Human tumor necrosis factor alpha antibody glucocorticoid conjugate

Abstract

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

Description

Technical Field

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

Background Art

[0002] Rheumatoid arthritis (RA) is a debilitating chronic autoimmune disease that attacks joints, most commonly the joints of the hands, wrists, and knees, and usually attacks many joints at once. In RA, the inner lining of the joint becomes inflamed, causing damage to joint tissue that can lead to stiffness, swelling, instability (lack of balance), deformity, and chronic pain. Current treatments use therapeutic agents such as, for example, non-steroidal anti-inflammatory drugs (NSAIDs), corticosteroids, disease modifying anti-rheumatic drugs (DMARDs), methotrexate, tofacitinib, etanercept, adalimumab, infliximab, golimumab, and certolizumab. Disadvantages of such treatments include, for example, off-target toxicity and the generation of anti-drug antibodies.

[0003] Anti-drug antibodies can be non-neutralizing antibodies that bind to anti-TNFα therapeutic agents simultaneously with TNFα, or they can be neutralizing antibodies that reduce the effective concentration of anti-TNFα therapeutic antibodies in serum and / or compete with TNFα for the antigen-binding site (paratope), thus inhibiting the mechanism of action 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 90 percent of anti-TNFα drug antibodies are neutralizing and can be cross-reactive with other anti-TNFα antibody therapeutic agents (van Schie KA, et al, 2015). Thus, in some cases, patients who generate anti-drug antibodies against anti-TNFα antibodies have been reported to have a reduced clinical response to these therapeutic agents and / or adverse events such as infusion-related reactions characterized by symptoms such as fever, itching, bronchospasm, or cardiovascular collapse during or on the first day after drug administration (Atiqi, S., Front Immunol., 2020, 26(11):312). Therefore, there is still a great need for new agents that provide improved and effective treatment of inflammatory and / or autoimmune diseases such as RA and minimize or eliminate the drawbacks of currently approved treatments.

[0004] WO 2017 / 210471 discloses certain glucocorticoid receptor agonists (GCs), antibodies, and immunoconjugates thereof. WO 2018 / 089373 discloses novel steroids, protein conjugates thereof, and methods of treating diseases, disorders, and conditions including administering the steroids and conjugates. To date, there are no approved human TNFα antibody GC conjugates for the treatment of diseases. SUMMARY OF THE INVENTION

[0005] The present disclosure provides certain novel human TNFα antibody GC conjugates in which the antibody binds to human ITNFα. The present disclosure further provides compositions comprising the novel anti-human TNFα antibody GC conjugates, as well as methods of using such anti-human TNFα antibody GC conjugates and their compositions. In addition, the present disclosure provides certain novel anti-human TNFα antibody GC conjugates useful in the treatment of autoimmune and inflammatory diseases such as rheumatoid arthritis. The present disclosure further provides certain novel anti-human TNFα antibody GC conjugates useful in the treatment of autoimmune 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 exhibit favorable development profiles such as favorable physicochemical properties (e.g., low viscosity, or aggregation, good thermal stability) to facilitate development, manufacture, and formulation. Accordingly, certain anti-human TNFα antibody GC conjugates as disclosed herein have one or more of the following properties: 1) binds to human TNFα with desired potency, 2) binds to human membrane TNFα and is taken up intracellularly, 3) binds to cynomolgus and / or canine TNFα with desired potency, 4) inhibits soluble and membrane human TNFα-induced apoptosis, 5) regulates both TNFR- and glucocorticoid receptor-mediated cytokine expression in vitro (e.g., inhibits IL-13, IL-6, GM-CSF, induces IL-10), 6) inhibits TNFR-mediated cytokine expression (e.g., CXCL1) in vivo, 7) induces ADCC activity, 8) exhibits low cross-reactivity or no cross-reactivity in vivo and in vitro to anti-drug antibodies to other anti-TNFα therapeutics (e.g., adalimumab), 9) significantly inhibits tissue and / or polyarthritis joint inflammation in vivo, 10) significantly inhibits joint inflammation in vivo in adalimumab-resistant mice, or 11) has a favorable development profile, e.g., acceptable viscosity, solubility, and aggregation, good stability, and / or an acceptable pharmacokinetic profile to facilitate development, manufacture, and / or formulation.

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

Chemical formula

Chemical formula

[0007] In a further embodiment, the present disclosure provides a conjugate of formula Ia:

Chemical formula

Chemical formula

[0008] In a further embodiment, the present disclosure is of formula Ib:

Chemical formula

Chemical formula

[0009] In a further embodiment, the present disclosure is of formula Ic:

Chemical formula

Chemical formula

[0010] In a further embodiment, the present disclosure provides a conjugate of formula Id:

Chemical formula

[0011] In a further embodiment, the present disclosure provides a conjugate of formula Ie: [Chemical formula] wherein Ab is an antibody that binds to human TNFα, Ab comprises a heavy chain variable region (VH) and a light chain variable region (VL), VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, HCDR1 comprises SEQ ID NO: 1 or 22, HCDR2 comprises SEQ ID NO: 2 or 23, HCDR3 comprises SEQ ID NO: 3, 13, or 30, LCDR1 comprises SEQ ID NO: 4, 14, 31, or 43, LCDR2 comprises SEQ ID NO: 5, LCDR3 comprises SEQ ID NO: 6, 15, 32, or 44, [Chemical formula] and provides a conjugate wherein n is from 1 to 5.

[0012] In a further embodiment, the present disclosure provides a conjugate of formula If: [Chemical formula] wherein Ab is an antibody that binds to human TNFα, Ab comprises a heavy chain variable region (VH) and a light chain variable region (VL), VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, HCDR1 comprises SEQ ID NO: 1 or 22, HCDR2 comprises SEQ ID NO: 2 or 23, HCDR3 comprises SEQ ID NO: 3, 13, or 30, LCDR1 comprises SEQ ID NO: 4, 14, 31, or 43, LCDR2 comprises SEQ ID NO: 5, LCDR3 comprises SEQ ID NO: 6, 15, 32, or 44, [Chemical formula] and n is from 1 to 5, providing a conjugate.

[0013] In some embodiments, the conjugate of formula I, an antibody that binds to human TNFα ("antibody, Ab") is Ab1, and Ab1 comprises a heavy chain variable region (VH) and a light chain variable region (VL). VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3. 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, Ab1 comprises a VH comprising SEQ ID NO: 7 and a VL comprising SEQ ID NO: 8. In some embodiments, Ab1 comprises a heavy chain (HC) comprising SEQ ID NO: 9 and a light chain (LC) comprising SEQ ID NO: 10.

[0014] In some embodiments, the conjugate of formula I, wherein the antibody that binds to human TNFα ( "Ab") is Ab2, Ab2 comprises a heavy chain variable region (VH) and a light chain variable region (VL), VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, 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.

[0015] In some embodiments, the conjugate of formula I, wherein the antibody that binds to human TNFα ( "Ab") is Ab3, Ab3 comprises a heavy chain variable region (VH) and a light chain variable region (VL), VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, 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.

[0016] In some embodiments, the conjugate of Formula I, wherein the antibody that binds to human TNFα (the "Ab") is Ab4, and Ab4 comprises a heavy chain variable region (VH) and a light chain variable region (VL), VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, 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.

[0017] In some embodiments, the conjugate of Formula I, wherein the antibody that binds to human TNFα (the "Ab") is Ab5, and Ab5 comprises a heavy chain variable region (VH) and a light chain variable region (VL), VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, 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.

[0018] In some embodiments, the conjugate of formula I, wherein the antibody that binds to human TNFα (the "Ab") is Ab6, Ab6 comprises a heavy chain variable region (VH) and a light chain variable region (VL), VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, 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.

[0019] In some embodiments, the conjugate of formula I, wherein the antibody that binds to human TNFα (the "Ab") comprises a heavy chain variable region (VH) and a light chain variable region (VL), VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, 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 the amino acid residues QQYDXaa 5 comprises LPLT, and Xaa of SEQ ID NO: 44 5 is asparagine or lysine.

[0020] In some embodiments, the conjugate of formula I, an antibody that binds to human TNFα ("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, VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, 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 is amino acid residue QASQGIXaa 7 comprises NYLN, and Xaa of SEQ ID NO: 43 7 is serine or arginine.

[0021] In some embodiments, the conjugate of formula I, wherein the anti-human TNFα antibody is a fully human antibody. In a further embodiment, the anti-human TNFα antibody has a human IgG1 isotype.

[0022] 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 present in the heavy chain variable region (VH). In some embodiments, the modification is present in the light chain variable region (VL). In some embodiments, the modification is present in both VH and VL. In a further embodiment, the modified human IgG1 VH and / or VL provides a desirable viscosity profile and / or immunogenic risk profile to the anti-human TNFα antibody of the present disclosure.

[0023] In a further embodiment, the conjugate of Formula I is a conjugate having a modified human IgG1 constant domain comprising an engineered cysteine residue for use in the production of an antibody conjugate (also referred to as a bioconjugate) (see International Publication No. WO 2018 / 232088 (A1)). 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 cysteine at amino acid residue 124 (EU numbering) within the heavy chain constant domain 1 (CH1), or cysteine at amino acid residue 378 (EU numbering) within the heavy chain constant domain 2 (CH2). In a further embodiment, the anti-human TNFα antibody comprises cysteine at amino acid residue 124 (EU numbering) within the CH1 domain and cysteine at amino acid residue 378 (EU numbering) within the CH2 domain.

[0024] In some embodiments, the conjugate of formula I, wherein the anti-human TNFα antibody has low cross-reactivity or no cross-reactivity with an anti-drug antibody against another anti-TNFα therapeutic agent (e.g., adalimumab, infliximab, golimumab, certolizumab, or etanercept) or its conjugate. In certain embodiments, the conjugate of formula I, wherein the anti-human TNFα antibody has low cross-reactivity or no cross-reactivity with an anti-drug antibody against adalimumab. In such embodiments, certain conjugates of formula I can be used to treat patients who have developed anti-drug antibodies against pre-treatment with 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 against other anti-TNFα therapeutic agents from pre-treatment with such other anti-TNFα therapeutic agents, and thus have a reduced clinical response or adverse reaction to other anti-TNFα therapeutic agents. In such embodiments, the conjugate of formula I, wherein the anti-human TNFα antibodies have sufficiently different amino acid sequences and nucleic acid sequences such that they have low cross-reactivity or no cross-reactivity with anti-drug antibodies against other anti-TNFα therapeutic agents. In certain embodiments, the conjugate of formula I, wherein the anti-human TNFα antibody of the present disclosure has sufficiently different CDR amino acid sequences such that it has low cross-reactivity or no cross-reactivity with an anti-drug antibody against other anti-TNFα therapeutic agents. In some embodiments, the other anti-TNFα therapeutic agent is adalimumab, infliximab, golimumab, certolizumab, or etanercept, or a conjugate thereof.

[0025] In some embodiments, the present disclosure provides a nucleic acid encoding the HC or LC, or VH or VL of a novel antibody that binds to anti-human TNFα, or a vector comprising such a nucleic acid.

[0026] 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.

[0027] In some embodiments, a nucleic acid encoding a heavy or light chain of an antibody that binds to human TNFα is provided. In some embodiments, a nucleic acid comprising a sequence encoding SEQ ID NO: 9, 10, 18, 19, 25, 28, 35, or 36 is provided. In some embodiments, a nucleic acid comprising a sequence encoding an antibody heavy chain comprising SEQ ID NO: 9, 18, 25, or 35 is provided. For example, the nucleic acid can comprise the sequence of SEQ ID NO: 11, 20, 26, or 37. In some embodiments, a nucleic acid comprising a sequence encoding an antibody light chain comprising SEQ ID NO: 10, 19, 28, or 36 is provided. For example, the nucleic acid can comprise the sequence of SEQ ID NO: 12, 21, 29, or 38.

[0028] In some embodiments of the present disclosure, a nucleic acid encoding VH or VL of an antibody that binds to human TNFα is provided. In some embodiments, a nucleic acid comprising a sequence encoding SEQ ID NO: 7, 8, 16, 17, 24, 27, 33, or 34 is provided. In some embodiments, a nucleic acid comprising a sequence encoding an antibody VH comprising SEQ ID NO: 7, 16, 24, or 33 is provided. In some embodiments, a nucleic acid comprising a sequence encoding an antibody VL comprising SEQ ID NO: 8, 17, 27, or 34 is provided.

[0029] Some embodiments of the present disclosure provide a vector comprising a nucleic acid sequence encoding an antibody heavy or light chain. For example, such a vector can 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.

[0030] Also provided herein is a vector comprising a nucleic acid sequence encoding antibody VH or VL. For example, such a vector can 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.

[0031] Also provided herein are vectors 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.

[0032] 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: 28. In some embodiments, the composition comprises 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.

[0033] 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.

[0034] In one embodiment, n is from 2 to 5.

[0035] In one embodiment, n is from 3 to 5.

[0036] In one embodiment, n is from 3 to 4.

[0037] In one embodiment, n is about 4.

[0038] In one embodiment, n is about 3.

[0039] In one embodiment, n is about 2.

