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CN116457023A - antibody-TLR agonist conjugates, methods and uses thereof - Google Patents

antibody-TLR agonist conjugates, methods and uses thereof Download PDF

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Publication number
CN116457023A
CN116457023A CN202180071626.XA CN202180071626A CN116457023A CN 116457023 A CN116457023 A CN 116457023A CN 202180071626 A CN202180071626 A CN 202180071626A CN 116457023 A CN116457023 A CN 116457023A
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China
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compound
antibody
substituted
cancer
linker
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CN202180071626.XA
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Chinese (zh)
Inventor
S-J·门
B·里昂
康名超
N·克努森
S·坂村
田丰
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Ambrx Inc
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Ambrx Inc
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Priority claimed from PCT/US2021/047009 external-priority patent/WO2022040596A1/en
Publication of CN116457023A publication Critical patent/CN116457023A/en
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Abstract

Disclosed herein are TLR agonist compounds, antibody-TLR agonist conjugates, pharmaceutical compositions, and methods of using such compounds or conjugates as therapeutic agents for treating diseases or disorders such as cancer.

Description

antibody-TLR agonist conjugates, methods and uses thereof
Citation of related application
The present application claims the benefit of U.S. provisional application Ser. No. 63/068,342, filed 8/20/2020, and U.S. provisional application Ser. No. 63/118,365, filed 11/25/2020, the contents of which are incorporated herein by reference in their entirety.
Sequence listing
The present application contains a sequence listing that has been submitted in ASCII format and is hereby incorporated by reference in its entirety. An ASCII copy created at 8.12 of 2021 is named ambx_0234_00pct_st25.Txt and is 80,620 bytes in size.
Background
Targeting molecules or polypeptides, such as antibodies and fragments thereof, as well as TLR agonist compounds, can be conjugated together to produce a TLR agonist conjugate (TC). TC may be suitable for treating diseases.
Incorporated by reference
All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
Disclosure of Invention
The present invention relates to targeting polypeptides having one or more non-naturally encoded amino acids conjugated to agonist compounds of a TLR, including but not limited to TLR7 and/or TLR8. Such conjugates are referred to herein as TLR agonist conjugates (TCs). The TCs of the present invention include targeted biomolecules or polypeptides and TLR agonist compounds conjugated together by site-specific conjugation using non-naturally encoded amino acids to produce novel biological TLR agonist conjugates (BTCs). The targeting biomolecule or polypeptide may be a tumor targeting biomolecule or polypeptide.
In additional embodiments, the invention also relates to TCs that are further conjugated to water-soluble polymers that form stable dimers or multimers. The present invention provides novel TCs designed, engineered or constructed to enhance, increase or improve their pharmacokinetic and therapeutic profile. The TCs of the present invention are designed to provide additional target specificity by blocking TLR exposure at unintended target sites using PEG mask and prodrug designs, for example where the PEG mask or prodrug cleavage release activity payload at the tumor microenvironment further enhances specificity. In some embodiments, the TC design comprises a hydrophilic drug-linker or a payload-linker design. In some embodiments, TC involves inclusion of PEG shielding. In some embodiments, the TC design comprises a PEG mask comprising one or more linear or branched PEG molecules. In some embodiments, the TC design comprises a prodrug approach with a proteolytic cleavable linker design. In some embodiments, the TC design comprises a proteolytic cleavable linker design and PEG shielding.
In one aspect, the present disclosure provides a compound of formula (I):
or a pharmaceutically acceptable salt, solvate, stereoisomer or tautomer thereof, wherein
A is CH or N;
x is O-R1, NH-R1, S-R1 or H;
YY is-ONH 2 、-N 3 -OH, maleimide, -COOH or-C (=o) CH 2 Y1, wherein Y1 is a halide;
each of L1 and L2 is independently (CH) 2 ) m 、(CH 2 ) m C(＝O)、(CH 2 ) m -NH(CH 2 ) n 、(CH 2 )m-C(＝O)NH(CH 2 ) n 、(CH 2 ) m -OC(＝O)-NH-(CH 2 ) n 、(CH 2 ) m -NHC(＝O)-NH-(CH 2 ) n 、(CH 2 ) m -NH、(CH 2 ) m -NHC(＝O)、(CH 2 ) m -NHC(＝O)-(CH 2 ) n -NHC(＝O)-(CH 2 ) p 、C(＝O)-(CH 2 ) n 、C 3 -C 8 Heterocycle or absent; wherein each of m, n and p is independently an integer from 0 to 12;
r1 is H, C 1 -C 12 Alkyl, substituted C 1 -C 12 Alkyl, oxygen-containing C 1 -C 12 Alkyl, C 3 -C 8 Heterocycloalkyl, substituted C 3 -C 8 Heterocycloalkyl, C 3 -C 8 Cycloalkyl, substituted C 3 -C 8 Cycloalkyl, -N 3 Terminally substituted C 1 -C 12 Alkyl, (CH) 2 ) q -(OCH 2 CH 2 ) r OMe, wherein each of q and r is independently an integer from 0 to 12;
r2 is C 1 -C 6 Alkylene, C 1 -C 12 Substituted alkylene, C 3 -C 8 Cycloalkylene, C 3 -C 8 Substituted cycloalkylene, arylene, substituted C 6 -C 10 Arylene, 5-12 membered heteroarylene containing 1-3 heteroatoms, substituted 5-12 membered heteroarylene containing 1-3 heteroatoms, or (OCH) 2 CH 2 ) ss Or a combination thereof, or R2 is absent; wherein ss is an integer from 1 to 12, wherein each heteroatom is independently N, O or S;
r3 is the side chain of an amino acid, C 1 -C 6 Alkylene, C 1 -C 6 Substituted alkylene, C 3 -C 8 Cycloalkylene, C 3 -C 8 Heterocycloalkylene, substituted C 3 -C 8 Cycloalkylene, arylene, substituted arylene, 5-12 membered heteroarylene containing 1-3 heteroatoms, substituted 5-12 membered heteroarylene containing 1-3 heteroatoms, C containing amino groups 1 -C 12 Alkylene group, carbonyl group-containing C 1 -C 12 Alkylene, oxygen-containing C 1 -C 12 Alkylene, -N 3 Terminal C 1 -C 6 Alkylene, -CCH terminal C 1 -C 6 Alkylene, -SH terminal C 1 -C 6 Alkylene, -OH terminal C 1 -C 6 Alkylene, nitrogen-containing C 1 -C 6 Alkylene, -OPO 3 H 2 Terminal C 1 -C 6 Alkylene, -OPO 3 H 2 Terminal arylene, glucuronide terminal C 1 -C 6 Alkylene, -N 3 Terminal arylene, acetylene terminal arylene, amine terminal arylene, (CH) 2 ) S 、(CH 2 ) S -C(＝O)、(CH 2 ) s -NH(CH 2 ) t 、(CH 2 ) S -C(＝O)NH(CH 2 ) t 、(CH 2 ) S -OC(＝O)-NH-(CH 2 ) t 、(CH 2 ) S -NHC(＝O)-NH-(CH 2 ) t Or a combination thereof; or R3 is absent; wherein each s and t is independently an integer from 0 to 6;
r4 is H, C 3 -C 8 Cycloalkyl, C 3 -C 8 Heterocycloalkyl, C 3 -C 8 Substituted heterocycloalkyl, aryl, substituted aryl, (CH) 2 ) u -(OCH 2 CH 2 ) v OMe, di/tri branched (CH) 2 ) u -(OCH 2 CH 2 ) v OMe or a combination thereof; or R4 is absent; wherein each u and v is independently an integer from 1 to 48.
In some embodiments, R4 comprises a PEG moiety. In some embodiments, the PEG moiety is linear, branched, or multi-armed. In some embodiments, R4 comprises (CH 2 ) u -(OCH 2 CH 2 ) v OMe. In some embodiments, v is an integer from 1 to 48, u is an integer from 1 to 12, and ss is independently an integer from 1 to 12. In some embodiments, v is an integer from 1 to 12, u is an integer from 1 to 12, and ss is independently an integer from 1 to 12. In some embodiments, R3 comprises a linker. In some embodiments, the linker comprises-ONH 2 Terminal or maleimide terminal or COOH terminal or haloacetyl terminal, each terminal having (CH) 2 )m-(OCH 2 CH 2 ) n-, wherein each of m and n is independently an integer from 1 to 12. In some embodiments, the PEG moiety has a molecular weight of 0.1kDa to 100kDa or 1kDa to 100kDa. In some embodiments, the PEG moiety has a molecular weight of 0.1kDa to 50kDa or 1kDa to 50kDa. In some embodiments, a is CH.
In some embodiments, the compound or salt thereof is selected from table 4. In some embodiments, compound 185, compound 186, compound 187, compound 188, compound 189, compound 190, compound 191, compound 213, compound 214, compound 216, compound 217, compound 218, compound 219, compound 220, compound 221, compound 222, compound 223, compound 224, compound 230, compound 233, compound 235, compound 238, compound 239, compound 240, compound 242, compound 244, compound 245, compound 246, compound 248, compound 251, compound 252, compound 253, compound 254, compound 255, compound 256, compound 257, compound 258, compound 259, compound 260, compound 261, compound 263, compound 265, compound 266, compound 267, compound 268, compound 269, compound 272, compound 275, 278, compound 279, compound 281, compound 283, compound 285, compound 287, compound 302, compound 303, or compound 299, in accordance with table 4.
The compounds are selected from the following group of compounds: 3-amino-N- (2- (1- (4- ((4-amino-6-butoxy-2-oxo-2, 3-dihydro-1H-imidazo [4,5-c ] pyridin-1-yl) methyl) benzyl) piperidin-4-yl) ethyl) benzamide (185); n- (2- (1- (4- ((4-amino-6-butoxy-2-oxo-2, 3-dihydro-1H-imidazo [4,5-c ] pyridin-1-yl) methyl) benzyl) piperidin-4-yl) ethyl) -4- (2-aminoethyl) benzamide (186); 4-amino-N- (2- (1- (4- ((4-amino-6-butoxy-2-oxo-2, 3-dihydro-1H-imidazo [4,5-c ] pyridin-1-yl) methyl) benzyl) piperidin-4-yl) ethyl) benzamide (187); 3-amino-N- (2- (1- (4- ((4-amino-6-butoxy-2-oxo-2, 3-dihydro-1H-imidazo [4,5-c ] pyridin-1-yl) methyl) benzyl) piperidin-4-yl) ethyl) -4-fluorobenzamide (188); n- (2- (1- (4- ((4-amino-6-butoxy-2-oxo-2, 3-dihydro-1H-imidazo [4,5-c ] pyridin-1-yl) methyl) benzyl) piperidin-4-yl) ethyl) -4- (2- (2- (aminooxy) acetamido) ethyl) benzamide (189); 6-amino-9- (4- ((4- (4-aminophenyl) piperidin-1-yl) methyl) benzyl) -2-butoxy-7H-purin-8 (9H) -one; 6-amino-9- (4- ((1 '- (3- (2- (aminoxy) ethoxy) propionyl) -4,4' -bipiperidin-1-yl) methyl) benzyl) -2-butoxy-7H-purin-8 (9H) -one (191); n- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -4-hydroxybenzoamide (213); n- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -3- (4-hydroxyphenyl) propionamide (214); (S) -2-amino-N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -6- (2- (aminooxy) acetamido) hexanamide (216); (S) -N- (5-amino-6- (1 '- (4- ((4-amino-6-butoxy-2-oxo-2, 3-dihydro-1H-imidazo [4,5-c ] pyridin-1-yl) methyl) benzyl) -4,4' -bipiperidin-1-yl) -6-oxohexyl) -2- (aminooxy) acetamide (217); 5-amino-N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) nicotinamide (218); 5-amino-N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) pyrazine-2-carboxamide (219); 6-amino-2-butoxy-9- (4- ((1 '- (4-hydroxybenzoyl) - [4,4' -bipiperidin ] -1-yl) methyl) benzyl) -7, 9-dihydro-8H-purin-8-one (220); (S) -2-amino-N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -3- (4-aminophenyl) propanamide (221); 6-amino-9- (4- ((1 '- (5-aminopyrazine-2-carbonyl) -4,4' -bipiperidin-1-yl) methyl) benzyl) -2-butoxy-7H-purin-8 (9H) -one (222); (S) -2-amino-N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -3- (4- (azidomethyl) phenyl) acrylamide (223); (S) -2-amino-N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -6-azidohexanamide (224); n- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -4- ((S) -2- (2- (aminooxy) acetamido) -3-methylbutanoylamino) propionylamino) benzamide (230); (S) -N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -2- ((S) -2-amino-3-methylbutanoylamino) propionylamino) -6- (2- (aminooxy) acetamido) hexanamide (233); (S) -N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -6- (2- (aminooxy) acetamido) -2-PEG 24-acylaminohexanamide (235); ((S) -1- ((2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) amino) -6- (2- (aminooxy) acetamido) -1-oxohex-2-yl) carbamic acid 4- ((S) -2- ((S) -3-methyl-2-PEG 24-acylaminobutyrylamino) propionylamino) benzyl ester (238); ((S) -1- ((2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) amino) -6- (2- (aminooxy) acetamido) -1-oxohex-2-yl) carbamic acid 4- ((S) -2-acetamido-3-methylbutanoylamino) propionylamino) benzyl ester (239); (S) -2-amino-N- (2- (1- (4- ((4-amino-6-butoxy-2-oxo-2, 3-dihydro-1H-imidazo [4,5-c ] pyridin-1-yl) methyl) benzyl) piperidin-4-yl) ethyl) -3-azidopropionamide (240); (S) -2-amino-N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -3- (4- ((4- ((aminooxy) methyl) -1H-1,2, 3-triazol-1-yl) methyl) phenyl) propanamide (242); (S) -2-amino-N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -3- (4- ((aminooxy) methyl) -1H-1,2, 3-triazol-1-yl) propanamide (244); (S) -2-amino-N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -3-hydroxypropionamide (245); (S) -2-amino-N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -3- (4-hydroxyphenyl) propanamide (246); (S) -2-amino-N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -6- (4- ((aminooxy) methyl) -1H-1,2, 3-triazol-1-yl) hexanamide (248); (S) -N1- (1- ((2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) amino) -6- (2- (aminooxy) acetamido) -1-oxohex-2-yl) -N5- (PEG 48) -glutarate-ide (251); (S) -2-PEG8-N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -6- (2- (aminooxy) acetamido) hexanamide (252); (S) -N1- (1- ((2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) amino) -6- (2- (aminooxy) acetamido) -1-oxohex-2-yl) -N5-mPEG4- (PEG 4) 3-glutarate-amide (253); (S) -2-PEG4-N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -6- (2- (aminooxy) acetamido) hexanamide (254); (S) -N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -6- (2- (aminooxy) acetamido) -2-PEG 12-acylaminohexanamide (255); (S) -N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -6- (2- (aminooxy) acetamido) -2-PEG 37-acylaminohexanamide (256); (S) -N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -6- (2- (aminooxy) acetamido) -2- (4-phenylbutyrylamino) hexanamide (257); (S) -N- (1- (2- (1- (4- ((4-amino-6-butoxy-2-oxo-2, 3-dihydro-1H-imidazo [4,5-c ] pyridin-1-yl) methyl) benzyl) piperidin-4-yl) ethylamino) -6- (2- (aminooxy) acetamido) -1-oxohex-2-yl) oleic acid amide (258); (S) -N- (1- ((2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) amino) -6- (2- (aminooxy) acetamido) -1-oxohex-2-yl) octanamide (259); (S) -N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -6- (2- (aminooxy) acetamido) -2-dPEG4- (m-dPEG 8) 3-acylaminohexanamide (260); (S) -N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -6- (2- (aminooxy) acetamido) -2-dPEG4- (m-dPEG 12) 3-acylaminohexanamide (261); (S) -6-amino-N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -2- (2- (aminooxy) acetamido) hexanamide (263); (S) -N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -2- (2- (aminooxy) acetamido) -6-PEG 24-acylaminohexanamide (265); (S) -N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -2- (2- (aminooxy) acetamido) -6-PEG 8-acylaminohexanamide (266); (S) -N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -2- (2- (aminooxy) acetamido) -6- (PEG 37) hexanamide (267); (S) -N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -2- (2- (aminooxy) acetamido) -6- (dPEG 4- (m-dPEG 8) 3) hexanamide (268); (S) -N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -2- (2- (aminooxy) acetamido) -3- (4-hydroxyphenyl) propanamide (269); (9- (4- ((4- (2- (2- (aminoxy) acetylamino) ethyl) piperidin-1-yl) methyl) benzyl) -2-butoxy-8-oxo-8, 9-dihydro-7H-purin-6-yl) carbamic acid butyl ester (272); n- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -3- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) propanamide (273); (S) -N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -2- (2- (aminooxy) acetamido) -3- (4- (((2S, 3R,4S,5S, 6R) -3,4, 5-trihydroxy-6- (hydroxymethyl) tetrahydro-2H-pyran-2-yl) oxy) phenyl) propanamide (275); (R) -6-amino-N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -2- (2- (aminooxy) acetamido) hexanamide (278); (R) -N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -2- (2- (aminooxy) acetamido) -6-PEG 24-acylaminohexanamide (279); (S) -4- (3- ((2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) amino) -2- (2- (aminooxy) acetamido) -3-oxopropyl) dihydrogen phosphate (281); (R) -N1- (6- ((2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) amino) -5- (2- (aminooxy) acetamido) -6-oxohexyl) -N5- (dPEG 4) - (mPEG 8) 3-glutaramide (282); (R) -N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -2- (2- (aminooxy) acetamido) -6- (PEG 8) amidohexanamide (283); n- (9- (4- ((4- (2-aminoethyl) piperidin-1-yl) methyl) benzyl) -2-butoxy-8-oxo-8, 9-dihydro-7H-purin-6-yl) hexanamide (284); n- (9- (4- ((4- (2-aminoethyl) piperidin-1-yl) methyl) benzyl) -2-butoxy-8-oxo-8, 9-dihydro-7H-purin-6-yl) acetamide (285); n- (9- (4- ((4- (2- (2- (aminoxy) acetamido) ethyl) piperidin-1-yl) methyl) benzyl) -2-butoxy-8-oxo-8, 9-dihydro-7H-purin-6-yl) hexanamide (286); n- (2- (1- (4- ((6-acetamido-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -2- (aminooxy) acetamide (287); n- (9- (4- ((4- (2- (aminoxy) ethyl) piperidin-1-yl) methyl) benzyl) -2-butoxy-8-oxo-8, 9-dihydro-7H-purin-6-yl) acetamide (296); 6-amino-9- (4- ((4- (2- (aminoxy) ethyl) piperidin-1-yl) methyl) benzyl) -2-butoxy-7H-purin-8 (9H) -one (297); n- (9- (4, 4' -bipiperidin-1-ylmethyl) benzyl) -2-butoxy-8-oxo-8, 9-dihydro-7H-purin-6-yl) acetamide (299); n- (9- (4- ((1 '- (2- (aminoxy) acetyl) -4,4' -bipiperidin-1-yl) methyl) benzyl) -2-butoxy-8-oxo-8, 9-dihydro-7H-purin-6-yl) acetamide (300); n- (9- (4- ((4- (2-aminoethyl) piperidin-1-yl) methyl) benzyl) -2-butoxy-8-oxo-8, 9-dihydro-7H-purin-6-yl) -3- (2- (2-methoxyethoxy) ethoxy) acrylamide (301); n- (9- (4- ((4- (2- (2- (aminoxy) acetamido) ethyl) piperidin-1-yl) methyl) benzyl) -2-butoxy-8-oxo-8, 9-dihydro-7H-purin-6-yl) -3- (2- (2-methoxyethoxy) ethoxy) propanamide (302); n- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -1- (aminooxy) -3,6,9, 12-tetraoxapentadecane-15-amide (303) or N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7H-purin-9 (8H) -yl) methyl) benzyl) piperidin-4-yl) ethyl) -3- (2- (aminooxy) acetamido) acrylamide (304).
In another aspect, the present disclosure provides a compound of formula (II):
or a pharmaceutically acceptable salt, solvate, stereoisomer or tautomer thereof, wherein
A is CH or N;
x is O-R1, NH-R1, S-R1 or H;
YY is H, -ONH 2 、-N 3 -OH, maleimide, -COOH or-C (=o) CH 2 Y1, wherein Y1 is a halide;
each of L1 and L2 is independently (CH) 2 ) m 、(CH 2 ) m C(＝O)、(CH 2 ) m -NH(CH 2 ) n 、(CH 2 ) m -C(＝O)NH(CH 2 ) n 、(CH 2 ) m -OC(＝O)-NH-(CH 2 ) n 、(CH 2 ) m -NHC(＝O)-NH-(CH 2 ) n 、(CH 2 ) m -NH、(CH 2 ) m -NHC(＝O)、(CH 2 ) m -NHC(＝O)-(CH 2 ) n -NHC(＝O)-(CH 2 ) p 、C(＝O)-(CH 2 ) n Arylene, substituted arylene, 5-12 membered heteroarylene containing 1-3 heteroatoms, substituted 5-12 membered heteroarylene containing 1-3 heteroatoms, C containing 1-3 heteroatoms 3 -C 8 Heterocycle or absent; wherein each of m, n, and p is independently an integer from 0 to 6, wherein each heteroatom is independently N, O or S;
l3 is C (=O), -CH (R5) -, - (AA) i -or arylene, or a combination thereof, or L3 is absent; wherein each AA is independently an amino acid, wherein i is an integer from 1 to 6;
r5 is NH-L4-Y2 or CH 2 -L4-Y2, wherein Y2 is H or absent;
l4 is C (=o), C (=o) O-, -OC (=o) -, -C (CH) 2 O) 3 -、-C(CH 2 CH 2 O) 3 -、-(AA) j -, arylene, substituted arylene, C 3 -C 8 Cycloalkylene, C 3 -C 8 Substituted cycloalkylene, arylene, substituted arylene, 5-12 membered heteroarylene containing 1-3 heteroatoms, substituted 5-12 membered heteroarylene containing 1-3 heteroatoms, 5-12 membered heterocycloalkylene containing 1-3 heteroatoms, substituted 5-12 membered heterocycloalkylene containing 1-3 heteroatoms, C 1 -C 12 Alkylene, -O-, -NH-, -S-, substituted C 1 -C 12 Alkylene, - (CH) 2 ) s -(OCH 2 CH 2 ) t -(CH 2 ) u -、(CH 2 ) s -(OCH 2 CH 2 ) t -OMe、-N 3 、-SH、-OH、-NH 2 、-OPO 3 H 2 Glucuronide, acetylene, or a combination thereof, or L4 is absent; wherein each AA is independently an amino acid, wherein j is an integer from 1 to 6, wherein each of S and u is independently an integer from 0 to 12, wherein t is independently an integer from 0 to 48, wherein each heteroatom is independently N, O or S;
r1 is H, C 1 -C 12 Alkyl, substituted C 1 -C 12 Alkyl, oxygen-containing C 1 -C 12 Alkyl, C 3 -C 8 Heterocycloalkyl, substituted C 3 -C 8 Heterocycloalkyl, C 3 -C 8 Cycloalkyl, substituted C 3 -C 8 Cycloalkyl, -N 3 Terminally substituted C 1 -C 12 Alkyl, (CH) 2 ) q -(OCH 2 CH 2 ) r -OMe; wherein each of q and r is independently an integer from 0 to 12;
r2 is C 1 -C 6 Alkylene, C 1 -C 12 Substituted alkylene, C 3 -C 8 Cycloalkylene, C 3 -C 8 Substituted cycloalkylene, arylene, substituted arylene, 5-12 membered heteroarylene containing 1-3 heteroatoms, substituted 5-12 membered heteroarylene containing 1-3 heteroatoms, 5-12 membered heterocycloalkylene containing 1-3 heteroatoms, substituted 5-12 membered heterocycloalkylene containing 1-3 heteroatoms, or (OCH) 2 CH 2 ) r Or a combination thereof, or R2 is absent; wherein r is an integer from 1 to 12, wherein each heteroatom is independently N, O or S;
r3 is H OR-C (=o) R6, -C (=o) OR6;
R6 is C 1 -C 12 Alkyl, substituted aryl, CH 3 -(CH 2 ) s -(OCH 2 CH 2 ) t -(CH 2 ) u -wherein each of s, t and u is independently an integer from 0 to 12.
In some embodiments, the compound comprises a PEG moiety. In some embodiments, the PEG moiety is linear, branched, or multi-armed. In some embodiments, L3 is-CH (R5) -, where R5 is NH-L4-Y2 or CH 2 -L4-Y2, wherein Y2 is absent, wherein L4 comprises (CH 2 ) s -(OCH 2 CH 2 ) t OMe, wherein s is an integer from 1 to 12, wherein t is an integer from 1 to 48. In some embodiments, t is an integer from 1 to 12. In some embodiments, YY is-ONH 2 Maleimide, -COOH or-C (=o) CH 2 Y1, wherein Y1 is a halide. In some embodiments, R2 is (CH 2 ) m (OCH 2 CH 2 ) r Wherein each of m and r is independently an integer from 1 to 12. In some embodiments, the PEG moiety has a molecular weight of 0.1kDa to 100kDa or 1kDa to 100kDa. In some embodiments, the PEG moiety has a molecular weight of 0.1kDa to 50kDa or 1kDa to 50kDa. In some embodiments, a is CH.
In some embodiments, the invention provides an immunoconjugate comprising a) an antibody or antibody fragment; b) A TLR agonist comprising a compound conjugated to the antibody or antibody fragment, wherein the TLR agonist comprises the compound or derivative of the compound of any one of claims 1-21, wherein the derivative of the compound is conjugated to the antibody or antibody fragment directly or through linker XX, through moiety YY of the compound, wherein linker XX is a hydrophilic linker, a cleavable linker, or a non-cleavable linker. In some embodiments, linker XX comprises alkylene, alkenylene, alkynylene, polyether, polyester, polyamide, polyamino acid, polypeptide, cleavable peptide, or aminobenzyl carbamate, or a combination thereof.
In some embodiments, the antibody or antigen fragment binds to an antigen of a cell. In some embodiments, the antibody or antibody fragment binds to a cell surface target or a tumor cell target. In some embodiments, the antibody or antibody fragment comprises an Fc fusion protein. In some embodiments, the antibody or antibody fragment is monospecific, bispecific or multispecific. In some embodiments, the antibody or antibody fragment binds to a target selected from the group consisting of: HER2, HER3, B7-H3, connexin-4, PD-1, PDL-1, EGFR, TROP2, FOLR1, PSMA, BCMA, FLT3, VEGFR, CTLA-4, epCAM, MUC1, MUC16, naPi2B, c-Met, GPC3, ENPP3, TIM-3, VISTA, VEGF, blocking protein 18.2, FGFR2, FOLR1, STEAP1, mesothelin, 5T4, CEA, CA9, cadherin 6, ROR1, LIV-1, LILRB-1, LRP-1, SLC34A2, SLC39A6, SLC44A4, LY6E, DLL3, ePhA2, TGFbR, PRLR, GPNMB, SLITRK6, SIRPa, CD3, CD19, CD20, CD22, CD24, CD25, CD30, CD33, CD37, CD38, CD44, CD47, CD52, CD56, CD70, CD79B, CD96, CD97, CD99, CD123, CD179, CD223, and CD 223. In some embodiments, the antibody or antibody fragment is an anti-HER 2, anti-CD 70, or anti-PSMA, or anti-TROP 2 antibody or fragment.
In some embodiments, an anti-HER 2 antibody or antibody fragment comprises a) a heavy chain variable region selected from SEQ ID NOs 1, 2, 3, 4, 16, 17, or 18; and b) a light chain variable region selected from SEQ ID NO. 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. In some embodiments, the antibody or antibody fragment comprises one or more Fc mutations. In some embodiments, the antibody or antibody fragment comprises one or more non-naturally encoded amino acids incorporated into the heavy chain, the light chain, or both the heavy and light chains. In some embodiments of the present invention, in some embodiments, one or more non-naturally encoded amino acids are p-acetylphenylalanine, p-nitrophenylalanine, p-sulfotyrosine, p-carboxyphenylalanine, O-nitrophenylalanine, m-nitrophenylalanine, p-boranylphenylalanine, O-boranylphenylalanine, m-boranylphenylalanine, p-aminophenylalanine, O-aminophenylalanine, m-aminophenylalanine, p-acylphenylalanine, O-acylphenylalanine, m-acylphenylalanine, p-OMe phenylalanine, O-OMe phenylalanine, m-OMe phenylalanine, p-sulfophenylalanine, O-sulfophenylalanine, m-sulfophenylalanine, 5-nitroHis, 3-nitroTyr, 2-nitroTyr, nitro-substituted Leu nitro-substituted His, nitro-substituted De, nitro-substituted Trp, 2-nitroTrp, 4-nitroTrp, 5-nitroTrp, 6-nitroTrp, 7-nitroTrp, 3-aminotyrosine, 2-aminotyrosine, O-sulfotyrosine, 2-sulfophenylalanine, 3-sulfophenylalanine, ortho-carboxyphenylalanine, meta-carboxyphenylalanine, para-acetyl-L-phenylalanine, para-propargyl-phenylalanine, O-methyl-L-tyrosine, L-3- (2-naphthyl) alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAc beta-serine, L-Dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, para-azido-L-phenylalanine, para-acyl-L-phenylalanine, para-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonotyrosine, para-iodophenylalanine, para-bromophenylalanine, para-amino-L-phenylalanine, isopropyl-L-phenylalanine, and para-propargyloxy-L-phenylalanine. In some embodiments, the one or more non-naturally encoded amino acids is p-acetyl-phenylalanine, 4-azido-L-phenylalanine, p-azidoethoxyphenylalanine, or p-azidomethyl-phenylalanine. In some embodiments, the one or more non-naturally encoded amino acids are site-specifically incorporated.
In some embodiments, the TLR agonist is a TLR7 agonist, a TLR8 agonist, or a TLR7/TLR8 dual agonist. In some embodiments, the TLR agonist comprises one or more PEG molecules.
In some embodiments, one or more PEG molecules are linear, branched, multi-armed. In some embodiments, one or more PEG molecules are between 0.1kDa and 100 kDa. In some embodiments, one or more PEG molecules are between 0.1kDa and 50 kDa.
In some embodiments, the linker is a bifunctional or multifunctional linker. In some embodiments, the linker is conjugated to one or more non-naturally encoded amino acids incorporated into the antibody or antibody fragment. In some embodiments, the linker is a hydrophilic linker, a cleavable linker, or a non-cleavable linker.
In one embodiment, the invention provides a method of treating a subject or patient suffering from a disease or disorder, the method comprising administering to the subject or patient a therapeutically effective amount of a conjugate of formula (I) or formula (II). In some embodiments, the disease or condition is an autoimmune disease, a chronic inflammatory disease, or cancer. In some embodiments, the cancer is breast cancer, small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, gastrointestinal pancreatic tumor, cervical cancer, esophageal cancer, colon cancer, colorectal cancer, cancer or tumor of epithelial origin, renal cancer, brain cancer, glioblastoma, pancreatic cancer, myelogenous leukemia, thyroid cancer, endometrial cancer, lymphoma, pancreatic cancer, head and neck cancer, or skin cancer. In some embodiments, the method of treatment further comprises administering another therapeutic agent. In some embodiments, the other therapeutic agent is a chemotherapeutic agent, hormonal agent, anti-tumor agent, immunostimulant, immunomodulator, immunotherapeutic agent, or combination thereof.
In some embodiments, the present disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of an immunoconjugate described above and a pharmaceutically acceptable carrier or excipient. In some embodiments, the present disclosure provides a pharmaceutical composition comprising a compound described above or an immunoconjugate described above for use as a medicament. In some embodiments, the present disclosure provides the use of an immunoconjugate described above in the manufacture of a medicament
The invention provides a method of inhibiting or reducing the growth of a tumor or cancer comprising contacting the tumor with an effective amount of a TC of the invention to stimulate the immune system of a patient in the vicinity of the tumor. The invention provides a method of inhibiting or reducing the growth of a tumor or cancer comprising contacting the tumor with an effective amount of a pegylated TC, or a stable dimer or multimer of a TC of the invention. In one embodiment, TC is non-pegylated or mono-pegylated. In one embodiment, TC is a dimer ethylene glycol. In one embodiment, TC has more than one and/or different TLR agonist molecules attached thereto. In one embodiment, TC has more than one and/or the same TLR agonist molecule attached thereto. Another embodiment of the invention provides a method of modulating an immune response to a tumor cell using a TC of the invention. In certain embodiments, TC is co-administered with at least one chemotherapeutic agent and/or at least one immunotherapeutic agent. The chemotherapeutic agent may be selected from the group consisting of: temozolomide, gemcitabine, doxorubicin, cyclophosphamide, paclitaxel, cisplatin, fluoropyrimidine, taxane, anthracyclines, lapatinib, capecitabine, letrozole, pertuzumab, docetaxel, IFN- α.
In some embodiments, TC comprises a targeting polypeptide, including but not limited to an Antigen Binding Polypeptide (ABP) comprising one or more non-naturally encoded amino acids. In some embodiments, the ABP comprises an intact antibody heavy chain. In some embodiments, the ABP comprises an intact antibody light chain. In some embodiments, the ABP comprises a variable region of an antibody light chain. In some embodiments, ABP comprises a variable region of an antibody heavy chain. In some embodiments, the ABP comprises at least one CDR of an antibody light chain. In some embodiments, the ABP comprises at least one CDR of an antibody heavy chain. In some embodiments, the ABP comprises at least one CDR of a light chain and at least one CDR of a heavy chain. In some embodiments, ABP comprises Fab. In some embodiments, the ABP comprises two or more Fab. In some embodiments, ABP comprises (Fab') 2. In some embodiments, the ABP comprises two or more (Fab') 2. In some embodiments, the ABP comprises an scFv. In some embodiments, the ABP comprises two or more scFv. In some embodiments, the ABP comprises a minibody. In some embodiments, the ABP comprises two or more minibodies. In some embodiments, the ABP comprises a diabody. In some embodiments, the ABP comprises two or more diabodies. In some embodiments, the ABP comprises a light chain variable region and a heavy chain variable region. In some embodiments, ABP comprises a complete light chain and a complete heavy chain. In some embodiments, the ABP comprises one or more Fc domains or portions thereof. In some embodiments, ABP comprises a combination of any of the above embodiments. In some embodiments, ABPs comprise a homodimer, a heterodimer, a homomultimer, or a heteromultimer of any of the above embodiments. In some embodiments, the ABP comprises a polypeptide that binds to a binding partner, wherein the binding partner comprises an antigen, polypeptide, nucleic acid molecule, polymer, or other molecule or substance. In some embodiments, ABP is associated with a non-antibody scaffold molecule or substance. In some embodiments, the antigen is a tumor antigen.
Toll-like receptors (TLRs) detect a wide range of conserved pathogen-associated molecular patterns (PAMPs). The TLRs play an important role in sensing invading pathogens and subsequently initiating innate immune responses. There are 10 known members of the human TLR family, which are type I transmembrane proteins with an extracellular leucine-rich domain and a cytoplasmic tail containing a conserved Toll/Interleukin (IL) -1 receptor (TIR) domain. Within this family, TLR3, TLR7, TLR8 and TLR9 are located in the endosome. TLR7 and TLR8 can be activated by binding to a specific small molecule ligand (i.e., TLR7 agonist or TLR8 agonist) or its natural ligand (i.e., single stranded RNA, ssRNA). Upon binding of an agonist to TLR7 or TLR8, its dimerized form of the receptor is thought to undergo a structural change, resulting in subsequent recruitment of adaptor proteins at its cytoplasmic domain, including myeloid differentiation primary response gene 88 (MyD 88). Upon initiation of the receptor signaling cascade through the MyD88 pathway, cytoplasmic transcription factors such as interferon regulatory factor 7 (IRF-7) and nuclear factor κB (NF- κB) are activated. These transcription factors then translocate to the nucleus and initiate transcription of various genes, such as IFN- α and other antiviral cytokine genes. TLR7 is expressed primarily on plasmacytoid cells and B cells. An alteration in immune cell responsiveness may result in a decrease in the innate immune response of a cancer patient. Thus, agonist-induced activation of TLR7 and/or TLR8 conjugated to a targeting moiety (such as an antibody or fragment thereof) may represent a novel approach for treating cancer. Treatment with TCs comprising TLR7 or TLR8 agonists represents a promising solution to provide higher efficacy and better tolerability. Suitable TLR7 and/or TLR8 agonists for use in the present invention to prepare TC are found in the following U.S. patents, each of which is incorporated herein by reference: U.S. patent No. 6,825,350; U.S. patent No. 6,656,389; U.S. patent No. 6,656,398; U.S. patent No. 6,683,088; U.S. patent No. 6,756,382; U.S. patent No. 6,825,350; U.S. patent No. 6,667,312; U.S. patent No. 6,677,347; U.S. patent No. 7,598,382; U.S. patent No. 8,673,932.
In some embodiments, TCs comprise a targeting polypeptide that further comprises an amino acid substitution, addition, or deletion that increases the compatibility of the TC polypeptide with a pharmaceutical preservative (e.g., m-cresol, phenol, benzyl alcohol) when compared to the compatibility of a corresponding wild-type TC without substitution, addition, or deletion. This increased compatibility will enable the preparation of preserved pharmaceutical formulations that maintain the physicochemical properties and biological activity of the protein during storage.
In some embodiments, one or more engineered bonds are created with one or more unnatural amino acids. Intramolecular bonds can be produced in a variety of ways, including but not limited to, two amino acids in a protein reacting under suitable conditions (one or both amino acids can be unnatural amino acids); two amino acids (each of which may be naturally encoded or non-naturally encoded) are reacted under suitable conditions with linkers, polymers or other molecules, etc.
In some embodiments, one or more amino acid substitutions in the TC polypeptide may be with one or more naturally occurring or non-naturally occurring amino acids. In some embodiments, the amino acid substitution in TC may be with a naturally occurring or non-naturally occurring amino acid, provided that at least one substitution is with a non-naturally encoded amino acid. In some embodiments, one or more amino acid substitutions in the TC polypeptide may be with one or more naturally occurring amino acids, and additionally, at least one substitution is with a non-naturally encoded amino acid. In some embodiments, the TC polypeptide may be an antibody or antibody fragment. In some embodiments, the TC polypeptide may be a tumor targeting polypeptide.
In some embodiments, the non-naturally encoded amino acid comprises a carbonyl, acetyl, aminooxy, hydrazino, semicarbazide, azide, or alkyne group.
In some embodiments, the non-naturally encoded amino acid comprises a carbonyl group. In some embodiments, the non-naturally encoded amino acid has the following structure:
wherein n is 0 to 10; r is R 1 Is alkyl, aryl, substituted alkyl or substituted aryl; r is R 2 Is H, alkyl, aryl, substituted alkyl, and substituted aryl; and R is 3 Is H, an amino acid, a polypeptide or an amino terminal modification; and R is 4 Is H, an amino acid, a polypeptide or a carboxyl terminal modification group.
In some embodiments, the non-naturally encoded amino acid comprises an aminooxy group. In some embodiments, the non-naturally encoded amino acid comprises a hydrazide group. In some embodiments, the non-naturally encoded amino acid comprises a hydrazino group. In some embodiments, the non-naturally encoded amino acid residue comprises a semicarbazide group.
In some embodiments, the non-naturally encoded amino acid residue comprises an azide group. In some embodiments, the non-naturally encoded amino acid has the following structure:
wherein n is 0 to 10; r is R 1 Is alkyl, aryl, substituted alkyl, substituted aryl, or absent; x is O, N, S or absent; m is 0 to 10; r is R 2 Is H, an amino acid, a polypeptide or an amino terminal modification; and R is 3 Is H, an amino acid, a polypeptide or a carboxyl terminal modification group.
In some embodiments, the non-naturally encoded amino acid comprises an alkyne group. In some embodiments, the non-naturally encoded amino acid has the following structure:
wherein n is 0 to 10; r is R 1 Is alkyl, aryl, substituted alkyl or substituted aryl; x is O, N, S or absent; m is 0 to 10; r is R 2 Is H, an amino acid, a polypeptide or an amino terminal modification; and R is 3 Is H, an amino acid, a polypeptide or a carboxyl terminal modification group.
In some embodiments, the polypeptide is a TC comprising a non-naturally encoded amino acid linked to a water soluble polymer. In some embodiments, the water-soluble polymer comprises a polyethylene glycol moiety. In some embodiments, TC comprises a non-naturally encoded amino acid and one or more post-translational modifications, linkers, polymers, or bioactive molecules.
The invention also provides an isolated nucleic acid comprising a polynucleotide encoding a targeting polypeptide of TC, and the invention provides an isolated nucleic acid comprising a polynucleotide that hybridizes to a polynucleotide under stringent conditions. The invention also provides an isolated nucleic acid comprising a polynucleotide encoding a targeting polypeptide, wherein the polynucleotide comprises at least one selector codon. It will be apparent to one of ordinary skill in the art that many different polynucleotides may encode any of the polypeptides of the invention.
In some embodiments, the selector codon is selected from the group consisting of: amber, ocher, opal, unique, rare, five and four base codons.
The invention also provides methods of preparing TC polypeptides linked to a water-soluble polymer or to one or more TC polypeptides to form homodimers or homomultimers. In some embodiments, the method comprises contacting an isolated TC polypeptide comprising a non-naturally encoded amino acid with a water soluble polymer or linker comprising a moiety that reacts with the non-naturally encoded amino acid. In some embodiments, the non-naturally encoded amino acid incorporated into the TC polypeptide is reactive with a water soluble polymer or linker and is not reactive with any of the 20 common amino acids. In some embodiments, the non-naturally encoded amino acid incorporated into the TC polypeptide is reactive with a linker, polymer, or biologically active molecule, but not with any of the 20 common amino acids.
In some embodiments, the TC polypeptide attached to the water soluble polymer or linker is prepared by reacting a TC polypeptide comprising a carbonyl-containing amino acid with a poly (ethylene glycol) molecule or a linker comprising an aminooxy, hydrazino, or semicarbazide group. In some embodiments, the aminooxy, hydrazino, hydrazono, or semicarbazide group is attached to the poly (ethylene glycol) molecule or linker through an amide bond. In some embodiments, the aminooxy, hydrazino, hydrazono, or semicarbazide group is linked to the poly (ethylene glycol) molecule or linker through a urethane linkage.
In some embodiments, the TC polypeptide attached to the water soluble polymer is prepared by reacting a poly (ethylene glycol) molecule or a linker comprising a carbonyl group with a polypeptide comprising a non-naturally encoded amino acid comprising an aminooxy, hydrazino, or semicarbazide group.
In some embodiments, the TC polypeptide attached to the water soluble polymer or linker is prepared by reacting a TC comprising an alkyne-containing amino acid with a poly (ethylene glycol) molecule comprising an azide moiety. In some embodiments, the azide or alkyne group is linked to the poly (ethylene glycol) molecule or linker through an amide linkage.
In some embodiments, the TC polypeptide attached to the water soluble polymer or linker is prepared by reacting a TC polypeptide comprising an azide-containing amino acid with a poly (ethylene glycol) molecule comprising an alkyne moiety. In some embodiments, the azide or alkyne group is linked to the poly (ethylene glycol) molecule or linker through an amide linkage.
In some embodiments, the molecular weight of the poly (ethylene glycol) molecule or linker is between about 0.1kDa and about 100 kDa. In some embodiments, the molecular weight of the poly (ethylene glycol) molecule or linker is between 0.1kDa and 50 kDa. In some embodiments, the poly (ethylene glycol) molecule or linker is a branched polymer or branched linker. In some embodiments, the molecular weight of each branch of the poly (ethylene glycol) branched polymer or branched linker is between 1kDa and 100kDa, or between 1kDa and 50 kDa.
In some embodiments, the water-soluble polymer attached to the TC polypeptide comprises a polyalkylene glycol moiety. In some embodiments, the non-naturally encoded amino acid residue incorporated into TC comprises a carbonyl group, an aminooxy group, a hydrazide group, a hydrazine group, a semicarbazide group, an azide group, or an alkyne group. In some embodiments, the non-naturally encoded amino acid residue incorporated into the TC polypeptide comprises a carbonyl moiety and the water soluble polymer comprises an aminooxy, hydrazide, hydrazine, or semicarbazide moiety. In some embodiments, the non-naturally encoded amino acid residue incorporated into the TC polypeptide comprises an alkyne moiety and the water soluble polymer comprises an azide moiety. In some embodiments, the non-naturally encoded amino acid residue incorporated into the TC polypeptide comprises an azide moiety and the water soluble polymer comprises an alkyne moiety. The invention also provides compositions comprising a TC polypeptide comprising a non-naturally encoded amino acid and a pharmaceutically acceptable carrier. In some embodiments, the non-naturally encoded amino acid is linked to a water soluble polymer.
The invention also provides a cell comprising a polynucleotide encoding a targeting polypeptide comprising a TC of a selector codon. In some embodiments, the cell comprises an orthogonal RNA synthetase and/or an orthogonal tRNA for substituting an unnatural coding amino acid into a targeting polypeptide to TC.
The invention also provides methods of making targeting polypeptides comprising TCs that do not naturally encode amino acids. In some embodiments, the method comprises culturing a cell comprising one or more polynucleotides encoding a targeting polypeptide of TC, an orthogonal RNA synthetase, and/or an orthogonal tRNA under conditions that allow expression of the targeting polypeptide of TC or a variant thereof; purifying the TC polypeptide from the cells and/or the culture medium.
The invention also provides methods of increasing the therapeutic half-life, serum half-life or circulation time of TC. The invention also provides methods of modulating TC immunogenicity. In some embodiments, the method comprises replacing any one or more amino acids of a naturally occurring targeting polypeptide of TC with a non-naturally encoded amino acid and/or attaching the targeting polypeptide to a linker, polymer, water soluble polymer, or biologically active molecule.
The invention also provides a method of treating a patient in need of such treatment with an effective amount of a TC molecule of the invention. In some embodiments, the method comprises administering to the patient a therapeutically effective amount of a pharmaceutical composition comprising TC comprising the non-naturally encoded amino acid and a pharmaceutically acceptable carrier. In some embodiments, the non-naturally encoded amino acid is linked to a water soluble polymer. In some embodiments, TC is glycosylated. In some embodiments, the TC is not glycosylated.
The invention also provides TCs comprising a water-soluble polymer or a linker attached to TC at a single amino acid by a covalent bond. In some embodiments, the water-soluble polymer comprises a poly (ethylene glycol) moiety. In some embodiments, the amino acid covalently linked to the water-soluble polymer or linker is a non-naturally encoded amino acid present in the targeting polypeptide of TC.
The present invention provides a TC polypeptide comprising at least one linker, polymer or biologically active molecule, wherein said linker, polymer or biologically active molecule is linked to the polypeptide by a functional group that non-naturally encodes an amino acid that is incorporated into a targeting polypeptide of TC in a ribosomal manner. In TC conjugates, PEG or other water soluble polymer, another TC, polypeptide, or biologically active molecule may be conjugated directly to TC through a linker. In one embodiment, the linker is long enough to allow flexibility and allow dimer formation. In one embodiment, the linker is at least 3 amino acids or 18 atoms in length to allow for dimer formation. In some embodiments, the polypeptide is linked to a linker to allow for the formation of multimers. In some embodiments, the linker is a bifunctional linker. In some embodiments, the compositions and/or TCs of the present invention may comprise a plurality of linkers. In other embodiments, each linker may include one or more compounds attached. The linker may also comprise alkylene, alkenylene, alkynylene, polyether, polyester, polyamide groups, and polyamino acids, polypeptides, cleavable peptides, or aminobenzyl carbamates. In some embodiments, the linkers may be the same or different linkers. Suitable linkers include, for example, cleavable linkers and non-cleavable linkers. Suitable cleavable linkers include, for example, peptide linkers cleavable by intracellular proteases, such as lysosomal proteases or endosomal proteases. The cleavable linker may comprise a valine-citrulline (Val-Cit) linker, or a valine-alanine (Val-Ala) peptide, or valine-lysine (Val-Lys) or valine-arginine (Vla-Arg) or an analog of any one of Val-Cit, val-Ala, val-Lys or Val-Arg. In some embodiments, the linker may be a dipeptide linker, such as a valine-citrulline or phenylalanine-lysine linker. The valine-citrulline or valine-alanine containing linker can contain maleimide or succinimide groups. The valine-citrulline or valine-alanine containing linker may contain a para-aminobenzyl alcohol (PABA) group or para-aminobenzyl carbamate (PABC). Other suitable linkers include linkers that are hydrolyzable at a pH of less than 5.5, such as hydrazone linkers. Additional suitable cleavable linkers include disulfide linkers. In some embodiments, cleavable linkers can include linkers that cleave at tumor microenvironments, such as tumor-infiltrating T cells. In some embodiments, the non-cleavable linker includes, but is not limited to, a maleimidocaproyl linker. The maleimidocaproyl linker may comprise N-maleimidomethyl cyclohexane-1-carboxylate, a succinimidyl group, a pentafluorophenyl group, and/or one or more PEG molecules, but is not limited thereto. In some embodiments, any of the compositions, compounds, or salts thereof of the invention may be linked to the polypeptide by means of a linker. In some embodiments, any of the compounds disclosed herein in tables 3, 4, 5, 6, and 7, or salts thereof, can be linked to the polypeptide by means of a linker. In some embodiments, the polypeptide is a targeting polypeptide or a biological targeting polypeptide or a tumor targeting polypeptide. In some embodiments, the targeting polypeptide is an antibody or antibody fragment.
In some embodiments, the TC polypeptide is monopegylated. The invention also provides a TC comprising a linker, polymer or biologically active molecule linked to one or more non-naturally encoded amino acids, wherein said non-naturally encoded amino acids are incorporated in ribosomal fashion at preselected sites in a polypeptide.
In some embodiments, the invention provides a composition comprising one or more targeting polypeptides having incorporated one or more non-naturally encoded amino acids, wherein at least one of the polypeptides is linked to a TLR agonist molecule through a linker that is covalently bonded to the non-natural amino acid of the polypeptide.
In another embodiment, the invention provides a composition wherein one or more targeting polypeptides are the same or different targeting polypeptides. In another embodiment, the invention provides a composition wherein one or more targeting polypeptides bind to a cell surface target, or a tumor cell target, or a cancer cell target. In another embodiment, the one or more targeting polypeptides are monospecific, bispecific or multispecific targeting polypeptides.
In other embodiments, the monospecific, bispecific or multispecific targeting polypeptide comprises a drug conjugate or checkpoint inhibitor. Any suitable immune checkpoint inhibitor is contemplated for use with the compositions or TCs of the present invention. In some embodiments, the immune checkpoint inhibitor reduces the expression or activity of one or more immune checkpoint proteins. In another embodiment, the immune checkpoint inhibitor reduces the interaction between one or more immune checkpoint proteins and their ligands. Inhibitory nucleic acids that reduce the expression and/or activity of immune checkpoint molecules may also be used in the present invention. In some embodiments, the immune checkpoint inhibitor is CTLA4, TIGIT, glucocorticoid-induced TNFR-related protein (GITR), inducible T cell costimulatory factor (ICOS), CD96, poliovirus receptor-related 2 (PVRL 2), PD-1, PD-L2, LAG-3, B7-H4, killer Immunoglobulin Receptor (KIR), OX40-L, indoleamine 2, 3-dioxygenase 1 (IDO-1), indoleamine 2, 3-dioxygenase 2 (IDO-2), CEACAM1, CD272, TEVI3, adenosine A2A receptor, and VISTA protein. In some embodiments, the immune checkpoint inhibitor is an inhibitor of CTLA4, PD-1, or PD-L1.
In another embodiment, the targeting polypeptide comprises an antibody or antibody fragment. In other embodiments, the targeting polypeptide is an antibody or antibody fragment that binds to a cellular antigen. In another embodiment, the targeting polypeptide is an antibody or antibody fragment that binds to a target selected from the group consisting of: HER2, HER3, PD-1, PDL-1, EGFR, TROP2, PSMA, VEGFR, CTLA-4, epCAM, MUC1, MUC16, c-met, GPC3, ENPP3, TIM-1, FOLR1, STEAP1, mesothelin, 5T4, CEA, CA9, cadherin 6, ROR1, SLC34A2, SLC39A6, SLC44A4, LY6E, DLL3, ePhA2, GPNMB, slittk 6, CD3, CD 19, CD22, CD24, CD25, CD30, CD33, CD38, CD44, CD47, CD52, CD56, CD70, CD96, CD97, CD99, CD117, CD123, CD179, CD223 and CD276. In some embodiments, the targeting polypeptide comprises an antibody or antibody fragment that binds to HER 2. In another embodiment, the targeting polypeptide is trastuzumab.
In another embodiment, the antibody or antibody fragment comprises IgG, fab, (Fab') 2, fv, or single chain Fv (scFv). In some embodiments, the antibody or antibody fragment comprises one or more Fab, (Fab') 2, fv, or single chain Fv (scFv) mutations. In some embodiments, the antibody or antibody fragment comprises one or more Fc mutations. In other embodiments, the antibody or antibody fragment comprises one to six Fc mutations. In some embodiments, the antibody or antibody fragment comprises two or more Fc mutations. In other embodiments, the antibody or antibody fragment comprises three or more Fc mutations. In some embodiments, the antibody or antibody fragment comprises four or more Fc mutations. In other embodiments, the antibody or antibody fragment comprises five or more Fc mutations. In other embodiments, the antibody or antibody fragment comprises six Fc mutations.
In another embodiment, the antibody or antibody fragment comprises one or more non-naturally encoded amino acids incorporated into the heavy chain, the light chain, or both the heavy and light chains. In another embodiment, the antibody or antibody fragment comprises one or more non-naturally encoded amino acids incorporated into the heavy and light chains. In another embodiment, the antibody or antibody fragment comprises one or more non-naturally encoded amino acids incorporated into the heavy chain, the light chain, or both the heavy and light chains, and further comprises one or more Fc mutations. In another embodiment, the antibody or antibody fragment comprises one or more non-naturally encoded amino acids incorporated into each of the heavy and light chains, the antibody or antibody fragment further comprising one or more Fc mutations. In another embodiment, the antibody or antibody fragment comprises one or more non-naturally encoded amino acids incorporated into the heavy chain, the light chain, or both the heavy and light chains, and further comprises at least two Fc mutations. In another embodiment, the antibody or antibody fragment comprises one or more non-naturally encoded amino acids incorporated into each of the heavy and light chains, the antibody or antibody fragment further comprising at least two Fc mutations. In another embodiment, the antibody or antibody fragment comprises one or more non-naturally encoded amino acids incorporated into the heavy chain, the light chain, or both the heavy and light chains, and further comprises at least three Fc mutations. In another embodiment, the antibody or antibody fragment comprises one or more non-naturally encoded amino acids incorporated into each of the heavy and light chains, the antibody or antibody fragment further comprising at least three Fc mutations. In another embodiment, the antibody or antibody fragment comprises one or more non-naturally encoded amino acids incorporated into the heavy chain, the light chain, or both the heavy and light chains, and further comprises at least four Fc mutations. In another embodiment, the antibody or antibody fragment comprises one or more non-naturally encoded amino acids incorporated into each of the heavy and light chains, the antibody or antibody fragment further comprising at least four Fc mutations. In another embodiment, the antibody or antibody fragment comprises one or more non-naturally encoded amino acids incorporated into the heavy chain, the light chain, or both the heavy and light chains, and further comprises at least five Fc mutations. In another embodiment, the antibody or antibody fragment comprises one or more non-naturally encoded amino acids incorporated into each of the heavy and light chains, the antibody or antibody fragment further comprising at least five Fc mutations. In another embodiment, the antibody or antibody fragment comprises one or more non-naturally encoded amino acids incorporated into the heavy chain, the light chain, or both the heavy and light chains, and further comprises at least six Fc mutations. In another embodiment, the antibody or antibody fragment comprises one or more non-naturally encoded amino acids incorporated into each of the heavy and light chains, the antibody or antibody fragment further comprising at least six Fc mutations.
In another embodiment, the targeting polypeptide comprises one or more non-naturally encoded amino acids selected from the group consisting of: para-acetylphenylalanine, para-nitrophenylalanine, para-sulfophenylalanine, para-carboxyphenylalanine, ortho-nitrophenylalanine, meta-nitrophenylalanine, para-boronylphenylalanine, ortho-boronylphenylalanine, meta-boronylphenylalanine, para-aminophenylalanine, ortho-aminophenylalanine, meta-aminophenylalanine, ortho-acylphenylalanine, para-OMe phenylalanine, ortho-OMe phenylalanine, meta-OMe phenylalanine, para-sulfophenylalanine, ortho-sulfophenylalanine, meta-sulfophenylalanine, 5-nitroHis, 3-nitroTyr, 2-nitroTyr, nitro-substituted Leu, nitro-substituted His, nitro-substituted De, nitro-substituted Trp 2-nitroTrp, 4-nitroTrp, 5-nitroTrp, 6-nitroTrp, 7-nitroTrp, 3-aminotyrosine, 2-aminotyrosine, O-sulfotyrosine, 2-sulfophenylalanine, 3-sulfophenylalanine, orthocarboxyphenylalanine, metacarboxyphenylalanine, para-acetyl-L-phenylalanine, para-propargyl-phenylalanine, O-methyl-L-tyrosine, L-3- (2-naphthyl) alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcβ -serine, L-Dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, para-azido-L-phenylalanine, para-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-iodophenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, p-propargyloxy-L-phenylalanine, 4-azido-L-phenylalanine, p-azidoethoxyphenylalanine, and p-azidomethyl-phenylalanine. In another embodiment, the unnatural amino acid is selected from the group consisting of: p-acetyl-phenylalanine, 4-azido-L-phenylalanine, p-azidoethoxyphenylalanine or p-azidomethyl-phenylalanine. In other embodiments, the non-naturally encoded amino acid is site-specifically incorporated into one or more targeting polypeptides.
In another embodiment, the TLR agonist is a TLR7 agonist, a TLR8 agonist, or a TLR7/TLR8 dual agonist. In other embodiments, the TLR agonist is a TLR agonist comprising a molecular structure according to formula (I) or formula (II) of fig. 1. In another embodiment, the TLR agonist is any one selected from the group of structures of tables 3, 4, 5, 6, 7 according to the invention.
In other embodiments, the targeting polypeptide is conjugated to one or more linkers, polymers, or bioactive molecules. In some embodiments, the targeting polypeptide is conjugated directly or indirectly to one or more linkers, polymers, or bioactive molecules. In some embodiments, one or more of the linkers is a cleavable or non-cleavable linker.
In some embodiments, the one or more linkers are 0.1kDa to 50kDa. In other embodiments, the one or more linkers are 0.1kDa to 10kDa. In other embodiments, one or more of the linkers or polymers are linear, branched, multimeric, or dendritic. In another embodiment, the one or more linkers or polymers are difunctional or polyfunctional linkers or difunctional or polyfunctional polymers.
In other embodiments, the one or more polymers are water soluble polymers. In other embodiments, the water-soluble polymer is polyethylene glycol (PEG). In some embodiments, the molecular weight of PEG is between 0.1kDa and 100 kDa. In other embodiments, the molecular weight of PEG is between 0.1kDa and 50 kDa. In other embodiments, the molecular weight of PEG is between 0.1kDa and 40 kDa. In other embodiments, the molecular weight of PEG is between 0.1kDa and 30kDa. In other embodiments, the molecular weight of PEG is between 0.1kDa and 20 kDa. In other embodiments, the molecular weight of PEG is between 0.1kDa and 10 kDa. In some embodiments, the molecular weight of the poly (ethylene glycol) molecule is between about 0.1kDa and about 100 kDa. In some embodiments, the molecular weight of the poly (ethylene glycol) molecule is between 0.1kDa and 50 kDa. In some embodiments, the molecular weight of the poly (ethylene glycol) molecule is between 1kDa and 25kDa, or between 2kDa and 22kDa, or between 5kDa and 20 kDa. For example, the molecular weight of the poly (ethylene glycol) polymer can be about 5kDa, or about 10kDa, or about 20kDa, or about 30kDa. For example, the molecular weight of the poly (ethylene glycol) polymer may be 5kDa, or 10kDa, or 20kDa, or 30kDa. In some embodiments, the poly (ethylene glycol) molecule is branched PEG. In some embodiments, the poly (ethylene glycol) molecule is branched 5K PEG. In some embodiments, the poly (ethylene glycol) molecule is branched 10K PEG. In some embodiments, the poly (ethylene glycol) molecule is branched 20K PEG. In some embodiments, the poly (ethylene glycol) molecule is linear PEG. In some embodiments, the poly (ethylene glycol) molecule is linear 5K PEG. In some embodiments, the poly (ethylene glycol) molecule is linear 10K PEG. In some embodiments, the poly (ethylene glycol) molecule is linear 20K PEG. In some embodiments, the poly (ethylene glycol) molecule is linear 30KPEG. In some embodiments, the molecular weight of the poly (ethylene glycol) polymer is the average molecular weight. In certain embodiments, the average molecular weight is a number average molecular weight (Mn). Average molecular weight may be determined or measured using GPC or SEC, SDS/PAGE analysis, RP-HPLC, mass spectrometry, or capillary electrophoresis.
In another embodiment, at least one linker, polymer or biologically active molecule is attached to at least one non-naturally encoded amino acid. In some embodiments, the linker is PEG. In other embodiments, the linker is PEG having a molecular weight between 0.1kDa and 50 kDa. In other embodiments, the linker is PEG having a molecular weight between 0.1kDa and 40 kDa. In other embodiments, the linker is PEG having a molecular weight between 0.1kDa and 30 kDa. In other embodiments, the linker is PEG having a molecular weight between 0.1kDa and 20 kDa. In other embodiments, the linker is PEG having a molecular weight between 0.1kDa and 10 kDa. In other embodiments, the linker is PEG having a molecular weight between 0.1kDa and 5 kDa.
In another embodiment, the targeting polypeptide comprises one or more amino acid substitutions, additions or deletions that increase the stability or solubility of the composition. In another embodiment, the targeting polypeptide comprises one or more amino acid substitutions, additions or deletions that enhance/reduce ADCP or ADCC activity. In another embodiment, the targeting polypeptide comprises one or more amino acid substitutions, additions or deletions that increase the pharmacokinetics of the composition. In other embodiments, the compositions comprise one or more amino acid substitutions, additions or deletions that increase expression of the targeting polypeptide in a recombinant host cell or synthesized in vitro.
In another embodiment, the non-naturally encoded amino acid is reactive with a linker, polymer or biologically active molecule, but is not reactive with any of the 20 common amino acids in the polypeptide. In another embodiment, the non-naturally encoded amino acid comprises a carbonyl, aminooxy, hydrazino, hydrazono, semicarbazide, azide, or alkyne group. In other embodiments, the non-naturally encoded amino acid comprises a carbonyl group.
In another embodiment, the targeting polypeptide is linked to a cytotoxic or immunostimulant. In another embodiment, TC or BTC of the invention is linked to a cytotoxic or immunostimulant. In another embodiment, the targeting polypeptide comprises a cytotoxic agent or an immunostimulant. In another embodiment, TC or BTC of the invention comprises a cytotoxic or immunostimulant.
In another embodiment, the invention provides a TLR agonist conjugate (TC) comprising an anti-HER 2 antibody or antibody fragment conjugated to a TLR agonist comprising a structure according to any of the structures of fig. 1, wherein the TLR agonist is conjugated to the antibody or antibody fragment by covalent bonding to a linker incorporating one or more non-naturally encoded amino acids in the antibody or antibody fragment. In another embodiment, the TLR agonist is a TLR7 agonist, a TLR8 agonist, or a TLR7/TLR8 dual agonist. In another embodiment, the TLR agonist comprises a structure according to formula (I) or formula (II) of fig. 1. In another embodiment, the TLR agonist comprises a structure according to formula I or formula II, further comprising a linker.
In another embodiment, an anti-HER 2 antibody or antibody fragment comprises one or more non-naturally encoded amino acids incorporated into the heavy chain, the light chain, or both the heavy and light chains. In another embodiment, the one or more non-naturally encoded amino acids are selected from the group consisting of: para-acetylphenylalanine, para-nitrophenylalanine, para-sulfophenylalanine, para-carboxyphenylalanine, ortho-nitrophenylalanine, meta-nitrophenylalanine, para-boronylphenylalanine, ortho-boronylphenylalanine, meta-boronylphenylalanine, para-aminophenylalanine, ortho-aminophenylalanine, meta-aminophenylalanine, ortho-acylphenylalanine, para-OMe phenylalanine, ortho-OMe phenylalanine, meta-OMe phenylalanine, para-sulfophenylalanine, ortho-sulfophenylalanine, meta-sulfophenylalanine, 5-nitroHis, 3-nitroTyr, 2-nitroTyr, nitro-substituted Leu, nitro-substituted His, nitro-substituted De, nitro-substituted Trp 2-nitroTrp, 4-nitroTrp, 5-nitroTrp, 6-nitroTrp, 7-nitroTrp, 3-aminotyrosine, 2-aminotyrosine, O-sulfotyrosine, 2-sulfophenylalanine, 3-sulfophenylalanine, orthocarboxyphenylalanine, metacarboxyphenylalanine, para-acetyl-L-phenylalanine, para-propargyl-phenylalanine, O-methyl-L-tyrosine, L-3- (2-naphthyl) alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcβ -serine, L-Dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, para-azido-L-phenylalanine, para-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-iodophenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, p-propargyloxy-L-phenylalanine, 4-azido-L-phenylalanine, p-azidoethoxyphenylalanine, and p-azidomethyl-phenylalanine. In other embodiments, the unnatural amino acid is a p-acetyl-phenylalanine, a 4-azido-L-phenylalanine, a p-azidomethyl-phenylalanine, or a p-azidoethoxyphenylalanine.
In another embodiment, the anti-HER 2 antibody or antibody fragment further comprises one or more mutations in the Fc region. In another embodiment, the anti-HER 2 antibody or antibody fragment further comprises two or more mutations in the Fc region. In another embodiment, the anti-HER 2 antibody or antibody fragment further comprises three or more mutations in the Fc region. In another embodiment, the anti-HER 2 antibody or antibody fragment further comprises four or more mutations in the Fc region. In another embodiment, the anti-HER 2 antibody or antibody fragment further comprises five or more mutations in the Fc region. In another embodiment, the anti-HER 2 antibody or antibody fragment further comprises six or more mutations in the Fc region. In another embodiment, the anti-HER 2 antibody or antibody fragment further comprises six mutations in the Fc region.
In another embodiment, one or more of the linkers is a cleavable or non-cleavable linker. In other embodiments, one or more of the linkers is a bifunctional or multifunctional linker.
In other embodiments, the TLR agonist is a TLR agonist comprising a molecular structure according to fig. 1, which further comprises a polyethylene glycol (PEG) shield to enhance or modify the hydrophilicity of TCs of the invention. In some embodiments, the PEG mask is linear PEG. In other embodiments, the linear PEG is PEG4, PEG8, PEG12, PEG24, or PEG48. In other embodiments, the linear PEG is PEG4. In other embodiments, the linear PEG is PEG8. In other embodiments, the linear PEG is PEG12. In other embodiments, the linear PEG is PEG24. In other embodiments, the linear PEG is PEG48. In some embodiments, the PEG mask is branched PEG. In other embodiments, the branched PEG is (PEG 4) nn 、(PEG8) nn 、(PEG12) nn 、(PEG24) nn Or (PEG 48) nn . In the case of a further embodiment of the present invention,branched PEG is (PEG 4) nn . In other embodiments, the branched PEG is (PEG 8) nn . In other embodiments, the branched PEG is (PEG 12) nn . In other embodiments, the branched PEG is (PEG 24) nn . In other embodiments, the branched PEG is (PEG 48) nn . In some embodiments, nn is an integer greater than 1. In some embodiments, nn is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or greater. In some embodiments, nn is 2. In some embodiments, nn is 3. In some embodiments, nn is 4. In some embodiments, nn is 5. In some embodiments, nn is 6. In some embodiments, nn is 7. In some embodiments, nn is 8. In some embodiments, nn is 9. In some embodiments, nn is 10. In some embodiments, TC PEG shielding improves or enhances the pharmacokinetic or therapeutic profile of the drug or payload. In another embodiment, the TLR agonist is any one selected from the group of structures of tables 3, 4, 5, 6, 7 according to the invention.
In another embodiment, a TLR agonist comprising a structure according to formula I or formula II further comprises a PEG mask.
In another embodiment, an anti-HER 2 antibody or antibody fragment comprises the amino acid sequence of any of SEQ ID NOs 1-18. In another embodiment, an anti-HER 2 antibody or antibody fragment comprises the amino acid sequences of at least two of SEQ ID NOs 1-18. In another embodiment, an anti-HER 2 antibody or antibody fragment comprises a) SEQ ID No. 1, 2, 3, 4, 16, 17 or 18; and b) any of SEQ ID NOs 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15. In another embodiment, an anti-HER 2 antibody or antibody fragment comprises a) the heavy chain of SEQ ID No. 1, 2, 3, 4, 16, 17 or 18; and b) the light chain of any one of SEQ ID NOs 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15. In another embodiment, an anti-HER 2 antibody or antibody fragment comprises a) SEQ ID No. 1; and b) any of SEQ ID NOs 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15. In another embodiment, an anti-HER 2 antibody or antibody fragment comprises a) SEQ ID NO. 2; and b) any of SEQ ID NOs 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15. In another embodiment, an anti-HER 2 antibody or antibody fragment comprises a) SEQ ID NO 3; and b) any of SEQ ID NOs 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15. In another embodiment, an anti-HER 2 antibody or antibody fragment comprises a) SEQ ID NO. 4; and b) any of SEQ ID NOs 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15. In another embodiment, an anti-HER 2 antibody or antibody fragment comprises a) SEQ ID NO. 16; and b) any of SEQ ID NOs 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15. In another embodiment, an anti-HER 2 antibody or antibody fragment comprises a) SEQ ID NO. 17; and b) any of SEQ ID NOs 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15. In another embodiment, an anti-HER 2 antibody or antibody fragment comprises a) SEQ ID NO. 18; and b) any of SEQ ID NOs 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15. In another embodiment, an anti-HER 2 antibody or antibody fragment comprises a mutation in the heavy chain disclosed in table 9A; and b) any of SEQ ID NOs 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15. In another embodiment, the anti-HER 2 antibody or antibody fragment comprises SEQ ID NO. 1 and SEQ ID NO. 5. In another embodiment, the anti-HER 2 antibody or antibody fragment comprises SEQ ID NO. 1 and SEQ ID NO. 6. In another embodiment, the anti-HER 2 antibody or antibody fragment comprises SEQ ID NO. 1 and SEQ ID NO. 7. In another embodiment, the anti-HER 2 antibody or antibody fragment comprises SEQ ID NO. 1 and SEQ ID NO. 8. In another embodiment, the anti-HER 2 antibody or antibody fragment comprises SEQ ID NO. 1 and SEQ ID NO. 9. In another embodiment, the anti-HER 2 antibody or antibody fragment comprises SEQ ID NO. 1 and SEQ ID NO. 10. In another embodiment, the anti-HER 2 antibody or antibody fragment comprises SEQ ID NO. 1 and SEQ ID NO. 11. In another embodiment, the anti-HER 2 antibody or antibody fragment comprises SEQ ID NO. 1 and SEQ ID NO. 12. In another embodiment, the anti-HER 2 antibody or antibody fragment comprises SEQ ID NO. 1 and SEQ ID NO. 13. In another embodiment, the anti-HER 2 antibody or antibody fragment comprises SEQ ID NO. 1 and SEQ ID NO. 14. In another embodiment, the anti-HER 2 antibody or antibody fragment comprises SEQ ID NO. 1 and SEQ ID NO. 15. In another embodiment, the anti-HER 2 antibody or antibody fragment comprises SEQ ID NO. 2 and SEQ ID NO. 5. In another embodiment, the anti-HER 2 antibody or antibody fragment comprises SEQ ID NO. 2 and SEQ ID NO. 6. In another embodiment, the anti-HER 2 antibody or antibody fragment comprises SEQ ID NO. 2 and SEQ ID NO. 7. In another embodiment, the anti-HER 2 antibody or antibody fragment comprises SEQ ID NO. 2 and SEQ ID NO. 8. In another embodiment, the anti-HER 2 antibody or antibody fragment comprises SEQ ID NO. 2 and SEQ ID NO. 9. In another embodiment, the anti-HER 2 antibody or antibody fragment comprises SEQ ID NO. 2 and SEQ ID NO. 10. In another embodiment, the anti-HER 2 antibody or antibody fragment comprises SEQ ID NO. 2 and SEQ ID NO. 11. In another embodiment, the anti-HER 2 antibody or antibody fragment comprises SEQ ID NO. 2 and SEQ ID NO. 12. In another embodiment, the anti-HER 2 antibody or antibody fragment comprises SEQ ID NO. 2 and SEQ ID NO. 13. In another embodiment, the anti-HER 2 antibody or antibody fragment comprises SEQ ID NO. 2 and SEQ ID NO. 14. In another embodiment, the anti-HER 2 antibody or antibody fragment comprises SEQ ID NO. 2 and SEQ ID NO. 15. In another embodiment, the anti-HER 2 antibody or antibody fragment comprises SEQ ID NO. 3 and SEQ ID NO. 5. In another embodiment, the anti-HER 2 antibody or antibody fragment comprises SEQ ID NO. 3 and SEQ ID NO. 6. In another embodiment, the anti-HER 2 antibody or antibody fragment comprises SEQ ID NO. 3 and SEQ ID NO. 7. In another embodiment, the anti-HER 2 antibody or antibody fragment comprises SEQ ID NO. 3 and SEQ ID NO. 8. In another embodiment, the anti-HER 2 antibody or antibody fragment comprises SEQ ID NO. 3 and SEQ ID NO. 9. In another embodiment, the anti-HER 2 antibody or antibody fragment comprises SEQ ID NO. 3 and SEQ ID NO. 10. In another embodiment, the anti-HER 2 antibody or antibody fragment comprises SEQ ID NO. 3 and SEQ ID NO. 11. In another embodiment, the anti-HER 2 antibody or antibody fragment comprises SEQ ID NO. 3 and SEQ ID NO. 12. In another embodiment, the anti-HER 2 antibody or antibody fragment comprises SEQ ID NO. 3 and SEQ ID NO. 13. In another embodiment, the anti-HER 2 antibody or antibody fragment comprises SEQ ID NO. 3 and SEQ ID NO. 14. In another embodiment, the anti-HER 2 antibody or antibody fragment comprises SEQ ID NO. 3 and SEQ ID NO. 15. In another embodiment, the anti-HER 2 antibody or antibody fragment comprises SEQ ID NO. 4 and SEQ ID NO. 5. In another embodiment, the anti-HER 2 antibody or antibody fragment comprises SEQ ID NO. 4 and SEQ ID NO. 6. In another embodiment, the anti-HER 2 antibody or antibody fragment comprises SEQ ID NO. 4 and SEQ ID NO. 7. In another embodiment, the anti-HER 2 antibody or antibody fragment comprises SEQ ID NO. 4 and SEQ ID NO. 8. In another embodiment, the anti-HER 2 antibody or antibody fragment comprises SEQ ID NO. 4 and SEQ ID NO. 9. In another embodiment, the anti-HER 2 antibody or antibody fragment comprises SEQ ID NO. 4 and SEQ ID NO. 10. In another embodiment, the anti-HER 2 antibody or antibody fragment comprises SEQ ID NO. 4 and SEQ ID NO. 11. In another embodiment, the anti-HER 2 antibody or antibody fragment comprises SEQ ID NO. 4 and SEQ ID NO. 12. In another embodiment, the anti-HER 2 antibody or antibody fragment comprises SEQ ID NO. 4 and SEQ ID NO. 13. In another embodiment, the anti-HER 2 antibody or antibody fragment comprises SEQ ID NO. 4 and SEQ ID NO. 14. In another embodiment, the anti-HER 2 antibody or antibody fragment comprises SEQ ID NO. 4 and SEQ ID NO. 15. In another embodiment, the invention provides an anti-HER 2 antibody or antibody fragment wherein the non-naturally encoded amino acid is site-specifically incorporated at position 114 according to Kabat numbering. In another embodiment, the invention provides an anti-HER 2 antibody or antibody fragment, wherein the antibody or antibody fragment comprises an Fc mutation according to table 9A.
In another embodiment, the invention provides a TLR agonist conjugate (TC) comprising an anti-HER 2 antibody or antibody fragment conjugated to a TLR agonist comprising a structure according to fig. 1, wherein the TLR agonist is conjugated to the antibody or antibody fragment by a linker covalently bound to one or more non-naturally encoded amino acids incorporated into the antibody or antibody fragment, the TC further comprising a chemotherapeutic or immunotherapeutic agent. In another embodiment, the invention provides a TLR agonist conjugate (TC) comprising an anti-HER 2 antibody or antibody fragment conjugated to a TLR agonist selected from any one of the compounds of tables 3-7, wherein the TLR agonist is conjugated to the antibody or antibody fragment by a linker covalently bonded to one or more non-naturally encoded amino acids incorporated into the antibody or antibody fragment. In another embodiment, the invention provides a TLR agonist conjugate (TC) comprising an anti-HER 2 antibody or antibody fragment conjugated to a TLR agonist of each of the compounds selected from tables 3-7, wherein the TLR agonist is conjugated to the antibody or antibody fragment by a linker covalently bonded to one or more non-naturally encoded amino acids incorporated into the antibody or antibody fragment, the TC further comprising a chemotherapeutic or immunotherapeutic agent.
In another embodiment, the invention provides a TLR agonist conjugate (TC) comprising an anti-HER 2 antibody or antibody fragment conjugated to a TLR agonist comprising a structure according to fig. 1, wherein the TLR agonist is conjugated to the antibody or antibody fragment by a linker covalently bound to one or more non-naturally encoded amino acids incorporated into the antibody or antibody fragment, the TC further comprising a drug conjugate. In other embodiments, the drug conjugate is an antibody drug conjugate. In another embodiment, the invention provides a TLR agonist conjugate (TC) comprising an anti-HER 2 antibody or antibody fragment conjugated to a TLR agonist selected from any one of the compounds of tables 3-7, wherein the TLR agonist is conjugated to the antibody or antibody fragment by a linker covalently bonded to one or more non-naturally encoded amino acids incorporated into the antibody or antibody fragment. In another embodiment, the invention provides a TLR agonist conjugate (TC) comprising an anti-HER 2 antibody or antibody fragment conjugated to a TLR agonist of each of the compounds selected from tables 3-7, wherein the TLR agonist is conjugated to the antibody or antibody fragment by a linker covalently bonded to one or more non-naturally encoded amino acids incorporated into the antibody or antibody fragment, the TC further comprising a drug conjugate. In other embodiments, the drug conjugate is an antibody drug conjugate. In other embodiments, TC further comprises a cytokine or cytotoxin.
In another embodiment, the invention provides a method of treating a subject or patient suffering from a cancer or disease or condition or indication or disorder, the method comprising administering to the subject or patient a therapeutically effective amount of a composition or TC of the invention. In certain embodiments, the tumor or cancer is a HER2 positive tumor or cancer. In certain embodiments, the tumor, cancer, indication, disease, disorder, or condition is a HER2 positive tumor, cancer, indication, disease, disorder, or condition. In certain embodiments, the tumor or cancer is selected from the group consisting of: colon cancer, ovarian cancer, breast cancer, melanoma, lung cancer, glioblastoma, prostate cancer, bladder cancer, cervical cancer, pancreatic cancer, renal cancer, esophageal cancer, vaginal cancer, gastric cancer, and leukemia.
In another embodiment, the invention provides a method of treating a subject or patient suffering from a cancer or disease or condition, the method comprising administering to the subject or patient a therapeutically effective amount of a composition or TC of the invention, the composition or TC of the invention further comprising a chemotherapeutic or immunotherapeutic agent. In certain embodiments, TC is co-administered with at least one chemotherapeutic agent. The chemotherapeutic agent may be selected from the group consisting of: temozolomide, gemcitabine, doxorubicin, cyclophosphamide, paclitaxel, cisplatin, fluoropyrimidine, taxane, anthracyclines, lapatinib, capecitabine, letrozole, pertuzumab, docetaxel, IFN- α.
In another embodiment, the invention provides a method of treating a subject or patient suffering from a cancer or disease or disorder, the method comprising administering to the subject or patient a therapeutically effective amount of a composition or TC of the invention, the composition or TC of the invention further comprising an antibody drug conjugate, a cytotoxic agent, or a checkpoint inhibitor.
In another embodiment, the invention provides a method of killing a cell comprising contacting the cell with a TC of the invention. In other embodiments, the cell is a tumor cell or a cancer cell. In certain embodiments, the tumor cell or cancer cell is a colon cancer cell, an ovarian cancer cell, a breast cancer cell, a melanoma cell, a lung cancer cell, a glioblastoma cell, a prostate cancer cell, a bladder cancer cell, a cervical cancer cell, a pancreatic cancer cell, a kidney cancer cell, an esophageal cancer cell, a vaginal cancer cell, a gastric cancer cell, or a leukemia cancer cell. In certain embodiments, the tumor or cancer is a HER2 positive tumor or cancer. In certain embodiments, the tumor, cancer, indication, disease, disorder or condition to be treated is a HER2 positive tumor, cancer, indication, disease, disorder or condition.
The invention provides a method of inhibiting or reducing the growth of a tumor or cancer comprising contacting the tumor with an effective amount of a TC of the invention to stimulate the immune system of a patient in the vicinity of the tumor. The invention provides a method of inhibiting or reducing the growth of a tumor or cancer comprising contacting the tumor with an effective amount of a pegylated TC, or a stable dimer or multimer of a TC of the invention. In one embodiment, TC is non-pegylated or mono-pegylated. In one embodiment, TC is a dimer ethylene glycol. In one embodiment, TC has more than one and/or different TLR agonist molecules attached thereto. Another embodiment of the invention provides a method of modulating an immune response to a tumor cell using a TC of the invention.
In some embodiments, the invention provides methods of treating cancer using TC. In some embodiments, TCs of the present invention may be used to treat or prevent cancer-related diseases, disorders, and conditions, including conditions directly or indirectly associated with cancer, e.g., angiogenesis and precancerous conditions, such as dysplasia. In some embodiments, the tumor is a liquid tumor or a solid tumor. In some embodiments, the condition to be treated is cancer. The cancer may be, but is not limited to, breast cancer, brain cancer, pancreatic cancer, skin cancer, lung cancer, liver cancer, gall bladder cancer, colon cancer, ovarian cancer, prostate cancer, uterine cancer, bone cancer, and blood cancer (leukemia) cancer or a cancer or disease or condition associated with any of these cancers. Cancer is a cancer that begins with epithelial cells, which are cells that cover the body surface, produce hormones, and make up glands. By way of non-limiting example, cancers include breast cancer, pancreatic cancer, lung cancer, colon cancer, colorectal cancer, rectal cancer, kidney cancer, bladder cancer, stomach cancer, prostate cancer, liver cancer, ovarian cancer, brain cancer, vaginal cancer, vulvar cancer, uterine cancer, oral cancer, penile cancer, testicular cancer, esophageal cancer, skin cancer, fallopian tube cancer, head and neck cancer, gastrointestinal stromal cancer, adenocarcinoma, skin or intraocular melanoma, anal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, urinary tract cancer, renal pelvis cancer, ureter cancer, endometrial cancer, cervical cancer, pituitary cancer, central Nervous System (CNS) tumors, primary CNS lymphomas, brain stem glioma, and spinal axis tumors. In some cases, the cancer is a skin cancer, such as basal cell carcinoma, squamous cell carcinoma, melanoma, non-melanoma, or actinic (solar) keratosis. In some embodiments, the invention also relates to a method of treating acute leukemia in a mammal, the method comprising administering to the mammal a therapeutically effective amount of TC of the invention. The invention also provides a method of inhibiting proliferation of acute leukemia blasts comprising administering to a mammal suffering from acute leukemia a therapeutically effective amount of TC of the invention.
