Glucocorticoid antibody drug conjugates

Abstract

Disclosed herein are antibody-drug-conjugates (ADC) that allow for delivery and release of glucocorticoid receptor modulators into desired tissues. The disclosed ADCs include a peptide that facilitates the release of a glucocorticoid receptor modulator by legumain. The antibody-drug-conjugates are useful as therapeutic agents for treating various autoimmune disorders, inflammatory disorders, and blood cancers. Pharmaceutical compositions which include the ADCs are also disclosed, as are methods of treating autoimmune disorders, inflammatory disorders, and blood cancers by administering a therapeutically effective amount of an antibody-drug-conjugate disclosed herein under conditions effective to treat said disorders.

Description

GLUCOCORTICOID ANTIBODY DRUG CONJUGATESCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority of US provisional application 63 / 724,465, filed November 25, 2024, the entire disclosure of which is hereby incorporated herein by reference.GOVERNMENT RIGHTS STATEMENT

[0002] This invention was made with government support under R01 GM144450 awarded by National Institutes of Health. The government has certain rights in the invention.FIELD OF THE INVENTION

[0003] The invention relates to the targeted delivery of glucocorticoids. In particular, the present invention includes compounds and antibody-drug-conjugates (ADC) that allow for delivery and release of the glucocorticoids into desired antigen expressing cells and tissues. Compounds of the present invention are thus useful as therapeutic agents for treating various inflammatory diseases, autoimmune disorders, and cancers.BACKGROUND OF THE INVENTION

[0004] Antibody-drug conjugates (ADCs) are antibodies loaded with drug payloads that allow for the delivery of those drugs to a desired cell type. The specificity of antibodies to a specific cell type results in more targeted delivery of the drugs with fewer negative side effects due to reduced exposure of the often cytotoxic drugs in healthy cells.

[0005] Glucocorticoids are broadly useful for a variety of diseases and disorders, including the treatment of inflammation, autoimmune diseases, allergies, and some cancers. However, glucocorticoids are often associated with significant adverse effects such as bone loss, hormone issues, weight gain, digestive issues, insomnia, mood changes and glucose elevation.

[0006] Fluticasone is a glucocorticoid that is on the market for the treatment of various inflammatory disorders. Fluticasone is administered nasally, topically, or orally, depending on the disorder being treated. However, as with other glucocorticoids, fluticasone can also cause adverse symptoms, such as nausea and vomiting, headaches, dizziness, shortness of breath, diarrhea, and nosebleeds.

[0007] The present disclosure is directed to overcoming these and other deficiencies in the art.SUMMARY OF THE INVENTION

[0008] Briefly, the present invention provides glucocorticoid receptor modulators and antibody-drug-conjugates (ADC) that allow for delivery and release of the glucocorticoid receptor modulators into desired tissues.

[0009] The present invention provides, in a first aspect, a compound of the Formula (I):OZ1-X1-A°-A1-A2-A3A4^O-GC(I)wherein:Z1is a conjugation handle;X1is a bond or is a spacer unit selected from branched or unbranched C1-C12alkyl, a PEG selected from PEG1 to PEG12, " 1-12?,A0is absent or is a natural or non-natural amino acid;A1is selected from Asn, Ala, Gly, Vai, and He;A2is selected from Asn, Cit, and Ala;A3is Gly or Ala;A4is selected from L-Pro, D-Pro, alpha-methyl-Pro, Hydroxy Proline, and homoproline; andGC is a glucocorticoid receptor modulator with a steroidal structure attached via the C-l 1 hydroxyl.

[0010] The present invention provides, in a second aspect, an antibody-drug conjugate of Formula (A):OAb-Z-X1A° A1A2A3A4^O-GC(A),wherein:Ab is an antibody;Z is a conjugation moiety;X1is a bond or is a spacer unit selected from branched or unbranched C1-C12 oalkyl, a PEG selected from PEG1 to PEG12, 1-12oA0is absent or is a natural or non-natural amino acid;A1is selected from Asn, Ala, Gly, Vai, and He;A2is selected from Asn, Cit, and Ala;A3is Gly or Ala;A4is selected from L-Pro, D-Pro, alpha-methyl-Pro, Hydroxy Proline, and homoproline; andGC is a glucocorticoid receptor modulator with a steroidal structure attached via the C-l 1 hydroxyl.

[0011] The present invention provides, in a third aspect, a pharmaceutical composition comprising a compound of the Formula (I) disclosed herein and a pharmaceutically acceptable carrier, diluent, or excipient.

[0012] The present invention provides, in a fourth aspect, a pharmaceutical composition comprising an antibody-drug conjugate of Formula (A) disclosed herein and a pharmaceutically acceptable carrier, diluent, or excipient.

[0013] The present invention provides, in a fifth aspect, a method for treating inflammation in a subject. The method includes administering a therapeutically effective amount of a compound of Formula (I) described herein under conditions effective to treat inflammation.

[0014] The present invention provides, in a sixth aspect, a method for treating inflammation in a subject. The method includes administering a therapeutically effective amount of an antibody-drug conjugate of Formula (A) described herein under conditions effective to treat inflammation.

[0015] The present invention provides, in a seventh aspect, a method for an autoimmune disease or disorder in a subject. The method includes administering a therapeutically effectiveamount of a compound of Formula (I) described herein under conditions effective to treat an autoimmune disease or disorder.

[0016] The present invention provides, in an eighth aspect, a method for treating a tumor or abnormal cell proliferation in a subject. The method includes administering a therapeutically effective amount of an antibody-drug conjugate of Formula (A) described herein under conditions effective to treat an autoimmune disease or disorder.

[0017] The present invention provides, in a ninth aspect, a method for treating a tumor or abnormal cell proliferation in a subject. The method includes administering a therapeutically effective amount of a compound of Formula (I) described herein under conditions effective to treat a tumor or abnormal cell proliferation. In some aspects, the abnormal cell proliferation is a blood cancer.

[0018] The present invention provides, in a tenth aspect, a method for treating a tumor or abnormal cell proliferation in a subject. The method includes administering a therapeutically effective amount of an antibody-drug conjugate of Formula (A) described herein under conditions effective to treat a tumor or abnormal cell proliferation. In some aspects, the abnormal cell proliferation is a blood cancer.

[0019] The present invention provides, in an eleventh aspect, a method for inhibiting or reducing rejection of a tissue or organ transplant in a subject. The method includes administering a therapeutically effective amount of a compound of Formula (I) described herein under conditions effective to inhibit or reduce rejection of a tissue or organ transplant. In some aspects, the organ is a kidney.

[0020] The present invention provides, in an eleventh aspect, a method for inhibiting or reducing rejection of a tissue or organ transplant in a subject. The method includes administering a therapeutically effective amount of an antibody-drug conjugate of Formula (A) described herein under conditions effective to inhibit or reduce rejection of a tissue or organ transplant. In some aspects, the organ is a kidney.

[0021] These, and other objects, features and advantages of this invention will become apparent from the following detailed description of the various aspects of the invention taken in conjunction with the accompanying drawings.BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIG. 1 is an illustration of the chemical design and key features of one embodiment of the ADC technology disclosed herein.

[0023] FIG. 2 shows the stability of ADCs disclosed herein in human and mouse plasma.

[0024] FIG. 3 illustrates the lysosomal stability of ADCs disclosed herein and the payload release in tritosomes.

[0025] FIG. 4 demonstrates luciferase induction in MDA-kb2 cell line by ADCs disclosed herein, in contrast to prior generation ADCs.

[0026] FIG. 5 shows the results of testing of inhibition of NF -Kb activation and cytokine release by glucocorticoid ADCs disclosed herein.

[0027] FIG. 6 illustrates the results of testing of glucocorticoid ADCs disclosed herein in a contact hypersensitivity mouse model.DETAILED DESCRIPTION OF THE INVENTION

[0028] Antibody-drug conjugate technology has been developed that allows for the release of ultrapotent glucocorticoids in antigen expressing cells. This technology may be useful for the treatment of autoimmune diseases, inflammatory disorders, or in the treatment of various leukemias and lymphomas.

[0029] A stable linker system has been developed that undergoes self-immolation to release these ultrapotent glucocorticoids, including fluticasone derivatives. The ADCs disclosed herein rely on a two-step process to release the warhead. The first cleavage event is facilitated by a lysosomal enzyme such as cathepsin or legumain. Both cathepsin and legumain (also known as asparaginyl endopeptidase) are proteases known to be overexpressed in a variety of cancers and are widely found in the lysosomes of most tumor cells. The disclosed ADCs include peptides that facilitate the cleavage by legumain or cathepsin.

[0030] There are several unique features of this ADC technology that render it useful, including for in vivo studies and clinical translation:• Incorporation of highly polar peptide linkers that imparts excellent biophysical properties, allowing up to 8 drugs per antibody without any meaningful aggregation.This is particularly interesting because glucocorticoids are very hydrophobic molecules that traditionally have been difficult to conjugate without aggregation. In some cases these peptides are asparagine-containing peptides that are cleaved by legumain.• The use of a “traceless” self-immolative linker that is stable in mouse plasma and human plasma for up to nine days. The drug is linked via an ester bond to the sterically hindered 11 -hydroxyl on the C-ring, making the resulting conjugate highly resistant to esterase-mediated degradation. It has been found, surprisingly, that this attachment at C-l 1 results in high plasma stability (see Figure 2) as compared with previous technology in which the attachment was at a different position (e.g., C-21). For example, a related bioconjugate attached via the C-21 position resulted in 100% payload release after just 4 days in mouse serum. (Bioorg Med Chem Lett. 2022 Nov 1:75:128953. doi: 10.1016 / j.bmcl.2022.128953)• The ability to deliver highly potent glucocorticoids that deliver a strong pharmacological response even when limited by low numbers of cell surface antigens.

[0031] A series of ADC linker-payloads have been developed that release ultra-potent glucocorticoids. Some of these glucocorticoids are fluticasone or related compounds. In some cases, the antibody directly targets the diseased or healthy cells, such as leukocytes, and delivers the glucocorticoid directly to the targeted cells This includes antibodies such as anti-CD38, anti-TNF, anti-CDlla, anti-CD20, anti-CD19, anti-CD79, and the like. Targeted cells include B-cell, T-cells, macrophages, monocytes, neutrophils, eosinophils, dendritic cells, and the like. In other cases, indirect targeting is used, that is, the antibody targets a tissue associated with a disease and the glucocorticoid diffuses to nearby cells and exerts its antiinflammatory potential.

[0032] In some embodiments, the compound is of Formula (I):OZ1-X1-A° A1-A2A3A4-^O-GC (I)wherein:Z1is a conjugation handle;X1is a bond or is a spacer unit selected from branched or unbranched C1-C12A0is absent or is a natural or non-natural amino acid;A1is selected from Asn, Ala, Gly, Vai, and He;A2is selected from Asn, Cit, and Ala;A3is Gly or Ala;A4is selected from L-Pro, D-Pro, alpha-methyl-Pro, Hydroxy Proline, and homoproline; andGC is a glucocorticoid receptor modulator with a steroidal structure attached via the C-l 1 hydroxyl.

[0033] In some embodiments, the antibody-drug conjugate is of Formula (A):0Ab-Z-X1A° A1A2A3A4^O-GC (A)wherein:Ab is an antibody;Z is a conjugation moiety;X1is a bond or is a spacer unit selected from branched or unbranched C1-C12 oalkyl, a PEG selected from PEG1 to PEG12, 1-12oA0is absent or is a natural or non-natural amino acid;A1is selected from Asn, Ala, Gly, Vai, and He;A2is selected from Asn, Cit, and Ala;A3is Gly or Ala;A4is selected from L-Pro, D-Pro, alpha-methyl-Pro, Hydroxy Proline, and homoproline; andGC is a glucocorticoid receptor modulator with a steroidal structure attached via the C-l 1 hydroxyl.

[0034] In some embodiments, Z1is an amine. In other embodiments, Z1is a Michael acceptor. In other embodiments, Z1comprises an alkyne. In some embodiments, Z1is an a, P-unsaturated carbonyl. In other embodiments, Z1is selected from:In some embodiments, Z1isThese examples of conjugation handles are not meant to be exhaustive, as those skilled in the art could readily envision a variety of other useful conjugation handles.

[0035] In some instances of Zl, the NH moiety shown next to the “squiggly line”O0Br. I, CL Ols. OMs. A.. A N I Hconnection point, such as inand NHAc,may come from the next connecting element, such as X1, A0or A1. For example, the NH may arise from the amino acid represented as A0or A1. This delineation can be seen in Examples 14 and 15, for instance. The person of skill will understand that there will not be two NH moieties in a row between Zl and its next connection, i.e., the depiction of Zl together with an amino group from A0or A1is not intended to represent an acyl hydrazide.

[0036] In some embodiments, X1is a bond. In other embodiments, X1is a spacer unit selected from branched or unbranched C1-C12 alkyl, a branched or unbranched PEG selectedfrom PEG1 to PEG12,. Insome embodiments, X1is embodiments, X1is a branched or unbranched C1-C12 alkyl. In some embodiments, X1is aPEG selected from PEG1 to PEG12. In other embodiments, X1is1-12. In someembodiments, X1is. In other embodiments, X1is

[0037] In some embodiments, A0is absent. In other embodiments, A0is a natural amino acid. In some embodiments, A0is a non-natural amino acid. In other embodiments, A0is Gly, Ser, Glu, or Asp. In still other embodiments, A0is Gly.

[0038] In some embodiments, A1is selected from Asn, Ala, Gly, Vai, and He. In other embodiments, A1is Asn or Ala. In some embodiments, A1is Asn. In other embodiments, A1is Ala.

[0039] In some embodiments, A2is selected from Asn, Cit, and Ala. In other embodiments, A2is Asn.

[0040] In some embodiments, A3is Gly or Ala. In other embodiments, A3is Gly. In some embodiments, A3is Ala.

[0041] In some embodiments, A4is selected from L-Pro, D-Pro, alpha-methyl-Pro, Hydroxy Proline, and homoproline. In other embodiments, A4is L-Pro. In some embodiments, A4is D-Pro. In other embodiments, A4is alpha-methyl-Pro. In some embodiments, A4is Hydroxy Proline. In other embodiments, A4is homoproline.

[0042] In some embodiments, A1and A2are both Asn. In some embodiments,A4isGly-Asn-Asn-Gly-L-Pro. In other embodiments, A0-A1-A2’ A3'4isVal-Cit-Gly-Pro.

[0043] In some embodiments, GC is a glucocorticoid receptor modulator with a steroidal structure attached via the C-l 1 hydroxyl. In other embodiments, GC is selected from fluticasone furoate, fluticasone propionate, halobetasol, Clobetasol propionate, dexamethasone, prednisolone, amcinonide, beclomethasone dipropionate, betamethasone dipropionate, budesonide, flunisolide, fludrocortisone, methylprednisolone, triamcinolone, ciclesonide, flunisolide, fluocinolone acetonide, fluocinonide, fluorometholone, flurandrenolide, halcinonide, halobetasol propionate, mometasone furoate, triamcinolone acetonide, and beclomethasone. In some embodiments, GC is fluticasone furoate or fluticasone propionate. In other embodiments, GC is fluticasone furoate. In some embodiments, GC is fluticasone propionate. In other embodiments, GC is halobetasol. In some embodiments, GC is Clobetasol propionate. In other embodiments, GC is dexamethasone.

[0044] In some embodiments, Ab is an antibody. In some embodiments, Ab is anti-cMET, anti-IL-31R, anti-BCMA, anti-CSF-lR, anti-HER3, anti-Her2, anti-Trop2, anti-CD38, anti-TNF, anti -CD Ila, anti-CD20, anti-CD19, anti-CD79, anti -DLL, anti-PDl, anti-PD-L1, anti-tissue factor, anti-MASP-2, anti-GPRC5D, anti-FRa, anti-CTLA-4, anti-IL36R, anti-LAG3, anti-gplOO, anti-IFNARl, anti-GD2, anti-IL6R, anti-IGF-lR, anti-Nectin4, anti-VEGFR2, anti-IL-23, anti-IFNgamma, anti-CGRP, anti-CD22, anti-CCR4, anti-CGRP-R, anti-CD4, anti-FGF23, anti-CLDN18.2, anti-IL-5R, anti-PDGFRa, anti-IL-5, anti-IL17a, anti-EGFR, anti-PCSK9, anti-a4p7, anti-IL6, anti-CD30, anti-BLyS, anti-RANK-L, anti-ILip, anti-EPCAM, anti-a4-integrin, anti-CD52, anti-CD33, anti-IL2R, anti-CCLll, anti-CD3, anti-CD27, anti-CD28, anti-CD40, anti-CD51, anti-CD123, anti-CD147, anti-CD152, anti-CEACAM5, anti-DLL4, anti-FGFR2, anti -fibronectin, anti-Folate receptor, anti-glypican, anti-GUCY2C, anti-MIF, anti-MSLN, anti-MUCl, anti-RORl, anti-SLAMF7, anti-STREAP1, anti-VISTA, anti-IL-4Ra, anti-BDCA2, anti-CXCR4, anti-CD163, anti-MSRl, and anti-CD74or anti-TIGIT. In some embodiments, Ab is an anti-Her2 antibody. In other embodiments, Ab is an anti-Trop2 antibody. In some embodiments, Ab is anti-CD38, anti-TNF, anti-CDlla, anti-CD20, anti-CD19, or anti-CD79.

[0045] In some embodiments, Z is a conjugation moiety that attaches to an antibody. A conjugation moiety as described herein includes a moiety that attaches Z as described herein to a moiety of an antibody. The attachment is typically accomplished through reaction with a nucleophilic residue such as a cysteine or lysine, or through an electrophilic residue such as glutamine, aspartic acid, or glutamic acid. Those skilled in the art may envision otherconjugation strategies, including the addition using click-chemistry handles, glycosyl moieties, aldehydes, ketones, and the like. In other embodiments, Z isOIn still other embodiments,Z is 0 These examples of conjugation moieties are not meant to be exhaustive, as those skilled in the art could readily envision a variety of other useful conjugation moieties.

[0046] For simplicity, when an antibody is attached to Z (as in Formula A) and Z is°, the person of skill will understand that, even though Z is formally a succinimide attached to the antibody through a sulfur, it will still be referred to as a maleimide (e.g., mp = maleimidopropionyl; me = maleimidocaproyl). Moreover, a person skilled in the art will understand that the succinimide ring may hydrolyze (“ring open”) during manufacturing, storage, or administration. As explained in the description of Z1 above, the person of skill will understand that there will not be two NH moieties in a rowbetween Z and its next connection, e.g., the depiction of Z together with an amino group from A0or A1is not intended to represent an acyl hydrazide.

[0047] In one aspect, the present invention provides a pharmaceutical composition comprising an ADC described herein and a pharmaceutically acceptable carrier, diluent, or excipient. In one embodiment, the pharmaceutical composition further comprises a therapeutically effective amount of a chemotherapeutic agent that is separate from the ADC.

[0048] In one aspect, the present invention provides a pharmaceutical composition comprising a compound described herein and a pharmaceutically acceptable carrier, diluent, or excipient. In one embodiment, the pharmaceutical composition further comprises a therapeutically effective amount of a chemotherapeutic agent.

[0049] In one aspect, the present invention provides a method for treating inflammation in a subject. The method includes administering a therapeutically effective amount of an ADC or a compound described herein under conditions effective to treat inflammation. In some embodiments, the administering is performed on a subject having respiratory inflammation; this includes, but is not limited to, chronic obstructive pulmonary disease (COPD), emphysema, asthma, rhinitis, and chronic rhinosinusitis. In some embodiments, the administering is performed on a subject having dermatological inflammation; this includes, but is not limited to, atopic dermatitis, eczema, and psoriasis. In some embodiments, the administering is performed on a subject having inflammation of the digestive tract, which includes, but is not limited to, inflammatory bowel disease (IBD).

[0050] In one aspect, the present invention provides a method for treating an autoimmune disease or disorder in a subject. The method includes administering a therapeutically effective amount of an ADC or a compound described herein under conditions effective to treat an autoimmune disease or disorder. In some embodiments, the administering is performed on a subject having rheumatoid arthritis or lupus, as non-limiting examples of an autoimmune disease or disorder.

[0051] In one aspect, the present invention provides a method for treating a blood cancer in a subject. The method includes administering a therapeutically effective amount of an ADC or a compound described herein under conditions effective to treat a blood cancer. Non-limiting examples of blood cancer include leukemia, lymphoma, and myeloma.

[0052] In one aspect, the present invention provides a method for treating a tumor or abnormal cell proliferation in a subject. The method includes administering a therapeutically effective amount of an ADC or a compound described herein under conditions effective to treat a tumor or abnormal cell proliferation. In some embodiments, the administering is performed on a subject having cancer. In some embodiments, the administering is performed on a subject having a tumor. In some embodiments, the tumor or abnormal cell proliferation is cancer. In some embodiments, the cancer is bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, kidney cancer, lung cancer, esophageal cancer, ovarian cancer, prostate cancer, pancreatic cancer, skin cancer, gastric cancer, testicular cancer, biliary cancer, colorectal cancer, endometrial cancer, head and neck cancer, medullary thyroid cancer, renal cancer, eye cancer, neuroblastoma, Mycosis fungoides, glial tumor, other brain tumor, spinal cord tumor, liver cancer, leukemia, lymphoma, or any combination thereof. In some embodiments, the tumor is selected from the group consisting of fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing’s tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms’ tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma. In some embodiments, the administering is performed in vitro.

