Human tumor necrosis factor alpha antibody glucocorticoid conjugates

Abstract

The present disclosure provides human tumor necrosis factor alpha antibody glucocorticoid receptor agonist conjugates and methods of treating autoimmune and inflammatory diseases using the conjugates.

Description

Technical Field

[0001] The present disclosure provides a human tumor necrosis factor alpha antibody glucocorticoid receptor agonist conjugate, a method of using the conjugate to treat autoimmune and inflammatory diseases (such as rheumatoid arthritis, psoriatic arthritis, Crohn's disease, ulcerative colitis, plaque psoriasis and ankylosing spondylitis), a method for preparing the conjugate, and a pharmaceutical composition comprising the human TNF alpha antibody glucocorticoid conjugate. Background Art

[0002] Rheumatoid arthritis (RA) is a debilitating chronic autoimmune disease that attacks joints, most commonly the joints in the hands, wrists, and knees, and often many joints at once. In RA, the membrane covering the joints becomes inflamed, causing damage to the joint tissue, which can lead to stiffness, swelling, instability (lack of balance), deformity, and chronic pain. Current treatments use, for example, nonsteroidal anti-inflammatory drugs (NSAIDS), corticosteroids, anti-rheumatic drugs (DMARDs) to alleviate the condition, antibody therapeutics (such as methotrexate, tofacitinib, etanercept, adalimumab, infliximab, golimumab, and certolizumab pegol). The disadvantages of this type of treatment include, for example, the generation of non-target toxicity and anti-drug antibodies.

[0003] Anti-drug antibodies can be non-neutralizing antibodies that bind to TNFα simultaneously with the anti-TNFα antibody therapeutic, or they can be neutralizing antibodies that reduce the effective concentration of anti-TNFα therapeutic antibodies in serum and / or compete with TNFα for antigen binding sites (paratopes) to inhibit the working mechanism of anti-TNFα therapeutic antibodies. (van Schie KA et al., Annals of the Rheumatic Diseases, 2015, 74: 311-314). For example, studies have shown that more than ninety percent of anti-TNFα drug antibodies are neutralizing and may cross-react with other anti-TNFα antibody therapeutics. (van Schie KA et al., 2015). Therefore, in some cases, it is reported that patients who develop anti-drug antibodies against anti-TNFα antibodies have reduced clinical responses and / or adverse events to these therapeutics during drug administration or within the first day after drug administration, such as infusion-related reactions characterized by symptoms such as fever, itching, bronchospasm or cardiovascular failure (Atiqi, S., Front Immunol., 2020, 26 (11): 312). Thus, there remains a significant need for new agents that provide improved, effective treatments of inflammatory and / or autoimmune diseases, such as RA, and that minimize or eliminate the shortcomings of currently approved treatments.

[0004] WO2017 / 210471 discloses certain glucocorticoid receptor agonists (GC), antibodies and their immunoconjugates. WO2018 / 089373 discloses new steroids, protein conjugates thereof and methods for treating diseases, disorders and conditions, the method comprising administering steroids and conjugates. To date, there are no approved human TNFα antibody GC conjugates for treating diseases. Summary of the invention

[0005] The present disclosure provides certain novel human TNFα antibody GC conjugates, wherein the antibody is bound to human TNFα. The present disclosure also provides compositions comprising novel anti-human TNFα antibody GC conjugates and methods of using such anti-human TNFα antibody GC conjugates and compositions thereof. In addition, the present disclosure provides certain novel anti-human TNFα antibody GC conjugates that can be used to treat autoimmune diseases and inflammatory diseases (such as rheumatoid arthritis). The present disclosure also provides certain novel anti-human TNFα antibody GC conjugates, which can be used to treat autoimmune diseases and inflammatory diseases in patients who have developed anti-drug antibodies to other anti-TNFα therapeutics (e.g., adalimumab). Certain anti-human TNFα antibody GC conjugates disclosed herein present good developability characteristics, such as good physico-chemical properties (e.g., low viscosity or aggregation, good thermal stability), to facilitate development, manufacturing and formulation. Thus, certain anti-human TNFα antibody GC conjugates provided herein have one or more of the following properties: 1) bind to human TNFα with desired potency, 2) bind to human membrane TNFα and be internalized into cells, 3) bind to rhesus monkey and / or canine TNFα with desired potency, 4) inhibit apoptosis induced by soluble and membrane human TNFα, 5) modulate both TNFR and glucocorticoid receptor-mediated cytokine expression in vitro (e.g., inhibit IL-13, IL-6, GM-CSF, induce IL-10), 6) inhibit TNFR-mediated cell proliferation in vivo, and 7) inhibit TNFR-mediated cell death. cytokine expression (e.g., CXCL1), 7) induce ADCC activity, 8) show low to no cross-reactivity to anti-drug antibodies directed against other anti-TNFα therapeutics (e.g., adalimumab) in vivo and in vitro, 9) significantly inhibit tissue and / or polyarthritis joint inflammation in vivo, 10) significantly inhibit joint inflammation in adalimumab refractory mice in vivo, or 11) have good developability characteristics, such as acceptable viscosity, solubility and aggregation, good stability and / or acceptable pharmacokinetic characteristics to facilitate development, manufacturing and / or formulation.

[0006] Thus, in one embodiment, the present disclosure provides a conjugate of Formula I:

[0007]

[0008] Wherein Ab is an antibody that binds to human tumor necrosis factor alpha ("anti-human TNFα antibody"), wherein Ab comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein:

[0009] HCDR1 comprises SEQ ID NO: 1 or 22;

[0010] HCDR2 comprises SEQ ID NO: 2 or 23;

[0011] HCDR3 comprises SEQ ID NO: 3, 13 or 30;

[0012] LCDR1 comprises SEQ ID NO: 4, 14, 31 or 43;

[0013] LCDR2 comprises SEQ ID NO:5; and

[0014] LCDR3 comprises SEQ ID NO: 6, 15, 32 or 44;

[0015] yes:

[0016]

[0017] And n is 1-5.

[0018] In further embodiments, the present disclosure provides a conjugate of Formula Ia:

[0019]

[0020] Wherein Ab is an antibody that binds to human TNFα, wherein Ab comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH comprises heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3, and VL comprises light chain complementary determining regions LCDR1, LCDR2 and LCDR3, wherein:

[0021] HCDR1 comprises SEQ ID NO: 1 or 22;

[0022] HCDR2 comprises SEQ ID NO: 2 or 23;

[0023] HCDR3 comprises SEQ ID NO: 3, 13 or 30;

[0024] LCDR1 comprises SEQ ID NO: 4, 14, 31 or 43;

[0025] LCDR2 comprises SEQ ID NO:5; and

[0026] LCDR3 comprises SEQ ID NO: 6, 15, 32 or 44;

[0027] yes:

[0028]

[0029] And n is 1-5.

[0030] In further embodiments, the present disclosure provides a conjugate of Formula Ib:

[0031]

[0032] Wherein Ab is an antibody that binds to human TNFα, wherein Ab comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH comprises heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3, and VL comprises light chain complementary determining regions LCDR1, LCDR2 and LCDR3, wherein:

[0033] HCDR1 comprises SEQ ID NO: 1 or 22;

[0034] HCDR2 comprises SEQ ID NO: 2 or 23;

[0035] HCDR3 comprises SEQ ID NO: 3, 13 or 30;

[0036] LCDR1 comprises SEQ ID NO: 4, 14, 31 or 43;

[0037] LCDR2 comprises SEQ ID NO:5; and

[0038] LCDR3 comprises SEQ ID NO: 6, 15, 32 or 44;

[0039] yes:

[0040]

[0041] And n is 1-5.

[0042] In further embodiments, the present disclosure provides a conjugate of Formula Ic:

[0043]

[0044] Wherein Ab is an antibody that binds to human TNFα, wherein Ab comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH comprises heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3, and VL comprises light chain complementary determining regions LCDR1, LCDR2 and LCDR3, wherein:

[0045] HCDR1 comprises SEQ ID NO: 1 or 22;

[0046] HCDR2 comprises SEQ ID NO: 2 or 23;

[0047] HCDR3 comprises SEQ ID NO: 3, 13 or 30;

[0048] LCDR1 comprises SEQ ID NO: 4, 14, 31 or 43;

[0049] LCDR2 comprises SEQ ID NO:5; and

[0050] LCDR3 comprises SEQ ID NO: 6, 15, 32 or 44;

[0051] yes:

[0052]

[0053]

[0054] And n is 1-5.

[0055] In further embodiments, the present disclosure provides a conjugate of Formula Id:

[0056]

[0057] Wherein Ab is an antibody that binds to human TNFα, wherein Ab comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH comprises heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3, and VL comprises light chain complementary determining regions LCDR1, LCDR2 and LCDR3, wherein:

[0058] HCDR1 comprises SEQ ID NO: 1 or 22;

[0059] HCDR2 comprises SEQ ID NO: 2 or 23;

[0060] HCDR3 comprises SEQ ID NO: 3, 13 or 30;

[0061] LCDR1 comprises SEQ ID NO: 4, 14, 31 or 43;

[0062] LCDR2 comprises SEQ ID NO:5; and

[0063] LCDR3 comprises SEQ ID NO: 6, 15, 32 or 44;

[0064] yes:

[0065]

[0066] And n is 1-5.

[0067] In further embodiments, the present disclosure provides a conjugate of Formula Ie:

[0068]

[0069] Wherein Ab is an antibody that binds to human TNFα, wherein Ab comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH comprises heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3, and VL comprises light chain complementary determining regions LCDR1, LCDR2 and LCDR3, wherein:

[0070] HCDR1 comprises SEQ ID NO: 1 or 22;

[0071] HCDR2 comprises SEQ ID NO: 2 or 23;

[0072] HCDR3 comprises SEQ ID NO: 3, 13 or 30;

[0073] LCDR1 comprises SEQ ID NO: 4, 14, 31 or 43;

[0074] LCDR2 comprises SEQ ID NO:5; and

[0075] LCDR3 comprises SEQ ID NO: 6, 15, 32 or 44;

[0076] yes:

[0077]

[0078] And n is 1-5.

[0079] In further embodiments, the present disclosure provides a conjugate of Formula If:

[0080]

[0081] Wherein Ab is an antibody that binds to human TNFα, wherein Ab comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH comprises heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3, and VL comprises light chain complementary determining regions LCDR1, LCDR2 and LCDR3, wherein:

[0082] HCDR1 comprises SEQ ID NO: 1 or 22;

[0083] HCDR2 comprises SEQ ID NO: 2 or 23;

[0084] HCDR3 comprises SEQ ID NO: 3, 13 or 30;

[0085] LCDR1 comprises SEQ ID NO: 4, 14, 31 or 43;

[0086] LCDR2 comprises SEQ ID NO:5; and

[0087] LCDR3 comprises SEQ ID NO: 6, 15, 32 or 44;

[0088] yes:

[0089]

[0090] And n is 1-5.

[0091] In some embodiments, the conjugate of Formula I, wherein the antibody ("Ab") that binds to human TNFα is Abl, wherein Abl comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH comprises heavy chain complementary determining regions HCDR1, HCDR2, and HCDR3, and VL comprises light chain complementary determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 2, HCDR3 comprises SEQ ID NO: 3, LCDR1 comprises SEQ ID NO: 4, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 6. In some embodiments, Abl comprises a VH comprising SEQ ID NO: 7 and a VL comprising SEQ ID NO: 8. In some embodiments, Abl comprises a heavy chain (HC) comprising SEQ ID NO: 9 and a light chain (LC) comprising SEQ ID NO: 10.

[0092] In some embodiments, the conjugate of Formula I, wherein the antibody ("Ab") that binds to human TNFα is Ab2, wherein Ab2 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH comprises heavy chain complementary determining regions HCDR1, HCDR2, and HCDR3, and VL comprises light chain complementary determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 2, HCDR3 comprises SEQ ID NO: 13, LCDR1 comprises SEQ ID NO: 14, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 15. In some embodiments, Ab2 comprises a VH comprising SEQ ID NO: 16 and a VL comprising SEQ ID NO: 17. In some embodiments, Ab2 comprises a heavy chain (HC) comprising SEQ ID NO: 18 and a light chain (LC) comprising SEQ ID NO: 19.

[0093] In some embodiments, the conjugate of Formula I, wherein the antibody ("Ab") that binds to human TNFα is Ab3, wherein Ab3 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH comprises heavy chain complementary determining regions HCDR1, HCDR2, and HCDR3, and VL comprises light chain complementary determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO: 22, HCDR2 comprises SEQ ID NO: 23, HCDR3 comprises SEQ ID NO: 13, LCDR1 comprises SEQ ID NO: 4, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 6. In some embodiments, Ab3 comprises a VH comprising SEQ ID NO: 24 and a VL comprising SEQ ID NO: 8. In some embodiments, Ab3 comprises a heavy chain (HC) comprising SEQ ID NO: 25 and a light chain (LC) comprising SEQ ID NO: 10.

[0094] In some embodiments, the conjugate of Formula I, wherein the antibody ("Ab") that binds to human TNFα is Ab4, wherein Ab4 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH comprises heavy chain complementary determining regions HCDR1, HCDR2, and HCDR3, and VL comprises light chain complementary determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO: 22, HCDR2 comprises SEQ ID NO: 23, HCDR3 comprises SEQ ID NO: 13, LCDR1 comprises SEQ ID NO: 14, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 15. In some embodiments, Ab4 comprises a VH comprising SEQ ID NO: 24 and a VL comprising SEQ ID NO: 17. In some embodiments, Ab4 comprises a heavy chain (HC) comprising SEQ ID NO: 25 and a light chain (LC) comprising SEQ ID NO: 19.

[0095] In some embodiments, the conjugate of Formula I, wherein the antibody ("Ab") that binds to human TNFα is Ab5, wherein Ab5 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH comprises heavy chain complementary determining regions HCDR1, HCDR2, and HCDR3, and VL comprises light chain complementary determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO: 22, HCDR2 comprises SEQ ID NO: 23, HCDR3 comprises SEQ ID NO: 13, LCDR1 comprises SEQ ID NO: 14, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 6. In some embodiments, Ab5 comprises a VH comprising SEQ ID NO: 24 and a VL comprising SEQ ID NO: 27. In some embodiments, Ab5 comprises a heavy chain (HC) comprising SEQ ID NO: 25 and a light chain (LC) comprising SEQ ID NO: 28.

[0096] In some embodiments, the conjugate of Formula I, wherein the antibody ("Ab") that binds to human TNFα is Ab6, wherein Ab6 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH comprises heavy chain complementary determining regions HCDR1, HCDR2, and HCDR3, and VL comprises light chain complementary determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 2, HCDR3 comprises SEQ ID NO: 30, LCDR1 comprises SEQ ID NO: 31, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 32. In some embodiments, Ab6 comprises a VH comprising SEQ ID NO: 33 and a VL comprising SEQ ID NO: 34. In some embodiments, Ab6 comprises a heavy chain (HC) comprising SEQ ID NO: 35 and a light chain (LC) comprising SEQ ID NO: 36.

[0097] In some embodiments, the conjugate of Formula I, wherein the antibody ("Ab") that binds to human TNFα comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH comprises heavy chain complementary determining regions HCDR1, HCDR2, and HCDR3, and VL comprises light chain complementary determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO: 22, HCDR2 comprises SEQ ID NO: 23, HCDR3 comprises SEQ ID NO: 13, LCDR1 comprises SEQ ID NO: 14, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 44. In some embodiments, SEQ ID NO: 44 comprises amino acid residues QQYDXaa5LPLT, wherein Xaa5 of SEQ ID NO: 44 is asparagine or lysine.

[0098] In some embodiments, the conjugate of Formula I, wherein the antibody ("Ab") that binds to human TNFα comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH comprises heavy chain complementary determining regions HCDR1, HCDR2, and HCDR3, and VL comprises light chain complementary determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO: 22, HCDR2 comprises SEQ ID NO: 23, HCDR3 comprises SEQ ID NO: 13, LCDR1 comprises SEQ ID NO: 43, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 6. In some embodiments, SEQ ID NO: 43 comprises amino acid residues QASQGIXaa7NYLN, wherein Xaa7 of SEQ ID NO: 43 is serine or arginine.

[0099] In some embodiments, the conjugate of Formula I, wherein the anti-human TNFα antibody is a fully human antibody. In additional embodiments, the anti-human TNFα antibody has a human IgG1 isotype.

[0100] In some embodiments of the present disclosure, the conjugate of Formula I, wherein the anti-human TNFα antibody has a modified human IgG1. In some embodiments, the modification is in the heavy chain variable region (VH). In some embodiments, the modification is in the light chain variable region (VL). In some embodiments, the modification is in VH and VL. In other embodiments, the modified human IgG1 VH and / or VL provide the anti-human TNFα antibody of the present disclosure with a desired viscosity profile and / or immunogenicity risk profile.

[0101] In another embodiment, a conjugate of Formula I, wherein the anti-human TNFα antibody has a modified human IgG1 constant domain comprising an engineered cysteine ​​residue, is used to produce an antibody conjugate (also referred to as a bioconjugate) (see WO2018 / 232088 Al). More specifically, in such embodiments of the present disclosure, the anti-human TNFα antibody comprises an engineered cysteine ​​residue in the IgG1 heavy chain. In such embodiments, the anti-human TNFα antibody comprises a cysteine ​​at amino acid residue 124 (EU numbering) in the heavy chain constant domain 1 (CH1), or a cysteine ​​at amino acid residue 378 (EU numbering) in the heavy chain constant domain 2 (CH2). In another embodiment, the anti-human TNFα antibody comprises a cysteine ​​at amino acid residue 124 (EU numbering) in the CH1 domain and a cysteine ​​at amino acid residue 378 (EU numbering) in the CH2 domain.

