Novel camptothecin derivatives and conjugates thereof

Abstract

The present disclosure provides novel camptothecin derivatives and their conjugates thereof. The present disclosure further provides to pharmaceutical composition comprising the novel camptothecin derivatives or their conjugates thereof, the generation method of the conjugates, and pharmaceutical uses of the novel camptothecin derivatives and their conjugates thereof.

Description

NOVEL CAMPTOTHECIN DERIVATIVES AND CONJUGATES THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to US Provisional Application No. 63 / 387,737, filed on December 16, 2022. The disclosure of the prior application is considered part of the disclosure of this application and is incorporated in its entirety into this application.REFERENCE TO THE SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (SeqListing-BIONECURE- 005.xml; Size: 3.51 Kilobytes; Production Date: December 14, 2023) is herein incorporated by reference in its entirety.FIELD OF THE INVENTION

[0003] The present disclosure relates to the pharmaceutical filed, specifically to camptothecin derivatives, conjugates thereof.BACKGROUND OF THE INVENTION

[0004] Today, cancer remains a major cause of death worldwide despite the numerous advanced diagnostic and therapeutic methods that have been developed. The major barrier to successful treatments and prevention of cancer lies in the fact that many cancers still fail to respond to the current chemotherapeutic and immunotherapy intervention, and many individuals suffer a recurrence or death, even after aggressive therapy.

[0005] Camptothecin (CPT) (Figure 1) was originally isolated from the bark of Camptotheca acuminata, a tree native to the rocky slopes of north China isolated by Wall et al. [Wall ME, Wani MC, Cook CE, Palmer KH, McPhail AT, Sim GA. Plant antitumor agents. I. The isolation and structure of camptothecin, a novel alkaloidal leukemia and tumor inhibitor from Camptotheca acuminata (J Am Chem Soc 1966; 88: 3888-3890). CPT has been demonstrated to be effective against a broad spectrum of tumors. Its molecular target has been firmly established to be DNA topoisomerase I (Topo I) that changes the topological state of duplex DNAby single-strand breakage and religation. [Chen AY, Liu LF. DNA topoisomerases: essential enzymes and lethal targets. Anna Rev Pharmacol Toxicol 1994; 34: 191-218; Hsiang YH, Hertzberg R, Hecht S, Liu LF. Camptothecin induces protein-linked DNA breaks via mammalian DNA topoisomerase I. J Biol Chem 1985; 260: 14873-14878. Hsiang YH, Liu LF. Identification of mammalian DNA topoisomerase I as an intracellular target of the anticancer drug camptothecin. Cancer Res 1988; 48: 1722-1726], Biochemical studies in vitro have revealed that CPT binds at the interface between Topo I and DNA and inhibits specifically the religation step in the cleavage / religation reaction [Hsiang YH, Hertzberg R, Hecht S, Liu LF. Camptothecin induces protein-linked DNA breaks via mammalian DNA topoisomerase I. J Biol Chem 1985; 260: 14873- 14878. Svejstrup JQ, Christiansen K, Gromova II, Anderson AH, Westergaard 0. New technique for uncoupling the cleavage and relegation reactions of eukaryotic topoisomerase I: the mode of action of camptothecin at a specific recognition site. J Mol Biol 1991; 222: 669-678],

[0006] CPT has attracted much attention because of a very good spectrum of its antitumor activity against experimental animal tumor models, such as L1210 leukemia in mice and Walker 256 sarcoma in rats. However, its clinical development failed due to the reversible bone marrow depression and hemorrhagic cystitis, which are the major dose-limiting toxicity [Muggia FM, Creaven PJ, Hansen HH, Cohen MH, Selawry OS. Phase I clinical trial of weekly and yearly treatment with camptothecin (NSC-100880); correlation with preclinical studies. Cancer Chemother Res 1972; 56; 515-521. Schappi U, Fleischmann RW, Cooney DA. Toxicity of camptothecin (NSC-100880). Cancer Cemother Rep Part 1974; 35: 25-36], Later, efforts directed at finding new camptothecin derivatives with higher anticancer activity and less toxicity led to the discovery of a potent and safer camptothecin derivative, named irinotecan or CPT-11 (Figure 1). As one of the prominent anti-neoplastic drugs widely used in clinical practice today, CPT-11 is a water-soluble pro-drug and undergoes carboxylesterase-mediated hydrolysis to form SN-38, a potent Topo I inhibitor [Bencharit S, Morton CL, Howard-Williams EL, Danks MK, Potter PM, Redinbo MR. Structural insights into CPT-11 activation by mammalian carboxyesterases. Nat Struct Biol 2002; 9: 337-342. Andoh T, Ishii K, Suzuki Y, Ikegami Y, Kusunoki Y, Takemoto Y, st al. Characterization of mammalian mutant with a camptothecin resistant DNA topoisomerase I. Proc Natl Acad Sci USA 1987; 84: 5565-5569],

[0007] Antibody-drug conjugates (ADCs) combine the binding specificity of an antibody with the potency of drugs such as, for example, cytotoxic agents, anticancer and immunosuppressive drugs. The use of ADCs allows the target-specific delivery of drugs which, if administered as unconjugated drugs, may result in unacceptable levels of toxicity to normal cells.The mechanism of an ADC is to recognize and bind to specific antigen through the antibodies, trigger a series of reactions, and then enter the cytoplasm through the endocytosis, where the highly cytotoxic drug is dissociated from the antibody after the degradation by lysosomal enzymes to kill cancer cells. Compared with the traditional chemotherapy which causes damage to both cancer cells and normal tissues indiscriminately, targeting drug delivery can make the drug act on cancer cells directly and reduce the damage to normal cells.

[0008] Recently, with the FDA approval of Trodelvy® and Enhertu®, two camptothecin derivatives, SN-38 and DXd, have been validated as ADC payloads. SN-38 is a relatively weak toxin, while the linker used by Trodelvy® is unstable. Interstitial lung disease (ILD) is a serious adverse effect associated with DXd ADCs. (Thomas M. Cardillo, Serengulam V. Govindan, Robert M. Sharkey, Preeti Trisal, and David M. Goldenberg, Clin Cancer Res; 17(10); 3157-3169. Yusuke Ogitani, Tetsuo Aida, Katsunobu Hagihara, Junko Yamaguchi, Chiaki Ishii, Naoya Harada, Masako Soma, Hiromi Okamoto, Masataka Oitate, Shingo Arakawa, Takehiro Hirai, Ryo Atsumi, Takashi Nakada, Ichiro Hayakawa, Yuki Abe, and Toshinori Agatsuma, Clin Cancer Res; 22(20); 5097-5108).

[0009] There still exists a great need for campothecin derivatives for use as ADC payloads for use in the treatment or to prevent recurrence of cancers and / or immunological disorders.SUMMARY OF THE INVENTION

[0010] The present disclosure provides novel camptothecin derivatives, conjugates thereof, and pharmaceutical composition comprising the novel camptothecin derivatives or the conjugates thereof. The present disclosure also provides the generation method of the conjugates, and pharmaceutical uses of the novel camptothecin derivatives and their conjugates thereof.

[0011] In one aspect, the present disclosure provides a camptothecin (CPT) derivative represented by the following structure formula (A),a pharmaceutically acceptable salt stereoisomer, enantiomer, or deuterated counterpart thereof; wherein X is selected from -C(=O)-(CH2)n1-O-N(R2)R3, -C(=O)-(CH2)n1-N(OR2)R3, -C(=O)- -S-R3;Ri, R2, R3and Rceach independently is a hydrogen atom, or Ci-Cs alkyl;R4 and R5 each independently represents a hydrogen atom, a deuterium atom, halogen, halogenated alkyl, deuterated alkyl, alkoxy, hydroxyl, amino, nitro, cyano, hydroxyalkyl, heterocyclic Ci-Cs alkyl or 3-6 membered heterocyclic or cycloalyl ring; or R.s and R5 together with the carbon atom form a 3-6 membered cyclic ring;Raand Rbeach independently represents a hydrogen atom, a deuterium atom, halogen, halogenated alkyl, deuterated alkyl, alkoxy, hydroxyl, amino, nitro, cyano, hydroxyalkyl, heterocyclic CrC® alkyl or 3-6 membered heterocyclic or cycloalyl ring; or Raand Rbtogether with the carbon atom form a 3-6 membered cyclic ring; n1is 1 , 2, 3, 4 or 5; n2is 2, 3, 4 or 5; n3is 1 , 2, 3, 4 or 5.

[0012] In one aspect, the present disclosure provides camptothecin derivative conjugate comprising at least one of the camptothecin derivatives, the pharmaceutically acceptable salts, stereoisomers, enantiomers, or deuterated counterparts thereof disclosed herein; or the pharmaceutically acceptable salt of the disclosed camptothecin derivative conjugate.

[0013] In one aspect, the present disclosure provides camptothecin derivative conjugate comprising (1) at least one of the camptothecin derivatives, the pharmaceutically acceptable salts, stereoisomers, enantiomers, or deuterated counterparts thereof disclosed herein; (2) a linker; and / or (3) a targeting agent; or the pharmaceutically acceptable salt of the disclosed camptothecin derivative conjugate.

[0014] In one aspect, the present disclosure provides a method of making the camptothecin derivative conjugate or the pharmaceutically acceptable salt thereof disclosed herein, comprising reacting a targeting agent and / or a linker with a camptothecin derivative. In some embodiments, the camptothecin derivative used in the method of making the camptothecin derivative conjugate or the pharmaceutically acceptable salt thereof is the camptothecin derivative disclosed herein.

[0015] In one aspect, the present disclosure provides a pharmaceutical composition, comprising the camptothecin derivative, a pharmaceutically acceptable salt, stereoisomer, enantiomer, or deuterated counterpart thereof disclosed herein, the camptothecin derivative conjugate or the pharmaceutically acceptable salt thereof disclosed herein, and a pharmaceutically acceptable diluent, carrier, or excipient.

[0016] In one aspect, the present disclosure provides a method of treating or preventing a disease, comprising administering to a subject in need thereof a therapeutically effective amount of the camptothecin derivative, a pharmaceutically acceptable salt, stereoisomer, enantiomer, or deuterated counterpart thereof disclosed herein, the camptothecin derivative conjugate or the pharmaceutically acceptable salt thereof disclosed herein, or the pharmaceutical composition disclosed herein.

[0017] In one aspect, the present disclosure relates to an antibody drug conjugate (ADC) comprising an antibody (e.g., a cell binding antibody) chemically linked to a camptothecin derivative represented by the following formula (I1):Ab-(L-D)n(I’) or a pharmaceutically acceptable salt thereof;wherein n is an integer of about 1 to about 12;Ab is an antibody or antigen binding fragment thereof;D is a camptothecin derivative;L is a linker. and the camptothecin derivative is selected from:L is a linker represented by the following formula (Ha and lib):L1-L2-L3(Ha)U-L2(lib) wherein L1is a linker moiety attached to the antibody, and U comprises a reacted functional group selected from maleimide, bromoacetyl, iodoacetyl, thiol, amino, alkyl bromide, alkyl iodide, allenamide, carboxyl, and NHS ester;L2is a linker moiety comprising 2-4 AA peptides, and optionally comprising spacers, preferably PEG spacers;L3 is a linker moiety connected to camptothecin derivative, and L3comprises any one of the following:R1 is a hydrogen atom or Ci-Cs alkyl;R2is a hydrogen atom or C1-C5 alkyl;Rs is a hydrogen atom,R4 and R5 each independently represents a hydrogen atom, a deuterium atom, halogen, halogenated alkyl, deuterated alkyl, alkoxy, hydroxyl, amino, nitro, cyano, hydroxyalkyl, heterocyclic, CrCs alkyl or 3-6 member heterocyclic or cycloalyl ring; or R4and R5 together with the carbon atom form a 3-6 member cyclic ring;Raand Rbeach independently represents a hydrogen atom, a deuterium atom, halogen, halogenated alkyl, deuterated alkyl, alkoxy, hydroxyl, amino, nitro, cyano, hydroxyalkyl, heterocyclic, CrCe alkyl or 3-6 member heterocyclic or cycloalyl ring; or Rsand Rbtogether with the carbon atom form a 3-6 member cyclic ring;Rcis a hydrogen atom or Ci-Ce alkyl; n1is 1 , 2, 3, 4 or 5; n2is 2, 3, 4 or 5;n3is 1, 2, 3, 4 or 5;Y is -C(~O)-Rs, -C(=0)-0R6, -C(=O)-NHR6, RS is CI-CB alkyl;Z is NHR7, R7is CrC6alkyl, -CH2-CH2-OH, -CH2-CH2-OCH3, or -CH2-CH2-N(CH3)2 W is NH or O.

[0018] In one embodiment, L1is selected from and -C(=O)-(CH2)n1~R8-and Rs is alkylene-aryl-alkylene, aryl, 3-7 memberedheterocyclyl, 3-7 membered cycloalkyl, heteroaryl, -alkylene-heteroaryl-alkylene-.

[0019] In one embodiment, L is selected from

[0020] In an aspect of the disclosure, L2is a dipeptide, tripeptide or tetrapeptide comprising naturally occurring and non-naturally occurring amino acids.

[0021] In an aspect of the disclosure, L2is selected from: gly-gly, gly-gly-gly, phe-lys, val-ala, val-cit, gly-gly-phe-gly (GGFG)(SEQ ID NO: 1), val-cit-gly, val-gln-gly, val-glu-gly, phe- lys-giy, glu-val-aia, glu-val-cit, p-aia-gly-phe-gly (AGFG)(SEQ ID NO: 2), and gly-gly-phe-gly-gly (GGFGG)(SEQ ID NO: 3), where the amino acid sequence is in either orientation.

[0022] In one embodiment, the camptothecin derivative-linker is selected fromz is an integer between 1-6; and X1is a reacted functional group selected from maleimide, bromoacetyl, iodoacetyl, thick amino, alkyl bromide, alkyl iodide, allenamide, carboxyl, and NHS ester.In one embodiment, the camptothecin derivative is

[0024] In one embodiment the reactive functional group is

[0025] In one embodiment -L-X comprises

[0026] In one embodiment, the camptothecin derivative-linker is selected from:ĶI

[0027] Another aspect of the disclosure is a pharmaceutical composition including a Formula I' ADC compound, a mixture of Formula T ADC compounds, or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable diluent, carrier, or excipient.

[0028] Another aspect provides a pharmaceutical combination comprising a FormulaI’ ADC compound and a second compound having anticancer properties or other therapeutic effects.

[0029] Another aspect is a method for killing or inhibiting the proliferation of tumor ceils or cancer cells comprising treating the cells with an amount of an antibody-drug conjugate of Formula I’, or a pharmaceutically acceptable salt or solvate thereof, being effective to kill or inhibit the proliferation of the tumor cells or cancer cells.

[0030] Another aspect is a method of treating cancer comprising administering to a patient a therapeutically effective amount of a pharmaceutical composition including a Formula !’ ADC.

[0031] In various embodiments, the disclosure relates to antibody drug conjugates wherein x is about 1 to about 8. In various embodiments, x is about 4 to about 7. In various embodiments, x is about 4. In various embodiments, x is about 6, In various embodiments, x is about 7.

[0032] In various embodiments, the disclosure relates to derivatized camptothecin derivatives wherein L1comprises pyrroline-dione. In various embodiments of the disclosure, the heterocyclyl ring is selected from saturated or unsaturated 4-6 membered nitrogen containing heterocyclic rings. Examples of saturated heterocyclic radicals include saturated 3 to 6- membered heteromonocylic group containing 1 to 4 nitrogen atoms [e.g. pyrrolidinyl, imidazolidinyl, piperidine, piperazinyl]; saturated 3 to 6-membered heteromonocyclic group containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms [e.g. morpholinyl]; saturated 3 to 6- membered heteromonocyclic group containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms [e.g., thiazolidinyl]. Examples of unsaturated heterocyclic radicals, also termed "heteroaryl" radicals, include unsaturated 5 to 6 membered heteromonocyclyl group containing 1 to 4 nitrogen atoms, for example, pyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, pyrimidyl, pyrazinyl, pyridazinyl, triazolyl [e.g., 4H-1 ,2,4-triazolyl, 1 H-1 ,2,3-triazolyl, 2H-1 ,2,3-triazolyl]; unsaturated condensed heterocyclic group containing 1 to 5 nitrogen atoms, for example, indolyl, isoindolyl, indolizinyl, benzimidazolyl, quinolyl, isoquinolyl, indazolyl, benzotriazolyl, tetrazolopyridazinyl [e.g., tetrazolo [1 ,5-b]pyridazinyl]; unsaturated 5- to 6-membered heteromonocyclic group containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms, for example, oxazolyl, isoxazolyl, oxadiazolyl [e.g., 1 ,2,4-oxadiazolyl, 1 ,3,4-oxadiazolyl, 1 ,2,5-oxadiazolyl]; unsaturated condensed heterocyclic group containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms [e.g. benzoxazolyl, benzoxadiazolyl]; and unsaturated 5 to 6-membered heteromonocyclicgroup containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms, for example, thiazolyl, thiadiazolyl [e.g., 1 ,2,4-thiadiazolyl. In various embodiments of the disclosure, the cyclic alkyl ring, also known as a cycloalkyl ring, is a saturated cyclic alkyl group derived by the removal of one hydrogen atom from a single carbon atom of a parent cycloalkane. Typical cycloalkyl groups include, but are not limited to, groups derived from cyclopropane, cyclobutane, cyclopentane, cyclohexane, and cycloheptane, and the like.

[0033] In various embodiments, the ADCs comprise an L that is a cleavable linker. In various embodiments, the ADC comprises an antibody that is conjugated with a camptothecin derivative through a linker that is peptidase cathepsin sensitive. In various embodiments, the ADC comprises an antibody conjugated with a camptothecin derivative via a iinker that is peptidase cathepsin sensitive, in various embodiments, the ADC is an anti-Her2 antibody conjugated with camptothecin derivatives, wherein the camptothecin derivative is linked to an anti-Her2 antibody via a linker that is not acid labile. In various embodiments, the ADC comprises an antibody conjugated with a camptothecin derivative via a linker that does not contain a disulfide bond. In various embodiments, the ADC comprises an antibody conjugated with camptothecin derivatives via a linker that provides stability during circulation while being able to release the drug once inside the cells. Such linkers are contemplated to provide stability to the conjugated molecule prior to endocytosis, such as during circulation, to prevent premature degradation of the linker and release of the toxic drug, thus minimize the toxic effect of the drug.

[0034] In various embodiments there is provided one or more of a set of ADCs of Formulas I’ wherein L and L1are cysteine reactive linkers.

[0035] In various embodiments there is provided one or more of a set of compounds of Formula !’ and II wherein L and L2are cleavable linkers.In various embodiments, the number of bonds formed between the drug-linker and cysteine residue on the antibody, such as anti-Her2 antibody, is from 3 to 8. In various embodiments, the number of such bonds is at least 2, or alternatively at least 4, or 5. In various embodiments, the number of such formed bonds is no more than 8, or alternatively no more than 7, 6, 5, or 4. In various embodiments, each antibody, such as anti-Her2 antibody, on average, is conjugated with about 4-7 drug molecules through cysteines.

[0036] Another aspect of the disclosure relates to pharmaceutical compositions of the cell binding agent conjugates of formulae I’ and a pharmaceutically acceptable carrier, additive or diluent thereof.

[0037] In various embodiments, the ADC constructs of the present disclosure comprise an Ab that is a targeting moiety, such as an antibody, a multispecific antibody, a bispecific antibody, or antibody fragment, capable of binding to a tumor associated antigen (TAA), a tissue- specific antigen, a cell surface molecule, extracellular matrix protein or protease(s), or any post- translational modification residue(s). In various embodiments, the ADC constructs of the present disclosure comprise an Ab that is a targeting moiety that exhibits binding affinity to a diseased cell or tissue.

[0038] In various embodiments, the antibody, multispecific antibody, bispeclfic antibody, or antibody fragment is capable of binding to a TAA selected from the group consisting of: tumor- associated calcium signal transducer 2 (also known as Trop-2), Her2, Her3, Her4, EGF, EGFR, CD2, CD3, CD5, CD7, CD13, CD19, CD20, CD21 , CD23, CD30, CD33, CD34, CD38, CD46, CD55, CD59, CD69, CD70, CD71 , CD97, CD117, CD123, CD127, CD134, CD137, CD138, CD146, CD147, CD152, CD154, CD174, CD195, CD200, CD205, CD212, CD223, CD227, CD253, CD272, CD274, CD276, CD278, CD279, CD309, CD319, CD326, CD340, DR6, Kv1.3, 5E10, MUC1 , uPA, MAGE3, MUC16, KLK3, K-ras, Mesothelin, p53, Survivin, G250, PSMA, Endoplasmin, BCMA, GPNMB, EphA2, EphB2, TMEFF2, Integrin beta 6, 5T4, CAS, IGF-1 R, Axl, B7H3, B7H4, CDH6, HAVCR1, STEAP-1 , STEAP-2, UPK2, CLDN18.2, CLDN6, CLDN9, c-Met, MICA / B, LIV-1, ROR1 , ADAM9, STn, Globo H, SSEA-4, MG7-Ag, Fucosyl GM-1, DLK-1, CEACAM5.

[0039] In various embodiments, the ADC comprises a TAA binding Ab selected from the group consisting of a fully human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, an antigen-binding antibody fragment, a Fab, a Fab', a Fab2, a Fab'2, a IgG, a IgM, a IgA, a IgE, a scFv, a dsFv, a dAb, a nanobody, a unibody, and an diabody. In various embodiments, the antibody is a chimeric antibody. In various embodiments, the antibody is a humanized monoclonal antibody. In various embodiments, the antibody is a fully human monoclonal antibody.

[0040] In another aspect, the present disclosure provides a pharmaceutical composition comprising the isolated ADC constructs in admixture with a pharmaceutically acceptable earner.

[0041] In another aspect, the disclosure provides uses of the ADC constructs for the preparation of a medicament for the treatment of cancer.

[0042] In another aspect, the present disclosure provides a method for treating cancer or cancer metastasis in a subject comprising administering a therapeutically effective amount of the pharmaceutical compositions of the disclosure to a subject in need thereof. In one embodiment,the subject is a human subject, in various embodiments, the cancer is selected from pancreatic cancer, gastric cancer, liver cancer, breast cancer, ovarian cancer, colorectal cancer, melanoma, leukemia, myelodysplastic syndrome, lung cancer, prostate cancer, brain cancer, bladder cancer, head-neck cancer, or rhabdomyosarcoma or any cancer.

[0043] In various embodiments, the subject previously responded to treatment with an anti-cancer therapy, but, upon cessation of therapy, suffered relapse (hereinafter “a recurrent cancer). In various embodiments, the subject has a resistant or refractory cancer.

[0044] In another aspect, the present disclosure provides a method for treating cancer or cancer metastasis in a subject comprising administering a therapeutically effective amount of the pharmaceutical compositions of the disclosure in combination with a second therapy selected from the group consisting of: cytotoxic chemotherapy, immunotherapy, small molecule kinase inhibitor targeted therapy, surgery, radiation therapy, stem cell transplantation, cell therapies including CAR-T, CAR-NK, IPS induced CAR-T or IPS induced CAR-NK and vaccine such as Bacille Calmette-Guerine (BCG). In various embodiments, the combination therapy may comprise administering to the subject a therapeutically effective amount of immunotherapy, including, but are not limited to, treatment using depleting antibodies to specific tumor antigens; treatment using antibody-drug conjugates; treatment using agonistic, antagonistic, or blocking antibodies to costimulatory or co-lnhibitory molecules (immune checkpoints) such as CTLA-4, PD-1, PD-L1 , CD40, OX-40, CD137, GITR, LAG3, TIM-3, Siglec 7, Sigtec 8, Siglec 9, Siglec 15 and VISTA; treatment using bispecific T cell engaging antibodies (BITE®) such as blinatumomab: treatment involving administration of biological response modifiers such as IL-12, IL-21, GM-CSF, IFN-alpha, IFN-p and IFN-y; treatment using therapeutic vaccines such as sipuleucel-T; treatment using dendritic cell vaccines, or tumor antigen peptide vaccines; treatment using chimeric antigen receptor (CAR)-T cells; treatment using CAR-NK cells; treatment using tumor infiltrating lymphocytes (TILs); treatment using adoptively transferred anti-tumor T cells (ex vivo expanded and / or TCR transgenic); treatment using TALL-104 cells; and treatment using immunostimulatory agents such as Toll-like receptor (TLR) agonists CpG and imiquimod; and treatment using vaccine such as BCG; wherein the combination therapy optionally provides increased effector cell killing of tumor cells, i.e. , a synergy exists between the ADC constructs and the immunotherapy when co-administered.

[0045] In another aspect, there are provided novel compounds described herein as well as methods of making thereof.BRIEF DESCRIPTION OF THE DRAWINGS

[0646] Figure 1 depicts the structures of CPT, CPT-11 , SN-38, exatecan and DXd.

[0047] Figure 2 depicts conjugation procedure for camptothecin derivative with reduced mAb.

[0048] Figure 3A depicts the line graphs depicting the percent of inhibition for SK-BR-3 breast cancer ceils with increasing concentrations of DXd, T-363, Herceptin-Dxd, and Herceptin- 36309 in an in vitro cytotoxicity assay. Figure 3B depicts the line graphs depicting the percent of inhibition for NCI-N87 gastric cancer cells with increasing concentrations of DXd, T-363, Herceptin-Dxd, and Herceptin-36309 in an in vitro cytotoxicity assay. Figure 3C depicts the line graphs depicting the percent of inhibition for MDA-MB-468 breast cancer cells with increasing concentrations of DXd, T-363, Herceptin-Dxd, and Herceptin-36309 in an in vitro cytotoxicity assay. Figure 3D depicts the iine graphs depicting the percent of inhibition for BxPC-3 pancreatic cancer cells with increasing concentrations of DXd, T-363, Herceptin-Dxd, and Herceptin-36309 in an in vitro cytotoxicity assay. Cell viability is shown as relative percentage of inhibition to the untreated controls. Representative figures from three independent experiments are shown, and the values indicate the mean + SEM. The circle with dotted line represents Dxd, the square with solid line represents T-363, the triangle with dotted line represents Herceptin-Dxd, and the diamond with solid line represents Herceptin-36309 (ADC-25).

[0049] Figure 4 depicts line graphs depicting the percent of inhibition for BT-474 breast cancer cells with increasing concentrations of DXd, T-363, Herceptin-Dxd, and Herceptin-363-11 in an in vitro cytotoxicity assay. Cell viability is shown as relative percentage of inhibition to the untreated controls. Representative figures from three independent experiments are shown, and the values indicate the mean + SEM. The circle with dotted line represents Dxd, the square with solid line represents T-363, the triangle with dotted line represents Herceptin-Dxd, and the diamond with solid iine represents Herceptin-363-11 (ADC-4).

[0050] Figure 5 depicts line graphs depicting the percent of inhibition for NCI-N87 gastric cancer cells with increasing concentrations of DXd, T-363, Herceptin-Dxd, and Herceptin-363-11 in an in vitro cytotoxicity assay. Cell viability is shown as relative percentage of inhibition to the untreated controls. Representative figures from three independent experiments are shown, and the values indicate the mean + SEM. The circle with dotted line represents Dxd, the square with solid iine represents T-363, the triangle with dotted line represents Herceptin-Dxd, and the diamond with solid line represents Herceptin-363-11.

[0051] Figure 6 depicts line graphs depicting the percent of inhibition for SK-BR-3 breast cancer ceiis with increasing concentrations of DXd, T-363, Herceptin-Dxd, and Herceptin-363-11 in an in vitro cytotoxicity assay. Ceil viability is shown as reiative percentage of inhibition to the untreated controls. Representative figures from three independent experiments are shown, and the values indicate the mean + SEM. The circle with doted line represents Dxd, the square with solid line represents T-363, the triangle with dotted line represents Herceptin-Dxd, and the diamond with solid line represents Herceptin-363-11.

[0052] Figure 7 depicts line graphs depicting the percent of inhibition for BxPC-3 pancreatic cancer cells with increasing concentrations of DXd, T-363, Herceptin-Dxd, and Herceptin-363-11 in an in vitro cytotoxicity assay. Cell viability is shown as relative percentage of inhibition to the untreated controls. Representative figures from three independent experiments are shown, and the values indicate the mean + SEM. The circle with dotted line represents Dxd, the square with solid line represents T-363, the triangle with dotted line represents Herceptin-Dxd, and the diamond with solid line represents Herceptin-363-11.

[0053] Figure 8 depicts line graphs depicting the percent of inhibition for NCI-H292 lung cancer cells with increasing concentrations of DXd, T-363, Herceptin-Dxd, and Herceptin-363-11 in an in vitro cytotoxicity assay. Cell viability is shown as relative percentage of inhibition to the untreated controls. Representative figures from three independent experiments are shown, and the values indicate the mean + SEM. The circle with dotted line represents Dxd, the square with solid line represents T-363, the triangle with dotted line represents Herceptin-Dxd, and the diamond with solid line represents Herceptin-363-11.

[0054] Figure 9 depicts line graphs depicting the percent of inhibition for MDA-MB-468 breast cancer cells with increasing concentrations of DXd, T-363, Herceptin-Dxd, and Herceptin- 363-11 in an in vitro cytotoxicity assay. Cell viability is shown as relative percentage of inhibition to the untreated controls. Representative figures from three independent experiments are shown, and the values indicate the mean + SEM. The circle with dotted line represents Dxd, the square with solid line represents T-363, the triangle with dotted line represents Herceptin-Dxd, and the diamond with solid line represents Herceptin-363-11.

[0055] Figure 10A depicts the percent of inhibition of HER2-postive SK-BR-3 cells, and HER2-negative MDA-MB-468 cells treated with indicated concentrations of Herceptin-Dxd for 5 days, and the percent of inhibition of MDA-MB-468 cells in the SK-BR-3 and MDA-MB-468 coculture treated with indicated concentrations of Herceptin-Dxd for 5 days. Figure 10B depicts the percent of inhibition of HER2-postive SK-BR-3 cells, and HER2-negative MDA-MB-468 cellstreated with indicated concentrations of Herceptin-363- 11 for 5 days, and the percent of inhibition of MDA-MB-468 cells in the SK-BR-3 and MDA-MB-468 co-culture treated with indicated concentrations of Herceptin-363-11 for 5 days. Each point represents the mean and SD (n = 3). The black column represents SK-BR-3, the blank column represents MDA-MB-468, and the grey column represents coculture.

[0056] Figure 11 depicts the percentage of remaining total antibody and Herceptin-363- 11 in human plasma at indicated time points. The circle with solid line represents total antibody, and the square with dotted line represents ADC-4 (Herceptin-363-11).

[0057] Figure 12 depicts the mean tumor volume over time in female athymic mice bearing HCC-1569 HER2~positive subcutaneous xenografts treated with vehicle control or ADC- 4. Five mice per group were dosed intravenously when their tumors reached a volume of -150 mm3. Single dose of ADC-4 was administered at 1 or 5 mg / kg. Vehicle control mice were used as negative controls. The solid circle with solid line represents vehicle control, the blank circle with dotted line represents ADC-4 at 1 mg / kg, and the blank circle with solid line represents ADC-4 at 5mg / kg.

[0058] Figure 13A depicts the mean tumor volume over time in female athymic mice bearing JIMT-1 HER2-positive subcutaneous xenografts treated with vehicle control or ADC-5. Figure 13B depicts the mean tumor volume over time in female athymic mice bearing NCI-N87 HER2-positive subcutaneous xenografts treated with vehicle control or ADC-5. Five mice per group were dosed intravenously when their tumors reached a volume of -200 mm3. Single dose of ADC-5 was administered at 1 or 5 mg / kg. Vehicle control mice were used as negative controls. The solid circle with solid line represents vehicle control, the blank circle with dotted line represents ADC-5 at 1 mg / kg, and the blank circle with solid line represents ADC-5 at 5mg / kg.

[0059] Figure 14A depicts the mean tumor volume over time in female athymic mice bearing JIMT-1 HER2-positive subcutaneous xenografts treated with vehicle control or ADC-14. Figure 14B depicts the mean tumor volume over time in female athymic mice bearing NCI-N87 HER2-positive subcutaneous xenografts treated with vehicle control or ADC-14. Figure 14C depicts the mean tumor volume over time in female athymic mice bearing HCC-1569 HER2- positive subcutaneous xenografts treated with vehicle control or ADC-14. Five mice per group were dosed intravenously when their tumors reached a volume of about 150-200 mm3. Single dose of ADC-14 was administered at 1 or 5 mg / kg. Vehicle control mice were used as negative controls. The solid circle with solid line represents vehicle control, the blank circle with dotted line represents ADC-5 at 1 mg / kg, and the blank circle with solid line represents ADC-5 at 5mg / kg.

[0060] Figure 15 depicts the mean tumor volume in femaie athymic mice bearing NCI* N87 HER2-positive subcutaneous xenografts treated with vehicle control, reference ADC or ADC*22. Five mice per group were dosed intravenously when their tumors reached a volume of about 200 mm3. Single dose of reference ADC or ADC-22 was administered at 5 mg / kg. Vehicle control mice were used as negative controls. The circle represents vehicle control, the square represents reference ADC at 5mg / kg, and the triangle represents ADC-22 at 5mg / kg.

[0061] Figure 16 depicts the mean tumor volume in female athymic mice bearing NCI- N87 HER2-positive subcutaneous xenografts treated with vehicle control, reference ADC or ADC-23. Five mice per group were dosed intravenously when their tumors reached a volume of about 150-200 mm3. Single dose of reference ADC or ADC-23 was administered at 2.5mg / kg or 5 mg / kg. Vehicle control mice were used as negative controls. The circle represents vehicle control, the inverted triangle represents reference ADC at 2.5 mg / kg, the diamond represents reference ADC at 5mg.kg, the square represents ADC-23 at 2.5mg / kg, and the triangle represents ADC-23 at 5mg / kg.

[0062] Figure 17.A depicts the body weight over time in naive CD-1 mice treated with blank control, PBS, or ADC-14 (160mg / kg). The solid circle with solid line represents blank control, the blank circle with solid line represents PBS, and the blank circle with dotted line represents ADC- 14. The arrow pointing at the X-axis represents the day of dosing. Figure 17B depicts the red blood cell count, white blood cell count, neutrophil count and lymphocyte count in naive CD-1 mice treated with blank control, PBS, or ADC-14 (160mg / kg). Five mice were randomized into each group based on body weight. Single dose of ADC- 14 at 160 mg / kg was administered on day 0. Body weight was measured on day 0, 3, 5, 7 post dosing. Whole blood was collected via orbital bleeding on day 3 and day 7 for automated red blood cells, white blood cells, neutrophils and lymphocytes determinations.

[0063] Figure 18A depicts the body weight over time in nai ve CD-1 mice treated with blank control, PBS, or ADC-19 (160mg / kg). The solid circle with solid line represents blank control, the blank circle with solid line represents PBS, and the blank circle with dotted line represents ADC- 19. The arrow pointing at the X-axis represents the day of dosing. Figure 18B depicts the red blood cell count, white blood cell count, neutrophil count and lymphocyte count in naive CD-1 mice treated with blank control, PBS, or ADC-19 (160mg / kg). Five mice were randomized into each group based on body weight. Single dose of ADC- 19 at 160 mg / kg was administered on day 0. Body weight was measured on day 0, 3, 5, 7 post dosing. Whole blood was collected via orbitalbleeding on day 3 and day 7 for automated red blood cells, white blood cells, neutrophils and lymphocytes determinations.

[0064] Figure 19A depicts the body weight over time in nai ve CD-1 mice treated with blank control, PBS, or ADC-20 (160mg / kg). The solid circle with solid line represents blank control, the blank circle with solid line represents PBS, and the blank circle with dotted line represents ADC-20. The arrow pointing at the X-axis represents the day of dosing. Figure 19B depicts the red blood cell count, white blood cell count, neutrophil count and lymphocyte count in naive CD-1 mice treated with blank control, PBS, or ADC-20 (160mg / kg). Five mice were randomized into each group based on body weight. Single dose of ADC-20 at 160 mg / kg was administered on day 0. Body weight was measured on day 0, 3, 5, 7 post dosing. Whole blood was collected via orbital bleeding on day 3 and day 7 for automated red blood cells, white blood cells, neutrophils and lymphocytes determinations.

[0065] Figure 20A depicts the body weight over time in naive CD-1 mice treated with blank control, PBS, or ADC-21 (160mg / kg). The solid circle with solid line represents blank control, the blank circle with solid line represents PBS, and the blank circle with dotted line represents ADC-21. The arrow pointing at the X-axis represents the day of dosing. Figure 20B depicts the red blood cell count, white blood cell count, neutrophil count and lymphocyte count in naive CD-1 mice treated with blank control, PBS, or ADC-21 (160mg / kg). Five mice were randomized into each group based on body weight. Single dose of ADC-21 at 160 mg / kg was administered on day 0. Body weight was measured on day 0, 3, 5, 7 post dosing. Whole blood was collected via orbital bleeding on day 3 and day 7 for automated red blood cells, white blood cells, neutrophils and lymphocytes determinations.

