RU2812919C2 - Cd40l antagonist and methods of its use - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: following is provided: methods of treating or preventing an autoimmune disease, comprising administering the CD40L-specific Tn3 scaffold protein at a dose of 500 mg to 3,000 mg to a subject in need thereof.

EFFECT: inventions are used for the treatment or prevention of such diseases as focal segmental glomerulosclerosis, systemic sclerosis, myositis, antiphospholipid syndrome, autoimmune hepatitis, lupus nephritis, idiopathic thrombocytopenic purpura, Sjogren's syndrome, IgG-related disease, erythematous lupus.

14 cl, 26 dwg, 2 tbl, 15 ex

Description

BACKGROUND OF THE ART

[001] The CD40/CD40L pathway plays an important role in controlling the humoral immune response and is involved in the pathogenesis of several autoimmune diseases. CD40 is constitutively expressed on a variety of antigen-presenting cells, including dendritic cells (DCs), macrophages, and B cells ( 1 ), and can also be expressed on cells other than hematopoietic cells.

[002] Expression of the CD40 ligand, CD40L (also known as CD154), is highly regulated and is primarily found on activated CD4+ T cells (2). CD40/CD40L interactions between B cells and activated T cells are required for the generation of effective humoral responses to T-dependent antigens (3–5). The CD40/CD40L axis drives B cell proliferation, differentiation, and isotype switching in vitro ( 6 – 9 ). In vivo, CD40 signaling is required for germinal center (GC) formation, mediation of somatic hypermutation, and generation of memory B cells and long-lived plasma cells (10–13). Defects in CD40 or CD40L in humans result in the development of X-linked hyperimmunoglobulin syndrome, a disorder characterized by isotype class switching disorder that manifests as high serum levels of IgM with low or no detectable levels of IgG, IgA, or IgE and increased susceptibility to infections (14-16).

[003] Clinical trials of compounds targeting CD40L have demonstrated the potential benefits of targeting the CD40 pathway in autoimmune disease. In a phase II study, a humanized anti-CD40L 5c8 antibody, BG9588, provided significant reductions in proteinuria and anti-dsDNA antibody titers in patients with proliferative lupus nephritis (17). Additional studies have shown that treatment with a CD40L-specific binding molecule reduces the number of circulating CD38 ^highs. Ig-secreting cells as well as peripheral GC B cells present in patients with active SLE (18, 19). Anti-CD40L monoclonal antibody (mAb) treatments have also been shown to induce robust responses in a subset of patients with immune thrombocytopenia (ITP) (20).

[004] Although clinical trials showed potential for treatment with CD40L mAbs, their programs were stopped due to the occurrence of thromboembolic adverse events. Although not precisely defined, one possible explanation for these unexpected safety concerns is that FcγRIIa (or CD32a) is expressed on human platelets but not on mouse platelets (21). CD40L is also highly expressed on activated platelets ( 22 ), where simultaneous antibody-mediated binding to both CD40L and FcγRIIa on adjacent cells can lead to platelet aggregation. Mouse models provide evidence that FcγRIIa plays a role in the development of thrombocytopenia induced by molecules that specifically bind CD40L. In mice transgenic for human FcγRIIa, mAb to CD40L caused shock and thrombocytopenia (23). This effect was not observed in either wild-type mice or transgenic mice injected with an aglycosylated version of the antibody unable to bind FcγR.

[005] To provide targeting of CD40L, but without the potential complications associated with mAbs, the CD40L-specific scaffold protein Tn3 was produced (24, 25). Tn3 proteins are derived from the third domain of human tenascin-C fibronectin type III and can be given target-specific binding properties by engineering techniques (26, 27). Fusion of the divalent CD40L-specific protein Tn3 with human serum albumin (HSA) resulted in the molecule, i.e. VIB4920, which was able to bind human CD40L and prevent its interaction with the CD40 receptor. Consistent with this disruption of CD40L/CD40 interaction, VIB4920 was able to effectively inhibit human B cell activation and differentiationin vitro by blocking signal transduction events involving CD40.