[0053] Glucocorticoids, including those described herein, are widely used in the treatment of a variety of inflammatory and autoimmune diseases. Glucocorticoids exert their effects primarily through intracellular glucocorticoid receptors, which function as ligand-activated transcription factors. Upon entering a cell, the glucocorticoid molecule diffuses across the plasma membrane and binds to its cytosolic receptor, inducing a conformational change that allows the receptorligand complex to translocate into the nucleus. There, it binds to specific DNA sequences known as glucocorticoid response elements, either up-regulating the transcription of anti-inflammatory genes or down-regulating the transcription of pro-inflammatory genes. Due to this robust andwell-characterized anti-inflammatory mechanism, it is anticipated that the glucocorticoidbioconjugates of the present invention will have broad utility in many inflammation and immune-related disorders and conditions. Below are described a variety of disease states amenable to treatment with the compounds of the present invention.

[0054] Systemic lupus erythematosus: Glucocorticoids constitute a fundamental component of the therapeutic regimen for systemic lupus erythematosus (SLE) due to their capacity to rapidly suppress inflammatory activity and modulate immune system dysfunction. In clinical practice, these agents — most commonly prednisone, methylprednisolone, and related corticosteroids — are employed to achieve prompt control of lupus exacerbations involving the skin, joints, kidneys, central nervous system, hematologic system, or other organ systems. The dosing of glucocorticoids is determined by the severity of the disease manifestations, ranging from low-dose administration for mild disease activity to moderate- or high-dose therapy for more significant inflammatory involvement. In circumstances wherein the patient exhibits severe or organ-threatening manifestations, clinicians may administer high-dose intravenous “pulse” methylprednisolone for short durations in order to provide rapid immunosuppression and mitigate the risk of permanent tissue injury. Contemporary treatment strategies emphasize reduction to the lowest effective dose of glucocorticoids, or discontinuation when clinically feasible, due to the well-documented adverse consequences associated with prolonged corticosteroid exposure. Such risks include metabolic disturbances, increased susceptibility to infection, bone density loss, ocular complications, cardiovascular effects, and various systemic or psychological effects.Accordingly, compounds of the present invention may be useful for alleviating many of the clinical manifestations of lupus while minimizing the risk of adverse events classically associated with glucocorticoid therapy. (See Ruiz-Irastorza et al., Rheumatology, 51(7), July 2012, pp 1145-1153 (https: / / doi.org / 10.1093 / rheumatology / ker410); Apostolopoulos et al., Rheumatology, 56 (suppl_l), April 2017, pp i 114— i 122 (https: / / doi.org / 10.1093 / rheumatology / kew406); and Martin-Iglesias et &\, Medilerr J Rheumatol. 2024 Jun 30; 35(Suppl 2), pp 342-353(doi: 10.31138 / mjr.230124.uos).)

[0055] Rheumatoid arthritis: Glucocorticoids serve as an established therapeutic option in the management of rheumatoid arthritis, principally due to their capacity to provide prompt mitigation of inflammatory activity and symptomatic relief while longer-acting diseasemodifying therapies take effect. In clinical practice, these agents are employed to attenuate synovial inflammation, reduce joint swelling, and prevent short-term functional decline,particularly during periods of acute disease exacerbation. Their use is generally circumscribed to the lowest effective dose and shortest feasible duration, consistent with prevailing standards of care and risk-management principles, given the potential for significant adverse effects associated with prolonged exposure. Accordingly, compounds described herein may be useful for alleviating many of the clinical manifestations of rheumatoid arthritis while minimizing the risk of adverse events classically associated with glucocorticoid therapy. (See Conn D., Seminars in Arthritis and Rheumatism, 51(1), February 2021, pp 15-19 (https: / / doi. Org / 10.1016 / j.semarthrit.2020.09.016); Hua C et al., RMD Open. 2020;6:e000536 (https: / / doi. Org / 10.l 136 / rmdopen-2017-000536); Ferreira et al., Rheumatic Disease Clinics of North America, 42(1), 2016, pp 33-46 (DOI: 10.1016 / j.rdc.2015.08.006 ).)

[0056] Multiple sclerosis: Glucocorticoids are employed in the treatment of multiple sclerosis primarily to manage acute relapses by rapidly reducing inflammation and stabilizing the bloodbrain barrier, thereby diminishing the severity and duration of neurologic symptoms. High-dose intravenous methylprednisolone is typically administered over a short course to expedite recovery from relapse-associated deficits, although it does not alter the long-term progression of the disease. Their use is thus limited to episodic intervention rather than chronic therapy, and clinicians balance their short-term benefits against the risk of adverse effects, employing glucocorticoids only when clinically warranted to restore neurologic function and reduce acute inflammatory activity.Accordingly, compounds described herein may be useful for managing flare-up events of multiple sclerosis while minimizing the risk of adverse events classically associated with glucocorticoid therapy. (See Reichardt, H. M. et al., Expert Review of Neurotherapeutics, 6(11), 2006, pp 1657- 1670 (https: / / doi. Org / 10.1586 / 14737175.6.ll.1657); Frohman, E. M. et al., Neurotherapeutics, 4, 2007, pp 618-626 (https: / / doi. Org / 10.1016 / j.nurt.2007.07.008).)

[0057] Crohn’s disease: Glucocorticoids are utilized in the treatment of Crohn’s disease primarily for the induction of remission during periods of active inflammation, as they provide rapid and effective suppression of the immune-mediated processes driving intestinal injury.Agents such as prednisone or budesonide are commonly administered to reduce abdominal pain, diarrhea, and other inflammatory symptoms, but their role is limited to short-term management because they do not promote mucosal healing or prevent long-term disease progression.Accordingly, glucocorticoids are not considered appropriate for maintenance therapy, and their use is strategically confined to acute flares, with subsequent transition to steroid-sparing agents to minimize the significant adverse effects associated with prolonged corticosteroid exposure.Accordingly, compounds of the present invention may be useful for managing acute flares associated with Crohn’s disease, while minimizing the risk of adverse events classically associated with glucocorticoid therapy. (See d / 77. J Gastroenterology 97(4):p 803-823, April 2002, DOI: 10.1111 / j.l572-0241.2002.05596.x; https: / / doi.0rg / lO.llll / j.1365-2036.2007.03379.x.)

[0058] Transplant rejection: Glucocorticoids play a critical role in the prevention and management of transplant rejection by providing rapid, broad immunosuppression that dampens T-cell activation and inflammatory cytokine production, both central drivers of graft injury. They are routinely included in perioperative immunosuppressive regimens and are often administered in high-dose “pulse” form to treat acute rejection episodes, where swift suppression of immune activity is essential to preserving graft function. Although effective in reversing or reducing the severity of rejection, glucocorticoids are used judiciously due to their well-recognized adverse effect profile, and long-term graft maintenance typically relies on other immunosuppressive agents to minimize cumulative steroid toxicity. Accordingly, compounds disclosed herein may be useful for prevention of allograft rejection while minimizing the risk of adverse events classically associated with glucocorticoid therapy. (See De Lucena, D. D., & Rangel, E. B. (2018). Glucocorticoids use in kidney transplant setting. Expert Opinion on Drug Metabolism & Toxicology, 1023-1041, https: / / doi.org / 10.1080 / 17425255.2018.1530214; Dashti-Khavidaki, World J Transplant 2021; 11(11): 443-465 [PMID: 34868896 DOI:10.5500 / wjt.vll.il 1.443].)

[0059] Cancer: Glucocorticoids are employed in the treatment of cancer for their potent antiinflammatory, immunosuppressive, and cytotoxic effects on certain malignant cell types, particularly lymphoid cancers. In hematologic malignancies such as leukemias and lymphomas, they can induce apoptosis of lymphocytes and thereby serve as integral components of multiagent chemotherapy regimens. In spite of their utility, the use of glucocorticoids must be carefully balanced against the well-known risks of prolonged corticosteroid exposure.Accordingly, compounds disclosed herein may be useful for the treatment of hematological malignancies while minimizing the risk of adverse events classically associated with glucocorticoid therapy. (See Pufall, M. A. (2015). Glucocorticoids and Cancer. In: Wang, JC., Harris, C. (eds) Glucocorticoid Signaling. Advances in Experimental Medicine and Biology, vol 872. Springer, New York, NY. https: / / doi.org / 10.1007 / 978-l-4939-2895-8_14; Blood (2023) 142 (Supplement 1): 2986, https: / / doi.org / 10.1182 / blood-2023-174325.)Abbreviations and Definitions

[0060] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this disclosure belongs. A comprehensive list of abbreviations utilized by organic chemists (i.e., persons of ordinary skill in the art) appears in the first issue of each volume of the Journal of Organic Chemistry. The list, which is typically presented in a table entitled “Standard List of Abbreviations” is incorporated herein by reference. In the event that there is a plurality of definitions for terms cited herein, those in this section prevail unless otherwise stated.

[0061] The following abbreviations and terms have the indicated meanings throughout:Ac = acetylADC = antibody drug conjugateAq = aqueousBoc = t-butyloxy carbonylBu = butylc- = cycloDAR = drug-antibody ratioDCM = dichloromethane = methylene chloride = CH2Cl2DMA = dimethylacetamideDMF = N, N-dimethylformamideDPBS = Dulbecco’s phosphate buffered salineeq. Or equiv. = equivalent(s)Et = ethylh or hr = hour(s)HATU = hexafluorophosphate azabenzotriazole tetramethyl uronium HOBt = hydroxybenzotriazoleIRF = Interferon Regulatory Factorme = maleimidocaproylmCPBA = meta-Chloroperoxybenzoic acidMe = methylmin. = minute(s)mp = maleimidopropionylPAB = 4-aminobenzylPABC = p-aminobenzylcarbamatePg = protecting groupPh = phenylPNGase F = Peptide: N-glycosidase FPNP = p-nitrophenolRT = room temperaturesat’d or sat. = saturatedSEAP = secreted embryonic alkaline phosphataseSTD = standard deviationt- or tert = tertiaryTCEP = tris(2-carboxyethyl)phosphineTFA = trifluoroacetic acidTHF = tetrahydrofuranTosyl = p-toluenesulfonylTPPMS = triphenylphosphine meta sulfonic acidUPLC = ultra performance liquid chromatography

[0062] Throughout this specification the terms and substituents retain their definitions.Substituents (e.g., An) are generally defined when introduced and retain that definition throughout the specification and in all independent claims. The descriptions of the elements below refer to any of the compound of Formula (I) or the ADC of Formula (A), unless specifically noted. That is, if an element is present in Formula (I) and Formula (A) — for instance, A3— then the description of that element is germane to any one of Formula (I) and Formula (A). However, if, for instance, an element is not present in Formula (I) — e.g., element “Z” — then the descriptions of element Z are only germane to Formula (A).

[0063] As used herein, the terms “comprising” and “including” or grammatical variants thereof are to be taken as specifying the stated features, integers, steps or components but do not preclude the addition of one or more additional features, integers, steps, components or groups thereof. This term encompasses the terms “consisting of’ and “consisting essentially of.”

[0064] The phrase “consisting essentially of’ or grammatical variants thereof when used herein are to be taken as specifying the stated features, integers, steps or components but do not preclude the addition of one or more additional features, integers, steps, components or groups thereof, but only if the additional features, integers, steps, components or groups thereof do not materially alter the basic and novel characteristics of the claimed composition or method.

[0065] For purposes of the present disclosure, the term “antibody” ( or “Ab” or “AB”) herein is used in the broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies e.g., bispecific antibodies), genetically engineered forms of the antibodies, and combinations thereof. In certain aspects, the antibody or other such targeting molecule acts to deliver a drug to the particular target cell population with which the antibody or other targeting molecule interacts. In one embodiment, “Ab” comprises an antibody. While some specific examples ofantibodies (i.e., “Ab”) are disclosed herein, antibodies that can successfully be used are not limited to these examples, as the person of skill will understand.

[0066] The term “antibody,” which is used interchangeably with the term “immunoglobulin,” includes full length (i.e., naturally occurring or formed by normal immunoglobulin gene fragment recombinatorial processes) immunoglobulin molecules (e.g., an IgG antibody).Antibody fragments, which again may be naturally occurring or synthetic in nature, may also be included. Accordingly, the term “antibody fragment” includes a portion of an antibody such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv and the like. Regardless of structure, an antibody fragment binds with the same antigen that is recognized by the full-length antibody.Antibodies may be naturally or artificially glycosylated or deglycosylated or aglycosyl.Methods of making and screening antibody fragments are well-known in the art.

[0067] Naturally occurring antibodies typically have two identical heavy chains and two identical light chains, with each light chain covalently linked to a heavy chain by an interchain disulfide bond and multiple disulfide bonds further link the two heavy chains to one another. Individual chains may fold into domains having similar sizes (110-125 amino acids) and structures, but different functions. The light chain can comprise one variable domain (VL) and / or one constant domain (CL). The heavy chain can also comprise one variable domain (VH) and / or, depending on the class or isotype of antibody, three or four constant domains (CHI, CH2, CH3, and CH4). The variable region binds to and interacts with a target antigen. The variable region includes a complementary determining region (CDR) that recognizes and binds to a specific binding site on a particular antigen. The constant region may be recognized by and interact with the immune system (see, e.g, Janeway et al., IMMUNOBIOLOGY, 5th Ed., Garland Science (New York 2001), which is hereby incorporated by reference in its entirety). An antibody can be of any type or class (e.g., IgG, IgE, IgM, IgD, and IgA) or subclass (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2). In humans, the isotypes are IgA, IgD, IgE, IgG, and IgM, with IgA and IgG further subdivided into subclasses or subtypes (IgAl -2 and IgGl -4). The antibody can be derived from any suitable species. In some embodiments, the antibody is of human or murine origin. An antibody can be, for example, human, humanized or chimeric. “Antibody,” as used herein, includes heavychain antibodies, which are antibodies that lack light chains and only contain two heavy chains. Non-limiting examples of such heavy-chain antibodies include immunoglobulin new antigen receptor (IgNAR) and heavy chain IgG (hcIgG).

[0068] Generally, the variable domains show considerable amino acid sequence variability from one antibody to the next, particularly at the location of the antigen-binding site. Three regions, called hyper-variable or complementarity-determining regions (CDRs), are found in each of VL and VH, which are supported by less variable regions called framework variable regions. Antibodies include IgG monoclonal antibodies as well as antibody fragments or engineered forms. These are, for example, Fv fragments, or proteins wherein the CDRs and / or variable domains of the exemplified antibodies are engineered as single-chain antigenbinding proteins.

[0069] Single chain antibodies lack some or all of the constant domains of the whole antibodies from which they are derived. Therefore, they can overcome some of the problems associated with the use of whole antibodies. For example, single-chain antibodies tend to be free of certain undesired interactions between heavy-chain constant regions and other biological molecules. Additionally, single-chain antibodies are considerably smaller than whole antibodies and can have greater permeability than whole antibodies, allowing singlechain antibodies to localize and bind to target antigen-binding sites more efficiently.Furthermore, the relatively small size of single-chain antibodies makes them less likely to provoke an unwanted immune response in a recipient than whole antibodies.

[0070] Fab (Fragment, antigen binding) refers to the fragments of the antibody consisting of the VL, CL, VH, and CHI domains. Those generated following papain digestion simply are referred to as Fab and do not retain the heavy chain hinge region. Following pepsin digestion, various Fabs retaining the heavy chain hinge are generated. Those fragments with the interchain disulfide bonds intact are referred to as F(ab')2, while a single Fab' results when the disulfide bonds are not retained. F(ab')2 fragments have higher avidity for antigen than the monovalent Fab fragments.

[0071] Fc (Fragment crystallization) is the designation for the portion or fragment of an antibody that comprises paired heavy chain constant domains. In an IgG antibody, for example, the Fc comprises CH2 and CH3 domains. The Fc of an IgA or an IgM antibody further comprises a CH4 domain. The Fc is associated with Fc receptor binding, activation of complement mediated cytotoxicity and antibody-dependent cellular-cytotoxicity (ADCC). For antibodies such as IgA and IgM, which are complexes of multiple IgG-like proteins, complex formation requires Fc constant domains.

[0072] Finally, the hinge region separates the Fab and Fc portions of the antibody, providing for mobility of Fabs relative to each other and relative to Fc, as well as including multiple disulfide bonds for covalent linkage of the two heavy chains.

[0073] Antibody “specificity” refers to selective recognition of an antibody for a particular epitope of an antigen. The term “epitope” includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor or otherwise interacting with a molecule. Epitopic determinants generally consist of chemically active surface groupings of molecules such as amino acids or carbohydrate or sugar side chains and generally have specific three-dimensional structural characteristics, as well as specific charge characteristics. An epitope may be “linear” or “conformational.” In a linear epitope, all of the points of interaction between the protein and the interacting molecule (such as an antibody) occur linearly along the primary amino acid sequence of the protein. In a conformational epitope, the points of interaction occur across amino acid residues on the protein that are separated from one another, i.e., noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. Antibodies that recognize the same epitope can be verified in a simple immunoassay showing the ability of one antibody to block the binding of another antibody to a target antigen. As described herein, the phrases “specifically binds” and “specific binding” refer to antibody binding to a predetermined antigen.

[0074] Useful polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of immunized animals. Useful monoclonal antibodies are homogeneous populations of antibodies to a particular antigenic determinant (e.g., a cancer cell antigen, a viral antigen, a microbial antigen, a protein, a peptide, a carbohydrate, a chemical, nucleic acid, or fragments thereof). A monoclonal antibody (mAb) to an antigen-of-interest can be prepared by using any technique known in the art which provides for the production of antibody molecules by continuous cell lines in culture.

[0075] 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 directedagainst a single antigenic site. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.

[0076] Monoclonal antibodies may be murine, human, humanized, or chimeric. A humanized antibody is a recombinant protein in which the CDRs of an antibody from one species; e.g., a rodent, rabbit, dog, goat, horse, or chicken antibody (or any other suitable animal antibody), are transferred into human heavy and light variable domains. The constant domains of an antibody molecule are derived from those of a human antibody. Methods for making humanized antibodies are well known in the art. Chimeric antibodies preferably have constant regions derived substantially or exclusively from human antibody constant regions and variable regions derived substantially or exclusively from the sequence of the variable region from a mammal other than a human. The chimerization process can be made more effective by also replacing the variable regions — other than the hyper-variable regions or the complementarity — determining regions (CDRs), of a murine (or other non-human mammalian) antibody with the corresponding human sequences. The variable regions other than the CDRs are also known as the variable framework regions (FRs).

[0077] The term “monoclonal antibodies” specifically includes “chimeric” antibodies in which a portion of the heavy and / or light chain is identical to or homologous with the corresponding sequence of antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical to or homologous with the corresponding sequences of antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.

[0078] Useful monoclonal antibodies include, but are not limited to, human monoclonal antibodies, humanized monoclonal antibodies, antibody fragments, or chimeric monoclonal antibodies. Human monoclonal antibodies may be made by any of numerous techniques known in the art (e.g., Teng et al., “Construction and Testing of Mouse— Human Heteromyelomas for Human Monoclonal Antibody Production,” Proc. Natl. Acad. Sci. USA 80:7308-12 (1983); Kozbor et al., “The Production of Monoclonal Antibodies From Human Lymphocytes,” Immunology Today 4:72-79 (1983); and Olsson et al., “Human-Human Monoclonal Antibody -Producing Hybridomas: Technical Aspects,” Meth. Enzymol. 92:3-16 (1982), all of which are hereby incorporated by reference in their entirety).

[0079] Antibodies may have residues artificially introduced into their backbone in order to facilitate conjugation. This may include cysteine mutations (doi: 10.1208 / sl2248-017-0083-7), cysteine insertions (https: / / doi.org / 10.1021 / acs.molpharmaceut.6b00995), addition of enzymatically recognized motifs (https: / / doi. Org / 10.1016 / j.chembiol.2013.01.010), and the incorporation of non-natural amino acids (doi: 10.1038 / srepl7196). The antibody can also be a bispecific antibody. Methods for making bispecific antibodies are known in the art and are discussed herein.

[0080] An “intact antibody” as described herein includes one which comprises an antigenbinding variable region as well as a light chain constant domain (CL) and heavy chain constant domains, CH1, CH2, CH3 and CH4, as appropriate for the antibody class. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof.

[0081] An intact antibody may have one or more “effector functions,” which refers to those biological activities attributable to the Fc region (e.g., a native sequence Fc region or amino acid sequence variant Fc region) of an antibody. Examples of antibody effector functions include complement dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis. See WO 2014 / 068443 to Pfizer Inc., which is hereby incorporated by reference in its entirety.

[0082] The term “variable” in the context of an antibody refers to certain portions of the variable domains of the antibody that differ extensively in sequence and are used in the binding and specificity of each particular antibody for its particular antigen. This variability is concentrated in three segments called “hypervariable regions” in the light chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FRs). The variable domains of native heavy and light chains each comprise four FRs connected by three hypervariable regions.

