Prodrugs for ADC-conjugated topoisomerase I inhibitors and methods of use thereof

Abstract

Described herein are protein-drug conjugates and compositions thereof that are useful, for example, for target-specific delivery of therapeutic moieties, such as camptothecin analogs and / or derivatives. In certain embodiments, provided are specific and effective methods for producing protein-drug constructs (e.g., antibody-drug conjugates) using a combination of a transglutaminase and a 1, 3-cycloaddition technique. Provided are camptothecin analogs, antibody-drug conjugates, and compositions comprising a glutaminyl modified antibody and a camptothecin analog payload.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This patent application claims the benefit of U.S. Provisional Application Nos. 63 / 472,064, filed June 9, 2023, and 63 / 434,230, filed December 21, 2022, the disclosures of each of which are incorporated herein by reference in their entireties. Technical Field

[0003] The present disclosure relates to protein-drug conjugates (e.g., antibody-drug conjugates), pharmaceutical compositions, and methods for treating diseases using the protein-drug conjugates and pharmaceutical compositions. Also provided are specific and efficient methods for generating protein-drug constructs using a combination of transglutaminase and 1,3-cycloaddition technology. More specifically, the present disclosure relates to prodrugs of topoisomerase I inhibitors for ADC conjugation and methods of using the same.

[0004] Sequence Listing

[0005] This application contains a sequence listing that has been submitted electronically in XML file format and is hereby incorporated 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 Art

[0006] Proliferative diseases are characterized by the uncontrolled growth and spread of abnormal cells. If the spread is not controlled, then death may result. Abnormal proliferation (such as cancer) is caused by both external factors (such as tobacco, chemicals, radiation and infectious organisms) and internal factors (genetic mutations, immune system conditions, mutations produced by metabolism). These causal factors can work together or work in sequence to cause or promote abnormal proliferation. Cancer is treated by surgery, radiation, chemotherapy, hormones and immunotherapy. However, more effective antiproliferative drugs are needed.

[0007] Ideal antiproliferative therapy will enable the targeted delivery of highly cytotoxic agents to tumor cells, and leave normal cells unaffected. Traditional chemotherapeutic treatments are limited because of the toxic side effects produced by the effects of drugs on non-cancerous cells. Various methods of targeted drug delivery have been attempted, including the conjugation of tumor-targeting probes (such as antibodies or growth factors) with toxins (such as pseudomonas or diphtheria toxins), which prevent the synthesis of proteins and cells. However, side effects include the reaction of the immune system due to the non-human components of the conjugate. In addition, due to elimination from circulation and schematic degradation, uptake by the reticuloendothelial system (RES) and accumulation in non-targeted organs and tissues by filtration through the kidneys, the half-life of drug conjugates is limited.

[0008] Another approach uses passive drug carriers (such as polymers, liposomes, and polymeric micelles) to exploit the hyperpermeability of the vascular endothelium of tumor tissue. Due to enhanced permeability and retention mechanisms, polymeric drugs and macromolecules accumulate within solid tumors. However, obstacles to using such targeted delivery include rapid removal of foreign particles from the blood, and technical barriers in obtaining highly standardized pharmaceutically acceptable drug delivery systems with the necessary specificity and selectivity for binding to tumor cells.

[0009] Protein conjugates, such as antibody conjugates, utilize the selective binding of a binding agent to deliver a payload to a target within a subject's tissue. The payload can be a therapeutic moiety capable of taking action at the target.

[0010] Several techniques are available for conjugating linkers and payloads to antibodies. Many conjugates are prepared by non-selective covalent attachment to cysteine ​​or lysine residues in antibodies. This non-selective technique can result in a heterogeneous mixture of products with conjugation at different sites, and the amount of conjugation varies for each antibody. Therefore, there is a need in the art to provide methods and techniques for site-selective antibody conjugation.

[0011] There is a need in the art for additional safe and effective anti-tumor targeting agents for use in monotherapy and combination therapy that can bind to various antigens to provide enhanced treatment of diseases such as cancer. In certain embodiments, the present disclosure satisfies these needs and provides other advantages.

[0012] The foregoing discussion is presented merely to provide a better understanding of the nature of the problems faced in the art and should not be construed in any way as an admission of prior art, nor should the citation of any reference herein be construed as an admission that such reference constitutes "prior art" to the present application. Summary of the Invention

[0013] Various non-limiting aspects and embodiments of the present disclosure are described below.

[0014] In one aspect, the present disclosure provides an antibody-drug conjugate comprising an antibody or an antigen-binding fragment thereof and a compound having formula (I)

[0015]

[0016] or a pharmaceutically acceptable salt thereof,

[0017] where R 1 、R 2 、R 3 and R 4 are independently hydrogen, C 1-5 alkyl or aryl;

[0018] AA is a natural or unnatural amino acid;

[0019] p is an integer from 1 to 6, and

[0020] The point of attachment to the antibody or the antigen-binding fragment thereof, directly or via a linker, is indicated.

[0021] In one embodiment, the compound of formula (I) comprises

[0022]

[0023] In one embodiment, the antibody or the antigen-binding fragment thereof is combined with a compound having a structure according to formula (II)

[0024]

[0025] or a pharmaceutically acceptable salt thereof, wherein R 1 、R 2 、R 3 and R 4 are independently hydrogen or C 1-5 alkyl;

[0026] A is a click chemistry adduct;

[0027] W is NH, O, CO, CH2, phenyl, or a combination of two or more thereof;

[0028] AA is a natural or unnatural amino acid;

[0029] m is an integer from 0 to 8;

[0030] n is 0 or 1;

[0031] p is an integer from 1 to 6, and

[0032] The point of attachment to the antibody or the antigen-binding fragment thereof, directly or via a linker, is indicated.

[0033] In one embodiment, the click chemistry adduct is the product of a copper-free click chemistry reaction selected from the group consisting of: (a) strain-promoted azide / dibenzocyclooctynamine (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.

[0034] In one embodiment, the click chemistry adduct comprises a triazole or a diazine.

[0035] In one embodiment, the click chemistry adduct is selected from the group consisting of:

[0036] and any regioisomer or enantiomer thereof, wherein R' is H or C 1-3 alkyl and Z is C or N.

[0037] In one embodiment, AA comprises a natural amino acid selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, threonine, lysine, asparagine, glutamine, aspartic acid, and glutamic acid.

[0038] In one embodiment, AA comprises an unnatural amino acid selected from the group consisting of an R-amino acid, an N-methyl amino acid,

[0039] In one embodiment, the compound of formula (II) comprises

[0040]

[0041]

[0042] In one embodiment, the compound of formula (II) comprises

[0043]

[0044] In another aspect, the present disclosure provides an antibody-drug conjugate having a structure according to formula (III):

[0045]

[0046] or a pharmaceutically acceptable salt thereof, wherein

[0047] Ab is an antibody or its antigen-binding fragment;

[0048] R 1 、R 2 、R 3 and R 4 are independently hydrogen or C 1-5 alkyl;

[0049] A is a click chemistry adduct;

[0050] LL is a linker or bond connecting the Ab and the A;

[0051] AA is a natural or unnatural amino acid;

[0052] m is an integer from 0 to 8;

[0053] n is 0 or 1;

[0054] p is an integer from 1 to 6;

[0055] And q is an integer from 1 to 10.

[0056] In another aspect, the present disclosure provides an antibody-drug conjugate having a structure according to Formula (IVa or IVb):

[0057]

[0058] or a pharmaceutically acceptable salt thereof, wherein

[0059] Ab is an antibody or its antigen-binding fragment;

[0060] R is the side chain of any natural or unnatural amino acid;

[0061] And n is an integer from 1 to 5.

[0062] In another aspect, the present disclosure provides an antibody-drug conjugate having a structure according to Formula (IVc, IVd, IVe, IVf, IVg, IVh, IVi, IVj, or IVk)

[0063]

[0064] (SEQ ID NOS 2115 and 2115, respectively),

[0065]

[0066] (respectively

[0067] SEQ ID NOS 2116 and 2116),

[0068] or a pharmaceutically acceptable salt thereof, wherein

[0069] Ab is an antibody or its antigen-binding fragment;

[0070] R is the side chain of any natural or unnatural amino acid;

[0071] And n is an integer from 1 to 5.

[0072] In one embodiment, the antibody or the antigen-binding fragment thereof comprises Gln295 and / or Gln297, and wherein the drug payload is conjugated to the antibody or antigen-binding fragment via the side chain of Gln295 and / or Gln297.

[0073] In one embodiment, the antibody or its antigen-binding fragment is selected from anti-HER2 antibody, anti-STEAP2 antibody, anti-MET antibody, anti-EGFRVIII antibody, anti-MUC16 antibody, anti-PRLR antibody, anti-PSMA antibody, anti-FGFR2 antibody, anti-FOLR1 antibody, anti-HER2 / HER2 bispecific antibody, anti-MET / MET bispecific antibody or its antigen-binding fragment.

[0074] In one embodiment, the antibody or antigen-binding fragment thereof is an anti-HER2 / HER2 bispecific antibody.

[0075] In one embodiment, the anti-HER2 / HER2 bispecific antibody comprises:

[0076] a first antigen binding domain (D1); and

[0077] a second antigen-binding domain (D2);

[0078] wherein D1 specifically binds to the first epitope of human HER2; and

[0079] Among them, D2 specifically binds to the second epitope of human HER2.

[0080] In one embodiment, the antibody and linker-drug payload are site-specifically conjugated using transglutaminase.

[0081] In one embodiment, the transglutaminase is a microbial transglutaminase.

[0082] In another aspect, the present disclosure provides a pharmaceutical composition comprising the antibody-drug conjugate according to any one of the above embodiments, formulated together with one or more pharmaceutically acceptable diluents, excipients and / or additives.

[0083] In another aspect, the present disclosure provides a composition comprising a population of the antibody-drug conjugate according to any one of the above embodiments, the composition having a drug-antibody ratio (DAR) of about 0.5 to about 30.0.

[0084] In one embodiment, the composition has a DAR of about 1.0 to about 2.5.

[0085] In one embodiment, the composition has a DAR of about 2.

[0086] In one embodiment, the composition has a DAR of about 3.0 to about 4.5.

[0087] In one embodiment, the composition has a DAR of about 4.

[0088] In one embodiment, the composition has a DAR of about 6.5 to about 8.5.

[0089] In one embodiment, the composition has a DAR of about 8.

[0090] In another aspect, the present disclosure provides a method for treating cancer in a subject in need thereof, comprising the step of administering to the subject a therapeutically effective amount of the antibody-drug conjugate according to any one of the above embodiments, or the pharmaceutical composition according to the above embodiments.

[0091] In another aspect, the present disclosure provides a method for making a linker-payload compound having a formula selected from the group consisting of the following (D') to (N'):

[0092]

[0093]

[0094]

[0095] or a pharmaceutically acceptable salt thereof,

[0096] where R 1 、R 2 、R 3 and R 4 are independently hydrogen or C 1-5 alkyl;

[0097] B is selected from the group consisting of: W is NH, O, CO, CH2, phenyl, or a combination of two or more thereof; and R 5 、R 6 、R 7 and R 8 is independently hydrogen, -NH2 or the side chain of any natural or unnatural amino acid,

[0098] The method comprises the steps of exposing a payload having an amino group to an activated intermediate having p-nitrophenyl carbonate in the presence of a base and a coupling catalyst to obtain the linker-payload compounds (D') to (G'), wherein the coupling catalyst is 4-hydroxy-2-methylquinoline (MeHYQ).

[0099] In another aspect, the present disclosure provides a method for producing a linker-payload compound having formula (D-1)

[0100]

[0101] (D-1), or a pharmaceutically acceptable salt thereof,

[0102] where R 1 、R 2 、R 3 and R 4 are independently hydrogen or C 1-5 alkyl, and

[0103] R 5 and R 6 is independently hydrogen, -NH2 or the side chain of any natural or unnatural amino acid,

[0104] The method comprises the steps of exposing a drug payload having an amino group to an activated intermediate having p-nitrophenyl 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).

[0105] In one embodiment, the activated intermediate with p-nitrophenyl carbonate has a structure according to Formula II:

[0106]

[0107] The present disclosure also relates to a method for preparing a linker-payload compound having formula (D-1)

[0108]

[0109] or a pharmaceutically acceptable salt thereof,

[0110] where R 1、R 2 、R 3 and R 4 are independently hydrogen or C 1-5 alkyl, and

[0111] R 5 and R 6 is independently hydrogen, -NH2 or the side chain of any natural or unnatural amino acid,

[0112] The method comprises:

[0113] (a) providing a compound of formula (I-1) having the following structure:

[0114]

[0115] in

[0116] X is selected from the group consisting of: as well as

[0117] (b) reacting the compound of formula (I-1) with a compound of formula (PI):

[0118]

[0119] in

[0120] R is H or PG; and

[0121] PG is a suitable protecting group;

[0122] reaction to produce the compound of formula (D-1).

[0123] In one embodiment, the compound of formula (D-1) has the following structure:

[0124]

[0125] In one embodiment, the step (b) of reacting the compound of formula (I-1) with the compound of formula (PI) further comprises reacting the compound of formula (PI), wherein R is PG, with a protecting group removing agent before reacting with the compound of formula (I-1).

[0126] In one embodiment, the PG is selected from the group consisting of allyloxycarbonyl (Alloc), benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), and 9-fluorenylmethoxycarbonyl (Fmoc).

[0127] In one embodiment, the compound of formula (I-1) has the following structure:

[0128]

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

[0130]

[0131] In one embodiment, the method for making a linker-payload compound having formula (D-1) further comprises the steps of providing a compound of formula (V) having the following structure:

[0132] and forming the compound of formula (I-1) from the compound of formula (V) prior to step (a).

[0133] In one embodiment, the step of forming the compound of formula (I-1) comprises reacting the compound of formula (V) with a compound of formula (VIa) or formula (VIb):

[0134]

[0135] wherein X' is a halogen, reacts to produce the compound of formula (I-1).

[0136] In one embodiment, the method further comprises providing a compound of formula (VII) having the following structure:

[0137]

[0138] Among them PG 1 is a suitable protecting group protecting group, and the compound of formula (V) is formed from the compound of formula (VII).

[0139] In one embodiment, the PG 1 Selected from the group consisting of allyloxycarbonyl (Alloc), benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc) and 9-fluorenylmethoxycarbonyl (Fmoc).

[0140] In one embodiment, the compound of formula (VII) has the following structure:

[0141]

[0142] In one embodiment, the step of forming the compound of formula (V) comprises reacting the compound of formula (VII) with a compound of formula (VIII):

[0143] to produce the compound of formula (V).

[0144] In one embodiment, the method further comprises the steps of providing a compound of formula (IX) having the following structure:

[0145] and forming the compound of formula (VII) from the compound of formula (IX).

[0146] In one embodiment, the compound of formula (IX) has the following structure:

[0147]

[0148] In one embodiment, the step of forming the compound of formula (VII) comprises reacting the compound of formula (IX) with a compound of formula (X):

[0149] to produce the compound of formula (VII).

[0150] In one embodiment, the method further comprises the steps of providing a compound of formula (XI) having the following structure:

[0151] and forming the compound of formula (IX) from the compound of formula (XI).

[0152] In one embodiment, the compound of formula (XI) has the following structure:

[0153]

[0154] In one embodiment, the step of forming the compound of formula (IX) comprises reacting the compound of formula (XI) with a compound of formula (XII):

[0155] to produce the compound of formula (IX).

[0156] In one embodiment, the method further comprises providing a compound of formula (XIII) having the following structure: as well as

[0157] The compound of formula (VIII) is formed from the compound of formula (XIII).

[0158] In one embodiment, the step of forming the compound of formula (VIII) comprises reacting the compound of formula (XIII) with a compound of formula (XII):

[0159] to produce the compound of formula (VIII).

[0160] In one embodiment, the method further comprises the step of providing a compound of formula (XIV) having the following structure:

[0161]

[0162] in

[0163] R a is a halogen; and

[0164] R b It is C 1-6 Alkyl, and

[0165] The compound of formula (XIII) is formed from the compound of formula (XIV).

[0166] In one embodiment, R a It's bromine.

[0167] In one embodiment, the compound of formula (XIV) has the following structure:

[0168]

[0169] In one embodiment, the step of forming the compound of formula (XIII) comprises reacting the compound of formula (XIV) with a base to produce the compound of formula (XIII).

[0170] 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).

[0171] In one embodiment, the method further comprises the step of providing a compound of formula (XV) having the following structure:

[0172] as well as

[0173] The compound of formula (XIV) is formed from the compound of formula (XV).

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

[0175]

[0176] In one embodiment, the step of forming the compound of formula (XIV) comprises reacting the compound of formula (XV) with a compound of formula (XVI):

[0177] to produce the compound of formula (XIV).

[0178] In one embodiment, the method further comprises the steps of providing a compound of formula (XVII) having the following structure:

[0179] and forming the compound of formula (XV) from the compound of formula (XVII).

[0180] In one embodiment, the step of forming the compound of formula (XV) comprises reacting the compound of formula (XVII) with a brominating agent to produce the compound of formula (XVII).

[0181] In one embodiment, the brominating agent is CHBr3.

[0182] In one embodiment, the method further comprises the step of providing a compound of formula (XVIII) having the following structure:

[0183] and forming the compound of formula (PI) from the compound of formula (XVIII).

[0184] In one embodiment, the compound of formula (XVIII) has the following structure:

[0185]

[0186] In one embodiment, the step of forming the compound of formula (PI) comprises reacting the compound of formula (XVIII) with a compound of formula (XIX):

[0187] to produce the compound of formula (PI).

[0188] In one embodiment, the method further comprises the steps of providing a compound of formula (XX) having the following structure:

[0189] and forming the compound of formula (XVIII) from the compound of formula (XX).

[0190] In one embodiment, the compound of formula (XX) has the following structure:

[0191]

[0192] In one embodiment, the step of forming the compound of formula (XVIII) comprises reacting the compound of formula (XX) with a compound of formula (XXI):

[0193] to produce the compound of formula (XVIII).

[0194] In one embodiment, the method further comprises the steps of providing a compound of formula (XXII) having the following structure:

[0195] and forming the compound of formula (XX) from the compound of formula (XXII).

[0196] In one embodiment, the compound of formula (XXII) has the following structure:

[0197]

[0198] The present disclosure also relates to a method for preparing a compound of formula (I-1):

[0199]

[0200] or a pharmaceutically acceptable salt thereof,

[0201] in

[0202] X is selected from the group consisting of:

[0203] R 1 、R 2 、R 3 and R 4 are independently hydrogen or C 1-5 alkyl, and

[0204] R 5 and R 6 is independently hydrogen, -NH2 or the side chain of any natural or unnatural amino acid,

[0205] The method comprises:

[0206] (a) providing a compound of formula (V) having the following structure:

[0207] as well as

[0208] (b) forming the compound of formula (I-1) from the compound of formula (V).

[0209] In one embodiment, the compound of formula (I-1) has the following structure:

[0210]

[0211] In one embodiment, step (b) of forming the compound of formula (I-1) comprises reacting the compound of formula (V) with a compound of formula (VIa) or formula (VIb):

[0212]

[0213] wherein X' is a halogen, reacts to produce the compound of formula (I-1).

[0214] In one embodiment, the method further comprises the steps of providing a compound of formula (VII) having the following structure:

[0215]

[0216] Among them PG 1 is a suitable protecting group protecting group, and

[0217] The compound of formula (V) is formed from the compound of formula (VII).

[0218] In one embodiment, the compound of formula (VII) has the following structure:

[0219]

[0220] In one embodiment, the step of forming the compound of formula (V) comprises reacting the compound of formula (VII) with a compound of formula (VIII):

[0221] to produce the compound of formula (V).

[0222] In one embodiment, the method further comprises the steps of providing a compound of formula (IX) having the following structure:

[0223] and forming the compound of formula (VII) from the compound of formula (IX).

[0224] In one embodiment, the compound of formula (IX) has the following structure:

[0225]

[0226] In one embodiment, the step of forming the compound of formula (VII) comprises reacting the compound of formula (IX) with a compound of formula (X):

[0227] to produce the compound of formula (VII).

[0228] In one embodiment, the method further comprises the steps of providing a compound of formula (XI) having the following structure:

[0229] and forming the compound of formula (IX) from the compound of formula (XI).

[0230] In one embodiment, the compound of formula (XI) has the following structure:

[0231]

[0232] In one embodiment, the step of forming the compound of formula (IX) comprises reacting the compound of formula (XI) with a compound of formula (XII):

[0233] to produce the compound of formula (IX).

[0234] The present disclosure also relates to a method for preparing a compound of formula (XVIII):

[0235]

[0236] or a pharmaceutically acceptable salt thereof,

[0237] where R 1 、R 2 、R 3 and R 4 are independently hydrogen or C 1-5 alkyl, and

[0238] R 5 and R 6 is independently hydrogen, -NH2 or the side chain of any natural or unnatural amino acid. The method comprises:

[0239] (a) providing a compound of formula (XX) having the following structure:

[0240] as well as

[0241] (b) forming the compound of formula (XVIII) from the compound of formula (XX).

[0242] In one embodiment, the compound of formula (XVIII) has the following structure:

[0243]

[0244] In one embodiment, the compound of formula (XX) has the following structure:

[0245]

[0246] In one embodiment, the step of forming the compound of formula (XVIII) comprises reacting the compound of formula (XX) with a compound of formula (XXI):

[0247] to produce the compound of formula (XVIII).

[0248] In one embodiment, the method further comprises the steps of providing a compound of formula (XXII) having the following structure:

[0249] and forming the compound of formula (XX) from the compound of formula (XXII).

[0250] In one embodiment, the compound of formula (XXII) has the following structure:

[0251]

[0252] The present disclosure also relates to a method for preparing a compound of formula (D-1):

[0253]

[0254] or a pharmaceutically acceptable salt thereof,

[0255] where R 1 、R 2 、R 3 and R 4 are independently hydrogen or C 1-5 alkyl, and

[0256] R 5 and R 6 is independently hydrogen, -NH2 or the side chain of any natural or unnatural amino acid. The method comprises:

[0257] (a) providing a compound of formula (I-1) having the following structure:

[0258]

[0259] in

[0260] X is selected from the group consisting of: as well as

[0261] (b) reacting the compound of formula (I-1) with a compound of formula (PI):

[0262]

[0263] in

[0264] R is H or PG; and

[0265] PG is a suitable protecting group,

[0266] reaction to produce the compound of formula (D-1).

[0267] In one embodiment, the compound of formula (D-1) has the following structure:

[0268]

[0269] In one embodiment, the compound of formula (I-1) has the following structure:

[0270]

[0271] In one embodiment, the step (b) of reacting the compound of formula (I-1) with the compound of formula (PI) further comprises reacting the compound of formula (PI), wherein R is PG, with a protecting group removing agent before reacting with the compound of formula (I-1).

[0272] In one embodiment, the PG is selected from the group consisting of allyloxycarbonyl (Alloc), benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), and 9-fluorenylmethoxycarbonyl (Fmoc).

[0273] In one embodiment, the compound of formula (PI) has the following structure:

[0274]

[0275] In one embodiment, the method further comprises the step of providing a compound of formula (XVIII) having the following structure:

[0276] and forming the compound of formula (PI) from the compound of formula (XVIII).

[0277] In one embodiment, the compound of formula (XVIII) has the following structure:

[0278]

[0279] In one embodiment, the step of forming the compound of formula (PI) comprises reacting the compound of formula (XVIII) with a compound of formula (XIX):

[0280] to produce the compound of formula (PI).

[0281] The present disclosure also relates to a method for preparing a compound of formula (D-1):

[0282]

[0283] or a pharmaceutically acceptable salt thereof,

[0284] where R1 、R 2 、R 3 and R 4 are independently hydrogen or C 1-5 alkyl, and

[0285] R 5 and R 6 is independently hydrogen, -NH2 or the side chain of any natural or unnatural amino acid,

[0286] The method comprises:

[0287] (a) providing a compound of formula (XXIII):

[0288] as well as

[0289] (b) reacting the compound of formula (XXIII) with a compound having the following structure:

[0290] The reaction is carried out in the presence of an activating agent and a base to produce the compound of formula (D-1).

[0291] In one embodiment, the compound of formula (D-1) has the following structure:

[0292]

[0293] In one aspect, the present disclosure provides a compound of formula (I-1):

[0294]

[0295] or a pharmaceutically acceptable salt thereof,

[0296] in

[0297] X is selected from the group consisting of:

[0298] R 1 、R 2 、R 3 and R 4 are independently hydrogen or C 1-5 Alkyl, and R 5 and R 6 is independently hydrogen, -NH2, or the side chain of any natural or unnatural amino acid.

[0299] In one embodiment, the compound of formula (I-1) has the following structure:

[0300]

[0301] In one aspect, the present disclosure provides a compound of formula (XVIII):

[0302]

[0303] or a pharmaceutically acceptable salt thereof,

[0304] where R 1 、R 2 、R 3 and R 4 are independently hydrogen or C 1-5 alkyl, and

[0305] R 5 and R 6 is independently hydrogen, -NH2, or the side chain of any natural or unnatural amino acid.

[0306] In one embodiment, the compound of formula (XVIII) has the following structure:

[0307]

[0308] In another aspect, the present disclosure provides a linker-payload compound of formula (D),

[0309] or a pharmaceutically acceptable salt thereof,

[0310] where R 1 、R 2 、R 3 and R 4 are independently hydrogen or C 1-5 alkyl, and

[0311] R 5 and R 6 is independently hydrogen, -NH2, or the side chain of any natural or unnatural amino acid.

[0312] In another aspect, the present disclosure provides a linker-payload compound having a formula selected from the group consisting of (D') to (N') below:

[0313]

[0314]

[0315]

[0316] or a pharmaceutically acceptable salt thereof,

[0317] where R 1 、R 2 、R 3 and R4 are independently hydrogen or C 1-5 alkyl;

[0318] B is selected from the group consisting of: W is NH, O, CO, CH2, phenyl, or a combination of two or more thereof; and R 5 、R 6 、R 7 and R 8 is independently hydrogen, -NH2 or the side chain of any natural or unnatural amino acid,

[0319] The method comprises the steps of exposing a payload having an amino group to an activated intermediate having p-nitrophenyl carbonate in the presence of a base and a coupling catalyst to obtain the linker-payload compounds (D') to (G'), wherein the coupling catalyst is 4-hydroxy-2-methylquinoline (MeHYQ).

[0320] In one embodiment, the structure is selected from the group consisting of:

[0321]

[0322]

[0323]

[0324]

[0325]

[0326] In one embodiment, the structure is selected from the group consisting of:

[0327]

[0328] These and other aspects of the disclosure will become apparent to those skilled in the art after reading the following detailed description of the disclosure, including the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0329] Figure 1 Schematic diagram illustrating the two-step site-specific generation of Dxd-ADCs according to embodiments of the present disclosure. The first step is to conjugate 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 to conjugate the antibody-L1-B to one or more linker 2-payloads (L2P).

[0330] Figure 2A and 2Bare schematic diagrams illustrating certain non-limiting embodiments of the present disclosure. Figure 2A Schematic diagram of the two-step site-specific generation of a Dxd-ADC with a DAR of 2 times n times m and a glutamine residue at position 295 according to an embodiment of the present disclosure. Figure 2B Schematic diagram of the two-step site-specific generation of a Dxd-ADC with a DAR of 4 times n times m and glutamine residues at positions 295 and 297 according to an embodiment of the present disclosure.

[0331] Figure 3A Figure 2 is a schematic diagram illustrating the two-step site-specific generation of a specific embodiment of a Dxd-ADC according to the present disclosure. The first step involves conjugating a linear first linker 1 (L1-B') containing one azide moiety (-N3) to glutamine residues at positions 295 and 297 of an antibody via an MTG-mediated conjugation reaction, generating an antibody (Ab-(N3)4) having a linker containing four azides attached thereto. The second step involves attaching 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 Describes the application Figure 3A Schematic diagram of an ADC having a DAR of 2 or 4 and an exemplary aminoazide linker according to embodiments of the present disclosure is depicted in FIG.

[0332] Figure 4A Figure 1 is a schematic diagram illustrating the two-step site-specific generation of a specific embodiment of a Dxd-ADC according to the present disclosure. The first step involves conjugating a branched first linker 1 (L1-B') comprising two azide moieties (-N3) to glutamine residues at positions 295 and 297 of an antibody via an MTG-mediated conjugation reaction, generating an antibody (Ab-(N3)8) having a linker comprising eight azides attached thereto. The second step involves attaching 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 Describes the application Figure 4A Schematic diagram of an ADC and an exemplary branched alkylamine azide linker of an embodiment of the present disclosure depicted in FIG.

[0333] Figure 5 Schematic diagram of a two-step antibody-drug conjugation according to an embodiment of the present disclosure. Step 1: Site-specific conjugation of handle-functionalized amines to antibodies, generating drug conjugates containing 2, 4, or 8 handles per antibody. Here, AL = unbranched handle-functionalized amine, BL = branched handle-functionalized amine. Step 2: Click reaction between the handle-functionalized antibody and the linker-payload (LP) to generate a site-specific ADC.

[0334] Figure 6 An exemplary conjugation procedure according to the present disclosure is depicted.

