Conjugates, methods for their preparation and uses

Abstract

Provided is a conjugate, a preparation method and use thereof. The conjugate of Formula 1, M-[(L1) a -(L2) b -(D) c 1, wherein M is a biopolymer having a nucleophilic functional group, M is connected to L1 by using the nucleophilic functional group of M, D is a functional molecule, L2 is a linker, and L1 is a compound of Formula I.

Description

Background Art

[0001] Protein modification can endow proteins with new properties and functions. Common protein modification strategies involve modifying amino acid residues on proteins. Since lysine and cysteine have high affinity reactivity, protein modification usually targets these two amino acids.

[0002] In 2019, three antibody-drug conjugates, namely Fam-trastuzumab Deruxtecan (used to treat unresectable or metastatic HER2-positive breast cancer), Polatuzumab Vedotin (used to treat relapsed / refractory diffuse large B-cell lymphoma), and Enfortumab Vedotin (used to treat locally advanced or metastatic urothelial carcinoma), were approved by the FDA. This was achieved by modifying the reduced inter-chain disulfide bonds on the antibody, and the linker was maleimide. It is well known that the adduct of maleimide and thiol is unstable and prone to thiol exchange reactions under physiological conditions. Antibody-drug conjugates (ADCs) synthesized through the addition reaction between maleimide and thiol can lead to premature drug release, which may seriously affect the stability and efficacy of ADCs.

[0003] Although chemists have achieved results in improving the stability of maleimide and finding new stable linkers, the problem has not been overcome. Therefore, there is an urgent need to develop a linker that meets the preparation requirements of conjugates, especially for balancing activity and stability. Summary of the Invention

[0004] This application provides a possible linker, which is expected to be applied to the preparation of conjugates (such as ADCs). This linker can react with the nucleophilic functional groups of biomacromolecules (such as the -SH of cysteine) with relatively high efficiency and / or chemoselectivity. In this application, this linker and / or the conjugate containing this linker and / or prepared through this linker can remain stable in vitro and in vivo environments. For example, the conjugate (such as ADC) described in this application can kill target cells more effectively and / or efficiently because the conjugate (such as ADC) described in this application has better stability and / or safety (especially at relatively high concentrations). In this application, the described conjugate can solve the balance problem between activity and stability.

[0005] On the one hand, this application provides a conjugate of Formula 1, M-[(L1) a -(L2) b -(D) c 1, where L1 is a compound of Formula I, I, R is -F or -OH, where M is a biological macromolecule, and M is linked to L1 using a nucleophilic functional group of M, L2 is a linker, and L2 is linked to R1, R3 or R2, D is a functional molecule, a is an integer from 1 to 10, b and c are each independently an integer from 0 to 10, provided that b and c are not both 0, where R1 is H, an optionally substituted alkyl or an optionally substituted aryl, where R 1' is H or its isotope, where R2 is H, an optionally substituted alkyl or an optionally substituted aryl, where R3 is H, an optionally substituted alkyl or an optionally substituted aryl, optionally, the C connecting R1 and the C connecting R2 form a ring.

[0006] In certain embodiments, the nucleophilic functional group of M is selected from: -SH, -NH2, -SeH, -OH and

[0007] On the one hand, the present application provides a conjugate of formula 2, M-S-[(L1)a-(L2)b-(D)c]2, where M-S is a biological macromolecule having cysteine, and M-S is linked to L1 using the cysteine, L1 is a compound of formula I, I, R is -F or -OH, L2 is a linker, L2 is linked to R1, R3 or R2, D is a functional molecule, a is an integer from 1 to 10, b and c are each independently an integer from 0 to 10, provided that b and c are not both 0, where R1 is H, an optionally substituted alkyl or an optionally substituted aryl, where R 1' is H or its isotope, where R2 is H, an optionally substituted alkyl or an optionally substituted aryl, where R3 is H, an optionally substituted alkyl or an optionally substituted aryl, optionally, the C connecting R1 and the C connecting R2 form a ring.

[0008] In certain embodiments, in the conjugate of formula 1 and / or the conjugate of formula 2, M is selected from the group consisting of: protein, DNA, RNA and virus.

[0009] In certain embodiments, in the conjugate of formula 1 and / or the conjugate of formula 2, M is a biological macromolecule expressed on the cell surface.

[0010] In certain embodiments, in the conjugate of formula 1 and / or the conjugate of formula 2, M is an antigen-binding protein or a fragment thereof.

[0011] In certain embodiments, in the conjugate of formula 1 and / or the conjugate of formula 2, M is a monoclonal antibody, a chimeric antibody, a humanized antibody, a human engineered antibody, a human antibody, a single-chain antibody scFv or an antibody fragment.

[0012] In certain embodiments, in the conjugate of Formula 1 and / or the conjugate of Formula 2, L2 is selected from the group consisting of: a cleavable linker, a non-cleavable linker, a hydrophilic linker, a hydrophobic linker, a charged linker, an uncharged linker, and a dicarboxylic acid linker.

[0013] In certain embodiments, in the conjugate of Formula 1 and / or the conjugate of Formula 2, L2 is selected from the group consisting of: VC-PAB, N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl 4-(2-pyridyldithio)valerate (SPP), N-succinimidyl 4-(2-pyridyldithio)butyrate (SPDB), N-succinimidyl-4-(2-pyridyldithio)-2-sulfo-butyrate (sulfo-SPDB), N-succinimidyl iodoacetate (SIA), N-succinimidyl (4-iodoacetyl)aminobenzoate (SIAB), maleimide PEG NHS, N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC), N-sulfo-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (sulfo-SMCC), or 2,5-dioxopyrrolidin-1-yl 17-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5,8,11,14-tetraoxo-4,7,10,13-tetraazapentadecan-1-oate (CX1-1).

[0014] In certain embodiments, in the conjugate of Formula 1 and / or the conjugate of Formula 2, D has a biological function.

[0015] In certain embodiments, in the conjugate of Formula 1 and / or the conjugate of Formula 2, D and / or its derivative is capable of inhibiting the growth of tumor cells.

[0016] In certain embodiments, in the conjugate of Formula 1 and / or the conjugate of Formula 2, D is a drug.

[0017] In certain embodiments, in the conjugate of Formula 1 and / or the conjugate of Formula 2, D is selected from the group consisting of: V-ATPase inhibitors, pro-apoptotic agents, Bcl2 inhibitors, MCL1 inhibitors, HSP90 inhibitors, IAP inhibitors, mTor inhibitors, microtubule stabilizers, microtubule destabilizers, auristatins, dolastatins, maytansinoids, MetAP (methionine aminopeptidase), protein CRM1 nuclear export inhibitors, DPPIV inhibitors, proteasome inhibitors, mitochondrial phosphoryl transfer reaction inhibitors, protein synthesis inhibitors, kinase inhibitors, CDK2 inhibitors, CDK9 inhibitors, kinesin inhibitors, HDAC inhibitors, DNA damaging agents, DNA alkylating agents, DNA intercalating agents, DNA minor groove binders, DHFR inhibitors, nucleoside analogs, HDAC inhibitors; anthracyclines; NAMPT inhibitors; hydrophilic prodrugs; SN-38 glucuronide, etoposide phosphate; nitrogen mustards, proteasome inhibitors, cytokines, Toll-like receptor agonists, and STING agonists.

[0018] In certain embodiments, in the conjugate of Formula 1 and / or the conjugate of Formula 2, D is MMAE or a derivative thereof; melphalan or a derivative thereof; lenalidomide or a derivative thereof; IL-2 or a derivative thereof; novel interleukin-2 / 15 or a derivative thereof; T785 or a derivative thereof; or MSA-2 or a derivative thereof.

[0019] In certain embodiments, in the conjugate of Formula 1 and / or the conjugate of Formula 2, R 1' is -H.

[0020] In certain embodiments, in the conjugate of Formula 1 and / or the conjugate of Formula 2, R1 is -H.

[0021] In certain embodiments, in the conjugate of Formula 1 and / or the conjugate of Formula 2, R3 is -H.

[0022] In certain embodiments, in the conjugate of Formula 1 and / or the conjugate of Formula 2, R2 is an optionally substituted phenyl.

[0023] In certain embodiments, in the conjugate of Formula 1 and / or the conjugate of Formula 2, R2 is wherein R4 is selected from the group consisting of: -OH, -PO3H2, -SeH, -SH, optionally substituted alkyl-OH, optionally substituted alkyl-halogen, optionally substituted alkyl-N3, -B(OH)2, -halogen, -OTf, optionally substituted alkyl-NH2, -O-optionally substituted alkyl-C≡CH, -CO-NH-C≡CH-optionally substituted alkyl.

[0024] In certain embodiments, in the conjugate of Formula 1 and / or the conjugate of Formula 2, R2 is R4 is selected from the group consisting of: -OH, -PO3H2, -SeH, -SH, -CH2OH, -CH2Br, -CH2N3, -B(OH)2, -Br, -OTf, -CH2NH2, -Cl, -OCH2C≡CH or -CO-NH-C≡CH.

[0025] In certain embodiments, in the conjugate of Formula 1 and / or the conjugate of Formula 2, R2 is wherein R4 is -O-(CH2)n1-COO-R5, n1 is an integer from 1 to 10, and wherein R5 is selected from the group consisting of: and H.

[0026] In certain embodiments, in the conjugate of Formula 1 and / or the conjugate of Formula 2, R2 is wherein R4 is -O-(CH2)n2-CO-NH-R6, n2 is an integer from 1 to 10, wherein R6 is -(CH2)n3-CO-R7, n3 is an integer from 1 to 10, and wherein R7 is selected from the group consisting of:

[0027] In certain embodiments, in the conjugate of Formula 1 and / or the conjugate of Formula 2, R2 is wherein R4 is -O-(CH2)n2-CO-NH-R6, n2 is an integer from 1 to 10, wherein R6 is -(CH2)n4-R8, n4 is an integer from 1 to 10, and wherein R8 is selected from the group consisting of:

[0028] In certain embodiments, in the conjugate of Formula 1 and / or the conjugate of Formula 2, R2 is wherein R4 is -O-(CH2)n2-CO-NH-R6, n2 is an integer from 1 to 10, wherein R6 is -(CH2CH2-O)n5-(CH2)n6-NH-CO-O-R9, n5 is an integer from 1 to 10, n6 is an integer from 1 to 10, and wherein R9 is selected from the group consisting of H and

[0029] In certain embodiments, in the conjugate of Formula 1 and / or the conjugate of Formula 2, R2 is wherein R4 is -(OCH2CH2)n7-O-(CH2)n8-R 10 , n7 is an integer from 1 to 10, n8 is an integer from 1 to 10, and wherein R 10 is selected from the group consisting of: -COOH, -NH2 and

[0030] In certain embodiments, in the conjugate of Formula 1 and / or the conjugate of Formula 2, R2 is wherein R4 is -(OCH2CH2)n7-O-(CH2)n8-R 10 , n7 is an integer from 1 to 10, n8 is an integer from 1 to 10, wherein R 10 is selected from the group consisting of: -COOH, -NH2 and wherein R 11 is selected from the group consisting of: optionally substituted alkyl-halogen, optionally substituted alkyl-N and O-optionally substituted alkyl.

[0031] In certain embodiments, in the conjugate of Formula 1 and / or the conjugate of Formula 2, R2 is wherein R4 is -(OCH2CH2)n7-O-(CH2)n8-R 10 , n7 is an integer from 1 to 10, n8 is an integer from 1 to 10, wherein R 10 is selected from the group consisting of: -COOH, -NH2 and wherein R 11 is selected from the group consisting of: -CF3, -CN and -OCH3.

[0032] In certain embodiments, in the conjugate of Formula 1 and / or the conjugate of Formula 2, R2 is optionally substituted alkyl-CH=CH-R 12 , wherein R 12 is wherein R 13 is -CH2N3.

[0033] In certain embodiments, in the conjugate of Formula 1 and / or the conjugate of Formula 2, the ring is an optionally substituted cycloolefin, or an optionally substituted aryl-cycloolefin.

[0034] In certain embodiments, in the conjugate of Formula 1 and / or the conjugate of Formula 2, the ring is selected from the group consisting of:

[0035] In certain embodiments, in the conjugate of Formula 1 and / or the conjugate of Formula 2, L1 is selected from the group consisting of:

[0036]

[0037]

[0038] In certain embodiments, in the conjugate of Formula 1 and / or the conjugate of Formula 2, the conjugate is selected from:

[0039]

[0040]

[0041] On the other hand, the present application provides a conjugate of Formula 3, (L1) a -(L2) b -(D) c 3, wherein L1 is a compound of Formula III, L2 is a linker, and L2 is linked to R1, R3 or R2, D is a functional molecule, a is an integer from 1 to 10, b and c are each independently an integer from 0 to 10, provided that b and c are not both 0, wherein R1 is H, optionally substituted alkyl or optionally substituted aryl, wherein R2 is H, optionally substituted alkyl or optionally substituted aryl, wherein R3 is H, optionally substituted alkyl or optionally substituted aryl, optionally, the C connecting R1 and the C connecting R2 form a ring.

[0042] In certain embodiments, in the conjugate of Formula 3, L2 is selected from the group consisting of: cleavable linkers, non-cleavable linkers, hydrophilic linkers, hydrophobic linkers, charged linkers, uncharged linkers, and dicarboxylic acid linkers.

[0043] In certain embodiments, in the conjugate of Formula 3, L2 is selected from: VC-PAB, N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl 4-(2-pyridyldithio)valerate (SPP), N-succinimidyl 4-(2-pyridyldithio)butyrate (SPDB), N-succinimidyl-4-(2-pyridyldithio)-2-sulfo-butyrate (sulfo-SPDB), N-succinimidyl iodoacetate (SIA), N-succinimidyl (4-iodoacetyl)aminobenzoate (SIAB), maleimide PEG NHS, N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC), N-sulfo-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (sulfo-SMCC), or 2,5-dioxopyrrolidin-1-yl 17-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5,8,11,14-tetraoxo-4,7,10,13-tetraazadecane-1-carboxylate (CX1-1).

[0044] In certain embodiments, in the conjugate of Formula 3, D has a biological function.

[0045] In certain embodiments, in the conjugate of Formula 3, D and / or its derivatives are capable of inhibiting the growth of tumor cells.

[0046] In certain embodiments, in the conjugate of Formula 3, D is a drug.

[0047] In certain embodiments, in the conjugate of Formula 3, D is selected from the group consisting of: V-ATPase inhibitors, apoptosis promoters, Bcl2 inhibitors, MCL1 inhibitors, HSP90 inhibitors, IAP inhibitors, mTor inhibitors, microtubule stabilizers, microtubule destabilizers, auristatins, dolostatins, maytansinoids, MetAP (methionyl aminopeptidase), protein CRM1 nuclear export inhibitors, DPPIV inhibitors, proteasome inhibitors, mitochondrial phosphoryl transfer reaction inhibitors, protein synthesis inhibitors, kinase inhibitors, CDK2 inhibitors, CDK9 inhibitors, kinesin inhibitors, HDAC inhibitors, DNA damaging agents, DNA alkylating agents, DNA intercalating agents, DNA minor groove binders, DHFR inhibitors, nucleoside analogs, HDAC inhibitors; anthracyclines; NAMPT inhibitors; hydrophilic prodrugs; SN-38 glucuronide, etoposide phosphate; nitrogen mustards, proteasome inhibitors, cytokines, Toll-like receptor agonists, and STING agonists.

[0048] In certain embodiments, in the conjugate of Formula 3, D is MMAE or a derivative thereof; melphalan or a derivative thereof; lenalidomide or a derivative thereof; IL-2 or a derivative thereof; novel interleukin-2 / 15 or a derivative thereof; T785 or a derivative thereof; or MSA-2 or a derivative thereof.

[0049] In certain embodiments, in the conjugate of Formula 3, R1 is -H.

[0050] In certain embodiments, in the conjugate of Formula 3, R3 is -H.

[0051] In certain embodiments, in the conjugate of Formula 3, R2 is an optionally substituted phenyl.

[0052] In certain embodiments, in the conjugate of Formula 3, R2 is wherein R4 is selected from the group consisting of: -OH, -PO3H2, -SeH, -SH, optionally substituted alkyl-OH, optionally substituted alkyl-halogen, optionally substituted alkyl-N3, -B(OH)2, -halogen, -OTf, optionally substituted alkyl-NH2, -O-optionally substituted alkyl-C≡CH, -CO-NH-C≡CH-optionally substituted alkyl.

[0053] In certain embodiments, in the conjugate of Formula 3, R2 is wherein R4 is selected from the group consisting of: -OH, -PO3H2, -SeH, -SH, -CH2OH, -CH2Br, -CH2N3, -B(OH)2, -Br, -OTf, -CH2NH2, -Cl, -OCH2C≡CH or -CO-NH-C≡CH.

[0054] In certain embodiments, in the conjugate of Formula 3, R2 is wherein R4 is -O-(CH2)n1-COO-

[0055] R5, and n1 is an integer from 1 to 10, wherein R5 is selected from the group consisting of: and H.

