Tripeptide linkers and methods of use thereof

Abstract

Provided herein are peptide linkers that can be used to prepare drug conjugates, drug conjugates prepared using these linkers, and compositions and methods of treatment thereof.

Description

[Technical field]

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 291,918, filed December 20, 2021, the entire contents of which are incorporated herein by reference.

[0002] I. Field of the Invention The present disclosure relates to the fields of medicine, pharmacology, chemistry, and oncology. In particular, compounds, compositions, diagnostic methods, treatment methods, and synthetic methods related to antibody-drug conjugates (ADCs) are disclosed. [Background technology]

[0003] II. Description of Related Art Targeted drug delivery has received increasing attention as a means to improve drug efficacy while reducing toxicity to healthy tissues. In particular, antibody-drug conjugates (ADCs), which are monoclonal antibodies (mAbs) linked to pharmacologically active molecules (payloads) via chemical linkers, are one of the most promising classes for the treatment of cancer and other diseases, with remarkable and sustained therapeutic effects. The clinical success of this drug class has been demonstrated with 11 U.S. Food and Drug Administration (FDA)-approved ADCs for a wide range of hematological malignancies and solid tumors, as well as over 100 candidates in clinical trials. Despite recent advances in ADC chemistry (e.g., novel linkers, payloads, conjugation methods), cancer biology, and clinical management, ADC-based treatments are often associated with a variety of side effects, including myelosuppression and hepatotoxicity. Therefore, ADC technologies that can minimize the risk of adverse effects are highly desirable in order to implement effective cancer treatments without compromising the quality of life of patients.

[0004] ADC linkers are key components that influence overall efficacy and safety profiles. Cleavable linkers are used for approximately 80% of ADCs to efficiently liberate conjugated payloads in target cancer cells and enhance ADC potency. Among them, cathepsin-sensitive valine-citrulline (VCit) and similar dipeptide linkers that link payloads with or without p-aminobenzyloxycarbonyl (PABC) are the most commonly used industry-standard technology for over 40 ADCs, including ADCETRIS®, POLIVY®, PADCEV®, and ZYNLONTA® (Figure 1A). However, their sensitivity to extracellular carboxylesterase 1c (Ces1c) in rodent plasma causes premature payload release in the circulation, complicating preclinical evaluation using rodent models. More importantly, the instability of this valine-based linker is likely associated with a high frequency of dose-limiting antigen-independent toxicity in clinical applications. In Phase II and III trials of ADCETRIS®, an ADC loaded with monomethylauristatin E (MMAE) via a VCit linker, neutropenia (16-22% of patients) or hepatotoxicity (7% of patients) were common side effects leading to dosing delays or treatment discontinuation. Thus, there is an unmet need to develop improved chemical linkers, such as for ADCs. Summary of the Invention

[0005] overview In some aspects, the present disclosure provides peptide linkers that can be used to prepare drug conjugates, drug conjugates prepared using these linkers, and compositions and methods of treatment thereof.

[0006] In some aspects, the present disclosure provides a compound of the formula: TIFF2025500942000001.tif26128, or a pharma- ceutically acceptable salt thereof; During the ceremony: X1 is a covalent bond, alkanediyl (C≦12) , or substituted alkanediyl (C≦12) and; R1 is hydrogen, -ZR6, -(OCH2CH2) n ZR6, or substituted -(OCH2CH2) n ZR6, where: n is 0 to 50; and R6 is hydrogen, hydroxy, aminohydroxy, amino, mercapto, hydroxylamino, hydrazino, or azido; or Alkyl (C≦12) , alkenyl (C≦12) , alkynyl (C≦12) , alkyl hydrazine (C≦12) or substituted versions of any of these groups; Polyglycine containing 1 to 6 glycine units; or formula: Substructure of TIFF2025500942000002.tif49143 (In the formula: A1 and A2 are each independently absent or arenediyl. (C≦12) , substituted arenediyl (C≦12) , Heteroarenediyl (C≦12) , or substituted heteroarenediyl (C≦12) and a fused arene (C≦12) , substituted arenes (C≦12) , Heteroarenes (C≦12) , or substituted heteroarenes (C≦12) Forming; A3 is a covalent bond, O, alkanediyl (C≦8) , substituted alkanediyl (C≦8) , alkoxydiyl (C≦8) , substituted alkoxydiyl (C≦8) and A4 and A5 each independently represent a covalent bond, an alkanediyl (C≦8) , substituted alkanediyl (C≦8) , Arendjiil (C≦8) , or substituted arenediyl (C≦8) More selected; R d , Re , R e ′, and R h each independently represents hydrogen, halo, thioether, selenoether, sulfate, tosylate, mesylate, aryl (C≦8) , or substituted aryl (C≦8) More selected; R f is a halo; R g amine, hydrazine, alkylamino (C≦8) , substituted alkylamino (C≦8) , dialkylamino (C≦8) , substituted dialkylamino (C≦8) , alkyl hydrazine (C≦8) or a substituted alkylhydrazine (C≦8) and; X4 and X5 are each independently O, N, C(O), CH2, or X4 and X5 are alkanediyl. (C≦8) or substituted alkanediyl (C≦8) and taken together form a fused cycloalkane group of 3 to 8 ring atoms; R7 is hydrogen, hydroxy, amino, or oxo; R8 is carboxy; or Alkyl (C≦12) , amide (C≦12) , aryl (C≦12) , Heteroaryl (C≦12) , -C(O)OR 11 , -C(O)NR 11 R 11 ', or a substituted version of any of these groups, where: R 11 and R 11 ' are each independently hydrogen; or Alkyl (C≦12) , aryl (C≦12) or a substituted form of either of these groups; R9 and R 10 are each independently hydroxy, amino, halo; or Alkyl (C≦12) , aryl (C≦12)or one of these groups is substituted) and; Z is a covalent bond, alkanediyl (C≦12) , -C(O)-alkanediyl (C≦12) , -C(O)-alkanediyl (C≦12) -C(O)NH-, or a substituted form of any of these groups; R2 is hydrogen, alkyl (C≦12) , substituted alkyl (C≦12) , aryl (C≦12) , Heteroaryl (C≦12) , Aralkyl (C≦12) , heteroaralkyl (C≦12) , Acyl (C≦12) , substituted acyl (C≦12) or a monovalent amino protecting group; W is a covalent bond or a multivalent polymer having 2 to 21 linkage points; n is 1 to 20, except that when W is a covalent bond, n is 1, and when W is a multivalent polymer, n is less than or equal to one less than the number of attachment points; Each X is independently a covalent bond, an alkanediyl (C≦12) , substituted alkanediyl (C≦12) , one or more amino acid residues, or an oligomeric peptide; Each X2 is independently an alkanediyl (C≦12) or substituted alkanediyl (C≦12) and; Each R3 is independently hydroxy, or amino; or Alkoxy (C≦12) , acyloxy (C≦12) , alkylamino (C≦12) , dialkylamino (C≦12) , amide (C≦12) , Heteroaryl (C≦12) or a substituted version of any of these groups; or -X6-C(O)R 12 (where: X6 is O, -NR b - or covalently bonded; R b is hydrogen, alkyl(C≦6) , substituted alkyl (C≦6) or a monovalent amino protecting group; R 12 is hydroxy or amino; or Alkoxy (C≦12) , alkylamino (C≦12) , dialkylamino (C≦12) or a substituted form of any of these groups; or -A3SO2NR 13 R 13 ′, -A3P(O)(OH)OR 14 , or -A3SO2OR 14 ′ (where: A3 is O, -NR c - or covalently bonded; R c is hydrogen, alkyl (C≦6) , substituted alkyl (C≦6) , aryl (C≦12) , Heteroaryl (C≦12) , Aralkyl (C≦12) , heteroaralkyl (C≦12) or a monovalent amino protecting group; R 13 , R 13 ′, R 14 , and R 14 ' are each independently hydrogen, alkyl, (C≦12) , cycloalkyl (C≦12) , aryl (C≦12) , Heteroaryl (C≦12) , Aralkyl (C≦12) , heteroaralkyl (C≦12) or a substituted version of any of these groups. and; m is 0 or 1; each R4 is independently the side chain moiety of glycine or valine; and Each R4' is hydrogen, alkyl, (C≦12) , substituted alkyl (C≦12) , aryl (C≦12) , Heteroaryl (C≦12) , Aralkyl (C≦12) , heteroaralkyl (C≦12) , Acyl (C≦12) , substituted acyl(C≦12) or a monovalent amino protecting group; each R5 is independently a side chain moiety of glycine, alanine, ornithine, lysine, arginine, citrulline, asparagine, glutamine, or an amino-protected form thereof; Each R5' is hydrogen, alkyl, (C≦12) , substituted alkyl (C≦12) , aryl (C≦12) , Heteroaryl (C≦12) , Aralkyl (C≦12) , heteroaralkyl (C≦12) , Acyl (C≦12) , substituted acyl (C≦12) or a monovalent amino protecting group; Each Q independently represents a group of the formula: Based on TIFF2025500942000003.tif7128, During the ceremony: Each R 15 are independently hydrogen, -R 16 , or -C(O)-R 16 where: R 16 is a therapeutic or imaging agent; Each X7 is independently a covalent bond, O, S, -NH-, or an alkanediyl (C≦12) , substituted alkanediyl (C≦12) , -(OCH2CH2) p - or substituted - (OCH2CH2) p -(where: p is 0 to 50); or formula: Based on TIFF2025500942000004.tif17128, During the ceremony: R a and R a ' are each independently hydrogen, alkyl, (C≦12) , substituted alkyl (C≦12) , aryl (C≦12) or heteroaryl (C≦12) and; X8 is O or -NR 17 R 17 ′-, where: R 17 and R17 ' are each independently an alkyl (C≦12) or substituted alkyl (C≦12) and; R 19 is hydrogen, a sugar or a sugar derivative; and X9 is a covalent bond, O, S, -NH-, -(OCH2CH2) q - or substituted - (OCH2CH2) q -(where: q is between 0 and 50); or formula: Based on TIFF2025500942000005.tif12128, During the ceremony: Y1 is O or S; Y2 is a covalent bond, O, S, -NH-, or -NR 18 -where: R 18 is an alkyl (C≦12) or substituted alkyl (C≦12) and X 10 is a covalent bond, alkyl (C≦12) , substituted alkyl (C≦12) , O, S, -NH-, -(CH2CH2O) r -, substitution -(CH2CH2O) r -, -(CH2CH2NR 20 ) r - or substituted -(CH2CH2NR 20 ) r - and; r is 0 to 50; R 20 is hydrogen, alkyl (C≦12) , or substituted alkyl (C≦12) and; With the proviso that when R4 is valine, then R4' is not hydrogen.

[0007] In some embodiments, the compound is defined as follows: TIFF2025500942000006.tif26128 (in the formula: X1 is a covalent bond, alkanediyl (C≦12) , or substituted alkanediyl(C≦12) and; R1 is hydrogen, -ZR6, -(OCH2CH2) n ZR6, or substituted -(OCH2CH2) n ZR6, where: n is 0 to 50; and R6 is hydrogen, hydroxy, aminohydroxy, amino, mercapto, hydroxylamino, hydrazino, or azido; or Alkyl (C≦12) , alkenyl (C≦12) , alkynyl (C≦12) , alkyl hydrazine (C≦12) or substituted versions of any of these groups; Polyglycine containing 1 to 6 glycine units; or formula: Sub-structure of TIFF2025500942000007.tif74128 (In the formula: A1 and A2 are each independently absent or arenediyl. (C≦12) , substituted arenediyl (C≦12) , Heteroarenediyl (C≦12) , or substituted heteroarenediyl (C≦12) and a fused arene (C≦12) , substituted arenes (C≦12) , Heteroarenes (C≦12) , or substituted heteroarenes (C≦12) Forming; A3 is a covalent bond, O, alkanediyl (C≦8) , substituted alkanediyl (C≦8) , alkoxydiyl (C≦8) , substituted alkoxydiyl (C≦8) and A4 and A5 each independently represent a covalent bond, an alkanediyl (C≦8) , substituted alkanediyl (C≦8) , Arendjiil (C≦8) , or substituted arenediyl (C≦8) More selected; R d , R e , R e ′, and R heach independently represents hydrogen, halo, thioether, selenoether, sulfate, tosylate, mesylate, aryl (C≦8) , or substituted aryl (C≦8) More selected; R f is a halo; R g amine, hydrazine, alkylamino (C≦8) , substituted alkylamino (C≦8) , dialkylamino (C≦8) , substituted dialkylamino (C≦8) , alkyl hydrazine (C≦8) or a substituted alkylhydrazine (C≦8) and; X4 and X5 are each independently O, N, C(O), CH2, or X4 and X5 are alkanediyl. (C≦8) or substituted alkanediyl (C≦8) and taken together form a fused cycloalkane group of 3 to 8 ring atoms; R7 is hydrogen, hydroxy, amino, or oxo; R8 is carboxy; or Alkyl (C≦12) , amide (C≦12) , aryl (C≦12) , Heteroaryl (C≦12) , -C(O)OR 11 , -C(O)NR 11 R 11 ', or a substituted version of any of these groups, where: R 11 and R 11 ' are each independently hydrogen; or Alkyl (C≦12) , aryl (C≦12) or a substituted form of either of these groups; R9 and R 10 are each independently hydroxy, amino, halo; or Alkyl (C≦12) , aryl (C≦12) or one of these groups is substituted) and; Z is a covalent bond, alkanediyl (C≦12) , -C(O)-alkanediyl (C≦12) , -C(O)-alkanediyl (C≦12) -C(O)NH-, or a substituted form of any of these groups; R2 is hydrogen, alkyl (C≦12) , substituted alkyl (C≦12) , Acyl (C≦12) , substituted acyl (C≦12) or a monovalent amino protecting group; W is a covalent bond or a multivalent polymer having 2 to 21 linkage points; n is 1 to 20, except that when W is a covalent bond, n is 1, and when W is a multivalent polymer, n is less than or equal to one less than the number of attachment points; Each X is independently a covalent bond, an alkanediyl (C≦12) , substituted alkanediyl (C≦12) , one or more amino acid residues, or an oligomeric peptide; Each X2 is independently an alkanediyl (C≦12) or substituted alkanediyl (C≦12) and; Each R3 is independently hydroxy, or amino; or Alkoxy (C≦12) , acyloxy (C≦12) , alkylamino (C≦12) , dialkylamino (C≦12) , amide (C≦12) , Heteroaryl (C≦12) or a substituted version of any of these groups; or -X6-C(O)R 12 (where: X6 is O, -NR b - or covalently bonded; R b is hydrogen, alkyl (C≦6) , substituted alkyl (C≦6) or a monovalent amino protecting group; R 12 is hydroxy or amino; or Alkoxy (C≦12) , alkylamino(C≦12) , dialkylamino (C≦12) or a substituted form of any of these groups; or -A3SO2NR 13 R 13 ′, -A3P(O)(OH)OR 14 , or -A3SO2OR 14 ′ (where: A3 is O, -NR c - or covalently bonded; R c is hydrogen, alkyl (C≦6) , substituted alkyl (C≦6) or a monovalent amino protecting group; R 13 , R 13 ′, R 14 , and R 14 ' are each independently hydrogen, alkyl, (C≦12) , cycloalkyl (C≦12) , aryl (C≦12) , Heteroaryl (C≦12) , Aralkyl (C≦12) , heteroaralkyl (C≦12) or a substituted version of any of these groups. and; m is 0 or 1; each R4 is independently the side chain moiety of glycine or valine; and Each R4' is hydrogen, alkyl, (C≦12) , substituted alkyl (C≦12) , Acyl (C≦12) , substituted acyl (C≦12) or a monovalent amino protecting group; each R5 is independently a side chain moiety of glycine, alanine, ornithine, lysine, arginine, citrulline, asparagine, glutamine, or an amino-protected form thereof; Each R5' is hydrogen, alkyl, (C≦12) , substituted alkyl (C≦12) , Acyl (C≦12) , substituted acyl (C≦12) or a monovalent amino protecting group; Each Q independently represents a group of the formula: Based on TIFF2025500942000008.tif7128, During the ceremony: Each R 15 are independently hydrogen, -R 16 , or -C(O)-R 16 where: R 16 is a therapeutic or imaging agent; Each X7 is independently a covalent bond, O, S, -NH-, or an alkanediyl (C≦12) , substituted alkanediyl (C≦12) , -(OCH2CH2) p - or substituted - (OCH2CH2) p -(where: p is 0 to 50); or formula: Based on TIFF2025500942000009.tif17128, During the ceremony: R a and R a ' are each independently hydrogen, alkyl, (C≦12) , or substituted alkyl (C≦12) and; X8 is O or -NR 17 R 17 ′-, where: R 17 and R 17 ' are each independently an alkyl (C≦12) or substituted alkyl (C≦12) and; R 19 is hydrogen, a sugar or a sugar derivative; and X9 is a covalent bond, O, S, -NH-, -(OCH2CH2) q - or substituted - (OCH2CH2) q -(where: q is between 0 and 50); or formula: Based on TIFF2025500942000010.tif12128, During the ceremony: Y1 is O or S; Y2 is O, S, -NH-, or -NR 18-where: R 18 is an alkyl (C≦12) or substituted alkyl (C≦12) and X 10 is a covalent bond, alkyl (C≦12) , substituted alkyl (C≦12) , O, S, -NH-, -(CH2CH2O) r -, substitution -(CH2CH2O) r -, -(CH2CH2NR 20 ) r - or substituted -(CH2CH2NR 20 ) r - and; r is 0 to 50; R 20 is hydrogen, alkyl (C≦12) , or substituted alkyl (C≦12) and; provided that when R4 is valine, R4' is not hydrogen. or a pharma- ceutically acceptable salt thereof.

[0008] In some embodiments, the compound is defined as follows: TIFF2025500942000011.tif26128(in the formula: R1 is hydrogen, alkyl (C≦12) , substituted alkyl (C≦12) , -ZR6, -(OCH2CH2) n ZR6, or substituted -(OCH2CH2) n ZR6, where: n is 0 to 50; and R6 is hydrogen, hydroxy, aminohydroxy, amino, mercapto, hydroxylamino, hydrazino, or azido; or Alkyl (C≦12) , alkenyl (C≦12) , alkynyl (C≦12) , alkyl hydrazine (C≦12) or substituted versions of any of these groups; Polyglycine containing 1 to 6 glycine units; or formula: Substructure of TIFF2025500942000012.tif53149 (In the formula: A1 and A2 are each independently absent or arenediyl. (C≦12) , substituted arenediyl (C≦12) , Heteroarenediyl (C≦12) , or substituted heteroarenediyl (C≦12) and a fused arene (C≦12) , substituted arenes (C≦12) , Heteroarenes (C≦12) , or substituted heteroarenes (C≦12) Forming; A3 is a covalent bond, O, alkanediyl (C≦8) , substituted alkanediyl (C≦8) , alkoxydiyl (C≦8) , substituted alkoxydiyl (C≦8) and A4 and A5 each independently represent a covalent bond, an alkanediyl (C≦8) , substituted alkanediyl (C≦8) , Arendjiil (C≦8) , or substituted arenediyl (C≦8) More selected; R d , R e , R e ′, and R h each independently represents hydrogen, halo, thioether, selenoether, sulfate, tosylate, mesylate, aryl (C≦8) , or substituted aryl (C≦8) More selected; R f is a halo; R g amine, hydrazine, alkylamino (C≦8) , substituted alkylamino (C≦8) , dialkylamino (C≦8) , substituted dialkylamino (C≦8) , alkyl hydrazine (C≦8) or a substituted alkylhydrazine (C≦8) and; X4 and X5 are each independently O, N, C(O), CH2, or X4 and X5 are alkanediyl.(C≦8) or substituted alkanediyl (C≦8) and taken together form a fused cycloalkane group of 3 to 8 ring atoms; R7 is hydrogen, hydroxy, amino, or oxo; R8 is carboxy; or Alkyl (C≦12) , amide (C≦12) , aryl (C≦12) , Heteroaryl (C≦12) , -C(O)OR 11 , -C(O)NR 11 R 11 ', or a substituted version of any of these groups, where: R 11 and R 11 ' are each independently hydrogen; or Alkyl (C≦12) , aryl (C≦12) or a substituted form of either of these groups; R9 and R 10 are each independently hydroxy, amino, halo; or Alkyl (C≦12) , aryl (C≦12) or one of these groups is substituted) and; Z is a covalent bond, alkanediyl (C≦12) , -C(O)-alkanediyl (C≦12) , -C(O)-alkanediyl (C≦12) -C(O)NH-, or a substituted form of any of these groups; R2 is hydrogen, alkyl (C≦12) , substituted alkyl (C≦12) , Acyl (C≦12) , substituted acyl (C≦12) or a monovalent amino protecting group; W is a covalent bond or a multivalent polymer having 2 to 21 linkage points; n is 1 to 20, except that when W is a covalent bond, n is 1, and when W is a multivalent polymer, n is less than or equal to one less than the number of attachment points; Each X is independently a covalent bond, an alkanediyl (C≦12) , substituted alkanediyl (C≦12) , one or more amino acid residues, or an oligomeric peptide; Each X2 is independently an alkanediyl (C≦12) or substituted alkanediyl (C≦12) and; Each R3 is independently hydroxy, or amino; or Alkoxy (C≦12) , acyloxy (C≦12) , alkylamino (C≦12) , dialkylamino (C≦12) , amide (C≦12) , Heteroaryl (C≦12) or a substituted version of any of these groups; or -X6-C(O)R 12 (where: X6 is O, -NR b - or covalently bonded; R b is hydrogen, alkyl (C≦6) , substituted alkyl (C≦6) or a monovalent amino protecting group; R 12 is hydroxy or amino; or Alkoxy (C≦12) , alkylamino (C≦12) , dialkylamino (C≦12) or a substituted form of any of these groups; or -A3SO2NR 13 R 13 ′, -A3P(O)(OH)OR 14 , or -A3SO2OR 14 ′ (where: A3 is O, -NR c - or covalently bonded; R c is hydrogen, alkyl (C≦6) , substituted alkyl (C≦6) or a monovalent amino protecting group; R 13 , R 13 ′, R 14 , and R 14' are each independently hydrogen, alkyl, (C≦12) , cycloalkyl (C≦12) , aryl (C≦12) , Heteroaryl (C≦12) , Aralkyl (C≦12) , heteroaralkyl (C≦12) or a substituted version of any of these groups. and; m is 0 or 1; each R4 is independently the side chain moiety of glycine or valine; and Each R4' is hydrogen, alkyl, (C≦12) , substituted alkyl (C≦12) , Acyl (C≦12) , substituted acyl (C≦12) or a monovalent amino protecting group; each R5 is independently a side chain moiety of glycine, alanine, ornithine, lysine, arginine, citrulline, asparagine, glutamine, or an amino-protected form thereof; Each R5' is hydrogen, alkyl, (C≦12) , substituted alkyl (C≦12) , Acyl (C≦12) , substituted acyl (C≦12) or a monovalent amino protecting group; Each Q independently represents a group of the formula: Based on TIFF2025500942000013.tif7128, During the ceremony: Each R 15 are independently hydrogen, -R 16 , or -C(O)-R 16 where: R 16 is a therapeutic or imaging agent; Each X7 is independently a covalent bond, O, S, -NH-, or an alkanediyl (C≦12) , substituted alkanediyl (C≦12) , -(OCH2CH2) p - or substituted - (OCH2CH2) p -(where: p is 0 to 50); or formula: Based on TIFF2025500942000014.tif17128, During the ceremony: R a and R a ' are each independently hydrogen, alkyl, (C≦12) , or substituted alkyl (C≦12) and; X8 is O or -NR 17 R 17 ′-, where: R 17 and R 17 ' are each independently an alkyl (C≦12) or substituted alkyl (C≦12) and; R 19 is hydrogen, a sugar or a sugar derivative; and X9 is a covalent bond, O, S, -NH-, -(OCH2CH2) q - or substituted - (OCH2CH2) q -(where: q is between 0 and 50); or formula: Based on TIFF2025500942000015.tif12128, During the ceremony: Y1 is O or S; Y2 is O, S, -NH-, or -NR 18 -where: R 18 is an alkyl (C≦12) or substituted alkyl (C≦12) and X 10 is a covalent bond, alkyl (C≦12) , substituted alkyl (C≦12) , O, S, -NH-, -(CH2CH2O) r -, substitution -(CH2CH2O) r -, -(CH2CH2NR 20 ) r - or substituted -(CH2CH2NR 20 ) r - and; r is 0 to 50; R 20 is hydrogen, alkyl (C≦12) , or substituted alkyl (C≦12) is) or a pharma- ceutically acceptable salt thereof.

[0009] In some embodiments, the compound is defined as follows: TIFF2025500942000016.tif28128(in the formula: R1 is hydrogen, alkyl (C≦12) , substituted alkyl (C≦12) , -ZR6, -(OCH2CH2) n ZR6, or substituted -(OCH2CH2) n ZR6, where: n is 0 to 50; and R6 is hydrogen, hydroxy, aminohydroxy, amino, mercapto, hydroxylamino, hydrazino, or azido; or Alkyl (C≦12) , alkenyl (C≦12) , alkynyl (C≦12) , alkyl hydrazine (C≦12) or substituted versions of any of these groups; Polyglycine containing 1 to 6 glycine units; or formula: Substructure of TIFF2025500942000017.tif54147 (In the formula: A1 and A2 are each independently absent or arenediyl. (C≦12) , substituted arenediyl (C≦12) , Heteroarenediyl (C≦12) , or substituted heteroarenediyl (C≦12) and a fused arene (C≦12) , substituted arenes (C≦12) , Heteroarenes (C≦12) , or substituted heteroarenes (C≦12) Forming; A3 is a covalent bond, O, alkanediyl (C≦8) , substituted alkanediyl (C≦8) , alkoxydiyl (C≦8) , substituted alkoxydiyl (C≦8) and A4 and A5 each independently represent a covalent bond, an alkanediyl(C≦8) , substituted alkanediyl (C≦8) , Arendjiil (C≦8) , or substituted arenediyl (C≦8) More selected; R d , R e , R e ′, and R h each independently represents hydrogen, halo, thioether, selenoether, sulfate, tosylate, mesylate, aryl (C≦8) , or substituted aryl (C≦8) More selected; R f is a halo; R g amine, hydrazine, alkylamino (C≦8) , substituted alkylamino (C≦8) , dialkylamino (C≦8) , substituted dialkylamino (C≦8) , alkyl hydrazine (C≦8) or a substituted alkylhydrazine (C≦8) and; X4 and X5 are each independently O, N, C(O), CH2, or X4 and X5 are alkanediyl. (C≦8) or substituted alkanediyl (C≦8) and taken together form a fused cycloalkane group of 3 to 8 ring atoms; R7 is hydrogen, hydroxy, amino, or oxo; R8 is carboxy; or Alkyl (C≦12) , amide (C≦12) , aryl (C≦12) , Heteroaryl (C≦12) , -C(O)OR 11 , -C(O)NR 11 R 11 ', or a substituted version of any of these groups, where: R 11 and R 11 ' are each independently hydrogen; or Alkyl (C≦12) , aryl (C≦12) or a substituted form of either of these groups; R9 and R 10 are each independently hydroxy, amino, halo; or Alkyl (C≦12) , aryl (C≦12) or one of these groups is substituted) and; Z is a covalent bond, alkanediyl (C≦12) , -C(O)-alkanediyl (C≦12) , -C(O)-alkanediyl (C≦12) -C(O)NH-, or a substituted form of any of these groups; R2 is hydrogen, alkyl (C≦12) , substituted alkyl (C≦12) , Acyl (C≦12) , substituted acyl (C≦12) or a monovalent amino protecting group; W is a covalent bond or a multivalent polymer having 2 to 21 linkage points; n is 1 to 20, except that when W is a covalent bond, n is 1, and when W is a multivalent polymer, n is less than or equal to one less than the number of attachment points; Each X2 is independently an alkanediyl (C≦12) or substituted alkanediyl (C≦12) and; Each R3 is independently hydroxy, or amino; or Alkoxy (C≦12) , acyloxy (C≦12) , alkylamino (C≦12) , dialkylamino (C≦12) , amide (C≦12) , Heteroaryl (C≦12) or a substituted version of any of these groups; or -X6-C(O)R 12 (where: X6 is O, -NR b - or covalently bonded; R b is hydrogen, alkyl (C≦6) , substituted alkyl (C≦6) or a monovalent amino protecting group; R 12is hydroxy or amino; or Alkoxy (C≦12) , alkylamino (C≦12) , dialkylamino (C≦12) or a substituted form of any of these groups; or -A3SO2NR 13 R 13 ′, -A3P(O)(OH)OR 14 , or -A3SO2OR 14 ′ (where: A3 is O, -NR c - or covalently bonded; R c is hydrogen, alkyl (C≦6) , substituted alkyl (C≦6) or a monovalent amino protecting group; R 13 , R 13 ′, R 14 , and R 14 ' are each independently hydrogen, alkyl, (C≦12) , cycloalkyl (C≦12) , aryl (C≦12) , Heteroaryl (C≦12) , Aralkyl (C≦12) , heteroaralkyl (C≦12) or a substituted version of any of these groups. and; m is 0 or 1; each R4 is independently the side chain moiety of glycine or valine; and Each R4' is hydrogen, alkyl, (C≦12) , substituted alkyl (C≦12) , Acyl (C≦12) , substituted acyl (C≦12) or a monovalent amino protecting group; each R5 is independently a side chain moiety of glycine, alanine, ornithine, lysine, arginine, citrulline, asparagine, glutamine, or an amino-protected form thereof; Each R5' is hydrogen, alkyl, (C≦12) , substituted alkyl (C≦12) , Acyl (C≦12) , substituted acyl (C≦12) or a monovalent amino protecting group; Each Q independently represents a group of the formula: Based on TIFF2025500942000018.tif7128, During the ceremony: Each R 15 are independently hydrogen, -R 16 , or -C(O)-R 16 where: R 16 is a therapeutic or imaging agent; Each X7 is independently a covalent bond, O, S, -NH-, or an alkanediyl (C≦12) , substituted alkanediyl (C≦12) , -(OCH2CH2) p - or substituted - (OCH2CH2) p -(where: p is 0 to 50); or formula: Based on TIFF2025500942000019.tif17128, During the ceremony: R a and R a ' are each independently hydrogen, alkyl, (C≦12) , or substituted alkyl (C≦12) and; X8 is O or -NR 17 R 17 ′-, where: R 17 and R 17 ' are each independently an alkyl (C≦12) or substituted alkyl (C≦12) and; R 19 is hydrogen, a sugar or a sugar derivative; and X9 is a covalent bond, O, S, -NH-, -(OCH2CH2) q - or substituted - (OCH2CH2) q -(where: q is between 0 and 50); or formula: Based on TIFF2025500942000020.tif12128, During the ceremony: Y1 is O or S; Y2 is O, S, -NH-, or -NR 18 -where: R 18 is an alkyl (C≦12) or substituted alkyl (C≦12) and X 10 is a covalent bond, alkyl (C≦12) , substituted alkyl (C≦12) , O, S, -NH-, -(CH2CH2O) r -, substitution -(CH2CH2O) r -, -(CH2CH2NR 20 ) r - or substituted -(CH2CH2NR 20 ) r - and; r is 0 to 50; R 20 is hydrogen, alkyl (C≦12) , or substituted alkyl (C≦12) is) or a pharma- ceutically acceptable salt thereof.

