 to 6, and q is an integer of 1 to 10, or a pharmaceutically acceptable salt thereof.

[0345] In one embodiment, the click chemistry adduct is (a) Strain-promoted azide / dibenzocyclooctyne-amine (DBCO) click chemistry; (b) inverse electron demand Diels-Alder (IED-DA) tetrazine / trans -cyclooctene (TCO) click chemistry; (c) Inverse electron demand Diels-Alder (IED-DA) tetrazine / norbornene click chemistry, (d) Diels-Alder maleimide / furan click chemistry, (e) Staudinger ligation, and (f) The product of a copper-free click chemistry reaction selected from nitrile-oxide / norbornene cycloaddition click chemistry.

[0346] In one non-limiting embodiment, the click chemistry adduct is the product of a strain-promoted azide / dibenzocyclooctyne-amine (DBCO) click chemistry reaction. In another embodiment, the click chemistry adduct is the product of an inverse electron demand Diels-Alder (IED-DA) tetrazine / trans-cyclooctene (TCO) click chemistry reaction.

[0347] In one embodiment, the click chemistry adduct comprises a triazole. In another embodiment, the click chemistry adduct comprises a diazine.

[0348] In one embodiment, the click chemistry adduct is

[0349] [ka] wherein R' is selected from the group consisting of H or C 1-3 alkyl and Z is C or N.

[0350] In one embodiment, the click chemistry adduct is

[0351] [ka] is.

[0352] In one embodiment, R 1 is H.

[0353] In one embodiment, R 2 is H. In one embodiment, R 3 is H. In one embodiment, R 2 and R 3 are both H.

[0354] In one embodiment, R 4 is H. In another embodiment, R 4 is C 1-5 In one particular embodiment, R 4 is a C1 alkyl (methyl).

[0355] In one embodiment, m is 0. In another embodiment, m is 1. In another embodiment, m is 2. In another embodiment, m is 3. In another embodiment, m is 4. In another embodiment, m is 5. In another embodiment, m is 6. In another embodiment, m is 7. In another embodiment, m is 8.

[0356] In one particular embodiment, m is 4.

[0357] In one embodiment, n is 0. In another embodiment, n is 1.

[0358] In one embodiment, p is 1. In another embodiment, p is 2, i.e., [AA]2 is a peptide dimer of two amino acids. In another embodiment, p is 3. In another embodiment, p is 4. In another embodiment, p is 5. In another embodiment, p is 6. In any embodiment where p is greater than 1, the amino acids can be the same or different from one another. In one embodiment, p is 2 and the two amino acids are different from one another.

[0359] In one embodiment, p is 1 and the amino acid is a natural amino acid. In one embodiment, p is 1 and the natural amino acid is selected from glycine, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, threonine, lysine, asparagine, glutamine, aspartic acid, and glutamic acid. In one embodiment, p is 1 and the natural amino acid is selected from glycine, phenylalanine, threonine, lysine, glutamine, and glutamic acid.

[0360] In one embodiment, p is 1 and the amino acid is an unnatural amino acid. In one embodiment, p is 1 and the unnatural amino acid is an R-amino acid, an N-methyl amino acid,

[0361] [ka] is selected from the group consisting of:

[0362] In another embodiment, p is 2, i.e., [AA]2 is a peptide dimer of two amino acids. In one embodiment, p is 2 and both amino acids are glycine.

[0363] In one embodiment, LL is

[0364] [Table 3-1]

[0365] [Table 3-2]

[0366] [Table 3-3]

[0367] [Table 3-4] where (B') is the point of attachment to the click chemistry adduct A and n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0368] In one embodiment, LL is

[0369] [Table 4-1]

[0370] [Table 4-2] wherein n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0371] In one embodiment, LL is

[0372] [Table 5] wherein n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0373] In some embodiments, the linker comprises a self-immolative group. The self-immolative group, self-immolative linker, or self-immolative spacer can be any such group known to those skilled in the art. The self-immolative linker plays an important role in the cascade mechanism of release of the attached compound. In the case of a drug delivery system, it is defined as a covalent linking group that, upon stimulation, cleaves two bonds between the protector group and the drug. The stimulation can include, among others, an enzyme trigger, a chemical trigger such as pH, a redox system, 1,4-, 1,6-, 1,8-elimination, a photolytic trigger, or multiple triggers. The cascade reaction of the self-immolative structural construct allows for controlled release of the drug. In an exemplary embodiment, the self-immolative group is p-aminobenzyl (PAB) or a derivative thereof. Useful derivatives include p-aminobenzyloxycarbonyl (PABC). Those skilled in the art will recognize that the self-immolative group can perform a chemical reaction that releases the remaining atoms of the linker from the payload.

[0374] In one embodiment, q is 1. In another embodiment, q is 2. In another embodiment, q is 3. In another embodiment, q is 4. In another embodiment, q is 5. In another embodiment, q is 6. In another embodiment, q is 7. In another embodiment, q is 8. In another embodiment, q is 9. In another embodiment, q is 10.

[0375] In one embodiment, an antibody-drug conjugate having the structure:

[0376] [ka] or a pharmaceutically acceptable salt thereof, wherein: Ab is an antibody or antigen-binding fragment thereof; R is the side chain of any natural or unnatural amino acid; Presented herein is an antibody-drug conjugate, or a pharmaceutically acceptable salt thereof, wherein n is an integer from 1 to 5.

[0377] In another embodiment, an antibody-drug conjugate having the structure:

[0378] [ka]

[0379] [ka] or a pharmaceutically acceptable salt thereof, wherein: Ab is an antibody or antigen-binding fragment thereof; Presented herein is an antibody-drug conjugate, or a pharmaceutically acceptable salt thereof, wherein n is an integer from 1 to 5.

[0380] In one embodiment, an antibody-drug conjugate having the structure:

[0381] [ka]

[0382] [ka]

[0383] [ka] or a pharmaceutically acceptable salt thereof, wherein: Ab is an antibody or antigen-binding fragment thereof; Presented herein is an antibody-drug conjugate, or a pharmaceutically acceptable salt thereof, wherein n is an integer from 1 to 5.

[0384] In one aspect, provided herein is an antibody-drug conjugate having a structure according to formula (IVa or IVb):

[0385] [ka] or a pharmaceutically acceptable salt thereof, wherein: Ab is an antibody or antigen-binding fragment thereof; R is the side chain of any natural or unnatural amino acid; Presented herein is an antibody-drug conjugate, or a pharmaceutically acceptable salt thereof, wherein n is an integer from 1 to 5.

[0386] In another aspect, the disclosure provides an antibody-drug conjugate having a structure according to formula (IVc, IVd, IVe, IVf, IVg, IVh, IVi, IVj, or IVk):

[0387] [ka]

[0388] [ka] (SEQ ID NOs: 2115 and 2115, respectively),

[0389] [ka] (SEQ ID NOs: 2116 and 2116, respectively), or a pharmaceutically acceptable salt thereof, wherein: Ab is an antibody or antigen-binding fragment thereof; R is the side chain of any natural or unnatural amino acid; The present invention provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein n is an integer of 1 to 5.

[0390] In one embodiment, R is hydrogen.

[0391] In one embodiment, R is a side chain of a naturally occurring amino acid. In one embodiment, R comprises the side chain of a naturally occurring amino acid selected from glycine, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, threonine, lysine, asparagine, glutamine, aspartic acid, and glutamic acid. In one embodiment, R is a side chain of a naturally occurring amino acid selected from glycine, phenylalanine, threonine, lysine, glutamine, and glutamic acid.

[0392] In one embodiment, R is the side chain of an unnatural amino acid. In one embodiment, R is an R-amino acid, an N-methyl amino acid,

[0393] [ka] is the side chain of an unnatural amino acid selected from the group consisting of:

[0394] In one embodiment of any of the above, the antibody or antigen-binding fragment thereof comprises Gln295 and / or Gln297 (i.e., glutamine residues at positions 295 and / or 297), and a payload (e.g., a prodrug of DXd) is conjugated to the antibody or antigen-binding fragment via the side chains of Gln295 and / or Gln297, either directly or via a linker.

[0395] payload In certain embodiments, the payload of the present disclosure is a prodrug of a topoisomerase I inhibitor. In certain embodiments, the payload of the present disclosure is a camptothecin analogue and / or derivative.

[0396] [ka] Camptothecin (CPT), shown above, is a topoisomerase toxin. It was discovered in 1966 by M.E. Wall and M.C. Wani during a systematic screening of natural products for anticancer agents. It was isolated from the bark and stems of Camptotheca acuminata (Camptotheca, Happy tree), a tree native to China used as a cancer treatment in traditional Chinese medicine. Camptothecin demonstrated significant anticancer activity in preliminary clinical trials. However, due to its low solubility, synthetic and medicinal chemists have developed numerous syntheses of camptothecin and various derivatives with good results. Four camptothecin analogs (topotecan, irinotecan, belotecan, and deruxtecan (Dxd)) have been approved and are currently used in cancer chemotherapy.

[0397] Trastuzumab deruxtecan (T-Dxd) is an antibody-drug conjugate containing the human epidermal growth factor receptor 2 (HER2)-directed antibody trastuzumab and the topoisomerase I inhibitor conjugate deruxtecan (Dxd, a derivative of exatecan). It was approved for use in the United States in December 2019. Exatecan, shown below, is a camptothecin analog.

[0398] [ka]

[0399] In one embodiment, the payload of the present disclosure is a prodrug of deruxtecan (Dxd).

[0400] In certain embodiments, the payload of the present disclosure is a compound having the structure PI,

[0401] [ka] In the formula, R 1 , R 2 , R 3 , and R4 are independently hydrogen or C 1-5 is alkyl, AA is a natural or unnatural amino acid; p is an integer of 1 to 6, or a pharmaceutically acceptable salt thereof.

[0402] In one embodiment, p is 1. In another embodiment, p is 2, i.e., [AA]2 is a peptide dimer of two amino acids. In another embodiment, p is 3. In another embodiment, p is 4. In another embodiment, p is 5. In another embodiment, p is 6. In any embodiment where p is greater than 1, the amino acids can be the same or different from one another. In one embodiment, p is 2 and the two amino acids are different from one another.

[0403] In one embodiment, p is 1 and the amino acid is a natural amino acid. In one embodiment, p is 1 and the natural amino acid is selected from glycine, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, threonine, lysine, asparagine, glutamine, aspartic acid, and glutamic acid. In one embodiment, p is 1 and the natural amino acid is selected from glycine, phenylalanine, threonine, lysine, glutamine, and glutamic acid.

[0404] In one embodiment, p is 1 and the amino acid is an unnatural amino acid. In one embodiment, p is 1 and the unnatural amino acid is an R-amino acid, an N-methyl amino acid,

[0405] [ka] is selected from the group consisting of:

[0406] In another embodiment, p is 2, i.e., [AA]2 is a peptide dimer of two amino acids. In one embodiment, p is 2 and both amino acids are glycine.

[0407] In one embodiment, R 1 is H.

[0408] In one embodiment, R 2 is H. In one embodiment, R 3 is H. In one embodiment, R 2 and R 3 are both H.

[0409] In one embodiment, R 4 is H. In another embodiment, R 4 is C 1-5 In one particular embodiment, R 4 is a C1 alkyl (methyl).

[0410] In one embodiment, the compound of formula (I) is selected from the group consisting of the compounds in Table 1.

[0411] [Table 6-1]

[0412] [Table 6-2]

[0413] Certain characteristics of payloads according to the present disclosure are summarized in Table 2 below.

[0414] [Table 7]

[0415] The present disclosure also relates to pharmaceutical compositions comprising a therapeutically effective amount of a payload as described above, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0416] The present disclosure also provides linker-payload compounds having formulas (D')-(G'):

[0417] [ka]

[0418] [ka]

[0419] [ka] or a pharmaceutically acceptable salt thereof, comprising: In the formula, R 1 , R 2 , R 3 , and R 4 are independently hydrogen or C 1-5 is alkyl, B,

[0420] [ka] is selected from the group consisting of W is NH, O, CO, CH, phenyl, or a combination of two or more thereof; R 5 , R 6 , R 7 , and R 8 are independently hydrogen, —NH, or the side chain of any natural or unnatural amino acid; The method comprises exposing a payload having an amino group to an activated intermediate having para-nitro-phenyl carbonate in the presence of a base and a coupling catalyst to obtain the linker-payload compounds (D')-(G'), wherein the coupling catalyst is 4-hydroxy-2-methylquinoline (MeHYQ).

[0421] The present disclosure also provides a linker-payload compound having the formula (D-1):

[0422] [ka] A process for producing (D-1), or a pharmaceutically acceptable salt thereof, comprising: In the formula, R 1 , R 2 , R 3 , and R 4 are independently hydrogen or C 1-5 is alkyl, R 5 and R 6 are independently hydrogen, —NH, or the side chain of any natural or unnatural amino acid; The method comprises exposing a payload having an amino group to an activated intermediate having para-nitro-phenyl carbonate in the presence of a base and a coupling catalyst to obtain the linker-payload compound (D-1), wherein the coupling catalyst is 4-hydroxy-2-methylquinoline (MeHYQ).

[0423] In one embodiment, the payload having an amino group is a structure according to the formula PI:

[0424] [ka] In the formula, R 1 , R 2 , R 3 , and R 4 are independently hydrogen or C 1-5 is alkyl, AA is a natural or unnatural amino acid; and p is an integer from 1 to 6, or a pharmaceutically acceptable salt thereof.

[0425] In one embodiment, the amino group of the payload is the amino terminus of AA.

[0426] In one embodiment, the activated intermediate having para-nitro-phenyl carbonate has a structure according to Formula II.

[0427] [ka]

[0428] The present disclosure also provides a linker-payload compound having the formula (D-1):

[0429] [ka] or a pharmaceutically acceptable salt thereof, comprising: In the formula, R 1 , R 2 , R 3 , and R 4 are independently hydrogen or C 1-5 is alkyl, R 5 and R 6 are independently hydrogen, —NH, or the side chain of any natural or unnatural amino acid; The process in question is: (a) A compound of formula (I-1) having the structure:

[0430] [ka] During the ceremony, X is,

[0431] [ka] providing a compound selected from the group consisting of: (b) reacting a compound of formula (I-1) with a compound of formula (PI),

[0432] [ka] During the ceremony, R is H or PG; by reacting with a compound of formula (PI) where PG is a suitable protecting group, and producing a compound of formula (D-1).

[0433] In one embodiment, the compound of formula (D-1) has the following structure:

[0434] [ka]

[0435] In one embodiment, step (b) of reacting a compound of Formula (I-1) with a compound of Formula (PI), wherein R is PG, further comprises reacting the compound of Formula (PI), wherein R is PG, with a deprotecting agent prior to said reacting with the compound of Formula (I-1).

[0436] In one embodiment, PG is selected from the group consisting of allyloxycarbonyl (Alloc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), and 9-fluorenylmethoxycarbonyl (Fmoc).

[0437] In one embodiment, the deprotecting agent is selected from the group consisting of Pd(PPh) 3 , PhSiH 3 , H 2 , piperidine, and trifluoroacetic acid (TFA).

[0438] In one embodiment, the compound of formula (I-1) has the following structure:

[0439] [ka]

[0440] In one embodiment, the compound of formula (PI) has the structure:

[0441] [ka]

[0442] In one embodiment, the process for producing a linker-payload compound having formula (D-1) comprises reacting a compound of formula (V) having the following structure:

[0443] [ka] and, prior to step (a), forming a compound of formula (I-1) from the compound of formula (V).

[0444] In one embodiment, the step of forming a compound of formula (I-1) comprises reacting a compound of formula (V) with a compound of formula (VIa) or formula (VIb),

[0445] [ka] wherein X' is a halogen, to produce a compound of formula (I-1).

[0446] In one embodiment, the compound of formula (VIa) is

[0447] [ka] is selected from the group consisting of:

[0448] In one embodiment, the compound of formula (VIb) is

[0449] [ka] is.

[0450] In one embodiment, the process comprises providing a compound of formula (VII) having the structure:

[0451] [ka] In the ceremony, P.G. 1 is a suitable protecting group; and forming a compound of formula (V) from the compound of formula (VII).

[0452] In one embodiment, PG 1 is selected from the group consisting of allyloxycarbonyl (Alloc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), and 9-fluorenylmethoxycarbonyl (Fmoc).

[0453] In one embodiment, the compound of formula (VII) has the structure:

[0454] [ka]

[0455] In one embodiment, the step of forming a compound of formula (V) comprises reacting a compound of formula (VII) with a compound of formula (VIII):

[0456] [ka] to produce a compound of formula (V).

[0457] In one embodiment, the process comprises reacting a compound of formula (IX) having the following structure:

[0458] [ka] and forming a compound of formula (VII) from the compound of formula (IX).

[0459] In one embodiment, the compound of formula (IX) has the structure:

[0460] [ka]

[0461] In one embodiment, the step of forming a compound of formula (VII) comprises reacting a compound of formula (IX) with a compound of formula (X):

[0462] [ka] to form a compound of formula (VII).

[0463] In one embodiment, the process comprises reacting a compound of formula (XI) having the following structure:

[0464] [ka] and forming a compound of formula (IX) from the compound of formula (XI).

[0465] In one embodiment, the compound of formula (XI) has the structure:

[0466] [ka]

[0467] In one embodiment, the step of forming a compound of formula (IX) comprises reacting a compound of formula (XI) with a compound of formula (XII):

[0468] [ka] to produce a compound of formula (IX).

[0469] In one embodiment, the process comprises reacting a compound of formula (XIII) having the following structure:

[0470] [ka] and and forming a compound of formula (VIII) from the compound of formula (XIII).

[0471] In one embodiment, the step of forming a compound of formula (VIII) comprises reacting a compound of formula (XIII) with a compound of formula (XII):

[0472] [ka] to form a compound of formula (VIII).

[0473] In one embodiment, the process comprises providing a compound of formula (XIV) having the structure:

[0474] [ka] In the formula, R a is a halogen, R b But C 1-6 providing a compound of formula (XIV), which is alkyl; and forming a compound of formula (XIII) from the compound of formula (XIV).

[0475] In one embodiment, R a is bromine.

[0476] In one embodiment, the compound of formula (XIV) has the structure:

[0477] [ka]

[0478] In one embodiment, the step of forming the compound of formula (XIII) comprises reacting a compound of formula (XIV) with a base to produce a compound of formula (XIII).

[0479] In one embodiment, the base is selected from the group consisting of sodium methoxide (NaOMe), potassium tert-butoxide (t-BuOK), sodium hydride (NaH), and lithium diisopropylamide (LDA).

[0480] In one embodiment, the reaction between the compound of formula (XIV) and a base is carried out in a suitable solvent such as methanol (MeOH), tetrahydrofuran (THF), dimethylformamide (DMF), or a mixture thereof.

[0481] In one embodiment, the process comprises the steps of providing a compound of formula (XV) having the structure:

[0482] [ka] forming a compound of formula (XIV) from the compound of formula (XV).

[0483] In one embodiment, the compound of formula (XV) has the structure:

[0484] [ka]

[0485] In one embodiment, the step of forming a compound of formula (XIV) comprises reacting a compound of formula (XV) with a compound of formula (XVI):

[0486] [ka] to produce a compound of formula (XIV).

[0487] In one embodiment, the compound of formula (XV) is reacted with methyl glycolate in the presence of AgOTf to produce the compound of formula (XIV).

[0488] In one embodiment, the process comprises reacting a compound of formula (XVII) having the structure:

[0489] [ka] and forming a compound of formula (XV) from the compound of formula (XVII).

[0490] In one embodiment, the step of forming the compound of formula (XV) comprises reacting a compound of formula (XVII) with a brominating agent to produce a compound of formula (XVII).

[0491] In one embodiment, the brominating agent is CHBr3.

[0492] In one embodiment, the compound of formula (XVII) is reacted with CHBr3 in a non-polar solvent in the presence of a base such as potassium tert-butoxide (t-BuOK).

[0493] In one embodiment, the process comprises reacting a compound of formula (XVIII) having the following structure:

[0494] [ka] and forming a compound of formula (PI) from the compound of formula (XVIII).

[0495] In one embodiment, the compound of formula (XVIII) has the structure:

[0496] [ka]

[0497] In one embodiment, the step of forming a compound of formula (PI) comprises reacting a compound of formula (XVIII) with a compound of formula (XIX):

[0498] [ka] to produce a compound of formula (PI).

[0499] In one embodiment, the process comprises reacting a compound of formula (XX) having the following structure:

[0500] [ka] and forming a compound of formula (XVIII) from the compound of formula (XX).

[0501] In one embodiment, the compound of formula (XX) has the structure:

[0502] [ka]

[0503] In one embodiment, the step of forming a compound of formula (XVIII) comprises reacting a compound of formula (XX) with a compound of formula (XXI):

[0504] [ka] to produce a compound of formula (XVIII).

[0505] In one embodiment, the process comprises reacting a compound of formula (XXII) having the structure:

[0506] [ka] and forming a compound of formula (XX) from the compound of formula (XXII).

[0507] In one embodiment, the compound of formula (XXII) has the structure:

[0508] [ka]

[0509] The present disclosure also provides a compound of formula (I-1):

[0510] [ka] or a pharmaceutically acceptable salt thereof, comprising: During the ceremony, X is,

[0511] [ka] For the process, selected from the group consisting of The process is (a) providing a compound of formula (V) having the structure:

[0512] [ka] (b) forming a compound of formula (I-1) from a compound of formula (V).

[0513] In one embodiment, the compound of formula (I-1) has the following structure:

[0514] [ka]

[0515] In one embodiment, step (b) of forming a compound of Formula (I-1) comprises reacting a compound of Formula (V) with a compound of Formula (VIa) or Formula (VIb),

[0516] [ka] with a compound of formula (VIa) or (VIb), wherein X' is a halogen, The method includes producing a compound of formula (I-1).

[0517] In one embodiment, the compound of formula (VIa) is

[0518] [ka] is selected from the group consisting of:

[0519] In one embodiment, the compound of formula (VIb) is

[0520] [ka] is.

[0521] In one embodiment, the process comprises providing a compound of formula (VII) having the structure:

[0522] [ka] In the ceremony, P.G. 1 is a suitable protecting group; forming a compound of formula (V) from the compound of formula (VII).

[0523] In one embodiment, the compound of formula (VII) has the structure:

[0524] [ka]

[0525] In one embodiment, the step of forming a compound of formula (V) comprises reacting a compound of formula (VII) with a compound of formula (VIII):

[0526] [ka] to produce a compound of formula (V).

[0527] In one embodiment, the process comprises reacting a compound of formula (IX) having the following structure:

[0528] [ka] and forming a compound of formula (VII) from the compound of formula (IX).

[0529] In one embodiment, the compound of formula (IX) has the structure:

[0530] [ka]

[0531] In one embodiment, the step of forming a compound of formula (VII) comprises reacting a compound of formula (IX) with a compound of formula (X):

[0532] [ka] to form a compound of formula (VII).

[0533] In one embodiment, the process comprises reacting a compound of formula (XI) having the following structure:

[0534] [ka] and forming a compound of formula (IX) from the compound of formula (XI).

[0535] In one embodiment, the compound of formula (XI) has the structure:

[0536] [ka]

[0537] In one embodiment, the step of forming a compound of formula (IX) comprises reacting a compound of formula (XI) with a compound of formula (XII):

[0538] [ka] to produce a compound of formula (IX).

[0539] The present disclosure also provides a compound of formula (XVIII):

[0540] [ka] or a pharmaceutically acceptable salt thereof, comprising: In the formula, R 1 , R 2 , R 3 , and R 4 are independently hydrogen or C 1-5 is alkyl, R 5 and R 6 are independently hydrogen, -NH, or the side chain of any natural or unnatural amino acid. (a) providing a compound of formula (XX) having the structure:

[0541] [ka] (b) forming a compound of formula (XVIII) from a compound of formula (XX).

[0542] In one embodiment, the compound of formula (XVIII) has the structure:

[0543] [ka]

[0544] In one embodiment, the compound of formula (XX) has the structure:

[0545] [ka]

[0546] In one embodiment, the step of forming a compound of formula (XVIII) comprises reacting a compound of formula (XX) with a compound of formula (XXI):

[0547] [ka] to produce a compound of formula (XVIII).

[0548] In one embodiment, the process comprises reacting a compound of formula (XXII) having the structure:

[0549] [ka] and forming a compound of formula (XX) from the compound of formula (XXII).

[0550] In one embodiment, the compound of formula (XXII) has the structure:

[0551] [ka]

[0552] The present disclosure also provides a compound of formula (D-1):

[0553] [ka] or a pharmaceutically acceptable salt thereof, comprising: In the formula, R 1 , R 2 , R 3 , and R 4 are independently hydrogen or C 1-5 is alkyl, R 5 and R 6 are independently hydrogen, -NH, or the side chain of any natural or unnatural amino acid. (a) A compound of formula (I-1) having the structure:

[0554] [ka] During the ceremony, X is,

[0555] [ka] providing a compound selected from the group consisting of: (b) reacting a compound of formula (I-1) with a compound of formula (PI),

[0556] [ka] During the ceremony, R is H or PG; by reacting with a compound of formula (PI) where PG is a suitable protecting group, and producing a compound of formula (D-1).

[0557] In one embodiment, the compound of formula (D-1) has the following structure:

[0558] [ka]

[0559] In one embodiment, the compound of formula (I-1) has the following structure:

[0560] [ka]

[0561] In one embodiment, step (b) of reacting a compound of Formula (I-1) with a compound of Formula (PI), wherein R is PG, further comprises reacting the compound of Formula (PI), wherein R is PG, with a deprotecting agent prior to said reacting with the compound of Formula (I-1).

[0562] In one embodiment, PG is selected from the group consisting of allyloxycarbonyl (Alloc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), and 9-fluorenylmethoxycarbonyl (Fmoc).

[0563] In one embodiment, the deprotecting agent is selected from the group consisting of Pd(PPh) 3 , PhSiH 3 , H 2 , piperidine, and trifluoroacetic acid (TFA).

[0564] In one embodiment, the compound of formula (PI) has the structure:

[0565] [ka]

[0566] In one embodiment, the process comprises reacting a compound of formula (XVIII) having the following structure:

[0567] [ka] and forming a compound of formula (PI) from the compound of formula (XVIII).

