Conjugate of cytotoxic agents versus cell-binding molecules using branched linkers

Abstract

The conjugation of a cytotoxic drug to a cell-binding molecule using a side-chain linker is provided. The present invention provides a method for preparing a side-chain linkage of a conjugate of a cytotoxic molecule to a cell-binding ligand, as well as a method for using said conjugate in the targeted treatment of cancer, infections, and immunological disorders.

Description

Technology Field

[0001] The present invention relates to the conjugation of a cytotoxic agent to a cell-binding molecule using a branch linker to have better pharmacokinetics in the delivery of the conjugated compound, thereby causing more precise targeted treatment of abnormal cells. The present invention also relates to a branched-linkage method of conjugating a tubulisin analog molecule to a cell-binding ligand, as well as a method of using the conjugate in the targeted treatment of cancer, infections, and autoimmune diseases. The present invention also relates to a branched-linkage method of conjugating a cytotoxic molecule to a cell-binding ligand, as well as a method of using the conjugate in the targeted prevention or treatment of cancer, infections, and immunological disorders. Background Technology

[0002] Antibody-drug conjugates (ADCs), consisting of monoclonal antibodies (mAbs) linked to cytotoxic drugs through specialized linking molecules, are becoming one of the major biotherapeutics for the treatment of cancer, infections, autoimmune disorders, and other drug-resistant diseases (Lambert, JM and Berkenblit, A., Annu Rev Med. 2018, 69:191-207; Mariathasan, S. and Tan, M., Trends Mol Med. 2017, 23(2):135-149; Kern, JC et al. J. Am. Chem.Soc. 2016, 138, 1430-1445; Lee, H. et al, Bioconjug Chem. 2017, 28(4): 1084-1092). Antibodies containing structures larger than 100 s-hours generally have a much longer half-life than that of cytotoxic drugs in blood circulation. Therefore, once bound to the antibody, the potential for systemic exposure and toxicity from the cytotoxic drug is typically significantly reduced. Furthermore, ADCs can possess the ability to deliver and release cytotoxic agents more accurately at the tumor site or within target tumor cells. Consequently, the therapeutic window—specifically the selectivity and specificity of the cytotoxic drug against tumor-normal tissues—is significantly improved. Currently, there are five ADCs approved by the US FDA—gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, inotuzumab ozogamicin, and moxetumomab pasodotox—and more than 100 new promising agents are under development.

[0003] Among the descriptions of ADC complexes of cytotoxic drugs, releaseable linkers, antibodies, and conjugates comprising components at the antibody site, it was known that the linker not only significantly influences the efficacy, selectivity, and pharmacokinetics of the resulting ADC conjugate, but also enables it to overcome multidrug-resistant diseased cells overexpressing efflux transporter proteins (References [Zhao, RY et al (2011) J. Med. Chem. 54, 3606; Acchionea, M. et al (2012) mAbs, 4, 362; Doronina, S. et al, (2006) Bioconjugation Chemistry , [17, 114; Hamann, P. et al. (2005) Bioconjug Chem. 16, 346]). Therefore, an optimized linker is significantly important to improve the spectrum of the therapeutic potential and safety profiles of ADCs.

[0004] Therefore, because the linker for an ADC must be degradable, the conjugated cytotoxic drug can potentially be released into the bloodstream, thereby increasing systemic toxicity and reducing efficacy. This type of off-target toxicity, along with poor permeability / internalization on cancer cells, metabolic disturbance, and target specificity, has caused more than 40 ADC drugs to fail in clinical trials over the past 40 years. Off-target toxicity also hinders the widespread application of approved ADC drugs.

[0005] For example, in clinical trials, adotrastuzumab emtansine (T-DM1, Kadcyla®) with a stable (non-truncational) MCC linker demonstrated significant benefit to patients with HER2-positive metastatic breast cancer (mBC) or those who had already been treated for mBC or experienced a HER2 tumor recurrence within 6 months of adjuvant therapy (Peddi, P. and Hurvitz, S., Ther. Adv. Med. Oncol. 2014, 6(5), 202-209; Piwko C, et al, Clin Drug Investig. 2015, 35(8), 487-93; Lambert, J. and Chari, R., J. Med. Chem. 2014, 57, 6949-64). However, T-DM1 failed in clinical trials as a first-line treatment for patients with HER2-positive unresectable locally advanced or metastatic breast cancer and as a second-line treatment for HER2-positive advanced gastric cancer due to the small benefit to patients when comparing efficacy with incidental toxicity (References [Ellis, PA, et al, J. Clin. Oncol. 2015, 33, (suppl; abstr 507 of 2015 ASCO Annual Meeting); Shen, K. et al, Sci Rep. 2016; 6: 23262; de Goeij, BE and Lambert, JM Curr Opin Immunol 2016, 40, 14-23; Barrios, CH et al, J Clin Oncol 2016, 34, (suppl; abstr 593 of 2016 ASCO Annual Meeting]).

[0006] To address the problem of off-target toxicity, particularly to address the activity of ADC linker-payloads against target / target diseases, research and development in ADC chemistry and design are currently expanding beyond the active payload alone to the categories of linker-payload compartment and conjugate chemistry (Lambert, JM Ther Deliv 2016, 7, 279-82; Zhao, RY et al, 2011, J. Med. Chem. 54, 3606-23). Currently, numerous drug development companies and laboratories are focusing significantly on establishing novel and feasible specific conjugation linkers and methods for site-specific ADC conjugation, which will result in longer cyclic half-lives, higher efficacy, potentially reduced off-target toxicity, and a narrow range of in vivo pharmacokinetic (PK) characteristics of ADCs, as well as better batch-to-batch consistency in ADC production (Hamblett, KJ et al, Clin. Cancer Res. 2004, 10, 7063-70; Adem, YT et al, Bioconjugate Chem. 2014, 25, 656-664; Boylan, NJ Bioconjugate Chem. 2013, 24, 1008-1016; Strop, P., et al 2013 Chem. Biol. 20, 161-67; Wakankar, A. mAbs, 2011, 3, 161-172). This specific conjugation method is comparable to previously engineered cysteine ​​(references [Junutula, JR et al. Nat. Biotechnol. 2008, 26, 925-32; Junutula, JR, et al 2010 Clin. Cancer Res. 16, 4769]; U.S. Patents No. 8,309,300; No. 7,855,275; No. 7,521,541; No. 7,723,485, WO2008 / 141044), selenocysteine ​​(references [Hofer, T., et al. Biochemistry 2009, 48, 12047-57; Li, X., et al. Methods 2014, 65, 133-8]; U.S. Patent No. 8,916,159 (US National Cancer Institute)), cysteine ​​containing a tag with a perfluoroaromatic reagent (Reference [Zhang, C. et al. Nat. Chem. 2015, 8, 1-9]), thiolfucose (Reference [Okeley, NM, et al 2013 Bioconjugate Chem. 24, 1650]), non-natural amino acids (Reference [Axup, JY, et al, Proc. Nat. Acad. Sci. USA. 2012, 109, 16101-6; Zimmerman, ES, et al., 2014, Bioconjug. Chem. 25, 351-361; Wu, P., et al, 2009 Proc. Natl. Acad. Sci. 106, 3000-5; Rabuka, D., et al, Nat. Protoc. [2012, 7, 1052-67]; U.S. Patent No. 8,778,631 and U.S. Patent Application Publication No. 20100184135, WO2010 / 081110 (Sutro Biopharma); WO2006 / 069246, 2007 / 059312, U.S. Patents No. 7,332,571, 7,696,312 and 7,638,299 (Ambrx); WO2007 / 130453, U.S. Patents No. 7,632,492 and 7,829,659 (Allozyne), Conjugation to reduced intermolecular disulfides by re-bridging of dibromomaleimide (Jones, MW et al. J. Am. Chem. Soc. 2012, 134, 1847-52), bis-sulfone reagents (Badescu, G. et al. Bioconjug. Chem. 2014, 25, 1124-36; WO2013 / 190272, WO2014 / 064424 (PolyTherics Ltd), dibromorphyridadione (Literature [Maruani, A. et al. Nat. Commun.[2015, 6, 6645]), galactosyl- and sialyltransferase (reference [Zhou, Q. et al. Bioconjug. Chem. 2014, 25, 510-520]; U.S. Patent Application Publication No. 20140294867 (Sanofi-Genzyme), formylicin-synthesizing enzyme (FGE) (reference [Drake, PM et al. Bioconj. Chem. 2014, 25, 1331-41]; U.S. Patents No. 7,985,783; No. 8,097,701; No. 8,349,910 and U.S. Patent Application Publications No. 20140141025, No. 20100210543 (Redwood Bioscience), Phosphopanthetinyl transferases (PPTases) (Reference [Grunewald, J. et al. Bioconjug. Chem. 2015, 26, 2554-62]), sortase A (Reference [Beerli, RR, et al. PLoS One 2015, 10, e0131177]), genetically introduced glutamine tags having Streptoverticillium mobaraense transglutaminase (mTG) (Reference [Strop, P., Bioconj. Chem., 2014, 25, 855-62; Strop, P., et al., Chem. Biol. 2013, 20, 161-7]; U.S. Patent Patent No. 8,871,908 (Rinat-Pfizer) or having microbial transglutaminase (MTGase) (References [Dennler, P., et al, 2014, Bioconjug. Chem. 25, 569-78; Siegmund, V. et al. Angew. Chemie - Int. Ed. 2015, 54, 13420-4]; U.S. Patent Application Publication No. 20130189287 (Innate Pharma); U.S. Patent No. 7,893,019 (Bio-Ker SrlIt has been reported that it includes the incorporation of enzymes / bacteria that form isopeptide bonds—peptide bonds—formed on the outside of the protein main chain (Kang, HJ, et al. Science 2007, 318, 1625-8; Zakeri, B. et al. Proc. Natl. Acad. Sci. USA 2012, 109, E690-7; Zakeri, B. & Howarth, MJ Am. Chem. Soc. 2010, 132, 4526-7).

[0007] The inventors have disclosed several conjugation methods for re-bridging a pair of thiols of the inter-chain disulfide bonds of a native antibody using, for example, bromomaleimide and dibromomaleimide linkers (WO2014 / 009774), 2,3-disubstituted succinic acid / 2-unisubstituted / 2,3-disubstituted fumaric acid or maleic acid linkers (WO2015 / 155753, WO20160596228), acetylene dicarboxylic acid linkers (WO2015 / 151080, WO20160596228), or hydrazine linkers (WO2015 / 151081). ADCs prepared with these linkers and methods exhibited better therapeutic index windows than traditional non-selective conjugation via cysteine ​​or lysine residues on the antibody. In this specification, the inventors disclose the invention of a conjugate of a cytotoxic drug containing a long side chain linker. The long side chain linker prevents the antibody-drug conjugate from being hydrolyzed by hydrolytic enzymes, e.g., proteinase or esterase, thereby making the conjugate more stable during targeted delivery and minimizing exposure to non-target cells, tissues or organs in the circulatory system, resulting in a longer half-life of the conjugate in the bloodstream, lower off-target toxicity, and a wider therapeutic window.

[0008] The present invention provides a branched linkage of a cytotoxic agent to an antibody. The present invention also provides a method for conjugating a cytotoxic agent analog to an antibody using a side chain linker.

[0009] In one aspect of the present invention, a conjugate containing a side chain linkage is represented by the following chemical formula (I):

[0010]

[0011] During the meal,

[0012] " " represents a single bond; n is 1 to 30;

[0013] T is a cell-binding agent / cell-binding molecule selected from the group consisting of antibodies, single-stranded antibodies, antibody fragments binding to target cells, monoclonal antibodies, single-stranded monoclonal antibodies, monoclonal antibody fragments binding to target cells, chimeric antibodies, chimeric antibody fragments binding to target cells, domain antibodies, domain antibody fragments binding to target cells, adnectin mimicking antibodies, DARPin, lymphokines, hormones; vitamins; growth factors, colony-stimulating factors; or nutrient-transport molecules (transferrins) and albumin, polymers, dendrimers, liposomes, nanoparticles, vesicles, or binding peptides, proteins, and small molecules attached to a (viral) capsid;

[0014] L1 and L2 are preferably chains of atoms selected from C, N, O, S, Si, and P, having 0 to 500 atoms, which are covalently linked to W and V1 and V1 and V2. These atoms used to form L1 and L2 can be combined in any chemically relevant manner and, for example, can form alkylenes, alkenylenes and alkylenes, ethers, polyoxyalkylenes, esters, amines, imines, polyamines, hydrazines, hydrazones, amides, ureas, semicarbazides, carbazides, alkoxyamines, alkoxylamines, urethanes, amino acids, peptides, acyloxylamines, hydroxy acids, or combinations thereof. Preferably, L1 and L2 are the same or different and are O, NH, N, S, P, NNH, NHNH, N(R3), N(R3)N(R 3' ), CH, CO, C(O)NH, C(O)O, NHC(O)NH, NHC(O)O, chemical formula (OCH2CH2) p OR3 or (OCH2CH-(CH3)) p OR3 or NH(CH2CH2O) p R3 or NH(CH2CH(CH3)O) p R3 or N[(CH2CH2O) p R3]-[(CH2CH2O) p' R 3' ] or (OCH2CH2) p COOR3 or CH2CH2(OCH2CH2) p Polyethyleneoxy units of COOR3 (wherein p and p' are independently integers selected from 0 to about 1000) or a combination thereof; of C1-C8 Alkyl; C2-C8 heteroalkyl, alkylcycloalkyl, heterocycloalkyl; C3-C8 aryl, Ar-alkyl, heterocyclic, carboncyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; or (Aa) r(r is 1 to 12 (1 to 12 amino acid units), which is independently selected from natural or non-natural amino acids, or dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide, or dodecapeptide units of the same or different sequences;

[0015] W is generally a self-immolative spacer, a peptide-dyl unit, a hydrazone, a disulfide, a thioether, an ester, or an amide-linked stretcher unit; w is 1 or 2 or 3;

[0016] V1 and V2 are independently spacer units, and O, NH, S, C1-C8 alkyl, C2-C8 heteroalkyl, alkenyl or alkynyl, C3-C8 aryl, heterocyclic, carboncyclic, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroaralkyl, heteroalkylcycloalkyl or alkylcarbonyl or (Aa) r (r is 1 to 12 (1 to 12 amino acid units), which consists of natural or non-natural amino acids, or dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide, or dodecapeptide units of the same or different sequences); or (CH2CH2O) p Selected from (p is 0 to 1000); v1 and v2 are independently 0, 1, or 2, but v1 and v2 are not simultaneously 0; if v1 or v2 is 0, it means that one of the side chain fragments Q1 or Q2 does not exist;

[0017] Q1 and Q2 are independently represented by the following chemical formula (I-q1):

[0018]

[0019] During the meal, is a site connected to L1 or L2; G1 and G2 are independently OC(O), NHC(O), C(O), CH2, NH, OC(O)NH, NHC(O)NH, O, S, B, P(O)(OH), NHP(O)(OH), NHP(O)(OH)NH, CH2P(O)(OH)NH, OP(O)(OH)O, CH2P(O)(OH)O, NHS(O)2, NHS(O)2NH, CH2S(O)2NH, OS(O)2O, CH2S(O)2O, Ar, ArCH2, ArO, ArNH, ArS, ArNR1, (Aa) r (r is 1 to 12); and X1 and X2 are independently O, CH2, S, NH, N(R 12 ), + NH(R 12 ), + N(R 12 )(R 13 ), C(O), OC(O), OC(O)O, NHSO2NH, NHP(O)(NH)2, SO2NH, P(O)(NH)2, NHS(O)NH, NHP(O)(OH)(NH), OC(O)NH, NHC(O)NH; Y2 is O, NH, NR1, CH2, S, Ar; G3 is OH, SH, OR1, SR1, OC(O)R1, NHC(O)R 12 , C(O)R 12 , CH3, NH2, NR 12 , + NH(R 12 ), + N(R 12 )(R 13 ), C(O)OH, C(O)NH2, NHC(O)NH2, BH2, BR 12 R 13 , P(O)(OH)2, NHP(O)(OH)2, NHP(O)(NH2)2, S(O)2(OH), (CH2) q1 C(O)OH, (CH2) q1 P(O)(OH)2, C(O)(CH2) q1 C(O)OH, OC(O)(CH2) q1 C(O)OH, NHC(O)(CH2) q1C(O)OH, CO(CH2) q1 P(O)(OH)2, NHC(O)O(CH2) q1 C(O)OH, OC(O)NH(CH2) q1 C(O)OH, NHCO(CH2) q1 P(O)(OH)2, NHC(O)(NH)(CH2) q1 C(O)OH, CONH(CH2) q1 P(O)(OH)2, NHS(O)2(CH2) q1 C(O)OH, CO(CH2) q1 S(O)2(OH), NHS(O)2NH(CH2) q1 C(O)OH, OS(O)2NH(CH2) q1 C(O)OH, NHCO(CH2) q1 S(O)2(OH), NHP(O)(OH)(NH)(CH2) q1 C(O)OH, CONH(CH2) q1 S(O)(OH), OP(O)(OH)2, (CH2) q1 P(O)(NH)2, NHS(O)2(OH), NHS(O)2NH2, CH2S(O)2NH2, OS(O)2OH, OS(O)2OR1, CH2S(O)2OR1, Ar, ArR 12 , ArOH, ArNH2, ArSH, ArNHR 12 , or (Aa) q1 and; p 1, p2 and p3 are independently 0 to 100 but not simultaneously 0; q1 and q2 are independently 0 to 24; preferably, Q1 and Q2 are independently linear or branched C2-C 90 Polycarboxylic acid; C2-C 90 Polyalkylamine; C6-C 90 Oligosaccharides or polysaccharides; C6-C composed of quaternary ammonium cations and sulfonate anions 90Zwitterionic betaine or zwitterionic poly(sulfobetaine) (PSB); For example, poly(lactic acid / glycolic acid) (PLGA), poly(acrylate), chitosan, copolymer of N-(2-hydroxypropyl)methacrylamide, poly[2-(methacryloyloxy)ethylphosphorylcholine] (PMPC), poly-L-glutamic acid, poly(lactide-co-glycolide) (PLG), poly(lactide-co-glycolide), poly(ethylene glycol) (PEG), poly(propylene glycol) (PPG), poly(lactide-co-glycolide), poly(ethylene glycol)-modified peptide, poly(ethylene glycol)-modified lipid, poly(ethylene glycol)-modified alkylcarboxylic acid, poly(ethylene glycol)-modified alkylamine, poly(lactide)-co-glycolide, polysaccharocine, hyaluronic acid (HA) (glycosaminoglycan), heparin / heparan sulfate (HSGAG), chondroitin It is a biodegradable polymer composed of sulfate / dermatan sulfate (CSGAG), poly(ethylene glycol)-modified alkyl sulfate, poly(ethylene glycol)-modified alkyl phosphate, or poly(ethylene glycol)-modified alkyl quaternary ammonium; D is a cytotoxic agent independently selected from calicheamicin, camptothecin, maytansinoid, taxane, daunorubicin / doxorubicin, vinca alkaloid, auristatin, eribulin, pyrrolobenzodiazepine (PBD), duocarmycin, kinase inhibitors, MEK inhibitors, KSP inhibitors, nicotinamide phosphoribosyltransferase (NAMPT) inhibitors, immunotoxins, analogs thereof, or prodrugs.

[0020] In another aspect of the present invention, a conjugate containing a side chain linkage is represented by the following formulas (II) and (III):

[0021]

[0022] In the formula, D, W, L1, L2, Q1, Q2, V1, V2, v1, v2, n, and T are defined as in formula (I); w and w' are independently 1, 2, or 3; is a single bond, a double bond, or has no bond; D1 and D2 are identical or different, and are defined as identical to D.

[0023] In another aspect of the present invention, a side-chain linkage compound is represented by the following formula (IV), which can be readily reacted with a cell-binding molecule T to form a conjugate of formula (I):

[0024]

[0025] In the formula, D, W, w, L1, L2, Q1, Q2, V1, V2, v1, v2 and n are defined as in formula (I); Lv1 is a functional group as described below.

[0026] In another aspect of the present invention, the side-chain linkage compounds are represented by formulas (V) and (VI), which can readily react with a pair of sites on the cell-binding molecule T to form conjugates of formulas (II) and (III), respectively:

[0027]

[0028] In the formula, D, D1, D2, W, w, w', L1, L2, Q1, Q2, V1, V2, v1, v2, n is defined in the same way as above.

[0029] Lv1 and Lv2 are the same or different reactive groups capable of reacting with thiols, amines, carboxylic acids, selenos, phenols, or hydroxyl groups on cell-binding molecules, and Lv1 and Lv2 are independently OH; F; Cl; Br; I; nitrophenol; N-hydroxysuccinimide (NHS); phenol; dinitrophenol; pentafluorophenol; tetrafluorophenol; difluorophenol; mono-fluorophenol; pentachlorophenol; triplate; imidazole; dichlorophenol; tetrachlorophenol; 1-hydroxybenzotriazole; tosylate; mesylate; 2-ethyl-5-phenylisoxazolium-3'-sulfonate, an anhydride formed from itself or formed together with other anhydrides, e.g., acetyl anhydride, formylic anhydride; Alternatively, it is selected from intermediate molecules produced using a condensation reagent for a peptide coupling reaction or a Mitsunobu reaction. Examples of condensation reagents are EDC (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide), DCC (dicyclohexyl-carbodiimide), N,N'-diisopropylcarbodiimide (DIC), N-cyclohexyl-N'-(2-morpholino-ethyl)carbodiimide metho-p-toluenesulfonate (CMC or CME-CDI), 1,1'-carbonyldiimidazole (CDI), TBTU (O-(benzotriazole-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate), N,N,N',N'-tetramethyl-O-(1H-benzo-triazole-1-yl)-uronium hexafluorophosphate (HBTU), (Benzotriazole-1-yloxy)tris(dimethylamino)-phosphonium hexafluorophosphate (BOP), (Benzotriazole-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP), diethyl cyanophosphonate (DEPC), chloro-N,N,N',N'-tetra-methylformamidinium hexafluorophosphate, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), 1-[(dimethylamino)-(morpholino)methylene]-1H-[1,2,3]Triazolo[4,5-b]pyridin-1-ium 3-oxide-hexafluorophosphate (HDMA), 2-chloro-1,3-dimethyl-imidazolidinium hexafluorophosphate (CIP), chlorotripyrrolidinophosphonium hexafluorophosphate (PyCloP), fluoro-N,N,N',N'-bis(tetramethylene)-formamidinium hexafluorophosphate (BTFFH), N,N,N',N'-tetramethyl-S-(1-oxido-2-pyridyl)thiuronium hexafluorophosphate, O-(2-oxo-1(2H)pyridyl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TPTU), S-(1-oxido-2-pyridyl)-N,N,N',N'-tetramethylthiuronium tetrafluoroborate, O-[(ethoxycarbonyl)-cyanomethyleneamino]-N,N,N',N'-tetramethyluronium hexafluorophosphate (HOTU), (1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate (COMU), O-(benzotriazole-1-yl)-N,N,N',N'-bis(tetramethylene)-uronium hexafluorophosphate (HBPyU), N-benzyl-N'-cyclohexyl-carbodiimide (polymerized or unpolymerized), dipyrrolidino(N-succinimidyl-oxy)carbenium hexafluorophosphate (HSPyU), Chlorodipyrrolidinocarbenium hexafluorophosphate (PyClU), 2-chloro-1,3-dimethylimidazolidinium tetrafluoroborate (CIB), (benzotriazole-1-yloxy)dipiferidino-carbenium hexafluorophosphate (HBPipU), O-(6-chlorobenzotriazole-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TCTU), bromotris(dimethylamino)-phosphonium hexafluorophosphate (BroP), propylphosphonic acid anhydride (PPACA, T3P, ®), 2-Morrfolinoethyl isocyanide (MEI), N,N,N',N'-tetramethyl-O-(N-succinimidyl)uronium hexafluorophosphate (HSTU), 2-bromo-1-ethyl-pyridinium tetrafluoroborate (BEP), O-[(ethoxycarbonyl)cyano-methyleneamino]-N,N,N',N'-tetra-methyluronium tetrafluoroborate (TOTU), 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmofolinium chloride (MMTM, DMTMM), N,N,N',N'-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (TSTU), O-(3,4-dihydro-4-oxo-1,2,3-benzotriazine-3-yl)-N,N,N',N'-tetramethyluronium tetrafluoro-borate (TDBTU), 1,1'-(azodicarbonyl)-dipipiridine (ADD), di-(4-chlorobenzyl)azodicarboxylate (DCAD), di-tert-butyl azodicarboxylate (DBAD), diisopropyl azodicarboxylate (DIAD), and diethyl azodicarboxylate (DEAD). Additionally, Lv1 and Lv2 may be anhydrides formed by the acid itself or in combination with other C1-C8 acid anhydrides.

[0030] The present invention further relates to a method for preparing a cell-bound molecule-drug conjugate of formulas (I) and (II) and to an application of the conjugate of formulas (I) and (II). Brief explanation of the drawing

[0031] FIG. 1 is a diagram illustrating the synthesis of a component of a tubulosin analog containing a linker. FIG. 2 is a diagram illustrating the synthesis of the components of a linear linker. FIG. 3 is a diagram illustrating the synthesis of a tubulin analog having a side chain linker. FIG. 4 is a diagram illustrating the synthesis of a tubulin analog having a side chain linker. FIG. 5 is a diagram illustrating the synthesis of a fragment of a tubulisin analog containing a side chain linker. FIG. 6 is a diagram illustrating the synthesis of a conjugate of a tubulin analog containing a side chain linker. FIG. 7 is a diagram illustrating the synthesis of exatecan and a fragment of a side chain linker. FIG. 8 is a diagram illustrating the synthesis of a conjugate of exatecan containing a side chain linker. FIG. 9 is a diagram illustrating the synthesis of the components of a linear linker. FIG. 10 is a diagram illustrating the general synthesis of a drug-linker component containing a side chain linker. FIG. 11 is a diagram illustrating the synthesis of a drug containing a side chain linker and a conjugate of a metansinoid-linker component having a side chain linker. FIG. 12 is a diagram illustrating the synthesis of a conjugate of a meitansinoid and an exatecan containing a side chain linker. FIG. 13 is a diagram illustrating the synthesis of a conjugate of an MMAE analog containing a side chain linker. FIG. 14 is a diagram illustrating the synthesis of a conjugate of an MMAF analog containing a side chain linker. FIG. 15 is a diagram illustrating the synthesis of conjugable eribulin containing a side chain linker. FIG. 16 is a diagram illustrating the synthesis of eribulin containing a side chain linker and a conjugateable CBI-dimer containing a side chain linker. FIG. 17 is a diagram illustrating the synthesis of a CBI-dimer containing a side chain linker and a conjugate of a topotecan analog containing a side chain linker. FIG. 18 is a diagram illustrating the synthesis of a conjugate of a tubulisin analog containing a side chain linker and a conjugate of an MMAE analog containing a side chain linker. FIG. 19 is a diagram illustrating the synthesis of a conjugate of a tubulin analog containing a side chain linker. FIG. 20 is a diagram showing the comparison of the antitumor effects of conjugate compounds 49 (C-30), 51 (C-48), C-173, C-238, C-312, 132 (C-131), 135 (C-134), C-321 and C-322 and T-DM1 using a single injection of 6 mg / kg in an IV human gastric tumor N87 cell model. FIG. 21 illustrates an acute toxicity study of ADC conjugates 49 (C-30), 51 (C-48), C-173, C-238, C-312, 132 (C-131), 135 (C-134), C-321 and C-322 and T-DM1 by observing changes in mouse body weight (BW) on day 12. Specific details for implementing the invention

[0032] definition

[0033] "Alkyl" refers to a monovalent group derived from an alkane by the removal of one or two hydrogen atoms from an aliphatic hydrocarbon group or a carbon atom. This can be linear or branched, having C1-C8 (1 to 8 carbon atoms) within the chain. "Branched" means that one or more lower C alkyl groups, such as methyl, ethyl, or propyl, are attached to a linear alkyl chain. Exemplary alkyl groups include methyl, ethyl, n -profile, i -profile, n -butyl, t -butyl, n -Pentyl, 3-pentyl, octyl, nonyl, decyl, cyclopentyl, cyclohexyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, 3,3-dimethylpentyl, 2,3,4-trimethylpentyl, 3-methyl-hexyl, 2,2-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 3,5-dimethylhexyl, 2,4-dimethylpentyl, 2-methylheptyl, 3-methylheptyl, n -Heptyl, isoheptyl, n- Includes octyl and isooctyl. The C1-C8 alkyl group may be substituted or unsubstituted with one or more groups including but not limited to -C1-C8 alkyl, -O-(C1-C8 alkyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R')2, -NHC(O)R', -SR', -S(O)2R', -S(O)R', -OH, -halogen, -N3, -NH2, -NH(R'), -N(R')2 and -CN; wherein each R' is independently selected from -C1-C8 alkyl and aryl.

[0034] "Halogen" refers to fluorine, chlorine, bromine, or iodine atoms; preferably, fluorine and chlorine atoms.

[0035] "Heteroalkyl" refers to a C2-C8 alkyl in which 1 to 4 carbon atoms are independently replaced by heteroatoms from the group consisting of O, S and N.

[0036] "Carbon ring" refers to a saturated or unsaturated ring having 3 to 8 carbon atoms as a monocycle or 7 to 13 carbon atoms as a bicycle. A monocyclic carbon ring has 3 to 6 ring atoms, more typically 5 or 6 ring atoms. A bicyclic carbon ring has 7 to 12 ring atoms arranged as a bicycle [4,5], [5,5], [5,6], or [6,6] system, or 9 or 10 ring atoms arranged as a bicycle [5,6] or [6,6] system. Representative C3-C8 carbon rings include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentadienyl, cyclohexyl, cyclohexenyl, 1,3-cyclohexadienyl, 1,4-cyclohexadienyl, cycloheptyl, 1,3-cycloheptadienyl, 1,3,5-cycloheptatrienyl, cyclooctyl, and cyclooctadienyl.

[0037] "C3-C8 carbon ring" refers to a saturated or unsaturated non-aromatic carbon ring of 3-, 4-, 5-, 6-, 7-, or 8-members. The C3-C8 carbon ring may be substituted or unsubstituted with one or more groups including but not limited to -C1-C8 alkyl, -O-(C1-C8 alkyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R')2, -NHC(O)R', -SR', -S(O)R', -S(O)2R', -OH, -halogen, -N3, -NH2, -NH(R'), -N(R')2, and -CN; wherein each R' is independently selected from -C1-C8 alkyl and aryl.

[0038] "Alkenyl" refers to an aliphatic hydrocarbon group containing a carbon-carbon double bond that may be linear or branched and has 2 to 8 carbon atoms in the chain. Exemplary alkenyl groups include ethenyl, propenyl, n-butenyl, i-butenyl, 3-methylbut-2-enyl, n-pentenyl, hexylenyl, heptenyl, and octenyl.

[0039] "Alkynyl" refers to an aliphatic hydrocarbon group containing a carbon-carbon triple bond that may be linear or branched and has 2 to 8 carbon atoms in the chain. Exemplary alkynyl groups include etynyl, propynyl, n -butynyl, 2-butynyl, 3-methylbutynyl, 5-pentynyl, n - Includes pentyle, hexylyl, heptyl, and octyle.

[0040] "Alkylene" refers to a saturated, branched, straight-chain, or cyclic hydrocarbon radical having 1 to 18 carbon atoms and having two monovalent radical centers derived by removing two hydrogen atoms from two identical or different carbon atoms of a parent alkane. Typical alkylene radicals include, but are not limited to, methylene (-CH2-), 1,2-ethyl (-CH2CH2-), 1,3-propyl (-CH2CH2CH2-), 1,4-butyl (-CH2CH2CH2CH2-), etc.

[0041] "Alkenylene" refers to an unsaturated, branched, straight-chain, or cyclic hydrocarbon radical having 2 to 18 carbon atoms and having two monovalent radical centers derived by removing two hydrogen atoms from two identical or different carbon atoms of a parent alkene. Typical alkenylene radicals include, but are not limited to, 1,2-ethylene (-CH=CH-).

[0042] "Alkynylene" refers to an unsaturated, branched, straight-chain, or cyclic hydrocarbon radical having 2 to 18 carbon atoms and having two monovalent radical centers derived by removing two hydrogen atoms from two identical or different carbon atoms of a parent alkyne. Typical alkynylene radicals include, but are not limited to, acetylene, propargyl, and 4-pentylene.

[0043] "Aryl" or "Ar" refers to an aromatic or heteroaromatic group composed of one or several rings containing 3 to 14 carbon atoms, preferably 6 to 10 carbon atoms. The term "heteroaromatic group" refers to one or several carbons on the aromatic group, preferably 1, 2, 3, or 4 carbon atoms are replaced with O, N, Si, Se, P, or S, preferably O, S, and N. The term aryl or Ar also refers to an aromatic group in which one or more H atoms are independently replaced by -R', -halogen, -OR', or -SR', -NR'R", -N=NR', -N=R', -NR'R", -NO2, -S(O)R', -S(O)2R', -S(O)2OR', -OS(O)2OR', -PR'R", -P(O)R'R", -P(OR')(OR"), -P(O)(OR')(OR"), or -OP(O)(OR')(OR"), where R', R" are independently H, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, arylalkyl, carbonyl, or pharmaceutical salt.

[0044] "Heterocycle" refers to a ring system in which one to four ring carbon atoms are independently replaced by heteroatoms from the group of O, N, S, Se, B, Si, and P. Preferred heteroatoms are O, N, and S. Heterocycles are also described in the literature [The Handbook of Chemistry and Physics, 78th Edition, CRC Press, Inc., 1997-1998, pp. 225 to 226], the disclosure thereof is incorporated by reference. Desirable non-aromatic heterocyclic formulas include epoxy, aziridinyl, tyranyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, oxiranyl, tetrahydrofuranyl, dioxolanyl, tetrahydropyranyl, dioxaneyl, dioxolanyl, piperidinyl, piperazineyl, morpholinyl, pyranyl, imidazolineyl, pyrrolinyl, pyrazolidinyl, thiazolidinyl, tetrahydrothiopyranyl, dithianyl, thiomopolinyl, dihydropyranyl, tetrahydropyranyl, dihydropyranyl, tetrahydropyridyl, dihydropyridyl, tetrahydropyrimidinyl, dihydrothiopyranyl, azepanyl, as well as fusion systems produced from condensation with phenyl groups.

[0045] The term "heteroaryl" or aromatic heterocycle refers to an aromatic hetero, mono-, bicyclic, or polycyclic ring of 3 to 14, preferably 5 to 10 members. Examples include pyrrolyl, pyridyl, pyrazolyl, thienyl, pyrimidinyl, pyrazinyl, tetrazolyl, indolyl, quinolinyl, purinyl, imidazolyl, thienyl, thiazolyl, benzothiazolyl, furanyl, benzofuranyl, 1,2,4-thiadiazolyl, isothiazolyl, triazolyl, tetrazolyl, isoquinolyl, benzothienyl, isobenzofuryl, pyrazolyl, carbazolyl, benzimidazolyl, isoxazolyl, pyridyl- N It includes oxides, as well as fusion systems generated from condensation with phenyl groups.

[0046] "Alkyl," "cycloalkyl," "alkenyl," "alkynyl," "aryl," "heteroaryl," "heterocyclic," etc. also refer to the corresponding "alkylene," "cycloalkylene," "alkenylene," "alkynylene," "arylene," "heteroarylene," and "heterocyclene" formed by the removal of two hydrogen atoms.

[0047] "Arylalkyl" refers to a carbon atom, typically at the terminal or sp² 3 It refers to an acyclic alkyl radical in which one of the hydrogen atoms bonded to a carbon atom is replaced by an aryl radical. Typical arylalkyl groups include benzyl, 2-phenylethane-1-yl, 2-phenylethene-1-yl, naphthylmethyl, 2-naphthylethane-1-yl, 2-naphthylethene-1-yl, naphthobenzyl, 2-naphthophenylethane-1-yl, etc.

[0048] "Heteroarylalkyl" refers to carbon atoms, typically terminal or sp² 3 It refers to an acyclic alkyl radical in which one of the hydrogen atoms bonded to a carbon atom is replaced by a heteroaryl radical. Examples of heteroarylalkyl groups are 2-benzimidazolylmethyl and 2-furylethyl.

[0049] Examples of "hydroxyl protecting groups" are methoxymethyl ether, 2-methoxyethoxymethyl ether, tetrahydropyranyl ether, benzyl ether, p -Methoxybenzyl ether, trimethylsilyl ether, triethylsilyl ether, triisopropylsilyl ether, t - Butyldimethylsilyl ether, triphenylmethylsilyl ether, acetate ester, substituted acetate ester, pivaloate, benzoate, methanesulfonate and p - Contains toluenesulfonate.

[0050] "Leaving group" refers to a functional group that can be substituted by another functional group. Such leaving groups are widely known in the relevant technical field, examples of which are halides (e.g., chlorides, bromides, and iodides), methanesulfonyl (mesil), p- Includes toluenesulfonyl (tosyl), trifluoromethylsulfonyl (triplate), and trifluoromethylsulfonate. A preferred leaving group is selected from nitrophenol; N-hydroxysuccinimide (NHS); phenol; dinitrophenol; pentafluorophenol; tetrafluorophenol; difluorophenol; monofluorophenol; pentachlorophenol; triplate; imidazole; dichlorophenol; tetrachlorophenol; 1-hydroxybenzotriazole; tosylate; mesylate; 2-ethyl-5-phenylisoxazolium-3'-sulfonate, anhydrides formed by itself or formed together with other anhydrides, e.g., acetyl anhydride, formyl anhydride; or intermediate molecules produced using a condensation reagent for a peptide coupling reaction or a Mitsunobu reaction.

[0051] The following abbreviations may be used herein and have the definitions provided: Boc, tert-butoxycarbonyl; BroP, bromotrispirolidinophosphonium hexafluorophosphate; CDI, 1,1'-carbonyldiimidazole; DCC, dicyclohexylcarbodiimide; DCE, dichloroethane; DCM, dichloromethane; DEAD, diethylazodicarboxylate; DIAD, diisopropylazodicarboxylate; DIBAL-H, diisobutyl-aluminum hydride; DIPEA or DEA, diisopropylethylamine; DEPC, diethylphosphorocyanidate; DMA, N,N-dimethylacetamide; DMAP, 4-(N,N-dimethylamino)pyridine; DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide; DTPA, diethylenetriaminepentaacetic acid; DTT, dithiothreitol; EDC, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; ESI-MS, electron spray mass spectrometry; EtOAc, ethyl acetate; Fmoc, N-(9-fluorenylmethoxycarbonyl); HATU, O-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate; HOBt, 1-hydroxybenzotriazole; HPLC, high-pressure liquid chromatography; NHS, N-hydroxysuccinimide; MeCN, acetonitrile; MeOH, methanol; MMP, 4-methylmorpholine; PAB, p-aminobenzyl; PBS, phosphate-buffered brine (pH 7.0 to 7.5); pH, Phenyl; phe, L-phenylalanine; PyBrop, Bromo-tris-pyrrolidino-phosphonium hexafluorophosphate; PEG, Polyethylene glycol; SEC, Size exclusion chromatography; TCEP, Tris(2-carboxyethyl)phosphine; TFA, Trifluoroacetic acid; THF, Tetrahydrofuran; Val, Valine. TLC, Thin film chromatography; UV, Ultraviolet light.

[0052] The "amino acid(s)" may be natural and / or non-natural amino acids, preferably alpha-amino acids. Natural amino acids are those encoded by the genetic code, and these are alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine, tryptophan, and valine. Non-natural amino acids are derived in the form of proteogenic amino acids. Examples include hydroxyproline, lanthionine, 2-aminoisobutyric acid, dehydroalanine, gamma-aminobutyric acid (a neurotransmitter), ornithine, citrulline, beta-alanine (3-aminopropanoic acid), gamma-carboxyglutamate, selenocysteine ​​(present in most eukaryotes as well as many non-eukaryotes, but not directly encoded by DNA), pyrrolicine (found only in some mackerel and one bacterium), N-formylmethionine (often the initial amino acid of proteins in bacteria, mitochondria, and chloroplasts), 5-hydroxytryptophan, L-dihydroxyphenylalanine, triiodothyronine, L-3,4-dihydroxyphenylalanine (DOPA), and O-phosphoserine. The term amino acid also includes amino acid analogs and mimics. An analog is a compound having the same general formula H2N(R)CHCO2H as a natural amino acid, except that the R group is not found in the natural amino acid. Examples of analogs include homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Preferably, an amino acid mimic is a compound having a structural formula different from the general chemical formula of an alpha-amino acid but acting in a similar manner. The term "non-natural amino acid" is intended to denote the "D" stereochemical form, while the natural amino acid is the "L" form. When 1 to 8 amino acids are used in this patent application, the amino acid sequence is preferably a cleavage recognition sequence for a protease.Numerous cleavage recognition sequences are known in the relevant art (see, e.g., Matayoshi et al. Science 247: 954 (1990); Dunn et al. Meth. Enzymol. 241: 254 (1994); Seidah et al. Meth. Enzymol. 244: 175 (1994); Thornberry, Meth. Enzymol. 244: 615 (1994); Weber et al. Meth. Enzymol. 244: 595 (1994); Smith et al. Meth. Enzymol. 244: 412 (1994); and Bouvier et al. Meth. Enzymol. 248: 614 (1995)]; the disclosures thereof are incorporated herein by reference). In particular, such sequences are selected from the group consisting of Val-Cit, Ala-Val, Ala-Ala, Val-Val, Val-Ala-Val, Lys-Lys, Ala-Asn-Val, Val-Leu-Lys, Cit-Cit, Val-Lys, Ala-Ala-Asn, Asp-Lys, Asp-Glu, Glu-Lys, Lys, Cit, Ser, and Glu.

[0053] A "glycoside" is a molecule in which a sugar group is bonded to another group via an anomeric carbon through a glycosidic bond. Glycosides can be linked by O-(O-glycoside), N-(glycosylamine), S-(thioglycoside), or C-(C-glycoside) glycosidic bonds. Its core empirical formula is C m (H2O) n(In the formula, m may differ from n, and m and n are less than 36). In this specification, glycosides include glucose (dextrose), fructose (levulose), allose, altrose, mannose, gloss, iodose, galactose, talose, galactosamine, glucosamine, sialic acid, N-acetylglucosamine, sulfoquinobose (6-deoxy-6-sulfo-D-glucopyranose), ribose, arabinose, xylose, lixose, sorbitol, mannitol, sucrose, lactose, maltose, trehalose, maltodextrin, raffinose, glucocuronic acid (glucuronide), and stachyose. This may be D-type or L-type, a 5-membered cyclic furanose form, a 6-membered cyclic furanose form, or an acyclic form, an α-isomer (the -OH of the anomalous carbon below the plane of the carbon atom in the Haworth projection) or a β-isomer (the -OH of the anomalous carbon above the plane of the Haworth projection). Monosaccharides, disaccharides, polyols, or oligosaccharides containing 3 to 6 sugar units are used herein.

[0054] As used herein, the term “antibody” refers to a full-length immunoglobulin molecule or an immunologically active portion of a full-length immunoglobulin molecule, i.e., a molecule or a portion thereof containing an antigen-binding site that binds immunospecifically to an antigen of a target of interest, and such targets include, but are not limited to, cancer cells or cells that produce autoimmune antibodies associated with autoimmune diseases. The immunoglobulins disclosed herein may be any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules. Immunoglobulins may be derived from any species. However, preferably, immunoglobulins are of human, murine, or rabbit origin. Antibodies useful for the present invention are preferably monoclonal, polyclonal, monoclonal, bispecific human, humanized or chimeric antibodies, single-strand antibodies, Fv, Fab fragment, F(ab') fragment, F(ab')2 fragment, fragments produced by a Fab expression library, anti-idiotype (anti-Id) antibody, CDR, and any epitope-binding fragments thereof (these bind immunospecifically to cancer cell antigens, viral antigens, or microbial antigens), but are not limited to these.

[0055] "Enantiomers," also known as "optical isomers," are one of two stereoisomers that are non-overlapping (non-identical) mirror images of each other, just as a left hand and a right hand are identical except that they are opposite along a single axis (hands cannot simply appear identical due to a change in orientation). A single chiral atom or similar structural feature within a compound causes the compound to have two possible structures that do not overlap with each other's mirror images. The presence of multiple chiral features within a given compound increases the number of possible geometric forms, although some perfect mirror-image pairs may exist. A pure enantiomer compound refers to a sample containing only one chiral molecule within the detection limit. When existing in a symmetric environment, enantiomers have identical chemical and physical properties, except for the ability to rotate plane-polarized light (+ / -) in opposite directions, even though they are equal in quantity (polarization can be considered an asymmetric medium). This is sometimes referred to as an optical isomer for this reason. A mixture of an optically active isomer and its enantiomer of the same amount is referred to as racemic and has a one-sided polarization of zero net rotation, because the positive rotation of each (+) form is exactly canceled out by the negative rotation of the (-) form. Enantiomer components usually have different chemical reactions than other enantiomer substances. Since many biological molecules are enantiomers, the effects of the two enantiomers on biological organisms are sometimes distinctly different. In drugs, for example, usually only one of the drug's enantiomers is responsible for the desired physiological effect, while the other enantiomer is less active, inactive, or sometimes even causes side effects. Due to these findings, it is possible to develop drugs composed of only one enantiomer ("pure enantiomer") to improve pharmacological efficacy and sometimes eliminate some side effects.