[0010] In some embodiments, the compound is defined as follows: TIFF2025500942000021.tif28128(in the formula: R1 is hydrogen, alkyl (C≦12) , substituted alkyl (C≦12) , -ZR6, -(OCH2CH2) n ZR6, or substituted -(OCH2CH2) n ZR6, where: n is 0 to 50; and R6 is hydrogen, hydroxy, aminohydroxy, amino, mercapto, hydroxylamino, hydrazino, or azido; or Alkyl (C≦12) , alkenyl (C≦12) , alkynyl (C≦12) , alkyl hydrazine (C≦12) or substituted versions of any of these groups; Polyglycine containing 1 to 6 glycine units; or formula: Substructure of TIFF2025500942000022.tif54147 (In the formula: A1 and A2 are each independently absent or arenediyl. (C≦12) , substituted arenediyl (C≦12) , Heteroarenediyl (C≦12) , or substituted heteroarenediyl (C≦12) and a fused arene (C≦12) , substituted arenes (C≦12) , Heteroarenes (C≦12) , or substituted heteroarenes (C≦12) Forming; A3 is a covalent bond, O, alkanediyl (C≦8) , substituted alkanediyl (C≦8) , alkoxydiyl (C≦8) , substituted alkoxydiyl (C≦8) and A4 and A5 each independently represent a covalent bond, an alkanediyl (C≦8) , substituted alkanediyl (C≦8) , Arendjiil (C≦8) , or substituted arenediyl (C≦8) More selected; R d , R e , R e ′, and R h each independently represents hydrogen, halo, thioether, selenoether, sulfate, tosylate, mesylate, aryl (C≦8) , or substituted aryl (C≦8) More selected; R f is a halo; R g amine, hydrazine, alkylamino (C≦8) , substituted alkylamino (C≦8) , dialkylamino (C≦8) , substituted dialkylamino (C≦8) , alkyl hydrazine (C≦8) or a substituted alkylhydrazine (C≦8) and; X4 and X5 are each independently O, N, C(O), CH2, or X4 and X5 are alkanediyl.(C≦8) or substituted alkanediyl (C≦8) and taken together form a fused cycloalkane group of 3 to 8 ring atoms; R7 is hydrogen, hydroxy, amino, or oxo; R8 is carboxy; or Alkyl (C≦12) , amide (C≦12) , aryl (C≦12) , Heteroaryl (C≦12) , -C(O)OR 11 , -C(O)NR 11 R 11 ', or a substituted version of any of these groups, where: R 11 and R 11 ' are each independently hydrogen; or Alkyl (C≦12) , aryl (C≦12) or a substituted form of either of these groups; R9 and R 10 are each independently hydroxy, amino, halo; or Alkyl (C≦12) , aryl (C≦12) or one of these groups is substituted) and; Z is a covalent bond, alkanediyl (C≦12) , -C(O)-alkanediyl (C≦12) , -C(O)-alkanediyl (C≦12) -C(O)NH-, or a substituted form of any of these groups; R2 is hydrogen, alkyl (C≦12) , substituted alkyl (C≦12) , Acyl (C≦12) , substituted acyl (C≦12) or a monovalent amino protecting group; W is a covalent bond or a multivalent polymer having 2 to 21 linkage points; n is 1 to 20, except that when W is a covalent bond, n is 1, and when W is a multivalent polymer, n is less than or equal to one less than the number of attachment points; Each X2 is independently an alkanediyl (C≦12) or substituted alkanediyl (C≦12) and; Each R3 is independently hydroxy, or amino; or Alkoxy (C≦12) , acyloxy (C≦12) , alkylamino (C≦12) , dialkylamino (C≦12) , amide (C≦12) , Heteroaryl (C≦12) or a substituted version of any of these groups; or -X6-C(O)R 12 (where: X6 is O, -NR b - or covalently bonded; R b is hydrogen, alkyl (C≦6) , substituted alkyl (C≦6) or a monovalent amino protecting group; R 12 is hydroxy or amino; or Alkoxy (C≦12) , alkylamino (C≦12) , dialkylamino (C≦12) or a substituted form of any of these groups; or -A3SO2NR 13 R 13 ′, -A3P(O)(OH)OR 14 , or -A3SO2OR 14 ′ (where: A3 is O, -NR c - or covalently bonded; R c is hydrogen, alkyl (C≦6) , substituted alkyl (C≦6) or a monovalent amino protecting group; R 13 , R 13 ′, R 14 , and R 14 ' are each independently hydrogen, alkyl, (C≦12) , cycloalkyl (C≦12) , aryl (C≦12) , Heteroaryl (C≦12) , Aralkyl(C≦12) , heteroaralkyl (C≦12) or a substituted version of any of these groups. and; m is 0 or 1; each R4 is independently the side chain moiety of glycine or valine; each R5 is independently a side chain moiety of glycine, alanine, ornithine, lysine, arginine, citrulline, asparagine, glutamine, or an amino-protected form thereof; Each Q independently represents a group of the formula: Based on TIFF2025500942000023.tif7128, During the ceremony: Each R 15 are independently hydrogen, -R 16 , or -C(O)-R 16 where: R 16 is a therapeutic or imaging agent; Each X7 is independently a covalent bond, O, S, -NH-, or an alkanediyl (C≦12) , substituted alkanediyl (C≦12) , -(OCH2CH2) p - or substituted - (OCH2CH2) p -(where: p is 0 to 50); or formula: Based on TIFF2025500942000024.tif17128, During the ceremony: R a and R a ' are each independently hydrogen, alkyl, (C≦12) , or substituted alkyl (C≦12) and; X8 is O or -NR 17 R 17 ′-, where: R 17 and R 17 ' are each independently an alkyl (C≦12) or substituted alkyl (C≦12) and; R 19 is hydrogen, a sugar or a sugar derivative; and X9 is a covalent bond, O, S, -NH-, -(OCH2CH2) q - or substituted - (OCH2CH2) q -(where: q is between 0 and 50); or formula: Based on TIFF2025500942000025.tif12128, During the ceremony: Y1 is O or S; Y2 is O, S, -NH-, or -NR 18 -where: R 18 is an alkyl (C≦12) or substituted alkyl (C≦12) and X 10 is a covalent bond, alkyl (C≦12) , substituted alkyl (C≦12) , O, S, -NH-, -(CH2CH2O) r -, substitution -(CH2CH2O) r -, -(CH2CH2NR 20 ) r - or substituted -(CH2CH2NR 20 ) r - and; r is 0 to 50; R 20 is hydrogen, alkyl (C≦12) , or substituted alkyl (C≦12) is) or a pharma- ceutically acceptable salt thereof.

[0011] In some embodiments, X1 is a covalent bond. In some embodiments, X is a covalent bond. In some embodiments, R4' is hydrogen. In some embodiments, R5' is hydrogen.

[0012] In some embodiments, W is a covalent bond. In other embodiments, W is a multivalent polymer having 1 to 5 attachment points. In some embodiments, n is 1. In other embodiments, n is 2, 3, 4, or 5.

[0013] In some embodiments, X2 is alkanediyl. (C≦12) In some embodiments, R is -X-C(O)R 12 where X6 is O, -NR b - or a covalent bond; R b is hydrogen, alkyl (C≦6) , substituted alkyl (C≦6) or a monovalent amino protecting group; R 12 is hydroxy or amino; or alkoxy (C≦12) , alkylamino (C≦12) , dialkylamino (C≦12) or a substituted version of any of these groups. In some embodiments, X6 is a covalent bond. In some embodiments, R 12 is hydroxy.

[0014] In some embodiments, m is 0. In other embodiments, m is 1. In some embodiments, each R4 is the side chain or glycine. In some embodiments, each R4 is the side chain or valine. In some embodiments, each R5 is the side chain or citrulline.

[0015] In some embodiments, Q is TIFF2025500942000026.tif7128. In some embodiments, X7 is represented by the formula: Based on TIFF2025500942000027.tif17128, During the ceremony: R a and R a ' are each independently hydrogen, alkyl, (C≦12) , or substituted alkyl (C≦12) and; X8 is O or -NR 17 R 17 ′-, where: R 17 and R 17 ' are each independently an alkyl (C≦12) or substituted alkyl(C≦12) and; R 19 is hydrogen, a sugar or a sugar derivative; and X9 is a covalent bond, O, S, -NH-, -(OCH2CH2) q - or substituted - (OCH2CH2) q -(where: q is between 0 and 50); or formula: Based on TIFF2025500942000028.tif12128, During the ceremony: Y1 is O or S; Y2 is O, S, -NH-, or -NR 18 -where: R 18 is an alkyl (C≦12) or substituted alkyl (C≦12) and X 10 is a covalent bond, alkyl (C≦12) , substituted alkyl (C≦12) , O, S, -NH-, -(CH2CH2O) r -, substitution -(CH2CH2O) r -, -(CH2CH2NR 20 ) r - or substituted -(CH2CH2NR 20 ) r - and; r is 0 to 50; R 20 is hydrogen, alkyl (C≦12) , or substituted alkyl (C≦12) It is.

[0016] In some embodiments, R a is hydrogen. In some embodiments, R a In some embodiments, R′ is hydrogen. 19 is hydrogen. In some embodiments, X8 is O. In some embodiments, X9 is a group of the formula: Based on TIFF2025500942000029.tif12128, During the ceremony: Y1 is O or S; Y2 is O, S, -NH-, or -NR 18 -where: R 18 is an alkyl (C≦12) or substituted alkyl (C≦12) and X 10 is a covalent bond, alkyl (C≦12) , substituted alkyl (C≦12) , O, S, -NH-, -(CH2CH2O) r -, substitution -(CH2CH2O) r -, -(CH2CH2NR 20 ) r - or substituted -(CH2CH2NR 20 ) r - and; r is 0 to 50; R 20 is hydrogen, alkyl (C≦12) , or substituted alkyl (C≦12) It is.

[0017] In some embodiments, Y is O. In some embodiments, Y is -NR 18 In some embodiments, R 18 is an alkyl (C≦12) In some embodiments, X is methyl. 10 is a covalent bond. In some embodiments, R 15 -R 16 or -C(O)-R 16 In some embodiments, R 16 is a therapeutic agent. In some embodiments, the therapeutic agent is a chemotherapeutic agent. In other embodiments, R 16 is an imaging agent. In some embodiments, the imaging agent is a radioactive tracer. In other embodiments, the imaging agent is a fluorescent molecule. In some embodiments, R 15 is hydrogen.

[0018] In another aspect, the present disclosure provides a method for producing a method for manufacturing a semiconductor device comprising: (A) a compound described herein; (B) a linker; and (C) Cell targeting group The present invention provides a drug conjugate comprising:

[0019] In some embodiments, the cell targeting group is an antibody. In other embodiments, the cell targeting group is an antibody fragment. In other embodiments, the cell targeting group is a protein. In other embodiments, the cell targeting group is a small molecule. In some embodiments, the small molecule is a receptor-specific ligand molecule. In some embodiments, the linker is a non-covalent bond formed by hydrogen bonding, nucleobase pairing, electrostatic interactions, pi-stacking, van der Waals interactions, or dipole-dipole interactions. In some embodiments, the linker is a covalent bond. In other embodiments, the linker is a monovalent spacer comprising one attachment point. In some embodiments, the linker is a multivalent spacer comprising 2-21 attachment points.

[0020] In some embodiments, the drug conjugates are formed by condensation, 1,3-dipolar cycloaddition, Diels-Alder reaction, hetero Diels-Alder reaction, Michael reaction, nucleophilic substitution reaction, non-aldol type carbonyl reaction, addition to carbon-carbon multiple bonds, oxidation reaction, enzymatic amino acid or peptide modification, transpeptidation, and / or click reaction.

[0021] In some embodiments, the linker is a group W in the compound. In some embodiments, the linker has the formula: TIFF2025500942000030.tif26128, or a pharma- ceutically acceptable salt thereof; During the ceremony: A6, A7, A8, and A9 each independently represent an alkanediyl group. C1-12 , Arendjiil C1-12 , Heteroarenediyl C1-12 , cycloalkanediyl C1-12 , heterocycloalkanediyl C1-12 or a substituted form thereof, or a side chain group of a canonical amino acid; X11 , Y3, and Z1 each independently represent a covalent bond, -[O(CH2) q ]-, -[O(CHW1´) q ]-, or -[O(CW1´W1´´) q ]- and; where: W1' and W1'' are each independently amino, hydroxy, halo, mercapto, alkyl, C1-12 , cycloalkyl C1-12 , alkenyl C1-12 , alkynyl C1-12 , aryl C1-12 , Aralkyl C1-12 , Heteroaryl C1-12 , heteroaralkyl C1-12 , heterocycloalkyl C1-12 , Acyl C1-12 , acyloxy C1-12 , alkylamino C1-12 or substituted forms thereof; q is 1 to 3; a, b, c, and d are each independently 0 to 12; e and f are each independently 0, 1, 2, or 3; R 21 , R 22 , and R 23 are each independently hydrogen, -NH2, or -NHR 24 , -NR 24 R 25 , -N3, heteroaryl (C≦12) , substituted heteroaryl (C≦12) , -Arendijyl (C≦12) -heteroaryl (C≦12) , substituted arenediyl (C≦12) -heteroaryl (C≦12) or a conjugating group; where: R 24 and R 25 are each independently an alkyl C1-12 , cycloalkyl C1-12 , alkenyl C1-12 , alkynyl C1-12 , aryl C1-12, Aralkyl C1-12 , Heteroaryl C1-12 , heteroaralkyl C1-12 , heterocycloalkyl C1-12 , Acyl C1-12 , acyloxy C1-12 , alkylamino C1-12 or a substituted form thereof, or a monovalent amino protecting group; or R 24 and R 25 taken together and is a divalent amino protecting group; However, R 21 , R 22 , and R 23 At least one of R is -NH or a group containing -NH, and 21 , R 22 , and R 23 At least one of is -N3, heteroaryl (C≦12) , or -Arendjiil (C≦12) -heteroaryl (C≦12) It is.

[0022] In some embodiments, A6 is alkanediyl C1-12 or substituted alkanediyl C1-12 In some embodiments, A6 is alkanediyl. C1-12 In some embodiments, A is alkanediyl. C1-12 or substituted alkanediyl C1-12 In some embodiments, A8 is alkanediyl. C1-12 In some embodiments, A is alkanediyl. C1-12 or substituted alkanediyl C1-12 In some embodiments, A9 is alkanediyl. C1-12 In some embodiments, A7 is alkanediyl. C1-12 or substituted alkanediyl C1-12 In some embodiments, A7 is alkanediyl. C1-12 , for example CH2CH2CH2CH2.

[0023] In some embodiments, X 11 -[O(CH2) q In some embodiments, Y3 is -[O(CH2) q In some embodiments, Z1 is -[O(CH2) q ]-. In some embodiments, q is 1, 2, or 3. In some embodiments, q is 2. In some embodiments, a is 2, 3, or 4. In some embodiments, a is 3. In some embodiments, b is 2, 3, or 4. In some embodiments, b is 3. In some embodiments, c is 2, 3, or 4. In some embodiments, c is 3. In some embodiments, d is 1, 2, or 3. In some embodiments, d is 1. In some embodiments, e is 1 or 2. In some embodiments, e is 1. In other embodiments, e is 2. In some embodiments, f is 0 or 1. In some embodiments, f is 0. In other embodiments, f is 1.

[0024] In some embodiments, R 21 is NH2. In other embodiments, R 21 is N3. In some embodiments, R 22 is N3. In some embodiments, R 23 is N3. In other embodiments, R 23 is hydrogen. In other embodiments, R 23 is heteroaryl (C≦12) or substituted heteroaryl (C≦12) In some embodiments, R 23 is heteroaryl (C≦12) , e.g., tetrazine or 3-methyltetrazine. 23 Is -Arengeiil (C≦12) -heteroaryl (C≦12) or substituted arenediyl (C≦12) -heteroaryl (C≦12) In some embodiments, R 23 Ha-Arengeiil (C≦12)-heteroaryl (C≦12) In some embodiments, R 23 is 4-tetrazyl-phenyl or 4-(3-methyltetrazyl)-phenyl.

[0025] In some embodiments, the polyvalent linker is It is further defined as TIFF2025500942000031.tif23796.

[0026] In some embodiments, the compound is administered as a chemotherapeutic agent. 16 In some embodiments, the chemotherapeutic agent is auristatin E (AE), auristatin F (AF), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), dolastatin, maytansine, duocarmycin, tubulysin, calicheamicin, pyrobenzodiazepine dimer, anthracycline, paclitaxel, vinblastine, amanitin, eribulin, or a derivative of any of these molecules. In some embodiments, the chemotherapeutic agent is MMAE or MMAF. In some embodiments, the antibody whose antigen is a tumor associated antigen. In some embodiments, the linker is linked to a second antibody or a second compound. In some embodiments, the second antibody is different from said antibody. In some embodiments, the second compound is different from said compound.

[0027] In yet another aspect, the present disclosure provides a pharmaceutical composition comprising the compound or drug conjugate described and excipient.In some embodiments, the compound is formulated for administration orally, intraadipose, intraarterial, intraarticular, intracranial, intradermal, intralesional, intramuscular, intranasal, intraocular, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrarectal, intrathecal, intratracheal, intratumoral, intraumbilical, intravaginal, intravenous, intraventricular, intravesicular, intravitreal, liposomal, topical, mucosal, parenteral, rectal, subconjunctival, subcutaneous, sublingual, topical, transbuccal, transdermal, vaginal, by catheter, by lavage, by continuous infusion, by injection, by inhalation, by injection, by local delivery, or by local perfusion.

[0028] In yet another aspect, the disclosure provides a method of treating a disease or disorder in a patient, comprising administering to the patient a therapeutically effective amount of a compound, drug conjugate, or pharmaceutical composition described herein. In some embodiments, the disease or disorder is cancer. In some embodiments, the cancer is carcinoma, sarcoma, lymphoma, leukemia, melanoma, mesothelioma, multiple myeloma, or seminoma. In some embodiments, the cancer is cancer of the bladder, blood, bone, brain, breast, central nervous system, cervix, colon, endometrium, esophagus, gallbladder, gastrointestinal tract, reproductive organs, genitourinary tract, head, kidney, larynx, liver, lung, muscle tissue, neck, oral or nasal mucosa, ovary, pancreas, prostate, skin, spleen, small intestine, large intestine, stomach, testis, or thyroid. In other embodiments, the disease or disorder is a microbial infection. In other embodiments, the disease or disorder is an autoimmune disease. In other embodiments, the disease or disorder is associated with inflammation. In other embodiments, the disease or disorder is diabetes. In other embodiments, the disease or disorder is a neurological disorder.

[0029] In some embodiments, the method further comprises administering a second therapy. In some embodiments, the patient is a mammal. In some embodiments, the patient is a human. In other embodiments, the patient is a mouse, a rat, a dog, or a monkey. In some embodiments, the compound, drug conjugate, or composition is administered once. In other embodiments, the compound, drug conjugate, or composition is administered two or more times.

[0030] In yet another aspect, the present disclosure provides for the use of the compound, drug conjugate, or pharmaceutical composition in the preparation of a medicament for the treatment of a disease or disorder. In some aspects, the present disclosure may also provide a composition, compound, or drug conjugate for use in the preparation of a medicament.

[0031] The terms "comprise" (and any form of including, such as "comprises" and "comprising"), "have" (and any form of having, such as "has" and "having"), "contain" (and any form of including, such as "contains" and "containing"), and "include" (and any form of including, such as "includes" and "including") are open-ended linking verbs. As a result, a method, composition, kit, or system that "comprises," "has," "contains," or "includes" one or more recited steps or elements has those recited steps or elements, but is not limited to having only those steps or elements; it may have (i.e., cover) elements or steps that are not recited. Similarly, an element of a method, composition, kit, or system that "comprises," "has," "contains," or "includes" one or more recited features has those features, but is not limited to having only those features; it may have features that are not recited.

[0032] Any embodiment of any of the methods, compositions, kits, and systems may consist of or consist essentially of the recited steps and / or features rather than comprising / comprising / containing / having the recited steps and / or features. Thus, in any of the claims, the terms "consisting of" or "consisting essentially of" may be used in place of any of the open-ended linking verbs set forth above in order to change the scope of a given claim from that which would otherwise use an open-ended linking verb.

[0033] Use of the term "or" in the claims is used to mean "and / or," unless expressly indicated to refer to alternatives only or unless the alternatives are mutually exclusive, although the disclosure supports a definition that refers to alternatives only as well as "and / or."

[0034] Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples, while showing specific embodiments of the present disclosure, are given for illustrative purposes only, since various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art from this detailed description. It should be noted that just because a particular compound belongs to a particular general formula, it does not mean that it cannot also belong to another general formula. [Brief description of the drawings]

[0035] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description. [Figure 1]Figure 1A-1C: Structure and stability profile of cleavable peptide linkers. (Figure 1A) VCit-based ADC linker. The VCit linker is unstable in mouse circulation due to its sensitivity to the extracellular carboxylesterase Ces1c. The VCit linker is also unstable to human neutrophil elastase-mediated degradation. This instability often causes premature payload release, leading to reduced efficacy in preclinical rodent models and safety concerns including neutropenia and hepatotoxicity in humans. (Figure 1B) EVCit-based ADC linker. The previously developed EVCit linker is stable in human and mouse plasma. However, this linker cannot withstand neutrophil elastase-mediated degradation, resulting in the same safety concerns as for the VCit linker. (Figure 1C) EGCit-based ADC linker. This study demonstrates that the EGCit linker can resist degradation in circulation and cleavage mediated by human neutrophil proteases while simultaneously releasing the payload in a traceless manner upon intracellular cleavage. [Figure 2-1]Figures 2A-2E: Incorporation of glycine at P2 and glutamic acid at P3 provides high resistance to undesired degradation. (Figure 2A) ESI-MS-based peptide mapping of cleavage sites in the presence of human neutrophil elastase. Cleavage of the amide bond between valine and citrulline was observed within the VCit (1) and EVCit (2) linkers. (Figure 2B) Structures of small molecule P2 probes containing EGCit (3a), EACit (3b), ELCit (3c), EICit (3d), EV(Me)Cit (3e), or GCit (3f). The pyrene group was used as a surrogate for the hydrophobic ADC payload. (Figure 2C-2E) Stability of probes 1, 2, and 3a-f in the presence of human neutrophil elastase (Figure 2C), in undiluted BALB / c mouse plasma (Figure 2F), or in human plasma (Figure 2E) at 37 °C. (1) light purple diamond; (2) green triangle; (3a) magenta square; (3b) black open circle; (3c) cyan asterisk; (3d) orange open triangle; (3e) purple inverted triangle; (3f) light green open diamond. All assays were performed at least three times in technical duplicates, and representative data from replicates are shown (n = 2). Data are presented as mean ± SEM. PABC, p-aminobenzyloxycarbonyl. [Figure 2-2] See description of Figure 2-1. [Figure 3-1]Figures 3A-3J: EGCit linkers increase ADC hydrophilicity and cell killing potency with efficient intracellular payload release. (Figure 3A) Construction of ADCs (4a-e) via MTGase-mediated branched linker conjugation and subsequent strain-promoted azide-alkyne cycloaddition (yellow sparks: MMAE). (Figure 3B) Deconvoluted ESI-mass spectrum of EGCit ADC 4c. Asterisks (*) indicate fragment ions detected in ESI-MS analysis. (Figure 3C) Overlay of five HIC traces (VCit ADC 4a: light purple; EVCit ADC 4b: green; EGCit ADC 4c: magenta; EV(N-Me)Cit ADC 4d: purple; GCit ADC 4e: light green) under physiological conditions (phosphate buffered saline, pH 7.4). (Figures 3D-3I) Cell killing efficacy in breast cancer cell lines KPL-4 (Figure 3D), SK-BR-3 (Figure 3E), BT-474 (Figure 3F), JIMT-1 (Figure 3G), MDA-MB-453 (Figure 3H), and MDA-MB-231 (Figure 3I). Unconjugated N297A anti-HER2 mAb (black circle), VCit ADC 4a (light purple diamond), EVCit ADC 4b (green triangle), EGCit ADC 4c (magenta square), EV(N-Me)Cit 4d (purple inverted triangle), GCit ADC 4e (light green open diamond), non-cleavable ADC 4f (cyan open circle), and non-targeting EGCit ADC 5 (isotype control, black open rectangle with dotted curve) were tested. (Figure 3J) ESI-MS-based quantification of free MMAE released from ADC 4a–c in KPL-4 cells after 24 h incubation at 37 °C. All assays were performed in quadruplicates (Figures 3D–3I) or triplicates (Figure 3J). Data are presented as mean ± SEM. For statistical analysis, one-way ANOVA with Dunnett post-hoc test was used (control: EGCit ADC 4c). BCN, bicyclo[6.1.0]nonine; DAR, drug-antibody ratio; MMAE, monomethylauristatin E; MTGase, microbial transglutaminase; PEG, polyethylene glycol. [Figure 3-2]See description of Figure 3-1. [Figure 4-1] Figure 4A-4I: EGCit ADCs are stable in plasma and tolerate human differentiating neutrophils derived from bone marrow. (Figure 4A-4C) Stability in undiluted mouse plasma (Figure 4A), cynomolgus monkey plasma (Figure 4B), and human plasma at 37 °C. VCit ADC 4a (light purple diamonds), EGCit ADC 4b (green triangles), and EGCit ADC 4c (magenta squares) were tested. (Figure 4D) ESI-MS traces of ADC 4b,c after incubation with human neutrophil elastase for 24 h at 37 °C. EGCit ADC 4c did not undergo cleavage, while VCit ADC 4b underwent linker degradation and lost part of the payload. (Figure 4E) Study timeline for differentiation of human bone marrow HSPCs into neutrophils and subsequent treatment with ADC 4b,c. After 3 days of proliferation (day 0), HSPCs were treated with growth factors for 7 days and differentiated into CD15+ and CD66b+ granulocytes / neutrophils. (Figure 4F-4G) Flow cytometry before (day 0, Figure 4F) and after (day 7, Figure 4G). (Figure 4H-4I) Effect of ADCs (vehicle, dark grey; VCit ADC 4a, light purple; EGCit ADC 4c, magenta) on the population of human neutrophils (Figure 4H) and on the viability of total hematopoietic cells (Figure 4I) compared to that of vehicle control (n = 3). All assays were performed in triplicate. Data are presented as mean ± SEM. For statistical analysis, one-way ANOVA with Dunnett's post-hoc test was used (control: EGCit ADC 4c). HSPCs, hematopoietic stem and progenitor cells. [Figure 4-2] See description of Figure 4-1. [Figure 4-3] See description of Figure 4-1. [Diagram 5]Figures 5A-5D: EGCit linker has the potential to minimize antigen-independent hepatotoxicity of ADCs. (Figures 5D-5D) Blood chemistry parameters (ALT (Figure 5A), AST (Figure 5B), ALKP (Figure 5C), and BUN (Figure 5D)) measured 5 days after ADC injection in female CD-1® mice. Mice were injected with a single dose of vehicle control (n = 4), EGCit ADC 4c (magenta squares, n = 4), ENHERTU® (purple inverted triangles, n = 3), or KADCYLA® (light purple squares, n = 3) at 80 mg kg-1. Data are presented as mean (bars) ± SEM. For statistical analysis, Welch's t-test (two-tailed, unpaired, unequal variance) was used. Crude P values ​​were adjusted by the Holm-Bonferroni method to control for family-wise error rate in multiple comparisons. ALKP, alkaline phosphatase; ALT, alanine transaminase; AST, aspartate transaminase; BUN, blood urea nitrogen. [Figure 6-1]Figures 6A-6F: EGCit ADCs exert improved antitumor efficacy in various xenograft models compared to conventional ADCs. (Figures 6A-6D) Antitumor activity (Figures 6A, 6C) and survival benefit (Figures 6B, 6D) in orthotopic xenograft mouse models of human breast cancer. KPL-4 model (Figures 6A-6B): A single dose of vehicle control (black circles), KADCYLA® (light purple squares), ENHERTU® (purple inverted triangles), EVCit-MMAE ADC 4b (green triangles), EGCit-MMAE ADC 4c (magenta squares) or EGCit-DuoDM ADC 6 (cyan circles) was administered intravenously at 1 mg kg-1 to tumor-bearing female NSG mice with a mean tumor volume of approximately 100 mm3 (n = 5). JIMT-1 / MDA-MB-231 4:1 mixed model (Figure 6C-6D): Eight days after implantation (indicated by black arrows), female NU / J mice were intravenously administered a single dose of ENHERTU® (3 mg kg-1, purple inverted triangles, n = 5) or EGCit-MMAE / F DAR 4+2 dual drug ADC 7a (1 mg kg-1, magenta open squares, n = 6). Note: Tumor volume and survival curve data shown here for vehicle control (black circle with dotted curve, n = 4) and EGCit dual drug ADC 7b (1 mg kg-1, green open triangle with dotted curve, n = 5) were generated previously. Data are presented as mean (bar) ± SEM. (Figure 6E-6F) Study schedule (Figure 6E) and post-treatment survival curves (Figure 6F) in U87ΔEGFR-luc orthotopic xenograft model. U87ΔEGFR-luc cells were intracranially implanted into male and female NSG mice. Five days after implantation, mice were intravenously administered a single dose of vehicle control, anti-EGFRvIII VCit-MMAE ADC (8a, 5 mg kg-1, lilac), or anti-EGFRvIII EGCit-MMAE ADC (8b, 5 mg kg-1, magenta). n = 6 for vehicle and 8a; n = 7 for 8b. All animals were killed at predefined humane endpoints and counted as dead, except for those that were confirmed dead or achieved complete remission.For statistical analysis of tumor volume data, the Welch t test (two-tailed, unpaired, unequal variance) was used. Kaplan-Meier survival curve statistics were analyzed with the log-rank (Mantel-Cox) test. Crude P values ​​were adjusted by the Holm-Bonferroni method to control for family-wise error rates in multiple comparisons. DuoDM, duocarmycin DM. [Figure 6-2] See description of Figure 6-1. [Figure 7] Figures 7A-7B: Stability of probes S1a-d in the presence of human neutrophil elastase (Figure 7A) or in undiluted BALB / c mouse plasma (Figure 7B) at 37 °C. (S1a) Green open triangles; (S1b) Cyan open squares; (S1c) Purple asterisks; (S1d) Black crosses. All assays were performed in duplicate. Data are presented as mean ± SEM. [Figure 8] Figures 8A-8C: SEC analysis of ADCs 4a, c, d before and after incubation in PBS (pH 7.4) at 37 °C for 28 days. VCit ADC 4a (Figure 8A), EGCit ADC 4c (Figure 8B), and EV(N-Me)Cit ADC 4d (Figure 8C). [Figure 9A]Figures 9A-9D: Gating strategy and CD15 / CD66b expression in differentiated hematopoietic cells. (Figure 9A) Gating strategy. Scatter, single cell selection, and CD15 / CD66b negative areas were set using unstained cells from the vehicle control group. ULTRACOMP EBEADS™ Compensation Beads (Invitrogen) labeled with CD15-APC and / or CD66b-FITC were also used to set voltage and gating parameters to obtain accurate fluorescent signals. Isotype control IgG-APC and -FITC treated cells were used to confirm the specificity of CD15-APC and CD66b-FITC antibodies. The same gating strategy was also used for flow cytometry shown in this figure (Figures 9B-9D). (Figures 9B-9D) Representative 2D histograms of differentiated hematopoietic cells following 7 days of treatment with vehicle control (Figure 9B), 200 nM EGCit-MMAE ADC 4b (Figure 9C), and 200 nM EGCit-MMAE ADC 4c (Figure 9D). All experiments were performed in triplicate. Data were acquired using an LSR II flow cytometer (BD Biosciences) and Diva acquisition software (version 8.0.1, BD Biosciences) and analyzed using FlowJo analysis software (v10.8.1, FlowJo, LLC). [Figure 9B] See legend to Figure 9A. [Figure 9C] See legend to Figure 9A. [Figure 9D] See legend to Figure 9A. [Figure 10]Figures 10A-10D: Tolerability and hematology analyses. (Figure 10A) Body weight change following administration of a single dose (80 mg kg-1) of vehicle control (n = 4), KADCYLA® (n = 6), ENHERTU® (n = 7), or EGCit ADC 4c (n = 7) to female CD-1® mice. No mice showed acute symptoms or reached the predefined humane endpoint during the 5 days of monitoring. (Figures 10B-10D) Red blood cell (RBC, Figure 10B), platelet (PLT, Figure 10C), and neutrophil (NEUT, Figure 10D) counts 5 days after injection of a single dose (80 mg kg-1) of vehicle control (n = 4), EGCit ADC 4c (n = 4), ENHERTU® (n = 3), or KADCYLA® (n = 3). Dotted lines (high and low) represent the 95% confidence interval for each parameter in healthy CD-1® mice (data from Charles River Laboratories). Data are presented as mean (bars) ± SEM. [Figure 11]Figures 11A-11D: Additional in vitro cytotoxicity data. (Figures 11A-11B) Cytotoxicity of anti-HER2 EGCit-DuoDM ADC (DAR 4, 6, cyan circles) and anti-HER2 EGCit-MMAE / F dual drug ADC (DAR 4+2, 7a, magenta open squares) in KPL-4 (Figure 11A) and JIMT-1 (Figure 11B). (Figure 11C) Cytotoxicity of anti-EGFRvIII VCit-MMAE ADC (DAR 4, 8a, lilac triangles) and anti-EGFRvIII EGCit-MMAE ADC (DAR 4, 8b, magenta squares) in U87ΔEGFR-luc. (FIG. 11D) Cytotoxicity of anti-EGFRvIII EGCit-PABC-DuoDM ADC (DAR 4, S21a, orange squares) and anti-EGFRvIII EGCit-PABQ-DuoDM ADC (DAR 4, S21b, purple diamonds) in U87ΔEGFR-luc. Concentrations (nM) are based on antibody dose without normalization to each DAR. All assays were performed in quadruplicate. Data are presented as mean + / - SEM (n = 4 for 6 and 7a; n = 3 for 8a,b and S21a,b). [Figure 12]Figures 12A-12E: Body weight change and antitumor activity in various xenograft models. (Figures 12A-12B) Body weight change (Figure 12A) and tumor volume change (Figure 12B) during treatment of KPL-4 tumor-bearing mice with each ADC at 1 mg kg-1. At tumor volumes of approximately 100 mm3, mice were intravenously injected with a single dose of vehicle control (black circle), KADCYLA® (light purple square), ENHERTU® (purple inverted triangle), EVCit-MMAE ADC 4b (green triangle), EGCit-MMAE ADC 4c (magenta square) or EGCit-DuoDM ADC 6 (cyan circle). (Figures 12C-12D) Body weight change (Figure 12C) and tumor volume change (Figure 12D) during treatment of JIMT-1 / MDA-MB-231 mixed tumor-bearing mice. Mice were intravenously injected with a single dose of ENHERTU® (3 mg kg-1, purple inverted triangles, n = 5) or EGCit-MMAE / F DAR 4+2 dual drug ADC 7a (1 mg kg-1, magenta open squares, n = 6). Note: Tumor volume and survival curve data shown here for vehicle control (black circles with dotted curves, n = 4) and EGCit dual drug ADC 7b (1 mg kg-1, green open triangles with dotted curves, n = 5) were generated previously. e Body weight change during treatment of orthotopic U87ΔEGFR-luc tumor-bearing mice with each ADC at 5 mg kg-1. Mice were intravenously administered a single dose of vehicle control, anti-EGFRvIII VCit-MMAE ADC 8a (light purple), or anti-EGFRvIII EGCit-MMAE ADC 8b (magenta). For vehicle and 8a n = 6; for 8b n = 7. Data are presented as mean + / - SEM. [Figure 13-1]Synthesis of pyrene probe. Reagents and conditions: (a) Fmoc-citrulline, DIPEA, DMF, room temperature, 2 hours; (b) p-aminobenzyl alcohol, EEDQ, DCM / MeOH = 4:1, room temperature, overnight; (c) bis(4-nitrophenyl) carbonate, DMAP, DMF, room temperature, 2 hours; (d) 20% TFA / DCM, room temperature, 40 minutes (for S3a) or 1N-HCl / ACN, room temperature, 1-3 hours (for S3b-e and S3g,h); (e) sarcosine-pyrene, DMAP, DIPEA, DMF, 37°C, 4 hours. [Figure 13-2] See description of Figure 13-1. [Figure 13-3] See description of Figure 13-1. [Figure 14] Synthesis of BCN-MMAE modules (a-c). Reagents and conditions: (a) p-aminobenzyl alcohol, EEDQ, DCM / MeOH = 4:1, room temperature, overnight; (b) bis(4-nitrophenyl) carbonate, DMAP, DMF, room temperature, 2 hours; (c) 20% TFA / DCM, room temperature, 40 minutes (for S5a and S5b); (d) MMAE, HOAt, DIPEA, DMF, 37 °C, overnight; (e) 50% diethylamine / DMF, room temperature, 1 hour; (f) BCN-NHS, DIPEA, DMF, room temperature, 3 hours. [Figure 15] Synthesis of BCN-EVCit-MMAE module S8. Reagents and conditions: (a) 20% TFA / DCM, room temperature, 1 h; (b) 50% diethylamine / DMF, room temperature, 1 h; (c) BCN-NHS, DIPEA, DMF, room temperature, overnight. [Figure 16] Synthesis of non-cleavable-MMAE module S10. Reagents and conditions: (a) Boc-peg4-acid, HATU, DIPEA, DMF, room temperature, 1 hour; (b) 50% TFA / DCM, room temperature, 30 minutes; (c) BCN-NHS, DIPEA, DMF, room temperature, 30 minutes. [Figure 17]Synthesis of TCO-EGCit-MMAF module S12. Reagents and conditions: (a) 20% TFA / DCM, room temperature, 50 min; (b) MMAF, HOAt, DIPEA, DMF, 37°C, overnight; (c) 50% diethylamine / DMF, room temperature, 30 min; (d) TCO-NHS, DIPEA, DMF, room temperature, 3.5 h. [Figure 18] Synthesis of BCN-DuoDM module (S15). Reagents and conditions: (a) 4-nitrophenyl chloroformate, DIPEA, ACN, rt, 30 min; (b) t-butyl methyl(2-(methylamino)ethyl)carbamate, rt, 15 min; (c) 20% TFA / DCM, rt, 50 min (for S5a), 50% TFA / DCM, 0°C, 30 min (for S13); (d) Boc-deprotection S11, DIPEA, DMF, rt, 2 h; (e) 50% diethylamine / DMF, rt, 30 min; (f) BCN-NHS, DIPEA, DMF, rt, 1 h. [Figure 19] Synthesis of the BCN-DuoDM-glucuronide module (S20). Reagents and conditions: (a) BF3·Et2O, methyl-(2,3,4-tri-O-acetyl-α-D-glucopyranosyl trichloroacetimidate, DCM, MS(4Å), -20°C, 2 h, then BF3·Et2O, rt, 2 h; (b) 5-(2-dimethylaminoethoxy)indole-2-carboxylic acid, EDC·HCl, DMF, rt, 1.5 h; (c) thionyl chloride, DCM, DMF, 0°C, 2.5 h; (d) S17, DIPEA, NaI, DMF, rt, overnight; (e) 20%TFA / DCM, rt, 1 h; (f) LiOH·H2O, MeOH, rt, 1 h; (g) BCN-NHS, DIPEA, DMF, rt, 1.5 h. [Figure 20] HPLC and ESI-MS data for S3a. [Figure 21] HPLC and ESI-MS data for S3b. [Figure 22] HPLC and ESI-MS data for S3c. [Diagram 23]HPLC and ESI-MS data for S3d. [Figure 24] HPLC and ESI-MS data for S3e. [Diagram 25] HPLC and ESI-MS data for S3f. [Figure 26] HPLC and ESI-MS data for S3g. [Figure 27] HPLC and ESI-MS data for S3h. [Figure 28] S3i HPLC and ESI-MS data. [Figure 29] HPLC and ESI-MS data for S3j. [Diagram 30] HPLC and ESI-MS data for 3a. [Diagram 31] HPLC and ESI-MS data for 3b. [Diagram 32] HPLC and ESI-MS data for 3c. [Diagram 33] 3d HPLC and ESI-MS data. [Diagram 34] HPLC and ESI-MS data for 3e. [Diagram 35] HPLC and ESI-MS data for 3f. [Diagram 36] HPLC and ESI-MS data for S1a. [Figure 37] HPLC and ESI-MS data for S1b. [Figure 38] HPLC and ESI-MS data for S1c. [Figure 39] HPLC and ESI-MS data for S1d. [Diagram 40] HPLC and ESI-MS data for S5a. [Diagram 41] HPLC and ESI-MS data for S5b. [Diagram 42] HPLC and ESI-MS data for S5c. [Diagram 43] HPLC and ESI-MS data for S6a. [Diagram 44]HPLC and ESI-MS data for S6b. [Diagram 45] HPLC and ESI-MS data for S6c. [Figure 46] HPLC and ESI-MS data for S7a. [Figure 47] HPLC and ESI-MS data for S7b. [Figure 48] HPLC and ESI-MS data for S7c. [Figure 49] HPLC and ESI-MS data for S8. [Figure 50] HPLC and ESI-MS data for S9. [Figure 51] HPLC and ESI-MS data for S10. [Figure 52] HPLC and ESI-MS data for S11. [Figure 53] HPLC and ESI-MS data for S12. [Figure 54] HPLC and ESI-MS data for S13. [Figure 55] HPLC and ESI-MS data for S14. [Figure 56] HPLC and ESI-MS data for S15. [Figure 57] HPLC and ESI-MS data for S16. [Figure 58] HPLC and ESI-MS data for S17. [Figure 59] HPLC and ESI-MS data for S18. [Figure 60] HPLC and ESI-MS data for S19. [Figure 61] S20 HPLC and ESI-MS data. [Figure 62-1]Figures 62A-62F: ESI-mass spectra of VCit ADC 4a (Figure 62A), EVCit ADC 4b (Figure 62B), EV(N-Me)Cit 4d (Figure 62C), GCit ADC 4e (Figure 52D), non-cleavable ADC 4f (Figure 62E), isotype control EGCit ADC 5 (Figure 62F). Asterisks (*) indicate fragment ions detected in ESI-MS analysis. [Figure 62-2] See description of Figure 62-1. [Figure 63] Figures 63A-63B ESI-mass spectra of anti-HER2 EGCit DuoDM ADC 6 (Figure 63A) and anti-HER2 EGCit MMAE / F dual drug ADC 7a (Figure 63B). Asterisks (*) indicate fragment ions detected by ESI-MS analysis. [Figure 64-1] Figures 64A-64F ESI-mass spectra of aglycosylated N297A anti-EGFRvIII mAb (Figure 64A), mAb-linker conjugate (Figure 64B), anti-EGFRvIII VCit MMAE ADC 8a (Figure 64C), anti-EGFRvIII EGCit MMAE ADC 8b (Figure 64D), anti-EGFRvIII EGCit PABC-DuoDM ADC S21a (Figure 64E), and anti-EGFRvIII EGCit PABQ-DuoDM ADC S21b (Figure 64F). Asterisks (*) indicate fragment ions detected in ESI-MS analysis. [Figure 64-2] See description of Figure 64-1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] Description of Exemplary Embodiments Previous studies have shown that glutamic acid-valine-citrulline (EVCit) tripeptide linkers are stable in human and mouse plasma. However, as is the case with VCit linkers and other similar valine-based linkers, EVCit linkers have been shown to be susceptible to human neutrophil elastase-mediated degradation and inhibit the population of differentiating neutrophils (Figure 1B) (WO2018218004; incorporated herein by reference in its entirety). Thus, in certain embodiments, the present disclosure provides glutamic acid-glycine-citrulline (EGCit) tripeptide linkers. Replacing valine with a small glycine residue is a unique chemical modification that contradicts the general belief in the field that bulky amino acids such as valine are necessary to ensure effective payload release upon intracellular cleavage. These linkers can resolve or reduce clinical issues caused by linker instability (e.g., hepatotoxicity, neutropenia, thrombocytopenia, leukopenia, and pancytopenia) without compromising ADC therapeutic efficacy.

