Targeted treatment of prostate cancer and other tumors by antibody-drug conjugates

Abstract

The present invention relates to antibody-drug conjugates with pendant linkers containing affinity ligands for enhancing targeted treatment of prostate cancer and other tumors. The invention also relates to the preparation of such conjugates, pharmaceutical compositions and methods of treating cancer.

Description

[0001] Description

[0002] BACKGROUND

[0003] Prostate cancer is the second most common cancer globally and the most common cancer among men in 84 developed countries, with an estimated 1.414 million new cases and 375,304 deaths in 2020 (Sung H, 2021, CA Cancer J Clin. 71:209-49). Incidence is also rising in developing countries (Baade PD et al, 2009 Molecular Nutrition & Food Research 53(2): 171-184. doi: 10.1002 / mnfr.200700511).

[0004] Treatment options for prostate cancer include surgery and non-surgical and combined treatments. Generally, external beam radiation therapy, brachytherapy, cryosurgery, high-intensity focused ultrasound and prostatectomy are suitable for men whose cancer is still confined to the prostate. (“Prostate cancer - Diagnosis and treatment - Mayo Clinic”. www.mayoclinic.org). Metastatic prostate cancer is usually treated with hormone therapy and chemotherapy. Exceptions include local or metastatic radiation therapy which can be used for advanced tumors with limited metastasis (Dhondt B et al, 2019 World Journal of Urology, 37(12):2557-2564, doi: 10.1007 / s00345-018-2609-8). Hormonal therapy is used to treat some early-stage tumors. If initial treatment fails and the cancer progresses, cryotherapy (a procedure that freezes the tumor), hormone therapy and chemotherapy can be performed.

[0005] Non-surgical treatments for prostate cancer include radiation therapy, chemotherapy, hormone therapy, external beam radiation therapy, particle therapy, high-intensity focused ultrasound therapy, or some combination of treatments (Hong H et al. 2010 Amino Acids. 39(1): 11-27; Peyromaure M et al. 2009, Progres en Urologie (in French) 19(11):803-9. doi:10.1016 / j.purol.2009.04.010). When hormone therapy reduces testosterone levels, the persistent prostate cancer is called castration-resistant prostate cancer (CRPC) ("Castrate-resistant prostate cancer: In NCI Dictionary of Cancer Terms", National Institutes of Health National Cancer Institute, September 17, 2019, accessed September 17, 2019). Many early cancers require normal levels of testicular hormones to grow, but CRPC does not. The term CRPC arose because these cancers exhibit dependence on hormones, particularly testosterone, to activate the androgen receptor (Seruga B et al. 2011 Nature Reviews. Clinical Oncology 8(1): 12-23). The cancer chemotherapy drug docetaxel has been used to treat CRPC with a median survival of 2 to 3 months ("Prostate cancer (hormone-refractory) - docetaxel", National Institute for Health and Clinical Excellence, 2010-12-10, original on 2012-02-02, accessed 2011-07-04). The second-line chemotherapy drug is cabazitaxel (de Bono J S et al. 2010 Lancet, 376(9747): 1147-1154). The combination therapy of bevacizumab, docetaxel, thalidomide, and prednisone appears to be effective against CRPC (Ning, Y-M et al. 2010 J Clin Oncol 28(12):2070-6). The use of sipuleucel-T immunotherapy in CRPC appears to extend survival by 4 months (Kantoff PW et al. 2010, The New England Journal of Medicine, 363(15):411-422).However, on May 19, 2015, the marketing authorization for sipuleucel-T was withdrawn because a second sipuleucel-T trial in asymptomatic CRPC phase II / III, compared to placebo, failed to show a statistically significant improvement in time to disease progression (www.sciencedirect.com / topics / neuroscience / sipuleucel-T). Enzalutamide is another second-line hormonal drug with a 5-month survival advantage. Abiraterone and enzalutamide are currently being tested in CRPC patients who have not received chemotherapy before (Liu J M et al., 2020, Sci Rep. 10(1): 4240). Abiraterone acetate. GnRH antagonist therapy is the standard of care for high-risk metastatic castration-sensitive prostate cancer (mCSPC) (Koroki, Y and Taguri, M, 2022, Target Oncol. doi: 10.1007 / s11523-022-00929-3; Saad F et al., 2022, Lancet Oncol. (10): 1297-1307). Not all patients respond to androgen signal blocking drugs. Certain cells with stem cell-like characteristics are not affected (Qin J, et al., 2012, Cell Stem Cell. 10(5): 556-569; Maitland N J, Collins A T, 2008, J. Clinical Onc. 26(17): 2862-2870). Therefore, to improve the outcome of CRPC treatment, it is necessary to increase the dose or combine treatment with a synergistic androgen signal blocker (Attard G et al., 2011, Clinical Cancer Research. 17(7): 1649-1657). But even these combinations do not affect stem cell-like cells that do not have androgen signals (Rane J K et al., 2012, Nature Reviews. Urology. 9(10): 595-602). For metastatic prostate cancer patients who have already spread to the bone, doctors use various bone-modifying agents to prevent bone complications and support the formation of new bone mass. Zoledronic acid (a bisphosphonate) and denosumab (a RANK ligand inhibitor) appear to be effective drugs, but they produce frequent and serious adverse events (Jakob T et al., 2020, The Cochrane Database of Systematic Reviews. 2020(12): CD013020. doi: 10.1002 / 14651858).

[0006] Over the past decade, immunotherapy and poly (ADP-ribose) polymerase (PARP) inhibitors have proven effective for specific subsets of prostate cancer patients. At the same time, several trials have also demonstrated survival benefits achieved with novel androgen receptor signal inhibitors (ARSI) in non-castration-resistant metastatic disease and non-metastatic castration-resistant cancer patients. Among the emerging drugs, “antibody-drug conjugates” (ADCs) are a new class of compounds consisting of a cytotoxic drug (called “payload”) linked to a specific antibody capable of recognizing antigens expressed on the surface of cancer cells, which are very noteworthy because their clinical practice has changed the treatment outcome of other malignancies (in particular breast cancer) (Fan P and Xu K., 2022, Biochim Biophys Acta Rev Cancer, 1878(1): 188849; Chen N et al., Expert Rev Anticancer Ther., (12) 1325-1331). The therapeutic approach of ADCs guarantees minimal exposure of healthy tissues to cytotoxic drugs, expanding the therapeutic window of targeted therapies (Schwach J et al., 2022, Front Biosci (Landmark Ed), 27(8): 240. doi: 10.31083 / jfbl2708240; Marei H E et al., 2022, Cancer Cell Int. 22(1): 255). In the development of ADCs, prostate cancer-selective antigens have been identified as targets for imaging or therapeutic interventions. For prostate cancer, many researchers, including us, have focused on the prostate-specific membrane antigen (PSMA) (Bander N H et al., J Clin Oncol. 2005;23:4591-601; Milowsky M I et al., J Clin Oncol. 2004;22, 2522-31; Tagawa S T et al., Clin Cancer Res. 2013, 19:5182-91; Afshar-Oromieh A et al., Eur J Nucl Med Mol Imaging. 2017;44:1258-68), the prostate stem cell antigen (PSCA) (Morris M J et al., Ann Oncol. 2012;23:2714-9), the B7 homolog 3 protein (B7-H3, also known as CD276), the prostate six-transmembrane epithelial antigen 1 (STEAP1), the trophoblast cell antigen 2

[0007] (Trop2), CD46 (Rosellini M et al., Int J Mol Sci. 2021, 22(4): 1551, doi:

[0008] 10.3390 / ijms22041551), NCAM1 and carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5)

[0009] (Lee, J. K. et al. Proc Natl Acad Sci USA. 2018; 115(19): E4473-82; DeLucia, D. C. et al. Clin Cancer Res. 2021; 27(3): 759-74), is the best antigen targeted by ADCs (Mjaess G et al. 2022, Clin Genitourin Cancer. S1558-7673(22)00164-1. doi: 10.1016 / j.clgc.2022.07.009).

[0010] Prostate-specific membrane antigen (PSMA), also known as folate hydrolase 1 or N-acetyl-alpha-linked acidic dipeptidase: N-acetyl-L-aspartyl-L-glutamate peptidase I (NAALADase I), is an integral membrane protein with a molecular weight of 110 kDa, consisting of 750 amino acids, distributed in three domains, including an intracellular domain comprising 19 amino acids, a transmembrane domain consisting of 24 amino acids, and an extracellular domain comprising 707 amino acids (Jones, W. et al., Cancers, 2020, 12: 1367; Wang, F. et al., Prostate Cancer Prostatic Dis. 2022, 25(1): 11-26, doi: 10.1038 / s41391-021-00394-5). PSMA is active in the central nervous system, cleaving the neurotransmitter N-acetylaspartate-l-glutamate (NAAG) to N-acetylaspartate (NAA) and glutamate (Zhou J et al., Nat Rev Drug Discov. 2005; 4: 1015-26). In malignant tissues, PSMA is thought to be involved in angiogenesis, as increased expression of PSMA is found in the stroma near neovasculature of solid tumors (Conway R.E. et al., Mol Cell Biol. 2006; 26: 5310-24). High expression of PSMA is associated with tumor aggressiveness, androgen ablation and deprivation (Wright, G.L. et al., Urology, 1996, 48, 326-34). PSMA is highly expressed in prostate cancer cells and in the endothelial neovasculature of several solid human malignancies (Israeli R S et al., 1993, Cancer Res. 53: 227-30; Perner S et al., 2007, Hum Pathol. 38: 696-701; Trover, J. et al., 1995, Int. J. Cancer 62, 552-8; Ryu, Y.J. et al., BMC Cancer 2022, 22(1): 1278, doi: 10.1186 / s12885-022-10375-z). PSMA high expression is an independent biomarker of poor prognosis throughout the course of prostate cancer and surgery (Hupe M C et al., 2018, Front Oncol. 8: 623; Bostwick D G et al., 1998, Cancer 82: 2256-61; Minner S et al., 2011, Prostate 71: 281-8). Normal human tissues, including the prostate epithelium, small intestine, renal tubules and salivary glands, have much lower levels of PSMA expression than prostate cancer (Silver DA et al., Clin Cancer Res. 1997, 3: 81-85).Therefore, it has become an ideal target for prostate cancer specific diagnosis and precision therapy (Barve A. et al., J. Control. Release., 2014, 187: 118-32.). Clinical studies of PSMA-targeted small molecules or antibodies labeled with radionuclides or cytostatic agents have been ongoing (He, Y. et al., Signal Transduction and Targeted Therapy (2022) 7: 198; Wang, F. et al., Prostate Cancer Prostatic Dis. 2022, 25(1): 11-26). 177 Lutetium 177 (Lu)-PSMA-617 (Pluvicto TM , lutetium Lu 177 vipivotide tetraxetan) clinical success, it was approved by the US FDA in 2022 for the treatment of metastatic castration-resistant prostate cancer (mCRPC). Many other PSMA-targeted radiopharmaceuticals are currently under development, including those containing 177 Lutetium 131 Iodide 131 (I) and 67 Copper 67 (Cu). Targeted PSMA with alpha emitters can include 225 Actinium 225 (Ac), 227 Thorium 227 (Th) and 212 Lead 212 (Pb), but these drugs still have salivary and kidney toxicity problems (Sartor, O. and Baghian, A., Front Med (Lausanne), 2022, 9: 1060922, doi: 10.3389 / fmed.2022.1060922). Therefore, some of these radioligands are coupled with monoclonal antibodies such as J591 and TLX591 to target PSMA. In addition, PSMA inhibition leads to phosphoinositide-3 kinase (PI3k) and serine / threonine kinase (AKT) signaling pathway blockade, both of which are significant in cancer cell proliferation (Olson W.C., Front. Biosci. Landmark. 2014, 19: 12-33). Several PSMA-targeted antibody-drug conjugates have also been evaluated in the treatment of castration-resistant metastatic tumors. MLN2704, developed by Millennium Pharmaceuticals (a subsidiary of Takeda Pharmaceuticals Co) and ImmunoGen Inc, is an ADC that links a deimmunized anti-PSMAextThe monoclonal antibody MLN591 was conjugated to the traditional anti-microtubule drug maytansine-1 (DM1) and showed preclinical activity, which was later evaluated in a phase I study (Galsky, M.D., et al., J Clin Oncol. 2008; 26(13):2147-54; Henry, M.D., et al., Cancer Res. 2004; 64:7995-8001). Unfortunately, the clinical trial of MLN2704 (NCT00070837) showed unfavorable safety, with peripheral neuropathy being the most serious toxicity, which was attributed to the release of free DM-1 from the labile disulfide linker (Niaz M.O., et al., Cureus. 2020 doi: 10.7759 / cureus.7147; Galsky M.D., et al., J. Clin. Oncol. 2008, 26:2147-54; Milowsky M.I., et al., Urol. Oncol. 2016, 34:530.e15-530.e21. doi: 10.1016 / j.urolonc.2016.07.005). But the trial demonstrated the potential of PSMA-targeted ADCs for the treatment of mCRPC. Progenics Pharmaceuticals’ anti-PSMA-ADC, using a fully human IgG1 anti-PSMA monoclonal antibody, linked to monomethyl auristatin E (MMAE) via a valine-citrulline linker, showed some activity in phase I and II trials in subjects with metastatic castration-resistant prostate cancer (mCRPC) treated with abiraterone / enzalutamide (abi / enz) in terms of prostate-specific antigen (PSA) decline, circulating tumor cell (CTC) conversion / reduction, and radiographic assessments (Petrylak, D.P., et al., Prostate, 2020, 80(1):99-108). But clinically significant treatment-related adverse events (AEs) included neutropenia and neuropathy, which were off-target toxicities of the free MMAE payload, hindering further clinical investigation of the ADC as a monotherapy. MEDI3726 (ADCT-401) from Medimmune / AstraZeneca is a PSMA-ADC comprising an engineered version of the anti-PSMA IgG1 kappa antibody (J591) specifically conjugated to a pyrrolobenzodiazepine (PBD) dimer (SG3199) via a pegylated, cleavable VA-PABC linker (Cho S, et al., Mol Cancer Ther. 2018 Oct; 17(10):2176-2186).The Phase I trial dose escalation plan for MEDI3726 was delayed due to treatment-related adverse events (TRAEs) (deBono, J.S. et al. Clin Cancer Res. 2021; 27(13): 3602-9). ARX517 is an anti-PSMA antibody drug conjugate (ADC) that incorporates synthetic amino acids (SAAs) into the antibody to conjugate MMAF via Ambrx’s short polyethylene glycol amine linker (AS269) in a site-specific manner, currently in a Phase I study (APEX-01; NCT04662580) to evaluate the safety, pharmacokinetics (PK), and anti-tumor activity of the drug in patients with PSMA-expressing solid tumors. 5D3-DM1 from Johns Hopkins University School of Medicine is an anti-PSMA-ADC that uses a traditional non-cleavable DM1-MCC payload / linker complex, successfully controlled the growth of PSMA(+) tumors without causing systemic toxicity in preclinical evaluations (Huang, C.T. et al. Mol Pharm. 2020; 17(9): 3392-3402). BIND-014 from Epic Sciences, Inc and Bind Therapeutics, Inc is a docetaxel-encapsulated nanoparticle and a hydrophilic polyethylene glycol corona modified with a small molecule PSMA-targeting ligand, clinical trials were evaluated in metastatic castration-resistant prostate cancer (mCRPC) patients (NCT01812746) showing anti-tumor activity and tolerability in chemotherapy-naive mCRPC patients (Autio, K.A. et al. JAMA Oncol. 2018, 4(10): 1344-51). There are several other PSMA-ADCs, including our DXC010 and HDP-103 from Heidelberg Pharma, also in preclinical studies.

[0011] B7-H3 (B7 homolog 3 protein, also known as CD276) is a member of the B7 ligand family, a type I transmembrane protein of 316 amino acids in length, with the extracellular domain consisting of two immunoglobulin constant (IgC) and variable (IgV) domains (Duan H. and Huang M. Int. J. Data Min. Bioinform. 2012, 6:292-303). It is overexpressed on differentiated malignant cells and cancer initiating cells, with limited heterogeneity, at high frequency (60% of 25,000 tumor samples) in many different types of cancer, such as prostate cancer, non-small cell lung cancer (NSCLC), melanoma, bladder cancer, breast cancer, clear cell kidney cancer, and head and neck squamous cell carcinoma (HNSCC), but rarely detected in normal tissues (U. Malapelle, Int J Mol Sci. 2022 Dec;23(24): 16077). In non-malignant tissues, B7-H3 plays a major inhibitory role in adaptive immunity, suppressing T cell activation and proliferation. In malignant tissues, B7-H3 suppresses tumor antigen-specific immune responses, leading to pro-tumor effects. B7-H3 also has non-immune pro-tumor functions, such as promoting migration and invasion, angiogenesis, chemotherapy resistance, and endothelial mesenchymal transition, as well as affecting tumor cell metabolism. Studies have shown that the expression of B7-H3 promotes the progression of prostate cancer in vivo by reducing the apoptosis of myeloid-derived suppressor cells (Zhou Y. et al. Technol. Cancer Res. Treat. 2020, 19: 1533033820971649). In addition, B7-H3 overexpression is associated with an increased risk of prostate cancer progression (Bonk S. et al. Pathol. Int. 2020, 70, 733-42). Therefore, the expression of B7-H3 in tumors has been shown to be associated with poor prognosis. Therefore, based on the latest knowledge of molecular biology and the progress of antibody engineering, it is possible to target B7-H3 through several mechanisms. Among them, antibody-drug conjugates, monoclonal antibody-mediated cytotoxicity, and bispecific antibodies with CD3 are therapeutic approaches being studied in clinical I / II phase trials for solid tumors (https: / / www.clinicaltrials.gov / ).These molecules include MGC018 (an ADC coupling a humanized B7-H3 monoclonal antibody to the alkylating agent prodrug seco-Duocarmycin hydroxybenzamide azaindole (DUBA) via a cleavable linker), DS-7300a (an ADC coupling a humanized anti-B7-H3 IgGl monoclonal antibody (MABX-9001a) to the topoisomerase I inhibitor Dxd via a cleavable linker), MGA271 (Enoblituzumab, an Fc-optimized humanized IgGl monoclonal antibody binding to B7-H3), and MGD009 (Obrindatamab, a humanized bispecific DART molecule recognizing B7-H3 and CD3). Ongoing clinical trials have demonstrated the antitumor activity and safety of these molecules, but the frequency of treatment-emergent adverse events (TEAEs) is high. Therefore, there is a great need for improved drug design targeting B7-H3 (Kontos F et al. Clin Cancer Res. 2021, 27(5): 1227-35).

[0012] Prostate six transmembrane epithelial antigen 1 (STEAP1) is a full membrane protein containing 339 amino acids, composed of 4 new cell surface markers that are highly expressed in prostate cancer and several other cancers, with limited expression in normal tissues, making it a potential target for ADC-based therapy (Hubert R.S. et al. Proc. Natl. Acad. Sci. USA. 1999, 96: 14523-8; Pia M Challita-Eid et al. Cancer Res. 2007, 67(12): 5798-805, doi: 10.1158 / 0008-5472.CAN-06-3849; Moreaux, J et al. Biochem Biophys Res Commun. 2012, 429: 148-55; Gomes, I.M. et al. Mol Cancer Res. 2012, 10: 573-8), monoclonal antibodies, DNA vaccines and small non-coding RNA drugs (Barroca-Ferreira J et al. Curr Cancer Drug Targets. 2018; 18: 222-30). The exact function of STEAP1 has not been determined, it seems to be an ion channel or transporter that plays a role in cell adhesion, possibly related to tumor proliferation and invasiveness (Hubert R.S. et al. Proc. Natl. Acad. Sci. USA. 1999, 96: 14523-8). Studies have shown that in the STEAP heterotrimer, STEAP1 binds to the intracellular NADPH binding domain of another member of the STEAP family, STEAP4, facilitating the reduction of iron (III) (Oosterheert W. et al. J. Biol. Chem. 2020, 295: 9502-12). Knockout of the STEAP1 gene was associated with inhibition of cell viability and proliferation, and enhancement of apoptosis in the LnCaP prostate cancer cell line (Gomes, I.M. et al. Med. Oncol. 2018, 35, doi: 10.1007 / s12032-018-1100-0). In addition, targeting STEAP1 by specific single-chain antibodies blocks gap junctions, resulting in an 80-90% reduction in communication between prostate cancer cells (Esmaeili S.-A. et al. Anticancer Agents Med. Chem. 2017, 18: 1674-9). Expression of STEAP1 can be used as a biomarker for poor prognosis in prostate cancer (Ihlaseh-Catalano SM et al. Histopathology, 2013; 63: 678-685).DSTP3086S is a humanized IgGl anti-STEAP1 monoclonal antibody (MSTP2109A) ADC conjugated to the potent anti-mitotic agent monomethyl auristatin E (MMAE). In a Phase I clinical trial, DSTP3086S showed acceptable safety and anti-tumor activity, demonstrating the potential benefit of treating metastatic castration-resistant prostate cancer expressing STEAP1 with a targeted STEAP1 antibody-drug conjugate (Danila D.C. et al., J. Clin. Oncology, 2019, 37(36), 3518-27). In addition, anti-Steap1 antibody-radioisotope conjugates have been used in preclinical studies. 111 In or 89 Zr-MSTP2109A showed a correlation between STEAP1 expression, radiolabeled antibody tumor uptake and ADC efficacy. 89 Zr-DFO-MSTP2109A has been used to detect anti-androgen therapy-induced changes in STEAP1 (Doran M G et al., J Nucl Med. 2014; 55, 2045-9).

[0013] Trophoblast cell surface antigen 2 (TROP2), also known as EGP-1, GA733-1, and M1S1, also known as tumor-associated calcium signal transducer 2 (TACSTD2), is a cell membrane-bound glycoprotein that can act as a transmembrane transducer of intracellular (IC) calcium signals. It is expressed in many normal tissues, including epidermis, breast, cervix, cornea, lung, liver, pancreas, prostate, trophoblast cells, or urothelium, but is overexpressed in a variety of tumors, such as pancreatic cancer, ovarian cancer, prostate cancer, and breast cancer (Shvartsur A. and Bonavida B., Genes Cancer 2014, 6: 84-105; Wen Y. et al., Ann. Transl. Med. 2022, 10(24): 1403). TROP2 plays an important role in tumor cell proliferation, apoptosis, and invasion, thereby affecting the prognosis and treatment of tumor patients (Wu B et al., Exp Ther Med 2017; 14: 1947-52). In many malignant tumors, TROP2 is up-regulated in invasive prostate cancer, and its expression can promote the alpha5beta1 integrin-dependent pro-metastatic signaling pathway in cancer cells (Trerotola M. et al., Oncotarget. 2015, 6: 14318-28). In addition, the expression of Trop2 has been confirmed to be related to the neuroendocrine differentiation of prostate cancer cells (Hsu E.C., et al., Proc. Natl. Acad. Sci. USA. 2020, 117: 2032-42), which makes it resistant to standard treatment and associated with poor prognosis (Ge R., et al., Ann. Oncol. 2020, 31: 470-9; Santoni M., et al., Biochim. Biophys. Acta Rev. Cancer. 2014, 1846: 630-7). As a transmembrane protein overexpressed in a variety of tumors, Trop2 has become a promising immunotherapy target (Goldenberg D.M. et al., Oncotarget. 2018, 9(48): 28989-29006; Lin H et al., Int. J. Cancer 2014, 134: 1239-49).Monoclonal antibodies (mAbs), bispecific antibody engagers, antibody-drug conjugates (ADCs), virus-like particles (VLPs), and antibody-drug combinations with traditional chemotherapy, immunotherapy, radioimmunotherapy, photodynamic therapy, and nanoparticles have rapidly evolved over the past two decades targeting TROP2 (Bignotti, E. et al., Int. J. Gynecol. Cancer 2011, 21: 1613-21; Kaplon, H. et al., MAbs 2020, 12: 1703531; Sahota S, Vahdat LT. Expert Opin. Biol. Ther. 2017, 17: 1027-31). The TROP2-ADC from Immunomedics, sacituzumab govitecan (IMMU-132), covalently links an irinotecan active metabolite (SN-38) to a monoclonal Trop2 antibody (hRS7) via a hydrolysable CL2A linker. hRS7-SN-38 treatment of tumor-bearing mice in five different tumor models significantly inhibited tumor growth. After showing clinical activity, sacituzumab govitecan received accelerated approval from the US FDA in April 2020 for the treatment of metastatic triple-negative breast cancer (Syed Y.Y, Drugs 2020, 80: 1019-25). Currently, the ADC is undergoing approximately 20 clinical trials to improve treatment against a variety of tumors (according to https: / / www.clinicaltrials.gov; Wen, Y. et al., Ann. Transl. Med. 2022, 10(24): 1403), including an ongoing phase II trial in mCRPC (NCT03725761). The site-specifically conjugated TROP2-ADC, RN927C (Pfizer’s PF-06664178) is a humanized anti-TROP2 hlgG1 antibody that is specifically conjugated to the microtubulin inhibitor payload dolastatin 10 analog (PF-06380101) at the C-terminus of the antibody heavy chain using a cleavable AcLys-VC-PABC linker via an enzymatic process. RN927C showed potent cell killing in a variety of tumor cell lines and patient-derived xenograft tumor models, including pancreatic cancer and triple-negative breast cancer (Strop, P. et al., Mol. Cancer Ther. (2016) 15(11): 2698-708).Datopotamab deruxtecan (Dato-DXd, DS-1062a, from Daiichi Sankyo) is another TROP2-targeting ADC, conjugated to the potent DNA topoisomerase I inhibitor (DXd) through a tetrapeptide (GPGG)-based linker (Okajima, D. et al., Mol Cancer Ther. 2021, 20(12):2329-40). Dato-DXd showed potent activity against TROP2-expressing tumors and acceptable safety in preclinical models by delivering the payload effectively into tumors. Dato-DXd is currently in clinical trials in patients with triple-negative breast cancer and other TROP2-expressing tumors (NCT03401385 and NCT04612751). So far, several Trop2-ADCs are in clinical evaluation, including our Trop2-tubulysin B analog ADC (DAC002 or JS108) (NCT046012857), SKB264 by Kolon (NCT04152499), FDA018 by Fudan-Zhangjiang Bio (NCT05174637).

[0014] CD46 is a transmembrane glycoprotein that is a complement regulator that inactivates C3b and C4b (Cardone J. et al., Clin. Exp. Immunol. 2011, 164:301-11). CD46 is upregulated by IFNy + IL-10 - CD4 + T cells replace IFNy + IL-10 +CD46 is a key player in downregulating Th1 responses (Cardone J. et al., Clin. Exp. Immunol. 2011, 164, 301-11). Deficiency in CD46 reduces surface expression of C3b and / or C4b and its inactivation capacity, leading to uncontrolled complement activation and systemic microthrombosis (Cardone J. et al., Clin. Exp. Immunol. 2011, 164:301-11). Based on the high expression of CD46 in prostate cancer tissues and CRPC and low expression in normal tissues (Elvington M. et al., Antibodies, 2020, 9:59. doi: 10.3390 / antib9040059), CD46 is an ideal target for ADC drugs (Su Y. et al., JCI Insight, 2018, 3:el21497. doi: 10.1172 / jci.insight.121497). A CD46-ADC (FOR46) has been shown to effectively and selectively kill adenocarcinoma and NEPC cells in vitro and in vivo (Su Y. et al., JCI Insight, 2018, 3:el21497. doi: 10.1172 / jci.insight.121497). This ADC (FOR46) is in a phase 1 clinical trial to evaluate safety and efficacy in mCRPC patients (NCT03575819).

[0015] Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) is a cell surface glycoprotein from the carcinoembryonic antigen family involved in cell adhesion, differentiation, proliferation, and survival (Taheri M et al., J Biol Chem. 2000;275:26935-43). This antigen is highly expressed in multiple epithelial tumors, such as colorectal, lung, and gastric adenocarcinoma. CEACAM5 is abundantly expressed in neuroendocrine prostate cancer (NEPC) with minimal overlap with prostate-specific membrane antigen, prostate stem cell antigen, and trophoblast cell surface antigen 2 expression compared to other mCRPC subtypes (DeLucia D.C. et al., Clin Cancer Res. 2021;27(3):759-74). Tusamitamab ravtansine (SAR408701) from Sanofi / ImmunoGen is a first-in-class humanized monoclonal CEACAM5 antibody conjugated to the potent maytansinoid derivative DM4 via an N-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB) linker, with a good safety profile, with its dose-limiting toxicity (DLT) being reversible dose-related keratopathy, with a maximum tolerated dose of 100 mg / m 2Q2W (Gazzah, A. et al., Ann Oncol., 2022, 33(4):416-25). The anti-CEACAM5-SN38 ADC, Labetuzumab govitecan by Immunomedics Inc. was shown to be able to induce DNA damage in CEACAM5 + induced DNA damage in prostate cancer cell lines and showed anti-tumor effects in CEACAM5 + CRPC xenograft models, including chemotherapy-resistant NEPC, showed significant anti-tumor responses (DeLucia D.C. et al., Clin Cancer Res. 2021;27(3):759-74).

[0016] In addition, TF-ADC (Tisotumab vedotin, de Bono J.S. et al., 2019, Lancet Oncol 7, 383-93) and DLL3-ADC (Ravalpituzumab tesirine, Rova-T, Mansfield A.S. et al., 2021, NPJ Precis Oncol, 5, 74, (NCT02709889)) are also in clinical trials for the treatment of prostate cancer. Tisotumab vedotin (Tivdak TM ) is an antibody-drug conjugate that couples a fully human monoclonal antibody specific for tissue factor (TF-011) to a protease-cleavable linker to monomethyl auristatin E (MMAE), which has been engineered to target tumors expressing tissue factor. Based on the results of a phase II clinical trial, tisotumab vedotin has received accelerated approval in the United States for the treatment of adult patients with recurrent or metastatic cervical cancer who have experienced disease progression on or after platinum-based chemotherapy. DLL3 (Delta-like protein 3) is highly expressed in solid tumors, including neuroendocrine cancers / tumors (NEC / NET), melanoma, small cell lung cancer (SCLC), medullary thyroid cancer (MTC), and glioblastoma (GBM). Ravalpituzumab tesirine (Rova-T) is an anti-DLL3 antibody conjugate that couples an antibody to a DNA minor groove binder, tesirine (pyrrolobenzodiazepine (PBD) dimer), via a protease-cleavable linker.

[0017] The glutamate urea small molecule (Glu-urea) is an inhibitor of PMSA folate hydrolase I and can bind specifically to PSMA and be endocytosed into PSMA-positive cells (Leamon, C.P. et al., 2019, Bioconjugate Chem. 30, 1805-1813). PluvictoTM (177Lu-PSMA-617, now known as lutetium Lu 177 vipivotide tetraxetan) uses a glutamate urea small molecule as a targeted delivery vehicle, linked to the radioisotope Lu177. On March 23, 2022, the U.S. FDA approved it for the treatment of adult patients with prostate-specific membrane antigen (PSMA)-positive metastatic castration-resistant prostate cancer (mCRPC) who have received androgen receptor (AR) pathway inhibition and chemotherapy with platinum-based chemotherapy (Keam S J. 2022, Mol Diagn Ther. 26(4):467-475). In addition to PSMA, the G-protein coupled neurotensin receptor (NTR) and its ligand neurotensin peptide (NT) have recently been implicated to play an important role in a variety of cancers, especially prostate and lung cancer (Morgat C., et al., 2014, J. Nucl. Med. 55, 1650-7; Valerie, N.C.K., et al., 2011, Cancer Res. 71, 6817; Souazé, F., et al., 2006, Cancer Res. 66, 6243; Alifano M., et al., 2010, Clin. Cancer Res. 16, 4401). NTR was found to be overexpressed in androgen-independent human prostate tissues, thus providing a potential target for prostate cancer diagnosis and treatment (Sehgal I., et al., 1994, Proc. Natl. Acad. Sci. USA. 91, 4673; Lee L.-F., et al., 2001, Mol. Cell. Biol. 21, 8385; Almeida T.A., et al., 2010, Peptides 31, 242-7). In addition, NTR1 has also been reported to be expressed in neuroendocrine prostate cancers that are low in PSMA expression (Hashimoto K., et al., 2015, Lab. Invest. 95, 283-95; Zhu S., et al., 2019 Oncogene 38(24): 4875-84). Clearly, NTR1 is complementary to PSMA and can be another important biomarker for prostate cancer.

[0018] The gastrin-releasing peptide receptor (GRPR) is a member of the bombesin (BBN or BN) G protein-coupled receptor family that is aberrantly overexpressed in several malignancies, including breast, prostate, pancreatic, lung, and central nervous system tumors (Liu S. et al., Bioconjug Chem. 1997, 8(5):621-36; Cornelio D.B. et al., (2007) Ann Oncol 18, 1457-66; Weber H.C., Curr Opin Endocrinol Diabetes Obes. 2009; 16(1): 66-71). In addition, it mediates non-histaminergic itch and pathological itch in mice. BBN is an amphibian neuropeptide, pGlu-Gln-Arg-Leu-[(Gly-Asn-Gln-)Trp-Ala-Val-Gly-His-Leu-Met-NH2], consisting of 14 amino acids (Maina T. et al., J Nucl Med. 2005, 46(5):823-30; Smith C.J. et al., Nucl Med Biol. 2003, 30(8):861-8), which was first isolated from the skin of the frog in 1970 (Erspamer V. et al., J Pharm Pharmacol. 1970, 22(11):875-6). GRP is a 26 / 27 amino acid mammalian regulatory peptide with the sequence Ala-Pro-Val-Ser-Val-Gly-Gly-Thr-Val-Leu-Ala-Lys-Met-Try-Pro-Arg-[(Gly-Asn-His-)Trp-Ala-Val-Gly-His-Leu-Met-NH2]. GRP and BBN share a homologous 7-amino acid amidated C-terminal region (-Trp-Ala-Val-Gly-His-Leu-Met-NH2) that is necessary for high-affinity binding to and signal transduction by the GRPR (Smith C.J. et al., Nucl Med Biol. 2005, 32(7):733-40; Ananias H.J. et al., Curr Pharm Des. 2008; 14(28):3033-47).In addition to the release of gastrin, GRP and BBN-like peptides produce a wide range of other biological responses in various tissues and act as potential growth factors for normal cells and cancer cells (Smith C.J. et al., Nucl Med Biol. 2003, 30(8): 861-8; Smith C.J. et al., Nucl Med Biol. 2005, 32(7): 733-40; Ananias H.J. et al., Curr Pharm Des. 2008, 14(28): 3033-47). The BBN receptor family has four members, including three mammalian receptors: GRPR (BB2 or BRS2, 384 amino acids), neuromedin B receptor (NMBR, BB1 or BRS1, 390 amino acids), and BN-like receptor 3 (BB3, BRS3 or orphan, 399 amino acids) (Smith C.J. et al., Nucl Med Biol. 2005, 32(7): 733-40; Ischia J. et al., Biofactors 2009, 35(1): 69-75; Maina T. et al., Cancer Imaging 2006; 6: 153-7); the fourth receptor (BB4) is only found in amphibians. GRPR is the only receptor in the family that is well characterized. GRPR is a glycosylated, 7-transmembrane G protein-coupled receptor that, upon binding to its ligands, elicits a complex intracellular cascade. It is normally present in non-neuroendocrine tissues of the breast and pancreas, as well as in neuroendocrine cells of the brain, gastrointestinal tract, lung, and prostate (Weber H.C., Curr Opin Endocrinol Diabetes Obes. 2009, 16(1): 66-71). Notably, GRPR is overexpressed in prostate cancer as well as in breast, lung, pancreatic, ovarian, renal, and gastrointestinal tumors. GRPR has been reported to be expressed at high density in prostate intraepithelial neoplasia and primary carcinoma, while it is mostly negative in normal prostate tissue and most benign prostatic hyperplasia (Yang Y.S. et al., Nucl Med Biol. 2006, 33(3): 371-80; Liu S. et al., Bioconjug Chem. 1997, 8(5): 621-36; Zhang X. et al., J Nucl Med. 2006, 47(3): 492-501; Schroeder R.P. et al., Methods, 2009, 48(2): 200-4). Several BBN peptides have been labeled with various radioisotopes for the diagnosis and treatment of GRPR-positive prostate lesions, e.g. 99m Tc, 177 Lu, 67 Ga and 111In-labeled for single photon emission computed tomography (SPECT), and 64 Cu, 68 Ga and 18 F-labeled for positron emission tomography (PET). Published BBN derivatives can generally be classified as truncated BBN (6-14 or 7-14) or full-length BN (1-14) analogs (Yang Y.S. et al., Nucl Med Biol. 2006, 33(3): 371-80; Zhang X. et al., J Nucl Med. 2006, 47(3): 492-501; Schroeder R.P. et al., Methods, 2009, 48(2): 200-4; Chen X. et al., J Nucl Med., 2004, 45(8): 1390-7; Hohne A. et al., Bioconjug Chem., 2008, 19(9): 1871-9; Li Z.B. et al., J Nucl Med., 2008, 49(3): 453-61; Prasanphanich A.F. et al., Nucl Med Biol., 2009, 36(2): 171-81; Santos-Cuevas C.L. et al., Int J Pharm., 2009, 375(1-2): 75-83). It is well known that truncated BN analogs are generally more stable than the full-length tetradecapeptide and still bind well to the GRPR.

[0019] Neurotensin receptor 1 (NTR1) is overexpressed in many cancer types, including prostate cancer. Neurotensin is a 13-amino acid peptide consisting of pGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu that can act as a neurotransmitter and a hormone (Morgat C. et al., J Nucl Med, 2014, 55:1650-7), exhibiting high affinity (nM) to the receptor (Sarret P and Kitabgi P., Encycl. Neurosci., 2010, 1021-34). In the absence of androgens, neurotensin and neurotensin receptors (NTSRs) are a substitute for androgens in the growth of advanced prostate cancer (Sehgal I et al., Proc Natl Acad Sci USA, 1994; 91 :4673-7). Neurotensin effects are mediated through 3 receptor subtypes: NTSR1 (neurotensin receptor 1) and NTSR2 (high- and low-affinity receptors, respectively), which are GPCRs, and NTSR3 (sortilin), which has a single transmembrane domain. NTSR1 is considered a promising cancer target, which is mainly localized in colonic peripheral tissues (Wu Z et al., Front Endocrinol (Lausanne), 2012, 3:184). In particular, NTSR1 is expressed in prostate cancer cells but not in normal prostate epithelial cells (Valerie N C et al., Cancer Res., 2011, 71:6817-26). In cell culture, NTSR1 expression increases with the tumorigenic potential of cancer cells (Taylor R M et al., Prostate., 2012; 72:523-532). NTSR1 has also been reported to be associated with radiotherapy resistance (Valerie N C et al., Cancer Res., 2011, 71:6817-26). The C-terminal region (8-13) of neurotensin is responsible for activating neurotensin receptors (White J.F. et al., Nature, 2012, 490:508-513). Based on the neurotensin (NT) peptide, various radiopharmaceuticals targeting NTR1 have been developed for diagnostic and radiotherapeutic applications (Alshoukr F et al., Bioconjug Chem., 2011, 22:1374-85; García-Garayoa E et al., Eur J Nucl Med Mol Imaging, 2009, 36:37-47; García-Garayoa E et al., Nucl Med Biol., 2001, 28:75-84; Sparr C et al., Chem Biodivers., 2013, 10:2101-21).

[0020] Neuropeptide-Y (NPY) receptors can influence tumor pathogenesis in tumors and are expressed in specific stages of carcinogenesis or tumor progression and in selective subtypes of tumors. It has been reported that neuropeptide-Y receptor gene and protein are expressed in prostate cancer cells and NPY has a role in regulating tumor growth (Ruscica M et al., Endocrinology, 2006, 147: 1466-73; Massoner P et al., PLoS ONE, 2013, 8: e55207, 32). However, there is no data on the expression of neuropeptide-Y receptors in tissues of patients with different stages of prostate cancer. To date, neuropeptide-Y receptors are considered as potential targets for cancer imaging and therapy (Morgat C, et al., J Nucl Med. 2014, 55: 1650-7).

[0021] In addition, prostate-specific membrane antigen (PSMA) and gastrin-releasing peptide receptor (GRPR) are both used as targets for prostate cancer (PCa) in nuclear medicine, and both can also be targets for breast cancer (BCa) and other tumors (Liolios C. et al., Mol Pharm, 2022, 19(7): 2231-47). GRPR is present in 62% of invasive breast cancers (Gugger M et al., Am J Pathol., 1999, 155: 2067-76), and neuropeptide-Y (NPY) receptors are present in 85% of breast cancers (Morgat C., J Nucl Med, 2014, 55: 1650-7). In addition, cell-penetrating peptides (CPPs), such as human calcitonin-derived peptides and lactoferrin (Duchardt F. et al., J. Biol. Chem., 2009, 284(52), 36099-108), and synthetic peptidomimetic ligands, including peptides containing arginine-glycine-aspartic acid (RGD) sequences (Peng L. et al., Nature Chem. Biol., 2006, 2(7), 381-9), can be activators of cell adhesion.

[0022] Due to the heterogeneity of receptor expression in prostate tumors, targeting multiple receptors can have an advantage over targeting a single receptor. Here, we disclose an antibody-drug conjugate with branched linkers containing glutamic acid urea small molecule groups, or / and affinity ligands for bombesin receptors / neurotensin receptors (including neuropeptide-Y receptors), and / or cell-penetrating peptides to supplement the affinity of ADCs to tumor cells, thereby enhancing targeted therapy for prostate cancer and other tumors. The invention continues to apply the specific conjugation method (PCT / CN2022 / 129122 and PCT / CN2021 / 128453) to construct these ADCs. Further disclosed are the preparation of conjugates, pharmaceutical compositions, drug screening and treatment methods. SUMMARY

[0024] The present invention provides an antibody-drug conjugate (ADC) with branched affinity ligands, wherein a group of glutamic acid urea small molecules, and / or affinity ligands for bombesin receptors / neurotensin receptors (including gastrin-releasing peptide receptors and neuropeptide-Y receptors), and / or cell-penetrating peptides are located at the end of the linker branch, which can supplement the affinity of ADCs to tumor cells, thereby enhancing the treatment of tumors, especially enhancing the targeted therapy of prostate cancer. The structure of the preferred ADC is represented as:

[0025]

[0026] wherein,

[0027] D1 and D2 are a cytotoxic substance; mAb is an antibody; n is 1-20;

[0028] L1, L2, La1, La2, Lb1, Lb2, Lc1, Lc2, Ld1, Ld2, Ld3, Ld4, Ld5, Ld6 are linkers, independently selected from O, NH, S, N, NH-NH, N-N, N(R3), N(R3)N(R3'), C(=O)N, C(=O)NH, C(=O)N-N, C1-C8 alkyl; C2-C8 heteroalkyl, alkylcycloalkyl, heterocycloalkyl; C3-C8 aryl, arylalkyl, heterocycle, carbocycle, cycloalkyl, heteroalkyl, cycloalkyl, alkylcarbonyl, heteroaryl; or ester, ether or amide of 1-8 carbon atoms; or 1-8 natural or unnatural amino acids described in the definition; or as (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) pR3, or (OCH2CH2) p COOR3, or CH2CH2(OCH2CH2) p polyoxyethylene units of COOR3, or CH2CH2(OCH2CH2)p, wherein p and p' are independently integers from 0 to about 1000, or combinations thereof; wherein R3and R3' are independently H, C(=O)H, C(=O)CH3, C1-C8alkyl; or combinations thereof;

[0029] E1is a linker connecting two reactive groups Lv1and Lv2, preferably reactive with a thiol, amino, phenol, ketone, aldehyde, alkyne, hydroxyl, or carboxylic acid group in an antibody. E1is selected from CH, CH2, CH-CH, NH, NHNH, N(R3), N(R3)N(R3'), N=N, N-N, P, P(=O), S, Si, C2-C8alkyl, heteroalkyl, alkylcycloalkyl, heterocycloalkyl; C3-C8aryl, Ar-alkyl, heterocycle, carbocycle, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; a peptide containing 1-4 amino acid units, preferably from aspartic acid, glutamic acid, arginine, histidine, lysine, serine, threonine, asparagine, glutamine, cysteine, selenocysteine, tyrosine, phenylalanine, glycine, proline, tryptophan, alanine; more detailed structures are described in the specification of the present invention.

[0030] m1, m2, m3, m4, m5, m6, m7, m8, m9, m 10 , m 11 and m 12 are independently 1-10; in addition, m2, m3, m8, m9 and / or m 10 may be 0, in which case Ld2-A2, Ld3-A3, Ld5-A5and / or Ld6-A6may be omitted;

[0031] A1, A2, A3, A4, A5and A6are respectively small molecules of glutamic acid urea or its analogues, or / and affinity ligands of bombesin receptors / neurotensin receptors (including neuropeptide-Y receptors) and / or cell-penetrating peptides. Detailed structures are described in the specification of the present invention.

[0032] wherein Lv1'and Lv2'are independently functional groups reactive with an amino acid or binding protein on an antibody. Detailed structures are described in the specification of the present invention.

[0033] The present application also provides an antibody-drug conjugate (ADC) against a specific prostate antigen (PSA) comprising a monoclonal antibody or antigen binding fragment thereof, a cytotoxin, and a linker containing an affinity ligand, such as 2-[3-(1,3-dicarboxypropyl)ureido]-pentanedioic acid (DUPA), a urea-based glutamic acid isodimer, glutamic acid-ureido-lysine, or a 2-(phosphonomethyl)pentanedioic acid analog, and / or an affinity ligand for a bombesin receptor / neurotensin receptor (including a neuropeptide-Y receptor) and / or a cell-penetrating peptide, and / or an affinity peptide that can bind to a protein called Programmed Death Ligand-1 (PD-L1 or CD274), which is expressed on tumor cells and tumor-infiltrating immune cells, the affinity peptide blocks its interaction with PD-1 and B7.1 receptors. In further embodiments, the antigen binding protein is conjugated to a toxin, such as a tubulysin analog, a camptothecin (CPT) analog, a PBD dimer, an auristatin analog, a duocarmycin analog, or an anthracycline analog.

[0034] Further, the present application provides a composition comprising the foregoing antibody-drug conjugate and a pharmaceutically acceptable carrier, and a method of killing prostate tumor cells expressing PSA by contacting the prostate cancer cells with the ADC.

[0035] In another aspect of the present application, there is provided a method of treating a human patient having a prostate-related disease or disorder, such as an antibody-mediated or plasma cell-mediated disease, or a plasma cell malignancy, such as a prostate-specific membrane antigen (PSMA) cell-related, the method comprising administering to said patient a therapeutically effective amount of an ADC as described herein.

[0036] In yet another aspect of the present application, there is provided a method of treating a human patient having papillary thyroid carcinoma (PTC) and other solid tumors, the method comprising administering to said patient a therapeutically effective amount of an ADC as described herein.

[0037] BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 . Synthesis of a camptothecin (CPT) compound moiety and linker is shown.

[0039] Figure 2 . Synthesis of a CPT analog with DUPA-containing linker is shown.

[0040] Figure 3 . Synthesis of a CPT analog with DUPA-containing linker and ADC is shown.

[0041] Figure 4 . Synthesis of a CPT analog with DUPA-containing linker and ADC is shown.

[0042] Figure 5 . Synthesis of CPT analogs and ADCs containing DUPA-containing linkers is shown.

[0043] Figure 6 . Synthesis of CPT analogs and ADCs containing DUPA-containing linkers is shown.

[0044] Figure 7 . Synthesis of ADCs containing DUPA, CPT analogs, and double-stranded linkers is shown.

[0045] Figure 8 . Synthesis of ADCs containing double DUPA, double-stranded linkers is shown.

[0046] Figure 9 . Synthesis of CPT analogs and ADCs containing one DUPA-containing linker is shown.

[0047] Figure 10 . Synthesis of CPT analogs and ADCs containing double DUPA-containing linkers is shown.

[0048] Figure 11 . Synthesis of double DUPA-containing linkers is shown.

[0049] Figure 12 . Synthesis of ADC linkers containing double CPT payloads and double DUPA is shown.

[0050] Figure 13 . Synthesis of ADCs containing double CPT payloads and double DUPA and linkers components with triple DUPA is shown.

[0051] Figure 14 . Synthesis of linkers components with triple DUPA is shown.

[0052] Figure 15 . Synthesis of CPT-ADCs containing triple DUPA-containing linkers is shown.

[0053] Figure 16 . Synthesis of linkers components with DUPA and tubulysin analogs is shown.

[0054] Figure 17 . Synthesis of tubulysin B analog-ADCs with DUPA is shown.

[0055] Figure 18 . Synthesis of payloads / linker complexes with tubulysin B analogs, CPT analogs, and DUPA ligands is shown.

[0056] Figure 19 . Synthesis of ADCs with two different payloads, a tubulysin B analog and a CPT analog, and a DUPA ligand is shown.

