US-12622944-B2 - Compositions containing, methods involving, and uses of non-natural amino acid linked dolastatin derivatives

Abstract

Disclosed herein are non-natural amino acids and dolastatin analogs that include at least one non-natural amino acid, and methods for making such non-natural amino acids and polypeptides. The dolastatin analogs can include a wide range of possible functionalities, but typically have at least one oxime, carbonyl, dicarbonyl, and/or hydroxylamine group. Also disclosed herein are non-natural amino acid dolastatin analogs that are further modified post-translationally, methods for effecting such modifications, and methods for purifying such dolastatin analogs. Typically, the modified dolastatin analogs include at least one oxime, carbonyl, dicarbonyl, and/or hydroxylamine group. Further disclosed are methods for using such non-natural amino acid dolastatin analogs and modified non-natural amino acid dolastatin analogs, including therapeutic, diagnostic, and other biotechnology use.

Inventors

• Zhenwei Miao
• Ying Sun
• Agnieszka Szydlik
• Delia Ianina Lopez De Valenta
• Kyle ATKINSON
• Sandra Biroc
• Timothy Buss
• Melissa Neal
• Vadim Kraynov
• Robin Marsden
• Jason PINKSTAFF
• Lillian Skidmore

Assignees

• AMBRX, INC.

Dates

Publication Date

20260512

Application Date

20220428

Priority Date

20120524

Claims (5)

1. 1 . An antibody drug conjugate comprising a trastuzumab antibody conjugated to a dolastatin, wherein the conjugation occurs via a non-naturally occurring amino acid in the antibody as depicted in Formula (VIII) or (IX), wherein the non-naturally occurring amino acid is located at position 121 of the antibody: wherein: A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, —O—, —O-(alkylene or substituted alkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O) k — where k is 1, 2, or 3, —S(O) k -(alkylene or substituted alkylene)-, —C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene or substituted alkylene)-, —C(O) N(R′)—, —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—, —S(O) k N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S) N(R′)—, —N(R′)S(O) k N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N=, —C(R′) 2 —N═N—, and —C(R′) 2 —N(R′)—N(R′)—, wherein each R′ is independently H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R 1 is H, an amino protecting group, resin, at least one amino acid, polypeptide, or polynucleotide; R 2 is OH, an ester protecting group, resin, at least one amino acid, polypeptide, or polynucleotide; wherein at least one of R 1 and R 2 is a polypeptide, wherein the polypeptide