[0056] In certain embodiments, in the conjugate of Formula 3, R2 is wherein R4 is -O-(CH2)n2-CO-NH-R6, n2 is an integer from 1 to 10, wherein R6 is -(CH2)n3-CO-R7, n3 is an integer from 1 to 10, wherein R7 is selected from the group consisting of:

[0057] In certain embodiments, in the conjugate of Formula 3, R2 is wherein R4 is -O-(CH2)n2-CO-NH-R6, n2 is an integer from 1 to 10, wherein R6 is -(CH2)n4-R8, n4 is an integer from 1 to 10, wherein R8 is selected from the group consisting of:

[0058] In certain embodiments, in the conjugate of Formula 3, R2 is wherein R4 is -O-(CH2)n2-CO-NH-R6, n2 is an integer from 1 to 10, wherein R6 is -(CH2CH2-O)n5-(CH2)n6-NH-CO-O-R9, n5 is an integer from 1 to 10, n6 is an integer from 1 to 10, wherein R9 is selected from the group consisting of H and

[0059] In certain embodiments, in the conjugate of Formula 3, R2 is wherein R4 is -(OCH2CH2)n7-O-(CH2)n8-R 10 , n7 is an integer from 1 to 10, n8 is an integer from 1 to 10, wherein R 10 is selected from the group consisting of: -COOH, -NH2, and

[0060] In certain embodiments, in the conjugate of Formula 3, R2 is wherein R4 is -(OCH2CH2)n7-O-(CH2)n8-R 10 , n7 is an integer from 1 to 10, n8 is an integer from 1 to 10, wherein R 10 is selected from: -COOH, -NH2, and wherein R 11 is selected from the group consisting of: optionally substituted alkyl-halogen, optionally substituted alkyl-N, and O-optionally substituted alkyl.

[0061] In certain embodiments, in the conjugate of Formula 3, R2 is wherein R4 is -(OCH2CH2)n7-O-(CH2)n8-R 10 , n7 is an integer from 1 to 10, n8 is an integer from 1 to 10, wherein R 10 is selected from the group consisting of: -COOH, -NH2, and wherein R 11 is selected from the group consisting of: -CF3, -CN, and -OCH3.

[0062] In certain embodiments, in the conjugate of Formula 3, R2 is optionally substituted alkyl-CH=CH-R 12 , wherein R 12 is wherein R 13 is -CH2N3.

[0063] In certain embodiments, in the conjugate of Formula 3, the ring is an optionally substituted cycloolefin, or an optionally substituted aryl-cycloolefin.

[0064] In certain embodiments, in the conjugate of Formula 3, the ring is selected from:

[0065] In certain embodiments, in the conjugate of Formula 3, L1 is selected from the group consisting of:

[0066]

[0067]

[0068]

[0069] In certain embodiments, in the conjugate of Formula 3, the conjugate is selected from:

[0070]

[0071]

[0072] On the other hand, the present application provides a method for preparing a conjugate, the method comprising the following steps: By conjugating the conjugate of Formula 3: 3 with M to obtain the conjugate of Formula 1: Wherein M is a biological macromolecule, and M is linked to L1 using a nucleophilic functional group of M, L2 is a linker, and L2 is linked to R1, R3 or R2 in Formula 1, D is a functional molecule, a is an integer from 1 to 10, b and c are each independently an integer from 0 to 10, provided that b and c are not both 0, wherein R1 is H, optionally substituted alkyl or optionally substituted aryl, wherein R 1' is H or an isotope thereof, wherein R2 is H, optionally substituted alkyl or optionally substituted aryl, wherein R3 is H, optionally substituted alkyl or optionally substituted aryl, optionally, the C connecting R1 and the C connecting R2 form a ring.

[0073] In certain embodiments, in the method, the nucleophilic functional group of M is selected from the group consisting of: -SH, -NH2, -SeH, -OH and

[0074] A method for preparing a conjugate, the method comprising the steps of: by conjugating a conjugate of Formula 3: with M to obtain a conjugate of Formula 2: R is -OH or -F, wherein M-S is a biological macromolecule having cysteine, M-S is linked to L1 using the cysteine, L2 is a linker, and L2 is linked to R1, R3 or R2 in Formula 3, D is a functional molecule, a is an integer from 1 to 10, b and c are each independently an integer from 0 to 10, provided that b and c are not both 0, wherein R1 is H, optionally substituted alkyl or optionally substituted aryl, wherein R 1' is H or an isotope thereof, wherein R2 is H, optionally substituted alkyl or optionally substituted aryl, wherein R3 is H, optionally substituted alkyl or optionally substituted aryl, optionally, the C connecting R1 and the C connecting R2 form a ring.

[0075] In certain embodiments, in the method, M is selected from: proteins, DNA, RNA and viruses.

[0076] In certain embodiments, in the method, M is a biological macromolecule expressed on the cell surface.

[0077] In certain embodiments, in the method, M is an antigen-binding protein or a fragment thereof.

[0078] In certain embodiments, in the method, M is a monoclonal antibody, a chimeric antibody, a humanized antibody, a human engineered antibody, a human antibody, a single-chain antibody scFv or an antibody fragment.

[0079] In certain embodiments, in the method, M contains a functional group for nucleophilic addition reaction.

[0080] In certain embodiments, in the method, L2 is selected from the group consisting of: cleavable linkers, non-cleavable linkers, hydrophilic linkers, hydrophobic linkers, pre-charged linkers, uncharged linkers, and dicarboxylic acid linkers.

[0081] In certain embodiments, in the method, L2 is selected from the group consisting of: VC-PAB, N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl 4-(2-pyridyldithio)valerate (SPP), N-succinimidyl 4-(2-pyridyldithio)butyrate (SPDB), N-succinimidyl-4-(2-pyridyldithio)-2-sulfo-butyrate (sulfo-SPDB), N-succinimidyl iodoacetate (SIA), N-succinimidyl (4-iodoacetyl)aminobenzoate (SIAB), maleimide PEG NHS, N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC), N-sulfo-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (sulfo-SMCC), or 2,5-dioxopyrrolidin-1-yl 17-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5,8,11,14-tetraoxo-4,7,10,13-tetraazadec-1-anoate (CX1-1).

[0082] In certain embodiments, in the method, D has a biological function.

[0083] In certain embodiments, in the method, D and / or its derivatives are capable of inhibiting the growth of tumor cells.

[0084] In certain embodiments, in the method, D is a drug.

[0085] In certain embodiments, in the method, D is selected from the group consisting of: V-ATPase inhibitors, pro-apoptotic agents, Bcl2 inhibitors, MCL1 inhibitors, HSP90 inhibitors, IAP inhibitors, mTor inhibitors, microtubule stabilizers, microtubule destabilizers, auristatins, dolastatins, maytansines, MetAP (methionine aminopeptidase), protein CRM1 nuclear export inhibitors, DPPIV inhibitors, proteasome inhibitors, mitochondrial phosphoryl transfer reaction inhibitors, protein synthesis inhibitors, kinase inhibitors, CDK2 inhibitors, CDK9 inhibitors, kinesin inhibitors, HDAC inhibitors, DNA damaging agents, DNA alkylating agents, DNA intercalating agents, DNA minor groove binders, and DHFR inhibitors, nucleoside analogs, HDAC inhibitors; anthracyclines; NAMPT inhibitors; hydrophilic prodrugs; SN-38 glucuronide, etoposide phosphate; nitrogen mustards, proteasome inhibitors, cytokines, Toll-like receptor agonists, and STING agonists.

[0086] In certain embodiments, in the method, D is MMAE or a derivative thereof; melphalan or a derivative thereof; lenalidomide or a derivative thereof; IL-2 or a derivative thereof; novel interleukin-2 / 15 or a derivative thereof; T785 or a derivative thereof; or MSA-2 or a derivative thereof.

[0087] In certain embodiments, in the method, R1 is -H.

[0088] In certain embodiments, in the method, R3 is -H.

[0089] In certain embodiments, in the method, R2 is an optionally substituted phenyl.

[0090] In certain embodiments, in the method, R2 is wherein R4 is selected from the group consisting of: -OH, -PO3H2, -SeH, -SH, optionally substituted alkyl-OH, optionally substituted alkyl-halogen, optionally substituted alkyl-N3, -B(OH)2, -halogen, -OTf, optionally substituted alkyl-NH2, -O-optionally substituted alkyl-C≡CH, -CO-NH-C≡CH-optionally substituted alkyl.

[0091] In certain embodiments, in the method, R2 is wherein R4 is selected from the group consisting of: -OH, -PO3H2, -SeH, -SH, -CH2OH, -CH2Br, -CH2N3, -B(OH)2, -Br, -OTf, -CH2NH2, -Cl, -OCH2C≡CH or -CO-NH-C≡CH.

[0092] In certain embodiments, in the method, R2 is wherein R4 is -O-(CH2)n1-COO-R5, n1 is an integer from 1 to 10, and R5 is selected from the group consisting of: and H.

[0093] In certain embodiments, in the method, R2 is wherein R4 is -O-(CH2)n2-CO-NH-R6, n2 is an integer from 1 to 10, and R6 is -(CH2)n3-CO-R7, n3 is an integer from 1 to 10, and R7 is selected from the group consisting of:

[0094] In certain embodiments, in the method, R2 is wherein R4 is -O-(CH2)n2-CO-NH-R6, n2 is an integer from 1 to 10, and R6 is -(CH2)n4-R8, n4 is an integer from 1 to 10, and R8 is selected from the group consisting of:

[0095] In certain embodiments, in the method, R2 is wherein R4 is -O-(CH2)n2-CO-NH-R6, n2 is an integer from 1 to 10, and R6 is -(CH2CH2-O)n5-(CH2)n6-NH-CO-O-R9, n5 is an integer from 1 to 10, n6 is an integer from 1 to 10, and R9 is selected from the group consisting of H and

[0096] In certain embodiments, in the method, R2 is wherein R4 is -(OCH2CH2)n7-O-(CH2)n8-R 10 , n7 is an integer from 1 to 10, n8 is an integer from 1 to 10, and R 10 is selected from the group consisting of -COOH, -NH2, and

[0097] In certain embodiments, in the method, R2 is wherein R4 is -(OCH2CH2)n7-O-(CH2)n8-R 10 , n7 is an integer from 1 to 10, n8 is an integer from 1 to 10, and R 10 is selected from the group consisting of -COOH, -NH2, and wherein R 11 is selected from the group consisting of optionally substituted alkyl-halogen, optionally substituted alkyl-N, and O-optionally substituted alkyl.

[0098] In certain embodiments, in the method, R2 is Wherein R4 is -(OCH2CH2)n7-O-(CH2)n8-R 10 , n7 is an integer from 1 to 10, n8 is an integer from 1 to 10, wherein R 10 is selected from the group consisting of: -COOH, -NH2 and wherein R 11 is selected from the group consisting of: -CF3, -CN and -OCH3.

[0099] In certain embodiments, in the method, R2 is an optionally substituted alkyl-CH=CH-R 12 , wherein R 12 is wherein R 13 is -CH2N3.

[0100] In certain embodiments, in the method, the ring is an optionally substituted cycloolefin, or an optionally substituted aryl-cycloolefin.

[0101] In certain embodiments, in the method, the ring is selected from:

[0102] In certain embodiments, the method is carried out at a temperature in the range of about 16 °C to about 37 °C.

[0103] In certain embodiments, the method is carried out at a pH in the range of about 7.4 to about 8.

[0104] In certain embodiments, the method is carried out with a catalyst.

[0105] In certain embodiments, the method further comprises the step of purifying the conjugate of formula 3.

[0106] On the other hand, the present application provides a compound of formula III, or a pharmaceutically acceptable salt thereof:

[0107] Wherein R1 is selected from the group consisting of: -OH, -PO3H2, -SeH, -SH, optionally substituted alkyl-OH, optionally substituted alkyl-halogen, optionally substituted alkyl-N3, -B(OH)2, -halogen, -OTf, optionally substituted alkyl-NH2, -O-optionally substituted alkyl-C≡CH, -CO-NH-C≡CH-optionally substituted alkyl.

[0108] In certain embodiments, R1 is selected from the group consisting of: -OH, -PO3H2, -SeH, -SH, -CH2OH, -CH2Br, -CH2N3, -B(OH)2, -Br, -OTf, -CH2NH2, -Cl, -OCH2C≡CH or -CO-NH-C≡CH.

[0109] In certain embodiments, the compound is selected from:

[0110] On the other hand, the present application provides a compound of formula IV, or a pharmaceutically acceptable salt thereof: wherein R1 is selected from the group consisting of: NH-(CH2)n1-CO-R2, -OH, NH-(CH2) n2 -R3, NH-(CH2CH2-O) n3 -(CH2)n4-NH-CO-O-R4, or wherein n1, n2, n3, or n4 is independently an integer from 1 to 10, and wherein R2 is selected from the group consisting of: wherein R3 is selected from the group consisting of: wherein R4 is

[0111] In certain embodiments, R1 is selected from the group consisting of: NH-(CH2)2-CO-R2, -OH, NH-(CH2)-R3, NH-(CH2CH2-O)3-(CH2)2-NH-CO-O-R4.

[0112] In certain embodiments, the compound is selected from the group consisting of:

[0113] On the other hand, the present application provides a compound of formula V, or a pharmaceutically acceptable salt thereof: wherein R1, R2 and R4 are any substituents, and wherein R3 is selected from the group consisting of: H, optionally substituted alkyl-F3, optionally substituted alkyl-N or O-optionally substituted alkyl, and wherein R5 is selected from the group consisting of: -COOH, -NH2 and

[0114] In certain embodiments, R1 is H.

[0115] In certain embodiments, R2 is H.

[0116] In certain embodiments, R4 is H.

[0117] In certain embodiments, R3 is selected from the group consisting of: H, CF3, CN and OCH3.

[0118] In certain embodiments, the compound is selected from:

[0119]

[0120] On the other hand, the present application provides a compound of formula VI, or a pharmaceutically acceptable salt thereof:

[0121]

[0122] On the other hand, the present application provides a pharmaceutical composition comprising the conjugate of the present application and a pharmaceutically acceptable carrier.

[0123] On the other hand, the present application provides a method for modulating the tumor microenvironment of a subject, the method comprising administering to the subject the conjugate of the present application, or the pharmaceutical composition of the present application.

[0124] On the other hand, the present application provides a method for modulating the immune response of a subject, the method comprising administering to the subject the conjugate of the present application, or the pharmaceutical composition of the present application.

[0125] On the other hand, the present application provides a method for preventing and / or treating a disease in a subject in need thereof, the method comprising administering to the subject the conjugate of the present application, or the pharmaceutical composition of the present application.

[0126] In certain embodiments, the disease includes tumors and / or autoimmune diseases.

[0127] On the other hand, the present application provides a diagnostic reagent comprising the conjugate of the present application.

[0128] In certain embodiments, the diagnostic reagent is labeled.

[0129] In certain embodiments, the label is selected from: radiolabels, fluorophores, chromophores, imaging agents, and metal ions.

[0130] Based on the following specific embodiments, those skilled in the art will readily appreciate other aspects and advantages of the present application. Only exemplary embodiments of the present application are shown and described in the specific embodiments. As will be recognized, the present application can have other different embodiments, and several details thereof can be modified in various obvious aspects, but all without departing from the present disclosure. Therefore, the drawings and the description are regarded as exemplary rather than restrictive in nature.

[0131] Incorporated by reference

[0132] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and separately indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0133] The novel features of the invention are set forth with particularity in the appended claims. The features and advantages of the invention will be better understood from the following detailed description of exemplary embodiments that illustrate the principles of the invention, along with the accompanying drawings, in which (also referred to herein as "figure" and "FIG."):

[0134] Figure 1 A reaction process for screening candidate Michael acceptors as linkers is shown.

[0135] Figure 2 The chemical structure of Michael acceptor candidates is shown.

[0136] Figure 3 The chemical structure of modified Michael acceptor candidates is shown.

[0137] Figure 4 A reaction process for screening modified candidate Michael acceptors as linkers is shown.

[0138] Figure 5 The chemical structure of Michael acceptor candidates is shown.

[0139] Figure 6 A reaction process for screening modified candidate Michael acceptors as linkers is shown.

[0140] Figure 7 The chemical structure of a previously reported linker is shown.

[0141] Figure 8 A reaction process between sfGFP E124C and a previously reported linker is shown.

[0142] Figure 9 The reaction result between sfGFP E124C and a previously reported linker is shown.

[0143] Figure 10 The competitive experiment result between the linker of this application and a previously reported linker is shown.

[0144] Figure 11 a to Figure 11 c show the stability of the linker of this application.

[0145] Figure 12 a to Figure 12c shows the generation of conjugates using the linker of the present application and verifies the stability of the conjugates.

[0146] Figure 13 a to Figure 13 c shows the mass spectrometry results of the conjugates obtained through the linker of the present application, demonstrating the stability in serum.

[0147] Figures 14a to 14c Shows the steps of generating conjugates using the linker of the present application.

[0148] Figures 15a to 15c Shows the mass spectrometry results of the conjugate with a DAR of 3.2 obtained through the linker of the present application.

[0149] Figure 16 a to Figure 16 b shows the mass spectrometry results of the conjugate with a DAR of 3.8 obtained through the linker of the present application.

[0150] Figure 17 a to Figure 17 a to h show the results of the tumor cell killing assay of the conjugates obtained through the linker of the present application.

[0151] Figure 18 Shows the LC-MS chromatogram and mass spectrum of SSF-PEG4-vc-PAB-MMAE 1.

[0152] Figure 19 Shows the LC-MS chromatogram of SSF-PEG4-GGFG-Dxd 3.

[0153] Figure 20 Shows the process of linker stability study.

[0154] Figure 21 Shows the hydrolysis stability results of MA 5 and MA 2.

[0155] Figure 22 A to Figure 22 B shows the comparison of MA 2 with previously reported stable Cys-specific labeling reagents.

[0156] Figure 23 Shows the MS / MS spectrum of the GFP fragment modified with MA 2.

[0157] Figure 24 A to Figure 24 G show the Cys-specific modification of different proteins using SSF.

[0158] Figure 25 A to Figure 25 A to D show the results of the anti-tumor activity of the conjugates obtained through the linker of the present application.