In another embodiment, TCs disclosed herein can be used to modulate immune responses. Modulation of an immune response may include stimulating, activating, increasing, enhancing, or upregulating an immune response. Modulation of an immune response may include suppressing, inhibiting, preventing, reducing, or downregulating the immune response.
In another embodiment, the invention provides a pharmaceutical composition comprising a therapeutically effective amount of a composition of the invention or TC and a pharmaceutically acceptable carrier or excipient.
In another embodiment, the invention provides the use of the composition of the invention in the manufacture of a medicament.
In another embodiment, the invention provides an immunostimulatory antibody conjugate (ISAC) comprising a TLR agonist according to any of the formulae in figure 1. In another embodiment, the invention provides an immunostimulatory antibody conjugate (ISAC) comprising a TLR agonist of any of the compounds according to tables 3, 4, 5, 6, 7. In another embodiment, the invention provides a pegylated ISAC, wherein the TLR agonist comprises a compound selected from the group of compounds of tables 3, 4, 5, 6, 7, which also comprises a PEG mask. In other embodiments, the invention provides ISACs wherein the TLR agonist comprises a compound selected from the group of compounds of table 4. In another embodiment, the invention provides a pegylated ISAC, wherein the TLR agonist comprises a compound selected from the group consisting of: also included are PEG-masked compounds of Table 4.
In another embodiment, the present invention provides a salt of any one of the compounds having the structure of fig. 1. In another embodiment, the invention provides a salt of any one of the compounds of tables 3, 4, 5, 6, 7. In another embodiment, the invention provides a salt of any one of the compounds of table 4. In another embodiment, the invention provides a pharmaceutical composition or salt thereof of a composition, compound, and TC according to the disclosure. In other embodiments, the pharmaceutical composition or salt further comprises a pharmaceutically acceptable excipient.
Drawings
FIG. 1 depicts the general structure of a TLR agonist
Figure 2 depicts TLR7 activity of various TLR7 agonists with PEG molecules.
Figures 3A-3B depict TLR7 activity of various TLR7 agonists with linear PEG (figure 3A) and branched PEG (figure 3B).
FIG. 4 depicts an SKBR3-RAWBlue co-culture in vitro assay for HER2 ISAC.
Figure 5 depicts the effect of the Fc region on HER2 ISAC activity.
FIG. 6 depicts the effect of conjugation site on HER2 ISAC activity
Figures 7A-7C show the in vitro activity of the AXC-879 derivative (figure 7A), additional HER2 ISAC (figure 7B) and the AXC-879 derivative with branching modification (figure 7C) in a RAW-Blue co-culture assay.
Fig. 8A-8C show in vitro activity comparisons of AXC-879 derivatives with PEG shielding (fig. 8A) (fig. 8B) and other AXC-879 derivatives with PEG shielding (fig. 8C) in RAW-Blue co-culture assays.
FIGS. 9A-9B show in vitro activity comparisons of AXC-879 derivatives having a D-Lys block or an L-Lys block in RAW-Blue co-culture assays.
FIG. 10 shows a comparison of ADCC activity between HER2 mAb and HER2-AXC879 ISAC in a PBMC co-culture assay.
FIG. 11 shows a comparison of HLA-DR marker induction of myeloid cells between HER2 mAb and HER2-AXC879 ISAC in a PBMC co-culture assay.
FIG. 12 shows a comparison of CD86/DC-SIGN+ biscationic cell induction between HER2 mAb and HER2-AXC879 ISAC in a PBMC co-culture assay.
Figures 13A-13C show ADCC effects of HER2ISAC with prodrug design in (figure 13A) HER2 high SKBR3/PBMC co-culture assay, (figure 13B) HER2 low HCC1806/PBMC co-culture assay and (figure 13C) HER2 negative MDA-MB-468/PBMC co-culture assay.
Figures 14A-14B show cytokine induction of TNF- α by HER2-AXC879 in (figure 14A) HER2 high N87/PBMC co-culture assay and (figure 14B) HER2 negative MDA-MB-468/PBMC co-culture assay.
Figures 15A-15D show cytokine induction of ifnγ by HER2ISAC in HER2 high SKBR3/PBMC co-culture assay (figure 15A) and HER2 low HCC1806/PBMC co-culture assay (figure 15B); and cytokine induction of TNF- α by HER2ISAC in HER2 high SKBR3/PBMC co-culture assay (fig. 15C) and HER2 low HCC1806/PBMC co-culture assay (fig. 15D).
Figures 16A-16D show cytokine induction of ifnγ by HER2 ISACs with prodrug design in HER2 high SKBR3/PBMC co-culture assay (figure 16A), HER2 low HCC1806/PBMC co-culture assay (figure 16B), HER2 negative MDA-MB-468/PBMC co-culture assay (figure 16C) and PBMC (figure 16D).
Figures 17A-17D show cytokine induction of TNF- α by HER 2ISAC with prodrug design in HER2 high SKBR3/PBMC co-culture assay (figure 17A), HER2 low HCC1806/PBMC co-culture assay (figure 17B), HER2 negative MDA-MB-468/PBMC co-culture assay (figure 17C) and PBMC (figure 17D).
Fig. 18A-18C show AXC879 and other antibodies targeting different tumor antigens TROP-2 (fig. 18A), PSMA (fig. 18B) and CD70 (fig. 18C).
FIGS. 19A-19B show Trop2 expression levels on different cell lines (FIG. 19A) and Trop2-AXC879 expression levels on different tumor cell lines (FIG. 19B).
FIGS. 20A-20B show in vitro activity of additional Trop2 ISACs in Trop2 positive HCC1806 (FIG. 20A) and Trop2 negative HCC1395 (FIG. 20B) cell lines as determined by Raw-Blue co-culture.
FIGS. 21A-21B show TNF- α cytokine induction of Trop2ISAC in Trop2 positive SKBR3 (FIG. 21A) and Trop2 negative HCC1395 (FIG. 21B) cell lines as determined by PBMC co-culture.
Fig. 22A-22B show that Trop2-AXC879 shows enhanced ADCC effect compared to unconjugated Trop2 antibody in Trop2 positive BxPC-3 (fig. 22A) and shows non-specific killing in Trop2 negative HCC1395 (fig. 22A) in PBMC co-culture assay.
FIG. 23 shows the PK profile of HER2-AXC879 after a single dose in C57/B6 mice.
Figures 24A-24C show the in vivo efficacy of HER2-AXC879 in the MC38-hHER2 allograft model (figure 24A), and the individual tumor growth curves of HER2-AXC863 group (figure 24B) and HER2-AXC879 group (figure 24C).
Figure 25 shows the in vivo efficacy synergy between HER2-AXC879 and anti-PD-1 antibodies.
Fig. 26A-26B show PK profiles of HER2-AXC879 (fig. 26A) and HER2-AXC863 (fig. 26B) after repeated dosing in C57/B6 mice.
FIG. 27 shows dose titration of HER2-AXC879 in MC38-hHER2 allograft model.
Figure 28 shows in vivo efficacy comparisons of different HER2 ISACs in MC38-hHER2 allograft models.
Fig. 29A-29B show re-challenge of MC38-hHER2 tumor (fig. 29A) and MC38 parental tumor (fig. 29B) in ISAC treated tumor completely resolved mice and untreated mice.
Fig. 30A-30B show in vivo efficacy of Trop2-AXC879 (fig. 30A) and body weight change over time (fig. 30B) in JIMT-1 xenograft models.
Detailed Description
Disclosed herein are TCs comprising a targeting moiety such as an antibody and one or more TLR agonists. TLR agonists may also comprise one or more linkers. The TCs of the present invention may comprise TLR agonists linked to an unnatural amino acid in the targeting moiety. Also included are methods for preparing such TCs comprising unnatural amino acids incorporated into targeting moiety polypeptides.
In certain embodiments, a pharmaceutical composition comprising any of the described compounds and a pharmaceutically acceptable carrier, excipient, or binder is provided.
In other or alternative embodiments are methods for detecting the presence of a polypeptide in a patient, the method comprising administering a polypeptide comprising at least one heterocycle-containing unnatural amino acid, and the resulting heterocycle-containing unnatural amino acid polypeptide modulates the immunogenicity of the polypeptide relative to a homologous naturally-occurring amino acid polypeptide.
It is to be understood that the methods and compositions described herein are not limited to the particular methodologies, protocols, cell lines, constructs, and reagents described herein, and as such, may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the methods and compositions described herein which will be limited only by the appended claims.
As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention described herein belongs. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the invention described herein, the preferred methods, devices, and materials are now described.
All publications and patents mentioned herein are incorporated by reference in their entirety for the purpose of describing and disclosing, for example, the constructs and methods described in the publications, which might be used in connection with the invention described herein. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. No admission is made that the inventors described herein are entitled to antedate such disclosure by virtue of prior invention or for any other reason.
The term "aldol-based linkage" or "mixed aldol-based linkage" refers to the acid-catalyzed or base-catalyzed condensation of an enolate/enol of one carbonyl compound with another carbonyl compound, which may be the same or different, to produce the β -hydroxycarbonyl compound, aldol.
As used herein, the term "affinity tag" refers to a tag that binds, reversibly or irreversibly, to another molecule to modify, destroy, or form a compound with the molecule. For example, affinity tags include enzymes and their substrates, or antibodies and their antigens.
The terms "alkoxy", "alkylamino" and "alkylthio" (or thioalkoxy) are used in their conventional sense and refer to those alkyl groups attached to the molecule through an oxygen atom, amino group or sulfur atom, respectively.
The term "alkyl" by itself or as part of another molecule, unless otherwise indicated, means a straight or branched chain, or cyclic hydrocarbon group, or a combination thereof, which may be fully saturated, monounsaturated or polyunsaturated and may include divalent and polyvalent groups, having the indicated number of carbon atoms (i.e., C 1 -C 10 Meaning one to ten carbons). Examples of saturated hydrocarbon groups include, but are not limited to, groups such as: methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl) methyl, cyclopropylmethyl, homologs and isomers thereof (e.g., n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like). Unsaturated alkyl is alkyl having one or more double or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2- (butadienyl), 2, 4-pentadienyl, 3- (1, 4-pentadienyl), ethynyl, 1-propynyl and 3-propynyl, 3-butynyl and higher homologs and isomers. Unless otherwise indicated, the term "alkyl" is also intended to include those alkanes defined in more detail herein Radical derivatives such as "heteroalkyl", "haloalkyl" and "homoalkyl".
The term "alkylene" by itself or as part of another molecule means a divalent group derived from an alkane, e.g., (-CH) 2 -) n Wherein n may be from 1 to about 24. By way of example only, such groups include, but are not limited to, groups having 10 or fewer carbon atoms, such as structure-CH 2 CH 2 -and-CH 2 CH 2 CH 2 CH 2 -. "lower alkyl" or "lower alkylene" is a shorter chain alkyl or alkylene group, typically having eight or fewer carbon atoms. Unless otherwise indicated, the term "alkylene" is also intended to include those groups described herein as "heteroalkylene".
The term "amino acid" refers to naturally occurring amino acids and non-natural amino acids, as well as amino acid analogs and amino acid mimics that function in a manner similar to naturally occurring amino acids. Naturally encoded amino acids are the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine), pyrrolysine and selenocysteine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, by way of example only, an alpha-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group. Such analogs can have modified R groups (e.g., norleucine) or can have modified peptide backbones while still retaining the same basic chemical structure as a naturally occurring amino acid. Non-limiting examples of amino acid analogs include homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium.
Amino acids may be represented herein by their names, their commonly known three-letter symbols, or by the one-letter symbols recommended by the IUPAC-IUB biochemical nomenclature committee. Alternatively, nucleotides may be represented by their commonly accepted single-letter codes.
"amino terminal modification" refers to any molecule that can be attached to a terminal amino group. For example, such terminal amine groups may be at the ends of polymeric molecules, wherein such polymeric molecules include, but are not limited to, polypeptides, polynucleotides, and polysaccharides. Terminal modifying groups include, but are not limited to, various water-soluble polymers, peptides or proteins. By way of example only, the terminal modifying group includes polyethylene glycol or serum albumin. The terminal modifying groups can be used to modify the therapeutic properties of the polymer molecule, including but not limited to increasing the serum half-life of the peptide.
By "antibody" herein is meant a protein consisting essentially of one or more polypeptides encoded by all or part of an antibody gene. Immunoglobulin genes include, but are not limited to, kappa, lambda, alpha, gamma (IgG 1, igG2, igG3, and IgG 4), delta, epsilon, and mu constant region genes, as well as a wide variety of immunoglobulin variable region genes. Antibodies herein are intended to include full length antibodies and antibody fragments, and include antibodies that naturally occur in any organism or that are engineered (e.g., are variants).
By "antibody fragment" is meant any form of antibody other than the full length form. Antibody fragments herein include antibodies that are smaller components present in full length antibodies, as well as antibodies that have been engineered. Antibody fragments include, but are not limited to Fv, fc, fab and (Fab') 2, single chain Fv (scFv), diabodies, triabodies, tetrabodies, bifunctional hybrid antibodies, CDR1, CDR2, CDR3, combinations of CDRs, variable regions, framework regions, constant regions, heavy chains, light chains, and variable regions, as well as alternative scaffold non-antibody molecules, bispecific antibodies, and the like (Maynad & Georgiou,2000, annu. Rev. Biomed. Eng.2:339-76; hudson,1998, curr. Opin. Biotechnol.9:395-402). Another functional substructure is a single chain Fv (scFv) which consists of variable regions of the immunoglobulin heavy and light chains, covalently linked by a peptide linker (S-zHu et al, 1996,Cancer Research,56,3055-3061). These small (Mr 25,000) proteins generally retain specificity and affinity for antigens in a single polypeptide and can provide convenient building blocks for larger antigen-specific molecules. Unless specifically stated otherwise, the statement and claims using the term "antibodies" explicitly include "antibody fragments (antibody fragment/antibody fragments)".
As used herein, "antibody-drug conjugate" or "ADC" refers to an antibody molecule or fragment thereof that is covalently bound to one or more bioactive molecules. The bioactive molecule can be conjugated to the antibody through a linker, polymer, or other covalent bond.
As used herein, the term "aromatic" or "aryl" refers to a closed-loop structure having at least one ring with a conjugated pi electron system and including carbocyclic aryl and heterocyclic aryl (or "heteroaryl" or "heteroaromatic") groups. Carbocyclic or heterocyclic aromatic groups may contain 5 to 20 ring atoms. The term includes covalently linked monocyclic or fused ring polycyclic (i.e., sharing adjacent pairs of carbon atoms) groups. The aromatic group may be unsubstituted or substituted. Non-limiting examples of "aromatic" or "aryl" groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, anthracenyl, and phenanthrenyl. The substituents of each of the above mentioned aromatic and heteroaromatic ring systems are selected from the group of acceptable substituents described herein.
For brevity, the term "aromatic" or "aryl" when used in combination with other terms (including, but not limited to, aryloxy, arylthio, aralkyl) includes aryl and heteroaryl rings as defined above. Thus, the term "aralkyl" or "alkaryl" is intended to include those groups in which an aryl group is attached to an alkyl group (including, but not limited to, benzyl, phenethyl, pyridylmethyl, and the like), including those alkyl groups in which an alkyl carbon atom (including, but not limited to, methylene) has been replaced by a heteroatom, by way of example only, by an oxygen atom. Examples of such aryl groups include, but are not limited to, phenoxymethyl, 2-pyridyloxymethyl, 3- (1-naphthyloxy) propyl, and the like.
As used herein, the term "arylene" generally refers to a divalent aryl group. Non-limiting examples of "arylene" include phenylene, naphthylene, fluorenylene, azulenylene, anthrylene, phenanthrylene, pyrenylene, biphenylene, and terphenylene. Arylene also refers to a bicyclic or tricyclic carbocyclic ring in which one of the rings is aromatic and the other rings may be saturated, partially unsaturated, or aromatic, such as dihydronaphthylene, indenylene, indanylene, or tetrahydronaphthylene (tetrahydronaphthylene). In certain embodiments, arylene groups may be optionally substituted with one or more substituents.
As used herein, the term "heteroarylene" generally refers to a divalent monocyclic aryl or divalent polycyclic aryl radical containing at least one aromatic ring, wherein at least one aromatic ring contains one or more heteroatoms independently selected from O, S and N in the ring. Each ring of the heteroarylene group may contain one or two O atoms, one or two S atoms, and/or one or four N atoms, provided that the total number of heteroatoms in each ring is four or less, and each ring contains at least one carbon atom. In certain embodiments, the heteroarylene has 5 to 20, 5 to 15, or 5 to 10 ring atoms. Examples of monocyclic heteroarylene groups include, but are not limited to, furanylene, imidazolylene, isothiazolylene, isoxazolylene, oxadiazolylene, oxazolylene, pyrazinylene, pyrazolylene, pyridazinylene, pyridinyl, pyrimidinylene, pyrrolylene, thiadiazolylene, thiazolylene, thiophenylene, tetrazolylene, triazinylene, and triazolylene. Examples of bicyclic heteroarylenes include, but are not limited to, benzofuranylene, benzimidazole, benzisoxazole, benzopyranylene, benzothiadiazole, benzothiazolylene, benzothiophenylene, benzotriazole, benzoxazolylene, furanylpyridylene, imidazopyridine, imidazothiazole, indolizinylene, indolylene, indazolyl, isobenzofuranylene, isobenzothienyl, isoindolyl, isoquinolylene, isothiazolylene, naphthyridine, oxazolopyridinylene, phthalazinylene, pteridine, purinylene, pyridopyridinyl, pyrrolopyridinylene, quinolinylene, quinoxalinylene, quinazolinylene, thiadiazolopyrimidinyl, and thienopyridinyl. Examples of tricyclic heteroarylenes include, but are not limited to, acriylene, benzindolyene, carbazolylene, dibenzofuranylene, pyridyl, phenanthroline, phenanthridine, phenazinylene, phenothiazinylene, phenoxazinylene, and oxaanthrylene. In certain embodiments, heteroarylene may also be optionally substituted.
"bifunctional polymer", also referred to as a "bifunctional linker", refers to a polymer comprising two functional groups capable of specifically reacting with other moieties to form covalent bonds or non-covalent bonds. Such moieties may include, but are not limited to, pendant groups on natural or unnatural amino acids or peptides containing such natural or unnatural amino acids. The other moieties that can be attached to the bifunctional linker or the bifunctional polymer can be the same or different moieties. By way of example only, the bifunctional linker may have a functional group that reacts with a group on the first peptide and another functional group that reacts with a group on the second peptide, thereby forming a conjugate comprising the first peptide, the bifunctional linker, and the second peptide. Many procedures and linker molecules for attaching various compounds to peptides are known. See, for example, european patent application No. 188,256; U.S. patent nos. 4,671,958, 4,659,839, 4,414,148, 4,699,784;4,680,338; and 4,569,789, which are incorporated herein by reference in their entirety. "multifunctional polymer" also referred to as "multifunctional linker" refers to a polymer that contains two or more functional groups capable of reacting with other moieties. Such moieties may include, but are not limited to, natural or unnatural amino acids or pendant groups (including, but not limited to, pendant amino acid groups) on peptides containing such natural or unnatural amino acids to form covalent or noncovalent linkages. The difunctional or polyfunctional polymer may be of any desired length or molecular weight and may be selected to provide a particular desired spacing or conformation between one or more molecules attached to the compound and the molecule to which it is bound or the compound.
As used herein, the term "bioavailability" refers to the rate and extent at which a substance or active portion thereof is delivered from a pharmaceutical dosage form and becomes available at the site of action or in the systemic circulation. Increased bioavailability refers to increasing the rate and extent at which a substance or active portion thereof is delivered from a pharmaceutical dosage form and becomes available at the site of action or in the systemic circulation. For example, an increase in bioavailability may be expressed as an increase in the concentration of the substance or active portion thereof in the blood when compared to other substances or active portions. Methods for assessing increased bioavailability are known in the art and can be used to assess the bioavailability of any polypeptide.
As used herein, the term "bioactive molecule," "bioactive moiety" or "bioactive agent" means any substance that can affect any physical or biochemical characteristic of a biological system, pathway, molecule, or organism-related interaction, including but not limited to viruses, bacteria, phages, transposons, prions, insects, fungi, plants, animals, and humans. Specifically, as used herein, bioactive molecules include, but are not limited to, any substance intended to diagnose, cure, alleviate, treat, or prevent a disease in a human or other animal, or otherwise enhance physical and mental health of a human or animal. Examples of bioactive molecules include, but are not limited to, peptides, proteins, enzymes, small molecule drugs, hard drugs, soft drugs, prodrugs, carbohydrates, inorganic atoms or molecules, dyes, lipids, nucleosides, radionuclides, oligonucleotides, toxins, cells, viruses, liposomes, microparticles, and micelles. Classes of bioactive agents suitable for use in the methods and compositions described herein include, but are not limited to, drugs, prodrugs, radionuclides, imaging agents, polymers, antibiotics, fungicides, antiviral agents, anti-inflammatory agents, antitumor agents, cardiovascular agents, anxiolytic agents, hormones, growth factors, steroid agents, microbial toxins, and the like.
By "modulating biological activity" is meant increasing or decreasing the reactivity of a polypeptide, altering the selectivity of a polypeptide, increasing or decreasing the substrate selectivity of a polypeptide. Analysis of the modified biological activity can be performed by comparing the biological activity of the non-native polypeptide to the biological activity of the native polypeptide.
As used herein, the term "biological material" refers to materials of biological origin, including but not limited to materials obtained from bioreactors and/or by recombinant methods and techniques.
As used herein, the term "biophysical probe" refers to a probe that can detect or monitor a structural change in a molecule. Such molecules include, but are not limited to, proteins, and "biophysical probes" may be used to detect or monitor interactions of proteins with other macromolecules. Examples of biophysical probes include, but are not limited to, spin labels, fluorophores, and photoactivating groups.
As used herein, the term "biosynthetic" refers to any method that utilizes a translation system (cellular or non-cellular) that includes the use of at least one of the following components: polynucleotides, codons, tRNA and ribosomes. For example, unnatural amino acids can be "biosynthetically incorporated" into unnatural amino acid polypeptides using methods and techniques described in WO 2002/085923, which is incorporated herein by reference in its entirety. In addition, methods for selecting useful unnatural amino acids that can be "biosynthetically incorporated" into unnatural amino acid polypeptides are described in WO 2002/085923, which is incorporated herein by reference in its entirety.
As used herein, the term "biotin analogue" or also referred to as "biotin mimetic" is any molecule other than biotin that binds with high affinity to avidin and/or streptavidin.
As used herein, the term "carbonyl" refers to a group containing a moiety selected from the group consisting of-C (O) -, -S (O) 2-, and-C (S) -including, but not limited to, a group containing at least one ketone group, and/or at least one aldehyde group, and/or at least one ester group, and/or at least one carboxylic acid group, and/or at least one thioester group. Such carbonyl groups include ketones, aldehydes, carboxylic acids, esters and thioesters. In addition, such groups may be part of a linear, branched, or cyclic molecule.
The term "carboxy-terminal modifying group" refers to any molecule that can be attached to a terminal carboxy group. For example, such terminal carboxyl groups may be at the ends of polymeric molecules, wherein such polymeric molecules include, but are not limited to, polypeptides, polynucleotides, and polysaccharides. Terminal modifying groups include, but are not limited to, various water-soluble polymers, peptides or proteins. By way of example only, the terminal modifying group includes polyethylene glycol or serum albumin. The terminal modifying groups can be used to modify the therapeutic properties of the polymer molecule, including but not limited to increasing the serum half-life of the peptide.
As used herein, the term "chemically cleavable group" is also referred to as "chemically labile" and refers to a group that breaks or cleaves upon exposure to an acid, base, oxidizing agent, reducing agent, chemical initiator, or free radical initiator.
As used herein, "co-folding" refers to a refolding process, reaction or method using at least two molecules that interact with each other and result in the conversion of an unfolded or incorrectly folded molecule to a correctly folded molecule. By way of example only, a "co-fold" uses at least two polypeptides that interact with each other and result in the conversion of an unfolded or incorrectly folded polypeptide to a native, correctly folded polypeptide. Such polypeptides may contain natural amino acids and/or at least one unnatural amino acid.
As used herein, "conjugate" refers to a polypeptide that is linked (e.g., covalently linked) directly or through a linker to a compound or compound-linker described herein, e.g., a compound or salt according to any of the structures of fig. 1 or any of the structures of tables 3-7. "targeting moiety" refers to a structure that has selective affinity for a target molecule relative to other non-target molecules. The targeting moiety of the invention binds to a target molecule. The targeting moiety may include, for example, an antibody, peptide, ligand, receptor, or binding portion thereof. The target biomolecule may be a biological receptor or other structure of a cell, such as a tumor antigen. As used herein, the terms "conjugate of the invention," "targeting moiety conjugate," "targeting moiety-active molecule conjugate," or "TC" refer to a targeting polypeptide or portion, analog, or derivative thereof that binds to a target, or portion or analog thereof (including but not limited to TLR7 and/or TLR8 agonists), conjugated to a biologically active molecule that is present on a cell or subunit thereof. As used herein, the term "tumor targeting moiety conjugate," "tumor targeting moiety-bioactive molecule conjugate," or "BTC" refers to a tumor targeting polypeptide or portion, analog, or derivative thereof that binds to a target, or portion or analog thereof (including but not limited to TLR7 and/or TLR8 agonists), conjugated to a bioactive molecule that is present on a tumor cell or subunit thereof. Unless otherwise indicated, the terms "compounds of the invention" and "compositions of the invention" are used as alternatives to the term "conjugates of the invention".
The term "conservatively modified variants" applies to both natural and unnatural amino acids, as well as to both natural and unnatural nucleic acid sequences, and combinations thereof. With respect to particular nucleic acid sequences, "conservatively modified variants" refers to those natural and unnatural nucleic acids that encode identical or substantially identical natural and unnatural amino acid sequences, or where natural and unnatural nucleic acids do not encode natural and unnatural amino acid sequences as substantially identical sequences. For example, a large number of functionally identical nucleic acids encode any given protein due to the degeneracy of the genetic code. For example, codons GCA, GCC, GCG and GCU both encode the amino acid alanine. Thus, at each position where alanine is specified by a codon, the codon can be changed to any of the corresponding codons described without changing the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one type of conservatively modified variations. Thus, for example, each native or non-native nucleic acid sequence herein encoding a native or non-native polypeptide also describes each possible silent variation of the native or non-native nucleic acid. One of ordinary skill in the art will recognize that each codon in a natural or unnatural nucleic acid (except AUG, which is typically the only codon for methionine, and TGG, which is typically the only codon for tryptophan) can be modified to obtain a functionally identical molecule. Thus, each silent variation of a natural or unnatural nucleic acid which encodes a natural or unnatural polypeptide is implied in each described sequence.
With respect to amino acid sequences, individual substitutions, deletions or additions which alter, add or delete a single natural or unnatural amino acid or a small percentage of natural or unnatural amino acids in the encoded sequence in a nucleic acid, peptide, polypeptide or protein sequence are "conservatively modified variants" where the alteration results in the deletion of an amino acid, addition of an amino acid, or substitution of a natural or unnatural amino acid with a chemically similar amino acid. Conservative substitutions that provide functionally similar natural amino acids are well known in the art. Such conservatively modified variants are variants other than and not exclusive of polymorphic variants, interspecies homologs, and alleles of the methods and compositions described herein.
Conservative substitutions that provide functionally similar amino acids are known to those of ordinary skill in the art. The following eight groups each contain amino acids that may be conservative substitutions of each other: 1) Alanine (a), glycine (G); 2) Aspartic acid (D), glutamic acid (E); 3) Asparagine (N), glutamine (Q); 4) Arginine (R), lysine (K); 5) Isoleucine (I), leucine (L), methionine (M), valine (V); 6) Phenylalanine (F), tyrosine (Y), tryptophan (W); 7) Serine (S), threonine (T); and 8) cysteine (C), methionine (M). (see, e.g., cright on, proteins: structures and Molecular Properties (W HFreeman & Co.; version 2 (month 12 1993).
Unless otherwise indicated, the terms "cycloalkyl" and "heterocycloalkyl" by themselves or in combination with other terms, represent cyclic versions of "alkyl" and "heteroalkyl," respectively. Thus, cycloalkyl or heterocycloalkyl includes saturated, partially unsaturated, and fully unsaturated ring bonds. In addition, for heterocycloalkyl, the heteroatom may occupy the position where the heterocycle is attached to the remainder of the molecule. Heteroatoms may include, but are not limited to, oxygen, nitrogen, or sulfur. Examples of cycloalkyl groups include, but are not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkyl groups include, but are not limited to, 1- (1, 2,5, 6-tetrahydropyridinyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothiophen-2-yl, tetrahydrothiophen-3-yl, 1-piperazinyl, 2-piperazinyl, and the like. In addition, the term encompasses polycyclic structures including, but not limited to, bicyclic and tricyclic ring structures. Similarly, the term "heterocycloalkylene" by itself or as part of another molecule means a divalent group derived from a heterocycloalkyl group, and the term "cycloalkylene" by itself or as part of another molecule means a divalent group derived from a cycloalkyl group.
As used herein, the term "cyclodextrin" refers to a cyclic carbohydrate consisting of at least six to eight glucose molecules in a cyclic form. The outside of the ring contains a water-soluble group; at the center of the ring is a relatively nonpolar cavity that can hold small molecules.
As used herein, the term "cytotoxicity" refers to a compound that damages cells.
As used herein, "denaturing agent" refers to any compound or material that will cause the polymer to unfold reversibly. Merely by way of example, a "denaturing agent" may cause the reversible unfolding of proteins. The strength of the denaturant (denaturant) will be determined by the nature and concentration of the particular denaturant (denaturant). For example, denaturants (denaturants) include, but are not limited to, chaotropes, detergents, organic water miscible solvents, phospholipids, or combinations thereof. Non-limiting examples of chaotropic agents include, but are not limited to, urea, guanidine, and sodium thiocyanate. Non-limiting examples of detergents may include, but are not limited to, strong detergents such as sodium dodecyl sulfate or polyoxyethylene ether (e.g., tween or Triton detergents), sarkosyl, mild nonionic detergents (e.g., digitonin), mild cationic detergents such as N- [1- (2, 3-dioleyloxy) -propyl-N, N-trimethylammonium, mild ionic detergents such as sodium cholate or sodium deoxycholate, or zwitterionic detergents including, but not limited to, sulfobetaines (Zwittergent), 3- (3-cholamidopropyl) dimethylamino-1-propane sulfate (CHAPS), and 3- (3-cholamidopropyl) dimethylamino-2-hydroxy-1-propane sulfonate (CHAPSO). Non-limiting examples of organic water miscible solvents include, but are not limited to, acetonitrile, lower alkanols (especially C2-C4 alkanols such as ethanol or isopropanol) or lower alkanediols (C2-C4 alkanediols such as ethylene glycol) that may be used as denaturants. Non-limiting examples of phospholipids include, but are not limited to, naturally occurring phospholipids (such as phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and phosphatidylinositol) or synthetic phospholipid derivatives or variants (such as dihexanoyl phosphatidylcholine or diheptanoyl phosphatidylcholine).
As used herein, the term "diamine" refers to a group/molecule comprising at least two amine functional groups including, but not limited to, hydrazino, amidino, imino, 1-diamino, 1, 2-diamino, 1, 3-diamino, and 1, 4-diamino. In addition, such groups may be part of a linear, branched, or cyclic molecule.
As used herein, the term "detectable label" refers to a label that can be observed using analytical techniques including, but not limited to, fluorescence, chemiluminescence, electron spin resonance, ultraviolet/visible absorption spectroscopy, mass spectrometry, nuclear magnetic resonance, and electrochemical methods.
As used herein, the term "dicarbonyl" is intended to mean a compound containing a compound selected from the group consisting of-C (O) -, -S (O) 2 -and-C (S) -groups of at least two moieties of the group consisting of, but not limited to, 1, 2-dicarbonyl, 1, 3-dicarbonyl and 1, 4-dicarbonyl, and groups containing at least one ketone group, and/or at least one aldehyde group, and/or at least one ester group, and/or at least one carboxylic acid group, and/or at least one thioester group. Such dicarbonyl groups include diketones, ketoaldehydes, ketoacids, ketoesters and ketothioesters. In addition, such groups may be part of a linear, branched, or cyclic molecule. The two moieties in the dicarbonyl may be the same or different and may include substituents that would produce an ester, ketone, aldehyde, thioester, or amide at either of the two moieties, just to name a few.
As used herein, the term "drug" refers to any substance used to prevent, diagnose, alleviate, treat, or cure a disease or condition.
As used herein, the term "effective amount" refers to a sufficient amount of an agent or compound that is administered that will alleviate to some extent one or more symptoms of the disease or disorder being treated. The result may be a reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. For example, the agent or compound administered includes, but is not limited to, a natural amino acid polypeptide, a non-natural amino acid polypeptide, a modified natural amino acid polypeptide, or a modified non-amino acid polypeptide. Compositions containing such natural amino acid polypeptides, unnatural amino acid polypeptides, modified natural amino acid polypeptides, or modified unnatural amino acid polypeptides can be administered for prophylactic, therapeutic, enhancing, and/or therapeutic treatment. Techniques such as dose escalation studies may be used to determine the appropriate "effective" amount in any individual case.
The term "enhancement" refers to increasing or prolonging the effectiveness or duration of a desired effect. For example, an "enhancing" the effect of a therapeutic agent refers to the ability to increase or prolong the effect of the therapeutic agent in efficacy or duration during treatment of a disease, disorder, or condition. As used herein, "an effective enhancing amount" refers to an amount sufficient to enhance the effect of a therapeutic agent in treating a disease, disorder, or condition. When used in a patient, the amount effective for such use will depend on the severity and course of the disease, disorder or condition, previous therapy, the patient's health condition and response to the drug, and the diagnosis of the attending physician.
As used herein, the term "eukaryote" refers to organisms belonging to the phylogenetic domain of eukaryotes, including but not limited to animals (including but not limited to mammals, insects, reptiles, birds, etc.), ciliates, plants (including but not limited to monocots, dicots, and algae), fungi, yeasts, flagellates, microsporidians, and protists.
As used herein, the term "fatty acid" refers to a carboxylic acid having a hydrocarbon side chain of about C6 or longer.
As used herein, the term "fluorophore" refers to a molecule that emits a photon upon excitation and thus fluoresces.
As used herein, the terms "functional group", "active moiety", "activating group", "leaving group", "reaction site", "chemically reactive group" and "chemically reactive moiety" refer to a molecular moiety or unit that undergoes a chemical reaction. The terms are somewhat synonymous in the chemical arts and are used herein to refer to the portion of a molecule that performs some function or activity and is reactive with other molecules.
The term "halogen" includes fluorine, chlorine, iodine and bromine.
As used herein, the term "haloacyl" refers to acyl groups containing a halogen moiety, including but not limited to-C (O) CH 3 、-C(O)CF 3 、-C(O)CH 2 OCH 3 Etc.
As used herein, the term "haloalkyl" refers to an alkyl group containing a halogen moiety, including but not limited to-CF 3 and-CH 2 CF 3 Etc.
As used herein, the term "heteroalkyl" refers to a straight or branched or cyclic hydrocarbon group or combination thereof, consisting of an alkyl group and at least one heteroatom selected from the group consisting of O, N, si and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. Heteroatoms O, N and S and Si may be placed at any internal position of the heteroalkyl group or at the position where the alkyl group is attached to the remainder of the molecule. Examples include, but are not limited to, -CH 2 -CH 2 -O-CH 3 、-CH 2 -CH 2 -NH-CH 3 、-CH 2 -CH 2 -N(CH 3 )-CH 3 、-CH 2 -S-CH 2 -CH 3 、-CH 2 -CH 2 ,-S(O)-CH 3 、-CH 2 -CH 2 -S(O) 2 -CH 3 、-CH＝CH-O-CH 3 、-Si(CH 3 ) 3 、-CH 2 -CH＝N-OCH 3 and-ch=ch-N (CH 3 )-CH 3 . In addition, up to two heteroatoms may be contiguous, such as, for example, -CH 2 -NH-OCH 3 and-CH 2 -O-Si(CH 3 ) 3 。
The term "heterocycle-based linkage" or "heterocycle linkage" refers to a moiety formed by the reaction of a dicarbonyl group with a diamine group. The resulting reaction product is a heterocycle, including heteroaryl or heterocycloalkyl. The resulting heterocyclic group serves as a chemical linkage between the unnatural amino acid or unnatural amino acid polypeptide and another functional group. In one embodiment, the heterocyclic linkages include nitrogen-containing heterocyclic linkages, including by way of example only pyrazole linkages, pyrrole linkages, indole linkages, benzodiazepine Zhuo Jianlian, and pyrazolone linkages.
Similarly, the term "heteroalkylene" refers to a divalent group derived from a heteroalkyl group, such as, but not limited to, -CH 2 -CH 2 -S-CH 2 -CH 2 -and-CH 2 -S-CH 2 -CH 2 -NH-CH 2 -. For heteroalkylene groups, the same or different heteroatoms may also occupy one or both chain ends (including, but not limited to, alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, aminooxyalkylene, and the like). Still further, for alkylene and heteroalkylene linking groups, the direction of the formula written without passing through the linking group implies the orientation of the linking group. For example, formula-C (O) 2 R' -represents-C (O) 2 R '-and-R' C (O) 2 -。
As used herein, the term "heteroaryl" or "heteroaromatic" refers to an aryl group containing at least one heteroatom selected from N, O and S; wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen atoms may optionally be quaternized. Heteroaryl groups may be substituted or unsubstituted. Heteroaryl groups may be attached to the remainder of the molecule through heteroatoms. Non-limiting examples of heteroaryl groups include 1-pyrrolyl, 2-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolinyl and 6-quinolinyl.
As used herein, the term "homoalkyl" refers to an alkyl group that is a hydrocarbyl group.
As used herein, the term "identical" refers to two or more sequences or subsequences that are the same. In addition, as used herein, the term "substantially identical" refers to two or more sequences having a percentage of identical sequence units (sequential units) when compared and aligned for maximum correspondence over a comparison window or designated area, as measured using a comparison algorithm or by manual alignment and visual inspection. For example only, two or more sequences may be "substantially identical" if the sequence units are about 60% identical, about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, or about 95% identical over a designated region. Such percentages are used to describe "percent identity" of two or more sequences. Sequence identity may be present over a region of at least about 75-100 sequence units in length, over a region of about 50 sequence units in length, or, in the unspecified case, over the entire sequence. The definition also refers to the complement of the test sequence. By way of example only, two or more polypeptide sequences are identical when the amino acid residues are identical, and two or more polypeptide sequences are "substantially identical" if the amino acid residues are about 60% identical, about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, or about 95% identical over the designated region. Identity may be present over a region of at least about 75 to about 100 nucleic acids in length, over a region of about 50 nucleic acids in length, or, where unspecified, over the entire sequence of the polypeptide sequence. In addition, by way of example only, two or more polynucleotide sequences are identical when the nucleic acid residues are identical, and two or more polynucleotide sequences are "substantially identical" if the nucleic acid residues are about 60% identical, about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, or about 95% identical over a designated region. Identity may be present over a region of at least about 75 to about 100 nucleic acids in length, over a region of about 50 nucleic acids in length, or, where unspecified, over the entire sequence of the polynucleotide sequence.
For sequence comparison, one sequence is typically used as a reference sequence, which is compared to the test sequence. When using a sequence comparison algorithm, the test sequence and reference sequence are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters may be used or alternative parameters may be specified. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the program parameters.
As used herein, the term "immunogenicity" refers to the response of an antibody to administration of a therapeutic agent. Immunogenicity of therapeutic unnatural amino acid polypeptides can be obtained using quantitative and qualitative assays for detecting antibodies to unnatural amino acid polypeptides in biological fluids. Such assays include, but are not limited to, radioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELISA), light-emitting immunoassays (LIA), and Fluorescent Immunoassays (FIA). An immunogenicity assay for a therapeutic unnatural amino acid polypeptide involves comparing the antibody response after administration of the therapeutic unnatural amino acid polypeptide with the antibody response after administration of the therapeutic natural amino acid polypeptide.
As used herein, the term "isolated" refers to the separation and removal of a target component from a non-target component. The isolated material may be in a dry or semi-dry state, or in a solution state, including but not limited to an aqueous solution. The isolated component may be in a homogeneous state or the isolated component may be part of a pharmaceutical composition comprising additional pharmaceutically acceptable carriers and/or excipients. Analytical chemistry techniques including, but not limited to, polyacrylamide gel electrophoresis or high performance liquid chromatography can be used to determine purity and uniformity. In addition, a component of interest is described herein as substantially purified when the component is isolated and is the predominant species present in the formulation. As used herein, the term "purified" may refer to a target component that is at least 85% pure, at least 90% pure, at least 95% pure, at least 99% pure, or higher. By way of example only, a nucleic acid or protein is "isolated" when such nucleic acid or protein is free of at least some of the cellular components with which it is associated in nature, or the nucleic acid or protein has been concentrated to a level above its in vivo or in vitro production concentration. Further, for example, a gene is isolated when separated from an open reading frame flanking the gene and encoding a protein other than the gene of interest.
As used herein, the term "label" refers to a substance that is incorporated into a compound and is readily detected, whereby its physical distribution can be detected and/or monitored.
As used herein, the term "linkage" or "linker" refers to a bond or chemical moiety formed by a chemical reaction between a functional group of the linker and another molecule. Such linkages may include, but are not limited to, covalent and non-covalent linkages, and such chemical moieties may include, but are not limited to, esters, carbonates, imine phosphates, hydrazones, acetals, orthoesters, peptide linkages, and oligonucleotide linkages. Hydrolytically stable linkages means that the linkages are substantially stable in water and do not react with water at suitable pH values, including but not limited to under physiological conditions for extended periods of time, and possibly even indefinitely. Hydrolytically unstable or degradable linkages means that the linkages are degradable in water or aqueous solutions (including, for example, blood). Enzymatically labile or degradable linkages means that the linkages are degradable by one or more enzymes. By way of example only, PEG and related polymers may include degradable linkages in the polymer backbone or in the linker groups between the polymer backbone and one or more terminal functional groups of the polymer molecule. Such degradable linkages include, but are not limited to, ester linkages formed from the reaction of PEG carboxylic acid or activated PEG carboxylic acid with an alcohol group on a bioactive agent, wherein such ester groups are typically hydrolyzed under physiological conditions to release the bioactive agent. Other hydrolytically degradable linkages include, but are not limited to, carbonate linkages; imine linkages resulting from the reaction of an amine and an aldehyde; phosphate linkages formed by the reaction of an alcohol with a phosphate group; hydrazone linkage of the reaction product of hydrazide and aldehyde; acetal linkage of the reaction product of aldehyde and alcohol; orthoester linkage of the reaction product of formate and alcohol; peptide linkages formed from amine groups and carboxyl groups of peptides, including but not limited to, at the ends of polymers such as PEG; and linkages from phosphoramidite groups and 5' hydroxyl groups of oligonucleotides, including but not limited to oligonucleotide linkages formed at the ends of the polymer. Linkers include, but are not limited to, short linear, branched, multi-arm, or dendrimers, such as polymers. In some embodiments of the invention, the linker may be branched. In other embodiments, the linker may be a bifunctional linker. In some embodiments, the linker may be a trifunctional linker. Many different cleavable linkers are known to those of skill in the art. See U.S. patent No. 4,618,492;4,542,225 and 4,625,014. Mechanisms for releasing agents from these linker groups include, for example, irradiation of photolabile bonds and acid-catalyzed hydrolysis. U.S. patent No. 4,671,958, for example, includes a description of an immunoconjugate comprising a linker that is cleaved in vivo by proteolytic enzymes of the patient's complement system. The length of the linker may be predetermined or selected according to the desired spatial relationship between the polypeptide and the molecule to which it is attached. In view of the numerous methods reported for linking various radiodiagnostic compounds, radiotherapeutic compounds, drugs, toxins and other agents to antibodies, one skilled in the art will be able to determine the appropriate method for linking a given agent or molecule to a polypeptide.
As used herein, the term "modified" refers to the presence of an alteration to a natural amino acid, a non-natural amino acid, a natural amino acid polypeptide, or a non-natural amino acid polypeptide. Such alterations or modifications may be obtained by post-synthesis modification of natural amino acids, unnatural amino acids, natural amino acid polypeptides or unnatural amino acid polypeptides, or by co-translation, or by post-translational modification of natural amino acids, unnatural amino acids, natural amino acid polypeptides or unnatural amino acid polypeptides. The form "modified or unmodified" means that the natural amino acid, unnatural amino acid, natural amino acid polypeptide or unnatural amino acid polypeptide in question is optionally modified, i.e. the natural amino acid, unnatural amino acid, natural amino acid polypeptide or unnatural amino acid polypeptide in question can be modified or unmodified.
As used herein, the term "modulated serum half-life" refers to a positive or negative change in the circulatory half-life of a modified biologically active molecule relative to its unmodified form. For example, modified bioactive molecules include, but are not limited to, natural amino acids, unnatural amino acids, natural amino acid polypeptides, or unnatural amino acid polypeptides. For example, serum half-life is measured by taking blood samples at different time points after administration of a bioactive molecule or modified bioactive molecule and determining the concentration of the molecule in each sample. The correlation of serum concentration with time allows calculation of serum half-life. For example, the modulated serum half-life may be an increase in serum half-life, which may improve dosing regimens or avoid toxic effects. Such serum increases may be at least about two times, at least about three times, at least about five times, or at least about ten times. Methods for assessing increased serum half-life of a polypeptide can be performed.
As used herein, the term "modulated therapeutic half-life" refers to a positive or negative change in the half-life of a therapeutically effective amount of a modified biologically active molecule relative to its unmodified form. For example, modified bioactive molecules include, but are not limited to, natural amino acids, unnatural amino acids, natural amino acid polypeptides, or unnatural amino acid polypeptides. For example, the therapeutic half-life is measured by measuring the pharmacokinetic and/or pharmacodynamic properties of the molecule at different time points after administration. The increased therapeutic half-life may allow for a particularly beneficial dosing regimen, a particularly beneficial total dose, or avoid undesirable effects. For example, the increased therapeutic half-life may be due to increased potency, increased or decreased binding of the modified molecule to its target, increased or decreased another parameter or mechanism of action of the unmodified molecule, or increased or decreased decomposition of the molecule by an enzyme (e.g., protease). Methods for assessing the increase in therapeutic half-life of any polypeptide are well known to the skilled artisan.
"unnatural amino acid" refers to an amino acid that is not one of the 20 common amino acids or pyrrolysine or selenocysteine. Other terms that may be used synonymously with the term "unnatural amino acid" are "unnatural encoded amino acid", "unnatural amino acid", "non-naturally occurring amino acid", as well as various hyphenated and non-hyphenated versions thereof. The term "unnatural amino acid" includes, but is not limited to, an amino acid that occurs naturally by modification of naturally encoded amino acids (including but not limited to 20 common amino acids or pyrrolysine and selenocysteine), but does not itself incorporate itself into an ever-growing polypeptide chain through a translation complex. Examples of such amino acids include, but are not limited to, N-acetylglucosamine-L-serine, N-acetylglucosamine-L-threonine, and O-phosphotyrosine. In addition, the term "unnatural amino acid" includes, but is not limited to, amino acids that do not occur naturally and that are obtainable synthetically or that are obtainable by modification of unnatural amino acids. In some embodiments, the unnatural amino acid comprises a lysine analog, e.g., N6-azidoethoxy-L-lysine (AzK), N6-propargylethoxy-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO-lysine, methyltetrazine lysine, or allyloxycarbonyl lysine. In some embodiments, the unnatural amino acid comprises a sugar moiety. Examples of such amino acids include N-acetyl-L-glucosamine-L-serine, N-acetyl-L-galactosamine-L-serine, N-acetyl-L-glucosamine-L-threonine, N-acetyl-L-glucosamine-L-asparagine, and O-mannosamine-L-serine. Examples of such amino acids also include examples in which naturally occurring N-linkages or O-linkages between amino acids and sugars are replaced by covalent bonds that are unusual in nature, including but not limited to olefins, oximes, thioethers, amides, and the like. Examples of such amino acids also include sugars that are not common in naturally occurring proteins, such as 2-deoxy-glucose, 2-deoxy galactose, and the like. Specific examples of unnatural amino acids include, but are not limited to, p-acetyl-L-phenylalanine, p-propargyloxyphenyl alanine, O-methyl-L-tyrosine, L-3- (2-naphthyl) alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcβ -serine, L-Dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-iodophenylalanine, p-bromophenyl alanine, p-amino-L-phenylalanine, p-propargyloxy-L-phenylalanine, 4-azido-L-phenylalanine, p-azidoethoxyphenylalanine, p-azidomethyl-phenylalanine, and the like. In some embodiments, the unnatural amino acid is selected from the group consisting of: p-acetyl-phenylalanine, 4-azido-L-phenylalanine, p-azidoethoxyphenylalanine or p-azidomethyl-phenylalanine.
As used herein, the term "nucleic acid" refers to deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides and polymers thereof in either single-or double-stranded form. By way of example only, such nucleic acids and nucleic acid polymers include, but are not limited to: (i) Analogs of natural nucleotides that have similar binding properties to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides; (ii) Oligonucleotide analogs, including but not limited to PNA (peptide nucleic acids), DNA analogs used in antisense technology (phosphorothioates, phosphoramidates, etc.); (iii) Conservatively modified variants thereof (including but not limited to degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. For example, degenerate codon substitutions may be achieved by generating sequences in which a third position of one or more selected (or all) codons is substituted with mixed bases and/or deoxyinosine residues (Batzer et al, nucleic Acid Res.19:5081 (1991); ohtsuka et al, J.biol. Chem.260:2605-2608 (1985); and Rossolini et al, mol. Cell. Probes 8:91-98 (1994)).
As used herein, the term "oxidizing agent" refers to a compound or material that is capable of removing electrons from an oxidized compound. For example, oxidizing agents include, but are not limited to, oxidized glutathione, cystine, cystamine, oxidized dithiothreitol, oxidized erythritol, and oxygen. A variety of oxidizing agents are suitable for use in the methods and compositions described herein.
As used herein, the term "pharmaceutically acceptable" refers to materials (including but not limited to salts, carriers, or diluents) that do not abrogate the biological activity or properties of the compound, and that are relatively non-toxic, i.e., that can be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which they are contained.
As used herein, the term "polyalkylene glycol" or "poly (alkylene glycol)" refers to a straight or branched chain polymeric polyether polyol. Such polyalkylene glycols include, but are not limited to, polyethylene glycol, polypropylene glycol, polytetramethylene glycol, and derivatives thereof. Other exemplary embodiments are listed, for example, in the commercial supplier catalog, such as catalog "Polyethylene Glycol and Derivatives for Biomedical Applications" of Shearwater Corporation (2001). By way of example only, such polymeric polyether polyols have average molecular weights between about 0.1kDa and about 100 kDa. For example, such polymeric polyether polyols include, but are not limited to, between about 100Da and about 100,000Da or greater. The molecular weight of the polymer may be between about 100 and about 100,000Da, including but not limited to about 100,000Da, about 95,000Da, about 90,000Da, about 85,000Da, about 80,000Da, about 75,000Da, about 70,000Da, about 65,000Da, about 60,000Da, about 55,000Da, about 50,000Da, about 45,000Da, about 40,000Da, about 35,000Da, about 30,000Da, about 25,000Da, about 20,000Da, about 15,000Da, about 10,000Da, about 9,000Da, about 8,000Da, about 7,000Da, about 6,000Da, about 5,000Da, about 4,000Da, about 3,000Da, about 2,000Da, about 1,000Da, about 900Da, about 800Da, about 700Da, about 600Da, about 500Da,400Da, about 300Da, about 200Da, and about 100Da. In some embodiments, the molecular weight of the polymer is between about 100Da and about 50,000 Da. In some embodiments, the molecular weight of the polymer is between about 100Da and about 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 1,000da and about 40,000 da. In some embodiments, the molecular weight of the polymer is between about 2,000da to about 50,000 da. In some embodiments, the molecular weight of the polymer is between about 5,000da and about 40,000 da. In some embodiments, the molecular weight of the polymer is between about 10,000da and about 40,000 da. In some embodiments, the poly (ethylene glycol) molecule is a branched polymer. The molecular weight of the branched PEG may be between about 1,000da and about 100,000da, including but not limited to about 100,000da, about 95,000da, about 90,000da, about 85,000da, about 80,000da, about 75,000da, about 70,000da, about 65,000da, about 60,000da, about 55,000da, about 50,000da, about 45,000da, about 40,000da, about 35,000da, about 30,000da, about 25,000da, about 20,000da, about 15,000da, about 10,000da, about 9,000da, about 8,000da, about 7,000da, about 6,000da, about 5,000da, about 4,000da, about 3,000da, about 2,000da, and about 1,000da. In some embodiments, the branched PEG has a molecular weight between about 1,000da and about 50,000 da. In some embodiments, the branched PEG has a molecular weight between about 1,000da and about 40,000 da. In some embodiments, the branched PEG has a molecular weight between about 5,000da and about 40,000 da. In some embodiments, the branched PEG has a molecular weight between about 5,000da and about 20,000 da. In other embodiments, the branched PEG has a molecular weight between about 2,000da to about 50,000 da.
As used herein, the term "polymer" refers to a molecule consisting of repeating subunits. Such molecules include, but are not limited to, polypeptides, polynucleotides or polysaccharides or polyalkylene glycols.
The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. That is, the description for polypeptides applies equally to the description for peptides and the description for proteins, and vice versa. The term applies to naturally occurring amino acid polymers as well as to amino acid polymers in which one or more amino acid residues are unnatural amino acids. In addition, such "polypeptides", "peptides" and "proteins" include amino acid chains of any length, including full length proteins, in which the amino acid residues are linked by covalent peptide bonds.
The term "post-translational modification" refers to any modification of a natural or unnatural amino acid that occurs after translational incorporation into a polypeptide chain. Such modifications include, but are not limited to, co-translational in vivo modifications, co-translational in vitro modifications (such as in a cell-free translation system), post-translational in vivo modifications, and post-translational in vitro modifications.
As used herein, the term "prodrug" or "pharmaceutically acceptable prodrug" refers to an agent that is converted to the parent drug in vivo or in vitro, wherein the agent does not abrogate the biological activity or properties of the drug, and is relatively non-toxic, i.e., the material can be administered to an individual without causing an undesirable biological effect or interacting in a deleterious manner with any of the components of the composition in which it is contained. Prodrugs are typically prodrugs that, after administration to a subject and subsequent absorption, are converted to the active or more active substance by some process, such as by conversion of the metabolic pathway. Some prodrugs have chemical groups present on the prodrug that reduce their activity and/or impart solubility or some other property to the drug. Once the chemical groups are cleaved and/or modified from the prodrug, the active agent is produced. The prodrug is converted to the active drug in vivo by an enzymatic or non-enzymatic reaction. Prodrugs can provide improved physicochemical properties such as better solubility, enhanced delivery properties such as specific targeting to specific cells, tissues, organs or ligands, and improved therapeutic value of the drug. Benefits of such prodrugs include, but are not limited to: (i) easy administration compared to the parent drug; (ii) Prodrugs can be made bioavailable by oral administration, whereas the parent drug is not; (iii) The solubility of the prodrug in the pharmaceutical composition is also improved compared to the parent drug. Prodrugs include pharmacologically inactive or reduced activity derivatives of active agents. Prodrugs can be designed to modulate the amount of the drug or bioactive molecule that reaches the desired site of action by manipulating properties of the drug, such as physiochemical, biopharmaceutical or pharmacokinetic properties. Non-limiting examples of prodrugs are non-natural amino acid polypeptides which are administered in the form of esters ("prodrugs") to facilitate transport across the cell membrane (where water solubility is detrimental to migration), but once inside the cell (where water solubility is beneficial), are then metabolically hydrolyzed to carboxylic acids (active entities). Prodrugs can be designed as reversible drug derivatives to act as modifiers to enhance drug transport to site-specific tissues.
As used herein, the term "prophylactically effective amount" refers to an amount of a composition containing at least one unnatural amino acid polypeptide or at least one modified unnatural amino acid polypeptide that is applied prophylactically to a patient, which will alleviate one or more symptoms of the disease, disorder, or condition being treated to some extent. In such control applications, such amounts may depend on the health condition, weight, etc. of the patient. Determination of such therapeutically effective amounts by routine experimentation (including, but not limited to, dose escalation clinical trials) is considered well within the skill of the art.
As used herein, the term "protected" refers to the presence of a "protecting group" or moiety that prevents a chemically reactive functional group from reacting under certain reaction conditions. The protecting group will vary depending on the type of chemically reactive group being protected. By way of example only, (i) if the chemically reactive group is an amine or a hydrazide, the protecting group may be selected from t-butoxycarbonyl (t-Boc) and 9-fluorenylmethoxycarbonyl (Fmoc); (ii) If the chemically reactive group is a thiol, the protecting group may be an ortho-pyridyl disulfide; (iii) If the chemically reactive group is a carboxylic acid, such as butyric acid or propionic acid, or a hydroxyl group, the protecting group may be benzyl or alkyl, such as methyl, ethyl or t-butyl.
By way of example only, the blocking/protecting group may be selected from:
in addition, protecting groups include, but are not limited to, including photolabile groups such as Nvoc and MeNvoc, as well as other protecting groups known in the art. Other protecting groups are described in Greene and Wuts, protective Groups in Organic Synthesis, 3 rd edition, john Wiley & Sons, new York, NY,1999, which is incorporated herein by reference in its entirety.
The term "recombinant host cell" also referred to as a "host cell" refers to a cell comprising an exogenous polynucleotide, wherein the method for inserting the exogenous polynucleotide into the cell includes, but is not limited to, direct uptake, transduction, f-mating, or other methods known in the art for producing a recombinant host cell. By way of example only, such exogenous polynucleotides may be non-integrating vectors, including but not limited to plasmids, or may be integrated into the host genome.
As used herein, the term "redox active agent" refers to a molecule that oxidizes or reduces another molecule, whereby the redox active agent is reduced or oxidized. Examples of redox activators include, but are not limited to, ferrocene, quinone, ru 2+/3+ Complex, co 2+/3+ Complexes and Os 2+/3+ A complex.
As used herein, the term "reducing agent" refers to a compound or material that is capable of adding electrons to a reduced compound. For example, reducing agents include, but are not limited to, dithiothreitol (DTT), 2-mercaptoethanol, dithioerythritol, cysteine, cysteamine (2-aminoethanethiol), and reduced glutathione. By way of example only, such reducing agents may be used to maintain sulfhydryl groups in a reduced state and reduce intramolecular or intermolecular disulfide bonds.
As used herein, "refolding" describes any process, reaction, or method of converting an incorrectly folded or unfolded state into a native or correctly folded conformation. Merely by way of example, refolding converts a disulfide bond containing polypeptide from an incorrectly folded or unfolded state to a native or correctly folded conformation relative to the disulfide bond. Such disulfide bond containing polypeptides may be natural amino acid polypeptides or non-natural amino acid polypeptides.
As used herein, the term "safety" or "safety profile" refers to side effects that may be associated with drug administration relative to the number of drug administrations. For example, drugs that have been administered multiple times and that have only minor or no side effects are said to have an excellent safety profile. Methods for assessing the safety profile of any polypeptide are known in the art.
As used herein, the phrase "selectively hybridizes" or "specifically hybridizes" refers to a molecule that binds, duplexes, or hybridizes to a particular nucleotide sequence under stringent hybridization conditions when that sequence is present in a complex mixture, including but not limited to total cell or library DNA or RNA.
The phrase "stringent hybridization conditions" refers to hybridization of sequences of DNA, RNA, PNA or other nucleic acid mimics or combinations thereof under conditions of low ionic strength and high temperature. For example, under stringent conditions, a probe will hybridize to its target sequence in a complex mixture of nucleic acids (including, but not limited to, total cells or library DNA or RNA), but not to other sequences in the complex mixture. Stringent conditions are sequence-dependent and will be different in different circumstances. For example, longer sequences hybridize specifically at higher temperatures. Stringent hybridization conditions include, but are not limited to: (i) About 5-10 ℃ below the thermal melting point (Tm) of the particular sequence at a defined ionic strength and pH; (ii) A salt concentration of about 0.01M to about 1.0M at about ph7.0 to about ph8.3 and a temperature of at least about 30 ℃ for short probes (including but not limited to about 10 to about 50 nucleotides) and at least about 60 ℃ for long probes (including but not limited to more than 50 nucleotides); (iii) adding destabilizing agents including, but not limited to, formamide; (iv) 50% formamide, 5 XSSC and 1% SDS are incubated at 42℃or 5 XSSC, about 1% SDS are incubated at 65℃and washed in 0.2 XSSC, and about 0.1% SDS are incubated at 65℃for about 5 minutes to about 120 minutes. By way of example only, detection of selective or specific hybridization includes, but is not limited to, a positive signal of at least twice background. A detailed guide to nucleic acid hybridization is found in Tijssen, laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993).
As used herein, "subject" refers to an animal that is the subject of treatment, observation, or experiment. By way of example only, the subject may be, but is not limited to, a mammal, including, but not limited to, a human.
As used herein, the term "substantially purified" refers to a target component that may be substantially or essentially free of other components that normally accompany or interact with the target component prior to purification. By way of example only, a target component may be "substantially purified" when a formulation of the target component contains less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% (by dry weight) of contaminating components. Thus, a "substantially purified" target component may have a purity level of about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or more. By way of example only, a natural amino acid polypeptide or a non-natural amino acid polypeptide may be purified from a natural cell or host cell in the case of recombinantly produced natural or non-natural amino acid polypeptides. For example, a formulation of a natural or unnatural amino acid polypeptide can be "substantially purified" when the formulation contains less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% (by dry weight) of contaminating material. For example, when the natural or unnatural amino acid polypeptide is recombinantly produced by a host cell, the natural or unnatural amino acid polypeptide can be present at about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% or less of the dry weight of the cell. For example, when the natural or unnatural amino acid polypeptide is recombinantly produced by a host cell, the natural or unnatural amino acid polypeptide can be present in the culture medium at about 5g/L, about 4g/L, about 3g/L, about 2g/L, about 1g/L, about 750mg/L, about 500mg/L, about 250mg/L, about 100mg/L, about 50mg/L, about 10mg/L, or about 1mg/L or less of the dry weight of the cell. For example, a "substantially purified" natural amino acid polypeptide or non-natural amino acid polypeptide can have a purity level of about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more, as determined by suitable methods, including, but not limited to, SDS/PAGE analysis, RP-HPLC, SEC, and capillary electrophoresis.
The term "substituent" also referred to as a "non-interfering substituent" refers to a group that can be used to replace another group on a molecule. Such groups include, but are not limited to, 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, substituted phenyl, tolyl, xylyl, 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 Alkylsulfinyl, C 1 -C 10 Alkylsulfonyl, - (CH) 2 ) m -O-(C 1 -C 10 Alkyl) (wherein m is 1 to 8), aryl, substituted alkoxy, fluoroalkyl, heterocyclyl, substituted heterocyclyl, nitroalkyl, -NO 2 、-CN、-NRC(O)-(C 1 -C 10 Alkyl), -C (O) - (C) 1 -C 10 Alkyl group, C 2 -C 10 Alkylthioalkyl, -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 group)-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) (wherein each m is 1 to 8), -C (O) NR 2 、-C(S)NR 2 、-SO 2 NR 2 、-NRC(O)NR 2 、-NRC(S)NR 2 Salts thereof, and the like. Each R group in the foregoing list includes, but is not limited to, H, alkyl or substituted alkyl, aryl or substituted aryl or alkylaryl. Where substituents are represented by their common formula written left to right, they likewise encompass chemically identical substituents formed by right to left written structures, e.g., -CH 2 O-is equivalent to-OCH 2 -。
By way of example only, substituents for alkyl and heteroalkyl groups (including those groups referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) include, but are not limited to: -OR, =o, =nr, =n-OR, -NR 2 -SR, -halogen, -SiR 3 、-OC(O)R、-C(O)R、-CO 2 R、-CONR 2 、-OC(O)NR 2 、-NRC(O)R、-NRC(O)NR 2 、-NR(O) 2 R、-NR-C(NR 2 )＝NR、-S(O)R、-S(O) 2 R、-S(O) 2 NR 2 、-NRSO 2 R, -CN and-NO 2 . Each R group in the foregoing list includes, but is not limited to, hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl (including but not limited to aryl substituted with 1-3 halogens), substituted or unsubstituted alkyl, alkoxy, or thioalkoxy, or aralkyl. When two R groups are attached to the same nitrogen atom, they may combine with the nitrogen atom to form a 5-, 6-, or 7-membered ring. For example, -NR 2 Is intended to include, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl.
Specifically, substituents for aryl and heteroaryl include, but are not limited to, -OR, =o, =nr, =n-OR, -NR 2 -SR, -halogen, -SiR 3 、-OC(O)R、-C(O)R、-CO 2 R、-CONR 2 、-OC(O)NR 2 、-NRC(O)R、-NRC(O)NR 2 、-NR(O) 2 R、-NR-C(NR 2 )＝NR、-S(O)R、-S(O) 2 R、-S(O) 2 NR 2 、-NRSO 2 R、-CN、-NO 2 、-R、-N 3 、-CH(Ph) 2 Fluoro (C) 1 -C 4 ) Alkoxy and fluoro (C) 1 -C 4 ) Alkyl in an amount ranging from zero to the total number of openings on the aromatic ring system; and wherein each R group in the foregoing list includes, but is not limited to, hydrogen, alkyl, heteroalkyl, aryl, and heteroaryl.
As used herein, the term "therapeutically effective amount" refers to an amount of a composition comprising at least one unnatural amino acid polypeptide and/or at least one modified unnatural amino acid polypeptide administered to a patient who has had a disease, disorder, or condition, sufficient to cure or at least partially arrest or alleviate to some extent one or more symptoms of the disease, disorder, or condition being treated. The effectiveness of such compositions depends on a number of conditions, including but not limited to the severity and course of the disease, disorder or condition, previous therapies, the health condition and response of the patient to the drug, and the diagnosis of the treating physician. By way of example only, a therapeutically effective amount may be determined by routine experimentation, including but not limited to, up-dosing clinical trials.
As used herein, the term "thioalkoxy" refers to a thioalkyl group attached to a molecule through an oxygen atom.
As used herein, the term "toxic moiety" or "toxic group" refers to a compound that can cause injury, interference, or death. Toxic moieties include, but are not limited to, auristatin, DNA minor groove binder, DNA minor groove alkylating agent, enediyne, lexitropsin, sesquicomycin (duocarmycin), taxane, puromycin, TLR agonist, maytansinoids, vinca alkaloids, AFP, MMAF, MMAE, AEB, AEVB, auristatin E, paclitaxel, docetaxel, CC-1065, SN-38, topotecan, morpholino doxorubicin, rhizoxin (rhizoxin), cyanomorpholino doxorubicin, TLR agonist-10, echinomycin, combretastatin (combretastatin), calicheamicin (chalazimycin), maytansine, DM-1, spindle mycin, podophyllotoxin (podophyllotoxin) (e.g., etoposide, teniposide, etc.), baccatin and derivatives thereof, antimicrotubule agents macrolides (cryptophycin), combretastatin, auristatin E, vincristine, vinblastine, vindesine, vinorelbine, VP-16, camptothecine, epothilone A, epothilone B, nocodazole (nocodazole), colchicine (colchicine), colchicine (colcimid), estramustine (estramustine), cimadodine (cemadtin), discodermolide (discodermolide) maytansine, sarcandol (eleutherobin), dichloromethyldiethylamine (mechlorethamine), cyclophosphamide, melphalan, carmustine, lomustine, semustine, streptozotocin (streptozocin), chlorouremycin (chlorozotocin), uratemustine (uracil mustard), nitrogen mustard (chloroethane), ifosfamide, chlorambucil, pipobroman (pipobroman), tremelline, triethylthiophosphamine, busulfan, dacarbazine and temozolomide, cytarabine, cytosine arabinoside, fluorouracil, fluorouridine, 6-thioguanine, 6-mercaptopurine, penstatin, 5-fluorouracil, methotrexate, 10-propargyl-5, 8-diazepate, 5, 8-diazepate tetrahydrofolate, folinic acid, fludarabine phosphate, jelutamine, gemcitabine, ara-C, paclitaxel, docetaxel, deoxymitomycin, mitomycin-C, L-asparaginase, azathioprine, buconazole, antibiotics (e.g., anthracyclines, gentamicin, cefalotin, vancomycin, telavancin, daptomycin, azithromycin, erythromycin, cefalotin, zidime roxithromycin, furazolidone, amoxicillin, ampicillin, carbenicillin, flucloxacillin, methicillin, penicillin, ciprofloxacin, moxifloxacin, ofloxacin, doxycycline, minocycline, terramycin, tetracycline, streptomycin, rifabutin, ethambutol, rifaximin, etc.), antiviral agents (e.g., abacavir (abacavir), acyclovir (acyclovir), an Puli-mer (ampligen), cidofovir, delavirdine (delavirdine), didanosine (didanosine), efavirenz (efavirenz), entecavir, phosphazene (fosfonet), ganciclovir, ibatabine (ibarabine), imunovir, iodine, inosine, lopinavir (pinavir), metirane Sha Zong (metazazole), nevirapine, virapine (nevirapine), oseltamivir, penciclovir, stavudine (stavudine), trifluoracene, taruva (truvada), valacyclovir (valaciclovir), zanamivir and the like), daunomycin hydrochloride, daunorubicin, rubicin, secomycin (cerubidine), idarubicin (idarubicin), doxorubicin, epirubicin and morpholino derivatives, phenoxazosin bicyclic peptides (e.g., actinomycin), basal glycopeptides (e.g., bleomycin), anthraquinones (e.g., plicamycin, mithramycin), anthracenedione (e.g., mitoxantrone), aziridopyrrolindoldione (e.g., mitomycin), macrocyclic immunosuppressants (e.g., cyclosporine, FK-506, tacrolimus) Praeparata (prograf), rapamycin, and the like), noveltrombone (navlbene), CPT-11, anastrozole, letrozole, capecitabine (capecitabine), lei Luosa-fene (reloxafine), cyclophosphamide, ifosfamide, droloxifene, allocolchicine, halftones B, colchicine derivatives, maytansine, rhizobiacin, paclitaxel derivatives, docetaxel, thiocolchicine, tritylcysteine, vinblastine sulfate, vincristine sulfate, cisplatin, carboplatin, hydroxyurea, N-methylhydrazine, epipodophyllotoxin, procarbazine, mitoxantrone, folinic acid, and tegafur (tegafur). "taxane" includes paclitaxel, as well as any active taxane derivative or prodrug.
As used herein, the term "treating" includes alleviating, reducing or ameliorating the symptoms of a disease or disorder, preventing additional symptoms, ameliorating or preventing the underlying metabolic cause of the symptoms, inhibiting the disease or disorder (e.g., arresting the development of the disease or disorder), alleviating the disease or disorder, causing regression of the disease or disorder, alleviating the symptoms of a disorder or halting the disease or disorder caused by the disease or disorder. The term "treatment" includes, but is not limited to, prophylactic and/or therapeutic treatment.
As used herein, the term "water-soluble polymer" refers to any polymer that is soluble in an aqueous solvent. Such water-soluble polymers include, but are not limited to, polyethylene glycol propionaldehyde, mono-C thereof 1 -C 10 Alkoxy or aryloxy derivatives (descriptionIn U.S. Pat. No. 5,252,714, incorporated herein by reference), monomethoxy-polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, polyamino acids, divinyl ether maleic anhydride, N- (2-hydroxypropyl) -methacrylamide, dextran derivatives (including dextran sulfate), polypropylene glycol, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols, heparin fragments, polysaccharides, oligosaccharides, glycans, cellulose and cellulose derivatives (including but not limited to methylcellulose and carboxymethylcellulose), serum albumin, starch and starch derivatives, polypeptides, polyalkylene glycols and derivatives thereof, copolymers of polyalkylene glycols and derivatives thereof, polyvinyl ethyl ether, and alpha-beta-poly [ (2-hydroxyethyl) -DL-asparagine, and the like, or mixtures thereof. By way of example only, coupling of such water-soluble polymers to a natural amino acid polypeptide or a non-natural polypeptide may result in variations including, but not limited to, the following: increased water solubility, increased or modulated serum half-life, increased or modulated therapeutic half-life relative to unmodified form, increased bioavailability, modulated biological activity, prolonged circulation time, modulated immunogenicity, modulated physical binding characteristics (including but not limited to aggregation and multimeric formation), altered receptor binding, activity modulator or other targeting polypeptide binding, altered binding to one or more binding partners, and altered dimerization or multimerization of the targeting polypeptide receptor. In addition, such water-soluble polymers may or may not have their own biological activity and may serve as linkers for linking the targeting polypeptide to other substances, including but not limited to one or more targeting polypeptides or one or more biologically active molecules.
Conventional methods of mass spectrometry, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniques and pharmacology within the skill of the art are employed unless otherwise indicated.
The compounds presented herein (including, but not limited to, unnatural amino acids, unnatural amino acid polypeptides, modified unnatural amino acid polypeptides, and reagents for producing the foregoing compounds) include isotopically labeled compounds that are listed in the various formulae and structures presented hereinThose compounds are the same, but in fact one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number commonly found in nature. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine and chlorine, such as respectively 2 H、 3 H、 13 C、 14 C、 15 N、 18 O、 17 O、 35 S、 18 F、 36 Cl. Certain isotopically-labeled compounds described herein, for example, are incorporated into radioisotopes (such as 3 H and 14 c) Is suitable for use in drug and/or matrix tissue distribution assays. Furthermore, the use of isotopes (such as deuterium, i.e 2 H) The substitution performed may provide certain therapeutic advantages resulting from greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements.
Some of the compounds herein (including, but not limited to, unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides, as well as reagents for producing the foregoing compounds) have asymmetric carbon atoms and may therefore exist in enantiomeric or diastereoisomeric forms. They can be separated into their individual diastereomers on the basis of their physicochemical differences by known methods, for example by chromatography and/or fractional crystallization. Enantiomers may also be separated as follows: the enantiomeric mixture is converted to a diastereomeric mixture by reaction with an appropriate optically active compound (e.g., an alcohol), the diastereomers are separated, and the individual diastereomers are converted (e.g., hydrolyzed) to the corresponding pure enantiomers. All such isomers, including diastereomers, enantiomers, and mixtures thereof, are considered as part of the compositions described herein.
In additional or other embodiments, the compounds described herein (including, but not limited to, unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides, as well as reagents for producing the foregoing compounds) are used in the form of prodrugs. In still other embodiments, the compounds described herein (including, but not limited to, unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides, as well as reagents for producing the foregoing compounds) are metabolized upon administration to organisms that need to produce metabolites that are then used to produce the desired effects, including the desired therapeutic effects. In other or further embodiments are active metabolites of unnatural amino acids and "modified or unmodified" unnatural amino acid polypeptides.
The methods and formulations described herein include the use of unnatural amino acids, unnatural amino acid polypeptides, and modified N-oxides, crystalline forms (also referred to as polymorphs) or pharmaceutically acceptable salts of unnatural amino acid polypeptides. In certain embodiments, the unnatural amino acid, unnatural amino acid polypeptide, and modified unnatural amino acid polypeptide can exist as tautomers. All tautomers are included within the scope of the unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides presented herein. In addition, the unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides described herein can exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents (such as water, ethanol, etc.). The unnatural amino acids, unnatural amino acid polypeptides, and solvated forms of modified unnatural amino acid polypeptides presented herein are also considered disclosed herein.
Some of the compounds herein (including, but not limited to, unnatural amino acids, unnatural amino acid polypeptides and modified unnatural amino acid polypeptides, as well as reagents for producing the foregoing compounds) can exist in several tautomeric forms. All such tautomeric forms are considered to be part of the compositions described herein. Furthermore, for example, all enol-ketone forms of any of the compounds herein (including, but not limited to, unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides, as well as reagents for producing the foregoing compounds) are considered as part of the compositions described herein.
Some of the compounds herein (including, but not limited to, unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides, as well as reagents for producing any of the foregoing compounds) are acidic and can form salts with pharmaceutically acceptable cations. Some of the compounds herein (including, but not limited to, unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides, as well as reagents for producing the foregoing compounds) can be basic, and thus can form salts with pharmaceutically acceptable anions. All such salts (including di-salts) are within the scope of the compositions described herein, and the salts can be prepared by conventional methods. For example, salts can be prepared by contacting acidic and basic entities in aqueous, non-aqueous, or partially aqueous media. Recovering the salt by using at least one of the following techniques: filtration, precipitation with a non-solvent followed by filtration, evaporation of the solvent, or in the case of aqueous solutions, lyophilization.
When acidic protons present in the parent unnatural amino acid polypeptide are replaced with metal ions (e.g., alkali metal ions, alkaline earth metal ions, or aluminum ions); or upon complexation with an organic base, may form a pharmaceutically acceptable salt of the unnatural amino acid polypeptides disclosed herein. In addition, salt forms of the disclosed unnatural amino acid polypeptides can be prepared using salts of starting materials or intermediates. The unnatural amino acid polypeptides described herein can be prepared as pharmaceutically acceptable acid addition salts (which are one pharmaceutically acceptable salt form) by reacting the free base form of the unnatural amino acid polypeptide described herein with a pharmaceutically acceptable inorganic or organic acid. Alternatively, the unnatural amino acid polypeptides described herein can be prepared as pharmaceutically acceptable base addition salts (which are pharmaceutically acceptable salt forms) by reacting the free acid form of the unnatural amino acid polypeptide described herein with a pharmaceutically acceptable inorganic or organic base.
Pharmaceutically acceptable salt forms include, but are not limited to: (1) Acid addition salts with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or acid addition salts formed with organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3- (4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1, 2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 2-naphthalenesulfonic acid, 4-methylbicyclo- [2.2.2] oct-2-ene-1-carboxylic acid, glucoheptonic acid, 4' -methylenebis- (3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, t-butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like; (2) When the acidic protons present in the parent compound are replaced with metal ions (e.g., alkali metal ions, alkaline earth metal ions, or aluminum ions); or a salt formed upon complexation with an organic base. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. Acceptable inorganic bases include aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, and the like.
The corresponding counter ion of the pharmaceutically acceptable salt of the unnatural amino acid polypeptide can be analyzed and identified using various methods, including, but not limited to, ion exchange chromatography, ion chromatography, capillary electrophoresis, inductively coupled plasma, atomic absorption spectrometry, mass spectrometry, or any combination thereof. In addition, such non-natural amino acid polypeptides can be tested for therapeutic activity of pharmaceutically acceptable salts using the techniques and methods described in the examples.
It is to be understood that reference to salts includes solvent addition forms or crystalline forms thereof, particularly solvates or polymorphs. Solvates contain stoichiometric or non-stoichiometric amounts of solvent and can be formed during crystallization processes using pharmaceutically acceptable solvents such as water, ethanol, and the like. Hydrates are formed when the solvent is water, or alcoholates are formed when the solvent is an alcohol. Polymorphs include different crystal packing arrangements of the same elemental composition of the compound. Polymorphs typically have different X-ray diffraction patterns, infrared spectra, melting points, densities, hardness, crystal shapes, optical and electrical properties, stability and solubility. Various factors such as recrystallization solvent, crystallization rate, and storage temperature may lead to a single crystal form predominating.
Screening and characterization of pharmaceutically acceptable salts, polymorphs, and/or solvates of the unnatural amino acid polypeptide can be accomplished using a variety of techniques, including, but not limited to, thermal analysis, x-ray diffraction, spectroscopy, vapor adsorption, and microscopy. Thermal analysis methods address thermochemical degradation or thermophysical processes, including but not limited to polymorphic transformations, and such methods are used to analyze relationships between polymorphs, determine weight loss, find glass transition temperatures, or for excipient compatibility studies. Such methods include, but are not limited to, differential Scanning Calorimetry (DSC), modulated differential scanning calorimetry (MDCS), thermogravimetric analysis (TGA), and thermogravimetric and infrared analysis (TG/IR). X-ray diffraction methods include, but are not limited to, single crystal and powder diffractometers and synchrotron radiation sources. Various spectroscopic techniques are used including, but not limited to, raman, FTIR, UVIS and NMR (liquid and solid). Various microscopy techniques include, but are not limited to, polarized light microscopy, scanning Electron Microscopy (SEM) with energy dispersive X-ray analysis (EDX), ambient scanning electron microscopy with EDX (in a gas or water vapor atmosphere), infrared microscopy, and raman microscopy.
While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Many alterations, modifications and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.
TLR agonist linker derivatives
In one level, described herein are tools (methods, compositions, techniques) for producing and using targeting polypeptides comprising at least one unnatural amino acid or TC or analog of a modified unnatural amino acid with a carbonyl, dicarbonyl, oxime, or hydroxylamine group. Such targeting polypeptides comprising TCs of unnatural amino acids can contain other functional groups, including but not limited to polymers; a water-soluble polymer; derivatives of polyethylene glycol; a second protein or polypeptide analog; an antibody or antibody fragment; and any combination thereof. It should be noted that the various foregoing functionalities do not mean that a member of one functional group cannot be classified as a member of another functional group. In practice, there will be overlap as the case may be. By way of example only, the water-soluble polymer overlaps in scope with the polyethylene glycol derivative, but the overlap is not complete, so that both functional groups are mentioned above.
In one aspect are methods for selecting and designing TLR agonist linker derivatives modified using the methods, compositions and techniques described herein, as well as targeting polypeptides. New TLR agonist linker derivatives and targeting polypeptides can be designed, either de novo or based on the interest of the researcher, including, for example, only as part of a high throughput screening process (in which case, many polypeptides can be designed, synthesized, characterized, and/or tested). Novel TLR agonist linker derivatives and targeting polypeptides can also be designed based on the structure of the known or partially characterized polypeptides. Merely by way of example, TLR agonists have been the subject of intensive research by the scientific community; new compounds can be designed based on the structure of TLR agonists. The principle of selecting which amino acids to substitute and/or modify is described herein separately. The choice of which modification to use is also described herein and can be used to meet the needs of the experimenter or end user. Such needs may include, but are not limited to, manipulating the therapeutic effect of the polypeptide, improving the safety profile of the polypeptide, modulating the pharmacokinetics, pharmacology, and/or pharmacodynamics of the polypeptide, such as, for example only, increasing water solubility, bioavailability, increasing serum half-life, increasing therapeutic half-life, modulating immunogenicity, modulating biological activity, or extending circulation time. Further, by way of example only, such modifications include providing additional functional groups to the polypeptide, incorporating antibodies, and any combination of the foregoing.