[0056] Isotopes are variants of a specific chemical element that differ in the number of neutrons. All isotopes of a given element have the same number of protons within their respective atoms. Each atomic number identifies a specific element but does not identify an isotope; atoms of a given element can have a varying range in the number of neutrons. The number of nucleons (both protons and neutrons) within the nucleus is the atomic mass number, and each isotope of a given element has a different mass number. For example, carbon-12, carbon-13, and carbon-14 are three isotopes of the element carbon, having mass numbers of 12, 13, and 14, respectively. The atomic number of carbon is 6, which means that all carbon atoms have 6 protons, so the neutron numbers of these isotopes are 6, 7, and 8, respectively. Hydrogen atoms are light hydrogen ( 1 H), deuterium ( 2 H) and tritium ( 3It has three isotopes of hydrogen (H), where deuterium is twice the mass of light hydrogen and tritium is three times the mass of light hydrogen. Isotope substitution can be used to determine the mechanisms of chemical reactions and dynamic isotope effects. Using isotope substitution, one can study metabolic changes of substances within the body (e.g., by metabolic enzymes, e.g., cytochrome P450 or glucuronosyltransferase), as well as how the body affects specific xenobiotics / chemicals after administration through mechanisms of absorption and distribution, and the effects and pathways of drug metabolites. This study is called pharmacokinetics (PK). Isotope substitution can be used to study the biochemical and physiological effects of drugs. These effects may include manifested ones in animals (including humans), microorganisms, or combinations of organisms (e.g., infections). This study is called pharmacodynamics (PD). These effects may include manifested in animals (including humans), microorganisms, or a combination of organisms (e.g., infections). Both together affect the administration, benefits, and side effects of the drug. Radioisotopes may contain stable (non-radioactive) or unstable elements. Radioisotope substitution of a drug may result in different therapeutic efficacy compared to the original drug.

[0057] "Pharmaceuticalally" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not cause harmful, allergic, or other inappropriate reactions when appropriately administered to animals or humans.

[0058] “Pharmaceuticalally acceptable solvate” or “solvate” refers to an association of one or more solvent molecules and the disclosed compound. Examples of solvents forming a pharmaceutically acceptable solvate include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine.

[0059] "Pharmaceuticalally acceptable excipients" include any carrier, diluent, adjuvant, or vehicle, e.g., preservatives or antioxidants, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, solvents, dispersion media, coating agents, antibacterial and antifungal agents, isotonic agents, and absorption retardants. The use of such media and agents for pharmaceutically active substances is widely known in the relevant art. Any conventional media or agent is considered for use in therapeutic compositions except where it is immiscible with the active ingredient. Additional active ingredients may also be incorporated into the composition as suitable therapeutic combinations.

[0060] As used herein, “pharmaceutical salt” refers to a derivative of the disclosed compound in which the parent compound is modified by preparing its acid or base salt. Pharmaceutically acceptable salts include, for example, conventional nontoxic salts or quaternary ammonium salts of the parent compound formed from nontoxic inorganic or organic acids. For example, such conventional nontoxic salts include salts prepared from inorganic acids, e.g., hydrochloric acid, bromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, etc.; and organic acids, e.g., acetic acid, propionic acid, succinic acid, tartaric acid, citric acid, methanesulfonic acid, benzenesulfonic acid, glucuronic acid, glutamic acid, benzoic acid, salicylic acid, toluenesulfonic acid, oxalic acid, fumaric acid, maleic acid, lactic acid, etc. Additional addition salts include ammonium salts, e.g., tromethamine, meglumine, eporamine, etc., metal salts, sodium, potassium, calcium, zinc, or magnesium.

[0061] The pharmaceutical salts of the present invention can be synthesized from parent compounds containing a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acidic or basic form of these compounds with a stoichiometric amount of a suitable base or acid in water, an organic solvent, or a mixture of both. Generally, a non-aqueous medium, such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile, is preferred. A list of suitable salts is provided in the literature [Remington's Pharmaceutical Sciences, 17 th It is found in [ed., Mack Publishing Company, Easton, PA, 1985, p. 1418], the disclosure thereof is incorporated by reference.

[0062] "Administering" or "administration" refers to any mode of transferring, delivering, introducing, or transporting a pharmaceutical drug or other agent to a subject. Such modes include oral administration, local contact, intravenous, intraperitoneal, intramuscular, intralesional, intranasal, subcutaneous, or intrathecal administration. The use of a device or equipment for the administration of the agent is also considered in the present invention. Such devices may utilize active or passive transport and may be slow-release or rapid-release delivery devices.

[0063] In the context of cancer, the term "treating" includes any or all of the prevention of growth of tumor cells or cancer cells, prevention of replication of tumor cells or cancer cells, reduction of the total tumor load, and improvement of one or more symptoms associated with the disease.

[0064] In the context of autoimmune diseases, the term "therapeutic" includes any or all of the prevention of replication of cells associated with an autoimmune disease state, including but not limited to cells capable of producing autoimmune antibodies, reduction of the autoimmune antibody load, and improvement of one or more symptoms of the autoimmune disease.

[0065] In the context of infectious diseases, the term "treating" includes any or all of the prevention of the growth, proliferation, or replication of pathogens causing the infectious disease and the improvement of one or more symptoms of the infectious disease.

[0066] Examples of "mammals" or "animals" include, but are not limited to, humans, rats, mice, guinea pigs, monkeys, pigs, goats, cattle, horses, dogs, cats, birds, and poultry.

[0067] The novel conjugate disclosed herein uses a bridge linker. Examples of some suitable linkers and their synthesis are provided. 1 Inner pages 26 It is built in.

[0068] Conjugate of cell-binding agent-cytotoxic molecules via side chain linkages

[0069] In one aspect of the present invention, a conjugate containing a side chain linkage is represented by the following formulas (I), (II) and (III):

[0070]

[0071] During the meal,

[0072] represents a single bond; is a single bond or a double bond or is absent; n is 1 to 30; w and w' are independently 1, 2 or 3;

[0073] T is a cell-binding agent (cell-binding agent) / cell-binding molecule selected from the group consisting of antibodies, single-stranded antibodies, antibody fragments binding to target cells, monoclonal antibodies, single-stranded monoclonal antibodies, monoclonal antibody fragments binding to target cells, chimeric antibodies, chimeric antibody fragments binding to target cells, domain antibodies, domain antibody fragments binding to target cells, antibody-mimicking adnectin, DARPin, lymphokines, hormones; vitamins; growth factors, colony-stimulating factors; or nutrient-transport molecules (transferrins), and / or albumin, polymers, dendrimers, liposomes, nanoparticles, vesicles, or cell-binding peptides, proteins, or small molecules attached to a (viral) capsid;

[0074] L1 and L2 are preferably chains of atoms selected from C, N, O, S, Si, and P, having 0 to 500 atoms, which are covalently linked to W and V1 and V1 and V2. These atoms used to form L1 and L2 can be combined in any chemically relevant manner and, for example, can form alkylenes, alkenylenes and alkylenes, ethers, polyoxyalkylenes, esters, amines, imines, polyamines, hydrazines, hydrazones, amides, ureas, semicarbazides, carbazides, alkoxyamines, alkoxylamines, urethanes, amino acids, peptides, acyloxylamines, hydroxy acids, or combinations thereof. Preferably, L1 and L2 are the same or different and are O, NH, N, S, P, NNH, NHNH, N(R3), N(R3)N(R 3' ), CH, CO, C(O)NH, C(O)O, NHC(O)NH, NHC(O)O, chemical formula (OCH2CH2) p OR3 or (OCH2CH-(CH3)) p OR3 or NH(CH2CH2O) p R3 or NH(CH2CH(CH3)O) p R3 or N[(CH2CH2O) p R3]-[(CH2CH2O) p' R3' ] or (OCH2CH2) p COOR3 or CH2CH2(OCH2CH2) p Polyethyleneoxy units of COOR3 (wherein p and p' are independently integers selected from 0 to about 1000) or a combination thereof; of C1-C8 Alkyl; C2-C8 heteroalkyl, alkylcycloalkyl, heterocycloalkyl; C3-C8 aryl, Ar-alkyl, heterocyclic, carboncyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; or (Aa) r (r is 1 to 12 (1 to 12 amino acid units), which is independently selected from natural or non-natural amino acids, or dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide, or dodecapeptide units of the same or different sequences;

[0075] W is C1-C 18 , generally a self-sacrificial spacer, a peptidedyl unit, a hydrazone, a disulfide, a thioether, an ester, or an amide-linked stretcher unit;

[0076] V1 and V2 are independently spacer units, and O, NH, S, C1-C8 alkyl, C2-C8 heteroalkyl, alkenyl or alkynyl, C3-C8 aryl, heterocyclic, carboncyclic, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroaralkyl, heteroalkylcycloalkyl or alkylcarbonyl or (Aa) r (r is 1 to 12 (1 to 12 amino acid units), which consists of natural or non-natural amino acids, or dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide, or dodecapeptide units of the same or different sequences); or (CH2CH2O) pSelected from (p is 0 to 1000); v1 and v2 are independently 0, 1, or 2, but v1 and v2 are not simultaneously 0; if v1 or v2 is 0, it means that one of the side chain fragments Q1 or Q2 does not exist;

[0077] Q1 and Q2 are independently represented by the following chemical formula (I-q1):

[0078]

[0079] During the meal, is a site connected to L1 or L2; G1 and G2 are independently OC(O), NHC(O), C(O), CH2, NH, OC(O)NH, NHC(O)NH, O, S, B, P(O)(OH), NHP(O)(OH), NHP(O)(OH)NH, CH2P(O)(OH)NH, OP(O)(OH)O, CH2P(O)(OH)O, NHS(O)2, NHS(O)2NH, CH2S(O)2NH, OS(O)2O, CH2S(O)2O, Ar, ArCH2, ArO, ArNH, ArS, ArNR1 or (Aa) q1 and; G3 is OH, SH, OR 12 , SR 12 ,OC(O)R 12 , NHC(O)R 12 , C(O)R 12 , CH3, NH2, NR 12 , + NH(R 12 ), + N(R 12 )(R 12' ), C(O)OH, C(O)NH2, NHC(O)NH2, BH2, BR 12 R 12' , P(O)(OH)2, NHP(O)(OH)2, NHP(O)(NH2)2, S(O)2(OH), (CH2) q1 C(O)OH, (CH2) q1 P(O)(OH)2, C(O)(CH2) q1 C(O)OH, OC(O)(CH2) q1C(O)OH, NHC(O)(CH2) q1 C(O)OH, CO(CH2) q1 P(O)(OH)2, NHC(O)O(CH2) q1 C(O)OH, OC(O)NH(CH2) q1 C(O)OH, NHCO(CH2) q1 P(O)(OH)2, NHC(O)(NH)(CH2) q1 C(O)OH, CONH(CH2) q1 P(O)(OH)2, NHS(O)2(CH2) q1 C(O)OH, CO(CH2) q1 S(O)2(OH), NHS(O)2NH(CH2) q1 C(O)OH, OS(O)2NH(CH2) q1 C(O)OH, NHCO(CH2) q1 S(O)2(OH), NHP(O)(OH)(NH)(CH2) q1 C(O)OH, CONH(CH2) q1 S(O)(OH), OP(O)(OH)2, (CH2) q1 P(O)(NH)2, NHS(O)2(OH), NHS(O)2NH2, CH2S(O)2NH2, OS(O)2OH, OS(O)2OR1, CH2S(O)2OR 12 , Ar, ArR 12 , ArOH, ArNH2, ArSH, ArNHR 12 or (Aa) q1 And; (Aa) q1 is a peptide containing natural or non-natural amino acids of the same or different sequences; X1 and X2 are independently O, CH2, S, S(O), NHNH, NH, N(R 12 ), + NH(R 12 ), + N(R 12 )(R 12' ), C(O), OC(O), OC(O)O, OC(O)NH, NHC(O)NH and; Y2 is O, NH, NR 12 , CH2. S, NHNH, Ar and; p 1,p2 and p3 are independently 0 to 100, but not simultaneously 0; q1 and q2 are independently 0 to 24; R 12 , R 12', R 13 및 R 13' is independently H, C1~C8 alkyl; C2~C8 heteroalkyl or heterocyclic; C3~C8 aryl, Ar-alkyl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, carbonic or alkylcarbonyl;

[0080] Preferably, Q1 and Q2 are independently C2-C 100 Polycarboxylic acid; C2-C 90 Polyalkylamine; C6-C 90 Oligosaccharides or polysaccharides; C6-C composed of quaternary ammonium cations and / or sulfonate anions 100 Zwitterionic betaine or zwitterionic poly(sulfobetaine) (PSB); For example, poly(lactic acid / glycolic acid) (PLGA), poly(acrylate), chitosan, copolymer of N-(2-hydroxypropyl)methacrylamide, poly[2-(methacryloyloxy)ethylphosphorylcholine] (PMPC), poly-L-glutamic acid, poly(lactide-co-glycolide) (PLG), poly(lactide-co-glycolide), poly(ethylene glycol) (PEG), poly(propylene glycol) (PPG), poly(lactide-co-glycolide), poly(ethylene glycol)-modified peptide, poly(ethylene glycol)-containing amino acid or peptide, poly(ethylene glycol)-modified lipid, poly-glycine, poly-N-methyl-glycine, poly(ethylene glycol)-modified alkylcarboxylic acid, poly(ethylene glycol)-modified alkylamine, poly(lactide-co-glycolide, C6-C composed of hyaluronic acid (HA) (glycosaminoglycan), heparin / heparan sulfate (HSGAG), chondroitin sulfate / dermatan sulfate (CSGAG), poly(ethylene glycol)-modified alkyl sulfate, poly(ethylene glycol)-modified alkyl phosphate, or poly(ethylene glycol)-modified alkyl quaternary ammonium. 100 It is a biodegradable polymer.

[0081] The exemplary structures of Q1 and Q2 are shown below:

[0082]

[0083]

[0084]

[0085] During the meal, R 25 and R 25' is H; HC(O), CH3C(O), CH3C(NH), NHCH3, COOH, CONH2, CONHCH3, C1-C 18 Alkyl, C1-C 18 Alkyl, Alkyl-Y1-SO3H, C1-C 18 Alkyl-Y1-PO3H2, C1-C 18 Alkyl-Y1-CO2H, C1-C 18 Alkyl-Y1-N + R 12 R 13 R 13 'R 14 , C1-C 18 Alkyl-Y1-CONH2, C2-C 18 Alkylene, C2-C 18 Ester, C2-C 18 Ether, C2-C 18 Amine, C2-C 18 Alkyl carboxyamide, C3-C 18 Aryl, C3-C 18 Cyclic alkyl, C3-C 18 Complex cyclic formula, 1 to 24 amino acids; C2-C 18 Geology, C2-C 18 fatty acids or C2-C 18Independently selected from fatty ammonium lipids; X1 and X2 are independently selected from NH, N(R1'), O, CH2, S, C(O), S(O), S(O2), P(O)(OH), NHNH, CH=CH, Ar, or (Aa)q1 (q1 is 0 to 24 (0 to 24 amino acids), and q1 being 0 means non-existent); X1, X2, X3, X4, Y1, Y2, and Y3 are NH, N(R 12 '), O, C(O), CH2, S, S(O), NHNH, C(O), OC(O), OC(O)O, OC(O)NH, NHC(O)NH, Ar or Ar or (Aa)q1 independently selected, and X1, X2, X3, X4, Y1, Y2, and Y3 may not exist independently; p1, p2, and p3 are independently 0 to 100 but not simultaneously 0; q1, q2, and q3 are independently 0 to 24; R 12 , R 13 , R 13 ' and R 14 ' is independently selected from H and C1-C6 alkyls; Aa is a natural or non-natural amino acid; Ar or (Aa)q1 is a peptide of the same or different sequence; q1 being 0 means that (Aa)q1 is not present;

[0086] D is a cytotoxic agent independently selected from caliceamycin, metansinoids, camptothecin, taxane, anthracycline (daunorubicin / doxorubicin), vinca alkaloid, auristatin, eribulin, (pyrrolo)benzodiazepine (PBD), CC-106 / duocamycin, tubulicin, amatoxin (e.g., amanitin), protein kinase inhibitors, MEK inhibitors, KSP inhibitors, nicotinamide phosphoribosyltransferase (NAMPT) inhibitors, immunotoxins, analogs or prodrugs of the above compounds; D1 and D2 are identical or different, and are defined as identical to D;

[0087] Caliceamicin and its related endi-in antibiotics are referenced in the literature [Nicolaou, KC et al, Science 1992, 256, 1172-1178; Proc. Natl. Acad. Sci USA. 1993, 90, 5881-8)], U.S. Patents No. 4,970,198; No. 5,053,394; No. 5,108,912; No. 5,264,586; No. 5,384,412; No. 5,606,040; No. 5,712,374; No. 5,714,586; No. 5,739,116; No. 5,770,701; No. 5,770,710; No. 5,773,001; No. 5,877,296; It is described in headings 6,015,562; 6,124,310; and 8,153,768. The structure of caliceamisin is preferably the following chemical formula or isotopes of their chemical elements or pharmaceutically acceptable salts, hydrates or hydrated salts; or polymorphic crystal structures; or optical isomers, racemic mixtures, diastereomers or stereoisomers:

[0088]

[0089] During the meal, is the part connected to W.

[0090] Meitansinol and its analogs, including meitansinoids, are U.S. Patents No. 4,256,746, No. 4,361,650, No. 4,307,016, No. 4,294,757, No. 4,294,757, No. 4,371,533, No. 4,424,219, No. 4,331,598, No. 4,450,254, No. 4,364,866, No. 4,313,946, No. 4,315,929, No. 4,362,663, No. 4,322,348, No. 4,371,533, No. 4,424,219, No. 5,208,020, No. 5,416,064, No. 5,208,020, It is described in Patent Nos. 5,416,064, 6,333,410, 6,441,163, 6,716,821, 7,276,497, 7,301,019, 7,303,749, 7,368,565, 7,411,063, 7,851,432 and 8,163,888. The structure of the meitansinoid is preferably given by the following chemical formula:

[0091]

[0092] During the meal, is the part connected to W.

[0093] Camptothecin (CPT) and its derivatives, which are topoisomerase inhibitors that induce DNA damage and lead to apoptosis by preventing DNA religation, are described in the literature [Shang, XF et al, Med Res Rev. 2018, 38(3):775-828; Botella, P. and Rivero-Buceta, E. J Control Release. 2017, 247: 28-54; Martino, E. et al, Bioorg Med Chem Lett. 2017, 27(4):701-707; Lu, A., et al, Acta Pharmacol Sin 2007, 28(2): 307-314]. It includes SN-38, topotecan, irinotecan (CPT-11), silatecan (DB-67, AR-67), cocitecan (BNP-1350), etirinotecan, exatecan, rutotecan, gimatecan (ST1481), belotecan (CKD-602), rubitecan and several others (reference [Shang, XF et al, Med Res Rev. 2018, 38(3):775-828]). To date, three CPT analogs—topotecan, irinotecan, and belotecan—have been approved and are used in cancer chemotherapy (Palakurthi, S., Expert Opin Drug Deliv. 2015;12(12):1911-21; Shang, XF et al, Med Res Rev. 2018, 38(3):775-828), and both SN-38 and exatecan have been successfully used as payloads for ADC conjugates in clinical trials (Ocean, AJ et al, Cancer. 2017, 123(19): 3843-3854; Starodub, AN, et al, Clin Cancer Res. 2015, 21(17): 3870-8; Cardillo, TM, et al, Bioconjug Chem. 2015, 26(5): 919-31; Ogitani, Y. et al, Bioorg Med Chem Lett.[2016, 26(20): 5069-5072; Takegawa, N. et al, Int J Cancer. 2017 OCt 15;141(8):1682-1689], U.S. Patents No. 7,591,994; No. 7,999,083; No. 8,080,250; No. 8,268,317; U.S. Patent Applications No. 20130090458; No. 20140099258; No. 20150297748; No. 20160279259).

[0094] The structure of camptothecin (CPT) is the following chemical formula or an isotope of one or more chemical elements of these compounds, or a pharmaceutically acceptable salt, hydrate, or hydrated salt; or a polymorphic crystal structure; or an optical isomer, racemic mixture, diastereomer, or enantiomer:

[0095]

[0096] During the meal, R 1, R2 and R4 are independently selected from H, F, Cl, Br, CN, NO2, C1-C8 alkyl; O-C1-C8 alkyl; NH-C1-C8 alkyl; C2-C8 heteroalkyl, alkylcycloalkyl, heterocycloalkyl; C3-C8 aryl, Ar-alkyl, heterocyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; or esters, ethers, amides, carbonates, ureas, or carbamates having 2 to 8 carbon atoms; and R3 is H, OH, NH2, C1-C8 alkyl; O-C1-C8 alkyl; NH-C1-C8 alkyl; C2-C8 heteroalkyl, alkylcycloalkyl, heterocycloalkyl; or are esters, ethers, amides, carbonates, ureas, or carbamates of 2 to 8 carbon atoms; or R1R2, R2R3, and R3R4 independently form 5 to 7 carbon-linked, heterocyclic, heterocycloalkyl, aromatic, or heteroaromatic ring systems.

[0097] The structure of camptothecin is preferably the following chemical formula or an isotope of one or more chemical elements of these compounds, or a pharmaceutically acceptable salt, hydrate, or hydrated salt; or a polymorphic crystal structure; or an optical isomer, racemic mixture, diastereomer, or enantiomer:

[0098]

[0099]

[0100]

[0101] During the meal, is the part connected to W; P 1 is H, OH, NH2, COOH, C(O)NH2,OCH2OP(O)(OR 18 )2,OC(O)OP(O)(OR 18 )2, OPO(OR 18 )2, NHPO(OR 18 )2,OC(O)R 18 , OP(O)(OR 18 )OP(O)(OR 18 )2, OC(O)NHR 18 , OC(O)N(C2H4)2NCH3, OSO2(OR 18 ), O-(C4-C 12 -glycoside), OC(O)N(C2H4)2CH2N(C2H4)2CH3, C1-C8 linear or branched alkyl or heteroalkyl; C2-C8 linear or branched alkenyl, alkynyl, alkylcycloalkyl, heterocycloalkyl; C3-C8 linear or branched aryl, Ar-alkyl, heterocyclic, carboncyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; carbonate(-C(O)OR 17 ), carbamate(-C(O)NR 17 R 18 ) and; R 17 and R 18is independently H, linear or branched alkyl or heteroalkyl; C2-C8 linear or branched alkenyl, alkynyl, alkylcycloalkyl, heterocycloalkyl; C3-C8 linear or branched aryl, Ar-alkyl, heterocyclic, carbonocyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; carbonate(-C(O)OR 17 ), carbamate(-C(O)NR 17 R 18 )am.

[0102] Taxanes including paclitaxel (Taxol), cytotoxic natural products and dodetaxel (Taxotere), semi-synthetic derivatives and analogs thereof that are desirable for conjugation are [K C. Nicolaou et al., J. Am. Chem. Soc. 117, 2409-20, (1995); Ojima et al, J. Med. Chem. 39:3889-3896 (1996); 40:267-78 (1997); 45, 5620-3 (2002); Ojima et al., Proc. Natl. Acad. Sci., 96:4256-61 (1999); Kim et al., Bull. Korean Chem. Soc., 20, 1389-90 (1999); Miller, et al. J. Med. Chem., 47, 4802-5 (2004)]; exemplified in U.S. Patents No. 5,475,011; No. 5,728,849; No. 5,811,452; No. 6,340,701; No. 6,372,738; No. 6,391,913; No. 6,436,931; No. 6,589,979; No. 6,596,757; No. 6,706,708; No. 7,008,942; No. 7,186,851; No. 7,217,819; No. 7,276,499; No. 7,598,290; and No. 7,667,054. The structure of the taxicane is preferably the following chemical formula:

[0103]

[0104]

[0105] During the meal, is a site connected to W; Ar and Ar' are independently aryl or heteroaryl.

[0106] Anthracyclines are mammalian DNA topoisomerase II inhibitors capable of stabilizing enzyme-DNA complexes, where DNA strands are cleaved and covalently linked to proteins. These anticancer agents have played a significant role in treating various forms of solid tumors and acute leukemia over the past few decades. However, anthracyclines cause cardiovascular morbidity and mortality (Reference [Sagi, JC, et al, Pharmacogenomics. 2016, 17(9), 1075-87; McGowan, JV, et al, Cardiovasc Drugs Ther. 2017, 31(1), 63-75]). Therefore, to improve the specific activity of these molecules while reducing cardiotoxicity, researchers actively use the conjugation of anthracyclines to cell-binding molecules as a general approach to improve the therapeutic index of these drugs (Mollaev, M. et al, Int J Pharm. 2018 Dec 29. pii: S0378-5173(18) 30991-8; Rossin, R., et al, Bioconjug Chem. 2016, 27(7):1697-706; Dal Corso, A., et al, J Control Release. 2017, 264:211-218]). The structure of the anthracycline is preferably of the following chemical formula:

[0107]

[0108]

[0109] During the meal, is a connecting part.

[0110] Vinca alkaloids are a set of anti-mitotic and anti-microtubule alkaloid agents that act by inhibiting the ability of cancer cells to divide. Vinca alkaloids include vinblastine, vincristine, vindecin, leurosin, vinorelbine, catalantine, vindolin, vincaminol, vineridine, minovincin, methoxyminovincin, minovincinin, vincadiformin, desoxibincaminol, vincamazine, vincamine, vinpocetin, and vinburnin. The structure of the vinca alkaloid is preferably vinblastine, and vincristine has the following chemical formula:

[0111]

[0112]

[0113] Auristatin or dolastatin analogs are preferred in the conjugate containing the bis-linker of the present patent. Auristatin, which is a synthetic analog of dolastatin (e.g., auristatin E (AE), auristatin EB (AEB), auristatin EFP (AEFP), monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), auristatin F phenylenediamine (AFP), and phenylalanine variants of MMAE), is referenced in the literature [Int. J. Oncol. 15: 367-72 (1999); Molecular Cancer Therapeutics, vol. 3, No. 8, pp. 921-32 (2004)]; U.S. Application No. 11134826, Publication No. 20060074008, No. 2006022925.U.S. Patents No. 4414205, 4753894; 4764368; 4816444; 4879278; 4943628; No. 4978744, No. 5122368, No. 5165923, No. 5169774, No. 5286637, No. 5410024, No. 5521284, No. 5530097, No. 5554725, No. 5585089, No. 5599902, No. 5629197, No. 5635483, No. 5654399, No. 5663149, No. 5665860, No. 5708146, No. 5714586, No. 5741892, No. 5767236, No. 5767237, No. 5780588, No. 5821337, No. 5840699, No. 5965537, No. 6004934, No. 6033876, No. 6034065, No. 6048720, No. 6054297, No. 6054561, No. 6124431, No. 6143721, No. 6162930, No. 6214345, No. 6239104, No. 6323315, No. 6342219, No. 6342221, No. 6407213, No. 6569834, No. 6620911, No. 6639055, No. 6884869, No. 6913748, No. 7090843, No. 7091186, No. 7097840, It is described in Nos. 7098305, 7098308, 7498298, 7375078, 7462352, 7553816, 7659241, 7662387, 7745394, 7754681, 7829531, 7837980, 7837995, 7902338, 7964566, 7964567, 7851437, and 7994135.The structure of the auristatin analog is preferably one of the following chemical formulas (Ih-01), (Ih-02), (Ih-03), (Ih-04), (Ih-05), (Ih-06) and (Ih-07) or isotopes of one or more chemical elements of these compounds, or pharmaceutically acceptable salts, hydrates, or hydrated salts; or polymorphic crystal structures; or optical isomers, racemic mixtures, diastereomers, or enantiomers.

[0114]

[0115]

[0116] During the meal, R 1 , R 2 , R 3 , R 4 and R 5 is independently H; C1-C8 linear or branched alkyl, aryl, heteroaryl, heteroalkyl, alkylcycloalkyl, ester, ether, amide, amine, heterocycloalkyl, or acyloxylamine; or a peptide containing 1 to 8 amino acids, or formula (OCH2CH2) p having Polyethyleneoxy unit, (OCH2CH(CH3)) p (where p is an integer from 1 to about 5000) and 2 R's, i.e., R' 1 R 2 , R 2 R 3 , R 1 R 3 or R 3 R 4can form a cyclic ring of 3 to 8 members of an alkyl, aryl, heteroaryl, heteroalkyl, or alkylcycloalkyl group; X3 is H, CH3, or X1'R1', wherein X1' is NH, N(CH3), NHNH, O, or S, and R1' is H or a C1-C8 linear or branched alkyl, aryl, heteroaryl, heteroalkyl, alkylcycloalkyl, or acyloxylamine; and R3' is H or a C1-C6 linear or branched alkyl; Z3' is H, COOR1, NH2, NHR1, OR1, CONHR1, NHCOR1, OCOR1, OP(O)(OM1)(OM2), OCH2OP(O)(OM1)(OM2), OSO3M1, R1 or O-glycoside (glucoside, galactoside, mannnoside, glucuronoside / glucuronide, alloside, fructoside, etc.), NH-glycoside, S-glycoside or CH2-glycoside; M1 and M2 are independently H, Na, K, Ca, Mg, NH4, NR1R2R3; Y1 and Y2 are independently the linking site In the case of being connected to O, NH, NHNH, NR5, S, C(O)O, C(O)NH, OC(O)NH, OC(O)O, NHC(O)NH, NHC(O)S, OC(O)N(R1), N(R1)C(O)N(R2), C(O)NHNHC(O) and C(O)NR1 or; or connection part In cases not connected to OH, NH2, NHNH2, NHR5, SH, C(O)OH, C(O)NH2, OC(O)NH2, OC(O)OH, NHC(O)NH2, NHC(O)SH, OC(O)NH(R1), N(R1)C(O)NH(R2), C(O)NHNHC(O)OH and C(O)NHR1 and; R 12 is OH, NH2, NHR1, NHNH2, NHNHCOOH, O-R1-COOH, NH-R1-COOH, NH-(Aa) n COOH, O(CH2CH2O) p CH2CH2OH, O(CH2CH2O) pCH2CH2NH2, NH(CH2CH2O) p CH2CH2NH2, NR1R1', NHOH, NHOR1, O(CH2CH2O) p CH2CH2COOH, NH(CH2CH2O) p CH2CH2COOH, NH-Ar-COOH, NH-Ar-NH2, O(CH2CH2O) p CH2CH2NH-SO3H, NH(CH2CH2O) p CH2CH2NHSO3H, R1-NHSO3H, NH-R1-NHSO3H, O(CH2CH2O) p CH 2- CH2NHPO3H2, NH(CH2CH2O) p CH2CH2NHPO3H2, OR1, R1-NHPO3H2, R1-OPO3H2, O(CH2CH2O) p CH2CH2OPO3H2, OR1-NHPO3H2, NH-R1-NHPO3H2, NH(CH2CH2NH) p CH 2- CH2NH2, NH(CH2CH2S) p CH2CH2NH2, NH(CH2CH2NH) p CH2CH2OH, NH(CH2CH2S) p CH 2- CH2OH , NH-R1-NH2 or NH(CH2CH2O) p CH2CH2NHPO3H2, wherein Aa is 1 to 8 identical or different amino acids; p is 1 to 5000; and R 1, R 2, R 3, R 4, R 5, R5' , Z1, Z2 and n are defined as above.

[0117] Eribulin, which binds predominantly to a small number of high-affinity sites at the plus end of existing microtubules, possesses both cytotoxic and non-cytotoxic action criteria. Its cytotoxic effect is related to anti-mitotic activity, in which apoptosis in cancer cells is induced following prolonged and irreversible mitotic blockade (Kuznetsov, G. et al, Cancer Research. 2004, 64 (16): 5760-6.; Towle, M. J, et al, Cancer Research. 2010, 71 (2): 496-505). In addition to its cytotoxicity, which is an anti-microlysis-based mechanism, preclinical studies in human breast cancer models have demonstrated that eribulin also exerts complex effects on the biology of surviving cancer cells and residual tumors, which appear to be unrelated to its anti-mitotic effect. Eribulin has been approved by the US FDA for the treatment of metastatic breast cancer that has received at least two prior chemotherapy regimens for terminal disease, including both anthracycline and taxane chemotherapy, as well as for the treatment of liposarcoma (a specific type of soft tissue sarcoma) that is surgically unresectable (non-resectable) or late (metastatic). Eribulin has been used as a payload for ADC conjugates (US20170252458). The structure of eribulin is preferably the following chemical formula, Eb01:

[0118]

[0119] Inhibitors of nicotinamide phosphoribosyltransferase (NAMPT) are ADC payloads of interest due to their significantly potent activity through their unique mechanism (Literature [Sampath D, et al, Pharmacol Ther 2015[; 151, 16-31]). NAMPT regulates nicotinamide adenine dinucleotide (NAD) levels in cells, where NAD acts as an essential redox cofactor supporting energy and anabolic metabolism. NAD has several essential roles in metabolism. It acts as a coenzyme in redox reactions, as a donor of the ADP-ribose moiety in the ADP-ribosylation reaction, and as cyclic ADP-ribose, a precursor of the second messenger molecule, as well as as a host for bacterial DNA ligases and NAD + It acts as a group of enzymes called sirtuins that remove acetyl groups from proteins using [it]. In addition to these metabolic functions, NAD + It is known as an adenine nucleotide that can be released from cells spontaneously and by regulated mechanisms (Smyth L. M, et al, J. Biol. Chem. 2004, 279 (47), 48893-903; Billington R. A, et al, Mol Med. 2006, 12, 324-7]), and therefore can have an important extracellular role (Billington R. A, et al, Mol Med. 2006, 12, 324-7) .In the presence of NAMPT inhibitors, NAD levels drop below the levels required for metabolism, leading to an energy crisis and consequently causing apoptosis. To date, clinical NAMPT inhibitor candidates FK-866, CHS-828, and GMX-1777 have been clinically tested, but each experienced dose-limiting toxicity before any empirical response occurred (Holen K., et al, Invest New Drugs 2008, 26, 45-51; Hovstadius, P., et al, Clin Cancer Res 2002, 8, 2843-50; Pishvaian, MJ, et al, J Clin Oncol 2009, 27, 3581). Therefore, using ADCs for the targeted delivery of NAMPT inhibitors can achieve a much broader therapeutic index by avoiding systemic toxicity. The structure of the NAMPT inhibitor is preferably the following chemical formulas NP01, NP02, NP03, NP04, NP05, NP06, NP07, NP08, and NP09 or isotopes of one or more chemical elements of these compounds, or pharmaceutically acceptable salts, hydrates, or hydrated salts; or polymorphic crystal structures; or optical isomers, racemic mixtures, diastereomers, or enantiomers:

[0120]

[0121]

[0122] During the meal, is the same as above; X5 is F, Cl, Br, I, OH, OR1, R1, OPO3H2, OSO3H, NHR1, OCOR1, NHCOR1.

[0123] A preferred cytotoxic agent according to the present invention, a benzodiazepine dimer and an analog thereof (e.g., a dimer of pyrrolobenzodiazepine (PBD) or (tomycin), indolinobenzodiazepine, imidazobenzothiadiazepine or oxazolidinobenzodiazepine) is related to the art, U.S. Patents No. 8,163,736, 8,153,627, 8,034,808, 7,834,005, 7,741,319, 7,704,924, 7,691,848, 7,678,787, 7,612,062, 7,608,615, 7,557,099, and 7,528,128 No. 7,528,126, No. 7,511,032, No. 7,429,658, No. 7,407,951, No. 7,326,700, No. 7,312,210, No. 7,265,105, No. 7,202,239, No. 7,189,710, No. 7,173,026, No. 7,109,193, No. 7,067,511, No. 7,064,120, No. 7,056,913, No. 7,049,311, No. 7,022,699, No. 7,015,215, No. 6,979,684, No. 6,951,853, No. 6,884,799, No. 6,800,622, No. 6,747,144, No. 6,660,856, No. 6,608,192, No. 6,562,806, No. 6,977,254, No. 6,951,853, No. 6,909,006, No. 6,344,451; No. 5,880,122; No. 4,935,362; No. 4,764,616; No. 4,761,412; No. 4,723,007; No. 4,723,003; No. 4,683,230; No. 4,663,453; No. 4,508,647; No. 4,464,467; No. 4,427,587; No. 4,000,304; illustrated in U.S. Patent Application Publications No. 20100203007, No. 20100316656, and No. 20030195196.Examples of structures of antibody-benzodiazepine dimer conjugates through the linker of the present invention are shown below as PB01, PB02, PB03, PB04, PB05, PB06, PB07, PB08, PB09, PB10, PB11, PB12, PB13, PB14, PB15, and PB16 or isotopes of one or more chemical elements of these compounds, or pharmaceutically acceptable salts, hydrates, or hydrated salts; or polymorphic crystal structures; or optical isomers, racemics, diastereomers, or enantiomers.

[0124]

[0125]

[0126]

[0127]

[0128] During the meal, X 1, X 2, Y1, Y2, R 4, R 5, R5' , Z1, Z 2 및 n is defined as above; preferably X 1, X 2, Y1 and Y2 are independently O, N, NH, NHNH, NR5, S, C(O)O, C(O)NH, OC(O)NH, OC(O)O, NHC(O)NH, NHC(O)S,OC(O)N(R1), N(R1)C(O)N(R1), CH , C(O)NHNHC(O) and C(O)NR1 and; R 1 , R 2 , R 3 , R 1' , R 2' and R 3'is independently H; F; Cl; =O; =S; OH; SH; C1-C8 linear or branched alkyl, aryl, alkenyl, heteroaryl, heteroalkyl, alkylcycloalkyl, ester (COOR5 or -OC(O)R5), ether (OR5), amide (CONR5), carbamate (OCONR5), amine (NHR5, NR5R5'), heterocycloalkyl or acyloxylamine (-C(O)NHOH, -ONHC(O)R5); or a peptide containing 1 to 20 natural or non-natural amino acids, or formula (OCH2CH2) p polyethyleneoxy unit of, (OCH2CH(CH3)) p (where p is an integer from 1 to about 5000) and 2 R's, i.e., R' 1 R 2 , R 2 R 3 , R 1 R 3 , R 1' R 2' , R 2' R 3' or R 1' R 3' can independently form a cyclic ring of 3 to 8 members of an alkyl, aryl, heteroaryl, heteroalkyl, or alkylcycloalkyl group; X3 and Y3 are independently N, NH, CH2, or CR5, wherein R in the formula 4, R5, R6, R 12 and R 12' is independently H, OH, NH2, NH(CH3), NHNH2, COOH, SH, OZ3, SZ3, F, Cl, or C1-C8 linear or branched alkyl, aryl, heteroaryl, heteroalkyl, alkylcycloalkyl, acyloxylamine; Z3 is H, OP(O)(OM1)(OM2), OCH2OP(O)(OM1)(OM2), OSO3M1, or O-glycoside (glucoside, galactoside, mannnoside, glucuronoside / glucuronide, alloside, fructoside, etc.), NH-glycoside, S-glycoside, or CH2-glycoside; M1 and M2 are independently H, Na, K, Ca, Mg, NH4, NR1R2R3.

[0129] CC-1065 analogs and Duokamycin analogs are also preferred for use in conjugates containing the bis-bridge linkage of the present patent. CC-1065 analogs and Duokamycin analogs, as well as their synthesis, are described, for example, in the literature [Warpehoski, et al, J. Med. Chem. 31:590-603(1988); D. Boger et al., J. Org. Chem; 66; 6654-61, 2001]; U.S. Patents No. 4,169,888, No. 4,391,904, No. 4,671,958, No. 4,816,567, No. 4,912,227, No. 4,923,990, No. 4,952,394, No. 4,975,278, No. 4,978,757, No. 4,994,578, No. 5,037,993, No. 5,070,092, No. 5,084,468, No. 5,101,038, No. 5,117,006, No. 5,137,877, No. 5,138,059, No. 5,147,786, No. 5,187,186, No. 5,223,409, No. 5,225,539, No. 5,288,514, No. 5,324,483, No. 5,332,740 No. 5332837, No. 5334528, No. 5403484, No. 5427908, No. 5475092, No. 5495009, No. 5530101, No. 5545806, No. 5547667, No. 5569825, No. 5571698, No. 5573922, No. 5580717, No. 5585089, No. 5585499, No. 5587161, No. 5595499, No. 5606017, No. 5622929, No. 5625126, No. 5629430, No. 5633425, No. 5641780, No. 5660829, No. 5661016, No. 5686237, No. 5693762, No. 5703080, No. 5712374, No. 5714586, No. 5739116, No. 5739350, No. 5770429, No. 5773001, No. 5773435, No. 5786377, No. 5786486, No. 5789650, No. 5814318, No. 5846545, No. 5874299, No. 5877296, No. 5877397, No. 5885793, No. 5939598, No. 5962216,No. 5969108, No. 5985908, No. 6060608, No. 6066742, No. 6075181, No. 6103236, No. 6114598, No. 6130237, No. 6132722, No. 6143901, No. 6150584, No. 6162963, No. 6172197, No. 6180370, No. 6194612, No. 6214345, No. 6262271, No. 6281354, No. 6310209, No. 6329497, No. 6342480, No. 6486326, No. 6512101, No. 6521404, No. 6534660, No. 6544731, No. 6548530, No. 6555313, No. 6555693, No. 6566336, No. 6,586,618, No. 6593081, No. 6630579, No. 6,756,397, No. 6759509, No. 6762179, No. 6884869, No. 6897034, No. 6946455, No. 7,049,316, No. 7087600, No. 7091186, No. 7115573, No. 7129261, No. 7214663, No. 7223837, No. 7304032, No. 7329507, It is described in Patents No. 7,329,760, No. 7,388,026, No. 7,655,660, No. 7,655,661, No. 7,906,545 and No. 8,012,978. Examples of structures of antibody-CC-1065 analog conjugates through the linker of the present patent are shown in CC01, CC02, CC03, CC04, CC05, CC06 and CC07 below:,

[0130]

[0131]

[0132] During the meal, X 1, X 2, Y1 and Y2 are the connection points In the case of being connected to, independently O, NH, NHNH, NR5, S, C(O)O, C(O)NH, OC(O)NH, OC(O)O, NHC(O)NH, NHC(O)S,OC(O)N(R1), N(R1)C(O)N(R2), C(O)NHNHC(O) and C(O)NR1 or; or connection part In cases not connected to OH, NH2, NHNH2, NHR1, SH, C(O)OH, C(O)NH2, OC(O)NH2, OC(O)OH, NHC(O)NH2, NHC(O)SH, OC(O)NH(R1), N(R1)C(O)NH(R2), C(O)NHNHC(O)OH and C(O)NHR1; Z3 is H, PO(OM1)(OM2), SO3M1, CH2PO(OM1)(OM2), CH3N(CH2CH2)2NC(O)-, O(CH2CH2)2NC(O)-, R1 or a glycoside; R 1, R 2, R 3, M 1, M2 and n are defined as above.

[0133] Tubulicin and analogs thereof preferred for conjugation in the present invention are widely known in the art and can be isolated from natural sources according to known methods or can be prepared by synthesis according to known methods (e.g., Balasubramanian, R., et al. J. Med. Chem., 2009, 52, 238-40; Pando, O., et al. J. Am. Chem. Soc., 2011, 133, 7692-5; Reddy, JA, et al. Mol. Pharmaceutics, 2009, 6, 1518-25; Raghavan, B., et al. J. Med. Chem., 2008, 51, 1530-33; Patterson, AW, et al. J. Org. Chem., 2008, 73, 4362-9; Pando, O., et al. Org. Lett., 2009, 11 (24), 5567-9; Wipf, P., et al. Org. Lett., 2007, 9 (8), 1605-7; Peltier, H.M., et al. J. Am. Chem. Soc., 2006, 128, 16018-9; Chandrasekhar, S., et al J. Org. Chem., 2009, 74, 9531-4; Liu, Y., et al. Mol. Pharmaceutics, 2012, 9, 168-75; Friestad, G.K., et al. Org. Lett., 2009, 11, 1095-8; Kubicek, K., et al., Angew Chem Int Ed Engl, 2010.49: 4809-12; Chai, Y., et al., Chem Biol, 2010, 17: 296-309; Ullrich, A., et al., Angew Chem Int Ed Engl, 2009, 48, 4422-5; Sani, M., et al. Angew Chem Int Ed Engl, 2007, 46, 3526-9; Domling, A., et al., Angew Chem Int Ed Engl, 2006, 45, 7235-9; Patent applications: Zanda, M., et al, Can. Pat. Appl. CA 2710693 (2011); Chai, Y., et al. Eur. Pat. Appl. 2174947 (2010)], WO 2010034724; Leamon, C. et al. WO2010033733, WO 2009002993; Ellman, J. et al. PCT WO2009134279; WO 2009012958, U.S. Application Publications No. 20110263650, No. 20110021568; Matschiner, G. et al. WO2009095447; Vlahov, I. et al. WO2009055562, WO 2008112873; Low, P. et al. WO2009026177; Richter, W. WO2008138561; Kjems, J. et al. WO 2008125116; Davis, M. et al. WO2008076333; Diener, J. et al. U.S. application 20070041901, WO2006096754; Matschiner, G. et al. WO2006056464; Vaghefi, F. et al. WO2006033913; Doemling, A. et al. German patent DE102004030227, WO2004005327, WO2004005326, WO2004005269; (US Patent Application Publication 20040249130 by Stanton, M. et al.; German Patents DE10254439, DE10241152, DE10008089 by Hoefle, G. et al.; WO2002077036 by Leung, D. et al.; German Patent DE19638870 by Reichenbach, H. et al.; US20120129779 by Wolfgang, R. et al.; US Application 20110027274 by Chen, H.). A preferred structure of tubulisin for the conjugation of cell binding molecules is described in patent application PCT / IB2012 / 053554.Examples of structures of antibody-tubulisin analog conjugates via a linker are Tb01, Tb02, Tb03, Tb04, Tb05, Tb06, Tb07, Tb08, Tb09, and T10 shown below, or isotopes of one or more chemical elements of these compounds, or pharmaceutically acceptable salts, hydrates, or hydrated salts; or polymorphic crystal structures; or optical isomers, racemic mixtures, diastereomers, or enantiomers:.