[0066] Figure 21A depicts the body weight over time in naive CD-1 mice treated with blank control, PBS, or ADC-22 (160mg / kg). The solid circle with solid line represents blank control, the solid triangle with solid line represents PBS, and the blank circle with dotted line represents ADC-22. The arrow pointing at the X-axis represents the day of dosing. Figure 21 B depicts the red blood cell count, white blood cell count, neutrophil count and lymphocyte count in naive CD-1 mice treated with blank control, PBS, or ADC-22 (160mg / kg). Five mice were randomized into each group based on body weight. Single dose of ADC-22 at 160 mg / kg was administered on day 0. Body weight was measured on day 0, 2, 4, 7 post dosing. Whole blood was collected via orbital bleeding on day 3 and day 7 for automated red blood cells, white blood cells, neutrophils and lymphocytes determinations.

[0067] Figure 22A depicts the body weight over time in naive CD-1 mice treated with blank control, PBS, or ADC-23 (160mg / kg). The solid circle with solid line represents blank control, the solid triangle with solid line represents PBS, and the blank circle with dotted line represents ADC- 23. The arrow pointing at the X-axis represents the day of dosing. Figure 22B depicts the red blood cell count, white blood cell count, neutrophil count and lymphocyte count in naive CD-1 mice treated with blank control, PBS, or ADC-23 (160mg / kg). Five mice were randomized into each group based on body weight. Single dose of ADC-23 at 160 mg / kg was administered on day 0. Body weight was measured on day 0, 2, 4, 7 post dosing. Whole blood was collected via orbital bleeding on day 3 and day 7 for automated red blood cells, white blood cells, neutrophils and lymphocytes determinations.

[0068] Figure 23.A depicts the body weight over time in nai ve CD-1 mice treated with blank control, PBS, or ADC-12 (160mg / kg). The solid circle with solid line represents blank control, the solid triangle with solid line represents PBS, and the blank circle with dotted line represents ADC- 12. The arrow pointing at the X-axis represents the day of dosing. Figure 23B depicts the red blood cell count, white blood cell count, neutrophil count and lymphocyte count in naive CD-1 mice treated with blank control, PBS, or ADC-12 (160mg / kg). Five mice were randomized into each group based on body weight. Single dose of ADC- 12 at 160 mg / kg was administered on day 0. Body weight was measured on day 0, 2, 5 post dosing. Whole blood was collected via orbital bleeding on day 2 and day 5 for automated red blood cells, white blood cells, neutrophils and lymphocytes determinations.MODE(S) OF CARRYING OUT THE INVENTION

[0069] The present disclosure provides novel camptothecin derivatives, and CPT derivative conjugates comprising at least one camptothecin derivative. In various embodiments, the present disclosure provides CPT derivatives, CPT derivative conjugates and methods relating to the use of such CPT derivatives or conjugates to treat a disease, such as cancer. The targeting agent in the conjugates, binds to e.g., a tumor associated antigen (TAA) on the cancer cell. In various embodiments, the targeting agent is conjugated to a novel camptothecin derivative and / or a linker, and the conjugate exerts a cytotoxic, cytostatic, or immunosuppressive effect on the antigen expressing cells to treat or prevent recurrence of the antigen expressing cancers or immunological disorders. Importantly, the CPT derivative conjugates of the present disclosure have superior drug / targeting agent ratios (such as DARs for ADCs), demonstrate improvedsolubility, enhanced CMC characteristics, and increased therapeutic efficacy particulary against high antigen expressing tumors while sparing the normal tissues expressing low or no level of antigen. Moreover, the CPT derivative conjugates provide for the targeting of broader patient populations and patients having a refractory cancer or who previously responded to treatment with an anti-cancer therapy, but, upon cessation of therapy, suffered relapse (hereinafter “a recurrent cancer”).Definitions

[0070] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those commonly used and well known in the art. The methods and techniques of the present disclosure are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012), incorporated herein by reference. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclature used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those commonly used and well known in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of subjects.

[0071] As used herein, the term “alkyl” refers to a fully saturated branched or unbranched hydrocarbon moiety having up to 20 carbon atoms. Examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n- nonyl, n-decyl and the like.

[0072] As used herein, ther term "heterocyclyl”, “heterocycloalkyi” or “heterocyclo” referes to a saturated or unsaturated non-aromatic ring or ring system, and contains at least one heteroatom selected from 0, S, and N. The heterocyclyl can be attached at a heteroatom, a carbon atom, or both.

[0073] As used herein, the term “aryl” refers to an aromatic hydrocarbon group having 6- 20 carbon atoms in the ring portion. Typically, aryl is monocyclic, bicyclic or tricyclic aryl having 6-20 carbon atoms. Furthermore, the term "ary!" as used herein, refers to an aromatic moiety which can be a single aromatic ring, or multiple aromatic rings that are fused together. Non-limiting examples include phenyl, naphthyl or tetrahydronaphthyl, each of which may optionally be substituted with 1-4 substituents, such as alkyl, trifluoromethyl, cycloalkyl, halogen, hydroxy, alkoxy, acyl, alkyl-C(O)-O-, aryl-O-, heteroaryl-O-, amino, thiol, alkyl-S-, ary!-S- nitro, cyano, carboxy, alkyl-O~C(O)-, carbamoyl, alkyl-S(O)-, sulfonyl, sulfonamide, phenyl, and heterocyclyl.

[0074] As used herein, “cyclic alkyl” or “cycloalkyl” refers to a saturated or unsaturated monocyclic, bicyclic or tricyclic hydrocarbon groups of 3-12 carbon atoms. Unless otherwise provided, cycloalkyl refers to cyclic hydrocarbon moiety having between 3 and 9 ring carbon atoms or between 3 and 7 ring carbon atoms, each of which can be optionally substituted with one, or two, or three, or more substituents independently selected from the group consisting of alkyl, halo, oxo, hydroxy, alkoxy, alkyl-C(O)--, acylamino, carbamoyl, alkyl-NH— -, (alkyl)2N-, thiol, alkyl-S-, nitro, cyano, carboxy, alkyl-O--C(O)-, sulfonyl, sulfonamido, sulfamoyl, and heterocyclyl.

[0075] As used herein, the term "optionally substituted" unless otherwise specified refers to a group that is unsubstituted or is substituted with one or more, typically 1 , 2, 3 or 4, suitable non-hydrogen substituents.

[0076] The point of attachment of a given moiety to the parent structure can be readily determined by one of skill in art. Thus, although the point of attachment may not be explicitly shown, it would be evident to the skilled artisan based on common general knowledge in the chemical arts.

[0077] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. In various embodiments, "peptides", "polypeptides", and "proteins" are chains of amino acids whose alpha carbons are linked through peptide bonds. The terminal amino acid at one end of the chain (amino terminal) therefore has a free amino group, while the terminal amino acid at the other end of the chain (carboxy terminal) has a free carboxyl group. As used herein, the term "amino terminus" (abbreviated N-terminus) refers to the free □-amino group on an amino acid at the amino terminal of a peptide or to the D-amino group (imino group when participating in a peptide bond) of an amino acid at any other location within the peptide. Similarly, the term "carboxy terminus" refers to the free carboxyl group on the carboxy terminus of a peptide or the carboxyl group of an amino acid at any other location within the peptide. Peptides also include essentially any polyamino acid including, but not limited to, peptide mimetics such as amino acids joined by an ether bond as opposed to an amide bond.

[0078] Polypeptides of the disclosure include polypeptides that have been modified in any way and for any reason, for example, to: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinities, and (5) confer or modify other physicochemical or functional properties. For example, single or multiple amino acid substitutions (e.g., conservative amino acid substitutions) may be made in the naturally occurring sequence (e.g., in the portion of the polypeptide outside the domain(s) forming intermolecular contacts). A "conservative amino acid substitution" refers to the substitution in a polypeptide of an amino acid with a functionally similar amino acid. A “nonconservative amino acid substitution” refers to the substitution of a member of one of these classes for a member from another class. In making such changes, according to various embodiments, the hydropathic index of amino acids may be considered. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8): phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0079] The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is understood in the art (see, for example, Kyte et al., 1982, J. Mol. Biol. 157:105-131). It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, in various embodiments, the substitution of amino acids whose hydropathic indices are within + 2 is included. In various embodiments, those that are within + 1 are included, and in various embodiments, those within + 0.5 are included.

[0080] It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity, particularly where the biologically functional protein or peptide thereby created is intended for use in immunological embodiments, as disclosed herein. In various embodiments, the greatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e. , with a biological property of the protein.

[0081] The following hydrophilicity values have been assigned to these amino acid residues: arginine (+3.0): lysine (+3.0); aspartate (+3.0.+- .1); glutamate (+3.0.+-.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 +-.1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5) and tryptophan (-3.4). In making changes based upon similar hydrophilicity values, in various embodiments, the substitution of amino acids whose hydrophilicity values are within + 2 is included, in various embodiments, those that are within + 1 are included, and in various embodiments, those within + 0.5 are included.

[0082] In some embodiments, the amino acids found in a linker is a non-conservative amino acid substitution. This class generally includes corresponding D-amino acids, homo-amino acids, N-alkyl amino acids, beta amino acids and other non-naturally occurring amino acids. The non-conservative amino acid substitutions still fall within the descriptions identified for the equivalent amino acid substitutions above [e.g. polar, nonpolar, etc.]. Examples of non- conservative amino acids are provided below.

[0083] Non limiting examples for alanine non-conservative amino acids are: D-alanlne [Dala, (dA), a], / V-Acetyl~3~(3,4-dimethoxyphenyl)-D-alanine, A / -Me-D-Ala-OH, M-Me-Ala-OH, H- P-Ala-]3-naphthalene, L-(-)-2-Amino-3-ureidopropionic acid, (R)-(+)-a-Allylalanine, (S)-(-)-a- Allylalanine, D-2-Aminobutyric acid, L-2-Aminobutyric acid, DL-2-Aminobutyric acid, 2- Aminoisobutyric acid, a-Aminoisobutyric acid, (S)-(+)-2-Amino-4-phenylbutyric acid ethyl ester, Benzyl a-aminoisobutyrate, Abu-OH, Aib-OH, p-(9-anthry!)-A!a-OH, p-(3-benzothienyl)-Ala-OH, p-(3-benzothienyl)-D-Ala-OH, Cha-OH, Cha-OMe, p-(2-furyl)-Ala-OH, p-(2-furyl)-D-Ala-OH, p- iodo-Ala-OBzl, g-iodo-D-Ala-OBzl, 3-iodo-D-Ala-OMe, p-iodo-Ala-OMe, 1-Nal-OH, D-1-Nal-OH, 2-Nal-OH, D-2-Nal-OH, (R)-3-(2-naphthyl)-p-Ala-OH, (S)-3-(2-naphthyl)-p-Ala-OH, p-phenyl- Phe-OH, 3-(2-pyridyl)-Ala-OH, 3-(3-pyridyl)-Ala-OH, 3-(3-pyridyl)-D-Ala-OH, (S)-3-(3-pyridyl)-p- Ala-OH, 3-(4-pyridyl)-Ala-OH, 3-(4-pyridyl)-D-Ala-OH, p-(2-quinolyl)-Ala-OH, 3-(2-quinolyl)-DL- Ala-OH, 3-(3-quinolyl)-DL-Ala-OH, 3-(2-quinoxalyl)-DL-Ala-OH, j3-(4-thiazolyl)-Ala-OH, p-(2- thienyl)-Ala-OH, |3-(2-thienyl)-D-Ala-OH, |3-(3-thienyl)-Ala-OH, 3-(3-thienyl)-D-Ala-OH, 3-Chloro- D-alanine methyl ester, fV-[(4-Chlorophenyl)sulfonyl]-p-alanine, 3-Cyclohexyl-D-alanine, 3- Cyclopentyl-DL-alanine, (-)-3-(3,4-Dihydroxyphenyl)-2-methyl-L-aianine, 3,3-Diphenyl-D- alanine, 3,3-Diphenyl-L-alanine, A / -[(S)-(+)-1-(Ethoxycarbonyl)-3-phenylpropyl]-L-alanine, A / -[1- (S)-(+)-Ethoxycarbonyl-3-phenylpropyl]-L-alanyl carboxyanhydride, N-(3-fluorobenzyl)alanine,A / -(3-!ndo!ylacetyl)-L~alanine, Methyl ( / ?S)-2-(aminomethyl)’3’phenylpropionate, 3-(2-Oxo-1,2- dihydro-4-quinolinyl)alanine, 3-(1-Pyrazolyl)-L-alanine, 3-(2-Pyridyl)-D-alanine, 3-(2-Pyridyl)-L- alanine, 3-(3-Pyridyl)-L-alanine, 3-(4-Pyridyl)-D-alanine, 3-(4-Pyridyl)-L-alanine, 3-(2-Quinolyl)- DL-aianine, 3-(4-Quino!yl)-DL-alanine, D-styrylalanine, L-styrylalanine, 3-(2-Thienyl)-L-aianine, 3-(2-Thienyl)-DL-alanine, 3-(2-Thienyl)-DL-alanine, 3,3,3-Trifluoro-DL-alanine, A / -Methyl-L- alanine, 3-Ureidopropionic acid, Aib-OH, Cha-OH, Dehydro-Ala-OMe, dehydro-Ala-OH, D-2- Nal-OH, p-Ala-ONp, p-Homoaia-OH, p-D-Homoala-OH, p-Alanine, p-Aianine ethyl ester, p- Alanine methyl ester, (S)- diphenyl-p-Homoa!a-OH, (R)-4-(4-pyridyl)-p-Homoala-OH, (S)-4-(4- pyridyl)-p~Homoala-OH, p-Ala-OH, (S)-dipheny!-p-Homoala-OH, L-p-Homoalanine, (R)-4-(3- pyridyl)-p-Homoala-OH, a-methyl-a-naphthylalanine [Manap], N-methyl-cyclohexylalanine [Nmchexa], cyclohexylalanine [Chexa], N-methyl-cyclopentylalanine [Nmcpen], cyclopentylalanine [Cpen], N-methyl-a-naphthylalanine [Nmanap], a-naphthylalanine [Anap], L- N-methylalanine [Nmala], D-N-methylalanine [Dnmala], o-methyl-cyclohexylalanine [Mchexa], a- methyl-cyclopentylalanine [Mcpen]. Each possibility represents a separate embodiment.

[0084] Non limiting examples for arginine non-conservative amino acids are: homoarginine (hArg), N-methyl arginine (NMeArg), citruline, 2-amino-3-guanidinopropionic acid, N-iminoethyl-L-ornithine, Nw-monomethyl-L-arginine, Nw-nitro-L-arginine, D-arginine, 2-amino- 3-ureidopropionic acid, Nw.w-dimethyl-L-arginine, Nw-Nitro-D-arginine, L-a-methylarginine [Marg], D-a-methylarginine [Dmarg], L-N-methylarginine [Nmarg], D-N-methylarginine [Dnmargj, p-Homoarg-OH, L-Homoarginine, N-(3-guanidinopropyl)glycine [Narg], and D- arginine [Darg, (dR), r]. Each possibility represents a separate embodiment.

[0085] Non limiting examples for asparagine non-conservative amino acids are: L-a- methylasparagine [Masn], D-a-methylasparagine [Dmasn], L-N-methylasparagine [Nmasn], D- N-methylasparagine [Dnmasn], N-(carbamylmethyl)g!ycine [Nasn] and D-asparagine [Dasn, (dN), n]. Each possibility represents a separate embodiment.

[0086] Non limiting examples for aspartic acid non-conservative amino acids are: L-a- methylaspartate [Maspj, D-a-methylaspartate [Dmasp], L-N-methylaspartic acid [Nmasp], D-N- methylasparatate [Dnmasp], N-(carboxymethyl)glycine [Nasp] and D-aspartic acid [Dasp, (dD), d]. Each possibility represents a separate embodiment.

[0087] Non limiting examples for cysteine non-conservative amino acids are: L-Cysteic acid, L-Cysteinesulfinic acid, D-Ethionine, S-(2-Thiazolyl)-L-cysteine, DL-Homocysteine, L- Homocysteine, L-Homocystine, L-a-methylcysteine [Mcys], D-a-methylcysteine [Dmcys], L-N-methylcysteine [Nmcys], D-N-mefhylcysteine [Dnmcys], N-(thiomethyi)g!ycine [Ncys] and D- cysteine [Deys, (dC), cl. Each possibility represents a separate embodiment

[0088] Non limiting examples for glutamic acid non-conservative amino acids are: y- Carboxy-DL-glutamic acid, 4-Fluoro-DL-glutamic acid, p-Glutamic acid, L-p-Homoglutamic acid, L-a-methylglutamate [Mglu], D-a-methyl glutamic acid [Dmglu], L-N-methylglutamic acid [Nmglu], D-N-methylglutamate [Dnmglu], N-(2-carboxyethyl)glycine [Nglu], and D-glutamic acid [Dglu, (dE), e]. Each possibility represents a separate embodiment.

[0089] Non limiting examples for glutamine non-conservative amino acids are: Cit-OH, D-Citrulline, Thio-L-citrulline, p-GIn-OH, L-p-Homoglutamine, L-a-methylglutamine [Mgln], D-a- methylglutamine [Dmgln], L-N-methylglutamine [Nmgln], D-N-methylglutamine [Dnmgln], N-(2- carbamylethyljglycine [Ngln], and D-glutamine [Dgln, (dQ), q]. Each possibility represents a separate embodiment.

[0090] Non limiting examples for glycine non-conservative amino acids are: tBu-Gly- OH , D-Allylglycine, A / -[Bis(methylthio)methylene]glycine methyl ester, Chg-OH, D-Chg-OH, D- cyclopropyiglycine, L-cyclopropylglycine, ( / ?)-4-fiuorophenylglycine, (S)-4-fluorophenylglycine, iminodiacetic acid, (2-indanyl)-Gly-OH, (±)-u-phosphonoglycine trimethyl ester, D- propargylglycine, propargyl-Gly-OH, ( / ?)-2~thienylglycine, (S)-2-thienylglycine, (R)-3- thienylglycine, (S)-3-thienylglycine, 2-(4-trifluoromethyl-phenyl)-DL-glycine, (2S,3R,4S)-a- (Carboxycyclopropyl)glycine, A / -(Chloroacetyl)g!ycine ethyl ester, (S)-(+)-2-chlorophenylglycine methyl ester, N-(2-chlorophenyl)-N-(methylsulfonyl)glycine, D-a-Cyclohexyiglycine, L-a- Cyclopropylglycine, Di-fert-butyl-iminodicarboxylate, Ethyl acetamidocyanoacetate, N-(2- fluorophenyi)-N-(methylsulfonyl) glycine, N-(4-fluorophenyl)-N-(methylsulfonyl)glycine, / V-(2- Furfurylideneacetyljglycine methyl ester, A / -(2-Furoyl)g!ycine, A / -(2-Hydroxyethyl)iminodiacetic acid, / V-(4-Hydroxyphenyl)glycine, Iminodiacetic acid, AFLauroylsarcosine sodium salt, L-a- Neopentylglycine, A / -(Phosphonomethyl)glycine, D-Propargylglycine, L-C-Propargylglycine, Sarcosine, A / , / V-Dimethylglycine, A / ,fV-Dimethylglycine ethyl ester, D-Chg-OH, a- Phosphonoglycine trimethyl ester, N-cyclobutylglycine [Ncbut], L-a-methylethylglycine [Metg], N- cycloheptylglycine [Nchep], L-a-methyl-i-butylglycine [Mtbug], N-methylglycine [Nmgly], L-N- methyl-ethylglycine [Nmetg], L-ethylglycine [Etg], L-N-methyl-t-butylglycine [Nmtbug], L-t- butylglycine [Tbug], N-cyclohexylglycine [Nchex], N-cyclodecylglycine [Ncdec], N- cyclododecylglycine [Ncdod], N-cyclooctylglycine [Ncoct], N-cyclopropylglycine [Ncpro], N- cycloundecylglycine [Ncund], N-(2-aminoethyl)giycine [Naeg], N-(N-(2,2-diphenylethyl) diphenylethyl)glycine [Nnbhm], N-(2,2- carbamylmethyl-glycine [Nbhm], N-(N-(3,3-diphenylpropyl) diphenylpropyl)glycine [Nnbhe] and N-(3,3- carbamylmethyl-glycine [Nbhe], Each possibility represents a separate embodiment.

[0091] Non limiting examples for histidine non-conservative amino acids are: L-a- methylhistidine [Mhis], D-a-methylhistidine [Dmhis], L- N-methylhistidine [Nmhis], D-N- methylhistidine [Dnmhis], N-(imidazolylethyl)glycine [Nhls], and D-histidine [Dhis, (dH). h], Each possibility represents a separate embodiment.

[0092] Non limiting examples for isoleucine non-conservative amino adds are: N- Methyl-L-isoleucine [Nmile], A / -(3-indoiylacetyl)-L-isoieucine. allo-lle-OH, D-allo-lsoleucine, L-p- Homoisoleucine, L-a-methyiisoleucine [Mile], D-a-methylisoleucine [Dmile], D-N- methylisoieucine [Dnmilej, N-(1 -methylpropyi)glycine [Nile], and D-isoleucine [Dile, (dD), ij. Each possibility represents a separate embodiment.

[0093] Non limiting examples for leucine non-conservative amino acids are: D-leuine [Dleu, (dL), I], Cycloleucine, DL-leucine, / V-Formyl-Leu-OH, D-tert-Leucine, L-tert-Leucine, DL- tert-Leucine, L-tert-Leucine methyl ester, 5,5,5-Trifluoro-DL-leucine, D-p-Leu-OH, L-p-Leucine, DL-p-Leucine, L-p-Homoleucine, DL-p-Homoleucine, L-N-methyl-leucine [Nmleu], D-N-methyl- leucine [Dnmleu], L-a-methyl-leucine [Mieu], D-a-methyl-leucine [Dmleu], N-(2- methylpropyl)glycine [Nleu], D-leucine [Dleu, I], D-Norleuclne, L-Norleucine, DL-Norieucine, L-N- methylnorleucine [Nmnle] and L-norleucine [Nle], Each possibility represents a separate embodiment.

[0094] Non limiting examples for lysine non-conservative amino acids are: DL-5- Hydroxylysine, (5R)-5-Hydroxy-L-lysine, p-Lys-OH, L-p-HomolysIne, L-a-methyl-lysine [Mlys], D- a-methyl-lysine [Dmlys], L-N-methyl-lysine [Nmlys], D-N-methyl-lysine [Dnmlys], N-(4- aminobutyl)glycine [Niys], and D-lysine [Dlys, (dK), k]. Each possibility represents a separate embodiment.

[0095] Non limiting examples for methionine non-conservative amino acids are: L-p- Homomethionine, DL-p-Homomethionine, L-a-methylmethionine [Mmet], D-a-methylmethionine [Dmmet], L-N-methylmethionine [Nmmet], D-N-methylmethionine [Dnmmet], N-(2- methylthioethyl)glycine [Nmet], and D-methionine [Dmet, (dM), m]. Each possibility represents a separate embodiment.

[0096] Non limiting examples for phenylalanine non-conservative amino acids are: A / - Acetyl-2-fluoro-DL-phenylalanine, Af-Acetyl-4-fluoro-DL-phenylalanine, 4-Amino-L- phenylalanine, 3-[3,4-bis(trifluoromethyl)phenyl]-L-alanine, Bpa-OH, D-Bpa-OH, 4-tert-butyl- Phe-OH, 4-fert-butyl-D-Phe-OH. 4-(amino)-L-phenylalanine. rac-p2-homophenylalanine, 2-methoxy-L’phenyialanine. (S)-4-methoxy~p~Phe-OH, 2-nitro-Lphenyiaianine, pentafluoro-D- phenylalanine, pentafluoro-L-phenyiaianine, Phe(4-Br)-OH. D-Phe(4-Br)-OH, Phe(2-CF3)-OH, D-Phe(2-CF3)-OH, Phe(3-CF3)-OH, D-Phe(3-CF3)-OH, Phe(4-CF3)-OH, D-Phe(4-CF3)-OH, Phe(2-CI)-OH, D-Phe(2-CI)-OH, Phe(2,4-Cb)-OH, D-Phe(2,4-Cb)-OH, D-Phe(3-CI)-OH, Phe(3,4-Ch)-OH, Phe(4-Ci)-OH, D-Phe(4-CI)-OH, Phe(2-CN)-OH, D-Phe(2-CN)-OH, D-Phe(3- CNJ-OH, Phe(4-CN)-OH, D-Phe(4-CN)-OHSPhe(2-Me)-OH, D-Phe(2-Me)-OH, Phe(3-Me)-OH, D-Phe(3-Me)-OH, Phe(4-Me)-OH, Phe(4-NH2)-OH, Phe(4-NO2)-OH, Phe(2-F)-OH, D-Phe(2-F)- OH, Phe(3-F)-OH, D-Phe(3-F)-OH, Phe(3,4-F2)-OH, D-Phe(3,4-F2)-OH, Phe(3,5-F2)-OH, Phe(4- F)~OH, D-Phe(4-F)-OH, Phe(4-i)-OH, D-3,4,5-trif!uorophenylalanine, p-Bromo-DL- phenyialanine, 4-Bromo-L-phenylaianine, p-phenyi-D-phenylalanine, 4-Chloro-L-phenyialanine, DL-2-Difluorophenylaianine, DL-3,5-Difluorophenyialanine, 3,4-Dihydroxy-L-phenyiaSanine, 3- (3,4-Dimethoxyphenyl)-L-aianine, A / -[(9H-Fluoren-9-ylmethoxy)carbonyl]-2-methoxy-L- phenylalanine, Q-Fluoro-DL-phenylalanine, m-Fiuoro-L-phenylaSanine, m-Fluoro-DL- phenylalanine, p-Fluoro-L-phenylalanine, p-Fluoro-DL-phenylalanine, 4-Fiuoro-D-phenylalanine, 2-f!uoro-L- phenylalanine methyl ester, p-fiuoro-DL-Phe-OMe, D-3-bromophenylaianine, D-4- bromophenylaianine, L-p-(6-chloro-4-pyridinyl)aianine, D-3,5-dif!uorophenylaianine, L-3- fiuorophenylalanine, L-4-fluorophenylaianine, L-p-(1H-5-indoiyl)aianine, 2-nitro-L-phenylalanine, pentafiuoro-L-phenyiaianine, phe(3-br)-oh, Phe(4-Br)-OH, Phe(2-CF3)~OH, D-Phe(2-CF3)-OH, Phe(3-CF3)-OH, D-Phe(3-CF3)-OH, Phe(4-CF3)-OH, D-Phe(4-CF3)-OH, Phe(2-Ci)-OH, D-Phe(2- CI)-OH, Phe(2,4-C!2)-OH, D-Phe(2,4-CI2)-OH, Phe(3,4-CI2)-OH, D-Phe(3,4-CI2)-OH, Phe(4-Ci)- OH, D-Phe(4-CI)-OH, Phe(2-CN)-OH, D-Phe(2-CN)-OH, D-Phe(3-CN)-OH, Phe(4-CN)-OH, Phe(2-Me)-OH, Phe(3-Me)-OH, D-Phe(3-Me)-OH, Phe(4-NO2)-OH, D-Phe(4-NO2)-OH, D- Phe(2-F)-OH, Phe(3-F)-OH, D-Phe(3-F)~OH, Phe(3,4-F2)-OH, Phe(3,5-F2)-OH, D-Phe(4-F)-OH, Phe(4-i)-OH, D-Phe(4-!)-OH, 4-(phosphonomethyl)-Phe-OH, L-4-trifluoromethylphenylalanine, 3,4.5-trifluoro-D-phenylalanine, L"3,4,54rifluorophenylalanine, 6-Hydroxy-DL-DOPA, 4~ (Hydroxymethyl)-D-phenylalanine. W-(34ndolylacetyl)-L-phenylalanine. p-lodo-D-phenylalanine, 4-lodo-L-phenylaianine, a-Methyi-D-phenylalanine, a-Methyi-L-phenylaianine, a-Methyi-DL- phenylalanine, a-Methyl-DL-phenylaianine methyl ester, 4-Nitro-D-phenylaianine, 4-Nitro-L- phenylalanine, 4-Nitro-DL-phenylalanine, (S)-(+)-4-Nitrophenylaianine methyl ester, 2- (Trifluoromethyl)-D-phenylalanine, 2-(Trifluoromethyl)-L-phenylalanine, 3-(Trifluoromethyl)-D- phenylaianine, 3-(Trifluoromethyl)-L-phenyla!anine, 4-(Trifluoromethyl)-D-phenylalanine, 3,3',5~ TrHodo-L-thyronine, (R)-4-bromo-|3-Phe-OH, N-Acetyi-DL-p-phenyialanine, (S)-4-bromo-p-Phe- OH, (R)-4-chioro-p-Homophe-OH, (S)-4-ch!oro-p-Homophe-OH, (R)-4-chloro-p-Phe-OH, (S)-4-chloro-P’Phe-OH, (S)-2-cyano~p~Homophe-OH, (R)- 4-cyano-p-Homophe-OH, (S)-4-cyano-p~ Homophe-OH, ( / ?)-3-cyano-p-Phe-OH, (R)-4-cyano-p-Phe-OH, (S)-4-cyano-p-Phe-OH, (R)-3,4- dimethoxy-p-Phe-OH, (SJ-S^-dimethoxy-p-Phe-OH, ( / ^)-4-fluoro-p-Phe-OH, (S)-4-f!uoro-p-Phe- OH, (S)-4-iodo-p-Homophe-OH, (S)-3-cyano-p-Homophe-OH, (S)-3,4-dif!uoro-p-Homophe-OH, (R )-4-fluoro-p-Homophe-OH, (S)-p2-homophenyialanine!(R)-3-methoxy-p-Phe-OH, (S)-3- methoxy-p-Phe-OH, (R)-4-methoxy-p-Phe-OH, (S)-4-methyl-p-Homophe-OH, (R)-2-methyl-p- Phe-OH, (S)-2-methyl-p-Phe-OH, (R)-3-methyl-p-Phe-OH, (S)-3-methyi-p-Phe-OH, (K)-4- methyl-p-Phe-OH, (S)-4-methyl-p-Phe-OH, p-Phe-OH, D-p-Phe-OH, (S)-2-(trifluoromethyl)-p- Homophe-OH, (S)-2-(trifluoromethyl)-p-Homophe-OH1(S)-3-(trifluoromethyl)-p-Homophe-OH1(R )-4-(trifluoromethyi)-p-Homophe-OH, (S)-2-(trifluoromethyl)-p-Phe-OH, ( / ?)-3-(trifluoromethyl)- P-Phe-OH, (S)-3-(trifluoromethyl)-p-Phe-OH, (R)-4-(trifluoromethyl)-p-Phe-OH, (S)-4- (trif!uoromethyl)-p-Phe-OH, p-Homophe-OH, D-P-Homophe-OH, (S)-2-methyl-p-Homophe-OH, (S)-3-methyl-p-Homophe-OH, p-Phe-OH, p-D-Phe-OH, (S)-3-(trifluoromethy!)-p-Homophe-OH, L-p-Homophenylalanine, DL-p-Homophenylalanine, DL-p-Phenylalanine, DL- homophenylalanine methyl ester, D-Homophenylalanine, L-Homophenylalanine, DL- Homophenylaianine, D-Homopheny!a!anine ethyl ester, (R)-p2-homaphenylalanlne, L-a-methyl- homophenylalanine [Mhphe], L-a-methylphenylalanine [Mphe], D-a-methylphenylalanine [Dmphe], L-N-methyl- homophenylalanine [Nm phe], L-homophenylalanine [Hphe], L-N- methylphenylalanine [Nmphe], D-N-methylphenylalanine [Dnmphe], N-benzylglycine [Nphe] and D-phenylalanine [Dphe, (dF), f]. Each possibility represents a separate embodiment.

[0097] Non limiting examples for proline non-conservative amino acids are: homoproline (hPro), (4-hydroxy)Pro (4HyP), (3-hydroxy)Pro (3HyP), gamma-benzyl-proline, gamma-(2- fluoro-benzyl)-proline, gamma-(3-fluoro-benzyl)-proline, gamma-(4-fluoro-benzyl)-proline, gamma-(2-chloro-benzyl)- proline, gamma-(3-chloro-benzyl)-proline, gamma-(4-chloro-benzyl)- proline, gamma-(2-bromo-benzyl)-proline, gamma-(3-brorno-benzyl)-proline, gamma-(4-brorno- benzyl)-proline, gamma-(2-methyl-benzyl)-proline, gamma-(3-methyl-benzyl)-proline, gamma- (4-methyl-benzyl)-proline, gamma-(2-nitro-benzyl)-proline, gamma-(3-nitro-benzyl)-proline, gamma-(4-nitro”benzyl)-proline, gamma-(l-naphthalenylmethyl)- proline, gamma-(2- naphthalenylmefhylj-proline, gamma-(2,4"dichloro”benzyl)-proline, gamma"(3,4-dichloro- benzylj-proline, gamma-(3,4-difluoro-benzyl)-proline, gamma-(2-trifluoro-methyl-benzy!)-proline, gamma-(3-trifluoro-methyl-benzyl)-proline, gamma-(4-trifluoro-methy!-benzyl)-proline. gamma- (2-cyano-benzyl)-proline, gamma-(3-cyano-benzyl)-proline, gamma-(4-cyano-benzyl)-proline, gamma-(2-iodo-benzyl)-proline, gamma-(3-iodo-benzyl)-proline, gamma-(4-iodo-benzyl)-proline!gamma-(3-phenykallyl-benzyl)-proline, gamma-(3-phenyl-propyFbenzyl)-proline, gamma~(4~tert~ butyl-benzyl)-proline, gamma-benzhydryl-proline, gamma-(4-biphenyl-methyl)-proline!gamma- (4-thiazolyl-!Yiethyl)-proline, gamma-(3-benzothienyl-methyl)-proline, gamma-(2-thienyl-methyl)- proline, gamma-(3-thienyl-methyl)- proline, gamma-(2-furanyl-methyl)-proline, gamma-(2- pyridinyl-methylj-proline, gamma-(3-pyridinyl-methyl)-proline, gamma-(4~pyridinyl~methyl)- proiine, gamma-allyl-proline, gamma-propynyl-proline, alpha-modified-proiine residues , pipecolic acid, azetidine-3-carboxylicacid, L-P-Homoproline, L-p3-homoproline, L-p- Homohydroxyproline, hydroxyproline [Hyp], L-D-methylproline [Mpro], D-D-methylproline [Dmpro], L-N-methylproline [Nmpro], D-N-methylproline [Dnmpro], and D-proline [Dpro, (dP), p].. Each possibility represents a separate embodiment.

[0098] Non limiting exampies for serine non-conservative amino acids are: (2R,3S)-3- phenylisoserine, D-cycloserine, L-!soserine, DL-lsoserine, DL-3-Phenylserine, L-p-Homoserine, D-Homoserine, D-Homoserine, L-3-Homoserine, L-homoserine, L-u-methylserine [Mser], D-a- methylserine [Dmser], L-N-methylserine [Nmser], D-N-methylserine [Dnmser], D-serine [Dser, (dS), s], N”(hydroxymethyl)giycine [Nser] and phosphoserine [pSer]. Each possibility represents a separate embodiment.

[0099] Non limiting examples for threonine non-conservative amino acids are: L-a / / o- Threonine, D-Thyroxine, L-p-Homothreonine, L-a-methylthreonine [Mthr], D-a-methylthreonine [Dmthr], L-N-methylthreonine [Nmthr], D-N-methylthreonine [Dnmthr], D-threonine [Dthr, (dT), t], N-(1 -hydroxyethyljglycine [Nthr] and phosphothreonine [pThr], Each possibility represents a separate embodiment.

[0100] Non limiting examples for tryptophan non-conservative amino acids are: 5- Fluoro-L-tryptophan, 5-Fluoro-DL-tryptophan, 5-Hydroxy-L-tryptophan, 5-Methoxy-DL- tryptophan, L-abrine, 5-Methyl-DL-tryptophan, H-Tpi-OMe. p-Homotrp-OMe, L-p- Homotryptophan, L-o-methyltryptophan [Mtrp], D-a-methyltryptophan [Dmtrp], L-N- methyltryptophan [Nmtrp], D-N-methyltryptophan [Dnmtrp], N-(3-indolylethyl)glycine [Nhtrp], D- tryptophan [Dtrp, (dW), wj. Each possibility represents a separate embodiment.

[0101] Non limiting examples for tyrosine non-conservative amino acids are: 3,5 diiodotyrosine (3,5-dlTyr), 3,5 diBromotyrosine (3,5-dBTyr), homotyrosine, D-tyrosine, 3-amino- L-tyrosine, 3-amino-D-tyrosine, 3- iodo- L- tyrosine, 3- iodo- D- tyrosine, 3-methoxy-L-tyrosine, 3-methoxy-D-tyrosine, L-thyroxine, D-thyroxine, L-thyronine, D-thyronine, O-methyl-L-tyrosine, O-methyl-D-tyrosine, D-thyronine, O-ethyl-L-tyrosine, O-ethyl-D-tyrosine, 3,5,3'-triiodo-L- thyronine, 3,5,3'-triiodo-D-thyronine, 3,5-diiodo-L-thyronine, 3,5-diiodo-D-thyronine, D-meta-tyrosine, L-meta-tyrosine, D-ortho- tyrosine, L-ortho-tyrosine, phenylalanine, substituted phaenylalanine, N-nitro phenylalanine, p-nitro phenylalanine, 3-chloro-Dtyr-oh, Tyr(3,5-dil), 3- Chloro-L-tyrosine, Tyr(3-NO2)-OH , Tyr(3,5-dil)-OH, A / -Me-Tyr-OH, a-Methyl-DL-tyrosine, 3- Nitro-L-tyrosine, DL-o-Tyrosine, p-Homotyr-OH, (R)-p-Tyr-OH, (S)-p-Tyr-OH, L-a-methyltyrosine [Mtyr], D-a-methyltyrosine [Dmtyr], L-N-methyltyrosine [Nmtyr], D-N-methyltyrosine [Dnmtyr], D- tyrosine [Dtyr, (dY), y], O-methyl-tyrosine, and phosphotyrosine [pTyr], Each possibility represents a separate embodiment.