[0102] In some embodiments, the conjugate of Formula I, wherein the anti-human TNFα has low to no cross-reactivity to anti-drug antibodies against other anti-TNFα therapeutic agents (e.g., adalimumab, infliximab, golimumab, certolizumab or etanercept) or their conjugates. In a specific embodiment, the conjugate of Formula I, wherein the anti-human TNFα antibody has low to no cross-reactivity to anti-drug antibodies against adalimumab. In such embodiments, certain conjugates of Formula I can be used to treat patients who have developed anti-drug antibodies to other anti-TNFα therapeutic agents (e.g., adalimumab) as defined herein. In further embodiments, certain conjugates of Formula I can be used to treat patients who have developed anti-drug antibodies to such other anti-TNFα therapeutic agents from previous treatment with other anti-TNFα therapeutic agents, and therefore have reduced clinical responses or adverse reactions to other anti-TNFα therapeutic agents. In such embodiments, the conjugates of Formula I, wherein the anti-human TNFα antibodies have sufficiently different amino acid sequences and nucleotide sequences such that they have low to no cross-reactivity to anti-drug antibodies directed against other anti-TNFα therapeutics. In specific embodiments, the conjugates of Formula I, wherein the anti-human TNFα antibodies of the present disclosure have sufficiently different CDR amino acid sequences such that they have low to no cross-reactivity to anti-drug antibodies directed against other anti-TNFα therapeutics. In some embodiments, the other anti-TNFα therapeutic is adalimumab, infliximab, golimumab, certolizumab pegol or etanercept or a conjugate thereof.

[0103] In some embodiments, the present disclosure provides nucleic acids encoding HC or LC, or VH or VL of novel antibodies that bind to anti-human TNFα, or vectors comprising such nucleic acids.

[0104] In some embodiments, the present disclosure provides a nucleic acid comprising the sequence of SEQ ID NO: 11, 12, 20, 21, 26, 29, 37 or 38.

[0105] In some embodiments, nucleic acids encoding heavy or light chains of antibodies that bind to anti-human TNFα are provided. In some embodiments, nucleic acids comprising sequences encoding SEQ ID NO:9, 10, 18, 19, 25, 28, 35, or 36 are provided. In some embodiments, nucleic acids comprising sequences encoding heavy chains of antibodies comprising SEQ ID NO:9, 18, 25, or 35 are provided. For example, the nucleic acid may comprise a sequence of SEQ ID NO:11, 20, 26, or 37. In some embodiments, nucleic acids comprising sequences encoding light chains of antibodies comprising SEQ ID NO:10, 19, 28, or 36 are provided. For example, the nucleic acid may comprise a sequence of SEQ ID NO:12, 21, 29, or 38.

[0106] In some embodiments of the present disclosure, nucleic acids encoding VH or VL of antibodies that bind to anti-human TNFα are provided. In some embodiments, nucleic acids comprising a sequence encoding SEQ ID NO: 7, 8, 16, 17, 24, 27, 33 or 34 are provided. In some embodiments, nucleic acids comprising a sequence encoding an antibody VH comprising SEQ ID NO: 7, 16, 24 or 33 are provided. In some embodiments, nucleic acids comprising a sequence encoding an antibody VL comprising SEQ ID NO: 8, 17, 27 or 34 are provided.

[0107] Some embodiments of the present disclosure provide vectors comprising a nucleic acid sequence encoding an antibody heavy chain or light chain. For example, such vectors may comprise a nucleic acid sequence encoding SEQ ID NO: 9, 18, 25, or 35. In some embodiments, the vector comprises a nucleic acid sequence encoding SEQ ID NO: 10, 19, 28, or 36.

[0108] Also provided herein are vectors comprising a nucleic acid sequence encoding an antibody VH or VL. For example, such vectors may comprise a nucleic acid sequence encoding SEQ ID NO: 7, 16, 24, or 33. In some embodiments, the vector comprises a nucleic acid sequence encoding SEQ ID NO: 8, 17, 27, or 34.

[0109] Also provided herein is a vector comprising a first nucleic acid sequence encoding an antibody heavy chain and a second nucleic acid sequence encoding an antibody light chain. In some embodiments, the vector comprises a first nucleic acid sequence encoding SEQ ID NO:9, 18, 25 or 35 and a second nucleic acid sequence encoding SEQ ID NO:10, 19, 28 or 36. In some embodiments, the vector comprises a first nucleic acid sequence encoding SEQ ID NO:9 and a second nucleic acid sequence encoding SEQ ID NO:10. In some embodiments, the vector comprises a first nucleic acid sequence encoding SEQ ID NO:18 and a second nucleic acid sequence encoding SEQ ID NO:19. In some embodiments, the vector comprises a first nucleic acid sequence encoding SEQ ID NO:25 and a second nucleic acid sequence encoding SEQ ID NO:10. In some embodiments, the vector comprises a first nucleic acid sequence encoding SEQ ID NO:25 and a second nucleic acid sequence encoding SEQ ID NO:19. In some embodiments, the vector comprises a first nucleic acid sequence encoding SEQ ID NO:25 and a second nucleic acid sequence encoding SEQ ID NO:28. In some embodiments, the vector comprises a first nucleic acid sequence encoding SEQ ID NO:35 and a second nucleic acid sequence encoding SEQ ID NO:36.

[0110] Also provided herein is a composition comprising a first vector comprising a nucleic acid sequence encoding an antibody heavy chain, and a second vector comprising a nucleic acid sequence encoding an antibody light chain. In some embodiments, the composition comprises a first vector comprising a nucleic acid sequence encoding SEQ ID NO:9, 18, 25 or 35 and a second nucleic acid sequence encoding SEQ ID NO:10, 19, 28 or 36. In some embodiments, the composition comprises a first vector comprising a nucleic acid sequence encoding SEQ ID NO:9 and a second vector comprising a nucleic acid sequence encoding SEQ ID NO:10. In some embodiments, the composition comprises a first vector comprising a nucleic acid sequence encoding SEQ ID NO:18 and a second vector comprising a nucleic acid sequence encoding SEQ ID NO:19. In some embodiments, the composition comprises a first vector comprising a nucleic acid sequence encoding SEQ ID NO:25 and a second vector comprising a nucleic acid sequence encoding SEQ ID NO:10. In some embodiments, the composition comprises a first vector comprising a nucleic acid sequence encoding SEQ ID NO:25 and a second vector comprising a nucleic acid sequence encoding SEQ ID NO:19. In some embodiments, the composition comprises a first vector comprising a nucleic acid sequence encoding SEQ ID NO:25 and a second vector comprising a nucleic acid sequence encoding SEQ ID NO:19. In some embodiments, the composition includes a first vector comprising a nucleic acid sequence encoding SEQ ID NO: 25 and a second vector comprising a nucleic acid sequence encoding SEQ ID NO: 28. In some embodiments, the composition includes a first vector comprising a nucleic acid sequence encoding SEQ ID NO: 35 and a second vector comprising a nucleic acid sequence encoding SEQ ID NO: 36.

[0111] Also provided herein is a composition comprising a vector comprising a nucleic acid sequence encoding an antibody heavy chain and a nucleic acid sequence encoding an antibody light chain. In some embodiments, the composition comprises a vector comprising a nucleic acid sequence encoding SEQ ID NO: 9, 18, 25 or 35 and a second nucleic acid sequence encoding SEQ ID NO: 10, 19, 28 or 36.

[0112] In an embodiment, n is 2-5.

[0113] In an embodiment, n is 3-5.

[0114] In an embodiment, n is 3-4.

[0115] In an embodiment, n is about 4.

[0116] In an embodiment, n is about 3.

[0117] In an embodiment, n is about 2.

[0118] As used herein, "GC" in the formula:

[0119]

[0120] refers to suitable glucocorticoid receptor agonist payloads and include the following Formulae IIa, IIb and IIc:

[0121]

[0122]

[0123] As used herein, the "L" in the formula

[0124]

[0125] Refers to a suitable linker group that connects Ab to GC. Suitable linkers known to those of ordinary skill in the art include, for example, cleavable and non-cleavable linkers. More specifically, suitable linkers "L" include the following formulas IIIa to IIIf:

[0126]

[0127]

[0128] In an embodiment, the present disclosure provides a glucocorticoid receptor agonist payload-linker of Formula IV:

[0129]

[0130] In an embodiment, the present disclosure provides a glucocorticoid receptor agonist payload-linker of Formula IVa:

[0131]

[0132] In an embodiment, the disclosure provides a glucocorticoid receptor agonist payload-linker of Formula IVb:

[0133]

[0134] In an embodiment, the present disclosure provides a glucocorticoid receptor agonist payload-linker of Formula IVc:

[0135]

[0136] In an embodiment, the present disclosure provides a glucocorticoid receptor agonist payload-linker of Formula IVd:

[0137]

[0138] In an embodiment, the present disclosure provides a compound of Formula V:

[0139]

[0140] In further embodiments, the present disclosure provides compounds of Formula Va:

[0141]

[0142] In an embodiment, the disclosure also provides a method for treating an autoimmune disease in a subject in need thereof, the method comprising administering an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof to the subject. In an embodiment, the disclosure also provides a method for treating an inflammatory disease in a subject in need thereof, the method comprising administering an effective amount of a conjugate of Formula I or a drug or salt thereof to the subject. In certain embodiments, the autoimmune disease or inflammatory disease is, for example, rheumatoid arthritis (RA), psoriatic arthritis (PsA), Crohn's disease (CD), ulcerative colitis, plaque psoriasis (PS), ankylosing spondylitis (AS), juvenile idiopathic arthritis, suppurative hidradenitis, uveitis, non-infectious intermediate uveitis, posterior uveitis, panuveitis, Behcet's disease or polymyalgia rheumatica (PMR). In an embodiment, the disclosure also provides a method for treating rheumatoid arthritis in a subject in need thereof, the method comprising administering an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof to the subject. In an embodiment, the disclosure also provides a method for treating psoriatic arthritis in a subject in need thereof, the method comprising administering an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof to the subject. In an embodiment, the disclosure also provides a method for treating Crohn's disease in a subject in need thereof, the method comprising administering an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof to the subject. In an embodiment, the disclosure also provides a method for treating ulcerative colitis in a subject in need thereof, the method comprising administering an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof to the subject. In an embodiment, the disclosure also provides a method for treating plaque psoriasis in a subject in need thereof, the method comprising administering an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof to the subject. In an embodiment, the disclosure also provides a method for treating ankylosing spondylitis in a subject in need thereof, the method comprising administering an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof to the subject. In some embodiments, the subject to whom an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof is administered receives prior treatment with other anti-TNFα therapeutic agents, and wherein the subject produces anti-drug antibodies to the other anti-TNFα therapeutic agents. In such embodiments, the other anti-TNFα therapeutic agent is selected from the group consisting of adalimumab, infliximab, golimumab, certolizumab pegol or their conjugates. In yet other embodiments, certain conjugates of Formula I as disclosed herein or pharmaceutically acceptable salts thereof have low to no cross-reactivity to anti-drug antibodies to at least four or more of the other anti-TNFα therapeutic agents selected from the group consisting of adalimumab, infliximab, golimumab, certolizumab pegol or their conjugates.In yet other embodiments, certain conjugates of Formula I as disclosed herein, or pharmaceutically acceptable salts thereof, have low to no cross-reactivity to anti-drug antibodies directed against at least three or more of the other anti-TNFα therapeutic agents selected from the group consisting of adalimumab, infliximab, golimumab, certolizumab pegol or their conjugates. In yet other embodiments, certain conjugates of Formula I as disclosed herein, or pharmaceutically acceptable salts thereof, have low to no cross-reactivity to anti-drug antibodies directed against at least two or more of the other anti-TNFα therapeutic agents selected from the group consisting of adalimumab, infliximab, golimumab, certolizumab pegol or their conjugates. In yet other embodiments, certain conjugates of Formula I as disclosed herein, or pharmaceutically acceptable salts thereof, have low to no cross-reactivity to anti-drug antibodies directed against adalimumab or its conjugates.

[0143] In an embodiment, the present disclosure also provides a conjugate of formula I or a pharmaceutically acceptable salt thereof for therapy. In an embodiment, the present disclosure provides a conjugate of formula I or a pharmaceutically acceptable salt thereof for treating autoimmune diseases. In an embodiment, the present disclosure provides a conjugate of formula I or a pharmaceutically acceptable salt thereof for treating inflammatory diseases. In certain embodiments, autoimmune diseases or inflammatory diseases are, for example, rheumatoid arthritis (RA), psoriatic arthritis (PsA), Crohn's disease (CD), ulcerative colitis, plaque psoriasis (PS), ankylosing spondylitis (AS), juvenile idiopathic arthritis, suppurative hidradenitis, uveitis, non-infectious intermediate uveitis, posterior uveitis, panuveitis, Behcet's disease or polymyalgia rheumatica (PMR). In an embodiment, the present disclosure provides a conjugate of formula I or a pharmaceutically acceptable salt thereof for treating rheumatoid arthritis. In an embodiment, the present disclosure provides a conjugate of formula I or a pharmaceutically acceptable salt thereof for treating psoriatic arthritis. In an embodiment, the present disclosure provides a conjugate of Formula I or a pharmaceutically acceptable salt thereof for treating Crohn's disease. In an embodiment, the present disclosure provides a conjugate of Formula I or a pharmaceutically acceptable salt thereof for treating ulcerative colitis. In an embodiment, the present disclosure provides a conjugate of Formula I or a pharmaceutically acceptable salt thereof for treating plaque psoriasis. In an embodiment, the present disclosure provides a conjugate of Formula I or a pharmaceutically acceptable salt thereof for treating ankylosing spondylitis. In some embodiments, the subject to whom an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof is administered receives prior treatment with other anti-TNFα therapeutic agents, and wherein the subject produces anti-drug antibodies to other anti-TNFα therapeutic agents. In such embodiments, another anti-TNFα therapeutic agent is selected from the group consisting of adalimumab, infliximab, golimumab, certolizumab pegol or their conjugates. In yet other embodiments, certain conjugates of Formula I as disclosed herein, or pharmaceutically acceptable salts thereof, have low to no cross-reactivity to anti-drug antibodies directed against at least four or more of other anti-TNFα therapeutics selected from the group consisting of adalimumab, infliximab, golimumab, certolizumab pegol, or conjugates thereof. In yet other embodiments, certain conjugates of Formula I as disclosed herein, or pharmaceutically acceptable salts thereof, have low to no cross-reactivity to anti-drug antibodies directed against at least three or more of other anti-TNFα therapeutics selected from the group consisting of adalimumab, infliximab, golimumab, certolizumab pegol, or conjugates thereof.In yet other embodiments, certain conjugates of Formula I as disclosed herein, or pharmaceutically acceptable salts thereof, have low to no cross-reactivity to anti-drug antibodies directed against at least two or more of the other anti-TNFα therapeutic agents selected from the group consisting of adalimumab, infliximab, golimumab, certolizumab pegol, or conjugates thereof. In yet other embodiments, certain conjugates of Formula I as disclosed herein, or pharmaceutically acceptable salts thereof, have low to no cross-reactivity to anti-drug antibodies directed against adalimumab or its conjugates.

[0144] In an embodiment, the present disclosure also provides the use of a conjugate of Formula I or a pharmaceutically acceptable salt thereof for the manufacture of a drug for the treatment of an autoimmune disease. In an embodiment, the present disclosure also provides the use of a conjugate of Formula I or a pharmaceutically acceptable salt thereof for the manufacture of a drug for the treatment of an inflammatory disease. In certain embodiments, an autoimmune disease or an inflammatory disease is, for example, rheumatoid arthritis (RA), psoriatic arthritis (PsA), Crohn's disease (CD), ulcerative colitis, plaque psoriasis (PS), ankylosing spondylitis (AS), juvenile idiopathic arthritis, suppurative hidradenitis, uveitis, non-infectious intermediate uveitis, posterior uveitis, panuveitis, Behcet's disease or polymyalgia rheumatica (PMR). In an embodiment, the present disclosure provides the use of a conjugate of Formula I or a pharmaceutically acceptable salt thereof for the manufacture of a drug for the treatment of rheumatoid arthritis. In an embodiment, the present disclosure provides the use of a conjugate of Formula I or a pharmaceutically acceptable salt thereof for the manufacture of a drug for the treatment of psoriatic arthritis. In an embodiment, the present disclosure provides the use of a conjugate of Formula I or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for treating Crohn's disease. In an embodiment, the present disclosure provides the use of a conjugate of Formula I or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for treating ulcerative colitis. In an embodiment, the present disclosure provides the use of a conjugate of Formula I or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for treating plaque psoriasis. In an embodiment, the present disclosure provides the use of a conjugate of Formula I or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for treating ankylosing spondylitis. In some embodiments, the subject to whom an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof is administered receives prior treatment with other anti-TNFα therapeutic agents, and wherein the subject produces anti-drug antibodies to other anti-TNFα therapeutic agents. In such embodiments, another anti-TNFα therapeutic agent is selected from the group consisting of adalimumab, infliximab, golimumab, certolizumab pegol or their conjugates. In yet other embodiments, certain conjugates of Formula I as disclosed herein, or pharmaceutically acceptable salts thereof, have low to no cross-reactivity to anti-drug antibodies directed against at least four or more of other anti-TNFα therapeutics selected from the group consisting of adalimumab, infliximab, golimumab, certolizumab pegol, or conjugates thereof. In yet other embodiments, certain conjugates of Formula I as disclosed herein, or pharmaceutically acceptable salts thereof, have low to no cross-reactivity to anti-drug antibodies directed against at least three or more of other anti-TNFα therapeutics selected from the group consisting of adalimumab, infliximab, golimumab, certolizumab pegol, or conjugates thereof.In yet other embodiments, certain conjugates of Formula I as disclosed herein, or pharmaceutically acceptable salts thereof, have low to no cross-reactivity to anti-drug antibodies directed against at least two or more of the other anti-TNFα therapeutic agents selected from the group consisting of adalimumab, infliximab, golimumab, certolizumab pegol, or conjugates thereof. In yet other embodiments, certain conjugates of Formula I as disclosed herein, or pharmaceutically acceptable salts thereof, have low to no cross-reactivity to anti-drug antibodies directed against adalimumab or its conjugates.

[0145] Nucleic acids of the present disclosure can be expressed in host cells, for example, after the nucleic acids have been operably connected to expression control sequences. Expression control sequences capable of expressing nucleic acids operably connected thereto are well known in the art. Expression vectors may include sequences encoding one or more signal peptides that promote secretion of polypeptides from host cells. Expression vectors containing nucleic acids of interest (for example, nucleic acids encoding heavy or light chains of antibodies) can be transferred to host cells by well-known methods (for example, stable or transient transfection, transformation, transduction or infection). In addition, the expression vector may include one or more selection markers, such as tetracycline, neomycin and dihydrofolate reductase, to help detect host cells transformed with the desired nucleic acid sequence.