[0057] Figure 20 . Synthesis of CPT-ADCs with duplex linkers containing DUPA is shown.

[0058] Figure 21 . Synthesis of CPT-ADCs with one DUPA ligand is shown.

[0059] Figure 22 . Synthesis of CPT-ADCs with one DUPA ligand is shown.

[0060] Figure 23 . Synthesis of CPT-ADCs with one DUPA ligand is shown.

[0061] Figure 24 . Synthesis of CPT-ADCs with one DUPA ligand is shown.

[0062] Figure 25 . Synthesis of CPT payloads with one DUPA ligand is shown.

[0063] Figure 26 . Synthesis of CPT payloads with one DUPA ligand and CPT-ADCs is shown.

[0064] Figure 27 . Synthesis of CPT-ADCs with one DUPA ligand and dual CPT payloads with duplex linkers containing dual DUPA ligands is shown.

[0065] Figure 28 . Synthesis of CPT-ADCs and CPT payload / linker complexes containing dual payloads, dual DUPA ligands, duplex linkers is shown.

[0066] Figure 29 . Synthesis of CPT-ADCs with dual payloads and dual DUPA ligands, duplex linkers, and tubulysin components with DUPA ligands is shown.

[0067] Figure 30 . Synthesis of dual payload components with dual DUPA ligands, duplex linkers is shown.

[0068] Figure 31 . Synthesis of ADCs with dual DUPA ligands, dual linkers, and two different payloads is shown.

[0069] Figure 32 Synthesis of a linker component containing a DUPA ligand and a TAT peptide is shown, as well as synthesis of a CPT-ADC with the linker component.

[0070] Figure 33 Synthesis of a CPT-ADC containing a DUPA ligand and a TAT peptide is shown.

[0071] Figure 34 Synthesis of a linker component containing a DUPA ligand and a TAT peptide is shown.

[0072] Figure 35 Synthesis of a CPT-ADC containing a DUPA ligand and a TAT peptide is shown.

[0073] Figure 36 Synthesis of a dual CPT payload / linker component is shown.

[0074] Figure 37 Synthesis of a dual CPT payload / component containing a DUPA ligand and a TAT peptide is shown.

[0075] Figure 38 Synthesis of a dual CPT payload / linker complex containing a dual DUPA ligand and a dual TAT peptide is shown.

[0076] Figure 39 Synthesis of a CPT-ADC of a dual stranded linker containing a dual DUPA ligand, a dual TAT peptide, and four CPT payloads is shown.

[0077] Figure 40 Synthesis of a linker component containing a DUPA ligand and a TAT peptide is shown.

[0078] Figure 41 Synthesis of a CPT payload containing a DUPA ligand and a TAT peptide is shown.

[0079] Figure 42 Synthesis of four CPT payloads of a dual stranded linker containing a dual DUPA ligand and a dual TAT peptide is shown.

[0080] Figure 43 Synthesis of a CPT payload / dual stranded linker complex, each dual stranded linker containing four payloads, two DUPA ligands, and two TAT peptides is shown.

[0081] Figure 44 Synthesis of a CPT-ADC of a dual stranded linker containing four CPT payloads, a dual DUPA ligand, and a dual TAT peptide is shown.

[0082] Figure 45 . The affinity of Steap 1 antibody (vandortuzumab) and its conjugates to C4-2B prostate cancer cells was shown. The results showed that the affinity of Steap 1 antibody conjugated with conventional payload / linker complex (vc-MMAE or GGFG-Dxd) was lower than that of naked Steap 1 antibody. However, after conjugation with the affinity ligand in the payload / linker complex of the present application, the affinity of the conjugate was superior to or at least consistent with that of naked antibody.

[0083] Figure 46 . The change of tumor volume of PC3-4H7 prostate cancer cell xenograft mice after continuous single dose (2 mg / Kg) administration of Steap 1 ADC (C060, C084, C078, vcMMAE, C144, C158, C443, C200, C486, DAR values as shown in the figure) treatment compared with PBS buffer (control). The figure shows that 9 kinds of conjugates all have anti-tumor activity, and the order of anti-tumor activity is: C060 < C084 < C078 < vcMMAE < C144 < C158 < C443 < C200 < C486. It is also shown that the affinity ligand in the payload / linker complex of the present application can improve the anti-tumor activity of the drug in vivo.

[0084] Figure 47 . The change of tumor volume of PC3-4H7 prostate cancer cell xenograft mice after continuous single dose (2 mg / Kg) administration of B7H3 ADC (GGFG-Dxd, C060, C054, C084, C078, C112, C144, C158, C443, DAR values as shown in the figure) treatment compared with PBS buffer (control). The figure shows that 9 kinds of conjugates all have anti-tumor activity, and the order of anti-tumor activity is: GGFG-Dxd < C060 < C054 < 084 < C078 < C112 < C144 < C158 < C443. It is also shown that the affinity ligand in the payload / linker complex of the present application can improve the anti-tumor activity of the drug in vivo. Using the same type of payload, the conjugate containing the affinity ligand in the payload / linker complex of the present application has better anti-tumor activity than the conventional GGFG-Dxd conjugate.

[0085] Figure 48Figure 6 shows the change in tumor volume in NCI-N87 gastric cancer cell xenograft mice after treatment with a single dose (2 mg / Kg) of Trop2 ADCs (GGFG-Dxd, C144, C420, C422, C484, C482, DAR as indicated) compared to PBS buffer (control) and paclitaxel (15 mg / Kg once a week for three weeks). The figure shows that all six conjugates have anti-tumor activity, with the order of anti-tumor activity being: GGFG-Dxd < C144 < C420 < 422 < C484 < C482. It is also shown that the affinity ligand in the payload / linker complex of the application can improve the anti-tumor activity of the drug in vivo, and that conjugates containing the affinity ligand in the payload / linker complex of the application have better anti-tumor activity than the conventional GGFG-Dxd conjugate. SUMMARY

[0086] DEFINITIONS

[0087] "Alkyl" refers to an aliphatic hydrocarbon group or univalent radical derived from an alkane by the removal of one or two hydrogen atoms. It can be straight-chained or branched, having C1-C8 (1-8 carbon atoms) in the chain. "Branched" refers to the straight-chained alkyl group having one or more lower alkyl groups, such as methyl, ethyl, or propyl, attached to it. Exemplary alkyl groups include methyl, ethyl, n-propyl, isopropyl, 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-octyl, and iso-octyl. C1-C8 alkyl groups can be unsubstituted or substituted with one or more groups, including but not limited to C1-C8 alkyl, -0-(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.

[0088] "Halogen" refers to a fluorine, chlorine, bromine, or iodine atom; preferably a fluorine and chlorine atom.

[0089] "Heteroalkyl" refers to a C2-C8 alkyl group in which 1 to 4 carbon atoms are independently replaced with a heteroatom selected from O, S, and N.

[0090] "Carbocyclic" refers to a saturated or unsaturated monocyclic ring containing 3 to 8 carbon atoms, or a saturated or unsaturated bicyclic ring containing 7 to 13 carbon atoms. Monocyclic carbocyclic rings have 3 to 6 ring atoms, typically 5 or 6 ring atoms. Bicyclic carbocyclic rings have 7 to 12 ring atoms, forming a [4,5], [5,5], [5,6], or [6,6] bicyclic ring system, or 9 or 10 ring atoms, forming a [5,6] or [6,6] bicyclic ring system. Representative C3-C8 carbocyclic 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.

[0091] "C3-C8 carbocyclic" can be unsubstituted or substituted by 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.

[0092] "Alkenyl" refers to a straight or branched chain aliphatic group containing a carbon-carbon double bond, containing 2-8 carbon atoms. Exemplary alkenyl groups include ethenyl, propenyl, n-butenyl, isobutenyl, 3-methylbut-2-enyl, n-pentenyl, hexenyl, heptenyl, octenyl.

[0093] "Alkynyl" refers to a straight or branched chain aliphatic group containing a carbon-carbon triple bond, containing 2-8 carbon atoms. Exemplary alkynyl groups include ethynyl, propynyl, n-butynyl, 2-butynyl, 3-methylbutynyl, 5-pentynyl, n-pentynyl, hexynyl, heptynyl, and octynyl.

[0094] "Alkylene" refers to a saturated branched or straight chain or cyclic hydrocarbon radical containing 1-18 carbon atoms, and having two valences by virtue of removal of two hydrogen atoms from the same or two different carbon atoms of the parent alkane. Typical alkylene groups include, but are not limited to: methylene (-CH2-), 1,2-ethyl (-CH2CH2-), 1,3-propyl (-CH2CH2CH2-), 1,4-butyl (-CH2CH2CH2CH2-), and the like.

[0095] "Alkylene" refers to an unsaturated branched or straight chain or cyclic hydrocarbon radical containing 2 to 18 carbon atoms and having two valences, produced by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent alkene. Typical alkylene groups include, but are not limited to, 1,2-ethylene (-CH=CH-).

[0096] "Alkynylene" refers to an unsaturated branched or straight chain or cyclic hydrocarbon radical containing 2 to 18 carbon atoms and having two valences, produced by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent alkyne. Typical alkynylene groups include, but are not limited to, acetylene, propargyl, and 4-pentynylene.

[0097] "Aryl" or "Aromatic" refers to an aromatic or heteroaromatic group consisting of one or more rings containing three to fourteen carbon atoms, preferably six to ten carbon atoms. The term "heteroaromatic group" refers to a group in which one or more carbons, most preferably one, two, three, or four carbon atoms, of an aromatic group are replaced by oxygen (O), nitrogen (N), silicon (Si), selenium (Se), phosphorus (P), or sulfur (S), preferably oxygen, sulfur, and nitrogen. The term "aryl" or "aromatic" also refers to an aryl group in which one or more hydrogen atoms are independently replaced by -R', halogen, -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"), to produce an aryl group. R' and R" are independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, aralkyl, carbonyl, or a pharmaceutically acceptable salt thereof.

[0098] "Heterocycle" refers to a ring structure in which one to four ring carbon atoms are independently replaced with a heteroatom such as O, N, S, Se, B, Si, or P. Preferred heteroatoms are O, N, and S. Heterocyclic compounds are also described in The Handbook of Chemistry and Physics, 78th Edition, CRC Press, Inc., 1997-1998, p. 225 to 226, which is incorporated herein by reference. Preferred non-aromatic heterocycles include epoxide, aziridinyl, thiiranyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, oxiranyl, tetrahydrofuranyl, dioxolanyl, tetrahydropyranyl, dioxanyl, piperidinyl, piperazinyl, morpholinyl, pyranyl, imidazolinyl, pyrazolinyl, thiazolidinyl, tetrahydrothiopyranyl, dithianyl, thiomorpholinyl, dihydropyranyl, tetrahydropyranyl, dihydropyridyl, tetrahydropyridyl, dihydrothiopyranyl, azepinyl, and fused ring systems formed by the condensation of the foregoing groups with phenyl.

[0099] The term "heteroaryl" or "aromatic heterocycle" refers to an aromatic heterocycle containing 3 to 14, preferably 5 to 10 atoms, and containing a single, double, or multiple ring. Examples include pyrrolyl, pyridyl, pyrazolyl, thienyl, pyrimidinyl, pyrazinyl, tetrazolyl, indolyl, quinolyl, purinyl, imidazolyl, thiazolyl, benzothiazolyl, furanyl, benzofuranyl, 1,2,4-thiadiazolyl, isothiazolyl, triazolyl, tetrazolyl, isoquinolyl, benzothienyl, isobenzofuranyl, pyrazolyl, carbazolyl, benzimidazolyl, isoxazolyl, pyridyl-N-oxide, and fused ring systems formed by the condensation of the foregoing groups with phenyl.

[0100] "Alkyl," "cycloalkyl," "alkenyl," "alkynyl," "aryl," "heteroaryl," "heterocycle," and the like, also contain the corresponding "alkylene," "cycloalkylene," "alkenylene," "alkynylene," "arylene," "heteroarylene," "heterocyclene," and the like, which are formed by the removal of one hydrogen atom from the respective groups.

[0101] "Arylalkyl" refers to a noncyclic alkyl group in which one of the hydrogen atoms bonded to a carbon atom (usually the terminal or sp 3 carbon atom) is replaced with an aryl group. Typical arylalkyl groups include benzyl, 2-phenyleth-1-yl, 2-phenylethen-1-yl, naphthylmethyl, 2-naphthyleth-1-yl, 2-naphthyleth-1-yl, naphthobenzyl, 2-naphthylphenyl-1-yl, and the like.

[0102] "Heteroarylalkyl" refers to a noncyclic alkyl group in which one of the hydrogen atoms bonded to a carbon atom (usually the terminal or sp 3The hydrogen atom bonded to a carbon atom) is replaced by a heteroaryl group. Examples of heteroarylalkyl groups include 2-benzimidazolylmethyl and 2-furylethyl.

[0103] Examples of the “hydroxy-protecting group” include methoxymethyl ether, 2-methoxyethoxymethyl ether, tetrahydropyranyl ether, benzyl ether, p-methoxybenzyl ether, trimethylsilyl ether, triethylsilyl ether, triisopropylsilyl ether, tert-butyldimethylsilyl ether, triphenylmethylsilyl ether, acetate, substituted acetate, pivalate, benzoate, methanesulfonate and p-toluenesulfonate.

[0104] "Leaving group" refers to a functional group that can be replaced by another functional group. Such leaving groups are well known in the art, and examples include halides (e.g., chloride, bromide, and iodide), mesyl, tosyl, triflate, and trifluoromethanesulfonate. Preferred leaving groups are selected from nitrophenol; N-hydroxysuccinimide (NHS); phenol; dinitrophenol; pentafluorophenol; tetrafluorophenol; difluorophenol; monofluorophenol; pentachlorophenol; trifluoromethanesulfonate; imidazole; dichlorophenol; tetrachlorophenol; 1-hydroxybenzotriazole; toluenesulfonate; mesylate; 2-ethyl-5-phenylisoxazole-3'-sulfonate, anhydrides formed by themselves or with other anhydrides, such as acetic anhydride, formic anhydride; or intermediate molecules generated from condensation reagents for peptide coupling reactions or Mitsunobu reactions.

[0105] The following abbreviations are employed in the present invention and are defined with their meanings: Boc, tert-butyloxycarbonyl; BroP, bromotetradecylphosphonium hexafluorophosphate; CDI, 1,1'- carbonyldiimidazole; DCC, dicyclohexylcarbodiimide; DCE, dichloroethane; dichloromethane, methylene chloride; DIAD, diisopropyl azodicarboxylate; DIBAL-H, diisobutylaluminum hydride; DIPEA diisopropylethylamine; DEPC, diethyl cyanophosphonate; DMA, N,N-dimethylacetamide; DMAP, 4-(N,N-dimethylamino)pyridine; DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide; DTT, dithiothreitol; EDC, l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; ESI-MS, electrospray mass spectrometry; ethyl acetate, ethyl acetate; HATU, O-(7-azabenzotriazol-l-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate; HOBt, 1-hydroxybenzotriazole; HPLC, high performance liquid chromatography; NHS, N-hydroxysuccinimide; MMP, 4-methylmorpholine; PAB, p-aminobenzoic acid; PBS, phosphate buffered saline (pH 7.0-7.5); PEG, polyethylene glycol; SEC, size exclusion chromatography; TCEP, tris(2-carboxyethyl)phosphine; TFA, trifluoroacetic acid; THF, tetrahydrofuran; Val, valine.

[0106] "Amino acid" can be natural or non-natural, preferably an alpha-amino acid. Natural amino acids can be encoded by the genetic code and 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 derivatives of the proteinogenic amino acids and include hydroxyproline, lanthionine, 2-amino isobutyric acid, dehydroalanine, gamma-aminobutyric acid (a neurotransmitter), ornithine, citrulline, beta-alanine (3-amino propionic acid), gamma-carboxyglutamate, selenocysteine (found in many non-eukaryotic and most eukaryotic cells, but not directly encoded by DNA), pyrrolysine (found only in some archaea and one bacterium), N-formylmethionine (usually the first amino acid in 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 mimetics. An analog is a compound having the same general structure H2N(R)CHCO2H as a natural amino acid, where R is in a natural amino acid. Examples of analogs include homoserine, norleucine, methionine-sulfoxide, and methionine methylsulfonium. More preferred are amino acid mimetics, which are compounds having a different chemical structure from an alpha-amino acid, but which act in a similar manner. Natural amino acids are predominantly in the "L" stereochemical configuration, and "non-natural amino acids" are also used to represent amino acids in the "D" configuration. When one to eight amino acids are used in this application, their sequence is preferably one that is recognized by a proteolytic enzyme. Many proteolytic enzyme recognition sequences are known in the art, and can be found in 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); which are incorporated herein by reference.In particular, selected from the group consisting of Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Ala-Asn-Val, Ala-Val-Lys, Ala-Val-Glu, Val-Leu-Lys, Cit-Cit, Val-Lys, Ala-Ala-Asn, Gly-Gly, Gly-Gly-Gly, Ala-Ala-Ala, Ala-Ala-Ala-Glu, Ala-Val-Arg, Ala-Val-Arg-Arg, Ala-Ala-Arg, Ala-Ala-Arg-Arg, Gly-Gly-Phe-Gly, Lys, Cit, Ser, and Glu. In addition to the 20 standard L- or 20 D-amino acids, a number of uncommon amino acids are listed here using their letter codes: aminobutyric acid (Abu), aminoisobutyric acid (Aib), alpha-cyclohexylalanine (Cha), citrulline (Cit), diaminopropionic acid (Dap), hydroxylysine (Hyl), hydroxyproline (Hyp), norleucine (Nle), norvaline (Nva), ornithine (O), penicillamine (Pen), pyroglutamic acid (Pyr), sarcosine (Sar), statine (Sta). Examples of single code modified amino acids are as follows: asparagine-EDANS (D-EDANS), cysteine-3-nitro-2-pyridinesulfenyl (C-NPys), glutamic acid-EDANS (E-EDANS), glycine N-methylation (G-NMe), leucine N-methylation (L-NMe), serine phosphorylation (pS), threonine phosphorylation (pT), tyrosine phosphorylation (pY), tyrosine O-methylation (Y-OMe), 3-nitrotyrosine (Y-NO2), tyrosine sulfation (sY), lysine 5-carboxyfluorescein (K-5-FAM), lysine 5-carboxytetramethylrhodamine (K-5-TAMRA), lysine acetylation (K-Ac), lysine biotinylation (K-biotin), lysine-DABCYL (K-DABCYL), lysine-dansyl (K-DANSYL), lysine-DNP (K-Dnp), lysine-MCA (K-Mca), lysine methylation (K-Me), lysine dimethylation (K-Me2), lysine trimethylation (K-Me3).

[0107] "Pharmaceutically" or "pharmaceutically acceptable" means that the molecular entity and compositions, as the case can be, when administered to an animal or human, do not produce an adverse, allergic or other untoward reaction.

[0108] "Pharmaceutically acceptable solvate" or "solvate" means a compound of the present disclosure in combination with one or more solvent molecules. Examples of solvents forming pharmaceutically acceptable solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine.

[0109] "Pharmaceutically acceptable excipient" includes any carrier, diluent, adjuvant or other, e.g., preservative or antioxidant, filler, disintegrant, wetting agent, emulsifying agent, suspending agent, solvent, dispersion medium, coating agent, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. These media and agents are those used in the pharmaceutical art to formulate pharmaceutical active substances. Any conventional medium or agent, unless incompatible with the active ingredient, can be considered for use in the therapeutic composition. Supplementary active ingredients can also be incorporated into the composition, as appropriate, to provide suitable therapeutic combinations.

[0110] In the present patent application, "pharmaceutically acceptable salts" refer to derivatives of the disclosed compounds obtained by making acid or base salts of the parent compound. Pharmaceutically acceptable salts include conventional non-toxic salts or quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, tartaric, citric, methanesulfonic, benzenesulfonic, glucouronic, glutamic, benzoic, salicylic, toluenesulfonic, oxalic, fumaric, maleic, lactic, and the like. Further addition salts include ammonium salts, such as those of trimethylamine, meglumine, glycerol, and the like, metal salts, such as sodium, potassium, calcium, zinc, or magnesium salts.

[0111] The pharmaceutically acceptable salts of the present application can be synthesized from the parent compound that contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by using eq. of the appropriate base or acid in a water or organic solvent, or a mixture thereof, to give the desired salt. Generally, ethyl ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred nonaqueous media. Lists of suitable salts are found in "Remington's Pharmaceutical Sciences," by Mack Publishing Company (1985), the disclosure of which is hereby incorporated by reference.

[0112] "Administering" or "administration" means transferring, delivering, introducing or transporting a drug or other pharmaceutical agent to a subject in any way. These ways include oral administration, topical contact, intravenous, intraperitoneal, intramuscular, intralesional, intranasal, subcutaneous, or intrathecal administration. The present application also contemplates the use of devices or appliances to administer the pharmaceutical agent. Such devices can use active or passive modes of transport, and can be slow release or rapid release delivery devices.

[0113] The abbreviations and chemical names of the biological buffers are as follows:

[0114] ACES (N-(2-acetamido)-2-aminoethanesulfonic acid) buffer system at pH 6.1-7.5 (pKa = 6.88).

[0115] ADA (N-(2-acetamido)iminodiacetic acid, N-(carbamoylmethyl)iminodiacetic acid) buffer system at pH 6.0-7.2 (pKa = 6.65).

[0116] AMPD (2-amino-2-methyl-1,3-propanediol) buffer system at pH 7.8-9.7.

[0117] AMPSO (N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid).

[0118] BES (N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid).

[0119] Bicine (bis(N,N-bis(2-hydroxyethyl)glycine), bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane) buffer system at pH 5.8-7.2 (pKa = 8.35).

[0120] BisTris (bis-(2-hydroxyethyl)amino-tris(hydroxymethyl)methane).

[0121] BisTris propane (1,3-bis[tris(hydroxymethyl)methylamino]propane).

[0122] DIPSO (N,N-bis(2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid) buffer system at pH 7.0-8.2.

[0123] Gly-Gly (diglycine, glycyl-glycine) buffer system at pH 7.5-8.9 (pKa = 8.30).

[0124] HEBPS (N-(2-hydroxyethyl)piperazine-N'-(4-butanesulfonic acid)) is a homolog of HEPES and EPPS with a higher pKa (pKa = 8.30) buffer system at pH 7.6-9.0.

[0125] HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 2-morpholinoethanesulfonic acid, 2-(4-morpholino)ethanesulfonic acid, 2-(N-morpholino)ethanesulfonic acid, morpholine-4-ethanesulfonic acid hydrate) buffer system at pH 6.8-8.2; pKa 7.45-7.65 at 20°C

[0126] HEPPS or EPPS (3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid hydrate, 4-(2-hydroxyethyl)piperazine-1-(2-hydroxypropanesulfonic acid) hydrate) buffer system at pH 7.3-8.7 (pKa = 8.00 / piperazine ring).

[0127] HEPPSO (4-(2-hydroxyethyl)piperazine-1-(2-hydroxypropanesulfonic acid) hydrate).

[0128] MES (2-(N-morpholino)ethanesulfonic acid monohydrate) buffer system at pH 5.2-7.1 (pKa 6.16).

[0129] MOBS (4-morpholinebutanesulfonic acid, 3-(N-morpholino)butanesulfonic acid hemisodium salt) is a homolog of MES and MOPS with a higher pKa, buffer system at pH 6.9-8.3 (pKa 7.6).

[0130] MOPS (4-morpholinepropanesulfonic acid sodium salt).

[0131] MOPSO (β-hydroxy-4-morpholinepropanesulfonic acid, 3-morpholino-2-hydroxypropanesulfonic acid).

[0132] PIPES (piperazine-1, 4-bis(2-ethanesulfonic acid), buffer system at pH 6.1-7.5 (pKa = 6.80).

[0133] POPSO (piperazine-1, 4-bis(2-hydroxypropanesulfonic acid) dihydrate).

[0134] TAPS ([(2-hydroxy-1, 1-bis(hydroxymethyl)ethyl)amino]-1-propanesulfonic acid).

[0135] TAPSO (2-hydroxy-3-[tris(hydroxymethyl)methylamino]-1-propanesulfonic acid).

[0136] TES (2-[(2-hydroxy-1, 1-bis(hydroxymethyl)ethyl)amino]ethanesulfonic acid).

[0137] Tricine (piperazine-N, N'-bis[2-hydroxypropanesulfonic acid]), buffer system at pH 7.4-8.8 (pKa 8.16).

[0138] The term "antibody" herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (so long as they exhibit the desired antigen-binding activity), antibody- containing fusion proteins, and any other configuration of immunoglobulin molecules that contain an antigen recognition site. Antibodies include antibodies of any class, such as IgG, IgA, or IgM (or sub-class thereof), and the antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant region of its heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chains of different classes of immunoglobulins have different constant region sequences, and the amino acid sequences of the variable regions of the heavy chains differ among the subclasses. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. "Antibody fragments" refer to molecules other than intact antibodies that comprise a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. "Humanized" antibodies refer to antibodies that contain amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, humanized antibodies will comprise substantially all of at least one, and typically two, variable domains containing all or substantially all of the CDRs and optionally all or substantially all of the FRs corresponding to those of a non-human antibody. Humanized antibodies optionally can comprise at least a portion of an antibody constant region from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that is humanized by, for example, the grafting of an antigen-binding site of a non-human antibody onto a human antibody. The terms "variable region" or "variable domain" refer to the domain of an antibody heavy or light chain that is involved in binding the antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures and each domain comprises four framework regions (FRs) connected by three hypervariable regions (HVRs). (See, e.g., Kindt et al. Kuby Immunology, 6th Ed., W.H. Freeman and Co., page 91 (2007).) The HVRs are primarily responsible for binding the antigen, while the FRs are more highly conserved regions between the HVRs.(See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991)).

[0139] In the present patent application, "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that can be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present application can be made by the hybridoma method first described by Kohler and Milstein, Nature 256:495, 1975, or can be made by recombinant DNA methods, such as described in U.S. Patent 4,816,567. Furthermore, the monoclonal antibodies can also be isolated from phage libraries using the techniques described in McCafferty et al., Nature 348:552-554, 1990.

[0140] In the present patent application, "humanized" antibodies refer to non-human (e.g., murine) antibodies that are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (e.g., Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) which contain minimal sequence of non-human immunoglobulin sequences. Preferably, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains containing all or substantially all of the CDR regions that correspond to those of a non-human species (donor antibody) and all or substantially all of the FR regions are those of a human immunoglobulin (recipient antibody). The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Preferably, the Fc region of the antibody has been modified as described in WO 99 / 58572. Other forms of humanized antibodies, having one or more CDRs (CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 or CDR H3) altered with respect to the original antibody, are also referred to as "derivatized" from one or more CDRs of the original antibody.

[0141] In the present patent application, "human antibody" means an antibody comprising an amino acid sequence corresponding to an amino acid sequence of an antibody produced by a human and / or an antibody produced by any technique known to those skilled in the art or disclosed in the present patent application for the production of a human-derived antibody. Human antibodies include antibodies comprising at least one human heavy chain polypeptide or at least one human light chain polypeptide. One example is an antibody composed of murine light chain and human heavy chain polypeptides. Human-derived antibodies can be produced using various techniques known in the art. In one embodiment, the human antibody is selected from a phage library expressing human antibodies (Vaughan et al., Nature Biotechnology, 14:309-314, 1996; Sheets et al., Proc. Natl. Acad. Sci. (USA) 95:6157-6162, 1998; Hoogenboom and Winter, J. Mol. Biol., 227:381, 1991; Marks et al., J. Mol. Biol., 222:581, 1991). Human antibodies can also be produced by immunizing animals, in which the endogenous immunoglobulin genes of the animal, such as a mouse, are partially or completely inactivated, and human immunoglobulin genes are introduced into the endogenous loci of the animal. This method is described in U.S. Patents 5545807; 5545806; 5569825; 5625126; 5633425; and 5661016. Alternatively, human antibodies can be produced by immortalizing human B lymphocytes producing antibodies targeting the antigen of interest (such B lymphocytes can be recovered from individuals or single cell clones of cDNA, and can be immunized in vitro). See, e.g., Cole et al. Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77, 1985; Boerner et al., J. Immunol., 147(1):86-95, 1991; and U.S. Patent 5750373.

[0142] The term "chimeric antibody" refers to an antibody whose variable region sequences are derived from one species and whose constant region sequences are derived from another species, such as an antibody whose variable region sequences are derived from a mouse antibody and whose constant region sequences are derived from a human antibody.

[0143] The terms "polypeptide," "oligopeptide," "peptide," and "protein" are used interchangeably herein to refer to a chain of amino acids of any length, preferably of relatively short length (e.g., 10-100 amino acids). The chain can be straight or branched, can contain modified amino acids, and / or can be interrupted by non-amino acids. The term also encompasses an amino acid chain that has been modified, e.g., by disulfide formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as coupling with a labeling component. The definition also includes polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. It is understood that a polypeptide can exist as a single chain or associated chains.

[0144] A "monovalent antibody" contains one antigen binding site per molecule (e.g., IgG or Fab). In some cases, a monovalent antibody can have multiple antigen binding sites, but the binding sites should be from different antigens.

[0145] A "monospecific antibody" contains two identical antigen binding sites per molecule (e.g., IgG), both of which bind to the same epitope on an antigen. Thus, they compete with each other for binding to an antigen molecule. Most antibodies found in nature are monospecific. In some cases, a monospecific antibody can also be a monovalent antibody (e.g., Fab).

[0146] A "divalent antibody" contains two antigen binding sites per molecule (e.g., IgG). In some cases, the two binding sites have the same antigen specificity. However, a divalent antibody can be bispecific.

[0147] A "bispecific" or "dual specific" antibody is a hybrid antibody with two different antigen binding sites. The two antigen binding sites of a bispecific antibody bind to two different epitopes, which can be on the same or different protein targets.

[0148] A "bifunctional" antibody refers to an antibody with the same antigen binding site (i.e., the same amino acid sequence) on both arms, but each binding site can recognize two different antigens.

[0149] A "heteromultimer," "heteromultimeric complex," or "heteromultimeric polypeptide" is a molecule comprising at least a first polypeptide and a second polypeptide, wherein the second polypeptide differs from the first polypeptide by at least one amino acid residue. A heteromultimer can comprise a "heterodimer" formed by the first and second polypeptides or a higher order tertiary structure formed when there are more polypeptides in addition to the first and second polypeptides.

[0150] An "heterodimer", "heterodimeric protein", "heterodimeric complex", or "heteromultimeric polypeptide" is a molecule composed of molecules of a first polypeptide and a second polypeptide, wherein the amino acid sequence of the second polypeptide differs from the amino acid sequence of the first polypeptide by at least one amino acid residue.

[0151] In the present patent application, the meaning of "hinge region", "hinge sequence" and other variants is well known in the art and is described in, for example, Janeway et al., ImmunoBiology: the immune system in health and disease, (Elsevier Science Ltd., NY) (4th ed., 1999); Bloom et al., Protein Science (1997), 6:407-415; Humphreys et al., J. Immunol. Methods (1997), 209:193-202.

[0152] In the present patent application, "immunoglobulin-like hinge region", "immunoglobulin-like hinge sequence" and variants thereof refer to the hinge region and hinge sequence of an immunoglobulin-like or antibody-like molecule (e.g., an immunoadhesin). In some embodiments, the immunoglobulin-like hinge region can be from or derived from any IgGl, IgG2, IgG3, or IgG4 subtype, or from IgA, IgE, IgD or IgM, including chimeric forms thereof, such as a chimeric IgGl / 2 hinge region.

[0153] The term "immune effector cell" or "effector cell" in the present patent application refers to a cell within the natural repertoire of the human immune system that can be activated to affect the viability of a target cell. The viability of a target cell can include the ability of the cell to survive, proliferate, and / or interact with other cells.

[0154] Antibodies in the present application can be prepared by techniques well known in the art, such as recombinant techniques, phage display techniques, synthetic techniques or combinations of such techniques, or other techniques known in the art (see, e.g., Jayasena S.D., Clin. Chem., 45:1628-50, 1999 and Fellouse F.A., et al., J. Mol. Biol., 373(4):924-40, 2007).

[0155] In the present patent application, the term "cytotoxic agent" refers to a substance that inhibits or prevents the function of cells and / or causes destruction or death of cells. Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., At211, 1131, 1125, Y90, In111, Re186, Re188, Sm153, Bi212, P32, Pb212, Zr89, F18 and radioactive isotopes of Lu, e.g. Lu177); chemotherapeutic agents or drugs (e.g., tubulysins, maytansinoids, auristatins, DNA minor groove binding agents (e.g., PBD dimers), duocarmycins, topoisomerase inhibitors, RNA polymerase inhibitors, DNA alkylating agents, methotrexate, adozelesin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitory agents; enzymes, and fragments thereof, such as a ribonuclease, a deoxyribonuclease and a

[0156] A "linker" is a chemical moiety that is a covalent bond or a chain of atoms that covalently links an antibody to a drug moiety. In various embodiments, linkers include divalent radicals such as alkyl diradicals, aryl diradicals, heteroaryl diradicals, moieties such as: -(CR2)nO(CR2)n-, repeating units of alkyleneoxy (e.g., polyethyleneoxy, PEG, poly methyleneoxy), and alkylamino (e.g., polyethyleneamino); and diesters and amides, including succinates, succinamides, diglycolates, malonates, and adipamides. In various embodiments, a linker can comprise one or more amino acid residues, such as valine, phenylalanine, lysine, and ornithine.

[0157] In the present specification and claims, the words "comprise" "comprising", "include" and "including" are used in their open-ended, non-limiting sense to mean that the described features, integers, components, or steps are present, but not excluding the presence of one or more other features, integers, components, steps or groups thereof. The novel conjugates disclosed in the present application application use a bridging linker. Examples of some suitable linkers and their synthesis are described in the specification Examples 1-297.

[0158] Antibody drug conjugates targeting prostate cancer

[0159] The present invention provides an antibody-drug conjugate that enhances the affinity of the conjugate to tumor cells and kills them, particularly prostate tumor cells. The antibody-drug conjugate (ADC) contains a branched linker with a set of glutamic acid urea small molecules or / and an affinity peptide (such as neurotensin peptide) at the end of the branched chain that can supplement the affinity of the ADC to tumor cells, thereby enhancing the therapeutic effect on tumors, particularly in the targeted therapy of prostate cancer, or the tissue surrounding prostate cancer cells, or metastatic prostate tumor cells. The structure of the ADC of the present invention is shown below:

[0160]

[0161] wherein,

[0162] D1 and D2 are cytotoxic agents; mAb is an antibody; n is 1-20;

[0163] L1, L2, La1, La2, Lb1, Lb2, Lc1, Lc2, Ld1, Ld2, Ld3, Ld4, Ld5, Ld6 are linkers, independently selected from O, NH, S, N, NH-NH, N-N, N(R3), N(R3)N(R3'), C(=O)N, C(=O)NH, C(=O)N-N, C1-C8 alkyl; C2-C8 heteroalkyl, alkylcycloalkyl, heterocycloalkyl; C3-C8 aryl, Ar-alkyl, heterocycle, carbocycle, cycloalkyl, heteroalkyl, cycloalkyl, alkylcarbonyl, heteroaryl; or an ester, ether or amide of 1-8 carbon atoms; or 1-8 natural or unnatural amino acids as described in the definition; or a polyoxyethylene unit of (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 COOR3, wherein p and p' are independently selected from an integer from 0 to about 1000, or a combination thereof; wherein R3and R 3’ are independently H, C(=O)H, C(=O)CH3, C1-C8 alkyl; or a combination thereof;

[0164] E1 is a linker connecting the two reactive groups Lv1 and Lv2. E1 is selected from CH, CH2, CH-CH, NH, NHNH, N(R3), N(R3)N(R3'), N=N, N-N, P, P(=0), S, Si, C2-C8alkyl, heteroalkyl, alkylcycloalkyl, heterocycloalkyl; C3-C8aryl, aryl-alkyl, heterocycle, carbocycle, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; a peptide containing 1-4 amino acid units, preferably selected from aspartic acid, glutamic acid, arginine, histidine, lysine, serine, threonine, asparagine, glutamine, cysteine, selenocysteine, tyrosine, phenylalanine, glycine, proline, tryptophan, alanine; or one of the following structures:

[0165] wherein is the attachment site; X1, X2, X3, X4, X5or X6are independently selected from NH, NHNH, N(R3), N(R3)N(R3'), O, S, C1-C6alkyl, R3and R3'are H when alkyl;

[0166] m1, m2, m3, m4, m5, m6, m7, m8, m9, m 10 , m 11 and m 12 are independently 1-10; furthermore, m2, m3, m8, m9and / or m 10 may be 0, in which case Ld2-A2, Ld3-A3, Ld5-A5and / or Ld6-A6may be omitted;

[0167] A1, A2, A3, A4, A5and A6are independently selected from:

[0168]

[0169]

[0170]

[0171]

[0172] wherein is the site of attachment to Ld1, Ld2, Ld3, Ld4, Ld5or Ld6; Ra is Ar, most preferably selected from: Rb is OH, COOH, COOCH3, CH3OH, CH3NH2, CONH2;

[0173] wherein Lv 1' and Lv 2' independently have the following structure:

[0174]

[0175]

[0176]

[0177]

[0178]

[0179] wherein is the site for attachment of a drug or the site for attachment of linker L1 or L2; "#" is the site for attachment of S (sulfhydryl), O (phenol), NH (amino), CHO (aldehyde), C(=O) (ketone), C(O)(NH) (amide), and C(O)(OH) (carboxylate) of an antibody; wherein R1, X 1' and X 2' are as described above; X is O, NH, S, CH2; the connecting bond between the two atoms means that it can connect either of the two atoms, and Ar is an aromatic group.

[0180] In some embodiments, the mAb is an antibody, preferably a humanized monoclonal antibody, more preferably an antibody that specifically binds to human PSMA, B7H3, STEAP1, CD46, TROP2, and CEACAM5 antigens, and is capable of delivering the attached drug to the inside of cells expressing these antigens.

[0181] The present application also provides an antibody-drug conjugate (ADC) comprising a monoclonal antibody or an antigen-binding fragment thereof, conjugated to a cytotoxin via a linker comprising a glutamic acid urea small molecule, such as 2-[3-(1,3-dicarboxypropyl)ureido]-pentanedioic acid (DUPA), urea-based glutamic acid isodimer, 2-(phosphonomethyl)-pentanedioic acid (PMPA), phosphoramide, glutamic acid-ureido-lysine or 2-(phosphonomethyl)pentanedioic acid analogue groups, directly targeting prostate antigens (PSA) of tumor cells, and / or a bombesin receptor (gastrin-releasing peptide receptor (GRPR), neurotensin receptor (including neurotensin receptor 1 (NTR1) and neuropeptide Y receptor) affinity ligand, and / or a cell-penetrating peptide, and / or an affinity peptide that can bind to a protein called programmed death-ligand-1 (PD-L1 or CD274) that is expressed on tumor cells and tumor-infiltrating immune cells, the affinity peptide blocking its interaction with PD-1 and B7.1 receptors. The affinity for the receptor is at least EC 50 <10 μΜ, preferably EC 50 <100 nM, more preferably EC 50 <50 nM. In further embodiments, the antigen-binding protein is conjugated to a toxin, such as a tubulysin analogue, a camptothecin (CPT) analogue, a PBD dimer, an anthracycline or an auristatin analogue.

[0182] In some embodiments, the cell-penetrating peptide (CPP) used in the present application can be screened from the CPP database (http: / / crdd.osdd.net / raghava / cppsite) or from known publications for sequences of less than 100 amino acids, or modified from known peptide sequences by replacing one or several amino acids, followed by a redundancy check. Preferred CPPs are linear or cyclic peptides containing less than 50 amino acids, preferably less than 20 natural or non-natural amino acids, more preferably less than 15 amino acids and containing one, two or several arginines and / or lysines. The CPP is more preferably a cyclic peptide, in particular a cyclic peptide of less than 8 amino acids. The selected peptides are usually further analyzed to filter out ambiguous peptides with unfavorable chemical modifications. The amphiphilicity is predicted by the online server AMPHIPASEE ( https: / / npsa-prabi.ibcp.fr / cgibin / npsa_ automat.pl?page= / NPSA / npsa_amphipaseek.html ).

[0183] AMPHIPASEE provides a score between 0 and 5 for each residue of a given peptide sequence. The higher the score, the higher the amphiphilicity, and vice versa (0 = low, 5 = high). The hydropathy value is calculated using the online server K-Me3 ( https: / / www.peptide2.com / N_peptide_hydrophobicity_hydrophilicity.php ). The Innovagen Peptide Solubility Calculator ( https: / / pepcalc.com / ) and CPPpred http: / / bioware.ucd.ie / ~compass / biowareweb / Server_pages / cpppred.php ) to calculate solubility and cell penetrability to a certain standard. The CPP score gives a range of 0-1, where peptides with a score >0.5 are better cell penetrants. The efficiency of CPP penetration into cells can be measured in several different ways (Lee H-M et al., Nature Communications Biology, 2021, 4:205; Penedo M. et al., Scientific Reports, 2021, 11:7756 and references cited therein). In general, a better CPP should be able to endocytose (transport) more than 40% of the ligand bound to the cell within 2 hours, or help endocytose 40% of the ADC bound to the cell, across the cell membrane.

[0184] In some embodiments, the present application provides antigen binding antibody-drug conjugates that bind to a target on a membrane, capable of endocytosis. In further embodiments, an immunoconjugate comprising an antigen binding protein of the present application and a cytotoxic agent is provided. In further embodiments, the antigen binding protein has ADCC effector function, e.g., the antigen binding protein has enhanced ADCC effector function. In one such embodiment, an antigen binding antibody / protein or antibody fragment is provided for use in an ADC targeting prostate cancer, specifically binds to PSMA, STEAP1, B7H3, CD46, TROP2, CEACAM5, TF and DLL3 antigens, e.g., specifically binds to human PSMA, STEAP1, B7H3, CD46, TROP2, CEACAM5, TF and DLL3 antigens / receptors.

[0185] In further embodiments, an antigen binding protein or fragment for use in an ADC targeting prostate cancer, specifically binds to PSMA, STEAP1, B7H3, CD46, TROP2, CEACAM5, TF and DLL3, wherein the antigen binding protein or fragment has binding to FcyRIIIA and mediates FcgRIIIA-mediated effector function, or has enhanced FcyRIIIA-mediated effector function. In one embodiment of the present application, the antigen binding protein as described herein is capable of endocytosis.

[0186] In one aspect of the present application, an antigen binding protein according to the present application is provided that binds to non-membrane bound PSMA, STEAP1, B7H3, CD46, TROP2, CEACAM5, TF and DLL3, e.g., to PSMA, STEAP1, B7H3, CD46, TROP2, CEACAM5, TF and DLL3 in serum.

[0187] In one aspect of the application, the antibody / protein for the antibody-drug conjugate of the application is provided, preferably from an antibody having affinity to PSMA, STEAP1, B7H3, CD46, TROP2, CEACAM5, TF or DLL3 antigen. Antibody sequence information can be found in the known public domain, such as patent databases of WIPO, USPTO, Espacenet, CNIPA, JPO, etc. Antibody information is shown as follows:

[0188] PSMA antibody and its sequence information can be found in, but not limited to, the following patents: WO1997035616, WO2000014257, WO2001009192, WO2002096460, WO2003064606, WO2005123129, WO2006076525, WO2006089231, WO2006110745, WO2007002222, WO2008153802, WO2009046294, WO2009130575, WO2010027513, WO2010037836, WO2011121110, WO2012016188, WO2013185117, WO2013188740, WO2014057113, WO2014057114, WO2014127365, WO2014178878, WO2014198223, WO2015052532, WO2016111344, WO2016145139, WO2016166299, WO2017087603, WO2017121905, WO2017134158, WO2017137953, WO2017180713, WO2017212250, WO2018033749, WO2018193103, WO2018218875, WO2019092452, WO2019173324, WO2019191728, WO2019245991, WO2020025564, WO2020181094, WO2020212947, WO2020212949, WO2021000018, WO2021038571, WO2021092019, WO2021096968, WO2021098834, WO2021142039, WO2021190583, WO2022194742, WO2022238522, WO2023019240, WO2023026235, WO2023026236.

[0189] STEAP1 antibodies and their sequence information can be found, but are not limited to, the following patents: WO2020153467, WO2020018695, WO2018184966, WO2016205176. Vandortuzumab is a well-known STEAP1 antibody, and its sequence information is as follows:

[0190] Vandortuzumab heavy chain:

[0191] EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWVRQAPGKGLEWVGYISNSGSTSYNPSLKSRFTISRDTSKNTLYLQMNSLRAEDTAVYYCARERNYDYDDYYYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0192] Vandortuzumab qing light chain:

[0193] DIQMTQSPSSLSASVGDRVTITCKSSQSLLYRSNQKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYNYPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0194] Light chain variable domain or comprising the amino acid sequence:

[0195] EIVLTQSPATLSLSPGERATLSCRASSSVSYMHWFQQKPGQAPRLLIYSTSNLASGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQRRSFPYTFGQGTKLEIK;

[0196] a light chain variable domain or comprising the amino acid sequence:

[0197] DIVLTQTPLSLSVTPGQPASISCKASQSVDYDGDSFMNWYLQKPGQPPQLLIYVASNLESGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQSNEEPPTFGQGTKLEIK;

[0198] a heavy chain variable domain or comprising the amino acid sequence:

[0199] QVQLVQSGAEVKKPGASVKVSCKASGYTFSTYWIEWVRQAPGQRLEWMGEILPGSGNTDFNEKFQGRVTFTADTSSDTAYMELSSLRSEDTAVYYCTRWGYYGTRGYFNVWGQGTLVTVSS;

[0200] a heavy chain variable domain or comprising the amino acid sequence:

[0201] QVQLVQSGAEVKKPGASVKVSCKASGYTFSTYWIEWVRQAPGQRLEWMGEILPGSGQTDFNEKFQGRVTFTADTSSDTAYMELSSLRSEDTAVYYCTRWGYYGTRGYFNVWGQGTLVTVSS;

[0202] a heavy chain variable domain comprising the amino acid sequence:

[0203] EIQLVQSGAEVKKPGATVKISCKASGYTFTNYGMNWVQQAPGQGLEWMGWMNTYTGEPTYADKFQGRVTFTLDTSARTVYMELSSLRSEDTAVYFCARAGGQLRPGAMDYWGQGTMVTVSS;

[0204] B7H3 antibodies and their sequence information can be found, but not limited to, the following patents: WO2018116219, WO2010096734, WO2016033225, WO2016106004, WO2016207103, WO2016207104, WO2019024911, WO2020063673, WO2020103100, WO2021081052, WO2021136571, WO2021168379, WO2021190586, WO2021244721, WO2022001020, WO2022126689, WO2022167052, WO2022232392, WO2022257893, WO2023060137, WO2023272924, WO2023274384.

[0205] Well-known B7H3 antibodies include Enoblituzumab, Ifinatamab, Mirzotamab, Obrindatamab, Omburtamab, and Vobramitamab, sequence information as follows:

[0206] Enoblituzumab heavy chain:

[0207] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISSDSSAIYYADTVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCGRGRENIYYGSRLDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELVGGPSVFLLPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTLRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPLVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0208] Enoblituzumab light chain:

[0209] DIQLTQSPSFLSASVGDRVTITCKASQNVDTNVAWYQQKPGKAPKALIYSASYRYSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQYNNYPFTFGQGTKLEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0210] Ifinatamab heavy chain:

[0211] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYVMHWVRQAPGQGLEWMGYINPYNDDVKYNEKFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARWGYYGSPLYYFDYWGQGTLSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQVTVSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0212] Ifinatamab light chain:

[0213] EIVLTQSPATLSLSPGERATLSCRASSRLIYMHWYQQKPGQAPRPLIYATSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWNSNPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0214] Mirzotamab heavy chain:

[0215] EVQLQESGPGLVKPSETLSLTCAVTGYSITSGYSWHWIRQFPGNGLEWMGYIHSSGSTNYNPSLKSRISISRDTSKNQFFLKLSSVTAADTAVYYCAGYDDYFEYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0216] Mirzotamab light chain:

[0217] DIQMTQSPSSLSASVGDRVTITCKASQNVGFNVAWYQQKPGKSPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFAEYFCQQYNWYPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0218] Obrindatamab heavy chain:

[0219] DIQLTQSPSFLSASVGDRVTITCKASQNVDTNVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNNYPFTFGQGTKLEIKGGGSGGGGEVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGGCGGGEVAALEKEVAALEKEVAALEKEVAALEKGGGDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0220] Obrindatamab light chain:

[0221] QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPWTPARFSGSLLGGKAALTITGAQAEDEADYYCALWYSNLWVFGGGTKLTVLGGGGSGGGGEVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISSDSSAIYYADTVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCGRGRENIYYGSRLDYWGQGTTVTVSSGGCGGGKVAALKEKVAALKEKVAALKEKVAALKE;

[0222] Omburtamab heavy chain:

[0223] QVQLQQSGAELVKPGASVKLSCKASGYTFTNYDINWVRQRPEQGLEWIGWIFPGDGSTQYNEKFKGKATLTTDTSSSTAYMQLSRLTSEDSAVYFCARQTTATWFAYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK;

[0224] Omburtamab light chain:

[0225] DIVMTQSPATLSVTPGDRVSLSCRASQSISDYLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC;

[0226] Vobramitamab heavy chain:

[0227] EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLEWVATINSGGSNTYYPDSLKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHDGGAMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0228] Vobramitamab light chain:

[0229] DIQMTQSPSSLSASVGDRVTITCRASESIYSYLAWYQQKPGKAPKLLVYNTKTLPEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYGTPPWTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0230] CD46 antibodies and their sequence information can be found, without limitation, in the following patents: WO2002018948, WO2003032814, WO2008007648, WO2013104728, WO2016040683, WO2018089807, WO2018187074, WO2021015571, WO2021143958, WO2021143959, WO2021257542, WO2022032020, WO2022150512, WO2022150517.