[0102] Non limiting examples for valine non-conservative amino acids are: 3-Fluoro-DL- valine, 4,4,4,4,,4',4'-Hexafluoro-DL-valine, D-valine [Dval, (dV), v], / V-Me-Val-OH [Nmval], N- Me-Val-OH, L-a-methylvaline [Mval], D-a-methylvaline [Dmval], (R)-(+)-a-Methylvaline, (S)-(~)- o-Methylvaline and D-N-methylvaiine [Dnmval]. Each possibility represents a separate embodiment.

[0103] Other non-natural amino acids that may be substituted as non-conservative replacements include: Ornithine and its modifications : D-Ornithine [Dorn], L-Ornithine [Orn], DL- Ornithine, L-a-methylornithine [Morn], D-a-methylornithine [Dmorn], L-N-methylornithine [Nmorn], D-N-methylornithine [Dnmorn] and N-(3-aminopropyl)glycine [Norn], Each possibility represents a separate embodiment.

[0104] Alicyclic amino acids : L-2,4-Diaminobutyric acid, L~2,3~Diaminopropionic Acid, N-Me-Aib-OH, (R)-2-(amino)-5-hexynoic acid, piperidine-2-carboxylic acid, aminonorbornylcarboxylate [Norb], alpha-aminobutyric acid [Abu], aminocyclopropane-carboxylate [Cpro], (c / s)-3-Aminobicyclo[2.2.1]heptane-2-carboxylic acid, exo-cis-3-Aminobicyclo[2.2.1]hept-5-ene- 2-carboxylic acid, 1 -Am ino-1 -cyclobutanecarboxylic acid, c / s-2-Aminocycloheptanecarboxylic acid, 1 -Aminocyclohexanecarboxylic acid, c / s-2-Aminocyclohexanecarboxylic acid, trans-2- Aminocyclohexanecarboxylic acid, c / s-6-Amino-3-cyclohexene-1-carboxylic acid, 2-(1- Aminocyclohexyl)acetic acid, c / s-2-Amino~1 -cyclooctanecarboxylic acid, c / s-2-Amino-3- cyclooctene-1-carboxylic acid, (1R,2S)-(-)-2-Amino-1 -cyclopentanecarboxylic acid, (1 S,2R)-(+)- 2-Amino-1-cyclopentanecarboxylic acid, c / s-2-Amino-1-cyclopentanecarboxylic acid, 2-(1- Aminocyclopentyljacetic acid, c / s-2-Amino-2-methylcyclohexanecarboxylic acid, c / s-2-Amino-2- methylcyclopentanecarboxylic acid, 3-Amino-3-(4-nitrophenyl)propionic acid, 3- Azetidinecarboxylic acid, amchc-oh, 1-aminocyclobutane carboxylic acid, 1- (amino)cyclohexanecarboxylic acid, crs-2-(amino)-cyclohexanecarboxylic acid, frans-2-(amino)- cyclohexanecarboxylic acid, c / s-4-(amino)cyclohexanecarboxylic acid, trans-4- (amino)cyclohexanecarboxylic acid, (±)-c / s-2-(amino)-3-cyclohexene-1 -carboxylic acid, (±)-c / s-6-(amino)-3-cyclohexene-1-carboxylic acid, 2-(1-aminocyclohexyl)acetic add, c / s-[4- (amino)cyclohexyl]acetic add, 1-(amino)cyclopentanecarboxylic add, (±)-c / s-2- (amino)cyclopentanecarboxy!ic add, (1R,4S)-(+)-4-(amino)-2-cyclopentene-1“Carboxylic add, (±)-c / s-2-(amino)-3-cydopentene-1-carboxy!ic add, 2-(1-aminocyclopentyl)acetic acid, 1- (amino)cyclopropanecarboxylic add, Ethyl 1 -aminocyclopropanecarboxylate, 1 ,2-trans-achec- oh, 1-(amino)cyclobutanecarboxylic add, 1-(amino)cyclohexanecarboxylic acid, ds-2-(amino)- cyclohexanecarboxylic acid, frans-2-(amino)cyclohexanecarboxylic acid, c / s-4- (amino)cyciohexanecarboxylic add, fra / ?s-4-(amino)cyclohexanecarboxylic add, c / s-[4- (amino)cyclohexyl]acetic add, 1-(amino)cyclopentanecarboxylic add, (1R,4SX+)-4~(amino)-2- cyclopentene-1-carboxylic add, (1 S,4f?)-(-)-4-(amino)-2-cyclopentene-1-carboxylic acid, 1- (amino)cyclopropanecarboxylic acid, frans-4-(aminomethyl)cyclohexanecarboxylic add, p-Dab- OH, 3-Amino-3-(3-bromophenyl)propionic add, 3-Aminobutanoic acid, c / s-2- Amino-3- cydopentene-1-carboxylic add, DL-3-Aminoisobutyric add, (f?)-3-Amino-2-phenylpropionic add, (±)-3-(amino)-4-(4-biphenylyl)butyric acid, c / s-3-(amino)cydohexanecarboxylic acid, (1 S,3R)-(+)- 3-(amino)cyclopentanecarboxylic acid, (2 / ?,3 / ?)-3-(amino)-2-hydroxy-4-phenylbutyric acid, (2S,3R )-3-(amino)-2-hydroxy-4-phenyibutyric add, 2-(aminomethyl)pheny!acetic acid, (R)-3- ( amino)-2-methylpropionic acid, (S)-3-(amino)-2-methylpropionic add, 0R)-3-(amino)-4-(2- naphthyl)butyric add, (S)-3-(amino)-4-(2-naphthyl)butyric acid, (R)-3-(amino)-5-phenylpentanoic add, (R)-3-(amino)-2-phenylpropionic add, Ethyl 3-(benzylamino)propionate, ds-3- (amino)cyclohexanecarboxylic acid, (S)-3-(amino)-5-hexenoic acid, ( / ?)-3-(amino)-2- methylpropionic add, (S)-3-(amino)-2-methylpropionic acid, (R)-3-(amino)-4-(2-naphthyl)butyric acid, (S)-3-(amino)-4-(2-naphthyl)butyric add, ( / ?)-(-)-Pyrrolidine-3-carboxylic acid, (S)-(+)- Pyrrolidine-3-carboxylic acid, N-methyl- y -aminobutyrate [Nmgabu], y-aminobutyric add [Gabu], N-methy!- a-amino- a- methyl butyrate [Nmaabu], a-amino- a-methylbutyrate [Aabu], N-methyl- a- aminoisobutyrate [Nmaib], a-aminoisobutyric acid [Aib], a-methyl-y-aminobutyrate [Mgabu]. Each possibility represents a separate embodiment.

[0105] Phenyl glycine and its modifications: Phg-OH, D-Phg-OH, 2-(piperazino)-2-(3,4- dimethoxyphenyl)acetic add, 2-(piperazino)-2-(2-fluorophenyl)acetic acid, 2-(4-piperazino)-2-(3- fluorophenyl)acetic acid, 2-(4-piperazino)-2-(4-methoxyphenyl)acetic acid, 2-(4-piperazino)-2-(3- pyridyl)acetic add, 2-(4-piperazino)-2-[4-(trifluoromethyl)phenyl]acetic acid, L-(+)-2- Chlorophenylglycine, (±)-2-Chlorophenylglycine, (±)-4-Chlorophenylglycine, (R)-(-~)-2-(2,5- Dihydrophenyl)glycine, (R)-(”)-AP(3,5-Dinitrobenzoyl)-a~phenylglycine, (S)-(+)-A / -(3,5- Dinitrobenzoyl)-a-phenylglycine, 2,2-Diphenylglycine, 2-FSuoro-DL-a-phenylg!ycine, 4-Fluoro-D-Q-phenylglycine. 4-Hydroxy-D-phenylglycine, 4-Hydroxy-L-phenylglycine, 2-Phenylglycine, D- (-)-a-Phenylglycine, D“(-)-a-Phenylglycine, DL-a-Phenylglycine, L-(+)-a-Phenylglycine, N- Phenylglycine, (f?)-(~)-2-Phenyiglycine methyl ester, (S)-(+)-2-Phenylglycine methyl ester, 2- Pheny Ig lyci nonitrile hydrochloride, a-Phenylglycinonitrile, 3-(T rifluoromethyl)-DL-phenylglycine, and 4-(Trifluoromethyl)-L-phenylglycine. Each possibility represents a separate embodiment.

[0106] Penicillamine and its modifications: N-Acetyl-D-penicillamine, D-Penicillamine, L- Penicillamine [Pen], DL-Penicillamine. a -methylpenicillamine [Mpen], N-methylpenicillamine [Nmpen], Each possibility represents a separate embodiment.

[0107] p-Homopyrrolidine. Each possibility represents a separate embodiment.

[0108] Aromatic amino acids: 3-Acetamidobenzoic acid, 4-Acetamidobenzoic acid, 4- Acetamido-2-methylbenzoic acid, / V-Acetylanthranilic acid, 3-Aminobenzoic acid, 3- Aminobenzoic acid hydrochloride, 4~Aminobenzoic acid, 4-Aminobenzoic acid, 4-Aminobenzoic acid, 4-Aminobenzoic acid, 4-Aminobenzoic acid, 4-Aminobenzoic acid, 2-Aminobenzophenone-2'-carboxylic acid, 2-Amino-4-bromobenzoic acid, 2-Amino-5- bromobenzoic acid, 3-Amino-2-bromobenzoic acid, 3-Amino-4-bromobenzoic acid, 3-Amino-5- bromobenzoic acid, 4-Amino-3-bromobenzoic acid, 5-Amino-2-bromobenzoic acid, 2-Amino-3- bromo-5-methylbenzoic acid, 2-Amino-3-chlorobenzoic acid, 2-Amino-4-chlorobenzoic acid, 2- Amino-5-chlorobenzoic acid, 2-Amino-5-chlorobenzoic acid, 2-Amino-6-chlorobenzoic acid, 3- Amino-2-chlorobenzoic acid, 3-Amino-4-chlorobenzoic acid, 4-Amino-2-chlorobenzoic acid, 4- Amino-3-chlorobenzoic acid, 5-Amino-2-chlorobenzoic acid, 5-Amino-2-chlorobenzoic acid, 4- Amino-5-chloro-2-methoxybenzoic acid, 2-Amino-5-chloro-3-methylbenzoic acid, 3-Amino-2,5- dichlorobenzoic acid, 4-Amino-3,5-dichlorobenzoic acid, 2-Amino-4,5-dimethoxybenzoic acid, 4-(2-Aminoethyl)benzoic acid hydrochloride, 2-Amino-4-fluorobenzoic acid, 2-Amino-5- fluorobenzoic acid, 2-Amino-6-fluorobenzoic acid, 4-Amino-2-fluorobenzoic acid, 2-Amino-5- hydroxybenzoic acid, 3-Amino-4-hydroxybenzoic acid, 4-Amino-3-hydroxybenzoic acid, 2- Amino-5-iodobenzoic acid, 5-Aminoisophthalic acid, 2-Amino-3-methoxybenzoic acid, 2-Amino- 4-methoxybenzoic acid, 2-Amino-5-methoxybenzoic acid, 3-Amino-2-methoxybenzoic acid, 3- Amino-4-methoxybenzoic acid, 3-Amino-5-methoxybenzoic acid, 4-Amino-2-methoxybenzoic acid, 4-Amino-3-methoxybenzoic acid, 5-Amino-2-methoxybenzoic acid, 2-Amino-3- methylbenzoic acid, 2-Amino-5-methylbenzoic acid, 2-Amino-6-methylbenzoic acid, 3- (Aminomethyl)benzoic acid, 3-Amino-2-methylbenzoic acid, 3-Amino-4-methylbenzoic acid, 4- (Aminomethyl)benzoic acid, 4-Amino-2-methylbenzoic acid, 4-Amino-3-methylbenzoic acid, 5- Amino-2-methylbenzoic acid, 3-Amino-2-naphthoic acid, 6-Amino-2-naphthoic acid, 2-Amino-3-nitrobenzoic add, 2"Amino-5~nitrobenzoic acid, 2-Amino-5-nitrobenzoic add, 4-Amino-3~ nitrobenzoic add, 5-Amino-2-nitrobenzoic acid, 3-(4-Aminophenyi)propionic add, 3- Aminophthalic add, 4-Aminophthalic add, 3-Aminosalicylic acid, 4-Aminosalicylic add, 5- Aminosalicyiic add, 5-Aminosalicylic add, 2-Aminoterephthalic add, 2-Amino-3, 4,5,6- tetrafiuorobenzoic add, 4-Amino-2,3,5,6-tetrafluorobenzoic acid, (R)-2-Amino-1 , 2,3,4- tetrahydronaphthalene-2-carboxylic acid, (S)-2-Amino-1 ,2,3,4-tetrahydro-2- naphthalenecarboxylic acid, 2-Amino-3-(trifluoromethyl)benzoic acid, 2-Amino-3- (trifluoromethyl)benzoic add, 3-Amino-5-(trifluoromethyl)benzoic acid, 5-Amino-2,4,6- triiodoisophthalic acid, 2-Amino-3,4,5-trimethoxybenzoic add, 2-Anilinophenylacetic acid, 2-Abz- OH, 3-Abz-OH, 4-Abz-OH, 2-(aminomethyl)benzoic acid, 3-(aminomethyl)benzoic acid, 4- (aminomethyl)benzoic acid, tert- Butyl 2-aminobenzoate, tert-Butyl 3-aminobenzoate, tert-Butyl 4-aminobenzoate, 4-(Butyiamino)benzoic add, 2,3-Diaminobenzoic acid, 3,4-Diaminobenzoic add, 3,5-Diaminobenzoic add, 3,5-Diaminobenzoic add, 3, 5-Dichloroanthranilic add, 4- (Diethylamino)benzoic add, 4,5-Difluoroanthranilic acid, 4-(Dimethylamino)benzoic add, 4- (Dimethylamino)benzoic add, 3,5-Dimethylanthranilic add, 5-Fluoro-2-methoxybenzoic acid, 2- Abz-OH, 3-Abz-OH, 4-Abz-OH, 3-(aminomethyl)benzoic acid, 4-(aminomethyl)benzoic acid, 4- (2-hydrazino)benzoic acid, 3-Hydroxyanthranilic add, 3-Hydroxyanthranilic acid, Methyl 3- aminobenzoate, 3-(Methylamino)benzoic acid, 4-(Methylamino)benzoic add, Methyl 2-amino-4- chlorobenzoate, Methyl 2-amino-4,5-dimethoxybenzoate, 4-Nitroanthranilic acid, N- Phenylanthranilic acid, A / -Phenylanthranilic acid, and Sodium 4-aminosalicylate. Each possibility represents a separate embodiment.

[0109] Other amino adds: (S)-a-Amino-y-butyrolactone, DL-2-Aminocaprylic add, 7- Aminocephalosporanic add , 4-Aminocinnamic acid, (S)-(+)-a-Aminocyclohexanepropionic acid,(R)-Amino-(4-hydroxyphenyl)acetic acid methyl ester, 5-Aminolevulinic acid, 4-Amino-nicotinic add, 3-Aminophenylacetic acid, 4-Aminophenylacetic add, 2-Amino-2-phenylbutyric acid, 4-(4- Aminophenyl)butyric acid, 2-(4-Aminophenylthio)acetlc add, DL-a-Amino-2-thlopheneacetic acid, 5-Aminovaleric add, 8-Benzyl (S)-2-aminooctanedioate, 4-(amino)-1-methylpyrrole-2- carboxylic acid, 4-(amino)tetrahydrothiopyran"4-carboxylic acid , (1 R,3S,4S)-2- azabicyclo[2.2.1]heptane"3-carboxylic add , bazetidine-2-carboxylic acid, azetidine-3- carboxy lie acid, 4-(amino)piperidine-4-carboxy!ic acid, diaminoacetic add, Inp-OH, (R)-Nip-OH,(S)-4~oxopiperidine~2-carboxylic acid, 2-(4-piperazino)-2-(4-fluorophenyl)acetic acid, 2-(4- piperazino)-2-phenylacetic acid, 4-piperidineacetaldehyde, 4-piperidylacetic acid, (-)-L- thioproline, Tle-OH, 3-piperidinecarboxylic acid, L-(+)-Canavanine, (±)-Carnitine, Chlorambucil,2,6-Diaminopimelic acid, meso-2,3-Diaminosuccinic acid, 4~(Dimethylamino)cinnamic acid, 4- (Dimethylamino)phenylacetic acid , Ethyl (S)- / V-Boc-piperidine-3-carboxylate, Ethyl piperazinoacetate , 4-[2-(amino)ethyl]piperazin-1-ylacetic acid, (R)-4-(amino)"5-phenylpentanoic acid, (S)-azetidine-2-carboxy!ic acid, azetidine-3-carboxylic acid, guvacine, Inp-OH, (R)-Nip-OH, DL-Nip-OH, 4-phenyl-piperidine-4-carboxylic acid, 1 -piperazineacetic acid, 4-piperidineacefc acid, (R)-piperidine-2-carboxylic acid, (S)-piperidine-2-carboxylic acid, (S)-1 , 2,3,4- tetrahydronorharmane-3-carboxylic acid, Tic-OH, D-Tic-OH, Iminodiacetic acid, lndoline-2- carboxylic acid, DL-Kynurenine, L-aziridine-2-carboxyiate, Methyl 4~aminobutyrate, (S)-2- Piperazinecarboxylic acid, 2-(1-Piperazinyl)acetic acid, (R)-(~ )-3-Piperidinecarboxylic acid, 2- Pyrrolidone-5-carboxylic acid, (R)-(+)-2-Pyrrolidone-5-carboxylic acid, (R)-1 ,2,3,4-Tetrahydro-3- isoquinolinecarboxylic acid, (S)-1 ,2,3,4-Tetrahydro-3-isoquinolinecarboxylic acid, L-4- Thiazolidinecarboxylic acid, (4R)-(“)-2-Thioxo-4-thiazolidinecarboxylic acid, hydrazinoacetic acid, and 3,3',5-Triiodo-L-thyronine. Each possibility represents a separate embodiment.

[0110] The terms "polypeptide variant" and “polypeptide mutant” as used herein refers to a polypeptide that comprises an amino acid sequence wherein one or more amino acid residues are inserted into, deleted from and / or substituted into the amino acid sequence relative to the parent polypeptide sequence. In various embodiments, the number of amino acid residues to be inserted, deleted, or substituted can be, e.g., at least 1 , at least 2, at least 3, at least 4, at least 5, at least 10, at least 25, at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, at least 350, at least 400, at least 450 or at least 500 amino acids in length. Variants of the present disclosure include fusion proteins.

[0111] A "derivative" of a polypeptide is a polypeptide that has been chemically modified, e.g., conjugation to another chemical moiety such as, for example, polyethylene glycol, albumin (e.g., human serum albumin), phosphorylation, and glycosylation.

[0112] The term “tumor associated antigen” (TAA) refers to, e.g., cell surface antigens that are selectively expressed by cancer cells or over-expressed in cancer cells relative to most normal cells. The terms "TAA variant" and “TAA mutant” as used herein refers to a TAA that comprises an amino acid sequence wherein one or more amino acid residues are inserted into, deleted from and / or substituted into the amino acid sequence relative to another TAA sequence.

[0113] The term "anti-TAA antagonist antibody" (interchangeably termed "anti-TAA antibody") refers to an antibody that is able to bind to TAA and inhibit TAA biological activity and / or downstream pathway(s) mediated by TAA signaling. The term "antibody" is used herein to referto a protein comprising one or more polypeptides substantially or partially encoded by immunoglobulin genes or fragments of immunoglobulin genes. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as subtypes of these genes and myriad of immunoglobulin variable region genes. Light chains (LC) are classified as either kappa or lambda. Heavy chains (HC) are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., lgG1, lgG2, IgG 3, lgG4, lgA1 and lgA2) or subclass. The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The antibody is able to specifically bind to a certain antigen, such as TAA.

[0114] The terms "an antigen-binding fragment" and “antigen-binding protein” as used herein means any protein that binds a specified target antigen. "Antigen-binding fragment" includes but is not limited to antibodies and binding parts thereof, such as immunologically functional fragments. An exemplary antigen-binding fragment of an antibody is the heavy chain and / or light chain CDR(s), or the heavy and / or light chain variable region.

[0115] Antibodies exist as intact immunoglobulins or as a number of well characterized fragments. Such fragments include Fab fragments, Fab' fragments, Fab2, F(ab)'2fragments, single chain Fv proteins (“scFv”) and disulfide stabilized Fv proteins (“dsFv”), that bind to the target antigen. A scFv protein is a fusion protein in which a light chain variable region of an immunoglobulin and a heavy chain variable region of an immunoglobulin are bound by a linker, while in dsFvs, the chains have been mutated to introduce a disulfide bond to stabilize the association of the chains. While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, as used herein, the term antibody encompasses e.g., monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g,, bispecific antibodies) formed from at least two intact antibodies, human antibodies, humanized antibodies, camelised antibodies, chimeric antibodies, single-chain Fvs (scFv), single-chain antibodies, single domain antibodies, domain antibodies, Fab fragments, F(ab')2 fragments, antibody fragments that exhibit the desired biological activity, disulfide-linked Fvs (sdFv), intrabodies, and epitope-binding fragments or antigen binding fragments of any of the above.

[0116] Bispecific antibodies or fragments can be of several configurations. For example, bispedfic antibodies may resemble single antibodies (or antibody fragments) but have two different antigen binding sites (variable regions). In various embodiments bispedfic antibodies of the present disclosure can have binding specificities for two different epitopes of one antigen or two individual antigens. In various embodiments the antibodies and fragments can also be heteroantibodies. Heteroantibodies are two or more antibodies, or antibody binding fragments (e.g., Fab) linked together, each antibody or fragment having a different specificity.

[0117] The term "monoclonal antibody" as used herein 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 may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigen. Furthermore, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" is not to be construed as requiring production of the antibody by any specific method.

[0118] The term “chimeric antibody” as used herein refers to an antibody which has framework residues from one species, such as human, and CDRs (which generally confer antigen binding) from another species, such as a murine antibody that specifically binds targeted antigen.

[0119] The term "human antibody”, as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3. However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[6120] The term “humanized antibody” as used herein refers to an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin. A humanized antibody binds to the same antigen as the donor antibody that provides the CDRs. The acceptor framework of a humanized immunoglobulin or antibody may have a limited number of substitutions by amino acids taken from the donor framework. Humanized or other monoclonal antibodies can have additional conservative amino acid substitutions which have substantially no effect on antigen binding or other immunoglobulin functions. In various embodiments, the framework regions arechosen from human germline exon XH, JH, VK and JK sequences. For example, acceptor sequences for humanization of FR of a VH domain can be chosen from genuine VH exons VH 1- 18 (Matsuda et ai., Nature Genetics 3:88-94, 1993) or VH1-2 (Shin et ai., EMBO J. 10:3641-3645, 1991) and for the hinge region (JH), exon JH-6 (Mattila et a!., Eur. J. Immunol. 25:2578-2582, 1995). In other examples, germline VK exon B3 (Cox et al., Eur. J. Immunol. 24:827-836, 1994) and JK exon JK-1 (Hieter et al., J. Biol. Chem. 257:1516-1522, 1982) can be chosen as acceptor sequences for VLdomain humanization.

[0121] An antigen binding protein, including an antibody, "specifically binds" to an antigen if it binds to the antigen with a high binding affinity as determined by a dissociation constant (KD, or corresponding Kb, as defined below) value of at least 1 x 10‘sM, or at least 1 x 10’7M, or at least 1 x 10’8M, or at least 1 x 10’9M, or at least 1 x 10’wM, or at least 1 x 10’11M. An antigen binding protein that specifically binds to the human antigen of interest may be able to bind to the same antigen of interest from other species as well, with the same or different affinities. The term "KD" as used herein refers to the equilibrium dissociation constant of a specific antibody-antigen interaction.

[0122] The term "pharmaceutical composition" refers to a composition suitable for pharmaceutical use in an animal. A pharmaceutical composition comprises a pharmacologically effective amount of an active agent and a pharmaceutically acceptable carrier. "Pharmacologically effective amount" refers to that amount of an agent effective to produce the intended pharmacological result. "Pharmaceutically acceptable carrier" refers to any of the standard pharmaceutical carriers, vehicles, buffers, and excipients, such as a phosphate buffered saline solution, 5% aqueous solution of dextrose, and emulsions, such as an oil / water or water / oil emulsion, and various types of wetting agents and / or adjuvants. Suitable pharmaceutical carriers and formulations are described in Remington's Pharmaceutical Sciences, 21st Ed. 2005, Mack Publishing Co, Easton. A “pharmaceutically acceptable salt" is a salt that can be formulated into a compound for pharmaceutical use including, e.g., metal salts (sodium, potassium, magnesium, calcium, etc.) and salts of ammonia or organic amines.

[0123] The terms "treat", "treating" and "treatment" refer to a method of alleviating or abrogating a biological disorder and / or at least one of its attendant symptoms. As used herein, to “alleviate" a disease, disorder or condition means reducing the severity and / or occurrence frequency of the symptoms of the disease, disorder, or condition. As used herein, "treatment" is an approach for obtaining beneficial or desired clinical results. For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, any one or more of: alleviationof one or more symptoms, diminishmenf of extent of disease, preventing or decaying spread (e.g., metastasis, for exampie metastasis to the lung or to the lymph node) of disease, preventing or delaying recurrence of disease, delay or slowing of disease progression, amelioration of the disease state, and remission (whether partial or total). Also encompassed by "treatment" is a reduction of pathological consequence of a proliferative disease. The methods of the disclosure contemplate any one or more of these aspects of treatment.

[0124] The term "effective amount" or “therapeutically effective amount” as used herein refers to an amount of a compound or composition sufficient to treat a specified disorder, condition or disease such as ameliorate, palliate, lessen, and / or delay one or more of its symptoms In reference to cancers or other unwanted cell proliferation, an effective amount comprises an amount sufficient to: (i) reduce the number of cancer cells; (ii) reduce tumor size; (ill) inhibit, retard, slow to some extent and preferably stop cancer cell infiltration into peripheral organs; (iv) inhibit (i.e. , slow to some extent and preferably stop) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay occurrence and / or recurrence of tumor; and / or (vii) relieve to some extent one or more of the symptoms associated with the cancer. An effective amount can be administered in one or more administrations.

[0125] The terms "patient," "individual," and "subject" may be used interchangeably and refer to a mammal, preferably a human or a non-human primate, but also domesticated mammals (e.g., canine or feline), laboratory mammals (e.g., mouse, rat, rabbit, hamster, guinea pig), and agricultural mammals (e.g., equine, bovine, porcine, ovine).The term "proliferative disease" includes tumor disease (including benign or cancerous) and / or any metastases. A proliferative disease may include hyperproliferative conditions such as hyperplasias, fibrosis (especially pulmonary, but also other types of fibrosis, such as renal fibrosis), angiogenesis, psoriasis, atherosclerosis and smooth muscle proliferation in the blood vessels, such as stenosis or restenosis following angioplasty. In some embodiments, the proliferative disease is cancer. In some embodiments, the proliferative disease is a non-cancerous disease. In some embodiments, the proliferative disease is a benign or malignant tumor.

[0126] "Linker" refers to a molecule that joins two other molecules, either covalently, or through ionic, van der Waals or hydrogen bonds. A linker could be cleavable or non-cleavable. A "cleavable linker" refers to a linker that can be degraded or otherwise severed to separate the two components connected by the cleavable linker. Cleavable linkers are generally cleaved by enzymes, typically peptidases, proteases, nucleases, lipases, and the like. Cleavable linkers mayalso be cleaved by environmental cues, such as, for example, changes in temperature, pH, salt concentration, etc.

[0127] In this application, the use of the singular includes the plural unless specifically stated otherwise. In this application, the use of "or" means "and / or" unless stated otherwise. Furthermore, the use of the term "including", as well as other forms, such as "includes" and "included", is not limiting. Also, terms such as "element" or "component" encompass both elements and components comprising one unit and elements and components that comprise more than one subunit unless specifically stated otherwise.Camptothecin (CPT) Derivative

[0128] In one aspect, the present disclosure provides novel camptothecin derivatives, a pharmaceutically acceptable salt, stereoisomer, enantiomer, or deuterated counterpart thereof The CPT derivative is represented by the following structure formula (A),wherein X is selected from -C(=:O)-(CH2)n1-O-N(R2)R3, -C(=O)-(CH2)nCN(OR2)R3!-C(=O)- (CH2)n1-O-(CH2)n1-N(R2)R3,-C Or(C(RH(R5;)n1-N(R2)R3, -S(=O)2-CH2-(CH2)n1-O-N(R2)R3, - S(=O)rCH2-(CH2)n1~N(OR2)R3, -S(=O)2~CH2-(CH2)n1-N(R2)R3, -C(=O)-O~(CH2)n2-O-N(R2)R3, - C(-O)-O-(CH2)n2-N(OR2)R3>-C(=O)-O-(CH2)n2-N(R2)R3, -C(=O)-NH-(CH2)n2-O-N(R2)R3, - C(=O)-NH-(CH2)n2-N(OR2)R3, -C(=O)-N(R2)-(CH2)n2-N(R2)R3, -C(=O)-NH-(CH2)n2-O-R3, -C(=O)- NH-(CH2)n2-S-R3, -C(=O)-O-(CH2)n2-O-R3, -C(=O)-S-(CH2)n2-O-R3, -C(=O)~S-(CH2)n2-S*R31- C(=O)“O"(CH2)n2"S"R3, -C(=O)-(C(R4)(R5))n!-N(R2)-C(=O)-(C(Ra)(Rb))n3-N(Rc)-R3, -C(=O)- (C(R4)(R5))n1-N(R2)-C(=O>(C(Ra)(Rb))nW-R3, -C(-O)-(C(R4)(R5))n1-N(R2)-C(=O)^C(Ra)(Rb))n3^ S-R3:R1, R2, R3and Rceach independently is a hydrogen atom, or CrCs alkyl;R4 and Rs each independently represents a hydrogen atom, a deuterium atom, halogen, halogenated alkyl, deuterated alkyl, alkoxy, hydroxyl, amino, nitro, cyano, hydroxyalkyl, heterocyclic Ci-Cs alkyl or 3-6 membered heterocyclic or cycloalyl ring; or R4 and Rs together with the carbon atom form a 3-6 membered cyclic ring;Raand Rbeach independently represents a hydrogen atom, a deuterium atom, halogen, halogenated alkyl, deuterated alkyl, alkoxy, hydroxyl, amino, nitro, cyano, hydroxyalkyl, heterocyclic CrCe alkyl or 3-6 membered heterocyclic or cycloalyl ring; or Raand R3together with the carbon atom form a 3-6 membered cyclic ring; n1is 1 , 2, 3, 4 or 5; n2is 2, 3, 4 or 5; n3is 1, 2, 3, 4 or 5.

[0129] In some embodiments, the camptothecin derivative is represented by the structure formula (A), wherein X is selected from -C(~O)-(CH2)n1-O-N(R2)R3, -C(=O)-(CH2)n1~ N(OR2)R3, -C(=O)-(CH2)n1-O-(CH2)n1-N(R2)R3, -C(=O)-(C(R4)(R5))n1-N(R2)R3, -S(=O)2-CH2- (CH2)n1-O-N(R2)R3, -S(=O)2-CH2-(CH2)n1-N(OR2)R3, -S(=O)2-CH2-(CH2)n1-N(R2)R3, -C(=O)-O- (CH2)n2-O-N(R2)R3, -C(=O)-O-(CH2)n2-N(OR2)R3, -C^O)-O-(CH2)n2-N(R2)R3, -C(=O)-NH- (CH2)n2-O-N(R2)R3, -C(=O)-NH-(CH2)n2-N(OR2)R3, -C^O)-N(R2)-(CH2)n2-N(R2)R31-C(=O)-NH- (CH2)n2-O-R3, -C(~O)-NH-(CH2)n2-S-R3)-C(“O)-O~(CH2)n2-O-R3)-C(~O)-S-(CH2)n2-O-R3, - C(=O)-S-(CH2)n2-S-R3, -C(=O)-O-(CH2)n2-S-R3, preferably X is selected from -C(=O)-(CH2)n1-O-N(R2)R3!-C(=O)-(CH2)n1-O-(CH2)n1-N(R2)R3, - C(=O)-(C(R4)(R5))n1-N(R2)R3, -S(=O)2-CH2-(CH2)n1-O-N(R2)R3, -S(=O)2-CH2-(CH2)n1-N(R2)R3, - C(=O)-O-(CH2)n2-O-N(R2)R3, -C(=O)-O-(CH2)n2-N(R2)R3, -C(=O)-NH-(CH2)n2-O-N(R2)R3>- C(=O)-N(R2)-(CH2)n2-N(R2)R3;R1is a hydrogen atom, or Ci-Cs alkyl;R2is a hydrogen atom;R3is a Ci-Cs alkyl;R4 and R5 each independently represents a hydrogen atom, a deuterium atom, halogen, hydroxyl, amino, nitro, cyano.

[0130] In some embodiments, the camptothecin derivative is represented by the structure formula (A), wherein X is selected from -C(=O)-(CH2)nkO-N(R2)R3, -C(=O)-(CH2)n1- N(OR2)R3, -C(=O)-(CH2)n1-O-(CH2)n1-N(R2)R3, -C(=O)-(C(R4)(R5))n1-N(R2)R3, -S(=O)2-CH2- (CH2)n1-O-N(R2)R3, -S(=O)2-CH2-(CH2)n!-N(OR2)R3, -S(=O)2-CH2-(CH2)n1-N(R2)R3, -C(=O)-O- (CH2)n2-O-N(R2)R3, -C(=O)-O-(CH2)n2-N(OR2)R3!-C(=O)-O-(CH2)n2-N(R2)R3, -C(=O)-NH-R1Is a hydrogen atom, or CrCs alkyl;R2is a hydrogen atom;R3is a CrCg alkyl;R4and R5each independently represents a hydrogen atom, a deuterium atom, halogen, halogenated alkyl, deuterated alkyl, alkoxy, hydroxyl, amino, nitro, cyano, hydroxyalkyl, heterocyclic CrCe alkyl or 3-6 membered heterocyclic or cycioalyl ring; or R4and R5together with the carbon atom form a 3-6 membered cyclic ring.

[0131] In some embodiments, the camptothecin derivative is represented by the structure formula (A), wherein X is selected from -C(=O)-(CH2)n1-O-N(R2)R3, -C(=O)-(CH2)n1- N(OR2)R3, -C(=O)-(CH2)n1-O-(CH2)n1-N(R2)R3, -C(=O)-(C(R4)(R5))n!-N(R2)R3, -S(=O)2-CH2- (CH2)n1-O-N(R2)R3, -S(=O)rCH2-(CH2)n1-N(OR2)R3, -S(=O)2-CH2-(CH2)n1-N(R2)R3, -C(=O)-O- (CH2)n2-O-N(R2)R3, -C(=O)-O-(CH2)n2-N(OR2)R3!-C(=O)-O-(CH2)n2-N(R2)R3, -C(=O)-NH- (CH2)n2-O-N(R2)R3, -C(=O)-NH-(CH2)n2-N(OR2)R3, -C(=O)-N(R2)-(CH2)n2-N(R2)R3, -C(=O)-NH- (CH2)n2-O-R3, -C(=O)-NH-(CH2)n2-S-R3, -C(=O)-O-(CH2)n2-O-R3, -C(=O)-S-(CH2)n2-O-R3, - C(=O)-S-(CH2)n2~S-R3, -C(=O)-O-(CH2)n2-S-R3, preferably X is selected from -C(=O)-(CH2)n1-O- N(R2)R31-C(=O)-(CH2)n1-O-(CH2)n1-N(R2)R3, -C(-O)-(C(R4)(R5))n1-N(R2)R3, -S(=O)2-CH2- (CH2)n1-O-N(R2)R3, -S(=O)2-CH2-(CH2)n1-N(R2)R3, -C(=O)-O-(CH2)n2-O-N(R2)R3, -C(=O)-O- (CH2)n2-N(R2)R3, -C(=O)-NH-(CH2)n2-O-N(R2)R3, -C(=O)-N(R2)-(CH2)n2-N(R2)R3;R> is a hydrogen atom, or Ci-Cs alkyl;R2and R3each is a hydrogen atom;R4and Rs each independently represents a hydrogen atom, a deuterium atom, halogen, halogenated alkyl, deuterated alkyl, alkoxy, hydroxyl, amino, nitro, cyano, hydroxyalkyl, heterocyclic CrCs alkyl or 3-6 membered heterocyclic or cycioalyl ring; or R4and Rs together with the carbon atom form a 3-6 membered cyclic ring.