[006] There is a need in the art for a new therapeutic agent that has a significant effect on the humoral immune response and that can be used to treat autoimmune and/or inflammatory conditions. There is also a need in the art for inducing immune tolerance to replacement therapy in patients in need thereof.

[007] VIB4920 has now been found to provide reductions in clinical symptoms and other disease markers when administered to patients suffering from an autoimmune/inflammatory disease or disorder. Specifically, administration of VIB4920 to subjects with rheumatoid arthritis (RA) at specific doses resulted in statistically significant reductions in rheumatoid factor (RF) autoantibody titers, Vectra DA biomarker score, and disease activity as measured by DAS28-CRP. compared to placebo.

BRIEF DESCRIPTION OF THE INVENTION

[008] The disclosure provides a method of suppressing a B cell and T cell mediated immune response in a subject. The method includes the steps of administering a dose of VIB4920 ranging from 500 mg to 3000 mg to a subject in need thereof and causing suppression of the B cell and T cell mediated immune response.

[009] The disclosure also provides a method of treating an autoimmune disease or disorder. The method includes the steps of administering a dose of VIB4920 ranging from 500 mg to 3000 mg to a subject in need thereof, thereby providing treatment for the autoimmune disease or disorder.

[0010] The disclosure further provides a method of reducing the disease activity score of RA in a patient being treated for RA. The method includes the steps of administering VIB4920 to a patient and causing a reduction in the patient's RA disease activity score. The measure of reduction in disease activity representing RA may include one or more of the following: DAS28-CRP, Clinical Disease Activity Index (CDAI), number of tender joints, number of swollen joints, patient global assessment, or physician global assessment. VIB4920 can be administered at a dose ranging from about 500 mg to 3000 mg.

[0011] The disclosure also provides a method for reducing the level of RF autoantibodies in a patient being treated for RA. The method includes the steps of administering VIB4920 at a dose of about 500 mg to 3000 mg to a patient and causing the level of RF autoantibodies in the patient to decrease.

[0012] The disclosure further provides a method of reducing a biomarker score in a patient while being treated for RA. The method includes the steps of administering from about 500 mg to 3000 mg of VIB4920 to a patient and causing a reduction in the patient's biomarker score. In such a method, the biomarker score may be one or more of a plasma cell (PC) gene signature occurrence, a Vectra-DA score, or a serum C-reactive protein (CRP) level.

[0013] The disclosure also provides a method for reducing the PC gene signature score in a patient in need thereof. The method includes the steps of administering VIB4920 to a patient in need thereof and causing a reduction in the patient's PC gene signature score. The patient in need of this may be a patient being treated for systemic lupus erythematosus, rheumatoid arthritis, myositis, antiphospholipid syndrome, autoimmune hepatitis, Sjogren's disease or other autoimmune or inflammatory conditions, as well as a patient with transplantation and graft-versus-host disease. . VIB4920 administered to a patient in need thereof may be at a dose ranging from about 500 mg to 3000 mg.

[0014] The disclosure further provides a method of reducing the level of autoantibodies in a patient when being treated for an autoimmune disorder or alloantibodies in the case of a transplant. The method includes the steps of administering VIB4920 to a patient in need thereof and causing a reduction in the level of autoantibodies or alloantibodies in the patient. In such a method, the patient is being treated for an autoimmune disease characterized by the presence of autoantibodies, or the patient is being treated to prevent transplant rejection. The patient is administered VIB4920 at a dose ranging from about 500 mg to 3000 mg.

[0015] The disclosure also provides a method for reducing the severity of inflammation in a patient. The method includes the steps of administering VIB4920 to a patient in need thereof and causing the patient's inflammation to be reduced. The patient may be a patient being treated for an inflammatory disease or disorder, or may be receiving prophylactic treatment for an expected inflammation in response to an organ or tissue graft. VIB4920 can be administered at a dose ranging from about 500 mg to 3000 mg.

[0016] The disclosure further provides a method of inducing immune tolerance to replacement therapy in a patient. The method includes the steps of administering VIB4920 to a patient in need of replacement therapy and causing the patient to induce immune tolerance to the replacement therapy. VIB4920 can be administered at a dose ranging from about 1000 mg to 3000 mg.