[0231] CD46 antibodies can also have the following sequences:

[0232] CDR VH1 :

[0233] QVQLVQSGGGVVQPGRSLRLACAASGLTVNNYAMHWVRQAPGKGLEWVAVISYDGNNKYYADSVKGRFTISRDNSKNTLYLQ MNSLRAEDTAVYYCAKGGGYFDLWGRGTLVTVSS;

[0234] CDR VL1 :

[0235] QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNNNRPSGVPDRFSGSKSGTSASLAITGLQAEDEA DYYCSSYTSGTWLFGGGTKLTVL

[0236] or CDR VH2 :

[0237] QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWLSFISYDGDEKYYADSVKGRFTISRDNSKNTLYLQM NSLRAEDTAVYWCAKASGYGMGILDYWGQGTLVTVSS;

[0238] or CDR VL2:

[0239] SSELTQDPAVSVALGQTVRITCQGDSLRSYYVSWFQQKPGQAPVFVMYGQNNRPSGISERFSGSSSGNTASLIITGAQAEDEADY YCHSRDSSGTHLRVFGGGTKLTVL

[0240] or CDR VH3:

[0241] QVQLVQSGGGVVQPGRSLRLACAASGFTVNNYAMHWVRQAPGKGLEWVAVISYDGNNKYYADSVKGRFTISRDNSKNTLYLQ MNSLRAEDTAVYYCAKGGGYFDLWGRGTLVTVSS;

[0242] or CDR VL3:

[0243] QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGDNNRPSGVPDRFSGSKSGTSASLAITGLQAEDEA DYYCSSYTSGTWLFGGGTKLTVL

[0244] TROP2 antibodies and sequence information thereof can be found, without limitation, in the following patents: WO1989007270, WO2021066869, WO1996024844, WO2007102869, WO2008144891, WO2011026026, WO2011145744, WO2011155579, WO2012105219, WO2013068946, WO2013077458, WO2013082254, WO2014092804, WO2015047510, WO2015098099, WO2015126548, WO2015186812, WO2016172427, WO2016201300, WO2017139623, WO2017189279, WO2018102212, WO2018156634, WO2018183041, WO2018187074, WO2018190379, WO2018190382, WO2020094670, WO2020191092, WO2020228604, WO2020240467, WO2020249063, WO2021027851, WO2021067403, WO2021068949, WO2021136274, WO2021136483, WO2021147993, WO2021188896, WO2021190480, WO2021214223, WO2021225892, WO2021247908, WO2021259162, WO2022010797, WO2022078424, WO2022095851, WO2022126593, WO2022143670, WO2022152308, WO2022159984, WO2022170619, WO2022170740, WO2022171192, WO2022222992, WO2023001248, WO2023009189, WO2023015322, WO2023046003, WO2023060277, WO2023060283, WO2023273595, WO2023274365.

[0245] Well-known Trop2 antibodies include Datopotamab, Sacituzumab, sequence information as follows:

[0246] Datopotamab heavy chain:

[0247] QVQLVQSGAEVKKPGASVKVSCKASGYTFTTAGMQWVRQAPGQGLEWMGWINTHSGVPKYAEDFKGRVTISADTSTSTAYLQLSSLKSEDTAVYYCARSGFGSSYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0248] Datopotamab light chain:

[0249] DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0250] Sacituzumab heavy chain:

[0251] QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0252] Sacituzumab heavy chain:

[0253] DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0254] CEACAM5 antibodies and their sequence information can be found, without limitation, in the following patents: WO2014092804, WO2015069430, WO2018187074, WO2019009388, WO2020145228, WO2020161214, WO2020244526, WO2020244528, WO2021067403, WO2021214221, WO2021214222, WO2021214223, WO2021214227, WO2022037002, WO2022101165, WO2022116079, WO2022267936, WO2023041065.

[0255] Well-known CEACAM5 antibodies include Cergutuzumab, altumomab, arcitumomab, cibisatamab, labetuzumab, tusamitamab, sequence information as follows:

[0256] Cergutuzumab heavy chain:

[0257] QVQLVQSGAEVKKPGASVKVSCKASGYTFTEFGMNWVRQAPGQGLEWMGWINTKTGEATYVEEFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCARWDFAYYVEAMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0258] Cergutuzumab light chain:

[0259] DIQMTQSPSSLSASVGDRVTITCKASAAVGTYVAWYQQKPGKAPKLLIYSASYRKRGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCHQYYTYPLFTFGQGTKLEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0260] or Cergutuzumab heavy chain:

[0261] QVQLVQSGAEVKKPGASVKVSCKASGYTFTEFGMNWVRQAPGQGLEWMGWINTKTGEAT YVEEFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCARWDFAYYVEAMDYWGQGTTVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCR DELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSAPASSSTKKTQLQLE HLLLDLQMILNGINNYKNPKLTRMLTAKFAMPKKATELKHLQCLEEELKPLEEVLNGAQSK NFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT

[0262] or Cergutuzumab light chain:

[0263] DIQMTQSPSSLSASVGDRVTITCKASAAVGTYVAWYQQKPGKAPKLLIYSASYRKRGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCHQYYTYPLFTFGQGTKLEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0264] Cibisatamab heavy chain:

[0265] QVQLVQSGAEVKKPGASVKVSCKASGYTFTEFGMNWVRQAPGQGLEWMGWINTKTGEAT YVEEFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCARWDFAYYVEAMDYWGQGTTVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSEVQL LESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYA DSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVT VSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECDKTHTCPPCP APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVY TLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0266] Cibisatamab heavy chain:

[0267] QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVLSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC;

[0268] or Cibisatamab heavy chain:

[0269] QVQLVQSGAEVKKPGASVKVSCKASGYTFTEFGMNWVRQAPGQGLEWMGWINTKTGEATYVEEFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCARWDFAYYVEAMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0270] or Cibisatamab heavy chain:

[0271] DIQMTQSPSSLSASVGDRVTITCKASAAVGTYVAWYQQKPGKAPKLLIYSASYRKRGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCHQYYTYPLFTFGQGTKLEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0272] Labetuzumab heavy chain:

[0273] EVQLVESGGGVVQPGRSLRLSCSASGFDFTTYWMSWVRQAPGKGLEWIGEIHPDSSTINY APSLKDRFTISRDNAKNTLFLQMDSLRPEDTGVYFCASLYFGFPWFAYWGQGTPVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0274] Labetuzumab light chain:

[0275] DIQLTQSPSSLSASVGDRVTITCKASQDVGTSVAWYQQKPGKAPKLLIYWTSTRHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYSLYRSFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0276] Tusamitamab heavy chain:

[0277] EVQLQESGPGLVKPGGSLSLSCAASGFVFSSYDMSWVRQTPERGLEWVAYISSGGGITYAPSTVKGRFTVSRDNAKNTLYLQMNSLTSEDTAVYYCAAHYFGSSGPFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG;

[0278] Tusamitamab light chain:

[0279] DIQMTQSPASLSASVGDRVTITCRASENIFSYLAWYQQKPGKSPKLLVYNTRTLAEGVPS RFSGSGSGTDFSLTISSLQPEDFATYYCQHHYGTPFTFGSGTKLEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0280] TF antibodies and sequence information thereof can be found, but not limited to, the following patents: WO1990008956, WO1991016350, WO1992004047, WO1992012429, WO1993013211, WO1993017045, WO1993020186, WO1995021243, WO1996025178, WO1997023509, WO1998010787, WO1998010792, WO1998054195, WO1999021577, WO1999033878, WO1999051743, WO1999062556, WO2000042856, WO2000074634, WO2001024626, WO2001062298, WO2002078738, WO2003063798, WO2003070275, WO2004007557, WO2004039842, WO2005000896, WO2005004793, WO2005030961, WO2005072126, WO2005118646, WO2007066823, WO2007131171, WO2008137382, WO2009025743, WO2009042917, WO2009046274, WO2009111889, WO2010033196, WO2010066803, WO2010131235, WO2011157741, WO2012100262, WO2012125559, WO2012174529, WO2013085021, WO2014140240, WO2015007880, WO2015075201, WO2015115656, WO2016084912, WO2016137108, WO2016153276, WO2017028823, WO2018036117, WO2018036243, WO2019087994, WO2019089973, WO2019102435, WO2019104385, WO2019136309, WO2019173523, WO2019183253, WO2019217455, WO2019217457, WO2020037024, WO2020092210, WO2020226907, WO2020244540, WO2021003399, WO2021037197, WO2021089794, WO2021090272, WO2021094917, WO2021158110,WO2021163299, WO2021188737, WO2021200131, WO2022002940, WO2022011324, WO2022034605, WO2022054009, WO2022104186, WO2022117060, WO2022118282, WO2023277763.

[0281] DLL3 antibodies and sequence information thereof can be found, but not limited to, the following patents: WO2011093097, WO2015031693, WO2015031698, WO2015127407, WO2017021349, WO2017031458, WO2017201442, WO2019195408, WO2021155380, WO2021173307, WO2022153194, WO2022153195, WO2022240688, WO2023006084, WO2023278585.

[0282] The antigen binding antibody / protein of the present application can include the antibody heavy chain variable region and light chain variable region of the present application, configured as a natural antibody or a functional fragment or its equivalent structure. Therefore, the antigen binding protein of the present application can include the VH region of the antibody of the present application, paired with a suitable light chain, configured as a full-length antibody, a (Fab')2 fragment, a Fab fragment or its equivalent (such as scFV, diabody, triabody or tetrabody, Tandabs, etc.). The antibody can be IgG1, IgG2, IgG3 or IgG4, IgM, IgA, IgE, IgD or modified variants thereof. The constant region of the antibody heavy chain can be selected accordingly. The light chain constant region can be kappa or lambda structure. In addition, the antigen binding protein can include all kinds of modifications, such as IgG dimer, Fc mutant that no longer binds Fc receptor or mediates C1q binding. The antigen binding protein can also be a chimeric antibody as described in WO86 / 001533, which includes an antigen binding region and a non-immunoglobulin region.

[0283] The constant region is selected according to the required function, for example, IgG1 can achieve the corresponding lysis function by binding complement and / or mediating ADCC (antibody-dependent cellular cytotoxicity).

[0284] In one aspect, the antibody-like protein is an antigen binding protein or antigen binding protein fragment or antibody fragment comprising one or more CDRs according to the application, or one or two heavy or light chain variable regions according to the application. In one embodiment, the antigen binding protein binds to a primate antigen of PSMA, STEAP1, B7H3, CD46, TROP2, TF, DLL3 and CEACAM5. In one embodiment, the antigen binding protein binds to a non-human primate antigen of PSMA, STEAP1, B7H3, CD46, TROP2, TF, DLL3 and CEACAM5, for example cynomolgus PSMA, STEAP1, B7H3, CD46, TROP2, TF, DLL3 and CEACAM5 antigen.

[0285] In another aspect, the antibody-like protein is from a dAb, Fab, Fab', F(ab')2, Fv, single domain antibody, diabody, triabody, tetrabody, minibody, microbody, full length antibody (polyclonal antibody, monoclonal antibody, antibody dimer, antibody multimer), multispecific antibody (selected from a bispecific antibody, a trispecific antibody or a tetraspecific antibody); single chain antibody, antibody fragment that binds to a target cell, monoclonal antibody, single chain monoclonal antibody, monoclonal antibody fragment that binds to a target cell, chimeric antibody, chimeric antibody fragment that binds to a target cell, domain antibody, domain antibody fragment that binds to a target cell, surface modified antibody, surface modified single chain antibody, surface modified antibody fragment that binds to a target cell, humanized antibody or surface antibody, humanized single chain antibody, humanized antibody fragment that binds to a target cell, anti-Id antibody, CDR construct, antibody precursor, precursor fragment, small immunizing protein (SIP), lymphokine, hormone, vitamin, growth factor, colony stimulating factor, nutrient transport molecule, large molecular mass protein, fusion protein, kinase inhibitor, gene targeting agent, nanoparticle or polymer modified with an antibody or large molecular weight protein, vitamin (including folate), macromolecular peptide, polymeric micelle, liposome, lipoprotein-based drug carrier, nanoparticulate drug carrier, dendrimer, and particle that coats or links a cell binding ligand or protein of the foregoing.

[0286] In one aspect of the application, the antibody-like protein is a humanized or chimeric antibody, in another aspect, the antibody is humanized. In another aspect, the antibody is a monoclonal antibody.

[0287] In yet another aspect of the application, the antibody and conjugate are capable of targeting tumor cells, virus-infected cells, microbe-infected cells, parasite-infected cells, autoimmune disease cells, activated tumor cells, bone marrow cells, activated T cells, B cells or melanocytes affecting, or any dysfunctional cell expressing any 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, CD13, 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, CD21b9, 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, CRIPTO, CR, CR1, CRGF, CRIPTO, CXCR5, LY64, TDGF1, 4-1BB, APO2, ASLG659, BMPR1B, 4-1BB, 5AC, 5T4 (Trophoblast glycoprotein, TPBG, WNT-activated inhibitor 1 or WAIF1), adenocarcinoma antigen, AGS-5, AGS-22M6, activin receptor kinase 1, AFP, AKAP-4, ALK, alpha integrin, alpha v beta 6, aminopeptidase N, amyloid beta, androgen receptor, Angiopoietin 2, Angiopoietin 3, Annexin A1, Anthrax protective antigen, Anti-metastatic protein receptor, AOC3 (VAP-1), B7-H3, Bacillus anthracis, BAFF (B-cell activating factor), BCMA, B-cell lymphoma cell, bcr-abl, bombesin, BORIS, C5, C242 antigen, CA125 (carbohydrate antigen 125, MUC16), CA-IX (or CAIX, carbonic anhydrase 9), CALLA, CanAg, Canine lupus erythematosus IL31, Carbonic anhydrase IX, Cardiac myosin, CCL11 (C-C motif chemokine 11), CCR4 (C-C chemokine receptor 4), CCR5, CD3E (epsilon), CEA (carcinoembryonic antigen), CEACAM3, CEACAM5 (carcinoembryonic antigen), CFD (Factor D), Ch4D5, Cholecystokinin 2 (CCK2R), CLDN18 (Claudin-18), Clusterin A, cMet, CRIPTO, FCSF1R (colony stimulating factor 1 receptor), CSF2 (colony stimulating factor 2, granulocyte-macrophage colony stimulating factor (GM-CSF)), CTLA4 (cytotoxic T-lymphocyte-associated protein 4), CTAA16.88 tumor antigen, CXCR4, C-X-C chemokine receptor 4, Cyclo-ADP- ribosylase, Cyclin B1, CYP1B1, Cytomegalovirus, Cytomegalovirus glycoprotein B, Dabigatran, DLL3 (Delta-like 3), DLL4 (Delta-like 4), DPP4 (dipeptidyl-peptidase 4), DR5 (Death receptor 5), E. coli shiga toxin type-1, E. coli shiga toxin type-2, ED-B, EGFL7 (EGF-like domain protein 7), EGFR, EGFRII, EGFRvIII, Endoglin, Endothelin B receptor, Endotoxin, EpCAM (epithelial cell adhesion molecule), EphA2, Episialin, ERBB2 (epidermal growth factor receptor 2), ERBB3,ERG (TMPRSS2 ETS fusion gene), E. coli, ETV6-AML, FAP (fibroblast activation protein alpha), FCGR1, alpha-fetoprotein, fibrillin II beta chain, fibronectin extra domain-B, FOLR (folate receptor), folate receptor alpha, folate hydrolase, Fos-related antigen 1, F protein of respiratory syncytial virus, Frizzled receptor, Ganglioside GM1, GD2 ganglioside, G-28 (cell surface antigen glycolipid), GD3 idiotype, GloboH, Glypican 3, N-glycolylneuraminic acid, GM3, GMCSF receptor alpha chain, growth differentiation factor 8, GP100, GPNMB (transmembrane glycoprotein NMB), GUCY2C (guanylate cyclase 2C), guanylate cyclase C (GC-C), intestinal guanylate cyclase, guanylate cyclase 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 / scatter factor), HHGFR, HIV-1, histone complex, HLA-DR (human leukocyte antigen), HLA-DR10, HLA-DRB, HMWMAA, human chorionic gonadotropin, HNGF, human scatter 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, IgE Fc region, IGHE, interleukins (including IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-6R, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-17, IL-17A, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-27, or IL-28), IL31RA, ILGF2 (insulin-like growth factor 2), integrin (alpha4, alphaIIb beta3, alpha v beta3, alpha4 beta7, alpha5 beta1, alpha6 beta4, alpha7 beta7, alphaII beta3, alpha5 beta5, alpha v beta5), interferon gamma inducible protein, ITGA2, ITGB2, KIR2D, Kappa Ig, LCK, Le, Legumain, 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 (macrophage migration inhibitory factor, or glycosyl inhibiting factor (GIF)), MS4A1 (membrane-spanning 4-domains, subfamily A, member 1), MSLN (mesothelin), MUC1 (mucin 1, cell surface associated (MUC1) or polymorphic epithelial mucin (PEM)), MUC1-KLH, MUC16 (CA125), MCP1 (monocyte chemoattractant protein 1), MelanA / MART1, ML-IAP, MPG, MS4A1, MYCN, myelin associated glycoprotein, Myostatin, NA17, NARP-1, NCA-90 (granulocyte antigen), Nectin-4 (ASG-22ME), NGF, neural apoptosis regulated proteinase 1, NOGO-A, Notch receptor, Nucleostemin, Neu oncogene product, NY-BR-1, NY-ESO-1, OX-40, OxLDL (oxidized low density lipoprotein), OY-TES1, P21, p53 nonmutant, P97, PAP, anti-(N-glycolylneuraminic acid) antibody binding site, PAX3, PAX5, PCSK9, PDCD1 (PD-1, programmed cell death protein 1), PDGF-R alpha (alpha platelet-derived growth factor receptor), PDGFR-beta, PDL-1, PLAC1, PLAP-like testicular alkaline phosphatase, platelet-derived growth factor receptor beta, sodium phosphate cotransporter, PMEL 17, polysialic acid, proteinase 3 (PR1), prostate cancer, PS (phosphatidylserine), prostate cancer cells, Pseudomonas aeruginosa, PSMA, PSA, PSCA, Rabies virus glycoprotein, RHD (Rh polypeptide 1 (RhPI)), Rhesus factor, RANKL, RhoC, Ras mutation, RGS5, ROBO4, Respiratory Syncytial Virus, RON, ROR1, sarcoma translocation breakpoints, SART3, Sclerostin, SLAMF7 (SLAM member 7), Selectin P, SDC1 (syndecan 1), Systemic Lupus Erythematosus (a), Somatomedin C, SIP (sphingosine-1-phosphate), Somatostatin, Sperm protein 17, SSX2, STEAP1 (six-transmembrane epithelial antigen of the prostate 1), STEAP2, STn, TAG-72 (tumor-associated glycoprotein), Survivin, T cell receptor, T cell transmembrane proteins, TEM1 (tumor endothelial marker 1), TENB2, Tenascin C (TN-C), TGF-alpha, TGF-beta (transforming growth factor beta), TGF-beta 1, TGF-beta 2 (transforming growth factor 2), Tie (CD202b), Tie2, TIM-1 (CDX-014), Tn, TNF, TNF-alpha, TNFRSF8, TNFRSF10B (tumor necrosis factor receptor superfamily member 10B),TNFRSF13B (tumor necrosis factor receptor superfamily member 13B), TPBG (trophoblast glycoprotein), TRAIL-R1 (TNF-related necrosis-inducing ligand receptor 1), TRAILR2 (death receptor 5 (DR5)), tumor-associated calcium signal transducer 2, tumor-specific glycosylated MUC1, TWEAK receptor, TYRP1 (glycoprotein 75), TRP-1 (Trop1), TRP-2 (Trop2), tyrosinase, VCAM-1, VEGF, VEGF-A, VEGF-2, VEGFR-1, VEGFR-2, vimentin, WT1, XAGE1, cells expressing insulin growth factor receptor, or cells expressing epidermal growth factor receptor.

[0288] In a further aspect of the application, the antibody and conjugate is capable of targeting a lymphoma cell, a myeloma cell, a renal carcinoma cell, a breast carcinoma cell, a prostate carcinoma cell, an ovarian carcinoma cell, a colorectal carcinoma cell, a gastric carcinoma cell, a squamous carcinoma cell, a small cell lung carcinoma cell, a non-small cell lung carcinoma cell, a testicular carcinoma cell, a malignant cell, or any cell whose growth and division rate is not regulated, accelerated leading to cancer.

[0289] In another aspect, the antibody-like protein binds to a human antigen with high affinity, for example, the antigen binding protein binds to a human antigen with an affinity of 20 nM or less or an affinity of 15 nM or less or an affinity of 5 nM or less or an affinity of 1000 pM or less or an affinity of 500 pM or less or an affinity of 400 pM or less, or 300 pM or less, for example, about 120 pM, when measured by Biacore or ForteBio. In a further embodiment, the antigen binding protein binds to a human antigen when measured by Biacore between about 100 pM to about 500 pM or between about 100 pM to about 400 pM, or between about 100 pM to about 300 pM. In one embodiment of the application, the antigen binding protein binds to an antigen with an affinity of less than 150 pM.

[0290] In one such embodiment, this is measured by Biacore or ForteBio.

[0291] In another aspect, the antigen binding protein / antibody binds to a human antigen in a cellular and in an assay, wherein the IC50 of the antigen binding protein 50 between about 1 nM and about 500 nM, or between about 1 nM and about 100 nM, or between about 1 nM and about 50 nM, or between about 1 nM and about 25 nM, or between about 5 nM and about 15 nM. In a further embodiment of the application, the antigen binding protein binds to an antigen and neutralizes the antigen in a cellular and in an assay, wherein the IC50 of the antigen binding protein 50 is about 10 nM.

[0292] Antibody-like proteins, preferably antibodies of the present application, can be produced by transfecting a host cell with an expression vector comprising the coding sequences of the antigen binding proteins of the present application. The expression vector or recombinant plasmid is produced by operably linking these coding sequences of the antigen binding proteins with conventional regulatory sequences capable of controlling replication, expression and / or secretion in the host cell. The regulatory sequences include promoter sequences, such as the CMV promoter, and signal sequences that can be derived from other known antibodies. Similarly, a second expression vector can be produced having DNA sequences encoding the light or heavy chain of the complementary antigen binding protein. In certain embodiments, this second expression vector is identical to the first expression vector except for the coding sequences and a selectable marker, so as to ensure as much as possible that each polypeptide chain is functionally expressed. Alternatively, the heavy and light chain coding sequences of the antigen binding protein can reside on a single vector.

[0293] The selected host cell is co-transfected with the first and second vectors (or simply by transfection with a single vector) by conventional techniques to create a transfected host cell of the present application containing the recombinant or synthetic light and heavy chains. The transfected cell is then cultured in conventional media to produce the engineered antigen binding protein of the present application. The antigen binding protein, including the binding of the recombinant heavy and / or light chains, is screened from the culture by appropriate assays, such as ELISA or RIA. Similar conventional techniques can be used to construct other antigen binding proteins.

[0294] Suitable vectors for cloning and subcloning steps in the methods and constructs of the present application can be selected by one of skill in the art. For example, the traditional pUC series of cloning vectors can be used. One of these vectors, pUC19, is available from supply companies such as Amersham Bioscience (Buckinghamshire, UK) or Kings- rey (Nanjing, China). In addition, any vector that can be easily replicated, has a rich repertoire of cloning sites and selectable genes (e.g., antibiotic resistance), and is easy to manipulate can be used for cloning. Thus, the choice of cloning vector is not a limiting factor of the present application.

[0295] Expression vectors can also be characterized by the genes for which they are suitable for expression of heterologous DNA sequences, for example, the mammalian dihydrofolate reductase gene (DHFR). Other vector sequences include poly A signal sequences, such as those from bovine growth hormone (BGH) and betaglopro promoter sequences. Expression vectors useful herein can be synthesized by techniques well known to those skilled in the art.

[0296] Components of these vectors, such as replicons, selectable genes, enhancers, promoters, signal sequences, etc., can be obtained from commercial or natural sources or synthesized by known procedures for directing expression and / or secretion of the recombinant DNA products in the selected host. Various types of other suitable expression vectors known in the art for mammalian, bacterial, insect, yeast, and fungal expression can also be used for this purpose.

[0297] The present application also includes cell lines transfected with recombinant plasmids containing the coding sequences for the antigen binding proteins of the present application. Host cells for cloning and other manipulation of these cloning vectors are also conventional. However, cells from various E. coli strains can be used to replicate the cloning vectors of the present application and for other steps in the construction of the antigen binding proteins.

[0298] Suitable host cells or cell lines for expressing the antigen binding proteins of the present application include mammalian cells such as NSO, Sp2 / 0, CHO (e.g., DG44), COS, HEK, fibroblast (e.g., 3T3), and myeloma cells, for example it can be expressed in CHO or myeloma cells. Human cells can be used, thereby enabling the molecule to be modified by the human glycosylation pattern.

[0299] Alternatively, other eukaryotic cell lines can be used. Selection of appropriate mammalian host cells and methods for transformation, culturing, amplification, screening, and product production and purification are known in the art. See, e.g., Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, Second Edition. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

[0300] Bacterial cells can prove to be suitable host cells for expression of the recombinant Fabs or other embodiments of the present application (see, e.g., Pluckthun, A., Immunol. Rev., 130: 151-188 (1992)). However, because proteins expressed in bacterial cells tend to exist in an unfolded or misfolded form or non-glycosylated form, any recombinant Fab produced in bacterial cells must be screened to retain antigen binding capacity. If the molecule expressed from the bacterial cells is produced in a properly folded form, then the bacterial cells will be an ideal host, or in other embodiments, the molecule can be expressed in a bacterial host and then subsequently refolded. For example, various E. coli strains are known in the biotechnology art as host cells for expression. This approach can also employ various strains of Bacillus subtilis, Streptomyces, other bacilli, and the like.

[0301] When desired, yeast cell strains known to those skilled in the art can also be used as host cells, as well as insect cells, e.g. Drosophila and Lepidoptera, and viral expression systems. See, for example, Miller et al., Genetic Engineering, 8:277-298, Plenum Press (1986) and McGuire, S. et al., Trends Genet. (2004) 20, 384-391 and references cited therein.

[0302] The general methods of constructing vectors, transfection methods required to produce the host cells of the application, and culture methods required to produce the antigen binding proteins of the application from such host cells can all be routine techniques. Typically, the culture methods of the application are serum-free culture methods, usually by culturing serum-free cells in suspension. Likewise, once produced, the antigen binding proteins of the application can be purified from the cell culture contents according to standard procedures in the art, including ammonium precipitation, affinity columns, column chromatography, gel electrophoresis, etc. These techniques are within the skill of the art and do not limit the application. For example, methods of altering antibody production are described in WO 99 / 058679 and WO 96 / 016990. Another method of expressing antigen binding proteins can utilize expression from transgenic animals, as described in U.S. Patent 4,873,316. This relates to an expression system using the animal casein promoter, when transgenically incorporated into a mammal, the female can produce the desired recombinant protein in its milk.

[0303] In another embodiment of the application, there is provided a method of producing an antibody of the application, the method comprising the steps of culturing a host cell transformed or transfected with a vector encoding a light chain and / or a heavy chain of an antibody of the application and recovering the antibody produced thereby.

[0304] According to the application, there is provided a method of producing an antibody of the application, the antibody binding and neutralizing the activity of a human antigen, the method comprising the steps of: providing a first vector encoding a heavy chain of the antibody; providing a second vector encoding a light chain of the antibody; transforming a mammalian host cell (e.g. CHO) with said first and second vectors; culturing the host cell of step (c) under conditions conducive to the secretion of the antibody from said host cell into said culture medium; recovering the antibody secreted by step (d).

[0305] Once expressed by the desired method, the antibody is then examined for in vitro activity by using appropriate assays. Currently, a routine ELISA assay format is employed to assess the qualitative and quantitative binding of the antibody to the antigen. In addition, other in vitro assays can be used to verify the neutralizing effect prior to subsequent human clinical studies to assess the persistence of the antibody in vivo, despite the usual clearance mechanisms.

[0306] The dosage and duration of treatment are related to the relative duration of the molecules of the invention (antibodies and antibody-drug conjugates) in the circulation of humans, and can be adjusted by those skilled in the art depending on the condition being treated and the general health of the patient. It is expected that repeated dosing (e.g., once a week or once every two weeks or once every 3 weeks or once every 4 weeks) over a longer period of time (e.g., 4 to 6 months) can be required to achieve maximal therapeutic effect.

[0307] In one embodiment of the invention, a recombinantly transformed, transfected or transduced host cell is provided that includes at least one expression cassette, e.g., an expression cassette that includes a polynucleotide encoding a heavy chain of an antigen binding protein described herein, and further includes a polynucleotide encoding a light chain of an antigen binding protein described herein or wherein there are two expression cassettes and the 1.sup.st encodes a light chain and the second encodes a heavy chain. For example, in one embodiment, a first expression cassette includes a polynucleotide encoding a heavy chain of an antigen binding protein that includes a constant region or antigen binding fragment thereof that is linked to a constant region described herein, and further includes a second cassette that includes a polynucleotide encoding a light chain of an antigen binding protein that includes a constant region or antigen binding fragment thereof that is linked to a constant region described herein, e.g., a first expression cassette includes a polynucleotide encoding a heavy chain and a second expression cassette that includes a polynucleotide encoding a light chain.

[0308] In another embodiment of the invention, a stably transformed host cell is provided whose vector includes one or more expression cassettes encoding an antibody heavy chain and / or light chain that includes a constant region or antigen binding fragment thereof that is linked to a constant region described herein. For example, such a host cell can include a first vector encoding a light chain and a second vector encoding a heavy chain, e.g., a first vector encoding a heavy chain and a second vector encoding a light chain.

[0309] In another embodiment of the invention, a host cell described herein is provided wherein the cell is a eukaryotic cell, e.g., a mammalian cell. Examples of such cell lines include CHO or NSO.

[0310] In another embodiment of the invention, a method for producing an antibody that includes a constant region or antigen binding fragment thereof that is linked to a constant region described herein is provided, the method including the step of culturing a host cell in a culture medium, e.g., a serum-free culture medium.

[0311] In another embodiment of the invention, a method of the invention described herein is provided wherein the antibody is further purified to at least 95% or greater (e.g., 98% or greater) relative to the antibody contained in a serum-free culture medium.

[0312] In yet another embodiment, a pharmaceutical composition comprising an antigen binding protein and a pharmaceutically acceptable carrier is provided.

[0313] In another embodiment of the application, a kit of parts comprising the inventive composition described herein and instructions for use is provided.

[0314] The therapeutic agents of the present application can be administered by any appropriate route of delivery of the agent to the host. The antigen binding proteins and pharmaceutical compositions of the present application are particularly suitable for parenteral administration, i.e., subcutaneous (s.c.), intrathecal, intraperitoneal, intramuscular (i.m.), or intravenous (i.v.). In one such embodiment, the antigen binding proteins of the present application are administered by intravenous or subcutaneous injection.

[0315] The therapeutic agents of the present application can be prepared as pharmaceutical compositions comprising an effective amount of the antigen binding proteins of the present application as active ingredients in a pharmaceutically acceptable carrier. In one embodiment, the prophylactic agents of the present application comprise an aqueous suspension or solution of the antigen binding proteins in a form suitable for injection. In one embodiment, the suspension or solution is buffered at physiological pH. In one embodiment, compositions for parenteral administration will include solutions of the antigen binding proteins of the present application dissolved in a pharmaceutically acceptable carrier or a mixture thereof. In one embodiment, the carrier is an aqueous carrier. A variety of aqueous carriers can be employed, e.g., 0.9% physiological saline, 0.3% glycine, etc. These solutions can be sterile and generally free of particulate matter. These solutions can be sterilized by conventional, well-known sterilization techniques, e.g., filtration. The compositions can contain pharmaceutically acceptable auxiliary substances as required, such as pH adjusting and buffering agents, etc. In such pharmaceutical formulations, the concentration of the antigen binding proteins of the present application can vary from less than about 0.5%, usually or at least about 1% up to about 15 or 20% by weight, and will be selected primarily based on liquid volumes, viscosities, etc., according to the particular mode of administration selected, mainiy based on the particular formulation.

[0316] Thus, the pharmaceutical compositions of the application for intravenous infusion can be prepared to contain about 250 ml of sterile Ringer's solution, and about 1 to 30 mg, or 5 mg to about 25 mg of the antigen binding protein of the application per ml of Ringer's solution. Actual methods for preparing parenterally administrable compositions are well known or apparent to those skilled in the art and are described in more detail, for example, in Remington's Pharmaceutical Science, 15thEd., Mack Publishing Company, Easton, PA. For the preparation of intravenously administrable antigen binding protein formulations of the application, see Parkins D. and Lasmar U. "The formulation of Biopharmaceutical products", Pharm. Sci. Tech. Today, 3 (2000) 129-137; Wang, W "Instability, stabilisation and formulation of liquid protein pharmaceuticals", Int. J. Pharm 185 (1999) 129-188; Jorgensen, L. et al., "Recent trends in stabilising peptides and proteins in pharmaceutical formulation - considerations in the choice of excipients" Expert Opin Drug Deliv 6 (2009) 1219-1230; Akers, MJ "Effects of types of sugar on stabilization of Protein in the dried state", J. Pharm Sci 91 (2002) 2283-2300; Imamura, K et al., "Effects of types of sugar on stabilization of Protein in the dried state", J Pharm Sci 92 (2003) 266-274; Izutsu, K kojima, S. "Excipient crystallinity and its protein-structure-stabilizing effect during freeze-drying", J. Pharm.Johnson, R, et al., "Mannitol-sucrose mixtures - versatile formulations for protein lyophilization", J. Pharm. Sci., 91 (2002) 914-922; Kerwin B. "Polysorbates 20 and 80 used in the formulation of protein biotherapeutics: structure and degradation pathways" J. Pharm Sci. 97 (2008) 2924-2935; Ha, E., et al., "Peroxide formation in polysorbate 80 and protein stability", J. Pharm Sci, 91 (2002), 2252-2264, and He, F., et al., "Effect of sugar molecules on the viscosity of high concentration monoclonal antibody solutions" Pharm Res. 28 (2011) 1552-1560; the entire contents of which are incorporated herein by reference.

[0317] In one embodiment, the antibody of the application is present in a unit dosage form when in a pharmaceutical formulation. A therapeutically effective dose is determined by one skilled in the art. Suitable doses can be calculated in relation to the body weight of the patient, for example suitable doses can be in the range of about 0.1 to about 200 mg / kg, about 1 to about 20 mg / kg, about 10 to about 20 mg / kg. For example about 1 to about 15 mg / kg, about 5 to about 15 mg / kg. For effective treatment of, for example, multiple myeloma, SLE or IPT, suitable doses can be in the range of about 0.1 to about 2000 mg, such as about 0.1 to about 500 mg, such as about 500 mg, about 0.1 mg to about 150 mg, or about 0.1 to about 80 mg, or about 0.1 to about 60 mg, or about 0.1 to about 40 mg, or such as about 1 to about 100 mg, or about 1 to about 50 mg of the antigen binding protein of the application, which can be administered parenterally, for example subcutaneously, intravenously or intramuscularly. If desired, the administration can be repeated at appropriate intervals selected by the physician.

[0318] The antigen binding proteins described herein can be stored lyophilized and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective for conventional immunoglobulins and can employ known peroxidation and reconstitution techniques.

[0319] In another aspect of the application, there is provided an antigen binding protein as described herein for use in medicine.

[0320] In one aspect of the application, there is provided an antigen binding protein according to the application for use in the treatment of rheumatoid arthritis, type 1 diabetes, multiple sclerosis or psoriasis, wherein the method comprises the step of administering to the patient a therapeutically effective amount of an antigen binding protein as described herein.

[0321] In one embodiment of the application, there is provided a method for treating human cancer comprising administering to the human population an antigen binding protein that specifically binds to PSMA, STEAP1, B7H3, TROP2, CD46, TF, DLL3 and CEACAM5 antigens. In certain cases, the antigen binding protein is part of an immunoconjugate.

[0322] The term "antibody-drug conjugate (ADC)" as used herein refers to a molecule comprising a monoclonal antibody (mAb) linked via a chemical linker to a cytotoxic agent, typically a small molecule drug with high systemic toxicity. The ADC of the present application is represented by the following formula:

[0323]

[0324] where D1 and D2 are small molecule cytotoxins or functional small molecules, generally referred to as payloads; L1 and L2 are functional linkers with affinity ligands; and mAb is a monoclonal antibody. In some embodiments, the ADC can comprise a small molecule cytotoxin that has been chemically modified to comprise a linker with an affinity ligand, or the linker with an affinity ligand is part of the payload known as a traceless linker. Linkers are typically used to couple cytotoxins to antibodies or antigen binding fragments thereof. Upon binding to a target antigen on the cell surface, the ADC is endocytosed and transported into the lysosome, where the cytotoxin is released by proteolysis of the cleavable linker (e.g., by cathepsin B found in the lysosome) or proteolytic degradation of the antibody, if linked to the cytotoxin by a non-cleavable linker. The cytotoxin is then transported from the lysosome into the cytoplasm or nucleus, where it binds to the target according to its mechanism of action.

[0325] The antibody-drug conjugates described herein can include whole antibodies or antibody fragments. A whole antibody typically consists of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide. Each heavy chain comprises 1 N-terminal variable region (VH) and 3 C-terminal constant regions (CHI, CH2, and CH3), and each light chain comprises 1 N-terminal variable region (VL) and 1 C-terminal constant region (CL). The variable regions of each pair of light and heavy chains form the antigen binding site of the antibody. The VH and VL regions have the same general structure, each consisting of four framework regions whose sequences are relatively conserved. The framework regions are connected by three complementarity determining regions (CDRs). The three CDRs, referred to as CDR1, CDR2, and CDR3, form the "hypervariable region" of the antibody, which is responsible for antigen binding.

[0326] An ADC can comprise an antigen-binding fragment of an antibody. The terms "antibody fragment," "antigen-binding fragment," "functional fragment of an antibody," and "antigen-binding portion" are used interchangeably herein to refer to one or more fragments or portions of an antibody that retain the ability to specifically bind to an antigen. An antibody fragment can include, for example, one or more CDRs, a variable region (or a portion thereof), a constant region (or a portion thereof), or combinations thereof. Examples of antibody fragments include, but are not limited to, (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CHI domains; (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (iv) a single chain Fv (scFv), which is a monovalent molecule consisting of the two domains of a Fv fragment (i.e., VL and VH) connected by a synthetic linker that enables the two domains to associate to form a single polypeptide chain (see, e.g., Kabat EA, Wu TT., J Immunol. 1991, 147(5): 1709-19), and (v) a diabody, which is a dimer of polypeptide chains, wherein each polypeptide chain includes a VH connected to a VL by a peptide linker that is too short to allow pairing between the VHand VL on the same polypeptide chain, thereby driving the pairing between complementary domains on different VHand VL polypeptide chains to produce a dimeric molecule having two functional antigen binding sites (see, e.g., Hudson PJ, Kortt AA, J Immunol Methods. 1999, 231(1-2): 177-89; Holliger P, Winter G. Cancer Immunol Immunother. 1997, 45(3-4): 128-30).

[0327] A monoclonal antibody or antigen-binding fragment thereof directed against an antigen may have any suitable binding affinity for the antigen or its epitope. The term "affinity" refers to the equilibrium constant for reversible binding of two agents, expressed as the dissociation constant (K D The affinity of an antibody or antigen-binding fragment thereof for a target antigen or epitope can be measured using any method known in the art. For example, these methods include fluorescence activated cell sorting (FACS), surface plasmon resonance (e.g., Biacore TM 、ProteOn TM ), biolayer interferometry (BLI, such as Octet), kinetic exclusion (such as KinExA TM ), separable microbeads (e.g., magnetic beads), antigen screening and / or ELISA (see, e.g., JR Crowther, Methods Mol Biol. 2000, 149: III-IV, 1-413). It is known in the art that the binding affinity of a particular antibody will depend on the method used to analyze the binding affinity.

[0328] The affinity of a binding agent for a ligand, such as the affinity of an antibody for an epitope, can be from about 1 picomolar (pM) to about 1 micromolar (1 μM) (e.g., from about 1 picomolar (pM) to about 1 nanomolar (nM), or from about 1 nM to about 1 micromolar (μM)). In one embodiment, a monoclonal antibody or antigen-binding fragment thereof can bind to an antigen with a Kd of less than or equal to 100 nanomolar (e.g., 100 nM, about 90 nM, about 80 nM, about 70 nM, about 60 nM, about 50 nM, about 40 nM, about 30 nM, about 20 nM, or about 10 nM, or a range defined by any two of the foregoing values).

[0329] In another embodiment, the monoclonal antibody can bind to an antigen with a Kd of less than or equal to 10 nanomolar (e.g., about 9 nM, about 8 nM, about 7 nM, about 6 nM, about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM, about 0.9 nM, about 0.8 nM, about 0.7 nM, about 0.6 nM, about 0.5 nM, about 0.4 nM, about 0.3 nM, about 0.2 nM, about 0.1 nM, about 0.05 nM, about 0.02 nM, about 0.01 nM, about 0.001 nM, or a range defined by any two of the foregoing values).

[0330] In another embodiment, the monoclonal antibody can bind to an antigen with a Kd of less than or equal to 200 pM (e.g., about 190 pM, about 175 pM, about 150 pM, about 125 pM, about 110 pM, about 100 pM, about 90 pM, about 80 pM, about 70 pM, about 60 pM, about 50 pM, about 40 pM, about 30 pM, about 25 pM, about 20 pM, about 15 pM, about 10 pM, about 5 pM, about 1 pM, or a range defined by any two of the foregoing values).

[0331] In one embodiment, the affinity of the antibody or antigen-binding fragment thereof is about 90 nM, about 80 nM, about 70 nM, about 60 nM, about 50 nM, about 40 nM, about 30 nM, or a range defined by any two of the foregoing values, e.g., about 50 nM to about 70 nM, about 55 nM to about 65 nM, or about 58 nM to about 62 nM, as measured by surface plasmon resonance (SPR).

[0332] In one embodiment, the affinity of the antibody or antigen-binding fragment thereof for a membrane-bound antigen is less than or equal to 10 nanomolar (e.g., about 9 nM, about 8 nM, about 7 nM, about 6 nM, about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM, about 0.9 nM, about 0.8 nM, about 0.7 nM, about 0.6 nM, about 0.5 nM, about 0.4 nM, about 0.3 nM, about 0.2 nM, about 0.1 nM, about 0.05 nM, about 0.02 nM, about 0.01 nM, about 0.001 nM, or a range defined by any two of the foregoing values), as measured in a FACS assay.

[0333] The antigen-binding portion or fragment of a monoclonal antibody can be of any size, so long as the portion binds to an antigen. In this regard, the antigen-binding portion or fragment of a monoclonal antibody to an antigen preferably comprises about 5 to 35 amino acids (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or a range defined by any two of the foregoing values).

[0334] In one embodiment, the antibody-drug conjugate comprises a variable region of a monoclonal antibody. In this regard, the ADC can include a light chain variable region, a heavy chain variable region, or a light chain variable region and a heavy chain variable region of a monoclonal antibody.

[0335] Monoclonal antibodies or antigen-binding fragments thereof can be conjugated to a cytotoxin using any suitable method known in the art, including site-specific or non-site specific conjugation methods. Traditional antibody conjugation strategies typically rely on random (i.e., non-specific) conjugation of the payload to the antibody and antigen-binding fragments thereof through lysine or cysteine. Thus, in certain aspects, the antibody or antigen-binding fragment thereof is conjugated to a cytotoxic agent randomly, e.g., by partially reducing the antibody or antibody fragment, and then reacting with the desired agent with or without an attached linker moiety. For example, the antibody or antigen-binding fragment thereof can be reduced using dithiothreitol (DTT), TCEP, thioerythritol, or a similar reducing agent. Then, in the presence of dimethyl sulfoxide (DMSO) or DMA, the cytotoxic agent (with or without a linker moiety) can be added to the reduced antibody or antibody fragment in a molar excess dose. After conjugation, an excess of free cysteine can be added to quench unreacted reagents. In the presence of DMSO or DMA, the cytotoxic agent can be added directly to the antibody or antibody fragment in a molar excess with or without a linker moiety having an amino-reactivity or phenol-reactivity or other-reactivity group (e.g., NHS, PFP) to form the conjugate. The reaction mixture can then be purified by chromatography or by exchanging the buffer into phosphate buffered saline (PBS).

[0336] The terms "cytotoxin" and "cytotoxic agent" refer to any molecule which inhibits or prevents the function of cells and / or causes destruction of cells (cell death) and / or brings about an anti-proliferative effect. The cytotoxin or cytotoxic agent of an ADC is also referred to in the art as the "payload" of the ADC. Multiple classes of cytotoxic agents known in the art have potential utility in ADC molecules and can be used in the ADCs described herein. Such cytotoxic agents include, for example, anti-microtubule agents (e.g., tubulysins, auristatins, and maytansinoids), DNA minor groove binders (e.g., pyrrolobenzodiazepines (PBDs) or indolinobenzodiazepines (IGNs) and dimers thereof), RNA polymerase II inhibitors (e.g., amatoxins), DNA topoisomerase I inhibitors (e.g., camptothecins), and DNA alkylating agents (e.g., duocarmycins, CC-1065, pyrrolobenzodiazepine dimers or indolinobenzodiazepine pseudodimers). Examples of specific cytotoxic agents that can be used in the ADCs described herein include, but are not limited to: tubulysins, amatoxins, auristatins, calicheamicins, camptothecins, daunorubicin, doxorubicin, duocarmycins, dolastatins, enediynes, lexitropsins, taxanes, puromycins, maytansinoids, vinca alkaloids, and pyrrolobenzodiazepines (PBDs). More specifically, the cytotoxic agent can be, for example, a tubulysin, an auristatin (AFP, MMAF, MMAE, AEB, AEVB, E), paclitaxel, docetaxel, CC-1065 (duocarmycin, DC1, DC4, CBI-dimer), a camptothecin (SN-38, topotecan), a morpholino-doxorubicin, a rhizoxin, a cyanomorpholino-doxorubicin, dolastatin-10, echinomycin, combrestatins, galiotoxins, a maytansine (DM1, DM4, DM21), vinblastine, methotrexate, a netropsin, or a derivative or analog thereof. Cytotoxins suitable for use in ADCs are also described, for example, in PCT Application: PCT / CN2021 / 128453.