[0335] Figure 7A Three methods for the preparation of antibody-drug conjugates according to the present disclosure are depicted. For methods 1 and 2, the handle may be bivalent or multivalent. An amine handle may be conjugated to an antibody via transglutaminase-mediated conjugation to produce an Ab-handle; another portion of the handle of the Ab-handle may be click-reacted with a linker-payload to generate an ADC. In the case where the handle has a diene, the linker-payload has a dienophile, and vice versa. For Figure 7B 3, the linker-payload can be conjugated directly to the antibody; LL contains an amine portion that can be conjugated to the antibody via transglutaminase-mediated conjugation; LL containing a portion reactive with cysteine-SH can be conjugated to antibody-cystine via Michael addition.

[0336] Figure 8 is a graph showing linker-ProDXd LP1 (SEQ ID NO: 2121) in mouse whole blood.

[0337] Figure 9 A schematic method for the preparation of liver S9 and liver microsomes from hepatocytes is shown. DETAILED DESCRIPTION

[0338] Detailed embodiments of the present disclosure are disclosed herein; however, it should be understood that the disclosed embodiments are merely illustrative of the various forms in which the present disclosure may be implemented. Additionally, each example provided in conjunction with the 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 to apply the present disclosure in various ways.

[0339] definition

[0340] Unless defined otherwise, 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.

[0341] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents 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 one skilled in the art upon reading this disclosure and so forth.

[0342] The terms "treat" or "treatment" of a condition, disorder, or condition include: (1) preventing, delaying, or reducing the incidence and / or likelihood of the appearance of at least one clinical or subclinical symptom of a condition, disorder, or condition that develops in a subject who may be suffering from or susceptible to the condition, disorder, or condition but has not yet experienced or displayed clinical or subclinical symptoms of the condition, disorder, or condition; or (2) inhibiting the condition, disorder, or condition, i.e., preventing, reducing, or delaying the development of the disease or its recurrence or at least one clinical or subclinical symptom thereof; or (3) alleviating the disease, i.e., causing regression of the condition, disorder, or condition or at least one of its clinical or subclinical symptoms. The benefit to the subject being treated is statistically significant, or at least perceptible to the patient or physician. In some embodiments, treatment includes methods in which cells are ablated in a manner that indirectly affects the disease. In certain embodiments, treatment includes depleting immune cells as a hematopoietic conditioning regimen prior to therapy.

[0343] 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 preferred embodiments, the subject is a human.

[0344] As used herein, the term "effective" as applied to dosage or amount refers to an amount of a compound or pharmaceutical composition sufficient to produce the desired activity upon administration to a subject in need thereof. Note that when a combination of active ingredients is administered, an effective amount of the combination may or may not include the amount of each ingredient that would be effective if administered alone. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the specific drug or drugs employed, the mode of administration, and the like.

[0345] As 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, acid-derived salts, base-derived salts, organic salts, inorganic salts, amines, and alkali metal or alkaline earth metal salts, including but not limited to calcium salts, magnesium salts, potassium salts, sodium salts, hydrochlorides, hydrobromides, sulfates, nitrates, phosphates, acetates, propionates, glycolates, pyruvates, oxalates, maleates, malonates, succinates, fumarates, tartrates, citrates, benzoates, cinnamates, mandelates, methanesulfonates, ethanesulfonates, p-toluenesulfonates, salicylates, etc. In some instances, the payload described herein (e.g., rifamycin analogs described herein) includes a tertiary amine, wherein the nitrogen atom in the tertiary amine is an atom by which the payload is bound to a joint or joint-spacer. In this case, the combination with the tertiary amine of the payload produces a quaternary amine in the joint-payload molecule. The positive charge on the quaternary amine can be balanced by a counterion (e.g., chlorine, bromine, iodine, or any other suitable charged moiety, such as those described herein).

[0346] 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.

[0347] “Comprising” or “containing” or “including” means that at least the recited 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 such other compounds, materials, particles or method steps have the same function as the recited ones.

[0348] The compounds of the present disclosure include compounds generally described herein and are further described by the classes, subclasses, and species disclosed herein. Unless otherwise indicated, as used herein, the following definitions shall apply. 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 edition. In addition, the general principles of organic chemistry are described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999; and "March's Advanced Organic Chemistry", 5th edition, eds.: Smith, MB and March, J., John Wiley & Sons, New York: 2001, the entire contents of which are hereby incorporated by reference.

[0349] As used herein, the term "alkyl" is given its ordinary meaning in the art and may include saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl groups (alicyclic), alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups. In certain embodiments, a straight-chain or branched-chain alkyl group has from about 1 to 20 carbon atoms in its backbone (e.g., C1-C2 for a straight chain). 20 ; For the branched chain, it is C2–C 20 ), and alternatively about 1 to 10 carbon atoms, or about 1 to 6 carbon atoms. In some embodiments, the cycloalkyl ring has about 3 to 10 carbon atoms in its ring structure, wherein such ring is monocyclic or bicyclic, and alternatively has about 5, 6 or 7 carbon atoms in the ring structure. In some embodiments, the alkyl group can be a lower alkyl group, wherein the lower alkyl group includes 1 to 4 carbon atoms (e.g., C1-C4 for straight chain lower alkyl).

[0350] As used herein, the term "alkenyl" refers to an alkyl group as defined herein having one or more double bonds.

[0351] As used herein, the term "alkynyl" refers to an alkyl group as defined herein having one or more triple bonds.

[0352] The term "aryl", used alone or as part of a larger moiety as in "aralkyl", "aralkyloxy" or "aryloxyalkyl", refers to a monocyclic or bicyclic ring system having a total of five to fourteen ring members, wherein at least one ring in the system is aromatic and wherein each ring in the system contains 3 to 7 ring members. The term "aryl" can 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, binaphthyl, anthracenyl, etc., which may carry one or more substituents. As used herein, groups in which an aromatic ring is fused to one or more non-aromatic rings, such as indanyl, phthalimidyl, naphthimidyl, phenanthridinyl, or tetrahydronaphthyl, are also included within the scope of the term "aryl".

[0353] 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).

[0354] The term "halogen" means F, Cl, Br or I; the term "halide" refers to a halogen group or substituent, ie, -F, -Cl, -Br or -I.

[0355] The term "click chemistry" refers to a class of biocompatible small molecule reactions commonly used in bioconjugation that allows a selected substrate to be attached to a specific biomolecule. Click chemistry is not a single specific reaction, but rather describes a way of generating products that mimics examples found in nature, where substances are generated by connecting small modular units. Click chemistry is not limited to biological conditions: the concept of a "click" reaction can be used in chemical proteomics applications, pharmacological applications, and various biomimetic-type applications. Specific non-limiting examples of click chemistry reactions include:

[0356] (a) Strain-promoted azide / dibenzocyclooctynamine (DBCO) click chemistry.

[0357] (b) Inverse electron demand Diels-Alder (IED-DA) tetrazine / trans-cyclooctene (TCO) click chemistry;

[0358] (c) Inverse electron demand Diels-Alder (IED-DA) tetrazine / norbornene click chemistry;

[0359] (d) Diels-Aldermaleimide / furan click chemistry;

[0360] (e) Staudinger connection; and

[0361] (f) Nitrile oxide / norbornene cycloaddition click chemistry.

[0362] The term "adduct," e.g., "adduct of group B" or "click chemistry adduct" of the present disclosure encompasses any moiety comprising the product of an addition reaction, e.g., an addition reaction of group B or a click chemistry addition reaction, independent of the synthetic steps employed to produce the moiety.

[0363] The term "covalent attachment" means the formation of a covalent bond, i.e., a chemical bond involving the sharing of one or more electron pairs between two atoms. Covalent bonding may include different interactions, including but not limited to σ-bonding, π bonding, metal-to-metal bonding, chelate interactions, bent bonds, and three-center two-electron bonds. When a first group is referred to as "capable of co-attaching" to a second group, this means that the first group is capable of forming a covalent bond with the second group, directly or indirectly, for example, by using a catalyst or under specific reaction conditions. Non-limiting examples of groups that can co-attach to each other can 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).

[0364] As described herein, the compounds of the present disclosure may contain "optionally substituted" moieties. In general, the term "substituted", whether or not preceded by the term "optionally", means that one or more hydrogens of the designated moiety are replaced by a suitable substituent. Unless otherwise indicated, an "optionally substituted" group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with one or more substituents selected from a designated group, the substituents may be the same or different at each position. The substituent combinations contemplated by the present disclosure are preferably substituent combinations that enable the formation of stable or chemically feasible compounds. As used herein, the term "stable" refers to a compound that does not substantially change when subjected to conditions that allow its production, detection, and in certain embodiments, its recovery, purification, and use for one or more purposes disclosed herein.

[0365] Unless otherwise indicated, structures depicted herein are also meant to encompass all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) forms of the structure; for example, R and S configurations for each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present compounds are within the scope of the present disclosure.

[0366] Unless otherwise stated, all tautomeric forms of the disclosed compounds are within the scope of the disclosure.

[0367] 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, structures having a structure other than the replacement of a hydrogen by a deuterium or tritium or by a 11 C- or 13 C- or 14 Compounds of the present structures in which a C-enriched carbon replaces a carbon other than carbon are within the scope of the present disclosure.

[0368] It should also be understood that reference to one or more method steps does not exclude the presence of additional method steps or intervening method steps between those steps explicitly identified. Similarly, it should be understood that reference to one or more components in an apparatus or system does not exclude the presence of additional components or intervening components between those explicitly identified components.

[0369] Unless otherwise indicated, all crystalline forms of the compounds of the present disclosure and their salts are 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 hemihydrates, monohydrates, dihydrates, etc. In some embodiments, the compounds of the present disclosure are anhydrous and non-solvated. "Anhydrous" means that the crystalline form of the compound does not substantially contain bound water in the lattice structure, that is, the compound does not form a crystalline hydrate.

[0370] As used herein, "crystalline form" means certain lattice configurations of a crystalline substance. Different crystalline forms of the same substance typically have different lattices (e.g., unit cells) that are attributed to different physical properties that are characteristic of each crystalline form. In some cases, different lattice configurations have different water or solvent contents. Different 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), solid-state NMR, etc., further contribute to the identification of the crystalline form and to determining stability and solvent / water content.

[0371] Crystalline forms of a substance include solvated (e.g., hydrated) and non-solvated (e.g., anhydrous) forms. A hydrated form is a crystalline form that includes water in the crystal lattice. A hydrated form can be a stoichiometric hydrate, wherein the water in the crystal lattice is present at a certain water / molecule ratio, such as a hemihydrate, a monohydrate, a dihydrate, etc. A hydrated form can also be non-stoichiometric, wherein the water content is variable and depends on external conditions such as humidity.

[0372] In some embodiments, the compounds of the present disclosure are substantially isolated. "Substantially isolated" means that a particular compound is at least partially separated 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 any substance that is not a substantially isolated compound, including, for example, other crystalline forms and other substances.

[0373] Certain groups, moieties, substituents, and atoms are depicted with wavy lines. A wavy line may intersect or limit one or more bonds. A wavy line indicates an atom through which a group, moiety, substituent, or atom is bonded. For example, a phenyl group substituted with a propyl group is depicted as: Has the following structure:

[0374] The expression "HER2" or "human epidermal growth factor receptor 2" refers to a member of the human epidermal growth factor receptor family. The protein is also known as NEU; NGL; HER2; TKR1; CD340; HER-2; MLN 19; HER-2 / neu. HER2 may refer to the amino acid sequence as shown in NCBI Accession No. NP_004439.2. Amplification or overexpression of this oncogene has been shown to play an important role in the development and progression of certain aggressive types of breast cancer. In recent years, the protein has become an important biomarker and therapeutic target for approximately 30% of breast cancer patients. Unless expressly specified as being from a non-human species, all references to proteins, polypeptides, and protein fragments herein are intended to refer to the human version of the corresponding protein, polypeptide, or protein fragment. Thus, the expression "HER2" is intended to refer to human HER2 unless specified as being from a non-human species, for example, "mouse HER2," "monkey HER2," etc.

[0375] The phrase "antibody that binds to HER2" or "anti-HER2 antibody" includes antibodies and antigen-binding fragments thereof that specifically recognize HER2.

[0376] The phrase "anti-HER2 / HER2" antibody, e.g., "anti-HER2 / HER2 bispecific antibody" includes antibodies and antigen-binding fragments thereof that specifically recognize two different HER2 epitopes. In some embodiments, the bispecific antibody and antigen-binding fragment thereof comprise a first antigen-binding domain (D1) and a second antigen-binding domain (D2), the first antigen-binding domain specifically binding to a first epitope of human HER2, and the second antigen-binding domain specifically binding to a second epitope of human HER2.

[0377] As used herein, the expression "STEAP2" refers to the six-transmembrane epithelial antigen of the prostate 2. STEAP2 is an integral six-transmembrane spanning protein that is highly expressed in prostate epithelial cells and is a cell surface marker for prostate cancer. For example, STEAP2 is expressed at significant levels in the LNCaP prostate cell line (Porkka et al. Lab Invest 2002, 82:1573-1582). STEAP2 (UniProtKB / Swiss-Prot: Q8NFT2.3) is a 490 amino acid protein encoded by the STEAP2 gene located in chromosome region 7q21 in humans. See, for example, the amino acid sequence of human STEAP2 shown in Tables 5 and 6.

[0378] As used herein, “antibodies that bind to STEAP2” or “anti-STEAP2 antibodies” include antibodies that specifically recognize STEAP2 and antigen-binding fragments thereof.

[0379] 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 expressions "MET," "c-Met," and the like refer to human transmembrane receptor tyrosine kinase.

[0380] The phrase "anti-MET / MET" antibody, such as "anti-MET / MET bispecific antibody," includes antibodies and antigen-binding fragments thereof that specifically recognize two different MET epitopes. In some embodiments, the bispecific antibody and antigen-binding fragment 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.

[0381] Amino acid abbreviations used in this disclosure are those accepted by the United States Patent and Trademark Office as set forth in 37 CFR §1.822(B)(J).

[0382] The term "protein" means any amino acid polymer having more than about 20 amino acids covalently linked via amide bonds. As used herein, "protein" includes recombinant proteins used in biotherapeutic proteins, 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, etc. Proteins can be produced using a recombinant cell-based production system, such as an insect baculovirus system, a yeast system (e.g., Pichia sp.), a mammalian system (e.g., CHO cells and CHO derivatives, such as CHO-K1 cells).

[0383] 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 naturally occurring in humans.

[0384] The terms "nonnatural" (also spelled non-natural and nonnatural) amino acids and "nonnatural amino acid side chains" refer to amino acids and their side chains, respectively, that do not naturally occur in the subject organism (e.g., humans). Such non-natural amino acids can be synthesized or produced naturally in different environments (e.g., in different organisms). Non-limiting examples of non-natural amino acids can include D-amino acids, homoamino acids, β-homoamino acids, N-methyl amino acids, α-methyl amino acids, and amino acids found in, for example, microbial peptides, such as citrulline (Cit), hydroxyproline (Hyp), norleucine (Nle), 3-nitrotyrosine, nitroarginine, ornithine (Orn), naphthylalanine (Nal), Abu, DAB, methionine sulfoxide, or methionine sulfone.

[0385] Unless expressly designated as being from a non-human species, all references herein to proteins, polypeptides, and protein fragments are intended to refer to the human versions of the corresponding proteins, polypeptides, or protein fragments. Thus, the expression "STEAP2" refers to human STEAP2 unless designated as being from a non-human species, e.g., "mouse STEAP2," "monkey STEAP2," etc.

[0386] The amino acid sequences of antibodies can be numbered using any known numbering scheme, including those described by Kabat et al. ("Kabat" numbering system); Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948 ("Chothia" numbering system); MacCallum et al., 1996, J. Mol. Biol. 262:732-745 ("Contact" numbering system); Lefranc et al., Dev. Comp. Immunol., 2003, 27:55-77 ("IMGT" numbering system); and Honegge and Pluckthun, J. Mol. Biol., 2001, 309:657-70 ("AHo" numbering system). Unless otherwise indicated, the numbering scheme used herein is the Kabat numbering scheme. However, the choice of numbering scheme is not intended to imply differences in sequences where they do not exist, and sequence positions can be readily confirmed by one skilled in the art by inspection of the amino acid sequence of one or more antibodies. Unless otherwise indicated, the "EU numbering scheme" (eg, as reported in Kabat et al., supra) is generally used when referring to residues in the antibody heavy chain constant region.

[0387] The term "glutaminyl-modified antibody" refers to an antibody having at least one covalent bond from a glutamine side chain to a primary amine compound of the present disclosure. In specific embodiments, the primary amine compound is linked by an amide bond on the glutamine side chain. In certain embodiments, the glutamine is endogenous glutamine. In other embodiments, the glutamine is an endogenous glutamine that is reactive through polypeptide engineering (e.g., via amino acid deletion, insertion, substitution, or mutation on the polypeptide). In further embodiments, the glutamine is a polypeptide engineered with a tag containing the acyl donor glutamine (e.g., a glutamine-containing peptide tag, a Q-tag, or a TG enzyme recognition tag).

[0388] The term "TG enzyme recognition tag" refers to an amino acid sequence comprising an acceptor glutamine residue and, when incorporated into (e.g., attached to) a polypeptide sequence under suitable conditions, is recognized by the TG enzyme and cross-linked by the reaction between the amino acid side chain and the reaction partner in the amino acid sequence by the TG enzyme. The recognition tag may be a peptide sequence that is not naturally present in the polypeptide comprising the TG enzyme recognition tag. In certain embodiments, the TG enzyme recognition tag comprises at least one Gln. In certain embodiments, the TG enzyme recognition tag comprises an amino acid sequence XXQX, wherein X is any amino acid (e.g., conventional amino acids Leu, Ala, Gly, Ser, Val, Phe, Tyr, His, Arg, Asn, Glu, Asp, Cys, Gln, Ile, Met, Pro, Thr, Lys, or Trp or unconventional amino acids). In some embodiments, the tag comprising the acyl donor glutamine comprises an amino acid sequence selected from the group consisting of 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), LLQLLQGA (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), NO: 1950), SLLQG (SEQ ID NO: 1951), LLQLQ (SEQ ID NO: 1952), LLQLLQ (SEQ ID NO: 1953) and LLQGR (SEQ ID NO: 1954). See, for example, WO2012059882, the entire contents of which are incorporated herein.

[0389] As used herein, the term "antibody" means any antigen-binding molecule or molecular complex comprising at least one complementary determining region (CDR) that specifically binds to or interacts with a specific antigen. The term "antibody" includes immunoglobulin molecules comprising 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 comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain (CL1). The VH and VL regions can be further subdivided into hypervariable regions referred to as complementary determining regions (CDRs), which are interspersed with more conservative regions referred to as framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In various embodiments, the FRs of the antibody (or its antigen-binding portion) may be identical to human germline sequences or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs.

[0390] As used herein, the term "antibody" also includes the antigen-binding fragment of a complete antibody molecule. As used herein, the term "antigen-binding portion thereof," "antigen-binding fragment" of an antibody, etc., includes any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. The antigen-binding fragment of an antibody can, for example, be obtained using any suitable standard technique, such as proteolytic digestion or recombinant genetic engineering techniques, which are derived from full antibody molecules and which relate to manipulation and expression of DNA encoding variable and optionally constant domains of an antibody. Such DNA is known and / or can be easily obtained from, for example, 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 arrange one or more variable and / or constant domains into a suitable configuration, or to introduce codons, produce cysteine ​​residues, modify, add or delete amino acids, etc.

[0391] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues that mimic the 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. As used herein, other engineered molecules such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, terabodies, minibodies, nanobodies (e.g., monovalent nanobodies, divalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains are also encompassed within the expression "antigen-binding fragment."

[0392] The antigen-binding fragment of an antibody generally comprises at least one variable domain. The variable domain can have any size or amino acid composition and will generally comprise at least one CDR adjacent to or within the frame of one or more framework sequences. In an antigen-binding fragment having a VH domain associated with a VL domain, the VH and VL domains can be positioned in any suitable arrangement relative to each other. 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.

[0393] In certain embodiments, the 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 can be found within the antigen-binding fragments of an antibody of the present specification 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 can be directly linked to each other or can be linked by a complete or partial hinge or linker region. The hinge region can be composed of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that create a flexible or semi-flexible connection between adjacent variable and / or constant domains in a single polypeptide molecule.

[0394] Furthermore, antigen-binding fragments of the antibodies of the present description may include homodimers or heterodimers (or other multimers) of any of the variable and constant domain configurations listed herein non-covalently associated with each other and / or with one or more monomeric VH or VL domains (e.g., via disulfide bonds).

[0395] As with full antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). A multispecific antigen-binding fragment of an antibody will typically include at least two different variable domains, each of which is capable of specifically binding to a separate antigen or a different epitope on the same antigen. Conventional techniques available in the art can be used to make any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, suitable for use in the context of the antigen-binding fragments of the antibodies of this specification.

[0396] The antibody of this specification sheets can work by complement dependent cytotoxicity (CDC) or antibody dependent cell-mediated cytotoxicity (ADCC). " Complement dependent cytotoxicity " (CDC) refers to the cracking of the antibody of this specification sheets to the cell expressing the antigen in the presence of complement. " Antibody dependent cell-mediated cytotoxicity " (ADCC) refers to the reaction of cell mediation, wherein the non-specific cytotoxic cells (for example, natural killer (NK) cells, neutrophils and macrophages) expressing Fc receptors (FcR) recognize the binding antibody on target cells and thus cause target cell lysis. CDC and ADCC can be measured using well known in the art and available assays. (see, for example, U.S. Patent number 5,500,362 and 5,821,337, and Clynes et al. (1998) Proc.Natl.Acad.Sci.(USA)95:652-656). The constant region of the antibody is important in the ability of antibody fixation complement and mediation cell dependent cytotoxicity. Thus, the isotype of the antibody can be selected based on whether antibody-mediated cytotoxicity is desired.

[0397] In certain embodiments, the antibodies of the present description (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. The human antibodies of the present description may include amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo), for example, in the CDR region, and specifically in the CDR3 region. However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (such as a mouse) have been transplanted onto human framework sequences.

[0398] In certain embodiments, antibody can be recombinant human antibody.As used herein, term " recombinant human antibody " is intended to include all human antibodies prepared, expressed, produced or separated by recombinant means, such as using the antibody (described further below) expressed by the recombinant expression vector transfected into the host cell, the antibody (described further below) separated from the combined human antibody library of restructuring, the antibody (described further below) separated from the transgenic animal (e.g., mouse) for human immunoglobulin genes (see, e.g., Taylor et al. (1992) Nucl.Acids Res.20:6287-6295) or prepared, expressed, produced or separated by any other means, these methods are related to human immunoglobulin gene sequences being spliced ​​onto other DNA sequences.Such recombinant human antibodies have variable regions and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies have undergone in vitro mutagenesis (or, when animals transgenic for human Ig sequences are used, in vivo somatic mutagenesis) and thus, 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, may not naturally exist within the human antibody germline repertoire in vivo.

[0399] Human antibodies can exist in two forms related to hinge heterogeneity. In one form, the immunoglobulin molecule includes a stable four-chain construct of about 150 to 160 kDa, in which the dimer is held together by an interchain heavy chain disulfide bond. In the second form, the dimer is not connected by an interchain disulfide bond and forms a molecule of about 75 to 80 kDa, which is composed of a light chain and a heavy chain (half antibody) covalently coupled. Even after affinity purification, these forms are extremely difficult to separate. The frequency of the second form occurring in various complete IgG isotype forms is based on, but is not limited to, the structural differences related to the hinge region isotype of the antibody. The single amino acid substitution in the hinge region of the human IgG4 hinge can significantly reduce the appearance of the second form (Angal et al. (1993) Molecular Immunology 30:105) to the level typically observed with the human IgG1 hinge. The present description encompasses antibodies having one or more mutations in the hinge, CH2, or CH3 regions, which may be desirable, for example, in production to improve yield of the desired antibody form.

[0400] The antibody of this specification can be an isolated antibody or a purified antibody. As used herein, "isolated antibody" or "purified antibody" means 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 tissues or cells in which the antibody naturally occurs or is naturally produced is a "isolated antibody" for the purposes of this specification. For example, an antibody that has been purified from at least one component of a reaction or reaction sequence is a "purified antibody" or is produced by a purified antibody. Isolated antibodies also include in situ antibodies within recombinant cells. Isolated antibodies are antibodies that have undergone at least one purification or separation step. According to certain embodiments, isolated antibodies or purified antibodies can be substantially free of other cellular substances and / or chemicals.

[0401] Compared to the corresponding germline sequence derived from the antibody, the antibodies disclosed herein can include one or more amino acid substitutions, insertions and / or deletions in the framework and / or CDR regions of the heavy chain variable domain and the light chain variable domain. By comparing the amino acid sequences disclosed herein with the germline sequences available from, for example, public antibody sequence databases, such mutations can be easily determined. This specification includes antibodies and antigen-binding fragments derived from any amino acid sequence disclosed herein, wherein one or more amino acids in the framework and / or CDR regions are mutated into the corresponding residues of the germline sequence of the derived antibody, or mutated into the corresponding residues of another human germline sequence, or mutated into the conservative amino acid substitutions of the corresponding germline residues (such sequence changes are collectively referred to as "germline mutations" in this article). Starting from the heavy chain and light chain variable region sequences disclosed herein, those of ordinary skill in the art can easily produce many antibodies and antigen-binding fragments including one or more single 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 residues found in the original germline sequence of the derived antibody. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., only the mutated residues found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the mutated residues found in CDR1, CDR2, or CDR3. In other embodiments, one or more of the framework and / or CDR residues are mutated to the corresponding residue of a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antibody was originally derived).

[0402] Furthermore, the antibodies of the present disclosure may contain any combination of two or more germline mutations within the framework and / or CDR regions, for example, wherein certain individual residues are mutated into corresponding residues of a particular germline sequence, while certain other residues different from the original germline sequence are maintained or mutated into corresponding residues of different germline sequences. Once obtained, antibodies and antigen-binding fragments containing one or more germline mutations may be easily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or exciting biological properties (as the case may be), reduced immunogenicity, improved drug-to-antibody ratios (DARs) of antibody-drug conjugates, and the like. Antibodies and antigen-binding fragments obtained in this general manner are encompassed within the present disclosure.

[0403] The term "aglycosylated antibody" refers to an antibody that does not contain a glycosylation sequence that may interfere with a transglutamination reaction, for example, an antibody that does not have a carbohydrate group at N297 on one or more heavy chains. In a specific embodiment, the antibody heavy chain has an N297 mutation. In other words, the antibody is mutated to no longer have an asparagine residue at position 297 (according to the EU numbering system disclosed by Kabat et al.). In a specific embodiment, 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 glutamine residues at sites 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 a combination of mutations that result in a lack of glycosylation.

[0404] The term "deglycosylated antibody" refers to an antibody in which sugar groups have been removed to facilitate transglutaminase-mediated conjugation. Sugars 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 accomplished enzymatically, including but not limited to, via PNGase.

[0405] 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 a paratope. A single antigen can have more than one epitope. Thus, different antibodies can bind to different regions on the antigen and can have different biological effects. Epitopes can be conformational or linear. Conformational epitopes are produced by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear epitopes are epitopes produced by adjacent amino acid residues in a polypeptide chain. In some cases, an epitope may comprise a portion of a sugar, phosphoryl, or sulfonyl group on the antigen.

[0406] As used herein, the term "conjugated protein" or "conjugated antibody" refers to a protein or antibody covalently linked to one or more chemical moieties. The chemical moieties may include amine compounds of the present disclosure. Linkers (LL) and payloads (P) suitable for use with the present disclosure are described in detail herein. In certain embodiments, the conjugated antibody comprising a therapeutic moiety is an antibody-drug conjugate (ADC), also referred to as an antibody-payload conjugate or an antibody-linker-payload conjugate.

[0407] The term "drug to antibody ratio" or (DAR) is the average number of conjugated therapeutic moiety (eg, drug) to binding agent of the disclosure.

[0408] The term "linker-antibody ratio" or (LAR), also denoted as the lower case L, is, in some embodiments, the average number of conjugated reactive primary amine compounds to the binding agents of the present disclosure. Such binding agents, such as antibodies, can be conjugated to primary amine compounds comprising, for example, suitable azides or alkynes. The resulting binding agents functionalized with azides or alkynes can then react with therapeutic moieties comprising the corresponding azides or alkynes via a 1,3-cycloaddition reaction.

[0409] 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 the antibody or antibody-drug conjugate provided herein. Suitable pharmaceutically acceptable amounts include, but are not limited to, from about 0.001% to about 10%, and any amount therebetween, such as about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% of an antibody or antibody-drug conjugate provided herein.

[0410] The phrase "reaction pH" refers to the pH of the reaction after all reaction components or reactants have been added.

[0411] As discussed below, the terms "substantial identity" or "substantially identical" when referring to nucleic acids or fragments thereof mean that, when optimally aligned with another nucleic acid (or its complementary strand) by appropriate nucleotide insertions or deletions, the nucleotide bases have at least about 95%, and more preferably at least about 96%, 97%, 98% or 99% nucleotide sequence identity as measured by any well-known sequence identity algorithm (such as FASTA, BLAST or gap). In some cases, a nucleic acid molecule having substantial identity to a reference nucleic acid molecule can encode a polypeptide having an amino acid sequence that is identical or substantially similar to that of the polypeptide encoded by the reference nucleic acid molecule.