[0040] As used herein, the formula:

Chemical formula

Chemical formula

[0041] As used herein, the formula:

Chemical formula

Chemical formula

[0042] In one embodiment, the present disclosure provides a glucocorticoid receptor agonist payload-linker represented by formula IV:

Chemical formula

[0043] In one embodiment, the present disclosure provides a glucocorticoid receptor agonist payload-linker represented by formula IVa:

Chemical formula

[0044] In one embodiment, the present disclosure provides a compound of formula IVb:

Chemical formula

[0045] In one embodiment, the present disclosure provides a compound of formula IVc:

Chemical formula

[0046] In one embodiment, the present disclosure provides a compound of formula IVd:

Chemical formula

[0047] In one embodiment, the present disclosure provides a compound of formula V:

Chemical formula

[0048] In a further embodiment, the present disclosure provides a compound of formula Va:

Chemical formula

[0049] In one embodiment, the present disclosure also provides a method for treating an inflammatory disease in a subject in need of treatment, the method comprising administering to the patient an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof. In one embodiment, the present disclosure also provides a method for treating an inflammatory disease in a subject in need of treatment, the method comprising administering to the patient an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof. In certain embodiments, the autoimmune 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, hidradenitis suppurativa, uveitis, non-infectious intermediate, posterior, panuveitis, Behçet's disease, or polymyalgia rheumatica (PMR). In one embodiment, the present disclosure further provides a method for treating rheumatoid arthritis in a subject in need of treatment, the method comprising administering to the patient an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof. In one embodiment, the present disclosure further provides a method for treating psoriatic arthritis in a subject in need of treatment, the method comprising administering to the patient an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof. In one embodiment, the present disclosure further provides a method for treating Crohn's disease in a subject in need of treatment, the method comprising administering to the patient an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof. In one embodiment, the present disclosure further provides a method for treating ulcerative colitis in a subject in need of treatment, the method comprising administering to the patient an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof. In one embodiment, the present disclosure further provides a method for treating plaque psoriasis in a subject in need of treatment, the method comprising administering to the patient an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof.In one embodiment, the present disclosure further provides a method of treating ankylosing spondylitis in a subject in need thereof, the method comprising administering to the patient an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof. In some embodiments, the subject being administered an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof has been pre-treated with another anti-TNFα therapeutic agent, and the subject has developed anti-drug antibodies against the other anti-TNFα therapeutic agent. In such embodiments, the other anti-TNFα therapeutic agent is selected from the group consisting of adalimumab, infliximab, golimumab, certolizumab, or conjugates thereof. In yet further embodiments, a particular conjugate of Formula I or a pharmaceutically acceptable salt thereof as disclosed herein has low cross-reactivity or no cross-reactivity with anti-drug antibodies against at least 4 or more of the other anti-TNFα therapeutic agents selected from the group consisting of adalimumab, infliximab, golimumab, certolizumab, or conjugates thereof. In yet further embodiments, a particular conjugate of Formula I or a pharmaceutically acceptable salt thereof as disclosed herein has low cross-reactivity or no cross-reactivity with anti-drug antibodies against at least 3 or more of the other anti-TNFα therapeutic agents selected from the group consisting of adalimumab, infliximab, golimumab, certolizumab, or conjugates thereof. In yet further embodiments, a particular conjugate of Formula I or a pharmaceutically acceptable salt thereof as disclosed herein has low cross-reactivity or no cross-reactivity with anti-drug antibodies against at least 2 or more of the other anti-TNFα therapeutic agents selected from the group consisting of adalimumab, infliximab, golimumab, certolizumab, or conjugates thereof. In still further embodiments, a particular conjugate of Formula I or a pharmaceutically acceptable salt thereof as disclosed herein has low cross-reactivity or no cross-reactivity with anti-drug antibodies against adalimumab or a conjugate thereof.

[0050] In one embodiment, the present disclosure further provides a conjugate of formula I or a pharmaceutically acceptable salt thereof for use in therapy. In one embodiment, the present disclosure provides a conjugate of formula I or a pharmaceutically acceptable salt thereof for use in the treatment of inflammatory diseases. In one embodiment, the present disclosure provides a conjugate of formula I or a pharmaceutically acceptable salt thereof for use in the treatment of inflammatory diseases. In certain embodiments, the autoimmune or inflammatory disease is, for example, rheumatoid arthritis (RA), psoriatic arthritis (PsA), Crohn's disease (CD), ulcerative colitis, psoriasis vulgaris (PS), ankylosing spondylitis (AS), juvenile idiopathic arthritis, hidradenitis suppurativa, uveitis, non-infectious intermediate, posterior, panuveitis, Behçet's disease, or polymyalgia rheumatica (PMR). In one embodiment, the present disclosure provides a conjugate of formula I or a pharmaceutically acceptable salt thereof for use in the treatment of rheumatoid arthritis. In one embodiment, the present disclosure provides a conjugate of formula I or a pharmaceutically acceptable salt thereof for use in the treatment of psoriatic arthritis. In one embodiment, the present disclosure provides a conjugate of formula I or a pharmaceutically acceptable salt thereof for use in the treatment of Crohn's disease. In one embodiment, the present disclosure provides a conjugate of formula I or a pharmaceutically acceptable salt thereof for use in the treatment of ulcerative colitis. In one embodiment, the present disclosure provides a conjugate of formula I or a pharmaceutically acceptable salt thereof for use in the treatment of psoriasis vulgaris. In one embodiment, the present disclosure provides a conjugate of formula I or a pharmaceutically acceptable salt thereof for use in the treatment of ankylosing spondylitis. In some embodiments, the subject being administered an effective amount of the conjugate of formula I or a pharmaceutically acceptable salt thereof has been pre-treated with another anti-TNFα therapeutic agent and the subject has developed anti-drug antibodies against the other anti-TNFα therapeutic agent. In such embodiments, the other anti-TNFα therapeutic agent is selected from the group consisting of adalimumab, infliximab, golimumab, certolizumab, or conjugates thereof.In yet further embodiments, a conjugate of a particular formula I or a pharmaceutically acceptable salt thereof, as disclosed herein, has low cross-reactivity or no cross-reactivity with anti-drug antibodies against at least four or more of other anti-TNFα therapeutic agents selected from the group consisting of adalimumab, infliximab, golimumab, certolizumab, or conjugates thereof. In yet further embodiments, a conjugate of a particular formula I or a pharmaceutically acceptable salt thereof, as disclosed herein, has low cross-reactivity or no cross-reactivity with anti-drug antibodies against at least three or more of other anti-TNFα therapeutic agents selected from the group consisting of adalimumab, infliximab, golimumab, certolizumab, or conjugates thereof. In yet further embodiments, a conjugate of a particular formula I or a pharmaceutically acceptable salt thereof, as disclosed herein, has low cross-reactivity or no cross-reactivity with anti-drug antibodies against at least two or more of other anti-TNFα therapeutic agents selected from the group consisting of adalimumab, infliximab, golimumab, certolizumab, or conjugates thereof. In still further embodiments, a conjugate of a particular formula I or a pharmaceutically acceptable salt thereof, as disclosed herein, has low cross-reactivity or no cross-reactivity with anti-drug antibodies against adalimumab or a conjugate thereof.

[0051] In one 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 medicament for the treatment of autoimmune diseases. In one 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 medicament for the treatment of inflammatory diseases. In certain embodiments, the autoimmune disease or inflammatory disease is, for example, rheumatoid arthritis (RA), psoriatic arthritis (PsA), Crohn's disease (CD), ulcerative colitis, psoriasis vulgaris (PS), ankylosing spondylitis (AS), juvenile idiopathic arthritis, hidradenitis suppurativa, uveitis, non-infectious intermediate, posterior, panuveitis, Behçet's disease, or polymyalgia rheumatica (PMR). In one 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 the treatment of rheumatoid arthritis. In one 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 the treatment of psoriatic arthritis. In one 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 the treatment of Crohn's disease. In one 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 the treatment of ulcerative colitis. In one 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 the treatment of psoriasis vulgaris. In one 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 the treatment of ankylosing spondylitis. In some embodiments, the subject being administered an effective amount of a conjugate of Formula I or a pharmaceutically acceptable salt thereof has been pre-treated with another anti-TNFα therapeutic agent, and the subject has developed anti-drug antibodies against the other anti-TNFα therapeutic agent. In such embodiments, the other anti-TNFα therapeutic agent is selected from the group consisting of adalimumab, infliximab, golimumab, certolizumab, or conjugates thereof.In yet further embodiments, a conjugate of a particular formula I or a pharmaceutically acceptable salt thereof, as disclosed herein, has low cross-reactivity or no cross-reactivity with anti-drug antibodies against at least four or more of other anti-TNFα therapeutic agents selected from the group consisting of adalimumab, infliximab, golimumab, certolizumab, or conjugates thereof. In yet further embodiments, a conjugate of a particular formula I or a pharmaceutically acceptable salt thereof, as disclosed herein, has low cross-reactivity or no cross-reactivity with anti-drug antibodies against at least three or more of other anti-TNFα therapeutic agents selected from the group consisting of adalimumab, infliximab, golimumab, certolizumab, or conjugates thereof. In yet further embodiments, a conjugate of a particular formula I or a pharmaceutically acceptable salt thereof, as disclosed herein, has low cross-reactivity or no cross-reactivity with anti-drug antibodies against at least two or more of other anti-TNFα therapeutic agents selected from the group consisting of adalimumab, infliximab, golimumab, certolizumab, or conjugates thereof. In yet other embodiments, a conjugate of a particular formula I or a pharmaceutically acceptable salt thereof, as disclosed herein, has low cross-reactivity or no cross-reactivity with anti-drug antibodies against adalimumab or a conjugate thereof.

[0052] The nucleic acids of the present disclosure can be expressed in a host cell, for example, after the nucleic acid is operably linked to an expression control sequence. Expression control sequences capable of expressing the operably linked nucleic acid are well known in the art. The expression vector may include a sequence encoding one or more signal peptides that facilitate the secretion of the polypeptide from the host cell. An expression vector containing a nucleic acid of interest (e.g., a nucleic acid encoding a heavy or light chain of an antibody) can be introduced into a host cell by well-known methods, such as stable or transient transfection, transformation, transduction, or infection. Additionally, the expression vector may include one or more selectable markers, such as tetracycline, neomycin, and dihydrofolate reductase, to assist in the detection of host cells transformed with the desired nucleic acid sequence.

[0053] In another aspect, provided herein are cells, such as host cells, comprising the nucleic acids, vectors, or nucleic acid compositions described herein. The host cell can be a cell that has been stably or transiently transfected, transformed, transduced, or infected with one or more expression vectors that express all or a portion of the antibodies described herein. In some embodiments, the host cell can be stably or transiently transfected, transformed, transduced, or infected with an expression vector that expresses the HC and LC polypeptides of the antibody of the present disclosure. In some embodiments, the host cell can be stably or transiently transfected, transformed, transduced, or infected with a first vector that expresses the HC polypeptide of the antibody described herein and a second vector that expresses the LC polypeptide. Such host cells, such as mammalian host cells, can express an antibody that binds to anti-human TNFα as described herein. Mammalian host cells known to be capable of expressing an antibody include CHO cells, HEK293 cells, COS cells, and NS0 cells.

[0054] In some embodiments, a cell, such as a host cell, comprises 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.

[0055] In some embodiments, a cell, e.g., a host cell, comprises 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.

[0056] The present disclosure further provides a method for generating an antibody that binds to anti-human TNFα described herein by culturing the above host cell, e.g., a mammalian host cell, under conditions such that the antibody is expressed and recovering the expressed antibody from the culture medium. The medium in which the antibody is secreted can be purified by conventional techniques. Various methods of protein purification can be employed, such methods being known in the art, for example, as described in Deutscher, Methods in Enzymology 182:83-89 (1990), and Scopes, Protein Purification: Principles and Practice, 3rd Edition, Springer, NY (1994).

[0057] The present disclosure provides a method for generating a conjugate, comprising contacting a compound of the present disclosure with an anti-human TNFα antibody.

[0058] The present disclosure provides a method for generating a conjugate, comprising contacting a compound of Formula IV with an anti-human TNFα antibody. The present disclosure provides a method for generating a conjugate, comprising contacting a compound of Formula IVa with an anti-human TNFα antibody. The present disclosure provides a method for generating a conjugate, comprising contacting a compound of Formula IVb with an anti-human TNFα antibody. The present disclosure provides a method for generating a conjugate, comprising contacting a compound of Formula IVc with an anti-human TNFα antibody. The present disclosure provides a method for generating a conjugate, comprising contacting a compound of Formula IVd with an anti-human TNFα antibody. In some embodiments, the conjugate generated is a conjugate of Formula I.

[0059] The present disclosure provides a method for generating a conjugate, comprising the following steps: (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 reagent; and (c) contacting a compound of the present disclosure with the anti-human TNFα antibody to generate a conjugate.

[0060] The present disclosure provides a method for generating a conjugate, comprising the following steps: (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 reagent; and (c) contacting a compound of the formula:

Chemical formula

[0061] 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.

[0062] The present disclosure further provides an antibody or an antigen-binding fragment thereof produced by any of the methods described herein.

[0063] In one embodiment, the present disclosure further provides a pharmaceutical composition comprising a conjugate of Formula I or a pharmaceutically acceptable salt thereof, or an antibody, nucleic acid, or vector described herein, together with one or more pharmaceutically acceptable carriers, diluents, or excipients. In one embodiment, the present disclosure further provides a pharmaceutical composition comprising a conjugate of Formula I or a pharmaceutically acceptable salt thereof, together with one or more pharmaceutically acceptable carriers, diluents, or excipients. In one embodiment, the present disclosure further provides a pharmaceutical composition comprising a conjugate of Formula I together with one or more pharmaceutically acceptable carriers, diluents, or excipients. In one embodiment, the present disclosure further provides a method for preparing a pharmaceutical composition comprising mixing a conjugate of Formula I or a pharmaceutically acceptable salt thereof with one or more pharmaceutically acceptable carriers, diluents, or excipients. In one embodiment, the present disclosure also encompasses novel intermediates and methods for the synthesis of conjugates of Formula I. BRIEF DESCRIPTION OF THE DRAWINGS

[0064]

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Fig. 5A

Fig. 5B

Fig. 5C

Fig. 6A

Fig. 6B

Fig. 6C

Fig. 7

MODE FOR CARRYING OUT THE INVENTION

[0065] As used herein, the term "TNFα" refers to soluble and membrane TNFα, and any native mature TNFα resulting from the processing of intracellular TNFα precursor proteins, unless otherwise indicated. This term includes TNFα from any vertebrate source, including mammals such as dogs, primates (e.g., humans, cynomolgus monkeys or rhesus monkeys), and rodents (e.g., mice and rats), unless otherwise indicated. This term also includes naturally occurring variants of TNFα, such as splice variants or allelic variants. An example of the amino acid sequence of anti-human TNFα is known in the art and is, for example, NCBI accession number NP_000585 (SEQ ID NO: 39). An example of the amino acid sequence of cynomolgus monkey TNFα is also known in the art and is, for example, UniProt reference sequence P79337 (SEQ ID NO: 42). An example of the amino acid sequence of rhesus monkey TNFα is also known in the art and is, for example, UniProt reference sequence P48094 (SEQ ID NO: 40). An example of the amino acid sequence of dog TNFα is also known in the art and is, for example, GenBank accession number CAA64403 (SEQ ID NO: 41). The term "TNFα" as used herein is used to collectively refer to all known human TNFα isoforms and polymorphic forms. The SEQ ID NOs used herein are based on the mature protein without the signal peptide.

[0066] As used herein, the terms "TNFR" or "TNF receptor" refer to any native mature TNFR, such as TNFR1 (also known as p55 or p60) or TNFR2 (also known as p75 or p80), unless otherwise specified. This term includes TNFR from any vertebrate source, including mammals such as dogs, primates (e.g., humans and cynomolgus or rhesus monkeys), and rodents (e.g., mice and rats), unless otherwise indicated. This term also includes naturally occurring variants of TNFR, such as splice variants or allelic variants. An example of the amino acid sequence of human TNFR1 is known in the art and is, for example, GenBank accession number: AAA61201 (SEQ ID NO: 45). An example of the amino acid sequence of human TNFR2 is known in the art and is, for example, NCBI accession number: NP_001057 (SEQ ID NO: 46). The term "TNFR" is used herein to collectively refer to all known human TNFR isoforms and polymorphic forms.