[0083] The phrase “hypervariable region” as used herein includes the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region generally comprises amino acid residues from a “complementarity determining region” or “CDR” (e.g., residues 24-34 (LI), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35 (Hl ), 50-65 (H2) and 95-102 (L3) in the heavy chain variable domain (Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, Fifth Edition, National Institute ofHealth (Bethesda, Md. 1991), which is hereby incorporated by reference in its entirety); and / or those residues from a “hypervariable loop” (e.g., residues 26-32 (LI), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (Hl), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk, “Canonical Structures For the Hypervariable Regions of Immunoglobulins,” J. Mol. Biol. 196:901-17 (1987), which is hereby incorporated by reference in its entirety). FR residues are those variable domain residues other than the hypervariable region residues as herein defined.

[0084] A “single-chain Fv” or “scFv” antibody fragment may include the VH and VL domains of an antibody, where these domains are present in a single polypeptide chain. Typically, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun in THE PHARMACOLOGY OF MONOCLONAL ANTIBODIES, vol.113, Rosenburg and Moore eds., SpringerVerlag (New York 1994) pp. 269-315, which is hereby incorporated by reference in its entirety).

[0085] The term “diabody” includes small antibody fragments with two antigen-binding sites, which fragments comprise a variable heavy domain (VH) connected to a variable light domain (VL) in the same polypeptide chain. By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP 0404097 to BEHRINGWERKE AG; WO 93 / 11161 to Enzon, Inc.; and Hollinger et al., “‘Diabodies’: Small Bivalent and Bispecific Antibody Fragments,” Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), all of which are hereby incorporated by reference in their entirety.

[0086] Completely human antibodies are useful and can be produced using transgenic mice that are incapable of expressing endogenous immunoglobulin heavy and light chains genes, but which can express human heavy and light chain genes. The transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the present disclosure. Monoclonal antibodies directed against the antigen can be obtained using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For anoverview of this technology for producing human antibodies, see Lonberg and Huszar, “Human Antibodies From Transgenic Mice,” Int. Rev. Immunol. 13:65-93 (1995), which is hereby incorporated by reference in its entirety. For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., U. S. Pat. Nos. 5,625,126 to Lonberg et al.; 5,633,425 to Lonberg et al.; 5,569,825 to Lonberg et al.; 5,661,016 to Lonberg et al.; 5,545,806 to Lonberg et al., all of which are hereby incorporated by reference in their entirety.

[0087] Completely human antibodies that recognize a selected epitope can be generated using a technique referred to as “guided selection.” In this approach a selected non-human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope. See, e.g., Jespers et al., “Guiding the Selection of Human Antibodies From Phage Display Repertoires to a Single Epitope of an Antigen,” Biotechnology 12:899-903 (1994), which is hereby incorporated by reference in its entirety. Human antibodies can also be produced using various techniques known in the art, including phage display libraries (see, e.g., Hoogenboom and Winter, “By-Passing Immunisation.Human Antibodies From Synthetic Repertoires of Germline VH Gene Segments Rearranged In Vitro,” J. Mol. Biol. 227:381 (1991); Marks et al., “By-Passing Immunization. Human Antibodies From V-gene Libraries Displayed on Phage,” J. Mol. Biol. 222:581 (1991); Quan and Carter, “The rise of monoclonal antibodies as therapeutics,” In ANTI-IGE AND ALLERGIC DISEASE, Jardieu and Fick, eds., Marcel Dekker (New York, N. Y., 2002) Chapter 20, pp. 427-469), all of which are hereby incorporated by reference in their entirety.

[0088] “Humanized” forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of thehypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., “Replacing the Complementarity-Determining Regions in a Human Antibody With Those From a Mouse,” Nature 321:522-25 (1986); Riechmann et al., “Reshaping Human Antibodies For Therapy,” Nature 332:323-329 (1988); and Presta, L. “Antibody Engineering,” Curr. Op. Struct. Biol.2:593-596 (1992), all of which are hereby incorporated by reference in their entirety.

[0089] Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are useful antibodies. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as for example, those having a variable region derived from a murine monoclonal and human immunoglobulin constant regions (see, e.g., U. S. Pat. No. 4,816,567 to Cabilly et al.; and U. S. Pat. No.4,816,397 to Boss et al., which are incorporated herein by reference in their entirety).Humanized antibodies are antibody molecules from non-human species having one or more complementarity determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule (see, e.g., U. S. Pat. No. 5,585,089 to Queen et al., which is incorporated herein by reference in its entirety). Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in International Publication No. WO 87 / 02671 to Int Genetic Eng; European Patent Publication No. 0 184 187 to Teijin Ltd; European Patent Publication No. 0 171 496 to Japan Res Dev Corp; European Patent Publication No. 0 173 494 to Univ Leland Stanford Junior; International Publication No. WO 86 / 01533 to Celltech Ltd; U. S. Pat. No. 4,816,567 to Cabilly et al.; Berter et al., “Escherichia coli Secretion of an Active Chimeric Antibody Fragment,” Science 240:1041-1043 (1988); Liu et al., “Chimeric Mouse-Human IgGl Antibody That Can Mediate Lysis of Cancer Cells,” Proc. Natl. Acad. Sci. USA 84:3439-3443 (1987); Liu et al., “Production of a Mouse-Human Chimeric Monoclonal Antibody to CD20 With Potent Fc-Dependent Biologic Activity,” J. Immunol. 139:3521-3526 (1987); Sun et al., “Chimeric Antibody With Human Constant Regions and Mouse Variable Regions Directed Against Carcinoma-Associated Antigen 17-1 A,” Proc. Natl. Acad. Sci. USA 84:214-218 (1987); Nishimura et al., “Recombinant Human-Mouse Chimeric Monoclonal Antibody Specific for Common Acute Lymphocytic LeukemiaAntigen,” Cancer. Res. 47:999-1005 (1987); Wood et al., “The Synthesis and In Vivo Assembly of Functional Antibodies in Yeast,” Nature 314:446-449 (1985); and Shaw et al., “Mouse / Human Chimeric Antibodies to a Tumor-Associated Antigen: Biologic Activity of the Four Human IgG Subclasses,” J. Natl. Cancer Inst. 80:1553-1559 (1988); Morrison, S. L., “Transfectomas Provide Novel Chimeric Antibodies,” Science 229:1202-1207 (1985); U. S. Pat. No. 5,225,539 to Winter; Jones et al., “Replacing the Complementarity-Determining Regions in a Human Antibody With Those From a Mouse,” Nature 321:552-525 (1986);Verhoeyan et al., “Reshaping Human Antibodies: Grafting an Antilysozyme Activity,” Science 239:1534 (1988); and Beidler et al., “Cloning and High Level Expression of a Chimeric Antibody With Specificity For Human Carcinoembryonic Antigen,” J. Immunol. 141:4053-4060 (1988), all of which are hereby incorporated by reference in their entirety.

[0090] As described herein, “isolated” includes separated from other components of (a) a natural source, such as a plant or animal cell or cell culture, or (b) a synthetic organic chemical reaction mixture. As used herein, “purified” means that when isolated, the isolate contains at least 95%, and in another aspect at least 98%, of a compound (e.g., a conjugate) by weight of the isolate.

[0091] An “isolated” antibody is one which has been identified and separated and / or recovered from component of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the antibody may be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and in some embodiments more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody may include the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, an isolated antibody may be prepared by at least one purification step.

[0092] In other embodiments, the antibody is a fusion protein of an antibody, or a functionally active fragment thereof, for example in which the antibody is fused via a covalent bond (e.g., a peptide bond), at either the N-terminus or the C-terminus to an amino acid sequence of another protein (or portion thereof, preferably at least 10, 20 or 50 amino acid portion of the protein)that is not from an antibody. In one embodiment, the antibody or fragment thereof is covalently linked to the other protein at the N-terminus of the constant domain.

[0093] Antibodies include analogs and derivatives that are either modified, i.e., by the covalent attachment of any type of molecule as long as such covalent attachment permits the antibody to retain its antigen binding immunospecificity. For example, derivatives and analogs of the antibodies include those that have been further modified, e.g, by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, linkage to a cellular antibody unit or other protein. Any of numerous chemical modifications can be carried out by known techniques including, but not limited to, specific chemical cleavage, acetylation, formylation, and metabolic synthesis in the presence of tunicamycin. Additionally, the analog or derivative may contain one or more unnatural amino acids.

[0094] Antibodies may have modifications (e.g, substitutions, deletions or additions) in amino acid residues that interact with Fc receptors. In particular, antibodies may have modifications in amino acid residues identified as involved in the interaction between the anti-Fc domain and the FcRn receptor (see, e.g., International Publication No. WO 97 / 34631, which is incorporated herein by reference in its entirety).

[0095] In one embodiment, Ab i.e., the antibody) is a tumor targeting antibody, a bispecific antibody, a monoclonal antibody, a chimeric antibody, or a humanized antibody.

[0096] Antibodies immunospecific for a cancer cell antigen can be obtained commercially or produced by any method known to one of skill in the art such as, e.g., chemical synthesis or recombinant expression techniques. The nucleotide sequence encoding antibodies immunospecific for a cancer cell antigen can be obtained, e.g., from the GenBank database or a database like it, literature publications, or by routine cloning and sequencing.

[0097] In one embodiment, Ab (i.e., the antibody) is selected from the group consisting of anti-Her2, anti-CD20, anti-CD38, anti-CD19, anti-CD79, anti-TNF, anti-IL-6 receptor, anti-VEGRF2, anti-DLL3, anti-Nectin4, anti-CD33, anti-CD79b, anti-CDlla, anti-BCMA, anti-PSMA, anti-CD22, anti-Trop2, anti-FRa, anti-EpCAM, anti-mesothelin, anti-LIVl, anti-cMET, anti-IL-31R, anti-CSF-lR, anti-HER3, anti-DLL, anti-PDl, anti-PD-Ll, anti-tissue factor, anti-MASP-2, anti-GPRC5D, anti-FRa, anti-CTLA-4, anti-IL36R, anti-LAG3, anti-gplOO, anti-IFNARl, anti-GD2, anti-IL6R, anti-IGF-lR, anti-Nectin4, anti-IL-23, anti-IFNgamma, anti-CGRP, anti-CCR4, anti-CGRP-R, anti-CD4, anti-FGF23, anti-CLDN18.2, anti-IL-5R, anti-PDGFRcc, anti-IL-5, anti-IL17a, anti-EGFR, anti-PCSK9, anti-a4p7, anti-IL6, anti-CD30, anti- BLyS, anti-RANK-L, anti-ILlp, anti-EPCAM, anti-a4-integrin, anti-CD52, anti-IL2R, anti-CCL11, anti-CD3, anti-CD27, anti-CD28, anti-CD40, anti-CD51, anti-CD123, anti-CD147, anti-CD152, anti-CEACAM5, anti-DLL4, anti-FGFR2, anti-fibronectin, anti -Folate receptor, anti -gly pi can, anti-GUCY2C, anti-MIF, anti-MSLN, anti-MUCl, anti-RORl, anti-SLAMF7, anti-STREAPI, anti-VISTA, anti-IL-4Ra, anti-BDCA2, anti-CXCR4, anti-CD163, anti-MSRl, and anti-CD74, or anti-TIGIT, oregovomab, edrecolomab, cetuximab, a humanized monoclonal antibody to the vitronectin receptor (otvPs), alemtuzumab, a humanized anti-HLA-DR antibody for the treatment of non-Hodgkin’s lymphoma, 1311 Lym-1, a murine anti-HLA-DrlO antibody for the treatment of non-Hodgkin’s lymphoma, a humanized anti-CD2 mAb for the treatment of Hodgkin’s Disease or non-Hodgkin’s lymphoma, labetuzumab, bevacizumab, ibritumomab tiuxetan, ofatumumab, panitumumab, rituximab, tositumomab, ipilimumab, gemtuzumab, humanized monoclonal antibody to the oncofecal protein receptor 5T4, MI / 70 (antibody to CD 11b receptor), anti-MRCl, anti GCC, anti CD32, and other antibodies.

[0098] In one embodiment, known antibodies for the treatment of cancer may be used.Antibodies immunospecific for a cancer cell antigen can be obtained commercially or produced by any method known to one of skill in the art such as, e.g., recombinant expression techniques. The nucleotide sequence encoding antibodies immunospecific for a cancer cell antigen can be obtained, e.g., from the GenBank database or a database like it, the literature publications, or by routine cloning and sequencing. Examples of antibodies available for the treatment of cancer include, but are not limited to, Oregovomab or OVAREX® which is a murine antibody for the treatment of ovarian cancer; Edrecolomab or panorex which is a murine IgG2a antibody for the treatment of colorectal cancer; Cetuximab (e.g., ERBITUX®) which is an anti-EGFR IgG chimeric antibody for the treatment of epidermal growth factor positive cancers, such as head and neck cancer; vitaxin, which is a humanized antibody for the treatment of sarcoma; Alemtuzumab or CAMPATH- 1H, which is a humanized IgGl antibody for the treatment of chronic lymphocytic leukemia (CLL); ONCOL YM, which is a radio labeled murine anti-HLA-DrlO antibody for the treatment of non-Hodgkin’s lymphoma;ALLOMUNE (Bio Transplant, CA) which is a humanized anti-CD2 mAb for the treatment of Hodgkin's Disease or non-Hodgkin’s lymphoma; and CEA-Cide (Immunomedics, NJ) which is a humanized anti -CEA antibody for the treatment of colorectal cancer.

[0099] The terms “protein,” “polypeptide,” and “peptide” may be referred to interchangeably herein. The terms may be distinguished as follows. A protein typically refers to the end product of transcription, translation, and post-translation modifications in a cell.

[0100] A polypeptide may include a protein or a peptide. A peptide, in contrast to a protein, typically is a short polymer of amino acids, of a length typically of 100 or less amino acids.

[0101] The term “peptide” or “polypeptide” as used herein refers to proteins and fragments thereof. Peptides may include amino acid sequences. Those sequences may be written left to right in the direction from the amino to the carboxy terminus. In accordance with standard nomenclature, amino acid residue sequences are denominated by either a three letter or a single letter code as indicated as follows: Alanine (Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic Acid (Asp, D), Citrulline (Cit), Cysteine (Cys, C), Glutamine (Gin, Q), Glutamic Acid (Glu, E), Glycine (Gly, G), Histidine (His, H), Isoleucine (He, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y), and Valine (Vai, V). It is to be understood that, even if not specifically indicated, Alanine includes beta-alanine.

[0102] The peptides of the immunotherapy compounds may be derived from nature, or may, alternatively be designed de nova. A peptide is said to be “derivable from a naturally occurring amino acid sequence” if it can be obtained by fragmenting a naturally occurring sequence, or if it can be synthesized based upon knowledge of the sequence of the naturally occurring amino acid sequence or of the genetic material (DNA or RNA) that encodes this sequence.

[0103] The peptides of the immunotherapy compounds may or may not share substantial homology or identity with naturally occurring proteins or portions thereof (e.g., peptides). The immunotherapy compound may or may not include peptides with “substantial similarity” with naturally occurring proteins or portions thereof (e.g., peptides). A peptide with substantial similarity includes peptides with at least 70% or greater sequence homology or identity with a peptide having the same number of amino acid residues as the reference peptide. In some instances, a peptide with substantial similarity includes peptides with at least 75% or greater, or 80% or greater, or 85% or greater, or 90% or greater, or 92% or greater, or 95% or greater, or 97% or greater, or 99% or greater sequence homology or identity with a peptide having the same number of amino acid residues as the reference peptide.

[0104] The terms loading or “drug loading” or “payload loading” refer to the average number of payloads (“payload” and “payloads” are used interchangeably herein with “drug” and “drugs”) per antibody in an ADC molecule. Drug loading may range from 1 to 50 drugs per antibody. This is sometimes referred to as the DAR, or drug to antibody ratio.Compositions of the ADCs described herein typically have DAR’s of from 1-25, and in certain embodiments, from 1-8, from 2-8, from 2-6, from 2-5 and from 2-4. Typical DAR values include 2, 4, 6, 8, and 10. The average number of drugs per antibody, or DAR value, may be characterized by conventional means such as UV / visible spectroscopy, mass spectrometry, ELISA assay, and HPLC. The quantitative DAR value may also be determined. In some instances, separation, purification, and characterization of homogeneous ADCs having a particular DAR value may be achieved by means such as reverse phase HPLC or electrophoresis. DAR may be limited by the number of attachment sites on the antibody. For example, where the attachment is a cysteine thiol, an antibody may have only one or several cysteine thiol groups, or may have only one or several sufficiently reactive thiol groups through which a linker unit may be attached. In some embodiments, the cysteine thiol is a thiol group of a cysteine residue that forms an interchain disulfide bond. In some embodiments, the cysteine thiol is a thiol group of a cysteine residue that does not form an interchain disulfide bond. Typically, fewer than the theoretical maximum of drug moieties are conjugated to an antibody during a conjugation reaction. An antibody may contain, for example, many lysine residues that do not react with a linker or linker intermediate. Only the most reactive lysine groups may react with a reactive linker reagent.

[0105] Generally, antibodies do not contain many, if any, free and reactive cysteine thiol groups which may be linked to a drug via a linker. Most cysteine thiol residues in the antibodies exist as disulfide bridges and must be reduced with a reducing agent such as dithiothreitol (DTT). The antibody may be subjected to denaturing conditions to reveal reactive nucleophilic groups such as lysine or cysteine. The loading (drug / antibody ratio) of an ADC may be controlled in several different manners, including: (i) limiting the molar excess of drug- linker relative to the antibody, (ii) limiting the conjugation reaction time or temperature, and (iii) partial or limiting reductive conditions for cysteine thiol modification. Where more than one nucleophilic group reacts with a drug-linker then the resulting product is a mixture of ADCs with a distribution of one or more drugs moieties per antibody. The average number of drugs per antibody may be calculated from the mixture by, for example, dual ELISA antibody assay, specific for antibody and specific for the drug. Individual ADCsmay be identified in the mixture by mass spectroscopy, and separated by HPLC, e.g., hydrophobic interaction chromatography.

[0106] In certain embodiments, the present disclosure relates to any of the aforementioned antibody drug conjugates and attendant definitions, wherein the antibody drug conjugate comprises between 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 compounds of the present disclosure, or any number of compounds therein.

[0107] In certain embodiments, the present disclosure relates to any of the aforementioned antibody drug conjugates and attendant definitions, wherein the antibody drug conjugate comprises 3 or 4 compounds of the present disclosure.

[0108] An amino acid “derivative” includes an amino acid having substitutions or modifications by covalent attachment of a parent amino acid, such as, e.g., by alkylation, glycosylation, acetylation, phosphorylation, and the like. Further included within the contemplated meaning of “derivative” is, for example, one or more analogs of an amino acid with substituted linkages, as well as other modifications known in the art.

[0109] A “natural amino acid” refers to arginine, glutamine, phenylalanine, tyrosine, tryptophan, lysine, glycine, alanine, histidine, serine, proline, glutamic acid, aspartic acid, threonine, cysteine, methionine, leucine, asparagine, isoleucine, and valine, unless otherwise indicated by context.

[0110] A “non-natural amino acid” (or “unnatural amino acid”) refers to an amino acid that does not occur naturally in a protein and / or that is not encoded genetically in a naturally-occurring organism. A “non-natural amino acid” may be synthetically obtained or may be obtained by modification of a natural or non-natural amino acid. Some non-natural amino acids incorporate functional groups to enhance solubility and polarity. Such groups may include PEG, phosphates, sulfates, and the like.

[0111] A self-immolative spacer as described herein includes covalent assemblies tailored to correlate the cleavage of two chemical bonds after activation of a protective part in a precursor: Upon stimulation, the protective moiety is removed, which generates a cascade of disassembling reactions leading to the temporally sequential release of smaller molecules. See Alouane et al., “Self-Immolative Spacers: Kinetic Aspects, Structure-Property Relationships, and Applications,” Angewandte Chemie 54(26):7492-7509 (2015), which ishereby incorporated by reference in its entirety. Self-immolative spacers were created to address limitations for drug delivery, and have gained wide interest in medicinal chemistry, analytical chemistry, and material science. See Alouane et al., “Self-Immolative Spacers: Kinetic Aspects, Structure-Property Relationships, and Applications,” Angewandte Chemie 54(26:7492-7509 (2015), which is hereby incorporated by reference in its entirety.

[0112] The term “Michael acceptor” refers to an electrophile moiety that can take part in a Michael reaction, wherein a new covalent bond is formed between a portion ofthe Michael acceptor moiety and the donor moiety. A Michael acceptor moiety generally will possess a) an alkene or an alkyne bond and b) a C=O, C=ONR, SONR, SO2NR, or NO2 group, wherein R is hydrogen, an aryl group or an unsubstituted or substituted alkyl or cycloalkyl moiety.

[0113] The phrase “substantial amount” includes a majority, z.e., greater than 50% of a population, of a mixture or a sample.

[0114] The term “intracellular metabolite” refers to a compound resulting from a metabolic process or reaction inside a cell on an antibody-drug conjugate (ADC). The metabolic process or reaction may be an enzymatic process such as proteolytic cleavage of a peptide linker of the ADC. Intracellular metabolites include, but are not limited to, antibodies and free drug which have undergone intracellular cleavage after entry, diffusion, uptake, or transport into a cell.