[0159] Figure 26 A to Figure 26 B shows a comparison of the stability of MA 2 and maleimide in an aqueous buffer.

[0160] Figure 27 A to Figure 27 H shows the preparation of a conjugate containing SSF-ssDNA obtained by the linker of the present application and its application in single-cell sequencing.

[0161] Figure 28 Shows the deconvolution intact protein MS of the conjugate obtained by the linker of the present application.

[0162] Figure 29 Shows the deconvolution intact protein MS of the conjugate obtained by the linker of the present application.

[0163] Figure 30 Shows the results of the cell viability assay of the cell line (N87). Detailed Description

[0164] Although various embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. Many variations, changes, and alternatives can be envisioned by those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein can be employed.

[0165] As used herein, the term "conjugate" generally refers to any substance formed by joining together separate moieties. In a conjugate, the separate moieties can be joined to each other at one or more active sites. Additionally, the separate moieties can be associated or linked to each other covalently or non-covalently and exhibit different stoichiometric molar ratios. Conjugates can include peptides, polypeptides, proteins, prodrugs that are metabolized in vivo to active agents, polymers, nucleic acid molecules, small molecules, binders, mimetics, synthetic drugs, inorganic molecules, organic molecules, and radioisotopes. For example, a conjugate can contain a drug and an antigen-binding protein and can be an antibody-drug conjugate ADC.

[0166] As used herein, the term "ADC" generally refers to the conjugation of an antigen-binding protein with a drug. The conjugation can be a covalent bond or a non-covalent interaction such as through electrostatic forces. Various linkers can be employed to form the immunoconjugate. Additionally, the immunoconjugate can be provided in the form of a fusion protein, which can be expressed from a polynucleotide encoding the immunoconjugate. As used herein, a "fusion protein" refers to a protein produced by joining two or more genes or gene fragments that originally encoded separate proteins (including peptides and polypeptides).

[0167] As used herein, the term "biomacromolecule" generally refers to biomolecules such as nucleic acids, proteins, antibodies, carbohydrates, polysaccharides, lipids, and the like.

[0168] As used herein, the term "linker" generally refers to a chemical moiety or bond that attaches two or more molecules. A linker can be any molecular component capable of joining or linking two or more scaffolds. A linker can be a molecule whose function is to act as a flexible linker between modules in a scaffold or a molecule with additional functions. In the present disclosure, a linker can be used to attach fucose or a fucose derivative to an active moiety. Linkers of different lengths allow the attachment of fucose or a fucose derivative at different distances from the active moiety.

[0169] As used herein, the term "functional molecule" generally refers to any molecule that is a component of a conjugate of the present application and can play a role in the function of the conjugate.

[0170] As used herein, the term "biological function" generally refers to any activity or process carried out by a functional molecule of the present application in biology. For example, a biological function can include any activity or process carried out by a functional molecule in vitro and / or in vivo, and a biological function can include any activity or process carried out by a conjugate containing a functional molecule in vitro and / or in vivo.

[0171] As used herein, the term "functional group" generally refers to a biomacromolecule group capable of participating in an addition reaction (e.g., for a nucleophilic addition reaction). In the present application, a nucleophilic addition reaction can be a chemical addition reaction in which a nucleophile forms a sigma bond with an electron-deficient species. The nucleophilic addition reaction can enable a carbonyl group to be converted into various functional groups. For example, a nucleophilic functional group of a biomacromolecule can be -SH, -NH2, -SeH, -OH or

[0172] As used herein, the term "addition reaction" generally refers to an organic reaction in which two or more molecules combine to form a larger molecule (an adduct). The addition reaction can include electrophilic addition and nucleophilic addition. The addition reaction can be limited to compounds having multiple bonds, such as molecules having a carbon-carbon double bond (alkene) or a triple bond (alkyne), and compounds having a ring, which rings are also considered unsaturated ring unsaturation points. For example, molecules containing a carbon-hetero double bond (such as a carbonyl (C=O) group or an imine (C=N) group) can undergo an addition reaction.

[0173] As used herein, the term "antigen-binding protein" generally refers to a polypeptide molecule that specifically binds to an antigenic determinant. For example, an antigen-binding protein can be directed to a target site, e.g., can be attached to a tumor stroma with a specific type of tumor cell or antigenic determinant (e.g., an effector moiety or a second antigen-binding moiety). It is possible. Further, as defined herein, an antigen-binding protein can comprise an antibody and fragments thereof. For example, an antigen-binding protein can include an antibody antigen-binding domain, which includes an antibody heavy chain variable region and an antibody light chain variable region. For example, an antigen-binding protein can comprise an antibody constant region as further defined herein and known in the art. Effective heavy chain constant regions can include five subtypes: α, δ, ε, γ, or μ. Effective light chain constant regions can include two subtypes: κ and λ. Effective light chain constant regions can include one of two subtypes: κ and λ.

[0174] As used herein, the term "antibody" generally refers to a polypeptide or protein complex that specifically binds to an epitope or its mimetic. Antibodies include intact antibodies or binding fragments thereof that compete with the intact antibody for specific binding and include chimeric antibodies, humanized antibodies, fully human antibodies, and bispecific antibodies. Binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, and single-chain antibodies. In certain embodiments, an antibody is referred to as an immunoglobulin and includes various classes and subtypes, such as IgA (IgA1 and IgA2), IgD, IgE, IgM, and IgG (IgG1, IgG3, and IgG4), etc. In certain embodiments, as used herein, the term "antibody" refers to polyclonal or monoclonal antibodies and functional fragments thereof. Antibodies include modified or derivatized antibody variants that retain the ability to specifically bind an epitope. Antibodies are capable of selectively binding to a target antigen or epitope. Antibodies can include, but are not limited to, polyclonal antibodies, monoclonal antibodies (mAbs), humanized and other chimeric antibodies, single-chain antibodies (scFvs), Fab fragments, F(ab')2 fragments, and disulfide-linked Fvs (sdFv) fragments. In certain embodiments, the antibody is from any source, such as a mouse or a human, including chimeric antibodies thereof. In certain embodiments, the antibody is humanized.

[0175] As used herein, the term "derivative" generally refers to a compound expected to exhibit similar (e.g., physical, and / or chemical and / or biological) activities as those exhibited by a parent compound. For example, a derivative can be a precursor, metabolite, salt, and / or ester of the parent compound.

[0176] As used herein, the term "drug" generally refers to any agent that is harmful to cell growth and proliferation and can be used to reduce, inhibit, or destroy cells or malignancies. For example, a drug can include toxins. For example, a drug can include chemotherapeutic agents.

[0177] As used herein, the term "cytokine" generally refers to a molecule that mediates and / or regulates biological or cellular functions or processes (e.g., immunity, inflammation, and hematopoiesis). In this application, cytokines can also include "lymphokines", "chemokines", "monokines", and "interleukins". Examples of cytokines can include, but are not limited to, GM-CSF, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IFN-α, IFN-β, IFN-γ, MIP-1α, MIP-1β, TGF-β, TNF-α, and TNF-β. For example, cytokines can include IL-2, IL-7, IL-10, IL-12, IL-15, IFN-α, and IFN-γ. For example, cytokines can be human cytokines. As used herein, the term "cytokine" can also refer to Sauve et al., Proc Natl Acad Sci USA 88, 4636-40 (1991); Hu et al., Blood 101, 4853-4861 (2003) and U.S. Patent Application Publication No. 2003 / 0124678; Shanafelt et al., Nature Biotechnol 18, 1197-1202 (2000); Heaton et al., Cancer Res 53, 2597-602 (1993) and U.S. Patent No. 5,229,109; wild-type cytokines such as the IL-2 mutants described in U.S. Patent Application Publication No. 2007 / 0036752; International Publication No. 2008 / 0034473; International Publication No. 2009 / 061853; PCT Patent Application PCT / EP2012 / 051991. Including cytokine variants containing one or more amino acid mutations in the corresponding amino acid sequences. In addition, cytokine variants such as IL-15 variants are also described herein. For example, cytokines can be mutated to eliminate glycosylation.

[0178] As used herein, the term "aryl" generally refers to a hydrocarbon ring system having carbon atoms with a hydrocarbon ring radical (i.e., a monocyclic hydrocarbon ring) or two to four fused rings, the cyclic hydrocarbon ring may be aromatic with 5 or 6 carbon atoms, and each ring forming the hydrocarbon ring system may be aromatic and independently have 5 or 6 carbon atoms. For example, examples of aryl groups may include phenyl, naphthyl (i.e., naphthalene), and anthracenyl. For example, aryl may preferably include phenyl.

[0179] As used herein, the term "alkyl" generally refers to at least one carbon atom (e.g., 1 to 20 carbon atoms. "1 to 20 carbon atoms" may refer to straight-chain and / or branched-chain groups of alkyl groups having up to 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., including up to 20 carbon atoms), and saturated aliphatic (i.e., non-aromatic) acyclic hydrocarbons (i.e., groups composed of carbon and hydrogen atoms) that do not include adjacent carbon-carbon double bonds or carbon-carbon triple bonds. For example, alkyl may contain 1 to 10 carbon atoms. For example, alkyl may contain 1 to 6 carbon atoms.

[0180] As used herein, the term "drug" generally refers to any substance that alters the physiological functions of a subject. In the present application, a drug may include any compound having the required biological activity and reactive functionality and that can be used to prepare the conjugates of the present application. The required biological activity may include activities that can be used for the diagnosis, cure, reduction, treatment, or prevention of diseases in humans or other animals. Thus, as long as they have the necessary reactive functional groups, these compounds can be associated with the term "drug" as mentioned in the official "Chinese Pharmacopoeia" (e.g., in the official "Homeopathic Pharmacopoeia", or in the official "National Formulary", or any revised version thereof). Exemplary drugs are described in the "Physicians' Desk Reference" (PDR) of the United States and the Orange Book maintained by the U.S. Food and Drug Administration (FDA). New drugs may be continuously discovered and developed, and the present application also incorporates these new drugs into the "drugs" of the drug conjugates of the present application.

[0181] As used herein, the term "Toll-like receptor agonist" generally refers to any agonist of the Toll-like receptor. In the present application, the Toll-like receptor can be recognized by TLR, and TLR can activate the immune cell response. TLR may include TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, and TLR13. For example, Toll-like receptor agonists may include vaccine adjuvants in anti-tumor therapies because the vaccine adjuvants can activate immune cells and trigger inflammation.

[0182] As used herein, the term "STING agonist" generally refers to an agent that can bind to STING and activate STING. For example, activation of STING activity can include stimulation of inflammatory cytokines, including interferons, such as type I interferons (including IFN-α, IFN-β), type III interferons (e.g., CXCL9, CCL4, CXCL11, CCL5, CCL3 or CCL8). The activity of a STING agonist can also include stimulation of TANK-binding kinase (TBK) 1 phosphorylation, interferon regulatory factor (IRF) activation (e.g., IRF3 activation), secretion of interferon-γ-induced protein (IP-10) or other inflammatory proteins and cytokines. The activity of a STING agonist can be determined, for example, by the ability of a compound to stimulate activation of the STING pathway, which is detected using interferon stimulation assays, reporter gene assays (e.g., hSTING wt assay or THP-1 dual assay), TBK1 activation assays, IP-10 assays or other assays known to those of skill in the art. The activity of a STING agonist can also be determined by the ability of a compound to increase the transcriptional level of a gene encoding a protein activated by STING or the STING pathway. For example, such activity can be detected using an RNAseq assay.

[0183] As used herein, the term "pharmaceutically acceptable carrier" generally refers to non-API (API means active pharmaceutical ingredient) used to form a pharmaceutical product, such as disintegrants, binders, fillers and lubricants. Pharmaceutically acceptable carriers can meet the established government standards (including those promulgated by the US Food and Drug Administration and the European Food and Drug Administration), and are generally safe for human administration. For example, pharmaceutically acceptable carriers can include sterile aqueous or non-aqueous solutions, dispersions, suspensions, emulsions and / or sterile injectable solutions or dispersions only before use.

[0184] The term "tumor" generally refers to a malignant tumor characterized by disordered or uncontrolled cell growth. For example, tumors can include primary malignant tumors (e.g., those in which the cells have not migrated to sites outside the original tumor site in a subject) and secondary malignant tumors (e.g., those caused by metastasis, where tumor cells migrate to a secondary site different from the original tumor site). Tumors can include solid tumors and / or non-solid tumors.

[0185] The term "tumor microenvironment" generally refers to the complex surrounding microenvironment of tumor cells. For example, the tumor microenvironment can include surrounding blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, various signaling molecules, and / or the extracellular matrix (ECM). For example, cancer stem cells and other molecules that contribute to tumor development and progression may be present in the tumor microenvironment. Thus, during treatment, targeting and manipulating the cells and factors in the tumor microenvironment can help control malignancies and achieve positive health outcomes.

[0186] The term "immune response" generally refers to the defense of a subject against foreign substances and / or pathogens. An immune response may result in an immune reaction, for example, the recognition and binding of an antigen by its specific antibody or by previously sensitized lymphocytes.

[0187] The term "autoimmune disease" generally refers to any disease and / or disorder caused by an immune-mediated attack on the subject's own organs. Examples of autoimmune diseases can include rheumatoid arthritis, systemic lupus erythematosus (lupus), inflammatory bowel disease (IBD), multiple sclerosis (MS), type 1 diabetes, Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, psoriasis, Graves' disease, Hashimoto's thyroiditis, myasthenia gravis, and / or vasculitis.

[0188] As used herein, the term "treatment" generally refers to ameliorating a disease or disorder (i.e., slowing or halting or reducing the development of a disease (such as a tumor) or at least one of its clinical symptoms). For example, treatment can include alleviating or improving at least one physical parameter, including those that may not be recognized by the patient.

[0189] As used herein, the term "prevention" generally refers to prophylactic treatment of a disease or disorder; or delaying the onset or progression of a disease or disorder.

[0190] As used herein, unless otherwise specified herein or clearly contradicted by the context, the terms "a," "an," and "the" and similar terms used in the context of this application (especially in the context of the claims) shall be construed to cover the singular and the plural.

[0191] Conjugate

[0192] In one aspect, the present application provides a conjugate of Formula 1, M-[(L1) a -(L2) b -(D) c 1, wherein L1 is a compound of Formula I, R is -F or -OH, where M is a biological macromolecule, and M is linked to L1 using a nucleophilic functional group of M, L2 is a linker, and L2 is linked to R1, R3 or R2, D is a functional molecule, a is an integer from 1 to 10, b and c are each independently an integer from 0 to 10, provided that b and c are not both 0, where R1 is H, optionally substituted alkyl or optionally substituted aryl, where R 1' is H or its isotope, where R2 is H, optionally substituted alkyl or optionally substituted aryl, where R3 is H, optionally substituted alkyl or optionally substituted aryl, optionally, the C connecting R1 and the C connecting R2 form a ring.

[0193] For example, the nucleophilic functional group of M can be selected from: -SH, -NH2, -SeH, -OH and

[0194] On the other hand, the present application provides a conjugate of formula 2, M-S-[(L1)a-(L2)b-(D)c]2, where M-S is a biological macromolecule having cysteine, M-S is linked to L1 using the cysteine, L1 is a compound of formula I, R is -F or -OH, L2 is a linker, L2 is linked to R1, R3 or R2, D is a functional molecule, a is an integer from 1 to 10, b and c are each independently an integer from 0 to 10, provided that b and c are not both 0, where R1 is H, optionally substituted alkyl or optionally substituted aryl, where R 1' is H or its isotope, where R2 is H, optionally substituted alkyl or optionally substituted aryl, where R3 is H, optionally substituted alkyl or optionally substituted aryl, optionally, the C connecting R1 and the C connecting R2 form a ring.

[0195] For example, M can be selected from: proteins, DNA, RNA and viruses.

[0196] For example, M can be a biological macromolecule expressed on the cell surface.

[0197] For example, M can be an antigen-binding protein or a fragment thereof.

[0198] For example, M can be a monoclonal antibody, a chimeric antibody, a humanized antibody, a human engineered antibody, a human antibody, a single-chain antibody scFv or an antibody fragment.

[0199] For example, M can contain a functional group for nucleophilic addition reaction.

[0200] For example, L2 can be selected from the group consisting of: a cleavable linker, a non-cleavable linker, a hydrophilic linker, a hydrophobic linker, a charged linker, an uncharged linker and a dicarboxylic acid linker.

[0201] For example, L2 can be selected from the group consisting of VC-PAB, N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl 4-(2-pyridyldithio)valerate (SPP), N-succinimidyl 4-(2-pyridyldithio)butyrate (SPDB), N-succinimidyl-4-(2-pyridyldithio)-2-sulfo-butyrate (sulfo-SPDB), N-succinimidyl iodoacetate (SIA), N-succinimidyl (4-iodoacetyl)aminobenzoate (SIAB), maleimide PEG NHS, N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC), N-sulfo-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (sulfo-SMCC), or 2,5-dioxopyrrolidin-1-yl 17-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5,8,11,14-tetraoxo-4,7,10,13-tetraazadecane-1-carboxylate (CX1-1).

[0202] For example, D can have a biological function.

[0203] For example, D and / or its derivatives can be capable of inhibiting the growth of tumor cells.

[0204] For example, D can be a drug.