[0146] In another aspect, cells, such as host cells, comprising nucleic acids, vectors or nucleic acid compositions described herein are provided herein. The host cell may be a cell stably or transiently transfected, transformed, transduced or infected with one or more expression vectors expressing all or a portion of an antibody described herein. In some embodiments, the host cell may be stably or transiently transfected, transformed, transduced or infected with expression vectors expressing the HC and LC polypeptides of the antibodies disclosed herein. In some embodiments, the host cell may be stably or transiently transfected, transformed, transduced or infected with a first vector expressing the HC polypeptide of an antibody described herein and a second vector expressing the LC polypeptide of an antibody described herein. Such host cells (e.g., mammalian host cells) may express antibodies that bind to anti-human TNFα as described herein. Mammalian host cells known to be capable of expressing antibodies include CHO cells, HEK293 cells, COS cells and NS0 cells.

[0147] In some embodiments, a cell (eg, a host cell) includes a vector comprising a first nucleic acid sequence encoding SEQ ID NO: 9, 18, 25, or 35 and a second nucleic acid sequence encoding SEQ ID NO: 10, 19, 28, or 36.

[0148] In some embodiments, the cell (e.g., a host cell) includes a first vector comprising a nucleic acid sequence encoding SEQ ID NO:9, 18, 25, or 35, and a second vector comprising a nucleic acid sequence encoding SEQ ID NO:10, 19, 28, or 36.

[0149] The present disclosure also provides a method for producing an antibody that binds to an anti-human TNFα described herein by culturing the above-mentioned host cell, such as a mammalian host cell, under conditions such that the antibody is expressed, and recovering the expressed antibody from the culture medium. The culture medium in which the antibody has been secreted can be purified by conventional techniques. Various protein purification methods can be used, and such methods are known in the art and described, for example, in Deutscher, Methods in Enzymology 182: 83-89 (1990) and Scopes, Protein Purification: Principles and Practice, 3rd Edition, Springer, NY (1994).

[0150] The present disclosure provides a method of producing a conjugate, the method comprising contacting a compound of the present disclosure with an anti-human TNFα antibody.

[0151] The present disclosure provides a method for producing a conjugate, the method comprising contacting a compound of formula IV with an anti-human TNFα antibody. The present disclosure provides a method for producing a conjugate, the method comprising contacting a compound of formula IVa with an anti-human TNFα antibody. The present disclosure provides a method for producing a conjugate, the method comprising contacting a compound of formula IVb with an anti-human TNFα antibody. The present disclosure provides a method for producing a conjugate, the method comprising contacting a compound of formula IVc with an anti-human TNFα antibody. The present disclosure provides a method for producing a conjugate, the method comprising contacting a compound of formula IVd with an anti-human TNFα antibody. In some embodiments, the conjugate produced is a conjugate of formula I.

[0152] The present disclosure provides a method for producing a conjugate, the method comprising the steps of:

[0153] (a) reducing an anti-human TNFα antibody with a reducing agent, wherein the anti-human TNFα antibody comprises one or more engineered cysteine ​​residues;

[0154] (b) oxidizing the anti-human TNFα antibody with an oxidizing agent; and

[0155] (c) contacting a compound of the present disclosure with an anti-human TNFα antibody to produce a conjugate.

[0156] The present disclosure provides a method for producing a conjugate, the method comprising the steps of:

[0157] (a) reducing an anti-human TNFα antibody with a reducing agent, wherein the anti-human TNFα antibody comprises one or more engineered cysteine ​​residues;

[0158] (b) oxidizing the anti-human TNFα antibody with an oxidizing agent; and

[0159] (c) making a compound of the formula

[0160]

[0161] Contacting with anti-human TNFα antibody to produce a conjugate.

[0162] In some embodiments, the reducing agent is dithiothreitol. In some embodiments, the oxidizing agent is dehydroascorbic acid. In some embodiments, the reducing agent is dithiothreitol and the oxidizing agent is dehydroascorbic acid.

[0163] The present disclosure also provides antibodies or antigen-binding fragments thereof produced by any of the methods described herein.

[0164] In an embodiment, the present disclosure also provides a pharmaceutical composition comprising a conjugate of Formula I described herein or a pharmaceutically acceptable salt thereof or an antibody, a nucleic acid or a vector and one or more pharmaceutically acceptable carriers, diluents or excipients. In an embodiment, the present disclosure also provides a pharmaceutical composition comprising a conjugate of Formula I or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable carriers, diluents or excipients. In an embodiment, the present disclosure also provides a pharmaceutical composition comprising a conjugate of Formula I and one or more pharmaceutically acceptable carriers, diluents or excipients. In an embodiment, the present disclosure also provides a method for preparing a pharmaceutical composition, the method comprising mixing a conjugate of Formula I or a pharmaceutically acceptable salt thereof with one or more pharmaceutically acceptable carriers, diluents or excipients. In an embodiment, the present disclosure also includes novel intermediates and methods for synthesizing conjugates of Formula I. DETAILED DESCRIPTION

[0165] Unless otherwise indicated, the term "TNFα" as used herein refers to soluble and / or membrane TNFα, as well as any natural mature TNFα produced by processing of TNFα precursor protein in cells. Unless otherwise indicated, the term includes TNFα from any vertebrate source, including mammals such as canines, primates (e.g., humans and cynomolgus monkeys or rhesus monkeys) and rodents (e.g., mice and rats). The term also includes naturally occurring TNFα variants, such as splice variants or allelic variants. Exemplary amino acid sequences of anti-human TNFα are known in the art, such as NCBI accession number: NP_000585 (SEQ ID NO: 39). Exemplary amino acid sequences of cynomolgus monkey TNFα are also known in the art, such as UniProt reference sequence P79337 (SEQ ID NO: 42). Exemplary amino acid sequences of rhesus monkey TNFα are also known in the art, such as UniProt reference sequence P48094 (SEQ ID NO: 40). Exemplary amino acid sequences of canine TNFα are also known in the art, such as GenBank Accession No. CAA64403 (SEQ ID NO: 41). The term human "TNFα" is used herein to collectively refer to all known anti-human TNFα isoforms and polymorphic forms. The sequence numbering used herein is based on the mature protein without the signal peptide.

[0166] Unless otherwise indicated, the term "TNFR" or "TNF receptor" as used herein refers to any naturally occurring mature TNFR, such as TNFR1 (also known as p55 or p60) or TNFR2 (also known as p75 or p80). Unless otherwise indicated, the term includes TNFRs from any vertebrate source, including mammals such as canines, primates (e.g., humans and cynomolgus or rhesus monkeys) and rodents (e.g., mice and rats). The term also includes naturally occurring TNFR variants, such as splice variants or allelic variants. Exemplary amino acid sequences of human TNFR1 are known in the art, such as GenBank Accession No.: AAA61201 (SEQ ID NO: 45). Exemplary amino acid sequences of human TNFR2 are known in the art, such as NCBI Accession No.: NP_001057 (SEQ ID NO: 46). The term "TNFR" is used herein to collectively refer to all known human TNFR isoforms and polymorphic forms.

[0167] As used herein, the term "anti-drug antibody" or "ADA" refers to an antibody formed in a mammal by an immune response to a therapeutic agent administered to the mammal. In some embodiments of the present disclosure, the anti-drug antibodies formed against the therapeutic agent can neutralize the effect of the therapeutic agent, thereby changing the pharmacokinetic (PK) and / or pharmacodynamic (PD) properties of the therapeutic agent, interfering with the effect of the therapeutic agent, and / or reducing the efficacy, and / or reducing the clinical response to the therapeutic agent. Anti-drug antibodies against therapeutic agents can also lead to adverse immune responses in patients, making the patient likely not a candidate for further treatment with the therapeutic agent. Examples of adverse immune responses include, but are not limited to, infusion-related reactions, characterized by symptoms during drug administration or within the first day after drug administration, such as fever, itching, bronchospasm, or cardiovascular failure (Atiqi, S., Front Immunol., 2020, 26 (11): 312).

[0168] As used herein, the term "low to no binding" to anti-drug antibodies refers to the binding of anti-human TNFα antibody glucocorticoid conjugates or anti-human TNFα antibodies of the present disclosure to anti-drug antibodies for other anti-TNFα therapeutic agents, wherein such binding is determined to be below the cutoff point of the assay for measuring binding or within a predetermined variable range of the assay. In such methods, the cutoff point is a predetermined threshold for identifying positive binding to anti-drug antibodies. In some embodiments, the predetermined variability of the assay is less than about 20% higher than the cutoff point of the assay. In such embodiments, the binding of anti-human TNFαAb GC conjugates or anti-human TNFα antibodies of the present disclosure to anti-drug antibodies for other therapeutic agents (e.g., adalimumab) is considered to be low binding, and the binding is less than about 20% above the cutoff point of the assay. In some embodiments, the binding of anti-human TNFαAbGC conjugates or anti-human TNFα antibodies of the present disclosure to anti-drug antibodies for other therapeutic agents (e.g., adalimumab) is considered to be no binding, and the binding is at or below the cutoff point of the assay.

[0169] The term "other anti-TNFα therapeutic agents" refers to agents that bind to TNFα and inhibit TNF receptor-mediated reactions, excluding the conjugates or anti-human TNFα antibodies described herein. Such agents may include, but are not limited to, antibodies or conjugates thereof, antibody fragments, or antigen-binding fragments, which contain at least a portion of an antibody that retains the ability to interact with an antigen, such as Fab, Fab', F(ab')2, Fv fragments, scFv, scFab, disulfide-linked Fv (sdFv), Fd fragments, or linear antibodies, which may, for example, be fused to an Fc region or an IgG heavy chain constant region. In some embodiments, other anti-TNFα therapeutic agents may be, for example, adalimumab, infliximab, golimumab, and certolizumab pegol and / or their conjugates.

[0170] As used herein, the term "antibody" refers to an immunoglobulin molecule that binds an antigen. Embodiments of antibodies include monoclonal antibodies, polyclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, bispecific or multispecific antibodies, or conjugated antibodies. These antibodies may belong to any class (e.g., IgG, IgE, IgM, IgD, IgA) and any subclass (e.g., IgG1, IgG2, IgG3, IgG4). Embodiments of the present disclosure also include antibody fragments or antigen binding fragments, and the term "antibody fragment or antigen binding fragment" includes at least a portion of an antibody that retains the ability to interact with an antigen, such as, for example, Fab, Fab', F(ab')2, Fv fragments, scFv, scFab, disulfide-linked Fv (sdFv), Fd fragments, or linear antibodies, which may, for example, be fused to Fc regions or IgG heavy chain constant regions.

[0171] An exemplary antibody is an immunoglobulin G (IgG) type antibody, which is composed of four polypeptide chains: two heavy chains (HC) and two light chains (LC) cross-linked by interchain disulfide bonds. The amino terminal portion of each of the four polypeptide chains contains a variable region of about 100 to 125 or more amino acids that is primarily responsible for antigen recognition. The carboxyl terminal portion of each of the four polypeptide chains contains a constant region that is primarily responsible for effector function. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain constant region refers to the region of the antibody that contains the Fc region and CH1 domain of the antibody heavy chain. Each light chain is composed of a light chain variable region (VL) and a light chain constant region. IgG isotypes can be further divided into subclasses (e.g., IgG1, IgG2, IgG3, and IgG4). The numbering of amino acid residues in the constant region is based on the EU index as in Kabat. Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Bethesda, MD: USDept. of Health and Human Services, Public Health Service, National Institutes of Health (1991). The terms EU index number or EU number are used interchangeably herein.

[0172] VH and VL regions can be further subdivided into hypervariable regions known as complementary determining regions (CDRs), which are interspersed with more conservative regions known as framework regions (FRs). CDRs are exposed to the surface of the protein and are important regions of the antigen-binding specificity of antibodies. Each VH and VL is composed of three CDRs and four FRs, which are arranged in the following order from amino-terminal to carboxyl-terminal: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In this article, the three CDRs of the heavy chain are referred to as "HCDR1, HCDR2 and HCDR3", and the three CDRs of the light chain are referred to as "LCDR1, LCDR2 and LCDR3". These CDRs contain most of the residues that form specific interactions with the antigen. The assignment of amino acid residues to CDRs can be performed according to well-known schemes, including those described in Kabat (Kabat et al., "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991)), Chothia (Chothia et al., "Canonical structures for the hypervariable regions of immunoglobulins," Journal of Molecular Biology, 196, 901-917 (1987); Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins," Journal of Molecular Biology, 273, 927-948 (1997)), North (North et al., "A New Clustering of Antibody CDR Loop Conformations," Journal of Molecular Biology, 274, 937-951 (1998)), and the like. Biology, 406, 228-256 (2011)) or IMGT (International Immunogenetics Database available at www.imgt.org; see Lefranc et al., Nucleic Acids Res. 1999; 27:209-212). The combination of IMGT and North CDR definitions is used for the exemplified anti-human TNFα antibodies as described herein.

[0173] As used herein, the term "Fc region" refers to the region of an antibody that includes the CH2 and CH3 domains of the antibody heavy chain. Optionally, the Fc region may include a portion or the entire hinge region of the antibody heavy chain. Biological activities such as effector functions are attributable to the Fc region, which vary with the antibody isotype. Examples of antibody effector functions include Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), C1q binding, complement-dependent cytotoxicity (CDC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0174] As used herein, the term "epitope" refers to the amino acid residues in an antigen that are bound by an antibody. An epitope can be a linear epitope, a conformational epitope, or a hybrid epitope. The term "epitope" can be used to refer to a structural epitope. According to some embodiments, a structural epitope can be used to describe an antigen region covered by an antibody (e.g., the footprint of an antibody when it binds to an antigen). In some embodiments, a structural epitope can be described as an amino acid residue of an antigen within a specified proximity of an amino acid residue of an antibody (e.g., within a specified number of angstroms). The term "epitope" can also be used to refer to a functional epitope. According to some embodiments, a functional epitope can be used to describe an amino acid residue of an antigen that interacts with an amino acid residue of an antibody in a manner that contributes to the binding energy between an antigen and an antibody. An epitope can be determined according to different experimental techniques (also referred to as "epitope mapping techniques"). It should be understood that the determination of an epitope can vary based on the different epitope mapping techniques used, and can also vary with the different experimental conditions used, such as due to conformational changes or cleavage of an antigen induced by specific experimental conditions. Epitope mapping techniques are known in the art (e.g., Rockberg and Nilvebrant, Epitope Mapping Protocols: Methods in Molecular Biology, Humana Press, 3rd ed. 2018; Holst et al., Molecular Pharmacology 1998, 53(1): 166-175), and include, but are not limited to, X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, site-directed mutagenesis, material exchange mutagenesis, alanine scanning mutagenesis, steric hindrance mutagenesis, hydrogen-deuterium exchange (HDX), and cross-blocking assays.

[0175] As used herein, unless otherwise indicated, the term "binding" refers to the ability of a protein or molecule to form a chemical bond or attractive interaction with another protein or molecule that brings the two proteins or molecules into proximity as determined by common methods known in the art.

[0176] As used herein, the term "nucleic acid" refers to a polymer of nucleotides, including molecules containing single-stranded and / or double-stranded nucleotides, such as DNA, cDNA and RNA molecules, incorporating natural, modified and / or analog nucleotides. The polynucleotides of the present disclosure may also include substrates incorporated therein, for example, by DNA or RNA polymerase or synthesis reactions.

[0177] Embodiments of the present disclosure include conjugates in which a polypeptide (e.g., an anti-human TNFα antibody) is conjugated to one or more drug moieties, such as 2 drug moieties, 3 drug moieties, 4 drug moieties, 5 drug moieties, or more drug moieties. As described herein, the drug moiety can be conjugated to the polypeptide at one or more sites in the polypeptide. In certain embodiments, the conjugate has an average drug to antibody ratio (DAR) (molar ratio) in the range of 2 to 5, or 3 to 5, or 3 to 4. In some embodiments, the conjugate has an average DAR of about 3. In certain embodiments, the conjugate has an average DAR of about 4. The so-called average means the arithmetic mean.

[0178] As used herein, it is understood that the conjugates of Formula I encompass conjugates of Formulas Ia, Ib, Ic, Id, Ie and If, and all references herein to the conjugates of Formula I are understood to include conjugates of Formulas Ia, Ib, Ic, Id, Ie and If. It will also be understood by those skilled in the art that the conjugates of Formula I including the conjugates of Formulas Ia, Ib, Ic, Id, Ie and If may also be referred to as anti-human TNFα antibody glucocorticoid conjugates ("anti-human TNFα Ab GC conjugates").

[0179] The anti-human TNFα antibody GC conjugates of the present disclosure can be formulated as pharmaceutical compositions that are administered by any route that makes the conjugate bioavailable, including, for example, intravenous or subcutaneous administration. Such pharmaceutical compositions can be prepared using techniques and methods known in the art (see, for example, Remington: The Science and Practice of Pharmacy, edited by A. Adejare, 23rd edition, published in 2020, Elsevier Science).

[0180] As used herein, the terms "treating", "treatment" or "to treat" include inhibiting, slowing down, stopping, controlling, delaying or reversing the progression or severity of existing symptoms or disorders, or improving existing symptoms or disorders, but do not necessarily mean completely eliminating existing symptoms or disorders. Treatment includes administering proteins or nucleic acids or vectors or compositions to treat symptoms or disorders in patients (especially humans).

[0181] As used herein, the term "inhibits" or "inhibiting" refers to, for example, reducing, lowering, slowing, decreasing, stopping, destroying, eliminating, antagonizing or blocking a biological response or activity, but does not necessarily mean complete elimination of a biological response.

[0182] As used herein, the term "subject" refers to mammals, including but not limited to humans, chimpanzees, apes, monkeys, cows, horses, sheep, goats, pigs, rabbits, dogs, cats, rats, mice, guinea pigs, etc. Preferably, the subject is a human.