[0115] The terms “intracellularly cleaved” and “intracellular cleavage” refer to a metabolic process or reaction inside a cell on an ADC or the like, whereby the covalent attachment, e.g., the linker, between the drug moiety and the antibody is broken, resulting in the free drug, or other metabolite of the conjugate dissociated from the antibody inside the cell. The cleaved moieties of the ADC are thus intracellular metabolites.

[0116] The term “bioavailability” refers to the systemic availability ( / .<., blood / plasma levels) of a given amount of a drug administered to a patient. Bioavailability indicates measurement of both the time (rate) and total amount (extent) of drug that reaches the general circulation from an administered dosage form.

[0117] The term “cytotoxic activity” refers to a cell-killing, a cytostatic or an antiproliferative effect of an ADC or an intracellular metabolite of said ADC. Cytotoxic activitymay be expressed as the IC50 value, which is the concentration (molar or mass) per unit volume at which half the cells survive.

[0118] A “disorder” is any condition that would benefit from treatment with a drug compound or antibody-drug conjugate. This includes chronic and acute disorders or diseases including those pathological conditions which predispose a mammal to the disorder in question. Non-limiting examples of disorders to be treated herein include benign and malignant cancers; leukemia and lymphoid malignancies, neuronal, glial, astrocytal, hypothalamic and other glandular, macrophagal, epithelial, stromal and blastocoelic disorders; and inflammatory, angiogenic and immunologic disorders.

[0119] The terms “cancer” and “cancerous” refer to or describe the physiological condition or disorder in mammals that is typically characterized by unregulated cell growth. A “tumor” comprises one or more cancerous cells.

[0120] As used herein, the terms “cell,” “cell line,” and “cell culture” are used interchangeably and all such designations include progeny. The words “transformants” and “transformed cells” include the primary subject cell and cultures or progeny derived therefrom without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where distinct designations are intended, it will be clear from the context.

[0121] The terms “subject” and “patient,” are used interchangeably and, as used herein, include both humans and other animals, particularly mammals. Thus, the methods are applicable to both human therapy and veterinary applications. Examples of a “subject” include, but are not limited to, a human, rat, mouse, guinea pig, monkey, pig, goat, cow, horse, dog, cat, bird, and fowl. In some embodiments, the subject is a mammal, for example, a primate. In some embodiments, the subject is a human. In one embodiment, the subject is an infant, a juvenile, or an adult.

[0122] The terms “treat” or “treatment,” unless otherwise indicated by context, refer to therapeutic treatment and prophylactic measures to prevent relapse, wherein the object is to inhibit or slow down (lessen) an undesired physiological change or disorder, such as the development or spread of cancer or a viral infection.

[0123] For purposes of the present disclosure, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already having the condition or disorder as well as those prone to have the condition or disorder.

[0124] In the context of cancer, the term “treating” includes any or all of inhibiting growth of tumor cells, cancer cells, or of a tumor; inhibiting replication of tumor cells or cancer cells; lessening of overall tumor burden or decreasing the number of cancerous cells; and ameliorating one or more symptoms associated with the disease.

[0125] Treatment can involve administering an ADC or a compound described herein to a patient diagnosed with a disease, and may involve administering the compound to a patient who does not have active symptoms. Conversely, treatment may involve administering the compositions to a patient at risk of developing a particular disease, or to a patient reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease may not have been made.

[0126] The terms “administer,” “administering” or “administration” in reference to a dosage form of the invention refers to the act of introducing the dosage form into the system of subject in need of treatment. When a dosage form of the invention is given in combination with one or more other active agents (in their respective dosage forms), “administration” and its variants are each understood to include concurrent and / or sequential introduction of the dosage form and the other active agents. Administration of any of the described dosage forms includes parallel administration, co-administration or sequential administration. In some situations, the therapies are administered at approximately the same time, e.g., within about a few seconds to a few hours of one another.

[0127] A “therapeutically effective” amount of the compounds described herein is typically one which is sufficient to achieve the desired effect and may vary according to the nature and severity of the disease condition, and the potency of the compound. It will be appreciated that different concentrations may be employed for prophylaxis than for treatment of an active disease. A therapeutic benefit is achieved with the amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement isobserved in the patient, notwithstanding that the patient may still be afflicted with the underlying disorder. In the case of cancer, a therapeutically effective amount of a drug may reduce the number of cancer cells; reduce the tumor size; inhibit ( / .<., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit ( / .<., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and / or relieve to some extent one or more of the symptoms associated with the cancer. To the extent the drug may inhibit the growth of and / or kill existing cancer cells, it may be cytostatic and / or cytotoxic. For cancer therapy, efficacy can, for example, be measured by assessing the time to disease progression (TTP) and / or determining the response rate (RR).

[0128] As such, the therapeutic effect can be a decrease in the severity of symptoms associated with the disorder and / or inhibition (partial or complete) of progression of the disorder, or improved treatment, healing, prevention or elimination of a disorder, or sideeffects. The amount needed to elicit the therapeutic response can be determined based on the age, health, size, and sex of the subject. Optimal amounts can also be determined based on monitoring of the subject’s response to treatment. The term “treatment” or “treat” may include effective inhibition, suppression or cessation of symptoms so as to prevent or delay the onset, retard the progression, or ameliorate the symptoms of a condition.

[0129] Throughout this specification the terms and substituents retain their definitions. Substituents (e.g., Rn) are generally defined when introduced and retain that definition throughout the specification and in all independent claims.

[0130] Ci to C20 hydrocarbon includes alkyl, cycloalkyl, polycycloalkyl, alkenyl, alkynyl, aryl, and combinations thereof, containing from 1 to 20 carbon atoms, inclusive. Nonlimiting examples include ethyl, benzyl, phenethyl, cyclohexylmethyl, camphoryl and naphthyl ethyl. Hydrocarbon refers to any substituent comprised of hydrogen and carbon as the only elemental constituents.

[0131] Alkyl is a subset of hydrocarbon. Unless otherwise specified, alkyl (or alkylene) is intended to include linear or branched saturated hydrocarbon structures and combinations thereof. In some embodiments, alkyl refers to alkyl groups from 1 to 20 carbon atoms, or from 1 to 10 carbon atoms, or from 1 to 8 carbon atoms, or from 1 to 6 carbon atoms, or from 1 to 5 carbon atoms, or from 1 to 4 carbon atoms. Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, n-butyl, s-butyl, t-butyl and the like.

[0132] Cycloalkyl is a subset of hydrocarbon and includes cyclic hydrocarbon groups of from 3 to 8 carbon atoms. Examples of cycloalkyl groups include cy-propyl, cy-butyl, cy-pentyl, norbomyl and the like.

[0133] As used herein, the term “optionally substituted” may be used interchangeably with “unsubstituted or substituted.” The term “substituted” refers to the replacement of one or more hydrogen atoms in a specified group with a specified radical. For example, “substituted aryl” or “substituted heteroaryl” refers to aryl or heteroaryl wherein one or more H atoms in each residue are replaced with halogen, haloalkyl, alkyl, alkoxy, or haloalkoxy.

[0134] The compounds described herein may contain asymmetric centers and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms which may be defined in terms of absolute stereochemistry as (R)- or (5 -. The present invention is meant to include all such possible diastereomers as well as their racemic and optically pure forms. Optically active (A)- and (5)- isomers may be prepared using homo-chiral synthons or homo-chiral reagents, or optically resolved using conventional techniques. When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended to include both (E)- and (Z)-geometric isomers. Likewise, all tautomeric forms are intended to be included.

[0135] The graphic representations of racemic, ambiscalemic and scalemic or enantiomerically pure compounds used herein are a modified version of the denotations taken from Maehr J. Chem. Ed. 62, 114-120 (1985): simple lines provide no information about stereochemistry and convey only connectivity; solid and broken wedges are used to denote the absolute configuration of a chiral element; solid and broken bold lines are geometric descriptors indicating the relative configuration shown but not necessarily denoting racemic character; and wedge outlines and dotted or broken lines denote enantiomerically pure compounds of indeterminate absolute configuration. For example, the graphic representationindicates either, or both, of the two trans:trans enantiomers:in any ratio, from pure enantiomers to racemates. The graphic representation:indicates a single enantiomer of unknown absolute stereochemistry, i.e., it could be either of the two preceding structures, as a substantially pure single enantiomer. And, finally, the representation:indicates a pure (R, R, S) absolute configuration. For the purpose of the present disclosure, a “pure” or “substantially pure” enantiomer is intended to mean that the enantiomer is at least 95% of the configuration shown and 5% or less of other enantiomers. Similarly, a “pure” or “substantially pure” diastereomer is intended to mean that the diastereomer is at least 95% of the relative configuration shown and 5% or less of other diastereomers. In some embodiments, the purity of the compound is at least 99%.

[0136] In any of these possibilities, compounds can be a single stereoisomer or a mixture. If a mixture, the mixture will most commonly be racemic, but it need not be. Substantially pure single stereoisomers of biologically active compounds such as those described herein often exhibit advantages over their racemic mixture.

[0137] Enantiomerically pure means greater than 80 e.e., and preferably greater than 90 e.e. For the purpose of the present disclosure, a “pure” or “substantially pure” stereoisomer is intended to mean that the stereoisomer is at least 95% of the configuration shown and 5% orless of other stereoisomers, or at least 97% of the configuration shown and 3% or less of other stereoisomers, or at least 99% of the configuration shown and 1% or less of other stereoisomers.

[0138] It may be found upon examination that certain species and genera are not patentable to the inventors in this application. In this case, the exclusion of species and genera in applicants' claims are to be considered artifacts of patent prosecution and not reflective of the inventors' concept or description of their invention, which encompasses all members of the genus that are not in the public’s possession.

[0139] As used herein, the recitation of “compound” may also be used in reference to an “antibody-drug conjugate”- unless expressly further limited. In some embodiments, the term “compound of formula” has the same meaning as “antibody-drug conjugate of formula,” and refers to antibody-drug conjugate, or a pharmaceutically acceptable salt thereof.

[0140] As used herein, and as would be understood by the person of skill in the art, the recitation of “compound” or “antibody-drug conjugate”- unless expressly further limited - is intended to include salts of that compound or antibody-drug conjugate. In a particular embodiment, the term “compound of formula” or “antibody-drug conjugate of formula” refers to the compound or antibody-drug conjugate, or a pharmaceutically acceptable salt thereof.

[0141] The term "pharmaceutically acceptable salt" refers to salts prepared from pharmaceutically acceptable non-toxic acids or bases including inorganic acids and bases and organic acids and bases. When the compounds of the present invention are basic, salts may be prepared from pharmaceutically acceptable non-toxic acids including inorganic and organic acids. Suitable pharmaceutically acceptable acid addition salts for the compounds of the present invention include acetic, adipic, alginic, ascorbic, aspartic, benzenesulfonic (besylate), benzoic, boric, butyric, camphoric, camphorsulfonic, carbonic, citric, ethanedisulfonic, ethanesulfonic, ethylenediaminetetraacetic, formic, fumaric, glucoheptonic, gluconic, glutamic, hydrobromic, hydrochloric, hydroiodic, hydroxynaphthoic, isethionic, lactic, lactobionic, laurylsulfonic, maleic, malic, mandelic, methanesulfonic, mucic, naphthylenesulfonic, nitric, oleic, pamoic, pantothenic, phosphoric, pivalic, polygalacturonic, salicylic, stearic, succinic, sulfuric, tannic, tartaric acid, teoclatic, p-toluenesulfonic, and the like. When the compounds contain an acidic side chain, suitable pharmaceutically acceptable base addition salts for the compounds of the present invention include, but are not limited to, metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from lysine, arginine, N, N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,ethylenediamine, meglumine (N-methylglucamine) and procaine. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium cations and carboxylate, sulfonate and phosphonate anions attached to alkyl having from 1 to 20 carbon atoms.

[0142] Also provided herein is a pharmaceutical composition comprising an ADC or a compound disclosed above, or a pharmaceutically acceptable salt form thereof, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0143] While it may be possible for the compounds disclosed herein to be administered as the raw chemical, it is preferable to present them as a pharmaceutical composition. According to a further aspect, the present invention provides a pharmaceutical composition comprising a compound of Formula (I) or an ADC of Formula (A), or a pharmaceutically acceptable salt thereof, together with one or more pharmaceutically carriers thereof and optionally one or more other therapeutic ingredients. The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. In one embodiment, the pharmaceutically acceptable carrier is selected from the group consisting of a liquid filler, a solid filler, a diluent, an excipient, a solvent, and an encapsulating material.

[0144] Pharmaceutically acceptable carriers (e.g., additives such as diluents, immunostimulants, adjuvants, antioxidants, preservatives and solubilizing agents) are nontoxic to the cell or subject being exposed thereto at the dosages and concentrations employed.Examples of pharmaceutically acceptable carriers include water, e.g., buffered with phosphate, citrate and another organic acid. Representative examples of pharmaceutically acceptable excipients that may be useful in the present disclosure include antioxidants such as ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; adjuvants (selected so as to avoid adjuvant-induced toxicity, such as a (3-glucan as described in U. S. Pat. No. 6,355,625, which is hereby incorporated by reference in its entirety, or a granulocyte colony stimulating factor (GCSF)); hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt forming counterions such as sodium; and / or nonionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®.

[0145] In one embodiment, the composition may further comprise an adjuvant. Suitable adjuvants are known in the art and include, without limitation, flagellin, Freund’s complete or incomplete adjuvant, aluminum hydroxide, lysolecithin, pluronic polyols, polyanions, peptides, oil emulsion, dinitrophenol, iscomatrix, and liposome polycation DNA particles.

[0146] The formulations include those suitable for parenteral (including subcutaneous, intradermal, intramuscular, intravenous and intraarticular), rectal, nasal, and topical (including dermal, buccal, sublingual and intraocular) administration. The most suitable route may depend upon the condition and disorder of the recipient. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing into association a compound disclosed herein or a pharmaceutically acceptable salt thereof ("active ingredient") with the carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both and then, if necessary, shaping the product into the desired formulation.

[0147] Formulations for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient. Formulations for parenteral administration also include aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents. The formulations may be presented in unit-dose of multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid carrier, for example saline, phosphate-buffered saline (PBS) or the like, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

[0148] The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products that contain information about the indication(s), usage, dosage, administration, contraindications, and / or warnings concerning the use of such therapeutic products.

[0149] It will be recognized that the compounds of this invention can exist in radiolabeled form, i.e., the compounds may contain one or more atoms containing an atomic mass or mass number different from the atomic mass or mass number usually found in nature.Radioisotopes of hydrogen, carbon, phosphorous, fluorine, and chlorine include2H,3H,13C,14C,15N,35S,18F, and36C1, respectively. Compounds that contain those radioisotopes and / or other radioisotopes of other atoms are within the scope of this invention. Tritiated, i.e.,3H, and carbon-14, i.e.,14C, radioisotopes are particularly preferred for their ease in preparation and detectability. Compounds that contain isotopesnC,13N,15O and18F are well suited for positron emission tomography. Radiolabeled ADCs or compounds of this invention and prodrugs thereof can generally be prepared by methods well known to those skilled in the art. Conveniently, such radiolabeled compounds can be prepared by carrying out the procedures disclosed in the Examples and Schemes by substituting a readily available radiolabeled reagent for a non-radiolabeled reagent.

[0150] Preparation of compounds can involve the protection and deprotection of various chemical groups. The need for protection and deprotection, and the selection of appropriate protecting groups, can be readily determined by one skilled in the art. Suitable groups for that purpose are discussed in standard textbooks in the field of chemistry, such as Protective Groups in Organic Synthesis by T. W. Greene and P. G. M. Wuts [John Wiley & Sons, New York, 1999], in Protecting Group Chemistry, 1stEd., Oxford University Press, 2000; and in March ’s Advanced Organic chemistry: Reactions, Mechanisms, and Structure, 5thEd., Wiley-Interscience Publication, 2001.

[0151] EXAMPLES

[0152] Example 1: Preparation of 6S,9R,10S,13S,16R,17R)-6.,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-17-((furan-2-carbonyl)oxy)-10,13,16-trimethyl-3-oxo-6,7.,8.,9,10,ll,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-ll-yl (3-(2,5-dioxo-2.,5-dihvdro-lH-pyrrol-l-yl)propanoyl)glvcyl-L-asparaginyl-L-asparaginylglvcyl-L-prolinate (mpGlyAsnAsnGlyPro Fluticasone furoate or mpGNNGP-FF):

[0153] Step 1: Preparation of 1 -(tert-butyl) 2-((6S,9R,10S,llS,13S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)- 17-((furan-2-carbonyl)oxy)- 10,13,16-trimethyl-3 -oxo- 6.7.8.9.10.11.12.13.14.15.16.17-dodecahydro-3H-cyclopenta[a]phenanthren-ll-yl) (2S)-pyrrolidine-l,2-dicarboxylate: Fluticasone furoate (55 mg, 102 pmol) was dissolved in 1 mL di chloromethane (DCM). To this solution, (tert-butoxycarbonyl)-L-proline (87.9 mg, 4 eq, 408 pmol) was added, followed by the addition of DMAP (2.5 mg, 0.2 eq, 20.4 pmol) while stirring. After 5 minutes, DCC (105 mg, 5 eq, 511 pmol) was added, and the reaction mixture was stirred at room temperature for 72 hours, with progress monitored via LCMS. Once complete, the DCM was evaporated, and the reaction was worked up using 20 mL ethyl acetate and 20 mL water. The ethyl acetate layer was washed three times with water, collected in a 50 mL beaker, and dried over magnesium sulfate to remove any residual water. After filtering, the ethyl acetate layer was air-dried and stored in a desiccator overnight. The final product was obtained as a white solid (55 mg, 77% yield), with M+H = 736.2 and a retention time (RT) of 4.13 minutes.

[0154] Step 2: (6S,9R,10S,llS,13S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)- 17-((furan-2-carbonyl)oxy)- 10,13,16-trimethyl-3 -oxo- 6.7.8.9.10.11.12.13.14.15.16.17-dodecahydro-3H-cyclopenta[a]phenanthren-ll-yl L-prolinate._The product of step 1 (55 mg) was dissolved in 1.6ml DCM, then treated with 0.4 ml TFA. The reaction was stirred for 30 minutes. The solvents were removed by air and the product dried under vacuum to give 41.5mg (87%) of the title compound as a slightly yellowish solid. M+H =636.2, RT= 2.81minutes.

[0155] Step 3: 6S,9R,10S,13S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-17-((furan-2-carbonyl)oxy)-10,13,16-trimethyl-3-oxo-6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-ll-yl (3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanoyl)glycyl-L-asparaginyl-L-asparaginylglycyl-L-prolinate [mpGlyAsnAsnGlyPro-Fluticasone Furoate]: The product of step 2 (41.5 mg, 1 Eq, 65.3 pmol) was dissolved in 2ml DMF and treated with (3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanoyl)glycyl-L-asparaginyl-L-asparaginylglycine [mpGlyAsnAsnGly-OH, prepared by GenScript] (92.4 mg, 2.5 Eq, 181 pmol) and DIPEA (33.8 mg, 45.5 pL, 4 Eq, 261 pmol) and HATU (99.3 mg, 4 Eq, 261 pmol). The reaction was monitored by LCMS. The final product was purified by prep-HPLC to give a white solid (8.5mg, 12%). RT=3.10, m / z for C51H59F3N8O16S (M+H)+=1129.4,. 'HNMR: 8.18 (t, J=5.68Hz, 1H), 8.11 (d, J=7.4Hz, 1H), 8.06(d, J=7.7Hz, 1H), 7.83 (t, J=5.5Hz, 1H), 7.35(s, 1H), 7.22(s,lH) 6.99(s, 2H), 6.88(s,lH), 6.85-6.77(m, 2H),6.35 (dd, J=10.2, 1.9, 1H), 6.15(s, 1H), 5.93 (d, J=50Hz, 2H), 5.74-5.52 (m, 1H), 5.29-5.20 (m, 1H), 4.54(dd, J= 14.1Hz, 7.0Hz, 3H), 4.39-4.32(m,lH), 3.99-3.91(m,lH) 3.80-3.72(m,lH), 3.68(d, 5.6Hz, 3H), 3.62(t, J=7.1, 3H), 3.50-3.40 (m,lH), 3.39-3.27(m,2H), 2.46-2.39(m, 8H), 2.24-2.13(m, 3H), 2.02(s,lH), 1.99(s, 1H), 1.96-1.85(m, 4H), 1.55(t, J=12.5Hz, 1H), 1.40(d, J=11.8Hz, 1H), 1.34(s, 3H), 1.03(t, J=7.7Hz, 3H), 0.91(d, J=7.1,3H).

[0156] Analytical HPLC was performed using a ACQUITY UPLC equipped with a BEH C18 (1.7 pm, 2.1* 50mm). Solvents were 0.01% formic acid in water (A) and 0.01% formic acid in acetonitrile (B). The flow rate was 0.800 ml / minute with a 5 minute analysis time. MS detection was performed using a QDa mass spectrometer (ESI) and UV detection was performed at 220 and 254 nm.