BRIEF DESCRIPTION OF THE FIGURES

[0017] In FIG. 1A-1G show biochemical characterization of human CD40L-specific Tn3 clones, including biochemical characterization of VIB4920, a bivalent HSA-fused clone 342. In fig. 1A demonstrates the ability of a number of human CD40L-specific Tn3 clones to inhibit CD40-CD40L interactions as measured by Proteon. Percent inhibition is shown over a range of Tn3 concentrations. The average value for wells with parallel samples is shown. In fig. 1B demonstrates the ability of Tn3 proteins, which specifically bind CD40L, to inhibit CD40L-mediated NFkB signaling. HEK293 cells expressing CD40R and luciferase as a reporter for NFκB were stimulated with recombinant CD40L overnight in the presence of Tn3 proteins, which specifically bind CD40L. The percentage inhibition of luciferase activity is shown. Data represent the average of parallel sample wells. In fig. Figure 1C demonstrates inhibition of CD86 upregulation by Tn3-based constructs at various concentrations. Human PBMCs were stimulated with recombinant CD40L followed by preincubation with the CD40L-specific binding protein Tn3, and CD86 expression levels were assessed by flow cytometry. The average value for wells with parallel samples is shown. In fig. 1D shows the results of testing the indicated Tn3 molecules for their ability to inhibit CD40-CD40L interactions in an ELISA assay. Data represent the average of parallel sample wells. In fig. 1E shows data obtained from a screen of clone 342 for binding to a panel of related TNF family members, including Fas, TNF-alpha, TNF-beta and OX40L. Clone 342 was found to selectively bind CD40L. In fig. 1F proposes the structure of VIB4920, derived from crystallization data of Tn3 342 and the published crystal structure of HSA ( 1 ). In fig. 1G schematic representation of the CD40/CD40L and 342/CD40L structures superimposed on the common CD40L molecule. CD40L is shown in green, Tn3 is shown in magenta, and the CD40 receptor is shown in cyan.

[0018] In FIG. 2A - 2D show the structural characterization of human CD40L-specific Tn3 clone 342. Fig. 2A is a schematic representation of the trimeric structure of 342/CD40L. The extracellular domain of CD40L is shown in green; 342 - in purple. In fig. 2B shows the interface between 342 and CD40L. CD40L and 342 fragments are shown as green and purple tubes, respectively. Amino acids involved in the formation of hydrogen bonds are indicated with sticks. Hydrogen bonds are shown as black dotted lines with corresponding distances (Å). Bonds with a distance of no more than 3.5 Å are shown. In fig. 2C and FIG. 2D illustrates the electrostatic potential at the interface of the interacting surfaces of (Fig. 2C) CD40L and (Fig. 2D) CD40L-specific Tn3 342. The molecules are rotated approximately 90 degrees to show the contact surface and are translucent to provide visualization of the amino acids involved in formation of hydrogen bonds. Red color indicates a negatively charged surface, and blue color indicates a positive charge.

