GLUCOCORTICOID RECEPTOR AGONIST AND IMMUNOCYTES OF THIS RECEPTOR

Abstract

This document provides glucocorticoid receptor agonist immunoconjugates, glucocorticoid receptor agonists, and methods for using them.

Description

GLUCOCORTICOID RECEPTOR AGONIST AND IMMUNOCYTES OF THIS RECEPTOR RELATED APPLICATIONS This application claims priority over U.S. Provisional Application No. 62 / 593.776 filed on December 1, 2017, and U.S. Provisional Application No. 62 / 595.054 filed on December 5, 2017, each of which is incorporated in its entirety by reference. LIST OF SEQUENCES This application contains a list of sequences that has been submitted electronically in ASCII format and is incorporated herein in its entirety by reference. This ASCII copy, created on November 28, 2018, is named A103017_1490WO_SL.txt and has a size of 14,758 bytes. TECHNICAL FIELD Tumor necrosis factor alpha (TNFα) plays a fundamental role in the pathophysiology of several human disorders, and anti-TNFα agents have clinically validated therapeutic utility in the treatment of autoimmune and inflammatory disorders, such as rheumatoid arthritis, psoriasis, and inflammatory bowel disease. Despite their success in the clinic, anti-TNFα biologics are still limited in terms of the maximum efficacy they can achieve in patients, making it necessary to identify and develop more potent and effective treatments. Patients treated with anti-TNFα biologics may also develop a response. IF-2019-03555061-APN-ANP#INPI Page 1 of 93 immunogenic to the treatment, thus limiting its effectiveness. Therefore, anti-TNFα therapies with lower immunogenicity and high efficacy would be useful for further controlling the disease. Synthetic glucocorticoid receptor agonists are a potent class of low-molecular-weight molecules used to treat inflammatory disorders, but their usefulness in the chronic management of disease is limited due to serious side effects. Drugs with improved efficacy and a longer duration of action compared to anti-TNF antibodies, and with minimal adverse effects, need to be developed. SUMMARY This disclosure provides glucocorticoid receptor agonist immunoconjugates useful for treating autoimmune diseases. In one respect, the present disclosure provides an antibody-drug conjugate comprising: (a) an anti-TNFα antibody comprising a heavy chain exposed as SEQ ID NO: 3 and a light chain exposed as SEQ TD NO: 4; and (b) a glucocorticoid receptor agonist comprising a radical represented by the formula: IF-2019-03555061-APN-ANP#INPI Page 2 of 93 and where the antibody is conjugated to the glucocorticoid receptor agonist via a connector represented by the formula: either In one, it provides a with the formula: HO O . realization, the present disclosure antibody-drug conjugate in accordance where A is the antibody and n is an integer of IIOs. In one respect, the present disclosure provides an antibody-drug conjugate comprising: (a) an anti-TNEAα antibody comprising a heavy chain as shown in SEQ ID NO: 3 and a light chain as shown in SEQ ID NO: 4; and (b) a glucocorticoid receptor agonist comprising a radical represented by the formula: IF-2019-03555061-APN-ANP#INPI Page 3 of 93 and where the antibody is conjugated to the glucocorticoid receptor agonist via a connector In one embodiment, the present disclosure provides an antibody-drug conjugate according to the formula: 10. In one embodiment, the present disclosure provides the antibody-drug conjugate of any preceding embodiment, wherein the DAR of the compound is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the present IF-2019-03555061-APN-ANP#INPI Page 4 of 93. The present disclosure provides the antibody-drug conjugate of any prior embodiment, where the DAR of the compound is 4, e.g., n in the prior antibody-drug conjugate formula is equal to 4. In one embodiment, the present disclosure provides the antibody-drug conjugate of any prior embodiment, where the DAR of the compound is 2, e.g., n in the prior antibody-drug conjugate formula is equal to 2. In one embodiment, this disclosure provides a method for preparing the antibody-drug conjugate of any preceding embodiment, comprising the step of conjugating the antibody with the glucocorticoid receptor agonist. In one embodiment, this disclosure provides the method of the preceding embodiment, further comprising the step of introducing a PO4 moiety into the glucocorticoid receptor agonist before conjugating the antibody with the glucocorticoid receptor agonist. In one embodiment, this disclosure provides the method of any preceding embodiment, wherein the conjugation comprises partially reducing the antibody and alkylating the partially reduced antibody with a compound of IF-2019-03555061-APN-ANP#INPI Page 5 of 93 In one embodiment, this disclosure provides a pharmaceutical composition comprising the antibody-drug conjugate of any preceding embodiment and a pharmaceutically acceptable carrier. In one embodiment, this disclosure provides the pharmaceutical composition of any of the preceding embodiments, comprising a drug-to-antibody ratio (DAR) of 1-10. In a preferred embodiment, the present disclosure provides an antibody-drug conjugate according to the formula: where A is adalimumab and n is 4. In another preferred embodiment, the present disclosure provides an antibody-drug conjugate according to the formula: IF-2019-03555061-APN-ANP#INPI Page 6 of 93 where A is adalimumab and n is 2. In a preferred embodiment, this disclosure provides the pharmaceutical composition of any prior embodiment, comprising a drug-to-antibody ratio (DAR) of 2.0. In a preferred embodiment, the present disclosure; provides the pharmaceutical composition of any preceding embodiment, comprising a drug-to-antibody ratio (DAR) of 4.0. In one embodiment, the present disclosure provides a method for treating a selected condition of rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, plaque psoriasis, ulcerative colitis, adult Crohn's disease, Crohn's disease; pediatric, uveitis, hidradenitis suppurativa and juvenile idiopathic arthritis in a subject, comprising administering an effective amount of the antibody-drug conjugate of any preceding embodiment or composition; pharmaceutical of any prior achievement to the subject. In one embodiment, the present disclosure provides the antibody-drug conjugate of any prior embodiment or the pharmaceutical composition of any prior embodiment for use in the treatment of a selected condition of rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, plaque psoriasis, ulcerative colitis, adult Crohn's disease, pediatric Crohn's disease, uveitis, hidradenitis suppurativa, and juvenile idiopathic arthritis. In one embodiment, this disclosure provides for the use of the antibody-drug conjugate of 7 IF-2019-03555061-APN-ANP#INPI i Page 7 of 93 any prior embodiment or the pharmaceutical composition of any prior embodiment for the preparation of a medicament to treat a selected condition of rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, plaque psoriasis, ulcerative colitis, adult Crohn's disease, pediatric Crohn's disease, uveitis, hidradenitis suppurativa and juvenile idiopathic arthritis. In one embodiment, the present disclosure provides a kit comprising: (a) a package comprising the antibody-drug conjugate of any prior embodiment or the pharmaceutical composition of any prior embodiment; and (b) a data sheet or package insert in or associated with the one or more packages, wherein the data sheet or package insert indicates that the antibody-drug conjugate or pharmaceutical composition is used to treat a selected condition from rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, plaque psoriasis, ulcerative colitis, adult Crohn's disease, pediatric Crohn's disease, uveitis, hidradenitis suppurativa, and juvenile idiopathic arthritis. In one embodiment, the present disclosure provides a method for delivering a glucocorticoid receptor agonist to a TNFα-expressing cell, comprising the step of contacting the cell with the antibody-drug conjugate of any preceding embodiment. In one embodiment, the present disclosure provides a method for determining the anti-inflammatory activity of an antibody-drug conjugate comprising: (a) contacting a cell expressing IF-2019-03555061-APN-ANP#INPI Page 8 of 93 TNFn with the antibody-drug conjugate of any preceding embodiment; and (b) determine the release of proinflammatory cytokines from the cell compared to a control cell. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 provides a chromatographic resolution of BrAc-Gly-Glu-glucocorticoid receptor modulator (GRM)-PO4, as carried out and described in Example 7. As shown, the ADC is a homogeneous mixture of ADCs containing ADCs with two attached drug-connector molecules, and ADCs with four attached drug-connector molecules. Figure 2 shows data from unconvulsed MS of adalimumab conjugated with BrAc-Gly-Glu-glucocorticosteroidP04, as performed and described in Example 7. As shown, conjugation is achieved. Figure 3 provides a graph demonstrating the efficacy of a high and low dose of ADC1 compared with anti-TNFa mAb (high dose) or vehicle in a mouse model of collagen-induced arthritis (CIA), as performed and described in Example 7. As shown, a single dose of the ADC1 anti-TNFa glucocorticosteroid showed a prolonged duration of action by improving paw inflammation for ~28 days compared with anti-TNFa mAb or vehicles alone. Figure 4 is a graph of the concentration (µg / ml) for open- and closed-ring ADCs in macaques over time, as performed and described in Example 7. As shown, the ring form 9 IF-2019-03555061-APN-ANP#INPI Page 9 of 93 closed is susceptible to experiencing the reverse Michael reaction and subsequent loss of the drug connector in vivo, DETAILED DESCRIPTION This document provides glucocorticoid receptor agonist immunoconjugates, glucocorticoid receptor Me agonists, and methods for their preparation and use. The present document provides an antibody-drug conjugate according to the formula: where A is adalumimab and n is 4. As shown below in Example 7, this ADC (i.e., the following ADC4) exhibits in vitro activity, plasma stability, and minimal aggregation. Methods for preparing and using ADC4 are also provided. I. Definitions To facilitate understanding of this disclosure, several terms and phrases are defined below. The term anti-TNFα protein refers to proteins that are able to (i) bind to TNFα and (ii) inhibit the IF-2019-03555061-APN-ANP#INPI Page 10 of 93. Binding of soluble TNFα to cell surface TNF receptors (p55 and / or p75) and / or lysing cells expressing TNFα receptors or TNFα on the surface in vitro in the presence of complement. In some embodiments, the anti-TNF antibody can bind to TNFα on the cell surface and be internalized. For example, US patent 2014 / 0294813, which is incorporated herein in its entirety by reference, discloses anti-TNF antibodies that exhibit cellular internalization by binding to human cell surface TNF. Anti-TNFα proteins include, for example, anti-TNFα antibodies (e.g., adalimumab, infliximab, and golimumab). Anti-TNFα antibodies are actively internalized by binding to transmembrane TNF in monocyte-derived dendritic cells and rapidly enter lysosomes where they are degraded. (Deora et al. MABS, 2017, Vol. 9, No. 4, 680-694). The term antibody, as used herein, is intended to refer to immunoglobulin molecules comprising four polypeptide chains, two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CHI, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain, CL. The VH and VL regions may be further subdivided into 11-regions IF-2019-03555061-APN-ANP#INPI Page 11 of 93 hypervariability, which are called complementarity-determining regions (CDRs), between which are interspersed regions that are more conserved, called scaffold regions (FRs). Each VH and VL comprises three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The term "anti-TNFα antibody" or "TNFα-binding antibody" refers to an antibody capable of binding to TNFα, e.g., with sufficient affinity to make the antibody useful as a therapeutic agent by specifically targeting TNFα. The degree of binding of an anti-TNFα antibody to a non-TNFα protein, with no apparent relation to it, may be less than approximately 10% of the antibody's binding to TNFα, as measured, e.g., by radioimmunoassay (RIA). In certain embodiments, a TNFα-binding antibody has a dissociation constant (Kd) of <1 μM, <100 nM, <10 nM, <1 nM, or <0.1 nM. The term 'immunoconjugate,' 'conjugate,' 'antibody-drug conjugate,' or 'ADC,' as used herein, refers to a compound or a derivative thereof that is conjugated to a protein, such as a cell-binding agent (e.g., an anti-TNFα antibody). Such immunoconjugates can be defined by the generic formula: (SM-LQ)n~A, where SM = radical derived from a low-molecular-weight glucocorticoid receptor agonist, e.g., a glucocorticosteroid, L = linker, Q = heterobifunctional group or is absent, and A = a protein (e.g., an antibody), and n = 1–10. Immunoconjugates 12 IF-2019-03555061-APN-ANP#INPI Page 12 of 93 can also be defined by the generic formula in reverse order: A-(QL-SM)n. In this disclosure, the term "connector" refers to a chemical residue capable of linking the anti-TNFcx protein (e.g., anti-antibody) to a glucocorticosteroid. Connectors may be cleaved (a cleavable connector), thereby allowing the release of the glucocorticosteroid. For example, such cleavable connectors may be cleaved by a peptidase, under conditions in which the glucocorticosteroid and / or antibody remains active. In particular, the cleavable linker component disclosed herein comprises a peptide consisting of two to three amino acid residues (a dipeptide or tripeptide), and specifically consists of dipeptides and tripeptides selected from the group consisting of alanine-alanine (Ala-Ala), glycine-glutamic acid (Gly-Glu), glutamic acid-alanine-alanine (Glu-Ala-Ala), and glycine-lysine (Gly-Lys). The peptide enables cleavage of the linker by the action of a protease, thereby allowing the release of the glucocorticosteroid upon exposure to intracellular proteases, such as lysosomal enzymes (Doronina et al. (2003) Nat. Biotechnol. 