[0134]

[0135]

[0136] In the formula, X1 and Y1 are independently O, NH, NHNH, NR5, S, C(O)O, C(O)NH, OC(O)NH, OC(O)O, NHC(O)NH, NHC(O)S,OC(O)N(R1), N(R1)C(O)N(R1), CH , C(O)NHNHC(O) and C(O)NR1; mAb is an antibody, preferably a monoclonal antibody; and R 12 is OH, NH2, NHR1, NHNH2, NHNHCOOH, O-R1-COOH, NH-R1-COOH, NH-(Aa) n COOH, O(CH2CH2O) p CH2CH2OH, O(CH2CH2O) p CH2CH2NH2, NH(CH2CH2O) p CH2CH2NH2, NR1R1', NHOH, NHOR1, O(CH2CH2O) p CH2CH2COOH, NH(CH2CH2O) p CH2CH2COOH, NH-Ar-COOH, NH-Ar-NH2, O(CH2CH2O) p CH2CH2NHSO3H, NH(CH2CH2O) p CH2CH2NHSO3H, R1-NHSO3H, NH-R1-NHSO3H, O(CH2CH2O) p CH2CH2NHPO3H2, NH(CH2CH2O) pCH2CH2NHPO3H2, OR1, R1-NHPO3H2, R1-OPO3H2, O(CH2CH2O) p CH2CH2OPO3H2, OR1-NHPO3H2, NH-R1-NHPO3H2, NH(CH2CH2NH) p CH2CH2NH2, NH(CH2CH2S) p CH2CH2NH2, NH(CH2CH2NH) p CH2CH2OH, NH(CH2CH2S) p CH2CH2OH , NH-R1-NH2 or NH(CH2CH2O) p CH2CH2NHPO3H2, wherein Aa is 1 to 8 amino acids; n and m1 are independently 1 to 20; p is 1 to 5000; preferably R1, R1', R2, R3 and R4 are independently H, C1-C8 linear or branched alkyl, amide or amine; C2-C8 aryl, alkenyl, alkynyl, heteroaryl, heteroalkyl, alkylcycloalkyl, ester, ether, heterocycloalkyl or acyloxylamine; or an amino acid containing 1 to 8 amino acids or formula (OCH2CH2) p polyethyleneoxy unit having (OCH2CH(CH3)) p (wherein p is an integer from 1 to about 5000); and two Rs, i.e., R1R2, R2R3, R1R3, or R3R4, can form a cyclic ring of 3 to 8 members of an alkyl, aryl, heteroaryl, heteroalkyl, or alkylcycloalkyl group; and X3 is H, CH 3, CH2CH 3,C3H7 or X1'R1', wherein X1' is NH, N(CH3), NHNH, O, or S; R1' is H or a C1-C8 linear or branched alkyl, aryl, heteroaryl, heteroalkyl, alkylcycloalkyl, or acyloxylamine; R3' is H or a C1-C6 linear or branched alkyl; Z3 is H, COOR1, NH2, NHR1, OR1, CONHR1, NHCOR1, OCOR1, OP(O)(OM1)(OM2), OCH2OP(O)(OM1)(OM2), OSO3M1, R1, O-glycoside (glucoside, galactoside, mannnoside, glucuronoside / glucuronide, alloside, fructoside, etc.), NH-glycoside, S-glycoside, or CH2-glycoside; M1 and M2 are independently H, Na, K, Ca, Mg, NH4, NR1R2R3.

[0137] Amatoxins, a subgroup of at least 10 toxic compounds first discovered in some genera of toxic mushrooms, most notably Amanita phalloides and several other mushroom species, are also preferred for the conjugation of this patent. These 10 types of amatoxins, named α-amanitine, β-amanitine, γ-amanitine, ε-amanitine, amanulin, amanulinic acid, amaninamide, amanin, and pro-amanulin, are rigid bicyclic peptides synthesized as 35-amino acid proteins, from which the final 8 amino acids are cleaved by prolyl oligopeptidase (Litten, W. 1975 Scientific American 232 (3): 90-101; HE Hallen, et al 2007 Proc. Nat. Aca. Sci. USA 104, 19097-101; K. Baumann, et al, 1993 Biochemistry 32 (15): 4043-50; Karlson-Stiber C, Persson H. 2003, Toxicon 42 (4): 339-49; Horgen, PA et al. 1978 Arch. Microbio. 118 (3): 317-9]). Amatoxins induce cell death by inhibiting RNA polymerase II (Pol II) and halt gene transcription and protein biosynthesis (Brodner, OG and Wieland, T. 1976 Biochemistry, 15(16): 3480-4; Fiume, L., Curr Probl Clin Biochem, 1977, 7: 23-8; Karlson-Stiber C, Persson H. 2003, Toxicon 42(4): 339-49; Chafin, DR, Guo, H. & Price, DH 1995 J. Biol. Chem. 270 (32): 19114-19; Wieland (1983) Int. J. Pept. Protein Res. 22(3): 257-76).Amatoxins are derived from collected Amanita palloides mushrooms (reference [Yocum, RR 1978 Biochemistry 17(18): 3786-9; Zhang, P. et al, 2005, FEMS Microbiol. Lett.252(2), 223-8]), from fermentation using basidiomycetes (reference [Muraoka, S. and Shinozawa T., 2000 J. Biosci. Bioeng. 89(1): 73-6]), from fermentation using A. fissa (reference [Guo, XW, et al, 2006 Wei Sheng Wu Xue Bao 46(3): 373-8]), or from strains belonging to the species Galerina fasciculata or Galerina helvoliceps It can be produced from culture (WO / 1990 / 009799, JP11137291). However, the yield from such isolation and fermentation is very low (less than 5 mg / L of culture). Several preparation methods for amatoxins and their analogs have been reported over the past 30 years (Literature [WE Savige, A. Fontana, Chem. Commun. 1976, 600-1; Zanotti, G., et al, Int. J Pept Protein Res, 1981. 18(2): 162-8; Wieland, T., et al, Eur. J. Biochem. 1981, 117, 161-4; PA Bartlett, et al, Tetrahedron Lett. 1982, 23, 619-22; Zanotti, G., et al., Biochim Biophys Acta, 1986. 870(3): 454-62; Zanotti, G., et al., Int. J. Peptide Protein Res. 1987, 30, 323-9; Zanotti, G., et al., Int. J. Peptide Protein Res. 1987, 30, 450-9; Zanotti, G., et al., Int J Pept Protein Res, 1988. 32(1): 9-20; G. Zanotti, T. et al., Int. J. Peptide Protein Res. 1989, 34, 222-8; Zanotti, G., et al., Int J Pept Protein Res, 1990. 35(3): 263-70; Mullersman, JE and JF Preston, 3rd, Int J Pept Protein Res, 1991. 37(6): 544-51; Mullersman, JE, et al, Int J Pept Protein Res, 1991. 38(5): 409-16; Zanotti, G., et al, Int J Pept Protein Res, 1992. 40(6): 551-8; Schmitt, W. et al, J. Am. Chem. Soc. 1996, 118, 4380-7; Anderson, MO, et al, J. Org. Chem., 2005, 70(12): 4578-84; JP May, et al, J. Org. Chem. 2005, 70, 8424-30; F. Brueckner, P. Cramer, Nat. Struct. Mol. Biol. 2008, 15, 811-8; JP May, DM Perrin, Chem. Time. J. 2008, 14, 3404-9; JP May, et al, Chem. Time. J. 2008, 14, 3410-17; Q. Wang, et al, Eur. J. Org. Chem. 2002, 834-9; May, JP and DM Perrin, Biopolymers, 2007. 88(5): 714-24; May, JP, et al., Chemistry, 2008. 14(11): 3410-7; S. De Lamo Marin, et al, Eur. J. Org. Chem. 2010, 3985-9; Pousse, G., et al., Org Lett, 2010.[12(16): 3582-5; Luo, H., et al., Chem Biol, 2014. 21(12): 1610-7; Zhao, L., et al., Chembiochem, 2015. 16(10): 1420-5]), most of these preparation methods were by partial synthesis. Due to its potent efficacy and unique mechanism of cytotoxicity, amatoxins have been used as payloads for conjugation (References [Fiume, L., Lancet, 1969. 2 (7625): 853-4; Barbanti-Brodano, G. and L. Fiume, Nat New Biol, 1973. 243(130): 281-3; Bonetti, E., M. et al, Arch Toxicol, 1976. 35(1): p. 69-73; Davis, MT, Preston, JF Science 1981, 213, 1385-1388; Preston, JF, et al, Arch Biochem Biophys, 1981. 209(1): 63-71; H. Faulstich, et al, Biochemistry 1981, 20, 6498-504; Barak, LS, et al., Proc Natl Acad Sci USA, 1981. 78(5): 3034-8; Faulstich, H. and L. Fiume, Methods Enzymol, 1985. 112: 225-37; Zhelev, Z., A. et al, Toxicon, 1987. 25(9): 981-7; Khalacheva, K., et al, Eksp Med Morfol, 1990. 29(3): 26-30; U. Bermbach, H. Faulstich, Biochemistry 1990, 29, 6839-45; Mullersman, J.E. and J.F. Preston, Int. J. Peptide Protein Res. 1991, 37, 544-51; Mullersman, J.E. and J.F.Preston, Biochem Cell Biol, 1991. 69(7): 418-27; J. Anderl, H. Echner, H. Faulstich, Beilstein J. Org. Chem. 2012, 8, 2072-84; Moldenhauer, G., et al, J. Natl. Cancer Inst. 2012, 104, 622-34; A. Moshnikova, et al; Biochemistry 2013, 52, 1171-8; Zhao, L., et al., Chembiochem, 2015. 16(10): 1420-5; Zhou, B., et al., Biosens Bioelectron, 2015. 68: 189-96]; WO2014 / 043403, US20150218220, EP 1661584). The inventors have been studying the conjugation of amatoxins for some time. Examples of structures of antibody-amatoxin conjugates via linkers are the structures of Am01, Am02, and Am03 below, or isotopes of one or more chemical elements of these compounds, or pharmaceutically acceptable salts, hydrates, or hydrated salts; or polymorphic crystal structures; or optical isomers, racemics, diastereomers, or enantiomers are preferred:.

[0138]

[0139] In the formula, X1 and Y1 are independently O, NH, NHNH, NR5, S, C(O)O, C(O)NH, OC(O)NH, OC(O)O, NHC(O)NH, NHC(O)S, OC(O)N(R1), N(R1)C(O)N(R1), CH , C(O)NHNHC(O) and C(O)NR1; R7, R8 and R9 are independently H, OH, OR1, NH2, NHR1, C1-C6 alkyl or are absent; Y2 is O, O2, NR1, NH or is absent; R 10 Silver is CH2, O, NH, NR 1,NHC(O), NHC(O)NH, NHC(O)O, OC(O)O, C(O), OC(O), OC(O)(NR1), (NR1)C(O)(NR1), C(O)R1 or not; R 11 OH, NH2, NHR1, NHNH2, NHNHCOOH, O-R1-COOH, NH-R1-COOH, NH-(Aa) r COOH, O(CH2CH2O) p CH2CH2OH, O(CH2CH2O) p CH2CH2NH2, NH(CH2CH2O) p CH2CH2NH2, NR1R1', O(CH2CH2O) p CH2CH2COOH, NH(CH2CH2O) p CH2CH2COOH, NH-Ar-COOH, NH-Ar-NH2, O(CH2CH2O) p CH2CH2NHSO3H, NH(CH2CH2O) p CH2CH2NHSO3H, R1-NHSO3H, NH-R1-NHSO3H, O(CH2CH2O) p CH2CH2NHPO3H2, NH(CH2CH2O) p CH2CH2NHPO3H2, OR1, R1-NHPO3H2, R1-OPO3H2, O(CH2CH2O) p CH2CH2OPO3H2, OR1-NHPO3H2, NH-R1-NHPO3H2, or NH(CH2CH2O) p CH2CH2NHPO3H2, but among the formulas, (Aa) r is 1 to 8 amino acids; n and m1 are independently 1 to 20; p is 1 to 5000; and R1 and Ar are defined as in formula (I).

[0140] Protein kinase inhibitors can regulate protein function by blocking the action of enzymes that add phosphate (PO4) groups to serine, threonine, or tyrosine amino acids on proteins. Protein kinase inhibitors can be used to treat diseases caused by overactive protein kinases (including mutant or overexpressed kinases) in cancer or to regulate cell function to overcome other disease-inducing factors. The structure of the protein kinase inhibitor is preferably selected from adavosertip, afatinib, axitinib, bafetinib, bosutinib, cobimetinib, crizotinib, cabozantinib, dasatinib, entrectinib, erdafitinib, erlotinib, erlotinib, fostamatinib, gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, mubritinib, nilotinib, pazopanib, pegaptanib, ponatinib, levastinib, regorafenib, ruxolitinib, sorafenib, sunitinib, SU6656, tofacitinib, vandetanib, and vemurafenib, having the following chemical formulas PK01 to PK29:

[0141]

[0142]

[0143]

[0144]

[0145]

[0146] MEK inhibitors inhibit MEK1 and / or MEK2, which are mitogen-activated protein kinases that are usually overactive in some cancers. MEK inhibitors are used, in particular, for the treatment of BRAF-mutated melanoma and KRAS / BRAF-mutated colorectal cancer, breast cancer, and non-small cell lung cancer (NSCLC). MEK inhibitors are selected from PD0325901, selumetinib (AZD6244), cobimetinib (XL518), refametinib, trametinib (GSK1120212), fimacertib, binimetinib (MEK162), AZD8330, RO4987655, RO5126766, WX-554, E6201, GDC-0623, PD-325901, and TAK-733. Preferred MEK inhibitors are selected from trametinib (GSK1120212), cobimetinib (XL518), binimetinib (MEK162), and selumetinib having the following chemical formulas:

[0147]

[0148] Among the formulas, Z5 is O, NH, NHNH, NR5, S, C(O)O, C(O)NH, OC(O)NH, OC(O)O, NHC(O)O, NHC(O)NH, NHC(O)S, OC(O)N(R1), N(R1)C(O)N(R2), C(O)NHNHC(O) and C(O)NR 1; Selected from.

[0149] The proteinase inhibitor used as a payload is preferably selected from carfilzomib, clindamycin, retapamulin, and indibulin as shown in the following structural formula:

[0150]

[0151] In this specification, an immunotoxin is a macromolecular drug, usually a cytotoxic protein derived from bacteria or plant proteins, such as diphtheria toxin (DT), cholera toxin (CT), trichosanthin (TCS), dianthin, Pseudomonas exotoxin A (ETA'), erythropoietic toxin, diphtheria toxin, AB toxin, type III exotoxin, etc. It may also be a highly toxic bacterial pore-forming protoxin that requires proteolytic processing for activation. Examples of such protoxins are proaerolysin and its genetically modified form, topsalysin. Topsalysin is a modified recombinant protein engineered to be selectively activated by enzymes in the prostate, leading to localized cell death and tissue destruction without damaging neighboring tissues and nerves. In this specification, an immunotoxin is preferably through amino acids having free amino, thiol, or carboxylic acid groups; More preferably, it is preferably joined through the side chain linker of the present application via the N-terminal amino acid.

[0152] Additionally, W, L 1, L 2, V 1 및V2 may be composed of one or more linker components selected from 6-maleimidocaproyl ("MC"), maleimidopropane oil ("MP"), valine-citrulline ("val-cit" or "vc"), alanine-phenylalanine ("ala-phe" or "af"), p-aminobenzyloxy-carbonyl ("PAB"), 4-thiopentanoate ("SPP"), 4-(N-maleimidomethyl)cyclohexane-1 carboxylate ("MCC"), (4-acetyl)aminobenzoate ("SIAB"), 4-thio-butyrate ("SPDB"), and 4-thio-2-hydroxysulfonyl-butyrate (2-sulfo-SPDB). Natural amino acids are preferably aspartic acid, glutamic acid, arginine, histidine, lysine, serine, threonine, asparagine, glutamine, cysteine, selenocysteine, tyrosine, phenylalanine, glycine, proline, tryptophan, and alanine;

[0153]

[0154]

[0155]

[0156] and selected from L- or D-, natural or non-natural peptides containing 1 to 20 amino acids; is a portion of the linkage; preferably X 2, X 3, X 4, X5 or X6 is NH; NHNH; N(R 12 ); N(R 12 )N(R 12' ); O; S; C1-C6 alkyl; C2-C6 heteroalkyl, alkylcycloalkyl, heterocycloalkyl; C3-C8 aryl, Ar-alkyl, heterocyclic, carboncyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; CH2OR 12 , CH2SR 12 , CH2NHR 12, or independently selected from 1 to 8 amino acids; R 12 및 R 12' is independently H; C1-C8 alkyl; C2-C8 hetero-alkyl, alkylcycloalkyl, heterocycloalkyl; C3-C8 aryl, Ar-alkyl, heterocyclic, carbon-cyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; or an ester, ether, or amide having 1 to 8 carbon atoms; or formula (OCH2CH2) p polyethyleneoxy units or (OCH2CH(CH3)) p (wherein p is an integer from 0 to about 1000) or a combination of the above;

[0157] W, L 1, L2V1 and V2 may also independently contain self-sacrificial or non-self-sacrificial components, peptide units, hydrazone bonds, disulfides, esters, oximes, amides, or thioether bonds. The self-sacrificial unit includes, but is not limited to, para-aminobenzylcarbamoyl (PAB) groups, e.g., 2-aminoimidazole-5-methanol derivatives, heterocyclic PAB analogs, beta-glucuronides, and aromatic compounds electronically similar to ortho or para-aminobenzylacetals;

[0158] Preferably, the self-sacrificial linker component has one of the following structures:

[0159]

[0160] In the formula, (*) atoms are additional spacers or releaseable linker units or attachment points of cytotoxic agents and / or binding molecules (CBA); X 1 , Y 1 , Z 2 and Z 3 is independently NH, O, or S; Z 1 H, NHR1, OR1, SR independently 1,COX1R1, wherein X1 and R1 are as defined above; v is 0 or 1; and U 1 is independently H, OH, C1~C6 alkyl, (OCH2CH2) n, F, Cl, Br, I, OR5, SR5, NR5R5', N=NR5, N=R 5, NR5R5' , NO 2, SOR5R5', SO2R5, SO3R 5, OSO3R5, PR5R5', POR5R5' , PO2R5R5', OPO(OR5)(OR5') or OCH2PO(OR5(OR5'), wherein R5 and R5' are independently selected from H, C1~C8 alkyl; C2~C8 alkenyl, alkynyl, heteroalkyl or amino acid; C3~C8 aryl, heterocyclic, carbocyclic, cycloalkyl, heterocycloalkyl, heteroaralkyl, alkylcarbonyl or glycoside; or pharmaceutical cation salts;

[0161] W, L 1, L2V1 and V2 may also independently contain a non-self-sacrificial linker component having one of the following structures:

[0162]

[0163]

[0164] In the formula, (*) atoms are additional spacers or releaseable linkers, cytotoxic agents and / or attachment points of binding molecules; X 1 , Y 1 , U 1 , R5, R5' are as defined above; r is 0 to 100; and m and n are independently 0 to 20.

[0165] Additionally, preferably, W, L 1, L2V 1 및V2 may be an independently releaseable linker component. The term releaseable refers to a linker comprising at least one bond that can be broken under physiological conditions, e.g., pH-unstable, acid-unstable, base-unstable, oxidatively unstable, metabolically unstable, biochemically unstable, or enzyme-unstable bond. It is recognized that these physiological conditions resulting in bond breaking do not necessarily involve biological or metabolic processes, but instead may involve standard chemical reactions, e.g., hydrolysis or substitution reactions, e.g., disulfide bond exchange reactions with intracellular thiols, such as endosomes having a pH lower than the cytoplasmic pH, and / or glutathione-rich millimolar ranges within malignant cells.

[0166] W, L 1, Examples of liberable components of L2V1 and V2 include, but are not limited to, the following independently:

[0167] -(CR 15 R 16 ) m (Aar)r(CR 17 R 18 ) n (OCH2CH2) t -, -(CR 15 R 16 ) m (CR 17 R 18 ) n (Aa) r (OCH2CH2) t -, -(Aa) r -(CR 15 R 16 ) m (CR 17 R 18 ) n (OCH2CH2) t -, -(CR 15 R 16 ) m (CR 17 R 18 ) n (OCH2CH2) r (Aa) t-, -(CR 15 R 16 ) m (CR 17 =CR 18 )(CR 19 R 20 ) n (Aa) t (OCH2CH2) r -, -(CR 15 R 16 ) m (NR 11 CO)(Aa) t (CR 19 R 20 ) n- (OCH2CH2) r -, -(CR 15 R 16 ) m (Aa) t (NR 21 CO)(CR 19 R 20 ) n (OCH2CH2) r -, -(CR 15 R 16 ) m (OCO)(Aa) t -(CR 19 R 20 ) n (OCH2CH2) r -, -(CR 15 R 16 ) m (OCNR 17 )(Aa) t (CR 19 R 20 ) n (OCH2CH2) r -, -(CR 15 R 16 ) m -(CO)(Aa) t- (CR 19 R 20 ) n (OCH2CH2) r -, -(CR 15 R 16 ) m (NR 21 CO)(Aa) t (CR 19 R 20) n (OCH2CH2) r -, -(CR 15 R 16 ) m- (OCO)(Aa) t (CR 19 R 20 ) n- (OCH2CH2) r -, -(CR 15 R 16 ) m (OCNR 17 )(Aa) t (CR 19 R 20 ) n -(OCH2CH2) r -, -(CR 15 R 16 ) m (CO)(Aa) t (CR 19 R 20 ) n- (OCH2CH2) r -, -(CR 15 R 16 ) m -phenyl-CO(Aa) t- (CR 17 R 18 ) n -, -(CR 15 R 16 ) m -Furyl-CO(Aa) t (CR 17 R 18 ) n -, -(CR 15 R 16 ) m -Oxazolyl-CO(Aa) t (CR 17 R 18 ) n -, -(CR 15 R 16 ) m -Thiazolyl-CO(Aa) t (CCR 17 R 18 ) n -, -(CR 15 R 16 ) t -Tianil-CO(CR 17 R18 ) n -, -(CR 15 R 16 ) t -Imidazolyl-CO-(CR 17 R 18 ) n -, -(CR 15 R 16 ) t -Morpolino-CO(Aa) t- (CR 17 R 18 ) n -, -(CR 15 R 16 ) t -Piperazino-CO(Aa) t (CR 17 R 18 ) n -, -(CR 15 R 16 ) t -N-methylpiperazine-CO(Aa) t (CR 17 R 18 ) n -, -(CR 15 R 16 ) m -(Aa) t phenyl-, -(CR 15 R 16 ) m -(Aa) t Furil-, -(CR 15 R 16 ) m -Oxazolil(Aa) t -, -(CR 15 R 16 ) m -Thiazolyl(Aa) t -, -(CR 15 R 16 ) m -Tianil-(Aa) t -, -(CR 15 R 16 ) m -Imidazolil(Aa) t -, -(CR 15 R 16 ) m -Morpolino-(Aa) t -, -(CR 15 R16 ) m -피페라지노-(Aa) t -, -(CR 15 R 16 ) m -N-메틸피페라지노-(Aa) t -, -K(CR 15 R 16 ) m (Aa)r(CR 17 R 18 ) n (OCH2CH2) t -, -K(CR 15 R 16 ) m (CR 17 R 18 ) n (Aa) r (OCH2CH2) t -, -K(Aa) r -(CR 15 R 16 ) m (CR 17 R 18 ) n (OCH2CH2) t -, -K(CR 15 R 16 ) m (CR 17 R 18 ) n (OCH2CH2) r (Aa) t -, -K(CR 15 R 16 ) m (CR 17 =CR 18 )(CR 19 R 20 ) n (Aa) t (OCH2CH2) r , -K(CR 15 R 16 ) m (NR 11 CO)(Aa) t -(CR 19 R 20 ) n (OCH2CH2) r -, -K(CR 15 R 16 )m (Aa) t (NR 21 CO)(CR 19 R 20 ) n (OCH2CH2) r -, -K(CR 15 R 16 ) m (O-CO)(Aa) t (CR 19 R 20 ) n (OCH2CH2) r -, -K(CR 15 R 16 ) m (OCNR 17 )(Aa) t (CR 19 R 20 ) n (OCH2CH2) r -, -K(CR 15 R 16 ) m (CO)(Aa) t- (CR 19 R 20 ) n (OCH2CH2) r -, -K(CR 15 R 16 ) m (NR 21 CO)(Aa) t (CR 19 R 20 ) n -(OCH2CH2) r -, -K(CR 15 R 16 ) m- (OCO)(Aa) t (CR 19 R 20 ) n (OCH2CH2) r -, -K(CR 15 R 16 ) m (OCNR 17 )(Aa) t -(CR 19 R 20 ) n (OCH2CH2) r -, -K-(CR 15R 16 ) m (CO)(Aa) t (CR 19 R 20 ) n (OCH2CH2) r -, -K(CR 15 R 16 ) m -phenyl-CO(Aa) t (CR 17 R 18 ) n -, -K-(CR 15 R 16 ) m -Furyl-CO(Aa) t (CR 17 R 18 ) n -, -K(CR 15 R 16 ) m -Oxazolyl-CO(Aa) t (CR 17 R 18 ) n -, -K(CR 15 R 16 ) m -Thiazolyl-CO(Aa) t- (CR 17 R 18 ) n -, -K(CR 15 R 16 ) t -Tianil-CO(CR 17 R 18 ) n -, -K(CR 15 R 16 ) t Imidazolyl-CO-(CR 17 R 18 ) n -, -K(CR5R6) t Morpolino-CO(Aa) t -(CR 17 R 18 ) n -, -K(CR 15 R 16 ) t -Piperazino-CO(Aa) t- (CR 17 R 18 ) n-, -K(CR 15 R 16 ) t -N-methylpiperazine-CO(Aa) t (CR 17 R 18 ) n -, -K(CR 15 R 16 ) m -(Aa) t Phenyl, -K-(CR 15 R 16 ) m- (Aa) t Furyl-, -K(CR 15 R 16 ) m -Oxazolil-(Aa) t -, -K(CR 15 R 16 ) m -Thiazolyl(Aa) t -, -K(CR 15 R 16 ) m -Tianil-(Aa) t -, -K(CR 15 R 16 ) m -Imidazolil(Aa) t -, -K(CR 15 R 16 ) m -Morpolino(Aa) t -, -K(CR 15 R 16 ) m Piperazino (Aa) t G, -K(CR 15 R 16 ) m -N-methylpiperazino(Aa) t -(Effect, m, Aa, n, R 13 , R 14 and R 15 is described above; t and r are independently from 0 to 100 in this specification; R 16 , R 17 , R 18, R 19 and R 20is independently selected from H; halide; C1–C8 alkyl or heteroalkyl, C2–C8 aryl, alkenyl, alkynyl, ether, ester, amine or amide, and C3–C8 aryl, which is one or more halides, CN, NR 12 R 12' , CF3, OR 12 , aryl, heterocycle, S(O)R 12 , SO2R 12, -CO2H, -SO3H, -OR 12 , -CO2R 12 , -CONR 12 , -PO2R 12 R 13 , -PO3H or P(O)R 12 R 12' R 13 Selectively substituted by; K is NR 12 , -SS-, -C(=O)-, -C(=O)NH-, -C(=O)O-, -C=NH-O-, -C=N-NH-, -C(=O)NH-NH-, O, S, Se, B, Het(C3-C 12 A heterocyclic or heteroaromatic ring having; or a peptide containing 1 to 20 amino acids).

[0168] More preferably, W, L 1, L2V 1 및 V2's The component is independently a linear alkyl having 1 to 6 carbon atoms, or chemical formula (OCH2CH2) p It is a polyethyleneoxy unit having (p is 1 to 5000) or a peptide containing 1 to 4 units of amino acids (L or D type) or a combination of the above.

[0169] Alternatively, W, Q1, Q2, L1, L2, V1 or Any one or more of V2 may not exist independently, but Q1 and Q2 do not exist simultaneously.

[0170] Generally, in another aspect, where V1 and / or V2 are connected to the cell-binding molecule, T, or where L1 and / or L2 are directly connected to T (where V1 and V2 does not exist), the junction linkage may have one or more of the following structures:

[0171]

[0172]

[0173]

[0174] During the meal, R 20 and R 21 is independently C1–C8 alkyl; C2–C8 heteroalkyl or heterocyclic; C3–C8 aryl, Ar-alkyl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, carbonyl or alkylcarbonyl; or (CH2CH2O) p C2-C having the chemical formula (p is defined above) 100 It is polyethylene glycol; p is as defined above or does not exist.

[0175] In another additional aspect, Q1 and Q2 are preferably C2-C 18 Lipids or C2-C 18 fatty acids or C2-C 18It is selected from polyalkylene glycols containing fatty ammonium lipids. The polyalkylene glycols not only help make the conjugate more hydrophilic during preparation but also prevent the conjugate linker from being hydrolyzed by hydrolytic enzymes, e.g., proteinase or esterase. The lipids can help the conjugate bind to albumin in mammalian blood, and subsequently cause the conjugate to gradually dissociate from this complex during blood circulation. Thus, the side chain linker of the present application makes the conjugate more stable during circulation. Polyalkylene glycols herein include, but are not limited to, poly(ethylene glycol (PEG), poly(propylene glycol), and copolymers of ethylene oxide and propylene oxide; PEG is particularly preferred, and monofunctional activated hydroxyPEG (e.g., hydroxyl PEG activated at a single end, e.g., reactive ester of hydroxyPEG-monocarboxylic acid, hydroxyPEG-monoaldehyde, hydroxyPEG-monoamine, hydroxyPEG-monohydrazide, hydroxyPEG-monocarbazate, hydroxyl PEG-monoiodoacetamide, hydroxyl PEG-monomaleimide, hydroxyl PEG-monoorthopyridyl disulfide, hydroxyPEG-monooxime, hydroxyPEG-monophenyl carbonate, hydroxyl PEG-monophenyl glyoxal, hydroxyl PEG-monothiazolidene-2-thione, hydroxyl PEG-monothioester, hydroxyl PEG-monothiol, hydroxyl PEG-monotriazine, and hydroxyl PEG-monovinylsulfone) are more particularly preferred. The polyalkylene glycol has a molecular weight of about 10 daltons to about 200 kDa, preferably about 88 Da to about 40 kDa; the two branched chains each have a molecular weight of about 88 Da to about 40 kDa; more preferably, the two branches each have a molecular weight of about 88 Da to about 20 kDa. In a specific embodiment, the polyalkylene glycol is poly(ethylene) glycol and has a molecular weight of about 10 kDa; about 20 kDa; or about 40 kDa.In specific embodiments, PEG is PEG 10 kDa (linear or branched), PEG 20 kDa (linear or branched), or PEG 40 kDa (linear or branched). A number of U.S. patents, e.g., U.S. Patent No. 5,428,128; No. 5,621,039; No. 5,622,986; No. 5,643,575; No. 5,728,560; No. 5,730,990; No. 5,738,846; No. 5,811,076; No. 5,824,701; No. 5,840,900; No. 5,880,131; No. 5,900,402; No. 5,902,588; No. 5,919,455; Disclosed in Patent Nos. 5,951,974; 5,965,119; 5,965,566; 5,969,040; 5,981,709; 6,011,042; 6,042,822; 6,113,906; 6,127,355; 6,132,713; 6,177,087 and 6,180,095 are linear or branched "non-antigenic" PEG polymers and derivatives or conjugates thereof.

[0176] The cell-binding agent / cell-binding molecule, T, may be any currently known or known class of molecules that bind to, form a complex with, or react with a moiety of a cell population sought to be therapeutically or otherwise biologically modified. Preferably, the cell-binding agent / cell-binding molecule is an immunotherapy protein, antibody, single-strand antibody; antibody fragment binding to a target cell; monoclonal antibody; single-strand monoclonal antibody; or monoclonal antibody fragment binding to a target cell; chimeric antibody; antibody fragment binding to a chimeric target cell; domain antibody; antibody fragment binding to a domain target cell; antibody-mimicking adnectin; DARPin; lymphokine; hormone; vitamin; growth factor; colony-stimulating factor; or nutrient-transporting molecule (transferrin); It is a binding peptide having more than 4 amino acids, or a protein, or an antibody, or an albumin, polymer, dendrimer, liposome, nanoparticle, vesicle, or small cell-binding molecule or ligand attached to a (viral) capsid.

[0177] Examples of chemical formulas (I), (II), and (III) are those shown below or one or more isotopes of the chemical elements of these compounds, pharmaceutically acceptable salts, hydrates, or hydrated salts; or polymorphic crystal structures; or optical isomers, racemic mixtures, diastereomers, or enantiomers:

[0178]

[0179]

[0180]

[0181]

[0182]

[0183]

[0184]

[0185]

[0186]

[0187]

[0188]

[0189]

[0190]

[0191]

[0192]

[0193]

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203]

[0204]

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

[0212]

[0213]

[0214]

[0215]

[0216]

[0217]

[0218]

[0219]

[0220]

[0221]

[0222]

[0223]

[0224]

[0225]

[0226] Among the formulas, X8 is O, S, NH, NHNH, NHR 12 , SR 12 , SSR 12 , SSCH(CH3)R 12 , SSC(CH3)2R 12 , or R 12 It becomes; R 1 , R 2 , R 3 , R 4 , R 5 , R4, R5, R7, R8, R9, R 10 , X1, X2, X3, X4, X5, 6, Y1, Y2, Y3, Y5, R 12 , R 12' , R 13 , R13 ', R 25 , R 25 ', p1, p2, q1, q2, m, m1, n and mAb are described as above; Aa is a natural or non-natural amino acid; r is 0 to 12; (Aa)r is a peptide containing amino acids of the same or different sequences when r is greater than 2; r being 0 means that (Aa)r is not present.

[0227] In another aspect of the present invention, the side-chain linkage compound is represented by formulas (IV), (V), and (VI), which can be readily reacted with cell-binding molecule T or modified cell-binding molecule T to form conjugates of formulas (I), (II), and (III), respectively:

[0228]

[0229] In the formula, D, D1, D2, W, w, w', L1, L2, Q1, Q2, V1, V2, v1, v2, and n are defined as in the above chemical formula (I);

[0230] Lv1 and Lv2 are reactive groups capable of independently reacting with thiols, amines, carboxylic acids, selenos, phenols, or hydroxyl groups on cell-bound molecules. These reactive groups include halides (e.g., fluorides, chlorides, bromides, and iodides), methanesulfonyl (mesyl), toluenesulfonyl (tosyl), trifluoromethylsulfonyl (triplate), trifluoromethylsulfonate, nitrophenoxyl, N-succinimidyloxyl (NHS), phenoxyl; dinitrophenoxyl; Pentafluorophenoxyl, tetrafluorophenoxyl, trifluorophenoxyl, difluorophenoxyl, monofluorophenoxyl, pentachlorophenoxyl, 1H-imidazole-1-yl, chlorophenoxyl, dichlorophenoxyl, trichlorophenoxyl, tetrachlorophenoxyl, N-(benzotriazole-yl)oxyl, 2-ethyl-5-phenylisoxazolium-3'-sulfonyl, phenyloxadiazole-sulfonyl (-sulfon-ODA), 2-ethyl-5-phenylisoxazolium-yl, phenyloxadiazole-yl (ODA), oxadiazole-yl, unsaturated carbon (double or triple bond between carbon-carbon, carbon-nitrogen, carbon-sulfur, carbon-phosphorus, sulfur-nitrogen, phosphorus-nitrogen, oxygen-nitrogen, or carbon-oxygen), or intermediate molecules produced using condensation reagents for the Mitsunobu reaction, but with these It is not limited to. Examples of condensation reagents include EDC (N-(3-dimethyl-aminopropyl)-N'-ethylcarbodiimide), DCC (dicyclohexyl-carbodiimide), N,N'-diisopropyl-carbodiimide (DIC), N-cyclohexyl-N'-(2-morpholino-ethyl)carbodiimide metho-p-toluenesulfonate (CMC or CME-CDI), 1,1'-carbonyldiimidazole (CDI), TBTU (O-(benzotriazole-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate), N,N,N',N'-tetramethyl-O-(1H-benzotriazole-1-yl)-uronium hexafluorophosphate (HBTU), (Benzotriazole-1-yloxy)tris-(dimethylamino)-phosphonium hexafluorophosphate (BOP), (Benzotriazole-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP),Diethyl cyanophosphonate (DEPC), chloro-N,N,N',N'-tetramethylformamidinium hexafluorophosphate, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), 1-[(dimethylamino)(morpholino)methylene]-1H-[1,2,3]triazolo[4,5-b]pyridin-1-um 3-oxide hexafluorophosphate (HDMA), 2-chloro-1,3-dimethyl-imidazolidinium hexafluorophosphate (CIP), chlorotripyrrolidinophosphonium hexafluorophosphate (PyCloP), fluoro-N,N,N',N'-bis(tetramethylene)formamidinium Hexafluorophosphate (BTFFH), N,N,N',N'-tetramethyl-S-(1-oxido-2-pyridyl)thiuronium hexafluorophosphate, O-(2-oxo-1(2H)pyridyl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TPTU), S-(1-oxido-2-pyridyl)-N,N,N',N'-tetramethylthiuronium tetrafluoroborate, O-[(ethoxycarbonyl)-cyanomethyleneamino]-N,N,N',N'-tetramethyluronium hexafluorophosphate (HOTU), (1-cyano-2-ethoxy-2-oxoethylideneamino-oxy)dimethylamino-morpholino-carbenium hexafluorophosphate (COMU), O-(benzotriazole-1-yl)-N,N,N',N'-bis(tetramethylene)uronium hexafluorophosphate (HBPyU), N-benzyl-N'-cyclohexyl-carbodiimide (polymerized or unpolymerized), dipyrrolidino(N-succinimidyl-oxy)carbenium hexafluorophosphate (HSPyU), chlorodipyrrolidinocarbenium hexafluorophosphate (PyClU), 2-chloro-1,3-dimethylimidazolidinium tetrafluoroborate (CIB), (benzotriazole-1-yloxy)dipiferidino-carbenium hexafluorophosphate (HBPipU), O-(6-chlorobenzotriazole-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TCTU), Bromotris(dimethylamino)-phosphonium hexafluorophosphate (BroP),Propylphosphonic acid anhydride (PPACA, T3P, ® ), 2-Morrfolinoethyl isocyanide (MEI), N,N,N',N'-tetramethyl-O-(N-succinimidyl)uronium hexafluorophosphate (HSTU), 2-bromo-1-ethyl-pyridinium tetrafluoro-borate (BEP), O-[(ethoxycarbonyl)cyano-methyleneamino]-N,N,N',N'-tetra-methyluronium tetrafluoroborate (TOTU), 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmofolinium chloride (MMTM, DMTMM), N,N,N',N'-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (TSTU), O-(3,4-dihydro-4-oxo-1,2,3-benzotriazine-3-yl)-N,N,N',N'-tetramethyluronium tetrafluoro-borate (TDBTU), 1,1'-(azodicarbonyl)-dipipiridine (ADD), di-(4-chlorobenzyl)-azodicarboxylate (DCAD), di-tert-butyl azodicarboxylate (DBAD), diisopropyl azodicarboxylate (DIAD), and diethyl azodicarboxylate (DEAD). Additionally, Lv1 and Lv2 may be anhydrides formed by the acid itself or in combination with other C1-C8 acid anhydrides.

[0231] Preferably, Lv1 and Lv2 are independently halides (e.g., fluoride, chloride, bromide, and iodide), methanesulfonyl (mesyl), toluenesulfonyl (tosyl), trifluoromethylsulfonyl (triplate), trifluoromethylsulfonate, nitrophenoxyl, N-succinimidyloxyl (NHS), phenoxyl; dinitrophenoxyl; Pentafluorophenoxyl, tetrafluorophenoxyl, trifluorophenoxyl, difluorophenoxyl, monofluorophenoxyl, pentachlorophenoxyl, 1H-imidazole-1-yl, chlorophenoxyl, dichlorophenoxyl, trichlorophenoxyl, tetrachlorophenoxyl, N-(benzotriazole-yl)oxyl, 2-ethyl-5-phenylisoxazolium-3'-sulfonyl, phenyloxadiazole-sulfonyl (-sulfon-ODA), 2-ethyl-5-phenylisoxazolium-yl, phenyloxadiazole-yl (ODA), oxadiazole-yl, unsaturated carbon (double or triple bond between carbon-carbon, carbon-nitrogen, carbon-sulfur, carbon-phosphorus, sulfur-nitrogen, phosphorus-nitrogen, oxygen-nitrogen, or carbon-oxygen), or selected from one of the following structures:

[0232]

[0233]

[0234] In the formula, X1' is F, Cl, Br, I, or Lv3; X2' is O, NH, N(R1), or CH2; R3 is independently H, aromatic, heteroaromatic, or aromatic group, where one or more H atoms are independently replaced by -R1, -halogen, -OR1, -SR1, -NR1R2, -NO2, -S(O)R1, -S(O)2R1, or -COOR1; Lv3 is F, Cl, Br, I, nitrophenol; N-hydroxysuccinimide (NHS); phenol; dinitrophenol; pentafluorophenol; tetrafluorophenol; difluorophenol; monofluorophenol; pentachlorophenol; triplate; imidazole; dichlorophenol; tetrachlorophenol; 1-hydroxybenzotriazole; tosylate; mesylate; 2-ethyl-5-phenylisoxazolium-3'-sulfonate, an anhydride formed from itself or formed together with other anhydrides, e.g., acetyl anhydride, formyl anhydride; or a leaving group selected from an intermediate molecule produced as a condensation reagent for a peptide coupling reaction or a Mitsunobu reaction.

[0235] Examples of chemical formulas (IV), (V), and (VI) are the following compounds or one or more isotopes of the chemical elements of these compounds, pharmaceutically acceptable salts, hydrates, or hydrated salts; or polymorphic crystal structures; or optical isomers, racemic mixtures, diastereomers, or enantiomers:

[0236]

[0237]

[0238]

[0239]

[0240]

[0241]

[0242]

[0243]

[0244]

[0245]

[0246]

[0247]

[0248]

[0249]

[0250]

[0251]

[0252]

[0253]

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[0260]

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[0262]

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[0264]

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[0270]

[0271]

[0272]

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[0274] delete

[0275]

[0276] delete

[0277]

[0278] delete

[0279]

[0280]

[0281]

[0282]

[0283]

[0284] Among the formulas, X8 is O, S, NH, NHNH, NHR 12 , SR 12 , SSR 12 , SSCH(CH3)R 12 , SSC(CH3)2R 12 , or R 12 and; R 1 , R 2 , R 3 , R 4 , R 5 , R4, R5, R7, R8, R9, R 10 , X1, X2, X3, X4, X5, 6, Y1, Y2, Y3, Y5, R 12 , R 12' , R13 , R 13 ', R 25 , R 25 ', Z2, Z3, p. p1, p2, p3, q1, q 2, Lv 1, Lv2, Lv 3, Lv 3' , m, m1, n and mAb are described as above; Aa is a natural or non-natural amino acid; r is 0 to 12; (Aa)r is a peptide containing amino acids of the same or different sequences when r is greater than 2; r being 0 means that (Aa)r is not present.

[0285] Preferably, Lv 1, Lv 2, Lv3 and Lv 3' reacts with the thiol of the cell-bonding agent / cell-bonding molecule. The thiol is more preferably a pair of sulfur atoms reduced from the interchain disulfide bonds of the cell-bonding agent by a reducing agent selected from dithiothreitol (DTT), dithioerythritol (DTE), L-glutathione (GSH), tris(2-carboxyethyl)phosphine (TCEP), 2-mercaptoethylamine (β-MEA), and / or beta-mercaptoethanol (β-ME, 2-ME). The thiol of the cell-bonding agent / cell-bonding molecule may also be generated via Traut's reagent or thiolactone, wherein the Traut's reagent or thiolactone reacts with the amine of the cell-bonding agent / cell-bonding molecule to form a thiol, and then Lv 1, Lv 2, Lv3 or Lv 3' Reacts simultaneously or sequentially.