is the trastuzumab antibody, wherein amino acid position 121 of the trastuzumab antibody is substituted with the non-naturally encoded amino acid; R 3 and R 4 are each independently H, halogen, lower alkyl, or substituted lower alkyl, or R 3 and R 4 or two R 3 groups optionally form a cycloalkyl or a heterocycloalkyl; Z has the structure of: R 5 is H, COR 8 , C 1 -C 6 alkyl or thiazole; R 8 is OH; R 6 is OH or H; Ar is phenyl or pyridine; R 7 is C 1 -C 6 alkyl or hydrogen; and L is a linker selected from the group consisting of -alkylene-, -alkylene-C(O)—, -(alkylene-O) n -alkylene-, -(alkylene-O) n -alkylene-C(O)—, -(alkylene-O) n —(CH 2 ) n′ —NHC(O)—(CH 2 ) n″ —C(Me) 2 -S—S—(CH 2 ) n″ —NHC(O)-(alkylene-O) n″ -alkylene-, -(alkylene-O) n -alkylene-W—, -alkylene-C(O)—W—, -(alkylene-O) n -alkylene-U-alkylene-C(O)—, and -(alkylene-O) n -alkylene-U-alkylene-; wherein: W has the structure of: U has the structure of: and each n, n′, n″, n′″ and n″ is independently an integer from 0 to 20; wherein substituted means substituted with one or more substituents independently selected from the group consisting of halo, C 1 -C 10 alkyl, C 2 -C 10 alkenyl, C 2 -C 10 alkynyl, C 1 -C 10 alkoxy, C 5 -C 12 aralkyl, C 3 -C 12 cycloalkyl, C 4 -C 12 cycloalkenyl, phenyl, toluolyl, xylenyl, biphenyl, C 2 -C 12 alkoxyalkyl, C 5 -C 12 alkoxyaryl, C 5 -C 12 aryloxyalkyl, C 7 -C 12 oxyaryl, C 1 -C 6 alkylsulfonyl, C 1 -C 10 alkylsulfonyl, —(CH 2 ) m —O—(C 1 -C 10 alkyl), aryl, fluoroalkyl, heterocyclic radical, nitroalkyl, —NO 2 , —CN, —NR″C(O)—(C 1 -C 10 alkyl), —C(O)—(C 1 -C 10 alkyl), C 2 -C 10 alkthioalkyl, —C(O)O—(C 1 -C 10 alkyl), —OH, —SO 2 , ═S, —COOH, —NR″ 2 , carbonyl, —C(O)—(C 1 -C 10 alkyl)-CF 3 , —C(O)—CF 3 , —C(O)NR″ 2 , —(C 1 -C 10 aryl)-S—(C 6 -C 10 aryl), —C(O)—(C 6 -C 10 aryl), —(CH 2 ) m —O—(CH 2 ) m —O—(C 1 -C 10 alkyl), —C(O)NR″ 2 , —C(S)NR″ 2 , —SO 2 NR″ 2 , —NR″C(O)NR″ 2 and —NR″C(S)NR″ 2 ; wherein each R″ group is independently H, alkyl, aryl or alkaryl; and each m is from 1 to 8; or a pharmaceutically acceptable salt or solvate thereof.
2. 2 . The compound of claim 1 , wherein: A is arylene; B is absent; Ar is phenyl; L is -(alkylene-O) n -alkylene-; R is alkyl; R 1 and R 2 is the trastuzumab antibody; R 3 and R 4 are H; R 5 is COR 8 ; R 6 is H; R 7 is C 1 -C 6 alkyl; and R 8 is OH.
3. 3 . The compound of claim 2 , wherein the compound is a compound of Formula (VIII).
4. 4 . The compound of claim 2 , wherein the compound is a compound of Formula (IX).
5. 5 . A pharmaceutical composition comprising a compound of claim 1 and a pharmaceutically acceptable carrier, excipient or binder.