21:778-784). In this disclosure, the term glucocorticosteroid refers to a naturally occurring or synthetic spheroid hormone that interacts with glucocorticoid receptors, and specific glucocorticosteroids are disclosed herein in detail. A “glucocorticosteroid radical” is derived from the 13 IF-2019-03555061-APN-ANP#INPI Page 13 of 93. Removal of one or more hydrogen atoms from a parent glucocorticosteroid. The removal of the hydrogen atom or atoms allows the parent glucocorticosteroid to be attached to a linker. In this disclosure, the hydrogen atom is removed from any suitable -NH2 group of the parent glucocorticosteroid. In particular, the radical of a glucocorticosteroid is a monovalent radical derived from the removal of a hydrogen atom from a parent glucocorticosteroid. In this disclosure, the term heterobifunctional group refers to a chemical residue that connects the linker and the anti-TNFα protein (e.g., antibody). Heterobifunctional groups are characterized by having different reactive groups at each end of the chemical residue. The term drug-to-antibody ratio or DAR refers to the number of SM (e.g., a radical derived from a low-molecular-weight glucocorticoid receptor agonist, e.g., a glucocorticosteroid) bound to A (e.g., an antibody). Thus, in the immunoconjugate having the generic formula (SM-LQ)nA, the DAR is defined by the drug load per antibody-drug conjugate, e.g., n. When referring to a compound having the formula (SM-LQ)nA that represents an individual immunoconjugate, the compound's DAR term refers to the number of SMs attached to the individual A (e.g., drug loading on as an integer from 1 to 10). IF-2019-03555061-APN-ANP#INPI Page 14 of 93 When referring to a compound having the formula (SM-LQ)nA representing a population of immunoconjugates, the population DAR term refers to the average number of SMs bound to the A's (e.g., drug loading on as a whole number or fraction from 1 to 10 + 0.5, + 0.4, + 0.3, + 0.2, + 0.1). The term "subject" refers to human beings, non-human primates, and similar beings who are to be the recipient of a particular treatment. Normally, the terms "subject" and "patient" are used interchangeably herein when referring to a human subject. The term pharmaceutical formulation refers to a preparation in a form that allows the biological activity of the active ingredient to be effective, and which does not contain additional components that are unacceptably toxic to the subject to whom the formulation will be administered. The formulation may be sterile. An effective amount of an immunoconjugate, as disclosed herein, is an amount sufficient to accomplish a specifically stated purpose. An effective amount can be determined in relation to the stated purpose. The term therapeutically effective amount refers to a quantity of an immunoconjugate effective in treating a disease or disorder in a subject or mammal. A prophylactically effective amount refers to a quantity effective in achieving the desired prophylactic outcome. IF-2019-03555061-APN-ANP#INPI Page 15 of 93 Terms such as "treating," "treatment," "to treat," "relieving," or "to relieve" refer to therapeutic measures that cure, slow, or lessen one or more symptoms of, and / or delay or stop the progression of, a diagnosed condition or pathological disorder (therapeutic treatment). Therefore, those who need therapeutic treatment include those who have already been diagnosed with the disorder or are suspected of having it. Prophylactic or preventive measures refer to measures that prevent the development of a specific condition or pathological disorder (prophylactic treatment). Therefore, those who need prophylactic treatment include those who are prone to developing the disorder and those in whom it is desired to prevent the disorder. II. Proteins for binding with glucocorticoid receptor agonists This disclosure provides immunoconjugates containing glucocorticoid receptor agonists bound to proteins, e.g., antibodies. In some embodiments, the antibody is human, humanized, chimeric, or murine. In some embodiments, the protein, e.g., antibody, can bind to a target on the surface of a cell and be internalized. This disclosure also provides immunoconjugates containing glucocorticoid receptor agonists bound to anti-TNFα proteins. In certain embodiments, the anti-TNFα proteins are antibodies that bind to TNFα (e.g., soluble TNFα and / or 16 IF-2019-03555061-APN-ANP#INPI Page 16 of 93 TNFα membrane-bound). In certain embodiments, anti-TNFα proteins are TNF receptor proteins; soluble, e.g., soluble TNF receptor proteins fused to a heavy-chain constant domain. In some embodiments, the anti-TNFα protein, e.g., anti-TNFα antibody, binds to TNFα on the surface of a cell and is internalized. For example, the publication of U.S. patent application no. 2014 / 0294813, incorporated herein by reference, discloses anti-TNFα proteins that exhibit cellular internalization upon binding to human cell-surface TNFα. In certain embodiments, the antibodies bind to' Human and / or mouse TNFα. The complete amino acid sequence for membrane-bound human TNFai i is: MSTESMIRDVELAEEALPKKTGGPQGSRRCLFLSLFSFLIVAGATTLFCLLHFG / VTGPQREEFPRDLSLISPLAQAVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLi ANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSA IKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIi AL (SEQ ID NO: 1) . Soluble human TNFa contains the; amino acids 77-233 of the SEQ ID NO:1. The sequence of; The complete amino acid profile for membrane-bound murine TNFα is: MSTESMIRDVELAEEALPQKMGGFQNSRRCLCLSLFSFLLVAGATTLFCLLNFG' i VIGPQRDEKFPNGLPLISSMAQTLTLRSSSQNSSDKPVAHWANHQVEEQLEWLSQRAN: ALLANGMDLKDNQLWPADGLYLVYSQVLFKGQGCPDYVLLTHTVSRFAISYQEKVNLLJ SAVKSPCPKDTPEGAELKPWYEPIYLGGVFQLEKGDQLSAEVNLPKYLDFAESGQVYFGi VIAL (SEQ ID NO: 2) . Soluble murine TNFa contains amino acids 80-235 of SEQ ID NO:2.17 IF-2019-03555061-APN-ANP#INPI Page 17 of 93 In some embodiments, the anti-TNFα antibody binds to human TNFα. In some embodiments, the anti-TNFα antibody binds to murine TNFα. In certain embodiments, the anti-TNFα antibody exerts one or more of the following effects: neutralizes the cytotoxicity of human TNFα in an in vitro L92 9 assay with a CT50 of 1X10-7M or less; blocks the interaction of TNFα with the cell surface receptors p55 and p75; and / or lyses cells expressing TNF on the surface in vitro in the presence of complement. In certain embodiments, the anti-TNFα antibody does not bind to TNF-β. Anti-TNFα antibodies include, for example, adalimumab, which is a recombinant human antibody. The amino acid sequences corresponding to the CDRs and variable regions of adalimumab are described in U.S. Patent No. 6,258,562 with reference to antibody D2E7, i.e., SEQ ID Nos. 1 to 8. The international nonproprietary name (INN) adalimumab is provided on the QMS INN list page: Year 2000, List 44 (WHO Drug Information (2000) Vol. 14 (3)). In certain embodiments, an anti-TNFα antibody comprises adalimumab sequences, e.g., the complementarity-determining regions (CDRs), the heavy chain variable domain (HV), and / or the light chain variable domain (LV). Example sequences are provided in Table 1. IF-2019-03555061-APN-ANP#INPI Page 18 of 93 Table 1: Example sequences of regions of the adalimumab antibody Antibody region Amino acid sequence Heavy chain EVCLVESGGGLVCPGRSLRLSCAASGFTFDDYAMHWVR CAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAK NSLYLCMNSLRAEDTAVYYCAKVSYLSTASSLDYWGCG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLCSSGLYSLSSVV TVPSSSLGTCTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKKPDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTPREECYNSTY RWSVLTVLHCDWLNGKEYKCKVSNKALPAPIEKTISK AKGCPREPCVYTLPPSRDELTKNCVSLTCLVKGFYPSD IAVEWESNGCPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWCCGNVFSCSVMHEALHNHYTCKSLSLSPGK (SEC ID NO: 3) Light Chain DICMTCSPSSLSASVGDRVTITCRASCGIRNYLAWYCC KPGKAPKLLIYAASTLCSGVPSRFSGSGSGTDFTLTIS SLCPEDVATYYCCRYNRAPYTFGCGTKVEIKRTVAAPS VFIFPPSDECLKSGTASVVCLLNNFYPREAKVCWKVDN ALCSGNSCESVTECDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHCGLSSPVTKSFNRGEC (SEQ ID NO:4) Heavy chain variable region EVCLVESGGGLVCPGRSLRLSCAASGFTFDDYAMHWVR CAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAK NSLYLCMNSLRAEDTAVYYCAKVSYLSTASSLDYWGCG TLVTVSS(SEQ ID NO:5) Light chain variable regionDICMTCSPSSLSASVGDRVTTTCRASCGIRNYLAWYCC KPGKAPKLLIYAASTLCSGVPSRFSGSGSGTDFTLTIS IF-2019-03555061-APN-ANP#INPI Page 19 of 93 SLCPEDVATYYCCRYNRAPYTFGCGTKVEIK ID NO: 6) (SEC VH-CDR1 DYAMH (SEC ID NO:7) or GFTFDDYAMH ID NO:8) (SEC VH-CDR2 AITWNSGHIDYADSVEG (SEO ID NO:9) VH-CDR3 VSYLSTASS (SEO ID NO:10) VSYLSTASSLDY (SEO ID NO:11) or VL-CDR1 RASCGIRNYLA (SEO ID NO:12) VL-CDR2 AASTLCS (SEQ ID NO:13) VL-CDR3 CRYNRAPYT (SEQ ID NO:14) In certain embodiments, the anti-TNFa antibody comprises the CDRs of SEQ ID NOs: 3 and 4. In some embodiments, the CDRs comprise SEQ ID NOs: 7 or 8, 9, 10 or 11, 12, 13 and 14. In certain embodiments, the anti-TNFa antibody comprises the heavy chain of SEQ ID NO:3 and / or the light chain of SEQ ID NO:4. This disclosure also covers variants and equivalents that are substantially homologous to the anti-TNFα antibodies described herein. These may contain, for example, conservative substitution mutations, i.e., the substitution of one or more amino acids for similar amino acids. For example, conservative substitution refers to the substitution of one amino acid for another within the same general class, such as an acidic amino acid for another acidic amino acid, a basic amino acid for another basic amino acid, or a neutral amino acid for another neutral amino acid. The intended effects of conservative amino acid substitution are well known in the art. IF-2019-03555061-APN-ANP#INPI Page 20 of 93 The isolated anti-TNFα antibodies described herein can be produced by any suitable method known to the art. Such methods range from direct methods for protein synthesis to the construction of a DNA sequence encoding isolated polypeptide sequences and expressing such sequences in a suitable transformed host. In some embodiments, a DNA sequence is constructed using recombinant technology by isolating or synthesizing a DNA sequence encoding a naturally occurring protein of interest. Optionally, the sequence can be mutagenized by site-directed mutagenesis to provide functional analogues thereof. See, e.g., Zoeller et al., Proc. Nat. I Acad. Sci. USA 81: 5662–5666 (1984) and U.S. Patent No. 4,588,585. In some embodiments, a DNA sequence encoding an antibody of interest will be constructed by chemical synthesis using an oligonucleotide synthesizer. Such oligonucleotides can be designed based on the amino acid sequence of the desired polypeptide and by selecting those codons that are favored in the host cell in which the recombinant polypeptide of interest will be produced. Standard methods can be applied to synthesize an isolated polynucleotide sequence encoding an isolated polypeptide of interest. In certain embodiments, recombinant expression vectors are used to amplify and express DNA encoding anti-TNFα antibodies. A wide variety of host / vector combinations can be employed.21 IF-2019-03555061-APN-ANP#INPI Page 21 of 93. Expression. Useful expression vectors for eukaryotic hosts include, for example, vectors comprising expression control sequences for SV40, bovine papillomavirus, adenovirus, and cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids, such as Escherichia coli plasmids, including pCR1, pBR322, pMB9, and their derivatives, plasmids from a wider diversity of hosts, such as M13, and single-stranded DNA filamentous phages. Suitable host cells for the expression of anti-TNFα antibodies include prokaryotes, yeast, insect cells, or higher eukaryotes under the control of appropriate promoters. Prokaryotes include Gram-negative or Gram-positive organisms, for example, E. coli or bacilli. Higher eukaryotic cells include established cell lines of mammalian origin. Cell-free translation systems could also be employed. Cloning and expression vectors suitable for use with bacterial, fungal, yeast, and mammalian cell hosts have been described by Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, NY, 1985). Additional information regarding methods for protein