[0037] Further provided herein is a drug conjugate, such as for targeted therapy, comprising the chemical linker. The drug conjugate may be an antibody-drug conjugate. The chemical linker contains a glutamic acid-glycine-citrulline (EGCit) tripeptide sequence, which provides significantly improved circulation stability, long-term stability in both rodent and primate plasma, resistance to degradation mediated by neutrophil elastase, and allows traceless drug release upon internalization into target cells. It has also been demonstrated that EGCit-based ADCs targeting HER2 or EGFR show significantly improved therapeutic efficacy and safety profile in mouse models of human breast cancer and glioblastoma compared to conventional ADCs, including VCit-based ADCs KADCYLA® (T-DM1) and ENHERTU® (DS-8201). In particular, EGCit-based ADCs have been shown to be effective in treating HER2- or EGFR-resistant HER2 ... -1showed no discernible hepatotoxicity in healthy mice. These findings indicated that the EGCit linker technology not only ensures a smooth transition from preclinical trials to human evaluation, but also provides a broadly applicable solution to significantly widen the therapeutic window of targeted drug delivery systems, including ADCs. The EGCit linker and other linkers with similar chemical compositions can provide a general approach to create truly effective and safe ADCs and other drug delivery systems for cancer therapy as well as the treatment of other diseases. The EGCit linker does not depend on a specific antibody structure or drug, and therefore this technology can be used to construct any type of ADC. In addition, the EGCit linker is more hydrophilic than the VCit linker, which may reduce the risk of protein aggregation. Thus, the present linker may lead to new, effective, and safe ADCs for treating cancer and other diseases.

[0038] Further provided herein is a method for preparing drug conjugates, such as ADCs, using this linker, which allows for the incorporation of one or more therapeutic compounds.By using MTGase, antibody can be coupled with the amine that is composed of pendant diazide functionality or azide-tetrazine heterobifunctionality.The azide and tetrazine moieties can then be further functionalized by cycloaddition to incorporate chemical payloads.

[0039] I. Delivery of Therapeutic Agents via Drug Conjugates A. Therapeutic Agents Any number of drugs can be used or modified to be used as the reactive partner for conjugating to the linker of the present disclosure.Examples of drugs include small molecules, peptide drugs, oligonucleotides, antibodies, and their fragments.Therefore, the present disclosure provides drug-antibody conjugates.

[0040] As used herein, "small molecule drug" refers to a compound, e.g., an organic compound, that exhibits a pharmaceutical activity of interest and generally has a molecular weight of 800 Da or less, or 2000 Da or less, but can include molecules up to 5 kDa and can be as large as 10 kDa.An inorganic small molecule refers to a molecule that does not contain carbon atoms, and an organic small molecule refers to a compound that contains at least one carbon atom.

[0041] As used herein, "peptide drug" refers to an amino acid-containing polymeric compound, and is meant to include naturally occurring and non-naturally occurring peptides, oligopeptides, cyclic peptides, polypeptides, and proteins, as well as peptidomimetics.Peptide drugs can be obtained by chemical synthesis or produced from genetically encoded sources (e.g., recombinant sources).Peptide drugs can range in molecular weight, from 200Da to 10kDa or more.

[0042] As used herein, "oligonucleotide" refers to a therapeutic agent that includes one or more nucleobases, such as mRNA, siRNA, or other polynucleotide agents, that can be used to treat a condition, such as a disease or disorder. These oligonucleotides may be naturally occurring or synthetically prepared. These oligonucleotides may be siRNA, miRNA, tRNA, pri-miRNA, messenger RNA (mRNA), clustered regularly interspaced short palindromic repeats (CRISPR)-associated nucleic acids, single guide RNA (sgRNA), CRISPR-RNA (crRNA), transactivating crRNA (tracrRNA), plasmid DNA (pDNA), transfer RNA (tRNA), antisense oligonucleotide (ASO), guide RNA, double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), single-stranded RNA (ssRNA), and double-stranded RNA (dsRNA). These nucleic acids may be chemically modified to reduce their ability to be degraded in vivo.

[0043] It is contemplated that a drug conjugate such as an ADC constructed from the compounds of the present disclosure can be used to deliver one or more drugs. In some embodiments, the drug is a cancer chemotherapeutic agent. For example, if the polypeptide is an antibody (or a fragment thereof) with specificity for tumor cells, the antibody can be modified as described herein to include modified amino acids, which can then be conjugated to a cancer chemotherapeutic agent. Cancer chemotherapeutic agents include non-peptide (i.e., non-proteinaceous) compounds that reduce the proliferation of cancer cells, including cytotoxic agents and cytostatic agents. Non-limiting examples of chemotherapeutic agents include alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, plant (vinca) alkaloids, and steroid hormones.

[0044] Suitable cancer chemotherapeutic agents include dolastatin and its active analogs and derivatives; and auristatin and its active analogs and derivatives.See, for example, WO 96 / 33212, WO 96 / 14856, and US 6,323,315.For example, dolastatin 10 or auristatin PE can be included in the antibody-drug conjugate of the present disclosure.Suitable cancer chemotherapeutic agents also include maytansinoids and its active analogs and derivatives (see, for example, EP 1,391,213; and Liu et al. 1996); and duocarmycins and its active analogs and derivatives (including, for example, synthetic analogs KW-2189 and CBI-TMI).

[0045] The agent that acts to reduce cell proliferation is known in the art and is widely used.Such agent includes alkylating agents such as nitrogen mustard, nitrosourea, ethyleneimine derivatives, alkylsulfonates, and triazenes, including but not limited to mechlorethamine, cyclophosphamide (Cytoxan™), melphalan (L-sarcolysin), carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU), streptozocin, chlorozotocin, uracil mustard, chlormethine, ifosfamide, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan, dacarbazine, and temozolomide.

[0046] Antimetabolites include folic acid analogs, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors, including, but not limited to, cytarabine, (CYTOSAR-U), cytosine arabinoside, fluorouracil (5-FU), floxuridine (FudR), 6-thioguanine, 6-mercaptopurine (6-MP), pentostatin, 5-fluorouracil (5-FU), methotrexate, 10-propargyl-5,8-dideazafolate (PDDF, CB3717), 5,8-dideazatetrahydrofolic acid (DDATHF), leucovorin, fludarabine phosphate, pentostatin, and gemcitabine.

[0047] Suitable natural products and their derivatives (e.g., vinca alkaloids, antitumor antibiotics, enzymes, lymphokines, and epipodophyllotoxins) include, but are not limited to, Ara-C, paclitaxel (Taxol®), docetaxel (Taxotere®), deoxycoformycin, mitomycin-C, L-asparaginase, azathioprine; brequinar; alkaloids, such as vincristine, vinblastine, vinorelbine, vindesine, and the like; podophyllotoxins, such as etoposide, teniposide, and the like; antibiotics, such as anthracyclines, cyclosporines, and the like; Examples of such anti-inflammatory drugs include cyclosporine, daunorubicin hydrochloride (daunomycin, rubidomycin, cerbidine), idarubicin, doxorubicin, epirubicin and morpholino derivatives; phenoxyzombis cyclopeptides such as dactinomycin; basic glycopeptides such as bleomycin; anthraquinone glycosides such as plicamycin (mithramycin); anthracenediones such as mitoxantrone; azirinopyrroloindoleziones such as mitomycin; macrocyclic immunosuppressants such as cyclosporine, FK-506 (tacrolimus, prograf), rapamycin and the like.

[0048] Other antiproliferative cytotoxic agents are navelbene, CPT-11, anastrazole, letrazole, capecitabine, reloxafme, cyclophosphamide, ifosamide, and droloxafme.

[0049] Microtubule affecting agents with antiproliferative activity are also suitable for use, including, but not limited to, allocolchicine (NSC 406042), halichondrin B (NSC 609395), colchicine (NSC 757), colchicine derivatives (e.g., NSC 33410), dolstatin 10 (NSC 376128), maytansine (NSC 153858), rhizoxin (NSC 332598), paclitaxel (Taxol®), Taxol® derivatives, docetaxel (Taxotere®), thiocolchicine (NSC 361792), tritylcysteine, vinblastine sulfate, vincristine sulfate, natural and synthetic epothilones, including but not limited to epothilone A, epothilone B, discodermolide; estramustine, nocodazole, and the like.

[0050] Hormonal regulators and steroids (including synthetic analogs) suitable for use include, but are not limited to, adrenocorticosteroids, such as prednisone, dexamethasone, and the like; estrogens and pregestins, such as hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate, estradiol, clomiphene, tamoxifen, and the like; and adrenal cortical suppressants, such as aminoglutethimide; 17a-ethynyl estradiol; ol; diethylstilbestrol, testosterone, fluoxymesterone, dromostanolone propionate, testolactone, methylprednisolone, methyl-testosterone, prednisolone, triamcinolone, chlorotrianisene, hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesterone acetate, leuprolide, flutamide (Drogenil), toremifene (Fareston) and Zoladex®. Estrogen stimulates proliferation and differentiation; therefore, compounds that bind to the estrogen receptor are used to block this activity. Corticosteroids can inhibit T cell proliferation.

[0051] Other suitable chemotherapeutic agents include metal complexes such as cisplatin (cis-DDP), carboplatin, etc.; ureas such as hydroxyurea; and hydrazines such as N-methylhydrazine; epidophyllotoxins; topoisomerase inhibitors; procarbazine; mitoxantrone; leucovorin; tegafur, etc. Other antiproliferative agents of interest include immunosuppressants such as mycophenolic acid, thalidomide, desoxyspergualin, azasporin, leflunomide, mizoribine, azaspirane (SKF 105685); Iressa® (ZD 1839, 4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-(3-(4-morpholinyl)propoxy)quinazoline), etc. Topoisomerase I inhibitors such as irinotecan, topotecan, SN-38, exatecan (part of deruxtecan) and other analogues RNA polymerase inhibitors (II and III) such as α-amanitin.

[0052] Taxanes are suitable for use. "Taxane" includes paclitaxel, as well as any active taxane derivative or prodrug. "Paclitaxel" (which should be understood herein to include analogs, formulations and derivatives such as, for example, docetaxel, TAXOL™, TAXOTERE™ (a formulation of docetaxel), the 10-desacetyl analog of paclitaxel, and the 3'N-desbenzoyl-3'Nt-butoxycarbonyl analog of paclitaxel) can be readily prepared utilizing techniques known to those skilled in the art (WO 94 / 07882, WO 94 / 07881, WO 94 / 07880, WO 94 / 07876, WO 93 / 23555, WO 93 / 10076; U.S. Patent Nos. 5,294,637; 5,283,253; 5,279,949; 5,274,137; 5,202,448; 5,200,534; 5,229,529; and EP 590,267), or from a variety of commercial sources including, for example, Sigma Chemical Co., St. Louis, Mo. (T7402 from Taxus brevifolia; or T-1912 from Taxus yannanensis).

[0053] Paclitaxel should be understood to refer not only to the common chemically available forms of paclitaxel, but also to analogues and derivatives (e.g., Taxotere™ docetaxel, as described above) and paclitaxel conjugates (e.g., paclitaxel-PEG, paclitaxel-dextran, or paclitaxel-xylose).

[0054] The term "taxane" also includes various known derivatives, including both hydrophilic and hydrophobic derivatives.Taxane derivatives include, but are not limited to, the galactose and mannose derivatives described in International Patent Application WO 99 / 18113; the piperazino and other derivatives described in WO 99 / 14209; the taxane derivatives described in WO 99 / 09021, WO 98 / 22451, and US Patent No. 5,869,680; the 6-thio derivatives described in WO 98 / 28288; the sulfenamide derivatives described in US Patent No. 5,821,263; and the taxol derivatives described in US Patent No. 5,415,869.It further includes the prodrugs of paclitaxel, including, but are not limited to, those described in WO 98 / 58927; WO 98 / 13059; and US Patent No. 5,824,701.

[0055] Biological response modifiers suitable for use include, but are not limited to, (1) inhibitors of tyrosine kinase (RTK) activity; (2) inhibitors of serine / threonine kinase activity; (3) tumor-associated antigen antagonists, such as antibodies that specifically bind to tumor antigens; (4) apoptosis receptor agonists; (5) interleukin-2; (6) IFN-α; (7) IFN-γ; (8) colony-stimulating factors; and (9) inhibitors of angiogenesis.

[0056] B. Methods for Modifying Drugs to Include Reactive Partners Drugs to be conjugated to polypeptides can be modified to incorporate reactive partners for reaction with the polypeptide. When the drug is a peptide drug, the reactive moiety (e.g., aminooxy or hydrazide) can be placed at the N-terminal region, N-terminus, C-terminal region, C-terminus, or at an internal position relative to the peptide. For example, one method involves synthesizing a peptide drug with an aminooxy group. In this example, the peptide is synthesized from a Boc-protected precursor. The amino group of the peptide can be reacted with a compound containing a carboxylic acid group and an oxy-N-Boc group. As an example, the amino group of the peptide is reacted with 3-(2,5-dioxopyrrolidin-1-yloxy)propanoic acid. Other variations on the compound containing a carboxylic acid group and an oxy-N-protecting group can include different numbers of carbons in the alkylene linker and the substituents on the alkylene linker. The reaction between the amino group of the peptide and the compound containing a carboxylic acid group and an oxy-N-protecting group occurs through standard peptide ligation chemistry.Examples of peptide coupling reagents that can be used include, but are not limited to, DCC (dicyclohexylcarbodiimide), DIC (diisopropylcarbodiimide), di-p-toluoylcarbodiimide, BDP (1-benzotriazole diethylphosphate-1-cyclohexyl-3-(2-morpholinylethyl)carbodiimide), EDC (1-(3-dimethylaminopropyl-3-ethyl-carbodiimide hydrochloride), cyanuric fluoride, cyanuric chloride, TFFH (tetramethylphenyl ether), tetramethylphenyl ether, ... (O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate), DPPA (diphenylphosphorazidate), BOP (benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate), HBTU (O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate), TBTU (O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium tetrafluoroborate), t), TSTU (O-(N-succinimidyl)-N,N,N',N'-tetramethyluronium tetrafluoroborate), HATU ((N-[(dimethylamino)-1-H-1,2,3-triazolo[4,5,6]-pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide), BOP-C1 (bis(2-oxo-3-oxazolidinyl)phosphinic chloride), PyBOP ((1-H-1,2,3-benzotriazole-1 -yloxy)-tris(pyrrolidino)phosphonium tetrafluorophosphate), BrOP (bromo tris(dimethylamino)phosphonium hexafluorophosphate), DEPBT (3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one), PyBrOP (bromo tris(pyrrolidino)phosphonium hexafluorophosphate). As non-limiting examples, HOBt and DIC can be used as peptide linking reagents.

[0057] Deprotection to expose aminooxy functionality is performed on peptides containing N-protecting groups. Deprotection of N-oxysuccinimide groups occurs, for example, according to standard deprotection conditions for cyclic amino groups. Deprotection conditions can be found in Greene and Wuts, Protective Groups in Organic Chemistry, 3rd Ed., 1999, John Wiley & Sons, NY, and Harrison et al. Specific deprotection conditions include hydrazine reagents, amino reagents, or sodium borohydride. Deprotection of Boc protecting groups occurs with TFA. Other reagents for deprotection include, but are not limited to, hydrazine, methylhydrazine, phenylhydrazine, sodium borohydride, and methylamine. Products and intermediates can be purified by conventional means, such as HPLC purification.

[0058] Those skilled in the art will recognize that factors such as pH and steric hindrance (i.e., accessibility of amino acid residues to react with the reactive partner of interest) are important. Modification reaction conditions to provide optimal conjugation conditions are well within the skill of the art and are routine in the art. When conjugation is performed with a polypeptide present in or on a living cell, conditions are selected to be physiologically compatible. For example, the pH can be temporarily lowered for a time sufficient to allow the reaction to occur, but within a period of time tolerated by the cell (e.g., about 30 minutes to 1 hour). Physiological conditions for performing modification of polypeptides on cell surfaces can be similar to those used in ketone-azide reactions in the modification of cells bearing cell surface azides (see, e.g., US 6,570,040).

[0059] The small molecule compounds that contain or are modified to contain nucleophilic groups that serve as reactive partners in the compounds or conjugates disclosed herein are also contemplated for use as drugs in the polypeptide-drug conjugates of the present disclosure.General methods are known in the art for the chemical synthesis schemes and conditions useful for synthesizing compounds of interest (see, for example, Smith and March, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Fifth Edition, Wiley-Interscience, 2001; or Vogel, A Textbook of Practical Organic Chemistry, Including Qualitative Organic Analysis, Fourth Edition, New York: Longman, 1978).

[0060] C. Peptide Drugs In some cases, the conjugate comprises a covalently attached peptide. Suitable peptides include, but are not limited to, cytotoxic peptides, angiogenic peptides, angiogenesis inhibitor peptides, B cell activating peptides, T cell activating peptides, antiviral peptides, peptides that inhibit viral fusion, peptides that enhance the production of one or more lymphocyte populations, antimicrobial peptides, growth factors, growth hormone releasing factors, vasoactive peptides, anti-inflammatory peptides, peptides that control glucose metabolism, antithrombotic peptides, antinociceptive peptides, vasodilatory peptides, platelet aggregation inhibitors, analgesics, and the like.

[0061] In some embodiments, peptides can be chemically synthesized to include groups reactive with amino acid residues or modified amino acid residues of a polypeptide. Suitable synthetic peptides have a length of 5 amino acids to 100 amino acids, or more than 100 amino acids; for example, suitable peptides have a length of 5 amino acids (aa) to 10 aa, 10 aa to 15 aa, 15 aa to 20 aa, 20 aa to 25 aa, 25 aa to 30 aa, 30 aa to 40 aa, 40 aa to 50 aa, 50 aa to 60 aa, 60 aa to 70 aa, 70 aa to 80 aa, 80 aa to 90 aa, or 90 aa to 100 aa.

[0062] In some embodiments, the peptides can be modified to include a nucleophile-containing moiety (e.g., an aminooxy or hydrazide moiety) and reacted, for example, with an fGly-containing polypeptide to obtain a conjugate in which the polypeptide and peptide are linked by a hydrazone bond or an oxime bond, respectively. Exemplary methods for synthesizing peptides, such as synthetic peptides that contain reactive groups reactive with amino acid residues or modified amino acid residues of a polypeptide, are described above.

[0063] Suitable peptides include, but are not limited to, hLF-11 (an 11 amino acid N-terminal fragment of lactoferrin), an antimicrobial peptide; granulysin, an antimicrobial peptide; plectasin (NZ21 14; SAR 215500), an antimicrobial peptide; viral fusion inhibitors such as Fuzeon (enfuvirtide), TRI-1249 (T-1249; see, e.g., Matos et al., 2010), TRI-2635 (T-2635; see, e.g., Eggink et al., 2009), T651, and TRI-1144; C5a receptor inhibitors such as PMX-53, JPE-1375, and JSM-7717; POT-4, human complement factor C3 inhibitor; Pancreate (INGAP-derived sequence, HIP-human proislet protein somatostatin; somatostatin analogues, e.g., DEBIO 8609 (Sanvar), octreotide, octreotide (C2L), octreotide QLT, octreotide LAR, Sandostatin LAR, SomaLAR, somatuline (Lanreotide), see e.g., Deghenghi et al., 2001; TH9507 (tesamorelin, growth hormone releasing factor); POL7080 (protegrin analogs, antimicrobial peptides); relaxin; corticotropin releasing factor agonists, e.g. urotensin, sauvagine, etc.; heat shock protein derivatives, e.g. DiaPep277; human immunodeficiency virus entry inhibitors; heat shock protein 20 mimetics, e.g. AZX100; thrombin receptor activating peptides, e.g. TP508 (Crysalin); urocortin 2 mimetics (e.g. CRF2 agonists), e.g. urocortin-2; immunostimulants, e.g. zadaxin (thymalfasin; thymosin-al), see, e.g. Sjogren (2004) J. Gastroenterol. Hepatol. 19:S69; hepatitis C virus (HCV) entry inhibitor E2 peptides, e.g. HCV3; atrial natriuretic peptides, e.g. HANP (Sun 4936;Carperitide;Annexin peptides;Defensins (antimicrobial peptides), e.g. hBD2-4;Defensins (antimicrobial peptides), e.g. hBD-3;Defensins (antimicrobial peptides), such as PMX-30063; histatins (antimicrobial peptides), such as histatin-3, histatin-5, histatin-6, and histatin-9; histatins (antimicrobial peptides), such as PAC-113; indolicidins (antimicrobial peptides), such as MX-594AN (Omniganin; CLSOOl); indolicidins (antimicrobial peptides), such as Omnigard (MBI-226; CPI-226); antimicrobial peptides, such as insect cecropins; antimicrobial peptides, such as lactoferrin (talactoferrin); LL-37 / cathelicidin derivatives (antimicrobial peptides), such as P60.4 (OP-145); magainins (antimicrobial peptides), such as pexiganan (MSI-78; Suponex); protegrins (antimicrobial peptides), such as IB-367 (Iseganan); agan peptides; beta-natriuretic peptides, such as Natrecor, or Noratak (nesiritide), or uralitide; bivalarudin (Angiomax), thrombin inhibitors; C-peptide derivatives; calcitonins, such as miacalcin (Fortical); enkephalin derivatives; erythropoiesis stimulating peptides, such as hematide; gap junction regulators, such as danegaptide (ZP1609); gastrin releasing peptide; ghrelin; glucagon-like peptides; glucagon Gon-like peptide 2 analogues, such as ZP1846 or ZP1848; glucosaminyl muramyl dipeptides, such as GMDP; glycopeptide antibiotics, such as oritabacin; teicoplanin derivatives, such as dalbavacin; gonadotropin releasing hormone (GnRH), such as Zoladex (Lupon) or triptorelin; histone deacetylase (HDAC) inhibitors depsipeptides, such as PM02734 (Irvalec); integrins, such as eptifibatide; insulin analogues, such as Humulog; kahalalide depsipeptides, such as PM02734; kallikrein inhibitors, such as carbitol (ecallantide); antibiotics, such as telavancin; lipopeptides, such as cubicin or MX-2401;Luteinizing hormone releasing hormone (LHRH), e.g. goserelin; LHRH synthetic decapeptide agonist analogues, e.g. Treistal (triptorelin pamoate); LHRH, e.g. Eligard; M2 protein channel peptide inhibitors; metreleptin; melanocortin receptor agonist peptides, e.g. bremalanotide / PT-141; melanocortin; muramyl tripeptides, e.g. mepact (mifamurtide); myelin basic protein peptides, e.g. MBP 8298 (zircotide); N-type voltage-gated calcium channel inhibitors such as ziconotide (prialt); parathyroid hormone peptides; parathyroid analogs such as 768974; peptide hormone analogs such as UGP281; prostaglandin F2-a receptor inhibitors such as PDC31; protease inhibitors such as PPL-100; surfacsin; thrombospondin-1 (TSP-1) mimetics such as CVX-045 or ABT 510; vasoactive intestinal peptide; vasopressin; Y2R agonist peptides such as RG7089; obinepeptide; and TM30339.

[0064] II. Linker In some aspects, the present disclosure provides linkers that can be used to link one or more different drug and / or cell targeting moieties to a drug molecule.

[0065] In other aspects, the linker may further comprise one or more spacer groups. The spacer group may comprise an amino acid sequence that is recognized by peptidase in vivo and leads to the cleavage of the cell targeting moiety from the drug. In some embodiments, the peptidase is an endosomal or lysosomal peptidase. In other embodiments, the peptidase is an extracellular peptidase, such as matrix metalloprotease, thimeto oligopeptidase, or CD10. One example is the amino acid sequence valine-citrulline, which is cleavable by cathepsin B. Other non-limiting examples of peptide sequences that are cleavable include valine-alanine, valine-lysine, valine-ornithine, phenylalanine-alanine, phenylalanine-lysine, and phenylalanine-ornithine. In addition, it is contemplated that other functional moieties may be added to the spacer group that may be used to achieve decoupling of the drug and the cell targeting moiety, including but not limited to hydrazone, disulfide bond, or ester. It is also contemplated that the spacer group may further comprise one or more self-immolative groups. As used herein, a self-immolative group is a group that undergoes decomposition once cleaved by a single functional group. Self-immolative groups are well known in the context of ADCs and are taught by Carl et al., 1981; WO 81 / 01145; Dubowchik et al., 1999; U.S. Pat. No. 6,214,345; Told et al., 2002; Doronina et al., 2003 (errata, p. 941); U.S. Pat. No. 7,691,962; US 2008 / 0279868; WO 2008 / 083312; U.S. Pat. No. 7,375,078 B2; US 2003 / 0096743; Burke et al., 2017; Staben et al., 2016, which are incorporated by reference in their entireties. One particular example of a self-immolative group is para-aminobenzyl alcohol, para-aminobenzyloxycarbonyl, or para-aminobenzyldialykylammonium.

[0066] III. Compounds and Formulations Thereof A. Compound The compounds provided by the present disclosure are shown, for example, in the summary section above, and in the examples and claims below. They can be made using the methods outlined in the examples section. The ADCs described herein can be synthesized, for example, according to the methods described in the examples section below. These methods can be further modified and optimized using the principles and techniques of organic chemistry applied by those skilled in the art. For example, such principles and techniques are taught in March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure (2007), which is incorporated herein by reference.

[0067] The ADCs described herein may contain one or more asymmetrically substituted carbon or nitrogen atoms and may be isolated as optically active or racemic.Therefore, unless a specific stereochemistry or isomer is specifically indicated, all chiral, diastereomeric, racemic, epimeric, and all geometric isomers of a chemical formula are intended.Compounds may occur as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures, and individual diastereomers.In some embodiments, single diastereomers are obtained.The chiral centers of the compounds of the present disclosure may have S or R configuration.

[0068] The chemical formulas used to represent the ADCs described herein typically show only one of possibly several different tautomers.For example, many types of ketone groups are known to exist in equilibrium with the corresponding enol groups.Similarly, many types of imine groups exist in equilibrium with enamine groups.Regardless of which tautomer is shown for a given compound, and regardless of which tautomer is most predominant, all tautomers of a given chemical formula are intended.

[0069] Additionally, the ADCs described herein, whether used in the indications described herein or otherwise, may have the advantage that they may be more effective, less toxic, longer acting, more potent, produce fewer side effects, be more readily absorbed, and / or have a better pharmacokinetic profile (e.g., higher oral bioavailability and / or lower clearance), and / or possess other useful pharmacological, physical, or chemical properties than compounds known in the prior art.

[0070] Furthermore, the atoms constituting the ADCs described herein are intended to include all isotopic forms of such atoms.As used herein, isotopes include atoms with the same atomic number but different mass numbers.By way of general example and without limitation, hydrogen isotopes include tritium and deuterium, and carbon isotopes include tetrahydrofuran and tetrahydrofuran. 13 C and 14 C is an example.