[0412] When applied to polypeptides, the term "substantial similarity" or "substantially similar" means that two peptide sequences share at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity, when optimally aligned, such as by the programs gAP or BESTFIT, using default gap weights. Preferably, residue positions that are not identical differ by conservative amino acid substitutions. A "conservative amino acid substitution" is an amino acid substitution in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Typically, conservative amino acid substitutions will not substantially change the functional properties of the protein. In the case where two or more amino acid sequences differ from each other by conservative substitutions, the percentage of sequence identity or degree of similarity can be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol.Biol.24:307-331. Examples of amino acid groups with side chains of 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.

[0413] Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443-1445. A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0414] Sequence similarity, also referred to as sequence identity, of polypeptides is usually measured using sequence analysis software. Protein analysis software uses similarity metrics assigned to various substitutions, deletions, and other modifications (comprising conservative amino acid substitutions) to match similar sequences. For example, gCG software contains programs such as gap and Bestfit, which can be used together with default parameters to determine sequence homology or sequence identity between closely related polypeptides (such as homologous polypeptides from different organism species) or between wild-type proteins and their mutant proteins. See, for example, gCG version 6.1. FASTA, which is a program in gCG version 6.1, can also be used to compare polypeptide sequences using default or recommended parameters. FASTA (such as FASTA2 and FASTA3) provides comparison and sequence identity percentages (Pearson (2000) the same) of the best overlapping region between the query sequence and the search sequence. When comparing the sequence of this specification with a database containing a large number of sequences from different organisms, another specific algorithm is the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. See, eg, Altschul et al. (1990) J. Mol. Biol. 215:403-410; and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402.

[0415] Protein-drug conjugate compounds

[0416] In accordance with the foregoing objectives and other objectives, the present disclosure provides protein-drug conjugate compounds, e.g., antibody-drug conjugate compounds, precursors and intermediates thereof, pharmaceutical compositions, and methods for treating certain diseases of subjects in need of such treatment. According to the present disclosure, provided herein are protein-drug conjugate compounds comprising a glutaminyl-modified binding agent conjugated to a primary amine compound attached to a therapeutic moiety (e.g., a camptothecin analog moiety), as described herein. Also provided are specific and effective methods 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, provided herein are protein-drug conjugate compounds comprising a prodrug of a topoisomerase I inhibitor, e.g., a prodrug of Dxd.

[0417] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to a compound of formula (I)

[0418]

[0419] or a pharmaceutically acceptable salt thereof,

[0420] where R 1、R 2 、R 3 and R 4 are independently hydrogen, C 1-5 alkyl or aryl;

[0421] AA is a natural or unnatural amino acid;

[0422] p is an integer from 1 to 6, and

[0423] The point of attachment to the antibody or the antigen-binding fragment thereof, directly or via a linker, is indicated.

[0424] In one embodiment, the compound of formula (I) is directly conjugated to an antibody or antigen-binding fragment thereof.

[0425] In another embodiment, the compound of formula (I) is conjugated to the antibody or antigen-binding fragment thereof via a bivalent linker.

[0426] 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 in which p is greater than one, the amino acids may be the same or different. In one embodiment, p is 2, and the two amino acids are different.

[0427] 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, glutamic acid (glutamine) and glutamic acid (glutamic acid).

[0428] 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 selected from the group consisting of: R-amino acids, N-methyl amino acids,

[0429] 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.

[0430] In one embodiment, R 1 It’s H.

[0431] In one embodiment, R2 is H. In one embodiment, R 3 is H. In one embodiment, R 2 and R 3 All are H.

[0432] In one embodiment, R 4 is H. In another embodiment, R 4 It is C 1-5 In a particular embodiment, R 4 It is a C1 alkyl (methyl) group.

[0433] In one embodiment, the compound of formula (I) is referred to as the payload.

[0434] In one embodiment, the compound of formula (I) comprises a compound selected from the group consisting of:

[0435]

[0436] It is conjugated to the antibody or antigen-binding fragment via the amino group.

[0437] In one embodiment, the antibody or the antigen-binding fragment thereof is combined with a compound having a structure according to formula (II)

[0438]

[0439] or a pharmaceutically acceptable salt thereof, wherein R 1 、R 2 、R 3 and R 4 are independently hydrogen or C 1-5 alkyl;

[0440] A is a click chemistry adduct;

[0441] W is NH, O, CO, CH2, phenyl, or a combination of two or more thereof;

[0442] AA is a natural or unnatural amino acid;

[0443] m is an integer from 0 to 8;

[0444] n is 0 or 1;

[0445] p is an integer from 1 to 6, and

[0446] The point of attachment to the antibody or the antigen-binding fragment thereof, directly or via a linker, is indicated.

[0447] In one embodiment, the click chemistry adduct is the product of a copper-free click chemistry reaction selected from:

[0448] (a) Strain-promoted azide / dibenzocyclooctynamine (DBCO) click chemistry.

[0449] (b) Inverse electron demand Diels-Alder (IED-DA) tetrazine / trans-cyclooctene (TCO) click chemistry;

[0450] (c) Inverse electron demand Diels-Alder (IED-DA) tetrazine / norbornene click chemistry;

[0451] (d) Diels-Aldermaleimide / furan click chemistry;

[0452] (e) Staudinger connection; and

[0453] (f) Nitrile oxide / norbornene cycloaddition click chemistry.

[0454] In one non-limiting embodiment, the click chemistry adduct is the product of a strain-promoted azide / dibenzocyclooctynamine (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.

[0455] In one embodiment, the click chemistry adduct comprises a triazole. In another embodiment, the click chemistry adduct comprises a diazine.

[0456] In one embodiment, the click chemistry adduct is selected from the group consisting of:

[0457] and any regioisomer or enantiomer thereof, wherein R' is H or C 1-3 alkyl and Z is C or N.

[0458] In one embodiment, the click chemistry adduct is

[0459] In one embodiment, R 1 It’s H.

[0460] In one embodiment, R 2 is H. In one embodiment, R 3 is H. In one embodiment, R 2 and R 3 All are H.

[0461] In one embodiment, R 4 is H. In another embodiment, R4 It is C 1-5 In a particular embodiment, R 4 It is a C1 alkyl (methyl) group.

[0462] In one embodiment, W is O. In one embodiment, W is NH. In one embodiment, W is CO. In one embodiment, W is CH2. In one embodiment, W is phenyl. In one embodiment, W is OCH2. In one embodiment, W is -OCH2-CO-NH-. In one embodiment, W is -O-CO-NH-. In one embodiment, W is

[0463] 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.

[0464] In a particular embodiment, m is 4.

[0465] In one embodiment, n is 0. In another embodiment, n is 1.

[0466] 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 in which p is greater than one, the amino acids may be the same or different. In one embodiment, p is 2, and the two amino acids are different.

[0467] 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, glutamic acid (glutamine) and glutamic acid (glutamic acid).

[0468] 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 selected from the group consisting of: R-amino acids, N-methyl amino acids,

[0469] 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.

[0470] In one embodiment, the compound of formula (II) comprises a compound having a structure selected from the group consisting of:

[0471]

[0472]

[0473] In one embodiment, the compound of formula (II) comprises

[0474]

[0475] In one aspect, presented herein is an antibody-drug conjugate having a structure according to formula (III)

[0476]

[0477]

[0478] or a pharmaceutically acceptable salt thereof, wherein

[0479] Ab is an antibody or its antigen-binding fragment;

[0480] R 1 、R 2 、R 3 and R 4 are independently hydrogen or C 1-5 alkyl;

[0481] A is a click chemistry adduct;

[0482] W is NH, O, CO, CH2, phenyl, or a combination of two or more thereof;

[0483] LL is a linker or bond connecting the Ab and the A;

[0484] AA is a natural or unnatural amino acid;

[0485] m is an integer from 0 to 8;

[0486] n is 0 or 1;

[0487] p is an integer from 1 to 6;

[0488] And q is an integer from 1 to 10.

[0489] In one embodiment, the click chemistry adduct is the product of a copper-free click chemistry reaction selected from:

[0490] (a) Strain-promoted azide / dibenzocyclooctynamine (DBCO) click chemistry.

[0491] (b) Inverse electron demand Diels-Alder (IED-DA) tetrazine / trans-cyclooctene (TCO) click chemistry;

[0492] (c) Inverse electron demand Diels-Alder (IED-DA) tetrazine / norbornene click chemistry;

[0493] (d) Diels-Aldermaleimide / furan click chemistry;

[0494] (e) Staudinger connection; and

[0495] (f) Nitrile oxide / norbornene cycloaddition click chemistry.

[0496] In one non-limiting embodiment, the click chemistry adduct is the product of a strain-promoted azide / dibenzocyclooctynamine (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.

[0497] In one embodiment, the click chemistry adduct comprises a triazole. In another embodiment, the click chemistry adduct comprises a diazine.

[0498] In one embodiment, the click chemistry adduct is selected from the group consisting of:

[0499] Where R' is H or C 1-3 alkyl and Z is C or N.

[0500] In one embodiment, the click chemistry adduct is

[0501] In one embodiment, R 1 It’s H.

[0502] In one embodiment, R 2 is H. In one embodiment, R 3 is H. In one embodiment, R 2 and R 3 All are H.

[0503] In one embodiment, R 4 is H. In another embodiment, R 4 It is C 1-5 In a particular embodiment, R 4It is a C1 alkyl (methyl) group.

[0504] 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.

[0505] In a particular embodiment, m is 4.

[0506] In one embodiment, n is 0. In another embodiment, n is 1.

[0507] 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 in which p is greater than one, the amino acids may be the same or different. In one embodiment, p is 2, and the two amino acids are different.

[0508] 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, glutamic acid (glutamine) and glutamic acid (glutamic acid).

[0509] 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 selected from the group consisting of: R-amino acids, N-methyl amino acids,

[0510] 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.

[0511] In one embodiment, LL is a divalent or multivalent linker selected from the group consisting of:

[0512]

[0513]

[0514]

[0515]

[0516]

[0517] wherein (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.

[0518] In one embodiment, LL is a divalent or multivalent linker selected from the group consisting of:

[0519]

[0520]

[0521] wherein n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0522] In one embodiment, LL is a divalent or multivalent linker selected from the group consisting of:

[0523]

[0524] wherein n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0525] In certain embodiments, the joint includes a self-eliminating group. Self-eliminating groups, self-eliminating joints or self-eliminating spacers can be any such groups known to those skilled in the art. Self-eliminating joints play an important role in the cascade mechanism of the release of connected compounds. It is defined as a covalent group that, after stimulation, has the effect of cutting two bonds between the protecting group and the drug in the case of a drug delivery system. Stimulation can especially include enzyme triggers, chemical triggers (such as pH, redox systems, 1,4-, 1,6-, 1,8-elimination), photodegradable triggers, multiple triggers. A series of reactions of the self-eliminating structural construct allows the release of controlled drugs. In an exemplary embodiment, the self-eliminating group is p-aminophenyl (PAB) or a derivative thereof. Useful derivatives include p-aminobenzyloxycarbonyl (PABC). Skilled technicians will recognize that self-eliminating groups can undergo chemical reactions that release the remaining atoms of the joint from the payload.

[0526] 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.

[0527] In one embodiment, an antibody-drug conjugate is presented herein having the structure

[0528]

[0529] or a pharmaceutically acceptable salt thereof, wherein

[0530] Ab is an antibody or its antigen-binding fragment;

[0531] R is the side chain of any natural or unnatural amino acid; and

[0532] n is an integer from 1 to 5.

[0533] In another embodiment, an antibody-drug conjugate is presented herein having the structure

[0534]

[0535]

[0536]

[0537] or a pharmaceutically acceptable salt thereof, wherein

[0538] Ab is an antibody or an antigen-binding fragment thereof; and

[0539] n is an integer from 1 to 5.

[0540] In one embodiment, an antibody-drug conjugate is presented herein having the structure

[0541]

[0542] or a pharmaceutically acceptable salt thereof, wherein

[0543] Ab is an antibody or an antigen-binding fragment thereof; and

[0544] n is an integer from 1 to 5.

[0545] In one aspect, presented herein is an antibody-drug conjugate having a structure according to Formula (IVa or IVb):

[0546]

[0547] or a pharmaceutically acceptable salt thereof, wherein

[0548] Ab is an antibody or its antigen-binding fragment;

[0549] R is the side chain of any natural or unnatural amino acid;

[0550] And n is an integer from 1 to 5.

[0551] In another aspect, the present disclosure provides an antibody-drug conjugate having a structure according to Formula (IVc, IVd, IVe, IVf, IVg, IVh, IVi, IVj, or IVk)

[0552]

[0553]

[0554]

[0555] or a pharmaceutically acceptable salt thereof, wherein

[0556] Ab is an antibody or its antigen-binding fragment;

[0557] R is the side chain of any natural or unnatural amino acid;

[0558] And n is an integer from 1 to 5.

[0559] In one embodiment, R is hydrogen.

[0560] In one embodiment, R is a side chain of a natural amino acid. In one embodiment, R is a side chain of a natural amino acid selected from the group consisting of 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 natural amino acid selected from the group consisting of glycine, phenylalanine, threonine, lysine, glutamine, and glutamic acid.

[0561] In one embodiment, R is a side chain of an unnatural amino acid. In one embodiment, R is a side chain of an unnatural amino acid selected from the group consisting of: an R-amino acid, an N-methyl amino acid,

[0562] In one embodiment of any of the above, the antibody or antigen-binding fragment thereof comprises Gln295 and / or Gln297 (i.e., a glutamine residue in position 295 and / or 297), and the payload (e.g., a prodrug of DXd) is conjugated to the antibody or antigen-binding fragment directly or via a linker through the side chains of Gln295 and / or Gln297.

[0563] Payload

[0564] 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 analog and / or derivative.

[0565]

[0566] Camptothecin (CPT), shown above, is a topoisomerase poison. It was discovered by ME Wall and MC Wani in 1966 during a systematic screening of natural products for anticancer drugs. It is isolated from the bark and stem of Camptotheca acuminata (Camptotheca, Happy tree), a tree native to China used as a cancer treatment in traditional Chinese medicine. In preliminary clinical trials, camptothecin showed significant anticancer activity. However, it has low solubility, so synthetic and medicinal chemists have developed many syntheses of camptothecin and various derivatives to improve the benefits of the chemical and achieve good results. Today, the following four camptothecin analogs have been approved and are used in cancer chemotherapy: topotecan, irinotecan, belotecan, and delunotecan (Dxd).

[0567] Trastuzumab deruxtecan (T-Dxd) is an antibody-drug conjugate consisting of the human epidermal growth factor receptor 2 (HER2)-directed antibody trastuzumab and the topoisomerase I inhibitor conjugate deruxtecan (Dxd, a derivative of exitecan). It was approved for use in the United States in December 2019. Exitecan, shown below, is a camptothecin analog.

[0568]

[0569] Exotecan, left; and delutec (Dxd), right

[0570] In one embodiment, the payload of the present disclosure is a prodrug of delutec (Dxd).

[0571] In certain embodiments, the payload of the present disclosure is a compound having structure PI:

[0572]

[0573] where R 1 、R 2 、R 3 and R 4 are independently hydrogen or C 1-5 alkyl;

[0574] AA is a natural or unnatural amino acid; and p is an integer from 1 to 6, or a pharmaceutically acceptable salt thereof.

[0575] 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 in which p is greater than one, the amino acids may be the same or different. In one embodiment, p is 2, and the two amino acids are different.

[0576] 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, glutamic acid (glutamine) and glutamic acid (glutamic acid).

[0577] 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 selected from the group consisting of: R-amino acids, N-methyl amino acids,

[0578] 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.

[0579] In one embodiment, R 1 It’s H.

[0580] In one embodiment, R 2 is H. In one embodiment, R 3 is H. In one embodiment, R 2 and R 3 All are H.

[0581] In one embodiment, R 4 is H. In another embodiment, R 4 It is C 1-5In a particular embodiment, R 4 It is a C1 alkyl (methyl) group.

[0582] In one embodiment, the compound of formula (I) is selected from the group consisting of the compounds of Table 1.

[0583] Table 1. Structures of EXT, DXd, and prodrugs of DXd according to embodiments of the present disclosure

[0584]

[0585]

[0586]

[0587] Certain characteristics of payloads according to the present disclosure are summarized in Table 2 below.

[0588] Table 2. SAR of DXd prodrugs (R1, R2, R3 = H)

[0589]

[0590]

[0591]

[0592] The present disclosure also relates to a pharmaceutical composition 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.

[0593] The present disclosure also relates to a method for producing a linker-payload compound having formula (D') to (G')

[0594]

[0595] or a pharmaceutically acceptable salt thereof,

[0596] where R 1 、R 2 、R 3 and R 4 are independently hydrogen or C 1-5 alkyl;

[0597] B is selected from the group consisting of:

[0598] W is NH, O, CO, CH2, phenyl, or a combination of two or more thereof; and

[0599] R5 、R 6 、R 7 and R 8 is independently hydrogen, -NH2 or the side chain of any natural or unnatural amino acid,

[0600] The method comprises the steps of exposing a payload having an amino group to an activated intermediate having p-nitrophenyl carbonate in the presence of a base and a coupling catalyst to obtain the linker-payload compounds (D') to (G'), wherein the coupling catalyst is 4-hydroxy-2-methylquinoline (MeHYQ).

[0601] The present disclosure also relates to a method for preparing a linker-payload compound having formula (D-1)

[0602]

[0603] (D-1), or a pharmaceutically acceptable salt thereof,

[0604] where R 1 、R 2 、R 3 and R 4 are independently hydrogen or C 1-5 alkyl, and

[0605] R 5 and R 6 is independently hydrogen, -NH2 or the side chain of any natural or unnatural amino acid,

[0606] The method comprises the following steps: exposing a payload having an amino group to an activated intermediate having p-nitrophenyl 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).

[0607] In one embodiment, the payload having an amino group has a structure according to Formula PI:

[0608]

[0609] where R 1 、R 2 、R 3 and R 4 are independently hydrogen or C 1-5 alkyl;

[0610] AA is a natural or unnatural amino acid; and

[0611] p is an integer from 1 to 6, or a pharmaceutically acceptable salt thereof.

[0612] In one embodiment, the amino group of the payload is the amino terminus of AA.

[0613] In one embodiment, the activated intermediate with p-nitrophenyl carbonate has a structure according to Formula II:

[0614]

[0615] The present disclosure also relates to a method for preparing a linker-payload compound having formula (D-1)

[0616]

[0617] or a pharmaceutically acceptable salt thereof,

[0618] where R 1 、R 2 、R 3 and R 4 are independently hydrogen or C 1-5 alkyl, and

[0619] R 5 and R 6 is independently hydrogen, -NH2 or the side chain of any natural or unnatural amino acid,

[0620] The method comprises:

[0621] (a) providing a compound of formula (I-1) having the following structure:

[0622]

[0623] in

[0624] X is selected from the group consisting of: as well as

[0625] (b) reacting the compound of formula (I-1) with a compound of formula (PI):

[0626]

[0627] in

[0628] R is H or PG; and

[0629] PG is a suitable protecting group;

[0630] reaction to produce the compound of formula (D-1).

[0631] In one embodiment, the compound of formula (D-1) has the following structure:

[0632]

[0633] In one embodiment, the step (b) of reacting the compound of formula (I-1) with the compound of formula (PI) further comprises reacting the compound of formula (PI), wherein R is PG, with a protecting group removing agent before reacting with the compound of formula (I-1).

[0634] In one embodiment, the PG is selected from the group consisting of allyloxycarbonyl (Alloc), benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), and 9-fluorenylmethoxycarbonyl (Fmoc).

[0635] In one embodiment, the protecting group removing agent is selected from the group consisting of Pd(PPh)3, PhSiH3, H2, piperidine and trifluoroacetic acid (TFA).

[0636] In one embodiment, the compound of formula (I-1) has the following structure:

[0637]

[0638] In one embodiment, the compound of formula (PI) has the following structure:

[0639]

[0640] In one embodiment, the method for making a linker-payload compound having formula (D-1) further comprises the steps of providing a compound of formula (V) having the following structure:

[0641] and forming the compound of formula (I-1) from the compound of formula (V) prior to step (a).

[0642] In one embodiment, the step of forming the compound of formula (I-1) comprises reacting the compound of formula (V) with a compound of formula (VIa) or formula (VIb):

[0643]

[0644] wherein X' is a halogen, reacts to produce the compound of formula (I-1).

[0645] In one embodiment, the compound of formula (VIa) is selected from the group consisting of:

[0646] In one embodiment, the compound of formula (VIb) is

[0647] In one embodiment, the method further comprises providing a compound of formula (VII) having the following structure:

[0648]

[0649] Among them PG 1 is a suitable protecting group protecting group, and the compound of formula (V) is formed from the compound of formula (VII).

[0650] In one embodiment, the PG 1 Selected from the group consisting of allyloxycarbonyl (Alloc), benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc) and 9-fluorenylmethoxycarbonyl (Fmoc).

[0651] In one embodiment, the compound of formula (VII) has the following structure:

[0652]

[0653] In one embodiment, the step of forming the compound of formula (V) comprises reacting the compound of formula (VII) with a compound of formula (VIII):

[0654] to produce the compound of formula (V).

[0655] In one embodiment, the method further comprises the steps of providing a compound of formula (IX) having the following structure:

[0656] and forming the compound of formula (VII) from the compound of formula (IX).

[0657] In one embodiment, the compound of formula (IX) has the following structure:

[0658]

[0659] In one embodiment, the step of forming the compound of formula (VII) comprises reacting the compound of formula (IX) with a compound of formula (X):

[0660] to produce the compound of formula (VII).

[0661] In one embodiment, the method further comprises the steps of providing a compound of formula (XI) having the following structure:

[0662] and forming the compound of formula (IX) from the compound of formula (XI).

[0663] In one embodiment, the compound of formula (XI) has the following structure:

[0664]

[0665] In one embodiment, the step of forming the compound of formula (IX) comprises reacting the compound of formula (XI) with a compound of formula (XII):

[0666] to produce the compound of formula (IX).

[0667] In one embodiment, the method further comprises providing a compound of formula (XIII) having the following structure: as well as

[0668] The compound of formula (VIIII) is formed from the compound of formula (XIII).

[0669] In one embodiment, the step of forming the compound of formula (VIII) comprises reacting the compound of formula (XIII) with a compound of formula (XII):

[0670] to produce the compound of formula (VIII).

[0671] In one embodiment, the method further comprises the step of providing a compound of formula (XIV) having the following structure:

[0672]

[0673] where R a It is a halogen;

[0674] R b It is C 1-6 alkyl; and forming a compound of formula (XIII) from the compound of formula (XIV).

[0675] In one embodiment, R a It's bromine.

[0676] In one embodiment, the compound of formula (XIV) has the following structure:

[0677]

[0678] In one embodiment, the step of forming the compound of formula (XIII) comprises reacting the compound of formula (XIV) with a base to produce the compound of formula (XIII).

[0679] 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).

[0680] In one embodiment, the reaction between the compound of formula (XIV) and the base is carried out in a suitable solvent such as methanol (MeOH), tetrahydrofuran (THF), dimethylformamide (DMF), or a mixture thereof.

[0681] In one embodiment, the method further comprises the step of providing a compound of formula (XV) having the following structure:

[0682] as well as

[0683] The compound of formula (XIV) is formed from the compound of formula (XV).

[0684] In one embodiment, the compound of formula (XV) has the following structure:

[0685]

[0686] In one embodiment, the step of forming the compound of formula (XIV) comprises reacting the compound of formula (XV) with a compound of formula (XVI):

[0687] to produce the compound of formula (XIV).

[0688] 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).

[0689] In one embodiment, the method further comprises the steps of providing a compound of formula (XVII) having the following structure:

[0690] and forming the compound of formula (XV) from the compound of formula (XVII).

[0691] In one embodiment, the step of forming the compound of formula (XV) comprises reacting the compound of formula (XVII) with a brominating agent to produce the compound of formula (XVII).

[0692] In one embodiment, the brominating agent is CHBr3.

[0693] In one embodiment, the compound of formula (XVII) is reacted with CHBr 3 in the presence of a base such as potassium tert-butoxide (t-BuOK) in a non-polar solvent.

[0694] In one embodiment, the method further comprises the step of providing a compound of formula (XVIII) having the following structure:

[0695] and forming the compound of formula (PI) from the compound of formula (XVIII).

[0696] In one embodiment, the compound of formula (XVIII) has the following structure:

[0697]

[0698] In one embodiment, the step of forming the compound of formula (PI) comprises reacting the compound of formula (XVIII) with a compound of formula (XIX):

[0699] to produce the compound of formula (PI).

[0700] In one embodiment, the method further comprises the steps of providing a compound of formula (XX) having the following structure:

[0701] and forming the compound of formula (XVIII) from the compound of formula (XX).

[0702] In one embodiment, the compound of formula (XX) has the following structure:

[0703]

[0704] In one embodiment, the step of forming the compound of formula (XVIII) comprises reacting the compound of formula (XX) with a compound of formula (XXI):

[0705] to produce the compound of formula (XVIII).

[0706] In one embodiment, the method further comprises the steps of providing a compound of formula (XXII) having the following structure:

[0707] and forming the compound of formula (XX) from the compound of formula (XXII).

[0708] In one embodiment, the compound of formula (XXII) has the following structure:

[0709]

[0710] The present disclosure also relates to a method for preparing a compound of formula (I-1):

[0711]

[0712] or a pharmaceutically acceptable salt thereof,

[0713] in

[0714] X is selected from the group consisting of:

[0715] The method comprises:

[0716] (a) providing a compound of formula (V) having the following structure:

[0717] as well as

[0718] (b) forming the compound of formula (I-1) from the compound of formula (V).

[0719] In one embodiment, the compound of formula (I-1) has the following structure:

[0720]

[0721] In one embodiment, step (b) of forming the compound of formula (I-1) comprises reacting the compound of formula (V) with a compound of formula (VIa) or formula (VIb):

[0722]

[0723] wherein X′ is a halogen,

[0724] reaction to produce the compound of formula (I-1).

[0725] In one embodiment, the compound of formula (VIa) is selected from the group consisting of:

[0726] In one embodiment, the compound of formula (VIb) is

[0727] In one embodiment, the method further comprises the steps of providing a compound of formula (VII) having the following structure:

[0728]

[0729] Among them PG 1 is a suitable protecting group protecting group, and

[0730] The compound of formula (V) is formed from the compound of formula (VII).

[0731] In one embodiment, the compound of formula (VII) has the following structure:

[0732]

[0733] In one embodiment, the step of forming the compound of formula (V) comprises reacting the compound of formula (VII) with a compound of formula (VIII):

[0734] to produce the compound of formula (V).

[0735] In one embodiment, the method further comprises the steps of providing a compound of formula (IX) having the following structure:

[0736] and forming the compound of formula (VII) from the compound of formula (IX).

[0737] In one embodiment, the compound of formula (IX) has the following structure:

[0738]

[0739] In one embodiment, the step of forming the compound of formula (VII) comprises reacting the compound of formula (IX) with a compound of formula (X):

[0740] to produce the compound of formula (VII).

[0741] In one embodiment, the method further comprises the steps of providing a compound of formula (XI) having the following structure:

[0742] and forming the compound of formula (IX) from the compound of formula (XI).

[0743] In one embodiment, the compound of formula (XI) has the following structure:

[0744]

[0745] In one embodiment, the step of forming the compound of formula (IX) comprises reacting the compound of formula (XI) with a compound of formula (XII):

[0746] to produce the compound of formula (IX).

[0747] The present disclosure also relates to a method for preparing a compound of formula (XVIII):

[0748]

[0749] or a pharmaceutically acceptable salt thereof,

[0750] where R 1 、R 2 、R 3 and R 4 are independently hydrogen or C 1-5 alkyl, and

[0751] R 5 and R 6 is independently hydrogen, -NH2 or the side chain of any natural or unnatural amino acid. The method comprises:

[0752] (a) providing a compound of formula (XX) having the following structure:

[0753] as well as

[0754] (b) forming the compound of formula (XVIII) from the compound of formula (XX).

[0755] In one embodiment, the compound of formula (XVIII) has the following structure:

[0756]

[0757] In one embodiment, the compound of formula (XX) has the following structure:

[0758]

[0759] In one embodiment, the step of forming the compound of formula (XVIII) comprises reacting the compound of formula (XX) with a compound of formula (XXI):

[0760] to produce the compound of formula (XVIII).

[0761] In one embodiment, the method further comprises the steps of providing a compound of formula (XXII) having the following structure:

[0762] and forming the compound of formula (XX) from the compound of formula (XXII).

[0763] In one embodiment, the compound of formula (XXII) has the following structure:

[0764]

[0765] The present disclosure also relates to a method for preparing a compound of formula (D-1):

[0766]

[0767] or a pharmaceutically acceptable salt thereof,

[0768] where R 1 、R 2 、R 3 and R 4 are independently hydrogen or C 1-5 alkyl, and

[0769] R 5 and R 6 is independently hydrogen, -NH2 or the side chain of any natural or unnatural amino acid. The method comprises:

[0770] (a) providing a compound of formula (I-1) having the following structure:

[0771]

[0772] in

[0773] X is selected from the group consisting of: as well as

[0774] (b) reacting the compound of formula (I-1) with a compound of formula (PI):

[0775]

[0776] in

[0777] R is H or PG; and

[0778] PG is a suitable protecting group,

[0779] reaction to produce the compound of formula (D-1).