[0183] As used herein, the term "effective amount" refers to the amount or dosage of the conjugate of the present invention or its pharmaceutically acceptable salt, which provides the desired effect in the subject receiving diagnosis or treatment after single or multiple administration to the subject. As used herein, the term "effective amount" further refers to the amount or dosage of the conjugate of the present invention or its pharmaceutically acceptable salt, which will induce the desired biological or medical response of the subject, for example, reducing or inhibiting protein activity, or improving symptoms, alleviating symptoms, slowing or delaying disease progression, or preventing diseases, etc. In a non-limiting embodiment, the term "effective amount" refers to the necessary amount (in terms of dosage and for time period and for administration mode) or dosage of the conjugate of the present invention or its pharmaceutically acceptable salt when administered to a subject, which can effectively at least partially alleviate, inhibit, prevent and / or improve the condition, disorder or disease to achieve the desired therapeutic result. The effective amount is also the amount in which any toxic or harmful effect or dosage of the conjugate of the present invention or its pharmaceutically acceptable salt of the present invention is exceeded by the beneficial effect.

[0184] The effective amount can be determined by one skilled in the art by using known techniques and by observing the results obtained under similar circumstances. In determining the effective amount for a patient, the attending diagnostician will consider many factors, including, but not limited to: the type of patient; the size, age and general health of the patient; the specific disease or disorder involved; the extent or severity of the disease or disorder; the response of the individual patient; the specific conjugate being administered; the mode of administration; the bioavailability characteristics of the administered preparation; the dosage regimen selected; the use of concomitant medications; and other relevant circumstances.

[0185] Included within the scope of the present invention are pharmaceutically acceptable salts of the conjugates of Formula I. Pharmaceutically acceptable salts of the conjugates of the present invention (such as the conjugates of Formula I) can be formed under standard conditions known in the art. See, for example, Berge, SM et al., "Pharmaceutical Salts," Journal of Pharmaceutical Sciences, 66: 1-19, (1977).

[0186] Table 1: Abbreviations and definitions

[0187] the term definition ACN Acetonitrile aq Aqueous solution cP Centipoise DCM Dichloromethane DIPEA N,N-Diisopropylethylamine DMF N,N-Dimethylformamide DMSO Dimethyl sulfoxide dppf 1,1'-Bis(diphenylphosphino)ferrocene ES / MS Electrospray mass spectrometry EtOAc Ethyl acetate HATU Azabenzotriazole tetramethyl urea hexafluorophosphate HPLC High performance liquid chromatography LDA Lithium diisopropylamide MeOH Methanol MS Mass spectrometry MTBE Methyl tert-butyl ether m / z Mass-to-charge ratio NMR Nuclear Magnetic Resonance Pet Ether Petroleum ether rt Room temperature satd saturation THF Tetrahydrofuran

[0188] The conjugates or salts thereof of the present invention can be easily prepared by a variety of procedures known to those of ordinary skill in the art, some of which are described in the preparation and in the following examples. It is recognized by those of ordinary skill in the art that the specific synthetic steps of each route described can be combined in different ways, or combined with steps from different schemes, to prepare the conjugates or salts thereof of the present invention. The product of each step can be recovered by conventional methods well known in the art, including extraction, evaporation, precipitation, chromatography, filtration, trituration and crystallization. Unless otherwise stated, all substituents are as previously defined. Reagents and starting materials are readily available to those of ordinary skill in the art. The following preparations, examples and determinations further illustrate the present disclosure, but should not be construed as limiting the scope of the present disclosure in any way. BRIEF DESCRIPTION OF THE DRAWINGS

[0189] Figure 1 In vitro ADCC activity of exemplified anti-human TNFα Ab1 GC conjugates is shown.

[0190] Figure 2 In vitro CDC activity of exemplified anti-human TNFα Ab1 GC conjugates is shown.

[0191] Figure 3 It was shown that the exemplified anti-human TNFα antibody Ab6 had significantly low binding to anti-drug antibodies against adalimumab formed in cynomolgus monkeys hyperimmunized with adalimumab.

[0192] Figure 4 The exemplary anti-human TNFα antibody Ab6 was shown to have significantly low binding to anti-drug antibodies against adalimumab formed in human patients treated with adalimumab.

[0193] FIG. 5A to FIG. 5C Shown are DSC thermograms of an exemplary anti-human TNFα Ab1 GC conjugate in PBS, pH 7.2 (5A), acetate, pH 5 (5B), and histidine, pH 6 (5C).

[0194] FIG. 6A to FIG. 6C Shown is a comparison of the efficacy of 1 mg / kg (6A), 3 mg / kg (6B), and 10 mg / kg (6C) of anti-human TNFα Ab1 GC conjugate, anti-human TNFα Ab1, and exemplary anti-human TNFα antibody conjugates in a humanized mouse model of contact hypersensitivity.

[0195] Figure 7Anti-human TNFα Ab2 GC conjugate was shown to block disease progression in a human TNFα transgenic mouse polyarthritis model as measured by clinical scores in both adalimumab naive and adalimumab treated mice, showing that anti-human TNFα Ab2 GC conjugate did not generate significant anti-drug antibody responses and had low to no cross-reactivity to anti-drug antibodies against adalimumab.

[0196] Preparation 1

[0197] 6-Bromo-2-fluoro-3-methoxybenzaldehyde

[0198]

[0199] Two reactions were carried out in parallel. At -78 ° C, in 30 minutes, LDA (2M, 730 mL) was slowly added to a solution of 4-bromo-2-fluoro-1-methoxybenzene (250 g, 1.2 mol) in THF (1500 mL). After another 30 minutes, at -78 ° C, in 30 minutes, DMF (140 mL, 1.8 mol) was slowly added. After 1 hour, the two reactions were combined, and the mixture was diluted with citric acid aqueous solution (2000 mL) and extracted with EtOAc (1500 mL × 2). The combined organic layer was washed with saturated NaCl aqueous solution (1000 mL) and concentrated under reduced pressure to obtain a residue. The residue was ground with petroleum ether (1000 mL) at room temperature within 12 hours to obtain the title compound (382 g, 67% yield). ES / MS m / z 233.9 (M+H).

[0200] Preparation 2

[0201] 2-Fluoro-3-methoxy-6-methylbenzaldehyde

[0202]

[0203] Three reactions were carried out in parallel. 6-bromo-2-fluoro-3-methoxybenzaldehyde (120g, 5.3mol), methylboric acid (47g, 7.9mol), Pd(dppf)Cl2 (12g, 0.02mol) and Cs2CO3 (340g, 1.1mol) were added to a mixture of 1,4-dioxane (600mL) and water (120mL). The mixture was stirred at 120°C. After 12 hours, the three reactions were combined, and the mixture was diluted with saturated NH4Cl aqueous solution (1000mL) and extracted with MTBE (1500mL×2). The combined organic layer was washed with saturated NaCl aqueous solution (1000mL) and concentrated under reduced pressure to obtain a residue. The residue was purified by silica gel chromatography, eluted with 40:1 petroleum ether:EtOAc to obtain the title compound (180g, 59%). ES / MS m / z 169.3 (M+H).

[0204] Preparation 3

[0205] 2-Fluoro-3-hydroxy-6-methylbenzaldehyde

[0206]

[0207] 2-Fluoro-3-methoxy-6-methylbenzaldehyde (175 g, 1.0 mol) was added to DCM (1050 mL). BBr3 (200 mL, 2.1 mol) was slowly added to the solution at 0 °C. The reaction was stirred at room temperature. After 1 hour, the mixture was diluted with saturated NaHCO3 aqueous solution (1000 mL) until pH = 7-8, and then extracted with MTBE (1500 mL × 2). The combined organic layers were washed with saturated NaCl aqueous solution (1000 mL) and concentrated under reduced pressure to obtain the title compound (110 g, 68%). ES / MS m / z 154.9 (M+H).

[0208] Preparation 4

[0209] tert-Butyl N-[3-[(2-fluoro-3-formyl-4-methyl-phenoxy)methyl]phenyl]carbamate

[0210]

[0211] 2-Fluoro-3-hydroxy-6-methylbenzaldehyde (130g, 0.84mol), (3-(bromomethyl)phenyl)carbamic acid tert-butyl ester (200g, 0.70mol) and potassium carbonate (350g, 2.5mol) were added to acetonitrile (780mL) at room temperature, and then heated to 50°C. After 5 hours, the reaction was diluted with water (600mL) and extracted with EtOAc (800mL×2). The combined organic layer was washed with saturated NaCl aqueous solution (800mL) and concentrated under reduced pressure to obtain a residue. The residue was purified by silica gel chromatography, eluted with 50:1 petroleum ether:EtOAc to obtain a crude product. The crude product was ground with MTBE (500mL) at room temperature for 30 minutes to obtain the title compound (103g, 32%). ES / MS m / z 382.1(M+Na).

[0212] Preparation 5

[0213] (6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(3-((3-aminobenzyl)oxy)-2- Fluoro-6-methylphenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a, 12,12a,12b-Dodecahydro-4H-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxol-4-one

[0214]

[0215] Perchloric acid (70% aqueous solution, 4.8 mL) was added to a suspension of (8S,9S,10R,11S,13S,14S,16R,17S)-11,16,17-trihydroxy-17-(2-hydroxyacetyl)-10,13-dimethyl-7,8,9,11,12,14,15,16-octahydro-6H-cyclopenta[a]phenanthrene-3-one (4.4 g, 12 mmol, also known as "16α-hydroxyprednisolone") and tert-butyl N-[3-[(2-fluoro-3-formyl-4-methyl-phenoxy)methyl]phenyl]carbamate (4.0 g, 11 mmol, Preparation 4) in acetonitrile (110 mL) at -10°C and warmed to room temperature. After 1 hour, DMF (10 mL) was added to the suspension at room temperature. After 18 hours, the reaction was quenched with saturated NaHCO3 aqueous solution and extracted with 9:1 DCM:isopropanol. The organic layers were combined, dried over MgSO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by reverse phase chromatography, eluted with 1:1 NH4HCO3 aqueous solution (10mM+5% MeOH):ACN to give the title compound, peak 1 (1.72g, 25%). ES / MS m / z 618.6 (M+H). 1H NMR(400.13MHz,d6-DMSO)δ0.93-0.87(m,6H),1.40(s,3H),1.71-1.60(m,1H),1.89-1.76(m,4H),2.1 8-2.12(m,2H),2.29(s,4H),4.23-4.17(m,1H),4.32-4.30(m,1H),4.50-4.43(m,1H),4.81(d,J=3.2Hz ,1H),4.98-4.95(m,3H),5.16-5.10(m,3H),5.61(s,1H),5.95(s,1H),6.18-6.15(m,1H),6.53-6.48(m ,2H),6.58(s,1H),6.90-6.86(m,1H),6.99(t,J=7.7Hz,1H),7.12(t,J=8.5Hz,1H),7.33-7.30(m,1H).

[0216] Preparation 6

[0217] (6aR,6bS,7S,8aS,8bS,10S,11aR,12aS,12bS)-10-(3-((3-aminobenzyl)oxy)-2- Fluoro-6-methylphenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a, 12,12a,12b-dodecahydro-4H-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxol-4-one (hereinafter referred to as Also known as GC1)

[0218]

[0219] From Preparation 5, the residue was purified by reverse phase chromatography eluting with 1:1 aqueous NH4HCO3 (10 mM + 5% MeOH):ACN to give the title compound, Peak 2 (1.24 g, 18%). ES / MS m / z 618.6 (M+H). 1 H NMR (400.13 MHz, d6-DMSO) 1H NMR(400.13MHz, DMSO):0.88(s,3H),1.24-1.12(m,2H),1.40(s,3H),1.69-1.56(m,1H),1.91-1.76(m,4H),2.08-2.01(m ,2H),2.22(s,3H),2.39-2.29(m,1H),3.18(d,J=5.2Hz,1H),4.12-4.00(m,1H),4.37-4.30(m,2H),4.79(d,J=3.1Hz,1H) ,5.00-4.93(m,2H),5.10-5.06(m,3H),5.31(d,J=6.7Hz,1H),5.95(s,1H),6.18(dd,J=1.8,10.1Hz,1H),6.34(s,1H),6. 53-6.48(m,2H),6.58(s,1H),6.87(d,J=8.5Hz,1H),6.99(t,J=7.7Hz,1H),7.09(t,J=8.5Hz,1H),7.33(d,J=10.1Hz,1H).

[0220] Preparation 7

[0221] (3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl)-L-alanyl-L-alanine

[0222]

[0223] To a solution of N-succinimidyl 3-maleimidopropionate (5.0 g, 19 mmol) and L-alanyl-L-alanine (3.4 g, 21 mmol) in DMF (25 mL) was added DIPEA (3.1 mL, 18 mmol), and the mixture was stirred at room temperature overnight. The reaction mixture was concentrated under reduced pressure to give a residue, which was purified by silica gel chromatography, eluting with 2% acetic acid in EtOAc to give the title compound (4.0 g, 69%). ES / MS m / z 312.3 (M+H).

[0224] Preparation 8

[0225] 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((S)-1-(((S)-1-((3-((2-fluoro-3- ((6aR,6bS,7S,8aS,8bS,10S,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl 4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2′,1′:4,5]indeno[1, 2-d][1,3]dioxol-10-yl)-4-methylphenoxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino 1-(2 ...

[0226]

[0227] To (6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(3-((3-aminobenzyl)oxy)-2-fluoro-6-methylphenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno [1,2-d][1,3]dioxol-4-one (24g, 39mmol, see Preparation 6) and 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propionyl)-L-alanyl-L-alanine (15g, 47mmol, see Preparation 7) in a solution of DMF (250mL) were added 2,6-lutidine (11mL, 97mmol) and then HATU (17g, 43mmol). The mixture was stirred at 0-5°C for 5 minutes, then the cooling bath was removed and the mixture was stirred for 2 hours. The mixture was diluted with EtOAc. The organic solution was washed with three parts of water, one part of a saturated aqueous NaCl solution, dried over Na2SO4 (MeOH was added to help dissolve), filtered and evaporated to give a crude product. The crude product was purified by silica gel chromatography using a gradient of 1% to 10% MeOH of DCM to give the title compound (24g, 68%). ES / MS m / z 911.4 (M+H). 1 H NMR(400.13MHz,DMSO):d 9.88(s,1H),8.20(d,J=7.1Hz,1H),8.11(d,J=7.2Hz,1H),7.68(s,1H),7.60-7.58(m,1H),7.34-7.29(m,2H),7.14-7.09(m, 2H),7.00(s,2H),6.89(d,J=8.4Hz,1H),6.34(s,1H),6.18(dd,J=1.8,10.0Hz,1H),5.95(s,1H),5.76(s,1H),5.31(d,J=6.8 Hz,1H),5.13-5.04(m,3H),4.78(d,J=3.1Hz,1H),4.41-4.30(m,4H),4.10-4.00(m,1H),3.61(t,J=7.3Hz,2H),2.42-2.31(m ,3H),2.22(s,3H),2.11-2.01(m,2H),1.91-1.78(m,5H),1.40(s,3H),1.31(d,J=7.2Hz,3H),1.19-1.11(m,5H),0.88(s,3H).

[0228] Preparation 9

[0229] 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((S)-1-(((S)-1-((3-((2-fluoro-3- ((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethoxy 4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2′,1′:4,5]indeno[1, 2-d][1,3]dioxol-10-yl)-4-methylphenoxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino (2-(1-oxypropane-2-yl)acrylamide

[0230]

[0231] In a manner analogous to the procedure described in Preparation 8, the compound of Preparation 9 was prepared from (6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(3-((3-aminobenzyl)oxy)-2-fluoro-6-methylphenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one (see Preparation 5) and 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl)-L-alanyl-L-alanine (see Preparation 7). ES / MS m / z 911.4(M+H). 1HNMR(500.11MHz,DMSO):d 9.88(s,1H),8.23-8.20(m,1H),8.11(d,J=7.2Hz,1H),7.69(s,1H),7.59(d,J=8.0Hz,1H),7.33-7.28(m,2H),7.15-7.08(m, 2H),7.00(s,2H),6.91-6.89(m,1H),6.17(dd,J=1.7,10.1Hz,1H),5.94(s,1H),5.61(s,1H),5.16-5.12(m,3H),4.98-4.96( m,1H),4.81(d,J=3.1Hz,1H),4.49-4.36(m,6H),3.61(t,J=7.3Hz,2H),2.41(t,J=7.3Hz,2H),2.30-2.29(m,4H),2.17-2.15 (m,2H),1.88-1.77(m,4H),1.69-1.61(m,1H),1.40(s,3H),1.31(d,J=7.2Hz,3H),1.18(d,J=7.2Hz,3H),0.93-0.87(m,6H).

[0232] Example

[0233] Example 1. Production of anti-human TNFα antibody glucocorticoid conjugate (anti-human TNFα Ab GC conjugate)

[0234] Example 1a: Generation and engineering of anti-human TNFα antibodies .

[0235] Antibody production: In order to develop human-specific antibodies TNFα, transgenic mice with human immunoglobulin variable regions were immunized with recombinant human TNFα. Human TNFα was screened and tested for cross-reactivity with other TNFα substances. Antibodies that cross-react with human and crab monkey TNFα were cloned, expressed and purified by standard procedures, and neutralization was tested in TNFα-induced cytotoxicity assays. Antibodies were selected and engineered in their CDRs, variable domain framework regions, and IgG isotypes to improve binding affinity and developable properties, such as stability, solubility, viscosity, hydrophobicity, and aggregation.

[0236] The amino acid sequence of human TNFα is provided in SEQ ID NO:39, and the amino acid sequence of cynomolgus monkey TNFα is provided in SEQ ID NO:42.

[0237] Anti-human TNFα antibodies can be synthesized and purified by known methods. If two vectors are used, or a single vector system encoding heavy and light chains, a predetermined HC:LC vector ratio can be used to transiently or stably transfect a suitable host cell (such as Chinese hamster ovary cells (CHO)) with an expression system for secreting antibodies. Commonly used techniques can be used to purify clarified culture media that have secreted antibodies.

[0238] Antibody Engineering for Improved Viscosity: The parental TNFα antibody lineage was found to have high viscosity when concentrated. Viscosity is a key developability criterion for assessing the feasibility of delivering therapeutic antibodies, such as by automatic injectors. Mutagenesis analysis of antibodies requires a balance between improving biophysical properties and maintaining desired affinity and potency without increasing the risk of immunogenicity. Computer modeling of the parental antibody is used to identify charge imbalance regions consisting of six complementary determining regions (CDRs) in the surface. Antibodies produced by mutagenesis are screened for TNFα binding, and antibodies that retain or improve target binding (as determined by ELISA) and have desired viscosity and other developable properties compared to the parental mAb are selected for further development.