[0286]

[0287] The present invention further relates to a method for preparing cell-binding molecule-amatoxin analog conjugates of formulas (I), (II), and (III), as well as a method for applying conjugates of formulas (I), (II), and (III).

[0288] Preparation of conjugates of drug-to-cell binding molecules via side chain linkages

[0289] The synthesis route for preparing a conjugate of a cell-binding molecule versus an amatoxin analog of the present invention and for preparing a conjugate through a side chain linkage is illustrated in FIGS. 1 to 19 and in the example section.

[0290] Conjugates of formulas (I), (II), and (III) can be prepared through intermediate compounds of formulas (IV), (V), and (VI), respectively. Generally, compounds of formulas (IV), (V), and (VI) are synthesized to have maleimido, Lv1, and Lv2 functional groups that can be easily reacted with cell-binding molecules and modified cell-binding molecules. The synthesis of compounds of formulas (IV), (V), and (VI) and the preparation of some of formulas of formulas (I), (II), and (III) are illustrated in the structural formulas of FIGS. 1 to 19.

[0291] To synthesize a conjugate of formula (I), the functional group Lv1 on formula (IV) is generally reacted with one, two, or more residues of a cell-binding molecule in an aqueous medium at 0 to 60°C and pH 5 to 9 (with or without 0 to 30% water-miscible organic solvent, e.g., DMA, DMF, ethanol, methanol, acetone, acetonitrile, THF, isopropanol, dioxane, propylene glycol, or ethylenediol), and then purified by dialysis or chromatography to form a conjugate compound of formula (I). Some of the residues of the cell-binding molecule (reactors for conjugation) may be obtained through protein manipulation.

[0292] Conjugates of formulas (II) and (III) can also be obtained by forming conjugate molecules by reacting the functional groups Lv1 and Lv2 of the linkers of formulas (V) and (VI) with a pair of free thiols produced by the reduction of two or more residues of a cell-bonding molecule, preferably the disulfide bond of the cell-bonding molecule, in an aqueous medium at 0 to 60°C and pH 5 to 9 (with or without the addition of 0 to 30% water-miscible organic solvent). The thiol pair is a pair of preferred disulfide bonds reduced from the interchain disulfide bonds of the cell-bonding agent by a reducing agent selected from dithiothreitol (DTT), dithioerythritol (DTE), L-glutathione (GSH), tris(2-carboxyethyl)phosphine (TCEP), 2-mercaptoethylamine (β-MEA), and / or beta-mercaptoethanol (β-ME, 2-ME) in an aqueous medium of pH 4 to 9 (with or without the addition of 0 to 30% water-miscible (miscible) organic solvent).

[0293] Independently disulfide, thiol, thioester, maleimido, halo-substituted maleimido, haloacetyl, azide, 1-phosphorus (yne), ketone, aldehyde, alkoxyamino, triplate, carbonylimidazole, tosylate, mesylate, 2-ethyl-5-phenylisoxazolium-3'-sulfonate, or carboxylic acid ester of nitrophenol, N-hydroxysuccinimide (NHS), phenol; One, two, or more groups of the cell-binding molecule / agent may be reacted simultaneously or sequentially with one, two, or more groups of formulas (IV), (V), and (VI), which may be dinitrophenol, pentafluorophenol, tetrafluorophenol, difluorophenol, monofluorophenol, pentachlorophenol, dichlorophenol, tetrachlorophenol, 1-hydroxybenzotriazole, anhydride, or hydrazide group, or other acid ester derivative, in an aqueous medium at 0 to 60°C and pH 4 to 9.5 (with or without the addition of 0 to 30% water-miscible organic solvent), and after column purification or dialysis, conjugates of formulas (I), (I), and (III) may be produced. Thus, the reactive groups of Lv1 and Lv2 of formulas (IV), (V), and (VI) react with the cell-binding molecule modified in different ways. For example, a disulfide-containing linkage within a cell-bonding agent-amatoxin analog conjugate of formula (I) is achieved by disulfide exchange between Lv1 and Lv2 having a free thiol group and a disulfide bond within the modified cell-bonding agent, or by disulfide exchange between a free thiol group within the modified cell-bonding agent and a disulfide bond of Lv1 and / or Lv2. To facilitate the disulfide exchange reaction, the disulfide group is generally a group such as disulfanylpyridine, disulfanyl-nitropyridine, disulfanyl-nitrobenzene, disulfanyl-nitrobenzoic acid, or disulfanyl-dinitrobenzene.Thioether-linked linkages within conjugates of formulas (I), (II), and (III) are achieved, respectively, by the reaction of a modified cell-bonding agent or a maleimido or haloacetyl or ethylsulfonyl on a free thiol group on a modified cell-bonding agent or a compound of formulas (IV), (V), and (VI) with a compound of formulas (IV), (V), and (VI); Accordingly, a linkage containing the binding of an acid-unstable hydrazone within the conjugate can be achieved by the reaction of a hydrazide moiety on a cell-binding molecule or a drug of formulas (IV), (V), and (VI) modified by a method known in the art (e.g., see references [P. Hamann et al., Cancer Res. 53, 3336-34, 1993; B. Laguzza et al., J. Med. Chem., 32; 548-55, 1959; P. Trail et al., Cancer Res., 57; 100-5, 1997] with the carbonyl group of the drug or cell-binding molecule of formulas (IV), (V), and (VI); Therefore, linkages containing triazole bonds within the conjugate can be achieved through click chemistry (Huisgen cyclization addition reaction) (Lutz, JF. et al, 2008, Adv. Drug Del. Rev.60, 958-70; Sletten, EM et al 2011, AccChem. Research 44, 666-76)) by the reaction of the 1-in group of the drug or cell-binding molecule of formulas (IV), (V), and (VI) with the azido moiety on the opposite side. Linkages containing oxime bonds in oxime-linked conjugates are achieved by the reaction of the ketone or aldehyde group of the drug or cell-binding molecule of formulas (IV), (V), and (VI) with the oxylamine group on the opposite side, respectively. Thiol-containing cell-binding molecules are formed in an aqueous buffer at a pH of 5.5 to 9.A thioether linkage conjugate of formulas (I), (II), and (III) can be provided by reacting a drug molecule linker of formulas (IV), (V), and (VI) having a maleimido, or haloacetyl, or ethylsulfonyl substituent at 0. A thiol-containing cell-binding molecule can be disulfide-exchanged with a drug linker of formulas (IV), (V), and (VI) having a pyridyldithio moiety to provide a conjugate having a disulfide-binding linkage. A cell-binding molecule having a hydroxyl group or a thiol group can be reacted with a drug linker of formulas (IV), (V), and (VI) having an alpha halide of a halogen, particularly a carboxylate, in the presence of a weak base, for example, at pH 8.0 to 9.5 to provide a modified drug having an ether or thiol ether linkage. An ester linkage can be provided by condensing a hydroxyl or amino group on a cell-binding molecule with a crosslinking drug linker of formulas (IV), (V), and (VI) having a carboxyl group in the presence of a dehydrating agent, e.g., EDC or DCC. A conjugate can be provided through an amide linkage by condensing a cell-binding molecule containing an amino group with a group of NHS, imidazole, nitrophenol carboxyl ester; N-hydroxysuccinimide (NHS); phenol; dinitrophenol; pentafluorophenol; tetrafluorophenol; difluorophenol; monofluorophenol; pentachlorophenol; triplate; imidazole; dichlorophenol; tetrachlorophenol; 1-hydroxybenzotriazole; tosylate; mesylate; 2-ethyl-5-phenylisoxazolium-3'-sulfonate on a drug linker of formulas (IV), (V), and (VI).

[0294] Synthetic conjugates can be purified by standard biochemical means such as gel filtration, adsorption chromatography, and ion exchange or dialysis on Sephadex G25 or Sephacryl S300 columns. In some cases, small molecules such as cell-binding agents conjugated with small molecule drugs (e.g., folic acid, melanocyte-stimulating hormone, EGF, etc.) can be purified by chromatography, e.g., HPLC, high-pressure column chromatography, or ion exchange chromatography.

[0295] To achieve a higher yield of the conjugation reaction for a cell-binding molecule, preferably a pair of free thiols on an antibody, and formulas (I), (II), or (III), it may be required to add a low percentage of a water-miscible organic solvent or phase transfer agent to the reaction mixture. First, a crosslinking reagent (linker) of formulas (IV), (V), or (VI) can be dissolved in a polar organic solvent that is miscible with water, for example, different alcohols, such as methanol, ethanol and propanol, acetone, acetonitrile, tetrahydrofuran (THF), 1,4-dioxane, dimethylformamide (DMF), dimethylacetamide (DMA), or dimethyl sulfoxide (DMSO), at a high concentration, for example, 1 to 800 mM. Meanwhile, a cell-binding molecule, e.g., an antibody, dissolved at a concentration of 1 to 50 mg / ml in an aqueous buffer at a pH of 4.0 to 9.5, preferably 6.0 to 8.5, was treated with 0.5 to 20 equivalents of TCEP or DTT for 20 minutes to 48 hours. After reduction, DTT can be removed by SEC chromatography purification. TCEP may be selectively removed by SEC chromatography or may remain in the reaction mixture for the next step reaction without further purification, but preferably, TCEP is neutralized with an azide compound, e.g., 4-azidobenzoic acid, 4-(azidomethyl)benzoic acid, or azido-polyethylene glycolyl (e.g., 2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethanol). In addition, the reduction of antibodies or other cell-binding agents to TCEP can be carried out with a drug-linker molecule of the present chemical formula (IV), (V), or (VI), and cross-linking to the cell-binding molecule can be achieved simultaneously with the reduction of TCEP.

[0296] An aqueous solution for modifying the cell-binding agent is buffered to a pH of 4 to 9, preferably 6.0 to 8.0, and may contain any non-nucleophilic buffer salt useful in this pH range. Typical buffers include phosphate, acetate, triethanolamine HCl, HEPES, and MOPS buffers, which may contain additional components such as cyclodextrin, hydroxypropyl-β-cyclodextrin, polyethylene glycol, sucrose, and salts, e.g., NaCl and KCl. After adding a drug-linker of formula (IV), (V), or (VI) to a solution containing the reduced cell-binding molecule, the reaction mixture is incubated at a temperature of 0 to 50°C, preferably 15 to 37.5°C. The progress of the reaction can be monitored by measuring a decrease in absorption at a specific UV wavelength, e.g., 252 nm, or by measuring an increase in absorption at a specific UV wavelength, e.g., 280 nm, or other appropriate wavelengths. After the reaction is complete, the isolation of the modified cell-binding agent can be performed by conventional methods, for example, using gel filtration chromatography, ion exchange chromatography, adsorption chromatography, or column chromatography on silica gel or alumina, crystallization, thin film chromatography for purification, ion exchange chromatography, or HPLC.

[0297] The degree of modification can be evaluated by measuring the absorbance of nitropyridine thione, dinitropyridine dithione, pyridine thione, carboxylamidopyridine dithione, and dicarboxyl-amidopyridine dithione groups emitted via UV spectrum. In the case of conjugates without chromophore groups, modification or conjugation reactions can be monitored by LC-MS, preferably HPLC-MS / MS, UPLC-QTOF mass spectrometry, or capillary electrophoresis (CE-MS). The side-chain cross-linkers described herein have various functional groups capable of reacting with any cell-binding molecule, particularly modified cell-binding molecules having suitable substituents. For example, modified cell-binding molecules having amino or hydroxyl substituents can react with drugs having N-hydroxysuccinimide (NHS) esters, and modified cell-binding molecules having thiol substituents can react with drugs having maleimido or haloacetyl groups. Additionally, through protein manipulation, enzymatic reactions, or chemical modification, modified cell-binding molecules having carbonyl (ketone or aldehyde) substituents can react with drugs having hydrazides or alkoxyamines. Those skilled in the art can easily determine which modified drug-linker to use based on the known reactivity of available functional groups on the modified cell-binding molecules.

[0298] Cell-binding agent

[0299] The cell-binding molecule T or Cb or mAb comprising the conjugate and modified cell-binding agent of the present invention may be any currently known or known class of molecules that bind to, form a complex with, or react with a moiety of a cell population sought to be therapeutically or otherwise biologically modified.

[0300] Cell binding molecules / cell binding agents include high molecular weight proteins, e.g., antibodies, antibody-like proteins, full-length antibodies (polyclonal antibodies, monoclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), etc.); single-stranded antibodies; antibody fragments, e.g., Fab, Fab', F(ab')2, F v,[Parham, J. Immunol. 131, 2895-902 (1983)], fragments produced by Fab expression libraries, anti-idiotype (anti-Id) antibodies, CDRs, diabodies, tribodies, tetrabodies, miniantibodies, small immune proteins (SIPs), and epitope-binding fragments of any of the above that are immune-specifically bound to cancer cell antigens, viral antigens, microbial antigens, or proteins produced by the immune system capable of recognizing and binding to specific antigens or exhibiting desired biological activity (Miller et al. (2003) J.of Immunology 170: 4854-61); interferons (e.g., types I, II, III); peptides; lymphokines, e.g., IL-2, IL-3, IL-4, IL-5, IL-6, IL-6R, IL-10, IL-11, IL-16, IL-17, GM-CSF, interferon-gamma (IFN-γ); hormones, e.g., insulin, TRH (thyroid-releasing hormone), MSH (melanocyte-stimulating hormone), steroid hormones, e.g., androgens and estrogens, melanocyte-stimulating hormone (MSH); Growth factors and colony-stimulating factors, e.g., epidermal growth factor (EGF), granulocyte macrophage colony-stimulating factor (GM-CSF), transforming growth factor (TGF), e.g., TGFα, TGFβ, insulin and insulin-like growth factors (IGF-I, IGF-II), G-CSF, M-CSF and GM-CSF [Burgess, Immunology Today, 5, 155-8 (1984)]; vaccinia growth factor (VGF); fibroblast growth factor (FGF); smaller molecular weight proteins, polypeptides, peptides and peptide hormones, e.g., bombesin, gastrin, gastrin-releasing peptide; platelet-derived growth factors; interleukins and cytokines, e.g., interleukin-2 (IL-2), interleukin-6 (IL-6), leukemia inhibitors, granulocyte macrophage colony-stimulating factor (GM-CSF); Vitamins, e.g., folates; apoproteins and glycoproteins, e.g., transferrin [Reference [O'Keefe et al, 260 J. Biol. Chem. 932-7 (1985)]]; glyco-binding proteins or lipoproteins, e.g., lectins; cellular nutrient-transport molecules; and small molecule inhibitors, e.g., prostate-specific membrane antigen (PSMA) inhibitors and small molecule tyrosine kinase inhibitors (TKIs), non-peptides or any other cell-binding molecules or substances, e.g., bioactive polymers (Reference [Dhar, et al, Proc. Natl. Acad. Sci.2008, 105, 17356-61)]; fusion protein; kinase inhibitor; gene-targeting agent; bioactive dendrimer(Literature [Lee, et al, Nat. Biotechnol. 2005, 23, 1517-26; Almutairi, et al; Proc. Natl. Acad. Sci. 2009, 106, 685-90]); nanoparticle(Literature [Liong, et al, ACS Nano, 2008, 2, 1309-12; Medarova, et al, Nat. Med. 2007, 13, 372-7; Javier, et al, Bioconjugate Chem. 2008, 19, 1309-12)]; Liposomes (Medinai, et al, Curr. Phar. Des. 2004, 10, 2981-9]); viral capsids (Flenniken, et al, Viruses Nanotechnol. 2009, 327, 71-93]) are included, but are not limited to these.

[0301] Generally, monoclonal antibodies are preferred as cell-surface binders where appropriately available. And the antibodies may be derived from murine, human, humanized, chimera, or other species.

[0302] The production of antibodies used in the present invention includes in vivo or in vitro procedures, or a combination thereof. Methods for producing polyclonal anti-receptor peptide antibodies are widely known in the art, for example, in U.S. Patent No. 4,493,795 (Nestor et al.). Monoclonal antibodies are typically produced by fusing mouse splenocytes and myeloma cells immunized with the target antigen (Koehler, G.; Milstein, C. (1975). Nature 256: 495-7). Detailed procedures are described in the “Antibody—Laboratory Manual” (Harlow and Lane, eds., Cold Spring Harbor Laboratory Press, New York (1988)), which is incorporated herein by reference. In particular, monoclonal antibodies are produced by immunizing mice, rats, hamsters, or any other mammals with the target antigen of interest, such as intact target cells, antigens isolated from target cells, whole viruses, attenuated whole viruses, and viral proteins. Splenocytes are typically fused with myeloma cells using polyethylene glycol (PEG) 6000. The fused hybrid is selected based on its sensitivity to HAT (hypoxanthine-aminopterin-thymine). Hybridomas that produce monoclonal antibodies useful for carrying out the present invention are identified by their ability to respond immunely to specific receptors or inhibit receptor activity on target cells.

[0303] The monoclonal antibody used in the present invention can be prepared by initiating a monoclonal hybridoma culture comprising a nutrient medium containing a hybridoma that secretes antibody molecules of appropriate antigen specificity. The culture is maintained under conditions and for a sufficient time for the hybridoma to secrete antibody molecules into the medium. Subsequently, the antibody-containing medium is collected. Subsequently, antibody molecules may be further isolated by well-known techniques such as protein-A affinity chromatography; anion, cation, hydrophobic, or size exclusion chromatography (particularly affinity and sizing column chromatography for proteins and subsequent specific antigens); centrifugation, differential solubility, or other standard techniques for protein purification.

[0304] Media useful for the preparation of these compositions are widely known in the relevant art and are commercially available, and include synthetic culture media. An exemplary synthetic medium is Dulbecco's minimum essential medium (DMEM; literature [Dulbecco et al., Virol. 8, 396(1959)]), which is supplemented with 4.5 g / ℓ glucose, 0 to 20 mM glutamine, 0 to 20% fetal bovine serum, other heavy metals such as Cu, Mn, Fe, Zn, added in amounts of several ppm or in the form of salts, and antifoaming agents, such as polyoxyethylene-polyoxypropylene block copolymer.

[0305] Additionally, antibody-producing cell lines may be generated by techniques other than fusion, such as directly transforming B lymphocytes with oncogenic DNA or transfection with an oncovirus, e.g., Epstein-Barr virus (EBV, also called human herpes virus 4 (HHV-4)) or Kaposi sarcoma-associated herpes virus (KSHV) (see U.S. Patents No. 4,341,761; 4,399,121; 4,427,783; 4,444,887; 4,451,570; 4,466,917; 4,472,500; 4,491,632; 4,493,890). Monoclonal antibodies can also be produced via anti-receptor peptides or peptides containing carboxyl termins as described, which are widely known in the relevant art (see [Niman et al., Proc. Natl. Acad. Sci. USA, 80: 4949-53 (1983); Geysen et al., Proc. Natl. Acad. Sci. USA, 82: 178-82 (1985); Lei et al. Biochemistry 34(20): 6675-88, (1995)]). Typically, anti-receptor peptides or peptide analogs are used alone or conjugated to an immunogenic carrier as immunogens to produce anti-receptor peptide monoclonal antibodies.

[0306] In addition, there are numerous other widely known techniques for preparing monoclonal antibodies as binding molecules in the present invention. Methods for preparing fully human antibodies are particularly useful. One method is phage display technology, which can be used to select various human antibodies that specifically bind to an antigen using an affinity enhancement method. Phage display is fully described in the literature, and the construction and screening of phage display libraries are widely known in the relevant art field (see, for example, literature [Dente et al, Gene. 148(1):7-13 (1994); Little et al, Biotechnol Adv. 12(3): 539-55 (1994); Clackson et al., Nature 352: 264-8 (1991); Huse et al., Science 246: 1275-81 (1989)]).

[0307] Monoclonal antibodies derived from other non-human species, such as mice, via hybridoma technology can be humanized to evade human anti-mouse antibodies when injected into humans. More common methods of antibody humanization include complementarity determining region transplantation and resurfacing. Such methods have been extensively described (e.g., U.S. Patents No. 5,859,205 and 6,797,492; see Liu et al, Immunol Rev. 222: 9-27 (2008); Almagro et al, Front Biosci. 13: 1619-33 (2008); Lazar et al, mol Immunol. 44(8): 1986-98 (2007); Li et al, Proc. Natl. Acad. Sci. US A. 103(10): 3557-62 (2006)], each incorporated herein by reference). Fully human antibodies may also be produced by immunizing transgenic mice, rabbits, monkeys, or other mammals carrying a substantial portion of human immunoglobulin heavy and light chains as immunogens. Examples of such mice are the Xenomouse (Abgenix / Amgen), HuMAb-mouse (Medarex / BMS), and Velocimouse (Regeneron) (see, for example, U.S. Patents No. 6,596,541, 6,207,418, 6,150,584, 6,111,166, 6,075,181, 5,922,545, 5,661,016, 5,545,806, 5,436,149, and 5,569,825). In human therapy, the variable and human constant regions of murine can also be fused to constructs called "chimeric antibodies," which have much lower immunogenicity in humans than murine mAbs (References [Kipriyanov et al, mol Biotechnol. 26: 39-60 (2004); Houdebine, Curr Opin Biotechnol.13: 625-9 (2002)], each incorporated herein by reference). Additionally, site-directed mutagenesis in the variable region of an antibody can produce antibodies with higher affinity and specificity for the antigen (see [Brannigan et al, Nat Revmol Cell Biol. 3: 964-70, (2002)); Adams et al, J Immunol Methods. 231: 249-60 (1999)]), and exchange of the constant region of an mAb can improve the ability to mediate binding and cytotoxicity effector functions.

[0308] Immunospecific antibodies against malignant cell antigens may also be obtained commercially or manufactured by any method known to those skilled in the art, such as chemical synthesis or recombinant expression technology. The nucleotide sequence encoding the immunospecific antibody against malignant cell antigens may be obtained commercially, for example, from gene databases or similar databases, literature publications, or conventional cloning and sequencing.

[0309] Apart from antibodies, peptides or proteins that bind to, block, target, or interact in some other way with epitopes or corresponding receptors on targeted cells may be used as binding molecules. These peptides or proteins may be any peptide or protein having an affinity for the epitope or corresponding receptor and do not necessarily have to be of the immunoglobulin family. Such peptides may be isolated using techniques similar to those used for phage display antibodies (see [Szardenings, J Recept Signal transduct Res. 2003, 23(4): 307-49]). The use of peptides from such random peptide libraries may be similar to that of antibodies and antibody fragments. The binding molecules of the peptides or proteins may be conjugated or linked to large molecules or materials, such as albumin, polymers, liposomes, nanoparticles, and dendrimers, provided that such attachment allows the peptides or proteins to maintain their antibody-binding specificity.

[0310] Examples of antibodies used for drug conjugation via these prophylactic linkers to treat cancer, autoimmune diseases, and / or infectious diseases include 3F8 (anti-GD2), avagovomab (anti-CA-125), absiximab (anti-CD41 (integrin alpha-IIb), adalimumab (anti-TNF-α), adecatumumab (anti-EpCAM, CD326), apelimomab (anti-TNF-α), aputuzumab (anti-CD20), alacizumab pegol (anti-VEGFR2), ALT518 (anti-IL-6), alemtuzumab (Campath, MabCampath, anti-CD52), altomonab (anti-CEA), anatumomab (anti-TAG-72), anleukinzumab (IMA-638, anti-IL-13), apolizumab (anti-HLA-DR), and arcitumomab (anti-CEA). Aselizumab (anti-L-Selectin (CD62L), Atlizumab (Tocilizumab, Actemra, Roactemra, anti-IL-6 receptor), Atorolimab (anti-Resus factor), Bapineuzumab (anti-beta-amyloid), Basiliximab (Simulect, anti-CD25 (α chain of IL-2 receptor), Barbituximab (anti-phosphatidylserine), Vectumab (LymphoScan, anti-CD22), Belimumab (Benlysta, LymphoStat-B, anti-BAFF), Benralizumab (anti-CD125), Bertilimumab (anti-CCL11 (Eotaxin-1), Becilesomab (Scintimun, anti-CEA-associated antigen), Bevacizumab (Avastin, anti-VEGF-A), Visiromab (FibriScint, anti-fibrin II beta chain), Vivatuzumab (anti-CD44 v6), blinatumomab (BiTE, anti-CD19), brentuximab (cAC10, anti-CD30 TNFRSF8), briakinumab (anti-IL-12, IL-23), canakinumab (Ilaris, anti-IL-1), cantuzumab (C242, anti-CanAg), capromab, catumaxomab (Removab, anti-EpCAM, anti-CD3), CC49 (anti-TAG-72), cedelizumab (anti-CD4), sertolizumab pegol (Cimzia, anti-TNF-alpha), cetuximab (Erbitux, IMC-C225, anti-EGFR), citatuzumab vogatox (anti-EpCAM),Siksutumab (anti-IGF-1), clinoliximab (anti-CD4), clivartuzumab (anti-MUC1), conatumumab (anti-TRAIL-R2), CR6261 (anti-influenza and hemagglutinin), darsetuzumab (anti-CD40), daclizumab (Zenapax, anti-CD25 (α-chain of IL-2 receptor)), daratumumab (anti-CD38 (cyclic ADP ribose hydrolase)), denosumab (Prolia, anti-RANKL), detumomab (anti-B lymphoma cells), dorlimomab, dorlixizumab, ecromeximab (anti-GD3 ganglioside), eculizumab (Soliris, anti-C5), edovacomab (anti-endotoxin), edrecoromab (Panorex, MAb17-1A, anti-EpCAM), Epalizumab (Raptiva, anti-LFA-1 (CD11a), Efungumab (Mycograb, anti-Hsp90), Elotuzumab (anti-SLAMF7), Elcilimomab (anti-IL-6), Enlimomab Pegol (anti-ICAM-1 (CD54)), Epitumomab (anti-episialin), Efratuzumab (anti-CD22), Erlizumab (anti-ITGB2 (CD18)), Ertumaxomab (Rexomun, anti-HER2 / neu, CD3), Etalacizumab (Abegrin, anti-integrin αvβ3), Exvivirumab (anti-hepatitis B surface antigen), Panolesomab (NeutroSpec, anti-CD15), Paralimomab (anti-interferon receptor), Parletuzumab (anti-folic acid receptor 1), Felbizumab (anti-respiratory syncytial virus), pezakinumab (anti-IL-22), pizitumab (anti-IGF-1 receptor), pontorizumab (anti-IFN-γ), poavirumab (anti-rabies virus glycoprotein), presolimumab (anti-TGB-β), galiximab (anti-CD80), gantenerumab (anti-beta-amyloid), gavilimomab (anti-CD147(asigin)), gemtuzumab (anti-CD33), zilentuximab (anti-carbonic anhydrase 9), glembatumumab (CR011, anti-GPNMB), golimumab (Simponi, anti-TNF-α), gomiliximab (anti-CD23(IgE receptor)), ibalizumab (anti-CD4), ibritumomab (anti-CD20), igobomab (Indimacis-125, Anti-CA-125),Imilomab (Myoscint, anti-cardiac myosin), Infliximab (Remicade, anti-TNF-α), Intetumumab (anti-CD51), Inolimomab (anti-CD25 (α chain of IL-2 receptor), Inotuzumab (anti-CD22), Ipilimumab (anti-CD152), Iratumumab (anti-CD30 (TNFRSF8)), Keliximab (anti-CD4), Labetuzumab (CEA-Cide, anti-CEA), Lebrikizumab (anti-IL-13), Remalesomab (anti-NCA-90 (granulocyte antigen)), Lerdelimumab (anti-TGF beta 2), Lexatumumab (anti-TRAIL-R2), Livivirumab (anti-hepatitis B surface antigen), Lintuzumab (anti-CD33), Lucatumumab (anti-CD40), Lumiliximab (anti-CD23 (IgE receptor), Mapatumumab (anti-TRAIL-R1), Maslimomab (anti-T-cell receptor), Matuzumab (anti-EGFR), Mepolizumab (BosaTria, anti-IL-5), Metelimumab (anti-TGF beta 1), Milatuzumab (anti-CD74), Minletumomab (anti-TAG-72), Mitumomab (BEC-2, anti-GD3 ganglioside), Morolimumab (anti-Ressus factor), Matavizumab (Numax, anti-Respiratory Syncytial Virus), Muromonab-CD3 (Orthoclon OKT3, anti-CD3), Nacolomab (anti-C242), Naptumomab (anti-5T4), Natalizumab (Tysabri, anti-integrin α4), Nevacumab (anti-endotoxin), Necitumumab (anti-EGFR), Nerilimomab (anti-TNF-α), Nimotuzumab (Theracim, Theraloc, anti-EGFR), Nofetumomab, Ocrelizumab (anti-CD20), Odulimomab (Apolimomab, anti-LFA-1(CD11a)), Ofatumumab (Arzerra, anti-CD20), Olatumab (anti-PDGF-Rα), Omalizumab (Xolair, anti-IgE Fc region), Oportuzumab (anti-EpCAM), Oregovomab (OVaRex, anti-CA-125), Otelixizumab (anti-CD3), Paginabaximab (anti-lipoteicoic acid), Palibizumab (Synagis, Abbosynagis, anti-respiratory syncytial virus), Paninumunab (Vectibix, ABX-EGF, anti-EGFR),Panovacumab (anti-Pseudomonas areluginosa), fascolizumab (anti-IL-4), femtumomab (Theragyn, anti-MUC1), pertuzumab (Omnitarg, 2C4, anti-HER2 / neu), fecelizumab (anti-C5), fintumomab (anti-adenocarcinoma antigen), priliximab (anti-CD4), pitumumab (anti-vimentin), PRO 140 (anti-CCR5), lacotumomab (1E10, anti-(N-glycolylneuraminic acid (NeuGc, NGNA)-ganglioside GM3)), rapivirumab (anti-rabies virus glycoprotein), ramucirumab (anti-VEGFR2), ranibizumab (Lucentis, anti-VEGF-A), laxivacumab (anti-anthrax toxin, protective antigen), Legavirumab (anti-cytomegalovirus glycoprotein B), reslizumab (anti-IL-5), rirotumumab (anti-HGF), rituximab (MabThera, rituxanab, anti-CD20), lovatumumab (anti-IGF-1 receptor), lontalizumab (anti-IFN-α), lovelizumab (LeukArrest, anti-CD11, Cd18), luplizumab (Antova, anti-CD154(CD40L)), satumomab (anti-TAG-72), cevirumab (anti-cytomegalovirus), cibrotuzumab (anti-FAP), cipalimumab (anti-IFN-α), siltuximab (anti-IL-6), siplizumab (anti-CD2), (Smart)MI95 (anti-CD33), solanezumab (anti-beta-amyloid), Sonepsizumab (anti-spingosin-1-phosphate), Sontuzumab (anti-episialin), Stamulumab (anti-myostatin), Sulesomab (LeukoScan, (anti-NCA-90 (granulocyte antigen), Tacatuzumab (anti-alpha-fetoprotein), Tadocizumab (anti-integrin αIIbβ3), Talizumab (anti-IgE), Tanezumab (anti-NGF), Tapritumomab (anti-CD19), Tepivazumab (Aurexis, (anti-clumping factor A), Telimomab, Tenatumomab (anti-tenacin C), Teneliximab (anti-CD40), Teflizumab (anti-CD3), TGN1412 (anti-CD28), Tisilimumab (Tremelimumab, (anti-CRLA-4), Tigatuzumab (anti-TRAIL-R2), TNX-650 (anti-IL-13), tocilizumab (atlizumab, Actemra, Roactemra,(anti-IL-6 receptor), toralizumab (anti-CD154 (CD40L)), tocitumomab (anti-CD20), trastuzumab (Herceptin, (anti-HER2 / neu), tremelimumab (anti-CTLA-4), tucotuzumab selmoleukin (anti-EpCAM), tuvirumab (anti-Hepatitis B virus), urtoxazumab (Escherichia coli), ustekinumab (Stelara, anti-IL-12, IL-23), bapaliximab (anti-AOC3 (VAP-1)), vedolizumab, (anti-integrin α4β7), veltuzumab (anti-CD20), bepalimomab (anti-AOC3 (VAP-1)), bicilizumab (Nuvion, anti-CD3), vitaxin (anti-vascular integrin avb3), Voloxiximab (anti-integrin α5β1), Botumumab (HumaSPECT, anti-tumor antigen CTAA16.88), Zalutumumab (HuMax-EGFr, anti-EGFR), Janolimumab (HuMax_CD4, anti-CD4), Ziralimumab (anti-CD147 (basigin)), Zolimab (anti-CD5), Etanercept (Enbrel®), Alefacept (Amevive®), Batacept (Orencia®), Rilonacept (Arcalyst), 14F7 [anti-IRP-2 (iron protein 2)], 14G2a (anti-GD2 ganglioside, from the National Cancer Institute for melanoma and solid tumors), J591 (anti-PSMA, from Weill Cornell Medical School for prostate cancer), 225.28S [anti-HMW-MAA (high molecular weight melanoma-associated antigen), for melanoma, Sorin Radiofarmaci SRL (Milan, Italy)], COL-1 (anti-CEACAM3, CGM1, for colorectal and gastric cancer, National Cancer Institute), CYT-356 (Oncoltad®, for prostate cancer), HNK20 (for respiratory syncytial virus, OraVax Inc.)), ImmuRAIT (for NHL, from Immunomedics), Lym-1 (anti-HLA-DR10, for cancer, Peregrine Pharm.)),MAK-195F (anti-TNF (tumor necrosis factor; TNFA, TNF-alpha: TNFSF2), from Abbott / Knoll for septic toxic shock), MEDI-500 [T10B9, anti-CD3, TRαβ (T cell receptor alpha / beta), complex [from MedImmune Inc. for graft-versus-host disease], Circular SCAN [anti-TAG 72 (tumor-associated glycoprotein 72), from Neoprobe Corp. for breast, colorectal, and rectal cancer], Avicidin (anti-EPCAM) (epithelial cell adhesion molecule), anti-TACSTD1 (tumor-associated calcium signal transducer 1), anti-GA733-2 (gastrointestinal tumor-associated protein 2), anti-EGP-2 (epithelial glycoprotein 2); anti-KSA; KS1 / 4 antigen; M4S; Tumor antigen 17-1A; CD326 [from NeoRx Corp. for colorectal, ovarian, prostate, and NHL]; Lymposide (Immunomedics, NJ, USA), SMART ID10 (Protein Design Lab), Oncorim (Techniclone Inc., California, USA), Allomun (BioTransplant, California, USA), Anti-VEGF (Genentech, California, USA); CEAcide (Immunomedics, NJ, USA), IMC-1C11 (ImClone, NJ, USA), and Cetuximab (ImClone, NJ, USA), including but not limited to these.

[0311] Other antibodies acting as cell-binding molecules / ligands are antibodies against the following antigens: Aminopeptidase N (CD13), Annexin A1, B7-H3 (CD276, various cancers), CA125 (ovary), CA15-3 (carcinoma), CA19-9 (carcinoma), L6 (carcinoma), Lewis Y (carcinoma), Lewis X (carcinoma), Alpha-fetoprotein (carcinoma), CA242 (colorectal), Placental alkaline phosphatase (carcinoma), Prostate-specific antigen (prostate), Prostatic acid phosphatase (prostate), Epidermal Growth Factor (carcinoma), CD2 (Hodgkin's disease, NHL lymphoma, multiple myeloma), CD3 (epsilon T-cell lymphoma, lung cancer, breast cancer, gastric cancer, ovarian cancer, autoimmune diseases, malignant ascites), CD19 (B-cell malignancy), CD20 (non-Hodgkin lymphoma), CD22 (leukemia, lymphoma, multiple myeloma, SLE), CD30 (Hodgkin lymphoma), CD33 (leukemia, autoimmune diseases), CD38 (multiple myeloma), CD40 (lymphoma, multiple myeloma, leukemia (CLL)), CD51 (metastatic melanoma, sarcoma), CD52 (leukemia), CD56 (small cell lung cancer, ovarian cancer, Markel cell carcinoma and liquid tumors, multiple myeloma), CD66e (cancer), CD70 (metastatic renal cell carcinoma and non-Hodgkin lymphoma), CD74 (multiple myeloma), CD80 (lymphoma), CD98 (cancer), mucin (carcinoma), CD221 (solid tumor), CD227 (breast cancer, ovarian cancer), CD262 (NSCLC and other cancers), CD309 (ovarian cancer), CD326 (solid tumor), CEACAM3 (colorectal, gastric cancer), CEACAM5 (carcinoembryonic antigen; CEA, CD66e)(breast cancer, colorectal cancer, and lung cancer), DLL3 (Delta-like-3), DLL4 (Delta-like-4), EGFR (Epidermal Growth Factor Receptor, various cancers), CTLA4 (melanoma), CXCR4 (CD184, Heme-oncology, solid tumors), Endoglin (CD105, solid tumors), EPCAM (epidermal cell adhesion molecule, bladder cancer, head cancer, cervical cancer, colon cancer, NHL prostate cancer, and ovarian cancer), ERBB2 (Epidermal Growth Factor Receptor 2; lung cancer, breast cancer,Prostate cancer), FCGR1 (autoimmune diseases), FOLR (folic acid receptor, ovarian cancer), GD2 ganglioside (cancer), G-28 (cell surface antigen glycolipid, melanoma), GD3 idiotype (cancer), heat shock protein (cancer), HER1 (lung cancer, gastric cancer), HER2 (breast, lung, and ovarian cancer), HLA-DR10 (NHL), HLA-DRB (NHL, B-cell leukemia), human chorionic gonadotropin (carcinoma), IGF1R (insulin-like growth factor 1 receptor, solid tumors, blood cancer), IL-2 receptor (interleukin 2 receptor, T-cell leukemia and lymphoma), IL-6R (interleukin 6 receptor, multiple myeloma, RA, Castleman disease, IL6-dependent tumors), integrins (αvβ3, α5β1, α6β4, αllβ3 in the case of various cancers, α5β5, αvβ5), MAGE-1 (carcinoma), MAGE-2 (carcinoma), MAGE-3 (carcinoma), MAGE 4 (carcinoma), anti-transferrin receptor (carcinoma), p97 (melanoma), MS4A1 (membrane-spanning 4-domain subfamily and member 1, non-Hodgkin B-cell lymphoma, leukemia), MUC1 or MUC1-KLH (breast cancer, ovarian cancer, cervical cancer, bronchial cancer and gastrointestinal cancer), MUC16(CA125) (ovarian cancer), CEA (colorectal), gp100 (melanoma), MART1 (melanoma), MPG (melanoma), MS4A1 (membrane-spanning 4-domain subfamily A, small cell lung cancer, NHL), nucleolin, Neu oncogene product (carcinoma), P21 (carcinoma), anti-(N-glycolylneuraminic acid) Paratop (breast cancer, melanoma), PLAP-like testicular alkaline phosphatase (ovarian cancer, testicular cancer), PSMA (prostate tumor), PSA (prostate), ROBO4, TAG 72 (tumor-associated glycoprotein 72, AML, gastric cancer, colorectal cancer, ovarian cancer), T cell transmembrane protein (cancer), Tie (CD202b), TNFRSF10B (tumor necrosis factor receptor superfamily member 10B, cancer), TNFRSF13B (tumor necrosis factor receptor superfamily member 13B, multiple myeloma, NHL, other cancers, RA and SLE),Includes, but is not limited to, TPBG (trophoblastic glycoprotein, renal cell carcinoma), TRAIL-R1 (tumor necrosis apoptosis-inducing ligand receptor 1, lymphoma, NHL, colorectal cancer, lung cancer), VCAM-1 (CD106, melanoma), VEGF, VEGF-A, and VEGF-2 (CD309) (various cancers). Some other tumor-associated antigens recognized by antibodies have been reviewed in the literature [Gerber, et al, mAbs 1:3, 247-53 (2009); Novellino et al, Cancer Immunol Immunother. 54(3), 187-207 (2005); Franke, et al, Cancer Biother Radiopharm. 2000, 15, 459-76].