[0780] In one embodiment, the compound of formula (D-1) has the following structure:

[0781]

[0782] In one embodiment, the compound of formula (I-1) has the following structure:

[0783]

[0784] In one embodiment, the step (b) of reacting the compound of formula (I-1) with the compound of formula (PI) further comprises reacting the compound of formula (PI), wherein R is PG, with a protecting group removing agent before reacting with the compound of formula (I-1).

[0785] In one embodiment, the PG is selected from the group consisting of allyloxycarbonyl (Alloc), benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), and 9-fluorenylmethoxycarbonyl (Fmoc).

[0786] In one embodiment, the protecting group removing agent is selected from the group consisting of Pd(PPh)3, PhSiH3, H2, piperidine and trifluoroacetic acid (TFA).

[0787] In one embodiment, the compound of formula (PI) has the following structure:

[0788]

[0789] In one embodiment, the method further comprises the step of providing a compound of formula (XVIII) having the following structure:

[0790] and forming the compound of formula (PI) from the compound of formula (XVIII).

[0791] In one embodiment, the compound of formula (XVIII) has the following structure:

[0792]

[0793] In one embodiment, the step of forming the compound of formula (PI) comprises reacting the compound of formula (XVIII) with a compound of formula (XIX):

[0794] to produce the compound of formula (PI).

[0795] The present disclosure also relates to a method for preparing a compound of formula (D-1):

[0796]

[0797] or a pharmaceutically acceptable salt thereof,

[0798] where R 1 、R 2 、R 3 and R 4 are independently hydrogen or C 1-5 alkyl, and

[0799] R 5 and R 6 is independently hydrogen, -NH2 or the side chain of any natural or unnatural amino acid,

[0800] The method comprises:

[0801] (a) providing a compound of formula (XXIII):

[0802]

[0803] as well as

[0804] (b) reacting the compound of formula (XXIII) with a compound having the following structure:

[0805] In the presence of an activating agent and a base, the compound of formula (D-1) is reacted to produce the compound. In one embodiment, the compound of formula (D-1) has the following structure:

[0806]

[0807] In one aspect, the present disclosure provides linker-payload compounds of formula (D) to (G),

[0808]

[0809]

[0810]

[0811] or a pharmaceutically acceptable salt thereof, wherein B is selected from the group consisting of: R 1 、R 2 、R 3 and R 4 are independently hydrogen or C 1-5 Alkyl, and R 5 、R 6 、R 7 and R 8 is independently hydrogen, -NH2, or the side chain of any natural or unnatural amino acid.

[0812] In one embodiment, R 1 、R 2 、R 3 and R 4 Each is hydrogen.

[0813] In one embodiment, R 6 It’s H.

[0814] In one embodiment, R 5 is selected from the group consisting of hydrogen and a side chain of alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, threonine, lysine, asparagine, glutamine, aspartic acid, and glutamic acid. 5A side chain selected from hydrogen and the group consisting of phenylalanine, threonine, lysine, glutamine, and glutamic acid.

[0815] In one embodiment, R 7 is H. In one embodiment, R 7 It is the side chain of glutamic acid.

[0816] In one embodiment, R 8 is H. In one embodiment, R 8 It is -CH2-SO3H.

[0817] In one embodiment, the present disclosure provides a linker-payload having a structure selected from the group of Table 3 below.

[0818] Table 3. Structure of linker-ProDXd

[0819]

[0820]

[0821]

[0822]

[0823]

[0824] Table 4 below provides further characterization of non-limiting examples of linker-payloads according to the present disclosure.

[0825] Table 4. List of linkers-ProDXd with corresponding payloads

[0826]

[0827]

[0828] In one aspect, the present disclosure provides a compound of formula (I-1):

[0829]

[0830] or a pharmaceutically acceptable salt thereof,

[0831] in

[0832] X is selected from the group consisting of:

[0833] R 1 、R 2 、R 3 and R 4 are independently hydrogen or C1-5 alkyl, and

[0834] R 5 and R 6 is independently hydrogen, -NH2, or the side chain of any natural or unnatural amino acid.

[0835] In one embodiment, the compound of formula (I-1) has the following structure:

[0836]

[0837] In one aspect, the present disclosure provides a compound of formula (XVIII):

[0838]

[0839] or a pharmaceutically acceptable salt thereof,

[0840] where R 1 、R 2 、R 3 and R 4 are independently hydrogen or C 1-5 alkyl, and

[0841] R 5 and R 6 is independently hydrogen, -NH2, or the side chain of any natural or unnatural amino acid.

[0842] In one embodiment, the compound of formula (XVIII) has the following structure:

[0843]

[0844] Therapeutic formulations and administration

[0845] The present disclosure provides pharmaceutical compositions comprising the protein-drug conjugates of the present disclosure.

[0846] In one aspect, the present disclosure provides a composition comprising a population of protein-drug compositions according to the present disclosure having a drug-antibody ratio (DAR) of about 0.5 to about 14.0.

[0847] In one embodiment, the composition has a DAR of about 1.0 to about 2.5.

[0848] In one embodiment, the composition has a DAR of about 2.

[0849] In one embodiment, the composition has a DAR of about 3.0 to about 4.5.

[0850] In one embodiment, the composition has a DAR of about 4.

[0851] In one embodiment, the composition has a DAR of about 6.5 to about 8.5.

[0852] In one embodiment, the composition has a DAR of about 8.

[0853] In one embodiment, the composition has a DAR of about 10 to about 14.

[0854] In one embodiment, the composition has a DAR of about 12.

[0855] The compositions of the present disclosure are formulated with suitable carriers, excipients, and other agents that provide improved transfer, delivery, tolerability, etc. Many suitable formulations can be found in the formulary known to all pharmaceutical chemists: 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 TM , Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semisolid gels, and semisolid mixtures containing carbowax. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52:238-311.

[0856] The dosage of the protein-drug conjugate administered to the patient can vary according to the patient's age and size, target disease, condition, route of administration, etc. The appropriate dosage is usually calculated based on body weight or body surface area. When the protein-drug conjugate of the present invention is used for therapeutic purposes in adult patients, it may be advantageous to administer the protein-drug conjugate of the present invention intravenously, usually at a single dose of about 0.01 to about 20 mg / kg body weight, more preferably about 0.02 to about 7 mg / kg body weight, about 0.03 to about 5 mg / kg body weight, or about 0.05 to about 3 mg / kg body weight. Depending on the severity of the condition, the frequency and duration of treatment can be adjusted. The effective dosage and schedule for administering the protein-drug conjugate can be determined empirically; for example, patient progress can be monitored by regular assessments, and the dosage can be adjusted accordingly. In addition, interspecies scaling of dosage can be performed using methods well known in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8: 1351).

[0857] Various delivery systems are known and can be used to administer the pharmaceutical compositions of the present disclosure, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis (see, for example, Wu et al., 1987, J. Biol. Chem. 262: 4429-4432). Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition can be administered by any convenient route, such as by infusion or bolus injection, absorbed through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be administered with other biologically active agents. Administration can be systemic or local.

[0858] The pharmaceutical compositions of the present disclosure can be delivered subcutaneously or intravenously using a standard needle and syringe. Additionally, with respect to subcutaneous delivery, pen-type delivery devices are readily applicable for delivering the pharmaceutical compositions of the present disclosure. Such pen-type delivery devices can be reusable or disposable. Reusable pen-type delivery devices typically utilize a replaceable cartridge containing the pharmaceutical composition. Once all of the pharmaceutical composition within 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-type delivery device can then be reused. In disposable pen-type delivery devices, there is no replaceable cartridge. Instead, the disposable pen-type delivery device is pre-filled with the pharmaceutical composition held in a reservoir within the device. Once the pharmaceutical composition in the reservoir is emptied, the entire device is discarded.

[0859] Many reusable pen and auto-injector delivery devices are available for subcutaneous delivery of the pharmaceutical compositions of the present disclosure. Examples include, but are not limited to, AUTOPEN TM (Owen Mumford, Inc., Woodstock, UK), DISSETRONIC TM Pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75 / 25 TM Pen, HUMALOG TM Pen, HUMALIN 70 / 30 TM Pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN TM I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR TM (Novo Nordisk, Copenhagen, Denmark), BDTM Pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN TM 、OPTIPEN PRO TM 、OPTIPEN STARLET TM and OPTICLIK TM (Sanofi-Aventis, Frankfurt, Germany), to name a few. Examples of disposable pen delivery devices for subcutaneous delivery of the pharmaceutical compositions of the present disclosure include, but are not limited to, SOLOSTAR TM Pen (Sanofi-Aventis), FLEXPEN TM (Novo Nordisk) and KWIKPEN TM (Eli Lilly), SURECLICK™ autoinjector (Amgen, Thousand Oaks, CA), PENLET™ (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and the HUMIRA™ pen (Abbott Labs, Abbott Park IL), to name a few.

[0860] In some cases, the pharmaceutical composition can be delivered in a controlled 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 Pres., Boca Raton, Florida. In yet another embodiment, a controlled release system can be placed near the target of the composition, thus requiring only a portion of the systemic dose (see, for example, Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled release systems are discussed in the review of Langer, 1990, Science 249: 1527-1533.

[0861] Injectable preparations can include dosage forms for intravenous, subcutaneous, intradermal and intramuscular injections, infusions, etc. These injectable preparations can be prepared by publicly known methods. For example, the above-mentioned antibody or its salt can be dissolved, suspended or emulsified in a conventional sterile aqueous medium or an oily medium for injection to prepare an injectable preparation. As an aqueous medium for injection, for example, there is physiological saline, an isotonic solution containing glucose and other adjuvants, etc., which can be used in combination with a suitable solubilizing agent (such as alcohol (for example, ethanol), a polyol (for example, propylene glycol, polyethylene glycol), a nonionic surfactant [for example, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc.). As an oily medium, for example, sesame oil, soybean oil, etc. are used, which can be used in combination with a solubilizing agent (such as benzyl benzoate, benzyl alcohol, etc.). The injection thus prepared is preferably filled in a suitable ampoule.

[0862] Advantageously, the pharmaceutical composition for oral or parenteral use is prepared into a dosage form in a unit dose suitable for a certain dose of the active ingredient. Such unit dose dosage forms include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the above-mentioned antibody contained is generally about 5 mg to about 500 mg per unit dosage form; especially in the case of injection form, it is preferred that the content of the above-mentioned antibody is about 5 mg to about 100 mg, and for other dosage forms, about 10 mg to about 250 mg.

[0863] Protein-drug conjugates, linker-payloads, and therapeutic uses of payloads

[0864] In another aspect, protein-drug conjugates (eg, ADCs disclosed herein) are particularly useful for the treatment, prevention, and / or amelioration of diseases, disorders, or conditions requiring such treatment.

[0865] In one embodiment, the present 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 disclosure (e.g., an antibody-drug conjugate, linker-payload and / or payload) or a composition comprising any compound according to the disclosure.

[0866] In one embodiment, a protein-drug conjugate, e.g., an ADC disclosed herein, can be used to treat cancer. In one embodiment, a protein-drug conjugate (e.g., an ADC disclosed herein) can be used to treat a cancer selected from the group consisting of breast cancer, ovarian cancer, prostate cancer, lung cancer, liver cancer, or brain cancer. In one embodiment, a protein-drug conjugate (e.g., an ADC disclosed herein) can be used to treat HER2+ breast cancer. In one embodiment, a protein-drug conjugate (e.g., an ADC disclosed herein) can be used to treat prostate cancer.

[0867] 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: breast cancer cell, ovarian cancer cell, prostate cancer cell, lung cancer cell, liver cancer cell, or brain cancer cell.

[0868] In certain embodiments, the present disclosure provides a method for selectively delivering into a cell a compound having the structure PI:

[0869]

[0870] where R 1 、R 2 、R 3 and R 4 are independently hydrogen or C 1-5 alkyl;

[0871] AA is a natural or unnatural amino acid; and p is an integer from 1 to 6, or a pharmaceutically acceptable salt thereof.

[0872] In one aspect, the present disclosure provides a method for selectively targeting an antigen on a cell surface with a compound. In one embodiment, the method for selectively targeting an antigen on a cell surface with a compound comprises linking the compound to a 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: breast cancer cell, ovarian cancer cell, prostate cancer cell, lung cancer cell, liver cancer cell, or brain cancer cell.

[0873] In certain embodiments, the present disclosure provides a method for selectively targeting an antigen on the surface of a cell with a compound having the structure PI:

[0874]

[0875] where R 1 、R 2 、R 3 and R 4 are independently hydrogen or C 1-5 alkyl;

[0876] AA is a natural or unnatural amino acid; and p is an integer from 1 to 6, or a pharmaceutically acceptable salt thereof.

[0877] In certain embodiments of any of the above methods, the compound having structure PI is selected from the group consisting of:

[0878]

[0879]

[0880]

[0881] or a pharmaceutically acceptable salt thereof.

[0882] binder

[0883] In one embodiment, the effectiveness of the protein-drug conjugate embodiments described herein depends on the selectivity of the binding agent to its binding partner. In one embodiment of the present disclosure, the binding agent is any molecule capable of binding to a given binding partner with a certain 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.

[0884] Suitable binding agents of the present disclosure include proteins that bind to a binding partner, wherein the binding agent comprises one or more glutamine residues. Suitable binding agents include, but are not limited to, antibodies, lymphocytes, hormones, growth factors, viral receptors, interleukins, or any other cell-binding or peptide-binding molecule or substance.

[0885] 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, miniantibodies, diabodies, triabodies, etc.). The antibodies herein can be humanized using the methods described in the following documents: 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.

[0886] In the present disclosure, an antibody can be any antibody deemed suitable by a practitioner skilled in the art. In some embodiments, the antibody comprises at least one glutamine residue in at least one polypeptide chain sequence. In certain embodiments, the antibody comprises one or more Gln295 residues. In certain embodiments, the antibody comprises two heavy chain polypeptides, each having a Gln295 residue. In further embodiments, the antibody comprises one or more glutamine residues at sites other than Gln295 in the heavy chain. Such antibodies can be isolated from natural sources or engineered to contain one or more glutamine residues. Techniques for engineering glutamine residues into antibody polypeptide chains are within the skill of a practitioner skilled in the art. In certain embodiments, the antibody is non-glycosylated.

[0887] The antibody can 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.

[0888] 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.

[0889] 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 a scFv (sFv) fragment. In some aspects, the antibody fragment is a scFv-Fc fragment.

[0890] In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a polyclonal antibody.

[0891] 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.

[0892] The antibodies have binding specificity for any antigen deemed suitable by one skilled in the art. In certain embodiments, the antigen is a transmembrane molecule (e.g., a receptor) or a growth factor. Exemplary antigens include, but are not limited to, molecules such as renin, growth hormones including human growth hormone and bovine growth hormone; growth hormone-releasing factor; parathyroid hormone; thyroid stimulating hormone; lipoproteins; α1-antitrypsin; insulin A chain; insulin B chain; proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; coagulation factors such as factor vmc, factor IX, tissue factor (TF) and von Willebrand factor; anticoagulant factors such as protein C; atrial natriuretic factor; pulmonary surfactant; plasminogen activators such as urokinase or human urine or tissue plasminogen activator (t-PA); bombesin; thrombin; hematopoietic growth factor cytotoxic T-lymphocyte-associated antigen (CTLA), such as CTLA-4; inhibins; activins; vascular endothelial growth factor (VEGF); receptors for hormones or growth factors; proteins A or D; rheumatoid factor; neurotrophic factors, such as Such as bone-derived neurotrophic factor (BDNF), neurotrophin-3, neurotrophin-4, neurotrophin-5 or neurotrophin-6 (NT-3, NT4, NT-5 or NT-6) or nerve growth factor, such as 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 factor (TGF), such as TGF-α and TGF-β, including TGF-β1, TGF-β2, TGF-β3, TGF-β4 or TGF-β5; pancreatic islet growth factor (PGF) insulin-like growth factor-1 and insulin-like growth factor-2 (IGF-1 and IGF-2); des(I-3)-IGF-1 (brain IGF-1), insulin-like growth factor binding protein, EpCAM, gD3, FLT3, PSMA, PSCA, MUC1, MUC16, STEAP, STEAP2, CEA, TENB2, EphA receptor, EphB receptor, folate receptor, FOLRI, mesothelin, cripto, αvβ6, integrin, VEGF, VEGFR, EGFR, transferrin receptor, lRTA1, lRTA2, lRTA3, lRTA4, lRTA5;CD proteins such as 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, or a combination thereof disclosed in U.S. Publication No. 2008 / 0171040 or U.S. Publication No. 2008 / 0305044 (incorporated by reference in their entireties). Antibodies that bind to one or more tumor-associated antigens or cell surface receptors; erythropoietin; osteoinductive factors; immunotoxins; bone morphogenetic proteins (BMPs); interferons, such as interferon-α, interferon-β, and interferon-γ; colony-stimulating factors (CSFs), such as M-CSF, gM-CSF, and g-CSF; interleukins (ILs), such as IL-1 to IL-10; superoxide dismutase; T cell receptors; surface membrane proteins; decay-accelerating factors; viral antigens, such as, for example, a portion of the HIV envelope; transport proteins; homing receptors; adhesins; regulatory proteins; integrins, such as CD11a, CD11b, CD11c, CDI8, 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, EGFRVIII, endoglin, Epcam, EphA2, ErbB2 / HER2, ErbB3 / 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. MAGE-2, MAGE-3, MAGE-4, MAGE-6 and MAGE-12), MART-1, mesothelin, mL-IAP, Muc1, Muc16(CA-125), MET, MUM1, NA17, NG EP, NY-BR1, NY-BR62, NY-BR85, NY-ESO1, OX40, p15, p53, PAP, PAX3, PAX5, PCTA-1, PDGFR-α, PDGFR-β, PDGF-A, PD GF-B, PDGF-C, PDGF-D, PLAC1, PRLR, PRAME, PSCA, PSGR, PSMA (FOLH1), RAGE protein, Ras, RGS5, Rho, SART-1, SART-3, STEAP1, STEAP2, STn, survivin, TAG-72, TGF-β, TMPRSS2, Tn, TNFRSF17, TRP-1, TRP-2, tyrosinase and urothelial protein-3, as well as fragments of any of the polypeptides listed herein.

[0893] 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.

[0894] In some embodiments, antigens also include, but are not limited to, hematological targets such as CD22, CD30, CD33, CD79a, and CD79b.

[0895] Some embodiments herein are targets particularly useful for therapeutic or diagnostic purposes. In one embodiment, a binding agent is prepared to interact and bind to an antigen defined as a tumor antigen, wherein these antigens include antigens specific for a certain type of tumor or antigens shared, overexpressed, or modified on a particular type of tumor. Examples include: α-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, such as BAGE-1, gAGE, gnTV, KK-LC-1, MAGE-A2, NA88-A, TRP2-INT2. In some embodiments, the antigen is PRLR or HER2. In some embodiments, the antibody binds to STEAP2, MUC16, EGFR, EGFRVIII, FGR2, or PRLR.

[0896] In some embodiments, the antigen includes HER2. In some embodiments, the antigen includes STEAP2. In some embodiments, the antigen includes MET. In some embodiments, the antigen includes EGFRVIII. In some embodiments, the antigen includes MUC16. In some embodiments, the antigen includes PRLR. In some embodiments, the antigen includes PSMA. In some embodiments, the antigen includes FGFR2.

[0897] In some embodiments, 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.

[0898] 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.

[0899] Anti-HER2 antibodies suitable for protein-drug conjugates

[0900] In some embodiments, the antibody is an anti-HER2 antibody. In some embodiments, the antibody is trastuzumab, pertuzumab (2C4) or magetuximab (MGAH22). In some embodiments, the antibody is trastuzumab. According to certain embodiments, the protein-drug conjugate (e.g., ADC according to the present disclosure) includes an anti-HER2 antibody. In some embodiments, the anti-HER2 antibody may include those described in WO 2019 / 212965 A1.

[0901] 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.

[0902] In certain embodiments, the D1 and D2 domains of the anti-HER2 / HER2 bispecific antibody are non-competitive with each other. The non-competitiveness between D1 and D2 for binding to HER2 means that the individual 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.

[0903] In certain embodiments, D1 and D2 bind to different (eg, non-overlapping or partially overlapping) epitopes on HER2.

[0904] In one non-limiting embodiment, the present disclosure provides a protein-drug conjugate comprising a bispecific antigen binding molecule comprising:

[0905] a first antigen binding domain (D1); and

[0906] a second antigen-binding domain (D2);

[0907] wherein D1 specifically binds to the first epitope of human HER2; and

[0908] Among them, D2 specifically binds to the second epitope of human HER2.

[0909] Anti-HER2 / HER2 bispecific antibodies can be constructed using the antigen-binding domains of two independent monospecific anti-HER2 antibodies.For example, the set of monoclonal monospecific anti-HER2 antibodies can be produced using standard methods as known in the art.Therefore, the single antibody produced can be tested in pairs to assess the cross competitiveness of each other for HER2 protein.If two different anti-HER2 antibodies can be combined with HER2 simultaneously (that is, not competing with each other), then single anti-HER2 / HER2 bispecific antibodies can be engineered from the antigen-binding domains of the first anti-HER2 antibody and from the antigen-binding domains of the second non-competitive anti-HER2 antibody according to the disclosure.

[0910] According to the present disclosure, bispecific antigen binding molecules can be single multifunctional polypeptides, or they can be multimeric complexes of two or more polypeptides covalently or non-covalently associated with each other. As can be clearly seen from the present disclosure, any antigen-binding constructs with two independent non-identical epitopes that simultaneously bind to HER2 molecules are considered as bispecific antigen binding molecules. Any bispecific antigen binding molecules as described herein or variants thereof can be constructed using standard molecular biology techniques (e.g., recombinant DNA and protein expression technology), as known to those of ordinary skill in the art.

[0911] On the other hand, the present disclosure provides a pharmaceutical composition comprising a recombinant human antibody or a fragment thereof that specifically binds to HER2 and a pharmaceutically acceptable carrier. In a non-limiting embodiment, the antibody can bind to two separate epitopes on the HER2 protein, i.e., the antibody is a HER2 / HER2 bispecific antibody. In a related aspect, the present disclosure is characterized in that a composition 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. Other combination therapies and co-formulations relating to the anti-HER2 / HER2 bispecific antibodies of the present disclosure are disclosed elsewhere herein.

[0912] On the other hand, the present disclosure provides a method for targeting / killing tumor cells expressing HER2 using the anti-HER2 / HER2 bispecific antibodies of the present disclosure, wherein the method includes administering a therapeutically effective amount of a pharmaceutical composition comprising an anti-HER2 / HER2 antibody of the present disclosure to a subject in need thereof. In some cases, anti-HER2 / HER2 antibodies (or their antigen-binding fragments) can be used to treat breast cancer, or they can be modified to be more cytotoxic by methods including, but not limited to, modified Fc domains to increase ADCC (see, for example, Shield et al. (2002) JBC 277:26733), radioimmunotherapy, antibody-drug conjugates, or other methods for improving the efficiency of tumor ablation.

[0913] The present disclosure also includes the use of anti-HER2 antibodies of the present disclosure in the manufacture of a medicament for treating a disease or disorder (e.g., cancer) associated with a cell expressing HER2 or caused by a cell expressing HER2. On the one hand, the present disclosure relates to a compound comprising an anti-HER2 antibody or antigen-binding fragment or a HER2 / HER2 bispecific antibody as disclosed herein, for medical use. On the one hand, the present disclosure relates to a compound comprising an antibody-drug conjugate (ADC) as disclosed herein, for medical use.

[0914] In yet another aspect, the present disclosure provides bispecific anti-HER2 / HER2 antibodies for use in diagnostic applications, such as, for example, imaging agents.

[0915] Anti-STEAP2 antibodies suitable for protein-drug conjugates

[0916] In some embodiments, the antibody is an anti-six-transmembrane epithelial antigen of the prostate 2 (STEAP2), i.e., an anti-STEAP2 antibody. STEAP2, which acts as a shuttle between the Golgi complex and the plasma membrane, is a metalloreductase that reduces iron and copper, thereby facilitating the import of these two metals into the cell. STEAP2 is primarily localized to epithelial cells of the prostate. STEAP2 is also expressed in normal heart, brain, pancreas, ovary, skeletal muscle, breast, testis, uterus, kidney, lung, trachea, colon, and liver. STEAP2 is overexpressed in cancerous tissues, including prostate, bladder, cervical, lung, colon, kidney, breast, pancreatic, stomach, uterine, and ovarian tumors (Gomes, IM et al., 2012, Mol. Cancer Res. 10:573-587; Challita-Eid-PM et al., 2003, WO 03 / 087306; Emtage, PCR, 2005, WO 2005 / 079490).

[0917] In one aspect, suitable anti-STEAP antibodies are those disclosed in US2018 / 0104357. Tables 5 and 6 herein list exemplary anti-STEAP2 antibodies according to the present disclosure. Table 5 lists the amino acid sequence identifiers of the heavy chain variable region (HCVR) and light chain variable region (LCVR) and heavy chain complementary determining regions (HCDR1, HCDR2, and HCDR3) and light chain complementary determining regions (LCDR1, LCDR2, and LCDR3) of exemplary anti-STEAP2 antibodies. Table 6 lists the sequence identifiers of nucleic acid molecules encoding the HCVR, LCVR, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of exemplary anti-STEAP2 antibodies.

[0918] The present disclosure provides antibodies or antigen-binding fragments thereof comprising a HCVR comprising an amino acid sequence selected from any one 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.

[0919] The present disclosure also provides antibodies or antigen-binding fragments thereof comprising a LCVR comprising an amino acid sequence selected from any one 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.

[0920] The present disclosure also provides antibodies or antigen-binding fragments thereof, comprising a HCVR and LCVR amino acid sequence pair (HCVR / LCVR), wherein the amino acid sequence pair comprises any one of the HCVR amino acid sequences listed in Table 5 paired with any one 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 a HCVR / LCVR amino acid sequence pair contained within any one 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 NO: 250 / 258 (e.g., H2M11162N).

[0921] The present disclosure also provides antibodies or antigen-binding fragments thereof, comprising a heavy chain CDR1 (HCDR1) comprising an amino acid sequence selected from any one 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.

[0922] 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 one 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.

[0923] 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 one 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.

[0924] The present disclosure also provides antibodies or antigen-binding fragments thereof, comprising a light chain CDR1 (LCDR1) comprising an amino acid sequence selected from any one 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.

[0925] The present disclosure also provides antibodies or antigen-binding fragments thereof, comprising a light chain CDR2 (LCDR2) comprising an amino acid sequence selected from any one 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.

[0926] The present disclosure also provides antibodies or antigen-binding fragments thereof, comprising a light chain CDR3 (LCDR3) comprising an amino acid sequence selected from any one 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.

[0927] The present disclosure also provides antibodies or antigen-binding fragments thereof, comprising a HCDR3 and LCDR3 amino acid sequence pair (HCDR3 / LCDR3), wherein the amino acid sequence pair comprises any one of the HCDR3 amino acid sequences listed in Table 5 paired with any one 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 a HCDR3 / LCDR3 amino acid sequence pair contained within any one 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 NO: 256 / 264 (e.g., H2M11162N).

[0928] 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 within 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).

[0929] In related embodiments, the present disclosure provides antibodies or antigen-binding fragments thereof comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within a HCVR / LCVR amino acid sequence pair as defined by any of the exemplary anti-STEAP2 antibodies listed in Table 5. For example, the present disclosure includes antibodies or antigen-binding fragments thereof comprising a HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence set contained within a HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NO: 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 the specified HCVR and / or LCVR amino acid sequences disclosed herein. Exemplary conventions that can be used to identify CDR boundaries include, for example, the Kabat definition, the Chothia definition, and the AbM definition. In general, the Kabat definition is based on sequence variability, the Chothia definition is based on the position of the structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, for example, 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 can also be used to identify CDR sequences within antibodies.

[0930] 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 one of the HCVR amino acid sequences listed in Table 5; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any one 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.

[0931] The present disclosure also provides nucleic acid molecules encoding any one of the LCVR amino acid sequences listed in Table 5; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any one 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.

[0932] The present disclosure also provides nucleic acid molecules encoding any one of the HCDR1 amino acid sequences listed in Table 5; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any one of the HCDR1 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.

[0933] The present disclosure also provides nucleic acid molecules encoding any one of the HCDR2 amino acid sequences listed in Table 5; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any one of the HCDR2 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.

[0934] The present disclosure also provides nucleic acid molecules encoding any one of the HCDR3 amino acid sequences listed in Table 5; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any one of the HCDR3 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.

[0935] The present disclosure also provides nucleic acid molecules encoding any one of the LCDR1 amino acid sequences listed in Table 5; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any one of the LCDR1 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.

[0936] The present disclosure also provides nucleic acid molecules encoding any one of the LCDR2 amino acid sequences listed in Table 5; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any one of the LCDR2 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.

[0937] The present disclosure also provides nucleic acid molecules encoding any one of the LCDR3 amino acid sequences listed in Table 5; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any one of the LCDR3 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.