[0568] In one embodiment, the compound of formula (XVIII) has the structure:

[0569] [ka]

[0570] In one embodiment, the step of forming a compound of formula (PI) comprises reacting a compound of formula (XVIII) with a compound of formula (XIX):

[0571] [ka] to produce a compound of formula (PI).

[0572] The present disclosure also provides a compound of formula (D-1):

[0573] [ka] or a pharmaceutically acceptable salt thereof, comprising: In the formula, R 1 , R 2 , R 3 , and R 4 are independently hydrogen or C 1-5 is alkyl, R 5 and R 6 are independently hydrogen, —NH, or the side chain of any natural or unnatural amino acid; The process in question is: (a) providing a compound of formula (XXIII);

[0574] [ka] (b) reacting a compound of formula (XXIII) with a compound having the following structure in the presence of an activating agent and a base:

[0575] [ka] to produce a compound of formula (D-1).

[0576] In one embodiment, the compound of formula (D-1) has the following structure:

[0577] [ka]

[0578] In one aspect, the present disclosure provides a linker-payload compound of formula (D)-(G):

[0579] [ka]

[0580] [ka] or a pharmaceutically acceptable salt thereof, In the formula, B is

[0581] [ka] is selected from the group consisting of R 1 , R 2 , R 3 , and R 4 are independently hydrogen or C 1-5 is alkyl, R 5 , R 6 , R 7 , and R 8 are independently hydrogen, —NH 2 , or the side chain of any natural or unnatural amino acid, or a pharmaceutically acceptable salt thereof.

[0582] In one embodiment, R 1 , R 2 , R 3 , and R 4 are each hydrogen.

[0583] In one embodiment, R 6 is H.

[0584] In one embodiment, R 5 is selected from hydrogen and the side chains of alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, threonine, lysine, asparagine, glutamine, aspartic acid, and glutamic acid. 5 is selected from hydrogen and the side chains of phenylalanine, threonine, lysine, glutamine, and glutamic acid.

[0585] In one embodiment, R7 is H. In one embodiment, R 7 is the side chain of glutamic acid.

[0586] In one embodiment, R 8 is H. In one embodiment, R 8 is -CH2-SO3H.

[0587] In one embodiment, the present disclosure provides a linker-payload having a structure selected from the group in Table 3 below.

[0588] [Table 8-1]

[0589] [Table 8-2]

[0590] [Table 8-3]

[0591] [Table 8-4]

[0592] Table 4 below provides further characterization of non-limiting examples of linker-payloads according to the present disclosure.

[0593] [Table 9-1]

[0594] [Table 9-2]

[0595] In one aspect, the present disclosure provides a compound of formula (I-1):

[0596] [ka] or a pharmaceutically acceptable salt thereof, During the ceremony, X is,

[0597] [ka] is selected from the group consisting of R 1 , R 2 , R 3 , and R 4 are independently hydrogen or C 1-5 is alkyl, R 5 and R 6 are independently hydrogen, —NH 2 , or the side chain of any natural or unnatural amino acid, or a pharmaceutically acceptable salt thereof.

[0598] In one embodiment, the compound of formula (I-1) has the following structure:

[0599] [ka]

[0600] In one aspect, the present disclosure provides a compound of formula (XVIII):

[0601] [ka] or a pharmaceutically acceptable salt thereof, In the formula, R 1 , R 2 , R 3 , and R 4 are independently hydrogen or C 1-5 is alkyl, R 5 and R 6are independently hydrogen, —NH 2 , or the side chain of any natural or unnatural amino acid, or a pharmaceutically acceptable salt thereof.

[0602] In one embodiment, the compound of formula (XVIII) has the structure:

[0603] [ka]

[0604] Therapeutic Formulations and Administration The present disclosure provides pharmaceutical compositions comprising the protein-drug conjugates of the present disclosure.

[0605] In one aspect, the present disclosure provides a composition comprising a population of protein-drug conjugates according to the present disclosure, having a drug-to-antibody ratio (DAR) of from about 0.5 to about 14.0.

[0606] In one embodiment, the composition has a DAR of about 1.0 to about 2.5.

[0607] In one embodiment, the composition has a DAR of about 2.

[0608] In one embodiment, the composition has a DAR of about 3.0 to about 4.5.

[0609] In one embodiment, the composition has a DAR of about 4.

[0610] In one embodiment, the composition has a DAR of about 6.5 to about 8.5.

[0611] In one embodiment, the composition has a DAR of about 8.

[0612] In one embodiment, the composition has a DAR of about 10 to about 14.

[0613] In one embodiment, the composition has a DAR of about 12.

[0614] The compositions of the present disclosure are formulated with suitable carriers, excipients, and other agents that improve transport, delivery, tolerability, etc. Many suitable formulations can be found in formularies known to all pharmacists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (such as LIPOFECTIN™, Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsion carbowax (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al., "Compendium of excipients for parenteral formulations," PDA (1998) J Pharm Sci Technol 52:238-311.

[0615] The dose of a protein-drug conjugate administered to a patient may vary depending on the patient's age and size, the target disease, condition, route of administration, and the like. Suitable doses are typically calculated based on body weight or body surface area. When the protein-drug conjugate of the present disclosure is used for therapeutic purposes in adult patients, it may be advantageous to administer the protein-drug conjugate of the present disclosure intravenously at a single dose of typically about 0.01 to about 20 mg / kg body weight, more preferably about 0.02 to about 7, about 0.03 to about 5, or about 0.05 to about 3 mg / kg body weight. The frequency and duration of treatment can be adjusted depending on the severity of the condition. Effective dosages and schedules for administering a protein-drug conjugate are determined empirically; for example, the patient's progress can be monitored by periodic evaluation, and the dosage can be adjusted accordingly. Furthermore, interspecies scaling of dosages can be performed using methods well known in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

[0616] Various delivery systems, such as liposomes, microparticles, encapsulation in microcapsules, recombinant cells capable of expressing mutant viruses, and receptor-mediated endocytosis, are known and can be used to administer the pharmaceutical compositions of the present disclosure (see, for example, Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Introduction methods include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions can be administered by any convenient route, for example, by infusion or bolus injection, or by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal, and intestinal mucosa), and can be administered together with other biologically active agents. Administration can be systemic or local.

[0617] The pharmaceutical composition of the present disclosure can be delivered subcutaneously or intravenously using a standard needle and syringe. In addition, for subcutaneous delivery, a pen delivery device is easily used to deliver the pharmaceutical composition of the present disclosure. Such a pen delivery device can be reusable or disposable. Reusable pen delivery devices generally utilize a replaceable cartridge containing the pharmaceutical composition. Once the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. In disposable pen delivery devices, there is no replaceable cartridge. Rather, disposable pen delivery devices are pre-filled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is empty of the pharmaceutical composition, the entire device is discarded.

[0618] Numerous reusable pen delivery devices and autoinjector-type delivery devices have application in the subcutaneous delivery of the pharmaceutical compositions of the present disclosure. Examples include the AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), the DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), the HUMALOG MIX75 / 25™ pen, the HUMALOG™ pen, the HUMALIN 70 / 30™ pen (Eli Lilly and Co., Indianapolis, IN), the NOVOPEN™ I, II, and III (Novo Nordisk, Copenhagen, Denmark), the NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), the BD™ pen (Becton Dickinson, Franklin Lakes, NJ), the OPTIPEN™, the OPTIPEN PRO™, the OPTIPEN™, to name a few. Examples of disposable pen delivery devices that have applications in the subcutaneous delivery of pharmaceutical compositions of the present disclosure include, but are not limited to, the SOLOSTAR pen (Sanofi-Aventis), FLEXPEN (Novo Nordisk), and KWIKPEN (Eli Lilly), the SURECLICK™ auto-injector (Amgen, Thousand Oaks, CA), PENLET™ (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and the HUMIRA™ pen (Abbott Labs, Abbott Park, IL), to name a few.

[0619] In certain circumstances, pharmaceutical compositions can be delivered in a sustained-release system. In one embodiment, a pump can be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, a polymeric material can be used; see Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Press, Boca Raton, Florida. In yet another embodiment, a sustained-release system can be placed near the target of the composition, thereby requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other sustained-release systems are discussed in the review by Langer, 1990, Science 249:1527-1533.

[0620] Injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal, and intramuscular injections, drip infusions, and the like. These injectable preparations may be prepared by publicly known methods. For example, injectable preparations may be prepared by dissolving, suspending, or emulsifying the antibody or its salt in a sterile aqueous or oily medium conventionally used for injections. Aqueous media for injection include, for example, saline, isotonic solutions containing glucose, and other adjuvants, which may be used in combination with suitable solubilizers such as alcohols (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)]. Oily media include, for example, sesame oil and soybean oil, which may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. Injections prepared in this manner are preferably filled into appropriate ampoules.

[0621] Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared into a suitable unit dosage form to accommodate the dose of the active ingredient. Examples of such unit dosage forms include tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the antibody contained is generally about 5 to about 500 mg per dosage form in a unit dose. In particular, for injections, the antibody is preferably contained in an amount of about 5 to about 100 mg, and for other dosage forms, it is preferably contained in an amount of about 10 to about 250 mg.

[0622] Therapeutic Uses of Protein-Drug Conjugates, Linker-Payloads, and Payloads In another aspect, the protein-drug conjugates, e.g., ADCs, disclosed herein are useful for, inter alia, the treatment, prevention, and / or amelioration of diseases, disorders, or conditions in need of such treatment.

[0623] In one embodiment, the invention provides a method of treating a condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound according to the present disclosure (e.g., an antibody-drug conjugate, a linker-payload, and / or a payload), or a composition comprising any compound according to the present disclosure.

[0624] In one embodiment, the protein-drug conjugates, e.g., ADCs, disclosed herein are useful for treating cancer. In one embodiment, the protein-drug conjugates, e.g., ADCs, disclosed herein are useful for treating a cancer selected from the group consisting of breast cancer, ovarian cancer, prostate cancer, lung cancer, liver cancer, or brain cancer. In one embodiment, the protein-drug conjugates, e.g., ADCs, disclosed herein are useful for treating HER2+ breast cancer. In one embodiment, the protein-drug conjugates, e.g., ADCs, disclosed herein are useful for treating prostate cancer.

[0625] In one aspect, the present disclosure provides a method for selectively delivering a compound to a cell. In one embodiment, the method for selectively delivering a compound to a cell comprises linking the compound to a targeting antibody. In one embodiment, the compound is a payload as described above. In one embodiment, the cell is a mammalian cell. In one embodiment, the cell is a human cell. In one embodiment, the cell is a cancer cell. In one embodiment, the cancer cell is selected from the group consisting of a breast cancer cell, an ovarian cancer cell, a prostate cancer cell, a lung cancer cell, a liver cancer cell, or a brain cancer cell.

[0626] In certain embodiments, the present disclosure provides a method for administering to a cell a compound having the structure PI,

[0627] [ka] In the formula, R 1 , R 2 , R 3 , and R 4 are independently hydrogen or C 1-5 is alkyl, AA is a natural or unnatural amino acid; A method for selectively delivering a compound, or a pharmaceutically acceptable salt thereof, wherein p is an integer from 1 to 6 is provided.

[0628] In one aspect, the present disclosure provides a method for selectively targeting an antigen on the surface of a cell using a compound. In one embodiment, the method for selectively targeting an antigen on the surface of a cell using a compound comprises linking the compound to a targeting antibody. In one embodiment, the compound is a payload as described above. In one embodiment, the cell is a mammalian cell. In one embodiment, the cell is a human cell. In one embodiment, the cell is a cancer cell. In one embodiment, the cancer cell is selected from the group consisting of a breast cancer cell, an ovarian cancer cell, a prostate cancer cell, a lung cancer cell, a liver cancer cell, or a brain cancer cell.

[0629] In certain embodiments, the present disclosure provides a method for detecting an antigen on the surface of a cell using a compound having the structure PI,

[0630] [ka] In the formula, R 1 , R 2 , R 3 , and R 4 are independently hydrogen or C 1-5 is alkyl, AA is a natural or unnatural amino acid; A method of selective targeting using a compound, or a pharmaceutically acceptable salt thereof, wherein p is an integer from 1 to 6 is provided.

[0631] In certain embodiments of any of the above methods, the compound having the structure PI is selected from the group consisting of:

[0632] [Table 10-1]

[0633] [Table 10-2] or a pharmaceutically acceptable salt thereof.

[0634] Binder In one embodiment, the efficacy of the protein-drug conjugate embodiments described herein depends on the selectivity of the binding agent to bind to the binding partner. In one embodiment of the present disclosure, the binding agent is any molecule that can bind to a given binding partner with some degree of specificity. In one embodiment, the binding agent is in a mammal where the interaction can result in therapeutic use. In an alternative embodiment, the binding agent is in vitro where the interaction can result in diagnostic use. In some aspects, the binding agent can bind to a cell or cell population.

[0635] Suitable binding agents of the present disclosure include proteins that bind to binding partners, where the binding agent comprises one or more glutamine residues. Suitable binding agents include, but are not limited to, antibodies, lymphokines, hormones, growth factors, viral receptors, interleukins, or any other cell- or peptide-binding molecule or substance.

[0636] In one embodiment, the binding agent is an antibody. In certain embodiments, the antibody is selected from a monoclonal antibody, a polyclonal antibody, an antibody fragment (Fab, Fab', and F(ab)2, a minibody, a diabody, a triabody, etc.). The antibodies herein can be humanized using the methods described in U.S. Patent No. 6,596,541 and U.S. Publication No. 2012 / 0096572, each of which is incorporated by reference in its entirety. In certain embodiments of the protein-drug conjugate compounds of the present disclosure, the BA is a humanized monoclonal antibody. For example, the BA can be a monoclonal antibody that binds to HER2, MET, or STEAP2. In certain embodiments of the protein-drug conjugate compounds of the present disclosure, the BA is a bispecific antibody, such as an anti-HER2 / HER2 bispecific antibody or an anti-MET / MET bispecific antibody.

[0637] In the present disclosure, the antibody may be any antibody deemed suitable by those skilled in the art. In some embodiments, the antibody contains at least one glutamine residue in at least one polypeptide chain sequence. In certain embodiments, the antibody contains one or more gln295 residues. In certain 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 engineered to contain one or more glutamine residues. Techniques for engineering glutamine residues into antibody polypeptide chains are within the skill of those skilled in the art. In certain embodiments, the antibody is aglycosylated.

[0638] The antibody may be in any form known to those skilled in the art. In certain embodiments, the antibody comprises a light chain. In certain embodiments, the light chain is a kappa light chain. In certain embodiments, the light chain is a lambda light chain.

[0639] In certain embodiments, the antibody comprises a heavy chain. In some aspects, the heavy chain is IgA. In some aspects, the heavy chain is IgD. In some aspects, the heavy chain is IgE. In some aspects, the heavy chain is IgG. In some aspects, the heavy chain is IgM. In some aspects, the heavy chain is IgG1. In some aspects, the heavy chain is IgG2. In some aspects, the heavy chain is IgG3. In some aspects, the heavy chain is IgG4. In some aspects, the heavy chain is IgA1. In some aspects, the heavy chain is IgA2.

[0640] In some embodiments, the antibody is an antibody fragment. In some aspects, the antibody fragment is an Fv fragment. In some aspects, the antibody fragment is a Fab fragment. In some aspects, the antibody fragment is a F(ab')2 fragment. In some aspects, the antibody fragment is a Fab' fragment. In some aspects, the antibody fragment is an scFv (sFv) fragment. In some aspects, the antibody fragment is an scFv-Fc fragment.

[0641] In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a polyclonal antibody.

[0642] 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.

[0643] The antibody can have binding specificity for any antigen deemed suitable by one of skill in the art. In certain embodiments, the antigen is a transmembrane molecule (e.g., a receptor) or a growth factor. Exemplary antigens include molecules such as renin; growth hormones, including human growth hormone and bovine growth hormone; growth hormone-releasing factor; parathyroid hormone; thyroid-stimulating hormone; lipoproteins; alpha-1-antitrypsin; insulin A chain; insulin B chain; proinsulin; follicle-stimulating hormone; calcitonin; luteinizing hormone; glucagon; clotting factors, such as factor vmc, factor IX, tissue factor (TF), and von Willebrand factor; anticoagulants, such as protein C; atrial natriuretic factor; pulmonary surfactant; plasminogen activators, such as urokinase or human urinary or tissue-type plasminogen activator (t-PA); bombesin; thrombin; hematopoietic growth factors; tumor necrosis factors α and β; enkephalinase; RANTES (normally expressed and secreted, regulated upon T cell activation); human macrophage inflammatory protein (MIP-I-α); serum albumins, such as human serum albumin; mueria inhibitor; and relaxin. A chain; relaxin B chain; prorelaxin; mouse gonadotropin-related peptide; microbial proteins such as betalactamase; DNase; 19E; cytotoxic T lymphocyte-associated antigen (CTLA) such as CTLA-4; inhibin; activin; vascular endothelial growth factor (VEGF); hormone or growth factor receptor; protein A or D; rheumatoid factor; neurotrophic factors such as bone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, N) nerve growth factors such as T4, NT-5, or NT-6), or NGF-β; platelet-derived growth factor (PDGF); fibroblast growth factors such as aFGF and bFGF; fibroblast growth factor receptor 2 (FGFR2), epidermal growth factor (EGF); transforming growth factors (TGFs) such as TGF-α and TGF-β, including TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5; insulin-like growth factor-1 and -2 (IGF-1 and IGF-2);des(I-3)-IGF-l (brain IGF-l), 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, Crypto, alpha v beta 6, integrin, VEGF, VEGFR, EGFR, transferrin receptor, lRTAI, lRTA2, lR TA3, lRTA4, lRTA5; CD2, CD3, CD4, CD5, CD6, CD8, CDII, CDI4, CDI9, CD20, CD21, CD22, CD25, CD26, CD28, CD30, CD33, CD36 , CD37, CD38, CD40, CD44, CD52, CD55, CD56, CD59, CD70, CD79, CD80, CD81, CD103, CD105, CD134, CD137, CD138, CDI52, etc. CD proteins, or antibodies that bind to one or more tumor-associated antigens or cell surface receptors disclosed in U.S. Publication No. 2008 / 0171040 or U.S. Publication No. 2008 / 0305044, incorporated by reference in their entireties; erythropoietin; bone morphogenetic factors; immunotoxins; bone morphogenetic proteins (BMPs); interferons, such as interferon-alpha, -beta, and -gamma; colony-stimulating factors (CSFs), e.g., M-CSF, gM-CSF, and g-CSF; interleukins (ILs), e.g., IL-1 through IL-10; superoxide dismutase; T cell receptors; surface membrane proteins; decay-accelerating factors; viral antigens, such as portions of the HIV envelope; transport proteins; homing receptors; addressins; regulatory proteins; integrins, such as CD11a, CD11b, CD11c, CD18, ICAM, VLA-4, and VCAM;Tumor-associated antigens, such as AFP, ALK, B7H4, BAGE protein, β-catenin, brc-abl, BRCA1, BORIS, CA9 (carbonic anhydrase IX), caspase-8, CD20, CD40, CD123, CDK4, CEA, CLEC12A, c-kit, cMET, CTLA4, cyclin-B1, CYP1B1, EGFR, EGF-VIII, endoglin, Epcam, EphA2, ErbB2 / HER2, Erb B3 / HER3, ErbB4 / HER4, ETV6-AML, Fra-1, FOLR1, gAGE proteins (e.g., gAGE-1, -2), gD2, gD3, globoH, glypican-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, and -12), MART-1, mesothelin, mL-IAP, Muc1, Muc16(CA-125), MET, MUM1, NA17, NGEP, NY-BR1, NY-BR62, NY-BR85, NY-ES O1, OX40, p15, p53, PAP, PAX3, PAX5, PCTA-1, PDGFR-α, PDGFR-β, PDGF-A, PDGF-B, PDGF-C, PDGF-D, PLAC1, PRLR, PRAME, These include, but are not limited to, PSCA, PSGR, PSMA (FOLH1), RAGE protein, Ras, RGS5, Rho, SART-1, SART-3, STEAP1, STEAP2, STn, survivin, TAG-72, TGF-β, TMPRSS2, Tn, TNFRSF17, TRP-1, TRP-2, tyrosinase, and uroplakin-3, as well as fragments of any of the polypeptides listed herein;

[0644] Exemplary antigens also include, but are not limited to, BCMA, SLAMF7, B7H4, gPNMB, UPK3A, and LGR5. Exemplary antigens also include, but are not limited to, MUC16, PSMA, STEAP2, and HER2.

[0645] In some embodiments, antigens also include, but are not limited to, blood targets, such as CD22, CD30, CD33, CD79a, and CD79b.

[0646] Some embodiments herein are target-specific for therapeutic or diagnostic applications. In one embodiment, the binding agent is prepared to interact with and bind to antigens defined as tumor antigens, including antigens specific to a type of tumor or antigens shared, overexpressed, or modified on a particular type of tumor. Examples include alpha-actinin-4 associated with lung cancer, ARTC1 associated with melanoma, BCR-ABL fusion protein associated with chronic myeloid leukemia, B-RAF, CLPP, or Cdc27 associated with melanoma, CASP-8 associated with squamous cell carcinoma, and hsp70-2 associated with renal cell carcinoma, as well as the following shared tumor-specific antigens: BAGE-1, gAGE, gnTV, KK-LC-1, MAGE-A2, NA88-A, and TRP2-INT2. In some embodiments, the antigen is PRLR or HER2. In some embodiments, the antibody binds to STEAP2, MUC16, EGFR, EGF VIII, FGR2, or PRLR.

[0647] In some embodiments, the antigen comprises HER2. In some embodiments, the antigen comprises STEAP2. In some embodiments, the antigen comprises MET. In some embodiments, the antigen comprises EGFRVIII. In some embodiments, the antigen comprises MUC16. In some embodiments, the antigen comprises PRLR. In some embodiments, the antigen comprises PSMA. In some embodiments, the antigen comprises FGFR2.

[0648] In some embodiments, the BA is an anti-HER2 antibody, an anti-STEAP2 antibody, an anti-MET antibody, an anti-EGFRVIII antibody, an anti-MUC16 antibody, an anti-PRLR antibody, an anti-PSMA antibody, or an anti-FGFR2 antibody, an anti-HER2 / HER2 bispecific antibody, an anti-MET / MET bispecific antibody, or an anti-FOLR1 antibody, or an antigen-binding fragment thereof.

[0649] In some embodiments, the BA targets a cancer selected from the group consisting of breast cancer, ovarian cancer, prostate cancer, lung cancer, liver cancer, or brain cancer.

[0650] Anti-HER2 antibodies suitable for protein-drug conjugates In some embodiments, the antibody is an anti-HER2 antibody. In some embodiments, the antibody is trastuzumab, pertuzumab (2C4), or margetuximab (MGAH22). In some embodiments, the antibody is trastuzumab. According to certain embodiments, a protein-drug conjugate, e.g., an ADC, according to the present disclosure comprises an anti-HER2 antibody. In some embodiments, the anti-HER2 antibody may comprise an antibody described in WO2019 / 212965A1.

[0651] 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.

[0652] In certain embodiments, the D1 and D2 domains of the anti-HER2 / HER2 bispecific antibody are non-competitive with each other. Non-competition between D1 and D2 for binding to HER2 means that the respective monospecific antigen-binding proteins from which D1 and D2 are derived do not compete with each other for binding to human HER2. Exemplary antigen-binding protein competition assays are known in the art.

[0653] In certain embodiments, D1 and D2 bind to different (eg, non-overlapping or partially overlapping) epitopes on HER2.

[0654] In one non-limiting embodiment, the present disclosure provides a protein-drug conjugate comprising a bispecific antigen-binding molecule, a first antigen-binding domain (D1); and a second antigen-binding domain (D2), D1 specifically binds to the first epitope of human HER2; An antibody-drug conjugate is provided, wherein D2 specifically binds to a second epitope of human HER2.

[0655] An anti-HER2 / HER2 bispecific antibody may be constructed using the antigen-binding domains of two separate monospecific anti-HER2 antibodies. For example, a collection of monoclonal monospecific anti-HER2 antibodies may be produced using standard methods known in the art. The individual antibodies thus produced may be tested pairwise against each other for cross-competition for HER2 protein. If two different anti-HER2 antibodies can simultaneously bind to HER2 (i.e., do not compete with each other), the antigen-binding domain from a first anti-HER2 antibody and the antigen-binding domain from a second non-competing anti-HER2 antibody may be engineered into a single anti-HER2 / HER2 bispecific antibody according to the present disclosure.

[0656] According to the present disclosure, a bispecific antigen-binding molecule can be a single multifunctional polypeptide, or a multimeric complex of two or more polypeptides that are covalently or non-covalently linked to each other.As will become clear from the present disclosure, any antigen-binding construct that has the ability to simultaneously bind to two distinct, non-identical epitopes of the HER2 molecule is considered a bispecific antigen-binding molecule.Any of the bispecific antigen-binding molecules or variants thereof described herein can be constructed using standard molecular biology techniques (e.g., recombinant DNA and protein expression techniques), which will be known to those skilled in the art.

[0657] In another aspect, the disclosure provides a pharmaceutical composition comprising a recombinant human antibody or fragment thereof that specifically binds to HER2 and 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 related aspect, the disclosure features a composition that is a combination of an anti-HER2 / HER2 antibody and a second therapeutic agent. In one embodiment, the second therapeutic agent is any agent that is advantageously combined with an anti-HER2 / HER2 antibody. Additional combination therapies and combination formulations that include the anti-HER2 / HER2 bispecific antibodies of the disclosure are disclosed elsewhere herein.

[0658] In another aspect, the present disclosure provides a therapeutic method for targeting / killing HER2-expressing tumor cells using an anti-HER2 / HER2 bispecific antibody of the present disclosure, the therapeutic method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising an anti-HER2 / HER2 antibody of the present disclosure. In some cases, the anti-HER2 / HER2 antibody (or antigen-binding fragment thereof) can be used to treat breast cancer or may be modified to enhance cytotoxicity by methods including, but not limited to, an engineered Fc domain that increases ADCC (see, e.g., Shield et al. (2002) JBC277:26733), radioimmunotherapy, antibody-drug conjugates, or other methods to enhance the efficiency of tumor resection.