[0239] Antibody Engineering to Reduce Immunogenicity Risk: Anti-human TNFα antibodies were tested in the MHC-associated peptide proteomics (MAPPS) assay to determine immunogenicity risk. Briefly, major histocompatibility complex (MHC) binding peptides of antibodies with specific CDR sequences were identified. CDR libraries with mutations identified as potentially reducing immunogenicity were constructed and screened for TNFα binding. Antibodies were screened and selected to optimize low immunogenicity risk while balancing the desired binding affinity to TNFα and other desired developable properties.

[0240] Table 2a, Table 2b and Table 3 show exemplary anti-human TNFα antibody sequences optimized for low viscosity, acceptable immunogenicity risk and other desirable developable properties, while retaining desired binding potency to human TNFα.

[0241] Table 2a: CDR amino acid sequences of exemplary anti-human TNFα Abs

[0242]

[0243]

[0244] Table 2b: CDR amino acid consensus sequences of exemplary anti-human TNFα Abs

[0245]

[0246] Table 3: Amino acid sequences of exemplary anti-human TNFα Abs

[0247]

[0248] Example 1b. Generation of anti-human TNFα Ab1 GC conjugate, wherein n is 4

[0249]

[0250] wherein n is 4; and

[0251] Ab is Ab1.

[0252] The exemplified anti-human TNFα antibody Ab1 (see Table 2a, Table 2b and Table 3) was first reduced at 37°C for 2 hours or at about 21°C for >16 hours in the presence of a 40-fold molar excess of dithiothreitol (DTT). This initial reduction step was used to remove various capping groups, including cysteine ​​and glutathione, which were bound to the engineered cysteines at positions 124 and 378 of the heavy chain during expression. After the reduction step, the sample was purified by a desalting column to remove unbound caps as well as the reducing agent. A subsequent 2-hour oxidation step was performed at room temperature (about 21°C) in the presence of a 10-fold molar excess of dehydroascorbic acid (DHAA) to reform the native interchain disulfide and hinge disulfide pairs between the light and heavy chains. After the 2 h oxidation step, 4-8 molar equivalents of the glucocorticoid receptor agonist payload-linker ("GC-L"), 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((S)-1-(((S)-1-((3-((2-fluoro-3-((6aR,6bS,7S,8aS,8bS,10S,11aR,12aS prepared in Preparation 8, were added using a 10 mM stock solution in DMSO. ,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)-4-methylphenoxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)propanamide. The sample is then incubated at room temperature for 30-60 minutes to allow efficient conjugation of GC-L with engineered cysteine. A subsequent polishing step (such as size exclusion chromatography (SEC) or tangential flow filtration (TFF)) is then used to buffer exchange the sample into a suitable formulation buffer and remove DMSO and any excess linker-payload.

[0253] Drug to Antibody Ratio (DAR) Assessment: To evaluate the average number of linkers-payloads present on the final conjugates, two analytical methods were used: 1) reverse phase (RP) HPLC and 2) time of flight (TOF) mass spectrometry. Both methods require an initial sample reduction step that includes the addition of dithiothreitol (DTT) to a final concentration of approximately 10 mM, followed by incubation at 42°C for 5 minutes.

[0254] Reverse phase HPLC method: 10 μg to 30 μg of reduced anti-human TNFα antibody Ab1 GC conjugate sample was injected onto a Phenyl 5PW, 4.6 mm × 7.5 cm, 10 μm column (Tosh Part # 0008043). A buffer consisted of 0.1% trifluoroacetic acid (TFA) in water, and B buffer consisted of 0.1% trifluoroacetic acid (TFA) in acetonitrile (ACN). Prior to sample injection, the column was equilibrated in 20% B buffer, followed by a gradient of 28% B to 40% B in approximately 8.5 column volumes. The average DAR was determined by calculating the contribution of each individual DAR substance by multiplying the fractional percentage by the DAR number of each contributing substance. Since this value is based on a partially reduced sample and represents only half of the molecule, this number is multiplied by 2 to account for the complete antibody GC conjugate. The DAR calculation for the anti-human TNFα Ab1 GC conjugate of Example 1b is provided in Table 4.

[0255] Table 4: Percentage of fractions of each DAR species from partially reduced samples against human TNFα Ab1 GC conjugation Quantification of the average DAR of a species .

[0256]

[0257] Time-of-flight mass spectrometry method: 8 μg of the partially reduced sample was injected onto a Poroshell 300sb-C3 2.1×2.5 mm, 5 μM column (Agilent Part #821075-924). Buffer A consisted of 0.1% trifluoroacetic acid (TFA) in water, while buffer B consisted of 0.1% trifluoroacetic acid (TFA) in acetonitrile (ACN). Prior to sample injection, the column was equilibrated in 0% B buffer, followed by a gradient of 10% B to 80% B in approximately 28 column volumes. The average DAR was determined by calculating the contribution of each individual DAR substance by multiplying the fractional percentage by the DAR number of each contributing substance. Since this value is based on a partially reduced sample and represents only half of the molecule, this number is multiplied by 2 to account for the complete antibody GC conjugate. The DAR calculations for the anti-human TNFαAb1 GC conjugate of Example 1b are provided in Table 5.

[0258] Table 5: Use of fractional percentages based on total ion counts from time-of-flight mass spectrometry to target human TNFα Quantification of average DAR of Ab1 GC conjugates .

[0259]

[0260]

[0261] Example 1c. Production of anti-human TNFα Ab1 GC conjugate, wherein n is 3

[0262]

[0263] wherein n is 3; and

[0264] Ab is Ab1.

[0265] The conjugate of Example 1c was prepared in a manner similar to the procedure described in Example 1b, using a lower molar ratio of GC-L, 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((S)-1-(((S)-1-((3-((2-fluoro-3-((6aR,6bS,7S,8aS,8bS,10S,11aR,12aS,12bS)-7-hydroxy-8b- (2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)-4-methylphenoxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)propanamide with Ab1. For example, using a corresponding GC-L:Ab1 molar ratio of 3.2:1 produced a final DAR of about 3.

[0266] Example 1d. Generation of anti-human TNFα Ab2 GC conjugate, wherein n is 4

[0267]

[0268] wherein n is 4; and

[0269] Ab is Ab2.

[0270] The conjugate of Example 1d was prepared essentially in a manner similar to the procedure described in Example 1b, using Ab2 instead of Abl.

[0271] Example 1e. Generation of anti-human TNFα Ab2 GC conjugate, where n is 3

[0272]

[0273] wherein n is 3; and

[0274] Ab is Ab2.

[0275] The conjugate of Example 1e was prepared essentially in a manner similar to the procedure described in Example 1c, using Ab2 instead of Abl.

[0276] Example 1f. Sulfosuccinimide Hydrolysis: Conjugates Where n is 4 The sulfosuccinimide ring of Formula Ie can be hydrolyzed under conditions well known in the art, as shown in Scheme 2 below (see, for example, WO 2017 / 210471, paragraph 001226) to provide a ring-opened product of Formula If.

[0277] Solution 2

[0278]

[0279] Furthermore, the above-mentioned thiosuccinimide ring of the conjugate of Formula Ie can undergo at least partial hydrolysis in vivo and under standard or well-known formulation conditions to provide the ring-opened product of Formula If.

[0280] Example 2. Binding efficacy of anti-human TNFα Ab1 GC conjugate and anti-human TNFα antibody

[0281] Example 2a. Elisa Binding: The binding potency of the exemplary anti-human TNFα Ab1 GC conjugate and anti-human TNFα antibody of Example 1b to human, rhesus and / or canine TNFα proteins was tested in an antigen down-modulation ELISA format. Briefly, 384-well high binding plates (Greiner Bio-one #781061) were diluted with 20 μL per well of 1 μg / mL human TNFα (Syngene), 2 μg / mL rhesus TNFα (R&D Systems, catalog number 1070-RM) or 2 μg / mL canine TNFα (R&D Systems, catalog number 1507-CT / CF) in carbonate buffer (0.015 M Na2CO3 and 0.035 M NaHCO3) at pH 9.3 and stored overnight at 4°C. The next day, the plate was blocked with 80 μL of casein (Thermo Fisher Pierce, catalog number 37528) at room temperature for 1 hour, the blocking buffer was removed, and 20 μL of titrated anti-human TNFα antibody expressed in CHO cells and anti-human TNFα Ab1 GC conjugate (starting concentration of 20 μg / mL, diluted and titrated 3 times in casein, down 8 points) was added to the plate. The plate was incubated at 37°C for 90 minutes and then washed three times in PBS / 0.1% Tween. 20 μL of secondary antibody reagent goat-anti-human-κ-AP (Southern Biotech, catalog number 2060-04) diluted 1:1500 was added to the plate and incubated at 37°C for 45 minutes. The plate was washed 3 times as above, and 20 μL of alkaline phosphatase substrate solution diluted to 1:35 in molecular grade water was added to each well. Once color developed (approximately 15-30 minutes), the plate was read at 560 nM OD on a Molecular Device Spectramax plate reader and data acquired using Softmax Pro 4.7 software. Data analysis was performed in GraphPad Prism.

[0282] The results in Table 6a show that the exemplified anti-human TNFα Ab1 GC conjugate of Example 1b binds human TNFα with the desired potency, which is comparable to the unconjugated anti-human TNFα Ab1.

[0283] The representative results in Table 6b show that the anti-human TNFα antibodies Abl, Ab2, Ab3, Ab4, Ab5, and Ab6 cross-react with human, rhesus monkey, and canine TNFα.

[0284] Table 6a. Binding EC values ​​of the exemplary anti-human TNFα Ab1GC conjugate of Example 1b to human TNFα 50

[0285] <![CDATA[Human TNFα EC 50 (μg / mL)]]> Ab1 0.280 Ab1 GC conjugate 0.325

[0286] Table 6b. Binding EC values ​​of exemplary anti-human TNFα antibodies to human, rhesus monkey and canine TNFα 50

[0287]

[0288] Example 2b. Cell surface binding: To evaluate the binding of the exemplified anti-human TNFα Ab1GC conjugate of Example 1b to cells expressing live membrane TNFα, a set of mutations previously demonstrated to allow expression of biologically active TNFα on the cell surface was used to inactivate the known TNFα cleavage site in the absence of TNFα cleavage (Mueller et al., 1999). The non-cleavable TNFα construct was stably transfected into Chinese hamster ovary (CHO) cells. As shown by flow cytometry, these cells expressed membrane-bound TNFα.

[0289] CHO cells transfected with TNFα were incubated with the exemplified anti-human TNFαAb1 GC conjugates at concentrations ranging from 600nM to 0.0304nM (three-fold dilution) in FACS buffer (PBS containing 2% FBS) at 4°C for 30 minutes. According to the manufacturer's protocol, secondary detection was performed with goat anti-human IgG F(ab')2 labeled with AlexaFluor-647 (Thermo#A20186) to demonstrate cell binding. Transfected CHO cells incubated with the exemplified anti-human TNFαAb1 GC conjugates were washed with FACS buffer and then stained with 2μg / mL of AlexaFluor-647 labeled goat anti-human IgG F(ab')2 in FACS buffer at 4°C for 30 minutes. The stained cells were washed, resuspended in FACS buffer, and analyzed on a BD LSRFortessa cell analyzer. Staining was repeated twice. A human IgG1 isotype control antibody was used as a negative control. Anti-human TNFαAb1 was used as a positive control.

[0290] Flow cytometry data were analyzed using FlowJo (v10.8.0) to obtain the mean fluorescence intensity (MFI) of AlexaFluor-647 for each test sample. EC was obtained by fitting a nonlinear regression (4PL curve) to the plotted MFI data using GraphPad Prism 9. 50 value.

[0291] The results in Table 7 show that conjugation of GC to anti-human TNFα Ab1 did not significantly affect the binding of anti-human TNFα Ab1 GC conjugate to membrane-expressed TNFα.

[0292] Table 7. Binding of the exemplary anti-human TNFα Ab1 GC conjugate of Example 1b to membrane human TNFα

[0293] <![CDATA[Binding to Membrane TNFα EC 50 (nM)]]> hIgG1 Isotype Control not applicable Ab1 4.258 Ab1 GC conjugate 5.292

[0294] Example 3: In vitro functional characterization of anti-human TNFα Ab GC conjugate

[0295] Example 3a. Internalization: The ability of the exemplified anti-human TNFα Ab1 GC conjugate and anti-human TNFα antibody of Example 1b to bind membrane-bound TNFα and internalize into human CD14+ monocytes derived from dendritic cells (DC) from different healthy human donors was evaluated. CD14+ monocytes were isolated from peripheral blood mononuclear cells (PBMCs), cultured, and differentiated into immature dendritic cells (with IL-4 and GM-CSF), all using standard protocols. To obtain mature DCs, cells were treated with 1 μg / mL LPS (lipopolysaccharide) for 4 hours.

[0296] The exemplified anti-human TNFαAb1 GC conjugate and anti-human TNFα antibody were diluted at 8 μg / mL in complete RPMI medium and mixed with equal volumes of detection probe Fab-TAMRA-QSY7 diluted to 5.33 μg / mL in complete RPMI medium, then incubated at 4°C in the dark for 30 minutes to form a complex, then added to immature and mature DC cultures, and incubated at 37°C in a CO2 incubator for 24 hours. The cells were washed with 2% FBS PBS and resuspended in 100 μL 2% FBS PBS containing CytoxGreen live / dead dye. Data were collected on BD LSR Forttessa X-20 and analyzed in FlowJo. Live single cells were gated and the percentage of TAMRA fluorescence-positive cells was recorded as a readout. In order to allow comparison of molecules with data generated from different donors, a normalized internalization index was used. The internalization signal was normalized to the IgG1 isotype (normalized internalization index = 0) and the internal positive control PC (normalized internalization index = 100) using the following calculation:

[0297]

[0298] Where X TAMRA , IgG1 isotype TAMRA and PC TAMRA are the percentages of the TAMRA-positive population for test molecule X, IgG1 isotype and PC, respectively.

[0299] The results in Table 8a show that the anti-human TNFαAb1 GC conjugate is internalized into dendritic cells in vitro after binding to TNFα expressed on mature dendritic cells, with an internalization index comparable to that of unconjugated anti-human TNFαAb1. This indicates that the conjugation of glucocorticoid to anti-human TNFαAb1 does not affect the internalization function of anti-human TNFαAb1.

[0300] Representative results from different donors in Table 8b show that anti-human TNFα antibodies Abl, Ab2, Ab3, Ab4, Ab5 and Ab6 are internalized into immature and mature dendritic cells after binding to TNFα on the cell surface.

[0301] Table 8a: Internalization of the exemplary anti-human TNFα Ab1 GC conjugate of Example 1b into dendritic cells

[0302] Mature dendritic cell internalization index Control hIgG1 0 Ab1 44.5 Ab1 GC conjugate 39.5

[0303] Table 8b: Internalization of exemplary anti-human TNFα antibodies into dendritic cells

[0304] Immature dendritic cell internalization index Mature dendritic cell internalization index Ab1 19.4 26.7 Ab2 21.9 38.3 Ab3 20.0 35.2 Ab4 33.6 43.1 Ab5 54.6 52.7

[0305] Example 3b. Inhibition of soluble and membrane TNFα-induced apoptosis: The exemplified anti-human TNFα Ab1 GC conjugate and anti-human TNFα antibodies of Example 1b were evaluated for inhibition of soluble and membrane TNFα-induced apoptosis in an in vitro cell-based assay.

[0306] Inhibition of soluble TNFα-induced apoptosis: In vitro evaluation of the ability of the exemplified anti-human TNFα Ab1GC conjugate and anti-human TNFα antibody to inhibit soluble TNFα-induced L929 cell apoptosis assay. L929 mouse fibrosarcoma cells naturally express TNF receptors. When combined with actinomycin D, TNFα induces classical apoptosis in these cells, resulting in rapid cell death due to excessive formation of reactive oxygen intermediates that can be rescued by TNFα neutralization. Briefly, L929 were cultured in assay medium (1×DMEM medium, 10% FBS, 1% Pen-Strep, 1% MEM essential amino acids, 1% L-glutamine, 1% sodium pyruvate). On the day of the assay, cells were washed with 1×PBS (Ca-free). ++ or Mg ++ ) and detached from the culture flask with 0.25% trypsin + EDTA. Trypsin was inactivated with assay medium. L929 cells were centrifuged at 1500 rpm for 5 minutes at room temperature. The cell pellet was resuspended in assay medium and 1×10 4L929 cells (in 100 μL) were added to a 96-well plate and placed in a tissue culture incubator (37°C, 95% relative humidity, 5% CO2) overnight. The conjugate / TNFα / actinomycin D mixture (TNFαAb1 GC conjugate and antibody added at 15 μg / mL to 0.0005 μg / mL, with three-fold dilutions) was transferred to a 96-well plate with L929 adherent cells and incubated (37°C, 95% relative humidity, 5% CO2) for 18 hours.

[0307] To determine the number of viable cells, the assay medium was removed and the MTS-tetrazolium substrate mix was added to the wells (100 μL) (where mitochondrial dehydrogenases in metabolically active cells reduce the MTS-tetrazolium to a colored formazan product). The plates were placed in an incubator (37°C, 95% relative humidity, 5% CO2) for 2 hours. Cell death was determined by reading the plates at 490 nm on a microplate reader (BiotekCytation 5 Imaging Multimode Reader). Results are expressed as the concentration at which 50% of TNFα-induced cytotoxicity is inhibited by the exemplified anti-human TNFα Ab1 GC conjugate or antibody (IC 50 ), calculated using a 4-parameter sigmoidal fit of the data (GraphPad Prism 9).