[0938] The present disclosure also provides nucleic acid molecules encoding HCVR, wherein the HCVR comprises a set of three CDRs (ie, HCDR1-HCDR2-HCDR3), wherein the HCDR1-HCDR2-HCDR3 amino acid sequence set is defined as any one of the exemplary anti-STEAP2 antibodies listed in Table 5.

[0939] The present disclosure also provides nucleic acid molecules encoding LCVRs, wherein the LCVR comprises a set of three CDRs (ie, LCDR1-LCDR2-LCDR3), wherein the LCDR1-LCDR2-LCDR3 amino acid sequence set is defined as any one of the exemplary anti-STEAP2 antibodies listed in Table 5.

[0940] The present disclosure also provides nucleic acid molecules encoding both HCVR and LCVR, wherein the HCVR comprises the amino acid sequence of any one of the HCVR amino acid sequences listed in Table 5, and wherein the LCVR comprises the amino acid sequence of any one of the LCVR amino acid sequences listed in Table 5. In certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any one 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, and a polynucleotide sequence selected from any one 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. In certain embodiments according to this aspect of the disclosure, the nucleic acid molecule encodes the HCVR and LCVR, wherein both the HCVR and LCVR are derived from the same anti-STEAP2 antibody listed in Table 5.

[0941] The present disclosure also provides recombinant expression vectors capable of expressing a polypeptide comprising a heavy chain variable region or a light chain variable region of an anti-STEAP2 antibody. For example, the present disclosure includes recombinant expression vectors comprising any of the nucleic acid molecules mentioned above, i.e., nucleic acid molecules encoding any of the HCVR, LCVR, and / or CDR sequences listed in Table 5. Also 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 host cells under conditions that allow for the production of antibodies or antibody fragments, and recovering the antibodies and antibody fragments thus produced.

[0942] The present disclosure includes anti-STEAP2 antibodies with modified glycosylation patterns. In some embodiments, for example, antibodies modified to remove undesirable glycosylation sites or to lack fucose moieties present on oligosaccharide chains can be used to increase antibody-dependent cellular cytotoxicity (ADCC) function (see Shield et al. (2002) JBC 277:26733). In other applications, modifications of galactosylation can be performed to modify complement-dependent cytotoxicity (CDC).

[0943] 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 comprising an anti-STEAP2 antibody in combination with 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 co-formulations involving the anti-STEAP2 antibodies of the present disclosure are disclosed elsewhere herein.

[0944] In another aspect, the present disclosure provides therapeutic methods for targeting / killing STEAP2-expressing tumor cells using the anti-STEAP2 antibodies of the present disclosure, wherein the therapeutic methods comprise administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising the anti-STEAP2 antibodies of the present disclosure. In some cases, anti-STEAP2 antibodies (or antigen-binding fragments thereof) can be used to treat prostate cancer, or can be modified to be more cytotoxic by methods including, but not limited to, modified Fc domains to increase ADCC (see, e.g., Shield et al. (2002) JBC 277:26733), radioimmunotherapy, antibody-drug conjugates, or other methods for improving the efficiency of tumor ablation.

[0945] The present disclosure also includes the use of the anti-STEAP2 antibodies of the present disclosure in the manufacture of a medicament for treating a disease or disorder associated with or caused by cells expressing STEAP2 (e.g., cancer). In one aspect, the present disclosure relates to a compound comprising an anti-STEAP2 antibody or antigen-binding fragment or a STEAP2xCD3 bispecific antibody as disclosed herein, for use in medicine. In one aspect, the present disclosure relates to a compound comprising an antibody-drug conjugate (ADC) as disclosed herein, for use in medicine.

[0946] In yet another aspect, the present disclosure provides monospecific anti-STEAP2 antibodies for use in diagnostic applications, such as, for example, imaging agents.

[0947] In another aspect, the present disclosure provides a therapeutic method for stimulating T cell activation using an anti-CD3 antibody or antigen-binding portion of an antibody of the present disclosure, wherein the therapeutic method comprises administering a therapeutically effective amount of a pharmaceutical composition comprising the antibody.

[0948] In another aspect, the present disclosure provides an isolated antibody or antigen-binding fragment thereof that binds to C4-2 cells expressing STEAP2 with an EC50 of less than 50 nM as measured by FACS analysis. In another aspect, the present disclosure provides an isolated antibody or antigen-binding fragment thereof that binds to and is internalized by C4-2 cells expressing STEAP2.

[0949] The present disclosure further provides an antibody or antigen-binding fragment that competes for binding to human STEAP2 with a reference antibody comprising a HCVR / LCVR amino acid sequence pair as shown in Table 5. In another aspect, the present disclosure provides an antibody or antigen-binding fragment that competes for binding to human STEAP2 with a reference antibody comprising a HCVR / LCVR amino acid sequence pair selected from the group consisting of: SEQ ID NO: NO: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.

[0950] Furthermore, the present disclosure provides an antibody or antigen-binding fragment, wherein the antibody or antigen-binding fragment thereof binds to the same epitope on human STEAP2 as a reference antibody comprising the HCVR / LCVR amino acid sequence pair as shown in Table 5. In another aspect, the antibody or antigen-binding fragment binds to the same epitope on human STEAP2 as a reference antibody comprising a HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NO: 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.

[0951] 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 as shown in Table 5; and a CDR of a light chain variable region (LCVR) having an amino acid sequence as shown in Table 5. In another aspect, 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 NO: 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 aspect, the isolated antibody or antigen-binding fragment comprises a HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domain selected from the group consisting of: SEQ ID NO: 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; 100-1 02-104-60-62-64; 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; 188-190-192-19 6-198-200; 204-206-208-212-214-216; 220-222-224-228-230-232; 236-238-240-244-246-248; 252-254-256-260-262-264; 268-270-272-276-278-280; 284-286-288-292-294 -296; 300-302-304-308-310-312; 316-318-320-324-326-328; 332-334-336-340-342-344; 348-350-352-356-358-360; 364-366-368-372-374-376; and 380-382-384-388-390-392.

[0952] In another aspect, the present 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 NO: 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 light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of SEQ ID NO: 314. NO: 10; 26; 42; 58; 114; 130; 146; 162; 178; 194; 210; 226; 242; 258; 274; 290; 306; 322; 338; 354; 370; and 386. In a further 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 NO: 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.

[0953] According to another aspect, the present disclosure provides an antibody-drug conjugate comprising an anti-STEAP2 antibody or antigen-binding fragment thereof as described above and a therapeutic agent (e.g., an anti-tumor agent, such as a camptothecin analog, such as Dxd). In some embodiments, the antibody or antigen-binding fragment and the anti-tumor agent are covalently attached via a linker, as discussed above. In various embodiments, the anti-STEAP2 antibody or antigen-binding fragment can be any anti-STEAP2 antibody or fragment described herein.

[0954] Amino acid and nucleic acid sequences of the heavy and light chain variable regions of anti-STEAP2 antibodies

[0955] Table 5 shows the amino acid sequence identifiers of 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 shown in Table 6.

[0956] Table 5. Amino acid sequence identifiers of anti-STEAP2 antibodies

[0957]

[0958] Table 6. Nucleic acid sequence identifiers of anti-STEAP2 antibodies

[0959]

[0960]

[0961] Anti-MET antibodies suitable for protein-drug conjugates

[0962] In some embodiments, the antibody is an anti-MET antibody. According to certain embodiments, the protein-drug conjugate (e.g., an ADC according to the present disclosure) comprises an anti-MET antibody. In some embodiments, the anti-MET antibody may include those described in US 2018 / 0134794.

[0963] In some embodiments, the antibody is an anti-MET / MET bispecific antibody comprising a first antigen-binding domain (D1) and a second antigen-binding domain (D2), the first antigen-binding domain specifically binding to a first epitope of human MET, and the second antigen-binding domain specifically binding to a second epitope of human MET. In some embodiments, the anti-MET / MET bispecific antibodies may include those described in US2018 / 0134794.

[0964] In certain embodiments, the D1 and D2 domains of the anti-MET / MET bispecific antibody are non-competitive with each other. Non-competitiveness between D1 and D2 for binding to MET means that the individual 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.

[0965] In certain embodiments, D1 and D2 bind to different (eg, non-overlapping or partially overlapping) epitopes on MET.

[0966] In one non-limiting embodiment, the present disclosure provides a protein-drug conjugate comprising a bispecific antigen binding molecule comprising:

[0967] a first antigen binding domain (D1); and

[0968] a second antigen-binding domain (D2);

[0969] wherein D1 specifically binds to the first epitope of human MET; and

[0970] D2 specifically binds to the second epitope of human MET.

[0971] Anti-MET / MET bispecific antibodies can be constructed using the antigen-binding domains of two separate monospecific anti-MET antibodies. For example, a collection of monoclonal monospecific anti-MET antibodies can be produced using standard methods known in the art. Thus, the generated single antibodies can be tested in pairs to assess their cross-competitiveness against MET proteins. If two different anti-MET antibodies are capable of binding to MET simultaneously (i.e., not competing with each other), the antigen-binding domain from the first anti-MET antibody and the antigen-binding domain from the second non-competitive anti-MET antibody can be engineered into a single anti-MET / MET bispecific antibody according to the present disclosure.

[0972] 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 covalently or non-covalently associated with each other. As is apparent from the present disclosure, any antigen binding construct having two separate non-identical epitopes that simultaneously bind to a MET molecule is considered a bispecific antigen binding molecule. Any bispecific antigen binding molecule described herein or variants thereof can be constructed using standard molecular biology techniques (e.g., recombinant DNA and protein expression techniques), as known to those of ordinary skill in the art.

[0973] The bispecific antigen-binding molecule may be referred to herein as a "MET / MET bispecific antibody," "MET xMET bispecific antibody," "MET / MET," "MET x MET," or other related terms, and comprises a first antigen-binding domain (D1) that specifically binds to a first epitope of human MET and a second antigen-binding domain (D2) that specifically binds to a second epitope of human MET. In some embodiments, the first epitope of human MET comprises amino acids 192 to 204 of SEQ ID NO: 2109. In some embodiments, the second epitope of human MET comprises amino acids 305 to 315 and 421 to 455 of SEQ ID NO: 2109. In some embodiments, the first epitope of human MET comprises amino acids 192 to 204 of SEQ ID NO: 2109; and the second epitope of human MET comprises amino acids 305 to 315 and 421 to 455 of SEQ ID NO: 2109.

[0974] Exemplary antigen-binding domains (D1 and D2) provided herein that can be included in a MET x MET bispecific antigen-binding molecule include antigen-binding domains derived from any anti-MET antibody disclosed herein. For example, the present disclosure includes a MET x MET bispecific antigen-binding molecule comprising the D1 or D2 antigen-binding domain, the bispecific antigen-binding molecule comprising a HCVR comprising an amino acid sequence selected from any one 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.

[0975] Also provided herein are MET x MET bispecific antigen binding molecules comprising a D1 or D2 antigen binding domain, comprising a LCVR comprising an amino acid sequence selected from any one 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.

[0976] Provided herein are MET x MET bispecific antigen binding molecules comprising a D1 or D2 antigen binding domain, the bispecific antigen binding molecule comprising a HCVR and LCVR amino acid sequence pair (HCVR / LCVR) comprising any one of the HCVR amino acid sequences listed in Table 7 paired with any one of the LCVR amino acid sequences listed in Table 7. According to certain embodiments, the present invention provides MET x MET bispecific antigen binding molecules comprising a D1 or D2 antigen binding domain, the bispecific antigen binding molecule comprising a HCVR / LCVR amino acid sequence pair contained within any one of the exemplary anti-MET antibodies listed in Table 7.

[0977] Also provided herein are MET x MET bispecific antigen binding molecules comprising a D1 or D2 antigen binding domain, comprising a heavy chain CDR1 (HCDR1) comprising an amino acid sequence selected from any one 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 thereto.

[0978] Also provided are MET x MET bispecific antigen binding molecules comprising a D1 or D2 antigen binding domain, comprising a heavy chain CDR2 (HCDR2) comprising an amino acid sequence selected from any one 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.

[0979] Also provided are MET x MET bispecific antigen binding molecules comprising a D1 or D2 antigen binding domain, the bispecific antigen binding molecules comprising a heavy chain CDR3 (HCDR3) comprising an amino acid sequence selected from any one 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.

[0980] Also provided herein are MET x MET bispecific antigen binding molecules comprising a D1 or D2 antigen binding domain, comprising a light chain CDR1 (LCDR1) comprising an amino acid sequence selected from any one 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.

[0981] Also provided are MET x MET bispecific antigen binding molecules comprising a D1 or D2 antigen binding domain, comprising a light chain CDR2 (LCDR2) comprising an amino acid sequence selected from any one 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.

[0982] Also provided are MET x MET bispecific antigen binding molecules comprising a D1 or D2 antigen binding domain, comprising a light chain CDR3 (LCDR3) comprising an amino acid sequence selected from any one 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.

[0983] Also provided are MET x MET bispecific antigen-binding molecules comprising a D1 or D2 antigen-binding domain, the bispecific antigen-binding molecules comprising a HCDR3 and LCDR3 amino acid sequence pair (HCDR3 / LCDR3), the amino acid sequence pair comprising any one of the HCDR3 amino acid sequences listed in Table 7 paired with any one of the LCDR3 amino acid sequences listed in Table 7. According to certain embodiments, the present disclosure provides antibodies or antigen-binding fragments thereof comprising a HCDR3 / LCDR3 amino acid sequence pair contained within any one of the exemplary anti-MET antibodies listed in Table 7.

[0984] Also provided are MET x MET bispecific antigen binding molecules comprising a D1 or D2 antigen binding domain comprising a set of six CDRs contained within any of the exemplary anti-MET antibodies listed in Table 7 (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3).

[0985] In related embodiments, the present disclosure provides a MET x MET bispecific antigen binding molecule comprising a D1 or D2 antigen binding domain, which bispecific antigen binding molecule comprises a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within a HCVR / LCVR amino acid sequence pair as defined for any of the exemplary anti-MET antibodies listed in Table 7.

[0986] The MET x MET bispecific antigen binding molecules provided herein may comprise a D1 antigen binding domain derived from any anti-MET antibody of Table 7 and a D2 antigen binding domain derived from any other anti-MET antibody of Table 7.

[0987] As non-limiting illustrative examples, the present disclosure includes a MET x MET bispecific antigen binding molecule comprising a D1 antigen binding domain and a D2 antigen binding domain, wherein the D1 antigen binding domain comprises the HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 2012 / 2092 or a set of heavy and light chain CDRs (HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) comprising SEQ ID NOs: 2014-2016-2018-2094-2096-2098, and wherein the D2 antigen binding domain comprises the HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 2036 / 2092 or a set of heavy and light chain CDRs (HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) comprising SEQ ID NOs: 2014-2016-2018-2094-2096-2098. NO: 2038-2040-2042-2094-2096-2098. An exemplary MET x MET bispecific antibody having these sequence characteristics is the bispecific antibody designated H4H14639D, also known as bispecific antibody No. 2076, which comprises D1 derived from H4H13306P2 and P2 and D2 derived from H4H13312.

[0988] Amino acid and nucleic acid sequences of the heavy and light chain variable regions of anti-MET and MET / MET antibodies

[0989] Table 7 shows the amino acid sequence identifiers for the heavy and light chain variable regions and CDRs of selected anti-MET antibodies described herein. (As described above, all anti-MET antibodies disclosed herein have identical light chain variable regions and, therefore, identical light chain CDR sequences.) The corresponding nucleic acid sequence identifiers are shown in Table 8.

[0990] Table 7. Amino acid sequence identifiers

[0991]

[0992] Table 8. Nucleic acid sequence identifiers

[0993]

[0994]

[0995] Antibodies are generally referred to herein according to the following nomenclature: an Fc prefix (e.g., "H4H"), followed by a numerical identifier (e.g., "13290," "13291," "13295," etc.), followed by a "P2" suffix, as shown in Tables 7 and 8. Thus, according to this nomenclature, an antibody may be referred to herein as, for example, "H4H13290P2," "H4H13291P2," "H4H13295P2," etc. The prefixes in the antibody nomenclature used herein indicate the specific Fc region isotype of the antibody. In particular, an "H4H" antibody has a human IgG4 Fc (all variables are fully human, as indicated by the first "H" in the antibody nomenclature). As will be understood by one of ordinary skill in the art, an antibody with a particular Fc isotype can be converted into an antibody with a different Fc isotype (e.g., an antibody with a mouse IgG4 Fc can be converted into an antibody with a human IgG1, etc.), but regardless, the variable domains (including the CDRs) indicated by the numerical identifiers shown in Tables 7 and 8 will remain the same, and the binding properties are expected to be the same or substantially similar regardless of the nature of the Fc domain.

[0996] Antibody conjugation

[0997] The techniques and linkers for conjugation to residues of antibodies or antigen-binding fragments are known in the art. Exemplary amino acid attachments that can be used in the context of this aspect include, for example, lysine (see, e.g., US 5,208,020; US 2010 / 0129314; Hollander et al., Bioconjugate Chem., 2008, 19:358-361; WO 2005 / 089808; US 5,714,586; US 2013 / 0101546; and US 2012 / 0585592), cysteine ​​(see, e.g., US 2007 / 0258987; WO 2013 / 055993; WO 2013 / 055990; WO 2013 / 053873; WO 2013 / 053872; WO 2011 / 130598; US 2013 / 0101546; and US 2012 / 0585592). 7,750,116), selenocysteine ​​(see, e.g., WO 2008 / 122039; and Hofer et al., Proc. Natl. Acad. Sci., USA, 2008, 105:12451-12456), formylglycine (see, e.g., Carrico et al., Nat. Chem. Biol., 2007, 3:321-322; Agarwal et al., Proc. Natl. Acad. Sci., USA, 2013, 110:46-51; and Rabuka et al., Nat. Protocols, 2012, 10:1052-1067), unnatural amino acids (see, e.g., WO 2013 / 068874 and WO 2012 / 166559), and acidic amino acids (see, e.g., WO 2012 / 05982). Lysine conjugation can also be carried out by NHS (N-hydroxysuccinimide). Linkers can also be conjugated to cysteine ​​residues (including cysteine ​​residues of cleaved interchain disulfide bonds) by forming a carbon bridge between thiols (see, for example, US 9,951,141 and US 9,950,076). Linkers can also be conjugated to antigen-binding proteins via: attachment to carbohydrates (see, for example, US 2008 / 0305497; WO 2014 / 065661; and Ryan et al., Food & Agriculture Immunol., 2001, 13: 127-130) and disulfide linkers (see, for example, WO 2013 / 085925; WO 2010 / 010324; WO 2011 / 018611; and Shaunak et al., Nat. Chem. Biol., 2006, 2: 312-313).Site-specific conjugation techniques can also be used to directly conjugate specific residues of antibodies or antigen-binding proteins (see, e.g., Schumacher et al. J Clin Immunol (2016) 36(Suppl 1): 100). In specific embodiments discussed in more detail below, site-specific conjugation techniques include glutamine conjugation via transglutaminase (see, e.g., Schibli, Angew Chemie Inter ed. 2010, 49, 9995).

[0998] Payloads according to the present disclosure that are linked through lysine and / or cysteine ​​(eg, via maleimide or amide conjugation) are included within the scope of the present disclosure.

[0999] In some embodiments, the protein-drug conjugates of the present disclosure are produced according to a two-step process, wherein 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).

[1000] In some embodiments, the protein-drug conjugates of the present disclosure are produced according to a two-step process, wherein 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).

[1001] In some embodiments, the protein-drug conjugates of the present disclosure are produced according to a two-step process, wherein step 1 is a transglutaminase-mediated site-specific conjugation and step 2 is a payload conjugation reaction (eg, a 1,3-cycloaddition reaction).

[1002] Step 1: Transglutaminase-mediated site-specific conjugation

[1003] In some embodiments, proteins (e.g., antibodies) can be modified according to known methods to provide proteins modified with glutaminyl groups. Techniques for conjugating antibodies and primary amine compounds are known in the art. Site-specific conjugation techniques are employed herein to guide conjugation to glutamine using glutamine conjugation via transglutaminase (see, e.g., Schibli, Angew Chemie Inter ed. 2010, 49, 9995).

[1004] The compounds containing primary amines of the present disclosure (e.g., linker L1) can be conjugated to one or more glutamine residues of a binding agent (e.g., a protein, such as an antibody) via a transglutaminase-based chemoenzymatic conjugation (see, e.g., Dennler et al., Protein Conjugate Chem. 2014, 25, 569-578; and WO 2017 / 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. In short, in some embodiments, in the presence of the enzyme transglutaminase, a binding agent having a glutamine residue (e.g., gln295, i.e., a Q295 residue) is treated with the above-mentioned primary amine-containing linker LL. In certain embodiments, the binding agent is non-glycosylated. In certain embodiments, the binding agent is deglycosylated.

[1005] In certain embodiments, the binding agent (e.g., a protein, such as an antibody) comprises at least one glutamine residue in at least one polypeptide chain sequence. In certain embodiments, the binding agent comprises two heavy chain polypeptides, each having a gln295 residue. In further embodiments, the binding agent comprises one or more glutamine residues at sites other than gln295 of the heavy chain.

[1006] In some embodiments, binding agents, such as antibodies, can be prepared by site-directed mutagenesis to insert glutamine residues into the site without producing disabled antibody function or binding. For example, antibodies carrying one or more Asn297Gln (N297Q) mutations as described herein are included herein. In some embodiments, antibodies with gln295 residues and / or N297Q mutations contain one or more other naturally occurring glutamine residues in their variable regions, which can be obtained by transglutaminase and therefore can be conjugated to a linker or linker-payload. For example, exemplary naturally occurring glutamine residues can be found at Q55 of the light chain. In this case, the binding agent, such as the antibody, conjugated via transglutaminase can have an LAR value higher than expected (e.g., LAR higher than 4). Any such antibody can be isolated from natural or artificial sources.

[1007] 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 from 1 to 8. In some embodiments, the LAR is from 1 to 6. In certain embodiments, the LAR is from 2 to 4. In some cases, the LAR is from 2 to 3. In some cases, the LAR is from 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.

[1008] Step 2: Payload conjugation reaction

[1009] In certain embodiments, according to the linker LL of the present disclosure, it comprises at least one reactive group capable of further reacting after transglutamination. In these embodiments, the protein (e.g., antibody) modified by glutaminyl can further react with a reactive payload compound or a reactive linker-payload compound (e.g., a linker-payload compound as disclosed herein) to form a protein-payload conjugate. More specifically, the reactive linker-payload compound may comprise a reactive group capable of reacting with the reactive group of linker LL via a click chemistry reaction to form a click chemistry adduct. In certain embodiments, the reactive group according to the present disclosure comprises a portion 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 internal straight-chain alkyne). In certain embodiments, the reactive group comprises a tetrazine. In certain embodiments, the reactive group comprises a straight-chain olefin. In certain embodiments of the present disclosure, the reactive group is compatible with the binding agent and transglutamination reaction conditions.

[1010] In one embodiment, the glutamine residue Gln is naturally present in the CH2 or CH3 domain of BA. In another embodiment, the glutamine residue Gln is introduced into BA by modifying one or more amino acids. In one embodiment, Gln is Q295 or N297Q.

[1011] In one embodiment, the transglutaminase is a microbial transglutaminase (MTG). In one embodiment, the transglutaminase is a bacterial transglutaminase (BTG).

[1012] Anti-HER2 antibody-drug conjugates

[1013] In certain embodiments, protein-drug conjugates (e.g., ADCs disclosed herein) are particularly useful for treating, preventing, and / or ameliorating any disease or disorder associated with or mediated by HER2 expression or activity, or that can be treated by binding to HER2 without competing with modified LDL, or / and promoting HER2 receptor internalization and / or reducing the number of cell surface receptors.

[1014] Protein-drug conjugates of the present disclosure (and therapeutic compositions comprising the same) are particularly useful for treating any disease or disorder for which stimulation, activation and / or targeted immune response will be beneficial. In particular, anti-HER2 protein-drug conjugates of the present disclosure (including both monospecific anti-HER2 antibodies and bispecific anti-HER2 / HER2 antibodies) can be used to treat, prevent and / or improve any disease or disorder associated with HER2 expression or activity or the proliferation of HER2+ cells or mediated by HER2 expression or activity or the proliferation of HER2+ cells. The mechanism of action of the disclosed therapeutic method is achieved, including killing HER2-expressing cells in the presence of effector cells, such as by CDC, apoptosis, ADCC, phagocytosis or by a combination of two or more of these mechanisms. Cells expressing HER2 that can be inhibited or killed using protein-drug conjugates of the present disclosure include, for example, breast tumor cells.

[1015] In one embodiment, the protein-drug conjugates of the present disclosure (and therapeutic compositions and dosage forms comprising the same) comprise a bispecific antigen binding molecule comprising:

[1016] a first antigen binding domain (D1); and

[1017] a second antigen-binding domain (D2);

[1018] wherein D1 specifically binds to the first epitope of human HER2; and

[1019] Among them, D2 specifically binds to the second epitope of human HER2.

[1020] In one embodiment above, D1 and D2 do not compete with each other for binding to human HER2.

[1021] The protein-drug conjugates of the present disclosure can be used to treat primary and / or metastatic tumors that occur, for example, in the prostate, bladder, cervix, lung, colon, kidney, breast, pancreas, stomach, uterus and / or ovary. In certain embodiments, the protein-drug conjugates of the present disclosure are used to treat one or more of the following cancers: prostate cancer, bladder cancer, cervical cancer, lung cancer, colon cancer, kidney cancer, breast cancer, pancreatic cancer, gastric cancer, uterine cancer and ovarian cancer. According to certain embodiments of the present disclosure, anti-HER2 antibodies or anti-HER2 / HER2 bispecific antibodies can be used to treat patients with IHC2+ or more breast cancer cells. According to other related embodiments of the present disclosure, a method is provided, comprising administering an anti-HER2 antibody or anti-HER2 / HER2 antibody as disclosed herein to a patient with IHC2+ or more breast cancer cells. Analytical / diagnostic methods known in the art (such as tumor scanning, etc.) can be used to determine whether a patient carries a castration-resistant tumor.

[1022] In certain embodiments, the present disclosure also includes methods for treating residual cancer in a subject.The term "residual cancer" means the presence or persistence of one or more cancer cells in a subject after treatment with an anti-cancer therapy.

[1023] Protein-drug conjugates of the present disclosure (and therapeutic compositions comprising the same) are particularly useful for treating any disease or disorder for which stimulation, activation and / or targeted immune response will be beneficial. In particular, protein-drug conjugates comprising anti-HER2 antibodies or anti-HER2 / HER2 antibodies of the present disclosure can be used to treat, prevent and / or improve any disease or disorder associated with HER2 expression or activity or the proliferation of HER2+ cells or mediated by HER2 expression or activity or the proliferation of HER2+ cells. The mechanism of action of the disclosed therapeutic method is achieved, including killing HER2-expressing cells in the presence of effector cells, such as by CDC, apoptosis, ADCC, phagocytosis or by a combination of two or more of these mechanisms. Cells expressing HER2 that can be inhibited or killed using protein-drug conjugates of the present disclosure include, for example, breast tumor cells.

[1024] According to certain aspects, the present disclosure provides a method for treating a disease or disorder (e.g., breast cancer) associated with HER2 expression, the method comprising administering to the subject one or more of an anti-HER2 protein-drug conjugate or an anti-HER2 / HER2 bispecific protein-drug conjugate described elsewhere herein after determining that the subject has breast cancer (e.g., IHC2+ breast cancer). For example, the present disclosure includes a method for treating breast cancer, the method comprising administering to the patient a protein-drug conjugate comprising an anti-HER2 antibody or antigen binding molecule or 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 receives hormone therapy (e.g., anti-androgen therapy).

[1025] In certain embodiments, the present disclosure also includes the use of the anti-HER2 antibodies of the present disclosure in the manufacture of a medicament for treating a disease or disorder (e.g., cancer) associated with or caused by cells expressing HER2. In one aspect, the present disclosure relates to a protein-drug conjugate comprising an anti-HER2 antibody or antigen-binding fragment or an anti-HER2 / HER2 bispecific antibody or antigen-binding fragment as disclosed herein for medical use. In one aspect, the present disclosure relates to a compound comprising an antibody-drug conjugate (ADC) as disclosed herein for medical use. Anti-STEAP2 Antibody-Drug Conjugate

[1026] In certain embodiments, protein-drug conjugates (e.g., ADCs disclosed herein) are particularly useful for treating, preventing, and / or ameliorating any disease or disorder associated with or mediated by STEAP2 expression or activity, or that can be treated by binding to STEAP2 without competing with modified LDL, or / and promoting STEAP2 receptor internalization and / or reducing the number of cell surface receptors.

[1027] The protein-drug conjugates of the present disclosure (and therapeutic compositions comprising the same) are particularly useful for treating any disease or disorder in which stimulating, activating, and / or targeting an immune response would 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 proliferation of STEAP2+ cells. The mechanism of action for achieving the therapeutic methods of the present disclosure includes killing STEAP2-expressing cells in the presence of effector cells, for example, via 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.

