Anti-human VISTA antibody and its use

Abstract

The present invention provides an anti-VISTA antibody-drug conjugate that can be used to target and deliver anti-inflammatory agents such as steroids to immune cells such as bone marrow cells. [Solution] The present invention provides an antibody-drug conjugate (ADC) comprising an antibody or antigen-binding fragment ("A") having a specific amino acid sequence and including an antigen-binding region that specifically binds to human VISTA, a cleavable or non-cleavable linker ("L"), and at least one small molecule anti-inflammatory agent ("AI"), optionally "Q", i.e., a "hetero-bifunctional group" or "hetero-trifunctional group", which is a chemical moiety optionally used to link the linker to the anti-VISTA antibody or antibody fragment and at least one small molecule anti-inflammatory agent ("AI").

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims priority to U.S. Provisional Application No. 62 / 013,878 filed on 22 April 2020, U.S. Provisional Application No. 63 / 013,887 filed on 22 April 2021, U.S. Provisional Application No. 63 / 134,811 filed on 7 January 2021, and U.S. Provisional Application No. 63 / 138,958 filed on 19 January 2021, each of which is incorporated herein by reference. (Field of Invention) The present invention relates to an antibody-drug conjugate (ADC) comprising an anti-human VISTA (V-region immunoglobulin-containing suppressor (1) for T cell activation) antibody or an anti-VISTA antigen-binding antibody fragment having a short serum half-life (approximately 24-27 hours or less in human VISTA knock-in rodents), and an anti-inflammatory agent, such as a steroid like dexamethasone or budesonide, or other steroids known in the art, or one of the novel steroid compounds disclosed herein. The present invention also relates to the use of such ADCs and novel steroids for the treatment of autoimmune and inflammatory conditions. The present invention further relates to a method for using such ADCs to selectively deliver anti-inflammatory agents, such as steroids, particularly small molecule anti-inflammatory agents such as glucocorticoid receptor agonists like dexamethasone, dexamethasone budesonide, or other steroids known in the art, or one of the novel steroid compounds disclosed herein, to target immune cells such as monocytes, neutrophils, T cells, Tregs, and particularly bone marrow cells, thereby reducing potential toxicity to non-target cells, thereby mitigating adverse side effects and / or enhancing the potency of these anti-inflammatory agents. [Background technology]

[0002] VISTA is an NCR ligand, and its most closely related phylogenetically is PD-L1. VISTA shares homology with PD-L1 but exhibits a unique expression pattern limited to the hematopoietic compartment. Specifically, VISTA is expressed as CD11b highIt is constitutively and highly expressed in bone marrow cells, and CD4 + and CD8 + It is expressed at lower levels in T cells. Similar to PD-L1, VISTA is a ligand that significantly suppresses the immune system, and like PD-L1, blocking VISTA makes it possible to develop therapeutic immunity against cancer in preclinical oncological models. Blocking VISTA suppresses the immune system, particularly CD8 + and CD4 + While mediated T-cell immunity is enhanced, treatment with a soluble Ig fusion protein of the extracellular domain of VISTA (VISTA-Ig) has been shown to suppress immunity and halt the progression of several mouse models of autoimmune diseases. Based on the above, the use of antagonist anti-VISTA antibodies to promote T-cell immunity and treat conditions such as cancer and infections in which this is effective has been reported. Conversely, the use of agonist anti-VISTA antibodies to inhibit T-cell immunity and treat conditions such as autoimmune, allergic, and inflammatory conditions in which this is therapeutically effective has been reported. Unfortunately, some anti-VISTA antibodies, including those used in human clinical trials, have very short serum half-lives, which is generally undesirable for the treatment of chronic conditions such as cancer or autoimmune diseases because it requires very frequent administration, which is inconvenient and costly for patients. Furthermore, the potential use of anti-VISTA antibodies and VISTA fusion proteins for delivering payloads, such as chemotherapeutic agents, to cancer cells or tumor sites has been suggested.

[0003] Synthetic glucocorticoid receptor agonists (e.g., dexamethasone, prednisolone, budesonide, beclomethasone, betamethasone, cortisol, cortisone acetate, 16-α-hydroxyprednisolone, dexamethasone, difluorazone, flumetazone, flunisolide, fluocinolone acetonide, fluticasone propionate, ciclesonide, methylprednisolone, prednisone, mometasone, triamcinolone acetonide, etc.) are a potent class of small molecules used to treat inflammation and related disorders. While these compounds are highly effective in suppressing inflammation associated with various conditions such as autoimmune diseases, inflammatory diseases, cancer, and infections, their usefulness in the chronic treatment of these diseases is limited due to severe side effects.

[0004] Based on the above, several approaches have been explored to avoid undesirable toxicity while maintaining the anti-inflammatory effects of synthetic glucocorticoids (Rosen, J and Miner, JN Endocrine Reviews 26:452-64 (2005)). In particular, antibody-drug conjugates (ADCs) have been developed in which such compounds are bound to antibodies that target antigens expressed by immune cells, such as CD40, CD163, CD74, PRLR, and TNF. Nevertheless, in the field of autoimmune and inflammatory diseases, there is still a need for the development of improved anti-inflammatory therapies and improved anti-inflammatory drugs that are more potent, have longer-lasting effects, and / or have reduced side effects compared to existing therapies for the treatment of such conditions. [Overview of the project]

[0005] The present invention aims to provide therapeutic methods for treating or preventing inflammation and related disorders by providing novel steroids and ADCs, particularly novel steroids and ADCs comprising anti-human VISTA antibodies or anti-human VISTA antibody fragments.

[0006] The present invention aims to provide a novel antibody-drug conjugate (ADC) comprising an anti-VISTA antibody or antibody fragment that has a very short serum half-life under physiological conditions (about pH 7.5) as defined herein, which is 1 to 72 hours, 1 to 32 hours, 1 to 16 hours, 1 to 8 hours, 1 to 4 hours or 1 to 2 hours in human VISTA knock-in rodents, or about 3 to 4 days or less in cynomolgus monkeys, wherein the anti-VISTA antibody or antibody fragment is conjugated to an anti-inflammatory drug that must be internalized into cells to exert efficacy, for example, a small-molecule anti-inflammatory drug, for example, a glucocorticoid receptor agonist or another steroid, such as dexamethasone, prednisolone, budesonide, beclomethasone, betamethasone, cortisol, cortisone acetate, 16-α-hydroxy prednisolone, dexamethasone, difluorasone, flumethasone, flunisolide, fluocinolone acetonide, fluticasone propionate, ciclesonide, methylprednisolone, prednisone, prednisolone, mometasone, triamcinolone acetonide or a novel steroid disclosed herein.

[0007] As shown below, the subject ADC has a unique combination of advantages over conventional ADCs, such as those targeting CD74, CD163, TNF, and PRLR, in targeting and directing the internalization of anti-inflammatory agents, particularly steroids, into immune cells; this is due to the combination of the advantages of VISTA as an ADC target and the specific properties of the anti-VISTA antibody contained in the subject ADC (binding to VISTA-expressing immune cells at physiological pH and having a very short pK). In particular, the ADC in question binds to immune cells that express VISTA at very high densities, and despite their very short PK, is effective for a long period (inducing anti-inflammatory activity), and is therefore well-suited for treating chronic inflammatory or autoimmune diseases, and long-term repeated administration is therapeutically justified; because the ADC in question targets a wide range of immune cells, including neutrophils, bone marrow cells, T cells, and endothelial cells, it may also be used to treat inflammatory or autoimmune diseases involving any or all of these types of immune cells; because the ADC in question has a rapid onset of action, it may also be used in acute treatment, and because the ADC in question does not bind to B cells, it is therefore less immunosuppressive than free steroids; the ADC in question acts on Treg cells, which are key immune cells involved in the potency of steroids. Furthermore, the subject ADC acts on both quiescent and activated immune cells, and as a result is activated (inducing anti-inflammatory activity) in both the active and remission phases of inflammatory and autoimmune states; the subject ADC acts on neutrophils (these immune cells are important in acute inflammation); due to the high turnover of VISTA cell surface, the subject ADC internalizes immune cells very rapidly and constitutively; because the subject ADC has a very short half-life (PK) and binds only to immune cells, the subject ADC is less likely to cause target-related toxicity and undesirable peripheral steroid exposure (low nonspecific loss effect); and because anti-VISTA antibodies possessing silent IgG do not exhibit immune function (do not block the biological activity of VISTA), the biological activity (anti-inflammatory effect) of the subject ADC is entirely attributable to the anti-inflammatory payload (steroid).

[0008] A more specific object of the present invention is to provide novel antibody-drug conjugates (ADCs) comprising anti-VISTA antibodies or antibody fragments having a serum half-life of ~72 hours, 1~32 hours, 1~16 hours, 1~8 hours, 1~4 hours, or 1~2 hours ± 0.5 hours in human VISTA knock-in rodents or approximately 3.5, 3, 2.5, or 2.3 days ± 0.5 days in primates (cynomolgus monkeys), and novel ADCs comprising an anti-inflammatory agent, such as a synthetic glucocorticoid receptor agonist such as dexamethasone, prednisolone, or budesonide, or a novel steroid disclosed herein, which, upon administration, result in the release and internalization of the anti-inflammatory agent, such as a synthetic glucocorticoid receptor agonist or derivative such as dexamethasone, prednisolone, or budesonide, to target immune cells.

[0009] A more specific object of the present invention is to provide an antibody-drug conjugate (ADC) comprising an antibody or antigen-binding fragment ("A") containing an antigen-binding region that specifically binds to the human V-domain Ig suppressor (human VISTA) for T cell activation, a cleavable or non-cleavable linker ("L"), and at least one small molecule anti-inflammatory agent ("AI"), optionally "Q", i.e., a "hetero-bifunctional group" or "hetero-trifunctional group" which is a chemical moiety optionally used to link the linker to the anti-VISTA antibody or antibody fragment and at least one small molecule anti-inflammatory agent ("AI"), wherein the ADC is represented by the following formula: "A-(QL-AI)" n " or "(AI-LQ) n -A" In the formula, "n" is at least 1, and when administered to a target requiring it, the antibody or ADC, or a composition containing them, is preferentially delivered to immune cells expressing VISTA, optionally monocytes or myeloid cells, thereby functionally internalizing the low molecular weight anti-inflammatory agent in the immune cells under physiological conditions (approximately pH 7.5). Preferably, the anti-VISTA antibody or antigen-binding fragment, when used in vivo, has a short in vivo serum half-life in serum at physiological pH (approximately pH 7.5), optionally having an in vivo serum half-life of 72 hours or less, 1 to 32 hours, 1 to 16 hours, 1 to 8 hours, 1 to 4 hours, or 1 to 2 hours ± 0.5 hours in human VISTA knock-in rodents in serum at physiological pH (approximately pH 7.5), or approximately 3.5, 3, 2.5, or 2.3 days ± 0.5 days in primates (cynomolgus monkeys) under physiological conditions (approximately pH 7.5).

[0010] A more specific object of the present invention is to provide an antibody-drug conjugate (ADC) that, when administered to a target requiring it, preferentially delivers to one or more immune cells expressing VISTA, optionally monocytes, myeloid cells, T cells, Tregs, NK cells, neutrophils, dendritic cells, macrophages, and endothelial cells, resulting in the functional internalization of a small molecule anti-inflammatory agent to one or more of the aforementioned immune cells; in this case, the anti-human VISTA antibody or antibody fragment has a pK of up to 40 hours in human VISTA knock-in rodents.

[0011] A more specific object of the present invention is to provide the above-mentioned antibody-drug conjugate (ADC) in which AI contains a glucocorticosteroid.

[0012] A more specific object of the present invention is to provide the above antibody-drug conjugate (ADC) comprising one of the following glucocorticosteroids: [ka]

[0013] A more specific object of the present invention is to provide the above-mentioned antibody-drug conjugate (ADC), wherein the glucocorticosteroid comprises 16-α-hydroxyprednisolone, dexamethasone, diflurazone, flumethasone, flunisolide, fluocinolone acetonide, fluticasone propionate, ciclesonide, methylprednisolone, prednisone, prednisolone, mometasone, triamcinolone acetonide, or a derivative thereof.

[0014] A more specific object of the present invention is to provide the above-mentioned antibody-drug conjugate (ADC) having a maximum pK of 3.5 to 4 days at physiological pH in cynomolgus monkeys or humans.

[0015] A more specific object of the present invention is to provide the above-mentioned antibody-drug conjugate (ADC) having a pK of approximately 2.8 days or approximately 2.5 days ± 0.5 days at physiological pH in cynomolgus monkeys or humans.

[0016] A more specific object of the present invention is to provide the above-mentioned antibody-drug conjugate (ADC) having a maximum pK of 6 to 12 hours at physiological pH in human VISTA rodents.

[0017] A more specific object of the present invention is to provide an antibody-drug conjugate (ADC) that contains a linker which cleaves upon internalization of the ADC into VISTA-expressing immune cells, and optionally into one or more of the following: T cells, Tregs, NK cells, neutrophils, monocytes, myeloid cells, dendritic cells, macrophages, and endothelial cells, thereby releasing a therapeutically effective amount of anti-inflammatory agent into the immune cells and inducing anti-inflammatory activity.

[0018] A more specific object of the present invention is to provide an antibody-drug conjugate (ADC) in which the anti-VISTA antibody or antigen-binding fragment has an in vivo serum half-life of about 2 days or less at physiological pH (about pH 7.5) in primates, and optionally in cynomolgus monkeys.

[0019] A more specific object of the present invention is to provide an antibody-drug conjugate (ADC) in which the anti-VISTA antibody or antigen-binding fragment has an in vivo serum half-life of 70 hours, 60 hours, 50 hours, 40 hours, 30 hours, 24 hours, 22-24 hours, 20-22 hours, 18-20 hours, 16-18 hours, 14-16 hours, 12-14 hours, 10-12 hours, 8-10 hours, 6-8 hours, 4-6 hours, 2-4 hours, 1-2 hours, 0.5-1.0 hours, or 0.1-0.5 hours in human VISTA knock-in rodents.

[0020] A more specific object of the present invention is to provide an antibody-drug conjugate (ADC) that, when used in vivo in human VISTA knock-in rodents, or humans or non-human primates, optionally cynomolgus monkeys, has a pK / pD ratio of at least 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1 or higher.

[0021] A more specific object of the present invention is to provide an antibody-drug conjugate (ADC) in which the PD of the ADC is at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, 2-3 weeks, 3-4 weeks, 4-5 weeks, 5-6 weeks, or longer in human VISTA knock-in rodents, or in humans or non-human primates, optionally in cynomolgus monkeys.

[0022] A more specific object of the present invention is to provide the above-mentioned antibody-drug conjugate (ADC) wherein the anti-human VISTA antibody includes an Fc region in which FcR binding is impaired.

[0023] A more specific object of the present invention is to provide the antibody-drug conjugate (ADC) described above, wherein the anti-human VISTA antibody comprises a human IgG1, IgG2, IgG3, or IgG4 Fc region in which FcR binding is impaired.

[0024] A more specific object of the present invention is to provide the above-mentioned antibody-drug conjugate (ADC) wherein the anti-human VISTA antibody contains a human IgG1 Fc region in which FcR binding is impaired.

[0025] A more specific object of the present invention is to provide the above-described antibody-drug conjugate (ADC) comprising a human or non-human primate constant region or Fc region modified to impair or eliminate binding to at least two native human Fcγ receptors.

[0026] A more specific object of the present invention is to provide the above antibody-drug conjugate (ADC) comprising a human or non-human primate constant region or Fc region modified to impair or eliminate binding to any one, two, three, four, or all five of the following FcRs: hFcγRI(CD64), FcγRIIA or hFcγRIIB(CD32 or CD32A), and FcγRIIIA(CD16A) or FcγRIIIB(CD16B).

[0027] A more specific object of the present invention is to provide the above-mentioned antibody-drug conjugate (ADC) comprising a human IgG2κ backbone having the V234A / G237A / P238S / H268A / V309L / A330S / P331S silencing mutation in the Fc region.

[0028] 19. An antibody-drug conjugate (ADC) according to any one of the above claims, comprising a human IgG1 / κ backbone having an L234A / L235A silencing mutation in the Fc region, or optionally a mutation that impairs complement (C1Q) binding.

[0029] A more specific object of the present invention is to provide the above-mentioned antibody-drug conjugate (ADC) comprising a human IgG1 / κ backbone having L234A / L235A silencing mutations and E269R and E233A mutations in the Fc region.

[0030] A more specific object of the present invention is to provide an antibody-drug conjugate (ADC) such that the binding of an anti-VISTA antibody or antigen-binding fragment to VISTA-expressing immune cells does not directly agonize or antagonize the VISTA-mediated effect on immunity.

[0031] A more specific object of the present invention is to provide the above-mentioned antibody-drug conjugate (ADC) comprising a human IgG2 Fc region in which endogenous FcR binding is not impaired.

[0032] A more specific object of the present invention is to provide the above-mentioned antibody-drug conjugate (ADC) containing a natural (unmodified) human IgG2 Fc region.

[0033] A more specific object of the present invention is to provide the above-mentioned antibody-drug conjugate (ADC), wherein the anti-VISTA antibody or antigen-binding fragment has a KD in the range of 0.0001 nM to 10.0 nM, 0.001 to 1.0 nM, and 0.01 to 0.7 or less, as measured by surface plasmon resonance (SPR) at 24°C or 37°C.

[0034] A more specific object of the present invention is to provide the above-mentioned antibody-drug conjugate (ADC), wherein the anti-VISTA antibody or antigen-binding fragment has a KD of 0.13 to 0.64 nM as measured by surface plasmon resonance (SPR) at 24°C or 37°C.

[0035] A more specific object of the present invention is to provide the above-described antibody-drug conjugate (ADC) in which the linker is a cleavable peptide.

[0036] A more specific object of the present invention is to provide the antibody-drug conjugate (ADC) described above, wherein the linker is selected from any of the linkers generally and specifically described herein.

[0037] A more specific object of the present invention is to provide an antibody-drug conjugate (ADC) in which the anti-inflammatory agent comprises a steroid, optionally a glucocorticoid receptor agonist, and optionally dexamethasone, prednisolone, or budesonide, or any of the aforementioned functional derivatives, i.e., the derivative induces anti-inflammatory activity upon internalization into VISTA-expressing immune cells.

[0038] A more specific object of the present invention is to provide the above-mentioned antibody-drug conjugate (ADC) having a drug-antibody ratio in the range of 1:1 to 10:1.

[0039] A more specific object of the present invention is to provide the above-mentioned antibody-drug conjugate (ADC) having a drug-antibody ratio in the range of 2-8:1, 4-8:1, or 6-8:1.

[0040] A more specific object of the present invention is to provide the above-mentioned antibody-drug conjugate (ADC) having a drug-antibody ratio of 8:1 (n=8).

[0041] A more specific object of the present invention is to provide the antibody-drug conjugate (ADC) described above, which internalizes one or more of the following: monocytes, myeloid cells, T cells, Tregs, macrophages, and neutrophils.

[0042] A more specific object of the present invention is to provide the above-mentioned antibody-drug conjugate (ADC) that does not internalize B cells to a degree that allows them to sense it.

[0043] A more specific object of the present invention is to provide the above-mentioned antibody-drug conjugate (ADC) that, when administered to a subject in need, enhances the efficacy associated with anti-inflammatory agents, such as steroids, optionally glucocorticoid receptor agonists, and optionally dexamethasone, prednisolone, or budesonide, and / or reduces adverse side effects, compared to administering the same dose of the anti-inflammatory agent in its naked (unconjugated) form.

[0044] A more specific object of the present invention is to provide an antibody-drug conjugate (ADC) comprising an anti-inflammatory agent, optionally a steroid or glucocorticoid receptor agonist, and optionally dexamethasone, prednisolone, or budesonide, or any of the aforementioned functional derivatives, conjugated to an antibody or antigen-binding fragment via interchain disulfides.

[0045] A more specific object of the present invention is to provide the antibody-drug conjugate (ADC) described above, comprising an esterase-sensitive linker and dexamethasone or budenoside or another corticosteroid or functional derivative as an anti-inflammatory agent.

[0046] A more specific object of the present invention is to provide the antibody-drug conjugate (ADC) described above, comprising a cleavage linker that is sensitive to one or more of the following: acid-induced cleavage, photo-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage.

[0047] A more specific object of the present invention is to provide the above-described antibody-drug conjugate (ADC), wherein the linker is an esterase-cleaving linker.

[0048] A more specific object of the present invention is to provide the antibody-drug conjugate (ADC) described above, comprising a non-cleaving linker that is substantially resistant to one or more of the following: acid-induced cleavage, photo-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage.

[0049] A more specific object of the present invention is to provide an antibody-drug conjugate (ADC) wherein the anti-VISTA antigen-binding fragment contained in the ADC comprises a Fab, F(ab')2, or scFv antibody fragment.

[0050] A more specific object of the present invention is to provide the above-mentioned antibody-drug conjugate (ADC), in which the anti-VISTA antibody or antibody fragment contained therein is: (i) V of sequence numbers 100, 101 and 102H the CDRs of SEQ ID NOs: 103, 104 and 105 and the V L CDR; comprises (ii) the V of SEQ ID NOs: 110, 111 and 112 H the CDRs of SEQ ID NOs: 113, 114 and 115 and the V L CDR; comprises (iii) the V of SEQ ID NOs: 120, 121 and 122 H the CDRs of SEQ ID NOs: 123, 124 and 125 and the V L CDR; comprises (iv) the V of SEQ ID NOs: 130, 131 and 132 H the CDRs of SEQ ID NOs: 133, 134 and 135 and the V L CDR; comprises (v) the V of SEQ ID NOs: 140, 141 and 142 H the CDRs of SEQ ID NOs: 143, 144 and 145 and the V L CDR; comprises (vi) the V of SEQ ID NOs: 150, 151 and 152 H the CDRs of SEQ ID NOs: 153, 154 and 155 and the V L CDR; comprises (vii) the V of SEQ ID NOs: 160, 161 and 162 H the CDRs of SEQ ID NOs: 163, 164 and 165 and the V L CDR; comprises (viii) the V of SEQ ID NOs: 170, 171 and 172 H the CDRs of SEQ ID NOs: 173, 174 and 175 and the V L CDR; comprises (ix) the V of SEQ ID NOs: 180, 181 and 182 H the CDRs of SEQ ID NOs: 183, 184 and 185 and the V L CDR; comprises (x) the V of SEQ ID NOs: 190, 191 and 192 H the CDRs of SEQ ID NOs: 193, 194 and 195 and the V L CDR; comprises (xi) the V of SEQ ID NOs: 200, 201 and 202 H the CDRs of SEQ ID NOs: 203, 204 and 205 and the V L CDR; comprises (xii) V of sequence numbers 210, 211 and 212 H CDR and sequence numbers 213, 214 and 215 L Includes CD-R; (xiii) V of sequence numbers 220, 221 and 222 H CDR and sequence numbers 223, 224 and 225 L Includes CD-R; (xiv) V of sequence numbers 230, 231 and 232 H CDR and sequence numbers 233, 234 and 235 L Includes CD-R; (xv) V of sequence numbers 240, 241 and 242 H CDR and V of Sequence IDs 243, 244 and 245 L Includes CD-R; (xvi) V of sequence numbers 250, 251 and 252 H CDR and Sequence IDs 253, 254 and 255 L Includes CD-R; (xvii) VH CDRs of sequence numbers 260, 261 and 262 and VH CDRs of sequence numbers 263, 264 and 265 L Includes CD-R; (xviii) V of sequence numbers 270, 271 and 272 H CDR and V of Sequence IDs 273, 274 and 275 L Includes CD-R; (xix) V of sequence numbers 280, 281 and 282 H CDR and V of Sequence IDs 283, 284 and 285 L Includes CD-R; (xx) V of sequence numbers 290, 291 and 292 H CDR and V of Sequence IDs 293, 294 and 295 L Includes CD-R; (xxi) V of sequence numbers 300, 301 and 302 H CDR and V of Sequence IDs 303, 304 and 305 L Includes CD-R; (xxii) V of sequence numbers 310, 311 and 312 H CDR and V of Sequence IDs 313, 314 and 315L Includes CD-R; (xxiii) V of sequence numbers 320, 321 and 322 H CDR and sequence numbers 323, 324 and 325 L Includes CD-R; (xxiv) V of sequence numbers 330, 331 and 332 H CDR and V of Sequence IDs 333, 334 and 335 L Includes CD-R; (xxv) V of sequence numbers 340, 341 and 342 H CDR and V of Sequence IDs 343, 344 and 345 L Includes CD-R; (xxvi) V of sequence numbers 350, 351 and 352 H CDR and V of Sequence IDs 353, 354 and 355 L Includes CD-R; (xxvii) V of sequence numbers 360, 361 and 362 H CDR and V of Sequence IDs 363, 364 and 365 L Includes CD-R; (xxviii) V of sequence numbers 370, 371 and 372 H CDR and V of Sequence IDs 373, 374 and 375 L Includes CD-R; (xxix) V of sequence numbers 380, 381 and 382 H CDR and V of Sequence IDs 383, 384 and 385 L Includes CD-R; (xxx) V of sequence numbers 390, 391 and 392 H CDR and V of Sequence IDs 393, 394 and 395 L Includes CD-R; (xxxi) V of sequence numbers 400, 401 and 402 H CDR and V of Sequence IDs 403, 404 and 405 L Includes CD-R; (xxxii) V of sequence numbers 410, 411 and 412 H CDR and V of Sequence IDs 413, 414 and 415 L Includes CD-R; (xxxiii) V of sequence numbers 420, 421 and 422 H CDR and V of Sequence IDs 423, 424 and 425 L Includes CD-R; (xxxiv) V of sequence numbers 430, 431 and 432 H CDR and V of Sequence IDs 433, 434 and 435 L Includes CD-R; (xxxv) V of sequence numbers 440, 441 and 442 H CDR and V of Sequence IDs 443, 444 and 445 L Includes CD-R; (xxxvi) V of sequence numbers 450, 451 and 452 H CDR and V of Sequence IDs 453, 454 and 455 L Includes CD-R; (xxxvii) V of sequence numbers 460, 461 and 462 H CDR and V of Sequence IDs 463, 464 and 465 L Includes CD-R; (xxxviii) V of sequence numbers 470, 471 and 472 H CDR and V of Sequence IDs 473, 474 and 475 L Includes CD-R; (xxxix) V of sequence numbers 480, 481 and 482 H CDR and V of Sequence IDs 483, 484 and 485 L Includes CD-R; (xl) V of sequence numbers 490, 491 and 492 H It contains CDRs and VL CDR polypeptides with sequence numbers 493, 494, and 495; (xli) V of sequence numbers 500, 501 and 502 H The CDR contains the VL CDR polypeptides of sequence numbers 503, 504, and 505; (xlii) V of sequence numbers 510, 511 and 512 H It contains CDRs and VL CDR polypeptides of sequence numbers 513, 514, and 515; (xliii) V of sequence numbers 520, 521 and 522 Hcomprises CDRs and the VL CDR polypeptides of SEQ ID NOs: 523, 524 and 525; (xliv) V of SEQ ID NOs: 530, 531 and 532 H comprises CDRs and the VL CDR polypeptides of SEQ ID NOs: 533, 534 and 535; (xlv) V of SEQ ID NOs: 540, 541 and 542 H comprises CDRs and the VL CDR polypeptides of SEQ ID NOs: 543, 544 and 545; (xlvi) V of SEQ ID NOs: 550, 551 and 552 H comprises CDRs and the VL CDR polypeptides of SEQ ID NOs: 553, 554 and 555; (xlvii) V of SEQ ID NOs: 560, 561 and 562 H comprises CDRs and V of SEQ ID NOs: 563, 564 and 565 L comprises CDRs; (xlviii) V of SEQ ID NOs: 570, 571 and 572 H comprises CDRs and V of SEQ ID NOs: 573, 574 and 575 L comprises CDRs; (xlix) V of SEQ ID NOs: 580, 581 and 582 H comprises CDRs and V of SEQ ID NOs: 583, 584 and 585 L comprises CDRs; (l) V of SEQ ID NOs: 590, 591 and 592 H comprises CDRs and V of SEQ ID NOs: 593, 594 and 595 L comprises CDRs; (li) V of SEQ ID NOs: 600, 601 and 602 H comprises CDRs and V of SEQ ID NOs: 603, 604 and 605 L comprises CDRs; (lii) V of SEQ ID NOs: 610, 611 and 612 H comprises CDRs and V of SEQ ID NOs: 613, 614 and 615 L comprises CDRs; (liii) V of SEQ ID NOs: 620, 621 and 622 H comprises CDRs and V of SEQ ID NOs: 623, 624 and 625 L comprises CDRs; (liv) V of sequence numbers 630, 631 and 632 H CDR and V of Sequence IDs 633, 634 and 635 L Includes CD-R; (lv) V of sequence numbers 640, 641 and 642 H CDR and V of Sequence IDs 643, 644 and 645 L Includes CD-R; (lvi) V of sequence numbers 650, 651 and 652 H CDR and V of Sequence IDs 653, 654 and 655 L Includes CD-R; (lvii) V of sequence numbers 660, 661 and 662 H CDR and V of Sequence IDs 663, 664 and 665 L Includes CD-R; (lviii) V of sequence numbers 670, 671 and 672 H CDR and V of Sequence IDs 673, 674 and 675 L Includes CD-R; (lix) V of sequence numbers 680, 681 and 682 H CDR and V of Sequence IDs 683, 684 and 685 L Includes CD-R; (lx) V of sequence numbers 690, 691 and 692 H CDR and V of Sequence IDs 693, 694 and 695 L Includes CD-R; (lxi) V of sequence numbers 700, 701 and 702 H CDR and V of Sequence IDs 703, 704 and 705 L Includes CD-R; (lxii) V of sequence numbers 710, 711 and 712 H CDR and V of Sequence IDs 713, 714 and 715 L Includes CD-R; (lxiii) V of sequence numbers 720, 721 and 722 H CDR and V of Sequence IDs 723, 724 and 725 L Includes CD-R; (lxiv) V of sequence numbers 730, 731 and 732 HCDR and V of Sequence IDs 733, 734 and 735 L Includes CD-R; (lxv) V of sequence numbers 740, 741 and 742 H CDR and V of Sequence IDs 743, 744 and 745 L Includes CD-R; (lxvi) V of sequence numbers 750, 751 and 752 H CDR and V of Sequence IDs 753, 754 and 755 L Includes CD-R; (lxvii) V of sequence numbers 760, 761 and 762 H CDR and V of Sequence IDs 763, 764 and 765 L Includes CD-R; (lxviii) V of sequence numbers 770, 771 and 772 H CDR and V of Sequence IDs 773, 774 and 775 L Includes CD-R; (lxix) V of sequence numbers 780, 781 and 782 H CDR and V of Sequence IDs 783, 784 and 785 L Includes CD-R; (lxx) V of sequence numbers 790, 791 and 792 H CDR and V of Sequence IDs 793, 794 and 795 L Includes CD-R; (lxxi) V of sequence numbers 800, 801 and 802 H CDR and V of Sequence IDs 803, 804 and 805 L Includes CD-R; (lxxii) V of sequence numbers 810, 811 and 812 H CDR and sequence numbers 813, 814 and 815 L This is a VISTA antibody or antibody fragment containing CDR.

[0051] A more specific object of the present invention is to provide the above-mentioned ADC, wherein the anti-VISTA antibody or antibody fragment contained herein contains the same CDR as one of VSTB92, VSTB56, VSTB95, VSTB103, and VSTB66. A more specific object of the present invention is to provide an antibody-drug conjugate (ADC) wherein the anti-VISTA antibody or antibody fragment contained herein is an anti-VISTA antibody or antibody fragment containing VH polypeptides and VL polypeptides having at least 90%, 95%, or 100% sequence identity with respect to the VH polypeptide and VL polypeptide of an antibody containing the following VH polypeptide and VL polypeptide, and furthermore, the CDR is unmodified: (i) Identity V of Sequence ID No. 106 H Polypeptide and V of SEQ ID NO: 108 L Antibodies containing polypeptides; (ii) V of Sequence ID 116 H Polypeptide and V of SEQ ID NO: 118 L Antibodies containing polypeptides; (iii) V of sequence number 126 H Polypeptide and V of SEQ ID NO: 128 L Antibodies containing polypeptides; (iv) V of sequence number 136 H Polypeptide and V of SEQ ID NO: 138 L Antibodies containing polypeptides; (v) V of sequence number 146 H Polypeptide and V of SEQ ID NO: 148 L Antibodies containing polypeptides; (vi) V of sequence number 156 H Polypeptides and the V of SEQ ID NO: 158 L Antibodies containing polypeptides; (vii) V of sequence number 166 H Polypeptide and V of SEQ ID NO: 168 L Antibodies containing polypeptides; (viii) V of sequence number 176 H Polypeptide and V of SEQ ID NO: 178 L Antibodies containing polypeptides; (ix) V of sequence number 186 H Polypeptide and V of SEQ ID NO: 188 L Antibodies containing polypeptides; (x) V of sequence number 196 H Polypeptides and the V of SEQ ID NO: 198L Antibodies containing polypeptides; (xi) V of sequence number 206 H Polypeptide and V of SEQ ID NO: 208 L Antibodies containing polypeptides; (xii) V of sequence number 216 H Polypeptide and V of SEQ ID NO: 218 L Antibodies containing polypeptides; (xiii) V of sequence number 226 H Polypeptide and V of Sequence ID No. 228 L Antibodies containing polypeptides; (xiv) V of sequence number 236 H Polypeptide and V of SEQ ID NO: 238 L Antibodies containing polypeptides; (xv) V of sequence number 246 H Polypeptide and V of SEQ ID NO: 248 L Antibodies containing polypeptides; (xvi) V of sequence number 256 H Polypeptide and V of SEQ ID NO: 258 L Antibodies containing polypeptides; (xvii) V of sequence number 266 H Polypeptide and V of SEQ ID NO: 268 L Antibodies containing polypeptides; (xviii) V of sequence number 276 H Antibodies containing polypeptides and the VL polypeptide of SEQ ID NO: 278; (xix) V of sequence number 286 H Polypeptide and V of SEQ ID NO: 288 L Antibodies containing polypeptides; (xx) V of sequence number 296 H Polypeptide and V of SEQ ID NO: 298 L Antibodies containing polypeptides; (xxi) V of sequence number 306 H Polypeptide and V of SEQ ID NO: 308 L Antibodies containing polypeptides; (xxii) V of sequence number 316 H Polypeptide and V of SEQ ID NO: 318 L Antibodies containing polypeptides; (xxiii) V of sequence number 326 H Polypeptide and V of SEQ ID NO: 328 L Antibodies containing polypeptides; (xxiv) V of sequence number 336 H Polypeptide and V of SEQ ID NO: 338 L Antibodies containing polypeptides; (xxv) V of sequence number 346 H Polypeptide and V of SEQ ID NO: 348 L Antibodies containing polypeptides; (xxvi) V of sequence number 356 H Polypeptide and V of SEQ ID NO: 358 L Antibodies containing polypeptides; (xxvii) V of sequence number 366 H Polypeptide and V of SEQ ID NO: 368 L Antibodies containing polypeptides; (xxviii) V of sequence number 376 H Polypeptide and V of SEQ ID NO: 378 L Antibodies containing polypeptides; (xxix) V of sequence number 386 H Polypeptide and V of SEQ ID NO: 388 L Antibodies containing polypeptides; (xxx) V of sequence number 396 H Polypeptide and V of SEQ ID NO: 398 L Antibodies containing polypeptides; (xxxi) V of sequence number 406 H Polypeptide and V of SEQ ID NO: 408 L Antibodies containing polypeptides; (xxxii) V of sequence number 416 H Polypeptide and V of SEQ ID NO: 418 L Antibodies containing polypeptides; (xxxiii) V of sequence number 426 H Polypeptide and V of SEQ ID NO: 428 L Antibodies containing polypeptides; (xxxiv) V of sequence number 436 H Polypeptide and V of SEQ ID NO: 438 L Antibodies containing polypeptides; (xxxv) V of sequence number 446 H Polypeptide and V of SEQ ID NO: 448 L Antibodies containing polypeptides; (xxxvi) V of sequence number 456 H Polypeptide and V of SEQ ID NO: 458 L Antibodies containing polypeptides; (xxxvii) V of sequence number 466 H Polypeptide and V of SEQ ID NO: 468 L Antibodies containing polypeptides; (xxxviii) V of sequence number 476 H Polypeptide and V of SEQ ID NO: 478 L Antibodies containing polypeptides; (xxxix) V of sequence number 486 H Polypeptide and V of SEQ ID NO: 488 L Antibodies containing polypeptides; (xl) V of sequence number 496 H Polypeptide and V of SEQ ID NO: 498 L Antibodies containing polypeptides; (xli) V of sequence number 506 H Polypeptides and the V of SEQ ID NO: 508 L Antibodies containing polypeptides; (xlii) V of sequence number 516 H Polypeptides and the V of SEQ ID NO: 518 L Antibodies containing polypeptides; (xliii) V of sequence number 526 H Polypeptide and V of SEQ ID NO: 528 L Antibodies containing polypeptides; (xliv) V of sequence number 536 H Polypeptides and the V of SEQ ID NOs: 533, 534 and 535 L Antibodies containing polypeptides; (xlv) V of sequence number 546 H Polypeptide and V of SEQ ID NO: 548 L Antibodies containing polypeptides; (xlvi) V of sequence number 556 H Polypeptides and the V of SEQ ID NO: 558 LAntibodies containing polypeptides; (xlvii) V of sequence number 566 H Polypeptide and V of SEQ ID NO: 568 L Antibodies containing polypeptides; (xlviii) V of sequence number 576 H Polypeptides and the V of SEQ ID NO: 578 L Antibodies containing polypeptides; (xlix) V of sequence number 586 H Polypeptides and the V of SEQ ID NO: 588 L Antibodies containing polypeptides; (l) V of sequence number 596 H Polypeptides and the V of SEQ ID NO: 598 L Antibodies containing polypeptides; (li) V of sequence number 606 H Polypeptide and V of SEQ ID NO: 608 L Antibodies containing polypeptides; (lii) V of sequence number 616 H Polypeptide and V of SEQ ID NO: 618 L Antibodies containing polypeptides; (liii) V of sequence number 626 H Polypeptide and V of SEQ ID NO: 628 L Antibodies containing polypeptides; (liv) V of sequence number 636 H Polypeptide and V of SEQ ID NO: 638 L Antibodies containing polypeptides; (lv) V of sequence number 646 H Polypeptide and V of SEQ ID NO: 648 L Antibodies containing polypeptides; (lvi) V of sequence number 656 H Polypeptide and V of SEQ ID NO: 658 L Antibodies containing polypeptides; (lvii) V of sequence number 666 H Polypeptide and V of SEQ ID NO: 668 L Antibodies containing polypeptides; (lviii) V of sequence number 676 H Polypeptide and V of SEQ ID NO: 678 LAntibodies containing polypeptides; (lix) V of sequence number 686 H Polypeptide and V of SEQ ID NO: 688 L Antibodies containing polypeptides; (lx) V of sequence number 696 H Polypeptide and V of SEQ ID NO: 698 L Antibodies containing polypeptides; (lxi) V of sequence number 706 H Polypeptides and the V of SEQ ID NO: 708 L Antibodies containing polypeptides; (lxii) V of sequence number 716 H Polypeptide and V of SEQ ID NO: 718 L Antibodies containing polypeptides; (lxiii) V of sequence number 726 H Polypeptide and V of SEQ ID NO: 728 L Antibodies containing polypeptides; (lxiv) V of sequence number 736 H Polypeptide and V of SEQ ID NO: 738 L Antibodies containing polypeptides; (lxv) V of sequence number 746 H Polypeptide and V of SEQ ID NO: 748 L Antibodies containing polypeptides; (lxvi) V of sequence number 756 H Polypeptides and the V of SEQ ID NO: 758 L Antibodies containing polypeptides; (lxvii) V of sequence number 766 H Polypeptide and V of SEQ ID NO: 768 L Antibodies containing polypeptides; (lxviii) V of sequence number 776 H Polypeptide and V of SEQ ID NO: 778 L Antibodies containing polypeptides; (lxix) V of sequence number 786 H Polypeptide and V of SEQ ID NO: 788 L Antibodies containing polypeptides; (lxx) V of sequence number 796 H Polypeptides and the V of SEQ ID NO: 798 LAntibodies containing polypeptides; (lxxi) V of sequence number 806 H Polypeptide and V of SEQ ID NO: 808 L Antibodies containing polypeptides; and (lxxii) V of sequence number 816 H Polypeptide and V of SEQ ID NO: 818 L Antibodies containing polypeptides.