[0312] A more preferred antibody that is a cell-binding agent may be any agent capable of functioning against tumor cells, virus-infected cells, microbial-infected cells, parasitic-infected cells, autoimmune cells, activated cells, bone marrow cells, activated T-cells, B cells, or melanocytes. More specifically, the cell binder may be any agonist / molecule capable of functioning for any one of the following antigens or receptors: CD1, CD1a, CD1b, CD1c, CD1d, CD1e, CD2, CD3, CD3d, CD3e, CD3g, CD4, CD5, CD6, CD7, CD8, CD8a, CD8b, CD9, CD10, CD11a, CD11b, CD11c, CD11d, CD12w, CD14, CD15, CD16, CD16a, CD16b, CDw17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD32a, CD32b, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42, CD42a, CD42b, CD42c, CD42d, CD43, CD44, CD45, CD46, CD47, CD48, CD49b, CD49c, CD49c, CD49d, CD49f, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CD60, CD60a, CD60b, CD60c, CD61, CD62E, CD62L, CD62P, CD63, CD64, CD65, CD65s, CD66, CD66a, CD66b, CD66c, CD66d, CD66e, CD66f, CD67, CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD75, CD75s, CD76, CD77, CD78, CD79, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CD85, CD85a, CD85b, CD85c, CD85d, CD85e, CD85f, CD85g, CD85g, CD85i, CD85j,CD85k, CD85m, CD86,CD87,CD88,CD89,CD90,CD91,CD92, CD93, CD94, CD95,CD96,CD97, CD98, CD99, CD100,CD101, CD102, CD103, CD104,CD105,CD106,CD107, CD107a, CD107b, CD108, CD109,CD110, CD111,CD112, CD113, CD114, CD115,CD116,CD117, CD118, CD119, CD120,CD120a, CD120b, CD121,CD121a, CD121b, CD122,CD123, CD123a,CD124, CD125, CD126,CD127, CD128, CD129, CD130, CD131,CD132, CD133,CD134,CD135,CD136, CD137, CD138, CD139,CD140,CD140a, CD140b, CD141,CD142,CD143,CD144,CD145,CDw145, CD146, CD147,CD148,CD149, CD150,CD151,CD152,CD153, CD154,CD155,CD156,CD156a, CD156b, CD156c, CD156d, CD157, CD158,CD158a,CD158b1,CD158b2,CD158c,CD158d,CD158e1,CD158e2,CD158f2,CD158g,CD158h,CD158i,CD158j,CD158k,CD159,CD159a,CD159b,CD159c,CD160, CD161,CD162, CD163,CD164,CD165, CD166,CD167, CD167a, CD167b, CD168,CD169,CD170,CD171,CD172, CD172a, CD172b, CD172g, CD173,CD174,CD175, CD175s, CD176,CD177, CD178,CD179,CD179a,CD179b,CD180,CD181,CD182, CD183,CD184,CD185, CD186,CDw186,CD187, CD188,CD189,CD190,CD191,CD192,CD193,CD194,CD195, CD196,CD197, CD198,CD199,CDw198,CDw199,CD200,CD201,CD202, CD202(a,b),CD203,CD203c,CD204,CD205, CD206,CD207, CD208,CD209,CD210,CDw210a,CDw210b,CD211,CD212, CD213,CD213a1,CD213a2,CD214,CD215, CD216,CD217, CD218,CD218a,CD218,CD219b,CD220,CD221,CD222, CD223,CD224,CD225, CD226,CD227, CD228,CD229,CD230,CD231,CD232, CD233,CD234,CD235, CD235a,CD235b,CD236,CD237, CD238,CD239,CD240,CD240ce,CD240d,CD241,CD242, CD243,CD244,CD245, CD246,CD247, CD248,CD249,CD250,CD251,CD252, CD253,CD254,CD255, CD256,CD257, CD258,CD259,CD260,CD261,CD262, CD263,CD264,CD265, CD266,CD267, CD268,CD269,CD270,CD271,CD272, CD273,CD274,CD275, CD276,CD277, CD278,CD279,CD281,CD282, CD283,CD284,CD285, CD286,CD287, CD288,CD289,CD290,CD291,CD292, CD293,CD294,CD295, CD296,CD297, CD298,CD299,CD300,CD300a,CD300b,CD300c,CD301,CD302, CD303,CD304,CD305, CD306,CD307, CD307a, CD307b, CD307c, CD307d, CD307e, CD307f, CD308,CD309,CD310,CD311,CD312, CD313,CD314,CD315, CD316,CD317, CD318,CD319,CD320,CD321,CD322,CD323, CD324, CD325, CD326, CD327, CD328, CD329, CD330, CD331, CD332, CD333, CD334, CD335, CD336, CD337, CD338, CD339, CD340, CD341, CD342, CD343, CD344, CD345, CD346, CD347, CD348, CD349, CD350, CD351, CD352, CD353, CD354, CD355, CD356, CD357, CD358, CD359, CD360, CD361, CD362, CD363, CD364, CD365, CD366, CD367, CD368, CD369, CD370, CD371, CD372, CD373, CD374, CD375, CD376, CD377, CD378, CD379, CD381, CD382, CD383, CD384, CD385, CD386, CD387, CD388, CD389, CRIPTO, CR, CR1, CRGF, CRIPTO, CXCR5, LY64, TDGF1, 4-1BB, APO2, ASLG659, BMPR1B, 4-1BB, 5AC, 5T4 (trophoblast glycoprotein, TPBG, 5T4, Wnt-activated inhibitor 1 or WAIF1), adenocarcinogen, AGS-5, AGS-22M6, activin receptor-like kinase 1, AFP, AKAP-4, ALK, Alpha Integrin, Alpha v Beta 6, Amino-Peptidase N, Amyloid Beta, Androgen Receptor, Angiopoietin 2, Angiopoietin 3, Annexin A1, Bacillus anthracis toxin-protective antigen, Anti-transferrin Receptor, AOC3 (VAP-1), B7-H3, Bacillus anthracis, BAFF (B-cell activating factor), B-lymphoma cell, bcr-abl, Bombesin, BORIS, C5, C242 antigen, CA125 (Carbohydrate Antigen 125, MUC16), CA-IX (or CAIX, Carbonic Anhydrase 9), CALLA, CanAg, Canis lupus familiaris IL31, Carbonic Anhydrase IX, Cardiac Myosin, CCL11 (CC Motif Chemokine 11),CCR4 (CC Chemokine Receptor Type 4, CD194), CCR5, CD3E (Epsilon), CEA (Carcinoembryonic Antigen), CEACAM3, CEACAM5 (Carcinoembryonic Antigen), CFD (Factor D), Ch4D5, Cholecystokinin 2 (CCK2R), CLDN18 (Claudin-18), Clumping Factor A, CRIPTO, FCSF1R (Colony Stimulating Factor 1 Receptor, CD115), CSF2 (Colony Stimulating Factor 2, Granulocyte Macrophage Colony Stimulating Factor (GM-CSF)), CTLA4 (Cytotoxic T-Lymphocyte Associated Protein 4), CTAA16.88 Tumor Antigen, CXCR4 (CD184), CXC Chemokine Receptor Type 4, Cyclonal ADP Ribose Hydrolyzer, Cyclin B1, CYP1B1, Cytomegalovirus, Cytomegalovirus Glycoprotein B, Dabigatran, DLL3 (delta-like ligand 3), DLL4 (delta-like ligand 4), DPP4 (dipeptidyl peptidase 4), DR5 (apoptosis receptor 5), E. E. coli Shiga toxin-1, E. E. coli Shiga Toxin Type-2, ED-B, EGFL7 (EGF-like Domain-Containing Protein 7), EGFR, EGFRII, EGFRvIII, Endoglin (CD105), Endothelin B Receptor, Endotoxin, EpCAM (Epidermal Adhesion Molecule), EphA2, Episialin, ERBB2 (Epidermal Growth Factor Receptor 2), ERBB3, ERG (TMPRSS2 ETS Fusion Gene), E. coli Escherichia, ETV6-AML, FAP (Fibroblast Activating Protein Alpha), FCGR1, Alpha-Fetoprotein, Fibrin II, Beta Chain, Fibronectin Extra Domain-B, FOLR (Folate Receptor), Folate Receptor Alpha, Folate Hydrolyzase, Phos-Related Antigen 1, F Protein of Respiratory Syncytial Virus, Frizzled Receptor, Fucosyl GM1, GD2 Ganglioside, G-28 (Cell Surface Antigen Glycolipid), GD3 Idiotype, GloboH, Glypican 3, N-glycolylneuraminic acid, GM3, GMCSF receptor α-chain, Growth Differentiation Factor 8, GP100, GPNMB (Transmembrane Glycoprotein NMB),GUCY2C (Guanylate Cyclistase 2C, Guanylyl Cyclistase C (GC-C), Intestinal Guanylate Cyclistase, Guanylate Cyclistase-C Receptor, Heat-Stable Enterotoxin Receptor (hSTAR)), Heat Shock Protein, Hemagglutinin, Hepatitis B Surface Antigen, Hepatitis B Virus, HER1 (Human Epidermal Growth Factor Receptor 1), HER2, HER2 / neu, HER3 (ERBB-3), IgG4, HGF / SF (Hepatocyte Growth Factor / Scattering Factor), HHGFR, HIV-1, Histone Complex, HLA-DR (Human Leukocyte Antigen), HLA-DR10, HLA-DRB, HMWMAA, Human Chorionic Gonadotropin, HNGF, Human Scattering Factor Receptor Kinase, HPV E6 / E7, Hsp90, hTERT, ICAM-1 (Intercellular Adhesion Molecule 1), Idiotype, IGF1R (IGF-1, Insulin-like Growth Factor 1 Receptor), IGHE, IFN-γ, Influenza Hemagglutinin, IgE, Fc domain, IGHE, IL-1, IL-2 Receptor (Interleukin 2 Receptor), IL-4, IL-5, IL-6, IL-6R (Interleukin 6 Receptor), IL-9, IL-10, IL-12, IL-13, IL-16, IL-17, IL-17A, IL-20, IL-22, IL-23, IL31RA, ILGF2 (Insulin-like Growth Factor 2), Integrin (α4, αIIbβ3, αvβ3, α4β7, α5β1, α6β4, α7β7, αllβ3, α5β5, αvβ5), Interferon Gamma-derived Protein, ITGA2, ITGB2, KIR2D, LCK, Le, Legumine, Lewis-Y Antigen, LFA-1 (Lymphocyte Function-Associated Antigen 1, CD11a), LHRH, LINGO-1, Lipoteichoic Acid, LIV1A, LMP2, LTA, MAD-CT-1, MAD-CT-2, MAGE-1, MAGE-2, MAGE-3, MAGE A1, MAGE A3, MAGE 4, MART1, MCP-1, MIF (Macromacillary Migration Inhibitor, or Glycosylation-Inhibitor (GIF)),MS4A1 (Membrane-spanning 4-domain subfamily A member 1), MSLN (Mesothelin), MUC1 (Mucin 1, Cell Surface-associated (MUC1) or Polymorphic Epithelial Mucin (PEM)), MUC1-KLH, MUC16 (CA125), MCP1 (Monocyte Chemotactic Protein 1), MelanA / MART1, ML-IAP, MPG, MS4A1 (Membrane-spanning 4-domain subfamily A), MYCN, Myelin-associated glycoprotein, Myostatin, NA17, NARP-1, NCA-90 (Granulocyte Antigen), Nectin-4 (ASG-22ME), NGF, Neuronal Apoptosis-regulating Proteinase 1, NOGO-A, Notch receptor, Nucleolin, Neu oncogene product, NY-BR-1, NY-ESO-1, OX-40, OxLDL (oxidized low-density lipoprotein), OY-TES1, P21, p53 non-mutant, P97, Page4, PAP, paratop of anti-(N-glycolylneuraminic acid), PAX3, PAX5, PCSK9, PDCD1 (PD-1, programmed cell death protein 1, CD279), PDGF-Rα (alpha-type platelet-derived growth factor receptor), PDGFR-β, PDL-1, PLAC1, PLAP-like testicular alkaline phosphatase, platelet-derived growth factor receptor beta, phosphate-sodium co-receptor, PMEL 17, polysialic acid, proteinase 3 (PR1), prostate carcinoma, PS (phosphatidylserine), prostate carcinoma cell, Pseudomonas aeruginosa, PSMA, PSA, PSCA, rabies virus glycoprotein, RHD (Rh polypeptide 1 (RhPI), CD240), rhesus factor, RANKL, RhoC, Ras mutant, RGS5, ROBO4, respiratory syncytial virus, RON, sarcoma translocation breakpoint, SART3, sclerostin, SLAMF7 (SLAM family member 7), selectin P, SDC1 (syndecan 1), sLe(a), somatomedin C, SIP (sphingosine-1-phosphate), somatostatin, sperm protein 17, SSX2, STEAP1 (prostate-1 6-transmembrane epithelial antigen),STEAP2, STn, TAG-72 (Tumor-associated glycoprotein 72), Survivin, T-cell receptor, T-cell transmembrane protein, TEM1 (Tumor endothelial marker 1), TENB2, Tenacin C (TN-C), TGF-α, TGF-β (Transforming growth factor beta), TGF-β1, TGF-β2 (Transforming growth factor-beta 2), TIE (CD202b), TIE2, TIM-1 (CDX-014), Tn, TNF, TNF-α, TNFRSF8, TNFRSF10B (Tumor necrosis factor receptor superfamily member 10B), TNFRSF13B (Tumor necrosis factor receptor superfamily member 13B), TPBG (Trophageal sublayer glycoprotein), TRAIL-R1 (Tumor necrosis apoptosis-inducing ligand receptor 1), TRAILR2 (Apoptosis receptor 5 (DR5)), Cells expressing tumor-associated calcium signal transducer 2, tumor-specific glycosylation of MUC1, TWEAK receptor, TYRP1 (glycoprotein 75), TROP-2, TRP-2, tyrosinase, VCAM-1 (CD106), VEGF, VEGF-A, VEGF-2 (CD309), VEGFR-1, VEGFR2, or vimentin, WT1, XAGE 1, or any insulin growth factor receptor or any epidermal growth factor receptor.

[0313] In another specific embodiment, the cell-binding molecule may be a ligand or receptor agonist selected from the following: folate derivatives (binding to folate receptors, proteins overexpressed in ovarian cancer and other malignancies) (Reference [Low, PS et al 2008, Acc. Chem. Res. 41, 120-9]); glutamate urea derivatives (binding to prostate-specific membrane antigens, surface markers of prostate cancer cells) (Reference [Hillier, SM et al, 2009, Cancer Res. 69, 6932-40]); Somatostatin (also known as growth hormone-inhibiting hormone (GHIH) or somatotropin-releasing-inhibiting factor (SRIF)) or somatotropin-releasing-inhibiting hormone) and analogs thereof, e.g., octreotide (Sandostatin) and lanreotide (Somatuline) (particularly, neuroendocrine tumors, GH-producing pituitary adenomas, paragangliomas, non-functional pituitary adenomas, pheochromocytomas) (Reference [Ginj, M., et al, 2006, Proc. Natl. Acad. Sci. USA 103, 16436-41]); Somatostatin receptor subtypes (sst1, sst2, sst3, sst4, and sst5) in GH-secreting pituitary adenomas (Reference [Reubi JC, Landolt, AM 1984 J. Clin. Endocrinol Metab 59: 1148-51; Reubi JC, Landolt AM 1987 J Clin Endocrinol Metab 65: 65-73; Moyse E, et al, J Clin Endocrinol Metab 61: 98-103]), gastrointestinal pancreatic tumors (Reference [Reubi JC, et al, 1987 J Clin Endocrinol Metab 65: 1127-34; Reubi, J.C, et al, 1990 Cancer Res 50: 5969-77]), pheochromocytoma (Literature [Epel-baum J, et al 1995 J Clin Endocrinol Metab 80:1837-44; Reubi JC, et al, 1992 J Clin Endocrinol Metab 74: 1082-9]), neuroblastoma (Literature [Prevostg, 1996 Neuroendocrinology 63:188-197; Moertel, C. L, et al 1994 Am J Clin Path 102:752-756]), medullary thyroid carcinoma (Literature [Reubi, J. C, et al 1991 Lab Invest 64:567-573]), small cell lung cancer (Literature [Sagman U, et al, 1990 Cancer 66:2129-2133]), meningioma, medulloblastoma or glioma (Revisi JC, et al 1986 J Clin Endocrinol Metab 63: 433-8; Reubi JC, et al 1987 Cancer Res 47: 5758-64; Fruhwald, M. C, et al 1999 Pediatr Res 45: 697-708]), breast carcinoma (Revisi JC, et al 1990 Int J Cancer 46: 416-20; Srkalovicg, et al 1990 J Clin Endocrinol Metab 70: 661-669]), lymphoma (Revisi JC, et al 1992, Int J Cancer 50: 895-900]), renal cells Cancer (Reference [Reubi JC, et al 1992, Cancer Res 52: 6074-6078]), mesenchymal tumor (Reference [Reubi JC, et al 1996 Cancer Res 56: 1922-31]), prostate (Reference [Reubi JC, et al 1995, J. Clin.Endocrinol Metab 80: 2806-14; et al 1989, Prostate 14:191-208; Halmos G, et al J. Clin. Endo-crinol Metab 85: 2564-71]), ovary (reference [Halmos, G, et al, 2000 J Clin Endocrinol Metab 85: 3509-12; Reubi JC, et al 1991 Am J Pathol 138:1267-72]), stomach (reference [Reubi JC, et al 1999, Int J Cancer 81: 376-86; Miller, G. V, 1992 Br J Cancer 66: 391-95]), hepatocellular (reference [Kouroumalis E, et al 1998 Gut 42: 442-7; Reubi JC, et al 1999 Gut 45: 66-774]) and nasopharyngeal carcinoma (reference [Loh K. S, et al, 2002 [Virchows Arch 441: 444-8]); specific to aromatic sulfonamides (specific to carbonic anhydrase IX) (markers of hypoxia and renal cell carcinoma) (reference [Neri, D., et al, Nat. Rev. Drug Discov. 2011, 10, 767-7]); pituitary adenylate cyclase-activating peptide (PACAP) (PAC1) for pheochromocytoma and paraganglioma; vasoactive intestinal peptides (VIP) and their receptor subtypes (VPAC1, VPAC2); α-melanocyte-stimulating hormone (α-MSH) receptors; Cholecystokinin (CCK) / gastrin receptors and their receptor subtypes (CCK1 (formerly CCK-A) and CCK2; bombesin (Pyr-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2) / gastrin-releasing peptide (GRP) and their receptor subtypes (BB1, GRP receptor subtype (BB2), BB3 and BB4) (Reference [Ohlsson, B., et al, 1999, Scand. J.Gastroenterology 34 (12): 1224-9; Weber, HC, 2009, Cur. Opin. Endocri. Diab. Obesity 16(1): 66-71, Gonzalez N, et al, 2008, Cur. Opin. Endocri. Diab. Obesity 15(1), 58-64]); neurotensin receptors and their receptor subtypes (NTR1, NTR2, NTR3); Substrate P receptors and their receptor subtypes (e.g., NK1 receptors for glial tumors (reference [Hennig IM, et al 1995 Int. J. Cancer 61, 786-792]); neuropeptide Y (NPY) receptors and receptor subtypes (Y1-Y6); homing peptides include RGD (Arg-Gly-Asp) and NGR (Asn-Gly-Arg), which are dimeric and polymeric cyclic RGD peptides (e.g., cRGDfV) (reference [Laakkonen P, Vuorinen K. 2010, Integr Biol (Camb). 2(7-8): 326-337; Chen K, Chen X. 2011, Theranostics. 1:189-200; Garanger E, et al, Anti-Cancer Agents Med Chem. 7(5): 552-558; Kerr, JS et al, AntiCancer Research, 19(2A), 959-968; Thumshirn, g, et al, 2003 Chem. Eur. J. 9, 2717-2725]) and TAASGVRSMH or LTLRWVGLMS (chondroitin sulfate proteoglycan NG2 receptor) and F3 peptide (31-amino acid peptide binding to cell surface-expressed nucleolin receptor) (Literature [Zitzmann, S., 2002 Cancer Res., 62, 18, pp. 5139-5143, Temminga, K., 2005, Drug Resistance Updates, 8, 381-402; P. Laakkonen and K.Vuorinen, 2010 Integrative Biol, 2(7-8), 326-337; MA Burg, 1999 Cancer Res., 59(12), 2869-2874; K. Porkka, et al 2002, Proc. Nat. Acad. Sci. USA 99(11), 7444-9]); cell-penetrating peptides (CPP) (Literature [Nakase I, et al, 2012, J. Control Release.[159(2), 181-188]); peptide hormones, e.g., luteinizing hormone-releasing hormone (LHRH) agonists and antagonists and gonadotropin-releasing hormone (GnRH) agonists act by targeting the production of follicle hormone (FSH) and luteinizing hormone (LH), as well as testosterone, e.g., buserelin (Pyr-His-Trp-Ser-Tyr-D-Ser(OtBu)-Leu-Arg-Pro-NHEt), gonadorelin (Pyr-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2), goserelin (Pyr-His-Trp-Ser-Tyr-D-Ser(OtBu)-Leu-Arg-Pro-AzGly-NH2), Histrelin (Pyr-His-Trp-Ser-Tyr-D-His(N-Benzyl)-Leu-Arg-Pro-NHEt), Leuprolide (Pyr-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt), Naparellin (Pyr-His-Trp-Ser-Tyr-2Nal-Leu-Arg-Pro-Gly-NH2), Triptorelin (Pyr-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH2), Naparellin, Deslorelin, Avarelix (Ac-D-2Nal-D-4-Chlorophe-D-3-(3-Pyridyl)Ala-Ser-(N-Me)Tyr-D-Asn-Leu-IsopropylLys-Pro-DAla-NH2), Cetrorelix (Ac-D-2Nal-D-4-chloro-Phe-D-3-(3-pyridyl)Ala-Ser-Tyr-D-Cit-Leu-Arg-Pro-D-Ala-NH2), degarellix (Ac-D-2Nal-D-4-chloroPhe-D-3-(3-pyridyl)Ala-Ser-4-aminoPhe(L-hydroorotyl)-D-4-aminoPhe(carbamoyl)-Leu-isopropylLys-Pro-D-Ala-NH2) and ganirelix (Ac-D-2Nal-D-4-chloroPhe-D-3-(3-pyridyl)Ala-Ser-Tyr-D-(N9,N10-diethyl)-homoArg-Leu-(N9, N10-Diethyl)-HomoArg-Pro-D-Ala-NH2)(Literature [Thundimadathil, J., J.Amino Acids, 2012, 967347, doi:10.1155 / 2012 / 967347; Boccon-Gibod, L.; et al, 2011, Therapeutic Advances in Urology 3(3): 127-140; Debruyne, F., 2006, Future Oncology, 2(6), 677-696; Schally A.V; Nagy, A. 1999 Eur J Endocrinol 141:1-14; KoppanM, et al 1999 Prostate 38:151-158]); and pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), C-type lectins, and NLRs (NLRs), ranging in size from small molecules (imiquimod, guanisine, and adenosine analogs) to large complex biomacromolecules, e.g., lipopolysaccharides (LPS), nucleic acids (CpG DNA, polyI:C), and lipopeptides (Pam3CSK4) (references [Kasturi, SP, et al, 2011, Nature 470, 543-547; Lane, T., 2001, JR Soc. Med. 94, 316; Hotz, C., and Bourquin, C., 2012, Oncoimmunology 1, 227-228; Dudek, AZ, et al, 2007, Clin. Cancer Res. 13, 7119-25]) (references [Fukata, M., et al, 2009, Semin. Immunol. 21, 242-253; Maisonneuve, C., et al, 2014, Proc. Natl. Acad. Sci. USA 111, 1-6; Botos, I., et al, 2011, Structure 19, 447-459; Means, T.K., et al, 2000, Life Sci.68, 241-258]); calcitonin receptor, a 32-amino acid neuropeptide significantly involved in regulating calcium levels through effects on osteoclasts and kidneys (Reference [ZaidiM, et al, 1990 Crit Rev Clin Lab Sci 28, 109-174; Gorn, AH, et al 1995 J Clin Invest 95:2680-91]); Integrin receptors and their receptor subtypes (e.g., αVβ1, αVβ3, αVβ5, αVβ6, α6β4, α7β1, αLβ2, αIIbβ3, etc.) that play an important role in angiogenesis and are expressed on the surface of various cells, particularly osteoclasts, endothelial cells, and tumor cells (Literature [Ruoslahti, E. et al, 1994 Cell 77, 477-8; Albelda, SM et al, 1990 Cancer Res., 50, 6757-64]). Short peptides, GRGDSPK and cyclic RGD pentapeptides, e.g., cyclo(RGDfV)(L1) and their derivatives [cyclo(-N(Me)R-GDfV), cyclo(R-Sar-DfV), cyclo-(RG-N(Me)D-fV), cyclo(RGD-N(Me)fV), cyclo(RGDf-N(Me)V-)(silengitide)] have exhibited high binding affinity for integrin receptors (Reference [Dechantsreiter, MA et al, 1999 J. Med. Chem. 42, 3033-40, Goodman, SL, et al, 2002 J. Med. Chem. 45, 1045-51]).

[0314] Cell-binding molecules / ligands or cell receptor agonists may be Ig-based and non-Ig-based protein scaffold molecules. Ig-based scaffolds include nanobodies (derivatives of VHH (camellid Ig)) (reference [Muyldermans S., 2013 Annu Rev Biochem. 82, 775-97]); domain antibodies (dAbs, derivatives of VH or VL domains) (reference [Holt, L. J, et al, 2003 Trends Biotechnol. 21, 484-90]); bispecific T cell agonists (BiTE, bispecific diabodies) (reference [Baeuerle, P. A, et al, 2009 Curr. Opin.mol. Ther. 11, 22-30]); They may be selected from dual-affinity retargeting antibodies (DART, bispecific diabodies) (Moore PA P, et al. 2011, Blood 117(17), 4542-51]); quadrivalent tandem antibodies (TandAb, dimeric bispecific diabodies) (Cochlovius, B, et al. 2000, Cancer Res. 60(16):4336-4341]), but are not limited to these. Non-Ig scaffolds are anticalin (derivatives of lipocalin) (References [Skerra A. 2008, FEBS J., 275(11): 2677-83; Besteg, et al, 1999 Proc. Nat. Acad. USA. 96(5):1898-903; Skerra, A. 2000 Biochim Biophys Acta, 1482(1-2): 337-50; Skerra, A. 2007, Curr Opin Biotechnol. 18(4): 295-304; Skerra, A. 2008, FEBS J. 275(11):2677-83]); Adnectin (FN3 (fibronectin)) (References [Koide, A, et al, 1998 J.mol. Biol., 284(4):1141-51; Batori V, 2002, Protein Eng. 15(12): 1015-20; Tolcher, A. W, 2011, Clin.Cancer Res. 17(2): 363-71; Hackel, B. J, 2010, Protein Eng. Des. Sel. 23(4): 211-19]); Designed Ancrin repeat proteins (DARPin) (derivatives of Ancrin repeat (AR) proteins) (Reference [Boersma, YL, et al, 2011 Curr Opin Biotechnol. 22(6): 849-57]), e.g., DARPin C9, DARPin Ec4, and DARPin E69_LZ3_E01 (Reference [Winkler J, et al, 2009 Mol Cancer Ther. 8(9), 2674-83; Patricia MK. M., et al, Clin Cancer Res. 2011; 17(1):100-10; Boersma Y. L, et al, 2011 J. Biol. Chem. 286(48), 41273-85]); Avimer (domain A / low-density lipoprotein (LDL) receptor) (reference [Boersma Y. L, 2011 J. Biol. Chem. 286(48): 41273-41285; Silverman J, et al, 2005 Nat. Biotechnol., 23(12):1556-61]) may be selected from, but is not limited to, these.

[0315] Examples of small molecule structures of cell-binding molecules / ligands or cell receptor agonists of this patent application are as follows: LB01 (folic acid), LB02 (PMSA ligand), LB03 (PMSA ligand), LB04 (PMSA ligand), LB05 (somatostatin), LB06 (somatostatin), LB07 (octreotide, somatostatin analog), LB08 (lanreotide, somatostatin analog), LB09 (vapreotide (Sanvar), somatostatin analog) as shown in the structural formulas below, LB10 (CAIX ligand), LB11 (CAIX ligand), LB12 (gastrin-releasing peptide receptor (GRPr), MBA), LB13 (luteinizing hormone-releasing hormone (LH-RH) ligand and GnRH), LB14 (luteinizing hormone-releasing Hormones (LH-RH) and GnRH ligands), LB15 (GnRH antagonist, Avarelix), LB16 (cobalamin, vitamin B12 analog), LB17 (cobalamin, vitamin B12 analog), LB18 (cyclic RGD pentapeptide for αvβ3 integrin receptors), LB19 (heterogeneous-divalent peptide ligand for VEGF receptors), LB20 (Neuromedin B), LB21 (bombesin for G-protein coupled receptors), LB22 (TLR2 for Toll-like receptors), LB23 (for androgen receptors), LB24 (silengitide / cyclo(-RGDfV-) for αv integrin receptors), LB23 (fludrocortisone), LB25 (rifabutin analog), LB26 (rifabutin analog), LB27 (Rifabutin analog), LB28 (Fludrocortisone), LB29 (Dexamethasone), LB30 (Fluticasone propionate), LB31 (Beclomethasone dipropionate), LB32 (Triamcinolone acetonide), LB33 (Prednisone), LB34 (Prednisolone), LB35 (Methylprednisolone), LB36 (Betamethasone), LB37 (Irinotecan analog), LB38 (Crizotinib analog), LB39 (Bortezomib analog), LB40 (Carfilzomib analog), LB41 (Carfilzomib analog), LB42 (Leuprolide analog),LB43 (triptorelin analog), LB44 (clindamycin), LB45 (liraglutide analog), LB46 (semaglutide analog), LB47 (letapamulin analog), LB48 (indibulin analog), LB49 (vinblastine analog), LB50 (lixisenatide analog), LB51 (osimertinib analog), LB52 (nucleoside analog), LB53 (erlotinib analog) and LB54 (lapatinib analog):,

[0316]

[0317]

[0318]

[0319]

[0320]

[0321]

[0322]

[0323]

[0324]

[0325]

[0326] During the meal, " " is a portion for connecting the side chain linker of the present patent; X4 and Y1 are independently O, NH, NHNH, NR1, S, C(O)O, C(O)NH, OC(O)NH, OC(O)O, NHC(O)NH, NHC(O)S, OC(O)N(R1), N(R1)C(O)N(R1), CH 2, C(O)NHNHC(O) and C(O)NR1; X1 is H, CH2, OH, O, C(O), C(O)NH, C(O)N(R1), R1, NHR1, NR 1, C(O)R1 or C(O)O; X5 is H, CH3, F, or Cl; M1 and M2 are independently H, Na, K, Ca, Mg, NH4, N(R12 R 12' R 13 R 13' ) and; R 12, R 12', R 13 a and R 13' is defined in chemical formula (I).

[0327] In another embodiment, the ligand may be used as a payload for a conjugate to a cell-binding molecule (e.g., an antibody) through the side chain linker of the present application for targeted treatment or prevention of cancer, infection, and autoimmune diseases.

[0328] In another additional embodiment, one, two, or more DNA, RNA, mRNA, small interfering RNA (siRNA), microRNA (miRNA), and PIWI interacting RNA (piRNA) may preferably be conjugated to a cell-binding molecule through the bis-linker of this patent. It is known that small RNAs (siRNA, miRNA, piRNA) and long non-coding antisense RNAs are responsible for epigenic changes within cells (Reference [Goodchild, J (2011), Methodsin molecular biology (Clifton, NJ). 764: 1-15]). In this specification, DNA, RNA, mRNA, siRNA, miRNA, or piRNA may be single or double-stranded having 3 to 10 million nucleotide units, some of the nucleotides thereof may be in a non-natural (synthetic) form, e.g., oligonucleotides having a phosphothioate linkage, such as in formivirsen, or nucleotides may be linked to a phosphothioate linkage rather than a phosphodiester linkage of natural RNA and DNA, and the sugar portion is deoxyribose in the central portion of the molecule and 2'-O-methoxyethyl-modified ribose at both ends, e.g., miformersen or peptide nucleic acid (PNA), morpholino, phosphothioate, thiophosphoramidate, or 2'-O-methoxyethyl (MOE), 2'-O-methyl, 2'-fluoro, lock nucleic acid (LNA), or bicyclic nucleic acid of ribose sugar (BNA). Alternatively, oligonucleotides prepared from nucleic acids are modified to remove the 2'-3' carbon bond from the sugar ring (Whitehead, KA; et al (2011), Annual Review of Chemical and Biomolecular Engineering 2: 77-96; Bennett, CF; Swayze, EE (2010), Annu. Rev. Pharmacol. Toxicol. 50: 259-29). Preferably, the length range of the oligonucleotides is approximately 8 to over 200 nucleotides. Examples of nucleotide structures for conjugates are shown below:

[0329]

[0330] Among the food, X1, is the same as chemical formula (I) or the one defined above; is a single or double strand of DNA, RNA, mRNA, siRNA, miRNA, or piRNA; Y is preferably O, S, NH, or CH2.

[0331] Applications of composites

[0332] The cell-binding ligand-drug conjugate via the side chain linker of the present invention is used for targeted cancer therapy. Target cancers include adrenocortical carcinoma, anal cancer, bladder cancer, brain tumors (adult, brainstem cell tumor, juvenile, cerebellar astrocytoma, cerebral astrocytoma, ependymoma, medulloblastoma, primitive neuroectodermal and pineal tumors, visual pathway and hypothalamic glioma), breast cancer, carcinoid tumor, gastrointestinal, unknown primary carcinoma, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, extrahepatic cholangiocarcinoma, tumors of the Ewing family (PNET), extracranial embryonic cell tumors, ocular cancer, intraocular melanoma, gallbladder cancer, gastric cancer (stomach), embryonic cell tumors, extragonadal, gestational trophoblastic tumor, head and neck cancer, hypopharyngeal cancer, parotid cell carcinoma, renal cancer (renal cell carcinoma), laryngeal cancer, leukemia (acute lymphocytic, acute myeloid, chronic lymphocytic, chronic myeloid, pilosa), lip and oral cancer, liver cancer, lung cancer (non-small cell, small cell, Lymphoma (AIDS-related, central nervous system, cortical T-cell, Hodgkin's disease, non-Hodgkin's disease, malignant mesothelioma, melanoma, Merkel cell carcinoma, metastatic squamous head carcinoma and other plasma cell neoplasms with latent primary multiple myeloma, mycosis fungoides, myelodysplastic syndrome, spinal proliferative disorder, nasopharyngeal cancer, neuroblastoma, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer (epithelial, germ cell tumor, low malignancy potential tumor), pancreatic cancer (exocrine, islet cell carcinoma), sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pheochromocytoma, pituitary cancer, plasma cell neoplasm, prostate cancer, rhabdomyosarcoma, rectal cancer, renal cell carcinoma (kidney cancer), renal pelvic and pneumonia (transitional cell), salivary gland cancer, Sézary syndrome, skin cancer, skin cancer (cutaneous T-cell lymphoma, Kaposi's sarcoma, melanoma), small intestine cancer, It includes, but is not limited to, soft tissue sarcomas, gastric cancer, testicular cancer, thymoma (malignant), thyroid cancer, urethral cancer, uterine cancer (sarcoma), cancers specific to childhood, vaginal cancer, Vulvar cancer, and Wilms tumor.

[0333] In another specific embodiment, the cell-linked-drug conjugate of the present invention is used according to a composition and method for the treatment or prevention of autoimmune diseases. Autoimmune diseases include achlorhydra-activated chronic infection, acute disseminated encephalomyelitis, acute hemorrhagic leukemia, Addison's disease, agammaglobulinemia, alopecia areata, sclerosis, ankylosing spondylitis, anti-GBM / TBM nephritis, antiphospholipid syndrome, antisynthetase syndrome, arthritis, atopic allergy, atopic dermatitis, autoimmune aplastic anemia, autoimmune cardiomyopathy, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune peripheral neuropathy, autoimmune pancreatitis, autoimmune polydactyly syndrome types I, II, and III, autoimmune progesterone dermatitis, autoimmune thrombocytopenic purpura, autoimmune uveitis, Vallo's disease / valley concentric sclerosis, Bachet's syndrome, and Buerger's disease. Vickersstaff encephalitis, Blau syndrome, bullous pemphigoid, Castleman disease, Sargagas disease, chronic fatigue immune disorder, chronic inflammatory demyelinating polyneuropathy, chronic relapsing multifocal osteomyelitis, chronic Lyme disease, chronic obstructive pulmonary disease, Chuk-Strauss syndrome, scar-like pemphigoid, celiac disease, Cogan syndrome, cold agglutination disorder, complement component II deficiency, cranial arteritis, CREST syndrome, Crohn's disease (a type of idiopathic inflammatory bowel disease), Cushing's syndrome, cutaneous leukocytoclastic vasculitis, Dego's disease, Durkum's disease, herpetic dermatitis, dermatomyositis, type 1 diabetes, extensive systemic scleroderma, Dressler syndrome, discoid lupus erythematosus, eczema, endometriosis, entrapmentitis-associated arthritis, eosinophilic fasciitis, acquired epidermobulosis, erythema nodosum, essential mixed cryoglobulinemia, Evan Syndrome, progressive ossifying fibrodysplasia, fibromyalgia, fibromyositis, fibrotic Aveolitis, gastritis, gastrointestinal pemphigus, giant cell arteritis, glomerulonephritis, Goodpasture syndrome, Graves' disease, Guillain-Barré syndrome (GBS), Hashimoto's encephalitis, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Scholllein purpura,Herpes zoster during pregnancy, hidradenitis suppurativa, Hyuges syndrome (see Antiphospholipid syndrome), hypogammaglobulinemia, idiopathic inflammatory demyelinating disease, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (see Autoimmune thrombocytopenic purpura), IgA nephropathy (also Buerger's disease), inclusion body myositis, inflammatory demyelinating polyneuropathy, interstitial cystitis, irritable bowel syndrome (IBS), juvenile idiopathic arthritis, juvenile rheumatoid arthritis, Kawasaki disease, Lambert-Eaton myasthenia gravis, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, linear IgA disease (LAD), amyotrophic lateral sclerosis (ALS), lupus erythematosus, Majid syndrome, Meniere's disease, microscopic polyvasculitis, Miller-Fisher syndrome, mixed connective tissue disease, sclerosis, Mucha-Haberman disease, Merkle-Wells syndrome, multiple myeloma, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (also Debick's disease), neuromyalgia, pemphigus scarletis, oculomotor-myoclonus syndrome, Odd's thyroiditis, relapsing rheumatism, PANDAS (Streptococcus-associated pediatric immunoneuropsychiatric disorder), adrenal neoplasmic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria, Parry-Romberg syndrome, Parsonage-Turner syndrome, intermediate uveitis, pemphigus, pemphigus vulgaris, pernicious anemia, perivenous encephalomyelitis, POEMS syndrome, polyarteritis nodosum, polymyalgia, polymyositis, primary biliary cirrhosis, primary sclerosing cholangitis, progressive inflammatory neuropathy, psoriasis, psoriatic arthritis, pyoderma gangrenosa, pure red blood cells Aplasia, Rasmussen encephalitis, Raynaud's phenomenon, recurrent polychondritis, Reiter's syndrome, Restless legs syndrome, retroperitoneal fibrosis, rheumatoid arthritis, rheumatic fever, sarcoidosis, schizophrenia, Schmidt syndrome, Schnitzler syndrome, scleritis, scleroderma, Sjögren's syndrome, spondyloarthritis, adhesive blood syndrome, Still's disease, rigid man syndrome, subacute bacterial endocarditis (SBE), Sosask syndrome, Sweet syndrome, Sydenam's chorea, sympathetic ophthalmitis, Takayasu's arteritis, temporal arteritis (including giant cell arteritis),Includes, but is not limited to, Tolosa-Hunt syndrome, transverse myelitis, ulcerative colitis (a type of idiopathic inflammatory bowel disease), undifferentiated connective tissue disease, undifferentiated spondyloarthropathy, vasculitis, leukoplakia, Wegener's granulomatosis, Wilson's syndrome, and Wiskott-Aldrich syndrome.

[0334] In another specific embodiment, the binding molecule used for the conjugate via the side-chain linker of the present invention for the treatment or prevention of autoimmune diseases comprises: an anti-elastin antibody; Abys against epithelial cell antibodies; an anti-basement membrane collagen type IV protein antibody; an anti-nuclear antibody; anti-ds DNA; anti-ss DNA, anti-cardiolipin antibodies IgM, IgG; anti-celiac antibody; anti-phospholipid antibodies IgK, IgG; anti-SM antibody; anti-mitochondrial antibody; thyroid antibody; microsome antibody, T-cell antibody; thyroglobulin antibody, anti-SCL-70; anti-Jo; anti-U.sub.1RNP; anti-La / SSB; anti-SSA; anti-SSB; anti-Perital cell antibody; anti-histone; anti-RNP; C-ANCA; P-ANCA; anti-kinetochore; anti-fibrillarin and anti-GBM antibodies, anti-ganglioside antibody; anti-desmogane 3 antibody; anti-p62 antibody; Anti-sp100 antibody; anti-mitochondrial (M2) antibody; rheumatoid factor antibody; anti-MCV antibody; anti-topoisomerase antibody; anti-neutrophil cytoplasmic (cANCA) antibody may be, but are not limited to, these.

[0335] In a specific preferred embodiment, the binding molecule for the conjugate in the present invention may bind to both the receptor and the receptor complex expressed on activated lymphocytes associated with autoimmune diseases. Receptors or receptor complexes are members of the immunoglobulin gene superfamily (e.g., CD2, CD3, CD4, CD8, CD19, CD20, CD22, CD28, CD30, CD33, CD37, CD38, CD56, CD70, CD79, CD79b, CD90, CD125, CD137, CD138, CD147, CD152 / CTLA-4, PD-1, or ICOS), members of the TNF receptor superfamily (e.g., CD27, CD40, CD95 / Fas, CD134 / OX40, CD137 / 4-1BB, INF-R1, TNFR-2, RANK, TACI, BCMA, osteopropegerin, Apo2 / TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4, and APO-3), integrins, cytokine receptors, It may include chemokine receptors, major histocompatibility proteins, lectins (C-type, S-type, or I-type), or complement control proteins.

[0336] In another specific embodiment, a useful cell-binding ligand that is immune-specific to a viral or microbial antigen is a humanized or human monoclonal antibody. As used herein, the term “viral antigen” includes, but is not limited to, any viral peptide, polypeptide protein capable of eliciting an immune response (e.g., HIV gp120, HIV nef, RSV F glycoprotein, influenza virus neuraminidase, influenza virus hemagglutinin, HTLV tax, herpes simplex virus glycoproteins (e.g., gB, gC, gD and gE) and hepatitis B surface antigen). As used herein, the term “microbial antigen” includes, but is not limited to, any microbial peptide, polypeptide, protein, sugar, polysaccharide, or lipid molecule capable of eliciting an immune response (e.g., bacterial, fungal, pathogenic protozoal, or yeast polypeptides, e.g., LPS and capsular polysaccharide 5 / 8). Examples of antibodies available for use against viral or microbial infections include, but are not limited to, palivizumab, a humanized anti-respiratory syncytial virus monoclonal antibody for the treatment of RSV infection; PRO542, a CD4 fusion antibody for the treatment of HIV infection; ostavir, a human antibody for the treatment of hepatitis B virus; PROTVIR, a humanized IgG.sub.1 antibody for the treatment of cytomegalovirus; and anti-LPS antibodies.

[0337] The cell-linked molecule-drug conjugate through the side-chain linker of the present invention can be used for the treatment of infectious diseases. These infectious diseases include Acinetobacter infection, actinomycosis, African sleeping sickness (African trypanosomiasis), AIDS (Acquired Immunodeficiency Syndrome), amoebiasis, anaplasmosis, anthrax, Arcanobacterium haemolyticum infection, Argentine hemorrhagic fever, ascariasis, aspergillosis, astrovirus infection, babesiosis, Bacillus cereus infection, bacterial pneumonia, bacterial vaginosis, Bacteroides infection, balantidialism, Baylisascaris infection, BK virus infection, melanocyticulosis, Blastocystis hominis infection, blastomycosis, Bolivian hemorrhagic fever, Borrelia infection, botulism (and Infantile botulism), Brazilian hemorrhagic fever, brucellosis, Burkholderia infection, Buruli ulcer, Calicivirus infection (Norovirus and Sapovirus), Campylobacteriosis, Candidiasis (Moniliasis; thrush), Cat-scratch disease, Cellulitis, Chagas disease (American sleeping sickness), Soft ulcer, Chickenpox, Chlamydia, Chlamydophila pneumoniae infection, Cholera, Chromoblastomycosis, Clonorchiasis, Clostridium difficile infection,Coccidioidomycosis, Colorado tick fever, common cold (acute viral rhinopharyngitis; acute rhinitis), Creutzfeldt-Jakob disease, Crimean-Congo hemorrhagic fever, Cryptococcosis, Cryptosporidiosis, Cutaneous larva migrans, Cysticercosis, Cytomegalovirus infection, Dengue fever, Dinuclear amoebiasis, Diphtheria, Diphyllobothrium latum, Guinea guinea, Ebola hemorrhagic fever, Echinococosis, Ehrlichiosis, Enterobiasis (pinworm infection), Enterococcus infection, Enterovirus infection, Epidemic typhus, Erythema infectiosum (Fifth disease), Roseola, Hypertrichosis, Epilepsy, Fatal familial insomnia, Filamentosarcosis, Food poisoning by Clostridium perfringens, Free-living amebic infection, Fusobacterium infection, Gas necrosis (Clostridial myonecrosis), Geotrichum, Gerstmann-Straussler-Scheinker syndrome, Giardiasis, Pulmonariasis, Gnathostomiasis, Gonorrhea, Inguinal granuloma (Donovaniosis), Group A Streptococcal infection, Group B Streptococcus infection, Haemophilus influenzae infection, hand, foot, and mouth disease (hand,Foot and mouse disease (HFM), Hantavirus pulmonary syndrome, Helicobacter pylori infection, Hemolytic-uremic syndrome, Hemorrhagic fever with renal syndrome, Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis D, Hepatitis E, Herpes simplex, Histoplasmosis, Hookworm infection, Human bocavirus infection, Human ewingii ehrlichiosis, Human granulocytic anaplasmosis, Human metapneumovirus infection, Human monocytic ehrlichiosis, Human papilloma infection, Human parainfluenza virus infection, Hymenolepiasis, Epstein-Barr virus infectious mononucleosis (Mono), Influenza, sporozoosis, Kawasaki disease, keratitis, Kingella kingae infection, rickets, Lassa fever, Legionellosis (Veteran's disease), Legionellosis (Pontiac fever), leishmaniasis, Hansen's disease, leptospirosis, listeriosis, Lyme disease (Lyme borreliosis), lymphocytic filariasis (epithelial disease), lymphocytic choriomeningitis, malaria, Marburg hemorrhagic fever, measles, melioidosis (Whitmore's disease), meningitis, meningococcal disease, Yokogawa fluke infection, microsporizoosis, molluscum contagiosum, mumps, typhus (infectious typhus), Mycoplasma pneumonia, mycosis, mycosis, neonatal conjunctivitis (newborn Conjunctivitis), (New) Variant Creutzfeldt-Jakob disease (vCJD, nvCJD), Nocardiosis, Onchocerciasis (filariasis),Paracoccidioidomycosis (South American blastomycosis), Paragonimiasis, Pasteurella, Head lice, Body lice, Pubic lice (Crab lice), Pelvic inflammatory disease, Pertussis (Whooping cough), Black death, Pneumococcal infection, Pneumocystis pneumonia, Pneumonia, Polio, Prevotella infection, Primary amoebic meningoencephalitis, Progressive multifocal leukencephalopathy, Psittacosis, Q fever, Rabies, Brachydactyly, Respiratory syncytial virus infection, Rhinosporidiosis, Rhinovirus infection, Rickettsia infection, Rickettsial smallpox, Rift Valley fever, Rocky Mountain spotted fever, Rotavirus infection, Rubella, Salmonellosis, SARS (Severe Acute Respiratory Syndrome), scabies, schistosomiasis, sepsis, bacterial dysentery (Shigellosis), herpes zoster (Shingles), smallpox (Variola), sporotrichum, Staphylococcus food poisoning, Staphylococcus infection, strongyloidiasis, syphilis, tapeworm infection, tetanus (Lockjaw), tinea barbae (Barber's itch), tinea capitis (Ringworm of the scalp), tinea corporis (Ringworm of the body), tinea cruris (Jock itch), tinea manuum (Ringworm of the hand), tinea melanosis, athlete's foot (Tinea pedis), onychomycosis (Tinea unguium), tinea versicolor (Tinea versicolor, Pityriasis versicolor), toxocariasis (ocular larval migration), toxocariasis (visceral larval migration), toxoplasmosis, trichinosis, trichomoniasis, whipworm infection, pulmonary tuberculosis,Includes, but is not limited to, hare disease, Ureaplasma infection, Venezuelan encephalitis, Venezuelan hemorrhagic fever, viral pneumonia, West Nile fever, albinism (white ringworm), pseudomycosis infection, jesiniasis, yellow fever, and zygomycosis.