[0157] The gradient is shown below:Time Flow (ml / minute) %A %B0 0.800 90 100.80 0.800 90 104.40 0.800 10 904.50 0.800 10 904.55 0.800 90 105.00 0.800 90 10

[0158] For exceptionally polar compounds (i.e., Example 4), the gradient was adjusted as shown.Time Flow (ml / minute) %A %B0 0.800 90 101.00 0.800 50 504.00 0.800 0 1004.40 0.800 0 1004.60 0.800 90 105.00 0.800 90 10

[0159] Example 2. Preparation of (6S,9R,10S,13S,16R,17R)-6,9-difluoro-17- (((fluoromethyl)thio)carbonyl)-10,13,16-trimethyl-3-oxo-17-(propionyloxy)- 6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-ll-yl L- prolinate [mpGlyAsnAsnGlyPro Fluticasone Propionate or mpGNNGP-FP]

[0160] Step 1: Preparation of 1 -(tert-butyl) 2-(6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)- 10,13,16-trimethyl-3 -oxo- 17-(propionyloxy)- 6.7.8.9.10.11.12.13.14.15.16.17-dodecahydro-3H-cyclopenta[a]phenanthren-ll-yl) (2S)-pyrrolidine-l,2-dicarboxylate. Fluticasone propionate (52mg, 1 Eq, 104 pmol) was dissolved in 1ml DCM, then treated with (tert-butoxycarbonyl)-L-proline (89.4 mg, 4 Eq, 416 pmol) and N, N-dimethylpyridin-4-amine (2.5 mg, 0.2 Eq, 20.8 pmol). The reaction was stirred for 5 minutes then DCC (107 mg, 5 Eq, 107 pmol) was added. The solution was stirred for 72hrs at RT and monitored by LCMS. The solvent was removed and the residue was partitioned between 20ml ethyl acetate and 20ml water. The ethyl acetate layer was washed three times with water, then was collected in a 50ml beaker. After drying over MgSO4, the ethyl acetate layer was dried by air, then placed in the desiccator overnight. The final yield was 39mg (80%) of a white solid. M+H= 698.3, RT= 4.13 minutes.

[0161] Step 2: (6S,9R,10S,llS,13S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)- 10,13,16-trimethyl-3 -oxo- 17-(propionyloxy)- 6.7.8.9.10.11.12.13.14.15.16.17-dodecahydro-3H-cyclopenta[a]phenanthren-ll-yl L-prolinate. The product of step 1 (39 mg) was dissolved in 1.6ml DCM and then 0.4ml TFA was added. The reaction was stirred for 30 minutes, then was dried by air. The yield was (43.2 mg, 52 pmol, 88 %), slightly yellowish solid. M+H= 598.2, RT= 2.58 minutes.

[0162] Step 3: (6S,9R,10S,13S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-10,13,16-trimethyl-3-oxo-17-(propionyloxy)-6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-ll-yl (3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanoyl)glycyl-L-asparaginyl-L-asparaginylglycyl-L-prolinate [mpGlyAsnAsnGlyPro-fluticasone propionate]. The product of step 2 (43.2 mg, 1 Eq, 72.3 pmol) was dissolved in 2ml DMF, then treated with (3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanoyl)glycyl-L-asparaginyl-L-asparaginylglycine [mpGlyAsnAsnGly-OH, prepared by GenScript] (92.4 mg, 2.5 Eq, 181 pmol), DIPEA (37.4 mg, 50.4 pL, 4 Eq, 289 pmol), and HATU (110 mg, 4 Eq, 289 pmol)were added to the solution. The reaction was monitored by LCMS and the final product was purified by prep-HPLC to give a white solid (4.9 mg, 4.5 pmol, 6.2 %) RT= 3.08, m / z: for C49H61F3N8015S; (M+H)+= 1091.4.1HNMR: 8.18 (t, J=5.68Hz, 1H), 8.11 (d, J=7.4Hz, 1H), 8.06(d, J=7.7Hz, 1H), 7.83 (t, J=5.5Hz, 1H), 7.35(s, 1H), 7.22(s,lH) 6.99(s, 2H), 6.88(s,lH), 6.85-6.77(m, 2H), 6.35 (dd, J=10.2, 1.9, 1H), 6.15(s, 1H), 5.93 (d, J=50Hz, 2H), 5.74-5.52 (m, 1H), 5.29-5.20 (m, 1H), 4.54(dd, J= 14.1Hz, 7.0Hz, 3H), 4.39-4.32(m,lH), 3.99-3.91(m,lH) 3.80-3.72(m,lH), 3.68(d, 5.6Hz, 3H), 3.62(t, J=7.1, 3H), 3.50-3.40 (m,lH), 3.39-3.27(m,2H), 2.46-2.39(m, 8H), 2.24-2.13(m, 3H), 2.02(s,lH), 1.99(s, 1H), 1.96-1.85(m, 4H), 1.55(t, J=12.5Hz, 1H), 1.40(d, J=11.8Hz, 1H), 1.34(s, 3H), 1.03(t, J=7.7Hz, 3H), 0.9 l(d, J=7.1,3H).

[0163] Example 3. Preparation of (8S,9R,10S, HS,13S,14S,16S,17R)-17-(2-chloroacetyl)-9-fluoro-10,13,16-trimethyl-3-oxo-17-(propionyloxy)-6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-ll-yl L-prolinate [mpGlyAsnAsnGlyPro-Clobetasole Propionate or mpGNNGP-CP].Step 2□3 i

[0164] Step 1. 1 -(tert-butyl) 2-((8S,9R,10S,llS,13S,14S,16S,17R)-17-(2-chloroacetyl)-9-fluoro-10,13,16-trimethyl-3-oxo-17-(propionyloxy)-6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-l 1-yl) (S)-pyrrolidine-l,2-dicarboxylate: Clobetasol propionate (24.2 mg, 1 Eq, 51.8 pmol)was dissolved in 1ml DCM then treated with (tert-butoxycarbonyl)-L-proline (44.6 mg, 4 Eq, 207 pmol) and N, N-dimethylpyridin-4-amine (1.27 mg, 0.2 Eq, 10.4 pmol). The mixture was stirred for 5 minutes, then DCC (53.5 mg, 5 Eq, 259 pmol) was added, the reaction was stirred at RT and monitored by LCMS. Upon completion, the DCM was dried by air and the reaction was partitioned between 20ml ethyl acetate and 20ml water. The ethyl acetate layer was washed three times with water, then was collected in 50ml beaker, dried over MgSO4, and concentrated to dryness. The product was placed in the desiccator overnight. The final product was white in color (26.5 mg, 39.9 pmol, 77.0 %). RT= 4.22, (M+H)+=664.3.

[0165] Step 2. Preparation of (8S,9R,10S,llS,13S,14S,16S,17R)-17-(2-chloroacetyl)-9-fluoro-10,13,16-trimethyl-3-oxo-17-(propionyloxy)-6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-ll-yl L-prolinate: The product of step 1 (26.5 mg, 1 Eq, 39.9 pmol) was dissolved in 1.6ml DCM, then treated with 0.4ml TFA. The mixture was stirred for 30 minutes at RT, then the TFA and DCM were evaporated to give a yellowish solid (17.6 mg, 31.2 pmol, 78.2 %). RT= 2.65, (M+H)+=564.3.

[0166] Step 3. Preparation of (8S,9R,10S,llS,13S,14S,16S,17R)-17-(2-chloroacetyl)-9-fluoro-10,13,16-trimethyl-3-oxo-17-(propionyloxy)-6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-ll-yl (3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanoyl)glycyl-L-asparaginyl-L-asparaginylglycyl-L-prolinate: The product of step 2 (17.6 mg, 1 Eq, 31.2 pmol) was dissolved in 2ml DMF and treated with (3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanoyl)glycyl-L-asparaginyl-L-asparaginylglycine [mpGlyAsnAsnGlyPro-OH, prepared by GenScript] (31.9 mg, 2 Eq, 62.4 pmol), DIPEA (8.07 mg, 10.9 pL, 2 Eq, 62.4 pmol), and HATU (23.7 mg, 2 Eq, 62.4 pmol). The reaction was stirred at RT and monitored by LCMS. The final product was purified by prep-HPLC to give white solid (17.3 mg, 16.4 pmol, 52.4 %). RT=3.00, m / z for C49H62C1FN8015 (M+H)+is 1057.6. [NMR in progress]

[0167] Example 4: Preparation of (6S,8S,9R,10S, HS,13S,14S,16S,17R)-17-(2-chloroacetyl)-6,9-difluoro-10,13,16-trimethyl-3-oxo-17-(propionyloxy)-6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-ll-yl (3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanoyl)glycyl-L-asparaginyl-L-asparaginylglycyl-L-prolinate [mpGlyAsnAsnGlyPro-Halobetasol propionate or mpGNNGP-HP]:

[0168] Step 1: 1 -(tert-butyl) 2-((6S,8S,9R,10S,llS,13S,14S,16S,17R)-17-(2-chloroacetyl)-6,9-difluoro-10,13,16-trimethyl-3-oxo-17-(propionyloxy)-6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-ll-yl) (S)-pyrrolidine-l,2-dicarboxylate:Halobetasole propionate (40 mg, 1 Eq, 82 pmol)was dissolved in 1ml DCM then treated with (tert-butoxycarbonyl)-L-proline (71 mg, 4 Eq, 0.33 mmol), and DMAP (2.0 mg, 0.2 Eq, 16 pmol). The reaction was stirred for 5 minutes, then treated with DCC (85 mg, 5 Eq, 0.41 mmol) was added. The reaction was kept stirring at RT for 72 hrs. Upon completion, the solvent was removed and the reaction was partitioned between 20ml ethyl acetate and 20ml water. The ethyl acetate layer was washed three times with water, then was collected in a 50ml beaker, dried over MgSO4, and dried to give the product as a white solid (42 mg, 62 pmol, 75 %). RT= 2.71 [nonpolar HPLC method], (M+H)+is 682.4.

[0169] Step 2. (6S,8S,9R,10S,l lS,13S,14S,16S,17R)-17-(2-chloroacetyl)-6,9-difluoro- 10.13.16-trimethyl-3-oxo-17-(propionyloxy)-6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-ll-yl L-prolinate: The product of step 1 (42 mg, 1 Eq, 62 pmol) was dissolved in 1.6ml DCM and 0.4ml TFA. The mixture was stirred for 30 minutes, then the solvents were removed providing the final product as a yellowish solid. (27.5 mg, 47.2 pmol, 77 %). RT= 1.42 (non-polar method), (M+H)+is 582.4.

[0170] Step 3. (6S,8S,9R,10S,l lS,13S,14S,16S,17R)-17-(2-chloroacetyl)-6,9-difluoro- 10.13.16-trimethyl-3-oxo-17-(propionyloxy)-6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-ll-yl (3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanoyl)glycyl-L-asparaginyl-L-asparaginylglycyl-L-prolinate [mpGlyAsnAsnGlyPro-Halobetasol propionate]: The product of step 2 (10 mg, 2 Eq, 17 pmol) was dissolved in 0.5ml DMF and treated with (3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanoyl)glycyl-L-asparaginyl-L-asparaginylglycine [mpGlyAsnAsnGlyPro-OH, prepared by Genscript] (18 mg, 2 Eq, 34 pmol). HATU (13 mg, 2 Eq, 34 pmol), and DIPEA (8.9 mg, 12 pL, 4 Eq, 69 pmol) were added. The reaction was stirred at RT and monitored by LCMS. The final product was purified by prep-HPLC, to give white solid. (4 mg, 4 pmol, 20 %). RT= 3.03, m / z: for C49H61C1F2N8015; (M+H)+= 1075.4. 'H NMR: 8.18 (t, J=5.68Hz, 1H), 8.11 (d, J=7.6Hz, 1H), 8.04(d, J=8.0Hz, 1H), 7.83 (t, J=5.3Hz, 1H), 7.35(s, 1H), 7.19(s,lH) 6.99(s, 2H), 6.88(s,lH), 6.80(s, 2H), 6.77(s,lH), 6.33 (dd, J=10.2, 1.9, 1H), 6.15(s, 1H), 5.71-5.52(m,lH), 5.32-5.25 (m, 1H), 4.53(dd, J= 14.1Hz, 7.0Hz, 3H), 4.38-4.33(m,lH), 4.14(dd, J=15.5Hz, 15.5Hz,2H) 3.97-3.89(m,lH), 3.49-3.45(m,2H), 3.38-3.29(m,2H), 2.46-ES‘ 01 punoduiOQ0d ‘lOi punoduiogd‘001 punoduiogd:8UIMO[[OJ aip ‘oj pgjiuiq jou 9JP jnq ‘gpnpui uopugAui 9ip jo spunoduioo |Puopippy [ I £ I o](HE ‘S)98 0 ‘(HP ‘S£=£ ‘l)Z0 I ‘(HP‘zHP£=£ ‘P)0£ I ‘(HE! ‘(Hl‘ui)8P I-p9 I ‘(Hl‘ui)S9 I -LL \ ‘(HS‘ui)68 Z-I0 Z ‘(HI ‘^SO -PI ‘(Hl)9I Z-EZ Z ‘(HE‘UI)OE Z;-£E Z; ‘(H8 Z O V9008ESS0Compound 106, and0Compound 107.Example # Z1 XI A0A1A2A3A4 Glucocorticoid (GC) Example 1 mp bond GlyAsnAsnGlyPro Fluticasone Furoate Example 2 mp bond GlyAsnAsnGlyPro Fluticasone Propionate Example 3 mp bond GlyAsnAsnGlyPro Clobetasole Propionate Example 4 mp bond GlyAsnAsnGlyPro Halobetasol propionate Compound 100 BrAc bond GlyAsnAsnGlyPro Fluticasone Furoate Compound 101 BrAc bond AsnAsnGlyPro Fluticasone Furoate Compound 102 mp bond GlyAsnAsnGly(aMe-Pro) Fluticasone Furoate Compound 103 BrAc bond GlyAsnAsnGlyPro Fluticasone Propionate Compound 104 BrAc bond AsnAsnGlyPro Fluticasone Propionate Compound 105 mp bond GlyAsnAsnGly(aMe-Pro) Fluticasone Propionate Compound 106 mp bond GlyAlaAlaGlyPro Fluticasone Furoate Compound 107 mp bond GlyAlaAlaGlyPro Fluticasone Propionate Example 10 mp bond GluAsnAsnGlyPro Fluticasone Furoate Example 11 mp bond AsnAsnGlyPro Fluticasone Furoate Example 12 mp PEG4 AsnAsnGlyPro Fluticasone Furoate Example 13 mp bond SerAsnAsnGlyPro Fluticasone Furoate Example 14 BrAc bond AsnAsnGlyPro Fluticasone Furoate Example 15 BrAc PEG6 AsnAsnGlyPro Fluticasone Furoate Example 16 BrAc bond GluAsnAsnGlyPro Fluticasone Furoate Example 17 mp bond GlyAsnAsnAlaPro Fluticasone Furoate Example 18 mp bond GlyAsnAsnGlyPro Beclomethasone Dipropionate Example 19 BrAc bond GluAsnAsnAlaPro Fluticasone Furoate Compound 106 BrAc bond AspAsnAsnGlyPro Fluticasone Furoate Compound 107 BrAc bond AspAsnAsnAlaPro Fluticasone Furoate Compound 108 BrAc PEG4 AspAsnAsnGlyPro Fluticasone Furoate Compound 109 BrAc PEG4 AspAsnAsnAlaPro Fluticasone FuroateExample # Z1 XI A0A1A2A3A4 Glucocorticoid (GC) Compound 110 BrAc PEG4 GluAsnAsnAlaPro Fluticasone Furoate Compound 111 BrAc PEG4 GluAsnAsnAlaPro Fluticasone Furoate Compound 112 BrAc PEG4 SerAsnAsnGlyPro Fluticasone Furoate Compound 113 BrAc bond SerAsnAsnAlaPro Fluticasone Furoate Compound 114 BrAc PEG4 SerAsnAsnAlaPro Fluticasone Furoate Compound 115 AcLys bond GluAsnAsnAlaPro Fluticasone Furoate Compound 116 AcLys bond AsnAsnAlaPro Fluticasone Furoate Compound 117 AcLys bond GlyAsnAsnAlaPro Fluticasone Furoate Compound 118 BrAc bond AlaAlaGlyPro Fluticasone Furoate Compound 119 BrAc bond GluAlaAlaGlyPro Fluticasone Furoate Compound 120 BrAc bond AspAlaAlaGlyPro Fluticasone Furoate Compound 121 BrAc PEG4 AlaAlaGlyPro Fluticasone Furoate Compound 122 BrAc PEG4 GluAlaAlaGlyPro Fluticasone Furoate Compound 123 BrAc PEG4 AspAlaAlaGlyPro Fluticasone Furoate Compound 124 mp bond AsnAsnGlyPro Halobetasol propionate Compound 125 BrAc bond AsnAsnGlyPro Halobetasol propionate Compound 126 AcLys bond AsnAsnGlyPro Halobetasol propionate Compound 127 BrAc PEG4 SerAsnAsnAlaPro Halobetasol propionate Compound 128 BrAc PEG6 AsnAsnGlyPro Halobetasol propionate Compound 129 mp bond GluAsnAsnGlyPro Halobetasol propionate Compound 130 BrAc bond AspAsnAsnGlyPro Halobetasol propionate Compound 131 BrAc bond AspAsnAsnAlaPro Halobetasol propionate Compound 132 BrAc PEG4 AspAsnAsnGlyPro Halobetasol propionate Compound 133 BrAc bond AlaAlaGlyPro Halobetasol propionate Compound 134 BrAc bond GluAlaAlaGlyPro Halobetasol propionate Compound 135 BrAc bond AspAlaAlaGlyPro Halobetasol propionate Compound 136 BrAc PEG4 AlaAlaGlyPro Halobetasol propionate Compound 137 BrAc PEG4 GluAlaAlaGlyPro Halobetasol propionate Compound 138 BrAc PEG4 AspAlaAlaGlyPro Halobetasol propionateAs indicated supra, these compounds may also be used in reference to an antibody-drug conjugate, or a pharmaceutically acceptable salt thereof. That is, while compounds of Formula (I) are shown above, i.e., without an attached antibody, these compounds with the addition of an antibody (i.e., ADCs of Formula (A)) are also included and can be made by the syntheses and methods described herein.

[0172] Example 5: ADC synthesis and characterization: Conjugation of mpGNNGP-FF to anti-HER2: 50pl of 20mg / mL anti-HER2 was placed into an Eppendorf tube (Img). The volume was completed to 0.9ml with PBS / 5mM EDTA and then TCEP (4 pL, 5 mmolar, 6 Eq, 0.02 pmol) was added. The reaction was placed at 37°C and incubated for 2hrs at which point 8 eq of mpGlyAsnAsnGlyPro-FF (lOuL of 5mM DMA stock) was added to 92 ul DMA and then added to the reduced antibody. The tube was incubated at RT for 3 hours then an LCMS was taken to confirm loading. The reaction was buffer exchanged into PBS using a 30kd spin filtration device. The material was resuspended in 0.9ml DPBS. A 20uL aliquot was taken for LCMS and treated with lOuL of 0.5M TCEP immediately prior to analysis. The final protein concentration was measured by nanodrop and aggregation was determined by SEC. Loading and aggregation are shown in the table below. The same procedures were repeated for Anti-CD38, Anti -RS V, Anti-TNFa.

[0173] (Note the number in parenthesis below is the number of payloads added to the corresponding unmodified chain)Antibody Linker payload DAR AggreLC major Expected HC major Expected gation peak (m / z) mass shift peak (m / z) mass shift LC (Da) (Da) Anti-HER2 mpGNNGP-FF 5 <1% 23444 (+0) 1129 53987 (+3) 3387 Anti-HER2 mpGNNGP-FP 4 <1% 23442 (+0) 1091 51688 (+1) 1091 Anti-CD38 mpGNNGP-FF 3.7 1.2% 23378 (+0) 1129 54021 (+3) 3387 Anti-CD38 mpGNNGP-FP 2.9 1.3% 23378 (+0) 1091 51729 (+1) 3266 Anti-TNFa mpGNNGP-FF 7.2 5.1% 24546 (+1) 1129 54046 (+3) 3387 Anti-CDlla mpGNNGP-FF 8 5.9% 24546 (+1) 1129 54725 (+3) 3387 Anti-RSV mpGNNGP-FF 3.6 <1% 23151 (+0) 1129 53026 (+2) 2258Anti-RSV mpGNNGP-FP 6.8 <1% 23153 (+0) 1091 54247 (+3) 3270

[0174] SEC was performed using an Agilent 1200 HPLC configured with a BioSep- SEC-e3000 (300*7.80 mm) column. The solvent was 10% Acetonitrile in PBS at ImL / minute with a 20 minute run time. UV detection was employed (220, 254, 280 nm).