[0052] A more specific object of the present invention is to provide the above-mentioned antibody-drug conjugate (ADC), wherein the anti-VISTA antibody or antibody fragment contains the same variable region as one of VSTB92, VSTB56, VSTB95, VSTB103, and VSTB66.

[0053] A more specific object of the present invention is to provide the above-mentioned antibody-drug conjugate (ADC), wherein the anti-VISTA antibody or antibody fragment comprises a human IgG2κ backbone having the V234A / G237A / P238S / H268A / V309L / A330S / P331S silencing mutation in the Fc region.

[0054] A more specific object of the present invention is to provide the above-mentioned antibody-drug conjugate (ADC) comprising an anti-VISTA antibody or antibody fragment containing a human IgG1 / κ backbone having an L234A / L235A silencing mutation in the Fc region.

[0055] A more specific object of the present invention is to provide the antibody-drug conjugate (ADC) described above, wherein AI, L, or Q is conjugated to an anti-VISTA antibody or antigen-binding fragment via an interchain disulfide.

[0056] A pharmaceutical composition comprising at least one therapeutically effective amount of any of the aforementioned antibody-drug conjugates (ADCs) and a pharmaceutically acceptable carrier.

[0057] The above composition, which can be administered via an optional intravenous, intramuscular, subarachnoid, or subcutaneous injection route.

[0058] The above composition, which can be administered subcutaneously.

[0059] An apparatus comprising the above composition, which provides subcutaneous administration selected from the group consisting of syringes, injection devices, infusion pumps, injection pens, needleless devices, auto-injectors, and subcutaneous patch delivery systems.

[0060] The above-mentioned device for delivering a fixed dose of an anti-inflammatory agent, such as a steroid, such as a glucocorticoid receptor agonist, optionally dexamethasone, prednisolone, or budesonide, or a functional derivative thereof, to a patient.

[0061] A kit comprising the above-mentioned device, and further comprising instructions for informing the patient of the method of administering the ADC composition contained therein, and the drug regimen.

[0062] A method for treatment and / or prevention comprising administering at least one antibody-drug conjugate (ADC) or one of the compositions described above to a patient in need thereof, wherein the composition may be present in the apparatus described above.

[0063] The above-mentioned therapeutic and / or prophylactic methods, used for the treatment of allergies, autoimmunity, transplantation, gene therapy, inflammation, GVHD, or sepsis, or for the treatment or prevention of inflammatory, autoimmune, or allergic side effects associated with any of the above conditions in human subjects.

[0064] The above-mentioned treatment and / or preventive methods for patients being treated who have a condition selected from rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis, ankylosing spondylitis, adult Crohn's disease, childhood Crohn's disease, ulcerative colitis, psoriasis vulgaris, hidradenitis suppurativa, uveitis, Behçet's disease, spondyloarthropathy, or psoriasis.

[0065] The above treatment and / or preventive measures include one or more of the following for the patient: (i) Conditions that can be effectively treated primarily with high doses of steroids, and, on an optional basis, polymyalgia rheumatica and / or giant cell arteritis (the patient has been or is being treated with high doses of steroids on an optional basis); (ii) Conditions with complications that limit the use of steroids, optionally: diabetes mellitus, non-alcoholic steatohepatitis (NASH), morbid obesity, avascular necrosis / osteonecrosis (AVN), glaucoma; steroid-induced hypertension, severe skin fragility, and / or osteoarthritis; (iii) Conditions for which safe long-term therapeutic agents are available but initiation of several months with high-dose steroids is desirable, optionally including AAV, polymyositis, dermatomyositis, lupus, inflammatory lung disease, autoimmune hepatitis, inflammatory bowel disease, immune thrombocytopenia, autoimmune hemolytic anemia, and gout patients for whom initiation of several months with high-dose steroids is therapeutically justified; (iv) dermatological conditions requiring short-term / long-term treatment for which there is no effective alternative to treatment or duration and / or steroid administration, optionally Stevens-Johnson syndrome, other severe rash conditions, conditions including widespread contact dermatitis, PG, LCV, other severe immune-related dermatological conditions, etc. (v) Conditions treated with high-dose corticosteroids for erythema / recurrence, optionally including COPD, asthma, lupus, gout, and pseudogout; (vi) Immune-related neurological disorders such as small fibrous neuropathy, MS (subset), chronic inflammatory demyelinating polyneuropathy, and myasthenia gravis; (vii) Optional hematological / oncological indications for which high doses of steroids are therapeutically justified or may be effective; (viii) Ophthalmic conditions, optional, such as uveitis, iritis, scleritis, etc. (ix) Conditions associated with persistent or very long-term adrenal insufficiency or secondary adrenal insufficiency, optionally iatrogenic Addison's disease crisis; (x) A condition that is often treated with long-term low-dose steroids, and optionally with lupus, RA, psA, vasculitis, etc.; and (xi) Special classes of patients, e.g., pregnant / lactating women, pediatric patients, and optionally patients with growth disorders or cataracts.

[0066] The above treatment and / or preventive methods, in which the patient is further treated with another active drug.

[0067] The above therapeutic and / or prophylactic method further involves treating the patient with an immunosuppressive antibody or fusion protein selected from one or more immunosuppressive antibodies or fusion proteins targeting CTLA4, PD-1, PDL-1, LAG-3, TIM-3, BTLA, B7-H4, B7-H3, VISTA, and / or an agonist antibody or fusion protein targeting one or more CD40, CD137, OX40, GITR, CD27, CD28, or ICOS.

[0068] A more specific object of the present invention is to provide a novel antibody-drug conjugate (ADC) as described above, wherein the drug-antibody ratio is in the range of 1:1 to 10:1.

[0069] A more specific object of the present invention is to provide a novel antibody-drug conjugate (ADC) according to any of the above, wherein the drug-antibody ratio is in the range of 2-8:1, 4-8:1, or 6-8:1.

[0070] A more specific object of the present invention is to provide a novel antibody-drug conjugate (ADC) as described above, having a drug-antibody ratio of 8:1 (n=8).

[0071] Another object of the present invention is to provide a novel antibody-drug conjugate (ADC) described above that, when administered to a subject in need, enhances the efficacy of an anti-inflammatory agent, such as a steroid, optionally a glucocorticoid receptor agonist, and optionally dexamethasone, prednisolone, or budesonide, and / or reduces adverse side effects compared to an anti-inflammatory agent administered in the same dose in a naked (uncomplexed) form.

[0072] Another object of the present invention is to provide a novel ADC (ADC) according to any of the above, wherein the antibody or antigen-binding fragment in the antibody-drug conjugate (ADC) contains an epitope to which any of the anti-human VISTA antibodies having the sequence of Figure 10 is bound, or competes with or binds to an overlapping VISTA epitope.

[0073] Another specific object of the present invention is to provide a method for contacting immune cells, such as human immune cells, with ADCs according to the present invention in vitro or in vivo, for example, by injecting the contacted cells into a human subject, such as a subject having an autoimmune or inflammatory condition or other pathological condition as specified above, after contact with such ADCs, where AI or steroid administration may be therapeutically desirable but potentially toxic and / or contraindicated due to other safety or clinical concerns. [Brief explanation of the drawing]

[0074] [Figure 1] The peptide mapping of the control VISTA antibody 767-IgG1.3 by trypsin digestion is shown. Determined sequences for 767-IgG1.3 (identified trypsin peptides are underlined) (A) Light chain (coverage 85.6%) (B) Heavy chain (coverage 76.1%).

[0075] [Figure 2] The sequence of 767-IgG1.3 determined using Lys-C digestion is shown. In the figure, the Lys-C peptide is underlined (A) light chain (coverage 63.3%) (B) heavy chain (coverage 76.3%).

[0076] [Figure 3] This includes the results of binding experiments confirming that the synthesized control antibody 767-IgG1.3 and INX200 exhibit opposite pH-dependent binding characteristics.

[0077] [Figure 4]This includes results from binding studies demonstrating that complexation of DAR8 and linker A does not affect the binding of VISTA to (A) INX200, (B) INX201, or (C) 767-IgG1.3.

[0078] [Figure 5] This report includes the results of ConA experiments in which female hVISTA knock-in animals were administered various naked anti-VISTA antibodies and Dex-conjugated anti-VISTA antibodies, in which changes in G-CSF in peripheral blood were detected 6 hours after ConA. Plasma concentrations (SEM; n=5 / group) were measured using mouse 7-plex (dosage: DEX-0.2 = 0.2 mg / kg, DEX-2 = 2 mg / kg, INX210 and INX210A (10 mg / kg), [INX210A 0.2 mg / kg dex payload provided]).

[0079] [Figure 6] This report includes results from ConA studies in which male hVISTA knock-in animals were administered various naked anti-VISTA antibodies and Dex-conjugated anti-VISTA antibodies. In the experiments shown in the figures, cytokine levels in peripheral blood changed 6 hours after ConA administration. Plasma concentrations were measured using a mouse 7-plex (SEM; n=10 / group, standard one-way ANOVA compared to the ConA-only group) (dosage: Dex 0.2 or 5 mg / kg, INX210 and INX210A 10 mg / kg).

[0080] [Figure 7] The results of ConA experiments, in which animals were administered various naked anti-VISTA antibodies and anti-VISTA antibodies conjugated with Dex, and cytokine changes in peripheral blood were detected 6 hours after ConA administration, are shown. Plasma concentrations were measured using an ELISA assay (SD; n=6 / group; one-way ANOVA compared to the ConA-only group) (dosage: Dex 0.02, 0.2 or 2 mg / kg, INX200A 10, 5 and 1 mg / kg).

[0081] [Figure 8]This includes the arrangement and legend of the variable weight region, variable light chain region, and steady region of INX200, INX201, and INX210.

[0082] [Figure 9] For example, exemplary budenoside derivatives that can be conjugated to anti-VISTA antibodies and anti-VISTA antibody fragments via the linker described herein are shown.

[0083] [Figure 10] This includes sequence listings and epitope information for exemplary anti-human VISTA antibodies VSTB49-VSTB116 (which have short serum half-lives in rodents and primates under physiological conditions (pH approximately 7.5)), including CDRs, variable heavy chain sequences, variable light chain sequences, framework sequences, and constant domains.

[0084] [Figure 11] This includes exemplary steroid structures derived from those disclosed in Example 3.

[0085] [Figure 12] The examples include sequences of exemplary anti-VISTA antibodies and control antibodies disclosed in the examples.

[0086] [Figure 13] This includes binding studies of INX200, 767-IgG1.3, and human IgG1si. The median fluorescence intensity measured for monocytes incubated with serial dilutions (0–333 nM) of the tested antibodies is shown; the black dashed line corresponds to the autofluorescence of unstained cells; n=1.

[0087] [Figure 14] This study shows which portion of the anti-VISTA antibody (INX200) is internalized by immune cells. The intracellular pool of cell-bound antibodies was plotted over a 60-minute time course; fluorescence at each data point was normalized relative to the fluorescence of INX200 at time 0; mean ± standard deviation. n=2 donors.

[0088] [Figure 15] This report includes results from experiments evaluating the internalization rate of INX200 antibodies. The internalization rate of INX200 antibodies was assessed in monocytes over a 60-minute time course; anti-CD45 antibodies were not incorporated at any point in time; results are shown as mean ± standard deviation, n=2 donors.

[0089] [Figure 16] Includes PK studies of INX200, INX200A, and human IgG1. Plasma concentrations of antibodies at annotated time points (SD; n=5 / group) in hVISTA KI mice.

[0090] [Figure 17] Includes PK studies of 767-IgG1.3, 767-IgG1.3A, and human IgG1. Plasma concentrations of antibodies at annotated time points (SD; n=5 / group) in hVISTA KI mice.

[0091] [Figure 18] This report includes the results of experiments evaluating the efficacy of the ADC complex according to the present invention. The experiments evaluated FKBP5 transcriptional activation in peritoneal resident macrophages and splenic monocytes after treatment with Dex (left) and ADC INX201J (right). The effect of Dex (left) was evaluated 4 and 24 hours after a single intraperitoneal injection of 2 mg / kg. The effect of ADC (right) was analyzed 24, 48, 72, and 96 hours after a single intraperitoneal injection of 10 mg / kg delivering a 0.2 mg / kg GC payload. FKBP5 transcriptional levels were measured by real-time PCR and shown as a Log2 factor change relative to the PBS control group. Four mice per group were pooled together to generate sufficient material for RNA preparation.

[0092] [Figure 19] This study includes in vivo experimental results demonstrating that Dex treatment prevents ex vivo induction of pro-inflammatory cytokines in PRM. The Dex effect was evaluated 2 hours after a single intraperitoneal injection of 2 mg / kg; IL-6 and TNFα were assessed in the cell supernatant (collected at 1 hour) using mouse 32-plex (n=4 mice / group; independent t-test).

[0093] [Figure 20] This study includes results from experiments evaluating the in vivo effects of INX201J or Dex treatment on TNFα during PRM. The results indicate that INX201J or Dex treatment prevents ex vivo induction of TNFα in PRM. In these experiments, the effect of Dex was evaluated 2 hours after single intraperitoneal injections of 2 and 0.2 mg / kg; the effect of INX201J was evaluated 1 day (d-1), 2 days (d-2), and 4 days (d-4) after injection of 10 mg / kg (equivalent to a 0.2 mg / kg payload). Cell supernatant was collected 2 hours later. TNFα was measured using ELISA (n=4 mice / group; standard one-way ANOVA compared to the PBS-only group).

[0094] [Figure 21] This report includes the results of experiments evaluating the long-term effects of exemplary ADCs according to the present invention. The results indicate that all ADCs tested induced long-term effects on the ex vivo induction of TNFα and IL-6 in PRM. The effect of Dex was evaluated 2 hours after a single intraperitoneal injection of 2 mg / kg; the effects of INX201J, INX231J, INX234J, and INX240J were evaluated 4 days (-4) and 7 days (-7) after a single intraperitoneal injection of 10 mg / kg. Cell supernatant was collected 2 hours later. TNFα and IL-6 were measured using ELISA (n=4 mice / group; standard one-way ANOVA compared to the PBS-only group).

[0095] [Figure 22]This report includes the results of experiments evaluating the efficacy of exemplary ADC conjugates according to the present invention, namely INX231J, INX234J, and INX240J. The results show that the INX231J, INX234J, and INX240J ADCs have comparable efficacy in preventing the ex vivo induction of TNFα and IL-6 in PRM. The effect of Dex was evaluated 2 hours after a single intraperitoneal injection of 2 mg / Kg; the effects of INX231J, INX234J, and INX240J were evaluated 7 days after a single intraperitoneal injection of 10, 3, or 1 mg / Kg (0.2, 0.06, and 0.02 mg / Kg GC payloads). Cellular supernatant was collected 2 hours later. TNFα and IL-6 were measured using ELISA (see Methods section) (n=4 mice / group, except for the PBS group which was n=1 for technical reasons; standard one-way ANOVA compared to the PBS-only group).

[0096] [Figure 23] This study includes experimental results comparing the efficacy of INX201J, INX201P, INX231J, INX234J, and INX240J ADCs, which demonstrate comparable efficacy in preventing ex vivo induction of TNFα and IL-6 in PRM. The effects of INX201J, INX201P, INX231J, INX234J, INX240J, and Dex were evaluated 7 days after a single intraperitoneal injection; ADCs were administered at 10 mg / kg (0.2 mg / kg GC payload), and Dex at 2 mg / kg. Cell supernatant was collected 2 hours later. TNFα and IL-6 were measured using ELISA (n=4 mice / group, except for the PBS and Dex groups which had n=3 for technical reasons; standard one-way ANOVA compared to the PBS-only group).

[0097] [Figure 24]INX201J, INX231P, INX234P, and INX240P ADCs exhibit comparable efficacy in preventing ex vivo induction of TNFα in PRM. In experiments, the effect of ADCs was evaluated 7 days after a single intraperitoneal injection; ADCs were administered at 10 mg / kg (0.2 mg / kg GC payload). Cell supernatant was collected 2 hours later. TNFα and IL-6 were measured using ELISA (see Methods section) (n=4 mice / group; standard one-way ANOVA compared to PBS-only group, SEM).

[0098] [Figure 25] INX231P, INX231R, INX233P, and INX234P exhibit comparable efficacy in preventing ex vivo induction of TNF and IL-6 in PRM. In experiments, the effect of ADC was evaluated 7 days after a single intraperitoneal injection; ADC was administered at 10 mg / kg (0.2 mg / kg GC payload). Cellular supernatant was collected at 24 hours. TNFα and IL-6 were measured using ELISA (see Methods section) (n=4 mice / group; standard one-way ANOVA compared to the PBS-only group, SEM).

[0099] [Figure 26] Evaluation of the efficacy of GC linker payloads of INX R, INX O, INX S, INX V, and INX W against INX P conjugated to INX231 or INX201 in preventing ex vivo induction of TNFα and IL-6 in PRM. In the experiment, the effect of ADC was evaluated 7 days after a single intraperitoneal injection; ADC was administered with a 0.2 mg / kg GC payload. Cell supernatant was collected at 24 hours. TNFα and IL-6 were measured using ELISA (see Methods section) (n=4 mice / group; standard one-way ANOVA compared to the PBS-only group, SEM).

[0100] [Figure 27]IL-12p40 levels change in peripheral blood 2 hours (left) and 4 hours (right) after LPS administration. Plasma concentrations measured using mouse multiplex; dose: Dex (squares) were administered 0.02, 0.2, 2, and 5 mg / kg 2 hours prior to LPS stimulation, and INX201J (circles) were administered 2 or 17 hours prior to a 10 mg / kg LPS injection providing 0.2 mg / kg GC. The PBS-only group (gray triangles) shows baseline cytokine levels without stimulation; PBS + LPS (black triangles) (SEM; n=5 / group, excluding cases of technical failures excluded from analysis; standard one-way ANOVA compared to the PBS + LPS group).

[0101] [Figure 28] Changes in cytokine levels in peripheral blood 2 hours after LPS stimulation. Plasma concentrations measured using mouse 5-plex; dose: Dex was administered at 0.002, 0.02, 0.2, and 2 mg / Kg (square) 2 hours prior to LPS stimulation, or at 2 mg / Kg (black square) 17 hours prior to LPS stimulation. INX201J (circle) was administered at 0.02, 0.06, and 0.2 mg / Kg GC payload 17 hours prior to LPS injection. The PBS-only group (gray triangle) shows baseline cytokine levels without stimulation; PBS + LPS (black triangle) (SEM; n=5 / group, excluding cases of technical failures excluded from analysis; standard one-way ANOVA compared to the PBS + LPS group).

[0102] [Figure 29]Changes in TNFα in peripheral blood 2 hours after LPS. Plasma TNFα concentrations measured using ELISA; Dosage: Dex was administered at 0.2 and 2 mg / kg 2 hours prior to LPS stimulation (square), and INX201J (circle) was administered at 0.06 and 0.2 mg / kg GC payloads 17 hours prior to LPS injection. The PBS group (black triangle) received PBS 2 hours prior to LPS, and the IgG1siJ (G1siJ) group (triangle) received human IgG1 silently conjugated to GC at a 0.2 mg / kg payload 17 hours prior to LPS. (SEM; n=5 / group, excluding cases of technical failure from analysis; standard one-way ANOVA compared to the PBS group).

[0103] [Figure 30] This chart shows the changes in TNFα in peripheral blood 2 hours after LPS administration. Plasma TNFα concentrations were measured by ELISA; Dosage: Dex was administered at 0.2 and 2 mg / kg 2 hours prior to LPS stimulation (squares), while INX201J (circles) and INX201N (inverted triangles) were administered 17 hours prior to LPS injection with a 0.2 mg / kg GC payload. The PBS group received PBS 2 hours prior to LPS (black triangles). (SEM; n=5 / group, excluding cases of technical failures from the analysis; standard one-way ANOVA compared to the PBS group).

[0104] [Figure 31] The graph shows the changes in TNFα (left) and IL-12p40 (right) in peripheral blood 2 hours after LPS administration. Cytokine plasma concentrations were measured by ELISA; dosage: PBS (black circles), INX201J (squares), INX231J (triangles), INX234J (diamonds), and INX201P (inverted triangles) were administered as a 0.2 mg / kg GC payload 17 hours prior to LPS injection (SEM; n=5 / group; standard one-way ANOVA compared to the PBS group).

[0105] [Figure 32]The image shows the changes in TNFα (left) and IL-12p40 (right) in peripheral blood 2 hours after LPS administration. Cytokine plasma concentrations were measured by ELISA; Dosage: PBS (black triangle), INX201J (circle), INX201O (square), and INX201P (diamond) were administered 17 hours prior to LPS injection with a 0.2 mg / kg GC payload (SEM; n=5 / group, excluding cases of technical failure from the analysis; standard one-way ANOVA compared to the PBS group).

[0106] [Figure 33] The changes in TNFα (right) and IL-12p40 (left) in peripheral blood 2 hours after LPS administration are shown. Cytokine plasma concentrations were measured by ELISA; Dosage: PBS, INX201J (circular), INX201O (square), and INX201P (diamond) were administered 17 hours prior to LPS injection with a 0.2 mg / kg GC payload (SEM; n=5 / group, excluding cases of technical failure from the analysis; standard one-way ANOVA compared to the PBS group (black triangle)).

[0107] [Figure 34] The changes in TNFα (right) and IL-12p40 (left) in peripheral blood 2 hours after LPS administration are shown. Cytokine plasma concentrations were measured by ELISA; all ADCs and PBS were administered 20 hours prior to LPS injection with a 0.2 mg / kg GC payload (INX231P (square), INX231R (triangle), INX233P (diamond)) (SEM; n=5 / group, excluding cases of technical failure from the analysis; standard one-way ANOVA compared to the PBS group (black circles)).

[0108] [Figure 35]The graph shows the changes in TNFα (right) and IL-12p40 (left) in peripheral blood 2 hours after LPS administration. Cytokine plasma concentrations were measured by ELISA; all ADCs and PBS were administered 20 hours prior to LPS injection with a 0.2 mg / kg GC payload (INX231P (black square), INX231R (black triangle), INX201O (black diamond), INX231S (circle), INX231V (square), INX231W (triangle)) (SEM; n=4 / group, excluding INX231S which was excluded from the analysis due to two technical failures; the standard one-way ANOVA compared to the PBS group (black circle) showed non-significant data).

[0109] [Figure 36] This shows the FKBP5 transcriptional activation in peritoneal macrophages 4 days after ADC treatment. ADC was administered intraperitoneally on day 0, delivering a 0.2 mg / kg GC payload; PRMs were separated on day 3. FKBP5 transcription levels were measured by real-time PCR and expressed as a log-2 factor change compared to the PBS control group (SEM, standard one-way ANOVA compared to the PBS group, n=4).

[0110] [Figure 37] This includes the results of experiments detecting VISTA expression in various cell types. As shown in the figure, VISTA is highly expressed in hepatic endothelial cells. CD45-CD31+ non-immune endothelial cells isolated from the liver of hVISTA knock-in mice and stained with anti-human VISTA (red line, shifted to the right) or unstained (solid gray line).

[0111] [Figure 38]This report includes results from experiments detecting FKBP5 transcriptional activation in the adrenal glands, brain, liver, and spleen after INX201J injection. As shown in the figure, the effect of INX201J was measured 20 hours after a single intraperitoneal injection of 0.3, 3, and 10 mg / Kg (delivering payloads of 0.006, 0.06, and 0.2 mg / Kg, respectively). The effect of Dex was measured 2 hours after a single intraperitoneal injection of 0.2 or 2 mg / Kg. FKBP5 transcriptional levels were measured by real-time PCR and shown as a log-2 multiplier change relative to the mean of the PBS control group (n=4 mice / group; standard one-way ANOVA compared to the PBS-only group).

[0112] [Figure 39] INX-SM-3, INX-SM-4, and INX-SM-1 inhibit the production of IL-1β (left) and IL-6 (right). Cytokine levels were measured at 24 hours in human PBMCs incubated with 1 ng / mL LPS and serial dilutions (1000–1 nM) of steroid payloads. Untreated controls were plotted on a logarithmic scale with the x-axis <1 nM; n=1 donor, standard deviation plotted based on technical two-point measurements.

[0113] [Figure 40] INX-SM-1, INX-SM-3, INX-SM-4, and INX-SM-6 inhibit IL-1β production. Cytokine levels were measured at 24 hours in human PBMCs incubated with 1 ng / mL LPS and serial dilutions (1000–1 nM) of steroid payloads. Untreated controls were plotted on a logarithmic scale with the x-axis <1 nM; n=1 donor, standard deviation plotted based on technical two-point measurements.

[0114] [Figure 41]INX-SM-9, INX-SM-31, and INX-SM-35 inhibit the production of IL-1β (top) and IL-6 (bottom). Cytokine levels were measured at 24 hours in human PBMCs incubated with 1 ng / mL LPS and serial dilutions (1000–0.2 nM) of steroid payloads. Untreated controls were plotted on a logarithmic scale with the x-axis <0.2 nM; n=2 donors, representative donors shown. Standard deviations were plotted based on technical two-point measurements.

[0115] [Figure 42] INX-SM-32 inhibits the production of IL-1β (top) and IL-6 (bottom). Cytokine levels were measured at 24 hours in human PBMCs incubated with 1 ng / mL LPS and serial dilutions (500–1 nM) of steroid payloads, and untreated controls were plotted on a logarithmic scale with the x-axis <1 nM; n=2. Representative donors are shown. Standard deviations were plotted based on technical two-point measurements.

[0116] [Figure 43] INX-SM-10 exhibits robust inhibition of IL-1β (top) and IL-6 (bottom) production. INX-SM-33 exhibits moderate inhibition of cytokine production. Cytokine levels were measured at 24 hours in human PBMCs incubated with 1 ng / mL LPS and serial dilutions (1000–0.5 nM) of steroid payloads, and untreated controls were plotted on a logarithmic scale with the x-axis <0.5 nM; n=1 donor, standard deviation plotted based on technical two-point measurements.

[0117] [Figure 44] INX-SM-2 and INX-SM-7 exhibit inhibition of IL-1β. Mean cytokine levels were measured at 24 hours in human PBMCs incubated with 1 ng / mL LPS and serial dilutions (1000–0.16 nM) of steroid payloads, and the untreated control was plotted on a logarithmic scale with the x-axis <0.16 nM; n=1, standard deviation plotted based on technical two-point measurements.

[0118] [Figure 45] This study demonstrates that efficacy is enhanced by halogenation at both C6 and C9, but not by C9 alone. Mean cytokine levels were measured at 24 hours in human PBMCs incubated with 1 ng / mL LPS and serial dilutions (1000–0.16 nM) of steroid payloads, and the untreated control was plotted on a logarithmic scale with the x-axis <0.16 nM; n=1, standard deviation plotted based on technical two-point measurements.

[0119] [Figure 46] This includes experimental results comparing the PK properties of the exemplary antibody INX200 of the present invention with those of human IgG1. As shown in the figure, the plasma concentrations of the antibody at annotated time points in hVISTA KI mice are (SD; n=5 / group).

[0120] [Figure 47] This includes the results of experiments comparing the PK properties of 767-IgG1.3 and human IgG1. As shown in the figure, the plasma concentrations of the antibody at the annotated time points in hVISTA KI mice are (SD; n=5 / group).

[0121] [Figure 48] This includes experimental results comparing the PK values ​​of other exemplary anti-VISTA antibodies according to the present invention, namely INX231, INX234, INX237, and INX240. As shown in the figure, these are the plasma concentrations (SD; n=5 / group) of the antibodies at annotated time points in hVISTA KI mice. The graph on the left shows the y and x axes in Log10; on the other hand, in the graph on the right, only the y axis is Log10.

[0122] [Figure 49] This includes experimental results comparing the PK values ​​of exemplary anti-VISTA antibodies according to the present invention, namely INX901, INX904, INX907, and INX908. Plasma concentrations of the antibodies at annotated time points (SD; n=5 / group) in hVISTA KI mice.

[0123] [Figure 50] This includes experimental results comparing the PK values ​​of different ADCs according to the present invention, namely INX201J, INX231J, INX234J, and INX240J. Plasma concentrations of antibodies at annotated time points (SD; n=4 / group) in hVISTA KI mice.

[0124] [Figure 51] This includes the results of an experiment analyzing the effects of long-term treatment with the exemplary VISTA Ab ADC complex INX201J and dexamethasone on corticosterone levels. The figure shows changes in plasma corticosterone levels. (SEM, one-way ANOVA, n=8 except for the PBS control group n=6 in the graph on the right).

[0125] [Figure 52] This shows the number of peripheral blood-derived Ag-specific CD8 T cells 6 days post-immunization in Experiment 1 of Example 12. (SEM, one-way ANOVA, n=5).

[0126] [Figure 53] The graph shows the number of peripheral blood-derived Ag-specific CD8 T cells 6 days post-immunization in Experiment 2 of Example 12. The graph on the left shows the PBS control group, which includes all samples, and the graph on the right shows the PBS control group with one outlier excluded (SEM, one-way ANOVA, n=5 excluding naive samples; one sample was excluded as an immunization failure in the 0.2 mg / kg Dex group).

[0127] [Figure 54]The number of peripheral blood-derived Ag-specific CD8 T cells on day 6 post-immunization in Experiment 3 of Example 12 is shown. Due to technical problems during the procedure, several samples had to be excluded in this experiment: PBS group n=3, 2 mg / Kg Dex n=2, 0.2 mg / Kg Dex n=3, INX201J D-1 n=5, INX201J D-7 n=2, INX231J D-7 n=3, INX234J D-7 n=5, INX240J D-7 n=4 (SEM, one-way ANOVA, D=day).

[0128] [Figure 55] The number of peripheral blood-derived Ag-specific CD8 T cells on day 6 post-immunization in Experiment 3 of Example 12 is shown. For technical reasons, two samples were excluded from the PBS, INX231P, and INX234P groups; in all other groups, n=5 (SEM, one-way ANOVA).

[0129] [Figure 56] The changes in absolute cell count in peripheral blood under two experimental schedules are shown. OVA challenges were performed on days 14–18 and 21–25 (SEM, one-way ANOVA, n=10, excluding the naive group of n=5).

[0130] [Figure 57] This shows the changes in peripheral blood immunoglobulin production under two experimental schedules: OVA challenges on days 14-18 (Part 1) and days 21-25 (Part 2) (SEM, one-way ANOVA, n=10, excluding the naive group of n=5).

[0131] [Figure 58] This shows the changes in immune infiltration in BAL under two experimental schedules: OVA challenges on days 14-18 (Part 1) and days 21-25 (Part 2); A) Changes in bone marrow infiltration; B) Lymphocyte infiltration (SEM, one-way ANOVA, n=10, 2 samples were censored in the control group, 3 in both the Dex and INX201J groups; n=5 in the naive group).

[0132] [Figure 59] The changes in BAL cytokine levels in two experimental schedules are shown: OVA challenges on days 14-18 (Part 1) and days 21-25 (Part 2) (SEM, one-way ANOVA, n=10, 2 samples were censored in the control group, 3 in both the Dex and INX201J groups; n=5 in the naive group).

[0133] [Figure 60] The scoring for lung disease in Part 1 of the study is shown. (SEM, one-way ANOVA, n=10, excluding the naive group of n=5).

[0134] [Figure 61] This image shows FKBP5 transcriptional activation in spleen (left) and blood (right) cells after INX231J injection. The effects of INX231J and hIgG1siJ (gray) were measured 20 hours after a single intravenous injection of 5 mg / kg (delivering a 0.1 mg / kg payload). The effect of Dex was measured 2 hours after a single intraperitoneal injection of 2 mg / kg. FKBP5 transcriptional levels were measured by real-time PCR and shown as a log-2 multiplier change relative to the mean of the PBS control group (n=4 mice / group; standard one-way ANOVA compared to the PBS-only group).

[0135] [Figure 62] This study demonstrates FKBP5 transcriptional activation after INX231P injection in C57Bl / 6 mice. The effect of INX231P was measured 20 hours after a single intraperitoneal injection of 10 mg / kg (delivering a 0.2 mg / kg payload). The effect of Dex was measured 2 hours after a single intraperitoneal injection of 2 mg / kg. FKBP5 transcription levels were measured by real-time PCR and expressed as a log-2 multiplier change relative to the mean of the PBS control group (n=4 mice / group; standard one-way ANOVA compared to the PBS-only group).

[0136] [Figure 63]This report includes experimental results demonstrating FKBP5 transcriptional activation after INX231P injection in C57Bl / 6 or hVISTA KI mice. The INX231P effect was measured 20 hours after a single intraperitoneal injection of 10 mg / kg (delivering a 0.2 mg / kg payload). The Dex effect was measured 2 hours after a single intraperitoneal injection of 2 mg / kg. FKBP5 transcription levels were measured by real-time PCR and expressed as a log-2 multiplier change relative to the mean of the PBS control group (n=4 mice / group; standard one-way ANOVA compared to the PBS-only group).

[0137] [Figure 64] This study includes experimental results demonstrating that in vivo Dex treatment reduces the ex vivo monocyte inflammatory response to LPS. Mice were intraperitoneally injected with PBS or Dex at 2 mg / kg or 0.2 mg / kg. Two hours later, splenic monocytes were isolated, cultured, and subjected to LPS stimulation at 0, 10, and 100 ng / ml. The supernatant at 24 hours was analyzed with Luminex 32-plex (n=5 mice / group, however, samples 1, 2, 3 and 4, 5 were pooled into two samples).

[0138] [Figure 65] This study includes experimental results demonstrating that in vivo treatment with INX231P affects the ex vivo monocyte inflammatory response to LPS. Mice were intraperitoneally injected with PBS or Dex at 2 mg / kg 2 hours, 2 days, or 6 days prior to cell isolation; and with INX231P and INX901 at 10 mg / kg 1 day, 3, and 7 days prior to cell isolation. After isolation, splenic monocytes were cultured and subjected to LPS stimulation at 0 or 10 ng / ml (only 10 ng / ml is shown). The supernatant at 24 hours was analyzed by ELISA (n=4 mice / group; one-way ANOVA compared with the PBS-treated group was performed only on the day 1 (D1) sample).

[0139] [Figure 66]This study includes experimental results demonstrating that in vivo treatment with INX231P affects the ex vivo monocyte inflammatory response to LPS. Mice were intraperitoneally injected with PBS or Dex at 2 mg / kg two hours before cell isolation; and with INX231P and INX901 at 10 mg / kg 24 hours before cell isolation. Splenic monocytes were cultured and stimulated with LPS at 10 and 100 ng / ml. The supernatant at 24 hours was analyzed by ELISA (n=4 mice / group; a standard one-way ANOVA was performed for each LPS dose compared to the PBS-treated group).

[0140] [Figure 67] This study demonstrates FKBP5 transcriptional activation in B cells or monocytes. Cells were treated with a 20 nM free J payload or an equimolar payload conjugated with INX201 (INX201J) or an isotype control (huIgG1si J). Transcriptional levels were analyzed as technical two-point measurements.

[0141] [Figure 68] This shows FKBP5 transcriptional activation in monocytes. Cells were treated with increased doses of INX201J (0-100 nM payload). A 0 payload represents treatment with uncomplexed INX201 antibody alone, equivalent to the dose of INX201J in a 100 nM payload. Transcriptional levels were analyzed as technical two-point measurements.

[0142] [Figure 69] This study demonstrates FKBP induction in Tregs from two donors treated with either 20 nM INX-SM-3 (free payload) or a molar equivalent of the INX231P payload (complexed payload). Samples were prepared and analyzed as single samples. The purity of the isolated Tregs was ≥75% as assessed by flow cytometry.

[0143] [Figure 70]This shows FKBP5 induction in Tregs from one donor treated with a 20 nM payload equivalent of INX201J, compared to a 20 nM payload equivalent of huIgG1siJ. Samples were analyzed using technical two-point measurements. The purity of the separated Tregs was ≥75% as assessed by flow cytometry.

[0144] [Figure 71] Based on reported "transcripts per million units" (TPM), this summarizes the consensus RNA expression levels of various immune cells reported for VISTA and other ADC targets (CD40, TNFα, CD74, CD163 (PRLR)). TPM < 10 represents (minimum expression / no expression "-"), TPM 10-100 represents (low expression / moderate expression "+"), and TPM > 100 represents (high expression "++").

[0145] [Figure 72] This summarizes the quantification of antigen densities for VISTA, CD74, CD163, and mTNFα in identified cell populations. A) Monocytes express VISTA, CD74, and CD163; B) B cells express CD74; C) CD4+ T cells, D) CD4+ T reg, and E) CD8+ T cells express VISTA (mean ± standard deviation, n=5 donors).