[0071] The ADCs herein may also exist in prodrug form. If desired, the compounds used in some methods of the present disclosure may be delivered in prodrug form, since prodrugs are known to enhance many desirable properties of pharmaceuticals (e.g., solubility, bioavailability, manufacturability, etc.). Thus, the present disclosure contemplates prodrugs of the compounds of the present disclosure, and methods of delivering the prodrugs. Prodrugs of the ADCs described herein can be prepared by modifying functional groups present in the compounds, such that the modifications are cleaved either by routine manipulation or in vivo to the parent compound. Thus, prodrugs include, for example, compounds described herein with a hydroxy, amino, or carboxy group attached to any group that is cleaved to form a hydroxy, amino, or carboxylic acid, respectively, when the prodrug is administered to a subject.

[0072] It should be recognized that the particular anion or cation forming part of any salt form of the compounds provided herein is not critical, so long as the salt, as a whole, is pharmacologically acceptable. Further examples of pharma-ceutically acceptable salts and methods of their preparation and use are provided in Handbook of Pharmaceutical Salts: Properties, and Use (2002), which is incorporated herein by reference.

[0073] Those skilled in the art of organic chemistry will understand that many organic compounds can form complexes with the solvents in which they are reacted or from which they are precipitated or crystallized. These complexes are known as "solvates". For example, complexes with water are known as "hydrates". Solvates of the ADCs described herein are within the scope of this disclosure. Those skilled in the art of organic chemistry will also understand that many organic compounds can exist in multiple crystal forms. For example, the crystal form can vary from solvate to solvate. Thus, all crystal forms of the ADCs or precursors described herein are within the scope of this disclosure.

[0074] B. Preparation In some embodiments of the present disclosure, drug conjugates, such as ADCs, are included in pharmaceutical formulations. Materials for use in preparing microspheres and / or microcapsules are biodegradable / bioerodible polymers, such as polygalactin, poly-(isobutylcyanoacrylate), poly(2-hydroxyethyl-L-glutamine), and poly(lactic acid). Biocompatible carriers that can be used when formulating controlled release parenteral formulations are carbohydrates (e.g., dextran), proteins (e.g., albumin), lipoproteins, or antibodies. Materials for use in implants can be non-biodegradable (e.g., polydimethylsiloxane), or biodegradable (e.g., poly(caprolactone), poly(lactic acid), poly(glycolic acid), or poly(orthoester), or combinations thereof).

[0075] Formulations for oral use include tablets containing the active ingredient (e.g., an ADC herein) mixed with non-toxic pharmaceutically acceptable excipients. Such formulations are known to those skilled in the art. Excipients may be, for example, inert diluents or fillers (e.g., sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starches such as potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate, or sodium phosphate); granulating and disintegrating agents (e.g., cellulose derivatives such as microcrystalline cellulose, starches such as potato starch, croscarmellose sodium, alginates, or alginic acid); binders (e.g., sucrose, glucose, sorbitol, gum arabic, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethylene glycol); and lubricants, glidants, and antiadhesives (e.g., magnesium stearate, zinc stearate, stearic acid, silica, hydrogenated vegetable oils, or talc). Other pharma- ceutically acceptable excipients can be colorants, flavoring agents, plasticizers, humectants, buffering agents, and the like.

[0076] The tablet may be uncoated or may be coated by known techniques, optionally to delay disintegration and absorption in the gastrointestinal tract, thereby providing a sustained action over a longer period of time. The coating may be adapted to release the active drug in a predetermined pattern (e.g., to achieve a controlled release formulation) or to not release the active drug until after passing through the stomach (enteric coating). The coating may be a sugar coating, a film coating (e.g., based on hydroxypropylmethylcellulose, methylcellulose, methylhydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose, acrylate copolymers, polyethylene glycol, and / or polyvinylpyrrolidone), or an enteric coating (e.g., based on methacrylic acid copolymers, cellulose acetate phthalate, hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose acetate succinate, polyvinyl acetate phthalate, shellac, and / or ethylcellulose). In addition, a time-delay material, such as, for example, glyceryl monostearate or glyceryl distearate, may be utilized.

[0077] IV. Indications A. Hyperproliferative Disorders Hyperproliferative disease may be associated with any disease that causes cells to start reproducing uncontrollably, but a prototypical example is cancer. One of the key elements of cancer is that the normal apoptotic cycle of cells is interrupted, and therefore agents that interrupt cell proliferation are important therapeutic agents for treating these diseases. In this disclosure, the ADCs described herein may be used to cause a reduction in cell number, and thus can potentially be used to treat various types of cancer cell lines. In some aspects, it is expected that the ADCs described herein can be used to treat virtually any malignant tumor.

[0078] Cancer cells that may be treated with the compounds of the present disclosure include, but are not limited to, cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal, gums, head, kidney, liver, lung, nasopharynx, cervix, ovary, prostate, skin, stomach, pancreas, testis, tongue, cervix, or uterus. In addition, the cancer may be of the following histological types, but is not limited to: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; mixed hepatocellular and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma of adenomatous polyps; adenocarcinoma, familial polyposis; solid tumors. ;Carcinoid tumor, malignant;Bronchiolo-alveolar adenocarcinoma;Papillary adenocarcinoma;Chromophobe carcinoma;Eosinophilic carcinoma;Eosinophilic adenocarcinoma;Basophilic carcinoma;Clear cell adenocarcinoma;Granular cell carcinoma;Follicle adenocarcinoma;Papillary follicular adenocarcinoma;Nonencapsulated sclerosing carcinoma;Adrenal cortical carcinoma;Endometrial carcinoma;Cutaneous adnexal carcinoma;Apocrine gland carcinoma;Sebaceous gland carcinoma;Ear wax gland carcinoma;Mucoepithelial carcinoma;Cystadenocarcinoma;Papillary cystadenoma;Papillary serous cystadenoma;Mucous cystadenoma;Mucous adenocarcinoma;Signet ring cell carcinoma;Invasive ductal carcinoma;Medullary carcinoma;Lobular carcinoma;Inflammatory carcinoma;Pa Jett's disease, breast;Acinic cell carcinoma;Adenosquamous carcinoma;Adenocarcinoma with squamous metaplasia;Thymoma, malignant;Ovarian stromal tumor, malignant;Theca cell tumor, malignant;Granulosa cell tumor, malignant;Male germinoma, malignant;Sertoli cell carcinoma;Leydig cell tumor, malignant;Lipocytoma, malignant;Paraganeuroma, malignant;Extramammary paraganglioma, malignant;Pheochromocytoma;Hemangiocytoma;Malignant melanoma;Amelanotic melanoma;Superficial spreading melanoma;Malignant melanoma of giant pigmented nevus;Epithelioid cell melanoma;Blue nevus, malignant;Sarcoma;Fibrosarcoma;Fibrohistiocyte tumor, malignant;myxosarcoma;liposarcoma;leiomyosarcoma;rhabdomyosarcoma;embryonal rhabdomyosarcoma;alveolar rhabdomyosarcoma;stromal sarcoma;mixed tumor, malignant;mixed Müllerian tumor;nephroblastoma;hepatoblastoma;carcinosarcoma;mesenchymal cell tumor, malignant;Brenner tumor, malignant;phyllodes tumor, malignant;synovial sarcoma;mesothelioma, malignant;dysgerminoma;embryonal carcinoma;teratoma, malignant;ovarian goiter, malignant;choriocarcinoma;mesonephroma, malignant;angiosarcoma;hemangioendothelioma, malignant;Kaposi's sarcoma;hemangiopericytoma, malignant;lymphangiosarcoma;osteosarcoma;parosteal osteosarcoma;chondrosarcoma;Chondroblastoma, malignant;Mesenchymal chondrosarcoma;Giant cell tumor of bone;Ewing's sarcoma;Odontogenic tumor, malignant;Ameloblastic odontosarcoma;Ameloblastoma, malignant;Ameloblastic fibrosarcoma;Pinealoma, malignant;Chordoma;Glioma, malignant;Ependymoma;Astrocytoma;Protoplasmic astrocytoma;Fibrous astrocytoma;Astroblastoma;Glioblastoma;Oligodendroglioma;Oligodendroglioma;Primitive neuroectodermal tumor;Cerebellar sarcoma;Ganglioblastoma;Neuroblastoma;Retinoblastoma;Olfactory nerve tumor;Meningioma, malignant;Neurofibrosarcoma;Neurilemoma, malignant;Granular Cell tumor, malignant; malignant lymphoma; Hodgkin's disease; paragranulomatous; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specific non-Hodgkin's lymphoma; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphocytic leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia. In some aspects, the tumor may include osteosarcoma, angiosarcoma, rhabdomyosarcoma, leiomyosarcoma, Ewing's sarcoma, glioblastoma, neuroblastoma, or leukemia.;

[0079] B. Microbial infections The compositions of the present disclosure may provide antimicrobial effects to target microorganisms and can be used to treat diseases or infections associated with target microorganisms.Antimicrobial effects include inhibiting or killing the growth of target microorganisms or interfering with the biological functions of target microorganisms.In general, the compositions of the present disclosure can be used to treat diseases or infections anywhere in a host, including, for example, any tissue or the surface of an implant.In one embodiment, the compositions are used to specifically kill or inhibit bacterial target microorganisms in bodily fluids (e.g., blood, sputum).

[0080] The compositions of the present disclosure are effective against bacteria including gram-positive and gram-negative cocci, gram-positive and gram-negative linear, curved and spiral / vibrio-like and branched rods, sheathed bacteria, sulfur-oxidizing bacteria, sulfur or sulfate-reducing bacteria, spirochetes, actinomycetes and related genera, slime molds, mycoplasmas, rickettsiae and chlamydiae, cyanobacteria, archaea, fungi, parasites, viruses and algae. For example, target microorganisms of the present disclosure include, but are not limited to, Escherichia coli, Candida, Salmonella, Staphylococcus, and Pseudomonas, Campylobacter jejuni, Candida albicans, Candida krusei, Chlamydia trachomatis, Clostridium difficile, Cryptococcus neoformans, Haempohilus influenzae, Helicobacter pylori, Moraxella catarrhalis, Neisseria gonorrhoeae, and the like. gonorrhoeae, Pseudomonas aeroginosa, Salmonella typhimurium, Shigella disenteriae, Staphylococcus aureus, and Streptococcus pneumoniae. Additionally, the composition may be used to treat chronic skin ulcers, infected acute wounds or burns, infected skin eczema, impetigo, atopic dermatitis, acne, otitis externa, vaginal infections, seborrheic dermatitis, oral infections, periodontitis, conjunctivitis, or pneumonia.

[0081] The compositions of the present disclosure may be effective against gram-negative bacteria. Gram-positive and Gram-negative cocci include Aerococcus, Enterococcus, Halococcus, Leuconostoc, Micrococcus, Mobiluncus, Moraxella catarrhalis, Neisseria (including N. gonorrheae and N. meningitidis), Pediococcus, Peptostreptococcus, Staphylococcus species (including S. aureus, methicillin-resistant S. aureus, coagulase-negative S. aureus, and S. saprophyticus), Streptococcus species (including S. pyogenes, S. agalactiae, S. bovis, S. pneumoniae, S. mutans, S. mutans, S. sanguis, S. equi, S. equinus, S. thermophilus, S. morbillorum, S. hansenii, S. pleomorphus, and S. parvulus, and Veillonella.

[0082] Gram-positive and Gram-negative linear, curved, spiral / vibrio-like and branched rods include Acetobacter, Acinetobacter, Actinobacillus equuli, Aeromonas, Agrobacterium, Alcaligenes, Aquaspirillum, Arcanobacterium haemolyticum, Bacillus species (including B. cereus and B. anthracis), Bacteroides species (including B. fragilis), Bartonella, Bordetella species (including B. pertussis), Brochothrix, Brucella, Burkholderia cepacia, Calymmatobacterium granulomatis, granulomatis, Campylobacter spp. (including C. jejuni), Capnocytophaga, Caulobacter, Chromobacterium violaceum, Citrobacter, Clostridium spp. (including C. perfringens, C. tetani, and C. difficile), Comamonas, Curtobacterium, Edwardsiella, Eikenella, Enterobacter, Erwinia, Erysipelothrix, Escherichia spp. (including E. coli), Flavobacterium spp. (including E. meninosepticum), Francisella spp. (including E. tularensis), Fusobacterium (including E. nucleatum), nucleatum), Gardnerella spp. (including G. vaginalis), Gluconobacter, Haemophilus spp. (including H. influenzae and H. ducreyi), Hafnia, Helicobacter (including H. pylori), Herpetosiphon, Klebsiella spp. (including K. pneumoniae), Kluyvera, Lactobacillus, Legionella spp. (including E.pneumophila), Leptotrichia, Listeria spp. (including E. monocytogenes), Microbacterium, Morganella, Nitrobacter, Nitrosomonas, Pasteurella spp. (including P. multocida), Pectinatus, Porphyromonas gingivalis, Proteus spp. (including E. mirabilis), Providencia, Pseudomonas spp. (including E. aeruginosa, P. mallei, P. pseudomallei, and E. solanacearum), Ranella, Renibacterium salmoninarum, salmoninarum, Salmonella, Serratia, Shigella, Spirillum, Streptobacillus spp. (including S. moniliformis), Vibrio spp. (including V. cholerae and V. vulnificus), Wallinella, Xanthobacter, Xenorhabdus, Yersinia spp. (including Y. pestis and Y. enterocolitica), Xanthomonas and Zymomonas.

[0083] Clinical diseases or infections caused by gram-positive and / or gram-negative bacteria treatable with the present disclosure include abscesses, bacteremia, contamination of peritoneal dialysis fluid, endocarditis, pneumonia, meningitis, osteomyelitis, cellulitis, pharyngitis, otitis media, sinusitis, scarlet fever, arthritis, urinary tract infections, laryngotracheitis, erysipelas, gas gangrene, tetanus, typhoid, acute gastroenteritis, bronchitis, epiglottitis, plague, septicemia, chancroid, wound and burn infections, cholera, glanders, periodontitis, genital infections, sinusitis, granuloma groin, legionnaires' disease, paratyphoid, bacillary dysentery, brucellosis, diphtheria, whooping cough, botulism, toxic shock syndrome, mastitis, rheumatic fever, cystic fibrosis, eye infections, dental plaque, and dental caries. Other uses include swine erysipelas, peritonitis, abortion, encephalitis, anthrax, nocardiosis, pericarditis, mycetoma, peptic ulcer, melioidosis, HaverhiU fever, tularemia, moko disease, galls (e.g., crowns, stems and leaves), hairy roots, bacterial rot, spotted bacterial disease, brown spot bacterial disease, bacterial wilt, bacterial fin rot, edema, columnaris disease, pasteurellosis, furunculosis, enteric red mouth disease, vibriosis of fish, and medical equipment stains.

[0084] The compounds and compositions of the disclosure may be used to treat or prevent infection with influenza viruses, cytomegaloviruses, avian leukemia sarcoma viruses, Rous sarcoma viruses, mammalian murine leukemia viruses type C, feline leukemia viruses, simian sarcoma viruses, type B mouse mammary tumor viruses, type D Mason-Pfizer monkey viruses, simian AIDS viruses, human T-cell leukemia viruses, simian T-cell leukemia viruses, bovine leukemia viruses, human immunodeficiency viruses, simian immunodeficiency viruses, feline immunodeficiency viruses, visna / maedi viruses, equine infectious anemia viruses, caprine arthritis encephalitis viruses, spuma viruses, foamy viruses, endogenous retroviruses, papilloma viruses, respiratory syncytial viruses, polio viruses, pox viruses, measles viruses, arbol viruses, coxsackie viruses, herpes viruses, hantaviruses, hepatitis viruses, baculoviruses, mumps viruses, circoviruses, arenaviruses, rotaviruses, Colorado tick fever CTF viruses, Eyach viruses, virus), Langat virus, Powassan virus, Omsk hemorrhagic fever virus, Crimean-Congo hemorrhagic fever virus, Yellow fever virus, Encephalitis virus, St. Louis encephalitis virus, Venezuelan equine encephalitis virus, Western equine encephalitis virus, Chikungunya virus, Japanese encephalitis virus, West Nile virus, Kyasanur Forest disease virus, Dengue virus, California encephalitis virus, Adenovirus, Korean hemorrhagic fever virus, Hantavirus, Argentine hemorrhagic fever virus, Junin virus, Aujeszky's disease virus, Pseudorabies virus, Herpes virus, Chikungunya virus, Cowpox virus, Ebola virus, Ganjam virus virus), herpes simplex virus, Lassa fever virus, looping virus, lymphocytic choriomeningitis virus, Marburg virus, milker's nodule virus, Newcastle disease virus, Omsk hemorrhagic fever virus, orf virus, parvovirus, poliovirus, pseudorabies, rabies virus, Rift Valley fever virus, Russian spring-summer encephalitis virus, Sabia virus, vaccinia virus, vesicular stomatitis virus, western equine encephalitis virus, or yellow fever virus.

[0085] C. Autoimmune diseases The compounds and compositions of the present disclosure may be effective in treating or preventing autoimmune diseases, including, but not limited to, Addison's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, antiphospholipid syndrome (APS), autoimmune hepatitis, autoimmune inner ear disease (AIED), axonal and neuronal neuropathy (AMAN), Behcet's disease, bullous pemphigoid, Castleman's disease (CD), celiac disease, Chagas' disease, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic relapsing multifocal osteomyelitis (CRMO), Churg-Strauss, cicatricial pemphigoid / benign mucositis, and rheumatoid arthritis (HA). Chronic pemphigoid, Cogan's syndrome, cold agglutinin disease, congenital heart block, Coxsackie myocarditis, CREST syndrome, Crohn's disease, dermatitis herpetiformis, dermatomyositis, Devic's disease (neuromyelitis optica), discoid lupus, Dressler's syndrome, endometriosis, eosinophilic esophagitis (EoE), eosinophilic fasciitis, erythema nodosum, essential mixed cryoglobulinemia, Evans syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Goodpasture's syndrome, granulomatosis with polyangiitis, Graves' disease, Guillain-Barré syndrome , Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schönlein purpura (HSP), herpes gestationis or pemphigoid of gestationis (PG), hypogammaglobulinemia, IgA nephropathy, IgG4-related sclerosing disease, inclusion body myositis (IBM), interstitial cystitis (IC), juvenile arthritis, juvenile diabetes mellitus (type 1 diabetes), juvenile myositis (JM), Kawasaki disease, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, ligneous conjunctivitis, linear immunoglobulin A disease (LAD), lupus, chronic Lyme disease, Meniere's disease, microscopic polyangiitis (MPA), mixed connective tissue MCTD, Mooren's ulcer, Mucha-Habermann disease, multiple sclerosis (MS), myasthenia gravis, myositis, narcolepsy, neuromyelitis optica, neutropenia, ocular cicatricial pemphigoid, optic neuritis, relapsing rheumatism (PR), PANDAS (Streptococcal childhood autoimmune neuropsychiatric disorders), paraneoplastic cerebellar degeneration (PCD), paroxysmal nocturnal hemoglobinuria (PNH), Parry-Romberg syndrome, pars planitis (peripheral uveitis), Parsonage-Turner syndrome, pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia (PA),POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, skin changes), polyarteritis nodosa, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, progesterone dermatitis, psoriasis, psoriatic arthritis, pure red cell aplasia (PRCA), pyoderma gangrenosum, Raynaud's phenomenon, reactive arthritis, reflex sympathetic dystrophy, Reiter's syndrome, relapsing polychondritis, restless legs syndrome (RLS), retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis (RA), monkeys coidosis, Schmidt syndrome, scleritis, scleroderma, Sjögren's syndrome, sperm and testicular autoimmunity, stiff-body syndrome (SPS), subacute bacterial endocarditis (SBE), Susac syndrome, sympathetic ophthalmia (SO), Takayasu's arteritis, temporal arteritis / giant cell arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome (THS), transverse myelitis, type 1 diabetes mellitus, ulcerative colitis (UC), undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vitiligo, and Wegener's granulomatosis (now called granulomatosis with polyangiitis (GPA)).

[0086] D. Inflammation Inflammation is a biological process that provides resistance to infectious or parasitic organisms and repair of damaged tissue. Inflammation is generally characterized by localized vasodilation, redness, swelling and pain, recruitment of leukocytes to the site of infection or injury, production of inflammatory cytokines such as TNF-α and IL-1, and production of reactive oxygen or nitrogen species such as hydrogen peroxide, superoxide and peroxynitrite. In the later stages of inflammation, tissue remodeling, angiogenesis and scar formation (fibrosis) may occur as part of the wound healing process. Under normal circumstances, the inflammatory response is controlled and temporary, and will systematically resolve if the infection or injury is properly treated. However, acute inflammation can become excessive and life-threatening if the control mechanisms are deactivated. Alternatively, inflammation can become chronic and cause cumulative tissue damage or systemic complications.

[0087] Many serious and intractable human diseases involve dysregulation of inflammatory processes, including diseases such as cancer, atherosclerosis, and diabetes, which were not traditionally viewed as inflammatory states. In cancer, inflammatory processes are associated with tumor formation, progression, metastasis, and resistance to therapy. Atherosclerosis, long viewed as a lipid metabolism disorder, is now understood to be an inherently inflammatory state, with activated macrophages playing a key role in the formation and eventual rupture of atherosclerotic plaques. Activation of inflammatory signaling pathways has also been found to play a role in the development of insulin resistance and peripheral tissue damage associated with diabetic hyperglycemia.

[0088] Chronic organ failure, such as kidney failure, heart failure, liver failure, and chronic obstructive pulmonary disease, is closely related to the presence of chronic oxidative stress and inflammation, which leads to the development of fibrosis and the eventual loss of organ function.Oxidative stress in vascular endothelial cells lining large and small blood vessels can lead to endothelial dysfunction and is considered to be an important contributing factor in the development of systemic cardiovascular disease, diabetic complications, chronic kidney disease, and other forms of organ failure, as well as several other age-related diseases, including degenerative central nervous system disease and degenerative retinal disease.

[0089] Many other disorders, including inflammatory bowel disease, inflammatory skin diseases, mucositis associated with radiation therapy and chemotherapy, eye diseases such as uveitis, glaucoma, macular degeneration, and various forms of retinopathy, transplant failure and transplant rejection, ischemia-reperfusion injury, chronic pain, degenerative conditions of bone and joints, including osteoarthritis and osteoporosis, asthma and cystic fibrosis, seizure disorders, and neuropsychiatric conditions, including schizophrenia, depression, bipolar disorder, post-traumatic stress disorder, attention deficit disorder, autism spectrum disorder, and eating disorders such as anorexia nervosa, involve oxidative stress and inflammation in affected tissues.Dysregulation of inflammatory signaling pathways is believed to be a major factor in the pathology of muscle wasting diseases, including muscular dystrophies and various forms of cachexia.

[0090] A variety of life-threatening acute disorders also involve dysregulation of inflammatory signaling, including acute organ failure involving the pancreas, kidney, liver or lung, myocardial infarction or acute coronary syndrome, stroke, septic shock, trauma, severe burns, and anaphylaxis.

[0091] Many complications of infectious diseases also involve dysregulation of inflammatory response.Inflammatory response can kill invading pathogens, but excessive inflammatory response can be quite destructive and in some cases can be the primary source of damage in infected tissue.In addition, excessive inflammatory response can lead to systemic complications due to overproduction of inflammatory cytokines such as TNF-α and IL-1.This is thought to be a factor in the deaths caused by severe influenza, severe acute respiratory syndrome, and sepsis.

[0092] V. Cell Targeting Moieties In some aspects, the present disclosure provides compounds that are conjugated directly or via a linker to cell targeting moieties. In some embodiments, the conjugation of a compound to a cell targeting moiety increases the effectiveness of the compound in treating a disease or disorder. The cell targeting moiety according to the embodiment can be, for example, an antibody, a growth factor, a hormone, a peptide, an aptamer, a small molecule such as a hormone, an imaging agent, or a cofactor, or a cytokine. It has been demonstrated that the gp240 antigen is expressed in various melanomas, but not in normal tissues. Thus, in some embodiments, the compounds of the present disclosure can be used in conjugation with an antibody against a specific antigen that is expressed by cancer cells but not in normal tissues.

[0093] In certain further embodiments, it is envisaged that the cancer cell targeting moiety binds to multiple types of cancer cells.For example, the 8H9 monoclonal antibody and the single chain antibody derived therefrom bind to a glycoprotein expressed in breast cancer, sarcoma and neuroblastoma (Onda et al., 2004).Another example is the cell targeting agent described in US Patent Publication No. 2004 / 0005647 and Winthrop et al. (2003) that binds to MUC-1, an antigen expressed in various cancer types.Thus, it will be understood that in certain embodiments, the cell targeting construct according to the embodiment may be targeted to multiple cancer or tumor types.

[0094] In addition, certain cell surface molecules are highly expressed in tumor cells, including hormone receptors such as human chorionic gonadotropin receptor and gonadotropin releasing hormone receptor (Nechushtan et al., 1997). Therefore, the corresponding hormones may be used as cell-specific targeting moieties in cancer therapy. In addition, cell targeting moieties that may be used include cofactors, sugars, drug molecules, imaging agents, or fluorescent dyes. Many cancerous cells are known to overexpress folate receptors, and therefore folic acid or other folate derivatives may be used as conjugates to induce cell-specific interactions between the conjugates of the present disclosure and cells (Campbell et al., 1991;Weitman et al., 1992).

[0095] Numerous cell surface receptors have been identified in hematopoietic cells of various lineages, and ligands or antibodies specific to these receptors may be used as cell-specific targeting moieties. IL-2 may also be used as cell-specific targeting moieties in chimeric proteins to target IL-2R+ cells. Alternatively, other molecules such as B7-1, B7-2 and CD40 may be used to specifically target activated T cells (The Leucocyte Antigen Facts Book, 1993, Barclay et al. (eds.), Academic Press). In addition, B cells express CD19, CD40 and IL-4 receptors, and may be targeted by moieties that bind to these receptors, such as CD40 ligand, IL-4, IL-5, IL-6 and CD28. Removal of immune cells such as T cells and B cells is particularly useful for treating lymphoma.

[0096] Other cytokines that can be used to target specific cell subsets include interleukins (IL-1 to IL-15), granulocyte colony-stimulating factor, macrophage colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, leukemia inhibitory factor, tumor necrosis factor, transforming growth factor, epidermal growth factor, insulin-like growth factor, and / or fibroblast growth factor (Thompson (ed.), 1994, The Cytokine Handbook, Academic Press, San Diego). In some aspects, the targeting polypeptide is a cytokine that binds to the Fn14 receptor, such as TWEAK (see, for example, Winkles 2008; Zhou et al., 2011 and Burkly et al., 2007, which are incorporated herein by reference).

[0097] Those skilled in the art will appreciate that certain of the following proteins are known: erythropoietin (four helix bundle) (e.g., EPO (erythropoietin), IL-2 (T cell growth factor), IL-3 (multi-colony CSF), IL-4 (BCGF-1, BSF-1), IL-5 (BCGF-2), IL-6 IL-4 (IFN-β2, BSF-2, BCDF), IL-7, IL-8, IL-9, IL-11, IL-13 (P600), G-CSF, IL-15 (T cell growth factor), GM-CSF (granulocyte macrophage colony stimulating factor), OSM (OM, oncostatin M), and LIF (leukemia inhibitory factor)); interferons (e.g., IFN-γ, IFN-α, and IFN-β); immunoglobulin superfamily (e.g., , B7.1 (CD80), and B7.2 (B70, CD86); the TNF family (e.g., TNF-α (cachectin), TNF-β (lymphotoxin, LT, LT-α), LT-β, ​​CD40 ligand (CD40L), Fas ligand (FasL), CD27 ligand (CD27L), CD30 ligand (CD30L), and 4-1BBL); and those not assigned to a particular family (e.g., TGF-β, IL 1α, IL-1β, IL-1 RA, IL-10 (cytokine synthesis inhibitor F), IL-12 (NK cell stimulatory factor), MIF, IL-16, IL-17 (mCTLA-8), and / or IL-18 (IGIF, interferon-γ inducer)). Additionally, the Fc portion of an antibody heavy chain may be used to target cells that express Fc receptors (eg, using the Fc portion of an IgE antibody to target mast cells and basophils).

[0098] Moreover, in some aspects, cell targeting moiety can be peptide sequence or cyclic peptide.For example, the cell targeting peptide and tissue targeting peptide that can be used according to the embodiment are provided in, for example, U.S. Patent No. 6,232,287; No. 6,528,481; No. 7,452,964; No. 7,671,010; No. 7,781,565; No. 8,507,445; and No. 8,450,278, each of which is incorporated herein by reference.

[0099] Thus, in some embodiments, cell targeting moiety is antibody or avimer.Antibody and avimer can be made against virtually any cell surface marker, thus providing a method of targeting GrB delivery to virtually any cell population of interest.These antibodies can also be used as fragments.Furthermore, antibody can be developed in one animal and then humanized, or developed using human model.The method for making antibody that can be used as cell targeting moiety is detailed below.The method for making avimer that binds to a given cell surface marker is detailed in US Patent Application Publication No. 2006 / 0234299 and US Patent Application Publication No. 2006 / 0223114, each of which is incorporated herein by reference.

[0100] It is further envisioned that the compounds described herein may be combined with nanoparticles or other nanomaterials.Some non-limiting examples of nanoparticles include metal nanoparticles, such as gold or silver nanoparticles, or polymeric nanoparticles, such as poly-L-lactic acid or poly(ethylene)glycol polymers.Nanoparticles and nanomaterials that can be combined with the compounds of the present invention include those described in U.S. Patent Application Publication Nos. 2006 / 0034925, 2006 / 0115537, 2007 / 0148095, 2012 / 0141550, 2013 / 0138032, and 2014 / 0024610, and PCT Publication Nos. WO 2008 / 121949, 2011 / 0053435, and 2014 / 0087413, each of which is incorporated herein by reference.

[0101] VI. Treatment A. Pharmaceutical Formulations and Routes of Administration When clinical use is envisaged, it will be necessary to prepare pharmaceutical composition in a form suitable for intended use.In some embodiments, such a formulation is envisaged that comprises the ADC of the present disclosure.Generally, this involves preparing a composition that is essentially free of pyrogens and essentially free of other impurities that may be harmful to humans or animals.

[0102] It will generally be desirable to use appropriate salts and buffers to stabilize the delivery vector and allow uptake by the target cells. Buffers will also be used when recombinant cells are introduced into a patient. Aqueous compositions of the present disclosure include an effective amount of the vector for the cells, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such compositions are also referred to as inocula. The phrase "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that do not cause adverse, allergic, or other untoward reactions when administered to animals or humans. As used herein, "pharmaceutically acceptable carriers" include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the vectors or cells of the present disclosure, its use in therapeutic compositions is contemplated. Supplementary active ingredients may also be incorporated into the compositions.

[0103] The active compositions of the present disclosure may include classical pharmaceutical preparations.The administration of these compositions according to the present disclosure may be carried out through any common route, so long as the target tissue is available through that route.Such routes include oral, nasal, buccal, rectal, vaginal or topical.Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intratumoral, intraperitoneal or intravenous injection.Such compositions will usually be administered as pharma-ceutically acceptable compositions as described above.

[0104] The active compound can also be administered parenterally or intraperitoneally.The solution of the active compound as a free base or a pharmacologically acceptable salt can be prepared in water appropriately mixed with a surfactant such as hydroxypropylcellulose.Dispersions can be prepared in glycerol, liquid polyethylene glycol and their mixtures as well as in oil.Under normal storage and use conditions, these preparations contain a preservative to prevent the growth of microorganisms.

[0105] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be protected against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial activity can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is preferable to include an isotonic agent, for example, sugar or sodium chloride. Prolonged absorption of the injectable composition can be brought about by the use of agents that delay absorption, for example, aluminum monostearate and gelatin in the composition.

[0106] Sterile injection solution is prepared by incorporating the required amount of active compound in suitable solvent together with some of the other components mentioned above as necessary, and then sterilizing by filtration.Generally, dispersion is prepared by incorporating various sterilized active components into a sterile vehicle that contains basic dispersion medium and other components required from above.In the case of sterile powder for preparing sterile injection solution, the preferred preparation method is vacuum drying and freeze-drying technology, which produces powder of active component and any additional desired components from its solution that has been previously sterilized and filtered.

[0107] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. The use of such media and agents for pharmaceutically active substances is well known in the art. Except where any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic composition is envisaged. Supplementary active ingredients can also be incorporated into the composition.

[0108] For oral administration, the ADCs described herein may be incorporated with excipients and used as non-digestible mouthwashes and dentifrices. Mouthwashes may be prepared by incorporating the active ingredient in the required amount in a suitable solvent, for example, sodium borate solution (Dobell's solution). Alternatively, the active ingredient may be incorporated in a germicidal antiseptic wash containing sodium borate, glycerin and potassium bicarbonate. The active ingredient may also be dispersed in dentifrices, including gels, pastes, powders and slurries. The active ingredient may be added in a therapeutically effective amount to a paste dentifrice, which may contain water, binders, abrasives, flavorings, foaming agents and humectants.

[0109] The compositions of the present disclosure may be formulated in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of protein), inorganic acids, such as hydrochloric acid or phosphoric acid, or organic acids, such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. Salts formed with free carboxyl groups may also be derived from inorganic bases, such as, for example, sodium, potassium, ammonium, calcium hydroxide or ferric hydroxide, or from organic bases, such as isopropylamine, trimethylamine, histidine, procaine, etc.

[0110] Once formulated, the solution will be administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. The formulations are easily administered in a variety of dosage forms, such as injectable solutions, drug release capsules, and the like. For parenteral administration in aqueous solution, for example, the solution must be suitably buffered if necessary, and the liquid diluent must first be rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, the sterile aqueous media that can be used will be known to those skilled in the art in light of this disclosure. For example, a dose can be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of subcutaneous infusion fluid or injected at the proposed infusion site (see, for example, "Remington's Pharmaceutical Sciences," 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The human responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety, and purity standards as required by FDA's Division of Biological Standards and Quality Control of the Office of Compliance and Biologics Quality.