TLR agonist linker derivatives and targeting polypeptides having or that can be modified to contain an oxime, carbonyl, dicarbonyl, or hydroxylamine group are also described herein. Aspects include methods for producing, purifying, characterizing, and using such TLR agonist linker derivatives and targeting polypeptides.
The TLR agonist linker derivative or targeting polypeptide may contain at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or ten or more carbonyl or dicarbonyl groups, oxime groups, hydroxylamine groups, or protected forms thereof. The TLR agonist linker derivatives or targeting polypeptides may be the same or different, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more different sites may be present in the derivative comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more different reactive groups.
As described herein, the present disclosure provides a targeting polypeptide coupled to another molecule having the formula "targeting polypeptide-L-M," wherein L is a linking group or a chemical bond and M is any other molecule, including but not limited to another targeting polypeptide. In some embodiments, L is stable in vivo. In some embodiments, L is hydrolyzable in vivo. In some embodiments, L is metastable in vivo.
The targeting polypeptide and M can be linked together by L using standard linkers and procedures known to those skilled in the art. In some aspects, the targeting polypeptide and M are fused directly and L is a bond. In other aspects, the targeting polypeptide and M are fused via a linking group L. For example, in some embodiments, the targeting polypeptide and M are linked together by a peptide bond, optionally by a peptide or amino acid spacer. In some embodiments, the targeting polypeptide and M are linked together by chemical conjugation, optionally via a linking group (L). In some embodiments, L is conjugated directly to each of the targeting polypeptide and M.
Chemical conjugation may occur by reacting a nucleophilic reactive group of one compound with an electrophilic reactive group of another compound. In some embodiments, when L is a bond, the targeting polypeptide is conjugated to M by reacting the nucleophilic reactive moiety on the targeting polypeptide with the electrophilic reactive moiety on Y, or by reacting the electrophilic reactive moiety on the targeting polypeptide with the nucleophilic reactive moiety on M. In embodiments, when L is a group linking together the targeting polypeptide and M, the targeting polypeptide and/or M may be conjugated to L by reacting the nucleophilic reactive moiety on the targeting polypeptide and/or M with the electrophilic reactive moiety on L, or by reacting the electrophilic reactive moiety on the targeting polypeptide and/or M with the nucleophilic reactive moiety on L. Non-limiting examples of nucleophilic reactive groups include amino groups, thiol groups, and hydroxyl groups. Non-limiting examples of electrophilic reactive groups include carboxyl groups, acid chlorides, anhydrides, esters, succinimidyl esters, haloalkanes, sulfonates, maleimidos, haloacetyl groups, and isocyanates. In embodiments where the targeting polypeptide and M are conjugated together by reaction of the carboxylic acid with an amine, an activator may be used to form an activated ester of the carboxylic acid.
The activated ester of a carboxylic acid may be, for example, N-hydroxysuccinimide (NHS), tosylate (Tos), mesylate, triflate, carbodiimide or hexafluorophosphate. In some embodiments, the carbodiimide is 1, 3-Dicyclohexylcarbodiimide (DCC), 1' -Carbonyldiimidazole (CDI), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), or 1, 3-diisopropylcarbodiimide (dic). In some embodiments, the hexafluorophosphate is selected from the group consisting of: benzotriazol-1-yl-oxy-tris (dimethylamino) phosphonium hexafluorophosphate (BOP), benzotriazol-1-yl-oxy-tripyrrolidinylphosphonium hexafluorophosphate (PyBOP), 2- (1H-7-azabenzotriazol-1-yl) -1, 3-tetramethyluronium Hexafluorophosphate (HATU) and o-benzotriazol-N, N, N ', N' -tetramethyluronium Hexafluorophosphate (HBTU).
In some embodiments, the targeting polypeptide comprises a nucleophilic reactive group (e.g., an amino group, thiol group, or hydroxyl group of a lysine, cysteine, or serine side chain) capable of conjugation to an electrophilic reactive group on M or L. In some embodiments, the targeting polypeptide comprises an electrophilic reactive group (e.g., carboxylate group of Asp or Glu side chain) capable of conjugation to a nucleophilic reactive group on M or L. In some embodiments, the targeting polypeptide is chemically modified to comprise a reactive group capable of direct conjugation to M or L. In some embodiments, the targeting polypeptide is modified at the N-terminus or C-terminus to comprise a natural or unnatural amino acid with a nucleophilic side chain. In exemplary embodiments, the N-terminal or C-terminal amino acid of the targeting polypeptide is selected from the group consisting of: lysine, ornithine, serine, cysteine and homocysteine. For example, the N-terminal or C-terminal amino acids of the targeting polypeptide can be modified to include lysine residues. In some embodiments, the targeting polypeptide is modified at the N-terminal or C-terminal amino acid to comprise natural or unnatural amino acids with electrophilic side chains, e.g., asp and Glu. In some embodiments, the internal amino acids of the targeting polypeptide are substituted with natural or unnatural amino acids with nucleophilic side chains as previously described herein. In exemplary embodiments, the internal amino acid of the substituted targeting polypeptide is selected from the group consisting of: lysine, ornithine, serine, cysteine and homocysteine. For example, an internal amino acid of the targeting polypeptide may be substituted with a lysine residue. In some embodiments, an internal amino acid of the targeting polypeptide is substituted with a natural or unnatural amino acid having an electrophilic side chain, e.g., asp and Glu.
In some embodiments, M comprises a reactive group capable of direct conjugation to a targeting polypeptide or L. In some embodiments, M comprises a nucleophilic reactive group (e.g., amine, thiol, hydroxyl) capable of conjugation to an electrophilic reactive group on the targeting polypeptide or L. In some embodiments, M comprises an electrophilic reactive group (e.g., carboxyl, activated form of carboxyl, compound with leaving group) capable of conjugation to a nucleophilic reactive group on the targeting polypeptide or L. In some embodiments, M is chemically modified to comprise a nucleophilic reactive group capable of conjugation to an electrophilic reactive group on the targeting polypeptide or L. In some embodiments, M is chemically modified to comprise an electrophilic reactive group capable of conjugation to a nucleophilic reactive group on the targeting polypeptide or L.
In some embodiments, conjugation may be by organosilanes, such as aminosilanes treated with glutaraldehyde; carbonyl Diimidazole (CDI) activated silanol groups; or using dendritic polymers. Various dendrimers are known in the art and include poly (amidoamine) (PAMAM) dendrimers, which are synthesized by a divergent process starting from ammonia or ethylenediamine initiator core reagents; a subclass of PAMAM dendrimers based on tri-amino ethylene-imine cores; radial lamellar poly (amidoamine-silicone) dendrimers (PAMAMOS), which are inverted single-molecule micelles consisting of hydrophilic, nucleophilic Polyamidoamine (PAMAM) interiors and hydrophobic silicone (OS) exteriors; poly (propylene imine) (PPI) dendrimers, which are typically primary amine-terminated polyalkylamines, with the dendrimer internally consisting of a number of tertiary tri-propenamines; poly (acrylamide) (POPAM) dendrimers; diaminobutane (DAB) dendrimers; amphiphilic dendritic polymers; a micelle dendrimer which is a single molecular micelle of a water-soluble hyperbranched polyphenylene; polylysine dendrimers; and dendrimers based on a polyphenyl methyl ether hyperbranched backbone.
In some embodiments, conjugation may be by olefin metathesis. In some embodiments, M and the targeting polypeptide, M and L, or both the targeting polypeptide and L comprise an alkene or alkyne moiety capable of undergoing metathesis. In some embodiments, a suitable catalyst (e.g., copper, ruthenium) is used to accelerate the metathesis reaction. Suitable methods for conducting olefin metathesis reactions are described in the art. See, e.g., schafmeister et al, J.am.chem.Soc.122:5891-5892 (2000); walensky et al, science 305:1466-1470 (2004); and Blackwell et al, angew, chem., int. Ed.37:3281-3284 (1998).
In some embodiments, conjugation may be performed using click chemistry. The "click reaction" is widely and easily performed, uses only off-the-shelf reagents, and is insensitive to oxygen and water. In some embodiments, the click reaction is a cycloaddition reaction between an alkyne group and an azide group to form a triazole group. In some embodiments, the click reaction uses a copper or ruthenium catalyst. Suitable methods for performing click reactions are described in the art. See, e.g., kolb et al, drug Discovery Today 8:1128 (2003); kolb et al Angew.chem.int.ed.40:2004 (2001); rostovtsev et al Angew.chem.int.ed.41:2596 (2002); tomoe et al, J.org.chem.67:3057 (2002); manetsch et al, J.am.chem.Soc.126:12809 (2004); lewis et al, angew.chem.int.ed.41:1053 (2002); speers, J.am.chem.Soc.125:4686 (2003); chan et al org. Lett.6:2853 (2004); zhang et al, J.am.chem.Soc.127:15998 (2005); and Waser et al, J.Am.chem.Soc.127:8294 (2005).
Indirect conjugation by high affinity specific binding partners, such as streptavidin/biotin or avidin/biotin or lectin/carbohydrate, is also contemplated.
In some embodiments, the targeting polypeptide and/or M is functionalized with an organic derivatizing agent to comprise a nucleophile or electrophile. The derivatizing agent is capable of reacting with the N-terminal or C-terminal residue of the targeted amino acid and the functional group on M on the selected side chain or targeted polypeptide. Reactive groups on the targeting polypeptide and/or M include, for example, aldehyde, amino, ester, thiol, α -haloacetyl, maleimido, or hydrazino groups. Derivatizing agents include, for example, maleimidobenzoyl sulfosuccinimidyl ester (conjugated via a cysteine residue), N-hydroxysuccinimide (conjugated via a lysine residue), glutaraldehyde, succinic anhydride, or other agents known in the art. Alternatively, the targeting polypeptide and/or M may be indirectly linked to each other by an intermediate carrier, such as a polysaccharide or polypeptide carrier. Examples of polysaccharide carriers include aminodextran. Examples of suitable polypeptide carriers include polylysine, polyglutamic acid, polyaspartic acid, copolymers thereof, and mixed polymers of these amino acids and other amino acids, such as serine, to impart desired solubility characteristics to the resulting loaded carrier.
Cysteinyl residues most commonly react with a-haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues are also derived by reaction with bromotrifluoroacetone, α -bromo- β - (5-imidazolyl) propionic acid, chloroacetyl phosphate, N-alkyl maleimide, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuric benzoic acid, 2-chloromercuric-4-nitrophenol or chloro-7-nitrobenzo-2-oxa-1, 3-diazole.
Histidyl residues are derived by reaction with diethyl pyrocarbonate at pH 5.5-7.0, as the agent is relatively specific for histidyl side chains. P-bromobenzoyl methyl bromide is also useful; the reaction is preferably carried out in 0.1M sodium dimethylarsinate at pH 6.0.
Lysyl and amino terminal residues are reacted with succinic acid or other carboxylic anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysyl residues. Other suitable reagents for derivatizing the α -amino group containing residue include imidoesters such as methyl picolinate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methyliso urea, 2, 4-pentanedione, and transamidase-catalyzed reactions with glyoxylate.
Arginyl residues are modified by reaction with one or several conventional reagents, including phenylglyoxal (phenylglyoxyl), 2, 3-butanedione, 1, 2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires that the reaction be carried out under basic conditions because guanidine functionality has a high pKa. In addition, these reagents can react with lysine groups and arginine epsilon-amino groups.
Specific modifications of tyrosyl residues can be made by reaction with aromatic diazonium compounds or tetranitromethane, with particular attention being paid to the incorporation of spectroscopic tags into tyrosyl residues. Most commonly, N-acetylimidazole and tetranitromethane are used to form O-acetyltyrosyl species and 3-nitro derivatives, respectively.
The pendant carboxyl groups (aspartyl or glutamyl) are selectively modified by reaction with a carbodiimide (R-n=c=n-R '), where R and R' are different alkyl groups, such as 1-cyclohexyl-3- (2-morpholinyl-4-ethyl) carbodiimide or 1-ethyl-3- (4-azocation-4, 4-dimethylpentyl) carbodiimide. In addition, aspartyl and glutamyl residues can be converted to aspartyl and glutamyl residues by reaction with ammonium ions.
Other modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl groups of seryl or threonyl residues, methylation of alpha-amino groups of lysine, arginine and histidine side chains (T.E.Creaton, proteins: structure and Molecular Properties, W.H.Freeman & Co., san Francisco, pages 79-86 (1983)), deamidation of asparagine or glutamine, acetylation of N-terminal amines and/or amidation or esterification of C-terminal carboxylic acid groups.
Another type of covalent modification involves chemical or enzymatic coupling of a glycoside to a peptide. The saccharide may be linked to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups, such as those of cysteine, (d) free hydroxyl groups, such as those of serine, threonine or hydroxyproline, (e) aromatic residues, such as those of tyrosine or tryptophan, or (f) amido groups of glutamine. These methods are described in WO 1987/05330 and Aplin and Wriston, CRC crit. Rev. Biochem, pages 259-306 (1981).
In some embodiments, L is a bond. In these embodiments, the targeting polypeptide and M are conjugated together by reacting a nucleophilic reactive moiety on the targeting polypeptide with an electrophilic reactive moiety on M. In alternative embodiments, the targeting polypeptide and M are conjugated together by reacting an electrophilic reactive moiety on the targeting polypeptide with a nucleophilic moiety on M. In an exemplary embodiment, L is an amide bond that is formed when an amine on the targeting polypeptide (e.g., epsilon-amine of a lysine residue) reacts with a carboxyl group on M. In alternative embodiments, the targeting polypeptide and/or M is derivatized with a derivatizing agent prior to conjugation.
In some embodiments, L is a linking group. In some embodiments, L is a bifunctional linker and comprises only two reactive groups prior to conjugation to the targeting polypeptide and M. In embodiments where both the targeting polypeptide and M have electrophilic reactive groups, L comprises two identical or two different nucleophilic groups (e.g., amine, hydroxyl, thiol) prior to conjugation to the targeting polypeptide and M. In embodiments where both the targeting polypeptide and M have nucleophilic reactive groups, L comprises two identical or two different electrophilic groups (e.g., carboxyl, activated form of carboxyl, compound with leaving group) prior to conjugation to the targeting polypeptide and M. In embodiments where one of the targeting polypeptides or M has a nucleophilic reactive group and the other of the targeting polypeptides or M has an electrophilic reactive group, L comprises one nucleophilic reactive group and one electrophilic reactive group prior to conjugation to the targeting polypeptide and M.
L may be any molecule having at least two reactive groups capable of reacting with each of the targeting polypeptide and M (prior to conjugation to the targeting polypeptide and M). In some embodiments, L has only two reactive groups and is difunctional. L (prior to conjugation to the peptide) can be represented by formula VI:
Wherein A and B are independently nucleophilic or electrophilic reactive groups. In some embodiments, a and B are both nucleophilic groups or are both electrophilic groups. In some embodiments, one of a or B is a nucleophilic group and the other of a or B is an electrophilic group. Non-limiting combinations of A and B are shown in Table 1 below.
Table 1: non-limiting combination of nucleophilic groups and electrophilic groups
In some embodiments, a and B may include alkene and/or alkyne functional groups suitable for alkene metathesis reactions. In some embodiments, a and B comprise moieties suitable for click chemistry (e.g., alkene, alkyne, nitrile, azide). Other non-limiting examples of reactive groups (a and B) include pyridyldithiols, aryl azides, bisazides, carbodiimides, and hydrazides.
In some embodiments, L is hydrophobic. Hydrophobic linkers are known in the art. See, e.g., bioconjugate Techniques, g.t. hermanson (Academic Press, san Diego, CA, 1996), which is incorporated by reference in its entirety. Suitable hydrophobic linking groups known in the art include, for example, 8-hydroxyoctanoic acid and 8-mercaptooctanoic acid. The hydrophobic linking group comprises at least two reactive groups (a and B) as described herein and as shown below prior to conjugation to the peptide of the composition:
In some embodiments, the hydrophobic linking group comprises a maleimido or iodoacetyl group and a carboxylic acid or activated carboxylic acid (e.g., NHS ester) as reactive groups. In these embodiments, the maleimido or iodoacetyl group can be coupled to a thiol moiety on the targeting polypeptide or M, and the carboxylic acid or activated carboxylic acid can be coupled to an amine on the targeting polypeptide or M with or without the use of a coupling agent. The carboxylic acid may be coupled with the free amine using any coupling agent known to those skilled in the art, such as DCC, DIC, HATU, HBTU, TBTU and other activators described herein. In particular embodiments, the hydrophilic linking group comprises an aliphatic chain of 2 to 100 methylene groups, wherein a and B are carboxyl groups or derivatives thereof (e.g., succinic acid). In other embodiments, L is iodoacetic acid.
In some embodiments, the linking group is hydrophilic, such as a polyalkylene glycol. Prior to conjugation to the peptide of the composition, the hydrophilic linking group comprises at least two reactive groups (a and B) as described herein and as shown below:
in a specific embodiment, the linking group is polyethylene glycol (PEG). In certain embodiments, the PEG has a molecular weight of about 100 daltons to about 10,000 daltons, e.g., about 500 daltons to about 5000 daltons. In some embodiments, the PEG has a molecular weight of about 10,000 daltons to about 40,000 daltons.
In some embodiments, the hydrophilic linking group comprises a maleimido or iodoacetyl group and a carboxylic acid or activated carboxylic acid (e.g., NHS ester) as reactive groups. In these embodiments, the maleimido or iodoacetyl group can be coupled to a thiol moiety on the targeting polypeptide or M, and the carboxylic acid or activated carboxylic acid can be coupled to an amine on the targeting polypeptide or M with or without the use of a coupling agent. The carboxylic acid may be coupled with an amine, such as DCC, DIC, HATU, HBTU, TBTU and other activators described herein, using any suitable coupling agent known to those skilled in the art. In some embodiments, the linking group is maleimido-polymer (20-40 kDa) -COOH, iodoacetyl-polymer (20-40 kDa) -COOH, maleimido-polymer (20-40 kDa) -NHS, or iodoacetyl-polymer (20-40 kDa) -NHS.
In some embodiments, the linking group is comprised of an amino acid, dipeptide, tripeptide, or polypeptide, wherein the amino acid, dipeptide, tripeptide, or polypeptide comprises at least two activating groups as described herein. In some embodiments, the linking group (L) comprises a moiety selected from the group consisting of: amino, ether, thioether, maleimido, disulfide, amide, ester, thioester, alkene, cyclic olefin, alkyne, triazolyl, carbamate, carbonate, cleavable cathepsin B and hydrazone.
In some embodiments, L comprises an atomic chain of 1 to about 60 or 1 to 30 atoms or more, 2 to 5 atoms, 2 to 10 atoms, 5 to 10 atoms, or 10 to 20 atoms long. In some embodiments, the chain atoms are all carbon atoms. In some embodiments, the chain atoms in the linker backbone are selected from the group consisting of C, O, N and S. The chain atoms and linkers may be selected according to their intended solubility (hydrophilicity) in order to provide more soluble conjugates. In some embodiments, L provides a functional group that undergoes cleavage by an enzyme or other catalyst or hydrolysis conditions found in the target tissue or organ or cell. In some embodiments, the length of L is long enough to reduce the likelihood of steric hindrance.
In some embodiments, L is stable in a biological fluid, such as blood or a blood fraction. In some embodiments, L is stable in serum for at least 5 minutes, e.g., less than 25%, 20%, 15%, 10% or 5% of the conjugate is cleaved when incubated in serum for 5 minutes. In other embodiments, L is stable in serum for at least 10, or 20, or 25, or 30, or 60, or 90, or 120 minutes, or 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, or 24 hours. In these embodiments, L does not contain a functional group capable of undergoing hydrolysis in vivo. In some exemplary embodiments, L is stable in serum for at least about 72 hours. Non-limiting examples of functional groups that are not capable of significant hydrolysis in vivo include amides, ethers, and thioethers. For example, the following compounds do not undergo significant hydrolysis in vivo.
In some embodiments, L is hydrolyzable in vivo. In these embodiments, L comprises a functional group capable of undergoing hydrolysis in vivo. Non-limiting examples of functional groups that are capable of undergoing hydrolysis in vivo include esters, anhydrides, and thioesters. For example, the following compound is capable of undergoing hydrolysis in vivo because it contains an ester group:
in some exemplary embodiments, L is unstable and undergoes substantial hydrolysis in plasma at 37 ℃ within 3 hours, and is fully hydrolyzed within 6 hours. In some exemplary embodiments, L is unstable.
In some embodiments, L is metastable in vivo. In these embodiments, L comprises a functional group (e.g., an acid labile, reduction labile, or enzyme labile functional group) that is capable of being cleaved, optionally in vivo, chemically or enzymatically over a period of time. In these embodiments, L may comprise, for example, a hydrazone moiety, a disulfide moiety, or a cathepsin-cleavable moiety. While L is metastable, and not intending to be bound by any particular theory, the targeting polypeptide-L-M conjugate is stable in the extracellular environment, e.g., stable in serum for the period of time described above, but unstable in the intracellular environment or conditions mimicking the intracellular environment, so it is cleaved upon entry into the cell. In some embodiments, when L is metastable, L is stable in serum for at least about 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 42, or 48 hours, such as at least about 48, 54, 60, 66, or 72 hours, or about 24-48, 48-72, 24-60, 36-48, 36-72, or 48-72 hours.
In another embodiment, the polymer derivative of the present invention comprises a polymer backbone having the structure:
X—CH 2 CH 2 O--(CH 2 CH 2 O) n --CH 2 CH 2 -O-(CH 2 ) m -W-N = n=n and, wherein:
w is an aliphatic or aromatic linking moiety comprising 1 to 10 carbon atoms;
n is 1 to about 4000; x is a functional group as described above; m is between 1 and 10.
The azide-containing polymer derivatives of the present invention may be prepared by a variety of methods known in the art and/or disclosed herein. In one method, as shown below, a water-soluble polymer backbone having an average molecular weight of about 800Da to about 100,000Da, a first end bonded to a first functional group and a second end bonded to a suitable leaving group is reacted with an azide anion (which may be paired with any of a number of suitable counter ions, including sodium, potassium, t-butylammonium, and the like). The leaving group undergoes nucleophilic displacement and is partially replaced by an azide to give the desired azide-containing polymer;
X-Polymer-LY+N 3 - →XPolymer-L N 3
As shown, the polymer backbone suitable for use in the present invention has the formula X-polymer-LY, wherein the polymer is poly (ethylene glycol) and X is a functional group that does not react with an azide group and Y is a suitable leaving group. Examples of suitable functional groups include, but are not limited to, hydroxyl, protected hydroxyl, acetal, alkenyl, amine, aminoxy, protected amine, protected hydrazide, protected thiol, carboxylic acid, protected carboxylic acid, maleimide, dithiopyridine, and vinylpyridine, and ketone. Examples of suitable leaving groups include, but are not limited to, chloride, bromide, iodide, mesylate, triflate monomethoxy (tresylate), and tosylate.
In another method of preparing the azide-containing polymer derivatives of the present invention, a linker bearing an azide functional group is contacted with a water-soluble polymer backbone having an average molecular weight of about 800Da to about 100,000Da, wherein the linker bears a chemical functional group that will selectively react with the chemical functional group on the polymer to form an azide-containing polymer derivative product, wherein the azide is separated from the polymer backbone by the linker.
An exemplary reaction scheme is shown below:
x-polymer-y+n-linker-n=n=n→' PG-X-polymers the linker-n=n=n, wherein:
the polymer is poly (ethylene glycol) and X is a capping group such as an alkoxy group or a functional group as described above; and Y is a functional group that does not react with azide functional groups but will effectively and selectively react with N functional groups.
Examples of suitable functional groups include, but are not limited to, if N is an amine, then Y is a carboxylic acid, carbonate, or active ester; if N is a hydrazide or aminooxy moiety, then Y is a ketone; if N is a nucleophile, then Y is a leaving group. Purification of the crude product may be accomplished by known methods including, but not limited to, precipitation of the product followed by chromatography if desired.
In the case of polymeric diamines, more specific examples are shown below, wherein one amine is protected with a protecting group moiety, such as t-butyl-Boc, and the resulting mono-protected polymeric diamine is reacted with a linking moiety carrying an azide functionality:
BocHN-Polymer-NH 2 +HO 2 C-(CH 2 ) 3 -N＝N＝N
In this case, the amine groups may be coupled to the carboxylic acid groups using a variety of activators such as thionyl chloride or carbodiimide reagents and N-hydroxysuccinimide or N-hydroxybenzotriazole to create amide linkages between the monoamine polymer derivative and the azide-bearing linker moiety. After successful amide bond formation, the resulting N-t-butyl-Boc protected azide-containing derivatives can be used directly to modify biologically active molecules, or can be further processed to install other useful functional groups. For example, the N-t-Boc group may be hydrolyzed by treatment with a strong acid to form an omega-amino-polymer-azide. The resulting amine can be used as a synthetic handle to mount other useful functional groups, such as maleimide groups, activated disulfides, activated esters, etc., to produce valuable heterobifunctional reagents.
Heterobifunctional derivatives are particularly useful when it is desired to attach a different molecule to each end of the polymer. For example, an omega-N-amino-N-azido polymer will allow a molecule with an activated electrophilic group, such as an aldehyde, ketone, activated ester, activated carbonate, etc., to be attached to one end of the polymer and a molecule with an ethynyl group to the other end of the polymer.
In another embodiment of the invention, A is an aliphatic linker having 1 to 10 carbon atoms or a substituted aryl ring having 6 to 14 carbon atoms. X is a functional group that does not react with an azide group, and Y is a suitable leaving group.
The plurality of targeting polypeptides may be joined by a linker polypeptide, wherein the linker polypeptide is optionally 6-14, 7-13, 8-12, 7-11, 9-11 or 9 amino acids in length. Other linkers include, but are not limited to, small polymers, such as PEG, which may be multi-armed, allowing multiple targeted polypeptide molecules to be linked together. The plurality of targeting polypeptides and the modified targeting polypeptide may be linked to each other by their N-terminal head-to-head configuration through the use of such linkers or through direct chemical bonding between the respective N-termini of each polypeptide. For example, two targeting polypeptides may be linked by a chemical bond between their N-terminal amino groups or modified N-terminal amino groups to form a dimer. Furthermore, linker molecules designed to contain multiple chemical functional groups to bond to the N-terminus of each targeting polypeptide may be used for multiple targeting polypeptides each joined at their respective N-terminus. In addition, multiple targeting polypeptides may be linked by linkages between amino acids other than the N-terminal amino acid or the C-terminal amino acid. Examples of covalent bonds that can be used to form dimers and multimers of the targeting polypeptides described herein include, but are not limited to, disulfide bonds or sulfhydryl or thiol bonds. In addition, certain enzymes, such as sortases, can be used to form a covalent bond between the targeting polypeptide and the linker, including at the N-terminus of the targeting polypeptide.
The linker may have a wide range of molecular weights or molecular lengths. A larger or smaller molecular weight linker may be used to provide the desired spatial relationship or conformation between the targeting polypeptide and the linked entity or between the linked entity and its binding partner, if any. Linkers having longer or shorter molecular lengths may also be used to provide the desired space or flexibility between the targeting polypeptide and the linked entity or between the linked entity and its binding partner. The joints may include, but are not limited to, the following:
in some embodiments, the present invention provides a water-soluble bifunctional linker having a dumbbell structure comprising: a) An azide, alkyne, hydrazine, hydrazide, hydroxylamine, or carbonyl-containing moiety on at least a first end of the polymer backbone; and b) at least a second functional group on a second end of the polymer backbone. The second functional group may be the same as or different from the first functional group. In some embodiments, the second functional group is not reactive with the first functional group. In some embodiments, the present invention provides water-soluble compounds comprising at least one arm of a branched molecular structure. For example, the branched molecular structure may be dendritic.
In exemplary embodiments, the polymer is linked to the targeting polypeptide or modified targeting polypeptide by a linker. For example, a linker may comprise one or two amino acids that bind at one end to a polymer, such as an albumin binding moiety, and at the other end to any available position on the polypeptide backbone. Additional exemplary linkers include hydrophilic linkers, such as chemical moieties containing at least 5 non-hydrogen atoms, wherein 30% -50% of the chemical moieties are N or O. Additional exemplary linkers that can link a polymer to a targeting polypeptide or modified targeting polypeptide are disclosed in U.S.2012/0295847 and WO/2012/168430, each of which is hereby incorporated by reference in its entirety.
Optionally, a plurality of targeting polypeptides or modified targeting polypeptide molecules may be joined by a linker polypeptide, wherein the linker polypeptide is optionally 1, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12 amino acids and longer in length, wherein optionally the N-terminus of one targeting polypeptide is fused to the C-terminus of the linker polypeptide and the N-terminus of the linker polypeptide is fused to the N-terminus of another targeting polypeptide. Other exemplary linker polypeptides that may be used are disclosed in WO/2013/004607, which is hereby incorporated by reference in its entirety.
As used herein, the terms "electrophile", and the like refer to an atom or group of atoms that can accept electron pairs to form a covalent bond. As used herein, "electrophilic group" includes, but is not limited to, halide-containing, carbonyl-containing, and epoxide-containing compounds. Common electrophiles may be halides such as thiophosgene, glycerol dichloropropanol, phthaloyl chloride, succinyl chloride, chloroacetyl chloride, chlorosuccinyl chloride, and the like; ketones such as chloroacetone, bromoacetone, and the like; aldehydes such as glyoxal and the like; isocyanates such as hexamethylene diisocyanate, toluene diisocyanate, m-xylylene diisocyanate, cyclohexylmethane-4, 4-diisocyanate, and the like; and derivatives of these compounds.
As used herein, the terms "nucleophilic group", "nucleophile", and the like refer to an atom or group of atoms having an electron pair capable of forming a covalent bond. This type of group may be an ionizable group that reacts as an anionic group. As used herein, "nucleophilic group" includes, but is not limited to, hydroxyl, primary amine, secondary amine, tertiary amine, and thiol.
Table 2 provides various starting electrophiles and nucleophiles that can be conjugated to produce the desired functional groups. The information provided is illustrative and not limiting of the synthetic techniques described herein.
Table 2: examples of covalent bonding and precursors thereof
Typically, carbon electrophiles are vulnerable to attack by complementary nucleophiles (including carbon nucleophiles), wherein the attack nucleophile brings electron pairs to the carbon electrophile to form new bonds between the nucleophile and the carbon electrophile.
Non-limiting examples of carbon nucleophiles include, but are not limited to, alkyl, alkenyl, aryl, and alkynyl grignard reagents, organolithium, organozinc, alkyl-tin reagents, alkenyl-tin reagents, aryl-tin reagents, and alkynyl-tin reagents (organotin), alkyl-borane reagents, alkenyl-borane reagents, aryl-borane reagents, and alkynyl-borane reagents (organoboranes and organoborates); these carbon nucleophiles have the advantage of being kinetically stable in water or polar organic solvents. Other non-limiting examples of carbon nucleophiles include phosphoylide, enolic, and enolate reagents; these carbon nucleophiles have the advantage of being relatively easy to produce from precursors well known to those skilled in the art of synthetic organic chemistry. When used in combination with a carbon nucleophile, a new carbon-carbon bond is created between the carbon nucleophile and the carbon electrophile.
Non-limiting examples of non-carbon nucleophiles suitable for coupling with the carbon electrophiles include, but are not limited to, primary and secondary amines, thiols, coloured alkoxides and sulfides, alcohols, alkoxides, azides, semicarbazides, and the like. When these non-carbon nucleophiles are used in combination with carbon electrophiles, heteroatom linkages (C-X-C) are typically created where X is a heteroatom, including but not limited to oxygen, sulfur, or nitrogen.
In some cases, the polymers used in the present invention terminate at one end with a hydroxyl or methoxy group, i.e., X is H or CH 3 ("methoxy PEG"). Alternatively, the polymer may terminate with a reactive group, thereby forming a difunctional polymer. Typical reactive groups may include those commonly used to react with functional groups found in 20 common amino acids (including but not limited to maleimide groups, activated carbonates (including but not limited to p-nitrophenyl esters), activated esters (including but not limited to N-hydroxysuccinimide, p-nitrophenyl esters) and aldehydes), and functional groups inert to 20 common amino acids but specifically reactive with complementary functional groups (including but not limited to azide groups, alkyne groups). It should be noted that the other end of the polymer represented by Y in the above formula will be linked directly or indirectly to the targeting polypeptide via naturally occurring or non-naturally encoded amino acids. For example, Y may be an amide, carbamate, or urea linked to an amine group of the polypeptide (including but not limited to lysine or the N-terminal epsilon amine). Alternatively, Y may be a maleimide linkage linked to a thiol group (including but not limited to a thiol group of cysteine). Alternatively, Y may be linked to residues that are not normally available through the 20 common amino acids. For example, azide groups on the polymer can react with alkyne groups on the targeting polypeptide to form Huisgen [3+2 ] ]Cycloaddition products. Alternatively, alkyne groups on the polymer can react with azide groups present in the targeted polypeptide to form a similar product. In some embodiments, strong nucleophiles (including but not limited to hydrazine, hydrazide, hydroxylamine, semicarbazide) can interact with aldehydes present in the targeted polypeptideThe groups or ketone groups react to form hydrazones, oximes or semicarbazides, which in some cases may be further reduced by treatment with a suitable reducing agent, as the case may be. Alternatively, strong nucleophiles may be incorporated into the targeting polypeptide by non-naturally encoded amino acids and used to preferentially react with ketone or aldehyde groups present in the water soluble polymer.
Any molecular weight of the polymer may be used as desired, including but not limited to about 100 daltons (Da) to 100,000Da or higher as desired (including but not limited to sometimes 0.1-50kDa or 10-40 kDa). The molecular weight of the polymer may be in a wide range including, but not limited to, between about 100Da and about 100,000Da or greater. The polymer may be between about 100 and about 100,000Da, including but not limited to 100,000Da, 95,000Da, 90,000Da, 85,000Da, 80,000Da, 75,000Da, 70,000Da, 65,000Da, 60,000Da, 55,000Da, 50,000Da, 45,000Da, 40,000Da, 35,000Da, 30,000Da, 25,000Da, 20,000Da, 15,000Da, 10,000Da, 9,000Da, 8,000Da, 7,000Da, 6,000Da, 5,000Da, 4,000Da, 3,000Da, 2,000Da, 1,000Da, 900Da, 800Da, 700Da, 600Da, 500Da, 400Da, 300Da, 200Da, and 100Da. In some embodiments, the polymer is between about 100Da and about 50,000 Da. Branched polymers may also be used, including but not limited to polymer molecules having a molecular weight of each strand ranging from 1 to 100kDa (including but not limited to 1 to 50kDa or 5 to 20 kDa). The molecular weight of each chain of the branched polymer may be, including but not limited to, between about 1,000Da and about 100,000Da or greater. The molecular weight of each chain of the branched polymer may be between about 1,000da and about 100,000da, including but not limited to 100,000da, 95,000da, 90,000da, 85,000da, 80,000da, 75,000da, 70,000da, 65,000da, 60,000da, 55,000da, 50,000da, 45,000da, 40,000da, 35,000da, 30,000da, 25,000da, 20,000da, 15,000da, 10,000da, 9,000da, 8,000da, 7,000da, 6,000da, 5,000da, 4,000da, 3,000da, 2,000da, and 1,000da. In some embodiments, the molecular weight of each chain of the branched polymer is between about 1,000da and about 50,000 da. In some embodiments, the molecular weight of each chain of the branched polymer is between about 1,000da and about 40,000 da. In some embodiments, the molecular weight of each chain of the branched polymer is between about 5,000da and about 40,000 da. In some embodiments, the molecular weight of each chain of the branched polymer is between about 5,000da and about 20,000 da. A broad range of polymer molecules are described in, but not limited to, shearwater Polymers, inc. Catalogue, nektar Therapeutics catalogue, which is incorporated herein by reference.
In some embodiments, the present invention provides azide-containing and acetylene-containing polymer derivatives comprising a water-soluble polymer backbone having an average molecular weight of about 800Da to about 100,000 Da. The polymer backbone of the water-soluble polymer may be poly (ethylene glycol). However, it should be understood that a variety of water-soluble polymers including, but not limited to, poly (ethylene glycol) and other related polymers including poly (dextran) and polypropylene glycol are also suitable for use in the practice and use of the term PEG or poly (ethylene glycol) in the present invention are intended to encompass and include all such molecules. The term PEG includes, but is not limited to, any form of polyethylene glycol, including difunctional PEG, multi-arm PEG, derivatized PEG, bifurcated PEG, branched PEG, pendent PEG (i.e., PEG having one or more functional groups pendent to the polymer backbone or related polymers), or PEG having degradable linkages therein.
In addition to these forms of polymers, polymers having weak linkages or degradable linkages in the backbone can also be prepared. For example, polymers having readily hydrolyzable ester linkages in the polymer backbone may be prepared. As shown below, this hydrolysis results in cleavage of the polymer into lower molecular weight fragments: polymer-CO 2 -Polymer-H 2 O.fwdarw.Polymer-CO 2 H+HO-Polymer
Many polymers are also suitable for use in the present invention. In some embodiments, water-soluble polymer backbones having from 2 to about 300 termini are particularly useful in the present invention. Examples of suitable polymers include, but are not limited to, other poly (alkylene glycols) such as polypropylene glycol ("PPG"), copolymers thereof (including, but not limited to, copolymers of ethylene glycol and propylene glycol), terpolymers thereof, mixtures thereof, and the like. Although the molecular weight of each chain of the polymer backbone may vary, it is typically in the range of about 800Da to about 100,000Da, typically in the range of about 6,000Da to about 80,000 Da. The molecular weight of each chain of the polymer backbone can be between about 100Da and about 100,000Da, including but not limited to 100,000Da, 95,000Da, 90,000Da, 85,000Da, 80,000Da, 75,000Da, 70,000Da, 65,000Da, 60,000Da, 55,000Da, 50,000Da, 45,000Da, 40,000Da, 35,000Da, 30,000Da, 25,000Da, 20,000Da, 15,000Da, 10,000Da, 9,000Da, 8,000Da, 7,000Da, 6,000Da, 5,000Da, 4,000Da, 3,000Da, 2,000Da, 1,000Da, 900Da, 800Da, 700Da, 600Da, 500Da, 400Da, 300Da, 200Da and 100Da. In some embodiments, the molecular weight of each chain of the polymer backbone is between about 100Da and about 50,000 Da. In some embodiments, the molecular weight of each chain of the polymer backbone is between about 100Da and about 40,000 Da. In some embodiments, the molecular weight of each strand of the polymer backbone is between about 1,000da and about 40,000 da. In some embodiments, the molecular weight of each strand of the polymer backbone is between about 5,000da and about 40,000 da. In some embodiments, the molecular weight of each strand of the polymer backbone is between about 10,000da and about 40,000 da.
In one feature of this embodiment of the invention, the intact polymer-conjugate is minimally degraded upon administration prior to hydrolysis, such that hydrolysis of the cleavable bond effectively controls the slow rate of release of the active targeting polypeptide into the blood stream, as opposed to enzymatic degradation of the targeting polypeptide prior to its release into the systemic circulation.
Suitable physiologically cleavable linkages include, but are not limited to, esters, carbonates, carbamates, sulfates, phosphates, acyloxyalkyl ethers, acetals and ketals. Such conjugates should have a physiologically cleavable bond that is stable upon storage and administration. For example, a targeting polypeptide linked to a polymer or modified targeting polypeptide should maintain its integrity at the time of manufacture of the final pharmaceutical composition, when dissolved in an appropriate delivery vehicle (if used), and when administered regardless of route. Any of the cleavable linkers disclosed herein may be linked to a drug, payload, targeting polypeptide, or modified targeting polypeptide of the invention. Illustrative examples of connections through cleavable linkers include, but are not limited to:
in some embodiments of the invention, the linker may be a non-cleavable linker linked to the drug, payload, targeting polypeptide, or modified targeting polypeptide. Illustrative examples of connections through non-cleavable linkers include, but are not limited to:
The invention also includes phosphate-based linkers, the stability of which is tunable for intracellular delivery of the drug conjugates disclosed in US 2017/0182181, which is incorporated herein by reference. The phosphate-based linker comprises a mono-, di-, tri-or tetraphosphate group (phosphate group) covalently linked to the distal end of the linker arm and to the reactive functional group, said linker arm comprising a tuning element, optionally a spacer element, in the distal to proximal direction. The phosphate group of the phosphate-based linker can be conjugated to a payload and the reactive functional group can be conjugated to a cell-specific targeting ligand (such as an antibody). The general structure of phosphate-based linkers is: phosphate group-tuning element-optional spacer element-functional reactive group. Phosphate-based linkers conjugated to payloads have the general structure: payload-phosphate group-tuning element-optional spacer element-functional reactive group, and has the general structure when conjugated to a targeting ligand: payload-phosphate group-tuning element-optional spacer element-targeting ligand. These phosphate-based linkers have differential and tunable stability in blood compared to the intracellular environment (e.g., lysosomal compartments). The rate at which phosphate groups are cleaved in the intracellular environment to release the payload in its natural or active form may be influenced by the structure of the tuning element, with other influences being mediated by the substitution of the phosphate groups and whether the phosphate groups are mono-, di-, tri-or tetraphosphates. Furthermore, these phosphate-based linkers provide the ability to construct conjugates, such as antibody-drug conjugates, that have a reduced propensity to form aggregates as compared to conjugates that use linkers other than the phosphate-based linkers disclosed herein to conjugate the same payload to an antibody or targeting ligand.
Structure and synthesis of TLR agonist linker derivatives: electrophilic groups and nucleophilic groups
TLR agonist derivatives having a linker containing a hydroxylamine (also referred to as aminooxy) group allow for reaction with various electrophilic groups, including but not limited to with PEG or other water-soluble polymers, to form conjugates. As with hydrazines, hydrazides, and semicarbazides, the enhanced nucleophilicity of the aminooxy groups enables them to efficiently and selectively react with various molecules containing carbonyl or dicarbonyl groups, including but not limited to ketones, aldehydes, or other functional groups having similar chemical reactivity. See, e.g., shao, J. And Tam, J., J.Am.chem.Soc.117:3893-3899 (1995); H.Hang and C.Bertozzi, acc.Chem.Res.34 (9): 727-736 (2001). However, the result of the reaction with hydrazine groups is the corresponding hydrazone, whereas oximes are typically produced by the reaction of aminoxy groups with carbonyl-or dicarbonyl-containing groups, such as ketones, aldehydes or other functional groups having similar chemical reactivity. In some embodiments, TLR agonist derivatives having a linker comprising an azide, alkyne, or cycloalkyne allow for linking of the molecule by a cycloaddition reaction (e.g., 1, 3-dipolar cycloaddition, azide-alkyne Huisgen cycloaddition, etc.). (described in U.S. patent No. 7,807,619, incorporated herein by reference to the extent that it is relative to the reaction).
Thus, in certain embodiments described herein are TLR agonist derivatives having a linker comprising hydroxylamine, an aldehyde, a protected aldehyde, a ketone, a protected ketone, a thioester, an ester, a dicarbonyl, a hydrazine, an amidine, an imine, a diamine, a ketone-amine, a ketone-alkyne, and an enedione hydroxylamine group, a hydroxylamine-like group (which has similar reactivity and is structurally similar to a hydroxylamine group), a masked hydroxylamine group (which can be easily converted to a hydroxylamine group), or a protected hydroxylamine group (which has similar reactivity to a hydroxylamine group when deprotected). In some embodiments, the TLR agonist derivative having a linker comprises an azide, alkyne, or cycloalkyne.
Such TLR agonist linker derivatives or targeting polypeptides may be in salt form, or may be incorporated into unnatural amino acid polypeptides, polymers, polysaccharides, or polynucleotides, and optionally post-translational modifications.
In certain embodiments, the compounds of formula (I) to (VII) are stable in aqueous solution under mildly acidic conditions for at least 1 month. In certain embodiments, the compounds of formula (I) to (VII) are stable under mildly acidic conditions for at least 2 weeks. In certain embodiments, the compounds of formula (I) to (VII) are stable under mildly acidic conditions for at least 5 days. In certain embodiments, such acidic conditions are pH 2 to 8.
The methods and compositions provided and described herein include polypeptides comprising an unnatural amino acid having at least one carbonyl or dicarbonyl group, an oxime group, a hydroxylamine group, or a protected or masked form thereof. The introduction of at least one reactive group into a TLR agonist linker derivative or targeting polypeptide may allow for the use of conjugation chemistry involving specific chemical reactions, including but not limited to reactions with one or more targeting polypeptides, but not with common amino acids. Once incorporated, the targeting polypeptide of the TC side chain may also be modified by chemical methods utilizing specific functional groups or substituents described herein or applicable to TLR agonist linker derivatives or the presence of the targeting polypeptide.
The TLR agonist linker derivatives and methods and compositions described herein provide conjugates of a substance having a variety of functional groups, substituents, or moieties with other substances (including but not limited to polymers, water-soluble polymers, derivatives of polyethylene glycol, second proteins or polypeptides or polypeptide analogs, antibodies or antibody fragments, and any combination thereof).
In certain embodiments, TLR agonist linker derivatives, targeting polypeptides, TCs, linkers and agents described herein, including compounds of formula (I) through formula (VII), are stable in aqueous solution under mildly acidic conditions, including but not limited to pH 2 to 8. In other embodiments, such compounds are stable under mildly acidic conditions for at least one month. In other embodiments, such compounds are stable under mildly acidic conditions for at least 2 weeks. In other embodiments, such compounds are stable under mildly acidic conditions for at least 5 days.
In another aspect of the compositions, methods, techniques and strategies described herein are methods for studying or using any of the foregoing "modified or unmodified" unnatural amino acid targeting polypeptides. By way of example only, such aspects include therapeutic, diagnostic, assay-based, industrial, cosmetic, plant biology, environmental, energy production, consumer and/or military uses that would benefit from targeting polypeptides comprising "modified or unmodified" non-natural amino acid polypeptides or proteins.
The present invention provides TC molecules comprising at least one unnatural amino acid. In certain embodiments of the invention, TCs having at least one unnatural amino acid include at least one post-translational modification. In one embodiment of the present invention, in one embodiment, at least one post-translational modification comprises a molecule (including but not limited to a label, dye, linker, another TC polypeptide, polymer, water-soluble polymer, polyethylene glycol derivative, photocrosslinker, radionuclide, cytotoxic compound, drug, affinity label, photoaffinity label, active compound, resin, second protein or polypeptide analog, antibody or antibody fragment, metal chelator, cofactor, fatty acid, carbohydrate, polynucleotide, DNA, RNA, antisense polynucleotide, saccharide, cyclodextrin, inhibitory ribonucleic acid, biological material, nanoparticle, spin label, fluorophore, metal-containing moiety, radioactive moiety, novel functional group, group that interacts covalently or non-covalently with other molecules, photocage moiety, actinic radiation excitable moiety, photoisomerization moiety) biotin, biotin derivatives, biotin analogues, moieties incorporating heavy atoms, chemically cleavable groups, photo cleavable groups, elongated side chains, carbon linked sugars, redox active agents, amino thio acids, toxic moieties, isotopically labeled moieties, biophysical probes, phosphorescent groups, chemiluminescent groups, electron dense groups, magnetic groups, intercalating groups, chromophores, energy transfer agents, bioactive agents, detectable labels, small molecules, quantum dots, nanoemitters, radionucleotides, radioactive emitters, neutron capture agents, or any combination of the foregoing or any other desired compound or substance) are attached to at least one unnatural amino acid comprising a first reactive group using chemical methods known to one of ordinary skill in the art as suitable for the particular reactive group, the first reactive group is an alkynyl moiety (including but not limited to p-propargyloxyphenylalanine in unnatural amino acids, where propargyl is sometimes also referred to as an ethynyl group) and the second reactive group is an azido moiety and uses [3+2] cycloaddition chemistry. In another example, the first reactive group is an azido moiety (including but not limited to p-azido-L-phenylalanine in unnatural amino acids or pAZ as sometimes referred to herein) and the second reactive group is an alkynyl moiety. In certain embodiments of the modified TCs of the present invention, at least one unnatural amino acid (including but not limited to a unnatural amino acid with a keto-functional group) is used that comprises at least one post-translational modification, where the at least one post-translational modification comprises a sugar moiety. In certain embodiments, the post-translational modification is performed in vivo in a eukaryotic cell or a non-eukaryotic cell. A linker, polymer, water-soluble polymer, or other molecule may connect the molecule to the polypeptide. In another embodiment, the linker attached to TC is long enough to allow formation of dimers. The molecule may also be directly linked to the polypeptide.
In certain embodiments, the TC protein comprises at least one post-translational modification made in vivo by one host cell, wherein the post-translational modification is not typically made by another host cell type. In certain embodiments, the protein comprises at least one post-translational modification made in vivo by a eukaryotic cell, wherein the post-translational modification is not typically made by a non-eukaryotic cell. Examples of post-translational modifications include, but are not limited to, glycosylation, acetylation, acylation, lipid-modification, palmitoylation, palmitate addition, phosphorylation, glycolipid-linkage modification, and the like.
In some embodiments, TC comprises one or more non-naturally encoded amino acids for glycosylation, acetylation, acylation, lipid-modification, palmitoylation, palmitate addition, phosphorylation, or glycolipid-linkage modification of a polypeptide. In some embodiments, TC comprises one or more non-naturally encoded amino acids for glycosylation of a polypeptide. In some embodiments, TC comprises one or more naturally encoded amino acids for glycosylation, acetylation, acylation, lipid-modification, palmitoylation, palmitate addition, phosphorylation, or glycolipid-linkage modification of a polypeptide. In some embodiments, TC comprises one or more naturally encoded amino acids for glycosylation of a polypeptide.
In some embodiments, TCs comprise one or more non-naturally encoded amino acid additions and/or substitutions that enhance glycosylation of the polypeptide. In some embodiments, TC comprises one or more deletions that enhance glycosylation of the polypeptide. In some embodiments, TCs comprise one or more non-naturally encoded amino acid additions and/or substitutions that enhance glycosylation at different amino acids in the polypeptide. In some embodiments, TC comprises one or more deletions that enhance glycosylation at different amino acids in the polypeptide. In some embodiments, TC comprises one or more non-naturally encoded amino acid additions and/or substitutions that enhance glycosylation at the non-naturally encoded amino acid in the polypeptide. In some embodiments, TC comprises one or more non-naturally encoded amino acid additions and/or substitutions that enhance glycosylation of naturally encoded amino acids in a polypeptide. In some embodiments, TCs comprise one or more naturally encoded amino acid additions and/or substitutions that enhance glycosylation at different amino acids in the polypeptide. In some embodiments, TC comprises one or more non-naturally encoded amino acid additions and/or substitutions that enhance glycosylation of naturally encoded amino acids in a polypeptide. In some embodiments, TC comprises one or more non-naturally encoded amino acid additions and/or substitutions that enhance glycosylation at the non-naturally encoded amino acid in the polypeptide.
In one embodiment, the post-translational modification includes the inclusion of an oligosaccharide (including, but not limited to, wherein the oligosaccharide comprises (GlcNAc-Man) 2 -Man-GlcNAc-GlcNAc, etc.) through GThe lcNAc-asparagine linkage is linked to asparagine. In another embodiment, post-translational modification includes linking an oligosaccharide (including but not limited to Gal-GalNAc, gal-GlcNAc, etc.) to serine or threonine through a GalNAc-serine, galNAc-threonine, glcNAc-serine or GlcNAc-threonine linkage. In certain embodiments, the proteins or polypeptides of the invention may comprise secretion or localization sequences, epitope tags, FLAG tags, polyhistidine tags, GST fusions, and the like. Examples of secretion signal sequences include, but are not limited to, prokaryotic secretion signal sequences, eukaryotic secretion signal sequences that are 5' -optimized for bacterial expression, novel secretion signal sequences, pectate lyase secretion signal sequences, omp a secretion signal sequences, and phage secretion signal sequences. Examples of secretion signal sequences include, but are not limited to, STII (prokaryotes), fd GIII and M13 (phages), bg12 (yeast), and signal sequences bla derived from transposons. Any such sequence may be modified to provide the desired result of the polypeptide, including, but not limited to, replacing one signal sequence with a different signal sequence, replacing a leader sequence with a different leader sequence, and the like.
The protein or polypeptide of interest may contain at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or ten or more unnatural amino acids. The unnatural amino acids can be the same or different, e.g., there can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different sites in the protein that comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different unnatural amino acids. In certain embodiments, at least one but less than all of the specific amino acids present in the naturally occurring version of the protein are substituted with unnatural amino acids.
The present invention provides methods and compositions based on TCs comprising at least one non-naturally encoded amino acid. The introduction of at least one non-naturally encoded amino acid into TC may allow applications involving specific chemical reactions, including but not limited to conjugation chemistry that react with one or more non-naturally encoded amino acids, but not with the common 20 amino acids. In some embodiments, TCs comprising non-naturally encoded amino acids are attached to a water soluble polymer, such as polyethylene glycol (PEG) or a linker, through side chains of the non-naturally encoded amino acids. The present invention provides a highly efficient method for selectively modifying proteins with PEG derivatives or TLR linker derivatives involving the selective incorporation of non-genetically encoded amino acids, including but not limited to those amino acids containing functional groups or substituents not found in 20 naturally incorporated amino acids, including but not limited to ketone, azide or acetylene moieties, which enter the protein in response to a selector codon and subsequently modify these amino acids with PEG derivatives of appropriate reactivity. Once incorporated, the amino acid side chains can then be modified by chemical methods known to those of ordinary skill in the art as being suitable for the particular functional groups or substituents present in the non-naturally encoded amino acid. A wide variety of known chemical methods are suitable for use in the present invention to incorporate water-soluble polymers into proteins. Such methods include, but are not limited to, the generation of Huisgen [3+2] cycloaddition reactions with, respectively, including, but not limited to, acetylene or azide derivatives (see, e.g., padwa, A., comprehensive Organic Synthesis, vol. 4, (1991) pp. Trost, B.M., pergamon, oxford, pp. 1069-1109, and Huisgen, R. 1,3-Dipolar Cycloaddition Chemistry, (1984) Padwa, A., wiley, new York, pp. 1-176).
Since the Huisgen [3+2] cycloaddition method involves cycloaddition rather than nucleophilic substitution reaction, proteins can be modified with extremely high selectivity. By adding a catalytic amount of a Cu (I) salt to the reaction mixture, the reaction can be carried out at room temperature under aqueous conditions with excellent regioselectivity (1, 4>1, 5). See, e.g., tornoe et al, (2002) J.org.chem.67:3057-3064; and Rostvtsev, et al, (2002) Angew.chem.int.ed.41:2596-2599; WO 03/101972. Molecules that may be added to the proteins of the present invention by [3+2] cycloaddition include virtually any molecule having suitable functional groups or substituents, including but not limited to azido or acetylene derivatives. These molecules may be added to unnatural amino acids having an ethynyl group, including but not limited to p-propargyloxyphenylalanine or azido groups (including but not limited to p-azidophenylalanine), respectively.
Five-membered rings produced by Huisgen [3+2] cycloaddition are generally irreversible in a reducing environment and stable against hydrolysis in an aqueous environment for a prolonged period of time. Thus, the physical and chemical properties of a variety of substances can be modified with the active PEG derivatives or TLR-linker derivatives of the invention under harsh aqueous conditions. More importantly, since the azide and acetylene moieties are specific to each other (e.g., do not react with any of the 20 common gene-encoded amino acids), the protein can be modified at one or more specific sites with extremely high selectivity.
The invention also provides water-soluble and hydrolytically stable derivatives of PEG derivatives or TLR-linker derivatives, and related hydrophilic polymers having one or more acetylene or azide moieties. PEG polymer derivatives containing acetylene moieties are highly selective for coupling with azide moieties selectively incorporated into proteins in response to selector codons. Similarly, PEG polymer derivatives containing azide moieties are highly selective for coupling with acetylene moieties that are selectively incorporated into proteins in response to a selector codon. More specifically, azide moieties include, but are not limited to, alkyl azides, aryl azides, and derivatives of these azides. Derivatives of alkyl azide and aryl azide may include other substituents as long as acetylene specific reactivity is maintained. Acetylene moieties include alkyl and aryl acetylenes and their respective derivatives. Derivatives of alkyl and aryl acetylenes may include other substituents, so long as azide-specific reactivity is maintained.
The present invention provides conjugates of a substance having a variety of functional groups, substituents or moieties with other substances, including but not limited to labels; a dye; a polymer; a water-soluble polymer; derivatives of polyethylene glycol; a photocrosslinking agent; a radionuclide; a cytotoxic compound; a drug; an affinity tag; a photoaffinity tag; an active compound; a resin; a second protein or polypeptide analog; an antibody or antibody fragment; a metal chelator; a cofactor; a fatty acid; a carbohydrate; a polynucleotide; DNA; RNA; an antisense polynucleotide; a saccharide; a water-soluble dendritic polymer; cyclodextrin; inhibitory ribonucleic acid; a biological material; a nanoparticle; spin labeling; a fluorophore, a metal-containing moiety; a radioactive portion; novel functional groups; groups that interact covalently or non-covalently with other molecules; a light cage portion; an actinic radiation excitable moiety; a photoisomerization moiety; biotin; derivatives of biotin; biotin analogues; incorporation of heavy atom moieties; a chemically cleavable group; a photocleavable group; an elongated side chain; a carbon-linked sugar; a redox active agent; an aminothiopropionic acid; a toxic moiety; isotopically labeled moieties; a biophysical probe; phosphorescent groups; a chemiluminescent group; an electron dense group; a magnetic group; an intercalating group; a chromophore; an energy transfer agent; a bioactive agent; a detectable label; a small molecule; a quantum dot; a nano-emitter; a radionucleotide; a radioactive emitter; a neutron capture agent; or any combination of the above, or any other desired compound or substance. The invention also includes conjugates of a substance having an azide or acetylene moiety with a PEG polymer derivative having a corresponding acetylene or azide moiety. For example, a PEG polymer containing azide moieties may be coupled to a biologically active molecule at a position in a protein containing a non-genetically encoded amino acid bearing an acetylene functional group. Linkages coupling PEG to bioactive molecules include, but are not limited to, huisgen [3+2] cycloaddition products.
PEG can be used to modify the surface of biological materials, which is well documented in the art (see, e.g., U.S. Pat. No. 6,610,281;Mehvar,R,J.Pharm Sci, 3 (1): 125-136 (2000), which is incorporated herein by reference). The invention also includes a biomaterial comprising a surface having one or more reactive azide or acetylene sites and one or more azide-containing or acetylene-containing polymers of the invention coupled to the surface by a Huisgen [3+2] cycloaddition bond. Biological materials and other substances may also be coupled with azide or acetylene activated polymer derivatives by linkages other than azide linkages or acetylene linkages, such as linkages comprising carboxylic acid, amine, alcohol, or thiol moieties, to leave the azide or acetylene moiety available for subsequent reactions.
The present invention includes a method of synthesizing the azide-containing and acetylene-containing polymers of the present invention. In the case of azide-containing PEG derivatives, the azide may be directly bonded to a carbon atom of the polymer. Alternatively, the azide-containing PEG derivative may be prepared by attaching a linker having an azide moiety at one end to a conventionally activated polymer such that the resulting polymer has an azide moiety at its end. In the case of PEG derivatives containing acetylene, the acetylene may be directly bonded to a carbon atom of the polymer. Alternatively, acetylene-containing PEG derivatives may be prepared by attaching a linker having an acetylene moiety at one end to a conventionally activated polymer such that the resulting polymer has an acetylene moiety at its end.
More specifically, in the case of azide-containing PEG derivatives, a water-soluble polymer having at least one active hydroxyl moiety undergoes a reaction to produce a substituted polymer having more reactive moieties thereon, such as mesylate, triflate monomethoxy, tosylate, or halogen leaving groups. The preparation and use of PEG derivatives or TLR-linker derivatives containing a sulfonyl halide, halogen atom and other leaving groups are known to those of ordinary skill in the art. The resulting substituted polymer then undergoes a reaction to replace the more reactive portion of the azide moiety at the end of the polymer. Alternatively, a water-soluble polymer having at least one active nucleophilic or electrophilic moiety is reacted with a linker having an azide at one end, such that a covalent bond is formed between the PEG polymer and the linker, and the azide moiety is located at the end of the polymer. Nucleophilic and electrophilic moieties, including amines, thiols, hydrazides, hydrazines, alcohols, carboxylic esters, aldehydes, ketones, thioesters, and the like, are known to one of ordinary skill.
More specifically, in the case of acetylene containing PEG derivatives, a water soluble polymer having at least one active hydroxyl moiety undergoes a reaction to displace a halogen or other activated leaving group from a precursor containing an acetylene moiety. Alternatively, a water-soluble polymer having at least one active nucleophilic or electrophilic moiety is reacted with a linker having acetylene at one end, such that a covalent bond is formed between the PEG polymer and the linker, and the acetylene moiety is located at the end of the polymer. The use of halogen moieties, activated leaving groups, nucleophilic and electrophilic moieties in the context of organic synthesis, and the preparation and use of PEG derivatives or TLR-linker derivatives are well known to practitioners in the art.
The invention also provides methods of selectively modifying proteins by adding other substances to the modified proteins, including but not limited to water soluble polymers such as PEG and PEG derivatives or TLR-linker derivatives, linkers or other TC polypeptides containing azide or acetylene moieties. The azide and acetylene containing PEG derivatives or TLR-linker derivatives can be used to modify the properties of surfaces and molecules where biocompatibility, stability, solubility and lack of immunogenicity are important, while providing a more selective method of attaching PEG derivatives or TLR-linker derivatives to proteins than previously known in the art.
General recombinant nucleic acid methods for use in the present invention
In many embodiments of the invention, nucleic acids encoding target polypeptides of interest TC will be isolated, cloned and often altered using recombinant methods. Such embodiments are useful, including but not limited to, for protein expression or during the production of variants, derivatives, expression cassettes, or other sequences derived from TC targeting polypeptides. In some embodiments, the sequence encoding a polypeptide of the invention is operably linked to a heterologous promoter.
The nucleotide sequence encoding the targeting polypeptide comprising a non-naturally encoded amino acid TC may be synthesized on the basis of the amino acid sequence of the parent polypeptide and then altered to effect the introduction (i.e., incorporation or substitution) or removal (i.e., deletion or substitution) of the relevant amino acid residue. The nucleotide sequence may be conveniently modified by site-directed mutagenesis according to conventional methods. Alternatively, the nucleotide sequence may be prepared by chemical synthesis, including but not limited to by using an oligonucleotide synthesizer, wherein the oligonucleotides are designed based on the amino acid sequence of the desired polypeptide, and preferably those codons are selected that are advantageous in the host cell in which the recombinant polypeptide is produced. For example, several small oligonucleotides encoding the desired polypeptide moiety may be synthesized and assembled by PCR, ligation, or ligation chain reaction. See, e.g., barany, et al, proc. Natl. Acad. Sci.88:189-193 (1991); U.S. Pat. No. 6,521,427, incorporated herein by reference.
The present invention utilizes conventional techniques in the field of recombinant genetics. Basic articles that disclose the general methods used in the present invention include Sambrook et al Molecular Cloning, ALaboratory Manual (3 rd edition 2001); kriegler, gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al, eds., 1994).
The invention also relates to eukaryotic host cells, non-eukaryotic host cells and organisms for in vivo incorporation of unnatural amino acids via orthogonal tRNA/RS pairs. Host cells are genetically engineered (including but not limited to transformed, transduced or transfected) with a polynucleotide of the invention or a construct including a polynucleotide of the invention, including but not limited to a vector of the invention (which may be, for example, a cloning vector or an expression vector).
Several well known methods of introducing a target nucleic acid into a cell are available, any of which can be used in the present invention. These methods include: fusion of recipient cells with DNA-containing bacterial protoplasts, electroporation, ballistic bombardment, and viral vector infection (discussed further below), and the like. Bacterial cells can be used to amplify the number of plasmids containing the DNA constructs of the invention. The bacteria grow to log phase and plasmids within the bacteria can be isolated by a variety of methods known in the art (see, e.g., sambrook). In addition, kits for purifying plasmids from bacteria are commercially available (see, e.g., easyPrep TM 、FlexiPrep TM All from Pharmacia Biotech; strataClean TM From Stratagene; QIAprep TM From Qiagen). The isolated and purified plasmid is then further manipulated to produce additional plasmids for use in transfecting cells or incorporating the relevant vector to infect an organism. Typical vectors contain transcriptional and translational terminators useful for regulating expression of a particular target nucleic acidTranscription and translation promoter sequences and promoters. The vector optionally comprises a universal expression cassette containing at least one independent terminator sequence, sequences allowing replication of the cassette in eukaryotes or prokaryotes or both (including but not limited to shuttle vectors), and selection markers for prokaryotic and eukaryotic systems. The vector is suitable for replication and integration in prokaryotes, eukaryotes, or both. See Gillam&Smith, gene 8:81 (1979); roberts et al, nature,328:731 (1987); schneider, E., et al, protein expr. Purif.6 (1): 10-14 (1995); ausubel, sambrook, berger (all supra). The list of bacteria and phages that can be used for cloning is provided by, for example, ATCC, for example, the list of bacteria and phages (1992) Gsouthern a et al (code) ATCC published by ATCC. Other basic procedures for sequencing, cloning and other aspects of molecular biology and basic theoretical considerations are also found in Watson et al (1992) Recombinant DNA Second Edition Scientific American Books, NY. In addition, substantially any nucleic acid (and almost any labeled nucleic acid, whether standard or non-standard) may be customized or ordered from various commercial sources, such as Midland Certified Reagent Company (Midland, TX, mcrc.com accessible on the world wide web), the Great American Gene Company (Ramona, CA, genco.com accessible on the world wide web), expressGen inc (Chicago, IL, expressgen.com accessible on the world wide web), operon Technologies inc (Alameda, CA), and many other companies.
Selecting codons
The selector codons of the invention amplify the genetic codon framework of the protein biosynthesis machinery. For example, selector codons include, but are not limited to, unique three base codons, nonsense codons (such as stop codons, including but not limited to, amber codons (UAG), ocher codons, or opal codons (UGA)), unnatural codons, four or more base codons, rare codons, and the like. It will be apparent to one of ordinary skill in the art that the number of selector codons that can introduce a desired gene or polynucleotide into a single polynucleotide encoding at least a portion of a TC can be varied widely, including but not limited to one or more, two or more, three or more, 4, 5, 6, 7, 8, 9, 10 or more.
In one embodiment, the method involves the use of a selector codon, which is a stop codon for the incorporation of one or more unnatural amino acids in vivo. For example, an O-tRNA that recognizes a stop codon, including but not limited to UAG, is produced and aminoacylated by an O-RS with the desired unnatural amino acid. Such O-tRNA is not recognized by the aminoacyl-tRNA synthetase of the natural host. Conventional site-directed mutagenesis can be used to introduce a stop codon at the target site of the target polypeptide, including, but not limited to, TAG. See, e.g., sayers, J.R., et al (1988), 5'-3'Exonucleases in phosphorothioate-based oligonucleotide-directed mutagenesis, nucleic Acids Res,16:791-802. When the O-RS, O-tRNA and nucleic acid that encodes the polypeptide of interest are combined in vivo, an unnatural amino acid is incorporated in response to the UAG codon to produce a polypeptide that contains the unnatural amino acid at the indicated position.
Unnatural amino acids can be incorporated in vivo without significantly perturbing the eukaryotic host cell. For example, because the suppression efficiency of a UAG codon depends on competition between an O-tRNA (including but not limited to an amber suppressor tRNA) and a eukaryotic release factor (including but not limited to eRF), which binds to a stop codon and initiates release of a growth peptide from the ribosome, the suppression efficiency can be modulated by, including but not limited to, increasing the expression level of the O-tRNA and/or the suppressor tRNA.
Unnatural amino acids can also be encoded with rare codons. For example, the rare arginine codon AGG has been shown to be effective in inserting Ala through an alanine acylated synthetic tRNA when the arginine concentration is reduced in an in vitro protein synthesis reaction. See, e.g., ma et al, biochemistry,32:7939 (1993). In this case, the synthetic tRNA competes with the naturally occurring tRNAArg, which is present as a minor species in E.coli. Some organisms do not use all triplet codons. Unassigned codon AGA in Micrococcus luteus (Micrococcus luteus) has been used to insert amino acids in vitro transcription/translation extracts. See, e.g., kowal and Oliver, nucleic. Acid. Res.,25:4685 (1997). The components of the invention can be generated to use these rare codons in vivo.
Selector codons also include extended codons including, but not limited to, four or more base codons, such as four, five, six or more base codons. Examples of four base codons include, but are not limited to AGGA, CUAG, UAGA, CCCU and the like. Examples of five base codons include, but are not limited to AGGAC, CCCCU, CCCUC, CUAGA, CUACU, UAGGC and the like. One feature of the present invention includes the use of extended codons based on frameshift suppression. Four or more base codons may be inserted into the same protein including, but not limited to, one or more unnatural amino acids. For example, in the presence of a mutated O-tRNA, including but not limited to a special frameshift suppressor tRNA with an anticodon loop (e.g., with an anticodon loop of at least 8-10 nt), four or more base codons are read as a single amino acid. In other embodiments, the anticodon loop may be decoded, including but not limited to at least one four base codon, at least one five base codon, or at least one six base codon or more. Since there are 256 possible four base codons, four or more base codons can be used to encode multiple unnatural amino acids in the same cell. See Anderson et al, (2002) Exploring the Limits of Codon and Anticodon Size, chemistry and Biology,9:237-244; magliry, (2001) Expanding the Genetic Code: selection of Efficient Suppressors of Four-base Codons and Identification of "Shift" Four-base Codons with a Library Approach in Escherichia coli, J.mol.biol.307:755-769.
For example, four base codons have been used to incorporate unnatural amino acids into proteins using in vitro biosynthetic methods. See, e.g., ma et al, (1993) Biochemistry,32:7939; and Hohsaka et al, (1999) J.am.chem.Soc.,121:34.CGGG and AGGU were used to incorporate both NBD derivatives of 2-naphthylalanine and lysine with two chemically acylated frameshift suppressor trnas into streptavidin in vitro. See, e.g., hohsaka et al, (1999) j.am.chem.soc.,121:12194. In one in vivo study Moore et al examined the ability of tRNALeu derivatives with NCUA anti-codons to suppress UAGN codons (N could be U, A, G or C) and found that the tetrad UAGA could be decoded with 13% to 26% efficiency by tRNALeu with UCUA anti-codons, with little decoding in the 0 or-1 boxes. See Moore et al, (2000) j.mol.biol.,298:195. In one embodiment, the invention may use extended codons based on rare codons or nonsense codons, which may reduce missense read-through and frameshift suppression of other undesired sites.
The selector codon can also include one of the natural three base codons for a given system, where the endogenous system does not use (or uses little) the natural base codon. For example, this includes systems that lack tRNA that recognizes the natural three base codon, and/or systems where the three base codon is a rare codon.
The selector codon optionally includes unnatural base pairs. These unnatural base pairs further extend the existing genetic alphabet. One extra base pair increases the number of triplet codons from 64 to 125. The characteristics of the third base pair include stable selective base pairing, efficient enzymatic incorporation of DNA by a polymerase with high fidelity, and efficient continuous primer extension after synthesis of nascent unnatural base pairs. Descriptions of unnatural base pairs that can be suitable for use in methods and compositions include, for example, hirao et al, (2002) An unnatural base pair for incorporating amino acid analogues into protein, nature Biotechnology,20:177-182. See also Wu, Y.et al, (2002) J.am.chem.Soc.124:14626-14630. Other related publications are listed below.
For in vivo use, the unnatural nucleoside is membrane permeable and is phosphorylated to form the corresponding triphosphate. In addition, the increased genetic information is stable and is not destroyed by cellular enzymes. Previous efforts by Benner and others have utilized a different pattern of hydrogen bonding than the typical Watson-Crick pair, with the most notable example being the iso-C: iso-G pair. See, e.g., switzer et al, (1989) j.am.chem.soc.,111:8322; and Picccirili et al, (1990) Nature,343:33; kool, (2000) Curr.Opin.chem.biol.,4:602. These bases are often mismatched to some extent with the natural base and cannot be enzymatically replicated. Kool and colleagues demonstrate that hydrophobic stacking interactions between bases can replace hydrogen bonding to drive base pair formation. See Kool, (2000) curr. Opin. Chem. Biol.,4:602; and Guckian and Kool, (1998) Angew.chem.int.ed.Engl.,36,2825). To develop unnatural base pairs that meet all of the above requirements, schultz, romesberg and colleagues systematically synthesized and studied a range of unnatural hydrophobic bases. PICS was found to be more stable against natural base pairs by itself and can be efficiently incorporated into DNA by the Klenow fragment of E.coli DNA polymerase I (KF). See, e.g., mcMinn et al, (1999) J.am.chem.Soc.,121:11585-6; and Ogawa et al, (2000) j.am.chem.soc.,122:3274. The 3mn:3mn self-pair can be synthesized by KF with sufficient efficiency and selectivity to satisfy biological functions. See, e.g., ogawa et al, (2000) j.am.chem.soc.,122:8803. However, both bases act as chain terminators for further replication. Recently, a mutant DNA polymerase has evolved that can be used to replicate PICS self-pairs. In addition, the 7AI self-pair can be replicated. See, e.g., tae et al, (2001) J.am.chem.Soc.,123:7439. A novel metal base pair, dipic: py, was also developed that forms a stable pair when bound to Cu (II). See Meggers et al, (2000) j.am.chem.soc.,122:10714. Because the extension codons and unnatural codons are essentially orthogonal to the natural codons, the methods of the invention can take advantage of this property to generate orthogonal tRNA's for them.
Translation bypass systems can also be used to incorporate unnatural amino acids into desired polypeptides. In the translational bypass system, a large sequence is incorporated into a gene, but is not translated into a protein. The sequence contains a structure that can serve as a cue for inducing ribosome skipping and restoring insertion into downstream translation.
Nucleic acid molecules encoding target polypeptides, such as TC, can be readily mutated to introduce cysteines at any desired position in the polypeptide. Cysteine is widely used to introduce reactive molecules, water-soluble polymers, proteins, or a variety of other molecules into a protein of interest. Methods suitable for incorporating cysteines into a desired position of a polypeptide are known to those of ordinary skill in the art, such as those described in U.S. patent No. 6,608,183 (which is incorporated herein by reference) and standard mutagenesis techniques.
III. non-naturally encoded amino acids
A very wide variety of non-naturally encoded amino acids are suitable for use in the present invention. Any number of non-naturally encoded amino acids may be introduced into TC. In general, the introduced unnatural encoded amino acids are essentially chemically inert to the 20 common genetically encoded amino acids (i.e., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine). In some embodiments, the non-naturally encoded amino acid includes side chain functional groups that react efficiently and selectively with functional groups not found in the 20 common amino acids (including, but not limited to, azido, keto, aldehyde, and aminoxy groups) to form stable conjugates. For example, a targeting polypeptide comprising a non-naturally encoded amino acid TC containing an azido functional group may be reacted with a polymer (including but not limited to poly (ethylene glycol) or alternatively, a second polypeptide or linker containing an alkyne moiety) to form a stable conjugate due to the selective reaction of azide with alkyne functional groups to form a Huisgen [3+2] cycloaddition product.
The general structure of the α -amino acid is shown below (formula I):
non-naturally encoded amino acids are generally any structure having the formula above, wherein the R group is any substituent other than the ones used in the twenty natural amino acids, and are suitable for use in the present invention. Because the non-naturally encoded amino acids of the invention are generally different from the natural amino acids only in side chain structure, the non-naturally encoded amino acids form amide bonds with other amino acids (including but not limited to naturally or non-naturally encoded) in the same manner they form in naturally occurring polypeptides. However, non-naturally encoded amino acids have side chain groups that distinguish them from natural amino acids. For example, R optionally includes alkyl-, aryl-, acyl-, keto-, azido-, hydroxy-, hydrazine, cyano-, halo-, hydrazide, alkenyl, alkynyl, ether, thiol, seleno-, sulfonyl-, boric acid, borate, phospho, phosphono, phosphine, heterocycle, enone, imine, aldehyde, ester, thioacid, hydroxylamine, amino, and the like, or any combination thereof. Other non-naturally occurring target amino acids that may be suitable for use in the present invention include, but are not limited to, amino acids comprising photoactivatable cross-linkers, spin-labeled amino acids, fluorescent amino acids, metal-binding amino acids, metal-containing amino acids, radioactive amino acids, amino acids with novel functional groups, amino acids that interact covalently or non-covalently with other molecules, photocage and/or photoisomerization amino acids, amino acids comprising biotin or biotin analogues, glycosylated amino acids (such as sugar-substituted serine), other carbohydrate-modified amino acids, keto-containing amino acids, amino acids comprising polyethylene glycol or polyethers, heavy atom-substituted amino acids, chemically cleavable and/or photocleavable amino acids, amino acids with extended side chains compared to natural amino acids (including, but not limited to polyethers or long chain hydrocarbons, including but not limited to, greater than about 5 or greater than about 10 carbons), carbon-linked sugar-containing amino acids, redox-active amino acids, amino-thio acids, and amino acids comprising one or more toxic moieties.
Exemplary non-naturally encoded amino acids that may be suitable for use in the present invention and for reaction with the water soluble polymer include, but are not limited to, those having carbonyl, aminooxy, hydrazine, hydrazide, semicarbazide, azide, and alkyne reactive groups. In some embodiments, the non-naturally encoded amino acid comprises a sugar moiety. Examples of such amino acids include N-acetyl-L-glucosamine-L-serine, N-acetyl-L-galactosamine-L-serine, N-acetyl-L-glucosamine-L-threonine, N-acetyl-L-glucosamine-L-asparagine, and O-mannosamine-L-serine. Examples of such amino acids also include examples in which naturally occurring N-linkages or O-linkages between amino acids and sugars are replaced by covalent bonds that are unusual in nature, including but not limited to olefins, oximes, thioethers, amides, and the like. Examples of such amino acids also include sugars that are not common in naturally occurring proteins, such as 2-deoxy-glucose, 2-deoxy galactose, and the like.
Many of the non-naturally encoded amino acids provided herein are commercially available, for example, from Sigma-Aldrich (St.Louis, MO, USA), novabiochem (a division of EMD Biosciences, darmstadt, germany) or Peptech (Burlington, mass., USA). Those amino acids that are not commercially available are optionally synthesized as provided herein or using standard methods known to those of ordinary skill in the art. For organic synthesis techniques see, e.g., festendon and festendon Organic Chemistry, (1982, second edition, willard Grant Press, boston mass.); advanced Organic Chemistry of March (third edition, 1985,Wiley and Sons,New York); and Advanced Organic Chemistry by Carey and Sundberg (third edition, section A and section B,1990,Plenum Press,New York). See also U.S. Pat. nos. 7,045,337 and 7,083,970, which are incorporated herein by reference. In addition to unnatural amino acids containing novel side chains, unnatural amino acids useful in the invention optionally comprise modified backbone structures, including, but not limited to, those shown in formulas II and III:
Wherein Z generally comprises OH, NH 2 SH, NH-R 'or S-R'; x and Y, which may be the same or different, typically comprise S or O, and optionally R and R', which may be the same or different, are typically selected from the same list of components as the above-described unnatural amino acid of formula I and the R group of hydrogen. For example, the unnatural amino acids of the invention optionally comprise substitutions in the amino or carboxyl groups as shown in formulas II and III. Unnatural amino acids of this type include, but are not limited to, alpha-hydroxy acids, alpha-thio acids, alpha-aminothiocarboxylic acid esters, including, but not limited to, side chains having side chains corresponding to the twenty common natural amino acids or unnatural side chains. In addition, the substitution on the α -carbon optionally includes, but is not limited to L, D or α - α -disubstitutedAmino acids such as D-glutamic acid, D-alanine, D-methyl-O-tyrosine, aminobutyric acid, and the like. Other structural substitutions include cyclic amino acids such as proline analogs and 3, 4, 6, 7, 8 and 9 membered ring proline analogs, β and γ amino acids such as substituted β -alanine and γ -aminobutyric acid.
Many unnatural amino acids are based on natural amino acids, such as tyrosine, glutamine, phenylalanine, and the like, and are suitable for use in the present invention. Tyrosine analogs include, but are not limited to, para-substituted tyrosine, ortho-substituted tyrosine, and meta-substituted tyrosine, wherein the substituted tyrosine comprises, but is not limited to, keto (including, but not limited to, acetyl), benzoyl, amino, hydrazine, hydroxylamine, thiol, carboxyl, isopropyl, methyl, C 6 -C 20 Linear or branched hydrocarbons, saturated or unsaturated hydrocarbons, O-methyl, polyether groups, nitro groups, alkynyl groups, and the like. In addition, polysubstituted aryl rings are also contemplated. Glutamine analogs suitable for use in the present invention include, but are not limited to, alpha-hydroxy derivatives, gamma-substituted derivatives, cyclic derivatives, and amide substituted glutamine derivatives. Exemplary phenylalanine analogs that can be used in the present invention include, but are not limited to, para-substituted phenylalanine, ortho-substituted phenylalanine, and meta-substituted phenylalanine, wherein the substituents include, but are not limited to, hydroxy, methoxy, methyl, allyl, aldehyde, azide, iodide, bromide, ketone (including, but not limited to, acetyl), benzoyl, alkynyl, and the like. Specific examples of unnatural amino acids that can be used in the present invention include, but are not limited to, p-acetyl-L-phenylalanine, O-methyl-L-tyrosine, L-3- (2-naphthyl) alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcβ -serine, L-Dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-iodophenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, and p-propargyloxy-phenylalanine, and the like. The structures of various unnatural amino acids that can be adapted for use in the invention Examples are provided, for example, in WO 2002/085923 entitled "In vivo incorporation of unnatural amino acids". See also Kiick et al, (2002) Incorporation of azides into recombinant proteins for chemoselective modification by the Staudinger ligation, PNAS 99:19-24, incorporated herein by reference for additional methionine analogs. International application No. PCT/US06/47822, entitled "Compositions Containing, methods Involving, and Users of Non-natural Amino Acids and Polypeptides," which is incorporated herein by reference, describes reductive alkylation and reductive amination of aromatic amine moieties, including but not limited to para-amino-phenylalanine.