[0146] [Figure 73] This shows the quantification of antigen densities for VISTA, CD74, CD163, and mTNFα in identified cell populations in human blood. A) Monocytes express VISTA, CD74, and CD163; B) B cells express CD74; C) Neutrophils express VISTA; D) CD4+ T cells, E) CD4+ T reg, and F) CD8+ T cells express VISTA (mean ± standard deviation, n=3). [Modes for carrying out the invention]

[0147] This specification provides an ADC comprising an anti-VISTA antibody or antibody fragment (this antibody or antibody fragment has a very short serum half-life under physiological conditions (pH approximately 7.5), generally having a serum half-life of less than 72 hours, 1 to 32 hours, 1 to 16 hours, 1 to 8 hours, 1 to 4 hours, or 1 to 2 hours ± 0.5 hours in human VISTA knock-in rodents, or approximately 3.5, 3, 2.5, or 2.3 days ± 0.5 days in primates (cynomolgus monkeys)), and a small molecule anti-inflammatory drug that requires intracellular integration for efficacy, such as a glucocorticoid receptor agonist such as a glucocorticosteroid (which is optionally bound via a linker, such as a peptide or non-peptide linker that may be cleavable under certain conditions, such as an esterase-cleavable dipeptide linker, which is optionally bound directly or indirectly to the antibody via a heterobifunctional group or heterotrifunctional group). Such ADCs, when administered to a target in need, deliver such anti-inflammatory drugs to target immune cells, such as monocytes, T cells, neutrophils, Tregs, CD8 T cells, CD4 T cells, or myeloid cells, and the glucocorticosteroids or other anti-inflammatory drugs induce a desired inhibitory effect on inflammation without or inducing adverse side effects, such as toxicity to non-target cells, substantially reducing them. Furthermore, methods for producing such ADCs and methods for using them are provided, particularly for use in the treatment of autoimmune and inflammatory conditions as previously identified.

[0148] More specifically, the present invention provides a novel antibody-drug conjugate (ADC) comprising an anti-VISTA antibody or antibody fragment having a very short serum half-life under physiological conditions (approximately pH 7.5), and an anti-inflammatory agent, such as a small molecule anti-inflammatory drug, such as a glucocorticoid receptor agonist like dexamethasone, prednisolone, or budesonide, or one of the other steroids disclosed herein.

[0149] More specifically, the present invention provides novel antibody-drug conjugates (ADCs) comprising an anti-VISTA antibody or antibody fragment having a serum half-life in rodents of up to approximately 72 hours, 1–32 hours, 1–16 hours, 1–8 hours, 1–4 hours, or 1–2 hours ± 0.5 hours in human VISTA knock-in rodents, or approximately 3.5, 3, 2.5, or 2.3 days ± 0.5 days in primates (cynomolgus monkeys), and an anti-inflammatory agent, such as a synthetic glucocorticoid receptor agonist such as dexamethasone, prednisolone, or budesonide, which, upon administration, results in the release and internalization of the anti-inflammatory agent, such as a synthetic glucocorticoid receptor agonist such as dexamethasone, prednisolone, or budesonide, or other glucocorticoids or derivatives, to target immune cells.

[0150] More specifically, we provide an antibody-drug conjugate (ADC) comprising an antibody or antigen-binding fragment ("A") containing an antigen-binding region that specifically binds to the human V-domain Ig suppressor (human VISTA) for T cell activation, a cleavable or non-cleavable linker ("L"), and at least one small molecule anti-inflammatory agent ("AI"), optionally "Q", i.e., a "hetero-bifunctional group" or "hetero-trifunctional group" which is an optionally used chemical moiety to link the linker to the anti-VISTA antibody or antibody fragment and at least one small molecule anti-inflammatory agent ("AI"), wherein the ADC is represented by the following formula: "A-(QL-AI)" n " or "(AI-LQ) n -A" In the formula, "n" is at least 1, and when administered to a target requiring it, the antibody or ADC, or a composition containing them, is preferentially delivered to immune cells expressing VISTA, optionally monocytes or myeloid cells, thereby functionally internalizing the low molecular weight anti-inflammatory agent in the immune cells under physiological conditions (approximately pH 7.5). Preferably, the anti-VISTA antibody or antigen-binding fragment, when used in vivo, has a short in vivo serum half-life in human VISTA knock-in rodents at physiological pH (approximately pH 7.5) of 1 to 72 hours, 1 to 32 hours, 1 to 16 hours, 1 to 8 hours, 1 to 4 hours, or 1 to 2 hours ± 0.5 hours in primates (cynomolgus monkeys) at physiological conditions (approximately pH 7.5).

[0151] Furthermore, the present invention provides a novel steroid (glucocorticoid agonist) that generally comprises the following general structure: [ka] formula 1 In the formula, X or Z may be phenyl, a 3-6 membered heterocycle, a cycloalkyl, a spiroalkyl, a spiroheterocycloalkyl, [1.1.1]bicyclopentane, bicyclo[2.2.2]octane, or cubane, each of which may be substituted with 1-4 heteroatoms independently selected from N, S, and O, and optionally 1-4 C 1-3 Further substituted with alkyl; The connection from X to Z may occupy any available position on X and Z; Y may be CHR1, O, S, or NR1; E may be CH2 or O; G may be CH2 or NR1; R1 may be H, a lower or branched alkyl, aryl, or heteroaryl having 1 to 8 carbon atoms. If an aryl or heteroaryl ring is substituted, the substituent may be alkyl, haloalkyl, halogen, biphenyl, nitro, nitrile, -OH, -O-alkyl, -NH2, alkylamino, dialkylamino, thiol, thioalkyl, guanidine, urea, carboxylate, alkoxyl, carboxamide, carboxylic acid ester, alkyl-C(O)O-, alkylamino-C(O)-, and dialkylamino-C(O)-; If R1=H, R2 may be H, a lower or branched alkyl, aryl, or heteroaryl having 1 to 8 carbon atoms. If an aryl or heteroaryl ring is substituted, the substituent may be alkyl, haloalkyl, halogen, biphenyl, nitro, nitrile, -OH, -O-alkyl, -NH2, alkylamino, dialkylamino, thiol, thioalkyl, guanidine, urea, carboxylate, alkoxyl, carboxamide, carboxylic acid ester, alkyl-C(O)O-, alkylamino-C(O)-, and dialkylamino-C(O)-; If R1 is H, a lower or branched alkyl or heteroaryl with 1 to 8 carbon atoms, then R2 is [(C=O)CH2(W)NHC=O] m -The functional group may be selected from VJ, and W may be H or [(CH2) n R3] n It may also be a branched alkyl chain terminated at R3 or 1 to 13 units of polyethylene glycol group OCH2CH2O; R3 may be H, or selected from OH, O-alkyl, NH2, NH-alkyl, N-dialkyl, SH, S-alkyl, guanidine, urea, carboxylic acid, carboxamide, carboxylic acid ester, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, wherein the substituent may be alkyl, haloalkyl, halogen, biphenyl, nitro, nitrile, -OH, -O-alkyl, -NH2, alkylamino, dialkylamino, thiol, thioalkyl, guanidine, urea, carboxylic acid, alkoxyl, carboxamide, carboxylic acid ester, alkyl-C(O)O-, alkylamino-C(O)-, and dialkylamino-C(O)-; The substituent NR1R2 may occupy any available position on Z; R2 is also C(=O)OCH2-p-aminophenyl[(C=O)CH(W)NHC=O] m -It may also be VJ, and W is H or [(CH2) n R3] n It may also be a branched alkyl chain terminated at R3, 1 to 13 units of polyethylene glycol group OCH2CH2O or C(=O)OCH2-p-aminophenyl-VJ; V may be selected from an alkyl chain having 1 to 8 carbon atoms, a polyethylene glycol group OCH2CH2O having 1 to 13 units, or a lower or branched alkyl, aryl, or heteroaryl ring having 1 to 8 carbon atoms. If an aryl or heteroaryl ring is substituted, the substituent may be an amino acid sequence of 1 to 3 residues selected from alkyl, haloalkyl, halogen, biphenyl, nitro, nitrile, -OH, -O-alkyl, -NH2, alkylamino, dialkylamino, thiol, thioalkyl, guanidine, urea, carboxylate, alkoxyl, carboxamide, carboxylic acid ester, alkyl-C(O)O-, alkylamino-C(O)-, dialkylamino-C(O)-, and Gly, Asn, Asp, Gln, Leu, Lys, Ala, βAla, Phe, Val, or Cit; J is a reactive group selected from -NH2, N3, thio, cyclooctin, -OH, -CO2H, and trans-cyclooctin; [ka] In the formula, R 32 is Cl, Br, F, mesylate or tosylate, and R 33 is Cl, Br, I, F, OH, -ON-succinimidyl, -O-(4-nitrophenyl), -O-pentafluorophenyl, or -O-tetrafluorophenyl, and R 34 is H, Me, or tetrazine-H or Me; Q may be H, P(O)OR4, R4 may be H or a lower 1-10 alkyl group, C(O)R6, and R6 may be a lower or branched alkyl group having 1-8 carbon atoms, or [(C=O)NR4CH n NR4(C=O)OCH m ] m -VVJ, n=1-8, m=1-6, R4=H, alkyl or branched alkyl; A1 and A2 may be H or halogens and, unless otherwise specified, include all possible stereoisomers; furthermore, linker "L" may include one or any non-cleavable or cleavable linker, including any of those known in the art and illustrated herein (see definition of linker, linkers identified in the section on exemplary embodiments of this application, and linkers used in the synthesis of steroid linker payloads and ADCs embodied in Example 3 below).

[0152] Furthermore, the present invention provides an ADC and steroid linker payload comprising the novel steroid of Formula 1 described above, a composition comprising the same, and its use for treating / preventing inflammation in a target area and for treating any condition or disorder acutely, chronically or transiently associated with inflammation, such as inflammatory diseases, autoimmune diseases, infections, cancer, and other conditions disclosed below.

[0153] Under such general understanding, unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention pertains. Methods and materials similar to or equivalent to those described herein may be used in the present invention or the testing of the present invention, but preferred methods and materials are described herein. Materials, methods, and examples are merely illustrative and not intended to limit the scope. The nomenclature and their testing methods and experimental techniques used in relation to analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein are well known and commonly used in the art. Standard techniques may be used in chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and delivery, as well as in the treatment of patients.

[0154] I. Definition

[0155] To facilitate understanding of this disclosure, several terms and phrases are defined below.

[0156] In this description and as used in the following claims, unless the context explicitly indicates otherwise, the meanings of “a,” “an,” and “the” include multiple referents.

[0157] In this disclosure, the terms “glucocorticosteroid” or “steroid” refer to natural or synthetic steroid hormones that interact with glucocorticoid receptors. Non-exclusive exemplary glucocorticosteroids include, in particular, those described in WO2009 / 069032, US20180126000, and WO05 / 028495. Non-exclusive examples of glucocorticosteroids include: [ka]

[0158] Other glucocorticosteroids are described in WO2009 / 069032. Specific examples of glucocorticosteroids include 16-α-hydroxyprednisolone, dexamethasone, difluorazone, flumethasone, flunisolide, fluocinolone acetonide, fluticasone propionate, ciclesonide, methylprednisolone, prednisone, prednisolone, mometasone, triamcinolone acetonide, and novel steroids of formula 1 disclosed herein.

[0159] A "glucocorticosteroid derivative" is a compound induced by the addition or removal of one or more atoms or functional groups to facilitate the binding of the "glucocorticosteroid derivative" to another part, such as a linker and / or an antibody or antibody fragment. Generally, this addition or removal does not eliminate the activity of the "glucocorticosteroid derivative," i.e., its ability to induce anti-inflammatory activity upon internalization by immune cells. Specifically, "glucocorticosteroid derivatives" include "glucocorticosteroid radicals" or "glucocorticosteroid radicals."

[0160] A "glucocorticosteroid radical" is generated by removing one or more atoms, namely hydrogen atoms, from a parent glucocorticosteroid to facilitate its attachment to another part, generally a linker. For example, a hydrogen atom may be removed from any suitable -NH2 group of the parent glucocorticosteroid; from any suitable -OH group of the parent glucocorticosteroid; from any suitable -SH group; from any suitable -N(H)- group; or from any suitable -CH3, -CH2-, or -CH= group of the parent glucocorticosteroid.

[0161] In this disclosure, the terms “heterobifunctional group” or “heterotrifunctional group” refer to a chemical moiety (in the general formula of the ADCs disclosed herein, “Q”) that may be optionally used to link a linker with an anti-VISTA antibody or antibody fragment. Heterobifunctional groups and heterotrifunctional groups are characterized by having different reactive groups at both ends of the chemical moiety. Non-limiting exemplary heterobifunctional groups are disclosed in U.S. Patent Application Publication No. 20180126000, incorporated herein by reference, and are further illustrated in the exemplary embodiments section and in Example 3 of this application.

[0162] Heterodifunctional and heterotrifunctional groups are well known in the art for the specific formation of protein complexes and antibody-drug conjugates (ADCs). These groups are characterized by having different reactive groups at both ends of the chemical moiety. Non-limiting exemplary heterodifunctional groups include: [ka] Examples include: [ka] That is the case.

[0163] As used herein, the terms "antibody" and "antibodies" are technical terms and may be used interchangeably herein, and refer to molecules that contain an antigen-binding site that specifically binds to an antigen.

[0164] The term “antibody” means an immunoglobulin molecule that recognizes and binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combination thereof, via at least one antigen recognition site within the variable region of the immunoglobulin molecule. As used herein, the term “antibody” includes intact polyclonal antibodies, intact monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, antibody-containing fusion proteins, and any other modified immunoglobulin molecules, insofar as the antibody exhibits the desired biological activity. An antibody may be any five major classes of immunoglobulins, namely IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes) (IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), based on the identity of heavy chain constant domains called α, δ, ε, γ, and μ, respectively. Different classes of immunoglobulins have different known subunit structures and three-dimensional structures. Antibodies can be naked antibodies or conjugated with other molecules such as toxins or radioisotopes. As used herein, the term “antibody” includes both bispecific and multispecific antibodies.

[0165] The term "antibody fragment" refers to a portion of an intact antibody. "Antigen-binding fragment" refers to a portion of an intact antibody that binds to an antigen. Antigen-binding fragments may include the antigen-determining variable region of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, and single-chain antibodies. "Antigen-binding fragments" may be bispecific or multispecific antigen-binding fragments.

[0166] A “blocking” antibody or “antagonist” antibody is an antibody that inhibits or reduces the biological activity of the antigen to which it binds, such as VISTA. In some embodiments, the blocking antibody or antagonist antibody substantially or completely inhibits the biological activity of the antigen. The biological activity may be reduced by 10%, 20%, 30%, 50%, 70%, 80%, 90%, 95%, or 100%.

[0167] A "promoting" antibody, "enhancing" antibody, or "agonist" antibody is an antibody that enhances or increases the biological activity of the antigen to which it binds, for example, VISTA. In some embodiments, a blocking antibody or antagonist antibody substantially or completely inhibits the biological activity of the antigen. The biological activity may be reduced by 10%, 20%, 30%, 50%, 70%, 80%, 90%, 95%, or 100%.

[0168] The term “anti-VISTA antibody” or “antibody that binds to VISTA” refers to an antibody that specifically binds to VISTA, generally human VISTA, and has sufficient affinity to be useful in targeting VISTA-expressing immune cells. The degree of binding of an anti-VISTA antibody to unrelated non-VISTA proteins may be less than about 10% of the binding of the antibody to VISTA, for example, when measured by radioimmunoassay (RIA). In certain embodiments, antibodies that bind to VISTA have a dissociation constant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, or ≤0.1 nM. The exemplary anti-VISTA antibodies and fragments included in the subject ADC contain the same CDR and / or the same variable heavy and light chain polypeptides as VSTB94 or VSTB49-116, i.e., have the sequences shown in Figures 8, 10, and 12, respectively.

[0169] A “monoclonal” antibody or its antigen-binding fragment refers to a homogeneous population of antibodies or antigen-binding fragments involved in the highly specific recognition and binding of a single antigenic determinant, i.e., an epitope. This is in contrast to polyclonal antibodies, which typically contain different antibodies against different antigenic determinants. The term “monoclonal” antibody or its antigen-binding fragment encompasses intact, full-length monoclonal antibodies as well as antibody fragments (e.g., Fab, Fab', F(ab')2, Fv), single-chain (scFv) variants, fusion proteins containing antibody moieties, and any other immunoglobulin molecules containing antigen recognition sites. Furthermore, “monoclonal” antibody or its antigen-binding fragment refers to antibodies and their antigen-binding fragments produced by any method, including but not limited to hybridomas, phage selection, recombinant expression, and transgenic animals.

[0170] The term "humanized" antibody or its antigen-binding fragment refers to a non-human (e.g., mouse) antibody or antigen-binding fragment that is a specific immunoglobulin chain, chimeric immunoglobulin, or fragment thereof containing minimal non-human (e.g., mouse) sequences. Typically, a humanized antibody or its antigen-binding fragment is a human immunoglobulin in which residues derived from its complementarity-determining region (CDR) are replaced with residues derived from the CDR of a non-human species (e.g., mouse, rat, rabbit, hamster) that has the desired specificity, affinity, and function ("CDR graft") (Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988)). In some cases, the Fv framework region (FR) of human immunoglobulin is replaced with corresponding residues in an antibody or fragment derived from a non-human species that has the desired specificity, affinity, and function. Humanized antibodies or their antigen-binding fragments can be further modified by substitution of additional residues within the Fv framework region and / or the replaced non-human residues to improve and optimize the specificity, affinity, and / or function of the antibody or its antigen-binding fragment. Generally, a humanized antibody or its antigen-binding fragment contains at least one, usually two or three, variable domains, including all or substantially all of the CDR region corresponding to the non-human immunoglobulin, while all or substantially all of the FR region is from the human immunoglobulin consensus region. The humanized antibody or its antigen-binding fragment may also contain at least a portion of the immunoglobulin constant region or domain (Fc), which is usually from human immunoglobulin. Examples of methods used to produce humanized antibodies are described in U.S. Patent No. 5,225,539, Roguska et al., Proc. Natl. Acad. Sci., USA, 91(3):969-973 (1994), and Roguska et al., Protein Eng. 9(10):895-904 (1996). In some embodiments, the “humanized antibody” is a surface reforming antibody.

[0171] The "variable region" of an antibody refers to either the variable region of the antibody light chain, the variable region of the antibody heavy chain, or a combination thereof. The variable regions of the heavy and light chains each consist of four framework regions (FRs) connected by three complementarity-determining regions (CDRs), also known as hypervariable regions. The CDRs within each chain are held together in close proximity by the FRs and, together with CDRs from other chains, contribute to the formation of the antibody's antigen-binding site. At least two techniques exist for determining CDRs: (1) methods based on interspecies sequence changes (i.e., Kabat et al. Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda Md.)), and (2) methods based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al (1997) J. Molec. Biol. 273:927-948). Furthermore, in this art, these two methods are sometimes used in combination to determine CDRs.

[0172] The Kabat numbering system is generally used when referring to residues within the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Unless otherwise specified, the numbering system used herein is the Kabat numbering system.

[0173] Kabat amino acid position numbering refers to the numbering system used for the heavy-chain or light-chain variable domains of antibody composition in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to shortenings or insertions of FR or CDR residues in the variable domain. For example, a heavy-chain variable domain may contain a single amino acid insertion after H2 residue 52 (Kabat-following residue 52a), as well as residues inserted after heavy-chain FR residue 82 (e.g., Kabat-following residues 82a, 82b, and 82c). The Kabat numbering of residues can be determined for a given antibody by alignment of the antibody sequence homology region with a “standard” Kabat numbering sequence. Chothia, on the other hand, refers to the location of the structural loop (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). When numbered using Kabat numbering rules, the end of the Chothia CDR-H1 loop differs between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places insertions at H35A and H35B; if neither 35A nor 35B exists, the loop ends at 32; if only 35A exists, the loop ends at 33; and if both 35A and 35B exist, the loop ends at 34). The AbM hypervariable region is a compromise between Kabat's CDR and Chothia's structural loop and is used by Oxford Molecular's AbM antibody modeling software.

[0174] In certain embodiments, the CDR of an antibody or its antigen-binding fragment may be determined according to the Chothia numbering scheme, which points to the position of the structural loop of the immunoglobulin (see, for example, Chothia C & Lesk AM, (1987), J Mol Biol 196:901-917; Al-Lazikani B et al., (1997) J Mol Biol 273:927-948; Chothia C et al., (1992) J Mol Biol 227:799-817; Tramontano A et al., (1990) J Mol Biol 215(1):175-82; and U.S. Patent No. 7,709,226). Typically, when using the Kabat numbering rules, the Chothia CDR-H1 loop is located at heavy chain amino acids 26-32, 33, or 34; the Chothia CDR-H2 loop is located at heavy chain amino acids 52-56; the Chothia CDR-H3 loop is located at heavy chain amino acids 95-102; the Chothia CDR-L1 loop is located at light chain amino acids 24-34; the Chothia CDR-L2 loop is located at light chain amino acids 50-56; and the Chothia CDR-L3 loop is located at light chain amino acids 89-97. When numbered using the Kabat numbering rules, the end of a Chothia CDR-H1 loop will differ between H32 and H34 depending on the loop length (this is because the Kabat numbering scheme places insertions at H35A and H35B; if neither 35A nor 35B exists, the loop ends at 32; if only 35A exists, the loop ends at 33; and if both 35A and 35B exist, the loop ends at 34).

[0175] In certain embodiments, the CDRs of an antibody or its antigen-binding fragment may be determined according to an IMGT numbering system such as that described in Lefranc MP, (1999) The Immunologist 7:132-136 and Lefranc MP et al., (1999) Nucleic Acids Res 27:209-212. According to the IMGT numbering scheme, VH-CDR1 is at positions 26-35, VH-CDR2 is at positions 51-57, VH-CDR3 is at positions 93-102, VL-CDR1 is at positions 27-32, VL-CDR2 is at positions 50-52, and VL-CDR3 is at positions 89-97.

[0176] In certain embodiments, the CDR of an antibody or its antigen-binding fragment may be determined according to MacCallum RM et al., (1996) J Mol Biol 262:732-745. See also, for example, Martin A. "Protein Sequence and Structure Analysis of Antibody Variable Domains," in Antibody Engineering, Kontermann and Dubel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001).

[0177] In certain embodiments, the CDR of an antibody or its antigen-binding fragment may be determined according to an AbM numbering scheme, which represents a compromise between Kabat's CDR and Chothia's structural loop, and is used by Oxford Molecular's AbM antibody modeling software (Oxford Molecular Group, Inc.).

[0178] The "constant region" of an antibody refers to either the constant region of the antibody light chain or the constant region of the antibody heavy chain, either alone or in combination.

[0179] The term "human" antibody means an antibody produced by a human, or an antibody having an amino acid sequence corresponding to a human-produced antibody manufactured using any technique known in the art. This definition of a human antibody includes intact or full-length antibodies, fragments thereof, and / or antibodies containing at least one human heavy chain and / or light chain polypeptide, such as an antibody containing a mouse light chain and a human heavy chain polypeptide.

[0180] The term "chimeric" antibody refers to an antibody in which the amino acid sequence of the immunoglobulin molecule originates from two or more species. Typically, both the light and heavy chain variable regions correspond to the variable regions of an antibody from one mammalian species (e.g., mouse, rat, rabbit) with desired specificity, affinity, and function, while its constant region is homologous to the sequence of an antibody from another species (usually human), thus avoiding the induction of an immune response in that species.

[0181] The terms “epitope” and “antigenic determinant” are used interchangeably herein and refer to a portion of an antigen that a particular antibody can recognize and specifically bind to. When the antigen is a polypeptide, epitopes can be formed from both adjacent amino acids and non-adjacent amino acids paralleled by the protein’s tertiary folding. Epitopes formed from adjacent amino acids are usually retained during protein denaturation, while epitopes formed by tertiary folding are usually lost during protein denaturation. Epitopes typically contain at least three, more commonly at least five or eight to ten, amino acids in their unique spatial structure. Preferred epitopes on VISTA to which exemplary anti-VISTA antibodies can bind are identified in Figure 10.

[0182] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" as used herein refers to the intrinsic binding affinity that reflects the 1:1 interaction between the members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for its partner Y can generally be expressed by a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including the methods described herein. Low-affinity antibodies generally tend to bind slowly to antigens and dissociate easily, while high-affinity antibodies generally tend to bind more quickly to antigens and remain bound for longer. Various methods for measuring binding affinity are known in the art, and any of them can be used for the purposes of this disclosure. Such methods include surface plasmon resonance (BIAcore), ELISA, Kinexa Biosensor, scintillation proximity assay, ORIGEN immunoassay (IGEN), fluorescence quenching, fluorescence transfer, and / or yeast display. Alternatively, binding affinity may be screened using a suitable bioassay. In this application, the Kd of exemplary anti-VISTA antibodies contained in exemplary ADCs was determined by surface plasmon resonance (SPR) using a ProteOn instrument.

[0183] As used herein to refer to binding affinity, “or better” refers to a stronger bond between a molecule and its binding partner. As used herein, “or better” refers to a stronger bond represented by a smaller Kd value. For example, in an antibody with an affinity for an antigen of “0.6 nM or better,” the affinity of the antibody for the antigen is <0.6 nM, i.e., 0.59 nM, 0.58 nM, 0.57 nM, etc., or any value less than 0.6 nM.

[0184] "Specifically binding" generally means that an antibody binds to an epitope via its antigen-binding domain, and that this binding requires a certain degree of complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" to an epitope when it binds to that epitope via its antigen-binding domain more readily than it would to a random, unrelated epitope. The term "specificity" is used herein to limit the relative affinity with which a particular antibody binds to a particular epitope. For example, antibody "A" may be considered to have higher specificity to a given epitope than antibody "B," or it may be said that antibody "A" binds to epitope "C" with higher specificity than it has to epitope "D" which is related to epitope "C."

[0185] "Preferential binding" means that an antibody specifically binds to a particular epitope more readily than it would to related, similar, homologous, or analogous epitopes. Therefore, an antibody that "preferentially binds" to a given epitope is more likely to bind to that epitope than to its related epitope, even if such an antibody may cross-react with the related epitope.

[0186] An antibody is said to "competitively inhibit" the binding of a reference antibody to a given epitope if it preferentially binds to that epitope, or a duplicate epitope, to such an extent that it blocks to some degree the binding of a reference antibody to that epitope. Competitive inhibition can be measured by any method known in the art, for example, a competitive ELISA assay. An antibody may be said to competitively inhibit the binding of a reference antibody to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

[0187] In this specification, "isotype" refers to an antibody class encoded by a heavy chain constant region gene (e.g., IgM, IgG1, IgG3, IgG4, or IgG4).

[0188] As used herein, "K-assoc" or "Ka" broadly refers to the association rate of a particular antibody-antigen interaction, while as used herein, the terms "Kdiss" or "Kd" refer to the dissociation rate of a particular antibody-antigen interaction.

[0189] As used herein, the term "KD" refers to the dissociation rate obtained from the Kd to Ka ratio (i.e., Kd / Ka) and expressed as molar concentration (M). The KD value for an antibody can be determined using well-established methods in the art, such as plasmon resonance (BIAcore®), ELISA, and KINEXA. Preferred methods for determining the KD of an antibody are those using surface plasmon resonance, preferably using a biosensor system such as the BIAcore® system, or by ELISA. These methods are typically carried out at 25°C or 37°C. Antibodies for therapeutic use generally have a KD of 50 nM or less, or more commonly 1 nM or less, at 25°C or 37°C, as determined by surface plasmon resonance.

[0190] In this specification, the term "Kd" refers to the ratio of the equilibrium dissociation constant between an antibody and its antigen, i.e., Koff / Kon. The binding constant (Kon) is used to characterize the rate at which an antibody binds to its target. In this specification, the Kd of an antibody was determined by surface plasmon resonance (SPR) using a Proteon instrument.

[0191] In this specification, the term "PK" refers to the in vivo half-life, i.e., the duration (time) during which half of an antibody or antibody fragment or antibody-drug conjugate (ADC), preferably the anti-VISTA or antibody fragment of the present invention (i.e., comprising an anti-VISTA antibody or antibody fragment that binds to VISTA-expressing cells at physiological pH) and an anti-inflammatory agent (AI) (AI is a small molecule that requires intracellular integration for its potency (anti-inflammatory activity), and is generally a steroid) remains in the peripheral circulation in the serum. The PK can be determined in vivo in subjects administered with the antibody or antibody fragment or ADC, for example, human VISTA knock-in rodents or primates (e.g., humans or cynomolgus monkeys). As described later, the anti-VISTA antibodies contained in the subject ADC generally have short PKs, i.e., about 2.3 ± 0.7 days in cynomolgus monkeys, and generally at most about 2.5 days, and more generally just a few hours or less, in human VISTA knock-in rodents.

[0192] In this specification, the term "PD" refers to the duration (time) during which administration of an antibody-drug conjugate (ADC), preferably an ADC according to the present invention (i.e., an ADC comprising an anti-VISTA antibody or antibody fragment that binds to VISTA-expressing cells at physiological pH) and an anti-inflammatory agent (AI) (AI is a small molecule that requires intracellular integration for efficacy (anti-inflammatory activity) and usually includes steroids) induces efficacy (anti-inflammatory activity). The PD of a steroid can be determined by various assays. For example, the PD of a VISTA ADC according to the present invention can be determined in vitro using VISTA-expressing immune cells contacted with the ADC, or in vivo in a subject to which the ADC dose has been administered, such as a rodent or primate (e.g., human or cynomolgus monkey). Because the subject ADCs bind to various immune cells (e.g., T cells, Tregs, monocytes, macrophages, neutrophils), and furthermore, because these ADCs internalize anti-VISTA antibody ADCs in various ways based on relative VISTA expression, and furthermore, because the turnover rates of such VISTA-expressing immune cells vary, the PD values ​​measured in vitro using various types of VISTA-expressing immune cells vary considerably. Since macrophages are present in circulation and (surprisingly) induce anti-inflammatory activity several weeks after ADC administration, generally, PD in this specification is expressed based on the duration of anti-inflammatory activity induced by these cells.

[0193] In this specification, the term PK / PD ratio refers to the PK / PD value of the ADC according to the present invention, determined in vitro or in vivo in immune cells of a particular species or in animal models, for example, in human VISTA knock-in rodents or primates (e.g., humans or cynomolgus monkeys). [As shown below, the PK / PD ratio of the ADC according to the present invention has been shown to be remarkably high, namely at least 14:1 in VISTA knock-in rodents. Furthermore, since the expression of VISTA by different immune cells of rodents, humans, and primates is very similar, and furthermore, drug metabolism generally occurs much faster in rodents than in humans and non-human primates, it is expected that equivalent or better PK / PD ratios will be obtained in humans and non-human primates. While I do not wish to be bound by this theory, it is thought that the ADC in question internalizes a very large amount of a certain type of VISTA-expressing cells, because the surface VISTA expression of these immune cells is dense, which apparently produces a "depot effect," namely, the depot of internalized ADCs is metabolized very slowly, thereby providing a remarkably long-lasting release of therapeutically effective (anti-inflammatory) amounts of anti-inflammatory agents (e.g., steroids).

[0194] "Onset of efficacy" refers to the time it takes for the efficacy of a therapeutic agent, such as a steroid or ADC complex, to begin in vivo. In this invention, this can be detected in subjects administered with the steroid or ADC complex according to the present invention using known in vivo assays that detect the anti-inflammatory effect of steroids. As disclosed below, the ADC according to the present invention has been shown to have a rapid onset of efficacy, i.e., onset of efficacy in human VISTA knock-in rodents, i.e., onset of efficacy in about 2 hours.

[0195] As used herein, the terms “substantially similar” or “substantially identical” represent a sufficiently high degree of similarity between two numerical values ​​(generally one relating to the antibody of this disclosure and the other to the reference / comparative antibody) so that a person skilled in the art would consider the difference between the two values ​​to be of little biological and / or statistical significance with respect to the biological characteristics measured by the said value (e.g., the Kd value). The difference between the two values ​​may be less than about 50%, less than about 40%, less than about 30%, less than about 20%, or less than about 10%, depending on the value relating to the reference / comparative antibody.

[0196] "Isolated" polypeptides, antibodies, polynucleotides, vectors, cells, or compositions are polypeptides, antibodies, polynucleotides, vectors, cells, or compositions in a form not found in nature. Isolated polypeptides, antibodies, polynucleotides, vectors, cells, or compositions include those purified to such an extent that they are no longer found in nature. In some embodiments, isolated antibodies, polynucleotides, vectors, cells, or compositions are substantially pure.

[0197] As used herein, “substantially pure” means a material that is at least 50% pure (i.e., free from contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0198] The terms “immune complex,” “complex,” “antibody-drug complex,” or “ADC” as used herein refer to an anti-VISTA antibody or a fragment thereof linked to a compound or derivative thereof, an anti-inflammatory agent, such as a glucocorticosteroid agonist, and generally a linker mediating between them, with the general formula: (AI-LQ) nIt can be represented as -A, where AI = anti-inflammatory agent, generally a low molecular weight glucocorticoid receptor agonist, e.g., glucocorticosteroid (may include steroids according to formula 1), L = linker, Q = heterobifunctional group, heterotrifunctional group, or absent, and A = anti-VISTA antibody or VISTA-binding fragment that preferentially binds to human VISTA at physiological pH and generally has the aforementioned short pK, where n is an integer greater than 1, optionally 1 to 10. The immune complex is represented by the general formula in reverse: A-(QL-AI) n It can also be defined by:

[0199] In this disclosure, the term “linker” means any chemical moiety that can link an antibody or antibody fragment (e.g., an antigen-binding fragment) or functional equivalent to an anti-inflammatory drug, generally a glucocorticosteroid receptor agonist, such as a glucocorticosteroid. Linkers may be prone to cleavage (“cleavable linkers”), thereby facilitating the release of anti-inflammatory drugs such as glucocorticosteroids. For example, such cleavable linkers may be prone to acid-induced cleavage, photo-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage under conditions that allow glucocorticosteroids and / or antibodies to maintain activity before or after internalization into immune cells such as monocytes or bone marrow cells. Alternatively, linkers may be substantially resistant to cleavage (“non-cleavable linkers”).

[0200] Non-cleavable linkers include any chemical moiety that allows anti-inflammatory agents, such as glucocorticosteroids (glucocorticosteroid agonists), to be bound to antibodies in a stable covalent manner, and do not fall into the categories listed above for cleavable linkers. Therefore, non-cleavable linkers are substantially resistant to acid-induced cleavage, photo-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage. Furthermore, non-cleavage refers to the ability of chemical bonds in or adjacent to the linker to withstand cleavage induced by acids, photo-cleaving agents, peptidases, esterases, or chemical or physiological compounds that cleave disulfide bonds, provided that the glucocorticosteroid and / or antibody do not lose their activity before or after internalization into immune cells such as monocytes or bone marrow cells.

[0201] Some cleavable linkers are cleaved by peptidases ("peptidase-cleavable linkers"). Only certain peptides are readily cleaved intracellularly or extracellularly (see, e.g., Trout et al., 79 Proc. Natl. Acad. Sci. USA, 626-629 (1982) and Umemoto et al., 43 Int. J. Cancer, 677-684 (1989)). Furthermore, peptides consist of an α-amino acid unit and a peptide bond (chemically an amide bond between the carboxylate of one amino acid and the amino group of a second amino acid). Other amino bonds, such as the bond between the carboxylate and the α-amino acid group of lysine, are understood not to be peptide bonds and are considered non-cleavable.

[0202] Some linkers are cleaved by esterases ("esterase-cleavable linkers"). Only certain esters can be cleaved by esterases present inside or outside the cell. Esters are formed by the condensation of carboxylic acids and alcohols. Simple esters are esters produced from simple alcohols, such as aliphatic alcohols, as well as microcyclic and microaromatic alcohols.

[0203] In some embodiments, the cleavable linker component may include a peptide containing 1 to 10 amino acid residues. In these embodiments, the peptide enables protease cleavage of the linker, thereby facilitating the release of anti-inflammatory agents, such as glucocorticosteroids, upon exposure to intracellular proteases, such as lysosomal enzymes (Doronina et al. (2003) Nat. Biotechnol. 21:778-784). Exemplary peptides include, but are not limited to, dipeptides, tripeptides, tetrapeptides, and pentapeptides. Exemplary dipeptides include, but are not limited to, alanine-alanine (ala-ala), valine-citrulline (vc or val-cit), alanine-phenylalanine (af or ala-phe), phenylalanine-lysine (fk or phe-lys), phenylalanine-homolynise (phe-homolys), and N-methyl-valine-citrulline (Me-val-cit). Examples of tripeptides include, but are not limited to, glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly), as well as specific linkers identified in the “Exemplary Embodiments” section and embodied in Example 3 of this application.

[0204] Peptides may contain native and / or unnatural amino acid residues. The term “native amino acids” refers to Ala, Asp, Cys, Glu, Phe, Gly, His, He, Lys, Leu, Met, Asn, Pro, Gin, Arg, Ser, Thr, Val, Trp, and Tyr. “Unnatural amino acids” (i.e., amino acids that do not exist in nature) include, without limitation, homoserine, homoarginine, citrulline, phenylglycine, taurine, iodotyrosine, selenocysteine, norleucine ("Nle"), norvaline ("Nva"), β-alanine, L- or D-naphthalene, and ornithine ("Orn"). Peptides may be designed and optimized for enzymatic cleavage by specific enzymes, such as tumor-associated proteases, cathepsins B, C, and D, or plasmin proteases.

[0205] Amino acids include both natural and unnatural D-form amino acids. "D-" indicates an amino acid with a "D" (dextrorotatory) configuration, as opposed to the configuration in the natural ("L-") amino acid. Natural and unnatural amino acids are commercially available (Sigma Chemical Co., Advanced Chemtech) or can be synthesized using methods known in the art.

[0206] The term "drug-antibody ratio" or "DAR" refers to the number of radicals derived from glucocorticosteroids such as dexamethasone or budesonide bound to an anti-inflammatory agent or functional derivative (i.e., a small molecule glucocorticoid receptor agonist, e.g., A (anti-VISTA antibody or its antigen-binding fragment)). Therefore, the general formula is (AI-LQ). n In immune complexes having -A or vice versa, DAR is defined by the variable "n".

[0207] Formulas representing individual immune complexes (AI-LQ) n When referring to compounds having -A, DAR refers to the number of inflammatory agents or functional derivatives (e.g., small molecule glucocorticoid receptor agonists, e.g., glucocorticosteroids, e.g., dexamethasone or budesonide or a novel steroid of formula 1 linked to a specific A (e.g., n is an integer from 1 to 10)).

[0208] Formula representing multiple immune complexes (AI-LQ) nWhen referring to compounds having -A, DAR refers to the average number of anti-inflammatory agents or functional derivatives (e.g., low molecular weight glucocorticoid receptor agonists, e.g., glucocorticosteroids, e.g., dexamethasone or budesonide, or radicals derived from novel steroids of formula 1 (e.g., n is an integer or fraction between 1 and 10) linked to A). Thus, as an example, formula (AI-LQ) includes a first immune complex having 3 AI per A and a second immune complex having 4 AI per A. n Compounds containing -A will have a DAR of 3.5 (i.e., "n").