[0205] For example, D can be selected from the group consisting of V-ATPase inhibitors, apoptosis promoters, Bcl2 inhibitors, MCL1 inhibitors, HSP90 inhibitors, IAP inhibitors, mTor inhibitors, microtubule stabilizers, microtubule destabilizers, auristatins, dolastatins, maytansinoids, MetAP (methionyl aminopeptidase), protein CRM1 nuclear export inhibitors, DPPIV inhibitors, proteasome inhibitors, mitochondrial phosphoryl transfer reaction inhibitors, protein synthesis inhibitors, kinase inhibitors, CDK2 inhibitors, CDK9 inhibitors, kinesin inhibitors, HDAC inhibitors, DNA damaging agents, DNA alkylating agents, DNA intercalating agents, DNA minor groove binders, DHFR inhibitors, nucleoside analogs, HDAC inhibitors; anthracyclines; NAMPT inhibitors; hydrophilic prodrugs; SN-38 glucuronide, etoposide phosphate; nitrogen mustards, proteasome inhibitors, cytokines, Toll-like receptor agonists, and STING agonists.

[0206] For example, D can be MMAE or its derivatives; melphalan or its derivatives; lenalidomide or its derivatives; IL-2 or its derivatives; novel interleukin-2 / 15 or its derivatives; T785 or its derivatives; or MSA-2 or its derivatives.

[0207] For example, R1’ It can be -H. For example, R1 is -H . For example, R3 is -H.

[0208] For example, R2 can be an optionally substituted phenyl group.

[0209] For example, R2 can be wherein R4 can be selected from the group consisting of: -OH, -PO3H2, -SeH, -SH, optionally substituted alkyl-OH, optionally substituted alkyl-halogen, optionally substituted alkyl-N3, -B(OH)2, -halogen, -OTf, optionally substituted alkyl-NH2, -O-optionally substituted alkyl-C≡CH, -CO-NH-C≡CH-optionally substituted alkyl.

[0210] For example, R2 can be wherein R4 can be selected from the group consisting of: -OH, -PO3H2, -SeH, -SH, -CH2OH, -CH2Br, -CH2N3, -B(OH)2, -Br, -OTf, -CH2NH2, -Cl, -OCH2C≡CH or -CO-NH-C≡CH.

[0211] For example, R2 can be wherein R4 can be -O-(CH2)n1-COO-R5, n1 can be an integer from 1 to 10, wherein R5 can be selected from the group consisting of and H.

[0212] For example, R2 can be wherein R4 can be -O-(CH2)n2-CO-NH-R6, n2 can be an integer from 1 to 10, wherein R6 can be -(CH2)n3-CO-R7, n3 can be an integer from 1 to 10, wherein R7 can be selected from the group consisting of

[0213] For example, R2 is wherein R4 can be -O-(CH2)n2-CO-NH-R6, n2 can be an integer from 1 to 10, wherein R6 can be -(CH2)n4-R8, n4 can be an integer from 1 to 10, wherein R8 can be selected from the group consisting of

[0214]

[0215] For example, R2 can be wherein R4 can be -O-(CH2)n2-CO-NH-R6, n2 can be an integer from 1 to 10, wherein R6 can be -(CH2CH2-O)n5-(CH2)n6-NH-CO-O-R9, n5 can be an integer from 1 to 10, n6 can be an integer from 1 to 10, wherein R9 can be selected from the group consisting of: H and

[0216] For example, R2 can be wherein R4 can be -(OCH2CH2)n7-O-(CH2)n8-R 10 , n7 can be an integer from 1 to 10, n8 can be an integer from 1 to 10, wherein R 10 can be selected from the group consisting of: -COOH, -NH2 and

[0217] For example, R2 can be wherein R4 can be -(OCH2CH2)n7-O-(CH2)n8-R 10 , n7 can be an integer from 1 to 10, n8 can be an integer from 1 to 10, wherein R 10 can be selected from the group consisting of: -COOH, -NH2 and wherein R 11 can be selected from the group consisting of: optionally substituted alkyl-halogen, optionally substituted alkyl-N and O-optionally substituted alkyl.

[0218] For example, R2 can be wherein R4 can be -(OCH2CH2)n7-O-(CH2)n8-R 10 , n7 can be an integer from 1 to 10, n8 can be an integer from 1 to 10, wherein R 10 can be selected from the group consisting of: -COOH, -NH2 and wherein R 11 can be selected from the group consisting of: -CF3, -CN and -OCH3.

[0219] For example, R2 can be optionally substituted alkyl-CH=CH-R 12 , wherein R 12 can be wherein R 13 can be -CH2N3.

[0220] For example, the ring can be an optionally substituted cycloolefin, or an optionally substituted aryl-cycloolefin.

[0221] For example, the ring can be selected from the group consisting of:

[0222] For example, L1 can be selected from the following group:

[0223]

[0224]

[0225] For example, the conjugate can be selected from the following group:

[0226]

[0227]

[0228] For example, the conjugate of Formula 1 or Formula 2 can be as follows:

[0229] wherein R is -F or -OH.

[0230] On the other hand, the present application provides a conjugate of Formula 3, (L1) a -(L2) b -(D) c 3, wherein L1 is a compound of Formula III, L2 is a linker, and L2 is connected to R1, R3 or R2, D is a functional molecule, a is an integer from 1 to 10, b and c are each independently an integer from 0 to 10, provided that b and c are not both 0, wherein R1 is H, optionally substituted alkyl or optionally substituted aryl, wherein R2 is H, optionally substituted alkyl or optionally substituted aryl, wherein R3 is H, optionally substituted alkyl or optionally substituted aryl, optionally, the C connecting R1 and the C connecting R2 form a ring.

[0231] In certain embodiments, in the conjugate of Formula 3, L2 is selected from: cleavable linkers, non-cleavable linkers, hydrophilic linkers, hydrophobic linkers, charged linkers, uncharged linkers and dicarboxylic acid linkers.

[0232] For example, L2 can be selected from: VC-PAB, N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl 4-(2-pyridyldithio)valerate (SPP), N-succinimidyl 4-(2-pyridyldithio)butyrate (SPDB), N-succinimidyl-4-(2-pyridyldithio)-2-sulfo-butyrate (sulfo-SPDB), N-succinimidyl iodoacetate (SIA), N-succinimidyl (4-iodoacetyl)aminobenzoate (SIAB), maleimide PEG NHS, N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC), N-sulfo-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (sulfo-SMCC), or 2,5-dioxopyrrolidin-1-yl 17-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5,8,11,14-tetraoxo-4,7,10,13-tetraazapentadecan-1-oate (CX1-1).

[0233] For example, D can have a biological function. For example, D and / or its derivatives can be capable of inhibiting the growth of tumor cells. For example, D can be a drug.

[0234] For example, D can be selected from the group consisting of: V-ATPase inhibitors, apoptosis promoters, Bcl2 inhibitors, MCL1 inhibitors, HSP90 inhibitors, IAP inhibitors, mTor inhibitors, microtubule stabilizers, microtubule destabilizers, auristatins, dolastatins, maytansines, MetAP (methionyl aminopeptidase), protein CRM1 nuclear export inhibitors, DPPIV inhibitors, proteasome inhibitors, mitochondrial phosphoryl transfer reaction inhibitors, protein synthesis inhibitors, kinase inhibitors, CDK2 inhibitors, CDK9 inhibitors, kinesin inhibitors, HDAC inhibitors, DNA damaging agents, DNA alkylating agents, DNA intercalating agents, DNA minor groove binders, DHFR inhibitors, nucleoside analogs, HDAC inhibitors; anthracyclines; NAMPT inhibitors; hydrophilic prodrugs; SN-38 glucuronide, etoposide phosphate; nitrogen mustards, proteasome inhibitors, cytokines, Toll-like receptor agonists, and STING agonists. For example, D can be MMAE or its derivatives; melphalan or its derivatives; lenalidomide or its derivatives; IL-2 or its derivatives; novel interleukin-2 / 15 or its derivatives; T785 or its derivatives; or MSA-2 or its derivatives.

[0235] For example, R1 can be -H.

[0236] For example, R3 can be -H.

[0237] For example, R2 can be an optionally substituted phenyl.

[0238] For example, R2 can be wherein R4 can be selected from the group consisting of: -OH, -PO3H2, -SeH, -SH, optionally substituted alkyl-OH, optionally substituted alkyl-halogen, optionally substituted alkyl-N3, -B(OH)2, -halogen, -OTf, optionally substituted alkyl-NH2, -O-optionally substituted alkyl-C≡CH, -CO-NH-C≡CH-optionally substituted alkyl.

[0239] For example, R2 can be wherein R4 can be selected from the group consisting of: -OH, -PO3H2, -SeH, -SH, -CH2OH, -CH2Br, -CH2N3, -B(OH)2, -Br, -OTf, -CH2NH2, -Cl, -OCH2C≡CH or -CO-NH-C≡CH.

[0240] For example, R2 can be wherein R4 can be -O-(CH2)n1-COO-R5, n1 is an integer from 1 to 10, wherein R5 can be selected from the group consisting of: and H.

[0241] For example, R2 can be wherein R4 can be -O-(CH2)n2-CO-NH-R6, n2 can be an integer from 1 to 10, wherein R6 can be -(CH2)n3-CO-R7, n3 can be an integer from 1 to 10, wherein R7 can be selected from the group consisting of:

[0242] For example, R2 is wherein R4 can be -O-(CH2)n2-CO-NH-R6, n2 is an integer from 1 to 10, wherein R6 can be -(CH2)n4-R8, n4 can be an integer from 1 to 10, wherein R8 can be selected from the group consisting of:

[0243] For example, R2 can be wherein R4 can be -O-(CH2)n2-CO-NH-R6, n2 is an integer from 1 to 10, wherein R6 can be -(CH2CH2-O)n5-(CH2)n6-NH-CO-O-R9, n5 can be an integer from 1 to 10, n6 can be an integer from 1 to 10, wherein R9 can be selected from the group consisting of H and

[0244] For example, R2 can be wherein R4 can be -(OCH2CH2)n7-O-(CH2)n8-R 10, n7 can be an integer from 1 to 10, n8 can be an integer from 1 to 10, where R 10 can be selected from the following group: -COOH, -NH2 and

[0245] For example, R2 can be where R4 can be -(OCH2CH2)n7 - O - (CH2)n8 - R 10 , n7 can be an integer from 1 to 10, n8 can be an integer from 1 to 10, where R 10 can be selected from the following group: -COOH, -NH2 and where R 11 can be selected from the following group: optionally substituted alkyl - halogen, optionally substituted alkyl - N and O - optionally substituted alkyl.

[0246] For example, R2 can be where R4 can be -(OCH2CH2)n7 - O - (CH2)n8 - R 10 , n7 can be an integer from 1 to 10, n8 can be an integer from 1 to 10, where R 10 can be selected from the following group: -COOH, -NH2 and where R 11 can be selected from the following group: -CF3, -CN and -OCH3.

[0247] For example, R2 can be optionally substituted alkyl - CH=CH - R 12 , where R 12 can be where R 13 can be -CH2N3.

[0248] For example, the ring can be an optionally substituted cycloolefin, or an optionally substituted aryl - cycloolefin.

[0249] For example, the ring can be selected from the following group:

[0250] For example, the L1 can be selected from the following group:

[0251]

[0252] For example, the conjugate can be selected from the following group:

[0253]

[0254] Method

[0255] On the other hand, the present application provides a method for preparing a conjugate, the method comprising the following steps: by conjugating a conjugate of formula 3: with M to obtain a conjugate of formula 1: where M is a biopolymer, and M is linked to L1 using a nucleophilic functional group of M, L2 is a linker, and L2 is linked to R1, R3 or R2 in formula 1, D is a functional molecule, a is an integer from 1 to 10, b and c are each independently integers from 0 to 10, provided that b and c are not both 0, wherein R1 is H, optionally substituted alkyl or optionally substituted aryl, wherein R 1' is H or its isotope, wherein R2 is H, optionally substituted alkyl or optionally substituted aryl, wherein R3 is H, optionally substituted alkyl or optionally substituted aryl, optionally, the C connecting R1 and the C connecting R2 form a ring.

[0256] For example, the nucleophilic functional group of M can be selected from the group consisting of: -SH, -NH2, -SeH, -OH and

[0257] On the other hand, the present application provides a method for preparing a conjugate, the method comprising the following steps: by conjugating a conjugate of formula 3: with M to obtain a conjugate of formula 2: R is -OH or -F, wherein M-S is a biopolymer having cysteine, M-S is linked to L1 using the cysteine, L2 is a linker, and L2 is linked to R1, R3 or R2 in formula 3, D is a functional molecule, a is an integer from 1 to 10, b and c are each independently integers from 0 to 10, provided that b and c are not both 0, wherein R1 is H, optionally substituted alkyl or optionally substituted aryl, wherein R 1' is H or its isotope, wherein R2 is H, optionally substituted alkyl or optionally substituted aryl, wherein R3 is H, optionally substituted alkyl or optionally substituted aryl, optionally, the C connecting R1 and the C connecting R2 form a ring.

[0258] In the present application, the method can be carried out at a temperature in the range of about 16 °C to about 37 °C. For example, the method can be carried out at a temperature of at least about 16 °C, at least about 17 °C, at least about 18 °C, at least about 19 °C, at least about 20 °C, at least about 21 °C, at least about 22 °C, at least about 23 °C, at least about 24 °C, at least about 25 °C, at least about 26 °C, at least about 27 °C, at least about 33 °C, at least about 34 °C, at least about 35 °C, at least about 36 °C, or at least about 37 °C.

[0259] In the present application, the method can be carried out at a pH in the range of about 7.4 to about 8. For example, the method can be carried out at a pH of at least about 7.4, at least about 7.5, at least about 7.6, at least about 7.7, at least about 7.8, at least about 7.9 or at least about 8.0.

[0260] In the present application, the method can be carried out using a catalyst. For example, the catalyst can comprise Pd(OAc)2.

[0261] In the present application, the method can further comprise the step of purifying the conjugate of Formula 3.

[0262] In the present application, the conjugation in the method can include an addition reaction (e.g., it can be a nucleophilic addition reaction), which can belong to "click chemistry". For example, the -SH of M can participate in the addition reaction in the method.

[0263] Compound

[0264] On the other hand, the present application provides a compound of Formula III, or a pharmaceutically acceptable salt thereof: wherein R1 is selected from the group consisting of: -OH, -PO3H2, -SeH, -SH, optionally substituted alkyl-OH, optionally substituted alkyl-halogen, optionally substituted alkyl-N3, -B(OH)2, -halogen, -OTf, optionally substituted alkyl-NH2, -O-optionally substituted alkyl-C≡CH, -CO-NH-C≡CH-optionally substituted alkyl.

[0265] For example, R1 can be selected from the group consisting of: -OH, -PO3H2, -SeH, -SH, -CH2OH, -CH2Br, -CH2N3, -B(OH)2, -Br, -OTf, -CH2NH2, -Cl, -OCH2C≡CH or -CO-NH-C≡CH.

[0266] In certain cases, the compound of Formula III can be one of the compounds in Table 1.

[0267] Table 1

[0268]

[0269]

[0270] On the other hand, the present application provides a compound of Formula IV, or a pharmaceutically acceptable salt thereof: wherein R1 is selected from the group consisting of: NH-(CH2)n1-CO-R2, -OH, NH-(CH2) n2 -R3, NH-(CH2CH2-O) n3-(CH2)n4-NH-CO-O-R4, or wherein n1, n2, n3, or n4 is independently an integer from 1 to 10, and wherein R2 is selected from the group consisting of: wherein R3 is selected from the group consisting of: wherein R4 is

[0271] For example, R1 can be selected from the group consisting of: NH-(CH2)2-CO-R2, -OH, NH-(CH2)-R3, NH-(CH2CH2-O)3-(CH2)2-NH-CO-O-R4.

[0272] In some cases, the compound of formula IV can be one of the compounds in Table 2.

[0273] Table 2

[0274]

[0275]

[0276] On the other hand, the present application provides a compound of formula V, or a pharmaceutically acceptable salt thereof: wherein R1, R2, and R4 are arbitrary substituents, wherein R3 is selected from the group consisting of: H, optionally substituted alkyl-F3, optionally substituted alkyl-N or O-optionally substituted alkyl, and wherein R5 is selected from the group consisting of: -COOH, -NH2, and

[0277] For example, R1 can be H. For example, R2 can be H. For example, R4 can be H.

[0278] For example, R3 can be selected from: H, CF3, CN, and OCH3.

[0279] In some cases, the compound of formula V can be one of the compounds in Table 3.

[0280] Table 3

[0281]

[0282] On the other hand, the present application provides a compound of formula VI, or a pharmaceutically acceptable salt thereof:

[0283]

[0284] As used herein, the term "Formula III" (or Formula IV, Formula V, Formula VI) may also be defined to include all forms of the compounds of "Formula III" (or Formula IV, Formula V, Formula VI), including hydrates, solvates, isomers, crystalline and amorphous forms, cocrystals, polymorphs, and their metabolites. For example, a compound of Formula VI or a pharmaceutically acceptable salt thereof may exist in unsolvated and solvated forms. When the solvent or water is tightly bound, the complex will have a defined stoichiometry independent of humidity. However, when the solvent or water is weakly bound, as in channel solvates and hygroscopic compounds, the water / solvent content will depend on humidity and drying conditions. In such cases, non-stoichiometry will be the norm.