[0067] As used herein, the terms "anti-drug antibody" or "anti-drug antibody, ADA" refer to antibodies formed in a mammal in response to an immune response against a therapeutic agent administered to the mammal. In some embodiments of the present disclosure, the anti-drug antibody formed against a therapeutic agent can neutralize the effect of the therapeutic agent and thus change the pharmacokinetic (PK) and / or pharmacodynamic (PD) properties of the therapeutic agent, interfere with the effect of the therapeutic agent, and / or reduce the efficacy and / or decrease the clinical response to the therapeutic agent. Anti-drug antibodies against a therapeutic agent can also lead to harmful immune reactions in a patient, as a result of which the patient may no longer be a candidate for further treatment with that therapeutic agent. Examples of harmful immune reactions include, but are not limited to, infusion-related reactions characterized by symptoms such as fever, itching, bronchospasm, or cardiovascular collapse on the first day after drug administration (Atiqi, S., Front Immunol., 2020, 26(11):312).

[0068] As used herein, the term "low or no binding" to an anti-drug antibody refers to the binding of the anti-human TNFα antibody glucocorticoid conjugate or anti-human TNFα antibody of the present disclosure to an anti-drug antibody against other anti-TNFα therapeutic agents, and such binding is determined to be below the cut-off point of the assay used to measure the binding or within a predetermined variability range of the assay. In such a method, the cut-off point is a predetermined threshold used to identify positive binding to an anti-drug antibody. In some embodiments, the predetermined variability of the assay is less than about 20% above the cut-off point of the assay. In such embodiments, binding of the anti-human TNFα Ab GC conjugate or anti-human TNFα antibody of the present disclosure to an anti-drug antibody against other therapeutic agents (e.g., adalimumab) that is less than about 20% above the cut-off point of the assay is considered low binding. In some embodiments, binding of the anti-human TNFα Ab GC conjugate or anti-human TNFα antibody of the present disclosure to an anti-drug antibody against other therapeutic agents (e.g., adalimumab) that is below the cut-off point of the assay is considered no binding.

[0069] The term "other anti-TNFα therapeutic agents" refers to agents that bind to TNFα and inhibit TNF receptor-mediated responses and do not include the conjugates or anti-human TNFα antibodies described herein. Such agents include, but are not limited to, antibodies or conjugates thereof, antibody fragments or antigen-binding fragments, including at least a portion of an antibody that retains the ability to interact with an antigen, such as Fab, Fab’, F(ab’)2, Fv fragment, scFv, scFab, disulfide-bonded Fv (sdFv), Fd fragment, or linear antibody, which may be fused, for example, to an Fc region or IgG heavy chain constant region. In some embodiments, other anti-TNFα therapeutic agents may be, for example, adalimumab, infliximab, golimumab, and certolizumab, and / or conjugates thereof.

[0070] As used herein, the term "antibody" refers to an immunoglobulin molecule that binds to an antigen. Embodiments of antibodies include monoclonal antibodies, polyclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, bispecific or multispecific antibodies, or conjugated antibodies. The antibody may be of any class (e.g., IgG, IgE, IgM, IgD, IgA) and any subclass (e.g., IgG1, IgG2, IgG3, IgG4). Embodiments of the 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 Fab, Fab’, F(ab’)2, Fv fragment, scFv, scFab, disulfide-bonded Fv (sdFv), Fd fragment, and linear antibodies, which may be fused, for example, to an Fc region or an IgG heavy-chain constant region.

[0071] An exemplary antibody is an immunoglobulin G (IgG) type antibody composed of four polypeptide chains: two heavy chains (HC) and two light chains (LC) cross-linked via 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 involved in antigen recognition. The carboxy-terminal portion of each of the four polypeptide chains contains a constant region that is primarily involved in effector functions. 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 includes the Fc region and the 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. The IgG isotype 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 edition, Bethesda, MD: U.S. Dept. of Health and Human Services, Public Health Service, National Institutes of Health (1991). The term EU index numbering or EU numbering is used interchangeably herein.

[0072] The VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). The CDRs are exposed on the surface of the protein and are important regions of the antibody for antigen-binding specificity. Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In this specification, 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". The CDRs contain most of the residues that form specific interactions with the antigen.The assignment of amino acid residues to CDRs is according to 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, 406, 228-256 (2011)), or IMGT (the international ImMunoGeneTics database, available at www.imgt.org, Lefranc et al., Nucleic Acids Res. 1999;27:209-212). The combination of the IMGT and North CDR definitions was used for the exemplary anti-human TNFα antibodies as described herein.

[0073] 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 due to the Fc regions that vary by 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 receptor), and B cell activation.

[0074] As used herein, the term "epitope" refers to the amino acid residues of 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 with respect to a structural epitope. A structural epitope, according to some embodiments, can be used to describe the region of an antigen covered by an antibody (e.g., the footprint of the antibody when bound to the antigen). In some embodiments, a structural epitope can describe the amino acid residues of an antigen that are within a particular proximity (e.g., within a particular number of angstroms) of the amino acid residues of the antibody. The term "epitope" can also be used with respect to a functional epitope. A functional epitope, according to some embodiments, can be used to describe the amino acid residues of an antigen that interact with the amino acid residues of an antibody in a manner that contributes to the binding energy between the antigen and the antibody. An epitope can be determined according to different experimental techniques, also referred to as "epitope mapping techniques". It is understood that the determination of an epitope can vary based on the different epitope mapping techniques used and can also vary depending on the different experimental conditions used, for example, due to conformational changes or cleavage of the antigen induced by particular experimental conditions. Epitope mapping techniques are known in the art (e.g., Rockberg and Nilvebrant, Epitope Mapping Protocols: Methods in Molecular Biology, Humana Press, 3 rd 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, species exchange mutagenesis, alanine scanning mutagenesis, steric hindrance mutagenesis, hydrogen-deuterium exchange (HDX), and cross-blocking assays.

[0075] As used herein, the term "bind" is intended to mean the ability of a protein or molecule to form a chemical bond or an attractive interaction with another protein or molecule, resulting in the proximity of two proteins or molecules as determined by common methods known in the art.

[0076] As used herein, the term "nucleic acid" refers to a polymer of nucleotides including single-stranded and / or double-stranded nucleotide-containing molecules such as DNA, cDNA, and RNA molecules incorporating natural nucleotides, modified nucleotides, and / or nucleotide analogs. The polynucleotides of the present disclosure may also contain, for example, substrates incorporated therein by DNA or RNA polymerase or synthetic reactions.

[0077] 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, e.g., two drug moieties, three drug moieties, four drug moieties, five drug moieties, or more drug moieties. The drug moieties can be conjugated to the polypeptide at one or more sites in the polypeptide as described herein. In certain embodiments, the conjugate has an average drug-to-antibody ratio (DAR) (molar ratio) in the range of 2-5, or 3-5, or 3-4. In some embodiments, the conjugate has an average DAR of about 3. In certain embodiments, the conjugate has an average DAR of about 4. Average means arithmetic mean.

[0078] As used herein, it is understood that the conjugate of Formula I includes the conjugates of Formulae Ia, Ib, Ic, Id, Ie, and If, and all references herein to the conjugate of Formula I should be read as including the conjugates of Formulae Ia, Ib, Ic, Id, Ie, and If. It is further understood by those skilled in the art that the conjugate of Formula I, including the conjugates of Formulae Ia, Ib, Ic, Id, Ie, and If, may be referred to as an anti-human TNFα antibody glucocorticoid conjugate (“anti-human TNFα Ab GC conjugate”).

[0079] The anti-human TNFα antibody GC conjugates of the present disclosure can be formulated as pharmaceutical compositions that are administered by any route that allows the conjugate to be absorbed and utilized in the body, 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, A. Adejare, Editor, 23 nd Edition, published 2020, Elsevier Science).

[0080] As used herein, the terms “treating,” “treatment,” or “for treating” include, but do not necessarily indicate complete elimination of, existing symptoms or disorders, such as suppressing, slowing down, halting, controlling, delaying, or reversing the progression or severity of an existing symptom or disorder, or improving an existing symptom or disorder. Treatment includes administration of a protein or nucleic acid or vector or composition for treating a patient's, particularly a human's, symptoms or disorder.

[0081] As used herein, the term “inhibiting” or “inhibition” refers to, for example, reducing, decreasing, slowing down, lessening, stopping, destroying, suppressing, antagonizing, or blocking a biological response or activity, but does not necessarily indicate complete elimination of the biological response.

[0082] 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.

[0083] As used herein, the term "effective amount" refers to the amount or dosage of a conjugate of the present disclosure or a pharmaceutically acceptable salt thereof that, when administered to a subject once or multiple times, provides a desired effect to the subject during diagnosis or treatment. The term "effective amount" as used herein further refers to the amount of a conjugate of the present disclosure or a pharmaceutically acceptable salt thereof that induces a desired biological or medical response in a subject, for example, induces a decrease or inhibition of the activity of a protein, or improves symptoms, alleviates conditions, decelerates or delays the progression of a disease, or prevents a disease, etc. In non-limiting embodiments, the term "effective amount" refers to the necessary amount (dosage and duration and means of administration) of a conjugate or a pharmaceutically acceptable salt thereof that is effective to at least partially alleviate, inhibit, prevent, and / or improve a condition, or a disorder or disease in order to achieve a desired therapeutic result when administered to a subject. The effective amount is also an amount such that the beneficial effect outweighs the toxic or harmful effects of the conjugate of the present disclosure or the pharmaceutically acceptable salt thereof.

[0084] The effective amount can be determined by one of ordinary skill in the art using known techniques and by observing the results obtained under similar circumstances. In determining the effective amount for a patient, the attending physician will consider factors such as the species of the patient; its size, age, and general health; the specific disease or disorder involved; the degree or involvement 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 preparation being administered; the selected dosing regimen; the use of concomitant medications; and other relevant circumstances, but are not limited thereto.

[0085] Included within the scope of the present invention are pharmaceutically acceptable salts of the conjugate of Formula I. Pharmaceutically acceptable salts of the conjugates of the present invention, such as the conjugate of Formula I, can be formed under standard conditions known in the art. See, e.g., Berge, S.M., et al., "Pharmaceutical Salts," Journal of Pharmaceutical Sciences, 66:1-19, (1977).

[0086] Table 1: Abbreviations and Definitions [Table 1]

[0087] The conjugates or salts thereof of the present disclosure can be readily prepared by a variety of procedures known to those skilled in the art, some of which are illustrated in the following preparations and examples. Those skilled in the art will recognize that they can combine the specific synthetic steps for each of the described routes in different ways or combine them with steps from different schemes to prepare the conjugates or salts thereof of the present disclosure. The product of each step can be recovered by conventional methods well known in the art, including extraction, evaporation, precipitation, chromatography, filtration, milling, and crystallization. All substituents are as defined, unless otherwise indicated. Reagents and starting materials are readily available to those skilled in the art. The following preparations, examples, and assays further illustrate the present disclosure but should in no way be construed as limiting the scope of the present disclosure.

[0088] Brief Description of the Drawings Figure 1 shows the in vitro ADCC activity for an exemplary anti-human TNFα Ab1 GC conjugate. Figure 2 shows the in vitro CDC activity for an exemplary anti-human TNFα Ab1 GC conjugate. Figure 3 shows that the exemplary anti-human TNFα antibody Ab6 has significantly lower binding to anti-drug antibodies against adalimumab formed in cynomolgus monkeys hyperimmunized with adalimumab. Figure 4 shows that the exemplary anti-human TNFα antibody Ab6 has significantly lower binding to anti-drug antibodies against adalimumab formed in human patients treated with adalimumab. Figure 5A shows DSC thermograms for an exemplary anti-human TNFα Ab1 GC conjugate in PBS, pH 7.2 (5A), acetate, pH 5 (5B), and histidine, pH 6 (5C). Figure 5B shows DSC thermograms for an exemplary anti-human TNFα Ab1 GC conjugate in PBS, pH 7.2 (5A), acetate, pH 5 (5B), and histidine, pH 6 (5C). Figure 5C shows DSC thermograms for an exemplary anti-human TNFα Ab1 GC conjugate in PBS, pH 7.2 (5A), acetate, pH 5 (5B), and histidine, pH 6 (5C). Figure 6A shows a comparison of the efficacy of an anti-human TNFα Ab1 GC conjugate, anti-human TNFα Ab1, and an exemplary anti-human TNFα antibody conjugate at 1 mg / kg (6A), 3 mg / kg (6B), and 10 mg / kg (6C) in a humanized mouse model of contact hypersensitivity. Figure 6B shows a comparison of the efficacy of an anti-human TNFα Ab1 GC conjugate, anti-human TNFα Ab1, and an exemplary anti-human TNFα antibody conjugate at 1 mg / kg (6A), 3 mg / kg (6B), and 10 mg / kg (6C) in a humanized mouse model of contact hypersensitivity. Figure 6C shows a comparison of the efficacy of an anti-human TNFα Ab1 GC conjugate, anti-human TNFα Ab1, and an exemplary anti-human TNFα antibody conjugate at 1 mg / kg (6A), 3 mg / kg (6B), and 10 mg / kg (6C) in a humanized mouse model of contact hypersensitivity. Figure 7 shows that the anti-human TNFα Ab2 GC conjugate halted disease progression as measured by clinical score in both adalimumab-naïve mice and adalimumab-treated mice, and that the anti-human TNFα Ab2 GC conjugate did not generate a significant anti-drug antibody response and had low or no cross-reactivity to anti-drug antibodies against adalimumab.

Example

[0089] Preparation 1 6-Bromo-2-fluoro-3-methoxybenzaldehyde

Chem.

[0090] Preparation 2 2-Fluoro-3-methoxy-6-methylbenzaldehyde

Chem.

[0091] Preparation 3 2-Fluoro-3-hydroxy-6-methylbenzaldehyde

Chemical formula

[0092] Preparation 4 tert-Butyl N-[3-[(2-fluoro-3-formyl-4-methyl-phenoxy)methyl]phenyl]carbamate

Chemical formula

[0093] Preparation 5 (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

Chemical Structure

[0094] Preparation 6 (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 (also referred to herein as GC1)

Chemical Structure

[0095] Preparation 7 (3-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl)-L-alanyl-L-alanine

Chem.

[0096] Preparation 8 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 (also referred to herein as "GC-L")

Chem.