[0209] The term "subject" refers to any animal (e.g., mammal) that is the subject of a particular treatment, including but not limited to humans, non-human primates, and rodents. In this specification, the terms "subject" and "patient" are used interchangeably to refer to a human subject.

[0210] The term "pharmaceutical preparation" refers to a preparation in which the biological activity of the active ingredient is effective, and which does not contain additional ingredients that are unacceptably toxic to the recipient. The preparation may be sterile.

[0211] The “effective dose” of an ADC or glucocorticoid receptor agonist disclosed herein is an amount sufficient to perform the purpose specifically stated. The “effective dose” may be determined in relation to the purpose described.

[0212] The term "therapeutic effective dose" refers to the amount of immune complex or glucocorticoid receptor agonist that is effective in "treating" a disease or disorder in a subject or mammal. "Preventive effective dose" refers to the amount that is effective in achieving a desired preventive outcome.

[0213] Terms such as "treat," "cure," "treat," "alleviate," or "reduce" refer to therapeutic measures that cure, slow, reduce, and / or halt the progression of the symptoms of a diagnosed condition or disorder. Therefore, people who need treatment include those who have already been diagnosed with or are suspected of having a disorder. Preventive or protective measures refer to measures that prevent and / or delay the onset of a target condition or disorder. Therefore, people who need preventive or protective measures include those who are prone to having a disorder and those who should be prevented from having a disorder.

[0214] As used herein, “polynucleotide” or “nucleic acid” refers to a polymer of nucleotides of any length, including DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or analogs thereof, or any substrate that can be incorporated into the polymer by DNA or RNA polymerase. Polynucleotides may include modified nucleotides such as methylated nucleotides and their analogs. If present, modifications to the nucleotide structure may be conferred before or after the assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. Polynucleotides may be further modified after polymerization, for example, by complexing with labeling components. Other types of modifications include, for example, "caps" that substitute one or more naturally occurring nucleotides with analogs, internucleotide modifications, such as those by non-charged bonds (e.g., methylphosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and charged bonds (e.g., phosphorothioates, phosphorodithioates, etc.), such as those involving the pendant portion of proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those by insertors (e.g., acridine, psoralen, etc.), those by chelating agents (e.g., metals, radioactive metals, boron, metal oxides, etc.), those involving alkylating agents, those by modified bonds (e.g., α-anomeric nucleic acids, etc.), and the unmodified form of polynucleotides. Furthermore, any of the hydroxyl groups normally present in sugars can be substituted with, for example, phosphonic acid groups or phosphate groups, protected with standard protecting groups, or activated to prepare additional binding to additional nucleotides, or complexed with a solid support. The 5' and 3' terminal OH groups can be phosphorylated or substituted with amines or organic capping groups of 1 to 20 carbon atoms. Other hydroxyls can also be derivatized to standard protecting groups.Polynucleotides may also include analogous forms of ribose or deoxyribose sugars generally known in the art, such as 2'-O-methyl-, 2'-O-allyl, 2'-fluoro- or 2'-azid-ribose, carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars, such as arabinose, xylose, or lyxose, pyranose sugars, furanose sugars, sedoheptulose, acyclic analogs, and debasalized nucleoside analogs, such as methylriboside. One or more phosphodiester bonds may be replaced with alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which the phosphate is replaced by P(O)S ("thioate"), P(S)S ("dithioate"), (O)NR2 ("amidate"), P(O)R, P(O)OR', CO, or CH2 ("formacetal"), where each R or R' is independently H, or a substituted or unsubstituted alkyl (1-20C) (optionally including an ether (--O--) link), aryl, alkenyl, cycloalkyl, cycloalkenyl, or aralgyl. Not all linkings in the polynucleotide need to be identical. The foregoing description applies to all polynucleotides referred to herein, including RNA and DNA.

[0215] The term "vector" refers to a construct that can deliver and selectively express one or more genes or sequences of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensers, liposome-encapsulated DNA or RNA expression vectors, and certain eukaryotic cells, such as producer cells.

[0216] The terms “polypeptide,” “peptide,” and “protein” are used herein interchangeably to refer to polymers of amino acids of any length. The polymers may be linear or branched, may contain modified amino acids, or may be interrupted by non-amino acids. These terms also encompass amino acid polymers modified naturally or by intervention, e.g., disulfide bond formation, glycosylation, lipidization, acetylation, phosphorylation, or any other operation or modification, e.g., conjugation with a labeling component. Also included in this definition are polypeptides comprising one or more analogues of amino acids (including, e.g., non-natural amino acids), and other modifications known in the Art. Since the polypeptides of this disclosure are antibody-based, it will be understood that in certain embodiments, the polypeptides may exist as single-chain or conjugated chains.

[0217] The term “identical” or “percentage of identity” in relation to two or more nucleic acids or polypeptides refers to two or more sequences or subsequences that are identical or have a certain percentage of the same nucleotide or amino acid residues when compared and aligned (with gaps introduced as necessary) to obtain the greatest possible match, without considering conservative amino acid substitutions as part of sequence identity. The percentage of identity can be measured using sequence comparison software or algorithms, or by visual inspection. Various algorithms and software that can be used to obtain alignment of amino acid or nucleotide sequences are known in the art. One such non-restrictive example of a sequence alignment algorithm is the algorithm described in Karlin et al., Proc. Natl. Acad. Sci., 87:2264-2268 (1990), modified in Karlin et al., Proc. Natl. Acad. Sci., 90:5873-5877 (1993), and incorporated into the NBLAST and XBLAST programs (Altschul et al., Nucleic Acids Res., 25:3389-3402 (1991)). In certain embodiments, Gapped BLAST may be used, as described in Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997). BLAST-2, WU-BLAST-2 (Altschul et al., Methods in Enzymology, 266:460-480 (1996)), ALIGN, ALIGN-2 (Genentech, South San Francisco, Calif), or Megalign (DNASTAR) are additional publicly available software programs that can be used to align sequences. In certain embodiments, the percentage of identity between two nucleotide sequences is determined using the GAP program of GCG software (e.g., using the NWSgapdna.CMP matrix and gap weights of 40, 50, 60, 70, or 90 and length weights of 1, 2, 3, 4, 5, or 6).In certain alternative embodiments, the percentage of identity between two amino acid sequences can be determined using the GAP program within the GCG software package, which incorporates the algorithm of Needleman and Wunsch (J. Mol. Biol. (48): 444-453 (1970)) (e.g., using either the Blossum 62 matrix or the PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, or 5). Alternatively, in certain embodiments, the percentage of identity between nucleotides or amino acid sequences is determined using the algorithm of Myers and Miller (CABIOS, 4: 11-17 (1989)). For example, the percentage of identity can be determined using the ALIGN program (version 2.0) with PAM120, which includes a residue table with a gap length penalty of 12 and a gap penalty of 4. Appropriate parameters for maximum alignment by specific alignment software can be determined by those skilled in the art. In certain embodiments, the default parameters of the alignment software are used. In certain embodiments, the ratio "X" of the identity of the first amino acid sequence to the amino acids of the second sequence is calculated as 100 × (Y / Z), where Y is the number of amino acid residues scored as identical in the alignment of the first and second sequences (by visual inspection or alignment by a specific sequence alignment program), and Z is the total number of residues in the second sequence. If the length of the first sequence is longer than that of the second sequence, the ratio of the identity of the first sequence to the second sequence will be longer than the ratio of the identity of the second sequence to the first sequence.

[0218] As a non-limiting example, whether any particular polynucleotide has a certain percentage of sequence identity with respect to a reference sequence (e.g., at least 80% identical, at least 85% identical, at least 90% identical, and in some embodiments, at least 95%, 96%, 97%, 98%, or 99% identical) can, in certain embodiments, be determined using the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis53711). Bestfit uses the Smith and Waterman local homology algorithm (Advances in Applied Mathematics 2:482 489(1981)) to find the optimal segment of homology between two sequences. When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for example, 95% identical to a reference sequence in this disclosure, the parameters are set such that the percentage of identity is calculated over the entire length of the reference nucleotide sequence, and that a gap of up to 5% homology in the total number of nucleotides in the reference sequence is permitted.

[0219] In some embodiments, two nucleic acids or polypeptides of the Disclosure are substantially identical, meaning that when compared and aligned to obtain maximum agreement using a sequence comparison algorithm or by visual inspection, they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments, at least 95%, 96%, 97%, 98%, and 99% nucleotide or amino acid residue identity. The identity can exist over a region of the sequence whose length is at least about 10, about 20, about 40–60 residues, or any integer value between them, and over a region longer than 60–80 residues, for example, at least about 90–100 residues, and in some embodiments, the sequences are substantially identical over the entire length of the sequences being compared, such as the coding region of a nucleotide sequence.

[0220] A "conservative amino acid substitution" is a substitution in which one amino acid residue is replaced by another amino acid residue having a similar side chain. The family of amino acid residues having similar side chains is defined in the art and includes basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, the substitution of tyrosine with phenylalanine is a conservative substitution. In some embodiments, conserved substitutions in the sequences of the polypeptides and antibodies of this disclosure do not invalidate the binding of the antibody to an antigen(s), such as VISTA, which includes an amino acid sequence. Methods for identifying conserved nucleotide and amino acid substitutions that do not preclude antigen binding are well known in the art (e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).

[0221] As used herein, “substantially pure” means a material that is at least 50% pure (i.e., free from contaminants), more preferably at least 90% pure, more preferably at least 95% pure, more preferably at least 98% pure, and more preferably at least 99% pure.

[0222] "Host cells" include individual cells or cell cultures that may or may have been recipients of a vector(s) for incorporating polynucleotide inserts. Host cells include offspring of a single host cell, which may not necessarily be completely identical (in morphology or genomic DNA complement) to the original parent cell due to spontaneous, accidental, or intentional mutations. Host cells include cells that have been transfected in vivo with the polynucleotide(s) of the present invention.

[0223] The term “Fc region” is used to define the C-terminal region of an immunoglobulin heavy chain. The “Fc region” can be either a native sequence Fc region or a variant Fc region. While the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is typically defined as extending from the amino acid residue at position Cys226, or from Pro230, to its carboxyl terminus. The numbering of residues in the Fc region is based on EU index numbering, as in Kabat's study (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, Md., 1991). The Fc region of immunoglobulins generally contains two constant domains, CH2 and CH3.

[0224] As used herein, “Fc receptor” and “FcR” refer to receptors that bind to the Fc region of an antibody. Preferred FcRs are naturally occurring human FcRs. Furthermore, preferred FcRs are those that bind to IgG antibodies (gamma receptors) and include FcγRI, FcγRII, and FcγRII subclass receptors, which include allele variants and alternative splicing forms of these receptors. FcγRII receptors include FcγRIIA (“activating receptor”) and FcγRIIB (“inhibitory receptor”), which have similar amino acid sequences, differing primarily in their cytoplasmic domains. FcR is outlined in Ravetch and Kinet, 1991, Ann. Rev. Immunol., 9:457-92; Capel et al., 1994, ImmunoMethods, 4:25-34; and de Haas et al., 1995, J. Lab. Clin. Med., 126:330-41. "FcR" also includes FcRn, a neonatal receptor responsible for the transfer of maternal IgG to the fetus (Guyer et al., 1976, J. Immunol., 117:587; and Kim et al., 1994, J. Immunol., 24:249).

[0225] "Complement-dependent cytotoxicity" and "CDC" refer to the lysis of a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule (e.g., an antibody) conjugated with an alloantigen. To evaluate complement activation, a CDC assay, such as that described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), may be performed.

[0226] A “functional Fc region” possesses at least one “effector function” of a native sequence Fc region. Exemplary “effector functions” include C1q binding; complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and downregulation of cell surface receptors (e.g., B cell receptors; BCRs). Such effector functions generally require the combination of an Fc region with a binding domain (e.g., an antibody-variable domain), and such antibody effector functions can be evaluated using a variety of assays known in the art.

[0227] A “natural sequence Fc region” or “endogenous FcR” contains an amino acid sequence identical to the amino acid sequence of a naturally occurring Fc region. A “variant Fc region” contains an amino acid sequence different from the amino acid sequence of a natural sequence Fc region by at least one amino acid modification, while still retaining at least one effector function of the natural sequence Fc region. Preferably, a variant Fc region has at least one amino acid substitution compared to the natural sequence Fc region or the Fc region of the parent polypeptide, for example, about 1 to about 10 amino acid substitutions in the natural sequence Fc region or the Fc region of the parent polypeptide, and preferably about 1 to about 5 amino acid substitutions. The variant Fc regions described herein preferably have at least about 80% sequence identity with the natural sequence Fc region and / or the Fc region of the parent polypeptide, most preferably at least about 90% sequence identity with them, and more preferably at least about 95%, at least about 96%, at least about 97%, at least about 98%, and at least about 99% sequence identity with them.

[0228] As used herein, “antibody-dependent cell-mediated cytotoxicity” and “ADCC” refer to a cell-mediated response in which nonspecific cytotoxic cells expressing the Fc receptor (FcR) (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize antibodies bound to target cells, subsequently causing lysis of the target cells. The ADCC activity of a molecule of interest can be evaluated using in vitro ADCC assays, such as those described in U.S. Patent No. 5,500,362 or No. 5,821,337. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and NK cells. Alternatively, or in addition, the ADCC activity of a molecule of interest may be evaluated in vivo using animal models, such as the model disclosed in Clynes et al., 1998, PNAS USA 95:652-656.

[0229] In this disclosure, the term "halo" means -Cl, -F, -Br, or -I, either by itself or as part of another group. For example, halo is -Cl or -F.

[0230] In this disclosure, the term "hydroxy" means -OH when used by itself or as part of another group.

[0231] In this disclosure, the terms “thiol” or “sulfhydryl” refer to -SH when used by themselves or as part of another group.

[0232] In this disclosure, the term "alkyl" means, when used by itself or as part of another group, 1 to 12 carbon atoms, i.e., C 1-12 Alkyl, or a specified number of carbon atoms, i.e., C1 alkyl such as methyl, C2 alkyl such as ethyl, C3 alkyl such as propyl or isopropyl), C such as methyl, ethyl, propyl, or isopropyl 1-3 This refers to unsubstituted linear or branched aliphatic hydrocarbons containing alkyl groups. For example, alkyl groups are C 1-10It is alkyl. In another example, alkyl is C 1-6 It is alkyl. In another example, alkyl is C 1-4 It is alkyl. In another example, alkyl is a linear C1-10 alkyl. In yet another example, alkyl is a branched C 3-10 It is alkyl. In another example, alkyl is a linear C 1-6 It is alkyl. In another example, alkyl is a branched chain C 3-6 It is alkyl. In another example, alkyl is a linear C 1-4 It is alkyl. In another example, alkyl is a branched chain C 3-4 It is alkyl. In another example, alkyl is a linear or branched C chain. 3-4 It is alkyl. Non-definitive exemplary C 1-10 Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, iso-butyl, 3-pentyl, hexyl, heptyl, octyl, nonyl, and decyl. (Non-limiting exemplary C) 1-4 Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, and iso-butyl.

[0233] In this disclosure, the term “optionally substituted alkyl” means an alkyl group that, when used by itself or as part of another group, is either unsubstituted or substituted with one, two, or three substituents independently selected from the group consisting of nitro, hydroxy, cyano, haloalkoxy, aryloxy, alkylthio, sulfonamide, alkylcarbonyl, arylcarbonyl, alkylsulfonyl, arylsulfonyl, carboxy, carboxamide, alkoxycarbonyl, thiol, --N(H)C(=O)NH2, and -N(H)C=NH)NH2, which are optionally substituted with an aryl substituent and optionally substituted with a heteroaryl substituent. For example, an optionally substituted alkyl group is substituted with two substituents. In another example, an optionally substituted alkyl group is substituted with one substituent. In yet another example, an optionally substituted alkyl group is unsubstituted. Examples of non-exclusive substituted alkyl groups include -CH2OH, -CH2SH, -CH2Ph, -CH2(4-OH)Ph, -CH2(imidazolyl), -CH2CH2CO2H, -CH2CH2SO2CH3, -CH2CH2COPh, and -CH2OC(=O)CH3.

[0234] In this disclosure, the term "cycloalkyl" means, when used by itself or as part of another group, a group having 3 to 12 carbon atoms, i.e., C 3-12 A cycloalkyl refers to an unsubstituted, saturated or partially unsaturated cyclic aliphatic hydrocarbon containing one to three rings having a specified number of carbon atoms, for example, containing one or two double bonds. In one example, a cycloalkyl has two rings. In another example, a cycloalkyl has one ring. In yet another example, a cycloalkyl is saturated. In yet another example, a cycloalkyl is unsaturated. In yet another example, a cycloalkyl has C 3-8 It is a cycloalkyl. In another example, a cycloalkyl is C 3-6These are cycloalkyl groups. The term "cycloalkyl" means that the group includes a ring -CH2- substituted with -C(=O)-. Non-exclusive exemplary cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, norbornyl, decalin, adamantyl, cyclohexenyl, cyclopentenyl, and cyclopentanone.

[0235] In this disclosure, when used by itself or as part of another group, the term “optionally substituted cycloalkyl” means a cycloalkyl that is either unsubstituted or substituted with one, two, or three substituents independently selected from the group consisting of halo, nitro, cyano, hydroxy, alkylcarbonyloxy, cycloalkylcarbonyloxy, amino, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, aryloxy, aralkyloxy, alkylthio, carboxamide, sulfonamide, alkylcarbonyl, arylcarbonyl, alkylsulfonyl, arylsulfonyl, carboxy, carboxyalkyl, optionally substituted alkyl, optionally substituted cycloalkyl, alkenyl, alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclo, alkoxyalkyl, (amino)alkyl, (carboxamide)alkyl, (heterocyclo)alkyl, and -OC(=O)-amino. The term "optionally substituted cycloalkyl" includes cycloalkyl groups having an optionally substituted condensed aryl group, e.g., phenyl, or an optionally substituted condensed heteroaryl group, e.g., pyridyl. Optionally substituted cycloalkyl groups having an optionally substituted condensed aryl group or an optionally substituted condensed heteroaryl group can be bonded to the rest of the molecule at any available carbon atoms on the cycloalkyl ring. In one example, the optionally substituted cycloalkyl group is substituted with two substituents. In another example, the optionally substituted cycloalkyl group is substituted with one substituent. In yet another example, the optionally substituted cycloalkyl group is unsubstituted.

[0236] In this disclosure, the term "aryl," when used by itself or as part of another group, refers to an unsubstituted monocyclic or bicyclic aromatic ring system having 6 to 14 carbon atoms, i.e., C 6-14 This refers to aryl groups. Non-exclusive exemplary aryl groups include phenyl (abbreviated as "Ph"), naphthyl, phenanthryl, anthrasyl, indenyl, azlenyl, biphenyl, biphenylenyl, and fluorenyl groups. For example, the aryl group is phenyl or naphthyl.

[0237] In this disclosure, when used as itself or as part of another group, the term “optionally substituted aryl” means either unsubstituted or aryl that is substituted with 1 to 5 substituents independently selected from the group consisting of halo, nitro, cyano, hydroxy, thiol, amino, alkylamino, dialkylamino, optionally substituted alkyl, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, aryloxy, aralkyloxy, alkylthio, carboxamide, sulfonamide, alkylcarbonyl, arylcarbonyl, alkylsulfonyl, haloalkylsulfonylcycloalkylsulfonyl, (cycloalkyl)alkylsulfonyl, arylsulfonyl, heteroarylsulfonyl, heterocyclosulfonyl, carboxy, carboxyalkyl, optionally substituted cycloalkyl, alkenyl, alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclo, alkoxycarbonyl, alkoxyalkyl, (amino)alkyl, (carboxamide)alkyl, and (heterocyclo)alkyl.

[0238] In one example, an arbitrarily substituted aryl is an arbitrarily substituted phenyl. In another example, an arbitrarily substituted phenyl has four substituents. In yet another example, an arbitrarily substituted phenyl has three substituents. In yet another example, an arbitrarily substituted phenyl has two substituents. In yet another example, an arbitrarily substituted phenyl has one substituent. In yet another example, an arbitrarily substituted phenyl is unsubstituted. Examples of non-limiting exemplary substituted aryl groups include 2-methylphenyl, 2-methoxyphenyl, 2-fluorophenyl, 2-chlorophenyl, 2-bromophenyl, 3-methylphenyl, 3-methoxyphenyl, 3-fluorophenyl, 3-chlorophenyl, 4-methylphenyl, 4-ethylphenyl, 4-methoxyphenyl, 4-fluorophenyl, 4-chlorophenyl, 2,6-di-fluorophenyl, 2,6-di-chlorophenyl, 2-methyl, 3-methoxyphenyl, 2-ethyl, 3-methoxyphenyl, 3,4-di-methoxyphenyl, 3,5-di-fluorophenyl, 3,5-dimethylphenyl, 3,5-dimethoxy, 4-methylphenyl, 2-fluoro-3-chlorophenyl, 3-chloro-4-fluorophenyl, and 4-(pyridine-4-ylsulfonyl)phenyl. The term optionally substituted aryl includes phenyl groups having optionally substituted condensed cycloalkyl groups or optionally substituted condensed heterocyclo groups. An optionally substituted phenyl having an optionally substituted condensed cycloalkyl group or an optionally substituted condensed heterocyclo group can be bonded to the rest of the molecule at any available carbon atoms on the phenyl ring.

[0239] In this disclosure, the term “alkenyl,” used either by itself or as part of another group, refers to an alkyl group containing one, two, or three carbon-carbon double bonds. In one example, an alkenyl has one carbon-carbon double bond. In another example, an alkenyl has C 2-6 It is an alkenil. In another example, an alkenil is C 2-4 These are alkenyl groups. Non-exclusive exemplary alkenyl groups include ethenyl, propenyl, isopropenyl, butenyl, sec-butenyl, pentenyl, and hexenyl.

[0240] In this disclosure, the term “optionally substituted alkenyl” as used herein, either by itself or as part of another group, means an alkenyl that is either unsubstituted or substituted with one, two, or three substituents independently selected from the group consisting of halo, nitro, cyano, hydroxy, amino, alkylamino, dialkylamino, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, aryloxy, aralkyloxy, alkylthio, carboxamide, sulfonamide, alkylcarbonyl, arylcarbonyl, alkylsulfonyl, arylsulfonyl, carboxy, carboxyalkyl, optionally substituted alkyl, optionally substituted cycloalkyl, alkenyl, alkynyl, optionally substituted aryl, heteroaryl, and optionally substituted heterocyclo.

[0241] In this disclosure, the term “alkynyl,” used either by itself or as part of another group, refers to an alkyl group containing one to three carbon-carbon triple bonds. In one example, an alkynyl has one carbon-carbon triple bond. In another example, an alkynyl has C 2-6 In another example, alkynyl is C 2-4 These are alkynyl groups. Non-exclusive exemplary alkynyl groups include ethynyl, propynyl, butynyl, 2-butynyl, pentynyl, and hexynyl groups.

[0242] In this disclosure, the term “optionally substituted alkynyl” as used herein, either by itself or in part, means an alkynyl that is either unsubstituted or substituted with one, two, or three substituents independently selected from the group consisting of halo, nitro, cyano, hydroxy, amino, alkylamino, dialkylamino, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, aryloxy, aralkyloxy, alkylthio, carboxamide, sulfonamide, alkylcarbonyl, arylcarbonyl, alkylsulfonyl, arylsulfonyl, carboxy, carboxyalkyl, optionally substituted alkyl, cycloalkyl, alkenyl, alkynyl, optionally substituted aryl, optionally substituted heteroaryl, and heterocyclo.

[0243] In this disclosure, the term “haloalkyl” means an alkyl group substituted with one or more fluorine, chlorine, bromine, and / or iodine atoms, either by itself or as part of another group. In one example, the alkyl group is substituted with one, two, or three fluorine and / or chlorine atoms. In another example, the haloalkyl group is C 1-4 These are haloalkyl groups. Examples of non-exclusive haloalkyl groups include fluoromethyl, 2-fluoroethyl, difluoromethyl, trifluoromethyl, pentafluoroethyl, 1,1-difluoroethyl, 2,2-difluoroethyl, 2,2,2-trifluoroethyl, 3,3,3-trifluoropropyl, 4,4,4-trifluorobutyl, and trichloromethyl groups.

[0244] In this disclosure, the term “alkoxy” means, when used by itself or as part of another group, an optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted alkenyl, or optionally substituted alkynyl, bonded to a terminal oxygen atom. For example, an alkoxy is an optionally substituted alkyl bonded to a terminal oxygen atom. For example, an alkoxy group is a C bonded to a terminal oxygen atom. 1-6 It is alkyl. In another example, an alkoxy group is a C bonded to the terminal oxygen atom.1-4 It is an alkyl group. Non-limiting exemplary alkoxy groups include methoxy, ethoxy, and tert-butoxy.

[0245] In this disclosure, the term “alkylthio” means an optionally substituted alkyl group bonded to a terminal sulfur atom, either by itself or as part of another group. For example, an alkylthio group is C 1-4 These are alkylthio groups. Non-exclusive exemplary alkylthio groups include -SCH3 and -SCH2CH3-.

[0246] In this disclosure, the term “haloalkoxy” refers to a haloalkyl group bonded to a terminal oxygen atom, whether used as a whole or as part of another group. Non-exclusive exemplary haloalkoxy groups include fluoromethoxy, difluoromethoxy, trifluoromethoxy, and 2,2,2-trifluoroethoxy.

[0247] In this disclosure, the term “heteroaryl” means an unsubstituted monocyclic and bicyclic aromatic ring system having 5 to 14 ring atoms, i.e., a 5 to 14-membered heteroaryl, in which at least one carbon atom of one of the rings is substituted with a heteroatom independently selected from the group consisting of oxygen, nitrogen, and sulfur. In one example, the heteroaryl contains 1, 2, 3, or 4 heteroatoms independently selected from the group consisting of oxygen, nitrogen, and sulfur. In one example, the heteroaryl has 3 heteroatoms. In another example, the heteroaryl has 2 heteroatoms. In another example, the heteroaryl has 1 heteroatom. In another example, the heteroaryl is a 5 to 10-membered heteroaryl. In another example, the heteroaryl is a 5 to 6-membered heteroaryl. In another example, the heteroaryl has 5 ring atoms, for example, thienyl, i.e., a 5-membered heteroaryl having 4 carbon atoms and 1 sulfur atom. In another example, a heteroaryl group has six ring atoms, for example, pyridyl, which is a six-membered heteroaryl having five carbon atoms and one nitrogen atom. Non-restrictive exemplary heteroaryl groups include thienyl, benzo[b]thienyl, naphtho[2,3-b]thienyl, thianthrenyl, furyl, benzofuryl, pyranyl, isobenzofuranyl, benzoxazonyl, clomenyl, xanthenyl, 2H-pyrrolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridadinyl, isoindolyl, 3H-indolyl, and indolyl Examples include indazolyl, prinyl, isoquinolyl, quinolyl, phthalazinyl, naphthilidinyl, sinnolinyl, quinazolinyl, pteridinyl, 4aH-carbazolyl, carbazolyl, β-carbolinyl, phenanthiazolyl, acridinyl, pyrimidinyl, phenanthrolinyl, phenadinyl, thiazolyl, isothiazolyl, phenothiazolyl, isoxazolyl, flazanil, and phenoxadinyl.For example, heteroaryls include thienyl (e.g., thien-2-yl and thien-3-yl), furyl (e.g., 2-furyl and 3-furyl), pyrrolyl (e.g., 1H-pyrrole-2-yl and 1H-pyrrole-3-yl), imidazolyl (e.g., 2H-imidazole-2-yl and 2H-imidazole-4-yl), pyrazolyl (e.g., 1H-pyrazole-3-yl, 1H-pyrazole-4-yl, and 1H-pyrazole-5-yl), pyridyl (e.g., pyridine-2-yl, pyridine-3-yl, and pyridine-4-yl), pyrimidinyl (e.g., pyrimidine-2-yl, The selection is made from the group consisting of pyrimidine-4-yl and pyrimidine-5-yl), thiazolyl (e.g., thiazole-2-yl, thiazole-4-yl, and thiazole-5-yl), isothiazolyl (e.g., isothiazole-3-yl, isothiazole-4-yl, and isothiazole-5-yl), oxazolyl (e.g., oxazole-2-yl, oxazole-4-yl, oxazole-5-yl), isoxazolyl (e.g., isoxazole-3-yl, isoxazole-4-yl, and isoxazole-5-yl), and indazolyl (e.g., 1H-indazole-3-yl). The term "heteroaryl" also means that it may include possible N-oxides. A non-limiting exemplary N-oxide is pyridyl N-oxide.

[0248] In one example, a heteroaryl is a five-membered or six-membered heteroaryl. In one example, a heteroaryl is a five-membered heteroaryl, that is, a heteroaryl is a monocyclic aromatic ring system having five ring atoms, in which at least one carbon atom of the ring is substituted with a heteroatom independently selected from nitrogen, oxygen, and sulfur. Non-limiting exemplary five-membered heteroaryl groups include thienyl, furyl, pyrrolyl, oxazolyl, pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, and isoxazolyl. In another example, a heteroaryl is a six-membered heteroaryl, for example, a heteroaryl is a monocyclic aromatic ring system having six ring atoms, in which at least one carbon atom of the ring is substituted with a nitrogen atom. Non-limiting exemplary six-membered heteroaryl groups include pyridyl, pyrazinyl, pyrimidinyl, and pyridadinyl.

[0249] In this disclosure, when used as itself or as part of another group, the term “optionally substituted heteroaryl” means a heteroaryl that is either unsubstituted or substituted with one, two, three, or four substituents independently selected from the group consisting of halo, nitro, cyano, hydroxy, amino, alkylamino, dialkylamino, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, aryloxy, aralkyloxy, alkylthio, carboxamide, sulfonamide, alkylcarbonyl, arylcarbonyl, alkylsulfonyl, haloalkylsulfonyl, cycloalkylsulfonyl, (cycloalkyl)alkylsulfonyl, arylsulfonyl, heteroarylsulfonyl, carboxy, carboxyalkyl, optionally substituted alkyl, optionally substituted cycloalkyl, alkenyl, alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclo, alkoxyalkyl, (amino)alkyl, (carboxamide)alkyl, and (heterocyclo)alkyl. In one example, an optionally substituted heteroaryl has one substituent. In another example, an optionally substituted heteroaryl is unsubstituted. Any available carbon or nitrogen atom can be substituted. The term optionally substituted heteroaryl includes groups having an optionally substituted condensed cycloalkyl or optionally substituted condensed heterocyclo group. Optionally substituted heteroaryls having an optionally substituted condensed cycloalkyl or optionally substituted condensed heterocyclo group can be bonded to the rest of the molecule with any available carbon atoms on the heteroaryl ring.

[0250] In this disclosure, the term “heterocyclo” means, when used by itself or as part of another group, a cyclic group comprising 1, 2, or 3 rings having 3 to 14 ring members, i.e., 3 to 14-membered heterocyclo, wherein at least one carbon atom of one of the rings is substituted with a heteroatom, and which is unsubstituted, saturated or partially unsaturated, and includes, for example, one or two double bonds. Each heteroatom is independently selected from the group consisting of oxygen, sulfur including sulfoxides and sulfones, and / or nitrogen atoms (which may be oxidized or quaternized). The term “heterocyclo” also includes groups in which the ring -CH2- is replaced with -C(=O)-, such as cyclic ureido groups like 2-imidazolidinone and cyclic amide groups like β-lactam, γ-lactam, δ-lactam, ε-lactam, and piperazine-2-one. The term "heterocyclo" also includes groups having optionally substituted condensed aryl groups, such as indolinyl or chroman-4-yl. In one embodiment, the heterocyclo group is C 4-6 A heterocyclo is a four-membered, five-membered, or six-membered cyclic group containing one ring and one or two oxygen and / or nitrogen atoms. In one embodiment, the heterocyclo group contains one ring and one nitrogen atom. 4-6 Heterocycloidal groups are heterocycloidal groups that can be optionally bonded to the rest of a molecule through available carbon or nitrogen atoms. Non-restrictive exemplary heterocycloidal groups include azetidinyl, dioxanyl, tetrahydropyranyl, 2-oxopyrrolidine-3-yl, piperazine-2-one, piperazine-2,6-dione, 2-imidazolidinone, piperidinyl, morpholinyl, piperazinyl, pyrrolidinyl, and indolinyl.

[0251] In this disclosure, as used by itself or as part of another group, the term “optionally substituted heterocyclo” means a heteroaryl that is either unsubstituted or substituted with one, two, three, or four substituents independently selected from the group consisting of halo, nitro, cyano, hydroxy, amino, alkylamino, dialkylamino, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, aryloxy, aralkyloxy, alkylthio, carboxamide, sulfonamide, alkylcarbonyl, cycloalkylcarbonyl, alkoxycarbonyl, CF3C(=O)-, arylcarbonyl, alkylsulfonyl, arylsulfonyl, carboxy, carboxyalkyl, alkyl, optionally substituted cycloalkyl, alkenyl, alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclo, alkoxyalkyl, (amino)alkyl, (carboxamide)alkyl, or (heterocyclo)alkyl. The substitution may occur at any available carbon or nitrogen atom, or both.

[0252] In this disclosure, the term "amino" as used by itself or as part of another group means formula NRO a R b It refers to the radical, and in the formula, R a and R b Each of these is independently selected from the group consisting of hydrogen, optionally substituted alkyl, and aralkyl, or R a and R b These groups combine to form 3- to 8-membered, optionally substituted heterocycloids. Non-limiting exemplary amino groups include -NH2 and -N(H)(CH3).

[0253] In this disclosure, the term "carboxamide," used either by itself or as part of another group, refers to the formula -C(=O)NR a R b It refers to the base of, and in the formula, R a and R bEach is independently selected from the group consisting of hydrogen, optionally substituted alkyl, hydroxyalkyl, optionally substituted aryl, optionally substituted heterocyclo, and optionally substituted heteroaryl, or R a and R b These, together with the nitrogen to which they are bonded, form a 3- to 8-membered optionally substituted heterocyclo group. In one embodiment, R a and R b Each is independently a hydrogen atom or an optionally substituted alkyl group. In one embodiment, R a and R b These, together with the nitrogen to which they are bonded, form a 3- to 8-membered, optionally substituted heterocyclo group. Non-exclusive exemplary carboxyamide groups include -CONH2, -CON(H)CH3, and -CON(CH3)2.

[0254] In this disclosure, the term “alkoxycarbonyl” means a carbonyl group substituted with an alkoxy group, i.e., -C(=O)-, when used by itself or as part of another group. In one embodiment, the alkoxy is C 1-4 It is an alkoxy group. Non-exclusive exemplary alkoxycarbonyl groups include -C(=O)OMe, -C(=O)OEt, and -C(=O)OtBu.

[0255] In this disclosure, the term "carboxyl" means the radical of formula -CO2H, whether used by itself or as part of another group.

[0256] In this disclosure, the terms “autodestructive group” or “autodestructive linker” refer to all or part of a cleavable linker comprising a bifunctional chemical moiety that can covalently link two spaced chemical moieties to a normally stable tripartite molecule, which can release one of the chemical moieties detached from the tripartite molecule by enzymatic cleavage; and, following enzymatic cleavage, can spontaneously cleave from the rest of the molecule to release the other detached chemical moiety, such as a glucocorticosteroid. In some embodiments, the autodestructive linker comprises a p-aminobenzyl unit. In some such embodiments, the p-aminobenzyl alcohol is bonded to the amino acid unit via an amide bond, and a carbamate, methylcarbamate, or carbonate is formed between the benzyl alcohol and the drug (Hamann et al. (2005) Expert Opin. Ther. Patents (2005) 15:1087-1103). In some embodiments, the self-destructing linker is p-aminobenzyloxycarbonyl (PAB). (See Example 3 and the Exemplary Embodiments section of this application.)

[0257] In this disclosure, the terms “protecting group” or “PG” refer to a group that blocks, or protects, a functional group, such as an amine functional group, while a reaction is taking place on another functional group or part of a molecule. Those skilled in the art will be familiar with the selection, bonding, and cleavage of amine protecting groups and will understand that many different protecting groups are known in the art and that the suitability of one protecting group or another depends on the particular planned synthetic scheme. A paper on this subject, e.g., Wuts, PGM; Greene, TW, “Greene’s Protective Groups in Organic Synthesis”, 4th Ed., J. Wiley & Sons, NY, 2007, is available for reference. Suitable protecting groups include carbobenzyloxy (Cbz), tert-butyloxycarbonyl (BOC), 9-fluorenylmethyloxycarbonyl (FMOC), and benzyl (Bn) groups. In one embodiment, the protecting group is a Boc group.

[0258] As used in this disclosure and claims, the singular forms "a," "an," and "the" include the plural form unless the context explicitly indicates otherwise.

[0259] Whenever an embodiment is described in this specification using the word “including,” it is understood that similar embodiments, otherwise described using the terms “consisting of” and / or “essentially consisting of,” are also provided.

[0260] In this specification, the term "and / or" as used in phrases such as "A and / or B" is intended to include both "A and B", "A or B", and "A" and "B". Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to include each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0261] As used herein, “autoimmunity” or “autoimmune disease or condition” broadly refers to and includes diseases or disorders that arise from and attack the body’s own tissues, coisoliths or signs thereof, or conditions arising therefrom. In this specification, autoimmune conditions include inflammatory or allergic conditions, chronic diseases characterized by a host immune response to autoantigens potentially associated with tissue destruction, such as rheumatoid arthritis characterized by inflammation, and / or steroids are effective treatments.

[0262] As used herein, “immune cells” broadly refers to cells of hematopoietic origin that play a role in the immune response. Immune cells include, but are not limited to, lymphocytes, such as B cells and T cells; natural killer cells; dendritic cells, and myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.

[0263] As used herein, “immune-related disorders (or conditions or conditions)” should be understood to encompass any disease disorder or condition selected from the group including, but not limited to, autoimmune diseases, inflammatory disorders, and graft rejection, such as acute and chronic rejection of organ transplants, allogeneic stem cell transplants, autologous stem cell transplants, and bone marrow transplants, as well as graft-versus-host diseases.

[0264] As used herein, “inflammatory disease,” “inflammatory condition,” and / or “inflammation” refer broadly to chronic or acute inflammatory diseases, and expressly include inflammatory autoimmune diseases and inflammatory allergic conditions. These conditions include, for example, inflammatory abnormalities characterized by dysregulation of the immune response to harmful stimuli such as pathogens, damaged cells, or irritants. Inflammatory diseases underlie a wide variety of human diseases. Non-immune diseases caused by inflammatory processes include cancer, atherosclerosis, and ischemic heart disease. Examples of inflammation-related disorders include: chronic prostatitis, glomerulonephritis, hypersensitivity, pelvic inflammatory disease, reperfusion injury, sarcoidosis, vasculitis, interstitial cystitis, hypocomplementary urticarial vasculitis, pericarditis, myositis, anti-synthetase syndrome, scleritis, macrophage activation syndrome, Behçet's syndrome, PAPA syndrome, Blau syndrome, gout, adult and juvenile Still's disease, cryopyrin-associated periodic fever syndromes, Macklewells syndrome, and familial cold-induced autoitis. Examples include inflammatory skin diseases selected from the group consisting of symptomatic syndromes, neonatal multiorgan inflammatory diseases, familial Mediterranean fever, chronic infantile neurological, cutaneous and joint syndromes, systemic juvenile idiopathic arthritis, hyper-IgD syndrome, Schnitzler syndrome, TNF receptor-associated periodic syndromes (TRAPSPs), gingivitis, periodontitis, hepatitis, cirrhosis, pancreatitis, myocarditis, vasculitis, gastritis, gout, gouty arthritis, as well as psoriasis, atopic dermatitis, eczema, rosacea, urticaria, and acne.