[0019] In FIG. 3A-3G demonstrate how VIB4920 inhibits CD40 signaling and human B cell activation but does not induce platelet aggregation in ex vivo studies. In fig. 3A shows the percentage of VIB4920 inhibition of the luciferase signal reflecting NFκB levels in engineered HEK29 cells stimulated with CD40L overnight. Data represents the average of parallel sample wells. One of two independent studies is shown. In fig. 3B demonstrates how VIB4920 and CD40L mAb were able to inhibit the increase in CD86 expression in stimulated human PBMCs that were stimulated. Human PBMCs were stimulated with recombinant human megaCD40L and the percentage of CD19+/CD86+ cells was measured by flow cytometry after 24 hours. Data represent the average of parallel sample wells. In fig. Human 3C B cells were stimulated with IL-21 and megaCD40L in the presence of control or a molecule that specifically binds CD40L (mAb or Tn3 VIB4920). The level of B cell expansion was quantified at day 3. The dotted line represents ATP levels in unstimulated cells. Data shown are the mean and SD of triplicate wells and are illustrative of two independent experiments. In fig. 3D and fig. 3E demonstrates the effect of a CD40L-specific binding molecule (mAb or VIB4920 Tn3) on human B cells when left unstimulated (nil) or stimulated with IL-21, anti-IgM, and megaCD40L. The number of PCs was quantified on day 7. Specifically, in FIG. 3D demonstrates the effect of these molecules on day 7 regarding the percentage of IgD-CD38 ^{is high.} PC on day 7. FIG. 3E demonstrates the effect of the indicated molecules at the indicated concentrations on the number of PCs on day 7. Data shown are the mean and SD of triplicate wells and are illustrative of two independent experiments. ****=p <0.0001 by two-tailed unpaired Student's t-test. In fig. 3F and FIG. 3G demonstrates the effect of molecules that specifically bind CD40L (mAb or Tn3 VIB4920) on washed human platelets incubated with previously formed immune complexes; platelet aggregation or lack thereof was measured within 12-14 minutes. In fig. Figure 3F shows the percentage of aggregation. Where indicated, platelets were preincubated with anti-CD32a antibody for 5 minutes before addition of immune complex. Adenosine diphosphate (ADP) was used as a positive control for aggregation. In fig. 3G shows the percentage of aggregation after incubation of platelets with immune complexes with VIB4920 (Tn3) or anti-CD40L mAb (5c8) at the indicated concentrations. Data are illustrations of two independent experiments.

[0020] FIG. 4A and 4B. A mouse substitute for CD40L-specific Tn3 demonstrated potent neutralizing activity in vivo in response to immunization. As in fig. 4A and FIG. 4B mice were immunized with sheep red blood cells (SRBC) on day 0 and control or CD40L specific binding Tn3 (M31-MSA) were administered daily from day 9 to day 13. FIG. 4A shows the percentage of germinal center B cells in the spleen and lymph node calculated by flow cytometry at day 14. The points represent individual animals and the data are illustrative of two independent studies. ****=p <0.0001 by two-tailed unpaired Student's t-test. In fig. 4B shows the level of ovine red blood cell IgG production as quantified in serum on day 14. Data are the mean and SEM of four animals per group (n=1 study).

[0021] FIG. 5A and 5B. Study design for a phase 1a clinical trial to evaluate the safety of VIB4920 in healthy volunteers. In fig. Figure 5A demonstrates the study cohorts for the phase 1a study. In fig. Figure 5B shows the dosing and immunization strategy for the Phase 1a study.

[0022] FIG. 6A and 6B. In a phase 1a study in healthy volunteers, VIB4920 demonstrated a preferential PK profile. In fig. 6A shows circulating levels of VIB4920 determined by ELISA at the indicated time points. The dotted line represents the lower limit of assay sensitivity. Error bars represent standard deviation from the mean, which was not calculated for groups with N=2 subjects. In fig. 6B demonstrates soluble CD40L levels, as assessed by ELISA, across all dosing cohorts at the indicated time points. The dotted line represents the lower limit of detection for the assay.

[0023] FIG. 7. VIB4920 inhibits anti-drug antibody (ADA) variants at high doses in a phase 1a study in healthy volunteers. The presence of ADA was determined using ELISA. Each subject within each cohort is depicted as a separate line. Subjects with high abundance of ADA variants (median titer >480) are indicated by a magenta line; subjects with low abundance of ADA variants (median titer <480) are indicated by a dark blue line; subjects with undetectable ADA variant content are indicated by a light blue line.

[0024] FIG. 8A-8C. VIB4920 inhibits B cell proliferation and TDARs in healthy human volunteers in a dose-dependent manner. Healthy volunteers were immunized with KLH 14 days before treatment with placebo or VIB4920 and repeated 15 days after dosing. In fig. 8A shows anti-KLH IgG titers in healthy volunteers at multiple time points and at different doses of VIB4920. In fig. 8B shows IgM titers to KLH in healthy volunteers at multiple time points and at different doses of VIB4920. IgG and IgM titers were measured using ELISA. Fig. 8C is a dose-response model of anti-KLH IgG inhibition on day 43.