[0097] Preparation 9 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-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

Chemical Structure

[0098] Example 1. Generation of an anti-human TNFα antibody glucocorticoid conjugate (anti-human TNFα Ab GC conjugate) Example 1a: Generation and manipulation of an anti-human TNFα antibody Antibody Generation: To generate antibodies specific to human TNFα, transgenic mice having human immunoglobulin variable regions were immunized with recombinant human TNFα. Screening was performed using human TNFα, and cross-reactivity with other TNFα species was tested. Antibodies that were cross-reactive with both human and cynomolgus monkey TNFα were cloned, expressed, purified by standard procedures, and tested for neutralization in a TNFα-induced cytotoxicity assay. Antibodies were selected and their CDRs, variable domain framework regions, and IgG isotypes were engineered to improve binding affinity and developability characteristics such as stability, solubility, viscosity, hydrophobicity, and aggregation.

[0099] The amino acid sequence of human TNFα is provided as SEQ ID NO: 39, and the amino acid sequence of cynomolgus monkey TNFα is provided as SEQ ID NO: 42.

[0100] Anti-human TNFα antibodies can be synthesized and purified by well-known methods. When two vectors are used in a suitable host cell, e.g., Chinese hamster ovarian cell (CHO), an expression system for secreting an antibody using a predetermined HC:LC vector ratio or an antibody using a single vector system encoding both the heavy and light chains can be transiently or stably transfected. The clarified medium in which the antibody is secreted can be purified using commonly employed techniques.

[0101] Antibody engineering for improving viscosity: The parental TNFα antibody system was found to have high viscosity upon concentration. Viscosity is an important development criterion, for example, for evaluating the feasibility of delivering therapeutic antibodies by self-injectors. Mutagenesis analysis of the antibody was required to balance improving biophysical properties without increasing immunogenic risk and retaining desirable affinity and potency. In silico modeling of the parental antibody was used to identify regions of charge imbalance on the surface consisting of six complementarity-determining regions (CDRs). Antibodies generated from mutagenesis were screened for TNFα binding, and those antibodies that retained or improved target binding (determined by ELISA) compared to the parental mAb and had desirable viscosity and other development properties were selected for further development.

[0102] Antibody engineering for reducing the risk of immunogenicity: Anti-human TNFα antibodies were tested in a MHC-associated peptide proteomics (MAPPS) assay to determine the immunogenic risk. Briefly, major histocompatibility complex (MHC) binding peptides were identified for antibodies with specific CDR sequences. A CDR library with mutations that were identified as potentially reducing immunogenicity was constructed and screened for TNFα binding. Antibodies were screened and selected to optimize for low immunogenic risk while balancing maintaining desirable binding affinity for TNFα and other desirable development properties.

[0103] Tables 2a, 2b, and 3 show exemplary anti-human TNFα antibody sequences optimized for low viscosity, acceptable immunogenic risk, and other desirable development properties while retaining desirable binding efficacy to human TNFα.

[0104] Table 2a: CDR amino acid sequences of exemplary anti-human TNFα Abs [Table 2-1]

[0105] Table 2b: CDR Amino Acid Sequences of Exemplary Anti-Human TNFα Abs [Table 2-2]

[0106] Table 3: Amino Acid Sequences of Exemplary Anti-Human TNFα Abs [Table 3]

[0107] Example 1b. Generation of Anti-Human TNFα Ab1-GC Conjugate (where n is 4) [Chemical formula] (where n is 4 and Ab is Ab1) An exemplary anti-human TNFα Ab1 (see Tables 2a, 2b, and 3) was first reduced in the presence of a 40-fold molar excess of dithiothreitol (DTT) at 37 °C for 2 hours or for over 16 hours at about 21 °C. This first reduction step was used to remove various capping groups containing cysteine and glutathione that bind to engineered cysteines at positions 124 and 378 of the heavy chain during expression. After the reduction step, the sample was purified through a desalting resin to remove unbound caps as well as the reducing agent. The subsequent 2-hour oxidation step was carried out at room temperature (about 21 °C) in the presence of a 10-fold molar excess of dehydroascorbic acid (DHAA) to reform the native interchain disulfides between the light and heavy chains as well as the hinge disulfide pairs. After the 2-hour oxidation step, 4 - 8 molar equivalents of a 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,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]inden[1,2-d][1,3]dioxol-10-yl)-4-methylphenoxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)propanamide prepared in Preparation 8, was added using a 10 mM stock solution dissolved in DMSO. The sample was then incubated at room temperature for 30 - 60 minutes to allow for efficient conjugation of GC-L to the engineered cysteines.Subsequently, subsequent finishing steps such as size exclusion chromatography (SEC) or tangential flow filtration (TFF) were used to buffer exchange the sample into an appropriate formulation buffer and remove DMSO and any excess linker - payload.

[0108] Drug - to - antibody ratio (DAR) assessment: To evaluate the average number of linker - payloads present on the final conjugate, two analytical methods were used: 1) reverse phase (RP) HPLC and 2) time of flight (TOF) mass spectrometry. Both methods required an initial sample reduction step that included adding dithiothreitol (DTT) to a final concentration of approximately 10 mM and then incubating at 42 °C for 5 minutes.

[0109] Reverse phase HPLC method: 10 - 30 μg of the reduced anti - human TNFα antibody Ab1GC conjugate sample was injected onto a Phenyl 5PW, 4.6 mm×7.5 cm, 10 μM column (Tosh Part#0008043). Buffer A was composed of 0.1% trifluoroacetic acid (TFA) in water, while buffer B was composed of 0.1% trifluoroacetic acid (TFA) in acetonitrile (ACN). The column was equilibrated in 20% buffer B before sample injection, followed by a gradient of 28% - 40% buffer B over approximately 8.5 column volumes. The average DAR was determined by calculating the contribution from each individual DAR species from the fraction percentages multiplied by the number of DARs for each contributing species. Since this value is based on a partially reduced sample and represents only half of the molecule, it was then multiplied by 2 to account for the intact antibody GC conjugate. The DAR calculations for the anti - human TNFα Ab1 GC conjugate of Example 1b are provided in Table 4.

[0110] Table 4: Quantification of the average DAR for anti-human TNFα Ab1 GC conjugate using the fraction percentages for each DAR species from the partially reduced sample. [Table 4]

[0111] Time-of-flight mass spectrometry: 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 was composed of 0.1% trifluoroacetic acid (TFA) in water, while buffer B was composed of 0.1% trifluoroacetic acid (TFA) in acetonitrile (ACN). The column was equilibrated in 0% B buffer prior to sample injection, followed by a gradient of 10% B - 80% B over approximately 28 column volumes. The average DAR was determined by calculating the contribution from each individual DAR species from the fraction percentage multiplied by the number of DARs for the contributing species. This value is based on the partially reduced sample and represents only half of the molecule, so it was then multiplied by 2 to account for the intact antibody GC conjugate. The DAR calculations for the anti-human TNFα Ab1 GC conjugate of Example 1b are provided in Table 5.

[0112] Table 5: Quantification of the average DAR for anti-human TNFα Ab1 GC conjugate using the fraction percentages based on the total ion counts from time-of-flight mass spectrometry. [Table 5]

[0113] Example 1c. Generation of anti-human TNFα Ab1 GC conjugate (where n is 3) [Chemical formula] (where n is 3 and Ab is Ab1) The conjugate of Example 1c was prepared 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 to Ab1 during the conjugation step. For example, the use of a 3.2:1 molar ratio of the corresponding GC-L:Ab1 resulted in a final DAR of approximately 3.

[0114] Example 1d. Generation of an anti-human TNFα Ab2 GC conjugate (wherein n is 4)

Chemical formula

[0115] Example 1e. Generation of an anti-human TNFα Ab2 GC conjugate (wherein n is 3)

Chemical formula

[0116] Example 1f. Thiosuccinimide hydrolysis: The thiosuccinimide ring of conjugate formula Ie (wherein n is 4) 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 obtain a ring-opened product of formula If.

[0117] Scheme 2

Chemical formula

[0118] Example 2. Binding potency of anti-human TNFα Ab1 GC conjugate and anti-human TNFα antibody Example 2a. ELISA binding: The binding potencies of the exemplary anti-human TNFα Ab1 GC conjugate and anti-human TNFα antibody of Example 1b to human, cynomolgus monkey, and / or canine TNFα protein were tested in an antigen-down ELISA format. Briefly, a 384-well high-binding plate (Greiner Bio-one #781061) was coated with carbonate buffer pH 9.3 (0.015 M Na 2 CO 3 and 0.035 M NaHCO 3) Diluted with [diluent] to 1 μg / mL of human TNFα (Syngene), 2 μg / mL of cynomolgus monkey TNFα (R&D Systems, catalog number 1070-RM), or 2 μg / mL of canine TNFα (R&D Systems, catalog number 1507-CT / CF), and coated at 20 μL per well and stored overnight at 4°C. The next day, the plate was blocked with 80 μL of casein (Thermo Fisher Pierce, catalog number 37528) for 1 hour at room temperature, 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 at 20 μg / mL diluted with casein, titrated 3-fold, 8-point down) were added to the plate. The plate was incubated at 37°C for 90 minutes and then washed 3 times in PBS / 0.1% Tween. 20 μL of secondary antibody reagent goat anti-human kappa-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 1:35 with molecular grade water was added to each well. When color developed (about 15 - 30 minutes), the plate was read at 560 nM OD using a Molecular Device Spectramax plate reader, and data was acquired using Softmax Pro 4.7 software. Data analysis was performed with GraphPad Prism.

[0119] The results in Table 6a show that the exemplary anti-human TNFα Ab1 GC conjugate of Example 1b binds to human TNFα with a desired potency comparable to that of the unconjugated anti-human TNFα Ab1.

[0120] Representative results in Table 6b show that anti-human TNFα antibodies Ab1, Ab2, Ab3, Ab4, Ab5, and Ab6 cross-react with human, cynomolgus monkey, and canine TNFα.

[0121] Table 6a. Binding EC of exemplary anti-human TNFα Ab1 GC conjugate of Example 1b to human TNFα 50 [Table 6-1]

[0122] Table 6b. Binding EC of exemplary anti-human TNFα antibodies to human, cynomolgus monkey, and canine TNFα 50 [Table 6-2]

[0123] Example 2b. Cell surface binding: To evaluate the binding of the exemplary anti-human TNFα Ab1 GC conjugate of Example 1b to living membrane TNFα-expressing cells, a series of mutations that had previously been demonstrated to allow the expression of bioactive TNFα on the cell surface in the absence of TNFα cleavage (Mueller et al. 1999) were used to inactivate the known cleavage sites of TNFα. A non-cleavable TNFα construct was stably transfected into Chinese hamster ovary (CHO) cells. These cells express membrane-bound TNFα as shown by flow cytometry.

[0124] TNFα-transfected CHO cells were incubated with the exemplary anti-human TNFα Ab1 GC conjugate at concentrations ranging from 600 nM to 0.0304 nM (3-fold dilutions) in FACS buffer (PBS containing 2% FBS) at 4°C for 30 minutes. Cell binding was demonstrated by secondary detection using goat anti-human IgG F(ab’) 2 labeled with AlexaFluor-647 (Thermo #A20186) according to the manufacturer's protocol. Transfected CHO cells incubated with the exemplary anti-human TNFα Ab1 GC conjugate were washed with FACS buffer and then incubated with 2 μg / mL AlexaFluor-647-labeled goat anti-human IgG F(ab’) 2It was stained. The stained cells were washed, resuspended in FACS buffer, and analyzed using a BD LSRFortessa Cell Analyzer. The staining was performed in duplicate. A human IgG1 isotype control antibody was used as a negative control. Anti-human TNFα Ab1 was used as a positive control.

[0125] FlowJo (v10.8.0) was used to analyze the flow cytometry data to obtain the mean fluorescence intensity (MFI) of AlexaFluor-647 for each test sample. EC 50 values were obtained by fitting a non-linear regression (4PL curve) to the plotted MFI data using GraphPad Prism9.

[0126] The results in Table 7 show that the conjugation of GC to anti-human TNFα Ab1 does not significantly affect the binding of the anti-human TNFα Ab1 GC conjugate to membrane-expressed TNFα.

[0127] Table 7. Binding of exemplary anti-human TNFα Ab1 GC conjugates of Example 1b to human TNFα

Table 7

[0128] Example 3: In Vitro Functional Characterization of Anti-Human TNFα Ab GC Conjugates Example 3a. Internalization: The exemplary anti-human TNFα Ab1 GC conjugate and anti-human TNFα antibody of Example 1b were evaluated for their ability to bind to membrane-bound TNFα and internalize into human CD14+ monocytes derived from dendritic cells (DC) from different healthy human donors. CD14+ monocytes were isolated from peripheral blood mononuclear cells (PBMC), cultured, and differentiated into immature dendritic cells (using IL-4 and GM-CSF), and all standard protocols were used. To obtain mature DC, the cells were treated with 1 μg / mL of LPS (lipopolysaccharide) for 4 hours.

[0129] The exemplary anti-human TNFα Ab1 GC conjugate and anti-human TNFα antibody were diluted at 8 μg / mL in complete RPMI medium and mixed in equal volume with the detection probe Fab-TAMRA-QSY7 diluted to 5.33 μg / mL in complete RPMI medium, then incubated in the dark at 4 °C for 30 minutes for complex formation, and then added to immature and mature DC cultures, and CO 2 incubated in an incubator at 37 °C for 24 hours. The cells were washed with 2% FBS PBS and resuspended in 100 μL of 2% FBS PBS containing the Cytox Green live / dead dye. Data were collected on a BD LSR Fortessa X-20 and analyzed with FlowJo. Live single cells were gated, and the percentage of TAMRA-fluorescent positive cells was recorded as the readout. To enable comparison of molecules with data generated from different donors, a normalized internalization index was used. The internalization signal was normalized against the IgG1 isotype (normalized internalization index = 0) and an internal positive control PC (normalized internalization index = 100) using the following equation:

Equation

[0130] The results in Table 8a show that the anti-human TNFα Ab1 GC conjugate internalized into dendritic cells in vitro upon binding to TNFα expressed on mature dendritic cells with an internalization index comparable to that of unconjugated anti-human TNFα Ab1. This indicates that the conjugation of glucocorticoid to anti-human TNFα Ab1 does not affect the internalization function of anti-human TNFα Ab1.

[0131] Representative results from different donors in Table 8b show that anti-human TNFα antibodies Ab1, Ab2, Ab3, Ab4, Ab5, and Ab6 internalize into immature and mature dendritic cells upon binding to TNFα on the cell surface.