[0338] The conjugate of the present invention is Acinetobacter baumannii, Actinomyces israelii, Actinomyces gerencseriae, Propionibacterium propionicus, Trypanosoma brucei, HIV (Human Immunodeficiency Virus), Entamoeba histolytica, Anaplasma genus, Bacillus anthracis, Arcanobacterium haemolyticum, Junin virus, Ascaris lumbricoides, Aspergillus genus, Astroviridae family, Babesia genus, Bacillus Bacillus cereus, multiple bacteria, Bacteroides genus, Balantidium coli, Baylisascaris genus, BK virus, Piedraia hortae, Blastocystis hominis, Blastomyces dermatitides, Machupo virus, Borrelia genus, Clostridium botulinum, Sabia, Brucella genus, usually Burkholderia cepacia and other Burkholderia species, Mycobacterium ulcerans,Caliciviridae family, Campylobacter genus, common Candida albicans and other Candida species, Bartonella henselae, Group A Streptococcus and Staphylococcus, Trypanosoma cruzi, Haemophilus ducreyi, Varicella zoster virus (VZV), Chlamydia trachomatis, Chlamydophila pneumoniae, Vibrio cholerae, Fonsecaea pedrosoi, Clonorchis sinensis, Clostridium difficile difficile), Coccidioides immitis and Coccidioides posadasii, Colorado tick fever virus, rhinovirus, coronavirus, CJD prion, Crimin-Congo hemorrhagic fever virus, Cryptococcus neoformans, Cryptosporidium genus, Ancylostoma braziliense; Multiple parasites, Cyclospora cayetanensis, Taenia solium, Cytomegalovirus, Dengue virus (DEN-1, DEN-2, DEN-3, and DEN-4) - Flavivirus, Intestinal amoeba (Dientamoeba fragilis), Corynebacterium diphtheriae, Diphyllobothrium,Dracunculus medinensis, Ebola virus, Echinococcus genus, Ehrlichia genus, Enterobius vermicularis, Enterococcus genus, Enterovirus genus, Rickettsia prowazekii, Parvovirus B19, Human Herpesvirus 6 and Human Herpesvirus 7, Fasciolopsis buski, Fasciola hepatica and Fasciola gigantica, FFI prion, Filarioidea superfamily, Clostridium perfringens, Fusobacterium Genus (Fusobacterium genus), Clostridium perfringens; Other Clostridium species, Geotrichum candidum, GSS prions, Giardia intestinalis, Burkholderia mallei, Gnathostoma spinigerum and Gnathostoma hispidum, Neisseria gonorrhoeae, Klebsiella granulomatis, Streptococcus pyogene, Streptococcus agalactiae, Haemophilus influenzae, enteroviruses, primarily Coxsackie A virus and Enterovirus 71, Sin Nombre virus,Helicobacter pylori, Escherichia coli O157:H7, Bunyaviridae family, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Herpes simplex virus 1, Herpes simplex virus 2, Histoplasma capsulatum, Ancylostoma duodenale and Necator americanus, Haemophilus influenzae, Human bocavirus, Ehrlichia ewingii, Anaplasma phagocytophilum, Human metapneumovirus, Ehrlichia chaffeensis, Human papillomavirus, Human parainfluenza virus, small tapeworm (Hymenolepis nana) and rat tapeworm (Hymenolepis diminuta), Epstein-Barr virus, Orthomyxoviridae family, Isosspora belli, Kingella kingae, Klebsiella pneumoniae, Klebsiella ozaenas, Klebsiella rhinoscleromotis, Kru prion, Lassa virus, Legionella pneumophila, Legionella pneumophila, Leishmania genus, Mycobacterium Mycobacterium leprae and Mycobacterium lepromatosis,Leptospira genus, Listeria monocytogenes, Borrelia burgdorferi and other Borrelia species, Wuchereria bancrofti and Brugia malayi, Lymphocytic choriomeningitis virus (LCMV), Plasmodium genus, Marburg virus, Measles virus, Burkholderia pseudomallei, Neisseria meningitides, Metagonimus yokagawai, Microsporidia phylum, Molluscum contagiosum virus (MCV), Mumps virus, Rickettsia typhi, Mycoplasma pneumoniae, multiple species of bacteria (Actinomycetoma) and fungi (Eumycetoma), parasitic dipterous fly larvae, Chlamydia trachomatis and Neisseria gonorrhoeae, vCJD prion, Nocardia asteroides and other Nocardia species, Onchocerca volvulus, Paracoccidioides brasiliensis, Paragonimus westermani and other Paragonimus species, Pasteurella genus, Pediculus humanus capitis,Body lice (Pediculus humanus corporis), pubic lice (Phthirus pubis), Bordetella pertussis, Yersinia pestis), Streptococcus pneumoniae, Pneumocystis jirovecii, Poliovirus, Prevotella genus, Naegleria fowleri, JC virus, Chlamydophila psittaci, Coxiella burnetii, rabies virus, Streptobacillus moniliformis and Spirillum minus, respiratory syncytial virus, Rhinosporidium seeberi, Rhinovirus, Rickettsia genus, Rickettsia akari, Rift Valley fever virus, Rickettsia rickettsii, Rotavirus, Rubella virus, Salmonella genus, SARS coronavirus, Sarcoptes scabiei, Schistosoma genus, Shigella genus, Varicella zoster virus, Variola major or Variola minor, Sporothrix schenckii, Staphylococcus genus, Staphylococcus aureus, Streptococcus Pyogenes (Streptococcus pyogenes), Strongyloides stercoralis,Treponema pallidum, Taenia genus, Clostridium tetani, Trichophyton genus, Trichophyton tonsurans, Trichophyton genus, Epidermophyton floccosum, Trichophyton rubrum and Trichophyton mentagrophytes, Trichophyton rubrum, Hortaea werneckii, Trichophyton genus, Malassezia genus, Toxocara canis or Toxocara cati, Toxoplasma Toxoplasma gondii, Trichinella spiralis, Trichomonas vaginalis, Trichuris trichiura, Mycobacterium tuberculosis, Francisella tularensis, Ureaplasma urealyticum, Venezuelan equine encephalitis virus, Vibrio cholerae, Guanarito virus, West Nile virus, Trichosporon beigelii, Yersinia pseudotuberculosis, Yersinia enterocolitica, Yellow fever virus,Mucorales order (Mucormycosis) and Entophthorales order (Entomophthoramycosis), Pseudomonas aeruginosa, Campylobacter (Vibrio) embryo, Aeromonas hydrophila, Edwardsiella tarda, Yersinia pestis, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Salmonella typhimurium, Treponema pertenue, Treponema carateneum, Borrelia vincentii, Borrelia burgdorferi, Leptospira icterohemorrhagiae, Pneumocystis carinii, Brucella abortus, Brucella suis, Brucella melitensis, Mycoplasma spp., Rickettsia prowazeki, Rickettsia tsutsugumushi, Chlamydia spp.; pathogenic fungi (Aspergillus fumigatus, Candida albicans, Histoplasma Capsulatum (Histoplasma capsulatum); protozoa (Amoeba histolytica, Trichomonas tenas,Trichomonas hominis, Tryoanosoma gambiense, Trypanosoma rhodesiense, Leishmania donovani, Leishmania tropica, Leishmania braziliensis, Pneumocystis pneumonia, Plasmodium vivax, Plasmodium falciparum, Plasmodium malaria); Or it is additionally desirable to be able to counter pathogenic strains including, but not limited to, Helminith (Schistosoma japonicum, Schistosoma mansoni, Schistosoma haematobium and hookworm).

[0339] Further conjugates of the present invention are intended for the treatment of viral diseases, and include, but are not limited to, the following pathogenic viruses: Poxyiridae, Herpesviridae, Adenoviridae, Papovaviridae, Enteroviridae, Picornaviridae, Parvoviridae, Reoviridae, Retroviridae, influenza virus, parainfluenza virus, mumps, measles, respiratory syncytial virus, rubella, Arboviridae, Rhabdoviridae, Arenaviridae, non-A / non-B hepatitis virus, Rhinoviridae, Coronaviridae, Rotoviridae, oncoviruses [e.g., HBV (hepatocellular carcinoma), HPV (cervical cancer, anal cancer), Kaposi's sarcoma-associated herpes virus (Kaposi's sarcoma), Epstein-Barr virus (nasopharyngeal carcinoma, Burkitt lymphoma, primary central nervous system lymphoma), MCPyV (Merkel cell carcinoma), SV40 (Simian virus 40), HCV (hepatocellular carcinoma), HTLV-I (adult T-cell leukemia / lymphoma)], immune-induced viruses: [e.g., Human Immunodeficiency Virus (AIDS)]; Central nervous system viruses: [e.g., JCV (progressive multifocal leukocytosis), MeV (subacute sclerosing meningitis), LCV (lymphocytic choriomeningitis), Arbovirus encephalitis, Orthomyxoviridae (possible) (lethargic encephalitis), RV (rabies), herpesvirus meningitis, Ramsay Hunt syndrome type II; poliovirus (poliomyelitis, post-poliomiasis syndrome), HTLV-I (tropical hard paralysis)]; cytomegalovirus (cytomegalovirus retinitis, HSV (herpetic keratitis)); cardiovascular viruses [e.g., CBV (pericarditis, myocarditis)];Respiratory / Acute Viral Nasopharyngitis / Viral Pneumonia: [Epstein-Barr Virus (EBV Infection / Infectious Mononucleosis), Cytomegalovirus; SARS coronavirus (Severe Acute Respiratory Syndrome), Orthomyxoviriformes: Influenza A / B / C (Influenza / Avian Influenza), Paramyxovirus: Human Parainfluenza Virus (Parainfluenza), RSV (Human Respiratory Syntypal Virus, hMPV)]; Digestive system viruses [MuV (Mumps), Cytomegalovirus (Cytomegalovirus esophagitis); Adenovirus (Adenovirus infection); Rotavirus, Novovirus, Astrovirus, Coronavirus; HBV (Hepatitis B), CBV, HAV (Hepatitis A), HCV (Hepatitis C), HDV (Hepatitis D), HEV (Hepatitis E), HGV (Hepatitis G)]; Genitourinary viruses [e.g., BK virus, MuV (Mumps)].;

[0340] Depending on additional purposes, the present invention also relates to a pharmaceutical composition comprising a conjugate of the present invention, together with a pharmaceutically acceptable carrier, diluent, or excipient, for the treatment of cancer, infection, or autoimmune disorder. Methods for treating cancer, infection, and autoimmune disorder may be performed in vitro, in vivo, or in vitro. Examples of in vitro use include the treatment of cell cultures to kill all cells except for the target variant that does not express the target antigen, or to kill variants that express the untarget antigen. Examples of in vitro use include the step of treating hematopoietic stem cells (HSCs) before performing transplantation (HSCT) on the same patient to kill diseased cells or malignant cells. For example, in vitro treatment to remove tumor cells or lymphocytes from bone marrow prior to autologous transplantation in the treatment of cancer or autoimmune disease, or in vitro treatment to remove T cells and other lymphocytes from allogeneic bone marrow prior to transplantation to prevent graft-versus-host disease, may be performed as follows. Bone marrow is collected from a patient or other individual and then incubated in a medium containing serum to which the conjugate of the present invention is added in a concentration range of about 1 pM to 0.1 mM at about 37°C for about 15 minutes to about 48 hours. The precise concentration conditions and incubation time (=dose) are easily determined by a skilled clinician. After incubation, the bone marrow cells are washed with a medium containing serum and returned to the patient by IV infusion according to known methods. In situations where the patient receives other treatment, such as a course of ablative chemotherapy or total body irradiation, between the collection of bone marrow and the re-infusion of the treated cells, the treated bone marrow cells are frozen in liquid nitrogen using standard medical equipment.

[0341] Chemotherapy drugs / cytotoxic agents for use as payloads for synergistic effects or for conjugates having linkers

[0342] A chemotherapy drug that may be used as a payload for the conjugation of the present invention or in combination with the conjugate of the present invention for synergistic treatment is a small molecule drug comprising a cytotoxic agent. The term “small molecule drug” is used herein broadly to refer to organic, inorganic, or organometallic compounds that may have a molecular weight of, for example, 100 to 2500, more suitably 200 to 2000. Small molecule drugs are particularly, for example, WO05058367A2 and U.S. Patent No. 4,956,303 and literature [Chessum, N., et al, Prog Med Chem. 2015, 54: 1-63; Eder, J., et al, Nat Rev Drug Discov. 2014, 13(8): 577-87; Zhang, M.-Q., et al, Curr Opin Biotechnol. As in [2007, 18(6): 478-88], the relevant art field is well characterized, and all of these are incorporated herein by reference. Drugs include known drugs and drugs that may be known drugs.

[0343] The disclosed cytotoxic drugs include, but are not limited to, the following:

[0344] (1) Chemotherapy agents: a) Alkylating agents: e.g., nitrogen mustard: chlorambucil, chlornafazine, cyclophosphamide, dacarbazine, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, mannomerstin, mitobronitol, melphalan, mitolactol, pipovoromin, novelichine, phenesterine, prednimustine, thiotepa, trophosphamide, uracil mustard; CC-1065 (including synthetic analogs of adezelesin, caselesin and bisezelesin thereof); duocamycin (including synthetic analogs, KW-2189 and CBI TMI); Benzodiazepine dimers (e.g., pyrrolobenzodiazepine (PBD) or tomymycin dimers, indolinobenzodiazepine, imidazobenzothiadiazepine, or oxazolidinobenzodiazepine dimers); nitrosoureas: (camustine, lomustine, chlorozotosine, potemustine, nimustine, ranimustine); alkylsulfonates: (busulfan, threosulfan, improsulfan, and piposulfan); triazenes: (dacarbazine); platinum-containing compounds (carboplatin, cisplatin, oxaliplatin); aziridines, e.g., benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylamellamines, including altretamine, triethylenemelamine, triethylenephosphamide, triethylenethiophosphamide, and trimethylolomellamine; b) Plant alkaloids: e.g., vinca alkaloids: (vincristine, vinblastine, vindecin, vinorelbine, nabelbine); taxoids: (paclitaxel, docetaxol) and their analogs, metansinoids (DM1, DM2, DM3, DM4, metansine, and ansamitosine) and their analogs, cryptophysin (particularly cryptophysin 1 and cryptophysin 8); epotillone, eleuterobin, discodermolid, bryostatin, dolostatin, auristatin, mamatoxin, cephalostatin; pancratistatin; sarcodictin; spongestatin;c) DNA topoisomerase inhibitors: e.g. [Epipodophilinse: (9-aminocamptothecin, camptothecin, crinatol, daunomycin, etoposide, etoposide phosphate, irinotecan, mitoxantrone, novantrone, retinoic acid (retinol), tenifoside, topotecan, 9-nitrocamptothecin (RFS 2000)); mitomycin (mitomycin C)]; d) Anmetanosides: e.g., {[Anti-folicates: DHFR inhibitors: (methotrexate, trimetrexate, denopterin, pteropterin, aminopterin (4-aminopteric acid) or other folic acid analogs); IMP dehydrogenase inhibitors: (mycophenolic acid, thiazofurine, ribavirin, EICAR); Ribonucleotide reductase inhibitors: (hydroxyurea, deferoxamine)]; [pyrimidine analogs: Uracil analogs: (ancitabine, azacitidine, 6-azauridine, capecitabine (Xeloda), camoper, cytarabine, dideoxyuridine, doxyfluridine, enositabine, 5-fluorouracil, florouridine, latitrexed (Tomudex)); cytosine analogs: (cytarabine, cytosine arabinoside, fludarabine); purine analogs: (azathioprine, fludarabine, mercaptopurine, thiaminephrine, thioguanine)]; folic acid supplements, e.g., proline acid}; e) Hormonal therapy: e.g., {receptor antagonists: [anti-estrogens: (megestrol, raloxifene, tamoxifen); LHRH agonists: (goscrclin, leuprolide acetate); anti-androgens: (bicalutamide, flutamide, calosterone, dromostanolone propionate, epithiostanol, goserelin, leuprolide, mefitiostan, nilutamide, testolactone, trilostan, and other androgen inhibitors)]; retinoids / deltoids: [Vitamin D3 analogs: (CB 1093, EB 1089, KH 1060, cholecalciferol, ergocalciferol); photodynamic therapy: (vertporfin, phthalocyanine, photosensitizer Pc4, demethoxyhypoquellin A);Cytokines: (Interferon-alpha, Interferon-gamma, Tumor Necrosis Factor (TNF), Human proteins containing the TNF domain)]}; f) Kinase inhibitors: e.g., BIBW 2992 (anti-EGFR / Erb2), imatinib, gefitinib, pegaptanib, sorafenib, dasatinib, sunitinib, erlotinib, nilotinib, lapatinib, axitinib, pazopanib, vandetanib, E7080 (anti-VEGFR2), mubritinib, ponatinib (AP24534), bafetinib (INNO-406), bosutinib SKI-606), cabozantinib, bismodezip, iniparib, ruxolitinib, CYT387, axitinib, tibozanib, sorafenib, bevacizumab, cetuximab, trastuzumab, ranibizumab, panitumumab, ispinesisib; g) Poly(ADP-ribose) polymerase (PARP) inhibitors: e.g., olaparib, niraparib, iniparib, talazoparib, veliparib, CEP 9722 (Cephalon), E7016 (Eisai), BGB-290 (BeiGene), or 3-aminobenzamide;

[0345] h) Antibiotics, e.g., enediin antibiotics (e.g., caliceamicin, particularly, caliceamicin γ1, δ1, α1 or β1 (e.g., literature [ J. Med. Chem.,[Reference: 39 (11), 2103-2117 (1996), Angew Chem Intl. Ed. Engl. 33:183-186 (1994)]); dynemycins including dynemycin A and deoxydynemycin; esperamicin, kedarcidin, C-1027, maduropeptide, as well as neocarzinostatin chromophores and related chromoprotein enedyin antibiotic chromophores); aclaxinomycin, actinomycin, otramycin, azacerin, bleomycin, cactinomycin, carrabicin, caminophylline; Cromomycin, Dactinomycin, Daunorubicin, Detorubicin, 6-Diazoo-5-oxo-L-norleucine, Doxorubicin, Morfollino-Doxorubicin, Cyanomorpholino-Doxorubicin, 2-Pyrrolino-Doxorubicin and Deoxydoxorubicin, Epirubicin, Esorubicin, Idarubicin, Marcelomycin, Nitomycin, Mycophenolic acid, Nogalamycin, Olivomycin, Peflomycin, Popperomycin, Puromycin, Quellamycin, Rhodorubicin, Streptonigreen, Streptozosin, Tubercidin, Uvenimex, Gynostatin, Rhodorubicin;i) Others: e.g., polyketides (acetogenins), particularly, bulatacin and bulatacinone, gemcitabine, epoxomycin (e.g., carfilzomib), bortezomib, thalidomide, lenalidomide, pomalidomide, tosedostat, zibrestat, PLX4032, STA-9090, Stimuvax, allovectin-7, Xegeva, Provenge, Yervoy, protein lipidation inhibitors (e.g., lovastatin), dopaminergic neurotoxins (e.g., 1-methyl-4-phenylpyridinium ion), cell cycle inhibitors (e.g., staurosporine), actinomycin (e.g., actinomycin D, dactinomycin), bleomycin (e.g., bleomycin A2, bleomycin B2, peplomycin), anthracyclines (e.g., daunorubicin, doxorubicin (adriamycin), idarubicin, epirubicin, pirarubicin, zorubicin, emtoxantrone, MDR inhibitors (e.g., verapamil), Ca2+ ATPase inhibitors (e.g., topsigargin), histone deacetylase inhibitors (vorinostat, romidepsin, panobinostat, valproic acid, mosetinostat (MGCD0103), velinostat, PCT-24781, entinostat, SB939, resminostat, zibinostat, AR-42, CUDC-101, sulforaphane, tricostatin A); topsigargin, celecoxib, glitazone, epigallocatechin gallate, disulfiram, Salinosporamide A; anti-adrenergics, e.g., aminoglutothymide, mithotan, trilostan; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; arabinoside, bestabucil; arsentren; edatlaxate; depopamine; demecolsin; diaziquone; eflonitin (DFMO), l-formitin; elliptinium acetate, etoglucid; gallium nitrate, kasaitosine, hydroxyurea; ibandronate, lentinan; ronidamine; mithoguazone; mitoxantrone; mophidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxic acid; lyzoxin; sizopyran; spirogermanium; Tenuazonic acid; triaziquone;2,2',2"-trichlorotriethylamine; trichotesene (particularly T-2 toxin, verucarin A, loridin A, and anguidin); urethanes, siRNA, antisense drugs; and nucleases.;

[0346] 2) Anti-autoimmune disease agents include, but are not limited to, cyclosporine, cyclosporine A, aminocaproic acid, azathioprine, bromocriptine, chlorambucil, chloroquine, cyclophosphamide, corticosteroids (e.g., ammonide, betamethasone, budesonide, hydrocortisone, flunisolide, fluticasone propionate, flucotolone danazol, dexamethasone, triamcinolone acetonide, beclomethasone dipropionate), DHEA, enanacept, hydroxychloroquine, infliximab, meroxicam, methotrexate, mofetil, mycophenylate, prednisone, sirolimus, and tacrolimus.

[0347] 3) Anti-infectious agents include, but are not limited to, the following: a) Aminoglycosides: Amikacin, Astromycin, Gentamicin (Netylmycin, Cisomycin, Isefamicin), Hygromycin B, Kanamycin (Amikacin, Arbekacin, Bekanamycin, Dibekacin, Tobramycin), Neomycin (Pramycetin, Paromomycin, Ribostamycin), Netylmycin, Spectinomycin, Streptomycin, Tobramycin, Verdamycin; b) Amphenicol: Azidafenicol, Chloramphenicol, Flofenicol, Thiamphenicol; c) Ansamicin: Zeldanamycin, Herbimycin; d) Carbapenems: Viapenem, Doripenem, Ertapenem, Imipenem / Cilastatin, Meropenem, Panipenem; e) Cephem: Carbacepem (Loracarbef), Cepacetril, Cefaclor, Cefradyn, Cefadroxyl, Cephalonium, Cephaloridine, Cephalotine or Cephalosin, Cephalexin, Cephaloglycin, Cefamandol, Cefapyrin, Cefatrizine, Cefazaflu, Cefazedone, Cefazolin, Cefubuperazone, Cefcapen, Cefdaloxim, Cefepim, Cefminox, Cefoxitin, Cefprozil, Cefroxadine, Ceftezol, Cefuroxim, Cefixim, Cefdinier, Cefditoren, Cefepim, Ceftamet, Cefmenoxim, Cefodizyme, Sefofenid, Cefoperazone, Sephoranide, Cefotaxim, Cefotiam, Cefozofran, Cephalexin, Cefpimizole, Cefpyramide, Cefpirom, Cefpodoxim, Cefprozil, Cefquinome, cefsulodin, ceftazidime, cefteramm, ceftibuten, ceftiolene, ceftizoxime, ceftobiprol, ceftriaxone, cefuroxime, cefuzonam, cefamycin (cefoxitin, cefoctetane, cefmethazole), oxacepem (flomoxef, latamoxef); f) Glycopeptides: bleomycin, vancomycin (oritavancine, telavancine), teicoplanin (dalbavancine), lamoplanin; g) Glycylcyclines: e.g., tigecycline; g) β-lactamase inhibitors: fenam (sulbactam, tazobactam), clavam (clavulanic acid); i) Lincosamides: clindamycin, lincomycin; j) Lipopeptides: daptomycin, A54145, calcium-dependent antibody (CDA);k) Macrolides: Azithromycin, Setromycin, Clarithromycin, Diristromycin, Erythromycin, Flurithromycin, Yosamicin, Ketolid (Telithromycin, Setromycin), Midecamicin, Myocamicin, Oleandomycin, Rifamycin (Rifampicin, Rifampin, Rifabutin, Ripapeptin), Rokitamicin, Roxithromycin, Spectinomycin, Spiramycin, Tacrolimus (FK506), Troleandomycin, Telithromycin; l) Monobactams: Aztreonam, Tigemonam; m) Oxazolidinones: Linezolid; n) Penicillin: Amoxicillin, Ampicillin (Pivampicillin, Hetacillin, Bacampicillin, Metampicillin, Talampicillin), Azidocillin, Azolocillin, Benzylpenicillin, Benzathine Benzylpenicillin, Benzathine Phenoxymethylpenicillin, Clomethocillin, Procaine Benzylpenicillin, Carbenicillin (Carindacillin), Cloxacillin, Dicloxacillin, Epicillin, Flucloxacillin, Mesillinam (Pibmesillinam), Mezolocillin, Methicillin, Nafcillin, Oxacillin, Phenamethicillin, Penicillin, Penethicillin, Phenoxymethylpenicillin, Piperacillin, Propicillin, Sulbenicillin, Temocillin, Ticarcillin; o) Polypeptide: Bacitracin, Colistin, Polymyxin B; p) Quinolones: Allatrofloxacin, Valofloxacin, Ciprofloxacin, Clinafloxacin, Danofloxacin, Difloxacin, Enoxacin, Enrofloxacin, Floxacin, Garenoxacin, Gatifloxacin, Gemifloxacin, Grepafloxacin, Carnotrobafloxacin, Levofloxacin, Lomefloxacin, Marbofloxacin, Moxifloxacin, Nadifloxacin, Norfloxacin, Orbifloxacin, Ofloxacin, Pefloxacin, Trovafloxacin, Grepafloxacin, Sitafloxacin, Spafloxacin, Temafloxacin, Tosufloxacin, Trovafloxacin; q) Streptogramin: Pristinamycin, Quinupristine / Dalfopristin; r) Sulfonamides: Maffenide, Prontosil, Sulfacetamide, Sulfamethisol, Sulfanylimide, Sulfasalazine, Sulf-Isoxazole, Trimethoprim, Trimethoprim-Sulfamethoxazole (Co-Trimoxazole); s) Steroid antimicrobial agents: Fusidic acid;t) Tetracyclines: Doxycycline, Chlortetracycline, Chlormocycline, Demeclocycline, Limecycline, Meclocycline, Metacycline, Minocycline, Oxytetracycline, Phenimepicycline, Roliteteracycline, Tetracycline, Glycylcycline (e.g., including Tigecycline); u) Other types of antibiotics: Annonacin, Asphenamine, Bactofrenol inhibitors (Bacitracin), DADAL / AR inhibitors (Cycloserine), Dictiostatin, Discodermolid, Eleutherobin, Epotillon, Ethambutol, Etoposide, Paropenem, Fusidic acid, Furazolidone, Isoniazide, Raulimalide, Metronidazole, Mupirocin, Mycolactone, NAM synthesis inhibitors (e.g., Fosfomycin), Nitrofurantoin, Paclitaxel, Platensimicin, Pyrazinamide, Quinupristin / Dalfopristin, Rifampicin (Rifampin), Tazobactam, Tinidazole, Uvaricin.;

[0348] 4) Antiviral drugs: a) Influx / fusion inhibitors: afraviroc, maraviroc, vicriviroc, gp41 (enfuvirtide), PRO 140, CD4 (ibalizunab); b) Integrase inhibitors: raltegravir, elvitegravir, globoidnan A; c) Maturation inhibitors: bevirimat, vibecon; d) Neuraminidase inhibitors: oseltamivir, zanamivir, peramivir; e) Nucleosides and nucleotides: Abacavir, Acyclovir, Adefovir, Amdoxovir, Apricitabine, Brivudine, Cidofovir, Clevudine, Dexelbusitabine, Didanosine (ddI), Elbusitabine, Emtricitabine (FTC), Entecavir, Pamcyclovir, Fluorouracil (5-FU), 3'-fluoro-substituted 2',3'-Dideoxynucleoside analogs (e.g., 3'-fluoro-2',3'-Dideoxythymidine (FLT) and 3'-fluoro-2',3'-Dideoxyguanosine (FLG)), Formivirsen, Ganciclovir, Idoxuridine, Lamivudine (3TC), 1-nucleosides (β-1-thymidine and β-1,2'-Deoxycytidine), Pencyclovir, Lasivir, Ribavirin, Stampedine, Stavudine (d4T), Taribavirin (Viramidine), Telbivudine, Tenofovir, Trifluridine, Valacyclovir, Valgancyclovir, Zalcitabine (ddC), Zidovudine (AZT); f) Non-nucleoside: Amantadine, Arteviridine, Caplavirin, Diarylpyrimidine (Etravirin, Rilpivirin), Delavirdine, Docosanol, Emivirin, Efavirenz, Foscarnet (Phosponoformic Acid), Imiquimod, Interferon Alpha, Virilide, Rhodenosine, Methisazone, Nevirapine, NOV-205, Pegylated Interferon Alpha, Podophyllotoxin, Rifampicin, Rimantadine, Resiquimod (R-848), Tromantadine; g) Protease inhibitors: Amprenavir, atazanavir, boceprevir, darunavir, fosamprenavir, indinavir, lopinavir, nelfinavir, pleconaryl, ritonavir, saquinavir, telaprevir (VX-950), tiplanavir;h) Other types of antiviral drugs: Abzyme, Arbidol, Calanolide a, Seragenin, Cyanovirin-n, Diarylpyrimidine, Epigallocatechin Gallate (EGCG), Foscarnet, Griffithsin, Taribavirin (Viramidine), Hydroxyurea, KP-1461, Miltefosin, Pleconaryl, Portmanteau inhibitors, Ribavirin, Celicyclib.;

[0349] 5) Radioisotopes for radiation therapy. Examples of radioisotopes (radionuclides) are 3 H, 11 C, 14 C, 18 F, 32 P, 35 S, 64 Cu, 68 Ga, 86 Y, 99 Tc, 111 In, 123 I, 124 I, 125 I, 131 I, 133 Xe, 177 Lu, 211 At or 213It is Bi. Radioisotope-labeled antibodies may be useful for receptor-targeting imaging experiments or, for example, for targeted therapy using antibody-radioisotope conjugates (Wu et al (2005) Nature Biotechnology 23(9): 1137-46). Cell-binding molecules, e.g., antibodies, can be labeled with ligand reagents that bind to, chelate with, or otherwise complex with radioisotope metals using techniques described in the literature [Current Protocols in Immunology, Volumes 1 and 2, Coligen et al, Ed. Wiley-Interscience, New York, Pubs. (1991)]. Chelating ligands capable of complexing with metal ions include DOTA, DOTP, DOTMA, DTPA, and TETA (Macrocyclics, Dallas, Texas).

[0350] 6). Another cell-linked molecule-drug conjugate as a synergistic therapy. Desirable synergistic conjugates include tubulisin analogs, metansinoid analogs, taxanoid (taxane) analogs, CC-1065 analogs, daunorubicin and doxorubicin compounds, amatoxin analogs, benzodiazepine dimers (e.g., dimers of pyrrolobenzodiazepine (PBD), tomycin, antramycin, indolinobenzodiazepine, imidazobenzothiadiazepine, or oxazolidinobenzodiazepine), caliceamicin and enedyin antibiotic compounds, actinomycin, azacerin, bleomycin, epirubicin, tamoxifen, idarubicin, dolastatin, and auristatin (e.g., monomethylauristatin E, MMAE, MMAF, auristatin PYE, auristatin TP, auristatin 2-AQ, 6-AQ, EB). It may be a conjugate having cytotoxic agents of (AEB) and EFP(AEFP)), Duokamycin, Zeldanamycin or Methotrexate, Thiotepa, Vindecin, Vincristine, Hemiasterin, Nazumamide, Microginine, Radiosumin, Alterobacterin, Microsclerodermin, Theonellamide, Esperamicin, PNU-159682; and analogs and derivatives thereof.

[0351] 7) Pharmaceutically acceptable salts, acids, or derivatives of any of the above drugs.

[0352] In another additional embodiment, an immunotoxin may be conjugated to a cell-binding molecule through the linker of the present invention. As specified herein, an immunotoxin is a macromolecular drug, typically a cytotoxic protein derived from bacteria or plant proteins, such as diphtheria toxin (DT), cholera toxin (CT), trichosanthin (TCS), dianthin, Pseudomonas exotoxin A (ETA'), erythrogenic toxin, diphtheria toxin, AB toxin, type III exotoxin, etc. It may also be a highly toxic pore-forming protoxin that requires proteolytic processing for activation. Examples of such protoxins are proaerolysin and its genetically modified form, topsallysin. Topsallysin is a modified recombinant protein engineered to be selectively activated by enzymes within the prostate, inducing localized apoptosis and tissue destruction without damaging neighboring tissues and nerves.

[0353] In another synergistic immunotherapy, antibodies of immune checkpoint inhibitors, TCR (T cell receptor) T cells or CAR (chimeric antigen receptor) T cells or B cell receptors (BCR), natural killer (NK) cells or cytotoxic cells or anti-CD3, CD4, CD8, CD16 (FcγRIII), CD19, CD20, CD22, CD25, CD27, CD30, CD33, CD37, CD38, CD40, CD40L, CD45RA, CD45RO, CD56, CD57, CD57 bright Antibodies of CD70, CD79, CD79b, CD123, CD125, CD138, TNFβ Fas ligand, MHC class I molecules (HLA-A, B, C), VEGF, or NKR-P1 are preferred for use with the conjugate of this patent for synergistic therapy.

[0354] Formulations and Applications

[0355] The conjugate of the present application is suitable for being formulated into a liquid or for being reconstituted into a liquid formulation after freeze-drying. The conjugate in the liquid formulation or the formulated freeze-dried powder may constitute 0.01% to 99% by weight as the main component in the formulation. Generally, a liquid formulation containing a conjugate active ingredient at a concentration of 0.1 g / ℓ to 300 g / ℓ for delivery to a patient without high levels of antibody agglutination may comprise one or more polyols (e.g., sugars), a buffer of pH 4.5 to 7.5, a surfactant (e.g., polysorbate 20 or 80), an antioxidant (e.g., ascorbic acid and / or methionine), an isotonic agent (e.g., mannitol, sorbitol or NaCl), a chelating agent, e.g., EDTA; a metal complex (e.g., a Zn-protein complex); a biodegradable polymer, e.g., a polyester; a preservative (e.g., benzyl alcohol) and / or free amino acids.

[0356] Buffers suitable for use in the formulation include organic acid salts, e.g., sodium, potassium, ammonium, or trihydroxyethylamino salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid; and buffers of Tris, tromethamine hydrochloride, sulfate, or phosphate, but are not limited thereto. Additionally, amino acid components may also be used as buffers. Such amino acid components include, but are not limited to, arginine, glycine, glycylglycine, and histidine. Arginine buffers include arginine acetate, arginine chloride, arginine phosphate, arginine sulfate, arginine succinate, etc. In one embodiment, the arginine buffer is arginine acetate. Examples of histidine buffers include histidine chloride-arginine chloride, histidine acetate-arginine acetate, histidine phosphate-arginine phosphate, histidine sulfate-arginine sulfate, histidine succinate-arginine succinate, etc. The formulation of the buffer has a pH of 4.5 to 7.5, preferably about 4.5 to about 6.5, more preferably about 5.0 to about 6.2. In some embodiments, the concentration of the organic acid salt in the buffer is about 10 mM to about 500 mM.

[0357] "Polyols," which may be optionally included in the formulation, are substances having multiple hydroxyl groups. Polyols can be used to stabilize excipients and / or isotonic agents in both liquid and lyophilized formulations. Polyols can protect biopharmaceuticals from both physical and chemical degradation pathways. Preferably, the excluded cosolvent increases the effective surface tension of the solvent at the protein interface, thereby ensuring that the most energy-preferred structural stereolocation has the smallest surface area. Polyols include sugars (reducing and non-reducing sugars), sugar alcohols, and sugar acids. "Reducing sugars" are those containing a hemiacetal group capable of reducing metal ions or covalently reacting with lysine and other amino groups within proteins, while "non-reducing sugars" are those that do not possess the characteristics of reducing sugars. Examples of reducing sugars are fructose, mannose, maltose, lactose, arabinose, xylose, ribose, rhamnose, galactose, and glucose. Non-reducing sugars include sucrose, trehalose, sorbose, melesitose, and raffinose. Sugar alcohols are selected from mannitol, xylitol, erythritol, maltitol, lactitol, erythritol, threitol, sorbitol, and glycerol. Sugar acids include L-gluconates and metal salts thereof. The polyol in the liquid formulation or the formulated lyophilized solid may be 0.0% to 20% by weight. Preferably, non-reducing sugars at a concentration of 0.1% to 15%: sucrose or trehalose are selected in the formulation, wherein trehalose is preferred over sucrose due to the solution stability of trehalose.

[0358] The surfactant in the formulation is optionally a polysorbate (polysorbate 20, polysorbate 40, polysorbate 65, polysorbate 80, polysorbate 81, polysorbate 85, etc.); a poloxamer (e.g., poloxamer 188, poly(ethylene oxide)-poly(propylene oxide), poloxamer 407, or polyethylene-polypropylene glycol, etc.); Triton; sodium dodecyl sulfate (SDS); sodium laurel sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl-, or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; Lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g., lauroamidopropyl); myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodium methyl oleyl-taurate; dodecyl betaine, dodecyl dimethylamine oxide, cocamidopropyl betaine and cocoamphoglycinate; and MONAQUAT TM It is selected from a series (e.g., isostearyl ethylimidonium ethossulfate); polyethyl glycol, polypropyl glycol, and copolymers of ethylene and propylene glycol (e.g., Pluronic, PF68, etc.). A preferred surfactant is a polyoxyethylene sorbitan fatty acid ester, e.g., polysorbate 20, 40, 60, or 80 (Tween 20, 40, 60, or 80). The concentration of the surfactant in the formulation is in the range of 0.0% to about 2.0%. In certain embodiments, the surfactant concentration is about 0.01% to about 0.2%. In one embodiment, the surfactant concentration is about 0.02%.

[0359] The “preservative” in the formulation is optionally a compound that essentially reduces bacterial activity therein. Examples of possible preservatives include octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chloride in which the alkyl group is a long-chain compound), and benzethonium chloride. Other types of preservatives include aromatic alcohols, e.g., phenol, butyl, and benzyl alcohols; alkyl parabens, e.g., methyl or propyl parabens; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol. The amount of the preservative in the liquid formulation or the formulated lyophilized powder may be 0.0% by weight to 5.0% by weight. In one embodiment, the preservative herein is benzyl alcohol.

[0360] Free amino acids suitable as bulk material, isotonic agent, or osmotic pressure regulator in the formulation are selected from, but are not limited to, one or more of, arginine, cystine, glycine, lysine, histidine, ornithine, isoleucine, leucine, alanine, glycine glutamic acid, or aspartic acid. The inclusion of basic amino acids, namely arginine, lysine, and / or histidine, is preferred. If the composition contains histidine, it may act as both a buffer and a free amino acid; however, if a histidine buffer is used, it is typical to include a histidine buffer and lysine to include non-histidine free amino acids, for example. Amino acids may be present in D-form and / or L-form, but the L-form is typical. Amino acids may be present as any suitable salt, for example, a hydrochloride, e.g., arginine-HCl. The amino acid content in the liquid formulation or the formulated lyophilized powder may be 0.0% by weight to 30% by weight.

[0361] The formulation may optionally include methionine, glutathione, cysteine, cystine, or ascorbic acid as an antioxidant at a concentration of up to about 5 mg / ml in the liquid formulation or at a concentration of 0.0% to 5.0% by weight in the formulated freeze-dried powder; the formulation may optionally include a metal chelating agent, e.g., EDTA, EGTA, etc., at a concentration of up to about 2 mM in the liquid formulation or at a concentration of 0.0% to 0.3% by weight in the formulated freeze-dried powder.

[0362] The final formulation can be adjusted to a desired pH using a buffer modifier (e.g., an acid, e.g., HCl, H2SO4, acetic acid, H3PO4, citric acid, etc., or a base, e.g., NaOH, KOH, NH4OH, ethanolamine, diethanolamine or triethanolamine, sodium phosphate, potassium phosphate, trisodium citrate, tromethamine, etc.), and the formulation must be controlled to be "isotonic," which means that the formulation of interest has an osmotic pressure essentially the same as human blood. An isotonic formulation generally has an osmotic pressure of about 250 to 350 mOsm. Isotonicity can be measured, for example, using a vapor pressure or ice-freezing type osmometer. The isotonic agent is selected from mannitol, sorbitol, sodium acetate, potassium chloride, sodium phosphate, potassium phosphate, trisodium citrate, or NaCl. Generally, buffer salts and isotonic agents can account for 30% by weight of the formulation.

[0363] Other excipients that may be useful in the liquid or lyophilized formulations of this patent application are, for example, fucose, cellobiose, maltotriose, melivise, octulose, ribose, xylitol, arginine, histidine, glycine, alanine, methionine, glutamic acid, lysine, imidazole, glycylglycine, mannosylglycerate, Triton X-100, Pluronic F-127, cellulose, cyclodextrin, (2-hydroxypropyl)-β-cyclodextrin, dextran (10, 40 and / or 70 kD), polydextrose, maltodextrin, picol, gelatin, hydroxypropylmethicone, sodium phosphate, potassium phosphate, ZnCl2, zinc, zinc oxide, sodium citrate, trisodium citrate, tromethamine, copper, fibronectin, heparin, human serum albumin, It includes protamine, glycerin, glycerol, EDTA, metacresol, benzyl alcohol, phenol, polyhydric alcohol, or polyalcohol, and hydrogenated forms of carbohydrates having a carbonyl group reduced to a primary or secondary hydroxyl group.

[0364] Other excipients considered for use in the aqueous pharmaceutical compositions of this patent application include, for example, flavorings, antimicrobial agents, sweeteners, antioxidants, antistatic agents, lipids, e.g., phospholipids or fatty acids, steroids, e.g., cholesterol, protein excipients, e.g., serum albumin (human serum albumin), recombinant human albumin, gelatin, casein, salt-forming counterions, e.g., sodium, etc. Such and additional known pharmaceutical excipients and / or additives suitable for use in the formulations of the present invention are known in the relevant art, for example, in the literature [The Handbook of Pharmaceutical Excipients, 4 th edition, Rowe et al., Eds., American Pharmaceuticals Association (2003); and Remington: the Science and Practice of Pharmacy, 21 thIt is listed in edition, Gennaro, Ed., Lippincott Williams & Wilkins (2005).

[0365] A pharmaceutical container or vessel is used to hold a pharmaceutical formulation of any of the conjugates of this patent application. The vessel is a vial, bottle, pre-filled syringe, or pre-filled or auto-injection syringe. Liquid formulations may be freeze-dried or drum-dried in the form of a cake or powder in borosilicate vials or soda-lime glass vials. Solid powders may also be prepared by efficient spray drying and subsequently held in vials or pharmaceutical containers for storage and distribution.

[0366] In a further embodiment, the present invention provides a method for preparing a formulation comprising: (a) freeze-drying a formulation comprising a conjugate, an excipient, and a buffer system; and (b) reconstituting the freeze-dried mixture of step (a) in a reconstitution medium so that the reconstituted formulation is stable. The formulation of step (a) may further comprise one or more excipients and stabilizers selected from the group comprising bulking agents, salts, surfactants, and preservatives as described above. As the reconstitution medium, some diluted organic acids or water, namely sterile water or bacteriostatic water for injection (BWFI), may be used. The reconstitution medium may be selected from the group consisting of water, namely sterile water or bacteriostatic water for injection (BWFI), or acetic acid, propionic acid, succinic acid, sodium chloride, magnesium chloride, acidic solution of sodium chloride, acidic solution of magnesium chloride, and acidic solution of arginine (in an amount of about 10 to about 250 mM).

[0367] The liquid pharmaceutical formulation of the conjugate of the present patent application must exhibit various predefined characteristics. One of the major issues with liquid drug products is stability, as proteins / antibodies tend to form soluble and insoluble aggregates during storage. Additionally, various chemical reactions may occur in solution (deamideation, oxidation, clipping, isomerization, etc.), which can lead to increased levels of product degradation and / or loss of biological activity. Preferably, the conjugate in liquid or lyophilized form must exhibit a shelf life of more than 6 months at 25°C. More preferably, the conjugate in liquid or lyophilized form must exhibit a shelf life of more than 12 months at 25°C. Most preferably, the liquid formulation must exhibit a shelf life of 24 to 36 months at 2 to 8°C, and the lyophilized formulation must exhibit a shelf life of approximately preferably up to 60 months at 2 to 8°C. Both the liquid formulation and the lyophilized formulation must exhibit a half-life of at least 2 years at -20°C or -70°C.

[0368] In certain embodiments, the formulation is stable after freezing (e.g., -20°C, or -70°C) and thawing of the formulation, e.g., 1, 2, or 3 cycles of freezing and thawing. Stability is evaluated by the drug / antibody (protein) ratio and aggregate formation (e.g., by UV, size exclusion chromatography, measurement of turbidity, and / or visual inspection); evaluation of charge heterogeneity using cation exchange chromatography, image capillary isoelectric focusing (icIEF), or capillary zone electrophoresis; amino-terminal or carboxy-terminal sequence analysis; mass spectrometry analysis or matrix-assisted laser desorption ionization / time-of-flight mass spectrometry (MALDI / TOF MS) or HPLC-MS / MS; SDS-PAGE for comparison of reduced antibodies and intact antibodies; Instability may be evaluated qualitatively and / or quantitatively in various different ways, including peptide map (e.g., tryptic or LYS--C) analysis; evaluation of the biological activity or antigen-binding function of the antibody, etc. Instability may include any one or more of the following: agglutination, deamideation (e.g., Asn deamideation), oxidation (e.g., Met oxidation), isomerization (e.g., Asp isomerization), clipping / hydrolysis / fragmentation (e.g., hinge region fragmentation), succinimide formation, uncoupled cysteine(s), N-terminal elongation, C-terminal processing, glycosylation difference, etc.

[0369] A stable conjugate must also "maintain biological activity" in a pharmaceutical formulation, and, for example, when determined by antigen binding assays and / or in vitro cytotoxicity assays, the biological activity of the conjugate must have a difference of about 20%, preferably about 10% (within the margin of error of the assay), of the biological activity shown at the time the pharmaceutical formulation was manufactured, over a given time, for example, 12 months.