[0132] In some embodiments, the camptothecin derivative is represented by the structure formula (A), wherein X is selected from -C(=O)-(C(R4)(R5))n1-N(R2)-C(=O)-(C(R4)(R5))n1"N(R2)"C(™O)-(C(Ra)(Rb))n3-S-R3, preferably X is selected from -C(=O)-R1is a hydrogen atom, or CrCs alkyl;R2, RS, and Rceach independently is a hydrogen atom or CrCs alkyl:R4and R5each independently represents a hydrogen atom, a deuterium atom, halogen, halogenated alkyl, deuterated alkyl, alkoxy, hydroxyl, amino, nitro, cyano, hydroxyalkyl, heterocyclic CrCs alkyl or 3-6 membered heterocyclic or cycloalyl ring; or R.;and R5together with the carbon atom form a 3-6 membered cyclic ring;R3and Rbeach independently represents a hydrogen atom, a deuterium atom, halogen, halogenated alkyl, deuterated alkyl, alkoxy, hydroxyl, amino, nitro, cyano, hydroxyalkyl, heterocyclic CrCs alkyl or 3-6 membered heterocyclic or cycloalyl ring; or Raand Rbtogether with the carbon atom form a 3-6 membered cyclic ring.

[0133] In some embodiments, the camptothecin derivative is selected from any one of the following In Table 1:Table 1Camptothecin Derivative Conjugates

[0134] in one aspect, the present disclosure provides camptothecin derivative conjugates, and the pharmaceutically acceptable salt thereof. The camptothecin derivative conjugates comprising at least one of the camptothecin derivatives, the pharmaceutically acceptable salts, stereoisomers, enantiomers, or deuterated counterparts disclosed herein.

[0135] In one aspect, the present disclosure provides camptothecin derivative conjugates, and the pharmaceutically acceptable salt thereof. The camptothecin derivative conjugate comprises (1) at least one of the camptothecin derivatives, the pharmaceutically acceptable salts, stereoisomers, enantiomers, or deuterated counterparts thereof disclosed herein; (2) a linker; and / or (3) a targeting agent.

[0136] In some embodiments, the linker is selected from at least one of the group consisting of cleavable and non-cleavable linkers, including but not limited to peptidase cathepsin sensitive linkers, acid sensitive linkers, glutathione sensitive linkers, sulfatase sensitive liners, lysosomal protease-sensitive linkers, beta-glucuronide linkers, phosphatase sensitive linkers, pyrophosphatase sensitive linkers.

[0137] In some embodiments, the linker attaches a targeting agent to one or more drug moieties (such as CPT derivatives) through covalent bond(s). The linker is a bifunctional or multifunctional moiety which can be used to link one or more drug moieties and a targeting agent to form a conjugate, for instance a camptothecin derivative conjugate. The linker may be stableoutside a cell, i.e. extracellular, or it may be cleavable by enzymatic activity, hydrolysis, or other metabolic conditions. CPT derivative conjugates can be conveniently prepared using a linker having reactive functionality for attaching to the CPT derivatives and to the targeting agent. For example, a cysteine thiol, or an amine, e g. N- terminus or amino acid side chain such as lysine, of the targeting agent can form a bond with a functional group of a linker reagent, CPT derivatives or CPT derivative-linker reagent.

[0138] The linkers are preferably stable outside the target cell. Before being internalized into a cell, the CPT derivative conjugates are preferably stable and remain intact, i.e. CPT derivatives remain linked to the targeting agent. An effective linker will: (I) maintain the specific binding properties of the targeting agent; (ii) allow intracellular delivery of the conjugate or drug moiety; (ill) remain stable and intact, i.e. not cleaved, until the conjugate has been delivered to its targeted site; and (iv) maintain a cytotoxic, cell-killing effect or a cytostatic effect of the camptothecin derivative. Stability of the CPT derivative conjugates may be measured by standard analytical techniques such as mass spectroscopy, HPLC, and the separation / analysis technique LC / MS. The linker may be immolative.

[0139] Covalent attachment of the targeting agent and the drug moiety, such as CPT derivatives, requires the linker to have two reactive functional groups. Bivalent linker reagents which are useful to attach two or more functional or biologically active moieties, such as peptides, nucleic acids, drugs, toxins, antibodies, haptens, and reporter groups are known, and methods have been described their resulting conjugates (Hermanson, G.T. (1996) Bioconjugate Techniques; Academic Press: New York, p 234-242).

[0140] In some embodiment, the linker may be substituted with groups which modulate solubility or reactivity. For example, a sulfonate substituent may increase water solubility of the reagent and facilitate the coupling reaction of the linker reagent with the targeting agent or the drug moiety or facilitate the coupling reaction of Targeting agent-Linkerwith Drug moiety, or Drug- moiety-Linker with Targeting agent, depending on the synthetic route employed to prepare the CPT derivative conjugates.

[0141] In some embodiments, a Linker has a reactive nucleophilic group which is reactive with an electrophilic group present on a targeting agent, such as an antibody. Useful electrophilic groups on a targeting agent include, but are not limited to, aldehyde and ketone carbonyl groups. The heteroatom of a nucleophilic group of a Linker can react with an electrophilic group on a targeting agent and form a covalent bond to the targeting agent. Useful nucleophilic groups on a Linker include, but are not limited to, hydrazide, oxime, amino, hydroxyl, hydrazine,thiosemicarbazone, hydrazine carboxylate, and aryihydrazide. The electrophilic group on a targeting agent provides a convenient site for attachment to a Linker.

[0142] Nucleophilic groups on a targeting agent include but are not limited to: (i) N- termina! amine groups, (ii) side chain amine groups, e.g. lysine, (Hi) side chain thiol groups, e.g. cysteine, and (iv) sugar hydroxyl or amino groups where the targeting agent is glycosylated. Amine, thiol, and hydroxyl groups are nucleophilic and capable of reacting to form covalent bonds with electrophilic groups on linker moieties and linker reagents including: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; (ill) aldehydes, ketones, carboxyl, and maleimide groups. Certain targeting agent have reducible interchain disulfides, i.e. cysteine bridges. Targeting agent may be made reactive for conjugation with linker reagents by treatment with a reducing agent such as DTT (dithiothreitol). Each cysteine bridge will thus form, theoretically, two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into targeting agents through the reaction oflysines with 2- iminothiolane (Traut's reagent) resulting in conversion of an amine into a thiol. Reactive thiol groups may be introduced into the targeting agent, such as antibodies, (or fragment thereof) by introducing one, two, three, four, or more cysteine residues (e.g., preparing mutant antibodies comprising one or more non-native cysteine amino acid residues). US 2007 / 0092940 teaches engineering antibodies by introduction of reactive cysteine amino acids.

[0143] Nucleophilic groups on a drug moiety, such as CPT derivatives include, but are not limited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groups capable of reacting to form covalent bonds with electrophilic groups on linker moieties and linker reagents including: (I) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimide groups.

[0144] Linkers can be peptidic, comprising one or more amino acid units. Peptide linker reagents may be prepared by solid phase or liquid phase synthesis methods (E. Schroder and K. Lubke, The Peptides, volume 1 , pp 76-136 (1965) Academic Press) that are well known in the field of peptide chemistry, including t-BOC chemistry (Geiser et al "Automation of solid-phase peptide synthesis" in Macromolecular Sequencing and Synthesis, Alan R. Liss, Inc., 1988, pp. 199-218) and Fmoc / HBTU chemistry (Fields, G. and Noble, R. (1990) "Solid phase peptide synthesis utilizing 9-fluoroenylmethoxycarbonyl amino acids", Int. J. Peptide Protein Res, 35:161- 214), on an automated synthesizer such as the Rainin Symphony Peptide Synthesizer (Protein Technologies, Inc., Tucson, AZ), or Model 433 (Applied Biosystems, Foster City, CA).

[0145] Exemplary amino add linkers include a dipeptide, a tripeptide, a tetrapeptide or a pentapeptide. Exemplary dipeptides include: valine-citruliine, alanine- phenylalanine. Exemplary tripeptides include: glycine-valine-citrulline and glycine-glycine-glycine. Exemplary tetrapeptides include: glycine-glycine-valine-citrulline and glycine-gSycine-phenylaianine-citrulline. Amino acid residues which comprise an amino acid linker component include those occurring naturally, as well as minor amino acids and non-naturally occurring amino acid analogs, such as citrulline. Amino acid linker components can be designed and optimized in their selectivity for enzymatic cleavage by a particular enzymes, for example, a tumor-associated protease, cathepsin B, C and D, or a plasmin protease.

[0146] Targeting agent indicates any moiety that can bind to a particular target, including but not limited to antibody or antigen binding fragment thereof, peptide, RNA, and DNA molecule. In some embodiments, the CPT derivative conjugates of the present disclosure comprise a targeting agent selected from antibody or antigen binding fragment thereof, peptide, RNA, and DNA molecule.

[0147] In some embodiments, the targeting agent is capable of binding to a tumor associated antigen (TAA), a tissue-specific antigen, a cell surface molecule, extracellular matrix protein or protease(s), or any post-translational modification residue(s). In various embodiments, the CPT derivative conjugates of the present disclosure comprise a targeting agent that exhibits binding affinity to a diseased cell or tissue.

[0148] In some embodiments, the targeting agent is capable of binding to a tumor associated antigen (TAA) selected from Trop-2, Her2, Her3, Her4, EGF, EGFR, CD2, CD3, CD5, CD7, CD13, CD19, CD20, CD21 , CD23, CD30, CD33, CD34, CD38, CD46, CD55, CD59, CD69, CD70, CD71 , CD97, CD117, CD123, CD127, CD134, CD137, CD138, CD146, CD147, CD152, CD154, CD174, CD195, CD200, CD205, CD212, CD223, CD227, CD253, CD272, CD274, CD276, CD278, CD279, CD309, CD319, CD326, CD340, DR6, Kv1.3, 5E10, MUC1 , uPA, MAGE3, MUC16, KLK3, K-ras, Mesothelin, p53, Survivin, G250, PSMA, Endoplasmin, BCM A, GPNMB, EphA2, EphB2, TMEFF2, Integrin beta 6, 5T4, CA9, IGF-1 R, Axl, B7H3, B7H4, CDH6, HAVCR1 , STEAP-1, STEAP-2, UPK2, CLDN18, CLDN6, CLDN9, c-Met, MICA, LIV-1, ROR1 , ADAM9, Stn, DLK-1 and CEACAM-5.

[0149] In a preferred embodiment, the targeting agent is an antibody or a binding fragment thereof, for example anti-Her2 antibody (such as Herceptin) or a binding fragment thereof, or an anti-Trop-2 antibody or a binding fragment thereof.

[0150] In various embodiments, the CPT derivative conjugates comprise a TAA binding antibody or antigen binding fragment thereof selected from the group consisting of a fully human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, an antigen-binding antibody fragment, a Fab, a Fab', a Fab2, a Fab’s, a IgG, a IgM, a IgA, a IgE, a scFv, a dsFv, a dAb, a nanobody, a unibody, and an diabody. In various embodiments, the antibody is a chimeric antibody, in various embodiments, the antibody is a humanized monoclonal antibody, in various embodiments, the antibody is a fully human monoclonal antibody.

[0151] In various embodiments, the targeting agent is a multipecific antibody, such as a bispecific antibody.

[0152] In some embodiments, the camptothecin derivative conjugate is represented by the following formula (I),Targeting agent-(L-D)n(I), wherein n is an integer of 1 to 24;D represents at least one of the camptothecin derivatives, the pharmaceutically acceptable salts, stereoisomers, enantiomers, or deuterated counterparts thereof disclosed herein;L is a linker comprising a peptide moiety of 2-8 amino acids represented by the following formula (Ila and lib),L1-L2-L3- (Ila)L1-L2- (lib) wherein L1is a linker moiety attached to the targeting agent, and L1comprises a reacted functional group selected from maleimide, bromoacetyl, iodoacetyl, thiol, amino, alkyl bromide, alkyl iodide, allenamide, carboxyl, and NHS ester;L2Is a linker moiety comprising 2-8 amino acid peptides, and optionally spacers, preferably PEG spacer;L3is a linker moiety connected to the camptothecin derivative, and L3comprises at least one of the following,

[0153] In one embodiment the reactive functional group is

[0154] In some embodiments, wherein the L1is selected from ”C(=O)-(CH2)ni-!and - C(=O)-(CH2)n1-Rs-[O-(CH2)ni]ni"N(R2)"C(=O)-)-(CH2)nr; Rs is alkylene-aryl-alkylene, aryl, 3-7 membered heterocyclyl, 3-7 membered cycloalkyl, heteroaryl, -alkylene-heteroaryl-alkylene-; n1is 1 , 2, 3, 4 or 5.

[0155] In some embodiments, L is selected from any one of the following,wherein n = 1-12, preferably 1-8.

[0156] In some embodiments, L2is a dipeptide, tripeptide or tetrapeptide comprising naturally occurring and non-naturally occurring amino acids.

[0157] In some embodiments, L2is selected from: gly-gly, g!y-gly-gly, phe-lys, val-ala, val-cit, giy-gly-phe-giy (GGFG)(SEQ ID NO: 1), val-cit-gly, val-gln-gly, val-glu-gly, phe-lys-gly, glu-val-ala, glu-val-cit, ^-ala-gly-phe-gly (AGFG)(SEQ ID NO: 2), and giy-gly-phe-gly-gly (GGFGG)(SEQ ID NO: 3), where the amino acid sequence is in either orientation.

[0158] In some embodiments, the camptothecin derivative conjugate comprises any one of the following structures,

[0159] in some embodiments, the camptothecin derivative conjugate or the pharmaceuticaiiy acceptable salt thereof disclosed herein has a ratio of the camptothecin derivative to the targeting agent from 4 to 12.

[0160] In some embodiments, the camptothecin derivative conjugate or the pharmaceutically acceptable salt thereof disclosed herein comprises any one of the following structures provided in Table 2:Table 2

[0161] In accordance with some of the methods described herein, the CPT derivative conjugate or CPT derivative is internalized by targeted tumor cells or by activated immune cells, where the CPT derivative conjugate or CPT derivative exerts a cytotoxic, cytostatic, or immunosuppressive effect on the antigen expressing cells to treat or prevent recurrence of the antigen expressing cancers or immunological disorders. In certain embodiments, the CPT derivative conjugate or CPT derivative is not internalized, and the anti-Target Ab is effective to deplete or inhibiting target antigen-expressing cells by binding to the cell membrane. In certainembodiments, the CPT derivative conjugate or CPT derivative can be targeted to a biological molecule in a cell (e.g. , an inflammatory agent) and accumulate at or adjacent cells secreting or binding the biological molecule, where the therapeutic drug moiety exerts an effect (e.g., a cytotoxic, cytostatic, or immunosuppressive effect).

[0162] Importantly, the CPT derivative conjugate of the present disclosure have superior drug / targeting agent ratios (such as DARs for ADCs), demonstrate improved solubility, enhanced CMC characteristics, and increased therapeutic efficacy against high antigen expressing tumor cells while having less effect on low or no antigen expressing ceils i.e. normal cells. Moreover, the CPT derivative conjugate or CPT derivative provide for the targeting of broader patient populations and patients with a refractory cancer or who previously responded to treatment with an anti-cancer therapy, but, upon cessation of therapy, suffered relapse (hereinafter “a recurrent cancer”).

[0163] In In one aspect, the present disclosure is directed to an ADC of the following formula (S’):Ab-(L-D)nor a pharmaceutically acceptable salt thereof; wherein n is an integer of about 1 to about 12;Ab is an antibody or antigen binding fragment thereof;D is a camptothecin derivative;L is a bivalent linker. the camptothecin derivative is selected from:X is selected from -C(=O)-(CH2)n!-O-N(R2)~, ~C(=O)-(CH2)n1-N(OR2)-, -C(=O)-(CH2)n1-O- (CH2)n1-N(R2)~s-C(=O)-(C(R4)(R5))n1-N(R2)-, -S(=O)2-CH2-(CH2)n1-O-N(R2)-,-S(=O)2-CH2- (CH2)n1-N(OR2)-, -S(=O)2-CH2-(CH2)n1-N(R2)-, -C(=O)-O-(CH2)n2-O-N(R2)-, -C(=O)-O-(CH2)n2- N(OR2)-:"C(=O)-O"(CH2)n2“N(R2)“, -C(-O)-NH-(CH2)n2-O-N(R2)-:-C(=O)-NH-(CH2)n2-N(OR2)-, and -C("O)”N(R2)”(CH2)n2"N(R2)-;-C(=:O)-NH-(CH2)ri2-O"l-C(=:O)-NH-(CH2)ri2-S-;-Cf^O)-©- (CH2)n2-O-,-C(=O)-S-(CH2)n2-O-1-C(=O)-S-(CH2)n2-S-1-C(=O)-O-(CH2)n2-S-:-C(=O)- (C(R4)(R5))n1-N(R2)-C(=O)-(C(Ra)(Rb))n3*N(Rc)-,-C(=O)-(C(R4)(R5))ni*N(R2)-C(=O)- (C(Ra)(Rb))n3~O-rC(=O)-(C(R4)(R5))n1-N(R2;hC(-O)-(C(Ra)(Rb))n3-S-lL is a bivalent linker comprising a peptide moiety of 2-4 amino acid represented by the following formula (Ila and lib):L1-L2(lib) wherein L1is a linker attached to the antibody comprising a reacted functional group selected from maleimide, bromoacetyl, iodoacetyl, thiol, amino, alkyl bromide, alkyl iodide, allenamide, carboxyl, and NHS ester;L2is 2-4 AA peptide moiety;L3is a moiety connected to camptothecin derivative that is:Ri is a hydrogen atom or CrCs alkyl;R2is a hydrogen atom or CrCs alkyl;Rs is a hydrogen atom, CrCs alkyl or -C(=O)-(CH2)n3-OH;R4and R5each independently represents a hydrogen atom, a deuterium atom, halogen, halogenated alkyl, deuterated alkyl, alkoxy, hydroxyl, amino, nitro, cyano, hydroxyalkyl, heterocyclic, CrCs alkyl or 3-6 member heterocyclic or cycloalyl ring; or R4and R5together with the carbon atom form a 3-6 member cyclic ring;Raand Rbeach independently represent a hydrogen atom, a deuterium atom, halogen, halogenated alkyl, deuterated alkyl, alkoxy, hydroxyl, amino, nitro, cyano, hydroxyalkyl, heterocyclic, CrCs alkyl or 3-6 member heterocyclic or cycloalyl ring; or Raand Rbtogether with the carbon atom form a 3-6 member cyclic ring;Rcis a hydrogen atom or CrCs alkyl; n1is 1 , 2, 3, 4 or 5; n2is 2, 3, 4 or 5; n3is 1 , 2, 3, 4 or 5;Y is -C(=O)-R6, -C(=O)-OR6, -C(=O)-NHR6, R6is CrC6alkyl:Z is NHR7, R7is C1-C6 alkyl, -CH2-CH2-OH, -CH2-CH2-OCH3, or -CH2-CH2-N(CH3)2;W is NH or 0.

[0164] Accordingly, drug moiety reagents include the structures:wherein R2 and Rc each independently is hydrogen atom or Ci-Ce alkyl, R3 and R4 each independently is a hydrogen atom, a deuterium atom, halogen, C1-C6 alkyl or a 3-6 member heterocyclic or cycloalyl ring; or R3 and R4 together with the carbon atom form a 3-6 member cyclic ring, Raand Rbeach independently is a hydrogen atom, a deuterium atom, halogen, C1-C6 alkyl or a 3-6 member heterocyclic or cycioalyi ring; or Raand Rbtogether with the carbon atom form a 3-6 member, ni and n? each independently is 0, 1 , 2, 3 or 4.

[0165] Embodiments of drug-linker reagents include:wherein Ri is H or Ci-Cs alkyl, n is 0, 1 , 2, 3, or 4;R2 and R3 are independently an amino add side chain selected from hydrogen, methyl, isopropyl, isobutyi, sec-butyl, benzyl, p-hydroxybenzyl, -CH2OH. -CH(OH)CH3, - CH2CH2SCH3, -CH2CONH2, -CH2COOH, -CH2CH2CONH2, -CH2CH2COOH, - (CH2)3NHC(=NH)NH2, -(CH2J3NH2, -(CH2)3NHCOCH3, ~(CH2)3NHCHO, - (CH2)4NHC(=NH)NH2, -(CH2)4NH2, -(CH2)4NHCOCH3, -(CH2)4NHCHO, -(CH 2) 3NHCONH2, -( CH2) 4NHCONH2, - CH2CH2CH(OH)CH2NH2, 2-pyridylmethyl-, 3- pyridylmethyl-, 4-pyridylmethyl-. phenyl and cyclohexyl;R4 and R5 each independently is a hydrogen atom, a deuterium atom, halogen, C-i-Cg alkyl or a 3-6 member heterocyclic or cycloalyl ring; or R4 and Rs together with the carbon atom form a 3- 6 member cyclic ring.Preparation Method

[0166] The present disclosure provides a method of making the camptothecin derivative conjugate or the pharmaceutically acceptable salt thereof disclosed herein, comprising reacting a targeting agent and / or a linker with a camptothecin derivative.Pharmaceutical Composition

[0167] The present disclosure provides a pharmaceutical composition, comprising the camptothecin derivative, a pharmaceutically acceptable salt, stereoisomer, enantiomer, or deuterated counterpart thereof disclosed herein, the camptothecin derivative conjugate or the pharmaceutically acceptable salt thereof disclosed herein, and a pharmaceutically acceptable diluent, carrier, or excipient.

[0168] Pharmaceutically acceptable excipients and carriers are well known and understood by those of ordinary skill and have been extensively described (see, e.g., Remington's Pharmaceutical Sciences, 18th Edition, A. R. Gennaro, ed., Mack Publishing Company, 1990). The pharmaceutically acceptable carriers may be included for purposes of modifying, maintaining or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. Such pharmaceutical compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the polypeptide. Suitable pharmaceutically acceptable carriers include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogensulfite); buffers (such as borate, bicarbonate, Tris-HCI, citrates, phosphates, other organic acids); bulking agents (such as mannitol or glycine), chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, betacyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides and other carbohydrates (such as glucose, mannose, or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring; flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; saltforming counter ions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethyleneglycol) ; sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides (preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants.

[0169] The primary vehicle or carrier in a pharmaceutical composition may be either aqueous or non-aqueous in nature. For example, a suitable vehicle or carrier may be water for injection, physiological saline solution or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. Other exemplary pharmaceutical compositions comprise Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which may further include sorbitol or a suitable substitute thereof. In one embodiment of the present disclosure, compositions may be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents (Remington's Pharmaceutical Sciences, supra) in the form of a lyophilized cake or an aqueous solution. Further, the therapeutic composition may be formulated as a lyophilizate using appropriate excipients such as sucrose. The optimal pharmaceutical composition will be determined by one of ordinary skill in the art depending upon, for example, the intended route of administration, delivery format, and desired dosage.

[0170] The pharmaceutical compositions of the disclosure are typically suitable for parenteral administration. As used herein, "parenteral administration" of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a patient and administration of the pharmaceutical composition through the breach in the tissue, thus generally resulting in the direct administration into the blood stream, into muscle, or into an internal organ. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non- surgical wound, and the like. In various embodiments, the pharmaceutical composition is formulated for parenteral administration via a route selected from, e.g., subcutaneous injection, intraperitoneal injection, intramuscular injection, intrasternal injection, intravenous injection, intraarterial injection, intrathecal injection, intraventricular injection, intraurethral injection, intracranial injection, intrasynovial injection or via infusions.

[0171] When parenteral administration is contemplated, the therapeutic pharmaceutical compositions may be in the form of a pyrogen-free, parenterally acceptable aqueous solution comprising the desired ADC in a pharmaceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water in which a polypeptide is formulated as a sterile, isotonic solution, properly preserved. In various embodiments, pharmaceutical formulations suitable for injectable administration may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionaily, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Optionally, the suspension may aiso contain suitable stabilizers or agents to increase the solubility of the compounds and ailow for the preparation of highly concentrated solutions. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampoules or in multi-dose containers containing a preservative. Other parentally administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form, or in a liposomal preparation. Formulations for parenteral administration may be formulated to be immediate and / or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release.

[0172] Any method for formulating and administering peptides, proteins, antibodies, and immunoconjugates accepted in the art may suitably be employed for administering the CPT derivatice conjugates of the present disclosure.Pharmaceutical Uses

[0173] The present disclosure provides a method of treating or preventing a disease, comprising administering to a subject in need thereof a therapeutically effective amount of the camptothecin derivative, a pharmaceutically acceptable salt, stereoisomer, enantiomer, or deuterated counterpart thereof disclosed herein, the camptothecin derivative conjugate or the pharmaceutically acceptable salt thereof disclosed herein, or the pharmaceutical composition disclosed herein.

[0174] In some embodiments, the method of treating or preventing a disease further comprising administering a second therapy to the subject.

[0175] When the camptothecin derivative, a pharmaceutically acceptable salt,stereoisomer, enantiomer, or deuterated counterpart thereof disclosed herein, the camptothecin derivative conjugate or the pharmaceutically acceptable salt thereof disclosed herein, or the pharmaceutical composition disclosed herein is administered with a second therapy, the second therapy selected from the group consisting of: cytotoxic chemotherapy, immunotherapy, small molecule kinase inhibitor targeted therapy, surgery, radiation therapy, stem cell transplantation, cell therapies including CAR-T, CAR-NK, IPS induced CAR-T or IPS induced CAR-NK and vaccine such as Bacille Calmette-Guerine (BCG). In various embodiments, the combination therapy may comprise administering to the subject a therapeutically effective amount of immunotherapy, including, but are not limited to, treatment using depleting antibodies to specific tumor antigens; treatment using antibody-drug conjugates; treatment using agonistic, antagonistic, or blocking antibodies to co-stimulatory or co-inhibitory molecules (immune checkpoints) such as CTLA-4, PD-1 , PD-L1, CD40, OX-40, CD137, GITR, LAG3, TIM-3, Siglec 7, Siglec 8, Siglec 9, Siglec 15 and VISTA; treatment using bispecific T cell engaging antibodies (BiTE®) such as blinatumomab: treatment involving administration of biological response modifiers such as IL-12, IL-21, GM-CSF, IFN-alpha, IFN-p and IFN-y; treatment using therapeutic vaccines such as sipuleucel-T; treatment using dendritic cell vaccines, or tumor antigen peptide vaccines; treatment using chimeric antigen receptor (CAR)-T cells; treatment using CAR-NK cells: treatment using tumor infiltrating lymphocytes (TILs); treatment using adoptively transferred anti-tumor T cells (ex vivo expanded and / or TOR transgenic); treatment using TALL-104 cells; and treatment using immunostimulatory agents such as Toll-like receptor (TLR) agonists CpG and imiquimod; and treatment using vaccine such as BCG; wherein the combination therapy optionally provides increased effector cell killing of tumor cells, i.e., a synergy exists between the ADC constructs and the immunotherapy when co-administered.

[0176] When the camptothecin derivative, a pharmaceutically acceptable salt, stereoisomer, enantiomer, or deuterated counterpart thereof disclosed herein, the camptothecin derivative conjugate or the pharmaceutically acceptable salt thereof disclosed herein, or the pharmaceutical composition disclosed herein is administered with a second therapy, they are administered either concomitantly or sequentially.

[0177] The list of exemplary chemotherapeutic agent includes, but is not limited to, daunorubicin, dactinomycin, doxorubicin, bleomycin, mitomycin, nitrogen mustard, chlorambucil, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, bendamustine, cytarabine (CA), 5-fluorouracil (5-FU), floxuridine (5-FUdR), methotrexate (MTX), colchicine, vincristine, vinblastine, etoposide, teniposide, cisplatin, carboplatin, oxaliplatin, pentostatin, cladribine,cytarabine, gemcitabine, pralatrexate, mitoxantrone, diethyistiibestrol (DES), fluradabine, ifosfamide, hydroxyureataxanes (such as paclitaxel and doxetaxel) and / or anthracycline antibiotics, as well as combinations of agents such as, but not limited to, DA-EPOCH, CHOP, CVP or FOLFOX.

[0178] In some embodiments, the disease is selected from any one of the following, a proliferative disorder, an autoimmune disorder, destructive bone disorder, infectious disease, viral disease, fibrotic disease, neurodegenerative disorder, pancreatitis or kidney disease.

[0179] In some embodiments, the proliferative disorder is a cancer, preferably, a cancer selected from the group consisting of pancreatic cancer, gastric cancer, liver cancer, breast cancer, ovarian cancer, colorectal cancer, melanoma, leukemia, myelodysplastic syndrome, lung cancer, prostate cancer, brain cancer, bladder cancer, head-neck cancer, and rhabdomyosarcoma.

[0180] The present disclosure provides use of the camptothecin derivative, a pharmaceutically acceptable salt, stereoisomer, enantiomer, or deuterated counterpart thereof disclosed herein, the camptothecin derivative conjugate or the pharmaceutically acceptable salt thereof disclosed herein, or the pharmaceutical composition disclosed herein in the preparation of a medicament in the treating or preventing of a disease.

[0181] The present disclosure provides the camptothecin derivative, a pharmaceutically acceptable salt, stereoisomer, enantiomer, or deuterated counterpart thereof disclosed herein, the camptothecin derivative conjugate or the pharmaceutically acceptable salt thereof disclosed herein, or the pharmaceutical composition disclosed herein for use in the preparation of a medicament for treating or preventing of a disease.

[0182] In one aspect, the present disclosure relates to a method of treating a proliferative disease (such as cancer) in an individual, comprising administering to the individual a therapeutically effective amount of the camptothecin derivative, a pharmaceutically acceptable salt, stereoisomer, enantiomer, or deuterated counterpart thereof disclosed herein, the camptothecin derivative conjugate or the pharmaceutically acceptable salt thereof disclosed herein, or the pharmaceutical composition disclosed herein. In some embodiments, cancers include recurrent, resistant, or refractory cancers, at surprisingly low doses.

[0183] Examples of tumor cell lines derived from human tumors and available for use in the in vitro and in vivo studies include, but are not limited to, leukemia cell lines (e.g., CCRF-CEM, HL-60(TB), K-562, MOLT-4, RPM 1 -8226, SR, P388 and P388 / ADR, H292, MV-4-11); non-small cell lung cancer cell lines (e.g., A549 / ATCC, EKVX, HOP-62, HOP-92, NCI-H226, NCI-H23, NCI-H322M, NCI-H460, NCI-H522 and LXFL 529); small cell lung cancer cell lines (e.g., DMS 114 and SHP-77); colon cancer cell lines (e.g., COLO 205, HCC-2998, HCT-116, HCT-15, HT29, KM12, SW-620, DLD-1 and KM20L2); central nervous system (CNS) cancer cell lines (e.g., SF- 268, SF-295, SF-539, SNB-19, SNB-75, U251, SNB-78 and XF 498); melanoma cell lines (e.g., LOX I MVI, MALME-3M, M14, SK-MEL-2, SK-MEL-28, SK-MEL-5, UACC-257, UACC-62, RPMI- 7951 and M19-MEL); ovarian cancer cell lines (e.g., IGROV1, OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8 and SK-OV-3); renal cancer cell lines (e.g., 786-0, A498, ACHN, CAKI-1 , RXF 393, SN12C, TK-10, UO-31, RXF-631 and SN12K1); prostate cancer cell lines (e.g., PC-3 and DU- 145); breast cancer cell lines (e.g., MCF7, NCI / ADR-RES, MDA-MB-231 / ATCC, HS 578T, MDA- MB-435, BT-549, T-47D and MDA-MB-468); and thyroid cancer cell lines (e.g., SK-N-SH).

[0184] In various embodiments, the methods described herein may be used in combination with other conventional anti-cancer therapeutic approaches directed to treatment or prevention of proliferative disorders, such approaches including, but not limited to chemotherapy, small molecule kinase inhibitor targeted therapy, surgery, radiation therapy, and stem cell transplantation. For example, such methods can be used in prophylactic cancer prevention, prevention of cancer recurrence and metastases after surgery, and as an adjuvant of other conventional cancer therapy. The present disclosure recognizes that the effectiveness of conventional cancer therapies (e.g., chemotherapy, radiation therapy, phototherapy, immunotherapy, and surgery) can be enhanced through use of the fusion molecules described herein.Target Antigens and Exemplary Antibodies

[0185] Tumor antigens expressed on the cell membrane are potential targets in immunotherapy, with the ideal tumor antigen absent on normal cells and overexpressed on the tumor cell surface. The CRT derivative conjugates used in the methods of the present disclosure may comprise a targeting agent, such as an antibody, or antigen binding antibody fragment, a multispecific antibody (such as bispecific antibody) specific to any of the tumor associated antigens described in the art, including any biosimilar, biogeneric, follow-on biologic, or follow-on protein version of any TAA described in the art. The TAA can be any peptide, polypeptide, protein, nucleic acid, lipid, carbohydrate, or small organic molecule, or any combination thereof, against which the skilled artisan wishes to induce an immune response.

[0186] In various embodiments, the TAA, TAA variant, or TAA mutant contemplated for use in the combination methods of the present disclosure is selected from, or derived from, the list provided in Table 3.Table 3Tumor Associated Antigen (TAA)

[0187] In various embodiments, the TAA has an amino acid sequence that shares an observed homology of, e.g., at least about 75%, at least about 80%, at least about 85%, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% with any one of the sequences disclosed in Table 3.

[0188] In various embodiments, the CPT derivative conjugates of the present disclosure utilize a targeting agent, such as an antibody or antigen-binding fragment thereof that is a polyclonal antibody, a monoclonal antibody or antigen-binding fragment thereof, a recombinant antibody, a diabody, a chimerized or chimeric antibody or antigen-binding fragment thereof, a humanized antibody or antigen-binding fragment thereof, a fully human antibody or antigenbinding fragment thereof, a CDR-grafted antibody or antigen-binding fragment thereof, a single chain antibody, an Fv, an Fd, an Fab, an Fab', or an F(ab')2, and synthetic or semi-synthetic antibodies. The antibody or antigen binding fragment thereof could be mono-specific (specifically binds to one antigen), bispecific (specifically binds to two antigens), or multispecific antibody (specifically binds to more than two antigens).

[0189] In various embodiments, antibodies contemplated for use in the CPT derivative conjugates of the present disclosure include but are not limited to LL1 (anti-CD74), LL2 or RFB4 (anti~CD22), velfuzumab (hA20, anti~CD20), rituxumab (anti-CD20), obinutuzumab (GA101. anti- CD20), lambrolizumab (anti-PD-1 receptor), nivolumab (anti-PD-1 receptor), ipilimumab (anti- CTLA-4), RS7 (anti-epithelial glycoprotein-1 (EGP-1, also known as TROP-2)), PAM4 or KC4 (both anti-mucin), MN-14 (anti-carcinoembryonic antigen (CEA, also known as CD66e or CEACAM5), MN-15 or MN-3 (anti-CEACAM6), Mu-9 (anti-colon-specific antigen-p), Immu 31 (an anti-alpha-fetoprotein), R1 (anti-IGF-1 R), A19 (anti-CD19), TAG-72 (e.g., CC49), Tn, J591 or HuJ591 (anti-PSMA (prostate-specific membrane antigen)), AB-PG1-XG1-026 (anti-PSMA dimer), D2 / B (anti-PSMA), G250 (an anti-carbonic anhydrase IX MAb), L243 (anti-HLA-DR) alemtuzumab (anti-CD52), bevacizumab (anti-VEGF), cetuximab (anti-EGFR), gemtuzumab (anti-CD33), ibritumomab tiuxetan (anti-CD20); panitumumab (anti-EGFR); tositumomab (anti- CD20); PAM4 (aka clivatuzumab, anti-mucin);trastuzumab (anti-ErbB2); and anti-CTLA4 antibodies include ipilimumab (Bristol-Myers Squibb) and tremelimumab (PFIZER). Such antibodies are known in the art (e.g., U.S. Pat. Nos. 5,686,072; 5,874,540; 6,107,090; 6,183,744; 6,306,393; 6,653,104; 6,730.300; 6,899,864; 6,926,893; 6,962,702; 7,074,403; 7,230,084; 7,238,785; 7,238,786; 7,256,004; 7,282,567; 7,300,655; 7,312,318; 7,585,491 ; 7,612,180; 7,642,239; and U.S. Patent Application Publ. Nos, 20050271671 ; 20060193865; 20060210475; 20070087001 ; the Examples section of each incorporated herein by reference.) Specific known antibodies of use include hPAM4 (U.S. Pat. No. 7,282,567), hA20 (U.S. Pat No. 7,251 ,164), hA19 (U.S. Pat. No. 7,109,304), hlMMU-31 (U.S. Pat. No. 7,300,655), hLL1 (U.S. Pat. No. 7,312,318,), hLL2 (U.S. Pat. No. 7,074,403), hMu-9 (U.S. Pat. No. 7,387,773), hL243 (U.S. Pat. No. 7,612,180), hMN-14 (U.S. Pat. No. 6,676,924), hMN-15 (U.S. Pat. No. 7,541 ,440), hR1 (U.S. patentapplication Ser No. 12 / 772,645), hRS7 (U.S. Pat. No. 7,238,785), hMN-3 (U.S. Pat. No. 7,541 ,440), AB-PG1-XG1-026 (U.S. patent application Ser. No. 11 / 983,372, deposited as ATCC PTA-4405 and PTA-4406) and D2 / B (WO 2009 / 130575) the text of each recited patent or application is incorporated herein by reference.