[0132] Example 8a: Internalization of the exemplary anti-human TNFα Ab1 GC conjugate of Example 1b into dendritic cells [Table 8-1]

[0133] Table 8b: Internalization of exemplary anti-human TNFα antibodies into dendritic cells [Table 8-2]

[0134] Example 3b. Inhibition of soluble and membrane TNFα-induced apoptosis: The inhibition of soluble and membrane TNFα-induced apoptosis by the exemplary anti-human TNFα Ab1 GC conjugate and anti-human TNFα antibody of Example 1b was evaluated in an in vitro cell-based assay.

[0135] Inhibition of Soluble TNFα-Induced Apoptosis: The ability of exemplary anti-human TNFα Ab1 GC conjugates and anti-human TNFα antibodies to inhibit the soluble TNFα-induced L929 apoptosis assay was evaluated in vitro. 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 the excessive formation of reactive oxygen intermediates that can be rescued by TNFα neutralization. Briefly, L929 was 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 rinsed with 1× PBS (Ca ++ and Mg ++ free) and detached from the culture flask using 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 4 L929 cells (in 100 μL) were added to a 96-well plate and placed in a tissue culture incubator (37°C, 95% relative humidity, 5% CO 2 ) overnight. Next, the conjugate / TNFα / actinomycin-D mixture (TNFα Ab1 GC conjugates and antibodies were added at 3-fold dilutions from 15 μg / mL to 0.0005 μg / mL) was transferred to the 96-well plate containing adherent L929 cells and incubated for 18 hours (37°C, 95% relative humidity, 5% CO 2 ).

[0136] To determine the number of viable cells, the assay medium was removed and an MTS-tetrazolium substrate mixture was added to the wells (100 μL) (mitochondrial dehydrogenase enzymes in metabolically active cells reduce MTS-tetrazolium to a colored formazan product). The plate was placed in an incubator (37°C, 95% relative humidity, 5% CO 2) It was left for 2 hours. Cell death was determined by reading the plate at 490 nm on a microplate reader (Biotek Cytation 5 Imaging Multi-Mode Reader). The results are expressed as the concentration (IC 50 ) at which 50% of TNFα-induced cytotoxicity is inhibited by an exemplary anti-human TNFα Ab1 GC conjugate or antibody, and were calculated using a four-parameter sigmoid fit (GraphPad Prism9) of the data.

[0137] Inhibition of membrane TNFα-induced apoptosis: To evaluate the ability of the exemplary anti-human TNFα Ab1 GC conjugate and anti-human TNFα antibody of Example 1b to inhibit membrane TNFα-induced apoptosis, a non-cleavable TNFα construct was stably transfected into Chinese hamster ovary (CHO) cells to generate cell surface (membrane) TNFα-expressing CHO cells. The non-cleavable TNFα construct was generated using a known mutation at the cleavage site of TNFα that allows for the expression of bioactive TNFα on the cell surface in the absence of TNFα cleavage (Mueller et.al. 1999). Incubation of L929 cells with CHO cells expressing non-cleavable human TNFα resulted in rapid L929 cell death. To determine whether the exemplary anti-human TNFα Ab1 GC conjugate and anti-human TNFα antibody were able to neutralize the observed apoptosis, a dose range of 15 μg / mL to 0.0005 μg / mL (3-fold dilutions) was evaluated. 100 μL / well of the exemplary anti-human TNFα Ab1 GC conjugate or anti-human TNFα antibody at each concentration was added in duplicate to plates containing 500 CHO TNFα transfectant cells / well + 6.25 μg / mL of actinomycin D. The mixture was incubated at room temperature for 30 minutes and then added to the L929 cell plates. 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.

[0138] The results in Table 9a show that the exemplary anti-human TNFα Ab1 GC conjugate of Example 1b inhibited soluble human TNFα (IC 50 of approximately 0.104 μg / mL) and membrane human TNFα (IC 50 of approximately 0.306 μg / mL)-induced apoptosis of L929 cells in vitro, which is comparable to that of anti-human TNFα Ab1. This indicates that conjugation of GC to the antibody does not affect the biological activity of the antibody. The negative control hIgG1 isotype did not inhibit TNFα-induced apoptosis as expected.

[0139] Representative results in Table 9b show that the exemplary anti-human TNFα antibodies Ab1, Ab2, Ab3, Ab4, Ab5, and Ab6 inhibited both soluble and membrane human TNFα-induced apoptosis of L929 cells in vitro in a dose-dependent manner.

[0140] Table 9a. The exemplary anti-human TNFα Ab1 GC conjugate of Example 1b inhibits soluble and membrane human TNFα-induced apoptosis of L929 cells

Table 9-1

[0141] Table 9b. Exemplary anti-human TNFα antibodies inhibit membrane and soluble human TNFα-induced apoptosis of L929 cells

Table 9-2

[0142] Example 3c. In Vitro Human T Cell Cytokine Expression Assay: To evaluate the functional activity of the exemplary anti-human TNFα Ab1 GC conjugate of Example 1b and the anti-human TNFα Ab2 GC conjugate of Example 1d against disease-related cells, human primary T cells were stimulated in vitro and co-treated with the conjugates. The activity of an exemplary anti-human TNFα Ab GC conjugate from U.S. Patent Application Publication No. 2020 / 338208 was also evaluated. Human primary CD3 + T cells were isolated from freshly purified human PBMCs by immunomagnetic negative selection according to the manufacturer's protocol (Human T Cell Isolation Kit, Stemcell Technologies #17951). Flow cytometry staining was used to assess cell purity on a BD LSRFortessa Cell Analyzer. T cells were CD3 + (anti-human CD3-APC, Fisher Scientific #17-0047-42) as confirmed using additional phenotyping for CD4 (anti-human CD4-eFluor-450, Fisher Scientific #48-0036-42) and CD8 (anti-human CD8a-FITC, BioLegend #301006) T cell subsets. 2×10 5 cells of 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). The cells were 1×10 5Stimulated with human T-activator anti-CD3 / CD28 Dynabeads (Thermo Fisher #11132D) and treated with each of exemplary anti-human TNFα Ab1 GC conjugate, anti-human TNFα Ab2 GC conjugate, or exemplary anti-human TNFα Ab GC conjugate from US Patent Application Publication No. 2020 / 338208 at 200 nM to 0.0914 nM (3-fold dilution) in duplicate plates per donor. Human IgG1 isotype control antibody was used as a negative control. The control contained unconjugated anti-human TNFα Ab1 and free GC. Then, the assay plates were incubated at 37 °C with 5% CO 2 for 72 hours. The cell culture supernatant was collected at 72 hours and frozen at -80 °C. Cytokine levels were measured from the thawed cell culture supernatant using a custom U-PLEX human biomarker multiplex assay (Mesoscale Discovery #K15067L) with detection antibodies specific for 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 such that 0% inhibition was equal to the mean concentration of the cytokine in the stimulated untreated control wells and 100% inhibition was equal to the mean concentration of the cytokine in the unstimulated control wells. The normalization parameter for IL-10 was set such that 0% induction was equal to the mean concentration of the cytokine in the stimulated untreated control wells and 100% induction was equal to the mean concentration of the cytokine in the 200 nM free GC-treated group. The normalized IC 50 values were obtained by fitting a non-linear regression (4PL curve) to the normalized data. Statistical analysis was performed using GraphPad Prism9.

[0143] The results in Table 10 show that the anti-human TNFα Ab GC conjugates of Example 1b and Example 1d significantly inhibited the cytokines IL-13, IL-6, and GM-CSF and significantly induced the cytokine IL-10. Additionally, the anti-human TNFα Ab GC conjugates of Example 1b and Example 1d inhibited the cytokines IL-13, IL-6, and GM-CSF and induced the cytokine IL-10 at a higher rate than the anti-human TNFα Ab1 or the exemplary anti-TNFα Ab GC conjugate disclosed in US Patent Application Publication No. 2020 / 338208 in this in vitro assay. In particular, the results show that the anti-human TNFα Ab1 GC conjugate of Example 1b and the anti-human TNFα Ab2 GC conjugate of Example 1d regulate cytokine expression via both the TNFα antibody and the glucocorticoid.

[0144] Table 10. Effects of the anti-human TNFα Ab1 GC conjugate of Example 1b and the anti-human TNFα Ab2 GC conjugate of Example 1d on T cell cytokine release [Table 10] Statistics: Tukey's multiple comparison test, * = p < 0.05 compared to isotype, ^ = p < 0.05 compared to Ab1, † = p < 0.05 compared to the exemplary anti-human TNFα Ab conjugate from US Patent Application Publication No. 2020 / 338208.

[0145] Example 4. Effector function activity of exemplary anti-human TNFα Ab GC conjugates Example 4a. Human Fcγ receptor binding. The binding affinity of the exemplary anti-human TNFα Ab1 GC conjugate of Example 1b for human Fcγ receptors was evaluated by surface plasmon resonance (SPR) analysis. A Series S CM5 chip (Cytiva P / N BR100530) was prepared using the manufacturer's EDC / NHS amine coupling method (Cytiva P / N BR100050). Briefly, the surfaces of all four flow cells (FC) 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 buffer at pH 4.5 and immobilized to approximately 4000 RU on all four FCs by injection at a flow rate of 10 μL / min for 7 minutes. Unreacted sites were blocked by injection of ethanolamine at 10 μL / min for 7 minutes. Any non-covalently associated proteins were removed using 2 × 10 μL injections of glycine at pH 1.5. The running buffer was 1×HBS-EP+ (TEKNOVA, P / N H8022). The FcγR extracellular domains (ECDs) - FcγRI (CD64), FcγRIIA_131R, and FcγRIIA_131H (CD32a), FcγRIIIA_158V, FcγRIIIA_158F (CD16a), and FcγRIIb (CD32b) were generated from stable CHO cell expression and purified using IgG sepharose and size exclusion chromatography. For FcγRI binding, test molecules (including the anti-human TNFα Ab1 GC conjugate of Example 1b and a human IgG1 isotype control antibody) were diluted to 2.5 μg / mL in the running buffer, and approximately 150 RU of each antibody was captured on FCs 2-4 (RU capture). Since FC1 was the reference FC, no antibody was captured on FC1. The FcγRI ECD was diluted to 200 nM in the running buffer and then serially diluted 2-fold in the running buffer to 0.78 nM. Duplicate injections of each concentration were injected into all FCs at 40 μL / min for 120 seconds, followed by a 1200-second dissociation phase.Regeneration was performed by injecting 15 μL of 10 mM glycine at pH 1.5 into all FCs at 30 μL / min. Reference-subtracted data were collected as FC2 - FC1, FC3 - FC1, and FC4 - FC1, and measurements were obtained at 25 °C. Affinity (K D ) was calculated using either steady-state equilibrium analysis with Scrubber 2 Biacore evaluation software or the "1:1 (Langmuir) binding" model of BIA evaluation. For binding of FcγRIIa, FcγRIIb, and FcγRIIIa, the test molecule was diluted to 5 μg / mL in the running buffer, and each antibody at approximately 500 RU was captured on FC2 - 4. FC1 was the reference FC. The Fcγ receptor ECD was diluted to 10 μM in the running buffer and then serially diluted 2-fold in the running buffer to 39 nM. Duplicate injections of each concentration were injected into all FCs at 40 μL / min for 60 s, followed by a 120-s dissociation phase. Regeneration was performed by injecting 15 μL of 10 mM glycine at pH 1.5 into all FCs at 30 μL / min. Reference-subtracted data were collected as FC2 - FC1, FC3 - FC1, and FC4 - FC1, and measurements were obtained at 25 °C. Affinity (K D ) was calculated using steady-state equilibrium analysis with Scrubber 2 Biacore® evaluation software. Each receptor was assayed at least twice.

[0146] The results in Table 11 show that the binding affinity (K D ) of the exemplary anti-human TNFα Ab1 GC conjugate of Example 1b for the human FcγRI, FcγRIIa, FcγRIIb, and FcγRIIIa receptor ECDs is comparable to that of the human IgG1 isotype control antibody.

[0147] Table 11. Binding Affinity of the Exemplary Anti-human TNFα Ab1 GC Conjugate of Example 1b for Human Fcγ Receptors

Table 11

[0148] Example 4b. Antibody-Dependent Cellular Cytotoxicity (ADCC): The in vitro ADCC assay of the exemplary anti-human TNFα Ab1 GC conjugate of Example 1b was evaluated in a reporter gene-based ADCC assay.

[0149] For the reporter gene-based ADCC assay, a CHO cell line (Eli Lilly and Co.) co-expressing membrane-bound TNFα and CD20 was used as the target cell line, and Jurkat cells expressing functional FcγRIIIa (V158)-NFAT-Luc (Eli Lilly and Company) were used as the effector cell line. All test molecules and cells were diluted in assay medium containing RPMI-1640 (without phenol red) supplemented with 0.1 mM non-essential amino acid (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 3× concentration of 20 nM and then serially diluted 7 times at a ratio of 1:4. 50 μL / well of each antibody was dispensed in triplicate into a white opaque-bottom 96-well plate (Costar, #3917). A CD20 antibody was used as a positive control. Next, Daudi target cells were added to the plate at 5×10 4 cells / well in a 50 μL aliquot and incubated at 37 °C for 1 hour. Next, Jurkat V158 cells were added to the wells at 1.5×10 5Added per cell / well and incubated at 37 °C for 4 hours, followed by the addition of 100 μL / well of One-Glo luciferase substrate (Promega, #E8130). The contents of the plate were mixed using a plate shaker at low speed, incubated at room temperature for 5 minutes, and the luminescence signal was read using a BioTek microplate reader (BioTek Instruments) with an integration of 0.2 cps. The data was analyzed using GraphPad Prism9, and the relative luminescence unit (RLU) for each antibody concentration was plotted in a scatter format of antibody concentration vs. RLU. The results were representative of two independent experiments.

[0150] The results in Figure 1 show that the exemplary anti-human TNFα Ab1 GC conjugate of Example 1b had moderate ADCC activity when compared to the positive control.