production, including antibody production, can be found in U.S. Patent Publication No. 2008 / 0187954 and U.S. Patent Nos. 6,413,746 and 6,660.501, and International Patent Publication No. WO 04009823. IF-2019-03555061-APN-ANP#INPI Page 22 of 93 III. Immunoconjugates containing glucocorticoid receptor agonists Immunoconjugates containing glucocorticoid receptor agonists are also provided. In some embodiments, an immunoconjugate binds to the Fe gamma receptor. In some embodiments, an immunoconjugate is active in the transmembrane TNFα GRE indicator assay (as used herein, the transmembrane TNFα GRE indicator assay refers to the assay used below in Example 7). In some embodiments, an immunoconjugate exhibits reduced immunogenicity (reduced immune response against anti-drug antibodies [ADAs]) compared to the protein in the immunoconjugate (e.g., the antibody) alone. In one embodiment, a compound having Formula Ia is hereby disclosed: (SM-LQ)nA Ia, where: A is an anti-tumor necrosis factor (TNF) a antibody, an anti-TNFα monoclonal antibody, or adalimumab; L is a connector; It is a heterobifunctional group; or Q is absent; n is 1-10; and SM is a monovalent radical of a glucocorticosteroid that has any one of: (1) Formula II-a: IF-2019-03555061-APN-ANP#INPI Page 23 of 93 In another embodiment, a compound having Formula Ia is disclosed herein, wherein SM is a monovalent radical of a glucocorticosteroid having any one of Formulas II-a, II-b, I1-c, or II-d, wherein L is a scintisole linker comprising a dipeptide or tripeptide, Q is a heterobifunctional group or Q is absent, and n is 1-10. In particular, L comprises a IF-2019-03555061-APN-ANP#INPI Page 24 of 93 dipeptide or tripeptide selected from the group consisting of alanine-alanine (Ala-Ala), glycine-glutamic acid (Gly-Glu), glutamic acid-alanine-alanine (Glu-Ala-Ala) and glycine-lysine (Gly-Lys). In another embodiment, a compound having Formula Ia is disclosed herein, where SM is a monovalent radical of a glucocorticosteroid having any one of Formulas II-a, II-b, II-c and II-d, where Q is a heterobifunctional group represented by: or HmQ-l where m is 0 or 1. In another embodiment, m is 0 and Q is represented by: In another realization, m is 1 and 0 is represented by: In another embodiment, a compound having Formula Ia is hereby disclosed, wherein SM is a monovalent radical of a glucocorticosteroid having any one of Formulas II-a, II-b, Tl-c and II-d, wherein LQ- is any one of the chemical structures of the Table 2: Table 2 IF-2019-03555061-APN-ANP#INPI Page 25 of 93 In another embodiment, a compound having Formula Ia is hereby disclosed, e.g., a compound having Formula I-a where SM is a monovalent radical of a glucocorticosteroid having any one of Formulas IX-a, II-b, II-c, and IT-d, where n is 2-8. In another embodiment, n is 1-5. In another embodiment, n is 2-5. In another embodiment, n is 1. In another embodiment, n is 2. 6 IF-2019-03555061-APN-ANP#INPI Page 26 of 93 another realization, n is 3. In another realization, n is 4. In another realization, n is 5. In another realization, n is 6. In another realization, n is 7. In another realization, n is 8. In another embodiment, a compound having Formula Ia is hereby disclosed, e.g., a compound having Formula Ia, where SM is a monovalent radical of a glucocorticosteroid having any one of Formulas II-a, II-b, II-c and II-d, where A is an antibody. In another embodiment, a compound having Formula Ia is hereby disclosed, e.g., a compound having Formula Ia, wherein SM is a monovalent radical of a glucocorticosteroid having any one of Formulas II-a, II-b, II-c and II-d, wherein the antibody is murine, chimeric, humanized or human. In another embodiment, a compound having Formula Ia is disclosed herein, e.g., a compound having Formula Ia, wherein SM is a monovalent radical of a glucocorticosteroid having any one of Formulas II-a, II-b, II-c and II-d, wherein A competitively inhibits the binding of an antibody selected from the group consisting of adalimumab, infliximab, certolizumab pegol and golimumab to TNFα. In another embodiment, a compound having Formula Ia is disclosed herein, e.g., a compound having Formula Ia, wherein SM is a monovalent radical of a glucocorticosteroid having any one of Formulas II-a, II-b, II-c and II-d, wherein A binds to the same TNFα epitope as an antibody selected from the group consisting of adalimumab, 27 IF-2019-03555061-APN-ANP#INPI Page 27 of 93 infliximab, certolizumab pegol, afelimomab, nerelimomab, ozoralizumab, placulumab and golimumab. In another embodiment, a compound having Formula Ia is disclosed, e.g., a compound having Formula Ia, where SM is a monovalent radical of a glucocorticosteroid having any one of Formulas II-a, II-b, II-c, and II-d, where A comprises the CDR1, CDR2, and CDR3 sequences of the heavy chain variable region of SEQ ID NO:7 or 8, SEQ ID NO:9, and SEQ ID NO:10 or 11, respectively, and the CDR1, CDR2, and CDR3 sequences of the light chain variable region of SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14, respectively. In another embodiment, a compound having Formula Ia is hereby disclosed, e.g., a compound having Formula Ia / where SM is a monovalent radical of a glucocorticosteroid having any one of Formulas II-a, II-b, II-c and II-d, wherein Ano is attached to IFN beta. In another embodiment, a compound having Formula Ia is hereby disclosed, e.g., a compound having Formula I-a, wherein SM is a monovalent form of a glucocorticosteroid having any one of Formulas II-a, II-b, II-c and II-d, wherein A binds to TNF beta. In another embodiment, a compound having Formula Ia is hereby disclosed, e.g., a compound having Formula Ia, where SM is a monovalent radical of a glucocorticosteroid having any one of Formulas II-a, II-b, II-c and II-d, where A IF-2019-03555061-APN-ANP#INPI Page 28 of 93 blocks the interaction of TNFα with the p55 and p75 receptors on the cell surface. In another embodiment, a compound having Formula Ia is hereby disclosed, e.g., a compound having Formula Ia, where SM is a monovalent form of a glucocorticosteroid having any one of Formulas II-a, II-b, II-c and II-d, where A lyses cells expressing TNF on the surface in vitro in the presence of complement. In another embodiment, a compound having Formula Ia is hereby disclosed, e.g., a compound having Formula Ia, where SM is a monovalent radical of a glucocorticosteroid having any one of Formulas II-a, II-b, II-c and II-d, where A is etanercept. In another embodiment, a compound having Formula Ia is hereby disclosed, e.g., a compound having Formula Ia, where SM is a monovalent radical of a glucocorticosteroid having any one of Formulas II-a, II-b, II-c and II-d, where A is adalimumab. In another embodiment, a compound having Formula Ia is hereby disclosed, which is any one of the chemical structures in Table 3: Table 3 Ί IF-2019-03555061-APN-ANP#INPI Page 29 of 93 Page 30 of 93 Page 31 of 93 IF-2019-03555061-APN-ANP#INPI Page 32 of 93 Page 33 of 93 or IF-2019-03555061-APN-ANP#INPI Page 34 of 93 1-5 and A is adalimumab. :¡ Ί Ί where n is In another realization, it is hereby made known a 35 IF-2019-03555061-APN-ANP#INPI i Ί Page 35 of 93 where A is adalumimab and n is 4. As shown below in Example 7, this ADC (i.e., the following ADC4) exhibits in vitro activity, plasma fetastability, and minimal aggregation. IV. Methods for preparing immunoconjugates and synthetic intermediates The general synthesis of different immunoconjugates from the disclosure involves reacting a low molecular weight (LMW) molecule with NH2 functional groups from any of the following Formulations III-a, III-b, III-c, or III-d with a linker portion and functionalizing the resulting compound to obtain an intermediate with bromoacetamide functional groups. The bromoacetamide-containing intermediate is then reacted with HS-A, where HS-A is an antibody, e.g. adilumimab, that has a limited number of reduced interchain disulfide bridges. (1) Formula III-a: Ill-a; ¢2) Formula III-b: IF-2019-03555061-APN-ANP#INPI Page 36 of 93 OH Ill-d. In another embodiment, a method for preparing a compound having Formula IVa is disclosed herein: IF-2019-03555061-APN-ANP#INPI Page 37 of 93 where: A is adalimumab; L is a connector; n is 1-10; and SM is a radical of a glucocorticosteroid that has any one of the Illd Formulas; Understanding the method: a) conjugate a compound that has the formula V: SM—L. Br O v with an anti-tumor necrosis factor (TNF) protein or a protein; and b) isolate the compound that has Formula IV-a. In some embodiments, the disclosed method further comprises purifying the compound of Formula IV-a. In certain embodiments, anion-exchange chromatography (AEC) is used, which (due to charged residues in the peptide portion of the linker in some embodiments and / or the phosphate group in the SM in some embodiments) can provide efficient separation of species with different DARs. In some embodiments, the disclosed method requires fewer synthetic steps than methods based on standard maleimide-based connector chemistry. In particular, methods based on standard maleimide-based connector chemistry may require 38 IF-2019-03555061-APN-ANP#INPI Page 38 of 93 A subsequent hydrolysis step involving the opening of the succinimide ring is carried out after the purification of the appropriate DAR species. As such, in certain embodiments, the disclosed method significantly shortens the conjugation protocol compared to standard maleimide-based connector chemistry. In another embodiment, a method for preparing a compound having Formula VI- is disclosed herein. where: A is adalimumab; 1-10 Understanding the method: a) conjugate a Br OH adalimumab with compound of O1· O1' partially Vitamin A Formula: VII-a reduced; and e.g., by chromatography, the compound that has the formula Vl-a. IF-2019-03555061-APN-ANP#INPI Page 39 of 93 In another embodiment, a method for preparing a compound having Formula IV-a or Formula VI-a, where n is 1-7, is disclosed herein. In another embodiment, n is: 1-5. In another embodiment, n is 2-4. In another embodiment, ni is 1. In another embodiment, n is 2. In another embodiment, n is 3. In another embodiment, n is 4. In another embodiment, n is 5. In another embodiment, n is 6. In another embodiment, n is 7. In another embodiment, n is 8. In another embodiment, a compound having Formula IV-a or VI-a is hereby disclosed, where: A is adalimumab; y' nes 1 -10 .< In another embodiment, a compound having Formula IV-a or VI-a, where n is 1-7, is hereby disclosed. In another realization, n is 1-5. In another realization, n is 2-i 4. In another realization, n is 1. In another realization, n is η 2. In another embodiment, n is 3. In another embodiment, n is: 4. In another embodiment, n is 5. In another embodiment, nes 6. In another realization, n is 7. In another realization, nes 8. Also provided herein are synthetic intermediates that are useful for the preparation of; immunoconjugates.i In one embodiment, the synthetic intermediate disclosed herein is a compound having any one of Formulas V or VII-a. VI. Methods of use and pharmaceutical compositions The following are provided conjugates that have; Formula Ia (e.g., which have the formulas shown in Table 3) that can be used in vitro or; IF-2019-03555061-APN-ANP#INPI Page 40 of 93 in vivo. Accordingly, compositions are also provided, e.g., pharmaceutical compositions for certain in vivo uses, comprising a glucocorticoid receptor conjugate or agonist having the desired degree of purity in a physiologically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA). The acceptable carriers, excipients, or stabilizers are non-toxic to the receptors at the dosages and concentrations employed. Compositions (e.g., pharmaceutical compositions) intended for in vivo administration may be sterile, which can be achieved by filtration through, e.g., sterile filtration membranes. Compositions (e.g., pharmaceutical compositions) intended for in vivo administration may include a preservative. The antibody-drug conjugates and / or pharmaceutical compositions comprising antibody-drug conjugates described herein may be useful for using a cell expressing TNFα on the surface (in vitro or in vivo) and / or for the treatment of diseases or disorders characterized by elevated TNFα (e.g., elevated TNFα in synovial fluid). In some embodiments, the antibody-drug conjugates and / or compositions are useful in inhibiting cytokine release (in vitro or in vivo) and / or for the treatment of autoimmune or inflammatory diseases. In some embodiments, the antibody-drug conjugates and / or compositions are used for 41 IF-2019-03555061-APN-ANP#INPI Page 41 of 93. The treatment of Crohn's disease (e.g., moderate to severely active Crohn's disease with involvement of the ileum and / or ascending colon, and / or the maintenance of clinical remission of moderate to severely active Crohn's disease with involvement of the ileum and / or ascending colon for up to 3 months). In some embodiments, the antibody-drug conjugates and / or compositions are used for the treatment of ulcerative colitis (e.g., for induction of remission in patients with moderate to severe active ulcerative colitis). In some embodiments, the