[1028] The protein-drug conjugates of the present disclosure can be used to treat primary and / or metastatic tumors that arise, for example, in the prostate, bladder, cervix, lung, colon, kidney, breast, pancreas, stomach, uterus, and / or ovary. In certain embodiments, the protein-drug conjugates of the present disclosure are used to treat one or more of the following cancers: prostate cancer, bladder cancer, cervical cancer, lung cancer, colon cancer, kidney cancer, breast cancer, pancreatic cancer, stomach cancer, uterine cancer, and ovarian cancer. Analytical / diagnostic methods known in the art (such as tumor scanning, etc.) can be used to determine whether a patient carries a castration-resistant tumor.

[1029] In certain embodiments, the present disclosure also includes methods for treating residual cancer in a subject.The term "residual cancer" means the presence or persistence of one or more cancer cells in a subject after treatment with an anti-cancer therapy.

[1030] According to certain aspects, the present disclosure provides methods for treating a disease or disorder associated with STEAP2 expression (e.g., prostate cancer), comprising administering to a subject one or more of the anti-STEAP2 protein-drug conjugates described elsewhere herein after determining that the subject has prostate cancer. For example, the present disclosure includes methods for treating prostate cancer, comprising administering to a patient a protein-drug conjugate comprising an anti-STEAP2 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).

[1031] In certain embodiments, the present disclosure also includes the use of the anti-STEAP2 antibodies of the present disclosure in the manufacture of a medicament for treating a disease or disorder associated with or caused by cells expressing STEAP2 (e.g., cancer). In one aspect, the present disclosure relates to a protein-drug conjugate comprising an anti-STEAP2 antibody or antigen-binding fragment as disclosed herein, for medical use. In one aspect, the present disclosure relates to a compound comprising an antibody-drug conjugate (ADC) as disclosed herein, for medical use.

[1032] Anti-MET antibody-drug conjugates

[1033] In certain embodiments, protein-drug conjugates (e.g., ADCs disclosed herein) are particularly useful for treating, preventing, and / or ameliorating any disease or disorder associated with or mediated by MET expression or activity, or that can be treated by binding to MET without competing with modified LDL, or / and promoting MET receptor internalization and / or reducing the number of cell surface receptors.

[1034] The protein-drug conjugates of the present disclosure (and therapeutic compositions comprising the same) are particularly useful for treating any disease or disorder in which stimulation, activation and / or targeting of an immune response would 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 proliferation of MET+ cells. The mechanism of action of the therapeutic methods of the present disclosure includes killing MET-expressing cells in the presence of effector cells, such as 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.

[1035] The protein-drug conjugates of the present disclosure can be used to treat primary and / or metastatic tumors that arise, for example, in the prostate, bladder, cervix, lung, colon, kidney, breast, pancreas, stomach, uterus, and / or ovary. In certain embodiments, the protein-drug conjugates of the present disclosure are used to treat one or more of the following cancers: prostate cancer, bladder cancer, cervical cancer, lung cancer, colon cancer, kidney cancer, breast cancer, pancreatic cancer, stomach cancer, uterine cancer, and ovarian cancer. Analytical / diagnostic methods known in the art (such as tumor scanning, etc.) can be used to determine whether a patient carries a castration-resistant tumor.

[1036] In certain embodiments, the present disclosure also includes methods for treating residual cancer in a subject.The term "residual cancer" means the presence or persistence of one or more cancer cells in a subject after treatment with an anti-cancer therapy.

[1037] According to certain aspects, the present disclosure provides methods for treating a disease or disorder associated with MET expression (e.g., lung cancer), the method comprising administering to the subject one or more of the anti-MET or anti-MET / MET bispecific protein-drug conjugates described elsewhere herein after determining that the subject has lung cancer. For example, the present disclosure includes methods for treating lung cancer, the method comprising administering to the 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 receives hormone therapy (e.g., anti-androgen therapy).

[1038] For example, the anti-MET antibody-drug conjugates and MET x MET bispecific antibody-drug conjugates of the present disclosure can be used to treat tumors that express (or overexpress) MET. For example, the anti-MET antibody-drug conjugates and MET x MET bispecific antibody-drug conjugates can be used to treat primary and / or metastatic tumors arising in the brain and meninges, oropharynx, lungs and bronchial trees, gastrointestinal tract, male and female reproductive tract, muscle, bone, skin and its appendages, connective tissue, spleen, immune system, hematopoietic cells and bone marrow, liver and urinary tract, and special sense organs (such as the eye). In certain embodiments, the anti-MET antibody-drug conjugates and MET x MET 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, bile duct cancer, 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.

[1039] In certain embodiments, the present disclosure also includes the use of the anti-MET antibody-drug conjugate or MET x MET bispecific antibody-drug conjugate of the present disclosure in the manufacture of a medicament for treating a disease or disorder (e.g., cancer) associated with or caused by cells expressing MET. In one aspect, the present disclosure relates to a protein-drug conjugate comprising an anti-MET antibody-drug conjugate or MET x MET bispecific antibody-drug conjugate as disclosed herein, for medical use. In one aspect, the present disclosure relates to a compound comprising an antibody-drug conjugate (ADC) as disclosed herein, for medical use.

[1040] Combination therapies and preparations

[1041] The present disclosure provides such a method, the method includes administering a pharmaceutical composition comprising any one of exemplary protein-drug conjugates as described herein (e.g., antibody-drug conjugates), linkers-payloads and payloads in combination with one or more additional therapeutic agents. The exemplary additional therapeutic agents that can be combined or administered in combination with protein-drug conjugates (e.g., antibody-drug conjugates), linkers-payloads and payloads of the present disclosure include, for example, HER2 antagonists (e.g., anti-HER2 antibodies [e.g., trastuzumab] or small molecule inhibitors of HER2 or anti-HER2 antibody-drug conjugates or anti-HER2 / HER2 bispecific antibodies or anti-HER2 / HER2 bispecific antibodies-drug conjugates), EGFR antagonists (e.g., anti-EGFR antibodies [e.g., cetuximab or panitumumab] or small molecule inhibitors of EGFR [e.g., gefitinib or erlotinib]), another EGFR family member (such as HER2 / ErbB2, ErbB3 or ErbB4) ), antagonists of EGFRvIII (e.g., antibodies that specifically bind to EGFRvIII), cMET antagonists (e.g., anti-cMET antibodies), IGF1R antagonists (e.g., anti-IGF1R antibodies), B-raf inhibitors (e.g., vemurafenib, sorafenib, gDC-0879, PLX-4720), PDGFR-α inhibitors (e.g., anti-PDGFR-α antibodies), PDGFR-β inhibitors (e.g., anti-PDGFR-β antibodies), VEGF antagonists (e.g., VEGF-Trap, see e.g., US Pat. 7,087,411 (also referred to herein as “VEGF inhibitory fusion protein”), 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, such as 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), urothelial protein antagonists (e.g., anti-urothelial protein antibodies), etc.

[1042] Other agents that can be beneficially administered in combination with the protein-drug conjugates (e.g., antibody-drug conjugates), linkers-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 corresponding receptors. The pharmaceutical compositions of the present disclosure (e.g., pharmaceutical compositions comprising anti-HER2, anti-HER2 / HER2 bispecific, anti-MET, anti-MET / MET bispecific, or anti-STEAP2 protein-drug conjugates (e.g., antibody-drug conjugates as disclosed herein)) can also be administered as a combination comprising an agent selected from "ICE": ifosfamide (e.g., ), carboplatin (e.g., ), etoposide (e.g., VP-16); "DHAP": dexamethasone (e.g., ), cytarabine (e.g., Cytosine arabinoside, ara-C), cisplatin (e.g., ); and "ESHAP": etoposide (e.g.,

[1043] VP-16), methylprednisolone (e.g., ), high-dose cytarabine, cisplatin (e.g., ) is administered as part of a treatment regimen of one or more therapeutic combinations.

[1044] 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 an inhibitor of one or more of: HER2, VEGF, Ang2, DLL4, EGFR, ErbB2, ErbB3, ErbB4, EGFRvIII, cMet, IGF1R, B-raf, PDGFR-α, PDGFR-β, FOLH1 (PSMA), PRLR, STEAP1, STEAP2, TMPRSS2, MSLN, CA9, urothelial protein, or any of the above cytokines, wherein the inhibitor is an aptamer, an antisense molecule, a ribozyme, an siRNA, a peptibody, a nanobody, or an antibody fragment (e.g., a Fab fragment; a F(ab')2 fragment; a Fd fragment; a Fv fragment; a scFv; a dAb fragment; or other engineered molecules such as diabodies, triabodies, tetrabodies, minibodies, and minimal recognition units). The antigen binding molecules of the present invention can also be administered in combination with antiviral agents, antibiotics, analgesics, corticosteroids and / or NSAIDs and / or co-formulated. The antigen binding molecules of the present invention can also be administered as part of a treatment regimen that also includes radiation therapy and / or conventional chemotherapy.

[1045] One or more additional therapeutically active components may be administered just prior to, simultaneously with, or shortly after administration of the antigen binding molecules of the disclosure; (for purposes of this disclosure, such administration regimens are considered to be administration of an antigen binding molecule "in combination" with the additional therapeutically active components).

[1046] The present disclosure includes pharmaceutical compositions in which the protein-drug conjugates (e.g., antibody-drug conjugates), linker-payloads, and / or payloads of the present disclosure are co-formulated with one or more of one or more additional therapeutically active components as described elsewhere herein.

[1047] Administration regimen

[1048] 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 limited time period. The method according to this aspect of the present disclosure comprises sequentially administering 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) of the present disclosure, linker-payload, and / or payload to a subject. As used herein, "sequential administration" means administering 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 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 second 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), a linker-payload, and / or a payload, and optionally followed by one or more third 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), a linker-payload, and / or a payload.

[1049] The terms "initial dose," "secondary dose," and "tertiary dose" refer to the temporal sequence of administration of a protein-drug conjugate (e.g., anti-HER2, anti-HER2 / HER2 bispecific, anti-MET, anti-MET / MET bispecific, or anti-STEAP2 antibody-drug conjugate) of the present disclosure, a linker-payload, and / or a payload. Thus, the "initial dose" is the dose administered at the beginning of the treatment regimen (also referred to as the "baseline dose"); the "secondary dose" is the dose administered after the initial dose; and the "tertiary dose" is the dose administered after the second dose. The initial, second, and third doses may all contain the same amount of protein-drug conjugate (e.g., 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 may typically differ from one another in terms of the frequency of administration. In certain embodiments, however, during the course of treatment, the amount 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 contained in the initial, second, and third doses are different from each other (e.g., adjusted upward or downward, as appropriate). In certain embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered at the beginning of the treatment regimen as "loading doses," followed by subsequent doses (e.g., "maintenance doses") on a less frequent basis.

[1050] In an exemplary embodiment of the present disclosure, immediately after the previous dose 1 to 26 (e.g., 1, 1 1 / 2, 2, 2 1 / 2, 3, 3 1 / 2, 4, 4 1 / 2, 5, 5 1 / 2, 6, 6 1 / 2, 7, 7 1 / 2, 8, 8 1 / 2, 9, 9 1 / 2, 10, 10 1 / 2, 11, 11 1 / 2, 12, 12 1 / 2, 13, 13 1 / 2, 14, 14 1 / 2, 15, 15 1 / 2, 16, 16 1 / 2, 17, 17 1 / 2, 18, 18 1 / 2, 19, 19 1 / 2, 20, 20 1 / 2, 21, 21 1 / 2, 22, 22 1 / 2, 23, 23 1 / 2, 24, 24 1 / 2, 25, 25 1 / 2, 26, 26 1 As used herein, the phrase "immediately following the previous dose" means 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 that is administered to the patient prior to administration of the next dose in the sequence, in the absence of an intervening dose, in the order of multiple administrations.

[1051] The methods according to this aspect of the present disclosure can include administering to the patient any number of second and / or third 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. For example, in certain embodiments, only a single second dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) second doses are administered to the patient. Similarly, in certain embodiments, only a single third dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) third doses are administered to the patient.

[1052] In embodiments involving multiple second doses, each second dose can be administered at the same frequency as the other second doses. For example, each second dose can be administered to the patient 1 to 2 weeks after the previous dose. Similarly, in embodiments involving multiple third doses, each third dose can be administered at the same frequency as the other third doses. For example, each third dose can be administered to the patient 2 to 4 weeks after the previous dose. Alternatively, the frequency of administration of the second and / or third doses to the patient can vary over the course of the treatment regimen. The physician can also adjust the frequency of administration during the course of treatment, depending on the needs of the individual patient after clinical examination.

[1053] Examples

[1054] The following examples illustrate specific aspects of this specification. These examples should not be interpreted as limiting, as these examples only provide a specific understanding and practice of the embodiment and its various aspects.

[1055] abbreviation

[1056]

[1057]

[1058] General Methods

[1059] Example 1. Synthesis of payload

[1060] Two synthetic routes were designed to make the DXd prodrug shown in Scheme 1.

[1061] Route A uses exitecan to react with 4, see Scheme 2A.

[1062] • Route B uses DXd to react with 3, see Scheme 2B.

[1063] Scheme 1. Two methods for synthesizing ProDXd according to the present disclosure

[1064]

[1065] Scheme 2A. General Synthesis of ProDXd (Route A)

[1066]

[1067] Scheme 2B. General Synthesis of ProDXd (Route B)

[1068]

[1069] Scheme 2C. Synthesis of P8

[1070]

[1071] Scheme 2D. Synthesis of P10

[1072]

[1073] Example 2. Synthesis of linker-payload

[1074] Five synthetic pathways are summarized in Scheme 3 below, based on the final step of the pathway. All building blocks (A to F) have suitable reactive moieties that can be used in the reaction. The synthetic schemes for the building blocks and the final linker-payload are shown below.

[1075] • Route 1 uses fragment F with exitecan.

[1076] • Pathway 2 uses Fragment E with DXd.

[1077] • Route 3 uses fragment D with a prodrug or Fmoc-protected prodrug.

[1078] Route 4 uses fragment B with vcPABC-prodrug

[1079] Route 5 uses fragment A with PEG4-vcPABC-prodrug

[1080] Scheme 3. Building blocks and methods for linker-payload synthesis.

[1081]

[1082] Intermediates A and B are commercially available or are reported building blocks with functional groups that can be conjugated to antibodies via, for example, bioorthogonal ("click") reactions (Table A).

[1083] Table A. Linkers-Payloads with Universal Reactive Moiety A

[1084]

[1085]

[1086] Scheme 4A. General Synthesis of Linker-Payload (Pathway 1)

[1087]

[1088] [Conditions] Exotecan, HATU, DIPEA, DMF, 25°C, 16 hours. Scheme 4B. General Synthesis of Linker-Payload (Pathway 2)

[1089]

[1090] [Conditions] DXd, Tf2NH, 4AMS, THF, 20°C, 10 min.

[1091] Scheme 4C. General Synthesis of Linker-Payload (Pathway 3a)

[1092]

[1093] [Conditions] Intermediate D, coupling catalyst 4-hydroxy-2-methylquinoline (MeHYQ), DIPEA, DMF, room temperature, 2 hours

[1094] Scheme 4D. General Synthesis of Linker-Payload (Pathway 3b)

[1095]

[1096] [Conditions] Intermediate D, coupling catalyst 4-hydroxy-2-methylquinoline (MeHYQ), Et3N, DBU, DMF, 50°C, 6 hours.

[1097] Scheme 4E. General Synthesis of Linker-Payload (Pathway 4)

[1098]

[1099] [Conditions] Step 1: a) Fmoc-vcPAB-PNP, coupling reagent 4-hydroxy-2-methylquinoline (MeHYQ), DIPEA, DMF, room temperature, 4 h; b) Et2NH, DMF, room temperature, 2 h. Step 2: Intermediate B, HATU, DIPEA, DMF, room temperature, 4 h.

[1100] Scheme 4F. General Synthesis of Linker-Payload (Pathway 5)

[1101]

[1102] Scheme 5A. General Synthesis of EvcPAB-Linker-Payload

[1103]

[1104] Scheme 5B. General Synthesis of Branched Linker-Payloads LP13 and LP13C

[1105]

[1106] Scheme 5C. General Synthesis of Branched GGFG-Linker-Payload LP15 and LP15C ("GGFG" disclosed as SEQ ID NO: 2142)

[1107]

[1108] (SEQ ID NOs 2125 to 2126 and 2119 and 2119, or 2120 and 2120, respectively)

[1109] Scheme 5D. Synthesis of Carbonate-DXd LP16

[1110]

[1111] Scheme 5E. Synthesis of Linker-DXd LP17

[1112]

[1113] Example 3. Synthesis of key intermediates / structural units

[1114] Intermediate A was prepared according to Scheme 6 and described below.

[1115] Scheme 6. Synthesis of intermediate Aa

[1116]

[1117] [1]KO t Bu, CHBr3, hexane, -10℃ to 25℃, 16h;

[1118] [2] Methyl glycolate, AgOTf, DCM, 25°C, 1 h;

[1119] [3] 30% NaOMe in MeOH, DMSO, 25 °C, 2 h, 47% yield from A-1;

[1120] [4]DCC, HOSu, DCM, 0°C to 25°C, 16h, crude.

[1121] 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)

[1122] Intermediate B was prepared according to Scheme 7 and described below.

[1123] Scheme 7. Synthesis of Intermediate B

[1124]

[1125] The synthesis of intermediate B was reported in WO2019094395 and described in Scheme 7 above.

[1126] The synthesis of intermediates 4a to h is described in Scheme 2A above. Intermediate 4a is reported in WO2015146132 (Scheme 8). Alternatively, intermediate 4a was prepared by a 2-step synthesis in 45% overall yield without chromatographic purification.

[1127] Scheme 8. Synthesis of intermediate 4a.

[1128]

[1129] [1] Pb(OAc)4 (1.5 to 2.0 equiv), DMF, 25°C, 16 h, 80% yield (10 g);

[1130] [2] Benzyl glycolate, 1,2-dichloroethane, pyridinium p-toluenesulfonate (PPTS), 45°C to 50°C, 18 hours, 53% yield (0.16 g);

[1131] [3] Pd-C, H2, methanol, THF, 25°C, 16 hours, 67% yield (90 mg); the total yield of 4a in 3 steps was 28%.

[1132] Alternatively, 4a* was prepared on a larger scale by the following two-step procedure:

[1133] Scheme 8a. Large-scale synthesis of 4a

[1134]

[1135] *Where [Step 1] Cu(OAc)2 (0.30 equiv), Pb(OAc)4 (1.5 to 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°C to 50°C, 18 hours, 75% yield (0.96 kg).

[1136] Scheme 9. Two-step synthesis of compound 4

[1137]

[1138] [Conditions] Step 1, Cu(OAc)2 (0.30 equiv), Pb(OAc)4 (1.5 to 2.0 equiv), pyridine (2.0 equiv), THF, 25°C, 16 hours; Step 2, glycolic acid, 1,2-dichloroethane, pyridinium p-toluenesulfonate (PPTS), 45°C to 50°C, 18 hours.

[1139] Two synthetic routes were combined to make intermediate D in Scheme 10. All building blocks have suitable reactive moieties that can be used in the reaction. The synthetic schemes of the building blocks and the final intermediate D are shown below.

[1140] Scheme 10. Structural units of intermediate D.

[1141]

[1142] • Pathway Da is from A to A-PEG4 (B), to A-PEG4-vcPAB, to A-PEG4-vcPAB-PNP (D).

[1143] Pathway Db is from A to A-PEG4-vcPAB (B), then to A-PEG4-vcPAB-PNP (D)

[1144] Scheme 11A. Synthesis of Intermediate D (Route Da)

[1145]

[1146] Scheme 11B. Synthesis of Intermediate D (Route Db)

[1147]

[1148] [1]DCC, HOSu, DCM, 0℃ to 25℃, 2h;

[1149] [2] vcPAB, DMF, 0°C to 25°C, 16 h, 73% yield in 2 steps from Fmoc-amino-PEG4-acid (D-1);

[1150] [3] a) DBU, Et3N, DMF, 25°C, 16h, b) Intermediate Aa, 0°C to 25°C, 1h, 54% yield; or a) Et2NH, MeOH, rt, 1h, b) Intermediate Ad, HATU, Et3N, DMF, rt, 4h.

[1151] [4] PNP, DIPEA, DMAP, DMF, 0°C to 25°C, 4h, 37% yield.

[1152] Scheme 12. General Synthesis of Intermediate E

[1153]

[1154] [Step 1] a) Compound 2, DBU, Et3N, DMF, 25°C, 16h; b) Intermediate D, HOAt, DIPEA, 25°C, 4h;

[1155] [Step 2] Pb(OAc), HOAc, DMF, 25°C, 16h

[1156] Scheme 13. General Synthesis of Intermediate F

[1157]

[1158] [Step 1] a) Intermediate 4, triethylamine, DBU, DMF, 25°C, 16h, b) HOAt, Intermediate D, 25°C, 16h.

[1159] Scheme 14. Comprehensive synthesis of LP1*

[1160]

[1161] *Procedures and conditions

[1162] Step [1] KO t Bu, CHBr3, hexane, -10℃ to 25℃, 16h;

[1163] Step [2] methyl glycolate, AgOTf, DCM, 25°C, 1 h;

[1164] Step [3] 30% NaOMe in MeOH, DMSO, 25 °C, 2 h, 47% yield from A-1;

[1165] Step [4] DCC, HOSu, DCM, 0°C to 25°C, 16h, crude.

[1166] Step [5] DCC, HOSu, DCM, 0°C to 25°C, 2h;

[1167] Step [6] vcPAB, DMF, 0°C to 25°C, 16 h, 73% yield in 2 steps from Fmoc-amino-PEG4-acid (D-1);

[1168] Step [7] a) DBU, Et3N, DMF, 25°C, 16h, b) Intermediate A, 0°C to 25°C, 1h, 54% yield;

[1169] Step [8] PNP, DIPEA, DMAP, DMF, 0°C to 25°C, 4h, 37% yield.

[1170] Step [9] Cu(OAc)2 (0.30 eq), Pb(OAc)4 (1.5 to 2.0 eq), pyridine (2.0 eq), THF, 25°C, 16 hours, 60% yield (0.80 kg);

[1171] Step

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

[1172] Step

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

[1173] Step

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

[1174] Example 4. Conjugation

[1175] Site-specific ADC conjugation is shown in Figure 5 middle.

[1176] Step 1 is the site-specific conjugation of handle-functionalized amines to antibodies, resulting in drug conjugates containing 2, 4, or 8 handles per antibody. Here, AL = unbranched handle-functionalized amine, BL = branched handle-functionalized amine.

[1177] Step 2 is a click reaction between the handle-functionalized antibody and the linker-payload (LP) to generate a site-specific ADC.

[1178] Payload synthesis

[1179] Example 5. General Synthesis of ProDXd (Scheme 2A)

[1180] General procedure for the synthesis of compound 2s

[1181] To a stirred solution of Fmoc protected amino acid 1 (1 eq) in DCM (0.2 M) was added HOSu (2.2 eq) and EDCI (2.2 eq), and the reaction mixture was stirred at room temperature for 2 to 16 hours, monitored by LCMS. The mixture was diluted with DCM, washed with water (3x) and brine, dried over anhydrous sodium sulfate, and concentrated in vacuo. The residue was dissolved in DMF (0.2 M). The corresponding amino acid (R 3 NHCHR 4 To the mixture was added 4-[4-(4-(4-(4-oxo-3-oxo-1-yl)-1-oxo-4-oxo-1-yl)-1-oxo-4-oxo-1-yl)-1-oxo-4-oxo-1-oxo-2-nitropropane-2-yl)-4 ...

[1182] 2-[2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)acetamido]acetic acid (2a)

[1183]

[1184] Commercially available.

[1185] 2-[(2S)-3-[(tert-Butyldimethylsilyl)oxy]-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)propionamido]acetic acid (2b)

[1186]

[1187] Following the general procedure, compound 2b (0.50 g, 54% yield) was obtained as a white solid. ESI m / z: 499 (M+H) + .

[1188] 2-[(2S)-5-(Benzyloxy)-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-5-oxopentanamido]acetic acid (2c)

[1189]

[1190] Following the general procedure, compound 2c (0.65 g, 58% yield) was obtained as a white solid. ESI m / z: 517 (M+H) + .

[1191] 2-[(2S)-6-Azido-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)hexanamido]acetic acid (2d)

[1192]

[1193] Following the general procedure, compound 2d (0.66 g, 70% yield) was obtained as a white solid. ESI m / z: 452 (M+H) + .

[1194] 2-[(2S)-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-3-phenylpropionamido]acetic acid (2e)

[1195]

[1196] Following the general procedure, compound 2e (0.43 g, 37% yield) was obtained as a white solid. ESI m / z: 445 (M+H) + .

[1197] 2-[2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-N-methylacetamido]acetic acid (2f)

[1198]

[1199] Following the general procedure, compound 2f (2.6 g, 72% yield) was obtained as a white solid. ESI m / z: 369 (M+H) + .

[1200] 2-[2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-2-methylpropionamido]acetic acid (2 g)

[1201]

[1202] Following the general procedure, compound 2g (0.60 g, 50% yield) was obtained as a white solid. ESI m / z: 383 (M+H) + .

[1203] 2-[(2R)-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-3-phenylpropionamido]acetic acid (2h)

[1204]

[1205] Following the general procedure, compound 2e (0.43 g, 37% yield) was obtained as a white solid. ESI m / z: 445 (M+H) + .

[1206] General procedure for the synthesis of compounds 3a to h

[1207] In 10L reaction flask, add THF (0.25 to 0.30M) and compound 2 (1.0 equivalent) at 25 ℃ to 30 ℃, and in the suspension obtained, add pyridine (2.0 equivalent) at 25 ℃ to 30 ℃.After stirring the mixture and becoming clear, cupric acetate (0 or 0.3 equivalent) is added to the solution. The reaction mixture is cooled to 0 ℃ to 5 ℃, and lead acetate (IV) (1.5 equivalent) is added to the reaction mixture at 0 ℃ to 5 ℃. Then the mixture is stirred for one hour at 0 ℃ to 5 ℃, and then it is allowed to warm to 25 ℃ to 30 ℃. The reaction mixture is stirred for 16 hours at 25 ℃ to 30 ℃, until most of compound 2 is consumed by LCMS monitoring. The gained mixture is filtered through a short silica gel plug, and silica gel is washed with ethyl acetate (2x). The filtrate merged is diluted with ethyl acetate and water. After being carefully neutralized to pH 7 with sodium bicarbonate powder, the mixture is separated, and the organic layer is washed with brine, dried over anhydrous sodium sulfate, and concentrated in vacuo to give a brown crude product, which is dissolved in DCM (3 L). The mixture is filtered through a short silica gel plug, eluted with DCM (3x) until compound 3 is completely collected. The collected solution is concentrated. MTBE is added to the residue, and a white solid is precipitated at 25° C. to 30° C., which is collected by filtration. The solid is dried at 25° C. to 30° C. with nitrogen purging for more than 16 hours to give pure compound 3 as a white solid. Alternatively, the brown crude product is purified by reverse phase flash chromatography or preparative HPLC to give pure compound 3 as a white solid. [2-({[(9H-fluorene-9-yl)methoxy]carbonyl}amino)acetamido]methyl acetate (3a)

[1208]

[1209] Following the general procedure (catalyzed with copper acetate (0.3 eq)), 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 )δ8.96(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.

[1210] [(2S)-3-[(tert-Butyldimethylsilyl)oxy]-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)propionamido]methyl acetate (3b)

[1211]

[1212] 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 to 100% acetonitrile in aqueous formic acid (0.1%)). ESI m / z: 535 (M+Na) + .

[1213] Benzyl (4S)-4-{[(acetyloxy)methyl]carbamoyl}-4-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)butanoate (3c)

[1214]

[1215] 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 to 60% acetonitrile in aqueous formic acid (0.1%)). ESI m / z: 553 (M+Na) + .

[1216] [(2S)-6-Azido-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)hexanamido]methyl acetate (3d)

[1217]

[1218] 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 to 100% acetonitrile in aqueous formic acid (0.1%)). ESI m / z: 488 (M+Na) + .

[1219] [(2S)-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-3-phenylpropionamido]methyl acetate (3e)

[1220]

[1221] 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 to 100% acetonitrile in aqueous formic acid (0.1%)). ESI m / z: 481 (M+Na)+ . 1 H NMR (400MHz, DMSO) δ9.13(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),7.3 5-7.23(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.

[1222] [2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-N-methylacetamido]methyl acetate (3f)

[1223]

[1224] 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) + .

[1225] [2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-2-methylpropionamido]methyl acetate (3 g)

[1226]

[1227] 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 to 100% acetonitrile in aqueous formic acid (0.1%)). ESI m / z: 481 (M+Na) + .

[1228] [(2R)-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-3-phenylpropionamido]methyl acetate (3h)

[1229]

[1230] Obtained as a white solid (0.34 g, 80% yield) following the general procedure (catalyzed with copper acetate (0.3 equiv)) after purification by preparative HPLC (0 to 100% acetonitrile in aqueous formic acid (0.1%)). ESI m / z: 481 (M+Na) + .