[0190] In a particular embodiment, the CPT derivative conjugates of the present disclosure include at least one antibody or fragment thereof that binds to Her2.

[0191] Bispecific antibodies or fragments can be of several configurations. For example, bispecific antibodies may resemble single antibodies (or antibody fragments) but have two different antigen binding sites (variable regions).

[0192] In various embodiments bispecific antibodies of the present disclosure can have binding specificities for at least two different epitopes at least one of which is a tumor associate antigen. In various embodiments the antibodies and fragments can also be heteroantibodies. Heteroantibodies are two or more antibodies, or antibody binding fragments (e.g., Fab) linked together, each antibody or fragment having a different specificity.

[0193] The following examples are provided to more fully illustrate the disclosure but are not construed as limiting the scope thereof.Example 1. Synthesis of Compound 12 (363-11)

[0194] Step 1. To a solution of sarcosine (0.9 g, 10 mmol) in THF (10 mL) and water (10 mL) was added K2CO3 (2.07g, 15 mmoi) and allyl chioroformate (1.45 g, 12 mmoi). The mixture was stirred for 2-3 hrs. 1 N H Cl solution was added to the mixture to acidify to pH = 6. The resulting mixture was concentrated under vacuum to give a residue, which was dissolved in DCM / MeOH(v / v=3:1, 20 mL). The mixture was filtered and evaporated to give crude compound 1.

[0195] Step 2. HATU (72 mg, 0.45 mmol) was added to a solution of exatecan mesylate(20 mg, 0.38 mmoi), compound 1 (130 mg , 0.75 mmol) in DMF (8 mL). DIPEA (130 pL, 0.75 mmoi) was added to the mixture and stirred for 1 hr at room temperature. The mixture was diluted with DCM, washed with aqueous sodium bicarbonate and brine. The organic layer was dried over anhydrous NazSCX filtered, and concentrated by evaporation to give a residue, which was purified by flash chromatography on silica gel (eluent: MeOH / DCM = 0-10%) to give the desired compound 2 (180 mg, 81%).2 3 (T-363)

[0196] Step 3. To a solution of compound 2 (180 mg, 0.30 mmol) in DCM (10 mL) were added Pd(PPhs)4 (17.6 mg, 0.015 mmol) and pyrrolidine (44 mg, 0.61 mmoi). The mixture was stirred for 20 mins. The reaction was quenched with 5 mL of brine, and the mixture was extractedwith DCM (2x 10 mL). The organic layers were dried over anhydrous Na2SO-s, filtered, and evaporated to give a residue, which was purified by HPLC (eluent: ACN / H2O=5-80% for 25 mins) to provide the desired compound 3 (T-363) (40mg, 26%) as a solid.

[0197] Step 4. HATU (34 mg, 0.089 mmol) was added to a solution of 3 (30 mg, 0.059 mmol), N-Fmoc-Glyane (35 mg, 0.118 mmol) in DMF (5 mL). DIPEA (15 mg, 0.118 mmol) was added to the mixture and stirred for 1 hr at room temperature. The mixture was loaded to HPLC column for purification (eluent: ACN / H2O~5~80% for 25 mins) to give the desired compound 4 (35 785.8^ .4 5

[0198] Step 5. To a solution of compound 4 (35 mg, 0.044 mmol) in DMF (5 mL) was added piperidine (1 mL). The mixture was stirred for 10 mins and evaporated to give a residue, which was purified by HPLC (eluent: ACN / H2O=5-80% for 25 mins) to give the desired compound 5 (20 mg, 80%). LRMS: m / z = 564.2 [M+1]+. Molecular Formula: C29H30FN5O6; Molecular weight: 563.58.

[0199] Step 6. To a solution of 6-maleimidohexanoic acid (2.11g, 10 mmol) in DMF (20 mL) were added tert-butyl glycinate (1.31g, 10 mmol), HATU (4.56g, 12 mmol) and DIPEA (3.51 mL, 20 mmol). The mixture was stirred for 2 hrs. The mixture was diluted with ethyl acetate (100 mL) and washed with NaHCCh solution (2x10 mL), water (3x10 mL) and brine. The organic layers were dried over anhydrous NasSCU, filtered, and evaporated to give a residue, which was purified by silica gel chromatography (eluent: MeOH / DCM=3-10%) to provide the desired compound 6 (1.8 g, 56%).

[0200] Step 7. To a solution of compound 6 (1.8 g, 3.08 mmol) in DCM (10 mL) was added TFA (10 mL). The mixture was stirred for 2 hrs. The volatiles were removed under vacuum and the resulting residue was dissolved in 1 mL of ethyl acetate. Methyl tert-butyl ether was added to precipitate the product. The solid was filtered and washed with some methyl tert-butyl ether to give compound 7 (1 g, 68%) as a white solid.

[0201] Step 8. To a solution of compound 7 (1 g, 3.73 mmol) in DMF (10 mL) were added tert-butyl glycinate (0.5 g, 3.73 mmol), HATU (1.7 g, 4.47 mmol) and DIPEA (1.30 mL, 7.46 mmol). The mixture was stirred for2 hrs. The mixture was diluted with ethyl acetate (100 mL) and washedwith NaHCOs solution (2x10 mL), water (3x10 mL) and brine. The organic layers were dried over anhydrous NasSCX filtered, and evaporated to give the crude compound 8.

[0202] Step 9. To a solution of crude compound 8 in DCM (10 mL) was added TFA (10 mL). The mixture was stirred for 2 hrs. The volatiles were removed under vacuum and the resulting residue was dissolved in 1 mL of ethyl acetate. Methyl tert-butyl ether was added to precipitate the product. The solid was filtered and washed with some methyl tert-butyl ether to give compound 9 (0.5 g, 42% from 7) as a white solid.

[0203] Step 10. To a solution of compound 9 (0.5 g, 1.53 mmol) in DMF (15 mL) were added H-L-Phe-OtBu (0.4 g, 1.53 mmoi), HATU (0.87 g, 2.30 mmol) and DI PEA (0.49 g, 3.84 mmol). The mixture was stirred for 2hrs. The mixture was diluted with ethyl acetate (60 mL) and washed with NaHCOs solution (2x50 mL), water (20 mL) and brine. The organic layers were dried over anhydrous Na2SO4, filtered, and evaporated to give a residue, which was purified by flash chromatography on silica gel (eluent: EtOAc / hexane = 5-50%) to give the title compound 10 (0.4

[0204] Step 11. To a solution of 10 (0.4 g, 0.75 mmol) in DCM (20mL) was added TFA (10 mL). The mixture was stirred for 3 hrs. The volatiles were removed under vacuum and the resulting residue was dissolved in 1 mL of DCM. Ether was added to precipitate the product. The solid was filtered and washed with ether to give compound 11 (0.15 g, 42%) as a white solid. LRMS: m / z=473.22 [M+1]+. Molecular Formula: C23H28N4O7; Molecular Weight: 472.49.

[0205] Step 12. HATU (20 mg, 0.053 mmol) was added to a solution of 5 (20 mg, 0.035 mmol), 11 (34 mg, 0.071 mmol) in DMF (3 mL). DIPEA (12 pL, 0.071 mmol) was added to the mixture and stirred for 1 hr at room temperature. The mixture was loaded to HPLC column for purification (eluent: ACN / H2O=:5-80% for 25 mins) to give the title compound 12 (363-11) (12 mg, 34%). LRMS: m / z =1018.4 [M-M]+. Chemical Formula: C52HS6FN90i2; Molecular Weight: 1018.05.Example 2. Synthesis of compound 17Compound 1713

[0206] Step 1. To a solution of exatecan methanesulfonate (90 mg, 0.17 mmol) in dichloromethane (10 mL) was added triethylamine (51.6 mg, 0.51 mmol) and di-tert-butyl114ecarbonate (55.6 mg, 0.255 mmol). The resulting mixture was stirred at room temperature overnight. The reaction mixture was diluted with DCM (15 mL), washed with water and brine, dried over anhydrous Na2SO4, and concentrated in vacuo to give crude compound 13 (189 mg) as a white solid, which was used in next step without further purification.Chemical Formula: C29H30FN3O6; Molecular Weight: 535.57.13 14

[0207] Step 2. A solution of compound 13 (60 mg, 0.112 mmol) in N1,N1-dimethylethane- 1 ,2-diamine (1 mL) was stirred at 50°C for 0.5 hr. The reaction mixture was concentrated in vacuo to give crude compound 14 (75 mg, crude) as a yellow oil which was used in next step without further purification. LRMS: m / z“624.60[M+H]4. Chemical Formula: C33H42FN5O6; Molecular Weight: 623.73.14 15

[0208] Step 3. To a solution of crude compound 14 (75 mg, 0.112 mmol) in pyridine (2 mL) was added propionic anhydride (29 mg, 0.224 mmol). The resulting mixture was stirred at room temperature overnight. The reaction mixture was concentrated in vacuo to give a crude residue which was diluted with ethyl acetate (30 mL), washed with brine, dried over anhydrous Na2SO4, and concentrated in vacuo to give crude compound 15 (61 .9 mg) as a yellow solid which was used in next step without further purification. LRMS: m / z=680.70 [M+H] T Chemical Formula: C36H46FN5O7; Molecular Weight: 679.79.

[0209] Step 4. To a solution of crude compound 15 (30 mg, 0.044 mmol) in dichloromethane (2 mL) was added trifluoroacetic acid (TFA, 215 mg, 1.88 mmol), the resulting mixture was stirred at room temperature for 2 hrs. TLC showed starting material was consumed completely. The reaction mixture was concentrated in vacuo to give crude compound 16 (40 mg) as a yellow oil which was used in next step without further purification. LRMS: m / z=580.35 [M+H] T Chemical Formula: C31H38FN5O5; Molecular Weight: 579.67.

[0210] Step 5. To a solution of crude 16 (40 mg, 0.044 mmol) and DIPEA (17 mg, 0.132 mmol) in DMF (1.5 mL) was added 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1- yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl (4-nitrophenyl) carbonate (25 mg, 0.033 mmol). The resulting mixture was stirred at room temperature overnight. The reaction mixture was concentrated in vacuo at 28°C to remove DMF. The residue was dissolved in EtOAc (20 mL) and washed with water and brine. The aqueous layer was extracted with a mixture of MeOH in DCM (v / v=10%, 30 mLx5), the combined organic layers were dried over anhydrous NazSCL; and concentrated in vacuo. The residue was purified by prep-TLC (DCM / MeOH, 10 / 1) to give a crude material, which was further purified by prep-HPLC (eluent: ACN / H2O=5-80% for 25 mins) to afford the title compound 17 (1.6 mg, 4.1 % of yield) as a yellow solid. LRMS: m / z=1179.1 [M+Hj L Chemical Formula: CsoHyeFNnOis; Molecular Weight: 1178.33.Example 3. Synthesis of compound 21Compound 21

[0211] Step 1. A solution of compound 13 (189 mg, 0.35 mmol) in 2-methoxyethan-1- amine (2 mL) was stirred at 50GC for 0.5 hr. The reaction mixture was concentrated in vacuo to give the crude compound 18 (230 mg) as a yellow solid which was used in next step without further purification. LRMS: m / z=611.3 [M+H]+. Chemical Formula: C32H39FN4O7; Molecular Weight: 610.68.

[0212] Step 2. To a solution of compound 18 (230 mg, 0.17 mmol) in pyridine (1.5 mL) was added acetic anhydride (Ac2O, 45 mg, 0.44 mmol). The resulting mixture was stirred at room temperature overnight. The reaction mixture was concentrated at 28°C. The residue was diluted with DCM (30 mL), washed with water and brine, dried over anhydrous Na2SO4, and concentrated in vacuo to give a crude material which was purified through silica gel flash column chromatography (PE / EtOAc, from 1 / 1 to 1 / 3, then DCM / MeOH=50 / 1) to afford the title compound 19 (96.3 mg, 86 %) as a yellow solid. LRMS: m / z=653.35 [M+H]+, Chemical Formula: C34H41FN4O8; Molecular Weight: 652.72.

[0213] Step 3. To a solution of compound 19 (20 mg, 0.03 mmol) in DCM (2 mL) was added trifluoroacetic acid (TFA, 137 mg, 0.12 mmol), the resulting mixture was stirred at room temperature for 4 hrs. TLC showed starting material was consumed completely. The mixture was concentrated in vacuo to give the crude compound 20 (30 mg) as a yellow oil which was used in next step without further purification.Chemical Formula: C29H33FN4O6; Molecular Weight: 552.60.

[0214] Step 4. To a solution of compound 20 (27.5 mg. 0.05 mmol) and DIPEA (195 mg, 1.5 mmol) in DMF (2 mL) was added 4-((S)-2-((S)-2-(6"(2,5-dioxo-2!5“dihydrO"1H“pyrrol-1- yl)hexanamido)-3-methylbutanamido)-5-reidopentanamido)benzyl (4-nitrophenyl) carbonate (21 mg, 0.028 mmol). The resulting mixture was stirred at room temperature overnight. The reaction mixture was concentrated in vacuo to remove DMF (29°C). The crude was dissolved in semi-brine (20 mL) and extracted with a mixture of methanol in DCM (10%, 15 mLx6). The combined organic layer was dried over anhydrous NajSCh and concentrated in vacuo. The crude was purified byprep-TLC (DCM / MeOH™10 / 1) to give a product which was further purified by prep-HPLC (eluent: ACN / H2O=5-80% for 25 mins) to give the title compound 21 (4.0 mg, 11.5%) as a yellow solid. LRMS: m / z=1151.60 [M+H]+. Molecular Formula: CW-biFNwO-u; Molecular weight: 1151.24.Example 4, Synthesis of Compound 3022

[0215] Step 1. To a solution of benzyl 2-hydroxyacetate (1.1 g, 6.62 mmol) in DMF (15 mL) was added imidazole (1.35 g, 19.86 mmol) and ferf-butyldimethylsilyl chloride (TBSCI) (1.1 g, 7.28 mmol), then the resulting mixture was stirred at room temperature for 2 hrs. The reaction mixture was poured into water (110 mL) and extracted with ethyl acetate (30 mLx3). The combined organic layer was washed with saturated aqueous ammonium chloride solution, dried over anhydrous Na2SO4, and concentrated to give a crude which was purified through silica gel flash column chromatography (EtOAc / petroleum ether (PE)= 0% to 10%) to afford the compound 22 (1.75 g, 94%) as a colorless oil.1H NMR (400 MHz, CDCh): 67.37-7.31 (m, 5H), 5.18 (s, 2H), 4.29 (s, 2H), 0.91 (s, 9H), 0.096 (s, 6H) ppm.

[0216] Step 2. A mixture of compound 22 (1.75 g, 6.24 mmol) and palladium on carbon (10%. 525 mg) in methanol (30 mL) was stirred at room temperature under hydrogen atmosphere overnight. The catalyst was removed through filtration, and the filtrate was concentrated to give a crude which was purified through silica gel flash column chromatography (hexane / EtOAc = 3 / 1) to afford the title compound 23 (197 mg, 16.5%) as a colorless oil.1H NMR (400 MHz, CDCh): 58.77 (brs, 1 H), 4.23 (s, 2H), 0.93 (s, 9H), 0.14 (s, 6H) ppm.

[0217] Step 3. To a solution of exatecan mesylate (120 mg, 0.225 mmol) and compound 23 (128 mg, 0.675 mmol) in DMF (15 mL) was added DIPEA (175 mg, 1.35 mmol) and PyBOP (234 mg, 0.45 mmol). The resulting mixture was stirred at room temperature overnight. The reaction mixture was diluted with water (50 mL) and extracted with EtOAc (40 mL x 4). The combined organic layer was washed with brine, dried over anhydrous Na2SO4 and concentrated in vacuo to give the crude compound 24 (349 mg, impure) as a light-yellow solid. LRMS: m / z=608.20 [M+H] T Chemical Formula: C32H38FN3O6Si; Molecular Weight: 607.75.

[0218] Step 4. A solution of compound 24 (122 mg, 0.2 mmol) in 2-methoxyethan-1-amine (2 mL) was stirred at 50°C for 1 hr. The reaction mixture was concentrated in vacuo to give the crude compound 25 (145 mg) as a yellow oil which was used in next step without further purification. LRMS: m / z=683.30 [M+H] T Chemical Formula: Css^F^OySi; Molecular Weight: 682.87.

[0219] Step 5. To a solution of crude compound 25 (145 mg, 0.2 mmol) in dichloromethane (11 mL) was added 4-dimethylaminopyridine (DMAP, 156 mg, 1.27 mmol) and tert-butyl (2-((chlorocarbonyl)(methyi)amino)ethyl)(methyl)carbamate (213 mg, 0.85 mmol). The resulting mixture was stirred at room temperature overnight. The reaction mixture was concentrated in vacuo to give a crude residue which was purified through silica gel flash column chromatography (PE / EtOAc=1 / 1, then DCM / MeOH, from 50 / 1 to 30 / 1) to afford the compound 26 (60 mg, 31.5%) as a yellow solid. LRMS: m / z=897.45 [M+H]+. Chemical Formula: C45H55FNsOwSi; Molecular Weight: 897.13.

[0220] Step 6. To a solution of compound 26 (48 mg, 0.054 mmol) in DCM (4 mL) was added tetrabutylammonium fluoride (TBAF, 1M in tetrahydrofuran, 162 pL, 0.162 mmol) andacetic acid (13 mg, 0.216 mmol), the resulting mixture was stirred at room temperature for 4 hrs. TLC showed starting material was consumed completely The reaction mixture was used in next step without further working up. LRMS: (ES+): m / z=783.3 [M+H]+. Chemical Formula: C39H51FN6O10; Molecular Weight: 782.37.

[0221] Step 7. A solution of compound 28 in DCM (crude solution from previous step, 0.054mmol) was added trifluoroacetic acid (TFA, 0.1 mL), the resulting mixture was stirred at room temperature for 2 hrs. TLC showed starting material was consumed completely. The mixture was concentrated in vacuo to give the crude compound 29 as a yellow oil which was used in next step without further purification. LRMS: m / z=683.25 [M+H]+. Chemical Formula: CsdFkFNsOa; Molecular Weight: 682 75

[0222] Step 8. To a solution of crude compound 29 (200 mg) and DIPEA (156 mg, 1.21 mmol) in DMF (2 mL) was added 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1- yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl (4-nitrophenyl) carbonate (32 mg, 0.043 mmol). The resulting mixture was stirred at room temperature overnight. Thereaction mixture was concentrated in vacuo at 28°C to remove DMF to get crude, which was dissolved in DCM (20 mL), washed with water and brine, dried over anhydrous NazSO^ and concentrated. The crude residue was purified by prep-TLC (DCM / MeOH=10 / 1) to give the product with impurities which was further purified by prep-HPLC (eluent: ACN / H2O=:5-80% for 25 mins) to give the title compound 30 (3.1 mg, 5.6% of yield) as a yellow solid. LRMS: m / z=1281.65 [M+H] L Chemical Formula: CesHsiFN izOw; Molecular Weight: 1281.41.Example 5. Synthesis of compound 43

[0223] Step 1. To a solution of Fmoc-Ala-OH (3.11 g, 10 mmol) in DMF (15 mL) were added H-Gly-OtBu (1.31 g, 10 mmol), HATU (5.7 g, 15 mmol) and DIPEA (2.58 g, 20 mmol). The mixture was stirred for 2 hrs. The mixture was diluted with DCM (200 mL) and washed with NaHCOs solution (2x50 mL), water (20 mLx2) and brine. The organic layers were dried over anhydrous NazSCh, filtered, and evaporated to give a residue, which was purified by flash chromatography on silica gel (eluent: EtOAc / hexane = 10-50%) to give the compound 31 (3.1 g, 73%).

[0224] Step 2. To a solution of compound 31 (3.1 g, 7.3 mmol) in DCM (7.5 mL) was added TFA (7.5 mL). The mixture was stirred for 4 hrs. The mixture was evaporated to give an oil, which was precipitated with ether. The solid was filtered and washed with some ether. The white solid was further dried under high vacuum to give compound 32 (2.2g, 82%).

[0225] Step 3. To a solution of compound 32 (2.2 g, 6 mmol) in DMF (40 mL) were added CU(OAC)2 (0.40 g, 2.2 mmol), HOAc (0.77 mL, 13.6 mmol) and Pb(OAc)4 (3.0 g, 6.8 mmol). The resulting mixture was heated to 60°C for 15 mins, and then cooled to rt. The mixture was diluted with EtOAc (80 mL) and washed with water (3x 50mL) and brine. The mixture was dried over anhydrous Na2SCk filtered, and evaporated to give a residue, which was passed through a column (eluent EtOAc / n-Heptane ~ 10-75%) to provide the compound 33 (1.4 g, 61.3%). LRMS: m / z =383.1 [M-M]4-. Molecular Formula: C21H22N2O5; Molecular weight: 382.41.

[0226] Step 4. To a solution of compound 33 (1 g. 2.61 mmol) and benzyl glycolate (2.17 g, 13.1 mmol) in DCM (16 mL) was added 4 mL of TFA. The resulting mixture was stirred for 55 mins. The mixture was evaporated to give a residue, which was dissolved in DCM. The mixture was washed with saturated NaHCOs solution, water and brine. The mixture was dried over anhydrous Na2SO4, filtered, and evaporated to give a residue, which was purified by silica gel chromatography (eluent: EtOAc / n-Heptane ~ 30%-100%) to give the compound 34 (600 mg, 47%).

[0227] Step 5. To a solution of compound 34 (600 mg, 1.22 mmol) in 20 mL of DCM and 20 mL of ethanol was added Pd / C (10%, 60 mg). The mixture was stirred under a hydrogen atmosphere for 2 hrs. The mixture was filtered through celite, and the filtrate was evaporated to give the crude compound 35 (400mg, 81.7%).

[0228] Step 6. To a solution of compound 35 (50 mg, 0.125 mmol) and exatecan mesylate (50 mg, 0.094 mmol) in DMF (5 mL) were added HATU (54 mg, 0.141 mmol) and DIPEA (24 mg, 0.188 mmol). The mixture was stirred for 2 hrs. The mixture was diluted with DCM (60 mL) and washed with NaHCOs solution (2x30 mL), water (20 mLx2) and brine. The organic layers were dried over anhydrous Na2SO4, filtered, and evaporated to give a residue, which was purified by flash chromatography on silica gel (eluent: MeOH / DCM = 5-10%) to give the compound 36 (55mg, 72%).

[0229] Step 7. To a solution of compound 36 (55 mg. 0.067 mmol) in DMF (3 mL) was added piperidine (29 mg). The mixture was stirred for 1 hr and evaporated to give the crude title compound 37, which was used without purification in next step.38

[0230] Step 8. To a solution of 2-azidoacetic acid (505 mg, 5 mmol) and H-Val-O-t-Bu (1.04 g, 5 mmol) in DMF (10 mL) were added HATU (2.47 g, 6.5 mmol) and DIPEA (0.97 g, 7.5 mmol). The mixture was stirred for 2 hrs. The mixture was diluted with EtOAc (50 mL) and washed with water (20 mLx3), NaHCCb solution (2x30 mL), and brine. The organic layers were dried over anhydrous Na2SO4, filtered, and evaporated to give a residue, which was purified by flash chromatography on silica gel (eluent: EtOAc / hexane = 10-50%) to give the compound 38 (950mg, 74%).

[0231] Step 9. To a solution of compound 38 (0.95 g, 3.7 mmol) in 1,4-dioxane (8 mL) was added concentrated HCI (12N, 4 mL). The mixture was stirred for 3 hrs and evaporated to give the crude compound 39 (0.69 g) as a light-yellow oil, which was used without purification in next step.37 39 40

[0232] Step 10. To a solution of crude compound 39 (17.4 mg, 0.0871 mmol) and 37 (crude, 0.067 mmol) in DMF (2 mL) were added HATU (38.2 mg, 0.1 mmol) and DIPEA (17.3 mg, 0.134 mmol). The mixture was stirred for 2 hrs. The mixture was diluted with DCM (60 mL) and washed with water (20 mLx3), NaHCOs solution (2x30 mL), and brine. The organic layers were dried over anhydrous I^SO^ filtered, and evaporated to give a residue, which was purified by flash chromatography on silica gel (eluent: MeOH / DCM = 0-10%) to give the compound 40 (40 mg, 77%).

[0233] Step 11. A solution of compound 40 (40 mg, 0.051 mmol) in N1,N1-dimethylethane- 1 ,2-diamine (5 mL) was stirred at 55°C for 1 hr. The reaction mixture was concentrated in vacuo to give a yellow oil, which was dissolved in DCM (5 mL). To the mixture were added propionic anhydride (13.4 mg, 0.103 mmol) and DMAP (1.2 mg, 0.010 mmol). The mixture was stirred for 4 hrs, then diluted with DCM (40 mL) and washed with brine. The organic layer was dried over Na2SO4filtered, and evaporated to give a residue, which was purified by flash chromatography on silica gel (eluent: MeOH / DCM = 0-15%) to give compound 41 (20 mg, 42%).42

[0234] Step 12. To a solution of 3-maleimidopropionic acid (170 mg, 1 mmol) and propargyl-PEG2-amine (143 mg, 1 mmol) in DMF (5 mL) were added HATU (494 mg, 1.3 mmol) and DIPEA (194 mg, 1.5 mmol). The mixture was stirred for 30 mins. The mixture was diluted with EtOAc (50 mL) and washed with water (20 mLx3), NaHCCh solution (2x30 mL), and brine. The organic layers were dried over anhydrous Na2SO^, filtered, and evaporated to give a residue, which was purified by flash chromatography on silica gel (eluent EtOAc / hexane = 10-50%) to give compound 42 (200 mg, 68%).

[0235] Step 13. Compound 41 (20 mg, 0.021 mmol) and 42 (12.8 mg, 0.043 mmol) were dissolved in DMSO (1.4 mL) and water (0.34 mL). To this solution was added cuprous bromide (9.3 mg, 0.065 mmol) and acetic acid (1 pL). The reaction mixture was stirred for 1 hr, then diluted with DCM (50 mL) and separated. The organic layer was washed twice with half-saturated aqueous sodium chloride (10 mL) and dried over Na2SO4, filtered and evaporated under vacuumto give a residue, which was purified by preparative HPLC (eluent: ACN / H2O”5-80% for 25 mins) to give the title compound 43 (3 mg, 11%). LRMS: m / z =1214.5 [M+1]+. Molecular formula: CssHveFNoOis; Moiecular weight: 1214.39.Example 6. Synthesis of Compound 47 (36309)

[0236] Step 1. Compound 44 (150 mg, 0.39 mmol) and 4-aminobenzyl alcohol (68 mg, 0.55 mmol) were dissolved in anhydrous DCM (9 ml) and anhydrous IMeOH (3 ml). EEDQ (190 mg, 0.78 mmol) was added to the mixture, which was stirred overnight at room temperature. The reaction mixture was evaporated under vacuum to give a residue, which was purified by silica gel chromatography (eluent: MeOH / DCM=3-10%) to provide compound 45 (150 mg, 78.4%). LRMS: m / z =487.2 (M+H). Chemical Formula: C25H34N4O5; Molecular weight: 486.56.

[0237] Step 2. To a mixture of compound 45 (150 mg, 0.3 mmol) in 4 mL of DMF was added bis p-nitrophenyl carbonate (0.19 g, 0.6 mmol) and the reaction lasted for 16 h at room temperature, the reaction mixture was evaporated under vacuum to give a residue, which was purified by silica gel chromatography (eluent: MeOH / DCM=3-10%) to provide the title compound 46 (150 mg, 74.6%). LRMS: m / z = 652.2 (M+1); Chemical Formula: C32H37N5O10; Molecular weight: 651.66.3 47 (36309)

[0238] Step 3. To a solution of compound 3 (20 mg, 0.039 mmol) and compound 46 (51 mg, 0.079 mmol) in DMF (4 mL) were added HOAt (1 mg) and DIPEA (15.3 mg, 0.118 mmol). The resulting mixture was stirred for 4 hrs at room temperature. The reaction mixture was purified by prep-HPLC (eluent: ACN / H2O:=25-80% for 25 mins) to give the title compound 47 (36309) (15 mg, 38% of yield) as a yellow solid. LRMS: m / z=1019.4 [M+H]+. Chemical Formula: C53H59FN8O12; Molecular Weight: 1019.08.Example 7. Synthesis of Compound 53Compound 53

[0239] Step 1. To a solution of exatecan mesylate (200 mg, 0.38 mmol) and Fmoc-Aib- OH (250 mg, 0.75 mmol) in DMF (8 mL) were added HATU (214 mg, 0.56 mmol) and DIPEA (164 pL, 0.94 mmol). The mixture was stirred for 1 hr. The mixture was diluted with DCM (60 mL) and washed with water (20 mLx3), NaHCCh solution (2x30 mL), and brine. The organic layers were dried over anhydrous NazSCu, filtered, and evaporated to give the crude compound 48, which was used directly in next step.

[0240] Step 2. To a solution of crude compound 48 (0,38 mmol) in DMF (5 mL) was added piperidine (1 mL). The mixture was stirred for 1 hr and evaporated to give the crude titie compound 49, which was purified by pre-HPLC (eluent: ACN / H2O=25-80% for 25 mins) to give the title compound 49 (120 mg, 62% of yield) as a yellow solid. LRMS: m / z=:521.2 [M+H] +. Chemical Formula: C28H29FN4O5; Molecular Weight: 520.55.49 5051

[0241] Step 3. To a solution of 49 (120 mg, 0.23 mmol) and 50 (121 mg, 0.28 mmol) in DMF (10 mL) were added HATU (132 mg, 0.35 mmol) and DIPEA (100 pL, 0.58 mmol). The mixture was stirred for 1 hr. The mixture was purified by pre-HPLC (eluent: ACN / H2O=25-80% for 25 mins) to give the title compound 51 (150 mg, 69% of yield) as a yellow solid. LRMS: m / z=939.3 [M+H] +. Chemical Formula: CdsHssFNsOn; Molecular Weight: 939.051 52

[0242] Step 4. To a solution of compound 51 (150mg, 0.16mmol) in DCM (1mL) was added TEA (1mL). The mixture was stirred for 15mins, then evaporated to give a residue, which was dried under high vacuum to give the crude 52. LRMS: m / z=839.3 [M+Hf. Chemical Formula: C43H47FN8O9; Molecular Weight: 838,88

[0243] Step 5. To a solution of crude 52 (20mg, 0.024mmol) in DMF (5mL) were added Mal-PEG2-NHS (12mg, 0.029mmol) and DIEA (10pL, 0.06mmol). The mixture was stirred for 1 hr. The mixture was purified by pre-HPLC (eluent: ACN / H2O=25-80% for 25 mins) to give the title compound 53 (20mg, 74% of yield) as a solid. LRMS: m / z=1049.4 [M+H]+. Chemical Formula: C57H55FN1DO15; Molecular Weight: 1049.18.Example 8. Synthesis of Compound 54

[0244] Step 1. To a solution of 52 (20 mg, 0.024 mmol) and Mal-PEG4-acid (15 mg, 0.036 mmol) in DMF (10 mL) were added HATU (14 mg, 0.036 mmol) and DIPEA (11 pL, 0.063 mmol).The mixture was stirred for 1 hr. The mixture was purified by pre-HPLC (eluent: ACN / H2O™25-80% for 25 mins) to give the title compound 54 (15 mg, 51 % of yield) as a solid. LRMS: m / z=1237.3 [M+H]+. Chemical Formula: C61H73FN10O17; Molecular Weight: 1237.29.Example 9, Synthesis of Compound 5552 55

[0245] Step 1. To a solution of 52 (20 mg, 0.024 mmol) and Mal-PEG8-acid (17 mg, 0.029 mmol) in DMF (4 mL) were added HATU (14 mg, 0.036 mmol) and DIPEA (11 pL, 0.063 mmol). The mixture was stirred for 1 hr. The mixture was purified by pre-HPLC (eluent: ACN / H2O~25-80% for 25 mins) to give the title compound 55 (20 mg, 61 % of yield) as a solid. LRMS: m / z=1413.5 [M+H]+- Chemical Formula: C69H89FN 10021 ; Molecular Weight: 1413.50.Example 10. Synthesis of Compound 58

[0246] Step 1. To a solution of 11 (400 mg, 0.85 mmol) and L-alanine terf-butyl ester (111 mg, 0,85 mmol) in DMF (10 mL) were added HATU (38 mg, 1.0 mmol) and DIPEA (350 pL, 2.1 mmol). The mixture was stirred for 1 hr. The mixture was diluted with ethyl acetate. The mixture was washed with saturated NaHCOs solution and brine. The organic layer was dried over anhydrous Na2SO4, filtered and evaporated to give the crude compound 56.

[0247] Step 2. To a solution of 56 (0.496 g, 0.85 mmol) in DCM (10mL) was added TFA (10 mL). The mixture was stirred for 2 hrs. The volatiles were removed under vacuum and the resulting residue was dissolved in 1 mL of DCM. Ether was added to precipitate the product. The solid was filtered and washed with ether and dried under high vacuum to give compound S7 (0.30 g, 67%) as a white solid. LRMS: m / z~530.2 [M+1]+. Molecular Formula: C25H31N5O8; Molecular Weight: 529.54.58

[0248] Step 3. To a solution of 52 (20 mg, 0.038 mmol) and 57 (40 mg, 0.077 mmol) in DMF (4 mL) were added HATU (22 mg, 0.057 mmol) and DIPEA (20 pL, 0.096 mmol). The mixture was stirred for 1 hr. The mixture was purified by pre-HPLC (eluent: ACN / H2O=25-80% for25 mins) to give the title compound 58 (3 mg, 7.7% of yield) as a solid. LRMS: m / z=1032.3Chemical Formula: C53H58FN9O12; Molecular Weight: 1032.08.Example 11. Synthesis of Compound 80Compound 6060

[0249] Step 1. To a solution of 52 (20 mg, 0.024 mmol) and 57 (9.6 mg, 0.036 mmol) in DMF (4 mL) were added PyBOP (62 mg, 0.117 mmol) and DIPEA (12 pL, 0.069 mmol). The mixture was stirred for 4 hrs. The mixture was purified by pre-HPLC (eluent: ACN / H2O=25-80% for 25 mins) to give the title compound 60 (16 mg, 62% of yield) as a solid. LRMS: m / z- 1089.3 [M+H]+. Chemical Formula: C54H57FN10O12F; Molecular Weight: 1089.16.Example 12. Synthesis of Compound 85Compound 65

[0250] Step 1. To a solution of exatecan mesylate (200 mg, 0.38 mmol) and 1-((((9H- Fluoren-9-yl)methoxy)carbonyl)(methyl)amino)cyclopropanecarboxylic acid (250 mg, 0.75 mmol) in DMF (8 mL) were added HATU (214 mg, 0.56 mmol) and DIPEA (164 pL, 0.94 mmol). The mixture was stirred for 1 hr. The mixture was diluted with DCM (60 mL) and washed with water (20 mLx3), NaHCCh solution (2x30 mL), and brine. The organic layers were dried over anhydrous Na2SO4, filtered, and evaporated to give the crude compound 61 which was used directly in next step.61 62

[0251] Step 2. To a solution of crude compound 61 (0.38 mmol) in DMF (5 mL) was added piperidine (1 mL). The mixture was stirred for 1 hr and evaporated to give the crude title compound 62, which was purified by pre-HPLC (eluent: ACN / H2O~25-80% for 25 mins) to give the title compound 62 (160 mg, 80% of yield) as a solid. LRMS: m / z=533.2 [M+H]+. Chemical Formula: C29H29FN4O5; Molecular Weight: 532.56.