[0265] As used herein, “mammal” broadly refers to any and all warm-blooded vertebrates of the mammalian class, including humans, characterized by a hairy skin and, in females, milk-producing mammary glands for raising offspring. Examples of mammals include, but are not limited to, alpacas, armadillos, capybaras, cats, camels, chimpanzees, chinchillas, cattle, dogs, goats, gorillas, hamsters, horses, humans, lemurs, llamas, mice, non-human primates, pigs, rats, sheep, shrews, squirrels, tapirs, and voles. Mammals include, but are not limited to, Bovidae, Canidae, Equidae, Felidae, Muridae, Ampidae, Swineidae, Primates, and Rodents. Mammals also include any and all mammals listed in the World Mammals List maintained by the Smithsonian Museum of Natural History in Washington, D.C.

[0266] The terms “patient,” “subject,” “recipient,” “individual,” or “individual being treated” are used herein interchangeably and broadly refer to any animal requiring treatment to alleviate a disease condition or to prevent the onset or recurrence of a disease condition. “Patient,” as used herein, broadly refers to any animal having risk factors, medical history, susceptibility, symptoms, and signs, and having been previously diagnosed with the disease, at risk of the disease, or being part of a patient population of the disease. Patients may be clinical patients such as humans, or animal patients such as companion animals, livestock, farm animals, exotic animals, or zoo animals.

[0267] In this specification, “subject,” “patient,” or “individual” in relation to treatment or diagnosis includes any human or non-human animal. The term “non-human animal” includes all vertebrates, e.g., mammals and non-mammals, e.g., non-human primates, sheep, dogs, cats, horses, cattle, chickens, amphibians, and reptiles. That is, subjects suitable for treatment by the present invention include, but are not limited to, birds and mammals, and are preferably mammals. Any mammalian subject that requires treatment according to the present invention is suitable. Human subjects of both sexes and any developmental stage (i.e., neonates, infants, juveniles, adolescents, and adults) can be treated according to the present invention. The present invention may also be carried out in animal subjects, particularly mammals such as mice, rats, dogs, cats, cattle, goats, sheep, and horses, for veterinary purposes and for drug screening and drug development purposes. “Subject” is used synonymously with “individual” and “patient.”

[0268] As used herein, “treatment,” “therapy,” “to treat,” or “treatment” broadly means treating a disease, stopping or reducing the progression of a disease or its clinical symptoms, and / or alleviating a disease and causing regression of a disease or its clinical symptoms. Treatment encompasses providing prevention, treatment, restoration, reduction, mitigation, and / or relief of a disease, its signs, and / or symptoms. Treatment encompasses reducing signs and / or symptoms in patients with signs and / or symptoms of a disease in progress (e.g., inflammation, pain). Treatment also encompasses “prevention.” The term “reduced” broadly means a clinically significant reduction of signs and / or symptoms for therapeutic purposes. Treatment includes treating signs and / or symptoms of relapse or recurrence (e.g., inflammation, pain). Treatment includes, but is not limited to, preventing the onset of signs and / or symptoms, as well as reducing existing signs and / or symptoms, and eliminating existing signs and / or symptoms. Treatment includes treating chronic diseases ("maintenance") and acute diseases. For example, treatment includes treating signs and / or symptoms (e.g., inflammation, pain) or preventing their recurrence or relapse.

[0269] After defining the specific terms and phrases used in this application, the anti-VISTA antibody and antigen-binding antibody fragments included in the present invention, as well as their manufacturing and use methods, will be further described below.

[0270] The present invention relates to an ADC comprising an antibody or antibody fragment containing an antigen-binding region that binds to the V-domain Ig suppressor (VISTA) of T cell activation, wherein the antibody or fragment has a short serum half-life under physiological pH conditions (approximately pH 7.5), for example, the serum half-life of the antibody or fragment in rodents (human VISTA knock-in) is generally 1-72 hours, 1-32 hours, 1-16 hours, 1-8 hours, 1-4 hours, or 1-2 hours ± 0.5 hours under physiological conditions (approximately pH 7.5) in human VISTA knock-in rodents, or approximately 3.5, 3, 2.5, or 2.3 days ± 0.5 days in primates (cynomolgus monkeys), and this anti-human VISTA antibody or antibody fragment binds directly or indirectly via a linker to an anti-inflammatory agent, such as the aforementioned steroid or This refers to corticosteroid receptor agonists, or more specifically, dexamethasone, prednisolone, budesonide, beclomethasone, betamethasone, cortisol, cortisone acetate, 16-α-hydroxyprednisolone, dexamethasone, difluorazone, flumetazone, flunisolide, fluocinolone acetonide, fluticasone propionate, ciclesonide, methylprednisolone, prednisone, prednisolone, mometasone, triamcinolone acetonide, etc., or radicals derived therefrom, or novel steroids of formula 1 disclosed herein) or their functional derivatives or radicals, i.e., derivatives that, when administered to a subject, e.g., human or other mammal, are released from the contained ADC upon internalization into immune cells, thereby inducing a desired anti-inflammatory effect.

[0271] In particular, ADCs specifically bind to VISTA-expressing immune cells at physiological pH, and anti-inflammatory agents are released from the ADCs and internalized by target (immune) cells, such as neutrophils, monocytes, bone marrow cells, T cells, and other immune cells present in peripheral blood. Anti-inflammatory agents, such as corticosteroid receptor agonists, e.g., dexamethasone, prednisolone, budesonide, beclomethasone, betamethasone, cortisol, cortisone acetate, 16-α-hydroxyprednisolone, dexamethasone, difluorazone, flumetazone, flunisolide, fluocinolone acetonide, fluticasone propionate, ciclesonide, methylprednisolone, prednisone, prednisolone, mometasone, triamcinolone acetonide, etc., or radicals derived therefrom, or novel steroids of Formula 1 disclosed herein, or their functional derivatives or radicals, i.e., when the derivatives are released from the ADCs containing them upon internalization into immune cells, induce the desired anti-inflammatory effect. Such release may occur outside the target cell or after the internalization of the ADCs into the target immune cells. Most commonly, the cleavage and release of anti-inflammatory agents occur intracellularly. As mentioned above, the efficacy (anti-inflammatory activity) of the anti-inflammatory agents contained in the subject ADC is achieved only after such steroid compounds or ADCs containing them are internalized by immune cells.

[0272] In preferred embodiments, the anti-VISTA antibody or fragment comprises a silent, i.e., mutated Fc region that impairs FcR binding, such as silent IgG1, IgG2, IgG3, or IgG4, most commonly silent IgG2 or silent IgG1; or the antibody or fragment may lack an Fc region or comprise an Fc fragment that does not bind to FcR. Exemplary silent Fc regions are disclosed below. Thereafter, the ADC comprising the anti-VISTA antibody or fragment binds to and internalizes VISTA-expressing immune cells, while generally not inducing a modulating effect on VISTA, i.e., not agonizing or antagonizing the VISTA-mediated effect on immunity. Rather, the therapeutic effect induced by ADCs is due to the anti-inflammatory agent(s) conjugated to them, such as the aforementioned corticosteroid receptor agonists or corticosteroids, such as dexamethasone, prednisolone, budesonide, beclomethasone, betamethasone, cortisol, cortisone acetate, 16-α-hydroxyprednisolone, dexamethasone, difluorazone, flumethasone, flunisolide, fluocinolone acetonide, fluticasone propionate, ciclesonide, methylpre When a functional derivative or radical is included in an ADC, a functional glucocorticosteroid that induces a desired anti-inflammatory effect is released upon internalization into immune cells, such as donisolone, prednisone, mometasone, triamcinolone acetonide, or any novel steroid of Formula 1 disclosed herein, or any of the aforementioned functional derivatives or radicals, either alone or primarily due to these, i.e., when the derivative is included in the ADC, the desired anti-inflammatory effect is induced either alone or preferentially upon internalization of the anti-inflammatory agent(s) into the target immune cells.

[0273] Because the subject ADC selectively binds to immune cells, such as myeloid cells, T cells, neutrophils, and monocytes, the subject ADC is potent against many immune cells but will mitigate or prevent adverse side effects (which can occur when the drug is internalized in non-target cells) that can still be induced by many anti-inflammatory agents, such as corticosteroid receptor agonists, such as dexamethasone and other steroids. Furthermore, because the subject ADC selectively binds to naive and activated target VISTA-expressing immune cells, such as monocytes, macrophages, T cells, Tregs, CD4 T cells, CD8 T cells, neutrophils, and myeloid cells, the ADC may potentially facilitate the reduced use of corticosteroid receptor agonists, such as dexamethasone and other steroids previously identified herein. In addition, the subject ADC may be used to treat conditions in which any or all of these specific types of immune cells are involved in disease pathology.

[0274] In fact, the subject ADC has a unique combination advantage in targeting and directing the internalization of anti-inflammatory agents compared to previously reported ADCs, particularly those that affect the internalization of steroids into immune cells, such as those targeting CD74, CD163, TNF, and PRLR; this is due to the combination of the advantages of VISTA as an ADC target and the specific properties of the anti-VISTA antibody contained in the subject ADC (i.e., binding to VISTA-expressing immune cells at physiological pH and having a very short pK).

[0275] In particular, the subject ADC binds to immune cells that express VISTA at very high densities, and despite their very short PK, it remains effective for a long period (inducing anti-inflammatory activity), and is therefore well-suited for the long-term treatment of chronic inflammatory or autoimmune diseases, and repeated administration over a long period is therapeutically justified.

[0276] Furthermore, because the ADCs in this subject target a wide range of immune cells, including neutrophils, bone marrow cells, T cells, and endothelial cells, the ADCs in this subject may be used to treat diseases such as inflammatory or autoimmune diseases, as well as conditions associated with inflammation, such as heart disease, ARDS, cancer, and infections involving any or all of these types of immune cells. For example, the ADCs in this subject may be used to treat or prevent inflammation associated with bacterial or viral infections such as COVID-19, influenza virus, and pneumonia (viral or bacterial) infections.

[0277] Furthermore, the subject ADC has a rapid onset of action, for example, inducing anti-inflammatory activity within 2 hours of administration, and therefore may be used in acute treatment and may be particularly effective in treating / preventing inflammation associated with bacterial or viral infections such as COVID-19, influenza virus, and pneumonia (viral or bacterial) infections (these, if not treated promptly, can lead to cytokine storms, ARDS, and in the worst case, sepsis or septic shock).

[0278] Furthermore, unlike other ADC target antigens, VISTA is expressed only on immune cells; therefore, the subject ADC does not tend to internalize non-target cells.

[0279] Furthermore, since the subject ADC does not bind to B cells, it is less immunosuppressive than free steroids, which should be beneficial in subjects receiving repeated and / or long-term administration of the subject ADC, because chronic steroid use is associated with several cancers, infections, and other conditions, which are likely unintended consequences of long-term immunosuppression due to long-term steroid use.

[0280] Furthermore, because the subject ADC acts on Treg cells, which are important immune cells involved in steroid efficacy, it may be more broadly or specifically effective, particularly in the treatment of autoimmune or inflammatory conditions or inflammation involving Treg cells.

[0281] Furthermore, the subject ADC acts on both naive immune cells and activated immune cells (on which VISTA is constitutively expressed), and as a result, the subject ADC maintains its activity (induces anti-inflammatory activity) in both the active and remission phases of inflammatory and autoimmune pathological conditions.

[0282] Furthermore, since the subject ADC acts on neutrophils (these immune cells are important in acute inflammation), the subject ADC should be useful in treating inflammatory or autoimmune conditions characterized by acute inflammation and / or infrequent or sporadic inflammatory episodes.

[0283] Furthermore, due to the high metabolic turnover on the surface of VISTA cells, the subject ADCs internalize immune cells very rapidly and constitutively (within 30 minutes), which further indicates that the subject ADCs are suitable for treating inflammatory or autoimmune conditions characterized by acute inflammation and / or infrequent or sporadic inflammatory episodes.

[0284] Furthermore, the subject ADC has a very short half-life (PK) and binds only to immune cells; therefore, the subject ADC should not be less likely to target associated toxicity and undesirable peripheral steroid exposure (low nonspecific loss effect) compared to other ADCs, such as Humira, which contain antibodies with conventional (longer) PKs.

[0285] Furthermore, in some embodiments, the biological activity (anti-inflammatory effect) of the subject ADC is entirely attributable to the anti-inflammatory payload (steroid) contained therein, i.e., in that case the anti-VISTA antibody has a silent IgG region, such as a silent IgG1 or IgG2 Fc region, that does not exhibit immunological function (does not block VISTA biology).

[0286] Based at least on the combination of the aforementioned advantages, the subject ADC should be well-suited for both acute and chronic use, and is suitable for both therapeutic and preventive use, namely, reducing or inhibiting inflammation, preventing the onset of inflammation, extending the inactivity period of the disease, and for use in the treatment of countless different types of inflammatory and autoimmune diseases.

[0287] As mentioned above, the ADC in question contains an anti-VISTA antibody, which binds to immune cells expressing VISTA (generally human VISTA) under physiological pH conditions and has a short half-life or PK, as previously stated. Typically, these antibodies contain silent Fc cells or do not contain Fc cells, and the binding of the ADC to VISTA-expressing cells does not induce any effect on VISTA signaling or VISTA-mediated effects on immunity.

[0288] In contrast, in some embodiments, anti-VISTA antibodies contain functional IgG2 and promote VISTA signaling or VISTA-related functions, such as suppression of T cell proliferation and T cell activity, as well as suppression of certain pro-inflammatory cytokines. This may result in additive or synergistic effects on suppressing inflammation and / or autoimmunity.

[0289] Exemplary anti-VISTA antibodies and antibody fragments with CDRs and variable sequences, i.e., those containing fragments, have short serum half-lives under physiological pH conditions (approximately pH 7.5). For example, the serum half-lives of antibodies or fragments in cynomolgus monkeys or humans are generally about 2.3 days ± 0.7 days or less, and under physiological conditions (approximately pH 7.5), in rodents (human VISTA knock-in), they are generally 1–72 hours, 1–32 hours, 1–16 hours, 1–8 hours, 1–4 hours, or 1–2 hours ± 0.5 hours, or in primates (cynomolgus monkeys), approximately 3.5, 3, 2.5, or 2.3 days ± 0.5 days, which can be shown in Figures 8, 10, and 12.

[0290] Exemplary inflammatory agents that can be incorporated into the ADC of the present invention, i.e., anti-VISTA antibodies and anti-VISTA antibody fragments, and can be optionally further complexed by heterobifunctional groups, for example, via a linker, include steroids or corticosteroid receptor agonists, such as the common corticosteroids mentioned above, more specifically budesonide, beclomethasone, betamethasone, ciclesonide, cortisol, cortisone, cortisone acetate, 16-α-hydroxyprednisolone, dexamethasone, difluorazone, etamethazoneb, flumetazone, flunisolide, fluocinolone acetonide, fludrocortisone, and fluticasone propionate (Flovent). Examples include (trademark), Flonase (trademark), hydrocortisone, ciclesonide, methylprednisolone, prednisone, prednisolone, mometasone, Pulmicort, triamcinolone, triamcinolone acetonide, or other steroid compounds or derivatives thereof having anti-inflammatory or steroidal activity, in particular the novel steroid of Formula 1 according to the present invention, and functional derivatives, such as the budenoside derivative shown in Figure 9 and the steroid compound of Formula 1 shown in Figure 11, and previously identified corticosteroids disclosed in patent applications, and corticosteroids disclosed in this application, in particular in the section titled “Exemplary Embodiments” and Example 3. Preferred exemplary ADCs according to the present invention are disclosed in the following examples, in particular in Example 3.

[0291] The subject ADC is intended to be used to treat subjects, e.g., humans or non-human mammals, having any condition in which the reduction of inflammation is therapeutically guaranteed by the use of anti-inflammatory agents such as steroids. Such conditions may be associated with acute or chronic inflammation, e.g., sporadic or sudden inflammation. In some preferred embodiments, subjects have a condition in which an anti-inflammatory agent, e.g., a corticosteroid receptor agonist, is required in repeated doses and / or high doses and is administered under conventional conditions (i.e., the anti-inflammatory agent is naked or uncomplexed, and the drug may induce undesirable side effects such as toxicity to non-target cells). Such conditions include autoimmune and inflammatory conditions. Non-limiting examples of such conditions include treating or preventing inflammatory, autoimmune, or allergic side effects associated with allergies, autoimmunity, transplantation, gene therapy, inflammation, GVHD or sepsis, infections, cancer, or any of the aforementioned conditions in human subjects.

[0292] In some other preferred embodiments, the subject has an acute or chronic inflammatory condition or relapse (e.g., an inflammatory condition characterized by frequent or rarely recurring acute inflammatory episodes) for which rapid onset of efficacy is therapeutically desirable, and optionally, repeated doses and / or high doses of an anti-inflammatory agent, e.g., a corticosteroid receptor agonist, are therapeutically justified, and optionally, the agent is administered under conventional conditions (i.e., the anti-inflammatory agent is naked or unconjugated, and the drug may induce undesirable side effects, such as toxicity, in non-target cells). Such conditions include autoimmune and inflammatory conditions, cancer, and infectious conditions associated with inflammation, e.g., characterized by acute and / or severe inflammatory episodes.

[0293] Non-limiting examples of such conditions include treating or preventing inflammatory, autoimmune, or allergic side effects associated with any of the aforementioned conditions in human subjects, such as allergies, autoimmune conditions, transplantation, gene therapy, inflammation, cancer, GVHD or sepsis, infections (e.g., bacterial, viral, fungal, or parasitic), acute respiratory distress syndrome (ARDS), or any of the aforementioned conditions.

[0294] Other specific exemplary conditions in which the use of ADCs may be effective include rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis, ankylosing spondylitis, adult Crohn's disease, childhood Crohn's disease, ulcerative colitis, psoriasis vulgaris, hidradenitis suppurativa, uveitis, Behçet's disease, spondyloarthropathy, or psoriasis.

[0295] Other exemplary conditions and cases in which the use of the subject ADC may be therapeutically beneficial include: (i) Conditions that can be effectively treated primarily with high doses of steroids, and, on an optional basis, polymyalgia rheumatica and / or giant cell arteritis (the patient has been or is being treated with high doses of steroids on an optional basis); (ii) Conditions with complications that limit the use of steroids, optionally: diabetes mellitus, non-alcoholic steatohepatitis (NASH), morbid obesity, avascular necrosis / osteonecrosis (AVN), glaucoma; steroid-induced hypertension, severe skin fragility, and / or osteoarthritis; (iii) Conditions for which safe long-term therapeutic agents are available but initiation of several months with high-dose steroids is desirable, optionally including AAV, polymyositis, dermatomyositis, lupus, inflammatory lung disease, autoimmune hepatitis, inflammatory bowel disease, immune thrombocytopenia, autoimmune hemolytic anemia, and gout patients for whom initiation of several months with high-dose steroids is therapeutically justified; (iv) dermatological conditions requiring short-term / long-term treatment for which there is no effective alternative to treatment or duration and / or steroid administration, optionally Stevens-Johnson syndrome, other severe rash conditions, conditions including widespread contact dermatitis, PG, LCV, other severe immune-related dermatological conditions, etc. (v) Conditions treated with high-dose corticosteroids for erythema / recurrence, optionally including COPD, asthma, lupus, gout, and pseudogout; (vi) Immune-related neurological disorders such as small fibrous neuropathy, MS (subset), chronic inflammatory demyelinating polyneuropathy, and myasthenia gravis; (vii) Optional hematological / oncological indications for which high doses of steroids are therapeutically justified or may be effective; (viii) Ophthalmic conditions, optional, such as uveitis, iritis, scleritis, etc. (ix) Conditions associated with persistent or very long-term adrenal insufficiency or secondary adrenal insufficiency, optionally iatrogenic Addison's disease crisis; (x) A condition that is often treated with long-term low-dose steroids, and optionally with lupus, RA, psA, vasculitis, etc.; and (xi) Special classes of patients, e.g., pregnant / lactating women, pediatric patients, and optionally patients with growth disorders or cataracts.

[0296] Compositions containing the ADC of Formula 1 or novel glucocorticosteroids according to the present invention may be used alone or with other therapeutic agents, particularly other immunosuppressive molecules or other therapeutic agents used to treat autoimmune and inflammatory conditions, such as acquired immunodeficiency syndrome (AIDS), acquired splenic atrophy, acute anterior uveitis, acute disseminated encephalomyelitis (ADEM), acute gouty arthritis, acute necrotizing hemorrhagic leukoencephalitis, acute or chronic sinusitis, acute suppurative meningitis (and Other central nervous system inflammatory disorders), acute severe inflammation, Addison's disease, adrenal inflammatory disease, adult-onset diabetes (type II diabetes), adult-onset idiopathic hypoparathyroidism (AOIH), agammaglobulinemia, agranulocytosis, vasculitis including vasculitis, optional large vessel vasculitis, optional polymyalgia rheumatica and giant cell (Takayasu) arteritis, allergic conditions, allergic contact dermatitis, allergic dermatitis, allergic granulomatous vasculitis, allergic hypersensitivity disorders Allergic neuritis, allergic reactions, alopecia areata, alopecia totalis, Alport syndrome, alveolitis, optional allergic alveolitis or fibrotic alveolitis, Alzheimer's disease, amyloidosis, amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease), eosinophil-related disorders, optional eosinophilia, anaphylaxis, ankylosing spondylitis, telangiectasia, antibody-mediated glomerulonephritis, anti-GBM / anti-TBM glomerulonephritis, antigen-antibody complex-mediated diseases, anti-glomerular basement membrane Antibody diseases, antiphospholipid antibody syndrome, antiphospholipid syndrome (APS), aphthous stomatitis, aplastic anemia, arrhythmia, arteriosclerosis, arteriosclerotic disorders, arthritis, optionally rheumatoid arthritis, e.g., acute arthritis, or chronic rheumatoid arthritis, chronic progressive arthritis, osteoarthritis, ascariasis, aspergilloma, eosinophilic granuloma, aspergillosis, azoospermia (aspermiogenese), asthma, optionally bronchial asthma bronchiale, bronchial asthma), or autoimmune asthma, ataxia telangiectasia, ataxic sclerosis, atherosclerosis, autism, autoimmune angioedema, autoimmune aplastic anemia, autoimmune atrophic gastritis, autoimmune diabetes mellitus, autoimmune orchitis and oophoritis and other autoimmune diseases of the testes and ovaries, autoimmune disorders associated with collagen diseases, autoimmune autonomic dysfunction, autoimmune ear diseases, and optionally autoimmune inner ear diseases (AGED), autoimmune endocrine diseases, for example,Thyroiditis, for example, autoimmune thyroiditis, autoimmune bowel disease syndrome, autoimmune gonadal insufficiency, autoimmune hearing loss, autoimmune hemolysis, autoimmune hepatitis, autoimmune liver disease, autoimmune hyperlipidemia, autoimmune immunodeficiency, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune neutropenia, autoimmune pancreatitis, autoimmune polyglandular endocrine disorder, type I autoimmune polyglandular syndrome, autoimmune retinopathy, autoimmune thrombocytopenic purpura (ATP), autoimmune thyroid disease, autoimmune urticaria, autoimmune-mediated gastrointestinal disease, axonal and neuronal nerve disorders, Barlow's disease, Behçet's disease, benign familial and ischemia-reperfusion injury, benign lymphocytic vasculitis, Berger's disease (IgA nephropathy), bird stool, blindness, Beck's disease, bronchiolitis obliterans (non-transplant) vs. NSIP, bronchitis, bronchopneumonitis aspergillosis, Bruton's syndrome, bullous pemphigoid, Kaplan's syndrome, cardiomyopathy, cardiovascular ischemia, Cussleman syndrome, celiac disease, celiac sprout (gluten enteropathy), cerebellar degeneration, cerebral ischemia, diseases with angiogenesis, Chagas disease, channelopathy, epilepsy (optional), channelopathy of the CNS, chorioretinitis, choroiditis, autoimmune hematological disorders, chronic activity Hepatitis or autoimmune chronic active hepatitis, chronic contact dermatitis, chronic eosinophilic pneumonia, chronic fatigue syndrome, chronic hepatitis, chronic hypersensitivity pneumonitis, chronic inflammatory arthritis, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic refractory inflammation, chronic mucocutaneous candidiasis, chronic neuropathy, optional IgM polyneuropathy or IgM-mediated neuropathy, chronic obstructive airway disease, chronic inflammatory lung disease, chronic relapsing multifocal osteomyelitis (CRMO), chronic thyroiditis (Hashimoto's thyroiditis) or subacute thyroiditis, Churg-Strauss syndrome, scarring pemphigoid / benign mucosal pemphigoid, coronavirus-borne infection Diseases such as SARS-CoV-2 (COVID-19), SARS-CoV, MERS, SARS-CoV-2 and related side effects, CNS inflammatory disorders, CNS vasculitis, celiac disease, Cogan syndrome, cold agglutinin disease, colon polyps, colitis such as ulcerative colitis, ulcerative colitis, collagenous colitis, conditions including T-cell infiltration and chronic inflammatory responses, congenital heart block, congenital rubella infection, Coombs test positive anemia, coronary artery disease, coxsackie myocarditis, CREST syndrome (calcification, Raynaud's phenomenon), Crohn's disease, cryoglobulinemia, Cushing's syndrome,Ciliitis, optionally chronic cilitis, heteroiridochromic cilitis, iridocyclitis, or Fuchs' cilitis, cystic fibrosis, cytokine-induced toxicity, hearing loss, degenerative arthritis, demyelinating diseases, optionally autoimmune demyelinating diseases, demyelinating neuropathy, dengue fever, herpetiform dermatitis and atopic dermatitis, dermatitis, e.g., contact dermatitis, dermatomyositis, skin diseases with acute inflammatory components, Devic's disease (neuromyelitis optica), diabetic aortic disease, diabetes Diabetic nephropathy, diabetic retinopathy, Diamond-Blackfan anemia, diffuse interstitial pulmonary fibrosis, dilated cardiomyopathy, lupus discoid, diseases with leukocytosis, Dressler syndrome, Dupuytren's contracture, echovirus infection, eczema, e.g., allergic or atopic eczema, encephalitis, e.g., Rasmussen encephalitis and limbic and / or brainstem encephalitis, encephalomyelitis, optionally allergic encephalomyelitis or encephalomyelitis allergic a) and experimental allergic encephalomyelitis (EAE), intraarterial hyperplasia, endocarditis, endocrine ophthalmopathy, endometriosis, endocardial fibrosis, lens hypersensitive endophthalmitis, endophthalmitis, allergic enteritis, eosinophilic myalgia syndrome, eosinophilic fasciitis, epidemic keratoconjunctivitis, acquired epidermolysis bullosa (EBA), episclerosis, episcleritis, Epstein-Barr virus infection, erythema elevata, erythema multiforme, erythema nodosum leprosy, erythema nodosum, erythroblastosis fetus, esophageal motility disorders, essential mixed cryoglobulinemia, ethmoid bone, Evans syndrome, experimental allergic encephalomyelitis (EAE), factor VIII deficiency, farmer's lung, rheumatic fever, Felty syndrome, fibromyopathy , fibrotic alveolitis, filariasis, focal segmental glomerulosclerosis (FSGS), food poisoning, anterior gastric atrophy, giant cell arthritis (temporal arthritis), giant cell hepatitis, giant cell polymyalgia, glomerulonephritis, glomerulonephritis (GN) with or without nephrotic syndrome such as chronic or acute glomerulonephritis (e.g., primary GN), Goodpasture syndrome, gouty arthritis, granulocyte transfusion-associated syndrome, granulomatous diseases, e.g., lymphomatous granulomatosis, granulomatosis with polyangiitis (GPA), granulomatous uveitis, Graves' disease, Guillain-Barré syndrome, guttate psoriasis, paroxysmal hemoglobinuria, Hanmann-Rich disease, Hashimoto's disease, Hashimoto's encephalitis, Hashimoto's thyroiditis, hemochromatosis,Hemolytic anemia or immune hemolytic anemia, e.g., autoimmune hemolytic anemia (AIHA), hemolytic anemia, hemophilia A, Henoch-Schönlein purpura, herpes zoster of pregnancy, human immunodeficiency virus (HIV) infection, hyperalgesia, hypogammaglobulinemia, hypogonadism, hypoparathyroidism, idiopathic diabetes insipidus, idiopathic facial nerve paralysis, idiopathic hypothyroidism, idiopathic IgA nephropathy, idiopathic membranous nephropathy or idiopathic membranous nephropathy, idiopathic glomerulonephritis syndrome, idiopathic pulmonary fibrosis, idiopathic sprue, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, IgE-mediated diseases, anaphylactic acid (Anaphylactic) at the discretion of the patient. Laxity and allergic or atopic rhinitis, IgG4-related sclerosing disease, focal ileitis, immune complex nephritis, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T lymphocytes, immune-mediated GN, immunomodulatory lipoproteins, e.g., adult or acute respiratory distress syndrome (ARDS), inclusion body myositis, infectious arthritis, infertility due to anti-sperm antibodies, inflammation of all or part of the uvea, inflammatory bowel disease (IBD), inflammatory hyperproliferative skin disease, inflammatory myopathy, insulin-dependent diabetes mellitus (type 1), isletitis, interstitial cystitis, interstitial lung disease, interstitial lung Fibrosis, iritis, ischemic reperfusion injury, arthritis, juvenile arthritis, juvenile dermatomyositis, juvenile diabetes, juvenile-onset (type 1) diabetes, childhood insulin-dependent diabetes mellitus (IDDM), juvenile-onset rheumatoid arthritis, Kawasaki syndrome, keratoconjunctivitis sicca, kypanosomiasis, Lambert-Eaton syndrome, leishmaniasis, leukopenia, leukocyte adhesion deficiency, leukocytopenia, lichen planus, lichen sclerosing, woody conjunctivitis, linear IgA bullous dermatosis, linear IgA disease (LAD), Loeffler's syndrome, lupoid hepatitis, lupus (nephritis) , encephalitis, pediatric, non-renal, extrarenal, alopecia areata, lupus (SLE), disseminated lupus erythematosus, Lyme arthritis, Lyme disease, lymphocytic interstitial pneumonia, malaria, autoimmune infertility in men and women, maxillary vasculitis (including Kawasaki disease and polyarteritis nodosa), membranous or membranoproliferative gnoses (MPGN), e.g., type I and type II and rapidly progressive gnoses, membranous gnoses (membranous nephropathy), Meniere's disease, meningitis, microscopic colitis, microscopic polyangiitis, migraine, minimal change nephropathy, mixed connective tissue disease (MCTD), infectious mononucleosis, Mohren's ulcer, Mucher-Habermann disease,Multifocal motor neuropathy, multiple endocrine deficiency, sepsis, multiple organ injury syndromes such as those secondary to trauma or bleeding, multiple sclerosis (MS), e.g., spinal visual MS, multiple sclerosis, mumps, muscle disorders, myasthenia gravis, e.g., thymoma-associated myasthenia gravis, myasthenia gravis, myocarditis, myositis, narcolepsy, necrotizing enterocolitis, and panthallus colitis, and autoimmune inflammatory bowel disease, necrotizing, cutaneous, and or hypersensitivity vasculitis, neonatal lupus syndrome (NLE), nephrotic syndrome, neurological disorders, neuromyelitis optica (Devic's disease), neuromyelitis optica, neurogenic totonia, neutropenia, noncancerous lymphocytosis, nongranulomatous uveitis, nonmalignant thymoma, ocular and orbital inflammatory disorders, scarring pemphigoid of the eye, oophoritis, sympathetic ophthalmitis, ocular clonus-myoclonus ataxia (OMS), ocular clonus or ocular clonus Myoclonus ataxia (OMS), and sensory neuropathy, optic neuritis, granulomatous orchitis, osteoarthritis, relapsing rheumatoid arthritis, pancreatitis, pancytopenia, PANDAS (Streptococcal-associated childhood autoimmune neuropsychiatric disorder), paraneoplastic cerebellar degeneration, paraneoplastic syndromes, multiple paraneoplastic syndromes, e.g., paraneoplastic neurological syndrome, optionally Lambert-Eaton myasthenia gravis or Eaton-Lambert syndrome, leishmaniasis, etc. Parasitic diseases, paroxysmal nocturnal hemoglobinuria (PNH), Parry-Romberg syndrome, squamous cellulitis (peripheral uveitis), Personage-Turner syndrome, parvovirus infection, bullous pemphigoid, such as bullous pemphigoid and cutaneous pemphigoid, pemphigus (including pemphigus vulgaris), erythematous pemphigus, foliar pemphigoid, mucosal pemphigoid, pemphigus, peptic ulcer, periodic paralysis, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia Perniciosa), pernicious anemia, lens antigenic uveitis, pulmonary cirrhosis, POEMS syndrome, polyarteritis nodosa, primary chronic polyarthritis type I, II, and III, polychondritis (e.g., refractory or relapsing polychondritis), polyautoimmune diseases, polyglandular endocrine disorders, polyglandular syndromes, optional autoimmune polyglandular syndromes (or polyglandular endocrine disorder syndromes), polymyalgia rheumatica, polymyositis, polymyositis / dermatomyositis, polyneuropathy, acute polyradiculitis, postcardiotomy syndrome, posterior uveitis, or autoimmune uveitis, post-myocardial infarction syndrome,Post-pericardiotomy syndrome, post-streptococcal nephritis, post-vaccination syndrome, presenile dementia, primary biliary cirrhosis, primary hypothyroidism, primary idiopathic myxedema, primary lymphocytosis, such as monoclonal B-cell lymphocytosis, and, on an optional basis, benign monoclonal gamma globulin abnormalities and monoclonal gamma globulin abnormalities of unknown significance. Brin disorder, MGUS, primary myxedema, primary progressive MS (PPMS), and relapsing-remitting MS (RRMS), primary sclerosing cholangitis, progesterone dermatitis, progressive systemic sclerosis, proliferative arthritis, psoriasis, e.g., plaque psoriasis, psoriasis, psoriatic arthritis, alveolar proteinosis, pulmonary infiltrative eosinophilia, euerythrocytic anemia or erythrocytic aplasia (PRCA), pure red cell aplasia, suppurative or nonsuppurative sinusitis, pustular psoriasis and onychopsoriasis, pyelonephritis, pyoderma gangrenosum, Quervain's thyroiditis, Raynaud's phenomenon, reactive arthritis, recurrent miscarriage, decreased blood pressure response, reflex sympathetic dystrophy Rophy's disease, refractory sprue, Reiter's disease or syndrome, relapsing polychondritis, reperfusion injury of the cardiomyocyte or other tissue, reperfusion injury, respiratory distress syndrome, restless legs syndrome, retinal autoimmune disease, retroperitoneal fibrosis, Raynaud's syndrome, rheumatic diseases, rheumatic fever, rheumatism, rheumatoid arthritis, rheumatoid spondylitis, rubella virus infection, Sumpter syndrome, sarcoidosis, schistosomiasis, Schmidt syndrome, SCID and Epstein-Barr virus-related diseases, sclerosis, scleritis, candidia, scleroderma, systemic scleroderma (optional), sclerosing cholangitis, disseminated sclerosis, sclerosis, for example Systemic sclerosis, sensorineural hearing loss, seronegative spondyloarthritis, Sheehan's syndrome, Charmant syndrome, silicosis, Sjögren's syndrome, sperm and testicular autoimmunity, sphenoid sinusitis, Stevens-Johnson syndrome, Stiffman (or Stiffperson) syndrome, subacute bacterial endocarditis (SBE), subacute cutaneous lupus erythematosus, sudden hearing loss, Suzak syndrome, Sydenham chorea, sympathetic ophthalmitis, systemic lupus erythematosus (SLE) or systemic lupus erythematosus, cutaneous SLE, systemic necrotizing vasculitis, and ANCA-associated vasculitis, optionally, Churgoose Trauss's vasculitis or syndrome (CSS), tabes dorsalis, Takayasu's arteritis, telangiectasia, temporal arteritis / giant cell arteritis, thromboangiitis obliterans, thrombocytopenia, e.g., thrombotic thrombocytopenic purpura (TTP) and autoimmune or immune-mediated thrombocytopenia, e.g., idiopathic thrombocytopenic purpura (ITP), e.g., chronic or acute ITP, thrombocytopenic purpura (TTP), thyrotoxicosis, tissue damage, Trosa-Hunt syndrome, toxic epidermal necrolysis, toxic shock syndrome, transfusion reaction, transient infantile hypogammaglobulinemia, transverse myelitis.It may be used in combination with drugs used to treat myelitis, tropical pulmonary eosinophilia, tuberculosis, ulcerative colitis, undifferentiated connective tissue disease (UCTD), urticaria, and optionally chronic allergic urticaria and chronic idiopathic urticaria, such as chronic autoimmune urticaria, uveitis, anterior uveitis, retinal uveitis, valvular arthritis, vascular dysfunction, vasculitis, spondyloarthritis, bullous dermatosis, vitiligo, Wegener's granulomatosis (granulomatosis with polyangiitis (GPA)), Wiscott-Aldrich syndrome, or X-linked hyper-IgM syndrome.

[0297] The subject ADC and the novel corticosteroid of Formula 1 may be used for both the prevention and / or treatment of inflammation and inflammation-related diseases, such as autoimmune disorders, inflammatory disorders, infections, and cancer. A preferred use of the subject ADC is the treatment of chronic inflammation-related diseases. Quite unexpectedly, as shown in the examples, the subject ADC has been found to maintain efficacy over a long period (PD) compared to the half-life (PK) of the antibody it contains, despite the short pK of the anti-VISTA antibody (it has not been modified to bind to VISTA-expressing cells under physiological conditions, alter or optimize pH binding, or enhance half-life, i.e., typically about 2.3 days or less in cynomolgus monkeys and typically only a few hours in human VISTA-modified mice).

[0298] As shown herein, when evaluated in vitro and in vivo models, the ADC complex according to the present invention has been shown to provide a PK / PD ratio of at least 14:1 (in fact, the PK / PD ratio may be substantially higher because longer-term efficacy could not be evaluated as the rodents were euthanized when the PD was determined).