[0285] Compounds of "Formula III" (or Formula IV, Formula V, Formula VI) may have asymmetric carbon atoms. For example, the carbon-carbon bonds of a compound of Formula VI may be depicted herein using solid lines, solid wedges, or dashed wedges. The use of a solid line to depict a bond to an asymmetric carbon atom is intended to indicate inclusion of all possible stereoisomers at that carbon atom (e.g., a specific enantiomer, a racemic mixture, etc.). The use of a solid or dashed wedge to depict a bond to an asymmetric carbon atom is intended to indicate inclusion of only the depicted stereoisomer. The compounds of the present application may contain more than one asymmetric carbon atom. In these compounds, the use of a solid line to depict a bond to an asymmetric carbon atom is intended to indicate inclusion of all possible stereoisomers. For example, unless otherwise indicated, it is intended that the compounds of "Formula III" (or Formula IV, Formula V, Formula VI) may exist as enantiomers and diastereomers or as racemates and mixtures thereof. The use of a solid line to depict a bond to one or more asymmetric carbon atoms in a compound of "Formula III" (or Formula IV, Formula V, Formula VI), and the use of a solid or dashed wedge to depict a bond to other asymmetric carbon atoms in the same compound, is intended to indicate the presence of a mixture of diastereomers.

[0286] The compounds of the present application (e.g., compounds of "Formula III" (or Formula IV, Formula V, Formula VI)) may exist as clathrates or other complexes. Complexes such as clathrates, drug-host inclusion compounds, etc. are included within the scope of the present invention, wherein the drug and the host are present in stoichiometric or non-stoichiometric amounts as compared to the aforementioned solvates. Also included are complexes of "Formula III" (or Formula IV, Formula V, Formula VI) containing two or more organic and / or inorganic components, which may be in stoichiometric or non-stoichiometric amounts. The resulting complexes may be ionized, partially ionized, or non-ionized. For a review of such complexes, see Haleblian, J. Pharm. Sci., 64(8), 1269-1288 (August 1975).

[0287] Stereoisomers of “Formula III” (or Formula V, Formula V, Formula VI) include cis- and trans-isomers of the compounds of “Formula III” (or Formula V, Formula V, Formula VI), optical isomers such as R and S enantiomers, diastereoisomers, geometric isomers, rotational isomers, conformational isomers and tautomers, including compounds exhibiting more than one type of isomerism; and mixtures thereof (such as racemates and diastereoisomer pairs). Also included are acid addition salts or base addition salts, wherein the counterion has optical activity (e.g., D-lactate or L-lysine), or racemic nature (e.g., DL-tartrate or DL-arginine).

[0288] Drug Compositions and Uses

[0289] On the other hand, the present application provides a drug composition comprising the conjugate of the present application and a pharmaceutically acceptable carrier.

[0290] In the present application, the drug composition may comprise the conjugate of the present application presented with a pharmaceutically acceptable carrier. The carrier may be a solid product, a liquid, or both, and may be formulated with the compound into a unit dose composition (such as a tablet), which may contain 0.05% to 95% by weight of the active compound. Other pharmacologically active substances may also be present.

[0291] In the present application, the conjugate and / or drug composition of the present invention may be administered by any suitable route.

[0292] On the other hand, the present application provides a method for modulating the tumor microenvironment of a subject, the method comprising administering to the subject the conjugate of the present application, or the drug composition of the present application.

[0293] On the other hand, the present application provides a method for modulating the immune response of a subject, the method comprising administering to the subject the conjugate of the present application, or the drug composition of the present application.

[0294] On the other hand, the present application provides a method for preventing and / or treating a disease in a subject in need thereof, the method comprising administering to the subject the conjugate of the present application, or the drug composition of the present application.

[0295] The method may be an in vitro method, an ex vivo method or an in vivo method. For example, the conjugate of the present application may be administered in vitro to one or more cells. Again, the conjugate of the present application may be administered to a subject in need thereof.

[0296] In another aspect, the present application provides the conjugate of the present application for preventing and / or treating a disease in a subject in need thereof.

[0297] On the other hand, the present application provides a method for preparing a drug for treating a disease.

[0298] For example, the disease can be a tumor. For example, the tumor can be a solid tumor. For example, the tumor can be a non-solid tumor. For example, the solid tumor can include sarcoma and carcinoma. The sarcoma can refer to a tumor in blood vessels, bones, adipose tissue, ligaments, lymphatic vessels, muscles or tendons. The carcinoma can refer to a tumor formed in epithelial cells. It is contemplated that the solid tumor is a non-lymphoma solid tumor. For example, the solid tumor can be named according to the cell type forming it.

[0299] For example, the disease can include tumors and / or autoimmune diseases.

[0300] For example, autoimmune diseases can include glomerulonephritis, Goodpasture's syndrome, necrotizing vasculitis, lymphadenitis, periarteritis nodosa, systemic lupus erythematosus, rheumatoid arthritis, psoriatic arthritis, systemic lupus erythematosus, psoriasis, ulcerative colitis, systemic sclerosis, dermatomyositis / polymyositis, antiphospholipid antibody syndrome, scleroderma, pemphigus vulgaris, ANCA-associated vasculitis (such as Wegener's granulomatosis, microscopic polyangiitis), uveitis, Sjogren's syndrome, Crohn's disease, Reiter's syndrome, ankylosing spondylitis, Lyme arthritis, Guillain-Barré syndrome, Hashimoto's thyroiditis and cardiomyopathy.

[0301] Generally, the conjugate of the present application can be administered in an effective amount for treating the diseases described herein. The conjugate of the present application can be in the form of a pharmaceutical composition suitable for such a route and administered in a dose effective for the intended treatment, by any suitable route. The therapeutically effective dose of the conjugate required to treat the progression of a medical condition can be readily determined by those of ordinary skill in the art using preclinical and clinical methods familiar to the medical field. As used herein, the term "therapeutically effective amount" generally refers to the amount of the conjugate being administered that will, to some extent, relieve one or more symptoms of the disease being treated.

[0302] The dosage regimen of the conjugate and / or the composition containing the conjugate can be based on various factors, including the type, age, weight, sex and medical condition of the patient; the severity of the condition; the route of administration; and the activity of the particular compound employed. Therefore, the dosage regimen can vary widely.

[0303] Suitable subjects according to the present invention include mammalian subjects. For example, the subject can be a mammal, for example, the subject can be a human.

[0304] On the other hand, the present application provides a diagnostic reagent, which comprises the conjugate of the present application.

[0305] For example, the diagnostic reagent can be labeled.

[0306] For example, the label can be selected from: radioactive labels, fluorophores, chromophores, imaging agents, and metal ions.

[0307] Examples

[0308] The following examples are set forth to provide a complete disclosure and description to those of ordinary skill in the art of how to make and use the present invention. They are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the experiments below are all or the only experiments conducted. Efforts have been made to ensure the accuracy of the numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are by weight, molecular weights are weight-average molecular weights, temperatures are in degrees Celsius, and pressures are at or near atmospheric pressure. Standard abbreviations may be used, e.g., bp: one or more base pairs; kb: one thousand or thousands of bases; pl: one or several picoliters; s or sec: one or several seconds; min: one or several minutes; h or hr: one or several hours; aa: one or more amino acids; nt: one or more nucleotides; i.m.: intramuscularly; i.p.: intraperitoneally; s.c.: subcutaneously; and so on.

[0309] Silica gel column chromatography

[0310] Silica gel column chromatography was carried out using silica gel 60 (200 - 300 mesh). Analytical thin-layer chromatography (TLC) was carried out using silica gel (silica gel 60F254). TLC was carried out on pre-coated silica gel plates using short-wave ultraviolet light as the visualizing agent, KMnO4, and heat energy as the developing agents.

[0311] Synthesis of SSF-DNA

[0312] To an Eppendorf tube containing PBS (50 mM, pH 8.0) with 5'-NH2-20 / 59nt ssDNA (final concentration 50 μM, 1 equivalent), SSF-NHS (100 equivalents, 100 mM in DMF) and DMF were added. The reaction mixture was vortexed and shaken at 30 °C. After incubation overnight, the reaction mixture was analyzed by LC-MS.

[0313] Synthesis of Mal-DNA

[0314] To an Eppendorf tube containing PBS (50 mM, pH 8.0) with 5'-NH2-20 (final concentration 50 μM, 1 equivalent), SSF-NHS (100 equivalents, 100 mM in DMF) and DMF were added. The reaction mixture was vortexed and shaken at 30 °C. After incubation overnight, the reaction mixture was analyzed by LC-MS.

[0315] Hydrolytic Stability of SSF-DNA and Mal-DNA

[0316] SSF-20nt ssDNA / Mal-20nt ssDNA (final concentration of 100 μM) was shaken in PBS (50 mM, pH 7.4) at 37 °C for 48 h. 20 μL of the reaction mixture was analyzed by LC-MS.

[0317] Synthesis of Protein-DNA Conjugates

[0318] SSF / Mal-DNA (56 μL, 450 mM in H2O) and PBS (10 μL, pH = 8.0, 50 mM) were added to a solution of Nb-PD-L1 or other protein (50 μL, 100 μM, HEPES buffer). The reactants were incubated at 37 °C for 12 h to produce homogeneous protein-DNA conjugates.

[0319] LC-MS

[0320] LC-MS analysis of protein conjugation and protein-DNA (GFP-20nt ssDNA, neo2-20nt ssDNA): LC–MS was performed on an Xevo G2-S TOF mass spectrometer coupled to an Acquity high performance liquid chromatography (UPLC) system, using an Acquity UPLC Protein BEH C4 column (1.7 mm, 2.1 × 50 mm). Solvent A (water containing 0.1% formic acid) and solvent B (acetonitrile containing 0.1% formic acid) were used as the mobile phase at a flow rate of 0.5 ml / min. The gradient used was: isocratic 95% H2O for 2 min, then from 95% to 10% H2O in 4 min, then 10% H2O for 1 min, then from 10% to 95% H2O in 1 min, then 95% H2O for 2 min. The electrospray ionization source was operated in positive ion mode, with a capillary voltage of 2.0 kV and a cone voltage of 40 V for the capillary. Nitrogen was used as the desolvation gas with a total flow rate of 850 L / h. The total mass spectrum was reconstructed from the ion series using the MaxEnt algorithm (version 4.1, from Waters) preloaded on the MassLynx software according to the manufacturer's instructions. To obtain the ion series, the main peak of the chromatogram was selected for integration and further analysis.

[0321] LC-MS analysis of SSF-DNA, Mal-DNA, and Nb-PD-L1-ssDNA: LC–MS was performed on an Xevo G2-S TOF mass spectrometer coupled to an Acquity ultra performance liquid chromatography (UPLC) system, using an Acquity UPLC Protein BEH C8 column (1.7 mm, 2.1 × 50 mm). Solvent A (10 mM aqueous ammonium formate) and solvent B (100% methanol) were used as the mobile phase at a flow rate of 0.5 ml / min. The gradient used was: isocratic 95% H2O for 2 min, then from 95% to 5% H2O in 4 min, then 5% H2O for 1 min, then from 5% to 95% H2O in 1 min, then 95% H2O for 2.5 min. The electrospray ionization source was operated in the negative mode, with a capillary voltage of 2.0 kV and a cone voltage of 80 V. Nitrogen was used as the desolvation gas with a total flow rate of 850 L / h. The total mass spectrum was reconstructed from the ion series using the MaxEnt algorithm (version 4.1, from Waters) pre-installed on the MassLynx software according to the manufacturer's instructions. To obtain the ion series, the main peak of the chromatogram was selected for integration and further analysis.

[0322] LC-MS analysis of peptide-MA conjugation: LC–MS was performed on an Xevo SQDetector 2 mass spectrometer coupled to an Acquity ultra performance liquid chromatography (UPLC) system, using an Acquity UPLC BEH300 C18 column (1.7 mm, 2.1 × 50 mm). Solvent A (water containing 0.1% formic acid) and solvent B (acetonitrile) were used as the mobile phase at a flow rate of 0.4 ml / min. Method A: The gradient used was: isocratic 90% H2O for 2 min, then from 90% to 10% H2O in 5 min, then 10% H2O for 1 min, then from 10% to 90% H2O in 1 min, then 95% H2O for 1 min. Method B: The gradient used was: isocratic 90% H2O for 2 min, then from 90% to 70% H2O in 15 min, then from 70% to 10% H2O for 20 min, then from 10% to 90% H2O in 1 min, then 95% H2O for 2 min. The electrospray ionization source was operated in the positive ion mode, with a capillary voltage of 2.0 kV and a cone voltage of 40 V. Nitrogen was used as the desolvation gas with a total flow rate of 850 L / h. The total mass spectrum was reconstructed from the ion series using the MaxEnt algorithm (version 4.1, from Waters) pre-installed on the MassLynx software according to the manufacturer's instructions. To obtain the ion series, the main peak of the chromatogram was selected for integration and further analysis.

[0323] LC-MS / MS Analysis of Protein Conjugation: For in-gel digestion, the labeled GFP was first resolved by SDS-PAGE and the gel was stained with Coomassie Brilliant Blue. The GFP band was excised, cut into small pieces and transferred to a pre-cleaned microcentrifuge tube. The resulting gel pieces were desalted twice with 50% ACN in 25 mM ammonium bicarbonate (ABC) and then dehydrated in ACN. The gel pieces were rehydrated with 20 mM DTT in 25 mM ABC and incubated at 55 °C for 45 min. The gel pieces were washed with 25 mM ABC and dehydrated again with acetonitrile, then incubated in the dark at room temperature with 55 mM iodoacetamide in 25 mM ABC for 30 min. The treated gel pieces were washed with 25 mM ABC and dehydrated again in acetonitrile. Then, the gel pieces were rehydrated in trypsin solution (20 ng / μL) and incubated at 37 °C for 16 h. To extract tryptic peptides, the gel pieces were soaked in ACN / water / FA solution (v:v:v = 50:45:5), vortexed for 30 min. The solution was carefully removed and the extraction was repeated once. The extracts were combined and dried in a vacuum centrifuge. LC-MS / MS was performed on an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific) coupled to an Easy-nLC 1200 LC system. The peptide sample was loaded onto an analytical column (1.9 μm, C18, 250 mm * 75 μm inner diameter) and eluted with a 65 min gradient. The mass spectrometer was operated in data-dependent mode. Using the Orbitrap mass analyzer, full-scan spectra were acquired in the m / z range of 350 - 1500. MS / MS fragmentation was performed using the HCD mode. The normalized collision energy was 30 V. The raw data were analyzed by Pfind3 and searched against the bovine proteome in the UniProt database. Carbamidomethylation of cysteine was set as a fixed modification. Oxidation of methionine and modification of cysteine residues were set as variable modifications. When the score (PSM score, peptide spectrum match score) was higher than 26, peptides with 2b-1a or 2b+1a modification were considered to be correctly identified, and the modified sites were manually verified.

[0324] NMR

[0325] NMR experiments were measured on a Bruker AVANCE III-400 or 500 spectrometer and in deuterated chloroform (CDCl3). 1 1H NMR and 13 13C NMR spectra were recorded on 400 MHz or 500 MHz and 100 MHz or 125 MHz spectrometers, respectively. 191H NMR spectra were recorded on a 376 MHz or 470 MHz spectrometer. Chemical shifts were reported as δ values relative to internal TMS (δ 0.00 for 1H NMR), chloroform ( 1 δ 7.26 for 1H NMR), and chloroform ( 13 δ 77.00 for 13C NMR). The following abbreviations were used for multiplicities: s: singlet, d: doublet, dd: doublet of doublets, t: triplet, q: quartet, m: multiplet, br: broad signal in the proton spectrum. Mass spectra were measured by ESI-MS (LCQ Fleet, Thermo Fisher Scientific).

[0326] Cell barcoding and scRNA-seq

[0327] A cell mixture of Jurkat, A549, JIMT-1, and MDA-MB-231 was stained with Nb-PD-L1-59ntssDNA at 4 °C for 30 min. After washing and determining the cell number and cell viability, the cells were pooled and loaded onto a microwell chip targeting 20,000 cells on a Singleron (GEXSCOPE single-cell RNA-seq kit, Singleron Biotechnologies Co., Ltd., Nanjing, China). The scRNA-seq library was prepared according to the manufacturer's instructions (Singleron Biotechnologies Co., Ltd., Nanjing, China). After amplification, the cDNA and Nb-PD-L1-59ntssDNA tags were separated by SPRI size selection (SPRI at 0.6× and 1.4×), respectively. The Nb-PD-L1-59ntssDNA tag library was quantified (Qubit, Invitrogen) and amplified using primers SGR-bead-1 / SGR-tag-1, and indexed by additional PCR using primers SGR-bead-2 / SGR-tag-2. The final Nb-PD-L1-59ntssDNA tag library and transcriptome library were analyzed on a BioAnalyzer High Sensitivity DNA kit (Agilent) and sequenced on an Illumina NovaSeq 6000.

[0328] Primers for preparing the Nb-PD-L1-labeled library

[0329]

[0330] Protein sequence

[0331] Engineered Neo2 sequence: Engineered Neo2 consists of 124 amino acids and 1 free cysteine.

[0332] MLVNRICGKGIDGGSPKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKDEAEKAKRMKEWMKRIKTTASEDEQEEMANAIITILQSWIFSAVDHHHHHH(SEQ ID NO.5)

[0333] Engineered Nb-Pd-L1 sequence: The engineered Nb-Pd-L1 consists of 143 amino acids and 1 free cysteine.

[0334] MDQVQLQESGGGLVQPGGSLRLSCAASGKMSSRRCMAWFRQAPGKERERVAKLLTTSGSTYLADSVKGRFTISQNNAKSTVYLQMNSLKPEDTAMYYCAADSFEDPTCTLVTSSGAFQYWGQGTQVTVSSLPETGGCHHHHHH(SEQ ID NO.6)

[0335] GFP sequence: The engineered GFP consists of 243 amino acids and 3 free cysteines.