In another embodiment of the invention, TC polypeptides having one or more non-naturally encoded amino acids are covalently modified. Selective chemical reactions orthogonal to the various functions of biological systems are considered important tools in chemical biology. As a relatively new member of the field of synthetic chemistry, these bioorthogonal reactions motivate new strategies for compound library synthesis, protein engineering, functional proteomics, and cell surface chemical remodeling. Azide plays an important role as a unique bioconjugation chemical treatment agent. Staudinger ligation has been used with phosphines to label azido sugars metabolically introduced into cell glycoconjugates. The Staudinger ligation can be performed in vivo in living animals without causing physiological damage; however, the Staudinger reaction is not without responsibility. The necessary phosphines are susceptible to air oxidation and their optimization in terms of improving water solubility and improving reaction rates has proven to be a comprehensive challenge.
The azide group has another bio-orthogonal reaction mode: huisgen describes a [3+2] cycloaddition reaction with alkynes. In its classical form, the reaction has limited applicability in biological systems due to the elevated temperatures (or pressures) required for reasonable reaction rates. Sharpless and colleagues have overcome this obstacle and developed a copper (I) catalyzed version, known as "click chemistry", which can be readily performed in biological environments with a abundance of physiological temperatures and functions. This discovery makes possible the selective modification of viral particles, nucleic acids and proteins from complex tissue lysates. Unfortunately, the mandatory copper catalysts are toxic to bacteria and mammalian cells, thus eliminating the application where the cells must remain viable. The catalyst-free Huisgen cycloaddition of alkynes activated by electron withdrawing substituents is reported to occur at ambient temperature. However, these compounds undergo Michael reactions with biological nucleophiles.
In one embodiment, a composition is provided that includes a targeting polypeptide that includes a TC of an unnatural amino acid, such as p- (propargyloxy) -phenylalanine. Also provided are various compositions comprising p- (propargyloxy) -phenylalanine and including, but not limited to, proteins and/or cells. In one aspect, the composition comprising a p- (propargyloxy) -phenylalanine unnatural amino acid further comprises an orthogonal tRNA. The unnatural amino acid can be bound to the orthogonal tRNA (including, but not limited to, covalent binding), including, but not limited to, covalent binding to the orthogonal tRNA via an amino-acyl bond, covalent binding to the 3'OH or 2' OH of a terminal ribose sugar of the orthogonal tRNA, and the like.
Various advantages and manipulations of proteins are provided by chemical moieties that can incorporate unnatural amino acids into proteins. For example, the unique reactivity of the keto-functional group allows for selective modification of proteins with any of a number of hydrazine-or hydroxylamine-containing reagents in vitro and in vivo. For example, heavy atom unnatural amino acids can be used to phase X-ray structural data. Site-specific introduction of heavy atoms using unnatural amino acids also provides selectivity and flexibility in selecting heavy atom positions. For example, photoreactive unnatural amino acids, including but not limited to amino acids with benzophenone and aryl azide (including but not limited to phenyl azide) side chains, allow for efficient photocrosslinking of proteins in vivo and in vitro. Examples of photoreactive unnatural amino acids include, but are not limited to, para-azido-phenylalanine and para-benzoyl-phenylalanine. Proteins with photoreactive unnatural amino acids can then be optionally crosslinked by excitation of photoreactive groups that provide time control. In one example, the methyl groups of the unnatural amino groups can be isotopically-labeled, including but not limited to methyl groups, as probes for local structure and dynamics, including but not limited to using nuclear magnetic resonance and vibrational spectroscopy. For example, alkynyl or azido functional groups allow for the selective modification of proteins with molecules via [3+2] cycloaddition reactions.
Unnatural amino acids incorporated into polypeptides at the amino terminus can be represented by an R group and a different NH than is normally present in alpha-amino acids 2 The 2 nd reactive group of the group, the R group is any substituent other than the substituents used in the twenty natural amino acids. Similar unnatural amino acids can have a 2 nd reactive group at the C-terminus that is different from the COOH group typically present in alpha-amino acids.
The unnatural amino acids of the invention can be selected or designed to provide additional features that are not available in the twenty natural amino acids. For example, unnatural amino acids can optionally be designed or selected to modify, e.g., the biological properties of the protein into which they are incorporated. For example, the following properties may optionally be modified by including unnatural amino acids into proteins: toxicity, biodistribution, solubility, stability (e.g., thermal, hydrolytic, oxidative, resistance to enzymatic degradation, etc.), ease of purification and processing, structural properties, spectral properties, chemical and/or photochemical properties, catalytic activity, redox potential, half-life, ability to react with other molecules (e.g., covalent or non-covalent), and the like.
In some embodiments, the invention provides TCs linked to a water-soluble polymer, such as PEG, through an oxime linkage. Many types of non-naturally encoded amino acids are suitable for forming oxime linkages. Such non-naturally encoded amino acids include, but are not limited to, non-naturally encoded amino acids containing carbonyl, dicarbonyl, or hydroxylamine groups. Such amino acids are described in U.S. patent publication nos. 2006/0194256, 2006/0217532 and 2006/0217289, and WO 2006/069246 entitled "Compositions containing, methods involving, and uses of non-natural amino acids and polypeptides", all of which are incorporated herein by reference in their entirety. Non-naturally encoded amino acids are also described in U.S. patent No. 7,083,970 and U.S. patent No. 7,045,337, both of which are incorporated herein by reference in their entirety.
Some embodiments of the invention utilize TC polypeptides substituted with a p-acetylphenylalanine amino acid at one or more positions. The synthesis of p-acetyl- (+/-) -phenylalanine and m-acetyl- (+/-) -phenylalanine is described in Zhang, Z. Et al, biochemistry 42:6735-6746 (2003), which is incorporated by reference. Other carbonyl-or dicarbonyl-containing amino acids may be similarly prepared by one of ordinary skill in the art. Further, a non-limiting exemplary synthesis of unnatural amino acids included herein is described in U.S. patent No. 7,083,970, which is incorporated by reference in its entirety.
Amino acids having electrophilic reactive groups allow a variety of reactions to occur to attach molecules via nucleophilic addition reactions, and the like. Such electrophilic reactive groups include carbonyl groups (including keto and dicarbonyl groups), carbonyl-like groups (which have similar reactivity and structure to carbonyl groups (including keto and dicarbonyl groups)), masked carbonyl groups (which can be readily converted to carbonyl groups (including keto and dicarbonyl groups)), or protected carbonyl groups (which have similar reactivity to carbonyl groups (including keto and dicarbonyl groups) upon deprotection). Such amino acids include amino acids having the structure of formula (IV):
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- (alkylene)Alkyl or substituted alkylene) -, -S- (alkylene or substituted alkylene) -, -S (O) k - (wherein k is 1, 2 or 3), -S (O) k (alkylene or substituted alkylene) -, -C (O) - (alkylene or substituted alkylene) -, -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 ') -, wherein each R' is independently H, alkyl or substituted alkyl;
j is
R is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;
each R "is independently H, alkyl, substituted alkyl, or a protecting group, or when more than one R" group is present, two R "optionally form a heterocycloalkyl;
R 1 is optional and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and is also provided with
R 2 Is optional and when present is an OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide;
R 3 and R is 4 Each independently is H, halogen, lower alkyl or substituted lower alkyl, or R 3 And R is 4 Or two R 3 The groups optionally form cycloalkyl or heterocycloalkyl;
or-a-B-J-R groups together form a bicyclic or tricyclic cycloalkyl or heterocycloalkyl group containing at least one carbonyl group (including dicarbonyl), protected carbonyl group (including protected dicarbonyl), or masked carbonyl group (including masked dicarbonyl);
or-J-R groups together form a mono-or bicyclic cycloalkyl or heterocycloalkyl group comprising at least one carbonyl group (including dicarbonyl), protected carbonyl group (including protected dicarbonyl) or masked carbonyl group (including masked dicarbonyl);
With the proviso that when A is phenylene and each R 3 When H, B is present; and when A is- (CH) 2 ) 4 - -and each R 3 When H is used, B is not-NHC (O) (CH 2 CH 2 ) -; and when A and B are absent and each R 3 When H, R is not methyl.
In addition, amino acids having the structure of formula (V):
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- (alkylene or substituted alkylene) -, -S- (alkylene or substituted alkylene) -, -S (O) k - (wherein k is 1, 2 or 3), -S (O) k (alkylene or substituted alkylene) -, -C (O) - (alkylene or substituted alkylene) -, -C (S) - (alkylene or substituted alkylene)Alkylene) -, -N (R '), -NR ' - (alkylene or substituted alkylene) -, -C (O) N (R '), -CON (R ') - (alkylene or substituted alkylene) -, -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 ') -, wherein each R' is independently H, alkyl or substituted alkyl;
r is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;
R 1 is optional and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and is also provided with
R 2 Is optional and when present is an OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide;
provided that when a is phenylene, B is present; and when A is- (CH) 2 ) 4 When B is not-NHC (O) (CH 2 CH 2 ) -; and when a and B are absent, R is not methyl.
In addition, amino acids having the structure of formula (VI):
wherein:
b 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- (alkylene or substituted alkylene) -, -S- (alkylene or substituted alkylene) -, -S (O) k - (wherein k is 1, 2 or 3), -S (O) k (alkylene or substituted alkylene) -, -C (O) - (alkylene or substituted alkylene) -, -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 ') - (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 ') -, wherein each R' is independently H, alkyl or substituted alkyl;
r is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;
R 1 is optional and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and is also provided with
R 2 Is optional and when present is an OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide;
each R a Independently selected from the group consisting of: H. halogen, alkyl, substituted alkyl, -N (R') 2 、-C(O) k R '(where k is 1, 2 or 3), -C (O) N (R') 2 -OR' and-S (O) k R ', wherein each R' is independently H, alkyl or substituted alkyl.
In addition, the following amino acids are included:
Wherein such compounds are optionally amino protecting groups, carboxyl protecting groups or salts thereof. In addition, any of the following unnatural amino acids can be incorporated into an unnatural amino acid polypeptide. />
In addition, the following amino acids having the structure of formula (VII) are included:
wherein the method comprises the steps of
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- (alkylene or substituted alkylene) -, -S- (alkylene or substituted alkylene) -, -S (O) k - (wherein k is 1, 2 or 3), -S (O) k (alkylene or substituted alkylene) -, -C (O) - (alkylene or substituted alkylene) -, -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 ') -, wherein each R' is independently H, alkyl or substituted alkyl;
R is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;
R 1 is optional and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and is also provided with
R 2 Is optional and when present is an OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide;
each R a Independently selected from the group consisting of: H. halogen, alkyl, substituted alkyl, -N (R') 2 、-C(O) k R '(where k is 1, 2 or 3), -C (O) N (R') 2 -OR' and-S (O) k R ', wherein each R' is independently H, alkyl, or substituted alkyl; and n is a number from 0 to 8,
provided that when A is- (CH) 2 ) 4 When B is not-NHC (O) (CH 2 CH 2 )-。
In addition, the following amino acids are included:
wherein such compounds are optionally amino protecting groups, optionally carboxyl protecting groups, optionally amino protecting groups and carboxyl protecting groups, or salts thereof. In addition, any of these unnatural amino acids and the following unnatural amino acids can be incorporated into unnatural amino acid polypeptides.
In addition, the following amino acids having the structure of formula (VIII) are included:
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 heterocycloalkyl, substituted lower heterocycloalkyl, 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- (alkylene or substituted alkylene) -, -S- (alkylene or substituted alkylene) -, -S (O) k - (wherein k is 1, 2 or 3), -S (O) k (alkylene or substituted alkylene) -, -C (O) - (alkylene or substituted alkylene) -, -C (S) - (alkylene or substituted alkylene) -, -N (R ') -, NR' - (alkylene or substituted alkylene) -, orSubstituted 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 ') -, wherein each R' is independently H, alkyl or substituted alkyl;
R 1 is optional and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and is also provided with
R 2 Is optional and when present is an OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide.
In addition, the following amino acids having the structure of formula (IX) are included:
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- (alkylene or substituted alkylene) -, -S- (alkylene or substituted alkylene) -, -S (O) k - (wherein k is 1, 2 or 3), -S (O) k (alkylene or substituted alkylene) -, -C (O) - (alkylene or substituted alkylene) -, -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 ') -, wherein each R' is independently H, alkyl or substituted alkyl;
r is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;
R 1 is optional and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and is also provided with
R 2 Is optional and when present is an OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide;
wherein each R is a Independently selected from the group consisting of: H. halogen, alkyl, substituted alkyl, -N (R') 2 、-C(O) k R '(where k is 1, 2 or 3), -C (O) N (R') 2 -OR' and-S (O) k R ', wherein each R' is independently H, alkyl or substituted alkyl.
In addition, the following amino acids are included:
wherein such compounds are optionally amino protecting groups, optionally carboxyl protecting groups, optionally amino protecting groups and carboxyl protecting groups, or salts thereof. In addition, any of these unnatural amino acids and the following unnatural amino acids can be incorporated into unnatural amino acid polypeptides.
In addition, the following amino acids having the structure of formula (X) are included:
wherein B is optional and when present is a linker selected from the group consisting of: lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenyleneLower alkylene, substituted lower alkylene, -O- (alkylene or substituted alkylene) -, -S- (alkylene or substituted alkylene) -, -S (O) k - (wherein k is 1, 2 or 3), -S (O) k (alkylene or substituted alkylene) -, -C (O) - (alkylene or substituted alkylene) -, -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 ') -, wherein each R' is independently H, alkyl or substituted alkyl;
r is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;
R 1 is optional and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and is also provided with
R 2 Is optional and when present is an OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide;
each R a Independently selected from the group consisting of: H. halogen, alkyl, substituted alkyl, -N (R') 2 、-C(O) k R '(where k is 1, 2 or 3), -C (O) N (R') 2 -OR' and-S (O) k R ', wherein each R' is independently H, alkyl, or substituted alkyl; and n is 0 to 8.
In addition, the following amino acids are included:
Wherein the method comprises the steps ofSuch compounds are optionally amino protecting groups, optionally carboxyl protecting groups, optionally amino protecting groups and carboxyl protecting groups, or salts thereof. In addition, any of these unnatural amino acids and the following unnatural amino acids can be incorporated into unnatural amino acid polypeptides.
In addition to monocarbonyl structures, unnatural amino acids described herein can include groups such as dicarbonyl, dicarbonyl-like, masked dicarbonyl, and protected dicarbonyl.
For example, the following amino acids having the structure of formula (XI) are included:
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 heterocycloalkyl, substituted lower heterocycloalkyl, 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- (alkylene or substituted alkylene) -, -S- (alkylene or substituted alkylene) -, -S (O) k - (wherein k is 1, 2 or 3), -S (O) k (alkylene or substituted alkylene) -, -C (O) - (alkylene or substituted alkylene) -, -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 ') -, wherein each R' is independently H, alkyl or substituted alkyl;
r is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;
R 1 is optional and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and is also provided with
R 2 Is optional and when present is an OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide.
In addition, the following amino acids having the structure of formula (XII) are included:
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- (alkylene or substituted alkylene) -, -S- (alkylene or substituted alkylene) -, -S (O) k - (wherein k is 1, 2 or 3), -S (O) k (alkylene or substituted alkylene) -, -C (O) - (alkylene or substituted alkylene) -, -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 ') -, wherein each R' is independently H, alkyl or substitutedAlkyl of (a);
r is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;
R 1 is optional and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and is also provided with
R 2 Is optional and when present is an OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide;
wherein each R is a Independently selected from the group consisting of: H. halogen, alkyl, substituted alkyl, -N (R') 2 、-C(O) k R '(where k is 1, 2 or 3), -C (O) N (R') 2 -OR' and-S (O) k R ', wherein each R' is independently H, alkyl or substituted alkyl.
In addition, the following amino acids are included:
wherein such compounds are optionally amino protecting groups, optionally carboxyl protecting groups, optionally amino protecting groups and carboxyl protecting groups, or salts thereof. In addition, any of these unnatural amino acids and the following unnatural amino acids can be incorporated into unnatural amino acid polypeptides.
In addition, the following amino acids having the structure of formula (XIII) are included:
wherein 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- (alkylene or substituted alkylene) -, -S- (alkylene or substituted alkylene) -, -S (O) k - (wherein k is 1, 2 or 3), -S (O) k (alkylene or substituted alkylene) -, -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 ') - (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 ') -, wherein each R' is independently H, alkyl or substituted alkyl;
r is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;
R 1 is optional and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and is also provided with
R 2 Is optional and when present is an OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide;
each R a Independently selected from the group consisting of: H. halogen, alkyl, substituted alkyl, -N (R') 2 、-C(O) k R '(where k is 1, 2 or 3), -C (O) N (R') 2 -OR' and-S (O) k R ', wherein each R' is independently H, alkyl, or substituted alkyl; and n is 0 to 8.
In addition, the following amino acids are included:
wherein such compounds are optionally amino protecting groups, optionally carboxyl protecting groups, optionally amino protecting groups and carboxyl protecting groups, or salts thereof. In addition, any of these unnatural amino acids and the following unnatural amino acids can be incorporated into unnatural amino acid polypeptides.
In addition, the following amino acids having the structure of formula (XIV) are included:
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;
R is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;
R 1 is optional and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and is also provided with
R 2 Is optional and when present is an OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide;
X 1 c, S or S (O); and L is alkylene, substituted alkylene, N (R ') (alkylene), or N (R ') (substituted alkylene), wherein R ' is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.
In addition, the following amino acids having the structure of formulSup>A (XIV-A) are included:
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;
r is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;
R 1 Is optional and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and is also provided with
R 2 Is optional and when present is an OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide;
l is alkylene, substituted alkylene, N (R ') (alkylene) or N (R ') (substituted alkylene), wherein R ' is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl.
In addition, the following amino acids having the structure of formula (XIV-B) are included:
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;
r is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;
R 1 is optional and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and is also provided with
R 2 Is optional and when present is an OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide;
l is alkylene, substituted alkylene, N (R ') (alkylene) or N (R ') (substituted alkylene), wherein R ' is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl.
In addition, the following amino acids having the structure of formula (XV) are included:
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;
r is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;
R 1 is optional and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and is also provided with
R 2 Is optional and when present is an OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide;
X 1 C, S or S (O), and n is 0, 1, 2, 3, 4, or 5; and each CR 8 R 9 Each R on the radical 8 And R is 9 Independently selected from the group consisting of: H. alkoxy, alkylamine, halogen, alkyl, aryl, or any R 8 And R is 9 May together form =o or cycloalkyl, or adjacent R 8 Any of the groups may together form cycloalkyl.
In addition, the following amino acids having the structure of formulSup>A (XV-A) are included:
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;
r is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;
R 1 is optional and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and is also provided with
R 2 Is optional and when present is an OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide;
n is 0, 1, 2, 3, 4 or 5; and each CR 8 R 9 Each R on the radical 8 And R is 9 Independently selected from the group consisting of: H. alkoxy, alkylamine, halogen, alkyl, aryl, or any R 8 And R is 9 May together form =o or cycloalkyl, or adjacent R 8 Any of the groups may together form cycloalkyl.
In addition, the following amino acids having the structure of formula (XV-B) are included:
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;
r is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;
R 1 is optional and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and is also provided with
R 2 Is optional and when present is an OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide;
n is 0, 1, 2, 3, 4 or 5; and each CR 8 R 9 Each R on the radical 8 And R is 9 Independently selected from the group consisting of: H. alkoxy, alkylamine, halogen, alkyl, aryl, or any R 8 And R is 9 May together form =o or cycloalkyl, or adjacent R 8 Any of the groups may together form cycloalkyl.
In addition, the following amino acids having the structure of formula (XVI) are included:
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;
r is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;
R 1 is optional and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and is also provided with
R 2 Is optional and when present is an OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide;
X 1 C, S or S (O); and L is alkylene, substituted alkylene, N (R ') (alkylene), or N (R ') (substituted alkylene), wherein R ' is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.
In addition, the following amino acids having the structure of formula (XVI-A) are included:
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;
r is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;
R 1 is optional and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and is also provided with
R 2 Is optional and when present is an OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide;
l is alkylene, substituted alkylene, N (R ') (alkylene) or N (R ') (substituted alkylene), wherein R ' is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl.
In addition, the following amino acids having the structure of formula (XVI-B) are included:
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;
r is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;
R 1 is optional and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and is also provided with
R 2 Is optional and when present is an OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide;
l is alkylene, substituted alkylene, N (R ') (alkylene) or N (R ') (substituted alkylene), wherein R ' is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl.
In addition, amino acids having the structure of formula (XVII) are included:
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;
M is-C (R) 3 )-、 Wherein (a) represents bonding to the A group, (b) represents bonding to the respective carbonyl groups, R 3 And R is 4 Independently selected from H, halogen, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl, or R 3 And R is 4 Or two R 3 Radicals or two R 4 The groups optionally form cycloalkyl or heterocycloalkyl;
r is H, halogen, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;
T 3 is a bond, C (R) (R), O or S, and R is H, halogen, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;
R 1 is optional and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and is also provided with
R 2 Is optional and when present is an OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide.
In addition, amino acids having the structure of formula (XVIII):
wherein:
m is-C (R) 3 )-、 Wherein (a) represents bonding to the A group, (b) represents bonding to the respective carbonyl groups, R 3 And R is 4 Independent and independentIs selected from H, halogen, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl, or R 3 And R is 4 Or two R 3 Radicals or two R 4 The groups optionally form cycloalkyl or heterocycloalkyl;
r is H, halogen, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;
T 3 Is a bond, C (R) (R), O or S, and R is H, halogen, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;
R 1 is optional and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and is also provided with
R 2 Is optional and when present is an OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide;
each R a Independently selected from the group consisting of: H. halogen, alkyl, substituted alkyl, -N (R') 2 、-C(O) k R '(where k is 1, 2 or 3), -C (O) N (R') 2 -OR' and-S (O) k R ', wherein each R' is independently H, alkyl or substituted alkyl.
In addition, amino acids having the structure of formula (XIX):
wherein:
r is H, halogen, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl; and is also provided with
T 3 Is O or S.
In addition, amino acids having the structure of formula (XX):
wherein:
r is H, halogen, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl.
In addition, the following amino acids having the structure of formula (XXI) are included:
in some embodiments, polypeptides comprising unnatural amino acids are chemically modified to produce reactive carbonyl or dicarbonyl functionalities. For example, aldehyde functional groups useful for conjugation reactions can be generated from functional groups having adjacent amino and hydroxyl groups. When the biologically active molecule is a polypeptide, for example, the N-terminal serine or threonine (which may typically be present or may be exposed by chemical or enzymatic digestion) can be used to generate aldehyde functionality under mild oxidative cleavage conditions using periodate. See, for example, gaertner et al, bioconjug. Chem.3:262-268 (1992); geoghegan, K. & Stroh, J, bioconjug. Chem.3:138-146 (1992); gaertner et al, J.biol. Chem.269:7224-7230 (1994). However, the methods known in the art are limited to amino acids at the N-terminus of a peptide or protein.
In the present invention, unnatural amino acids with adjacent hydroxyl and amino groups can be incorporated into polypeptides as "masked" aldehyde functionalities. For example, 5-hydroxylysine has a hydroxyl group adjacent to epsilon amine. The reaction conditions used to produce the aldehyde typically involve adding a molar excess of sodium metaperiodate under mild conditions to avoid oxidation at other sites within the polypeptide. The pH of the oxidation reaction is typically about 7.0. A typical reaction involves adding about 1.5 molar excess of sodium metaperiodate to a buffered solution of the polypeptide followed by incubation in the dark for about 10 minutes. See, for example, U.S. patent No. 6,423,685.
The carbonyl or dicarbonyl functional group can be selectively reacted with a hydroxylamine-containing reagent in aqueous solution under mild conditions to form the corresponding oxime bond that is stable under physiological conditions. See, e.g., jencks, W.P., J.Am.Chem.Soc.81,475-481 (1959); shao, J. And Tam, J.P., J.Am.Chem.Soc.117:3893-3899 (1995). Furthermore, the unique reactivity of carbonyl or dicarbonyl groups allows for selective modification in the presence of other amino acid side chains. See, e.g., cornish, V.W., et al, J.Am.chem.Soc.118:8150-8151 (1996); geoghegan, K.F. & Stroh, J.G, bioconjug.Chem.3:138-146 (1992); mahal, L.K., et al Science 276:1125-1128 (1997).
A. Carbonyl reactive group
Amino acids having carbonyl reactive groups allow for attachment of molecules (including but not limited to PEG or other water-soluble molecules) by a variety of reactions such as nucleophilic addition reactions or aldol reactions.
Exemplary carbonyl-containing amino acids can be represented as follows:
wherein n is 0 to 10; r is R 1 Is alkyl, aryl, substituted alkyl or substituted aryl; r is R 2 Is H, alkyl, aryl, substituted alkyl, and substituted aryl; and R is 3 Is H, an amino acid, a polypeptide or an amino terminal modification; and R is 4 Is H, an amino acid, a polypeptide or a carboxyl terminal modification group. In some embodiments, n is 1, R 1 Is phenyl and R 2 Is a simple alkyl group (i.e., methyl, ethyl, or propyl) and the ketone moiety is in a para position relative to the alkyl side chain. In some embodiments, n is 1, R 1 Is phenyl and R 2 Is a simple alkyl group (i.e., methyl, ethyl, or propyl) and the ketone moiety is in a meta position relative to the alkyl side chain.
The synthesis of p-acetyl- (+/-) -phenylalanine and m-acetyl- (+/-) -phenylalanine is described in Zhang, Z. Et al, biochemistry 42:6735-6746 (2003), which is incorporated herein by reference. Other carbonyl-containing amino acids can be similarly prepared by one of ordinary skill in the art.
In some embodiments, polypeptides comprising non-naturally encoded amino acids are chemically modified to produce reactive carbonyl functional groups. For example, aldehyde functional groups useful for conjugation reactions can be generated from functional groups having adjacent amino and hydroxyl groups. When the biologically active molecule is a polypeptide, for example, the N-terminal serine or threonine (which may typically be present or may be exposed by chemical or enzymatic digestion) can be used to generate aldehyde functionality under mild oxidative cleavage conditions using periodate. See, e.g., gaertner, et al, bioconjug. Chem.3:262-268 (1992); geoghegan, K. & Stroh, J. & bioconjug. Chem.3:138-146 (1992); gaertner et al, J.biol. Chem.269:7224-7230 (1994). However, the methods known in the art are limited to amino acids at the N-terminus of a peptide or protein.
In the present invention, non-naturally encoded amino acids with adjacent hydroxyl and amino groups can be incorporated into polypeptides as "masked" aldehyde functionalities. For example, 5-hydroxylysine has a hydroxyl group adjacent to epsilon amine. The reaction conditions used to produce the aldehyde typically involve adding a molar excess of sodium metaperiodate under mild conditions to avoid oxidation at other sites within the polypeptide. The pH of the oxidation reaction is typically about 7.0. A typical reaction involves adding about 1.5 molar excess of sodium metaperiodate to a buffered solution of the polypeptide followed by incubation in the dark for about 10 minutes. See, for example, U.S. patent No. 6,423,685, which is incorporated herein by reference.
Carbonyl functions can be selectively reacted with reagents containing hydrazine, hydrazide, hydroxylamine or semicarbazide under mild conditions in aqueous solution to form the corresponding hydrazone, oxime or semicarbazide linkages, respectively, that are stable under physiological conditions. See, e.g., jencks, W.P., J.Am.Chem.Soc.81,475-481 (1959); shao, J. And Tam, J.P., J.Am.Chem.Soc.117:3893-3899 (1995). Furthermore, the unique reactivity of carbonyl groups allows for selective modification in the presence of other amino acid side chains. See, e.g., cornish, V.W., et al, J.Am.chem.Soc.118:8150-8151 (1996); geoghegan, K.F. & Stroh, J.G., bioconjug.Chem.3:138-146 (1992); mahal, L.K., et al Science 276:1125-1128 (1997).
B. Hydrazine, hydrazide or semicarbazide reactive groups
Non-naturally encoded amino acids containing nucleophilic groups (such as hydrazine, hydrazide, or semicarbazide) allow for reaction with various electrophilic groups (including but not limited to with PEG or other water-soluble polymers) to form conjugates.
Exemplary hydrazine, hydrazide or semicarbazide containing amino acids may be represented as follows:
wherein n is 0 to 10; r is R 1 Is alkyl, aryl, substituted alkyl or substituted aryl or is absent; x is O, N or S or is absent; r is R 2 Is H, an amino acid, a polypeptide or an amino terminal modification; and R is 3 Is H, an amino acid, a polypeptide or a carboxyl terminal modification group.
In some embodiments, n is 4, R 1 Is absent, and X is N. In some embodiments, n is 2, R 1 Absent, and X absent. In some embodiments, n is 1, R 1 Is phenyl, X is O, and the oxygen atom is located para to the aliphatic group on the aromatic ring.
Amino acids containing hydrazides, hydrazines, and semicarbazides are available from commercial sources. For example, L-glutamic acid-gamma-hydrazide is available from Sigma Chemical (St. Louis, MO). Other amino acids that are not commercially available can be prepared by one of ordinary skill in the art. See, for example, U.S. patent No. 6,281,211, which is incorporated herein by reference.
Polypeptides containing non-naturally encoded amino acids with hydrazide, hydrazine, or semicarbazide functionality can be efficiently and selectively reacted with a variety of molecules containing aldehydes or other functional groups with similar chemical reactivity. See, e.g., shao, J. And Tam, J. Am. Chem. Soc.117:3893-3899 (1995). The unique reactivity of the hydrazide, hydrazine and semicarbazide functions makes them significantly more reactive towards aldehydes, ketones and other electrophilic groups than nucleophilic groups present on 20 common amino acids, including but not limited to serine or threonine hydroxyl groups or lysine and N-terminal amino groups.
C. Amino acids containing aminooxy groups
Non-naturally encoded amino acids containing aminooxy (also known as hydroxylamine) groups allow for reaction with a variety of electrophilic groups, including but not limited to with PEG or other water-soluble polymers to form conjugates. As with hydrazines, hydrazides, and semicarbazides, the enhanced nucleophilicity of the aminooxy groups enables them to effectively and selectively react with a variety of molecules containing aldehydes or other functional groups having similar chemical reactivity. See, e.g., shao, J. And Tam, J., J.Am.chem.Soc.117:3893-3899 (1995); H.Hang and C.Bertozzi, acc.Chem.Res.34:727-736 (2001). However, the result of the reaction with hydrazine groups is the corresponding hydrazone, whereas oximes are generally produced by the reaction of aminoxy groups with carbonyl-containing groups, such as ketones.
Exemplary amino acids containing an aminooxy group can be represented as follows:
wherein n is 0 to 10; r is R 1 Is alkyl, aryl, substituted alkyl or substituted aryl or is absent; x is O, N, S or absent; m is 0 to 10; y=c (O) or absent; r is R 2 Is H, an amino acid, a polypeptide or an amino terminal modification; and R is 3 Is H, an amino acid, a polypeptide or a carboxyl terminal modification group. In some embodiments, n is 1, R 1 Is phenyl, X is O, m is 1 and Y is present. In some embodiments, n is 2, R 1 And X is absent, m is 0 and Y is absent.
Amino acids containing an oxy group can be prepared from readily available amino acid precursors (homoserine, serine and threonine). See, e.g., M.Carrasco and R.Brown, J.Org.Chem.68:8853-8858 (2003). Certain amino acids containing aminoxy groups, such as L-2-amino-4- (aminoxy) butanoic acid, have been isolated from natural sources (Rosenthal, G., life Sci.60:1635-1641 (1997)). Other amino acids containing aminooxy groups can be prepared by one of ordinary skill in the art.
D. Azide and alkyne reactive groups
The unique reactivity of azide and alkyne functionalities makes them extremely useful for the selective modification of polypeptides and other biomolecules. Organic azides, particularly aliphatic azides and alkynes, are generally stable to common reaction chemistry conditions. In particular, azide and alkyne functional groups are inert to the side chains (i.e., R groups) of the 20 common amino acids found in naturally occurring polypeptides. However, when brought into close proximity, the "spring-loaded" nature of the azide and alkyne groups emerges and the groups react selectively and efficiently by the Huisgen [3+2] cycloaddition reaction to the corresponding triazoles. See, e.g., chin J., et al, science 301:964-7 (2003); wang, q., et al, j.am.chem.soc.125,3192-3193 (2003); chin, J.W., et al, J.Am.chem.Soc.124:9026-9027 (2002).
Because Huisgen cycloaddition involves a selective cycloaddition reaction (see, e.g., padwa, A., COMPREHENSIVE ORGANIC SYNTHESIS, vol. 4, (Trost, B.M. eds., 1991), pages 1069-1109; huisgen, R.1,3-DlPOLAR CYCLOADDITION CHEMISTRY, (Padwa, A., eds., 1984), pages 1-176) rather than nucleophilic substitution, the incorporation of a non-naturally encoded amino acid with azide and alkyne-containing side chains allows the resulting polypeptide to be selectively modified at the position of the non-naturally encoded amino acid. Cycloaddition reactions involving azide-containing or alkyne-containing TCs can be performed at room temperature under aqueous conditions by adding Cu (II) in the presence of a reducing agent (including, but not limited to, a catalytic amount of CuSO 4 In the form of (2) in catalytic amounts by in situ reduction of Cu (II) to Cu (I). See, e.g., wang, q., et al, j.am.chem.soc.125,3192-3193 (2003); tornoe, c.w., et al, j.org.chem.67:3057-3064 (2002); rostovtsev, et al, angew.chem.int.ed.41:2596-2599 (2002). Exemplary reducing agents include, but are not limited to, ascorbate, metallic copper, quinine, hydroquinone, vitamin K, glutathione, cysteine Fe 2+ 、Co 2+ And an applied potential.
In some cases, when a Huisgen [3+2] cycloaddition reaction between an azide and an alkyne is desirable, TC comprises a non-naturally encoded amino acid comprising an alkyne moiety and the water soluble polymer to be attached to the amino acid comprises an azide moiety. Alternatively, the opposite reaction (i.e., with the azide moiety on the amino acid and the alkyne moiety present on the water soluble polymer) may also be performed.
The nitride functional groups may also be selectively reacted with the water-soluble polymer containing the aryl ester and appropriately functionalized with aryl phosphine moieties to produce amide linkages. The aryl phosphine group reduces the azide in situ and then the resulting amine effectively reacts with the adjacent ester linkage to form the corresponding amide. See, e.g., e.saxon and c.bertozzi, science 287,2007-2010 (2000). The azide-containing amino acid may be an alkyl azide (including but not limited to 2-amino-6-azido-1-hexanoic acid) or an aryl azide (p-azido-phenylalanine).
Exemplary water-soluble polymers containing aryl ester and phosphine moieties can be represented as follows:
wherein X may be O, N, S or absent, ph is phenyl, W is a water soluble polymer, and R may be H, alkyl, aryl, substituted alkyl, and substituted aryl. Exemplary R groups include, but are not limited to, -CH 2 、-C(CH 3 ) 3 -OR ', -NR ' R ', -SR ', -halogen, -C (O) R ', -CONR ' R ', -S (O) 2 R’、-S(O) 2 NR 'R', -CN and-NO 2 . R ', R ", R'" and R "" each independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl (including but not limited to aryl substituted with 1-3 halogens), substituted or unsubstituted alkyl, alkoxy or thioalkoxy, or aralkyl. When the compounds of the present invention include more than one R group, when more than one of these groups is present, for example, each R group is independently selected as each R ', R ", R'" and R "" group. When R 'and R' are attached to the same nitrogen atom, they may combine with the nitrogen atom to form a 5-, 6-, or 7-membered ring. For example, -NR' R "is intended to include, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl. From the above discussion of substituents, those skilled in the art will understand that the term "alkyl" is intended to include groups that include carbon atoms bonded to groups other than hydrogen groups, such as haloalkyl (e.g., -CF 3 and-CH 2 CF 3 ) And acyl groups (including but not limited to-C (O) CH 3 、-C(O)CF 3 、-C(O)CH 2 OCH 3 Etc.).
The azide functional groups can also be selectively reacted with a water-soluble polymer containing thioesters and appropriately functionalized with aryl phosphine moieties to produce amide linkages. The aryl phosphine group reduces the azide in situ and then the resulting amine reacts effectively with the thioester bond to form the corresponding amide. Exemplary water-soluble polymers containing thioester and phosphine moieties can be represented as follows:
wherein n is 1 to 10; x may be O, N, S or absent, ph is phenyl, and W is a water soluble polymer.
Exemplary alkyne-containing amino acids can be represented as follows:
wherein n is 0 to 10; r is R 1 Is alkyl, aryl, substituted alkyl or substituted aryl or is absent; x is O, N, S or absent; m is 0 to 10; r is R 2 Is H, an amino acid, a polypeptide or an amino terminal modification; and R is 3 Is H, an amino acid, a polypeptide or a carboxyl terminal modification group. In some embodiments, n is 1, R 1 Is phenyl, X is absent, m is 0 and the acetylene moiety is in a para position relative to the alkyl side chain. In some embodiments, n is 1, R 1 Is phenyl, X is O, m is 1 and propargyloxy is in the para position relative to the alkyl side chain (i.e., O-propargyl-tyrosine). In some embodiments, n is 1, R 1 And X is absent and m is 0 (i.e., propargylglycine).
Alkyne-containing amino acids are commercially available. For example, propargylglycine is commercially available from Peptech (Burlington, MA). Alternatively, alkyne-containing amino acids can be prepared according to standard methods. For example, p-propargyloxyphenylalanine can be synthesized, e.g., as described in Deiters, A., et al, J.Am.chem.Soc.125:11782-11783 (2003), and 4-alkynyl-L-phenylalanine can be synthesized, e.g., as described in Kayser, B., et al, tetrahedron 53 (7): 2475-2484 (1997). Other alkyne-containing amino acids can be prepared by one of ordinary skill in the art.
Exemplary azide-containing amino acids can be represented as follows:
wherein n is 0 to 10; r is R 1 Is alkyl, aryl, substituted alkyl, substituted aryl, or absent; x is O, N, S or absent; m is 0 to 10; r is R 2 Is H, an amino acid, a polypeptide or an amino terminal modification; and R is 3 Is H, an amino acid, a polypeptide or a carboxyl terminal modification group. In some embodiments, n is 1, R 1 Is phenyl, X is absent, m is 0 and the azide moiety is para to the alkyl side chain. In some embodiments, n is 0-4 and R 1 And X is absent, and m=0. In some embodiments, n is 1, R 1 Is phenyl, X is O, m is 2 and the-azidoethoxy moiety is in a para position relative to the alkyl side chain.
Azide-containing amino acids are available from commercial sources. For example, 4-azidophenylalanine is available from Chem-Impex International, inc. (Wood Dale, IL). For those non-commercially available azide-containing amino acids, azide groups can be prepared relatively easily using standard methods known to those of ordinary skill in the art, including but not limited to by displacement of suitable leaving groups (including but not limited to halides, mesylate, tosylate) or by opening of appropriately protected lactones. See, e.g., advanced Organic Chemistry by March (third edition, 1985,Wiley and Sons,New York).
E. Amino mercaptan reactive groups
The unique reactivity of beta-substituted aminothiol functionalities makes them very suitable for the selective modification of polypeptides and other biomolecules containing aldehyde groups by formation of thiazolidines. See, e.g., J.Shao and J.Tam, J.Am.Chem.Soc.1995,117 (14) 3893-3899. In some embodiments, the β -substituted aminothiol amino acid can be incorporated into a TC polypeptide and then reacted with a water soluble polymer comprising aldehyde functionality. In some embodiments, the water-soluble polymer, drug conjugate, or other payload may be coupled to a targeting polypeptide comprising a TC of a β -substituted aminothiol amino acid by formation of a thiazolidine.
F. Additional reactive groups
Additional reactive groups and non-naturally encoded amino acids that may be incorporated into the TC polypeptides of the present invention are described in the following patent applications, including but not limited to para-amino-phenylalanine, all of which are incorporated herein by reference in their entirety: U.S. patent publication No. 2006/0194256, U.S. patent publication No. 2006/0217532, U.S. patent publication No. 2006/0217289, U.S. provisional patent No. 60/755,338; U.S. provisional patent nos. 60/755,711; U.S. provisional patent nos. 60/755,018; international patent application No. PCT/US06/49397; WO 2006/069246; U.S. provisional patent No. 60/743,041; U.S. provisional patent nos. 60/743,040; international patent application No. PCT/US06/47822; U.S. provisional patent No. 60/882,819; U.S. provisional patent No. 60/882,500; and U.S. provisional patent No. 60/870,594. These applications also discuss reactive groups that may be present on PEG or other polymers, including but not limited to hydroxylamine (aminooxy) groups for conjugation.
Positions of unnatural amino acids in TC polypeptides
The methods and compositions described herein include incorporating one or more unnatural amino acids into a targeted polypeptide to make a TC of the invention. One or more unnatural amino acids can be incorporated at one or more specific positions that do not disrupt the activity of the targeted polypeptide. This can be accomplished by making "conservative" substitutions, including but not limited to substitution of hydrophobic amino acids with unnatural or natural hydrophobic amino acids, substitution of bulky amino acids with unnatural or natural bulky amino acids, substitution of hydrophilic amino acids with natural or unnatural hydrophilic amino acids, and/or insertion of unnatural amino acids into positions where activity is not desired.
Various biochemical and structural methods can be employed to select the desired site for substitution with unnatural amino acids within a targeting polypeptide for TC. In some embodiments, the unnatural amino acid is linked to the C-terminus of the TLR agonist derivative. In other embodiments, the unnatural amino acid is linked to the N-terminus of the TLR agonist derivative. Any position of the targeting polypeptide of TC is suitably selected to incorporate an unnatural amino acid, and the selection may be based on rational design or by random selection for any or no particular desired purpose. The selection of the desired site may be based on the production of a non-natural amino acid polypeptide (which may be further modified or remain unmodified) having any desired property or activity, including but not limited to receptor binding modulators, receptor activity modulators, modulators of binding to binding agent partners, modulators of binding partner activity, binding partner conformational modulators, dimer or multimer formation, no change in activity or property compared to the natural molecule, or manipulation of any physical or chemical property of the polypeptide, such as solubility, aggregation or stability. Alternatively, sites identified as critical to biological activity may also be good candidates for substitution with unnatural amino acids, again depending on the desired activity sought for the polypeptide. Another option is to simply make successive substitutions with unnatural amino acids at each position along the polypeptide chain and observe the effect on the activity of the polypeptide. Any means, technique or method for selecting a position for substitution with an unnatural amino acid into any polypeptide is suitable for use in the methods, techniques and compositions described herein.
The structure and activity of naturally occurring polypeptide mutants containing deletions can also be examined to determine protein regions that are likely to tolerate unnatural amino acid substitutions. Once residues that may not tolerate unnatural amino acid substitutions are eliminated, methods including, but not limited to, the three-dimensional structure of the relevant polypeptide and any relevant ligand or binding protein can be used to examine the effect of the proposed substitutions at each of the remaining positions. The X-ray crystallography and NMR structures of many polypeptides are available in the protein database (PDB, www.rcsb.org), which is a central database of three-dimensional structural data containing proteins and nucleic acid macromolecules that can be used to identify amino acid positions that can be substituted with unnatural amino acids. In addition, if three-dimensional structural data is not available, models can be made to study the secondary and tertiary structure of the polypeptide. Thus, the identity of the amino acid position that can be substituted with an unnatural amino acid can be readily obtained.
Exemplary sites for incorporation of unnatural amino acids include, but are not limited to, sites that are excluded from potential receptor binding domains, or regions for binding to binding proteins or ligands may be fully or partially exposed to solvents, have minimal or no hydrogen bonding interactions with nearby residues, may be least exposed to nearby reactive residues, and/or may be located in highly flexible regions, as predicted by the three-dimensional crystal structure of a particular polypeptide and its associated receptor, ligand, or binding protein.
A given position in a polypeptide may be substituted or incorporated with a variety of unnatural amino acids. For example, a particular unnatural amino acid can be selected for incorporation based on a preference for conservative substitutions for examination of the three-dimensional crystal structure of the polypeptide and its associated ligand, receptor, and/or binding protein.
In one embodiment, the methods described herein comprise incorporating an unnatural amino acid into a targeting polypeptide of TC, where the targeting polypeptide of TC comprises a first reactive group; and contacting the targeted polypeptide of TC with a molecule comprising a second reactive group (including but not limited to a second protein or polypeptide analog; an antibody or antibody fragment; and any combination thereof). In certain embodiments, the first reactive group is a hydroxylamine moiety and the second reactive group is a carbonyl or dicarbonyl moiety, thereby forming an oxime bond. In certain embodiments, the first reactive group is a carbonyl or dicarbonyl moiety and the second reactive group is a hydroxylamine moiety, thereby forming an oxime bond. In certain embodiments, the first reactive group is a carbonyl or dicarbonyl moiety and the second reactive group is an oxime moiety, whereby an oxime exchange reaction occurs. In certain embodiments, the first reactive group is an oxime moiety and the second reactive group is a carbonyl or dicarbonyl moiety, whereby an oxime exchange reaction occurs.
In some cases, a targeting polypeptide incorporating TC of an unnatural amino acid will be combined with other additions, substitutions, or deletions within the polypeptide to affect other chemical, physical, pharmacological, and/or biological traits. In some cases, other additions, substitutions, or deletions may increase the stability of the polypeptide (including, but not limited to, resistance to proteolytic degradation) or increase the affinity of the polypeptide for its suitable receptor, ligand, and/or binding protein. In some cases, other additions, substitutions, or deletions may be madeIncreasing the solubility of a polypeptide (including but not limited to when inEscherichia coliOr other host cell). In some embodiments, in addition to another site for incorporation of an unnatural amino acid, a site is selected for substitution with a naturally encoded or unnatural amino acid, with the aim of increasing the position of the polypeptide inEscherichia coliOr solubility after expression in other recombinant host cells. In some embodiments, the polypeptide comprises another addition, substitution, or deletion that modulates affinity for the relevant ligand, binding protein, and/or receptor, modulates (including, but not limited to, increases or decreases) receptor dimerization, stabilizes receptor dimers, modulates circulatory half-life, modulates release or bioavailability, facilitates purification, or improves or alters a particular route of administration. Similarly, the unnatural amino acid polypeptide can comprise a chemical or enzymatic cleavage sequence, a protease cleavage sequence, a reactive group, an antibody binding domain (including but not limited to FLAG or polyHis) or other affinity-based sequence (including but not limited to FLAG, polyHis, GST, etc.), or a linker molecule (including but not limited to biotin) that improves detection of the polypeptide (including but not limited to GFP), purification, transport through tissue or cell membranes, prodrug release or activation, size reduction, or other trait.
Exemplary anti-HER 2 antibodies as targeting moiety
The methods, compositions, strategies, and techniques described herein are not limited to a particular type, class, or family of targeting moiety polypeptides or proteins. Virtually any targeting moiety polypeptide can be designed or modified to include at least one "modified or unmodified" unnatural amino acid of a targeting polypeptide that comprises a TC described herein. By way of example only, the targeting moiety polypeptide may be homologous to a therapeutic protein selected from the group consisting of: alpha-1 antitrypsin, angiostatin, anti-hemolytic factor, antibodies, antibody fragments, monoclonal antibodies (e.g., bevacizumab, cetuximab, panitumumab, infliximab, adalimumab, basiliximab, darifenacin, omalizumab, utekumamab, etanercept, gemtuzumab alemtuzumab, rituximab, trastuzumab, nituzumab, palivizumab and acymumab), apolipoproteins, apoproteins (apoproteins), atrial natriuretic peptides (atrial natriuretic factor), atrial natriuretic polypeptide (atrial natriuretic polypeptide), atrial peptides (atrial peptides), C-X-C chemokines, T39765, NAP-2, ENA-78, gro-a gro-b, gro-C, IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG, calcitonin, C-kit ligand, CC chemokine, monocyte chemotactic protein-1, monocyte chemotactic protein-2, monocyte chemotactic protein-3, monocyte inflammatory protein-1α, monocyte inflammatory protein-iβ, RANTES, 1309, R83915, R91733, HCC1, T58847, D31065, T64262, CD40 ligand, C-kit ligand, collagen, colony Stimulating Factor (CSF), complement factor 5a, complement inhibitor, complement receptor 1, cytokine, epithelial neutrophil activating peptide-78, MIP-16, MCP-1, epidermal Growth Factor (EGF), epithelial neutrophil activating peptide, erythropoietin (EPO), EPO, exfoliating toxins, factor IX, factor VII, factor VIII, factor X, fibroblast Growth Factor (FGF), fibrinogen, fibronectin, tetralin, G-CSF, glp-1, GM-CSF, glucocerebrosidase, gonadotropin, growth factor receptor, growth hormone releasing factor, hedgehog, hemoglobin, hepatocyte growth factor (hGF), hirudin, human growth hormone (hGH), human serum albumin, ICAM-1 receptor, LFA-1 receptor, insulin-like growth factor (IGF), IGF-I, IGF-II, interferon (IFN), IFN-alpha, IFN-beta, IFN-gamma, interleukin (IL), IL-1 IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, keratinocyte Growth Factor (KGF), lactoferrin, leukemia inhibitory factor, luciferase, neurturin, neutrophil Inhibitory Factor (NIF), oncostatin M, osteoblast protein, oncogene product, paralin, parathyroid hormone (PTH), PD-ECGF, PDGF, peptide hormones, pleiotropic protein (pleotopin), protein A, protein G, pyrogenic exotoxin A, pyrogenic exotoxin B, pyrogenic exotoxin C, peptide YY (PYY), relaxin, renin, SCF, microglobulin, soluble complement receptor I, soluble I-CAM 1, soluble interleukin receptor, soluble TNF receptor, somatostatin, growth hormone, streptokinase, superantigen, staphylococcal enterotoxin, SEA, SEB, SEC, SEC2, SEC3, SED, SEE, steroid hormone receptor, superoxide dismutase, toxic shock syndrome toxin, thymosin alpha 1, tissue plasminogen activator, tumor Growth Factor (TGF), tumor necrosis factor alpha, tumor necrosis factor beta, tumor Necrosis Factor Receptor (TNFR), VLA-4 protein, VCAM-1 protein, vascular Endothelial Growth Factor (VEGF), urokinase, mos, ras, raf, met, p, tat, fos, myc, jun, myb, rel, estrogen receptor, progestin receptor, testosterone receptor, aldosterone receptor, LDL receptor, and corticosterone.
In one embodiment is a method of treating a solid tumor that overexpresses HER-2, the solid tumor being selected from the group consisting of: breast cancer, small cell lung cancer, ovarian cancer, endometrial cancer, bladder cancer, head and neck cancer, prostate cancer, gastric cancer, cervical cancer, uterine cancer, esophageal cancer, and colon cancer. In another embodiment, the solid tumor is breast cancer. In another embodiment, the solid tumor is ovarian cancer.
Accordingly, the following description of trastuzumab is provided for illustration purposes only and is by way of example, and not as a limitation on the scope of the methods, compositions, strategies, and techniques described herein. Furthermore, references to trastuzumab in this application are intended to use generic terms as an example of any antibody. Thus, it is to be understood that the modifications and chemistry described herein with respect to trastuzumab are equally applicable to any antibody or monoclonal antibody, including those specifically listed herein.
Trastuzumab is a humanized monoclonal antibody that binds to domain IV of the extracellular domain of HER2/neu receptor. The HER2 gene (also known as HER2/neu and ErbB2 genes) is amplified in 20% -30% of early breast cancers, causing them to be overexpressed. Furthermore, in cancer, HER2 may signal it to be overactive in the absence of mitogens reaching and binding to any receptors.
HER2 extends across the cell membrane and carries the signal from outside the cell into the cell. In healthy humans, a signaling compound called a mitogen reaches the cell membrane and binds externally to other members of the HER receptor family. Those bound receptors then bind to HER2 (dimerize), activating HER2. HER2 then signals the cell interior. The signals pass through different biochemical pathways. The paths include PI3K/Akt paths and MAPK paths. These signals promote vascular invasion, survival and growth (angiogenesis) of cells.
Cells treated with trastuzumab undergo arrest in the G1 phase of the cell cycle, and thus proliferate less. Trastuzumab has been proposed to induce certain effects by down-regulating HER2/neu leading to disruption of receptor dimerization and signaling through the downstream PI3K cascade. P27Kip1 is then not phosphorylated and is able to enter the nucleus and inhibit cdk2 activity, resulting in cell cycle arrest. In addition, trastuzumab represses angiogenesis by inducing anti-angiogenic factors and repressing pro-angiogenic factors. It is believed that the contribution of unregulated growth observed in cancer may be due to proteolytic cleavage of HER2/neu resulting in release of the extracellular domain. Trastuzumab has been shown to inhibit HER2/neu ectodomain cleavage in breast cancer cells.
Expression in non-eukaryotic and eukaryotic organisms
In order to obtain high levels of expression of cloned TC polynucleotides, polynucleotides encoding the targeting polypeptides of the TC polypeptides of the invention are typically subcloned into expression vectors containing a strong promoter for direct transcription, a transcription/translation terminator, and, if nucleic acids encoding proteins, a ribosome binding site for translation initiation. Suitable bacterial promoters are known to those of ordinary skill in the art and are described, for example, in Sambrook et al and Ausubel et al.
Bacterial expression systems for expressing the TC polypeptides of the present invention can be used, including but not limited to, escherichia coli, bacillus species (Bacillus sp.), pseudomonas fluorescens (Pseudomonas fluorescens), pseudomonas aeruginosa (Pseudomonas aeruginosa), pseudomonas putida (Pseudomonas putida), and Salmonella (Salmonella) (Palva et al, gene 22:229-235 (1983); mosbach et al, nature 302:543-545 (1983)). Kits for such expression systems are commercially available. Eukaryotic expression systems for mammalian cells, yeast and insect cells are known to those of ordinary skill in the art and are also commercially available. Where orthogonal tRNA and aminoacyl tRNA synthetases (as described above) are used to express TC polypeptides of the invention, host cells are selected for expression based on their ability to use orthogonal components. Exemplary host cells include gram-positive bacteria (including but not limited to Bacillus brevis, bacillus subtilis, or Streptomyces) and gram-negative bacteria (Escherichia coli, pseudomonas fluorescens, pseudomonas aeruginosa, pseudomonas putida), as well as yeast and other eukaryotic cells. Cells comprising an O-tRNA/O-RS pair can be used as described herein.
Eukaryotic or non-eukaryotic host cells of the invention provide the ability to synthesize proteins comprising a large number of useful amounts of unnatural amino acids. In one aspect, the composition optionally includes, but is not limited to, at least 10 micrograms, at least 50 micrograms, at least 75 micrograms, at least 100 micrograms, at least 200 micrograms, at least 250 micrograms, at least 500 micrograms, at least 1 milligram, at least 10 milligrams, at least 100 milligrams, at least one gram or more of a protein comprising an unnatural amino acid, or an amount that can be achieved by an in vivo protein production process (see recombinant protein production and purification provided herein for details). In another aspect, the protein is optionally present in the composition at a concentration including but not limited to the following in a volume including but not limited to a cell lysate, buffer, drug buffer, or other liquid suspension including but not limited to a volume including but not limited to about 1nl to about 100L or more: at least 10 micrograms of protein per liter, at least 50 micrograms of protein per liter, at least 75 micrograms of protein per liter, at least 100 micrograms of protein per liter, at least 200 micrograms of protein per liter, at least 250 micrograms of protein per liter, at least 500 micrograms of protein per liter, at least 1 milligram of protein per liter, or at least 10 milligrams of protein per liter or more. It is a feature of the present invention to produce proteins comprising at least one unnatural amino acid in large quantities in eukaryotic cells, including but not limited to, more than is typically possible by other methods, including but not limited to in vitro translation.
The nucleotide sequence encoding the targeting polypeptide of the TC polypeptide may or may not include a sequence encoding a signal peptide. When a polypeptide is to be secreted from the cell in which it is expressed, a signal peptide is present. Such signal peptide may be any sequence. The signal peptide may be prokaryotic or eukaryotic. Coloma, M (1992) J.Imm.methods 152:89.104 describe a signal peptide (murine Ig kappa light chain signal peptide) for mammalian cells. Other signal peptides include, but are not limited to, the alpha factor signal peptide from Saccharomyces cerevisiae (U.S. Pat. No. 4,870,008, incorporated herein by reference), the signal peptide of mouse salivary amylase (O.Hagenbuchle et al, nature 289,1981, pages 643-646), the modified carboxypeptidase signal peptide (L.A. Valls et al, cell 48,1987, pages 887-897), the Yeast BAR1 signal peptide (WO 87/02670, incorporated herein by reference) and the Yeast aspartic protease 3 (YAP 3) signal peptide (see M.Egel-Mitani et al, yeast 6,1990, pages 127-137).
Examples of suitable mammalian host cells are known to those of ordinary skill in the art. Such host cells may be Chinese Hamster Ovary (CHO) cells (e.g., CHO-K1; ATCC CCL-61), green monkey Cells (COS) (e.g., COS 1 (ATCC CRL-1650), COS 7 (ATCC CRL-1651)); mouse cells (e.g., NS/O), baby Hamster Kidney (BHK) cell lines (e.g., ATCC CRL-1632 or ATCC CCL-10), and human cells (e.g., HEK 293 (ATCC CRL-1573)), plant cells in tissue culture. These and other cell lines are available from public depositories such as the American type culture Collection (American Type Culture Collection) of Rockville, md. To provide improved glycosylation of TC polypeptides, mammalian host cells may be modified to express sialyltransferases, such as 1, 6-sialyltransferases, for example as described in U.S. Pat. No. 5,047,335, incorporated herein by reference.
Methods of introducing exogenous DNA into mammalian host cells include, but are not limited to, calcium phosphate mediated transfection, electroporation, DEAE-dextran mediated transfection, liposome mediated transfection, viral vectors, and the transfection methods described by Life Technologies Ltd, paisley, UK using Lipofectamin 2000 and Roche Diagnostics Corporation, indianapolis, USA using FuGENE 6. These methods are well known in the art and are described by Ausbel et al (ed.), 1996,Current Protocols in Molecular Biology,John Wiley&Sons,New York,USA. Mammalian cell culture may be performed according to established methods, for example, as disclosed in (Animal Cell Biotechnology, methods and Protocols, by Nigel Jenkins, 1999,Human Press Inc.Totowa,N.J, USA and Harrison Mass, and Rae IF, general Techniques of Cell Culture, cambridge University Press 1997).
I.Escherichia coli, pseudomonas species and other prokaryotesBacterial expression techniques are known to those of ordinary skill in the art. A variety of vectors are available for bacterial hosts. The vector may be a single copy or low or high multiple copy vector. Vectors may be used for cloning and/or expression. In view of the vast literature on vectors, the commercial availability of many vectors, and even manuals describing the vectors and their restriction patterns and features, extensive discussion is not necessary here. As is well known, vectors typically include markers that allow selection, which can provide resistance to cytotoxic agents, prototrophy, or immunity. There are typically multiple markers that provide different properties.
A bacterial promoter is any DNA sequence capable of binding to a bacterial RNA polymerase and initiating transcription of the coding sequence (e.g., structural gene) downstream (3') into mRNA. The promoter will have a transcription initiation region typically located near the 5' end of the coding sequence. The transcription initiation region typically includes an RNA polymerase binding site and a transcription initiation site. The bacterial promoter may also have a second domain, called an operator, which may overlap with the adjacent RNA polymerase binding site at the beginning of RNA synthesis. The operator allows for negative regulation (inducible) of transcription, as the gene repressor protein can bind to the operator, thereby inhibiting transcription of a particular gene. Constitutive expression may occur in the absence of a negative regulatory element, such as an operator. Alternatively, positive regulation may be achieved by gene-activated protein binding sequences, if present, which are typically located proximal (5') to the RNA polymerase binding sequence. An example of a gene-activated protein is catabolism-activated protein (CAP), which helps to initiate transcription of the lac operon in E.coli (Escherichia coli/E.coli) (see Raibaud et al, ANNU. REV. GENET. (1984) 18:173). Thus, the regulated expression may be positive or negative, thereby enhancing or reducing transcription.
The term "bacterial host" or "bacterial host cell" refers to a bacterium that can or has been used as a recipient for a recombinant vector or other transfer DNA. The term includes progeny of the original bacterial host cell that has been transfected. It will be appreciated that the progeny of a single parent cell need not be identical, in form or genomic or whole DNA complement, to the original parent, due to the accidental or deliberate mutation. Offspring of a parent cell that are sufficiently similar to the parent to be characterized by relevant properties, such as in the presence of a nucleotide sequence encoding a TC polypeptide, are included in the offspring referred to by the definition.
The selection of suitable host bacteria for expression of the TC polypeptide is known to those of ordinary skill in the art. In selecting bacterial hosts for expression, suitable hosts may include those that exhibit, inter alia, good inclusion body formation ability, low proteolytic activity, and overall robustness. Bacterial hosts are generally available from a variety of sources including, but not limited to, the university of california (Berkeley, CA) biophysical and medical physics system bacterial genetic library center; and American type culture Collection ("ATCC") (Manassas, va.). Industrial/pharmaceutical fermentation typically uses bacteria derived from strain K (e.g. W3110) or bacteria derived from strain B (e.g. BL 21). These strains are particularly useful because their growth parameters are well known and robust. In addition, these strains are non-pathogenic, which is commercially important for safety and environmental reasons. Other examples of suitable E.coli hosts include, but are not limited to, strains of BL21, DH10B or derivatives thereof. In another embodiment of the method of the invention, the E.coli host is a protease negative strain, including but not limited to OMP-and LON-. The host cell strain may be a Pseudomonas species including, but not limited to, pseudomonas fluorescens, pseudomonas aeruginosa, and Pseudomonas putida. Pseudomonas fluorescens biological variant 1, designated strain MB101, is known to be useful for recombinant production and for therapeutic protein production processes. Examples of Pseudomonas expression systems include those available from The Dow Chemical Company as host strains (Midland, MI, available at www.dow.com).
Once the recombinant host cell strain has been established (i.e., the expression construct has been introduced into a host cell and the host cell with the appropriate expression construct isolated), the recombinant host cell strain is cultured under conditions suitable for the production of the TC polypeptide. As will be apparent to those skilled in the art, the method of culturing the recombinant host cell strain will depend on the nature of the expression construct used and the nature of the host cell. Recombinant host strains are typically cultured using methods known to those of ordinary skill in the art. Recombinant host cells are typically cultured in a liquid medium containing assimilable carbon sources, nitrogen sources and inorganic salts, and optionally vitamins, amino acids, growth factors and other protein culture supplements known to those of ordinary skill in the art. The liquid medium used for host cell culture may optionally contain antibiotics or antifungals to prevent the growth of unwanted microorganisms and/or compounds (including but not limited to antibiotics) to select host cells containing the expression vector.
Recombinant host cells may be cultured in batch or continuous form, where the cells are harvested (in the case of TC polypeptide accumulation within the cell) or the culture supernatant is harvested in batch or continuous form. For the production of prokaryotic host cells, batch culture and cell harvest are preferred.
The TC polypeptides of the invention are typically purified after expression in a recombinant system. The TC polypeptide may be purified from the host cell or culture medium by a variety of methods known in the art. TC polypeptides produced in bacterial host cells may be poorly soluble or insoluble (in the form of inclusion bodies). In one embodiment of the invention, amino acid substitutions may be readily made in selected TC polypeptides to increase the solubility of recombinantly produced proteins using methods disclosed herein as well as methods known in the art. In the case of insoluble proteins, the proteins may be collected from the host cell lysate by centrifugation, and the cells may then be further homogenized. In the case of poorly soluble proteins, compounds including, but not limited to, polyethylenimine (PEI) may be added to induce precipitation of partially soluble proteins. The precipitated protein can then be conveniently collected by centrifugation. The recombinant host cells can be disrupted or homogenized using a variety of methods known to those of ordinary skill in the art to release inclusion bodies from the cells. Host cell disruption or homogenization may be performed using well known techniques including, but not limited to, enzymatic cell disruption, sonication, dunn homogenization, or high pressure release disruption. In one embodiment of the method of the invention, a high pressure release technique is used to disrupt escherichia coli host cells to release inclusion bodies of the TC polypeptide. When processing inclusion bodies of TC polypeptides, it may be advantageous to minimize repeated homogenization times to maximize inclusion body yield without loss due to factors such as solubilization, mechanical shear, or proteolysis.
Any of several suitable solubilizing agents known in the art may then be used to solubilize the insoluble or precipitated TC polypeptide. The TC polypeptide may be solubilized with urea or guanidine hydrochloride. The volume of dissolved TC polypeptide should be minimized so that mass production can be performed using easily manageable batches. This factor may be important in large-scale commercial environments where recombinant hosts may grow in batches in volumes of thousands of liters. In addition, in the production of TC polypeptides in large scale commercial environments, particularly for human pharmaceutical use, the use of irritating chemicals that may damage the machinery and the container or the protein product itself should be avoided as much as possible. In the methods of the invention, it has been demonstrated that the milder denaturant urea can be used to replace the milder denaturant guanidine hydrochloride to solubilize TC polypeptide inclusion bodies. The use of urea significantly reduces the risk of damage to stainless steel equipment used in the process of manufacturing and purifying TC polypeptides while effectively dissolving TC polypeptide inclusion bodies.
In the case of soluble targeting polypeptides of TC proteins, the targeting polypeptide of TC may be secreted into the periplasmic space or medium. In addition, soluble TCs may be present in the cytoplasm of the host cell. Concentration of soluble TC may be required before the purification step is performed. Standard techniques known to those of ordinary skill in the art may be used to concentrate the soluble targeting polypeptide from, for example, a cell lysate or culture medium. In addition, standard techniques known to those of ordinary skill in the art may be used to destroy host cells and release soluble TCs from the cytoplasmic or periplasmic space of the host cells.
In general, there is an occasional need to denature and reduce the expressed polypeptide and then refold the polypeptide into a preferred conformation. For example, guanidine, urea, DTT, DTE and/or chaperonin may be added to the target translation product. Methods of reducing, denaturing and renaturating proteins are known to those of ordinary skill in the art (see the above references and Debinski et al (1993) J.biol. Chem.,268:14065-14070; kreitman and Pastan (1993) bioconjug. Chem.,4:581-585; and Buchner et al (1992) Anal. Biochem., 205:263-270). For example, debinski et al describe denaturation and reduction of inclusion body proteins in guanidine-DTE. The protein may be refolded in a redox buffer containing (including but not limited to) oxidized glutathione and L-arginine. Refolding reagents can flow or otherwise move into contact with one or more polypeptides or other expression products, and vice versa.
In the case of prokaryotic production of TC polypeptides, the TC polypeptides thus produced may be misfolded and thus lack or have reduced biological activity. The biological activity of the protein can be restored by "refolding". In general, misfolded TC polypeptides are refolded by dissolving (wherein the TC polypeptide is also insoluble), unfolding and reducing the polypeptide chain using, for example, one or more chaotropic agents (e.g., urea and/or guanidine) and a reducing agent capable of reducing disulfide bonds (e.g., dithiothreitol, DTT, or 2-mercaptoethanol, 2-ME). At moderate concentrations of chaotropic agents, an oxidizing agent (e.g., oxygen, cystine, or cystamine) is then added, which allows the disulfide bond to reform. The TC polypeptides may be refolded using standard methods known in the art, such as those described in U.S. patent nos. 4,511,502, 4,511,503 and 4,512,922, which are incorporated herein by reference. TC polypeptides may also be co-folded with other proteins to form heterodimers or heteromultimers.
After refolding, the TC-targeted polypeptide can be further purified. Purification of TC may be accomplished using a variety of techniques known to those of ordinary skill in the art, including hydrophobic interaction chromatography, size exclusion chromatography, ion exchange chromatography, reversed phase high performance liquid chromatography, affinity chromatography, and the like, or any combination thereof. Additional purification may also include drying or precipitation steps to purify the protein.
After purification, the TC-targeted polypeptide can be exchanged into a different buffer and/or concentrated by any of a variety of methods known in the art, including, but not limited to, diafiltration and dialysis. TC provided as a single purified protein may aggregate and precipitate.
Purified targeting polypeptides of TC may be at least 90% pure (as measured by reverse phase high performance liquid chromatography, RP-HPLC, or sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE) or at least 95% pure, or at least 96% pure, or at least 97% pure, or at least 98% pure, or at least 99% or more pure. Regardless of the exact purity value of the purity of the targeting polypeptide of TC, the purity of the targeting polypeptide of TC is sufficient for use as a pharmaceutical product or for further processing, such as conjugation with a water soluble polymer (such as PEG).
Some TC molecules may be used as therapeutic agents in the absence of other active ingredients or proteins (except excipients, carriers and stabilizers, serum albumin, etc.), or they may be complexed with another protein or polymer.
It has been previously shown that unnatural amino acid sites can be specifically incorporated into proteins in vitro by adding a chemical aminoacylation-suppressing tRNA to a protein synthesis reaction programmed with a gene containing the desired amber nonsense mutation. Using these methods, strains that are auxotrophic for a particular amino acid can be used to replace many of the twenty common amino acids with structurally similar homologs, such as phenylalanine with fluorophenylalanine. See, e.g., noren, C.J., anthony-Call, griffith, M.C., schultz, P.G.A. general method for site-specific incorporation of unnatural amino acids into proteins, science,244:182-188 (1989); nowak, et al Science 268:439-42 (1995); bain, j.d., glabe, c.g., dix, t.a., chamberlin, a.r., diala, e.s. biosystemic site-specific Incorporation of a non-natural amino acid into a polypeptide, j.am Chem Soc,111:8013-8014 (1989); n. Budisk et al, FASEB J.13:41-51 (1999); ellman, j.a., mendel, d., anthony-Cahill, s., noren, c.j., schultz, p.g., biosystemic method for introducing unnatural amino acids site-specifically into proteins, methods in enz., volume 202, 301-336 (1992); and Mendel, D., cornish, V.W, & Schultz, P.G. site-Directed Mutagenesis with an Expanded Genetic Code, annu Rev Biophys. Biomol Structure.24, 435-62 (1995).
For example, suppressor tRNA is prepared that recognizes the stop codon UAG and is chemically aminoacylated with an unnatural amino acid. Conventional site-directed mutagenesis is used to introduce a stop codon TAG at a target site in a protein gene. See, e.g., sayers, J.R., schmidt, W.Eckstein, F.5'-3'Exonucleases in phosphorothioate-based oligonucleotide-directed mutagenesis, nucleic Acids Res,16 (3): 791-802 (1988). When the acylation suppressor tRNA and the mutant gene are combined in an in vitro transcription/translation system, an unnatural amino acid is incorporated in response to the UAG codon, thereby producing a protein containing the amino acid at the indicated position. Use [ use 3 H]Experiments with Phe and with alpha-hydroxy acids show that only the desired amino acid is incorporated at the position specified by the UAG codon and that said amino acid is not incorporated at any other position in the protein. See, e.g., noren, et al, supra; kobayashi et al, (2003) Nature Structural Biology 10 (6): 425-432; and Ellman, j.a., mendel, d., schultz, p.g., site-specific incorporation of novel backbone structures into proteins, science,255 (5041): 197-200 (1992).
The tRNA can be aminoacylated with the desired amino acid by any method or technique, including, but not limited to, chemical or enzymatic aminoacylation.
Aminoacylation can be accomplished by aminoacyl tRNA synthetases or by other enzyme molecules, including but not limited to ribozymes. The term "ribozyme" is interchangeable with "catalytic RNA". Cech and colleagues (Cech, 1987, science,236:1532-1539; mcCorkle et al, 1987,Concepts Biochem.64:221-226) demonstrate the presence of naturally occurring RNA that can act as a catalyst (ribozyme). However, although these natural RNA catalysts have only been shown to act on ribonucleic acid substrates for cleavage and splicing, recent developments in ribozyme artificial evolution have extended catalysis to a variety of chemical reactions. Studies have identified RNA molecules that catalyze an aminoacyl-RNA bond at their 3 '-end (2') themselves (Illangake kare et al, 1995Science 267:643-647), and RNA molecules that can transfer amino acids from one RNA molecule to another (Lohse et al, 1996,Nature 381:442-444).
U.S. patent application publication 2003/0228593, incorporated herein by reference, describes methods of constructing ribozymes and their use in aminoacylating tRNA's with naturally encoded and non-naturally encoded amino acids. Enzyme molecules (including but not limited to ribozymes) that can aminoacylate the substrate-immobilized form of the tRNA can achieve efficient affinity purification of the aminoacylated product. Examples of suitable substrates include agarose, agarose gel and magnetic beads. Chemistry and Biology 2003,10:1077-1084 and U.S. patent application publication 2003/0228593, incorporated herein by reference, describe the production and use of ribozymes in substrate-immobilized form for aminoacylation.
Chemical aminoacylation methods include, but are not limited to, those described by Hecht and colleagues (Hecht, S.M.Acc.Chem.Res.1992,25,545;Heckler,T.G; roeser, J.R; xu, c.; chang, P., hecht, S.M. biochemistry 1988,27,7254, hecht, S.M., alford, B.L., kuroda, Y., kitano, S.J., chem.1978,253, 4517) and Schultz, chamberlin, dougherty et al (Cornish, V.W., mendel, D., schultz, P.G.Angew.Chem.Int.Ed.Engl.1995,34,621;Robertson,S.A, ellman, J.A., schultz, P.G., J.chem.Soc.1991, 113,2722, noren, C.J., anthony-Cahill, S.J, griffith, M.C., schultz, P.G., science 1989,244,182, bain J.D., gle, C.G., dix, T.A., R.A., J., J.35, J., J.A., J., J.J., J., J,. Such methods or other chemical aminoacylation methods can be used to aminoacylate tRNA molecules.
Methods for producing catalytic RNAs can include generating separate pools of random ribozyme sequences, subjecting the pools to directed evolution, screening the pools for a desired aminoacylation activity, and selecting those ribozyme sequences that exhibit the desired aminoacylation activity.
A reconstructed translation system may also be used. Mixtures of purified translation factors have also been successfully used to translate mRNA into protein, as well as combinations of lysates or lysates supplemented with purified translation factors such as initiation factor-1 (IF-1), IF-2, IF-3 (α or β), elongation factor T (EF-Tu), or termination factor. The cell-free system may also be a coupled transcription/translation system in which DNA is introduced into the system, transcribed into mRNA and translated into mRNA as described in Current Protocols in Molecular Biology (F.M. Ausubel et al, wiley Interscience, 1993), which is hereby specifically incorporated by reference. RNA transcribed in eukaryotic transcription systems may be in the form of heteronuclear RNA (hnRNA) or 5 '-terminal cap (7-methylguanosine) and 3' -terminal poly-A tail mature mRNA, which may be an advantage in certain translation systems. For example, capped mRNA is efficiently translated in a reticulocyte lysate system.
Macromolecular polymers conjugated to TC polypeptides
Various modifications may be made to the unnatural amino acid polypeptides described herein using the compositions, methods, techniques, and strategies described herein. Such modifications include incorporation of other functional groups, including but not limited to tags, into the unnatural amino acid component of the polypeptide; a dye; a polymer; a water-soluble polymer; derivatives of polyethylene glycol; a photocrosslinking agent; a radionuclide; a cytotoxic compound; a drug; an affinity tag; a photoaffinity tag; an active compound; a resin; a second protein or polypeptide analog; an antibody or antibody fragment; a metal chelator; a cofactor; a fatty acid; a carbohydrate; a polynucleotide; DNA; RNA; an antisense polynucleotide; a saccharide; a water-soluble dendritic polymer; cyclodextrin; inhibitory ribonucleic acid; a biological material; a nanoparticle; spin labeling; a fluorophore, a metal-containing moiety; a radioactive portion; novel functional groups; groups that interact covalently or non-covalently with other molecules; a light cage portion; an actinic radiation excitable moiety; a photoisomerization moiety; biotin; derivatives of biotin; biotin analogues; incorporation of heavy atom moieties; a chemically cleavable group; a photocleavable group; an elongated side chain; a carbon-linked sugar; a redox active agent; an aminothiopropionic acid; a toxic moiety; isotopically labeled moieties; a biophysical probe; phosphorescent groups; a chemiluminescent group; an electron dense group; a magnetic group; an intercalating group; a chromophore; an energy transfer agent; a bioactive agent; a detectable label; a small molecule; a quantum dot; a nano-emitter; a radionucleotide; a radioactive emitter; a neutron capture agent; or any combination of the above, or any other desired compound or substance. As an illustrative, non-limiting example of the compositions, methods, techniques, and strategies described herein, the following description will focus on the addition of macromolecular polymers to unnatural amino acid polypeptides, with the understanding that the compositions, methods, techniques, and strategies described therein are also applicable (with appropriate modifications, if necessary, and as may be modified by those of skill in the art using the disclosure herein) to the addition of other functional groups, including, but not limited to, those listed above.
A variety of macromolecular polymers and other molecules may be attached to the TC polypeptides of the invention to modulate the biological properties of the TC polypeptides and/or to provide novel biological properties to the TC molecules. These macromolecular polymers may be linked to the TC polypeptide by naturally encoded amino acids, non-naturally encoded amino acids, or any functional substituent of a natural or non-natural amino acid, or any substituent or functional group added to a natural non-natural amino acid. The molecular weight of the polymer may be in a wide range including, but not limited to, between about 100Da and about 100,000Da or greater. The molecular weight of the polymer may be between about 100 and about 100,000Da, including but not limited to 100,000Da, 95,000Da, 90,000Da, 85,000Da, 80,000Da, 75,000Da, 70,000Da, 65,000Da, 60,000Da, 55,000Da, 50,000Da, 45,000Da, 40,000Da, 35,000Da, 30,000Da, 25,000Da, 20,000Da, 15,000Da, 10,000Da, 9,000Da, 8,000Da, 7,000Da, 6,000Da, 5,000Da, 4,000Da, 3,000Da, 2,000Da, 1,000Da, 900Da, 800Da, 700Da, 600Da, 500Da, 400Da, 300Da, 200Da and 100Da. In some embodiments, the molecular weight of the polymer is between about 100Da and about 50,000 Da. In some embodiments, the molecular weight of the polymer is between about 100Da and about 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 1,000da and about 40,000 da. In some embodiments, the molecular weight of the polymer is between about 5,000da and about 40,000 da. In some embodiments, the molecular weight of the polymer is between about 10,000da and about 40,000 da.
The present invention provides substantially homogeneous formulations of multimeric protein conjugates. As used herein, "substantially homogeneous" means that more than half of the total protein is observed for the multimeric protein conjugate molecule. The multimeric protein conjugates are biologically active and the "substantially homogeneous" pegylated TC polypeptide formulations of the invention provided herein are sufficiently homogeneous to show the advantages of homogeneous formulations, such as those formulations that are easy for clinical application in terms of predictability of inter-batch pharmacokinetics.
It is also possible to choose a mixture of the preparation of the multimeric protein conjugate molecules and the advantage provided herein is that the ratio of the single multimeric protein conjugates included in the mixture can be chosen. Thus, if desired, a mixture of various proteins having different numbers of linked polymer moieties (i.e., dimerized, trimerized, tetramerized, etc.) can be prepared, and the conjugates combined with a single multimeric protein conjugate prepared using the methods of the invention and having a predetermined ratio of the mixture of single multimeric protein conjugates.
The polymer selected may be water-soluble such that the protein to which it is attached does not precipitate in an aqueous environment such as a physiological environment. The polymer may be branched or unbranched. For therapeutic use of the final product formulation, the polymer will be pharmaceutically acceptable.
Examples of polymers include, but are not limited to, polyalkyl ethers and alkoxy-terminated analogs thereof (e.g., polyoxyethylene glycol, polyoxyethylene/propylene glycol and methoxy or ethoxy-terminated analogs thereof, especially polyoxyethylene glycol, the latter also known as polyethylene glycol or PEG); polyvinylpyrrolidone; polyvinyl alkyl ether; polyoxazolines, polyalkyloxazolines and polyhydroxyalkyl oxazolines; polyacrylamides, polyalkylacrylamides and polyhydroxyalkylacrylamides (e.g., polyhydroxypropylmethacrylamides and derivatives thereof); a polyhydroxyalkyl acrylate; polysialic acid and analogues thereof; a hydrophilic peptide sequence; polysaccharides and derivatives thereof, including dextran and dextran derivatives, such as carboxymethyl dextran, dextran sulfate, aminodextran; cellulose and its derivatives, such as carboxymethyl cellulose, hydroxyalkyl cellulose; chitin and its derivatives, such as chitosan, succinyl chitosan, carboxymethyl chitin and carboxymethyl chitosan; hyaluronic acid and derivatives thereof; starch; alginic acid; chondroitin sulfate; albumin; amylopectin and carboxymethyl amylopectin; polyamino acids and derivatives thereof, such as polyglutamic acid, polylysine, polyaspartic acid; maleic anhydride copolymers such as: styrene maleic anhydride copolymer, divinyl ethyl ether maleic anhydride copolymer; polyvinyl alcohol; copolymers thereof; a terpolymer thereof; mixtures thereof; and derivatives of the foregoing.
The ratio of polyethylene glycol molecules to protein molecules will vary, as will their concentration in the reaction mixture. In general, the optimal ratio (in terms of reaction efficiency, since there is a minimum excess of unreacted protein or polymer) may be determined by the molecular weight of the polyethylene glycol selected and the number of available reactive groups. With respect to molecular weight, generally the higher the molecular weight of the polymer, the fewer the number of polymer molecules that can be attached to the protein. Similarly, branching of the polymer should be considered in optimizing these parameters. In general, the higher the molecular weight (or more branches), the higher the polymer to protein ratio.
As used herein, and when considering PEG: TC polypeptide conjugate, the term "therapeutically effective amount" refers to an amount that provides a desired benefit to a patient. The amount varies from person to person and depends on a variety of factors including the overall physical condition of the patient and the root cause of the condition to be treated. The amount of TC polypeptide used in the therapy provides an acceptable rate of change and maintains the desired response at a beneficial level. One of ordinary skill in the art can readily determine a therapeutically effective amount of the compositions of the present invention using publicly available materials and procedures.
The water-soluble polymer may be in any structural form including, but not limited to, linear, forked, or branched. Typically, the water-soluble polymer is a poly (alkylene glycol), such as polyethylene glycol (PEG), although other water-soluble polymers may also be used. For example, PEG is used to describe certain embodiments of the invention.
Polyethylene glycols are well known water-soluble polymers which are commercially available or can be prepared by ring-opening polymerization of ethylene glycol according to methods known to those skilled in the art (Sandler and Karo, polymer Synthesis, academic Press, new York, vol. 3, pages 138-161). The term "PEG" is used broadly to encompass any polyethylene glycol molecule, regardless of size or modification of the PEG end, and may be expressed as linked to a TC polypeptide by the formula:
XO-(CH 2 CH 2 O) n -CH 2 CH 2 -Y
wherein n is 2 to 10,000 and X is H or a terminal modification, including but not limited to C 1-4 Alkyl, protecting group or terminal functional group.