antibody-drug conjugates and / or compositions are used for the treatment of rheumatoid arthritis (RA). In some embodiments, the antibody-drug conjugates and / or compositions are used for the treatment of juvenile idiopathic arthritis (JA).In some embodiments, the antibody-drug conjugates and / or compositions are used for the treatment of psoriatic arthritis (PsA). In some embodiments, the antibody-drug conjugates and / or compositions are used for the treatment of a spondyloarthropathy such as ankylosing spondylitis (AS) or axial spondyloarthritis (axSpA). In some embodiments, the antibody-drug conjugates and / or compositions are used for the treatment of adult Crohn's disease (CD). In some embodiments, the antibody-drug conjugates and / or compositions are used for the treatment of pediatric Crohn's disease. In some embodiments, the antibody-drug conjugates and / or compositions are used for the treatment of... IF-2019-03555061-APN-ANP#INPI Page 42 of 93 ulcerative colitis (UC). In some embodiments, antibody-drug conjugates and / or compositions are used for the treatment of plaque psoriasis (Ps). In some embodiments, antibody-drug conjugates and / or compositions are used for the treatment of hidradenitis suppurativa (HS). In some embodiments, antibody-drug conjugates and / or compositions are used for the treatment of uveitis. In some embodiments, antibody-drug conjugates and / or compositions are used for the treatment of Behçet's disease. In some embodiments, antibody-drug conjugates and / or compositions are used for the treatment of psoriasis, including plaque psoriasis. Some embodiments comprise the use of drug conjugates and / or pharmaceutical compositions for the preparation of a medicament to treat the diseases or disorders described herein. Some embodiments comprise methods for delivering a glucocorticoid receptor agonist to a TNFα-expressing cell. Such methods may include a step of contacting a TNFα-expressing cell with an antibody-drug conjugate as described herein. Some embodiments comprise an in vitro method for delivering a glucocorticoid receptor agonist to a TNFα-expressing cell. Methods for determining the anti-inflammatory activity of an antibody-drug conjugate are also provided. Such methods may include a step of contacting a TNFα-expressing cell with a conjugate of 43 IF-2019-03555061-APN-ANP#INPI Page 43 of 93 antibody-drug conjugate as described herein. Some embodiments comprise contacting a TNFκ-expressing cell with an antibody-drug conjugate as described herein and determining the reduction in the release of proinflammatory cytokines from the cell compared to a control cell. Some embodiments comprise an in vitro method for determining the anti-inflammatory activity of an antibody-drug conjugate. Some embodiments include selection methods (e.g., in vitro methods) that involve directly or indirectly exposing cells (e.g., TNFκ-expressing cells) to an antibody-drug conjugate and determining whether the antibody-drug conjugate modulates a cellular activity or function, as reflected, for example, by changes in cell morphology or viability, marker expression, differentiation or dedifferentiation, cellular respiration, mitochondrial activity, membrane integrity, maturation, proliferation, viability, apoptosis, or cell death. An example of a direct interaction is physical interaction, while an indirect interaction includes, for example, the action of a composition on an intermediate molecule that, in turn, acts on the reference entity (e.g., cell or cell culture). VII. Manufactured Articles The disclosure also includes pharmaceutical packaging and kits comprising one or more containers, where one container may comprise one or more doses of an antibody-drug conjugate or composition as described in 44 IF-2019-03555061-APN-ANP#INPI Page 44 of 93 describes herein. In certain embodiments, the package or kit contains a unit dosage, which means a predetermined amount of an antibody-drug composition or conjugate, with or without one or more additional agents. The kit may comprise one or more containers and a data sheet or package insert in, on, or associated with the container(s), indicating that the contained composition is used to treat the preferred pathological process. Suitable containers include, for example, bottles, vials, syringes, etc. The containers may be made of various materials such as glass or plastic. The container(s) may include a sterile access nozzle; for example, the container may be an intravenous solution bag or a vial with a stopper that can be pierced with a hypodermic injection needle. In some embodiments, the kit may contain a means of administering the antibody and any optional components to a patient, e.g., one or more needles or syringes (pre-filled or empty), an ophthalmic dropper, a pipette, or other similar device, from which the formulation can be injected or introduced into the subject or applied to an affected area of ​​the body. Disclosure kits will also typically include a means of containing vials or similar containers and other components confined in a small space for marketing purposes, such as, e.g., blow-molded plastic containers in which the desired vials and other devices are placed and retained. IF-2019-03555061-APN-ANP#INPI Page 45 of 93 Examples It is understood that the examples and realizations described herein are for illustrative purposes only, and in view of these, people skilled in the technique will come up with various modifications or changes, and these will be included within the nature and scope of this disclosure. The starting materials are commercially available and can be prepared using the procedures described herein, bibliographic procedures, or procedures that would be very familiar to an expert in: the field of organic chemistry. The names of the reagents / reactants provided are as follows: They are named on the commercial bottle or as they were generated according to IUPAC standards, ChemDraw Ultra 12.0. CambridgeSoft® Chemistry E-Notebook 11 or CambridgeSoft® AutoNom 2000. It is understood that the examples and implementations described herein are for illustrative purposes only and that, in view of these, persons skilled in the art will think of various modifications or changes, and these will be included within the nature and scope of this disclosure. Analytical methods for the synthesis and characterization of compounds Analytical data is included in the following procedures, in the illustrations of the procedures; general or in the example tables. Unless otherwise stated, all TH and 13C NMR data were collected on a Varian Mercury Plus 400 MHz or 46 Ω instrument IF-2019-03555061-APN-ANP#INPI Page 46 of 93 Bruker AVIII 300 MHz; chemical shifts are quoted in parts per million (ppm). HPLC analytical data are either detailed in the experimental section or referenced in the LC / MS and HPLC conditions table, using the method provided in Table 4. Table 4 Method Conditions The gradient was 1–90% B for 3.4 min, 90–100% B for 0.45 min, 100–1% B for 0.01 min, and then maintained at 1% B for 0.65 min (0.8 mL / min flow rate). Mobile phase A was 0.0375% TEA in H₂O, and mobile phase B was 0.018% TEA in MeCN. The column used for chromatography was a 2.0 x 50 mm Phenomenex Luna-C18 column (5 pm particles). Detection methods included DAD and ELSD, as well as positive-mode electrofoaming ionization. The abbreviations used in the following examples are: ACTH Adrenocorticotropic hormone HIC Hydrophobic interaction chromatography BrAc Bromoacetamide HPL C High-performance liquid chromatography CV Column volumes LCM S Liquid chromatography- IF-2019-03555061-APN-ANP#INPI Page 47 of 93 Mass spectrometry DAD Diode beam MeC N Acetonitrile DAR Drug-to-antibody ratio MEM Minimum essential media DBF Dibutyl flalate MeO H Methanol DCM Dichloromethane MS Mass spectrometry DMA Dimethylacetamide NEA A Non-essential amino acids DMF Dimethylformamide NMR Nuclear magnetic resonance DMSO Dimethyl sulfoxide PBS Phosphate-buffered saline DTT Dithiothreitol PE Petroleum ether EEDQ 2-Ethoxy-1-ethoxycarbonyl 2-Dihydroquinoline PIN P N-terminal propeptide of type 1 procollagen ELSD Evaporative light scattering detector TA Room temperature Eq Equivalents RPM I Roswell Park Memorial Institute EtOA c Ethyl acetate SEC Size exclusion chromatography IF-2019-03555061-APN-ANP#INPI Page 48 of 93 FBS Fetal bovine serum TCE P (tris(2-carboxyethyl)phosphine) Fmoc 9- Fluorenylmethyloxycarbonyl 0 TFA Trifluoroacetic acid Example 1: Synthesis of (2S,6aS,6bR,7S,8aS,8bS,IOS,llaR,12aS,12bS)-10-(4-(3aminobenzyl)phenyl)-2,6b-difluoro-7-hydroxy-8b-(2hydroxyaceti l)-6a,8a-dimethyl1,2,6a,6b,7,8,8a,8b,lia,12,12a,12b-dodecahydro-4Hnaphtho[2',1':4,5]indene[1,2-d][1,3]dioxol-4-one Step 1: Synthesis of 4-(bromomethyl)benzaldehyde Diisobutylaluminum hydride (153 mL, 153 mmol, 1 M in toluene) was added dropwise to a 0 °C solution of 4-(bromomethyl)benzonitrile (20 g, 102 mmol) in toluene (400 mL) over 1 hour. Two additional vials were prepared as described above. All three reaction mixtures were combined. 10% aqueous HCl (1.5 L) was added to the mixture. The mixture was extracted with DCM (3 x 500 mL). The organic layer was dried with Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluted with PE:EtOAc = 10:1) to obtain the title compound (50 g, 82% yield) as a white solid. H NMR (400MHz, CDCI3) δ 10.02 (s, 1H), 7.91 - 7.82 (m, 2H), 7.56 (d, J=1.9 Hz, 2H), 4.55 - 4.45 (m, 2H). IF-2019-03555061-APN-ANP#INPI Page 49 of 93 Step 2: Synthesis of 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline Cl 1^° \ Cí^NH2h2n To a solution of 3-bromoaniline (40 g, 233 mmol) in 1,4-dioxane (480 mL) were added 4 / 4,4',4',5,5,5',5'-tetramethyl-2,2'-bi(1,3,2-dioxaborolane) (94 g, 372 mmol), potassium acetate (45.6 g, 465 mmol), 2-dicyclohexylphosphino-2',4',6'-tri-i-propyl-1,1'-bitenyl (8.07 g, 13.95 mmol), and tris(dibenzylideneacetone)dipalladium (0) (8.52 g, 9.30 mmol). The resulting mixture was heated to °C for 4 hours in a nitrogen atmosphere. An additional vial was prepared as described above. The two reaction mixtures were combined and concentrated, and the residue was It was purified by silica gel column chromatography (eluted with PE:EtOAc = 10:1) to obtain the title compound (60 g, 55.4% yield) as a light yellow solid.1H NMR (400MHz, CDC13) δ 7.23 7.13 (m, 3H) , 6.80 (d, J=7.5 Hz, 1H) , 3.82 - 3.38 (m, 2H) , 1.34 (s, 12H). Step 3: Synthesis of tert-butyl (3-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)phenyl)carbamate 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (Example 1, Step 2) (30 g, 137 mmol) and di-tert-butyl dicarbonate (38.9 g, 178 mmol) were mixed in toluene (600 mL) at 100 °C for 24 hours. Another vial was prepared as described above. The two mixtures of IF-2019-03555061-APN-ANP#INPI Page 50 of 93 reaction were combined and the mixture was evaporated, dissolved in EtOAc (1.5 L), washed with 0.1 N HC1 (3 x 2 L) and brine (3 L), dried with Na2SO4, filtered and concentrated under reduced pressure to obtain the title compound (50 g, 57% yield) as a red solid.1H NMR (400MHz, CDC13) δ 7.63 (ma, 2H) , 7.48 (d, J=1.1 Hz, 1H), 7.37 - 7.28 (m, 1H), 1.52 (s, 9H), 1.34 (s, 12H). Step 4: Synthesis of tert-butyl (3—(4—formylbenzyl)phenyl)carbamate Boc A mixture of 4-(bromomethyl)benzaldehyde (Example 1, Step 1) (24.94 g, 125 mmol), 1,1'-bis(diphenylphosphino)ferrocenedichloropalladium(II) complex with DCM (13.75 g, 18.80 mmol), tert-butyl (3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)carbamate (from Example 1, Step 3) (20 g, 62.7 mmol) and potassium carbonate (43.3 g, 313 mmol) in tetrahydrofuran (400 mL) was heated to 80 °C for 12 hours. Another vial was prepared as described above. The two reaction mixtures were combined and diluted with water (500 mL). The aqueous mixture was extracted with EtOAc (3 x 500 mL). The organic layers were combined and dried with Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluted with PE:EtOAc = 10:1) to obtain the title compound (15 g, 38.4% yield) as a white solid. TH NMR (400 MHz, CDC13) δ 9.95 (s, 1H), 7.78 (d, J=7.9 Hz, 2H), 7.33 (d, J=1.9 Hz, 2H), 7.27 - 7.13 (m, 51. IF-2019-03555061-APN-ANP#INPI Page 51 of 93 3H) , 6.82 (d, J=7.1 Hz, 1H) , 6.47 (sa, 1H) , 4.00 (s, 2H) , 1.48 (s, 9H). Step 5: Summary of (6S, 8S, 9R, IOS, 11S, 13S,14S,16R,17S)-6,9-difluoro-11,16,17trihidroxy-17-(2-hidroxyacetyl)-10,13-dimethyl6,7,8,9,10,11,12,13,14,15,16,17-dodecahidro-3Hcyclopenta[a]phenanthren-3-ona If suspended (2S,6aS,6bR,7 S,8aS,8bS,llaR,12aS,12bS) 2,6b-difluoro-7-hidroxi-8b-(2-hidroxyacetyl)-6a,8a,10,10tetrameti1—1,2,6a,6b,7,8,8a,8b,Ila,12,12a,12b—dodecahidro— 4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-ona (20 g, 44.2 mmol) in 40% aqueous HBF4 (440 mL) and the mixture was stirred at 25 °C for 48 hours. After the reaction was complete, 2 L of water were added and the solid was collected by filtration. This solid was washed with water (1 L) and then with MeOH (200 mL) to obtain the title compound (11 g, 60.3% yield) as a white solid. NMR (400 MHz, DMSO-d6) δ 7.25 (d, J-10.1 Hz, 1H) , 6.28 (d, J-10.1 Hz, 1H), 6.10 (s, 1H) , 5.7 3 - 5.50 (m, 1H), 5.39 (sa, 1H) , 4.85 - 4.60 (m, 2H) , 4.50 (d, J=19.4 Hz, 1H) , 4.20 — 4.04 (m, 2H), 2.46 - 2.06 (m, 6H), 1.87 - 1.75 (m, 1H), 1.56 - 1.30 (m, 6H), 0.83 (s, 3H). Step 6: Synthesis of (2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-aminobenzyl)phenyl)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl52 IF-2019-03555061-APN-ANP#INPI Page 52 of 93 1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4Hnaphtho[2 ' , 1' : 4,5]indene[1,2-d][1,3]dioxol-4-one. A suspension of (6S,8S, 9R,IOS,11S,13S,14S,16R,17S) 6,9-difluoro-11,16,17-trihydroxy-17-(2-hydroxyacetyl)10,13-dimethyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro3H-cyclopenta[a]phenanthren-3-one (Example 1, Step 5) (4.4 g, 10.67 mmol) and MgSO4 (6.42 g, 53.3 mmol) in MeCN (100 mL) was stirred at 20 °C for 1 hour. A solution of tert-butyl (3-(4-formylbenzyl)phenyl)carbamate (Example 1, Step 4) (3.65 g, 11.74 mmol) was added to MeCN (100 mL) in one portion. Trifluoromethanesulfonic acid (9.01 mL, 53.3 mmol) was added dropwise while maintaining an internal temperature below 25 °C using an ice bath. After the addition, the mixture was stirred at 20 °C for 2 hours. Three additional vials were prepared as described above. All four reaction mixtures were combined and concentrated, and the residue was purified by HPLC Prep to obtain the title compound (4.5 g, 14.2% yield) as a yellow solid.LCMS (Method a, Table 4) tR= 2.65 min; MS m / z = 606.2 (M+H)+;1H NMR (400MHz, DMSO-d6) δ 7.44 - 7.17 (m, 5H) , 6.89 (t, J=7.7 Hz, 1H) , 6.44 - 6.25 (m, 4H) , 6.13 (sa, 1H) , 5.79 - 5.52 (m, 2H) , 5.44 (s, 1H), 5.17 - 4.89 (m, 3H) , 4.51 (d, J=19.4 Hz, 1H) , 4.25 - 4.05 (m, 2H) , 3.73 (s, 2H) , 3.17 (sa, 1H), 2.75 53. IF-2019-03555061-APN-ANP#INPI Page 53 of 93 2.55 (m, 1H) , 2.36 - 1.97 (m, 3H) , 1.76 - 1.64 (m, 3H) , 1.59 - 1.39 (m, 4H) , 0.94 - 0.78 (m, 3H) . HPLC Prep Method: Instrument: Gilson 281 semi-preparative HPLC system; Mobile phase: A: Formic acid / H20 = 0.01% v / v; B: MeCN; Column: Moon CIS 150*25 5 micrometers; Caudal: 25ml / min; Monitored wave length: 220 and 254 nm. T i m e 0.0 10.5 10.6 10.7 13.7 13.8 15.0 % of B 15 35 35 100 100 10 10 Example 2: Summary of (6aR,6bS,7S,8aS,8bS,10R,llaR,12aS,12bS)-10-(4-(3aminobencyl)phenyl)-7-hidroxi-8b-(2- hidroxyacetyl)-6a,8adimethyl-1,2,6a,6b,7,8,8a,8b,Ila,12,12a,12b-dodecahidro-4Hnaphtho[2 ' , 1':4,5]indeno[1,2-d][1,3]dioxol-4-ona. Example 2 was summarized in a similar procedure to Example 1 using (8S,9S,10R,11S,13S,14S,16R,17S)11,16,17-trihidroxi-17-(2-hidroxyacetyl)-10,13-di methyl6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3Hcyclopenta[a]phenanthren-3-ona. NMR (400MHz, DMSO-d6) δ 7.36 (d, J=7.9 Hz, 2H) , 7.31 (d, J=10.1 Hz, 1H) , 7.20 (d, J=7.9 Hz, 2H) , 6.8 9 (t, J=1.9 Hz, 1H) , 6.39 - 6.28 (m, 3H) , 6.16 (dd, J=1.5, 9.9 Hz, 1H) , 5.93 (s, 1H) , 5.39 (s, 1H) , 5.08 (t, J=5.7 Hz, 1H), 4.98 - 4.87 (m, 3H), 4.78 (d, J=3.1 Hz, 1H) , 4.49 (dd, J=6.2, 19.4 Hz, 1H), 4.29 (sa, 1H), 4.17 (dd, J=5.5, 19.6 54 IF-2019-03555061-APN-ANP#INPI Page 54 of 93 Hz, 1H), 3.74 (s, 2H), 2.61 - 2.53 (m, 1H), 2.36 - 2.26 (m, 1H), 2.11 (d, J=11.0 Hz, 1H), 2.07 (s, 1H), 2.02 (d, J=12.8 Hz, 1H), 1.83 - 1.54 (m, 5H), 1.39 (s, 3H), 1.16 - 0.96 (m, 2H), 0.85 (s, 3H). LCMS (Method a, Table 4) TR = 2.365 min; m / z = 570.2 (M+H)+. Example 3· Synthesis of (6aS,6bR,7S,8aS,8bS,10R,llaR,12aS,12bS)-10-¢4-(3aminobenzyl)phenyl)-6b-fluoro-7-hydroxy-8b-(2 hydroxyacetyl)-6a,8a-dimethyl1,2,6a,6b,7,8,8a,8b,Ila,12,12a,12b-dodecahydro-4Hnaphtho[2',11:4,5]indene[1,2-d][1,3]dioxol-4-one. Example 3 was synthesized in a procedure similar to Example 1 using (8S,9R,IOS,11S,13S,14S, 16R, 17S)-9fluoro-11,16,17-trihydroxy-17-(2-hydroxyacetyl)-10,13dimethyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3Hcyclopenta[a]phenanthren-3-one. ςΗ RMN (400MHz, DMSO-d6) δ 7.37 - 7.26 (m, 3H) , 7.21 (d, J=7.9 Hz, 2H) , 6.89 (t, J=7.7 Hz, 1H) , 6.43 - 6.30 (m, 3H) , 6.23 (d, J=10.1 Hz, 1H), 6.04 (s, 1H), 5.75 (s, 1H), 5.44 (s, 2H), 5.09 (t, J-5.7 Hz, 1H), 4.93 (sa, 3H), 4.50 (dd, J=6.2, 19.4 Hz, 1H), 4.28 - 4.09 (m, 2H), 3.74 (s, 2H), 2.73 - 2.54 (m, 2H), 2.35 (d, J=13.2 Hz, 1H), 2.25 2.12 (m, 1H), 2.05 (d, J=15.0 Hz, 1H), 1.92 - 1.77 (m, 1H), 1.74 - 1.58 (m, 3H), 1.50 (s, 3H), 1.45 - 1.30 (m, 1H), IF-2019-03555061-APN-ANP#INPI Page 55 of 93 0.87 (s, 3H). LCMS (Método a, Tab 4) TR - 2.68 min; m / z = 588.1 (M+H)+ Ejemplo 4: Síntesis de ácido (S)-4-(2-(2bromoacetamido)acetamido)-5-((3-(4((6aR,6bS,7S,8aS,8bS,10R,llaR,12aS,12bS)-7-hidroxi-6a,8adimetil-4-oxo-8b-(2-(fosfonooxi)ac etil)2,4,6a,6b,7,8,8a,8b,Ila,12,12a,12b-dodecahidro-lHnafto[2',1':4,5]indeno[1,2-d][1,3]dioxol-10il)bencil)fenil)amino)-5-oxopentanoico Step 1: Synthesis of (S)-2-(2-((((9H-fluoren-9yl)methoxy)carbonyl)amino)acetamido)-5-(tert-butoxy)-5 oxopentanoic acid. A mixture of 2-chlorotrityl chloride resin (30 g, 92 mmol), triethylamine (46.4 g, 458 mmol), and (S)-2((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)5-oxopentanoic acid (25.5 g, 60 mmol) in anhydrous DCM (200 mL) was bubbled through at 200°C for 8 hours. The mixture was filtered, and the resin was washed with DCM (2 x 200 mL), MeOH (2 x 200 mL), and DMF (2 x 200 mL). A piperine:DMF solution (1:4, 400 mL) was added to the resin, and the mixture was bubbled through with N2 for 8 minutes and then filtered. This operation was repeated five times to obtain complete removal of the Fmoc protecting group. The resin was washed with DMF (5 x 500 mL) to provide resin-bound (S)-2-amino-5-(tert-butoxy)-5-oxopentanoic acid. IF-2019-03555061-APN-ANP#INPI Page 56 of 93 A mixture of acid 2-(((9H-fluoren-9yl)methoxy)carbonyl)amino)acetic acid (13.38 g, 45.0 mmol), N,N-diisopropylethylamine (7.86 mL, 45 mmol), hydroxybenzotriazole (6.89 g, 45 mmol), 2-(6-chloro-1H-benzo[d][1,2,3]triazol-1-yl)-1,1,3,3-tetramethylisuronium hexafluorophosphate (V) (18.62 g, 45.0 mmol) in DMF (200 mL) was stirred at 20 °C for 30 min. Resin-bound (S)-2-amino-5-(tert-butoxy)-5-oxopentanoic acid was added to the mixture and the resulting mixture was bubbled with N2 at 25 °C for 1.5 hours. The mixture was filtered, and the resin was washed with DMF (4 x 500 mL) and DCM (2 x 500 mL). TFA at 1% / DCM (5 x 500 mL) was added to the mixture, and it was bubbled through with N2 for 5 minutes. The mixture was filtered, and the filtrate was added directly to a saturated NaHCO3 solution (200 mL). The combined mixture was separated, and the organic phase was washed with saturated aqueous citric acid solution (4 x 400 mL) and brine (2 x 300 mL).The final organic solution was dried with Na2SO4(20 g), filtered and concentrated under reduced pressure to provide the title compound (10 g, 20% yield) as a light yellow solid. NMR: (CDC13, 400 MHz) δ = 7.75 (d, J = 7.5 Hz, 2H), 7.59 (da, J = 7.5 Hz, 2H), 7.41 - 7.36 (m, 2H), 7.30 (t, J = 7.0 Hz, 2H), 5.82 (sa, 1H), 4.57 (da, J = 4.8 Hz, 1H), 4.38 (da, J = 7.5 Hz, 2H), 4.27 - 4.15 (m, 1H), 4.063.83 (m, 2H), 2.50 - 2.29 (m, 2H), 2.26 - 2.13 (m, 1H), 2.06 - 2.02 (m, 1H), 1.43 (s, 9H). Step 2: Synthesis of (S)-4-(2-((((9H-fluoren-9yl)methoxy)carbonyl)amino)acetamido)-5-((3-(4((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2 IF-2019-03555061-APN-ANP#INPI Page 57 of 93 hydroxyacetyl)-6a,8a-dimethyl-4-oxo tert-butyl 2,4,6a,6b,7,8,8a,8b,Ila,12,12a,12b-dodecahydro-lH naphtho[2',1':4,5]indene[1,2-d][1,3]dioxol-10yl)benzyl)phenyl)amino)-5-oxopentanoate A solution of (S)-2-(2-((((9H-fluoren-9yl)methoxy)carbonyl)amino)acetamido)-5-(tert-butoxy)-5-oxopentanoic acid (Example 4, Step 1) (424 mg, 0.878 mmol) in DMF (3.5 mL) was added (6aR,6bS,7S,8aS,8bS,10R,llaR,12aS,12bS)-10-(4-(3aminobenzyl)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,Sadimet i 1-1 ,2,6a,6b,7,8,Sa,8b,Ila,12,12a,12b—dodecahydro—4Hnaphtho[2',1':4,5]indene[1,2-d][1,3]dioxol-4-one (Example 2) (500 mg, 0.878 mmol) and triethylamine (0.3 mL, 2.63 mmol) at 25°C. The solution was cooled to 0 °C and then 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphinan trioxide (1.12 g, 1.755 mmol) was added. The reaction mixture was stirred for 12 hours at 25 °C. Completion of the reaction was confirmed by LC-MS. Fourteen additional vials were prepared as described above. All fifteen reaction mixtures were combined. The mixture was purified by reversed-phase column spectrometry to yield the title compound (5 g, 38.4% yield) as a yellow solid. Reversed-phase column method: Instrument: HPLC 58 IF-2019-03555061-APN-ANP#INPI Page 58 of 93 preparative Shimadzu LC-8A; Column: Phenomenex Luna Cl8 200*40 mm*10 pm; Mobile phase: A for H2O (0.05% TFA) and B for MeCN; Gradient: B from 30% to 100% in 30 min; Flow rate: 60 mi / min; Wavelength: 220 and 254 nm. LCMS (Method a, Table 4) TR = 1.34 min; m / z 1016.6 (M+H-18)+. Step 3: Synthesis of (S)-4-(2-((((9H-fluoren-9yl)methoxy)carbonyl)amino)acetamido)-5-((3—(4— ((6aR,6bS,7S,8aS,8bS,10R,llaR,12aS,12bS)-8b-(2-((di-tertbutoxyphosphoryl)oxy)acetyl)-7 -hydroxy-6a,8a-dimethyl-4-oxo2,4,6a,6b,7,8,8a,8b,lia,12,12a,12b-dodecahydro-lHnaphtho[2 ' , 1' : tert-butyl 4,5]indeno[1,2-d][1,3]dioxol-10yl)benzyl)phenyl)amino)-5-oxopentanoate. yl)methoxy)carbonyl)amino)acetamido)-5-((3-(4((6aR,6bS,7S,8aS,8bS,10R,llaR,12aS,12bS)-7-hydroxy-8b-(2hydroxyacetyl)-6a,8a-dimethyl-4-o xo2,4,6a,6b,7,8,8a,8b,lia,12,12a,12b-dodecahydro-lHnaphtho[21,1':4,5]indene[1,2-d][1,3]dioxol-10yl)benzyl)phenyl)amino)-5-oxopentanoate of tert-butyl (Example 4, Step 2) (400 mg, 0.387 mmol) in DMF (2.5 mL) IH-tetrazol (271 mg, 3.87 (1.16 g, 4.64 mmol) and di-tert-butyl diethylphosphoramidite (1.16 g, 4.64 mmol). The reaction was stirred at room temperature for 2.5 hours, then IF-2019-03555061-APN-ANP#INPI Page 59 of 93. The mixture was cooled to 0 °C. Hydrogen peroxide (241 mg, 2.127 mmol) was added to the resulting mixture, which was heated to TA and stirred for 1 hour. Afterward, LC-MS was used to confirm that the reaction was complete. Eleven additional vials were prepared as described above. All twelve reaction mixtures were combined. The mixture was purified by reversed-phase column spectrometry to yield the title compound (4.4 g, 64.2% yield) as a yellow solid. Reversed-phase column method: Instrument: Shimadzu LC-8A preparative HPLC; Column: Phenomenex Luna C18 200*40 mm*10 pm; Mobile phase: A for H2O and B for MeCN; Gradient: B from 50% to 100% in 30 min; Flow rate: 60 mL / min; Wavelength: 220 and 254 nm. LCMS (Method a, Table 4) TR = 1.41 min; m / z 1226.7 (M+H)+. Step 4: Synthesis of (S)-4-(2-aminoacetamido)-5-((3-(4((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-8b-(2-((di-tert-butoxyphosphoryl)oxy)acetyl)-7-hydroxy-6a,8a-dimethyl-4-oxo2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1Hnaphto[2 ' , 1':4,5]indeno[1,2-d][1,3]dioxol-10yl)benzyl)phenyl)amino)-5-oxopentanoate tert-butyl. To a solution of (3)-4-(2-((((9H-fluoren-9yl)methoxy)carbonyl)amino)acetamido)-5-((3-(4((6aR,6bS,73,8aS,8bS,10R,11R,12aS,12bS)-8b-(2-((di-tert60 IF-2019-03555061-APN-ANP#INPI Page 60 of 93 butoxyphosphoryl)oxy)acetyl)—7-hydroxy—6a, 8a—dimethyl 4 oxo 2,4,6a, 6b, 7,8, 8a, 8b,1a,12,12a,12b-dodecahydro-1Hnaphto[2', 1':4,5]indeno[1,2-d][1,3]dioxol-10yl)benzyl)phenyl)amino)-5-oxopentanoate tert-butyl (Example 4, Step 3) (1.1 g, 0.897 mmol) in MeCN (6 ml) was piperidine (0.75 mL, 7.58 mmol) added at 25 °C. The reaction was stirred at TA for 20 minutes, after which it was verified by LC / MS that the reaction was complete. Three additional vials were prepared as described above. All four reaction mixtures were combined. The mixture was concentrated to provide a residue, which was treated with PE (10 mL) with stirring for 2 hours. The resulting solid was collected by filtration and dried under reduced pressure to provide the title compound (3.8 g, 90% yield) as a yellow solid. LCMS (Method a, Table 4) TR = 1.16 min; m / z 1004.6 (M+H)+. Step 5: Synthesis of (S)-4-(2-(2 bromoacetamido)acetamido)-5-((3-(4((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-8b-(2-((di-tert-butoxyphosphoryl)oxy)acetyl·)-7-hydroxy-6a,8a-dimethyl-4-oxo2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1Hnaphto[21,11:4,5]indeno[1,2-d][1,3]dioxol-10yl)benzyl)phenyl)amino)-5-oxopentanoate tert-butyl. IF-2019-03555061-APN-ANP#INPI Page 61 of 93 EEDQ (172 mg, 0.697 mmol) was added to a solution of 2-bromoacetic acid (97 mg, 0.697 mmol) in DMF (2.5 mL) at room temperature. The mixture was stirred at room temperature for 1 hour. (S)4-(2-aminoacetamido)-5-((3-(4((6aR,6bS, 7S,8aS,8bS,10R,11aR,12aS,12bS)-8b-(2-((di-tert-butoxyphosphoryl)oxy)acetyl)-7-hydroxy-6a,8a-dimethyl-4-oxo2,4, 6a, 6b, 7,8, 8a, 8b, 11a, 12,12a,12b-dodecahydro-1Hnaphto[2',1':4,5]indeno[1,2-d][1,3]dioxol-10yl)benzyl)phenyl)amino)-5-oxopentanoate tert-butyl (Example 4, Step 4) (350 mg, 0.349 mmol) was added and the solution was The mixture was shaken for 2.5 hours, after which the reaction was confirmed to be complete by LC-MS. Seven additional vials were prepared as described above. All eight reaction mixtures were combined. The reaction mixture was diluted with DCM (100 mL), washed with aqueous HBr (1 M, 2 x 80 mL), aqueous NaHCCh (60 mL), and brine (60 mL).The organic layer was dried with Na2SO4, filtered and concentrated under reduced pressure to provide the title compound (2 g, 63.7% yield) as a yellow oil. LCMS (Method a, Table 4) TR = 1.30 min; m / z 1124.2, 1125.9 (M+H)+. Step 6: Synthesis of (S)-4-(2-typhoromoacetamido)acetamido)-5-((3-(4( (6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-6a,8-adimethyl-4-oxo-8b-(2-(phosphonooxy)acetyl)2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1Hnaphto[2',1':4,5]indeno[1,2-d][1,3]dioxol-10yl)benzyl)phenyl)amino)-5-oxopentanoic IF-2019-03555061-APN-ANP#INPI Page 62 of 93 >0 bromoacetamido)acetamido)-5-((3-(4((6aR, 6bS, 7S,8aS,8bS,10R,11aR,12aS,12bS)-8b-(2-( (di-tert-butoxyphosphoryl) oxy)acetyl)-7-hydroxy-6a,8a-dimethyl~4-oxo2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1Hnaphto[2',1':4,5]indeno[1,2-d][1,3]dioxol-10yl)benzyl)phenyl)amino)-5-oxopentanoate tert-butyl (Example 4, Step 5) (2 g, 1.778 mmol) in DCM (16 mL) TEA (8 mL, 104 mmol) and the resulting mixture was stirred at room temperature for 40 minutes, after which the reaction was confirmed to be complete by LC-MS. The solvent was removed under reduced pressure. The resulting residue was purified by HPLC Prep. The mobile phase was directly lyophilized to provide the title compound (640 mg, 35.3% yield) as a yellow solid. HPLC Prep Method: Instrument: Shimadzu LC-8A Preparative HPLC; Column: Phenomenex Luna C18 200*40 rrílO pm; Mobile Phase: A for H2O (0.09% de TFA) y B per MeCN; Gradient: B del 30% and 40% in 20 minutes; Caudal: 60 mL / min; Longitudinal size: 220 y 254 nm. XH RMN: (DMSO-d6, 400 MHz) δ = 9.88 (s, 1H) , 8.52 (s, 1H) , 8.24 (da, J = 8.4 Hz, 1H) , 7.46 (da, J = 7.9 Hz, 1H) , 7.42 (s, 1H) , 7.36 (da, J =7.9 Hz, 2H), 7.30 (da, J = 9.