[0299] While the applicants do not wish to be bound by their beliefs, it is theorized that Subject ADCs are delivered in very large quantities to target VISTA-expressing cells such as macrophages, T cells, and Tregs, as well as other VISTA-expressing immune cells, including immune cells with long cell turnover (weeks, months, or more). Essentially, the depot effect is thought to be made within certain types of immune cells, namely, because they surface-express VISTA to such a high degree that large quantities or a “depot” of Subject ADCs are internalized into VISTA-expressing immune cells such as macrophages and bone marrow cells. This depot, containing the internalized ADCs, is then slowly metabolized or cleaved within the immune cells, for example, by cellular enzymes. In vivo studies disclosed herein show that the metabolism or cleavage of internalized ADCs clearly occurs in rodents over a week, two weeks, or longer, thereby providing a gradual and prolonged release of a therapeutically effective steroid payload within the host immune cells. This occurs despite the fact that, by that point, there is no recognizable amount of ADC remaining in the serum (i.e., based on a PK where there is not enough ADC to be therapeutically significant) (due to the short PK of ADC and the antibodies within it).

[0300] Furthermore, while these observations are quite surprising, drug metabolism generally occurs much faster in rodents than in primates (and much slower in humans than in rodents); and since the expression levels of VISTA and the levels of immune cells expressing VISTA are similar in rodents, humans, and non-human primates, it is expected that the subject ADC will have a similar or better PK / PD ratio in humans and other primates. Therefore, the subject ADC should be well-suited for therapeutic applications where long-term efficacy is desired or required.

[0301] As mentioned above, another preferred use of the subject ADC and the novel corticosteroid of Formula 1 is for acute use, i.e., for the treatment of acute inflammation. As shown in the examples, the subject ADC has a rapid onset of action, for example, they induce anti-inflammatory effects as quickly as within 2 hours after administration. Furthermore, the acute use of the subject ADC is even more advantageous because it has been shown that the subject ADC effectively targets and internalizes neutrophils and induces anti-inflammatory effects. This is particularly useful in acute use because neutrophils are involved in the initial stages of the inflammatory response, and therefore the subject ADC is also suitable for the treatment of acute inflammatory signs.

[0302] Another preferred use of the subject ADC and novel corticosteroid of formula 1 is for maintenance therapy. Essentially, since VISTA is expressed on both activated and inactivated (naive) immune cells (thus VISTA is constitutively expressed), the subject ADC can be administered regularly over long periods, and such administration induces anti-inflammatory activity both when the target is in an active phase of the inflammatory response and when the target is in remission of the disease. This is therapeutically useful because many chronic autoimmune and inflammatory disorders are known to be characterized by an active phase or episode in which the patient experiences disease-related inflammation and other symptoms or pathological reactions, as well as a remission phase (i.e., remission / relapse or idiopathic) in which there are no symptoms of the disease, including inflammation and other symptoms, or disease-related pathological reactions are absent or not very severe. Because the subject ADC binds to both activated and deactivated immune cells, it may be able to more effectively maintain disease remission, i.e., the duration of remission should be longer, and / or it is expected that patients treated with the subject ADC may experience disease activity in a much milder form, as the anti-inflammatory effect of the subject ADC on target immune cells is maintained in both the active and remission phases.

[0303] Furthermore, the ADC in question should be suitable for long-term or chronic use because it does not affect non-target cells, i.e., non-immune cells. As shown in the example below, the ADC in question acts substantially only on immune cells and not on non-immune cells (some anti-inflammatory activity was detected in the liver, which is likely explained by the fact that the liver contains immune cells).

[0304] Furthermore, due to the short PK (but remarkably long PD) of the subject ADCs, the subject ADCs do not remain in the serum for long periods; that is, they rapidly bind to immune cells and are internalized by them, in which case the ADCs are efficiently and rapidly taken up in large quantities by the immune cells and slowly metabolized within these immune cells, thus delivering the anti-inflammatory payload and remaining effective for a long period. Therefore, because the subject ADCs are only present in the peripheral circulation for a short time, the subject ADCs have limited opportunities to interact with non-target cells compared to ADCs with longer PKs, compared to ADCs containing antibodies (conventional therapeutic antibodies).

[0305] Furthermore, since the efficacy of the ADC according to the present invention (in particular, the anti-VISTA ADC according to the present invention, which includes an Fc region modified to impair FcR and complement binding) is entirely due to the anti-inflammatory payload such as steroids, the subject ADC should be well-suited for long-term or chronic use. Essentially, in such cases, the anti-VISTA antibody provides only a targeting function; i.e., the anti-VISTA antibody promotes the binding and internalization of the ADC by target immune cells. However, the binding of such ADCs to VISTA-expressing immune cells does not modulate VISTA activity; i.e., the anti-VISTA antibody containing an Fc modified to eliminate Fc crosslinking does not antagonistize or agonize VISTA activity. This is in contrast to existing ADCs for delivering steroids (such as Humira ADCs), which include antibodies that induce biological effects upon binding to a target antigen. This should be useful from a dosing standpoint, as the efficacy of the ADC depends solely on the anti-inflammatory payload. Furthermore, VISTA agonist and antagonist antibodies are even more therapeutically useful because they can induce pro-inflammatory cytokine responses that may be undesirable in the context of drugs intended to alleviate inflammation.

[0306] The acute and chronic autoimmune and inflammatory indications for which ADC may be used are as previously stated, including acquired aplastic anemia, acquired hemophilia, acute disseminated encephalomyelitis (ADEM), acute hemorrhagic leukoencephalitis (AHLE) / Hearst disease, primary agammaglobulinemia, alopecia areata, ankylosing spondylitis (AS), anti-NMDA receptor encephalitis, antiphospholipid syndrome (APS), arteriosclerosis, autism spectrum disorder (ASD), autoimmune Addison's disease (AAD), and autoimmune autonomic dysfunction / autoimmune autonomic nervous system dysfunction. Pelvic floor syndrome (AAG), autoimmune encephalitis+, autoimmune gastritis, autoimmune hemolytic anemia (AIHA)+, autoimmune hepatitis (AIH)+, autoimmune hyperlipidemia, autoimmune hypophysitis / lymphophytitis+, autoimmune inner ear disease (AIED)+, autoimmune lymphoproliferative syndrome (ALPS)+, autoimmune myocarditis, autoimmune oophoritis+, autoimmune orchitis+, autoimmune pancreatitis (AIP) / immunoglobulin G4-related disease (IgG4-RD)+, autoimmune polyglandular syndrome type I, II, and III+, autoimmune prognathitis Gesterone dermatitis +, autoimmune sudden sensorineural hearing loss (SNHL) achalasia, Addison's disease, adult Still's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, antiphospholipid syndrome, autoimmune angioedema, autoimmune autonomic dysfunction, autoimmune encephalomyelitis, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune oophoritis, autoimmune orchitis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune urticaria, axonal and neuronal Patchy's disease (AMAN), Balo's disease, Behçet's disease, benign mucosal pemphigoid, bullous pemphigoid, Castleman disease (CD), celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic relapsing multifocal osteomyelitis (CRMO), Churg-Strauss syndrome (CSS) or eosinophilic granulomatosis (EGPA), scarring pemphigoid, Cogan's syndrome, cold agglutinin disease, congenital heart block, coxsackie myocarditis, CREST syndrome, type 1 diabetes, herpetiform dermatitis, dermatomyositis, Devic's disease (neuromyelitis optica). Lupus discoid, Dressler syndrome, endometriosis, eosinophilic esophagitis (EoE), eosinophilic fasciitis, erythema nodosum, essential mixed cryoglobulinemia, Evans syndrome, fibromyalgia, fibrous alveolitis, fibrous alveolitis,Giant cell myocarditis, glomerulonephritis, Goodpasture syndrome, granulomatosis with polyangiitis, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schönlein purpura (HSP), herpes zoster of pregnancy or bullous pemphigoid (PG), hidradenitis suppurativa (HS) (reverse acne), hypogammaglobulinemia, IgA nephropathy, IgG4-related sclerosis, immune thrombocytopenic purpura (ITP), inclusion body myositis (IBM), interstitial cystitis (IC), juvenile arthritis, juvenile diabetes mellitus (type 1 diabetes mellitus), juvenile myositis (JM), Kawasaki disease, Lambert-Eaton syndrome, leukocytosis Vasculitis, lichen planus, lichen sclerosing, woody conjunctivitis, linear IgA disease (LAD), lupus (including nephritis and cutaneous lesions), chronic Lyme disease, Meniere's disease, microscopic polyangiitis (MPA), mixed connective tissue disease (MCTD), Mollen's ulcer, Mucher-Habermann disease, multifocal motor neuropathy (MMN) or MMNCB, multiple sclerosis, myasthenia gravis, myelin oligodendrocyte glycoprotein antibody disorder, myositis, narcolepsy, neonatal lupus, neuromyelitis optica, neutropenia, ocular scarring pemphigoid, optic neuritis, ocular clonus-myoclonus ataxia (OMS) ), relapsing rheumatoid arthritis (PR), PANDAS, paraneoplastic cerebellar degeneration (PCD), paroxysmal nocturnal hemoglobinuria (PNH), hemifacial atrophy, ciliary body squamous cellulitis (peripheral uveitis), personality-Turner syndrome, pemphigus, peripheral neuropathy, perivenosis encephalomyelitis, pernicious anemia (PA), POEMS syndrome, polyarteritis nodosa, type I, type II, type III polyglandular syndrome, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, primary biliary cholangitis, primary sclerosing cholangitis, progesterone-induced dermatitis, psoriasis, psoriatic arthritis, pure red cell aplasia (PRCA) , pyoderma gangrenosum, Raynaud's phenomenon, reactive arthritis, reflex sympathetic dystrophy, relapsing polychondritis, restless legs syndrome (RLS), retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt syndrome, scleritis, scleroderma, Sjögren's syndrome, sperm / testicular autoimmunity, generalized rigidus syndrome (SPS), subacute bacterial endocarditis (SBE), Suzak syndrome, sympathetic ophthalmitis (SO), Takayasu's arteritis, temporal arteritis / giant cell arteritis, thrombocytopenic purpura (TTP), thyroid eye disease (TED), Tolosa-Hunt syndrome (THS), transverse myelitis, type 1 diabetes,Examples include undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vitiligo, and Vogt-Koyanagi-Harada syndrome.

[0307] Preferred indications for which ADC is therapeutically effective include severe asthma, giant cell arteritis, ANKA vasculitis, and IBD (colitis (e.g., ulcerative) and Crohn's disease). Naturally, it should be understood that the disease conditions identified herein are illustrative and not exhaustive.

[0308] The subject ADC may be used in combination with other therapeutic agents, and may be administered simultaneously or at different times in the same or different compositions. For example, the subject ADC may be administered in a therapeutic regimen that includes the administration of a PD-1 or PD-L1 agonist, CTLA4-Ig, cytokines, cytokine agonists or antagonists, or other immunosuppressive receptor agonists or antagonists.

[0309] Other examples of specific immunosuppressive molecules that may be used in combination with the ADC according to the present invention include antibodies that block costimulatory signals (e.g., against CD28 or ICOS), antibodies that activate suppressive signals via CTLA4, and / or antibodies against other immune cell markers (e.g., against CD40, CD40 ligand, or cytokines), fusion proteins (e.g., CTLA4-Fc or PD-1-Fc), and immunosuppressants (e.g., rapamycin, cyclosporine A, or FK506).

[0310] Modified Fc region of ADC according to the present invention As described above, in some preferred embodiments of the present invention, the ADC contains Fc which can be modified to include modifications within the Fc region that alter one or more functional properties of the antibody, such as complement fixation, Fc receptor binding, and / or antigen-dependent cell-mediated cytotoxicity. Furthermore, in some embodiments of the present invention, the ADC may be chemically modified (e.g., by allowing one or more chemical moieties to bind to the antibody) or modified to change its glycosylation, also in order to alter one or more functional properties of the antibody. Such embodiments are described further below. The numbering of the residues in the Fc region is the numbering of the Kabat EU index.

[0311] In one embodiment, the hinge region of CH1 is modified to change the number of cysteine ​​residues within the hinge region, for example, by increasing or decreasing them. This approach is further described in U.S. Patent No. 5,677,425 by Bodmer et al. Changing the number of cysteine ​​residues in the hinge region of CHI is used, for example, to facilitate the assembly of the light and heavy chains or to increase or decrease the stability of the antibody.

[0312] In another embodiment, mutations are introduced into the Fc hinge region of the antibody to further reduce its biological half-life. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc hinge fragment such that the antibody's staphylococcal protein A (SpA) binding is impaired compared to the natural Fc hinge domain SpA binding. This approach is described in more detail in U.S. Patent No. 6,165,745 by Ward et al.

[0313] In further embodiments, the effector function of the antibody is altered by modifying the Fc region by substituting at least one amino acid residue with a different amino acid residue. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320, and 322 can be substituted with a different amino acid residue so that the antibody has a modified affinity for the effector ligand but retains the antigen-binding ability of the parent antibody. The effector ligand whose affinity is altered may be, for example, the Fc receptor or the Cl component of complement. This approach is described in more detail by Winter et al., patents 5,624,821 and 5,648,260.

[0314] In another example, one or more amino acids selected from amino acid residues 329, 331, and 322 can be substituted with different amino acid residues so that the antibody alters C1q binding and / or reduces or eliminates complement-dependent cell-mediated cytotoxicity (CDC). This approach is described in more detail in U.S. Patent No. 6,194,551 by Idusogie et al.

[0315] In another example, altering one or more amino acid residues within positions 231-239 changes the antibody's ability to fix complement. This approach is further described in PCT publication WO94 / 29351 by Bodmer et al.

[0316] In yet another example, the Fc region of the ADC is modified to increase the affinity of the antibody to the Fγ receptor by modifying one or more amino acids at the following positions: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, ​​388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439. This approach is further described in PCT Publication WO00 / 42072 by Presta. Furthermore, binding sites for FcyRI, FcyRII, FcyRIII, and FcRn on human IgG1 have been mapped, and variants with improved binding have been described (see Shields, R. Let al. (2001) J. Biol. Chem. 276:6591-6604). Specific mutations at positions 256, 290, 298, 333, 334, and 339 have been shown to improve binding to FcγRIII. Additionally, the following combination mutants have been shown to improve FcγRIII binding: T256A / S298A, S298A / E333A, S298A / K224A, and S298A / E333A / K334A. Furthermore, mutations such as M252Y / S254T / T256E or M428L / N434S improve binding to FcRn and increase the circulating half-life of the antibody (see Chan CA and Carter PJ (2010) Nature Rev Immunol 10:301-316).

[0317] In yet another embodiment, the antibody in the ADC can be modified to invalidate Fab arm exchange in vivo. Specifically, this process involves the exchange of IgG4 halves (one heavy chain + one light chain) between other IgG4 antibodies, effectively yielding a functionally monovalent β-specific antibody. Mutations in the hinge region and constant domain of the heavy chain can invalidate this exchange (see Aalberse, RC, Schuurman J., 2002, Immunology 105:9-19).

[0318] In yet another embodiment, the glycosylation of the antibody in the ADC is modified. For example, a non-glycosylated antibody can be produced (i.e., the antibody lacks glycosylation). By altering glycosylation, for example, the affinity of the antibody to an antigen can be increased. Such carbohydrate modification can be achieved, for example, by altering one or more glycosylation sites in the antibody sequence. For example, one or more amino acid substitutions can be performed to remove one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. Such aglycosylation may increase the affinity of the antibody to an antigen. Such approaches are described in more detail in U.S. Patents 5,714,350 and 6,350,861 by Co et al.

[0319] Furthermore, or alternatively, antibodies within ADCs can be used to produce antibodies with modified glycosylation, such as low-fucosylated antibodies with reduced fucosyl residue levels or antibodies with increased branched GlcNac structures. Such altered glycosylation patterns have been shown to enhance the antibody's ADCC capability. Such carbohydrate modifications can be achieved, for example, by expressing antibodies in host cells having a modified glycosylation mechanism. Cells having a modified glycosylation mechanism have been described in the Art and can be used as host cells expressing recombinant antibodies according to at least some embodiments of the Invention to produce antibodies having modified glycosylation. For example, the cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene FUT8 ((1,6)fucosyltransferase), so antibodies expressed in the Ms704, Ms705, and Ms709 cell lines lack the fucose of their carbohydrates. The Ms704, Ms705, and Ms709 FUT8 cell lines are created by targeted disruption of the FUT8 gene in CHO / DG44 cells using two substitution vectors (see U.S. Patent Publication No. 20040110704 by Yamane et al. and Yamane-Ohnuki et al. (2004) Biotechnol Bioeng 87:614-22). As another example, Hanai et al. (EP1,176,195) describe cell lines having a functionally disrupted FUT8 gene (encoding fucosyltransferase), thereby reducing or eliminating 1,6-binding related enzymes, resulting in antibodies expressed in such cell lines exhibiting low fucosylation. Hanai et al. also describe cell lines with low or no enzymatic activity for adding fucose to N-acetylglucosamine bound to the Fc region of antibodies, such as the rat myeloma cell line YB2 / 0 (ATCC CRL 1662).Presta's PCT publication WO03 / 035835 describes the Lecl3 cell line, a variant CHO cell line in which the ability to bind fucose to Asn(297)-linked carbohydrates is reduced, and which results in low fucosylation of antibodies expressed in its host cells (see also Shields, R. Let al. (2002) J Biol. Chem. 277:26733-26740). Umana et al.'s PCT publication WO99 / 54342 describes a cell line modified to express glycoprotein-modifying glycosyltransferases (e.g., P(l,4)-N-acetylglucosaminyltransferase III (GnTIII)), resulting in antibodies expressed in the modified cell line showing an increase in bifurcated GlcNac structure, and consequently, increased ADCC activity of the antibodies (Umana et al. (1999) Nat. Biotech. 17:176-180). Alternatively, the fucose residues of the antibody may be cleaved using a fucosidase enzyme. For example, α-L-fucosidase removes fucosyl residues from the antibody (Tarentino, A et al. (1975) Biochem. 14:5516-23).

[0320] As described in the exemplary embodiments, the Fc region of the antibody is mutated to impair FcR binding and optionally to impair complement binding. These mutations include those found in those exemplary antibodies. These mutations include any or all of L234A / L235A and L234A / L235A / E269R / K322A (IgG1 Fc); and V234A / G237A / P238s.V309L / A330S / P331S (IgG2 Fc).

[0321] Nucleic acid molecule encoding ADC according to the present invention The present invention further provides nucleic acids encoding ADCs according to the present invention (in which case the anti-inflammatory agent in the ADC is a peptide). Nucleic acids may exist in whole cells, cell lysates, or in partially purified or substantially pure forms. Nucleic acids are “isolated” or “substantially pure” when purified from other cellular components or other contaminants, e.g., other cellular nucleic acids or proteins, by standard techniques including alkali / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis and other techniques well known in the art. See F. Ausubel, et al., ed. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. Nucleic acids according to at least some embodiments of the present invention may be, for example, DNA or RNA, and may or may not contain intron sequences. In preferred embodiments, the nucleic acid is a cDNA molecule.

[0322] Ex vivo use of ADC according to the present invention According to at least some embodiments, immune cells, such as monocytes or myeloid cells, T cells, and other hematopoietic cells, can be ex vivo contacted with the subject ADC to induce an anti-inflammatory response, and then the contacted cells can be injected, for example, into a patient with an allergic, autoimmune, or inflammatory condition for which reduction of inflammation is therapeutically desired, to modulate the immune response.

[0323] Exemplary Uses of Subjective ADCs and Pharmaceutical Compositions Containing Them for the Treatment of Autoimmune Diseases The ADC of Formula 1 and novel steroids described herein may be used to treat immune system-related disorders. Optionally, immune system-related conditions include previously identified autoimmune or inflammatory diseases such as transplant rejection, severe asthma, colitis or IBD, and graft-versus-host disease. Optionally, the treatment may be combined with another component useful for treating immune system-related conditions.

[0324] Therefore, the treatment of multiple sclerosis using the subject ADC may be used in combination with any known therapeutic agent or treatment for multiple sclerosis, such as, for example, those described herein at the discretion of the user.

[0325] Therefore, the treatment of rheumatoid arthritis or other arthritis conditions using the subject ADC may be used in combination with any known therapeutic agent or treatment for rheumatoid arthritis, such as, for example, those described herein by choice.

[0326] Therefore, the treatment of IBD using the subject ADC can be used in combination with any known therapeutic agent or treatment for IBD, such as, for example, those described herein at the discretion of the user.

[0327] Therefore, the treatment of psoriasis using the subject ADC can be used in combination with any known therapeutic agent or treatment for psoriasis, such as, for example, those described herein at the discretion of the user.

[0328] Therefore, the treatment of type 1 diabetes using the subject ADC can be used in combination with any known therapeutic agent or treatment for type 1 diabetes, such as, for example, those described herein at the discretion of the user.

[0329] Therefore, the treatment of uveitis using the subject ADC can be used in combination with any known therapeutic agent or treatment for uveitis, such as, for example, those described herein at the discretion of the user.

[0330] Therefore, the treatment of psoriasis using the subject ADC can be used in combination with any known therapeutic agent or treatment for psoriasis, such as, for example, those described herein at the discretion of the user.

[0331] Therefore, the treatment of Sjögren's syndrome using the subject ADC can be used in combination with any known therapeutic agent or treatment for Sjögren's syndrome, such as, for example, those described herein at the discretion of the user.

[0332] Therefore, the treatment of systemic lupus erythematosus using the subject ADC can be used in combination with any known therapeutic agent or treatment for systemic lupus erythematosus, such as, for example, those described herein at the discretion of the user.

[0333] Therefore, the treatment of GVHD using the subject ADC can be used in combination with any known therapeutic agent or treatment for GVHD, such as, for example, those described herein at the discretion of the user.

[0334] Therefore, the treatment of chronic or acute infections and / or associated hepatotoxicity, such as hepatitis, using the subject ADC may be used in combination with any known therapeutic agent or treatment for chronic or acute infections and / or associated hepatotoxicity, such as, for example, those optionally described herein.

[0335] Therefore, the treatment of chronic or acute severe asthma using the subject ADC can be used in combination with any known therapeutic agent or treatment for severe asthma, such as, for example, those described herein at the discretion of the user.

[0336] Therefore, the treatment of chronic or acute giant cell arteritis using the subject ADC can be used in combination with any known therapeutic agent or treatment for giant cell arteritis, such as, for example, those described herein at the discretion of the user.

[0337] Therefore, the treatment of chronic or acute ANKA vasculitis using the subject ADC can be used in combination with any known therapeutic agent or treatment for ANKA vasculitis, such as, for example, those described herein at the discretion of the user.

[0338] Therefore, the treatment of chronic or acute IBD (colitis and Crohn's disease) using the subject ADC can be used in combination with any known therapeutic agent or treatment for ANKA vasculitis, such as, for example, those described herein at the discretion of the user.

[0339] Even in this case, it should be understood that the disease conditions identified and the treatments proposed herein are illustrative and not exhaustive.

[0340] In the above treatment, preferably, the ADC according to the present invention is administered to a subject having one of the above or other autoimmune or inflammatory pathological conditions, thereby preventing or improving the symptoms of the disease.

[0341] Pharmaceutical composition In another aspect, the present invention provides compositions, such as pharmaceutical compositions, comprising one or a combination of ADCs of Formula 1 or novel steroids according to the present invention, and optionally another immunosuppressant or other active agent. Accordingly, the present invention features a pharmaceutical composition comprising a therapeutically effective amount of ADCs of Formula 1 or novel steroids according to the present invention. In particular, the present invention features a pharmaceutical composition comprising a therapeutically effective [anti-inflammatory] amount of at least one of Formula 1 or novel steroids according to the present invention.

[0342] The term "therapeutic dose" refers to the amount of the agent according to the present invention that is effective in treating a disease or disorder in a mammal. The therapeutic agents of the present invention can be provided to a target alone or as part of a pharmaceutical composition mixed with a pharmaceutically acceptable carrier. In many cases, the ADC according to the present invention is used in combination with other immunotherapeutic agents or other therapeutic agents that are useful in treating a particular pathological condition.

[0343] A composition is referred to as “pharmaceutically acceptable” if its administration is tolerable by the recipient patient. As used herein, “pharmaceutically acceptable carrier” includes any physiologically compatible solvent, dispersion medium, coating, antimicrobial and antifungal agents, isotonic agents and absorption retarders, etc. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epithelial administration (e.g., by injection or inhalation).

[0344] Such compositions include sterile water, buffered saline (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength, and optionally additives, such as surfactants and solubilizers (e.g., polysorbate 20, polysorbate 80), antioxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., thymerzol, benzyl alcohol), and fillers (e.g., lactose, mannitol). Non-aqueous solvents or vehicles may also be used, as detailed below.

[0345] Suitable aqueous and non-aqueous carriers that may be used in pharmaceutical compositions according to at least some embodiments of the present invention include water, ethanol, polyols (glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Appropriate fluidity can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Depending on the route of administration, the active compound, i.e., a monoclonal or polyclonal antibody and antigen-binding fragment and / or a complex containing the same, and / or an alternative skeleton or bispecific molecule, may be coated in the material to protect the compound from the action of acids and other natural conditions that may inactivate the compound. Pharmaceutical compounds according to at least some embodiments of the present invention may contain one or more pharmaceutically acceptable salts. A "pharmaceutically acceptable salt" is a salt that retains the desired biological activity of the parent compound without conferring any unwanted toxic effects (see, for example, Berge, SM, et al. (1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from non-toxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, and phosphorous acid, as well as those derived from non-toxic organic acids such as aliphatic mono- and di-carboxylic acids, phenyl-substituted alkanos, hydroxyalkanoics, aromatic acids, and aliphatic and aromatic sulfonic acids. Base addition salts include those derived from alkaline earth metals such as sodium, potassium, magnesium, and calcium, as well as those derived from non-toxic organic amines such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, and procaine.

[0346] Pharmaceutical compositions according to at least some embodiments of the present invention may also contain pharmaceutically acceptable antioxidants. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfite, sodium metabisulfite, and sodium sulfite; (2) oil-soluble antioxidants such as ascorbic acid palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, and α-tocopherol; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid.

[0347] These compositions may also contain adjuvants such as preservatives, humectants, emulsifiers, and dispersants. Prevention of the presence of microorganisms can be ensured by both the sterilization procedures described above and by the inclusion of various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, and sorbic acid. Inclusion of isotonic agents such as sugars and sodium chloride in the composition may also be desirable. Furthermore, sustained absorption of the injectable pharmaceutical form may be achieved by including absorption-delaying agents such as aluminum monostearate and gelatin.

[0348] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the immediate preparation of injectable sterile solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art. Unless any conventional media or agent is unsuitable for the active compound, its use in pharmaceutical compositions according to at least some embodiments of the present invention is intended. Complementary active ingredients may also be incorporated into the composition.

[0349] Therapeutic compositions generally must be sterile and stable under manufacturing and storage conditions. Compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations. Carriers may be solvents or dispersion media containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Adequate fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. In many cases, it is preferable to include isotonic agents, such as sugar, polyalcohols (e.g., mannitol, sorbitol, or sodium chloride), in the composition. Sustained absorption of injectable compositions can be achieved by including absorption-delaying agents in the composition, such as monostearate and gelatin. Sterile injectable solutions can be prepared by incorporating the required amount of the active compound into a suitable solvent having, if necessary, one or a combination of the components listed above, followed by microfiltration sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other required components from those listed above. For sterile powders for the preparation of sterile injection solutions, preferred preparation methods are vacuum drying and freeze-drying (lyophilization), which result in a powder in which the active ingredient is added to any additional desired components from its pre-sterilized filtered solution.

[0350] Sterile injectable solutions can be prepared by incorporating the required amount of the active compound into a suitable solvent having, if necessary, one or a combination of the components listed above, followed by microfiltration sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other required components from those listed above. For sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze-drying (lyophilization), which result in a powder in which the active component is added to any additional desired components from its pre-sterile filtered solution.

[0351] The compositions of the present invention can be administered by one or more routes of administration using one or more of the various methods known in the art. As will be understood by those skilled in the art, the route and / or form of administration will vary depending on the desired outcome. Preferred routes of administration of therapeutic agents according to at least some embodiments of the present invention include intravascular delivery (e.g., injection or infusion), intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, oral, enteral, rectal, pulmonary (e.g., inhalation), intranasal, topical (including percutaneous, buccal and sublingual), intravesical, intravitreous, intraperitoneal, intravaginal, intracerebral delivery (e.g., ventricular, intracerebral, and convective-enhancing diffusion), CNS delivery (e.g., subarachnoid, perispinal, and intraspinal), or parenteral (including subcutaneous, intramuscular, intravenous, and intradermal), transmucosal (e.g., sublingual administration), or implantation, or administration by other parenteral routes of administration, e.g., injection or infusion, or by other routes of delivery and / or forms of administration known in the art. As used herein, the term "parenteral administration" means a mode of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intra-arterial, subarachnoid, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions. In certain embodiments, proteins, therapeutic agents, or pharmaceutical compositions according to at least some embodiments of the present invention may be administered intraperitoneally or intravenously.

[0352] Alternatively, the ADC according to the present invention can be administered via a route other than parenteral administration, such as a topical, epithelial, or mucosal administration route, for example, intranasally, orally, intravaginally, rectally, sublingually, or topically.

[0353] The therapeutic compositions comprising the ADC according to the present invention can be administered by medical devices known in the art. For example, in preferred embodiments, therapeutic compositions according to at least some embodiments of the present invention can be administered using needle subcutaneous injection devices such as those disclosed in U.S. Patents 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556. Examples of well-known implants and modules useful for the present invention include: U.S. Patent No. 4,487,603 disclosing an implantable microinfusion pump for administering drugs at a controlled rate; U.S. Patent No. 4,486,194 disclosing a therapeutic device for administering drugs through the skin; U.S. Patent No. 4,447,233 disclosing a drug infusion pump for delivering drugs at a precise infusion rate; U.S. Patent No. 4,447,224 disclosing a variable flow rate implantable infusion device for continuous drug delivery; U.S. Patent No. 4,439,196 disclosing a permeable drug delivery system having multiple chamber compartments; and U.S. Patent No. 4,475,196 disclosing a permeable drug delivery system. These patents are incorporated herein by reference. Many other such implants, delivery systems, and modules are known to those skilled in the art.

[0354] In certain embodiments, ADCs may be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) ​​excludes many highly hydrophilic compounds. To ensure that therapeutic compounds according to at least some embodiments of the present invention cross the BBB (if desired), they may be formulated, for example, in liposomes. For methods of producing liposomes, see, for example, U.S. Patents 4,522,811; 5,374,548 and 5,399,331. Liposomes may contain one or more moieties that are selectively transported into specific cells or organs, thereby enhancing targeted drug delivery (see, for example, VVRanade (1989) J. Clin. Pharmacol. 29:685). Exemplary targeted moieties include folic acid or biotin (see, for example, U.S. Patent 5,416,016 by Low et al.). Examples include mannoside (Umezawa et al., (1988) Biochem. Biophys. Res.Commun. 153:1038); antibodies (PGBloeman et al. (1995) FEBS Lett. 357:140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180); surfactant protein A receptor (Briscoe et al. (1995) Am. J Physiol. 1233:134); pl20 (Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K. Keinanen; M. Laukkanen (1994) FEBS Lett. 346:123; J. J. Killion; and I. J. Fidler (1994) Immunomethods 4:273.

[0355] As used herein, “pharmaceutically acceptable carriers” include any physiologically compatible solvent, dispersion medium, coating, antimicrobial and antifungal agents, isotonic agents and absorption retarders, etc. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epithelial administration (e.g., by injection or inhalation). Depending on the route of administration, the active compound, i.e., a soluble polypeptide complex containing the external domain of the VISTA antigen, an antibody, an immune complex, an alternative skeleton, and / or a bispecific molecule, may be coated with a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound. Pharmaceutical compounds according to at least some embodiments of the present invention may include one or more pharmaceutically acceptable salts. A “pharmaceutically acceptable salt” means a salt that retains the desired biological activity of the parent compound and does not confer any unwanted toxic effects (see, for example, Berge, SM, et al. (1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from non-toxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, and phosphorous acid, as well as those derived from non-toxic organic acids such as aliphatic mono- and di-carboxylic acids, phenyl-substituted alkanes, hydroxyalkanoates, aromatic acids, and aliphatic and aromatic sulfonic acids. Base addition salts include those derived from alkaline earth metals such as sodium, potassium, magnesium, and calcium, as well as those derived from non-toxic organic amines such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, and procaine.

[0356] Pharmaceutical compositions according to at least some embodiments of the present invention may also contain pharmaceutically acceptable antioxidants. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfite, sodium metabisulfite, and sodium sulfite; (2) oil-soluble antioxidants such as ascorbic acid palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, and α-tocopherol; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid. Examples of suitable aqueous and non-aqueous carriers that may be used in pharmaceutical compositions according to at least some embodiments of the present invention include water, ethanol, polyols (glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Appropriate fluidity can be maintained, for example, by using coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants.

[0357] These compositions may also contain adjuvants such as preservatives, humectants, emulsifiers, and dispersants. Prevention of the presence of microorganisms can be ensured by both the sterilization procedures described above and by the inclusion of various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, and sorbic acid. Inclusion of isotonic agents such as sugars and sodium chloride in the composition may also be desirable. Furthermore, sustained absorption of the injectable pharmaceutical form may be achieved by including absorption-delaying agents such as aluminum monostearate and gelatin.

[0358] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the immediate preparation of injectable sterile solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art. Unless any conventional media or agent is unsuitable for the active compound, its use in pharmaceutical compositions according to at least some embodiments of the present invention is intended. Complementary active ingredients may also be incorporated into the composition.

[0359] Therapeutic compositions generally must be sterile and stable under manufacturing and storage conditions. Compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations. Carriers may be solvents or dispersion media containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Adequate fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. In many cases, it is preferable to include isotonic agents, such as sugar, polyalcohols (e.g., mannitol, sorbitol, or sodium chloride), in the composition. Sustained absorption of injectable compositions can be achieved by including absorption-delaying agents in the composition, such as monostearate and gelatin. Sterile injectable solutions can be prepared by incorporating the required amount of the active compound into a suitable solvent having, if necessary, one or a combination of the components listed above, followed by microfiltration sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other required components from those listed above. For sterile powders for the preparation of sterile injection solutions, preferred preparation methods are vacuum drying and freeze-drying (lyophilization), which result in a powder in which the active ingredient is added to any additional desired components from its pre-sterilized filtered solution.

[0360] Sterile injectable solutions can be prepared by incorporating the required amount of the active compound into a suitable solvent having, if necessary, one or a combination of the components listed above, followed by microfiltration sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other required components from those listed above. For sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze-drying (lyophilization), which result in a powder in which the active component is added to any additional desired components from its pre-sterile filtered solution.

[0361] The amount of active ingredient that can be combined with a carrier material to produce a single dosage form varies considerably depending on the target being treated and the specific mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form is usually the amount of the composition that produces the therapeutic effect. Generally, in combinations with pharmaceutically acceptable carriers, this amount is in the range of about 0.01 percent to about 99 percent, preferably about 0.1 percent to about 70 percent, and most preferably about 1 percent to about 30 percent, of 100 percent being the active ingredient.

[0362] The drug regimen is adjusted to provide the optimal desired response (e.g., therapeutic response). For example, a single bolus may be administered, multiple divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the urgent requirements of the treatment situation. It is particularly advantageous to formulate parenteral compositions into unit dosage forms to facilitate administration and equalize the dosage. As used herein, a unit dosage form refers to a physically separate unit suitable as a unit dose for treating a subject, each unit containing a predetermined amount of the active compound calculated to produce the desired therapeutic effect in relation to the required pharmaceutical carrier. The specifications of the unit dosage forms according to at least some embodiments of the present invention are determined by and directly depend on (a) the unique characteristics of the active compound and the specific therapeutic effect to be achieved, and (b) the inherent limitations in the art of formulating such active compounds to treat susceptibility in an individual.

[0363] With regard to the administration of ADCs disclosed herein, in some embodiments, the dose range generally includes an amount of ADC that delivers an equivalent or lesser amount of anti-inflammatory agent, e.g., a steroid such as dexamethasone, in terms of therapeutic effect compared to administering a specific anti-inflammatory agent, e.g., a steroid such as dexamethasone, via a conventional route to treat a particular pathological condition, i.e., when the steroid is administered in a naked or uncomplexed form. In exemplary embodiments, apart from reducing or eliminating the adverse side effects of AIs such as steroids, as can be expected based on results obtained so far, the ADCs of the present invention are more effectively delivered to the target immune cells of interest and have a less tendency to reach non-target cells, thereby reducing the required effective dose of steroid and / or reducing the impact on non-target cells. Therefore, the dose range generally includes an amount of ADC that delivers a reduced amount of anti-inflammatory agent, e.g., 10-90% of the amount of dexamethasone, in terms of therapeutic effect compared to administering an AI via a conventional route, i.e., when the steroid is administered in a naked or uncomplexed form.

[0364] The ADCs disclosed herein can be administered multiple times. The interval between single doses may be, for example, every 3-5 days, weekly, or bi-weekly. In some cases, the dose is adjusted to achieve the desired level of plasma steroid concentration. Determining an effective drug regimen for treatment or prophylaxis with the subject ADC should be relatively straightforward compared to other ADCs where internal antibodies induce biological or therapeutic effects, because the therapeutic activity of the subject ADC is entirely governed by its anti-inflammatory payload. (Essentially, the antibodies target and direct only the internalization of the subject ADC to specific immune cells.)

[0365] Alternatively, ADCs can be administered as sustained-release formulations, in which case less frequent administration is required. Dosage and frequency of administration may vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic administration, relatively low doses may be administered over a long period at relatively infrequent intervals. Some patients may continue treatment for the rest of their lives. In therapeutic administration, relatively high doses at relatively short intervals may be required until the progression of the disease is reduced or halted, and preferably until the patient shows partial or complete remission of the symptoms of the disease. Thereafter, the patient may be administered a prophylactic regimen. As mentioned above, the ADCs in question are preferred for such applications because they remain in peripheral circulation for a very short period, do not bind to non-immune cells, and do not induce toxicity in non-target cells to a degree that can be sensed.

[0366] The actual dose levels of the active ingredient in the pharmaceutical compositions of the present invention may vary considerably to obtain an amount of the active ingredient effective in achieving the desired therapeutic response with respect to a particular patient, composition, and mode of administration without being toxic to the patient. The selected dose level depends on a variety of pharmacokinetic factors, including the activity of the particular composition of the present invention used, the route of administration, the time of administration, the excretion rate of the particular compound used, the duration of treatment, other drugs, compounds, and / or substances used in combination with the particular composition used, the age, sex, weight, condition, overall health status, and prior medical history of the patient being treated, as well as similar factors well known in the medical field.