[0175] Example 6: Stability of the ADCs in human and mouse plasma: A volume of 134 pL of anti-CD38-mpGNNGP-FF (1.33 mg / mL stock solution) was mixed with 350 pL of mouse serum and 116 pL of phosphate-buffered saline (PBS), resulting in a final concentration of 300 pg / mL in a total volume of 600 pL. This procedure was repeated using human serum in place of mouse serum. For samples incubated in PBS, the volume was completed to 600 pL by PBS. In parallel, 133 pL of Anti-CD38-mpGNNP-FP (1.35 mg / mL stock solution) was processed under identical conditions. Aliquots of 60 pL were collected atspecific time points (days 0, 1, 3, 6, and 9) and stored at -80°C to halt any further reaction. Upon thawing, each sample was treated with 60 pL of acetonitrile (ACN) to precipitate serum proteins. The mixture was then centrifuged at 5000 rpm for 5 minutes, and 100 pL of the supernatant was carefully collected for LCMS analysis. Samples were analyzed using the Xevo-TQD liquid chromatography-tandem mass spectrometry (LC-MS / MS) system, employing multiple reaction monitoring (MRM) mode. The MRM transitions were tuned for the detection of the target compounds, with m / z 501.24 (daughter ions: 205.19, 275.31, 313.31) for fluticasone propionate and m / z 539.24 (daughter ions: 205.19, 275.27, 313.24) for fluticasone furoate. Quantification was performed against a standard curve prepared on the day of analysis. The results of this experiment are shown in FIG. 2. Less than 10% payload release was shown over the nine-day period, strongly suggesting good biological stability.

[0176] Example 7: Lysosmal stability of the ADCs: 30 pg of antibody-drug conjugates (ADCs: Anti-CD38mp_GNNGP_FF, FP) were buffer-exchanged into sodium acetate (NaOAc) buffer at pH 4.7, with a final volume of 60 pL at a concentration of 0.5 mg / mL. Separately, 40 pL of rat liver tritosomes (Xenotech) at a concentration of 2.5 mg / mL (100 pg) were activated by adding 40 pL of 2 mM DTT and 20 pL of EDTA in NaOAc buffer (pH 4.7). After incubating the mixture at 37°C for 30 minutes, 40 pL of the activated tritosomes were added to the 60 pL of each ADC. Aliquots (10 pL) were taken at intervals of 1, 5, 10, 15, 30, 60, 120, 180, and 240 minutes. After each aliquot was taken, 90 pL of acetonitrile (ACN) was immediately added, vortexed to precipitate the proteins, and stored at -80°C until analysis. Upon thawing, all samples were centrifuged for 5 minutes at 10000 rpm, and 80 pL of the supernatant from each sample was transferred into an LCMS vial containing 80 pL of PBS (pH 7.4). The samples were then incubated at 37°C for 24 hours to allow cyclization and payload release to occur. After the incubation period, 5 pL of 5% formic acid was added to halt further cyclization. The samples were subsequently analyzed using an LCMS MRM method for each payload (as outlined in Example 6) and the data were processed using a standard curve. The results of this experiment are shown in FIG. 3. As can be seen, the payloads are rapidly released in lysosomal extracts, achieving nearly full release by about 1 to 2 hours.

[0177] Example 8: Luciferase induction in MDA-kb2 cell line: The primary screening assay for the disclosed ADCs involves a reporter cell line (MDA-KB2) in which the glucocorticoid receptor (and androgen receptor) are linked to a firefly luciferase reporter enzyme. The cell line expresses modest amounts of HER2 (it is considered a TNBC cell line),allowing the use of a HER2 -targeting ADC as a proof of concept. Luciferase induction in MDA-kb2 is indicative of glucocorticoid release.

[0178] MDA-kb2 cells were diluted in L-15 cell media (cytiva) into a seeding suspension of -0.14 million cells per ml with lOOx pen / strep. Cells were seeded into a 96 well plate (90uL / well). The cells were incubated at 37C for 2 hours to allow them to adhere. After that, 10 uL of the ADCs solution (concentration range lOpg / ml-Opg / ml) 9points, 3x dilution factor was added to corresponding wells. The total volume in each well was 100 uL. The plate was incubated at 37C for 24 hours under atmospheric CO2 levels. The next day, culture media was discarded and the cells were rinsed twice with DPBS. Then, 25 uL of 2X passive lysis buffer (Promega) was added to each well with pipetting to allow for homogenous mixing. The plates were brough through 2 freeze-thaw cycles (-20°C) to ensure complete lysis of the cells.Subsequently, 25 uL of ATP solution was added to each well. Then samples were treated by the 25 uL 2mM D-luciferin solution (D-luciferin monopotassium salt, Fisher Scientific), and the luminescence at all wavelengths was read immediately for 0.1 seconds. Each concentration was measured in triplicate. The results are shown in FIG. 4 (top graph is fluticasone furoate, middle graph is fluticasone propionate, bottom graph is dexamethasone (reference technology)). The structures of the three graphed ADCs are also shown next to each graph and, as can be seen, the dexamethasone reference technology is attached at C-21, as compared with the ADCs of the invention, which are attached at C-l 1. ADCs of the invention promote significant luciferase signal at low ADC concentrations, while isotype control ADCs are far less active. No luciferase was observed with the control (naked) antibody groups. As can be seen, the HER2 -targeting ADCs exhibit -100-fold higher activity than analogous isotype control ADCs. Previous generation immune-suppressing ADC technology had only a very small window between targeted and non-targeted delivery, while the newly designed ADC technology exhibits a -100-fold window between the targeted and non-targeted ADC.

[0179] It has also been shown that the ADCs are stable in mouse and human plasma, losing less than 10% of the total payload over the course of 9 days of incubation (data not shown).

[0180] Example 9: Inhibition of NF-Kb by GC ADCs: THP-1 Dual cells (InVivogen) are a human monocyte-derived cell line modified to report on NF-KB / AP-1 and IRF pathways. This makes them ideal for studying immune responses and screening for modulators of these pathways (i.e. GCs). THP-1 dual cells were cultured in RPMI-1640 medium, supplemented with10% fetal bovine serum (FBS), 25 mM HEPES buffer, lx penicillin-streptomycin (lOOx stock), and lx Normocin (500x stock) for maintenance. For experiments, cells were seeded in a 96 well-plate at a density of 0.2 x 1OA6 cells / mL (120 pL / well) in RPMI medium supplemented with 10% human serum to block Fey receptor-mediated uptake of antibodies. Antibody-drug conjugates (ADCs) were prepared by diluting each ADC to a starting concentration of 100 pg / mL. A 3-fold serial dilution was performed in phosphate-buffered saline (PBS) to obtain a concentration gradient ranging from 100 pg / mL to 0 pg / mL. This ensured the concentrations were 10-fold higher than the final test concentrations. Subsequently, 15 pL of the corresponding ADC dilution was added to each well. All treatments were performed in triplicate. Plates were incubated at 37°C with 5% CO2 for 20 hours. After the 20-hour incubation period, cells were treated with Resiquimod (lOmM stock solution in DMSO) to a final concentration of 10 pM resiquimod (R848) per well, with 0.5% DMSO content. The plates were further incubated for 12 hours under the same conditions. The plates were then centrifuged at 1990 rpm for 10 minutes. The supernatant was collected, and secreted embryonic alkaline phosphatase (SEAP) levels, indicative of NF-KB activation, were measured. For SEAP quantification, 10 pL of the supernatant was transferred to 190 pL of Quanti-Blue solution (1% Quanti-Blue buffer, 1% Quanti-Blue reagent, and 98% distilled water). The reaction was incubated at 37°C, and absorbance was measured at 620 nm after 2 hours using a spectrophotometer. The results of this experiment are shown in FIG. 5. As can be seen, ADCs disclosed herein block NF-Kb activation in THPl-dual cells, while the naked mAb and isotype control have minimal effects. Moreover, the IL-8 cytokine release was assessed by ELISA. As shown in FIG. 5, the targeted ADCs (anti-TNF and anti-CD38) suppressed IL-8 release more effectively than the analogous isotype (non-targeted) control or unmodified antibody.

[0181] Example 10: Preparation of (S)-5-(((S)-4-amino-l-(((S)-4-amino-l-((2-((S)-2-((((6S,8S,9R,10S,llS,13S,14S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-17-((furan-2-carbonyl)oxy)-10,13,16-trimethyl-3-oxo-6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-ll-yl)oxy)carbonyl)pyrrolidin-l-yl)-2-oxoethyl)amino)-l,4-dioxobutan-2-yl)amino)-l,4-dioxobutan-2-yl)amino)-4-(3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanamido)-5-oxopentanoic acid (mpGluAsnAsnGly-Fluticasone Furoate) (mpGluAsnAsnGlyPro Fluticasone furoate or mpQNNGP-FF):Pfi fl tex?<y« a!j? -

[0182] Step 1: Preparation of 1 -(tert-butyl) 2-((6S,9R,10S,llS,13S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)- 17-((furan-2-carbonyl)oxy)- 10,13,16-trimethyl-3 -oxo- 6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-ll-yl) (2S)-pyrrolidine-l,2-dicarboxylate [ Boc-Pro-Fluticasone Furoate]: Fluticasone furoate (100 mg, 186 pmol) was dissolved in 2 mL dry di chloromethane (DCM). To this solution, (tertbutoxy carbonyl)-L-proline (160 mg, 4 eq, 743 pmol) was added, followed by the addition of DMAP (4.54 mg, 0.2 eq, 37.1 pmol) while stirring. After 5 minutes, DCC (160 mg, 4 eq, 743 pmol) was added, and the reaction mixture was stirred at room temperature for 24 hours, with progress monitored via LCMS. Once complete, the DCM was evaporated, and the product was re-dissolved in DMA. The mixture was centrifuged to remove dicyclohexylurea (DCC byproduct), and the clear solution was taken and purified by preparative RP-HPLC. The final product was obtained as a white solid (125.5 mg, 89.7 % yield), with M+H = 736.2 and a retention time (RT) of 2.74 minutes.

[0183] Step 2: 1 -(tert-butyl) 2-((6S,9R,10S,llS,13S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)- 17-((furan-2-carbonyl)oxy)- 10,13,16-trimethyl-3 -oxo- 6.7.8.9.10.11.12.13.14.15.16.17-dodecahydro-3H-cyclopenta[a]phenanthren-ll-yl) (2S)-pyrrolidine-l,2-dicarboxylate [Boc-Pro-Fluticasone Furoate] (125.5 mg) was dissolved in 1.6ml DCM, then treated with 0.4 ml TFA. The reaction was stirred for 30 minutes. The solvents were removed by air and the product dried under vacuum to give 99 mg (94 %) of the title compound as a slightly yellowish solid. M+H =636.1, RT= 1.43 minutes.

[0184] Step 3: (6S,9R,10S,llS,13S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)- 17-((furan-2-carbonyl)oxy)- 10,13,16-trimethyl-3 -oxo- 6.7.8.9.10.11.12.13.14.15.16.17-dodecahydro-3H-cyclopenta[a]phenanthren-ll-yl L-prolinate [H2N-Pro-Fluticasone Furoate] (99mg, leq, 155 pmol) was dissolved in 2ml DMF and treated with (3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanoyl)glycyl-L-asparaginyl-L-asparaginylglycine [Fmoc-AsnAsnGly-OH, prepared by GenScript] (81.8 mg, 1 Eq, 156 pmol) and DIPEA (80.5 mg, 109 pL, 4 Eq, 623 pmol) and HATU (237 mg, 4 Eq, 623 pmol). The reaction was monitored by LCMS. The final product was purified by preparative-HPLC to give a white solid (144mg, 80.9%). M+H = 1143.3, RT=2.21.

[0185] Step 4: (6S,9R,10S,llS,13S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-17-((furan-2-carbonyl)oxy)-10,13,16-trimethyl-3-oxo-6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-ll-yl (((9H-fluoren-9-yl)methoxy)carbonyl)-L-asparaginyl-L-asparaginylglycyl-L-prolinate [Fmoc-AsnAsnGlyPro-Fluticasone Furoate] (144mg, leq) was dissolved in 1.4 mL DMF then 0.4 mL piperidine was added. The mixture was stirred for 30 minutes then checked by LCMS. The product was purified by preparative-HPLC with a final yield of 99.6 mg (85.9 %) as a white solid. M+H = 921.2, RT= 1.46.

[0186] Step 5: (6S,9R,10S,llS,13S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)- 17-((furan-2-carbonyl)oxy)- 10,13,16-trimethyl-3 -oxo-6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-ll-yl L-asparaginyl-L-asparaginylglycyl-L-prolinate [ FhN-AsnAsnGlyPro-Fluticasone Furoate] (20mg, leq, 22 pmol) was dissolved in ImL DMF, then 5 -(tert-butyl) l-(2,5-dioxopyrrolidin-1-yl) (((9H-fluoren-9-yl)methoxy)carbonyl)-L-glutamate (23 mg, 2eq, 43 pmol) was added. After that DIPEA (11 mg (15 pl), 4 eq, 87 pmol) was added to the mixture. The reaction was stirred for 4 hours and checked by LCMS. The product was purified by preparative-HPLC to yield a white solid product (12.5 mg, 43%). M+H= 1329.3, RT= 3.01.

[0187] Step 6: (6S,9R,10S,llS,13S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-17-((furan-2-carbonyl)oxy)-10,13,16-trimethyl-3-oxo-6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-ll-yl ((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5-oxopentanoyl)-L-asparaginyl-L-asparaginylglycyl-L-prolinate (12.5mg, 1 eq, 9.41 pmol) was dissolved in 0.8 mL DMF then 0.2 mL piperidine was added. The reaction was stirred for 30 minutes. A sample was checked by LCMS and the final product was purified by preparative-HPLC with a yield of 10 mg (96%) as a white solid. M+H: 1107.02, RT = 2.2.

[0188] Step 7: (6S,9R,10S,llS,13S,16R,17R)-6,9-difluoro-17- (((fluoromethyl)thio)carbonyl)- 17-((furan-2-carbonyl)oxy)- 10,13,16-trimethyl-3 -oxo-6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-ll-yl ((S)-2-amino-5-(tert-butoxy)-5-oxopentanoyl)-L-asparaginyl-L-asparaginylglycyl-L-prolinate (lOmg, leq, 9.0 pmol) was dissolved in ImL DMF, then 2,5-dioxopyrrolidin-l-yl 3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanoate (4.8 mg, 2eq, 18 pmol) and N-ethyl-N-isopropylpropan-2-amine (2.3 mg, 2 eq, 18 pmol) were added. The reaction was stirred at room temperature for 30 minutes then checked by LCMS. The reaction was partitioned between ethyl acetate and water (10:10 ml), and then the ethyl acetate layer was washed twice with water. Then magnesium sulfate was added to remove residual water. The product was dried by air and then re-dissolved by DCM (2mL) and treated with 2mL of TFA. After ~lh, the solvents were removed by air andthe final product was purified by preparative HPLC with a yield of 4.5 mg (41 %). RT=3.01, m / z for C54H63F3N8O18S (M+H)+=1201.89. 'H NMR: 8.23-8.15 (m, 2H), 8.09 (d,j=7.35Hz, 1H), 8.06-7.99 (m, 1H) 7.86 (dt, j=50.09Hz, j= 5.56Hz, 1H), 7.44-7.24 (m, 3H), 6.99 (d, j=1.66Hz, 2H), 6.92 (d, j=10.98Hz,lH), 6.84-6.78 (m, 2H) 6.72 (q, j=1.75Hz, 2H), 6.39-6.32 (m,lH), 6.16 (s, 1H), 6.01 (s,lH), 5.89 (s, 1H), 5.74-5.53 (m, 1H), 5.37-5.22(m,lH), 4.64-4.50 (m, 3H), 4.49-4.43 (m, 2H), 4.39-4.31 (m, 4H), 4.24-4.11 (m, 6H), 3.69-3.53 (m, 3H), 3.51-3.36 (m, 2H), 2.47-2.27(m,3H), 2.35-2.26 (m,3H), 2.24-2.16 (m, 3H), 1.99-1.87 (m, 4H), 1.40-1.29 (m, 3H), 1.01 (s, 3H), 0.95 (d, j= 6.97Hz,3H).

[0189] For compounds of Examples 10-17 with molecular weights less than 1200 Da, analytical LCMS was performed as described in Example 1. For compounds with molecular masses exceeding 1200 Da, analytical HPLC was performed on a Waters ACQUITY HPLC system equipped with an XBridge C18 column (5 pm, 4.6 x 100 mm). The mobile phases consisted of 0.02% formic acid in water (A) and 0.02% formic acid in acetonitrile (B).Samples were eluted at a flow rate of 2.0 mL / min over a 5-minute run time, with the gradient shown below. Mass spectrometric detection was conducted using an Waters TQD electrospray ionization (ESI) detector, and UV absorbance was monitored at 220 and 254 nm.Time Flow (ml / minute) %A %B0 2.00 95.0 5.01.00 2.00 95.0 5.03.80 2.00 1.0 99.03.90 2.00 1.0 99.04.00 2.00 95.0 5.05.00 2.00 95.0 5.0

[0190] Example 11: (6S,8S,9R,10S,llS,13S,14S,16R,17R)-6,9-difluoro-17- (((fluoromethyl)thio)carbonyl)-17-((furan-2-carbonyl)oxy)-10,13,16-trimethyl-3-oxo- 6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-ll-yl (3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanoyl)-L-asparaginyl-L-asparaginylglycyl-L-prolinate (mpAsnAsnGlyPro-Fluticasone Furoate):

[0191] (6S,9R, 1 OS, 11 S, 13 S, 16R, 17R)-6,9-difluoro- 17-(((fl uoromethyl )thi o)carbonyl )- 17- ((furan-2-carbonyl)oxy)-10,13,16-trimethyl-3-oxo-6,7,8,9,10,ll,12,13,14,15,16,17- dodecahydro-3H-cyclopenta[a]phenanthren-ll-yl L-asparaginyl-L-asparaginylglycyl-L- prolinate [H2N-AsnAsnGlyPro, Example 10 step 4] (10 mg, leq, 11 pmol) was dissolved in 1 mL DMF. Then 2,5-dioxopyrrolidin-l-yl 3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l- yl)propanoate (5.8 mg, 2 eq, 22 pmol) and DIPEA (2.8 mg, 2 eq, 22 pmol) were added. The reaction was stirred at room temperature for 30 minutes and checked by LCMS. The product was purified by prep-HPLC, dried by air to give a final yield of 4.5 mg (39%), white solid.RT= 3.07. m / z for C49H56F3N7O15S (M+H)+= 1071.41. 'HNMR: 8.23 (d, j=7.54Hz, 1H), 8.17-8.07 (m, 1H), 8.04 (d, j=1.02Hz, 1H), 6.98(dt, j=40.33Hz, j= 5.35Hz, 1H), 7.38-7.24 (m, 3H), 6.99(s, 2H), 6.94-6.77 (m,3H) 6.72 (q, j=7.69Hz, 1H), 6.35(dt, j=10.34Hz, j=1.49Hz,lH), 6.16 (s, 1H), 6.01 (s,lH), 5.89 (s, 1H), 5.76-5.53 (m, 1H), 5.29(d,j=9.0,lH), 4.63-4.41 (m, 3H), 4.40-4.31 (m, 1H), 3.63-3.54 (m, 4H), 3.50-3.39 (m,3H) 2.89(s,lH), 2.73 (s,lH), 2.47-2.42 (m, 2H), 2.32-2.35 (m,3H), 2.34-2.27 (m, 3H), 2.24-2.14 (m, 1H), 2.04 (d, j=14.33Hz, 1H), 1.98-1.85(m,4H), 1.67-1.51 (m,2H), 1.36 (d,j= 3.14Hz, 3H) 1.01 (s,3H).

[0192] Example 12: (6S,8S,9R,10S, HS,13S,14S,16R,17R)-6,9-difluoro-17- (((fluoromethyl)thio)carbonyl)-17-((furan-2-carbonyl)oxy)-10,13,16-trimethyl-3-oxo- 6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-ll-yl (l-(2,5- dioxo-2,5-dihydro-lH-pyrrol-l-yl)-3-oxo-7, 10,13, 16-tetraoxa-4-azanonadecan-19-oyl)-L- asparaginyl-L-asparaginylglycyl-L-prolinate (mp-PEG4-AsnAsnGlyPro-FluticasoneFuroate):

[0193] (6S,9R, 1 OS, 11 S, 13 S, 16R, 17R)-6,9-difluoro- 17-(((fl uoromethyl )thi o)carbonyl )- 17- ((furan-2-carbonyl)oxy)-10,13,16-trimethyl-3-oxo-6,7,8,9,10,ll,12,13,14,15,16,17- dodecahydro-3H-cyclopenta[a]phenanthren-ll-yl L-asparaginyl-L-asparaginylglycyl-L- prolinate (15 mg, 1 eq, 16 pmol) [H^N-AsnAsnGlyPro, Example 10 step 4] was dissolved in 1 ml DMF, then 2,5-dioxopyrrolidin-l-yl l-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)-3-oxo- 7, 10,13, 16-tetraoxa-4-azanonadecan- 19-oate (17 mg, 2 eq, 33 pmol) and DIPEA (4.2 mg, 2 eq, 33 pmol) were added. The reaction was stirred for 30 minutes and the checked by LCMS. The final product was purified by preparative-HPLC and dried by air to yield colorless solid (14.5 mg, 67 %). RT = 2.85 m / z for C60H77F3N8O20S = 1319.66.1HNMR: 8.15-8.07 (m, 2H), 8.04 (d, j=1.04Hz, 1H), 8.00 (t, j=5.79Hz, 1H) 7.86 (dt, j=37.85Hz, j= 5.69Hz, 1H), 7.38-7.24 (m, 3H), 7.00 (s, 2H), 6.94-6.83 (m,3H), 6.81 (d, j= 10.38Hz, 2H) 6.72 (q, j=8.18Hz, 2H), 6.35(dt, j=10.34Hz, j=1.49Hz,lH), 6.16 (s, 1H), 6.02 (s,lH), 5.89 (s, 1H), 5.74-5.53 (m, 2H), 5.33-5.25(m,lH), 4.64-4.55 (m, 5H), 4.00-3.71 (m, 2H), 3.53-3.41 (m, 16H), 3.36 (t, j= 6.08Hz, 3H), 3.16 (q, j= 5.84Hz, 2H), 2.87-2.62(m,lH), 2.48-2.42 (m,2H), 2.42-2.36 (m, 3H), 2.33 (t, J=7.16,3H), 2.26-2.14 (m, 2H) 2.09-2.06 (m, 1H), 1.99-1.88 (m, 4H), 1.72-1.53 (m, 1H), 1.36(d, j= 2.90Hz,3H), 1.01 (s,2H), 0.95(d, j= 6.62Hz, 3H).