[0308] Inhibition of membrane TNFα-induced apoptosis: To evaluate the ability of the exemplified anti-human TNFα Ab1 GC conjugate and anti-human TNFα antibody of Example 1b to inhibit membrane TNFα-induced apoptosis, the non-cleavable TNFα construct was stably transfected into Chinese hamster ovary (CHO) cells to generate cell surface (membrane) CHO cells expressing TNFα. The non-cleavable TNFα construct was generated in the absence of TNFα cleavage, which has a known mutation at the cleavage site of TNFα, which allows biologically active TNFα to be expressed on the cell surface (Mueller et al., 1999). Incubation of L929 cells with CHO cells expressing human cleavable TNFα resulted in rapid L929 cell death. To determine whether the exemplified anti-human TNFα Ab1 GC conjugate and anti-human TNFα antibody can neutralize the observed apoptosis, a dose range of 15 μg / mL to 0.0005 μg / mL (with three-fold dilution) was evaluated. Each concentration of the exemplified anti-human TNFα Ab1 GC conjugate or anti-human TNFα antibody was added in duplicate at 100 μL / well to a plate containing 500 CHO TNFα transfected cells / well + 6.25 μg / mL actinomycin D. The mixture was incubated at room temperature for 30 minutes and then added to the L929 cell plate. A human IgG1 isotype control antibody was used as a negative control. L929 cell death was determined essentially as described for the soluble TNFα-induced apoptosis assay.

[0309] The results in Table 9a show that the exemplified anti-human TNFα Ab1 GC conjugate of Example 1b inhibited soluble human TNFα in vitro in a dose-dependent manner (IC of about 0.104 μg / mL) compared to anti-human TNFα Ab1. 50 ) and membrane human TNFα (IC of approximately 0.306 μg / mL 50 ) induced apoptosis in L929 cells. This indicates that conjugation of GC to the antibody does not affect the biological activity of the antibody. As expected, the negative control hIgG1 isotype did not inhibit TNFα-induced apoptosis.

[0310] The representative results in Table 9b show that the exemplified anti-human TNFα antibodies Abl, Ab2, Ab3, Ab4, Ab5 and Ab6 inhibited L929 cell soluble and membrane human TNFα-induced apoptosis in vitro in a dose-dependent manner.

[0311] Table 9a. The exemplified anti-human TNFα Ab1 GC conjugates of Example 1b inhibited the soluble and membrane expression of L929 cells. TNFα-induced apoptosis

[0312]

[0313] Table 9b. Exemplary anti-human TNFα antibodies inhibit apoptosis induced by membrane and soluble human TNFα in L929 cells

[0314]

[0315] Example 3c. In vitro human T cell cytokine expression assay: To evaluate the functional activity of the exemplified anti-human TNFαAb1 GC conjugate of Example 1b and the anti-human TNFαAb2 GC conjugate of Example 1d on disease-related cells, human primary T cells were stimulated in vitro and co-treated with the conjugates. The activity of the exemplary anti-human TNFαAb GC conjugate from US2020338208 was also evaluated. Human primary CD3 was isolated from freshly purified human PBMCs by immunomagnetic negative selection according to the manufacturer's protocol (Human T Cell Isolation Kit, Stemcell Technologies #17951). + T cells. Flow cytometry staining was used to assess cell purity on a BD LSRFortessa cell analyzer. Additional phenotyping of T cell subsets using CD4 (anti-human CD4-eFluor-450, Fisher Scientific #48-0047-42) and CD8 (anti-human CD8a-FITC, BioLegend #301006) was used to confirm that T cells were CD3 + (anti-human CD3-APC, Fisher Scientific #17-0036-42). 2×10 5 CD3+ T cells / well were seeded into 96-well flat bottom plates in assay medium (1× RPMI-1640 medium, 10% FBS, 1% non-essential amino acids, 1% sodium pyruvate, 1% Glutamax, 1% Pen-Strep, and 0.1% β-mercaptoethanol). 5 Cells were stimulated with human T activator anti-CD3 / CD28 Dynabeads (Thermo Fisher #11132D) and treated with each of the exemplified anti-human TNFαAb1 GC conjugates, anti-human TNFαAb2 GC conjugates, or exemplary anti-human TNFαAb GC conjugates from US2020338208 in duplicate plates per donor at 200 nM to 0.0914 nM (3-fold dilution). Human IgG1 isotype control antibody was used as a negative control. Controls included unconjugated anti-human TNFαAb1 and free GC. The assay plate was then incubated at 37 ° C with 5% CO2 for 72 hours. Cell culture supernatants were harvested at 72 hours and frozen at -80 ° C. Using customized U-PLEX Human Biomarker Multiplex Assays (Mesoscale Discovery # K15067L), cytokine levels were measured from thawed cell culture supernatants using detection antibodies specific to human IL-6, IL-10, IL-13 and GM-CSF. Activity was measured as inhibition of cytokines IL-6, IL-13, GM-CSF and induction of IL-10. For each individual donor, the detected cytokine levels (pg / mL) were converted to normalized "% inhibition" or "% induction" values. The normalization parameters for IL-6, IL-13 and GM-CSF were set to 0% inhibition equal to the average concentration of cytokines in the stimulated-untreated control wells, and 100% inhibition equal to the average concentration of cytokines in the unstimulated control wells. The standardized parameters for IL-10 were set to 0% induction equal to the average concentration of cytokines in the stimulated untreated control wells, and 100% induction equal to the average concentration of cytokines in the 200nM free GC treatment group. Normalized IC was obtained by fitting a nonlinear regression (4PL curve) to the normalized data. 50 Statistical analysis was performed using GraphPad Prism 9.

[0316] The results in Table 10 show that the anti-human TNFαAb GC conjugates of Example 1b and Example 1d significantly inhibited cytokines IL-13, IL-6, and GM-CSF, and significantly induced cytokine IL-10. In addition, in this in vitro assay, the anti-human TNFαAb GC conjugates of Example 1b and Example 1d inhibited cytokines IL-13, IL-6, and GM-CSF, and induced cytokine IL-10 at a percentage greater than that of the anti-human TNFαAb1 or exemplary anti-TNFαAb GC conjugates disclosed in US2020338208. In particular, the results show that the anti-human TNFαAb1 GC conjugate of Example 1b and the anti-human TNFαAb2 GC conjugate of Example 1d regulate cytokine expression via both TNFα antibodies and glucocorticoids.

[0317] Table 10. Anti-human TNFα Ab1 GC conjugate of Example 1b and anti-human TNFα Ab2 GC conjugate of Example 1d Effects on T cell cytokine release

[0318]

[0319] Statistics: Tukey's multiple comparison test, *=p<0.05 compared with isotype, ^=p<0.05 compared with Ab1, p<0.05 compared to the exemplary anti-human TNFα Ab conjugate from US2020338208.

[0320] Example 4. Effector function activity of exemplified anti-human TNFα Ab GC conjugates

[0321] Example 4a. Human Fcγ receptor binding. The binding affinity of the exemplified anti-human TNFα Ab1 GC conjugate of Example 1b to human Fcγ receptors was evaluated by surface plasmon resonance (SPR) analysis. Series S CM5 chips (Cytiva P / N BR100530) were prepared using the manufacturer's EDC / NHS amine coupling method (Cytiva P / N BR100050). Briefly, the surfaces of all 4 flow cells (FCs) were activated by injecting a 1:1 mixture of EDC / NHS at 10 μL / min for 7 minutes. Protein A (Calbiochem P / N 539202) was diluted to 100 μg / mL in 10 mM acetate, pH 4.5 buffer, and approximately 4000 RU were immobilized on all 4 FCs by injecting at a flow rate of 10 μL / min for 7 minutes. Unreacted sites were blocked by injecting ethanolamine at 10 μL / min for 7 minutes. Inject 2×10 μL of glycine, pH 1.5, for removing any non-covalently bound proteins. The running buffer is 1×HBS-EP+ (TEKNOVA, P / N H8022). FcγR extracellular domain (ECD)-FcγRI (CD64), FcγRIIA_131R and FcγRIIA_131H (CD32a), FcγRIIIA_158V, FcγRIIIA_158F (CD16a) and FcγRIIb (CD32b) were produced from stable CHO cells and purified using IgG Sepharose and size exclusion chromatography. For FcγRI binding, the test molecule (which includes the anti-human TNFαAb1 GC conjugate of Example 1b and the human IgG1 isotype control antibody) was diluted to 2.5 μg / mL in the running buffer, and about 150RU of each antibody (capture RU) was captured in FCs 2 to 4. FC1 is the reference FC, so there is no capture antibody in FC1. FcγRI ECD was diluted to 200nM in running buffer and then serially diluted two-fold to 0.78nM in running buffer. Duplicate injections of each concentration were injected into all FCs at 40μL / min for 120 seconds, followed by a dissociation period of 1200 seconds. Regeneration was performed by injecting 15μL of 10mM glycine, pH 1.5 at 30μL / min on all FCs. The reference-subtracted data were collected as FC2 FC1, FC3-FC1, and FC4-FC1, and measurements were obtained at 25°C. Affinity (K) was calculated using the "1:1 (Langmuir) binding" model in steady-state equilibrium analysis or BIA evaluation with Scrubber 2Biacore evaluation software. D). For FcγRIIa, FcγRIIb and FcγRIIIa binding, the test molecules were diluted to 5 μg / mL in running buffer and approximately 500 RU of each antibody were captured in FC 2 to 4. FC1 is the reference FC. The Fcγ receptor ECD was diluted to 10 μM in running buffer and then serially diluted 2-fold to 39 nM in running buffer. Duplicate injections of each concentration were injected into all FCs at 40 μL / min for 60 seconds, followed by a dissociation period of 120 seconds. Regeneration was performed by injecting 15 μL of 10 mM glycine, pH 1.5 at 30 μL / min on all FCs. The reference-subtracted data were collected as FC2-FC1, FC3-FC1 and FC4-FC1, and measurements were obtained at 25°C. Affinity (K) was calculated using steady-state equilibrium analysis with Scrubber2 Biacore evaluation software D ). Each receptor was assayed at least twice.

[0322] The results in Table 11 show the binding affinity (K) of the exemplified anti-human TNFα Ab1 GC conjugate of Example 1b to human FcγRI, FcγRIIa, FcγRIIb and FcγRIIIa receptor ECD. D ) is comparable to a human IgG1 isotype control antibody.

[0323] Table 11. Binding affinity of the exemplary anti-human TNFα Ab1GC conjugate of Example 1b to human Fcγ receptors

[0324]

[0325] Example 4b. Antibody-dependent cellular cytotoxicity (ADCC): In vitro ADCC assay of the exemplified anti-human TNFα Ab1 GC conjugate of Example 1b was evaluated using a reporter gene-based ADCC assay.

[0326] For the reporter gene-based ADCC assay, a CHO cell line co-expressing membrane-bound TNFα and CD20 (Eli Lilly and Co.) was used as the target cell line, and Jurkat cells expressing functional FcγRIIIa (V158)-NFAT-Luc (Eli Lilly) were used as the effector cell line. All test antibodies and cells were diluted in assay medium containing RPMI-1640 (phenol red-free) with 0.1 mM non-essential amino acids (NEAA), 1 mM sodium pyruvate, 2 mM L-glutamine, 500 U / mL penicillin-streptomycin and 0.1% w / v BSA. The test antibody was first diluted to a 3X concentration of 20 nM and then serially diluted 7 times at a 1:4 ratio. 50 μL / well of each antibody was aliquoted in triplicate in a white opaque bottom 96-well plate (Costar, #3917). The CD20 antibody was used as a positive control. Daudi target cells were then plated at 5×104 50 μL aliquots of 10 cells / well were added to the plate and incubated at 37°C for 1 hour. Next, Jurkat V158 cells were plated at 1.5 × 10 5 Cells / wells were added to wells with 50 μL aliquots and incubated at 37°C for 4 hours, followed by the addition of 100 μL / well One-Glo luciferase substrate (Promega, #E8130). Plate oscillator was used to mix the contents of the plate at low speed, incubated at room temperature for 5 minutes, and luminescent signals were read on a BioTek microplate reader (BioTek Instruments) using a 0.2cps integration. GraphPad Prism 9 was used to analyze data, and the relative luminescent units (RLU) of each antibody concentration were plotted in a scatter format of antibody concentration to RLU. The results represent two independent experiments.

[0327] Figure 1 The results in show that the exemplified anti-human TNFα Ab1GC conjugate of Example 1b has moderate ADCC activity compared to the positive control.

[0328] Example 4c. Complement-dependent cytotoxicity (CDC): An in vitro CDC assay of the exemplified anti-human TNFα Ab1 GC conjugate of Example 1b was performed using a CHO cell line (Eli Lilly) co-expressing membrane-bound TNFα and CD20. All test antibodies, complement, and cells were diluted in assay medium consisting of RPMI-1640 (without phenol red) with 0.1 mM non-essential amino acids (NEAA), 1 mM sodium pyruvate, 2 mM L-glutamine, 500 U / mL penicillin-streptomycin, and 0.1% w / v BSA. The test antibodies were first diluted to a 3X concentration of 200 nM and then serially diluted 7-fold at a 1:4 ratio. 50 μL / well of each antibody (including the CD20 positive control antibody) was aliquoted in triplicate in a white opaque bottom 96-well plate (Costar, #3917). 5×10 4Daudi target cells were added to each well, 50 μL / well, and incubated at 37°C for 1 hour. Next, human serum complement (Quidel, #A113) thawed quickly in a 37°C water bath was diluted 1:6 in the assay medium and added to the assay plate at 50 μL / well. The plate was incubated at 37°C for 2 hours, and then 100 μL / well CellTiter Glo substrate (Promega, #G7571) was added. The contents of the plate were mixed at low speed using a plate oscillator, incubated at room temperature for 5 minutes, and the luminescent signal was read on a BioTek microplate reader (BioTek Instruments) using a 0.2cps integration. GraphPad Prism 9 was used to analyze the data, and the relative luminescent units (RLU) of each antibody concentration were plotted in a scatter format of antibody concentration to RLU.

[0329] The results of two representative independent experiments (one of which is shown in Figure 2 Middle) shows that the exemplified anti-human TNFα Ab1 GC conjugate had marginal induction of CDC activity when compared to the positive control.

[0330] Example 5: Characterization of Exemplary Anti-Human TNFα Antibodies Binding to Anti-Drug Antibodies Against Adalimumab

[0331] Example 5a. Binding to cynomolgus anti-drug antibodies against adalimumab: The binding of the exemplified anti-human TNFα antibodies to anti-drug antibodies against adalimumab (anti-adalimumab antibodies) obtained from affinity purified hyperimmune monkey serum (AP-HIMS) of cynomolgus monkeys hyperimmunized with adalimumab was evaluated. Adalimumab-AffiGel10 was used to purify anti-adalimumab antibodies from cynomolgus monkeys hyperimmunized with adalimumab. Anti-adalimumab antibodies were detected using AP-HIMS titration in the ACE-Bridge assay. The assay was performed in accordance with FDA guidance on immunogenicity testing. Briefly, the streptavidin-coated 96-well plates (Pierce, 15500) were washed with 1X TBST (Boston BioProducts, IBB-181X) and coated with 30nM biotinylated adalimumab at 100 μL / well in TBST / 0.1% bovine serum albumin (BSA; Sigma, A7888) for 1 hour at room temperature. The plates were washed three times with TBST, and affinity-purified anti-adalimumab antibodies were diluted 1:10 with TBS (Fisher, BP2471-1), and added to the coated plates at 100uL / well and incubated overnight at 4°C. The next day, the plates were washed three times with TBST, and the captured anti-adalimumab antibodies were acid eluted for 5 minutes at room temperature using 65 μL / well of 300mM acetic acid (FisherScientific, A38-500). Then polypropylene 96-well plates (Corning, 3359) were loaded with 50 μL of biotinylated adalimumab and ruthenium-labeled adalimumab in neutralization buffer (0.375M Tris, 300mM NaCl, pH 9) at 1 μg / mL each. Next, 50 μL of acid-eluted samples were added to polypropylene plates containing a mixture and ADA in neutralization buffer, and allowed to bridge with labeled antibodies for 1 hour at room temperature. MSD Gold 96-well streptavidin plates (Mesoscale, L15SA-1) were washed and blocked with TBS+1% BSA at room temperature for 1 hour, then washed, and 80 μL of bridging samples were added to the plates for 1 hour. The plates were washed three times with TBST, and 150 μL / well of 2×MSD buffer (Mesoscale, R92TC-2) was added to the plates. Plates were read on an MSD SQ120 reader to provide a level 1 signal expressed as an electrochemiluminescent unit (EGUL).

[0332] The same AP-HIMS were also tested in the ACE-Bridge assay to detect antibodies against the exemplified anti-human TNFα antibody Ab6, essentially following the same method outlined above for adalimumab, but using biotin and ruthenium labeled Ab6. The resulting ECUL signal was then plotted as a function of the AP-HIMS concentration tested.

[0333] Figure 3 The results in show that the exemplified anti-human TNFα antibody Ab6 has low to no binding (maximum ECUL signal of 4000) to anti-drug antibodies against adalimumab purified from the serum of cynomolgus monkeys hyperimmunized with adalimumab, when compared to the binding of adalimumab to its own anti-drug antibodies (maximum ECUL signal of 4000). Specifically, the results show that the exemplified anti-human TNFα antibody Ab6 recognizes only about 10% of the anti-drug antibodies against adalimumab produced by cynomolgus monkeys, indicating that this binding may be due to shared sequences located away from the CDR region, such as the antibody constant region.

[0334] Example 5b. Binding to human patient anti-drug antibodies against adalimumab: Binding of exemplified anti-human TNFα antibodies to anti-drug antibodies against adalimumab (anti-adalimumab antibodies) in 21 patient serum samples obtained from patients treated with adalimumab enrolled in the study RA-BEAM was evaluated. 21 serum samples were collected after baseline and confirmed to have high ADA titers against adalimumab using essentially the same method as that used for the cynomolgus ADA evaluation. The 21 serum samples were then evaluated for binding to exemplified anti-human TNFα antibody Ab6 using essentially the same method as that used for the cynomolgus ADA evaluation.

[0335] Figure 4 The results in show that in 16 of the 21 patient samples tested, the exemplified anti-human TNFα antibody Ab6 had low to no binding (ECUL signal below the assay cutoff point (102ECUL)) to anti-drug antibodies directed against adalimumab. When determining immunogenicity, the cutoff point is the threshold used to identify samples that are "putatively positive" or contain anti-drug antibodies. Figure 4 As shown, five of the 21 samples had EGUL signals above the cutoff, but all were less than 20% above the cutoff and were therefore determined to be within the variability of the assay. The significantly low or no binding of anti-human TNFα antibody Ab6 to anti-drug antibodies against adalimumab in human patients treated with adalimumab indicates that Ab6 and adalimumab antibody sequences are sufficiently different that ADA generated by the tested human subjects against adalimumab (which is specific for an epitope present in adalimumab) is not shared by Ab6 and is therefore not significantly recognized by Ab6.