[0025] FIG. 9A-9C. VIB4920 inhibits B cell proliferation and plasma cell responses, causing a decrease in TDAR levels in healthy human subjects. In fig. 9A shows the detected frequency of circulating proliferating B cells (Ki67+ CD19+) as quantified by flow cytometry at various time points in volunteers receiving either placebo or high dose VIB4920 in the TDAR trial study. In fig. 9B shows the detected frequency of class-switched memory B cells (Ki67+ CD19+ IgD-CD27+) in circulation as quantified by flow cytometry at various time points in volunteers receiving either placebo or high dose VIB4920 in the TDAR trial study. In fig. 9C presents a score for the occurrence of the PC signature in whole blood, as assessed by Taqman PCR. For placebo and high dose VIB4920 groups, expression level values are shown as mean and standard error. * = P<0.05, ** = P<0.01 when compared with placebo, according to the Mann-Whitney U test.

[0026] FIG. 10. Phase 1b study design to evaluate VIB4920 in patients with RA. The arrow indicates the dose of VIB4920 or placebo.

[0027] FIG. 11. Demographic statistics and clinical characteristics of the RA patient cohorts in the phase 1b clinical trial for VIB4920.

[0028] FIG. 12. VIB4920 demonstrates an acceptable safety profile in patients with RA. Shown are the most common TEAEs occurring in at least 2 subjects in a phase 1b study of subjects with RA.

[0029] FIG. 13A-13C. VIB4920 demonstrates linear PK and dose-dependent reduction of ADA variants in a phase 1b study in patients with RA. In fig. 13A shows circulating VIB4920 concentrations determined by ELISA at the indicated time points. The dotted line represents the lower limit of assay sensitivity. The mean and standard error of the mean are shown. In fig. 13B shows the percentage of subjects with a positive ADA titer, determined by ELISA, for each administered dosage at any time point in the study. In fig. 13C shows the ADA titer determined by ELISA over time in subjects with detectable ADA levels.

[0030] FIG. 14A-14F. VIB4920 reduces disease index and autoantibody scores in patients with RA. In fig. 14A demonstrates the change in DAS28-CRP from baseline (mean and standard error are indicated) when assessed at the indicated time points for the indicated dose of VIB4920 or placebo. In fig. 14B demonstrates the change in CDAI from baseline (mean and standard error indicated) when assessed at the indicated time points for the indicated dose of VIB4920 or placebo. In fig. 14C demonstrates the change in patient global score from baseline (mean and standard error reported) when assessed at the indicated time points for the indicated dose of VIB4920 or placebo. In fig. 14D demonstrates the change in physician global assessment from baseline (mean and standard error reported) when assessed at the indicated time points for the indicated dose of VIB4920 or placebo. In fig. 14E demonstrates the change in Vectra DA score from baseline (mean and standard error reported) at the indicated time points for the indicated dose of VIB4920 or placebo. In fig. 14F shows the percentage reduction in RF antibody titers measured by ELISA at the indicated time points for each indicated dose of VIB4920 or placebo.

[0031] FIG. 15A-15B. VIB4920 provides a dose-dependent reduction in DAS28-CRP scores and RF autoantibodies in patients with RA. In fig. 15A demonstrates the difference in DAS28-CRP score at day 85 between placebo and the indicated doses of VIB4920. A linear dose-response relationship is shown; it was identified as the most suitable model for assessing the relationship between VIB4920 dose and reduction in disease activity. In fig. 15B shows the percentage reduction in RF autoantibodies compared to placebo at the indicated doses of VIB4920 on day 85. Emax model shown; it was identified as most suitable for assessing the relationship of VIB4920 dose to RF titers.

[0032] FIG. 16. VIB4920 provides improvement across DAS28 categories in treated RA patients. DAS28 categories at day 85 are shown. Fifty percent and seventy-five percent of patients with RA treated in the 1000 mg and 1500 mg groups, respectively, had low disease activity or remission at day 85.