[0151] Example 4c. Complement-dependent cytotoxicity (CDC): An in vitro CDC assay of the exemplary anti-human TNFα Ab1 GC conjugate of Example 1b was performed using a CHO cell line (Eli Lilly and Co.) co-expressing membrane-bound TNFα and CD20. All test antibodies, complement, and cells were diluted in an assay medium consisting of RPMI-1640 (without phenol red) containing 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 3× concentration of 200 nM and then serially diluted 7 times at a ratio of 1:4. 50 μL / well of each antibody (including the CD20 positive control antibody) was dispensed in triplicate into a white opaque-bottom 96-well plate (Costar, #3917). Daudi target cells were 5×10 4Cells / well were added at 50 μL / well and incubated at 37 °C for 1 hour. Next, human serum complement (Quidel, #A113) rapidly thawed in a 37 °C water bath was diluted 1:6 in assay medium and added to the assay plate at 50 μL / well. The plate was incubated at 37 °C for 2 hours, followed by the addition of 100 μL / well of CellTiter Glo substrate (Promega, #G7571). The plate contents were mixed using a plate shaker at low speed, incubated at room temperature for 5 minutes, and the luminescence signal was read using a BioTek microplate reader (BioTek Instruments) with an integration of 0.2 cps. The data were analyzed using GraphPad Prism9, and the relative light units (RLU) for each antibody concentration were plotted in a scatter format of antibody concentration vs. RLU.

[0152] The results of two representative independent experiments, one of which is shown in Figure 2, showed that the exemplary anti-human TNFα Ab1 GC conjugate had a slight induction of CDC activity when compared to the positive control.

[0153] Example 5: Characterization of Exemplary Anti-Human TNFα Antibodies That Bind to Anti-Drug Antibodies Against Adalimumab Example 5a. Binding to a cynomolgus anti-drug antibody against adalimumab: The binding of exemplary anti-human TNFα antibodies to anti-drug antibodies (anti-adalimumab antibodies) against adalimumab obtained from affinity purified hyperimmune monkey serum (AP-HIMS) from cynomolgus monkeys highly immunized with adalimumab was evaluated. Anti-adalimumab antibodies from adalimumab-highly immunized cynomolgus monkeys were purified using Adalimumab-AffiGel10. Anti-adalimumab antibodies were detected using titration of AP-HIMS in the ACE-Bridge assay. The assay was developed according to FDA guidance on immunogenicity testing. Briefly, streptavidin-coated 96-well plates (Pierce, 15500) were washed with 1×TBST (Boston BioProducts, IBB-181X) and coated with 30 nM 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 in TBS (Fisher, BP2471-1) and added to the coated plates at 100 μL / 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 at room temperature for 5 minutes using 65 μL / well of 300 mM acetic acid (Fisher Scientific, A38-500). Next, polypropylene 96-well plates (Corning, 3359) were loaded with 50 μL each of 1 μg / mL biotinylated adalimumab and ruthenium-labeled adalimumab in neutralization buffer (0.375 M Tris, 300 mM NaCl, pH 9). Then, 50 μL of the acid elution sample was added to the polypropylene plate containing the mixture in neutralization buffer and ADA and cross-linked to the labeled antibody for 1 hour at room temperature. MSD Gold 96-well streptavidin plates (Mesoscale, L15SA-1) were washed, blocked with TBS + 1% BSA for 1 hour at room temperature, then washed, and 80 μL of the cross-linking sample was added to the plate for 1 hour.The plate was washed three times with TBST, and 150 μL / well of 2× MSD buffer (Mesoscale, R92TC-2) was added to the plate. The plate was read with an MSD SQ120 reader to obtain a Tier1 signal expressed as electro chemiluminescent unit (ECLU).

[0154] The same AP-HIMS was also tested in the ACE-Bridge assay, and antibodies against the exemplary anti-human TNFα antibody Ab6 were detected essentially according to the same method as outlined above for adalimumab, but using biotin and ruthenium-labeled Ab6. The resulting ECLU signals were then plotted as a function of the concentration of the AP-HIMS tested.

[0155] The results in Figure 3 show that the exemplary anti-human TNFα antibody Ab6 had low or no binding to anti-drug antibodies (maximum ECLU signal 4000) against adalimumab purified from serum of cynomolgus monkeys highly immunized with adalimumab, when compared to the binding of adalimumab itself to anti-drug antibodies (maximum ECLU signal 40000). Specifically, the results show that the exemplary anti-human TNFα antibody Ab6 recognized only about 10% of the anti-drug antibodies against adalimumab produced by cynomolgus monkeys, suggesting that this binding is likely due to shared sequences located away from CDR regions such as the antibody constant region.

[0156] Example 5b. Binding to anti-drug antibodies in human patients against adalimumab: The binding of an exemplary anti-human TNFα antibody to anti-drug antibodies (anti-adalimumab antibodies) against adalimumab in 21 patient serum samples obtained from adalimumab-treated patients enrolled in the trial RA-BEAM was evaluated. The 21 serum samples were collected after baseline and were confirmed to have a high ADA titer against adalimumab by using a method essentially as described for cynomolgus ADA assessment. The 21 serum samples were then evaluated for binding to an exemplary anti-human TNFα antibody Ab6 by using a method essentially as described for cynomolgus ADA assessment.

[0157] The results in Figure 4 show that the exemplary anti-human TNFα antibody Ab6 had low or no binding to anti-drug antibodies against adalimumab in 16 of the 21 patient samples tested (ECLU signal was below the assay cut-off point (102 ECLU)). In the determination of immunogenicity, the cut-off point is the threshold used to identify "presumed positive" or anti-drug antibody-containing samples. As shown in Figure 4, 5 of the 21 samples had an ECLU signal above the cut-off point, but all were less than 20% above the cut-off point and were thus determined to be within the range of assay variability. The significantly low or no binding of the anti-human TNFα antibody Ab6 to anti-drug antibodies against adalimumab in human patients treated with adalimumab indicates that the ADA generated against adalimumab by the human subjects tested, for which the Ab6 and adalimumab antibody sequences are sufficiently different such that the epitopes present in adalimumab are specific, is not shared by Ab6 and is thus not recognized much by Ab6.

[0158] These results indicate the potential use of exemplary anti-human TNFα antibodies or conjugates for the treatment of subjects that develop a reduced clinical response or adverse reaction to anti-drug antibodies against other TNFα therapeutics such as adalimumab.

[0159] Example 6: Immunogenicity assessment Example 6a. MHC-Related Peptide Proteomics (MAPP) Assay: MAPP profiles MHC-II presented peptides on human dendritic cells pre-treated with an exemplary anti-human TNFα antibody. CD14+ monocytes were isolated from peripheral blood mononuclear cells (PBMCs), cultured, and differentiated into immature dendritic cells (containing IL-4 and GM-CSF) using standard protocols. On day 4, the exemplary antibody was added to the immature dendritic cells, and the fresh medium containing LPS was replaced after a 5-hour incubation to transform the cells into mature dendritic cells. The next day, the mature dendritic cells were lysed in RIPA containing protease inhibitors and DNAse. Immunoprecipitation of the MHC-II complex was performed using a biotinylated anti-MHC-II antibody conjugated to streptavidin beads. The bound complex was eluted and filtered. The isolated MHC-II peptides were analyzed by a mass spectrometer. Peptide identification was generated by an in-house proteomics pipeline using a search algorithm without enzymatic search parameters against a bovine / human database containing the test sequences added to the database. The peptides identified from the exemplary antibody were aligned against the parental sequences.

[0160] The results in Table 12 show that the exemplary anti-human TNFα antibody had varying degrees of presentation by MAPP. Ab1 demonstrated the lowest MAPP presentation with one non-germline cluster in 3 out of 10 donors tested.

[0161] Table 12. MAPP Analysis of Exemplary Anti-Human TNFα Antibodies [Table 12]

[0162] T cell proliferation assay: The ability of an exemplary anti-human TNFα antibody MAPP-derived peptide cluster 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 seeded at 4 × 10 6 cells / mL / well in AIM-V medium (Life Technologies, catalog number 12055-083) containing 5% CTS™ Immune Cell SR (Gibco, catalog number A2596101) and tested in triplicate with 2.0 mL containing the following different molecules: DMSO control, medium control, keyhole limpet hemocyanin (KLH; positive control), PADRE-X peptide (synthetic vaccine helper peptide, positive peptide control), or each anti-human TNFα antibody MAPP-derived peptide cluster (10 μM of each peptide). The cells were cultured and incubated at 37 °C for 7 days with 5% CO 2 2. On day 7, samples were stained with the following cell surface markers for flow cytometric viability detection using a BD LSRFortessa™ equipped with a high throughput sampler (HTS): anti-CD3, anti-CD4, anti-CD14, anti-CD19, and DAPI. Data were analyzed using FlowJo® software (FlowJo, LLC, TreeStar) and the Cellular Division Index (CDI) was calculated. Briefly, the CDI for each MAPP-derived peptide cluster was calculated by dividing the percentage of proliferating CFSE dim CD4+ T cells from peptide-stimulated wells by the percentage of proliferating CFSE dim CD4+ T cells in unstimulated wells. A CDI of 2.5 or greater was considered to represent a positive response. The percentage of donor frequency across all donors was evaluated.

[0163] The results in Tables 13a and 13b show that the LCDR1 (Table 13a) and HCDR3 (Table 13b) peptides for Ab2 induced T cell response frequencies in approximately 22.0% and 25% of the donors, respectively, indicating a significantly reduced immunogenic risk for Ab2 when compared to the positive controls. The KLH positive control induced T cell responses in 100% of the donors, and the PADRE-X (synthetic vaccine helper peptide) positive control induced T cell responses in 67% and 62.5% of the donors in two studies, respectively. This is within the expected range of this assay (positive donor frequency of 48.1% + 24.4).

[0164] Table 13a. Frequency of CD4+ T cell responses induced by MAPP-derived peptides in healthy donors.

Table 13-1

[0165] Table 13b. Frequency of CD4+ T cell responses induced by MAPP-derived peptides in healthy donors.

Table 13-2

[0166] Example 7. Biophysical properties of the exemplary anti-human TNFα Ab1 GC conjugate of Example 1b The biophysical properties of the exemplary anti-human TNFα Ab1 GC conjugate of Example 1b were evaluated for developmental potential.

[0167] 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. Using a VROC® initium (RheoSense), the viscosity of the conjugate at all three concentrations was measured at 15 °C using the average of nine repeated measurements. The results in Table 15 showed that the anti-human TNFα Ab1 GC conjugate of Example 1b had good viscosity profiles at 50 mg / mL (2 cP), 100 mg / mL (4.7 cP), and 125 mg / mL (8.2 cP). The viscosity of the exemplary anti-human TNFα Ab1 GC conjugate at 125 mg / mL (8.2 cP) was similar to that of the unconjugated anti-human TNFα Ab1 at 125 mg / mL (8.8 cP), indicating that conjugating the linker-payload to the four engineered cysteines on the surface of the exemplary anti-human TNFα Ab1 did not adversely affect the viscosity.

[0168] Example 7b. Thermal stability: Differential Scanning Calorimetry (DSC) was used to evaluate the stability of the anti-human TNFα Ab1 GC conjugate of Example 1b against thermal denaturation. The melting onset temperature (Tonset) and the thermal melting temperatures (TM1, TM2, and TM3) of the exemplary anti-human TNFα Ab1 GC conjugate in PBS, pH 7.2 buffer, acetate at pH 5, and histidine at pH 6 were obtained by data fitting and listed in Table 14. The thermograms of the three buffer compositions are shown in FIGS. 5A, 5B, and 5C. The thermal transitions of each domain were well-resolved, and the results in Table 14 showed that the anti-human TNFα Ab1 GC conjugate of Example 1b had good thermal stability.

[0169] Example 7c. Aggregation during temperature stress: The solution stability over time of an exemplary anti-human TNFα Ab1 GC conjugate was evaluated at approximately 100 mg / mL and 50 mg / mL in a common 5 mM histidine pH 6.0 buffer containing excipients. Samples were incubated at 5 °C and 35 °C for a period of 28 weeks. After incubation, size exclusion chromatography (SEC-HPLC) was used to analyze the percentage of high molecular weight (%HMW) species. The results in Table 15 show that the anti-human TNFα Ab1 GC conjugate of Example 1b has an acceptable aggregation profile over 4 weeks at either 5 °C or 35 °C.

[0170] Example 7d. Pharmacokinetics: The PK profile of the anti-human TNFα Ab 1GC conjugate of Example 1b in cynomolgus monkeys was found to have an acceptable development profile.

[0171] Table 14. Thermal stability (°C) of the exemplary anti-human TNFα Ab1 GC conjugate of Example 1b

Table 14

[0172] Table 15: Viscosity and high-concentration temperature holding stability for the exemplary anti-human TNFα Ab1 GC conjugate of Example 1b

Table 15

[0173] Example 8. In vivo function of the anti-human TNFα Ab GC conjugates of Example 1b, Example 1c, and Example 1d Example 8a. In vivo inhibition of human TNFα-induced CXCL1 cytokine The neutralization of TNFα-induced CXCL1 by the exemplary anti-human TNFα Ab1 GC conjugate and anti-human TNFα Ab1 of Example 1b was evaluated in vivo. Administration of human TNFα to C57 / B6 mice induces a rapid and transient increase in mouse plasma CXCL1 levels. This enables investigation of the neutralizing ability of the exemplary anti-human TNFα Ab1 and anti-human TNFα Ab1 GC conjugate in vivo.

[0174] Briefly, C57 / B6 mice (N = 8 / group) were administered subcutaneously (SC) with 0.3 mg / kg or 3 mg / kg of the exemplary anti-human TNFα Ab1 GC conjugate or anti-human TNFα Ab1, and 3 mg / kg of an unbound isotype control. Twenty-four hours after administration, the mice were challenged with human TNFα via intraperitoneal injection at a dose of 3 μg / mouse. Two hours after the human TNFα challenge, the mice were sacrificed, blood was collected, and the plasma was clarified by centrifugation. The plasma was analyzed using a commercially available MSD assay (MesoScale Discovery, P / N. number K152QTG-1) according to the manufacturer's instructions.

[0175] The results in Table 16 show that the exemplary anti-human TNFα Ab1 and anti-human TNFα Ab1 GC conjugate of Example 1b significantly inhibited human TNFα-induced plasma CXCL1 production in vivo, by approximately 80.5 and 81.6% at 3 mg / kg, and approximately 69.4 and 58.9% at 0.3 mg / kg, respectively, compared to isotype control-treated mice (p < 0.001, ANOVA, followed by Tukey's multiple comparison test). Importantly, at the tested doses, there was no significant difference between the anti-human TNFα Ab1 GC conjugate and anti-human TNFα Ab1. This indicates that the exemplary anti-human TNFα Ab1 neutralizes the biological effects induced by human TNFα in vivo, and conjugation to GC does not affect this function.

[0176] Table 16: In vivo inhibition of human TNFα-induced CXCL1 cytokine production by anti-human TNFα Ab1 GC conjugate of Example 1b [Table 16] Ordinary one-way analysis of variance, Tukey test, multiple comparisons

[0177] Example 8b. In vivo efficacy in type IV hypersensitivity of a fully humanized mouse model: Using a humanized mouse model of contact hypersensitivity, the in vivo activities of the anti-human TNFα Ab1 GC conjugates of Example 1b and Example 1c and anti-human TNFα Ab1 were determined.