[0370] For in vivo clinical use, the conjugate via the linker of the present invention will be supplied as a solution or as a lyophilized solid that can be redissolved in sterile water for injection. Examples of suitable protocols for administering the conjugate are as follows. The conjugate is provided as an IV bolus on a daily, weekly, bi-weekly, 3-weekly, once every 4 weeks for 8 to 54 weeks, or monthly. The bolus dose is provided in 50 to 1000 ml of saline solution to which human serum albumin (e.g., 0.5 to 1 ml of a concentrated solution of human serum albumin, 100 mg / ml) may be optionally added. The dosage will be about 50 µg to 20 mg / kg body weight / week IV (ranging from 10 µg to 200 mg / kg per injection). At 4 to 54 weeks after treatment, a second course of treatment may be provided to the patient. Specific clinical protocols regarding the route of administration, excipients, diluents, dosage, time, etc., can be determined by a skilled physician.

[0371] Examples of medical conditions that can be treated by in vivo or in vitro methods of killing selected cell populations include cancer, autoimmune diseases, transplant rejection, and any type of malignant condition of infection (viral, bacterial, or parasitic).

[0372] The amount of conjugate required to achieve the desired biological effect will depend on a number of factors, including the chemical characteristics, potency, bioavailability, type of disease, species to which the patient belongs, the patient's disease state, route of administration, required dosage, and all factors describing the delivery and therapy to be administered.

[0373] Generally, the conjugate via the linker of the present invention may be provided in an aqueous physiological buffer containing 0.1 to 10% w / v conjugate for parenteral administration. Typical dose ranges are 1 mg / kg body weight to 0.1 g / kg body weight on a weekly; bi-weekly; 3-weekly; or monthly basis; and preferred dose ranges are 0.01 mg / kg body weight to 20 mg / kg body weight on a weekly; bi-weekly; 3-weekly; or monthly basis, equivalent to human doses. The preferred dose of the drug to be administered will depend on variables such as the type and degree of progression of the disease or disorder, the general health status of the specific patient, the relative biological efficacy of the selected compound, the formulation of the compound, the route of administration (intravenous, intramuscular, etc.), the pharmacokinetic characteristics of the conjugate by the selected delivery route, the administration rate (bolus or continuous infusion), and the schedule (number of repetitions over a given time period).

[0374] The conjugate via the linker of the present invention may also be administered in a unit dose form, wherein the term “unit dose” means a single dose that is maintained as a physically and chemically stable unit dose comprising the active conjugate itself or a pharmaceutically acceptable composition as described below, which can be administered to a patient and can be easily handled and packaged. As such, a typical total daily unit / weekly unit / bi-weekly unit / monthly unit dose range is 0.01 to 100 mg / kg body weight. According to general guidelines, for humans, the unit dose ranges from 1 mg to 3000 mg per day, week, 2 weeks (bi-weekly), 3 weeks, or month. Preferably, the unit dose range is 1 to 500 mg administered 1 to 4 times per month, and more preferably 1 mg to 100 mg administered once a week, once every two weeks, or once every three weeks. The conjugate provided herein may be formulated in a pharmaceutical composition by mixing with one or more pharmaceutically acceptable excipients. These unit dose compositions may be prepared for oral administration, particularly in the form of tablets, simple capsules, or soft gel capsules; or nasal use, particularly in the form of powders, nasal drops, or aerosols; or topical use through the skin, for example, as ointments, creams, lotions, gels, sprays, or transdermal patches.

[0375] In another additional embodiment, a pharmaceutical composition comprising a therapeutically effective amount of a conjugate of formula (I), (II), or (III) or any conjugate described herein may be administered concurrently with other therapeutic agents, such as chemotherapy agents, radiotherapy agents, immunotherapy agents, agents for autoimmune disorders, anti-infective agents, or other conjugates, for the synergistically effective treatment or prevention of cancer, autoimmune disease, or infectious disease. The synergistic agent is preferably selected from one or some of the following drugs: abatacept, abiraterone acetate, Abraxane, acetaminophen / hydrocodone, acalabrutinib, aducanumab, adalimumab, ADXS31-142, ADXS-HER2, afatinib diamaleate, aldesleukin, alectinib, alemtuzumab, alitretinoin, adotrastuzumab emtansine, amphetamine / dextroamphetamine, anastrozole, aripiprazole, anthracycline, aripiprazole, atazanavir, atezolizumab, atorvastatin, avelumab, Axicabtagene ciloleucel, axitinib, velinostat, BCG (raw), bevacizumab, bexarotene, blinatumomab, bortezomib, Bosutinib, Brentuximab Vedotin, Brigatinib, Budesonide, Budesonide / Formoterol, Buprenofine, Cabazitaxel, Cabozantinib, Capmatinib, Capecitabine, Carfilzomib, Chimeric Antigen Receptor-Operated T (CAR-T) Cells, Celecoxib, Ceritinib, Cetuximab, Sidamide, Cyclosporine, Cinacalcet, Crizotinib, Cobimetinib, Cosentyx, Crizotinib, CTL019, Dabigatran, Dabrafenib, Dacarbazine, Daclizumab, Dacomotinib, Daptomycin, Daratumumab, Darbepoetin Alpha, Darunavir, Dasatinib, Denileukin Diptitox, Denosumab, Depacote, Dexlansoprazole, Dexmethylphenidate, Dexamethasone, Dignicab Cooling System, Dinutuximab, Doxycycline, Duloxetine, Duvelisib, Durvalumab, Elotuzumab, Emtricitabine / Rilpivirine / Tenofovir, Disoproxil Fumarate, Emtricitabine / Tenofovir / Efavirenz,Enoxaparin, Ensatinib, Enzalutamide, Epoetin Alpha, Erlotinib, Esomeprazole, Eszopiclone, Etanercept, Everolimus, Exemestane, Everolimus, Exenatide ER, Ezetimibe, Ezetimibe / Simvastatin, Fenofibrate, Filgrastim, Fingolimod, Fluticasone Propionate, Fluticasone / Salmeterol, Fulvestrant, Gajiva, Gefitinib, Glatiramer, Goserelin Acetate, Icotinib, Imatinib, Ibritumomab Tiuxetane, Ibrutinib, Idelalisib, Ifosfamide, Infliximab, Imiquimod, ImmuCyst, Immuno BCG, Iniparib, Insulin Aspart, Insulin Detemia, Insulin Glargine, Insulin Lispro, Interferon Alpha, Interferon Alpha-1b, Interferon Alpha-2a, Interferon Alpha-2b, Interferon Beta, Interferon Beta 1a, Interferon Beta 1b, Interferon Gamma-1a, Lapatinib, Ipilimumab, Ipratropium Bromide / Salbutamol, Ixazomib, Kanuma, Lanreotide Acetate, Lenalidomide, Lenaliomide, Lenvatinib Mesylate, Letrozole, Levothyroxine, Levothyroxine, Lidocaine, Linezolid, Liraglutide, Risdexamphetamine, LN-144, Loratinib, Memantine, Methylphenidate, Metoprolol, Mekinist, Mericitabine / Rilpivirine / Tenofovir, Modalfinil, Mometasone, Missidac-C, Necitumumab, Neratinib, Nilotinib, Niraparib, Nivolumab, Ofatumumab, Obinutuzumab, Olaparib, Olmesartan, Olmesartan / Hydrochlorothiazide, Omalizumab, Omega-3 Fatty Acid Ethyl Ester, Oncorin, Oseltamivir, Osimertinib, Oxycodone, Palbociclib, Palivizumab, Panitumumab, Panobinostat, Pazopanib, Pembrolizumab, PD-1 Antibody, PD-L1 Antibody, Pemetrexed, Fetuzumab, Pneumococcal Conjugate Vaccine, Pomalidomide, Pregabalin, Proscarvax, Propranolol, Quetiapine, Rabeprazole, Radium 223 Chloride, Raloxifene, Raltegravir, Ramucirumab, Ranibizumab, Regorafenib, rituximab, rivaoxaban,Romidepsin, Rosuvastatin, Ruxolitinib Phosphate, Salbutamol, Sabolitinib, Semaglutide, Ceveramer, Sildenafil, Siltuximab, Cipulecel-T, Sitagliptin, Sitagliptin / Metformin, Solifenacin, Solanezumab, Sonidex, Sorafenib, Sunitinib, Tacrolimus, Tacrimus, Tadalafil, Tamoxifen, Tafinlar, Talimogene laherparepvec, Talazoparib, Telaprevir, Talazoparib, Temozolomide, Temsirolimus, Tenofovir / Emtricitabine, Tenofovir Diisoproxil Fumarate, Testosterone Gel, Thalidomide, TICE BCG, Tiotropium Bromide, Tisagenlecleucel, toremifene, trametinib, trastuzumab, travectedin (ecteinascidin 743), trametinib, tremelimumab, trifluridine / tipiracil, tretinoin, Uro-BCG, ustekinumab, valsartan, veliparib, vandetanib, vemurafenib, venetoclax, vorinostat, ziv-aflivercept, zostavax and their analogs, derivatives, their pharmaceutically acceptable salts, carriers, diluents, or excipients, or combinations thereof.

[0376] The drug / cytotoxic agent used for conjugation via the branched linker of the present patent may be any analogue and / or derivative of the amatoxin described in the present patent. Those skilled in the art of drugs / cytotoxic agents will readily understand that each amatoxin described herein may be modified in such a way that the resulting compound still retains the specificity and / or activity of the starting compound. Those skilled in the art will also understand that many of these analogue or derivative compounds may be used instead of the drug analogue described herein. Accordingly, the drug analogue of the present invention comprises a number of analogues or derivatives that may not be described in detail herein.

[0377] All references cited in this specification and the following examples are explicitly included by reference in their entirety.

[0378] Examples

[0379] The invention is further described in the following examples, which are not intended to limit the scope of the invention. Unless otherwise specified, the cell lines described in the following examples were maintained in cultures according to conditions specified by the American Type Culture Collection (ATCC), the German Society of Microbiology and Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany) (DMSZ), or the Shanghai Cell Culture Institute of the Chinese Academy of Science. Cell culture reagents were obtained from Invitrogen, Inc. unless otherwise specified. All anhydrous solvents were obtained commercially and stored in Sure-seal bottles under nitrogen. PEG compounds were purchased from Biomatrik Inc. (Jiaxing, China). Topotecan, meitansinol, MMAE, MMAF, exatecan, eribulin, and their derivatives or active ingredients were obtained from several commercial sources, e.g., Chengdu Tianyuan Natural Product Co., Ltd (Chengdu, China); Brightgene Biomedical Co. (Suzhou, China), etc. Experimental animals were purchased from international model animal resource centers through GemPharmatech Co., Ltd (Lajing, China) and Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China); T-DM1 was purchased from Roche through a pharmacy in Hong Kong, China. All other reagents and solvents were purchased at the highest available grade and used without further purification.HPLC separation for purification was performed using a Varain PreStar HPLC. NMR spectra were recorded on a Bruker 500 MHz instrument. Chemical shifts (delta) were recorded in parts per million (ppm) relative to tetramethylsilane at 0.00, and coupling constants (J) were recorded in Hz. Mass spectrum data were acquired from a Waters Xevo G2 QTOF mass spectrometer equipped with a Waters Acquity UPLC separation module and an Acquity TUV detector. Generally, UPLC separation was performed on a C8 column using mobile phase A, 1% formic acid, and phase B, 100% CH3CN.

[0380] Example 1. tert-butyl 2,5,8,11,14,17,20,23,26-nonaoxaoctacosan-28-oate ( 1 Synthesis of )

[0381]

[0382] NaH (60%, 8.0 g, 200 mmol) was added to a solution of mPEG8-OH (38.4 g, 100 mmol) in THF (1.0 L). After stirring at rt for 30 minutes, tert-butyl 2-bromoacetate (48.8 g, 250 mmol) was added to the mixture and stirred at rt for 1 hour. The mixture was then poured into ice water, extracted with DCM, the organic layer was washed with brine, and dried over anhydrous sodium sulfate. The compound was purified by column chromatography (0% to 5% methanol / dichloromethane). 1 was obtained as a yellow oil (27.6 g, 59% yield). ESI MS m / z 499.40 ([M+H] + ).

[0383] Example 2. 2,5,8,11,14,17,20,23,26-nonaoxaoctacosan-28-acid( 2 Synthesis of )

[0384]

[0385] compound1 (29.4 g, 59.0 mmol) was dissolved in DCM (400 mL), and then formic acid (600 mL) was added. The resulting solution was stirred overnight at 25°C. All volatile substances were removed under vacuum, which yielded the title compound as a yellow oil (26.1 g, >100% yield). C 19 H 39 O 11 [M+H] + Calculated ESI m / z for: 443.24, measured: 443.25.

[0386] Example 3. Compound 3 Synthesis of.

[0387]

[0388] compound 2 A solution of (59.0 mM) was dissolved in DCM (600 mL), and (COCl)2 (100 mL) and DMF (41 g, 0.59 mM) were added. The resulting solution was stirred at rt for 4 hours. All volatile substances were removed under vacuum to obtain the title compound as a yellow oil. ESI MS m / z 461.38 ([M+H] + ).

[0389] Example 4. Compound 4 Synthesis of.

[0390]

[0391] ZL-Lys-OH (33.1 g, 118.0 ml), Na2CO3 (18.7 g, 177.1 ml), and NaOH (4.7 g, 118.0 ml) were dissolved in water (700 ml). The mixture was cooled to 0°C, and the compound in THF (20 ml) to this 3A solution of (59.0 mM) was added. The resulting mixture was stirred at rt for 1 hour. THF was removed under vacuum, the solution was concentrated, and HCl was added to the aqueous solution under ice cooling until pH 3 was reached. After extraction with DCM, the organic layer was washed with brine, dried over sodium sulfate, and concentrated to provide the title compound as a yellow oil (44 g, 99% yield). C 33 H 57 N2O 14 [M+H] + Calculated ESI m / z for: 705.40, measured: 705.39.

[0392] Example 5. Compound 5 Synthesis of.

[0393]

[0394] compound 4 (20 g, 28.4 mmol, 1.0 eq) was dissolved in 350 ml of anhydrous DCM and cooled in an ice bath. NHS (3.9 g, 34.1 mmol, 1.2 eq) and EDC (27 g, 142.0 mmol, 5.0 eq) were added sequentially. The reaction mixture was stirred overnight at rt, then washed with water (200 ml × 2) and brine (200 ml × 1), dried over anhydrous sodium sulfate, and concentrated. The residue was dissolved in a small amount of DCM, loaded onto a silica gel column, and eluted with 2:49:49 to 4:48:48 MeOH / EA / DCM. The product was obtained as a yellow oil (14.2 g, 62% yield). C 37 H 60 N3O 16 [M+H] + ESI m / z Calculated value for: 802.4, Measured value: 802.4.

[0395] Example 6. (2S,4R)-5-(3-amino-4-hydroxyphenyl)-4-((tert-butoxycarbonyl)-amino)-2-methylpentanoic acid (compound) 7 Synthesis of )

[0396]

[0397] (2S,4R)-4-((tert-butoxycarbonyl)amino)-5-(4-hydroxy-3-nitrophenyl)-2-methylpentanoic acid (compound) in 150 ml of methanol 6 A mixture of )(15 g, 0.041 mol, 1.0 eq) and palladium on carbon (2.0 g, 10 wt%) was stirred under an H2 balloon at rt for 4 hours. The catalyst was filtered and washed with methanol. The filtrate was concentrated to yield 13.8 g of crude material, which was used directly in the next step (yield >100%). C 17 H 27 N2O5[M+H] + ESI m / z Calculated value for: 339.2, Measured value: 339.2.

[0398] Example 7. Compound 9 Synthesis of .

[0399]

[0400] The crude product from the previous step (13.8 g, 0.041 mol, 1.0 eq) was dissolved in 2 ml of ethanol, and 0.2 ml of 0.1 M NaH2PO4 4, 2,5-dioxopyrrolidine-1-yl 4-(((benzyloxy)-carbonyl)amino)-butanoate (15.0 g, 0.054 mol, 1.1 eq) was added. The mixture was stirred overnight, concentrated, redissolved in DCM, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by a silica gel column (0-5% MeOH / DCM) to yield a yellow oil (14.9 g, 66% yield). C 29 H 40 N3O8[M+H] + ESI m / z calculated value for: 558.3, measured value: 558.3.

[0401] Example 8. Compound 10 Synthesis of.

[0402]

[0403] Compounds in 100 ml of methanol 9 (8.7 g, 15.08 mmol, 1.0 eq) and palladium on carbon (1.0 g, 10 wt%) were stirred overnight at rt under an H2 balloon. The catalyst was filtered and washed with methanol. The filtrate was evaporated under vacuum to yield 6.4 g of crude material, which was used directly in the next step (yield >100%). C 21 H 34 N3O6[M+H] + ESI m / z Calculated value for: 424.2, Measured value: 424.2.

[0404] Example 9. Compound 11 Synthesis of.

[0405]

[0406] Compound in a mixture of 40 ml of ethanol and 10 ml of 0.1 M NaH2PO4 10 (6.4g, 15.1mol, 1.0eq) of compound 5 It was added to (12.7 g, 15.9 mmol, 1.05 eq). The mixture was stirred overnight, concentrated, dissolved in DCM, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by a silica gel column (3-5% MeOH / DCM) to yield a white foam (11.7 g, 70% yield). C 54 H 88 N5O 19 [M+H] + Calculated ESI m / z for: 1110.6, measured: 1110.6.

[0407] Example 10. Compound 12 Synthesis of.

[0408]

[0409] Compounds in 5 ml of methanol 11A mixture of (4.2 g, 3.79 mmol, 1.0 eq) and palladium on carbon (0.4 g, 10 wt%) was stirred overnight at rt under an H2 balloon. The catalyst was filtered and washed with methanol. The filtrate was evaporated to yield 0.32 g of crude material, which was used directly in the next step (yield 87%). C 46 H 82 N5O 17 [M+H] + Calculated ESI m / z for: January 1997, measured: January 1997.

[0410] Example 11. Meso-2,3-bis(benzylamino)succinic acid (compound) 13 Synthesis of )

[0411]

[0412] Benzylamine (150 ml) was added dropwise to a solution of meso-2,3-dibromosuccinic acid (50 g, 181 ml) in EtOH (400 ml). After addition, the mixture was heated to 90°C and stirred overnight. The mixture was cooled to rt and diluted with water. 6N HCl was added until a pH of 4 was reached, yielding a white precipitate. The precipitate was filtered, rinsed with water, and dried to yield meso-2,3-bis(benzylamino)succinic acid (50 g, 152 ml, 84%).

[0413] Example 12. Synthesis of meso-2,3-diaminosuccinic acid.

[0414]

[0415] Pd / C (3g, 10 wt%) was added to a solution of meso-2,3-bis(benzylamino)succinic acid (18g, 55 mMol) in AcOH (100 ml) and HCl (100 ml), and the mixture was stirred at 50°C for 48 hours under a 1 atm hydrogen atmosphere. The catalyst was removed by filtration and washed with water. The filtrate was concentrated, and the residue was dissolved in 1N NaOH (200 ml). Acetic acid was added until the pH reached 5, yielding a white precipitate. The precipitate was filtered, rinsed with water, and dried to yield meso-2,3-diaminosuccinic acid (8.7g, >100%).

[0416] Example 13. Synthesis of meso-2,3-bis(((benzyloxy)carbonyl)amino)succinic acid

[0417]

[0418] Benzyl chloroformate (61 ml, 428 ml) was added dropwise at 0°C to a solution of meso-2,3-diaminosuccinic acid (31.74 g, 214 ml) in THF (220 ml) and 4 N NaOH (214 ml). After addition was complete, the mixture was heated to rt and stirred for 2 hours. The reaction mixture was diluted with water (1600 ml) and washed with ethyl acetate (2 × 1500 ml). The aqueous layer was separated and acidified with concentrated HCl until a pH of 2 was reached. The resulting solution was stirred for 1 hour and left to stand at 5°C to yield a white precipitate. The precipitate was filtered, rinsed with water, and dried to yield meso-2,3-bis(((benzyloxy)carbonyl)amino)succinic acid (52.2 g, 125 ml, 59%).

[0419] Example 14. Synthesis of dibenzyl ((3R,4S)-2,5-dioxotetrahydrofuran-3,4-diyl)dicarbamate.

[0420]

[0421] A solution of meso-2,3-bis(((benzyloxy)carbonyl)amino)succinic acid (5.0 g, 12 mmol) in Ac2O (37.5 mL) was refluxed for 20 minutes, cooled, and concentrated to provide the anhydrous form. The mixture of diastereomers was treated with CHCl3 (37 mL), the insoluble meso-isomer was filtered, and the filtrate was treated with petroleum ether to provide crystals of dibenzyl ((3R,4S)-2,5-dioxotetrahydrofuran-3,4-diyl)dicarbamate (racemic mixture, 2.0 g, 5 mmol, 42%).

[0422] Example 15. Compound 17 Synthesis of.

[0423]

[0424] To a mixture of Compound 16 (4.25 g, 10.68 mmol, 1.0 eq) and DMAP (13 mg, 0.11 mmol, 0.01 eq) in 20 ml of anhydrous DCM, of 10 ml of anhydrous DCM t A solution of butyl aminobutyrate (1.78 g, 11.21 mmol, 1.05 eq) was added. After the addition was complete, the compound 16 [Unclear text] was completely dissolved, and the reaction mixture was stirred overnight at [temperature]. The crude product was loaded onto a silica gel column and eluted with 3-5% MeOH / DCM. The fractions were combined and evaporated, and the residue was acid-treated with PE / DCM (1:1) to yield 3.3 g of a white solid (yield 55.9%). C 28 H 36 N3O9[M+H] + Calculated ESI m / z for: 558.2, measured: 558.2.

[0425] Example 16. Compound 18 Synthesis of.

[0426]

[0427] In a 500 ml flask, H2N-PEG4-CH2CH2CO2H (3.0 g, 11.3 ml, 1.0 eq) and K2CO3 (4.7 g, 33.93 ml, 3.0 eq) were dissolved in 50 ml of water and cooled in an ice bath. Boc2O (3.2 g, 14.7 ml, 1.3) in 50 ml of THF was added dropwise. The reaction mixture was heated to rt and stirred overnight. The mixture was adjusted to pH 4 to 5 using 1 N KHSO4, extracted with DCM (200 ml × 1, 100 ml × 3), washed with water (500 ml × 1) and brine (500 ml × 1), dried over anhydrous sodium sulfate, and concentrated. The residue was dissolved in a small amount of DCM, then loaded onto a silica gel column, eluted with 2-4% MeOH / DCM, fractions were combined and concentrated to yield 3.8 g of colorless oil (yield 93%). C 16 H 32 NO8[M+H] + ESI m / z calculated value for: 366.2, measured value: 366.2.

[0428] Example 17. Compound 19 Synthesis of

[0429]

[0430] In a 50 ml single-neck flask, BocHN-PEG4-CH2CH2CO2H (0.81 g, 2.22 ml, 1.0 eq), K2CO3 (0.92 g, 6.66 ml, 3.0 eq), and NaI (0.033 g, 0.222 ml, 0.1 eq) were mixed in 10 ml of DMF, cooled in an ice bath, BnBr (0.57 g, 3.33 ml, 1.5 eq) was added dropwise, the mixture was heated to rt, and stirred overnight. The mixture was diluted with 100 ml of water, extracted with DCM (100 ml × 2), washed with water (200 ml × 1) and brine (200 ml × 1), dried over anhydrous sodium sulfate, and concentrated. The residue was dissolved in a small amount of DCM, loaded onto a silica gel column, and eluted with 70-90% EA / PE to yield 0.69 g of colorless oil (69% yield). C 23 H 38 NO8[M+H] + Calculated ESI m / z for: 446.3, measured: 446.3.

[0431] Example 18. Compound 20 Synthesis of.

[0432]

[0433] A solution of BocHN-PEG4-CH2CH2CO2Bn (0.69 g, 1.5 mmol, 1.0 eq) in 6 mL of DCM and 3 mL of TFA was stirred at rt for 30 minutes. The solvent was removed, the residue was co-evaporated with DCM three times, and placed on a high vacuum pump. The crude product was used directly in the next step. C 18 H 30 NO6[M+H] + ESI m / z for: Calculated value: 356.2, Measured value: 356.2.

[0434] Example 19. Compound 21 Synthesis of.

[0435]

[0436] NHS (1.4 g, 12.5 mol, 1.2 eq) and EDC (10.0 g, 52.0 mmol, 5.0 eq) were added to a solution of BocHN-PEG4-CH2CH2CO2H (3.8 g, 10.4 mmol, 1.0 eq) in 50 ml of anhydrous DCM. The reaction mixture was stirred overnight at rt, then washed with water (50 ml × 2) and brine (100 ml × 1), dried over anhydrous sodium sulfate, and concentrated. The crude product was used directly in the next step. C 20 H 35 N2O 10 [M+H] + ESI m / z for: Calculated value: 463.2, Measured value: 463.2.

[0437] Example 20. Compound 22 Synthesis of.

[0438]

[0439] In a 300 ml flask, H2N-PEG4-CH2CH2CO2H (2.8 g, 10.4 ml, 1.0 eq) and K2CO3 (4.3 g, 31.2 ml, 3.0 eq) were dissolved in 40 ml of water and cooled in an ice bath. Then, the above crude substance NHS ester solution (3.8 g, 10.4 ml, 1.0 eq) in 40 ml of THF was added dropwise, the mixture was heated to rt, and stirred overnight. The mixture was adjusted to pH 4 to 5 using 1N KHSO4, extracted with DCM (150 ml × 1, 100 ml × 2), washed with water (200 ml × 1) and brine (200 ml × 1), dried over anhydrous sodium sulfate, and concentrated. The residue was dissolved in a small amount of DCM and loaded onto a silica gel column eluted with 4 to 6% MeOH / DCM to yield a colorless oil (5.18 g, 81% yield). C 27 H 53 N2O 13 [M+H] + Calculated ESI m / z for: 613.3, measured: 613.3.

[0440] Example 21. Compound23 Synthesis of.

[0441]

[0442] Dissolve H2N-PEG4-CH2CH2CO2Bn (crude product from the previous step) in 3 mL of DMF, cool in an ice / water bath, add DIPEA (0.78 g, 6.0 mmol, 4.0 eq) dropwise, and then the compound in 7 mL of DMF 22 A solution of (0.93 g, 1.5 mmol, 1.0 eq) and HATU (1.72 g, 4.5 mmol, 3.0 eq) were added. The reaction mixture was stirred in an ice bath for 2 hours, diluted with 100 ml of water, extracted with DCM (100 ml × 3), washed with 1 N KHSO4 (200 ml × 1), saturated sodium bicarbonate (200 ml × 1), and brine (200 ml × 1), dried over anhydrous sodium sulfate, and concentrated. The residue was dissolved in a small amount of DCM, loaded onto a silica gel column, and eluted with 0-5% MeOH / DCM. The fractions were combined and concentrated to yield 1.0 g of light yellow oil (71% yield). C 45 H 80 N3O 18 [M+H] + ESI m / z calculated value for: 950.5, measured value: 950.5.

[0443] Example 22. Synthesis of Compound 24.

[0444]

[0445] Compounds in 6 ml of DCM 23 A solution of (1.0 g, 1.03 ml, 1.0 eq) and 3 ml of TFA were stirred at rt for 1 hour. The solvent was removed, the residue was co-evaporated with DCM three times, and placed on a high vacuum pump.

[0446] The crude product was redissolved in 10 ml of DMF and cooled in an ice bath. To this, DIPEA (0.53 g, 4.12 ml, 4.0 eq), compound 17(0.56 g, 1.03 mmol, 1.0 eq) and HATU (1.17 g, 3.09 mmol, 3.0 eq) were added sequentially. After stirring in an ice bath for 1 hour, 100 ml of water was added, and the solid precipitated. The solid was collected by filtration, washed with water, dissolved in DCM, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was dissolved in a small amount of DCM, loaded onto a silica gel column, and eluted with 0-10% MeOH / DCM. The fractions were combined and concentrated to yield 0.93 g of light yellow foam (yield 65%). C 68 H 107 N8O 26 [M+H] + Calculated ESI m / z for: 1451.7, measured: 1451.7.

[0447] Example 23. Compound 25 Synthesis of.

[0448]

[0449] Compounds in 6 ml of DCM 24 A solution of (0.93 g, 0.67 mmol, 1.0 eq) and 3 mL of TFA were stirred at rt for 1 hour (completion of the reaction was monitored by LC-MS). The solvent was removed, the residue was co-evaporated with DCM three times, and placed on a high vacuum pump. The crude product was dissolved in a small amount of DCM, loaded onto a silica gel column, and then eluted with 15-20% MeOH / DCM. The fractions were combined and concentrated to yield 0.53 g of white foam (yield 60%) product. C 64 H 99 N8O 26 [M+H] + Calculated ESI m / z for: 1395.7, measured: 1395.7.

[0450] Example 24. Synthesis of Compound 26.

[0451]

[0452] Compounds in 10 ml of DCM 25 Pentafluorophenol (0.081 g, 0.44 ml, 1.1 eq) and EDC (0.38 g, 2.0 ml, 5.0 eq) were added to (0.53 g, 0.40 ml, 1.0 eq). The mixture was stirred overnight at rt, then washed with cold water (5 ml × 2) and brine (10 ml × 1), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was used directly in the next step. C 70 H 98 F5N6O 26 [M+H] + Calculated ESI m / z for: 1561.6, measured: 1561.6.

[0453] Example 25. Synthesis of Compound 27.

[0454]

[0455] The crude product (0.4 mmol, 1.0 eq) from the previous step was dissolved in 10 ml of DMF and cooled in an ice bath. To this, a compound 12 (0.39 g, 0.4 mmol, 1.0 eq) and DIPEA (0.15 g, 1.2 mmol, 3.0 eq) were added sequentially. After stirring in an ice bath for 1 hour, the reaction mixture was concentrated, redissolved in a small amount of DCM, loaded onto a silica gel column, and eluted with 0-20% MeOH / DCM to yield a colorless oil (0.53 g, 58% yield). C 110 H 176 N 11 O 40 [M+H] + ESI m / z calculated value for: 2291.2, measured value: 2291.2.

[0456] Example 26. Compound 28 Synthesis of.

[0457]

[0458] Compounds in 10 ml of methanol 27(0.53 g, 0.23 mmol, 1.0 eq) and anhydrous palladium carbon (0.1 g, 10% wt) were stirred overnight at rt under an H2 balloon. The mixture was filtered, and the filtrate was evaporated to yield 0.35 g of crude material, which was used directly in the next step (yield 80%). C 87 H 158 N 11 O 36 [M+H] + Calculated ESI m / z for: 1933.1, measured: 1933.1.

[0459] Example 27. Synthesis of Compound 29.

[0460]

[0461] The crude product from the previous step (0.35 g, 0.18 ml, 1.0 eq) was redissolved in a mixture of 3 ml of ethanol and 0.2 ml of 0.1 M NaH2PO4, and , N-(4-maleimidobutyryloxy)succinimide (0.20 g, 0.72 mmol, 4.0 eq) was added. The mixture was stirred overnight at rt, then concentrated, redissolved in DCM, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was dissolved in a small amount of DCM, loaded onto a silica gel column, and eluted with 0-20% MeOH / DCM to yield a colorless oil product (0.13 g, 33% yield). C 103 H 172 N 13 O 42 [M+H] + Calculated ESI m / z for: 2263.2, Measured: 2263.2

[0462] Example 28. Compound 30 Synthesis of.

[0463]

[0464] compound 29(0.13g, 0.0574mol, 1.0eq) was dissolved in 2ml of DCM and stirred with 2ml of TFA for 3 hours at rt. The solvent was removed, the residue was co-evaporated with DCM three times, and placed on a high vacuum pump.

[0465] The crude product was redissolved in DMF and cooled in an ice bath. Tub-pentafluorophenol (0.048 g, 0.0690 mmol, 1.0 eq) was added, followed by the addition of DIPEA (0.022 g, 0.172 mmol, 3.0 eq). The reaction mixture was stirred in an ice bath for 1 hour, and then the pH was adjusted to 4 to 5 using formic acid. The mixture was concentrated, redissolved in a small amount of DCM, loaded onto a silica gel column, and eluted with PE / EA and MeOH / DCM (both containing 0.1% formic acid). The fractions were combined and concentrated to yield 0.1 g of yellow foam (70% yield). The product was further purified by preparative HPLC (45-50% MeCN / H2O (containing 0.1% formic acid)). The fractions were combined and concentrated to yield a colorless oil (0.030 g, 20% yield). C 123 H 204 N 17 O 45 S [M+H] + Calculated ESI m / z for: 2671.4, measured: 2671.4.

[0466] Example 29. Synthesis of Compound 31.

[0467]

[0468] TEA (10.5g, 104mol, 4.0eq), DMAP (32mg, 0.26mol, 0.01eq), and TsCl (14.9g, 78mol, 3.0eq) were sequentially added over an ice bath to a solution of mPEG8-OH (10g, 26mol, 1.0eq) in 100ml of anhydrous DCM. The reaction mixture was stirred at 0°C for 10 minutes, then heated to rt and stirred overnight. The reaction mixture was washed with 1N HCl (100ml × 1), water (100ml × 1), and brine wash (100ml × 1), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was dissolved in a small amount of DCM, loaded onto a silica gel column, and eluted with EA / PE (5–100%) and 1–3% MeOH / DCM. The fractions were combined and concentrated to yield a yellow oil (11.6 g, 83% yield). C 24 H 43 O 11 S [M+H] + ESI m / z Calculated value for: 539.2, Measured value: 539.2.

[0469] Example 30. Compound 32 Synthesis of.

[0470]

[0471] Compounds in 20 ml of anhydrous DMF 31 A mixture of (11.6 g, 21.5 mmol, 1.0 eq) and dibenzylamine (5.5 g, 27.8 mmol, 1.5 eq) was heated to 100°C overnight while stirring. The reaction mixture was diluted with 300 ml of DCM, washed with water (300 ml × 3) and brine (300 ml × 1), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified on a silica gel column (50-100% EA / PE) to yield a bright yellow oil (8.2 g, 66% yield). C 31 H 50 NO8[M+H] + ESI m / z calculated value for: 564.3, measured value: 564.3.

[0472] Example 31. Compound 33 Synthesis of.

[0473]

[0474] Compounds in 100 ml of anhydrous methanol 32 A mixture of solutions of palladium (0.9 g, 10 wt%) on anhydrous carbon (8.6 g, 15.2 mmol, 1.0 eq) and catalytic converter (8.6 g, 15.2 mmol, 1.0 eq) was refluxed overnight under an H2 balloon. The catalyst was filtered and washed with methanol. The filtrate was evaporated to yield 5.3 g of colorless oil (yield 90%). C 17 H 38 NO8[M+H] + Calculated ESI m / z for: 384.3, measured: 384.3.

[0475] Example 32. Compound 34 Synthesis of.

[0476]

[0477] compound 17 (1.6g, 2.84mol, 1.0eq) and compound 33 (1.2g, 2.84mol, 1.0eq) was dissolved in 5ml of anhydrous DMF, to which HATU (3.2g, 8.52mol, 3.0eq) and DIPEA (1.5g, 11.36mol, 4.0eq) were sequentially added over an ice bath. The reaction mixture was stirred in an ice bath for 2 hours, then 150ml of water was added, and the mixture was extracted with DCM (150ml × 1, 100ml × 1). The organic phase was washed with 1 N HCl (200ml × 1), saturated sodium bicarbonate (200ml × 1), and brine (200ml × 1), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was dissolved in a small amount of DCM, loaded onto a silica gel column, and then eluted with 0-5% MeOH / DCM. The fractions were combined and concentrated to yield 2.29 g of a white solid (87% yield). C 45 H 71 N4O 16 [M+H] +ESI m / z calculated value for: 923.5, measured value: 923.5.

[0478] Example 33. Compound 35 Synthesis of.

[0479]

[0480] Compounds in a mixture of 5 ml DCM and 5 ml TFA 34 A solution of (2.29 g, 2.48 mmol, 1.0 eq) was stirred at rt for 3 hours. The solvent was removed, the residue was co-evaporated with DCM three times, the residue was dissolved in a small amount of DCM, loaded onto a silica gel column, and eluted with 5-8% MeOH / DCM. The fractions were combined and concentrated to yield 2.09 g of a white jelly solid (97% yield). C 41 H 63 N4O 16 [M+H] + ESI m / z calculated value for: 867.4, measured value: 867.4.

[0481] Example 34. Compound 36 Synthesis of.

[0482]

[0483] Compounds in 10 ml of DCM on the ice bath 35 Pentafluorophenol (0.35 g, 1.90 ml, 1.1 eq) and EDC (1.7 g, 8.66 ml, 5.0 eq) were added to (1.5 g, 1.73 ml, 1.0 eq). The reaction mixture was heated to rt and stirred for 5 hours, then washed with water (10 ml × 2) and brine (20 ml × 1), dried over anhydrous sodium sulfate, filtered, and concentrated to yield 1.07 g of crude product (60% yield). C 47 H 62 F5N4O 16 [M+H] + Calculated ESI m / z for: 1033.4, measured: 1033.4.

[0484] Example 35. Compound37 Synthesis of.

[0485]

[0486] To the above crude product (1.07g, 1.0mol, 1.0eq) in 10ml of DMF in a shaved ice bath, a compound 12 (0.92 g, 1.0 ml, 1.0 eq) and DIPEA (0.39 g, 3.0 ml, 3.0 eq) were added. The reaction mixture was stirred in an ice bath for 1 hour, the pH was adjusted to 4 to 5 using 1N HCl, diluted with EA (100 ml), and extracted with water (30 ml × 5). The aqueous phase was concentrated, then redissolved in a small amount of DCM, loaded onto a silica gel column, and eluted with 15-18% MeOH / DCM. The fractions were combined and concentrated to yield 0.88 g of colorless oil (51% yield). C 87 H 142 N9O 32 [M+H] + Calculated ESI m / z for: 1825.0, measured: 1825.0.

[0487] Example 36. Compound 38 Synthesis of.

[0488]

[0489] Compounds in 5 ml of methanol 37 A mixture of (0.88 g, 0.48 mmol, 1.0 eq) and palladium on carbon (0.1 g, 10 wt%) was stirred overnight at rt under an H2 balloon. The catalyst was filtered, and the filtered solution was concentrated to yield 0.75 g of crude material, which was used directly in the following reaction (yield 80%). C 71 H 130 N9O 28 [M+H] + Calculated ESI m / z for: 1556.9, measured: 1556.9.

[0490] Example 37. Synthesis of Compound 39.

[0491]

[0492] The crude product from the previous step (0.75 g, 0.48 ml, 1.0 eq) was dissolved in a mixture of 2 ml of ethanol and 0.2 ml of 0.1 M NaH2PO4. , N -(4-maleimidobutyryloxy)succinimide (0.54 g, 1.92 mmol, 4.0 eq) was added. The mixture was stirred overnight at rt, then concentrated, redissolved in DCM, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was dissolved in a small amount of DCM, loaded onto a silica gel column, and eluted with 0-20% MeOH / DCM to yield a colorless oil (0.26 g, yield 29%). C 87 H 144 N 11 O 34 [M+H] + Calculated ESI m / z for: 1887.0, measured: 1887.0.

[0493] Example 38. Compound 40 Synthesis of.

[0494]

[0495] Compounds in 3 ml of DCM and 1 ml of TFA 39 (0.26 g, 0.138 ml, 1.0 eq) was stirred at rt for 1 hour. The solvent was removed, the residue was co-evaporated with DCM three times, and placed on a high vacuum pump.

[0496] The crude product was redissolved in 5 ml of DMF and cooled in an ice bath. Tub-PFP (0.114 g, 0.166 mmol, 1.2 eq) and DIPEA (0.265 g, 2.07 mmol) were added. The reaction mixture was stirred in an ice bath for 1 hour, and then the pH was adjusted to 4 to 5 using formic acid. The mixture was concentrated, redissolved in a small amount of DCM, loaded onto a silica gel column, and eluted with PE / EA and MeOH / DCM (both containing 0.1% formic acid). The fractions were combined and concentrated to yield 0.2 g of a yellow foam product (63% yield). The product was further purified by preparative HPLC (45-50% MeCN / H2O (containing 0.1% formic acid)). The fractions were combined and concentrated to provide a colorless oil product (0.10 g, 23% yield). C 107 H 176 N 15 O 37 S [M+H] + ESI m / z calculated value for: 2295.2, measured value: 2295.2.

[0497] Example 39. Synthesis of Compound 41.

[0498]

[0499] EDC (1.91 g, 12.0 ml) and TEA (3.5 ml, 25.0 ml) were added to a solution of benzyl 11-aminoundecanoate (2.91 g, 10.0 ml) and Boc-Glu(OBzl)-OH (3.37 g, 10.0 ml) in DMF (50 ml). The reaction mixture was stirred at rt for 8 hours, diluted with water (100 ml), and extracted with EA (3 × 100 ml). The combined organic phase was washed once with 100 ml of brine, then dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by SiO2 column chromatography (EA / DCM, 1:15) to yield the title compound as a colorless oil (5.37 g, 88% yield).

[0500] Example 40. Synthesis of Compound 42.

[0501]

[0502] Compound in a mixture of 5 ml of DCM and 2 ml of TFA 41 (0.64 g, 1.05 mmol, 1.0 eq) was stirred at rt for 2 hours and then concentrated. The residue was co-evaporated with DCM three times and placed on a high vacuum pump. C 30 H 42 N2O5[M+H] + ESI m / z calculated value for: 511.3, measured value: 511.3.

[0503] The above crude product was redissolved in 3 ml of DMF and cooled in an ice bath. To this, a compound in 7 ml of DMF 22 A solution of (0.64 g, 1.05 ml, 1.0 eq) was added, followed by the addition of DIPEA (0.54 g, 4.20 ml, 4.0 eq) and HATU (1.2 g, 3.15 ml, 3.0 eq). The reaction mixture was stirred in an ice bath for 1 hour, then 100 ml of water was added, and the mixture was extracted with DCM (150 ml × 1, 100 ml × 1). The organic phase was washed with 1N KHSO4 (200 ml × 1), saturated sodium bicarbonate (200 ml × 1), and brine (200 ml × 1), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was dissolved in a small amount of DCM, loaded onto a silica gel column, and then eluted with 0-10% MeOH / DCM. The fractions were combined and concentrated to yield 0.94 g of light yellow oil (81% yield). C 57 H 92 N4O 17 [M+H] + Calculated ESI m / z for: 1104.6, measured: 1104.6.

[0504] Example 41 Synthesis of Compound 43.

[0505]

[0506] Compound in a mixture of 7 ml of DCM and 3 ml of TFA 42 (0.94 g, 0.458 mmol, 1.0 eq) was stirred and concentrated at rt for 2 hours. The residue was co-evaporated with DCM three times and then placed on a high vacuum pump. C 52 H 84 N4O 15 [M+H] + Calculated ESI m / z for: 1004.6, measured: 1004.6.

[0507] The above crude compound was redissolved in 10 ml of DMF and cooled in an ice bath, and the compound therein 17 (0.46 g, 0.85 mmol, 1.0 eq), DIPEA (0.44 g, 3.40 mmol, 4.0 eq), and HATU (0.97 g, 2.55 mmol, 3.0 eq) were added. The reaction mixture was stirred in an ice bath for 1 hour, then 100 mL of water was added, and the solid precipitated. The solid was collected by filtration, washed with water, dissolved in DCM, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was dissolved in a small amount of DCM, loaded onto a silica gel column, and eluted with 0-10% MeOH / DCM. The fractions were combined and concentrated to yield 1.13 g of a light yellow oil product (87% yield). C 80 H 120 N9O 24 [M+H] + Calculated ESI m / z for: 1590.8, measured: 1590.8.

[0508] Example 42. Synthesis of Compound 44.

[0509]

[0510] Compound in the mixture of 7 ml of DCM and 3 ml of TFA 43(1.13 g, 0.73 mmol, 1.0 eq) was stirred at rt for 3 hours and concentrated. The residue was co-evaporated with DCM three times, dissolved in a small amount of DCM, loaded onto a silica gel column, and eluted with 5-15% MeOH / DCM. The fractions were combined and concentrated to yield 0.85 g of a white foam product (78% yield). C 76 H 112 N9O 25 [M+H] + Calculated ESI m / z for: 1550.8, measured: 1550.8.

[0511] Example 43. Synthesis of Compound 45.

[0512]

[0513] Compounds in 10 ml of DCM on the ice bath 44 Pentafluorophenol (0.11 g, 0.63 ml, 1.1 eq) and EDC (0.55 g, 2.85 ml, 5.0 eq) were added to (0.85 g, 0.57 ml, 1.0 eq). The mixture was heated to rt, stirred overnight, then washed with ice water (10 ml × 2) and cold brine (20 ml × 1), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was used directly in the next step.

[0514] Example 44. Synthesis of Compound 46.

[0515]

[0516] To the above crude product (0.94g, 0.57mol, 1.0eq) in 10ml of DMF in a shaved ice bath, a compound 12(0.56 g, 0.57 mmol, 1.0 eq) and DIPEA (0.22 g, 1.71 mmol, 3.0 eq) were added. The mixture was stirred in an ice bath for 3 hours, concentrated, then redissolved in a small amount of DCM, loaded onto a silica gel column, and eluted with 12-20% MeOH / DCM. The fractions were combined and concentrated to yield 1.00 g of colorless oil (71% yield). C 122 H 189 N 12 O 39 [M+H] + Calculated ESI m / z for: 2446.3, measured: 2446.3.