[0252] Step 3. To a solution of 62 (80 mg, 0.15 mmol) in THF (20 mL) were added DIPEA (158 pL, 0.90 mmol) and 2-(Fmoc-amino)acetyl Chloride (189 mg, 0.60 mmol). The mixture was heated to 50°C and stirred for 3 hrs. The mixture was purified by pre-HPLC (eluent: ACN / H2O=25- 80% for 25 mins) to give the title compound 63 (50 mg, 41% of yield) as a solid. LRMS: m / z=812.2 [M+H]+. Chemical Formula: C46H42FN5O8; Molecular Weight: 811.85

[0253] Step 4. To a solution of compound 63 (50mg, 0.06 mmol) in DMF (5 mL) was added piperidine (1mL). The mixture was stirred for 1 hr and evaporated to give the crude title compound 64, which was purified by pre-HPLC (eluent: ACN / H2O=25-80% for 25 mins) to give the title compound 64 (20 mg, 55% of yield) as a solid. LRMS: m / z=590.3 [M+H]+. Chemical Formula: C31H32FNSO6; Molecular Weight: 589.6164 65

[0254] Step 5. To a solution of 64 (20 mg, 0.034 mmol) and 11 (24 mg, 0.05 mmol) in DMF (4 mL) were added HATU (22 mg, 0.05 mmol) and DI PEA (15 pL, 0.085 mmol). The mixture was stirred for 1 hr. The mixture was purified by pre-HPLC (eluent: ACN / H2O=25-80% for25 mins) to give the title compound 65 (7 mg, 20% of yield) as a solid. LRMS: m / z=1044.3 [M+Hj+. Chemical Formula: C54H58FN9O12; Molecular Weight: 1044.09.Example 13. Synthesis of Compound 68

[0255] Step 1. To a solution of 62 (80 mg, 0.15 mmol) and 50 (196 mg, 0.46 mmol) in THF (20 mL) were added HATU (170 mg, 0,46 mmol) and DIPEA (262 pL, 1.50 mmol). The mixture was heated to 50°C and stirred for 3 hrs. The mixture was purified by pre-HPLC (eluent: ACN / H2O:=25-80% for 25 mins) to give the title compound 63 (50 mg, 41% of yield) as a solid. LRMS: m / z=951.3 [M+H]+. Chemical Formula: C49H55FN8O11; Molecular Weight: 951.00

[0256] Step 2. To a solution of compound 66 (60mg, 0.063mmol) in DCM (5mL) was added TFA (5mL). The mixture was stirred for 15mins, then evaporated to give a residue, which was dried under high vacuum to give the crude 67. LRMS: m / z=851.3 [M+Hp. Chemical Formula: C44H47FNSO9; Molecular Weight: 850.8967 68

[0257] Step 3. To a solution of crude 67 (20mg, 0.024mmol) in DMF (5mL) were added Mal-PEG2-NHS (16mg. 0.047mmol) and DIEA (10pL, 0.06mmo!). The mixture was stirred for 1 hr. The mixture was purified by pre-HPLC (eluent: ACN / H2O=25-80% for 25 mins) to give the title compound 69 (10mg, 37% of yield) as a solid. LRMS: m / z=1161.4 [M+H]+. Chemical Formula: C5sH55FN10Oi5; Molecular Weight: 1161.19.Example 14. Synthesis of Compound 70Compound 70

[0258] Step 3. To a solution of crude 67 (20mg, 0.024mmol) in DMF (5mL) were added Mal-PEG4-acid (20mg, 0.047mmol), HATU (14 mg, 0.036 mmol) and DI PEA (10 pL, 0.059 mmol). The mixture was stirred for 1 hr. The mixture was purified by pre-HPLC (eluent: ACN / H2O:::25-80% for 25 mins) to give the title compound 70 (9mg, 31 % of yield) as a solid. LRMS: m / z=1249.4 [M+H]+- Chemical Formula: C82H73FN10O17; Molecular Weight: 1249.30.Exampie 15. Synthesis of Compound 73

[0259] Step 1. To a solution of exatecan mesylate (200 mg, 0.38 mmol) and Fmoc-Aia- OH (245 mg, 0.75 mmol) in DMF (8 mL) were added HATU (172 mg, 0.45 mmol) and DIPEA (155 pL, 0.94 mmol). The mixture was stirred for 1 hr. The mixture was diluted with DCM (60 mL) and washed with water (20 mLx3), NaHCOs solution (2x30 mL), and brine. The organic layers weredried over anhydrous Na2SO4. filtered, and evaporated to give a residue, which was purified by flash chromatography to give compound 71 (150mg, 54% of yield).

[0260] Step 2. To a solution of compound 71 (1b0mg, 0.20 mmol) in DMF (5 mL) was added piperidine (1mL). The mixture was stirred for 1 hr and evaporated to give the crude title compound 72, which was purified by pre-HPLC (eluent: ACN / H2O=25-80% for 25 mins) to give the title compound 72 (100 mg, 95% of yield) as a solid. LRMS: m / z=521.2 [MH- HP Chemical Formula: C28H2gFN40s; Molecular Weight: 520.55.72 73

[0261] Step 3. To a solution of 72 (30 mg, 0.057 mmol) and 57 (61 mg, 0.115 mmol) in DMF (4 mL) were added HATU (33 mg, 0.086 mmol) and DIPEA (25 pL, 0.144 mmol). The mixture was stirred for 1 hr. The mixture was purified by pre-HPLC (eluent: ACN / H2O=25-80% for25 mins) to give the title compound 73 (15 mg, 25.4% of yield) as a solid. LRMS: m / z=1032.4 [M+H]+. Chemical Formula: C53H53FN9O12; Molecular Weight: 1032.08.Example 16. Synthesis of Compound 74Compound 74

[0262] Compound 74 was synthesized following the same protocol as compound 73.LRMS: m / z=1032.4 [M+H]+. Chemical Formula: C53H58FN9O12; Molecular Weight: 1032.08.Example 17. Synthesis of Compound 79

[0263] Step 1. To a solution of exatecan mesylate (200 mg, 0.38 mmol) and 2-{[(9H- fluoren-9-ylmethoxy)carbonyl](methyl)amino}-2-methy!propano!C acid (250 mg, 0.75 mmol) in DMF (8 mL) were added HATU (172 mg, 0.45 mmol) and DI PEA (160 pL, 0.94 mmol). The mixture was stirred for 1 hr. The mixture was diluted with DCM (60 mL) and washed with water (20 mLx3),NaHCOs solution (2x30 mL), and brine. The organic layers were dried over anhydrous Na2SO4, filtered, and evaporated to give the crude compound 75, which was used directly in next step.

[0264] Step 2. To a solution of crude compound 75 (0.38 mmol) in DMF (5 mL) was added piperidine (1mL). The mixture was stirred for 1 hr and evaporated to give the crude title compound 76, which was purified by pre-HPLC (eluent: ACN / H2O25~80% for 25 mins) to give the title compound 76 (200 mg, 99% of yield) as a solid. LRMS: m / z~534.2 [M+H]f. Chemical Formula: C29H31FN4O5; Molecular Weight: 534.58.

[0265] Step 3. To a solution of 76 ( 100 mg, 0.18 mmol) in TH F (20 m L) were added DI PEA (158 pL, 0.90 mmol) and 2-(Fmoc-amino)acetyl Chloride (236 mg, 0.75 mmol). The mixture was heated to 50°C and stirred for 3 hrs. The mixture was purified by pre-HPLC (eluent: ACN / H2O25- 80% for 25 mins) to give the title compound 77 (50 mg, 30% of yield) as a solid. LRMS: m / z=814.3Chemical Formula: C^FNsOa; Molecular Weight: 813.8777 78

[0266] Step 4. To a solution of compound 77 (50mg, 0.06 mmol) in DMF (5 mL) was added piperidine (1mL). The mixture was stirred for 1 hr and evaporated to give the crude title compound 78, which was purified by pre-HPLC (eluent: ACN / H2O:=25-80% for 25 mins) to give the title compound 77 (20 mg, 55% of yield) as a solid. LRMS: m / z=592.3 [M+Hf. Chemical Formula: C3IH34FN5O6; Molecular Weight: 591.63

[0267] Step 5. To a solution of 78 (20 mg, 0.034 mmol) and 11 (32 mg, 0.068 mmol) in DMF (4 mL) were added HATU (20 mg, 0.051 mmol) and DI PEA (15 pL, 0.085 mmol). The mixture was stirred for 1 hr. The mixture was purified by pre-HPLC (eluent: ACN / H2O=:25-80% for25 mins) to give the title compound 79 (3 mg, 8.5% of yield) as a solid. LRMS: m / z=1046.4Chemical Formula: C54H60FN9O12; Molecular Weight: 1046.10.Example 18. Synthesis of Compound 84Compound 84

[0268] Step 1. To a solution of exatecan mesylate (200 mg, 0.38 mmol) and 1-(9H-fluoren-9-ylmethoxycarbonylamino)cyclopropanecarboxylic acid (183 mg, 0.56 mmol) in DMF (10 mL) were added HATU (214 mg, 0.56 mmol) and DIPEA (164 pL, 0.94 mmol). The mixture was stirred for 1 hr. The mixture was diluted with DCM (60 mL) and washed with water (20 ml_x3), NaHCOs solution (2x30 mL), and brine. The organic layers were dried over anhydrous I^SO^ filtered, and evaporated to give the crude compound 80, which was used directly in the next step.80 81

[0269] Step 2. To a solution of crude compound 80 (0.38 mmol) in DMF (5 mL) was added piperidine (1 mL). The mixture was stirred for 1 hr and evaporated to give the crude title compound 81 , which was purified by pre-HPLC (eluent: ACN / H2O=25-80% for 25 mins) to give the title compound 81 (180 mg, 92% of yield) as a yellow solid. LRMS: m / z=519.2 [M+H] +. Chemical Formula: C28H27N4O5,; Molecular Weight: 518.54.82

[0270] Step 3. To a solution of 81 (52 mg, 0.10 mmol) and 50 (52 mg, 0.12 mmol) in DMF (5 mL) were added HATU (57 mg, 0.15 mmol) and DI PEA (44 pL, 0.25 mmol). The mixture was stirred for 1 hr. The mixture was purified by pre-HPLC (eluent: ACN / H2O=25-80% for 25 mins) to give the title compound 82 (70 mg, 75% of yield) as a solid. LRMS: m / z=:937.3 [M+H]+. Chemical Formula: C48H53FN8O11; Molecular Weight: 936.98

[0271] Step 4. To a solution of compound 82 (70mg, 0.075mmol) in DCM (9mL) was added TFA (3mL). The mixture was stirred for 15mins, then evaporated to give a residue, which was dried under high vacuum to give the crude 83.Chemical Formula: C43H4SFNSO9: Molecular Weight: 836.86

[0272] Step 5. To a solution of crude 83 (15mg. 0.018mmol) in DMF (2mL) were added Mal-PEG2-NHS (12mg, 0.029mmol) and DIEA (8pL, 0.045mmol). The mixture was stirred for 1 hr. The mixture was purified by pre-HPLC (eluent: ACN / H2O=25-80% for 25 mins) to give the title compound 84 (15mg, 73% of yield) as a solid. LRMS: m / z=1047.4 [M+H]+. Chemical Formula: C57HS3FN10O15; Molecular Weight: 1047.16Exampie 19. Synthesis of Compound 89

[0273] Step 1. To a solution of exatecan mesylate (200 mg, 0.38 mmol) and Fmoc-D-Ala- OH (185 mg, 0.56 mmol) in DMF (10 mL) were added HATU (214 mg, 0.56 mmol) and DIPEA (164 pL, 0.94 mmol). The mixture was stirred for 1 hr. The mixture was diluted with DCM (60 mL) and washed with water (20 mLx3), NaHCOs solution (2x30 mL), and brine. The organic layerswere dried over anhydrous Na2SO4, filtered, and evaporated to give the crude compound 85, which was used directly in the next step.

[0274] Step 2. To a solution of crude compound 85 (0.38 mmol) in DMF (5 mL) was added piperidine (0.5mL). The mixture was stirred for 20 mins and evaporated to give the crude title compound 86, which was purified by pre-HPLC (eluent: ACN / H2O=25-80% for 25 mins) to give the title compound 86 (180 mg, 92% of yield) as a yellow solid. LRMS: m / z=519.2 [M+H] +. Chemical Formula: C27H27FN4O5; Molecular Weight: 518.54.

[0275] Step 3. To a solution of 86 (51 mg, 0.10 mmol) and 50 (52 mg, 0.12 mmol) in DMF (5 mL) were added HATU (57 mg, 0.15 mmol) and DI PEA (44 pL, 0.25 mmol). The mixture was stirred for 1 hr. The mixture was purified by pre-HPLC (eluent: ACN.4H2(>:25"80% for 25 mins) togive the title compound 87 (70 mg, 75% of yield) as a solid. LRMS: m / z™925.3 [M+H]7 ChemicalFormula: C47H53FN8O11; Molecular Weight: 924.97

[0276] Step 4. To a solution of compound 87 (70mg, 0.075mmol) in DCM (9mL) was added TFA (3mL). The mixture was stirred for 15mins, then evaporated to give a residue, which was dried under high vacuum to give the crude 88. LRMS: m / z=825.3 [M+Hp. Chemical Formula: C42H45FN8O9; Molecular Weight: 824.85.

[0277] Step 5. To a solution of crude 88 (15mg, 0.018mmol) in DMF (2mL) were added Mal-PEG2-NHS (12mg, 0.028mmol) and DIEA (8pL, 0.045mmol). The mixture was stirred for 1 hr. The mixture was purified by pre-HPLC (eluent: ACN / H2O=;25-80% for 25 mins) to give the title compound 89 (12mg, 58% of yield) as a solid. LRMS: m / z=1135.4 [M+H]+. Chemical Formula: C56H63FN10O15; Molecular Weight: 1135.15Example 20. Synthesis of Compound 9490

[0278] Step 1. To a solution of exatecan mesylate (150 mg, 0.28 mmol) and (R)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-2-cyclopropylacetic acid (100 mg, 0.28 mmol) in DMF (10 mL) were added HATU (161 mg, 0.42 mmol) and DIPEA (120 pL, 0.70 mmol). The mixture was stirred for 1 hr. The mixture was diluted with DCM (60 mL) and washed with water (20 mLx3), NaHCCh solution (2x30 mL), and brine. The organic layers were dried over anhydrous Na2SO-4, filtered, and evaporated to give the crude compound 90, which was used directly in the next step.

[0279] Step 2. To a solution of crude compound 90 (0.28 mmol) in DMF (5 mL) was added piperidine (0.5mL). The mixture was stirred for 20 mins and evaporated to give the crude title compound 91, which was purified by pre-HPLC (eluent: ACN / H2O=:25-80% for 25 mins) to givethe title compound 91 (130 mg, 87% of yield) as a yellow solid. LRMS: m / z~533.2Chemical Formula: C29H29FN4O5; Molecular Weight: 532.57.

[0280] Step 3. To a solution of 91 (53 mg, 0.10 mmol) and 50 (52 mg, 0.12 mmol) in DMF(5 mL) were added HATU (57 mg, 0.15 mmol) and DI PEA (44 pL, 0.25 mmol). The mixture was stirred for 1 hr. The mixture was purified by pre-HPLC (eluent: ACN / H2O=25-80% for 25 mins) to give the title compound 92 (70 mg, 74% of yield) as a solid. LRMS: m / z=951.3 [M+H]\ Chemical Formula: C49H55FN80II; Molecular Weight: 951.00

[0281] Step 4. To a solution of compound 92 (70mg, 0.075mmol) in DCM (9mL) was added TFA (3mL). The mixture was stirred for 10mins, then evaporated to give a residue, whichwas dried under high vacuum to give the crude 93. LRMS: m / z-851.3 [M+Hp . Chemical Formula: C44H47FN8O3; Molecular Weight: 850.89.93 94

[0282] Step 5. To a solution of crude 93 (15mg, 0.018mmol) in DMF (2mL) were added Mal-PEG2-NHS (12mg, 0.028mmol) and DIEA (8pL, 0.045mmo!). The mixture was stirred for 1 hr. The mixture was purified by pre-HPLC (eluent: ACN / H2O=25-80% for 25 mins) to give the title compound 94 (13mg, 64% of yield) as a solid. LRMS: m / z=1161.4 [M+H]+. Chemical Formula: C58H65FN10O15; Molecular Weight 1161.19.Example 21. Synthesis of Compound 95Compound 95

[0283] Compound 95 was synthesized from (S)-2-((((9H-Fluoren-9- yl)methoxy)carbonyl)amino)-2-cyclopropylacetic acid foilowing the same procedure as compound 94. LRMS: m / z=1161.4 [M+H]+. Chemical Formula: CSBHBSFNIOOIS; Molecular Weight: 1161.19.Example 22. Synthesis of Compound 9752 96

[0284] Step 1. To a solution of 52 (50 mg, 0.060 mmol) and Boc-N-amido-PEG4-acid (28 mg, 0.089 mmol) in DMF (6 mL) were added HATU (34 mg, 0.089 mmol) and DIPEA (26 pL, 0.15 mmol). The mixture was stirred for 1 hr. The mixture was purified by pre-HPLC (eluent: ACN / H2O=25-80% for 25 mins) to give the title compound 96 (50 mg, 76% of yield) as a solid.Chemical Formula: C55H38FN9O14; Molecular Weight: 1098.1896 97

[0285] Step 2. To a solution of compound 96 (50mg, 0.046mmol) in DCM (9mL) was added TFA (3mL). The mixture was stirred for 10mins, then evaporated to give a residue, which was dried under high vacuum to give the crude amine. To a solution of the crude amine (0.046 mmol) and 57 (18.3 mg, 0.068 mmol) in DMF (10 mL) were added PyBOP (118 mg, 0.228 mmol) and DIPEA (24 pL, 0.137 mmol). The mixture was stirred for 3 hrs. The mixture was was purified by pre-HPLC (eluent: ACN / H2O~25-80% for 25 mins) to give the title compound 97 (35 mg, 62% of yield) as a solid. LRMS: m / z" 1249.4 [M+H]+. Chemical Formula: C61iH70FN11O15; Molecular Weight: 1249.34.Exampie 23. Synthesis of Compound 10098

[0286] To a solution of 3 (100 mg, 0.20 mmol) and 50 (100 mg, 0.24 mmol) in DMF (5 mL) were added HATU (98 mg, 0.26 mmol) and DIPEA (86 pL, 0.49 mmol). The mixture was stirred for 1 hr. The mixture was purified by pre-HPLC (eluent: AC N / H 20=25-80% for 25 mins) to give the title compound 98 (130 mg, 71% of yield) as a solid. LRMS: m / z=925.3 [M+H]+. Chemical Formula: C49H55FN8O11; Molecular Weight: 925.00

[0287] Step 4. To a solution of compound 98 (130mg, 0.14mmol) in DCM (9mL) was added TFA (3mL). The mixture was stirred for 10mins, then evaporated to give a residue, which was dried under high vacuum to give the crude 99. LRMS: m / z;=825.3 [M+Hp. Chemical Formula: C42H45FN8O9: Molecular Weight: 824.85.99 100

[0288] Step 5. To a solution of crude 99 (65mg, 0.079mmol) in DMF (2mL) were added Mal-PEG2-acid (31mg, 0.094mmol), HATU (39mg, 0.102 mmol) and DIEA (34pL, 0.20 mmol). The mixture was stirred for 1 hr. The mixture was purified by pre-HPLC (eluent: ACN / H2O=:25-80% for 25 mins) to give the title compound 100 (52mg, 58% of yield) as a solid. LRMS: m / z=1135.4 [M+H]+. Chemical Formula: CssHesFNioOis; Molecular Weight: 1135.15.Example 24. Synthesis of Compound 101Compound 101

[0289] Step 5. To a solution of crude 100 (30mg, 0.036mmol) in DMF (2mL) were added Mal-PEG8-acid (32mg, 0.055mmol), HATU (21mg, 0.055 mmol) and DIEA (15pL, 0.09 mmol). The mixture was stirred for 1 hr. The mixture was purified by pre-HPLC (eluent: ACN / H2O:=25-80% for 25 mins) to give the title compound 101 (20mg, 40% of yield) as a solid. LRMS: m / z=1399.5 [M+H]4. Chemical Formula: C68H87FN1O21; Molecular Weight: 1399.47.Example 25. Synthesis of compound 102114Step 1. To a solution of exatecan mesylate (100 mg, 0.19 mmol) and cis-3-(Boc- amino)cyclobutanecarboxylic acid ( (49 mg, 0.23 mmol) in DMF (10 mL) were added HATU (107 mg, 0.28 mmol) and DIPEA (82 pL, 0.47 mmol). The mixture was purified by pre-HPLC (eluent: ACN / H2OA25"80% for 25 mins) to give the title compound 114 (85mg, 71% of yield). LRMS: m / z= 633.2 [M+H]+. Chemical Formula: C34H37FN4O7; Molecular Weight: 632.68.114 102

[0290] Step 2. To a solution of compound 114 (85mg, 0.134 mmol) in DCM (6 mL) was added TFA (3 mL). The mixture was stirred for 30 mins and evaporated to give the title compound 102 (60 mg, 84% of yield). LRMS: m / z=533.2 [M+H]+. Chemical Formula: C29H29FN4O5; Molecular Weight: 532.56.Example 26. Synthesis of compound 103Compound 103

[0291] Compound 103 was synthesized from cis-4-(Boc-amino)cyclohexanecarboxylic acid following the same procedure as compound 102. LRMS: m / z=561.2 [M+H]4. Chemical Formula: C31H33FN4O5; Molecular Weight: 560.61.Example 27. Synthesis of compound 107

[0292] Step 1 To a solution of exatecan mesylate (200 mg, 0.38 mmol) and Fmoc-D-Ala- OH (245 mg, 0.75 mmol) in DMF (8 mL) were added HATU (172 mg, 0.45 mmol) and DIPEA (155 pL, 0.94 mmol). The mixture was stirred for 1 hr. The mixture was diluted with DCM (60 ml) and washed with water (20 mLx3), NaHCOs solution (2x30 mL), and brine. The organic layers were dried over anhydrous NaaSCX filtered, and evaporated to give crude compound 104.

[0293] Step 2. To a solution of compound 104 (0.38 mmol) in DMF (5 mL) was added piperidine (1 mL). The mixture was stirred for 1 hr and evaporated to give the crude title compound 105, which was purified by pre-HPLC (eluent: ACN / H2O=25-80% for 25 mins) to give the title compound 105 (100 mg, 77% of yield) as a solid. LRMS: m / z=521.2 [M+H]+. Chemical Formula: C28H29FN4O5: Molecular Weight: 520.55.

[0294] Step 3. To a solution of compound 105 (120 mg, 0.23 mmol) and (R)-2-((((9H- Fluoren-9-yl)methoxy)carbonyl)amino)-2-cyclopropylacetic acid (93 mg, 0.28 mmol) in DMF (10 mL) were added HATU (131 mg, 0.35 mmol) and DIPEA (100 pL, 0.58 mmol). The mixture was stirred for 1 hr. The mixture was diluted with DCM (60 ml) and washed with water (20 mLx3), NaHCOs solution (2x30 mL), and brine. The organic layers were dried over anhydrous Na2SO4, filtered, and evaporated to give the crude compound 106, which was used directly in the next step.106 107

[0295] Step 4. To a solution of crude compound 106 (0.23 mmol) in DMF (5 mL) was added piperidine (0.5mL). The mixture was stirred for 20 mins and evaporated to give the crude title compound 107, which was purified by pre-HPLC (eluent: ACN / H2O“25-80% for 25 mins) to give the title compound 107 (120 mg, 85% of yield) as a yellow solid. LRMS: m / z~618.2 [M+H]+. Chemical Formula: C33H36FN5O6; Molecular Weight: 617.67.Example 28. Synthesis of compound 1093 108

[0296] Step 1. To a solution of compound 3 (120 mg, 0.23 mmol) and (R)-2-((((9H- Fluoren-9-yl)methoxy)carbonyl)amino)-2-cyclopropylacetic acid (93 mg, 0.28 mmol) in DMF (10 mL) were added HATU (135 mg, 0.35 mmol) and DIPEA (100 pL, 0.58 mmol). The mixture was stirred for 1 hr. The mixture was diluted with DCM (60 mL) and washed with water (20 mLx3), NaHCOs solution (2x30 mL), and brine. The organic layers were dried over anhydrous NajSCU, filtered, and evaporated to give the crude compound 108, which was used directly in the next step.108 109

[0297] Step 2. To a solution of crude compound 108 (0.23 mmol) in DMF (5 mL) was added piperidine (0.5mL). The mixture was stirred for 20 mins and evaporated to give the crude title compound 109, which was purified by pre-HPLC (eluent: ACN / H2O~25-80% for 25 mins) togive the title compound 109 (100 mg, 70% of yield) as a yellow solid.Chemical Formula: C32H34FN5O8; Molecular Weight: 603.64.Example 29. Synthesis of compound 111

[0298] Step 1. To a solution of exatecan mesylate (200 mg, 0.38 mmol) and (R)-2-((((9H- Fluoren-9-yl)methoxy)carbonyl) (methyl)amino)-2-cyclopropylacetic acid (159 mg, 0.45 mmol) in DMF (8 mL) were added HATU (215 mg, 0.56 mmol) and DIPEA (155 pL, 0.94 mmol). The mixture was stirred for 1 hr. The mixture was diluted with DCM (60 mL) and washed with water (20 mLx3), NaHCOa solution (2x30 mL), and brine. The organic layers were dried over anhydrous NazSCu, filtered, and evaporated to give the crude compound 110, which was used directly in the next step.110 111

[0299] Step 2. To a solution of compound 110 (0.38 mmol) in DIViF (5 mL) was added piperidine (1 mL). The mixture was stirred for 1 hr and evaporated to give the crude title compound 111 , which was purified by pre-HPLC (eluent: ACN / H2O=25-80% for 25 mins) to give the title compound 111 (150 mg, 73% of yield) as a solid. LRMS: m / z = 547.2 [M+H]+. Chemical Formula: C30H31FN4O5; Molecular Weight: 546.59.Example 30. Synthesis of compound 113

[0300] Step 1. To a solution of exatecan mesylate (200 mg, 0.38 mmol) and (R)-2-((((9H- Fluoren-9-yl)methoxy)carbonyl) (methyl)amino)-2-cyclopropylacetic acid (160 mg, 0.45 mmol) in DMF (8 mL) were added HATU (215 mg, 0.56 mmol) and DI PEA (155 pL, 0.94 mmol). The mixture was stirred for 1 hr. The mixture was diluted with DCM (60 mL) and washed with water (20 mLx3), NaHCCh solution (2x30 mL), and brine. The organic layers were dried over anhydrous hfeSCX filtered, and evaporated to give the crude compound 112, which was used directly in the next step.112 113

[0301] Step 2. To a solution of compound 112 (0.38 mmol) in DMF (5 mL) was added piperidine (1mL). The mixture was stirred for 1 hr and evaporated to give the crude title compound 113, which was purified by pre-HPLC (eluent: ACN / H2O=:25-80% for 25 mins) to give the title compound 113 (140 mg, 68% of yield) as a solid.Chemical Formula: C30H33FN4O5; Molecular Weight: 548.60.Example 31. Synthesis of compound 116Compound 116

[0302] Step 1. To a solution of compound 113 (25 mg, 0.046 mmol) and 22 (13 mg, 0.068 mmol) in DMF (1 mL) were added HATU (35 mg, 0.091 mmol) and DIPEA (24 pL, 0.137 mmol). The mixture was stirred for 1 hr. The mixture was purified by pre-HPLC (eluent: ACN / H2O=:25-80% for 25 mins) to give the title compound 115 (25 mg, 76% of yield) as a solid. LRMS: m / z ~ 721.3 [M+H]+. Chemical Formula: C38H49FN4O7SI; Molecular Weight: 720.90.115 116

[0303] Step 2. To a solution of compound 115 (25 mg, 0.035 mmol) in DCM (10 mL) were added TBAF (86 pL, 0.087 mmol) and acetic acid (173 pL, 0.173 mmol). The mixture was stirred overnight and evaporated to give a residue, which was purified by pre-HPLC (eluent: ACN / H2O:=25-80% for 25 mins) to give the title compound 116 (10 mg, 48% of yield) as a solid. LRMS: m / z= 607.2Chemical Formula: C32H35FN4O7; Molecular Weight: 606.64.Example 32. Synthesis of compound 118Compound 118113 117

[0304] Step 1. To a solution of compound 113 (100 mg:0.18 mmol) and Fmoc-L-Ala-OH (74 mg, 0.24 mmol) in DMF (1 mL) were added HATU (104 mg, 0.27 mmol) and DIPEA (80 pL, 0.46 mmol). The mixture was stirred for 1 hr. The mixture was purified by pre-HPLC (eluent: ACN / H2O=25“80% for 25 mins) to give the title compound 117 (120 mg, 78% of yield) as a solid. LRMS: m / z = 842.3 [M+H]+. Chemical Formula: C48H48FN5O8; Molecular Weight: 841.92.117 118

[0305] Step 2. To a solution of compound 117 (0.38 mmol) in DMF (5 mL) was added piperidine (1 mL). The mixture was stirred for 1 hr and evaporated to give the crude title compound 118, which was purified by pre-HPLC (eluent: ACN / H2O=25-80% for 25 mins) to give the title compound 118 (70 mg, 79% of yield) as a solid. LRMS: m / z~620.2 [M+H]+. Chemical Formula: C33H38FN5O6; Molecular Weight: 619.68.Example 33. Synthesis of compound 124119 120

[0306] Step 1. To a solution of 119 (250 mg, 0.45 mmol) in DMF (10 mL) was addedHATU (255 mg, 0.67 mmol). The mixture was stirred for 30 mins, then 4-aminobenzyl alcohol (77 mg, 0.63 mmol) and DIPEA (195 pL, 1.12 mmol) were added. The mixture was stirred for 2 hrs. The mixture was diluted with DCM (60 mL) and washed with water (20 mLx3), NaHCCh solution (2x30 mL), and brine. The organic layers were dried over anhydrous Na2SO4, filtered, and evaporated to give the crude compound 120, which was used directly in the next step.120 121

[0307] Step 2. To a solution of 120 (0.45 mmol) in DMF (10 mL) were added bis(4- nitrophenyl) carbonate (176 mg, 0.58 mmol) and DIPEA (155 pL, 0.90 mmol). The mixture was stirred for 3 hrs. The mixture was purified by pre-HPLC (eluent: ACN / H2O=25-80% for 25 mins) to give the title compound 121 (250 mg, 67% of yield) as a solid. LRMS: m / z=829.2 [M+H]+. Chemical Formula: C44H40N6O11; Molecular Weight: 828.82.3 121

[0308] Step 3. To a solution of 121 (108 mg, 0.13 mmol) in DMF (10 mL) were added 3 (51 mg, 0.1 mmol) and DI PE, A (35 pL, 0.2 mmol). The mixture was stirred for 3 hrs. The mixture was purified by pre-HPLC (eluent: ACN / H2O=:25-80% for 25 mins) to give the title compound 122 (80 mg, 67% of yield) as a solid. LRMS: m / z=1196.4 [M+H]+. Chemical Formula: CS5HS2FN90i3;Molecular Weight: 1196.24.122 123

[0309] Step 4. To a solution of compound 122 (80mg, 0.067 mmol) in DMF (5 mL) was added piperidine (0.1mL). The mixture was stirred for 20 mins and evaporated to give the crude title compound 123, which was purified by pre-HPLC (eluent: ACN / H2O=25-80% for 25 mins) to give the title compound 123 (50 mg, 77% of yield) as a solid. LRMS: m / z= 974.3 [M+H]+. Chemical Formula: C50H52FN9O11; Molecular Weight: 974.00.123 124

[0310] Step 5. To a solution of compound 123 (50 mg, 0.051 mmol) and Mal-PEG8-acid (40 mg, 0.067 mmol) in DMF (1 mL) were added HATU (29 mg, 0.077 mmol) and DIPEA (23 pL, 0.128 mmol). The mixture was stirred for 1 hr. The mixture was purified by pre-HPLC (eluent:ACN / H2O”25-80% for 25 mins) to give the title compound 124 (60 mg, 75% of yield) as a solid. LRMS: m / z= 1548.6Chemical Formula: C7SH94FN11O23; Molecular Weight: 1548.62.Example 34. Synthesis of compound 127125

[0311] Step 1. To a solution of 121 (73 mg, 0.087 mmol) in DMF (10 mL) were added 113 (40 mg, 0.073 mmol) and DI PEA (51 pL, 0.29 mmol). The mixture was stirred for 3 hrs. The mixture was purified by pre-HPLC (eluent: ACN / H2O=25-80% for 25 mins) to give the title compound 125 (80 mg, 67% of yield) as a solid. LRMS: m / z=1238.4 [M+H]+. Chemical Formula: C68H38FN9OI3; Molecular Weight: 1238.31.125 126

[0312] Step 2. To a solution of compound 125 (75mg, 0.061 mmol) in DMF (5 mL) was added piperidine (0.1mL). The mixture was stirred for 20 mins and evaporated to give the crude title compound 126, which was purified by pre-HPLC (eluent ACN / H2O=25-80% for 25 mins) to give the title compound 126 (50 mg, 81% of yield) as a solid. LRMS: m / z= 1016.4Chemical Formula: C53H58FN9O11Molecular Weight: 1016.08.126 127

[0313] Step 3. To a solution of compound 126 (50 mg, 0.049 mmol) and Mal-PEG8-acid (38 mg, 0.064 mmol) In DMF (1 mL) were added HATU (28 mg, 0.074 mmol) and DIPEA (22 pL, 0.123 mmol). The mixture was stirred for 1 hr. The mixture was purified by pre-HPLC (eluent: ACN / H2O=;25-80% for 25 mins) to give the title compound 127 (43 mg, 55% of yield) as a solid. LRMS: m / z= 1590.6[M+H]+. Chemical Formula: C79H100FN11O23; Molecular Weight: 1590.70.Example 35. Synthesis of compound 130Compound 130107 128

[0314] Step 1. To a solution of 107 (100 mg, 0.16 mmol) and 50 (85 mg, 0.19 mmol) in DMF (5 mL) were added HATU (92 mg, 0.24 mmol) and DIPEA (67 pL, 0.40 mmol). The mixture was stirred for 1 hr. The mixture was purified by pre-HPLC (eluent: ACN / H2O=25~80% for25 mins) to give the title compound 128 (120 mg, 72% of yield) as a solid. LRMS: m / z= 1036.4 [M+H]+. Chemical Formula: C53H62FN9O12; Molecular Weight: 1036.11128 129

[0315] Step 2. To a solution of compound 128 (120mg, 0.12mmol) in DCM (9mL) was added TFA (3mL). The mixture was stirred for 10mins, then evaporated to give a residue, which was dried under high vacuum to give the crude 129. LRMS: m / z= 936.3 [M+H]+. Chemical Formula: C48H54FN9O10; Molecular Weight: 935.99.

[0316] Step 3. To a solution of crude 129 (0.12mmol) in DMF (2mL) were added Mal- PEG8-acid (85mg, 0.14 mmol), HATU (68 mg, 0.18 mmol) and DIPEA (50pL, 0.30 mmol). The mixture was stirred for 1 hr. The mixture was purified by pre-HPLC (eluent: ACN / H2O=25-80% for25 mins) to give the title compound 130 (100 mg, 57% of yield) as a solid. LRMS: m / z=1510.6[M-!-H]+. Chemical Formula: C74H96FN11O22; Molecular Weight: 1510.61.Example 36. Synthesis of compound 132

[0317] Step 1. To a solution of exatecan mesylate (200 mg, 0.38 mmol) and (S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-2-cyclopropylacetic acid (190 mg, 0.56 mmol) in DMF (10 mL) were added HATU (215 mg, 0.56 mmol) and DIPEA (155 pL, 0.94 mmol). The mixture was stirred for 1 hr. The mixture was diluted with DCM (60 mL) and washed with water (20 mLx3), NaHCOs solution (2x30 mL), and brine. The organic layers were dried over anhydrous NasSCU, filtered, and evaporated to give the crude compound 131 , which was used directly in the next step.131 132

[0318] Step 2. To a solution of crude compound 131 (0.38 mmol) in DMF (5 mL) was added piperidine (0.5mL). The mixture was stirred for 20 mins and evaporated to give the crude title compound 132, which was purified by pre-HPLC (eluent: ACN / H2O=25-80% for 25 mins) to give the title compound 132 (180 mg, 92% of yield) as a yellow solid. LRMS: m / z=533.2 [M+H]T Chemical Formula: C29H29FN4O5; Molecular Weight: 532.57.Exampie 3 / . Synthesis of compound 135

[0319] Step 1. To a solution of 121 (60 mg, 0.72 mmol) in DMF (10 mL) were added 111 (33 mg, 0.60 mmol) and DIPEA (42 pL, 0.24 mmol). The mixture was stirred for 3 hrs. The mixture was purified by pre-HPLC (eluent: ACN / H2O=:25"80% for 25 mins) to give the title compound 133(50 mg, 67% of yield) as a solid. LRMS: m / z=1236.4 [M+H]1'. Chemical Formula: CesHssFNgOis;Molecular Weight: 1236.30.133 134

[0320] Step 2. To a solution of compound 133 (50mg, 0.040 mmol) in DMF (5 mL) was added piperidine (0.1mL). The mixture was stirred for 20 mins and evaporated to give the crude title compound 134, which was purified by pre-HPLC (eluent: ACN / H2O=25-80% for 25 mins) to give the title compound 134 (35 mg, 85% of yield) as a solid. LRMS: m / z™ 1014.4 [M+H]+. Chemical Formula: C53H56FN9O11; Molecular Weight: 1014.06.134 135

[0321] Step 3. To a solution of compound 134 (35 mg, 0.035 mmol) and Mal-PEG8-acid (27 mg, 0.045 mmol) in DMF (5 mL) were added HATU (26 mg, 0.069 mmol) and DIPEA (18 pL, 0.10 mmol). The mixture was stirred for 1 hr. The mixture was purified by pre-HPLC (eluent: ACN / H2O:=25-80% for 25 mins) to give the title compound 135 (40 mg, 73% of yield) as a solid. LRMS: m / z= 1588.6 [M+H]+. Chemical Formula: C79H98FN11O23; Molecular Weight: 1588.68.Example 38. Synthesis of compound 137Compound 137

[0322] Step 1. To a solution of compound 62 (25 mg, 0.046 mmol) and 22 (13 mg, 0.068 mmol) in DMF (1 mL) were added HATU (35 mg, 0.091 mmol) and DIPEA (24 pL, 0.137 mmol). The mixture was stirred for 1 hr. The mixture was purified by pre-HPLC (eluent: ACN / H2O=:25-80% for 25 mins) to give the title compound 136 (25 mg, 76% of yield) as a solid. LRMS: m / z ~ 721.3 [M+H]+. Chemical Formula: C38H49FN4O7SI; Molecular Weight: 720.90.136 137

[0323] Step 2. To a solution of compound 136 (25 mg, 0.035 mmol) in DCM (10 mL) were added TBAF (86 pL, 0.087 mmol) and acetic acid (173 pL, 0.173 mmol). The mixture was stirred overnight and evaporated to give a residue, which was purified by pre-HPLC (eluent: ACN / H2O:=25-80% for 25 mins) to give the title compound 137 (10 mg, 48% of yield) as a solid. LRMS: m / z= 607.2Chemical Formula: C32H35FN4O7; Molecular Weight: 606.64.Example 39. Synthesis of compound 141Compound 141

[0324] Step 1. To a solution of compound 138 (55 mg, 0.14 mmol) and 113 (60 mg, 0.11 mmol) in DMF (6 mL) were added HATU (62 mg, 0.16 mmol) and DIPEA (47 pL, 0.27 mmol). The mixture was stirred for 5 hrs. The mixture was purified by pre-HPLC (eluent: ACN / H2O=:25-80% for 25 mins) to give the title compound 139 (70 mg, 70% of yield) as a solid. LRMS: m / z ~ 915.3 [M+H]+. Chemical Formula: C5OH5IFN6OI0; Molecular Weight: 914.97.139 140

[0325] Step 2. To a solution of compound 139 (70mg, 0.077 mmol) in DMF (5 mL) was added piperidine (0.1mL). The mixture was stirred for 20 mins and evaporated to give the crude title compound 140, which was purified by pre-HPLC (eluent: ACN / H2O=25-80% for 25 mins) to give the title compound 140 (45 mg, 85% of yield) as a solid. LRMS: m / z= 693.2 [M+H]+. Chemical Formula: CssH^FNsOs; Molecular Weight: 692.73.11 140 141

[0326] Step 3. To a solution of compound 11 (50 mg, 0.084 mmol) and 140 (45 mg, 0.065 mmol) in DMF (6 mL) were added HATU (49 mg, 0.13 mmol) and DIPEA (34 pL, 0.19 mmol). The mixture was stirred for 30 mins. The mixture was purified by pre-HPLC (eluent: ACN / H2O=:25-80% for 25 mins) to give the title compound 141 (50 mg, 61% of yield) as a solid. LRMS: m / z = 1264.5 [M+H]+. Chemical Formula: C62H74FN11O17; Molecular Weight: 1264.31.Example 40. Synthesis of compound 144

[0327] Step 1. To a solution of compound 138 (43 mg, 0.12 mmol) and 62 (50 mg, 0.94 mmol) in DMF (6 mL) were added HATU (53 mg, 0.14 mmol) and DIPEA (39 pL, 0.23 mmol). The mixture was stirred for 30mins. The mixture was purified by pre-HPLC (eluent: ACN / H2O=25-80% for 25 mins) to give the title compound 142 (60 mg, 71 % of yield) as a solid. LRMS: m / z = 899.3 [M+H]L Chemical Formula: C^FkFNeOio; Molecular Weight: 898.93.142 143

[0328] Step 2. To a solution of compound 142 (60mg, 0.067 mmol) in DMF (5 mL) was added piperidine (0.1mL). The mixture was stirred for 20 mins and evaporated to give the crude title compound 143, which was purified by pre-HPLC (eluent: ACN / H2O25-80% for 25 mins) to give the title compound 143 (40 mg, 89% of yield) as a solid. LRMS: m / z= 677.2 [M+H]1. Chemical Formula: C34H37FN6O8; Molecular Weight: 676.69.