[0337] In general, chemotherapeutic drugs or functional compounds can also be conjugated to the antibodies of the present application. The chemotherapeutic drug or functional compound is selected from the group consisting of:

[0338] a) alkylating agents, nitrogen mustards: chlornaphazine, chloroambucil, cyclophosphamide, dacarbazine, estramustine, ifosfamide, mechlorethamine, megestrol, oxaliplatin, prednimustine, thiotepa, uramustine; CC-1065 (including adozelesin, carzelesin, bizelesin and synthetic analogs thereof); duocarmycin (including KW-2189 and CBI-TMI, and synthetic analogs thereof); benzodiazepine dimer (including dimers of pyrrolobenzodiazepine (PBD) or tomaymycin, indolobenzodiazepine, imidazobenzothiadiazepine or oxazolidinebenzodiazepine); nitrosoureas (carmustine, lomustine, streptozocin, fotemustine, nimustine, ranimustine); alkyl sulfonates (alseribine, elliptinium, sulfisoxazole and tiopanate); triazenes (dacarbazine); platinum-containing compounds (carboplatin, cisplatin, oxaliplatin); aziridines, benzodioxepines, carboquone, mechinostat and ureddopa; ethylenimines and methyl triazenes, including hexamethylmelamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and triethylenethylophosphoramide;

[0339] b) plant alkaloids: vinca alkaloids (vincristine, vinblastine, vindesine, vinorelbine, navelbine); taxoids (paclitaxel, docetaxel and analogs thereof); maytansinoids (DM1, DM2, DM3, DM4, maytansine, ansamitocin and analogs thereof); cryptophycin (in particular cryptophycin 1 and cryptophycin 8); epothilone, halichondrin, dolastatin, bryostatin, dinoflagridine, auristatin, tubulysin, ephalostatin; pancratistatin; sarcodictyin; spongothymidine;

[0340] c) DNA topoisomerase inhibitors, etoposide (9-aminocamptothecin, camptothecin, clintazene, dolastatin, etoposide, etoposide phosphate, irinotecan, mitoxantrone, mithramycin, retinoic acid (retinol), teniposide, topotecan, 9-nitrocamptothecin (RFS2000)); mitomycin (mitomycin C);

[0341] d) antimetabolites, antifolates, DHFR inhibitors (methotrexate, trimetrexate, dimethyl folic acid, pteropterin, aminopterin (4-aminobenzoic acid) or other folic acid analogs); IMP dehydrogenase inhibitors (mycophenolic acid, tiazofurin, ribavirin, EICAR); ribonucleotide reductase inhibitors (hydroxyurea, desferrioxamine); pyrimidine analogs, uracil analogs (ancitabine, azacitidine, 6-azauridine, capecitabine (Xeloda), carmofur, cytarabine, dideoxyuridine, deoxyfluridine, enocitabine, 5-fluorouracil, floxuridine, ratitrexed (Tomudex); cytosine analogs (cytarabine, cytosine arabinoside, fludarabine); purine analogs (azathioprine, fludarabine, mercaptopurine, thiamiprine, thioguanine); folic acid supplements, floxuridine; nicotinamide phosphoribosyltransferase (NAMPT) inhibitors;

[0342] e) hormonal therapy agents, receptor antagonists, antiestrogens (megestrol, raloxifene, tamoxifen), LHRH agonists (goserelin, leuprolide acetate); antiandrogens (bicalutamide, flutamide, casodex, dihydrotestosterone propionate, epitiostanol, goserelin, leuprolide, meptinidine, nilutamide, testolactone, triazolopyrimidine, and other androgen inhibitors); retinoids, vitamin D3 analogs (CB1093, EB1089, KH1060, cholecalciferol, ergocalciferol); photodynamic therapy agents (verteporfin, phthalocyanines, photosensitizer Pc4, desmethoxy-hypericin A); cytokines (interferon-alpha, interferon-gamma, tumor necrosis factor (TNF), human proteins containing TNF);

[0343] 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 (AP24534), bafetinib (INNO-406), bosutinib (SKI-606), cabozantinib, vismodegib, iniparib, ruxolitinib, CYT387, axitinib, tivozanib, sorafenib, bevacizumab, cetuximab, trastuzumab, ranibizumab, panitumumab, ipilimumab;

[0344] g) poly (ADP-ribose) polymerase (PARP) inhibitors, olaparib, niraparib, iniparib, talazoparib, veliparib, CEP 9722 (Cephalon’s), E7016 (Eisai’s), BGB-290 (Beijing Genomics Institute) or 3-aminobenzamide.

[0345] h) antibiotics such as enediyne antibiotics (calicheamicin, particularly calicheamicin gammal, deltal, alphal and beta 1, dynemicin, including dynemicin A and deoxymycin, esperamicins, caratamicin, C-1027, maduropeptin, new carzeri Austin and related chromoprotein enediyne antibiotics), aclacinomysins, actinomycin, anthramycin, azaserine, bleomycins, cactinomycin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino doxorubicin and deoxydoxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, nitomycin, mycophenolic acid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin;

[0346] i) polyketides (calanolide), particularly bullatacin and bullatacinone; gemcitabine, epothilones (such as cafiectin), bortezomib, thalidomide, lenalidomide, pomalidomide, tosedostat, zybrestat, PLX4032, STA-9090, Stimuvax, allovectin-7, Xegeva, Provenge, Yervoy, isoprenylation inhibitors (such as lovastatin), dopaminergic neurotoxins (such as staurosporine), actinomycin (such as dactinomycin, actinomycin), bleomycin (such as bleomycin A2, bleomycin B2, pepleomycin), anthracycline antibiotics (such as daunorubicin), amatoxins, doxorubicin (adriamycin), idarubicin, epirubicin, pirarubicin, zorubicin, mitoxantrone, MDR inhibitors (such as verapamil), Ca 2+ATPase inhibitors (e.g., thapsigargin), histone deacetylase inhibitors (vorinostat, romidepsin, panobinostat, valproic acid, Mocetinostat (MGCD0103), Belinostat, PCI-24781, entinostat, SB939, Resminostat, Givinostat, AR-42, CUDC-101, sulforaphane, trichostatin A); celecoxib, glitazones, epigallocatechin gallate, disulfiram, Salinosporamide A; antiadrenal drugs, aminoglutethimide, mitotane, trilostane, acetretin, aldo-phosphamide, aminolevulinic acid, amsacrine, araboside, bestrabucil, bisantrene, edatraxate, defofamine, melittin, diaziquone, difluoromethylornithine (DFMO), elfomithine, elliptinium acetate, etoglucid, gallium nitrate, cytosine, hydroxyurea, ibandronate, lentinan, lonidamide, mitoguazone, mitoxantrone, mopidamol, nitracine, pento statin, phenamet, pirarubicin, podophyllinic acid, 2-ethylhydrazine, procarbazine; propanediol pyrazon; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine), polyurethane, siRNA, antisense drugs and nucleolytic enzymes;

[0347] (2) Anti-autoimmune disease drugs: cyclosporin, cyclosporin A, aminohexanoic acid, azathioprine, bromocriptine, chlorambucil, chloroquine, cyclophosphamide, corticosteroids (including amcinonide, betamethasone, budesonide, hydrocortisone, flunisolide, fluticasone propionate, fluocortolone, dexamethasone, triamcinolone acetonide, beclomethasone dipropionate), DHEA, etanercept, hydroxychloroquine, infliximab, meloxicam, methotrexate, mycophenolate mofetil, prednisone, sirolimus, tacrolimus.

[0348] (3) Anti-infectious disease drugs, including:

[0349] a) Aminoglycosides: amikacin, arbekacin, gentamicin (neltamycin, sisomicin, isepamicin), hygromycin B, kanamycin (amikacin, arbekacin, aminodeoxykanamycin, dibekacin, tobramycin), neomycin (framycetin, paromomycin, ribostamycin), neltamycin, spectinomycin, streptomycin, tobramycin, methylosuomycin;

[0350] b) Amphenicols: azidamycin, chloramphenicol, florfenicol, thiamphenicol;

[0351] c) Ansamycins: geldanamycin, herbimycin;

[0352] d) Carbapenems: biapenem, doripenem, ertapenem, imipenem / cilastatin, meropenem, panipenem;

[0353] e) Cephem: carbacephem (loracabib), ceftriaxone, clomiphene, cephradine, cefdrolamine, cefuroxime, cephaloridine, cephalothin or cephalothin, cephalexin, cefuroxime, cefadroxil, cefamandole, cefpirin, hydroxylamine cephalosporin, fluazifop cephalosporin, cefotaxime, oxazolidinone, oxazolidinone cephalosporin, cefoperazone, cefcaprine, cefuroxime, cefepime, cefixime, cefoxitin, cefprozil, ceftriaxone, cefotaxime, cefotaxime, cefditoren, cephalothin, cefotaxime Ceftriaxone, cefotaxime, cefodizime, cefonicid, cefoperazone, cefoperazone, cefuroxime, cephalosporins, cefazolin, cefalexin, ceftriaxone, cefpiramide, cefpirome, cefpodoxime, cefprozil, cefquinolones, cefsulodin, ceftazidime, cefditoren, cefbutenone, ceftiolin, ceftizoxime, ceftobiprole, ceftriaxone, cefuroxime, cefazolin, cephalosporins (cefoxitin, cefotetan, cefazolin), oxocarbazone (floxacin, latamoxef);

[0354] f) Glycopeptides: bleomycin, vancomycin (oritavancin, telavancin), teicoplanin (dalbavancin), ramoplanin;

[0355] g) Glycylcyclines: such as tigecycline;

[0356] h) β-lactamase inhibitors: penicillins (sulbactam, tazobactam), oxopenicillins (clavulanic acid);

[0357] i) Lincosamides: clindamycin, lincomycin;

[0358] j) Lipopeptides: daptomycin, A54145, calcium-dependent antibiotic (CDA);

[0359] k) Macrolides: azithromycin, clotrimazole, clarithromycin, dirithromycin, erythromycin, fluramycin, josamycin, ketolides (telithromycin, cetrithromycin), midecamycin, micamycin, oleandomycin, rifamycins (isoniazid, rifampicin, rifabutin, rifapentine), ropitomycin, roxithromycin, spectinomycin, spiramycin, tacrolimus (FK506), troleandomycin, telithromycin;

[0360] l) Monocyclic amines: aztreonam, tigemonam;

[0361] m) Oxazolidinones: linezolid;

[0362] n) Penicillins: amoxicillin, ampicillin (blamox, heloxilin, bapenem, ampicillin, amoxil), azidocillin, azlocillin, benzylpenicillin, benzathine benzylpenicillin, phenoxymethylpenicillin, cloxacillin, procaine penicillin (meticiilin), mezlocillin, methicillin, nafcillin, oxacillin, propicillin, penicillin, phenoxymethylpenicillin, piperacillin, sulbenicillin, temocillin, ticarcillin;

[0363] o) Polypeptides: bacitracin, colistin, polymyxin B;

[0364] p) Quinolones: alatrofloxacin, balofloxacin, ciprofloxacin, clinafloxacin, danofloxacin, difloxacin, enoxacin, enrofloxacin, garenoxacin, gatifloxacin, gemifloxacin, grepafloxacin, kanofloxacin, levofloxacin, lomefloxacin, maiprofloxacin, moxifloxacin, nadifloxacin, norfloxacin, orbifloxacin, ofloxacin, perfloxacin, trovafloxacin, grepafloxacin, sitafloxacin, sparfloxacin, temafloxacin, tosufloxacin, trovafloxacin;

[0365] q) Streptogramins: pristinamycin, quinupristin / dalfopristin;

[0366] r) Sulfonamides: aminosulfamide, azulfidine, sulfadiazine, sulfisoxazole, sulfisoxazole, sulfamethoxazole, sulfamethoxazole (sulfamethoxazole), sulfamethoxazole (sulfamethoxazole);

[0367] s) Steroid antibacterial drugs: such as fusidic acid;

[0368] t) Tetracyclines: doxycycline, chlortetracycline, clomocycline, demeclocycline, ramocycline, meclocycline, metacycline, minocycline, terramycin, pumiliciclne, pyrrolidinyl tetracycline, tetracycline, glycylcyclines (such as tigecycline);

[0369] u) Other types of antibiotics: annocatin, arsphenamine, bacterial terpene alcohol inhibitors (bacitracin), DANAL / AR inhibitors (cyclamic acid), dictyostatin, discodermolide, eleutherobin, epothilone, ethambutol, etoposide, faropenem, fusidic acid, furazolium, isoniazid, laulimalide, metronidazole, mupirocin, NAM synthesis inhibitors (e.g. fosfomycin), allopurinol, paclitaxel, plinabulin, pyrazinamide, quinupristin / dalfopristin, rifampicin, sulbactam metronidazole, ulithiol;

[0370] (4) Antiviral drugs, including:

[0371] a) Invasion / fusion inhibitors: apricitabine, maraviroc, vicriviroc, gp41 (enfuvirtide), PRO 140, CD4 (ibalizumab);

[0372] b) Integrase inhibitors: raltegravir, elvite-gravir, globoidnan A;

[0373] c) Maturation inhibitors: bevirimat, vivecon;

[0374] d) Neuraminidase inhibitors: oseltamivir, zanamivir, peramivir;

[0375] e) Nucleosides and nucleotides: abacavir, aciclovir, adefovir, amdoxovir, aciclovir, brivudine, cidofovir, clafvirine, dexamethasone, didanosine (ddl), elvucitabine, emtricitabine (FTC), entecavir, famciclovir, flurazolidone (5-FU), 3'-fluoro-substituted 2',3'-dideoxynucleoside analogs such as 3'-fluoro-2',3'- dideoxythymidine (FLT) and 3'-fluoro-2',3'-dideoxyguanosine (FLG), fomivirsen, 9- guanine, iododeoxyuridine, lamivudine (3TC), 1-nucleosides (e.g. beta-1-thymidine and beta-1-2'-deoxycytidine), penciclovir, racivir, ribavirin, diethylthiambutene, stavudine (d4T), taribavirin (viramidine), telbivudine, tenofovir, trifluridine valaciclovir, valganciclovir, zalcitabine (ddC), zidovudine (AZT);

[0376] f) Non-nucleosides: amantadine, atiprimine, capravirine, diarylpyrimidines (etravirine, rilpivirine), delavirdine, docosanol, emivirine, efavirenz, foscarnet (phosphonoformic acid), imiquimod, pegylated interferon, lovirimide, lodenosine, meglumine antimonate, nevirapine, NOV-205, long-acting interferon alpha, podophyllotoxin, rifampicin, rimantadine, resiquimod (R-848), meglumine amantadine;

[0377] g) Protease inhibitors: amprenavir, atazanavir, boceprevir, darunavir, fosamprenavir, indinavir, lopinavir, nelfinavir, pleconaril, ritonavir, saquinavir, telaprevir (VX-950), tipranavir;

[0378] h) Other types of antiviral drugs: abzyme, arbidol, calanolide a, ceragenin, cypermethrin-n, diarylpyrimidine, epigallocatechin gallate (EGCG), foscarnet, griffithsin, taribavirin (viramidine), hydroxyurea, KP-1461, miltefosine, pleconaril, mixed inhibitors, ribavirin, seliciclib.

[0379] (5) A radioactive isotope that can be obtained from (radioactive nuclide) 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 213 Select from the group consisting of Bi.

[0380] (6) a chromophore molecule that can absorb a light, such as ultraviolet light, fluorescent light, infrared light, near infrared light, or visible light; a chromophore molecule including one or a subset of a group of yellow pigments, red pigments, iridescent pigments, white pigments, black pigments, and blue-green pigments, one or a subset of a group of fluorescent molecules (fluorescent chemicals that emit light after absorbing light), one or a subset of a group of visual light transduction molecules, one or a subset of a group of photon molecules, one or a subset of a group of luminescent molecules, and one or a subset of a group of fluorescein compounds. Non-protein organic fluorophores such as xanthene derivatives (fluorescein, rhodamine, Oregon green, eosin, and Texas red); cyanine derivatives (cyanine, indocyanine, oxonol, thiacarbocyanine, and merocyanine); squaraine derivatives and ring-substituted squaraines including Seta, SeTau, and Square dyes; naphthalene derivatives (naphthalene and fluoroscein derivatives); coumarin derivatives; oxadiazole derivatives (pyridyl oxadiazole, nitrobenzoxadiazole, and benzoxadiazole); anthracene derivatives (anthraquinones including DRAQ5, DRAQ7, and CyTRAK Orange); pyrene derivatives (Cascade Blue, etc.); oxazine derivatives (Nile red, Nile blue, cresyl violet, oxazine 170, etc.); acridine derivatives (lutein, acridine orange, acridine yellow, etc.); arylmethylamine derivatives (malachite green, crystal violet, malachite green); and tetrapyrrole derivatives (porphyrin, phthalocyanine, bilirubin). Any analogs and derivatives of the following fluorescent compounds: CF dyes (Biotium), DRAQ and CyTRAK probes (BioStatus), BODIPY (Invitrogen), Alexa Fluor (Invitrogen), DyLight Fluor (Thermo Scientific, Pierce), Atto and Tracy (SigmaAldrich), FluoProbes (Interchim), Abberior dyes (Abberior), DY and MegaStokes dyes (Dyomics), Sulfo Cy dyes (Cyandye), HiLyte Fluor (AnaSpec), Seta, SeTau, and Square dyes (Biosearch Technologies), SureLight dyes (APC, RPE PerCP, Phycobilisomes) (Columbia Biosciences), APC, APCXL, RPE, BPE (Phyco-Biotech), Allophycocyanin (APC), Aminomalcite, APC-Cy7 conjugates, BODIPY-FL, Cascade Blue, Cy2, Cy3, Cy3.5, Cy3B, Cy5, Cy5.5, Cy7, Fluorescein, FluorX, Hydroxycoumarin, Lissamine Rhodamine B, Lucifer Yellow, Me-Methoxycoumarin, NBD, Pacific Blue, Pacific Orange, PE-Cy5 conjugate, PE-R-Phycoerythrin (PE), Red 613, Seta-555-Azide, Seta-555-DBCO, Seta-555-NHS, Seta-580-NHS, Seta-680-NHS, Seta-APC-780, Seta-PerCP-680, Seta-R-PE-670, SeTau-380-NHS, SeTau-405-Maleimide, SeTau-405-NHS, SeTau-425-NHS, SeTau-647-NHS, Texas Red, TRITC, TruRed, X-Rhodamine, 7-AAD (7-Aminoactinomycin D, CG-selective), Acridine Orange, Chomomycin A3, CyTRAK Orange (Biostatus), DAPI, DRAQ5, DRAQ7, Ethidium Bromide, Hoechst 33258, Hoechst 33342, LDS 751, Mithramycin, Propidium Iodide (PI), SYTOX Blue, SYTOX Green, SYTOX Orange, Thiazole Orange, TO-PRO, Cyanine monomer, TOTO-1, TO-PRO-1, TOTO-3, TO-PRO-3, YOSeta-1, YOYO-1. Fluorescent compounds that can be linked to the linker of the present application for studying cells are selected from the following compounds or derivatives thereof: DCFH (2',7'-Dichlorodihydrofluorescein, oxidized form), DHR (Dihydro-Rhodamine 123, oxidized form, photo catalytic oxidation), Fluo-3 (AM ester, pH > 6), Fluo-4 (AM ester, pH 7.2), Indo-1 (AM ester, low / high calcium (Ca 2+)), SNARF (pH 6 / 9). Preferred fluorescent compounds are selected from the group consisting of Allophycocyanin (APC), AmCyanl (tetramer, Clontech), AsRed2 (tetramer, Clontech), B-phycoerythrin (BPE), CyPet, DsRed monomer (Clontech), DsRed2 ("RFP", Clontech), EBFP, EBFP2, ECFP, EGFP (weak dimer, Clontech), Emerald (weak dimer, Invitrogen), EYFP (weak dimer, Clontech), GFP (S65A mutation), GFP (S65C mutation), GFP (S65L mutation), GFP (Y66H mutation), GFP (Y66W mutation), GFPuv, HcRedl, J-Red, Katusha, Kusabira Orange (monomer, MBL), mCFP, mCherry (monomer, MBL), mKate (TagFP635, monomer, Evrogen), mKeima-Red (monomer, MBL), mKO, mOrange, mPlum, mRaspberry, mRFP1 (monomer, Tsien lab), mStrawberry, mTFPl, mTurquoise2, P3 (phycobilisome complex), Peridinin-chlorophyll-protein complex (PerCP), R-phycoerythrin (RPE), T-Sapphire, TagCFP (dimer, Evrogen), TagGFP (dimer, Evrogen), TagRFP (dimer, Evrogen), TagYFP (dimer, Evrogen), tdTomato (tandem dimer), Topaz, TurboFP602 (dimer, Evrogen), TurboFPP635 (dimer, Evrogen), TurbogFP (dimer, Evrogen), TurboRFP (dimer, Evrogen), TurboYFP (dimer, Evrogen), Venus, wild-type GFP type, YPet, Zsgreenl (tetramer, Clontech), ZsYellowl (tetramer, Clontech).

[0381] (7) a cell binding ligand or receptor agonist selected from the group consisting of a folate derivative, a glutamate urea derivative, a somatostatin and analogs thereof (selected from octreotide (Sandostatin) and lanreotide (Somatuline)), an arylsulfonamide, pituitary adenylate cyclase activating peptide (PACAP) (PAC1), vasoactive intestinal peptide (VIP / PACAP) (VPAC1, VPAC2), melanocyte-stimulating hormone (a-MSH), cholecystokinin (CCK) / gastrin receptor agonists, bombesin (selected from Pyr-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2) / gastrin releasing peptide (GRP). neurotensin receptor ligands (NTR1, NTR2, NTR3); substance P (NK1 receptor) ligands; neuropeptide Y (Y1-Y6); homing peptides including RGD (Arg-Gly-Asp), NGR (Asn-Gly-Arg), dimeric and multimeric cyclic RGD peptides (selected from cRGDfV), TAASGVRSMH and LTLRWVGLMS (chondroitin sulfate proteoglycan NG2 receptor ligands) and F3 peptides; cell penetrating peptides (CPPs);Peptide hormones are selected from the group consisting of luteinizing hormone releasing hormone (LHRH) agonists and antagonists, and gonadotropin releasing hormone (GnRH) agonists, acting by targeting follicle stimulating hormone (FSH) luteinizing hormone (LH), and testosterone production, such as buserelin (Pyr-His-Trp-Ser-Tyr-D-Ser(OtBu)-Leu-Arg-Pro-NHEt), goserelin (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), nafarelin (Pyr-His-Trp-Ser-Tyr-2Nal-Leu-Arg-Pro-Gly-NH2), triptorelin (Pyr-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH2), nafarelin, delolorin, abarelix (Ac-D-2Nal-D-4-chloroPhe-D-3-(3-pyridyl)Ala-Ser-(N-Me)Tyr-D-Asn-Leu-iso-propylLys-Pro-DAla-NH2), cetrorelix (Ac-D-2Nal-D-4-chloroPhe-D-3-(3-pyridyl)Ala-Ser-Tyr-D-Cit-Leu-Arg-Pro-D-Ala-NH2), degarelix (Ac-D-2Nal-D-4-chloroPhe-D-3-(3-pyridyl)Ala-Ser-4-aminoPhe(L-hydroorotyl)-D-4-aminoPhe(carba-moyl)-Leu-iso-propylLys-Pro-D-Ala-NH2), and degarelix (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); pattern recognition receptors (PRRs) selected from the group consisting of Toll-like receptor (TLRs) ligands, C-type lectins and nod-like receptors (NLRs) ligands; calcitonin receptor agonists; integrin receptors and their receptor subtypes (selected from the group consisting of α; V β1, αV β3, α V β5, α V β6, α6 β4, α7 β1, α L β2, α IIb β3) agonists (selected from the group consisting of GRGDSPK, cyclo(RGDfV)(L1) and derivatives thereof [cyclo(-N(Me)R-GDfV), cyclo(R-Sar-DfV), cyclo(RG-N(Me)D-fV), cyclo(RGD-N(Me)f-V), cyclo(RGDf-N(Me)V-)(cilengitide)]; single domain antibodies (derivatives of VHH (camelid Ig)); domain antibodies (dAbs, derivatives of VH or VL domains); bispecific T cell engagers (BiTEs, bispecific dimers); dual affinity retargeting (DARTs, bispecific dimers); tetravalent tandem antibodies (TandAbs, a dimerized bispecific dimer); Anticalins (derivatives of calcin); Adnectins (FN3 (fibronectin) number 10); designed ankyrin repeat proteins (DARPins); Avimers; EGF receptor and VEGF receptor agonists; a short antibody-like protein, siRNA or DNA molecule for immunotherapy.

[0382] (8) pharmaceutically acceptable salts, acids, derivatives, hydrates or hydrated salts; or crystal structures; or optical isomers, racemates, diastereomers or enantiomers of any of the foregoing drugs.

[0383] In another embodiment, the drug can be a polyalkylene glycol, which, when administered to a mammal, is used to extend the half-life of a cell binding molecule antibody, or antibody molecule. Polyalkylene glycols include, but are not limited to, polyethylene glycol (PEG), polypropylene glycol, and copolymers of ethylene oxide and propylene oxide; PEG is preferred, and monofunctionally activated hydroxyl PEG (e.g., single terminal activated hydroxyl PEG, including hydroxyl PEG- active ester, hydroxyl PEG-monocarboxaldehyde, hydroxyl PEG-monocarboxamide, hydroxyl PEG-monohydrazide, hydroxyl PEG-monohydrazine carboxylate, hydroxyl PEG- monoiodoacetamide, hydroxyl PEG-monomaleimide, hydroxyl PEG-o-pyridyl disulfide, hydroxyl PEG-monooxime, hydroxyl PEG-monophenyl carbonate, hydroxyl PEG- monophenyl glyoxal, hydroxyl PEG-monothiazolidine-2-thione, hydroxyl PEG- monothioester, hydroxyl PEG-monothiol, hydroxyl PEG-monotriazine, and hydroxyl PEG-monovinyl sulfone) is more preferred.

[0384] In certain embodiments, the polyalkylene glycol has a molecular weight of about 10 Da to about 200 kDa, preferably about 88 Da to about 40 kDa; has two branches, each branch having a molecular weight of about 88 Da to about 40 kDa; more preferably having two branches, each branch of about 88 Da to about 20 kDa. In a particular embodiment, the polyalkylene glycol is a polyethylene glycol having a molecular weight of about 10 kDa, 20 kDa, or 40 kDa. In particular embodiments, the PEG is PEG 10 kDa (linear or branched), PEG 20 kDa (linear or branched), or PEG 40 kDa (linear or branched). The preparation of linear or branched "non-antigenic" PEG polymers and derivatives or conjugates thereof are disclosed in U.S. Patents 5,428,128; 5,621,039; 5,622,986; 5,643,575; 5,728,560; 5,730,990; 5,738,846; 5,811,076; 5,824,701; 5,840,900; 5,880,131; 5,900,402; 5,902,588; 5,919,455; 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.

[0385] In another embodiment, D is more preferably an effective cytotoxic agent selected from the group consisting of tubulysins and analogs thereof, maytansine and analogs thereof, taxanes and analogs thereof, CC-1065 and analogs thereof, daunorubicin or doxorubicin and their analogs, amatoxins and analogs thereof, benzodiazepine dimers (e.g., dimers of pyrrolobenzodiazepine (PBD), tomaymycin, anthramycin, indolinobenzodiazepine, imidazobenzothiadiazepine, or oxazolidinobenzodiazepine) and analogs thereof, calicheamicin and enediyne antibiotic analogs, dactinomycin and analogs thereof, zinostatin and analogs thereof, bleomycin and analogs thereof, epirubicin and analogs thereof, tamoxifen and analogs thereof, idarubicin and analogs thereof, dolastatins and analogs thereof, auristatins (including monomethyl auristatin (MMAE), MMAF, auristatin PYE, auristatin TP, auristatin 2-AQ, 6-AQ, EB(AEB), and EFP(AEFP)) and analogs thereof, combretastatin, duocarmycin and analogs thereof, camptothecin, geldanamycin and analogs thereof, methotrexate and analogs thereof, thiotepa and analogs thereof, vindesine and analogs thereof, vincristine and analogs thereof, hemiasterlins and analogs thereof, nazumamide and analogs thereof, spliceostatin, a pladienolide, a crystallin and analogs thereof, radiosensitizers and analogs thereof, alterobactin and analogs thereof, a microsclerodermin and analogs thereof, theonellamide and analogs thereof, esperamicin and analogs thereof, PNU-159682 and analogs thereof, a protein kinase inhibitor, a MEK inhibitor, a KSP inhibitor, a nicotinamide phosphoribosyltransferase (NAMPT) inhibitor, an immunotoxin, a cellular receptor agonist, a cellular stimulatory molecule or intracellular signaling molecule, one, two or more of a DNA, RNA, mRNA, small interfering RNA (siRNA), microRNA (miRNA), and PIWI-interacting RNA (piRNA), and stereoisomers, isosteres, analogs, or derivatives thereof.

[0386] Tubulysins and analogs thereof are well known to those skilled in the art and can be isolated from natural sources according to known methods or prepared synthetically according to known methods (e.g. Balasubramanian R., et al., J. Med. Chem., 2009, 52, 238-40; Wipf P., et al., Org. Lett., 2004, 6, 4057-60; Pando O., et al., J. Am. Chem. Soc, 2011, 133, 7692-5; Reddy, J.A., et al., Mol. Pharmaceutics, 2009, 6, 1518-25; Raghavan B., et al., J. Med. Chem., 2008, 51, 1530-33; Patterson A.W., 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; Friestad, G.K., Org. Lett., 2004, 6, 3249-52; 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., Canadian patent application CA 2710693 (2011); Chai Y., et al., European patent application 2174947 (2010), WO 2010034724; Leamon, C. et al., WO 2010033733, WO 2009002993; Ellman, J., et al., PCT WO2009134279; WO 2009012958, US Patent Applications 20110263650, 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., US Patent Application 20070041901, WO2006096754; Matschiner G., et al., WO2006056464; Vaghefi F., et al., WO2006033913; Doemling A., German Patent Application DE102004030227, WO2004005327, WO2004005326, WO2004005269; Stanton M., et al., US Patent Application 20040249130; Hoefle G., et al., German Patent Applications DE10254439, DE10241152, DE10008089; Leung D., et al., WO2002077036; Reichenbach H., et al., German Patent Application DE19638870; Wolfgang R., US20120129779; Chen H., US Patent Application 20110027274. Preferred structures of tubulysins that can be conjugated to the cell binding molecules of the application are described in patent PCT / IB2012 / 053554.

[0387] Tubulysin analogs have the structure of formula (IV):

[0388]

[0389] or a pharmaceutically acceptable salt, hydrate or hydrated salt; or polymorphic structure; or optical isomer, racemate, diastereomer or enantiomer thereof;

[0390] wherein is one or two attachment sites independently attached to L1and / or L2; when two are simultaneously attached to L1and L2, R 1 and R 2 , or Z 2 and Z 3 are preferably double attachment sites;

[0391] wherein R 1 , R 2 , R 3 , and R 4 are independently H, C1-C8 alkyl, C2-C8 heteroalkyl or heterocycle, C3-C8 aryl, arylalkyl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, carbocyclic, or alkylcarbonyl; or R 1 R 2 , R 1 R 3 , R 2 R 3 , R 3 R 4 , R 5 R 6 , R 11 R 12 or R 13 R 14 form a 3-7 membered carbocyclic, cycloalkyl, heterocyclic, heterocycloalkyl, aryl, or heteroaryl ring system; R 1 and R 2 may independently be null when attached independently or simultaneously to L1or L2, Y 1 is N or CH;

[0392] wherein R 5 , R 6 , R 8 , R 10 and R 11 are independently H, or C1-C4 alkyl or heteroalkyl;

[0393] wherein R 7 is independently H, R 14 , -R 14 C(=O)X 1 R 15 ; or -R 14 X 1 R 15 ; X 1 is O, S, S-S, NH, CH2, or NR 14 ;

[0394] wherein R 9 is selected from H, OH, =O, -OR 14 , -OC(=O)R 14 , -OC(=O)NHR 14 , -OC(=O)NR 14 R 15 , OP(=O)(OR 14 )2, -OC(=O)NR 14 R 15 , or OR 14OP(=O)(OR 15 )2; when R 9 is attached to L1or L2, R 9 is -O-, -OC(=O)NH-, or -OC(=O)N(R 14 )-;

[0395] wherein R 11 is independently H, R 14 , -R 14 C(=O)R 15 , -R 14 C(=O)X 2 R 15 , wherein X 2 is -O-, -S-, -NH-, or -N(R 14 )-;

[0396] wherein R 12 is -COOH, -COSH, -CONH2, CONHNH2, CONHNHR 15 , -CONH(R 15 ), -COOR 15 , -R 15 COR 16 , -R 15 COOR 16 , -R 15 C(O)NH2, -R 15 C(O)NHR 16 , -COSR 15 , R 15 S(=O)2R 16 , -R 15 P(=O)(OR 17 )2, -R 15 OP(=O)(OR 17 )2, -COOCH2OP(=O)(OR 17 )2, -COX 2 SO2R 17 , -COOR 15 X 2 R 16 , tetrazole, imidazole, or triazole, X 2 is -O-, -S-, -NH-, -N(R 15 )-, -O-R 15 -, -S-R 15 -, CH2, or -NHR 15 -; when R 12 is attached to L1or L2, R 12 is -C(O)O-, -C(O)NH-, -C(=O)NHS(O)2R15 - or -C(=O)N(R 15 )-;

[0397] R 13 and R 14 are independently C1-C8alkyl, heteroalkyl; C2-C8alkenyl, alkynyl, heteroalkyl, heterocycloalkyl; C3-C8aryl, arylalkyl;

[0398] Z 2 and Z 3 are independently H, O, S, NH, N(R 15 ), NHNH, -OH, -SH, -NH2, NH, NHNH2, -NH(R 15 ), -OR 15 , CO, -COX 2 , -COX 2 R 16 , R 17 , F, Cl, Br, I, SR 16 , NR 16 R 17 , N=NR 16 , N=R 16 , NO2, SOR 16 R 17 , SO2R 16 , SO3R 16 , OSO3R 16 , PR 16 R 17 , POR 16 R 17 , PO2R 16 R 17 , OP(O)(OR 17 )2, OCH2OP(O)(OR 17 )2, OC(O)R 17 , OC(O)OP(O)(OR 17 )2, PO(OR 16 )(OR 17 ), OP(O)(OR 17 )OP(O)(OR 17 )2, OC(O)NHR 17 ; -O-(C4-C 12 sugar), -N-(C4-C 12 sugar); C1-C8alkyl, heteroalkyl; C2-C8alkenyl, alkynyl, heteroalkyl, heterocycloalkyl; C3-C8aryl, arylalkyl, carbocyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl, or an ester, ether or amide of 2-8 carbon atoms; or a peptide comprising 1-8 amino acids (NH(Aa)1～8 or CO(Aa) 1～8 , N-terminal or C-terminal 1-8 identical or different amino acids), or having a polyoxyethylene group of the formula (OCH2CH2) p or (OCH2CH(CH3)) p wherein p is an integer from 0 to about 1000, or a combination of the preceding groups; X 2 is O, S, S-S, NH, CH2, OH, SH, NH2, CHR 15 or NR 15 ;

[0399] R 15 , R 16 and R 17 are independently H, C1-C8 alkyl, heteroalkyl; C2-C8 alkenyl, alkynyl, heteroalkyl, heterocycloalkyl; C3-C8 aryl, arylalkyl, carbocyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl, alkylcarbonyl, or Na + , K + , Cs + , Li + , Ca 2+ , Mg + , Zn 2+ , N + (R 1 )(R 2 )(R 3 )(R 4 ), HN + (C2H5OH)3 salt;

[0400] Y 1 and Y 2 are independently N or CH; q is 0 or 1; when q = 0, Y 3 is defaulted, Y 4 , Y 5 , Y 6 and Y 7 are independently CH, N, NH, O, S, or N(R1), so Y 2 , Y 4 , Y 5 , Y 6 and Y 7 form a furan, pyrrole thiophene, thiazole, oxazole and imidazole, pyrazole, triazole, tetrazole, thiadiazole heteroaromatic ring; when q = 1, Y 3 , Y 4 , Y 5 , Y 6 and Y 7 are independently CH or N, Y 2 , Y 3 , Y 4 , Y5 , Y 6 and Y 7 form a benzene, pyridine, pyridazine, pyrimidine, pyrazine, triazine, tetrazine, pentazine aromatic ring;

[0401] Structural examples of tubulysin analogs are shown below:

[0402]

[0403]

[0404]

[0405]

[0406]

[0407]

[0408]

[0409]

[0410]

[0411]

[0412]

[0413]

[0414]

[0415] wherein R 20 is H; C1-C8 straight or branched chain alkyl or heteroalkyl, C2-C8 straight or branched chain alkenyl, alkynyl, alkylcycloalkyl, heterocycloalkyl, C3-C8 aryl, aralkyl, heterocyclyl, carbocyclyl, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl straight or branched chain; carbonate (-C(O)OR 17 ), carbamate (-C(O)NR 17 R 18 ); or carboxylate, ester, ether or amide of 1-8 carbons; or 1-8 amino acids; or polyethyleneoxy units of the formula (OCH2CH2) p or (OCH2CH(CH3)) p wherein p is an integer from 0 to about 1000; or R 20 default oxygen to carbon forms a ketone, or combinations of the foregoing;

[0416] Z 3and Z 3 independently H, OH, NH2, O, NH, COOH, COO, C(O), C(O), C(O)NH, C(O)NH2, R 18 , OCH2OP(O)(OR 18 )2, OC(O)OP(O)(OR 18 )2, OPO(OR 18 )2, NHPO(OR 18 )2, OP(O)(OR 18 )OP(O)(OR 18 )2, OC(O)R 18 , OC(O)NHR 18 , OSO2(OR 18 ), O-(C4-C 12 -glycoside), straight or branched chain alkyl or heteroalkyl; C2-C8straight or branched chain alkenyl, alkynyl, alkylcycloalkyl, heterocycloalkyl; C3-C8straight or branched chain aryl, aralkyl, heterocyclyl, carbocyclyl, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; carbonate (-C(O)OR 17 ), carbamate (-C(O)NR 17 R 18 ); R 17 and R 18 are independently H, straight or branched chain alkyl or heteroalkyl; C2-C8straight or branched chain alkenyl, alkynyl, alkylcycloalkyl, heterocycloalkyl; C3-C8straight or branched chain aryl, alkyl, heterocyclo, carbocyclo, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; carbonate (-C(O)OR 17 ), carbamate (-C(O)NR 17 R 18 );

[0417] R 19 is H, OH, NH2, OSO2(OR 18 ), XCH2OP(O)(OR 18 )2, XPO(OR 18 )2, XC(O)OP(O)(OR 18 )2, XC(O)R 18 , XC(O)NHR 18 , C1-C8alkyl or carboxylate; C2-C8alkenyl, alkynyl, alkylcycloalkyl, heterocycloalkyl; C3-C8aryl or alkylcarbonyl; or a pharmaceutically acceptable salt;

[0418] X is O, S, NH, NHNH, or CH2;

[0419] R 7 is as defined above; wherein the point of attachment In Formula IV-01-IV-79, the same as shown in Formula (IV).

[0420] Calicheamicin and its related enediyne antibiotics are described in: Nicolaou K.C. et al., Science 1992, 256, 1172-1178; Proc. Natl. Acad. Sci USA. 1993, 90, 5881-8; U.S. Patents 4970198; 5053394; 5108912; 5264586; 5384412; 5606040; 5712374; 5714586; 5739116; 5770701; 5770710; 5773001; 5877296; 6015562; 6124310; 8153768. Exemplary enediyne include, but are not limited to, calicheamicin, esperamicin, uncialamicin, dynemicin, and their derivatives. The structure of calicheamicin is preferably the following formula:

[0421]

[0422] or an elemental isotope substitute, or a pharmaceutically acceptable salt, hydrate or hydrated salt; or a polymorphic structure; or an optical isomer, racemate, diastereomer or enantiomer thereof;

[0423] wherein is the site of attachment to L1or L2;

[0424] Geldanamycin is a benzoquinone ansamycin antibiotic that binds to Hsp90 (heat shock protein 90) and has been used as an anti-tumor drug. Exemplary geldanamycins include, but are not limited to, 17-AAG (17-N-allylamino-17-demethoxygeldanamycin) and 17-DMAG (17-dimethylaminoethylamino-17-demethoxygeldanamycin), having the following molecular formula:

[0425]

[0426] wherein is the site of attachment to L1or L2;

[0427] Maytansine or its derivatives, maytansinoids, inhibit cell proliferation by inhibiting the polymerization of tubulin, thereby inhibiting microtubule formation during mitosis. See Remillard et al., Science 189: 1002-1005 (1975). Exemplary maytansines and maytansinoids include, but are not limited to, mertansines (DM1, DM4), maytansine and its derivatives, and ansamitocins. Maytansines are described in U.S. Patents 4,256,746; 4,361,650; 4,307,016; 4,294,757; 4,294,757; 4,371,533; 4,424,219; 4,331,598; 4,450,254; 4,364,866; 4,313,946; 4,315,929; 4,362,663; 4,322,348; 4,371,533; 4,424,219; 5,208,020; 5,416,064; 5,208,020; 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 maytansine is preferably as follows:

[0428]

[0429] wherein is the site of attachment to L1or L2;

[0430] Camptothecins (CPTs) and their derivatives are topoisomerase inhibitors that prevent DNA re-ligation, thus causing DNA damage and leading to apoptosis, described in Shang, X.F., 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), gimatecan (BNP-1350), Etirinotecan, Exatecan, Lurtotecan, Gimatecan (ST1481), Belotecan (CKD-602), Rubitecan (Rubitcan) and others (Shang, X.F., et al. Med Res Rev. 2018, 38(3):775-828). So far, three CPT analogs, topotecan, irinotecan and belotecan, have been approved for cancer chemotherapy (Palakurthi, S., Expert Opin Drug Deliv. 2015; 12(12): 1911-21; Shang, X.F., et al. Med Res Rev. 2018, 38(3):775-828), SN-38 and Exatecan are also used in clinical trials for ADC conjugates' payloads (Ocean, A.J., Cancer. 2017, 123(19):3843-3854; Starodub, A.N., et al. Clin Cancer Res. 2015, 21(17):3870-8; Cardillo, T.M., 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 7591994; 7999083; 8080250; 8268317; U.S. Patent Applications 20130090458, 20140099258, 20150297748, 20160279259).

[0431] The structure of camptothecin (CPT) is shown below:

[0432]

[0433] or one or more elemental isotope substitutions, or a pharmaceutically acceptable salt, hydrate or hydrated salt; or a polymorphic structure; or an optical isomer, racemate, diastereomer or enantiomer thereof; wherein R1, R2and R4are independently selected from H, F, Cl, Br, CN, NO2, C1-C8alkyl; O-C1-C8alkyl, NH-C1-C8alkyl; C2-C8heteroalkyl, alkylcycloalkyl, heterocycloalkyl; C3-C8aryl, arylalkyl, heterocycle, carbocycle, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; C2-C8esters, ethers, amides, carbonates, ureas or carbamates; R3is H, OH, NH2, C1-C8alkyl, O-C1-C8alkyl; NH-C1-C8alkyl; C2-C8heteroalkyl, alkylcycloalkyl, heterocycloalkyl; C2-C8esters, ethers, amides, carbonates, ureas or carbamates; or R1R2, R2R3and R3R4independently form a 5-7 membered carbocyclic, heterocyclic, heterocycloalkyl, aryl or heteroaryl ring system. Wherein is the site in the molecule that is attached to L1or L2.

[0434] Camptothecin is preferably selected from the structures:

[0435] SN-38,

[0436]

[0437] Topotecan analogs,

[0438] Irinotecan analogs,

[0439] Irinotecan analogs,

[0440] Siliretecans,

[0441] Cositecans,

[0442] Exitecan,

[0443] Lurtotecan,

[0444]

[0445] GI-149893 analogs,

[0446] Gimatecan analogs, Belotecan analogs, Rubitecan or IDEC-132 analogs, BN-80927 analogs, BN-80927 analogs,

[0447] or one or more elemental isotope substitutions, or a pharmaceutically acceptable salt, hydrate or hydrated salt; or polymorphic structures of these compounds; or optical isomers, racemates, diastereomers or enantiomers; wherein is the site of attachment to L1or L2; 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, O-(C1-C8branched or straight chain alkyl), C1-C8straight or branched chain alkyl or heteroalkyl, C2-C8straight or branched chain alkenyl, alkynyl, alkylcycloalkyl, heterocycloalkyl; C3-C8straight or branched chain aryl, arylalkyl, heterocycle, carbocycle, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl, carbonate (-C(O)OR 17 ), carbamate (-C(O)NR 17 R 18 ); R 17 and R 18independently H, straight or branched chain alkyl or heteroalkyl; C2-C8straight or branched chain alkenyl, alkynyl, alkylcycloalkyl, heterocycloalkyl; C3-C8straight or branched chain aryl, arylalkyl, heterocycle, carbocycle, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl, carbonate (-C(O)OR 17 ), carbamate (-C(O)NR 17 R 18 ); X is NH, O, S or CH2.

[0448] Combretastatins are natural phenols with a vascular disrupting effect in tumors. Exemplary combretastatins and derivatives thereof include, but are not limited to, combretastatin A-4 (CA-4), CA4-βGals, CA-4PD, CA4-NPs, and ombrabulin, having the following molecular formula:

[0449]

[0450] Taxanes, including the cytotoxic natural product paclitaxel (Taxol) and the semi-synthetic derivative docetaxel (Taxotere), and their analogs, are preferred for conjugation and can be referenced in the following: 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); U.S. Patents 5475011; 5728849; 5811452; 6340701; 6372738; 6391913; 6436931; 6589979; 6596757; 6706708; 7008942; 7186851; 7217819; 7276499; 7598290; 7667054. Taxanes preferably have the following structure:

[0451]

[0452] wherein is the site of attachment to L1or L2; Ar and Ar' are independently aryl or heteroaryl.

[0453] Anthrocyclics are mammalian DNA topoisomerase II inhibitors that stabilize the DNA-enzyme complex at the site of DNA strand cleavage and covalent linkage with the antibody. These anticancer agents have played an important role in the treatment of various forms of solid tumors and acute leukemias over the past several decades. However, anthrocyclics have been associated with morbidity and mortality from cardiovascular disease (Sagi, J.C., et al., Pharmacogenomics. 2016, 17(9), 1075-87; McGowan, J.V., et al., Cardiovasc Drugs Ther. 2017, 31(1), 63-75). Therefore, to enhance the specific activity of these molecules while reducing cardiotoxicity, researchers have conjugated anthrocyclics to cell-binding antibodies, or antibody-like molecules, 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). Exemplary anthrocyclics include, but are not limited to, daunorubicin, doxorubicin (i.e., adriamycin), epirubicin, idarubicin, valrubicin, and mitoxantrone. The structure of the anthrocyclics in this application are preferably selected from the following formulas:

[0454] Daunorubicin analogs,

[0455] Daunorubicin analogs,

[0456] Doxorubicin analogs,

[0457] Epirubicin analogs,

[0458] Idarubicin analogs, Mitoxantrone analogs, Pixantrone analogs, Losoxantrone analogs,

[0459] Amrubicin analogs,

[0460] wherein is the site of attachment to L1or L2.

[0461] Vinca alkaloids are a class of anti-mitotic and anti-microtubule alkaloids that act by inhibiting cancer cell division. Vinca alkaloids include vinblastine, vincristine, vindesine, anhydrovinblastine, vinorelbine, leurosidine, catharanthine, destomyrine, vindesine sulfate, vinleurosine, minovincine, methoxymino-vincine, catharicine, destomyrine, leurosidine, vindesine, vinburnine, vinerpine, vincamayine. Vinca alkaloids are preferably vinblastine, vincristine, having the following structural formula:

[0462] Vincristine,

[0463] Vincristine,

[0464] Vinblastine,

[0465] Vinblastine;

[0466] Rifabutin analogs,

[0467] Rifabutin analogs,

[0468] wherein is attached to the L1 or L2 site;

[0469] Dolastatins and their peptide analogs and derivatives, auristatins, are highly potent antimitotic agents that have been shown to have anticancer and antifungal activity. See, e.g., U.S. Patent No. 5,663,149 and Pettit et al., Antimicrob. Agents Chemother. 42:2961-2965, 1998. Exemplary dolastatins and auristatins include, but are not limited to, dolastatin 10, auristatin E (AE), auristatin EB (AEB), auristatin EFP (AEFP), MMAD (monomethyl auristatin D or monomethyl dolastatin 10), MMAF (monomethyl auristatin F or N-methylvaline-valine-dolaisoleuine-dolaproine-phenylalanine), MMAE (monomethyl auristatin E or N-methylvaline-valine-dolaisoleuine-dolaproine-norephedrine), 5-benzoylvaleric acid-AE ester (AEVB), auristatin F phenyldiamine (AFP), and other novel auristatin compounds.Auristatins are described in Int. J. Oncol. 15: 367-72 (1999); Molecular Cancer Therapeutics, vol. 3, No. 8, pp. 921-32 (2004); U.S. Patent Applications 11 / 134826, 20060074008, 2006022925, U.S. Patents 4414205, 4753894, 4764368, 4816444, 4879278, 4943628, 4978744, 5122368, 5165923, 5169774, 5286637, 5410024, 5521284, 5530097, 5554725, 5585089, 5599902, 5629197, 5635483, 5654399, 5663149, 5665860, 5708146, 5714586, 5741892, 5767236, 5767237, 5780588, 5821337, 5840699, 5965537, 6004934, 6033876, 6034065, 6048720, 6054297, 6054561, 6124431, 6143721, 6162930, 6214345, 6239104, 6323315, 6342219, 6342221, 6407213, 6569834, 6620911, 6639055, 6884869, 6913748, 7090843, 7091186, 7097840, 7098305, 7098308, 7498298, 7375078, 7462352, 7553816, 7659241, 7662387, 7745394, 7754681, 7829531, 7837980, 7837995, 7902338, 7964566, 7964567, 7851437, 7994135. The structure of the auristatin is preferably selected from the following structural formulas (Ih-01), (Ih-02), (Ih-03), (Ih-04), (Ih-05), (Ih-06), (Ih-07), (Ih-08), (Ih-09), (Ih-10), and (Ih-11):

[0470]

[0471]

[0472]

[0473] or one or more isotopic substitutions of elements, or pharmaceutically acceptable salts, hydrates or hydrated salts; or polymorphic structures of these compounds; or optical isomers, racemates, diastereomers or enantiomers; wherein R 1 , R 2 , R 3 , R 4 and R 5 are independently H; C1-C8 straight chain or branched alkyl, aryl, heteroaryl, heteroalkyl, alkylcycloalkyl, ester, ether, amide, amine, heterocycloalkyl or acyloxyamine; or a peptide comprising 1-8 amino acids, or a polyoxyethylene group having the formula (OCH2CH2) p or (OCH2CH(CH3)) p wherein p is an integer from 1 to about 1000. Two R 1 R 2 , R 2 R 3 , R 1 R 3 or R 3 R 4 may form a 3-8 membered cyclic ring of alkyl, aryl, heteroaryl, heteroalkyl or alkylcycloalkyl; Y1and Y2are 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; when not attached to a site , (independently attached to L1and / or L2) is 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; 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) pCH2CH2COOH, 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 1-8 identical or different amino acids; p is 1-5000; R1, R2, R3, R4, R5, R5', Z1, Z2, and n are as defined above.