Ί Hz, 1H), 7.23 - 7.17 (m, 3H), 6.90 (da, J = 6.8 Hz, 1H), 6.16 (da, J = 10.4 Hz, 1H), 5.93 (s, 1H), 5.47(s, IF-2019-03555061-APN-ANP#INPI Page 63 of 93 1H), 4.96 - 4.85 (m, 3H), 4.58 (d ad, J = 7.9, 18.7 Hz, 1H), 4.38 (da, J = 5.3 Hz, 1H), 4.29 (sa, 1H), 3.93 (s, (2H) , 3.89 (s, 2H) , 3.80 (sa, 2H) , 2.30 - 2.22 (m, 2H) , 2.16 - 1.91 (m, 4H), 1.85 - 1.62 (m, 6H), 1.39 (s, 3H), 1.00 (sa, 2H), 0.87 (s, 3H). LCMS (Método a, Tab 4) TR = 2.86 min; m / z 956.0, 958.0 (M+H)+. Example 5: Synthesis of dihydrogenofosphato of 2((2S,6aS,6bR,7S,8aS,8bS,10R,llaR,12aS,12bS)-10-(4-(3-( (S)6-amino-2-(2-(2bromoacetamido)acetamido)hexanamido)bencil)fenil)-2,6bdifluoro-7-hidroxi-6a,8a-dimetil-4-oxo1,2,4,6a,6b,7,8,8a,Ila,12,12a,12b-dodecahidro-8bHnafto[2 ' , 1' : 4,5]indeno[1,2-d][1,3]dioxol-8b-il)-2-oxoetilo. Step 1: Synthesis of ((S)-5-(2-((((9H-fluoren-9il)metoxi)carbonil)amino)acetamido)-6-((3-(4((2S,6aS,6bR,7S,8aS,8bS,10R,llaR,12aS,12bS)-2,6b-difluoro7-hidroxi-8b-(2-hidroxiacetil)-6a,8a-dimethyl-4-oxo2,4,6a,6b,7,8,8a,8b,Ila,12,12a,12b-dodecahidro-lHnafto[21,11:4,5]indeno[1,2-d][1,3]dioxol-10il)bencil)fenil)amino)-6-oxohexil)carbamato tert-butilo. A solution of N2-((((9H-fluoren-9il)metoxi)carbonil)glycil)-N6-(tert-butoxicarbonil)-lili sina (5.58 g, 8.26 mmol) in DMF (60 mL) a 0 C se añadieron of 2,4,6-trioxido 2,4,6-tripropil-l,3,5,2,4,6trioxatrifosfinano (10.51 g, 16.51 mmol) and trietilamina IF-2019-03555061-APN-ANP#INPI Page 64 of 93 (3.45 mL / 24.77 mmol). The resulting mixture was stirred at ΔT for 1 hour and ¢23,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-aminobenzyl)phenyl)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4Hnaphto[21,1':4,5]indene[1,2-d][1,3]dioxol-4-one (Example 1, Step 6) (5 g, 8.26 mmol) was added. The resulting mixture was stirred for 5 hours at room temperature, after which the reaction was confirmed to be complete by LC-MS. Six additional vials were prepared as described above. All seven reaction mixtures were combined. The reaction was purified by reversed-phase column spectrometry to provide the title compound (24 g, 24.62% yield) as a blank solid. Reversed-phase column method: Instrument: Shimadzu LC-8A HPLC prep; Column: Phenomenex Luna C18 200*40 mm*10 pm; Mobile phase: A for H2O (0.0.5% of TFA) and B for MeCN; Gradient: B from 30% to 100% in 30 min; Flow rate: 60 ml / min; Wavelength: 220 and 254 nm. LCMS (Method a, Table 4) TR = 1.29 min; m / z 1095.6 (M+H-18)+. Step 2: Synthesis of ( (S)-5-(2-( ( ( (9H-fluoren-9yl)methoxy)carbonyl)amino)acetamido)-6-((3-(4((2S,6aS,6bR,7S,8aS,8bS,10R,llaR,12aS,12bS)-8b-(2-((d itert-butoxyphosphoryl)oxy)acetyl)-2,6b-difluoro-7-hydroxy6a,8a-dimethyl—4—oxo—2,4,6a,6b,7,8,8a,8b,lia,12,12a,12b— dodecahydro-lH-naphtho[2',1':4,5]indene[1,2-d][1,3]dioxol-10yl)benzyl)phenyl)amino)-6-oxohexyl)carbamate tert-butyl. IF-2019-03555061-APN-ANP#INPI Page 65 of 93 To a solution of ((S)-5~¢2-((((9H-fluoren-9yl)methoxy)carbonyl)amino)acetamido)-6-((3-(4((2S,6aS,6bR,7S,8aS,8bS,10R,llaR,12aS,12bS))-2,6b-difluoro7-hydroxy-8b-(2-hydroxyacetyl )-6a,8a-dimethyl-4-oxo2,4,6a,6b,7,8,8a,8b,lia,12,12a,12b-dodecahydro-lHnaphtho[2',1':4,5]indene[1,2-d][1,3]dioxol-10yl)benzyl)phenyl)amino)-6-oxohexyl)carbamate tert-butyl (Example 5, Step 1) (3 g, 2.69 mmol) in DMF (30 mL) were mixed with 1H-tetrazol (1.888 g, 26.9 mmol) and di-tert-butyl diethylphosphoramidite (8.06 g, 32.3 mmol), and the reaction was stirred at room temperature for 3.5 hours. Hydrogen peroxide (224 mg, 1.97 mmol) was added to the reaction and stirred for 0.5 hours, after which the reaction was confirmed by LC-MS to be complete. Six additional vials were prepared as described above. All seven reaction mixtures were combined. The reaction was purified by reversed-phase column to yield the title compound (10 g, purity: 78%, 37).1% yield) as a blank solid. Reversed-phase column method: Instrument: Shimadzu LC-8A HPLC prep; Column: Phenomenex Luna C18 200*40mm*10 pm; Mobile phase: A for H2O and B for MeCN; Gradient: B from 50% to 100% in 30 min; Flow rate: 60 ml / min. Wavelength: 220 and 254 nm. 6 IF-2019-03555061-APN-ANP#INPI Page 66 of 93 LCMS (Method a, Table 4) TR = 1.42 min; m / z 1305.7 (M+H)+. Step 3: Synthesis of ((Ξ)-5-(2-aminoacetamido)-6-( (3(4-((2S,6aS,6bR,7S,8aS,8bS,10R,llaR,12aS,12bS)-8b-(2-((ditert-butoxyphosphoryl)oxy)acetyl)-2,6b-difluoro-7-hydroxy6a,8a-dimethyl-4~oxo- 2,4,6a,6b,7,8,8a,8b,Ila,12,12a,12bdodecahydro-lH-naphtho[2',1':4,5]indene[1,2-d][1,3]dioxol-10yl)benzyl)phenyl)amino)-6-oxohexyl)carbamate tert-butyl. To a solution of ((S)-5-(2-((((9H-fluoren-9 yl)methoxy)carbonyl)amino)acetamido)-6-((3-(4((2S,6aS,6bR,7S,8aS,8bS,10R,llaR,12aS,12bS))-8b-(2-((ditert-butoxyphosphoryl)oxy)acetyl)-2,6b-difluoro-7-hydroxy6 a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,Ila,12,12a,12bdodecahydro-lH-naphtho[2',1':4,5]indene[1,2-d][1,3]dioxol-10yl)benzyl)phenyl)amino)-6-oxohexyl)carbamate of tert-butyl (Ex Example 5, Step 2) (2.5 g, 1.969 mmol) in MeCN (10 mL) was mixed with piperidine (2 mL, 1.969 mmol), and the reaction was stirred at room temperature for 1 hour, after which it was verified by LC-MS that the reaction had ended. Three additional vials were prepared as described above. All four reaction mixtures were combined. The reaction was concentrated to give a crude product, which was stirred in PE (30 mL) for 2 hours. The resulting solid was collected by filtration and dried under reduced pressure to give the compound of the IF-2019-03555061-APN-ANP#INPI Page 67 of 93 title (7 g, purity: 83%, 70.4% yield) as a yellow solid. LCMS (Method a, Table 4) TR = 1.17 min; m / z 1083.5 (M+H)+. Step 4: Synthesis of ((S)—5— (2 — (2 — bromoacetamido)acetamido)-6-((3-(4((2S,6aS,6bR,7S,8aS,8bS,10R,llaR,12aS,12bS)-8b-(2-((ditert-butoxyphosphoryl)oxy)acetyl)-2,6b-difluoro-7-hydroxy6a,8 tert-butyl a-dimethi1-4-oxo-2,4,6a,6b,7,8,8a,8b,lia,12,12a,12bdodecahydro-lH-naphtho[2',1':4,5]indene[1,2-d][l,3]dioxol-1011)benzyl)phenyl)amino)-6-oxohexyl)carbamate. To a solution of 2-bromoacetic acid (0.929 g, 6.68 mol) in DMF (35 mL), 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (1.653 g, 6.68 mol) was added, and the resulting mixture was stirred at room temperature for 1 hour. The product from Step 3 of Example 5 (3.5 g, 3.34 mol) was added, and the resulting mixture was stirred at room temperature for 2 hours. The reaction was confirmed by LC-MS to be complete. The reaction was diluted with DCM (100 mL), washed with aqueous HBr (1 M, 2 x 80 mL), aqueous NaHCO3 (60 mL), and brine (60 mL). The organic layer was dried with Na2SO4, filtered and concentrated under reduced pressure to provide the title compound (2 g, 51.2% yield) as a yellow oil. IF-2019-03555061-APN-ANP#INPI Page 68 of 93 LCMS (Method a, Table 4) TR - 1.32 min; m / z 1205.5 (M+H)+. Step 5: Dihydrogen phosphate synthesis of 2((2S, 6aS,6bR,7S,8aS,8bS,10R,llaR,12aS,12bS)-10-(4-(3-(3)6-amino-2-(2-(2bromoacetamido)acetamido)hexanido)benzyl)phenyl)-2,6-bdifluoro-h hydroxy-6a,8a-dimethyl~4-oxo1,2,4,6a,6b,7,8,8a,Ila,12,12a,12b-dodecahydro-8bHnaphtho[2',1':4,5]indeno[1,2-d][1,3]dioxyl-2-oxoethyl) To a solution of ((Ξ)-5-(2-aminoacetamido)-6-((3-(4((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-8b-(2-((ditert-butoxyphosphoryl) oxy)acetyl)-2,6b-difluoro-7-hydroxy6a,8a-dimethyl1-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12bdodecahydro-1H-naphtho[21,1':4,5]indene[1,2-d][1,3]dioxol-10yl)benzyl)phenyl)amino)-6-oxohexyl)carbamate of tert-butyl (Example 5, Step 3) (2 g, 1.661 rrmol) in DCM (10 mL) was mixed with TEA (5 mL, 64.9 rmol), and the reaction was stirred at ΔT for 40 min, after which LC-MS confirmed that the reaction was complete. The solvent was removed under reduced pressure, and the crude product was purified by HPLC Prep. The mobile phase was lyophilized directly to provide the title compound (550 mg, 96.9% purity, 32.3% yield) as a whitish solid. HPLC Prep Method: Instrument: Shimadzu LC-8A HPLC Prep; Column: Phenomenex Luna C18 200*40 mm*10 pm; Mobile Phase: A IF-2019-03555061-APN-ANP#INPI Página 69 de 93 para H20 (0.09% de TFA) y B para MeCN; Gradiente: B del 30% al 40% en 20 min; Caudal: 60 ml / min; Longitud de onda: 220 y 254 nm. ςΗ RMN: (DMSO-d6, 400 MHz) δ ppm 0.90 (s, 3 H) 1.19 1.41 (m, 2 H) 1.43 - 1.62 (m, 7 H) 1.64 - 1.77 (m, 3H) 1.84 (d a, J=14.55 Hz, 1 H) 1.95 - 2.07 (m, 1 H) 2.18 2.36 (m, 3 H) 2.65 - 2.78 (m, 3 H) 3.71 - 3.86 (m, 3H) 3.89 (s, 2 H) 3.93 (s, 2 H) 4.20 (d a, J=9.48 Hz, 1 H) 4.33 - 4.41 (m, 1 H) 4.59 (d ad, J=18.41, 8.05 Hz, 1 H) 4.81(d ad, J=18.52, 8.60 Hz, 1 H) 4.94 (d, J=4.63 Hz, 1 H)5.50 (s, 1 H) 5.54 - 5.76 (m, 1 H) 6.13 (s, 1 H) 6.29(dd, J=10.14, 1.32 Hz, 1 H) 6.95 (d, J=7.72 Hz, 1 H) 7.15 -7.28 (m, 4 H) 7.30 - 7.41 (m, 3 H) 7.51 (d a, J=7.94 Hz,1 H) 7.72 (s a, 3 H) 8.21 (d a, J=7.72 Hz, 1 H) 8.54 (t, J=5.62 Hz, 1 H) 9.93 (d a, J=2.65 Hz, 1 H) LCMS (Método a, Tabla 4) TR = 2.31 min. Example 6: Synthesis of (S)-2-( (2-(2-bromoacetamido)ethyl)amino)-N-((S)—1—((3-(4((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyaceti)-6a,8a-dimethyl-4-oxo 2,4,6a,6b,7,8,8a,8b,lia,12,12a,12b-dodecahydro-lHnaphtho[2',1':4,5]indene[1,2-d][1,3]dioxol-10yl)benzylphenyl)amino)-l-oxopropan-2-yl)propanamide. The product of Example 6 can be synthesized from the coupling of N-(2-((((9H-fluoren-9yl)methoxy)carbonyl)amino)ethyl)-N-(tert-butoxycarbonyl)-Lalanyl-L-alanine (the product of steps Si and S2 below) with the amino product of Example 2, followed by 70 IF-2019-03555061-APN-ANP#INPI Page 70 of 93 Steps S4-S6: (1) deprotection of Fmoc, (2) coupling with 2-bromoacetic acid and (3) deprotection of Boc. Fmoc = fluorenylmethyloxycarbonyl; Boc = tert-butoxycarbonyl. General protocol for conjugation with cistern An approximate 5-20 mg / mL solution of the desired antibody was prepared in PBS buffer, pH 6 - 7.4. A preferred reducing agent, such as TCEP, was diluted or dissolved in solvents such as H2O, DMSO, DMA, or DMF to obtain a solution with a concentration range of between 1 and 25 mM. The antibodies (anti-hTNF hlgGl (D2E7) or anti-mTNF mlgG2a (8C11; McRae BL et al. J Crohns Colitis 10(1):6976 (2016)) were then partially reduced by adding approximately 2–3.5 equivalents of reducing agent, mixing briefly, and incubating overnight at 0–4 °C. Tris buffer, pH 8–8.5 (20–50 mM), was then added, followed by the drug-connector in DMSO or DMA (less than 15% total), and the mixture was incubated for 2–3 hours at room temperature. Excess drug-connector and organic solvent were then removed by purification. The samples of IF-2019-03555061-APN-ANP#INPI Page 71 of 93 The purified ADCs were then analyzed by SEC, HIC, and reduced mass spectroscopy. ADC Analytical Procedures The ADCs were analyzed by anion exchange chromatography (AEC) or hydrophobic interaction chromatography (HIC) to determine the degree of conjugation and the purity of the ADCs. AEC. Approximately 20 pg of ADC were placed on an Ultimate 3000 Dual LC system (Thermo Scientific) equipped with a 4 x 250 mm Propac WAX-10 column (Tosoh Bioscience, cat. 054999). The column was equilibrated with 100% buffer A and eluted using a linear gradient from 100% buffer A to 100% buffer B in 18 minutes at a flow rate of 1.0 mL / min, where buffer A is 20 mM MES, pH 6.7, and buffer B is 20 mM MES, 500 M sodium chloride, pH 6.7. HIC. 20 pg of ADC were placed on an Ultimate 3000 Dual LC system (Thermo Scientific) equipped with a 4.6 x 35 mm butyl-NPR column (Tosoh Bioscience, cat. 14947). The column was equilibrated with 100% buffer A and eluted using a linear gradient from 100% buffer A to 100% buffer B in 12 minutes at a flow rate of 0.8 mL / min, where buffer A is 25 mM sodium phosphate, 1.5 M ammonium sulfate, pH 7.0, and buffer B is 25 mM sodium phosphate, 25% isopropanol, pH 7.0. SEC. The size distributions of the ADCs were