[0329] Step 3. To a solution of compound 11 (42 mg, 0.071 mmol) and 143 (40 mg, 0.059 mmol) in DMF (8 mL) were added HATU (34 mg, 0.89 mmol) and DIPEA (25 pL, 0.15 mmol). The mixture was stirred for 30 mins. The mixture was purified by pre-HPLC (eluent: ACN / H2O:::25-80% for 25 mins) to give the title compound 144 (20 mg, 27% of yield) as a solid. LRMS: m / z = 1248.4 [M+H]T Chemical Formula: CsiHyoFNnOn; Molecular Weight: 1248.27.Example 41. Generation of ADCs

[0330] Some embodiments of the disclosure relate to camptothecin derivatives linked to antibodies via a cleavable linker to generate antibody-drug conjugates. Table 2 provides a list of camptothecin derivatives. The conjugation procedure is illustrated in Figure 2. The cysteine conjugation method used in the conjugation process produces more homogeneous ADCs compared to lysine conjugation method. In these examples, the anti-Her2 antibody, Herceptin, or anti-trop-2 antibody was conjugated to camptothecin derivatives to form ADCs and evaluated for their ability to inhibit the growth of multiple cancer cell lines expressing different levels of Her2. Camptothecin derivative stabilizes the topoisomerase I complex after it has broken the DNA chain for replication, preventing the DNA double helix from being resealed and thereby stopping the process of replication.41-1. Preparation Example of ADC -1

[0331] The anti-Trop-2 antibody in PBS buffer (10 mM, pH7) containing 5 mM EDTA (3 mg / ml, 0.004 pmol) was reduced by adding a solution of 5mM TCEP (Tris (2-carboxyethyl) phosphine) (0.0048 ml, 0.024 pmol). The antibody / TCEP solution was incubated at 28°C for 2 hrs. At the end of the reduction, the reaction solution was cooled down to 25°C and the excessive TCEP was removed by ultrafiltration using an amicon ultra filter. The solution of camptothecin derivative-linker, compound 43 in DMA (10 mg / ml, 0.04 pmol) was then added to the above reduced antibody solution. The reaction solution was incubated for 2 hr at 25°C while the reduced antibody was conjugated to the toxin-linker via the thiol maleimide ligation. After conjugation was completed, the reaction mixture was desalted and concentrated by ultrafiltration using an amicon ultra filter to yield anti-Trop-2 ADC-1 (0.6 mg / ml, 0.2 mg) in PBS (10 mM, pH 7) buffer. The biochemical properties of the resulting ADC were characterized using size-exclusion chromatography high pressure liquid chromatography (SEC-HPLC) to determine purity and aggregation content, and by using hydrophobic interaction chromatography HPLC (HIC-HPLC) to determine drug loading (DAR). The average number of conjugated drug molecules per antibody molecule is 4.5.41-2. Preparation Example of ADC -2

[0332] The anti-Trop-2 antibody in PBS buffer (10 mM, pH 7) containing 5 mM EDTA (3 mg / ml, 0.01 pmol) was reduced by adding a solution of 5mM TCEP (Tris (2-carboxyethyl) phosphine) (0.012 ml, 0.08 pmol). The antibody / TCEP solution was incubated at 28°C for 2 hrs. At the end of the reduction, the reaction solution was cooled down to 25°C and the excessive TCEP was removed by ultrafiltration using an amicon ultra filter. The solution of camptothecinderivative-linker, compound 21 in DMA (10 mg / ml. 0.12 pmol) was then added to the above reduced antibody solution. The reaction solution was incubated for 2 hr at 25°C while the reduced antibody was conjugated to the toxin-linker via the thiol maleimide ligation. After conjugation was completed, the reaction mixture was desalted and concentrated by ultrafiltration using an amicon ultra filter to yield anti-Trop-2 ADC-2 (4.6 mg / ml, 0.9 mg) in PBS (10 mM, pH 7) buffer. The biochemical properties of the resulting ADC were characterized using size-exclusion chromatography high pressure liquid chromatography (SEC-HPLC) to determine purity and aggregation content, and by using hydrophobic interaction chromatography HPLC (HIC-HPLC) to determine drug loading (DAR). The average number of conjugated drug molecules per antibody molecule is 5.3.41-3. Preparation Example of ADC -3

[0333] The anti-Her2 antibody Herceptin in PBS buffer (10 mM, pH 7) containing 5 mM EDTA (3 mg / ml, 0.01 pmol) was reduced by adding a solution of 5 mM TCEP (Tris (2-carboxyethyl) phosphine) (0.012 ml, 0.06 pmol). The antibody / TCEP solution was incubated at 28°C for 2 hrs. At the end of the reduction, the reaction solution was cooled down to 25°C and the excessive TCEP was removed by ultrafiltration using an amicon ultra filter. The solution of camptothecin derivative-linker, compound 30 in DMA (10 mg / ml, 0.1 pmol) was then added to the above reduced antibody solution. The reaction solution was incubated for 2 hr at 25°C while the reduced antibody was conjugated to the toxin-linker via the thiol maleimide ligation. After conjugation was completed, the reaction mixture was desalted and concentrated by ultrafiltration using an amiconultra filter to yield anti-Her2 ADC-3 (2.15 mg / ml, 0.9 mg) in PBS (10 mM, pH 7) buffer. The biochemical properties of the resulting ADC were characterized using size-exclusion chromatography high pressure liquid chromatography (SEC-HPLC) to determine purity and aggregation content, and by using hydrophobic interaction chromatography HPLC (HIC-HPLC) to determine drug loading (DAR). The average number of conjugated drug molecules per antibody molecule is 5.2.41-4. Preparation Example of ADC -4

[0334] The anti-Her2 antibody Herceptin in PBS buffer (20 mM, pH 7) containing 5 mM EDTA (3 mg / ml, 0.06 pmol) was reduced by adding a solution of 5 mM TCEP (Tris (2-carboxyethyl) phosphine) (0.24 ml, 12 pmol). The antibody / TCEP solution was incubated at 28°C for 2 hrs. At the end of the reduction, the reaction solution was cooled down to 25°C and the excessive TCEP was removed by ultrafiltration using an amicon ultra filter. The solution of camptothecin derivative- linker, compound 12 in DMA (10 mg / ml, 10 pmol) was then added to the above reduced antibody solution. The reaction solution was incubated for 2 hr at 25°C while the reduced antibody was conjugated to the toxin-linker via the thiol maleimide ligation. After conjugation was completed, the reaction mixture was desalted and concentrated by ultrafiltration using an amicon ultra filter to yield anti-Her2 ADC-4 (3.4 mg / ml, 5.1 mg) in PBS (10 mM, pH 7) buffer. The biochemical properties of the resulting ADC were characterized using size-exclusion chromatography high pressure liquid chromatography (SEC-HPLC) to determine purity and aggregation content, and by using hydrophobic interaction chromatography HPLC (HIC-HPLC) to determine drug loading (DAR). The average number of conjugated drug molecules per antibody molecule is 6.6.41-5. Preparation Example of ADC -5

[0335] The anti-Her2 antibody Herceptin in PBS buffer (10 mM, pH 7) containing 5 mM EDTA (3 mg / ml, 0.6 pmol) was reduced by adding a solution of 5 mM TCEP (Tris (2-carboxyethyl) phosphine) (4.2 ml, 21 pmol). The antibody / TCEP solution was incubated at 28°C for 2 hrs. At the end of the reduction, the reaction solution was cooled down to 25°C and the excessive TCEP was removed by ultrafiltration using an amicon ultra filter. The solution of camptothecin derivative- linker, compound 100 in DMA (10 mg / ml, 12 pmol) was then added to the above reduced antibody solution.The reaction solution was incubated for 2 hr at 25°C while the reduced antibody was conjugated to the toxin-linker via the thiol maleimide ligation. After conjugation was completed, the reaction mixture was desalted and concentrated by ultrafiltration using an amicon ultra filter to yield anti-Her2 ADC-5 (6.3 mg / ml, 56.7 mg) in PBS (10 mM, pH 6) buffer. The biochemical properties of the resulting ADC were characterized using size-exclusion chromatography high pressure liquid chromatography (SEC-HPLC) to determine purity and aggregation content, and by using hydrophobic interaction chromatography HPLC (HIC-HPLC) to determine drug loading (DAR). The average number of conjugated drug molecules per antibody molecule is 7.7.

[0336] The anti-Her2 antibody Herceptin in PBS buffer (10 mM. pH 7) containing 5 mM EDTA (5 mg / ml, 0.04 pmol) was reduced by adding a solution of 5 mM TCEP (Tris (2-carboxyethyl) phosphine) (0.2 ml, 1 pmol). The antibody / TCEP solution was incubated at 28°C for 2 hrs. At the end of the reduction, the reaction solution was cooled down to 25°C and the excessive TCEP was removed by ultrafiltration using an amicon ultra filter. The solution of camptothecin derivative- linker, compound 101 in DMA (10 mg / ml, 0.8 pmol) was then added to the above reduced antibody solution. The reaction solution was incubated for 2 hr at 25°C while the reduced antibody was conjugated to the toxin-linker via the thiol maleimide ligation. After conjugation was completed, the reaction mixture was desalted and concentrated by ultrafiltration using an amicon ultra filter to yield anti-Her2 ADC-6 (5.7 mg / ml, 2.9 mg) in PBS (10 mM, pH 7) buffer. The biochemical properties of the resulting ADC were characterized using size-exclusion chromatography high pressure liquid chromatography (SEC-HPLC) to determine purity and aggregation content, and by using hydrophobic interaction chromatography HPLC (HIC-HPLC) to determine drug loading (DAR). The average number of conjugated drug molecules per antibody molecule is 7.9.41 -7. Preparation Example of ADC -7

[0337] The anti-Her2 antibody Herceptin in His buffer (20 mM, pH 6) containing 5 mM EDTA (3 mg / ml, 0.06 pmol) was reduced by adding a solution of 5 mM TCEP (Tris (2-carboxyethyl) phosphine) (0.24 ml, 1 .2 pmol). The antibody / TCEP solution was incubated at 28°C for 2 hrs. At the end of the reduction, the reaction solution was cooled down to 25°C and the excessive TCEP was removed by ultrafiltration using an amicon ultra filter. The solution of camptothecin derivative- linker, compound 124 in DMA (10 mg / ml, 1.5 pmol) was then added to the above reduced antibody solution. The reaction solution was incubated for 2 hr at 25°C while the reduced antibody was conjugated to the toxin-linker via the thiol maleimide ligation. After conjugation was completed, the reaction mixture was desalted and concentrated by ultrafiltration using an amicon ultra filterto yield anti-Her2 ADC-7 (7.5 mg / ml, 4.5 mg) in His(20 mM, pH 6) buffer. The biochemical properties of the resulting ADC were characterized using size-exclusion chromatography high pressure liquid chromatography (SEC-HPLC) to determine purity and aggregation content, and by using hydrophobic interaction chromatography HPLC (HIC-HPLC) to determine drug loading (DAR). The average number of conjugated drug molecules per antibody molecule is 7.5.41-4. Preparation Example of ADC -8

[0338] The anti-Her2 antibody Herceptin in PBS buffer (10 mM, pH 7) containing 5 mM EDTA (3 mg / ml, 0.04 pmol) was reduced by adding a solution of 5 mM TCEP (Tris (2-carboxyethyl) phosphine) (0.2 ml, 1 pmol). The antibody / TCEP solution was incubated at 28°C for 2 hrs. At the end of the reduction, the reaction solution was cooled down to 25°C and the excessive TCEP was removed by ultrafiltration using an amicon ultra filter. The solution of camptothecin derivative- linker, compound 65 in DMA (10 mg / ml, 0.8 pmol) was then added to the above reduced antibody solution. The reaction solution was incubated for 2 hr at 25°C while the reduced antibody was conjugated to the toxin-linker via the thiol maleimide ligation. After conjugation was completed, the reaction mixture was desalted and concentrated by ultrafiltration using an amicon ultra filter to yield anti-Her2 ADC-8 (4.3 mg / ml. 1.9 mg) in PBS (10 mM, pH 7) buffer. The biochemical properties of the resulting ADC were characterized using size-exclusion chromatography high pressure liquid chromatography (SEC-HPLC) to determine purity and aggregation content, and by using hydrophobic interaction chromatography HPLC (HIC-HPLC) to determine drug loading (DAR). The average number of conjugated drug molecules per antibody molecule is 7.4.41-9. Preparation Example of ADC -9

[0339] The anti-Her2 antibody Herceptin in PBS buffer (10 mM, pH 7) containing 5 mM EDTA (4mg / ml, 0.03 pmol) was reduced by adding a solution of 5 mM TCEP (Tris (2-carboxyethyl) phosphine) (0.13 ml, 0.75 pmol). The antibody / TCEP solution was incubated at 28°C for 2 hrs. At the end of the reduction, the reaction solution was cooled down to 25°C and the excessive TCEP was removed by ultrafiltration using an amicon ultra filter. The solution of camptothecin derivative-linker, compound 68 in DMA (10 mg / ml, 0.6 pmol) was then added to the above reduced antibody solution. The reaction solution was incubated for 2 hr at 25°C while the reduced antibody was conjugated to the toxin-linker via the thiol maleimide ligation. After conjugation was completed, the reaction mixture was desalted and concentrated by ultrafiltration using an amicon ultra filter to yield ant--Her2 ADC-9 (9.1 mg / ml, 2.7 mg) in PBS (10 mM, pH 7) buffer. The biochemical properties of the resulting ADC were characterized using size-exclusion chromatography high pressure liquid chromatography (SEC-HPLC) to determine purity and aggregation content, and by using hydrophobic interaction chromatography HPLC (HIC-HPLC) to determine drug loading (DAR). The average number of conjugated drug molecules per antibody molecule is 6.7.41-10. Preparation Example of ADC -10

[0340] The anti-Her2 antibody Herceptin in PBS buffer (10 mM, pH 7) containing 5 mM EDTA (3 mg / ml, 0.02 pmol) was reduced by adding a solution of 5 mM TCEP (Tris (2-carboxyethyl)phosphine) (0.1 mi, 0.5 pmol). The antibody / TCEP solution was incubated at 28°C for 2 hrs. At the end of the reduction, the reaction solution was cooled down to 25°C and the excessive TCEP was removed by ultrafiltration using an amicon ultra filter. The solution of camptothecin derivative- linker, compound 73 in DMA (10 mg / ml, 0.4 pmol) was then added to the above reduced antibody solution. The reaction solution was incubated for 2 hr at 25°C while the reduced antibody was conjugated to the toxin-linker via the thiol maleimide ligation. After conjugation was completed, the reaction mixture was desalted and concentrated by ultrafiltration using an amicon ultra filter to yield anti-Her2 ADC-10 (3.0 mg / ml, 1.8 mg) in PBS (10 mM, pH 7) buffer. The biochemical properties of the resulting ADC were characterized using size-exclusion chromatography high pressure liquid chromatography (SEC-HPLC) to determine purity and aggregation content, and by using hydrophobic interaction chromatography HPLC (HIC-HPLC) to determine drug loading (DAR). The average number of conjugated drug molecules per antibody molecule is 7.4.41-11 . Preparation Example of ADC -11

[0341] The anti-Her2 antibody Herceptin in PBS buffer (10mM, pH 7) containing 5 mM EDTA (3 mg / ml, 0.04 pmol) was reduced by adding a solution of 5 mM TCEP (Tris (2-carboxyethyl) phosphine) (0.2 ml, 1 pmol). The antibody / TCEP solution was incubated at 28°C for 2 hrs. At the end of the reduction, the reaction solution was cooled down to 25°C and the excessive TCEP was removed by ultrafiltration using an amicon ultra filter. The solution of camptothecin derivative- linker, compound 79 in DMA (10 mg / ml, 0.8 pmol) was then added to the above reduced antibody solution. The reaction solution was incubated for 2 hr at 25°C while the reduced antibody was conjugated to the toxin-linker via the thiol maleimide ligation. After conjugation was completed, the reaction mixture was desalted and concentrated by ultrafiltration using an amicon ultra filter to yield anti-Her2 ADC-11 (5.7 mg / ml, 2.9 mg) in PBS (10mM, pH7) buffer. The biochemical properties of the resulting ADC were characterized using size-exclusion chromatography highpressure liquid chromatography (SEC-HPLC) to determine purity and aggregation content, and by using hydrophobic interaction chromatography HPLC (HIC-HPLC) to determine drug loading (DAR). The average number of conjugated drug molecules per antibody molecule is 7.5.41-12. Preparation Example of ADC -12

[0342] The anti-Her2 antibody Herceptin in His buffer (20 mM, pH 6) containing 5 mM EDTA (5 mg / mL 0.83 pmol) was reduced by adding a solution of 5 mM TCEP (Tris (2-carboxyethyl) phosphine) (2.5 ml, 12.5 pmol). The antibody / TCEP solution was incubated at 28°C for 2 hrs. At the end of the reduction, the reaction solution was cooled down to 25°C and the excessive TCEP was removed by ultrafiltration using an amicon ultra filter. The solution of camptothecin derivative- linker, compound 135 in DMA (10 mg / ml, 16.6 pmol) was then added to the above reduced antibody solution.The reaction solution was incubated for 2 hr at 25°C while the reduced antibody was conjugated to the toxin-linker via the thiol maleimide ligation. After conjugation was completed, the reaction mixture was desalted and concentrated by ultrafiltration using an amicon ultra filter to yield anti-Her2 ADC-12 (11.1 mg / ml, 55.5 mg) in His(20 mM.pH 6) buffer. The biochemical properties of the resulting ADC were characterized using size-exclusion chromatography high pressure liquid chromatography (SEC-HPLC) to determine purity and aggregation content, and by using hydrophobic interaction chromatography HPLC (HIC-HPLC) to determine drug loading (DAR). The average number of conjugated drug molecules per antibody molecule is 7.9.41-13. Preparation Example of ADC -13

[0343] The anti-Her2 antibody Herceptin in His buffer (20mM, pH 6) containing 5 mM EDTA (5 mg / ml, 0.8 pmol) was reduced by adding a solution of 5 mM TCEP (Tris (2-carboxyethyl) phosphine) (2.7 ml, 16.6 pmol). The antibody / TCEP solution was incubated at 28°C for 2 hrs. At the end of the reduction, the reaction solution was cooled down to 25°C and the excessive TCEP was removed by ultrafiltration using an amicon ultra filter. The solution of camptothecin derivative- linker, compound 127 in DMA (10 mg / ml, 20.7 pmol) was then added to the above reduced antibody solution.The reaction solution was incubated for 2 hr at 25°C while the reduced antibody was conjugated to the toxin-linker via the thiol maleimide ligation. After conjugation was completed, the reaction mixture was desalted and concentrated by ultrafiltration using an amicon ultra filter to yield anti-Her2 ADC-13 (11.3mg / ml, 83.8 mg) in His (20 mM, pH 6) buffer. The biochemical properties of the resulting ADC were characterized using size-exclusion chromatography high pressure liquid chromatography (SEC-HPLC) to determine purity and aggregation content, and by using hydrophobic interaction chromatography HPLC (HIC-HPLC) to determine drug loading (DAR). The average number of conjugated drug molecules per antibody molecule is 7.5.41-14. Preparation Exampie of ADC -14

[0344] The anti-Her2 antibody Herceptin in PBS buffer (10 mM, pH 7) containing 5 mM EDTA (3 mg / ml, 0.04 pmol) was reduced by adding a solution of 5 mM TCEP (Tris (2-carboxyethyl) phosphine) (0.16 ml, 0.8 pmol). The antibody / TCEP solution was incubated at 28°C for 2 hrs. Atthe end of the reduction, the reaction solution was cooled down to 25°C and the excessive TCEP was removed by ultrafiltration using an amicon ultra filter. The solution of camptothecin derivative- linker, compound 55 in DMA (10 mg / ml, 1 pmol) was then added to the above reduced antibody solution. The reaction solution was incubated for 2 hr at 25°C while the reduced antibody was conjugated to the toxin-linker via the thiol maleimide ligation. After conjugation was completed, the reaction mixture was desalted and concentrated by ultrafiltration using an amicon ultra filter to yield anti-Her2 ADC-14 (7.1 mg / ml, 4.3 mg) in PBS (10 mM, pH 7) buffer. The biochemical properties of the resulting ADC were characterized using size-exclusion chromatography high pressure liquid chromatography (SEC-HPLC) to determine purity and aggregation content, and by using hydrophobic interaction chromatography HPLC (HIC-HPLC) to determine drug loading (DAR). The average number of conjugated drug molecules per antibody molecule is 7.3.41-15. Preparation Example of ADC -15

[0345] The anti-Her2 antibody Herceptin in PBS buffer (10mM, pH 7) containing 5 mM EDTA (3 mg / ml, 0.04 pmol) was reduced by adding a solution of 5 mM TCEP (Tris (2-carboxyethyl) phosphine) (0.16ml, 0.8 pmol). The antibody / TCEP solution was incubated at 28°C for 2 hrs. At the end of the reduction, the reaction solution was cooled down to 25°C and the excessive TCEP was removed by ultrafiltration using an amicon ultra filter. The solution of camptothecin derivative- linker, compound 54 in DMA (10 mg / ml, 1 pmol) was then added to the above reduced antibody solution. The reaction solution was incubated for 2 hr at 25GC while the reduced antibody was conjugated to the toxin-linker via the thiol maleimide ligation. After conjugation was completed, the reaction mixture was desalted and concentrated by ultrafiltration using an amicon ultra filter to yield anti-Her2 ADC-15 (6.9 mg / ml, 3.4 mg) in PBS (10 mM,pH 7) buffer. The biochemical properties of the resulting ADC were characterized using size-exclusion chromatography high pressure liquid chromatography (SEC-HPLC) to determine purity and aggregation content, and by using hydrophobic interaction chromatography HPLC (HIC-HPLC) to determine drug loading (DAR). The average number of conjugated drug molecules per antibody molecule is 7.4.41-16. Preparation Exampie of ADC -16

[0346] The anti-Her2 antibody Herceptin in PBS buffer (10mM, pH 7) containing 5 mM EDTA (3 mg / ml, 0.04 pmol) was reduced by adding a solution of 5 mM TCEP (Tris (2-carboxyethyl) phosphine) (0.2 ml, 1 pmol). The antibody / TCEP solution was incubated at 28°C for 2 hrs. At the end of the reduction, the reaction solution was cooled down to 25°C and the excessive TCEP was removed by ultrafiltration using an amicon ultra filter. The solution of camptothecin derivative- linker, compound 58 in DMA (10 mg / ml, 0.8 pmol) was then added to the above reduced antibody solution. The reaction solution was incubated for 2 hr at 25°C while the reduced antibody was conjugated to the toxin-linker via the thiol maleimide ligation. After conjugation was completed, the reaction mixture was desalted and concentrated by ultrafiltration using an amicon ultra filter to yield anti-Her2 ADC-16 (5.7 mg / ml, 2.6 mg) in PBS (10 mM, pH 7) buffer. The biochemical properties of the resulting ADC were characterized using size-exclusion chromatography high pressure liquid chromatography (SEC-HPLC) to determine purity and aggregation content, and by using hydrophobic interaction chromatography HPLC (HIC-HPLC) to determine drug loading (DAR). The average number of conjugated drug molecules per antibody molecule is 7,3.41-17.Preparation Example of ADC -17

[0347] The anti-Her2 antibody Herceptin in PBS buffer (10 mM, pH 7) containing 5 mM EDTA (3 mg / ml, 0.04 pmol) was reduced by adding a solution of 5 mM TCEP (Tris (2-carboxyethyl) phosphine) (0.16ml, 0.8 pmol). The antibody / TCEP solution was incubated at 28°C for 2 hrs. At the end of the reduction, the reaction solution was cooled down to 25°C and the excessive TCEP was removed by ultrafiltration using an amicon ultra filter. The solution of camptothecin derivative- linker, compound 53 in DMA (10 mg / ml, 1 pmol) was then added to the above reduced antibody solution. The reaction solution was incubated for 2 hr at 25°C while the reduced antibody was conjugated to the toxin-linker via the thiol maleimide ligation. After conjugation was completed, the reaction mixture was desalted and concentrated by ultrafiltration using an amicon ultra filter to yield anti-Her2 ADC-17 (5.7mg / ml, 2.8 mg) in PBS (10 mM, pH 7) buffer. The biochemical properties of the resulting ADC were characterized using size-exclusion chromatography high pressure liquid chromatography (SEC-HPLC) to determine purity and aggregation content, and by using hydrophobic interaction chromatography HPLC (HIC-HPLC) to determine drug loading (DAR). The average number of conjugated drug molecules per antibody molecule is 7.5.41-18. Preparation Example of ADC -18

[0348] The anti-Her2 antibody Herceptin in PBS buffer (10 mM, pH 7) containing 5 mM EDTA (3 mg / ml, 0.04 pmol) was reduced by adding a solution of 5 mM TCEP (Tris (2-carboxyethyl) phosphine) (0.16 ml, 0.8 pmol). The antibody / TCEP solution was incubated at 28°C for 2 hrs. At the end of the reduction, the reaction solution was cooled down to 25°C and the excessive TCEP was removed by ultrafiltration using an amicon ultra filter. The solution of camptothecin derivative- linker, compound 60 in DMA (10 mg / ml, 1 pmol) was then added to the above reduced antibody solution. The reaction solution was incubated for 2 hr at 25°C while the reduced antibody was conjugated to the toxin-linker via the thiol maleimide ligation. After conjugation was completed, the reaction mixture was desalted and concentrated by ultrafiltration using an amicon ultra filter to yield anti-Her2 ADC-18 (9 mg / ml, 4.5 mg) in PBS (10 mM, pH 7) buffer. The biochemicalproperties of the resulting ADC were characterized using size-exclusion chromatography high pressure liquid chromatography (SEC-HPLC) to determine purity and aggregation content, and by using hydrophobic interaction chromatography HPLC (HIC-HPLC) to determine drug loading (DAR). The average number of conjugated drug molecules per antibody molecule is 7.7.41-19. Preparation Example of ADC -19

[0349] The anti-Her2 antibody Herceptin in PBS buffer (10 mM, pH 7) containing 5 mM EDTA (3 mg / ml. 0.04 pmol) was reduced by adding a solution of 5 mM TCEP (Tris (2-carboxyethy!) phosphine) (0.16 ml, 0.8 pmol). The antibody / TCEP solution was incubated at 28°C for 2 hrs. At the end of the reduction, the reaction solution was cooled down to 25°C and the excessive TCEP was removed by ultrafiltration using an amicon ultra filter. The solution of camptothecin derivative- linker, compound 84 in DMA (10 mg / ml, 1 pmol) was then added to the above reduced antibody solution. The reaction solution was incubated for 2 hr at 25°C while the reduced antibody was conjugated to the toxin-linker via the thiol maleimide ligation. After conjugation was completed, the reaction mixture was desalted and concentrated by ultrafiltration using an amicon ultra filter to yield anti-Her2 ADC-19 (5.5 mg / ml, 2.8 mg) in PBS (10 mM, pH 7) buffer. The biochemical properties of the resulting ADC were characterized using size-exclusion chromatography high pressure liquid chromatography (SEC-HPLC) to determine purity and aggregation content, and by using hydrophobic interaction chromatography HPLC (HIC-HPLC) to determine drug loading (DAR). The average number of conjugated drug molecules per antibody molecule is 7.7.41-20. Preparation Example of ADC -20

[0350] The anti-Her2 antibody Herceptin in PBS buffer (10 mM, pH 7) containing 5 mM EDTA (3 mg / ml, 0.04 pmol) was reduced by adding a solution of 5 mM TCEP (Tris (2-carboxyethyl) phosphine) (0.16 ml, 0.8 pmol). The antibody / TCEP solution was incubated at 28°C for 2 hrs. At the end of the reduction, the reaction solution was cooled down to 25°C and the excessive TCEP was removed by ultrafiltration using an amicon ultra filter. The solution of camptothecin derivative- linker, compound 89 in DMA (10 mg / ml, 1 pmol) was then added to the above reduced antibody solution. The reaction solution was incubated for 2 hr at 25°C while the reduced antibody was conjugated to the toxin-linker via the thiol maleimide ligation. After conjugation was completed, the reaction mixture was desalted and concentrated by ultrafiltration using an amicon ultra filter to yield anti-Her2 ADC-20 (4.6 mg / ml, 2.7 mg) in PBS (10 mM, pH 7) buffer. The biochemical properties of the resulting ADC were characterized using size-exclusion chromatography high pressure liquid chromatography (SEC-HPLC) to determine purity and aggregation content, and by using hydrophobic interaction chromatography HPLC (HIC-HPLC) to determine drug loading (DAR). The average number of conjugated drug molecules per antibody molecule is 7.7.41-21. Preparation Example of ADC -21

[0351] The anti-Her2 antibody Herceptin in PBS buffer (20 mM, pH 7) containing 5 mM EDTA (3 mg / ml, 0.04 pmol) was reduced by adding a solution of 5 mM TCEP (Tris (2-carboxyethyl) phosphine) (0.16 ml, 0.8 pmol). The antibody / TCEP solution was incubated at 28°C for 2 hrs. At the end of the reduction, the reaction solution was cooled down to 25°C and the excessive TCEP was removed by ultrafiltration using an amicon ultra filter. The solution of camptothecin derivative- linker, compound 94 in DMA (10 mg / ml, 1 pmol) was then added to the above reduced antibody solution. The reaction solution was incubated for 2 hr at 25°C while the reduced antibody was conjugated to the toxin-linker via the thiol maleimide ligation. After conjugation was completed, the reaction mixture was desalted and concentrated by ultrafiltration using an amicon ultra filter to yield anti-Her2 ADC-21 (5.2 mg / ml, 3.1 mg) in PBS (10 mM, pH 7) buffer. The biochemical properties of the resulting ADC were characterized using size-exclusion chromatography high pressure liquid chromatography (SEC-HPLC) to determine purity and aggregation content, and by using hydrophobic interaction chromatography HPLC (HIC-HPLC) to determine drug loading (DAR). The average number of conjugated drug molecules per antibody molecule is 7.6.41-22. Preparation Example of ADC -22

[0352] The anti-Her2 antibody Herceptin in His buffer (20 mM, pH6) containing 5 mM EDTA (3 mg / ml, 0.04 pmol) was reduced by adding a solution of 5 mM TCEP (Tris (2-carboxyethyl) phosphine) (0.16 ml, 0.8 pmol). The antibody / TCEP solution was incubated at 28°C for 2 hrs. At the end of the reduction, the reaction solution was cooled down to 25°C and the excessive TCEP was removed by ultrafiltration using an amicon ultra filter. The solution of camptothedn derivative- linker, compound 130 in DMA (10 mg / ml, 1 pmol) was then added to the above reduced antibody solution. The reaction solution was incubated for 2 hr at 25°C while the reduced antibody was conjugated to the toxin-linker via the thiol maleimide ligation. After conjugation was completed,the reaction mixture was desalted and concentrated by ultrafiltration using an amicon ultra filter to yield anti-Her2 ADC-22 (7.0 mg / ml, 3.5 mg) in His (20 mM, pH 6) buffer. The biochemical properties of the resulting ADC were characterized using size-exclusion chromatography high pressure liquid chromatography (SEC-HPLC) to determine purity and aggregation content, and by using hydrophobic interaction chromatography HPLC (HIC-HPLC) to determine drug loading (DAR). The average number of conjugated drug molecules per antibody molecule is 7.8.41-23. Preparation Example of ADC -23

[0353] The anti-Her2 antibody Herceptin in His buffer (20 mM, pH 6) containing 5 mM EDTA (5 mg / ml, 1 pmol) was reduced by adding a solution of 5 mM TCEP (Tris (2-carboxyethyl) phosphine) (3 ml, 15 pmol). The antibody / TCEP solution was incubated at 28°C for 2 hrs. At the end of the reduction, the reaction solution was cooled down to 25°C and the excessive TCEP was removed by ultrafiltration using an amicon ultra filter. The solution of camptothecin derivative- linker, compound 141 in DMA (10 mg / mM, 20 pmol) was then added to the above reduced antibody solution.The reaction solution was incubated for 2 hr at 25°C while the reduced antibody was conjugated to the toxin-linker via the thiol maleimide ligation. After conjugation was completed, the reaction mixture was desalted and concentrated by ultrafiltration using an amicon ultra filter to yield anti-Her2 ADC-23 ( 13.5 mg / ml, 108 mg) in His(20 mM, pH 6) buffer. The biochemical properties of the resulting ADC were characterized using size-exclusion chromatography high pressure liquid chromatography (SEC-HPLC) to determine purity and aggregation content, and by using hydrophobic interaction chromatography HPLC (HIC-HPLC) to determine drug loading (DAR). The average number of conjugated drug molecules per antibody molecule is 7.9.41-24. Preparation Example of ADC -24

[0354] The anti-Her2 antibody Herceptin in His buffer (10m M, pH 6) containing 5 mM EDTA (5 mg / ml, 0.04 pmol) was reduced by adding a solution of 5 mM TCEP (Tris (2-carboxyethyl) phosphine) (0.12 ml, 0.6 pmol). The antibody / TCEP solution was incubated at 28°C for 2 hrs. At the end of the reduction, the reaction solution was cooled down to 25°C and the excessive TCEP was removed by ultrafiltration using an amicon ultra filter. The solution of camptothecin derivative- linker, compound 144 in DMA (10 mg / ml, 0.8 pmol) was then added to the above reduced antibody solution. The reaction solution was incubated for 2 hr at 25°C while the reduced antibody was conjugated to the toxin-linker via the thiol maleimide ligation. After conjugation was completed, the reaction mixture was desalted and concentrated by ultrafiltration using an amicon ultra filter to yield anti-Her2 ADC-24 (8.6 mg / ml, 4.3 mg) in His (20 mM, pH 6) buffer. The biochemical properties of the resulting ADC were characterized using size-exclusion chromatography high pressure liquid chromatography (SEC-HPLC) to determine purity and aggregation content, and by using hydrophobic interaction chromatography HPLC (HIC-HPLC) to determine drug loading (DAR). The average number of conjugated drug molecules per antibody molecule is 7.3.41-25. Preparation Example of ADC -25

[0355] The anti-Her2 antibody Herceptin in PBS buffer (10 mM, pH 7) containing 5 mM EDTA (3 mg / ml, 0.02 pmol) was reduced by adding a solution of 5 mM TCEP (Tris (2-carboxyethyi) phosphine) (0.06 ml, 0.3 pmol). The antibody / TCEP solution was incubated at 28°C for 2 hrs. Atthe end of the reduction, the reaction solution was cooled down to 25°C and the excessive TCEP was removed by ultrafiltration using an amicon ultra filter. The solution of camptothecin derivative- linker, compound 47 in DMA (10 mg / ml, 0.36 pmol) was then added to the above reduced antibody solution. The reaction solution was incubated for 2 hr at 25°C while the reduced antibody was conjugated to the toxin-linker via the thiol maleimide ligation. After conjugation was completed, the reaction mixture was desalted and concentrated by ultrafiltration using an amicon ultra filter to yield anti-Her2 ADC- 14 (3.3 mg / ml, 0.98 mg) in PBS (10 mM, pH 7) buffer. The biochemical properties of the resulting ADC were characterized using size-exclusion chromatography high pressure liquid chromatography (SEC-HPLC) to determine purity and aggregation content, and by using hydrophobic interaction chromatography HPLC (HIC-HPLC) to determine drug loading (DAR). The average number of conjugated drug molecules per antibody molecule is 5.5.41-26. Preparation Example of reference ADC (Herceptin-Deruxtecan ADC)

[0356] The anti-Her2 antibody Herceptin in His buffer (20 mM, pH 6) containing 5 mM EDTA (3 mg / ml, 0.48 pmol) was reduced by adding a solution of 5 mM TCEP (Tris (2-carboxyethyl) phosphine) (1.92 ml, 9.6 pmol). The antlbody / TCEP solution was Incubated at 28°C for 2 hrs. At the end of the reduction, the reaction solution was cooled down to 25°C and the excessive TCEP was removed by ultrafiltration using an amicon ultra filter. The solution of deruxtecan in DMA (10 mg / ml, 12 pmol) was then added to the above reduced antibody solution. The reaction solution was incubated for 2 hr at 25°C while the reduced antibody was conjugated to the toxin-linker via the thiol maleimide ligation. After conjugation was completed, the reaction mixture was desalted and concentrated by ultrafiltration using an amicon ultra filter to yield reference ADC ( 9.2 mg / ml, 35.4 mg) in His(20 mM, pH 6) buffer. The biochemical properties of the resulting ADC were characterized using size-exclusion chromatography high pressure liquid chromatography (SEC- HPLC) to determine purity and aggregation content, and by using hydrophobic interactionchromatography HPLC (HIOHPL.C) to determine drug loading (DAR). The average number of conjugated drug molecules per antibody molecule is 7.7. Reference ADC with DAR 4.55 was prepared in the same procedure as DAR 7.7 ADC except for the ratio of deruxtecan / antibody used in the preparation was reduced to 10.Example 42. / n Vitro Cytotoxicity of camptothecin derivatives, and ADCs in Cancer Cell Lines Expressing Different Her2 Expression Levels

[0357] The in vitro cytotoxicity of free camptothecin derivatives, Herceptin ADCs, or anti- Trop-2 ADCs containing camptothecin derivatives were tested on SK-BR-3 breast cancer cells, NCI-N87 gastric cancer cells, BT-474 breast cancer cells, NCI-H292 lung cancer cells, BxPC pancreatic cancer cells, and MDA-MB-468 breast cancer cells along with reference Toxin DXd or reference ADC (Herceptin-Deruxtecan ADC). Briefly, al! cell lines were cultured in a suitable culture medium at 37°C in a humidified incubator atmosphere of 5% CO2. Cells were plated in 96-well flat bottom plates. Cell seeding number ranged from 3000 cells 7100 ul / well to 6,000 cells / 100 pl / well. Cells were allowed to adhere overnight at 37°C in a humidified atmosphere of 5% CO2. Free camptothecin derivatives or Herceptin-ADCs were prepared from stock solution and diluted into appropriated working concentration 24 hours after cell seeding. A serial ten-fold dilution for nine points was performed with culture medium. The final free camptothecin derivatives concentrations were ranging from 5,000 nM to 0.00005 nM and Herceptin-ADCs concentrations were ranging from 1 ,000 nM to 0.00001 nM. The cells were incubated for 3 or 5 days. Cell Counting Kit-8 solution (Dojindo China Co., Ltd, lot#PL701 ) was added to the wells for 1-4 hours at 37oC and the absorbance at 450 nm was measured using a Microplate Reader (SpectraMax M5, Molecular Devices) and SoftMax Pro5.4.1 software. Dose-response curves were generated and IC50 was calculated using GraphPad Prism 7 three-parameter curve fitting.