[0474] Hemiasterlin and analogs thereof (e.g., HTI-286) bind to tubulin, disrupting normal microtubule dynamics, and depolymerize tubulin in a stoichiometric number. The structure of maytansine is preferably of the formula:

[0475]

[0476] wherein R 1 , R 2 , R 3 , R 4 and R 5 are independently H; C1-C8 straight chain or branched alkyl, aryl, heteroaryl, heteroalkyl, alkylcycloalkyl, ester, ether, amide, amine, heterocycloalkyl, or acyloxyamine; or a peptide containing 1-8 amino acids, or a polyoxyethylene unit of the formula (OCH2CH2) p or (OCH2CH(CH3)) p wherein p is an integer from 1 to about 5000; further, R 2 R 3alkyl, aryl, heteroaryl, heteroalkyl, or alkylcycloalkyl rings.

[0477] Eribulin binds primarily to a small number of high-affinity sites on the plus end of microtubules, with both cytotoxic and noncytotoxic mechanisms of action. Its cytotoxic action is associated with its antimitotic activity, inducing apoptosis of cancer cells after a 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 cytotoxic and antimitotic-based mechanisms, preclinical studies in human breast cancer models have shown that Eribulin also has complex effects on the biology of surviving cancer cells and residual tumors that appear to be independent of its antimitotic action. Eribulin has been approved by the US FDA for the treatment of metastatic breast cancer in patients who have received at least two prior chemotherapy regimens for advanced disease, including anthracycline- and taxane-based chemotherapies, and for the treatment of liposarcoma, a soft tissue sarcoma, that cannot be removed by surgery (unresectable) or has progressed (metastatic). Eribulin has been used as an effective payload for ADC conjugates (US20170252458). Its preferred structure is as follows:

[0478]

[0479] is independently connected to a site of L1and / or L2;

[0480] Nicotinamide phosphoribosyltransferase inhibitors (NAMPT) can be ADC payloads because they have a unique mechanism of high activity (Sampath D et al. Pharmacol Ther 2015; 151, 16-31). NAMPT regulates the levels of nicotinamide adenine dinucleotide (NAD) in cells, and NAD is an important redox cofactor that maintains energy and anabolic metabolism. NAD has several important roles in metabolism. It acts as a coenzyme in redox reactions, as a donor of ADP-ribose moieties in ADP-ribosylation reactions, as a precursor of the second messenger molecule cyclic ADP-ribose, as a substrate for bacterial DNA ligases, and as a class of enzymes called Sirtuins that use NAD+ to remove acetyl groups from proteins. In addition to these metabolic functions, NAD+ is also released from cells as an adenine nucleotide, either spontaneously or through regulatory 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 thus can have important extracellular functions (Billington R.A., et al. Mol Med. 2006, 12, 324-7). When NAMPT inhibitors are present, the levels of NAD fall below that required for metabolism, resulting in an energy crisis and thus cell death. So far, NAMPT inhibitor drug candidates FK-866, CHS-828 and GMX-1777 have entered clinical trials, but each has encountered dose-limiting toxic effects before any objective remission has occurred (Holen K., et al. Invest New Drugs 2008, 26, 45-51; Hovstadius, P., et al. Clin Cancer Res 2002, 8, 2843-50; Pishvaian, M.J., et al. J Clin Oncol 2009, 27, 3581). Thus, targeted delivery of NAMPT inhibitors using ADCs can avoid systemic toxicity, resulting in a greater therapeutic index. The structure of the NAMPT inhibitor is preferably one of the following formulae NP01, NP02, NP03, NP04, NP05, NP06, NP07, NP08 and NP09:

[0481]

[0482] or one or more isotope substitutions of the elements, or a pharmaceutically acceptable salt, hydrate or hydrated salt; or polymorphic structures of these compounds; or optical isomers, racemates, diastereomers or enantiomers; wherein The same as the foregoing; X5 is F, Cl, Br, I, OH, OR1, R1, OPO3H2, OSO3H, NHR1, OCOR1, NHCOR1.

[0483] Benzodiazepine dimers and analogs thereof (e.g., pyrrolobenzodiazepine dimers of pyrrolobenzodiazepine (PBD) or (tomaymycin), indenobenzodiazepine (IGN) dimers, imidazobenzothiazepine dimers, or oxazolidinylbenzodiazepine dimers) contain one or more imine functional groups or their equivalents that are capable of binding to double stranded DNA. PBD and IGN molecules, which are based on the natural product anthramycin, interact with DNA in a sequence-selective manner, preferentially selecting purine- guanine-purine sequences. Preferred benzodiazepine dimers according to the present application are exemplified in the following documents: US Patents 8163736; 8153627; 8034808; 7834005; 7741319; 7704924; 7691848; 7678787; 7612062; 7608615; 7557099; 7528128; 7528126; 7511032; 7429658; 7407951; 7326700; 7312210; 7265105; 7202239; 7189710; 7173026; 7109193; 7067511; 7064120; 7056913; 7049311; 7022699; 7015215; 6979684; 6951853; 6884799; 6800622; 6747144; 6660856; 6608192; 6562806; 6977254; 6951853; 6909006; 6344451; 5880122; 4935362; 4764616; 4761412; 4723007; 4723003; 4683230; 4663453; 4508647; 4464467; 4427587; 4000304; US Applications 20100203007, 20100316656, 20030195196. Antibody-benzodiazepine dimers conjugates are exemplified as follows: PB01-PB30:

[0484]

[0485]

[0486]

[0487]

[0488]

[0489] or one or more isotopic substitutions of elements, or pharmaceutically acceptable salts, hydrates or hydrated salts; or polymorphic structures of these compounds; or optical isomers, racemates, diastereomers or enantiomers; wherein X1, X2, Y1, Y2, R5', Z1, Z2and n are defined as described above; preferably X1, X2, Y1and Y2are 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;

[0490] R 1 , R 2 , R 3 , R 1’ , R 2’ , and R 3’ are independently H, F, Cl, =O, =S, OH, SH, C1-C8 straight chain or branched benzyl, aryl, alkenyl, heteroaryl, heteroalkyl, alkylcycloalkyl, ester (COOR5or -OC(O)R5), ether (OR5), amide (CONR5), carbamate (OCONR5), amine (NHR5, NR5R5'), heterocycloalkyl, or acyloxyamine (-C(O)NHOH, -ONHC(O)R5), or a peptide containing 1-20 natural or unnatural amino acids, or a polyoxyethylene unit of the structure (OCH2CH2) p or (OCH2CH(CH3)) p wherein p is an integer from 1 to 5000. Two R groups, such as 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’ may independently form a 3-8 membered ring alkyl, aryl, heteroaryl, heteroalkyl or alkylcycloalkyl;

[0491] X3and Y3are independently N, NH, CH2or CR5, one of X3and Y3may be absent;

[0492] wherein R1, and R2are C1-C8 linear or branched alkyl, heteroalkyl; C3-C8 aryl, heteroaryl, alkylcycloalkyl, acyloxy, alkylaryl, alkylaryloxy, alkylaryl amino, alkylaryl mercapto; or 1-6 identical or different amino acid / peptide sequences (Ar)r, r = 1-6;

[0493] wherein R4, R5, R5', R6, R 12 and R 12 are independently H, OH, NH2, NH(CH3), NHNH2, COOH, SH, OZ3, SZ3, F, Cl, or C1-C8 linear or branched alkyl, aryl, heteroaryl, heteroalkyl, alkylcycloalkyl, acyloxy amine;

[0494] Z3is H, OP(O)(OM1)(OM2), OCH2OP(O)(OM1)(OM2), OSO3M1, or O-glycoside (glucoside, galactoside, mannoside, glucuronoside / glucuronic acid, alloside, fructoside, etc.), NH-glycoside, S-glycoside or CH2-glycoside; M1and M2are independently H, Na, K, Ca, Mg, NH4or NR1R2R3;

[0495] X6is CH, N, P(O)NH, P(O)NR1, CHC(O)NH, C3-C8 aryl, heteroaryl, alkylcycloalkyl, acyloxy, alkylaryl, alkylaryloxy, alkylaryl amino or Aa (amino acid, preferably selected from Lys, Phe, Asp, Glu, Ser, Thr, His, Cys, Tyr, Trp, Gin, Asn, Arg);

[0496] X and X' are independently CH2, or N, when the six-membered aromatic ring becomes a five-membered ring, X and / or X' can be O, S or NH;

[0497] Y 21 is Ms (mesyl), Ts (tosyl) or Tf (trifyl), SO3H, P(O)(OH)2, CH2(O)P(O)(OH)2, glycoside;

[0498] R 31 is H, C1-C8 alkyl or Ar, CF3; are as defined above.

[0499] CC-1065 analogs and duocarmycin analogs are preferred for conjugates of the present patent application. Examples of CC-1065 analogs and duocarmycin analogs and their synthesis are described in: Warpehoski, et al., J. Med. Chem. 31 :590-603 (1988); D. Boger et al., J. Org. Chem; 66; 6654-61, 2001; U.S. Patents: 4,169,888, 4,391,904, 4,671,958, 4,816,567, 4,912,227, 4,924,990, 4,952,394, 4,975,278, 4,978,757, 4,994,578, 5,037,993, 5,070,092, 5,084,468, 5,101,038, 5,117,006, 5,137,877, 5,138,059, 5,147,786, 5,187,186, 5,223,409, 5,225,539, 5,288,514, 5,324,483, 5,332,740, 5,332,837, 5,334,528, 5,403,484, 5,427,908, 5,475,092, 5,494,009, 5,530,101, 5,545,806, 5,547,667, 5,569,825, 5,571,698, 5,573,922, 5,580,889, 5,585,499, 5,587,161, 5,595,499, 5,606,017, 5,622,929, 5,625,126, 5,629,430, 5,633,425, 5,641,780, 5,660,829, 5,661,016, 5,686,237, 5,693,762, 5,703,080, 5,712,374, 5,714,586, 5,739,116, 5,739,350, 5,770,429, 5,773,001, 5,773,435, 5,786,377, 5,786,486, 5,789,050, 5,814,318, 5,846,545, 5,874,299, 5,877,296, 5,877,397, 5,885,793, 5,962,216, 5,969,108, 5,985,908, 6,060,608, 6,066,742, 6,075,181, 6,103,236, 6,114,598, 6,130,237, 6,132,722, 6,143,901, 6,150,584, 6,162,963, 6,172,197, 6,180,370, 6,194,612, 6,214,345, 6,262,271, 6,281,354, 6,310,209, 6,329,497, 6,342,480, 6,486,326, 6,512,101, 6,521,404, 6,534,660, 6,544,731, 6,548,530, 6,555,313, 6,565,693, 6,566,336, 6,586,618, 6,593,081, 6,630,579, 6,756,397, 6,759,509, 6,762,179,6884869, 6897034, 6946455, 7049316, 7087600, 7091186, 7115573, 7129261, 7214663, 7223837, 7304032, 7329507, 7329760, 7388026, 7655660, 7655661, 7906545, and 8012978. Structures of conjugates of antibodies-CC-1065 analogs of the application using linkers are exemplified by CC01, CC02, CC03, CC04, CC05, CC06, and CC07:

[0500]

[0501]

[0502] wherein when attached to a site X1, X2, Y1, and Y2are 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 when not attached to a linking site 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; Z3is H, PO(OM1)(OM2), SO3M1, CH2PO(OM1)(OM2), CH3N(CH2CH2)2NC(O)-, O(CH2CH2)2NC(O)-, R1, or a glycoside; wherein R1, R2, R3, M1, M2, and n are as defined above.

[0503] Amatoxins and analogs thereof are a subset of at least ten toxic compounds originally found in some poisonous Amanita mushrooms, most notably A. phalloides and several other mushrooms, and are also preferred for use in the conjugates of the present patent. These ten amatoxins, a-amanitin, β-amanitin, γ-amanitin, ε-amanitin, Amanullin, Amanullinic acid, Amaninamide, Amanin, Proamanullin, whose synthetic precursor is a 35-amino acid protein that is cleaved by prolyl oligopeptidase to yield a rigid bicyclic peptide of 8 amino acids (Litten, W. 1975 Scientific American 232(3): 90-101; H. E. 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, P. A. et al. 1978 Arch. Microbio. 118(3):317-9). Amatoxins kill cells by inhibiting RNA polymerase II (Pol II), shutting down gene transcription and protein biosynthesis (Brodner, O. G. 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, D. R., Guo, H. & Price, D. H. 1995 J. Biol. Chem. 270(32): 19114-19; Wieland (1983) Int. J. Pept. Protein Res. 22(3):257-76). Amatoxins can be produced from collected A. phalloides mushrooms (Yocum, R. R. 1978 Biochemistry 17(18):3786-9; Zhang, P. et al. 2005, FEMS Microbiol. Lett. 252(2), 223-8), or using basidiomycetes (Muraoka, S. and Shinozawa T. 2000 J. Biosci. Bioeng. 89(1):73-6) or A. fissa fermentation (Guo, X. W., et al., 2006 Wei Sheng Wu Xue Bao 46(3):373-8), or by cultivation of Galerina fasciculata or Galerina helvoliceps (WO / 1990 / 009799, JP 11137291). However, these isolations and fermentations have low yields (less than 5 mg / L of culture). Several preparations of amatoxins and analogs have been reported over the past three decades (W. E. 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; P. A. 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, J. E. and J. F. Preston, 3rd, Int J Pept Protein Res, 1991. 37(6): 544-51; Mullersman, J. E., 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, M. O., et al., J. Org. Chem., 2005, 70(12): 4578-84; J. P. May, et al., J. Org. Chem. 2005, 70, 8424-30; F.Brueckner, P. Cramer, Nat. Struct. Mol. Biol. 2008, 15, 811-8; J. P. May, D. M. Perrin, Chem. Eur. J. 2008, 14, 3404-9; J. P. May, et al., Chem. Eur. J. 2008, 14, 3410-17; Q. Wang, et al., Eur. J. Org. Chem. 2002, 834-9; May, J. P. and D. M. Perrin, Biopolymers, 2007. 88(5): 714-24; May, J. P., 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 which are partially synthetic methods. Due to their extreme potency and unique mechanism of cytotoxicity, amatoxins have been used as potent payloads for conjugates (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, M. T., Preston, J. F. Science 1981, 213, 1385-1388; Preston, J. F., et al., Arch Biochem Biophys, 1981. 209(1): 63-71; H. Faulstich, et al., Biochemistry 1981, 20, 6498-504; Barak, L. S., et al., Proc Natl Acad Sci U S A, 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:.

[0504] 189-96; WO2014 / 043403, US20150218220, EP 1661584). We have been working on amatoxins. Examples of amatoxins applied in the present application are preferably the following Am01, Am02 and Am03 structures:

[0505]

[0506] or one or more isotopic substitutes of chemical elements, or pharmaceutically acceptable salts, hydrates or hydrated salts; or polymorphic structures of these compounds; or optical isomers, racemates, diastereomers or enantiomers; wherein X1, and Y1are 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), CH2, CHNH, CH2O, C(O)NHNHC(O), and C(O)NR1; R7, R8, and R9are independently H, OH, OR1, NH2, NHR1, C1-C6alkyl, or null; Y2is O, O2, NR1, NH, or null; R 10 is CH2, O, NH, NR1, NHC(O), NHC(O)NH, NHC(O)O, OC(O)O, C(O), OC(O), OC(O)(NR1), (NR1)C(O)(NR1), C(O)R1or null; R 11is 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, NR1R2, O(CH2CH2O) p CH2CH2-COOH, NH(CH2CH2O) p CH2CH2COOH, NH-Ar-COOH, NH-Ar-NH2, O(CH2CH2O) p CH2CH2-NHSO3H, NH(CH2CH2O)

[0507] 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 CH2-CH2NHPO3H2, wherein (Aa) r denotes 1-8 amino acids; n and m1 are independently 1-20; p is 1-5000; R1, R2, and Ar are defined as the same throughout this patent application; are defined as described previously.

[0508] Spliceostatins and pladienolides are antitumor compounds that interact with the spliceosome SF3b, inhibiting splicing. Examples of spliceostatin include, but are not limited to, spliceostatin A, FR901464, and (2S,3Z)-5-{[(2R,3R,5S,6S)-6-{(2E,4E)-5-[(3R,4R,5R,7S)-7-(2-hydrazinyl-2-oxoethyl)-4-hydroxy-1,6-dioxypyrido[2,5]oct-5-yl]-3-pentyl-2,4-dien-1-yl}-2,5-dimethyltetrahydro-2H-pyrano-3-yl]amino}-5-oxo-3- en-2-yl acetate, the parent nucleus structure of which is as follows:

[0509]

[0510] Examples of Pladienolide include, but are not limited to, Pladienolide B, Pladienolide D, and E7107.

[0511] Protein kinase inhibitors can inhibit the activity of kinases that catalyze the phosphorylation of serine, threonine, or tyrosine residues on antibodies and modulate the function of proteins. Protein kinase inhibitors can be used to treat cancers due to overactive protein kinases, including mutated or overexpressed kinases, or to modulate cellular function to overcome other disease drivers. Protein kinase inhibitors are preferably Adavosertib, Afatinib, Axitinib, Baricitinib, Bosutinib, Cipatinib, Crizotinib, Cabozantinib, Dasatinib, Entospletinib, Erdafitinib, Erlotinib, Fotatinib, Gefitinib, Ibrutinib, Imatinib, Lapatinib, Lenvatinib, Mubritinib, Nilotinib, Pazopanib, Panatinib, Ponatinib, Rebastinib, Regorafenib, Ruxolitinib, Sorafenib, Sunitinib, SU6656, Tofacitinib, Vandetanib, Vemurafenib, Entospletinib, Palbociclib, Ribociclib, Abemaciclib, Dacomitinib, Neratinib, CO-1686, Osimertinib, AZD3759, Nazartinib (EGF816), having the structures shown below, PK01-PK40:

[0512] Adavosertib,

[0513] Afatinib,

[0514] Axitinib,

[0515] Baricitinib

[0516] Bosutinib,

[0517] Cipatinib,

[0518] Crizotinib,

[0519] Dasatinib,

[0520] Entospletinib,

[0521] Erdafitinib,

[0522] Erlotinib,

[0523] Fostamatinib,

[0524] Gefitinib,

[0525] Gefitinib,

[0526] Gefitinib,

[0527] Ibrutinib,

[0528] Imatinib,

[0529] Lapatinib,

[0530] Lenvatinib,

[0531] Mubritinib,

[0532] Nilotinib,

[0533] Pazopanib,

[0534] Panatinib,

[0535] Ruxolitinib,

[0536] Sorafenib,

[0537] Sunitinib,

[0538] SU6656,

[0539] Tofacitinib,

[0540] Vandetanib,

[0541] Vemurafenib,

[0542] Encorafenib;

[0543] Palbociclib analog,

[0544] Ribociclib,

[0545] Abemaciclib,

[0546] Dacomitinib,

[0547] Larotrectinib,

[0548] Rociletinib (CO-1686),

[0549] Osimertinib,

[0550] AZD3759,

[0551] Naziitinib (EGF816),

[0552] wherein Z5and Z5' are independently selected from 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)NR1.

[0553] MEK inhibitors can inhibit the mitogen-activated protein kinases MEK1 and / or MEK2 which are overactive in certain cancers. MEK inhibitors are particularly useful for the treatment of BRAF-mutated melanoma and KRAS / BRAF-mutated colorectal, breast and non-small cell lung cancer (NSCLC). The MEK inhibitor is selected from the group consisting of PD0325901, selumetinib (AZD6244), cobimetinib (XL518), refametinib, trametinib (GSK1120212), pimasertib, binimetinib (MEK162), AZD8330, RO4987655, RO5126766, WX-554, E6201, GDC-0623, PD-325901 and TAK-733. A preferred MEK inhibitor is trametinib (GSK1120212), cobimetinib (XL518), binimetinib (MEK162), selumetinib, having the following structures:

[0554] Trametinib,

[0555] Cabozantinib,

[0556] Binimetinib,

[0557] Sunitinib,

[0558] wherein Z5is selected from 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)NR1.

[0559] The protease inhibitor used as a payload of the conjugate is preferably selected from the group consisting of Carfilzomib, Clindamycin, Retaspimycin, Indibulin, having the following structures:

[0560] Carfilzomib,

[0561] Clindamycin,

[0562] Carmaphycin analogues,

[0563] An immunotoxin is a macromolecular drug, usually a cytotoxic protein derived from bacterial or plant proteins, such as diphtheria toxin (DT), cholera toxin (CT), ricin (TCS), amylase, Pseudomonas exotoxin A (ETA), abrin, diphtheria toxin, AB toxin, type III exotoxin, etc. It can also be a highly toxic bacterial pore-forming protoxin that requires proteolytic processing for activation. One example of such a protoxin is pre-aeruginosa and its genetically modified form topalysin. Topalysin is a modified recombinant protein that is engineered to be selectively activated by an enzyme in the prostate, resulting in local cell death and tissue destruction without damaging adjacent tissues and nerves. The immunotoxin in the present invention is preferably conjugated to an amino acid having a free amino group, a thiol group or a carboxylic acid group by the method in the present invention; and more preferably to an N-terminal amino acid.

[0564] In addition, cell receptor agonists, cell stimulating molecules or intracellular signaling molecules can also be conjugated as chemotherapeutic / functional compounds by the method of the present invention.

[0565] Cell binding ligands or receptor agonists are selected from the group consisting of: folate derivatives, glutamate urea derivatives, somatostatin and its analogues (selected from octreotide (Sandostatin) and lanreotide (Somatuline)), arylsulfonamides, pituitary adenylate cyclase activating peptide (PACAP) (PAC1), vasoactive intestinal peptide (VIP / PACAP) (VPAC1, VPAC2), melanocyte-stimulating hormone (a-MSH), cholecystokinin (CCK) / gastrin receptor agonists, bombesin (selected from Pyr-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2) / gastrin releasing peptide (GRP). neurotensin receptor ligands (NTR1, NTR2, NTR3); substance P (NK1 receptor) ligands; neuropeptide Y (Y1-Y6); homing peptides including RGD (Arg-Gly-Asp), NGR (Asn-Gly-Arg), dimeric and multimeric cyclic RGD peptides (selected from cRGDfV), TAASGVRSMH and LTLRWVGLMS (chondroitin sulfate proteoglycan NG2 receptor ligands) and F3 peptides; cell penetrating peptides (CPPs);Peptide hormones are selected from the group consisting of luteinizing hormone releasing hormone (LHRH) agonists and antagonists, and gonadotropin releasing hormone (GnRH) agonists, acting by targeting follicle stimulating hormone (FSH) luteinizing hormone (LH), and testosterone production, such as buserelin (Pyr-His-Trp-Ser-Tyr-D-Ser(OtBu)-Leu-Arg-Pro-NHEt), goserelin (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), nafarelin (Pyr-His-Trp-Ser-Tyr-2Nal-Leu-Arg-Pro-Gly-NH2), triptorelin (Pyr-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH2), nafarelin, delolorin, abarelix (Ac-D-2Nal-D-4-chloroPhe-D-3-(3-pyridyl)Ala-Ser-(N-Me)Tyr-D-Asn-Leu-iso-propylLys-Pro-DAla-NH2), cetrorelix (Ac-D-2Nal-D-4-chloroPhe-D-3-(3-pyridyl)Ala-Ser-Tyr-D-Cit-Leu-Arg-Pro-D-Ala-NH2), degarelix (Ac-D-2Nal-D-4-chloroPhe-D-3-(3-pyridyl)Ala-Ser-4-aminoPhe(L-hydroorotyl)-D-4-aminoPhe(carba-moyl)-Leu-iso-propylLys-Pro-D-Ala-NH2), and degarelix (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); pattern recognition receptors (PRRs) selected from the group consisting of Toll-like receptor (TLRs) ligands, C-type lectins and nod-like receptors (NLRs) ligands; calcitonin receptor agonists; integrin receptors and their receptor subtypes (selected from the group consisting of α; V β1, αV β3, α V β5, α V β6, α6 β4, α7 β1, α L β2, α IIb β3) agonists (selected from the group consisting of GRGDSPK, cyclo(RGDfV)(L1) and derivatives thereof [cyclo(-N(Me)R-GDfV), cyclo(R-Sar-DfV), cyclo(RG-N(Me)D-fV), cyclo(RGD-N(Me)f-V), cyclo(RGDf-N(Me)V-)(Cilengitide)]; Anticalins (derivatives of lipocalins); Adnectins (10 FN3 (fibronectin)); Designed Ankyrin Repeat Proteins (DARPins); Avimers; EGF receptor, or VEGF receptor agonists;

[0566] LB01 (Folic acid), LB02 (PMSA ligand), LB03 (PMSA ligand), LB04 (PMSA ligand), LB05 (Somatostatin), LB06 (Somatostatin), LB07 (Octreotide, somatostatin analogue), LB08 (Lanreotide, somatostatin analogue), LB09 (Vapreotide (Sanvar), somatostatin analogue), LB10 (CAIX ligand), LB11 (CAIX ligand), LB12 (Gastrin-releasing peptide receptor (GRPr), MBA), LB13 (Luteinizing hormone-releasing hormone (LH-RH) and GnRH ligand), LB14 (Luteinizing hormone-releasing hormone (LH-RH) and GnRH ligand), LB15 (GnRH antagonist, Abarelix), LB16 (Cobalamin, vitamin B12 analogue), LB17 (Cobalamin, vitamin B12 analogue), LB18 (Cyclic RGD pentapeptide for avb3 integrin receptor), LB19 (Heterobivalent peptide ligand for VEGF receptor), LB20 (Neuromedin B), LB21 (Bombesin, acting on G protein-coupled receptors), LB22 (TLR2, acting on Toll-like receptors), LB23 (acting on androgen receptors), LB24 (Cilengitide or cyclic (-RGDfV-) av integrin receptor, LB23 (Fludrocortisone), LB25 (Rifabutin analogue), LB26 (Rifabutin analogue), LB27 (Rifabutin analogue), LB28 (Fludrocortisone), LB29 (Dexamethasone), LB30 (Fluticasone propionate), LB31 (Beclometasone dipropionate), LB32 (Triamcinolone acetonide), LB33 (Prednisolone), LB34 (Prednisolone), LB35 (Methylprednisolone), LB36 (Betamethasone), LB37 (Irinotecan analogue), LB38 (Crizotinib analogue), LB39 (Bortezomib analogue), LB40 (Carfilzomib analogue), LB41 (Carfilzomib analogue), LB42 (Leuprolide analogue), LB43 (Triptorelin analogue), LB44 (Clindamycin), LB45 (Liraglutide analogue), LB46 (Vincristine analogue), LB47 (Retapamulin analogue), LB48 (Dinutuximab analogue), LB49 (Vinblastine analogue), LB50 (Lixisenatide peptide analogue), LB51 (Oxindanib analogue), LB52 (Nucleoside analogue), LB53 (Erlotinib analogue), and LB54 (Lapatinib analogue), the structures of which are shown below:

[0567] (Folic acid),

[0568] (Somatostatin),

[0569] Somatostatin,

[0570] Octreotide, somatostatin analogue,

[0571] Lanreotide, somatostatin analogue,

[0572] Pentetrexate (Sanvar), somatostatin analogue,

[0573] CAIX ligand,

[0574] CAIX ligand,

[0575]

[0576] Gastrin-releasing peptide receptor (GRPr), MBA,

[0577]

[0578] LB13 (Luteinizing hormone-releasing hormone (LH-RH) and gonadotropin-releasing hormone GnRH ligand),

[0579]

[0580] LB14 (Luteinizing hormone-releasing hormone (LH-RH) and gonadotropin-releasing hormone GnRH ligand),

[0581]

[0582] LB15 (GnRH antagonist, abarelix),

[0583] R 19 is 5' deoxyadenosyl, Me, OH, CN; LB16 (Cobalamin, vitamin B12 analogue),

[0584] R 19 is 5' deoxyadenosyl, Me, OH, CN; LB17 (Cobalamin, vitamin B12 analogue),

[0585]

[0586] LB18 (Cyclic RGD pentapeptide, acting on the alpha v beta3 integrin receptor),

[0587]

[0588] LB19 (hetero-bivalent peptide ligand conjugate, acting on vascular endothelial growth factor VEGF receptor),

[0589] (neuromedin B),

[0590]

[0591] LB21 (bombesin conjugate, acting on G protein-coupled receptor),

[0592] (TLR2 conjugate, acting on Toll-like receptor),

[0593] (androgen receptor),

[0594]

[0595] (cilengitide / cyclo(-RGDfV-) conjugate, acting on alpha v integrin receptor)

[0596] (rifabutin analog),

[0597] (rifabutin analog),

[0598] (rifabutin analog),

[0599] (fludrocortisone),

[0600] (dexamethasone),

[0601] (fluticasone propionate),

[0602] (beclomethasone dipropionate),

[0603] (triamcinolone acetonide),

[0604] (prednisone),

[0605] (prednisolone),

[0606] (methylprednisolone),

[0607] (difluprednate),

[0608] (irinotecan analogs),

[0609] (quizartinib analogs),

[0610] (bortezomib analogs) wherein Y5 is N, CH, C(Cl), C(CH3) or C(COOR1); R1 is H, C1-C6 alkyl, C3-C8 Ar;

[0611] (kafperomycin analogs),

[0612] (kafperomycin analogs),

[0613] (leuprolide analogs),

[0614] (triptorelin analogs),

[0615] (clindamycin),

[0616] (liraglutide analogs),

[0617] (semaglutide analogs),

[0618] (retapamulin analogs),

[0619] (dinburs analogs),

[0620] (vinblastine analogs),

[0621] (lixisenatide analogs),

[0622] (oxindin analogs),

[0623] (nucleoside analogs),

[0624] (erlotinib analogs),

[0625] (lapatinib analogs);

[0626] wherein X4, and Y1are 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), CH2, C(O)NHNHC(O), and C(O)NR1.

[0627] In another embodiment, one, two or more DNA, RNA, mRNA, small interfering RNA (siRNA), micro RNA (miRNA), and PIWI-interacting RNA (piRNA) are conjugated as a chemotherapeutic / functional compound by the method of the present application:

[0628]

[0629] wherein is the site of attachment of the branched linker of the present patent; is single or double stranded DNA, RNA, mRNA, siRNA, miRNA, or piRNA; X1, and Y 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), CH2, C(O)NHNHC(O), and C(O)NR1.

[0630] In another embodiment, L1, L2, La1, La2, Lb1, Lb2, Lc1, and Lc2linkers are the same or different, independently selected from O, NH, S, S-S, NHNH, N(R3), N(R3)N(R3'), C1-C8alkyl; C2-C8heteroalkyl, alkylcycloalkyl, heterocycloalkyl; C3-C8aryl, arylalkyl, heterocycle, carbocycle, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; C2-C8(2-8 carbon atoms) ester, ether, or amide; 1-8 natural or unnatural amino acids as described in the definition; structural formulae of (OCH2CH2) p (OCH2CH(CH3)) p (OCH2CH2) p OR3, (OCH2CH(CH3)) p OR3, NH(CH2CH2O) p R3, NH(CH2CH(CH3)O) p R3, N[(CH2CH2-O) p R3][(CH2CH2O) p R3’], (OCH2CH2) pCOOR3, or CH2CH2(OCH2CH2) p COOR3, or CH2CH2(OCH2CH2)

[0631] L1, L2, La1, La2, Lb1, Lb2, Lc1, and Lc2 independently can contain a self- immolative or non-self-immolative component, a peptide unit, a hydrazone bond, a disulfide, an ester, an oxime, an amide, or a thioether bond. Self- immolative units include, but are not limited to, aromatic compounds with electronic structures similar to that of p-aminobenzylcarbamoyl (PAB), such as derivatives of 2- aminoimidazole-5-carboxaldehyde, heterocyclic PAB analogs, β-glucuronides, and ortho or para aminobenzyl acetal.

[0632] Preferred self-immolative linker components have one of the following structures:

[0633]

[0634] wherein (*) is an additional spacer or cleavable linker unit, or a cytotoxic agent, and / or a site of attachment of an antibody; X 1 , Y 1 , Z 2 and Z 3 are independently NH, O, or S; Z 1 are independently H, NH, O, or S; v is 0 or 1; U 1 are independently H, OH, C1-C6 alkyl, (OCH2CH2) n F, Cl, Br, I, OR5, SR5, NR5R5', N=NR5, N=R5, NR5R5', NO2, SOR5R5', SO2R5, SO3R5, OSO3R5, PR5R5', POR5R5', PO2R5R5', OPO(OR5)(OR5'), or OCH2PO(OR5(OR5') wherein R5and R5' are as defined above; preferably R5and R5' are independently selected from H, C1-C8 alkyl, C2-C8 alkenyl, alkynyl or heteroalkyl, C3-C8 aryl, heterocycle, carbocycle, cycloalkyl, heterocycloalkyl, heteroarylalkyl, alkylcarbonyl, or glycoside; or a pharmaceutically acceptable cation salt.

[0635] Non-self-immolative linker components are one of the following structures:

[0636]

[0637]

[0638] wherein (*) is the point of attachment of an additional spacer R1or a cleavable linker unit, or a cytotoxic molecule and / or a cell binding molecule; X 1 , Y 1 , U 1 , R5, R5' are as defined above; r is 0-100; m and n are independently 0-6.

[0639] More preferably, L1, L2, La1, La2, Lb1, Lb2, Lc1and Lc2may independently be composed of one or more of the following linker components: 6-maleimidocaproyl ("MC"), maleimidopropanoyl ("MP"), valine-citrulline ("val-cit" or "vc"), alanine-phenylalanine ("ala-phe" or "af"), p-aminobenzyloxy carbonyl ("PAB"), 4-sulfopentanoyl ("SPP"), 4-(N-maleimidomethyl)cyclohexane-1- acyl ("MCC"), (4-acetyl)aminobenzyl ("SIAB"), 4-sulfobutyryl (SPDB), 4-sulfo-2- hydroxysulfonyl-butyryl (2-Sulfo-SPDB), or a natural or unnatural peptide containing 1-8 natural or unnatural amino acid units.

[0640] Further preferably, L1, L2, La1, La2, Lb1, Lb2, Lc1and Lc2are independently cleavable linkers. The term "cleavable" means that the linker contains at least one bond which can be broken under physiological conditions, such as pH, acid, base, oxidation, metabolic, biochemical or enzymatic labile bonds. It is understood that the bond breakage does not necessarily have to be a biological or metabolic process, but can be a standard chemical reaction such as a hydrolysis or substitution reaction. Examples of such physiological conditions are endosomes with a pH lower than the pH in the cell cytoplasm, and / or the ability to undergo disulfide exchange reactions with the millimolar concentrations of glutathione present in malignant cells.

[0641] Examples of cleavable linkers (L, L1or L2) include, but are not limited to:

[0642] -(CR5R6) m (Aa)r(CR7R8) n (OCH2CH2) t -, -(CR5R6) m (CR7R8) n (Aa) r (OCH2CH2) t -, -(Aa) r -(CR5R6) m (CR7R8)n (OCH2CH2) t -, -(CR5R6) m (CR7R8) n (OCH2CH2) r (Aa) t -, -(CR5R6) m- (CR7=CR8)(CR9R 10 ) n (Aa) t (OCH2CH2) r -, -(CR5R6) m (NR 11 CO)(Aa) t (CR9R 10 ) n- (OCH2CH2) r -, -(CR5R6) m (Aa) t (NR 11 CO)(CR9R 10 ) n (OCH2CH2) r -, -(CR5R6) m (OCO)(Aa) t (CR9R 10 ) n- (OCH2CH2) r -, -(CR5R6) m (OCNR7)(Aa) t (CR9R 10 ) n (OCH2CH2) r -, -(CR5R6) m (CO)(Aa) t- (CR9R 10 ) n (OCH2CH2) r -, -(CR5R6) m (NR 11 CO)(Aa) t (CR9R 10 ) n (OCH2CH2) r -, -(CR5R6) m- (OCO)(Aa) t (CR9R 10 ) n- (OCH2CH2) r -, -(CR5R6) m (OCNR7)(Aa) t(CR9R 10 ) n (OCH2CH2) r -、-(CR5R6) m (CO)(Aa) t (CR9R 10 ) n- (OCH2CH2) r -、-(CR5R6) m -phenyl-CO(Aa) t (CR7R8) n -、-(CR5R6) m -Furan-CO(Aa) t (CR7R8) n -、-(CR5R6) m -Oxazole-CO(Aa) t (CR7R8) n -、-(CR5R6) m Oxazole-CO-(Aa) t (CCR7R8) n -、-(CR5R6) t -Thiophene-CO(CR7R8) n -、-(CR5R6) t -imidazole-CO-(CR7R8) n -、-(CR5R6) t -morpholine-CO(Aa) t- (CR7R8) n -、-(CR5R6) t Piperazine-CO(Aa) t (CR7R8) n -、-(CR5R6) t -N-Methylpiperazine-CO(Aa) t- (CR7R8) n -、-(CR5R) m -(Aa) t Phenyl-, -(CR5R6) m -(Aa) t Furan-, -(CR5R6) m -Oxazole (Aa) t -、-(CR5R6) m -Oxazole (Aa) t -、-(CR5R6) m -thiophene-(Aa) t -、-(CR5R6) m -imidazole (Aa) t -、-(C R5R6) m- morpholine-(Aa) t -, -(CR5R6) m - piperazine-(Aa) t -, -(CR5R6) m - N-methylpiperazine-(Aa) t -, -K(CR5R6) m (Aa) r(CR7R8) n (OCH2CH2) t -, -K(CR5R6) m (CR7R8) n -(Aa) r (OCH2CH2) t -, -K(Aa) r (CR5R6) m (CR7R8) n (OCH2CH2) t -, -K(CR5R6) m (CR7R8) n -(OCH2CH2) r (Aa) t -, -K(CR5R6) m (CR7=CR8)(CR9R 10 ) n (Aa) t (OCH2CH2) r -, -K(CR5R6) m -(NR 11 CO)(Aa) t (CR9R 10 ) n (OCH2CH2) r -, -K(CR5R6) m (Aa) t (NR 11 CO)(CR9R 10 ) n (OCH2CH2) r -, -K(CR5R6) m (OCO)(Aa) t (CR9R 10 ) n- (OCH2CH2) r -, -K(CR5R6) m (OCNR7)(Aa) t (CR9R 10 ) n -(OCH2CH2) r -, -K(CR5R6) m(CO)(Aa) t- (CR9R 10 ) n (OCH2CH2) r -、-K(CR5R6) m (NR 11 CO)-(Aa) t (CR9R 10 ) n (OCH2CH2) r -、-K(CR5R6) m- (OCO)(Aa) t (CR9R 10 ) n (OCH2CH2) r -、-K(CR5R6) m (OCNR7)(Aa) t (CR9R 10 ) n (OCH2CH2) r -、-K(CR5R6) m (CO)(Aa) t (CR9R 10 ) n -(OCH2CH2) r -、-K(CR5R6) m -phenyl-CO(Aa) t (CR7R8) n -、-K-(CR5R6) m -Furan-CO(Aa) t- (CR7R8) n -、-K(CR5R6) m -Oxazole-CO(Aa) t (CR7R8) n -、-K(CR5R6) m -Oxazole-CO(Aa) t- (CR7R8) n -、-K(CR5R6) t -Thiophene-CO(CR7R8) n -、-K(CR5R6) t Imidazole-CO-(CR7R8) n -、-K(CR5R6) t Morpholine-CO(Aa) t (CR7R8) n -、-K(CR5R6) t Piperazine-CO(Aa) t- (CR7R8) n- K(CR5R6) t - N-methylpiperazine CO (Aa) t (CR7R8) n - K(CR5R) m (Aa) t phenyl, -K-(CR5R6) m- (Aa) t furan-, -K(CR5R6) m - oxazole (Aa) t - K(CR5R6) m - oxazole (Aa) t - K(CR5R6) m - thiophene- (Aa) t - K(CR5R6) m - imidazole (Aa) t - K(CR5R6) m - morpholine (Aa) t - K(CR5R6) m - piperazine- (Aa) t G, -K(CR5R6) m N-methylpiperazine (Aa) t -; wherein m, Aa, m, n, R3, R4, and R5 are as defined above; t and r are independently 0-100; R6, R7, and R8 are independently selected from H; halide; C1-C8 alkyl, aryl, alkenyl, alkynyl, ether, ester, amine or amide, optionally substituted with one or more halide, CN, NR1R2, CF3, OR1, Aryl, heterocycle, S(O)R1, SO2R1, -CO2H, -SO3H, -OR1, -CO2R1, -CONR1, -PO2R1R2, -PO3H, or P(O)R1R2R3; K is NR1, -SS-, -C(=O)-, -C(=O)NH-, -C(=O)O-, -C=NH-O-, -C=N-NH-, -C(=O)NH-NH-, O, S, Se, B, or C3-C6 heteroaryl.

[0643] Examples structures of components of linkers L1, L2, La1, La2, Lb1, Lb2, Lc1, and Lc2 independently comprise one or several of the following structures:

[0644] (including MC, 6-maleimidocaproyl), (including MP, maleimidopropanoyl), (including PAB, p-aminobenzyloxyamido), (valine-citrulline (VC)), (maleimidomethyl) cyclohexane-1 carboxylate (MCC), (4-acetyl) aminobenzoate, 4-thio-2-hydroxysulfonobutyrate, 2-sulfo-SPDB, 4-thiopentanoate (SPP), 4-thiobutyryl (SPDB), 4-(N-maleimidomethyl) cyclohexane-1 -carboxylate (MCC), maleimidethy lamino (ME), 4-thio-2-hydroxysulfonobutyryl (2-sulfo-SPDB), arylmercaptoether (PhSS), (4-acetyl) aminobenzoyl (SIAB), oxybenzyl sulfide, aminobenzyl sulfide, dioxobenzyl sulfide, diaminobenzyl sulfide, aminooxybenzyl sulfide, alkyloxyamino (AOA), ethyleneoxy (EO), disulfide, 4-methyl-4-dithiopentanoic acid (MPDP), triazole, alkylsulfonyl, alkylsulfonamide, sulfonedi sulfonamide, phosphorodiamide, alkylphosphoramide, phosphonic acid, N-methylalkylphosphoramide, N,N'-dimethylphosphorodiamide, alkylphosphorodiamide, hydrazine, acetamidine, oxime, diacetylhydrazine, aminoethyl amine, glycyl-glycyl-aminopropyl-amine, gly-gly-gly, gly-gly, gly-gly-gly-gly, Lys-gly, gly-gly-phe-gly,

[0645] ala-ala-ala-ala, ala-ala-ala, ala-ala, glu-gly, glu-lys, (VC),

[0646] ala-val-ala, (ala-phe), (lys-phe), or combinations thereof, wherein is a linkage site; X2, X3, X4, X5, or X6, independently, is selected from the group consisting of NH, NHNH, N(R 12 ), N(R 12 )N(R 12 ), O, S, C1-C6 alkyl, C2-C6 heteroalkyl, alkylcycloalkyl, heterocycloalkyl, C3-C8 aryl, arylalkyl, heterocycle, carbocycle, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl, CH2OR 12 , CH2SR 12 , CH2NHR 12 , or 1-8 amino acids; wherein R 12 and R 12 are independently H, C1-C8 alkyl, C2-C8 heteroalkyl, alkylcycloalkyl, heterocycloalkyl; C3-C8 aryl, arylalkyl, heterocycle, carbocycle, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; or C1-C8 ester, ether or amide; or a polyethylene glycol unit of the structure (OCH2CH2) p or (OCH2CH(CH3)) p , wherein p is an integer from 0 to about 1000.

[0647] In another embodiment, L1, L2, La1, La2, Lb1, Lb2, Lc1, and Lc2 configured in the structures of formula (I), (II), and (III) are selected from the following preferences, respectively:

[0648] wherein the structure of formula (I) preferably has the structure of formula (la); wherein the structure of formula (II) preferably has the structure of formula (lb) or (lc); wherein the structure of formula (III) The structure of the preferred structure of Formula (Id), (Ie), (If) or (Ig) is illustrated below;

[0649]

[0650]

[0651] wherein, is the site of attachment of the drug or linker L1or L2; "#" is the site of attachment of S (sulfhydryl), O (phenol), NH (amino), CHO (aldehyde), C(=O) (ketone), C(O)(NH) (amide) and C(O)(OH) (carboxylate) of the antibody; Aais L- or D- natural or unnatural amino acid; "@" is the site of attachment of Lc1or Lc2described in Formula (I), (II) and (III).

[0652] R1is H, C1-C8alkyl, OH, CH2OH, CH2CH2OH, NH2, SH, SCH3, CH2COOH, CH2CH2COOH, CH2CH2CH2CH2NH2, C6H5, CH2C6H5, CH2C6H4OH, CH(OH)CH3, CH2C(O)NH2, CH2CH2C(O)NH2, CH2CH2CH2NHC(=NH)NH2;

[0653] r is 0-12; when r is not 0, (Aa)r is the same or different amino acid or peptide unit;

[0654] m1= 1-18; m2= 1-100; m3= 1-8; m4= 0-8; m5= 1-8;

[0655] Y 7 is NH, OCH2NH, NHC(=O), NHNH, C(=O)NH, N(R1), SO2, P

[0656] (O)(OH), NHS(O)2, NHS(O)2NH, NHS(O)2NHC(O), NHS(O)

[0657] 2NHC(O)O, NHS(O)2NHC(O)NH, NHP(O)(OH), NHP(O)(OH)NH, OP(O)(OH)O, NHP(O)(OH)O, OP(O)(OH)NH, S, O, OP(O)(OH)OP(O)(OH)NH, NHP(O)

[0658] (OH)OP(O)(OH)O, OCH2CH2O, OCH2CH2NH, N(CH2CH2)2N, NHC6H4NH, CH2;

[0659] Y 8 is NHC(=O), NHS(O2), NH(SO), NHS(O2)NH, NHP(O)(OH)NH, C(O)NH, OC(O)NH, NHC(O)NH, C(O), N, NH, CH2, or CH;

[0660] Lv1' and Lv2' are independently selected from:

[0661]

[0662]

[0663]

[0664]

[0665]

[0666] wherein is the site of attachment to the linker component; "#" is the site of attachment of the S (sulfhydryl), O (phenol), NH (amino), CHO (aldehyde), C(=O) (ketone), C(O)(NH) (amide), and C(O)(OH) (carboxylate) moiety to Lv1' and Lv2' indicated in the formula; wherein R1, X1' and X2' are as described above; X is O, NH, S, CH2; the bond between the two atoms means that it can attach to either of the two atoms, Ar is an aromatic group.