[0517] Example 45. Synthesis of Compound 47.

[0518]

[0519] compound 46 (0.53 g, 0.23 mmol, 1.0 eq) was dissolved in 5 ml of methanol, palladium on anhydrous carbon (0.1 g, 10 wt%) was added, and the reaction mixture was stirred overnight at rt under an H2 balloon. The catalyst was filtered, and the filtrate was evaporated to yield 0.71 g of crude material (yield 87%). C 92 H 165 N 12 O 35 [M+H] + Calculated ESI m / z for: January 1997, measured: January 1997.

[0520] The above crude product (0.71g, 0.355mmol, 1.0eq) was redissolved in 2ml of ethanol and 0.2ml of 0.1M NaH2PO4, and N -(4-maleimidobutyryloxy)succinimide (0.40 g, 1.42 mmol, 4.0 eq) was added, the mixture was stirred overnight at rt, concentrated, purified on a preparative C-18 HPLC column, and eluted with 0-40% MeOH / H2O to yield a colorless oil (0.26 g, 33%). C 108 H 179 N 14O 41 [M+H] + Calculated ESI m / z for: 2328.2, measured: 2328.2.

[0521] Example 46. Compound 48 Synthesis of.

[0522]

[0523] compound 47 (0.26 g, 0.112 mmol, 1.0 eq) was stirred in a mixture of 2 mL of DCM and 2 mL of TFA at rt for 3 hours. The solvent was removed, the residue was co-evaporated with DCM three times, and placed on a high vacuum pump. C 103 H 171 N 14 O 39 [M+H] + Calculated ESI m / z for: 2228.2, measured: 2228.2.

[0524] The above crude product was redissolved in 5 ml of DMF and cooled in an ice bath. Tub-PFP (0.0.93 g, 0.134 mmol, 1.2 eq) was added, followed by DIPEA (0.043 g, 0.336 mmol, 3.0 eq). The reaction mixture was stirred in an ice bath for 1 hour, and then the pH was adjusted to 4 to 5 using formic acid. The mixture was concentrated, redissolved in a small amount of DCM, loaded onto a silica gel column, and eluted with PE / EA and MeOH / DCM (containing 0.1% formic acid). The fractions were combined and concentrated to yield 0.09 g of yellow foam. The product was dissolved in 50:50 MeOH / H2O (2 ml) and further purified by preparative HPLC (45-50% MeCN / H2O (containing 0.1% formic acid)). The fractions were combined and concentrated to yield a colorless oil (0.027 g, 15% yield). C 128 H 211 N 18 O 44 S [M+H] +Calculated ESI m / z for: 2736.4, measured: 2736.4.

[0525] Example 47. Synthesis of (S)-3,4-dimethyloxazolidine-2,5-dione (NCA).

[0526]

[0527] (S)-2-((tert-butoxycarbonyl)(methyl)amino) acid propane (5.0 g, 24.6 mmol) was dissolved in dichloromethane (95 mL) and stirred in an ice / water bath for 15 minutes. After cooling, triphosgene (8.7 g, 29.6 mmol, 1.2 eq) was added dropwise over approximately 5 minutes. After the addition was complete, the ice bath was removed, and the reaction was allowed to proceed at room temperature for an additional 4 hours. The solution was removed under pressure, and carbon tetrachloride (130 mL) was added to precipitate the product. The white precipitate was collected by filtration, and the remaining carbon tetrachloride was washed away. After a few minutes, the crystals were yellow; the yellow crystals were redissolved in a small amount of dichloromethane, and the yellow residue was removed by filtration. The filtrate (product) was then precipitated with carbon tetrachloride and filtered. The crystals were dried on a filter at ambient temperature for 3 hours to provide the title compound (2.3 g, 69% yield). ESI MS, 130.05 (M+1) + .

[0528] Example 48. Synthesis of May-NMA

[0529]

[0530] Meitansinol (200 mg, 0.354 mmol) was dissolved in DMF (5 mL) and THF (2.5 mL) and cooled in an ice / water bath. After a few minutes, DIPEA (0.25 mL, 1.42 mmol, 4 eq) and zinc trilate (6 eq) were added while stirring with a magnet, followed by the addition of NCA (183 mg, 1.42 mmol, 4 eq), and the reaction mixture was stirred under argon at room temperature for 17 hours. The reaction mixture was diluted with EtOAC (20 mL), treated with a solution (4.4 mL) of brine:saturated sodium bicarbonate (1:1), and the resulting solution was stirred for 10 minutes. The white precipitate was filtered, the resulting aqueous solution was re-extracted with EtOAC (20 mL × 2), and the organic layer was then washed with brine. The generated organic layer was concentrated to provide the crude product, which was used in the next step without further purification (210 mg, approximately 91% yield, 87% purity by HPLC). C 32 H 45 ClN3O9[M+H] + ESI m / z calculated value for: 650.28, measured value: 650.29.

[0531] Example 49. (S)-tert-butyl 34-(((benzyloxy)carbonyl)amino)-28,35-dioxo-2,5,8,11,14,17,20,23,26-nonaoxa-29,36-diazatetracontan-40-oate( 210 Synthesis of )

[0532]

[0533] in DMF (18 ml) at 0 ℃ tert -Butyl 4-aminobutanoate (1.03 g, 6.12 mmol) and compound 4HATU (2.32 g, 6.12 mmol) and TEA (1.2 mL, 8.34 mmol) were sequentially added to a mixture of (3.91 g, 5.56 mmol). The reaction mixture was stirred for 1 hour, then diluted with water (300 mL) and extracted with ethyl acetate (3 × 250 mL). The organic solution was washed with brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (32:1 dichloromethane / methanol) to obtain the title compound ( 210 )(5.10g, 99% yield) was provided. ESI MS m / z 846.50 ([M+H] + ).

[0534] Example 50. (S)-tert-butyl 34-amino-28,35-dioxo-2,5,8,11,14,17,20,23,26-nonaoxa-29,36-diazatetracontan-40-oate( 211 Synthesis of )

[0535]

[0536] compound 210 (1.0g, 1.18mol) and Pd / C (10 wt%, 0.10 g) were added to a hydrogenation bottle containing methanol (50ml), the mixture was shaken for 2 hours, filtered through Celite (filter aid), and the filtrate was concentrated to obtain a compound 211 Provided (0.93g, yield > 100%). ESI MS m / z 712.50 ([M+H] + ).

[0537] Example 51. (S)-tert-butyl 34-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)butaneamido)-28,35-dioxo-2,5,8,11,14,17,20,23,26-nonaoxa-29,36-diazatetracontan-40-oate( 212 Synthesis of )

[0538]

[0539] Compounds in 95% EtOH (50 ml) and NaH2PO4 solution (0.1 M, pH 5.0, 10 ml) 211 N-succinimidyl 4-maleimido-butyrate (0.50 g, 1.77 ml, 1.5 eq) was added to a solution of (0.93 g, 1.18 ml). The mixture was stirred overnight, then concentrated, diluted with water (50 ml), extracted with dichloromethane (80 ml × 3), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (25:1 dichloromethane / methanol) to provide the title compound as a bright yellow oil (0.82 g, 80%). ESI MS m / z 877.52 ([M+H] + ).

[0540] Example 52. (S)-34-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)butaneamido)-28,35-dioxo-2,5,8,11,14,17,20,23,26-nonaoxa-29,36-diazatetracontan-40-acid( 213 Synthesis of )

[0541]

[0542] compound 212 0.82 g (0.94 ml) was dissolved in HCOOH (50 ml) and stirred at room temperature for 1 hour. The mixture was concentrated, co-evaporated twice with toluene, the residue was placed under a vacuum pump, and the compound 213 (0.80g, crude product) was provided. ESI MS m / z 820.45 ([M+H] + ).

[0543] Example 53. (S)-2,5-dioxopyrrolidine-1-yl 34-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)butaneamido)-28,35-dioxo-2,5,8,11,14,17,20,23,26-nonaoxa-29,36-diazatetracontan-40-oate( 214 Synthesis of )

[0544]

[0545] NHS (0.12 g, 1.03 mmol) and EDC·HCl (0.27 g, 1.41 mmol) were added to a solution of Compound 213 (0.80 g, crude material, 0.94 mmol) in DMA (5.0 mL), and the reaction mixture was stirred at rt for 2 hours. It was then diluted with water (15 mL) and extracted with ethyl acetate (3 × 10 mL). The combined organic phase was washed with brine (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified using a silica gel column (10-50% ethyl acetate / petroleum ether) to yield a colorless oil compound (0.67 g, 78% yield). ESI MS m / z 918.55 ([M+H] + ).

[0546] Example 54. tert-butyl (2-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)ethyl)carbamate ( 215) Synthesis of .

[0547]

[0548] N A mixture of -Boc-ethylenediamine (5.6 ml, 35.4 mmol, 1.1 eq.) and saturated NaHCO3 (60 ml) is cooled to 0°C, and to this N -Methoxycarbonyl maleimide (5.00 g, 32.2 mmol, 1.0 eq.) was added all at once. After stirring at 0°C for 30 minutes, the reaction mixture was heated to rt and stirred for 1 hour. The precipitate was collected by filtration, washed with cold water, dissolved in ethyl acetate, washed with brine, dried over anhydrous sodium sulfate, and concentrated to yield a white solid (6.69 g, 87% yield). ESI MS m / z 241.12 ([M+H] + ).

[0549] Example 55. tert-butyl (2-(1,3-dioxo-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-2(3H)-yl)ethyl)carbamate ( 216 Synthesis of )

[0550]

[0551] Compound in toluene (120 ml) in a high-pressure tube 215 A solution of (6.00 g, 25.0 ml) and furan (18.0 ml) was heated to reflux and stirred for 16 hours. The colorless solution turned yellow during the reaction. The mixture was then cooled to rt and concentrated. The resulting white solid was acid-treated with ethyl ether to obtain the compound 216 (6.5g, 84% yield) was provided. ESI MS m / z 309.13 ([M+H] + ).

[0552] Example 57. 2-(2-aminoethyl)-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione hydrochloride ( 217 Synthesis of )

[0553]

[0554] Compound in dioxane (15 ml) 216 A solution of (9.93 g, 32.2 ml) was treated with concentrated HCl (15 ml) at rt for 3 hours. The reaction mixture was concentrated, the resulting solid was collected by filtration, and the filter cake was washed with ethyl acetate. The solid was dried overnight in an oven (50°C) to form the compound 217 (6.94g, 88% yield) was provided. ESI MS m / z 206.05 ([M+H] + ).

[0555] Example 58. Compound 218 Synthesis of .

[0556]

[0557] Compound in THF (10 ml) at -10℃ 217POCl3 (0.47 mL, 5 mmol) was added to a solution of (1.22 g, 5 mmol). After stirring for 10 minutes, 2,5,8,11,14,17,20,23,26-nonaoxaoctacosan-28-amine (2.14 g, 5 mmol) was added, followed by the addition of DIPEA (0.87 mL, 5 mmol). The reaction mixture was heated to 0°C, stirred for 3 hours, and then concentrated. The residue was diluted with dichloromethane (10 mL), filtered over Celite, and the filtrate was used directly in the next step. ESI MS m / z 716.29 ([M+H] + ).

[0558] Example 59. Methyl 4-(bis(2-hydroxyethyl)amino)-4-oxobutanoate ( 219 Synthesis of )

[0559]

[0560] Dimethyl succinate (20.0 g, 136.9 mmol) and dihydroxyethylamine (7.20 g, 68.7 mmol) in a mixture of anhydrous toluene (500 mL) and pyridine (50 mL) were heated at 150 °C for 28 hours. The mixture was concentrated, purified on a silica gel column, and eluted with 5-25% ethyl acetate / dichloromethane to yield the title compound (12.5 g, 83% yield). ESI MS m / z 242.42 [M + Na] + .

[0561] Example 60. Methyl 4-(bis(2-((methylsulfonyl)oxy)ethyl)amino)-4-oxobutanoate( 220 Synthesis of )

[0562]

[0563] Methanesulfonyl chloride (20.0 g, 175.4 mmol) was added to a solution of methyl 4-(bis(2-hydroxyethyl)amino)-4-oxobutanoate (12.0 g, 49.56 mmol) in anhydrous pyridine (350 mL). After stirring overnight, the mixture was concentrated, diluted with ethyl acetate (350 mL), washed with cold 1 M NaH2PO4 (2 × 300 mL), dried over MgSO4, filtered, and evaporated to yield a crude product (approx. 18.8 g, >100% yield). The crude product was used in the next step without further purification. ESI MS m / z 376.06 ([M+H] + ).

[0564] Example 61. 3,6-endoxo-Δ-tetrahydrophthalimide ( 221 Synthesis of )

[0565]

[0566] Furan (10.0 mL, 137.4 mmol) was added to a solution of maleimide (10.0 g, 103.0 mmol) in toluene (200 mL). The mixture was heated in a 1 L autoclave bomb at 100°C for 8 hours. The bomb was cooled to room temperature, the solid was rinsed with methanol, concentrated, and crystallized in ethyl acetate / hexane to yield 16.7 g (99%) of the title compound. 1H NMR (CDCl3): 11.12 (s, 1H), 6.68–6.64 (m, 2H), 5.18–5.13 (m, 2H), 2.97–2.92 (m, 2H). ESI MS m / z [M + Na] + 188.04.

[0567] Example 62. Methyl 4-((2-((3aR,4R,7S,7aS)-1,3-dioxo-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-2(3H)-yl)ethyl)(2-((4R,7S,7aS)-1,3-dioxo-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-2(3H)-yl)ethyl)amino)-4-oxobutanoate(222 Synthesis of )

[0568]

[0569] Methyl 4-(bis(2-((methylsulfonyl)oxy)ethyl)amino)-4-oxobutanoate in DMA (350 ml) 220 To a solution of 8.5 g (80% purity, 80%, approx. 20 mM), 3,6-endoxo-Δ-tetrahydrophthalimide (10.2 g, 61.8 mM), sodium carbonate (8.0 g, 75.5 mM), and sodium iodide (0.3 g, 2.0 mM) was added. The mixture was stirred overnight at room temperature, concentrated, diluted with ethyl acetate (350 mL), and washed with saturated NaHCO3 solution (300 mL), saturated NaCl solution (300 mL), and 1 M NaH2PO4 (300 mL). The organic layer was dried over sodium sulfate, filtered, evaporated, loaded onto a silica gel column, and eluted with 10-30% ethyl acetate / hexane to yield the title compound (7.9 g, 77% yield). ESI MS m / z [M + Na] + 536.4.

[0570] Example 63. 4-(bis(2-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)ethyl)amino)-4-oxobutanoic acid ( 223 Synthesis of )

[0571]

[0572] Compound in 1,2-dichloroethane (150 ml) 222(3.0 g, 5.8 mmol) and trimethyltanol (4.8 g, 26.4 mmol) were refluxed at 80 °C for 8 hours, then cooled to room temperature, and the residue was passed through a short silica gel column and eluted with dichloromethane / methanol to remove excess trimethyltin hydroxide. The pooled fractions were then combined, concentrated, diluted with DMA and toluene, heated to 120 °C, and stirred overnight. The mixture was loaded onto a silica gel column and eluted with 5-10% methanol / dichloromethane to yield the title compound (1.62 g, 76% yield). ESI MS m / z [M + Na] + 386.2.

[0573] Example 64. (S)-tert-butyl 34-(4-(bis(2-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)ethyl)amino)-4-oxobutanamido)-28,35-dioxo-2,5,8,11,14,17,20,23,26-nonaoxa-29,36-diazatetracontan-40-oate( 224 Synthesis of )

[0574]

[0575] Compounds in DMA (20 ml) 223 (1.62g, 4.20mol) and compound 211 EDC·HCl (0.81 g, 4.20 ml) was added to a solution of (2.71 g, 3.82 ml). The reaction mixture was stirred overnight at rt, then poured into water (50 ml) and extracted with ethyl acetate (3 × 40 ml). The combined organic phase was washed with brine (40 ml), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography (10-50% ethyl acetate / petroleum ether) to yield a colorless oil (3.20 g, 80% yield). ESI MS m / z 1057.85 ([M+H] + ).

[0576] Example 65. (S)-34-(4-(bis(2-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)ethyl)amino)-4-oxobutanamido)-28,35-dioxo-2,5,8,11,14,17,20,23,26-nonaoxa-29,36-diazatetracontan-40-acid( 225 Synthesis of )

[0577]

[0578] Compound in formic acid (10 ml) 224 A solution of (3.20 g, 3.03 ml) was stirred overnight at rt. The solution was then concentrated and co-evaporated three times with toluene to yield a colorless oil (3.00 g, crude), which was used without further purification. ESI MS m / z 1001.50 ([M+H] + ).

[0579] Example 66. (S)-2,5-dioxopyrrolidine-1-yl 34-(4-(bis(2-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)ethyl)amino)-4-oxobutanamido)-28,35-dioxo-2,5,8,11,14,17,20,23,26-nonaoxa-29,36-diazatetracontan-40-oate( 226 Synthesis of )

[0580]

[0581] Compounds in DMA (15.0 ml) 225NHS (0.38 g, 3.33 mmol) and EDC·HCl (0.87 g, 4.55 mmol) were added to a solution of (3.00 g, crude material, 3.03 mmol), and the reaction mixture was stirred at rt for 2 hours, then diluted with water (50 mL) and extracted with ethyl acetate (3 × 30 mL). The combined organic phase was washed with brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified using a silica gel column (10-50% ethyl acetate / petroleum ether) to yield a colorless oil (2.90 g, 90% yield). ESI MS m / z 1098.50 ([M+H] + ).

[0582] Example 67. 14-(benzyloxy)-14-oxotetradecanoic acid ( 227 Synthesis of ) .

[0583]

[0584] K2CO3 (1.1g, 8mol) and BnBr (1.36g, 8mol) were added to a solution of tetradecanediate (2.06g, 8mol) in DMF (30ml). The mixture was stirred overnight at rt, then concentrated, and purified by column chromatography (ethyl acetate / petroleum ether) to obtain the title compound. 227 (1.2g, 45% yield) was provided. ESI MS m / z 349.23 ([M+H] + ).

[0585] Example 68. tert-butyl 3-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)propaneoate ( 228 Synthesis of )

[0586]

[0587] Sodium (0.1 g) was added to a solution of 2,2'-(ethane-1,2-diylbis(oxy))diethanol (55.0 ml, 410.75 ml, 3.0 eq.) in anhydrous THF (200 ml). The mixture was stirred until the Na disappeared, and then tertButyl acrylate (20.0 mL, 137.79 mmol, 1.0 eq.) was added dropwise. The mixture was stirred overnight, and then the reaction was stopped with an HCl solution (20.0 mL, 1 N) at 0°C. THF was removed by rotary evaporation, brine (300 mL) was added, and the resulting mixture was extracted with ethyl acetate (3 × 100 mL). The organic layer was washed with brine (3 × 300 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to yield a colorless oil (30.20 g, 79.0% yield), which was used without further purification. MS ESI m / z 278.17 ([M+H] + ).

[0588] Example 69. tert-butyl 3-(2-(2-(2-(tosyloxy)ethoxy)ethoxy)ethoxy)propaneoate ( 229 Synthesis of )

[0589]

[0590] in anhydrous DCM (220 ml) at 0 ℃ tert TEA (30.0 mL, 217.0 mL, 2.0 eq.) was added to a solution of 3-butyl 3-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)propaneoate (30.20 g, 108.5 mmol, 1.0 eq.) and TsCl (41.37 g, 217.0 mmol, 2.0 eq.). The mixture was stirred overnight at room temperature, then washed with water (3 × 300 mL) and brine (300 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (3:1 hexane / ethyl acetate) to yield a colorless oil (39.4 g, 84.0% yield). MS ESI m / z 433.28 ([M+H] + ). [05...

Claims

Claim 1 Side-chain linked conjugate compounds selected from the following: ; ; ; ; ; ; ; ; and In the above, mAb is a monoclonal antibody, and n is 1 to 30. Claim 2 Side-chain linked conjugate compounds selected from the following: ; ; ; ; ; ; ; ; ; ; ; and In the above, mAb is a monoclonal antibody, and n is 1 to 30. Claim 3 A side-chain linkage compound selected from the following, which can be readily reacted with a monoclonal antibody mAb to form a conjugate compound defined in claim 1: ; ; ; ; ; ; ; ; and . Claim 4 Side chain-linkage compounds selected from the following, which can be readily reacted with a monoclonal antibody mAb to form a conjugate compound defined in each of the following paragraphs: ; ; ; ; ; ; ; ; ; ; ; and . Claim 5 delete Claim 6 delete Claim 7 delete Claim 8 delete Claim 9 delete Claim 10 delete Claim 11 delete Claim 12 delete Claim 13 delete Claim 14 delete Claim 15 A pharmaceutical composition for the treatment or prevention of cancer, comprising the conjugate compound of claim 1 or 2, a pharmaceutically acceptable salt thereof, or a combination thereof together with a carrier, a diluent, or an excipient. Claim 16 In claim 15, 0.01 wt% to 99 wt% of the side-chain linked conjugate compound; 0.0 wt% to 20.0 wt% of one or more polyols; 0.0 wt% to 2.0 wt% of one or more surfactants; 0.0 wt% to 5.0 wt% of one or more preservatives; 0.0 wt% to 30 wt% of one or more amino acids; 0.0 wt% to 5.0 wt% of one or more antioxidants; 0.0 wt% to 0.3 wt% of one or more metal chelating agents; 0.0 wt% to 30.0 wt% of one or more buffer salts for adjusting the pH of the formulation to pH 4.5 to 7.5; and comprises 0.0% to 30.0% by weight of one or more isotonic agents for adjusting the osmotic pressure to 250 to 350 mOsm when reconstituted for administration to a patient; wherein the polyol is fructose, mannose, maltose, lactose, arabinose, xylose, ribose, rhamnose, galactose, glucose, sucrose, trehalose, sorbose, melesitose, raffinose, mannitol, xylitol, erythritol, maltitol, lactitol, erythritol, threitol, sorbitol, glycerol, or L-gluconate or a metal salt thereof; and the surfactant is polysorbate 20, polysorbate 40, polysorbate 65, polysorbate 80, polysorbate 81, or polysorbate 85, poloxamer, poly(ethylene oxide)-poly(propylene oxide) or polyethylene-polypropylene glycol; Triton TM ; Sodium dodecyl sulfate (SDS); sodium laurel sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl-, or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (lauroamidopropyl); myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodium methyl oleyl-taurate; Dodecyl betaine, dodecyl dimethylamine oxide, cocamidopropyl betaine and cocoamphoglycinate; or isostearyl ethylimidonium ethosulfate; The preservative is polyethyl glycol, polypropyl glycol, or a copolymer of ethylene and propylene glycol; the preservative is benzyl alcohol, octadecyl dimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl and benzyl alcohol, alkyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, or m-cresol; the amino acid is arginine, cysteine, glycine, lysine, histidine, ornithine, isoleucine, leucine, alanine, glycine, glutamic acid, or aspartic acid; the antioxidant is ascorbic acid, glutathione, cystine, or methionine; the chelating agent is EDTA or EGTA; and the buffer salt is citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid. A pharmaceutical composition comprising: sodium, potassium, ammonium, or trihydroxyethylamino salt; Tris or tromethamine hydrochloride, phosphate, or sulfate; arginine, glycine, glycylglycine, or histidine having an anionic acetate, chloride, phosphate, sulfate, or succinate salt; and the isotonic agent being mannitol, sorbitol, or sodium acetate, potassium chloride, sodium phosphate, potassium phosphate, trisodium citrate, or sodium chloride. Claim 17 A kit comprising a pharmaceutical composition according to claim 15 held in a vial, bottle, pre-filled syringe or pre-filled auto-injection syringe, wherein the pharmaceutical composition is in the form of a liquid or a lyophilized solid. Claim 18 A side-chain linked conjugate compound of claim 1 or 2 having in vitro, in vivo, or in vitro apoptotic activity. Claim 19 A pharmaceutical composition according to claim 15, formulated to be administered simultaneously with a chemotherapy agent, a radiation therapy agent, an immunotherapy agent, a treatment for autoimmune disorders, an anti-infective agent, or a conjugate for the synergistic treatment or prevention of cancer. Claim 20 In claim 19, the conjugate for the chemotherapy agent, radiotherapy agent, immunotherapy agent, treatment for autoimmune disorders, anti-infective agent, or synergistic treatment or prevention of cancer comprises the following: (1) a) alkylating agent: nitrogen mustard: chlorambucil, chlornafazine, cyclophosphamide, dacarbazine, estramustine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, mannomerstin, mitobronitol, melphalan, mitolactol, pipovoromatin, novelichine, phenesterin, prednimustine, thiotepa, trophosphamide, uracil mustard; CC-1065 or adezelesin, caselesin and bisezelesin or synthetic analogs thereof; duocamycin synthetic analogs thereof, KW-2189, CBI-TMI or CBI dimer; Benzodiazepine dimers or pyrrolobenzodiazepine dimers, or tomymycin dimers, indolinobenzodiazepine dimers, imidazobenzothiadiazepine dimers, or oxazolidinobenzodiazepine dimers; nitrosoureas: including camustine, lomustine, chlorozotocin, potemustine, nimustine, or ranimustine; alkylsulfonates: including busulfan, threosulfan, improsulfan, or piposulfan; triazenes or dacarbazine; platinum-containing compounds: including carboplatin, cisplatin, or oxaliplatin; aziridine, benzodopa, carbokuone, meturedopa, or uredopa; a) ethyleneimines or methylamellamines including altretamine, triethylenemelamine, triethylenephosphoamide, triethylenethiophosphoamide, or trimethylolomellamine; b) plant alkaloids: vinca alkaloids: including vincristine, vinblastine, vindecin, vinorelbine, or nabelbine; taxoids: including paclitaxel, docetaxol and their analogs, metansinoids including DM1, DM2, DM3, DM4, DM5, DM6, DM7, metansine and ansamitosine and their analogs, and cryptophysin; epotillone, eleuterobin, discodermolid, bryostatin, dolostatin, auristatin, tubulisin, cephalostatin;c) DNA topoisomerase inhibitors: Epipodophyllins: including 9-aminocamptothecin, camptothecin, crinatol, daunomycin, etoposide, etoposide phosphate, irinotecan, mitoxantrone, novantrone, retinoic acid (or retinol), tenifoside, topotecan, 9-nitrocamptothecin or RFS 2000; or including mitomycin; d) Antimetabolites: {[Anti-folicates: DHFR inhibitors: including methotrexate, trimetrexate, denopterin, pteropterin, aminopterin (4-aminopteric acid) or folic acid; IMP dehydrogenase inhibitors: including mycophenolic acid, thiazopurines, ribavirin, or EICAR; ribonucleotide reductase inhibitors: including hydroxyurea or deferoxamine; including pyrimidine, uracil, ancitabine, azacitidine, 6-azauridine, capecitabine, camoper, cytarabine, dideoxyuridine, doxyfluridine, enositabine, 5-fluorouracil, floxuridine, or laetitrexed; including cytosine, cytarabine, cytosine arabinoside, or fludarabine; including purines, azathioprine, fludarabine, mercaptopurines, thiaminephrine, or thioguanine; including folic acid supplements or proline acid; e) Hormonal therapy: Receptor antagonists: Anti-estrogens: including megestrol, raloxifene, or tamoxifen; LHRH agonists: including goscrclin or leuprolide acetate); anti-androgens: including bicalutamide, flutamide, calosterone, dromostanolone propionate, epithiostanol, goserelin, leuprolide, mefitiostan, nilutamide, testolactone, or trilostan)]; retinoids / deltoids: [including vitamin D3, CB 1093, EB 1089, KH 1060, cholecalciferol, or ergocalciferol; photodynamic therapy: including vertophorfin, phthalocyanine, photosensitizer Pc4, or demethoxyhypoquerelin A; cytokines: including interferon-alpha, interferon-gamma, tumor necrosis factor (TNF), or human proteins containing a TNF domain;f) Kinase inhibitors: BIBW 2992, anti-EGFR / Erb2, imatinib, gefitinib, pegaptanib, sorafenib, dasatinib, sunitinib, erlotinib, nilotinib, lapatinib, axitinib, pazopanib, vandetanib, E7080, anti-VEGFR2, mubritinib, ponatinib, bafetinib, bosutinib, cabozantinib, bismodezip, iniparib, ruxolitinib, CYT387, axitinib, tibozanib, sorafenib, bevacizumab, cetuximab, trastuzumab, ranibizumab, panitumumab, or ispinesib; g) Poly(ADP-ribose) polymerase (PARP) inhibitors: olaparib, niraparib, iniparib, talazoparib, h) Antibiotics: Enedyin antibiotics: Caliceamycin, Caliceamycin γ1, δ1, α1 or β1; Dynemycin including Dynemycin A and Deoxydynemycin; Esperamicin, Kedarcidin, C-1027, Maduropeptide or Neocarzinostatin chromophore or related chromoprotein enedyin antibiotic chromophores including Aclasinomycin, Actinomycin, Otramycin, Azacerin, Bleomycin, Cactinomycin, Carabicin, Kaminomycin, Carzinophylline; Includes chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, morpholino-doxorubicin, cyanomofolino-doxorubicin, 2-pyrrolino-doxorubicin or deoxydoxorubicin, epirubicin, eribulin, esorubicin, idarubicin, marcelomycin, nitomycin, mycophenolic acid, nogalamycin, olibomycin, peflomycin, popperomycin, furomycin, quellamycin, rhodorubicin, streptonigreen, streptozosin, tubercidin, uvenimex, genostatin, or rhodorubicin;i) Polyketides, acetogenins, bulathacin or bulathacinone, gemcitabine, efoxomycin, carfilzomib, bortezomib, thalidomide, lenalidomide, pomalidomide, tosedostat, zibrestat, PLX4032, STA-9090, Stimuvax, allovectin-7, Xegeva, provenge; TM , Yervoy TM , isoprenelation inhibitors or lovastatin, dopaminergic neurotoxins or 1-methyl-4-phenylpyridinium ion, cell cycle inhibitors including staurosporine, actinomycin including actinomycin D or dactinomycin, amanitin, bleomycin A2, bleomycin B2, bleomycin including peplomycin, daunorubicin, doxorubicin, adriamycin, idarubicin, epirubicin, pirarubicin, zorubicin, emtoxantrone, MDR inhibitors or anthracyclines including verapamil, Ca2+ ATPase inhibitors or tapsigargin, vorinostat, romidepsin, panobinostat, valproic acid, mosetinostat (MGCD0103), bellinostat, PCT-24781, entinostat, Histone deacetylase inhibitors including SB939, resminostat, zibinostat, AR-42, CUDC-101, sulforaphane, or tricostatin A; tapsigargin, celecoxib, glitazone, epigallocatechin gallate, disulfiram, salinosporamide A; anti-adrenergics including aminoglutothymide, mitothane, or trilostan; aceglatone; aldophosphamide glycosides; aminolevulinic acid; amsacrine; arabinoside, bestabucil; vasantren; edatlaxate; depopamine; demecolsin; diaziquone; eflonitin, DFMO, l-formitin; elliptinium acetate, etoglucid; gallium nitrate, gasaitosine, hydroxyurea; ibandronate, lentinan; Ronidamine; mithoguazone; mitoxantrone; mophidamol; nitracrine; pentostatin; phenamet; pyrarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; lasozoxic acid; lyzoxin; sizopyran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichotesene including T-2 toxin, verucarin A, loridin A, or anguidin; including urethanes, siRNA, or antisense drugs;(2) Anti-autoimmune disease agents: corticosteroids including cyclosporine, cyclosporine A, aminocaproic acid, azathioprine, bromocriptine, chlorambucil, chloroquine, cyclophosphamide, ammonide, betamethasone, budesonide, hydrocortisone, flunisolide, fluticasone propionate, flucotolone danazole, dexamethasone, triamcinolone acetonide, or beclomethasone dipropionate, DHEA, enanacept, hydroxychloroquine, infliximab, meroxicam, methotrexate, mofetil, mycophenylate, prednisone, sirolimus, or tacrolimus; (3) anti-infectious disease agents comprising a) to u) below: a) aminoglycosides: amikacin, astromycin, a) Gentamicin, Netylmicin, Cisomicin, Isefamicin, Hygromycin B, Kanamycin, Amikacin, Arbekacin, Bekanamycin, Dibekacin, Tobramycin, Neomycin, Pramycetin, Paromomycin, Ribostamycin, Netylmicin, Spectinomycin, Streptomycin, Tobramycin, or Verdamycin; b) Amphenicol: Azidafenicol, Chloramphenicol, Flofenicol, or Thiamphenicol; c) Ansamicin: Zeldanamycin or Herbimycin; d) Carbapenems: Viapenem, Doripenem, Ertapenem, Imipenem, Cilastatin, Meropenem, or Panipenem;e) Cephem: Carbacepem, Loracarbep, Cepacetril, Cefaclor, Cefradine, Cefadroxyl, Cephalonium, Cephaloridine, Cephalotine or Cephalosin, Cephalexin, Cephaloglycin, Cefamandol, Cefapyrin, Cefatrizine, Cefazaflu, Cefazedone, Cefazolin, Cefubuperazone, Cefcapen, Cefdaloxim, Cefepim, Cefminox, Cefoxitin, Cefprozil, Cefroxadine, Ceftezol, Cefuroxim, Cefixim, Cefdinier, Cefditoren, Cefepim, Ceftamet, Cefmenoxim, Cefodizyme, Sefofenidone, Cefoperazone, Sephoranide, Cefotaxim, Cefotiam, Cefozofran, Cephalexin, Cefpimizole, Cefpyramide, Cefpirom, Cefpodoxim, Cefprozil, f) Glycopeptides: including cefquinome, cefsulodin, ceftazidime, cefteramm, ceftibuten, ceftiolene, ceftizoxime, ceftobiprol, ceftriaxone, cefuroxime, cefuzonam, cefamycin, cefoxitin, cefoctetane, cefmethazole, oxacepem, flomoxef, or latamoxef; g) Glycopeptides: including bleomycin, vancomycin, orritavancin, telavancin, teicoplanin, dalbavancin, or lamoplanin; g) Glycylcyclines: including tigecycline; h) β-lactamase inhibitors: including fenam, sulbactam, tazobactam, clavam, or clavulanic acid; i) Lincosamides: including clindamycin or lincomycin; j) Lipopeptides: including daptomycin, A54145, or calcium-dependent antibodies; k) Macrolides: including azithromycin, setromycin, clarithromycin, dilistromycin, erythromycin, flurithromycin, yosamicin, ketolid, telithromycin, setromycin, midecamycin, myocamycin, oleandomycin, rifamycin, rifampicin, rifampin, rifabutin, rifapeptin, rokitamicin, roxithromycin, spectinomycin, spiramycin, tacrolimus (FK506), troleandomycin, or telithromycin; l) Monobactams: including aztreonam or tigemonam; m) Oxazolidinones: including linezolid;n) Penicillin: including amoxicillin, ampicillin, pavamphicillin, hetacillin, bacampicillin, metamphicillin, talampicillin, azidocillin, azlocillin, benzylpenicillin, benzathine benzylpenicillin, benzathine phenoxymethylpenicillin, clomethocillin, procaine benzylpenicillin, carbenicillin, carindacillin, cloxacillin, dicloxacillin, epicillin, flucloxacillin, mesillinam, pibmesillinam, mezlocillin, methicillin, napcillin, oxacillin, phenamecillin, penicillin, pheneticillin, phenoxymethylpenicillin, piperacillin, propicillin, sulfenicillin, temocillin, or ticarcillin; o) Polypeptide: including bacitracin, colistin, or polymyxin B; p) Quinolones: Allatrofloxacin, Valofloxacin, Ciprofloxacin, Clinafloxacin, Danofloxacin, Difloxacin, Enoxacin, Enrofloxacin, Floxacin, Garenoxacin, Gatifloxacin, Gemifloxacin, Grepafloxacin, Carnotrovafloxacin, Levofloxacin, Lomefloxacin, Marbofloxacin, Moxifloxacin, Nadifloxacin, Norfloxacin, Orbifloxacin, Ofloxacin, Pefloxacin, Trovafloxacin, Grepafloxacin, Sitafloxacin, Spafloxacin, Temafloxacin, Tosufloxacin, or Trovafloxacin; q) Streptogramin: Pristinamycin or Quinupristine / Dalfopristin; r) Sulfonamides: including mafenide, prontosil, sulfacetamide, sulfamethisole, sulfanylimide, sulfasalazine, sulfisoxazole, trimethoprim, trimethoprim-sulfamethoxazole, or co-trimoxazole; s) Steroid antimicrobial agents: including fusidic acid; t) Tetracyclines: including doxycycline, chlortetracycline, clomocycline, demeclocycline, limecycline, meclocycline, metacycline, minocycline, oxytetracycline, phenimepicycline, roliteteracryline, tetracycline, glycylcycline, or tigecycline;u) Other antibiotics: annonacin, asphenamine, bactofrenol inhibitor, bacitracin, DADAL / AR inhibitor, cycloserine, dictiostatin, discodermolid, eleuterobin, epotillon, ethambutol, etoposide, paropenem, fusidic acid, furazolidone, isoniazid, raulimalide, metronidazole, mupirocin, mycolactone, NAM synthesis inhibitor, phosphomycin, nitrofurantoin, paclitaxel, platensymycin, pyrazinamide, quinupristin / dalfopristin, rifampicin, rifampin, tazobactam, tinidazole, or uvarisin; (4) Antiviral drugs comprising a) to h) below: a) Inflow / fusion inhibitors: afraviroc, maraviroc, vicriviroc, gp41, a) Including enfuvirtide, PRO 140, CD4, or ibalizunab; b) Integrase inhibitors: including raltegravir, elvitegravir, or globoidnan A; c) Maturation inhibitors: including bevirimat or vibecon; d) Neuraminidase inhibitors: including oseltamivir, zanamivir, or peramivir; e) Nucleosides and nucleotides: Abacavir, Acyclovir, Adefovir, Amdoxovir, Apricitabine, Brivudine, Cidofovir, Clevudine, Dexelbusitabine, Didanosine, ddI, Elbusitabine, Emtricitabine, FTC, Entecavir, Pamcyclovir, Fluorouracil, 5-FU, 3'-fluoro-substituted 2',3'-dideoxynucleoside analogs (3'-fluoro-2',3'-dideoxythymidine, FLT, 3'-fluoro-2',3'-dideoxyguanosine, FLG, Formivirsen, Ganciclovir, Idoxuridine, Lamivudine, 3TC, 1-nucleosides including β-1-thymidine and β-1,2'-deoxycytidine, Pencyclovir, Includes Lasivir, Ribavirin, Stampedine, Stavudine, d4T, Taribavirin, Viramidine, Telbivudine, Tenofovir, Trifluridine, Valacyclovir, Valgancyclovir, Zalcitabine, ddC, Zidovudine, or AZT;f) Non-nucleoside: amantadine, arteviridine, caplavirine, diarylpyrimidine, etravirine, rilpivirine, delavirdine, docosanol, emivirine, efavirenz, foscarnet, phosphonoformic acid, imiquimod, interferon alpha, viride, rhodenosine, methizazone, nevirapine, NOV-205, pegylated interferon alpha, podophyllotoxin, rifampicin, rimantadine, resiquimod, R-848, or tromantadine; g) Protease inhibitors: including amprenavir, atazanavir, boceprevir, darunavir, fosamprenavir, indinavir, lopinavir, nelfinavir, pleconaryl, ritonavir, saquinavir, telaprevir, VX-950, or tiplanavir; h) Other types of antiviral drugs: including abuzyme, arbidol, calanolide a, ceragenin, cyanovirine-n, diarylpyrimidine, epigallocatechin gallate, foscarnet, griphiscin, taribavirin, viramidine, hydroxyurea, KP-1461, miltefoscin, pleconaryl, portmanteau inhibitors, ribavirin, or seliciclib. (5) Pharmaceutically acceptable salts, acids, hydrates, or hydrated salts of any of the above drugs; or optical isomers, racemic mixtures, diastereomers, or enantiomers.; Claim 21 In paragraph 19, chemotherapy agents, radiotherapy agents, immunotherapy agents, agents for autoimmune disorders, anti-infectives, or conjugates for the synergistic treatment or prophylaxis of cancer are abatacept, abemaciclib, abiraterone acetate, Abraxane, acetaminophen / hydrocodone, acalabrutinib, aducanumab, adalimumab, ADXS31-142, ADXS-HER2, afatinib diamaleate, aldesleukin, alectinib, alemtuzumab, alitretinoin, adotrastuzumab emtansine, amphetamine / dextroamphetamine, anastrozole, aripiprazole, anthracycline, aripiprazole, atazanavir, atezolizumab, atorvastatin, avelumab, Axicabtagene ciloleucel, axitinib, velinostat, BCG (raw), Bevacizumab, Bexarotene, Blinatumomab, Bortezomib, Bosutinib, Brentuximab Vedotin, Brigatinib, Budesonide, Budesonide / Formoterol, Buprenorphine, Cabazitaxel, Cabozantinib, Capmatinib, Capecitabine, Carfilzomib, Chimeric Antigen Receptor-Manipulated T (CAR-T) Cells, Celecoxib, Ceritinib, Cetuximab, Sidamide, Cyclosporine, Cinacalcet, Crizotinib, Cobimetinib, Cosentyx, Crizotinib, CTL019, Dabigatran, Dabrafenib, Dacarbazine, Daclizumab, Dacomotinib, Daptomycin, Daratumumab, Darbepoetin Alpha, Darunavir, Dasatinib, Denileukin Diptitox, Denosumab, Depacote, Dexlansoprazole, Dexmethylphenidate, Dexamethasone, Dignicab Cooling System, Dinutuximab, Doxycycline, Duloxetine, Duvelisib, Durvalumab, Elotuzumab, Emtricibin / Rilpivirine / Tenofovir Disoproxil Fumarate, Emtricibin / Tenofovir / Efavirenz, Enoxaparin, Ensatinib, Enzalutamide, Epoetin Alpha, Erlotinib, Esomeprazole, Eszopiclone, Etanercept, Everolimus, Exemestane, Everolimus, Exenatide ER, Ezetimibe, Ezetimibe / Simvastatin, Fenofibrate, Filgrastim, Fingolimod, Fluticasone Propionate, Fluticasone / Salmeterol, Fulvestrant,Gajiva, Gefitinib, Glatiramer, Goserelin Acetate, Icotinib, Imatinib, Ibritumomab Tiuxetane, Ibrutinib, Idelalisib, Ifosfamide, Infliximab, Imiquimod, ImmuCyst, Immuno BCG, Iniparib, Insulin Aspart, Insulin Detemia, Insulin Glargine, Insulin Lispro, Interferon Alpha, Interferon Alpha-1b, Interferon Alpha-2a, Interferon Alpha-2b, Interferon Beta, Interferon Beta 1a, Interferon Beta 1b, Interferon Gamma-1a, Lapatinib, Ipilimumab, Ipratropium Bromide / Salbutamol, Ixazomib, Canuma, Lanreotide Acetate, Lenalidomide, Lenaliomide, Lenvatinib Mesylate, Letrozole, Levothyroxine, Levothyroxine, Lidocaine, Linezolid, Liraglutide, Risdexamphetamine, LN-144, Loratinib, Memantine, Methylphenidate, Metoprolol, Mekinnist, Mercitabine / Rilpivirine / Tenofovir, Modalfinil, Mometasone, Mycidac-C, Necitumumab, Neratinib, Nilotinib, Niraparib, Nivolumab, Ofatumumab, Obinutuzumab, Olaparib, Olmesartan, Olmesartan / Hydrochlorothiazide, Omalizumab, Omega-3 Fatty Acid Ethyl Ester, Oncorin, Oseltamivir, Osimertinib, Oxycodone, Palbociclib, Palivizumab, Panitumumab, Panobinostat, Pazopanib, Pembrolizumab, PD-1 Antibody, PD-L1 antibody, pemetrexed, fetuzumab, Pneumococcal conjugate vaccine, pomalidomide, pregabalin, proscarvax, propranolol, quetiapine, rabeprazole, radium 223 chloride, raloxifene, raltegravir, ramucirumab, ranibizumab, regorafenib, ribociclib, rituximab, rivaoxaban, romidepsin, rosuvastatin, ruxolitinib phosphate, salbutamol, savolitinib, semaglutide, severamer, sildenafil, siltuximab, cipulucel-T, sitagliptin, sitagliptin / metformin, solifenacin, solanezumab, sonidextrin, sorafenib, sunitinib, tacrolimus, tacrimus, tadalafil, Tamoxifen, Tafinlar,Talimogene laherparepvec, talazoparib, telaprevir, talazoparib, temozolomide, temsirolimus, tenofovir / emtricitabine, tenofovir diisoproxyl fumarate, testosterone gel, thalidomide, TICE BCG, tiotropium bromide, Tisagenlecleucel, toremifene, trametinib, trastuzumab, Travectedin (ecteinascidin 743), trametinib, tremelimumab, trifluridine / tipiracil, tretinoin, Uro-BCG, ustekinumab, valsartan, veliparib, vandetanib, vemurafenib, venetoclax, vorinostat, A pharmaceutical composition comprising zeb-aflivercept or zostavax or pharmaceutically acceptable salts thereof or combinations thereof.

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