[0358] Different camptothecin (CPT) derivatives and their in vitro cytotoxicity were listed in Table 4. ADCs and their in vitro cytotoxicity data were listed in Table 5.

[0359] Representatively, Herceptin-36309 had DAR of 5.5, Herceptin-363-11 had DAR of 6.2 and Herceptin-DXd (reference ADC) had DAR of 4.55 or 7.7. The ADCs were tested against BT-474 breast cancer cells, NCI-N87 gastric cancer cells and SK-BR-3 breast cancer cells representing high Her2 expression, and BxPC-3 pancreatic cancer cells, NCI-H292 lung cancer cells and MDA-MB-468 breast cancer cells with low / none level of Her2. In vitro cytotoxicity assay was performed. Free camptothecin derivatives were also tested in the same assay. Briefly, allcell lines were cultured in a suitable culture medium at 37°C in a humidified incubator atmosphere of 5% CO2. Cells were plated in 96-well flat bottom plates. Cell seeding number ranged from 500 cells / / 100 ul / well to 6,000 cells / 100 pl / well. Cells were allowed to adhere overnight at 37°C in a humidified atmosphere of 5% CO2. ADCs or free camptothecin derivatives were prepared from stock solution and diluted into appropriated working concentration 24 hours after ceil seeding. A serial ten-fold dilution for seven points was performed with culture medium. The final concentrations were ranging from 10,000 nM to 0.001 nM. The cells were incubated with ADCs for 5 days. Cell Counting Kit-8 solution (Dojindo China Co., Ltd, lot#PL701) was added to the wells for 1-4 hours at 37°C and the absorbance at 450 nm was measured using a Microplate Reader (SpectraMax M5, Molecular Devices) and SoftMax Pro5.4.1 software. Dose-response curves were generated and IC50 was calculated using GraphPad Prism 7 three-parameter curve fitting.

[0360] Figures 3A-3D shows the representative killing curves of free camptothecin derivatives (T-363, DXd) and ADCs containing 36309 or DXd in SK-BR-3, NCI-N87, MDA-MB- 468 and BxPC3 cells.

[0361] Figures 4-3 show the representative killing curves of free camptothecin derivatives (T-363, DXd) and ADCs containing 363-11 or DXd in BT-474 (Figure 4), NCI-N87 (Figure 5), SK- BR-3 (Figure 6), BxPC-3 (Figure 7), NCI-H292 (Figure 8) and MDA-MB-468 (Figure 9) cells. Overall, ADCs containing various camptothecin derivatives demonstrate specific and potent in vitro killing activities in Her2-expressing cells.

[0362] Table 6 and Table 7 summarize the IC50 values from in vitro cytotoxicity assays for camptothecin derivatives (T-363 and DXd) and representative ADCs. The ADC generated from Val-Ala-PABC linker-payload 36309 showed potent cytotoxicity in Her2 positive cells but has narrower therapeutic window compared to DXd as demonstrated by some non-specific killing of Herceptin-36309 in Her2 negative cells (Table 6). In addition, PABC is a hydrophobic moiety, which contributes to the aggregation issue observed with high DAR ADC of Herceptin-36309. Indeed, results demonstrate that Herceptin-363-11 and Herceptin-DXd induced potent cytotoxicity against BT-474, SK-BR-3 and NCI-N87 tumor cells that have high Her2 expression, with IC50 in the sub nM range. The killing activity of Herceptin-363- 11 was comparable or better than Herceptin-DXd. In the Her2 negative BxPC-3, MDA-MB-468 and NCI-H292 cells, IC50 of both ADCs was above 100 nM. On the other hand, the free toxin T-363 and DXd were able to kill all three cell lines with similar IC50s (Table 7). These in vitro cytotoxicity results suggest a wide therapeutic window for the 363-11 containing ADCs.Table 4Camptothedn derivatives and IC50Table 5CPT derivative-Linker-Antibody structures, and IC50 for ADCsTable 6IC50 for Dxd, T-363, Herceptin- Dxd, and Herceptin-36308Table 7IC50 for Dxd, T-363, Herceptin-Dxd, and Herceptin-363- 11Example 43. Bystander killing effect

[0363] The ability of an ADC to kill the neighboring antigen-negative cells surrounding the antigen-positive cells is called a bystander effect. To confirm whether Herceptin ADCs containing camptothecin derivatives induced bystander killing, a flow cytometry-based cell killing assay was carried out. HER2-positive cells NCI-N87 and -negative cells MDA-MB-468 in exponential growth phase were collected separately and counted and suspended with culture medium at adjusted cell density. Two types of cells were then seeded to the same well at a ratio of 1 :1 in a 24-well cell culture plate and incubated overnight at 37°C. Herceptin-ADCs were then added at a concentration of 10 nM per well. The cells were further incubated in the presence of ADCs for 5 days at 37°C. At the end of incubation period, cells were collected and transferred into a roundbottom 96-well plate and rinsed with PBS and resuspended in DPBS. The anti-Herceptin antibody was added and incubated for 60 min followed with goat anti-human IgG H&L (FITC) antibody for HER2-positive cell detection. Flow cytometry analysis was performed using a BD Fortessa flow cytometer, and HER2-positive and HER2-negative cell counts were analyzed using FlowJo software. Bystander killing effect was evaluated by the survival rate of antigen negative cells (such as HER2-negative cells), which was calculated as survived antigen negative cells after ADC treatment / total amount of antigen negative cells used. In some of the experiments, the procedure described below was used to evaluate the bystander killing effect of the ADC.

[0364] In order to confirm whether Herceptin-363-11 induced bystander killing, a transwell coculture cell killing assay was carried out. A cell culture insert was placed into the 24 well plate to create a chamber. Her2-positive SK-BR-3 cells were seeded in the top chamber, and Hemnegative MDA-MB-468 cells were placed in the bottom chamber. Chambers were separated by membrane that limits transit of cells, but not soluble factors, between chambers. Cells are incubated in the presence of ADCs. The concentrations of ADCs were 1 , 10, 100 nM. After 5 days in culture, the remaining MDA-MB-468 cells in the bottom chamber was analyzed by Cell Counting Kit-8 assay as described in Example 42 % of growth inhibition was calculated by compared to untreated samples. SK-BR-3 cells or MDA-MB-468 cells alone were also plated in the 24 well plate and treated with ADCs as comparison. As shown in Figure 10A-B, Herceptin-363-11 had strong growth inhibition against Her2-positive SK-BR-3 cells, but only induce minimal cell killing in Her2-negative MDA-MB-468 cells at the concentrations of 10 nM or above. However, when both cells were cocultured in a transwell system, Herceptin-363-11 showed significant growth inhibitory effect on MDA-MB-468 cells. As the ADC has Her2-specific cytotoxicity, MDA-MB-468 cell killing could be caused by the released T-363, suggesting the bystander killing effect ofHerceptin-363-11. Herceptin-DXd was included as a positive control. Herceptin-363-11 demonstrated similar bystander effect as compared to Herceptin-DXd.

[0365] The relevant bystander killing effect data is shown in Table 8.Table 8HER2 negative cells viability regarding various ADCsIt can be seen that ADCs including ADC-7, ADC-12, ADC-14, ADC-15, ADC-16, ADC-17, ADC- 18, ADC- 19, ADC-20, ADC-21, ADC-23 exhibit a strong bystander killing effect, whereas ADC-5, ADC-11 , and ADC-22 show a moderate or no bystander killing effect.Exampie 44. Plasma Stability of Herceptin-363-11

[0366] To assess the in vitro plasma stability of ADC, Herceptin-363-11 (0.1 mg / ml) was incubated in blank human plasma at 37°C for up to 96 hours. At each time point, samples were drawn and the remaining amount of total antibody (naked + conjugated) and conjugated antibody (ADC) were measured using a quantitative sandwich enzyme linked immunoassay (ELISA). In brief, to measure total antibody, human Her2 protein was coated onto the microplate to capture Herceptin antibodies. After removing the unbound antibodies, the bound total Herceptin antibody was detected with horseradish peroxidase (HRP) conjugated goat anti-human IgG Fc specific polycolonal antibody. For the determination of Herceptin-363-11 ADC only, a mouse anti-DXd antibody was added as the secondary antibody. The concentration of Herceptin-363-11 ADC wasthen detected with HRP conjugated goat anti-mouse lgG(H+L) antibody. Figure 11 shows the percentage of remaining total antibody and Herceptin-363-11 at different time points. The trend for Herceptin-363-11 was similar to that of total antibody. Herceptin-363-11 remained at 79% after 96 hours. These results indicate that Herceptin-363-11 is stable in human plasma.Example 45. / n Vivo Anti-tumor Activity

[0367] The anti-tumor activities of Herceptin ADCs containing camptothecin derivatives (ADC-4, ADC-5, ADC-14, ADC-22, ADC-23) was assessed using Her2-positive NCI-N87 cells, JIMT-1 cells and HCC1569 cells xenograft models in mouse subjects. Five million NCI-N87 cells, J I MT- 1 cells or HCC1569 cells were harvested from culture flasks, and implanted subcutaneously into the right flank of 6- to 7-week-old BALB / c nude mice (Shanghai SLAC Laboratory Animal Co., Ltd) or NSG mice (Shanghai Model Organisms Center, Inc). When turners reached a size of approximately 150-200 mm3, five mice from each tumor model were randomly assigned to different groups. The groups include Herceptin ADC groups, reference ADC group, and vehicle control group, where the vehicle control means the buffer used for diluting ADCs was injected and used as negative control. ADCs or vehicle control was administered intravenously via the lateral tail vein at the indicated doses. Tumor volumes were measured twice weekly with calipers. The tumor volumes were calculated using the formula: L x L x W2; where L = length and W = width.

[0368] The results are shown in Figure 12-16. Figure 12-16 depict in vivo tumor volume change after administering ADCs for JIMT-1 , NCI-N87, and HCC-1569 models. It can be seen that the tested ADCs (ADC-4, ADC-5, ADC-14, ADC-22, and ADC-23) all demonstrate a potent anti-tumor activity in human xenograft tumor models.Example 46. in vivo Tolerability and Safety

[0369] Heathy adult female CD-1 (ICR) mice were used to determine the toxicity profile of camptothecin derivatives ADCs. Mice were purchased from Charles River Laboratories (Beijing, China). After a week of adaptive feeding, mice were randomized into groups (0=5 per group) based on the body weight using serpentine arrangement: blank control, negative control (PBS groups), and 160 mg / kg administration groups (ADC groups). For negative and ADC administration groups, PBS or ADCs (ADC-12, ADC-14, ADC-19, ADC-20, ADC-21, ADC-22,ADC-23) diluted in PBS were administered intravenous injection via tail veins. For blank control group, no injection was performed. Whole blood was drawn through orbital bleeding at various timepoints (such as 3 and 7 days post administration, or at 2 and 5 days post administration) , and was transferred to EDTA-K2 tubes for further analysis. Whole blood counts were determined using XN-1000V automated hematology analyzer (Sysmex Asia Pacific Pte Ltd., Japan). Nine hematology parameters were analyzed: erythrocytes, hemoglobin, platelet, reticulocytes, white blood cells, lymphocytes, neutrophils, monocytes, eosinophils, and basophils. The body weight of the mice was monitored throughout the experiment as well.

[0370] The results are shown in Figures 17A-B to Figures 23A-B. Figures 17A-B to Figures 23A-B depict / n wVo tolerability and safety regarding various ADCs.

[0371] It can be seen that ADC-14, ADC-19, ADC-20, and ADC-21 show a slight decrease in body weight and WBC, neutrophil, and lymphocyte counts, swiftly recovering within 7 days, indicating great tolerability and safety, even at significantly high doses. ADC-22 and 23 show an excellent tolerability and safety without observing any changes in body weight and WBC counts even at very high doses.

[0372] All of the articles and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the articles and methods of this disclosure have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the articles and methods without departing from the spirit and scope of the disclosure. All such variations and equivalents apparent to those skilled in the art, whether now existing or later developed, are deemed to be within the spirit and scope of the disclosure as defined by the appended claims. All patents, patent applications, and publications mentioned in the specification are indicative of the levels of those of ordinary skill in the art to which the disclosure pertains. All patents, patent applications, and publications are herein incorporated by reference in their entirety for all purposes and to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference in its entirety for any and all purposes. The disclosure illustratively described herein suitably may be practiced in the absence of any element(s) not specifically disclosed herein. Thus, it should be understood that although the present disclosure has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope of this invention as defined by the appended ciaims.

Claims

What is claimed is:1 . A camptothecin (CPT) derivative represented by the foilowing structure formula (A),a pharmaceutically acceptable salt, stereoisomer, enantiomer, or deuterated counterpart thereof; wherein X is selected from -C(=O)-(CH2)n1-O-N(R2)R3, -C(=OHCH2)n1-N(OR2)R3, -C(=O)~ (CH2)n1-O-(CH2)n1-N(R2)R3, -C(=O)-(C(R4)(Rs))n1-N(R2)R3, ’S(=O)2”CH2~(CH2)n1-O-N(R2)R3, - S(=O)2-CH2-(CH2)n1~N(OR2)R3, -S(=O)2~CH2-(CH2)n1-N(R2)R3, -C(=O)-O-(CH2)n2-O-N(R2)R3, - C(=O)-O-(CH2)n2-N(OR2)R3!~C(=O)-O~(CH2)n2-N(R2)R3, -C(=O)-NH-(CH2)n2-O-N(R2)R3, - C(=O)-NH-(CH2)n2-N(OR2)R3, -C(=O)-N(R2)-(CH2)n2-N(R2)R3, -C(=O)-NH-(CH2)n2-O-R3, -C(=O)- NH-(CH2)n2"S-R3, -C(=O)-O~(CH2)n2-O-R3, -C(=O)-S-(CH2)n2-(>R3, -C(-O)-S-(CH2)n2-S-R3, - C(=O)-O-(CH2)n2-S-R3, -C(=O)-(C(R4)(R5))n1-N(R2)-C(=O)-(C(Ra)(Rb))n3-N(Rc)-R3, -C(=O)- (C(R4)(R5))n1-N(R2)-C(=O)-(C(Ra)(Rb))n3-O-R3, -C(=O)-(C(R4)(R5))n1-N(R2)-C(=O)-(C(Ra)(Rb))n3- S-R3;Ri, R2iR3and Rceach independently is a hydrogen atom, or CrCe alkyl;R4and R5each independently represents a hydrogen atom, a deuterium atom, halogen, halogenated alkyl, deuterated alkyl, alkoxy, hydroxyl, amino, nitro, cyano, hydroxyalkyl, heterocyclic CrCe alkyl or 3-6 membered heterocyclic or cycloalyl ring; or R4and R5together with the carbon atom form a 3-6 membered cyclic ring;Raand Rbeach independently represents a hydrogen atom, a deuterium atom, halogen, halogenated alkyl, deuterated alkyl, alkoxy, hydroxyl, amino, nitro, cyano, hydroxyalkyl, heterocyclic C1-C5 alkyl or 3-6 membered heterocyclic or cycloalyl ring; or Raand Rbtogether with the carbon atom form a 3-6 membered cyclic ring; n1is 1 , 2, 3, 4 or 5; n2is 2, 3, 4 or 5; n3is 1 , 2, 3, 4 or 5.

2. The camptothecin derivative, the pharmaceutically acceptable salt, stereoisomer, enantiomer, or deuterated counterpart thereof according to claim 1 , wherein X is selected from -C(=O)-(CH2)n1-O-N(R2)R3, -C(=O)-(CH2)n1-N(OR2)R3, -C(=O)-(CH2)n1-O-(CH2)n1-N(R2)R3, - C(=O)-(C(R4)(R5))n1-N(R2)R3, -S(=O)2-CH2-(CH2)n'i-O-N(R2)R3, -S(=O)2-CH2-(CH2)n1-N(OR2)R3l-S(^=Oh-CH2-(CH2jn1..N(R2)R3i-C(=O)-O-(CH2)n2-O-N(R2)R31-C(=O)-O-(CH2}n2-N(OR2)R3, - C(=O)-O-(CH2)n2-N(R2)R3)-C(=O)~NH-(CH2)n2-O-N(R2)R3, -C(=O)-NH~(CH2)n2-N(OR2)R3, - C(=O)-N(R2)-(CH2)n2-N(R2)R3, -C(=O)-NH-(CH2)n2~O-R3, -C(=O)-NH-(CH2)n2-S-R3, -C(=O)-O- (CH2)n2-O-R3, -C(=O)-S-(CH2)n2-O-R3, -C(=O)-S-(CH2)n2-S-R3, -C(=O)-O-(CH2)n2-S-R3!preferably X is selected from -C(™O)"(CH2)n1"O-N(R2)R31-C(=O)-(CH2)n1-O-(CH2)n1-N(R2)R3, - C(=O)-(C(R4)(Rs))n1-N(R2)R3, -S(=O)2-CH2-(CH2)n1-O-N(R2)R3l-S(=O)2-CH2-(CH2)n1-N(R2)R3l- C(=O)-O-(CH2)n2-O-N(R2)R3, -C(=O)-O~(CH2)n2-N(R2)R3, -C(=O>NH-(CH2)n2-O~N(R2)R3, - C(=O)-N(R2)-(CH2)n2-N(R2)R3;R2is a hydrogen atom;R3is a CrCg alkyl;R4and Rseach independently represents a hydrogen atom, a deuterium atom, halogen, hydroxyl, amino, nitro, cyano.

3. The camptothecin derivative, the pharmaceutically acceptable salt, stereoisomer, enantiomer, or deuterated counterpart thereof according to claim 1 , wherein X is selected from • C(=O)-(CH2)n1-O-N(R2)R3, -C(=O)-(CH2)n1-N(OR2)R3, -C(=O)-(CH2)n1-O-(CH2)n1-N(R2)R3, - C(=O)-(C(R4)(R5))ni-N(R2)R3, -S(=O)2-CH2-(CH2)n1-O-N(R2)R3, -S(=O)2-CH2-(CH2)n1-N(OR2)R3, -S(=O)2-CH2-(CH2)n1-N(R2)R3, -C(=O)-O-(CH2)n2-O-N(R2)R3, -C(=O)-O-(CH2)n2-N(OR2)R3, - C(=O)-O-(CH2)n2-N(R2)R3>-C(=O)-NH~(CH2)n2-O-N(R2)R3, -C(=O)-NH~(CH2)n2-N(OR2)R3, - C(=O)-N(R2)-(CH2)n2-N(R2)R3, -C(=O)-NH-(CH2)n2-O-R3, -C(=O)-NH-(CH2)n2-S-R3, -C(=O)-O- (CH2)n2-O-R3, -C(=O)-S-(CH2)n2-O-R3, -C(=O)-S-(CH2)n2-S-R31-C(=O)-O-(CH2)n2-S-R3, preferably X is selected from -C(=O)-(CH2)n1-O-N(R2)R31-C(=O)-(CH2)n1-O-(CH2)n'i-N(R2)R3, - C(-O)-(C(R4)(Rs))n1-N(R2)R3, -S(=O)2-CH2-(CH2)n1-O-N(R2)R3, -S(=O)2-CH2-(CH2)n1-N(R2)R3, - C(-O)-O-(CH2)n2-O-N(R2)R3, -C(-O)-O-(CH2)n2-N(R2)R3, -C(=O)-NH-(CH2)n2-O-N(R2)R3, - C(=O)-N(R2)-(CH2)n2-N(R2)R3;R2is a hydrogen atom;R3is a CrCs alkyl;R4 and Rs each independently represents a hydrogen atom, a deuterium atom, halogen, halogenated alkyl, deuterated alkyl, alkoxy, hydroxyl, amino, nitro, cyano, hydroxyalkyl, heterocyclic C1-C6alkyl or 3-6 membered heterocyclic or cycloalyl ring; or R4 and Rs together with the carbon atom form a 3-6 membered cyclic ring.

4. The camptothecin derivative, the pharmaceutically acceptable salt, stereoisomer, enantiomer, or deuterated counterpart thereof according to claim 1 , wherein X Is selected from - C(=O)-(CH2)n1-O-N(R2)R3>-C(=O)-(CH2)n1-N(OR2)R3, -C(=O)-(CH2)n1-O-(CH2)n1-N(R2)R3, - C(=O)-(C(R4)(R5))n1-N(R2)R3, -S(=O)2-CH2-(CH2)n1-O-N(R2)R3, -S(=O)2-CH2-(CH2)n1-N(OR2)R3, -S(=O)2-CH2-(CH2)n1-N(R2)R3, -C(=O)-O-(CH2)n2-O-N(R2)R3, -C(=O)-O-(CH2)n2-N(OR2)R3, - C(~O)-O-(CH2)n2-N(R2)R3, -C(=O)-NH-(CH2)n2-O-N(R2)R3, -C(=O)-NH-(CH2)n2-N(OR2)R3, - C(“O)"N(R2)-(CH2)n2-N(R2)R3, -C(=O)-NH-(CH2)n2-O-R3, -C(=O)-NH-(CH2)n2-S-R3, -C(=O)-O- (CH2)n2-O-R3, -C(=O)-S-(CH2)n2-O-R3, -C(=O)-S-(CH2)n2-S-R3, -C(=O)-O-(CH2)n2-S-R3, preferably X is selected from -C(=O)-(CH2)n1-O-N(R2)R3, -C(=:O)-(CH2)n1-O-(CH2)n1-N(R2)R3, - C(=O)-(C(R4)(R5))n1-N(R2)R3, -S(=O)2-CH2-(CH2)ni-O-N(R2)R3, -S(=O)2-CH2-(CK2)n1-N(R2)R3, - C(=O)-O-(CH2)n2-O-N(R2)R3l-C(=O)-O-(CH2)n2-N(R2)R3, -C(-O)-NH-(CH2)n2-O-N(R2)R3, - C(=O)-N(R2)-(CH2)n2-N(R2)R3;R2and R3each is a hydrogen atom;R4 and Rs each independently represents a hydrogen atom, a deuterium atom, halogen, halogenated alkyl, deuterated alkyl, alkoxy, hydroxyl, amino, nitro, cyano, hydroxyalkyl, heterocyclic Ci-C@ alkyl or 3-6 membered heterocyclic or cycloalyl ring; or R4 and R5 together with the carbon atom form a 3-6 membered cyclic ring.

5. The camptothecin derivative, the pharmaceutically acceptable salt, stereoisomer, enantiomer, or deuterated counterpart thereof according to claim 1 , wherein X is selected from - C(=O)-(C(R4)(R5))n1-N(R2)-C(=O)-(C(Ra)(Rb))n3-N(Rc)-R3, -C(=O)-(C(R4)(R5))n1-N(R2)-C(=O)- (C(Ra)(Rb))n3-O-R3, -C(=O)-(C(R4)(R5))ni-N(R2)-C(=O)-(C(Rs)(Rb))n3-S-R3, preferably X is selected from -C(-O)-(C(R4)(Rs))n1-N(R2)-C(=O)-(C(Ra)(Rb))n3-N(Rc)-R3, -C(=O)- (C(R4)(R5))n1-N(R2)-C(=O)-(C(Ra)(Rb))n3-O-R3;R2, R3, and Rceach independently is a hydrogen atom or CrCs alkyl;R4 and R5each independently represents a hydrogen atom, a deuterium atom, halogen, halogenated alkyl, deuterated alkyl, alkoxy, hydroxyl, amino, nitro, cyano, hydroxyalkyl,heterocyclic Ch-Cs alky! or 3-6 membered heterocyclic or cycloalyl ring; or R.s and Rs together with the carbon atom form a 3-6 membered cyclic ring;Raand Rb each independently represents a hydrogen atom, a deuterium atom, halogen, halogenated alkyl, deuterated alkyl, alkoxy, hydroxyl, amino, nitro, cyano, hydroxyalkyl, heterocyclic CrCs alkyl or 3-6 membered heterocyclic or cycloalyl ring; or Raand Rbtogether with the carbon atom form a 3-6 membered cyclic ring.

6. The camptothecin derivative, the pharmaceutically acceptable salt, stereoisomer, enantiomer, and deuterated counterpart thereof according to any one of claims 1 to 5, wherein the camptothecin derivative is selected from any one of the following:

7. A pharmaceutical composition, comprising the camptothecin derivative, a pharmaceutically acceptable salt, stereoisomer, enantiomer, or deuterated counterpart thereof according to any one of claims 1 to 6, and a pharmaceutically acceptable diluent, carrier, or excipient.

8. A camptothecin derivative conjugate comprising at least one of the camptothecin derivatives, the pharmaceutically acceptable salts, stereoisomers, enantiomers, or deuterated counterparts thereof according to any one of claims 1 to 6; or the pharmaceutically acceptable salt thereof.

9. The camptothecin derivative conjugate according to claim 8 comprising: (1) at least one of the camptothecin derivatives, the pharmaceutically acceptable salts, stereoisomers, enantiomers, or deuterated counterparts thereof of any one of claims 1-6; (2) a linker; and (3) a targeting agent? or the pharmaceutically acceptable salt thereof.

10. The camptothecin derivative conjugate or the pharmaceutically acceptable salt thereof according to claim 9, wherein the linker is selected from the group consisting of a cleavable linker and a non-cleavable linker, and wherein the targeting agent is selected from an antibody or antigen binding fragment thereof, peptide, RNA, and DNA molecule.

11. The camptothecin derivative conjugate or the pharmaceutically acceptable salt thereof according to any one of claims 9 to 10, wherein the linker is selected from the group consisting of peptidase cathepsin sensitive linkers, acid sensitive linkers, glutathione sensitive linkers, sulfatase sensitive liners, lysosomal protease-sensitive linkers, beta-glucuronide linkers, phosphatase sensitive linkers, and pyrophosphatase sensitive linkers.

12. The camptothecin derivative conjugate or the pharmaceutically acceptable salt thereof according to any one of ciaims 9 to 11 , wherein the targeting agent is capable of binding to a target selected from the group consisting of a tumor associated antigen (TAA), a tissue-specific antigen, a cell surface molecule, extracellular matrix protein or protease(s), and any post- translational modification residue(s).

13. The camptothecin derivative conjugate or the pharmaceutically acceptable salt thereof according to claim 12, wherein the target is a TAA selected from the group consisting of Trop-2, Her2, Her3, Her4, EGF, EGFR, CD2, CD3, CD5, CD7, CD13, CD19, CD20, CD21 , CD23, CD30, CD33, CD34, CD38, CD46, CD55, CD59, CD69, CD70, CD71 , CD97, CD117, CD123, CD127, CD134, CD137, CD138, CD146, CD147, CD152, CD154, CD174, CD195, CD200, CD205, CD212, CD223, CD227, CD253, CD272, CD274, CD276, CD278, CD279, CD309, CD319, CD326, CD340, DR6, Kv1.3, 5E10, MUC1, uPA, MAGE3, MUC16, KLK3, K-ras, Mesothelin, p53, Survivin, G250, PSMA, Endoplasmin, BCMA, GPNMB, EphA2, EphB2, TMEFF2, Integrin beta s, 5T4, CA9, IGF-1 R, Axl, B7H3, B7H4, CDH6, HAVCR1 , STEAP-1 , STEAP-2, UPK2, CLDN18, CLDN6, CLDN9, c-Met, MICA, LIV-1 , ROR1, ADAM9, Stn, DLK-1 and CEACAM-5,14. The camptothecin derivative conjugate or the pharmaceutically acceptable salt thereof according to any one of claims 12 to 13 wherein the targeting agent is selected from the group consisting of an anti-Her2 antibody or a binding fragment thereof, and an anti-Trop-2 antibody or a binding fragment thereof.

15. The camptothecin derivative conjugate or the pharmaceutically acceptable salt thereof according to any one of claims 9-14, wherein the targeting agent is a multispecific antibody.

16. The camptothecin derivative conjugate or the pharmaceutically acceptable salt thereof of any one of claims 8-15, wherein the camptothecin derivative conjugate is represented by the following formula (I),Targeting agent-(L-D)n(I), wherein n is an integer of 1 to 24;D represents at least one of the camptothecin derivatives, the pharmaceutically acceptable salts, stereoisomers, enantiomers, or deuterated counterparts thereof of any one of claims 1-6;L is a linker comprising a peptide moiety of 2-8 amino acids represented by the following formula (Ila and lib),L1-L2-L3- (Ha)U-L2- (Hb) wherein L1is a linker moiety attached to the targeting agent, and L1comprises a reacted functional group selected from maleimide, bromoacetyl, iodoacetyl, thiol, amino, alkyl bromide, alkyl iodide, allenamide, carboxyl, and NHS ester;L2is a linker moiety comprising 2-8 amino acid peptides, and optionally spacers, preferably PEG spacer;L3is a linker moiety connected to the camptothecin derivative, and L3comprises at least one of the following:

17. The camptothecin derivative conjugate or the pharmaceutically acceptable salt thereof according to claim 16, wherein the L1is selected from -C(=O)-(CH2)ni-, and -C(=;O)"(CH2)n1-R8- [O-(CH2)ni]ni-N(R2)-C(=;O)“ )-(CH2)ni~; Rs is alkylene-aryl-alkylene, aryl, 3-7 membered heterocyclyl, 3-7 membered cycloalkyl, heteroaryl, -alkylene-heteroaryl-alkylene-; n1is 1 , 2, 3, 4 or 5.

18. The camptothecin derivative conjugate or the pharmaceutically acceptable salt thereof according to any one of claims 16 to 17, wherein L is selected from any one of the following:

19. The camptothecin derivative conjugate or the pharmaceutically acceptable salt thereof according to any one of claims 16 to 18, L2is a dipeptide, tripeptide or tetrapeptide comprising naturally occurring and non-naturally occurring amino acids.

20. The camptothecin derivative conjugate or the pharmaceutically acceptable salt thereof according to any one of claims 16 to 19, wherein L2is selected from: gly-gly. gly-gly-giy, phe-lys, val-ala, val-cit, gly-gly-phe-gly (GGFG)(SEQ ID NO: 1), val-cit-gly, val-gln-gly, val-glu-gly, phe* lys-gly, glu-val-ala, glu-val-cit, p-ala-gly-phe-gly (AGFG)(SEQ ID NO: 2), and gly-gly-phe-gly-gly (GGFGG)(SEQ ID NO: 3), where the amino acid sequence is in either orientation.

21. The camptothecin derivative conjugate or the pharmaceutically acceptable sait thereof according to any one of claims 16 to 19, wherein the camptothecin derivative conjugate comprises any one of the following structures.

22. The camptothecin derivative conjugate or the pharmaceutically acceptable salt thereof according to any one of claims 16 to 21 , having a ratio of the camptothecin derivative to the targeting agent from 4 to 12.23, The camptothecin derivative conjugate or the pharmaceutically acceptable salt thereof according to claim 22, comprising any one of the following structures,24. A pharmaceutical composition comprising the camptothecin derivative conjugate or the pharmaceutically acceptable salt thereof according to any one of claims 8*23, and a pharmaceutically acceptable diluent, carrier, or excipient.

25. A method of making the camptothecin derivative conjugate or the pharmaceutically acceptable salt thereof according to any one of ciaims 8 to 23, comprising reacting a targeting agent and / or a linker with a camptothecin derivative.

26. A method of treating or preventing a disease, comprising administering to a subject in need thereof a therapeutically effective amount of the camptothecin derivative, a pharmaceutically acceptable salt, stereoisomer, enantiomer, or deuterated counterpart thereof according to any one of claims 1-6.

27. A method of treating or preventing a disease, comprising administering to a subject in need thereof a therapeutically effective amount of the camptothecin derivative conjugate or the pharmaceutically acceptable salt thereof according to any one of claims 8-23.

28. The method according to any one of claims 26 to 27, further comprising administering a second therapy to the subject,29. The method according to any one of claims 26 to 28, wherein the disease is selected from the group consisting of a proliferative disorder, an autoimmune disorder, destructive bone disorder, infectious disease, viral disease, fibrotic disease, neurodegenerative disorder, pancreatitis and kidney disease.

30. The method according to claim 29, wherein the proliferative disorder is a cancer selected from the group consisting of pancreatic cancer, gastric cancer, liver cancer, breast cancer, ovarian cancer, colorectal cancer, melanoma, leukemia, myelodysplastic syndrome, lung cancer, prostate cancer, brain cancer, bladder cancer, head-neck cancer, and rhabdomyosarcoma.

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