Description

CROSS REFERENCE This application is a continuing application of U.S. application Ser. No. 17/142,169, filed Jan. 5, 2021, which is a divisional application of U.S. application Ser. No. 15/702,682, filed Sep. 12, 2017, now U.S. Pat. No. 10,954,270, which is a divisional application of U.S. application Ser. No. 14/122,672, filed Sep. 17, 2015, now U.S. Pat. No. 9,796,754, which in turn is a U.S. National Stage entry under 35, U.S.C. § 371 of International Application No. PCT/US2012/039472, filed May 24, 2012, designating the United States of America and published in English on Dec. 6, 2012, which in turn claims priority to U.S. Provisional Application No. 61/491,146, filed May 27, 2011, the entire contents of each of which are hereby incorporated by reference in their entirety. BACKGROUND OF THE INVENTION The ability to incorporate non-genetically encoded amino acids (i.e., “non-natural amino acids”) into proteins permits the introduction of chemical functional groups that could provide valuable alternatives to the naturally-occurring functional groups, such as the epsilon —NH2 of lysine, the sulfhydryl —SH of cysteine, the imino group of histidine, etc. Certain chemical functional groups are known to be inert to the functional groups found in the 20 common, genetically-encoded amino acids but react cleanly and efficiently to form stable linkages with functional groups that can be incorporated onto non-natural amino acids. Methods are now available to selectively introduce chemical functional groups that are not found in proteins, that are chemically inert to all of the functional groups found in the 20 common, genetically-encoded amino acids and that may be used to react efficiently and selectively with reagents comprising certain functional groups to form stable covalent linkages. SUMMARY OF THE INVENTION Disclosed herein are toxic moieties with one or more linker(s), toxic groups linked to non-natural amino acids, and methods for making such non-natural amino acids and polypeptides. Some embodiments of the present invention describe a compound, or salt thereof, comprising Formula (I): wherein: Z has the structure of: R5 is H, COR8, C1-C6alkyl, or thiazole; R8 is OH or —NH-(alkylene-O)n—NH2; R6 is OH or H;Ar is phenyl or pyridine;R7 is C1-C6alkyl or hydrogen;Y is selected from the group consisting of an hydroxylamine, methyl, aldehyde, protected aldehyde, ketone, protected ketone, thioester, ester, dicarbonyl, hydrazine, amidine, imine, diamine, azide, keto-amine, keto-alkyne, alkyne, cycloalkyne, and ene-dione;L is a linker selected from the group consisting of -alkylene-, -alkylene-C(O)—, -(alkylene-O)n-alkylene-, -(alkylene-O)n-alkylene-C(O)—, -(alkylene-O)n—(CH2)n′—NHC(O)—(CH2)n″—C(Me)2-S—S—(CH2)n′″—NHC(O)-(alkylene-O)n″″-alkylene-, -(alkylene-O)n-alkylene-W—, -alkylene-C(O)—W—, -(alkylene-O)n-alkylene-U-alkylene-C(O)—, and -(alkylene-O)n-alkylene-U-alkylene-; W has the structure of: U has the structure of: or L is absent, Y is methyl, R5 is COR8, and R8 is —NH-(alkylene-O)n—NH2; andeach n, n′, n″, n′″ and n″″ are independently integers greater than or equal to one. In some embodiments, R5 is thiazole. In other embodiments, R6 is H. In certain embodiments, Ar is phenyl. In further or additional embodiments, R7 is methyl. In some embodiments, n is an integer from 0 to 20, 0 to 10 or 0 to 5. In some embodiments, a compound is described comprising Formula (II): In certain embodiments, L is -(alkylene-O)n-alkylene-. In specific embodiments, each alkylene is —CH2CH2—, n is equal to 3, and R7 is methyl. In other embodiments, L is -alkylene-. In specific embodiments, each alkylene is —CH2CH2— and R7 is methyl or hydrogen. In certain embodiments, L is -(alkylene-O)n-alkylene-C(O)—. In certain specific embodiments, each alkylene is —CH2CH2—, n is equal to 4, and R7 is methyl. In further or alternative embodiments, L is -(alkylene-O)n—(CH2)n′—NHC(O)—(CH2)n″—C(Me)2-S—S—(CH2)n′″—NHC(O)-(alkylene-O)n″″-alkylene-. In specific embodiments, each alkylene is —CH2CH2—, n is equal to 1, n′ is equal to 2, n″ is equal to 1, n′″ is equal to 2, n″″ is equal to 4, and R7 is methyl. In some embodiments, Y is azide. In other embodiments, Y is cyclooctyne. In specific embodiments, the cyclooctyne has a structure of: each R19 is independently selected from the group consisting of C1-C6 alkyl, C1-C6 alkoxy, ester, ether, thioether, aminoalkyl, halogen, alkyl ester, aryl ester, amide, aryl amide, alkyl halide, alkyl amine, alkyl sulfonic acid, alkyl nitro, thioester, sulfonyl ester, halosulfonyl, nitrile, alkyl nitrile, and nitro; andq is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11. Some embodiments of the present invention describe a compound, or salt thereof, comprising Formula (III), (IV), (V) or (VI): wherein: Z has the structure of: R5 is H, COR8, C1-C6alkyl, or thiazole; R8 is OH; R6 is OH or H;Ar is phenyl or pyridine;R7 is C1-C6alkyl or hy