In some cases, the PEG used in the present invention is terminated at one end with a hydroxyl or methoxy group, i.e., X is H or CH 3 ("methoxy PEG"). Alternatively, PEG may terminate with a reactive group, thereby forming a difunctional polymer. Typical reactive groups may include those typically used to react with functional groups found in 20 common amino acids (including but not limited to maleimide groups, activated carbonates (including but not limited to p-nitrophenyl esters), activated esters (including but not limited to N-hydroxysuccinimide, p-nitrophenyl esters) and aldehydes), and functional groups inert to 20 common amino acids but specifically reactive with complementary functional groups found in non-naturally encoded amino acids (including but not limited to azide groups, alkyne groups). It should be noted that the other end of PEG represented by Y in the above formula will pass through the sky Naturally occurring or non-naturally encoded amino acids are linked directly or indirectly to TC polypeptides. For example, Y may be an amide, carbamate, or urea linked to an amine group of the polypeptide (including but not limited to lysine or the N-terminal epsilon amine). Alternatively, Y may be a maleimide linkage linked to a thiol group (including but not limited to a thiol group of cysteine). Alternatively, Y may be linked to residues that are not normally available through the 20 common amino acids. For example, azide groups on PEG can react with alkyne groups on TC polypeptides to form Huisgen [3+2 ]]Cycloaddition products. Alternatively, alkyne groups on PEG can react with azide groups present in non-naturally encoded amino acids to form similar products. In some embodiments, strong nucleophiles (including but not limited to hydrazine, hydrazide, hydroxylamine, semicarbazide) may react with aldehyde or ketone groups present in the non-naturally encoded amino acid to form hydrazones, oximes, or semicarbazide, which may be further reduced in some cases by treatment with a suitable reducing agent. Alternatively, strong nucleophiles may be incorporated into the TC polypeptide by non-naturally encoded amino acids and used to preferentially react with ketone or aldehyde groups present in the water soluble polymer.
Any molecular weight of PEG may be used as desired, including but not limited to about 100 daltons (Da) to 100,000Da or higher as desired (including but not limited to sometimes 0.1-50kDa or 10-40 kDa). The molecular weight of PEG can be in a wide range including, but not limited to, between about 100Da and about 100,000Da or greater. The PEG may be between about 100 and about 100,000Da, including but not limited to 100,000Da, 95,000Da, 90,000Da, 85,000Da, 80,000Da, 75,000Da, 70,000Da, 65,000Da, 60,000Da, 55,000Da, 50,000Da, 45,000Da, 40,000Da, 35,000Da, 30,000Da, 25,000Da, 20,000Da, 15,000Da, 10,000Da, 9,000Da, 8,000Da, 7,000Da, 6,000Da, 5,000Da, 4,000Da, 3,000Da, 2,000Da, 1,000Da, 900Da, 800Da, 700Da, 600Da, 500Da, 400Da, 300Da, 200Da and 100Da. In some embodiments, PEG is between about 100Da and about 50,000 Da. In some embodiments, PEG is between about 100Da and about 40,000 Da. In some embodiments, PEG is between about 1,000da and about 40,000 da. In some embodiments, PEG is between about 5,000da and about 40,000 da. In some embodiments, PEG is between about 10,000da and about 40,000 da. Branched PEG's may also be used, including but not limited to PEG molecules having a molecular weight in the range of 1-100kDa (including but not limited to 1-50kDa or 5-20 kDa) per chain. The molecular weight of each strand of the branched PEG may be, including but not limited to, between about 1,000da and about 100,000da or greater. The molecular weight of each chain of branched PEG may be between about 1,000da and about 100,000da, including but not limited to 100,000da, 95,000da, 90,000da, 85,000da, 80,000da, 75,000da, 70,000da, 65,000da, 60,000da, 55,000da, 50,000da, 45,000da, 40,000da, 35,000da, 30,000da, 25,000da, 20,000da, 15,000da, 10,000da, 9,000da, 8,000da, 7,000da, 6,000da, 5,000da, 4,000da, 3,000da, 2,000da, and 1,000da. In some embodiments, the molecular weight of each strand of branched PEG is between about 1,000da and about 50,000 da. In some embodiments, the molecular weight of each strand of branched PEG is between about 1,000da and about 40,000 da. In some embodiments, the molecular weight of each strand of branched PEG is between about 5,000da and about 40,000 da. In some embodiments, the molecular weight of each strand of branched PEG is between about 5,000da and about 20,000 da. A broad range of PEG molecules are described in, but not limited to, shearwater Polymers, inc.
Typically, at least one terminus of a PEG molecule is available for reaction with a non-naturally encoded amino acid. For example, PEG derivatives or TLR linker derivatives carrying alkyne and azide moieties for reaction with amino acid side chains can be used to link PEG to non-naturally encoded amino acids as described herein. If the non-naturally encoded amino acid comprises an azide, the PEG typically contains an alkyne moiety to affect the formation of a [3+2] cycloaddition product or an activated PEG species (i.e., ester, carbonate) containing a phosphine group to affect the formation of an amide linkage. Alternatively, if the non-naturally encoded amino acid comprises an alkyne, then the PEG typically contains an azide moiety to affect the formation of the [3+2] huisgen cycloaddition product. If the non-naturally encoded amino acid comprises a carbonyl group, the PEG typically comprises a potent nucleophile (including but not limited to a hydrazide, hydrazine, hydroxylamine, or semicarbazide functional group) to affect the corresponding hydrazone, oxime, and semicarbazone linkages, respectively. In other alternatives, the reverse orientation of the above-described reactive groups may be used, i.e., the azide moiety in the non-naturally encoded amino acid may be reacted with an alkyne-containing PEG derivative.
In some embodiments, the TC polypeptide having a PEG derivative contains a chemical functionality that reacts with a chemical functionality present on a non-naturally encoded amino acid side chain.
In some embodiments, the present invention provides azide-containing and acetylene-containing polymer derivatives comprising a water-soluble polymer backbone having an average molecular weight of about 800Da to about 100,000 Da. The polymer backbone of the water-soluble polymer may be poly (ethylene glycol). However, it should be understood that a variety of water-soluble polymers including, but not limited to, poly (ethylene glycol) and other related polymers including poly (dextran) and polypropylene glycol are also suitable for use in the practice and use of the term PEG or poly (ethylene glycol) in the present invention are intended to encompass and include all such molecules. The term PEG includes, but is not limited to, any form of polyethylene glycol, including difunctional PEG, multi-arm PEG, derivatized PEG, bifurcated PEG, branched PEG, pendent PEG (i.e., PEG having one or more functional groups pendent to the polymer backbone or related polymers), or PEG having degradable linkages therein.
Polyethylene glycols are generally transparent, colorless, odorless, readily water-soluble, thermally stable, inert to many chemical agents, non-hydrolysing or deteriorating, and generally non-toxic. Poly (ethylene glycol) is considered biocompatible, that is, PEG can coexist with living tissue or organisms without causing injury. More specifically, PEG is substantially non-immunogenic, that is, PEG does not tend to produce an immune response in vivo. When attached to molecules having certain desired functions in the body (such as bioactive agents), PEG tends to mask the agents and may reduce or eliminate any immune response, thereby allowing the organism to tolerate the presence of the agents. PEG conjugates often do not produce a significant immune response or cause clotting or other adverse effects. Having the formula- -CH 2 CH 2 O--(CH 2 CH 2 O) n --CH 2 CH 2 PEG of which n is from about 3 to about 4000, typically from about 20 to about 2000, is suitable for use in the present invention. PEG having a molecular weight of about 800Da to about 100,000Da is particularly useful as the polymer backbone in some embodiments of the invention. The molecular weight of PEG can be in a wide range including, but not limited to, between about 100Da and about 100,000Da or greater. The molecular weight of the PEG may be between about 100Da and about 100,000Da, including but not limited to 100,000Da, 95,000Da, 90,000Da, 85,000Da, 80,000Da, 75,000Da, 70,000Da, 65,000Da, 60,000Da, 55,000Da, 50,000Da, 45,000Da, 40,000Da, 35,000Da, 30,000Da, 25,000Da, 20,000Da, 15,000Da, 10,000Da, 9,000Da, 8,000Da, 7,000Da, 6,000Da, 5,000Da, 4,000Da, 3,000Da, 2,000Da, 1,000Da, 900Da, 800Da, 700Da, 600Da, 500Da, 400Da, 300Da, 200Da and 100Da. In some embodiments, the molecular weight of PEG is between about 100Da and about 50,000 Da. In some embodiments, the molecular weight of PEG is between about 100Da and about 40,000 Da. In some embodiments, the molecular weight of PEG is between about 1,000da and about 40,000 da. In some embodiments, the molecular weight of PEG is between about 5,000da and about 40,000 da. In some embodiments, the molecular weight of PEG is between about 10,000da and about 40,000 da.
The polymer backbone may be linear or branched. Branched polymer backbones are generally known in the art. Typically, branched polymers have a central branched core portion and a plurality of linear polymer chains attached to the central branched core. PEG is typically used in branched form and can be prepared by adding ethylene oxide to various polyols such as glycerol, glycerol oligomers, pentaerythritol and sorbitol. The central branching moiety may also be derived from several amino acids, such as lysine. Branched poly (ethylene glycol) can be represented in general form as R (-PEG-OH) m Wherein R is derived from a core moiety such as glycerol, glycerol oligomer or pentaerythritol and m represents the number of arms. Multi-arm PEG molecules, such as those described in U.S. Pat. nos. 5,932,462;5,643,575;5,229,490;4,289,872; U.S. patent application 2003/0143596; WO 96/21469; and those described in WO93/21259 can also be used as polymersA backbone, each of which is incorporated by reference herein in its entirety.
Branched PEG may also be in the form of a forked PEG, consisting of PEG (- -YCHZ) 2 ) n There is still another branched form, the pendent PEG has a reactive group, such as a carboxyl group, along the PEG backbone rather than at the end of the PEG chain.
In addition to these forms of PEG, polymers with weak or degradable linkages in the backbone can also be prepared. For example, PEG having readily hydrolyzable ester linkages in the polymer backbone can be prepared. As shown below, this hydrolysis results in cleavage of the polymer into lower molecular weight fragments:
-PEG-CO 2 -PEG-+H 2 O→PEG-CO 2 H+HO-PEG-
those of ordinary skill in the art understand that the term poly (ethylene glycol) or PEG represents or includes all forms known in the art, including but not limited to those disclosed herein.
Many other polymers are also suitable for use in the present invention. In some embodiments, water-soluble polymer backbones having from 2 to about 300 termini are particularly useful in the present invention. Examples of suitable polymers include, but are not limited to, other poly (alkylene glycols) such as polypropylene glycol ("PPG"), copolymers thereof (including, but not limited to, copolymers of ethylene glycol and propylene glycol), terpolymers thereof, mixtures thereof, and the like. Although the molecular weight of each chain of the polymer backbone may vary, it is typically in the range of about 800Da to about 100,000Da, typically in the range of about 6,000Da to about 80,000 Da. The molecular weight of each chain of the polymer backbone can be between about 100Da and about 100,000Da, including but not limited to 100,000Da, 95,000Da, 90,000Da, 85,000Da, 80,000Da, 75,000Da, 70,000Da, 65,000Da, 60,000Da, 55,000Da, 50,000Da, 45,000Da, 40,000Da, 35,000Da, 30,000Da, 25,000Da, 20,000Da, 15,000Da, 10,000Da, 9,000Da, 8,000Da, 7,000Da, 6,000Da, 5,000Da, 4,000Da, 3,000Da, 2,000Da, 1,000Da, 900Da, 800Da, 700Da, 600Da, 500Da, 400Da, 300Da, 200Da and 100Da. In some embodiments, the molecular weight of each chain of the polymer backbone is between about 100Da and about 50,000 Da. In some embodiments, the molecular weight of each chain of the polymer backbone is between about 100Da and about 40,000 Da. In some embodiments, the molecular weight of each strand of the polymer backbone is between about 1,000da and about 40,000 da. In some embodiments, the molecular weight of each strand of the polymer backbone is between about 5,000da and about 40,000 da. In some embodiments, the molecular weight of each strand of the polymer backbone is between about 10,000da and about 40,000 da.
Those of ordinary skill in the art will recognize that the above list of substantially water-soluble backbones is by no means exhaustive, but merely illustrative, and that all polymeric materials having the above qualities are considered suitable for use in the present invention.
In some embodiments of the invention, the polymer derivative is "multifunctional", meaning that the polymer backbone has at least two ends, and possibly up to about 300 ends, functionalized or activated with functional groups. Polyfunctional polymer derivatives include, but are not limited to, linear polymers having two ends, each end bonded to a functional group that may be the same or different.
The term "protected" refers to a moiety in which a protecting group is present or which prevents a chemically reactive functional group from reacting under certain reaction conditions. The protecting group will vary depending on the type of chemically reactive group being protected. For example, if the chemically reactive group is an amine or a hydrazide, the protecting group may be selected from t-butoxycarbonyl (t-Boc) and 9-fluorenylmethoxycarbonyl (Fmoc). If the chemically reactive group is a thiol, the protecting group may be an ortho-pyridyl disulfide. If the chemically reactive group is a carboxylic acid, such as butyric acid or propionic acid, or a hydroxyl group, the protecting group may be benzyl or alkyl, such as methyl, ethyl or t-butyl. Other protecting groups known in the art may also be used in the present invention.
Specific examples of terminal functional groups in the literature include, but are not limited to, N-succinimidyl carbonate (see, e.g., U.S. Pat. Nos. 5,281,698, 5,468,478), amine (see, e.g., buckmann et al Makromol. Chem.182:1379 (1981), zalipsky et al Eur. Polym. J.19:1177 (1983)), hydrazide (see, e.g., andresz et al Makromol. Chem.179:301 (1978)), succinimidyl propionate and succinimidyl butyrate (see, e.g., olson et al, poly (ethylene glycol) Chemistry & Biological Applications, pages 170-181, harris & Zalippy, ACS, washington, D.C.,1997; see also U.S. Pat. No.5,672,662), succinimidyl succinate (see, e.g., abuchowski et al Cancer biochem. Biophys.7:175 (1984) and Joppich et al Makromol. Chem.180:1381 (1979), succinimidyl esters (see, e.g., U.S. Pat. No. 4,670,417), benzotriazole carbonates (see, e.g., U.S. Pat. No.5,650,234), glycidyl ethers (see, e.g., pitha et al Eur. J biochem.94:11 (1979), elling et al, biotech. Appl. Biochem.13:354 (1991), oxycarbonyl imidazoles (see, e.g., beauchamp, et al, anal. Biochem.131:25 (1983), tondelli et al J. Controlled Release 1:251 (1985)), p-nitrophenyl carbonates (see, e.g., veronese et al, appl. Biochem.94:11 (1985); and Sartre et al, appl.biochem.Biotech.,27:45 (1991)), aldehydes (see, e.g., harris et al J.Polym.Sci.chem.Ed.22:341 (1984), U.S. Pat. No.5,824,784, U.S. Pat. No.5,252,714), maleimides (see, e.g., goodson et al Biotechnology (NY):343 (1990), romani et al Chemistry of Peptides and Proteins:29 (1984)) and Kogan, synthetic Comm.22:2417 (1992)), ortho-pyridyl-disulfides (see, e.g., woghiren, et al bioconj.chem.4:314 (1993)), propenols (see, e.g., sawhney et al Macromolecules,26:581 (1993)), vinyl sulfones (see, e.g., U.S. Pat. No.5,900,461). All references and patents above are incorporated herein by reference.
PEGylation of TC polypeptides containing non-naturally encoded amino acids, such as para-azido-L-phenylalanine (i.e., the addition of any water-soluble polymers) is performed by any convenient method. For example, TC polypeptides are pegylated with alkyne-terminated mPEG derivatives. Briefly, at room temperature, excess solid mPEG (5000) -O-CH was stirred 2 -c≡ch is added to an aqueous solution of a TC polypeptide containing para-azido-L-Phe. Generally, pK is used for aqueous solutions a Buffer buffers near the pH at which the reaction is carried out (typically about pH 4-10)And (5) punching. Examples of suitable buffers for PEGylation at pH 7.5 include, but are not limited to HEPES, phosphate, borate, TRIS-HCl, EPPS, and TES. The pH is continuously monitored and adjusted if necessary. The reaction is typically allowed to continue for about 1 to 48 hours.
The reaction product is then subjected to hydrophobic interaction chromatography to bind the PEGylated TC polypeptide to free mPEG (5000) -O-CH 2 Any high molecular weight complex of c≡ch and pegylated TC polypeptide separates, when the unblocked PEG is activated at both ends of the molecule, thereby crosslinking the TC polypeptide molecule. The conditions during hydrophobic interaction chromatography are free mPEG (5000) -O-CH 2 The c≡ch flows through the column and any cross-linked pegylated TC polypeptide complex, which contains one TC polypeptide molecule conjugated to one or more PEG groups, elutes after the desired form. Suitable conditions vary depending on the relative size of the crosslinked complex to the desired conjugate and are readily determined by one of ordinary skill in the art. The eluate containing the desired conjugate is concentrated by ultrafiltration and desalted by diafiltration.
Substantially purified PEG-TC may be produced using the elution methods outlined above, wherein the PEG-TC produced has a purity level of at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, particularly a purity level of at least about 75%, 80%, 85%, and more particularly a purity level of at least about 90%, a purity level of at least about 95%, a purity level of at least about 99% or greater, as determined by suitable methods such as SDS/PAGE analysis, RP-HPLC, SEC, and capillary electrophoresis. If desired, the PEGylated TC polypeptide obtained from the hydrophobic chromatography can be further purified by one or more procedures known to those of ordinary skill in the art, including but not limited to affinity chromatography; anion or cation exchange chromatography (using, but not limited to, DEAE SEPHAROSE); silica chromatography; reversed phase HPLC; gel filtration (using a filter including but not limited to Sephadex G-75); hydrophobic interaction chromatography; size exclusion chromatography, metal chelate chromatography; ultrafiltration/diafiltration; precipitating with ethanol; precipitating ammonium sulfate; focusing the chromatogram; displacement chromatography; electrophoresis procedures (including but not limited to preparative isoelectric focusing), differential solubility (including but not limited to ammonium sulfate precipitation), or extraction. Apparent molecular weight can be estimated by GPC compared to globular protein standards (Preneta, AZ in PROTEIN PURIFICATION METHODS, A PRACTICAL APPROACH (Harris & Angal) IRL Press 1989, 293-306). The purity of the TC-PEG conjugate may be assessed by proteolytic degradation (including but not limited to trypsin cleavage) followed by mass spectrometry. Pepinsky RB., et al, J.Pharmcol. & exp. Ther.297 (3): 1059-66 (2001).
The water-soluble polymer linked to the amino acid of the targeting polypeptide of the TC polypeptide of the present invention may be further derivatized or substituted, but is not limited thereto.
Azide-containing PEG derivatives or TLR linker derivatives
In another embodiment of the invention, the targeting polypeptide of TC is modified with a PEG derivative containing an azide moiety that will react with an alkyne moiety present on a non-naturally encoded amino acid side chain. Generally, the average molecular weight of the PEG derivative or TLR linker derivative ranges from 1 kDa to 100kDa, and in some embodiments, from 10 kDa to 40kDa.
In some embodiments, the azide-terminated PEG derivative will have the following structure:
RO-(CH 2 CH 2 O) n -O-(CH 2 ) m -N 3
where R is a simple alkyl group (methyl, ethyl, propyl, etc.), m is 2-10 and n is 100-1,000 (i.e., average molecular weight between 5-40 kDa).
In another embodiment, the azide-terminated PEG derivative will have the following structure:
RO-(CH 2 CH 2 O) n -O-(CH 2 ) m -NH-C(O)-(CH 2 ) p -N 3
where R is a simple alkyl group (methyl, ethyl, propyl, etc.), m is 2-10, p is 2-10 and n is 100-1,000 (i.e., average molecular weight between 5-40 kDa).
In another embodiment of the invention, the targeting polypeptide comprising an alkyne amino acid containing TC is modified with a branched PEG derivative containing a terminal azide moiety, wherein the molecular weight of each strand of the branched PEG ranges from 10 to 40kDa and can be from 5 to 20kDa. For example, in some embodiments, the azide-terminated PEG derivative will have the following structure:
[RO-(CH 2 CH 2 O) n -O-(CH 2 ) 2 -NH-C(O)] 2 CH(CH 2 ) m -X-(CH 2 ) p N 3
Where R is a simple alkyl group (methyl, ethyl, propyl, etc.), m is 2-10, p is 2-10 and n is 100-1,000, and X is optionally O, N, S or carbonyl (c=o), in each case either present or absent.
Alkyne-containing PEG derivative or TLR joint derivative
In another embodiment of the invention, the targeting polypeptide of TC is modified with a PEG derivative containing an alkyne moiety that will react with an azide moiety present on a non-naturally encoded amino acid side chain.
In some embodiments, the alkyne-terminal PEG derivative will have the following structure:
RO-(CH 2 CH 2 O) n -O-(CH 2 ) m C≡CH
where R is a simple alkyl group (methyl, ethyl, propyl, etc.), m is 2-10 and n is 100-1,000 (i.e., average molecular weight between 5-40 kDa).
In another embodiment of the invention, the targeting polypeptide comprising alkyne-containing non-naturally encoded amino acid TC is modified with a PEG derivative containing a terminal azide or terminal alkyne moiety linked to the PEG backbone through an amide linkage.
In some embodiments, the alkyne-terminal PEG derivative will have the following structure:
RO-(CH 2 CH 2 O )n -O-(CH 2 ) m -NH-C(O)-(CH 2 ) p -C≡CH
where R is a simple alkyl group (methyl, ethyl, propyl, etc.), m is 2-10, p is 2-10 and n is 100-1,000.
In another embodiment of the invention, the targeting polypeptide comprising an azide amino acid containing TC is modified with a branched PEG derivative containing a terminal alkyne moiety, wherein the molecular weight of each strand of the branched PEG ranges from 10 to 40kDa and can be from 5 to 20kDa. For example, in some embodiments, the alkyne-terminal PEG derivative will have the following structure:
[RO-(CH 2 CH 2 O) n -O-(CH 2 ) 2 -NH-C(O)] 2 CH(CH 2 ) m -X-(CH 2 ) p C≡CH
where R is a simple alkyl group (methyl, ethyl, propyl, etc.), m is 2-10, p is 2-10 and n is 100-1,000, and X is optionally O, N, S or carbonyl (c=o), or absent.
Phosphine-containing PEG derivative or TLR joint derivative
In another embodiment of the invention, the targeting polypeptide of TC is modified with a PEG derivative containing an activated functional group (including but not limited to esters, carbonates) further comprising an aryl phosphine group to be reacted with azide moieties present on the non-naturally encoded amino acid side chains. Generally, the average molecular weight of the PEG derivative or TLR linker derivative ranges from 1 kDa to 100kDa, and in some embodiments, from 10 kDa to 40kDa.
In some embodiments, the PEG derivative will have the following structure:
wherein n is 1 to 10; x may be O, N, S or absent, ph is phenyl, and W is a water soluble polymer.
In some embodiments, the PEG derivative will have the following structure:
wherein X may be O, N, S or absent, ph is phenyl, W is a water soluble polymer, and R may be H, alkyl, aryl, substituted alkyl, and substituted aryl. Exemplary R groups include, but are not limited to, -CH 2 、-C(CH 3 ) 3 -OR ', -NR ' R ', -SR ', -halogen, -C (O) R ', -CONR ' R ', -S (O) 2 R’、-S(O) 2 NR 'R', -CN and-NO 2 . R ', R ", R'" and R "" each independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl (including but not limited to aryl substituted with 1-3 halogens), substituted or unsubstituted alkyl, alkoxy or thioalkoxy, or aralkyl. When the compounds of the present invention include more than one R group, when more than one of these groups is present, for example, each R group is independently selected as each R ', R ", R'" and R "" group. When R 'and R' are attached to the same nitrogen atom, they may combine with the nitrogen atom to form a 5-, 6-, or 7-membered ring. For example, -NR' R "is intended to include, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl. From the above discussion of substituents, those skilled in the art will understand that the term "alkyl" is intended to include groups that include carbon atoms bonded to groups other than hydrogen groups, such as haloalkyl (e.g., -CF 3 and-CH 2 CF 3 ) And acyl groups (including but not limited to-C (O) CH 3 、-C(O)CF 3 、-C(O)CH 2 OCH 3 Etc.).
Other PEG derivatives or TLR linker derivatives and general conjugation techniques
Other exemplary PEG molecules that may be linked to the TC polypeptide and pegylation methods include, but are not limited to, those described in the following: for example, U.S. patent publication No. 2004/0001838;2002/0052009;2003/0162949;2004/0013637;2003/0228274;2003/0220447;2003/0158333;2003/0143596; 2003/0104847; 2003/0105275;2003/0105224;2003/0023023;2002/0156047;2002/0099133;2002/0086939;2002/0082345; 2002/0074973; 2002/0052430;2002/0040076;2002/0037949;2002/0002250;2001/0056171;2001/0044526;2001/0021763; U.S. patent No. 6,646,110;5,824,778;5,476,653;5,219,564;5,629,384;5,736,625;4,902,502;5,281,698;5,122,614;5,473,034;5,516,673;5,382,657;6,552,167;6,610,281;6,515,100;6,461,603;6,436,386;6,214,966;5,990,237;5,900,461;5,739,208;5,672,662;5,446,090;5,808,096;5,612,460;5,324,844;5,252,714;6,420,339;6,201,072;6,451,346;6,306,821;5,559,213;5,747,646;5,834,594;5,849,860;5,980,948;6,004,573;6,129,912; WO 97/32607, EP 229,108, EP 402,378, WO 92/16555, WO 94/04193, WO 94/14758, WO 94/17039, WO 94/18247, WO 94/28024, WO 95/00162, WO 95/11924, WO 95/13090, WO 95/33490, WO 96/00080, WO 97/18832, WO 98/41562, WO 98/48837, WO 99/32134, WO 99/32139, WO 99/32140, WO 96/40791, WO 98/32466, WO 95/06058, EP 439 508, WO 97/03106, WO 96/21469, WO 95/13312, EP 921 131, WO 98/05363, EP 809 996, WO 96/41813, WO 96/07670, EP 605 963, EP 510 356, EP 400 472, EP 183 503 and EP 154 316, which are incorporated herein by reference. Any of the PEG molecules described herein may be used in any form, including but not limited to single chain, branched, multi-arm chain, mono-functional, bi-functional, multi-functional, or any combination thereof.
Additional polymers including, but not limited to, hydroxylamine (aminooxy) PEG derivatives or TLR-linker derivatives and PEG derivatives or TLR-linker derivatives are described in the following patent applications, which are incorporated herein by reference in their entirety: U.S. patent publication No. 2006/0194256, U.S. patent publication No. 2006/0217532, U.S. patent publication No. 2006/0217289, U.S. provisional patent No. 60/755,338; U.S. provisional patent nos. 60/755,711; U.S. provisional patent nos. 60/755,018; international patent application No. PCT/US06/49397; WO 2006/069246; U.S. provisional patent No. 60/743,041; U.S. provisional patent nos. 60/743,040; international patent application No. PCT/US06/47822; U.S. provisional patent No. 60/882,819; U.S. provisional patent No. 60/882,500; and U.S. provisional patent No. 60/870,594.
Glycosylation of TC polypeptides
Glycosylation can significantly affect the physical properties (e.g., solubility) of polypeptides, such as TC polypeptides, and can also be important in protein stability, secretion, and subcellular localization. Glycosylated polypeptides may also exhibit enhanced stability or may improve one or more pharmacokinetic properties, such as half-life. In addition, the solubility improvement may, for example, enable production of a formulation that is more suitable for pharmaceutical administration than a formulation comprising a non-glycosylated polypeptide.
The present invention includes the incorporation of one or more non-naturally encoded amino acid TC polypeptides bearing a sugar residue. The sugar residues may be natural (including but not limited to N-acetylglucosamine) or non-natural (including but not limited to 3-fluorogalactose). The sugar may be linked by an N-linked or O-linked glycoside (including but not limited to N-acetylgalactose-L-serine) or a non-natural linkage (including but not limited to an oxime or a corresponding C-linked or S-linked glycoside).
Sugar (including but not limited to glycosyl) moieties can be added to TC polypeptides in vivo or in vitro. In some embodiments of the invention, the targeting polypeptide comprising a carbonyl-containing non-naturally encoded amino acid TC is modified with an aminooxy-derived sugar to produce a corresponding glycosylated polypeptide linked by an oxime linkage. Once attached to the non-naturally encoded amino acid, the sugar may be further refined by treatment with glycosyltransferases and other enzymes to produce oligosaccharides that bind to TC polypeptides. See, e.g., H.Liu, et al J.am.chem.Soc.125:1702-1703 (2003).
In some embodiments of the invention, TC polypeptides comprising carbonyl-containing non-naturally encoded amino acids are modified directly with glycans of defined structure prepared as aminooxy derivatives. One of ordinary skill in the art will recognize that other functional groups, including azides, alkynes, hydrazides, hydrazines, and semicarbazides, may be used to link the sugar to the non-naturally encoded amino acid.
In some embodiments of the invention, the targeted polypeptide comprising an azide or alkyne-containing non-naturally encoded amino acid TC may then be modified by a cycloaddition reaction including, but not limited to, huisgen [3+2] with an azide or azide derivative, respectively. This method allows modification of proteins with extremely high selectivity.
TC dimer and multimer
The invention also provides combinations of TCs and TC analogs, such as homodimers, heterodimers, homomultimers, or heteromultimers (i.e., trimers, tetramers, etc.), in which TCs containing one or more unnatural encoded amino acids are bound to another TC or to any other polypeptide that is not TC, either directly to the polypeptide backbone or through a linker. Due to the increased molecular weight compared to the monomer, the TC dimer or multimer conjugates may exhibit new or desirable properties relative to the monomer TC, including but not limited to different pharmacology, pharmacokinetics, pharmacodynamics, modulated therapeutic half-life, or modulated plasma half-life. In some embodiments, the TC dimers of the invention will modulate TC receptor signaling. In other embodiments, the TC dimers or multimers of the invention will act as TC receptor antagonists, agonists or modulators.
In some embodiments, one or more TC molecules present in a TC containing a dimer or multimer comprise a non-naturally encoded amino acid linked to a water soluble polymer.
In some embodiments, including but not limited to, direct attachment to a TC polypeptide via an Asn-Lys amide linkage or a Cys-Cys disulfide linkage. In some embodiments, the TC polypeptide and/or linked non-TC molecule will comprise a different non-naturally encoded amino acid to promote dimerization, including but not limited to an alkyne in one non-naturally encoded amino acid of the first TC polypeptide and an azide in a second non-naturally encoded amino acid of the second molecule, are conjugated by Huisgen [3+2] cycloaddition. Alternatively, TC and/or a linked non-TC molecule comprising a ketone-containing non-naturally encoded amino acid may be conjugated to a second polypeptide comprising a hydroxylamine-containing non-naturally encoded amino acid and the polypeptide reacted by formation of the corresponding oxime.
Alternatively, the two TC polypeptides and/or the linked non-peptide TC molecules are linked by a linker. Any heterologous or homologous bifunctional linker may be used to link the two molecules, and/or linked non-peptide TC molecules, which may have the same or different primary sequences. In some cases, the linker used to link the TC and/or linked non-peptide TC molecules together may be a bifunctional PEG reagent. The linker may have a wide range of molecular weights or molecular lengths. A linker of greater or lesser molecular weight may be used to provide the desired spatial relationship or conformation between TC and the attached entity or between TC and its receptor or between the attached entity and its binding partner, if any. Linkers having longer or shorter molecular lengths may also be used to provide the required space or flexibility between the TC and the attached entity or between the attached entity and its binding partner (if any).
In some embodiments, the present invention provides a water-soluble bifunctional linker having a dumbbell structure comprising: a) An azide, alkyne, hydrazine, hydrazide, hydroxylamine, or carbonyl-containing moiety on at least a first end of the polymer backbone; and b) at least a second functional group on a second end of the polymer backbone. The second functional group may be the same as or different from the first functional group. In some embodiments, the second functional group is not reactive with the first functional group. In some embodiments, the present invention provides water-soluble compounds comprising at least one arm of a branched molecular structure. For example, the branched molecular structure may be dendritic.
In some embodiments, the invention provides a multimer comprising one or more TC polypeptides formed by reaction with a water-soluble activated polymer having the structure: r- (CH) 2 CH 2 O) n -O-(CH 2 ) m -X, wherein n is about 5 to 3,000, m is 2-10, X can be azide, alkyne, hydrazine, hydrazide, aminoxy, hydroxylamine, acetyl or carbonyl containing moiety, and R is a capping group, a functional group or a leaving group which can be the same as or different from X. R may be, for example, a functional group selected from the group consisting of: hydroxy, protected hydroxy, alkoxy, N-hydroxysuccinimide ester, 1-benzotriazole ester, N-hydroxysuccinimide carbonate, 1-benzotriazole carbonate, acetal, aldehyde hydrate, alkenyl, acrylate, methacrylate, acrylamide, reactive sulfone, amine, aminoxy, protected amine, hydrazide, protected thiol, carboxylic acid, protected carboxylic acid, isocyanate, isothiocyanate, maleimide, vinyl sulfone, dithiopyridine, Vinyl pyridine, iodoacetamide, epoxide, glyoxal, diketone, mesylate, tosylate and triflate monomethoxy, alkene and ketone.
Measurement of TC polypeptide Activity and affinity of TC polypeptide for HER2 target
TC polypeptide activity may be determined using standard or known in vitro or in vivo assays. The biological activity of TC may be assayed by suitable methods known in the art. Such assays include, but are not limited to, activation of TC response genes, receptor binding assays, antiviral activity assays, cytopathic effect inhibition assays, antiproliferative assays, immunomodulatory assays, and assays to monitor MHC molecule induction.
TC polypeptides may be analyzed for their ability to activate TC-sensitive signaling pathways. One example is the Interferon Stimulus Responsive Element (ISRE) assay. Cells constitutively expressing TC receptor were transiently transfected with ISRE-luciferase vector (pIRRE-luc, clontech). Following transfection, the cells are treated with a targeting polypeptide of TC. Various protein concentrations, e.g., 0.0001-10ng/mL, were tested to generate dose-response curves. If the TC polypeptide binds to and activates the TC receptor, the resulting signal transduction cascade induces luciferase expression. Luminescence can be measured in a number of ways, for example by using TopCount TM Or Fusion TM Microplate reader and Steady-Glo R Luciferase assay System (Promega).
TC polypeptides can be assayed for their ability to bind to TC receptors. For non-PEGylated or PEGylated TC polypeptides comprising unnatural amino acids, BIAcore may be used TM Biosensors (Pharmacia) measure the affinity of TC for its receptor. Suitable binding assays include, but are not limited to, BIAcore assay (Pearce et al Biochemistry 38:81-89 (1999)) and alpha Screen TM Assay (PerkinElmer).
Regardless of the method used to produce the TC polypeptide, a biological activity assay is performed on the TC polypeptide. In general, a biological activity test should provide an analysis of the desired result, such as an increase or decrease in biological activity (as compared to a modified TC), a different biological activity (as compared to a modified TC), a receptor or binding partner affinity analysis, a conformational or structural change in TC itself or its receptor (as compared to a modified TC), or a serum half-life analysis.
Measurement of potency, functional half-life in vivo and pharmacokinetic parameters
An important aspect of the invention is the extended biological half-life obtained by constructing TC with or without conjugation of the polypeptide to a water soluble polymer moiety. The rate at which TC serum concentration decreases after administration may make it important to assess biological responses to treatment with conjugated and unconjugated TC polypeptides and variants thereof. Conjugated and unconjugated TC polypeptides and variants thereof of the invention may also have an extended serum half-life after administration, e.g., subcutaneously or intravenously, making it possible to measure by, e.g., ELISA methods or by primary screening assays. ELISA or RIA kits from commercial sources, such as Invitrogen (Carlsbad, calif.), may be used. In vivo biological half-life measurements were performed as described herein.
The potency and functional in vivo half-life of a targeting polypeptide comprising a non-naturally encoded amino acid TC can be determined according to protocols known to those of ordinary skill in the art.
Pharmacokinetic parameters of TC polypeptides comprising non-naturally encoded amino acids can be assessed in normal Sprague-Dawley male rats (n=5 animals per treatment group). Animals will receive a single dose of 25 ug/rat iv or 50 ug/rat sc and will collect about 5-7 blood samples according to a predetermined time course, typically covering about 6 hours of TC polypeptide comprising non-naturally encoded amino acids not conjugated to the water soluble polymer and about 4 days of TC polypeptide comprising non-naturally encoded amino acids conjugated to the water soluble polymer. Pharmacokinetic data without TC of the non-naturally encoded amino acid can be directly compared to data obtained for TC polypeptides comprising the non-naturally encoded amino acid.
Administration and pharmaceutical compositions
The polypeptides or proteins of the invention (including but not limited to TC, synthetases, proteins comprising one or more unnatural amino acids, etc.) are optionally used for therapeutic purposes, including but not limited to in combination with a suitable pharmaceutical carrier. Such compositions comprise, for example, a therapeutically effective amount of the compound and a pharmaceutically acceptable carrier or excipient. Such carriers or excipients include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and/or combinations thereof. Formulations suitable for the mode of administration are prepared. In general, methods of administering proteins are known to those of ordinary skill in the art and may be applied to administer the polypeptides of the invention. The composition may be in a water-soluble form, such as in the form of a pharmaceutically acceptable salt, which is meant to include both acid addition salts and base addition salts.
Therapeutic compositions comprising one or more polypeptides of the invention are optionally tested according to methods known to those of ordinary skill in the art in one or more suitable in vitro and/or in vivo animal models of disease to confirm efficacy, tissue metabolism, and estimated dosages. In particular, dosages may be initially determined by activity, stability, or other suitable measure of non-natural and natural amino acid homologs herein, i.e., in a relevant assay (including, but not limited to, comparison of a targeted polypeptide modified to include TC of one or more non-natural amino acids to a natural amino acid TC polypeptide, and comparison of a targeted polypeptide modified to include TC of one or more non-natural amino acids to currently available TC therapies).
Administration is accomplished by any route commonly used to introduce molecules for eventual contact with blood or tissue cells. The unnatural amino acid polypeptides of the invention are administered in any suitable manner, optionally with one or more pharmaceutically acceptable carriers. Suitable methods of administering such polypeptides to a patient in the context of the present invention may be obtained, and although more than one route may be used to administer a particular composition, a particular route may generally provide a more direct and more effective effect and response than another route.
The pharmaceutically acceptable carrier is determined in part by the particular composition being administered and by the particular method used to administer the composition. Thus, there are a wide variety of suitable formulations for the pharmaceutical compositions of the present invention.
The TC polypeptides of the invention may be administered by any conventional route suitable for proteins or peptides, including but not limited to parenteral, e.g., injection, including but not limited to subcutaneous or intravenous or any other form of injection or infusion. The polypeptide composition may be administered by a variety of routes including, but not limited to: oral, intravenous, intraperitoneal, intramuscular, transdermal, subcutaneous, topical, sublingual or rectal means. Compositions comprising modified or unmodified non-natural amino acid polypeptides may also be administered via liposomes. Such routes of administration and appropriate formulations are generally known to those skilled in the art. The TC polypeptides may be used alone or in combination with other suitable components, such as a pharmaceutical carrier. The TC polypeptide may be used in combination with other agents or therapeutic agents.
TC polypeptides comprising unnatural amino acids, alone or in combination with other suitable components, can also be formulated into aerosol formulations (i.e., they can be "nebulized") for administration by inhalation. The aerosol formulation may be placed in a pressurized acceptable propellant, such as dichlorodifluoromethane, propane, nitrogen, and the like.
Formulations suitable for parenteral, such as by intra-articular (in the joints), intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes include aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents, solubilising agents, thickening agents, stabilisers and preservatives. Formulations of TC may be presented in unit-dose or multi-dose sealed containers, such as ampules and vials.
Parenteral and intravenous administration are preferred methods of administration. In particular, routes of administration (including, but not limited to, proteins commonly used for EPO, GH, G-CSF, GM-CSF, IFN (e.g., TC), interleukins, antibodies, FGF and/or any other drug delivery) that have been used for natural amino acid homolog therapeutics, along with the formulations currently in use, provide preferred routes of administration and formulations for the polypeptides of the invention.
In the context of the present invention, the dose administered to the patient is sufficient to produce a beneficial therapeutic response in the patient over time or other suitable activity, depending on the application. Such dosages are determined by the efficacy of the particular carrier or formulation, and the activity, stability or serum half-life of the unnatural amino acid polypeptide employed, and the condition of the patient, as well as the weight or surface area of the patient to be treated. The size of such doses is also determined by the presence, nature and extent of any adverse side effects associated with administration of a particular carrier, formulation, etc. in a particular patient.
In determining an effective amount of the vector or formulation to be administered in the treatment or prevention of a disease including, but not limited to, neutropenia, aplastic anemia, periodic neutropenia, idiopathic neutropenia, chdiak-Higashi syndrome, systemic Lupus Erythematosus (SLE), leukemia, myelodysplastic syndrome, and myelofibrosis, etc., a physician evaluates circulating plasma levels, formulation toxicity, and disease progression.
For example, a dose administered to a 70 kg patient is typically in a range corresponding to the therapeutic protein dose currently in use and is adjusted according to the altered activity or serum half-life of the relevant composition. The vectors or pharmaceutical formulations of the invention may be supplemented with therapeutic conditions by any known conventional therapy, including administration of antibodies, vaccines, cytotoxic agents, natural amino acid polypeptides, nucleic acids, nucleotide analogs, biological response modifiers, and the like.
For administration, the formulations of the invention are administered at any side effect observed by the LD-50 or ED-50 of the relevant formulation, and/or by different concentrations of the unnatural amino acid polypeptide, including, but not limited to, rates determined by the weight and overall health of the application to the patient. Administration may be accomplished in a single dose or in divided doses.
If a patient receiving an infusion of the formulation develops fever, chills or muscle pain, he/she receives an appropriate dose of aspirin, ibuprofen, acetaminophen or other pain/fever controlling drug. Patients infused with reactions such as fever, muscle pain and coldness were pre-dosed 30 minutes prior to future infusions with aspirin, acetaminophen or (including but not limited to) diphenhydramine. Frigid is used to treat more severe chills and muscle aches that do not react quickly to antipyretics and antihistamines. Cell infusion was slowed or stopped depending on the severity of the reaction.
The human form of the targeting polypeptide of TC of the invention may be administered directly to a mammalian subject. Administration is by any route commonly used for introducing TC polypeptides into a subject. TC polypeptide compositions according to embodiments of the invention include those suitable for oral, rectal, topical, inhalation (including but not limited to by aerosol), buccal (including but not limited to sublingual), vaginal, parenteral (including but not limited to subcutaneous, intramuscular, intradermal, intra-articular, intrapleural, intraperitoneal, intracerebral, intraarterial or intravenous), topical (i.e. skin and mucosal surfaces, including airway surfaces), pulmonary, intraocular, intranasal and transdermal administration, although the most suitable route in any given case will depend on the nature and severity of the condition being treated. Administration may be local or systemic. The formulations of the compounds may be presented in unit-dose or multi-dose sealed containers, such as ampules and vials. The TC polypeptides of the invention can be prepared in unit dose injectable forms (including but not limited to solutions, suspensions, or emulsions) in admixture with a pharmaceutically acceptable carrier. The TC polypeptides of the invention may also be administered by continuous infusion (using, including but not limited to, micropumps such as osmotic pumps), single bolus injection, or sustained release depot formulations.
Formulations suitable for administration include aqueous and non-aqueous solutions, isotonic sterile solutions (which may contain antioxidants, buffers, bacteriostats and solutes that render the formulation isotonic), and aqueous and non-aqueous sterile suspensions (which may include suspending agents, solubilizers, thickening agents, stabilizers and preservatives). Solutions and suspensions can be prepared from sterile powders, granules and tablets of the kind previously described.
Lyophilization is a technique commonly used to render proteins; the freeze drying is used to remove water from the protein formulation of interest. Lyophilization or freeze-drying is a method used to freeze a substance to be dried first and then remove ice or frozen solvent by sublimation in a vacuum environment. Excipients may be included in the pre-lyophilization formulation to enhance stability during the lyophilization process and/or to improve the stability of the lyophilized product upon storage. Pikal, M.Biopharm.3 (9) 26-30 (1990) and Arakawa et al pharm.Res.8 (3): 285-291 (1991).
Spray drying of drugs is also known to those of ordinary skill in the art. See, e.g., broadhead, J.et al, "The Spray Drying of Pharmaceuticals," Drug Dev. Ind. Pharm,18 (11 & 12), 1169-1206 (1992). In addition to small molecule drugs, various biological materials have been spray dried and include enzymes, serum, plasma, microorganisms and yeast. Spray drying is a useful technique because it can convert liquid pharmaceutical formulations into fine, dust-free or agglomerated powders in a one-step process. The basic technology comprises the following four steps: a) Atomizing the feed solution into a spray; b) Air-jet contact; c) Drying and spraying; and d) separating the dried product from the drying air. U.S. patent nos. 6,235,710 and 6,001,800 (which are incorporated herein by reference) describe the preparation of recombinant erythropoietin by spray drying.
The pharmaceutical compositions and formulations of the present invention may comprise a pharmaceutically acceptable carrier, excipient or stabilizer. The pharmaceutically acceptable carrier is determined in part by the particular composition being administered and by the particular method used to administer the composition. Thus, there are a wide variety of suitable formulations of the pharmaceutical compositions of the present invention (including optional pharmaceutically acceptable carriers, excipients or stabilizers) (see, e.g., remington's Pharmaceutical Sciences, 17 th edition, 1985).
Suitable carriers include, but are not limited to, buffers containing succinate, phosphate, borate, HEPES, citrate, histidine, imidazole, acetate, bicarbonate, and other organic acids; antioxidants, including but not limited to ascorbic acid; low molecular weight polypeptides, including but not limited to those of less than about 10 residues; proteins, including but not limited to serum albumin, gelatin, or immunoglobulins; hydrophilic polymers including, but not limited to, polyvinylpyrrolidone; amino acids including, but not limited to, glycine, glutamine, asparagine, arginine, histidine or histidineDerivatives, methionine, glutamic acid or lysine; monosaccharides, disaccharides, and other carbohydrates including, but not limited to, trehalose, sucrose, glucose, mannose, or dextrins; chelating agents including, but not limited to, EDTA and disodium edetate; divalent metal ions including, but not limited to, zinc, cobalt, or copper; sugar alcohols, including but not limited to mannitol or sorbitol; salt-forming counterions, including, but not limited to, sodium and sodium chloride; fillers such as microcrystalline cellulose, lactose, corn and other starches; an adhesive; sweeteners and other flavoring agents; a colorant; and/or nonionic surfactants including, but not limited to Tween TM (including but not limited to Tween 80 (polysorbate 80) and Tween 20 (polysorbate 20), pluronic TM And other pluronic acids, including but not limited to pluronic acid F68 (poloxamer 188), or PEG. Suitable surfactants include, for example, but are not limited to, polyethers based on poly (ethylene oxide-poly (propylene oxide) -poly (ethylene oxide), i.e., (PEO-PPO-PEO), or poly (propylene oxide-poly (ethylene oxide) -poly (propylene oxide), i.e., (PPO-PEO-PPO), or combinations thereof, PEO-PPO-PEO and PPO-PEO-PPO may be under the trade name Pluronics TM 、R-Pluronics TM 、Tetronics TM And R-Tetronics TM (BASF Wyandotte corp., wyandotte, mich.) commercially available and further described in U.S. patent No. 4,820,352, which is incorporated herein by reference in its entirety. Other ethylene/polypropylene block polymers may be suitable surfactants. The surfactant or combination of surfactants may be used to stabilize the pegylated TC against one or more pressures, including but not limited to, the pressure caused by agitation. Some of the above surfactants may be referred to as "swelling agents". Some may also be referred to as "tonicity adjusting agents". The antibacterial preservative can also be applied to the stability and antibacterial effect of the product; suitable preservatives include, but are not limited to, benzyl alcohol, benzalkonium chloride, m-cresol, methyl/propyl p-hydroxybenzoate, cresols, and phenol, or combinations thereof. U.S. patent No. 7,144,574, which is incorporated herein by reference, describes additional materials that may be suitable for use in the pharmaceutical compositions and preparations of the present invention, as well as other delivery formulations.
TC polypeptides of the invention (including those linked to water soluble polymers such as PEG) may also be administered by or as part of a slow release system. Sustained release compositions include, but are not limited to, semipermeable polymer matrices in the form of shaped articles, including, but not limited to, films or microcapsules. Sustained release matrices include those derived from biocompatible materials such as poly (2-hydroxyethyl methacrylate) (Langer et al, J.Biomed. Mater. Res.,15:267-277 (1981); langer, chem. Tech.,12:98-105 (1982), ethylene vinyl acetate (Langer et al, supra) or poly-D- (-) -3-hydroxybutyric acid (EP 133,988), polylactide (polylactic acid) (U.S. Pat. No. 3,773,919;EP 58,481), polyglycolide (polymers of glycolic acid), polylactide co-glycolide (copolymers of lactic acid and glycolic acid) polyanhydrides, copolymers of L-glutamic acid and gamma-ethyl-L-glutamic acid (Sidman et al, biopolymers,22,547-556 (1983), poly (orthoesters), polypeptides, hyaluronic acid, collagen, chondroitin sulfate, carboxylic acid, fatty acid, phospholipids, polysaccharides, nucleic acids, polyaminoacids, amino acids (such as phenylalanine), tyrosine, isoleucine, polynucleotides, polyethylene, polyvinylpyrrolidone and silicone and combinations including those containing the same, are also prepared by the methods described in U.S. patent No. 121:35:35, sc. Nature, sc. 11, sc. Et al, and Sc. Et al, and Pr. Acid. Et al, and 3:35, prov. Sc. Crohn. Et al, and Pr. Ach. Et al, prov. Scd., 77:4030-4034 (1980); EP 52,322; EP 36,676; U.S. patent No. 4,619,794; EP 143,949; U.S. patent No. 5,021,234; japanese patent application 83-118008; U.S. patent nos. 4,485,045 and 4,544,545; and EP 102,324. All references and patents cited are incorporated herein by reference.
Liposome-encapsulated TC polypeptides can be obtained, for example, by DE 3,218,121; eppstein et al, proc.Natl.Acad.Sci.U.S.A.,82:3688-3692 (1985); hwang et al, proc.Natl.Acad.Sci.U.S.A.,77:4030-4034 (1980); EP 52,322; EP 36,676; U.S. patent No. 4,619,794; EP 143,949; U.S. patent No. 5,021,234; japanese patent application 83-118008; U.S. patent nos. 4,485,045 and 4,544,545; and the process described in EP 102,324. The composition and size of liposomes are well known or can be readily determined empirically by one of ordinary skill in the art. Some examples of liposomes are described, for example, in Park JW, et al, proc.Natl. Acad.Sci.USA 92:1327-1331 (1995); lasic D and Papahadjopoulos D (eds.): MEDICAL APPLICATIONS OF LIPOSOMES (1998); drummond DC, et al, liposomal drug delivery systems for cancer therapy, teicher B (ed.): CANCER DRUG DISCOVERY AND DEVELOPMENT (2002); park JW, et al, clin.cancer Res.8:1172-1181 (2002); nielsen UB, et al, biochim. Biophys. Acta 1591 (1-3): 109-118 (2002); mamot C, et al, cancer Res.63:3154-3161 (2003). All references and patents cited are incorporated herein by reference.
In the context of the present invention, the dose administered to the patient should be sufficient to produce a beneficial response in the subject over time. Typically, the total pharmaceutically effective amount of the TC polypeptide of the present invention administered per dose parenterally is in the range of about 0.01 μg/kg/day to about 100 μg/kg or about 0.05mg/kg to about 1mg/kg of patient body weight, although this is at the discretion of the treatment. In particular aspects of the embodiments, the conjugate may be administered at a dose in the range of greater than 4 μg/kg/day to about 20 μg/kg/day. In still other aspects, the conjugate may be administered at a dose in the range of greater than 4 μg/kg/day to about 9 μg/kg/day. In still other aspects, the conjugate may be administered at a dose in the range of greater than 4 μg/kg/day to about 12.5 μg/kg/day. In a particular aspect, the conjugate may be administered at a dose at or below the maximum tolerated dose without undue toxicity. Furthermore, the conjugate may be administered at least twice a week or the conjugate may be administered at least three times a week, at least four times a week, at least five times a week, at least six times a week, or seven times a week. In a particular aspect, when the conjugate is administered more than once, the conjugate may be administered at a dose of greater than 4 μg/kg/day each time. In particular, the conjugate may be administered over a period of two weeks or more. In certain aspects, the growth of cells expressing interleukin-10 receptor may be inhibited by at least 50%, at least 65%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% as compared to a reference sample (i.e., a cell sample not contacted with a conjugate of the invention). In a particular aspect of the embodiment, the conjugate may be administered at a dose of about 5.3 μg/kg/day, or at a dose of about 7.1 μg/kg/day, or at a dose of about 9.4 μg/kg/day, or at a dose of about 12.5 μg/kg/day. The frequency of administration also depends on the judgment of the treatment and may be more frequent or less frequent than commercially available TC polypeptide products approved for use in humans. Generally, a targeting polypeptide of TC, a pegylated TC polypeptide, a conjugated TC polypeptide, or a pegylated conjugated TC polypeptide of the invention may be administered by any of the routes of administration described above.
Therapeutic use of TC of the invention
The TCs of the present invention are useful in the treatment of a wide range of disorders. The invention also includes methods of treating a mammal at risk of, suffering from, and/or already suffering from a cancer in response to TC, cd8+ T cell stimulation, and/or TC formulations. The administration of TC may have a short term effect, i.e. a direct beneficial effect on several clinical parameters observed, and this may occur within 12 or 24 hours after administration, on the other hand, a long term effect may also be produced, advantageously slowing down the progression of tumor growth, shrinking tumor size and/or increasing circulating cd8+ T cell levels, and TC of the present invention may be administered by any means known to the person skilled in the art and may be advantageously administered by infusion, e.g. by arterial, intraperitoneal or intravenous injection and/or infusion of a dose sufficient to obtain the desired pharmacological effect.
The dosage of TC may range from 10-200mg, or 40-80mg, of TC polypeptide per kilogram of body weight per treatment. For example, the dose of TC administered may be about 20-100mg TC polypeptide/kg body weight, administered as a bolus and/or infusion for a clinically necessary period of time, e.g., for a period ranging from a few minutes to a few hours, e.g., up to 24 hours. TC administration may be repeated one or several times, if necessary. Administration of TC may be combined with administration of other agents, such as chemotherapeutic agents. Furthermore, the present invention relates to a method for controlling and/or treating cancer comprising administering to a subject in need thereof an effective amount of TC.
The average amount of TC may vary and should be based on, among other things, recommendations and prescriptions of qualified doctors. The exact amount of TC is a matter of preference, affected by factors such as the exact type of condition being treated, the condition of the patient being treated, and other ingredients in the composition. The invention also provides for the administration of a therapeutically effective amount of another active agent. The amount to be administered can be readily determined by one of ordinary skill in the art based on TC therapy.
Examples
The following examples are provided to illustrate, but not limit, the claimed invention.
Example 1 general procedure for the Synthesis of TLR agonists
This example provides a general method for synthesizing TLR agonists of the invention.
All commercially available anhydrous solvents were used without further purification and stored under nitrogen atmosphere. TLC on Merck silica gel 60F254 plates with UV light and/or with KMnO 4 The aqueous staining was visualized. Chromatographic purification was performed on CombiFlash Rf from Teledyne ISCO using the conditions detailed in the experimental procedure. Analytical HPLC was performed on a Shimadzu system using a Phenomenex Gemini-NX C18.mu.m50X4.6mm column eluting with a linear gradient of acetonitrile in water containing 0.05% TFA at 1ml/min. (mobile phase A: 0.05% trifluoroacetic acid in water; mobile phase B: 0.05% trifluoroacetic acid in 90% Acetonitrile (ACN) in water) or Waters BEH 1.7 μm v2.1X50 mm column. Analytical method 1: 0% B in 1min, 0-50% B in 11min, 50-100% B in 0.5min, 100% B in 1.5min, 100-0% B in 1min, 0% B lasting for 2min; method 2: 10-20% B in 1min, 20-70% B in 11min, 70-100% B in 0.5min, 100% B lasting for 1.5min, 100-10% B in 1min, 10% B lasting for 2min; method 3: 0-40% B in 1min, 40-90% B in 11min, 90-100% B in 0.5min, 100% B lasting for 1.5min, 100-10% B in 1min, 10% B lasting for 2min; method 4: 5% B in 0.3min, 5% to 100% B in 0.3min to 1.5min, 100% B in 1.5min to 1.8min, and flow rate in 0.8ml/min to 1.1ml/min in 0min to 1.8 min.
Preparative HPLC was performed on a Shimadzu system using a Gemini-NX C18 5 μm 100X 30mm, 150X 30mm or 250X 50mm column, depending on the scale. Mass Spectra (MS) were recorded on a Shimadzu LCMS-2020 system and the data were processed using Shimadzu LabSolutions software. The Agilent 1260 affinity binary LC coupling 6230 Accidet-Mass TOFMS system was used for HR-ESI-TOF analysis. NMR spectroscopic data were collected on a 500MHz Bruker NMR spectrometer. Chemical shift (δ) is reported in ppm and is referenced to deuterium solvent signal. The coupling constant (J) is reported in hertz (Hz). Spin multiplexing is described as: s (singlet), br (broad), d (doublet), dd (doublet), t (triplet), q (quartet) or m (multiplet). The monomeric antibodies were pooled, filtered at 0.22. Mu.M, and stored at < 65℃until further use.
Abbreviations used in the examples include: CDI:1,1' -carbonyl diimidazole, DIEA: n, N-diisopropylethylamine, DCM: dichloromethane, DIAD: diisopropyl azodicarboxylate, DMF: dimethylformamide, dmmmt: 4- (4, 6-dimethoxy-1, 3, 5-triazin-2-yl) -4-methylmorpholinium tetrafluoroborate, etOAc: ethyl acetate, meOH: methanol, TFA: trifluoroacetic acid.
Example 2 synthesis of TLR agonists comprising the following structure-core 1:
in some embodiments, X is CH or N;
R 2 is C 1 To C 12 Alkylene, nitrogen-containing alkylene, aromatic ring, or-C (=nh) NH-, or a combination thereof;
R 3 is-H, C 1 To C 12 Alkyl, nitrogen-containing alkyl, aromatic ring or-C (=nh) NH 2 Or a combination thereof;
or R is 2 And R is 3 Ligating to form C 4 To C 8 A cycloalkylene group;
R 4 is C 1 To C 12 Alkyl, C 1 To C 12 Substituted alkyl, C 4 To C 8 Cycloalkyl, C 4 To C 8 Substituted cycloalkyl, aromatic ring, substituted aromatic ring, aromatic heterocycle, substituted aromatic heterocycle, -ONH 2 Terminal C 1 To C 12 Alkyl or a combination thereof; or R is 4 Absence of;
Z 1 is C 1 To C 6 Alkylene, C 3 To C 8 Cycloalkylene or C 3 To C 8 A nitrogen-containing heterocycle, or a combination thereof; and is also provided with
R 5 Is C 1 To C 12 Alkyl, C 1 To C 12 Substituted alkyl, oxygen-containing C 1 To C 12 Alkyl, C 4 To C 8 Cycloalkyl, C 4 To C 8 Substituted cycloalkyl or a combination thereof.
TLR agonists with a core 1 structure were synthesized as disclosed in the following schemes.
Tert-butyl 2- (2- (3-nitroquinolin-4-ylamino) ethoxy) ethylcarbamate (1): 4-chloro-3-nitroquinoline (1750 mg,8.39 mmol) was dissolved in DCM (30 mL) and treated with free amine (1800 mg,8.55 mmol) followed by TEA (2.29 mL,17.3 mmol). The reaction was kept at room temperature for 18H, then H was used 2 O (20 mL), brine (10 mL), over MgSO 4 Dried and concentrated in vacuo. The title compound (1) (3130 mg, 99%) was obtained as a yellow solid, MS M/z 399 (M+Na) + 。
Tert-butyl 2- (2- (3-aminoquinolin-4-ylamino) ethoxy) ethylcarbamate (2): the nitro compound (1) (3.12 g,8.29 mmol) was dissolved in THF (100 mL) and water (80 mL). Zinc (13.55 g,207.2 mmol) was added in one portion followed by NH 4 Cl (13.3 g,248.6 mmol). The suspension was stirred vigorously at room temperature for 1H (HPLC). After filtration, the cake was washed with THF (20 ml×2). NaCl was added to the filtrate until the aqueous phase was saturated. The liquid phase was collected and the THF layer was separated. The aqueous layer was extracted with THF/EA (50 ml/50 ml). The organic layers were combined, over MgSO 4 Dried, and concentrated to obtain a residue (2) (3.1 g,>100％)。MS m/z 347(M+H) + 。
2- (2- (2-butyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) ethoxy) ethylcarbamic acid tert-butyl ester (3): will beAmine compound (2) (3.1 g, crude,<8.95 mmol) and triethyl orthovalerate (3.1 mL,13.5 mmol) were suspended in toluene (200 mL) and heated to 110deg.C. Pyridine HCl (55 mg,0.48 mmol) was then added. The reaction was heated for 4h. The mixture was kept at room temperature for 48h. The liquid was decanted and the remaining solid/residue was stirred with toluene (20 mL x 2), combined with the liquid and concentrated. The residue was dissolved in DCM and purified by column chromatography (methanol in DCM, 0-10-20%,80g column) to obtain the target compound (3) (1.05 g, 2-step 30% from nitro compound 1). MS M/z 413 (M+H) + 。
1- (2- (2- (tert-Butoxycarbonylamino) ethoxy) ethyl) -2-butyl-1H-imidazo [4,5-c]Quinoline 5-oxide (4): compound 3 (1.05 g,2.54 mmol) was dissolved in DCM (20 mL) and treated with mCPBA (750 mg,2.83 mmol). The reaction was kept at room temperature for 4h. The mixture was treated with NaHCO 3 The saturated solution (15 mL. Times.3) was washed, dried and concentrated to give crude syrup (4) (900 mg, 83%) for the next step. MS M/z 429 (M+H) + 。
2- (2- (4-amino-2-butyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) ethoxy) ethylcarbamic acid tert-butyl ester (5): in a pressure tube, compound 4 (900 mg,2.18 mmol) was dissolved in dichloroethane (25 mL) and treated with concentrated ammonium hydroxide (28%, 1 mL) and the temperature was raised to 80 ℃. After cooling, tosyl chloride (470 mg,2.46 mmol) was slowly added to the mixture over 5 min. Concentrated ammonium hydroxide (0.5 mL) was added and the tube sealed. The tube was heated at 80℃for 4h. After cooling, the mixture was diluted with DCM (60 mL), washed with water (40 mL), dried and purified by silica gel column chromatography to give the target compound (5) (750 mg, 80%). MS M/z 428 (M+H) + 。
1- (2- (2-Aminoethoxy) ethyl) -2-butyl-1H-imidazo [4,5-c]Quinolin-4-amine (a): compound 5 (750 mg,1.75 mmol) was treated with 1.25M HCl in EtOH (20 mL) for 17h at room temperature. Next, the reaction was dried in vacuo and the residue was resuspended in EtOH/Et 2 O (1/10; 20 mL) and filtered. The solid was collected to obtain the target compound (a) (600 mg, 85%). HPLC (method 1): 5.8min, MS M/z 328 (M+H) + 。
4- (2- (4-amino-2-butyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) ethoxy) ethylcarbamoyl) piperazine-1-carboxylic acid tert-butyl ester (6): the HCl salt of compound a (100 mg,0.25 mmol) was dissolved in DCM (10 mL) and treated with TEA (68 μl,0 511 mmol). To the suspension was added tert-butyl 4- (chlorocarbonyl) piperazine-1-carboxylate (75 mg, 0.284 mmol). The reaction was held at room temperature for 17h and diluted with DCM/MeOH (4 mL/1 mL) and the solution was washed with brine. The organic phase was purified by column chromatography on silica gel to give pure product 6 (130 mg,0.24mmol, 96%). MS M/z 540 (M+H) + 。
N- (2- (2- (4-amino-2-butyl-1H-imidazo [4, 5-c))]Quinolin-1-yl) ethoxy) ethyl) piperazine-1-carboxamide (7): compound (6) (10 mg,0.018 mmol) was treated with HCl/EtOH (about 1.5M,1 mL) at room temperature for 1h, then at 60℃for 1h and dried in vacuo. The residue was washed with diethyl ether and dried to obtain the objective compound (7) (9 mg,0.018mmol, quantitative). MS M/z 440 (M+H) + 。
N- (2- (2- (4-amino-2-butyl-1H-imidazo [4,5-c ] quinolin-1-yl) ethoxy) ethyl) morpholine-4-carboxamide (8): compound 7 was prepared using a procedure similar to that described for 6 by reacting compound a with morpholine-4-carbonyl chloride to obtain the target compound 7 (7 mg, 42%). MS M/z 441 (M+H).
4- (2- (4-amino-2-butyl-1H-imidazo [4, 5-c)]Quinoline-1-yl) ethoxy) ethylcarbamoyl) piperazine-1-carboxylic acid 4- ((R) -2- (2- (aminooxy) acetamido) -3-methylbutanoylamino) -5-ureidovalerylamino) benzyl ester (9): compound 7 (22 mg,0.02 mmol) was dissolvedSolution in DCM (1 mL) and treatment with DIPEA (3.5. Mu.L, 0.02 mmol) followed by 2, 5-dioxopyrrolidin-1-yl 2- (tert-butoxycarbonylaminooxy) acetate (3.3 mg,0.01 mmol). The reaction was kept at room temperature for 17h. TFA (0.3 mL) was added to the mixture and stirred for 15min. After drying in vacuo, the residue was purified by prep HPLC to give compound 9 (15 mg,22% from 7). MS M/z 918 (M+H) + 。
2-butyl-1- (2- (2- (piperazine-1-carboxamido) ethoxy) ethyl) -1H-imidazo [4,5-c]Quinoline-4-ylcarbamic acid 4- ((R) -2- (2- (aminooxy) acetamido) -3-methylbutanoylamino) -5-ureidovalerylamino) benzyl ester (10)): compound 6 (65 mg,0.097 mmol) was dissolved in DMF (2 mL) and treated with DIPEA (34 μL,0.194 mmol) followed by Fmoc-VC-PAB-PNP (94 mg,0.116 mmol). The reaction was held at room temperature for 1h, and water (10 mL) was added. The solid was collected, washed with water (2 mL) and dried. The yellow solid was dissolved in DMF (2 mL) and treated with diethylamine (100. Mu.L, 0.97 mmol) at room temperature for 30min. The reaction mixture was purified by preparative LC to give intermediate Val-Cit-PAB-OCO- (Compound 6). The intermediate (11 mg,0.01 mmol) was dissolved in DCM (1 mL) and treated with DIPEA (3.5. Mu.L, 0.02 mmol) followed by 2, 5-dioxopyrrolidin-1-yl 2- (tert-butoxycarbonylaminooxy) acetate (3.3 mg,0.01 mmol). The reaction was kept at room temperature for 17h. TFA (0.3 mL) was added and the mixture stirred for 15min. After drying in vacuo, the residue was purified by preparative HPLC to give compound 10 (15 mg,16% from compound 6). MS M/z 918 (M+H) + 。
1- (2- (2-Aminoethoxy) ethyl) -2-butyl-1H-imidazo [4,5-c]Quinolin-4-ylcarbamic acid 4- ((R) -2- ((R) -2- (2- (aminooxy) acetamido) -3-methylbutanoylamino) -5-ureidopentanoylamino)) Benzyl ester (11): compound 11 was prepared using 5 as starting material in a similar procedure as described for 10 to give the target compound 11 (22 mg,21% from 5). MS M/z 806 (M+H) + 。
2-butyl-1- (2- (2- (piperazine-1-carboxamido) ethoxy) ethyl) -1H-imidazo [4,5-c]4- ((R) -2- ((2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) propionylamino) -3-methylbutanoylamino) -5-ureidovalerylamino) benzyl quinoline-4-ylcarbamate (12): compound 12 was prepared in analogy to the procedure described for 10 using 2, 5-dioxopyrrolidin-1-yl 5,3- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) propionate as starting material to obtain the target compound 12 (not treated with TFA) (15 mg,17% from 5). MS M/z 984 (M+H) + 。
Adipic acid-bis- (N- (2- (2- (4-amino-2-butyl-1H-imidazo [4, 5-c))]Quinolin-1-yl) ethoxy) ethyl) piperazine-1-carboxamide (13): to a solution of compound 7 (8 mg,0.018 mmol) and adipic acid (2 mg,0.07 mmol) in DMF (1 mL) was added EDC (3 mg,0.016 mmol), HOBt (1 mg,0.018 mmol) and DIEA (4. Mu.L, 0.23 mmol) at 23 ℃. After 24h, the mixture was purified by preparative LC and dried to give compound 13 (5 mg, 0.04 mmol, 23%). MS M/z 1217 (M+H) + 。
4- (4-amino-2-ethyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) butyl carbamate (14): compound 14 was prepared in a similar procedure to that described for 5 using Boc-l, 4-butanediamine and triethyl orthopropionate as starting materials to obtain the target compound 14 (420 mg,1.095mmol, 14)5% from starting material). MS M/z 384 (M+H) + 。
1- (4-Aminobutyl) -2-ethyl-1H-imidazo [4,5-c]Quinolin-4-amine-2 HCl (B): compound 14 (400 mg,1.043 mmol) was added to 4M HCl (10 mL,40 mmol) in DCM (0.5 mL) and dioxane at 23 ℃. After 1h LCMS showed the reaction was complete. The solvent was removed in vacuo and dried under a high vacuum pump for 6h to give compound B (400 mg,1.129mmol, 99%) as a pale yellow solid. MS M/z 284 (M+H) + 。
2- (4- (4-amino-2-ethyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) butylamino-acetic acid tert-butyl ester (15): to a solution of compound B (31 mg,0.109 mmol) in DCM (5 mL) was added tert-butyl bromoacetate (15 μl,0.102 mmol) followed by TEA (88 μl,0.681 mmol) at 23 ℃. After 24h, the mixture was purified by preparative LC to give compound 15 (4 mg,0.006mmol, 6%) as a yellow solid. MS M/z 398 (M+H) + 。
5-amino-N- (4- (4-amino-2-ethyl-1H-imidazo [4, 5-c) ]Quinolin-1-yl) butyl) picolinamide (16): to a solution of compound B (25 mg,0.071 mmol) and 5-aminopyridine-2-carboxylic acid (10 mg,0.072 mmol) in DMF (1 mL) was added HATU (20 mg,0.083 mmol) and DIEA (50 μl,0.287 mmol) at 23 ℃. After 1h, the mixture was purified by preparative LC and dried to give compound 16 (21 mg,0.033mmol, 46%) as a white solid. MS M/z 404 (M+H) + 。
N- (4- (4-amino-2-ethyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) butyl) -5,6, 7-trimethoxy-1H-indole-2-carboxamide (17) a: using chemical compoundsCompound 17 was prepared in analogy to the procedure described for 16, starting materials from compound B and 5,6, 7-trimethoxy-1 h-indole-2-carboxylic acid to give the title compound 17 (13 mg,0.017mmol, 44%). MS M/z517 (M+H) + 。
5-amino-N- (2- (2- (4-amino-2-butyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) ethoxy) ethyl) picolinamide (18): compound 18 was prepared using compound a and 5-aminopyridine-2-carboxylic acid as starting materials and in a similar procedure as described for 16 to give the target compound 18 (24 mg,0.036mmol, 82%) MS M/z 448 (m+h) + 。
3- (4- (N- (4- (4-amino-2-ethyl-1H-imidazo [4, 5-c))]Quinolin-1-yl) butyl) sulfamoyl) phenyl) methyl propionate (19): to a solution of compound B (14 mg,0.035 mmol) and 5-aminopyridine-2-carboxylic acid (6 mg,0.043 mmol) in DMF (1 mL) was added DIEA (40 μl,0.230 mmol) at 23 ℃. After 30min, the mixture was purified by preparative LC and dried to give compound 19 (15 mg,0.020mmol, 58%) as a pale yellow solid. MS M/z 510 (M+H) + 。
1- (4- (4-amino-2-ethyl-1H-imidazo [4,5-c ] quinolin-1-yl) butyl) -3- (3- (pyrrolidin-1-ylmethyl) benzyl) urea (20): to a solution of (3- (pyrrolidin-1-ylmethyl) phenyl) methylamine (19 mg,0.100 mmol) and nitrophenyl chloroformate (21 mg,0.104 mmol) in DMF (1 mL) was added DIEA (34. Mu.L, 0.195 mmol) at 23 ℃. After 10min, LCMS showed nitrophenol activation was complete. To this mixture was added compound B. After 2h, the mixture was purified by preparative LC and dried to give compound 20 (3 mg,0.006mmol, 6%) as a white solid.
N- (4- (4-amino-2-ethyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) butyl) pyrazine-2-carboxamide (21): compound 28 was prepared in a similar procedure as described for 16, using compound B and pyrazinecarboxylic acid as starting materials, to obtain the target compound 21 (11 mg,0.013mmol, 23%). MS M/z 390 (M+H) + 。
2- (4-amino-2-butyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) ethylcarbamic acid tert-butyl ester (22): compound 22 was prepared in a similar procedure as described for compound 5 using 4-chloro-3-nitroquinoline, tert-butyl 2-aminoethylcarbamate and triethyl orthovalerate as starting materials to give the target compound 22 (140 mg,0.365mmol, 33%). MS M/z 384 (M+H) + 。
1- (4-Aminobutyl) -2-ethyl-1H-imidazo [4,5-c]Quinolin-4-amine-3, hcl (C): compound C was prepared in a similar procedure to that described for a, using compound 22 as starting material, to give the target compound C (60 mg,0.169mmol, quantitative) MS M/z 284 (m+h) + 。
N- (2- (4-amino-2-butyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) ethyl) pyrazine-2-carboxamide (23): compound 23 was prepared in a similar procedure as described for 16, using compound C and pyrazinecarboxylic acid as starting materials, to obtain the target compound 23 (8 mg,0.09mmol, 29%). MS M/z 390 (M+H) + 。
1- (2- (4-amino-2-butyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) ethylamino) -5-guanidino-1-oxopent-2-ylcarbamic acid (S) -tert-butyl ester (24): compound 24 was prepared in a similar procedure as described for 16, using compound C and Boc-Arg-OH as starting materials, to obtain the target compound 24 (75 mg,0.098mmol, 79%). MS M/z 540 (M+H) + 。
(S) -2-amino-N- (2- (4-amino-2-butyl-1H-imidazo [4,5-c ] quinolin-1-yl) ethyl) -5-guanidinopentanamide, 3HCl (25): to compound 24 (60 mg,0.111 mmol) was added 4M HCl in dioxane (1 ml,4 mmol) at 23 ℃. After 2h, the reaction was dried in vacuo. The residue was dried under a high vacuum pump to obtain compound 25 (64 mg,0.110mmol, quantitative) as a white solid.