[0194] Example 13: (6S,9R,10S,llS,13S,16R,17R)-6,9-difluoro-17- (((fluoromethyl)thio)carbonyl)-17-((furan-2-carbonyl)oxy)-10,13,16-trimethyl-3-oxo- 6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-ll-yl (3-(2,5- dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanoyl)-L-seryl-L-asparaginyl-L- asparaginylglycyl-L-prolinate (mpSerAsnAsnGlyPro-FF or mpSNNGP-FF):HOBt PyBOP DIPEAStep 1O

[0195] Step 1: (6S,9R,10S,llS,13S,16R,17R)-6,9-difluoro-17- (((fluoromethyl)thio)carbonyl)- 17-((furan-2-carbonyl)oxy)- 10,13,16-trimethyl-3 -oxo- 6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-ll-yl L- asparaginyl-L-asparaginylglycyl-L-prolinate (20 mg, 1 eq, 22 pmol) [FhN-AsnAsnGlyPro, Example 10 step 4] was dissolved in DMF in 20 ml vial. Then benzotriazol- 1- yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) (45 mg, 4 eq, 87 pmol), 1- Hydroxybenzotriazole (HOBT) (3.2 mg, leq, 22 pmol), and DIPEA (11 mg, 15 pl, 4 eq, 87 pmol) were added to the vial. This mixture was stirred for 10 minutes, then (tert- butoxycarbonyl)-L-serine (18 mg, 4 eq, 87 pmol) was added. The reaction was stirred for 30 minutes and checked by LCMS. The product was partitioned between ethyl acetate and water, then the ethyl acetate layer was dried by air and this was taken directly to the next step (15.9 mg, 66% white solid).

[0196] Step 2: (6S,9R,10S,llS,13S,16R,17R)-6,9-difluoro-17- (((fluoromethyl)thio)carbonyl)- 17-((furan-2-carbonyl)oxy)- 10,13,16-trimethyl-3 -oxo- 6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-ll-yl (tert- butoxycarbonyl)-L-seryl-L-asparaginyl-L-asparaginylglycyl-L-prolinate [Boc- SerAsnAsnGlyPro-FF] (15.9mg) was dissolved in 1.6 ml DCM, then 0.4 ml of TFA was added. The mixture was stirred for 30 minutes at room temperature and the DCM and TFAlayer were removed by air. The final yield was slightly yellowish in color and it was taken directly to the next step. (11.5 mg, 79.5 %).

[0197] Step 3: (6S,9R,10S,llS,13S,16R,17R)-6,9-difluoro-17- (((fluoromethyl)thio)carbonyl)- 17-((furan-2-carbonyl)oxy)- 10,13,16-trimethyl-3 -oxo- 6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-ll-yl L-seryl-L- asparaginyl-L-asparaginylglycyl-L-prolinate [EEN-SerAsnAsnGlyPro-FF] (11.5mg, 1 eq,11.4 pmol) was dissolved in 1 ml DMF in a 4 ml vial. Then 3-(Maleimido)propionic acid N- hydroxysuccinimide ester (6 mg, 2 eq, 22.84 pmol) and DIPEA (2.95 mg, 2 eq, 22.84 pmol) were added to the vial. The reaction was stirred for 30 minutes, and then checked by LCMS.The final product was purified by preparative HPLC and dried by air to yield colorless solid (3.6 mg, 27 %). (M+H)+=l 158.81, RT = 2.96. 'HNMR: 8.21-8.12 (m, 1H) 8.08 (d, j=7.69, 1H), 8.04 (d, j=1.01, 1H), 8.01-7.93 (m, 1H), 7.79 (dt, j=20.20Hz, j=5.77, 1H), 7.37 (s, 1H), 7.30 (d, j=3.67Hz, 1H), 7.24 (s, 1H), 7.00 (d, j=1.42Hz, 2H), 6.93 (s, 1H), 6.82 (t, j=l 1.22Hz, 2H), 6.72 (q, j=1.75Hz, 1H), 6.35 (dt, j=10.12Hz, j=1.93Hz, 1H), 6.16 (s, 1H), 6.01 (s,lH), 5.89 (s, 1H), 5.73-5.53 (m, 1H), 5.32-5.21(m,lH), 4.61-4.43 (m, 3H), 4.40-4.22 (m, 3H),3.63-3.53 (m, 6H), 3.52-3.39 (m, 4H), 2.46-2.39(m,4H), 2.37-2.27 (m,3H), 2.25-2.14 (m, 2H), 1.99-1.87 (m, 4H), 1.79- 1.69 (m, 1H), 1.68-1.54 (m, 1H), 1.36 (d, j= 3.91, 4H), 1.01 (s, 3H), 0.95 (d, j= 7.08 Hz,3H).

[0198] Example 14: (6S,9R,10S,llS,13S,16R,17R)-6,9-difluoro-17- (((fluoromethyl)thio)carbonyl)-17-((furan-2-carbonyl)oxy)-10,13,16-trimethyl-3-oxo- 6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-ll-yl N4-(2- bromoacetyl)-L-asparaginyl-L-asparaginylglycyl-L-prolinate (BrAc-AsnAsnGlyPro-FF, or BrAc-NNGP-FF):DIPEA

[0199] (6S,9R, 1 OS, 11 S, 13 S, 16R, 17R)-6,9-difluoro- 17-(((fl uoromethyl )thi o)carbonyl )- 17- ((furan-2-carbonyl)oxy)-10,13,16-trimethyl-3-oxo-6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-ll-yl L-asparaginyl-L-asparaginylglycyl-L-prolinate (10.8 mg, 1 eq, 11.7 pmol) [H2N-AsnAsnGlyPro, Example 10 step 4] was dissolved in dry DCM and treated with 2-bromoacetyl bromide (9.47mg, 4eq, 4.06 pl, 46.9 pmol) and DIPEA (3.03 mg, 2 eq, 4.09 pl 23.5 pmol). The reaction was stirred for 30 minutes at room temperature, then quenched with 20 pl water. Then the mixture was evaporated by air and redissolved in DMF and purified by preparative-HPLC. The final yield was 2.6 mg (21 %) of a colorless product. C44H52BrF3N6O13S (M+H)+=1041.2, RT = 1.77.1HNMR: 8.51 (dd, j=7.67Hz, j=1.89, 1H), 8.22 (dd, j=7.85Hz, j=1.88Hz, 1H), 8.04 (d, j=1.44Hz, 1H) 7.86 (dt, j=57.15Hz, j= 5.19Hz, 1H), 7.44-7.24 (m, 3H), 6.94 (d, j=10.69Hz, 1H), 6.84-6.78 (m, 2H) 6.72 (q, j=1.75Hz, 2H), 6.35 (dt, j=10.12Hz, j=1.78Hz, 1H), 6.16 (s, 1H), 6.02 (s,lH), 5.89 (s, 1H), 5.73-5.53 (m, 1H), 5.32-5.26(m,lH), 4.60-4.47 (m, 2H), 4.40-4.32 (m, 2H), 3.63-3.53 (m, 3H), 3.50-3.38 (m, 3H), 2.46-2.36(m,3H), 2.35-2.26 (m,4H), 2.25-2.14 (m, 2H), 2.05(d, j=14.81Hz, 1H), 1.99-1.87 (m, 4H), 1.36 (d, j= 2.02, 4H), 1.02 (s, 3H), 0.95 (d, j= 6.66Hz,4H).

[0200] Example 15: (6S,9R,10S, HS,13S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-17-((furan-2-carbonyl)oxy)-10,13,16-trimethyl-3-oxo-6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-ll-yl (1-bromo-2-oxo-6,9,12,15,18,21-hexaoxa-3-azatetracosan-24-oyl)-L-asparaginyl-L-asparaginylglycyl-L-prolinate (BrAc-PEG6-AsnAsnGlyPro-FF, or BrAc-PEG6-NNGP-FF):Nft- -

[0201] Step 1: l-(9H-fluoren-9-yl)-3-oxo-2,7,10,13,16,19,22-heptaoxa-4-azapentacosan-25-oic acid (19 mg, 16 pL, 2 Eq, 33 pmol), DIPEA (4.2 mg, 5.7 pL, 2 Eq, 33 pmol), HOBt (2.5 mg, 1 Eq, 16 pmol), PyBOP (17 mg, 2 Eq, 33 pmol), and DIPEA (4.2 mg, 5.7 pL, 2 Eq, 33 pmol) were dissolved in dry DCM (2ml). The reaction was stirred for 15 minutes and then (6S,9R,10S,llS,13S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-17-((furan-2-carbonyl)oxy)-10,13,16-trimethyl-3-oxo-6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-ll-yl L-asparaginyl-L-asparaginylglycyl-L-prolinate (15 mg, 1 Eq, 16 pmol) [EEN-AsnAsnGlyPro, Example 10 step 4] was added. The mixture was stirred for 2 hrs, then an LCMS was taken which showed the reaction was finished. The product was purified by prep-HPLC. The product was dried by air to yield a colorless substance (10.5 mg, 44%).

[0202] Step 2: (6S,9R,10S,llS,13S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)- 17-((furan-2-carbonyl)oxy)- 10,13,16-trimethyl-3 -oxo-6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-ll-yl (1-(9H-fluoren-9-yl)-3 -oxo-2,7, 10,13,16,19,22-heptaoxa-4-azapentacosan-25-oyl)-L-asparaginyl-L-asparaginylglycyl-L-prolinate (10.5 mg, leq, 7.10 pmol) was dissolved in 0.7 ml DMF, then 0.3 ml piperidine was added. The reaction was stirred for 30 minutes, then checked by LCMS. The product was purified by prep-HPLC and dried by air with a final yield of 6.5 mg. (73 %).

[0203] Step 3: (6S,9R,10S,llS,13S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)- 17-((furan-2-carbonyl)oxy)- 10,13,16-trimethyl-3 -oxo-6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-ll-yl (1-amino-3,6,9, 12,15, 18-hexaoxahenicosan-21-oyl)-L-asparaginyl-L-asparaginylglycyl-L-prolinate (6.5 mg, 1 eq, 5.2 pmol) was dissolved in 2 ml DCM, then DIPEA (1.3 mg, 1.8 pL, 2 Eq, 10 pmol) and 2-bromoacetyl bromide (2.1 mg, 0.90 pL, 2 Eq, 10 pmol) were added. The reaction was stirred for 30 minutes, then checked by LCMS. The final product was purified by prep-HPLC with colorless solid and 2.4 mg (34%) yield, m / z for C59H81BrF3N7O20S (M+H)+= 1376.65, RT= 4.15. 'HNMR: 7.81 (t, j=0.74Hz, 1H), 7.24 (d, j=3.56Hz, 1H), 6.98(d, j=9.97Hz, 1H), 6.66-6.63 (m, 1H), 6.44-6.37(m, 1H), 6.35 (s,lH) 6.10-5.86 (m, 2H), 5.49(s,lH), 4.83-4.77 (m, 2H), 4.73-4.61 (m,lH), 4.58-4.48 (m, 1H), 4.15-3.98 (m, 2H), 1.96(s,2H), 3.81-3.70 (m, 1H), 3.69-3.61 (m, 22H), 3.58 (t, j= 5.21Hz,3H), 3.52-3.47 (m,2H) 3.36-3.34(m,2H), 3.18-3.12 (m,lH), 3.0 (d, j= 5483Hz, 2H), 2.85-2.76 (m,3H), 2.75-2.66 (m, 3H), 2.59-2.49 (m,2H), 2.48-2.35(m,3H), 2.33-2.23(m,lH), 2.17 (d,j= 14.6Hz, 1) 2.12-2.02(m,5H), 1.95 (s, 1H), 1.75-1.66 (m,lH), 1.51(s,lH), 1.45 (s,3H), 1.30(s,lH), 1.15 (s, 3H), 1.06(d, j=7.34, 3H).

[0204] Example 16: (S)-5-(((S)-4-amino-l-(((S)-4-amino-l-((2-((S)-2- ((((6S,8S,9R,10S,llS,13S,14S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-17- ((furan-2-carbonyl)oxy)-10,13,16-trimethyl-3-oxo-6,7,8,9,10,ll,12,13,14,15,16,17- dodecahydro-3H-cyclopenta[a]phenanthren-ll-yl)oxy)carbonyl)pyrrolidin-l-yl)-2- oxoethyl)amino)-l,4-dioxobutan-2-yl)amino)-l,4-dioxobutan-2-yl)amino)-4-(2- bromoacetamido)-5-oxopentanoic acid (BrAc-GluAsnAsnGlyPro-FF or BrAcQNNGP-FF):o a) DIPEA; b) TFA

[0205] (6S,9R,10S,llS,13S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-17- ((furan-2-carbonyl)oxy)-10,13,16-trimethyl-3-oxo-6,7,8,9,10,ll,12,13,14,15,16,17- dodecahydro-3H-cyclopenta[a]phenanthren-ll-yl ((S)-2-amino-5-(tert-butoxy)-5- oxopentanoyl)-L-asparaginyl-L-asparaginylglycyl-L-prolinate (lOmg, leq, 9.0 pmol) [H2N- AsnAsnGlyPro, Example 10 step 6] was dissolved in 2 ml DCM. Then 2-bromoacetyl bromide (6.9mg, 3.0 pl, 4eq, 34 pmol) and DIPEA (4.4 mg, 3.0 pl, 4eq, 34 pmol) wereadded. The reaction was stirred at room temperature for 30 minutes then checked by LCMS.The reaction was partitioned between ethyl acetate and water (10:10 ml), and then the ethyl acetate layer was washed twice with water. Magnesium sulfate was added to remove residual water. The product was dried by air and then re-dissolved in 2ml of DCM. Then 2 mL TFA was added and the rxn was stirred for ~lh. After completion of the reaction, DCM and TFA were removed by air and the final product was purified by preparative-HPLC with a yield of 1.2 mg (12%) white solid, m / z for C49H59BrF3N7O16S (M+H)+= 1170.2, RT= 3.16.

[0206] Example 17: (6S,8S,9R,10S,llS,13S,14S,16R,17R)-6,9-difluoro-17- (((fluoromethyl)thio)carbonyl)-17-((furan-2-carbonyl)oxy)-10,13,16-trimethyl-3-oxo- 6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-ll-yl (3-(2,5- dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanoyl)glycyl-L-asparaginyl-L-asparaginylalanyl- L-prolinate (mpGlyAsnAsnAlaPro-FF or mpGNNAP-FF):

[0207] Step 1: (6S,9R,10S,llS,13S,16R,17R)-6,9-difluoro-17- (((fl uoromethyl )thi o)carbonyl )- 17-((furan-2-carbonyl)oxy)- 10,13,16-trimethyl-3 -oxo- 6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-ll-yl L-prolinate (12 mg, 1 eq, 18.9 pmol) [Example 10, step 2] was dissolved in 2 ml DMF and treated with (((9H-fluoren-9-yl)methoxy)carbonyl)-L-asparaginyl-L-asparaginylalanine (Fmoc-GlyAsnAsnAla-OH) from Genscript (15.3mg, 1.5 eq, 28.3 pmol), PyBOP (39.3 mg, 4 eq, 75.5 pmol), HOBt (2.89 mg, 1 eq, 18.9 pmol), and DIPEA (9.76 mg, 13.2 pL, 4 eq, 75.5 pmol were added. The reaction continued stirring for 24 hours and then checked LCMS. The desired product was purified by preparative HPLC and the final yield was 11.5 mg (52.6%) white solid. (M+H)+= 1157.4, RT= 2.01.

[0208] Step 2: (6S,9R,10S,llS,13S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)- 17-((furan-2-carbonyl)oxy)- 10,13,16-trimethyl-3 -oxo- 6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-ll-yl (((9H-fluoren-9-yl)methoxy)carbonyl)-L-asparaginyl-L-asparaginylalanyl-L-prolinate was dissolved in 1.6 ml DMF and then 0.4 ml piperidine was added. The reaction was stirred for 30 minutes and checked by LCMS. The desired compound was purified by prep-HPLC with a yield of 4.2 mg (45%) and white solid. (M+H)+= 935.3, RT= 1.33.

[0209] Step 3: (6S,9R,10S,llS,13S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)- 17-((furan-2-carbonyl)oxy)- 10,13,16-trimethyl-3 -oxo- 6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-ll-yl L-asparaginyl-L-asparaginylalanyl-L-prolinate (4.2 mg, 1 eq, 4.5 pmol) was dissolved in 1 ml DMF, then (tert-butoxycarbonyl)glycine (3.1 mg, 4 eq, 18 pmol), PyBOP (9.4 mg, 4 eq, 18 pmol), HOBt (Img, 1.5 eq, 6.75 pmol), and DIPEA (3.2 mg, 3.1 pl, 4eq, 18 pmol) were added and the reaction was stirred at room temperature for two hours. The reaction was stirred for 30 minutes and checked by LCMS. The product was worked-up between ethyl acetate and water, then the ethyl acetate layer was dried by air. Then the product was dissolved in 0.8 ml DCM and 0.2 ml TFA was added. The reaction was stirred for 30 minutes and then checked by LCMS. DCM and TFA were removed by air and this was taken directly to the next step (2.8 mg, 57%), (M+H)+= 992.3, RT=1.33 minutes.

[0210] Step 4: (6S,8S,9R,10S,llS,13S,14S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)- 17-((furan-2-carbonyl)oxy)- 10,13,16-trimethyl-3 -oxo-6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-ll-yl glycyl-L- asparaginyl-L-asparaginylalanyl-L-prolinate (2.8 mg, 1 eq, 2.8 pmol) was dissolved in 1 ml DMF. Then 2,5-dioxopyrrolidin-l-yl 3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanoate (3 mg, 4 eq, 11 pmol) and DIPEA (1.2 mg, 2 pl, 4eq, 11 pmol) were added. The reaction was finished after one hour of stirring at room temperature and then the final linker payload was purified by prep-HPLC. (M+H)+= 1143.4, RT=1.59 minutes.

[0211] Example 18: (8S,9R,10S,llS,13S,14S,16S,17R)-9-chloro-10,13,16-trimethyl-3- oxo-17-(propionyloxy)-17-(2-(propionyloxy)acetyl)-6,7,8,9,10,ll,12,13,14,15,16,17- dodecahydro-3H-cyclopenta[a]phenanthren-ll-yl (3-(2,5-dioxo-2,5-dihydro-lH-pyrrol- l-yl)propanoyl)glycyl-L-asparaginyl-L-asparaginylglycyl-L-prolinate (mpGlyAsnAsnGlyPro-BD, or mpGlyAsnAsnGlyPro-Beclomethasone Dipropionate)oDIPEA HATUStep 3

[0212] Step 1: (8S,9R,10S,llS,13S,14S,16S,17R)-9-chloro-ll-hydroxy-10,13,16- trimethyl-3-oxo-17-(2-(propionyloxy)acetyl)-6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro- 3H-cyclopenta[a]phenanthren-17-yl propionate [Beclomethasone Dipropionate] (50mg, 1 eq, 96 pmol) was dissolved in 2 ml DCM. Then (tert-butoxycarbonyl)-L-proline (83mg, 4 eq,380 pmol), dicyclohexylmethanediimine (160mg, 8 eq, 0.77 mmol), and N, N- dimethylpyridin-4-amine (3.4mg, 0.3 eq, 28 pmol) were added. The reaction was stirred at room temperature for 24 hours, with progress monitored via LCMS. Once complete, theDCM was evaporated, and then product was re-dissolved in DMA. The mixture was centrifuged to remove dicyclohexylurea (DCC byproduct), and the clear solution was takenand purified by preparative RP-HPLC. The final product was obtained as a white solid (20 mg, 29 % yield), with (M+H)+= 718.3 and a retention time (RT) of 4.55 minutes.