[0367] Exemplary Embodiments The present invention provides an antibody-drug conjugate (ADC) comprising an antibody or antigen-binding fragment ("A") having an antigen-binding domain that specifically binds to the human V-domain Ig suppressor (human VISTA) for T cell activation, a cleavable and / or non-cleavable linker ("L"), and at least one small molecule anti-inflammatory agent ("AI"), optionally "Q", i.e., a "hetero-bifunctional group" or "hetero-trifunctional group" which is an optionally used chemical moiety to link the linker to an anti-VISTA antibody or antibody fragment and at least one small molecule anti-inflammatory agent ("AI") (typically a steroid), wherein the ADC is represented by the following formula: "A-(QL-AI)" n " or "(AI-LQ) n -A" In the formula, "n" is at least 1, and when an antibody or ADC, or a composition containing them, is administered to a target requiring it, it is preferentially delivered to immune cells expressing VISTA, optionally monocytes or myeloid cells, thereby functionally internalizing the low molecular weight anti-inflammatory agent in the immune cells under physiological conditions (approximately pH 7.5), preferably an anti-VISTA antibody or antigen-binding fragment, when used in vivo, in short in serum at physiological pH (approximately pH 7.5). It has a vivo serum half-life and, optionally, at physiological pH (approximately pH 7.5), in rodents (human VISTA knock-in mice or rats) it has an in vivo serum half-life of approximately 70 hours or less, approximately 60 hours or less, 50 hours or less, 40 hours or less, 30 hours or less, 24 hours or less, 22-24 hours or less, 20-22 hours or less, 18-20 hours or less, 16-18 hours or less, 14-16 hours or less, 12-14 hours or less, 10-12 hours or less, 8-10 hours or less, 6-8 hours or less, 4-6 hours or less, 2-4 hours or less, 1-2 hours or less, 0.5-1.0 hours or less, or 0.1-0.5 hours or less, and / or in primates (e.g., humans or cynomolgus monkeys) it has an in vivo serum half-life of approximately 3, 2.5, or 2.3 ± 0.7 days or less.

[0368] Exemplary cleavable and non-cleavable linkers that can be incorporated into the subject ADC have been previously identified herein and are well known in the art. Specific types of linkers that can be used in the ADC according to the present invention and examples of such types are further identified below.

[0369] As described above, the present invention aims to include any small molecule anti-inflammatory agent (AI) that requires intracellular integration for efficacy (anti-inflammatory activity) as an anti-inflammatory agent (AI) contained in the anti-VISTA ADC according to the present invention. In particular, the present invention includes synthetic glucocorticoid receptor agonists as AI (e.g., dexamethasone, prednisolone, budesonide, beclomethasone, betamethasone, cortisol, cortisone acetate, 16-α-hydroxyprednisolone, dexamethasone, difluorazone, flumetazone, flunisolide, fluocinolone acetonide, fluticasone propionate, ciclesonide, methylprednisolone, prednisone, prednisolone, mometasone, triamcinolone acetonide, etc.). As mentioned above, these steroid compounds are highly effective in suppressing inflammation associated with various pathological conditions such as autoimmune diseases, inflammatory diseases, cancer, and infections. However, their usefulness in the chronic treatment of these diseases is limited due to severe side effects, which are mitigated when they are incorporated into anti-VISTA ADCs according to the present invention.

[0370] In particular, the present invention comprises an anti-VISTA ADC according to the present invention, wherein AI comprises a steroid (glucocorticoid agonist) having the following general structure: [ka] In the formula, X or Z may be phenyl, a 3-6 membered heterocycle, a cycloalkyl, a spiroalkyl, a spiroheterocycloalkyl, [1.1.1]bicyclopentane, bicyclo[2.2.2]octane, or cubane, each of which may be substituted with 1-4 heteroatoms independently selected from N, S, and O, and optionally 1-4 C 1-3 Further substituted with alkyl; The connection from X to Z may occupy any available position on X and Z; Y may be CHR1, O, S, or NR1; E may be CH2 or O; G may be CH2 or NR1; R1 may be H, a lower or branched alkyl, aryl, or heteroaryl having 1 to 8 carbon atoms. If an aryl or heteroaryl ring is substituted, the substituent may be alkyl, haloalkyl, halogen, biphenyl, nitro, nitrile, -OH, -O-alkyl, -NH2, alkylamino, dialkylamino, thiol, thioalkyl, guanidine, urea, carboxylate, alkoxyl, carboxamide, carboxylic acid ester, alkyl-C(O)O-, alkylamino-C(O)-, and dialkylamino-C(O)-; If R1=H, R2 may be H, a lower or branched alkyl, aryl, or heteroaryl having 1 to 8 carbon atoms. If an aryl or heteroaryl ring is substituted, the substituent may be alkyl, haloalkyl, halogen, biphenyl, nitro, nitrile, -OH, -O-alkyl, -NH2, alkylamino, dialkylamino, thiol, thioalkyl, guanidine, urea, carboxylate, alkoxyl, carboxamide, carboxylic acid ester, alkyl-C(O)O-, alkylamino-C(O)-, and dialkylamino-C(O)-; If R1 is H, a lower or branched alkyl or heteroaryl with 1 to 8 carbon atoms, then R2 is [(C=O)CH2(W)NHC=O] m -The functional group may be selected from VJ, and W may be H or [(CH2) n R3] n It may also be a branched alkyl chain terminated at R3 or 1 to 13 units of polyethylene glycol group OCH2CH2O; R3 may be H, or selected from OH, O-alkyl, NH2, NH-alkyl, N-dialkyl, SH, S-alkyl, guanidine, urea, carboxylic acid, carboxamide, carboxylic acid ester, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, wherein the substituent may be alkyl, haloalkyl, halogen, biphenyl, nitro, nitrile, -OH, -O-alkyl, -NH2, alkylamino, dialkylamino, thiol, thioalkyl, guanidine, urea, carboxylic acid, alkoxyl, carboxamide, carboxylic acid ester, alkyl-C(O)O-, alkylamino-C(O)-, and dialkylamino-C(O)-; The substituent NR1R2 may occupy any available position on Z; R2 is also C(=O)OCH2-p-aminophenyl[(C=O)CH(W)NHC=O] m -It may also be VJ, and W is H or [(CH2) n R3] n It may also be a branched alkyl chain terminated at R3, 1 to 13 units of polyethylene glycol group OCH2CH2O or C(=O)OCH2-p-aminophenyl-VJ; V may be selected from an alkyl chain having 1 to 8 carbon atoms, a polyethylene glycol group OCH2CH2O having 1 to 13 units, or a lower or branched alkyl, aryl, or heteroaryl ring having 1 to 8 carbon atoms. If an aryl or heteroaryl ring is substituted, the substituent may be an amino acid sequence of 1 to 3 residues selected from alkyl, haloalkyl, halogen, biphenyl, nitro, nitrile, -OH, -O-alkyl, -NH2, alkylamino, dialkylamino, thiol, thioalkyl, guanidine, urea, carboxylate, alkoxyl, carboxamide, carboxylic acid ester, alkyl-C(O)O-, alkylamino-C(O)-, dialkylamino-C(O)-, and Gly, Asn, Asp, Gln, Leu, Lys, Ala, βAla, Phe, Val, or Cit; J is a reactive group selected from -NH2, N3, thio, cyclooctin, -OH, -CO2H, and trans-cyclooctin; [ka] In the formula, R 32 is Cl, Br, F, mesylate or tosylate, and R 33 is Cl, Br, I, F, OH, -ON-succinimidyl, -O-(4-nitrophenyl), -O-pentafluorophenyl, or -O-tetrafluorophenyl, and R 34 is H, Me, or tetrazine-H or Me; Q may be H, P(O)OR4, R4 may be H or a lower 1-10 alkyl group, C(O)R6, and R6 may be a lower or branched alkyl group having 1-8 carbon atoms, or [(C=O)NR4CH n NR4(C=O)OCH m ] m -VVJ, n=1-8, m=1-6, R4=H, alkyl or branched alkyl; A1 and A2 may be H or halogens, and unless otherwise specified, all possible stereoisomers are included.

[0371] I. Exemplary Linker As mentioned above, different linkers may be incorporated into the ADC according to the present invention. Such linkers are previously identified in the definition section that defines “linker”. Furthermore, exemplary linkers that may be incorporated into the ADC according to the present invention are shown below: A. Self-destructing linker ADC (I) [ka] During the ceremony, Ab=antibody L = Linker AA = single, double, or triple amino acid sequence [ka] REG is independently selected from the group consisting of hydrogen, alkyl, biphenyl, -CF3, -NO2, -CN, fluoro, bromo, chloro, alkoxyl, alkylamino, dialkylamino, alkyl-C(O)O-, alkylamino-C(O)-, and dialkylaminoC(O)-. (II) [ka] During the ceremony, Ab=antibody L = Linker AA = single, double, or triple amino acid sequence [ka] REG is independently selected from the group consisting of hydrogen, alkyl, biphenyl, -CF3, -NO2, -CN, fluoro, bromo, chloro, alkoxyl, alkylamino, dialkylamino, alkyl-C(O)O-, alkylamino-C(O)-, and dialkylaminoC(O)-. (III) [ka] During the ceremony, Ab=antibody L = Linker AA = single, double, or triple amino acid sequence, or absent. [ka] REG is independently selected from the group consisting of hydrogen, alkyl, biphenyl, -CF3, -NO2, -CN, fluoro, bromo, chloro, alkoxyl, alkylamino, dialkylamino, alkyl-C(O)O-, alkylamino-C(O)-, and dialkylaminoC(O)-. RT=AA or [ka] (IV) [ka] During the ceremony, Ab=antibody L = Linker AA = single, double, or triple amino acid sequence [ka] (V) [ka] During the ceremony, Ab=antibody L = Linker AA = single, double, or triple amino acid sequence, or absent. [ka] RT=AA or [ka] B. Amino Acid (AA) Linker (I) Sequences that are cleaved by cathepsin a. Single amino acid linker [ka] b. Dipeptide linker [ka] [ka] c. Tripeptide linker [ka] [ka] [ka] (I) Regmine cleavage linker [ka] During the ceremony, L = Linker Ab=antibody [ka]

[0372] II. Exemplary Antibody Complexation Strategies Various conjugation strategies can be used to conjugate the anti-VISTA antibody to the linker and payload (steroid or other anti-inflammatory compound). Detailed synthesis methods for generating exemplary ADCs and linker payloads are shown in the examples. Furthermore, exemplary conjugation strategies are shown below: (I) Payload-Linker-J In the formula, the payload is: [ka] Linker = Q, R1 or R2, J is a functional group suitable for reacting with complementary functional groups on Ab to form an antibody-drug complex. J is selected from the following: [ka] [ka] This indicates the connection point of J to the linker selected from Q, R1, or R2. In the formula, R 32 is Cl, Br, F, mesylate or tosylate, and R 33 is Cl, Br, I, F, OH, -ON-succinimidyl, -O-(4-nitrophenyl), -O-pentafluorophenyl, or -O-tetrafluorophenyl, and R 34 is H, Me, or pyridyl;

[0373] The -OH group can be esterified with the carboxyl group of the antibody, for example, the aspartic acid or glutamic acid side chain.

[0374] The -CO2H group can be esterified with the -OH group of the antibody or amidated with an amino group (e.g., on the lysine side chain);

[0375] The N-hydroxysuccinimide group is a functionally activated carboxyl group that can be conveniently amidated by reaction with an amino group (e.g., derived from lysine);

[0376] The maleimide group can be complexed with an -SH group on an antibody (e.g., derived from cysteine ​​or from chemical modifications of the antibody to introduce a sulfhydryl functional group) in the Michael addition reaction;

[0377] If the antibody does not have a cysteine ​​or -SH group available for complexation, a cysteine ​​surrogate can be created by introducing a free thiol (-SH) group by reacting the ε-amino group of the lysine residue side chain with 2-iminothiolane or N-succinimidyl-3-(2-pyridyldithio)propionate ("SPDP"). The thiol group can then react with maleimide or other nucleophilic receptor groups to result in complexation.

[0378] The antibody Ab can be modified with 4-(N-maleimidomethyl)cyclohexanecarboxylic acid N-hydroxysuccinimide ester ("SMCC") or its sulfonated variant sulfo-SMCC (both available from Sigma-Aldrich) to introduce a maleimide group. Then, complexation can be performed using a drug-linker compound having an -SH group on the linker.

[0379] In copper-free "click chemistry," an azide group (-N3) is added across the strained cyclooctin to form a 1,2,3-triazole ring. The azide can be located on the antibody and the cyclooctin on the drug linker moiety, or vice versa. The preferred cyclooctin group is dibenzocyclooctin (DBCO).

[0380] Non-natural amino acids can be introduced into antibodies, providing a function for complexing with the reactive functional group of the drug moiety. For example, the non-natural amino acid p-acetylphenylalanine can be incorporated into an antibody or other polypeptide. The ketone group of p-acetylphenylalanine can serve as a complexing site via oxime formation with the hydroxylamino group of the linker-drug moiety. Alternatively, the non-natural amino acid p-azidophenylalanine (or p-azidomethyl-1-phenylalanine) can be incorporated into an antibody to provide an azide functional group for complexing via click chemistry with DBCO, forming a 1,2,3-triazole ring.

[0381] Another example would be the incorporation of unnatural amino acids, including norbornene, transcyclooctene, or cyclopropene, strained alkenes that can undergo reverse electron-demanding Diels-Alder "click chemistry" reactions with tetrazine to form bicyclic diazine products.

[0382] Another complexation technique uses the enzyme transglutaminase (preferably bacterial transglutaminase or BTG derived from Streptomyces mobaraensis). BTG forms an amide bond between the carboxamide side chain of glutamine (amine acceptor) and an alkylene amino group (amine donor), which can be, for example, the ε-amino group or 5-amino-n-pentyl group of lysine. In a typical complexation reaction, the glutamine residue is located on the antibody, and the alkylene amino group is located on the linker-drug moiety.

[0383] III. Exemplary Antibody Complexes Using the detailed synthesis methods disclosed above and in the examples, an ADC complex according to the present invention may be optionally generated, comprising an anti-VISTA antibody (bound to human VISTA at physiological pH and having the previously defined short PK), one or more cleavable and / or non-cleavable linkers, and optionally one or more payloads (steroids or other anti-inflammatory compounds) conjugated to a self-destructing linker. Several exemplary ADC structures and complexation methods are shown below. (i) Preferred example [ka] [ka] [ka] [ka] This indicates a binding site to the antibody or its antigen-binding fragment. [ka] This indicates a binding site to an antibody or its antigen-binding fragment via a sulfur atom of a cysteine ​​residue; or a pharmaceutically acceptable salt thereof, tautomer, stereoisomer, and / or mixture thereof. [ka] This indicates a linking point to the linker or AA.

[0384] IV. Exemplary Payload-Linker Structure Various payloads (steroids or other anti-inflammatory compounds) can be generated by linking them to the linker using the detailed synthesis methods disclosed above and in the examples. Some exemplary payload-linker structures are shown below: (I) Payload-Linker-J During the ceremony, Linker-protease cleavage sequence (AA) J = Alkylamine [ka] [ka] [ka] [ka] During the ceremony, Linker-protease cleavage sequence (AA) J = Bromoacetyl [ka] [ka] During the ceremony, Linker-protease cleavage sequence (AA) J = Dibenzylcyclooctin [ka] [ka] [ka] [ka] During the ceremony, Linker-protease cleavage sequence (AA) J = Hydroxysuccinimide [ka] [ka] [ka] [ka] [ka] During the ceremony, Linker-protease cleavage sequence (AA) J = Maleimide [ka] [ka] [ka] [ka] During the ceremony, Linker-protease cleavage sequence (AA) J=Tetrazine [ka] [ka] [ka] [ka] [ka] During the ceremony, Linker-protease cleavage sequence (AA) J=TG complex [ka] [ka] [ka] (II) Payload-Linker-J During the ceremony, Linker = protease-cleaving sequence (AA) + self-destructing linker para-aminobenzyl (PAB) J = Alkylamine [ka] [ka] [ka] [ka] During the ceremony, Linker = protease-cleaving sequence (AA) + self-destructing linker para-aminobenzyl (PAB) J = Bromoacetyl [ka] [ka] [ka] [ka] During the ceremony, Linker = protease-cleaving sequence (AA) + self-destructing linker para-aminobenzyl (PAB) J = Dibenzocyclooctin [ka] [ka] [ka] [ka] During the ceremony, Linker = protease-cleaving sequence (AA) + self-destructing linker para-aminobenzyl (PAB) J = Hydroxysuccinimide [ka] [ka] [ka] [ka] During the ceremony, Linker = protease-cleaving sequence (AA) + self-destructing linker para-aminobenzyl (PAB) J = Maleimide [ka] [ka] [ka] [ka] During the ceremony, Linker = protease-cleaving sequence (AA) + self-destructing linker para-aminobenzyl (PAB) J=Tetrazine [ka] [ka] [ka] [ka] During the ceremony, Linker = protease-cleaving sequence (AA) + self-destructing linker para-aminobenzyl (PAB) J=amine [ka] [ka] [ka] [ka] (III) Payload-Linker-J During the ceremony, Linker = Glucuronidase-cleavable sugar (GlcA) + self-destructing linker para-aminobenzyl (PAB) J = Alkylamine [ka] [ka] [ka] [ka] [ka] During the ceremony, Linker = Glucuronidase-cleavable sugar (GlcA) + self-destructing linker para-aminobenzyl (PAB) J = Bromoacetyl [ka] [ka] [ka] [ka] [ka] During the ceremony, Linker = Glucuronidase-cleavable sugar (GlcA) + self-destructing linker para-aminobenzyl (PAB) J = Dibenzocyclooctin [ka] [ka] [ka] [ka] [ka] [ka] During the ceremony, Linker = Glucuronidase-cleavable sugar (GlcA) + self-destructing linker para-aminobenzyl (PAB) J = Hydroxysuccinimide [ka] [ka] [ka] [ka] [ka] [ka] During the ceremony, Linker = Glucuronidase-cleavable sugar (GlcA) + self-destructing linker para-aminobenzyl (PAB) J = Maleimide [ka] [ka] [ka] [ka] [ka] [ka] During the ceremony, Linker = Glucuronidase-cleavable sugar (GlcA) + self-destructing linker para-aminobenzyl (PAB) J=Tetrazine [ka] [ka] [ka] [ka] [ka] During the ceremony, Linker = Glucuronidase-cleavable sugar (GlcA) + self-destructing linker para-aminobenzyl (PAB) J=amine [ka] [ka] [ka] [ka] [ka] (IV) Payload-Linker-J Alternative linker-J bond site (C11-OH) to the payload. INX-SM-3 is used as an example payload. Alkoxyamine [ka] [ka] [ka] [ka] [ka] (IV) Payload-Linker-J Alternative linker-J junction site (C17) to the payload. INX-SM-3 is used as an example payload. [ka] [ka] [ka] [ka] [ka] [ka]

[0385] V. Exemplary payload-linker-Ab complex (wherein INX-SM-3 is the exemplary payload) Using the detailed synthesis methods disclosed above and in the examples, various ADC complexes may be generated comprising an antibody or antibody fragment that binds to an antigen expressed by immune cells, optionally an anti-VISTA antibody or fragment having the pH-binding / PK properties described herein, one or more linkers, and one or more payloads (steroids or other anti-inflammatory compounds). Several exemplary ADCs, including an exemplary steroid payload (INX-SM-3), are shown below: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0386] Therapeutic applications of the steroid payload of Equation 1 and ADCs containing it. Synthetic glucocorticoid agonists, such as dexamethasone, prednisolone, budesonide, beclomethasone, betamethasone, cortisol, cortisone acetate, 16-α-hydroxyprednisolone, dexamethasone, difluorazone, flumethasone, flunisolide, fluocinolone acetonide, fluticasone propionate, ciclesonide, methylprednisolone, prednisone, prednisolone, mometasone, triamcinolone acetonide, or steroids of formula 1 may be produced as described above. In exemplary embodiments, the internally contained antibody comprises an anti-human VISTA antibody or fragment that binds to immune cells at physiological pH and has a short PK. However, in ADCs where the steroid is one of formula 1, the antibody or antibody fragment in the ADC may bind to another antigen expressed on immune cells, preferably an antigen expressed only on immune cells.

[0387] For example, these ADCs may be used in prophylactic and / or therapeutic treatments for inflammatory and inflammation-related diseases, including previously disclosed autoimmune disorders, inflammatory disorders, and cancer. In this case as well, the preferred application of the subject ADCs, including the steroid-containing ADC of Formula 1, is aimed at the treatment of chronic inflammation-related diseases.

[0388] As described herein, the subject ADC has been found to maintain long-term (PD) efficacy against the antibody half-life (PK), even though the pK of the anti-VISTA antibody contained in the ADC that binds to VISTA-expressing cells under physiological conditions is short and has not been modified to alter or optimize pH binding, i.e., generally about 2.3 days or less in cynomolgus monkeys, and about 70 hours or less, about 60 hours or less, 50 hours or less, 40 hours or less, 30 hours or less, 24 hours or less, 22-24 hours or less, 20-22 hours or less, 18-20 hours or less, 16-18 hours or less, 14-16 hours or less, 12-14 hours or less, 10-12 hours or less, 8-10 hours or less, 6-8 hours or less, 4-6 hours or less, 2-4 hours or less, 1-2 hours or less, 0.5-1.0 hours or less, or 0.1-0.5 hours or less in human VISTA-modified mice.

[0389] As shown herein, the ADC complex according to the present invention has been shown to exhibit a PK / PD ratio of at least 14:1 when evaluated in an in vivo model. Again, the applicant does not wish to be bound by its own beliefs, but it has been theorized that the ADC of Subject is delivered in very large quantities to target VISTA-expressing cells such as macrophages, T cells, and Tregs, as well as other VISTA-expressing immune cells with long cell turnover (weeks, months or more). Essentially, an accumulation effect is thought to occur, i.e., large quantities of the ADC of Subject is internalized in VISTA-expressing immune cells, i.e., due to the very high expression of VISTA, the ADC is slowly metabolized or cleaved by, for example, cellular enzymes, resulting in the gradual release of a therapeutically effective steroid payload within the cell over a long period.

[0390] The present invention further includes the following embodiments. Embodiment (1) A glucocorticoid agonist compound having the structure of formula (1): [ka] In the formula, X or Z may be phenyl, a 3-6 membered heterocycle, a cycloalkyl, a spiroalkyl, a spiroheterocycloalkyl, [1.1.1]bicyclopentane, bicyclo[2.2.2]octane, or cubane, each of which may be substituted with 1-4 heteroatoms independently selected from N, S, and O, and optionally 1-4 C 1-3 Further substituted with alkyl; The connection from X to Z may occupy any available position on X and Z; Y may be CHR1, O, S, or NR1; E may be CH2 or O; G may be CH2 or NR1; The glucocorticoid agonist compound wherein R1 may be H, a lower or branched alkyl, aryl, or heteroaryl ring having 1 to 8 carbon atoms. If the aryl or heteroaryl ring is substituted, the substituent may be alkyl, haloalkyl, halogen, biphenyl, nitro, nitrile, -OH, -O-alkyl, -NH2, alkylamino, dialkylamino, thiol, thioalkyl, guanidine, urea, carboxylate, alkoxyl, carboxamide, carboxylic acid ester, alkyl-C(O)O-, alkylamino-C(O)-, and dialkylamino-C(O)-; If R1=H, R2 may be H, a lower or branched alkyl, aryl, or heteroaryl having 1 to 8 carbon atoms. If an aryl or heteroaryl ring is substituted, the substituent may be alkyl, haloalkyl, halogen, biphenyl, nitro, nitrile, -OH, -O-alkyl, -NH2, alkylamino, dialkylamino, thiol, thioalkyl, guanidine, urea, carboxylate, alkoxyl, carboxamide, carboxylic acid ester, alkyl-C(O)O-, alkylamino-C(O)-, and dialkylamino-C(O)-; If R1 is H, a lower or branched alkyl or heteroaryl with 1 to 8 carbon atoms, then R2 is [(C=O)CH2(W)NHC=O] m -The functional group may be selected from VJ, and W may be H or [(CH2) n R3] n It may also be a branched alkyl chain terminated at R3 or 1 to 13 units of polyethylene glycol group OCH2CH2O; R3 may be H, or selected from OH, O-alkyl, NH2, NH-alkyl, N-dialkyl, SH, S-alkyl, guanidine, urea, carboxylic acid, carboxamide, carboxylic acid ester, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, wherein the substituent may be alkyl, haloalkyl, halogen, biphenyl, nitro, nitrile, -OH, -O-alkyl, -NH2, alkylamino, dialkylamino, thiol, thioalkyl, guanidine, urea, carboxylic acid, alkoxyl, carboxamide, carboxylic acid ester, alkyl-C(O)O-, alkylamino-C(O)-, and dialkylamino-C(O)-; The substituent NR1R2 may occupy any available position on Z; R2 is also C(=O)OCH2-p-aminophenyl[(C=O)CH(W)NHC=O] m -It may also be VJ, and W is H or [(CH2) n R3] n It may also be a branched alkyl chain terminated at R3, 1 to 13 units of polyethylene glycol group OCH2CH2O or C(=O)OCH2-p-aminophenyl-VJ; V may be selected from an alkyl chain having 1 to 8 carbon atoms, a polyethylene glycol group OCH2CH2O having 1 to 13 units, or a lower or branched alkyl, aryl, or heteroaryl ring having 1 to 8 carbon atoms. If an aryl or heteroaryl ring is substituted, the substituent may be an amino acid sequence of 1 to 3 residues selected from alkyl, haloalkyl, halogen, biphenyl, nitro, nitrile, -OH, -O-alkyl, -NH2, alkylamino, dialkylamino, thiol, thioalkyl, guanidine, urea, carboxylate, alkoxyl, carboxamide, carboxylic acid ester, alkyl-C(O)O-, alkylamino-C(O)-, dialkylamino-C(O)-, and Gly, Asn, Asp, Gln, Leu, Lys, Ala, βAla, Phe, Val, or Cit; J is a reactive group selected from -NH2, N3, thio, cyclooctin, -OH, -CO2H, and trans-cyclooctin; [ka] In the formula, R 32 is Cl, Br, F, mesylate or tosylate, and R 33 is Cl, Br, I, F, OH, -ON-succinimidyl, -O-(4-nitrophenyl), -O-pentafluorophenyl, or -O-tetrafluorophenyl, and R 34 is H, Me, or tetrazine-H or Me; Q may be H, P(O)OR4, R4 may be H or a lower 1-10 alkyl group, C(O)R6, and R6 may be a lower or branched alkyl group having 1-8 carbon atoms, or [(C=O)NR4CH n NR4(C=O)OCH m ] m -VVJ, n=1-8, m=1-6, R4=H, alkyl or branched alkyl; A1 and A2 may be H or halogen. Unless otherwise specified, all possible stereoisomers are claimed. (2) A glucocorticoid agonist compound according to Example 1, selected from any of the glucocorticoid agonist compounds disclosed in Example 3. (3) A glucocorticoid agonist compound selected from the compounds shown in Figure 11. (4) A glucocorticoid agonist compound selected from the INX-SM compounds disclosed herein. (5) A glucocorticoid agonist compound selected from the following: [ka] In the formula, X or Z is an annular sp 3 It may also be the phenyl equivalent [1.1.1]bicyclopentane or bicyclo[2.2.2]octane, where Y may be CH2 or O; W1 is CH2CH2CO2H, and W2 is H. (6) A glucocorticoid agonist compound according to any of the above embodiments, which is directly or indirectly bound to at least one cleavable or non-cleavable peptide and / or non-peptide linker (steroid-linker payload). (7) A compound (steroid-linker payload) comprising at least one cleavable or non-cleavable linker ("L"), optionally a chemical moiety ("Q"), i.e., a "hetero-bifunctional group" or "hetero-trifunctional group"), which is optionally used to link the linker in the compound to an antibody or antibody fragment, and at least one anti-inflammatory agent ("AI"), wherein AI is a glucocorticoid agonist compound according to any of embodiments (1) to (5), which may be represented by the following structures: QL-AI or AI-LQ. (8) The steroid-linker payload according to (6) or (7), wherein the linker is selected from the linkers disclosed herein. (9) PAB and / or amino acids or peptides, optionally 1 to 12 amino acids, optionally dipeptides, tripeptides, quatrapeptides, pentapeptides, and optionally Gly, Asn, Asp, Gln, Leu, Lys, Ala, Phe, Cit, Val, Val-Cit, Val-Ala, Val-Gly, Val-Gln, Ala-Val, Cit-Cit, Lys-Val-Cit, Asp-Val-Ala, Ala-Ala-Asn, Asp A steroid-linker payload according to (6), (7), or (8), comprising at least one cleavable or non-cleavable linker, comprising -Val-Ala, Ala-Val-Cit, Ala-Asn-Val, βAla-Leu-Ala-Leu, Lys-Val-Ala, Val-Leu-Lys, Asp-Val-Cit, Val-Ala-Val, and Ala-Ala-Asn; or optionally selected from at least one of GlcA, PAB, and Glu-Gly. (10) A steroid-linker payload (according to any of the embodiments described above) comprising at least one cleavable linker, and / or a self-destructing linker, is directly or indirectly bound to a glucocorticoid agonist steroid compound. (11) A glucocorticoid agonist steroid compound or steroid-linker payload according to any of the above embodiments, selected from either a glucocorticoid agonist compound or a steroid-linker payload compound disclosed in Example 3. (12) A glucocorticoid agonist (payload)-linker complex selected from the following: (i) INX-SM-3-GluGly-alkoxyamine, INX-SM-4-GluGly-alkoxyamine, INX-SM-53-GluGly-alkoxyamine, INX-SM-54-GluGly-alkoxyamine, INX-SM-56-GluGly-alkoxyamine, INX-SM-98-GluGly-alkoxyamine, INX-SM-6-GluGly-alkoxyamine, INX-SM-2-GluGly-alkoxyamine, INX-SM-57-GluGly-alkoxyamine, INX-SM-31-GluGly-alkoxyamine, INX-SM-32-GluGly-alkoxyamine, INX-SM-10-GluGly-alkoxyamine, IINX-S M-40-GluGly-alkoxyamine, INX-SM-34-GluGly-alkoxyamine, INX-SM-28-GluGly-alkoxyamine, INX-SM-27-GluGly-alkoxyamine, INX-SM-35-GluGly-alkoxyamine, INX-SM-8-GluGly-alkoxyamine, INX-SM-7-GluGly-alkoxyamine, INX-SM-33-GluGly-alkoxyamine, or glucocorticoid agonist (payload)-linker complex (where Glu-Gly is substituted with a different cleavable peptide linker, and / or another INX-SM payload is substituted with the INX-SM payload contained therein); or (ii) INX-SM-53-GluGly-bromoacetyl, INX-SM-3-GluGly-bromoacetyl, INX-SM-54-GluGly-bromoacetyl, INX-SM-1-GluGly-bromoacetyl, INX-SM-4-GluGly-bromoacetyl, INX-SM-2-GluGly-bromoacetyl, INX-SM-47-GluGly-bromoacetyl, INX-SM-7-GluGly-bromoacetyl, INX-SM-8-GluGly-bromoacetyl, INX-SM-56-GluGly-bromoacetyl, INX-SM-32-GluGly-bromoacetyl, INX-SM-6-GluGly-bromoacetyl, INX-SM-10-GluGly-bromoacetyl, INX-SM-3 3-GluGly-bromoacetyl, INX-SM-31-GluGly-bromoacetyl, INX-SM-35-GluGly-bromoacetyl, INX-SM-9-GluGly-bromoacetyl, INX-SM-28-GluGly-bromoacetyl, INX-SM-27-GluGly-bromoacetyl, INX-SM-34-GluGly-bromoacetyl, INX-SM-35-GluGly-bromoacetyl, IINX-SM-40-GluGly-bromoacetyl, or glucocorticoid agonist (payload)-linker complex (where Glu-Gly is substituted with a different cleavable peptide linker, and / or another INX-SM payload is substituted with the INX-SM payload contained therein); (iii) INX-SM-53-GluGly-dibenzocyclooctin, INX-SM-1-GluGly-dibenzocyclooctin, INX-SM-4-GluGly-dibenzocyclooctin, INX-SM-54-GluGly-dibenzocyclooctin, INX-SM-7-GluGly-dibenzocyclooctin, INX-SM-8-GluGly-dibenzocyclooctin, INX-SM-2-GluGly-dibenzocyclooctin, INX-SM-57-GluGly-dibenzocyclooctin, INX-SM-40-GluGly-dibenzocyclooctin, INX-SM-34-GluGly-dibenzocyclooctin, INX-SM-28-GluGly-dibenzocyclooctin, INX-SM-27-GluGly-dibenzocyclooctin, INX-SM-35-G luGly-dibenzocyclooctin, INX-SM-9-GluGly-dibenzocyclooctin, INX-SM-10-GluGly-dibenzocyclooctin, INX-SM-31-GluGly-dibenzocyclooctin, INX-SM-32-GluGly-dibenzocyclooctin, INX-SM-33-GluGly-dibenzocyclooctin, INX-SM-56-GluGly-dibenzocyclooctin, INX-SM-6-GluGly-dibenzocyclooctin, INX-SM-3-GluGly-dibenzocyclooctin, or glucocorticoid agonist (payload)-linker complex (where GluGly is substituted with another cleavable peptide linker, and / or another INX-SM payload is substituted with the INX-SM payload contained therein); or (iv) INX-SM-1-GluGly-NHS ester; INX-SM-31-GluGly-NHS ester; INX-SM-32-GluGly-NHS ester; INX-SM-33-GluGly-NHS ester; INX-SM-53-GluGly-NHS ester; INX-SM-7-GluGly-NHS ester; INX-SM-8-GluGly-NHS ester; INX-SM-2-GluGly-NHS ester; INX-SM-56-GluGly-NHS ester; INX-SM-6-GluGly-NHS ester; INX-SM-54-GluGly-NHS ester; INX-SM-4-GluGly-NHS ester; INX-SM-53-GluGly-NHS ester; INX-SM-3-GluGly-NH S ester; INX-SM-9-GluGly-NHS ester; INX-SM-40-GluGly-NHS ester; INX-SM-34-GluGly-NHS ester; INX-SM-28-GluGly-NHS ester; INX-SM-34-GluGly-NHS ester; INX-SM-28-GluGly-NHS ester; INX-SM-27-GluGly-NHS ester; INX-SM-35-GluGly-NHS es...

Claims

1. An antibody or antigen-binding fragment ("A") containing an antigen-binding region that specifically binds to the human V-domain Ig suppressor (human VISTA) for T cell activation, At least one cleavable or non-cleavable linker ("L"), Optionally, "Q" is a "hetero-bifunctional group" or "hetero-trifunctional group" which is a chemical moiety optionally used to connect the linker with the anti-VISTA antibody or antibody fragment, and Preferably, a low molecular weight, at least one anti-inflammatory agent ("AI") which requires intracellular integration for efficacy (anti-inflammatory activity). An antibody-drug conjugate (ADC) comprising, The ADC is given by the following formula: 「A-(Q-L-AI) ｎ 」または「(AI-L-Q) ｎ - A It is expressed as follows, where "n" is at least 1, Furthermore, when the ADC is administered to a target requiring it, it is preferentially delivered to one or more immune cells expressing VISTA, optionally monocytes, myeloid cells, T cells, Treg cells, NK cells, neutrophils, dendritic cells, macrophages, and endothelial cells, resulting in the functional internalization of the small molecular weight anti-inflammatory agent to one or more of the immune cells; (i) The anti-human VISTA antibody or antibody fragment preferentially binds to VISTA-expressing cells at a physiological pH (approximately 7.5); (ii) The anti-human VISTA antibody or antibody fragment has a pK of up to 70 hours in human VISTA knock-in rodents. Antibody-drug conjugate (ADC).

2. The antibody-drug conjugate (ADC) according to claim 1, wherein the AI ​​comprises a glucocorticosteroid.

3. The antibody-drug conjugate (ADC) according to claim 1, wherein the glucocorticosteroid comprises one of the following: 【Chemistry 1】

4. The antibody-drug conjugate (ADC) according to claim 1, wherein the glucocorticosteroid comprises 16-α-hydroxyprednisolone, dexamethasone, diflurazone, flumethasone, flunisolide, fluocinolone acetonide, fluticasone propionate, ciclesonide, methylprednisolone, prednisone, prednisolone, mometasone, triamcinolone acetonide or a derivative thereof.

5. An antibody-drug conjugate (ADC) according to any of the prior claims, having a pK of up to 3.5 to 4 days at physiological pH in cynomolgus monkeys or humans, or an antibody-drug conjugate (ADC) according to any of the prior claims, having a pK of up to 2.8 days ± 0.5 days at physiological pH in cynomolgus monkeys or humans.

6. An antibody-drug conjugate (ADC) according to any of the prior claims, having a maximum pK of 6 to 12 hours in human VISTA rodents at physiological pH.

7. The antibody-drug conjugate (ADC) according to any of the prior claims, wherein the PK of the antibody is determined by ELISA using a PKsolver program of option and by performing non-compartmental analysis (NCA) after intravenous bolus administration as described in Example 10.

8. An antibody-drug conjugate (ADC) according to any of the prior claims, comprising a linker that cleaves upon internalization of the ADC into VISTA-expressing immune cells, optionally one or more of T cells, Treg cells, NK cells, neutrophils, monocytes, myeloid cells, dendritic cells, macrophages, and endothelial cells, thereby releasing a therapeutically effective amount of the anti-inflammatory agent into the immune cells and inducing anti-inflammatory activity.

9. The ADC according to any of the prior claims, wherein the anti-VISTA antibody or antigen-binding fragment has an in vivo serum half-life of approximately 2.3 days ± 0.7 days or less in primates, and optionally in cynomolgus monkeys, at physiological pH (approximately pH 7.5).

10. The ADC according to any of the prior claims, wherein the anti-VISTA antibody or antigen-binding fragment has an in vivo serum half-life of 70 hours, 60 hours, 50 hours, 40 hours, 30 hours, 24 hours, 22-24 hours, 20-22 hours, 18-20 hours, 16-18 hours, 14-16 hours, 12-14 hours, 10-12 hours, 8-10 hours, 6-8 hours, 4-6 hours, 2-4 hours, 1-2 hours, 0.5-1.0 hours, or 0.1-0.5 hours in human VISTA knock-in rodents.

11. The ADC according to any of the prior claims, wherein, when used in vivo in human VISTA knock-in rodents and / or humans or non-human primates, optionally in cynomolgus monkeys, the pK / pD ratio of the ADC is at least 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1 or higher.

12. The ADC according to any one of the prior claims, wherein the PD of the ADC is at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, 2 to 3 weeks, or longer in human VISTA knock-in rodents and / or humans or non-human primates, optionally cynomolgus monkeys.

13. The ADC according to any one of the prior claims, wherein the anti-human VISTA antibody includes an Fc region in which FcR binding is impaired.