1- (2- (4-amino-2-butyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) ethoxy) ethylamino) -5-guanidino-1-oxopent-2-ylcarbamic acid (S) -tert-butyl ester (26): compound 26 was prepared in a similar procedure as described for 16, using compound a and Boc-Arg-OH as starting materials, to give the title compound 26 (20 mg,0.019mmol, 59%). MS M/z584 (M+H) + 。
(S) -2-amino-N- (2- (4-amino-2-butyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) ethyl) -5-guanidinopentanamide (27): compound 27 was prepared in a similar procedure as described for 25, using compound 26 as starting material, to give the title compound 27 (14 mg,0.016mmol, quantitative). MS M/z 484 (M+H) + 。
N- (2- (2- (4-amino-2-butyl-1H-imidazo [4, 5-c))]Quinolin-1-yl) ethoxy) ethyl) pyrazine-2-carboxamide (28): use of Compound A and pyrazinecarboxylic acid as the active ingredientStarting material, compound 28 was prepared in a similar procedure as described for 16 to obtain the target compound 28 (1.3 mg,0.001mmol, 4%). MS M/z 434 (M+H) + 。
1- (2- (4-amino-2-butyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) ethoxy) ethyl) guanidine (29): to a solution of compound A (15 mg,0.038 mmol) and formamidiylthiomethyl ester bis (sulfate) (25 mg,0.090 mmol) in DMF (1 mL) and water (1 mL) was added TEA (50 μL,0.358 mmol) at 80deg.C. After 18h, the mixture was purified by preparative LC to give compound 29 (12 mg,0.017mmol, 45%). MS M/z 370 (M+H) + 。
1- (2- (4-amino-2-butyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) ethyl) guanidine (30): compound 30 was prepared in a similar procedure as described for 29, using compound C as starting material, to obtain the target compound 30 (10 mg,0.013mmol, 29%). MS M/z434 (M+H) + 。
1- (4- (4-amino-2-ethyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) butyl) guanidine (31): compound 31 was prepared in a similar procedure as described for 29 using compound B as starting material to obtain the target compound 31 (8 mg,0.010mmol, 29%). MS M/z 326 (M+H) + 。
(S) -2-acetamido-N- (2- (4-amino-2-butyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) ethyl) -5-guanidineDipentamide (32): compound 32 was prepared in a similar procedure as described for 16 using compound C and acetyl-arginine as starting materials to obtain the target compound 32 (18 mg,0.025mmol, 69%). MS M/z 482 (M+H) + 。
(S) -2-acetamido-N- (2- (2- (4-amino-2-butyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) ethoxy) ethyl) -5-guanidinopentamide (33): compound 33 was prepared in a similar procedure as described for 16, using compound a and acetyl-arginine as starting materials, to obtain the target compound 33 (4 mg,0.019mmol, 67%). MS M/z 526 (M+H) + 。
(S) -2-acetamido-N- (4- (4-amino-2-ethyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) butyl) -5-guanidinopentanamide (34): compound 34 was prepared in a similar procedure as described for 16, using compound B and acetyl-arginine as starting materials, to give the target compound 34 (5 mg, 0.0070 mmol, 30%). MS M/z 482 (M+H) + 。
N- (4- (4-amino-2-ethyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) butyl) benzamide (35): compound 35 was prepared in a similar procedure as described for 16, using compound B and benzoic acid as starting materials, to obtain the target compound 35 (8 mg,0.013mmol, 53%). MS M/z 388 (M+H) + 。
4-(4-(4-amino-2-ethyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) butylcarbamoyl) phenethylcarbamic acid tert-butyl ester (36): compound 36 was prepared in a similar procedure as described for 16 using compound B and 4- ((2-boc-amino) ethyl) benzoic acid as starting materials to obtain the target compound 36 (25 mg,0.033mmol, 38%). MS M/z 531 (M+H) + 。
N- (4- (4-amino-2-ethyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) butyl) -4-guanidinobutyramide (37): compound 37 was prepared in a similar procedure as described for 16, using compound B and 4-guanidinobutyric acid as starting materials, to obtain the target compound 37 (10 mg,0.016mmol, 45%). MS M/z 411 (M+H) + 。
N- (4- (4-amino-2-ethyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) butyl) -2,3,5, 6-tetrafluorobenzamide (38): compound 38 was prepared in a similar procedure as described for 16 using compound B and 2,3,5, 6-tetrafluorobenzoic acid as starting materials to obtain the target compound 38 (7 mg,0.010mmol, 36%). MS M/z 460 (M+H) + 。
4- (4-amino-2-butyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) butyl carbamate (39): compound 39 was prepared in a similar procedure as described for compound 5 using 4-chloro-3-nitroquinoline, tert-butyl 4-aminobutylcarbamate and triethyl orthovalerate as starting materials to obtain the target compound 39 (177 mg,0.430mmol,20% from starting material). MS M/z 412 (M+H) + 。
1- (4-Aminobutyl) -2-butyl-1H-imidazo [4,5-c]Quinolin-4-amine-3, hcl (D): compound D was prepared in a similar procedure as described for a, using compound 39 as starting material, to obtain the target compound D (180 mg,0.431mmol, quantitative). MS M/z 312 (M+H) + 。
N- (4- (4-amino-2-ethyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) butyl) -4-iodobenzamide (40): compound 40 was prepared in a similar procedure as described for 16, using compound B and 4-iodobenzoic acid as starting materials, to obtain the target compound 40 (15 mg,0.020mmol, 72%). MS M/z 514 (M+H) + 。
N- (4- (4-amino-2-ethyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) butyl) -4- (2-guanidinoethyl) benzamide (41): compound 41 was prepared in a similar procedure as described for 16, using compound B and 4- (2-guanidinoethyl) benzoic acid as starting materials, to obtain the target compound 41 (7 mg,0.009mmol, 29%) MS M/z 473 (m+h) + 。
N- (4- (4-amino-2-butyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) butyl) benzamide (42): compound 42 was prepared in a similar procedure as described for 16, using compound D and benzoic acid as starting materials, to obtain the target compound 42 (6 mg,0.012mmol, 38%). MS M/z 416 (M+H) + 。
4- (4- (4-amino-2-butyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) butylCarbamoyl) tert-butyl phenethylcarbamate (43): compound 43 was prepared in a similar procedure as described for 16 using compound D and 4- ((2-boc-amino) ethyl) benzoic acid as starting materials to obtain the target compound 43 (9 mg,0.010mmol, 38%). MS M/z 559 (M+H) + 。
4- (4-amino-2-butyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) butyl carbamate (44): compound 44 was prepared in a similar procedure as described for 5 using 4-chloro-3-nitro-l, 5-naphthyridine, tert-butyl 4-aminobutylcarbamate and triethyl orthovalerate as starting materials to obtain the target compound 44 (120 mg,0.159mmol,5.3% from starting material). MS M/z 413 (M+H) + 。
1- (4-Aminobutyl) -2-butyl-1H-imidazo [4,5-c][1,5]Naphthyridin-4-amine, 4HCl (E): compound E was prepared in a similar procedure as described for a, using compound 44 as starting material, to obtain the target compound E (145 mg, 0.298 mmol, quantitative). MS M/z313 (M+H) +
N- (4- (4-amino-2-butyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) butyl) pyrazine-2-carboxamide (45): compound 45 was prepared in a similar procedure as described for 16, using compound D and pyrazinecarboxylic acid as starting materials, to obtain the target compound 45 (4 mg, 0.04 mmol, 14%) MS M/z 418 (m+h) + 。
4-amino-N- (4- (4-amino-2-ethyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) butyl) -3-methoxybenzamide (46): using compound B and 4-amino-3-methoxybenzoic acid as starting materialsCompound 46 was prepared in a similar procedure as described for 16 to obtain the target compound 46 (12.1 mg,0.014mmol, 58%). MS M/z 433 (M+H) + 。
4-amino-N- (4- (4-amino-2-ethyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) butyl) benzamide (47): compound 47 was prepared in a similar procedure as described for 16, using compound B and 4-aminobenzoic acid as starting materials, to obtain the target compound 47 (6 mg, 0.0070 mmol, 30%). MS M/z 403 (M+H) + 。
4-amino-N- (4- (4-amino-2-butyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) butyl) benzamide (48): compound 48 was prepared in a similar procedure as described for 16, using compound D and 4-aminobenzoic acid as starting materials, to obtain the target compound 48 (0.4 mg,0.0005mmol, 2%). MS M/z 431 (M+H) + 。
4-amino-N- (4- (4-amino-2-butyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) butyl) -3-methoxybenzamide (49): compound 49 was prepared in a similar procedure as described for 16, using compound D and 4-amino-3-methoxybenzoic acid as starting materials, to obtain the target compound 49 (4.2 mg,0.005mmol, 21%). MS M/z 461 (M+H) + 。
2-acetyl-N- (4- (4-amino-2-butyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) butyl) benzoylAmine (50): compound 50 was prepared in a similar procedure as described for 16, using compound D and 2-acetyl benzoic acid as starting materials, to obtain the target compound 50 (7.6 mg,0.01mmol, 43%). MS M/z 458 (M+H) + 。
N- (4- (4-amino-2-butyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) butyl) -4- (2-aminoethyl) benzamide-4 HCl (51): compound 51 was prepared in a similar procedure as described for a, using compound 43 as starting material, to obtain the target compound 51 (10 mg,0.017mmol, quantitative). MS M/z 459 (M+H) +
N- (4- (4-amino-2-butyl-1H-imidazo [4, 5-c)][1,5]Naphthyridin-1-yl) butyl) benzamide (52): compound 52 was prepared in a similar procedure as described for 16, using compound E and benzoic acid as starting materials, to give the target compound 52 (1.5 mg,0.002mmol, 8%), MS M/z 417 (m+h) + 。
4- (4- (4-amino-2-butyl-1H-imidazo [4, 5-c)][1,5]Naphthyridin-1-yl) butylcarbamoyl) phenethylcarbamic acid tert-butyl ester (53): compound 53 was prepared in a similar procedure as described for 16, using compound E and 4- ((2-boc-amino) ethyl) benzoic acid as starting materials to give the target compound 53 (3.8 mg, 0.04 mmol, 16%), MS M/z560 (m+h) + 。
N-(4-(4-amino-2-butyl-1H-imidazo [4,5-c][1,5]Naphthyridin-1-yl) butyl) pyrazine-2-carboxamide (54): compound 54 was prepared in a similar procedure as described for 16, using compound D and pyrazinecarboxylic acid as starting materials, to obtain the target compound 54 (3 mg, 0.04 mmol, 20%), MS M/z 419 (m+h) + 。
N- (4- (4-amino-2-butyl-1H-imidazo [4, 5-c)][1,5]Naphthyridin-1-yl) butyl) -4-guanidinobutyramide (55): compound 55 was prepared in a similar procedure as described for 16, using compound E and 4-guanidinoformic acid as starting materials, to obtain the target compound 55 (2 mg,0.003mmol, 12%). MS M/z 440 (M+H) + 。
N- (4- (4-amino-2-butyl-1H-imidazo [4, 5-c)][1,5]Naphthyridin-1-yl) butyl) -4- (2-aminoethyl) benzamide, 4TFA (56): to compound 53 (3.6 mg, 0.006mmol) was added DCM (0.5 mL) and TFA (1 mL) at 23 ℃. After 20min, the reaction was dried under vacuum and then dried under high vacuum pump overnight to give the target compound 56 (7 mg,0.008mmol, quantitative). MS M/z 460 (M+H) + 。
2-acetyl-N- (4- (4-amino-2-butyl-1H-imidazo [4, 5-c)][1,5]Naphthyridin-1-yl) butyl) benzamide (57): compound 57 was prepared in a similar procedure as described for 16, using compound E and 2-acetyl benzoic acid as starting materials, to obtain the target compound 57 (5.2 mg,0.006mmol, 27%). MS M/z 459 (M+H) + 。
4-amino-N- (4- (4-amino-2-butyl-1H-imidazo [4, 5-c)][l,5]Naphthyridin-1-yl) butyl) -3-methoxybenzamide (58): compound 58 was prepared in a similar procedure as described for 16, using compound E and 4-amino-3-methoxybenzoic acid as starting materials, to obtain the target compound 58 ((4.3 mg,0.04mmol, 20%) MS M/z 462 (m+h) + 。
4-amino-N- (4- (4-amino-2-butyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) butyl) benzamide (59): compound 59 was prepared in a similar procedure as described for 16, using compound E and 4-aminobenzoic acid as starting materials, to obtain the target compound 59 (1.6 mg,0.002mmol, 8%) MS M/z 432 (m+h) + 。
N- (4- (4-amino-2-ethyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) butyl) -4- (dimethylamino) benzamide (60): compound 60 was prepared in a similar procedure as described for 16, using compound B and 4-dimethylaminobenzoic acid as starting materials, to give the target compound 60 (1 mg,0.001mmol, 6%). MS M/z 431 (M+H) + 。
(E) -N- (4- (4-amino-2-ethyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) butyl) -3- (4- (dimethylamino) phenyl) acrylamide (61): compound 61 was prepared in a similar procedure as described for 16, using compound B and 4-dimethylaminocinnamic acid as starting materials, to give the target compound 61 (2 mg,0.003mmol, 11%), MS M/z 457 (m+h) + 。
N- (4- (4-amino-2-butyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) butyl) -4- (dimethylamino) benzamide (62): compound 62 was prepared in a similar procedure as described for 16, using compound D and 4-dimethylaminobenzoic acid as starting materials, to give the target compound 62 (3.5 mg, 0.04 mmol, 20%), MS M/z 459 (m+h) + 。
(E) -N- (4- (4-amino-2-butyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) butyl) -3- (4- (dimethylamino) phenyl) acrylamide (63): compound 63 was prepared in a similar procedure as described for 16, using compound D and 4-dimethylaminocinnamic acid as starting materials, to give the target compound 63 (4.5 mg,0.005mmol, 25%). MS M/z 485 (M+H) +
N- (4- (4-amino-2-butyl-1H-imidazo [4, 5-c)][1,5]Naphthyridin-1-yl) butyl) -4- (dimethylamino) benzamide (64): compound 64 was prepared in a similar procedure as described for 16, using compound E and 4-dimethylaminobenzoic acid as starting materials, to give the target compound 64 (0.5 mg,0.001mmol, 7%), MS M/z 460 (m+h) + 。
(E) -N- (4- (4-amino-2-butyl-1H-imidazo [4, 5-c)][1,5]Naphthyridin-1-yl) butyl) -3- (4- (dimethylamino) phenyl) acrylamide (65): using compound E and 4-dimethylaminocinnamic acid as starting materials, the compound was prepared in a similar procedure to that described for 16Compound 65 to obtain the target compound 65 (0.5 mg,0.001mmol, 7%). MS M/z 486 (M+H) + 。
(E) -N- (2- (4-amino-2-butyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) ethyl) -3- (4- (dimethylamino) phenyl) acrylamide (66): compound 66 was prepared in a similar procedure as described for 16 using compound C and 4-dimethylaminocinnamic acid as starting materials to give the target compound 66 (3 mg, 0.04 mmol, 16%). MS M/z 457 (M+H) + 。
4- (2- (4-amino-2-butyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) ethylcarbamoyl-phenethylcarbamic acid tert-butyl ester (67): compound 67 was prepared in a similar procedure as described for 16 using compound C and 4- (2- (tert-butoxycarbonylamino) ethyl) benzoic acid as starting materials to obtain the target compound 67 (1.5 mg,0.002mmol, 11%). MS M/z 457 (M+H) + 。
N- (2- (4-amino-2-butyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) ethyl) -4- (2-aminoethyl) benzamide (68): compound 68 was prepared in a similar procedure as described for 56, using compound 67 as starting material, to obtain the target compound 68 (2.9 mg,0.003mmol, quantitative). MS M/z 431 (M+H) + 。
1- (4-Aminobutyl) -2-ethyl-1H-imidazo [4,5-c]Quinolin-4-amine (F): compound F was prepared in a similar procedure to that described for a using 4-chloro-3-nitro-1, 5-naphthyridine, tert-butyl 4-aminobutylcarbamate and triethyl orthoacetate as starting materials to obtain the target compound F (130 mg,0.316mmol,9% fromStarting materials). MS M/z 270 (M+H) + 。
N- (4- (4-amino-2-methyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) butyl) benzamide (69): compound 69 was prepared in a similar procedure as described for 16, using compound F and benzoic acid as starting materials, to give the target compound 69 (6.5 mg,0.008mmol, 42%). MS M/z 374 (M+H) + 。
N- (4- (4-amino-2-methyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) butyl) -4- (dimethylamino) benzamide (70): compound 70 was prepared in a similar procedure as described for 16, using compound F and 4-dimethylaminobenzoic acid as starting materials, to give the title compound 70 (6.5 mg,0.009mmol, 39%). MS M/z 417 (M+H) + 。
N- (4- (4-amino-2-methyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) butyl) -4- (pyrrolidin-1-yl) benzamide (71): compound 71 was prepared in a similar procedure as described for 16, using compound F and 4- (1-pyrrolidinylbenzoic acid) as starting materials, to obtain the target compound 71 (3.1 mg, 0.04 mmol, 18%), MS M/z 443 (m+h) + 。
N- (4- (4-amino-2-methyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) butyl) -4- (diethylamino) benzamide (72): compound F and 4- (diethylamino) benzoic acid were used as starting materials to match the class described for 16A similar procedure prepares compound 72 to obtain the target compound 72 (6.4 mg,0.008mmol, 41%), MS M/z 445 (M+H) + 。
3, 4-diamino-N- (4- (4-amino-2-ethyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) butyl) benzamide (73): compound 73 was prepared in a similar procedure as described for 16, using compound B and 3, 4-diaminobenzoic acid as starting materials, to obtain the target compound 73 (1.1 mg,0.001mmol, 4%). MS M/z 418 (M+H) + 。
N- (4- (4-amino-2-ethyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) butyl) -4- (pyrrolidin-1-yl) benzamide (74): compound 74 was prepared in a similar procedure as described for 16, using compound B and 4- (pyrrolidin-1-yl) benzoic acid as starting materials to give the target compound 74 (4.5 mg,0.005mmol, 19%). MS M/z 457 (M+H) + 。
N- (4- (4-amino-2-ethyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) butyl) -4- (methylamino) benzamide (75): compound 75 was prepared in a similar procedure as described for 16, using compound B and 4-methylaminobenzoic acid as starting materials, to obtain the target compound 75 (7.6 mg,0.009mmol, 33%). MS M/z 417 (M+H) + 。
4-amino-N- (4- (4-amino-2-ethyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) butyl) -3-fluorobenzamide (76): make the following stepsCompound 76 was prepared using compound B and 3-fluoro-4-aminobenzoic acid as starting materials in a similar procedure as described for 16 to obtain the target compound 76 (7 mg,0.008mmol, 31%). MS M/z 421 (M+H) + 。
N- (4- (4-amino-2-ethyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) butyl) -4- (dimethylamino) -3, 5-difluorobenzamide (77): compound 77 was prepared in a similar procedure as described for 16 using compound B and 4- (dimethylamino) -3, 5-difluorobenzoic acid as starting materials to obtain the target compound 77 (9.5 mg,0.010mmol, 41%). MS M/z 467 (M+H) + 。
N- (4- (4-amino-2-ethyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) butyl) -4- (dimethylamino) -3-fluorobenzamide (78): compound 78 was prepared in a similar procedure as described for 16, using compound B and 4- (dimethylamino) -3-fluorobenzoic acid as starting materials, to obtain the target compound 78 (12 mg,0.013mmol, 49%). MS M/z 449 (M+H) + 。
N- (4- (4-amino-2-ethyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) butyl) -4- (dimethylamino) -3-nitrobenzamide (79): compound 79 was prepared in a similar procedure as described for 16 using compound B and 4- (dimethylamino) -3-nitrobenzoic acid as starting materials to obtain the target compound 79 (11 mg,0.012mmol, 50%). MS M/z 476 (M+H) + 。
N- (4- (4-amino-2-ethyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) butyl) -4- (diethylamino) benzamide (80): compound 80 was prepared in a similar procedure as described for 16, using compound B and 4- (diethylamino) benzoic acid as starting materials, to obtain the target compound 80 (10 mg,0.01 mmol, 42%). MS M/z 459 (M+H) + 。
N- (4- (4-amino-2-ethyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) butyl) -2- (dimethylamino) benzamide (81): compound 81 was prepared in a similar procedure as described for 16 using compound B and 2- (diethylamino) benzoic acid as starting materials to give the title compound 81 (12.2 mg,0.014mmol, 57%). MS M/z 431 (M+H) + 。
4-amino-N- (4- (4-amino-2-ethyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) butyl) -3, 5-difluorobenzamide (82): compound 82 was prepared in a similar procedure as described for 16, using compound B and 4-amino-3, 5-difluorobenzoic acid as starting materials, to give the target compound 82 (10.2 mg,0.01 mmol, 49%). MS M/z 439 (M+H) + 。
N- (4- (4-amino-2-butyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) butyl) -4- (dimethylamino) -3-fluorobenzamide (83): compound 83 was prepared in a similar procedure as described for 16 using compound D and 4- (dimethylamino) -3-fluorobenzoic acid as starting materials to obtain the target compound 83 (9 mg,0.01 mmol, 50%). MS M/z 477 (M+H) + 。
N- (4- (4-amino-2-butyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) butyl) -4- (dimethylamino) -3, 5-difluorobenzamide (84): compound 84 was prepared in a similar procedure as described for 16 using compound D and 4- (dimethylamino) -3, 5-difluorobenzoic acid as starting materials to obtain the target compound 84 (7 mg,0.008mmol, 38%). MS M/z 495 (M+H) + 。
N- (4- (4-amino-2-butyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) butyl) -4- (dimethylamino) -3-nitrobenzamide (85): compound 85 was prepared in a similar procedure as described for 16, using compound D and 4- (dimethylamino) -3-nitrobenzoic acid as starting materials, to obtain the title compound 85 (10 mg,0.012mmol, 54%). MS M/z 504 (M+H) + 。
N- (4- (4-amino-2-butyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) butyl) -4- (pyrrolidin-1-yl) benzamide (86): compound 86 was prepared in a similar procedure as described for 16 using compound D and 4- (1-pyrrolidinylamino) benzoic acid as starting materials to obtain the target compound 86 (2 mg,0.002mmol, 11%). MS M/z 485 (M+H) + 。
4-amino-N- (4- (4-amino-2-butyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) butyl) -3-fluorobenzamide (87): compound 87 was prepared in a similar procedure as described for 16 using compound D and 4-amino-3-fluoro-benzoic acid as starting materialsTo obtain the objective compound 87 (6 mg,0.008mmol, 20%). MS M/z 449 (M+H) + 。
4-amino-N- (4- (4-amino-2-butyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) butyl) -3, 5-difluorobenzamide (88): compound 88 was prepared in a similar procedure as described for 16, using compound D and 4-amino-3, 5-difluoro-benzoic acid as starting materials, to obtain the target compound 88 (6 mg, 0.0070 mmol, 34%), MS M/z 467 (m+h) + 。
N- (4- (4-amino-2-ethyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) butyl) -4- (dimethylamino) benzamide (89): compound 89 was prepared in a similar procedure as described for 16 using compound B and 4-amino-3, 5-difluorobenzoic acid as starting materials to give the title compound 89 (10 mg,0.01 mmol, 27%), MS M/z 431 (m+h) + 。
5-amino-N- (4- (4-amino-2-ethyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) butyl) pyrazine-2-carboxamide (90): compound 90 was prepared in a similar procedure as described for 16 using compound B and 5-aminopyrazine-2-carboxylic acid as starting materials to give the title compound 90 (8 mg,0.009mmol, 37%). MS M/z 405 (M+H) + 。
4- (3-aminoquinolin-4-ylamino) butylcarbamic acid tert-butyl ester (91): using 4-chloro-3-nitroquinoline and 4-aminobutylcarbamic acid tert-butyl esterButyl ester compound 91 was prepared in a similar procedure to that described for 2 to obtain the target compound 91 (5050 mg,15.284mmol,97% from starting material). MS M/z 331 (M+H) + 。
4- (2- (ethoxymethyl) -1H-imidazo [4, 5-c)]Quinolin-1-yl) butyl carbamate tert-butyl carbamate (92): to a solution of tert-butyl 4- (3-aminoquinolin-4-ylamino) butylcarbamate (1070 mg,3.238 mmol) in anhydrous THF (12 mL) at 23℃was added triethylamine (885. Mu.L, 8.746 mmol) and 2-ethoxyacetyl chloride (500 mg,4.078 mmol). After 20h, the solvent was removed in vacuo. The residue was dissolved in DCM (50 mL), washed with saturated sodium bicarbonate (50 mL) and brine (50 mL), and dried over MgSO 4 Drying to obtain the crude intermediate (tert-butyl 4- (3- (2-ethoxyacetamido) quinolin-4-ylamino) butylcarbamate). The crude product was dissolved in MeOH (5 mL) followed by the addition of calcium oxide (0.5 g) in a sealed tube. The reaction mixture was heated at 120℃for 2.5h. After removal of CaO by filtration, the solvent was removed in vacuo and the residue was purified by preparative LC to give compound 92 (346 mg,0.868mmol, 27%), MS M/z 399 (M+H) + 。
1- (4-Aminobutyl) -2- (ethoxymethyl) -1H-imidazo [4,5-c]Quinolin-4-amine, 3HCl (G): compound G was prepared using compound 92 in a similar procedure as described for a to obtain the target compound G (5270 mg,0.595mmol,4% from starting material). MS M/z 312 (M+H) + 。
3-amino-N- (4- (4-amino-2-butyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) butyl) benzamide (93): compound 93 was prepared in a similar procedure as described for 16, using compound D and 3-aminobenzoic acid as starting materials, to give the target compound 93 (5 mg,0.006mmol, 19%) MS M/z 431 (m+h) + 。
3-amino-N- (4- (4-amino-2-butyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) butyl) -4-fluorobenzamide (94): to a solution of D (10 mg,0.024 mmol) and 3-amino-4-fluoro-benzoic acid (4 mg,0.026 mmol) in DMF (1 ml) was added 4- (4, 6-dimethoxy-1, 3, 5-triazin-2-yl) -4-methyl-morpholinium tetrafluoroborate (DMTMMT; 7mg,0.029 mmol) and DIEA (30. Mu.L, 0.172 mmol) at 23 ℃. After 10min, the mixture was purified by preparative LC to give the target compound 94 (5 mg,0.006mmol, 21%) MS M/z 449 (M+H) + 。
3-amino-N- (4- (4-amino-2- (ethoxymethyl) -1H-imidazo [4, 5-c) ]Quinolin-1-yl) butyl) -4-fluorobenzamide (95): compound 95 was prepared in a similar procedure as described for 94 using compound G and 3-amino-4-fluoro-benzoic acid as starting materials to obtain the target compound 95 (6 mg, 0.0070 mmol, 26%). MS M/z 451 (M+H) + 。
3-amino-N- (4- (4-amino-2- (ethoxymethyl) -1H-imidazo [4, 5-c)]Quinolin-1-yl) butyl) -4-fluorobenzamide (96): compound 96 was prepared in a similar procedure as described for 94 using compound G and 3-aminobenzoic acid as starting materials to obtain the target compound 96 (5 mg,0.006mmol, 19%). MS M/z 433 (M+H) + 。
3-amino-N- (4- (4-amino-2- (ethoxymethyl) -1H-imidazo [4, 5-c)]Quinolin-1-yl) butyl) benzamide (97): compound G and 4-amino-3-methoxybenzoic acid were used as starting materials as described for 94A similar procedure was followed to prepare compound 97 to afford the title compound 97 (5 mg,0.005mmol, 23%). MS M/z 463 (M+H) + 。
5-amino-N- (2- (4-amino-2-butyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) ethyl) pyrazine-2-carboxamide (98): compound 98 was prepared in a similar procedure as described for 16 using compound C and 5-amino-pyrazine-2-carboxylic acid as starting materials to give the title compound 98 (2.13 mg,0.002mmol, 11%). MS M/z 405 (M+H) + 。
4-amino-N- (4- (4-amino-2- (ethoxymethyl) -1H-imidazo [4, 5-c)]Quinolin-1-yl) butyl) -3-fluorobenzamide (99): compound 99 was prepared in a similar procedure as described for 94 using compound G and 3-fluoro-4-aminobenzoic acid as starting materials to obtain the target compound 99 (7.37 mg,0.008mmol, 37%). MS M/z 451 (M+H) + 。
4-amino-N- (4- (4-amino-2- (ethoxymethyl) -1H-imidazo [4, 5-c)]Quinolin-1-yl) butyl) -3, 5-difluorobenzamide (100): compound 100 was prepared in a similar procedure as described for 94 using compound G and 4-amino-3, 5-difluorobenzoic acid as starting materials to obtain the target compound 100 (9.7 mg,0.01 mmol, 51%), MS M/z 469 (m+h) + 。
Methyl-4-amino-3, 5-difluorobenzoate (101): to 4-amino groupTo a solution of 3, 5-difluorobenzoic acid (2087 mg,12.055 mmol) in acetonitrile (15 mL) was added thionyl chloride (12 mL) and then heated to 80 ℃. After 1h, the solvent was removed in vacuo. Toluene (10 mL) was added to the mixture followed by evaporation in vacuo. The residue was dissolved in anhydrous MeOH (5 mL). After 0.5h, the solvent was removed in vacuo. The residue was dissolved in DCM (20 mL), washed with saturated sodium bicarbonate (50 mL) and brine (50 mL), followed by MgSO 4 Is dried and filtered. The solvent was removed in vacuo to give compound 101 (1730 mg,9.244mmol, 77%), MS M/z 188 (M+H) + 。
1- (1H-imidazol-1-yl) -1-oxo-5-ureidopent-2-ylcarbamic acid (S) -tert-butyl ester (102): CDI (880 mg, 5.427 mmol) was added to a solution of Boc-Cit-OH (1150 mg,4.177 mmol) in DMF (5 mL) at room temperature and then heated to 60 ℃. After 2h CDI (220 mg,1.357 mmol) was added to the mixture. The reaction was stirred at 60℃for 1h. After 3h, the reaction was dried in vacuo. The residue was diluted with EtOAc (50 mL) and washed with water (50 mL), saturated sodium bicarbonate (20 mL) and brine (20 mL). The organic layer was dried over MgSO 4 Drying, followed by filtration and removal of the solvent in vacuo gave compound 102 (1465 mg,4.503mmol, crude), MS M/z 326 (M+H) + 。
(S) -4- (2- (tert-Butoxycarbonylamino) -5-ureidopentanoylamino) -3, 5-difluorobenzoic acid (103): to a solution of compound 103 (188 mg,0.578 mmol) and compound 102 (108 mg,0.577 mmol) in THF (2 mL) at 23℃was added 60% NaH (70 mg, 1.706 mmol). After 20h, 1mL of water was added and the mixture was stirred for 10min. The solvent was removed in vacuo and the residue was purified by prep LC to give compound 103 (17 mg,0.049mmol, 9%), MS M/z 345 (m+h) + 。
1- (4- (4- (4-amino-2-butyl-1H-imidazo [4, 5-c))]Quinolin-1-yl) butylcarbamoyl) -2, 6-difluorophenylamino) -1-oxo-5-ureidopent-2-ylcarbamic acid (S) -tert-butyl ester (104): to a solution of compound D (20 mg,0.044 mmol) and compound 103 (17 mg,0.040 mmol) in DMF (1 mL) was added HATU (16 mg,0.042 mmol) and DIEA (60. Mu.L, 0.344 mmol) at 23 ℃. After 20min, the mixture was purified by preparative LC to obtain the compound104(23mg，0.022mmol，55％)。MS m/z724(M+H) + 。
(S) -N- (4- (4-amino-2-butyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) butyl) -4- (2-amino-5-ureidovalerylamino) -3, 5-difluorobenzamide (105): to a solution of compound 104 (23 mg,0.022 mmol) in DCM (1 mL) was added TFA (1 mL) at 23 ℃. After 15min, the solvent was removed in vacuo. 10mL of toluene was added to the residue and evaporated again. The residue was dried on a high vacuum pump to obtain compound 105 (24 mg,0.022mmol, quantitative). MS M/z 624 (M+H) + 。
N- (4- (4-amino-2-butyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) butyl) -4- ((S) -2- (2- (aminoxy) acetylamino) -3-methylbutanoylamino) -5-ureidopentanoylamino) -3, 5-difluorobenzamide (106): HATU (9 mg,0.037 mmol) and DIEA (40. Mu.L, 0.023 mmol) were added to a solution of Fmoc-Aoa-Val-OH (11 mg,0.027 mmol) and compound 105 (24 mg,0.022 mmol) in DMF (1 mL) at 23 ℃. After 10min piperidine (50 μl, 5%) was added to the mixture. After 5min, the mixture was purified by preparative LC to give compound 106 (9 mg, 0.0070 mmol, 27%), MS M/z 796 (M+H) + 。
1- (1H-imidazol-1-yl) -1-oxopropan-2-ylcarbamic acid (S) -tert-butyl ester (107): compound 107 was prepared using Boc-alanine in a similar procedure as described for 102 to give the target compound 107 (730 mg,3.051mmol,75% crude). MS M/z 326 (M+H) + 。
(S) -4- (2- (tert-Butoxycarbonylamino) -5-ureidopentanoylamino) -3, 5-difluorobenzoic acid (108): compound 108 was prepared in a similar procedure as described for 103, using 107, to obtain the target compound 108 (17 mg,0.049mmol, 9%). MS M/z 431 (M+H) + 。
(S) -N- (4- (4-amino-2-butyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) butyl) -4- (2- (2- (aminooxy) acetamido) propionylamino) -3, 5-difluorobenzamide (109): using 108 and compound D,compound 109 was prepared in a similar procedure as described for 106 to obtain the target compound 109 (9 mg, 0.0070 mmol,31% from 108). MS M/z 796 (M+H) + 。
2- (3- (2- (1, 3-dioxoisoindolin-2-yloxy) ethoxy) propionylamino) -3-methylbutanoic acid (S) -tert-butyl ester (110): to a solution of 3- (2- (1, 3-dioxoisoindolin-2-yloxy) ethoxy) propionic acid (295 mg,1.056 mmol) and Val-OtBu-HCl (225 mg,1.078 mmol) in DCM (5 mL) was added DMTMMT (304 mg,1.260 mmol) and DIEA (460. Mu.L, 2.641 mmol) at 23 ℃. After 1.5h, the solution was diluted with EtOAC (100 mL) and washed with 1N HCl (100 mL), saturated sodium bicarbonate (100 mL) and brine (50 mL). The organic layer was dried over MgSO 4 Dried, filtered, and the solvent removed in vacuo. The residue was purified by flash chromatography to obtain compound 110 (379 mg,0.872mmol, 83%). MS M/z 435 (M+H) + 。
(S) -2- (3- (2- (1, 3-dioxoisoindolin-2-yloxy) ethoxy) propionylamino) -3-methylbutanoic acid (111): to a solution of compound 110 (379 mg,0.872 mmol) was added 4M HCl (5 mL,20 mmol) in dioxane at 23 ℃. After 20h, the solvent was removed in vacuo and dried using a high vacuum pump to give compound 111 (320 mg,0.846mmol, 97%). MS M/z 379 (M+H) + 。
N- (4- (4-amino-2-butyl-1H-imidazo [4, 5-c)]Quinolin-1-yl) butyl) -4- ((S) -2- (3- (2- (aminooxy) ethoxy) propionylamino) -3-methylbutanoylamino) -5-ureidovalerylamino) -3, 5-difluorobenzamide (112): to a solution of compound 111 (3 mg, 0.0071 mmol) and compound 105 (5 mg,0.005 mmol) in DMF (1 mL) was added DMTMMT (3 mg,0.012 mmol) and DIEA (20. Mu.L, 0.115 mmol) at 23 ℃. After 1.5h, hydrazine hydrate (2 μl,0.506 mmol) was added to the mixture. After 5min, the mixture was purified by preparative LC to give compound 112 (2 mg,0.002mmol, 21%). MS M/z 855 (M+H) + 。
(S) -N- (4- ((N- (2- (4-amino-2-butyl-1H-imidazo [4, 5-c)) ]Quinolin-1-yl) ethyl) carbamimidocarbamoyloxy) methyl) phenyl) -2- ((S) -2- (2- (aminooxy) acetamido) -3-methylbutanoylamino) -5-ureidovaleramide (113): compound 113 was prepared in a similar procedure as described for 106, using compound 30 as starting material, to obtain the target compound 113 (2 mg,0.001mmol,2% from compound 30). MS M/z 805 (M+H) + 。
Table 3-TLR agonist-Nuclear 1 Compounds
Example 3: synthesis of TLR agonists comprising the following representative structures-core 5 of formula (I) and formula (II) (fig. 1):
or a pharmaceutically acceptable salt, solvate, stereoisomer or tautomer thereof, wherein
A is CH or N;
x is O-R1, NH-R1, S-R1 or H;
YY is-ONH 2 、-N 3 -OH, maleimide, -COOH or-C (=o) CH 2 Y1, wherein Y1 is a halide;
each of L1 and L2 is independently (CH) 2 ) m 、(CH 2 ) m C(＝O)、(CH 2 ) m -NH(CH 2 ) n 、(CH 2 ) m -C(＝O)NH(CH 2 ) n 、(CH 2 ) m -OC(＝O)-NH-(CH 2 ) n 、(CH 2 ) m -NHC(＝O)-NH-(CH 2 ) n 、(CH 2 ) m -NH、(CH 2 ) m -NHC(＝O)、(CH 2 ) m -NHC(＝O)-(CH 2 ) n -NHC(＝O)-(CH 2 ) p 、C(＝O)-(CH 2 ) n 、C 3 -C 8 Heterocycle or absent; wherein each of m, n and p is independently an integer from 0 to 12;
r1 is H, C 1 -C 12 Alkyl, substituted C 1 -C 12 Alkyl, oxygen-containing C 1 -C 12 Alkyl, C 3 -C 8 Heterocycloalkyl, substituted C 3 -C 8 Heterocycloalkyl, C 3 -C 8 Cycloalkyl, substituted C 3 -C 8 Cycloalkyl, -N 3 Terminally substituted C 1 -C 12 Alkyl, (CH) 2 ) q -(OCH 2 CH 2 ) r OMe, wherein each of q and r is independently an integer from 0 to 12;
R2 is C 1 -C 6 Alkylene, C 1 -C 12 Substituted alkylene, C 3 -C 8 Cycloalkylene, C 3 -C 8 Substituted cycloalkylene, arylene, substituted C 6 -C 10 Arylene, 5-12 membered heteroarylene containing 1-3 heteroatoms, substituted 5-12 membered heteroarylene containing 1-3 heteroatoms, or (OCH) 2 CH 2 ) ss Or a combination thereof, or R2 is absent; wherein ss is an integer from 1 to 12, wherein each heteroatom is independently N, O or S;
r3 is the side chain of an amino acid, C 1 -C 6 Alkylene, C 1 -C 6 Substituted alkylene, C 3 -C 8 Cycloalkylene, C 3 -C 8 Heterocycloalkylene, substituted C 3 -C 8 Cycloalkylene, arylene, substitutedArylene, 5-12 membered heteroarylene containing 1-3 heteroatoms, substituted 5-12 membered heteroarylene containing 1-3 heteroatoms, C containing amino groups 1 -C 12 Alkylene group, carbonyl group-containing C 1 -C 12 Alkylene, oxygen-containing C 1 -C 12 Alkylene, -N 3 Terminal C 1 -C 6 Alkylene, -CCH terminal C 1 -C 6 Alkylene, -SH terminal C 1 -C 6 Alkylene, -OH terminal C 1 -C 6 Alkylene, nitrogen-containing C 1 -C 6 Alkylene, -OPO 3 H 2 Terminal C 1 -C 6 Alkylene, -OPO 3 H 2 Terminal arylene, glucuronide terminal C 1 -C 6 Alkylene, -N 3 Terminal arylene, acetylene terminal arylene, amine terminal arylene, (CH) 2 ) s 、(CH 2 ) s -C(＝O)、(CH 2 ) s -NH(CH 2 ) t 、(CH 2 ) s -C(＝O)NH(CH 2 ) t 、(CH 2 ) s -OC(＝O)-NH-(CH 2 ) t 、(CH 2 ) s -NHC(＝O)-NH-(CH 2 ) t Or a combination thereof; or R3 is absent; wherein each s and t is independently an integer from 0 to 6;
r4 is H, C 3 -C 8 Cycloalkyl, C 3 -C 8 Heterocycloalkyl, C 3 -C 8 Substituted heterocycloalkyl, aryl, substituted aryl, (CH) 2 ) u -(OCH 2 CH 2 ) v OMe, di/tri branched (CH) 2 ) u -(OCH 2 CH 2 ) v OMe or a combination thereof; or R4 is absent; wherein each u and v is independently an integer from 1 to 48
Or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein a is CH or N;
x is O-R1, NH-R1, S-R1 or H;
YY is H, -ONH 2 、-N 3 -OH, maleimide, -COOH or-C (=o) CH 2 Y1, wherein Y1 is a halide;
each of L1 and L2 is independently (CH) 2 ) m 、(CH 2 ) m C(＝O)、(CH 2 ) m -NH(CH 2 ) n 、(CH 2 ) m -C(＝O)NH(CH 2 ) n 、(CH 2 ) m -OC(＝O)-NH-(CH 2 ) n 、(CH 2 ) m -NHC(＝O)-NH-(CH 2 ) n 、(CH 2 ) m -NH、(CH 2 ) m -NHC(＝O)、(CH 2 ) m -NHC(＝O)-(CH 2 ) n -NHC(＝O)-(CH 2 ) p 、C(＝O)-(CH 2 ) n Arylene, substituted arylene, 5-12 membered heteroarylene containing 1-3 heteroatoms, substituted 5-12 membered heteroarylene containing 1-3 heteroatoms, C containing 1-3 heteroatoms 3 -C 8 Heterocycle or absent; wherein each of m, n, and p is independently an integer from 0 to 6, wherein each heteroatom is independently N, O or S;
l3 is C (=O), -CH (R5) -, - (AA) i -or arylene, or a combination thereof, or L3 is absent; wherein each AA is independently an amino acid, wherein i is an integer from 1 to 6;
r5 is NH-L4-Y2 or CH 2 -L4-Y2, wherein Y2 is H or absent;
l4 is C (=o), C (=o) O-, -OC (=o) -, -C (CH) 2 O) 3 -、-C(CH 2 CH 2 O) 3 -、-(AA) j -, arylene, substituted arylene, C 3 -C 8 Cycloalkylene, C 3 -C 8 Substituted cycloalkylene, arylene, substituted arylene, 5-12 membered heteroarylene containing 1-3 heteroatoms, substituted 5-12 membered heteroarylene containing 1-3 heteroatoms, 5-12 membered heterocycloalkylene containing 1-3 heteroatoms, substituted 5-12 membered heterocycloalkylene containing 1-3 heteroatoms, C 1 -C 12 Alkylene, -O-, -NH-, -S-, substituted C 1 -C 12 Alkylene group、-(CH 2 ) s -(OCH 2 CH 2 ) t -(CH 2 ) u -、(CH 2 ) s -(OCH 2 CH 2 ) t -OMe、-N 3 、-SH、-OH、-NH 2 、-OPO 3 H 2 Glucuronide, acetylene, or a combination thereof, or L4 is absent; wherein each AA is independently an amino acid, wherein j is an integer from 1 to 6, wherein each of S and u is independently an integer from 0 to 12, wherein t is independently an integer from 0 to 48, wherein each heteroatom is independently N, O or S;
r1 is H, C 1 -C 12 Alkyl, substituted C 1 -C 12 Alkyl, oxygen-containing C 1 -C 12 Alkyl, C 3 -C 8 Heterocycloalkyl, substituted C 3 -C 8 Heterocycloalkyl, C 3 -C 8 Cycloalkyl, substituted C 3 -C 8 Cycloalkyl, -N 3 Terminally substituted C 1 -C 12 Alkyl, (CH) 2 ) q -(OCH 2 CH 2 ) r -OMe; wherein each of q and r is independently an integer from 0 to 12;
r2 is C 1 -C 6 Alkylene, C 1 -C 12 Substituted alkylene, C 3 -C 8 Cycloalkylene, C 3 -C 8 Substituted cycloalkylene, arylene, substituted arylene, 5-12 membered heteroarylene containing 1-3 heteroatoms, substituted 5-12 membered heteroarylene containing 1-3 heteroatoms, 5-12 membered heterocycloalkylene containing 1-3 heteroatoms, substituted 5-12 membered heterocycloalkylene containing 1-3 heteroatoms, or (OCH) 2 CH 2 ) r Or a combination thereof, or R2 is absent; wherein r is an integer from 1 to 12, wherein each heteroatom is independently N, O or S;
r3 is H OR-C (=o) R6, -C (=o) OR6;
r6 is C 1 -C 12 Alkyl, substituted aryl, CH 3 -(CH 2 ) s -(OCH 2 CH 2 ) t -(CH 2 ) u -, wherein each of s, t and u is independently an integer of 0 to 12A number.
TLR agonists with a core 5 structure were synthesized as disclosed in the following schemes.
Tert-butyl 4- ((2, 6-dichloro-9H-purin-9-yl) methyl) benzyl carbamate, tert-butyl 4- ((2, 6-dichloro-7H-purin-7-yl) methyl) benzyl carbamate (114): to a solution of tert-butyl 4- (hydroxymethyl) phenylmethylcarbamate (1280 mg, 5.390 mmol) and 2, 6-dichloropurine (1050 mg, 5.554 mmol) in THF (10 mL) at 23℃was added PPh 3 (1560 mg,5.948 mmol). After 30min, DIAD (1600 μL,8.126 mmol) was added over 5min at 0deg.C. The mixture was stirred at 50 ℃. After 2h, the solvent was removed in vacuo. The residual mixture was diluted with EtOAc (100 mL) and washed with half-saturated sodium bicarbonate (100 mL) and brine (20 mL). The organic layer was dried over MgSO 4 Drying and filtering. The solvent was removed in vacuo. The residue was purified by flash chromatography to give compound 114 (2268 mg,<5.555mmol, with PPh 3 Crude mixture of (d). MS M/z 409 (M+H) + 。
4- ((6-amino-2-chloro-9H-purin-9-yl) methyl) benzyl carbamic acid tert-butyl ester (115): compound 114 (crude mixture of PPh3, 2268mg,<5.555 mmol) was placed in a pressure-resistant glass vessel equipped with a stirring rod. 7NNH in MeOH was added to the vessel 3 (12 mL,84 mmol). The tube was sealed and heated at 120 ℃. After 1h, the solvent was removed in vacuo and the residue was dissolved in DCM (100 mL). The precipitate was removed by filtration. The liquid was purified by flash chromatography to obtain compound 115 (1043 mg,2.682mmol,50% from 2, 6-dichloropurine). MS M/z 400 (M+H) + 。
4- ((6-amino-2-butoxy-9H-purin-9-yl) methyl) benzyl carbamic acid tert-butyl ester (116): compound 115 (1043 mg,2.682 mmol) was dissolved in 20% sodium n-butoxide (5 ml,10.4 mmol) at 23 ℃ under dry nitrogen, and the temperature was raised to 110 ℃. After 1.5h, 1ml of water was added to the mixture followed by the addition of Boc anhydride (170 mg,0.779 mmol). After 5 minutes of the time period of the reaction,the solvent was removed in vacuo. The residue was dissolved in DCM (30 ml), washed with half-saturated sodium bicarbonate (50 ml) and brine (50 ml), and dried over MgSO 4 Drying and filtering. The organic solvent was removed in vacuo. The residue was purified by flash chromatography to obtain compound 116 (560 mg,1.313mmol, 49%). MS M/z 427 (M+H) + 。
4- ((6-amino-8-bromo-2-butoxy-9H-purin-9-yl) methyl) benzyl carbamic acid tert-butyl ester (117): to a solution of compound 116 (560 mg,1.313 mmol) in DCM (10 mL) was added bromine (135. Mu.L, 0.507 mmol) at 23 ℃. After 10min, the reaction was dried in vacuo. The residue was dissolved in DCM (50 mL), washed with half-saturated sodium bicarbonate (50 mL) and brine (50 mL), and dried over MgSO 4 Drying and filtering. The solvent was removed in vacuo. The residue was purified by flash chromatography to give compound 117 (440 mg,0.871mmol, 66%) as a salt of HBr, MS M/z 506 (M+H) + 。
6-amino-9- (4- (aminomethyl) benzyl) -2-butoxy-7H-purin-8 (9H) -one (118): compound 117 (240 mg,0.410 mmol) was dissolved in 37% concentrated HCl solution (10 mL) and refluxed. After 4.5h, the solvent was removed in vacuo. To the residue were added water (10 mL) and MeOH (4 mL) by adding 28% NH 3 The solution (9 mL) was neutralized. The solvent was removed in vacuo. The residue was purified by preparative LC to obtain compound 118 (11 mg,0.019mmol, 5%). MS M/z 343 (M+H) + 。
N- (4- ((6-amino-2-butoxy-8-oxo-7H-purin-9 (8H) -yl) methyl) benzyl) -2- (aminoxy) acetamide (119): to a solution of compound 118 (5 mg, 0.0070 mmol) and 2, 5-dioxopyrrolidin-1-yl 2- (tert-butoxycarbonylaminooxy) acetate (3 mg, 0.010mmol) in DMF (1 mL) was added DIEA (5 μl,0.057 mmol) at 23deg.C. After 10min, the solvent was removed in vacuo. DCM (1 mL) and TFA (1 mL) were added to the residue at 23 ℃. After 10min, the mixture was purified by preparative LC to give compound 119 (3.6 mg,0.005mmol, 65%). MS M/z 416 (M+H) + 。
N- (4- ((6-amino-2-butoxy-8-oxo-7H-purin-9 (8H) -yl) methyl) benzyl) -3- (2- (aminooxy) ethoxy) propanamide (120): to a solution of 118 (5 mg, 0.0070 mmol) and 3- (2- (1, 3-dioxoisoindolin-2-yloxy) ethoxy) propionic acid (3 mg,0.01 mmol) in DMF (1 mL) was added DMTMMT (3 mg,0.012 mmol) and DIEA (8. Mu.L, 0.046 mmol) at 23 ℃. After 15min, hydrazine hydrate (3 μl,0.06 mmol) was added to the mixture. After 20min, the mixture was purified by preparative LC to give compound 120 (3.5 mg, 0.04 mmol, 59%). MS M/z 474 (M+H) + 。
2, 6-dichloro-9- (tetrahydro-2H-pyran-2-yl) -9H-purine (121): to a magnetically stirred solution of 2, 6-dichloropurine (2950 mg,15.608 mmol) in ethyl acetate (100 mL) was added benzenesulfonic acid (30 mg,0.19 mmol), and the mixture was heated to 50deg.C under dry nitrogen. 3, 4-dihydro-2H-pyran (2200. Mu.L, 26.153 mmol) was added to the stirred mixture over 1H at 50 ℃. The temperature was reduced to 23 ℃. After 1h, the mixture was treated with half saturated NaHCO 3 (50 ml) and brine (50 ml) over MgSO 4 Drying and filtering. The organic solvent was removed in vacuo and the residue was dried in a high vacuum pump to give compound 121 (4170 mg, 15.279 mmol, 98%), MS M/z 274 (M+H) + 。
2-chloro-9- (tetrahydro-2H-pyran-2-yl) -9H-purin-6-amine (122): compound 121 (4170 mg, 15.263 mmol) was placed in a pressure-resistant glass container equipped with a stirring rod. 7N NH in MeOH was added to the vessel 3 (12.84 mmol). The tube was sealed and heated at 110 ℃. After 3.5h, the mixture was cooled to room temperature and allowed to stand overnight. The precipitate was filtered and washed with MeOH (5 mL). The solid was dried on a high vacuum pump to obtain compound 122 (3450 mg,13.6mmol, 89%). MS M/z 254 (M+H) + 。
2-chloro-9- (tetrahydro-2H-pyran-2-yl) -9H-purin-6-amine (123): compound 122 (1746 mg,6.882 mmol) was placed in a pressure-resistant glass vessel equipped with a stirring barIs a kind of medium. To the vessel were added n-butylamine (7 mL,70.86 mmol) and DIEA (2.3 mL,13.25 mmol). The tube was sealed and heated at 150 ℃. After 5h, the mixture was cooled to room temperature and the solvent was removed in vacuo. The residue was dissolved in DCM (100 mL), washed with water (30 mL) and brine (50 mL), and concentrated over MgSO 4 Drying and filtering. The organic solvent was removed in vacuo. The residue (intermediate) was dissolved in MeOH (10 mL) and TFA (2 mL) and stirred overnight at 23 ℃. After 18h, the solvent was removed in vacuo. To the residue were added EtOAc (10 mL) and hexane (50 mL) to precipitate. The precipitate was collected by filtration and dried in a vacuum pump to give compound 123 as a 2TFA salt (1640 mg,3.777mmol, 55%). MS M/z 207 (M+H) + 。
N2-butyl-9- ((6-chloropyridin-3-yl) methyl) -9H-purine-2, 6-diamine (124): to a solution of compound 123 (1640 mg,3.777 mmol) and 2-chloro-5- (chloromethyl) pyridine (900 mg,5.556 mmol) in DMF (5 mL) was added K 2 CO 3 (2600 mg,18.813 mmol) and the mixture was stirred at 50℃under nitrogen. After 24h, ice water (100 mL) was added to the mixture and the precipitate was separated. The precipitate was dissolved in DCM (100 mL), washed with brine (50 mL), and dried over MgSO 4 Drying and filtering. The organic solvent was removed in vacuo. The residue was purified by flash chromatography (silica gel) using a gradient of 1% to 10% MeOH in DCM to give compound 124 (10200 mg,3.074mmol, 81%). MS M/z 332 (M+H) + 。
6-amino-2- (butylamino) -9- ((6-chloropyridin-3-yl) methyl) -7H-purin-8 (9H) -one (125): to a solution of compound 124 (10200 mg,3.074 mmol) in DCM (10 mL) was added bromine (250 μL,0.939 mmol) at 23 ℃. After 1.5h, the solvent was removed in vacuo. The residue was dried on a high vacuum pump. 8-bromo-N2-butyl-9- ((6-chloropyridin-3-yl) methyl) -9H-purine-2, 6-diamine, HBr (1500 mg,<3.074 mmol) of the crude intermediate was dissolved in 37% concentrated HCl solution (15 mL) and the solution was refluxed. After 8h, the solvent was removed in vacuo. To the residue were added water (10 mL) and MeOH (4 mL), and then by adding 28% NH 3 The solution (5 mL) was neutralized. The precipitated solid was separated by centrifuge (5 min,4000 rpm) and washed with MeOH (2 mL) and water (10 mL). The precipitate was dried to obtain compound 125 (1100 mg,2.421mmol，79％)。MS m/z 348(M+H) + 。
6-amino-9- ((6- (4- (2-aminoethyl) piperazin-1-yl) pyridin-3-yl) methyl) -2- (butylamino) -7H-purin-8 (9H) -one (126): a mixture of compound 125 (30 mg,0.086 mmol) and tert-butyl 2- (piperazin-1-yl) ethylcarbamate (26 mg,0.113 mmol) was heated at 140 ℃. After 20h, the mixture was cooled to 23 ℃. DCM (0.5 mL) and TFA (0.5 mL) were added to the residue. After 30 min. The solvent was removed in vacuo, and the residue was purified by preparative LC to give compound 126 (9 mg,0.010mmol, 12%) as a TFA salt. MS M/z 441 (M+H) + 。
N- (2- (4- (5- ((6-amino-2- (butylamino) -8-oxo-7H-purin-9 (8H) -yl) methyl) pyridin-2-yl) piperazin-1-yl) ethyl) -2- (aminooxy) acetamide (127): to a solution of compound 126 (9 mg, 0.010mmol) and 2, 5-dioxopyrrolidin-1-yl 2- (tert-butoxycarbonylaminooxy) acetate (2.5 mg, 0.0111 mmol) in DMF (1 mL) was added DIEA (10. Mu.L, 0.060 mmol) at 23 ℃. After 15min, the solvent was removed in vacuo. To the residue was added DCM (1 mL) and TFA (1 mL). After 5min, the solvent was removed in vacuo. The residue was purified by preparative LC to give compound 127 as TFA salt (8 mg,0.008mmol, 82%). MS M/z 514 (M+H) + 。
6-amino-9- ((6- (2- ((2-aminoethyl) (methyl) amino) ethylamino) pyridin-3-yl) methyl) -2- (butylamino) -7H-purin-8 (9H) -one (128): a mixture of compound 125 (30 mg,0.086 mmol) and 2,2' -diamino-N-methyldiethylamine (100. Mu.L, 0.853 mmol) was heated at 130 ℃. After 20h, the mixture was cooled to 23 ℃ and purified by preparative LC to give compound 128 (40 mg,0.045mmol, 52%). MS M/z 423 (M+H) + 。
N- (2- ((2- (5- ((6-amino-2- (butylamino) -8-oxo-7H-purin-9 (8H) -yl) methyl) pyridin-2-ylamino) ethyl) (methyl) amino) ethyl) -2- (aminooxy) acetamide (129): compound 128 was used as starting material to be similar to that described for 127Compound 129 was prepared to give the target compound 129 (13 mg,0.012mmol, 54%). MS M/z502 (M+H) + 。
NH2O-PEG3-Pr- (6-amino-9- ((6- (2- ((2-aminoethyl) (methyl) amino) ethylamino) pyridin-3-yl) methyl) -2- (butylamino) -7H-purin-8 (9H) -one) acetamide (130): to a solution of compound 128 (20 mg,0.023 mmol) and Phth-PEG4-OSu (10 mg,0.022 mmol) in DMF (1 mL) was added DIEA (50 μL,0.287 mmol) at 23 ℃. After 5min, hydrazine hydrate (10 μl) was added to the mixture at 23deg.C. After 5min, the mixture was purified by preparative LC to give compound 130 (20 mg,0.016mmol, 70%). MS M/z 692 (M+H) + 。
6-amino-2- (butylamino) -9- ((6- (2- ((2-hydroxyethyl) (methyl) amino) ethoxy) pyridin-3-yl) methyl) -7H-purin-8 (9H) -one (131): to a solution of compound 125 (124 mg,0.215 mmol) in DMF (4 mL) was added N-methyldiethanolamine (200. Mu.L, 1.007 mmol) and 60% NaH (350 mg,8.750 mmol) at 23 ℃. The mixture was stirred at 60 ℃ under dry nitrogen. After 3h, 1N HCl (4 mL) was added to the mixture and purified by preparative LC to give compound 131 (75 mg,0.085mmol, 39%). MS M/z 431 (M+H) + 。
2-butoxy-9H-purin-6-amine (132): compound 122 (690 mg,2.720 mmol) was dissolved in 20% sodium n-butoxide (8 mL,16.71 mmol) at 23℃under dry nitrogen. After the addition, the temperature was raised to 100 ℃. After 20h, the solvent was removed in vacuo. The residue was dissolved in DCM (30 mL), washed with half-saturated sodium bicarbonate (50 mL) and brine (50 mL), and dried over MgSO 4 Drying and filtering. The organic solvent was removed in vacuo. MeOH (5 mL) and TFA (1 mL) were added to the residue and stirred at 23 ℃. After 18h, the solvent was removed in vacuo. The residue was dissolved in DCM (30 mL), washed with sodium bicarbonate (50 mL) and brine (50 mL), and dried over MgSO 4 Drying and filtering. The organic solvent was removed in vacuo to give crude 2-butoxy-9H-purin-6-amine (1300 mg,2.987, quantitative). MS M/z 208 (M+H) + 。
N2-butyl-9- ((6-chloropyridin-3-yl) methyl) -9H-purine-2, 6-diamine (133): compound 133 was prepared in a similar procedure as described for 124, using compound 132 as starting material, to give the title compound 133 (460 mg,1.406mmol, 47%). MS M/z333 (M+H) + 。
N- (2- (4- (5- ((6-amino-2-butoxy-9H-purin-9-yl) methyl) pyridin-2-yl) piperazin-1-yl) ethyl) -2- (aminooxy) acetamide (134): compound 134 was prepared in a similar procedure as described for 127, using compound 133 as starting material, to obtain the target compound 134 (12 mg,0.012mmol,10% from compound 133). MS M/z 514 (M+H) + 。
6-amino-2-butoxy-9- ((6-chloropyridin-3-yl) methyl) -7H-purin-8 (9H) -one (135): to a solution of compound 133 (124 mg,0.373 mmol) in DCM (10 mL) was added bromine (30. Mu.L, 0.113 mmol) at 23 ℃. After 2h, the reaction was dried in vacuo. 8-bromo-2-butoxy-9- ((6-chloropyridin-3-yl) methyl) -9H-purin-6-amine, HBr (150 mg,<0.373mmol, crude) was dissolved in 3N HCl solution (15 mL) and refluxed. After 20h, the solvent was removed in vacuo. The mixture was purified by preparative LC to give compound 135 (47 mg,0.110mmol, 29%). MS M/z 349 (M+H) + 。
6-amino-9- ((6- (4- (2-aminoethyl) piperidin-1-yl) pyridin-3-yl) methyl) -2-butoxy-7H-purin-8 (9H) -one (136): compound 135 (46 mg,0.132 mmol) and 4- (2-boc-aminoethyl) -piperidine (120 mg,0.526 mmol) were mixed and the mixture was takenThe mixture was stirred at 140 ℃. After 25h, cooling to 23 ℃, DCM (1 mL) and TFA (1 mL) were added to the mixture. After 10min, the organic solvent was removed in vacuo. The mixture was purified by preparative LC to obtain compound 136 (11 mg,0.014mmol, 11%). MS M/z 441 (M+H) + 。
N- (2- (1- (5- ((6-amino-2-butoxy-8-oxo-7H-purin-9 (8H) -yl) methyl) pyridin-2-yl) piperidin-4-yl) ethyl) -3- (2- (aminooxy) ethoxy) propionamide (137): using compound 136 and 3- (2- (1, 3-dioxoisoindolin-2-yloxy) ethoxy) propionic acid as starting materials, compound 137 was prepared in a similar procedure to that described for 130 to give the title compound 137 (9 mg,0.010mmol, 70%), MS M/z 572 (M+H) + 。
9- (4- (2-aminoethyl) benzyl) -2-butoxy-9H-purin-6-amine (138): compound 122 (3330 mg,13.126 mmol) was dissolved in 20% sodium n-butoxide (25 mL) at 23 ℃ and under dry nitrogen and the temperature was raised to 100 ℃. After 1.5h, the solvent was removed in vacuo. The residue was dissolved in DCM (30 mL), washed with half-saturated sodium bicarbonate (50 mL) and brine (50 mL), and dried over MgSO 4 Drying and filtering. The organic solvent was removed in vacuo. The residue was purified by flash chromatography using a gradient of 1% to 4% MeOH in DCM to give compound 138 (2687 mg,9.224mmol, 70%). MS M/z 292 (M+H) + 。
8-bromo-2-butoxy-9- (tetrahydro-2H-pyran-2-yl) -9H-purin-6-amine (139): to a solution of compound 138 (2687 mg,9224 mmol) in DCM (50 ml) was added N-bromosuccinimide (2000 mg,11069 mmol) at 23 ℃. After 1h, saturated sodium thiosulfate (20 mL) was added to the mixture. The material was extracted with DCM (20 ml). The organic layer was washed with saturated sodium bicarbonate (50 mL) and brine (50 mL), and dried over MgSO 4 Drying and filtering. The organic solvent was removed in vacuo. The residue was purified by flash chromatography using a gradient of 20% to 70% EtOAc/hexanes to give compound 139 (2517 mg,6.799mmol, 74%). MS M/z 371 (M+H) + 。
2-butoxy-8-methoxy-9H-purin-6-amine (140): compound 139 (2517 mg,6.799 mmol) was dissolved in 25% sodium methoxide (20 mL,42 mmol) at 23℃under dry nitrogen. After the addition, the temperature was raised to 70 ℃. After 2.5h, the mixture was concentrated in vacuo, dissolved in EtOAc (100 mL), washed with water (100 mL) and brine (100 mL) and concentrated over MgSO 4 Drying and filtering. The organic layer was collected and evaporated in vacuo. To the residue were added MeOH (10 mL) and TFA (3 mL). After 48h TFA was added, the solvent was removed in vacuo. The mixture was purified by preparative LC to give compound 140 (706 mg,2.984, 44%). MS M/z 238 (M+H) + 。
(4- ((6-amino-2- (butylamino) -8-methoxy-9H-purin-9-yl) methyl) phenyl) methanol (141): to a solution of compound 140, tfa salt (25 mg,0.054 mmol) in DMF (2 mL) was added potassium carbonate (20 mg,0.524 mmol) and (4-hydroxymethyl) benzyl chloride (11 mg,0.070 mmol) and stirred at 50 ℃. After 2h, the solvent was concentrated. To the residue was added water, and the mixture was then extracted with DCM (50 mL). The organic layer was washed with water (10 mL) and brine (20 mL), then over MgSO 4 Drying and filtering. The solvent was removed in vacuo. The mixture was purified by preparative LC to give compound 141 as TFA salt (29 mg,0.042mmol, 77%). MS M/z 357 (M+H) + 。
6-amino-2-butoxy-9- (4- (chloromethyl) benzyl) -7H-purin-8 (9H) -one (142): to compound 141 (607 mg,1.037 mmol) was added dichloromethane (10 mL). Thionyl chloride (1000 μl) was added to the resulting suspension and the mixture was stirred at 50deg.C for 3 hours. Toluene (30 mL) was added to the mixture and the solvent was evaporated. Toluene (100 mL) was added again to the residue, and the solvent was distilled off and dried under reduced pressure to obtain compound 142 (402 mg,1.111mmol, quantitative). MS M/z 362 (M+H) + 。
Tert-butyl 2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethylcarbamate (143): to a solution of compound 142 (166 mg,0.384 mmol) and 4- (2-boc-aminoethyl) -piperidine (180 mg,0.788 mmol) in DMF (2 mL) was added DIEA (1000. Mu.L, 5.741 mmol) and the temperature was raised to 80DEG C. After 3.5h, the solvent was removed in vacuo. The mixture was purified by preparative LC to obtain compound 143 (205 mg,0.229mmol, 29%). MS M/z 554 (M+H) + 。
6-amino-9- (4- ((4- (2-aminoethyl) piperidin-1-yl) methyl) benzyl) -2-butoxy-7H-purin-8 (9H) -one (144): compound 143 (41 mg,0.052 mmol) was dissolved in DCM (2 mL) and TFA (1 mL). After 5min, the solvent was removed in vacuo. Toluene (5 ml) was added to the residue and evaporated in vacuo. The residue was dried on a high vacuum pump to give compound 144 as TFA salt (41 mg,0.052mmol, quantitative). MS M/z 454 (M+H) + 。
N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7H-purin-9 (8H) -yl) methyl) benzyl) piperidin-4-yl) ethyl) -2- (aminooxy) acetamide (145): compound 145 was prepared in a similar procedure as described for 127, using compound 144 as starting material, to obtain the target compound 145 (15 mg,0.015mmol, 87%). MS M/z 527 (M+H) + 。
N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7H-purin-9 (8H) -yl) methyl) benzyl) piperidin-4-yl) ethyl) -3- (2- (aminooxy) ethoxy) acrylamide (146): compound 146 was prepared in a similar procedure as described for 137 using compound 144 as starting material to obtain the target compound 146 (16 mg,0.015mmol, 87%). MS M/z 585 (M+H) + 。
Tert-butyl 2- (1- (4- ((6-amino-2-butoxy-8-oxo-7H-purin-9 (8H) -yl) methyl) benzyl) piperidin-4-yl) ethylcarbamate (147): compound 147 was prepared in a similar procedure as described for 143, using compound 142 and tert-butyl 6-aminohexylcarbamate as starting materials, to obtain the target compound 147 (23 mg,0.026mmol, 14%). MS M/z 542 (M+H) + 。
6-amino-9- (4- ((6-aminohexylamino) methyl) benzyl) -2-butoxy-7H-purin-8 (9H) -one (148): compound 148 was prepared in a similar procedure as described for 144 using compound 147 as starting material to obtain the target compound 148 (24 mg,0.027mmol, quantitative). MS M/z 442 (M+H) + 。
N- (6- (4- ((6-amino-2-butoxy-8-oxo-7H-purin-9 (8H) -yl) methyl) benzylamino) hexyl) -2- (aminooxy) acetamide (149): compound 149 was prepared in a similar procedure to that described for 127 using compound 148 as starting material to obtain the target compound 149 (7 mg,0.008mmol, 31%). MS M/z 515 (M+H) + 。
N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7H-purin-9 (8H) -yl) methyl) benzyl) piperidin-4-yl) ethyl) -3- (2- (aminooxy) ethoxy) propionamide (150): to a solution of compound 144 (14 mg,0.018 mmol) and N-Boc-N, 2-dimethyl-alanine (5.5 mg, 0.020mmol) in DMF (1 mL) was added DMTMMT (5 mg,0.021 mmol) and DIEA (20. Mu.L, 0.115 mmol) at 23 ℃. After 30min, the mixture was purified by preparative LC to give compound 150 (7 mg,0.007mmol, 37%). MS M/z653 (M+H) + 。
N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7H-purin-9 (8H) -yl) methyl) benzyl) piperidin-4-yl) ethyl) -2-methyl-2- (methylamino) acrylamide (151): compound 151 was prepared in a similar procedure as described for 144, using compound 150 as starting material, to obtain the target compound 151 (5.5 mg,0.005mmol, quantitative). MS M/z 553 (M+H) + 。
N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7H-purin-9 (8H) -yl) methyl) benzyl) piperidin-4-yl) ethan-yl)Radical) pivalamide (152): to a solution of compound 144 (14 mg,0.018 mmol) and trimethylacetyl chloride (2.8 μl,0.022 mmol) in DMF (1 mL) was added DIEA (20 μl,0.115 mmol) at 23 ℃. After 1h, the mixture was purified by preparative LC to give compound 152 (7 mg,0.008mmol, 36%). MS M/z538 (M+H) + 。
N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7H-purin-9 (8H) -yl) methyl) benzyl) piperidin-4-yl) ethyl) acetamide (153): to a solution of compound 144 (20 mg,0.022 mmol) and acetic anhydride (2.1 μl,0.021 mmol) in DMF (1 mL) was added DIEA (20 μl,0.115 mmol) at 23 ℃. After 1h, the mixture was purified by preparative LC to give compound 153 (8 mg,0.010mmol, 43%). MS M/z 496 (M+H) + 。
4-amino-N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7H-purin-9 (8H) -yl) methyl) benzyl) piperidin-4-yl) ethyl) -3, 5-difluorobenzamide (154): compound 154 was prepared in a similar procedure as described for 150, using compound 144 and 4-amino-3, 5-difluorobenzoic acid as starting materials, to obtain the target compound 154 (8 mg,0.008mmol, 38%). MS M/z 609 (M+H) + 。
N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7H-purin-9 (8H) -yl) methyl) benzyl) piperidin-4-yl) ethyl) isobutyramide (155): compound 155 was prepared in a similar procedure to that described for 150 using compound 144 and isobutyric acid as starting materials to obtain compound 155 (6 mg, 0.0070 mmol, 32%). MS M/z 524 (M+H) + 。
1, 7-bis (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7H-purin-9 (8H) -yl) methyl) benzyl) piperidin-4-yl) ethylamino) -1, 7-dioxohept-4-ylcarbamic acid tert-butyl ester (156): compound 156 was prepared in a similar procedure as described for 150 using compound 144 and 4- (N-Boc-amino) -1, 6-heptanedioic acid as starting materials to obtain the target compound 156 (15 mg,0.009mmol, 34%). MS M/z 1147 (M+H) + 。
4-amino-N1, N7-bis (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7H-purin-9 (8H) -yl) methyl) benzyl) piperidin-4-yl) ethyl) pimelic acid amide (157): compound 157 was prepared in a similar procedure as described for 144, using compound 156 as starting material, to give the target compound 157 (15 mg,0.01mmol, quantitative). MS M/z 1047 (M+H) + 。
N1, N7-bis (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7H-purin-9 (8H) -yl) methyl) benzyl) piperidin-4-yl) ethyl) -4- (2- (aminooxy) acetamido) pimelamide (158): compound 158 was prepared in a similar procedure to that described for 145 using compound 157 and 2, 5-dioxopyrrolidin-1-yl 2- (tert-butoxycarbonylaminooxy) acetate as starting materials to give the title compound 158 (5 mg,0.003mmol, 34%). MS M/z 1120 (M+H) + 。
(S) -2-amino-N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7H-purin-9 (8H) -yl) methyl) benzyl) piperidin-4-yl) ethyl) propionamide (173): to a solution of compound 144 (20 mg,0.022 mmol) and N-Boc-alanine (5 mg,0.026 mmol) in DMF (1 mL) was added DMTMT (6 mg,0.025 mmol) and DIEA (20. Mu.L, 0.115 mmol) at 23 ℃. After 10min, the solvent was removed in vacuo. To the residue was added DCM (1 ml) and TFA (1 ml). After 10min, the solvent was removed in vacuo and the residue was passed through Preparative LC purification to give compound 173 (8 mg, 0.09 mmol, 42%). MS M/z525 (M+H) + 。
(S) -2-amino-N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7H-purin-9 (8H) -yl) methyl) benzyl) piperidin-4-yl) ethyl) -5-guanidinopentanamide (174): compound 174 was prepared in a similar procedure as described for 173 using compound 144 and N-Boc-arginine as starting materials to give the target compound 174 (10 mg,0.01 mmol, 48%). MS M/z 610 (M+H) + 。
4- (2- (isobutylamino) ethyl) piperidine-1-carboxylic acid tert-butyl ester (175): 4- (2-aminoethyl) -1-Boc-piperidine (520 mg,2.278 mmol) and isobutyraldehyde (230. Mu.L, 3.190 mmol) were dissolved in methanol (10 ml) at 23 ℃. After 2h, sodium borohydride (142 mg,3.754 mmol) was added to the mixture. After 10min, the solvent was removed in vacuo. The residue was dissolved in DCM (100 mL) and saturated NaHCO 3 (50 mL) and brine (50 mL) over MgSO 4 Drying and filtering. The solvent was removed in vacuo. The residue was purified by preparative LC to give compound 175 (499 mg,1.755mmol, 55%) as a clear colorless solid. MS M/z285 (M+H) + 。
4- (2- (3- (2- (1, 3-dioxoisoindolin-2-yloxy) ethoxy) -N-isobutylpropionamido) ethyl) piperidine-1-carboxylic acid tert-butyl ester (176): to a solution of compound 175 (80 mg,0.201 mmol) and Phth-PEG1-COOH (56 mg,0.201 mmol) in EtOAc (10 ml) were added CMPI (62 mg,0.243 mmol) and DIEA (70. Mu.L, 0.402 mmol) at 23 ℃. After 3h, the precipitate was removed by filtration, and the filtrate was purified by flash chromatography to give compound 176 (65 mg,0.119mmol, 59%) as a white solid. MS M/z 546 (M+H) + 。
3- (2- (1, 3-dioxoisoindolin-2-yloxy)) Ethoxy) -N-isobutyl-N- (2- (piperidin-4-yl) ethyl) propionamide (177): compound 177 was prepared in a similar procedure as described for 144, using compound 176 as starting material, to obtain the target compound 177 (66 mg,0.118mmol, quantitative). MS M/z 446 (M+H) + 。
N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7H-purin-9 (8H) -yl) methyl) benzyl) piperidin-4-yl) ethyl) -3- (2- (aminooxy) ethoxy) -N-isobutylpropionamide (178): compound 178 was prepared using compound 177 and compound 142 as starting materials, following a procedure similar to that described for 143, with treatment of hydrazine hydrate (10 μl) described for 130 to give compound 178 (19 mg,0.019mmol, 7%). MS M/z 641 (M+H) + 。
6-amino-2-butoxy-9- (4- (piperidin-1-ylmethyl) benzyl) -7H-purin-8 (9H) -one (179): compound 179 was prepared in a similar procedure as described for 143, using compound 142 and piperidine as starting materials, to give compound 179 (31 mg,0.041mmol, 54%). MS M/z 411 (M+H) + 。
4-amino-N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7H-purin-9 (8H) -yl) methyl) benzyl) piperidin-4-yl) ethyl) -3-methoxybenzamide (180): compound 180 was prepared in a similar procedure as described for 150, using compound 144 and 4-amino-3-methoxybenzoic acid as starting materials, to obtain the title compound 180 (8 mg,0.008mmol, 39%). MS M/z 603 (M+H) + 。
(S) -N- (2- (1- (4- ((6-amino-2-butan) 2)oxy-8-oxo-7H-purin-9 (8H) -yl) methyl) benzyl piperidin-4-yl) ethyl) -2- (2- (aminooxy) acetamido) acrylamide (181): compound 181 was prepared in a similar procedure to that described for 127 using compound 173 and 2, 5-dioxopyrrolidin-1-yl 2- (tert-butoxycarbonylaminooxy) acetate as starting materials to give the title compound 181 (5 mg,0.005mmol, 58%). MS M/z 598 (M+H) + 。
(S) -N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7H-purin-9 (8H) -yl) methyl) benzyl) piperidin-4-yl) ethyl) -2- (2- (aminooxy) acetamido) -5-guanidinopentanamide (182): compound 182 was prepared in a similar procedure to that described for 127 using compound 174 and 2, 5-dioxopyrrolidin-1-yl 2- (tert-butoxycarbonylaminooxy) acetate as starting materials to give the title compound 182 (5 mg, 0.04 mmol, 42%). MS M/z 683 (M+H) + 。
9- (4, 4' -bipiperidin-1-ylmethyl) benzyl) -6-amino-2-butoxy-7H-purin-8 (9H) -one (183): compound 183 was prepared in a similar procedure to that described for 143 using compound 142 and 4,4' -bipiperidine as starting materials to give compound 183 (13 mg,0.016mmol, 7%). MS M/z 494 (M+H) + 。
6-amino-9- (4- ((1 '- (2- (aminooxy) acetyl) -4,4' -bipiperidin-1-yl) methyl) benzyl) -2-butoxy-7H-purin-8 (9H) -one (184): prepared in a similar procedure to that described for 127 using compound 183 and 2, 5-dioxopyrrolidin-1-yl 2- (tert-butoxycarbonylaminooxy) acetate as starting materialsCompound 184 to obtain the target compound 184 (5 mg,0.005mmol, 18%). MS M/z 567 (M+H) + 。
3-amino-N- (2- (1- (4- ((4-amino-6-butoxy-2-oxo-2, 3-dihydro-1H-imidazo [4, 5-c))]Pyridin-1-yl) methyl) benzyl) piperidin-4-yl) ethyl) benzamide (185): compound 185 was prepared in a similar procedure as described for 150, using compound 144 and 3-aminobenzoic acid as starting materials, to obtain the target compound 185 (8 mg, 0.09 mmol, 53%). MS M/z 572 (M+H) + 。
N- (2- (1- (4- ((4-amino-6-butoxy-2-oxo-2, 3-dihydro-1H-imidazo [4, 5-c))]Pyridin-1-yl) methyl) piperidin-4-yl) ethyl) -4- (2-aminoethyl) benzamide (186): compound 186 was prepared in a similar procedure as described for 173 using compound 144 and 4- (2-Boc-amino) ethylbenzoic acid as starting materials to give the target compound 186 (9 mg,0.010mmol, 58%). MS M/z 600 (M+H) + 。
4-amino-N- (2- (1- (4- ((4-amino-6-butoxy-2-oxo-2, 3-dihydro-1H-imidazo [4, 5-c))]Pyridin-1-yl) methyl) benzyl) piperidin-4-yl) ethyl) benzamide (187): compound 187 was prepared in a similar procedure as described for 150, using compound 144 and 4-aminobenzoic acid as starting materials to give the title compound 187 (8 mg, 0.09 mmol, 53%). MS M/z 572 (M+H) + 。
3-amino-N- (2- (1- (4- ((4-amino-6-butoxy-2-oxo-2, 3-dihydro-1H-imidazo [4, 5-c))]Pyridin-1-yl) methyl) benzyl) piperidin-4-yl) ethyl) -4-fluorobenzamide (188): compound 188 was prepared in a similar procedure as described for 150, using compound 144 and 3-amino-4-fluorobenzoic acid as starting materials, to obtain the target compound 188 (11 mg,0.01 mmol, 64%). MS M/z 590 (M+H) + 。
N- (2- (1- (4- ((4-amino-6-butoxy-2-oxo-2, 3-dihydro-1H-imidazo [4, 5-c))]Pyridin-1-yl) methyl) piperidin-4-yl) ethyl) -4- (2- (2- (aminooxy) acetamido) ethyl) benzamide (189): compound 189 was prepared in a similar procedure as described for 127 using compound 186 and 2, 5-dioxopyrrolidin-1-yl 2- (tert-butoxycarbonylaminooxy) acetate as starting materials to give the target compound 189 (11 mg,0.010mmol, 94%), MS M/z 673 (m+h) + 。
6-amino-9- (4- ((4- (4-aminophenyl) piperidin-1-yl) methyl) benzyl) -2-butoxy-7H-purin-8 (9H) -one (190): compound 190 was prepared in a similar procedure as described for 143, using compound 142 and 4- (4-aminophenyl) -piperidine as starting materials, to give compound 190 (3 mg, 0.04 mmol, 5%). MS M/z 502 (M+H) + 。
6-amino-9- (4- ((1' - (3- (2- (aminooxy) ethoxy) propionyl) -4, 4) ’ -bipiperidin-1-yl) methyl) benzyl) -2-butoxy-7H-purin-8 (9H) -one (191): compound 183 was used as starting materialMaterial, compound 191 was prepared in a similar procedure as described for 137 to give the title compound 191 (13 mg,0.012mmol, 48%). MS M/z 625 (M+H) + 。
N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -4-hydroxybenzoamide (213): compound 213 was prepared in a similar procedure as described for 150, using compound 144 and 4-hydroxybenzoic acid as starting materials, to obtain the target compound 213 (10 mg,0.01 mmol, 66%). MS M/z 573 (M+H) + 。
N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -3- (4-hydroxyphenyl) propanamide (214): compound 214 was prepared in a similar procedure as described for 150, using compound 144 and 3- (4-hydroxyphenyl) propionic acid as starting materials, to obtain the title compound 214 (9 mg,0.010mmol, 58%). MS M/z 602 (M+H) + 。
Boc-Lys (Boc-aminooxyacetyl) -OH (215); to 2, 5-dioxopyrrolidin-1-yl 2- (tert-butoxycarbonylaminooxy) acetate (399 mg, 1.284 mmol) and Boc-Lys-OH (335 mg,1.360 mmol) in DMF (5 mL) was added DIEA (750. Mu.L, 4.306 mmol) at 23 ℃. After 2h, the solvent was removed in vacuo. The residue was dissolved in EtOAc (50 mL) and washed with 1N HCl (50 mL) and brine (20 mL). The organic layer was dried over MgSO 4 Drying and then filtering. The solvent was removed in vacuo. The residue was purified by flash chromatography to give compound 215 (480 mg,1.144mmol, 83) as a white solid％)。MS m/z 420(M+H) + 。
(S) -2-amino-N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -6- (2- (aminooxy) acetamido) hexanamide (216): compound 216 was prepared in a similar procedure to that described for 173, using compound 144 and compound 215 as starting materials, to obtain the title compound 216 (6 mg,0.006mmol, 37%). MS M/z 655 (M+H) + 。
(S) -N- (5-amino-6- (1' - (4- ((4-amino-6-butoxy-2-oxo-2, 3-dihydro-1H-imidazo [4, 5-c))]Pyridin-1-yl) methyl) benzyl) -4,4' -bipiperidin-1-yl) -6-oxohexyl) -2- (aminoxy) acetamide (217): compound 217 was prepared in a similar procedure to that described for 173, using compound 183 and compound 215 as starting materials, to obtain the title compound 217 (6 mg,0.006mmol, 37%). MS M/z 696 (M+H) + 。
5-amino-N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) nicotinamide (218): compound 218 was prepared in a similar procedure as described for 150 using compound 144 and 5-aminonicotinic acid as starting materials to give the title compound 218 (1 mg,0.001mmol, 7%). MS M/z574 (M+H) + 。
5-amino-N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) pyrazine-2-carboxamide (219): compound 144 and 5-amino-pyrazine-carboxylic acid were used as starting materialsCompound 219 was prepared in a similar procedure as described for 150 to give the target compound 219 (10 mg,0.11mmol, 66%). MS M/z 575 (M+H) + 。
6-amino-2-butoxy-9- (4- ((1 '- (4-hydroxybenzoyl) - [4,4' -bipiperidine)]-1-yl) methyl) benzyl) -7, 9-dihydro-8H-purin-8-one (220): compound 220 was prepared in a similar procedure as described for 150, using compound 183 and 4-hydroxybenzoic acid as starting materials, to obtain the target compound 220 (10 mg,0.01 mmol, 69%). MS M/z 614 (M+H) + 。
(S) -2-amino-N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -3- (4-aminophenyl) propanamide (221): compound 221 was prepared in a similar procedure as described for 173, using compound 144 and Boc-L-4-aminophenylalanine as starting materials, to give the title compound 221 (15 mg,0.014mmol, 85%). MS M/z 616 (M+H) + 。
6-amino-9- (4- ((1 '- (5-aminopyrazine-2-carbonyl) -4,4' -bipiperidin-1-yl) methyl) benzyl) -2-butoxy-7H-purin-8 (9H) -one (222): compound 222 was prepared in a similar procedure as described for 150, using compound 183 and 5-amino-pyrazinecarboxylic acid as starting materials, to obtain the target compound 222 (11 mg,0.012mmol, 73%). MS M/z615 (M+H) + 。
(S) -2-amino-N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -3- (4- (azidomethyl) phenyl) acrylamide (223): compound 223 was prepared in a similar procedure as described for 173, using compound 144 and Boc-L-4-azidomethylphenylalanine as starting materials, to give the target compound 223 (11 mg,0.010mmol, 90%). MS M/z 656 (M+H) + 。
(S) -2-amino-N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -6-azidohexanamide (224): compound 224 was prepared in a similar procedure as described for 173 using compound 144 and Boc-L-azido lysine as starting materials to give the target compound 224 (12 mg,0.01 mmol, 93%). MS M/z 608 (M+H) + 。
(S) -methyl 4- (2- (tert-butoxycarbonylamino) propionylamino) benzoate (225): EDCI (960 mg,5.008 mmol) and HOBt (450 mg,3.330 mmol) were added to a solution of Boc-Ala-OH (625 mg,3.303 mmol) in DMF (5 ml) at 0deg.C. After 30min, methyl-4-aminobenzoate (500 mg,3.307 mmol) and DMAP (410 mg,3.356 mmol) were added to the mixture at 23 ℃. After 20h, the solvent was reduced to about 1ml by rotary evaporation and diluted with 50ml EtOAC. The solution was washed with 1N HCl (50 ml), saturated sodium bicarbonate (50 ml) and brine (50 ml), and dried over MgSO 4 Drying and filtering. The organic solvent was removed in vacuo. The residue was purified by flash chromatography to give compound 225 (180 mg,0.558mmol, 17%) as a white solid. MS M/z 323 (M+H) + 。
4- (2-aminopropionylamino) benzoic acid (S) -methyl ester (226): at 23 deg.C, toTo a solution of compound 225 (180 mg, 0.5538 mmol) in DCM (1 ml) was added TFA (1 ml). After 20min, the solvent was removed in vacuo. Toluene (5 ml) was added to the residue and evaporated in vacuo. The residue was dried overnight in a high vacuum pump to give compound 226 as a brown TFA salt (200 mg,<0.595mmol, quantitative). MS M/z 223 (M+H) + 。
Methyl 4- ((S) -2- (tert-butoxycarbonylamino) -3-methylbutanoylamino) propionylamino) benzoate (227): to a solution of compound 226 (200 mg,0.595 mmol) and Boc-Val-OH (130 mg,0.598 mmol) in DCM (10 ml) was added DMTMMT (170 mg, 0.704 mmol) and DIEA (350. Mu.L, 2.009 mmol) at 23 ℃. After 15min, the solvent was removed in vacuo and the residue was dissolved in EtOAc (50 ml), washed with 1N HCl (50 ml), saturated sodium bicarbonate (50 ml) and brine (20 ml). The organic layer was dried over MgSO 4 Drying and filtering. The solvent was removed by rotary evaporation. The residue was purified by flash chromatography to obtain compound 227 (202 mg,0.479mmol, 81%) as a white solid. MS M/z 422 (M+H) + 。
4- ((S) -2- (tert-butoxycarbonylamino) -3-methylbutanoylamino) propionylamino) benzoic acid (228): to a solution of compound 227 (202 mg,0.479 mmol) in MeOH (10 mL) and water (1 mL) was added LiOH (24 mg, 1.002mmol) at 23 ℃. After 24h, the solvent was removed in vacuo and the residue was dissolved in EtOAc (50 ml) and washed with 1N HCl (50 ml) and brine (20 ml). The organic layer was dried over MgSO 4 Drying and filtering. The solvent was removed by rotary evaporation. The residue was purified by flash chromatography to give compound 228 (96 mg,0.236mmol, 49%) as a white solid. MS M/z 408 (M+H) + 。
N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -4- ((S) -2-amino-3-methylbutanoylamino) propionylamino) benzamide (229): compound 229 was prepared in a similar procedure to that described for 173 using compound 144 and compound 228 as starting materials to give the title compound 229 (14 mg,0.012mmol, 97%). MS M/z 743 (M+H) + 。
N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9)H-purin-9-yl) methyl) piperidin-4-yl) ethyl) -4- ((S) -2- (2- (aminooxy) acetamido) -3-methylbutanoylamino) propionylamino) benzamide (230): to a solution of compound 229 (14 mg,0.012 mmol) and 2, 5-dioxopyrrolidin-1-yl 2- (tert-butoxycarbonylaminooxy) acetate (4 mg,0.014 mmol) in DMF (1 ml) was added DIEA (30 μl,0.172 mmol) at 23deg.C. After 10min, the solvent was removed in vacuo, and DCM (1 ml) and TFA (1 ml) were added to the residue. After 10min, the solvent was removed in vacuo. The residue was purified by preparative LC to obtain the target compound 230 (11 mg, 0.09 mmol, 74%). MS M/z 816 (M+H) + 。
5-amino-6- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7H-purin-9 (8H) -yl) methyl) benzyl) piperidin-4-yl) ethylamino) -6-oxohexylcarbamic acid (S) - (9H-fluoren-9-yl) methyl ester (231): compound 231 was prepared in a similar procedure as described for 173 using compound 144 and Boc-Lys (Fmoc) -OH as starting materials to give the title compound 231 (85 mg,00.074mmol, 67%). MS M/z 804 (M+H) + 。
((S) -1- (((S) -6-amino-1- ((2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) amino) -1-oxohex-2-yl) amino) -1-oxopropan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) carbamic acid tert-butyl ester (232): to a solution of compound 231 (24 mg,0.019 mmol) and Boc-Val-Ala-OH (6 mg,0.021 mmol) in DMF (1 ml) was added DMTMT (7 mg,0.029 mmol) and DIEA (30. Mu.L, 0.172 mmol) at 23 ℃. After 10min piperidine (100 μl) was added to the mixture. After 10min, the mixture was purified by preparative LC to give compound 232 (24 mg,0.018mmol, 96%) as a light brown solid.