[0659] The present disclosure also includes the use of an anti-HER2 antibody of the present disclosure in the manufacture of a medicament for the treatment of a disease or disorder (e.g., cancer) associated with or caused by HER2-expressing cells. In one aspect, the present disclosure relates to a compound comprising an anti-HER2 antibody or antigen-binding fragment, or a HER2 / HER2 bispecific antibody disclosed herein, for use in medical treatment. In one aspect, the present disclosure relates to a compound comprising an antibody-drug conjugate (ADC) disclosed herein, for use in medicine.

[0660] In yet another aspect, the present disclosure provides bispecific anti-HER2 / HER2 antibodies for diagnostic applications, such as, for example, as imaging reagents.

[0661] Anti-STEAP2 antibodies suitable for protein-drug conjugates In some embodiments, the antibody is an anti-six-transmembrane epithelial antigen 2 (STEAP2) of the prostate, i.e., an anti-STEAP2 antibody. STEAP2, which functions as a shuttle between the Golgi complex and the plasma membrane, is a metalloreductase that reduces iron and copper to promote their import into cells. STEAP2 is primarily localized in the epithelial cells of the prostate. STEAP2 is also expressed in normal heart, brain, pancreas, ovary, skeletal muscle, mammary gland, testis, uterus, kidney, lung, trachea, colon, and liver. STEAP2 is overexpressed in cancerous tissues including prostate, bladder, cervical, lung, colon, kidney, breast, pancreatic, gastric, uterine, and ovarian tumors (Gomes, IM et al., 2012, Mol. Cancer Res. 10:573-587; Challita-Eid-PM, et al., 2003, WO03 / 087306; Emtage, PCR, 2005, WO2005 / 079490).

[0662] In one embodiment, a suitable anti-STEAP antibody is one disclosed in US2018 / 0104357. Exemplary anti-STEAP2 antibodies according to the present disclosure are listed in Tables 5 and 6 herein. Table 5 lists the amino acid sequence identifiers for the heavy chain variable region (HCVR) and light chain variable region (LCVR), as well as the heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) and light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) of exemplary anti-STEAP2 antibodies. Table 6 lists the sequence identifiers for nucleic acid molecules encoding the HCVR, LCVR, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of exemplary anti-STEAP2 antibodies.

[0663] The present disclosure provides antibodies or antigen-binding fragments thereof comprising an HCVR comprising an amino acid sequence selected from any of the HCVR amino acid sequences listed in Table 5, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0664] The present disclosure also provides antibodies or antigen-binding fragments thereof comprising an LCVR comprising an amino acid sequence selected from any of the LCVR amino acid sequences listed in Table 5, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0665] The present disclosure also provides antibodies or antigen-binding fragments thereof, including an HCVR and LCVR amino acid sequence pair (HCVR / LCVR) comprising any of the HCVR amino acid sequences listed in Table 5 paired with any of the LCVR amino acid sequences listed in Table 5. According to certain embodiments, the present disclosure provides antibodies or antigen-binding fragments thereof comprising an HCVR / LCVR amino acid sequence pair included in any of the exemplary anti-STEAP2 antibodies listed in Table 5. In certain embodiments, the HCVR / LCVR amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 250 / 258 (e.g., H2M11162N).

[0666] The present disclosure also provides an antibody or antigen-binding fragment thereof comprising a heavy chain CDR1 (HCDR1) comprising an amino acid sequence selected from any of the HCDR1 amino acid sequences listed in Table 5, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0667] The present disclosure also provides antibodies or antigen-binding fragments thereof comprising a heavy chain CDR2 (HCDR2) comprising an amino acid sequence selected from any of the HCDR2 amino acid sequences listed in Table 5, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0668] The present disclosure also provides antibodies or antigen-binding fragments thereof comprising a heavy chain CDR3 (HCDR3) comprising an amino acid sequence selected from any of the HCDR3 amino acid sequences listed in Table 5, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0669] The present disclosure also provides an antibody or antigen-binding fragment thereof comprising a light chain CDR1 (LCDR1) comprising an amino acid sequence selected from any of the LCDR1 amino acid sequences listed in Table 5, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0670] The present disclosure also provides an antibody or antigen-binding fragment thereof comprising a light chain CDR2 (LCDR2) comprising an amino acid sequence selected from any of the LCDR2 amino acid sequences listed in Table 5, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0671] The present disclosure also provides an antibody or antigen-binding fragment thereof comprising a light chain CDR3 (LCDR3) comprising an amino acid sequence selected from any of the LCDR3 amino acid sequences listed in Table 5, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0672] The present disclosure also provides antibodies or antigen-binding fragments thereof comprising an HCDR3 and LCDR3 amino acid sequence pair (HCDR3 / LCDR3) comprising any of the HCDR3 amino acid sequences listed in Table 5 paired with any of the LCDR3 amino acid sequences listed in Table 5. According to certain embodiments, the present disclosure provides antibodies or antigen-binding fragments thereof comprising an HCDR3 / LCDR3 amino acid sequence pair included in any of the exemplary anti-STEAP2 antibodies listed in Table 5. In certain embodiments, the HCDR3 / LCDR3 amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 256 / 264 (e.g., H2M11162N).

[0673] The present disclosure also provides antibodies or antigen-binding fragments thereof comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained in any of the exemplary anti-STEAP2 antibodies listed in Table 5. In certain embodiments, the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence set is selected from the group consisting of SEQ ID NOs: 252-254-256-260-262-264 (e.g., H2M11162N).

[0674] In related embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within an HCVR / LCVR amino acid sequence pair defined by any of the exemplary anti-STEAP2 antibodies listed in Table 5. For example, the present disclosure includes an antibody or antigen-binding fragment thereof comprising a set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences contained within an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 250 / 258 (e.g., H2M11162N). Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within particular HCVR and / or LCVR amino acid sequences disclosed herein. Exemplary rules 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 diversity, the Chothia definition is based on the location of structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, e.g., Kabat, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989). Public databases are also available for identifying CDR sequences within antibodies.

[0675] The present disclosure also provides nucleic acid molecules encoding anti-STEAP2 antibodies or portions thereof. For example, the present disclosure provides nucleic acid molecules encoding any of the HCVR amino acid sequences listed in Table 5, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table 6, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0676] The present disclosure also provides nucleic acid molecules encoding any of the LCVR amino acid sequences listed in Table 5, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Table 6, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0677] The present disclosure also provides nucleic acid molecules encoding any of the HCDR1 amino acid sequences listed in Table 5, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR1 nucleic acid sequences listed in Table 6, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0678] The present disclosure also provides nucleic acid molecules encoding any of the HCDR2 amino acid sequences listed in Table 5, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR2 nucleic acid sequences listed in Table 6, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0679] The present disclosure also provides nucleic acid molecules encoding any of the HCDR3 amino acid sequences listed in Table 5, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR3 nucleic acid sequences listed in Table 6, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0680] The present disclosure also provides nucleic acid molecules encoding any of the LCDR1 amino acid sequences listed in Table 5, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR1 nucleic acid sequences listed in Table 6, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0681] The present disclosure also provides nucleic acid molecules encoding any of the LCDR2 amino acid sequences listed in Table 5, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR2 nucleic acid sequences listed in Table 6, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0682] The present disclosure also provides nucleic acid molecules encoding any of the LCDR3 amino acid sequences listed in Table 5, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR3 nucleic acid sequences listed in Table 6, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0683] The present disclosure also provides nucleic acid molecules encoding HCVRs, wherein the HCVRs comprise a set of three CDRs (i.e., HCDR1-HCDR2-HCDR3), wherein the HCDR1-HCDR2-HCDR3 amino acid sequence set is as defined by any of the exemplary anti-STEAP2 antibodies listed in Table 5.

[0684] The present disclosure also provides nucleic acid molecules encoding LCVRs, wherein the LCVRs comprise a set of three CDRs (i.e., LCDR1-LCDR2-LCDR3), wherein the LCDR1-LCDR2-LCDR3 amino acid sequence set is as defined by any of the exemplary anti-STEAP2 antibodies listed in Table 5.

[0685] The present disclosure also provides nucleic acid molecules encoding both an HCVR and an LCVR, wherein the HCVR comprises the amino acid sequence of any of the HCVR amino acid sequences listed in Table 5, and the LCVR comprises the amino acid sequence of any of the LCVR amino acid sequences listed in Table 5. In certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table 6, 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 nucleic acid sequences listed in Table 6, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments according to this aspect of the disclosure, the nucleic acid molecule encodes an HCVR and an LCVR, wherein the HCVR and LCVR are both derived from the same anti-STEAP2 antibody listed in Table 5.

[0686] The present disclosure also provides recombinant expression vectors capable of expressing polypeptides comprising the heavy chain variable region or light chain variable region of an anti-STEAP2 antibody. For example, the present disclosure includes recombinant expression vectors comprising any of the above-described nucleic acid molecules, i.e., nucleic acid molecules encoding any of the HCVR, LCVR, and / or CDR sequences set forth in Table 5. Also included within the scope of the present disclosure are host cells into which such vectors have been introduced, as well as methods for producing antibodies or portions thereof by culturing the host cells under conditions that allow the production of antibodies or antibody fragments, and recovering the antibodies and antibody fragments so produced.

[0687] The present disclosure includes anti-STEAP2 antibodies with altered glycosylation patterns. In some embodiments, antibodies modified to remove undesired glycosylation sites or lacking fucose moieties present on the oligosaccharide chains may be useful, for example, to increase antibody-dependent cellular 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).

[0688] In another aspect, the present disclosure provides a pharmaceutical composition comprising a recombinant human antibody or fragment thereof that specifically binds to STEAP2 and a pharmaceutically acceptable carrier. In a related aspect, the present disclosure features a composition that is a combination of an anti-STEAP2 antibody and a second therapeutic agent. In one embodiment, the second therapeutic agent is any agent that is advantageously combined with the anti-STEAP2 antibody. Additional combination therapies and formulations that include the anti-STEAP2 antibodies of the present disclosure are disclosed elsewhere herein.

[0689] In another aspect, the present disclosure provides a therapeutic method for targeting / killing tumor cells expressing STEAP2 using an anti-STEAP2 antibody of the present disclosure, the therapeutic method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising an anti-STEAP2 antibody of the present disclosure. In some cases, the anti-STEAP2 antibody (or antigen-binding fragment thereof) can be used to treat prostate cancer or may be modified to enhance cytotoxicity by methods including, but not limited to, an engineered Fc domain that increases ADCC (see, e.g., Shield et al. (2002) JBC277:26733), radioimmunotherapy, antibody-drug conjugates, or other methods to enhance the efficiency of tumor resection.

[0690] The present disclosure also includes the use of an anti-STEAP2 antibody of the present disclosure in the manufacture of a medicament for the treatment of a disease or disorder (e.g., cancer) associated with or caused by STEAP2-expressing cells. In one aspect, the present disclosure relates to a compound comprising an anti-STEAP2 antibody or antigen-binding fragment, or a STEAP2xCD3 bispecific antibody disclosed herein, for use in medical treatment. In one aspect, the present disclosure relates to a compound comprising an antibody-drug conjugate (ADC) disclosed herein, for use in medicine.

[0691] In yet another aspect, the present disclosure provides monospecific anti-STEAP2 antibodies for diagnostic applications, such as, for example, as imaging reagents.

[0692] In yet another aspect, the present disclosure provides a therapeutic method for stimulating T cell activation using an anti-CD3 antibody or antigen-binding portion of the antibody of the present disclosure, the therapeutic method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising the antibody.

[0693] In another aspect, the present disclosure provides an isolated antibody or antigen-binding fragment thereof 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 present disclosure provides an isolated antibody or antigen-binding fragment thereof that binds to and is internalized by STEAP2-expressing C4-2 cells.

[0694] The present disclosure further provides antibodies or antigen-binding fragments that compete with a reference antibody comprising a HCVR / LCVR amino acid sequence pair listed in Table 5 for binding to human STEAP2. In another aspect, the disclosure provides an antibody or antigen-binding fragment that competes for binding to human STEAP2 with a reference antibody comprising 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.

[0695] The present disclosure further provides antibodies or antigen-binding fragments thereof that bind to the same epitope on human STEAP2 as a reference antibody comprising the HCVR / LCVR amino acid sequence pair listed in Table 5. In another embodiment, the antibody or antigen-binding fragment binds to the same epitope on human STEAP2 as a reference antibody comprising 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.

[0696] The present disclosure further provides an isolated antibody or antigen-binding fragment thereof that binds to human STEAP2, wherein the antibody or antigen-binding fragment comprises a complementarity-determining region (CDR) of a heavy chain variable region (HCVR) having an amino acid sequence set forth in Table 5, and a CDR of a light chain variable region (LCVR) having an amino acid sequence set forth in Table 5. In another embodiment, the isolated antibody or antigen-binding fragment comprises the heavy and light chain CDRs of an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 58, 74 / 58, 82 / 58, 90 / 58, 98 / 58, 106 / 114, 122 / 130, 138 / 146, 154 / 162, 170 / 178, 186 / 194, 202 / 210, 218 / 226, 234 / 242, 250 / 258, 266 / 274, 282 / 290, 298 / 306, 314 / 322, 330 / 338, 346 / 354, 362 / 370, and 378 / 386.In yet another embodiment, the isolated antibodies or antigen-binding fragments are selected from the group consisting of SEQ ID NOs: 4-6-8-12-14-16, 20-22-24-28-30-32, 36-38-40-44-46-48, 52-54-56-60-62-64, 68-70-72-60-62-64, 76-78-80-60-62-64, 84-86-88-60-62-64, 92-94-96-60- 62-64, 100-102-104-60-62-64, 108-110-112-116-118-120, 124-126-128-132-134-136, 140-142-144-148-150-152, 156-158-160-164-166-168, 172-174-176-180-182-184, 188-190-192-196-198-200, 2 04-206-208-212-214-216, 220-222-224-228-230-232, 236-238-240-244-246-248, 252-254-256-260-262-264, 268-270-272-276-278-280, 284-286-288-292-294-296, 300-302-304-308-310-312, 316-3 and 380-382-384-388-390-392.

[0697] In another aspect, the disclosure provides an isolated antibody or antigen-binding fragment thereof that binds to human STEAP2, wherein the antibody or antigen-binding fragment comprises: (a) a heavy chain variable region (HCVR) having an amino acid sequence selected from the group consisting of 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 378; and (b) a heavy chain variable region (HCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 60, 62, 64, 66, 74, 82, 90, 98, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, 298, 314, 330, 346, 362, and 378. and a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of: 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, 290, 306, 322, 338, 354, 370, and 386. In a further aspect, the isolated antibody or antigen-binding fragment of claim 10, wherein the antibody or antigen-binding fragment comprises 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.

[0698] According to another aspect, the present disclosure provides an antibody-drug conjugate comprising the above-described anti-STEAP2 antibody or antigen-binding fragment thereof and a therapeutic agent (e.g., an anti-tumor agent, such as a camptothecin analog, e.g., Dxd). In some embodiments, the antibody or antigen-binding fragment and the anti-tumor agent are covalently linked via a linker, as described above. In various embodiments, the anti-STEAP2 antibody or antigen-binding fragment may be any of the anti-STEAP2 antibodies or fragments thereof described herein.

[0699] Anti-STEAP2 antibody heavy and light chain variable region amino acid and nucleic acid sequences Table 5 lists the amino acid sequence identifiers for the heavy and light chain variable regions and CDRs of selected anti-STEAP2 antibodies according to the present disclosure. The corresponding nucleic acid sequence identifiers are listed in Table 6.

[0700] [Table 11]

[0701] [Table 12]

[0702] Anti-MET antibodies suitable for protein-drug conjugates In some embodiments, the antibody is an anti-MET antibody. According to certain embodiments, a protein-drug conjugate, e.g., an ADC, according to the present disclosure comprises an anti-MET antibody. In some embodiments, the anti-MET antibody may comprise an antibody described in US2018 / 0134794.

[0703] In some embodiments, the antibody is an anti-MET / MET bispecific antibody comprising 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 those described in US2018 / 0134794.

[0704] In certain embodiments, the D1 and D2 domains of an anti-MET / MET bispecific antibody are non-competitive with each other. Non-competition between D1 and D2 for binding to MET means that the respective monospecific antigen-binding proteins from which D1 and D2 are derived do not compete with each other for binding to human MET. Exemplary antigen-binding protein competition assays are known in the art.

[0705] In certain embodiments, D1 and D2 bind to different (eg, non-overlapping or partially overlapping) epitopes on MET.

[0706] In one non-limiting embodiment, the present disclosure provides a protein-drug conjugate comprising a bispecific antigen-binding molecule, a first antigen-binding domain (D1); and a second antigen-binding domain (D2), D1 specifically binds to the first epitope of human MET; A protein-drug conjugate is provided in which D2 specifically binds to a second epitope of human MET.

[0707] Anti-MET / MET bispecific antibodies may be constructed using the antigen-binding domains of two separate, monospecific anti-MET antibodies. For example, a collection of monoclonal, monospecific anti-MET antibodies may be produced using standard methods known in the art. Individual antibodies thus produced may be tested pairwise against each other for cross-competition for MET protein. If two different anti-MET antibodies are capable of binding to MET simultaneously (i.e., do not compete with each other), the antigen-binding domain from a first anti-MET antibody and the antigen-binding domain from a second, non-competing anti-MET antibody may be engineered into a single anti-MET / MET bispecific antibody according to the present disclosure.

[0708] According to the present disclosure, a bispecific antigen-binding molecule can be a single multifunctional polypeptide, or a multimeric complex of two or more polypeptides that are covalently or non-covalently linked to each other. As will become clear from the present disclosure, any antigen-binding construct that has the ability to simultaneously bind to two distinct, non-identical epitopes of the MET molecule is considered a bispecific antigen-binding molecule. Any of the bispecific antigen-binding molecules or variants thereof described herein can be constructed using standard molecular biology techniques (e.g., recombinant DNA and protein expression techniques), which will be known to those skilled in the art.

[0709] A bispecific antigen-binding molecule comprising 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 may be referred to herein as a "MET / MET bispecific antibody," "METxMET bispecific antibody," "MET / MET," "METxMET," or other related term. 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.

[0710] Exemplary antigen-binding domains (D1 and D2) that can be included in the METxMET bispecific antigen-binding molecules provided herein include antigen-binding domains derived from any of the anti-MET antibodies disclosed herein. For example, the present disclosure includes METxMET bispecific antigen-binding molecules comprising a D1 or D2 antigen-binding domain that comprises an HCVR comprising an amino acid sequence selected from any of the HCVR amino acid sequences listed in Table 7, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0711] Also provided herein is a METxMET bispecific antigen binding molecule comprising a D1 or D2 antigen binding domain that comprises an LCVR comprising an amino acid sequence selected from any of the LCVR amino acid sequences listed in Table 7, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0712] Provided herein are METxMET bispecific antigen-binding molecules comprising a D1 or D2 antigen-binding domain comprising an HCVR and LCVR amino acid sequence pair (HCVR / LCVR), which comprises any of the HCVR amino acid sequences listed in Table 7 paired with any of the LCVR amino acid sequences listed in Table 7. According to certain embodiments, the present invention provides METxMET bispecific antigen-binding molecules comprising a D1 or D2 antigen-binding domain comprising an HCVR / LCVR amino acid sequence pair included in any of the exemplary anti-MET antibodies listed in Table 7.

[0713] Also provided herein is a METxMET bispecific antigen-binding molecule comprising a D1 or D2 antigen-binding domain that comprises a heavy chain CDR1 (HCDR1) comprising an amino acid sequence selected from any of the HCDR1 amino acid sequences listed in Table 7, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0714] Also provided are METxMET bispecific antigen binding molecules comprising a D1 or D2 antigen binding domain that comprises a heavy chain CDR2 (HCDR2) comprising an amino acid sequence selected from any of the HCDR2 amino acid sequences listed in Table 7, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0715] Also provided are METxMET bispecific antigen binding molecules comprising a D1 or D2 antigen binding domain that comprises a heavy chain CDR3 (HCDR3) comprising an amino acid sequence selected from any of the HCDR3 amino acid sequences listed in Table 7, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0716] Also provided are METxMET bispecific antigen binding molecules comprising a D1 or D2 antigen binding domain that comprises a light chain CDR1 (LCDR1) comprising an amino acid sequence selected from any of the LCDR1 amino acid sequences listed in Table 7, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0717] Also provided are METxMET bispecific antigen binding molecules comprising a D1 or D2 antigen binding domain that comprises a light chain CDR2 (LCDR2) comprising an amino acid sequence selected from any of the LCDR2 amino acid sequences listed in Table 7, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0718] Also provided are METxMET bispecific antigen binding molecules comprising a D1 or D2 antigen binding domain that comprises a light chain CDR3 (LCDR3) comprising an amino acid sequence selected from any of the LCDR3 amino acid sequences listed in Table 7, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0719] Also provided are METxMET bispecific antigen-binding molecules comprising a D1 or D2 antigen-binding domain comprising an HCDR3 and LCDR3 amino acid sequence pair (HCDR3 / LCDR3) comprising any of the HCDR3 amino acid sequences listed in Table 7 paired with any of the LCDR3 amino acid sequences listed in Table 7. According to certain embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof comprising an HCDR3 / LCDR3 amino acid sequence pair included in any of the exemplary anti-MET antibodies listed in Table 7.

[0720] Also provided are METxMET bispecific antigen binding molecules comprising a D1 or D2 antigen binding domain comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained in any of the exemplary anti-MET antibodies listed in Table 7.

[0721] In related embodiments, the present disclosure provides MET×MET bispecific antigen binding molecules comprising a D1 or D2 antigen binding domain that comprises a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained in an HCVR / LCVR amino acid sequence pair defined by any of the exemplary anti-MET antibodies listed in Table 7.

[0722] The METxMET bispecific antigen-binding molecules provided herein may comprise a D1 antigen-binding domain derived from any of the anti-MET antibodies in Table 7 and a D2 antigen-binding domain derived from any other anti-MET antibody in Table 7.

[0723] As a non-limiting illustrative example, the present disclosure includes a MET×MET bispecific antigen-binding molecule comprising a D1 antigen-binding domain and a D2 antigen-binding domain, wherein the D1 antigen-binding domain comprises the HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 2012 / 2092, or the set of heavy and light chain CDRs (HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) comprising SEQ ID NOs: 2014-2016-2018-2094-2096-2098, and the D2 antigen-binding domain comprises the HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 2036 / 2092, or the set of heavy and light chain CDRs (HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) comprising SEQ ID NOs: 2038-2040-2042-2094-2096-2098. An exemplary MET×MET bispecific antibody having these sequence characteristics is the bispecific antibody designated H4H14639D, also known as bispecific antibody number 2076, which contains D1 derived from H4H13306P2 and D2 derived from H4H13312P2.

[0724] Heavy and light chain variable region amino acid and nucleic acid sequences of anti-MET and MET / MET antibodies Table 7 lists the amino acid sequence identifiers for the heavy and light chain variable regions and CDRs of selected anti-MET antibodies described herein. (As noted above, all anti-MET antibodies of the present disclosure have the same light chain variable region and therefore the same light chain CDR sequences as well.) The corresponding nucleic acid sequence identifiers are listed in Table 8.

[0725] [Table 13]

[0726] [Table 14]

[0727] Antibodies are typically referred to herein according to the following nomenclature, as shown in Tables 7 and 8: an Fc prefix (e.g., "H4H") followed by a numerical identifier (e.g., "13290," "13291," "13295," etc.), followed by a "P2" suffix. Thus, according to this nomenclature, antibodies may be referred to herein as, for example, "H4H13290P2," "H4H13291P2," "H4H13295P2," etc. The prefixes in the antibody designations used herein indicate the antibody's particular Fc region isotype. In particular, an "H4H" antibody has a human IgG4 Fc (all variable regions are fully human, as indicated by the initial "H" in the antibody designation). As will be appreciated by those skilled in the art, an antibody with a particular Fc isotype may be converted to an antibody with a different Fc isotype (e.g., an antibody with a murine IgG4 Fc may be converted to an antibody with a human IgG1 Fc, etc.), but in each case the variable domains (including the CDRs) indicated by the numerical identifiers shown in Tables 7 and 8 will remain the same, and the binding characteristics are expected to be the same or substantially similar regardless of the nature of the Fc domain.

[0728] Antibody conjugation Techniques and linkers for conjugating to residues of antibodies or antigen-binding fragments are known in the art. Exemplary amino acid additions that may be used in the context of this embodiment include, for example, lysine (see, e.g., US 5,208,020, US 2010 / 0129314, Hollander et al., Bioconjugates Chem., 2008, 19:358-361, WO2005 / 089808, US5,714,586, US2013 / 0101546, and US2012 / 0585592), cysteine ​​(see, e.g., US2007 / 0258987, WO2013 / 055993, WO2013 / 055990, WO2013 / 053873, WO2013 / 053872, WO2011 / 130598, US2013 / 0101546, and US7,750,116), selenocysteine ​​(see, e.g., WO2008 / 122039 and Hofer et al. al., Proc. Natl. Acad. Sci., USA, 2008, 105: 12451-12456), formylglycine (e.g., see Carrico et al., Nat. Chem. Biol., 2007, 3: 321-322; Agarwal et al., Proc. Natl. Acad. Sci., USA, 2013, 110: 46-51; and Rabuka et al., Nat. Protocols, 2012, 10: 1052-1067), unnatural amino acids (e.g., see WO2013 / 068874 and WO2012 / 166559), and acidic amino acids (e.g., see WO2012 / 05982). Lysine conjugation can also proceed via NHS (N-hydroxysuccinimide). Linkers can also be conjugated to cysteine ​​residues, including those of cleaved interchain disulfide bonds, by forming a carbon bridge between the thiols (see, for example, US Pat. Nos. 9,951,141 and 9,950,076).Linkers can also be conjugated to antigen-binding proteins via attachment to carbohydrates (see, e.g., US2008 / 0305497, WO2014 / 065661, and Ryan et al., Food & Agriculture Immunol., 2001, 13:127-130) and disulfide linkers (see, e.g., WO2013 / 085925, WO2010 / 010324, WO2011 / 018611, and Shaunak et al., Nat. Chem. Biol., 2006, 2:312-313). Site-specific conjugation techniques may also be employed to directly conjugate to specific residues of antibodies or antigen-binding proteins (see, e.g., Schumacher et al., J Clin Immunol (2016) 36 (Suppl 1): 100). In certain embodiments described in more detail below, site-specific conjugation techniques include transglutaminase-mediated glutamine conjugation (see, e.g., Schibli, Angew Chemie Inter Ed. 2010, 49, 9995).