14. The ADC according to any one of the prior claims, wherein the anti-human VISTA antibody comprises a human IgG1, IgG2, IgG3, or IgG4 Fc region in which FcR binding is impaired.

15. The ADC according to any one of the prior claims, wherein the anti-human VISTA antibody comprises a human IgG1 Fc region in which FcR binding is impaired.

16. An antibody-drug conjugate (ADC) according to any of the prior claims, comprising a human or non-human primate constant region or Fc region modified to impair or eliminate binding to at least two native human Fcγ receptors.

17. An antibody-drug conjugate (ADC) according to any of the prior claims, comprising a human or non-human primate constant region or Fc region modified to impair or eliminate binding to any one, two, three, four, or all five of the following FcRs: hFcγRI (CD64), FcγRIIA or hFcγRIIB (CD32 or CD32A), and FcγRIIIA (CD16A) or FcγRIIIB (CD16B).

18. The antibody-drug conjugate (ADC) according to any of the prior claims, wherein the Fc region comprises a human IgG2κ backbone having the V234A / G237A / P238S / H268A / V309L / A330S / P331S silencing mutation.

19. The aforementioned Fc region has an L234A / L235A silencing mutation, and complement (C1) is optionally added. Ｑ An antibody-drug conjugate (ADC) according to any of the prior claims, comprising a human IgG1 / κ backbone having a mutation that impairs binding.

20. The antibody-drug conjugate (ADC) according to any of the prior claims, comprising a human IgG1 / κ skeleton having L234A / L235A silencing mutations and E269R and E233A mutations in the Fc region.

21. The ADC according to any of the prior claims, wherein the binding of the anti-VISTA antibody or antigen-binding fragment to VISTA-expressing immune cells does not directly agonize or antagonize the VISTA-mediated effect on immunity.

22. An antibody-drug conjugate (ADC) according to any of the prior claims, comprising a human IgG2 Fc region in which endogenous FcR binding is not impaired.

23. An antibody-drug conjugate (ADC) according to any of the prior claims, comprising a natural (unmodified) human IgG2 Fc region.

24. The ADC according to any of the prior claims, wherein the anti-VISTA antibody or antigen-binding fragment has a KD in the range of 0.0001 nM to 10.0 nM, 0.001 to 1.0 nM, or 0.01 to 0.7 or less, as measured by surface plasmon resonance (SPR) at 24°C or 37°C.

25. The ADC according to any one of the prior claims, wherein the anti-VISTA antibody or antigen-binding fragment has a KD of 0.02 to 0.64 nM as measured by surface plasmon resonance (SPR) at 24°C or 37°C.

26. The ADC according to any of the prior claims, wherein the linker includes any of the linkers disclosed in this application.

27. The ADC according to any of the prior claims, wherein the linker is a positively, negatively, or neutrally charged cleavable peptide, and optionally esterase-cleavable.

28. The ADC according to any of the prior claims, wherein the anti-inflammatory agent comprises a steroid, optionally a glucocorticoid receptor agonist, and optionally dexamethasone, prednisolone, or budesonide, or any of the functional derivatives thereof, i.e., the derivative induces anti-inflammatory activity upon internalization into VISTA-expressing immune cells.

29. The ADC according to any of the prior claims, wherein the drug-antibody ratio is in the range of 1:1 to 10:

1.

30. The ADC according to any one of the prior claims, wherein the drug-antibody ratio is in the range of 2 to 8:1, 4 to 8:1, or 6 to 8:

1.

31. The ADC according to any of the prior claims, wherein the drug-antibody ratio is 8:1 (n=8).

32. An ADC according to any of the prior claims, which internalizes one or more of monocytes, myeloid cells, T cells, Treg cells, macrophages, and neutrophils.

33. An ADC according to any of the prior claims, which does not internalize to the extent that it can sense B cells.

34. An ADC according to any of the prior claims, which, when administered to a target requiring it, enhances the efficacy and / or reduces adverse side effects associated with the anti-inflammatory agent, for example, a steroid, optionally a glucocorticoid receptor agonist, and optionally dexamethasone, prednisolone, or budesonide, compared to the same dose of the anti-inflammatory agent administered in its naked (uncomplexed) form.

35. The antibody-drug conjugate (ADC) according to any of the prior claims, wherein the anti-inflammatory agent, optionally a steroid or a glucocorticoid receptor agonist, and optionally dexamethasone, prednisolone, or budesonide, or any of the aforementioned functional derivatives, are conjugated to the antibody or antigen-binding fragment via interchain disulfides.

36. An antibody-drug conjugate (ADC) according to any of the prior claims, comprising an esterase-sensitive linker and dexamethasone or a functional derivative as the anti-inflammatory agent.

37. The antibody-drug conjugate (ADC) according to any of the prior claims, wherein the cleavageable linker is sensitive to one or more of the following: acid-induced cleavage, photo-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage.

38. The antibody-drug conjugate (ADC) according to any one of the prior claims, wherein the cleavable linker is an esterase-cleavable linker.

39. An antibody-drug conjugate (ADC) according to any of the prior claims, comprising a non-cleaving linker that is substantially resistant to one or more of the following: acid-induced cleavage, photo-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage.

40. The antibody-drug conjugate (ADC) according to any of the prior claims, wherein the anti-VISTA antigen-binding fragment contained in the ADC comprises a Fab, F(ab')2, or scFv antibody fragment.

41. The anti-VISTA antibody or antibody fragment contained in the antibody-drug conjugate (ADC) is: (i) V of sequence numbers 100, 101 and 102 Ｈ CDR and V of sequence numbers 103, 104 and 105 Ｌ Includes CD-R; (ii) V of sequence numbers 110, 111 and 112 Ｈ CDR and V of Sequence IDs 113, 114 and 115 Ｌ Includes CD-R; (iii) V of SEQ ID NOs: 120, 121 and 122 Ｈ CDRs and V of SEQ ID NOs: 123, 124 and 125 Ｌ CDRs; (iv) V of sequence numbers 130, 131 and 132 Ｈ CDR and V of Sequence IDs 133, 134 and 135 Ｌ Includes CD-R; (v) V of sequence numbers 140, 141 and 142 Ｈ CDR and V of Sequence IDs 143, 144 and 145 Ｌ Includes CD-R; (vi) V of sequence numbers 150, 151 and 152 Ｈ CDR and V of Sequence IDs 153, 154 and 155 Ｌ Includes CD-R; (vii) V of sequence numbers 160, 161 and 162 Ｈ CDR and V of sequence numbers 163, 164 and 165 Ｌ Includes CD-R; (viiii) V of sequence numbers 170, 171 and 172 Ｈ CDR and V of Sequence IDs 173, 174 and 175 Ｌ Includes CD-R; (ix) V of sequence numbers 180, 181 and 182 Ｈ CDR and V of Sequence IDs 183, 184 and 185 Ｌ Includes CD-R; (x) V of sequence numbers 190, 191 and 192 Ｈ CDR and V of Sequence IDs 193, 194 and 195 Ｌ Includes CD-R; (xi) V of sequence numbers 200, 201 and 202 Ｈ CDR and V of Sequence IDs 203, 204 and 205 Ｌ Includes CD-R; (xii) V of sequence numbers 210, 211 and 212 Ｈ CDR and V of Sequence IDs 213, 214 and 215 Ｌ Includes CD-R; (xiii) V of sequence numbers 220, 221 and 222 Ｈ CDR and V of Sequence IDs 223, 224 and 225 Ｌ Includes CD-R; (xiv) V of sequence numbers 230, 231 and 232 Ｈ CDR and V of Sequence IDs 233, 234 and 235 Ｌ Includes CD-R; (xv) V of sequence numbers 240, 241 and 242 Ｈ CDR and V of Sequence IDs 243, 244 and 245 Ｌ Includes CD-R; (xvi) V of sequence numbers 250, 251 and 252 Ｈ CDR and V of Sequence IDs 253, 254 and 255 Ｌ Includes CD-R; (xvii) VH CDR of sequence numbers 260, 261 and 262 and V of sequence numbers 263, 264 and 265 Ｌ Includes CD-R; (xviiii) V of sequence numbers 270, 271 and 272 Ｈ CDR and V of Sequence IDs 273, 274 and 275 Ｌ Includes CD-R; (xix) V of sequence numbers 280, 281 and 282 Ｈ CDR and V of Sequence IDs 283, 284 and 285 Ｌ Includes CD-R; (xx) V of sequence numbers 290, 291 and 292 Ｈ CDR and V of Sequence IDs 293, 294 and 295 Ｌ Includes CD-R; (xxi) V of sequence numbers 300, 301 and 302 Ｈ CDR and V of Sequence IDs 303, 304 and 305 Ｌ Includes CD-R; (xxii) V of sequence numbers 310, 311 and 312 Ｈ CDR and V of Sequence IDs 313, 314 and 315 Ｌ Includes CD-R; (xxiii) V of sequence numbers 320, 321 and 322 Ｈ CDR and V of Sequence IDs 323, 324 and 325 Ｌ Includes CD-R; (xxiv) V of sequence numbers 330, 331 and 332 Ｈ CDR and V of Sequence IDs 333, 334 and 335 Ｌ Includes CD-R; (xxv) V of sequence numbers 340, 341 and 342 Ｈ CDR and V of Sequence IDs 343, 344 and 345 Ｌ Includes CD-R; (xxvi) V of sequence numbers 350, 351 and 352 Ｈ CDR and V of Sequence IDs 353, 354 and 355 Ｌ Includes CD-R; (xxvii) V of sequence numbers 360, 361 and 362 Ｈ CDR and V of sequence numbers 363, 364 and 365 Ｌ Includes CD-R; (xxviiii) V of sequence numbers 370, 371 and 372 Ｈ CDR and V of Sequence IDs 373, 374 and 375 Ｌ Includes CD-R; (xxx) V of sequence numbers 380, 381 and 382 Ｈ CDR and V of Sequence IDs 383, 384 and 385 Ｌ Includes CD-R; (xxx) V of sequence numbers 390, 391 and 392 Ｈ CDR and V of sequence numbers 393, 394 and 395 Ｌ Includes CD-R; (xxxi) V of sequence numbers 400, 401 and 402 Ｈ CDR and V of sequence numbers 403, 404 and 405 Ｌ Includes CD-R; (xxxii) V of sequence numbers 410, 411 and 412 Ｈ CDR and V of sequence numbers 413, 414 and 415 Ｌ Includes CD-R; (xxxiii) V of sequence numbers 420, 421 and 422 Ｈ CDR and V of Sequence IDs 423, 424 and 425 Ｌ Includes CD-R; (xxxiv) V of sequence numbers 430, 431 and 432 Ｈ CDR and V of Sequence IDs 433, 434 and 435 Ｌ Includes CD-R; (xxxv) V of sequence numbers 440, 441 and 442 Ｈ CDR and V of Sequence IDs 443, 444 and 445 Ｌ Includes CD-R; (xxxvi) V of sequence numbers 450, 451 and 452 Ｈ CDR and V of Sequence IDs 453, 454 and 455 Ｌ Includes CD-R; (xxxvii) V of sequence numbers 460, 461 and 462 Ｈ CDR and V of sequence numbers 463, 464 and 465 Ｌ Includes CD-R; (xxxviiii) V of sequence numbers 470, 471 and 472 Ｈ CDR and V of Sequence IDs 473, 474 and 475 Ｌ Includes CD-R; (xxxix) V of sequence numbers 480, 481 and 482 Ｈ CDR and sequence numbers 483, 484 and 485 Ｌ Includes CD-R; (xl) V of sequence numbers 490, 491 and 492 Ｈ The CDR comprises the VL CDR polypeptide of Sequence IDs 493, 494, and 495; (xli) V of sequence numbers 500, 501 and 502 Ｈ The CDR comprises the VL CDR polypeptide of Sequence IDs 503, 504, and 505; (xlii) V of sequence numbers 510, 511 and 512 Ｈ The CDR comprises the VL CDR polypeptide of Sequence IDs 513, 514, and 515; (xliiii) V of sequence numbers 520, 521 and 522 Ｈ The CDR comprises the VL CDR polypeptide of Sequence IDs 523, 524, and 525; (xliv) V of sequence numbers 530, 531 and 532 Ｈ The CDR comprises the VL CDR polypeptide of Sequence IDs 533, 534, and 535; (xlv) V of sequence numbers 540, 541 and 542 Ｈ The CDR comprises the VL CDR polypeptide of Sequence IDs 543, 544, and 545; (xlvi) V of sequence numbers 550, 551 and 552 Ｈ The CDR comprises the VL CDR polypeptide of Sequence IDs 553, 554, and 555; (xlvii) V of sequence numbers 560, 561 and 562 Ｈ CDR and V of sequence numbers 563, 564 and 565 Ｌ Includes CD-R; (xlviii) V of SEQ ID NOs: 570, 571 and 572 Ｈ CDRs and V of SEQ ID NOs: 573, 574 and 575 Ｌ CDRs; (xlix) V of sequence numbers 580, 581 and 582 Ｈ CDR and V of Sequence IDs 583, 584 and 585 Ｌ Includes CD-R; (l) V of sequence numbers 590, 591 and 592 Ｈ CDR and V of Sequence IDs 593, 594 and 595 Ｌ Includes CD-R; (li) V of SEQ ID NOs: 600, 601 and 602 Ｈ CDRs and V of SEQ ID NOs: 603, 604 and 605 Ｌ CDRs; (lii) V of sequence numbers 610, 611 and 612 Ｈ CDR and V of sequence numbers 613, 614 and 615 Ｌ Includes CD-R; (liiii) V of sequence numbers 620, 621 and 622 Ｈ CDR and V of sequence numbers 623, 624 and 625 Ｌ Includes CD-R; (lv) V of sequence numbers 630, 631 and 632 Ｈ CDR and V of Sequence IDs 633, 634 and 635 Ｌ Includes CD-R; (lv) V of sequence numbers 640, 641 and 642 Ｈ CDR and V of sequence numbers 643, 644 and 645 Ｌ Includes CD-R; (lvi) V of sequence numbers 650, 651 and 652 Ｈ CDR and V of sequence numbers 653, 654 and 655 Ｌ Includes CD-R; (lvii) V of sequence numbers 660, 661 and 662 Ｈ CDR and V of sequence numbers 663, 664 and 665 Ｌ Includes CD-R; (lviiii) V of sequence numbers 670, 671 and 672 Ｈ CDR and V of Sequence IDs 673, 674 and 675 Ｌ Includes CD-R; (lix) V of sequence numbers 680, 681 and 682 Ｈ CDR and V of Sequence IDs 683, 684 and 685 Ｌ Includes CD-R; (lx) V of sequence numbers 690, 691 and 692 Ｈ CDR and V of sequence numbers 693, 694 and 695 Ｌ Includes CD-R; (lxi) V of sequence numbers 700, 701 and 702 Ｈ CDR and V of Sequence IDs 703, 704 and 705 Ｌ Includes CD-R; (lxii) V of sequence numbers 710, 711 and 712 Ｈ CDR and V of Sequence IDs 713, 714 and 715 Ｌ Includes CD-R; (lxiii) V of sequence numbers 720, 721 and 722 Ｈ CDR and V of Sequence IDs 723, 724 and 725 Ｌ Includes CD-R; (lxiv) V of sequence numbers 730, 731 and 732 Ｈ CDR and V of Sequence IDs 733, 734 and 735 Ｌ Includes CD-R; (lxv) V of sequence numbers 740, 741 and 742 Ｈ CDR and V of Sequence IDs 743, 744 and 745 Ｌ Includes CD-R; (lxvi) V of sequence numbers 750, 751 and 752 Ｈ CDR and V of Sequence IDs 753, 754 and 755 Ｌ Includes CD-R; (lxvii) V of sequence numbers 760, 761 and 762 Ｈ CDR and V of Sequence IDs 763, 764 and 765 Ｌ Includes CD-R; (lxviiii) V of sequence numbers 770, 771 and 772 Ｈ CDR and V of Sequence IDs 773, 774 and 775 Ｌ Includes CD-R; (lxix) V of sequence numbers 780, 781 and 782 Ｈ CDR and V of Sequence IDs 783, 784 and 785 Ｌ Includes CD-R; (lxx) V of sequence numbers 790, 791 and 792 Ｈ CDR and V of Sequence IDs 793, 794 and 795 Ｌ Includes CD-R; (lxxi) V of sequence numbers 800, 801 and 802 Ｈ CDR and V of Sequence IDs 803, 804 and 805 Ｌ Includes CD-R; (lxxii) V of sequence numbers 810, 811 and 812 Ｈ CDR and the V of sequence numbers 813, 814 and 815 Ｌ An ADC according to any of the prior claims, which is an antibody or antibody fragment containing a CDR.

42. The ADC according to any of the prior claims, wherein the anti-VISTA antibody or antibody fragment contained in the antibody-drug conjugate (ADC) contains the same CDR as any one of VSTB92, VSTB56, VSTB95, VSTB103, and VSTB66.

43. The anti-VISTA antibody or antibody fragment contained in the antibody-drug conjugate (ADC) is as follows: Ｈ Polypeptides and V Ｌ V has at least 90%, 95%, or 100% sequence identity with respect to the sequence of the polypeptide-containing antibody. Ｈ Polypeptides and V Ｌ An antibody or antibody fragment comprising a polypeptide, further comprising an unmodified CDR, according to any of the prior claims: (i) Identity V of Sequence ID No. 106 Ｈ Polypeptide and V of SEQ ID NO: 108 Ｌ Antibodies containing polypeptides; (ii) V of sequence number 116 Ｈ Polypeptide and V of SEQ ID NO: 118 Ｌ Antibodies containing polypeptides; (iii) V of sequence number 126 Ｈ Polypeptide and SEQ ID NO: 128 Ｌ Antibodies containing polypeptides; (iv) V of sequence number 136 Ｈ Polypeptide and SEQ ID NO: 138 Ｌ Antibodies containing polypeptides; (v) V of sequence number 146 Ｈ Polypeptide and V of SEQ ID NO: 148 Ｌ Antibodies containing polypeptides; (vi) V of sequence number 156 Ｈ Polypeptide and V of SEQ ID NO: 158 Ｌ Antibodies containing polypeptides; (vii) V of sequence number 166 Ｈ Polypeptide and V of SEQ ID NO: 168 Ｌ Antibodies containing polypeptides; (viiii) V of sequence number 176 Ｈ Polypeptide and V of SEQ ID NO: 178 Ｌ Antibodies containing polypeptides; (ix) V of sequence number 186 Ｈ Polypeptide and V of SEQ ID NO: 188 Ｌ Antibodies containing polypeptides; (x) V of sequence number 196 Ｈ Polypeptide and V of SEQ ID NO: 198 Ｌ Antibodies containing polypeptides; (xi) V of sequence number 206 Ｈ Polypeptide and V of SEQ ID NO: 208 Ｌ Antibodies containing polypeptides; (xii) V of sequence number 216 Ｈ Polypeptide and V of SEQ ID NO: 218 Ｌ Antibodies containing polypeptides; (xiii) V of sequence number 226 Ｈ Polypeptide and V of SEQ ID NO: 228 Ｌ Antibodies containing polypeptides; (xiv) V of sequence number 236 Ｈ Polypeptide and V of SEQ ID NO: 238 Ｌ Antibodies containing polypeptides; (xv) V of sequence number 246 Ｈ Polypeptide and V of SEQ ID NO: 248 Ｌ Antibodies containing polypeptides; (xvi) V of sequence number 256 Ｈ Polypeptide and V of Sequence ID No. 258 Ｌ Antibodies containing polypeptides; (xvii) V of sequence number 266 Ｈ Polypeptide and V of SEQ ID NO: 268 Ｌ Antibodies containing polypeptides; (xviiii) V of sequence number 276 Ｈ Antibodies containing polypeptides and the VL polypeptide of SEQ ID NO: 278; (xix) V of sequence number 286 Ｈ Polypeptide and V of SEQ ID NO: 288 Ｌ Antibodies containing polypeptides; (xx) V of sequence number 296 Ｈ Polypeptide and V of Sequence ID No. 298 Ｌ Antibodies containing polypeptides; (xxi) V of sequence number 306 Ｈ Polypeptide and V of Sequence ID No. 308 Ｌ Antibodies containing polypeptides; (xxii) V of sequence number 316 Ｈ Polypeptide and V of SEQ ID NO: 318 Ｌ Antibodies containing polypeptides; (xxiii) V of sequence number 326 Ｈ Polypeptide and V of SEQ ID NO: 328 Ｌ Antibodies containing polypeptides; (xxiv) V of sequence number 336 Ｈ Polypeptide and V of Sequence ID No. 338 Ｌ Antibodies containing polypeptides; (xxv) V of sequence number 346 Ｈ Polypeptide and V of SEQ ID NO: 348 Ｌ Antibodies containing polypeptides; (xxvi) V of sequence number 356 Ｈ Polypeptide and V of Sequence ID No. 358 Ｌ Antibodies containing polypeptides; (xxvii) V of sequence number 366 Ｈ Polypeptide and V of SEQ ID NO: 368 Ｌ Antibodies containing polypeptides; (xxviiii) V of sequence number 376 Ｈ Polypeptide and V of Sequence ID No. 378 Ｌ Antibodies containing polypeptides; (xxix) V of sequence number 386 Ｈ Polypeptide and V of SEQ ID NO: 388 Ｌ Antibodies containing polypeptides; (xxx) V of sequence number 396 Ｈ Polypeptide and V of SEQ ID NO: 398 Ｌ Antibodies containing polypeptides; (xxxi) V of sequence number 406 Ｈ Polypeptide and V of Sequence ID No. 408 Ｌ Antibodies containing polypeptides; (xxxii) V of sequence number 416 Ｈ Polypeptide and V of SEQ ID NO: 418 Ｌ Antibodies containing polypeptides; (xxxiii) V of sequence number 426 Ｈ Polypeptide and V of SEQ ID NO: 428 Ｌ Antibodies containing polypeptides; (xxxiv) V of sequence number 436 Ｈ Polypeptide and V of SEQ ID NO: 438 Ｌ Antibodies containing polypeptides; (xxxv) V of sequence number 446 Ｈ Polypeptide and sequence number 448 V Ｌ Antibodies containing polypeptides; (xxxvi) V of sequence number 456 Ｈ Polypeptide and V of SEQ ID NO: 458 Ｌ Antibodies containing polypeptides; (xxxvii) V of sequence number 466 Ｈ Polypeptide and V of SEQ ID NO: 468 Ｌ Antibodies containing polypeptides; (xxxviiii) V of sequence number 476 Ｈ Polypeptide and V of Sequence ID No. 478 Ｌ Antibodies containing polypeptides; (xxxix) V of sequence number 486 Ｈ Polypeptide and V of SEQ ID NO: 488 Ｌ Antibodies containing polypeptides; (xl) V of sequence number 496 Ｈ Polypeptide and V of Sequence ID No. 498 Ｌ Antibodies containing polypeptides; (xli) V of sequence number 506 Ｈ Polypeptide and V of Sequence ID No. 508 Ｌ Antibodies containing polypeptides; (xlii) V of sequence number 516 Ｈ Polypeptide and V of SEQ ID NO: 518 Ｌ Antibodies containing polypeptides; (xliiii) V of sequence number 526 Ｈ Polypeptide and V of SEQ ID NO: 528 Ｌ Antibodies containing polypeptides; (xliv) V of sequence number 536 Ｈ Polypeptides and the V of Sequence IDs 533, 534 and 535 Ｌ Antibodies containing polypeptides; (xlv) V of sequence number 546 Ｈ Polypeptide and V of SEQ ID NO: 548 Ｌ Antibodies containing polypeptides; (xlvi) V of sequence number 556 Ｈ Polypeptide and V of Sequence ID No. 558 Ｌ Antibodies containing polypeptides; (xlvii) V of sequence number 566 Ｈ Polypeptide and V of SEQ ID NO: 568 Ｌ Antibodies containing polypeptides; (xlviiii) V of sequence number 576 Ｈ Polypeptide and V of Sequence ID No. 578 Ｌ Antibodies containing polypeptides; (xlix) V of sequence number 586 Ｈ Polypeptide and V of Sequence ID No. 588 Ｌ Antibodies containing polypeptides; (l) V of sequence number 596 Ｈ Polypeptide and V of Sequence ID No. 598 Ｌ Antibodies containing polypeptides; (li) V of sequence number 606 Ｈ Polypeptide and V of SEQ ID NO: 608 Ｌ Antibodies containing polypeptides; (lii) V of sequence number 616 Ｈ Polypeptide and V of SEQ ID NO: 618 Ｌ Antibodies containing polypeptides; (liiii) V of sequence number 626 Ｈ Polypeptide and V of SEQ ID NO: 628 Ｌ Antibodies containing polypeptides; (liv) V of sequence number 636 Ｈ Polypeptide and V of SEQ ID NO: 638 Ｌ Antibodies containing polypeptides; (lv) V of sequence number 646 Ｈ Polypeptide and V of SEQ ID NO: 648 Ｌ Antibodies containing polypeptides; (lvi) V of sequence number 656 Ｈ Polypeptide and V of Sequence ID No. 658 Ｌ Antibodies containing polypeptides; (lvii) V of sequence number 666 Ｈ Polypeptide and V of SEQ ID NO: 668 Ｌ Antibodies containing polypeptides; (lviiii) V of sequence number 676 Ｈ Polypeptide and V of SEQ ID NO: 678 Ｌ Antibodies containing polypeptides; (lix) V of sequence number 686 Ｈ Polypeptide and V of SEQ ID NO: 688 Ｌ Antibodies containing polypeptides; (lx) V of sequence number 696 Ｈ Polypeptide and V of SEQ ID NO: 698 Ｌ Antibodies containing polypeptides; (lxi) V of sequence number 706 Ｈ Polypeptide and V of Sequence ID No. 708 Ｌ Antibodies containing polypeptides; (lxii) V of sequence number 716 Ｈ Polypeptide and V of SEQ ID NO: 718 Ｌ Antibodies containing polypeptides; (lxiii) V of sequence number 726 Ｈ Polypeptide and V of SEQ ID NO: 728 Ｌ Antibodies containing polypeptides; (lxiv) V of sequence number 736 Ｈ Polypeptide and V of Sequence ID No. 738 Ｌ Antibodies containing polypeptides; (lxv) V of sequence number 746 Ｈ Polypeptide and V of Sequence ID 748 Ｌ Antibodies containing polypeptides; (lxvi) V of sequence number 756 Ｈ Polypeptide and V of Sequence ID No. 758 Ｌ Antibodies containing polypeptides; (lxvii) V of sequence number 766 Ｈ Polypeptide and V of SEQ ID NO: 768 Ｌ Antibodies containing polypeptides; (lxviiii) V of sequence number 776 Ｈ Polypeptide and V of Sequence ID No. 778 Ｌ Antibodies containing polypeptides; (lxix) V of sequence number 786 Ｈ Polypeptide and V of Sequence ID 788 Ｌ Antibodies containing polypeptides; (lxx) V of sequence number 796 Ｈ Polypeptide and V of Sequence ID No. 798 Ｌ Antibodies containing polypeptides; (lxxi) V of sequence number 806 Ｈ Polypeptide and V of Sequence ID No. 808 Ｌ Antibodies containing polypeptides; and (lxxii) V of sequence number 816 Ｈ Polypeptide and V of SEQ ID NO: 818 Ｌ Antibodies containing polypeptides.

44. The ADC according to any of the prior claims, wherein the anti-VISTA antibody or antibody fragment includes the same variable region as one of VSTB92, VSTB56, VSTB95, VSTB103, and VSTB66.

45. The ADC according to any one of the prior claims, wherein the anti-VISTA antibody or antibody fragment comprises a human IgG2κ skeleton having a V234A / G237A / P238S / H268A / V309L / A330S / P331S silencing mutation in the Fc region.

46. The ADC according to any one of the prior claims, wherein the anti-VISTA antibody or antibody fragment comprises a human IgG1κ skeleton having an L234A / L235A silencing mutation in the Fc region.

47. The ADC according to any one of the prior claims, wherein the AI ​​or the L or Q is complexed with the anti-VISTA antibody or antigen-binding fragment via an interchain disulfide.

48. A pharmaceutical composition comprising at least one therapeutically effective amount of antibody-drug conjugate (ADC) as described in any of the prior claims, and a pharmaceutically acceptable carrier.

49. The composition according to claim 48, which can be administered via an optional intravenous, intramuscular, subarachnoid, or subcutaneous injection route.

50. The composition according to claim 48 or 49, which can be administered subcutaneously.

51. An apparatus for providing subcutaneous administration, comprising the composition according to claim 48, 49, or 50, and selected from the group consisting of a syringe, an injection device, an infusion pump, an injection pen, a needleless device, an auto-injector, and a subcutaneous patch delivery system.

52. The apparatus according to claim 51, which delivers a fixed dose of the anti-inflammatory agent, for example, a steroid, for example, a glucocorticoid receptor agonist, optionally dexamethasone, prednisolone, or budesonide, or a functional derivative thereof, to a patient.

53. A kit comprising the apparatus according to claim 51 or 52, further comprising a method for administering the ADC composition contained therein, and instructions for informing a patient of the drug regimen.

54. A method for treatment and / or prevention comprising administering at least one antibody-drug conjugate (ADC) or composition to a patient in need thereof, wherein the composition may be present in the apparatus described in any of the prior claims.

55. The method according to claim 54, for use in the treatment of allergies, autoimmunity, transplantation, gene therapy, inflammation, GVHD, or sepsis, or for treating or preventing inflammatory, autoimmune, or allergic side effects associated with any of the above conditions in human subjects.

56. The method according to claim 54 or 55, wherein the patient has a condition selected from rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis, ankylosing spondylitis, adult Crohn's disease, childhood Crohn's disease, ulcerative colitis, psoriasis vulgaris, hidradenitis suppurativa, uveitis, Behçet's disease, spondyloarthropathy, or psoriasis.

57. The aforementioned patient: (i) Conditions that can be effectively treated primarily with high doses of steroids, and optionally, polymyalgia rheumatica and / or giant cell arteritis (in which the patient has optionally been treated with or is currently being treated with high doses of steroids); (ii) Conditions with complications that limit the use of steroids, optionally: diabetes mellitus, non-alcoholic steatohepatitis (NASH), morbid obesity, avascular necrosis / osteonecrosis (AVN), glaucoma; steroid-induced hypertension, severe skin fragility, and / or osteoarthritis; (iii) Conditions for which safe long-term therapeutic agents are available but several months of induction with high-dose steroids are desirable, optionally including AAV, polymyositis, dermatomyositis, lupus, inflammatory lung disease, autoimmune hepatitis, inflammatory bowel disease, immune thrombocytopenia, autoimmune hemolytic anemia, and gout patients for whom several months of induction with high-dose steroids is therapeutically justified; (iv) Optional dermatological conditions requiring short-term / long-term treatment for which there is no effective alternative to treatment or duration and / or steroid administration; optional conditions including Stevens-Johnson syndrome, other severe rash conditions, widespread contact dermatitis, PG, LCV, and other severe immune-related dermatological conditions; (v) Conditions treated with high-dose corticosteroids for erythema / recurrence, optionally including COPD, asthma, lupus, gout, and pseudogout; (vi) Immune-related neurological disorders such as small fiber neuropathy, MS (subset), chronic inflammatory demyelinating polyneuropathy, and myasthenia gravis; (vii) Hematological / oncological indications in which high doses of steroids are optionally justified or may be effective as treatment; (viiii) Ophthalmic conditions, optional, such as uveitis, iritis, scleritis, etc. (ix) Conditions associated with permanent or very long-term adrenal insufficiency or secondary adrenal insufficiency, optionally iatrogenic Addison's disease crisis; (x) Conditions that are often treated with long-term low-dose steroids, and optionally with lupus, RA, PsA, vasculitis, etc.; and (xi) The method according to any one of claims 54 to 56, comprising one or more patients of a special class, e.g., pregnant / lactating women, pediatric patients, and optionally patients with growth disorders or cataracts.

58. The method according to any one of claims 54 to 57, further comprising treating the patient with another active agent.

59. The method according to any one of claims 54 to 58, further treating the patient with an immunomodulatory antibody or fusion protein selected from an immunosuppressive antibody or fusion protein targeting one or more of CTLA4, PD-1, PDL-1, LAG-3, TIM-3, BTLA, B7-H4, B7-H3, or VISTA, and / or an agonist antibody or fusion protein targeting one or more of CD40, CD137, OX40, GITR, CD27, CD28, or ICOS.

60. The aforementioned conditions are, at random selection, acquired aplastic anemia +, acquired hemophilia +, acute disseminated encephalomyelitis (ADEM) +, acute hemorrhagic leukoencephalitis (AHLE) / Hearst disease +, primary agammaglobulinemia +, alopecia areata +, ankylosing spondylitis (AS), anti-NMDA receptor encephalitis +, antiphospholipid syndrome (APS) +, arteriosclerosis, autism spectrum disorder (ASD), autoimmune Addison's disease (AAD) +, autoimmune autonomic dysfunction / autoimmune autonomic ganglion disorder (AAG), autoimmune encephalitis +, autoimmune Gastritis, autoimmune hemolytic anemia (AIHA)+, autoimmune hepatitis (AIH)+, autoimmune hyperlipidemia, autoimmune hypophysitis / lymphocytic hypophysitis+, autoimmune inner ear disease (AIED)+, autoimmune lymphoproliferative syndrome (ALPS)+, autoimmune heart Myositis, autoimmune oophoritis+, autoimmune orchitis+, autoimmune pancreatitis (AIP) / immunoglobulin G4-related disease (IgG4-RD)+, autoimmune polyglandular syndrome type I, II, and III+, autoimmune progesterone dermatitis+, autoimmune syndrome Sensorineural hearing loss (SNHL), achalasia, Addison's disease, Adult Still's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, antiphospholipid syndrome, autoimmune angioedema, autoimmune autonomic dysfunction, autoimmune encephalomyelitis, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune oophoritis, autoimmune orchitis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune urticaria, axonal and neuronal neuropathy (AMAN) ), Balo's disease, Behçet's disease, benign mucosal pemphigoid, bullous pemphigoid, Castleman disease (CD), celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic relapsing multifocal osteomyelitis (CRMO), Churg-Strauss syndrome (CSS) or eosinophilic granulomatosis (EGPA), scarring pemphigoid, Cogan syndrome, cold agglutinin disease, congenital heart block, coxsackie myocarditis, CREST syndrome, type 1 diabetes mellitus, herpetiform dermatitis, dermatomyositis, and Devic's disease (neuromyelitis optica). Lupus discoid, Dressler syndrome, endometriosis, eosinophilic esophagitis (EoE), eosinophilic fasciitis, erythema nodosum, essential mixed cryoglobulinemia, Evans syndrome, fibromyalgia, fibrous alveolitis, giant cell myocarditis, glomerulonephritis, Goodpasture syndrome, granulomatosis with polyangiitis, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schönlein purpura (HSP), herpes zoster of pregnancy or bullous pemphigoid (PG), hidradenitis suppurativa (HS) (reverse acne), hypogammaglobulinemia, IgA nephropathy, IgG4-related sclerosis, immunological Thrombocytopenic purpura (ITP), inclusion body myositis (IBM), interstitial cystitis (IC), juvenile arthritis, juvenile diabetes mellitus (type 1 diabetes mellitus), juvenile myositis (JM), Kawasaki disease, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosing, woody conjunctivitis, linear IgA disease (LAD), lupus (including nephritis and cutaneous infections), chronic Lyme disease, Meniere's disease, microscopic polyangiitis (MPA), mixed connective tissue disease (MCTD), Mohren's ulcer, Mucher-Habermann disease, multifocal motor neuropathy (MMN) or MMNCB, multiple sclerosis, myasthenia gravis, myelin oligo Dendrocyte glycoprotein antibody disorder, myositis, narcolepsy, neonatal lupus, neuromyelitis optica, neutropenia, ocular scarring pemphigoid, optic neuritis, ocular clonus-myoclonus ataxia (OMS), relapsing rheumatoid arthritis (PR), PANDAS, paraneoplastic cerebellar degeneration (PCD), paroxysmal nocturnal hemoglobinuria (PNH), hemifacial atrophy, ciliary body squamous cellulitis (peripheral uveitis), personality-Turner syndrome, pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia (PA), POEMS syndrome, polyarteritis nodosa, type I, type II, type III polyglandular syndromes Polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, primary biliary cholangitis, primary sclerosing cholangitis, progesterone dermatitis, psoriasis, psoriatic arthritis, pure red cell aplasia (PRCA), pyoderma gangrenosum, Raynaud's phenomenon, reactive arthritis, reflex sympathetic dystrophy, relapsing polychondritis, restless legs syndrome (RLS), retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt syndrome, scleritis, scleroderma, Sjögren's syndrome, sperm / testicular autoimmunity, generalized rigidus syndrome (SPS), subacute bacterial endocarditis (SBE), Suzac syndrome,The method according to any one of claims 54 to 59, for the treatment or prevention of acute or chronic inflammation, and associated autoimmune and inflammatory indications, including sympathetic ophthalmitis (SO), Takayasu's arteritis, temporal arteritis / giant cell arteritis, thrombocytopenic purpura (TTP), thyroid eye disease (TED), Toloser-Hunt syndrome (THS), transverse myelitis, type 1 diabetes mellitus, undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vitiligo, Vogt-Koyanagi-Harada syndrome, and the like.

61. The method according to any one of claims 54 to 60, for the treatment or prevention of acute or chronic inflammation, and related autoimmune and inflammatory indications, wherein the aforementioned condition optionally includes severe asthma, giant cell arteritis, ANKA vasculitis, and IBD (colitis and Crohn's disease).

62. The method according to any one of claims 54 to 59 for the treatment or prevention of a condition selected from rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis, ankylosing spondylitis, adult Crohn's disease, childhood Crohn's disease, ulcerative colitis, psoriasis vulgaris, hidradenitis suppurativa, uveitis, Behçet's disease, spondyloarthropathy, or psoriasis.

63. A method for internalizing a steroid into one or more of T cells, CD4 T cells, CD8 T cells, Treg cells, NK cells, neutrophils, monocytes, myeloid cells, dendritic cells, and macrophages, comprising administering an ADC described in any of the prior claims to a subject or bringing the subject into ex vivo contact with the cells.

64. The method according to claim 60, comprising running ex vivo and contacting a purified or concentrated composition containing immune cells, or a specific type of immune cell selected from CD4 T cells, CD8 T cells, Treg, NK cells, neutrophils, monocytes, myeloid cells, dendritic cells, and macrophage cells, with an ADC according to any of the prior claims ex vivo, and then introducing it to a patient in need.

65. A method for treating an inflammatory or autoimmune condition involving one or more T cells, Treg cells, NK cells, neutrophils, monocytes, myeloid cells, dendritic cells, and macrophages, comprising administering the ADC described in any of the prior claims to a subject in need of treatment.

Images