analyzed by size exclusion SEC using an Ultimate 3000 Dual LC system (Thermo Scientific) equipped with a 7.8 x 300 mm TSK-gel 3000SWXL column (Tosoh 72 IF-2019-03555061-APN-ANP#INPI Page 72 of 93 Bioscience, cat. 08541). Approximately 20 pg of ADC were placed on the column and eluted for 17 min using an isocratic gradient of 100 mM sodium sulfate, 100 mM sodium phosphate, pH 6.8, at a flow rate of 1.0 mL / min. MS. Reduced samples (10 pL) were injected into an Agilent 6550 QTof LC / MS system via a temperature-controlled (5°C) CTC autosampler. Sample elution was performed on a Waters C-4, 3.5 pm, 300 Å, 2.1 x 50 mm dH HPLC column. The mobile phases were: A: 0.1% formic acid in water, and B: 0.1% formic acid in MeCN; the flow rate was 0.45 mL / min; and the column compartment was maintained at 40°C. The HPLC gradient is as shown in Table 5: Table 5 Time (min) %A %B 0 95 5 0.6 95 5 1.1 10 90 2.2 10 90 2.4 95 5 3.5 95 5 Example 7: Preparation of adalimumab conjugated with a glucocorticosteroid to provide an antibody-drug conjugate An adalimumab-BrAc-G1y-Glu steroid-PCp ADC having a population DAR of 4.0 was prepared by a two-step chemical process: reduction of 73 IF-2019-03555061-APN-ANP#INPI Page 73 of 93 adalimumab disulfide followed by alkylation (conjugation) with bromoacetamidoglycine-glutamic acid- One hundred milligrams of adalimumab were reduced to a concentration of 20 mg / mL with diphenylphosphinoacetic acid (2.9–3.0 eq) at 0 °C overnight. The partially reduced adalimumab was then conjugated with Example 4 (10 eq) in DMSO for 3 hours at room temperature. A first buffer change was performed in the conjugation mixture using 20 mM Tris, 50 mM NaCl buffer, pH 7.8, using multiple NAP 25 desalination columns. The desalted ADC solution was purified by AEC to provide the DAR4 components of the ADC. AEC chromatography method: Instrument: Akta Pure; Column: 2X Hitrap Q HP 5 mL; Mobile phase: A for 20 mM Tris buffer, pH 7.8; B for 20 mM Tris buffer, 1 M NaCl, pH 7.8; Gradient: B from 0% to 25% in 60 min; Flow rate: 5 ml / min; Wavelength: 280 and 214 nm. Referring to Fig. 1, which shows a chromatographic resolution of the resulting ADC preparation, the ADC is a heterogeneous mixture containing antibodies that have two drug-connector molecules attached (DAR2 peak), four drug-connector molecules attached (DAR4 peak), depending on the number of disulfide bridges IF-2019-03555061-APN-ANP#INPI Page 74 of 93 reduced intercatenaries. The AEC conditions used in Fig. 1 were as follows: The column was a Propac™ WAX-10, 4 x 250 mm (Thermo Fisher Scientific, cat. 054999) and the column temperature was 37°C. The wavelength was 280 nm, the run time was 18 minutes, the injection quantity was 20 pg, and the flow rate was 1.0 mL / minute. Mobile phase A: 20 mM MES, pH 6.7; Mobile phase B: 20 ​​mM MES, 500 mM NaCl, pH 6.7. Gradient profile (Table 6): Table 6 Time (min) Mobile Phase A (%) Mobile Phase B (%) 0 75 25 14 5 95 16 0 100 18 0 100 Figure 2 shows a decongested mass spectrum of the purified ADC. This ADC has four connective drug molecules conjugated to each antibody. The peak on the left has a molecular weight of 24284.74 Da, resulting from a connective drug bound to a single light chain. The peak on the right has a molecular weight of 51513.96 Da, resulting from a connective drug bound to a single heavy chain. The ADCs in Tables 7 and 8 were prepared according to the procedure described above. Table 7: Mouse anti-TNFα antibody-drug conjugates. X refers to murine anti-TNFα antibody 8C11 IF-2019-03555061-APN-ANP#INPI Page 75 of 93 ID No. Structure DAR ADC1 '5 σ o xv 7 / XX o í X* ' / 'ΆΓ —t*lx > ♦·> \ / X X'M--RCJ q ¿ OSA 7 o U < 0 / / A x X >-X o Τ'......νί oc 11.. A.. <...“Α τ4 a.... Yes JL Q!. ZjT H jT CHj Λ Xn 0 θ JL 3 H i. XX JL J r 0 » i HA HD q^ HO^O 4 ADC3 0 Quirai K CH TT °“XXhXch3 Η H 1 Π £ J An J y JL Λ N Ass. A. A-, , af □ 0 i H o < HΌ A HG bH 4 Table 8: Humanpharmaceutical anti-TNFa antibody conjugates. X refers to the human anti-TNFa antibody adalimumab Company ID to Structure FROM R IF-2019-03555061-APN-ANP#INPI Page 76 of 93 Generation of human and mouse transmembrane TNF-alpha GRE indicator cell lines To create a parental cell line, K562 cells were seeded in a 6-well plate (Costar: 3516) with 2 mL of complete growth medium (RPMI, 10% FBS, 1% L-glutamine, 1% sodium pyruvate, and 1% MEM NEAA) at a density of 500,000 cells per well for 24 hours at 37°C and 5% CO2. The following day, 1.5 pg of 77 IF-2019-03555061-APN-ANP#INPI Page 77 of 93 pGL4.36[Luc2P / MMTV / Hygro] (Promega: E316), 1-5 µg of pG14.75[hRLuc / CMV] (Promega: E639A) and 3 pL of PLUS reagent (Invitrogen: 10964-021) were diluted in 244 pL of Opti-MEM (Gibco: 31985-070) and incubated at room temperature for 15 minutes. The pGL4.36[Iuc2P / MMTV / Hygro] vector contains an MMTV (murine mammary tumor virus long terminal repeat) LTR that drives transcription of the luc2P luciferase reporter gene in response to activation of various nuclear receptors such as the glucocorticoid receptor and the androgen receptor. The pGL4.75[hRíuc / CMV] vector encodes the luciferase marker gene hRluc (Renilla reniformis) and is designed for high expression and reduced abnormal transcription. After incubation, the diluted DNA solution was pre-incubated with a 1:1 Lipofectamine LTX solution (Invitrogen: 94756) (13.2 pL + 256.8 pL of Opti-MEM) and incubated at room temperature for 25 minutes to form DNA-Lipofectamine LTX complexes. After incubation, 500 pL of lipofectamine DNA complexes were added directly to the well containing the cells. K562 cells were translocated for 24 h at 37 °C, 5% CO2. After incubation, the cells were washed with 3 mT of PBS and selected on complete growth medium containing 125 pg / mL of hygromycin B (Invitrogen: 10687-010) for two weeks. K562 pGL4.36[Luc2P / MMTV / Hygro]_pGL4.75[hRLuc / CMV] cells were produced.” To create a murine transmembrane TNFalpha indicator GRE cell line, parental cells, K562 pGL4.36[Luc2P / MMTV / Hygro]_pGL4.75[hRLuc / CMV], were seeded 78 IF-2019-03555061-APN-ANP#INPI Page 78 of 93 in a 6-well plate (Costar: 3516) with 2 ml of complete growth medium (RPMI, 10% FBS, 1% L-glutamine, 1% Na Pyruvate and 1% MEM NEAA) at a density of 500,000 cells per well for 24 hours at 37°, 5% CO2. The next day, 3 pg of unlabeled mouse TNF-encoding mFL__TNFa DNA (Origene: MC208048) and 3 pL of PLUS reagent (Tnvitrogen: 10964-021) were diluted in 244 pL of Opti-MEM (Gibco: 31985-07 0) and incubated at TA for 15 minutes. After 1a incubation, the diluted DNA solution was pre-incubated with a 1:1 Lipofectamine LTX solution (Tnvitrogen: 94756) (13.2 pL + 256.8 pL of Opti-MEM) and incubated at room temperature for 25 minutes to form DNA-Lipofectamine LTX complexes. After 1a incubation, 500 pL of the DNA-lipofectamine complexes were added directly to the well containing the cells. Parental K562 pGL4.36[Luc2P / MMTV / Hygro]_pGL4.75[hRLuc / CMV] cells were translocated for 24 h at 37 °C, 5% CO2.After incubation, the cells were washed with 3 mL of PBS and selected with complete growth medium containing 125 pg / mL of hygromycin B (Invitrogen: 10687-010) and 250 pg / mL of G418 (Gibco: 10131-027) for two weeks. FL-TNFa GRE mouse K562 cells (pGL4.36[luc2P / MMTV / Hygro]) were produced. To create a human transmembrane TNF-alpha GRE indicator cell line, parental cells, K562 pGL4.36[Luc2P / MMTV / Hygro]_pGL4.75[hRLuc / CMV], were transjected with the pcDNA3.1(~) plasmid construct of the hTNF delta 1-12 C-Myc plasmid. This plasmid is pcDNA 3.1 (Thermofisher cat# V79020) encoding TNF. IF-2019-03555061-APN-ANP#INPI Page 79 of 93 transmembrane resistant to TNF (i.e., the SEQ ID NO:1 that lacks amino acids 77-88). (See Perez C et al. Cell 63 (2): 251-8 ¢1990) which studies transmembrane TNF resistant to TNF). These cell lines were subsequently used in the TNF-alpha indicator assays described in the following examples. Anti-TNF-alpha immunoconjugate activity in transmembrane TNF-alpha GRE indicator assays Parental K562 (pGL4.36[luc2P / MMTV / Hygro]) GRE cells and K562 mFL-TNF-α or hTNF delta 1-12 (pGL4.36[luc2P / MMTV / Hygro]) GRE cells were seeded in 96-well treated white plates for tissue culture (Costar: 3917) at a density of 50,000 cells per well in 50 pL of assay medium (RPMI, 1% CSFBS, 1% L-glutamine, 1% Na-pyruvate, and 1% MEIA). Cells were treated with 25 pL of murine or human anti-TNF-α antibody-drug conjugates serially diluted 3-fold in assay medium, spheroid compound, or medro alone and incubated for 48 hours at 37 °C, 5% CO2. After 48 hours of incubation, cells were treated with 75 pL of Dual-Glo Luciferase Assay System (PromegaE2 92 0) for 10 min and analyzed for luminescence using the Microbeta (PerkinElmer).The data were analyzed using a four-parameter curve fitting to generate CE50 values. The maximum % deactivation was normalized to 100 nM dexamethasone. The results for the use of the murine TNF-alpha cell line are shown below in Table 9 and the results for the use of the human TNF-alpha cell line are shown in Table 80. IF-2019-03555061-APN-ANP#INPI Page 80 of 93 below in Table 10. In Table 9 below, A refers to 8C11. In Table 10 below, A refers to adalimumab. The percentage (%) of monomer was determined by SEC as previously described (see ADC analytical procedures). Table 9: In vitro activity of murine anti-TNFa antibody-drug conjugate in a GRE indicator assay mouse transmembrane TNFα ADC % of mTNFa GRE CE50 monomer (pg / mL) mTNFa GRE (% max) K562 GRE CE50 (pg / mL) K562 GRE (% max) ADC1 99.9 0.06 150 >50 63 ADC2 99.3 0.39 164 >50 72 ADC3 100 0.04 104 3.9 84 Table 10: In vitro activity of human anti-TNFα antibody-drug conjugate in a human transmembrane TNFα GRE indicator assay ADC % of hTNFa GRE CE50 monomer (pg / mL) hTNFa GRE (% max) K562 GRE CE50 (pg / mL) K562 GRE (% max) ADC 4 99.9 0.03 118 >50 63 ADC 5 100 0.03 126 >50 28 ADC 6 100 0.05 97 >50 83 IF-2019-03555061-APN-ANP#INPI Page 81 of 93 Activity of anti-hTNF alpha immunoconjugates in the cytokine release assay in lipopolysaccharide-stimulated human PBMCs Primary human peripheral blood mononuclear cells (PBMCs) were acquired from Biological Specialty Corporation (cat# 214-00-10), washed in 50 mL of PBS, resuspended in FBS with 5% DMSO, divided into aliquots, and cryopreserved in liquid nitrogen until use. The PBMCs were thawed, resuspended in RPMI medium supplemented with 2% FBS and 1% penicillin / streptomycin, and seeded in a cell assay plate (Costar #3799). The cells were then incubated at 37°C and 5% CO2 for 4 hours, varying the concentration of anti-TNF ADC. The cells were then stimulated overnight with 100 ng / mL of LPS. The next day, the plate was centrifuged for five minutes at 1000 rpm, and 100 pL of supernatant medium were directly transferred to an additional 96-well plate and analyzed for IL-6 (MSB, #K151AKB) and IL-1 beta (MSD, #K151AGB) concentrations.Dose-response data were fitted to a sigmoidal curve using nonlinear regression, and IC50 values ​​were calculated using GraphPad 5.0 (GraphPad Software, Inc.). The results shown in Table 11 demonstrate that anti-TNF ADCs have potent activity in inhibiting the release of the proinflammatory cytokines IL-6 and IL-1β from activated primary immune cells. IF-2019-03555061-APN-ANP#INPI Page 82 of 93 Table 11. In vitro activity of anti-TNF-steroid ADCs in the cytokine release assay in stimulated PBMC cells ADC CI50 of IL-1 beta. (ng / mL) IL-6 IC50 (ng / mL) ADC4 44 265 Activity of an anti-mTNF-alpha immunoconjugate in the contact hypersensitivity model Anti-steroid TNFα-ADCs were evaluated in an acute contact hypersensitivity model, which involved stimulating acute skin inflammation using the delayed-type hypersensitivity (DTH) response (triggered by T lymphocytes) through the application of a sensitizing agent (fluorescein isothiocyanate (FITC)). The efficacy of the anti-steroid TNFα-ADCs was measured by their ability to reduce ear inflammation. The steroidal biomarkers corticosterone and type 1 procollagen N-terminal propeptide (P1NP) were included as readouts to assess the potential impact of anti-steroid TNFα-ADC treatment on the hypothalamic-pituitary-adrenal (HPA) axis and bone turnover, respectively. Ear inflammation On day 0, mice were anesthetized with general anesthesia and their abdomens were shaved. Using a micropipette, mice were sensitized by epicutaneous application of 400 pL of FITC solution (1.5% in 1:1 acetone:DBP solution) to the abdomen. Six days later, a dose of vehicle or therapeutic agent was administered to the mice 1 hour before exposure. IF-2019-03555061-APN-ANP#INPI Page 83 of 93 Ear to FITC. To expose the ear, mice were anesthetized with general anesthesia and their right ear was exposed to 20 pL of FITC. Twenty-four hours after exposure, the mice were anesthetized with general anesthesia and their ear thickness was measured with calipers. The difference between exposed and unexposed ears was calculated. Seventy-two hours after ear exposure, ACTH was injected into the mice at a concentration of Impk IP, and they bled to death 30 min after ACTH injection. Plasma was collected and analyzed for levels of P1NP, corticosterone, free spheroid, and large molecules. Quantification of free steroid