[0336] MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRICLKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEFVTAAGITHGGGGLEHHHHHH(SEQ ID NO.7)

[0337] Trastuzumab light chain:

[0338] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO.8);

[0339] Trastuzumab heavy chain:

[0340] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESLTQPEGKNNTTKPPKVSNKALPAPIEKTISKSRKQPREPQVYTLPPSREEMTKNQVSLTCLVKSLKSPK(SEQ ID NO.9)

[0341] The amino acid sequences of the light and heavy chains of KN026 can be referred to US2018 / 0291103, and the amino acid sequences of the light and heavy chains of KN046 can be referred to US20210095031A1.

[0342] DNA sequence

[0343] 20nt ssDNA: 5'-NH2-C6-AGC AGC ACA GAG GTC AGA TG(SEQ ID NO.10)

[0344] 59ntssDNA: 5'-NH2-C6-TGT CAA GAT GCT ACC GTT CAG AGC GCA AGA CAC TCCACA AAA AAA AAA AAA AAA AAA*A*A* phosphorothioate modification (SEQ ID NO.11)

[0345] Data analysis

[0346] Raw sequence reads were processed using the CeleScope pipeline (version 1.3.1) with default parameters (https: / / github.com / singleron-RD / CeleScope). The Nb-PD-L1-59ntssDNA tag library was processed using the CeleScope's new function barcode processing plugin ("teg"), which was inspired by previous scRNA-seq multiplexing algorithms. Then, gene expression matrices were analyzed using the R language.

[0347] Modifying GFP with MA

[0348] MA (1 μL, 20 mM in DMSO) and PBS (15 μL, pH = 7.4 / 9.0, 50 mM) were added to the GFP solution (4 μL, 250 μM, HEPES buffer). After incubation at 37 °C for 2 h, the solution was desalted to obtain GFP-MA.

[0349] Linker stability study

[0350] GSH (0.8 μL, 100 mM) and PBS (20 μL, pH = 8.0, 50 mM) were added to the GFP-linker solution (20 μL, 40 μM, PBS solution) at 37 °C and kept for 48 h( Figure 20 )

[0351] Example 1: Screening Michael acceptors as linkers

[0352] Michael acceptors are commonly used reagents for addition reactions with cysteine on antibodies. A new type of Michael acceptor has been studied so that it can react chemically with cysteine on antibodies effectively and chemoselectively. And after modifying the Michael acceptor with an antibody, no degradation occurred in the serum. Based on this, candidate Michael acceptors were used as reaction reagents, where green fluorescent protein was regarded as the template protein for screening Michael acceptors. Considering reactivity, chemoselectivity, and stability, Michael acceptors capable of reacting efficiently with cysteine were screened out.

[0353] The reaction process is shown in Figure 1 . And the chemical structures of the following Michael acceptor candidates are in Figure 2Shown in

[0354] Table 4. Screening of Michael acceptors

[0355] <![CDATA[Entry 1 > Michael acceptor Time (h) Addition number Conversion rate (%) 1 MA 1 2 <![CDATA[- 2 > >99 2 MA 2 2 2 >99 3 MA 3 2 1 60 4 MA 4 2 0 0 5 MA 5 2 0 0 6 MA 6 2 2 60 7 MA 7 2 2 <20 8 MA 8 2 0 60 9 MA 9 2 0 0

[0356] 1 Reaction conditions: sfGFP E124C (320 μM, 5 μL), Michael acceptor (6.4 μL, 5 mM), PBS buffer (40 μL, pH = 8), 37 °C, 2 h. 2 The addition number has no attribution.

[0357] And Michael acceptor MA 2 was named Reagent 1 or PhESF.

[0358] Example 2: Modification of the linker

[0359] Modify Reagent 1 in Example 1 (the chemical structures of the modified Michael acceptor candidates are shown in Figure 3 Shown in

[0360] When the benzene ring of Reagent 1 was modified with different substituents (Michael acceptors 1-1 to 1-4), the effects of different substituents on the addition reaction (e.g., addition activity) were investigated (Table 5). Michael acceptors 1-1 to 1-4 reacted with sfGFP-TEV. When the para position of the sulfonyl fluoride group was connected to the strong electron-withdrawing group trifluoromethoxy, the addition activity decreased significantly (Entry 4); when the electron-donating groups methoxy and hydroxymethyl were attached to the para position of the sulfonyl fluoride group and the electron-withdrawing group trifluoromethyl was attached to the meta position, the addition reaction activity did not change significantly (Entries 2-3, Entry 5). When the sulfonyl fluoride group was attached to allene 1-5, its addition reaction activity decreased significantly (Entry 6).

[0361] The reaction process can be seen in Figure 4 .

[0362] Table 5: Modification of sfGFP-TEV with Reagent 1 derivatives

[0363]

[0364]

[0365] Example 3: Modification of the linker

[0366] Using alkenylsulfonyl fluorides with different configurations (the chemical structures of the modified Michael acceptor candidates are shown in Figure 5As shown in [Figure number not provided], the effects of olefin sulfonyl fluorides with different configurations on the addition reaction were investigated (Table 6). When 5 equivalents of the Michael acceptor were reacted with sfGFP E124C, Michael acceptors 1, 1a, 1b, and 1a-1 all showed good chemoselectivity; while Michael acceptor 1b-1 reacted not only with cysteine but also with lysine. When 20 equivalents of the Michael acceptor were reacted with sfGFP E124C, only Michael acceptor 1 had good chemoselectivity.

[0367] The reaction process can be seen Figure 6 .

[0368] Table 6

[0369]

[0370] Example 4: Characteristics of the selected linker

[0371] 4.1 Selectivity

[0372] To further verify the reactivity, chemoselectivity, and stability of Michael acceptor 1, linkers reported in previous literature (e.g., linkers used in ADCs) were selected as comparative examples to demonstrate that Michael acceptor 1 has good reaction selectivity.

[0373] The chemical structures of the previously reported linkers are shown in Figure 7 . The reaction process of the reaction between sfGFP E124C and the previously reported linkers can be seen Figure 8 . The reaction results are shown in Figure 9 ; Reaction conditions: sfGFP E124C (320 μM, 5 μL), linker (5 mM, 6.4 μL), PBS buffer (40 μL, pH = 8), 37 °C, 2 h.

[0374] 4.2 Competition

[0375] To verify the differences in their reactivity, comparative examples were made in a competition experiment ( Figure 10 ), and the results showed that the reactivity of Michael acceptor 1 was moderate: this means that its reactivity was higher than that of alkenyl sulfonamides and alkynyl phosphates and lower than that of maleimide (5) and acrylamide carbonyl (4) (Table 7).

[0376] Table 7

[0377]

[0378]

[0379] 1Reaction conditions: sfGFP E124C (320 μM, 5 μL), 1 (5 mM, 3.2 μL), x (5 mM, 3.2 μL), PBS buffer (40 μL, pH = 8), 37 °C, 2 h.

[0380] 4.3 Stability

[0381] Using the highly reactive linkers 5 and 4 as controls, the stability of PhESF modified with sfGFP as a linker was verified. After sfGFP was modified, it was added to a buffer containing GSH and incubated at 37 °C for different times, and mass spectrometry analysis was performed. From the mass spectrometry results, it can be seen that both linkers 4 and 5 with modified sfGFP significantly underwent thiol exchange. However, under the same conditions, Michael acceptor 1 did not undergo thiol exchange ( Figure 11 a to Figure 11 c).

[0382] Example 5: Generation of conjugates containing a linker and an antibody

[0383] Modify an antibody (e.g., Herceptin) with Michael acceptor 1 ( Figure 12 a to Figure 12 b), and verify that the modification of the antibody does not affect its binding ability to the antigen ( Figure 12 c).

[0384] Example 6: Further modification of the linker

[0385] Michael acceptor 1 has no additional modifiable groups. Therefore, Michael acceptor 1 was modified to have an azide group, and then the modified Michael acceptor 1 was used to modify the antibody, and the stability of the conjugated antibody was tested in serum.

[0386] Mass spectrometry detection revealed that the modified antibody did not undergo elimination reaction after 7 days in serum ( Figure 13 a to Figure 13 C). In Figure 13 which, Figure 13 a shows Herceptin chemically selectively modified with N3-PhESF; Figure 13 b shows the results of the mass spectrometry of the antibody modified with N3-PhESF; Figure 13 c shows the results of the Herceptin-PhESF-N3 in the serum stability test.

[0387] Example 7: Generation of conjugates containing a linker and a drug

[0388] Use Michael acceptor 1 as a linker to synthesize an ADC. To improve the solubility of Michael acceptor 1, polyethylene glycol was modified on Michael acceptor 1 ( Figure 14a), and then conjugate the toxin MMAE to the cleavable vc-PAB( Figure 14b ), and finally perform a condensation reaction to obtain the ADC: PhESF-PEG4-MMAE( Figure 14c ).

[0389] Example 8: Generation of ADC

[0390] 8.1 DAR = 3.2

[0391] React PhESF-PEG4-MMAE obtained in Example 7 with the reduced antibody to synthesize an ADC (Herceptin-PhESF-PEG4-MMAE) with a DAR of approximately 3.2. As a comparative example, an ADC (Herceptin-Mal-MMAE) with a DAR of approximately 3.2 was also synthesized.

[0392] First, study the stability of the two ADCs in serum, and it was found that the DAR value of the ADC with Michael acceptor 1 as the linker did not decrease significantly after 7 days, while the DAR value of the ADC using maleimide decreased by 70%( Figures 15a to 15c ).

[0393] Figures 15a to 15c The results of the ADC stability test are shown. Figure 15a The results of the ADC stability test in human serum are shown; Figure 15b The mass spectrometry results of Herceptin-PhESF-PEG4-MMAE at different time points in serum are shown; Figure 15c The mass spectrometry results of Herceptin-PhESF-PEG4-MMAE at different time points in serum are shown.

[0394] 8.2 DAR = 3.8

[0395] The DAR value of the ADC with MMAE as the drug generally does not exceed 4. To enhance the killing effect, an ADC with a DAR value of approximately 3.8 was synthesized and verified by mass spectrometry( Figure 16 a to Figure 16 b). As a comparative example, an ADC (Herceptin-Mal-MMAE) with a DAR of approximately 3.8 was also synthesized.

[0396] Figure 16 a shows the structure and mass spectrometry results of Herceptin-PhESF-PEG4-MMAE; Figure 16 b shows the structure and mass spectrometry results of Herceptin-Mal-MMAE.

[0397] Example 9: Tumor cell killing assay

[0398] Tumor cell killing assays were performed using two ADCs (Herceptin-PhESF-PEG4-MMAE and Herceptin-Mal-MMAE prepared in Example 8). From the results, it can be seen that the two ADCs have similar killing efficiencies against HER2 + cells SKBR3, NCI-N87, and MDA-MB-435 ( Figure 17 a to Figure 17 c).

[0399] The IC 50 values of Herceptin-PhESF-PEG4-MMAE for the three cells were 18.3 ng / mL, 7.76 ng / mL, and 14.66 ng / mL, respectively, and the IC50 values of Herceptin-Mal-MMAE were 24.48 ng / mL, 10.94 ng / mL, and 12.03 ng / mL, respectively.

[0400] For HER - cells MDA-MB-231 and HER - cells MDA-MB-435, neither of the two ADCs showed significant killing effects ( Figure 17 d to Figure 17 e).

[0401] It was found that Herceptin-Mal-MMAE could significantly kill HER2 - cells at high concentrations, while Herceptin-PhESF-PEG4-MMAE did not show obvious killing effects at high concentrations ( Figure 17 f).

[0402] Then, a bystander lethality assay was performed on the ADC, and it was found that stable Michael acceptor 1 did not affect its bystander lethality ( Figure 17 g).

[0403] Figure 17 a to Figure 17 h shows the results of the tumor cell killing assay. Figure 17 a shows the killing results for SKBR3 cells, Figure 17 b shows the killing results for NCI-N87 cells; Figure 17 c shows the killing results for HER + MDA-MB-435 cells; Figure 17 d shows the killing results for HER - MDA-MB-435 cells; Figure 17 e shows the killing results for MDA-MB-231 cells; Figure 17 f shows the killing results for MDA-MB-231 cells with a relatively high concentration of ADC; Figure 17g shows the results of the ADC bystander killing assay, and Figure 17 h shows the killing results of the ADC at a concentration of 10 μg / mL against MDA-MB-231 cells.

[0404] Example 10: Chemical Synthesis and Analysis

[0405] Example 10.1: Synthesis of SSF-PEG4-PAB-MMAE 1

[0406]

[0407] Compound S2 was synthesized based on a previously disclosed procedure. 1 1H NMR (400 MHz, CDCl3): δ 7.52 (2H, d, J = 8.8 Hz), 6.68 (2H, d, J = 8.8 Hz), 4.07 (2H, t, J = 4.6 Hz), 4.00 (2H, s), 3.83 (2H, t, J = 4.6 Hz), 3.70 - 3.65 (12H, m), 1.46 (9H, s); 13 13C NMR (100 MHz, CDCl3): δ 169.68, 158.69, 138.17, 117.06, 82.92, 81.58, 70.83, 70.71, 70.60, 69.60, 69.03, 67.53, 28.12. HRMS m / z (ESI): C 20 H 31 IO7 [M + H] + Calculated: 511.1193, Found 511.1192.

[0408] tert-Butyl (E)-14-(4-(2-(fluorosulfonyl)vinyl)phenoxy)-3,6,9,12-tetraoxatetradecanoate (Compound S4):

[0409] An oven-dried reaction tube (20 mL) was charged with AgTFA (2.4 mmol, 1.2 equiv), Pd(OAc)2 (22 mg, 5 mol%), acetone (5 mL), S2 (1.02 g, 2 mmol), and vinyl S3 (440 mg, 4.0 mmol, 2 equiv). The resulting mixture was refluxed at 60 °C for 12 h. The crude product was purified by silica gel column chromatography to give S4. (403 mg, 82%). 11H NMR (400 MHz, CDCl3): δ 7.71 (1H, d, J = 15.2 Hz), 7.48 (2H, d, J = 8.8 Hz), 6.97 (2H, d, J = 8.8 Hz), 6.70 (1H, dd, J = 15.2 Hz, 2.4 Hz), 4.17 (2H, t, J = 4.6 Hz), 4.00 (2H, s), 3.86 (2H, t, J = 4.6 Hz), 3.72 - 3.64 (12H, m), 1.46 (9H, s); 13 13C NMR (100 MHz, CDCl3): 169.65, 162.50, 148.60, 131.08, 123.68, 115.43, 114.82, 114.54, 81.59, 70.87, 70.70, 70.58, 69.45, 69.00, 67.75, 28.09; 19 19F (376 MHz, CDCl3): δ +63.01; HRMS m / z (ESI): C 22 H 33 19FO9S [M + H]+ calculated: 493.1908, found 493.1904:

[0410] (E)-14-(4-(2-(Fluorosulfonyl)vinyl)phenoxy)-3,6,9,12-tetraoxatetradecanoic acid (Compound S5)

[0411]

[0412] An oven-dried reaction tube (20 mL) was charged with S4 (286 mg, 0.5 mmol), TFA (2 mL), and CH2Cl2 (2 mL). The resulting mixture was incubated at room temperature for 4 h. The crude product was purified by silica gel column chromatography to give S5 (192 mg, 90%). 1 1H NMR (400 MHz, CDCl3): δ 7.73 (1H, d, J = 15.2 Hz), 7.49 (2H, d, J = 8.8 Hz), 6.98 (2H, d, J = 8.8 Hz), 6.70 (1H, dd, J = 15.2 Hz, 2.4 Hz), 4.19 (2H, t, J = 4.6 Hz), 4.11 (2H, s), 3.89 (2H, t, J = 4.6 Hz), 3.75 - 3.71 (4H, m), 3.68 - 3.65 (8H, m); 1313C NMR (100 MHz, CDCl3): 171.77, 162.38, 148.61, 131.10, 123.77, 115.46, 114.87, 114.59, 71.13, 70.90, 70.58, 70.35, 70.23, 70.15, 69.44, 69.17, 67.53; 19 19F (376 MHz, CDCl3): δ +63.00; HRMS m / z (ESI): C 18 H 25 FO9S [M−H] - Calcd: 435.1125, found 435.1138.

[0413] SSF-PEG4-vc-PAB-MMAE 1

[0414]

[0415] An oven-dried reaction tube (20 mL) was charged with EDC (0.1 mmol, 2 equiv), DIPEA (0.15 mmol, 3 equiv), dry DMF (2 mL), S5 (22 mg, 0.05 mmol, 1 equiv) and S6 (67 mg, 0.06 mmol, 1.2 equiv). The resulting mixture was incubated at room temperature for 12 h. The crude product was purified by preparative HPLC to give 6 (26 mg, 34%). HRMS m / z (ESI): C 76 H 117 FN 10 O 20 S [M+H] + Calcd: 1541.8229, found 1541.8230. Product 1 was analyzed by LC-MS.

[0416] LC-MS chromatogram and mass spectrum of 1

[0417] The results are shown in Figure 18 .

[0418] Example 10.2: Synthesis of SSF-PEG4-GGFG-Dxd 3

[0419]

[0420] (E)-2-(4-(((S)-10-Benzyl-1-(((1R,9R)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15,18-hexaoxa-3,20,23,26,29-pentaoxa-5,8,11,14,17-pentaazatriacontan-31-yl)oxy)phenyl)ethen-1-sulfonyl fluoride (Compound SSF-PEG4-GGFG-Dxd 3)

[0421]

[0422] Add an oven-dried reaction tube (20 mL) containing EDC (0.04 mmol, 2 equiv), DIPEA (0.06 mmol, 3 equiv), dry DMF (2 mL), S7 (16.8 mg, 0.02 mmol, 1 equiv) and S5 (10.5 mg, 0.024 mmol, 1.2 equiv). Incubate the resulting mixture at room temperature for 12 h. The crude product was purified by preparative HPLC to give 3 (8.0 mg, 32%). HRMS m / z (ESI): C 76 H 117 FN 10 O 20 S[M+Na] + Calculated: 1281.4238, Found 1281.4196. Product 3 was analyzed by LC-MS.