(S) -N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -2- ((S) -2- ((S) -2-amino-3-methylbutanoylaminopropionamide) -6- (2- (aminooxy) acetamido) hexanamide(233): compound 233 was prepared in a similar procedure as described for 230 using compound 232 and 2, 5-dioxopyrrolidin-1-yl 2- (tert-butoxycarbonylaminoxy) acetate as starting materials to give the title compound 233 (22 mg,0.016mmol, 79%). MS M/z 825 (M+H) + 。
(S) -6-amino-N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -2-PEG 24-acylaminohexanamide (234): to a solution of compound 231 (11 mg,0.0109 mmol) and PEG24-NHS (12 mg, 0.010mmol) in DMF (1 ml) was added DIEA (12. Mu.L, 0.069 mmol) at 23 ℃. After 20min piperidine (50 μl) was added to the mixture. After 10min, the mixture was purified by preparative LC to give compound 234 (18 mg,0.008mmol, 96%) as a clear solid. MS M/z 1682 (M-H) + 。
(S) -N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -6- (2- (aminooxy) acetamido) -2-PEG 24-acylaminohexanamide (235): compound 235 was prepared in a similar procedure to that described for 230 using compound 234 and 2, 5-dioxopyrrolidin-1-yl 2- (tert-butoxycarbonylaminooxy) acetate as starting materials to give the title compound 235 (13 mg,0.006mmol, 66%). MS M/z 1753 (M-H) + 。
(S) -1- ((S) -1- ((S) -6-amino-1- (2- (1- (4- ((4-amino-6-butoxy-2-oxo-2, 3-dihydro-1H-imidazo [4, 5-c))]Pyridin-1-yl) methyl) benzyl) piperidin-4-yl) ethylamino) -1-oxohex-2-ylamino) -1-oxoprop-2-ylamino) -3-methyl-1-oxobut-2-ylcarbamic acid tert-butyl ester (236): prepared in a similar procedure as described for 232 using compound 231 and Boc-Val-Ala-PABC-PNP as starting materialsCompound 236 to obtain the target compound 236 (18 mg,0.012mmol, 71%). MS M/z 1001 (M+H) + 。
((S) -17- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) -1- (9H-fluoren-9-yl) -3,7, 14-trioxo-2, 5-dioxa-4,8,15-triazaheptadec-n-13-yl) carbamic acid 4- ((S) -2-amino-3-methylbutanoylamino) propionylamino) benzyl ester (237): compound 237 was prepared in a similar procedure as described for 173, using compound 236 and 2- (((9H-fluoren-9-yl) methoxy) carbonylaminooxy) acetic acid as starting materials, to obtain the target compound 237 (19 mg,0.012mmol, 93%). MS M/z 1197 (M+H) + 。
((S) -1- ((2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) amino) -6- (2- (aminooxy) acetamido) -1-oxohex-2-yl) carbamic acid 4- ((S) -2- ((S) -3-methyl-2-PEG 24-acylaminobutyrylamino) propionylamino) benzyl ester (238): compound 238 was prepared by a procedure similar to that described for 234 using compound 237 and PEG24-NHS as starting materials to obtain the target compound 238 (8 mg,0.003mmol, 27%) MS M/z 1037 (m+2h) + 。
((S) -1- ((2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) amino) -6- (2- (aminooxy) acetamido) -1-oxohex-2-yl) carbamic acid 4- ((S) -2- ((S) -2-acetamido-3-methylbutanoylamino) propionylamino) benzyl ester (239): compound 239 was prepared in a similar procedure as described for 234, using compound 237 and acetic anhydride as starting materials, to give the target compound 239 (6 mg, 0.04 mmol, 71%) MS M/z 1016 (m+h) + 。
(S) -2-amino-N- (2- (1- (4- ((4-amino-6-butoxy-2-oxo-2, 3-dihydro-1H-imidazo [4, 5-c))]Pyridin-1-yl) methyl) benzyl) piperidin-4-yl) ethyl) -3-azidopropionamide (240): compound 240 was prepared in a similar procedure as described for 173 using compound 144 and Boc-L-azidoalanine as starting materials to give the target compound 240 (10 mg,0.010mmol, 89%). MS M/z 566 (M+H) + 。
(S) -2- (tert-butoxycarbonylamino) -3- (4- ((4- ((tert-butoxycarbonylaminooxy) methyl) -1H-1,2, 3-triazol-1-yl) methyl) phenyl) propanoic acid (241): to a solution of Boc-L-azidomethyl-phenylalanine (120 mg,0.375 mmol) and tert-butyl prop-2-ynyloxy carbamate (70 mg, 0.169 mmol) in DCM (1 mL) was added CuBr (55 mg,0.383 mmol) at 23 ℃. After 2 hours, the mixture was purified by preparative LC to give compound 241 (9 mg,0.018mmol, 5%) as a white solid. MS M/z 492 (M+H) + 。
(S) -2-amino-N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -3- (4- ((4- ((aminooxy) methyl) -1H-1,2, 3-triazol-1-yl) methyl) phenyl) propanamide (242): to a solution of compound 144 (8 mg, 0.09 mmol) and compound 241 (9 mg,0.018mmol, crude) in DMF (1 mL) was added DMTMT (5 mg,0.021 mmol) and DIEA (12. Mu.L, 0.069 mmol) at 23 ℃. After 10min, the solvent was removed in vacuo, and DCM (1 mL) and TFA (1 mL) were added to the residue. After 10min LCMS showed deprotection was complete. The mixture was purified by preparative LC to give compound 242 (6 mg, 0.04 mmol, 71%), MS M/z 725 (M-H) + 。
(S) -2- (tert-Butoxycarbonylamino) -3- (4- ((1, 3-dioxoisoindolin-2-yloxy) methyl) -1H-)1,2, 3-triazol-1-yl) propionic acid (243): compound 243 was prepared in a similar procedure to that described for 242 using Boc-L-azido-Ala-OH and 2-prop-2-ynyloxy isoindoline-1, 3-dione as starting materials to give the title compound 243 (130 mg,0.238mmol, 84%). MS M/z 432 (M+H) + 。
(S) -2-amino-N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -3- (4- ((aminooxy) methyl) -1H-1,2, 3-triazol-1-yl) propanamide (244): to a solution of compound 144 (10 mg,0.01 mmol) and compound 243 (7 mg,0.013 mmol) in DMF (1 mL) was added DMTMMT (5 mg,0.021 mmol) and DIEA (12. Mu.L, 0.069 mmol) at 23 ℃. After 10min, the solvent was removed in vacuo, and DCM (1 mL) and TFA (1 mL) were added to the residue. After 15min, the solvent was removed in vacuo, and DCM (1 mL) and hydrazine hydrate (0.1 mL) were added to the residue. After 2h, the solvent was removed in vacuo and the residue was purified by preparative LC to give compound 244 (10 mg,0.010mmol, 89%) as a colorless transparent solid. MS M/z 637 (M+H) + 。
(S) -2-amino-N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -3-hydroxypropionamide (245): compound 245 was prepared in a similar procedure as described for 242 using Boc-L-Ser-OH and compound 144 as starting materials to obtain the target compound 245 (7 mg, 0.0070 mmol, 64%). MS M/z 541 (M+H) + 。
(S) -2-amino-N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -3- (4-hydroxyphenyl) propanamide (246): boc-L-Tyr-OH and Compound 144 were used as starting materials in a similar procedure as described for 242Compound 246 was prepared sequentially to obtain the target compound 246 (8 mg, 0.0070 mmol, 68%). MS M/z 617 (M+H) + 。
(S) -2- (tert-butoxycarbonylamino) -6- (4- ((1, 3-dioxoisoindolin-2-yloxy) methyl) -1H-1,2, 3-triazol-1-yl) hexanoic acid (247): compound 247 was prepared in a similar procedure to that described for 241 using Boc-L-azido-Lys-OH and 2-prop-2-ynyloxy isoindoline-1, 3-dione as starting materials to give the title compound 247 (57 mg,0.097mmol, 53%). MS M/z 474 (M+H) + 。
(S) -2-amino-N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -6- (4- ((aminooxy) methyl) -1H-1,2, 3-triazol-1-yl) hexanamide (248): compound 248 was prepared in a similar procedure as described for 244, using compound 144 and compound 247 as starting materials, to obtain the target compound 248 (10 mg,0.010mmol, 89%). MS M/z 679 (M+H) + 。
(S) - (2- ((5-amino-6- ((2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) amino) -6-oxohexyl) amino) -2-oxoethoxy) carbamic acid tert-butyl ester (249): to a solution of 2, 5-dioxopyrrolidin-1-yl 2- (((tert-butoxycarbonyl) amino) oxy) acetate (380 mg,1.318 mmol) and Fmoc-L-Lys-OH (444 mg,1.205 mmol) in DMF (5 mL) was added DIEA (660. Mu.L, 3.789 mmol) at 50deg.C. After 1h, the solvent was removed in vacuo, and the residue was dissolved in EtOAc (50 mL) and washed with 1N HCl (50 mL) and brine (20 mL). The organic layer was dried over MgSO 4 Dried and then filtered. The solvent was removed in vacuo. The residue was purified by flash chromatography using a MeOH/DCM gradient (0-10%) to obtain compound 249 (466 mg,0.860 m) mol，65％)。MS m/z 542(M+H) + 。
(S) - (2- ((5-amino-6- ((2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) amino) -6-oxohexyl) amino) -2-oxoethoxy) carbamic acid tert-butyl ester (250): to a solution of compound 144 (180 mg,0.198 mmol) and compound 249 (105 mg,0.194 mmol) in DMF (2 mL) was added DMTMMT (68 mg,0.282 mmol) and DIEA (200. Mu.L, 1.148 mmol) at 23 ℃. After 10min piperidine (100 μl) was added to the mixture. After 20min, LCMS showed deprotection was complete. The mixture was purified by preparative LC to give the title compound 250 (160 mg,0.146mmol, 74%). MS M/z 755 (M+H) + 。
(S) -N1- (1- ((2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) amino) -6- (2- (aminooxy) acetamido) -1-oxohex-2-yl) -N5- (PEG 48) -glutarate-amide (251): to a solution of compound 250 (10 mg, 0.09 mmol) and PEG48-NHCO- (CH 2) 3-TFP ester (22 mg, 0.09 mmol) in DMF (0.5 mL) was added DIEA (12. Mu.L, 0.069 mmol) at 23 ℃. After 10min, the solvent was removed in vacuo. To the residue was added DCM (1 mL) and TFA (1 mL). After 10min LCMS showed deprotection was complete. The solvent was removed in vacuo. The residue was purified by preparative LC to obtain the target compound 251 (19 mg,0.006mmol, 62%). MS M/z 1449 (M+2H) + 。
(S) -2-PEG8-N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -6- (2- (aminooxy) acetamido) hexanamide (252): compound 252 was prepared in a similar procedure as described for 251, using compound 250 and mPEG8-NHS as starting materials, to obtain the target compound 252 (9 mg,0.006mmol, 66%). MS M/z 1050 (M+H) + 。
(S) -N1- (1- ((2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) amino) -6- (2- (aminooxy) acetamido) -1-oxohex-2-yl) -N5-mPEG4- (PEG 4) 3-glutarate-amide (253): compound 253 was prepared in a similar procedure as described for 251 using compound 250 and mPEG4- (m-PEG 4) 3-NHS as starting materials to give the target compound 253 (20 mg,0.008mmol, 93%). MS M/z 952 (M+2H) + 。
(S) -2-PEG4-N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -6- (2- (aminooxy) acetamido) hexanamide (254): compound 254 was prepared in a similar procedure as described for 251, using compound 250 and mPEG4-NHS as starting materials, to give the title compound 254 (9 mg, 0.0070 mmol, 74%). MS M/z 873 (M+2H) + 。
(S) -N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -6- (2- (aminooxy) acetamido) -2-PEG 12-acylaminohexanamide (255): compound 255 was prepared in a similar procedure as described for 251 using compound 250 and mPEG12-NHS as starting materials to give the target compound 255 (12 mg, 0.0070 mmol, 78%). MS M/z 1224 (M-H) + 。
(S) -N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidine-4-Ethyl) -6- (2- (aminooxy) acetamido) -2-PEG 37-acylaminohexanamide (256): compound 256 was prepared in a similar procedure as described for 251 using compound 250 and mPEG37-NHS as starting materials to give the target compound 256 (21 mg,0.008mmol, 83%). MS M/z 1164 (M+2H) + 。
(S) -N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -6- (2- (aminooxy) acetamido) -2- (4-phenylbutyrylamino) hexanamide (257): compound 257 was prepared in a similar procedure as described for 251, using compound 250 and 4-phenylbutyric acid as starting materials, to give the title compound 257 (11 mg, 0.09 mmol, 96%). MS M/z 801 (M+H) + 。
(S) -N- (1- (2- (1- (4- ((4-amino-6-butoxy-2-oxo-2, 3-dihydro-1H-imidazo [4, 5-c))]Pyridin-1-yl) methyl) piperidin-4-yl) ethylamino) -6- (2- (aminooxy) acetamido) -1-oxohex-2-yl) oleamide (258): compound 258 was prepared in a similar procedure as described for 251, using compound 250 and oleoyl chloride as starting materials, to obtain the title compound 258 (11 mg,0.008mmol, 88%). MS M/z 920 (M+H) + 。
(S) -N- (1- ((2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) amino) -6- (2- (aminooxy) acetamido) -1-oxohex-2-yl) octanamide (259): compound 259 was prepared in a similar procedure as described for 242 using compound 250 and octanoic acid as starting materials toTarget compound 259 (10 mg,0.008mmol, 89%) was obtained. MS M/z 781 (M+H) + 。
(S) -N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -6- (2- (aminooxy) acetamido) -2-dPEG4- (m-dPEG 8) 3-acylaminohexanamide (260): compound 260 was prepared in a similar procedure as described for 251 using compound 250 and dPEG4- (m-dPEG 8) 3-NHS as starting materials to give the title compound 260 (21 mg, 0.0070 mmol, 80%). MS M/z 1217 (M+2H) + 。
(S) -N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -6- (2- (aminooxy) acetamido) -2-dPEG4- (m-dPEG 12) 3-acylaminohexanamide (261): compound 261 was prepared in a similar procedure as described for 261 using compound 250 and dPEG4- (m-dPEG 12) 3-NHS as starting materials to give the title compound 261 (25 mg, 0.0070 mmol, 80%). MS M/z 988 (M+3H) + 。
(S) - (5-amino-6- ((2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) amino) -6-oxohexyl) carbamic acid (9H-fluoren-9-yl) methyl ester (262): compound 262 was prepared in a similar procedure as described for 242 using compound 144 and Boc-L-Lys (Fmoc) -OH as starting materials to give the target compound 262 (85 mg,00.074mmol, 67%). MS M/z 804 (M+H) + 。
(S) -6-amino-N- (2- (1- (4- ((6-amino-2-butoxy-8-oxy)) oxy)-7, 8-dihydro-9H-purin-9-yl) methyl-piperidin-4-yl) ethyl) -2- (2- (aminooxy) acetamido) hexanamide (263): to a solution of compound 263 (15 mg,0.015 mmol) and Fmoc-aminooxyacetate (3 mg,0.023 mmol) in DMF (2 ml) was added DMTMMT (5 mg,0.021 mmol) and DIEA (15. Mu.L, 0.086 mmol) at 23 ℃. After 10min piperidine (0.1 mL) was added to the mixture. After 10min LCMS showed deprotection was complete. The mixture was purified by preparative LC to give the target compound 263 (15 mg,0.012mmol, 94%). MS M/z 655 (M+H) + 。
2- (6-amino-1- (2- (1- (4- ((4-amino-6-butoxy-2-oxo-2, 3-dihydro-1H-imidazo [4, 5-c))]Pyridin-1-yl) methyl) benzyl) piperidin-4-yl) ethylamino) -1-oxohex-2-ylamino) -2-oxoethoxycarbamic acid (S) -tert-butyl ester (264): compound 264 was prepared in a similar procedure to that described for 250 using compound 262 and 2, 5-dioxopyrrolidin-1-yl 2- (((tert-butoxycarbonyl) amino) oxy) acetate as starting materials to give the title compound 264 (108 mg,0.089mmol, 87%). MS M/z 755 (M+H) + 。
(S) -N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -2- (2- (aminooxy) acetamido) -6-PEG 24-acylaminohexanamide (265): compound 265 was prepared in a similar procedure as described for 251, using compound 264 and mPEG24-NHS as starting materials, to obtain the target compound 265 (16 mg, 0.0070 mmol, 88%). MS M/z 1753 (M-H) + 。
(S) -N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -2- (2- (aminooxy) acetamido) -6-PEG 8-acylaminohexanamide (266): use of compound 264 And mPEG8-NHS as starting material, compound 266 was prepared in a similar procedure as described for 251 to give the target compound 266 (12 mg,0.008mmol, 97%). MS M/z 1049 (M+H) + 。
(S) -N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -2- (2- (aminooxy) acetamido) -6- (PEG 37) hexanamide (267): compound 267 was prepared in a similar procedure as described for 251, using compound 264 and mPEG37-NHS as starting materials, to obtain the target compound 267 (10 mg, 0.04 mmol, 43%). MS M/z 1164 (M+2H) + 。
(S) -N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -2- (2- (aminooxy) acetamido) -6- (dPEG 4- (m-dPEG 8) 3) hexanamide (268): compound 268 was prepared using compound 264 and dPEG4- (m-dPEG 8) 3-NHS as starting materials in a similar procedure as described for 251 to obtain the target compound 268 (20 mg, 0.0070 mmol, 84%). MS M/z 1217 (M+2H) + 。
(S) -N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -2- (2- (aminooxy) acetamido) -3- (4-hydroxyphenyl) propanamide (269): compound 269 was prepared in a similar procedure to that described for 251 using compound 246 and 2, 5-dioxopyrrolidin-1-yl 2- (((tert-butoxycarbonyl) amino) oxy) acetate as starting materials to obtain the title compound 269 (6 mg,0.005mmol, 70%). MS M/z 689 (M) +H) + 。
Tert-butyl (2- (1- (4- ((2-butoxy-6- ((butoxycarbonyl) amino) -8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) carbamate (270): to a solution of compound 143 (255 mg,0.282 mmol) and n-butyl chloroformate (50 μl,0.366 mmol) in DMF (5 mL) was added DIEA (300 μl,1.722 mmol), and the temperature was raised to 80 ℃. After 30min, the mixture was diluted with dichloromethane (50 mL), washed with half-saturated sodium bicarbonate (50 mL) and brine (50 mL), and concentrated over MgSO 4 Drying and filtering. The solvent was removed in vacuo to give crude compound 270 (160 mg, crude) as a light brown solid. The crude product was used without further purification. MS M/z 654 (M+H) + 。
9- (4- ((4- (2-aminoethyl) piperidin-1-yl) methyl) benzyl) -2-butoxy-8-oxo-8, 9-dihydro-7H-purin-6-ylcarbamate (271): to a solution of compound 270 (160 mg, crude) in DCM (5 mL) was added TFA (1 mL) at 23 ℃. After 30min, the liquid was removed in vacuo, and the mixture was purified by preparative LC to give the title compound 271 (93 mg,0.104mmol, 42% in two steps) as a light brown solid. MS M/z 554 (M+H) + 。
(9- (4- ((4- (2- (2- (aminooxy) acetylamino) ethyl) piperidin-1-yl) methyl) benzyl) -2-butoxy-8-oxo-8, 9-dihydro-7H-purin-6-yl) carbamic acid butyl ester (272): compound 272 was prepared in a similar procedure to that described for 251 using compound 271 and 2, 5-dioxopyrrolidin-1-yl 2- (((tert-butoxycarbonyl) amino) oxy) acetate as starting materials to give the title compound 272 (40 mg,0.041mmol, 82%). MS M/z 627 (M+H) + 。
N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl)Phenyl) piperidin-4-yl) ethyl) -3- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) propanamide (273): to a solution of compound 144 (10 mg,0.01 mmol) and 2, 5-dioxopyrrolidin-1-yl 3- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) propionate (3 mg,0.012 mmol) in DMF (1 mL) was added DIEA (12. Mu.L, 0.069 mmol) at 23 ℃. After 10min, the mixture was purified by preparative LC to give compound 273 (10 mg,0.01 mmol, 96%) as a colorless transparent solid. MS M/z 605 (M+H) + 。
(S) -2-amino-N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -3- (4- (((2S, 3R,4S,5S, 6R) -3,4, 5-trihydroxy-6- (hydroxymethyl) tetrahydro-2H-pyran-2-yl) oxy) phenyl) propanamide (274): compound 274 was prepared in analogy to the procedure described for 242 using compound 144 and (S) -2- (tert-butoxycarbonylamino) -3- (4- ((2S, 3r,4S,5S,6 r) -3,4, 5-trihydroxy-6- (hydroxymethyl) tetrahydro-2H-pyran-2-yloxy) phenyl) propionic acid as starting materials to obtain the title compound 274 (47 mg,0.038mmol, 67%). MS M/z 779 (M+H) + 。
(S) -N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -2- (2- (aminooxy) acetamido) -3- (4- (((2S, 3R,4S,5S, 6R) -3,4, 5-trihydroxy-6- (hydroxymethyl) tetrahydro-2H-pyran-2-yl) oxy) phenyl) propanamide (275): compound 275 was prepared in a similar procedure to that described for 251 using compound 274 and 2, 5-dioxopyrrolidin-1-yl 2- (((tert-butoxycarbonyl) amino) oxy) acetate as starting materials to give the title compound 275 (22 mg,0.018mmol, 91%). MS M/z 852 (M+H) + 。
(R) - (5-amino-6- ((2- (1- (4- ((6-amino-2-butan)) 2-yl)Oxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) amino) -6-oxohexyl) carbamic acid tert-butyl ester (276): compound 276 was prepared in a similar procedure as described for 250 using compound 144 and Fmoc-D-Lys (Boc) -OH as starting materials to give the title compound 276 (110 mg,0.097mmol, 85%). MS M/z 682 (M+H) + 。
(R) - (2- ((6-amino-1- ((2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) amino) -1-oxohex-2-yl) amino) -2-oxoethoxy) carbamic acid (9H-fluoren-9-yl) methyl ester (277): compound 277 was prepared using compound 276 and 2- ((((((9H-fluoren-9-yl) methoxy) carbonyl) amino) oxy) acetic acid as starting materials in a similar procedure as described for 242 to obtain the target compound 277 (114 mg,0.086mmol, 88%). MS M/z 877 (M+H) + 。
(R) -6-amino-N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -2- (2- (aminooxy) acetamido) hexanamide (278): to a solution of compound 277 (14 mg,0.01 mmol) in DMF (0.5 mL) was added piperidine (100. Mu.L, 1.012 mmol) at 23 ℃. After 10min, the mixture was purified by preparative LC to give compound 278 (12 mg,0.01 mmol, quantitative) as a light brown solid. MS M/z 655 (M+H) + 。
(R) -N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -2- (2- (aminooxy) acetamido) -6-PEG 24-acylaminohexanamide (279): to a solution of compound 277 (20 mg,0.015 mmol) and mPEG24-NHS (18 mg,0.015 mmol) in DMF (1 mL) was added DIEA (20. Mu.L, 0.069 mmol) at 23 ℃. After 20min piperidine (100 μl,1.012 mmol) was added to the mixture. After 10min LCMS showed deprotection was complete. Volatiles were removed in vacuo and the residue was purified by preparative LC to give the target compound 279(28mg，0.013mmol，84％)。MS m/z 1755(M+H) + 。
(S) -4- (2-amino-3- ((2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) amino) -3-oxopropyl) phenyl dihydrogen phosphate (280): compound 280 was prepared in a similar procedure as described for 250 using compound 144 and Fmoc-1-Tyr (PO 3H 2) -OH as starting materials to give the title compound 280 (22 mg,0.019mmol, 30%). MS M/z 697 (M+H) + 。
(S) -4- (3- ((2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) amino) -2- (2- (aminooxy) acetamido) -3-oxopropyl) phenyl dihydrogen phosphate (281): compound 281 was prepared in a similar procedure as described for 251, using compound 280 and 2, 5-dioxopyrrolidin-1-yl 2- (((tert-butoxycarbonyl) amino) oxy) acetate as starting materials, to give the title compound 281 (15 mg,0.012mmol, 64%). MS M/z 770 (M+H) + 。
(R) -N1- (6- ((2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) amino) -5- (2- (aminooxy) acetamido) -6-oxohexyl) -N5- (dPEG 4) - (mPEG 8) 3-glutaramide (282): compound 282 was prepared in a similar procedure as described for 279 using compound 277 and dPEG4- (m-dPEG 8) 3-NHS as starting materials to obtain the target compound 282 (37 mg,0.013mmol, 86%). MS M/z 1217 (M+2H) + 。
(R) -N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -2- (2- (aminooxy) acetamido) -6- (PEG 8) amidohexanamide (283): compound 283 was prepared in a similar procedure as described for 279 using compound 277 and mPEG8-NHS as starting materials to obtain the target compound 283 (15 mg,0.010mmol, 66%). MS M/z 1049 (M+H) + 。
N- (9- (4- ((4- (2-aminoethyl) piperidin-1-yl) methyl) benzyl) -2-butoxy-8-oxo-8, 9-dihydro-7H-purin-6-yl) hexanamide (284): to a solution of tert-butyl 2- (1- (4- ((6-amino-2-butoxy-8-oxo-7H-purin-9 (8H) -yl) methyl) benzyl) piperidin-4-yl) ethylcarbamate compound 143 (66 mg,0.074 mmol) and n-hexanoyl chloride (30 μl,0.223 mmol) in DCM (1 mL) was added DIEA (100 μl,0.574 mmol) at 23 ℃. After 1.5h, TFA (1 mL) was added to the mixture. After 10min, volatiles were removed in vacuo and the residue was purified by preparative LC to give compound 284 (50 mg,0.050mmol, 50%) as a light brown solid. MS M/z 552 (M+H) + 。
N- (9- (4- ((4- (2-aminoethyl) piperidin-1-yl) methyl) benzyl) -2-butoxy-8-oxo-8, 9-dihydro-7H-purin-6-yl) acetamide (285): compound 285 was prepared in a similar procedure as described for 284, using compound 143 and acetyl chloride as starting materials, to give the title compound 285 (40 mg,0.048mmol, 99%). MS M/z 496 (M+H) +.
N- (9- (4- ((4- (2- (2- (aminooxy) acetamido) ethyl) piperidin-1-yl) methyl) benzyl)) -2-butoxy-8-oxo-8, 9-dihydro-7H-purin-6-yl) hexanamide (286): compound 286 was prepared in a similar procedure to that described for 251 using compound 284 and 2, 5-dioxopyrrolidin-1-yl 2- (((tert-butoxycarbonyl) amino) oxy) acetate as starting materials to give the title compound 286 (38 mg,0.035mmol, 79%). MS M/z 625 (M+H) + 。
N- (2- (1- (4- ((6-acetamido-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -2- (aminooxy) acetamide (287): compound 287 was prepared in a similar procedure to that described for 251 using compound 285 and 2, 5-dioxopyrrolidin-1-yl 2- (((tert-butoxycarbonyl) amino) oxy) acetate as starting materials to give the title compound 287 (31 mg,0.030mmol, 63%). MS M/z 569 (M+H) + 。
(4- ((2-methyl-7H-pyrrolo [2, 3-H)]Quinazolin-7-yl) methyl) phenyl) methanol (288): cesium carbonate (176 mg,5.43 mmol) was added to a solution of compound 165 (200 mg,1.09 mmol) and 4-chloromethylbenzyl alcohol (510.1 mg,3.26 mmol) in DMSO (14.4 mL) at 23 ℃. After 21 hours, the reaction mixture was poured into water (15 mL) and washed with diethyl ether (5 mL). The aqueous layer was then extracted with diethyl ether (3X 50 mL). The combined organic layers were washed with water (2X 100 mL) followed by brine (50 mL). The filtrate was concentrated in vacuo and the residue was purified by flash chromatography on silica gel to give the title compound 288 (31 mg,0.030mmol, 9%). MS M/z 305 (M+H) + 。
7- (4- (chloromethyl) benzyl) -7H-pyrrolo [2,3-H]Quinazolin-2-amine (289): to compound 288 (22.6 mg,0.074 mmol) was added dichloromethane (0.67 mL). Thionyl chloride (16 μl,0.22 mmol) was added to the resulting suspension, and the mixture was stirred at 50deg.C. 1 small After that time, toluene (30 mL) was added to the mixture, and the solvent was evaporated. Toluene (100 mL) was again added to the residue and the solvent evaporated. The residue was dried under reduced pressure to obtain the target compound 289 (24 mg,0.074mmol, 100%), which was used in the next step without further purification. MS M/z 323 (M+H) + 。
(2- (1- (4- ((2-amino-7H-pyrrolo [2, 3-H))]Quinazolin-7-yl) methyl) benzyl) piperidin-4-yl) ethyl) carbamic acid tert-butyl ester (290): compound 290 was prepared in a similar procedure as described for 143 using compound 289 and tert-butyl (2- (piperidin-4-yl) ethyl) carbamate as starting materials to give the title compound 290 (25 mg,0.049mmol, 65%). MS M/z 515 (M+H) + 。
7- (4- ((4- (2-aminoethyl) piperidin-1-yl) methyl) benzyl) -7H-pyrrolo [2,3-H]Quinazolin-2-amine (291): compound 291 was prepared in a similar procedure as described for 144 using compound 290 as starting material to obtain the target compound 291 (20 mg,0.04mmol, 78%). MS M/z 415 (M+H) + 。
N- (2- (1- (4- ((2-amino-7H-pyrrolo [2, 3-H))]Quinazolin-7-yl) methyl) piperidin-4-yl) ethyl) -2- (aminoxy) acetamide (292): compound 292 was prepared in a similar procedure as described for 127 using compound 291 and 2, 5-dioxocyclopentylacetate as starting materials to give the title compound 292 (3.7 mg,0.006mmol, 28%). MS M/z 488 (M+H) + 。
4- (2- (1, 3-dioxoisoindolin-2-yloxy) ethyl) piperidine-1-carboxylic acid tert-butyl ester (293): triphenylphosphine (505 mg,1.925 mmol) was added to a solution of l-Boc-4- (2-hydroxyethyl) piperidine (405 mg,1.766 mmol) and N-hydroxyphthalimide (309 mg,1.895 mmol) in THF (10 mL) at 23 ℃. After 30min, the temperature was reduced to 0 ℃ and DIAD (400 μl,2.032 mmol) was added to the mixture over 5 min. The mixture was stirred at 0 ℃ overnight. After 20h, trueThe solvent was removed empty and the residue was dissolved in EtOAc (50 mL), washed with 1N HCl (50 mL), saturated sodium bicarbonate (50 mL) and brine (50 mL), over MgSO 4 Drying and filtering. The organic solvent was removed in vacuo. The residue was purified by flash chromatography (SiO 2 ) Purification was performed to obtain the title compound 293 (650 mg,1.736mmol, 98%) as a white solid. MS M/z 375 (M+H) + 。
2- (2- (piperidin-4-yl) ethoxy) isoindoline-1, 3-dione, TFA (294): to a solution of compound 293 (650 mg,1.736 mmol) in DCM (2 mL) was added TFA (2 mL) at 23 ℃. After 10min, the liquid was removed in vacuo. The residue was dissolved in EtOAc (50 mL) and washed with saturated sodium bicarbonate (50 mL) and brine (50 mL), over MgSO 4 Drying and filtering. The organic solvent was removed in vacuo and the residue was dried by high vacuum pump to obtain the target compound 294 (650 mg,1.674mmol, 96%) as a transparent solid. MS M/z275 (M+H) + 。
2- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7H-purin-9 (8H) -yl) methyl) benzyl) piperidin-4-yl) ethoxy) isoindoline-1, 3-dione (295): to a solution of 6-amino-2-butoxy-9- (4- (chloromethyl) benzyl) -7H-purin-8 (9H) -one (compound 142) (90 mg,0.208 mmol) and compound 294 (90 mg,0.328 mmol) in DMF (2 mL) was added DIEA (150. Mu.L, 0.861 mmol) and the temperature was raised to 80 ℃. After 4h, the solvent was removed in vacuo. The mixture was purified by preparative LC to give the title compound 295 as a light brown solid (70 mg,0.074mmol, 36%). MS M/z 600 (M+H) + 。
N- (9- (4- ((4- (2- (aminoxy) ethyl) piperidin-1-yl) methyl) benzyl) -2-butoxy-8-oxo-8, 9-dihydro-7H-purin-6-yl) acetamide (296): to a solution of compound 295 (30 mg,0.032 mmol) and acetyl chloride (30 μl,0.383 mmol) in DCM (5 mL) was added DIEA (110 μl,0.632 mmol) at 23deg.C. After 1.5h, the liquid was removed in vacuo and MeOH (2 mL) plus hydrazine hydrate (20 μL,0.4 mmol) was added to the residue. After 10min, the solvent was removed in vacuo and the residue was purified by preparative LC to give the title compound 296 (4 mg,0.005mmol, 15%) as a colorless transparent solid. MS M/z512 (M+H) + 。
6-amino-9- (4- ((4- (2- (aminoxy) ethyl) piperidin-1-yl) methyl) benzyl) -2-butoxy-7H-purin-8 (9H) -one (297): to a solution of compound 295 (40 mg,0.042 mmol) in DCM (5 mL) was added hydrazine hydrate (200. Mu.L, 4 mmol) at 23 ℃. After 5min, the liquid was removed in vacuo and the residue was purified by preparative LC to give the title compound 297 as a light brown solid (33 mg,0.041mmol, 96%). MS M/z 470 (M+H) + 。
1'- (4- ((6-amino-2-butoxy-8-oxo-7H-purin-9 (8H) -yl) methyl) benzyl) -4,4' -bipiperidine-1-carboxylic acid tert-butyl ester (298): compound 298 was prepared in analogy to the procedure described for 295 using N-Boc-4,4' -bipiperidine and 6-amino-2-butoxy-9- (4- (chloromethyl) benzyl) -7, 9-dihydro-8H-purin-8-one (compound 142) as starting materials to give the title compound 298 as a light brown solid (50 mg,0.053mmol, 26%). MS M/z 594 (M+H) + 。
N- (9- (4, 4' -bipiperidin-1-ylmethyl) benzyl) -2-butoxy-8-oxo-8, 9-dihydro-7H-purin-6-yl) acetamide (299): compound 299 was prepared in a similar procedure as described for 284, using compound 298 and acetyl chloride as starting materials, to give the title compound 299 (29 mg,0.033mmol, 62%) as a light brown solid. MS M/z 536 (M+H) + 。
N- (9- (4- ((1 '- (2- (aminoxy) acetyl) -4,4' -bipiperidin-1-yl) methyl) benzyl) -2-butoxy-8-oxo-8, 9-dihydro-7H-purin-6-yl) acetamide (300): compound 300 was prepared in a similar procedure to that described for 251 using compound 299 and 2, 5-dioxopyrrolidin-1-yl 2- (((tert-butoxycarbonyl) amino) oxy) acetate as starting materials to give the title compound 300 as a light brown solid (12 mg,0.013mmol, 43%). MS M/z 609 (M+H) + 。
N- (9- (4- ((4- (2-aminoethyl) piperidin-1-yl) methyl) benzyl) -2-butoxy-8-oxo-8, 9-dihydro-7H-purin-6-yl) -3- (2- (2-methoxyethoxy) ethoxy) propanamide (301): compound 301 was prepared in a similar procedure to that described for 251 using compound 143 and 3- (2- (2-methoxyethoxy) ethoxy) propionyl chloride as starting materials to give the title compound 301 (62 mg,0.064mmol, 92%) as a light brown solid. MS M/z 628 (M+H) + 。
N- (9- (4- ((4- (2- (2- (aminoxy) acetamido) ethyl) piperidin-1-yl) methyl) benzyl) -2-butoxy-8-oxo-8, 9-dihydro-7H-purin-6-yl) -3- (2- (2-methoxyethoxy) ethoxy) propanamide (302): compound 302 was prepared in a similar procedure to that described for 251 using compound 301 and 2, 5-dioxopyrrolidin-1-yl 2- (((tert-butoxycarbonyl) amino) oxy) acetate as starting materials to give the title compound 302 as a light brown solid (32 mg,0.031mmol, 56%). MS M/z 701 (M+H) + 。
N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) methyl) benzyl) piperidin-4-yl) ethyl) -1- (aminooxy) -3,6,9, 12-tetraoxapentadecane-15-amide (303): to a solution of 6-amino-9- (4- ((4- (2-aminoethyl) piperidin-1-yl) methyl) benzyl) -2-butoxy-7, 9-dihydro-8H-purin-8-one (compound 144) (40 mg,0.044 mmol) in DMF (1 mL) was added 1- ((1, 3-dioxoisoindol-2-yl) oxy) -3,6,9, 12-tetraoxopentadecan-15-oic acid 2, 5-dioxopyrrolidin-1-yl ester (22 mg,0.043 mmol) and DIEA (40 μl,0.23 mmol) at 23 ℃. After 30min, hydrazine hydrate (100 μl,0.66 mmol) was added to the mixture. After 10min, the liquid was removed in vacuo and the residue was purified by preparative LC to give the title compound 303 (45 mg,0.038mmol, 87%) as a light brown solid. MS M/z 717 (M+H) + 。
3-amino-N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7H-purin-9 (8H) -yl) methyl) benzyl) piperidin-4-yl) ethyl) acrylamide (303): compound 303 was prepared in a similar procedure as described for 242 using Boc- β -alanine and compound 144 as starting materials to obtain the target compound 303 (28 mg,0.029mmol, 26%). MS M/z525 (M+H) + 。
N- (2- (1- (4- ((6-amino-2-butoxy-8-oxo-7H-purin-9 (8H) -yl) methyl) benzyl) piperidin-4-yl) ethyl) -3- (2- (aminooxy) acetamido) acrylamide (304): compound 304 was prepared in a similar procedure to that described for 251 using compound 303 and 2, 5-dioxopyrrolidin-1-yl 2- (((tert-butoxycarbonyl) amino) oxy) acetate as starting materials to give the title compound 304 (20 mg,0.019mmol, 66%). MS M/z 598 (M+H) + 。
Table 4-TLR agonist-core 5 compounds
Example 4: synthesis of TLR agonists comprising the structure-core 3:
core 3
In some embodiments, X is N or H; y is C or N;
R 1 is C 1 To C 12 Alkyl, substituted C 1 To C 12 Alkyl, oxygen-containing C 1 To C 12 Alkyl, heterocycle, substituted heterocycle or H
R 2 Is C 1 To C 12 Alkyl, C 1 To C 12 Substituted alkyl, C 4 To C 8 Cycloalkyl, aromatic ring, substituted aromaticRing, aromatic heterocycle, substituted aromatic heterocycle, -ONH 2 Terminal C 1 To C 12 Alkyl or H
TLR agonists with a core 3 structure were synthesized as disclosed in the following schemes.
4-nitro-1-tosyl-1H-indole (160): compound 159 (4-nitro-1H-indole) (2.43 g,15.0 mmol) was dissolved in THF (15 mL). Sodium hydride (900 mg,22.5 mmol) in THF (30 mL) was added dropwise to the suspension at 0deg.C. The solution was warmed to room temperature and stirred for an additional 1h. Next, tosyl chloride (3.0 g,15.75 mmol) in THF (15 mL) was slowly added and the reaction stirred overnight, the solution was taken up in NaHCO 3 With Et 2 O. The aqueous layer was extracted (3X 75 mL) and the combined organic layers were washed with brine, over MgSO 4 Dried and concentrated in vacuo. The resulting solid was absorbed in AcCN, sonicated and filtered. No solid (starting material) was used. The liquid was distilled off and the residue (160) (3.12 g) was used in the next step. No MS m/z NO2 was observed.
5-methyl-4-nitro-1- (benzenesulfonyl) -1H-indole (161): compound 160 (3.11 g,9.83 mmol) was dissolved in THF (98.3 ml) at-15 ℃. Methyl magnesium chloride (4.9 mL,14.75 mmol) was added and the solution stirred for 1h 45min. DDQ (3.79 g,16.71 mmol) was then added while maintaining the temperature below-10 ℃. The reaction was allowed to warm to room temperature and stirred overnight. The reaction was then diluted with DCM to quench the reaction, followed by rotary evaporation. The crude product was eluted with DCM for SiO 2 A plug. The eluate was dried and purified by column chromatography (hexane/DCM, 0-40%,40g column) to obtain the target compound 161 (2.06 g, 63% in two steps). No MS m/z NO2 was observed.
(E) N, N-dimethyl-2- (4-nitro-1- (benzenesulfonyl) -1H-indol-5-yl) ethylene-1-amine (162): 5-methyl-4-nitro-1- (benzenesulfonyl) -1H-indole (161) (0.83 g,2.63 mmol) was dissolved in DMF (26.3 ml). N, N-dimethylformamide dimethyl acetal (3.54 mL,26.3 mmol) was added and the reaction was heated at 115 ℃. The reaction was evaporated by rotary evaporator. The residue (compound 162) was used in the next reaction (0.98 g). No MS m/zNO2 was observed.
4-nitro-1- (benzenesulfonyl) -1H-indole-5-carbaldehyde (163): to a solution of compound 162 (0.98 g,2.6 mmol) in THF (13.2 mL) and water (13.2 mL) was added sodium metaperiodate (1.7 g,7.9 mmol) and stirred. The reaction was filtered and washed with EtOAc (50 mL). The organic layer was treated with NaHCO 3 Washing with MgSO 4 Dried, filtered and evaporated by rotary evaporator. The residue (compound 163,0.56 g) was dried on a vacuum pump and used in the next reaction. No MS m/z NO was observed 2 。
4-amino-1- (benzenesulfonyl) -1H-indole-5-carbaldehyde (164): to a solution of compound 163 (0.56 g,1.7 mmol) in MeOH (47 mL) was added Pd/C (0.03 g). The reaction was stirred under a hydrogen atmosphere (double balloon/1 atm). The reaction was filtered through celite and washed with MeOH. The solvent was dried in vacuo and the residue was purified by column chromatography (hexane/EtOAc, 0-50%,12g column) to give the target compound 164 (93 mg, 12% in three steps), MS M/z 301 (M+H) + 。
7H-pyrrolo [2,3-H]Quinazolin-2-amine (165): to a solution of compound 164 (0.092 g,0.31 mmol) in DMA (3.1 mL) was added guanidine carbonate (279 mg,3.09 mmol) and stirred at 150 ℃. LCMS showed the reaction was complete and the mixture was purified by prep LC to give the target compound 165 (6 mg, 11%), MS M/z 257 (m+h) + 。
1- (2-amino-7H-pyrrolo [2, 3-H)]Quinazolin-7-yl) -2-methylpropan-2-ol (166): to a solution of sodium hydride (60% dispersion in mineral oil, 2.2mg,0.054 mmol) was added 5mL of hexane at 0 ℃ and the solution was stirred. Hexane is removed to wash off mineral oil. Then, compound 165 (2 mg,0.01 mmol) in DMF (1.1 mL) was added dropwise to the solution and stirred for 1h. Next, oxidized isobutylene (1. Mu.L, 0.011 mmol) was added dropwise and the reaction was stirred. The reaction was filtered and the mixture was purified by prep LC to give the target compound 166 (1.5 mg, 23%), MS M/z 185 (m+h) + 。
Synthesis of compounds 288-292:
(4- ((2-methyl-7H-pyrrolo [2, 3-H)]Quinazolin-7-yl) methyl) phenyl) methanol (288): cesium carbonate (176 mg,5.43 mmol) was added to a solution of compound 165 (200 mg,1.09 mmol) and 4-chloromethylbenzyl alcohol (510.1 mg,3.26 mmol) in DMSO (14.4 mL) at 23 ℃. After 21 hours, the reaction mixture was poured into water (15 mL) and washed with diethyl ether (5 mL). The aqueous layer was extracted with diethyl ether (3X 50 mL). The combined organic layers were washed with water (2X 100 mL) followed by brine (50 mL). The filtrate was concentrated in vacuo and the residue was purified by flash chromatography on silica gel to give the title compound 288 (31 mg,0.030mmol, 9%). MS M/z 305 (M+H) + 。
7- (4- (chloromethyl) benzyl) -7H-pyrrolo [2,3-H]Quinazolin-2-amine (289): to compound 288 (22.6 mg,0.074 mmol) was added dichloromethane (0.67 mL). Thionyl chloride (16 μl,0.22 mmol) was added to the resulting suspension, and the mixture was stirred at 50deg.C. After 1 hour, toluene (30 ml) was added to the mixture, and the solvent was evaporated. Toluene (100 ml) was again added to the residue and the solvent was evaporated. The residue was dried under reduced pressure to obtain the target compound 289 (24 mg,0.074mmol, 100%), which was used in the next step without further purification. MS M/z 323 (M+H) + 。
(2- (1- (4- ((2-amino-7H-pyrrolo [2, 3-H))]Quinazolin-7-yl) methyl) benzyl) piperidin-4-yl) ethyl) carbamic acid tert-butyl ester (290): compound 290 was prepared in a similar procedure as described for 143 using compound 289 and tert-butyl (2- (piperidin-4-yl) ethyl) carbamate as starting materials to give the title compound 290 (25 mg,0.049mmol, 65%). MS M/z 515 (M+H) + 。
7- (4- ((4- (2-aminoethyl) piperidin-1-yl) methyl) benzyl) -7H-pyrrolo [2,3-H]Quinazolin-2-amine (291): compound 291 was prepared in a similar procedure as described for 144 using compound 290 as starting material to obtain the target compound 291 (20 mg,0.04mmol, 78%). MS (MS) m/z 415(M+H) + 。
N- (2- (1- (4- ((2-amino-7H-pyrrolo [2, 3-H))]Quinazolin-7-yl) methyl) piperidin-4-yl) ethyl) -2- (aminoxy) acetamide (292): compound 292 was prepared in a similar procedure as described for 127 using compound 291 and 2, 5-dioxocyclopentylacetate as starting materials to give the title compound 292 (3.7 mg,0.006mmol, 28%). MS M/z 488 (M+H) + 。
Table 5-TLR agonist-core 3 compounds
Each of the compounds AXC-967, AXC-968, AXC-969 and AXC-970 comprising a core 3 structure showed an EC of greater than 10,000nM 50 。
Example 5: synthesis of TLR agonists comprising the following representative structures-core 2:
core 2
In some embodiments, R 1 Or R is 2 Each connected to form C 4 To C 8 Cycloalkyl or independently-H, C 1 To C 12 Alkyl, nitrogen-containing alkyl, aromatic ring or-C (NH) NH 2 ；
R 3 Is C 1 To C 12 Alkyl, substituted C 1 To C 12 Alkyl, oxygen-containing C 1 To C 12 Alkyl, heterocycle substituted heterocycle or H.
TLR agonists with a core 2 structure were synthesized as disclosed in the following schemes.
2- (5, 6-diaminopyrimidin-4-ylamino) -2-oxoethylcarbamic acid tert-butyl ester (167): to a solution of pyrimidine-4, 5, 6-triamine (2050 mg,16.383 mmol) and Boc-Gly-OH (2880, 16.440 mmol) in DCM (50 ml) was added DCC (380 mg,18.417 mmol) and DMAP (80 mg, 0.015 mmol) at 23 ℃. After 3h, the precipitate was removed by filtration. The mixture was purified by preparative LC to give the title compound 167 (2458 mg,8.707mmol, 53%), MS M/z 283 (M+H) + 。
(6-amino-9H-purin-8-yl) methyl carbamic acid tert-butyl ester (168): to a solution of compound 167 (620 mg,2.196 mmol) in n-BuOH (20 ml) was added 25% NaOMe (2500 ul,11.570 mmol) in MeOH at 23 ℃. The temperature was raised to 70 ℃. After 1h, 6N HCl (1.83 ml,11 mmol) was added to the mixture in the ice bath and the mixture was diluted with EtOAc (50 ml). The mixture was washed with saturated sodium bicarbonate (50 ml) and brine (50 ml), and dried over MgSO 4 Drying and filtering. The mixture was purified by flash chromatography to give the target compound 168 (250 mg,0.946mmol, 43%), MS M/z 265 (M+H) + 。
(6-amino-9- (2-bromoethyl) -9H-purin-8-yl) methyl carbamic acid tert-butyl ester (169): to a solution of compound 168 (250 mg,0.946 mmol) in DMF (5 ml) was added dibromoethane (2100 mg,2.795 mmol) and CsCO at 23 ℃ 3 (2400 mg,1.842 mmol). After 2.5h, the mixture was diluted with 20ml of DCM and washed with saturated sodium bicarbonate (50 ml) and brine (50 m). The organic layer was dried over MgSO 4 Drying and filtering. The mixture was purified by preparative LC to give compound 169 (144 mg,0.545mmol, 58%), MS M/z372 (M+H) + 。
4-amino-8, 9-dihydropyrazino [1,2-e ]]Purine-7 (6H) -carboxylic acid tert-butyl ester (170): to a solution of sodium hydride (60% dispersion in mineral oil, 51.4mg, 1.284 mmol) was added 5mL of hexane. The solution was stirred and then hexane was removed to wash off the mineral oil. Compound 169 (159.2 mg,0.429 mmol) in DMF (2.9 mL) was added dropwise to the solution with stirring at 23 ℃. After 1h LCMS showed the reaction was complete. By making the following steps The mixture was purified with a Gemini NX,150X 30C 18 column using a 5% to 60% water/90% ACN 0.05% TFA gradient preparative LC for 20min. The product-containing fractions were combined and evaporated by rotary evaporator. The residue was dried on a high vacuum pump to obtain the target compound 170 (36.7 mg,0.05mmol, 11%), MS M/z 291 (M+H) + 。
6,7,8, 9-tetrahydropyrazino [1,2-e ]]Purin-4-amine (171): to a solution of 170 (35.7 mg,0.123 mmol) was added 1.2mL of DCM. Trifluoroacetic acid (45.7 μl,0.615 mmol) was added dropwise with stirring at 23 ℃. After 1h LCMS showed the reaction was complete. The mixture was evaporated by rotary evaporator with additional azeotropy using PhMe to give compound 171 (38.5 mg,0.05mmol, 41%), MS M/z 191 (m+h) + 。
7-benzyl-6, 7,8, 9-tetrahydropyrazino [1,2-e ]]Purin-4-amine (172): to a solution of 171 (10 mg,0.053 mmol) in DMF (1.1 mL) was added benzaldehyde (6.7. Mu.L, 0.066 mmol). DIEA (18.3 μl,0.105 mmol) was added and the reaction stirred for 15 min. Then, boron-pyridine complex (6.7 μl,0.067 mmol) was added and the reaction was stirred at 23 ℃ overnight. The mixture was purified by preparative LC using a Gemini NX,150x30 c18 column using a 5% to 60% water/90% ACN 0.05% TFA gradient for 20min. The product-containing fractions were combined and evaporated by rotary evaporator. The residue was dried on a high vacuum pump to obtain compound 172 (1.3 mg,0.002mmol, 3%), MS M/z 281 (M+H) + 。
Table 6-TLR agonist-core 2 compounds
Example 6: synthesis of TLR agonists comprising the following representative structures-core 4:
core 4
In some embodiments, R 1 Is C 1 To C 12 Alkyl, substituted C 1 To C 12 Alkyl, oxygen-containing C 1 To C 12 Alkyl, C 3 To C 8 Cycloalkyl, heterocycle, substituted heterocycle, halogen or H; r is R 2 Is C 1 To C 12 Alkyl, C 1 To C 12 Substituted alkyl, C 4 To C 8 Cycloalkyl, aromatic ring, substituted aromatic ring, aromatic heterocycle, substituted aromatic heterocycle, -ONH 2 -NH2, carbonyl, terminal C 1 To C 12 Alkyl or a combination thereof; or R is 2 Is H;
R 3 is C 1 To C 12 Alkyl, substituted C 1 To C 12 Alkyl, C containing oxygen/nitrogen/sulfur 1 To C 12 Alkyl, heterocycle, substituted heterocycle, cycloalkyl, substituted cycloalkyl, -N 3 -OH, terminal C 1 To C 12 Alkyl, terminally substituted C 1 To C 12 Alkyl or a combination thereof; or R is 3 Is H.
TLR-agonists with core 4 structure were synthesized as disclosed in the following schemes.
N- (4, 6-diaminopyrimidin-5-yl) valeramide (193): pyrimidine-4, 5, 6-triamine (1015 mg,8.112 mmol) was dissolved in N-methyl-2-pyrrolidone (10 mL) at 70 ℃. After the solution became clear, it was cooled to 23 ℃. To the mixture was added pentanoyl chloride (980 μl,8.127 mmol) and the temperature was raised to 50 ℃. After 20h, the temperature was reduced to 23 ℃, etOAc (50 ml, precipitate) was added to the mixture, and the precipitate was isolated by filtration. The solid was washed with EtOAc (10 ml) and acetone (10 ml) and dried to give compound 193 (1780 mg,7.237mmol, 90%) as a light brown solid. The product was used in the next step without further purification. MS M/z 210 (M+H) + 。
8-butyl-9H-purin-6-amine (194): to a solution of crude compound 193 (1780 mg, 7.2793 mmol) in n-BuOH (30 mL) was added sodium methoxide (1570 mg,29.063 mmol) at 23℃and heated toAnd (5) refluxing. After 1h, the solution was cooled to room temperature, neutralized with 6M HCl (3.2 ml), and brine (20 ml) was added to obtain a biphasic mixture. The organic layer was separated with MgSO 4 Dried, then concentrated in vacuo to give the title compound 194 (1148 mg,6.003mmol, 83%) as a pale brown solid. MS M/z 192 (M+H) + 。
4- (6-amino-8-butyl-9H-purin-9-yl) butylcarbamic acid tert-butyl ester (195): to a solution of N-Boc-amino-butanol (460 mg,8.032 mmol) and compound 194 (1420 mg,7.426 mmol) in THF (20 ml) at 0deg.C was added PPh 3 (2080 mg,7.930 mmol). After 30min, DIAD (2200 μL,11.174 mmol) was added to the mixture at 0deg.C for 5 min. After 3h, the solvent was removed in vacuo. The residue was diluted with DCM (100 ml) and washed with half-saturated sodium bicarbonate (100 ml) and brine (20 ml). The organic layer was dried over MgSO 4 Drying and filtering. The solvent was removed in vacuo. The residue was purified by flash chromatography to give compound 195 (1064 mg,2.935mmol, 37%) as a light brown solid. MS M/z363 (M+H) + 。
4- (6- (N-benzoylamino) -8-butyl-9H-purin-9-yl) butylcarbamic acid tert-butyl ester (196): to a solution of compound 195 (1064 mg,2.935 mmol) in DCM (10 ml) was added benzoyl chloride (700. Mu.L, 4.980 mmol) and TEA (900. Mu.L, 17.788 mmol) at 0deg.C, and the temperature was raised to 20deg.C. After 2.5h, the mixture was washed with saturated sodium bicarbonate (50 ml) and brine (50 ml), over MgSO 4 Drying and filtering. The solvent was removed in vacuo and the residue was purified by flash chromatography to give compound 196 (1650 mg,2.891mmol, 98%) as a light brown oil. MS M/z 571 (M+H) + 。
4- (6- (N-benzoylamino) -8-butyl-2-nitro-9H-purin-9-yl) butylcarbamic acid tert-butyl ester (197): to a solution of tetramethyl ammonium nitrate (780 mg,5.729 mmol) in DCM (10 ml) was added trifluoroacetic anhydride (1200. Mu.L, 17.118 mmol) at 23 ℃. After 1h, the mixture was cooled to 0deg.C, and a solution of compound 196 (1650 mg,2.891 mmol) in DCM (20 ml) was added. The temperature was raised to 23 ℃. After 2h, the mixture was diluted with DCM (20 ml), washed with half-saturated sodium bicarbonate (20 ml) and brine (20 ml), and concentrated in vacuogSO 4 Drying and filtering. The solvent was removed in vacuo and the residue was purified by flash chromatography to give compound 197 (1067 mg,1.733mmol, 60%) as a clear pale yellow solid. MS M/z 616 (M+H) + 。
9- (4-aminobutyl) -8-butyl-9H-purin-6-amine (198) and 6-amino-9- (4-aminobutyl) -8-butyl-9H-purin-2-ol (199): pd/C (10%, 0.1 g) was added to a solution of compound 197 (220 mg,0.357 mmol) in EtOH (20 ml) at 23℃and bubbled with hydrogen. After 18h LCMS showed that the denitrified compound was prepared. NaOMe (30 mg,0.6 mmol) was added and the mixture stirred for 4h. Next, TFA (3 ml) was added. After 20min, the solvent was removed in vacuo and the mixture was purified by preparative LC to give compound 198 (94 mg,0.156mmol, 44%) as a light brown solid, MS M/z 263 (m+h) + And compound 199 (0.024 mmol, 7%) as a light brown solid, MS M/z 279 (M+H) + 。
4-amino-N- (4- (6-amino-8-butyl-2-hydroxy-9H-purin-9-yl) butyl) -3, 5-difluorobenzamide (200): compound 200 was prepared in a similar procedure as described for 150, using compound 199 and 4-amino-3, 5-difluorobenzoic acid as starting materials, to give the title compound 200 (14 mg,0.018mmol, 61%) as a light brown solid, MS M/z 434 (m+h) + 。
4-amino-N- (4- (6-amino-8-butyl-9H-purin-9-yl) butyl) -3, 5-difluorobenzamide (201): compound 201 was prepared in a similar procedure as described for 150, using compound 198 and 4-amino-3, 5-difluoro-benzoic acid as starting materials to give the title compound 201 (13 mg,0.017mmol, 93%) as a light brown solid, MS M/z 418 (m+h) + 。
3-amino-N- (4- (6-amino-8-butyl-9H-purin-9-yl) butyl) benzamide (202): compound 202 was prepared in a similar procedure as described for 150, using compound 198 and 3-aminobenzoic acid as starting materials, to give the title compound 202 (10 mg,0.014mmol, 88%) as a light brown solid, MS M/z 382 (m+h) + 。
5-amino-N- (4- (6-amino-8-butyl-9H-purin-9-yl) butyl) nicotinamide (203): compound 203 was prepared in a similar procedure as described for 150, using compound 198 and 5-amino-nicotinic acid as starting materials, to give the title compound 203 (14 mg,0.019mmol, quantitative) as a light brown solid, MS M/z 383 (m+h) + 。
4- (2, 6-dichloro-9H-purin-9-yl) butylcarbamic acid tert-butyl ester (204): PPh was added to a solution of N-Boc-amino-butanol (1620 mg,8.560mmol and 2, 6-dichloropurine (1495 mg,7.910 mmol) in THF (10 ml) at 0deg.C 3 (2280 mg,8.693 mmol). After 30min, DIAD (2300. Mu.L, 11.681 mmol) was added over 5min at 0deg.C. The mixture was stirred at 50 ℃. After 6h, the solvent was removed in vacuo. The residue was diluted with EtOAc (100 ml) and washed with half-saturated sodium bicarbonate (100 ml) and brine (20 ml). The organic layer was dried over MgSO 4 Drying and filtering. The solvent was removed in vacuo. The residue was purified by flash chromatography to give compound 204 as a yellow oil (4500 mg,<12.492mmol，<100%). (about 20% of the impurities are PPh 3). MS M/z 361 (M+H) + 。
4- (6-amino-2-chloro-9H-purin-9-yl) butylcarbamic acid tert-butyl ester (205): compound 204 (PPh) 3 Is of (1)The mixture, 4500mg,<12.492 mmol) was placed in a pressure-resistant glass vessel equipped with a stirring rod. 7N NH in MeOH was added to the vessel 3 (12 mL,84 mmol). The tube was sealed and then heated at 120 ℃. After 30min, the solvent was removed in vacuo and the residue was dissolved in DCM (100 ml). The solution was washed with saturated sodium bicarbonate (100 ml) and brine (30 ml). The organic layer was dried over MgSO 4 Drying and filtering. The solvent was removed in vacuo. The residue was purified by flash chromatography to give compound 205 (1310 mg,3.844mmol, 37%) as a pale yellow solid. MS M/z 341 (M+H) + 。
4- (6-amino-2-butoxy-9H-purin-9-yl) butylcarbamic acid tert-butyl ester (206): to a solution of compound 205 (257 mg,0.754 mmol) in n-butanol (5 ml) was added sodium metal (90 mg, 2.458 mmol) at 23℃under dry nitrogen. The temperature was raised to 100 ℃. After 18h, the solvent was removed in vacuo. The residue was dissolved in DCM (50 ml) and washed with half-saturated sodium bicarbonate (50 ml) and brine (50 ml). The organic layer was dried over MgSO 4 Drying and filtering. The solvent was removed in vacuo. Compound 206 was obtained as a light brown crude solid (310 mg,<0.819mmol, quantitative). MS M/z 379 (M+H) + 。
4- (6-amino-8-bromo-2-butoxy-9H-purin-9-yl) butylcarbamic acid tert-butyl ester (207): to a solution of compound 206 (310 mg,0.819mmol, crude) in DCM (10 ml) was added bromine (150. Mu.L, 0.563 mmol) at 23 ℃. After 1h, the solvent was removed in vacuo. The mixture was purified by flash chromatography to obtain compound 207 (250 mg,0.547mmol, 67%) as a transparent pale yellow solid. MS M/z 458 (M+H) + 。
4- (6-amino-2-butoxy-8-methyl-9H-purin-9-yl) butylcarbamic acid tert-butyl ester (208): to a solution of compound 207 (82 mg, 0.178 mmol) in dry THF (5 ml) at 23℃was added 1M trimethylaluminum (360. Mu.L, 0.36 mmol) and PdCl in THF 2 (PPh 3 ) 2 (44 mg,0.063 mmol). The mixture was refluxed. After 20h, the mixture was diluted with 20ml of DCM and washed with half saturated sodium bicarbonate (20 ml) and brine (20 ml). The organic layer was dried over MgSO4 and filtered. The solvent was removed in vacuo. The residue was purified by flash chromatography to give the product as a pale mixtureCompound 208 (12 mg,0.031mmol, 17%) as a brown solid. MS M/z 393 (M+H) + 。
9- (4-aminobutyl) -2-butoxy-8-methyl-9H-purin-6-amine (209): to a solution of compound 208 (12 mg,0.031 mmol) in DCM (0.5 ml) was added trifluoroacetic acid (0.5 ml) at 23 ℃. After 1h, the solvent was removed in vacuo. The residue was dried overnight on a high vacuum pump to give compound 209 (15 mg,0.02mmol, quantitative) as a light brown solid. MS M/z 293 (M+H) + 。
4-amino-N- (4- (6-amino-2-butoxy-8-methyl-9H-purin-9-yl) butyl) -3, 5-difluorobenzamide (210): compound 210 was prepared in a similar procedure as described for 150, using compound 209 and 4-amino-3, 5-difluoro-benzoic acid as starting materials to give the title compound 210 (3.5 mg, 0.04 mmol, 22%) as a light brown solid, MS M/z469 (m+h) + 。
9- (4-aminobutyl) -8-bromo-2-butoxy-9H-purin-6-amine (211): compound 211 was prepared in a similar procedure as described for 209 using compound 207 to give the title compound 211 ((8 mg,0.01 mmol, quantitative), MS M/z 358 (m+h) as a light brown solid + 。
4-amino-N- (4- (6-amino-8-bromo-2-butoxy-9H-purin-9-yl) butyl) -3, 5-difluorobenzamide (212): compound 212 was prepared in a similar procedure as described for 150, using compound 211 and 4-amino-3, 5-difluoro-benzoic acid as starting materials, to give the title compound 212 (8 mg,0.009mmol, 82%) as a light brown solid, MS M/z 513 (m+h) + 。
Table 7-TLR agonist-core 4 compounds
Example 7: this example discloses various methods and techniques for use in the present invention.
Molecular cloning-CHO cell codon optimized antibody heavy and light chain cDNA sequences were obtained from commercial DNA synthesis service (IDT, san Diego, CA). The synthesized DNA fragment was digested with HindIII and EcoRI (both from New England Biolabs (NEB), ipswich, mass.) and purified by PCR purification kit (Qiagen, valencia, calif.). The digested antibody gene fragments were ligated into expression vectors by a quick connect kit (NEB) to generate constructs for expression of wild-type antibody heavy and light chains. The resulting plasmid was propagated in E.coli and verified by the DNA sequencing service (EtON).
Generation of amber codon-containing mutants-based on the crystal structure of the anti-HER 2 Fab, 10 different surface accessible sites located in the constant region of the light chain were selected for genetic incorporation of unnatural amino acids (e.g., p-acetyl-phenylalanine (pAF), or p-azido-phenylalanine or p-amino-phenylalanine). Those sites are not important for antigen-antibody binding. Each genetic codon of the selected site is then mutated to an amber codon (TAG) by site-directed mutagenesis to generate an expression plasmid for the antibody mutant. Primers were purchased from IDT. All site-directed mutagenesis experiments were performed according to the instruction manual (NEB) using the Q5 site-directed mutagenesis kit. The expression plasmid of the mutant was propagated in E.coli and verified by the DNA sequencing service (EtON). Table 8 provides a list of amber mutation sites in the heavy or light chain constant regions of the anti-HER 2 Fab and their Kabat numbering, as well as the corresponding amino acid sequences, SEQ ID NOS: 2, 3, 4 and 6 to 15.SEQ ID NOS.1 and 5 show the wild type heavy and light chains, respectively, of an anti-HER 2 Fab. The anti-HER 2 Fab includes the following heavy and light chain sequences: SEQ ID NO. 1 and SEQ ID NO. 5; SEQ ID NO. 1 and SEQ ID NO. 6; SEQ ID NO. 1 and SEQ ID NO. 7; SEQ ID NO. 1 and SEQ ID NO. 8; SEQ ID NO. 1 and SEQ ID NO. 9; SEQ ID NO. 1 and SEQ ID NO. 10; SEQ ID NO. 1 and SEQ ID NO. 11; SEQ ID NO. 1 and SEQ ID NO. 12; SEQ ID NO. 1 and SEQ ID NO. 13; SEQ ID NO. 1 and SEQ ID NO. 14; SEQ ID NO. 1 and SEQ ID NO. 15. The anti-HER 2 Fab includes the following heavy and light chain sequences: SEQ ID NO. 2 and SEQ ID NO. 5; SEQ ID NO. 2 and SEQ ID NO. 6; SEQ ID NO. 2 and SEQ ID NO. 7; SEQ ID NO. 2 and SEQ ID NO. 8; SEQ ID NO. 2 and SEQ ID NO. 9; SEQ ID NO. 2 and SEQ ID NO. 10; SEQ ID NO. 2 and SEQ ID NO. 11; SEQ ID NO. 2 and SEQ ID NO. 12; SEQ ID NO. 2 and SEQ ID NO. 13; SEQ ID NO. 2 and SEQ ID NO. 14; SEQ ID NO. 2 and SEQ ID NO. 15. The anti-HER 2 Fab includes the following heavy and light chain sequences: SEQ ID NO. 3 and SEQ ID NO. 5; SEQ ID NO. 3 and SEQ ID NO. 6; SEQ ID NO. 3 and SEQ ID NO. 7; SEQ ID NO. 3 and SEQ ID NO. 8; SEQ ID NO. 3 and SEQ ID NO. 9; SEQ ID NO. 3 and SEQ ID NO. 10; SEQ ID NO. 3 and SEQ ID NO. 11; SEQ ID NO. 3 and SEQ ID NO. 12; SEQ ID NO. 3 and SEQ ID NO. 13; SEQ ID NO. 3 and SEQ ID NO. 14; SEQ ID NO. 3 and SEQ ID NO. 15. The anti-HER 2 Fab includes the following heavy and light chain sequences: SEQ ID NO. 4 and SEQ ID NO. 5; SEQ ID NO. 4 and SEQ ID NO. 6; SEQ ID NO. 4 and SEQ ID NO. 7; SEQ ID NO. 4 and SEQ ID NO. 8; SEQ ID NO. 4 and SEQ ID NO. 9; SEQ ID NO. 4 and SEQ ID NO. 10; SEQ ID NO. 4 and SEQ ID NO. 11; SEQ ID NO. 4 and SEQ ID NO. 12; SEQ ID NO. 4 and SEQ ID NO. 13; SEQ ID NO. 4 and SEQ ID NO. 14; SEQ ID NO. 4 and SEQ ID NO. 15. In other embodiments, any of SEQ ID NOs 1, 2, 3, 4 may include the Fc mutations disclosed in Table 9A.
Table 8. Anti-HER 2 Fab Heavy (HC) and Light (LC) amino acid sequences with amber sites for unnatural amino acid incorporation. Also disclosed are all the sequences in the following table, wherein pAF is replaced by any other unnatural amino acid.
X represents an unnatural amino acid (nnAA);underline lineRepresents the Fc mutations in Table 9A
In addition to the amber mutation in the heavy chain at position 114, fc mutations were generated at different positions of the anti-HER 2 antibody or antibody fragment to improve pharmacokinetics and/or enhance antibody-dependent cell phagocytosis (ADCP) and/or antibody-dependent cellular cytotoxicity ADCC activity (tables 9A and 9B).
TABLE 9A-anti-HER 2 Fc mutations
Table 9B. Anti-HER 2 monoclonal antibody variants with amber sites for unnatural amino acid incorporation and additional mutations used. Also disclosed are all the sequences in the following table, wherein pAF is replaced by any other unnatural amino acid.
X represents an unnatural amino acid (nnAA)
The anti-HER 2 monoclonal antibodies used in the invention include the heavy chain of SEQ ID NO. 16, or SEQ ID NO. 17, or SEQ ID NO. 18, as well as any of the following light chain sequences: SEQ ID NO. 5; SEQ ID SEQ ID NO. 6; SEQ ID NO. 7; SEQ ID NO. 8; SEQ ID NO. 9; SEQ ID NO. 10; SEQ ID NO. 11; SEQ ID NO. 12; SEQ ID NO. 13; SEQ ID NO. 14; SEQ ID NO. 15. In other embodiments, the anti-HER 2 Fab used in the present invention comprises any of the heavy chain mutations disclosed in table 9A and any of the following light chain sequences: SEQ ID NO. 5; SEQ ID SEQ ID NO. 6; SEQ ID NO. 7; SEQ ID NO. 8; SEQ ID NO. 9; SEQ ID NO. 10; SEQ ID NO. 11; SEQ ID NO. 12; SEQ ID NO. 13; SEQ ID NO. 14; SEQ ID NO. 15.
Transient expression-the platform Cell line was maintained in EX-Cell 302 (Sigma) supplemented with 3mM L-glutamine (Gibco) and 3mM Glutamax (Gibco). Cells were passaged every 3-4 days at a seed density of 40 ten thousand cells/ml. Cells were seeded at 60 ten thousand cells/ml one day prior to transfection. On day 0, cells were transfected with antibody expression plasmids encoding light and heavy chains using a MaxCyte electroporation platform according to the instruction manual. After transfection, the cells were placed in 125ml shake flasks and incubated in a static incubator at 37℃for 30min. The transfected cells were then incubated at 3X 10 6 A density of/ml was inoculated into basal expression medium (50% dynamics-50% Excell 302 supplemented with 50. Mu.M MSX) in shake flasks. Transfected cells were incubated at 37℃with 5% CO 2 Incubate on an orbital shaker set at 140 rpm. 1mM pAF was added to the culture on day 1 along with 7g/L Cell Boost 5 (GE healthcare), 120 μg/L Long R3 IGF-1 (sigma) and 2mM Glutamax. The temperature in the incubator was changed from 37℃to 32 ℃. 7g/L Cell Boost 5 and 2mM Glutamax were added on day 3 and the supernatant collected on day 5. Glucose levels were monitored using a glucose meter, and when the glucose level in the medium was below 2g/L, additional glucose was added to the culture. Viable Cell count and viability were measured by Vi-Cell instrument. Productivity was measured by Octet using a protein G sensor.
Stable batch pool generation-expression plasmids were linearized using PvuI (NEB) digestion for 6 hours. After linearization, the DNA was purified using phenol extraction and dissolved in endotoxin-free water at a concentration of 2.5. Mu.g/. Mu.l. The plateau Cell line BB-117 was maintained in EX-Cell 302 supplemented with 3mM L-glutamine and 3mM Glutamax. Cells were passaged every 3-4 days at a seed density of 0.4X10 6 /ml. One day before transfection, at 0.6X10 6 Cells were inoculated per ml. On day 0, cells were transfected with linearized antibody expression plasmids using a MaxCyte electroporation platform according to the instruction manual. After transfection, the cells were placed in 125ml shake flasks and incubated in a static incubator at 37℃for 30min. Then 30ml of recovery medium (make-up50% Ex-302-50% CD-CHO filled with 3mM glutamine and 3mM Glutamax) was added to the flask and shaken overnight. The first day, transfected cells were counted, centrifuged, washed and resuspended in selection medium (50% Ex-302-50% CD-CHO with 50-100 μm MSX) to create a stable bulk pool. Viable cell number and viability were monitored and the medium was changed every 3-4 days until the viability of the stable batch pool returned to 90%. When the selection is completed, frozen cell stock is prepared and the resulting stable batch pool is used to generate material for fed-batch expression.
Fed-batch expression-on day 0, the previously generated antibodies were stabilised in a batch pool of 0.5X10 6 A density of/ml was inoculated into basal expression medium (50% dynamics-50% Excell 302 supplemented with 50. Mu. MMSX) in shake flasks. Transfected cells were incubated at 37℃with 5% CO 2 Incubate on an orbital shaker set at 140 rpm. On day 3, 0.5mM pAF was added to the culture along with 10g/L Cell Boost 4 (GE healthcare) and 0.52g/L Cell Boost 7b (GE healthcare). On day 5, 120. Mu.g/L Long R3 IGF-1 was added to the culture. Glucose levels were monitored using a glucose meter, and when the glucose level in the medium was below 2g/L, additional glucose was added to the culture. Viable Cell count and viability were measured by Vi-Cell instrument. On day 7, the supernatant was collected for purification. Productivity was measured by Octet using a protein G sensor.
Purification of nnAA-containing antibodies from the EuCODE expression system-clarified cell culture medium containing nnAA-containing target antibodies was loaded onto a protein a ProSep Ultra column (EMD Millipore) equilibrated in 20mM sodium phosphate, 100mM sodium chloride, pH 7.5. After loading, the column was washed with buffer a (20 mM sodium phosphate, 100mM sodium chloride, pH 7.5) followed by washing with wash buffer B (5 mM succinic acid, pH 5.8) to remove host cell contaminants. The target antibody was eluted from the column with elution buffer C (50 mM glycine, 10mM succinic acid, pH 3.2). The target antibodies were pooled and the pH was adjusted to pH 5.0 with 2.0M tris base. The target antibody was further purified by loading the conditioned protein a pool onto Capto SP Impres column (GE Healthcare) equilibrated in 30mM sodium acetate at pH 5.0. The target antibody was eluted from the column using a linear gradient to 100% buffer B (30 mM sodium acetate, 0.5M sodium chloride, pH 5.0) and fractions containing monomeric antibody were pooled, filtered through 0.22 μm and stored at +.65 ℃ until further use.
Site-specific conjugation of TLR agonist linker payload-antibody buffer containing nnAA (e.g. para-acetylphenylalanine) was exchanged into conjugation buffer (30 mM sodium acetate, pH 4.0) and concentrated to 10-20mg/mL. Finally 100mM acetic acid hydrazide was added to the antibody followed by 10 molar equivalents of hydroxylamine functionalized TLR agonist drug-linker. The conjugation reaction was incubated at 25-30℃for 18-20 hours, followed by purification through Capto SP Impres column (GE Healthcare) to remove excess reagents. The purified ADC buffer was replaced with formulation buffer (50 mM histidine, 100mM NaCl, 5% trehalose, pH 6.0) and stored at < 65℃until further use.
The TLR agonist drug-linker antibody conjugates of the invention are shown below:
as shown, the antibody is linked to a linker, which may be a cleavable amino acid linker or a non-cleavable linker, and the linker is linked to a drug or payload. The linker-drug may be one or more as shown by N, where N is an integer from 1 to 10. Further, the amount of the drug or payload may be one or more, or an integer greater than 1, and the drug may be 1 to 8.
Example 8: in vitro functional assays for small molecule TLR agonists
HEK-Blue TM hTLR7 cells in HEK-Blue TM Incubation in assay medium and stimulation with increasing concentrations of TLR7 or TLR8 or TLR7/8 agonist. After 24h incubation, NF-kB induced SEAP levels were determined by means of Quanti-Blue detection reagent (Invivogen, san Diego, calif.), and readings were taken at an OD of 655 nm. Determination of EC from dose-response curves using Prism software 50 . The following tables and figures illustrate the invention described in examples 1-6 hereinActivity of an exemplary TLR agonist. EC (EC) 50 Values less than 500nM indicate that the potency of the compound is higher than EC 50 Values between 50nM to 1. Mu.M or greater than 1. Mu.M to 3. Mu.M.
Use of EC in HEK-Blue hTLR7 reporter cell lines 50 2.08. Mu.M commercial TLR7 agonist Resimmod (Resiquimod) (R848), EC 50 Compound 1 and EC at 0.435. Mu.M 50 TLR7 agonist stimulation was performed for compound 2 at 0.153 μm (data not shown).
The activity of the exemplary TLR agonists disclosed in examples 1-6 above was determined. Table 10 shows EC compared to commercial controls DSR-6434, raschimod and Mo Tuo mod (Motolimod) 50 Values.
TABLE 10 Activity of exemplary TLR agonists
Numbering of compounds Compounds of formula (I) TLR7 EC 50 (nM)
N/A control-DSR-6434 9.203
N/A R848-raschimod 519.7
88 AXC-779 242.1
60 AXC-738 11.08
49 AXC-725 249.4
54 AXC-732 255.5
58 AXC-736 213.4
62 AXC-740 115.6
86 AXC-777 910.4
87 AXC-778 83.34
89 AXC-789 71.34
109 AXC-800 >5000
106 AXC-801 >5000
112 AXC-802 >5000
93 AXC-803 331.5
94 AXC-804 260.3
97 AXC-807 412.6
61 AXC-739 28.17
59 AXC-737 18.7
64 AXC-743 56.11
Table 11 shows TLR7 activity of selected TLR7 agonists. AXC-887 and AXC-877 show EC 50 Values below 10nM indicate that these compounds are very potent TLR7 agonists. AXC-887 showed measurable activity in the TLR8 reporter assay, wherein EC 50 3733nM, and EC of the commercial compound motomomod 50 1427nM. This suggests that ACX-887 is a TLR7/8 dual agonist.
TABLE 11 Activity of exemplary TLR agonists
Table 12 shows TLR7 activity of selected TLR7 agonists attached to the linker. AXC-879 showed the highest potency in different TLR agonist (payload) linkers.
TABLE 12 Activity of exemplary TLR agonists and linkers
Compounds of formula (I) TLR agonist payload+linker
EC 50 (nM)
DSR-6434 14.8
AXC-876 362.4
AXC-879 151.2
AXC-880 409.5
AXC-882 550.8
AXC-874 >10,000
AXC-875 >10,000
AXC-862 400.1
AXC-863 864.2
AXC-867 829.0
AXC-868 >5,000
AXC-869 >10,000
AXC-889 696.5
Table 13 shows TLR7 activity of additional TLR7 agonists and TLR7 agonists attached to the linker. AXC-895 showed the highest potency in the different payloads tested, while AXC-901 showed the highest potency in the different payload splices.
TABLE 13 Activity of exemplary TLR agonists and linkers
Table 14 shows TLR7 activity of additional TLR7 agonists and TLR7 agonists attached to the linker. All compounds tested have TLR7 agonist activity. AXC-894, AXC-903, AXC-904, AXC905 and AXC-906 tested the highest efficacy of the different payloads, where EC 50 The value was below 10nM.
TABLE 14 Activity of exemplary TLR agonists and linkers
Compounds of formula (I) TLR7 EC 50 (nM)
DSR-6434 10.5
AXC-903 4.3
AXC-907 51.8
AXC-894 2.1
AXC-909 71.9
AXC-904 8.1
AXC-905 2.6
AXC-906 5.4
AXC-908 11.5
AXC-860 119.5
AXC-910 2591.1
Table 15 compares TLR7 activity of different TLR7 agonists attached to the linker (drug linker or DL) and unnatural amino acids containing the final metabolite, e.g., pAF (DL-pAF). In all cases, the pAF containing the final metabolite showed higher potency than its payload with the drug linker each. AXC-879 showed the highest potency among the different TLR payload linkers.
TABLE 15 Activity of exemplary TLR agonist+linker in the presence or absence of unnatural amino acid (nnAA)
DL = drug linker; DL-pAF = pAF containing final metabolite
Example 9: site-specific conjugation of TC
Site-specific conjugation of TC was performed using analytical reversed phase HPLC as described in example 7. Analytical reversed phase HPLC chromatogram of unconjugated anti-HER 2 antibody with an unnatural amino acid (e.g. pAF) at amino acid position HA114 under reducing conditions. The anti-HER 2 antibody was conjugated to TLR agonists AXC-875 and AXC-880, respectively, at amino acid position HA114 (data not shown). Table 16 shows the drug-to-antibody ratio (DAR) of TLR conjugates from the standard conjugation conditions described above. Different DAR levels (0.3-2.0) can be seen between TLR conjugates, mainly due to the different nature of the relevant TLR linker-payload.
Table 16 drug-to-antibody ratio (DAR) of TLR conjugates as determined by RP-HPLC.
Example 10: in vitro co-culture assay using multiple tumor cells
RAW-Blue TM Cells (Invivogen, san Diego, calif.) were co-cultured with human tumor cells having different levels of HER2 expression at a 1:1E:T ratio, with a total of 100 tens of thousands of cells per well in 96-well plates. Various concentrations of small molecule TLR7 agonist and conjugated immunostimulatory antibody conjugate (ISAC) were added to the co-culture cell culture medium. After 24h incubation, the samples from RAW-Blue were assayed by means of the Quanti-Blue detection reagent (Invivogen, san Diego, calif.) TM NF-kB induced SEAP levels of cells, readings were taken at OD 655 nm. Dose-response curves were generated using Prism software.
Comparison of tumor-dependent ISAC activity of selected payload linkers when conjugated to anti-HER 2 antibodies. SKOV3 is a HER2 high expressing tumor cell line (data not shown); JIMT-1 is a HER2 mid/low expressing tumor cell line (data not shown); and a431 is a HER2 low expressing tumor cell line (data not shown). Small molecule TLR7 agonists exhibit potent TLR7 activity in the presence of all tumor cell lines. All TLR7 ISACs were inactive in the presence of HER2 low expression or expression tumor cell lines. ISACs with a payload-drug linker, AXC-863, showed potent dose-dependent activity only in the presence of HER 2-highly expressing tumor cell lines.