and endogenous corticosterone released The steroid calibration curve was prepared in mouse plasma with final concentrations ranging from 0.03 nM to 0.1 pM at eight different concentration levels. The corticosterone calibration curve, with a final corticosterone concentration ranging from 0.3 nM to 1 pM, was prepared in 70 mg / mL bovine serum albumin solution in PBS buffer. A 160 pL solution of MeCN with 0.1% formic acid was added to 40 pL of study plasma samples or calibration standards. The supernatants were diluted with distilled water, and 30 pL of the final sample solution was injected for LC / MS analysis. The quantification of free steroid and released corticosterone was performed on an AB Sciex 5500 triple quadrupole mass spectrometer connected to a Shimadzu AC20 HPLC system interconnected with an electronebulization ionization source operating in mode 84 IF-2019-03555061-APN-ANP#INPI Page 84 of 93 positive. An XBridge BEH C18, 2.1 x 30 column was used; mm, 3.5 pm, from Waters for chromatographic separation. Mobile phase A was formic acid at 0.1-s in water. Milli HPLC The mobile phase B was 0.1% formic acid in MeCN. A linear gradient from 2% to 98% mobile phase B was applied from 0.6 to 1.2 min. The total run time was 2.6 min with a flow rate of 0.8 mL / min. The mass spectrometer operated in positive MRM mode at a source temperature of 700°C. Quantification of P1NP in plasma P1NP quantification in plasma was performed on an LCMS platform based on trypsin-conq protein digestion. Partial precipitation of plasma samples was induced and completely reduced by adding a 0.1 M MeCN / ammonium bicarbonate / DTT mixture. The The supernatant was alkylated by adding iodoacetic acid. The alkylated proteins were digested with trypsin and the resulting tryptic peptides were analyzed by LC-MS. The calibration curve was generated using a peptide; A synthetic tryptic was added to horse serum (matrix, non-interfering substitute). A peptide was used; stable isotope-labeled flanking (3-6 amino acid extension at both ends of the tryptic peptide) as an internal standard added to the precipitation mixture of MeCN / DTT proteins to normalize both the efficiency of, ! digestion as well as LCMS injection. A Columnex Chromenta BB-C18 column, 2.1 x 150 mm, 5 pm, was used for the chromatographic separation. Mobile phase A was 0.1% formic acid in Milli-Q HPLC water, and mobile phase B was 0.1% formic acid in MeCN. 85 Ί IF-2019-03555061-APN-ANP#INPI Page 85 of 93 applied a linear gradient from 2% mobile phase B to 65% mobile phase B from 0.6 to 3 min. The total run time was 8 min with a flow rate of 0.45 mL / min. A Sciex 4000Q AB trap mass spectrometer in positive MRM mode was used to quantify the P1NP peptides, at a source temperature of 700°C. Results The results are shown below in Table 12: Table 12: Comparison of antimTNF alpha-steroid ADC activity on ear inflammation and steroidal biomarkers in the CHS model of inflammation ADC Ear inflammation (% inhibition in lOmpk ± SEM) P1NP (% inhibition in lOmpk ± SEM) Corticosterone (% inhibition in lOmpk ± SEM) ADC1 82.8 ± 2.3 32.6 ± 3.8 4.7 ± 10.5 Anti-mTNF-alpha immunoconjugate activity in collagen-induced arthritis The ability of an anti-mTNFa-steroid ADC (ADC1) to affect disease was evaluated in the arthritis model for collagen-induced arthritis (CIA). In these experiments, male DBA / 1J mice were obtained from Jackson Labs (Bar Harbor, ME). The mice were used at 6 to 8 weeks of age. All animals were maintained at constant temperature and humidity on a 12-hour light / dark cycle and fed with IF-2019-03555061-APN-ANP#INPI Page 86 of 93. Rodent food (Lab Diet 5010 PharmaServ, Framingham, MA) and water were provided ad libitum. AbbVie is accredited by AAALAC (International Association for Evaluation and Accreditation of Laboratory Animal Care), and all procedures were authorized by the Institutional Animal Care and Use Committee (IACUC) and supervised by a veterinarian. Body weight and condition were monitored, and animals were euthanized if they experienced a weight loss of >20%. Male DBA / J mice were immunized intradermally (id) at the base of the tail with 100 pL of emulsion containing 100 pg of bovine type II collagen (MD Biosciences) dissolved in 0.1 N acetic acid and 200 pg of heat-inactivated Mycobacterium tuberculosis H37Ra (Complete Adjuvant from Freund, Difeo, Laurence, KS). Twenty-one days post-collagen immunization, mice received a 1-mg IP booster dose of Zymosan A (Sigma, St. Louis, MO) in PBS. After the booster, mice were monitored 3 to 5 times per week for arthritis. Hind legs were assessed for limb swelling using a Dyer spring caliper (Dyer 310-115). Mice were enrolled in the study between days 24 and 28 upon showing the first clinical signs of the disease and were divided into groups of equivalent arthritic severity. Early therapeutic treatment began at the time of enrollment in the study. The animals were administered a single intraperitoneal (ip) dose of anti-mTNF mAb (high dose) or ADC 87 IF-2019-03555061-APN-ANP#INPI Page 87 of 93 of anti-mTNF steroid (high and low dose - mpk) in 0.9% saline solution. Blood for antibody exposure was collected via tail incision at 24 and 72 hours post-dose. Legs were collected at terminal time for histopathology. Blood was collected at terminal time via cardiac puncture for complete blood counts (Sysmex XT-20001V). Statistical significance was determined by ANOVA. The results are shown in Fig. 3 and demonstrate that a single dose of anti-TNFα-steroid ADC can show a prolonged duration of action through an improvement of paw inflammation for ~28 days compared to the anti-TNFα mAb or vehicle alone. ADC stability in plasma Although hydrolysis has been used to increase the in vivo stability of maleimide-based connectors, it generally requires exposure to basic pH, which can lead to antibody modifications (e.g., deamidation), increased heterogeneity, reduced yield, and the like. (Shen et al., Nature Biotechnology 30:184–189 (2012) (hydrolyzing maleimides avoids premature drug release and systemic drug exposure); Strop et al., Chemistry & Biology 20(2):161–167 (2013) (a similar study); Tumey et al., Bioconjugate Chem 25(10):1871–1880 (2014) (use of a proximal PEG chain to allow ring hydrolysis under basic conditions); Lyon et al., Nature Biotechnology 32:1059–1062 (2014) (processes to allow the creation of hydrolyzed succinimide); Christie et al., J Control Release 220(PtB):660–70 (December 28, 2015) 88 IF-2019-03555061-APN-ANP#INPI Page 88 of 93 (use of N-arylmaleimides to allow hydrolysis of the succinimide ring under basic conditions); Dovgan et al., Scientific Reports 6:1 (2016) (use of 2(maleimidomethyl)-1,3-dioxane to allow hydrolysis of the ring under slightly basic conditions for a prolonged period of time); and J Pharm Sci 2013: 102 (6) 1712-1723 (dependence of asparagine deamidation on buffer type, pH, and temperature). In contrast, typical conditions for conjugation using bromoacetamide are completed in a few hours, compared to several days for maleimide (conjugation and subsequent ring hydrolysis at basic pH). Furthermore, the 2-mercaptoacetamide formed during the reaction of the cistern with bromoacetamide is not susceptible to undergoing the reverse Michael reaction and allows a stable binding of the connector to the antibody as shown below in Table 13 for ADC4. Table 13 % of SM released from ADC in plasma after 4d at 37°C PBS1X buffer Mouse Rat Macaque Human BLC BLC BLC BLC BLC BLQ - Below the level of quantification ADC stability in solution To assess long-term stability, a biological product is subjected to an accelerated stress test. The protocol for this test is 100 mg / mL of the product. IF-2019-03555061-APN-ANP#INPI Page 89 of 93 biological in 15 mM histidine at 40 °C for 21 days. When ADC4 was subjected to this test, the percentage increase in aggregate was <5% compared to ADC203 of US Patent Application Publication No. 2018 / 012600, published May 10, 2018, which experienced an 18% increase (Table 14). This demonstrates the enhanced properties conferred to ADC4 by the Gly-Glu connector with the phosphate-type prodrug payload. ADC203 of US2018 / 0126000 is: • j Ί where n=4 and A refers to the human anti-TNFα antibody adalimumab. Table 14 ADC Monomer loss at 5°C for 21 days Monomer loss at 25°C for 21 days Monomer loss at 40°C for 21 days ADC203 1.43% 2.26% 17.56% ADC4 <0.5% <0.5% 3.46% IF-2019-03555061-APN-ANP#INPI Page 90 of 93 An additional benefit of the phosphate-type prodrug is that it allows the use of anion-exchange chromatography for DAR purification. This results in improved peak resolution compared to interaction chromatography, resulting in higher yields of hydrophobic ACD purified by In the formulation buffer, the hydrolyzed succinimide ring of ADC203 is in equilibrium with the closed-ring form. The closed-ring form is susceptible to undergoing the Michael reaction, resulting in subsequent loss of the drug-connector in vivo in macaques (Fig. 4). Equilibrium in Formulation buffer Under these conditions, the union will reform Ring open mAb mAb storage liquid Ring closed succinimide in the closed conformation more than 5% at 5 nominal open °C and more than 15% at 25 °C after 6 months (Table 15). Table 15 % of closed ring succinimide Time / Temp Light chain Heavy chain IF-2019-03555061-APN-ANP#INPI Page 91 of 93 3 Months / 5°C 2.2 4.6 6 Months / 5°C 3.2 6.9 3 Months / 25°C 10.1 13.3 6 Months / 25°C 16.6 24.1 3 Months / 40°C 2 6.2 33.7 3 Months / 40°C 26.8 39.0 It should be noted that the Detailed Description section, and not the Summary and Abstract sections, is intended to be used for interpreting the claims. The Summary and Abstract sections set forth one or more, but not all, embodiments of the present disclosure as envisioned by the inventor(s) and are therefore not intended to limit the present disclosure or the accompanying claims in any way. This disclosure has been described above with the help of functional building blocks that illustrate the implementation of specific functions and their relationships. The boundaries of these functional building blocks have been arbitrarily defined herein for the sake of clarity. Alternative boundaries may be defined provided that the specified functions and their relationships are properly realized. The above description of the specific achievements will so fully reveal the general nature of the disclosure that others may, applying knowledge within the competences of the technique, 92 IF-2019-03555061-APN-ANP#INPI Page 92 of 93. These specific embodiments may be easily modified and / or adapted for various applications without excessive experimentation and without departing from the general concept of this disclosure. Therefore, it is intended that such adaptations and modifications remain within the meaning and range of equivalents of the embodiments disclosed, based on the teachings and guidelines presented herein. It is understood that the phraseology or terminology herein is descriptive and not exhaustive, and that the terminology or phraseology of this descriptive document should be interpreted by the person skilled in the art in light of the teachings and guidelines. The scope and extent of this disclosure should not be limited by any of the illustrative embodiments described above, but should be defined solely in accordance with the following claims and their equivalents. INCORPORATION BY REFERENCE All publications, including published patents and applications, referenced in the Detailed Description are incorporated herein in their entirety by reference. It is hereby stated that, as of this date, the best method known to the applicant for putting the aforementioned invention into practice is the one that is clear from the present description of the invention. IF-2019-03555061-APN-ANP#INPI Page 93 of 93 Argentine Republic - National Executive Branch 2019 - Year of Exports Additional Signature Sheet Graphic Report Number: IF-2019-03555061 -APN-ANP#INPI CITY OF BUENOS AIRES Friday, January 18, 2019 Reference: 20180103524 The document was imported by the GEDO system with a total of 93 page(s). Digitally signed by GESTION DOCUMENTAL ELECTRONICA - GDE DN: cn=ELECTRONIC DOCUMENTARY MANAGEMENT - GDE, c=AR, o=MINISTRY OF MODERNIZATION, ou=SECRETARY OF ADMINISTRATIVE MODERNIZATION, serialNumber=CUIT 30715117564 Date: 2019.01.18 17:28:56 -03'00' Darío Julio Martin Mayares Administrative Advisor National Patent Administration National Institute of Industrial Property Digitally signed by GESTION DOCUMENTAL ELECTRONICA GDE DN: cn=GEST10N ELECTRONIC DOCUMENTARY - GDE, c=AR, o=MINISTRY OF MODERNIZATION, ou=SECRETARY OF ADMINISTRATIVE MODERNIZATION, serialNumber=CUIT 30715117564 Date: 2019.01.18 17:28:57 -03'00'

Claims

1. An antibody-drug conjugate characterized in that it comprises: (a) an anti-TNFα antibody comprising a heavy chain as shown in SEQ ID NO: 3 and a light chain as shown in SEQ ID NO: 4; and (b) a glucocorticoid receptor agonist comprising a radical represented by the formula: (follows formula 1) wherein the antibody is conjugated to the glucocorticoid receptor agonist via a linker represented by the formula: (follows formula 2). Five claims follow.