[0423] The results are shown in Figure 19 .

[0424] Example 10.3: Synthesis of MA 16-biotin

[0425]

[0426] (E)-2-(4-(Azidomethyl)phenyl)ethen-1-sulfonyl fluoride (Compound MA 16):

[0427] Under an inert gas atmosphere, diphenylphosphoryl azide (DPPA) (2 mmol, 1 equiv) was added dropwise to a solution of S9 (432 mg, 2 mmol, 1 equiv) in anhydrous DMF (2 mL). The mixture was cooled to 0 °C, and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (2 mmol, 1 equiv) was added dropwise. The resulting mixture was stirred at room temperature for 12 h and quenched with water. The solution was extracted with DCM. The organic layers were combined, dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to give MA 16 (120 mg, 25%). 1 1H NMR (400 MHz, CDCl3): δ 7.81 (1H, d, J = 15.4 Hz), 7.58 (2H, d, J = 6.8 Hz), 7.43 (2H, d, J = 6.8 Hz), 6.88 (1H, dd, J = 15.4 Hz, 2.8 Hz), 4.43 (2H, s); 13 13C NMR (100 MHz, CDCl3): 148.01, 140.31, 129.48, 128.93, 118.61, 118.33, 54.16; 19 19F (376 MHz, CDCl3): δ +62.32; HRMS m / z (ESI): C9H8FN3O2S [M + H] + Calculated: 242.0400, Found 242.0402.

[0428] (E)-2-(4-((4-(15-oxo-19-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-2,5,8,11-tetraoxa-14-azanonadecyl)-1H-1,2,3-triazol-1-yl)methyl)phenyl)ethene-1-sulfonyl fluoride (Compound MA 16-biotin)

[0429]

[0430] An oven-dried reaction tube (20 mL) was charged with CuSO4·5H2O (0.005 mmol, 0.1 equiv), BTTP (0.01 mmol, 0.2 equiv), ASC (0.02 mmol, 0.4 equiv), DMSO (1.5 mL), H2O (0.5 mL), and S10 (0.055 mmol, 1.1 equiv) and MA 16 (0.05 mmol, 1 equiv). The resulting mixture was reacted at 37 °C for 12 h. The crude product was purified by silica gel column chromatography to give MA 16-biotin (31 mg, 89%). 11H NMR (400 MHz, CDCl3): δ 7.80 (1H, s), 7.58 (1H, d, J = 15.2 Hz), 7.45 (1H, d, J = 8.0 Hz), 7.19 - 7.13 (3H, m), 5.40 (2H, s), 4.40 (2H, s), 4.22 - 4.19 (1H, m), 4.03 - 4.00 (1H, m), 3.38 - 3.33 (4H, m), 3.31 - 3.28 (6H, m), 3.23 - 3.20 (2H, m), 3.05 - 3.00 (3H, m), 2.92 - 2.87 (1H, m), 2.64 - 2.60 (1H, m), 2.42 - 2.39 (1H, m), 1.91 (2H, t, J = 7.4 Hz), 1.41 - 1.28 (4H, m), 1.16 - 1.10 (2H, m); 13 13C NMR (100 MHz, CDCl3): 174.83, 164.69, 147.83, 139.64, 131.68, 129.65, 128.57, 119.23, 118.95, 70.12, 70.01, 70.07, 69.82, 69.52, 69.12, 63.43, 62.10, 60.39, 55.57, 53.15, 39.61, 38.98, 35.28, 28.33, 28.06, 25.43; 19 19F (376 MHz, CDCl3): δ +59.81; HRMS m / z (ESI): C 30 H 43 FN6O8S2[M + H] + Calculated: 699.2946, Found: 699.2942.

[0431] Example 10.4: Synthesis of SSF - NHS

[0432]

[0433] Compound S10 was synthesized based on a previously reported procedure. 1H NMR (400 MHz, CDCl3): δ 7.54 (2H, d, J = 8.9 Hz), 6.66 (2H, d, J = 8.9 Hz), 3.92 (2H, t, J = 6.4 Hz), 2.40 (2H, t, J = 7.4 Hz), 1.83 - 1.74 (2H, m), 1.73 - 1.67 (2H, m), 1.56 - 1.78 (2H, m); HRMS m / z (ESI): C12H15IO3[M - H] - Calculated: 332.9988, Found: 332.9985.

[0434] tert-Butyl 6-(4-iodophenoxy)hexanoate (Compound S12):

[0435] An oven-dried reaction tube (20 mL) was charged with S11 (501 mg, 1.5 mmol, 1 equiv), tBuOH (5 equiv), and anhydrous DCM (2 mL). The mixture was cooled to 0 °C, and DMAP (0.1 equiv) was added dropwise. Then DCC (1.1 equiv) was added. The resulting mixture was stirred overnight at room temperature. The crude product was purified by silica gel column chromatography to give S12. (444.6 mg, 76%). 1 H NMR (400 MHz, CDCl3): δ 7.53 (2H, d, J = 8.9 Hz), 6.66 (2H, d, J = 8.9 Hz), 3.91 (2H, t, J = 6.4 Hz), 2.24 (2H, t, J = 7.4 Hz), 1.81 - 1.74 (2H, m), 1.68 - 1.60 (2H, m), 1.51 - 1.46 (2H, m), 1.44 (9H, s); HRMS m / z (ESI): C 16 H 23 IO3[M + H] + Calculated: 391.0770, Found: 391.0773.

[0436] tert-Butyl (E)-6-(4-(2-(fluorosulfonyl)vinyl)phenoxy)hexanoate (Compound S13):

[0437] An oven-dried reaction tube (20 mL) was charged with AgTFA (0.6 mmol, 1.2 equiv), Pd(OAc)2 (5.5 mg, 5 mol%), acetone (2 mL), S12 (1.02 g, 0.5 mmol), and vinyl S3 (220 mg, 1 mmol, 2 equiv). The resulting mixture was refluxed at 60 °C for 12 h. The crude product was purified by silica gel column chromatography to give S13. (163.7 mg, 88%). 1 H NMR (400 MHz, CDCl3): δ 7.74 (1H, d, J = 15.4 Hz), 7.49 (2H, d, J = 8.8 Hz) 6.93 (2H, d, J = 8.8 Hz), 6.68 (1H, dd, J = 2.8 Hz), 4.02 (2H, t, J = 6.4 Hz), 2.25 (2H, t, J = 7.4 Hz), 1.86 - 1.79 (2H, m), 1.70 - 1.63 (2H, m), 1.54 - 1.51 (2H, m), 1.44 (9H, s); 19 F (376 MHz, CDCl3): δ +63.07; HRMS m / z (ESI): C18 H 25 FO5S[M+H] + Calculated value: 373.1485, measured value 373.1486.

[0438] (E)-6-(4-(2-(Fluorosulfonyl)vinyl)phenoxy)hexanoic acid (Compound S14):

[0439] Add an oven-dried reaction tube (20 mL) containing S12 (93 mg, 0.25 mmol), TFA (2 mL), and DCM (2 mL). The resulting mixture was stirred at room temperature for 4 h. The crude product was purified by silica gel column chromatography to give S13 (72 mg, 92%). 1H NMR (400 MHz, CDCl3): δ 7.74 (1H, d, J = 15.4 Hz), 7.49 (2H, d, J = 8.8 Hz), 6.94 (2H, d, J = 8.8 Hz), 6.69 (1H, dd, J = 2.6 Hz), 4.02 (2H, t, J = 6.4 Hz), 2.41 (2H, t, J = 7.4 Hz), 1.91 - 1.81 (2H, m), 1.77 - 1.69 (2H, m), 1.58 - 1.52 (2H, m); 19 F(376 MHz, CDCl3): δ +63.05; HRMS m / z (ESI): C 14 H 17 FO5S[M-H] - Calculated value: 315.0702, measured value 315.0703.

[0440] 2,5-Dioxopyrrolidin-1-yl (E)-6-(4-(2-(fluorosulfonyl)vinyl)phenoxy)hexanoate (Compound SSF-NHS)

[0441] Add an oven-dried reaction tube (20 mL) containing S14 (63 mg, 0.2 mmol), N-hydroxysuccinimide (1.2 equiv), EDC (1.2 equiv), and CH2Cl2 (4 mL). The resulting mixture was stirred at room temperature for 12 h. After completion, the reaction was quenched with 20 mL of water and extracted 3 times with 20 mL of DCM. The combined organic phases were dried over anhydrous Na2SO4 and concentrated to give SSF-NHS (67 mg, 81%). 11H NMR (400 MHz, CDCl3): δ 7.74 (1H, d, J = 15.4 Hz), 7.49 (2H, d, J = 8.8 Hz), 6.94 (2H, d, J = 8.8 Hz), 6.69 (1H, dd, J = 15.4 Hz, 2.6 Hz), 4.03 (2H, t, J = 6.4 Hz), 2.84 (4H, s), 2.66 (2H, t, J = 7.4 Hz), 1.88 - 1.82 (4H, m), 1.64 - 1.59 (2H, m); 13 13C NMR (100 MHz, CDCl3): 169.17, 168.46, 162.70, 148.68, 131.12, 123.48, 115.31, 114.35, 67.87, 33.96, 30.84, 28.54, 25.60, 24.29; 19 19F (376 MHz, CDCl3): δ +63.06; HRMS m / z (ESI): C 18 H 20 19FNO7S [M + H] + Calculated: 436.0842, Found: 436.0827.

[0442] Example 10.5: Synthesis of SSF - OSO2F

[0443]

[0444] An oven - dried reaction tube (20 mL) was charged with SN38 (98.3 mg, 0.25 mmol), Et3N (3 equiv) and DCM (2 mL). The mixture was stirred at room temperature under a SO2F2 balloon for 24 h. The reaction was quenched by the addition of brine (30 mL) and extracted with DCM (30 mL). The organic layers were combined, dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography to afford SN38 - OSO2F as a yellow foam (100.5 mg, 85%). 1H NMR (400 MHz, DMSO): δ 8.37 (1H, d, J = 2.6 Hz), 8.33 (1H, d, J = 9.4 Hz), 7.66 (1H, s), 5.60 (1H, d, J = 16.4 Hz), 5.41 - 5.37 (3H, m), 3.29 - 3.27 (2H, m), 1.99 - 1.94 (2H, m), 1.43 (3H, t, J = 7.6 Hz), 1.01 (3H, t, J = 7.4 Hz).

[0445] In - vivo cytotoxicity study

[0446] At 37 °C, in the presence of 5% CO2, cells were seeded in a 96-well plate at 5,000 cells per well and maintained for 24 h. Serial dilutions of SN38 and SN38-OSO2F were added to the cells in complete growth medium, and the cells were incubated at 37 °C in the presence of 5% CO2 for 96 h. Cell viability was evaluated using the Cell Counting-Lite 2.0 luminescent cell viability assay (Vazyme, DD1101-01). Cell viability was plotted as a percentage of untreated cells. Each measurement was performed in triplicate.

[0447] Figure 30 The results of the cell viability assays of SN38 and SN38-OSO2F evaluated using the cell line (N87) are shown.

[0448] Step 10.7: Synthesis of SSF-PEG4-T785

[0449]

[0450] An oven-dried reaction tube (20 mL) was charged with EDC (0.1 mmol, 2 equiv), DIPEA (0.15 mmol, 3 equiv), dry DMF (2 mL), T785 (15.6 mg, 0.05 mmol, 1 equiv), and S5 (26 mg, 0.06 mmol, 1.2 equiv). The resulting mixture was incubated at room temperature for 12 h. The crude product was purified by preparative HPLC to give SSF-PEG4-T785 (15.6 mg, 24%). HRMS m / z (ESI): C 36 H 48 FN5O8S [M+H] + Calculated: 730.3286, Found: 730.3253.

[0451] Example 11: Synthesis of Antibody-Biotin Conjugates

[0452] Example 11.1: Synthesis of Trastuzumab-MA 16-Cy5.5 Conjugates

[0453] Cysteine-selective protein modification with MA 16

[0454] MA 16 (1 μL, 20 mM in DMSO) and PBS (15 μL, pH = 7.5, 50 mM) were added to the protein solution (4 μL, 250 μM, in PBS buffer). After incubation at 37 °C for 2 h, the solution was desalted to obtain the product before LC-MS analysis.

[0455] Reduction of the antibody

[0456] TCEP·HCl (2.6 μL, 25 mM in PBS) was added to the antibody (KN026, KN046, trastuzumab) solution (40 μL, 160 μM in PBS solution), and the resulting solution was incubated at 37 °C for 2 h. Before LC-MS analysis, the solution was desalted to obtain the reduced antibody.

[0457] MA 16-biotin (1.6 μL, 5 mM in DMSO) and PBS (34.5 μL, pH = 7.4, 50 mM) were added to the antibody solution (4 μL, 100 μM, in PBS buffer). After incubation at 37 °C for 2 h, the solution was desalted to obtain the antibody biotin conjugate before LC-MS analysis.

[0458] Synthesis of trastuzumab-MA 16-Cy5.5 conjugate

[0459] MA 16 (1.6 μL, 5 mM in DMSO) and PBS (34.5 μL, pH = 7.4, 50 mM) were added to the trastuzumab solution (4 μL, 100 μM, in PBS buffer). After incubation at 37 °C for 2 h, the solution was desalted to obtain trastuzumab-MA 16.

[0460] DBCO-Cy5.5 (0.8 μL, 5 mM in DMSO) and PBS (15 μL, pH = 7.4, 50 mM) were added to the trastuzumab-MA 16 solution (4 μL, 50 μM, in PBS buffer). After incubation at 37 °C for 12 h, the solution was desalted to obtain trastuzumab-MA 16-Cy5.5 before LC-MS analysis.

[0461] SDS–PAGE analysis of antibody conjugates

[0462] 2 μL of trastuzumab, trastuzumab-MA 16-Cy5.5, and trastuzumab-MA 16-biotin were mixed with 10 μL of ultrapure water and 4 μL of SDS–PAGE loading buffer containing 2-mercaptoethanol. The samples were heated to 95 °C or heated for 10 min and then completely loaded onto the SDS–PAGE gel.

[0463] Example 11.2: Synthesis of trastuzumab-1 (Herceptin-PhESF-MMAE)

[0464] 1 (i.e., 2.6 μL of SSF-PEG4-PAB-MMAE, 25 mM in DMSO), DMSO (17.4 μL) and PBS (280 μL, pH = 7.4, 50 mM) were added to the trastuzumab solution (100 μL, 130 μM, PBS buffer). After incubation at 25 °C for 2 h, the solution was desalted to obtain trastuzumab-1.

[0465] Example 11.3: Synthesis of Trastuzumab-2 (Herceptin-Mal-MMAE)

[0466] 2 (i.e., 1.8 μL of Mal-vc-PAB-MMAE, 25 mM in DMSO), DMSO (18.2 μL) and PBS (280 μL, pH = 7.4, 50 mM) were added to the trastuzumab solution (100 μL, 130 μM, PBS buffer). After incubation at 25 °C for 2 h, the solution was desalted to obtain trastuzumab-2. Mal-VC-PAB-MMAE was purchased from Shandong Meitai Pharmaceutical Co., Ltd.

[0467] Example 11.4: Synthesis of Trastuzumab-3 (Herceptin-PhESF-Dxd)

[0468] 3 (i.e., 1.5 μL of SSF-PEG4-GGFG-Dxd, 25 mM in DMSO), DMSO (0.5 μL) and PBS (10 μL, pH = 9.0, 50 mM) were added to the trastuzumab solution (10 μL, 130 μM, PBS buffer). After incubation at 37 °C for 12 h, the solution was desalted to obtain trastuzumab-3.

[0469] Figure 28 The deconvoluted intact protein MS of trastuzumab-3 is shown.

[0470] Step 11.5: Synthesis of Trastuzumab-T785

[0471] SSF-PEG4-T785 (13 μL, 10 mM in DMSO), DMSO (27 μL) and PBS (560 μL, pH = 9.0, 50 mM) were added to the trastuzumab solution (200 μL, 130 μM, PBS buffer). After incubation at 25 °C for 2 h, the solution was desalted to obtain trastuzumab-T785.

[0472] Figure 29 The deconvoluted intact protein MS of trastuzumab-T785 is shown.

[0473] Example 12: Analysis of MA 16-Related Conjugates

[0474] SDS-PAGE Analysis of Antibody Conjugates

[0475] 2 μL of trastuzumab, trastuzumab-MA 16-Cy5.5, and trastuzumab-MA 16-biotin were mixed with 10 μL of ultrapure water and 4 μL of SDS–PAGE loading buffer containing 2-mercaptoethanol. The samples were heated to 95 °C or heated for 10 min and then completely loaded onto the SDS–PAGE gel.

[0476] The results of modifying trastuzumab using detection probes of MA 16 derivatives are as Figure 24 shown.

[0477] Figure 24 A shows the synthetic scheme for attaching MA16-biotin and MA16-Cy5.5 to trastuzumab. Reaction conditions for trastuzumab-MA16-biotin: 20 μM trastuzumab, 400 μM MA16-biotin, PBS, 37 °C, 2 h. Reaction conditions for trastuzumab-MA16: 20 μM trastuzumab, 400 μM MA16, PBS, 37 °C, 2 h. Reaction conditions for trastuzumab-MA16-Cy5.5: 10 μM trastuzumab-MA16, 200 μM DBCO-Cy5.5, PBS, 37 °C, 12 h. Figure 24 B and Figure 24 C show SDS–PAGE gels, Western blots, and LC-MS analyses before and after the reaction of trastuzumab with MA16-biotin.