[0336] These results suggest the potential use of the exemplified anti-human TNFα antibodies or conjugates for treating subjects who have reduced clinical responses or adverse reactions to anti-drug antibodies to other TNFα therapeutics, such as adalimumab.

[0337] Example 6: Immunogenicity Assessment

[0338] Example 6a. MHC-associated peptide proteomics (MAPPs) assay: MAPPs profile peptides presented by MHC-II on human dendritic cells previously treated with the exemplified anti-human TNFα antibodies. CD14+ monocytes were isolated from peripheral blood mononuclear cells (PBMCs) using standard protocols, cultured, and differentiated into immature dendritic cells (with IL-4 and GM-CSF). The exemplified antibodies were added to the immature dendritic cells on day 4, and fresh medium containing LPS was replaced after 5 hours of incubation to convert the cells into mature dendritic cells. The next day, mature dendritic cells were lysed in RIPA buffer containing protease inhibitors and DNase. MHC-II complexes were immunoprecipitated using biotinylated anti-MHC-II antibodies coupled to streptavidin beads. The bound complexes were eluted and filtered. The isolated MHC-II peptides were analyzed by mass spectrometry. Peptide identifications were generated by an in-house proteomics pipeline, using a search algorithm without enzyme search parameters to search against a bovine / human database, with the test sequences appended to the database. Peptides identified from the exemplified antibodies were aligned against the parental sequences.

[0339] The results in Table 12 show that the exemplified anti-human TNFα antibodies have varying degrees of MAPPs presentation. Abl showed the lowest MAPPs presentation, with 1 non-germline cluster in 3 of 10 tested donors.

[0340] Table 12. MAPPs analysis of exemplary anti-human TNFα antibodies

[0341] Number of non-germline clusters in all donors Number of donors with >1 cluster Ab1 1 3 / 10 Ab2 2 6 / 10 Ab3 2 5 / 10 Ab4 3 5 / 10 Ab5 3 8 / 10

[0342] T cell proliferation assay: The ability of the exemplified anti-human TNFα antibody MAPPs-derived peptide clusters to activate CD4+ T cells by inducing cell proliferation was evaluated. CD8+ T cells were depleted from cryopreserved PBMCs from 10 healthy donors and labeled with 1 μM carboxyfluorescein diacetate succinimidyl ester (CFSE). CD8+ T cell-depleted PBMCs were incubated at 4×10 6 cells / mL / well were seeded in a 5% CTS TMImmune cell SR (Gibco, catalog number A2596101) in AIM-V medium (Life Technologies, catalog number 12055-083) and tested in triplicate in 2.0 mL containing different test molecules: DMSO control, medium control, keyhole limpet hemocyanin (KLH; positive control), PADRE-X peptide (synthetic vaccine adjuvant peptide, positive peptide control), or the corresponding anti-human TNFα antibody MAPPs-derived peptide clusters (10 μM per peptide). Cells were cultured and incubated at 37°C and 5% CO2 for 7 days. On day 7, samples were stained with the following cell surface markers: anti-CD3, anti-CD4, anti-CD14, anti-CD19, and DAPI to use a BD LSRFortessa equipped with a high-throughput sampler (HTS) TM Viability was measured by flow cytometry. Software (FlowJo, LLC, TreeStar) was used to analyze the data and calculate the cell division index (CDI). Briefly, the proliferation CFSE from the peptide-stimulated wells was dim The percentage of CD4+ T cells was divided by the proliferation of CFSE in the unstimulated wells dim The CDI of each MAPPs-derived peptide cluster was calculated using the percentage of CD4+ T cells. > A CDI of 2.5 was considered to represent a positive reaction. The donor frequency percentages were evaluated for all donors.

[0343] The results in Table 13a and Table 13b show that the LCDR1 (Table 13a) and HCDR3 (Table 13b) peptides of Ab2 induced T cell response frequencies in approximately 22.0% and 25% of donors, respectively, indicating that the immunogenicity risk of Ab2 was significantly reduced compared to the positive control. In both studies, the KLH positive control induced T cell responses in 100% of donors, and the PADRE-X (synthetic vaccine adjuvant peptide) positive control induced T cell responses in 67% and 62.5% of donors, respectively. This range falls within the expected range for this assay (48.1% + 24.4 positive donor frequencies).

[0344] Table 13a. Frequency of CD4+ T cell responses induced by MAPPs-derived peptides in healthy donors .

[0345]

[0346] Table 13b. Frequency of CD4+ T cell responses induced by MAPPs-derived peptides in healthy donors .

[0347]

[0348] Example 7. Biophysical properties of the exemplary anti-human TNFα Ab1 GC conjugate of Example 1b

[0349] The biophysical properties of the exemplified anti-human TNFα Ab1 GC conjugate of Example 1b were evaluated for developability.

[0350] Example 7a. Viscosity: Samples of the anti-human TNFα Ab1 GC conjugate of Example 1b were concentrated to approximately 50 mg / mL, 100 mg / mL, and 150 mg / mL in a common formulation buffer matrix at pH 6. The viscosity of the conjugates at all three concentrations was measured by initium (RheoSense) at 15°C using the average of 9 replicate measurements. The results in Table 15 show that the anti-human TNFαAb1 GC conjugate of Example 1b at 50 mg / mL (2 cP), 100 mg / mL (4.7 cP) and 125 mg / mL (8.2 cP) have good viscosity characteristics. The viscosity of the exemplified anti-human TNFαAb1 GC conjugate at 125 mg / mL (8.2 cP) is similar to the viscosity of the unconjugated anti-human TNFαAb1 at 125 mg / mL (8.8 cP), indicating that conjugating the linker-payload to the 4 engineered cysteines on the surface of the exemplified anti-human TNFαAb1 does not negatively affect the viscosity.

[0351] Example 7b. Thermal stability: Differential scanning calorimetry (DSC) was used to evaluate the stability of the anti-human TNFαAb1GC conjugate of Example 1b against thermal denaturation. The melting onset temperature (Tonset) and thermal melting temperatures (TM1, TM2 and TM3) of the exemplary anti-human TNFαAb1GC conjugate in PBS, pH 7.2 buffer, acetate, pH 5 and histidine, pH 6 were obtained by data fitting and are listed in Table 14. Figure 5A , Figure 5B and Figure 5C Thermograms of the three buffer compositions are depicted in . The thermal transitions of each domain were well resolved, and the results in Table 14 show that the anti-human TNFα Ab1 GC conjugate of Example 1b has good thermal stability.

[0352] Example 7c. Aggregation under temperature stress: The solution stability of the anti-human TNFαAb1 GC conjugate of Example 1b over time was evaluated at about 100 mg / mL and 50 mg / mL in a common 5 mM histidine pH 6.0 buffer with excipients. The samples were incubated at 5°C and 35°C for 28 days. After incubation, the percentage of high molecular weight species (% HMW) in the samples was analyzed using size exclusion chromatography (SEC-HPLC). The results in Table 15 show that the anti-human TNFαAb1 GC conjugate of Example 1b has an acceptable aggregation curve at 5°C or 35°C over a period of 4 weeks.

[0353] Example 7d. Pharmacokinetics: The PK profile of the anti-human TNFα Ab1 GC conjugate of Example 1b in cynomolgus monkeys was found to have an acceptable developability profile.

[0354] Table 14. Thermal stability of exemplary anti-human TNFα Ab1 GC conjugates of Example 1b (°C)

[0355]

[0356] Table 15: Viscosity and high concentration temperature stability of the exemplary anti-human TNFα Ab1 GC conjugate of Example 1b sex

[0357]

[0358] Example 8. In vivo function of the anti-human TNFα Ab GC conjugates of Example 1b, Example 1c and Example 1d

[0359] Example 8a. In vivo inhibition of human TNFα-induced CXCL1 cytokine :

[0360] The exemplified anti-human TNFαAb1 GC conjugate and anti-human TNFαAb1 of Example 1b were evaluated in vivo for neutralization of TNFα-induced CXCL1. Administration of human TNFα to C57 / B6 mice induced a rapid and transient increase in mouse plasma CXCL1 levels. This allowed the neutralization capacity of the exemplified anti-human TNFαAb1 and anti-human TNFαAb1 GC conjugate to be interrogated in vivo.

[0361] Briefly, C57 / B6 mice (N=8 / group) were subcutaneously (SC) administered with 0.3 mg / kg or 3 mg / kg doses of the exemplified anti-human TNFαAb1 GC conjugate or anti-human TNFαAb1 and 3 mg / kg of a non-binding isotype control. Twenty-four hours after dosing, mice were challenged with human TNFα at a dose of 3 μg / mouse via intraperitoneal injection. Two hours after human TNFα challenge, mice were sacrificed, blood was collected and clarified into plasma by centrifugation. Plasma was analyzed for mouse CXCL1 concentration using a commercial MSD assay (MesoScaleDiscovery, P / N.K152QTG-1) according to the manufacturer's instructions.

[0362] The results in Table 16 show that the exemplified anti-human TNFαAb1 and anti-human TNFαAb1 GC conjugate of Example 1b significantly inhibited human TNFα-induced plasma CXCL1 production in vivo relative to isotype control-treated mice (p<0.001, ANOVA, followed by Tukey's multiple comparison test), inhibiting about 80.5% and 81.6% at 3 mg / kg, respectively, and about 69.4% and 58.9% at 0.3 mg / kg, respectively. Importantly, there was no significant difference between anti-human TNFαAb1 GC conjugate and anti-human TNFαAb1 at the doses tested. This indicates that the exemplified anti-human TNFαAb1 neutralizes the biological effects induced by human TNFα in vivo, and conjugation to GC does not affect this function.

[0363] Table 16: Effect of the anti-human TNFα Ab1 GC conjugate of Example 1b on human TNFα-induced CXCL1 cytokine production In vivo inhibition

[0364]

[0365] Ordinary one-way ANOVA, Tukey test, multiple comparisons

[0366] Example 8b. In vivo efficacy in a fully humanized mouse model of type IV hypersensitivity: A humanized mouse model of contact hypersensitivity was used to determine the in vivo activity of the anti-human TNFα Ab1 GC conjugate and anti-human TNFα Ab1 of Example 1b and Example 1c.

[0367] Immunodeficient NOG mice (huNOG-EXL, Taconic) expressing human GM-CSF and human IL-3 to support myeloid development were transplanted with human CD34+ hematopoietic stem cells isolated from human umbilical cord blood at 6 weeks of age. 20-24 weeks after administration of stem cells, mice were evaluated for adequate human CD45 engraftment (>25% in the blood) and subjected to an oxazolidinone-induced contact hypersensitivity regimen. On day 0, mice grouped by weight were administered 1 mg / kg subcutaneously (SC) with anti-human TNFαAb1 GC conjugates of Example 1b (n=7), TNFαAb1 GC conjugates of Example 1c (n=7), anti-human TNFαAb1 (n=7), exemplary anti-human TNFαAb GC conjugates from US2020338208 (n=7), or control human IgG1 antibodies (n=7). On the first day, mice were anesthetized with 5% isoflurane, their abdomens were shaved, and 100 μL of 3% oxazolone in ethanol solution were applied to the shaved area. Mice were re-administered with 1 mg / kg SC on the 7th day, anesthetized, and then challenged with 2% oxazolone in ethanol solution to both ears 24 hours after administration (10 μL / side / ear). The dose challenge paradigm was repeated weekly; the dose of the test agent was increased to 3 mg / kg for attack 2 and to 10 mg / kg for attack 3. The inflammatory response was determined by the difference in ear thickness 24 hours before and after each attack using a Miltenyi Biotec electronic caliper. The P value between the groups was calculated by one-way ANOVA, followed by Tukey post hoc test, and was considered significant if <0.05 (GraphPad Prism).

[0368] Table 17 and FIG. 6A to FIG. 6C The results in show that the anti-human TNFαAb1 GC conjugates of Example 1b and Example 1c cause an excellent reduction in the in vivo inflammatory response from hapten-induced contact hypersensitivity reactions compared to both anti-human TNFαAb1 that reduced ear swelling only at 10 mg / kg attack 3 doses and the exemplary anti-human TNFαAb GC conjugate from US2020338208 at all 3 attacks (1 mg / kg, 3 mg / kg, and 10 mg / kg). In addition, the results show that conjugation of anti-human TNFαAb1 with 3 or 4 GC molecules (DAR) causes similar efficacy. These results indicate that anti-human TNFαAb1 GC conjugates effectively deliver glucocorticoids to inflamed tissues, and that glucocorticoids significantly eliminate the biological effects associated with type IV hypersensitivity reactions in humanized mouse models, indicating that this anti-inflammatory response can be elicited in human subjects.

[0369] Table 17: Anti-human TNFα Ab1 GC conjugates of Example 1b and Example 1c in type IV hypersensitivity humanized mice In vivo efficacy in models

[0370]

[0371] *p<0.05 vs isotype; ANOVA Tukey; ^p<0.05 vs Ab1; ANOVA Tukey

[0372] Example 8c. In vivo efficacy in a human TNFα transgenic mouse polyarthritis model: A human TNFα transgenic mouse polyarthritis model (Taconic, #1006) was used to evaluate the efficacy of the anti-human TNFαAb2 GC conjugate and anti-human TNFαAb2 of Example 1d as primary treatment in adalimumab naive mice and as secondary treatment in adalimumab-treated mice (i.e., adalimumab refractory mice) that produce anti-drug antibodies to adalimumab and have a reduced or lost response to adalimumab. This mouse model constitutively expresses human TNFα via a CMV promoter, resulting in progressive joint inflammation, primarily in the front and hind paws. Treatment with adalimumab attenuated disease progression for several weeks; however, this beneficial effect was attenuated due to the development of neutralizing anti-drug antibodies. To eliminate the potential of adalimumab to generate anti-drug antibodies directed against the human Fc portion of the antibody and thereby affect the activity of the anti-human TNFα Ab2 GC conjugate and anti-human TNFα Ab2 of Example 1d, all molecules were produced as chimeric species in which the antibody constant domains were replaced with those of a mouse IgG2a antibody.

[0373] At 13 weeks of age, once all mice showed moderate inflammation (score 4-9) in one or more paws, the mice were divided into 6 clinical score-matched groups of 8 mice each. Mice in each group were administered subcutaneously (SC) at 3 mg / kg per week with mIgG2a isotype control, human / mouse chimeric anti-human TNFαAb2 (referred to herein as "h / mAb2"), human / mouse chimeric anti-human TNFαAb2 GC conjugate (referred to herein as "h / mAb2-GC"), human / mouse chimeric adalimumab (referred to herein as "h / m-adalimumab"), h / m-adalimumab 2 doses for 9 weeks, then switched to human / mouse chimeric adalimumab GC conjugate (referred to herein as "h / m-adalimumab-GC"; prepared with engineered cysteine ​​and conjugated to GC-L as described in Example 1b), and h / m-adalimumab 2 doses, then switched to h / mAb2-GC for the duration of the study. The parameters for clinical scoring of each limb are as follows: 0 = no signs of deformity; 1 = mild deformity; 2 = moderate deformity; 3 = severe deformity / mild swelling; 4 = severe deformity / severe swelling / loss of function. Mice were scored twice a week and weighed routinely. Blood was collected on day 10 to quantify the level of antibody exposure and determine the extent of anti-drug antibody development. At the end of the experiment, mice were anesthetized with isoflurane, and blood and tissues were collected.

[0374] Figure 7 The results in show that compared with all other treatments, h / mAb2-GC and h / mAb2 completely prevented the progression of the disease after the start of treatment, and continued for the duration of 9 weeks of treatment, as measured by clinical scores. Importantly, this shows that h / mAb2-GC and h / mAb2 do not produce significant anti-drug antibody responses, so that it will neutralize and / or reduce the efficacy of the conjugate or antibody. The results also show that h / m-adalimumab can delay disease progression for about 2 weeks; however, around the 2nd week of the 9-week treatment, with the appearance of anti-drug antibodies to h / m-adalimumab, efficacy is lost. However, it is important that mice treated with h / m-adalimumab for 2 weeks and then switched to h / mAb2-GC maintained a significant elimination of disease progression, while mice treated with h / m-adalimumab for 2 weeks and then switched to h / m-adalimumab-GC showed an inflammatory response that reflected the h / m-adalimumab treatment group alone. These results indicate that the anti-human TNFα Ab2 GC conjugate has low to no cross-reactivity to anti-drug antibodies to adalimumab. These results indicate the potential use of the exemplified anti-human TNFα Ab conjugates in treating subjects who develop anti-drug antibodies to other anti-TNFα therapeutics, such as adalimumab, and have a diminished response to such treatment.