[0729] Payloads according to the present disclosure that are linked via lysines and / or cysteines, for example, via maleimide or amide conjugation, are included within the scope of the present disclosure.

[0730] In some embodiments, the protein-drug conjugates of the present disclosure are produced according to a two-step process, where step 1 is a lysine-based linker conjugation, e.g., with an NHS-ester linker, and step 2 is a payload conjugation reaction (e.g., a 1,3-cycloaddition reaction).

[0731] In some embodiments, the protein-drug conjugates of the present disclosure are produced according to a two-step process, where step 1 is a cysteine-based linker conjugation, e.g., with a maleimide linker, and step 2 is a payload conjugation reaction (e.g., a 1,3-cycloaddition reaction).

[0732] In some embodiments, the protein-drug conjugates of the present disclosure are produced according to a two-step process, where step 1 is a transglutaminase-mediated site-specific conjugation and step 2 is a payload conjugation reaction (e.g., a 1,3-cycloaddition reaction).

[0733] Step 1: Transglutaminase-mediated site-specific conjugation In some embodiments, proteins (e.g., antibodies) can be modified according to known methods to provide glutaminyl-modified proteins. Techniques for conjugating antibodies and primary amine compounds are known in the art. A site-specific conjugation technique is utilized herein to directly conjugate to glutamine using transglutaminase-mediated glutamine conjugation (see, for example, Schibli, Angew Chemie Inter Ed. 2010, 49, 9995).

[0734] A primary amine-containing compound (e.g., linker L1) of the present disclosure can be conjugated to one or more glutamine residues of a binder (e.g., a protein, e.g., an antibody) via transglutaminase-based chemoenzymatic conjugation (see, e.g., Dennler et al., Protein Conjugate Chem. 2014, 25, 569-578 and WO2017 / 147542). For example, in the presence of transglutaminase, one or more glutamine residues of an antibody can be coupled to a primary amine linker compound. Briefly, in some embodiments, a binder having a glutamine residue (e.g., gln295, i.e., Q295 residue) is treated with the above-described primary amine-containing linker LL in the presence of the enzyme transglutaminase. In certain embodiments, the binder is aglycosylated. In certain embodiments, the binder is deglycosylated.

[0735] In certain embodiments, a binding agent (e.g., a protein, e.g., an antibody) comprises at least one glutamine residue in at least one polypeptide chain sequence. In certain embodiments, a binding agent comprises two heavy chain polypeptides, each having a gln295 residue. In further embodiments, a binding agent comprises one or more glutamine residues at a site other than 295 in the heavy chain.

[0736] In some embodiments, binding agents, such as antibodies, can be prepared by site-directed mutagenesis to insert glutamine residues at the site without impairing antibody function or binding. For example, antibodies with the Asn297Gln (N297Q) mutation(s) described herein are included herein. In some embodiments, antibodies with the gln295 and / or N297Q mutations contain one or more additional naturally occurring glutamine residues in their variable regions, which may be accessible to transglutaminase and therefore can be conjugated to a linker or linker-payload. An exemplary naturally occurring glutamine residue can be found, for example, at Q55 in the light chain. In such cases, binding agents, such as antibodies, conjugated via transglutaminase may have a higher than expected LAR value (e.g., a LAR greater than 4). Any such antibodies can be isolated from natural or artificial sources.

[0737] In certain embodiments of the present disclosure, the linker-antibody ratio or LAR is 1, 2, 3, 4, 5, 6, 7, or 8 linker LL molecules per antibody. In some embodiments, the LAR is 1 to 8. In some embodiments, the LAR is 1 to 6. In certain 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, or about 1.5, or about 2, or about 2.5, or about 3, or about 3.5. In some embodiments, the LAR is 2. In some embodiments, the LAR is 4.

[0738] Step 2: Payload conjugation reaction In certain embodiments, a linker LL according to the present disclosure comprises at least one reactive group capable of further reaction after transglutamination. In these embodiments, a glutaminyl-modified protein (e.g., an antibody) can be further reacted with a reactive payload compound or a reactive linker-payload compound (e.g., a linker-payload compound disclosed herein) to form a protein-payload conjugate. More specifically, the reactive linker-payload compound can comprise a reactive group capable of reacting with a reactive group of the linker LL via a click chemistry reaction to form a click chemistry adduct. In certain embodiments, a reactive group according to the present disclosure comprises a moiety capable of undergoing a 1,3-cycloaddition reaction. In certain embodiments, the reactive group is an azide. In certain embodiments, the reactive group comprises an alkyne (e.g., a terminal alkyne or an internally strained alkyne). In certain embodiments, the reactive group comprises a tetrazine. In certain embodiments, the reactive group comprises a strained alkene. In certain embodiments of the present disclosure, the reactive group is compatible with the conjugation agent and transglutamination reaction conditions.

[0739] In one embodiment, the glutamine residue Gln is naturally present in the CH2 or CH3 domain of the BA. In another embodiment, the glutamine residue Gln is introduced into the BA by modifying one or more amino acids. In one embodiment, the Gln is Q295 or N297Q.

[0740] In one embodiment, the transglutaminase is a microbial transglutaminase (MTG). In one embodiment, the transglutaminase is a bacterial transglutaminase (BTG).

[0741] Anti-HER2 antibody-drug conjugate In certain embodiments, the protein-drug conjugates, e.g., ADCs, disclosed herein are useful for treating, preventing, and / or ameliorating, inter alia, any disease or disorder associated with or mediated by HER2 expression or activity, or treatable by binding to HER2 without competition with modified LDL, and / or promoting internalization of the HER2 receptor, and / or reducing the number of cell surface receptors.

[0742] The protein-drug conjugates of the present disclosure (and therapeutic compositions comprising them) are useful, inter alia, for treating any disease or disorder in which stimulating, activating, and / or targeting an immune response may be beneficial. In particular, anti-HER2 protein-drug conjugates, including both monospecific anti-HER2 antibodies and bispecific anti-HER2 / HER2 antibodies of the present disclosure, can be used to treat, prevent, and / or ameliorate any disease or disorder associated with or mediated by HER2 expression or activity or the proliferation of HER2+ cells. The mechanism of action achieved by the therapeutic methods of the present disclosure includes the killing of HER2-expressing cells in the presence of effector cells, for example, by CDC, apoptosis, ADCC, phagocytosis, or a combination of two or more of these mechanisms. HER2-expressing cells that can be inhibited or killed using the protein-drug conjugates of the present disclosure include, for example, breast tumor cells.

[0743] In one embodiment, the protein-drug conjugates of the present disclosure (and therapeutic compositions and dosage forms comprising them) are a first antigen-binding domain (D1); and a second antigen-binding domain (D2), D1 specifically binds to the first epitope of human HER2; D2 specifically binds to a second epitope on human HER2.

[0744] In one embodiment of the above, D1 and D2 do not compete with each other for binding to human HER2.

[0745] The protein-drug conjugates of the present disclosure can be used to treat primary and / or metastatic tumors occurring in, for example, the prostate, bladder, cervix, lung, colon, kidney, breast, pancreas, stomach, uterus, and / or ovary. In certain embodiments, the protein-drug conjugates of the present disclosure are used to treat one or more of prostate cancer, bladder cancer, cervical cancer, lung cancer, colon cancer, kidney cancer, breast cancer, pancreatic cancer, stomach cancer, uterine cancer, and ovarian cancer. According to certain embodiments of the present disclosure, anti-HER2 antibodies or anti-HER2 / HER2 bispecific antibodies are useful for treating patients with breast cancer cells that are IHC2+ or higher. According to other related embodiments of the present disclosure, methods are provided that include administering an anti-HER2 antibody or anti-HER2 / HER2 antibody disclosed herein to a patient with breast cancer cells that are IHC2+ or higher. Analytical / diagnostic methods known in the art, such as tumor scanning, can be used to determine whether a patient has a castration-resistant tumor.

[0746] In certain embodiments, the present disclosure also includes methods for treating residual cancer in a subject. The term "residual cancer" refers to the presence or persistence of one or more cancerous cells in a subject after treatment with an anti-cancer therapy.

[0747] The protein-drug conjugates of the present disclosure (and therapeutic compositions comprising them) are useful, inter alia, for treating any disease or disorder in which stimulating, activating, and / or targeting the immune response may be beneficial. In particular, protein-drug conjugates comprising the anti-HER2 antibody or anti-HER2 / HER2 antibody of the present disclosure can be used to treat, prevent, and / or ameliorate any disease or disorder associated with or mediated by HER2 expression or activity or the proliferation of HER2+ cells. The mechanism of action achieved by the therapeutic methods of the present disclosure includes the killing of 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. Cells expressing HER2 that can be inhibited or killed using the protein-drug conjugates of the present disclosure include, for example, breast tumor cells.

[0748] According to certain aspects, the present disclosure provides methods for treating a disease or disorder associated with HER2 expression (e.g., breast cancer), comprising administering to a subject one or more of the anti-HER2 protein-drug conjugates or anti-HER2 / HER2 bispecific protein-drug conjugates described elsewhere herein after the subject has been determined to have breast cancer (e.g., IHC2+ breast cancer). For example, the present disclosure includes methods for treating breast cancer, comprising administering to a patient an anti-HER2 antibody or antigen-binding molecule, or a protein-drug conjugate comprising an anti-HER2 / HER2 bispecific antibody or antigen-binding molecule, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or 4 weeks, 2 months, 4 months, 6 months, 8 months, 1 year, or more after the subject has received hormone therapy (e.g., antiandrogen therapy).

[0749] In certain embodiments, the present disclosure also includes the use of an anti-HER2 antibody of the present disclosure in the manufacture of a medicament for the treatment of a disease or disorder (e.g., cancer) associated with or caused by HER2-expressing cells. In one aspect, the present disclosure relates to a protein-drug conjugate comprising an anti-HER2 antibody or antigen-binding fragment, or an anti-HER2 / HER2 bispecific antibody or antigen-binding fragment, disclosed herein, for use in medicine. In one aspect, the present disclosure relates to a compound comprising an antibody-drug conjugate (ADC) disclosed herein, for use in medicine.

[0750] Anti-STEAP2 antibody-drug conjugate In certain embodiments, the protein-drug conjugates, e.g., ADCs, disclosed herein are useful for treating, preventing, and / or ameliorating, inter alia, any disease or disorder associated with or mediated by STEAP2 expression or activity, or treatable by binding to STEAP2 without competition with modified LDL, and / or promoting internalization of the STEAP2 receptor and / or reducing the number of cell surface receptors.

[0751] The protein-drug conjugates of the present disclosure (and therapeutic compositions comprising them) are useful, inter alia, for treating any disease or disorder in which stimulating, activating, and / or targeting the immune response may be beneficial. In particular, the anti-STEAP2 protein-drug conjugates of the present disclosure can be used to treat, prevent, and / or ameliorate any disease or disorder associated with or mediated by STEAP2 expression or activity or the proliferation of STEAP2+ cells. The mechanism of action achieved by the therapeutic methods of the present disclosure includes the killing of STEAP2-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. Cells expressing STEAP2 that can be inhibited or killed using the protein-drug conjugates of the present disclosure include, for example, prostate tumor cells.

[0752] The protein-drug conjugates of the present disclosure can be used to treat primary and / or metastatic tumors occurring in, for example, the prostate, bladder, cervix, lung, colon, kidney, breast, pancreas, stomach, uterus, and / or ovary. In certain embodiments, the protein-drug conjugates of the present disclosure are used to treat one or more of prostate cancer, bladder cancer, cervical cancer, lung cancer, colon cancer, kidney cancer, breast cancer, pancreatic cancer, stomach cancer, uterine cancer, and ovarian cancer. Analytical / diagnostic methods known in the art, such as tumor scanning, can be used to determine whether a patient has a castration-resistant tumor.

[0753] In certain embodiments, the present disclosure also includes methods for treating residual cancer in a subject. The term "residual cancer" refers to the presence or persistence of one or more cancerous cells in a subject after treatment with an anti-cancer therapy.

[0754] According to certain aspects, the present disclosure provides a method for treating a disease or disorder associated with STEAP2 expression (e.g., prostate cancer), comprising administering one or more of the anti-STEAP2 protein-drug conjugates described elsewhere herein to a subject after the subject has been determined to have prostate cancer. For example, the present disclosure includes a method for treating prostate cancer, comprising administering a protein-drug conjugate comprising an anti-STEAP2 antibody or antigen-binding molecule to a patient 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or 4 weeks, 2 months, 4 months, 6 months, 8 months, 1 year, or more after the subject has received hormone therapy (e.g., antiandrogen therapy).

[0755] In certain embodiments, the present disclosure also includes the use of an anti-STEAP2 antibody of the present disclosure in the manufacture of a medicament for the treatment of a disease or disorder (e.g., cancer) associated with or caused by STEAP2-expressing cells. In one aspect, the present disclosure relates to a protein-drug conjugate comprising an anti-STEAP2 antibody or antigen-binding fragment disclosed herein for use in medicine. In one aspect, the present disclosure relates to a compound comprising an antibody-drug conjugate (ADC) disclosed herein for use in medicine.

[0756] Anti-MET antibody-drug conjugate In certain embodiments, the protein-drug conjugates, e.g., ADCs, disclosed herein are useful for treating, preventing, and / or ameliorating, inter alia, any disease or disorder associated with or mediated by MET expression or activity, or treatable by binding to MET without competition with modified LDL, and / or promoting internalization of the MET receptor, and / or reducing the number of cell surface receptors.

[0757] The protein-drug conjugates of the present disclosure (and therapeutic compositions comprising the same) are useful, inter alia, for treating any disease or disorder in which stimulating, activating, and / or targeting the immune response may be beneficial. In particular, the anti-MET or anti-MET / MET bispecific protein-drug conjugates of the present disclosure can be used to treat, prevent, and / or ameliorate any disease or disorder associated with or mediated by MET expression or activity or the proliferation of MET+ cells. The mechanism of action by which the therapeutic methods of the present disclosure are achieved involves the killing of MET-expressing cells in the presence of effector cells, e.g., by CDC, apoptosis, ADCC, phagocytosis, or a combination of two or more of these mechanisms. Cells expressing MET that can be inhibited or killed using the protein-drug conjugates of the present disclosure include, for example, lung tumor cells.

[0758] The protein-drug conjugates of the present disclosure can be used to treat primary and / or metastatic tumors occurring in, for example, the prostate, bladder, cervix, lung, colon, kidney, breast, pancreas, stomach, uterus, and / or ovary. In certain embodiments, the protein-drug conjugates of the present disclosure are used to treat one or more of prostate cancer, bladder cancer, cervical cancer, lung cancer, colon cancer, kidney cancer, breast cancer, pancreatic cancer, stomach cancer, uterine cancer, and ovarian cancer. Analytical / diagnostic methods known in the art, such as tumor scanning, can be used to determine whether a patient has a castration-resistant tumor.

[0759] In certain embodiments, the present disclosure also includes methods for treating residual cancer in a subject. The term "residual cancer" refers to the presence or persistence of one or more cancerous cells in a subject after treatment with an anti-cancer therapy.

[0760] According to certain aspects, the present disclosure provides methods for treating a disease or disorder associated with MET expression (e.g., lung cancer), comprising administering to a subject one or more of the anti-MET or anti-MET / MET bispecific protein-drug conjugates described elsewhere herein after the subject has been determined to have lung cancer. For example, the present disclosure includes methods for treating lung cancer, comprising administering to a patient a protein-drug conjugate comprising an anti-MET or anti-MET / MET bispecific antibody or antigen-binding molecule 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or 4 weeks, 2 months, 4 months, 6 months, 8 months, 1 year, or more after the subject has received hormone therapy (e.g., anti-androgen therapy).

[0761] For example, the anti-MET antibody-drug conjugates and METxMET bispecific antibody-drug conjugates of the present disclosure are useful for treating tumors that express (or overexpress) MET. For example, the anti-MET antibody-drug conjugates and METxMET bispecific antibody-drug conjugates may be used to treat primary and / or metastatic tumors arising in the brain and meninges, oropharynx, lung and bronchial tree, gastrointestinal tract, male and female reproductive tract, muscle, bone, skin and appendages, connective tissue, spleen, immune system, blood-forming cells and bone marrow, liver and urinary tract, and special sensory organs such as the eye. In certain embodiments, anti-MET antibody-drug conjugates and METxMET bispecific antibody-drug conjugates are used to treat one or more of the following cancers: acute myeloid leukemia, adult T-cell leukemia, astrocytoma, bladder cancer, breast cancer, cervical cancer, cholangiocarcinoma, chronic myeloid leukemia, colorectal cancer, endometrial cancer, esophageal cancer, gastric cancer (e.g., gastric cancer with MET amplification), glioblastoma, head and neck cancer (e.g., head and neck squamous cell carcinoma [HNSCC]), Kaposi's sarcoma, kidney cancer, leiomyosarcoma, liver cancer, lung cancer (e.g., non-small cell lung cancer [NSCLC]), lymphoma, malignant glioma, malignant mesothelioma, melanoma, mesothelioma, MFH / fibrosarcoma, multiple myeloma, nasopharyngeal carcinoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, rhabdomyosarcoma, small cell lung cancer, synovial sarcoma, thyroid cancer, and Wilms' tumor.

[0762] In certain embodiments, the present disclosure also includes the use of an anti-MET antibody-drug conjugate or a METxMET bispecific antibody-drug conjugate of the present disclosure in the manufacture of a medicament for the treatment of a disease or disorder associated with or caused by MET-expressing cells (e.g., cancer). In one aspect, the present disclosure relates to a protein-drug conjugate comprising an anti-MET antibody-drug conjugate or a METxMET bispecific antibody-drug conjugate disclosed herein for use in medicine. In one aspect, the present disclosure relates to a compound comprising an antibody-drug conjugate (ADC) disclosed herein for use in medicine.

[0763] Combination Therapies and Formulations The present disclosure provides methods comprising administering a pharmaceutical composition comprising any of the exemplary protein-drug conjugates (e.g., antibody-drug conjugates), linker-payloads, and payloads described herein in combination with one or more additional therapeutic agents. Exemplary additional therapeutic agents that may be combined or administered in combination with the protein-drug conjugates (e.g., antibody-drug conjugates), linker-payloads, and payloads of the present disclosure include, for example, a HER2 antagonist (e.g., an anti-HER2 antibody [e.g., trastuzumab] or a small molecule inhibitor of HER2, or an anti-HER2 antibody-drug conjugate, or an anti-HER2 / HER2 bispecific antibody or anti-HER2 / HER2 bispecific antibody-drug conjugate), an EGFR antagonist (e.g., an anti-EGFR antibody [e.g., cetuximab or panitumumab] or a small molecule inhibitor of EGFR [e.g., gefitinib or erlotinib]), a HER2 / ErbB2, ErbB3, or other HER2 / ErbB2 inhibitors, as well as other HER2 / ErbB2 / ErbB3 inhibitors. or an antagonist of another EGFR family member such as ErbB4 (e.g., an anti-ErbB2, anti-ErbB3, or anti-ErbB4 antibody, or a small molecule inhibitor of ErbB2, ErbB3, or ErbB4 activity), an antagonist of EGFRvIII (e.g., an antibody that specifically binds to EGFRvIII), a cMET antagonist (e.g., an anti-cMET antibody), an IGF1R antagonist (e.g., an anti-IGF1R antibody), a B-raf inhibitor (e.g., vemurafenib, sorafenib, gDC-0879, PLX-4720), a PDGFR-α inhibitor (e.g., an anti-PDGFR-α antibody), a PDGFR-β inhibitor (e.g., an anti-PDGFR-β antibody), a VEGF antagonist (e.g., a VEGF trap, e.g., U.S. Pat. No. 7,087,087,411 (also referred to herein as "VEGF inhibitory fusion proteins"), anti-VEGF antibodies (e.g., bevacizumab), small molecule kinase inhibitors of VEGF receptors (e.g., sunitinib, sorafenib, or pazopanib), DLL4 antagonists (e.g., anti-DLL4 antibodies disclosed in US2009 / 0142354), Ang2 antagonists (e.g., anti-Ang2 antibodies disclosed in US2011 / 0027286, e.g., H1H685P). , FOLH1 (PSMA) antagonists, PRLR antagonists (e.g., anti-PRLR antibodies), STEAP1 or STEAP2 antagonists (e.g., anti-STEAP1 antibodies 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), uroplakin antagonists (e.g., anti-uroplakin antibodies), and the like.

[0764] Other agents that may be beneficially administered in combination with the protein-drug conjugates (e.g., antibody-drug conjugates), linker-payloads, and payloads of the present disclosure include cytokine inhibitors, including small molecule cytokine inhibitors and antibodies that bind to cytokines such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-11, IL-12, IL-13, IL-17, IL-18, or their respective receptors. Pharmaceutical compositions of the present disclosure (e.g., pharmaceutical compositions comprising an anti-HER2, anti-HER2 / HER2 bispecific, anti-MET, anti-MET / MET bispecific, or anti-STEAP2 protein-drug conjugate (e.g., an antibody-drug conjugate disclosed herein)) also include other anti-cancer drugs, such as: "ICE": ifosfamide (e.g., Ifex®), carboplatin (e.g., Paraplatin®), etoposide (e.g., Etopophos®, Toposar®, VePesid®, VP-16); "DHAP": dexamethasone (e.g., Dec adron®), cytarabine (e.g., Cytosar-U®, cytosine arabinoside, ara-C), cisplatin (e.g., Platinol®-AQ); and "ESHAP": etoposide (e.g., Etopophos®, Toposar®, VePesid®, VP-16), methylprednisolone (e.g., Medrol®), high-dose cytabine, cisplatin (e.g., Platinol®-AQ).

[0765] The present disclosure also includes therapeutic combinations comprising any of the protein-drug conjugates (e.g., antibody-drug conjugates), linker-payloads, and payloads mentioned herein and one or more inhibitors of HER2, VEGF, Ang2, DLL4, EGFR, ErbB2, ErbB3, ErbB4, EGFRvIII, cMet, IGF1R, B-raf, PDGFR-α, PDGFR-β, FOLH1 (PSMA), PRLR, STEAP1, STEAP2, TMPRSS2, MSLN, CA9, uroplakin, or any of the foregoing cytokines, wherein the inhibitor is an aptamer, antisense molecule, ribozyme, siRNA, peptibody, nanobody, or antibody fragment (e.g., a Fab fragment, F(ab')2 fragment, Fd fragment, Fv fragment, scFv, dAb fragment, or diabody, triabody, tetrabody, minibody, and minimal recognition unit). The antigen-binding molecules of the present disclosure may also be administered and / or co-formulated in combination with antiviral drugs, antibiotics, analgesics, corticosteroids, and / or NSAIDs. The antigen-binding molecules of the present disclosure may also be administered as part of a treatment regimen that also includes radiation therapy and / or conventional chemotherapy.

[0766] The additional therapeutically active ingredient(s) may be administered immediately before, simultaneously with, or immediately after administration of the antigen-binding molecule of the present disclosure (for purposes of the present disclosure, such an administration regimen is considered administration of the antigen-binding molecule "in combination" with the additional therapeutically active ingredient).

[0767] The present disclosure includes pharmaceutical compositions in which a protein-drug conjugate (e.g., antibody-drug conjugate), linker-payload and / or payload of the present disclosure is co-formulated with one or more of the additional therapeutically active ingredient(s) described elsewhere herein.

[0768] Dosing regimen According to certain embodiments of the present disclosure, multiple doses of a protein-drug conjugate (e.g., an anti-HER2, anti-HER2 / HER2 bispecific, anti-MET, anti-MET / MET bispecific, or anti-STEAP2 antibody-drug conjugate), linker-payload, and / or payload may be administered to a subject over a predetermined time course. Methods according to this aspect of the present disclosure include sequentially administering to a subject multiple doses of a protein-drug conjugate (e.g., an anti-HER2, anti-HER2 / HER2 bispecific, anti-MET, anti-MET / MET bispecific, or anti-STEAP2 antibody-drug conjugate), linker-payload, and / or payload of the present disclosure. As used herein, "sequentially administering" means that each dose of a protein-drug conjugate (e.g., an anti-HER2, anti-HER2 / HER2 bispecific, anti-MET, anti-MET / MET bispecific, or anti-STEAP2 antibody-drug conjugate), linker-payload, and / or payload is administered to a subject at different time points, e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks, or months). The present disclosure includes methods comprising sequentially administering to a patient a single initial dose of a protein-drug conjugate (e.g., an anti-HER2, anti-HER2 / HER2 bispecific, anti-MET, anti-MET / MET bispecific, or anti-STEAP2 antibody-drug conjugate), a linker-payload, and / or a payload, followed by one or more secondary doses of the protein-drug conjugate (e.g., an anti-HER2, anti-HER2 / HER2 bispecific, anti-MET, anti-MET / MET bispecific, or anti-STEAP2 antibody-drug conjugate), a linker-payload, and / or a payload, and optionally, then one or more tertiary doses of the protein-drug conjugate (e.g., an anti-HER2, anti-HER2 / HER2 bispecific, anti-MET, anti-MET / MET bispecific, or anti-STEAP2 antibody-drug conjugate), a linker-payload, and / or a payload.