[0478] Flow cytometry assay

[0479] Cells were suspended in flow cytometry buffer (PBS containing 2% FBS) containing trastuzumab-MA 16-biotin or control trastuzumab and incubated at 4 °C for 45 min. After washing twice with flow cytometry buffer, the cells were further incubated with APC-streptavidin (BioLegend, 405207) at 4 °C for 30 min, resuspended, washed twice with flow cytometry buffer, and analyzed using an Agilent flow cytometer (Agilent NovoCyte Quanteon). Isotype control antibody staining was used to determine the thresholds for positive and negative cells. Agilent Novoexpress was used for all flow data analysis.

[0480] Figure 24D shows flow cytometry analysis of trastuzumab-MA16-biotin stained cancer cells. NCI-N87 (HER2+) and MDA-MB-468 cells (HER2-) were incubated with trastuzumab-MA16-biotin, while the control group was treated with trastuzumab. After staining, the cells were further stained with SA-APC to detect biotin.

[0481] Figure 24 E and Figure 24 F shows the analysis of trastuzumab by fluorescence imaging (right), Coomassie staining (left), and LC-MS before and after modification with Cy5.5.

[0482] Fluorescence imaging

[0483] MDA-MB-231 and NCI-N87 cells were grown overnight at 37 °C on sterile glass coverslips or slides. After a brief wash with DPBS, the sections were incubated with 20 μg / mL trastuzumab-MA 16-Cy5.5 at 37 °C for 1 h. MDA-MB-231 cells were transfected with a plasmid encoding GFP. Then, the co-cultured cells were imaged at 40X magnification under a microscope.

[0484] Figure 24 G shows fluorescence imaging for specific cell surface HER2 detection enabled by trastuzumab-MA16-Cy5.5 (scale bar 50 μm).

[0485] Example 13: Analysis of Trastuzumab-Related Conjugates

[0486] Example 13.1: In Vivo Stability Study

[0487] In an Eppendorf, for each sample, 90 μL of human serum was mixed with 10 μL of trastuzumab-1 (20 mg / ml) or trastuzumab-2 respectively to obtain a final solution of 0.2 mg / mL ADC in human serum. The samples were incubated at 37 °C in humans and mice for 3 days and 7 days. The samples were incubated at 37 °C in mouse serum for 3 days. The samples at day 0 were directly further processed.

[0488] Example 13.2: In Vitro Cytotoxicity Study

[0489] At 37 °C, in the presence of 5% CO2, cells were seeded at 5,000 cells per well in a 96-well plate and maintained for 24 h. Serial dilutions of trastuzumab-1, trastuzumab-2, and trastuzumab were added to the cells in complete growth medium and incubated at 37 °C in the presence of 5% CO2 for 96 h. Cell viability was evaluated using the Cell Counting-Lite 2.0 luminescent cell viability assay (Vazyme, DD1101-01). Cell viability was plotted as a percentage of untreated cells. Each measurement was performed in triplicate.

[0490] Example 13.3: Bystander killing assay

[0491] A cell mixture of SKBR-3 and MDA-MB-231 was seeded at 1:1 per well in a 96-well plate, and MDA-MB-231 cells alone were seeded at the same density in a 96-well plate and maintained at 37 °C and 5% CO2 for 24 h. Then, 2 μg / mL trastuzumab, trastuzumab-1, and trastuzumab-2 were added and incubated at 37 °C with 5% CO2 for 96 h. MDA-MB-231 is a stable cell line overexpressing luciferase. Cell viability was evaluated using a dual-luciferase reporter assay kit (Vazyme, DL101-01).

[0492] Example 13.4: Nude mouse xenograft assay

[0493] All in vivo studies were conducted in accordance with the local guidelines of the Institutional Animal Care and Use Committee (approval number: IACUC-2101001). NCI-N87 cells (2 million) were subcutaneously inoculated into specific pathogen-free female nude mice. Tumor-bearing mice were randomly divided into a treatment group and a control group. When the average volume of the tumor reached approximately 100 mm 3 to 200 mm 3 , administration began on day 0. On day 0 and day 14, each substance, 1 mg / kg trastuzumab-1 or trastuzumab-2 and vehicle (PBS), was administered intravenously to the mice. Tumor volume was defined as 1 / 2 * length * width 2 , and tumor size was recorded every three days.

[0494] Results are shown in Figure 25 .

[0495] Figure 25 A shows the anti-tumor activity of ADC (5 mg / kg) in a BALB / c nude mouse NCI-N87 tumor xenograft model. Figure 25B shows the anti-tumor activity of ADC (1 mg / kg) in a NCI-N87 tumor xenograft model in BALB / c nude mice. Tumor volumes of seven mice per group are shown separately. Figure 25 C shows Figure 25 The Kaplan–Meier survival analysis of the study shown in B. Figure 25 D shows that neutropenia was observed in rats after a 20 mg / kg ADC dose. Four animals were given trastuzumab-1, trastuzumab-2, or vehicle, and blood samples were taken for hematological markers.

[0496] Example 13.5: Safety study

[0497] All in vivo studies were conducted in accordance with the local guidelines of the Institutional Animal Care and Use Committee (approval number: ZJCLA-IACUC-20040026). In female rats aged 12 to 14 weeks, ADC or PBS was administered intravenously at 20 mg / kg (four rats per dose group, randomly assigned). Four serum samples were taken for hematological analysis before and several days after dosing.

[0498] Example 14: Analysis of MA 2-related conjugates

[0499] Hydrolytic stability of MA 2 and MA 5

[0500] At 37 °C, MA 2 (i.e., 5 mM final concentration) or MA 5 (i.e., 5 mM final concentration) was shaken in 100 μL of 50 mM PBS (pH 9.0) for 48 h. Then, 1 μL of the reaction mixture and PBS (15 μL, pH = 7.4, 50 mM) were added to the GFP solution (4 μL, 250 μM, HEPES buffer), respectively. After incubation at 37 °C for 2 h, the solution was desalted to obtain the product before LC-MS analysis.

[0501] The hydrolytic stability results are shown in Figure 21 .

[0502] The stability results of SSF(MA 2) and maleimide in aqueous buffer are shown in Figure 26 .

[0503] Figure 26 A shows that 5'-maleimide-ssDNA was shaken at 37 °C for 48 h. Then, the reaction mixture was analyzed by LC-MS. 5'-maleimide-ssDNA was completely hydrolyzed to 5'-maleic acid-ssDNA. Figure 26Panel B shows the hydrolytic stability of SSF: 5'-maleimide-ssDNA was shaken at 37 °C for 48 h. The reaction mixture was then analyzed by LC-MS, and only the starting material was obtained.

[0504] Modification of GFP-TEV with MA 2 analogues

[0505] MA 2 or an MA 2 analogue (i.e., compound 1a, 1a-1, 1b, 1b-1) (1 μL, 20 mM in DMSO) and PBS (15 μL, pH = 7.4, 50 mM) were added to a GFP-TEV solution (4 μL, 250 μM in HEPES buffer). After incubation at 37 °C for 2 h, the solution was desalted to obtain the product prior to LC-MS analysis.

[0506] Kinetic experiments

[0507] MA 2 or a previous stable linker (i.e., compounds 2, 3, 6, 7) (1 μL, 5 mM in DMSO) or PBS (15 μL, pH = 7.4, 50 mM) was added to a GFP solution (4 μL, 250 μM, HEPES buffer). After incubation at 37 °C for different times (5 min, 10 min, 30 min, 60 min, 90 min, 120 min, and 240 min), the solution was desalted to obtain the product prior to LC-MS analysis.

[0508] Results are shown in Figure 22 in. Figure 22 MA 2 was compared with previously reported stable Cys-specific labeling reagents.

[0509] Figure 22 Panel A shows the chemical structures of the reported stable Cys-specific labeling reagents. The arrow points to the cysteine reaction site. Figure 22 Panel B shows the reaction kinetics of GFP (50 μM) with 5 equivalents of the labeling reagent.

[0510] Competitive experiments

[0511] MA 2 (0.5 μL, 20 mM in DMSO), 2-5 (0.5 μL, 20 mM in DMSO), and PBS (15 μL, pH = 7.4, 50 mM) were added to a GFP solution (4 μL, 250 μM, in HEPES buffer). After incubation at 37 °C for 2 h, the solution was desalted to obtain the product prior to LC-MS analysis.

[0512] The MS / MS spectral results of the GFP fragment modified with MA 2 are shown in Figure 23 in.

[0513] Figure 24Shows the Cys-specific modification of different proteins using SSF.

[0514] Figure 24 A shows the reaction scheme of MA2 with different proteins. Figure 24 B shows the deconvoluted intact protein MS of protein-MA6 conjugates, including neo2, Nb-PD-L1, GFP, KN046, trastuzumab, KN026.

[0515] Determination of the binding affinity of trastuzumab-MA 2 conjugate

[0516] In 200 μL of flow cytometry buffer (PBS containing 2% FBS), SKBR3 cells were incubated with different concentrations of trastuzumab-MA6 or trastuzumab on ice for 30 min. After binding, the cells were washed twice with PBS and further incubated with trastuzumab-Cy5.5 on ice for 30 min, resuspended and washed twice more with flow cytometry buffer, and analyzed using an Agilent flow cytometer (Agilent NovoCyte Quanteon).

[0517] Example 15: Conjugation integrity study of Nb-PD-L1-20ntssDNA in the presence of 10% human serum based on LC-MS / SDS-PAGE

[0518] A mixture of Nb-PD-L1-20ntssDNA (2 μL, 50 μM), human serum (2 μL), and PBS (16 μL, pH = 8.0) was incubated in the dark at 37 °C for 24 h, 48 h, and 72 h. The reaction mixture was analyzed by LC-MS and SDS-PAGE. SDS-PAGE: 10 μL of Nb-PD-L1-20nt ssDNA was mixed with 5 μL of SDS-PAGE loading buffer containing 2-mercaptoethanol. The sample was heated to 95 °C for 10 min and completely loaded onto the SDS-PAGE gel.

[0519] Figure 27 Shows the site-specific DNA-protein conjugates constructed by SSF-ssDNA and the application of Nb-PD-L1 ssDNA in single-cell RNA sequencing.

[0520] Figure 27 A shows the scheme for protein modification using SSF-ssDNA probes. Figure 27 B shows the deconvoluted mass spectra of DNA-protein conjugates constructed by 20nt SSF-ssDNA or 59nt SSF-ssDNA probes. Figure 27Panel C shows the conjugate integrity study of Nb-PD-L1-20nt ssDNA based on LC-MS in the presence of 10% human serum, as well as the deconvoluted mass spectra of samples collected at specific time points. Figure 27 Panel D shows the flow chart of Nb-PD-L1-ssDNA for CITE-seq, which is used to detect target cells at the single cell level using transcriptomics. Figure 27 Panel E shows the clustering of single cell expression profiles based on transcriptomics. Cyan: Jurkat; Red: A549; Green: JIMT-1; Purple: MDA-MB-231. Figure 27 Panel G shows the overlay on Figure 27 the relative intensity targeted by Nb-PD-L1-ssDNA on the UMAP projection shown in Panel F. Figure 27 Panel G shows the violin plot depicting the mRNA expression levels of PD-L1 (CD274) in four cell lines. Figure 27 Panel H shows the violin plot depicting the scaled (z-score) normalized UMI counts of the 59nt-ssDNA barcode (Nb-PD-L1 binding intensity) in four cell lines.

[0521] While the preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. The present invention is not limited by the specific examples provided in the specification. Although the present invention has been described with reference to the foregoing specification, the description and illustration of the embodiments herein are not restrictive. Many variations, changes and alternatives will occur to those skilled in the art without departing from the present invention. In addition, it should be understood that all aspects of the present invention are not limited to the specific recitations, configurations or relative proportions set forth herein, which depend on various conditions and variables. It should be understood that various alternatives of the embodiments of the present invention described herein may be employed to practice the present invention. Accordingly, it is contemplated that the present invention should equally cover any such alternatives, modifications, variations or equivalents. The appended claims are intended to define the scope of the present invention and thereby cover the methods and structures within the scope of these claims and their equivalents. Sequence Listing <110> Nanjing University <120> Conjugates and Their Preparation Methods and Uses <130> 0230-PA-004CN <160> 11 <170> PatentIn version 3.5 <210> 1 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> SGR-beads-1 sequence <400> 1 aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctc 49 <210> 2 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> SGR-tag-1 sequence <400> 2 tggagttcag acgtgtgctc ttccgatctg ttgtcaagat gctaccgttc agag 54 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> SGR-beads-2 sequence <400> 3 aatgatacgg cgaccaccga gatct 25 <210> 4 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> SGR-tag-2 sequence <400> 4 caagcagaag acggcatacg agattcgcct tagtgactgg agttcagacg tgtgctc 57 <210> 5 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> Engineered Neo2 <400> 5 Met Leu Val Asn Arg Ile Cys Gly Lys Gly Ile Asp Gly Gly Ser Pro 1 5 10 15 Lys Lys Lys Ile Gln Leu His Ala Glu His Ala Leu Tyr Asp Ala Leu 20 25 30 Met Ile Leu Asn Ile Val Lys Thr Asn Ser Pro Pro Ala Glu Glu Lys 35 40 45 Leu Glu Asp Tyr Ala Phe Asn Phe Glu Leu Ile Leu Glu Glu Ile Ala 50 55 60 Arg Leu Phe Glu Ser Gly Asp Gln Lys Asp Glu Ala Glu Lys Ala Lys 65 70 75 80 Arg Met Lys Glu Trp Met Lys Arg Ile Lys Thr Thr Ala Ser Glu Asp 85 90 95 Glu Gln Glu Glu Met Ala Asn Ala Ile Ile Thr Ile Leu Gln Ser Trp 100 105 110 Ile Phe Ser Ala Val Asp His His His His His His 115 120 <210> 6 <211> 143 <212> PRT <213> Artificial Sequence <220> <223> Engineered Nb-Pd-L1 <400> 6 Met Asp Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Lys Met Ser Ser 20 25 30 Arg Arg Cys Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Arg Val Ala Lys Leu Leu Thr Thr Ser Gly Ser Thr Tyr Leu Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asn Asn Ala Lys Ser Thr 65 70 75 80 Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Ala Ala Asp Ser Phe Glu Asp Pro Thr Cys Thr Leu Val Thr 100 105 110 Ser Ser Gly Ala Phe Gln Tyr Trp Gly Gln Gly Thr Gln Val Thr Val 115 120 125 Ser Ser Leu Pro Glu Thr Gly Gly Cys His His His His His His 130 135 140 <210> 7 <211> 243 <212> PRT <213> Artificial Sequence <220> <223> Engineered GFP <400> 7 Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val 1 5 10 15 Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Arg Gly Glu 20 25 30 Gly Glu Gly Asp Ala Thr Asn Gly Lys Leu Thr Leu Lys Phe Ile Cys 35 40 45 Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu 50 55 60 Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Arg 65 70 75 80 His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg 85 90 95 Thr Ile Ser Phe Lys Asp Asp Gly Thr Tyr Lys Thr Arg Ala Glu Val 100 105 110 Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Cys Leu Lys Gly Ile 115 120 125 Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn 130 135 140 Phe Asn Ser His Asn Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn Gly 145 150 155 160 Ile Lys Ala Asn Phe Lys Ile Arg His Asn Val Glu Asp Gly Ser Val 165 170 175 Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro 180 185 190 Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Val Leu Ser 195 200 205 Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val 210 215 220 Thr Ala Ala Gly Ile Thr His Gly Gly Gly Gly Leu Glu His His His 225 230 235 240 His His His <210> 8 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Trastuzumab light chain: <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 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> 9 <211> 453 <212> PRT <213> Artificial Sequence <220> <223> Trastuzumab heavy chain <400> 9 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 Ala 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 Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro 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 Leu Thr Gln Pro Glu Gly Lys Asn Asn Thr Thr Lys Pro Pro Lys Val 385 390 395 400 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ser 405 410 415 Arg Lys Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 420 425 430 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Ser 435 440 445 Leu Lys Ser Pro Lys 450 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 20ntssDNA <400> 10 agcagcacag aggtcagatg 20 <210> 11 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> 59ntssDNA <400> 11 tgtcaagatg ctaccgttca gagcgcaaga cactccacaa aaaaaaaaaa aaaaaaaaa 59

Claims

1. A conjugate having a structure selected from the group consisting of:

2. A conjugate having a structure selected from the group consisting of: and and wherein R is -F.

3. Use of a compound having a structure selected from the group consisting of in the preparation of a conjugate: Among them, The conjugate is an antibody-drug conjugate, and the compound reacts with the -SH of the cysteine of the antibody.

4. A pharmaceutical composition comprising the conjugate according to claim 1 or 2 and a pharmaceutically acceptable carrier.

5. Use of the conjugate according to claim 1 or 2, or the pharmaceutical composition according to claim 4 in the preparation of a drug for preventing and / or treating a disease in a subject in need thereof, the disease being a tumor or an autoimmune disease.

6. The use according to claim 5, wherein the tumor comprises a solid tumor and / or a non-solid tumor.

7. A diagnostic reagent comprising the conjugate according to claim 1 or 2.

8. The diagnostic reagent according to claim 7, wherein the diagnostic reagent is labeled.

9. The diagnostic reagent according to claim 8, wherein the label is selected from: a radioactive label, a fluorophore, a chromophore, an imaging agent, and a metal ion.

Images