[0667] More preferably, the following core linker structures have an affinity ligand (L1') in formula (I) (or an affinity ligand (L1") in formula (Ib')):

[0668] are preferably selected from:

[0669]

[0670] wherein Aa is an L- or D- natural or unnatural amino acid; A1 is an affinity ligand as defined above;

[0671] R1is H, C1-C8alkyl, OH, CH2OH, CH2CH2OH, NH2, SH, SCH3, CH2COOH, CH2CH2COOH, CH2CH2CH2CH2NH2, C6H5, CH2C6H5, CH2C6H4OH, CH(OH)CH3, CH2C(O)NH2, CH2CH2C(O)NH2, CH2CH2CH2NHC(=NH)NH2;

[0672] r is 0-12; when r is not 0, (Aa)rare the same or different amino acid or peptide units;

[0673] m1= 1-18; m2= 1-100; m3= 1-8; m4= 0-8; m5= 1-6; m7= 1-8;

[0674] Y 7 is NH, OCH2NH, NHC(=O), NHNH, C(=O)NH, N(R1), SO2, P(O)(OH), NHS(O)2, NHS(O)2NH, NHS(O)2NHC(O), NHS(O)2NHC(O)O, NHS(O)2NHC(O)NH, NHP(O)(OH), NHP(O)(OH)NH, OP(O)(OH)O, NHP(O)(OH)O, OP(O)(OH)NH, S, O, OP(O)(OH)OP(O)(OH)NH, NHP(O)(OH)OP(O)(OH)NH, NHP(O)(OH)OP(O)(OH)O, OCH2CH2O, OCH2CH2NH, N(CH2CH2)2N, NHC6H4NH, CH2;

[0675] Y 8 is NHC(=O), NH, O, NHS(O2), NH(SO), NHS(O2)NH, NHP(O)(OH)NH, C(O)O, C(O), OC(O)NH, C(O)NH, or Ar;

[0676] R 9 is (O=)CR1, (O=)CNHR1, NHC(=O), NH, O, NHS(O2), NH(SO), NHS(O2)NH, NHP(O)(OH)NH, or C(O)NH, R1(COCH2NH) m4 H, R1(Aa) r , (Aa)r, C(O), Ar, or wherein R3 is H, C1-C8 alkyl, ester, amide, aryl, ketone, alkyl acid, alkanol, alkyl amine, CH2C6H5, CH2C6H4OH, CH(OH)CH3, CH2C(O)NH2, CH2CH2C(O)NH2, CH2CH2CH2NHC(=NH)NH2; R1 is as defined above.

[0677] or the following core structure in formula (III) (referred to as L1" and L2" combined) (or respectively L1" and L2" combined in formula (IIIb')):

[0678] (L1" and L2" combined)

[0679] Preferred are the following formulae (Ib) and (Ic):

[0680]

[0681] wherein R1, Y 7 , Y 8 , R9, A1, Aa, r, m1, m2, m4, and m5 are as defined above.

[0682] In certain embodiments, examples of conjugates in formula (I), (II), and (III) are shown in the following figures:

[0683]

[0684]

[0685]

[0686]

[0687]

[0688]

[0689]

[0690]

[0691]

[0692]

[0693]

[0694]

[0695]

[0696]

[0697]

[0698] wherein

[0699]

[0700]

[0701]

[0702]

[0703] wherein

[0704]

[0705]

[0706]

[0707]

[0708]

[0709] wherein

[0710]

[0711]

[0712] wherein

[0713]

[0714]

[0715]

[0716] Raand Rbare the same as defined previously,

[0717]

[0718] wherein, D1and

[0719] D2and

[0720]

[0721]

[0722]

[0723] Raare the same as previously defined,

[0724]

[0725]

[0726]

[0727] wherein D1and

[0728] D2and wherein,

[0729]

[0730]

[0731] Raand Rbare the same as previously defined,

[0732]

[0733]

[0734] wherein D1and

[0735] D2and

[0736]

[0737]

[0738]

[0739]

[0740]

[0741] wherein,

[0742] wherein

[0743] A1, A2 and

[0744]

[0745] A1, A2 and

[0746]

[0747] A1 and

[0748]

[0749] A1 and

[0750]

[0751] A1 and

[0752]

[0753]

[0754] A1 and

[0755]

[0756] A1 and

[0757]

[0758] A1 and

[0759]

[0760] A1 and

[0761]

[0762] A1 and

[0763] A1 and

[0764]

[0765]

[0766] wherein

[0767]

[0768]

[0769] wherein

[0770] wherein

[0771]

[0772]

[0773] wherein

[0774] wherein

[0775]

[0776] wherein

[0777] wherein

[0778] wherein

[0779] wherein

[0780]

[0781] wherein

[0782] wherein

[0783] wherein

[0784] wherein

[0785]

[0786] wherein

[0787] wherein

[0788] wherein

[0789] wherein

[0790]

[0791] wherein

[0792] wherein

[0793] wherein

[0794] wherein

[0795]

[0796] wherein

[0797] wherein

[0798] wherein

[0799] wherein

[0800]

[0801] wherein

[0802] wherein

[0803] wherein

[0804] wherein

[0805]

[0806] wherein

[0807] wherein

[0808] wherein

[0809] wherein

[0810]

[0811] wherein

[0812] wherein

[0813] wherein

[0814] wherein

[0815]

[0816] wherein

[0817] wherein

[0818] wherein

[0819] wherein wherein the foregoing mAb is an antibody; n is 1-30, preferably 1-20, more preferably 2-8.

[0820] In certain embodiments, conjugates of formula (I), (II) and (III) are prepared by reacting an antibody with a compound having the following formula (IV), (V) and (VI), respectively:

[0821]

[0822] wherein D1, D2, L1, L2, La1, La2, Lb1, Lb2, Lc1, Lc2, Ld1, Ld2, Ld3, Ld4, Ld5, Ld6, A1, A2, A3, A4, A5, A6, E1, m1, m2, m3, m4, m5, m6, m7, m8, m9, m 10 , m 11 , and m 12 are defined as in formula (I), (II), (III);

[0823] Lv1and Lv2are a reactive group, independently or combined together, selected from the following structures:

[0824]

[0825] aryl-palladium complex,

[0826]

[0827] (ADPN),

[0828]

[0829]

[0830]

[0831]

[0832] wherein X1and X2are independently F, Cl, Br, I, OTf, OMs, OC6H4(NO2), OC6H3(NO2)2, OC6F5, OC6HF4, or Lv3; X2is O, NH, N(R1), or CH2; R3and R5are independently H, R1, an aromatic group, heteroaromatic or aromatic, wherein one or more H atoms are independently replaced by -R1, -halogen, -OR1, -SR1, -NR1R2, -NO2, -S(O)R1, -S(O)2R1, or -COOR1; Lv3and Lv3are independently leaving groups selected from F, Cl, Br, I, nitrophenoxide; N-hydroxysuccinimide (NHS); phenoxide; phenylmercapto, dinitrophenoxide; pentafluorophenoxy; tetrafluorophenoxy; difluorophenoxy; monofluorophenoxy; penta-chlorophenol; triflate; imidazole; dichlorophenoxy; tetrachlorophenoxy; 1-hydroxybenzotriazole; p-toluenesulfonic acid; methanesulfonic acid; 2-ethyl-5-phenylisoxazole-3'-sulfonic acid ester, anhydride formed by itself or with other anhydrides, such as acetic anhydride, formic anhydride; or intermediates of polypeptide coupling reactions using condensing reagents or intermediates of Mitsunobu reactions;

[0833] In formula (V) and (VI), selected from: 2-((alkyl or arylsulfonyl)methyl)acryloyl, (ADPN),

[0834]

[0835]

[0836]

[0837] wherein Lv3, Lv3, X1and X2are as described above; the bond between the two atoms means that it can be attached to either of the two atoms.

[0838] Examples of formula (IV), (V) and (VI) are as follows:

[0839]

[0840]

[0841]

[0842]

[0843]

[0844]

[0845]

[0846]

[0847]

[0848]

[0849]

[0850]

[0851]

[0852] wherein

[0853]

[0854]

[0855] wherein

[0856]

[0857]

[0858] wherein Ra is as defined above;

[0859] wherein Ra is as defined above;

[0860]

[0861]

[0862]

[0863] wherein

[0864]

[0865]

[0866]

[0867]

[0868] wherein

[0869]

[0870]

[0871]

[0872]

[0873] wherein

[0874] wherein Ra is as defined above;

[0875]

[0876]

[0877] Ra and Rb are as defined above;

[0878]

[0879]

[0880]

[0881] wherein

[0882]

[0883]

[0884]

[0885]

[0886] wherein

[0887]

[0888]

[0889]

[0890]

[0891] wherein

[0892]

[0893]

[0894] wherein Raand Rbare as defined above;

[0895]

[0896] wherein D1and

[0897] D2and

[0898]

[0899]

[0900] Raas defined above;

[0901]

[0902]

[0903]

[0904] wherein D1and

[0905] D2and wherein

[0906]

[0907]

[0908] and Rbare as defined above;

[0909]

[0910] wherein D1and

[0911] D2and

[0912] Raand Rbare as defined above;

[0913]

[0914]

[0915]

[0916]

[0917] wherein

[0918]

[0919] wherein

[0920] A1, A2and

[0921]

[0922] A1, A2and

[0923]

[0924] A1and

[0925]

[0926] A1and

[0927]

[0928] A1and

[0929]

[0930]

[0931] A1and

[0932]

[0933] A1 and

[0934]

[0935] A1 and

[0936]

[0937] A1 and

[0938]

[0939] A1 and

[0940]

[0941] A1 and

[0942]

[0943] wherein

[0944]

[0945]

[0946] wherein

[0947]

[0948]

[0949] wherein

[0950] wherein

[0951]

[0952] wherein

[0953] wherein

[0954]

[0955] wherein

[0956] wherein

[0957] wherein

[0958] wherein

[0959]

[0960] wherein

[0961] wherein

[0962] wherein

[0963] wherein

[0964]

[0965] wherein

[0966] wherein

[0967] wherein

[0968] wherein

[0969]

[0970] wherein

[0971] wherein

[0972] wherein

[0973] wherein

[0974]

[0975] wherein

[0976] wherein

[0977] wherein

[0978] wherein

[0979]

[0980] wherein

[0981] wherein

[0982] wherein

[0983] wherein

[0984]

[0985] wherein

[0986] wherein

[0987] wherein

[0988] wherein

[0989]

[0990] wherein

[0991] wherein

[0992] wherein

[0993] wherein

[0994]

[0995] wherein

[0996] wherein

[0997] wherein

[0998] wherein

[0999] In some embodiments, in the preparation of the conjugates of the present invention, the linker compounds having the affinity ligands of the following formula (VII), (VIII) or (IX) can be readily reacted first independently with the amino acids in the antibody, while reacting with the cytotoxic drug or cytotoxic drug / linker complex or subsequently condensed to form the conjugates of formula (I), (II) or (III); the linkers of formula (VII), (VIII) or (IX) shown in the following scheme can also be first reacted with the cytotoxic drug, while reacting with the amino acids in the antibody or subsequently condensed to form the conjugates of formula (I), (II) or (III):

[1000]

[1001] wherein, L1, L2, E1, Lv1and Lv2are the same as defined in formula (I), (II), (III), (IV), (V) and (VI) above; wherein Lv5and Lv6are independently selected from

[1002]

[1003] wherein X1is F, Cl, Br, I, OTs (tosylate), OTf (triflate), OMs (mesylate), OC6H4(NO2), OC6H3(NO2)2, OC6F5, OC6HF4, or Lv3; X2is O, NH, N(R1), or CH2; R3and R5are independently H, R1, aryl, heteroaryl, or an aryl group in which one or more H atoms are independently replaced with -R1, -halogen, -OR1, -SR1, -NR1R2, -NO2, -S(O)R1, -S(O)2R1, or -COOR1; Lv3and Lv3are leaving groups independently selected from F, Cl, Br, I, nitrophenoxide, N-hydroxysuccinimidyl (NHS), phenoxide, phenylmercapto, dinitrophenoxide, pentafluorophenoxy, tetrafluorophenoxy, difluorophenoxy, monofluorophenoxy, pentachlorophenoxy, triflate, imidazole, dichlorophenoxy, tetrachlorophenoxy, 1-hydroxybenzotriazole, tosylate, mesylate, 2-ethyl-5-phenylisoxazolium-3'-sulfonate, an anhydride formed by itself or with other anhydrides, such as acetic anhydride, formic anhydride, or an intermediate molecule resulting from a condensing reagent for peptide coupling reactions or Mitsunobu reactions; wherein functional groups Lv5and / or Lv6may also react with sulfhydryl groups in cytotoxic drugs, provided that they react at least one-fold faster or slower than Lv1or Lv2react with sulfhydryl groups in antibodies.

[1004] Examples of formula (VII), (VIII), and (IX) are as follows:

[1005]

[1006]

[1007]

[1008]

[1009]

[1010]

[1011] wherein

[1012]

[1013]

[1014]

[1015]

[1016]

[1017] wherein DU047, A1, A2and

[1018] A1and

[1019] A1and

[1020]

[1021]

[1022] In other embodiments, in the preparation of the conjugates of the present patent, the linker having the structure of Formula (X), (XI) or (XII) can first be reacted independently with the amino acids in the antibody, while or subsequently condensed with the binding ligand or binding ligand / linker complex, to form the conjugates of Formula (I), (II) or (III); the linker having the structure of Formula (X), (XI) or (XII) can also first be reacted with the cytotoxic drug, while or subsequently condensed with the amino acids in the antibody, to form the conjugates of Formula (I), (II) or (III):

[1023]

[1024]

[1025] wherein, D1, D2, L1, L2, E1, Lv1and Lv2are the same as defined in Formula (I), (II), (III), (IV), (V) and (VI) above; wherein Lv7, Lv8, Lv9, Lv 10 , Lv 11 and Lv 12 are independently selected from

[1026]

[1027] wherein X1' is F, Cl, Br, I, OTs (tosylate), OTf (triflate), OMs (mesylate), OC6H4(NO2), OC6H3(NO2)2, OC6F5, OC6HF4, or Lv3; X2' is O, NH, N(R1), or CH2; R3and R5are independently H, R1, aryl, heteroaryl, or an aryl group in which one or more H atoms are independently replaced with -R1, -halogen, -OR1, -SR1, -NR1R2, -NO2, -S(O)R1, -S(O)2R1, or -COOR1; Lv3and Lv3' are leaving groups independently selected from F, Cl, Br, I, nitrophenoxide, N-hydroxysuccinimidyl (NHS), phenoxide, phenylmercapto, dinitrophenoxide, pentafluorophenoxy, tetrafluorophenoxy, difluorophenoxy, monofluorophenoxy, pentachlorophenoxy, triflate, imidazole, dichlorophenoxy, tetrachlorophenoxy, 1-hydroxybenzotriazole, tosylate, mesylate, 2-ethyl-5-phenylisoxazolium-3'-sulfonate, an anhydride formed by itself or with other anhydrides, such as acetic anhydride, formic anhydride, or an intermediate molecule resulting from a condensing reagent for peptide coupling reactions or for Mitsunobu reactions; wherein functional groups Lv5and / or Lv6may also react with thiol or amino acid groups in the cytotoxic drug, provided that their reaction rate is at least one-fold faster or slower than the reaction rate of Lv1or Lv2with the thiol or amino acid groups in the antibody.

[1028] Examples of formula (X), (XI), and (XII) are as follows:

[1029]

[1030] wherein

[1031] D1is the same as DW001;

[1032] wherein D1=

[1033] m1and m2= 4, 6, or 8;

[1034] D1is the same as DW003;

[1035] D1is the same as DW003, m1= 4, 6, or 8;

[1036] D1is the same as DW003, m1= 4, 6, or 8;

[1037] D1 is the same as DW001, m1 = 4, 6 or 8;

[1038] D1 is the same as DW003;

[1039] D1 is the same as DW003;

[1040] D1 is the same as DW001;

[1041] D1 is the same as DW003;

[1042] D1 is the same as DW003, D2 =

[1043] D1 is the same as DW003;

[1044] D1 is the same as DW003, D2 is the same as DW013;

[1045] wherein

[1046] wherein D1 is the same as DW016;

[1047] wherein D1 is the same as DW016.

[1048] In some embodiments, during the preparation of the conjugates of the present application, the structure of formula (XIII), (XIV) or (XV) of the affinity ligand-containing linker can be readily reacted with a cytotoxic drug or a cytotoxic drug / linker complex to form the conjugates of formula (I), (II) or (III).

[1049]

[1050]

[1051] wherein L1, L2, La1, La2, Lb1, Lb2, Lc1, Lc2, Ld1, Ld2, Ld3, E1, A1, A2, A3, A4, A5, A6, n, m1, m2, m3, m4, m5, m6, m7, m8, m9, m 10 , m 11 , m 12 , mAb, Lv1', Lv2', Lv5 and Lv6 are the same as defined above.

[1052] Examples of formula (X), (XI) and (XII) are as follows:

[1053]

[1054]

[1055]

[1056] In some embodiments, in the preparation of conjugates of the present invention, linkers having the formula (XVI), (XVII) or (XVIII) shown below can be readily reacted with binding ligands or binding ligand / linker complexes to form conjugates of formula (I), (II) or (III).

[1057]

[1058] wherein L1, L2, La1, La2, Lb1, Lb2, Lc1, Lc2, Ld1, Ld2, Ld3, E1, n, m1, m2, m3, m4, m5, m6, m7, m8, m9, m 10 , m 11 , m 12 , mAb, Lv1', Lv2', Lv7, Lv8, Lv9, Lv 10 , Lv 11 and Lv 12 are as defined above.

[1059] Examples of formula (XVI), (XVII) and (XVIII) are as follows:

[1060]

[1061]

[1062] wherein D1and D3= D2and

[1063]

[1064] D2and

[1065] DZ011, wherein D1and

[1066] D2and

[1067] to distinguish between the reaction of Lv5 and / or Lv6 with the cytotoxic drug / cytotoxic drug linker complex, and the reaction of Lv1 and / or Lv2 with the amino acid in the antibody, and Lv7, Lv8, Lv9, Lv 10 , Lv 11 and Lv 12The reaction between the conjugate ligand / conjugate ligand linker complex, each step reaction of the compounds of formula (IV)-(IX) can be carried out in the same or different reaction vessels under different conditions. For example, the drug containing amino group can condense with the carboxylic acid group in the linker in the presence of condensing reagent such as EDC, TBTU or BrOP to give the modified drug / linker complex with amide bond. This condensation reaction can be carried out in a physiological buffer solution in which the carboxylic acid group at one end of the compound of formula (IV)-(IX) is activated by N-hydroxysuccinimidyl (NHS), pentafluorophenyl, dinitrophenyl ester, or carboxylic acid chloride, etc. groups which can react with the cytotoxic drug / cytotoxic drug linker complex, or the conjugate ligand / conjugate ligand linker complex, followed or simultaneously coupled with the thiol group of the antibody to produce the conjugate of formula (I), (II) or (III). In another example, the linker / payload complex of formula (IV), (V), (VI), (VII), (VIII) or (IX) has one end with a thiol-reactive group (e.g. maleimido, vinylsulfonyl, haloacetyl, acrylic, substituted propargyl) and the other end with a reactive group (e.g. hydroxysuccinimidyl (NHS), pentafluorophenyl, dinitrophenyl ester, amino, alkoxyamino, or a group that can undergo click chemistry (e.g. azide, alkyne, dibenzocyclooctyl, BCN ((1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethanol)) which can react with the cytotoxic drug / cytotoxic drug linker complex, or the conjugate ligand / conjugate ligand linker complex. The conjugate of formula (XIII), (XIV), (XV), (XVI), (XVII) or (XVIII) can be first reacted with the amino acid on the antibody in a buffer solution at pH 4.5 - 7.5, 2 °C - 40 °C (preferably 2 °C - 8 °C, more preferably 2 °C - 6 °C) with or without the addition of 0 - 30% water-miscible (miscible) organic solvent. Then, the cytotoxic drug / cytotoxic drug linker complex, or the conjugate ligand / conjugate ligand linker complex that matches the reactive group on the conjugate of formula (XIII), (XIV), (XV), (XVI), (XVII) or (XVIII) is added subsequently or simultaneously to give the conjugate of formula (I), (II) or (III). In the second step reaction, optionally, the conjugate of formula (XIII), (XIV), (XV), (XVI), (XVII) or (XVIII) can be purified before condensation with the cytotoxic drug / cytotoxic drug linker complex, or the conjugate ligand / conjugate ligand linker, and the condensation conditions of the second step can be adjusted to give a higher reaction rate, for example, the pH is adjusted to 6.5 - 8.0, and / or the temperature is adjusted to 20 - 45 °C.

[1068] In some embodiments, the antibody can be modified by a heterobifunctional crosslinker such as Formula (IV), (V), (VI) (VII), (VIII), or (IX) prior to conjugation with the cytotoxic drug / cytotoxic drug linker complex, or binding ligand / binding ligand linker complex, e.g., amine to sulfhydryl (succinimidyl (NHS) ester / maleimide, NHS ester / pyridyl dimethylthiole, NHS ester / haloacetyl), diazirine (SDA) to sulfhydryl, azido to sulfhydryl, alkynyl to sulfhydryl, sulfhydryl to sugar (maleimide / hydrazide, pyridyl dithiol / hydrazide, haloacetyl / hydrazide), hydroxyl to sulfhydryl (isocyanate / maleimide), sulfhydryl to DNA (maleimide / psoralen, pyridyl dithiol / psoralen, haloacetyl / psoralen), sulfhydryl to carboxyl (carbodiimide) prior to conjugation with the cytotoxic drug / cytotoxic drug linker complex, or binding ligand / binding ligand linker complex, during the conjugation process, in some embodiments.

[1069] The reactive groups in the cytotoxic drug / cytotoxic drug linker complex, or binding ligand / binding ligand linker complex, react with the modified antibody-linker conjugate of formula (XIII), (XIV), (XV), (XVI), (XVII), or (XVIII) in different ways to produce the final conjugate. For example, for conjugates linked by disulfide bonds, the first step, the linker of formula (IV), (V), (VI), (VII), (VIII), or (IX), is coupled to the antibody at 2°C to 8°C, pH 4.5 to 6.0, followed by a disulfide exchange reaction of the disulfide linkage (e.g., pyridyl disulfide moiety) on the linker with the cytotoxic drug / cytotoxic drug linker complex, or binding ligand / binding ligand linker complex, containing a free thiol group at 20°C to 40°C, pH 6.5 to 8.0. Excess reducing agent (e.g., TCEP or tris(3-hydroxypropyl)phosphine) in the reaction is preferably removed or quenched by the addition of an azide compound (e.g., 4-(azidomethyl)benzoic acid) prior to the addition of the cytotoxic drug / cytotoxic drug linker complex, or binding ligand / binding ligand linker complex, containing a free thiol group. For conjugates linked by thioether bonds, the antibody-linker conjugate of formula (XIII), (XIV), (XV), (XVI), (XVII), or (XVIII) is first reacted with the thiol-reactive group on the linker, which contains a maleimido, haloacetyl, ethylsulfonyl, or substituted propargyl group at both ends, with the thiols on the antibody which were reduced by the methods of this application at 2°C to 8°C, pH 4.5 to 6.0, to produce the antibody-linker conjugate, followed by reaction with the cytotoxic drug / cytotoxic drug linker complex, or binding ligand / binding ligand linker complex, containing a thiol group at 20°C to 40°C, pH 6.5 to 8.0, to produce the conjugate of formula (I), (II), or (III). If the same pH and / or temperature conditions are chosen for both steps, then more than four equivalents of the linker containing a disulfide-reactive group at both ends are used for the coupling. It should be noted that the preferred method for the synthesis of disulfide or thioether-linked conjugates is to first chemically synthesize the drug-linker complex containing a disulfide or thioether bond of formula (IV), (V), or (VI); and then react with the thiols on the protein (antibody) according to the methods of this application. The synthesis of conjugates linked by acid-labile hydrazone bonds can be achieved by the reaction of a carbonyl group with a hydrazide moiety on the linker according to methods known in the art (see, e.g., 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).Synthesis of conjugates with triazole linkage can be achieved by click chemistry through reaction of the 1 -alkyne group of the drug with the azido moiety in the linker (Huisgen cycloaddition) (Lutz J-F. et al., 2008, Adv. Drug Del. Rev., 60, 958-70; Sletten E.M. et al., 2011, Acc Chem. Research, 44, 666-76). Synthesis of oxime-linked conjugates is achieved by reaction of a modified antibody containing a ketone or aldehyde with a cytotoxic drug / cytotoxic drug linker complex or binding ligand / binding ligand linker complex containing an oxamido group. Cytotoxic drug / cytotoxic drug linker complexes or binding ligand / binding ligand linker complexes containing a hydroxyl or thiol group can be reacted with a modified linker containing a halogen, in particular an alpha halide of a carboxylate ester, such as formula (VII), (VIII), (IX), (X), (XI) or (XII), in the presence of a mild base, such as pH 8.0-9.5, to give a modified drug / linker complex containing an ether or thioether linkage, such as formula (IV), (V), (VI), (VII), (VIII) or (IX). Cytotoxic drug / cytotoxic drug linker complexes containing a hydroxyl group can be condensed with a linker containing a carboxyl group, such as formula (VII), (VIII), (IX), (X), (XI) or (XII), in the presence of a dehydrating agent such as EDC or DCC, to give an ester linkage, such as formula (IV), (V), (VI), (VII), (VIII) or (IX), and then the drug / linker complex is coupled to the antibody using the methods of the application to give a conjugate such as (I), (II), (III). Cytotoxic drug / cytotoxic drug linker complexes or binding ligand / binding ligand linker complexes containing an amino group can be reacted with an active ester, such as an NHS ester, imidazole ester, nitrophenoxide ester, N-hydroxysuccinimidyl ester (NHS), methylsulfonylphenoxide ester, dinitrophenoxide ester, pentafluorophenoxide ester, tetrafluorophenoxide ester, difluorophenoxide ester, monofluorophenoxide ester, pentachlorophenoxide ester, triflate ester, imidazole ester, dichlorophenoxide ester, tetrachlorophenoxide ester, 1 -hydroxybenzotriazole ester, tosylate ester, mesylate ester, 2-ethyl-5-phenylisoxazole-3'-sulfonate ester, on the formula (XIII), (XIV), (XV), (XVI), (XVII) or (XVIII), to give a conjugate linked through an amide linkage, such as formula (I), (II) or (III).Many conventional chemical and biochemical processes for antibody-drug conjugation are known in the art (see, e.g., Matsuda Y. and Mendelsohn B. A., Expert Opin Biol Ther., 2021, 21(7):963-975; Puthenveetil S., Methods Mol Biol., 2020, 2078:99-112; van Delft, F., and Lambert J. M. eds. Chemical Linkers in Antibody-Drug Conjugates (ADCs), Royal Soc. Chem. Pub., 22, Dec. 2021, ISBN 978-1-83916-263-3, doi:10.1039 / 9781839165153; Tumey L. N. ed. Antibody-Drug Conjugates, Methods and Protocols, Springer Pub., 2020, ISBN: 978-1-4939-9929-3; Khongorzul, P. et al., Mol Cancer Res. 2020, 18(1):3-19; and many references contained in these books and papers). More preferably in all of the above protocols, the conjugates of formula (I), (II) and (III) are directly conjugated to the antibody in water-based solution by formula (IV), (V) and (VI), and the compounds in formula (IV), (V) and (VI) are constructed by chemical synthesis.

[1070] In some embodiments, the antibody drug conjugate is preferably prepared by a homogeneous conjugation process, which includes the following three key steps:

[1071] (a) incubating the antibody in a buffer system (e.g., PBS, Mes, Bis-Tris, Bis-Tris Propane, Pipes, Aces, Mopso, Bes, Mops, Hepes, Tes, Pipps, Dipso, Tapso, Heppso, Tris-up, Tris-HCl, Tricine, Hepps, Gly-Gly, Bicine, Taps, Hepee, Acetates, Histidine, Citrates, MES or Borates, etc.) containing an effective amount of zinc cation-amino chelate / complex (Zn(NR1R2R3) m1 m2+ ) and a reducing agent (e.g., tris(2-carboxyethyl)phosphine (TCEP)), selectively reducing interchain disulfide bonds within the antibody, generating thiols;

[1072] (b) adding an effective amount of a cytotoxic drug linker of formula (IV), (V) or (VI) with a thiol-reactive group (e.g., a drug with a maleimide end) to react with the thiol groups produced in step (a); and

[1073] (c) adding an effective amount of an oxidizing agent (e.g., dehydroascorbic acid (DHAA)) to re-oxidize unreacted thiols, followed by purification of the resulting conjugate;

[1074] (d) Step (c) can also be replaced by adding an effective amount of cystine to quench excess linker or linker / payload complex with thiol-reactive groups (e.g., maleimides) while simultaneously or sequentially adding an azide compound (e.g., 4-(azidomethyl)-benzoic acid) or a disulfide compound (e.g., cystine) to quench unreacted reducing agent (e.g., TCEP or tris(hydroxypropyl)phosphine). The addition of cystine to quench unreacted reducing agent (e.g., TCEP) can form cysteine, which can simultaneously quench excess conjugated linker or linker / payload complex with thiol-reactive groups (e.g., maleimides) of formula (IV), (V) or (VI).

[1075] wherein Zn(NR1R2R3) m1 m2+ R1, R2and R3in the formula are independently selected from C1-C8alkyl; C2-C8heteroalkyl, alkylcycloalkyl, heterocycloalkyl; C3-C8aryl, aryl-alkyl, heterocycle, carbocycle, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; m1is selected from 1, 2, 3, 4, 5, 6, 7 or 8; m1may be 1, 2, 3 or 4.

[1076] In addition, (NR1R2R3) m1 Dimer, trimer, tetramer, pentamer or hexamer can be formed, which are covalently linked between N, R1, R2and R3; N, R1, R2or R3itself can form a heterocycle, carbocycle, diheterocycle or dicarbocycle.

[1077] The concentration of the zinc cation-amino chelate / complex (Zn(NR1R2R3) m1 m2+ ) used in step (a) is 0.01 mM to 1.0 mM, or 0.5 to 20 protein molar equivalents, which is dissolved in a water-soluble organic solvent selected from ethanol, methanol, propanol, propylene glycol, DMA, DMF, DMSO, THF, CH3CN, and then added to the reaction system.

[1078] The reducing agent is an organic phosphine, preferably tris(2-carboxyethyl)phosphine (TECP) or tris(hydroxypropyl)phosphine, at a concentration of 0.02 mM to 1.0 mM, or 1.0 to 20 protein molar equivalents in the reaction solution. The oxidizing agent added in step (c) can be DHAA, Fe 3+ , I2, Cu 2+ , Mn 3+ , MnO2, or Fe 3+ / I - mixtures. The concentration of the oxidizing agent used in the reaction solution is 0.02 mM to 1.0 mM, or 0.2 to 100 protein molar equivalents. The preferred pH for the coupling reaction is generally between about 5.0 and 8.0, more preferably about 5.5 to 7.5. The preferred temperature for the coupling reaction is generally between about -5 and about 40 °C, more preferably about 0 to 37 °C; optimally about 2 to 8 °C; further, about 2 to 6 °C. The preferred time for the coupling reaction is generally between about 15 minutes and about 48 hours, more preferably about 30 minutes to overnight (10-16 hours), more preferably about 2 hours to 6 hours. The optimal reaction conditions (e.g., pH, temperature, buffer, reactant concentrations) will of course depend on the antibody-like protein, payload / linker complex, reducing agent, and / or Zn(NR1R2R3) m1 m2+ .

[1079] In further embodiments, in a homogeneous coupling process, the linkage between the cysteine sites of the heavy-light chains of the resulting conjugate of formula (I), (II), or (III) to the antibody is greater than 75%, and the linkage between the cysteine sites of the heavy-heavy chains of the antibody (hinge region) is less than 15%. Typically, for formula (I), (II), or (III), when the drug / antibody ratio (DAR) is 4, the percentage distribution of the number of drugs in the antibody is: DO < 1%, D2 < 10%, D4 > 65%, D6 < 10%, D8 < 10%; for formula (III).

[1080] The resulting conjugate can be purified by standard biochemical means, such as Sephadex G25 or Sephacryl S300 column gel filtration, adsorption chromatography, ion (cationic or anionic) exchange chromatography, or dialysis (ultrafiltration (UF) and diafiltration (DF)). In some cases, the antibody small molecule conjugated to a small molecule drug (e.g., < 100 KD) can be purified by chromatography, such as HPLC, medium pressure column chromatography, or ion exchange chromatography.

[1081] Typically, the conjugate of formula (I), (II) or (III) is preferably prepared from the drug / linker complex of formula (IV), (V) or (VI) in a one-pot reaction. When the antibody is reduced to generate a thiol group that reacts with the thiol-reactive group at the end of the drug / linker complex of formula (IV), (V) or (VI), the Ellman reagent can be used to measure the amount of free thiol groups during the course of the reaction, and thus monitor the reduction of the disulfide bond and the conjugation of the thiol group. The conjugation reaction can be monitored using UV spectroscopy with a wavelength range of 190-390 nm, preferably 240-380 nm, more preferably 240-370 nm. Thus, the conjugation reaction can be performed and monitored in a quartz cuvette or Pyrex tube in a controlled temperature environment. The drug / protein (antibody) ratio (DAR) of the conjugate can also be obtained by calculating the concentration from the absorbance of the drug and protein in the wavelength range of 240-380 nm, or by measuring the hydrophobic interaction chromatography (HIC-HPLC) integrated area of each drug / protein fragment, or by capillary electrophoresis (CE), and / or LC-MS or LC-MS / MS or CE-MS (liquid chromatography (LC) or CE coupled with mass spectrometry (MS) to measure the LC or CE integrated area and MS peak intensity of each drug / protein fragment). It should also be noted that in the conjugation process of the present application, when the solubility of the drug or drug / linker complex in the water-based buffer solution is not good, up to 30% of an organic solvent that is miscible (miscible) with water, such as DMA, DMF, ethanol, methanol, acetone, acetonitrile, tetrahydrofuran, isopropanol, dioxane, propylene glycol or ethylene glycol, can be added as a co-solvent in the water-based buffer solution.

[1082] The pH of the aqueous buffer system for the antibody class protein modification reaction is between 4 and 9, preferably between 6.0 and 7.5, and can contain any non-nucleophilic buffer salt available in these pH ranges. Typical buffers include phosphate, acetate, triethanolamine hydrochloride, HEPES and MOPS buffers, which can contain other components such as cyclodextrin, sucrose and salts such as NaCl and KCl. The defined section lists other biological buffers that can be used in the conjugation process. The progress of the reaction can be measured by measuring the decrease in absorbance at a specific UV wavelength (e.g. 254 nm) or other suitable wavelength, or the increase in absorbance at a specific UV wavelength (e.g. 280 nm) or other suitable wavelength. After the reaction is complete, the separation of the modified cell-binding antibody can be performed in a conventional manner, for example using gel filtration chromatography or adsorption chromatography.

[1083] When the antibody is modified using a disulfide exchange reaction, the extent of modification can be assessed by measuring the UV spectral absorbance of the nitropyridinethione, dinitropyridinedithione, pyridinethione, carboxyaminopyridinedithione, or dicarboxyaminopyridinedithione group released. If the chromophore group is not present, the modification or conjugation reaction can be monitored by LC-MS (preferably UPLC-QTOF mass spectrometry), or capillary electrophoresis-mass spectrometry (CE-MS). The linker compound can be reacted with a drug, preferably a cytotoxic agent, having a suitable substituent. For example, a modified antibody with an amino or hydroxyl substituent can be reacted with a drug having an N-hydroxysuccinimide (NHS) ester, a modified antibody with a thiol substituent can be reacted with a drug having a maleimido or haloacetyl group. In addition, a modified antibody with a carbonyl (ketone or aldehyde) substituent can be reacted with a drug having a hydrazide or alkoxyamine substituent. One skilled in the art can readily determine which linker to use based on the reactivity of the available functional groups on the linker.

[1084] Formulations and uses

[1085] The conjugates of the present patent application are formulated as liquids, or in a form suitable for lyophilization, and reconstituted as a liquid formulation after lyophilization. The conjugates in a liquid formulation or formulated lyophilized powder are the major component of the formulation, and comprise from 0.01% to 99% by weight. In general, liquid formulations that can be administered to a patient without high levels of antibody aggregation comprise, in addition to the conjugate active ingredient, from 0.1 g / L to 300 g / L, one or more polyols (e.g., sugars), a buffer at a pH of 4.5 to 7.5, a surfactant (e.g., polysorbate 20 or 80), an antioxidant (e.g., ascorbic acid and / or methionine), a bulking agent (e.g., mannitol, sorbitol, or sodium chloride), a chelator (e.g., EDTA), a metal complex (e.g., zinc-protein complex), a biodegradable polymer (e.g., a polyester), a preservative (e.g., benzyl alcohol), and / or a free amino acid.

[1086] Suitable buffers for the formulations include, but are not limited to, organic acid salts such as sodium, potassium, ammonium, or trihydroxyethylamino salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid, tromethamine hydrochloride, sulfuric acid, or phosphoric acid buffers. In addition, amino acid cations can also be used as buffers. These amino acids include, but are not limited to, arginine, glycine, glycinylglycine, and histidine. Arginine buffers include arginine acetate, arginine chloride, arginine phosphate, arginine sulfate, arginine succinate, and the like. 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, and the like. The pH of the buffer is from 4.5 to pH 7.5, preferably from about 4.5 to about 6.5, more preferably from about 5.0 to about 6.2. In some embodiments, the concentration of the organic acid salt in the buffer is from about 10 mM to about 500 mM.

[1087] An optional "polyol" in the formulation is a substance having multiple hydroxyl groups. Polyols can be used as stabilizing excipients and / or isotonicity agents in liquid and lyophilized formulations. Polyols can protect biopharmaceuticals from physical and chemical degradation. Co-solvents that are preferably excluded increase the effective surface tension of solvents at protein interfaces, and the most energetically favorable structural conformation is that with the smallest surface area. Polyols include sugars (reducing and non-reducing sugars), sugar alcohols, and sugar acids. "Reducing sugars" are sugars that contain a hemiacetal group that is capable of reducing metal ions, or reacting with lysine and other amino groups in proteins. "Non-reducing sugars" are sugars that do not have the properties 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, mannose, and raffinose. Sugar alcohols are selected from the group consisting of mannitol, xylitol, erythritol, maltitol, lactitol, erythritol, threitol, sorbitol, and glycerol. Sugar acids include L-gluconate and metal salts thereof. The polyol content in the liquid formulation or lyophilized formulation is from 0.0% to 20% by weight. Non-reducing sugars, sucrose or trehalose, are preferred in the formulation at concentrations of about 0.1% to 15%, with trehalose being more preferred due to better solution stability.

[1088] The optional surfactant in the formulation can be selected from the group consisting of polysorbates (polysorbate 20, polysorbate 40, polysorbate 65, polysorbate 80, polysorbate 81, polysorbate 85, etc.); poloxamers (e.g., poloxamer 188, poly(ethylene oxide)-poly(propylene oxide), poloxamer 407, or polypropylene glycol-propylene glycol, etc.); Triton; sodium dodecyl sulfate (SDS); sodium lauryl sulfate; sodium octyl glycoside; dodecyl, myristyl, linoleyl, or stearyl sulfobetaine; dodecyl, myristyl, linoleyl, or stearyl sarcosine; linoleic, myristyl, or cetyl betaine; lauroamidopropyl, cocamidopropyl, linoleamidopropyl, myristamidopropyl, palmitamidopropyl, or isostearamidopropyl-betaine (e.g., lauroamidopropyl); myristamidopropyl, palmitamidopropyl, or isostearamidopropyl-dimethylamine; methyl cocoyl sodium or methyl oleyl taurate disodium; dodecyl betaine, dodecyl dimethyl amine oxide, cocamidopropyl betaine, and cocoamphoglycinate; MONAQUAT TM series (e.g., isostearyl ethyl imidonium ethyl sulfate); polyethylene glycol, polypropylene glycol, and copolymers of ethylene glycol and propylene glycol (e.g., Pluronic, PF68, etc.). A preferred surfactant is a polyoxyethylene sorbitol fatty acid ester, such as polysorbate 20, 40, 60, or 80 (Tween 20, 40, 60, or 80). The concentration of surfactant in the formulation ranges from 0.0% to about 2.0% by weight. In certain specific embodiments, the surfactant concentration is from about 0.01% to about 0.2%. In one embodiment, the surfactant concentration is about 0.02%.

[1089] The optional "preservative" in the formulation is a compound that can inhibit bacteria. Examples of preservatives include octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyl dimethylammonium chlorides where the alkyl groups are long alkyl groups), and benzethonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butyl and benzyl alcohol, alkyl esters of p-hydroxybenzoic acid such as methyl or propyl esters, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol. The amount of preservative in the liquid formulation or lyophilized powder ranges from 0.0% to 5.0% by weight. In one embodiment, the preservative used is benzyl alcohol.

[1090] The free amino acids in the formulation as bulking or tonicity or osmotic pressure adjusting agents are selected from, but not limited to, one or more of arginine, cystine, glycine, lysine, histidine, ornithine, isoleucine, leucine, alanine, glycine glutamic acid or aspartic acid. Preferred basic amino acids are arginine, lysine and / or histidine. If histidine is included in the composition, it can serve as a buffer and a free amino acid, but when a histidine buffer is used, a non-histidine free amino acid such as lysine is usually also included. The amino acids can be present in the D- and / or L-form, but the L-form is more common. The amino acids can be present in the form of any suitable salt, such as arginine hydrochloride. The amino acid content in the liquid formulation or lyophilized powder is 0.0% to 30% by weight.

[1091] Optionally, the formulation also includes methionine, glutathione, cysteine, cystine or ascorbic acid as an antioxidant, in a concentration of up to about 5 mg / mL in a liquid formulation, and in a content of 0.0% to 5.0% by weight in a lyophilized powder; optionally, the formulation includes a metal chelator, such as EDTA, EGTA, and the like, in a concentration of about 2 mM in a liquid formulation, and in a content of 0.0% to 0.3% by weight in a lyophilized powder.

[1092] The final formulation can be adjusted to the preferred pH with a buffer adjuster, such as an acid, including HC1, H2S04, acetic acid, H3P04, citric acid, and the like, or a base, such as NaOH, KOH, NH4OH, ethanolamine, diethanolamine or triethanolamine, sodium phosphate, potassium phosphate, trisodium citrate, tromethamine, and the like. The formulation should also be adjusted to be "isotonic", i.e., the target formulation has essentially the same osmotic pressure as human blood. Isotonic formulations typically have an osmotic pressure of 250 to 350 mOsm. Isotonicity can be measured using a vapor pressure or freezing point osmometer. The isotonic agent is selected from mannitol, sorbitol, sodium acetate, potassium chloride, sodium phosphate, potassium phosphate, trisodium citrate, or NaCl. Typically, the buffer salt and isotonic agent are included in the formulation at a level of up to 30% by weight.

[1093] Other excipients that can be useful in the liquid or lyophilized formulations herein include, for example, fucose, cellobiose, maltotriose, melibiulose, octulose, ribose, xylitol, arginine, histidine, glycine, alanine, methionine, glutamic acid, lysine, imidazole, glycine, mannosyl glycerate, Triton X-100, Puloronic F-127, cellulose, cyclodextrin, (2-hydroxypropyl)-beta-cyclodextrin, dextran (10, 40 and / or 70 kD), polydextrose, ficoll, gelatin, hydroxypropyl methyl, sodium phosphate, potassium phosphate, zinc chloride, zinc, zinc oxide, sodium citrate, trisodium citrate, tromethamine, copper, fibronectin, heparin, human serum albumin, protamine, glycerol, EDTA, m-cresol, benzyl alcohol, phenol, polyols, reduced carbohydrates in which a carbonyl group is reduced to a primary or secondary alcohol.

[1094] Other excipients that can be used in the liquid formulations herein include, for example, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, lipids such as phospholipids or fatty acid lipids, steroids such as cholesterol, protein excipients such as serum albumin (human serum albumin), recombinant human albumin, gelatin, casein, salt-forming counterions such as sodium, and the like. These and other known pharmaceutical excipients and / or additives suitable for use in the formulations of the present application are well known in the art, as listed in The Handbook of Pharmaceutical Excipients, 4thEdition, edited by Rowe et al., American Pharmaceutical Association; and Remington: the Science and Practice of Pharmacy, 21stEdition, edited by Gennaro et al., published by Lippincott Williams & Wilkins (2005).

[1095] Pharmaceutical containers or devices that hold the conjugate formulations herein include vials, bottles, pre-filled syringes, pre-filled or auto-injectors. The liquid formulations can be lyophilized or roller-dried in borosilicate or sodium calcium glass vials, or as cakes or powders. The solid powders can also be prepared by effective spray-drying and then packaged in vials or pharmaceutical containers for storage and distribution.

[1096] In yet further embodiments, the present application provides methods of preparing a formulation comprising the steps of: (a) lyophilizing a liquid comprising the conjugate, a bulking agent and a buffer system; and (b) reconstituting the lyophilized mixture of step (a) in a medium such that the reconstituted formulation is stable. The liquid of step (a) can further comprise a stabilizer and one or more bulking agents selected from the group consisting of the aforementioned fillers, salts, surfactants and preservatives. The reconstitution medium can be water, such as sterile water, bacteriostatic water for injection (BWFI), acetic acid, propionic acid, succinic acid, sodium chloride, magnesium chloride, acidic solutions of sodium chloride, magnesium chloride or arginine, in amounts of about 10 to about 250 mM.

[1097] Liquid formulations of the conjugates of the present patent application should have various set characteristics. One of the main issues to be considered is its stability, since proteins / antibodies often form soluble and insoluble aggregates during manufacturing and storage. In addition, various chemical reactions (deamidation, oxidation, clipping, isomerization, etc.) occur in solution, leading to increased levels of degradation products and / or loss of biological activity. The conjugate in the liquid or lyophilized formulation should preferably have a shelf life of more than 6 months at 25°C. More preferably, the conjugate in the liquid or lyophilized formulation should have a shelf life of more than 12 months at 25°C. Most preferably, the liquid formulation should have a shelf life of about 24 to 36 months at 2-8°C and the lyophilized powder should have a shelf life of up to about 60 months at 2-8°C. The liquid and lyophilized formulations should have a shelf life of at least two years at -20°C or -70°C.

[1098] In some embodiments, the formulation is stable after freezing (e.g., at -20°C or -70°C) and thawing, e.g., after 1, 2, or 3 freeze and thaw cycles. Stability can be assessed qualitatively and / or quantitatively in various ways, including assessing the specific activity of the drug / antibody (protein) and formation of aggregates (e.g., using UV, size exclusion chromatography, by measuring turbidity, and / or by visual inspection); assessing charge heterogeneity by using cation exchange chromatography, image capillary isoelectric focusing (icIEF), or capillary zone electrophoresis; amino terminal or carboxy terminal sequence analysis; mass spectrometry analysis, matrix assisted laser desorption ionization / time of flight mass spectrometry (MALDI / TOF MS), HPLC-MS / MS, or SDS-PAGE analysis to compare reduced and intact antibody; peptide mapping analysis (e.g., trypsin or LYS-C); assessing biological activity or antigen binding function of the antibody. Instability can be due to one or more of the following: aggregation, deamidation (e.g., Asn deamidation), oxidation (e.g., Met oxidation), isomerization (e.g., Asp isomerization), clipping / hydrolysis / breakage (e.g., hinge breakage), succinimide formation, unpaired cysteines, N-terminal extensions, C-terminal processing, glycosylation differences, etc.

[1099] The stabilized conjugate should "retain its biological activity" in the pharmaceutical formulation, e.g., the biological activity of the conjugate can be retained within 20%, preferably 10% (within the error of the assay) over a given period of time, e.g., 24 months, according to the methods of antigen binding assays and / or in vitro cytotoxicity assays.

[1100] For clinical in vivo use, the conjugate of the application will be provided in the form of a solution or a lyophilized solid that can be reconstituted in sterile water for injection. Examples of modes of administration of the conjugate are as follows: once a day, once a week, once every two weeks, once every three weeks, once every four weeks, or once a month, for 8-108 weeks, as an intravenous bolus. The administration is in 50 to 1000 mL of normal saline, optionally to which human serum albumin can be added (e.g., 0.5 to 1 mL of hu...