[0769] The terms "initial dose," "secondary dose," and "tertiary dose" refer to the time sequence of administration of a protein-drug conjugate (e.g., an anti-HER2, anti-HER2 / HER2 bispecific, anti-MET, anti-MET / MET bispecific, or anti-STEAP2 antibody-drug conjugate), linker-payload, and / or payload of the present disclosure. Thus, a "initial dose" is a dose administered at the beginning of a treatment regimen (also referred to as a "baseline dose"), a "secondary dose" is a dose administered after the initial dose, and a "tertiary dose" is a dose administered after the secondary dose. The initial, secondary, and tertiary doses may all contain the same amount of protein-drug conjugate (e.g., an anti-HER2, or anti-HER2 / HER2 bispecific, anti-MET, anti-MET / MET bispecific, or anti-STEAP2 antibody-drug conjugate), linker-payload, and / or payload, but generally may differ from one another with respect to administration frequency. However, in certain embodiments, the initial dose, secondary dose, and / or tertiary dose contain different amounts of protein-drug conjugate (e.g., anti-HER2, anti-HER2 / HER2 bispecific, anti-MET, anti-MET / MET bispecific, or anti-STEAP2 antibody-drug conjugate), linker-payload, and / or payload during the course of treatment (e.g., adjusted accordingly). In certain embodiments, two or more doses (e.g., 2, 3, 4, or 5) are administered as "loading doses" at the beginning of the treatment regimen, followed by subsequent doses (e.g., "maintenance doses") administered on a less frequent basis.

[0770] In one exemplary embodiment of the present disclosure, each secondary dose and / or tertiary dose is administered within 1 to 26 (e.g., 1, 1½, 2, 2½, 3, 3½, 4, 4½, 5, 5½, 6, 6½, 7, 7½, 8, 8½, 9, 9½, 10, 10½, 11, 11½, 12, 12½, 2, 24, 25, 25, 26, 26, or more) weeks. The phrase "immediately preceding dose" as used herein refers to the dose of protein-drug conjugate (e.g., anti-HER2, anti-HER2 / HER2 bispecific, anti-MET, anti-MET / MET bispecific, or anti-STEAP2 antibody-drug conjugate), linker-payload, and / or payload administered to a patient prior to administration of the next dose in the sequence with no intervening doses in a multiple administration series.

[0771] Methods according to this aspect of the disclosure can include administering any number of secondary and / or tertiary doses of protein-drug conjugate (e.g., anti-HER2, anti-HER2 / HER2 bispecific, anti-MET, anti-MET / MET bispecific, or anti-STEAP2 antibody-drug conjugate), linker-payload, and / or payload to the patient. For example, in certain embodiments, only a single secondary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered to the patient. Similarly, in certain embodiments, only a single tertiary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiary doses are administered to the patient.

[0772] In embodiments involving multiple secondary doses, each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 1-2 weeks after the immediately preceding dose. Similarly, in embodiments involving multiple tertiary doses, each tertiary dose may be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 2-4 weeks after the immediately preceding dose. Alternatively, the frequency with which the secondary and / or tertiary doses are administered to the patient may vary over the course of the treatment regimen. The administration frequency may also be adjusted by the physician during the course of treatment depending on the needs of the individual patient after clinical testing. [Example]

[0773] The following examples illustrate certain aspects of the present invention and should not be construed as limiting, as the examples merely provide a more specific understanding and practice of the embodiments and their various aspects.

[0774] [Table 15-1]

[0775] [Table 15-2]

[0776] General Method Example 1. Payload synthesis Two synthetic routes were designed to generate prodrugs of DXd as shown in Scheme 1. Route A used exatecan reacting with 4. See Scheme 2A. Route B used DXd reacting with 3. See Scheme 2B. Scheme 1. Two methods for synthesizing ProDXd according to the present disclosure

[0777] [ka] Scheme 2A. General Synthesis of ProDXd (Route A)

[0778] [ka] Scheme 2B. General Synthesis of ProDXd (Route B)

[0779] [ka] Scheme 2C. Synthesis of P8

[0780] [ka] Scheme 2D. Synthesis of P10

[0781] [ka]

[0782] Example 2. Linker-Payload Synthesis The five synthetic routes are summarized in Scheme 3 below, based on the final step of the route. All building blocks (A-F) have suitable reactive moieties to be used in the reaction. The synthetic schemes for the building blocks and the final linker-payload are shown below. Route 1 used fragment F with exatecan. • Route 2 used fragment E with DXd. Route 3 used fragment D with a prodrug or an Fmoc-protected prodrug. Route 4 used fragment B with a vcPABC-prodrug Route 5 used fragment A with a PEG4-vcPABC-prodrug Scheme 3. Building blocks and methods for the synthesis of linker-payloads.

[0783] [ka]

[0784] Intermediates A and B can be purchased commercially or reported building blocks bearing functional groups that can be conjugated to antibodies via, for example, bioorthogonal ("click") reactions (Table A).

[0785] [Table 16-1]

[0786] [Table 16-2] Scheme 4A. General Linker-Payload Synthesis (Route 1)

[0787] [ka]

[0788] Conditions: Exatecan, HATU, DIPEA, DMF, 25°C, 16 hours Scheme 4B. General Linker-Payload Synthesis (Route 2)

[0789] [ka]

[0790] [Conditions] DXd, Tf2NH, 4A MS, THF, 20℃, 10 minutes Scheme 4C. General Synthesis of Linker-Payload (Route 3a)

[0791] [ka]

[0792] [Conditions] Intermediate D, coupling catalyst 4-hydroxy-2-methylquinoline (MeHYQ), DIPEA, DMF, room temperature, 2 hours Scheme 4D. General Linker-Payload Synthesis (Route 3b)

[0793] [ka]

[0794] [Conditions] Intermediate D, coupling catalyst 4-hydroxy-2-methylquinoline (MeHYQ), Et3N, DBU, DMF, 50°C, 6 hours. Scheme 4E. General Linker-Payload Synthesis (Route 4)

[0795] [ka]

[0796] Conditions: Step 1: a) Fmoc-vcPAB-PNP, coupling reagent 4-hydroxy-2-methylquinoline (MeHYQ), DIPEA, DMF, room temperature, 4 hours; b) EtNH, DMF, room temperature, 2 hours. Step 2: Intermediate B, HATU, DIPEA, DMF, room temperature, 4 hours. Scheme 4F. General Linker-Payload Synthesis (Route 5)

[0797] [ka] Scheme 5A. General Synthesis of EvcPAB-Linker-Payload

[0798] [ka] Scheme 5B. General Synthesis of Branched Linker-Payload LP13 and LP13C

[0799] [ka] Scheme 5C. General Synthesis of Branched GGFG-Linker-Payload LP15 and LP15C ("GGFG" is disclosed as SEQ ID NO: 2142)

[0800] [ka] (SEQ ID NOs: 2125-2126, 2119 and 2119, or 2120 and 2120, respectively) Scheme 5D. Synthesis of Carbonate-DXd LP16

[0801] [ka] Scheme 5E. Synthesis of Linker-DXd LP17

[0802] [ka]

[0803] Example 3. Synthesis of Key Intermediates / Building Blocks Intermediate A was prepared according to Scheme 6 and the following description. Scheme 6. Synthesis of intermediate Aa

[0804] [ka] [1]KO t Bu, CHBr3, hexane, -10 to 25°C, 16 hours [2] Methyl glycolate, AgOTf, DCM, 25°C, 1 hour [3] 30% NaOMe in MeOH, DMSO, 25°C, 2 hours, 47% yield from A-1 [4] DCC, HOSu, DCM, 0~25℃, 16 hours, crude.

[0805] The synthesis of intermediate A-4 (COT) is reported in WO2010106245, and the synthesis of intermediate A is reported in WO2015143092, both of which are incorporated herein by reference in their entirety. (Scheme 6)

[0806] Intermediate B was prepared according to Scheme 7 and the following description. Scheme 7. Synthesis of Intermediate B

[0807] [ka]

[0808] The synthesis of intermediate B is reported in WO2019094395 and described in Scheme 7 above.

[0809] The synthesis of intermediates 4a-h is described above in Scheme 2A. Intermediate 4a is reported in WO2015146132 (Scheme 8). Alternatively, intermediate 4a was prepared by a two-step synthesis in 45% overall yield without chromatographic purification. Scheme 8. Synthesis of intermediate 4a.

[0810] [ka] [1]Pb(OAc)4 (1.5-2.0 equivalents), DMF, 25°C, 16 hours, 80% yield (10 g), [2] Benzyl glycolic acid, 1,2-dichloroethane, pyridinium p-toluenesulfonate (PPTS), 45-50°C, 18 hours, yield 53% (0.16 g), [3]Pd-C, H2, methanol, THF, 25°C, 16 hours, 67% yield (90 mg), 28% overall yield of 4a over three steps. Alternatively, 4a was prepared on a larger scale by the following two-step procedure*. Scheme 8a. Large-scale synthesis of 4a

[0811] [ka] Step 1: Cu(OAc)2 (0.30 equiv.), Pb(OAc)4 (1.5-2.0 equiv.), pyridine (2.0 equiv.), THF, 25°C, 16 hours, 60% yield (0.80 kg). Step 2: Glycolic acid, 1,2-dichloroethane, pyridinium p-toluenesulfonate (PPTS), 45-50°C, 18 hours, 75% yield (0.96 kg). Scheme 9. Two-step synthesis of compound 4

[0812] [ka]

[0813] Conditions: Step 1: Cu(OAc) (0.30 equiv.), Pb(OAc) (1.5-2.0 equiv.), pyridine (2.0 equiv.), THF, 25 °C, 16 h; Step 2: glycolic acid, 1,2-dichloroethane, pyridinium p-toluenesulfonate (PPTS), 45-50 °C, 18 h.

[0814] Two synthetic routes were summarized to produce intermediate D in Scheme 10. All building blocks have suitable reactive moieties to be used in the reaction. The synthetic scheme for the building blocks and final intermediate D is shown below. Scheme 10. Building blocks for intermediate D.

[0815] [ka] • Route Da was from A to A-PEG4 (B), A-PEG4-vcPAB, and A-PEG4-vcPAB-PNP (D). Route Db was from A with A-PEG4-vcPAB (B) to A-PEG4-vcPAB-PNP (D). Scheme 11A. Synthesis of Intermediate D (Route Da)

[0816] [ka] Scheme 11B. Synthesis of Intermediate D (Route Db)

[0817] [ka] [1]DCC, HOSu, DCM, 0~25℃, 2 hours, [2]vcPAB, DMF, 0-25°C, 16 hours. 73% yield in two steps from Fmoc-amino-PEG4-acid (D-1). [3] a) DBU, Et3N, DMF, 25°C, 16 hours, b) Intermediate Aa, 0-25°C, 1 hour, 54% yield or a) Et2NH, MeOH, room temperature, 1 hour, b) intermediate Ad, HATU, Et3N, DMF, room temperature, 4 hours. [4] PNP, DIPEA, DMAP, DMF, 0-25°C, 4 hours, 37% yield. Scheme 12. General synthesis of intermediate E

[0818] [ka] [Step 1] a) Compound 2, DBU, Et3N, DMF, 25°C, 16 hours, b) Intermediate D, HOAt, DIPEA, 25°C, 4 hours, [Step 2] Pb(OAc), HOAc, DMF, 25°C, 16 hours Scheme 13. General synthesis of intermediate F

[0819] [ka] [Step 1] a) Intermediate 4, triethylamine, DBU, DMF, 25°C, 16 hours, b) HOAt, Intermediate D, 25°C, 16 hours Scheme 14. Summary synthetic process of LP1*

[0820] [ka] *Procedures and conditions Step [1] KO tBu, CHBr3, hexane, -10 to 25°C, 16 hours Step [2] Methyl glycolate, AgOTf, DCM, 25°C, 1 hour, Step [3] 30% NaOMe in MeOH, DMSO, 25°C, 2 hours, 47% yield from A-1 Step [4] DCC, HOSu, DCM, 0-25°C, 16 hours, crude. Step [5] DCC, HOSu, DCM, 0-25℃, 2 hours, Step [6] vcPAB, DMF, 0-25°C, 16 hours. 73% yield in two steps from Fmoc-amino-PEG4-acid (D-1). Step [7] a) DBU, Et3N, DMF, 25°C, 16 hours, b) Intermediate A, 0-25°C, 1 hour, yield 54% Step [8] PNP, DIPEA, DMAP, DMF, 0–25°C, 4 hours, 37% yield. Step [9] Cu(OAc)2 (0.30 equiv.), Pb(OAc)4 (1.5-2.0 equiv.), pyridine (2.0 equiv.), THF, 25°C, 16 hours, 60% yield (0.80 kg). Step

[10] Glycolic acid, 1,2-dichloroethane, pyridinium p-toluenesulfonate (PPTS), 45-50°C, 18 hours, yield 75% (0.96 kg), Step

[11] Exatecan, HATU, DMF, 25°C, 3 hours, yield 86% (14g), Step

[12] Intermediate D, MeHYQ (4-methyl-2-hydroxyquinoline), Et3N, DBU, DMF, 25°C, 16 hours, yield 58% (13 g).

[0821] Example 4. Conjugation Site-specific ADC conjugation is shown in FIG.

[0822] Step 1 is the site-specific conjugation of a handle-functionalized amine with an antibody, generating drug conjugates containing 2, 4, or 8 handles per antibody, where AL = unbranched handle-functionalized amine and BL = branched handle-functionalized amine.

[0823] Step 2 is a click reaction between the handle-functionalized antibody and the linker-payload (LP) to generate a site-specific ADC.

[0824] Payload synthesis Example 5. General synthesis of ProDXd (Scheme 2A) General procedure for the synthesis of compound 2s To a stirred solution of Fmoc-protected amino acid 1 (1 equiv.) in DCM (0.2 M) was added HOSu (2.2 equiv.) and EDCI (2.2 equiv.), and the reaction mixture was stirred at room temperature for 2-16 h and monitored by LCMS. The mixture was diluted with DCM, washed with water (3 times) and brine, dried over anhydrous sodium sulfate, and concentrated in vacuo. The residue was dissolved in DMF (0.2 M). The solution was added with the corresponding amino acid (R 3 NHCHR 4 COOH (1.0 equiv.) and DIPEA (3.0 equiv.) were added, and the reaction mixture was stirred at room temperature for 1 h and monitored by LCMS. Volatiles were removed in vacuo, and the residual solution was directly purified by reverse-phase flash chromatography (0-100% acetonitrile in aqueous TFA (0.03%)) to give compound 2 (37-70% yield) as a white solid.

[0825] 2-[2-({[(9H-Fluoren-9-yl)methoxy]carbonyl}amino)acetamido]acetic acid (2a)

[0826] [ka] It is commercially available.

[0827] 2-[(2S)-3-[(tert-butyldimethylsilyl)oxy]-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)propanamido]acetic acid (2b)

[0828] [ka] Following the general procedure, compound 2b (0.50 g, 54% yield) was obtained as a white solid. ESI m / z: 499 (M+H). + .

[0829] 2-[(2S)-5-(benzyloxy)-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-5-oxopentanamido]acetic acid (2c)

[0830] [ka] Following the general procedure, compound 2c (0.65 g, 58% yield) was obtained as a white solid. ESI m / z: 517 (M+H) + .

[0831] 2-[(2S)-6-Azido-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)hexanamide]acetic acid (2d)

[0832] [ka] Following the general procedure, compound 2d (0.66 g, 70% yield) was obtained as a white solid. ESI m / z: 452 (M+H). + .

[0833] 2-[(2S)-2-({[(9H-Fluoren-9-yl)methoxy]carbonyl}amino)-3-phenylpropanamido]acetic acid (2e)

[0834] [ka] Following the general procedure, compound 2e (0.43 g, 37% yield) was obtained as a white solid. ESI m / z: 445 (M+H) + .

[0835] 2-[2-({[(9H-Fluoren-9-yl)methoxy]carbonyl}amino)-N-methylacetamido]acetic acid (2f)

[0836] [ka] Following the general procedure, compound 2f (2.6 g, 72% yield) was obtained as a white solid. ESI m / z: 369 (M+H). + .

[0837] 2-[2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-2-methylpropanamido]acetic acid (2g)

[0838] [ka] Following the general procedure, compound 2g was obtained as a white solid (0.60 g, 50% yield). ESI m / z: 383 (M+H). + .

[0839] 2-[(2R)-2-({[(9H-Fluoren-9-yl)methoxy]carbonyl}amino)-3-phenylpropanamido]acetic acid (2h)

[0840] [ka] Following the general procedure, compound 2e (0.43 g, 37% yield) was obtained as a white solid. ESI m / z: 445 (M+H) + .

[0841] General procedure for the synthesis of compounds 3a-h THF (0.25-0.30 M) and compound 2 (1.0 equiv.) were added to a 10 L reaction flask at 25-30 °C. To the resulting suspension, pyridine (2.0 equiv.) was added at 25-30 °C. After the mixture was stirred and clarified, copper acetate (0 or 0.3 equiv.) was added to the solution. The reaction mixture was cooled to 0-5 °C, and lead(IV) acetate (1.5 equiv.) was added to the reaction mixture at 0-5 °C. The mixture was then stirred at 0-5 °C for 1 h and then warmed to 25-30 °C. The reaction mixture was stirred at 25-30 °C for 16 h until most of compound 2 was consumed, which was monitored by LCMS. The resulting mixture was filtered through a short silica gel plug, and the silica gel was washed with ethyl acetate (twice). The combined filtrate was diluted with ethyl acetate and water. After carefully neutralizing to pH 7 with sodium bicarbonate powder, the mixture was separated, and the organic layer was washed with brine, dried over anhydrous sodium sulfate, and concentrated in vacuo to give a brown crude product, which was dissolved in DCM (3 L). The mixture was filtered through a short silica gel plug eluting with DCM (3 times) until compound 3 was completely collected. The collected solution was concentrated. MTBE was added to the residue, and a white solid precipitated at 25-30 °C, which was collected by filtration. The solid was dried with nitrogen blow at 25-30 °C for more than 16 hours to give pure compound 3 as a white solid. Alternatively, the brown crude product was purified by reverse-phase flash chromatography or preparative HPLC to give pure compound 3 as a white solid.

[0842] [2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)acetamido]methyl acetate (3a)

[0843] [ka] Following the general procedure (catalyzed with copper acetate (0.3 equiv.)), compound 3a (1.3 kg, 60% yield) was obtained as a white solid. ESI m / z: 391 (M+Na). + . 1 H NMR (400 MHz, DMSO d6)δ 8.96(t,J=6.8Hz,1H), 7.90(d,J=7.6Hz,2H), 7.72(d,J=7.2Hz,2H), 7.59(t,J=6.0Hz,1H), 7.43(t,J=7.2Hz, 2H), 7.34(t,J=7.2Hz,2H), 5.10(d,J=7.2Hz,2H), 4.36-4.19(m,3H), 3.66(d,J=6.0Hz,2H), 2.00(s,3H)ppm.

[0844] [(2S)-3-[(tert-butyldimethylsilyl)oxy]-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)propanamide]methyl acetate (3b)

[0845] [ka] Following the general procedure without copper acetate, compound 3b (0.19 g, 45% yield) was obtained as a white solid after purification by preparative HPLC (0-100% acetonitrile in aqueous formic acid (0.1%)). ESI m / z: 535 (M+Na). + .

[0846] Benzyl (4S)-4-{[(acetyloxy)methyl]carbamoyl}-4-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)butanoate (3c)

[0847] [ka] Following the general procedure without copper acetate, compound 3c (0.20 g, 30% yield) was obtained as a white solid after purification by reverse-phase flash chromatography (0-60% acetonitrile in aqueous formic acid (0.1%)). ESI m / z: 553 (M+Na). + .

[0848] [(2S)-6-Azido-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)hexanamide]methyl acetate (3d)

[0849] [ka] Following the general procedure without copper acetate, compound 3d (0.57 g, 84% yield) was obtained as a white solid after purification by preparative HPLC (0-100% acetonitrile in aqueous formic acid (0.1%)). ESI m / z: 488 (M+Na). + .

[0850] [(2S)-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-3-phenylpropanamide]methyl acetate (3e)

[0851] [ka] Following the general procedure without copper acetate, compound 3c (0.36 g, 81% yield) was obtained as a white solid after purification by preparative HPLC (0-100% acetonitrile in aqueous formic acid (0.1%)). ESI m / z: 481 (M+Na). + . 1 H NMR(400MHz,DMSO)δ 9.13(t,J=6.9Hz,1H), 7.88(d,J=7.5Hz,2H), 7.71(d,J=8.7Hz,1H), 7.67-7.58(m,2H), 7.46-7.36(m,2H), 7.35-7.23(m, 6H), 7.19(t,J=7.1Hz,1H), 5.18-5.04(m,2H), 4.32-4.21(m,1H), 4.21-4.07(m,3H), 3.05-2.73(m,2H), 2.00(s,3H)ppm.

[0852] [2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-N-methylacetamido]methyl acetate (3f)

[0853] [ka] Following the general procedure without copper acetate, compound 3f (1.65 g, 60% yield) was obtained as a white solid. ESI m / z: 405 (M+Na).+ .

[0854] [2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-2-methylpropanamide]methyl acetate (3g)

[0855] [ka] Following the general procedure (catalyzed with copper acetate (0.3 equiv.)), compound 3g (0.36 g, 81% yield) was obtained as a white solid after purification by preparative HPLC (0-100% acetonitrile in aqueous formic acid (0.1%)). ESI m / z: 481 (M+Na). + .

[0856] [(2R)-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-3-phenylpropanamide]methyl acetate (3h)

[0857] [ka] Following the general procedure (catalyzed with copper acetate (0.3 equiv.)), after purification by preparative HPLC (0-100% acetonitrile in aqueous formic acid (0.1%)), 0.34 g (80% yield) was obtained as a white solid. ESI m / z: 481 (M+Na). + .

[0858] General procedure for the synthesis of compounds 4a-h A reaction flask was charged with 1,2-dichloroethane (0.10–0.15 M), compound 3 (1.0 equiv.), glycolic acid (0.6 equiv.), and pyridinium p-toluenesulfonate (PPTS) (0.2 equiv.) at room temperature. The reaction mixture was heated to 45–50 °C and stirred for 1 h. Glycolic acid (0.6 equiv., 2 times) was added to the hot solution, once per hour. The mixture was then stirred at 45–50 °C for 16 h and monitored by LCMS. After cooling to 25–30 °C, the precipitate was filtered and collected. The solid was dissolved in aqueous sodium bicarbonate (3%) to obtain a pH 7–8 mixture at 5–10 °C. This mixture was washed with a mixture of ethyl acetate and THF (v / v = 1, 3 times). MTBE was added to the aqueous layer at 5–10 °C, and the mixture was acidified with saturated aqueous citric acid to a pH of 3–4, precipitating a large amount of solid. The mixture was filtered, and the cake was washed with water (once) and MTBE (twice) and dried under nitrogen blowing at 25-30 °C for 48 h to give wet compound 4 (75% yield) as a white solid containing 3% water according to HNMR. The product was again dried in vacuo for 48 h to give dry compound 4 (73% yield) as a white solid. Alternatively, the reaction mixture was directly purified by reverse-phase flash chromatography or preparative HPLC to give pure compound 4 as a white solid.

[0859] 2-{[2-({[(9H-Fluoren-9-yl)methoxy]carbonyl}amino)acetamido]methoxy}acetic acid (4a)

[0860] [ka] Following the general procedure, compound 4a (0.96 g, 80% yield) was obtained as a white solid. >99% in HPLC, ESI m / z: 407 (M+Na). + . 1 H NMR (400 MHz, DMSO d6)δ 12.53(br s,1H), 8.72(t,J=6.8Hz,1H), 7.90(d,J=7.2Hz,2H), 7.72(d,J=7.6Hz,2H), 7.59(t,J=6.4Hz,1H), 7.42(d,J=7.6 Hz,2H), 7.33(d,J=7.2Hz,2H), 4.60(d,J=6.8Hz,2H), 4.31-4.18(m,3H), 3.98(s,2H), 3.62(d,J=6.0Hz,2H)ppm.

[0861] 2-{[(2S)-3-[(tert-butyldimethylsilyl)oxy]-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)propanamido]methoxy}acetic acid (4b)

[0862] [ka] Following the general procedure, compound 4b (57 mg, 31% yield) was obtained as a yellow solid after purification by preparative HPLC (0-100% acetonitrile in aqueous TFA (0.05%)). ESI m / z: 551 (M+Na). + .

[0863] 2-{[(2S)-5-(benzyloxy)-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-5-oxopentanamido]methoxy}acetic acid (4c)

[0864] [ka] Following the general procedure, compound 4c (0.13 g, 65% yield) was obtained as a white solid after purification by preparative HPLC (0-90% acetonitrile in aqueous formic acid (0.1%)). ESI m / z: 569 (M+Na). + .

[0865] 2-{[(2S)-6-Azido-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)hexanamido]methoxy}acetic acid (4d)

[0866] [ka] Following the general procedure, compound 4d (0.30 g, 51% yield) was obtained as a white solid after purification by preparative HPLC (5-95% acetonitrile in aqueous ammonium bicarbonate (10 mM)). ESI m / z: 504 (M+Na). + .

[0867] 2-{[(2S)-2-({[(9H-Fluoren-9-yl)methoxy]carbonyl}amino)-3-phenylpropanamido]methoxy}acetic acid (4e)

[0868] [ka] Following the general procedure, compound 4e (0.14 g, 38% yield) was obtained as a white solid after purification by reverse-phase flash chromatography (0-25% acetonitrile in water). ESI m / z: 474 (M+Na). + .

[0869] 2-{[2-({[(9H-Fluoren-9-yl)methoxy]carbonyl}amino)-N-methylacetamido]methoxy}acetic acid (4f)

[0870] [ka] Following the general procedure, compound 4f (1.0 g, 50% yield) was obtained as a white solid after purification by reverse-phase flash chromatography (0-100% acetonitrile in aqueous TFA (0.01%)). ESI m / z: 421 (M+Na). + .

[0871] 2-{[2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-2-methylpropanamido]methoxy}acetic acid (4g)

[0872] [ka] Following the general procedure, compound 4g (0.10 g, 40% yield) was obtained as a white solid after purification by reverse-phase flash chromatography (0-100% acetonitrile in aqueous TFA (0.01%)). ESI m / z: 435 (M+Na). + .