[1231] General procedure for the synthesis of compounds 4a to h

[1232] 1,2-dichloroethane (0.10 to 0.15M), compound 3 (1.0 equivalent), glycolic acid (0.6 equivalent) and pyridinium p-toluenesulfonate (PPTS) (0.2 equivalent) were added to the reaction flask at room temperature. The reaction mixture was heated to 45°C to 50°C and stirred for one hour, and glycolic acid (0.6 equivalent 2x) was added twice to the hot solution, once per hour. The mixture was then stirred at 45°C to 50°C for 16 hours, monitored by LCMS. After cooling to 25°C to 30°C, the precipitate was filtered and collected. At 5°C to 10°C, the solid was dissolved in aqueous sodium bicarbonate (3%) to obtain a mixture of pH 7 to 8, which was washed with a mixed solvent of ethyl acetate and THF (v / v=1, 3x). MTBE was added to the aqueous layer at 5°C to 10°C, and acidified to pH 3 to 4 with saturated aqueous citric acid to precipitate a large amount of solid. The mixture is filtered, and the cake is washed with water (1x) and MTBE (2x), dried under a stream of nitrogen at 25°C to 30°C for 48 hours to obtain the wet compound 4 (75% yield) as a white solid, which contains 3% water according to HNMR. The product is dried again in a vacuum for 48 hours to obtain the dry compound 4 (73% yield) as a white solid. Alternatively, the reaction mixture is purified by reversed-phase flash chromatography or preparative HPLC to obtain the pure compound 4 as a white solid.

[1233] 2-{[2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)acetamido]methoxy}acetic acid (4a)

[1234]

[1235] Following the general procedure, compound 4a (0.96 g, 80% yield) was obtained as a white solid. >99% in HPLC,

[1236] ESI m / z:407 (M+Na) + . 1 H NMR (400 MHz, DMSO d6 )δ12.53(br s,1H),8.72(t,J=6.8Hz,

[1237] 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=7.6Hz,2H),7.3 3(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.

[1238] 2-{[(2S)-3-[(tert-Butyldimethylsilyl)oxy]-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)propionamido]methoxy}acetic acid (4b)

[1239]

[1240] Following the general procedure, compound 4b (57 mg, 31% yield) was obtained as a yellow solid after purification by preparative HPLC (0 to 100% acetonitrile in aqueous TFA (0.05%)). ESI m / z: 551 (M+Na) + .

[1241] 2-{[(2S)-5-(Benzyloxy)-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-5-oxopentanamido]methoxy}acetic acid (4c)

[1242]

[1243] Following the general procedure, compound 4c (0.13 g, 65% yield) was obtained as a white solid after purification by preparative HPLC (0 to 90% acetonitrile in aqueous formic acid (0.1%)). ESI m / z: 569 (M+Na) + .

[1244] 2-{[(2S)-6-Azido-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)hexanamido]methoxy}acetic acid (4d)

[1245]

[1246] Following the general procedure, compound 4d (0.30 g, 51% yield) was obtained as a white solid after purification by preparative HPLC (5% to 95% acetonitrile in aqueous ammonium bicarbonate (10 mM)). ESI m / z: 504 (M+Na) + .

[1247] 2-{[(2S)-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-3-phenylpropionamido]methoxy}acetic acid (4e)

[1248]

[1249] 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 to 25% acetonitrile in water). ESI m / z: 474 (M+Na) + .

[1250] 2-{[2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-N-methylacetamido]methoxy}acetic acid (4f)

[1251]

[1252] 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 to 100% acetonitrile in aqueous TFA (0.01%)). ESI m / z: 421 (M+Na) + .

[1253] 2-{[2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-2-methylpropionamido]methoxy}acetic acid (4 g)

[1254]

[1255] 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 to 100% acetonitrile in aqueous TFA (0.01%)). ESI m / z: 435 (M+Na) + .

[1256] 2-{[2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-3-methylpropionamido]methoxy}acetic acid (4h)

[1257]

[1258] 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 to 100% acetonitrile in aqueous TFA (0.01%)). ESI m / z: 474 (M+Na) + .

[1259] Synthesis of 2-{[2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)acetamido]methoxy}acetic acid (4a)

[1260] The large-scale synthesis of intermediate 4a is described in Scheme 8a.

[1261] [2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)acetamido]methyl acetate (3a)

[1262] At 25 ℃ to 30 ℃, THF (6.7L) and Fmoc-Gly-Gly-OH (2a) (0.67kg, 1.9mol) are added to a 10L reaction flask. At 25 ℃ to 30 ℃, pyridine (0.30kg, 3.8mol) is added to the suspension obtained. After the mixture is stirred and clarified, cupric acetate (0.10kg, 0.57mol) is added to the solution. The reaction mixture is cooled to 0 ℃ to 5 ℃, and at 0 ℃ to 5 ℃, lead acetate (IV) (1.7kg, 2.8mol) is added to the reaction mixture. The mixture is then stirred for one hour at 0 ℃ to 5 ℃, and then allowed to warm to 25 ℃ to 30 ℃. The reaction mixture is stirred for 16 hours at 25 ℃ to 30 ℃, until most of compound 2a is consumed, monitored by LCMS. The gained mixture is filtered by a short silica gel plug (200g), and silica gel is washed with ethyl acetate (1Lx2). The combined filtrate is diluted with ethyl acetate (10 L) and water (10 L). After being carefully neutralized to pH 7 with sodium bicarbonate powder, the mixture is separated, and the organic layer is washed with brine (5 Lx1), dried over anhydrous sodium sulfate, and concentrated in vacuo to obtain a brown crude product, which is 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 is filtered through a short silica gel plug (0.30 kg) and eluted with DCM (1 Lx3) until compound 3a is completely collected. The collected solution is concentrated to 2 L. MTBE (3 L) is added to the residue, and a white solid is precipitated at 25 ° C to 30 ° C, which is collected by filtration. The solid is dried at 25 ° C to 30 ° C with nitrogen purge for more than 16 hours, to obtain 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 )δ8.96(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.

[1263]

[1264] 2-{[2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)acetamido]methoxy}acetic acid (4a)

[1265] 1,2-dichloroethane (19L), compound 3a (0.96kg, 2.6mol), glycolic acid (0.12kg, 1.6mol) and pyridinium p-toluenesulfonate (PPTS) (0.13kg, 0.52mol) were added successively to a 50L jacketed reaction flask at 20°C to 25°C. The reaction mixture was stirred at 45°C to 50°C for one hour, and glycolic acid (0.12kg, 1.6mol) was added twice to the hot solution, once per hour. The mixture was then stirred at 45°C to 50°C for 16 hours, monitored by LCMS. After cooling to 25°C to 30°C, the precipitate was filtered and collected, which was merged with other batches (200g, 1.25kg) with similar LCMS. The combined solid is dissolved in aqueous sodium bicarbonate (0.44 kg in 15 L water) at 5 ° C to 10 ° C, providing a mixture with pH 7 to 8, which is washed with a mixed solvent of ethyl acetate and THF (v / v = 1, 4.0 Lx3). MTBE (5 L) is added to the aqueous layer at 5 ° C to 10 ° C, and it is acidified to pH 3 to 4 with saturated aqueous citric acid to precipitate a large amount of solid. The mixture is filtered, and the cake is washed with water (1 L) and MTBE (1 Lx2), dried under a stream of nitrogen at 25 ° C to 30 ° C for 48 hours, to obtain compound 4a (1.1 kg, 75% yield) (according to HNMR, containing 3% water) as a white solid. The product is dried again in a vacuum for 48 hours to obtain dry compound 4a (0.96 kg, 97% recovery yield from wet product) as a white solid. HPLC> 99%, ESI m / z: 407 (M + Na) + . 1 H NMR (400 MHz, DMSO d6 )δ12.53(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.

[1266] The product contains about 0.96% of an unknown contaminant, M / Z=617 (positive mode). This byproduct can be removed in the next step. Product C should be dried thoroughly because water will reduce the yield in the next step.

[1267]

[1268] General procedure for the synthesis of compounds 5a to h

[1269] To the solution of compound 4 (1.1 equivalents) in DMF (5 to 8mL / gram 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 a mixture of isotecan mesylate (1.0 equivalents) and DIPEA (2.0 equivalents) in DMF (10mL / gram isotecan) was added to the stirred solution. The reaction mixture was stirred at room temperature for 4 hours, monitored by LCMS. The resulting mixture was diluted with ethyl acetate and washed with salt water (2x). The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated in a vacuum. The residue was co-evaporated with ethyl acetate (4x) in a vacuum to provide a crude product 5, to which ethyl acetate was added. The suspension was refluxed for approximately 20 minutes, until it became clear, then naturally cooled to 25°C and left for half an hour. The white precipitate was collected by filtration, washed with ethyl acetate (2x), and dried in a vacuum to obtain 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.

[1270] (9H-fluoren-9-yl)methyl N-({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazepine[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]Tetracosyl-1,6(11),12,14,16,18,20(24)-hepten-23-yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamate (5a)

[1271]

[1272] 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.

[1273] (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-diazepine[14.7.1.02 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]Tetracosyl-1,6(11),12,14,16,18,20(24)-hepten-23-yl]carbamoyl}methoxy)methyl]carbamoyl}ethyl]carbamate (5b)

[1274]

[1275] 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 to 100% acetonitrile in aqueous TFA (0.05%)). ESI m / z: 946 (M+H) + .

[1276] (4S)-4-{[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazepine[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 Benzyl 1,6(11),12,14,16,18,20(24)-hepten-23-yl]carbamoyl}methoxy)methyl]carbamoyl}-4-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)butanoate (5c)

[1277]

[1278] 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 to 70% acetonitrile in aqueous formic acid (0.1%)). ESI m / z: 964 (M+H) + .

[1279] (4S)-4-{[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazepine[14.7.1.0 2 , 14 .0 4 , 13 .0 6 ,11 .0 20 , 24 ]Tetracosyl-1,6(11),12,14,16,18,20(24)-hepten-23-yl]carbamoyl}methoxy)methyl]carbamoyl}-4-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)butanoic acid (5ca)

[1280]

[1281] To a stirred solution of compound 5a (0.21 g, 0.22 mmol) in methanol (20 mL) was added palladium carbon (36 mg, containing 10% palladium) under nitrogen protection. The reaction mixture was stirred at room temperature for 4 hours under a hydrogen atmosphere and monitored by LCMS. The mixture was filtered through celite and the filtrate was concentrated in vacuo to give a 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) + .

[1282] (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-diazepine[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]Tetracosyl-1,6(11),12,14,16,18,20(24)-hepten-23-yl]carbamoyl}methoxy)methyl]carbamoyl}propyl]carbamate (5cb)

[1283]

[1284] To a solution of compound 5ca (50 mg, 57 μmol) in DMF (1 mL) was added ammonium chloride (3.0 mg, 57 μmol), HATU (32 mg, 85 μmol) and DIPEA (22 mg, 0.17 mmol), and the reaction mixture was stirred at room temperature for 3 hours, monitored by LCMS. The resulting mixture was directly purified by reverse phase flash chromatography (0 to 100% acetonitrile in aqueous formic acid (0.1%)) to afford compound 5cb (40 mg, 81% yield) as a white solid. ESI m / z: 873 (M+H) + .

[1285] (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-diazepine[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]Tetracosyl-1,6(11),12,14,16,18,20(24)-hepten-23-yl]carbamoyl}methoxy)methyl]carbamoyl}pentyl]carbamate (5d)

[1286]

[1287] Following the general procedure, compound 5d (85 mg, 91% yield) was obtained as a white solid after purification by preparative HPLC (5% to 95% acetonitrile in aqueous TFA (0.1%)). ESI m / z: 900 (M+H) + .

[1288] (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-diazepine[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]Tetracosyl-1,6(11),12,14,16,18,20(24)-hepten-23-yl]carbamoyl}methoxy)methyl]carbamoyl}pentyl]carbamate (5da)

[1289]

[1290] Palladium carbon (10 mg, containing 10% palladium) was added to a stirred solution of compound 5d (45 mg, 50 μmol) in methanol (20 mL) under nitrogen protection. The reaction mixture was stirred at room temperature for 2 hours under a hydrogen atmosphere, monitored by LCMS. The mixture was filtered through diatomaceous earth, and the filtrate was concentrated in a vacuum and the residue was purified by preparative HPLC (5% to 95% acetonitrile in aqueous TFA (0.03%)) to obtain compound 5da (38 mg, 86% yield) as a yellow solid. ESI m / z:873 (M+H) + .

[1291] (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-diazepine[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]Tetracosyl-1,6(11),12,14,16,18,20(24)-hepten-23-yl]carbamoyl}methoxy)methyl]carbamoyl}-2-phenylethyl]carbamate (5e)

[1292]

[1293] Following the general procedure, compound 5e (24 mg, 64% yield) was obtained as a white solid after purification by preparative HPLC (5% to 95% acetonitrile in aqueous TFA (0.1%)). ESI m / z: 892 (M+H) + .

[1294] (9H-fluoren-9-yl)methyl N-({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazepine[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]Tetracosyl-1,6(11),12,14,16,18,20(24)-hepten-23-yl]carbamoyl}methoxy)methyl](methyl)carbamoyl}methyl)carbamate (5f)

[1295]

[1296] Following the general procedure, compound 5f (50 mg, 61% yield) was obtained as a white solid after purification by preparative HPLC (5% to 95% acetonitrile in aqueous TFA (0.01%)). ESI m / z: 816 (M+H) + .

[1297] (9H-fluoren-9-yl)methyl N-(1-{[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazepine[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]tetracosyl-1,6(11),12,14,16,18,20(24)-hepten-23-yl]carbamoyl}methoxy)methyl]carbamoyl}-1-methylethyl)carbamate (5 g)

[1298]

[1299] 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 to 70% acetonitrile in aqueous TFA (0.01%)). ESI m / z: 830 (M+H) + .

[1300] (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-diazepine[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]Tetracosyl-1,6(11),12,14,16,18,20(24)-hepten-23-yl]carbamoyl}methoxy)methyl]carbamoyl}-2-phenylethyl]carbamate (5h)

[1301]

[1302] Following the general procedure, compound 5h (92 mg, 73% yield) was obtained as a yellow solid after purification by preparative HPLC (5% to 95% acetonitrile in aqueous TFA (0.1%)). ESI m / z: 892 (M+H) + .

[1303] Payload (ProDXd)

[1304] General procedure for obtaining de-Fmoc of payload (ProDXd)

[1305] To the solution of compound 5 (1.0 equivalent) in THF (20mL / gram 5), diethylamine (2mL / gram 5) is added, and reaction mixture is stirred at room temperature for 2 to 48 hours, until Fmoc is completely removed according to LCMS. Volatiles are completely removed in a vacuum and residue is diluted with water (5mL). Aqueous mixture is adjusted to pH 2 and washed with TBE (20mLx2) with the addition of aqueous TFA (10%). Then water layer is stirred at room temperature for 16 hours, until the open-ring form is monitored by LCMS and becomes lactone form. Gained aqueous mixture is freeze-dried, to obtain thick payload, it is passed through reversed-phase flash chromatography (0 to 100% acetonitrile in aqueous TFA (0.03%)) purification, to obtain the pure payload in solid, or by preparative HPLC (5% to 95% acetonitrile in aqueous formic acid (0.1%)) purification, to obtain the pure payload (free alkali) in solid.

[1306] P1

[1307] 2-amino-N-[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazepine[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]Tetracosyl-1,6(11),12,14,16,18,20(24)-hepten-23-yl]carbamoyl}methoxy)methyl]acetamide (P1)

[1308]

[1309] Following the general procedure, P1 (1.4 g, 77% yield) was obtained as a light yellow solid. ESI m / z: 580.3 (M+H) + . 1 HNMR (400 MHz, DMSOd6 )δ9.20(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.

[1310] P2

[1311] (2S)-2-amino-N-[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazepine[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]Tetracosyl-1,6(11),12,14,16,18,20(24)-hepten-23-yl]carbamoyl}methoxy)methyl]-3-hydroxypropionamide (P2)

[1312]

[1313] 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 hours until complete removal of Fmoc was monitored by LCMS. The resulting mixture was directly purified by reverse phase flash chromatography (0 to 100% acetonitrile in aqueous TFA (0.05%)) to give the de-Fmoc-product as a yellow solid (68 mg, ESI m / z: 724 (M+H) +) was dissolved in DMF (1 mL). Cesium fluoride (31 mg, 0.20 mmol) was added to the solution at 0°C. The mixture was stirred at room temperature for one hour and monitored by LCMS. The mixture was separated by reverse phase flash chromatography (0 to 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 )δ9.26(t,J=6.4Hz,1H),8.58(d,J=8.8Hz,1H),8.14(s,2H),7.79(d,J=10.8Hz,1H),7. 33(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.

[1314] P3

[1315] (2S)-2-amino-N-[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazepine[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]Tetracosyl-1,6(11),12,14,16,18,20(24)-hepten-23-yl]carbamoyl}methoxy)methyl]glutaramide (P3)

[1316]

[1317] Following the general procedure, P3 (20 mg, 49% yield) was obtained as a light yellow solid. ESI m / z: 651 (M+H) + .1 HNMR (400 MHz, DMSO d6 )δ8.95-8.80(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. 29-7.20(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.

[1318] P4

[1319] (4S)-4-amino-4-{[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazepine[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]Tetracosyl-1,6(11),12,14,16,18,20(24)-hepten-23-yl]carbamoyl}methoxy)methyl]carbamoyl}butanoic acid (P4)

[1320]

[1321] Following the general procedure, P4 (20 mg, 49% yield) was obtained as a light yellow solid. ESI m / z: 652 (M+H) + . 1 HNMR (400 MHz, DMSO d6)δ8.95-8.82(m,1H),8.55(d,J=9.2Hz,1H),8.30(s,1H),7.79(d,J=11.2H z,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 )δ-111(Ar-F)ppm.

[1322] P5

[1323] (2S)-2,6-diamino-N-[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazepine[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]Tetracosyl-1,6(11),12,14,16,18,20(24)-hepten-23-yl]carbamoyl}methoxy)methyl]hexanamide (P5)

[1324]

[1325] 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)δ9.33(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),7.3 4(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. 01(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.

[1326] P6

[1327] (2S)-2-amino-6-azido-N-[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazepine[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]Tetracosyl-1,6(11),12,14,16,18,20(24)-hepten-23-yl]carbamoyl}methoxy)methyl]hexanamide (P6)

[1328]

[1329] 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)δ8.92-8.78(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,2 H), 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.

[1330] P7

[1331] (2S)-2-amino-N-[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazepine[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]Tetracosyl-1,6(11),12,14,16,18,20(24)-hepten-23-yl]carbamoyl}methoxy)methyl]-3-phenylpropionamide (P7)

[1332]

[1333] 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)δ8.86-8.77(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),5.4 5-5.34(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.

[1334] P9

[1335] 2-amino-N-[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazepine[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]Tetracosyl-1,6(11),12,14,16,18,20(24)-hepten-23-yl]carbamoyl}methoxy)methyl]-N-methylacetamide (P9)

[1336]

[1337] 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)δ8.70(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.

[1338] P11

[1339] 2-amino-N-[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazepine[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]Tetracosyl-1,6(11),12,14,16,18,20(24)-hepten-23-yl]carbamoyl}methoxy)methyl]-2-methylpropionamide (P11)

[1340]

[1341] Following the general procedure, P11 (15 mg, 62% yield) was obtained as a white solid. ESI m / z: 608 (M+H) + . 1 HNMR (400 MHz, DMSO d6)δ9.17-9.14(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=4.0 Hz,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.

[1342] P12

[1343] (2R)-2-amino-N-[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazepine[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]Tetracosyl-1,6(11),12,14,16,18,20(24)-hepten-23-yl]carbamoyl}methoxy)methyl]-3-phenylpropionamide (P12)

[1344]

[1345] 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)δ9.29-9.26(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.

[1346] (9H-fluoren-9-yl)methyl N-({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazepine[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]Tetracosyl-1,6(11),12,14,16,18,20(24)-hepten-23-yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamate (Fmoc-ProDXd)(5a)

[1347]

[1348] [1] Exitecan (1.0 eq.), HATU (1.1 eq.), DMF, 25 °C, 3 h, 86% yield (2.6 g);

[1349] [2] Piperidine in DMF (v / v=1 / 4), 25°C, 1 hour, 77% yield (1.4 g).

[1350] Exitecan is commercially available.

[1351] To the yellow solution of intermediate 4a (9.55g, 24.86mmol) in dry DMF (60mL), HATU (9.45g, 24.86mmol) and DIPEA (2.91g, 22.6mmol) were added, and the mixture was stirred at 25°C for 15 minutes. Then, a mixed solution of isotecan mesylate (12.0g, 22.6mmol) and DIPEA (5.82g, 45.2mmol) in dry DMF (60mL) was added to the reaction mixture. The reaction solution was stirred at 25°C for 4 hours until isotecan mesylate was consumed by LCMS monitoring. The resulting solution was diluted with ethyl acetate (0.90L) and washed with brine (180mLx2). The organic phase was dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was co-evaporated in vacuo with ethyl acetate (180mLx4), and the residue (50g) was dissolved in ethyl acetate (400mL). The suspension was refluxed for 20 minutes until it became clear. The solution was allowed to stand and a white solid was precipitated. The suspension was refluxed for another hour, and then naturally cooled to 25 ° C and allowed to stand for half an hour. The white precipitate was collected by filtration and dried in a vacuum to obtain compound Fmoc-proDxd (5a) (14.2 g, 78.3% yield, >99% purity) as a white solid. The filtrate was concentrated and purified by a C18 column to provide (1.6 g, 8.8% yield, 97% purity). ESI m / z: 802.2 (M+H) + . 1 HNMR (400 MHz, DMSO d6 ): δ8.79(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, 1H),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 FNMR (376 MHz, DMSO d6 )δ-111.33ppm.

[1352]

[1353]

[1354] Example 6. Exemplary Synthesis of ProDXd from DXd (Scheme 2B)

[1355] Synthesis of P1(CP1190) from DXd

[1356] 2-amino-N-[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazepine[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]Tetracosyl-1,6(11),12,14,16,18,20(24)-hepten-23-yl]carbamoyl}methoxy)methyl]acetamide (P1)

[1357]

[1358] To a solution of DXd (62 mg, 0.13 mmol) in THF (HPLC grade, 5 mL) were added compound 3a (0.23 g, 0.63 mmol) and Molecular sieves, and the mixture was stirred at room temperature for 5 minutes. Tf2NH (0.18 g, 0.63 mmol) was then added to the mixture and the reaction mixture was stirred at room temperature for 10 minutes. Although DXd was still present according to LCMS, the reaction was quenched by aqueous TFA (0.1%, 0.05 mL). The mixture was directly separated by reverse phase flash chromatography (0 to 100% acetonitrile in aqueous TFA (0.1%)) to obtain DXd (40 mg, 65% recovery yield) and 5a (Fmoc-P1) (31 mg, ESI m / z: 803 (M+H) + ) was dissolved in DMF (1 mL). Diethylamine (0.1 mL) was added to the 5a solution and the reaction mixture was stirred at room temperature for one hour until Fmoc was completely removed according to LCMS. The resulting mixture was directly purified by preparative HPLC (5% to 95% acetonitrile in aqueous TFA (0.1%)) to give P1 (15 mg, 17% yield, TFA salt) as a light yellow solid. ESI m / z: 580.3 (M+H) + .

[1359] Example 7. Exemplary Synthesis of Diamino Acid-ProDXd (Scheme 2C)

[1360] P8

[1361] 2-amino-N-({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazepine[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]Tetracosyl-1,6(11),12,14,16,18,20(24)-hepten-23-yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)acetamide (P8)

[1362]

[1363] To a solution of N-Fmoc-glycine (16.2 mg, 0.054 mmol) in DMF (1 mL) was added HATU (30.9 mg, 0.081 mmol) and DIPEA (21 mg, 0.108 mmol), and the reaction mixture was stirred at room temperature for 15 minutes. 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 one hour, monitored by LCMS. Diethylamine (1 mL) was then added to the resulting mixture, and the mixture was stirred at room temperature for one hour until Fmoc was completely removed according to LCMS. The volatiles were removed in vacuo and the residual mixture was directly separated by preparative HPLC (5% to 95% acetonitrile in aqueous TFA (0.1%)) to obtain P8 (11 mg, 31% yield, TFA salt) as a light yellow solid. ESI m / z: 637 (M+H) + . 1 H NMR (400 MHz, DMSO d6)δ8.88(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.

[1364] Example 8. Synthesis of 2-[2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)ethanesulfonamido]acetic acid (2i)

[1365]

[1366] To a stirred solution of glycine tert-butyl ester (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 mixture was stirred at room temperature for 2 hours, monitored by LCMS. The resulting mixture was directly purified by reverse phase flash chromatography (0 to 60% acetonitrile in aqueous TFA (0.1%)) to give a white solid (0.23 g, ESI m / z: 483 (M+Na) + ) was dissolved in DCM (10 mL). TFA (1 mL) was added to the solution, and the reaction mixture was stirred at room temperature for 8 hours, 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) + .

[1367] Linker-payload synthesis

[1368] Example 9. Exemplary Synthesis of Linker-Payload via Route 1 (Scheme 4A)

[1369] Synthesis of LP1 (M2980) from the reaction of exitecan with intermediate Fa

[1370] {4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadeca-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-diazepine[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]Tetracosyl-1,6(11),12,14,16,18,20(24)-hepten-23-yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamate (LP1)

[1371]

[1372] To a stirred solution of compound F (see Example 26) (48 mg, 49 μmol, 88% purity) in DMF (1 mL) was added HATU (20 mg, 54 μmol) and DIPEA (13 mg, 98 μmol) at 25° C., and the mixture was stirred at 25° C. for 15 minutes. To a mixture of exitecan mesylate (26 mg, 49 μmol) in DMF (0.8 mL) at 0° C. was added DIPEA (6.3 mg, 49 μmol), and the exitecan solution was stirred at 25° C. for 15 minutes. The two solutions were mixed at 25° C. and the mixture was stirred at 25° C. for 16 hours, monitored by LCMS. The resulting mixture was directly purified by reverse phase flash chromatography (5 to 95% acetonitrile in aqueous TFA (0.1%) over 60 minutes at a flow rate of 75 mL / min) to afford LP1 as a white solid (35 mg, 51% yield, 98% purity in HPLC). ESI m / z: 1396 (M+H) + . 1 H NMR (400 MHz, DMSO d6)δ10.00(s,1H),8.80(t,J=6.4Hz,1H),8.51(d,J=8.8Hz,1H),8.13(d,J=7.6Hz,1H),7.90(d,J=8.8Hz,1H),7.79(d,J=10.8Hz,1H),7.65-7.50(m,3H),7.43(t,J=6.0Hz,1H),7.31(s,1H),7.27(d,J=8.8Hz,2H),6.54(s,1H),5.98(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 )δ174.77,169.55,168.95,167.68,167.16,161.10,158.63,157.39,154.93,150.50,148.30,146.11,143.38,138.89,137.01,134.57,129.97,123.39,122.06,121.87,119.88,117.41,117.30,108.08,99.18,95.12,90.49,70.73,70.55,68.18,68.11,67.89,67.20,66.23,65.31,63.69,55.98,51.52,47.95,43.14,41.97,40.04,36.42,34.34,32.25,28.95,28.72,27.63,26.25,25.18,24.23,22.21,18.38,17.55,16.47,9.22,6.10ppm。 19 F NMR(376MHz,DMSO d6)δ-74.132(0.3F,CF3CO2H),-111.314(1F)ppm.

[1373] Example 10. Exemplary Synthesis of Linker-Payload via Route 2 (Scheme 4B)

[1374] Synthesis of LP1 (M2980) from the reaction of DXd with intermediate Ea

[1375] {4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadeca-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-diazepine[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]Tetracosyl-1,6(11),12,14,16,18,20(24)-hepten-23-yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamate (LP1)

[1376]

[1377] To a stirred mixture of intermediate Ea (see Example 25) (50 mg, 52 μmol) in dry THF (5 mL) were added DXd (26 mg, 52 μmol) and The mixture was stirred at 20°C for 5 minutes, and then trifluoromethanesulfonimide (73 mg, 0.25 mmol) was added. The reaction mixture was stirred at 20°C for 10 minutes until most of the intermediate Ea was consumed as monitored by LCMS. The mixture was removed by filtration. The residue was purified by reverse phase flash chromatography (5% to 95% acetonitrile in aqueous TFA (0.1%)) to give LP1 (29 mg, 39% yield) as a light yellow solid. ESI m / z: 699.1 (M / 2+H) + .

[1378] Example 11. Synthesis of Linker-Payload via Route 3a (Scheme 4C)

[1379] General procedure for Linker-ProDXd via route 3a.

[1380] To a solution of Intermediate D (1.0 to 1.2 eq) in DMF (0.15 mM) was added HOBt (0.5 eq) or HOAt (0.5 eq), DIPEA (3.0 eq) and payload (1.0 eq), and the reaction mixture was stirred at room temperature for 2 h, monitored by LCMS. The resulting mixture was directly purified by reverse phase flash chromatography to afford Linker-ProDXd as a white solid.