[0111] B. Treatment Method In particular, compositions are disclosed herein that can be used to treat diseases or disorders, such as cancer, in a subject (e.g., a human subject). The compositions described above are preferably administered to a mammal (e.g., rodent, human, non-human primate, dog, cow, sheep, horse, cat, etc.) in an effective amount, i.e., an amount capable of producing a desired result in the treated subject (e.g., causing apoptosis of cancerous cells or killing microorganisms). The toxicity and therapeutic efficacy of the compositions utilized in the disclosed methods can be determined by standard pharmaceutical procedures. As is well known in the medical and veterinary arts, the dosage for any one animal depends on a number of factors, including the subject's size, body surface area, weight, age, the particular composition to be administered, the time and route of administration, general health, clinical symptoms of infection or cancer, and other drugs being administered concomitantly. The compositions described herein are typically administered in a dosage that induces the death of cancerous cells (e.g., induces apoptosis of cancer cells), as assayed by identifying a decrease in hematological parameters (complete blood count - CBC) or growth or proliferation of cancer cells. In some embodiments, the amount of ADC used to induce apoptosis of cancer cells is calculated to be about 0.01 mg to about 10,000 mg / day. In some embodiments, this amount is about 1 mg to about 1,000 mg / day. In some embodiments, these doses can be reduced or increased based on specific patient biological factors, such as increased or decreased metabolic degradation of the drug, or decreased uptake by the digestive tract when administered orally. Additionally, ADCs may be more efficient, and therefore smaller doses are required to achieve similar effects. Such doses are typically administered once a day for several weeks or until sufficient reduction of cancer cells is achieved.

[0112] The therapeutic methods (including prophylactic treatments) of the present disclosure generally include administering a therapeutically effective amount of the compositions described herein to a subject in need thereof, including a mammal, particularly a human. Such treatments are suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk of a disease, disorder, or symptoms thereof. The determination of a subject at "risk" can be made by any objective or subjective determination, such as by diagnostic testing or the opinion of the subject or health care provider (e.g., genetic testing, enzyme or protein markers, Markers (as defined herein), family history, etc.).

[0113] In some embodiments, the present disclosure provides a method for monitoring the progress of treatment. The method includes determining the change level of hematological parameters in a subject suffering from or susceptible to a disorder or symptom thereof related to cancer (e.g., leukemia) and / or cancer stem cell (CSC) analysis or diagnostic measurement (e.g., screening, assay) with cell surface proteins (e.g., can include but are not limited to CD34, CD38, CD90, and CD117) as diagnostic markers, wherein the subject is administered a therapeutic amount of the composition described herein. The level of the marker determined in the method can be compared to the known level of the marker in either healthy controls or other affected patients to determine the disease state of the subject. In a preferred embodiment, a second level of the marker in the subject is determined at a time later than the determination of the first level, and the two levels are compared to monitor the progress of the disease or the efficacy of the treatment. In certain preferred embodiments, the pre-treatment level of the marker in the subject is determined before starting treatment according to the methods described herein, and then this pre-treatment level of the marker can be compared to the level of the marker in the subject after treatment has begun to determine the efficacy of the treatment.

[0114] C. Combination Therapy It is envisaged that the drug conjugate described herein may be used in combination with one or more therapeutic methods or compounds that alleviate one or more of the side effects experienced by patients.Combining therapeutic modalities is common in medical therapy.The following is a general discussion of the therapeutic methods that can be used together with the therapeutic methods disclosed herein.

[0115] To treat disease or disorder using the disclosed method and composition, generally, subject is contacted with compound and at least one other therapy.These therapies are provided in a total amount effective to achieve the reduction in one or more disease parameters.This step may involve contacting cell / subject with both agents / therapies at the same time, for example, using a single composition or pharmaceutical preparation that contains both agents, or by contacting cell / subject with two separate compositions or preparations at the same time, where one composition contains compound and the other contains other agents.

[0116] Alternatively, the drug conjugates described herein may precede or follow the other treatment by intervals ranging from minutes to weeks. It is generally ensured that no significant time passes between each delivery time point so that the therapies can still exert a combined effect beneficial to the cell / subject. In such cases, it is contemplated to contact the cells with both modalities within about 12-24 hours of each other, within about 6-12 hours of each other, or with a delay of only about 1-2 hours. In some circumstances, it may be desirable to significantly extend the duration of treatment, whereby several days (2, 3, 4, 5, 6, or 7 days) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8 weeks) pass between each administration.

[0117] It is also contemplated that multiple administrations of either the compound or other therapies may be desirable. Various combinations may be used, as exemplified below, where a compound of the present disclosure is "A" and the other therapy is "B": TIFF2025500942000032.tif17128Other combinations are possible.

[0118] VII.Definition As used herein, "essentially free" with respect to a particular component is used herein to mean that none of the particular components are intentionally formulated into the composition and / or are present only as contaminants or only in trace amounts.Therefore, the total amount of the particular component resulting from any unintentional contamination of the composition is much less than 0.05%, preferably less than 0.01%.Most preferably, the composition is such that the amount of the particular component cannot be detected by standard analytical methods.

[0119] As used herein, "a" or "an" can mean one or more than one. As used in the claims, when used in conjunction with the word "comprising," the words "a" or "an" can mean one or more than one.

[0120] Use of the term "or" in the claims is used to mean "and / or," unless expressly indicated to refer to alternatives only or the alternatives are mutually exclusive, however, the present disclosure supports a definition that refers to alternatives only and "and / or." As used herein, "another" can mean at least a second, or more.

[0121] The term "about" generally means within the standard deviation of the stated value, as determined using standard analytical techniques to measure the stated value. The term may also be used to refer to plus or minus 5% of the stated value.

[0122] The phrase "effective amount" or "therapeutically effective" refers to a dosage of a drug or agent sufficient to produce a desired result, which may be a subjective or objective improvement in the recipient of the dosage, increased lung growth, increased lung repair, reduced tissue edema, increased DNA repair, reduced apoptosis, reduced tumor size, reduced rate of cancer cell growth, reduced metastasis, or any combination of the above.

[0123] As used herein, the term "antibody" refers to immunoglobulins, derivatives thereof that maintain specific binding ability, and proteins that have a binding domain that is homologous or largely homologous to an immunoglobulin binding domain. These proteins may be derived from natural sources or may be partially or fully synthetically produced. Antibodies may be monoclonal or polyclonal. Antibodies may be members of any immunoglobulin class, including any of the human classes: IgG, IgM, IgA, IgD, and IgE. Antibodies may be bispecific antibodies. In exemplary embodiments, the antibodies used with the methods and compositions described herein are derivatives of the IgG class. The term antibody also refers to antigen-binding antibody fragments. Examples of such antibody fragments include, but are not limited to, Fab, Faby, F(aby)2, scFv, Fv, dsFv diabody, and Fd fragments. Antibody fragments may be produced by any means. For example, antibody fragments can be enzymatically or chemically produced by fragmentation of an intact antibody, can be recombinantly produced from a gene encoding a partial antibody sequence, or can be fully or partially synthetically produced. Antibody fragments can optionally be single-chain antibody fragments. Alternatively, the fragments can include multiple chains linked together, for example, by disulfide bonds. The fragments can also optionally be multimolecular complexes. Functional antibody fragments will typically include at least about 10 amino acids, and more typically include at least about 200 amino acids.

[0124] "Subject" and "patient" refer to either human or non-human animals, such as primates, mammals, and vertebrates. In certain embodiments, the subject is a human.

[0125] As used herein, the terms "treat", "treatment", "treating" or "amelioration" when used in reference to a disease, disorder, or medical condition refer to therapeutic treatment of a condition, the purpose of which is to reverse, alleviate, improve, inhibit, slow down, or stop the progression or severity of a symptom or condition. The term "treat" includes reducing or alleviating at least one adverse effect or symptom of a condition. A treatment is generally "effective" if one or more symptoms or clinical markers are reduced. Alternatively, a treatment is "effective" if the progression of a condition is reduced or stopped. That is, "treatment" includes not only the improvement of a symptom or marker, but also the halting or at least slowing of the progression or worsening of a symptom that would be expected in the absence of treatment. Beneficial or desired clinical outcomes include, but are not limited to, alleviation of one or more symptoms, a reduction in the extent of a defect, a stabilized (i.e., not worsening) state of a tumor or malignancy, a delay or slowing of tumor growth and / or metastasis, and an increase in life span compared to that expected in the absence of treatment.

[0126] When used in the context of chemical groups, "hydrogen" means -H; "hydroxy" means -OH; "oxo" means =O; "carbonyl" means -C(=O)-; "carboxy" means -C(=O)OH (also written -COOH or -CO2H); "halo" means, independently, -F, -Cl, -Br, or -I; "amino" means -NH2; "hydroxyamino" means -NHOH; "nitro" means -NO2; and imino means =NH. "cyano" means -CN; "isocyanate" means -N=C=O; "azide" means -N; in the monovalent context, "phosphate" means -OP(O)(OH)2 or its deprotonated equivalent; in the divalent context, "phosphate" means -OP(O)(OH)O- or its deprotonated equivalent; "mercapto" means -SH; "thio" means =S; "sulfonyl" means -S(O)2-; and "sulfinyl" means -S(O)-.

[0127] In the context of chemical formulas, the symbol "-" denotes a single bond, "=" denotes a double bond, and "≡" denotes a triple bond. TIFF2025500942000033.tif4128 refers to any bond, if present, which may be either a single bond or a double bond. TIFF2025500942000034.tif4128 means a single bond or a double bond. Therefore, the formula For example, TIFF2025500942000035.tif10128 contains TIFF2025500942000036.tif10128 is included. It is also understood that one such ring atom does not form part of more than one double bond. It is further noted that the covalent bond symbol "-" does not indicate any preferred stereochemistry when linking one or two stereogenic atoms. Instead, it includes all stereoisomers as well as mixtures thereof. The symbol TIFF2025500942000037.tif4128 shows that the bond is drawn perpendicularly across the TIFF2025500942000038.tif7128), indicating the point of attachment of the group. It is noted that points of attachment are typically only identified in this fashion on larger groups to aid the reader in unambiguously identifying the point of attachment. TIFF2025500942000039.tif4128 represents a single bond, with the group attached to the thick end of the wedge "off the page". TIFF2025500942000040.tif4128 represents a single bond, with the group attached to the thick end of the wedge "into the page". TIFF2025500942000041.tif4128 refers to a single bond with undefined geometry around the double bond (e.g., either E or Z). Thus, both options, as well as combinations, are contemplated. Any undefined valence on an atom of a structure shown in this application implicitly represents a hydrogen atom bonded to that atom. A bold dot on a carbon atom indicates that the hydrogen bonded to that carbon is pointing out of the plane of the paper.

[0128] The variable symbol may be used as a "floating group" on the ring system, for example, in the formula: When depicted as the group "R" in TIFF2025500942000042.tif14128, the variable may replace any hydrogen atom attached to any ring atom, including drawn, implied, or explicitly defined hydrogens, so long as a stable structure is created. When depicted as the group "R" in TIFF2025500942000043.tif17128, the variable symbol may replace any hydrogen attached to any ring atom of any of the fused rings, unless otherwise noted. Replaceable hydrogens include depicted hydrogens (e.g., hydrogens attached to the nitrogen in the formula above), implied hydrogens (e.g., hydrogens in the formula above that are not shown but whose presence is understood), explicitly defined hydrogens, and any hydrogens whose presence depends on the identity of the ring atom (e.g., hydrogens attached to group X when X is equal to -CH-), so long as a stable structure is formed. In the depicted example, R may be in either the 5-membered or 6-membered ring of the fused ring system. In the formula above, the subscript "y" immediately following the parenthesized R represents a numerical variable. Unless otherwise noted, this variable may be 0, 1, 2, or any integer greater than 2, limited only by the maximum number of replaceable hydrogen atoms in the ring or ring system.

[0129] For chemical groups and compound classes, the number of carbon atoms in the group or class is as follows: "Cn" defines the exact number (n) of carbon atoms in the group / class. "C≦n" defines the maximum number (n) of carbon atoms that may be in the group / class, the minimum number being as small as possible for the group / class in question, e.g., the group "alkenyl (C≦8) " or class "Alkenes (C≦8) It is understood that the minimum number of carbon atoms in is 2. "Alkoxy" refers to an alkoxy group having 1 to 10 carbon atoms. (C≦10) "Cn-n'" defines both the minimum (n) and maximum number (n') of carbon atoms in the group. Thus, "alkyl (C2-10) " denotes an alkyl group having from 2 to 10 carbon atoms. These carbon number numbers may precede or follow the chemical group or class that they modify, and may or may not be in parentheses, and do not imply any change in meaning. Thus, "C5 olefin," "C5-olefin," "olefin (C5) " and "Olefin C5" are all synonymous. When any chemical group or class of compounds defined herein is modified with the term "substituted," any carbon atoms in the moiety that replace a hydrogen atom are not counted. Thus, methoxyhexyl has a total of seven carbon atoms, but a substituted alkyl (C1-6) is an example of: Unless otherwise stated, any chemical group or compound class recited in a claim without a carbon atom limit has a limit of 12 or less carbon atoms.

[0130] The term "saturated", when used to modify a compound or chemical group, means that the compound or chemical group has no carbon-carbon double bonds and no carbon-carbon triple bonds, except as noted below. When the term is used to modify an atom, it means that the atom is not part of any double or triple bonds. In the case of substituted forms of saturated groups, one or more carbon-oxygen or carbon-nitrogen double bonds may be present. Furthermore, it does not exclude carbon-carbon double bonds that may occur as part of keto-enol or imine / enamine tautomerism, if such bonds are present. When the term "saturated", when used to modify a solution of a substance, it means that no more of the substance can be dissolved in that solution.

[0131] The term "aliphatic", when used without the "substituted" modifier, indicates that the compound or chemical group so modified is an acyclic or cyclic, but non-aromatic, hydrocarbon compound or group. In an aliphatic compound / group, the carbon atoms may be linked together in a straight chain, branched chain, or non-aromatic ring (alicyclic). An aliphatic compound / group may be saturated (alkane / alkyl) linked by a single carbon-carbon bond, or unsaturated by one or more carbon-carbon double bonds (alkene / alkenyl) or one or more carbon-carbon triple bonds (alkyne / alkynyl).

[0132] The term "aromatic", when used to modify a compound or chemical group, refers to a planar unsaturated ring of atoms having 4n+2 electrons in a completely conjugated cyclic pi-system.

[0133] The term "alkyl" when used without the "substituted" modifier refers to a monovalent saturated aliphatic group with a carbon atom as the point of attachment, a straight or branched acyclic structure, and no atoms other than carbon and hydrogen. The groups -CH3(Me), -CH2CH3(Et), -CH2CH2CH3(n-Pr or propyl), -CH(CH3)2(i-Pr, i Pr or isopropyl), -CH2CH2CH2CH3(n-Bu), -CH(CH3)CH2CH3(sec-butyl), -CH2CH(CH3)2(isobutyl), -C(CH3)3(tert-butyl, t-butyl, t-Bu or tBu), and -CH2C(CH3)3 (neo-pentyl) are non-limiting examples of alkyl groups. The term "alkanediyl" when used without the "substituted" modifier refers to a divalent saturated aliphatic group having one or two saturated carbon atoms as points of attachment, a straight or branched acyclic structure, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. The groups -CH2- (methylene), -CH2CH2-, -CH2C(CH3)2CH2-, and -CH2CH2CH2- are non-limiting examples of alkanediyl groups. The term "alkylidene" when used without the "substituted" modifier refers to the divalent group =CRR', where R and R' are independently hydrogen or alkyl. Non-limiting examples of alkylidene groups include =CH2, =CH(CH2CH3), and =C(CH3)2. "Alkane" refers to the class of compounds having the formula H-R, where R is alkyl, as that term is defined above. When any of these terms are used with the "substituted" modifier, one or more hydrogen atoms are independently replaced with -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2. The following groups are non-limiting examples of substituted alkyl groups: -CH2OH, -CH2Cl, -CF3, -CH2CN, -CH2C(O)OH, -CH2C(O)OCH3, -CH2C(O)NH2, -CH2C(O)CH3, -CH2OCH3, -CH2OC(O)CH3, -CH2NH2, -CH2N(CH3)2, and -CH2CH2Cl. The term "haloalkyl" is a subset of substituted alkyl, limited to the replacement of hydrogen atoms with halo (i.e., -F, -Cl, -Br, or -I), such that there are no other atoms other than carbon, hydrogen, and halogen. The group -CH2Cl is a non-limiting example of a haloalkyl.The term "fluoroalkyl" is a subset of substituted alkyl, limited to the replacement of hydrogen atoms with fluoro, such that no other atoms other than carbon, hydrogen, and fluorine are present. The groups -CH2F, -CF3, and -CH2CF3 are non-limiting examples of fluoroalkyl groups.

[0134] The term "cycloalkyl", when used without the "substituted" modifier, refers to a monovalent saturated aliphatic group having a carbon atom as the point of attachment, the carbon atom forming part of one or more non-aromatic ring structures, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. Non-limiting examples include -CH(CH2)2 (cyclopropyl), cyclobutyl, cyclopentyl, or cyclohexyl (Cy). As used herein, the term does not exclude the presence of one or more alkyl groups (where the carbon number limit permits) attached to the carbon atom of the non-aromatic ring structure. The term "cycloalkanediyl", when used without the "substituted" modifier, refers to a divalent saturated aliphatic group having two carbon atoms as the point of attachment, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. Group TIFF2025500942000044.tif8128 is a non-limiting example of a cycloalkanediyl group. "Cycloalkane" refers to a class of compounds having the formula HR, where R is cycloalkyl as that term is defined above. When any of these terms are used with the "substituted" modifier, one or more hydrogen atoms are independently replaced with -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2.

[0135] The term "alkenyl" when used without the "substituted" modifier refers to a monovalent unsaturated aliphatic group having a carbon atom as the point of attachment, a linear or branched acyclic structure, at least one non-aromatic carbon-carbon double bond, no carbon-carbon triple bond, and no atoms other than carbon and hydrogen. Non-limiting examples include -CH=CH2 (vinyl), -CH=CHCH3, -CH=CHCH2CH3, -CH2CH=CH2 (allyl), -CH2CH=CHCH3, and -CH=CHCH=CH2. The term "alkenediyl" when used without the "substituted" modifier refers to a divalent unsaturated aliphatic group having two carbon atoms as the point of attachment, a linear or branched, a linear or branched acyclic structure, at least one non-aromatic carbon-carbon double bond, no carbon-carbon triple bond, and no atoms other than carbon and hydrogen. The groups -CH=CH-, -CH=C(CH3)CH2-, -CH=CHCH2-, and -CH2CH=CHCH2- are non-limiting examples of alkenediyl groups. It is noted that while alkenediyl groups are aliphatic, once linked at both ends, this does not prevent the group from forming part of an aromatic structure. The terms "alkene" and "olefin" are synonymous and refer to the class of compounds having the formula HR, where R is alkenyl as this term is defined above. Similarly, the terms "terminal alkene" and "α-olefin" are synonymous and refer to an alkene with only one carbon-carbon double bond, which bond is part of a vinyl group at the end of the molecule. When any of these terms are used in conjunction with the "substituted" modifier, one or more hydrogen atoms are independently replaced with -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2.The groups -CH=CHF, -CH=CHCl, and -CH=CHBr are non-limiting examples of substituted alkenyl groups.

[0136] The term "alkynyl", when used without the "substituted" modifier, refers to a monovalent unsaturated aliphatic group having a carbon atom as the point of attachment, a straight or branched chain acyclic structure, at least one carbon-carbon triple bond, and no atoms other than carbon and hydrogen. As used herein, the term alkynyl does not preclude the presence of one or more non-aromatic carbon-carbon double bonds. The groups -C≡CH, -C≡CCH3, and -CH2C≡CCH3 are non-limiting examples of alkynyl groups. "Alkyne" refers to the class of compounds having the formula HR, where R is alkynyl. When any of these terms are used in conjunction with the "substituted" modifier, one or more hydrogen atoms are independently replaced with -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2.

[0137] The term "aryl", when used without the "substituted" modifier, refers to a monovalent unsaturated aromatic group having an aromatic carbon atom as the attachment point, the carbon atom forming part of one or more aromatic ring structures, the ring atoms are all carbon, and the group does not consist of atoms other than carbon and hydrogen. When multiple rings are present, the rings may be fused or non-fused. Non-fused rings are linked by covalent bonds. As used herein, the term aryl does not exclude the presence of one or more alkyl groups (carbon number limitations permitting) attached to the first aromatic ring or any additional aromatic rings present. Non-limiting examples of aryl groups include monovalent groups derived from phenyl (Ph), methylphenyl, (dimethyl)phenyl, -CHCHCH(ethylphenyl), naphthyl, and biphenyl (e.g., 4-phenylphenyl). The term "arenediyl", when used without the "substituted" modifier, refers to a divalent aromatic group having two aromatic carbon atoms as attachment points, the carbon atoms forming part of one or more six-membered aromatic ring structures, the ring atoms are all carbon, and the monovalent group consists of no atoms other than carbon and hydrogen. As used herein, the term arenediyl does not exclude the presence of one or more alkyl groups (carbon number limitations permitting) attached to the first aromatic ring or to any additional aromatic rings present. When multiple rings are present, the rings may be fused or non-fused. Non-fused rings are linked by covalent bonds. Non-limiting examples of arenediyl include: TIFF2025500942000045.tif32132 is included. "Arene" refers to a class of compounds having the formula HR, where R is aryl, as that term is defined above. Benzene and toluene are non-limiting examples of arenes. When any of these terms are used with the "substituted" modifier, one or more hydrogen atoms are independently replaced with -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2.

[0138] The term "aralkyl," when used without the "substituted" modifier, refers to the monovalent group -alkanediyl-aryl, where the terms alkanediyl and aryl are each used in a manner consistent with the above definitions. Non-limiting examples are phenylmethyl (benzyl, Bn) and 2-phenyl-ethyl. When the term aralkyl is used with the "substituted" modifier, one or more hydrogen atoms from the alkanediyl and / or aryl group are independently replaced with -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2. Non-limiting examples of substituted aralkyls are (3-chlorophenyl)-methyl, and 2-chloro-2-phenyl-eth-1-yl.

[0139] The term "heteroaryl", when used without the "substituted" modifier, refers to a monovalent aromatic group having an aromatic carbon or nitrogen atom as the point of attachment, the carbon or nitrogen atom forming part of one or more aromatic ring structures, at least one of the ring atoms being nitrogen, oxygen or sulfur, and the heteroaryl group being composed of no atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen and aromatic sulfur. When multiple rings are present, the rings may be fused or non-fused. Non-fused rings are linked by covalent bonds. As used herein, the term heteroaryl does not exclude the presence of one or more alkyl or aryl groups (where carbon number limitations permit) attached to the aromatic ring or aromatic ring system. Non-limiting examples of heteroaryl groups include furanyl, imidazolyl, indolyl, indazolyl (Im), isoxazolyl, methylpyridinyl, oxazolyl, phenylpyridinyl, pyridinyl (pyridyl), pyrrolyl, pyrimidinyl, pyrazinyl, quinolyl, quinazolyl, quinoxalinyl, triazinyl, tetrazolyl, thiazolyl, thienyl, and triazolyl. The term "N-heteroaryl" refers to a heteroaryl group having a nitrogen atom as the attachment point. "Heteroarene" refers to the class of compounds having the formula HR, where R is heteroaryl. Pyridine and quinoline are non-limiting examples of heteroarenes. When these terms are used with the "substituted" modifier, one or more hydrogen atoms are independently replaced with -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2.

[0140] The term "heterocycloalkyl", when used without the "substituted" modifier, refers to a monovalent non-aromatic group having a carbon or nitrogen atom as the point of attachment, the carbon or nitrogen atom forming part of one or more non-aromatic ring structures, at least one of the ring atoms being nitrogen, oxygen or sulfur, and the heterocycloalkyl group being composed of no atoms other than carbon, hydrogen, nitrogen, oxygen and sulfur. When multiple rings are present, the rings may be fused or non-fused. As used herein, the term does not exclude the presence of one or more alkyl groups (where the carbon number limit permits) attached to the ring or ring system. The term also does not exclude the presence of one or more double bonds in the ring or ring system, provided that the resulting group remains non-aromatic. Non-limiting examples of heterocycloalkyl groups include aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, tetrahydrofuranyl, tetrahydrothiofuranyl, tetrahydropyranyl, pyranyl, oxiranyl, and oxetanyl. The term "N-heterocycloalkyl" refers to a heterocycloalkyl group having a nitrogen atom as the attachment point. N-pyrrolidinyl is such a non-limiting example. When these terms are used with the "substituted" modifier, one or more hydrogen atoms are independently replaced with -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2.

[0141] The term "acyl", when used without the "substituted" modifier, refers to the group -C(O)R, where R is hydrogen, alkyl, cycloalkyl, or aryl, as those terms are defined above. The groups -CHO, -C(O)CH3 (acetyl, Ac), -C(O)CH2CH3, -C(O)CH(CH3)2, -C(O)CH(CH2)2, -C(O)CH6H5, and -C(O)CH4CH3 are non-limiting examples of acyl groups. "Thioacyl" is defined in a similar manner, except that the oxygen atom of the group -C(O)R is replaced with a sulfur atom, and is -C(S)R. The term "aldehyde" corresponds to an alkyl group, as defined above, attached to a -CHO group. When any of these terms are used in conjunction with the "substituted" modifier, one or more hydrogen atoms (including the hydrogen atom directly bonded to the carbon atom of the carbonyl or thiocarbonyl group, if any) are independently replaced with -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2. The groups -C(O)CH2CF3, -CO2H (carboxyl), -CO2CH3 (methylcarboxyl), -CO2CH2CH3, -C(O)NH2 (carbamoyl), and -CON(CH3)2 are non-limiting examples of substituted acyl groups.

[0142] The term "alkoxy" when used without the "substituted" modifier refers to the group -OR, where R is alkyl as that term is defined above. Non-limiting examples include -OCH3 (methoxy), -OCH2CH3 (ethoxy), -OCH2CH2CH3, -OCH(CH3)2 (isopropoxy), -OC(CH3)3 (tert-butoxy), -OCH(CH2), -O-cyclopentyl, and -O-cyclohexyl. The terms "cycloalkoxy", "alkenyloxy", "alkynyloxy", "aryloxy", "aralkoxy", "heteroaryloxy", "heterocycloalkoxy" and "acyloxy", when used without the "substituted" modifier, refer to the group defined as -OR, where R is cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl, and acyl, respectively. The terms "alkylthio" and "acylthio", when used without the "substituted" modifier, refer to the group -SR, where R is alkyl and acyl, respectively. The term "alcohol" corresponds to an alkane, as defined above, in which at least one hydrogen atom has been replaced with a hydroxy group. The term "ether" corresponds to an alkane, as defined above, in which at least one hydrogen atom has been replaced with an alkoxy group. When any of these terms are used in conjunction with the "substituted" modifier, one or more hydrogen atoms are independently replaced with -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2.

[0143] The term "alkylamino" when used without the "substituted" modifier refers to the group -NHR, where R is alkyl as that term is defined above. Non-limiting examples include -NHCH3 and -NHCH2CH3. The term "dialkylamino" when used without the "substituted" modifier refers to the group -NRR', where R and R' can be the same or different alkyl groups, or R and R' together can represent an alkanediyl. Non-limiting examples of dialkylamino groups include -N(CH3)2 and -N(CH3)(CH2CH3). The terms "cycloalkylamino", "alkenylamino", "alkynylamino", "arylamino", "aralkylamino", "heteroarylamino", "heterocycloalkylamino", "alkoxyamino", and "alkylsulfonylamino", when used without the "substituted" modifier, refer to the group defined as -NHR, where R is cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl, alkoxy, and alkylsulfonyl, respectively. A non-limiting example of an arylamino group is -NHC6H5. The term "amido" (acylamino) when used without the "substituted" modifier refers to the group -NHR, where R is acyl, as that term is defined above. A non-limiting example of an amido group is -NHC(O)CH3. The term "alkylimino" when used without the "substituted" modifier refers to the divalent group =NR, where R is alkyl, as that term is defined above. When any of these terms are used with the "substituted" modifier, one or more hydrogen atoms bonded to the carbon atom are independently replaced with -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2. The groups -NHC(O)OCH3 and -NHC(O)NHCH3 are non-limiting examples of substituted amide groups.

[0144] The term "heteroarenediyl", when used without the "substituted" modifier, refers to a divalent aromatic group having two aromatic carbon atoms, two aromatic nitrogen atoms, or one aromatic carbon atom and one aromatic nitrogen atom as two points of attachment, which form part of one or more aromatic ring structures, at least one of the ring atoms being nitrogen, oxygen, or sulfur, and the divalent group being composed of no atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen, and aromatic sulfur. When multiple rings are present, the rings may be fused or non-fused. Non-fused rings are linked by covalent bonds. As used herein, the term heteroarenediyl does not exclude the presence of one or more alkyl or aryl groups (where carbon number limitations permit) attached to the aromatic ring or aromatic ring system. Non-limiting examples of heteroarenediyl groups include: Contains TIFF2025500942000046.tif15128.

[0145] The term "heterocycloalkanediyl", when used without the "substituted" modifier, refers to a divalent cyclic group having two carbon atoms, two nitrogen atoms, or one carbon atom and one nitrogen atom as two points of attachment, which form part of one or more ring structures, at least one of the ring atoms being nitrogen, oxygen, or sulfur, and the divalent group being composed of no atoms other than carbon, hydrogen, nitrogen, oxygen, and sulfur. When multiple rings are present, the rings may be fused or non-fused. Non-fused rings are linked by covalent bonds. As used herein, the term heterocycloalkanediyl does not exclude the presence of one or more alkyl groups (where the carbon number limit permits) attached to the ring or ring system. Also, the term does not exclude the presence of one or more double bonds in the ring or ring system, provided that the resulting group remains non-aromatic. Non-limiting examples of heterocycloalkanediyl groups include: Contains TIFF2025500942000047.tif14128.

[0146] The terms "alkylsulfonyl" and "alkylsulfinyl," when used without the "substituted" modifier, refer to the groups -S(O)R and -S(O)R, respectively, where R is alkyl, as that term is defined above. The terms "cycloalkylsulfonyl," "alkenylsulfonyl," "alkynylsulfonyl," "arylsulfonyl," "aralkylsulfonyl," "heteroarylsulfonyl," and "heterocycloalkylsulfonyl" are defined in an analogous manner. When any of these terms are used in conjunction with the "substituted" modifier, one or more hydrogen atoms are independently replaced with -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2.

[0147] The term "alkyl phosphate" when used without the "substituted" modifier refers to the group -OP(O)(OH)(OR), where R is alkyl as that term is defined above. Non-limiting examples of alkyl phosphate groups include -OP(O)(OH)(OMe) and -OP(O)(OH)(OEt). The term "dialkyl phosphate" when used without the "substituted" modifier refers to the group -OP(O)(OR)(OR'), where R and R' can be the same or different alkyl groups, or R and R' together can represent an alkanediyl. Non-limiting examples of dialkyl phosphate groups include -OP(O)(OMe), -OP(O)(OEt)(OMe), and -OP(O)(OEt). When any of these terms are used in conjunction with the "substituted" modifier, one or more hydrogen atoms are independently replaced with -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2.

[0148] An "excipient" is a pharma- ceutically acceptable substance that is formulated with the active ingredient of a medicament, pharmaceutical composition, formulation, or drug delivery system. Excipients may be used, for example, to stabilize the composition, bulk the composition (and thus are often called "bulking agents," "fillers," or "diluents" when used for this purpose), or to provide therapeutic enhancements to the active ingredient in the final dosage form, such as facilitating drug absorption, reducing viscosity, or enhancing solubility. Excipients include pharma- ceutically acceptable anti-adherents, binders, coatings, colorants, disintegrants, flavoring agents, glidants, lubricants, preservatives, adsorbents, sweeteners, and vehicles. The primary excipient that serves as a vehicle for carrying the active ingredient is usually referred to as the vehicle. Excipients may be used in the manufacturing process to aid in the handling of the active, for example, by promoting powder flow or non-adhesiveness, as well as to aid in vitro stability, such as preventing denaturation or aggregation during the expected shelf life. The suitability of an excipient will typically vary depending on the route of administration, the dosage form, the active ingredient, as well as other factors.

[0149] The term "hydrate," when used as a modifier to a compound, means that the compound has less than one (e.g., a hemihydrate), one (e.g., a monohydrate), or multiple (e.g., a dihydrate) water molecules bound to each compound molecule, such as in a solid form of the compound.

[0150] As used herein, "IC 50 The term "inhibitory dose" refers to an inhibitory dose that is 50% of the maximum response obtained. This quantitative measure indicates how much of a particular drug or other substance (inhibitor) is needed to inhibit a given biological, biochemical, or chemical process (or a component of the process, i.e., an enzyme, cell, cell receptor, or microorganism) by half.

[0151] An "isomer" of a first compound is a distinct chemical compound whose each molecule contains the same constituent atoms as the first compound, but differs in the arrangement of those atoms in three dimensions.

[0152] As used herein, the term "patient" or "subject" refers to a living mammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, or a transgenic species thereof. In some embodiments, the patient or subject is a primate. Non-limiting examples of human patients are adults, juveniles, infants, and fetuses.

[0153] As used herein generally, "pharmacologically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with the tissues, organs, and / or body fluids of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, within the scope of sound medical judgment, commensurate with a reasonable risk / benefit ratio.

[0154] "Pharmaceutically acceptable salts" refers to salts of the compounds of the present invention that are pharma- ceutically acceptable as defined above and have the desired pharmacological activity. Such salts include salts with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid; or salts with 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, 2-naphthalenesulfonic acid, 3-phenylpropionic acid, 4,4'-methylenebis(3-hydroxy-2-ene-1-carboxylic acid), 4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, acetic acid, aliphatic mono- and dicarboxylic acids, aliphatic sulfuric acid, aromatic sulfuric acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid, cyclopentanepropionic acid, ethanesulfonic acid, ethylenediaminetetraacetic ... Included among these are acid addition salts formed with organic acids such as sulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid, lauryl sulfuric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, muconic acid, o-(4-hydroxybenzoyl)benzoic acid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substituted alkanoic acid, propionic acid, p-toluenesulfonic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, tartaric acid, tert-butylacetic acid, and trimethylacetic acid. Pharmaceutically acceptable salts also include base addition salts that may be formed when acidic protons present are capable of reacting with inorganic or organic bases. Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide, and calcium hydroxide. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, etc. It should be understood that the particular anion or cation forming a part of any salt of this invention is not critical, so long as the salt, as a whole, is pharmacologically acceptable.Further examples of pharma- ceutically acceptable salts and their methods of preparation and use are provided in Handbook of Pharmaceutical Salts: Properties, and Use (PH Stahl & CG Wermuth eds., Verlag Helvetica Chimica Acta, 2002).