[0873] 2-{[(2R)-2-({[(9H-Fluoren-9-yl)methoxy]carbonyl}amino)-3-phenylpropanamido]methoxy}acetic acid (4h)

[0874] [ka] Following the general procedure, compound 4h (0.14 g, 38% yield) was obtained as a white solid after purification by reverse-phase flash chromatography (0-100% acetonitrile in aqueous TFA (0.01%)). ESI m / z: 474 (M+Na). + .

[0875] Synthesis of 2-{[2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)acetamido]methoxy}acetic acid (4a) The large scale synthesis of intermediate 4a is described in Scheme 8a.

[0876] [2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)acetamido]methyl acetate (3a) To a 10 L reaction flask, THF (6.7 L) and Fmoc-Gly-Gly-OH (2a) (0.67 kg, 1.9 mol) were added at 25–30 °C. To the resulting suspension, pyridine (0.30 kg, 3.8 mol) was added at 25–30 °C. After the mixture was stirred and clarified, copper acetate (0.10 kg, 0.57 mol) was added to the solution. The reaction mixture was cooled to 0–5 °C, and lead(IV) acetate (1.7 kg, 2.8 mol) was added to the reaction mixture at 0–5 °C. The mixture was then stirred at 0–5 °C for 1 h and then warmed to 25–30 °C. The reaction mixture was stirred at 25–30 °C for 16 h until most of 2a was consumed, which was monitored by LCMS. The resulting mixture was filtered through a short silica gel plug (200 g), and the silica gel was washed with ethyl acetate (1 L × 2). The combined filtrate was diluted with ethyl acetate (10 L) and water (10 L). After careful neutralization to pH 7 with sodium bicarbonate powder, the mixture was separated, and the organic layer was washed with brine (5 L × 1), dried over anhydrous sodium sulfate, and concentrated in vacuo to give a brown crude product. This was combined with the crude product from two other batches (0.60 kg batch and 0.80 kg batch) with similar LCMS and dissolved in DCM (3 L). The mixture was filtered through a short silica gel plug (0.30 kg) eluting with DCM (1 L × 3) until compound 3a was completely collected. The collected solution was concentrated to 2 L. To the residue, MTBE (3 L) was added, and a white solid precipitated at 25-30 °C, which was collected by filtration. The solid was dried with nitrogen blown at 25-30 °C for >16 h to give pure compound 3a (1.3 kg, 60% yield) as a white solid. ESI m / z: 254 (M+H). + . 1 H NMR (400 MHz, DMSO d6 )δ 8.96(t,J=6.8Hz,1H), 7.90(d,J=7.6Hz,2H), 7.72(d,J=7.2Hz,2H), 7.59(t,J=6.0Hz,1H), 7.43(t,J=7.2Hz, 2H), 7.34(t,J=7.2Hz,2H), 5.10(d,J=7.2Hz,2H), 4.36-4.19(m,3H), 3.66(d,J=6.0Hz,2H), 2.00(s,3H)ppm.

[0877] [Table 17]

[0878] 2-{[2-({[(9H-Fluoren-9-yl)methoxy]carbonyl}amino)acetamido]methoxy}acetic acid (4a) To a 50 L jacketed reaction flask, 1,2-dichloroethane (19 L), compound 3a (0.96 kg, 2.6 mol), glycolic acid (0.12 kg, 1.6 mol), and pyridinium p-toluenesulfonate (PPTS) (0.13 kg, 0.52 mol) were added sequentially at 20-25 °C. The reaction mixture was stirred at 45-50 °C for 1 h, and glycolic acid (0.12 kg, 1.6 mol) was added twice to the hot solution, once per hour. The mixture was then stirred at 45-50 °C for 16 h, which was monitored by LCMS. After cooling to 25-30 °C, the precipitate was collected by filtration and combined with another batch with a similar LCMS (200 g, 1.25 kg). The combined solids were dissolved in aqueous sodium bicarbonate (0.44 kg in 15 L of water) to obtain a mixture with a pH of 7–8 at 5–10 °C, which was then washed with a mixture of ethyl acetate and THF (v / v = 1, 4.0 L × 3). MTBE (5 L) was added to the aqueous layer at 5–10 °C, and the mixture was acidified with saturated aqueous citric acid to a pH of 3–4, precipitating a large amount of solid. The mixture was filtered, and the cake was washed with water (1 L) and MTBE (1 L × 2). It was then dried under nitrogen blowing at 25–30 °C for 48 h to obtain compound 4a (1.1 kg, 75% yield) as a white solid (containing 3% water according to HNMR). The product was again dried in vacuo for 48 h to obtain dry compound 4a (0.96 kg, 97% recycled yield from the wet product) as a white solid. >99% in HPLC, ESI m / z: 407 (M+Na). + . 1 H NMR (400 MHz, DMSO d6)δ 12.53(br s,1H), 8.72(t,J=6.8Hz,1H), 7.90(d,J=7.2Hz,2H), 7.72(d,J=7.6Hz,2H), 7.59(t,J=6.4Hz,1H), 7.42(d,J=7.6 Hz,2H), 7.33(d,J=7.2Hz,2H), 4.60(d,J=6.8Hz,2H), 4.31-4.18(m,3H), 3.98(s,2H), 3.62(d,J=6.0Hz,2H)ppm.

[0879] This product contained approximately 0.96% of an unknown contaminant and had an M / Z of 617 (positive mode). This by-product can be removed in the next step. Product C must be thoroughly dried because water reduces the yield in the next step.

[0880] [Table 18]

[0881] General procedure for the synthesis of compounds 5a-h To a solution of compound 4 (1.1 equivalents) in DMF (5–8 mL per gram of 4), HATU (1.1 equivalents) and DIPEA (1.0 equivalents) were added, and the reaction mixture was stirred at room temperature for 15 minutes. Then, to the stirred solution, a mixture of exatecan mesylate (1.0 equivalents) and DIPEA (2.0 equivalents) in DMF (10 mL per gram of exatecan) was added. The reaction mixture was stirred at room temperature for 4 hours and monitored by LCMS. The resulting mixture was diluted with ethyl acetate and washed with brine (twice). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was coevaporated in vacuo with ethyl acetate (four times) to give crude product 5, to which ethyl acetate was added. The suspension was refluxed for approximately 20 minutes until clear, then naturally cooled to 25°C and allowed to stand for 30 minutes. The white precipitate was collected by filtration, washed with ethyl acetate (twice), and dried in vacuo to give 5 as a white solid. Alternatively, the crude product 5 was purified by reverse phase flash chromatography to give pure compound 5 as a solid.

[0882] (9H-Fluoren-9-yl)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)-heptaen-23-yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamate (5a)

[0883] [ka] Following the general procedure, compound 5a (2.6 g, 87% yield) was obtained as a white solid. ESI m / z: 802.3 (M+H) + 95.2% by HPLC.

[0884] (9H-Fluoren-9-yl)methyl N-[(1S)-2-[(tert-butyldimethylsilyl)oxy]-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)-heptaen-23-yl]carbamoyl}methoxy)methyl]carbamoyl}ethyl]carbamate (5b)

[0885] [ka] Following the general procedure, compound 5b (0.12 g, 53% yield) was obtained as a white solid after purification by reverse-phase flash chromatography (0-100% acetonitrile in aqueous TFA (0.05%)). ESI m / z: 946 (M+H). + .

[0886] Benzyl(4S)-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)-heptaen-23-yl]carbamoyl}methoxy)methyl]carbamoyl}-4-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)butanoate (5c)

[0887] [ka] Following the general procedure, compound 5c (0.21 g, 74% yield) was obtained as a yellow solid after purification by reverse-phase flash chromatography (0-70% acetonitrile in aqueous formic acid (0.1%)). ESI m / z: 964 (M+H). + .

[0888] (4S)-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)-heptaen-23-yl]carbamoyl}methoxy)methyl]carbamoyl}-4-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)butanoic acid (5ca)

[0889] [ka] To a stirred solution of compound 5a (0.21 g, 0.22 mmol) in methanol (20 mL) was added palladium on carbon (36 mg, containing 10% palladium) under the protection of nitrogen. The reaction mixture was stirred at room temperature under a hydrogen atmosphere for 4 hours, which was monitored by LCMS. The mixture was filtered through Celite, and the filtrate was concentrated in vacuo to give crude compound 5ca (0.10 g, 53% yield) as a yellow solid, which was used in the next step without further purification. ESI m / z: 874 (M+H). + .

[0890] (9H-Fluoren-9-yl)methyl N-[(1S)-3-carbamoyl-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)-heptaen-23-yl]carbamoyl}methoxy)methyl]carbamoyl}propyl]carbamate (5cb)

[0891] [ka] To a solution of compound 5ca (50 mg, 57 μmol) in DMF (1 mL), ammonium chloride (3.0 mg, 57 μmol), HATU (32 mg, 85 μmol), and DIPEA (22 mg, 0.17 mmol) were added, and the reaction mixture was stirred at room temperature for 3 h, which was monitored by LCMS. The resulting mixture was directly purified by reverse-phase flash chromatography (0-100% acetonitrile in aqueous formic acid (0.1%)) to give compound 5cb (40 mg, 81% yield) as a white solid. ESI m / z: 873 (M+H). + .

[0892] (9H-Fluoren-9-yl)methyl N-[(1S)-5-azido-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)-heptaen-23-yl]carbamoyl}methoxy)methyl]carbamoyl}pentyl]carbamate (5d)

[0893] [ka] Following the general procedure, compound 5d (85 mg, 91% yield) was obtained as a white solid after purification by preparative HPLC (5-95% acetonitrile in aqueous TFA (0.1%)). ESI m / z: 900 (M+H). + .

[0894] (9H-Fluoren-9-yl)methyl N-[(1S)-5-amino-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)-heptaen-23-yl]carbamoyl}methoxy)methyl]carbamoyl}pentyl]carbamate (5da)

[0895] [ka] To a stirred solution of compound 5d (45 mg, 50 μmol) in methanol (20 mL) was added palladium on carbon (10 mg, containing 10% palladium) under the protection of nitrogen. The reaction mixture was stirred at room temperature under a hydrogen atmosphere for 2 hours, which was monitored by LCMS. The mixture was filtered through Celite, the filtrate was concentrated in vacuo, and the residue was purified by preparative HPLC (5-95% acetonitrile in aqueous TFA (0.03%)) to give compound 5da (38 mg, 86% yield) as a yellow solid. ESI m / z: 873 (M+H). + .

[0896] (9H-Fluoren-9-yl)methyl 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)-heptaen-23-yl]carbamoyl}methoxy)methyl]carbamoyl}-2-phenylethyl]carbamate (5e)

[0897] [ka] Following the general procedure, compound 5e (24 mg, 64% yield) was obtained as a white solid after purification by preparative HPLC (5-95% acetonitrile in aqueous TFA (0.1%)). ESI m / z: 892 (M+H). + .

[0898] (9H-Fluoren-9-yl)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)-heptaen-23-yl]carbamoyl}methoxy)methyl](methyl)carbamoyl}methyl)carbamate (5f)

[0899] [ka] Following the general procedure, compound 5f (50 mg, 61% yield) was obtained as a white solid after purification by preparative HPLC (5-95% acetonitrile in aqueous TFA (0.01%)). ESI m / z: 816 (M+H). + .

[0900] (9H-Fluoren-9-yl)methyl N-(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)-heptaen-23-yl]carbamoyl}methoxy)methyl]carbamoyl}-1-methylethyl)carbamate (5g)

[0901] [ka] Following the general procedure, compound 5g (0.17 g, 61% yield) was obtained as a white solid after purification by reverse-phase flash chromatography (0-70% acetonitrile in aqueous TFA (0.01%)). ESI m / z: 830 (M+H). + .

[0902] (9H-Fluoren-9-yl)methyl N-[(1R)-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)-heptaen-23-yl]carbamoyl}methoxy)methyl]carbamoyl}-2-phenylethyl]carbamate (5h)

[0903] [ka] Following the general procedure, compound 5h (92 mg, 73% yield) was obtained as a yellow solid after purification by preparative HPLC (5-95% acetonitrile in aqueous TFA (0.1%)). ESI m / z: 892 (M+H). + .

[0904] Payload (ProDXd) General procedure for de-Fmoc to obtain payload (ProDXd) To a solution of compound 5 (1.0 equiv.) in THF (20 mL per gram of 5), diethylamine (2 mL per gram of 5) was added, and the reaction mixture was stirred at room temperature for 2–48 h until complete removal of Fmoc according to LCMS. Volatiles were completely removed in vacuo, and the residue was diluted with water (5 mL). The aqueous mixture was adjusted to pH 2 by adding aqueous TFA (10%) and washed with MTBE (20 mL × 2). The aqueous layer was then stirred at room temperature for 16 h until the ring-opened form converted to the lactone form, which was monitored by LCMS. The resulting aqueous mixture was lyophilized to give the crude payload, which was purified by reverse-phase flash chromatography (0–100% acetonitrile in aqueous TFA (0.03%)) to give the pure payload (TFA salt) as a solid, or by preparative HPLC (5–95% acetonitrile in aqueous formic acid (0.1%)) to give the pure payload (free base) as a solid.

[0905] P1 2-amino-N-[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl}methoxy)methyl]acetamide (P1)

[0906] [ka] Following the general procedure, P1 (1.4 g, 77% yield) was obtained as a pale yellow solid. ESI m / z: 580.3 (M+H). + . 1 H NMR (400 MHz, DMSO d6)δ 9.20(t,J=6.4Hz,1H), 8.59(d,J=9.2Hz,1H), 8.07(br s,3H), 7.80(d,J=11.2Hz,1H), 7.33(s,1H), 6.57(s,1H), 5.64-5.57(m,1H), 5.43(s,2H), 5.30-5.09(m,2H), 4.77-4.69(m,2H) ), 4.07(s,2H), 3.67(s,2H), 3.28-3.09(m,2H), 2.40(s,3H), 2.26-2.12(m,2H), 1.93-1.80(m,2H), 0.88(t,J=7.2Hz,3H)ppm. 19 F NMR (376 MHz, DMSO d6 )δ-73(TFA), -111(Ar-F)ppm.

[0907] P2 (2S)-2-amino-N-[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl}methoxy)methyl]-3-hydroxypropanamide (P2)

[0908] [ka] To a solution of compound 5b (0.12 g, 0.12 mmol) in DMF (1 mL) was added diethylamine (0.1 mL), and the mixture was stirred at room temperature for 2 h until complete removal of Fmoc, which was monitored by LCMS. The resulting mixture was directly purified by retained-phase flash chromatography (0-100% acetonitrile in aqueous TFA (0.05%)) to give the de-Fmoc-product (68 mg, ESI m / z: 724 (M+H)) as a yellow solid. +) was obtained and dissolved in DMF (1 mL). To the solution, cesium fluoride (31 mg, 0.20 mmol) was added at 0 °C. The mixture was stirred at room temperature for 1 h, which was monitored by LCMS. The mixture was separated by reverse-phase flash chromatography (0-100% acetonitrile in aqueous TFA (0.05%)) to give P2 (17 mg, 22% yield) as a white solid. ESI m / z: 610 (M+H) + . 1 H NMR (400 MHz, DMSO d6 )δ 9.26(t,J=6.4Hz,1H), 8.58(d,J=8.8Hz,1H), 8.14(s,2H), 7.79(d,J=10.8Hz,1H), 7.3 3(s,1H), 6.56(s,1H), 5.60-5.53(m,2H), 5.42(s,2H), 5.20-5.18(m,2H), 4.80-4.76(m ,1H), 4.67-4.63(m,1H), 4.05(s,2H), 3.90-3.89(m,1H), 3.77-3.76(m,2H), 3.27-3.1 4(m,2H), 2.39(s,3H), 2.17-2.16(m,2H), 1.90-1.83(m,2H), 0.87(t,J=6.8Hz,3H)ppm. 19 F NMR (376 MHz, DMSO d6 )δ-74(TFA), -111(Ar-F)ppm.

[0909] P3 (2S)-2-amino-N-[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl}methoxy)methyl]pentanediamide (P3)

[0910] [ka] Following the general procedure, P3 (20 mg, 49% yield) was obtained as a pale yellow solid. ESI m / z: 651 (M+H). + . 1 H NMR (400 MHz, DMSO d6 )δ 8.95-8.80(m,1H),8.80(d,J=8.4Hz,1H),8.18(s,1H),7.81(d,J=11.2Hz,1H),7.31(s,1H),7.2 9-7.20(m,1H), 6.73(s,1H), 6.52(s,1H), 5.65-5.56(m,1H), 5.42(s,2H), 5.21(s,2H), 4.63(br s,2H), 4.01(s,1H), 3.25-3.13(m,2H), 3.06-2.90(m,2H), 2.40(s,3H), 2.25- 2.05(m,4H), 1.93-1.74(m,3H), 1.64-1.50(m,1H), 0.87(t,J=6.8Hz,1H)ppm. 19 F NMR (376 MHz, DMSO d6 )δ-111(Ar-F)ppm.

[0911] P4 (4S)-4-amino-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)-heptaen-23-yl]carbamoyl}methoxy)methyl]carbamoyl}butanoic acid (P4)

[0912] [ka] Following the general procedure, P4 (20 mg, 49% yield) was obtained as a pale yellow solid. ESI m / z: 652 (M+H).+ . 1 H NMR (400 MHz, DMSO d6 )δ 8.95-8.82(m,1H),8.55(d,J=9.2Hz,1H),8.30(s,1H),7.79(d,J=11.2Hz ,1H), 7.31(s,1H), 5.65-5.56(m,1H), 5.42(s,2H), 5.21(s,2H), 4.62(br s,2H), 4.00(s,2H), 3.25-3.10(m,4H), 3.06-2.90(m,2H), 2.32(s,3H), 2.27- 2.12(m,4H), 1.92-1.71(m,3H), 1.60-1.50(m,1H), 0.87(t,J=7.2Hz,3H)ppm. 19 F NMR (376 MHz, DMSO d6 )-111(Ar-F) ppm.

[0913] P5 (2S)-2,6-Diamino-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)-heptaen-23-yl]carbamoyl}methoxy)methyl]hexanamide (P5)

[0914] [ka] Following the general procedure, P5 (13 mg, 43% yield) was obtained as a white solid. ESI m / z: 651 (M+H). + . 1 H NMR (400 MHz, DMSO d6)δ 9.33(t,J=6.5Hz,1H), 8.61(d,J=8.8Hz,1H), 8.18(s,3H), 7.81(d,J=10.9Hz,1H), 7.72(s,2H), 7.34 (s,1H), 6.57(s,1H), 5.63-5.57(m,1H), 5.43(s,2H), 5.26-5.15(m,2H), 4.79-4.67(m,2H), 4.11-4.0 1(m,2H), 3.86-3.80(m,1H), 3.25-3.10(m,2H), 2.81-2.72(m,2H), 2.40(s,3H), 2.22-2.13(m,2H), 1 .92-1.83(m,2H), 1.80-1.71(m,2H), 1.60-1.50(m,2H), 1.40-1.31(m,2H), 0.88(t,J=7.3Hz,3H)ppm. 19 F NMR (376 MHz, DMSO d6 )δ-74(TFA), -111(Ar-F)ppm.

[0915] P6 (2S)-2-Amino-6-azido-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)-heptaen-23-yl]carbamoyl}methoxy)methyl]hexanamide (P6)

[0916] [ka] Following the general procedure, P6 (27 mg, 89% yield) was obtained as a white solid. ESI m / z: 677 (M+H). + . 1 H NMR (400 MHz, DMSO d6)δ 8.92-8.78(m,1H), 8.57(d,J=8.8Hz,1H), 8.23(s,1H), 7.77(d,J=10.9Hz,1H), 7.30(s ,1H), 6.54(s,1H), 5.66-5.54(m,1H), 5.41(s,2H), 5.19(s,2H), 4.60(d,J=1.8Hz,2H) , 4.00(s,2H), 3.26(t,J=6.8Hz,3H), 3.21-3.11(m,3H), 2.38(s,3H), 2.24-2.12(m,2H ), 1.92-1.80(m,2H), 1.54-1.41(m,3H), 1.36-1.24(m,3H), 0.87(t,J=7.3Hz,3H)ppm. 19 F NMR (376 MHz, DMSO d6 )δ-111(Ar-F)ppm.

[0917] P7 (2S)-2-amino-N-[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl}methoxy)methyl]-3-phenylpropanamide (P7)

[0918] [ka] Following the general procedure, P7 (15 mg, 62% yield) was obtained as a white solid. ESI m / z: 670 (M+H) + . 1 H NMR (400 MHz, DMSO d6)δ 8.86-8.77(m,1H), 8.56(d,J=8.8Hz,1H), 8.29(s,1H), 7.78(d,J=11.0Hz,1H), 7.29(s, 1H), 7.21(t,J=7.2Hz,2H), 7.12(t,J=8.5Hz,3H), 6.52(s,1H), 5.64-5.56(m,1H), 5.45 -5.34(m,2H), 5.25-5.12(m,2H), 4.58(s,2H), 3.96(s,2H), 3.25-3.08(m,4H), 2.85-2. 70(m,1H), 2.39(s,3H), 2.28-2.08(m,3H), 1.87-1.76(m,2H), 0.84(t,J=7.2Hz,3H)ppm. 19 F NMR (376 MHz, DMSO d6 )δ-111(Ar-F)ppm.

[0919] P9 2-amino-N-[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl}methoxy)methyl]-N-methylacetamide (P9)

[0920] [ka] Following the general procedure, P9 (22 mg, 60% yield) was obtained as a white solid. ESI m / z: 594 (M+H). + . 1 H NMR (400 MHz, DMSO d6)δ 8.70(d,J=8.9Hz,0.5H), 8.61(d,J=8.9Hz,0.5H), 8.07(s,2H), 7.82-7.78(m,1H), 7.33 (d,J=1.8Hz,1H), 6.55(d,J=2.4Hz,1H), 5.67-5.53(m,1H), 5.43(s,2H), 5.29-5.10(m, 2H), 4.96-4.87(m,2H), 4.20-3.90(m,4H), 3.19(d,J=6.6Hz,2H), 3.02(s,1.5H), 2.99( s,1.5H), 2.40(s,3H), 2.19-2.17(m,2H), 1.93-1.81(m,2H), 0.88(t,J=7.2Hz,3H)ppm. 19 F NMR (376 MHz, DMSO d6 )δ-73(TFA), -111(Ar-F)ppm.

[0921] P11 2-amino-N-[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl}methoxy)methyl]-2-methylpropanamide (P11)

[0922] [ka] Following the general procedure, P11 (15 mg, 62% yield) was obtained as a white solid. ESI m / z: 608 (M+H) + . 1 H NMR (400 MHz, DMSO d6)δ 9.17-9.14(m,1H), 8.56(d,J=8.0Hz,1H), 8.17(s,2H), 7.80(d,J=8.0Hz,1H), 7.33(s,1H), 6.56(s,1H), 5.63-5.68(m,1H), 5.42(s,2H), 5.21(s,2H), 4.73( d,J=4.0Hz,2H), 4.04(s,2H), 3.24-3.13(m,2H), 2.40(s,3H), 2.18(d,J=4.0H z,2H), 1.89-1.83(m,2H), 1.475(s,3H), 1.473(s,3H), 0.89-0.86(m,3H)ppm. 19 F NMR (376 MHz, DMSO d6 )δ-73(TFA), -111(Ar-F)ppm.

[0923] P12 (2R)-2-amino-N-[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl}methoxy)methyl]-3-phenylpropanamide (P12)

[0924] [ka] Following the general procedure, P12 (36 mg, 52% yield, TFA salt) was obtained as a white solid. ESI m / z: 670 (M+H). + . 1 H NMR (400 MHz, DMSO d6)δ 9.29-9.26(m,1H), 8.55(d,J=8.0Hz,1H), 8.19(s,3H), 7.80(d,J=12Hz,1H),7. 33-7.23(m,6H), 6.55(s,1H), 5.62-5.57(m,1H), 5.45-5.35(m,2H), 5.20(s,2H) , 4.69(d,J=8.0Hz,1H), 4.07-3.96(m,3H), 3.11-2.96(m,3H), 2.40(s,3H), 2.2 1-2.16(m,2H), 1.92-1.82(m,2H), 1.27-1.23(m,1H), 0.88(t,J=8.0Hz,3H)ppm. 19 F NMR (376 MHz, DMSO d6 )δ-73(TFA), -111(Ar-F)ppm.

[0925] (9H-Fluoren-9-yl)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)-heptaen-23-yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamate (Fmoc-ProDXd) (5a)

[0926] [ka] [1] Exatecan (1.0 equivalent), HATU (1.1 equivalent), DMF, 25°C, 3 hours, yield 86% (2.6 g), [2] Piperidine in DMF (v / v=1 / 4), 25°C, 1 hour, yield 77% (1.4g). Exatecan is commercially available. To a yellow solution of intermediate 4a (9.55 g, 24.86 mmol) in dry DMF (60 mL), HATU (9.45 g, 24.86 mmol) and DIPEA (2.91 g, 22.6 mmol) were added, and the mixture was stirred at 25 °C for 15 minutes. A mixed solution of exatecan mesylate (12.0 g, 22.6 mmol) and DIPEA (5.82 g, 45.2 mmol) in dry DMF (60 mL) was then added to the reaction mixture. The reaction mixture was stirred at 25 °C for 4 hours until the exatecan mesylate was consumed, which was monitored by LCMS. The resulting solution was diluted with ethyl acetate (0.90 L) and washed with brine (180 mL × 2). The organic phase was dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was co-evaporated with ethyl acetate (180 mL x 4) in vacuo, and the residue (50 g) was dissolved in ethyl acetate (400 mL). The suspension was refluxed for 20 minutes until it became clear. The solution was allowed to stand, and a white solid precipitated. The suspension was refluxed again for 1 hour, then naturally cooled to 25 °C and allowed to stand for 30 minutes. The white precipitate was collected by filtration and dried in vacuo to give compound Fmoc-proDxd (5a) (14.2 g, 78.3% yield, >99% purity) as a white solid. The filtrate was concentrated and purified by C18 column to give (1.6 g, 8.8% yield, 97% purity). ESI m / z: 802.2 (M+H) + . 1 H NMR (400 MHz, DMSO d6):δ 8.79(t,J=6.4Hz,1H), 8.50(d,J=9.6Hz,1H), 7.88(d,J=7.6Hz,2H), 7.77(d,J=10.8Hz,1H), 7.68(d,J=7 .2Hz,2H), 7.56(t,J=6.0Hz,1H), 7.39(t,J=7.6Hz,2H), 7.34(s,1H), 7.34-7.27(m,2H), 6.62-6.45(m,1 H), 5.66-5.34(m,3H), 5.25-5.16(m,2H), 4.70-4.57(m,2H), 4.30-4.12(m,3H), 4.01(s,2H), 3.74-3.54 (m,2H), 3.25-3.05(m,2H), 2.37(s,3H), 2.24-2.13(m,2H), 1.92-1.80(m,2H), 0.84(t,J=7.6Hz,3H)ppm. 19 F NMR (376 MHz, DMSO d6 )δ-111.33 ppm.