[0375] Sequence Listing

[0376] Ab1

[0377] SEQ ID NO: 1 HCDR1 of Ab1, Ab2 and Ab6

[0378] GYTFTGYYIH

[0379] SEQ ID NO:2 HCDR2 of Ab1, Ab2 and Ab6

[0380] WINPYTGGTNYAQKFQG

[0381] SEQ ID NO:3HCDR3 of Ab1

[0382] DLYGSSNYGGDV

[0383] SEQ ID NO:4 LCDR1 of Ab1 and Ab3

[0384] QASQGISNYLN

[0385] SEQ ID NO:5 LCDR2 of Ab1, Ab2, Ab3, Ab4, Ab5 and Ab6

[0386] DASNLET

[0387] SEQ ID NO:6 LCDR3 of Ab1, Ab3 and Ab5

[0388] QQYDKLPLT

[0389] SEQ ID NO: 7 VH of Ab1

[0390]

[0391] SEQ ID NO: 8 VL of Ab1 and Ab3

[0392]

[0393] SEQ ID NO: 9HC of Ab1

[0394]

[0395] SEQ ID NO: 10 LC of Ab1 and Ab3

[0396]

[0397] SEQ ID NO:11 HC DNA of Ab1

[0398]

[0399]

[0400] SEQ ID NO: 12 LC DNA of Ab1 and Ab3

[0401]

[0402]

[0403] Ab2

[0404] SEQ ID NO: 1 HCDR1 of Ab1, Ab2 and Ab6

[0405] GYTFTGYYIH

[0406] SEQ ID NO:2 HCDR2 of Ab1, Ab2 and Ab6

[0407] WINPYTGGTNYAQKFQG

[0408] SEQ ID NO: 13 HCDR3 of Ab2, Ab3, Ab4 and Ab5

[0409] DLYGSSNYGMDV

[0410] SEQ ID NO: 14 LCDR1 of Ab2, Ab4 and Ab5

[0411] QASQGIRNYLN

[0412] SEQ ID NO:5 LCDR2 of Ab1, Ab2, Ab3, Ab4, Ab5 and Ab6

[0413] DASNLET

[0414] SEQ ID NO:15 LCDR3 of Ab2 and Ab4

[0415] QQYDNLPLT

[0416] SEQ ID NO: 16 VH of Ab2

[0417]

[0418] SEQ ID NO: 17 VL of Ab2 and Ab4

[0419]

[0420] SEQ ID NO: 18 HC of Ab2

[0421]

[0422] SEQ ID NO: 19 LC of Ab2 and Ab4

[0423]

[0424] SEQ ID NO: 20 HC DNA of Ab2

[0425]

[0426] SEQ ID NO:21 LC DNA of Ab2 and Ab4

[0427]

[0428] Ab3

[0429] SEQ ID NO:22 HCDR1 of Ab3, Ab4 and Ab5

[0430] GYTFTGYYMH

[0431] SEQ ID NO:23 HCDR2 of Ab3, Ab4 and Ab5

[0432] WINPYTGGTKYAQKFQG

[0433] SEQ ID NO: 13 HCDR3 of Ab2, Ab3, Ab4 and Ab5

[0434] DLYGSSNYGMDV

[0435] SEQ ID NO:4 LCDR1 of Ab1 and Ab3

[0436] QASQGISNYLN

[0437] SEQ ID NO:5 LCDR2 of Ab1, Ab2, Ab3, Ab4, Ab5 and Ab6

[0438] DASNLET

[0439] SEQ ID NO:6 LCDR3 of Ab1, Ab3 and Ab5

[0440] QQYDKLPLT

[0441] SEQ ID NO: 24 VH of Ab3, Ab4 and Ab5

[0442]

[0443] SEQ ID NO: 8 VL of Ab1 and Ab3

[0444]

[0445] SEQ ID NO:25 HC of Ab3, Ab4 and Ab5

[0446]

[0447] SEQ ID NO: 10 LC of Ab1 and Ab3

[0448]

[0449] SEQ ID NO:26 HC DNA of Ab3, Ab4 and Ab5

[0450]

[0451] SEQ ID NO: 12 LC DNA of Ab1 and Ab3

[0452]

[0453] Ab4

[0454] SEQ ID NO:22 HCDR1 of Ab3, Ab4 and Ab5

[0455] GYTFTGYYMH

[0456] SEQ ID NO:23 HCDR2 of Ab3, Ab4 and Ab5

[0457] WINPYTGGTKYAQKFQG

[0458] SEQ ID NO: 13 HCDR3 of Ab2, Ab3, Ab4 and Ab5

[0459] DLYGSSNYGMDV

[0460] SEQ ID NO: 14 LCDR1 of Ab2, Ab4 and Ab5

[0461] QASQGIRNYLN

[0462] SEQ ID NO:5 LCDR2 of Ab1, Ab2, Ab3, Ab4, Ab5 and Ab6

[0463] DASNLET

[0464] SEQ ID NO:15 LCDR3 of Ab2 and Ab4

[0465] QQYDNLPLT

[0466] SEQ ID NO: 24 VH of Ab3, Ab4 and Ab5

[0467]

[0468] SEQ ID NO: 17 VL of Ab2 and Ab4

[0469]

[0470] SEQ ID NO:25 HC of Ab3, Ab4 and Ab5

[0471]

[0472] SEQ ID NO: 19 LC of Ab2 and Ab4

[0473]

[0474]

[0475] SEQ ID NO:26 HC DNA of Ab3, Ab4 and Ab5

[0476]

[0477]

[0478] SEQ ID NO:21 LC DNA of Ab2 and Ab4

[0479]

[0480] Ab5

[0481] SEQ ID NO:22 HCDR1 of Ab3, Ab4 and Ab5

[0482] GYTFTGYYMH

[0483] SEQ ID NO:23 HCDR2 of Ab3, Ab4 and Ab5

[0484] WINPYTGGTKYAQKFQG

[0485] SEQ ID NO: 13 HCDR3 of Ab2, Ab3, Ab4 and Ab5

[0486] DLYGSSNYGMDV

[0487] SEQ ID NO: 14 LCDR1 of Ab2, Ab4 and Ab5

[0488] QASQGIRNYLN

[0489] SEQ ID NO:5 LCDR2 of Ab1, Ab2, Ab3, Ab4, Ab5 and Ab6

[0490] DASNLET

[0491] SEQ ID NO:6 LCDR3 of Ab1, Ab3 and Ab5

[0492] QQYDKLPLT

[0493] SEQ ID NO: 24 VH of Ab3, Ab4 and Ab5

[0494]

[0495] SEQ ID NO: 27 VL of Ab5

[0496]

[0497] SEQ ID NO:25 HC of Ab3, Ab4 and Ab5

[0498]

[0499] SEQ ID NO: 28 LC of Ab5

[0500]

[0501] SEQ ID NO:26 HC DNA of Ab3, Ab4 and Ab5

[0502]

[0503]

[0504] SEQ ID NO: 29 LC DNA of Ab5

[0505]

[0506] Ab6

[0507] SEQ ID NO: 1 HCDR1 of Ab1, Ab2 and Ab6

[0508] GYTFTGYYIH

[0509] SEQ ID NO:2 HCDR2 of Ab1, Ab2 and Ab6

[0510] WINPYTGGTNYAQKFQG

[0511] SEQ ID NO: 30 HCDR3 of Ab6

[0512] DIYGSSNYGGDV

[0513] SEQ ID NO:31 LCDR1 of Ab6

[0514] QASQDISNYLN

[0515] SEQ ID NO:5 LCDR2 of Ab1, Ab2, Ab3, Ab4, Ab5 and Ab6

[0516] DASNLET

[0517] SEQ ID NO:32 LCDR3 of Ab6

[0518] QQYDTLPLT

[0519] SEQ ID NO:33 VH of Ab6

[0520]

[0521] SEQ ID NO:34 VL of Ab6

[0522]

[0523] SEQ ID NO:35 HC of Ab6

[0524]

[0525] SEQ ID NO: 36 LC of Ab6

[0526]

[0527] SEQ ID NO: 37 HC DNA of Ab6

[0528]

[0529]

[0530] SEQ ID NO: 38 LC DNA of Ab6

[0531]

[0532] SEQ ID NO: 39 Human TNFα protein

[0533] SEQ ID NO:40 Rhesus monkey TNFα protein

[0534] SEQ ID NO:41 Canine TNFα protein

[0535] SEQ ID NO:42 Cynomolgus monkey TNFα protein

[0536] SEQ ID NO:43 LCDR1 consensus sequence QASQGIXaa7NYLN

[0537] Where Xaa7 is serine or arginine

[0538] SEQ ID NO:44LCDR3 consensus sequenceQQYDXaa5LPLT

[0539] Wherein Xaa5 is asparagine or lysine SEQ ID NO: 45 Human TNFR1

[0540]

[0541] SEQ ID NO:46 Human TNFR2

[0542]

Claims

1. A conjugate of the formula: Wherein Ab is an antibody that binds to human TNFα, wherein Ab comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, and the VL comprises light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein: The HCDR1 comprises SEQ ID NO: 1 or 22; The HCDR2 comprises SEQ ID NO: 2 or 23; The HCDR3 comprises SEQ ID NO: 3, 13 or 30; The LCDR1 comprises SEQ ID NO: 4, 14, 31 or 43; The LCDR2 comprises SEQ ID NO:5; and The LCDR3 comprises SEQ ID NO: 6, 15, 32 or 44; in yes: And n is 1-5.

2. The conjugate according to claim 1, wherein yes:

3. The conjugate according to claim 1, wherein yes:

4. The conjugate according to claim 1, wherein yes:

5. The conjugate according to claim 1, wherein yes:

6. The conjugate according to claim 1, wherein yes:

7. The conjugate according to claim 1, wherein yes:

8. The conjugate according to claim 1, wherein yes:

9. The conjugate according to claim 1, wherein yes:

10. The conjugate according to any one of claims 1 to 9, wherein the antibody that binds to human TNFα comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, and the VL comprises light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein: The HCDR1 comprises SEQ ID NO: 1; The HCDR2 comprises SEQ ID NO: 2; The HCDR3 comprises SEQ ID NO: 3; The LCDR1 comprises SEQ ID NO:4; The LCDR2 comprises SEQ ID NO:5; and The LCDR3 comprises SEQ ID NO:

6.

11. The conjugate of claim 10, wherein the VH comprises SEQ ID NO: 7 and the VL comprises SEQ ID NO:

8.

12. The conjugate according to any one of claims 10 to 11, wherein the antibody comprises a heavy chain (HC) and a light chain (LC), wherein the HC comprises SEQ ID NO: 9 and the LC comprises SEQ ID NO:

10.

13. The conjugate according to any one of claims 1 to 9, wherein the antibody that binds to human TNFα comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, and the VL comprises light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein: The HCDR1 comprises SEQ ID NO: 1; The HCDR2 comprises SEQ ID NO: 2; The HCDR3 comprises SEQ ID NO: 13; The LCDR1 comprises SEQ ID NO: 14; The LCDR2 comprises SEQ ID NO:5; and The LCDR3 comprises SEQ ID NO:

15.

14. The conjugate of claim 13, wherein the VH comprises SEQ ID NO: 16 and the VL comprises SEQ ID NO:

17.

15. The conjugate of any one of claims 13 to 14, wherein the antibody comprises a heavy chain (HC) and a light chain (LC), wherein the HC comprises SEQ ID NO: 18 and the LC comprises SEQ ID NO:

19.

16. The conjugate of any one of claims 1 to 9, wherein the antibody that binds to human TNFα comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, and the VL comprises light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein: The HCDR1 comprises SEQ ID NO: 22; The HCDR2 comprises SEQ ID NO: 23; The HCDR3 comprises SEQ ID NO: 13; The LCDR1 comprises SEQ ID NO:4; The LCDR2 comprises SEQ ID NO:5; and The LCDR3 comprises SEQ ID NO:

6.

17. The conjugate of any one of claims 1 to 9, wherein the antibody that binds to human TNFα comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, and the VL comprises light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein: The HCDR1 comprises SEQ ID NO: 22; The HCDR2 comprises SEQ ID NO: 23; The HCDR3 comprises SEQ ID NO: 13; The LCDR1 comprises SEQ ID NO: 14; The LCDR2 comprises SEQ ID NO:5; and The LCDR3 comprises SEQ ID NO:

15.

18. The conjugate of any one of claims 1 to 9, wherein the antibody that binds to human TNFα comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, and the VL comprises light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein: The HCDR1 comprises SEQ ID NO: 22; The HCDR2 comprises SEQ ID NO: 23; The HCDR3 comprises SEQ ID NO: 13; The LCDR1 comprises SEQ ID NO: 14; The LCDR2 comprises SEQ ID NO:5; and The LCDR3 comprises SEQ ID NO:

6.

19. The conjugate of any one of claims 1 to 9, wherein the antibody that binds to human TNFα comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, and the VL comprises light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein: The HCDR1 comprises SEQ ID NO: 22; The HCDR2 comprises SEQ ID NO: 23; The HCDR3 comprises SEQ ID NO: 13; The LCDR1 comprises SEQ ID NO:43; The LCDR2 comprises SEQ ID NO:5; and The LCDR3 comprises SEQ ID NO:

6.

20. The conjugate of any one of claims 1 to 9, wherein the antibody that binds to human TNFα comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, and the VL comprises light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein: The HCDR1 comprises SEQ ID NO: 22; The HCDR2 comprises SEQ ID NO: 23; The HCDR3 comprises SEQ ID NO: 13; The LCDR1 comprises SEQ ID NO: 14; The LCDR2 comprises SEQ ID NO:5; and The LCDR3 comprises SEQ ID NO:

44.

21. The conjugate of claims 16 to 20, wherein the VH comprises SEQ ID NO: 24 and the VL comprises SEQ ID NO: 8, 17 or 27.

22. The conjugate of any one of claims 16 to 21, wherein the antibody comprises a heavy chain (HC) and a light chain (LC), wherein the HC comprises SEQ ID NO: 25 and the LC comprises SEQ ID NO: 10, 19 or 28.

23. The conjugate of any one of claims 1 to 9, wherein the antibody that binds to human TNFα comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, and the VL comprises light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein: The HCDR1 comprises SEQ ID NO: 1; The HCDR2 comprises SEQ ID NO: 2; The HCDR3 comprises SEQ ID NO: 30; The LCDR1 comprises SEQ ID NO:31; The LCDR2 comprises SEQ ID NO:5; and The LCDR3 comprises SEQ ID NO:

32.

24. The conjugate of claim 23, wherein the VH comprises SEQ ID NO: 33 and the VL comprises SEQ ID NO:

34.

25. The conjugate of any one of claims 23 to 24, wherein the antibody comprises a heavy chain (HC) and a light chain (LC), wherein the HC comprises SEQ ID NO: 35 and the LC comprises SEQ ID NO:

36.

26. The conjugate of any one of claims 1 to 11, 13 to 14, 16 to 21, 23 to 24, wherein the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises: cysteine ​​at amino acid residue 124 (EU numbering); Cysteine ​​at amino acid residue 378 (EU numbering); or Cysteine ​​at amino acid residue 124 (EU numbering) and Cysteine ​​at amino acid residue 378 (EU numbering).

27. The conjugate of any one of claims 1 to 11, 13 to 14, 16 to 21, 23 to 24, wherein the antibody comprises a heavy chain (HC) and a light chain (LC), wherein the HC is of human IgG1 isotype.

28. The conjugate according to any one of claims 1 to 27, wherein n is 2-5.

29. The conjugate according to any one of claims 1 to 27, wherein n is 3-5.

30. The conjugate according to any one of claims 1 to 27, wherein n is 3-4.

31. The conjugate of any one of claims 1 to 27, wherein n is about 4.

32. The conjugate of any one of claims 1 to 27, wherein n is about 3.

33. The conjugate of any one of claims 1 to 27, wherein n is about 2.

34. A pharmaceutical composition comprising the conjugate of any one of claims 1 to 33 or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable carriers, diluents or excipients.

35. A method of treating an autoimmune disease or an inflammatory disease in a subject in need thereof, the method comprising administering to the subject an effective amount of the conjugate according to any one of claims 1 to 33 or the pharmaceutical composition according to claim 34.

36. The method of claim 35, wherein the autoimmune disease or the inflammatory disease is selected from rheumatoid arthritis (RA), juvenile idiopathic arthritis, psoriatic arthritis (PsA), ankylosing spondylitis (AS), Crohn's disease (CD), ulcerative colitis (UC), plaque psoriasis (PS), hidradenitis suppurativa (HS), uveitis, non-infectious intermediate uveitis, posterior uveitis, panuveitis, Behcet's disease, or polymyalgia rheumatica (PMR).

37. The method of claim 35, wherein the subject to whom the effective amount of the conjugate is administered has received prior treatment with other anti-TNFα therapeutic agents, and wherein the subject develops anti-drug antibodies to the other anti-TNFα therapeutic agents.

38. The method of claim 37, wherein the other anti-TNFα therapeutic agent is selected from adalimumab, infliximab, golimumab, certolizumab pegol and etanercept, or a conjugate thereof.

39. The method of claim 38, wherein the conjugate has low to no cross-reactivity to anti-drug antibodies directed against adalimumab.

40. The conjugate of any one of claims 1 to 33, or the pharmaceutical composition of claim 34, for use in therapy.

41. The conjugate according to any one of claims 1 to 33, or the pharmaceutical composition according to claim 34, for use in the treatment of an autoimmune disease or an inflammatory disease.

42. The conjugate or pharmaceutical composition for use according to claim 41, wherein the autoimmune disease or the inflammatory disease is selected from rheumatoid arthritis (RA), juvenile idiopathic arthritis, psoriatic arthritis (PsA), ankylosing spondylitis (AS), Crohn's disease (CD), ulcerative colitis, plaque psoriasis (PS), hidradenitis suppurativa, uveitis, non-infectious intermediate uveitis, posterior uveitis, panuveitis, Behcet's disease or polymyalgia rheumatica (PMR).

43. Use of the conjugate according to any one of claims 1 to 33, or the pharmaceutical composition according to claim 34, in the manufacture of a medicament for treating an autoimmune disease or an inflammatory disease.

44. The use according to claim 43, wherein the autoimmune disease or the inflammatory disease is selected from rheumatoid arthritis (RA), juvenile idiopathic arthritis, psoriatic arthritis (PsA), ankylosing spondylitis (AS), Crohn's disease (CD), ulcerative colitis, plaque psoriasis (PS), hidradenitis suppurativa, uveitis, non-infectious intermediate uveitis, posterior uveitis, panuveitis, Behcet's disease or polymyalgia rheumatica (PMR).

45. The conjugate of any one of claims 1 to 33, wherein the antibody neutralizes human TNFα.

46. ​​The conjugate of any one of claims 1 to 33, wherein the antibody is an internalizing antibody.

47. A compound of the formula:

48. The compound according to claim 47, which is:

49. The compound according to claim 47, which is:

50. The compound according to claim 48, which is:

51. The compound according to claim 49, which is:

52. A compound of the formula:

53. The compound of claim 52, wherein the conjugate is:

54. A method of producing a conjugate, the method comprising contacting the compound according to claim 47 with an anti-human TNFα antibody.

55. The method of claim 54, wherein the conjugate comprises a conjugate according to any one of claims 1 to 33.

56. The method of claim 54, wherein the conjugate is produced according to the steps comprising: (a) reducing an anti-human TNFα antibody with a reducing agent, wherein the anti-human TNFα antibody comprises one or more engineered cysteine ​​residues; (b) oxidizing the anti-human TNFα antibody with an oxidizing agent; and (c) making the compound of the following formula and contacting with the anti-human TNFα antibody to produce the conjugate.

57. The method of claim 56, wherein the reducing agent is dithiothreitol and the oxidizing agent is dehydroascorbic acid.

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