[1381] LP1

[1382] {4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadeca-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-diazepine[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]Tetracosyl-1,6(11),12,14,16,18,20(24)-hepten-23-yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamate (LP1)

[1383]

[1384] 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% to 60% acetonitrile in aqueous formic acid (0.1%)). ESI m / z: 699.0 (M / 2+H) + . 1 H NMR (400 MHz, DMSO d6)δ10.00(s,1H),8.80(t,J=6.4Hz,1H),8.51(d,J=8.8Hz,1H),8.13(d,J=7. 6Hz,1H),7.90(d,J=8.8Hz,1H),7.79(d,J=10.8Hz,1H),7.65-7.50(m,3H), 7.43(t,J=6.0Hz,1H),7.31(s,1H),7.27(d,J=8.8Hz,2H),6.54(s,1H),5.9 8(t,J=5.2Hz,1H),5.63-5.57(m,1H),5.41(s,4H),5.21(s,2H),4.92(s,2H ),4.62(d,J=6.4Hz,2H),4.43-4.33(m,1H),4.31-4.17(m,2H),4.01(s,2H) ,3.86(d,J=14.4Hz,1H),3.75(d,J=14.8Hz,1H),3.67-3.46(m,15H),3.44- 3.39(m,2H),3.27-3.10(m,4H),3.06-2.90(m,2H),2.47-2.32(m,5H),2.26 -1.64(m,14H),1.63-1.52(m,3H),1.47-1.32(m,3H),0.90-0.80(m,9H)ppm. 19 F NMR (376 MHz, DMSO d6 )δ-111ppm.

[1385] LP2

[1386] {4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadeca-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-diazepine[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]Tetracosyl-1,6(11),12,14,16,18,20(24)-hepten-23-yl]carbamoyl}methoxy)methyl]carbamoyl}-2-hydroxyethyl]carbamate (LP2)

[1387]

[1388] Following the general procedure starting with 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% to 95% acetonitrile in aqueous TFA (0.1%)). ESI m / z: 714 (M / 2+H) + . 1 H NMR (400 MHz, DMSO d6 )δ10.00(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,3 H),7.35-7.24(m,3H),7.18(d,J=8.2Hz,1H),6.59-6.46(m,1H),5.99(s, 1H),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.

[1389] LP3

[1390] {4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadeca-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-diazepine[14.7.1.0 2 , 14 .04 , 13 .0 6 , 11 .0 20 , 24 ]Tetracosyl-1,6(11),12,14,16,18,20(24)-hepten-23-yl]carbamoyl}methoxy)methyl]carbamoyl}propyl]carbamate (LP3)

[1391]

[1392] Following the general procedure starting with payload P3 (14 mg, 22 μmol) catalyzed by HOAt, linker-payload LP3 (16 mg, 49% yield) was obtained as a white solid after purification by preparative HPLC (5% to 95% acetonitrile in aqueous TFA (0.1%)). ESI m / z: 734 (M / 2+H) + . 1 H NMR (400 MHz, DMSO d6 )δ10.00(s,1H),8.83(t,J=6.5Hz,1H),8.54(d,J=8.6Hz,1H),8.14(d,J=7 .0Hz,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),4.95-4.8 1(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. 53-3.44(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.

[1393] LP4

[1394] (4S)-4-{[({4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadeca-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-diazepine[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]Tetracosyl-1,6(11),12,14,16,18,20(24)-hepten-23-yl]carbamoyl}methoxy)methyl]carbamoyl}butanoic acid (LP4)

[1395]

[1396] Following the general procedure starting with 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% to 95% acetonitrile in aqueous TFA (0.1%)). ESI m / z: 735 (M / 2+H) + . 1 H NMR (400 MHz, DMSO d6)δ9.99(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.

[1397] LP5

[1398] {4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadeca-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-diazepine[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]Tetracosyl-1,6(11),12,14,16,18,20(24)-hepten-23-yl]carbamoyl}methoxy)methyl]carbamoyl}pentyl]carbamate (LP5)

[1399]

[1400] Following the general procedure starting from compound 5da (32 mg, 37 μmol) and intermediate D catalyzed by HOBt, Fmoc-LP5 (35 mg, 56% yield) was obtained as a white solid after purification by preparative HPLC (5% to 95% acetonitrile in aqueous TFA (0.1%)).

[1401] 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 hours until Fmoc was completely removed according to LCMS. The resulting mixture was directly purified by reverse phase flash chromatography (5% to 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 )δ10.00(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),5.2 0(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.

[1402] LP5C

[1403] {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-tetraoxopentadec-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-diazepine[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]Tetracosyl-1,6(11),12,14,16,18,20(24)-hepten-23-yl]carbamoyl}methoxy)methyl]carbamoyl}pentyl]carbamate (LP5C)

[1404]

[1405] Following a similar procedure to LP5 except that intermediate Dd was used instead of intermediate Da, linker-payload LP5C (15 mg, 17% yield) was obtained as a red solid after purification by preparative HPLC (5% to 95% acetonitrile in aqueous TFA (0.1%)). ESI m / z: 758 (M / 2+H) + .

[1406] LP7

[1407] {4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadeca-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-diazepine[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24]Tetracosyl-1,6(11),12,14,16,18,20(24)-hepten-23-yl]carbamoyl}methoxy)methyl]carbamoyl}-2-phenylethyl]carbamate (LP7)

[1408]

[1409] Following the general procedure starting with 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% to 95% acetonitrile in aqueous TFA (0.1%)). ESI m / z: 744 (M / 2+H) + . 1 H NMR (400 MHz, DMSO d6 )δ9.98(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,1 H),5.47-5.34(m,3H),5.26-5.14(m,2H),4.86-4.75(m,2H),4.70-4.5 7(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.

[1410] LP8

[1411] {4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadeca-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-diazepine[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]Tetracosyl-1,6(11),12,14,16,18,20(24)-hepten-23-yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamoyl]methyl}carbamate (LP8)

[1412]

[1413] Following the general procedure starting with 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% to 95% acetonitrile in aqueous TFA (0.1%)). ESI m / z: 727 (M / 2+H) + . 1 H NMR (400 MHz, DMSO d6)δ9.99(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),7.47-7.4 3(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.

[1414] LP9

[1415] {4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadeca-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-diazepine[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]Tetracosyl-1,6(11),12,14,16,18,20(24)-hepten-23-yl]carbamoyl}methoxy)methyl](methyl)carbamoyl}methyl)carbamate (LP9)

[1416]

[1417] Following the general procedure starting with 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% to 95% acetonitrile in aqueous TFA (0.1%)). ESI m / z: 706 (M / 2+H) + . 1 H NMR (400 MHz, DMSO d6 )δ9.99(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.

[1418] Example 12. Exemplary Synthesis of Linker-Payload via Route 3b (Scheme 4D)

[1419] Synthesis of LP1 from Fmoc-P1(5a) by reaction with intermediate D using HOBt

[1420] {4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadeca-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-diazepine[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24]Tetracosyl-1,6(11),12,14,16,18,20(24)-hepten-23-yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamate (LP1)

[1421]

[1422] 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 minutes. HOBt (0.14 g, 1.0 mmol) and Intermediate D (2.0 g, 2.1 mmol) were added to the reaction mixture, and the resulting clear solution was stirred at 25°C for 16 hours. The resulting mixture was poured into MTBE (150 mL), and the heterogeneous mixture was stirred at room temperature for 5 minutes. The MTBE layer, which contained most of the Fmoc-ene byproduct and the base, was then separated. The black oil at the bottom was diluted with DMF (20 mL), and the solution was purified by preparative HPLC (5% to 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)δ10.00(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 (376 MHz, DMSO d6 )δ-111(Ar-F)ppm.

[1423] Synthesis of LP1 from Fmoc-P1(5a) and intermediate D using MeHYQ (small scale)

[1424] {4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadeca-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-diazepine[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24]Tetracosyl-1,6(11),12,14,16,18,20(24)-hepten-23-yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamate (LP1)

[1425]

[1426] To a yellow solution of 5a (10 mg, 1 eq), intermediate D (12.5 mg, 1.05 eq) 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 hours and monitored by LCMS. After cooling to room temperature, the resulting mixture was poured into stirred MTBE (600 uL) at 0 ° C to 10 ° C and a brown oil appeared, which was collected after separation and removal of the MTBE layer. The oil was then purified by reverse phase flash chromatography (5% to 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) + .

[1427] Synthesis of LP1 from Fmoc-P1(5a) by reaction with intermediate D using MeHYQ (large scale)

[1428] {4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadeca-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-diazepine[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]Tetracosyl-1,6(11),12,14,16,18,20(24)-hepten-23-yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamate (LP1)

[1429]

[1430] 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 hours and monitored by LCMS. After cooling to room temperature, the resulting mixture was poured into stirred MTBE (600 mL) at 0 ° C to 10 ° C and a brown oil appeared, which was collected after separation and removal of the MTBE layer. The oil was then purified by reverse phase flash chromatography (5% to 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) + .

[1431] 1 H NMR (400 MHz, DMSO d6 )δ10.00(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 (376 MHz, DMSO d6 )δ-111(Ar-F)ppm.

[1432] 13 C NMR (100 MHz, DMSO d6 )δ172.36,171.11,170.65,170.61,170.31,169.20,168.68,162.70,160.23,158.90,156.58,156.43,152.14,149.8 9,147.79,147.65,144.99,140.48,138.59,136.24,131.52,128.56,125.08,123.65,123.45,121.50,118.83,109.81 ,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.

[1433]

[1434]

[1435] Certificate of Analysis for LP1 (1g batch):

[1436] Chemical structure:

[1437]

[1438] Compound information result Molecular formula <![CDATA[C 69 H 90 FN 11 Oh 19 ]]> MW (with salt, solvent) 1396.54 Salt / active substance ratio N / A Appearance White solid LC-MS (accurate mass) <![CDATA[[M+H] + =1396.7]]> HPLC purity 99.99％ Chiral HPLC purity 99ee%

[1439] Certificate of Analysis for LP1 (13g batch)

[1440] Chemical structure:

[1441]

[1442]

[1443]

[1444] Analytical tests and results:

[1445] entry standard HPLC purity >98％ Chiral HPLC purity ee>98%

[1446] Example 13. Exemplary 2-step synthesis of linker-payload via Route 4 (Scheme 4E)

[1447] LP1 was synthesized from P1 by reaction with Fmoc-vcPAB and then with intermediate Ba

[1448] {4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadeca-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-diazepine[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 ]Tetracosyl-1,6(11),12,14,16,18,20(24)-hepten-23-yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamate (LP1)

[1449]

[1450] [Step 1] vcPAB-P1

[1451] To a solution of compound Fmoc-vcPAB-PNP (0.36 g, 0.47 mmol, 1.0 equiv., commercial) 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 hours, monitored by LCMS. The resulting mixture was directly purified by reverse phase flash chromatography to afford compound Fmoc-vcPAB-P1 (0.22 mg, ESI m / z: 1207 (M+H)) as a yellow solid. + ) was dissolved in DMF (2 mL). Diethylamine (0.2 mL) was added to the solution, and the reaction mixture was stirred at room temperature for 2 hours, monitored by LCMS. The resulting mixture was directly purified by reverse flash chromatography to give vcPAB-P1 (0.16 g, 28% yield from P1) as a white solid. ESI m / z: 1085 (M+H) + .

[1452] [Step 2]: LP1

[1453] 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) was added HATU (83 mg, 0.22 mmol, 1.5 equiv.) and DIPEA (58 mg, 0.45 mmol, 3.0 equiv.), and the reaction mixture was stirred at room temperature for one hour, 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 hours, 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) + .

[1454] LP1A was synthesized from the reaction of P1 with Fmoc-vcPAB and then with intermediate Bb

[1455] {4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-[1-({[(4E)-cyclooct-4-en-1-yloxy]carbonyl}amino)-3,6,9,12-tetraoxapentadeca-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-diazepine[14.7.1.0 2 , 14 .0 4 , 13 .0 6...

Claims

1. An antibody-drug conjugate comprising an antibody or an antigen-binding fragment thereof and a compound having formula (I) or a pharmaceutically acceptable salt thereof, where 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, and The point of attachment to the antibody or the antigen-binding fragment thereof, directly or via a linker, is indicated.

2. The antibody-drug conjugate according to claim 1, wherein the compound of formula (I) comprises 3. The antibody-drug conjugate according to claim 1, wherein the antibody or the antigen-binding fragment thereof is conjugated to a compound having a structure according to formula (II): or a pharmaceutically acceptable salt thereof, wherein R 1 、R 2 、R 3 and R 4 are independently hydrogen or C 1-5 alkyl; A is a click chemistry adduct; W is NH, O, CO, CH2, 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, and The point of attachment to the antibody or the antigen-binding fragment thereof, directly or via a linker, is indicated.

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 the group consisting of: (a) strain-promoted azide / dibenzocyclooctynamine (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 antibody-drug conjugate according to any one of claims 3 to 5, wherein the click chemistry adduct is selected from the group consisting of: and any regioisomer or enantiomer thereof, wherein R' is H or C 1-3 alkyl and Z is C or N.

7. The antibody-drug conjugate of any one of claims 3 to 6, wherein AA comprises a natural amino acid selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, threonine, lysine, asparagine, glutamine, aspartic acid, and glutamic acid.

8. The antibody-drug conjugate of any one of claims 3 to 6, wherein AA comprises an unnatural amino acid selected from the group consisting of: an R-amino acid, an N-methyl amino acid, 9. The antibody-drug conjugate according to any one of claims 3 to 6, wherein the compound of formula (II) comprises 10. An antibody-drug conjugate having a structure according to formula (III) or a pharmaceutically acceptable salt thereof, wherein Ab is an antibody or its antigen-binding fragment; R 1 、R 2 、R 3 and R 4 are independently hydrogen or C 1-5 alkyl; A is a click chemistry adduct; LL is a linker or bond connecting the Ab and the 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 from 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) (SEQ ID NOs 2115 and 2115, respectively) or (SEQ ID NOs 2116 and 2116, respectively), or a pharmaceutically acceptable salt thereof, wherein Ab is an antibody or its antigen-binding fragment; R is the side chain of any natural or unnatural amino acid; And n is an integer from 1 to 5.

12. The antibody-drug conjugate of claim 11, wherein the antibody or the antigen-binding fragment thereof comprises Gln295 and / or Gln297, and wherein the drug payload is conjugated to the antibody or antigen-binding fragment via the side chains of Gln295 and / or Gln297.

13. The antibody-drug conjugate of claims 1 to 12, wherein the antibody or the antigen-binding fragment thereof is selected from anti-HER2 antibodies, anti-STEAP2 antibodies, anti-MET antibodies, anti-EGFRVIII antibodies, anti-MUC16 antibodies, anti-PRLR antibodies, anti-PSMA antibodies, anti-FGFR2 antibodies, anti-FOLR1 antibodies, anti-HER2 / HER2 bispecific antibodies, anti-MET / MET bispecific antibodies, or antigen-binding fragments 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 antibody-drug conjugate according to any one of claims 13 or 14, wherein the anti-HER2 / HER2 bispecific antibody comprises: a first antigen binding domain (D1); and a second antigen-binding domain (D2); wherein D1 specifically binds to the first epitope of human HER2; and Among them, D2 specifically binds to the second epitope of human HER2.

16. The antibody-drug conjugate of any one of claims 1 to 15, wherein the antibody and linker-drug payload are site-specifically conjugated 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 according to any one of claims 1 to 17, formulated together with one or more pharmaceutically acceptable diluents, excipients and / or additives.

19. A composition comprising a population of the antibody-drug conjugate of any one of claims 1 to 17, said composition having a drug-antibody ratio (DAR) of about 0.5 to about 30.

0.

20. The composition of claim 19, having a DAR of about 1.0 to about 2.

5.

21. The composition of claim 20, having a DAR of about 2.

22. The composition of claim 19, having a DAR of about 3.0 to about 4.

5.

23. The composition of claim 22, having a DAR of about 4.

24. The composition of claim 19, having a DAR of about 6.5 to about 8.

5.

25. The composition of claim 24, having a DAR of about 8.

26. A method for treating cancer in a subject in need thereof, the method comprising the steps of: A therapeutically effective amount of the antibody-drug conjugate according to any one of claims 1 to 17, or the pharmaceutical composition according to claim 18, is administered to the subject.

27. A method for producing a linker-payload compound having a formula selected from the group consisting of (D') to (N'): or a pharmaceutically acceptable salt thereof, where R 1 、R 2 、R 3 and R 4 are independently hydrogen or C 1-5 alkyl; B is selected from the group consisting of: W is NH, O, CO, CH2, phenyl, or a combination of two or more thereof; and R 5 、R 6 、R 7 and R 8 is independently hydrogen, -NH2 or a side chain of any natural or unnatural amino acid, the method comprising the steps of: A payload having an amino group is exposed to an activated intermediate having p-nitrophenyl carbonate in the presence of a base and a coupling catalyst, wherein the coupling catalyst is 4-hydroxy-2-methylquinoline (MeHYQ), to give the linker-payload compounds (D') to (G').

28. A method for producing a linker-payload compound having formula (D-1) (D-1), or a pharmaceutically acceptable salt thereof, where R 1 、R 2 、R 3 and R 4 are independently hydrogen or C 1-5 alkyl, and R 5 and R 6 is independently hydrogen, -NH2 or the side chain of any natural or unnatural amino acid, The method comprises the following steps: A drug payload having an amino group is exposed to an activated intermediate having p-nitrophenyl carbonate in the presence of a base and a coupling catalyst, wherein the coupling catalyst is 4-hydroxy-2-methylquinoline (MeHYQ), to give the linker-payload compound (D).

29. A linker-payload compound having formula (D), (D), or a pharmaceutically acceptable salt thereof, where R 1 、R 2 、R 3 and R 4 are independently hydrogen or C 1-5 alkyl, and R 5 and R 6 is independently hydrogen, -NH2, or the side chain of any natural or unnatural amino acid.

30. A linker-payload compound having a formula selected from the group consisting of (D') to (N') below: or a pharmaceutically acceptable salt thereof, where R 1 、R 2 、R 3 and R 4 are independently hydrogen or C 1-5 alkyl; B is selected from the group consisting of: W is NH, O, CO, CH2, phenyl, or a combination of two or more thereof; and R 5 、R 6 、R 7 and R 8 is independently hydrogen, -NH2 or the side chain of any natural or unnatural amino acid, The method comprises the following steps: A payload having an amino group is exposed to an activated intermediate having p-nitrophenyl carbonate in the presence of a base and a coupling catalyst, wherein the coupling catalyst is 4-hydroxy-2-methylquinoline (MeHYQ), to give the linker-payload compounds (D') to (G').

31. The linker-payload compound of claim 30, having a structure selected from the group consisting of:

32. A method for preparing a compound of formula (D-1): or a pharmaceutically acceptable salt thereof, where R 1 、R 2 、R 3 and R 4 are independently hydrogen or C 1-5 alkyl, and R 5 and R 6 is independently hydrogen, -NH2 or the side chain of any natural or unnatural amino acid, The method comprises: (a) providing a compound of formula (I-1) having the following structure: in X is selected from the group consisting of: as well as (b) reacting the compound of formula (I-1) with the compound of formula (PI): in R is H or PG; and PG is a suitable protecting group; reaction to produce the compound of formula (D-1).

33. The method of claim 32, wherein the compound of formula (D-1) has the following structure:

34. The method of claim 32, wherein the step (b) of reacting the compound of formula (I-1) with the compound of formula (PI) further comprises: Prior to said reaction with said compound of formula (I-1), said compound of formula (PI), wherein R is PG, is reacted with a protecting group removing agent.

35. The method of claim 32, wherein the PG is selected from the group consisting of allyloxycarbonyl (Alloc), benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), and 9-fluorenylmethoxycarbonyl (Fmoc).

36. The method of claim 32, wherein the compound of formula (I-1) has the following structure:

37. The method of claim 32, wherein the compound of formula (PI) has the following structure:

38. The method according to claim 32, further comprising the steps of: Provided are compounds of formula (V) having the following structure: as well as The compound of formula (I-1) is formed from the compound of formula (V) before step (a).

39. The method of claim 38, wherein the step of forming the compound of formula (I-1) comprises: The compound of formula (V) is reacted with a compound of formula (VIa) or formula (VIb): wherein X′ is a halogen, reaction to produce the compound of formula (I-1).

40. The method according to claim 38, further comprising the steps of: Provided is a compound of formula (VII) having the following structure: Among them PG 1 is a suitable protecting group, and The compound of formula (V) is formed from the compound of formula (VII).

41. The method of claim 40, wherein the compound of formula (VII) has the following structure:

42. The method of claim 40, wherein the step of forming the compound of formula (V) comprises: The compound of formula (VII) and the compound of formula (VIII): reaction to produce the compound of formula (V).

43. The method according to claim 40, further comprising the steps of: Provided are compounds of formula (IX) having the following structure: as well as The compound of formula (VII) is formed from the compound of formula (IX).

44. The method of claim 43, wherein the compound of formula (IX) has the following structure:

45. The method of claim 43, wherein the step of forming the compound of formula (VII) comprises: The compound of formula (IX) and the compound of formula (X): react to produce the compound of formula (VII).

46. ​​The method of claim 43, further comprising the steps of: Provided are compounds of formula (XI) having the following structure: as well as The compound of formula (IX) is formed from the compound of formula (XI).

47. The method of claim 46, wherein the compound of formula (XI) has the following structure:

48. The method of claim 46, wherein the step of forming the compound of formula (IX) comprises: The compound of formula (XI) and the compound of formula (XII): react to produce the compound of formula (IX).

49. The method of claim 42, further comprising the steps of: Provided are compounds of formula (XIII) having the following structure: as well as The compound of formula (VIII) is formed from the compound of formula (XIII).

50. The method of claim 49, wherein the step of forming the compound of formula (VIII) comprises: The compound of formula (XIII) and the compound of formula (XII): reaction to produce the compound of formula (VIII).

51. The method of claim 49, further comprising the steps of: Provided is a compound of formula (XIV) having the following structure: where R a It is a halogen; R b It is C 1-6 Alkyl; and The compound of formula (XIII) is formed from the compound of formula (XIV).

52. The method of claim 51, wherein the compound of formula (XIV) has the following structure:

53. The method of claim 51, wherein the step of forming the compound of formula (XIII) comprises: The compound of formula (XIV) is reacted with a base to produce the compound of formula (XIII).

54. The method of claim 53, wherein the base is selected from the group consisting of NaOMe, t-BuOK, NaH, and LDA.

55. The method of claim 51 , further comprising the steps of: Provided are compounds of formula (XV) having the following structure: as well as The compound of formula (XIV) is formed from the compound of formula (XV).

56. The method of claim 55, wherein the compound of formula (XV) has the following structure:

57. The method of claim 55, wherein the step of forming the compound of formula (XIV) comprises: The compound of formula (XV) and the compound of formula (XVI): react to produce the compound of formula (XIV).

58. The method of claim 55, further comprising the steps of: Provided is a compound of formula (XVII) having the following structure: as well as The compound of formula (XV) is formed from the compound of formula (XVII).

59. The method of claim 58, wherein the step of forming the compound of formula (XV) comprises: The compound of formula (XVII) is reacted with a brominating agent to produce the compound of formula (XVII).

60. The process of claim 59, wherein the brominating agent is CHBr3.

61. The method of claim 32, further comprising the steps of: Provided are compounds of formula (XVIII) having the following structure: as well as The compound of formula (PI) is formed from the compound of formula (XVIII).

62. The method of claim 61, wherein the compound of formula (XVIII) has the following structure:

63. The method of claim 61, wherein the step of forming the compound of formula (PI) comprises: The compound of formula (XVIII) is reacted with the compound of formula (XIX): react to produce the compound of formula (PI).

64. The method of claim 61, further comprising the steps of: Provided are compounds of formula (XX) having the following structure: as well as The compound of formula (XVIII) is formed from the compound of formula (XX).

65. The method of claim 64, wherein the compound of formula (XX) has the following structure: g。 66. The method of claim 64, wherein the step of forming the compound of formula (XVIII) comprises: The compound of formula (XX) and the compound of formula (XXI): react to produce the compound of formula (XVIII).

67. The method of claim 64, further comprising the steps of: Provided is a compound of formula (XXII) having the following structure: as well as The compound of formula (XX) is formed from the compound of formula (XXII).

68. The method of claim 67, wherein the compound of formula (XXII) has the structure:

69. A method for preparing a compound of formula (I-1): or a pharmaceutically acceptable salt thereof, in X is selected from the group consisting of: The method comprises: (a) providing a compound of formula (V) having the following structure: as well as (b) forming the compound of formula (I-1) from the compound of formula (V).

70. The method of claim 69, wherein step (b) of forming the compound of formula (I-1) comprises: The compound of formula (V) is reacted with a compound of formula (VIa) or formula (VIb): in X' is a halogen, reaction to produce the compound of formula (I-1).

71. The method of claim 69, further comprising the steps of: Provided is a compound of formula (VII) having the following structure: Among them PG 1 is a suitable protecting group protecting group, and The compound of formula (V) is formed from the compound of formula (VII).

72. The method of claim 71, wherein the compound of formula (VII) has the structure:

73. The method of claim 71, wherein the step of forming the compound of formula (V) comprises: The compound of formula (VII) and the compound of formula (VIII): reaction to produce the compound of formula (V).

74. The method of claim 71 , further comprising the steps of: Provided are compounds of formula (IX) having the following structure: as well as The compound of formula (VII) is formed from the compound of formula (IX).

75. The method of claim 74, wherein the compound of formula (IX) has the structure:

76. The method of claim 74, wherein the step of forming the compound of formula (VII) comprises: The compound of formula (IX) and the compound of formula (X): react to produce the compound of formula (VII).

77. The method of claim 74, further comprising the steps of: Provided are compounds of formula (XI) having the following structure: as well as The compound of formula (IX) is formed from the compound of formula (XI).

78. The method of claim 77, wherein the compound of formula (XI) has the structure:

79. The method of claim 77, wherein the step of forming the compound of formula (IX) comprises: The compound of formula (XI) and the compound of formula (XII): reaction to produce the compound of formula (IX).

80. A compound of formula (I-1): or a pharmaceutically acceptable salt thereof, in X is selected from the group consisting of:

81. A method for preparing a compound of formula (XVIII): or a pharmaceutically acceptable salt thereof, where R 1 、R 2 、R 3 and R 4 are independently hydrogen or C 1-5 alkyl, and R 5 and R 6 is independently hydrogen, -NH2 or the side chain of any natural or unnatural amino acid, The method comprises: (a) providing a compound of formula (XX) having the following structure: as well as (b) forming the compound of formula (XVIII) from the compound of formula (XX).

82. The method of claim 81, wherein the compound of formula (XVIII) has the following structure:

83. The method of claim 81, wherein the compound of formula (XX) has the following structure:

84. The method of claim 81, wherein the step of forming the compound of formula (XVIII) comprises: The compound of formula (XX) and the compound of formula (XXI): reaction to produce the compound of formula (XVIII).

85. The method of claim 81 , further comprising the steps of: Provided is a compound of formula (XXII) having the following structure: as well as The compound of formula (XX) is formed from the compound of formula (XXII).

86. The method of claim 85, wherein the compound of formula (XXII) has the structure:

87. A compound of formula (XVIII): or a pharmaceutically acceptable salt thereof, where R 1 、R 2 、R 3 and R 4 are independently hydrogen or C 1-5 alkyl, and R 5 and R 6 is independently hydrogen, -NH2, or the side chain of any natural or unnatural amino acid.

88. The compound of claim 87, wherein the compound has the structure:

89. A method for preparing a compound of formula (D-1): or a pharmaceutically acceptable salt thereof, where R 1 、R 2 、R 3 and R 4 are independently hydrogen or C 1-5 alkyl, and R 5 and R 6 is independently hydrogen, -NH2 or the side chain of any natural or unnatural amino acid, The method comprises: (a) providing a compound of formula (I-1) having the following structure: in X is selected from the group consisting of: as well as (b) reacting the compound of formula (I-1) with the compound of formula (PI): in R is H or PG; and PG is a suitable protecting group, reaction to produce the compound of formula (D-1).

90. The method of claim 89, wherein the compound of formula (D-1) has the following structure:

91. The method of claim 89, wherein the step (b) of reacting the compound of formula (I-1) with the compound of formula (PI) further comprises: Prior to said reaction with said compound of formula (I-1), said compound of formula (PI), wherein R is PG, is reacted with a protecting group removing agent.

92. The method of claim 89, wherein the PG is selected from the group consisting of allyloxycarbonyl (Alloc), benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), and 9-fluorenylmethoxycarbonyl (Fmoc).

93. The method of claim 91, wherein the protecting group removing agent is selected from the group consisting of Pd(PPh)3, PhSiH3, H2, piperidine and trifluoroacetic acid (TFA).

94. The method of claim 89, wherein the compound of formula (PI) has the structure:

95. The method of claim 89, further comprising the steps of: Provided are compounds of formula (XVIII) having the following structure: as well as The compound of formula (PI) is formed from the compound of formula (XVIII).

96. The method of claim 95, wherein the compound of formula (XVIII) has the structure:

97. The method of claim 95, wherein the step of forming the compound of formula (PI) comprises: The compound of formula (XVIII) is reacted with the compound of formula (XIX): react to produce the compound of formula (PI).

98. A method for preparing a compound of formula (D-1): or a pharmaceutically acceptable salt thereof, where R 1 、R 2 、R 3 and R 4 are independently hydrogen or C 1-5 alkyl, and R 5 and R 6 is independently hydrogen, -NH2 or the side chain of any natural or unnatural amino acid, The method comprises: (a) providing a compound of formula (XXIII): as well as (b) reacting the compound of formula (XXIII) with a compound having the following structure: The reaction is carried out in the presence of an activating agent and a base to produce the compound of formula (D-1).

99. The method of claim 98, wherein the compound of formula (D-1) has the following structure:

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