[0927] [Table 19]

[0928] Example 6. Exemplary synthesis of ProDXd from DXd (Scheme 2B) P1 (CP1190) synthesized from DXd 2-amino-N-[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl}methoxy)methyl]acetamide (P1)

[0929] [ka] To a solution of DXd (62 mg, 0.13 mmol) in THF (HPLC grade, 5 mL) was added compound 3a (0.23 g, 0.63 mmol) and 4 Å molecular sieves, and the mixture was stirred at room temperature for 5 min. Then, TfNH (0.18 g, 0.63 mmol) was added to the mixture, and the reaction mixture was stirred at room temperature for 10 min. According to LCMS, DXd still remained, but the reaction was quenched with aqueous TFA (0.1%, 0.05 mL). The mixture was directly separated by reverse-phase flash chromatography (0–100% acetonitrile in aqueous TFA (0.1%)) to give DXd (40 mg, 65% recovery yield) and 5a (Fmoc-P1) (31 mg, ESI m / z: 803 (M+H)). + ) was dissolved in DMF (1 mL). To the solution of 5a, diethylamine (0.1 mL) was added, and the reaction mixture was stirred at room temperature for 1 h until the Fmoc was completely removed according to LCMS. The resulting mixture was directly purified by preparative HPLC (5-95% acetonitrile in aqueous TFA (0.1%)) to give P1 (15 mg, 17% yield, TFA salt) as a pale yellow solid. ESI m / z: 580.3 (M+H) + .

[0930] Example 7. Exemplary synthesis of diamino acid-ProDXd (Scheme 2C) P8 2-amino-N-({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)acetamide (P8)

[0931] [ka] To a solution of N-Fmoc-glycine (16.2 mg, 0.054 mmol) in DMF (1 mL), HATU (30.9 mg, 0.081 mmol) and DIPEA (21 mg, 0.108 mmol) were added, and the reaction mixture was stirred at room temperature for 15 min. Compound P1 (30 mg, 0.054 mmol, TFA salt) was added to the stirred mixture, and the reaction mixture was stirred at room temperature for 1 h, which was monitored by LCMS. Diethylamine (1 mL) was then added to the resulting mixture, and the mixture was stirred at room temperature for 1 h until the Fmoc was completely removed according to LCMS. The volatiles were removed in vacuo, and the residual mixture was directly separated by preparative HPLC (5–95% acetonitrile in aqueous TFA (0.1%)) to give P8 (11 mg, 31% yield, TFA salt) as a pale yellow solid. ESI m / z: 637 (M+H). + . 1 H NMR (400 MHz, DMSO d6 )δ 8.88(t,J=6.4Hz,1H), 8.65(t,J=5.6Hz,1H), 8.54(d,J=8.8Hz,1H), 8.01(br s,3H), 7.80(d,J=10.4Hz,1H), 7.31(s,1H), 6.55(s,1H), 5.62-5.56(m,1H), 5.42(s,2H), 5.19(s,2H), 4.65(d,J=6.4Hz,2H), 4.01(s,2 H), 3.86(d,J=5.6Hz,2H), 3.67(s,2H), 3.24-3.09(m,2H), 2.39(s,3H), 2.24-2.11(m,2H), 1.94-1.79(m,2H), 0.87(t,J=7.2Hz,3H)ppm. 19 F NMR (376 MHz, DMSO d6 )δ-73(TFA), -111(Ar-F)ppm.

[0932] Example 8. Synthesis of 2-[2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)ethanesulfonamido]acetic acid (2i)

[0933] [ka] To a stirred solution of tert-butyl glycinate (0.42 g, 2.5 mmol) in DMF (8 mL) was added N-Fmoc-2-aminoethanesulfonyl chloride (0.83 g, 2.3 mmol) and DIPEA (0.88 g, 6.8 mmol) at 0 °C. The reaction was stirred at room temperature for 2 h and monitored by LCMS. The resulting mixture was directly purified by reverse-phase flash chromatography (0-60% acetonitrile in aqueous TFA (0.1%)) to give a white solid (0.23 g, ESI m / z: 483 (M+Na)). + ) was obtained and dissolved in DCM (10 mL). To the solution, TFA (1 mL) was added, and the reaction mixture was stirred at room temperature for 8 hours, which was monitored by LCMS. The mixture was concentrated in vacuo to give compound 2i (0.19 g, 21% yield) as a yellow solid, which was used in the next step without further purification. ESI m / z: 427 (M+Na) + , 405(M+H) + .

[0934] Linker-payload synthesis Example 9. Exemplary synthesis of linker-payload via Route 1 (Scheme 4A) LP1 (M2980), synthesized from exatecan, reacted with intermediate Fa. {4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadecan-15-amido}-3-methylbutanamido]pentanamido]phenyl}methyl N-({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamate (LP1)

[0935] [ka] To a stirred solution of compound F (see Example 26) (48 mg, 49 μmol, 88% purity) in DMF (1 mL), HATU (20 mg, 54 μmol) and DIPEA (13 mg, 98 μmol) were added at 25°C, and the mixture was stirred at 25°C for 15 min. To a mixture of exatecan mesylate (26 mg, 49 μmol) in DMF (0.8 mL), DIPEA (6.3 mg, 49 μmol) was added at 0°C, and the exatecan solution was stirred at 25°C for 15 min. The two solutions were mixed at 25°C, and the mixture was stirred at 25°C for 16 h, which was monitored by LCMS. The resulting mixture was directly purified by reverse-phase flash chromatography (5–95% acetonitrile in aqueous TFA (0.1%) for 60 min, flow rate 75 mL / min) to give LP1 (35 mg, 51% yield, 98% purity by HPLC) as a white solid. ESI m / z: 1396 (M+H) + . 1 H NMR (400 MHz, DMSO d6)δ 10.00(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)、5.98(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)、3.67-3.54(m,3H)、3.53-3.46(m,12H)、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。 13 C NMR(100MHz,DMSO d6 )δ 174.77、169.55、168.95、167.68、167.16、161.10、158.63、157.39、154.93、150.50、148.30、146.11、143.38、138.89、137.01、134.57、129.97、123.39、122.06、121.87、119.88、117.41、117.30、108.08、99.18、95.12、90.49、70.73、70.55、68.18、68.11、67.89、67.20、66.23、65.31、63.69、55.98、51.52、47.95、43.14、41.97、40.04、36.42、34.34、32.25、28.95、28.72、27.63、26.25、25.18、24.23、22.21、18.38、17.55、16.47、9.22、6.10ppm. 19 F NMR(376MHz,DMSO d6)δ-74.132(0.3F,CF3CO2H), -111.314(1F)ppm.

[0936] Example 10. Exemplary synthesis of linker-payload via Route 2 (Scheme 4B) LP1 (M2980) synthesized from DXd reacted with intermediate Ea {4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadecan-15-amido}-3-methylbutanamido]pentanamido]phenyl}methyl N-({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamate (LP1)

[0937] [ka] To a stirred mixture of intermediate Ea (see Example 25) (50 mg, 52 μmol) in dry THF (5 mL), DXd (26 mg, 52 μmol) and 4 Å molecular sieves were added. The mixture was stirred at 20° C. for 5 min, followed by the addition of trifluoromethanesulfonimide (73 mg, 0.25 mmol). The reaction mixture was stirred at 20° C. for 10 min until intermediate Ea was largely consumed, as monitored by LCMS. The 4 Å molecular sieves were removed by filtration, and the filtrate was concentrated in vacuo. The residue was purified by reverse-phase flash chromatography (5-95% acetonitrile in aqueous TFA (0.1%)) to give LP1 (29 mg, 39% yield) as a pale yellow solid. ESI m / z: 699.1 (M / 2+H). + .

[0938] Example 11. Linker-Payload Synthesis via Route 3a (Scheme 4C) General procedure for linker-ProDXd via route 3a. To a solution of intermediate D (1.0-1.2 equiv.) in DMF (0.15 mM), HOBt (0.5 equiv.) or HOAt (0.5 equiv.), DIPEA (3.0 equiv.), and payload (1.0 equiv.) were added. The reaction mixture was stirred at room temperature for 2 h and monitored by LCMS. The resulting mixture was directly purified by reverse-phase flash chromatography to give linker-ProDXd as a white solid.

[0939] LP1 {4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadecan-15-amido}-3-methylbutanamido]pentanamido]phenyl}methyl N-({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11.0 20 , 24 ]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamate (LP1)

[0940] [ka] Following the general procedure starting with payload P1 (0.85 g, 1.2 mmol) catalyzed by HOBt, linker-payload LP1 (1.1 g, 62% yield) was obtained as a white solid after purification by preparative HPLC (5-60% acetonitrile in aqueous formic acid (0.1%)). ESI m / z: 699.0 (M / 2+H). + . 1 H NMR (400 MHz, DMSO d6 )δ 10.00(s,1H), 8.80(t,J=6.4Hz,1H), 8.51(d,J=8.8Hz,1H), 8.13(d,J=7.6 Hz,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), 5.98 (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.

[0941] LP2 {4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadecan-15-amido}-3-methylbutanamido]pentanamido]phenyl}methyl N-[(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)-heptaen-23-yl]carbamoyl}methoxy)methyl]carbamoyl}-2-hydroxyethyl]carbamate (LP2)

[0942] [ka] Following the general procedure starting from payload P2 (18 mg, 29 μmol) catalyzed by HOAt, linker-payload LP2 (19 mg, 45% yield) was obtained as a white solid after purification by preparative HPLC (5-95% acetonitrile in aqueous TFA (0.1%)). ESI m / z: 714 (M / 2+H). + . 1 H NMR (400 MHz, DMSO d6)δ 10.00(s,1H), 8.82(t,J=6.7Hz,1H), 8.50(d,J=8.6Hz,1H), 8.14(d,J=7. 2Hz,1H), 7.89(d,J=8.6Hz,1H), 7.79(d,J=11.0Hz,1H), 7.66-7.54(m,3H ), 7.35-7.24(m,3H), 7.18(d,J=8.2Hz,1H), 6.59-6.46(m,1H), 5.99(s,1 H), 5.65-5.55(m,1H), 5.42(s,3H), 5.21(s,2H), 4.97-4.83(m,2H), 4.67- 4.57(m,2H), 4.41-4.34(m,1H), 4.30-4.21(m,2H), 4.00(s,2H), 3.99-3. 65(m,3H), 3.62-3.57(m,2H), 3.53-3.46(m,12H), 3.28-3.16(m,4H), 3.0 6-2.81(m,3H), 2.39(s,3H), 2.26-2.02(m,6H), 2.02-1.80(m,6H), 1.78- 1.66(m,3H), 1.62-1.52(m,3H), 1.48-1.22(m,8H), 0.92-0.77(m,9H)ppm.

[0943] LP3 {4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadecan-15-amido}-3-methylbutanamido]pentanamido]phenyl}methyl N-[(1S)-3-carbamoyl-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)-heptaen-23-yl]carbamoyl}methoxy)methyl]carbamoyl}propyl]carbamate (LP3)

[0944] [ka] Following the general procedure starting from HOAt-catalyzed payload P3 (14 mg, 22 μmol), linker-payload LP3 (16 mg, 49% yield) was obtained as a white solid after purification by preparative HPLC (5-95% acetonitrile in aqueous TFA (0.1%)). ESI m / z: 734 (M / 2+H). + . 1 H NMR (400 MHz, DMSO d6 )δ 10.00(s,1H), 8.83(t,J=6.5Hz,1H), 8.54(d,J=8.6Hz,1H), 8.14(d,J=7.0 Hz,1H), 7.89(d,J=8.7Hz,1H), 7.79(d,J=10.8Hz,1H), 7.66-7.51(m,3H), 7.44(d,J=7.4Hz,1H), 7.31-7.23(m,3H), 6.78(s,1H), 6.59-6.48(m,1H), 6.03-5.94(m,1H), 5.64-5.56(m,1H), 5.42(s,3H), 5.22(s,2H), 4.95-4.81 (m,2H), 4.68-4.56(m,2H), 4.42-4.34(m,1H), 4.31-4.19(m,2H), 4.01(s, 2H), 3.87(d,J=14.8Hz,2H), 3.75(d,J=14.8Hz,1H), 3.64-3.55(m,2H), 3.5 3-3.44(m,12H), 3.28-3.20(m,4H), 3.08-2.90(m,3H), 2.40(s,3H), 2.24- 2.02(m,8H), 2.01-1.50(m,16H), 1.46-1.29(m,4H), 0.90-0.76(m,9H)ppm.

[0945] LP4 (4S)-4-{[({4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadecan-15-amido}-3-methylbutanamido]pentanamido]phenyl}methoxy)carbonyl]amino}-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)-heptaen-23-yl]carbamoyl}methoxy)methyl]carbamoyl}butanoic acid (LP4)

[0946] [ka] Following the general procedure starting from payload P4 (16 mg, 25 μmol) catalyzed by HOAt, linker-payload LP4 (12 mg, 35% yield) was obtained as a white solid after purification by preparative HPLC (5-95% acetonitrile in aqueous TFA (0.1%)). ESI m / z: 735 (M / 2+H). + . 1 H NMR (400 MHz, DMSO d6)δ 9.99(s,1H), 8.89-8.82(m,1H), 8.53(d,J=8.5Hz,1H), 8.12(d,J=7.5Hz,1H), 7.88(d,J=8.9Hz,1H), 7.78(d,J=11.1Hz,1H), 7 .65-7.51(m,3H), 7.44(d,J=7.4Hz,1H), 7.33-7.22(m,2H), 6.53(s,1H), 6.04-5.95(m,1H), 5.64-5.54(m,1H), 5.42(s,3H), 5. 21(s,2H), 4.97-4.81(m,2H), 4.67-4.56(m,2H), 4.41-4.33(m,1H), 4.31-4.18(m,2H), 4.00(s,2H), 3.99-3.65(m,3H), 3.63-3 .55(m,2H), 3.54-3.42(m,12H), 3.27-3.17(m,4H), 3.09-2.87(m,3H), 2.39(s,3H), 2.27-1.13(m,28H), 0.98-0.64(m,9H)ppm.

[0947] LP5 {4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadecan-15-amido}-3-methylbutanamido]pentanamido]phenyl}methyl N-[(5S)-5-amino-5-{[({[(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 ]tetracos-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl}methoxy)methyl]carbamoyl}pentyl]carbamate (LP5)

[0948] [ka] Following the general procedure starting from compound 5da (32 mg, 37 μmol) with intermediate D catalyzed by HOBt, Fmoc-LP5 (35 mg, 56% yield) was obtained as a white solid after purification by preparative HPLC (5–95% acetonitrile in aqueous TFA (0.1%)). To a solution of Fmoc-LP5 (35 mg, 21 μmol) in DMF (2 mL) was added diethylamine (7.6 mg, 0.10 mmol), and the reaction mixture was stirred at room temperature for 2 h until complete removal of Fmoc according to LCMS. The resulting mixture was directly purified by reverse-phase flash chromatography (5-95% acetonitrile in aqueous TFA (0.01%)) to give LP5 (9.6 mg, 31% yield) as a white solid. ESI m / z: 734 (M / 2+H). + . 1 H NMR (400 MHz, DMSO d6 )δ 10.00(d,J=13.5Hz,1H), 9.19-9.04(m,1H), 9.01-8.87(m,1H), 8.65-8.55(m,1H), 8.20-8.08(m,1H), 7.92-7.86(m,1H), 7.85-7.75( m,1H), 7.71(s,1H), 7.63-7.54(m,2H), 7.33-7.15(m,3H), 6.54(s,1H), 6.06-5.97(m,1H), 5.63-5.56(m,1H), 5.45-5.39(m,2H), 5.20 (s,1H), 5.14-5.04(m,1H), 4.90(d,J=6.1Hz,2H), 4.69-4.57(m,2H), 4.40-4.33(m,1H), 4.31-4.18(m,2H), 4.10-3.96(m,2H), 3.99- 3.65(m,3H), 3.65-3.55(m,4H), 3.53-3.44(m,12H), 3.29-3.19(m,4H), 3.03-2.35(m,7H), 2.27-1.17(m,32H), 0.91-0.63(m,9H)ppm.

[0949] LP5C {4-[(2S)-5-(carbamoylamino)-2-[(2S)-3-methyl-2-(1-{2-[4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl]acetamido}-3,6,9,12-tetraoxapentadecan-15-amido)butanamido]pentanamido]phenyl}methyl N-[(5S)-5-amino-5-{[({[(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)-heptaen-23-yl]carbamoyl}methoxy)methyl]carbamoyl}pentyl]carbamate (LP5C)

[0950] [ka] Following a similar procedure as for LP5, except using intermediate Dd instead of intermediate Da, linker-payload LP5C (15 mg, 17% yield) was obtained as a red solid after purification by preparative HPLC (5-95% acetonitrile in aqueous TFA (0.1%)). ESI m / z: 758 (M / 2+H). + .

[0951] LP7 {4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadecan-15-amido}-3-methylbutanamido]pentanamido]phenyl}methyl N-[(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)-heptaen-23-yl]carbamoyl}methoxy)methyl]carbamoyl}-2-phenylethyl]carbamate (LP7)

[0952] [ka] Following the general procedure starting from payload P7 (17 mg, 25 μmol) catalyzed by HOAt, linker-payload LP7 (17 mg, 46% yield) was obtained as a white solid after purification by preparative HPLC (5-95% acetonitrile in aqueous TFA (0.1%)). ESI m / z: 744 (M / 2+H). + . 1 H NMR (400 MHz, DMSO d6 )δ 9.98(s,1H), 8.97(t,J=6.8Hz,1H), 8.54(d,J=8.7Hz,1H), 8.13(d,J=7.2Hz,1H), 7.89(d,J=8.8Hz,1H), 7.79(d,J=11.0Hz,1H), 7.62(t,J=5. 5Hz,1H), 7.55(d,J=8.5Hz,2H), 7.49(d,J=8.2Hz,1H), 7.30(s,1H), 7. 26-7.20(m,4H), 7.18-7.12(m,2H), 6.03-5.95(m,1H), 5.64-5.56(m,1H) ), 5.47-5.34(m,3H), 5.26-5.14(m,2H), 4.86-4.75(m,2H), 4.70-4.57 (m,2H), 4.41-4.33(m,1H), 4.31-4.12(m,4H), 3.99(s,2H), 3.99-3.65 (m,3H), 3.63-3.57(m,3H), 3.50-3.47(m,12H), 3.28-3.10(m,4H), 2.9 8-2.90(m,1H), 2.39(s,3H), 2.28-1.16(m,25H), 0.87-0.80(m,9H)ppm.

[0953] LP8 {4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadecan-15-amido}-3-methylbutanamido]pentanamido]phenyl}methyl N-{[({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamoyl]methyl}carbamate (LP8)

[0954] [ka] Following the general procedure starting from payload P8 (20 mg, 32 μmol) catalyzed by HOBt, linker-payload LP8 (14 mg, 29% yield) was obtained as a white solid after purification by preparative HPLC (5-95% acetonitrile in aqueous TFA (0.1%)). ESI m / z: 727 (M / 2+H). + . 1 H NMR (400 MHz, DMSO d6)δ 9.99(s,1H), 8.72-8.68(m,1H), 8.51(d,J=8.5Hz,1H), 8.19-8.12(m,2H), 7 .88(d,J=9.1Hz,1H), 7.80(d,J=11.2Hz,1H), 7.63-7.55(m,3H), 7.47-7.43 (m,1H), 7.31(s,1H), 7.28(d,J=8.3Hz,2H), 6.53(s,1H), 6.00-5.95(m,1H) , 5.62-5.58(m,1H), 5.42(d,J=4.9Hz,3H), 5.21(s,2H), 4.94(s,2H), 4.63(d ,J=6.4Hz,2H), 4.39-4.35(m,1H), 4.28-4.22(m,2H), 4.01(s,2H), 3.85(s, 1H), 3.77(s,1H), 3.74-3.70(m,2H), 3.66-3.57(m,4H), 3.52-3.39(m,14H) , 3.28-3.21(m,4H), 3.04-2.97(m,2H), 2.40(s,3H), 2.25-2.13(m,6H), 2.0 1-1.82(m,9H), 1.61-1.55(m,3H), 1.49-1.36(m,5H), 0.91-0.78(m,9H)ppm.

[0955] LP9 {4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadecan-15-amido}-3-methylbutanamido]pentanamido]phenyl}methyl N-({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl}methoxy)methyl](methyl)carbamoyl}methyl)carbamate (LP9)

[0956] [ka] Following the general procedure starting from payload P9 (6.0 mg, 10 μmol) catalyzed by HOBt, linker-payload LP9 (5.0 mg, 36% yield) was obtained as a white solid after purification by preparative HPLC (5-95% acetonitrile in aqueous TFA (0.1%)). ESI m / z: 706 (M / 2+H). + . 1 H NMR (400 MHz, DMSO d6 )δ 9.99(s,1H), 8.63-8.50(m,1H), 8.15-8.08(m,1H), 7.88-7.78(m,2H), 7.59-7.57(m,3H), 7.30-7 .22(m,4H), 6.52(s,1H), 5.98(s,1H), 5.61(s,1H), 5.42(br,4H), 5.22(s,2H), 4.88-4.82(m,4H), 4.42-4.20(m,4H), 4.09-3.74(m,8H), 3.59-3.49(m,13H), 3.25-3.23(m,4H), 3.00-2.88(m,6H), 2 .39-2.38(m,4H), 2.20-2.16(m,3H), 1.97-1.73(m,9H), 1.56-1.35(m,7H), 0.86-0.83(m,9H)ppm.

[0957] Example 12. Exemplary synthesis of linker-payload via route 3b (Scheme 4D) LP1 synthesized from Fmoc-P1 (5a) was reacted with intermediate D using HOBt. {4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadecan-15-amido}-3-methylbutanamido]pentanamido]phenyl}methyl N-({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .04 , 13 .0 6 , 11 .0 20 , 24 ]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamate (LP1)

[0958] [ka] To a yellow solution of 5a (1.7 g, 2 mmol) in DMF (17 mL) was added DBU (30 mg, 0.20 mmol) and triethylamine (0.40 g, 4.0 mmol) at 25 °C, and the mixture was stirred at 25 °C for 15 min. To the reaction mixture, HOBt (0.14 g, 1.0 mmol) and intermediate D (2.0 g, 2.1 mmol) were added, and the resulting clear solution was stirred at 25 °C for 16 h. The resulting mixture was poured into MTBE (150 mL), and the heterogeneous mixture was stirred at room temperature for 5 min. The MTBE layer, containing most of the Fmoc-ene by-product and base, was then separated. The bottom black oil was diluted with DMF (20 mL), and the solution was purified by preparative HPLC (5–95% acetonitrile in aqueous TFA (0.01%)) to give LP1 (1.5 g, 55% yield) as a white solid. ESI m / z: 1396 (M+H) + . 1 H NMR (400 MHz, DMSO d6)δ 10.00(t,J=6.4Hz,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), 7.65-5.50(m,3H) , 7.43(t,J=6.0Hz,1H), 7.31(s,1H), 7.27(d,J=8.8Hz,2H), 6.54(s,1H), 5.98( t,J=5.2Hz,1H), 5.63-5.57(m,1H), 5.41(s,4H), 5.21(s,2H), 4.92(s,2H), 4.6 2(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), 3.67-3.54(m,4H), 3.53-3.46(m,12H), 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(Ar-F)ppm.

[0959] LP1 synthesized from Fmoc-P1 (5a) was reacted with intermediate D using MeHYQ (small scale). {4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadecan-15-amido}-3-methylbutanamido]pentanamido]phenyl}methyl N-({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20, 24 ]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamate (LP1)

[0960] [ka] To a yellow solution of 5a (10 mg, 1 equiv.), intermediate D (12.5 mg, 1.05 equiv.), and 4-methyl-2-hydroxyquinoline (MeHYQ) (1.0 mg, 0.5 mmol) in DMF (130 uL) was added DBU (0.24 mg, 1.6 umol) and triethylamine (3.2 mg, 32 umol) at 25 °C. The clear solution was stirred at 50 °C for 1.5 h, which was monitored by LCMS. After cooling to room temperature, the resulting mixture was poured into MTBE (600 uL) stirred at 0-10 °C. A brown oil appeared, which was collected after separation and the MTBE layer was removed. The oil was then purified by reverse-phase flash chromatography (5-95% acetonitrile in aqueous TFA (0.01%)) to give LP1 (10 mg, 90% yield) as a white solid. ESI m / z: 699.0 (M / 2+H). + .

[0961] LP1 synthesized from Fmoc-P1 (5a) was reacted with intermediate D using MeHYQ (large scale). {4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadecan-15-amido}-3-methylbutanamido]pentanamido]phenyl}methyl N-({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamate (LP1)

[0962] [ka] To a yellow solution of Fmoc-ProDXd (5a) (13 g, 16 mmol), intermediate Da (16 g, 17 mmol), and 4-methyl-2-hydroxyquinoline (MeHYQ) (1.3 g, 8.2 mmol) in DMF (130 mL) was added DBU (0.24 g, 1.6 mmol) and triethylamine (3.2 g, 32 mmol) at 25 °C. The clear solution was stirred at 50 °C for 6 h, which was monitored by LCMS. After cooling to room temperature, the resulting mixture was poured into MTBE (600 mL) stirred at 0–10 °C. A brown oil appeared, which was collected after separation and the MTBE layer was removed. The oil was then purified by reverse-phase flash chromatography (5–95% acetonitrile in aqueous TFA (0.01%)) to give LP1 (13 g, 58% yield) as a white solid. ESI m / z: 698.8 (M / 2+H) + .