[0178] Immunodeficient NOG mice (huNOG-EXL, Taconic) expressing human GM-CSF and human IL-3 to support myeloid lineage development were transplanted with human CD34+ hematopoietic stem cells isolated from human umbilical cord blood at 6 weeks of age. Twenty to twenty-four weeks after stem cell administration, the mice were evaluated for sufficient human CD45 engraftment (blood > 25%) and subjected to the oxazolone-induced contact hypersensitivity protocol. On day 0, mice grouped by body weight were subcutaneously (SC) administered either the anti-human TNFα Ab1 GC conjugate of Example 1b (n = 7), the TNFα Ab1 GC conjugate of Example 1c (n = 7), anti-human TNFα Ab1 (n = 7), an exemplary anti-human TNFα Ab GC conjugate from US Patent Application Publication No. 2020 / 338208 (n = 7), or a control human IgG1 antibody (n = 7) at 1 mg / kg. On day 1, the mice were anesthetized with 5% isoflurane, their abdomens were shaved, and 100 μL of 3% oxazolone in ethanol was applied to the shaved area. On day 7, the mice were re-administered at 1 mg / kg SC, anesthetized, and then challenged with 2% oxazolone in ethanol in both ears (10 μL / side / ear) 24 hours after administration. The dose challenge paradigm was repeated weekly, and the dose of the test agent was increased to 3 mg / kg for challenge 2 and 10 mg / kg for challenge 3. The inflammatory response was determined by the difference in ear thickness before and 24 hours after each challenge using a Miltenyi Biotec electronic caliper. The P value between groups was calculated by one-way ANOVA followed by Tukey's post hoc test and considered significant if < 0.05 (GraphPad Prism).

[0179] The results in Table 17 and Figures 6A - 6C show that the anti - human TNFα Ab1 GC conjugates of Example 1b and Example 1c induced excellent in - vivo reduction of the inflammatory response from hapten - induced contact hypersensitivity reactions at all three challenges (1, 3, and 10 mg / kg), compared to both the anti - human TNFα Ab1 that attenuated ear swelling only at the dose of 10 mg / kg of challenge 3 and the exemplary anti - human TNFα Ab GC conjugate from US Patent Application Publication No. 2020 / 338208. Further, the results show that the conjugation of anti - human TNFα Ab1 to three or four GC molecules (DAR) induced similar efficacy. These results indicate that the anti - human TNFα Ab1 GC conjugate effectively delivered glucocorticoids to the inflamed tissue and significantly suppressed the biological effects associated with type IV hypersensitivity reactions in the humanized mouse model, indicating that this anti - inflammatory response can be induced in human subjects.

[0180] Table 17: In - vivo efficacy of the anti - human TNFα Ab1 GC conjugates of Example 1b and Example 1c in a type IV hypersensitivity humanized mouse model

Table 17

[0181] Example 8c. In Vivo Efficacy in a Human TNFα Transgenic Mouse Model of Arthritis: Using a human TNFα transgenic mouse model of arthritis (Taconic, #1006), the efficacy of the anti-human TNFα Ab2 GC conjugate and anti-human TNFα Ab2 of Example 1d was evaluated as a primary treatment in adalimumab-naïve mice and as a secondary treatment in adalimumab-treated mice that had generated anti-drug antibodies to adalimumab and had a reduced or lost or non-responsive to adalimumab, i.e., adalimumab non-responsive mice. This mouse model constitutively expresses human TNFα via the CMV promoter, which results in progressive joint inflammation that appears mainly in the front and hind paws. Treatment with adalimumab weakens the progression of the disease for a short period of a few weeks, however, the beneficial effect was due to the generation of neutralizing anti-drug antibodies. To rule out the possibility that adalimumab generates anti-drug antibodies against the human Fc portion of the antibody and thus affects the activity of the anti-human TNFα Ab2 GC conjugate and anti-human TNFα Ab2 of Example 1d, all molecules were generated as chimeric species in which the antibody constant domain was replaced with the constant domain of the mouse IgG2a antibody.

[0182] At 13 weeks of age, when all mice showed moderate inflammation (score 4 - 9) in one or more feet, the mice were divided into six clinical score-matched groups of 8 mice / group. Mice in each group were administered subcutaneously (SC) once a week for 9 weeks at 3 mg / kg with either mIgG2a isotype control, human / mouse chimeric anti-human TNFα Ab2 (referred to herein as "h / mAb2"), human / mouse chimeric anti-human TNFα Ab2 GC conjugate (referred to herein as "h / mAb2-GC"), human / mouse chimeric adalimumab (referred to herein as "h / m-adalimumab"), switching to h / m-adalimumab GC conjugate (prepared using engineered cysteine and conjugated to GC-L essentially as described in Example 1b and referred to herein as "h / m-adalimumab-GC") after two doses of h / m-adalimumab during the test period, or switching to h / mAb2-GC after two doses of h / m-adalimumab during the test period. The parameters of the clinical score for each limb were as follows: 0 = no evidence of distortion, 1 = mild distortion, 2 = moderate distortion, 3 = severe distortion / mild swelling, 4 = severe distortion / severe swelling / loss of function. Mice were scored twice a week and their body weights were measured regularly. Blood was collected on day 10 to quantify antibody exposure levels and determine the extent of antidrug antibody development. At the end of the experiment, the mice were anesthetized with isoflurane and blood and tissues were collected.

[0183] The results in Figure 7 show that h / mAb2-GC and h / mAb2 completely halted disease progression at the start of treatment, as measured by clinical score compared to all other treatments, and this was sustained over the 9-week treatment period. Importantly, this showed that h / mAb2-GC and h / mAb2 did not generate a significant anti-drug antibody response, and as a result, it did not neutralize and / or reduce the efficacy of the conjugate or antibody. The results also showed that h / m-adalimumab was able to delay disease progression by approximately 2 weeks; however, efficacy was lost concurrently with the appearance of anti-drug antibodies to h / m-adalimumab by approximately 2 weeks into the 9-week treatment. However, importantly, mice treated with h / m-adalimumab for 2 weeks and then switched to treatment with h / mAb2-GC maintained significant suppression of disease progression, while mice treated with h / m-adalimumab for 2 weeks and then switched to treatment with h / m-adalimumab-GC showed an inflammatory response that reflected the h / m-adalimumab treatment alone group. These results indicate that the anti-human TNFα Ab2 GC conjugate has low or no cross-reactivity to anti-drug antibodies to adalimumab. These results demonstrate the potential use of exemplary anti-human TNFα Ab conjugates in the treatment of subjects who have generated anti-drug antibodies to other anti-TNFα therapeutics such as adalimumab and whose response to that treatment has waned.

[0184] Sequence Listing Ab1 HCDR1 for SEQ ID NO:1 Ab1, Ab2, and Ab6 GYTFTGYYIH HCDR2 for SEQ ID NO:2 Ab1, Ab2, and Ab6 WINPYTGGTNYAQKFQG HCDR3 for SEQ ID NO:3 Ab1 DLYGSSNYGGDV LCDR1 for SEQ ID NO:4 Ab1 and Ab3 QASQGISNYLN LCDR2 for SEQ ID NO:5 Ab1, Ab2, Ab3, Ab4, Ab5, and Ab6 DASNLET LCDR3 for Sequence Numbers 6 Ab1, Ab3, and Ab5 QQYDKLPLT VH for Sequence Number 7 Ab1 QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYIHWVRQAPGQGLEWMGWINPYTGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDLYGSSNYGGDVWGQGTTVTVSS VL for Sequence Numbers 8 Ab1 and Ab3 DIQMTQSPSSLSASVGDRVTITCQASQGISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDKLPLTFGGGTKVEIK HC for Sequence Number 9 Ab1 QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYIHWVRQAPGQGLEWMGWINPYTGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDLYGSSNYGGDVWGQGTTVTVSSASTKGPCVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDICVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK LC for Sequence Numbers 10 Ab1 and Ab3 DIQMTQSPSSLSASVGDRVTITCQASQGISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDKLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC HC DNA for Sequence No. 11 Ab1 LC DNA for Array No. 12 Ab1 and Ab3 GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCGAGTCAGGGCATTAGCAACTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTACGATGCATCCAATTTGGAAACAGGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCAACATATTACTGTCAACAGTATGATAAGCTCCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGGACCGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGC

[0185] Ab2 HCDR1 for Array No. 1 Ab1, Ab2, and Ab6 GYTFTGYYIH HCDR2 for Array No. 2 Ab1, Ab2, and Ab6 WINPYTGGTNYAQKFQG HCDR3 for Array No. 13 Ab2, Ab3, Ab4, and Ab5 DLYGSSNYGMDV LCDR1 for Array No. 14 Ab2, Ab4, and Ab5 QASQGIRNYLN LCDR2 for Sequence Numbers 5 Ab1, Ab2, Ab3, Ab4, Ab5, and Ab6 DASNLET LCDR3 for Sequence Numbers 15 Ab2 and Ab4 QQYDNLPLT VH for Sequence Number 16 Ab2 QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYIHWVRQAPGQGLEWMGWINPYTGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDLYGSSNYGMDVWGQGTTVTVSS VL for Sequence Numbers 17 Ab2 and Ab4 DIQMTQSPSSLSASVGDRVTITCQASQGIRNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLPLTFGGGTKVEIK HC for Sequence Number 18 Ab2 QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYIHWVRQAPGQGLEWMGWINPYTGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDLYGSSNYGMDVWGQGTTVTVSSASTKGPCVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDICVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK LC for Sequence Numbers 19 Ab2 and Ab4 DIQMTQSPSSLSASVGDRVTITCQASQGIRNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC HC DNA for SEQ ID NO: 20 Ab2 LC DNA for Array No. 21 Ab2 and Ab4 GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCGAGTCAGGGCATTCGCAACTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTACGATGCATCCAATTTGGAAACAGGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCAACATATTACTGTCAACAGTATGATAACCTCCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGGACCGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGC

[0186] Ab3 HCDR1 for Array No. 22 Ab3, Ab4, and Ab5 GYTFTGYYMH HCDR2 for Array No. 23 Ab3, Ab4, and Ab5 WINPYTGGTKYAQKFQG HCDR3 for Array No. 13 Ab2, Ab3, Ab4, and Ab5 DLYGSSNYGMDV LCDR1 for Array No. 4 Ab1 and Ab3 QASQGISNYLN LCDR2 for Sequence Numbers 5 Ab1, Ab2, Ab3, Ab4, Ab5, and Ab6 DASNLET LCDR3 for Sequence Numbers 6 Ab1, Ab3, and Ab5 QQYDKLPLT VH for Sequence Numbers 24 Ab3, Ab4, and Ab5 QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPYTGGTKYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDLYGSSNYGMDVWGQGTTVTVSS VL for Sequence Numbers 8 Ab1 and Ab3 DIQMTQSPSSLSASVGDRVTITCQASQGISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDKLPLTFGGGTKVEIK HC for Sequence Numbers 25 Ab3, Ab4, and Ab5 QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPYTGGTKYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDLYGSSNYGMDVWGQGTTVTVSSASTKGPCVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDICVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK LC for Array No. 10 Ab1 and Ab3 DIQMTQSPSSLSASVGDRVTITCQASQGISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDKLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC HC DNA for Array No. 26 Ab3, Ab4, and Ab5 LC DNA for Array No. 12 Ab1 and Ab3 GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCGAGTCAGGGCATTAGCAACTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTACGATGCATCCAATTTGGAAACAGGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCAACATATTACTGTCAACAGTATGATAAGCTCCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGGACCGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGC

[0187] Ab4 HCDR1 for Array No. 22 Ab3, Ab4, and Ab5 GYTFTGYYMH HCDR2 for Array No. 23 Ab3, Ab4, and Ab5 WINPYTGGTKYAQKFQG HCDR3 for Array No. 13 Ab2, Ab3, Ab4, and Ab5 DLYGSSNYGMDV LCDR1 for Array No. 14 Ab2, Ab4, and Ab5 QASQGIRNYLN LCDR2 for Sequence Nos. 5 Ab1, Ab2, Ab3, Ab4, Ab5, and Ab6 DASNLET LCDR3 for Sequence Nos. 15 Ab2 and Ab4 QQYDNLPLT VH for Sequence Nos. 24 Ab3, Ab4, and Ab5 QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPYTGGTKYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDLYGSSNYGMDVWGQGTTVTVSS VL for Sequence Nos. 17 Ab2 and Ab4 DIQMTQSPSSLSASVGDRVTITCQASQGIRNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLPLTFGGGTKVEIK HC for Sequence Nos. 25 Ab3, Ab4 and Ab5 QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPYTGGTKYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDLYGSSNYGMDVWGQGTTVTVSSASTKGPCVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDICVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK LC for Array No. 19 Ab2 and Ab4 DIQMTQSPSSLSASVGDRVTITCQASQGIRNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC HC DNA for Array No. 26 Ab3, Ab4, and Ab5 LC DNA for Array No. 21 Ab2 and Ab4 GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCGAGTCAGGGCATTCGCAACTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTACGATGCATCCAATTTGGAAACAGGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCAACATATTACTGTCAACAGTATGATAACCTCCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGGACCGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGC

[0188] Ab5 HCDR1 for Array No. 22 Ab3, Ab4, and Ab5 GYTFTGYYMH HCDR2 for Array No. 23 Ab3, Ab4, and Ab5 WINPYTGGTKYAQKFQG HCDR3 for Array No. 13 Ab2, Ab3, Ab4, and Ab5 DLYGSSNYGMDV LCDR1 for Array No. 14 Ab2, Ab4, and Ab5 QASQGIRNYLN LCDR2 for Sequence Nos. 5 Ab1, Ab2, Ab3, Ab4, Ab5, and Ab6 DASNLET LCDR3 for Sequence Nos. 6 Ab1, Ab3, and Ab5 QQYDKLPLT VH for Sequence Nos. 24 Ab3, Ab4, and Ab5 QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPYTGGTKYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDLYGSSNYGMDVWGQGTTVTVSS VL for Sequence No. 27 Ab5 DIQMTQSPSSLSASVGDRVTITCQASQGIRNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDKLPLTFGGGTKVEIK HC for Sequence Nos. 25 Ab3, Ab4 and Ab5 QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPYTGGTKYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDLYGSSNYGMDVWGQGTTVTVSSASTKGPCVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDICVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK LC for Sequence No. 28 Ab5 DIQMTQSPSSLSASVGDRVTITCQASQGIRNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDKLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC HC DNA for Sequence Nos. 26 Ab3, Ab4, and Ab5 LC DNA for Array No. 29 Ab5 GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCGAGTCAGGGCATTCGCAACTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTACGATGCATCCAATTTGGAAACAGGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCAACATATTACTGTCAACAGTATGATAAGCTCCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGGACCGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGC

[0189] Ab6 HCDR1 for Array No. 1 Ab1, Ab2, and Ab6 GYTFTGYYIH HCDR2 for Array No. 2 Ab1, Ab2, and Ab6 WINPYTGGTNYAQKFQG HCDR3 for Array No. 30 Ab6 DIYGSSNYGGDV LCDR1 for Array No. 31 Ab6 QASQDISNYLN LCDR2 for Sequence Numbers 5 Ab1, Ab2, Ab3, Ab4, Ab5, and Ab6 DASNLET LCDR3 for Sequence Number 32 Ab6 QQYDTLPLT VH for Sequence Number 33 Ab6 QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYIHWVRQAPGQGLEWMGWINPYTGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDIYGSSNYGGDVWGQGTTVTVSS VL for Sequence Number 34 Ab6 DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDTLPLTFGGGTKVEIK HC for Sequence Number 35 Ab6 QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYIHWVRQAPGQGLEWMGWINPYTGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDIYGSSNYGGDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK LC for Sequence Number 36 Ab6 DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDTLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC HC DNA for SEQ ID NO: 37 Ab6 LC DNA for Array No. 38 Ab6 GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCGAGTCAGGACATTAGCAACTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTACGATGCATCCAATTTGGAAACAGGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCAACATATTACTGTCAACAGTATGATACCCTCCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGGACCGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGC Array No. 39 Human TNFα Protein MSTESMIRDVELAEEALPKKTGGPQGSRRCLFLSLFSFLIVAGATTLFCLLHFGVIGPQREEFPRDLSLISPLAQAVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL Array No. 40 Rhesus TNFα Protein MSTESMIRDVELAEEALPRKTAGPQGSRRCWFLSLFSFLLVAGATTLFCLLHFGVIGPQREEFPKDPSLISPLAQAVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELTDNQLVVPSEGLYLIYSQVLFKGQGCPSNHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINLPDYLDFAESGQVYFGIIAL SEQ ID NO: 41 Dog TNFα Protein MSTESMIRDVELAEEPLPKKAGGPPGSRRCFCLSLFSFLLVAGATTLFCLLHFGVIGPQREELPNGLQLISPLAQTVKSSSRTPSDKPVAHVVANPEAEGQLQWLSRRANALLANGVELTDNQLIVPSDGLYLIYSQVLFKGQGCPSTHVLLTHTISRFAVSYQTKVNLLSAIKSPCQRETPEGTEAKPWYEPIYLGGVFQLEKGDRLSAEINLPNYLDFAESGQVYFGIIAL SEQ ID NO: 42 Cynomolgus Monkey TNFα Protein MSTESMIQDVELAEEALPRKTAGPQGSRRCWFLSLFSFLLVAGAATLFCLLHFGVIGPQREEFPKDPSLISPLAQAVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALVANGVELTDNQLVVPSEGLYLIYSQVLFKGQGCPSNHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINLPDYLDFAESGQVYFGIIAL SEQ ID NO: 43 LCDR1 Consensus Sequence QASQGIXaa 7 NYLN wherein Xaa 7 is serine or arginine SEQ ID NO: 44 LCDR3 Consensus Sequence QQYDXaa 5 LPLT In the formula, Xaa5 is asparagine or lysine SEQ ID NO: 45 Human TNFR1 MGLSTVPDLLLPLVLLELLVGIYPSGVIGLVPHLGDREKRDSVCPQGKYIHPQNNSICCTKCHKGTYLYNDCPGPGQDTDCRECESGSFTASENHLRHCLSCSKCRKEMGQVEISSCTVDRDTVCGCRKNQYRHYWSENLFQCFNCSLCLNGTVHLSCQEKQNTVCTCHAGFFLRENECVSCSNCKKSLECTKLCLPQIENVKGTEDSGTTVLLPLVIFFGLCLLSLLFIGLMYRYQRWKSKLYSIVCGKSTPEKEGELEGTTTKPLAPNPSFSPTPGFTPTLGFSPVPSSTFTSSSTYTPGDCPNFAAPRREVAPPYQGADPILATALASDPIPNPLQKWEDSAHKPQSLDTDDPATLYAVVENVPPLRWKEFVRRLGLSDHEIDRLELQNGRCLREAQYSMLATWRRRTPRREATLELLGRVLRDMDLLGCLEDIEEALCGPAALPPAPSLLR SEQ ID NO: 46 Human TNFR2 MAPVAVWAALAVGLELWAAAHALPAQVAFTPYAPEPGSTCRLREYYDQTAQMCCSKCSPGQHAKVFCTKTSDTVCDSCEDSTYTQLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCRPGWYCALSKQEGCRLCAPLRKCRPGFGVARPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAIPGNASMDAVCTSTSPTRSMAPGAVHLPQPVSTRSQHTQPTPEPSTAPSTSFLLPMGPSPPAEGSTGDFALPVGLIVGVTALGLLIIGVVNCVIMTQVKKKPLCLQREAKVPHLPADKARGTQGPEQQHLLITAPSSSSSSLESSASALDRRAPTRNQPQAPGVEASGAGEARASTGSSDSSPGGHGTQVNVTCIVNVCSSSDHSSQCSSQASSTMGDTDSSPSESPKDEQVPFSKEECAFRSQLETPETLLGSTEEKPLPLGVPDAGMKPS

Claims

1. Formula: 【Chemical 1】 A conjugate represented by the formula, wherein Ab is an antibody that binds to human TNFα, Ab comprises a heavy chain variable region (VH) and a light chain variable region (VL), said VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, said VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, and said HCDR1 comprises SEQ ID NO: 1, said HCDR2 comprises SEQ ID NO: 2, said HCDR3 comprises SEQ ID NO: 3, said LCDR1 comprises SEQ ID NO: 4, said LCDR2 comprises SEQ ID NO: 5, said LCDR3 comprises SEQ ID NO: 6; or said HCDR1 comprises SEQ ID NO: 1, said HCDR2 comprises SEQ ID NO: 2, said HCDR3 comprises SEQ ID NO: 13, said LCDR1 comprises SEQ ID NO: 14, said LCDR2 comprises SEQ ID NO: 5, said LCDR3 comprises SEQ ID NO: 15; or said HCDR1 comprises SEQ ID NO: 22, said HCDR2 comprises SEQ ID NO: 23, said HCDR3 comprises SEQ ID NO: 13, said LCDR1 comprises SEQ ID NO: 4, said LCDR2 comprises SEQ ID NO: 5, said LCDR3 comprises SEQ ID NO: 6; or said HCDR1 comprises SEQ ID NO: 22, said HCDR2 comprises SEQ ID NO: 23, said HCDR3 comprises SEQ ID NO: 13, said LCDR1 comprises SEQ ID NO: 14, said LCDR2 comprises SEQ ID NO: 5, said LCDR3 comprises SEQ ID NO: 15; or said HCDR1 comprises SEQ ID NO: 22, said HCDR2 comprises SEQ ID NO: 23, said HCDR3 comprises SEQ ID NO: 13, said LCDR1 comprises SEQ ID NO: 14, said LCDR2 comprises SEQ ID NO: 5, said LCDR3 comprises SEQ ID NO: 6; or said HCDR1 comprises SEQ ID NO: 1, said HCDR2 comprises SEQ ID NO: 2, said HCDR3 comprises SEQ ID NO: 30, said LCDR1 comprises SEQ ID NO: 31, said LCDR2 comprises SEQ ID NO: 5, said LCDR3 comprises SEQ ID NO: 32, and [Chemical Formula 2] wherein n is from 1 to 5, the conjugate.

2. 【Fig. 3】 The conjugate according to claim 1, wherein

3. 【Fig. 4】 The conjugate according to claim 1, wherein

4. 【Fig. 5】 The conjugate according to claim 1, wherein

5. 【Fig. 6】 The conjugate according to claim 1, wherein

6. 【Fig. 7】 The conjugate according to claim 1, wherein

7. 【Fig. 8】 The conjugate according to claim 1, which is as described above.

8. 【Fig. 9】 The conjugate according to claim 1, which is as described above.

9. 【Fig. 10】 The conjugate according to claim 1, which is as described above.

10. The antibody that binds to human TNFα comprises a heavy chain variable region (VH) and a light chain variable region (VL), 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. 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. The conjugate according to any one of claims 1 to 9, wherein the LCDR3 comprises SEQ ID NO:

6.

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

8.

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

10.

13. The antibody that binds to human TNFα comprises a heavy chain variable region (VH) and a light chain variable region (VL), 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. 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. The conjugate according to any one of claims 1 to 9, wherein the LCDR3 comprises SEQ ID NO:

15.

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

17.

15. The conjugate according to claim 13, wherein the antibody comprises a heavy chain (HC) and a light chain (LC), the HC comprises SEQ ID NO: 18, and the LC comprises SEQ ID NO:

19.

16. The antibody that binds to human TNFα comprises a heavy chain variable region (VH) and a light chain variable region (VL), 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. The HCDR1 comprises SEQ ID NO:

22. The HCDR2 includes SEQ ID NO: 23, The HCDR3 includes SEQ ID NO: 13, The LCDR1 includes SEQ ID NO: 4, The LCDR2 includes SEQ ID NO: 5, The conjugate according to any one of claims 1 to 9, wherein the LCDR3 includes SEQ ID NO:

6.

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

15.

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

6.

19. The conjugate according to any one of claims 1 to 9, wherein the VH includes SEQ ID NO: 24 and the VL includes SEQ ID NO: 8, 17, or 27.

20. The conjugate according to any one of claims 1 to 9, wherein the antibody includes a heavy chain (HC) and a light chain (LC), the HC includes SEQ ID NO: 25, and the LC includes SEQ ID NO: 10, 19, or 28.

21. The antibody that binds to human TNFα includes a heavy chain variable region (VH) and a light chain variable region (VL). The VH includes heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL includes light chain complementarity determining regions LCDR1, LCDR2, and LCDR3. The HCDR1 includes SEQ ID NO: 1, The HCDR2 includes SEQ ID NO: 2, The HCDR3 includes SEQ ID NO: 30, The LCDR1 includes SEQ ID NO: 31, The LCDR2 includes SEQ ID NO: 5, The LCDR3 includes SEQ ID NO: 32, the conjugate according to any one of claims 1 to 9.

22. The VH includes SEQ ID NO: 33, and the VL includes SEQ ID NO: 34, the conjugate according to claim 21.

23. The antibody includes a heavy chain (HC) and a light chain (LC), the HC includes SEQ ID NO: 35, and the LC includes SEQ ID NO: 36, the conjugate according to claim 21.

24. The antibody includes a heavy chain and a light chain, and the heavy chain 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), the conjugate according to any one of claims 1 to 9.

25. The antibody includes a heavy chain (HC) and a light chain (LC), and the HC is of human IgG1 isotype, the conjugate according to any one of claims 1 to 9.

26. n is 2 to 5, the conjugate according to any one of claims 1 to 9.

27. n is 3 to 5, the conjugate according to any one of claims 1 to 9.

28. n is 3 to 4, the conjugate according to any one of claims 1 to 9.

29. n is 4, the conjugate according to any one of claims 1 to 9.

30. n is 3, the conjugate according to any one of claims 1 to 9.

31. n is 2, the conjugate according to any one of claims 1 to 9.

32. A pharmaceutical composition comprising the conjugate according to any one of claims 1 to 9 or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable carriers, diluents, or excipients.

33. A pharmaceutical composition for treating an autoimmune disease or an inflammatory disease in a subject in need of treatment, comprising the conjugate according to any one of claims 1 to 9.

34. The pharmaceutical composition according to claim 33, 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), psoriasis vulgaris (PS), hidradenitis suppurativa (HS), uveitis, non-infectious intermediate, posterior, panuveitis, Behçet's disease, or polymyalgia rheumatica (PMR).

35. The pharmaceutical composition according to claim 33, wherein the subject has received prior treatment with another anti-TNFα therapeutic agent and the subject has generated anti-drug antibodies against the other anti-TNFα therapeutic agent.

36. The pharmaceutical composition according to claim 35, wherein the other anti-TNFα therapeutic agent is selected from adalimumab, infliximab, golimumab, certolizumab, and etanercept, or conjugates thereof.

37. The pharmaceutical composition according to claim 36, wherein the conjugate has low cross-reactivity or no cross-reactivity with anti-drug antibodies against adalimumab.

38. A pharmaceutical composition comprising the conjugate according to any one of claims 1 to 9 for use in therapy, wherein the conjugate is used to be administered to a subject in need of therapy.

39. A pharmaceutical composition comprising the conjugate according to any one of claims 1 to 9 for use in the treatment of an autoimmune disease or an inflammatory disease, wherein the conjugate is used to be administered to a subject in need of treatment.

40. The pharmaceutical composition according to claim 39, 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, psoriasis vulgaris (PS), hidradenitis suppurativa, uveitis, non-infectious intermediate, posterior, panuveitis, Behçet's disease, or polymyalgia rheumatica (PMR).

41. Use of the conjugate according to any one of claims 1 to 9 in the manufacture of a medicament for the treatment of an autoimmune disease or an inflammatory disease.

42. The 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, psoriasis vulgaris (PS), hidradenitis suppurativa, uveitis, non-infectious intermediate, posterior, panuveitis, Behçet's disease, or polymyalgia rheumatica (PMR).

43. The conjugate according to any one of claims 1 to 9, wherein the antibody neutralizes human TNFα.

44. The conjugate according to any one of claims 1 to 9, wherein the antibody is an internalizing antibody.

45. A method for generating a conjugate, the method comprising contacting a compound represented by the formula: 【Chemical 11】 with an anti-human TNFα antibody, wherein the conjugate is the conjugate according to any one of claims 1 to 9.

46. The conjugate is (a) a step of reducing an anti-human TNFα antibody with a reducing agent, wherein the anti-human TNFα antibody contains one or more engineered cysteine residues, (b) a step of oxidizing the anti-human TNFα antibody with an oxidizing reagent, and (c) a step of contacting a compound represented by the formula: 【Chemical Formula 12】 with the anti-human TNFα antibody to generate the conjugate, and the method according to claim 45, which is generated according to the steps.

47. The method according to claim 46, wherein the reducing agent is dithiothreitol and the oxidizing reagent is dehydroascorbic acid.

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