Comparison of tumor-dependent ISAC activity of additional payload linkers when conjugated to anti-HER 2 antibodies. SKBR3 is a HER2 high expressing tumor cell line (data not shown) and HCC1806 is a HER2 very low expressing tumor cell line (data not shown). Small molecule TLR7 agonists exhibit potent TLR7 activity in the presence of all tumor cell lines. While all TLR7 ISACs and unconjugated anti-HER 2 antibodies showed no activity in the presence of a HER2 very low expressing tumor cell line. ISACs with a payload-drug linker, AXC-863, showed potent dose-dependent activity only in the presence of HER 2-highly expressing tumor cell lines.
Comparison of tumor-dependent ISAC activity of additional payload linkers when conjugated to anti-HER 2 antibodies. SKBR3 is a HER2 high expressing tumor cell line (data not shown) and HCC1806 is a HER2 very low expressing tumor cell line (data not shown). Small molecule TLR7 agonists exhibit potent TLR7 activity in the presence of all tumor cell lines. All TLR7 ISACs and unconjugated anti-HER 2 antibodies showed no activity in the presence of a HER2 very low expressing tumor cell line. All ISACs showed potent dose-dependent activity only in the presence of HER 2-highly expressing tumor cell lines (data not shown). ISACs with a payload drug linker, AXC-879, exhibited the highest HER 2-dependent TLR7 activity.
Comparison of tumor-dependent ISAC activity of additional payload linkers when conjugated to anti-HER 2 antibodies. SKBR3 is a HER2 high expressing tumor cell line (data not shown) and HCC1806 is a HER2 very low expressing tumor cell line (data not shown). Small molecule TLR7 agonists exhibit potent TLR7 activity in the presence of all tumor cell lines. Both TLR7 ISAC and unconjugated anti-HER 2 antibodies showed no activity in the presence of a HER2 very low expressing tumor cell line. All ISACs showed potent dose-dependent activity only in the presence of HER 2-highly expressing tumor cell lines. ISACs with a payload drug linker AXC-901 showed the highest HER 2-dependent TLR7 activity.
Comparison of tumor-dependent ISAC activity of the three (3) payload linkers conjugated to anti-HER 2 antibody in SKBR3 HER2 high expressing tumor cell line (data not shown) and HCC1806HER2 very low expressing tumor cell line (data not shown) indicated that HER2-AXC-879 had optimal ISAC activity compared to HER2-AXC-860 and HER2-AXC-910, and HER2-AXC-910 represents a known ISAC. Table 17 describes TLR agonist-linker structures used to generate the comparative HER2-AXC ISACs.
TABLE 17 TLR agonist structures
Example 11: drug-linker design/payload-linker design
The drug-linker or payload-linker is designed to enhance, augment or modify the pharmacokinetic and therapeutic profiles of the TLR agonist compositions and conjugates of the invention. The TCs of the present invention are designed to provide additional target specificity by blocking TLR exposure at unintended target sites using PEG mask and prodrug designs, e.g., where cleavage of the PEG mask or prodrug at the tumor microenvironment releases the active payload further enhancing specificity.
PEG shielding-a method of minimizing or masking the inherent hydrophobicity in a drug/payload linker by PEG or PEG shielding. PEG designs are incorporated into TCs of the present invention to improve or enhance drug-linker solubility by shielding TCs from unintended target interactions, allowing for better targeting efficiency, e.g., anti-tumor activity at the tumor microenvironment. The drug/payload linker design strategy of the present TC with PEG shielding using TLR agonist compound AXC-914 (denoted payload) is illustrated below:
examples of payloads with PEG mask designs:
Compound AXC-913:
examples of AXC-913 payloads with PEG mask design illustrated using PEG 24:
figures 2 and table 18 show TLR7 activity of TLR7 agonists using the PEG shielding approach.
TABLE 18 Activity of TLR agonists with PEG shielding
Compounds of formula (I) EC 50 (nM)
AXC-913 29.6
AXC-923 434
AXC-924 398
AXC-925 1591
AXC-926 824
Figures 3A-3B and table 19 show that for PEG maskTLR7 agonist drug-linker TLR7 activity in the presence of linear or branched PEG of the method. The use of AXC-913 payloads with PEG shielding, represented by the compounds AXC-939 (PEG 4), AXC-937 (PEG 8), AXC-940 (PEG 12) and AXC-936 (PEG 48), demonstrated linear PEG shielding activity. Similarly, AXC-913 payloads with branched PEG, e.g., represented by AXC-938, AXC-945 and AXC-946, respectively (PEG 4) 3 、(PEG8) 3 And (PEG 12) 3 Shows active EC under PEG shielding 50 。
TABLE 19 Activity of exemplary TLR agonist drug-linkers with PEG shielding
Compounds of formula (I) EC 50 (nM) Compounds of formula (I) EC 50 (nM) Compounds of formula (I) EC 50 (nM)
AXC-931 490 AXC-946 1408 AXC-962 452.6
AXC-932 156 AXC-947 337 AXC-963 19.4
AXC-933 40 AXC-949 760 AXC-964 10.3
AXC-934 10.3 AXC-950 443 AXC-965 48.3
AXC-935 40 AXC-951 842 AXC-966 65.5
AXC-936 764 AXC-952 1023 AXC-978 28.9
AXC-937 456 AXC-953 200.9 AXC-979 21.7
AXC-938 644 AXC-954 111.3 AXC-980 29.6
AXC-939 303 AXC-955 172.8 AXC-981 10.1
AXC-940 644 AXC-956 157.9 AXC-982 19.1
AXC-941 699 AXC-958 39.6 AXC-983 53.3
AXC-942 387 AXC-959 550.7 DSR-6434 14
AXC-943 410 AXC-960 266.8 AXC-863 320
AXC-945 232 AXC-961 856.1
Prodrugs-another approach for improving or enhancing the pharmacokinetic activity of TLR agonists of the invention involves proteolytic cleavable linker design in the absence or presence of PEG shielding.
Examples of drug linker/payload linker designs with proteolytic cleavable groups using exemplary AXC-914 and Boc, as well as Val-Cit-PABA groups. The Boc and Val-Cit-PABA groups may be replaced with any other proteolytically cleavable group known to those skilled in the art and disclosed herein.
Examples of drug linker/payload linker designs with proteolytic cleavable groups using exemplary AXC-913 and cleavable groups Val-Ala or Val-Ala-PABA groups with and without PEG 24. It should be noted that any proteolytically cleavable group can be used with PEG mask. Additional examples of TCs designed using proteolytic cleavable groups with or without PEG shielding are listed in table 4.
AXC-913
Representative exemplary AXC-913 and a cleavable group Val-Ala or Val-Ala-PABA groups in the presence and absence of PEG 24.
Example 12: activity of HER2 ISAC with PEG shielded linker
FIG. 4 shows an in vitro SKBR3-RAWBlue co-culture assay for HER2 ISAC. FIG. 4 shows tumor dependent ISAC activity of HER2-AXC879, HER2-AXC863 and HER 2-control TLR7 agonist conjugates. The data indicate that HER2-AXC879 is a more potent ISAC with respect to HER2 positive tumor-specific macrophage reporter cell activation.
The effect of the Fc region on HER2 ISAC activity was also determined. Figure 5 shows the effect of Fc modification on ISAC activity. The ADCC enhancement (ADE mutation, table 9A) did not show increased potency compared to HER2 ISAC with wild-type IgG1 Fc region, whereas the Fc null mutation (PVA mutation, table 9A) showed significantly reduced potency.
The effect of the conjugation site on HER2 ISAC activity was also determined. Figure 6 shows the effect of different conjugation sites on ISAC activity. Of all conjugation sites tested, heavy chain a114 (Kabat numbering, actual number a 121) showed the highest target-specific ISAC activity.
In SKBR3 and HCC1806 cell lines, RAWBlue co-culture in vitro assays were used to test the activity of various HER2 ISACs with PEG-shielded TLR7 agonist payloads. Figures 7-9 and tables 20A-20D show HER2 dependent activity of HER2 ISAC with PEG-shielded TLR7 agonist payload. In comparison to AXC-879, some PEG-screened TLR7 agonists exhibit reduced non-specific activity at high concentrations. FIGS. 7A-7B show in vitro activity of AXC-879 derivatives in the absence or presence of PEG shielding, respectively, in RAW-Blue co-culture assays. FIG. 7C shows the in vitro activity of AXC-879 derivatives with branching modification. Figures 8A-8C and figures 9A-9B show in vitro activity of various HER2 ISACs with PEG shielding. Tables 20A-20C show the activity of various HER2 ISACs with PEG shielding.
FIGS. 9A-9B show in vitro activity comparisons of AXC-879 derivatives with D-Lys block or L-Lys block with Table 20D.
TABLE 20A Activity of various HER2 TLR agonist payloads with PEG shielding
TABLE 20B Activity of various HER2 TLR agonist payloads with PEG shielding
TABLE 20C Activity of various HER2 TLR agonist payloads with PEG shielding
TABLE 20D Activity of various HER2 TLR agonist payloads with PEG shielding
Example 13: in vitro co-culture assay of tumor cells and reporter cells
Additional in vitro co-culture assays described in example 10 were performed with tumor cells and reporter cells. The data is provided in this embodiment.
In vitro co-culture assays with tumor cells and human PBMCs were also used. Briefly, fresh human PBMCs were isolated from human whole blood (purchased from ALLCELLS). A total of 250,000 PBMCs and 50,000 target cells (E: T ratio of 5:1) in RPMI-1640 medium with 4% low IgG serum were seeded in each well of a 96-well plate, and different concentrations of small molecule TLR 7 agonist and conjugated ISAC were added to the co-culture cell culture medium. After 24h or 48h incubation, the levels of the different cytokines were determined using the U-PLEX Multiplex assay system from MesoScale. Tumor cell killing was measured by LDH levels using the Cytotox96Non-Radioactive Cytotoxicity Assay kit (Promega). For dendritic cell maturation, 1X 10 will be 6 PBMC and 1X 10 5 Each target cell was incubated with 10nM HER2mAb, HER2-ISAC or isotype control-ISAC for 24 or 48h. Cells were collected and incubated with dendritic cell markers, HLA-DR, DC-SIGN/CD209 and CD86 antibodies for flow cytometry. Dose-response curves were generated using Prism software. The data is provided in this embodiment.
Figure 10 shows that HER2-AXC879 has enhanced ADCC-mediated tumor killing compared to unconjugated HER2mAb and AXC-879 isotype control in HER2 high tumor cell line SKBR 3. In the HER2 low tumor cell line HCC1806, HER2-AXC879 showed little tumor cell killing.
Figures 11-12 show that HER2-AXC879 induced higher HLA-DR, CD86 and CD209 expression levels on human PBMC when co-cultured with HER2 high tumor cell line SKBR3 compared to unconjugated HER2 mAb. FIG. 11 shows a comparison of HLA-DR marker induction of bone marrow cells between HER2mAb and HER2-AXC879 ISAC in a PBMC co-culture assay. FIG. 12 (panels A-D) shows a comparison of CD86/DC-SIGN+ biscationic cell induction between HER2mAb and HER2-AXC879 ISAC in a PBMC co-culture assay. FIG. 12, panel A shows untreated 1.5% HLA-DR+/DC-SIGN+/CD86+ cells. FIG. 12, panel B shows HER2mAb 18% HLA-DR+/DC-SIGN+/CD86+ cells. FIG. 12, panel C, shows HER-AXC87940.8% HLA-DR+/DC-SIGN+/CD86+ cells. FIG. 12, panel D shows PSMA-aXC879 isotype control 4.04% HLA-DR+/DC-SIGN+/CD86+ cells. The data indicate that HER2-ISAC promotes bone marrow cell maturation and activation in human PBMC.
Figures 13A-13C show the enhanced HER 2-dependent tumor cell killing activity of various HER2 ISACs relative to unconjugated HER2 mabs and their target-dependent cytotoxicity against HER2-AXC879 was compared in PBMC co-culture assays. HER2-AXC879 is the most active ISAC among all conjugates tested in the HER2 high expressing cell line (fig. 13A) and the HER2 low expressing cell line (fig. 13B). In the HER2 negative cell line MDA-MB-468 (fig. 13C), none of the HER2 ISACs showed specific tumor cell killing activity.
Figures 14A and 14B show induction of HER 2-dependent cytokines in human PBMC co-culture assays using HER2 highly expressing cell line NCI-N87 (figure 14A) and HER2 negative cell line MDA-MB-468 (figure 14B).
Figures 15-17 show additional HER 2-dependent cytokine induction in human PBMC co-culture assays using HER2 high expressing cell line SKBR3, HER2 low cell line HCC1806, HER2 negative cell line MDA-MB-468, and human PBMC alone. Two different pro-inflammatory cytokines, tnfα and ifnγ, were measured. HER2-AXC879 showed the highest efficacy among all HER2 ISACs tested. Compared to HER2-AXC879, HER2-AXC966 showed similar cytokine-induced activity in HER2 high SKBR3 cell line, but significantly lower cytokine-induced activity in HER2 low HCC1806 cell line. None of the HER2 ISACs induced measurable cytokines in HER2 negative MDA-MB-468 cell lines and human PBMC alone. Figure 15A shows induction of ifnγ cytokines by HER2 ISACs in HER2 high SKBR3/PBMC co-cultures. Figure 15B shows induction of ifnγ cytokines by HER2ISAC in HER2 low HCC1806/PBMC co-culture assay. Figure 15C shows induction of tnfα cytokines by HER2ISAC in a HER2 high SKBR3/PBMC co-culture assay. Figure 15D shows induction of tnfα cytokines by HER2ISAC in a HER2 low HCC1806/PBMC co-culture assay. Figure 16A shows induction of ifnγ cytokines by HER2 ISACs with prodrug design in HER2 high SKBR3/PBMC co-culture assays. Figure 16B shows induction of ifnγ cytokines by HER2 ISACs with prodrug design in HER2 low HCC1806/PBMC co-culture assay. Figure 16C shows induction of ifnγ cytokines by HER2 ISACs with prodrug design in HER2 negative MDA-MB-468/PBMC co-culture assay. Figure 16D shows induction of ifnγ cytokines in PBMCs alone by HER2 ISACs with prodrug design. Figure 17A shows induction of tnfα cytokines by HER2 ISACs with prodrug design in HER2 high SKBR3/PBMC co-culture assays. Figure 17B shows induction of tnfα cytokines by HER2ISAC with prodrug design in HER2 low HCC1806/PBMC co-culture assay. Figure 17C shows induction of tnfα cytokines by HER2ISAC with prodrug design in HER2 negative MDA-MB-468/PBMC co-culture assay. Figure 17D shows induction of tnfα cytokines by HER2 ISACs with prodrug design in PBMCs alone.
To test that the ISAC platform of the invention is suitable for use with other targets, RAWBlue in vitro co-culture assays were performed on other antibodies targeting different tumor antigens. FIGS. 18A-18C show the effects of an exemplary ISAC AXC879 using HCC1806 cells for TROP2ISAC (FIG. 18A), C4-2 cells for PSMA (FIG. 18B) and 786-O-cells for CD70 (FIG. 18C). In contrast to its unconjugated antibody, TROP2-AXC879, PSMA-aXC879 and CD70-AXC879 all showed target-dependent ISAC activity. The data indicate that the ISAC platform of the present invention can be used with other tumor targets.
TROP2 expression levels on different cell lines were tested. Figure 19A shows TROP2 target expression levels on different tumor cell lines. Figure 19B shows that in vitro potency of TROP2-AXC879 correlates with TROP2 expression levels on different tumor cell lines. The data indicate that TROP2-AXC879 ISAC activity is dependent on TROP2 target expression levels.
The ISAC activity of the additional TROP2 TLR7 agonist conjugate was compared to TROP2-AXC 879. Additional TROP2 ISACs were tested in the TROP2 positive HCC1806 cell line (fig. 20A) and the TROP2 negative HCC1395 cell line (fig. 20B) using a RAW-Blue co-culture assay. Additional studies were performed in the TROP2 positive SKBR3 cell line (fig. 21A) and TROP2 negative HCC1395 cell line (fig. 21B) using the PBMC co-culture assay described elsewhere herein. The data indicate that all ISACs tested were active, with TROP2-AXC879 exhibiting the highest activity. The induction of tnfα cytokine by TROP2-ISAC was higher in the case of TROP2-AXC879 in SKBR3 cells (fig. 21A) compared to HCC1395 cells (fig. 21B).
The ADCC effect of TROP2-AXC879 in the TROP2 positive BxPC-3 cell line was further assessed (fig. 22A) and killing of TROP2-AXC879 in the TROP2 negative HCC1395 cell line (fig. 22B) compared to unconjugated TROP2 mAb and AXC-879 isotype control (PSMA-AXC 879) using PBMC co-culture assay. The data indicate that TROP2-AXC879 exhibited enhanced ADCC-mediated tumor killing in TROP2 positive BxPC-3 cells and no tumor cell killing in TROP2 negative HCC1395 cells compared to unconjugated TROP2 antibody.
Table 21. Anti-TROP 2, anti-PSAMA and anti-CD 70 Heavy (HC) and Light (LC) amino acids with amber sites for unnatural amino acid incorporation. Also disclosed are all the sequences in the following table, wherein pAF is replaced by any other unnatural amino acid.
X represents an unnatural amino acid (nnAA)
anti-TROP 2 monoclonal antibodies for use in the present invention include the heavy chain of SEQ ID NO. 19 or SEQ ID NO. 21 and the light chain sequence of SEQ ID NO. 20. The anti-PSMA monoclonal antibodies used in the present invention include the heavy chain of SEQ ID NO. 22 or SEQ ID NO. 24 and the light chain sequence of SEQ ID NO. 23. The anti-CD 70 monoclonal antibodies used in the present invention include the heavy chain of SEQ ID NO. 25 or SEQ ID NO. 27 and the light chain sequence of SEQ ID NO. 26.
Example 14: PK study of HER2-AXC879
10 female C57BL/6J mice were divided into four groups and treated with a single dose (IV) of HER2-AXC879 at two dose levels (1 mg/kg and 5 mg/kg) to evaluate their pharmacokinetics. At the start of the experiment, animals will be bled prior to dose and then bled at the indicated time point after injection. Whole blood samples were collected from each mouse tail at the following time points: 2h, 6h, 24h, 48h, 72h, 4d, 7d, 10d, 14d, 21d and 28d. Blood samples were diluted 1:10 into casein blocking buffer and stored. All samples were then evaluated using the PK assay developed for total antibody measurement in diluted blood samples. As shown in FIG. 23, both the 1mg/kg and 5mg/kg groups were detectable at day 28. Cmax levels can be linearly extended approximately from 1mg/kg to 5mg/kg. PK data indicated a good linear PK profile for HER2-AXC 879.
Example 15: in vivo testing of anti-HER 2 TLR7 agonist conjugates in MC38-hHER2 colon tumor homology model
MC38-hHER2 cells, genetically modified by knocking out the mouse HER2 gene and overexpressing the human HER2 gene in MC38 cells, were cultured in DMEM+10% FBS for a minimum of 2 weeks. C57BL/6 mice were subcutaneously injected 1X 10 in the right anterior abdomen 6 Individual MC38-hHER2 cells and 0.1ml PBS. When the average tumor size reaches about 200-250mm 3 At this time, tumor-bearing mice were randomly divided into 7 groups. All therapies were administered Intravenously (IV) once a week for 4 weeks. Animals were monitored for tumor growth by caliper measurements and body weight twice weekly.
As shown in fig. 24A-24C and fig. 25, group average tumor volumes are plotted against time. In fig. 24A, the tumor volume of the control increased continuously over time, while three treatment groups with different TLR7 agonist payloads (administered once a week at 3mg/kg for 4 weeks) exhibited varying degrees of tumor growth inhibition. Among the three different HER2-TLR7 agonist conjugates, HER2-AXC879 showed the strongest anti-tumor activity compared to other groups such as HER2-AXC863 (fig. 24B) (fig. 24C). In the HER2-AXC879 group, 10 out of 10 mice showed complete tumor regression response after 3mg/kg dose treatment.
In FIG. 25, the synergistic effect between anti-PD 1 antibody and HER2-AXC879 was evaluated. Anti-mouse PD-1 antibody was administered once a week at 1mg/kg for 4 weeks, and HER2-AXC879 was administered once a week at 0.3mg/kg for 4 weeks. Both drugs were administered alone and in combination. The combination treatment group showed better tumor growth inhibition compared to each monotherapy group, and the differences were statistically significant (p-value < 0.05). This data demonstrates that HER2-AXC879 can be used in combination with an anti-PD-1 antibody for the treatment of cancer and may result in better efficacy than treatment with an anti-PD-1 antibody alone.
To further compare the difference between HER2-AXC879 and HER2-AXC863, the serum concentration of total HER2 antibody was measured after each dose injection. The data (fig. 26A and 26B) indicate that HER2-AXC879 can maintain high drug exposure and consistent serum concentrations during each treatment cycle, while HER2-AXC863 cannot maintain the same exposure levels during the second and third treatment cycles. This suggests that HER2-AXC879 has a more favorable PK profile than HER2-AXC 863.
To further investigate the dose-efficacy relationship of HER2-AXC879 and compare HER2-AXC879 with other HER2 ISACs, 1 x 10 was subcutaneously injected in the right anterior abdomen of C57BL/6 mice 6 Individual MC38-hHER2 cells and 0.1ml PBS. When the average tumor size reaches about 200-250mm 3 When tumor-bearing mice were randomly divided into different groups. All therapies were administered Intravenously (IV) once a week for 4 weeks. Animals were monitored for tumor growth by caliper measurements and body weight twice weekly.
As shown in fig. 27 and 28, group average tumor volumes are plotted against time. In figure 27, different doses of HER2-AXC879 exhibited different levels of tumor growth inhibition. At the 1mg/kg dose and higher, significant tumor growth inhibition and tumor regression were observed in most mice, while the 3mg/kg dose exhibited the highest level of tumor regression. This experiment shows that the anti-tumor efficacy of HER2-AXC879 is related to the dose level and HER2 receptor occupancy. Figure 28 shows that the tumor volumes of the control group and HER2-AXC966 increased continuously over time, while three treatment groups with different TLR7 agonist payloads (HER 2-AXC955, HER2-AXC879 and HER2-AXC 979) showed different degrees of tumor growth inhibition. Of the three different HER2-TLR7 agonist conjugates, HER2-AXC955 showed the strongest anti-tumor activity.
Example 16: re-challenge of ISAC treated mice with MC38-hHER2 and MC38 parental tumors
To determine whether HER2 ISAC treatment induced anti-tumor immune memory and long term protection of the same or similar tumors, mice previously treated with HER2 ISAC and showing complete tumor regression were grouped and treated with MC38-hHER2 (1×10) in the left anterior abdomen 28 days after the last drug administration 6 Individual cells) or MC38 parent tumor (5×10 5 Individual cells) are re-challenged. As a control, the first test mice that did not receive any prior treatment were vaccinated with the same amount of tumor cells.
Tumor growth curves in average tumor volume are shown in fig. 29A and 29B. MC38-hHER2 and MC38 failed to grow in previously treated mice with complete tumor regression, but grew rapidly in control first-trial mice. This data suggests that HER2 ISAC treatment produced long-term MC38-hHER2 and MC38 specific anti-tumor immune responses in mice.
Example 17: in vivo testing of anti-Trop 2 TLR7 agonist conjugates in JIMT-1 breast tumor xenograft models
JIMT-1 cells were cultured in rpmi+10% FBS for at least 2 weeks prior to implantation. Freshly harvested cells were suspended in PBS and mixed with Matrigel at 1:1. 70 female NSG mice were subcutaneously implanted 5X 10 in the right flank 6 0.1ml cell suspension per cell/mouse. When the tumor reaches about 180mm 3 When mice were divided into 5 groups of 10 animals each, a control group and a different therapeutic dose group (10 mg/kg, 3mg/kg and 1mg/kg for TROP2-HA114-AXC879 and 10mg/kg for RSV-HA114-AXC879 as isotype control). All therapies were administered Intravenously (IV) once a week for 4 weeks. Animals were monitored for tumor growth by caliper measurements and body weight twice weekly.
As shown in fig. 30A, group average tumor volumes are plotted against time. Tumor volumes of control group (gr.1) and isotype control group (gr.2) showed similar tumor growth. TROP2-AXC879 (Gr.3, 1 mpk) and TROP2-AXC879 (Gr.4, 3 mpk) showed marginal activity with TGI of 25% and 26%, respectively. TROP2-AXC879 at 10mpk (Gr.5) showed 59% TGI of the control. This suggests that TROP2 TLR7 agonist conjugates have significant tumor growth inhibitory activity even in immunodeficient NSG mice. Animal body weight was monitored twice weekly and plotted over time in figure 30B. When treated with TROP2-AXC879 and weekly doses up to 10mg/kg, the body weights of the control and treatment groups did not show any observable toxicity in this model. This indicates that TROP2-AXC879 is well tolerated in the NSG mouse model.
Example 18: treatment of breast cancer
Human clinical trial of safety and/or efficacy of trastuzumab-linked TLR agonist derivatives for breast cancer therapy
The purpose is as follows: the safety and pharmacokinetics of the administration compositions comprising trastuzumab-linked TLR agonist derivatives were compared.
Study design: the study was a phase I, single-center, open-label, randomized dose escalation study, followed by a phase II study on breast cancer patients. Patients should not be exposed to trastuzumab-linked TLR agonist derivatives prior to entering the study. Patients had not received treatment for their cancer within 2 weeks of the start of the trial. Treatment includes the use of chemotherapy, hematopoietic growth factors, and biological therapies (such as monoclonal antibodies). The patient must have recovered from all toxicities associated with previous treatments (grade 0 or grade 1). All subjects were evaluated for safety and all blood collections were collected as planned for pharmacokinetic analysis. All studies were performed with institutional ethical committee approval and patient consent.
Stage I: patients received i.v. trastuzumab-linked TLR agonist derivatives on days 1, 8 and 15 of each 28-day cycle. The dose of trastuzumab-linked TLR agonist derivatives may remain unchanged or be modified according to the toxicity assessment outlined below. The treatment was repeated every 28 days without unacceptable toxicity. A group of 3-6 patients received increasing doses of trastuzumab-linked TLR agonist derivatives until the Maximum Tolerated Dose (MTD) of trastuzumab-linked TLR agonist derivatives was determined. MTD is defined as the dose before 2 out of 3 patients or 2 out of 6 patients experience dose limiting toxicity. Dose-limiting toxicity was determined according to definitions and standards established by the national cancer institute (National Cancer Institute, NCI) adverse events general term (Common Terminology for Adverse Events, CTCAE) version 3.0 (8/9/2006).
Stage II: patients receive trastuzumab-linked TLR agonist derivatives as in phase I at the MTD determined in phase I. The treatment was repeated every 4 weeks for 2-6 time periods without disease progression or unacceptable toxicity. After completion of 2 time courses of study therapy, patients who obtained complete or partial responses may receive a further 4 time courses. After completion of 6 time course study therapy, patients who maintain disease stability for more than 2 months can receive a further 6 time courses as the disease progresses, provided they meet the original qualification criteria.
Serial blood samples were drawn by direct venipuncture before and after administration of trastuzumab-linked TLR agonist derivatives. Venous blood samples (5 mL) for determining serum concentration were obtained at about 10 minutes prior to dosing and at about the following times after dosing: day 1, day 8 and day 15. Each serum sample was split into two equal parts. All serum samples were stored at-20 ℃. Serum samples were transported on dry ice.
Pharmacokinetics: patients were plasma/serum sample collected for pharmacokinetic assessment prior to initiation of treatment and on days 1, 8 and 15. Pharmacokinetic parameters were calculated by a model independent method on a Digital Equipment Corporation VAX 8600 computer system using the latest version of the BIOAVL software. The following pharmacokinetic parameters were determined: peak serum concentration (C) max ) The method comprises the steps of carrying out a first treatment on the surface of the Time to peak serum concentration (t max ) The method comprises the steps of carrying out a first treatment on the surface of the Concentration-time Area Under Curve (AUC) calculated using linear trapezoidal rule from time zero to last blood collection time 0-72 ) The method comprises the steps of carrying out a first treatment on the surface of the And a terminal elimination half-life (t 1/2 ). The elimination rate constant is estimated by linear regression of consecutive data points in the end linear region of the log-linear concentration-time curve. Calculating the mean of pharmacokinetic parameters for each treatmentValues, standard Deviation (SD) and Coefficient of Variation (CV). The ratio of parameter mean values (preserved formulation/non-preserved formulation) was calculated.
Patient response to combination therapy: patient response was assessed via X-ray, CT scan and MRI, and imaging was performed before the beginning of the study and at the end of the first cycle, with additional imaging performed every four weeks or at the end of the subsequent cycle. The imaging modality is selected based on the type of cancer and feasibility/availability, and the same imaging modality is used for similar cancer types and throughout the study for each patient. The response rate was determined using RECIST criteria. (Therasse et al, J.Natl.cancer Inst.2000, 2 nd day; 92 (3): 205-16; http:// ctep. Cancer. Gov/forms/TherasseRECISTJNCI. Pdf). Patients also received cancer/tumor biopsies to assess changes in progenitor cell phenotype and clonogenic cell growth by flow cytometry, western blot, and IHC, and cytogenetic changes by FISH. Patients were followed periodically for 4 weeks after completion of study treatment.
Example 19: treatment of breast cancer
Human clinical trial of safety and efficacy of trastuzumab-linked TLR agonist derivatives for breast cancer therapy
The purpose is as follows: in women with metastatic breast cancer with HER2 overexpression, the efficacy and toxicity of TLR agonist derivatives linked by combining trastuzumab and paclitaxel followed by trastuzumab alone at disease progression were compared against the first line combination trastuzumab and paclitaxel.
Study design: the study was a randomized, multicentric study. Patients were stratified according to the degree of HER2/neu overexpression (2+ versus 3+), previous anthracycline-containing adjuvant therapy (no previous therapy versus previous therapy without radiotherapy versus previous therapy with radiotherapy versus left chest wall), estrogen receptor status (positive versus negative versus unknown), previous therapy (first line versus second/third line), and center. Patients were randomly assigned to one of two treatment groups. Group I: patients received 30-90 minutes weekly of trastuzumab-linked TLR agonist derivative IV. At the time of disease progression, the patient receives a combination of trastuzumab-linked TLR agonist derivative IV and paclitaxel IV as in group II. Group II: patients received 30-90 minutes weekly of trastuzumab-linked TLR agonist derivative IV. Paclitaxel was administered IV weekly for 1 hour for 3 weeks followed by rest for 1 week.
Treatment was continued in both groups without disease progression or unacceptable toxicity. Quality of life was assessed on day 1 of baseline and schedules 2, 3, 4, 5, 6, 8, 10 and 12. Patients were followed up on months 1,3 and 6, after which they were followed up every 6 months.
Example 20: treatment of bladder cancer
The purpose is as follows: acute toxicity of paclitaxel and radiation therapy was determined in patients treated for bladder muscle invasive transitional cell carcinoma prior to transurethral cystectomy with or without TLR agonist derivatives described herein.
Disease characteristics: histological or cytologically confirmed primary transitional cell carcinoma of the bladder (TCC); histological evidence of intrinsic myometrium infiltration; meets one of the following staging criteria: stage T2-4 a; NX, N0 or N1; and M0 disease or stage T1,3/3 disease and require definitive topical treatment; tumors are allowed to reach the prostatic urethra if the following criteria are met: the tumor is obviously completely resected; there was no evidence of interstitial infiltration of the prostate, no evidence of distant metastasis (by chest X-ray or CT scan and abdominal/pelvic CT scan); transurethral cystectomy (as thoroughly as possible diagnostic safety) was accepted within the past 3-8 weeks, including double co-diagnostic examination with tumor mapping; sufficient tumor tissue was available for HER2/neu analysis; are not candidates for radical cystectomy.
Study design: the study was a non-randomized, multicenter study. Patients were assigned to 1 of the 2 treatment groups based on HER2/neu status (HER 2/neu2+ or 3+ staining [ group 1] versus HER 2/neu0 or 1+ staining [ group 2 ]).
Group 1: patients received paclitaxel IV for 1 hour on days 1, 8, 15, 22, 29, 36 and 43 and trastuzumab-linked TLR agonist derivatives IV described herein for 90 minutes on day 1, and then 30 minutes on days 8, 15, 22, 29, 36 and 43. Patients also received one radiation therapy per day on days 1-5, 8-12, 15-19, 22-26, 29-33, 36-40, 43-47 and 50. Treatment is continued without disease progression or unacceptable toxicity.
Group 2: patients received paclitaxel and were subjected to radiation therapy as in group 1. After completion of study treatment, patients received follow-up within 4-5 weeks, once every 3 months for 1 year, once every 4 months for 1 year, once every 6 months for 3 years, and then once annually.
Example 21: treatment of ovarian cancer
Human clinical trial of the safety and efficacy of trastuzumab-linked TLR agonist derivatives described herein for ovarian cancer therapy
The purpose is as follows: the safety and efficacy of weekly IV doses of compositions comprising trastuzumab-linked TLR agonist derivatives described herein in women with HER2 overexpressing ovarian cancer were evaluated.
Study design: the study was a non-randomized, open-label, 11 week multicenter study. The present study will evaluate the safety profile of four once weekly IV doses, MTD, PK and immunogenicity of trastuzumab-linked TLR agonist derivatives. Patients are assigned to a single group. Patients receive one dose of trastuzumab-linked TLR agonist derivative weekly for 4 weeks. Trastuzumab-linked TLR agonist derivatives will be administered by IV infusion on study days 1, 8, 15 and 22. Urine samples will be collected on day 1 and day 22.
Serial blood samples were drawn by direct venipuncture before and after administration of trastuzumab-linked TLR agonist derivatives. Venous blood samples (5 mL) for determining serum concentration were obtained at about 10 minutes prior to dosing and at about the following times after dosing: days 1, 2, 4, 5, 8, 15, 22, 36, 43 and 50. Each serum sample was split into two equal parts. All blood samples were stored at-20 ℃. Blood samples were transported on dry ice.
Treatment is continued without disease progression or unacceptable toxicity. Quality of life was assessed on day 1 of baseline and schedules 2, 3, 4, 5, 6, 8, 10 and 12. Patients were followed on days 29, 36, 43 and 50. The patient will be asked for adverse events. The patient will undergo imaging scans and ECG to assess tumor size and cardiac function (day 43). At the end of the study, the patient will undergo physical examination on day 50. Patients with evidence of disease regression may receive continued treatment until evidence of disease progression is recorded.
It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications and changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, patent applications, and/or other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent application, and/or other document were individually indicated to be incorporated by reference for all purposes.
Sequence listing
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Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 7
<211> 214
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> light chain A112 mutation
<220>
<221> misc_feature
<222> (112)..(112)
<223> Xaa = unnatural amino acid (nnAA)
<400> 7
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Xaa
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 8
<211> 214
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> light chain S114 mutation
<220>
<221> misc_feature
<222> (114)..(114)
<223> Xaa = unnatural amino acid (nnAA)
<400> 8
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Xaa Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 9
<211> 214
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> light chain S121 mutation
<220>
<221> misc_feature
<222> (121)..(121)
<223> Xaa = unnatural amino acid (nnAA)
<400> 9
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Xaa Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 10
<211> 214
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> light chain S127 mutation
<220>
<221> misc_feature
<222> (127)..(127)
<223> Xaa = unnatural amino acid (nnAA)
<400> 10
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Xaa Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 11
<211> 214
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> light chain K149 mutation
<220>
<221> misc_feature
<222> (149)..(149)
<223> Xaa = unnatural amino acid (nnAA)
<400> 11
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Xaa Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 12
<211> 214
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> light chain S156 mutation
<220>
<221> misc_feature
<222> (156)..(156)
<223> Xaa = unnatural amino acid (nnAA)
<400> 12
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Xaa Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 13
<211> 214
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> light chain S168 mutation
<220>
<221> misc_feature
<222> (168)..(168)
<223> Xaa = unnatural amino acid (nnAA)
<400> 13
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Xaa Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 14
<211> 214
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> light chain S202 mutation
<220>
<221> misc_feature
<222> (202)..(202)
<223> Xaa = unnatural amino acid (nnAA)
<400> 14
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Xaa Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 15
<211> 214
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> light chain V205 mutation
<220>
<221> misc_feature
<222> (205)..(205)
<223> Xaa = unnatural amino acid (nnAA)
<400> 15
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Xaa Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 16
<211> 449
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> heavy chain A114/ADE variants
<220>
<221> misc_feature
<222> (121)..(121)
<223> Xaa = unnatural amino acid (nnAA)
<400> 16
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser Xaa Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Ala Gly
225 230 235 240
Pro Asp Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Glu Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly
<210> 17
<211> 449
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> heavy chain A114/G236A variants
<220>
<221> misc_feature
<222> (121)..(121)
<223> Xaa = unnatural amino acid (nnAA)
<400> 17
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser Xaa Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Ala Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Glu Ile
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly
<210> 18
<211> 449
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> heavy chain A114/PVA variants
<220>
<221> misc_feature
<222> (121)..(121)
<223> Xaa = unnatural amino acid (nnAA)
<400> 18
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser Xaa Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly
<210> 19
<211> 450
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Trop2 heavy chain 1 (HC 1) WT
<400> 19
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Thr Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Gly Met Asn Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Thr Glu Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Thr Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Gly Phe Gly Ser Ser Tyr Trp Tyr Phe Asp Val Trp Gly
100 105 110
Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
115 120 125
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
130 135 140
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
145 150 155 160
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
180 185 190
Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
195 200 205
Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
210 215 220
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
225 230 235 240
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
245 250 255
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
260 265 270
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
275 280 285
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
290 295 300
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
305 310 315 320
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
325 330 335
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
340 345 350
Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser
355 360 365
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
370 375 380
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
385 390 395 400
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
405 410 415
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
420 425 430
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
435 440 445
Pro Gly
450
<210> 20
<211> 214
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Trop2 light chain 3 (LC 3) WT
<400> 20
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Asp Val Ser Ile Ala
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala
65 70 75 80
Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln His Tyr Ile Thr Pro Leu
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 21
<211> 450
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Trop2 heavy chain 1 (HC 1) A114pAF (kabat numbering, actual
Position A121)
<220>
<221> misc_feature
<222> (122)..(122)
<223> Xaa = unnatural amino acid (nnAA)
<400> 21
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Thr Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Gly Met Asn Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Thr Glu Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Thr Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Gly Phe Gly Ser Ser Tyr Trp Tyr Phe Asp Val Trp Gly
100 105 110
Gln Gly Thr Met Val Thr Val Ser Ser Xaa Ser Thr Lys Gly Pro Ser
115 120 125
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
130 135 140
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
145 150 155 160
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
180 185 190
Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
195 200 205
Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
210 215 220
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
225 230 235 240
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
245 250 255
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
260 265 270
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
275 280 285
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
290 295 300
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
305 310 315 320
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
325 330 335
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
340 345 350
Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser
355 360 365
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
370 375 380
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
385 390 395 400
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
405 410 415
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
420 425 430
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
435 440 445
Pro Gly
450
<210> 22
<211> 444
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> PSMA heavy chain WT
<400> 22
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Tyr
20 25 30
Thr Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met
35 40 45
Gly Asn Ile Asn Pro Asn Asn Gly Gly Thr Thr Tyr Asn Gln Lys Phe
50 55 60
Glu Asp Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Gly Trp Asn Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr
100 105 110
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
115 120 125
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
130 135 140
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
145 150 155 160
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
165 170 175
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
180 185 190
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
195 200 205
Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys
210 215 220
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
225 230 235 240
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
245 250 255
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
260 265 270
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
275 280 285
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
290 295 300
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
305 310 315 320
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
325 330 335
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
340 345 350
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
355 360 365
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
370 375 380
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
385 390 395 400
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
405 410 415
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
420 425 430
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440
<210> 23
<211> 214
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> PSMA light chain WT
<400> 23
Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ala
20 25 30
Val Asp Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Leu
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 24
<211> 444
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> PSMA heavy chain A114pAF (kabat numbering, actual position A118)
<220>
<221> misc_feature
<222> (116)..(116)
<223> Xaa = unnatural amino acid (nnAA)
<400> 24
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Tyr
20 25 30
Thr Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met
35 40 45
Gly Asn Ile Asn Pro Asn Asn Gly Gly Thr Thr Tyr Asn Gln Lys Phe
50 55 60
Glu Asp Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Gly Trp Asn Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr
100 105 110
Val Ser Ser Xaa Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
115 120 125
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
130 135 140
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
145 150 155 160
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
165 170 175
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
180 185 190
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
195 200 205
Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys
210 215 220
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
225 230 235 240
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
245 250 255
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
260 265 270
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
275 280 285
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
290 295 300
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
305 310 315 320
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
325 330 335
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
340 345 350
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
355 360 365
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
370 375 380
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
385 390 395 400
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
405 410 415
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
420 425 430
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440
<210> 25
<211> 447
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> CD70 heavy chain WT
<400> 25
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Thr Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Tyr Met Asn Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met
35 40 45
Gly Ile Ile Asn Pro Tyr Asn Gly Gly Thr His Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Ser Gly Tyr Asp Leu Tyr Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
210 215 220
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
225 230 235 240
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
275 280 285
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
290 295 300
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
355 360 365
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
385 390 395 400
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
405 410 415
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
<210> 26
<211> 214
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> CD70 light chain WT
<400> 26
Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Lys Ala Ser Gln Asn Val Gly Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Ser Ala Phe Asn Arg Tyr Asn Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Pro Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Ser Thr Tyr Pro Leu
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 27
<211> 447
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> CD70 heavy chain A114pAF (kabat numbering, actual position A119)
<220>
<221> misc_feature
<222> (119)..(119)
<223> Xaa = unnatural amino acid (nnAA)
<400> 27
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Thr Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Tyr Met Asn Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met
35 40 45
Gly Ile Ile Asn Pro Tyr Asn Gly Gly Thr His Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Ser Gly Tyr Asp Leu Tyr Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser Xaa Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
210 215 220
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
225 230 235 240
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
275 280 285
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
290 295 300
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
355 360 365
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
385 390 395 400
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
405 410 415
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445

Claims (53)

1.一种式(I)化合物：1. A compound of formula (I): 或其药学上可接受的盐、溶剂化物、立体异构体或互变异构体，其中or a pharmaceutically acceptable salt, solvate, stereoisomer or tautomer thereof, wherein A是CH或N；A is CH or N; X是O-R1、NH-R1、S-R1或H；X is O-R1, NH-R1, S-R1 or H; YY是-ONH2、-N3、-OH、马来酰亚胺、-COOH或-C(＝O)CH2Y1，其中Y1是卤化物；YY is -ONH2 , -N3 , -OH, maleimide, -COOH, or -C(=O) CH2Y1 , wherein Y1 is a halide; L1和L2中的每一个独立地为(CH2)m、(CH2)mC(＝O)、(CH2)m-NH(CH2)n、(CH2)m-C(＝O)NH(CH2)n、(CH2)m-OC(＝O)-NH-(CH2)n、(CH2)m-NHC(＝O)-NH-(CH2)n、(CH2)m-NH、(CH2)m-NHC(＝O)、(CH2)m-NHC(＝O)-(CH2)n-NHC(＝O)-(CH2)p、C(＝O)-(CH2)n、C3-C8杂环或不存在；其中m、n和p中的每一个独立地为0至12的整数；Each of L1 and L2 is independently (CH 2 ) m , (CH 2 ) m C(=O), (CH 2 ) m -NH(CH 2 ) n , (CH 2 ) m -C(=O )NH(CH 2 ) n , (CH 2 ) m -OC(=O)-NH-(CH 2 ) n , (CH 2 ) m -NHC(=O)-NH-(CH 2 ) n , (CH 2 ) m -NH, (CH 2 ) m -NHC(=O), (CH 2 ) m -NHC(=O)-(CH 2 ) n -NHC(=O)-(CH 2 ) p , C( =O)-(CH 2 ) n , C 3 -C 8 heterocycle or absent; wherein each of m, n and p is independently an integer from 0 to 12; R1是H、C1-C12烷基、取代的C1-C12烷基、含氧C1-C12烷基、C3-C8杂环烷基、取代的C3-C8杂环烷基、C3-C8环烷基、取代的C3-C8环烷基、-N3末端取代的C1-C12烷基、(CH2)q-(OCH2CH2)r-OMe，其中q和r中的每一个独立地为0至12的整数；R1 is H, C 1 -C 12 alkyl, substituted C 1 -C 12 alkyl, oxygen-containing C 1 -C 12 alkyl, C 3 -C 8 heterocycloalkyl, substituted C 3 -C 8 hetero Cycloalkyl, C 3 -C 8 cycloalkyl, substituted C 3 -C 8 cycloalkyl, -N 3 terminally substituted C 1 -C 12 alkyl, (CH 2 ) q -(OCH 2 CH 2 ) r -OMe, wherein each of q and r is independently an integer from 0 to 12; R2是C1-C6亚烷基、C1-C12取代的亚烷基、C3-C8亚环烷基、C3-C8取代的亚环烷基、亚芳基、取代的C6-C10亚芳基、包含1-3个杂原子的5-12元亚杂芳基、包含1-3个杂原子的取代的5-12元亚杂芳基或(OCH2CH2)ss，或其组合，或R2不存在；其中ss是1至12的整数，其中每个杂原子独立地为N、O或S；R2 is C 1 -C 6 alkylene, C 1 -C 12 substituted alkylene, C 3 -C 8 cycloalkylene, C 3 -C 8 substituted cycloalkylene, arylene, substituted C 6 -C 10 arylene, 5-12 membered heteroarylene containing 1-3 heteroatoms, substituted 5-12 membered heteroarylene containing 1-3 heteroatoms or (OCH 2 CH 2 ) ss , or a combination thereof, or R2 is absent; wherein ss is an integer from 1 to 12, wherein each heteroatom is independently N, O, or S; R3是氨基酸的侧链、C1-C6亚烷基、C1-C6取代的亚烷基、C3-C8亚环烷基、C3-C8亚杂环烷基、取代的C3-C8亚环烷基、亚芳基、取代的亚芳基、包含1-3个杂原子的5-12元亚杂芳基、包含1-3个杂原子的取代的5-12元亚杂芳基、含有氨基的C1-C12亚烷基、含有羰基的C1-C12亚烷基、含氧C1-C12亚烷基、-N3末端C1-C6亚烷基、-CCH末端C1-C6亚烷基、-SH末端C1-C6亚烷基、-OH末端C1-C6亚烷基、含氮C1-C6亚烷基、-OPO3H2末端C1-C6亚烷基、-OPO3H2末端亚芳基、葡糖苷酸末端C1-C6亚烷基、-N3末端亚芳基、乙炔末端亚芳基、胺末端亚芳基、(CH2)s、(CH2)s-C(＝O)、(CH2)s-NH(CH2)t、(CH2)s-C(＝O)NH(CH2)t、(CH2)S-OC(＝O)-NH-(CH2)t、(CH2)s-NHC(＝O)-NH-(CH2)t或其组合；或R3不存在；其中每个s和t独立地为0至6的整数；R3 is the side chain of an amino acid, C 1 -C 6 alkylene, C 1 -C 6 substituted alkylene, C 3 -C 8 cycloalkylene, C 3 -C 8 heterocycloalkylene, substituted C 3 -C 8 cycloalkylene, arylene, substituted arylene, 5-12 membered heteroarylene containing 1-3 heteroatoms, substituted 5-12 containing 1-3 heteroatoms Elementary heteroarylene group, C 1 -C 12 alkylene group containing amino group, C 1 -C 12 alkylene group containing carbonyl group, C 1 -C 12 alkylene group containing oxygen, -N 3 terminal C 1 -C 6 Alkylene, -CCH terminal C 1 -C 6 alkylene, -SH terminal C 1 -C 6 alkylene, -OH terminal C 1 -C 6 alkylene, nitrogen-containing C 1 -C 6 alkylene , -OPO 3 H 2 terminal C 1 -C 6 alkylene, -OPO 3 H 2 terminal arylene, glucuronide terminal C 1 -C 6 alkylene, -N 3 terminal arylene, acetylene terminal Aryl, amine-terminated arylene, (CH 2 ) s , (CH 2 ) s -C(=O), (CH 2 ) s -NH(CH 2 ) t , (CH 2 ) s -C(=O )NH( CH2 ) t , ( CH2 ) S -OC(=O)-NH-( CH2 ) t , ( CH2 ) s -NHC(=O)-NH-( CH2 ) t , or combinations thereof ; or R3 does not exist; wherein each s and t is independently an integer from 0 to 6; R4是H、C3-C8环烷基、C3-C8杂环烷基、C3-C8取代的杂环烷基、芳基、取代的芳基、(CH2)u-(OCH2CH2)v-OMe、二/三支链(CH2)u-(OCH2CH2)v-OMe或其组合；或R4不存在；其中每个u和v独立地为1至48的整数。R4 is H, C 3 -C 8 cycloalkyl, C 3 -C 8 heterocycloalkyl, C 3 -C 8 substituted heterocycloalkyl, aryl, substituted aryl, (CH 2 ) u -( OCH 2 CH 2 ) v -OMe, di/triple branched (CH 2 ) u -(OCH 2 CH 2 ) v -OMe or combinations thereof; or R4 is absent; wherein each u and v independently range from 1 to 48 an integer of . 2.如权利要求1所述的化合物或其药学上可接受的盐、溶剂化物、立体异构体或互变异构体，其中R4包含PEG部分。2. The compound of claim 1, or a pharmaceutically acceptable salt, solvate, stereoisomer or tautomer thereof, wherein R4 comprises a PEG moiety. 3.如权利要求1或2所述的化合物或其药学上可接受的盐、溶剂化物、立体异构体或互变异构体，其中所述PEG部分是线性的、分支的或多臂的。3. The compound of claim 1 or 2, or a pharmaceutically acceptable salt, solvate, stereoisomer or tautomer thereof, wherein the PEG moiety is linear, branched or multiarmed . 4.如权利要求2所述的化合物或其药学上可接受的盐、溶剂化物、立体异构体或互变异构体，其中R4包含(CH2)u-(OCH2CH2)v-OMe。4. The compound of claim 2 or a pharmaceutically acceptable salt, solvate, stereoisomer or tautomer thereof, wherein R4 comprises (CH 2 ) u -(OCH 2 CH 2 ) v - O Me. 5.如权利要求4所述的化合物或其药学上可接受的盐、溶剂化物、立体异构体或互变异构体，其中v是1至48的整数，其中u是1至12的整数，并且其中ss独立地为1至12的整数。5. The compound of claim 4 or a pharmaceutically acceptable salt, solvate, stereoisomer or tautomer thereof, wherein v is an integer from 1 to 48, and wherein u is an integer from 1 to 12 , and wherein ss is independently an integer from 1 to 12. 6.如权利要求4所述的化合物或其药学上可接受的盐、溶剂化物、立体异构体或互变异构体，其中v是1至12的整数，其中u是1至12的整数，并且其中ss独立地为1至12的整数。6. The compound of claim 4 or a pharmaceutically acceptable salt, solvate, stereoisomer or tautomer thereof, wherein v is an integer from 1 to 12, and wherein u is an integer from 1 to 12 , and wherein ss is independently an integer from 1 to 12. 7.如权利要求1所述的化合物或其药学上可接受的盐、溶剂化物、立体异构体或互变异构体，其中R3包含接头。7. The compound of claim 1, or a pharmaceutically acceptable salt, solvate, stereoisomer or tautomer thereof, wherein R3 comprises a linker. 8.如权利要求7所述的化合物或其药学上可接受的盐、溶剂化物、立体异构体或互变异构体，其中所述接头包含-ONH2末端或马来酰亚胺末端或COOH末端或卤代乙酰基末端，每个末端均具有(CH2)m-(OCH2CH2)n-，其中m和n中的每一个独立地为1至12的整数。8. The compound or pharmaceutically acceptable salt thereof, solvate, stereoisomer or tautomer as claimed in claim 7, wherein said linker comprises -ONH 2 end or maleimide end or A COOH terminal or a haloacetyl terminal, each having (CH 2 )m-(OCH 2 CH 2 )n-, wherein each of m and n is an integer of 1 to 12 independently. 9.如权利要求2所述的化合物或其药学上可接受的盐、溶剂化物、立体异构体或互变异构体，其中所述PEG部分的分子量为0.1kDa至100kDa或1kDa至100kDa。9. The compound of claim 2, or a pharmaceutically acceptable salt, solvate, stereoisomer or tautomer thereof, wherein the PEG moiety has a molecular weight of 0.1 kDa to 100 kDa or 1 kDa to 100 kDa. 10.如权利要求2所述的化合物或其药学上可接受的盐、溶剂化物、立体异构体或互变异构体，其中所述PEG部分的分子量为0.1kDa至50kDa或1kDa至50kDa。10. The compound of claim 2, or a pharmaceutically acceptable salt, solvate, stereoisomer or tautomer thereof, wherein the PEG moiety has a molecular weight of 0.1 kDa to 50 kDa or 1 kDa to 50 kDa. 11.如权利要求1所述的化合物或其药学上可接受的盐、溶剂化物、立体异构体或互变异构体，其中A是CH。11. The compound of claim 1, or a pharmaceutically acceptable salt, solvate, stereoisomer or tautomer thereof, wherein A is CH. 12.如权利要求1所述的化合物或其药学上可接受的盐、溶剂化物、立体异构体或互变异构体，其中所述化合物选自表4。12. The compound of claim 1 or a pharmaceutically acceptable salt, solvate, stereoisomer or tautomer thereof, wherein the compound is selected from Table 4. 13.如权利要求1所述的化合物或其药学上可接受的盐、溶剂化物、立体异构体或互变异构体，其中所述化合物是表4中公开的化合物185、化合物186、化合物187、化合物188、化合物189、化合物190、化合物191、化合物213、化合物214、化合物216、化合物217、化合物218、化合物219、化合物220、化合物221、化合物222、化合物223、化合物224、化合物230、化合物233、化合物235、化合物238、化合物239、化合物240、化合物242、化合物244、化合物245、化合物246、化合物248、化合物251、化合物252、化合物253、化合物254、化合物255、化合物256、化合物257、化合物258、化合物259、化合物260、化合物261、化合物263、化合物265、化合物266、化合物267、化合物268、化合物269、化合物272、化合物273、化合物275、化合物278、化合物279、化合物281、化合物282、化合物283、化合物284、化合物285、化合物286、化合物287、化合物296、化合物297、化合物299、化合物300、化合物301、化合物302、化合物303或化合物304。13. The compound or its pharmaceutically acceptable salt, solvate, stereoisomer or tautomer as claimed in claim 1, wherein said compound is compound 185, compound 186, compound disclosed in table 4 187, Compound 188, Compound 189, Compound 190, Compound 191, Compound 213, Compound 214, Compound 216, Compound 217, Compound 218, Compound 219, Compound 220, Compound 221, Compound 222, Compound 223, Compound 224, Compound 230, Compound 233, Compound 235, Compound 238, Compound 239, Compound 240, Compound 242, Compound 244, Compound 245, Compound 246, Compound 248, Compound 251, Compound 252, Compound 253, Compound 254, Compound 255, Compound 256, Compound 257 , Compound 258, Compound 259, Compound 260, Compound 261, Compound 263, Compound 265, Compound 266, Compound 267, Compound 268, Compound 269, Compound 272, Compound 273, Compound 275, Compound 278, Compound 279, Compound 281, Compound 282, Compound 283, Compound 284, Compound 285, Compound 286, Compound 287, Compound 296, Compound 297, Compound 299, Compound 300, Compound 301, Compound 302, Compound 303, or Compound 304. 14.一种式(II)化合物：14. A compound of formula (II): 或其药学上可接受的盐、溶剂化物、立体异构体或互变异构体，其中or a pharmaceutically acceptable salt, solvate, stereoisomer or tautomer thereof, wherein A是CH或N；A is CH or N; X是O-R1、NH-R1、S-R1或H；X is O-R1, NH-R1, S-R1 or H; YY是H、-ONH2、-N3、-OH、马来酰亚胺、-COOH或-C(＝O)CH2Y1，其中Y1是卤化物；YY is H, -ONH2 , -N3 , -OH, maleimide, -COOH, or -C(=O) CH2Y1 , wherein Y1 is a halide; L1和L2中的每一个独立地为(CH2)m、(CH2)mC(＝O)、(CH2)m-NH(CH2)n、(CH2)m-C(＝O)NH(CH2)n、(CH2)m-OC(＝O)-NH-(CH2)n、(CH2)m-NHC(＝O)-NH-(CH2)n、(CH2)m-NH、(CH2)m-NHC(＝O)、(CH2)m-NHC(＝O)-(CH2)n-NHC(＝O)-(CH2)p、C(＝O)-(CH2)n、亚芳基、取代的亚芳基、包含1-3个杂原子的5-12元亚杂芳基、包含1-3个杂原子的取代的5-12元亚杂芳基、包含1-3个杂原子的C3-C8杂环或不存在；其中m、n和p中的每一个独立地为0至6的整数，其中每个杂原子独立地为N、O或S；Each of L1 and L2 is independently (CH 2 ) m , (CH 2 ) m C(=O), (CH 2 ) m -NH(CH 2 ) n , (CH 2 ) m -C(=O )NH(CH 2 ) n , (CH 2 ) m -OC(=O)-NH-(CH 2 ) n , (CH 2 ) m -NHC(=O)-NH-(CH 2 ) n , (CH 2 ) m -NH, (CH 2 ) m -NHC(=O), (CH 2 ) m -NHC(=O)-(CH 2 ) n -NHC(=O)-(CH 2 ) p , C( =O)-(CH 2 ) n , arylene, substituted arylene, 5-12 membered heteroarylene containing 1-3 heteroatoms, substituted 5-12 containing 1-3 heteroatoms Member heteroarylene, C 3 -C 8 heterocycle containing 1-3 heteroatoms or nonexistent; wherein m, n and p are each independently an integer from 0 to 6, wherein each heteroatom is independently Ground is N, O or S; L3是C(＝O)、-CH(R5)-、-(AA)i-或亚芳基，或其组合，或L3不存在；其中每个AA独立地为氨基酸，其中i是1至6的整数；L3 is C(=O), -CH(R5)-, -(AA) i- , or arylene, or a combination thereof, or L3 is absent; wherein each AA is independently an amino acid, wherein i is 1 to 6 an integer of R5是NH-L4-Y2或CH2-L4-Y2，其中Y2是H或不存在；R5 is NH-L4-Y2 or CH2 - L4-Y2, wherein Y2 is H or absent; L4是C(＝O)、C(＝O)O-、-OC(＝O)-、-C(CH2O)3-、-C(CH2CH2O)3-、L4 is C(=O), C(=O)O-, -OC(=O)-, -C(CH 2 O) 3 -, -C(CH 2 CH 2 O) 3 -, -(AA)j-、亚芳基、取代的亚芳基、C3-C8亚环烷基、C3-C8取代的亚环烷基、亚芳基、取代的亚芳基、包含1-3个杂原子的5-12元亚杂芳基、包含1-3个杂原子的取代的5-12元亚杂芳基、包含1-3个杂原子的5-12元亚杂环烷基、包含1-3个杂原子的取代的5-12元亚杂环烷基、C1-C12亚烷基、-O-、-NH-、-S-、取代的C1-C12亚烷基、-(CH2)s-(OCH2CH2)t-(CH2)u-、(CH2)s-(OCH2CH2)t-OMe、-N3、-SH、-OH、-NH2、-OPO3H2、葡糖苷酸、乙炔或其组合，或L4不存在；其中每个AA独立地为氨基酸，其中j是1至6的整数，其中s和u中的每一个独立地为0至12的整数，其中t独立地为0至48的整数，其中每个杂原子独立地为N、O或S；-(AA) j- , arylene, substituted arylene, C 3 -C 8 cycloalkylene, C 3 -C 8 substituted cycloalkylene, arylene, substituted arylene, including 5-12 membered heteroarylene containing 1-3 heteroatoms, substituted 5-12 membered heteroarylene containing 1-3 heteroatoms, 5-12 membered heteroarylene containing 1-3 heteroatoms Alkyl, substituted 5-12 membered heterocycloalkylene containing 1-3 heteroatoms, C 1 -C 12 alkylene, -O-, -NH-, -S-, substituted C 1 -C 12 Alkylene, -(CH 2 ) s -(OCH 2 CH 2 ) t -(CH 2 ) u -, (CH 2 ) s -(OCH 2 CH 2 ) t -OMe, -N 3 , -SH, -OH, -NH 2 , -OPO 3 H 2 , glucuronide, acetylene, or combinations thereof, or L4 is absent; wherein each AA is independently an amino acid, wherein j is an integer from 1 to 6, wherein s and u are Each of is independently an integer of 0 to 12, wherein t is independently an integer of 0 to 48, wherein each heteroatom is independently N, O, or S; R1是H、C1-C12烷基、取代的C1-C12烷基、含氧C1-C12烷基、C3-C8杂环烷基、取代的C3-C8杂环烷基、C3-C8环烷基、取代的C3-C8环烷基、-N3末端取代的C1-C12烷基、(CH2)q-(OCH2CH2)r-OMe；其中q和r中的每一个独立地为0至12的整数；R1 is H, C 1 -C 12 alkyl, substituted C 1 -C 12 alkyl, oxygen-containing C 1 -C 12 alkyl, C 3 -C 8 heterocycloalkyl, substituted C 3 -C 8 hetero Cycloalkyl, C 3 -C 8 cycloalkyl, substituted C 3 -C 8 cycloalkyl, -N 3 terminally substituted C 1 -C 12 alkyl, (CH 2 ) q -(OCH 2 CH 2 ) r -OMe; wherein each of q and r is independently an integer from 0 to 12; R2是C1-C6亚烷基、C1-C12取代的亚烷基、C3-C8亚环烷基、C3-C8取代的亚环烷基、亚芳基、取代的亚芳基、包含1-3个杂原子的5-12元亚杂芳基、包含1-3个杂原子的取代的5-12元亚杂芳基、包含1-3个杂原子的5-12元亚杂环烷基、包含1-3个杂原子的取代的5-12元亚杂环烷基或(OCH2CH2)r，或其组合，或R2不存在；其中r是1至12的整数，其中每个杂原子独立地为N、O或S；R2 is C 1 -C 6 alkylene, C 1 -C 12 substituted alkylene, C 3 -C 8 cycloalkylene, C 3 -C 8 substituted cycloalkylene, arylene, substituted Arylene, 5-12 membered heteroarylene containing 1-3 heteroatoms, substituted 5-12 membered heteroarylene containing 1-3 heteroatoms, 5-12 membered heteroarylene containing 1-3 heteroatoms 12-membered heterocycloalkylene, substituted 5-12 membered heterocycloalkylene containing 1-3 heteroatoms or (OCH 2 CH 2 ) r , or a combination thereof, or R is absent; wherein r is 1 to An integer of 12, wherein each heteroatom is independently N, O or S; R3是H或-C(＝O)R6、-C(＝O)OR6；R3 is H or -C(=O)R6, -C(=O)OR6; R6是C1-C12烷基、取代的烷基、取代的芳基、CH3-(CH2)s-(OCH2CH2)t-(CH2)u-，其中s、t和u中的每一个独立地为0至12的整数。R6 is C 1 -C 12 alkyl, substituted alkyl, substituted aryl, CH 3 -(CH 2 ) s -(OCH 2 CH 2 ) t -(CH 2 ) u -, where s, t and u Each of is independently an integer from 0 to 12. 15.如权利要求14所述的化合物或其药学上可接受的盐、溶剂化物、立体异构体或互变异构体，其中所述化合物包含PEG部分。15. The compound of claim 14, or a pharmaceutically acceptable salt, solvate, stereoisomer or tautomer thereof, wherein the compound comprises a PEG moiety. 16.如权利要求15所述的化合物或其药学上可接受的盐、溶剂化物、立体异构体或互变异构体，其中所述PEG部分是线性的、分支的或多臂的。16. The compound of claim 15, or a pharmaceutically acceptable salt, solvate, stereoisomer or tautomer thereof, wherein the PEG moiety is linear, branched or multiarmed. 17.如权利要求14所述的化合物或其药学上可接受的盐、溶剂化物、立体异构体或互变异构体，其中L3是-CH(R5)-，其中R5是NH-L4-Y2或CH2-L4-Y2，其中Y2不存在，其中L4包含(CH2)s-(OCH2CH2)t-OMe，其中s是1至12的整数，其中t是1至48的整数。17. The compound of claim 14, or a pharmaceutically acceptable salt, solvate, stereoisomer or tautomer thereof, wherein L3 is -CH(R5)-, wherein R5 is NH-L4- Y2 or CH2 -L4-Y2, where Y2 is absent, where L4 comprises ( CH2 ) s- ( OCH2CH2 ) t -OMe, where s is an integer from 1 to 12, and where t is an integer from 1 to 48 . 18.如权利要求17所述的化合物或其药学上可接受的盐、溶剂化物、立体异构体或互变异构体，其中t是1至12的整数。18. The compound of claim 17, or a pharmaceutically acceptable salt, solvate, stereoisomer or tautomer thereof, wherein t is an integer from 1 to 12. 19.如权利要求14所述的化合物或其药学上可接受的盐、溶剂化物、立体异构体或互变异构体，其中YY是-ONH2、马来酰亚胺、-COOH或-C(＝O)CH2Y1，其中Y1是卤化物。19. The compound of claim 14, or a pharmaceutically acceptable salt, solvate, stereoisomer or tautomer thereof, wherein YY is -ONH 2 , maleimide, -COOH or - C(=O) CH2Y1 , where Y1 is a halide. 20.如权利要求19所述的化合物或其药学上可接受的盐、溶剂化物、立体异构体或互变异构体，其中R2是(CH2)m(OCH2CH2)r，其中m和r中的每一个独立地为1至12的整数。20. The compound of claim 19, or a pharmaceutically acceptable salt, solvate, stereoisomer or tautomer thereof, wherein R2 is (CH 2 ) m (OCH 2 CH 2 ) r , wherein Each of m and r is an integer of 1 to 12 independently. 21.如权利要求14所述的化合物或其药学上可接受的盐、溶剂化物、立体异构体或互变异构体，其中A是CH。21. The compound of claim 14, or a pharmaceutically acceptable salt, solvate, stereoisomer or tautomer thereof, wherein A is CH. 22.如权利要求15所述的化合物或其药学上可接受的盐、溶剂化物、立体异构体或互变异构体，其中所述PEG部分的分子量为0.1kDa至100kDa或1kDa至100kDa。22. The compound of claim 15, or a pharmaceutically acceptable salt, solvate, stereoisomer or tautomer thereof, wherein the PEG moiety has a molecular weight of 0.1 kDa to 100 kDa or 1 kDa to 100 kDa. 23.如权利要求15所述的化合物或其药学上可接受的盐、溶剂化物、立体异构体或互变异构体，其中所述PEG部分的分子量为0.1kDa至50kDa或1kDa至50kDa。23. The compound of claim 15, or a pharmaceutically acceptable salt, solvate, stereoisomer or tautomer thereof, wherein the PEG moiety has a molecular weight of 0.1 kDa to 50 kDa or 1 kDa to 50 kDa. 24.一种免疫缀合物，其包含a)抗体或抗体片段；b)包含缀合至所述抗体或抗体片段的化合物的TLR激动剂，其中所述TLR激动剂包含根据权利要求1至23中任一项所述的化合物或其药学上可接受的盐、溶剂化物、立体异构体或互变异构体或所述化合物的衍生物，其中所述化合物的衍生物经由所述化合物的部分YY直接或经由接头XX缀合至所述抗体或所述抗体片段，其中所述接头XX是亲水性接头、可裂解接头或不可裂解接头。24. An immunoconjugate comprising a) an antibody or antibody fragment; b) a TLR agonist comprising a compound conjugated to said antibody or antibody fragment, wherein said TLR agonist comprises a compound according to claims 1 to 23 Any one of the compound or its pharmaceutically acceptable salt, solvate, stereoisomer or tautomer or derivative of the compound, wherein the derivative of the compound is Moiety YY is conjugated to said antibody or said antibody fragment directly or via a linker XX, wherein said linker XX is a hydrophilic linker, a cleavable linker or a non-cleavable linker. 25.如权利要求24所述的免疫缀合物，其中所述接头XX包含亚烷基、亚烯基、亚炔基、聚醚、聚酯、聚酰胺、聚氨基酸、多肽、可裂解肽或氨基苯甲基氨基甲酸酯或其组合。25. The immunoconjugate of claim 24, wherein the linker XX comprises an alkylene, alkenylene, alkynylene, polyether, polyester, polyamide, polyamino acid, polypeptide, cleavable peptide or Aminobenzyl carbamates or combinations thereof. 26.如权利要求24所述的免疫缀合物，其中所述抗体或所述抗体片段结合至细胞的抗原。26. The immunoconjugate of claim 24, wherein said antibody or said antibody fragment binds to an antigen of a cell. 27.如权利要求24所述的免疫缀合物，其中所述抗体或所述抗体片段结合至细胞表面靶标或肿瘤细胞靶标。27. The immunoconjugate of claim 24, wherein said antibody or said antibody fragment binds to a cell surface target or a tumor cell target. 28.如权利要求24所述的免疫缀合物，其中所述抗体或所述抗体片段包含Fc融合蛋白。28. The immunoconjugate of claim 24, wherein said antibody or said antibody fragment comprises an Fc fusion protein. 29.如权利要求24所述的免疫缀合物，其中所述抗体或抗体片段是单特异性、双特异性或多特异性的。29. The immunoconjugate of claim 24, wherein the antibody or antibody fragment is monospecific, bispecific or multispecific. 30.如权利要求24所述的免疫缀合物，其中所述抗体或抗体片段结合至选自由以下各项组成的组的靶标：HER2、HER3、B7-H3、连接蛋白-4、PD-1、PDL-1、EGFR、TROP2、FOLR1、PSMA、BCMA、FLT3、VEGFR、CTLA-4、EpCAM、MUC1、MUC16、NaPi2b、c-Met、GPC3、ENPP3、TIM-3、VISTA、VEGF、封闭蛋白18.2、FGFR2、FOLR1、STEAP1、间皮素、5T4、CEA、CA9、钙粘蛋白6、ROR1、LIV-1、LILRB-1、LRP-1、SLC34A2、SLC39A6、SLC44A4、LY6E、DLL3、ePhA2、TGFbR、PRLR、GPNMB、SLITRK6、SIRPa、CD3、CD19、CD20、CD22、CD24、CD25、CD30、CD33、CD37、CD38、CD44、CD47、CD52、CD56、CD70、CD79b、CD96、CD97、CD99、CD117、CD123、CD179、CD223和CD276。30. The immunoconjugate of claim 24, wherein the antibody or antibody fragment binds to a target selected from the group consisting of: HER2, HER3, B7-H3, connexin-4, PD-1 , PDL-1, EGFR, TROP2, FOLR1, PSMA, BCMA, FLT3, VEGFR, CTLA-4, EpCAM, MUC1, MUC16, NaPi2b, c-Met, GPC3, ENPP3, TIM-3, VISTA, VEGF, occludin 18.2 , FGFR2, FOLR1, STEAP1, Mesothelin, 5T4, CEA, CA9, Cadherin 6, ROR1, LIV-1, LILRB-1, LRP-1, SLC34A2, SLC39A6, SLC44A4, LY6E, DLL3, ePhA2, TGFbR, PRLR, GPNMB, SLITRK6, SIRPa, CD3, CD19, CD20, CD22, CD24, CD25, CD30, CD33, CD37, CD38, CD44, CD47, CD52, CD56, CD70, CD79b, CD96, CD97, CD99, CD117, CD123, CD179, CD223 and CD276. 31.如权利要求24所述的免疫缀合物，其中所述抗体或抗体片段是抗HER2、抗CD70或抗PSMA，或抗TROP2抗体或片段。31. The immunoconjugate of claim 24, wherein the antibody or antibody fragment is an anti-HER2, anti-CD70 or anti-PSMA, or an anti-TROP2 antibody or fragment. 32.如权利要求31所述的免疫缀合物，其中所述抗HER2抗体或抗体片段包含：a)选自SEQ ID NO:1、2、3、4、16、17或18的重链可变区；和b)选自SEQ ID NO:5、6、7、8、9、10、11、12、13、14或15的轻链可变区。32. The immunoconjugate of claim 31, wherein said anti-HER2 antibody or antibody fragment comprises: a) a heavy chain selected from SEQ ID NO: 1, 2, 3, 4, 16, 17 or 18 can be and b) a light chain variable region selected from SEQ ID NO:5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. 33.如权利要求24所述的免疫缀合物，其中所述抗体或抗体片段包含一个或多个Fc突变。33. The immunoconjugate of claim 24, wherein the antibody or antibody fragment comprises one or more Fc mutations. 34.如权利要求24所述的免疫缀合物，其中所述抗体或抗体片段包含掺入所述重链、轻链或所述重链和所述轻链两者中的一种或多种非天然编码氨基酸。34. The immunoconjugate of claim 24, wherein the antibody or antibody fragment comprises one or more of the heavy chain, light chain, or both of the heavy chain and the light chain incorporated Non-naturally encoded amino acids. 35.如权利要求24所述的免疫缀合物，其中所述一种或多种非天然编码氨基酸是对乙酰基苯丙氨酸、对硝基苯丙氨酸、对磺基酪氨酸、对羧基苯丙氨酸、邻硝基苯丙氨酸、间硝基苯丙氨酸、对硼酰基苯丙氨酸、邻硼酰基苯丙氨酸、间硼酰基苯丙氨酸、对氨基苯丙氨酸、邻氨基苯丙氨酸、间氨基苯丙氨酸、对酰基苯丙氨酸、邻酰基苯丙氨酸、间酰基苯丙氨酸、对OMe苯丙氨酸、邻OMe苯丙氨酸、间OMe苯丙氨酸、对磺基苯丙氨酸、邻磺基苯丙氨酸、间磺基苯丙氨酸、5-硝基His、3-硝基Tyr、2-硝基Tyr、硝基取代的Leu、硝基取代的His、硝基取代的De、硝基取代的Trp、2-硝基Trp、4-硝基Trp、5-硝基Trp、6-硝基Trp、7-硝基Trp、3-氨基酪氨酸、2-氨基酪氨酸、O-磺基酪氨酸、2-磺氧基苯丙氨酸、3-磺氧基苯丙氨酸、邻羧基苯丙氨酸、间羧基苯丙氨酸、对乙酰基-L-苯丙氨酸、对炔丙基-苯丙氨酸、O-甲基-L-酪氨酸、L-3-(2-萘基)丙氨酸、3-甲基-苯丙氨酸、O-4-烯丙基-L-酪氨酸、4-丙基-L-酪氨酸、三-O-乙酰基-GlcNAcβ-丝氨酸、L-Dopa、氟化苯丙氨酸、异丙基-L-苯丙氨酸、对叠氮基-L-苯丙氨酸、对酰基-L-苯丙氨酸、对苯甲酰基-L-苯丙氨酸、L-磷酸丝氨酸、膦酰基丝氨酸、膦酰基酪氨酸、对碘苯丙氨酸、对溴苯丙氨酸、对氨基-L-苯丙氨酸、异丙基-L-苯丙氨酸和对炔丙基氧基-L-苯丙氨酸。35. The immunoconjugate of claim 24, wherein the one or more non-naturally encoded amino acids are p-acetylphenylalanine, p-nitrophenylalanine, p-sulfotyrosine, p-carboxyphenylalanine, o-nitrophenylalanine, m-nitrophenylalanine, p-boronylphenylalanine, o-boronylphenylalanine, m-boronylphenylalanine, p-aminobenzene Alanine, o-aminophenylalanine, m-aminophenylalanine, p-acylphenylalanine, o-acylphenylalanine, m-acylphenylalanine, p-OMe phenylalanine, ortho-OMe phenylalanine Amino acid, m-OMe phenylalanine, p-sulfophenylalanine, o-sulfophenylalanine, m-sulfophenylalanine, 5-nitroHis, 3-nitroTyr, 2-nitro Tyr, nitro substituted Leu, nitro substituted His, nitro substituted De, nitro substituted Trp, 2-nitro Trp, 4-nitro Trp, 5-nitro Trp, 6-nitro Trp, 7-NitroTrp, 3-aminotyrosine, 2-aminotyrosine, O-sulfotyrosine, 2-sulfoxyphenylalanine, 3-sulfoxyphenylalanine, o-carboxyl Phenylalanine, m-carboxyphenylalanine, p-acetyl-L-phenylalanine, p-propargyl-phenylalanine, O-methyl-L-tyrosine, L-3-(2 -naphthyl)alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl- GlcNAcβ-serine, L-Dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-phenyl Formyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-iodophenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, iso Propyl-L-phenylalanine and p-propargyloxy-L-phenylalanine. 36.如权利要求35所述的免疫缀合物，其中所述一种或多种非天然编码氨基酸是对乙酰基-苯丙氨酸、4-叠氮基-L-苯丙氨酸、对叠氮基乙氧基苯丙氨酸或对叠氮基甲基-苯丙氨酸。36. The immunoconjugate of claim 35, wherein the one or more non-naturally encoded amino acids are p-acetyl-phenylalanine, 4-azido-L-phenylalanine, p- Azidoethoxyphenylalanine or p-azidomethyl-phenylalanine. 37.如权利要求35所述的免疫缀合物，其中所述一种或多种非天然编码氨基酸是位点特异性掺入的。37. The immunoconjugate of claim 35, wherein the one or more non-naturally encoded amino acids are site-specifically incorporated. 38.如权利要求24所述的免疫缀合物，其中所述TLR激动剂是TLR7激动剂、TLR8激动剂或TLR7/TLR8双重激动剂。38. The immunoconjugate of claim 24, wherein the TLR agonist is a TLR7 agonist, a TLR8 agonist, or a TLR7/TLR8 dual agonist. 39.如权利要求24所述的免疫缀合物，其中所述TLR激动剂包含一个或多个PEG分子或部分。39. The immunoconjugate of claim 24, wherein the TLR agonist comprises one or more PEG molecules or moieties. 40.如权利要求39所述的免疫缀合物，其中所述一个或多个PEG分子是线性的、分支的、多臂的。40. The immunoconjugate of claim 39, wherein the one or more PEG molecules are linear, branched, multi-armed. 41.如权利要求39所述的免疫缀合物，其中所述一个或多个PEG分子为0.1kDa至100kDa。41. The immunoconjugate of claim 39, wherein the one or more PEG molecules are 0.1 kDa to 100 kDa. 42.如权利要求39所述的免疫缀合物，其中所述一个或多个PEG分子为0.1kDa至50kDa。42. The immunoconjugate of claim 39, wherein the one or more PEG molecules are 0.1 kDa to 50 kDa. 43.如权利要求24所述的免疫缀合物，其中所述接头XX是双官能或多官能接头。43. The immunoconjugate of claim 24, wherein the linker XX is a bifunctional or multifunctional linker. 44.如权利要求24所述的免疫缀合物，其中所述接头XX缀合至掺入所述抗体或抗体片段中的一种或多种非天然编码氨基酸。44. The immunoconjugate of claim 24, wherein the linker XX is conjugated to one or more non-naturally encoded amino acids incorporated into the antibody or antibody fragment. 45.如权利要求24所述的缀合物，其中所述接头XX是亲水性接头、可裂解接头或不可裂解接头。45. The conjugate of claim 24, wherein the linker XX is a hydrophilic linker, a cleavable linker or a non-cleavable linker. 46.一种治疗患有疾病或疾患的受试者或患者的方法，其包括向所述受试者或患者施用治疗有效量的如权利要求24-45中任一项所述的免疫缀合物。46. A method of treating a subject or patient suffering from a disease or disorder, comprising administering a therapeutically effective amount of the immunoconjugate according to any one of claims 24-45 to said subject or patient things. 47.如权利要求46所述的方法，其中所述疾病或疾患是自身免疫疾病、慢性炎性疾病或癌症。47. The method of claim 46, wherein the disease or condition is an autoimmune disease, a chronic inflammatory disease, or cancer. 48.如权利要求47所述的方法，其中所述癌症是乳腺癌、小细胞肺癌、卵巢癌、前列腺癌、胃癌、胃肠胰腺肿瘤、宫颈癌、食管癌、结肠癌、结直肠癌、上皮细胞源癌症或肿瘤、肾癌、脑癌、胶质母细胞瘤、胰腺癌、骨髓性白血病、甲状腺癌、子宫内膜癌、淋巴瘤、胰腺癌、头颈癌或皮肤癌。48. The method of claim 47, wherein the cancer is breast cancer, small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, gastroenteropancreatic tumor, cervical cancer, esophageal cancer, colon cancer, colorectal cancer, epithelial Cancer or tumor of cell origin, kidney cancer, brain cancer, glioblastoma, pancreatic cancer, myelogenous leukemia, thyroid cancer, endometrial cancer, lymphoma, pancreatic cancer, head and neck cancer, or skin cancer. 49.如权利要求48所述的方法，其还包括施用另一治疗剂。49. The method of claim 48, further comprising administering another therapeutic agent. 50.如权利要求48所述的方法，其中所述另一治疗剂是化学治疗剂、激素剂、抗肿瘤剂、免疫刺激剂、免疫调节剂、免疫治疗剂或其组合。50. The method of claim 48, wherein the other therapeutic agent is a chemotherapeutic agent, a hormonal agent, an antineoplastic agent, an immunostimulatory agent, an immunomodulatory agent, an immunotherapeutic agent, or a combination thereof. 51.一种药物组合物，其包含治疗有效量的如权利要求24-45中任一项所述的免疫缀合物以及药学上可接受的载剂或赋形剂。51. A pharmaceutical composition comprising a therapeutically effective amount of the immunoconjugate of any one of claims 24-45 and a pharmaceutically acceptable carrier or excipient. 52.一种药物组合物，其包含如权利要求1-23中任一项所述的化合物或其药学上可接受的盐、溶剂化物、立体异构体或互变异构体或如权利要求24-45中任一项所述的免疫缀合物，所述药物组合物用作药物。52. A pharmaceutical composition comprising a compound as claimed in any one of claims 1-23 or a pharmaceutically acceptable salt, solvate, stereoisomer or tautomer thereof or as claimed in claim The immunoconjugate of any one of 24-45, the pharmaceutical composition for use as a medicament. 53.如权利要求24-45中任一项所述的免疫缀合物在制造药物中的用途。53. Use of the immunoconjugate according to any one of claims 24-45 in the manufacture of a medicament.
CN202180071626.XA 2020-08-20 2021-08-20 antibody-TLR agonist conjugates, methods and uses thereof Pending CN116457023A (en)

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* Cited by examiner, † Cited by third party
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CN117826532A (en) * 2024-03-05 2024-04-05 烟台舜康生物科技有限公司 Preparation method of photoresist without light initiator
WO2025146173A1 (en) * 2024-01-05 2025-07-10 信达生物制药(苏州)有限公司 Antibody, and immunoconjugate comprising same and tlr7/8 agonist and use thereof

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025146173A1 (en) * 2024-01-05 2025-07-10 信达生物制药(苏州)有限公司 Antibody, and immunoconjugate comprising same and tlr7/8 agonist and use thereof
CN117826532A (en) * 2024-03-05 2024-04-05 烟台舜康生物科技有限公司 Preparation method of photoresist without light initiator
CN117826532B (en) * 2024-03-05 2024-05-07 烟台舜康生物科技有限公司 Preparation method of photoresist without light initiator

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