Claims

1. Antibody drug conjugates with branched affinity ligands, as shown in formula (I), (II) and (III): wherein, D1 and D2 are cytotoxic drugs; mAb is an antibody or antibody-like protein; n is 1-20; L1, L2, La1, La2, Lb1, Lb2, Lc1, Lc2, Ld1, Ld2, Ld3, Ld4, Ld5, Ld6are linkers independently selected from O, NH, S, N, NH-NH, N-N, N(R3), N(R3)N(R3'), C(=O)N, C(=O)NH, C(=O)N-N, C1-C8alkyl; C2-C8heteroalkyl, alkylcycloalkyl, heterocycloalkyl; C3-C8aryl, arylalkyl, heterocycle, carbocycle, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; or ester, ether or amide of 1-8 carbon atoms; or 1-8 natural or non-natural amino acids as described in the definition; or polyethyleneoxy units of the structure (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 COOR3, wherein p and p' are independently selected from an integer from 0 to about 1000, or a combination thereof; wherein R3and R3' are H, C(=O)H, C(=O)CH3, C1-C8alkyl; or a combination of the two; Lv1' and Lv2' are functional groups independently linked to an amino acid of the antibody or antibody-like protein; Lv1' and Lv2' independently have the following structure: wherein is the site of attachment of the drug or the site of attachment of the linker L1or L2; "#" is the site of attachment of the antibody S (sulfhydryl), O (phenol), NH (amino), CHO (aldehyde), C(=O) (ketone), C(O)(NH) (amide), and C(O)(OH) (carboxylate); wherein R1, X1and X2are as described above; X is O, NH, S, CH2; the connecting bond "-" in between two atoms means that it can connect either of the two atoms, Ar is an aromatic group. E1 is a linker linking the two reactive groups Lv1 and Lv2. E1 is selected from CH, CH2, CH-CH, NH, NHNH, N(R3), N(R3)N(R3'), N=N, N-N, P, P(=0), S, Si, C2-C8 alkyl, heteroalkyl, alkylcycloalkyl, heterocycloalkyl; C3-C8 aryl, aryl-alkyl, heterocycle, carbocycle, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; a peptide containing 1-4 amino acid units, preferably selected from aspartic acid, glutamic acid, arginine, histidine, lysine, serine, threonine, asparagine, glutamine, cysteine, selenocysteine, tyrosine, phenylalanine, glycine, proline, tryptophan, alanine; m1, m2, m3, m4, m5, m6, m7, m8, m9, m 10 , m 11 and m 12 are each 1-10; further, m2, m3, m8, m9 and / or m 10 may be 0, in which case Ld2-A2, Ld3-A3, Ld5-A5 and / or Ld6-A6 can be omitted; A1, A2, A3, A4, A5 and A6 are affinity ligands, independently selected from small molecules of glutamic acid urea or its analogs, or / and affinity ligands of bombesin receptors / neurotensin receptors (including neuropeptide-Y receptors) and / or cell-penetrating peptides. A1, A2, A3, A4, A5 and A6 are independently selected from: wherein is a site attached to Ld1, Ld2, Ld3, Ld4, Ld5, or Ld6; Ra is Ar, preferably selected from the group consisting of: Rb is OH, COOH, COOCH3, CH3OH, CH3NH2, CONH2.

2. According to claim 1, the affinity ligands A1, A2, A3, A4, A5 and A6 are independently, and have affinity for 2-[3-(1,3-dicarboxypropyl)ureido]-pentanedioic acid (DUPA) receptors, bombesin receptors (including gastrin-releasing peptide receptor (GRPR) and neurotensin receptors (including neurotensin receptor 1 (NTR1) and neuropeptide-Y receptors) and / or cell-penetrating peptides (CPP). The affinity of the ligand for the receptor is EC50 < 100 nM. A CPP is a linear or cyclic peptide of less than 50 amino acids, containing one, two or more arginines or lysines, capable of endocytosing (transporting) more than 40% of the ligand bound on the cell, or helping to endocytose 40% of the ADC bound on the cell, crossing the cell membrane within 2 hours.

3. According to claim 1, the cytotoxic drugs D1 and D2 are independently selected from: (1) Chemotherapeutic drugs: a). alkylating agents selected from nitrogen mustards: chiorambucil, chloro-napaz, cyclophosphamide, dacarbazine, estramustine, ifosfamide, mechlorethamine, mechloroethanolamine hydrochloride, mechlorethamine oxide hydrochloride, melphalan, myleran, piposulfan, ranimustine, thioepa, uramustine; CC-1065 and its analogues adozelesin, carzelesin, bizelesin; duocarmycin and its synthetic analogues KW-2189 and CBI-TMI or CBI dimers; benzodiazepine dimers or pyrrolobenzodiazepine (PBD) dimers, tomaymycin dimers, indolinobenzodiazepine, imidazophenylthiazole-diazepine or oxazolidine-benzodiazepine dimers; nitrosoureas: including carmustine, lomustine, streptozocin, fotemustine, nimustine, ranimustine; alkyl sulfonates: including busulfan, treosulfan, sulfisoxazole and pipsulfan; triazenes or dacarbazine; platinum-containing compounds: carboplatin, cisplatin, oxaliplatin; aziridines, benzodiazepine, carboquone, meturedepa and urethan; ethylenimines and methyl triazenes, including hexamethylmelamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolmelamine; b) Plant alkaloids: selected from the group consisting of vinca alkaloids: including vincristine, vinblastine, vindesine, vinorelbine, desmethylvinblastine; taxols: including paclitaxel, docetaxel and analogs thereof; maytansinoids including DM1, DM2, DM3, DM4, DM5, DM6, DM7, maytansine, ansamitocin and analogues thereof; cryptophycins (including cryptophycin 1 and cryptophycin 8); epothilones, halichondrins, dolastatins, ecteinascidins, sarcodictyins, auristatins, a microtububule toxins, cephalostatin; pancratistatin; erbulins, a sar-codictyin; halichondrins; c). DNA topoisomerase inhibitors: selected from etoposide teniposide: including 9-aminocamptothecin, camptothecin, lurtotecan, duocarmycin, etoposide, etoposide phosphate, irinotecan, mitoxantrone, novobiocin, retinoic acid (retinol), teniposide, topotecan, 9-nitrocamptothecin or RFS2000; mitomycin and analogues thereof; d). antimetabolites: selected from {[antifolates: (DHFR inhibitors: including methotrexate, trimetrexate, dimethyl folic acid, palafoxin, aminopterin (4-aminobenzoic acid) or other folic acid analogues; IMP dehydrogenase inhibitors: (including mycophenolic acid, tiazofurin, ribavirin, EICAR); ribonucleotide reductase inhibitors: (including hydroxyurea, desferrioxamine)] [pyrimidine analogs, uracil analogs: (including ancitabine, azacitidine, 6-azauracil, capecitabine (Xeloda), carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, 5-fluorouracil, floxuridine, ratitrexed (Tomudex)); cytosine analogs: (including cytarabine, cytosine arabinoside, fludarabine); purine analogs (including azathioprine, fludarabine, mercaptopurine, thiamiprine, thioguanine)]; folate supplements, floxuridine; nicotinamide phosphoribosyltransferase (NAMPT) inhibitors; e). Hormonal therapy agents: selected from {receptor antagonists: [anti-estrogens: (including megestrol, raloxifene, tamoxifen), LHRH agonists: (including goserelin, leuprolide acetate); anti-androgens: (including bicalutamide, flutamide, casodex, dihydrotestosterone propionate, epitestosterone, goserelin, leuprolide, meptiiridine, nilutamide, testolactone, trilostane and other androgen inhibitors)]; retinoids: [vitamin D3 analogs: (including CB1093, EB1089, KH1060, cholecalciferol, ergocalciferol); photodynamic therapy agents: (including verteporfin, phthalocyanines, photosensitizer Pc4, des-methoxy- pyrromethene A); cytokines: (including interferon-alpha, interferon-gamma, tumor necrosis factor (TNF), human proteins containing TNF)]}; f). Kinase inhibitors selected from 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, vismodegib, iniparib, ruxolitinib, CYT387, axitinib, tivozanib, sorafenib, bevacizumab, cetuximab, trastuzumab, ranibizumab, panitumumab, ipatasertib; g). Poly (ADP-ribose) polymerase (PARP) inhibitors, olaparib, niraparib, iniparib, talazoparib, veliparib, CEP 9722 (Cephalon’s), E7016 (Eisai’s), BGB-290 (Beijing Genomics Institute) or 3-aminobenzamide. h) antibiotics such as enediyne antibiotics (calicheamicin, especially calicheamicin gammal, deltal, alpha 1 and beta 1, dynemicin, including dynemicin A and deoxymycin, esperamicins, catharanthine, C-1027, maduropeptin, neocarzatinostin and related chromoprotein enediyne antibiotics), aclacinomysins, actinomycin, anthramycin, azaserine, bleomycins, cactinomycin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino doxorubicin and deoxydoxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, nitomycin, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; i). polyketides (campothecins), in particular bullatacin and bullatacinone; gemcitabine, epoxide hydrolase (e.g. carzelesin), bortezomib, thalidomide, lenalidomide, pomalidomide, tosedostat, zybrestat, PLX4032, STA-9090, Stimuvax, allovectin-7, Xegeva, Provenge, Yervoy, isoprenylation inhibitors and lovastatin, dopaminergic neurotoxins and 1-methyl-4-phenylpyridinium ion, cell cycle inhibitors (e.g. staurosporine), dactinomycin (e.g. actinomycin D, actinomycin), bleomycin (e.g. bleomycin A2, bleomycin B2, pepleomycin), anthracyclines (e.g. daunorubicin), amatoxins, doxorubicin (adriamycin), idarubicin, epirubicin, pirarubicin, valrubicin, mitoxantrone, MDR inhibitors or verapamil, Ca 2+ ATPase inhibitors or thapsigargin, histone deacetylase inhibitors (vorinostat, romidepsin, panobinostat, valproic acid, Mocetinostat (MGCD0103), Belinostat, PCI-24781, entinostat, SB939, Resminostat, Givinostat, AR-42, CUDC-101, sulforaphane, trichostatin A); celecoxib, glitazones, epigallocatechin gallate, disulfiram, Salinosporamide A; anti-adrenal drugs, aminoglutethimide, mitotane, trilostane, acetretin, aldo-phosphamide, aminolevulinic acid, amsacrine, araboside, bestrabucil, bisantrene, edatraxate, defofamine, demecolcine, diaziquone, difluoromethylornithine (DFMO), elfomithine, elliptinium acetate, etoglucid, gallium nitrate, cytosine, hydroxyurea, ibandronate, lentinan, lonidamide, mitoguazone, mitoxantrone, mopidamol, nitracine, pentostatin, phenamet, pirarubicin, podophyllinic acid, 2-ethylhydrazine, procarbazine; pyrazidinopropane; rhizoxin; siccatives; spirogeals; thujaplicin; triazenes; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and an-guidine), polyurethanes, siRNA, antisense drugs and nucleolytic enzymes; (2) anti-autoimmune disease drugs: cyclosporine, cyclosporine A, aminoglutethimide, azathioprine, bromocriptine, chlorambucil, chloroquine, cyclophosphamide, corticosteroids (including amcinonide, betamethasone, budesonide, hydrocortisone, flunisolide, fluticasone propionate, flucloronide, dexamethasone, triamcinolone acetonide, beclomethasone dipropionate), DHEA, etanercept, hydroxychloroquine, infliximab, meloxicam, methotrexate, mycophenolate mofetil, prednisone, sirolimus, tacrolimus. (3) anti-infective disease drugs, including: a) aminoglycosides: amikacin, arbekacin, gentamicin (neltamycin, sisomicin, isepamicin), hygromycin B, kanamycin (amikacin, arbekacin, aminodeoxykanamycin, dibekacin, tobramycin), neomycin (framycetin, paromycin, ribostamycin), neltamycin, spectinomycin, streptomycin, tobramycin, methyprycin; b) amphenicols: azidamycin, chloramphenicol, florfenicol, thiamphenicol; c) ansamycins: geldanamycin, herbimycin; d) carbapenems: biapenem, doripenem, ertapenem, imipenem / cilastatin, meropenem, panipenem; e) cephalosporins: cefaclor, cefadroxil, cefaloglucin, cefalonium, cefaloridine, cefalotin, cefaparid, cefatamet, cefazedone, cefazaflur, cefazedone, cefazaflur, cefozopran, cefdinir, cefditoren, cefenit, cefepime, cefixime, cefmenoxime, cefmenoxime, cefmetazole, cefodizime, cefonicid, cefoperazone, ceforanide, cefotetan, cefotiam, cefpimizole, cefoperazone, ceforanide, cefotetan, cefotiam, cefpimizole, cefquinome, cefuroxime, cefuzonam, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephacloxacine, cephacetrile, cephaclo e). Cephalosporins: carbacephem (loracarbef), cephacetrile, clavulanic acid, cephradine, cefadroxil, cefalonium, cefaloridine, cefalotin or cephaloglycin, cephalexin, cephaloglycin, cephamandole, cephaloridine, cephalothin, cefamandole, cefaparole, cephapirin, cefoxitin, cefuroxime, cefpodoxime, cefprozil, cefuroxime axetil, cefixime, cefdinir, ceflupag, cefoperazone, ceforanide, cefotetan, cefotaxime, ceftazidime, ceftizoxime, ceftriaxone, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime, cefuroxime, cefixime, cefdinir, cefpodoxime ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ p). quinolones: alatrofloxacin, balofloxacin, ciprofloxacin, clinafloxacin, danofloxacin, difloxacin, enoxacin, enrofloxacin, garenoxacin, gatifloxacin, gemifloxacin, grepafloxacin, kanofloxacin, levofloxacin, lomefloxacin, mafofloxacin, moxifloxacin, nadifloxacin, norfloxacin, orbifloxacin, ofloxacin, perfloxacin, trovafloxacin, grepafloxacin, sitafloxacin, sparfloxacin, temafloxacin, tosufloxacin, trovafloxacin; q). streptogramins: pristinamycin, quinupristin / dalfopristin; r). sulfonamides: aminosulfamides, azosulfamides, sulfadiazine, sulfisoxazole, sulfisoxazole, sulfamethoxazole, sulfamethoxazole (sulfamethoxazole), sulfamethoxazole (sulfamethoxazole); s). steroid antibacterial drugs: such as fusidic acid; t). tetracyclines: doxycycline, chlortetracycline, demeclocycline, ramocycline, meclocycline, methacycline, metacycline, minocycline, terramycin, pumilicillin, tetracycline, glycylcyclines (such as tigecycline); u). other types of antibiotics: annedolide, arsenophanes, bacteriophage inhibitors (polymyxin), DANAL / AR inhibitors (cycloserine), dictyostatin, discodermolide, eleutherobin, epothilone, ethambutol, etoposide, faropenem, fusidic acid, furazolium, isoniazid, laulimalide, metronidazole, mupirocin, NAM synthesis inhibitors (e.g. fosfomycin), nitrofurantoin, paclitaxel, plinabulin, pyrazinamide, quinupristin / dalfopristin, rifampicin, sulbactam, thiazolidine, ulithiol; (4). antiviral drugs, including: a). entry / fusion inhibitors: apelisib, maraviroc, vicriviroc, gp41 (enfuvirtide), PRO 140, CD4 (ebolizumab); b). integrase inhibitors: raltegravir, elvite-gravir, globoidnan A; c). maturation inhibitors: bevirimat, vivecon; d). neuraminidase inhibitors: oseltamivir, zanamivir, peramivir; e) nucleosides and nucleotides: abacavir, aciclovir, adefovir, amantadine, amantadine, arbidol, cidofovir, cladrin, dexamethasone, didanosine (ddl), elvucitabine, emtricitabine (FTC), entecavir, famciclovir, floxuridine (5-FU), 3'-fluoro-substituted 2',3'-dideoxynucleoside analogs such as 3'-fluoro-2',3'- dideoxythymidine (FLT) and 3'-fluoro-2',3'-dideoxyguanosine (FLG), fomivirsen, 9- guanine, iododeoxyuridine, lamivudine (3TC), 1-nucleosides (e.g. beta-1-thymidine and beta-1-2'-deoxycytidine), penciclovir, racivir, ribavirin, rimantadine, stavudine (d4T), taribavirin (viramidine), telbivudine, tenofovir, trifluridine valaciclovir, valganciclovir, zalcitabine (ddC), zidovudine (AZT); f) non-nucleosides: amantadine, atovaquone, capravirine, diarylpyrimidines (etravirine, rilpivirine), delavirdine, docosanol, emivirine, efavirenz, foscarnet (phosphonoformic acid), imiquimod, peginterferon, lovirimide, lodenosine, meglumine antimonate, nevirapine, NOV-205, long-acting interferon alpha, podophyllotoxin, rifampicin, rimantadine, resiquimod (R-848), tromantadine; g) protease inhibitors: amprenavir, atazanavir, boceprevir, darunavir, fosamprenavir, indinavir, lopinavir, nelfinavir, pleconaril, ritonavir, saquinavir, telaprevir (VX-950), tipranavir; h) other types of antiviral drugs: abiciclovir, calanolide a, ceragenin, cyanovirin-n, diarylpyrimidines, epigallocatechin gallate (EGCG), foscarnet, griffithsin, tari-bavirin (viramidine), hydroxyurea, KP-1461, miltefosine, pleconaril, promisive inhibitors, ribavirin, seliciclib. (5) A radioisotope selected from the group consisting of (radionuclides) 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 213 Bi. (6). The chromophore molecule can absorb a light such as ultraviolet light, fluorescent light, infrared light, near infrared light, or visible light; the chromophore molecule includes one or a subcategory of a group of yellow pigments, red pigments, iridescent pigments, white pigments, black pigments, and blue-green pigments, one or a subcategory of a group of fluorescent molecules (fluorescent chemicals that emit light after absorbing light), one or a subcategory of a group of visual light transduction molecules, one or a subcategory of a group of photon molecules, one or a subcategory of a group of luminescent molecules, and one or a subcategory of a group of fluorescein compounds. Non-protein organic fluorophores such as xanthene derivatives (fluorescein, rhodamine, Oregon green, eosin, and Texas red); cyanine derivatives (cyanine, indocyanine, oxonol, thiacarbocyanine, and merocyanine); squaraine derivatives and ring-substituted squaraines including Seta, SeTau, and Square dyes; naphthalene derivatives (naphthalene and fluorosilicate derivatives); coumarin derivatives; oxadiazole derivatives (pyridyl oxadiazole, nitrobenzoxadiazole, and benzoxadiazole); anthracene derivatives (anthraquinones including DRAQ5, DRAQ7, and CyTRAK Orange); pyrene derivatives (Cascade Blue, etc.); oxazine derivatives (Nile red, Nile blue, cresyl violet, oxazine 170, etc.); acridine derivatives (lutein, acridine orange, acridine yellow, etc.); arylmethylamine derivatives (malachite green, crystal violet, malachite green); and tetrapyrrole derivatives (porphyrin, phthalocyanine, bilirubin). Any analogues and derivatives of the following fluorescent compounds: CF dyes (Biotium), DRAQ and CyTRAK probes (BioStatus), BODIPY (Invitrogen), Alexa Fluor (Invitrogen), DyLight Fluor (Thermo Scientific, Pierce), Atto and Tracy (SigmaAldrich), FluoProbes (Interchim), Abberior dyes (Abberior), DY and MegaStokes dyes (Dyomics), Sulfo Cy dyes (Cyandye), HiLyte Fluor (AnaSpec), Seta, SeTau, and Square dyes (Biosearch Technologies), SureLight dyes (APC, RPE PerCP, Phycobilisomes) (Columbia Biosciences), APC, APCXL, RPE, BPE (Phyco-Biotech), Allophycocyanin (APC), Aminomalcite, APC-Cy7 conjugates, BODIPY-FL, Cascade Blue, Cy2, Cy3, Cy3.5, Cy3B, Cy5, Cy5.5, Cy7, Fluorescein, FluorX, Hydroxycoumarin, Lissamine Rhodamine B, Lucifer Yellow, Me-Methoxycoumarin, NBD, Pacific Blue, Pacific Orange, PE-Cy5 conjugate, PE-R-Phycoerythrin (PE), Red 613, Seta-555-Azide, Seta-555-DBCO, Seta-555-NHS, Seta-580-NHS, Seta-680-NHS, Seta-APC-780, Seta-PerCP-680, Seta-R-PE-670, SeTau-380-NHS, SeTau-405-Maleimide, SeTau-405-NHS, SeTau-425-NHS, SeTau-647-NHS, Texas Red, TRITC, TruRed, X-Rhodamine, 7-AAD (7-Aminoactinomycin D, CG-selective), Acridine Orange, Chomomycin A3, CyTRAK Orange (Biostatus), DAPI, DRAQ5, DRAQ7, Ethidium Bromide, Hoechst 33258, Hoechst 33342, LDS 751, Mithramycin, Propidium Iodide (PI), SYTOX Blue, SYTOX Green, SYTOX Orange, Thiazole Orange, TO-PRO, Cyanine monomer, TOTO-1, TO-PRO-1, TOTO-3, TO-PRO-3, YOSeta-1, YOYO-1. Fluorescent compounds that can be linked to the linker of the present application for studying cells are selected from the following compounds or derivatives thereof: DCFH (2',7'-Dichlorodihydrofluorescein, oxidized form), DHR (Dihydro-Rhodamine 123, oxidized form, photo catalytic oxidation), Fluo-3 (AM ester, pH > 6), Fluo-4 (AM ester, pH 7.2), Indo-1 (AM ester, low / high calcium (Ca 2+)), SNARF (pH 6 / 9). Preferred fluorescent compounds are selected from the group consisting of: Allophycocyanin (APC), AmCyanl (tetramer, Clontech), AsRed2 (tetramer, Clontech), B-phycoerythrin (BPE), Cerulean, CyPet, DsRed monomer (Clontech), DsRed2 ("RFP"), EBFP, EBFP2, ECFP, EGFP (weak dimer), Emerald (weak dimer), EYFP (weak dimer), GFP (S65A mutation), GFP (S65C mutation), GFP (S65L mutation), GFP (Y66H mutation), GFP (Y66W mutation), GFPuv, HcRedl, J-Red, Katusha, Kusabira Orange (monomer, MBL), mCFP, mCherry, mCitrine, Mi-doriishi Cyan (weak dimer, MBL), mKate (TagFP635, monomer), mKeima-Red (monomer), mKO, mOrange, mPlum, mRaspberry, mRFP1 (monomer), mStrawberry, mTFPl, mTurquoise2, P3 (phycobilisome complex), Peridinin-chlorophyll-protein complex (PerCP), R-phycoerythrin (RPE), T-Sapphire, TagCFP (dimer), TagGFP (dimer), TagRFP (dimer), TagYFP (dimer), tdTomato (tandem dimer), Topaz, TurboFP602 (dimer), TurboFPP635 (dimer), TurbogFP (dimer), TurboRFP (dimer), TurboYFP (dimer), Venus, wild-type GFP type, YPet, Zsgreenl (tetramer), ZsYellowl (tetramer), and derivatives thereof. (7). The cell binding ligand or receptor agonist is selected from the group consisting of: folate derivatives, glutamate urea derivatives, somatostatin and its analogues (selected from octreotide (Sandostatin) and lanreotide (Somatuline)), arylsulfonamides, pituitary adenylate cyclase activating peptide (PACAP) (PAC1), vasoactive intestinal peptide (VIP / PACAP) (VPAC1, VPAC2), melanocyte-stimulating hormone (a-MSH), cholecystokinin (CCK) / gastrin receptor agonists, bombesin (selected from Pyr-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2) / gastrin releasing peptide (GRP). neurotensin receptor ligands (NTR1, NTR2, NTR3); substance P (NK1 receptor) ligands; neuropeptide Y (Y1-Y6); homing peptides including RGD (Arg-Gly-Asp), NGR (Asn-Gly-Arg), dimeric and multimeric cyclic RGD peptides (selected from cRGDfV), TAASGVRSMH and LTLRWVGLMS (chondroitin sulfate proteoglycan NG2 receptor ligands) and F3 peptides; cell-penetrating peptides (CPPs);Peptide hormones are selected from the group consisting of luteinizing hormone releasing hormone (LHRH) agonists and antagonists, and gonadotropin releasing hormone (GnRH) agonists, acting by targeting follicle stimulating hormone (FSH) luteinizing hormone (LH), and testosterone production, such as buserelin (Pyr-His-Trp-Ser-Tyr-D-Ser(OtBu)-Leu-Arg-Pro-NHEt), goserelin (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), nafarelin (Pyr-His-Trp-Ser-Tyr-2Nal-Leu-Arg-Pro-Gly-NH2), triptorelin (Pyr-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH2), nafarelin, delolorin, abarelix (Ac-D-2Nal-D-4-chloroPhe-D-3-(3-pyridyl)Ala-Ser-(N-Me)Tyr-D-Asn-Leu-iso-propylLys-Pro-DAla-NH2), cetrorelix (Ac-D-2Nal-D-4-chloroPhe-D-3-(3-pyridyl)Ala-Ser-Tyr-D-Cit-Leu-Arg-Pro-D-Ala-NH2), degarelix (Ac-D-2Nal-D-4-chloroPhe-D-3-(3-pyridyl)Ala-Ser-4-aminoPhe(L-hydroorotyl)-D-4-aminoPhe(carba-moyl)-Leu-iso-propylLys-Pro-D-Ala-NH2), and degarelix (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); pattern recognition receptors (PRRs) selected from the group consisting of Toll-like receptor (TLRs) ligands, C-type lectins and nod-like receptors (NLRs) ligands; calcitonin receptor agonists; integrin receptors and their receptor subtypes (selected from the group consisting of α; V β1, α V β3, α V β5, α V β6, α6 β4, α7 β1, α L β2, α IIb β3) agonists (selected from the group consisting of GRGDSPK, cyclo(RGDfV) (L1) and derivatives thereof [cyclo(-N(Me)R-GDfV), cyclo(R-Sar-DfV), cyclo(RG-N(Me)D-fV), cyclo(RGD-N(Me)f-V), cyclo(RGDf-N(Me)V-)(Cilengitide)]; single domain antibodies (derivatives of VHH (camelid Ig)); domain antibodies (dAbs, derivatives of VH or VL domains); bispecific T cell engagers (BiTEs, bispecific dimers); dual affinity retargeting (DARTs, bispecific dimers); tetravalent tandem antibodies (TandAbs, a dimerized bispecific dimer); Anticalins (derivatives of calcin); Adnectins (FN3 (fibronectin) number 10); designed ankyrin repeat proteins (DARPins); Avimers; EGF receptor and VEGF receptor agonists; a short antibody-like protein, siRNA or DNA molecule for immunotherapy. (8) pharmaceutically acceptable salts, acids, derivatives, hydrates or hydrated salts; or crystal structures; or optical isomers, racemates, diastereomers or enantiomers of any of the above drugs.

4. The cytotoxic drugs D1 and D2 according to claim 1 are independently selected from: tubulysin and analogs thereof, maytansine and analogs thereof, taxane and analogs thereof, CC-1065 and analogs thereof, daunorubicin or doxorubicin and their analogs, amatoxins and analogs thereof, benzodiazepine dimers (e.g., dimers of pyrrolobenzodiazepine (PBD), tomaymycin, anthramycin, indolinobenzodiazepine, imidazobenzothiadiazepine, or oxazolidinobenzodiazepine) and analogs thereof, calicheamicin and enediyne antibiotic analogs, dactinomycin and analogs thereof, netropsin and analogs thereof, bleomycin and analogs thereof, epirubicin and analogs thereof, tamoxifen and analogs thereof, idarubicin and analogs thereof, dolastatins and analogs thereof, auristatins (including monomethyl auristatin (MMAE), MMAF, auristatin PYE, auristatin TP, auristatin 2-AQ, 6-AQ, EB (AEB), and EFP (AEFP)) and analogs thereof, combretastatin, duocarmycin and analogs thereof, camptothecin, geldanamycin and analogs thereof, methotrexate and analogs thereof, thiotepa and analogs thereof, vindesine and analogs thereof, vincristine and analogs thereof, hemiasterlins and analogs thereof, nazuma-mide and analogs thereof, spliceostatin, a pladienolide, a microcrystalline protein and analogs thereof, radiosensitizers and analogs thereof, alterobactin and analogs thereof, a microsclerodermin and analogs thereof, theonellamide and analogs thereof, esperamicin and analogs thereof, PNU-159682 and analogs thereof, a protein kinase inhibitor, a MEK inhibitor, a KSP inhibitor, a nicotinamide phosphoribosyltransferase (NAMPT) inhibitor, an immunotoxin, a cellular receptor agonist, a cellular stimulatory molecule or intracellular signaling molecule, one, two or more of a DNA, RNA, mRNA, small interfering RNA (siRNA), microRNA (miRNA), and PIWI-interacting RNA (piRNA) and stereoisomers, isosteres, analogs, or derivatives thereof; wherein: (a) the tubulysin analog has the structure of Formula (IV): or a pharmaceutically acceptable salt, hydrate or hydrated salt; or a crystalline structure; or an optical isomer, racemate, diastereomer or enantiomer thereof. wherein is one or two linkers that can be independently attached to L1and / or L2; when two are simultaneously attached to L1and L2, R 1 and R 2 , or Z 2 and Z 3 are preferred dual attachment sites; wherein R 1 , R 1’ , R 2 , R 3 and R 4 are independently H, C1-C8 alkyl; C2-C8 heteroalkyl, heterocycloalkyl; C3-C8 aryl, arylalkyl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, carbocycloalkyl, or alkylcarbonyl; or R 1 R 2 , R 1 R 3 , R 2 R 3 , R 3 R 4 or a 3-7 membered carbocyclic, cycloalkyl, heterocyclic, heterocycloalkyl, aryl, or heteroaryl ring system formed; R 1 and R 2 may independently be null when attached independently or simultaneously to L1or L2, Y 1 is N or CH; wherein R 5 , R 6 , R 8 , R 10 and R 11 are independently H, or C1-C4 alkyl or heteroalkyl; wherein R 7 independently H, R 14 , -R 14 C(=O)X 1 R 15 ; or -R 14 X 1 R 15 ; X 1 is O, S, S-S, NH, CH2or NR 14 ; wherein R 9 is selected from H, OH, =0, -OR 14 , -OC(=0)R 14 , -OC(=0)NHR 14 , -OC(=0)NR 14 R 15 , OP(=0)(OR 14 )2, -OC(=0)NR 14 R 15 , or OR 14 OP(=0)(OR 15 )2; when R 9 is attached to L1or L2, R 9 is -0-, -OC(=0)NH- or -OC(=0)N(R 14 )-; wherein R 11 independently H, R 14 , -R 14 C(=O)R 15 , -R 14 C(=O)X 2 R 15 , wherein X 2 is -O-, -S-, -NH-, or -N(R 14 )-; wherein R 12 is -COOH, -COSH, -CONH2, CONHNH2, CONHNHR 15 15 15 15 16 15 16 15 15 16 15 15 16 15 17 15 17 17 2 17 15 2 16 tetrazole, imidazole, or triazole, X 2 is -O-, -S-, -NH-, -N(R 15 )-, -O-R 15 -, -S-R 15 -, CH2, or -NHR 15 -; when R 12 is attached to L1or L2, R 12 is -C(O)O-, -C(O)NH-, -C(=O)NHS(O)2R 15 -, or -C(=O)N(R 15 )-;​​​​​​​​​​​​​​​​​​​​​​ R 13 and R 14 are independently C1-C8alkyl, heteroalkyl; C2-C8alkenyl, alkynyl, heteroalkyl, heterocycloalkyl; C3-C8aryl, arylalkyl; Z 2 and Z 3 are independently H, O, S, NH, N(R 15 ), NHNH, -OH, -SH, -NH2, NH, NHNH2, -NH(R 15 ), -OR 15 , CO, -COX 2 , -COX 2 R 16 , R 17 , F, Cl, Br, I, SR 16 , NR 16 R 17 , N=NR 16 , N=R 16 , NO2, SOR 16 R 17 , SO2R 16 , SO3R 16 , OSO3R 16 , PR 16 R 17 , POR 16 R 17 , PO2R 16 R 17 , OP(O)(OR 17 )2, OCH2OP(O)(OR 17 )2, OC(O)R 17 , OC(O)OP(O)(OR 17 )2, PO(OR 16 )(OR 17 ), OP(O)(OR 17 )OP(O)(OR 17 )2, OC(O)NHR 17 ; -O-(C4-C 12 oside), -N-(C4-C 12 oside); C1-C8alkyl, heteroalkyl; C2-C8alkenyl, alkynyl, heteroalkyl, heterocycloalkyl; C3-C8aryl, arylalkyl, carbocyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl, or an ester, ether, or amide of 2-8 carbons; or a peptide comprising 1-8 amino acids (NH(Aa) 1～8 or CO(Aa) 1～8 , 1-8 identical or different amino acids at the N- or C-terminus), or a polyoxyethylene unit having the formula (OCH2CH2) p or (OCH2CH(CH3)) p wherein p is an integer from 0 to about 1000, or a combination of the foregoing radicals; X 2 is O, S, S-S, NH, CH2, OH, SH, NH2, CHR 15 or NR 15 ; R 15 , R 16 , and R 17 are independently H, C1-C8 alkyl, heteroalkyl; C2-C8 alkenyl, alkynyl, heteroalkyl, heterocycloalkyl; C3-C8 aryl, arylalkyl, carbocyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl, alkylcarbonyl, or Na + , K + , Cs + , Li + , Ca 2+ , Mg + , Zn 2+ , N + (R 1 )(R 2 )(R 3 )(R 4 ), HN + (C2H5OH)3 salt; Y 1 and Y 2 are independently N or CH; q is 0 or 1 ; when q = 0, Y 3 by default, Y 4 , Y 5 , Y 6 and Y 7 are independently CH, N, NH, O, S, or N(R1), so Y 2 , Y 4 , Y 5 , Y 6 and Y 7 form furan, pyrrole thiophene, thiazole, oxazole and imidazole, pyrazole, triazole, tetrazole, thiadiazole heteroaromatic rings; when q = 1, Y 3 , Y 4 , Y 5 , Y 6 and Y 7 are independently CH or N, Y 2 , Y 3 , Y 4 , Y 5 , Y 6 and Y 7 form benzene, pyridine, pyridazine, pyrimidine, pyrazine, triazine, tetrazine, pentazine aromatic rings; Examples of structures of tubulysin analogs are shown below: wherein R 20 is H; C1-C8 straight chain or branched alkyl or heteroalkyl, C2-C8 straight chain or branched alkenyl, alkynyl, alkylcycloalkyl, heterocycloalkyl, C3-C8 aryl, aralkyl, heterocyclyl, carbocyclyl, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl, straight chain or branched; carbonate (-C(O)OR 17 ), carbamate (-C(O)NR 17 R 18 ); or a carboxylate, ester, ether or amide of 1-8 carbons; or 1-8 amino acids; or a polyethyleneoxy unit having the formula (OCH2CH2) p or (OCH2CH(CH3)) p wherein p is an integer from 0 to about 1000; or R 20 default oxygen forms a ketone with carbon, or combinations of the above. Z 3 and Z 3 independently H, OH, NH2, O, NH, COOH, COO, C(O), C(O), C(O)NH, C(O)NH2, R 18 , OCH2OP(O)(OR 18 )2, OC(O)OP(O)(OR 18 )2, OPO(OR 18 )2, NHPO(OR 18 )2, OP(O)(OR 18 )OP(O)(OR 18 )2, OC(O)R 18 , OC(O)NHR 18 , OSO2(OR 18 ), O-(C4-C 12 -glycoside), straight or branched alkyl or heteroalkyl; C2-C8straight or branched alkenyl, alkynyl, alkylcycloalkyl, heterocycloalkyl; C3-C8straight or branched aryl, aralkyl, heterocyclyl, carbocyclyl, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; carbonate (-C(O)OR 17 ), carbamate (-C(O)NR 17 R 18 ); R 17 and R 18 are independently H, straight or branched alkyl or heteroalkyl; C2-C8straight or branched alkenyl, alkynyl, alkylcycloalkyl, heterocycloalkyl; C3-C8straight or branched aryl, alkyl, heterocyclo, carbocyclo, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; carbonate (-C(O)OR 17 ), carbamate (-C(O)NR 17 R 18 ); R 19 is H, OH, NH2, OSO2(OR 18 ), XCH2OP(O)(OR 18 )2, XPO(OR 18 )2, XC(O)OP(O)(OR 18 )2, XC(O)R 18 , XC(O)NHR 18 , C1-C8alkyl or carboxylate; C2-C8alkenyl, alkynyl, alkylcycloalkyl, heterocycloalkyl; C3-C8aryl or alkylcarbonyl; or a pharmaceutically acceptable salt; X is O, S, NH, NHNH, or CH2; R 7 as defined above; wherein the point of attachment in formulae IV-01 to IV-79 is the same as indicated in formula (IV); (b) the calicheamicin and related enediyne antibiotics have the following formula: or an elemental isotope substitution, or a pharmaceutically acceptable salt, hydrate or hydrated salt; or a polymorphic structure; or an optical isomer, racemate, diastereomer or enantiomer thereof; wherein is a site of attachment to L1or L2; (c) Geldanamycin is a benzoquinone ansamycin antibiotic, 17-AAG (17-N-allylamino-17-demethoxygeldanamycin) and 17-DMAG (17-dimethylaminoethylamino-17-demethoxygeldanamycin) having the following formula: wherein is a site of attachment to L1or L2; (d) Maytansine or its derivatives Maytansinoids have the following formula: (e) Camptothecins (CPTs) and their derivatives have the following formula: or one or more elemental isotope substitution, or a pharmaceutically acceptable salt, hydrate or hydrated salt; or a polymorphic structure; or an optical isomer, racemate, diastereomer or enantiomer thereof; wherein R1, R2and R4are independently selected from H, F, Cl, Br, CN, NO2, C1-C8alkyl; O-C1-C8alkyl, NH-C1-C8alkyl; C2-C8heteroalkyl, alkylcycloalkyl, heterocycloalkyl; C3-C8aryl, arylalkyl, heterocycle, carbocycle, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; or an ester, ether, amide, carbonate, urea or carbamate of 2-8 carbon atoms; R3 is H, OH, NH2, C1-C8 alkyl, O-C1-C8 alkyl; NH-C1-C8 alkyl; C2-C8 heteroalkyl, alkylcycloalkyl, heterocycloalkyl; C2-C8 ester, ether, amide, carbonate, urea, or carbamate; or R1R2, R2R3, and R3R4 independently form a 5-7 membered carbocyclic, heterocyclic, heterocycloalkyl, aryl, or heteroaryl ring system. Wherein is the site in the molecule to which L1or L2is attached. Camptothecins (CPTs) and their derivatives have the following formula: SN-38, Topotecan analogs, or one or more elemental isotope substitution, or a pharmaceutically acceptable salt, hydrate or hydrated salt; or a polymorphic structure of these compounds; or an optical isomer, racemate, diastereomer or enantiomer; wherein is a site of attachment to L1or L2; 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, O-(C1-C8branched or straight chain alkyl), C1-C8straight or branched chain alkyl or heteroalkyl, C2-C8straight or branched chain alkenyl, alkynyl, alkylcycloalkyl, heterocycloalkyl; C3-C8straight or branched chain aryl, arylalkyl, heterocycle, carbocycle, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl, carbonate (-C(O)OR 17 ), carbamate (-C(O)NR 17 R 18 ); R 17 and R 18 are independently H, straight or branched chain alkyl or heteroalkyl; C2-C8straight or branched chain alkenyl, alkynyl, alkylcycloalkyl, heterocycloalkyl; C3-C8straight or branched chain aryl, arylalkyl, heterocycle, carbocycle, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl, carbonate (-C(O)OR 17 ), carbamate (-C(O)NR 17 R 18 ); X is NH, O, S or CH2. (f) Combretastatins have the following formula: (g) Taxanes have the following formula: wherein is a site attached to L1or L2; Ar and Ar' are independently aryl or heteroaryl. (h) Anthracyclines have the following formula: wherein is a site of attachment to L1or L2. (i) Vinca alkaloids are selected from the group consisting of vinblastine, vincristine, vindesine, leurosine, vinorelbine, leurosidine, vincaleukoblastine, vinamycine, vindesine, vinleurosine, vinrosidine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vindesine, vincristine, vincristine, vinblastine, vinblastine; rifabutin analogs, rifabutin analogs, wherein is attached to the L1 or L2 site. ​ ​ ​ where R 1 、R 2 、R 3 、R 4 and R 5 are independently H; C1-C8 straight or branched chain alkyl, aryl, heteroaryl, heteroalkyl, alkylcycloalkyl, ester, ether, amide, amine, heterocycloalkyl or acyloxyamine; or a peptide comprising 1-8 amino acids, or having the formula (OCH2CH2) p or (OCH2CH(CH3)) p wherein p is an integer from 1 to about 1000. 1 R 2 、R 2 R 3 、R 1 R 3 or R 3 R 4 It can form a 3-8 membered ring of alkyl, aryl, heteroaryl, heteroalkyl or alkylcycloalkyl; Y1and Y2are 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, when attached to a site (independently attached to L1and / or L2); or 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, when not attached to a site ; 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 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 1-8 identical or different amino acids; p is 1-5000; R1, R2, R3, R4, R5, R5', Z1, Z2, and n are as defined above. ​ wherein R 1 , R 2 , R 3 , R 4 and R 5 are independently H; C1-C8 straight chain or branched alkyl, aryl, heteroaryl, heteroalkyl, alkylcycloalkyl, ester, ether, amide, amine, heterocycloalkyl, or acyloxyamine; or a peptide containing 1-8 amino acids, or a polyoxyethylene unit of the formula (OCH2CH2) p p p is an integer from 1 to about 5000; further, R 2 R 3 may form an alkyl, aryl, heteroaryl, heteroalkyl, or alkylcycloalkyl group of 3-8 rings. (l) Eribulin has the following molecular formula: are sites independently linked to L1 and / or L2. (m) Nicotinamide phosphoribosyltransferase inhibitors (NAMPT) have the following molecular formula NP01, NP02, NP03, NP04, NP05, NP06, NP07, NP08 and NP09: or an isotope of one or more elements, or a pharmaceutically acceptable salt, hydrate or hydrated salt; or polymorphic forms of these compounds; or optical isomers, racemates, diastereomers or enantiomers; wherein The same as the foregoing; X5 is F, Cl, Br, I, OH, OR1, R1, OPO3H2, OSO3H, NHR1, OCOR1, NHCOR1. (n) Benzodiazepine dimers and analogues thereof have the following molecular formula: or an isotope of one or more elements, or a pharmaceutically acceptable salt, hydrate or hydrated salt; or polymorphic structures of these compounds; or optical isomers, racemates, diastereomers or enantiomers; wherein Z1, Z2 and n are as defined above; X1, X2, 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; R 1 , R 2 , R 3 , R 1’ , R 2’ , and R 3’ are independently H, F, Cl, =0, =S, OH, SH, C1-C8 straight chain or branched benzyl, aryl, alkenyl, heteroaryl, heteroalkyl, alkylcycloalkyl, ester (COOR5 or -OC (O)R5), ether (OR5), amide (CONR5), carbamate (OCONR5), amine (NHR5, NR5R5'), heterocycloalkyl, or acyloxy amine (-C(O)NHOH, -ONHC(O)R5), or a peptide containing 1-20 natural or unnatural amino acids, or a polyoxyethylene group of the formula (OCH2CH2) p or (OCH2CH(CH3)) p wherein p is an integer from 1 to 5000. Two R groups, such as 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’ alkyl, aryl, heteroaryl, heteroalkyl, or alkylcycloalkyl groups which can independently form 3-8 membered rings; X3 and Y3 are independently N, NH, CH2 or CR5, one of X3 and Y3 can be null; wherein R1, and R2 are C1-C8 straight chain or branched alkyl, heteroalkyl; C3-C8 aryl, heteroaryl, alkylcycloalkyl, acyloxy, alkylaryl, alkylaryloxy, alkylaryl amino, alkylarylthio; or 1-6 identical or different amino acid / peptide sequences (Ar)r, r = 1-6; wherein R4, R5, R5', R6, R 12 and R 12 ' are independently H, OH, NH2, NH(CH3), NHNH2, COOH, SH, OZ3, SZ3, F, Cl, or a C1-C8 straight chain or branched alkyl, aryl, heteroaryl, heteroalkyl, alkylcycloalkyl, acyloxy amine; Z3 is H, OP(O)(OM1)(OM2), OCH2OP(O)(OM1)(OM2), OSO3M1, or O-glycoside (glucoside, galactoside, mannoside, glucuronoside / glucuronic acid, alloside, fructoside, etc.), NH-glycoside, S-glycoside or CH2-glycoside; M1 and M2 are independently H, Na, K, Ca, Mg, NH4 or NR1R2R3; X6 is CH, N, P(O)NH, P(O)NR1, CHC(O)NH, C3-C8 aryl, heteroaryl, alkylcycloalkyl, acyloxy, alkylaryl, alkylaryloxy, alkylaryl amino or Aa (amino acid, preferably selected from Lys, Phe, Asp, Glu, Ser, Thr, His, Cys, Tyr, Trp, Gin, Asn, Arg); wherein X and X' are independently CH2, or N, when the six-membered aromatic ring becomes a five-membered ring, X and / or X' can be O, S or NH; Y 21 Ms (mesyl), Ts (tosyl) or Tf (trifyl), SO3H, P(O)(OH)2, CH2(O)P(O)(OH)2, glycoside; R 31 is H, C1-C8alkyl or Ar, CF3; as previously described. (o) CC-1065 analogues and duocarmycin analogues have the following molecular formula CC01, CC02, CC03, CC04, CC05, CC06 and CC07: wherein when attached to a site X1, X2, Y1and Y2are 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 when not attached to a site of attachment 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; Z3is H, PO(OM1)(OM2), SO3M1, CH2PO(OM1)(OM2), CH3N(CH2CH2)2NC(O)-, O(CH2CH2)2NC(O)-, R1, or a glycoside; wherein R1, R2, R3, M1, M2, and n are as previously described. (p) Amatoxins and analogues thereof have the following molecular formula Am01, Am02 and Am03: or an isotope of one or more elements, or a pharmaceutically acceptable salt, hydrate or hydrated salt; or polymorphic forms of these compounds; or optical isomers, racemates, diastereomers or enantiomers; wherein X1and Y1are 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), CH2, CHNH, CH2O, C(O)NHNHC(O), and C(O)NR1; R7, R8, and R9are independently H, OH, OR1, NH2, NHR1, C1-C6alkyl, or null; Y2is O, O2, NR1, NH, or null; R 10 is CH2, O, NH, NR1, 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 null; R 11 is 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, NR1R2, O(CH2CH2O) p CH2CH2-COOH, NH(CH2CH2O) p CH2CH2COOH, NH-Ar-COOH, NH-Ar-NH2, O(CH2CH2O) p CH2CH2-NHSO3H, 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 CH2-CH2NHPO3H2, wherein (Aa) r denotes 1-8 amino acids; n and m1are independently 1-20; p is 1-5000; R1, R2, and Ar are as defined herein; The definitions are as previously described. (q) Spliceostatins and pladienolides are spliceostatin A, FR901464 and (2S,3Z)-5-{[(2R,3R,5S,6S)-6-{(2E,4E)-5-[(3R,4R,5R,7S)-7-(2-hydrazinyl-2-oxoethyl)-4-hydroxy-1,6-dioxopyrrolidin-5-yl]-3-pentyl-2,4-dien-1 -yl}-2,5-dimethyltetrahydro-2H-pyran-3-yl]amino}-5-oxo-3- en-2-yl acetate, the parent nucleus structure of which is Sp-01 : (r) Protein kinase inhibitors are preferably Adavosertib, Afatinib, Axitinib, Baricitinib, Bosutinib, Cipatinib, Crizotinib, Cabozantinib, Dasatinib, Encorafenib, Erdafitinib, Erlotinib, Fotatinib, Gefitinib, Ibrutinib, Imatinib, Lapatinib, Lenvatinib, Mubritinib, Nilotinib, Pazopanib, Panatinib, Ponatinib, Rebastinib, Regorafenib, Ruxolitinib, Sorafenib, Sunitinib, SU6656, Tofacitinib, Vandetanib, Vemurafenib, Entrectinib, Palbociclib, Ribo-ciclib, Abemaciclib, Dacomitinib, Larotrectinib, CO-1686, Osimertinib, AZD3759, Nazartinib (EGF816) having the following structures, PK01-PK40: Adavosertib, Afatinib, axitinib, Bafitinib, Bosutinib, Cometinib, Crizotinib, dasatinib, encorafenib, Erdafitinib, Erlotinib, Fostamatinib, Gefitinib, Gefitinib, Gefitinib, Ibrutinib, Imatinib, Lapatinib, Lenvatinib, Mobitinib, Nilotinib, Pazopanib, Ponatinib, Ruxolitinib, Sorafenib, Sunitinib, SU6656, Tofacitinib, Vandetinib, vemurafenib, encorafenib; Palbociclib analogs, Ribociclib, Abecilis, Dacomitinib, Neratinib, Rociletinib (CO-1686), Osimertinib, AZD3759, Naziatinib (EGF816), wherein Z5and Z5’are independently selected from 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)NR1. (s) MEK inhibitors are selected from PD0325901, Selumetinib (AZD6244), cobimetinib (XL518), Refametinib, Trametinib (GSK1120212), pimasertib, Binimetinib (MEK162), AZD8330, RO4987655, RO5126766, WX-554, E6201, GDC-0623, PD-325901 and TAK-733, structures of which are as follows: Qumikae Ni, Cobimetinib, Bimetinib, Selumetinib, wherein Z5is selected from 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)NR1. (t) Protease inhibitors are preferably selected from: Carfilzomib, Clindamycin, Retaspimycin, Indibulin, structures of which are as follows: Carfilzomib, Clindamycin, Carmaphycin analogs, (u) an immunotoxin selected from the group consisting of diphtheria toxin (DT), cholera toxin (CT), ricin (TCS), amylase, Pseudomonas exotoxin A (ETA), erythrogenic toxin, diphtheria toxin, AB toxin, type III exotoxin, pre-lysozyme and topalysin; (v) a cell receptor agonist or stimulatory molecule selected from the group consisting of: a folic acid derivative, a glutamate urea derivative, a somatostatin and analogs thereof (selected from octreotide (Sandostatin) and lanreotide (Somatuline)), an arylsulfonamide, pituitary adenylate cyclase-activating peptide (PACAP) (PAC1), vasoactive intestinal peptide (VIP / PACAP) (VPAC1, VPAC2), melanocyte-stimulating hormone (a-MSH), cholecystokinin (CCK) / gastrin receptor agonists, bombesin (selected from Pyr-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2) / gastrin-releasing peptide (GRP). neurotensin receptor ligands (NTR1, NTR2, NTR3); substance P (NK1 receptor) ligands; neuropeptide Y (Y1-Y6); homing peptides including RGD (Arg-Gly-Asp), NGR (Asn-Gly-Arg), dimeric and multimeric cyclic RGD peptides (selected from cRGDfV), TAASGVRSMH and LTLRWVGLMS (chondroitin sulfate proteoglycan NG2 receptor ligands) and F3 peptides; cell-penetrating peptides (CPPs);Peptide hormones are selected from the group consisting of luteinizing hormone releasing hormone (LHRH) agonists and antagonists, and gonadotropin releasing hormone (GnRH) agonists, acting by targeting follicle stimulating hormone (FSH) luteinizing hormone (LH), and testosterone production, such as buserelin (Pyr-His-Trp-Ser-Tyr-D-Ser(OtBu)-Leu-Arg-Pro-NHEt), goserelin (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), nafarelin (Pyr-His-Trp-Ser-Tyr-2Nal-Leu-Arg-Pro-Gly-NH2), triptorelin (Pyr-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH2), nafarelin, delolorin, abarelix (Ac-D-2Nal-D-4-chloroPhe-D-3-(3-pyridyl)Ala-Ser-(N-Me)Tyr-D-Asn-Leu- isopropyl Lys-Pro-DAla-NH2), cetrorelix (Ac-D-2Nal-D-4-chloroPhe-D-3-(3-pyridyl)Ala-Ser-Tyr-D-Cit-Leu-Arg-Pro-D-Ala-NH2), degarelix (Ac-D-2Nal-D-4-chloroPhe-D-3-(3-pyridyl)Ala-Ser-4-aminoPhe(L-hydroorotyl)-D-4-aminoPhe(carba-moyl)-Leu-isopropyl Lys-Pro-D-Ala-NH2), and degarelix (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); pattern recognition receptors (PRRs) selected from the group consisting of Toll-like receptor (TLRs) ligands, C-type lectins and Nod-like receptors (NLRs) ligands; calcitonin receptor agonists; integrin receptors and their receptor subtypes (selected from the group consisting of α V β1, α V β3, α V β5, α V β6, α6 β4, α7 β1, α L β2, α IIb β3) agonists (selected from the group consisting of GRGDSPK, cyclo(RGDfV) (L1) and derivatives thereof [cyclo(-N(Me)R-GDfV), cyclo(R-Sar-DfV), cyclo(RG-N(Me)D-fV), cyclo(RGD-N(Me)f-V), cyclo(RGDf-N(Me)V-)(cilengitide)]; Anticalins (derivatives of lipocalins); Adnectins (10 FN3 (fibronectin)); Designed Ankyrin Repeat Proteins (DARPins); Avimers; EGF receptor, or VEGF receptor agonists; a cell receptor agonist selected from the group consisting of: LB01 (folic acid), LB02 (PMSA ligand), LB03 (PMSA ligand), LB04 (PMSA ligand), LB05 (somatostatin), LB06 (somatostatin), LB07 (octreotide, somatostatin analogue), LB08 (lanreotide, somatostatin analogue), LB09 (vapreotide (Sanvar), somatostatin analogue), LB10 (CAIX ligand), LB11 (CAIX ligand), LB12 (gastrin releasing peptide receptor (GRPr), MBA), LB13 (luteinizing hormone releasing hormone (LH-RH) and GnRH ligand), LB14 (luteinizing hormone releasing hormone (LH-RH) and GnRH ligand), LB15 (GnRH antagonist, Abarelix), LB16 (cobalamin, vitamin B12 analogue), LB17 (cobalamin, vitamin B12 analogue), LB18 (cyclic RGD pentapeptide for avb3 integrin receptor), LB19 (heterobivalent peptide ligand for VEGF receptor), LB20 (neuromedin B), LB21 (bombesin, acting on G protein coupled receptor), LB22 (TLR2, acting on Toll-like receptor), LB23 (acting on androgen receptor), LB24 (cilengitide or cyclic (-RGDfV-) av integrin receptor, LB23 (fludrocortisone), LB25 (rifabutin analogue), LB26 (rifabutin analogue), LB27 (rifabutin analogue), LB28 (flurohydrocortisone), LB29 (dexamethasone), LB30 (fluticasone propionate), LB31 (beclometasone dipropionate), LB32 (triamcinolone acetonide), LB33 (prednisolone), LB34 (prednisolone), LB35 (methylprednisolone), LB36 (betamethasone), LB37 (irinotecan analogue), LB38 (crizotinib analogue), LB39 (bortezomib analogue), LB40 (carfilzomib analogue), LB41 (carfilzomib analogue), LB42 (leuprolide analogue), LB43 (triptorelin analogue), LB44 (clindamycin), LB45 (liraglutide analogue), LB46 (vinka alkaloid analogue), LB47 (retapamulin analogue), LB48 (dibutyl analogue), LB49 (vinblastine analogue), LB50 (lixivaptan peptide analogue), LB51 (oxindin analogue), LB52 (nucleoside analogue), LB53 (erlotinib analogue), and LB54 (lapatinib analogue), the structures of which are shown below: R 19 is 5' deoxyadenosyl, Me, OH, CN; R 19 is 5' deoxyadenosyl, Me, OH, CN; wherein Y5is N, CH, C(Cl), C(CH3), or C(COOR1); R1is H, C1-C6alkyl, C3-C8Ar; wherein X4, and Y1are 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), CH2, C(O)NHNHC(O), and C(O)NR1. (w) one, two or more of DNA, RNA, mRNA, small interfering RNA (siRNA), microRNA (miRNA), and PIWI-interacting RNA (piRNA) have a structure of: wherein is the point of attachment of the branched linker of the present patent; is single or double stranded DNA, RNA, mRNA, siRNA, miRNA, or piRNA; X1and Y 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), CH2, C(O)NHNHC(O), and C(O)NR1.