[0155] A "pharmaceutically acceptable carrier", "drug carrier", or simply "carrier" is a pharma-ceutically acceptable substance formulated with an active ingredient drug that is involved in carrying, delivering and / or transporting a chemical. Drug carriers may be used to improve drug delivery and efficacy, including, for example, controlled release technologies to regulate drug bioavailability, reduce drug metabolism, and / or reduce drug toxicity. Some drug carriers may enhance the efficacy of drug delivery to a particular target site. Examples of carriers include liposomes, microspheres (e.g., made of poly(lactic-co-glycolic acid)), albumin microspheres, synthetic polymers, nanofibers, protein-DNA complexes, protein conjugates, red blood cells, virosomes, and dendrimers.

[0156] A "pharmaceutical drug" (also called a drug, pharmaceutical agent, pharmaceutical preparation, pharmaceutical composition, pharmaceutical formulation, pharmaceutical product, medicinal product, medicine, medicament, or simply drug) is a drug used to diagnose, cure, treat, or prevent a disease. An active ingredient (AI) (defined above) is the ingredient in a pharmaceutical drug or pesticide that is biologically active. The similar terms pharmacoactive ingredient (API) and bulk active are also used for drugs, and the term active substance may be used for pesticide formulations. Some drug and pesticide products may contain more than one active ingredient. In contrast to active ingredients, inactive ingredients are usually called excipients (defined above) in the pharmaceutical context.

[0157] A "multivalent polymer" describes a linking group that contains two or more open points of valency that can be used to link different moieties together. Some non-limiting examples of multivalent polymers include polymers, dendrimers, dendrons, or fragments thereof, having side chains that can react with the moieties. In one embodiment, the multivalent polymer is a dendrimer or dendron.

[0158] "Prevention" or "preventing" includes (1) inhibiting the onset of a disease in a subject or patient who may be at risk and / or predisposed to the disease, but who has not yet experienced or exhibited any or all of the symptoms or symptomatology of the disease, and / or (2) delaying the onset of symptoms or symptomatology of a disease in a subject or patient who may be at risk and / or predisposed to the disease, but who has not yet experienced or exhibited any or all of the symptoms or symptomatology of the disease.

[0159] "Prodrug" refers to a compound that can be metabolically converted to an inhibitor of the invention in vivo. The prodrug itself may or may not have activity with respect to a given protein. For example, a compound containing a hydroxy group may be administered as an ester that is converted to a hydroxy compound by hydrolysis in vivo. Suitable esters that can be converted to a hydroxy compound in vivo include acetate, citrate, lactate, phosphate, tartrate, malonate, oxalate, salicylate, propionate, succinate, fumarate, maleate, methylene-bis-β-hydroxynaphthoate, gentisate, isethionate, di-p-toluoyltartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, cyclohexylsulfamate, quinate, and esters of amino acids. Similarly, a compound containing an amine group may be administered as an amide that is converted by hydrolysis in vivo to the amine compound.

[0160] "Amine protecting groups" are well understood in the art. Amine protecting groups are groups that prevent the reactivity of the amine group in a reaction that modifies some other part of the molecule and can be easily removed to generate the desired amine. Amine protecting groups can be found at least in Greene and Wuts, 1999, which is incorporated herein by reference.Some non-limiting examples of monovalent amino protecting groups include formyl, acetyl, propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, o-nitrophenoxyacetyl, α-chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl, and the like; sulfonyl groups, e.g., benzenesulfonyl, p-toluenesulfonyl, and the like; alkoxy- or aryloxycarbonyl groups (which form urethanes with the protected amine), e.g., benzyloxycarbonyl (Cbz), p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 3,5-dimethoxybenzyloxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl, 4-methoxy ...5-dimethoxybenzyloxycarbonyl, 2,5-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 3,5-dimethoxybenzyloxycarbonyl, 2,5-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, 3, 4,5-dimethoxybenzyloxycarbonyl, 3,4,5-trimethoxybenzyloxycarbonyl, 1-(p-biphenylyl)-1-methylethoxycarbonyl, α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxycarbonyl, t-butyloxycarbonyl (Boc), diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl (Alloc), 2,2,2-trimethoxybenzyloxycarbonyl, ... trichloroethoxycarbonyl, 2-trimethylsilylethyloxycarbonyl (Teoc), phenoxycarbonyl, 4-nitrophenoxycarbonyl, fluorenyl-9-methoxycarbonyl (Fmoc), cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl, etc.; aralkyl groups, such as benzyl, triphenylmethyl, benzyloxymethyl, etc.; and silyl groups, such as trimethylsilyl, etc. Additionally, the "amine protecting group" may be a divalent protecting group in which both hydrogen atoms on a primary amine are replaced with one protecting group.In such a situation, the amine protecting group may be phthalimide (phth) or a substituted derivative thereof, where the term "substituted" is as defined above. In some embodiments, the halogenated phthalimide derivative may be tetrachlorophthalimide (TCphth). As used herein, a "protected amino group" refers to a group having the formula PG. MA NH- or PG DA N- group, where PG MA is a monovalent amine protecting group, sometimes referred to as a "monovalently protected amino group"; PG DA is a divalent amine protecting group as described above, which may also be described as a "divalently protected amino group."

[0161] "Hydroxyl protecting group" is well understood in the art. A hydroxyl protecting group is a group that prevents the reactivity of a hydroxyl group in a reaction that modifies some other part of a molecule, and can be easily removed to generate the desired hydroxyl. Hydroxyl protecting groups can be found at least in Greene and Wuts, 1999, which is incorporated herein by reference.Some non-limiting examples of hydroxyl protecting groups include acyl groups, such as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, o-nitrophenoxyacetyl, α-chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl, and the like; sulfonyl groups, such as benzenesulfonyl, p-toluenesulfonyl, and the like; acyloxy groups, such as benzyloxycarbonyl (Cbz), p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 3,5-dimethoxybenzyloxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl, and the like. benzyloxycarbonyl, 3,4,5-trimethoxybenzyloxycarbonyl, 1-(p-biphenylyl)-1-methylethoxycarbonyl, α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxycarbonyl, t-butyloxycarbonyl (Boc), diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl (Alloc), 2,2,2-trichloroethoxy Examples of protected hydroxy groups include aryloxycarbonyl, 2-trimethylsilylethyloxycarbonyl (Teoc), phenoxycarbonyl, 4-nitrophenoxycarbonyl, fluorenyl-9-methoxycarbonyl (Fmoc), cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl, and the like; aralkyl groups, such as benzyl, triphenylmethyl, benzyloxymethyl, and the like; and silyl groups, such as trimethylsilyl, and the like. As used herein, a protected hydroxy group is represented by the formula PG. H O- group, where PG H is a hydroxyl protecting group as described above.

[0162] "Stereoisomers" or "optical isomers" are isomers of a given compound that have the same atoms bonded to the same other atoms but differ in the three-dimensional arrangement of those atoms. "Enantiomers" are stereoisomers of a given compound that are mirror images of each other, like a left and right hand. "Diastereomers" are stereoisomers of a given compound that are not mirror images. Chiral molecules contain a chiral center, also called a stereogenic center or stereogenic center, which is any point in a molecule that has groups such that the exchange of any two groups leads to a stereoisomer, but not necessarily an atom. In organic compounds, the chiral center is typically a carbon, phosphorus, or sulfur atom, although other atoms can be stereogenic centers in organic and inorganic compounds. Molecules can also have multiple stereocenters, resulting in many stereoisomers. In compounds whose stereoisomerism is due to tetrahedral stereogenic centers (e.g., tetrahedral carbon), the total number of hypothetical possible stereoisomers is 2. n n is the number of tetrahedral stereocenters. Molecules with symmetry often have fewer than the maximum number of stereoisomers possible. A 50:50 mixture of enantiomers is called a racemic mixture. Alternatively, a mixture of enantiomers can be enantiomerically enriched, such that one enantiomer is present in an amount greater than 50%. Typically, enantiomers and / or diastereomers can be resolved or separated using techniques known in the art. For any stereocenter or axis of chirality where the stereochemistry is not defined, it is contemplated that that stereocenter or axis of chirality can exist as its R form, S form, or a mixture of R and S forms, including racemic and non-racemic mixtures. As used herein, the phrase "substantially free of other stereoisomers" means that the composition contains ≦15%, more preferably ≦10%, even more preferably ≦5%, or most preferably ≦1% of another stereoisomer.

[0163] "Treatment" or "treating" includes (1) inhibiting a disease in a subject or patient experiencing or exhibiting the pathology or symptomology of the disease (e.g., halting further development of the pathology and / or symptomology), (2) ameliorating a disease in a subject or patient experiencing or exhibiting the pathology or symptomology of the disease (e.g., reversing the pathology and / or symptomology), and / or (3) making any measurable reduction in a disease in a subject or patient experiencing or exhibiting the pathology or symptomology of the disease.

[0164] As used herein, average molecular weight refers to the weight average molecular weight (Mw) determined by static light scattering.

[0165] A "repeating unit" is the simplest structural entity of a given material (e.g., framework and / or polymer, whether organic, inorganic, or organometallic). In the case of a polymer chain, the repeating units are linked together sequentially along the chain like beads on a necklace. For example, in polyethylene, -[-CH2CH2-] n In the formula, the repeating unit is -CH2CH2-. The subscript "n" represents the degree of polymerization, i.e., the number of repeating units linked together. If the value for "n" is left undefined or is absent, it simply specifies the polymeric nature of the material in addition to the repetition of the formula in the brackets. The concept of repeating units applies equally to where the connectivity between repeating units extends in three dimensions, e.g., metal organic frameworks, modified polymers, thermosetting polymers, etc.

[0166] The term "conjugating group" refers to a chemical group that can be coupled to a functional group of another compound to form a covalent bond under mild conditions and is stable under physiological conditions. In some embodiments, the conjugating group is S NCovalent bonds can be formed via the 2 reaction, the Diels-Alder reaction, or conjugate addition reactions. Examples of conjugating groups include, but are not limited to, diene groups (e.g., tetrazinyl), alkene groups (e.g., trans-cyclooctenyl and norbornyl), and -SH.

[0167] The term "canonical amino acid" refers to one of the 20 standard amino acids used in nature. Other amino acids include citrillune or ornithine. The term "side chain" of an amino acid refers to the R group on the particular amino acid. For example, the R group in glycine is a hydrogen atom, in alanine it is a methyl group, and in lysine it is 4-aminobutyl.

[0168] The above definitions supersede any conflicting definitions in any of the references incorporated herein by reference. However, the fact that a particular term is defined should not be taken as indicating that any undefined term is indefinite. Rather, all terms used are believed to describe the invention in terms that allow one skilled in the art to appreciate the scope and practice the invention. EXAMPLES

[0169] VIII. Examples The following examples are included to demonstrate preferred embodiments of the present disclosure. It should be recognized by those skilled in the art that the techniques disclosed in the following examples represent techniques that the inventors have found to work well in implementing the present disclosure, and therefore may be considered to constitute preferred modes for its implementation. However, those skilled in the art should recognize in light of the present disclosure that many changes can be made in the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the present disclosure.

[0170] Example 1 – Development and characterization of a tripeptide linker This study demonstrated that the glutamic acid-glycine-citrulline (EGCit) tripeptide linker has the potential to solve the clinical problems caused by linker instability without compromising ADC therapeutic efficacy (Figure 1C). The EGCit sequence provides long-term stability in both rodent and primate plasma and fully tolerates differentiating human neutrophils while retaining the ability to rapidly liberate free payload upon intracellular cleavage. It was also demonstrated that MMAE ADCs constructed with EGCit linkers exhibited improved antitumor activity in a panel of cancer cell lines and three different xenograft mouse models compared to conventional conjugates as well as the FDA-approved anti-HER2 ADCs, KADCYLA® (T-DM1) and ENHERTU® (DS-8201). Notably, EGCit-based ADCs were shown to be effective in treating pulmonary edema at doses of 80 mg kg -1 showed no discernible hepatotoxicity in healthy mice. These findings demonstrate that the EGCit linker technology not only ensures a smooth transition from preclinical trials to human evaluation, but also offers a broadly applicable solution to significantly broaden the therapeutic window of targeted drug delivery systems, including ADCs.

[0171] Incorporation of glycine at P2 and glutamic acid at P3 provided high resistance to unwanted degradationTo search for an alternative to the traditional VCit linker with improved in vivo stability and tolerability, we first investigated ways that could circumvent the linker instability that leads to neutropenia. It was shown that serine proteases secreted extracellularly from differentiating human neutrophils promote the release of MMAE from VCit-based ADCs and reduce the population of bone marrow neutrophils (Zhao et al., 2017). With this report in mind, the neutrophil elastase cleavage site was identified using a small molecule probe. VCit-PABC-pyrene probe 1 was incubated with human neutrophil elastase at 37 °C for 24 h. Pyrene fragments containing citrulline-PABC were detected as the main product by liquid chromatography (LC)-electrospray ionization mass spectrometry (ESI-MS) (Figure 2A). This result indicated that neutrophil elastase cleaves the amide bond between P1 valine and P2 citrulline. EVCit probe 2 also provided the same citrulline-containing fragment, suggesting that the glutamic acid at P3 does not affect neutrophil elastase-mediated linker degradation.

[0172] Based on this structural analysis, we screened various amino acids at the P2 position. A panel of pyrene probes containing EXCit-PABC units where X was glycine (3a), alanine (3b), leucine (3c), and isoleucine (3d) was prepared (Figure 2B). EV(N-Me)Cit-PABC-pyrene probe 3e and GCit dipeptide probe 3f were also prepared. All probes were then tested for stability against human neutrophil elastase-mediated degradation (Figure 2C). Surprisingly, EVCit probe 2 was degraded faster than VCit probe 1, indicating that P3 glutamate may increase linker susceptibility to elastase-mediated degradation. EACit and EICit probes 3b and 3d also showed complete degradation. These results were consistent with a previous study that demonstrated that human neutrophil elastase preferentially cleaves the N-terminal amide bond of valine, alanine, and isoleucine. (Fu et al., 2018). In contrast, little or no degradation was observed for probes containing EGCit (3a), ELCit (3c), EV(N-Me)Cit (3e), or GCit probe (3f). This observation suggested that the P2 amino acid affects reactivity with neutrophil elastase much more significantly than the P3 amino acid. EGCit and EV(N-Me)Cit probes 3a and 3e were further tested for resistance to cleavage by other abundant proteases secreted by human neutrophils: human proteinase 3 and cathepsin G. EGCit probe 3a was fully intact in the presence of both proteases, whereas EV(N-Me)Cit probe 3e was partially degraded by proteinase 3 (Table 1).

[0173] Next, probes 3a-f were tested for stability in undiluted BALB / c plasma (Figure 2D). EGCit, EACit, and EV(N-Me)Cit probes 3a, 3b, and 3e showed improved stability compared to EGCit probe 2. Notably, the EGCit probe 3a probe remained 40% intact after 4 days of incubation. GCit probe 3f was less stable than EGCit probe 3a, which is consistent with previous findings that glutamic acid at the P3 position enhances linker stability in mouse plasma. Furthermore, the EGCit probe 3a probe fully resisted degradation in monkey and human plasma (Figure 2E and Table 2). Finally, additional stability assays were performed for EFCit, E(N-Me)VCit, and V(N-Me)Cit probes. However, these probes showed instability to degradation mediated by neutrophil elastase or mouse plasma (Figure 7). Collectively, these findings indicate that the EGCit sequence provides complete stability against undesired proteolytic degradation leading to premature payload release.

[0174] The EGCit linker increases ADC hydrophilicity and cell-killing potency with efficient intracellular payload releaseWe next sought to explore how the P2 amino acids evaluated above affect ADC physicochemical properties, intracellular payload release upon cleavage, and antigen-specific cell killing potency. To this end, we constructed anti-HER2 ADCs using selected P2-modified cleavable linkers and previously developed conjugation techniques (Amani et al., 2017; Anami et al., 2020; Anami et al., 2017) (Figure 3A). First, a diazide branched linker was site-specifically attached to the side chain of glutamine 295 (Q295) within the N297A anti-HER2 mAb (derived from trastuzumab) by microbial transglutaminase (MTGase)-mediated transpeptidation. In parallel, VCit, EVCit, EGCit, EV(N-Me)Cit, and GCit linker-based modules containing bicyclo[6.1.0]nonyne (BCN) as a handle for strain-promoted azide-alkyne cycloaddition, polyethylene glycol (PEG), PABC as a self-immolative spacer, and MMAE were prepared. Finally, these payload modules were click-reacted with mAb-tetraazide to obtain homogeneous anti-HER2 ADCs 4a-e with a drug-to-antibody ratio (DAR) of 4. The homogeneity was confirmed by ESI-MS analysis (Figure 3B). SEC analysis confirmed that no significant degradation or aggregation occurred after incubating each ADC in phosphate-buffered saline (PBS, pH 7.4) at 37 °C for 28 days (Figure 2). Then, hydrophobic interaction chromatography (HIC) analysis was performed in physiological conditions (phosphate-buffered saline, pH 7.4) to evaluate the relative hydrophobicity of each ADC (Figure 2C). EGCit ADC 4c was the least hydrophobic of the ADCs tested. EVCit ADC 4b, EV(N-Me)Cit ADC 4d, and GCit ADC 4e had intermediate hydrophobicity. VCit ADC 4a was the most hydrophobic. The results suggested that incorporation of the smallest amino acid, glycine, at the P2 position and negatively charged glutamic acid at the P3 position could synergistically reduce ADC hydrophobicity at physiological pH.This feature is advantageous in the construction of ADCs, as hydrophobic ADCs often exhibit high aggregation rates and rapid clearance ( Lyon et al., 2015 ).

[0175] These ADCs were then evaluated for in vitro cytotoxicity in HER2-positive (KPL-4, SK-BR-3, BT-474, JIMT-1, MDA-MB-453) and -negative (MDA-MB-231) human breast cancer cell lines (Figure 3D-I). As controls, non-cleavable anti-HER2 MMAE ADC 4f (DAR4) and a non-targeting ADC (5, DAR 4) constructed using the BCN-EGCit-PABC-MMAE module were also prepared and tested. VCit ADC 4a, EVCit ADC 4b, and EGCit ADC 4c showed comparable cell killing potency in HER2-positive lines but not in HER2-negative MDA-MB-231 cells; under the assay conditions, EC 50 The values ​​ranged from 0.071 to 0.087 nM for KPL-4, 0.119 to 0.175 nM for SK-BR-3, 0.459 to 0.578 nM for BT-474, 0.088 to 0.114 nM for JIMT-1, and 0.183 to 0.254 nM for MDA-MB-453 (Table 3). These ADCs also showed similar maximum cell killing potency at high concentrations (Table 4). EV(N-Me)Cit ADC 4d had similar EC 50 However, the EC 50 The values ​​and cell viability at high concentrations were slightly or significantly higher than those of EGCit ADC 4c in other HER2-positive cell lines. GCit ADC 4e was as potent as EGCit 4c in KPL-4 and JIMT-1. However, EC 50 Increased EC values ​​and / or percentage of viable cells at maximum ADC concentration were observed in SK-BR-3 and MDA-MB-453. The non-cleavable anti-HER2 ADC 4f, lacking a peptide cleavable sequence within the linker scaffold, showed much higher EC in KPL-4 and JIMT-1 cells compared to the cleavable ADCs 4a-c. 50values. Furthermore, noncleavable ADC 4f and nontargeting EGCit ADC 5 showed very poor or almost no cell killing effect in either cell line. These findings highlight the importance of internalization and subsequent intracellular release of free MMAE for effective cell killing. The comparable potency of ADCs 4a–c also suggests that the EGCit linker has an enzymatic cleavage rate similar to that of the VCit and EVCit linkers. To further validate this point, we quantified free MMAE released from ADCs 4a–d in KPL-4 cells (Figure 3J). After treating KPL-4 cells with each ADC for 24 h, the MMAE concentration in the cell lysates was determined by high-resolution MS. As expected, approximately 80% of the conjugated MMAE was detected as free payload in VCit, EVCit, and EGCit ADCs 4a–c. In contrast, only 10.5% MMAE release was observed for the EV(N-Me)Cit ADC 4d, indicating that N-methylation of citrulline delayed the enzymatic linker cleavage required for payload release. Collectively, these results demonstrate that the hydrophilic EGCit sequence enables efficient traceless payload release upon ADC internalization in a wide range of cell types with various catabolic profiles, ensuring maximal ADC potency.

[0176] EGCit ADC is stable in plasma and tolerates bone marrow-derived human neutrophilsTo evaluate the ADC stability and safety profile, ADCs 4a-c were first tested for plasma stability. No significant degradation was observed in any of the ADCs after 28 days of incubation in undiluted human and monkey plasma at 37 °C (Figures 4A-4B and Tables 5-6). EVCit and EGCit ADCs 4b and 4c showed little linker cleavage after 14 days of incubation in undiluted BALB / c mouse plasma (Figure 4C and Table 7). In contrast, VCit ADC 4a lost approximately 74% of the conjugated MMAE after the same period. The stability of these ADCs was then evaluated in the presence of human neutrophil elastase (Figure 4D). EVCit ADC 4b underwent partial degradation, generating DAR 0-3 fragments, whereas EGCit ADC 4c completely resisted degradation. These results are consistent with previous studies using pyrene probes (Figures 2C-E).

[0177] To investigate the potential effects of ADCs on neutrophil production in human bone marrow, we performed ex vivo differentiation of hematopoietic stem and progenitor cells (HSPCs) into neutrophils, followed by treatment with ADCs (Figure 4E). reported that ADCs loaded with MMAE via a VCit linker significantly reduced the population of differentiating human neutrophils, while mature neutrophils were able to withstand the toxic effects (Zhao et al., 2018). Following the protocol with some modifications, HSPCs from a single donor were differentiated into granulocytes with growth factors over a period of 7 days. At this time point, the population of live cells expressing both granulocyte markers CD15 and CD66b increased from 0.7% to 71.3% (Figures 4F, 4G, and 9). Because neutrophils are the most abundant granulocyte type in humans (50–75% of total white blood cells), we considered these CD15 / CD66b double-positive cells to be a representative population of differentiating neutrophils. These cells were then treated with 200 nM of EVCit and EGCit ADCs 4b and 4c. Encouragingly, EGCit ADC 4c significantly reduced CD15 + / CD66 +EGCit ADC 4c caused only a slight decrease in the percentage of hematopoietic cells (Figures 4G and 9). In contrast, the neutrophil population was significantly reduced to 46% by treatment with EVCit ADC 4b. A notable difference was also observed in nonspecific toxicity to total hematopoietic cells between these two ADCs (Figure 4I). The percentage of total viable cells (including both neutrophils and other cells) compared to the vehicle group was sharply reduced to 8% by EVCit ADC 4b, whereas EGCit ADC 4c caused only moderate toxicity (relative viability: 45%). In a report by Zhao et al., such nonspecific toxicity was observed for high concentrations of ADC (approximately 50% cell death at 10 nM), regardless of linker cleavability or cell permeability of the payload (Zhao et al., 2018). Collectively, these results highlight the potential of the EGCit linker to reduce the risk of myelosuppression, especially neutropenia, caused by prematurely released ADC payloads.

[0178] EGCit linkers have the potential to minimize antigen-independent hepatotoxicity of ADCs To investigate whether the EGCit linker affects ADC safety at therapeutic doses, EGCit MMAE ADC 4c, EGCit MMA ENHERTU®, or KADCYLA® were administered at 80 mg kg -1An exploratory study was performed in healthy CD-1® IGS mice using ENHERTU® and KADCYLA®. No significant weight loss (>20%) or other severe clinical symptoms were observed in any of the treatment groups for 5 days after injection (Figure 10). Next, blood chemistry tests were performed by collecting serum at the end of the 5-day monitoring to evaluate potential hepatotoxicity (Figures 5A-5D). The following molecules related to liver function were quantified: alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALKP), and blood urea nitrogen (BUN). Increases in AST, ALT, and ALKP as well as decreases in BUN generally indicate liver damage. In the case of EGCit ADC, the values ​​of these parameters were comparable to those of untreated mice. In contrast, mice treated with ENHERTU® and KADCYLA® showed significantly elevated levels of AST and ALT as well as decreased levels of BUN. ALKP levels also appeared to be elevated with these two ADCs. Hematology analyses were also performed using whole blood samples drawn on day 5. No significant changes in red blood cell, platelet, and neutrophil counts were observed with the ADC or ENHERTU® compared to the untreated cohort, while KADCYLA® appeared to decrease platelets and increase neutrophils (Figure 10). Overall, these results suggest that the EGCit MMAE ADC has an improved safety profile compared to current ADCs.

[0179] EGCit ADCs exhibit improved antitumor efficacy in various xenograft models compared with conventional ADCs We next sought to evaluate EGCit-based ADCs for treatment efficacy in multiple xenograft mouse tumor models (Figures 6A-6F). In the KPL-4 inflammatory breast tumor model, orthotopically xenografted NOD-scid gamma (NSG) mice were treated with a single dose of our anti-HER2 ADC 4b or 4c, KADCYLA®, or ENHERTU® at 1 mg kg -1To explore the applicability of the EGCit linker technology, we also prepared the duocarmycin DM (DuoDM) ADC 6. This ADC has a subnanomolar in vitro EC 50 values ​​(0.116 nM for KPL-4 and 0.059 nM for JIMT-1), demonstrating that the EGCit linker could also efficiently release DuoDM (Figure 11 and Table 3). No significant toxicity associated with administration of ADCs 4b, 4c and 6 was observed over the course of the study, as assessed by monitoring significant weight loss and other clinical symptoms (Figure 12). Furthermore, these ADCs demonstrated notable percent tumor growth inhibition (%TGI at day 31: 107%, ADC 4b; 104%, ADC 4c; 94%, ADC 6) and survival benefit (animal deaths by day 70: 0 of 5, ADC 4b, c; 1 of 5, ADC 6) (Figures 6A-6B and 12). In contrast, only limited tumor growth inhibition was observed with KADCYLA® (47% TGI, P = 0.0097) and ENHERTU® (48% TGI, P = 0.0158) compared with EGCit ADC 4c. All animals treated with these FDA-approved ADCs were either dead or reached the predefined humane endpoint (>1,000 mm) by the end of the study. 3 Mice were sacrificed based on tumor size or >20% body weight loss (median survival: 35 days, KADCYLA®; 45 days, ENHERTU®).

[0180] Next, the clinical potential of the EGCit linker was tested in a dual-drug ADC format. It was demonstrated that dual-drug ADCs loaded with MMAE and MMAF can effectively treat low HER2 heterogeneous breast tumors with high drug resistance (Yamazaki et al., 2021). An EGCit-based MMAE / F DAR 4+2 ADC 7a was prepared. This ADC and ENHERTU® were tested in the JIMT-1 / MDA-MB-231 mixed tumor model established by the inventors, a model of refractory human breast cancer characterized by aggressive growth, heterogeneous HER2 expression, and moderate resistance to hydrophobic payloads such as MMAE (Yamazaki et al., 2021). A single dose of each ADC (ENHERTU®, 3 mg kg -1 ; ADC 7a, 1 mg kg -1 ) was administered intravenously to orthotopic tumor-bearing nude mice 8 days after implantation (mean tumor volume: 100–150 mm 3 ). No acute toxicity associated with ADC administration was observed for either ADC (Figure 12). The EGCit dual drug ADC 7a demonstrated a significant antitumor effect (112% TGI at day 31, Figures 6C and 12) and survival benefit (no animal deaths by day 70, Figure 6D). In contrast, at a dose of 3 mg kg -1 Even with increased dosing, ENHERTU® demonstrated only modest tumor growth inhibition in this HER2 heterogeneous tumor model (76% TGI at day 31, P = 0.0065, comparator: EGCit ADC 7a). Additionally, 4 of 5 mice had to be euthanized by the end of the study due to severe clinical symptoms, including ulceration.

[0181] Finally, we sought to establish the generalizability of our linker technology by testing it in an orthotopic glioblastoma multiforme (GBM) model (Figures 6E-6F). ADC-based systemic treatment of GBM has not been successful, as demonstrated by the recent failure of depatuxizumab mafodotin (Depatux-M, formerly called ABT-414) (Phillips et al., 2016) and AMG-595 (Hamblett et al., 2015) in clinical trials. Using this linker technology, we were able to construct a panel of homogenous anti-epidermal growth factor receptor variant III (EGFRvIII) ADCs from N297A depatuxizumab, including the conventional VCit ADC 8a, EGCit ADC 8b, and EGCit-DuoDM ADCs conjugated via PABC or p-aminobenzyl quaternary ammonium (PABQ) linkages (Staben et al., 2016). These ADCs were equally potent in EGFRvIII-positive U87ΔEGFR-luc GBM cells (Figure 11). ADCs 8a and b were then tested in an orthotopic U87ΔEGFR-luc model (Figure 6E). A single dose of each ADC was administered at 5 mg kg 5 days after implantation into intracranial tumor-bearing NSG mice. -1 The mice were intravenously injected at 100-200 mg / kg / day for 12 h. No acute toxicity associated with ADC administration was observed in any group (Figure 12). The short survival time of the untreated cohort (median survival: 16 days) demonstrated the extremely aggressive tumor growth in this model (Figure 6F). EGCit ADC 8b exerted remarkable therapeutic efficacy; median survival time was extended to >70 days, and 5 of 7 mice achieved complete remission without clinical symptoms at the end of the study. In contrast, VCit ADC 8a moderately extended median survival to 26 days (P = 0.001, vs. vehicle; P = 0.0006, vs. EGCit ADC 8b). All mice treated with VCit ADC 8a died or were euthanized by the end of the study.

[0182] Table 1. Stability of pyrene-based peptide probes in the presence of human neutrophil enzymes. Quantification was performed after 24 h of incubation (n = 2). Data are presented as mean ± SEM. TIFF2025500942000048.tif36150

[0183] Table 2: Stability of pyrene-based peptide probes in undiluted human, monkey, and mouse plasma. Quantification was performed after 48 and 96 hours of incubation (n = 2). Data are presented as mean ± SEM. TIFF2025500942000049.tif52150

[0184] Table 3. EC in human breast cancer cell lines (n = 4) and GBM cell lines (n = 3). 50 Values ​​in brackets are 95% confidence intervals. TIFF2025500942000050.tif160150

[0185] Table 4: Cell viability at maximum ADC concentration (n = 4). Values ​​in brackets are 95% confidence intervals. TIFF2025500942000051.tif95150

[0186] Table 5: Stability of ADC in undiluted human plasma. Quantification was performed after 7, 14, and 28 days of incubation (n = 3). Data are presented as mean ± SEM. TIFF2025500942000052.tif24128

[0187] Table 6: Stability of ADC in undiluted monkey plasma. Quantification was performed after 7, 14, and 28 days of incubation (n = 3). Data are presented as mean ± SEM. TIFF2025500942000053.tif20128

[0188] Table 7: Stability of ADC in undiluted mouse plasma. Quantification was performed after 4, 7, and 14 days of incubation (n = 3). Data are presented as mean ± SEM. TIFF2025500942000054.tif24128

[0189] (Table 8) Statistical significance. TIFF2025500942000055.tif254150 a Adjusted by Holm-Bonferroni test. *P < 0.05; **P < 0.01; ***P < 0.005; ns, not significant.

[0190] In this study, it was demonstrated that the enzymatically cleavable EGCit, tripeptide linker could solve the problem of neutropenia, a common clinical off-target toxicity issue derived from human neutrophil protease-mediated degradation of VCit- and similar dipeptide-MMAE ADCs, without hindering treatment efficacy. The linker cleavage site of human neutrophil elastase was identified using a small molecule-based pyrene probe. It was found that P2 modification of VCit and EVCit linkers by replacing valine with glycine could significantly improve the resistance of neutrophil protease-mediated degradation. The EGCit linker also had higher mouse plasma stability and less hydrophobicity compared with VCit and EVCit linkers. To show that EGCit-based ADCs have comparable potency to ADCs containing VCit or similar peptide linkers, EGCit ADCs were constructed with different antibodies and payloads to ensure broad applicability. In vitro results showed that EGCit ADCs had comparable in vitro cytotoxicity to VCit and EVCit ADCs in various breast and brain tumor lines. Comparable in vitro MMAE release among VCit, EVCit, and EGCit ADCs indicated that the EGCit sequence allows efficient traceless payload release upon ADC internalization in a wide range of cell types with various catabolic profiles. The in vivo treatment efficacy of EGCit ADCs was also investigated in KPL-4 with homogeneous HER2 antigen, mixed JIMT-1 / MDA-MB-231 breast cancer model with heterogeneous HER2 antigen, and aggressive U87ΔEGFR-luc brain tumor model. These three animal models with different antigens and antigen expression levels ensured maximum ADC treatment efficacy. Compared with non-cleavable linkers with high resistance to proteolysis, cleavable EGCit linkers are more preferable to use in ADCs. This is because the payload catabolism products of ADCs containing non-cleavable linker-payload structures following lysosomal degradation of the ADC consist of amino acid residues from the linker, payload, and antibody, and therefore the linker and payload structures need to be carefully designed so as not to compromise their efficacy.This is the reason why only limited payloads can be selected for non-cleavable linkers.

[0191] Example 2 – Materials and Methods antibody. Anti-HER2, anti-EGFRvIII, and isotype control mAbs with N297A mutation were expressed in-house. Other antibodies used in this study were purchased from commercial sources: mouse anti-MMAE / F mAb (LEV-MAF3) from Levena Biopharma; goat anti-human IgG Fab antibody (109-005-097) and goat anti-mouse IgG-HRP conjugate (115-035-071) from Jackson ImmunoResearch; mouse anti-human CD66b FITC conjugate (305104), mouse anti-human CD15 APC conjugate (301908), and mouse anti-human CD34 FITC conjugate (343503) from BioLegend.