[0213] Step 2: 1 -(tert-butyl) 2-((8S,9R, 10S, 1 IS, 13S,14S,16S, 17R)-9-chloro-10, 13,16-trimethyl-3-oxo-17-(propionyloxy)-17-(2-(propionyloxy)acetyl)-6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-ll-yl) (S)-pyrrolidine-l,2-dicarboxylate (20mg, 1 eq, 28 pmol) was dissolved in 1.6 ml DCM. Then 0.4 ml TFA was added and the reaction was stirred for 30 minutes. The reaction was checked by LCMS and then DCM and TFA were dried by air to yield 11.5mg (67%) yellowish solid. (M+H)+= 618.5, RT = 2.71.

[0214] Step 3: (8S,9R,10S,llS,13S,14S,16S,17R)-9-chloro-10,13,16-trimethyl-3-oxo-17-(propionyloxy)-17-(2-(propionyloxy)acetyl)-6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-l 1-yl L-prolinate (1 Img, leq, 18 pmol) was dissolved in DMF. Then (3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanoyl)glycyl-L-asparaginyl-L-asparaginylglycine (14mg, 1.5 eq, 27 pmol), HATU (27 mg, 4eq, 71 pmol), DIPEA (9.2 mg, 12 pL, 4eq, 71 pmol) were added and the reaction was stirred for 2 hours, then reaction was checked by LCMS. The final product was purified by preparative HPLC with a yield of 8.4 mg (76%) white solid, m / z for C52H67CIN8O17 = 1111.7, RT= 3.15, ' H NMR: 8.18 (t, j=5.85Hz, 1H), 8.09 (dd, j=6.00Hz and 16Hz, 2H), 7.85 (t, j=3.75Hz, 1H), 7.35 (s, 1H), 7.19 (s, 1H), 6.99 (s, 2H), 6.88 (s, 1H), 6.78 (d, j=10.23 Hz 2H), 6.25 (dd j=6.76Hz, j=1.95Hz, 1H), 6.03 (s 1H), 5.48 (s 1H), 4.70 (dj=10.82Hz 1H), 4.52 (m, 3H), 4.34 (d j=12.45 1H), 3.91 (ddj=3.4Hz and 12Hz 1H), 3.76 (dd j=3.6 and 12.9Hz 1H), 3.68 (d j=3.982H), 3.63-3.5 (m, 3H), 3.49-3.42 (m, 1H), 2.83-2.7 (m, 2H), 2.68-2.60 (m, 1H), 2.47-2.34 (m,9H), 2.23-2.09 (m, 4H), 2.03-1.82 (m, 5H), 1.59-1.47 (m, 2H), 1.42 (s 3H), 1.24 (d j=8.3Hz 4H), 1.08- 1.02 (m, 6H), 0.84 (s, 3H).

[0215] Example 19: Preparation of (4R)-5-(((2S)-4-amino-l-(((2S)-4-amino-l-((l-((2S)-2-((((6S,9R,10S,llS,13S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-17-((furan-2-carbonyl)oxy)-10,13,16-trimethyl-3-oxo-6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-ll-yl)oxy)carbonyl)pyrrolidin-l-yl)-l-oxopropan-2-yl)amino)-l,4-dioxobutan-2-yl)amino)-l,4-dioxobutan-2-yl)amino)-4-(2-bromoacetamido)-5-oxopentanoic acid (BrAc-GluAsnAsnAlaPro-Fluticasone Furoate or BrAcENNAP-FF).o

[0216] Step 1: (6S,9R,10S,llS,13S,16R,17R)-6,9-difluoro-17- (((fluoromethyl)thio)carbonyl)- 17-((furan-2-carbonyl)oxy)- 10,13,16-trimethyl-3 -oxo- 6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-ll-yl L- asparaginyl-L-asparaginylalanyl-L-prolinate (10 mg, 1 eq, 11 pmol) [example 17, step 2] was dissolved in 1ml DMF. Then 5 -(tert-butyl) l-(2,5-dioxopyrrolidin-l-yl) (((9H-fluoren-9- yl)methoxy)carbonyl)-L-glutamate ( 22mg, 4 eq, 43 pmol) and DIPEA (5.5 mg, 7.5 pL, 4 eq, 43 pmol) were added. Th reaction was stirred at room temperature for 2 hours and thenchecked by LCMS. The product was extracted between ethyl acetate and water (10 ml: 10ml), then washed three times with water. Then, sodium sulfate was added the ethyl acetatelayer to dry residual water, then filtered and dried by air. The yield was 9.7 mg (68%), white solid. (M+H)+= 1142.73, RT= 3.90.

[0217] Step 2: (6S,9R,10S,llS,13S,16R,17R)-6,9-difluoro-17- (((fluoromethyl)thio)carbonyl)- 17-((furan-2-carbonyl)oxy)- 10,13,16-trimethyl-3 -oxo- 6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-ll-yl ((R)-2- ((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5-oxopentanoyl)-L- asparaginyl-L-asparaginylalanyl-L-prolinate (9.7 mg, leq, 7.2 pmol) was dissolved in 0.8 ml DMF, then 0.2 ml piperidine was added. The reaction was stirred for 30 minutes and then checked by LCMS. The product was purified by preparative-HPLC, with a final yield of7.2mg (89%), white solid. (M+H)+=1120.67, RT = 2.78 minutes.

[0218] Step 3: (6S,9R,10S,llS,13S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)- 17-((furan-2-carbonyl)oxy)- 10,13,16-trimethyl-3 -oxo-6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-ll-yl ((R)-2-amino-5-(tert-butoxy)-5-oxopentanoyl)-L-asparaginyl-L-asparaginylalanyl-L-prolinate (7.2 mg, 1 eq, 6.4 pmol) was dissolved in 2 ml DCM. Then 2-bromoacetyl bromide (10 mg, 4.5 pL, lOmg, 51 pmol) and DIPEA (6.6 mg, 9.0 pL, 8eq, 51 pmol) were added. The reaction was stirred at room temperature for 1 hour and then checked by LCMS. Then, DCM, DIPEA, and 2-bromoacetyl bromide were removed by rotavap under pressure, then 2 ml DCM was added again to remove any remaining 2-bromoacetyl bromide and DIPEA, then removed by rotavap. This product was then dissolved in 1 ml DCM, then 1 ml TFA was added and the reaction was stirred for an hour and checked by LCMS. The DCM was removed and the product was dissolved in DMF and purified by preparative HPLC with a final yield of 3.9 mg (54%). m / z for CsoHeiBrFsNjOieS = 1184.52, RT = 3.05 minutes.Example 20: Preparation of (6S,9R,10S,13S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-17-((furan-2-carbonyl)oxy)-10,13,16-trimethyl-3-oxo-6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-ll-yl acetyl- L-lysyl-L-asparaginyl-L-asparaginyl-L-alanyl-L-prolinate (AcLysAsnAsnAlaPro-Fluticasone Furoate, Compound 116).

[0219] (6S,9R,10S,13S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-17-((furan-2-carbonyl)oxy)-10,13,16-trimethyl-3-oxo-6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-ll-yl L-asparaginyl-L-asparaginyl-L-alanyl-L-prolinate (Example 17, step 2) is dissolved in DMF and treated with N2-acetyl-N6-(tert-butoxycarbonyl)-L-lysine (1.5 eq), HATU (4 eq), and DIPEA (4 eq). The reaction is stirred for 24 hours then partitioned between ethyl acetate and water. The ethyl acetate layer is washed twice with water and dried over magnesium sulfate. Upon concentration, the residueis re-dissolved in DCM and treated with an equal volume of TFA. After Ih of stirring, the DCM and TFA are removed by air and the final product is purified by preparative-HPLC.

[0220] Example 21: ADCs synthesis and characterization: Mai eimide conjugations were performed as described for example 5. Bromoacetamide-based linkers were conjugated as follows. Anti-CD38 (48 uL of 20.8mg / mL) was placed in 0.5 mL Eppendorf tube. EDTA (2 pl, 0.1M) was added along with 30pl PBS. Finally, TCEP (20 pL, 5 mM, 15 Eq, 0.10 pmol) was added. The final concertation of the antibody during the reduction step was lOmg / mL. The reaction was incubated at 37°C for 2 hours at which point borate buffer (pH=8, 0.5 M, 20 pL) and 20 eq of BrAc-NNGP-FF (27 pL of 5mM DMA stock) were added. The reaction was kept at room temperature for 4 hrs. Buffer exchanged and ADC characterization was performed as in Example 5. The same procedure was employed for the other bromoacetamide conjugations shown below.Antibody Linker payload DAR Aggregation LC major Expected HC major Expected mass peak mass shift peak (m / z) shift (Da) (m / z) LC (Da)Anti-CD19 mpGNNGP-FF 5.6 < 5% 24150 1129 54035 (+3) 3387(+1)Anti-CD20 mpGNNGP-FF 5 <5% 24172 1129 53915 (+3) 3387(+1)Anti- mpGNNGP-FF 4.7 <5% 23732 (0) 1129 53632 (+3) 3387 CD79bAnti-CD19 MpGNNGP- 4.8 <5% 23020 1091 53938 (+3) 3270FP (+0)Anti-CD20 MpGNNGP- 4.6 <5% 24134 1091 53806 (+3) 3270FP (+1)Anti- MpGNNGP- 4.2 < 5% 23732 1091 53518 (+3) 3270 CD79b FP (+0)Anti-CD19 MpGNNGP- 5 <5% 23026 1074 53893 (+3) 3222Halobetasol (+0)Anti-CD20 MpGNNGP- 4.3 <5% 24118 1074 52682 (+2) 2148Halobetasol (+1)Anti- MpGNNGP- 5.3 <5% 23733 (0) 1074 53474 (+3) 3222 CD79b HalobetasolAnti-CD19 MpGNNGP- 5.9 <5% 24077 1056 53836 (+3) 3168Clobetasol (+1)Anti-CD20 MpGNNGP- 4.5 < 5% 24100 1056 52643 (+2) 2112Clobetasol (+1)Anti- MpGNNGP- 6.1 6.6% 24791 1056 53421 (+3) 3168 CD79b Clobetasol (+1)Anti-CD38 MpQNNGP- 4.9 5.8% 23389 1202 54266 (+3) 3606FF (+0)Anti-CD38 MpNNGP-FF 5.7 9.3% 24458 1072 53875 (+3) 3216(+1)Antibody Linker payload DAR Aggregation LC major Expected HC major Expected mass peak mass shift peak (m / z) shift (Da) (m / z) LC (Da)Anti-CD38 Mal-PEG4- 4.4 <5% 23388 1319 54622 (+3) 3957NNGP-FF (+0)Anti-CD38 MpSNNGP-FF 4.7 15.1% 23416 1158 54138 (+3) 3474(+0)Anti-CD38 BrAc-NNGP- 3.8 <5% 23384 961 53534 (+3) 2883FF (+0)Anti-CD38 BrAc-PEG6- 5.9 <5% 23384 1198 54540 (+3) 3594NNGP-FF (+0)Anti- TNFa MpQNNGP- 7.1 <5% 24629 1202 54286 (+3) 3606FF (+1)Anti- TNFa MpNNGP-FF 7.4 8.02% 24500 1072 53903 (+3) 3216(+1)Anti- TNFa Mal-PEG4- 7.0 <1% 24748 1319 54646 (+3) 3957NNGP-FF (+1)Anti- TNFa MpSNNGP-FF 3.8 15.1% 23416 1158 54114 (+3) 3474(+0)Anti-TNFa BrAc-NNGP- 7.1 <5% 24374 961 53534 (+3) 2883FF (+1)Anti- TNFa BrAc-PEG6- 6.2 <5% 23412 1198 54537 (+3) 3594NNGP-FF (+0)Anti-CD38 MpGNNAP- 4.0 TBD 23383 1142 54078 (+3) 3426FF (+1)Anti- TNFa MpGNNAP- 6.2 TBD 24555 1142 54077 (+3) 3426FF

[0221] Example 22: Efficacy of anti-CD38-GNNGP-FF and anti-TNFa-GNNGP-FF in contact hypersensitivity mouse model: Female C57BL / 6 mice (6-8 weeks old, the Jackson Laboratory, Bar Harbor, ME) were anesthetized and sensitized on day 0 by applying 400 pL of 1.5% fluorescein isothiocyanate (FITC; Sigma-Aldrich, St. Louis, MO) dissolved in a 1:1 mixture of acetone and dibutyl phthalate (Sigma-Aldrich) to the shaved abdomen. On day 6, mice received investigational compounds one hour before being challenged on the right ear with 20 pL of FITC solution. anti-CD38-NNGP-FF and anti-TNFa-NNGP-FF, isotype control ADC were administered intraperitoneally as a single dose (5 mg / kg) on day 6.Twenty -four hours post-challenge, mice were anesthetized and the thickness of both ears (challenged and unchallenged) was measured using a digital micrometer. To determine the change in ear thickness, the measurement of the unchallenged ear was subtracted from that of the challenged ear. The results shown in FIG. 6 demonstrate the efficacy of these treatments in attenuating inflammation in this model. As demonstrated by Marvin in the Journal of Medicinal Chemistry 202467 (11), 9495-9515 (DOI: 10.1021 / acs.jmedchem.4c00598), activity in a contact hypersensitivity model is demonstrative of immune suppression thatcorrelates with efficacy in other inflammatory diseases and disorders such as rheumatoid arthritis. These data show that the efficacy in this model is driven by the glucocorticoid, as evidenced by the reduced activity of the unmodified antibody. As described supra, glucocorticoids have broad therapeutic utility in a variety of disease states including, but not limited to, autoimmune disorders and transplant rejection, indicating the ADCs disclosed herein will also have utility in these disease areas.

[0222] While several aspects of the present invention have been described and depicted herein, alternative aspects may be effected by those skilled in the art to accomplish the same objectives. Accordingly, it is intended by the appended claims to cover all such alternative aspects as fall within the true spirit and scope of the invention.

Claims

CLAIMSWe claim:

1. A compound of the Formula (I):OZ1-X1-A°-A1-A2-A3-A4^O-GC(I)wherein:Z1is a conjugation handle;X1is a bond or is a spacer unit selected from branched or unbranched C1-C12 oalkyl, a PEG selected from PEG1 to PEG12,,A0is absent or is a natural or non-natural amino acid;A1is selected from Asn, Ala, Gly, Vai, and He;A2is selected from Asn, Cit, and Ala;A3is Gly or Ala;A4is selected from L-Pro, D-Pro, alpha-methyl-Pro, Hydroxy Proline, and homoproline; andGC is a glucocorticoid receptor modulator with a steroidal structure attached via the C-l 1 hydroxyl.

2. The compound of claim 1, wherein Z1is selected from an amine, a Michael acceptor,4. The compound of claim 1, wherein X1is a bond or is a spacer unit selected from branched or unbranched C1-C12 alkyl, a PEG selected from PEG1 to PEG12,5. The compound of claim 1, wherein X1is selected fromo6. The compound of claim 5, wherein X17. The compound of claim 1, wherein A0is Gly, Ser, Glu, or Asp.

8. The compound of claim 1, wherein A0is Gly.

9. The compound of claim 1, wherein A0is absent.

10. The compound of claim 1, wherein A1is Asn or Ala; and / or wherein A2is Asn;and / or wherein A3is Gly; and / or wherein A4is L-Pro.

11. The compound of claim 1, wherein A1and A2are both Asn.

12. The compound of claim 1, wherein GC is selected from fluticasone furoate, fluticasone propionate, halobetasol, Clobetasol propionate, dexamethasone, prednisolone, amcinonide, beclomethasone dipropionate, betamethasone dipropionate, budesonide, flunisolide, fludrocortisone, methylprednisolone, triamcinolone, ciclesonide, flunisolide, fluocinolone acetonide, fluocinonide, fluoromethoIone, flurandr enolide, halcinonide, halobetasol propionate, mometasone furoate, triamcinolone acetonide, and beclomethasone.

13. The compound of claim 1, wherein:A0is Gly;A1is Asn or Ala;A2is Asn;A3is Gly; andA4is L-Pro.

14. The compound of claim 1, wherein A0is is Gly, A1is Asn, A2is Asn, A3is Gly, and A4is L-Pro.

15. An antibody-drug conjugate of Formula (A):OAb-Z-X1A° A1A2A3A4^O-GC(A),wherein:Ab is an antibody;Z is a conjugation moiety;X1is a bond or is a spacer unit selected from branched or unbranched C1-C12 oalkyl, a PEG selected from PEG1 to PEG12, 1-12A0is absent or is a natural or non-natural amino acid;A1is selected from Asn, Ala, Gly, Vai, and He;A2is selected from Asn, Cit, and Ala;A3is Gly or Ala;A4is selected from L-Pro, D-Pro, alpha-methyl-Pro, Hydroxy Proline, and homoproline; andGC is a glucocorticoid receptor modulator with a steroidal structure attached via the C-l 1 hydroxyl.

16. The antibody-drug conjugate of claim 15, wherein Ab is selected from anti-cMET, anti-IL-31R, anti-BCMA, anti-CSF-lR, anti-HER3, anti-DLL, anti-PDl, anti-PD-Ll, anti-tissue factor, anti-MASP-2, anti-GPRC5D, anti-FRa, anti-CTLA-4, anti-IL36R, anti-LAG3, anti-gplOO, anti-IFNARl, anti-GD2, anti-IL6R, anti-IGF-lR, anti- Nectin4, anti-VEGFR2, anti-IL-23, anti-IFNgamma, anti-CGRP, anti-CD22, anti- CCR4, anti-CGRP-R, anti-CD4, anti-FGF23, anti-CLDN18.2, anti-IL-5R, anti- PDGFRa, anti -IL-5, anti-IL17a, anti-EGFR, anti-PCSK9, anti-a4|37, anti-IL6, anti- CD30, anti-BLyS, anti-RANK-L, anti-ILip, anti-EPCAM, anti-a4-integrin, anti- CD52, anti-CD33, anti-lL2R, anti-CCLll, anti-CD3, anti-CD27, anti-CD28, anti- CD40, anti-CD51, anti-CD123, anti-CD147, anti-CD152, anti-CEACAM5, anti- DLL4, anti-FGFR2, anti-fibronectin, anti-Folate receptor, anti -gly pi can, anti- GUCY2C, anti-MIF, anti-MSLN, anti-MUCl, anti-RORl, anti-SLAMF7, anti- STREAPl, and anti-TIGIT.

17. The antibody-drug conjugate of claim 15, wherein Ab is selected from anti-cMET, anti-IL-31R, anti-BCMA, anti-CSF-lR, anti-HER3, anti-DLL, anti-PDl, anti-PD-Ll, anti-tissue factor, anti-MASP-2, anti-GPRC5D, anti-FRa, anti-CTLA-4, anti-IL36R, anti-LAG3, anti-gplOO, anti-IFNARl, anti-GD2, anti-IL6R, anti-IGF-lR, anti- Nectin4, anti-VEGFR2, anti-IL-23, anti-IFNgamma, anti-CGRP, anti-CD22, anti-CCR4, anti-CGRP-R, anti-CD4, anti-FGF23, anti-CLDN18.2, anti-IL-5R, anti- PDGFRa, anti-IL-5, anti-IL17a, anti-EGFR, anti-PCSK9, anti-a4p7, anti-IL6, anti- CD30, anti-BLyS, anti-RANK-L, anti-ILip, anti-EPCAM, anti-a4-integrin, anti- CD52, anti-CD33, anti-IL2R, anti-CCLll, anti-CD3, anti-CD27, anti-CD28, anti- CD40, anti-CD51, anti-CD123, anti-CD147, anti-CD152, anti-CEACAM5, anti- DLL4, anti-FGFR2, anti -fibronectin, anti-Folate receptor, anti-glypican, anti- GUCY2C, anti-MIF, anti-MSLN, anti-MUCl, anti-RORl, anti-SLAMF7, anti- STREAP1, anti-VISTA, anti-IL-4Ra, anti-BDCA2, anti-CXCR4, anti-CD163, anti- MSR1, and anti-CD74, and anti-TIGIT.

18. The antibody-drug conjugate of claim 16 or 17, wherein Z is:

19. The antibody-drug conjugate of claim 16, whereinZ is20. A pharmaceutical composition comprising the compound of any one of claims 1 to 14, or the antibody-drug conjugate of any one of claims 15 to 19, and a pharmaceutically acceptable carrier, diluent, or excipient.

21. A method for treating inflammation in a subject, the method comprising administering to the subject a therapeutically effective amount of the compound of any one of claims 1 to 14, the antibody-drug conjugate of any one of claims 15 to 19, or the pharmaceutical composition of claim 20.

22. A method for treating an autoimmune disease or disorder in a subject, the method comprising administering to the subject a therapeutically effective amount of the compound of any one of claims 1 to 14, the antibody-drug conjugate of any one of claims 15 to 19, or the pharmaceutical composition of claim 20.

23. A method for treating a blood cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of the compound of any one of claims 1 to 14, the antibody-drug conjugate of any one of claims 15 to 19, or the pharmaceutical composition of claim 20.

24. A method for inhibiting or reducing rejection of a tissue or organ transplant in a subject, the method comprising administering to the subject a therapeutically effective amount of the compound of any one of claims 1 to 14, the antibody-drug conjugate of any one of claims 15 to 19, or the pharmaceutical composition of claim 20.

25. The method of claim 24, wherein said organ transplant is a kidney transplant.