5. The linker L1, L2, La1, La2, Lb1, Lb2, Lc1, and Lc2 of claim 1 independently contains: (a). The self-immolative linker component has one of the following structures: wherein (*) is the point of attachment of an additional spacer or cleavable linker unit, or a cytotoxic agent, and / or a therapeutic antibody; X 1 , Y 1 , Z 2 and Z 3 are independently NH, O, or S; Z 1 are independently H, NH, O or S; v is 0 or 1 ; U 1 are independently H, OH, C1-C6 alkyl, (OCH2CH2) n F, Cl, Br, I, OR5, SR5, NR5R5', N=NR5, N=R5, NR5R5', NO2, SOR5R5', SO2R5, SO3R5, OSO3R5, PR5R5', POR5R5', PO2R5R5', OPO(OR5)(OR5'), or OCH2PO(OR5(OR5') where R5and R5' are as defined above; preferably R5and R5' are independently selected from H, C1-C8 alkyl, C2-C8 alkenyl, alkynyl or heteroalkyl, C3-C8 aryl, heterocycle, carbocycle, cycloalkyl, heterocycloalkyl, heteroarylalkyl, alkylcarbonyl or glycoside; or a pharmaceutically acceptable cationic salt. (b). The non-self-immolative linker component has one of the following structures: wherein (*) is the point of attachment of an additional spacer R1or cleavable linker unit, or a cytotoxic molecule and / or a cell binding molecule; X 1 , Y 1 , U 1 , R5, R5' are as defined above; r is 0-100; m and n are independently 0-6. (c). One or more linker components are 6-maleimidocaproyl ("MC"), maleimidopropionyl ("MP"), valine-citrulline ("val-cit" or "vc"), alanine-phenylalanine ("ala-phe" or "af"), p-aminobenzyloxy carbonyl ("PAB"), 4-sulfopentanoyl ("SPP"), 4-(N-maleimidomethyl)cyclohexane-1- carboxyl ("MCC"), (4-acetyl)aminobenzyl ("SIAB"), 4-sulfobutyryl (SPDB), 4-sulfo-2- hydroxysulfonyl-butyryl (2-Sulfo-SPDB), or a natural or unnatural peptide containing 1-8 natural or unnatural amino acid units. (d). One or more cleavable linker components have the following structures: -(CR5R6) m (Aa)r(CR7R8) n (OCH2CH2) t -、-(CR5R6) m (CR7R8) n (Aa) r (OCH2CH2) t -、-(Aa) r -(CR5R6) m (CR7R8) n (OCH2CH2) t -、-(CR5R6) m (CR7R8) n (OCH2CH2) r (Aa) t -、-(CR5R6) m- (CR7=CR8)(CR9R 10 ) n (Aa) t (OCH2CH2) r -、-(CR5R6) m (NR 11 CO)(Aa) t (CR9R 10 ) n- (OCH2CH2) r -、-(CR5R6) m (Aa) t (NR 11 CO)(CR9R 10 ) n (OCH2CH2) r -、-(CR5R6) m (OCO)(Aa) t (CR9R 10 ) n- (OCH2CH2) r -、-(CR5R6) m (OCNR7)(Aa) t (CR9R 10 ) n (OCH2CH2) r -、-(CR5R6) m (CO)(Aa) t- (CR9R 10 ) n (OCH2CH2) r -、-(CR5R6) m (NR 11 CO)(Aa) t (CR9R 10 ) n (OCH2CH2) r -、-(CR5R6) m- (OCO)(Aa) t (CR9R 10 ) n- (OCH2CH2) r -、-(CR5R6) m (OCNR7)(Aa) t (CR9R 10 ) n (OCH2CH2) r -、-(CR5R6) m (CO)(Aa) t (CR9R 10 ) n- (OCH2CH2) r -、-(CR5R6) m -phenyl-CO(Aa) t (CR7R8) n -、-(CR5R6) m -Furan-CO(Aa) t (CR7R8) n -、-(CR5R6) m -Oxazole-CO(Aa) t (CR7R8) n -、-(CR5R6) m Oxazole-CO-(Aa) t (CCR7R8) n -、-(CR5R6) t -Thiophene-CO(CR7R8) n -、-(CR5R6) t -imidazole-CO-(CR7R8) n -、-(CR5R6) t -morpholine-CO(Aa) t- (CR7R8) n -、-(CR5R6) t Piperazine-CO(Aa) t (CR7R8) n -、-(CR5R6) t -N-Methylpiperazine-CO(Aa) t- (CR7R8) n -、-(CR5R) m -(Aa) t Phenyl-, -(CR5R6) m -(Aa) t Furan-, -(CR5R6) m -Oxazole (Aa) t -、-(CR5R6) m -Oxazole (Aa) t -、-(CR5R6) m -thiophene-(Aa) t -、-(CR5R6) m -imidazole (Aa) t -、-(C R5R6) m -morpholine-(Aa) t -、-(CR5R6) m -piperazine-(Aa) t -、-(CR5R6) m -N-Methylpiperazine-(Aa) t -、-K(CR5R6) m (Aa)r(CR7R8) n (OCH2CH2) t -、-K(CR5R6) m (CR7R8) n -(Aa) r (OCH2CH2) t -、-K(Aa) r (CR5R6) m (CR7R8) n (OCH2CH2) t -、-K(CR5R6) m (CR7R8) n -(OCH2CH2) r (Aa) t -、-K(CR5R6) m (CR7=CR8)(CR9R 10 ) n (Aa) t (OCH2CH2) r -、-K(CR5R6) m -(NR 11 CO)(Aa) t (CR9R 10 ) n (OCH2CH2) r -、-K(CR5R6) m (Aa) t (NR 11 CO)(CR9R 10 ) n (OCH2CH2) r -、-K(CR5R6) m (OCO)(Aa) t (CR9R 10 ) n- (OCH2CH2) r -、-K(CR5R6) m (OCNR7)(Aa) t (CR9R 10 ) n -(OCH2CH2) r -、-K(CR5R6) m (CO)(Aa) t- (CR9R 10 ) n (OCH2CH2) r -、-K(CR5R6) m (NR 11 CO)-(Aa) t (CR9R 10 ) n (OCH2CH2) r -、-K(CR5R6) m- (OCO)(Aa) t (CR9R 10 ) n (OCH2CH2) r -、-K(CR5R6) m (OCNR7)(Aa) t (CR9R 10 ) n (OCH2CH2) r -、-K(CR5R6) m (CO)(Aa) t (CR9R 10 ) n -(OCH2CH2) r -、-K(CR5R6) m -phenyl-CO(Aa) t (CR7R8) n -、-K-(CR5R6) m -Furan-CO(Aa) t- (CR7R8) n -、-K(CR5R6) m -Oxazole-CO(Aa) t (CR7R8) n -、-K(CR5R6) m -Oxazole-CO(Aa) t- (CR7R8) n -、-K(CR5R6) t -Thiophene-CO(CR7R8) n -、-K(CR5R6) t Imidazole-CO-(CR7R8) n -、-K(CR5R6) t Morpholine-CO(Aa) t (CR7R8) n -、-K(CR5R6) t Piperazine-CO(Aa) t- (CR7R8) n -、-K(CR5R6) t -N-MethylpiperazineCO(Aa) t (CR7R8) n -、-K(CR5R) m (Aa)tphenyl, -K-(CR5R6) m- (Aa) t Furan-, -K(CR5R6) m -Oxazole (Aa) t -、-K(CR5R6) m -Oxazole (Aa) t -、-K(CR5R6) m -thiophene-(Aa) t -、-K(CR5R6) m -imidazole (Aa) t -、-K(CR5R6) m -Morpholine (Aa) t -、-K(CR5R6) m -piperazine-(Aa) t G, -K(CR5R6) m N-Methylpiperazine (Aa) t -; wherein m, Aa, m, n, R3, R4, and R5 are as defined above; t and r are independently 0-100; R6, R7, and R8 are independently selected from H; halide; C1-C8 alkyl, aryl, alkenyl, alkynyl, ether, ester, amine or amide, optionally substituted with one or more halide, CN, NR1R2, CF3, OR1, Aryl, heterocycle, S(O)R1, SO2R1, -CO2H, -SO3H, -OR1, -CO2R1, -CONR1, -PO2R1R2, -PO3H or P(O)R1R2R3; K is NR1, -SS-, -C(=O)-, -C(=O)NH-, -C(=O)O-, -C=NH-O-, -C=N-NH-, -C(=O)NH-NH-, O, S, Se, B or C3-C6 heteroaryl. (e). One or more linker components have the following structural units: (lys-phe), or combinations thereof, wherein is a point of attachment; X2, X3, X4, X5, or X6, is independently selected from the group consisting of NH, NHNH, N(R 12 )N(R 12 )N(R 12 ), O, S, Ci-C6alkyl, C2-C6heteroalkyl, alkylcycloalkyl, heterocycloalkyl, C3-C8aryl, arylalkyl, heterocycle, carbocycle, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl, CH2OR 12 , CH2SR 12 , CH2NHR 12 , or 1-8 amino acids; wherein R 12 and R 12 are independently H, Ci-C8alkyl, C2-C8heteroalkyl, alkylcycloalkyl, heterocycloalkyl; C3-C8aryl, arylalkyl, heterocycle, carbocycle, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; or a Ci-C8ester, ether, or amide; or a polyethylene glycol unit of the structure (OCH2CH2) p or (OCH2CH(CH3)) p wherein p is an integer from 0 to about 1000.

6. According to claim 1, wherein the structure of formula (I) the structure of formula (II) and the structure of formula (III) further have the corresponding structures of formula (la), (lb), (Ic), (Id), (Ie), (If) and (Ig) as follows: wherein is the site of attachment of the drug or linker L1or L2; "#" is the site of attachment of S (sulfhydryl), O (phenol), NH (amino), CHO (aldehyde), C(=O) (ketone), C(O)(NH) (amide), and C(O)(OH) (carboxylate) of the antibody; Aais L- or D- natural or unnatural amino acid; "@" is the site of attachment of Lc1or Lc2described in formula (I), (II), and (III); R1is H, C1-C8alkyl, OH, CH2OH, CH2CH2OH, NH2, SH, SCH3, CH2COOH, CH2CH2COOH, CH2CH2CH2CH2NH2, C6H5, CH2C6H5, CH2C6H4OH, CH(OH)CH3, CH2C(O)NH2, CH2CH2C(O)NH2, CH2CH2CH2NHC(=NH)NH2; r is 0-12; when r is not 0, (Aa)r is the same or different amino acid or peptide unit; m1= 1-18; m2= 1-100; m3= 1-8; m4= 0-8; m5= 1-8; Y 7 is NH, OCH2NH, NHC(=0), NHNH, C(=0)NH, N(R1), SO2, P(O)(OH), NHS(O)2, NHS(O)2NH, NHS(O)2NHC(O), NHS(O)2NHC(O)O, NHS(O)2NHC(O)NH, NHP(O)(OH), NHP(O)(OH)NH, OP(O)(OH)O, NHP(O)(OH)O, OP(O)(OH)NH, S, O, OP(O)(OH)OP(O)(OH)NH, NHP(O)(OH)OP(O)(OH)NH, NHP(O)(OH)OP(O)(OH)O, OCH2CH2O, OCH2CH2NH, N(CH2CH2)2N, NHC6H4NH, CH2; Y 8 is NHC(=0), NHS(02), NH(SO), NHS(02)NH, NHP(0)(OH)NH, C(0)NH, OC(0)NH, NHC(0)NH, C(O), N, NH, CH2, or CH; Lv1’ and Lv2’ are independently selected from: wherein is the site of attachment to the linker component; "#" is the site of attachment of Lv1' and Lv2' indicated in the S (sulfhydryl), O (phenol), NH (amino), CHO (aldehyde), C(=O) (ketone), C(O)(NH) (amide), and C(O)(OH) (carboxylate) molecular formula of the antibody; wherein R1, X1', and X2' are as described above; X is O, NH, S, CH2; the bond "-" between the two atoms means that it can attach to either of the two atoms, Ar is an aromatic group.

7. The core linker structure (L1”) of claim 1, having an affinity ligand in formula (I) selected from formula (Ia'): wherein Aa is L- or D-natural or unnatural amino acid; A1 is an affinity ligand, defined the same as in claim 1; R1is H, C 1- C8alkyl, OH, CH2OH, CH2CH2OH, NH2, SH, SCH3, CH2COOH, CH2CH2COOH, CH2CH2CH2CH2NH2, C6H5, CH2C6H5, CH2C6H4OH, CH(OH)CH3, CH2C(O)NH2, CH2CH2C(O)NH2, CH2CH2CH2NHC(=NH)NH2; r is 0-12; when r is not 0, (Aa)r is the same or different amino acid or peptide unit; m1= 1-18; m2= 1-100; m3= 1-8; m4= 0-8; m5= 1-8; Y 7 is NH, OCH2NH, NHC(=0), NHNH, C(=0)NH, N(R1), SO2, P(O)(OH), NHS(O)2, NHS(O)2NH, NHS(O)2NHC(O), NHS(O)2NHC(O)O, NHS(O)2NHC(O)NH, NHP(O)(OH), NHP(O)(OH)NH, OP(O)(OH)O, NHP(O)(OH)O, OP(O)(OH)NH, S, O, OP(O)(OH)OP(O)(OH)NH, NHP(O)(OH)OP(O)(OH)NH, NHP(O)(OH)OP(O)(OH)O, OCH2CH2O, OCH2CH2NH, N(CH2CH2)2N, NHC6H4NH, CH2; Y 8 is NHC(=O), NH, O, NHS(O2), NH(SO), NHS(O2)NH, NHP(O)(OH)NH, C(O)O, C(O), OC(O)NH, C(O)NH, or Ar; R 9 is (O=)CR1, (O=)CNHR1, NHC(=O), NH, O, NHS(O2), NH(SO), NHS(O2)NH, NHP(O)(OH)NH, or C(O)NH, R1(COCH2NH) m4 H, R1(Aa) r , (Aa)r, C(O), Ar, or where R3is H, C1-C8alkyl, ester, amide, Ar, ketone, alkyl acid, alkanol, alkyl amine, CH2C6H5, CH2C6H4OH, CH(OH)CH3, CH2C(O)NH2, CH2CH2C(O)NH2, CH2CH2CH2NHC(=NH)NH2; R1is as defined above.

8. The core linker structure (L1" and L2" combined) according to claim 1, having an affinity ligand of formula (III), preferably from formula (lb) and (Ic): wherein R1, Y 7 , Y 8 , R9, A1, Aa, r, m1, m2, m4, and m5 are defined as in claim 7.

9. The antibody drug conjugate containing branched affinity ligand according to claim 1, having the following structures DX001-DX237, C031, C039, C054, C060, C078, C084, C084C, C112, C117, C144, C158, C193, C200, C207, C207C, C213, C218, C225, C230, C237, C257, C263, C266, C266C, C269, C269C, C289, C289C, C418, C420, C422, C427, C429, C431, C433, C441, C443, C480, C482, C484 and C486: wherein wherein wherein wherein Raand Rbare the same as defined above, wherein Ra is as defined above, wherein wherein Raand Rbare the same as defined above, wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein m1= 4, m2= 3; wherein the mAb is an antibody; n is 1-30.

10. The conjugates of formula (I), (II) and (III) according to claim 1 are prepared by the conjugation reaction of the antibody with the compounds having the following formula (IV), (V) and (VI) affinity ligands, respectively: wherein, D1, D2, L1, L2, La1, La2, Lb1, Lb2, Lc1, Lc2, Ld1, Ld2, Ld3, Ld4, Ld5, Ld6, A1, A2, A3, A4, A5, A6, E1, m1, m2, m3, m4, m5, m6, m7, m8, m9, m 10 , m 11 , and m 12 are defined as in claim 1; Lv1and Lv2are a reactive group, independently or combined together, selected from the following structures: aryl-palladium complex, wherein X1' and X2' are independently F, Cl, Br, I, OTf, OMs, OC6H4(NO2), OC6H3(NO2)2, OC6F5, OC6HF4, or Lv3; X2is O, NH, N(R1), or CH2; R3and R5are independently H, R1, an aromatic group, heteroaromatic or aromatic, wherein one or more H atoms are independently replaced by -R1, -halogen, -OR1, -SR1, -NR1R2, -NO2, -S(O)R1, -S(O)2R1, or -COOR1; Lv3and Lv3' are independently leaving groups selected from F, Cl, Br, I, nitrophenyloxy; N-hydroxysuccinimide (NHS); phenoxy; phenylmercapto, dinitrophenyloxy; pentafluorophenoxy; tetrafluorophenoxy; difluorophenoxy; monofluorophenoxy; pentachlorophenol; triflate; imidazole; dichlorophenoxy; tetrachlorophenoxy; 1-hydroxybenzotriazole; p-toluenesulfonic acid; methanesulfonic acid; 2-ethyl-5-phenylisoxazole-3'-sulfonic acid ester, an anhydride formed by itself or with other anhydrides, such as acetic anhydride, formic anhydride; or an intermediate of a polypeptide coupling reaction using condensing reagents or an intermediate of a Mitsunobu reaction.

11. Compounds of formula (V) and (IV) according to claim 10, wherein the linking structure ###00016### is ###00017### selected from the group consisting of: wherein Lv3, Lv3', X1' and X2' are as described above; the binding bond "- " in between the two atoms means that it can be connected to either of the two atoms.

12. The compound according to claim 10, having the structure as shown in DV001-DV318, 31, 39, 54, 60, 78, 84, 112, 117, 144, 158, 193, 200, 207, 213, 218, 225, 230, 237, 257, 263, 266, 269, 269, 418, 420, 422, 427, 429, 431, 433, 441, 443, 480, 482, 484: wherein wherein wherein Ra is as defined above; wherein Ra is as defined above, wherein wherein wherein Ra is as defined above; Raand Rbare as defined above, wherein wherein wherein wherein Raand Rbare as defined above; wherein Ra is as defined above; wherein Raand Rbare as defined above, wherein Ra is as defined above; wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein wherein m1= 4, m2= 3; 13. The conjugate according to claim 1, prepared by the conjugation of the affinity ligand-containing linker compound having formula (VII), (VIII) or (IX) with the amino acid in the antibody first, and then with the cytotoxic drug or cytotoxic drug / linker complex simultaneously or subsequently: wherein L1, L2, E1, Lv1and Lv2are the same as defined above in formula (I), (II), (III), (IV), (V) and (VI); wherein Lv5and Lv6are independently selected from: wherein X1' is F, Cl, Br, I, OTs (tosylate), OTf (triflate), OMs (mesylate), OC6H4(NO2), OC6H3(NO2)2, OC6F5, OC6HF4, or Lv3; X2' is O, NH, N(R1), or CH2; R3and R5are independently H, R1, aryl, heteroaryl, or aryl groups in which one or more H atoms are independently replaced with -R1, -halogen, -OR1, -SR1, -NR1R2, -NO2, -S(O)R1, -S(O)2R1, or -COOR1; Lv3and Lv3' are leaving groups independently selected from F, Cl, Br, I, nitrophenyloxy, N-hydroxysuccinimidyl (NHS), phenoxy, phenylmercapto, dinitrophenyloxy, pentafluorophenyloxy, tetrafluorophenyloxy, difluorophenyloxy, monofluorophenyloxy, pentachlorophenyloxy, triflate, imidazole, dichlorophenyloxy, tetrachlorophenyloxy, 1-hydroxybenzotriazole, tosylate, mesylate, 2-ethyl-5-phenylisoxazolium-3'-sulfonate, anhydride formed by itself, or anhydride formed with other anhydrides (acetic anhydride, formic anhydride); or an intermediate molecule resulting from reaction with a condensing reagent for peptide coupling reaction or Mitsunobu reaction; wherein functional groups Lv5and / or Lv6may also react with sulfhydryl groups in cytotoxic drugs, provided that the rate of reaction is at least one-fold faster or slower than the rate of reaction of Lv1or Lv2with sulfhydryl groups in antibodies.

14. The affinity ligand-containing linker compound according to claim 13, shown as follows: wherein wherein 15. The conjugate according to claim 1, prepared by the conjugation of the linker compound having formula (X), (XI) or (XII) with the amino acid in the antibody first, and then with the binding ligand or binding ligand / linker complex simultaneously or subsequently: wherein, D1, D2, L1, L2, E1, Lv1and Lv2are defined as above for formula (I), (II), (III), (IV), (V) and (VI); wherein Lv7, Lv8, Lv9, Lv 10 , Lv 11 and Lv 12 are independently selected from: wherein X1' is F, Cl, Br, I, OTs (tosylate), OTf (triflate), OMs (mesylate), OC6H4(NO2), OC6H3(NO2)2, OC6F5, OC6HF4, or Lv3; X2' is O, NH, N(R1), or CH2; R3and R5are independently H, R1, aryl, heteroaryl, or an aryl group in which one or more H atoms are independently replaced with -R1, -halogen, -OR1, -SR1, -NR1R2, -NO2, -S(O)R1, -S(O)2R1, or -COOR1; Lv3and Lv3' are leaving groups independently selected from F, Cl, Br, I, nitrophenoxide, N-hydroxysuccinimidyl (NHS), phenoxide, phenylmercapto, dinitrophenoxide, pentafluorophenoxy, tetrafluorophenoxy, difluorophenoxy, monofluorophenoxy, pentachlorophenoxy, triflate, imidazole, dichlorophenoxy, tetrachlorophenoxy, 1-hydroxybenzotriazole, tosylate, mesylate, 2-ethyl-5-phenylisoxazolium-3'-sulfonate, an anhydride formed by itself, or an anhydride formed with other anhydrides (acetic anhydride, formic anhydride); or an intermediate molecule resulting from a peptide coupling reaction or a condensing reagent reacting with Mitsunobu reaction; wherein functional groups Lv5and / or Lv6may also react with a thiol in a cytotoxic drug, provided that the rate of reaction is at least one-fold faster or slower than the rate of reaction of Lv1or Lv2with a thiol in an antibody.

16. The linker compound with affinity ligand according to claim 15 is shown below: D1 is the same as DW003 and D2 is the same as DW013; 17. The conjugate according to claim 1 is prepared by reacting a linker compound with affinity ligand having the formula (XIII), (VIX), or (XV) shown below with a cytotoxic drug or cytotoxic drug / linker complex: wherein D1 is the same as DW001; wherein m1and m2= 4, 6 or 8; D1 is the same as DW003; D1 is the same as DW003, m1 = 4, 6 or 8; D1 is the same as DW003, m1 = 4, 6 or 8; D1 is the same as DW001, m1 = 4, 6 or 8; D1 is the same as DW003; D1 is the same as DW003; D1 is the same as DW001; D1 is the same as DW003; D1 is the same as DW003; D1 is the same as DW003; 18. The linker compound with affinity ligand according to claim 17 is shown below: wherein wherein D1 is the same as DW016; wherein D1 is the same as DW016.

19. The conjugate according to claim 1 is prepared by reacting a linker compound having the formula (XVI), (XVII), or (XVIII) shown below with a binding ligand or binding ligand / linker complex: wherein L1, L2, La1, La2, Lb1, Lb2, Lc1, Lc2, Ld1, Ld2, Ld3, E1, A1, A2, A3, A4, A5, A6, n, m1, m2, m3, m4, m5, m6, m7, m8, m9, m 10 , m 11 , m 12 , m The definitions of mAb, Lv1', Lv2', Lv5, and Lv6 are the same as described above.

20. The linker compound according to claim 19 is shown below: ​ wherein L1, L2, La1, La2, Lb1, Lb2, Lc1, Lc2, Ld1, Ld2, Ld3, E1, n, m1, m2, m3, m4, m5, m6, m7, m8, m9, m 10 , m 11 , m 12 , mAb, Lv1', Lv2', Lv7, Lv8, Lv9, Lv 10 , Lv 11 and Lv 12 are defined as in the above claims. ​ wherein wherein 21. The antibody or antibody-like protein of claim 1, 9, 17 or 19 selected from the group consisting of: a dAb, a Fab, a Fab', a F(ab')2, a Fv, a single domain antibody, a diabody, a triabody, a tetrabody, a minibody, a microbody, a full length antibody (a polyclonal antibody, a monoclonal antibody, an antibody dimer, an antibody multimer), a multispecific antibody (selected from a bispecific antibody, a trispecific antibody or a tetraspecific antibody); a single chain antibody, an antibody fragment that binds to a target cell, a monoclonal antibody, a single chain monoclonal antibody, a monoclonal antibody fragment that binds to a target cell, a chimeric antibody, a chimeric antibody fragment that binds to a target cell, a domain antibody that binds to a target cell, a domain antibody fragment that binds to a target cell, a surface modified antibody, a surface modified single chain antibody or a surface modified antibody fragment that binds to a target cell, a humanized antibody or a surface antibody, a humanized single chain antibody or a humanized antibody fragment that binds to a target cell, an anti-idiotypic (anti-Id) antibody, a CDR antibody, a probody, a probody fragment, a small immunoprotein (SIP), a lymphokine, a hormone, a vitamin, a growth factor, a colony stimulating factor, a nutrient transport molecule, a large molecular mass protein, a fusion protein, a kinase inhibitor, a gene targeting agent, a nanoparticle or polymer modified with an antibody or large molecular weight protein; a vitamin (including folate); or a macromolecular peptide, a polymeric micelle, a liposome, a lipoprotein-based drug carrier, a nanoparticle drug carrier, a dendrimer and a particle coated or linked with a cell binding ligand or protein as described above.

22. The conjugate of claim 1, 9, 17 or 19 targeted to a prostate tumor or other tumor having PSMA, STEAP1, B7H3, CD46, TROP2, CEACAM5, TF or DLL3 antigen.

23. The conjugate of claim 1, 9, 17, or 19 is capable of targeting tumor cells, virus infected cells, microbe infected cells, parasite infected cells, autoimmune disease cells, activated tumor cells, bone marrow cells, activated T cells, B cells or melanocyte cells, or any dysfunctional cells expressing any 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, CD13, 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, CD21b9,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, CRIPTO, CR, CR1, CRGF, CRIPTO, CXCR5, LY64, TDGF1, 4-1BB, APO2, ASLG659, BMPR1B, 4-1BB, 5AC, 5T4 (Trophoblast glycoprotein, TPBG, WNT-activated inhibitor 1 or WAIF1), adenocarcinoma antigen, AGS-5, AGS-22M6, activin receptor kinase 1, AFP, AKAP-4, ALK, alpha integrin, alpha v beta 6, aminopeptidase N, amyloid beta, androgen receptor, Angiopoietin 2, Angiopoietin 3, Annexin A1, Anthrax protective antigen, Anti-metastatic protein receptor, AOC3 (VAP-1), B7-H3, Bacillus anthracis, BAFF (B-cell activating factor), BCMA, B-cell lymphoma cell, bcr-abl, bombesin, BORIS, C5, C242 antigen, CA125 (carbohydrate antigen 125, MUC16), CA-IX (or CAIX, carbonic anhydrase 9), CALLA, CanAg, Canine lupus erythematosus IL31, Carbonic anhydrase IX, Cardiac myosin, CCL11 (C-C motif chemokine 11), CCR4 (C-C chemokine receptor 4), CCR5, CD3E (epsilon), CEA (carcinoembryonic antigen), CEACAM3, CEACAM5 (carcinoembryonic antigen), CFD (Factor D), Ch4D5, Cholecystokinin 2 (CCK2R), CLDN18 (Claudin-18), Clusterin A, cMet, CRIPTO, FCSF1R (colony stimulating factor 1 receptor), CSF2 (colony stimulating factor 2, granulocyte-macrophage colony-stimulating factor (GM-CSF)), CTLA4 (cytotoxic T-lymphocyte-associated protein 4), CTAA16.88 tumor antigen, CXCR4, C-X-C chemokine receptor 4, Cyclo-ADP- ribosylase, Cyclin B1, CYP1B1, Cytomegalovirus, Cytomegalovirus glycoprotein B, Dabigatran, DLL3 (Delta-like 3), DLL4 (Delta-like 4), DPP4 (dipeptidyl-peptidase 4), DR5 (Death receptor 5), E. coli shiga toxin type-1, E. coli shiga toxin type-2, ED-B, EGFL7 (EGF-like domain protein 7), EGFR, EGFRII, EGFRvIII, Endoglin, Endothelin B receptor, Endotoxin, EpCAM (epithelial cell adhesion molecule), EphA2, Episialin,ERBB2 (epidermal growth factor receptor 2), ERBB3, ERG (TMPRSS2 ETS fusion gene), E. coli, ETV6-AML, FAP (fibroblast activation protein alpha), FCGR1, alpha-fetoprotein, fibrillin II beta chain, fibronectin extra domain-B, FOLR (folate receptor), folate receptor alpha, folate hydrolase, Fos-related antigen 1, F protein of respiratory syncytial virus, Frizzled receptor, Ganglioside GM1, GD2 ganglioside, G-28 (cell surface antigen glycolipid), GD3 idiotype, GloboH, Glypican 3, N-glycolylneuraminic acid, GM3, GMCSF receptor alpha chain, growth differentiation factor 8, GP100, GPNMB (transmembrane glycoprotein NMB), GUCY2C (guanylate cyclase 2C), guanylate cyclase C (GC-C), intestinal guanylate cyclase, guanylate cyclase 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 / scatter factor), HHGFR, HIV-1, histone complex, HLA-DR (human leukocyte antigen), HLA-DR10, HLA-DRB, HMWMAA, human chorionic gonadotropin, HNGF, human scatter 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, IgE Fc region, IGHE, interleukins (including IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-6R, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-17, IL-17A, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-27, or IL-28), IL31RA, ILGF2 (insulin-like growth factor 2), integrin (alpha4, alphaIIb beta3, alpha v beta3, alpha4 beta7, alpha5 beta1, alpha6 beta4, alpha7 beta7, alphaII beta3, alpha5 beta5, alpha v beta5), interferon gamma inducible protein, ITGA2, ITGB2, KIR2D, Kappa Ig, LCK, Le, Legumain, 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 Al, MAGE A3, MAGE 4, MART1, MCP-1, MIF (macrophage migration inhibitory factor, or glycosyl inhibiting factor (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 chemoattractant protein 1), MelanA / MART1, ML-IAP, MPG, MS4A1, MYCN, myelin associated glycoprotein, Myostatin, NA17, NARP-1, NCA-90 (granulocyte antigen), Nectin-4 (ASG-22ME), NGF, neural apoptosis regulated proteinase 1, NOGO-A, Notch receptor, Nucleostemin, Neu oncogene product, NY-BR-1, NY-ESO-1, OX-40, OxLDL (oxidized low density lipoprotein), OY-TES1, P21, p53 nonmutant, P97, PAP, anti-(N-glycolylneuraminic acid) antibody binding site, PAX3, PAX5, PCSK9, PDCD1 (PD-1, programmed cell death protein 1), PDGF-R alpha (alpha platelet-derived growth factor receptor), PDGFR-beta, PDL-1, PLAC1, PLAP-like testicular alkaline phosphatase, platelet-derived growth factor receptor beta, sodium phosphate cotransporter, PMEL 17, polysialic acid, proteinase 3 (PR1), prostate cancer, PS (phosphatidylserine), prostate cancer cells, Pseudomonas aeruginosa, PSMA, PSA, PSCA, Rabies virus glycoprotein, RHD (Rh polypeptide 1 (RhPI)), Rhesus factor, RANKL, RhoC, Ras mutation, RGS5, ROBO4, Respiratory Syncytial Virus, RON, ROR1, sarcoma translocation breakpoints, SART3, Sclerostin, SLAMF7 (SLAM member 7), Selectin P, SDC1 (syndecan 1), Systemic Lupus Erythematosus (a), Somatomedin C, SIP (sphingosine-1 -phosphate), Somatostatin, Sperm protein 17, SSX2, STEAP1 (six-transmembrane epithelial antigen of prostate 1), STEAP2, STn, TAG-72 (tumor-associated glycoprotein), Survivin, T cell receptor, T cell transmembrane proteins, TEM1 (tumor endothelial marker 1), TENB2, Tenascin C (TN-C), TGF-alpha, TGF-beta (transforming growth factor beta), TGF-beta 1, TGF-beta 2 (transforming growth factor 2), Tie (CD202b), Tie2, TIM-1 (CDX-014), Tn, TNF, TNF-alpha, TNFRSF8,TNFRSF10B (tumor necrosis factor receptor superfamily member 10B), TNFRSF13B (tumor necrosis factor receptor superfamily member 13B), TPBG (trophoblast glycoprotein), TRAIL-R1 (TNF-related necrosis-inducing ligand receptor 1), TRAILR2 (death receptor 5 (DR5)), tumor-associated calcium signal transducer 2, tumor-specific glycosylated MUC1, TWEAK receptor, TYRP1 (glycoprotein 75), TRP-1 (Trop1), TRP-2 (Trop2), tyrosinase, VCAM-1, VEGF, VEGF-A, VEGF-2, VEGFR-1, VEGFR-2, vimentin, WT1, XAGE1, cells expressing insulin growth factor receptor, or cells expressing epidermal growth factor receptor.

24. The tumor cell of claim 23 selected from a lymphoma cell, a myeloma cell, a renal cancer cell, a breast cancer cell, a prostate cancer cell, an ovarian cancer cell, a colorectal cancer cell, a gastric cancer cell, a squamous cancer cell, a small cell lung cancer cell, a non-small cell lung cancer cell, a testicular cancer cell, a malignant cell or any cell that grows and divides at an unregulated, accelerated rate resulting in cancer.

25. A pharmaceutical composition comprising a therapeutically effective amount of the conjugate compound of any one of claims 1 or 9 and a pharmaceutically acceptable salt, carrier, diluent or adjuvant, or a combination of conjugate compositions, for the treatment or prevention of cancer.

26. The pharmaceutical composition of claim 25 is a liquid or a lyophilized solid formulation having a weight of: 0.01-99% of one or more conjugates, which are one or more conjugates of any one or more of claims 1 or 9; 0.0-20.0% of one or more polyols; 0.0-2.0% of one or more surfactants; 0.0-5.0% of one or more preservatives; 0.0-30% of one or more amino acids; 0.0-5.0% of one or more antioxidants; 0.0-0.3% of one or more metal chelators; 0.0-30.0% of one or more buffer salts for adjusting the pH of the formulation to 4.5 to 7.5; 0.0-30.0% of one or more isotonic agents for adjusting the osmolarity to about 250 to 350 mOsm when reconstituted for administration to a patient; wherein said polyols are selected from the group consisting of fructose, mannose, maltose, lactose, arabinose, xylose, ribose, rhamnose, galactose, and glucose. Non-reducing sugars include sucrose, trehalose, sorbose, turanose, and raffinose. Sugar alcohols are selected from the group consisting of mannitol, xylitol, erythritol, maltitol, lactitol, erythritol, threitol, sorbitol, glycerol, or L-gluconate and its metal salts; wherein said surfactants are selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 65, polysorbate 80, polysorbate 81, or polysorbate 85, poloxamer, poly(ethylene oxide)-poly(propylene oxide), poly(ethylene)-poly(propylene), Triton; sodium dodecyl sulfate (SDS); sodium lauryl sulfate; sodium octyl glycoside; dodecyl, myristyl, linoleyl, or stearyl sulfobetaine; dodecyl, myristyl, linoleyl, or stearyl sarcosine; linoleic, myristyl, or cetyl betaine; lauryl, cocamidopropyl, linoleamidopropyl, myristamidopropyl, palmitamidopropyl, or isostearamidopropyl-betaine (e.g., cocamidopropyl); myristamidopropyl, palmitamidopropyl, or isostearamidopropyl-dimethylamine; sodium methyl cocoyl or disodium methyl oleyl taurate; dodecyl betaine, dodecyl dimethylamine oxide, cocamidopropyl betaine, and cocoamphoglycinate; or isostearyl ethyl imidonium ethyl sulfate; polyethylene glycol, polypropylene glycol, copolymers of ethylene glycol and propylene glycol; wherein said preservatives are selected from the group consisting of benzyl alcohol, octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, phenol, butyl and benzyl alcohol, alkyl paraben such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, or meta-cresol; wherein amino acids are selected from the group consisting of arginine, cystine, glycine, lysine, histidine, ornithine, isoleucine, leucine, alanine, glycine glutamic acid, or aspartic acid; wherein said antioxidants are selected from the group consisting of ascorbic acid, glutathione, cystine, or methionine; wherein chelators are selected from the group consisting of EDTA or EGTA; The buffering salt is selected from sodium, potassium, ammonium salt or trihydroxyethylamino salt of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid or phthalic acid; triethylamine or tromethamine hydrochloride, phosphate or sulfate; arginine, glycine, glycyglycine or histidine and anion acetate, chloride, phosphate, sulfate or succinate; The isotonic agent is selected from mannitol, sorbitol, sodium acetate, potassium chloride, sodium phosphate, potassium phosphate, trisodium citrate or sodium chloride.

27. The pharmaceutical composition of claim 25 or 26, packaged in a vial, a bottle, a pre-filled syringe or a pre-filled auto-injector in a liquid or a lyophilized solid form.

28. The conjugate of claim 1, 9, or the conjugate in the form of the pharmaceutical composition of claim 25 or 26, having in vitro, in vivo or ex vivo cell-killing activity.

29. The pharmaceutical composition of claim 25 or 26, wherein the pharmaceutical composition is administered simultaneously with a chemotherapeutic agent, radiotherapy, an immunotherapeutic agent, an autoimmune disease agent, an anti-infective agent or other conjugate, synergistically effective in treating or preventing cancer.

30. The compound for use in the synergistic treatment or prevention of cancer according to claim 29 is selected from one or several of the following drugs: Abatacept, Abemaciclib, Abiraterone acetate, Abraxane, Acetaminophen / hydrocodone, Acalabrutinib, Aducanumab, Adalimumab, ADXS31-142, ADXS-HER2, Afatinib dimaleate, Aldesleukin alectinib, Alemtuzumab, Alitretinoin, ado-trastuzumab emtansine, Amphetamines / dextroamphetamine, Anastrozole, Aripiprazole, Anthracyclines, Aripiprazole, Atazanavir, Atezolizumab, Atorvastatin, Avelumab, Axicabtagene ciloleucel, Brentuximab vedotin, Brigatinib, Budesonide, Budesonide / formoterol, Buprenorphine, Cabazitaxel, Cabozantinib, Capmatinib, Capecitabine, carfilzomib, Chimeric antigen receptor engineered T (CAR-T) cells, Celecoxib, Ceritinib, Cetuxib, Cetuximab, Crizotinib, Cobimetinib, Cosentyx, Crizotinib, CTL019, Dabigatran, Dabrafenib, Dacarbazine, Daratumumab, Dacomitinib, Daptomycin, Daratumumab, Darbepoetin alfa, Darunavir, Dasatinib, Denileukin diftitox, Denosumab, Depakote, Dexlansoprazole, Dexmedetomidine, Dexamethasone, Dinutuximab, Doxycycline, Duloxetine, Duvelisib, Durvalumab, Eculizumab, Eculizumab / acyclovir, Enoxaparin, Eintinib, Enzalutamide, Epoetin alfa, Erlotinib, Esomeprazole, Ezopiclone, Etanercept, Everolimus, Exemestane, Everolimus, Exenatide ER, Ezetimibe, Ezetimibe / simvastatin, Fenofibrate, Fexofenadine, Fingolimod, Fluticasone propionate, Fluticasone / salmeterol, Fulvestrant, Gazyva, Gefitinib, Glatiramer, Goserelin acetate, Icotinib, Imatinib, Ibrutinib, Ibrutinib, Idelalisib, Ifosfamide, Infliximab, Imiquimod, ImmuCyst, ImmunoBCG, Iniparib, insulin aspart, insulin detemir, insulin glargine, insulin lispro, alpha-interferon, alpha-1b interferon, alpha-2a interferon, alpha-2b interferon, beta-interferon, beta-1a interferon, beta-1b interferon, gamma-1a interferon, Lapatinib, Ipilimumab, ipratropium bromide / albuterol, isoxazole MIB, Canumax, Laneprostone acetate, Linodromide, Linamide, Linifanib mesylate, Letrozole, Levothyroxine, Levothyroxine, Lidocaine, Linzagolamide, Liraglutide, Lidexamethasone, LN-144, Larotrectinib, Memantine, Metyrapone, Metoprolol, Mekinist, Mekinist, Metastat / Ribavirin / Tenofovir, Modafinil, Mometasone, Mycidac-C, Nectumumab, Neratinib, Nilotinib, Niraparib, Nivolumab, Ofatumumab, Obidoximab, Olaparib, Olmesartan, Olmesartan / Hydrochlorothiazide, Omalizumab, Omega-3 fatty acid ethyl ester, Oncorine, Oseltamivir, Osimertinib, Oxycodone, Palbociclib, Palivizumab, Panitumumab, Panobinostat, Pazopanib, Pembrolizumab, PD-1 antibody, PD-L1 antibody, Pemetrexed, Pertuzumab, Pneumococcal conjugate vaccine, Pomalidomide, Pregabalin, ProscaVax, Propranolol, Quetiapine, Rabeprazole, Probanex 223 Radium Chloride, Raloxifene, Raloxifene, Ramucirumab, Ranibizumab, Regorafenib, Rituximab, Rivaroxaban, Romidepsin, Rosuvastatin, Ruxolitinib phosphate, Salbutamol, Savolitinib, Semaglutide, Sevelamer, Sildenafil, Siltuximab, Sipuleucel-T, Sitagliptin, Sitagliptin / metformin, Solifenacin, Solane-zumab, Sonidegib, Sorafenib, Sunitinib, Tacrolimus, Tacrimus, Tapaaral, Tacrimus tataparal, Tazolaparib, Temozolomide, Temsirolimus, Tenofovir / Emtricitabine, Tenofovir disoproxil fumarate, Testosterone gel, Thalidomide, TICE BCG, Tiotoxium bromide, Tisaglumide, Toremifene, Trametinib, Trastuzumab, Trabectedin (Ecteinascidin743), Trametinib, Tremelimumab, Trifluridine / Tipiracil, Tretinoin, Uro-BCG, Ustekinumab, Valsartan, Veliparib, Vandetanib, Vemurafenib, Venetoclax, Vorinostat, Ziv-aflibercept, Zostavax, and analogs, derivatives, pharmaceutically acceptable salts, carriers, diluents or adjuvants thereof, or combinations thereof.

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