[0192] Human neutrophil protease-mediated cleavage assay using a pyrene probe. Each test compound (10 mM in DMSO, 2 μL) was mixed with 97 μL of Tris-buffered saline (TBS, pH 7.4) and 1 μL of 1-pyrenemethylamine (10 mM in DMSO, internal standard). The mixture was incubated at 37° C. for 10 min. Human neutrophil elastase (40 ng μL -1 , 20 μL in TBS, MilliporeSigma), human proteinase 3 (250 ng μL -1 , 5 μL in TBS, MilliporeSigma) or human cathepsin G (330 ng μL -1, 5 μL in TBS, MilliporeSigma) was added to the test compound mixture in a 1:1 volume ratio, followed by incubation at 37°C. Aliquots (5 μL) were collected at each time point (0, 1, 3, 6, and 24 h for elastase, and 0 and 24 h for proteinase 3 and cathepsin G). Proteins were precipitated by adding cold acetonitrile (25 μL) containing 1% formic acid. The mixtures were then kept at -20°C for 30 min. Precipitated proteins were pelleted by centrifugation (15,000 × g, 4°C, 30 min) and the supernatant of each sample was analyzed by analytical HPLC for quantification (UV absorbance at 342 nm). The amount of each probe was normalized to the peak area of ​​the internal standard. All assays were performed at least three times with technical duplicates, and data shown are representative of replicates.

[0193] Plasma stability study using a pyrene probe. Each test compound (10 mM in DMSO, 2 μL) was mixed with 1 μL of 1-pyrenemethylamine (10 mM in DMSO, internal standard) and incubated at 37° C. for 10 min. Pooled healthy human plasma, cynomolgus monkey plasma, or BALB / c mouse plasma (197 μL, Innovative Research) was added to the mixture, followed by incubation at 37° C. Aliquots (5 μL) were collected at each time point (0, 48, and 96 h for human and cynomolgus monkey plasma; 0, 1, 6, 24, 48, and 96 h for BALB / c mouse plasma) and proteins were precipitated by adding 25 μL of cold acetonitrile containing 1% formic acid. The mixtures were then kept at −20° C. for 30 min. Precipitated proteins were separated by centrifugation (15,000 × g, 4° C., 30 min), and the supernatant of each sample was collected and analyzed for quantification by analytical HPLC as described above. All assays were performed at least three times in technical duplicates and data shown are representative of replicates.

[0194] MTGase-mediated antibody-linker conjugation. Anti-HER2 mAb with N297A mutation (1.06 mL, 10.5 mg mL in PBS) -1, 11.1 mg antibody) was incubated with a previously developed diazide branched linker (Anami et al., 2018; Anami et al., 2020; Anami et al., 2017) (37.1 μL of 100 mM stock in water, 50 equivalents) and an Activa TI® (275 μL of 40% solution in PBS, purchased from Ajinomoto, Modernist Pantry) for 16-20 h at room temperature. The reaction was monitored using a Thermo LC-MS system consisting of a Vanquish UHPLC and a Q Exactive™ Hybrid Quadrupole-Orbitrap™ mass spectrometer equipped with a MabPac RP column (2.1 × 50 mm, 4 μm, Thermo Scientific). The elution conditions were as follows: mobile phase A = water (0.1% formic acid); mobile phase B = acetonitrile (0.1% formic acid); gradient A:B = 75:25 to 45:55 over 3 min; flow rate = 0.25 mL min -1 The conjugated antibody was analyzed by SEC (Superdex 200 increase 10 / 300 GL, GE Healthcare, solvent: PBS, flow rate = 0.6 mL min -1 ) to give the antibody-linker conjugate [9.62 mg, 86% yield as determined by bicinchoninic acid (BCA) assay]. The other antibody-linker conjugates used in this study were prepared in the same manner.

[0195] Strain-promoted azide-alkyne cycloaddition for payload attachment. BCN-PEG3-EGCit-PABC-MMAE (18.9 μL of a 10 mM stock solution in DMSO, 2 equivalents per azide group) was dissolved in a solution of mAb-linker conjugate in PBS (1.70 mL, 2.1 mg mL -1) and the mixture was incubated at room temperature for 17 h. The reaction was monitored using a Thermo LC-MS system consisting of a Vanquish UHPLC and a Q Exactive™ Hybrid Quadrupole-Orbitrap™ mass spectrometer equipped with a MabPac RP column. The crude product was purified by SEC to obtain the ADC [3.0 mg, 85% yield determined by bicinchoninic acid (BCA) assay]. The analysis and purification conditions were the same as those described above. The average DAR values ​​were determined based on UV peak area and ESI-MS analysis. Other conjugates used in this study were prepared in a similar manner or according to previous reports (Anami et al., 2018; Anami et al., 2020; Anami et al., 2017; Yamazaki et al., 2021; Yamaguchi et al., 2021; Anami et al., 2020).

[0196] HIC analysis. Each ADC (1 mg mL -1 , 10 μL in PBS) was analyzed using an Agilent 1100 HPLC system equipped with a MAbPac HIC-Butyl column (4.6 × 100 mm, 5 μm, Thermo Scientific). The elution conditions were as follows: mobile phase A = 50 mM sodium phosphate (pH 7.4) containing ammonium sulfate (1.5 M) and 5% isopropanol; mobile phase B = 50 mM sodium phosphate (pH 7.4) containing 20% ​​isopropanol; gradient A:B = 99:1 to 1:99 over 30 min; flow rate = 0.5 mL min -1 .

[0197] Long-term stability testing. Each ADC (1 mg mL -1, 100 μL in PBS) were incubated at 37 °C. Aliquots (8 μL) were taken at 28 days and immediately stored at -80 °C until use. Samples were analyzed using an Agilent 1100 HPLC system equipped with a MAbPac SEC analytical column (4.0 × 300 mm, 5 μm, Thermo Scientific). Elution conditions were as follows: flow rate = 0.2 mL min -1 ;solvent = PBS.

[0198] Cell culture. U87ΔEGFR-luc was generated by lentiviral transduction of U87ΔEGFR cells (a gift from Dr. Balveen Kaur, UTHealth) with LENTIFECT™ lentiviral particles (GeneCopoeia, LP461-025) encoding firefly luciferase and puromycin resistance genes. Transduction was performed according to the manufacturer's instructions. JIMT-1 (AddexBio), SK-BR-3 (ATCC), and BT-474 (ATCC) were cultured in 10% EQUAFETAL® (Atlas Biologicals), GLUTAMAX® (2 mM, Gibco), sodium pyruvate (1 mM, Corning), and penicillin-streptomycin (Penicillin: 100 units mL -1 ;Streptomycin: 100μg mL -1 KPL-4 (provided by Dr. Junichi Kurebayashi, Kawasaki Medical School), MDA-MB-453 (ATCC), MDA-MB-231 (ATCC), and U87ΔEGFR-luc were cultured in RPMI 1640 (Corning) supplemented with 10% EQUAFETAL®, GLUTAMAX® (2 mM), and penicillin-streptomycin (Penicillin: 100 units mL -1 ;Streptomycin: 100μg mL -1All cells were cultured in DMEM (Corning) supplemented with 0.1% CO2. All cells were cultured at 37°C under 5% CO2 and passaged before full confluence for up to 20 passages. All cell lines were routinely tested for mycoplasma contamination.

[0199] Cell viability assay. Cells were seeded (5,000 cells per well in 50 μL culture medium) into culture-treated 96-well clear plates and incubated for 24 h at 37 °C under 5% CO2. Serially diluted samples (50 μL) were added to each well, and the plates were incubated at 37 °C for 72 h for KPL-4, SK-BR-3, MDA-MB-231, and U87ΔEGFR-luc cells, and for 96 h for JIMT-1, BT-474, and MDA-MB-453 cells. For DuoDM-ADC, all tested cell lines were incubated for 120 h. After replacing the old medium with 100 μL of fresh medium, WST-8 (1.5 mg mL -1 A mixture of 20 μL of 1-methoxy-5-methylphenazinium methylsulfate (1-methoxyPMS, 100 μM, Cayman Chemical) and 1-methoxy-5-methylphenazinium methylsulfate (1-methoxyPMS, 100 μM, Cayman Chemical) was added to each well and the plate was incubated at 37°C for 2 hours. After gentle agitation of the plate, the absorbance at 460 nm was recorded using a BioTek Synergy HTX plate reader. EC 50 Values ​​were calculated using Graph Pad Prism 8 software. All assays were performed in triplicate (U87ΔEGFR-luc cell line) or quadruplicate (other cell lines).

[0200] Determination of cellular MMAE concentration. KPL-4 cells were seeded into culture-treated 12-well plates (3 × 10 cells per well in 500 μL culture medium). 5 Cells) were incubated at 37°C under 5% CO2 for 24 hours. Each ADC (final concentration 4 μg mL -1, 26.7 nM) was added to each well and the plate was incubated at 37°C for 24 hours. After centrifugation of the plate (500 × g for 5 min at 4°C), the supernatant (450 μL) was collected and then the proteins were precipitated by adding MeOH (600 μL). The cells were lysed with MeOH (500 μL) and transferred to a microtube. An additional 100 μL of MeOH was added to rinse the plate and transferred to a microtube. The supernatant and cell lysate were centrifuged (15,000 × g for 10 min) and each supernatant was transferred to a microtube. These supernatants were then dried by airflow at 40°C. After drying, MS-grade water (30 μL) was added and the mixture was centrifuged (15,000 × g for 5 min). The supernatants were then analyzed by a Q EXACTIVE™ HYBRID QUADRUPOLE-ORBITRAP™ mass spectrometer. For quantification, a six-point standard curve was generated using serial dilutions of free MMAE (0.032-100 nM). Untreated cell lysates were used for the free MMAE standard curve. All assays were performed in triplicate.

[0201] Plasma stability study using ADC. [1] Stability in mouse plasma. -1 , 1.2 μL in PBS) to a final concentration of 1 μg mL -1ADCs were added to undiluted BALB / c mouse plasma (118.8 μL) until 1:1000. After incubation at 37°C for various times, aliquots (15 μL each) were taken and stored at -80°C until use. Samples were analyzed by sandwich ELISA assay. High-binding 96-well plates (Corning) were coated with goat anti-human IgG Fab antibody (500 ng per well). After coating overnight at 4°C, the plates were blocked with 100 μL of 2% BSA in PBS containing 0.05% Tween 20 (PBS-T) for 1 h at room temperature with agitation. The solution was then removed and each ADC sample (100 μL in PBS-T containing 1% BSA) was added to each well and the plates were incubated at room temperature for 2 h. Each well was washed three times with 100 μL of PBS-T before adding 100 μL of mouse anti-MMAE / F mAb (1:5,000). After 1 h incubation at room temperature, each well was washed three times with 100 μL of PBS-T and 100 μL of goat anti-mouse IgG-HRP conjugate (1:10,000) was added. After 1 h, the plate was washed three times with 100 μL of PBS-T and 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB) substrate (0.1 mg mL) in phosphate-citrate buffer / 30% H2O2 (1:0.0003 volume to volume, pH 5) was added. -1 After allowing color to develop for 10–30 min, 25 μL of 3N-HCl was added to each well, and the absorbance at 450 nm was then recorded using a plate reader (Biotek Cytation 5). Concentrations were calculated based on the standard curve.

[0202] [2] Stability in human and monkey plasma. Assays were performed similarly using human HER2 (100 ng per well, ACROBiosystems) for plate coating and mouse anti-MMAE / F mAb (1:5,000) and goat anti-mouse IgG-HRP conjugate (1:10,000) as secondary and tertiary detection antibodies, respectively. All assays were performed in triplicate.

[0203] Human neutrophil enzyme-mediated cleavage assay with ADC.Each ADC in TBS buffer (1 mg mL -1 , 30 μL) was incubated at 37 °C for 10 min. Preheated human neutrophil elastase (1 ng μL -1 , 30 μL, MilliporeSigma) was added, followed by incubation at 37 °C for 24 h. Samples were analyzed using a Thermo LC-MS system consisting of a Vanquish UHPLC and a Q Exactive™ Hybrid Quadrupole-Orbitrap™ mass spectrometer equipped with a MabPac RP column (2.1 × 50 mm, 4 μm, Thermo Scientific). Elution conditions were as follows: mobile phase A = water (0.1% formic acid); mobile phase B = acetonitrile (0.1% formic acid); gradient A:B = 75:25 to 45:55 over 3 min; flow rate = 0.25 mL min -1 NOTE: In this analysis, hinge cleavage of anti-HER2 mAb and ADC was observed.

[0204] Human neutrophil killing assay. The assay was performed according to the protocol reported by Zhao et al. (Zhao et al., 2018) with modifications. CD34 positive HSPCs isolated from bone marrow were purchased from AllCells (received as cryopreserved samples and stored in liquid nitrogen until use). Reagents purchased from StemCell Technologies for this assay were as follows: cell culture medium (STEMSPAN™ SFEM II, 09655), proliferation supplement (STEMSPAN™ CC100, 02690), interleukin 3 (IL-3), stem cell factor (SCF), Flt-3 ligand (Flt-3L), granulocyte macrophage colony-stimulating factor (GM-CSF), and granulocyte colony-stimulating factor (G-CSF). HSCs (2 × 10 4 cells mL -1) were expanded for 3 days in STEMSPAN™ SFEM II supplemented with CC100. Prior to differentiation, expression levels of granulocytic markers (CD66b and CD15) were measured using a BD LSR II flow cytometer (day 0, see next section for details). Expanded HSCs were cultured in SCF (50 ng mL -1 ), Flt-3L (100 ng mL -1 ), IL-3 (5 ng mL -1 ), GM-CSF (5 ng mL -1 ), and G-CSF (5 ng mL -1 ) for 4 days. On day 4, the cell culture medium was changed to IL-3 (5 ng mL -1 ) and G-CSF (30 ng mL -1 ) and the cells were incubated for another 3 days. On day 7, CD15 and CD66b of differentiated cells were measured by flow cytometry. The cell culture medium was replaced with STEMSPAN™ SFEM II supplemented with G-CSF (30 ng mL -1 ), 1% penicillin-streptomycin, and vehicle control or each ADC (200 nM) were replaced with STEMSPAN™ SFEM II supplemented with 1% ethanol, and the cell density was adjusted to 1 × 10 5 cells mL -1 After 7 days of incubation (day 14), cells were measured for CD15 and CD66b by flow cytometry. The effect of each ADC on the neutrophil population was expressed as the percentage of CD66b / CD15 double positive cells in the live cell population.

[0205] Flow cytometry.Cells were washed twice with cold staining buffer (1 mL for microtubes; 200 μL for microwell plates, FBS, BD Bioscience, Cat. No.: 554656) and collected by centrifugation (400 × g, 4 °C for 5 min). Cells were then resuspended in cold staining buffer (100 μL) in microtubes or microwell plates and mixed with 5 μL of fluorescently labeled antibody (mouse anti-human CD66b FITC conjugate, mouse anti-human CD15 APC conjugate, or mouse anti-human CD34 FITC conjugate). To set the voltage and gating parameters to obtain accurate fluorescent signals, a drop of ULTRACOMP EBEADS™ Compensation Beads (Invitrogen, Cat. No.: 01-2222-42) was also labeled with 5 μL of antibody separately from the cells. Cells and beads were incubated on ice in the dark for 20 min and then washed three times with 1 mL (for microtubes) or 200 μL (for microwell plates) of cold staining buffer to remove unbound antibodies. After centrifugation (400 × g, 4 °C for 5 min), the supernatant was carefully aspirated from the cell pellet or beads. Stained cells or beads were resuspended in 1 mL of cold staining buffer and transferred to tubes for flow cytometry analysis, and data were acquired with an LSR II flow cytometer and Diva acquisition software (BD Biosciences), gated on live cells with appropriate isotype-matched controls and unstained cells as negative controls. CD66b and CD15 percentages resulting from flow cytometry were normalized to untreated samples. All assays were performed in triplicate.

[0206] animal research.All procedures were approved by the Animal Welfare Committee of the University of Texas Health Science Center at Houston and were performed in accordance with institutional guidelines for the care and use of animals. All animals were housed under controlled conditions, i.e., 21-22 °C (± 0.5 °C), relative humidity 30-75% (± 10%), and a 12:12 light / dark cycle with lights on at 7.00 am. Food and water were available ad libitum for all animals.

[0207] Tolerability studies. Female 6- to 8-week-old CD-1® IGS mice (6- to 8-week-old, Charles River Laboratories, strain code: 022) were administered a single dose of each ADC (80 mg kg -1 ) was administered intraperitoneally. Body weight was monitored daily for 5 days. Humane endpoints were defined as 1) weight loss of more than 20% or 2) signs of severe distress; however, no mice met these criteria during the course of the study. Five days after injection, the mice were deeply anesthetized with isoflurane and whole blood was collected by cardiac puncture for subsequent hematology and blood chemistry analysis (vehicle, n = 4; KADCYLA®, n = 6; ENHERTU®, n = 7; EGCit ADC 4c, n = 10).

[0208] Blood chemistry and hematology analysis. Blood chemistry: Whole blood (400-600 μL) was collected using S-MONOVETTE® (1.1 mL syringe, Sarstedt) charged with serum gel and allowed to clot for 30-40 min at room temperature. After centrifugation at 2,000 × g for 10 min, the resulting serum sample (150 μL) was loaded onto a specialized NSAID 6-clip for identification of liver injury (IDEXX, Westbrook, ME) and analyzed using a Catalyst Dx Chemistry Analyzer (IDEXX).

[0209] Hematology.Whole blood (700-1,000 μL) was collected using S-MONOVETTE® (1.1 mL syringe, Sarstedt) filled with K3EDTA. Blood samples were mixed gently and thoroughly by inversion and stored on ice until analysis (<4 h). Each blood sample (500 μL) was analyzed using PROCYTE DX® (IDEXX).

[0210] In vivo xenograft mouse model of human breast cancer. KPL-4 model: Cells (1 × 10) suspended in 100 μL of 1:1 PBS / CULTREX® BME Type 3 (Trevigen) 7 Cells) were orthotopically injected into the inguinal mammary fat pad of female NSG mice (6-8 weeks old, purchased from The Jackson Laboratory, stock number: 005557, maintained by in-house breeding). Tumor volumes were approximately 100 mm 3 When t100 mg / kg was reached, mice were randomly assigned to six groups (n=5 for each group) and administered a single dose of each ADC (1 mg kg -1 ) or vehicle was administered intravenously to mice. Tumor volumes (0.52 × a × b 2 , a: major axis, b: minor axis) and body weight were monitored twice a week. 3 Mice were euthanized when the tumor size exceeded 100 μg / kg, tumor size exceeded 2 cm in diameter, a weight loss of more than 20% was observed, or the mice showed signs of distress. Such events were counted as death.

[0211] JIMT-1 / MDA-MB-231 mixed tumor model. 1 × 10 in 100 μL of 1:1 PBS / CULTREX® BME Type 3 (Trevigen) 7 JIMT-1 cells and 2.5 × 10 6A co-suspension of MDA-MB-231 cells was orthotopically injected into the inguinal mammary fat pad of female NU / J mice (6-8 weeks old, The Jackson Laboratory, stock number: 002019). Seven days after implantation, mice were randomly assigned to each group (n=5 for ENHERTU®; n=6 for EGCit-MMAE / F dual drug ADC 7a) and injected with sterile-filtered human IgG (30 mg kg ) in PBS. -1 The following day, subjects were administered a single dose of ENHERTU® (3 mg kg -1 ) or dual drug ADC 7a (1 mg kg -1 ) was intravenously administered to mice. Tumor volume (0.52 × a × b 2 , a: major axis, b: minor axis) and body weight were monitored twice a week. 3 Mice were euthanized when the tumor size exceeded 100 μg / mL, the tumor size exceeded 2 cm in diameter, or the mice showed signs of severe distress. Such events were counted as deaths.

[0212] Orthotopic xenograft mouse model of human GBM. Infect NSG mice (6–8 weeks old, male and female) with U87ΔEGFR-luc cells (1 × 10 5 NSG mice were stereotactically implanted with ketamine (67.5 mg kg -1 ) and dexmedetomidine (0.45 mg kg -1) was injected intraperitoneally and maintained at 37 °C on a heating pad until the completion of surgery. After shaving the scalp and treating with 10 μL of 0.25% bupivacaine supplemented with epinephrine (1:200,000), the anesthetized mouse was placed in a stereotaxic apparatus. After disinfecting the scalp with chlorhexidine and ethanol, a small incision was made and then a burr hose was drilled into the skull (1 mm anterior and 2 mm lateral to the bregma) of the right hemisphere. A 10 μL Hamilton syringe (model 701 N) was loaded with cells suspended in 2 μL of cold Hanks' balanced salt solution (HBSS) and slowly inserted (3.5 mm deep) into the right hemisphere through the burr hole. After a 1 min hold time, the cells were instilled for 5 min (0.4 μL min -1 After a 3-minute hold time, the needle was removed at 0.75 mm min -1 The incision was closed using GLUTURE® (Zoetis) and the mice were administered atipamezole (1 mg kg -1 , i.p.). Five days after implantation, brain tumor-bearing NSG mice were randomized and injected with the ADC (5 mg kg -1 Mice were intravenously injected with a single dose of either VCit ADC 8a (n = 6; EGCit ADC 8b (n = 7) or PBS (n = 6). Body weight was monitored every 3-4 days, and mice were euthanized if >20% weight loss or severe clinical symptoms were observed. Such events were counted as deaths.

[0213] Data reporting.No statistical analysis was performed before the experiment was performed, but the sample size was determined by following the method for similar experiments in the art previously reported. We did not use vehicle control for statistical analysis in xenograft breast cancer study. The researchers were blinded to the allocation during the experiment. For the determination of cellular MMAE concentration and human neutrophil killing, one-way ANOVA with Dunnett's post-hoc test was used for multiple comparisons. For blood chemistry and hematology analysis and xenograft tumor model study, Welch's t-test (two-tailed, unpaired, unequal variance) was used. Kaplan-Meier survival curve statistics were analyzed with the log-rank (Mantel-Cox) test. To control the family-wise error rate in multiple comparisons, the crude P-values ​​were adjusted by the Holm-Bonferroni method. Differences with adjusted P-values ​​less than 0.05 were considered statistically significant in all analyses. See Table 8 for all P-values.

[0214] Data availability. All data supporting the findings in this study are available within the paper, its Supplementary Information files, or from the corresponding author on reasonable request.

[0215] Example 3 – Synthesis Unless otherwise noted, all materials for chemical syntheses were purchased from commercial suppliers (Acros Organics, AnaSpec, Broadpharm, Chem-Impex International, Fisher Scientific, Levena Biopharma, Sigma Aldrich, TCI America, and others) and used as received. All anhydrous solvents were purchased and stored over activated molecular sieves under an argon atmosphere.

[0216] Analytical reversed-phase high-performance liquid chromatography (RP-HPLC) was performed using a Thermo LC-MS system consisting of a Vanquish UHPLC and an LTQ XL™ linear ion trap mass spectrometer equipped with a C18 reversed-phase column (ACCUCORE™ VANQUISH™ C18+ UHPLC column, 2.1 × 50 mm, 1.5 μm, Thermo Scientific). Standard analytical conditions for organic molecules were as follows: flow rate = 0.5 mL min -1 ; Solvent A = water containing 0.1% formic acid; Solvent B = acetonitrile containing 0.1% formic acid. Compounds were analyzed using a linear gradient and monitored with UV detection at 210 and 254 nm. Preparative HPLC was performed using a Breeze HPLC system (Waters) equipped with a C18 reversed-phase column (XBridge Peptide BEH C18 OBD Prep column, 130 Å, 5 μm, 19 × 150 mm, Waters). Standard purification conditions were as follows: flow rate = 20 mL min -1 Solvent A = water containing 0.05% trifluoroacetic acid (TFA), 0.1% formic acid, or 0.1% NH4OH; Solvent B = acetonitrile containing 0.05% TFA (standard conditions), 0.1% formic acid (FA conditions), or 0.1% NH4OH (basic conditions). Compounds were analyzed using a linear gradient and monitored with UV detection at 210 and 254 nm. In both cases, fractions were analyzed offline using either LC-MS system for purity confirmation, and fractions containing the desired product were lyophilized using a Labconco Freezone 4.5 Liter Benchtop Freeze Dry System. High-resolution mass spectra were acquired using a THERMO Q EXACTIVE™ HYBRID QUADRUPOLE-ORBITRAP™ mass spectrometer.

[0217] Fmoc solid phase peptide synthesis (Fmoc SPPS) for acetyl-capped compounds (S2a-j).2-Chlorotrityl chloride resin (600 mg, 0.96 mmol) and Fmoc-citrulline-OH (1.8 equiv.) were placed in a manual solid-phase reactor containing N,N-diisopropylethylamine (DIPEA, 3 equiv.) and DMF (3 mL) and stirred for 2 h. MeOH (600 μL) was added to the resin and stirred for 20 min. The solution was drained and the resin was washed with DMF (5×3 mL) and DCM (5×3 mL). To remove the Fmoc protecting group after each coupling, the resin was treated with 20% piperidine / DMF (5 mL) or 1M oxime in 20% piperidine / DMF (5 mL) for 20 min and washed with DMF (5×3 mL) and DCM (5×3 mL). N-Methylation 1For , the resin was treated with 2-nitrobenzenesulfonyl chloride (o-NBS-Cl, 4 equiv.) and collidine (10 equiv.) in NMP (2 mL) for 15 min and washed with NMP (2 × 1 mL) to temporarily protect the amine groups with o-NBS. After repeating the o-NBS protection twice, the resin was treated with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 3 equiv.) in NMP (1 mL) for 3 min, then with dimethyl sulfate (DMS, 10 equiv.) in NMP (1 mL) for 2 min. This step was repeated twice. The resin was then treated twice with 2-mercaptoethanol (10 equiv.) and DBU (5 equiv.) in NMP (2 mL) for 5 min to deprotect the o-NBS protecting groups. Fmoc-protected amino acids (4 equiv.) were preactivated with 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU, 4 equiv.) and DIPEA (6 equiv.) in DMF for 3 min, and the cocktail was added to the resin. The resin was stirred at room temperature for 1 h. Completion of the coupling was verified by Kaiser test. After each coupling step, the coupling cocktail was drained and the resin was washed with DMF (5×3 mL) and DCM (5×3 mL). After peptide elongation, the resin was treated with acetic anhydride (4 equiv.) and DIPEA (6 equiv.) in DMF for 1 h, then washed with DMF (5×3 mL) and DCM (5×3 mL). The resulting protected peptide resin was treated with a cocktail of 1% trifluoroacetic acid (TFA) / DCM for 1 h at room temperature. The solution was concentrated in vacuo and the crude peptides were precipitated with cold diethyl ether (20 mL) followed by centrifugation at 2,000 × g for 3 min (three times). The resulting crude peptides S2a-j were dried in vacuum and then used immediately in the next step without purification (Figure 13).

[0218] Ac-Glu(t-Bu)-Gly-Cit-PABC-PNP (S3a).To a solution of S2a (9.5 mg, 21 μmol) in DCM / MeOH (4:1, 1.25 mL) were added p-aminobenzyl alcohol (5.2 mg, 42 μmol) and EEDQ (10.4 mg, 42 μmol). After stirring overnight at room temperature in the dark, the solution was concentrated in vacuum and the crude peptide was precipitated with cold diethyl ether (20 mL) followed by centrifugation at 2,000 × g for 3 min (10 times). The resulting crude peptide was dried in vacuum and then used immediately in the next step without purification. Bis(2,4-dinitrophenyl)carbonate (27.4 mg, 90 μmol) and DMAP (4.4 mg, 36 μmol) were added to a solution of the crude peptide (10.2 mg, 18 μmol) in DMF (300 μL) and the mixture was stirred at room temperature for 2 h. The reaction was quenched with 3-N HCl / ACN at 0° C., and the crude product was then purified by preparative RP-HPLC to give analytically pure peptide S3a (6.0 mg, 39% for two steps). Purity was confirmed by LC-MS. White powder. HRMS (ESI) Calcd. For C 33 H 44 N7O 12 [M+H] + : 730.3043. Found: 730.3033. Peptides S3b–j were synthesized from S2b–j in a similar manner.

[0219] Ac-Glu(t-Bu)-Ala-Cit-PABC-PNP (S3b). 47.9 mg, 80% in 2 steps. White powder. HRMS (ESI) Calcd. For C 34 H 46 N7O 12 [M+H] + : 744.3199. Found: 744.3177.

[0220] Ac-Glu(t-Bu)-Leu-Cit-PABC-PNP (S3c). 8.1 mg, 29% in 2 steps. White powder. HRMS (ESI) Calcd. For C 37 H 52 N7O 12 [M+H] + : 786.3669. Found: 786.3647.

[0221] Ac-Glu(t-Bu)-Ile-Cit-PABC-PNP (S3d). 42 mg, 2ステップで66%. White powder. HRMS (ESI) Calcd. For C 37 H 52 N7O 12 [M+H] + : 786.3669. Found: 786.3644.

[0222] Ac-Glu(t-Bu)-Val-(N-Me)Cit-PABC-PNP (S3e). 6.4 mg, 2ステップで21%. White powder. HRMS (ESI) Calcd. For C 37 H 52 N7O 12 [M+H] + : 786.3669. Found: 786.3643.

[0223] Ac-Gly-Cit-PABC-PNP (S3f). 14.2 mg, 2ステップで31%. White powder. HRMS (ESI) Calcd. For C 24 H 29 N6O9[M+H] + : 545.1991. Found: 545.1965.

[0224] Ac-Glu(t-Bu)-Phe-Cit-PABC-PNP (S3g). 14.7 mg, 2ステップで44%. White powder. HRMS (ESI) Calcd. For C 40 H 50 N7O 12 [M+H] + : 820.3512. Found: 820.3495.

[0225] Ac-Glu(t-Bu)-(N-Me)Val-Cit-PABC-PNP (S3h). 14.1 mg, 2ステップで57%. White powder. HRMS (ESI) Calcd. For C 37 H 52 N7O 12 [M+H] + : 786.3669. Found: 786.3642.

[0226] Ac-(N-Me)Val-Cit-PABC-PNP (S3i). 9.2 mg, 2ステップで19%. White powder. HRMS (ESI) Calcd. For C 28H 37 N6O9[M+H] + : 601.2617. Found: 601.2591.

[0227] Ac-Val-(N-Me)Cit-PABC-PNP (S3j). 6.1 mg, 20% in 2 steps. White powder. HRMS (ESI) Calcd. For C 28 H 37 N6O9[M+H] + : 601.2617. Found: 601.2602.

[0228] Ac-Glu-Gly-Cit-PABC-sar-pyrene(3a). Compound S3a (6.0 mg, 8.2 μmol) was dissolved in 20% TFA / DCM (200 μL and 800 μL). After stirring at room temperature for 40 min, the solution was concentrated in vacuum and the crude peptide was precipitated with cold diethyl ether (20 mL) followed by centrifugation at 2,000 × g for 3 min (3 times). The resulting crude peptide was dried in vacuum and then used immediately in the next step without purification. The crude peptide was dissolved in DMF (300 μL) and sarcosine-pyrene was added to the solution. 2 (54.5μL, 9.0μmol, 50 mg mL in DMSO -1 ), DIPEA (2.9 μL, 16.4 μmol), and DMAP (10 μL, 10 mol%, 10 mg mL in DMF -1 ) was added. The mixture was stirred at 37° C. for 4 h and purified by preparative RP-HPLC under acidic conditions to give 3a (3.3 mg, 48% for two steps). White powder. HRMS (ESI) Calcd. For C 43 H 49 N8O 10 [M+H] + : 837.3566. Found: 837.3525. Peptides 3b–f and S1a–d were synthesized from S3b–j in a similar manner.

[0229] Ac-Glu-Ala-Cit-PABC-sar-pyrene(3b). 8.1 mg, 35% in 2 steps. White powder. HRMS (ESI) Calcd. For C 44 H 51 N8O 10 [M+H]+ : 851.3723. Found: 851.3683.

[0230] Ac-Glu-Leu-Cit-PABC-sar-pyrene(3c). 5.0 mg, 2ステップで59%. White powder. HRMS (ESI) Calcd. For C 47 H 57 N8O 10 [M+H] + : 893.4192. Found: 893.4163.

[0231] Ac-Glu-Ile-Cit-PABC-sar-pyrene(3d). 0.8 mg, 2ステップで3.5%. White powder. HRMS (ESI) Calcd. For C 47 H 57 N8O 10 [M+Na] + : 893.4192. Found: 893.4178.

[0232] Ac-Glu-Val-(N-Me)Cit-PABC-sar-pyrene(3e). 0.4 mg, 2ステップで5.5%. White powder. HRMS (ESI) Calcd. For C 47 H 57 N8O 10 [M+H] + : 893.4192. Found: 893.4183.

[0233] Ac-Gly-Cit-PABC-sar-pyrene(3f). 4.2 mg, 74%. White powder. HRMS (ESI) Calcd. For C 38 H 42 N7O7[M+H] + : 708.3140. Found: 708.3128.

[0234] Ac-Glu-Phe-Cit-PABC-sar-pyrene(S1a). 6.9 mg, 2ステップで58%. White powder. HRMS (ESI) Calcd. For C 50 H 55 N8O 10 [M+H] + : 927.4036. Found: 927.4027.

[0235] Ac-Glu-(N-Me)Val-Cit-PABC-sar-pyrene(S1b). 3.1 mg, 45% in 2 steps. White powder. HRMS (ESI) Calcd. For C 47 H 57 N8O 10 [M+H] + : 893.4192. Found: 893.4177.

[0236] Ac-(N-Me)Val-Cit-PABC-sar-pyrene(S1c). 9.0 mg, 79%. White powder. HRMS (ESI) Calcd. For C 42 H 50 N7O7[M+H] + : 764.3766. Found: 764.3738.

[0237] Ac-Val-(N-Me)Cit-PABC-sar-pyrene(S1d). 2.5 mg, 33%. White powder. HRMS (ESI) Calcd. For C 42 H 50 N7O7[M+H] + : 764.3766. Found: 764.3750.

[0238] Fmoc solid-phase peptide synthesis (Fmoc SPPS) for Fmoc-protected compounds (S4a~c). Fmoc-protected peptide compounds S4a-c were prepared as described above. The resulting protected peptide resin was treated with a cocktail of 1% trifluoroacetic acid (TFA) / DCM for 1 h at room temperature. The solution was concentrated in vacuum and the crude peptide was precipitated with cold diethyl ether (20 mL) followed by centrifugation at 2,000 × g for 3 min (3 times). The resulting crude peptides S4a-c were dried in vacuum and then used immediately in the next step without purification (Figure 14).