[0963] 1 H NMR (400 MHz, DMSO d6)δ 10.00(t,J=6.4Hz,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)、7.65-5.50(m,3H)、7.43(t,J=6.0Hz,1H)、7.31(s,1H)、7.27(d,J=8.8Hz,2H)、6.54(s,1H)、5.98(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)、3.67-3.54(m,4H)、3.53-3.46(m,12H)、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(376MHz,DMSO d6 )δ-111(Ar-F)ppm。

[0964] 13 C NMR(100MHz,DMSO d6)δ 172.36, 171.11, 170.65, 170.61, 170.31, 169.20, 168.68, 162.70, 160.23, 158.90, 156.58, 156.43, 152.14, 149.89, 147.79, 147.65, 144.99, 140.48, 138.59, 136.24, 131.52, 128.56, 125.08, 123.65, 123.45, 121.50, 118.83, 109.81, 109.58, 100.73, 96.67, 92.06, 72.30, 72.10, 69.74, 69.48, 68.76, 67.80, 66.87, 65.31, 57.48, 53.07, 49.54, 44.63, 43.53, 41.62, 35.88, 33.83, 30.54, 30.32, 29.19, 27.80, 26.77, 25.80, 23.72, 19.96, 19.13, 18.05, 10.86, 7.69ppm.

[0965] [Table 20] Certificate of Analysis for LP1 (1g lot): Chemical structure:

[0966] [Table 21] Certificate of Analysis for LP1 (13g lot) Chemical structure:

[0967] [Table 22] Analytical Tests and Results:

[0968] [Table 23]

[0969] Example 13. Exemplary two-step synthesis of linker-payload via Route 4 (Scheme 4E) LP1 synthesized from P1 using Fmoc-vcPAB and then reacted with intermediate Ba {4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadecan-15-amido}-3-methylbutanamido]pentanamido]phenyl}methyl N-({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamate (LP1)

[0970] [ka] [Step 1] vcPAB-P1 To a solution of compound Fmoc-vcPAB-PNP (0.36 g, 0.47 mmol, 1.0 equiv., commercially available) in DMF (2 mL) was added P1 (0.27 g, 0.47 mmol, 1.0 equiv.), HOAt (95 mg, 0.70 mmol, 1.5 equiv.), and DIPEA (0.12 mg, 0.94 mmol, 2.0 equiv.), and the reaction mixture was stirred at room temperature for 4 h, which was monitored by LCMS. The resulting mixture was directly purified by reverse-phase flash chromatography to give compound Fmoc-vcPAB-P1 (0.22 mg, ESI m / z: 1207 (M+H)) as a yellow solid. + ) was obtained and dissolved in DMF (2 mL). To the solution, diethylamine (0.2 mL) was added, and the reaction mixture was stirred at room temperature for 2 hours, which was monitored by LCMS. The resulting mixture was directly purified by reverse-phase flash chromatography to give vcPAB-P1 (0.16 g, 28% yield from P1) as a white solid. ESI m / z: 1085 (M+H)+ .

[0971] [Step 2]:LP1 To a solution of COT-PEG4-acid (intermediate Ba) (63 mg, 0.15 mmol, 1.0 equiv., synthesized according to WO2018089373) in DMF (2 mL), HATU (83 mg, 0.22 mmol, 1.5 equiv.) and DIPEA (58 mg, 0.45 mmol, 3.0 equiv.) were added. The reaction mixture was stirred at room temperature for 1 h, followed by the addition of vcPAB-P1 (0.16 g, 0.15 mmol, 1.0 equiv.). The reaction mixture was stirred at room temperature for 4 h and monitored by LCMS. The resulting mixture was directly purified by preparative HPLC to give LP1 (20 mg, 10% yield) as a white solid. ESI m / z: 1396 (M+H). + .

[0972] LP1A synthesized from P1 using Fmoc-vcPAB and then reacted with intermediate Bb {4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-[1-({[(4E)-cyclooct-4-en-1-yloxy]carbonyl}amino)-3,6,9,12-tetraoxapentadecan-15-amido]-3-methylbutanamido]pentanamido]phenyl}methyl N-({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl...

Claims

1. A compound having the formula (I): 【Chemistry 1】 or a pharmaceutically acceptable salt thereof, In the formula, R １ , R ２ , R ３ , and R ４ are independently hydrogen, C １－５ alkyl or aryl, AA is a natural or unnatural amino acid; p is an integer from 1 to 6, 【Chemistry 2】 represents a point of attachment to said antibody or said antigen-binding fragment thereof, either directly or via a linker.

2. The compound of formula (I) 【Transformation 3】 The antibody-drug conjugate of claim 1, comprising:

3. the antibody or antigen-binding fragment thereof having a structure according to formula (II), 【Chemistry 4】 or a pharmaceutically acceptable salt thereof, wherein R １ , R ２ , R ３ , and R ４ are independently hydrogen or C １－５ is alkyl, A is a click chemistry adduct; W is NH, O, CO, CH ２ , phenyl, or a combination of two or more thereof; AA is a natural or unnatural amino acid; m is an integer from 0 to 8; n is 0 or 1; p is an integer from 1 to 6, 【Transformation 5】 10. The antibody-drug conjugate of claim 1, wherein: represents a point of attachment to the antibody or antigen-binding fragment thereof, either directly or via a linker.

4. 4. The antibody-drug conjugate of claim 3, wherein the click chemistry adduct is the product of a copper-free click chemistry reaction selected from (a) strain-promoted azide / dibenzocyclooctyne-amine (DBCO) click chemistry, (b) inverse electron demand Diels-Alder (IED-DA) tetrazine / trans-cyclooctene (TCO) click chemistry, (c) inverse electron demand Diels-Alder (IED-DA) tetrazine / norbornene click chemistry, (d) Diels-Alder maleimide / furan click chemistry, (e) Staudinger ligation, and (f) nitrile-oxide / norbornene cycloaddition click chemistry.

5. The antibody-drug conjugate of claim 3 or 4, wherein the click chemistry adduct comprises a triazole or a diazine.

6. the click chemistry adduct 【Transformation 6】 and any positional isomer or entantiomer thereof, wherein R' is H or C １－３ The antibody-drug conjugate of any one of claims 3 to 5, wherein Z is alkyl and Z is C or N.

7. The antibody-drug conjugate of any one of claims 3 to 6, wherein AA comprises a naturally occurring amino acid selected from glycine, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, threonine, lysine, asparagine, glutamine, aspartic acid, and glutamic acid.

8. AA is an R-amino acid, an N-methyl amino acid, 【Transformation 7】 The antibody-drug conjugate of any one of claims 3 to 6, comprising an unnatural amino acid selected from the group consisting of:

9. The compound of formula (II) 【Chemistry 8-1】 【Chemistry 8-2】 The antibody-drug conjugate of any one of claims 3 to 6, comprising:

10. An antibody-drug conjugate having a structure according to formula (III): 【Chemistry 9】 or a pharmaceutically acceptable salt thereof, wherein: Ab is an antibody or antigen-binding fragment thereof; R １ , R ２ , R ３ , and R ４ are independently hydrogen or C １－５ is alkyl, A is a click chemistry adduct; LL is a linker or bond connecting said Ab and said A; AA is a natural or unnatural amino acid; m is an integer from 0 to 8; n is 0 or 1; p is an integer from 1 to 6, An antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein q is an integer of 1 to 10.

11. an antibody-drug conjugate having a structure according to formula (IVa, IVb, IVc, IVd, IVe, IVf, IVg, IVh, IVi, IVj, or IVk); 【Chemistry 10-1】 【Chemistry 10-2】 (SEQ ID NOs: 2115 and 2115, respectively), or 【Chemistry 11】 (SEQ ID NOs: 2116 and 2116, respectively); or a pharmaceutically acceptable salt thereof, wherein: Ab is an antibody or antigen-binding fragment thereof; R is the side chain of any natural or unnatural amino acid; An antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein n is an integer of 1 to 5.

12. 12. The antibody-drug conjugate of claim 11, wherein the antibody or antigen-binding fragment thereof comprises Gln295 and / or Gln297, and a drug payload is conjugated to the antibody or antigen-binding fragment via the side chains of Gln295 and / or Gln297.

13. 13. The antibody-drug conjugate of claims 1 to 12, wherein the antibody or antigen-binding fragment thereof is selected from 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-FGFR2 antibody, an anti-FOLR1 antibody, an anti-HER2 / HER2 bispecific antibody, an anti-MET / MET bispecific antibody, or an antigen-binding fragment thereof.

14. The antibody-drug conjugate of any one of claims 1 to 13, wherein the antibody or antigen-binding fragment thereof is an anti-HER2 / HER2 bispecific antibody.

15. the anti-HER2 / HER2 bispecific antibody a first antigen-binding domain (D1); and a second antigen-binding domain (D2), D1 specifically binds to the first epitope of human HER2; The antibody-drug conjugate of claim 13 or 14, wherein D2 specifically binds to a second epitope of human HER2.

16. 16. The antibody-drug conjugate of any one of claims 1 to 15, wherein the antibody and linker-drug payload are site-specifically conjugated by using transglutaminase.

17. The antibody-drug conjugate of claim 16, wherein the transglutaminase is a microbial transglutaminase.

18. A pharmaceutical composition comprising the antibody-drug conjugate of any one of claims 1 to 17 co-formulated with one or more pharmaceutically acceptable diluents, excipients, and / or additives.

19. 18. A composition comprising a population of antibody-drug conjugates of any one of claims 1 to 17, having a drug-to-antibody ratio (DAR) of from about 0.5 to about 30.

0.

20. 20. The composition of claim 19, having a DAR of about 1.0 to about 2.

5.

21. 21. The composition of claim 20 having a DAR of about 2.

22. 20. The composition of claim 19, having a DAR of about 3.0 to about 4.

5.

23. 23. The composition of claim 22 having a DAR of about 4.

24. 20. The composition of claim 19 having a DAR of about 6.5 to about 8.

5.

25. 25. The composition of claim 24 having a DAR of about 8.

26. 19. A method for treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the antibody-drug conjugate of any one of claims 1 to 17, or the pharmaceutical composition of claim 18.

27. a linker-payload compound having a formula selected from the group consisting of (D') to (N'): 【Chemistry 12-1】 【Chemistry 12-2】 or a pharmaceutically acceptable salt thereof, In the formula, R １ , R ２ , R ３ , and R ４ are independently hydrogen or C １－５ is alkyl, B, 【Chemistry 13】 is selected from the group consisting of W is NH, O, CO, CH ２ , phenyl, or a combination of two or more thereof; R ５ , R ６ , R ７ , and R ８ are independently hydrogen, —NH ２ or a side chain of any natural or unnatural amino acid, or a pharmaceutically acceptable salt thereof, comprising: The method comprises exposing a payload having an amino group to an activated intermediate having para-nitro-phenyl carbonate in the presence of a base and a coupling catalyst to obtain said linker-payload compounds (D') to (G'), wherein said coupling catalyst is 4-hydroxy-2-methylquinoline (MeHYQ).

28. A linker-payload compound having the formula (D-1): 【Chemistry 14】 (D-1), or a pharmaceutically acceptable salt thereof, In the formula, R １ , R ２ , R ３ , and R ４ are independently hydrogen or C １－５ is alkyl, R ５ and R ６ are independently hydrogen, —NH ２ or a side chain of any natural or unnatural amino acid, or a pharmaceutically acceptable salt thereof, comprising:

1. A process comprising the step of exposing a drug payload having an amino group to an activated intermediate having para-nitro-phenyl carbonate in the presence of a base and a coupling catalyst to obtain said linker-payload compound (D), wherein said coupling catalyst is 4-hydroxy-2-methylquinoline (MeHYQ).

29. A linker-payload compound of formula (D): 【Chemistry 15】 or a pharmaceutically acceptable salt thereof, In the formula, R １ , R ２ , R ３ , and R ４ are independently hydrogen or C １－５ is alkyl, R ５ and R ６ are independently hydrogen, —NH ２ or the side chain of any natural or unnatural amino acid, or a pharmaceutically acceptable salt thereof.

30. a linker-payload compound having a formula selected from the group consisting of (D') to (N'): 【Chemistry 16-1】 【Chemistry 16-2】 or a pharmaceutically acceptable salt thereof, In the formula, R １ , R ２ , R ３ , and R ４ are independently hydrogen or C １－５ is alkyl, B, 【Chemistry 17】 is selected from the group consisting of W is NH, O, CO, CH ２ , phenyl, or a combination of two or more thereof; R ５ , R ６ , R ７ , and R ８ are independently hydrogen, —NH ２ or the side chain of any natural or unnatural amino acid, The method comprises exposing a payload having an amino group to an activated intermediate having para-nitro-phenyl carbonate in the presence of a base and a coupling catalyst to obtain said linker-payload compounds (D') to (G'), wherein said coupling catalyst is 4-hydroxy-2-methylquinoline (MeHYQ), or a pharmaceutically acceptable salt thereof.

31. 31. The linker-payload compound of claim 30, having a structure selected from the group consisting of: Table 1-1 Table 1-2 Table 1-3 Table 1-4 Table 1-5

32. A compound of formula (D-1), [Chemistry 18] or a pharmaceutically acceptable salt thereof, In the formula, R １ , R ２ , R ３ , and R ４ are independently hydrogen or C １－５ is alkyl, R ５ and R ６ are independently hydrogen, —NH ２ or a side chain of any natural or unnatural amino acid, or a pharmaceutically acceptable salt thereof, comprising: The process comprises: (a) providing a compound of formula (I-1) having the structure: 【Chemistry 19】 During the ceremony, X is, 【Chemistry 20】 providing a compound selected from the group consisting of: (b) reacting the compound of formula (I-1) with a compound of formula (PI), 【Chemistry 21】 During the ceremony, R is H or PG; reacting with a compound of formula (PI) wherein PG is a suitable protecting group, and producing the compound of formula (D-1).

33. The compound of formula (D-1) has the following structure: 【Chemistry 22】 33. The process of claim 32, comprising:

34. The step (b) of reacting the compound of formula (I-1) with the compound of formula (PI) 33. The process of claim 32, further comprising reacting the compound of formula (P-I), wherein R is PG, with a deprotecting agent prior to said reacting with the compound of formula (I-1).

35. 33. The process of claim 32, wherein PG is selected from the group consisting of allyloxycarbonyl (Alloc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), and 9-fluorenylmethoxycarbonyl (Fmoc).

36. 33. The process of claim 32, wherein the compound of formula (I-1) has the following structure: 【Chemistry 23】

37. 33. The process of claim 32, wherein the compound of formula (PI) has the following structure: 【Chemistry 24】

38. providing a compound of formula (V) having the structure: 【Chemistry 25】 33. The process of claim 32, further comprising, before step (a), forming the compound of formula (I-1) from the compound of formula (V).

39. The step of forming the compound of formula (I-1) The compound of formula (V) is a compound of formula (VIa) or formula (VIb), 【Chemistry 26】 with a compound of formula (VIa) or (VIb), wherein X' is a halogen, The process according to claim 38, comprising producing the compound of formula (I-1).

40. A compound of formula (VII) having the structure: 【Chemistry 27】 In the formula, PG １ is a suitable protecting group; and forming the compound of formula (V) from the compound of formula (VII).

41. 41. The process of claim 40, wherein the compound of formula (VII) has the following structure: 【Chemistry 28】

42. said step of forming a compound of formula (V) Reacting the compound of formula (VII) with a compound of formula (VIII) 【Chemistry 29】 41. The process of claim 40, comprising producing the compound of formula (V).

43. providing a compound of formula (IX) having the structure: 【Transformation 30】 and forming the compound of formula (VII) from the compound of formula (IX).

44. 44. The process of claim 43, wherein the compound of formula (IX) has the following structure: 【Chemistry 31】

45. said step of forming the compound of formula (VII) reacting the compound of formula (IX) with a compound of formula (X), 【Chemistry 32】 44. The process of claim 43, comprising producing the compound of formula (VII).

46. providing a compound of formula (XI) having the structure: 【Transformation 33】 and forming the compound of formula (IX) from the compound of formula (XI).

47. 47. The process of claim 46, wherein the compound of formula (XI) has the following structure: 【Transformation 34】

48. said step of forming a compound of formula (IX) reacting the compound of formula (XI) with a compound of formula (XII) 【Chemistry 35】 47. The process of claim 46, comprising producing the compound of formula (IX).

49. providing a compound of formula (XIII) having the structure: 【Transformation 36】 and forming the compound of formula (VIII) from the compound of formula (XIII).

50. said step of forming the compound of formula (VIII) reacting the compound of formula (XIII) with a compound of formula (XII) 【Chemistry 37】 50. The process of claim 49, comprising producing the compound of formula (VIII).

51. A compound of formula (XIV) having the structure: 【Transformation 38】 In the formula, R ａ is a halogen, R ｂ But C １－６ providing a compound of formula (XIV), wherein X is alkyl; and forming the compound of formula (XIII) from the compound of formula (XIV).

52. 52. The process of claim 51 , wherein the compound of formula (XIV) has the following structure: 【Chemistry 39】

53. said step of forming the compound of formula (XIII) 52. The process of claim 51, comprising reacting the compound of formula (XIV) with a base to produce the compound of formula (XIII).

54. 54. The process of claim 53, wherein the base is selected from the group consisting of NaOMe, t-BuOK, NaH, and LDA.

55. providing a compound of formula (XV) having the structure: 【Chemistry 40】 and b. forming the compound of formula (XIV) from the compound of formula (XV).

56. 56. The process of claim 55, wherein the compound of formula (XV) has the following structure: 【Chemistry 41】

57. said step of forming a compound of formula (XIV) reacting the compound of formula (XV) with a compound of formula (XVI), 【Chemistry 42】 56. The process of claim 55, comprising producing the compound of formula (XIV).

58. providing a compound of formula (XVII) having the structure: 【Chemistry 43】 and forming the compound of formula (XV) from the compound of formula (XVII).

59. said step of forming a compound of formula (XV) 59. The process of claim 58, comprising reacting the compound of formula (XVII) with a brominating agent to produce the compound of formula (XVII).

60. The brominating agent is CHBr ３ 60. The process of claim 59, wherein

61. providing a compound of formula (XVIII) having the structure: 【Chemistry 44】 forming the compound of formula (P-I) from the compound of formula (XVIII).

62. 62. The process of claim 61 , wherein the compound of formula (XVIII) has the following structure: 【Chemistry 45】

63. said step of forming the compound of formula (PI) reacting said compound of formula (XVIII) with a compound of formula (XIX) 【Chemistry 46】 62. The process of claim 61, comprising producing the compound of formula (PI).

64. providing a compound of formula (XX) having the structure: 【Chemistry 47】 and forming the compound of formula (XVIII) from the compound of formula (XX).

65. 65. The process of claim 64, wherein the compound of formula (XX) has the following structure: g.

66. said step of forming said compound of formula (XVIII) reacting said compound of formula (XX) with a compound of formula (XXI), 【Chemistry 48】 65. The process of claim 64, comprising producing the compound of formula (XVIII).

67. providing a compound of formula (XXII) having the structure: 【Chemistry 49】 and forming the compound of formula (XX) from the compound of formula (XXII).

68. 68. The process of claim 67, wherein the compound of formula (XXII) has the following structure: [Transformation 50]

69. A compound of formula (I-1), 【Chemistry 51】 or a pharmaceutically acceptable salt thereof, During the ceremony, X is, 【Chemistry 52】 1. A process for the preparation of a compound selected from the group consisting of: The process comprises: (a) providing a compound of formula (V) having the structure: 【Chemistry 53】 (b) forming the compound of formula (I-1) from the compound of formula (V).

70. The step (b) of forming the compound of formula (I-1) The compound of formula (V) is a compound of formula (VIa) or formula (VIb), 【Chemistry 54】 During the ceremony, with a compound of formula (VIa) or (VIb) wherein X' is a halogen, 70. The process according to claim 69, comprising producing the compound of formula (I-1).

71. A compound of formula (VII) having the structure: 【Transformation 55】 In the formula, PG １ is a suitable protecting group; 70. The process of claim 69, further comprising forming the compound of formula (V) from the compound of formula (VII).

72. 72. The process of claim 71, wherein the compound of formula (VII) has the following structure: 【Transformation 56】

73. said step of forming a compound of formula (V) Reacting the compound of formula (VII) with a compound of formula (VIII) 【Chemistry 57】 72. The process of claim 71, comprising producing the compound of formula (V).

74. providing a compound of formula (IX) having the structure: 【Chemistry 58】 and forming the compound of formula (VII) from the compound of formula (IX).

75. 75. The process of claim 74, wherein the compound of formula (IX) has the following structure: 【Chemistry 59】

76. said step of forming the compound of formula (VII) reacting the compound of formula (IX) with a compound of formula (X), 【Transformation 60】 75. The process of claim 74, comprising producing the compound of formula (VII).

77. providing a compound of formula (XI) having the structure: 【Chemistry 61】 75. The process of claim 74, further comprising forming the compound of formula (IX) from the compound of formula (XI).

78. 78. The process of claim 77, wherein the compound of formula (XI) has the following structure: 【Transformation 62】

79. said step of forming a compound of formula (IX) reacting the compound of formula (XI) with a compound of formula (XII) 【Transformation 63】 78. The process of claim 77, comprising producing the compound of formula (IX).

80. A compound of formula (I-1), 【Chemistry 64】 or a pharmaceutically acceptable salt thereof, During the ceremony, X is, 【Transformation 65】 10. A compound selected from the group consisting of:

81. A compound of formula (XVIII), 【Chemical Formula 66】 or a pharmaceutically acceptable salt thereof, In the formula, R １ , R ２ , R ３ , and R ４ are independently hydrogen or C １－５ is alkyl, R ５ and R ６ are independently hydrogen, —NH ２ or a side chain of any natural or unnatural amino acid, or a pharmaceutically acceptable salt thereof, comprising: The process comprises: (a) providing a compound of formula (XX) having the structure: 【Transformation 67】 (b) forming said compound of formula (XVIII) from said compound of formula (XX).

82. 82. The process of claim 81, wherein the compound of formula (XVIII) has the following structure: 【Transformation 68】

83. 82. The process of claim 81, wherein the compound of formula (XX) has the following structure: 【Transformation 69】

84. said step of forming said compound of formula (XVIII) reacting said compound of formula (XX) with a compound of formula (XXI), 【Transformation 70】 82. The process of claim 81, comprising producing the compound of formula (XVIII).

85. providing a compound of formula (XXII) having the structure: 【Chemistry 71】 and forming the compound of formula (XX) from the compound of formula (XXII).

86. 86. The process of claim 85, wherein the compound of formula (XXII) has the following structure: 【Chemistry 72】

87. A compound of formula (XVIII), 【Transformation 73】 or a pharmaceutically acceptable salt thereof, In the formula, R １ , R ２ , R ３ , and R ４ are independently hydrogen or C １－５ is alkyl, R ５ and R ６ are independently hydrogen, —NH ２ or the side chain of any natural or unnatural amino acid, or a pharmaceutically acceptable salt thereof.

88. 88. The compound of claim 87, wherein the compound has the structure: 【Chemistry 74】

89. A compound of formula (D-1), 【Chemistry 75】 or a pharmaceutically acceptable salt thereof, In the formula, R １ , R ２ , R ３ , and R ４ are independently hydrogen or C １－５ is alkyl, R ５ and R ６ are independently hydrogen, —NH ２ or a side chain of any natural or unnatural amino acid, or a pharmaceutically acceptable salt thereof, comprising: The process comprises: (a) A compound of formula (I-1) having the following structure: 【Transformation 76】 During the ceremony, X is, 【Chemical 77】 providing a compound selected from the group consisting of: (b) reacting the compound of formula (I-1) with a compound of formula (PI), 【Transformation 78】 During the ceremony, R is H or PG; reacting with a compound of formula (PI) wherein PG is a suitable protecting group, and producing the compound of formula (D-1).

90. 90. The process of claim 89, wherein the compound of formula (D-1) has the following structure: 【Transformation 79】

91. The step (b) of reacting the compound of formula (I-1) with the compound of formula (PI) 90. The method of claim 89, further comprising reacting the compound of formula (P-I), wherein R is PG, with a deprotecting agent prior to said reacting with the compound of formula (I-1).

92. 90. The process of claim 89, wherein PG is selected from the group consisting of allyloxycarbonyl (Alloc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), and 9-fluorenylmethoxycarbonyl (Fmoc).

93. The deprotecting agent is Pd(PPh) ３ , PhSiH ３ , H ２ 92. The process of claim 91, wherein the amine is selected from the group consisting of piperidine and trifluoroacetic acid (TFA).

94. 90. The process of claim 89, wherein the compound of formula (PI) has the following structure: 【Chemistry 80】

95. providing a compound of formula (XVIII) having the structure: 【Chemistry 81】 forming the compound of formula (P-I) from the compound of formula (XVIII).

96. 96. The process of claim 95, wherein the compound of formula (XVIII) has the following structure: 【Chemistry 82】

97. said step of forming the compound of formula (PI) reacting said compound of formula (XVIII) with a compound of formula (XIX) 【Chemistry 83】 96. The process of claim 95, comprising producing the compound of formula (PI).

98. A compound of formula (D-1), 【Chemical 84】 or a pharmaceutically acceptable salt thereof, In the formula, R １ , R ２ , R ３ , and R ４ are independently hydrogen or C １－５ is alkyl, R ５ and R ６ are independently hydrogen, —NH ２ or a side chain of any natural or unnatural amino acid, or a pharmaceutically acceptable salt thereof, comprising: The process comprises: (a) providing a compound of formula (XXIII); 【Chemical 85】 (b) reacting the compound of formula (XXIII) with a compound having the following structure in the presence of an activating agent and a base: 【Chemical 86】 to produce the compound of formula (D-1).

99. 99. The process of claim 98, wherein the compound of formula (D-1) has the following structure: 【Chemistry 87】

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