[0239] Fmoc-peg3-Glu(t-Bu)-Gly-Cit-PABC-PNP (S5a).To a solution of crude S4a (158.6 mg, 192 μmol) in DCM / MeOH (4:1, 2.5 mL) was added p-aminobenzyl alcohol (70.8 mg, 576 μmol) and EEDQ (285 mg, 1.15 mmol). After stirring overnight at room temperature in the dark, the solution was concentrated in vacuum and the crude peptide was precipitated with cold diethyl ether (20 mL) followed by centrifugation at 2,000 × g for 3 min (10 times). The resulting crude peptide was dried in vacuum and then used immediately in the next step without purification.

[0240] Bis(2,4-dinitrophenyl)carbonate (55 mg, 183 μmol) and DMAP (8.9 mg, 73 μmol) were added to a solution of the crude peptide (34.1 mg, 36.5 μmol) in DMF (1 mL) and the mixture was stirred at room temperature for 2 h. The reaction was quenched with 3-N HCl / ACN at 0° C., and the crude product was then purified by preparative RP-HPLC to give analytically pure peptide S5a (25.1 mg, 63% for two steps). Purity was confirmed by LC-MS. White powder. HRMS (ESI) Calcd. For C 54 H 67 N8O 17 [M+H] + : 1099.4619. Found: 1099.4602. Peptides S5b and S5c were synthesized in a similar manner from S4b and S4c.

[0241] Fmoc-peg3-Glu(t-Bu)-Val-(N-Me)Cit-PABC-PNP (S5b). 8.2 mg, 17% in 2 steps. White powder. HRMS (ESI) Calcd. For C 58 H 75 N8O 17 [M+H] + : 1155.5245. Found: 1155.5240.

[0242] Fmoc-peg4-Gly-Cit-PABC-PNP (S5c). 7.3 mg, 47% in 2 steps. White powder. HRMS (ESI) Calcd. For C 47 H 56 N7O 15 [M+H] +: 958.3829. Found: 958.3811.

[0243] Fmoc-peg3-Glu-Gly-Cit-PABC-MMAE (S6a). Compound S5a (25.1 mg, 22.8 μmol) was dissolved in 20% TFA / DCM (200 μL and 800 μL). After stirring at room temperature for 40 min, the solution was concentrated in vacuum and the crude peptide was precipitated with cold diethyl ether (20 mL) followed by centrifugation at 2,000 × g for 3 min (3 times). The resulting crude peptide was dried in vacuum and then used immediately in the next step without purification. The crude peptide was dissolved in DMF (500 μL) and MMAE (18 mg, 25.1 μmol), DIPEA (7.9 μL, 45.6 μmol), and 1-hydroxy-7-azabenzotriazole (HOAt, 6.2 mg, 45.6 μmol) were added to the solution. The mixture was stirred at 37 °C for 4 h and purified by preparative RP-HPLC under acidic conditions to give S6a (20.9 mg, 57% for two steps). White powder. HRMS (ESI) Calcd. For C 83 H 121 N 12 O 21 [M+H] + : 1621.8764. Found: 1621.8722. Peptides S6b and S6c were synthesized in a similar manner from S5b and S5c.

[0244] Fmoc-peg3-Glu-Val-(N-Me)Cit-PABC-MMAE (S6b). 3.6 mg, 30% in 2 steps. White powder. HRMS (ESI) Calcd. For C 87 H 129 N 12 O 21 [M+H] + : 1667.9390. Found: 1667.9362.

[0245] Fmoc-peg4-Gly-Cit-PABC-MMAE (S6c). 6.5 mg, 56%. White powder. HRMS (ESI) Calcd. For C 80 H 118 N 11 O 19 [M+H] +: 1536.8600. Found: 1536.8584.

[0246] BCN-peg3-Glu-Gly-Cit-PABC-MMAE (S7a). Compound S6a (10.5 mg, 6.5 μmol) was dissolved in 50% diethylamine / DMF solution (800 μL) at room temperature. After 1 h, the solution was concentrated in vacuo and used in the next step without further purification. BCN-NHS (2.3 mg, 7.8 μmol, Berry&Associates) and DIPEA (2.3 μL, 13 μmol) were added to a solution of this crude mixture in DMF (400 μL) and the mixture was stirred at room temperature for 3 h. The crude product was purified by preparative RP-HPLC under basic conditions to give analytically pure peptide S7a (7.5 mg, 73% for two steps). Purity was confirmed by LC-MS. White powder. HRMS (ESI) Calcd. For C 79 H 123 N 12 O 21 [M+H] + : 1575.8920. Found: 1575.8909. Modules S7b and S7c were synthesized in a similar manner from S6b and S6c.

[0247] BCN-peg3-Glu-Val-(N-Me)Cit-PABC-MMAE (S7b). 1.2 mg, 35% in 2 steps. White powder. HRMS (ESI) Calcd. For C 83 H 131 N 12 O 21 [M+H] + : 1631.9546. Found: 1631.9529.

[0248] BCN-peg4-Gly-Cit-PABC-MMAE (S7c). 4.1 mg, 65% in 2 steps. White powder. HRMS (ESI) Calcd. For C 76 H 120 N 11 O 19 [M+H] + : 1490.8757. Found: 1490.8737.

[0249] BCN-peg3-Glu-Val-Cit-PABC-MMAE (S8).(Figure 15) Fmoc-peg3-Glu(t-Bu)-Val-Cit-PABC-MMAE (7.3 mg, 4.2 μmol, prepared as previously described) 3 was added to a 20% TFA / DCM solution at room temperature. After 1 h, the solution was concentrated in vacuum and the crude compound was precipitated with cold diethyl ether (20 mL) followed by centrifugation at 2,000 g for 3 min (3 times). The crude product was dissolved in a 50% diethylamine / DMF solution. After stirring at room temperature for 1 h, the solution was concentrated in vacuum and used in the next step without further purification. BCN-NHS (2.5 mg, 8.4 μmol) and DIPEA (2.2 μL, 12.6 μmol) were added to a solution of this crude mixture in DMF and the mixture was stirred at room temperature overnight. The crude product was purified by preparative RP-HPLC under basic conditions to give S8 (4.2 mg, 61% for 3 steps). Purity was confirmed by LC-MS. White powder. HRMS (ESI) Calcd. For C 82 H 128 N 12 O 21 Na2[M+2Na] 2+ : 831.4551. Found: 831.4577. (Figure 15).

[0250] Boc-peg4-MMAE (S9). Boc-peg4 acid (5.3 mg, 14.6 μmol) in DMF (150 μL) was mixed with HATU (11 mg, 29.1 μmol) and DIPEA (7.6 μL, 43.7 μmol) and stirred for 5 min to activate it. The mixture was then added to a solution of MMAE (7 mg, 9.7 μmol) in DMF (450 μL) and stirred at room temperature for 1 h. The crude product was purified by preparative RP-HPLC under acidic conditions to give analytically pure peptide S9 (13 mg, quantitative). Purity was confirmed by LC-MS. White powder. HRMS (ESI) Calcd. For C 55 H 97 NO 14 [M+H] + : 1065.7057. Found: 1065.7045.

[0251] BCN-peg4-MMAE (S10) (Figure 16). Compound S9 (13 mg, 12.2 μmol) was dissolved in 50% TFA / DCM solution (1 mL). After stirring at room temperature for 30 min, the solution was concentrated in vacuum and the crude compound was precipitated with cold diethyl ether (20 mL) followed by centrifugation at 2,000 g for 3 min (3 times). BCN-NHS (0.3 mg, 1.0 μmol) and DIPEA (0.25 μL, 1.4 μmol) were added to a solution of this crude mixture in DMF (500 μL) and the mixture was stirred at room temperature for 30 min. The crude product was purified by preparative RP-HPLC under FA conditions to give S10 (1.8 mg, 13% for two steps). Purity was confirmed by LC-MS. White powder. HRMS (ESI) Calcd. For C 61 H 101 NO 14 [M+H] + : 1141.7370. Found: 1141.7361.

[0252] Fmoc-peg3-Glu-Gly-Cit-PABC-MMAF (S11). Compound S5a (7.4 mg, 6.7 μmol) was dissolved in 20% TFA / DCM (200 μL and 800 μL). After stirring at room temperature for 50 min, the solution was concentrated in vacuum and the crude peptide was precipitated with cold diethyl ether (20 mL) followed by centrifugation at 2,000 × g for 3 min (3 times). The resulting crude peptide was dried in vacuum and then used immediately in the next step without purification. The crude peptide was dissolved in DMF (350 μL) and MMAF·TFA salt (7.4 mg, 8.7 μmol), DIPEA (2.3 μL, 13.4 μmol), and HOAt (1.8 mg, 13.4 μmol) were added to the solution. The mixture was stirred at 37 °C overnight and purified by preparative RP-HPLC under acidic conditions to give S11 (9.4 mg, 85% for two steps). White powder. HRMS (ESI) Calcd. For C 83 H 119 N 12 O 22 [M+H] + : 1635.8556, Found: 1635.8541.

[0253] BCN-peg3-Glu-Gly-Cit-PABC-MMAF (S12) (Figure 17). Compound S11 (4.7 mg, 2.9 μmol) was dissolved in 50% diethylamine / DMF solution (600 μL) at room temperature. After 30 min, the solution was concentrated in vacuo and used in the next step without further purification. BCN-NHS (1.1 mg, 3.8 μmol) and DIPEA (1 μL, 5.8 μmol) were added to a solution of this crude mixture in DMF (300 μL) and the mixture was stirred at room temperature for 3.5 h. The crude product was purified by preparative RP-HPLC under basic conditions to give analytically pure peptide S12 (1.5 mg, 33% for two steps). Purity was confirmed by LC-MS. White powder. HRMS (ESI) Calcd. For C 77 H 121 N 12 O 22 [M+H] + : 1565.8713, Found: 1565.8671.

[0254] Boc-protected duocarmycin DM(S13) (FIG. 18). To a solution of duocarmycin DM (DuoDM, 7.0 mg, 12.1 μmol) in dry ACN (800 μL) was added DIPEA (6.3 μL, 36.3 μmol) and 4-nitrophenyl chloroformate (4.9 mg, 24.2 μmol). After stirring at room temperature for 30 min, t-butyl methyl(2-(methylamino)ethyl)carbamate (12 μL, 60.5 μmol, AK Scientific) was added to the mixture and the mixture was stirred at room temperature for 15 min. The reaction was quenched with 2-N HCl / ACN at 0° C. and the crude product was purified by preparative RP-HPLC to give analytically pure compound S13 (8.0 mg, 84% for two steps). Purity was confirmed by LC-MS. White powder. HRMS (ESI) Calcd. For C 36 H 45 NO6Cl [M+H] + : 678.3053, Found: 678.3029.

[0255] Fmoc-peg3-Glu-Gly-Cit-PABC-DuoDM (S14).Compound S5a (10.4 mg, 9.5 μmol) was dissolved in 20% TFA / DCM (200 μL and 800 μL). After stirring at room temperature for 50 min, the solution was concentrated in vacuum and the crude peptide was precipitated with cold diethyl ether (20 mL) followed by centrifugation at 2,000 × g for 3 min (3 times). In parallel, compound S13 (8.0 mg, 10.1 μmol) was dissolved in 50% TFA / DCM (500 μL and 800 μL) at 0 °C and the mixture was stirred for 30 min. The solution was concentrated in vacuum and the crude compound was precipitated with cold diethyl ether (20 mL) followed by centrifugation at 2,000 × g for 3 min (3 times). The crude peptide and Boc-deprotected compound were dissolved in DMF (600 μL) and DIPEA (8.2 μL, 47.5 μmol) was added to the mixture at 0 °C. The mixture was stirred at room temperature for 2 h, and the reaction was quenched with 2-N HCl / ACN at 0° C. The crude product was purified by preparative RP-HPLC under acidic conditions to give S14 (8.7 mg, 58% for two steps). White powder. HRMS (ESI) Calcd. For C 75 H 90 N 12 O 18 Cl [M+H] + : 1481.6179, Found: 1481.6165.

[0256] BCN-peg3-Glu-Gly-Cit-PABC-DuoDM (S15). Compound S14 (8.7 mg, 5.5 μmol) was dissolved in 50% diethylamine / DMF solution (600 μL) at room temperature. After 30 min, the solution was concentrated in vacuo and used in the next step without further purification. BCN-NHS (2.1 mg, 7.2 μmol) and DIPEA (1.9 μL, 11 μmol) were added to a solution of this crude mixture in DMF (300 μL) and the mixture was stirred at room temperature for 1 h. The crude product was purified by preparative RP-HPLC under basic conditions to give analytically pure peptide S15 (3.5 mg, 44% for two steps). Purity was confirmed by LC-MS. White powder. HRMS (ESI) Calcd. For C 71 H 92 N 12 O 18 Cl [M+H] +: 1435.6336, Found: 1435.6329.

[0257] seco-CBI-β-glucuronide (S16). A suspension of Boc-seco-CBI (10 mg, 30 μmol), methyl-(2,3,4-tri-O-acetyl-α-D-glucopyranosyl) trichloroacetimidate (21.5 mg, 45 μmol, AmBeed), and molecular sieves 4 Å (50 mg) in DCM (1 mL) was stirred at room temperature for 30 min. The mixture was cooled to -20 °C and BF3·Et2O (3.3 μL, 15 μmol, ca. 48% BF3) in DCM (100 μL) was added dropwise. After stirring at -20 °C for 2 h, additional BF3·Et2O (20 μL, 90 μmol, ca. 48% BF3) in DCM (100 μL) was added dropwise. BF3) was added dropwise, then the mixture was allowed to warm to room temperature and stirred at room temperature for 2 h. The reaction was quenched by filtration through a pad of Celite. The crude product was purified by preparative RP-HPLC under FA conditions to give analytically pure compound S16 (13.5 mg, 82%). Purity was confirmed by LC-MS. Off-white powder. HRMS (ESI) Calcd. For C 26 H 29 NO 10 Cl [M+H] + : 550.1475, Found: 550.1464.

[0258] Duocarmycin DM-β-glucuronide (S17). Compound S16 (5.4 mg, 9.8 μmol) in DMF (400 μL) was added to 5-(2-dimethylaminoethoxy)indole-2-carboxylic acid (7.3 mg, 29.4 μmol, prepared as previously described) at room temperature. 4 and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC·HCl, 11.2 mg, 58.5 μmol), and then the reaction mixture was stirred at room temperature for 1.5 h. The crude product was purified by preparative RP-HPLC under FA conditions to give analytically pure compound S17 (5.9 mg, 77%). The purity was confirmed by LC-MS. Off-white powder. HRMS (ESI) Calcd. For C39 H 43 N3O 12 Cl [M+H] + : 780.2530, Found: 780.2494.

[0259] Fmoc-peg3-Glu(t-Bu)-Gly-Cit-PAB-Cl (S18). Fmoc-peg3-Glu(t-Bu)-Gly-Cit-PABOH (21.8 mg, 23 μmol, intermediate of S5a) in dry DMF (300 μL) was cooled to 0° C. and thionyl chloride (1.8 μL, 25 μmol) in dry DCM (100 μL) was added dropwise. After stirring at 0° C. for 1 h, additional thionyl chloride (1.8 μL, 25 μmol) in DCM (100 μL) was added dropwise. After 1 h, additional thionyl chloride (1.8 μL, 25 μmol) in DCM (100 μL) was added dropwise and then the mixture was stirred at 0° C. for another 30 min. The crude product was purified by preparative RP-HPLC to give peptide S18 (13.7 mg, approx. 70% pure). Note: Although a single peak was collected by preparative RP-HPLC, the chloride compound S18 was hydrolyzed to the starting material during lyophilization. Therefore, the purity of S18 decreased to about 70% after lyophilization. White powder. HRMS (ESI) Calcd. For C 47 H 63 N7O 12 Cl [M+H] + : 952.4218, Found: 952.4213.

[0260] Fmoc-peg3-Glu(t-Bu)-Gly-Cit-PAB-DuoDM-β-glucuronide (S19).Compound S18 (3.4 mg, 1.8 μmol, approximately 70% pure) in dry DMF (250 μL) was mixed with compound S17 (0.9 mg, 1.2 μmol), sodium iodide (0.1 mg, 0.6 μmol), and DIPEA (0.4 μL, 2.4 μmol), and the mixture was then stirred at room temperature for 6 hours. Additional compound S17 (1.2 mg, 1.5 μmol) and DIPEA (0.4 μL, 2.4 μmol) were added to the mixture. After 2 hours, additional compound S17 (0.6 mg, 0.75 μmol) was added to the mixture and stirred at room temperature overnight. The progress of the reaction was monitored by LC-MS, and starting material S18 was still remaining. The reaction mixture was warmed to 37° C., and additional compound S17 (0.5 mg, 0.64 μmol) was added. After 3 h, the crude product was purified by preparative RP-HPLC under acidic conditions to give analytically pure compound S19 (2.9 mg, 95%). Purity was confirmed by LC-MS. White powder. HRMS (ESI) Calcd. For C 86 H 104 N 10 O 24 Cl [M] + : 1695.6908, Found: 1695.6901.

[0261] BCN-peg3-Glu-Gly-Cit-PAB-DuoDM-β-glucuronide (S20).(Figure 19) Compound S19 (2.7 mg, 1.6 μmol) was dissolved in 20% TFA / DCM (200 μL and 800 μL). After stirring at room temperature for 1 h, the solution was concentrated in vacuum and the crude peptide was precipitated with cold diethyl ether (20 mL) followed by centrifugation at 2,000 × g for 3 min (3 times). The crude peptide was dissolved in MeOH (300 μL) and LiOH·H2O (2 mg, 48 μmol) in water (300 μL) was added to the mixture. After stirring at room temperature for 1 h, the mixture was cooled to 0 °C and quenched with formic acid (3 μL). The solution was then removed in vacuum and the crude peptide was dissolved in DMF (400 μL). BCN-NHS (0.6 mg, 2.1 μmol) and DIPEA (0.5 μL, 3.2 μmol) were added to the solution and the mixture was stirred at room temperature for 1.5 h. The crude product was purified by preparative RP-HPLC under basic conditions to give analytically pure peptide S20 (1.0 mg, 42% for 3 steps). Purity was confirmed by LC-MS. White powder. HRMS (ESI) Calcd. For C 71 H 90 N 10 O 21 Cl [M] + : 1453.5965, Found: 1453.5950.

[0262] Expression and purification of human monoclonal antibodies. Free style HEK-293 human embryonic kidney cells (Invitrogen) were transfected with mammalian expression vectors encoding human IgG1 kappa light chain and full-length heavy chain sequences (based on the variable sequences of trastuzumab and depatuxizumab). To produce aglycosylated mAbs, the N297A mutation was incorporated into the heavy chain constant region. Transfected HEK-293 cells were cultured for 7 days at 37°C with 8% CO2 in a humidified cell culture incubator with shaking at 150 rpm, after which the culture medium was harvested. Antibodies secreted into the culture medium were purified using Protein A resin (GE Healthcare).

[0263] All compositions and / or methods disclosed and claimed herein can be made and carried out without undue experimentation in light of the present disclosure. Although the compositions and methods of the present disclosure have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that modifications may be applied to the compositions and / or methods, and to the steps or order of steps of the methods described herein, without departing from the concept, spirit and scope of the present disclosure. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein, while still achieving the same or similar results. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the present disclosure, as defined by the appended claims.

[0264] V. References The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference: TIFF2025500942000056.tif19546TIFF2025500942000057.tif230128TIFF2025500942000058.tif230148TIFF2025500942000059.tif24128

Claims

1. Formula (I): or a pharmaceutically acceptable salt thereof, During the ceremony: X 1 is a covalent bond, alkanediyl (C≦12) , or substituted alkanediyl (C≦12) and R 1 is hydrogen, -ZR 6 , -(OCH 2 CH 2 )0-50ZR 6 , or substitution -(OCH 2 CH 2 )0-50ZR 6 and where: R6 is hydrogen, hydroxy, aminohydroxy, amino, mercapto, hydroxylamino, hydrazino, or azido; Alkyl (C≦12) , alkenyl (C≦12) , alkynyl (C≦12) , alkylhydrazine (C≦12) or substituted forms thereof; polyglycines containing 1 to 6 glycine units; or formula: Substructure of (In the formula: A 1 and A 2 are each independently absent or (C≦12) , substituted arenediyl (C≦12) , heteroarenediyl (C≦12) , or substituted heteroarenediyl (C≦12) and a fused arene (C≦12) , substituted arenes (C≦12) , heteroarenes (C≦12) , or substituted heteroarenes (C≦12) Forming; A 3 is a covalent bond, O, alkanediyl (C≦8) , substituted alkanediyl (C≦8) , alkoxydiyl (C≦8) , or substituted alkoxydiyl (C≦8) and A 4 and A 5 are each independently a covalent bond, an alkanediyl (C≦8) , substituted alkanediyl (C≦8) , Arendjiil (C≦8) , and substituted arenediyl (C≦8) Selected from: R d , R e , R e ′, and R h are each independently hydrogen, halo, thioether, selenoether, sulfate, tosylate, mesylate, aryl (C≦8) , or substituted aryl (C≦8) Selected from: R f is a halo; R g are amines, hydrazines, alkylaminos (C≦8) , substituted alkylamino (C≦8) , dialkylamino (C≦8) , substituted dialkylamino (C≦8) , alkylhydrazine (C≦8) , or substituted alkylhydrazine (C≦8) and X 4 and X 5 are each independently O, N, C(O), or CH 2 or X 4 and X 5 Alkanediyl (C≦8) or substituted alkanediyl (C≦8) and taken together form a fused cycloalkane group of 3 to 8 ring atoms; R 7 is hydrogen, hydroxy, amino, or oxo; R 8 is carboxy; or Alkyl (C≦12) , amide (C≦12) , aryl (C≦12) , heteroaryl (C≦12) , -C(O)OR 11 , -C(O)NR 11 R 11 ', or its substitutions, where: R 11 and R 11 ' are each independently hydrogen; or Alkyl (C≦12) , aryl (C≦12) or a substituted form thereof; R 9 and R 10 are each independently hydroxy, amino, or halo; or Alkyl (C≦12) , aryl (C≦12) , or its replacement) and x is 0 to 4, as valence allows; y is 0 to 4, as valence allows; z is 0 to 4; Z is a covalent bond, alkanediyl (C≦12) , -C(O)-alkanediyl (C≦12) , -C(O)-alkanediyl (C≦12) -C(O)NH- or substituted forms thereof; R 2 is hydrogen, alkyl (C≦12) , substituted alkyl (C≦12) , aryl (C≦12) , heteroaryl (C≦12) , aralkyl (C≦12) , heteroaralkyl (C≦12) , acyl (C≦12) , substituted acyl (C≦12) or a monovalent amino protecting group; W is a covalent bond or a multivalent polymer having 2 to 21 attachment points; n is 1 to 20, except that when W is a covalent bond, n is 1, and when W is a multivalent polymer, n is less than or equal to one less than the number of attachment points; Each X is independently a covalent bond, an alkanediyl (C≦12) , substituted alkanediyl (C≦12) , one or more amino acid residues, or an oligomeric peptide; each X 2 are independently alkanediyl (C≦12) or substituted alkanediyl (C≦12) and Each R 3 are independently hydroxy or amino; Alkoxy (C≦12) , acyloxy (C≦12) , alkylamino (C≦12) , dialkylamino (C≦12) , amide (C≦12) , heteroaryl (C≦12) or a substituted form thereof; or -X 6 -C(O)R 12 (where: X 6 -O, -NR b - or a covalent bond; R b is hydrogen, alkyl (C≦6) , substituted alkyl (C≦6) or a monovalent amino protecting group; R 12 is hydroxy or amino; or Alkoxy (C≦12) , alkylamino (C≦12) , dialkylamino (C≦12) , or a substitution thereof); or -OSO2NR13R13', -OP(O)(OH)OR14, -OSO2OR14', -NRc-SO2NR13R13', -NRc-P(O)(OH)OR14, -NRc-SO2OR14', -SO2NR13R13', -P(O)(OH)OR14, or -SO2OR14' (where: R c is hydrogen, alkyl (C≦6) , substituted alkyl (C≦6) , aryl (C≦12) , heteroaryl (C≦12) , aralkyl (C≦12) , heteroaralkyl (C≦12) or a monovalent amino protecting group; R 13 , R 13 ′, R 14 , and R 14 ' are each independently hydrogen, alkyl, (C≦12) , cycloalkyl (C≦12) , aryl (C≦12) , heteroaryl (C≦12) , aralkyl (C≦12) , heteroaralkyl (C≦12) or its replacement) and each m is independently 0 or 1; Each R 4 is independently the side chain moiety of glycine or valine; and Each R 4 ' is hydrogen, alkyl (C≦12) , substituted alkyl (C≦12) , aryl (C≦12) , heteroaryl (C≦12) , aralkyl (C≦12) , heteroaralkyl (C≦12) , acyl (C≦12) , substituted acyl (C≦12) or a monovalent amino protecting group; Each R 5 are independently the side chain moiety of glycine, alanine, ornithine, lysine, arginine, citrulline, asparagine, or glutamine, or an amino-protected form thereof; Each R 5 ' is hydrogen, alkyl (C≦12) , substituted alkyl (C≦12) , aryl (C≦12) , heteroaryl (C≦12) , aralkyl (C≦12) , heteroaralkyl (C≦12) , acyl (C≦12) , substituted acyl (C≦12) or a monovalent amino protecting group; Q is a group having the formula: is the basis of During the ceremony: R15 is hydrogen, -R 16 , or -C(O)-R 16 and where: R 16 is a therapeutic or imaging agent; X7 is a covalent bond, O, S, -NH-, or alkanediyl (C≦12) , substituted alkanediyl (C≦12) , -(OCH 2 CH 2 ) p - or substituted -(OCH 2 CH 2 ) p -(where: p is 0 to 50; or formula: is the basis of During the ceremony: R a and R a ' are each independently hydrogen, alkyl, (C≦12) , substituted alkyl (C≦12) , aryl (C≦12) , or heteroaryl (C≦12) and X 8 -O or -NR 17 R 17 ′-, where: R 17 and R 17 ' are each independently alkyl (C≦12) or substituted alkyl (C≦12) and R 19 is hydrogen, a sugar or a sugar derivative; and X 9 is a covalent bond, O, S, -NH-, -(OCH 2 CH 2 ) q - or substituted -(OCH 2 CH 2 ) q -(where: q is 0 to 50; or formula: is the basis of During the ceremony: Y 1 is O or S; Y 2 is a covalent bond, O, S, -NH-, or -NR 18 -where: R 18 is an alkyl (C≦12) or substituted alkyl (C≦12) and X 10 is a covalent bond, alkyl (C≦12) , substituted alkyl (C≦12) , O, S, -NH-, -(CH 2 CH 2 O) r -, substitution -(CH 2 CH 2 O) r -, -(CH 2 CH 2 NR 20 ) r - or substituted -(CH 2 CH 2 NR 20 ) r - and; r is between 0 and 50; R 20 is hydrogen, alkyl (C≦12) , or substituted alkyl (C≦12) and However, R 4 If is valine, R 4 ' is not hydrogen, the compound, or a pharmaceutically acceptable salt thereof; or the below described: or a pharmaceutically acceptable salt thereof.

2. The compound of claim 1, wherein the compound is of formula (II), (III), or (IV): ； ； 。

3. X 1 2. The compound of claim 1, wherein is a covalent bond.

4. R 4 2. The compound of claim 1, wherein ' is hydrogen.

5. R 5 2. The compound of claim 1, wherein ' is hydrogen.

6. 10. The compound of claim 1, wherein W is a multivalent polymer having 1 to 5 attachment points.

7. 2. The compound of claim 1, wherein n is 1.

8. X 2 Ga-CH 2 CH 2 2. The compound of claim 1, wherein:

9. R 3 Ga-X 6 -C(O)R 12 where: X 6 -O, -NR b - or a covalent bond; R b is hydrogen, alkyl (C≦6) , substituted alkyl (C≦6) or a monovalent amino protecting group; R 12 is hydroxy or amino; or alkoxy (C≦12) , alkylamino (C≦12) , dialkylamino (C≦12) 2. The compound of claim 1, wherein:

10. X 6 2. The compound of claim 1, wherein is a covalent bond.

11. R 12 2. The compound of claim 1, wherein is hydroxy.

12. 2. The compound of claim 1, wherein m is 1.

13. Each R 4 is the side chain of glycine.

14. Each R 5 2. The compound of claim 1, wherein is the side chain of citrulline.

15. X 7 But the formula: The basis of The compound of claim 1.

16. R 16 10. The compound of claim 1, wherein

17. 17. The compound of claim 16, wherein the therapeutic agent is a chemotherapeutic agent.

18. The compound of claim 17, wherein the chemotherapeutic agent is auristatin E, auristatin F, monomethylauristatin E, monomethylauristatin F, dolastatin, maytansine, duocarmycin, tublysin, calicheamicin, pyrobenzodiazepine dimer, anthracycline, paclitaxel, vinblastine, amanitin, or a derivative thereof.

19. The compound of claim 18, wherein the chemotherapeutic agent is monomethyl auristatin E or monomethyl auristatin F.

20. The compound of claim 1, wherein:

10. The compound of claim 1, which is:

21. (A) a drug moiety, wherein, prior to conjugation, the drug moiety corresponds to a compound of claim 1; (B) a linker; and (C) Cell targeting group A drug conjugate comprising:

22. 22. The drug conjugate of claim 21, wherein the cell targeting group is an antibody, an antibody fragment, a protein, or a small molecule.

23. (i) The linker is a non-covalent bond formed by hydrogen bonding, nucleobase pairing, electrostatic interactions, π-stacking, van der Waals interactions, or dipole-dipole interactions; (ii) the linker is a covalent bond; (iii) the linker is a monovalent spacer containing one point of attachment; or (iv) The drug conjugate of claim 21, wherein the linker is a multivalent spacer comprising 2 to 21 linkage points.

24. The method of claim 24, wherein the linker, prior to attachment, has the following formula (V): or a pharmaceutically acceptable salt thereof, During the ceremony: A 6 , A 7 , A 8 , and A 9 are each independently an alkanediyl C1-12 , Arendjiil C1-12 , heteroarenediyl C1-12 , cycloalkanediyl C1-12 , heterocycloalkanediyl C1-12 or a substituted form thereof, or a side chain group of a canonical amino acid; X 11 , Y 3 , and Z 1 are each independently a covalent bond, -[O(CH 2 ) q ]-, -[O(CHW 1 ´) q ]-, or -[O(CW 1 ´W 1 ´´) q ]- and; Where: W 1 ´ and W 1 ´´ are each independently amino, hydroxy, halo, mercapto, or alkyl C1-12 , cycloalkyl C1-12 , alkenyl C1-12 , alkynyl C1-12 , aryl C1-12 , aralkyl C1-12 , heteroaryl C1-12 , heteroaralkyl C1-12 , heterocycloalkyl C1-12 , acyl C1-12 , acyloxy C1-12 , or alkylamino C1-12 or substituted forms thereof; q is 1 to 3; a, b, c, and d are each independently 0 to 12; e and f are each independently 0, 1, 2, or 3; R 21 , R 22 , and R 23 are each independently hydrogen, -NH 2 , -NHR 24 , -NR 24 R 25 , -N 3 , heteroaryl (C≦12) , substituted heteroaryl (C≦12) , -Arendjiil (C≦12) -heteroaryl (C≦12) , substituted arenediyl (C≦12) -heteroaryl (C≦12) or a conjugating group; Where: R 24 and R 25 are each independently an alkyl C1-12 , cycloalkyl C1-12 , alkenyl C1-12 , alkynyl C1-12 , aryl C1-12 , aralkyl C1-12 , heteroaryl C1-12 , heteroaralkyl C1-12 , heterocycloalkyl C1-12 , acyl C1-12 , acyloxy C1-12 , alkylamino C1-12 or a substituted version thereof, or a monovalent amino protecting group; or R 24 and R 25 taken together and is a divalent amino protecting group; However, R 21 , R 22 , and R 23 At least one of the groups is -NH 2 or -NH 2 and R 21 , R 22 , and R 23 At least one of the 3 , heteroaryl (C≦12) , or -Arendjiil (C≦12) -heteroaryl (C≦12) That is, 22. The drug conjugate of claim 21.

25. A 6 is CH 2 25. The drug conjugate of claim 24, wherein A7 is CH2CH2CH2CH2, A8 is CH2, and A9 is CH2CH2.

26. X 11 , Y 3 , and Z 1 are each independently —[O(CH 2 ) q 25. The drug conjugate of claim 24, wherein:

27. R 21 Ga-NH 2 or -N 3 ; R 22 is —N 3 ; and 25. The drug conjugate of claim 24, wherein R23 is tetrazine, 3-methyltetrazine, 4-tetrazyl-phenyl, or 4-(3-methyltetrazyl)-phenyl.

28. Prior to attachment, the linker comprises:

22. The drug conjugate of claim 21, which corresponds to:

29. 29. A pharmaceutical composition comprising the drug conjugate of any one of claims 21 to 28 and an excipient.

30. 29. A pharmaceutical composition for treating a disease or disorder in a patient, comprising the drug conjugate of any one of claims 21 to 28.

31. The disease or disorder is: (A) Cancer; (B) It is a microbial infection; (C) an autoimmune disease; (D) associated with inflammation; (E) diabetes; or (F) a neurological disorder; 31. The pharmaceutical composition of claim 30.

32. 32. The pharmaceutical composition of claim 31, wherein the cancer is carcinoma, sarcoma, lymphoma, leukemia, melanoma, mesothelioma, multiple myeloma, or seminoma.

33. 32. The pharmaceutical composition of claim 31, wherein the cancer is cancer of the bladder, blood, bone, brain, breast, central nervous system, cervix, colon, endometrium, esophagus, gallbladder, gastrointestinal tract, reproductive organs, genitourinary tract, head, kidney, larynx, liver, lung, muscle tissue, neck, oral or nasal mucosa, ovary, pancreas, prostate, skin, spleen, small intestine, large intestine, stomach, testicle, or thyroid.

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