[0033] FIG. 17A-17C. Effect of VIB4920 on the number of tender/swollen joints and CRP in a phase 1b study in subjects with RA. In fig. 17A demonstrates the change from baseline in the number of tender joints in RA subjects at the indicated doses and time points. In fig. 17B demonstrates the change from baseline in the number of swollen joints in RA subjects at the indicated doses and time points. In fig. 17C demonstrates the change in RA subjects in the ratio of CRP levels at the indicated doses and at the indicated time points to baseline. The mean and standard error for each are shown.

[0034] FIG. 18. Amino acid sequence of the VIB4920 molecule.

[0035] FIG. 19A-19B. Amino acid sequences of CD40L-specific Tn3 clone 342 molecules.

[0036] FIG. 20. Amino acid sequence of the molecule of clone 342 of the divalent CD40L-specific Tn3.

[0037] FIG. 21A and 21B. Amino acid sequences of the CD40L-specific Tn3 clone 309 molecule.

[0038] FIG. 22A-22F. VIB4920 provided reductions in disease index scores and autoantibody levels in patients with RA during both the 12-week VIB4920 dosing period and the twelve-week follow-up period following the last VIB4920 dose. In fig. 22A demonstrates the change in DAS28-CRP from baseline (mean and standard error are indicated) when assessed at the indicated time points for the indicated dose of VIB4920 or placebo. In fig. 22B demonstrates the change in CDAI from baseline (mean and standard error are indicated) when assessed at the indicated time points for the indicated dose of VIB4920 or placebo. In fig. 22C demonstrates the change in patient global score from baseline (mean and standard error reported) when assessed at the indicated time points for the indicated dose of VIB4920 or placebo. In fig. 22D demonstrates the change in physician global assessment from baseline (mean and standard error reported) when assessed at the indicated time points for the indicated dose of VIB4920 or placebo. In fig. 22E demonstrates the change in Vectra DA score from baseline (mean and standard error reported) at the indicated time points for the indicated dose of VIB4920 or placebo. In fig. 22F shows the percentage reduction in RF antibody titers measured by ELISA at the indicated time points for each indicated dose of VIB4920 or placebo.

Fig. 23A-23C. VIB4920 affects the number of tender/swollen joints and CRP in patients with RA. The effects of VIB4920 were detectable in a phase 1b clinical trial in patients with RA during both the dosing phase and the 12-week post-dose observation period. In fig. 23A demonstrates the change from baseline in the number of tender joints in RA subjects at the indicated doses and time points. In fig. 23B demonstrates the change from baseline in the number of swollen joints in RA subjects at the indicated doses and time points. In fig. 23C demonstrated a change in RA subjects in the ratio of CRP levels at the indicated doses and at the indicated time points to baseline. The mean and standard error for each are shown.

DETAILED DESCRIPTION

[0039] Disclosed herein is VIB4920 and its utility in methods of suppressing B cell mediated immune responses, methods of treating autoimmune diseases or disorders, methods of reducing inflammation, methods of reducing autoantibody levels in a patient, methods of reducing disease activity score RA in a patient, methods of causing a reduction in the level of RF autoantibodies in a patient, methods of reducing a plasma cell gene signature score in a patient, and methods of inducing immune tolerance to replacement therapy in a patient.

[0040] If VIB4920 is used to treat an autoimmune disease or disorder, VIB4920 can be used to treat alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune adrenal diseases, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, Sjögren's syndrome , psoriasis, atherosclerosis, diabetic and other retinopathy, retrolental fibroplasia, age-related macular degeneration, neovascular glaucoma, hemangiomas, thyroid hyperplasia (including Graves' disease), transplantation and chronic inflammation of the cornea and other tissues, sepsis, rheumatoid arthritis, peritonitis, Crohn's disease , reperfusion injury, septic fever, endotoxin shock, cystic fibrosis, endocarditis, psoriasis, arthritis (eg, psoriatic arthritis), anaphylactic shock, ischemic organ disease, reperfusion injury, spinal cord injury and allograft rejection, autoimmune thrombocytopenia, Behçet's disease, bullous pemphigoid, cardiomyopathy, celiac enteropathic dermatitis, chronic fatigue and immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy , Churg-Strauss syndrome, bullous pemphigoid, CREST syndrome, cold agglutination syndrome, Crohn's disease, discoid lupus, primary mixed type cryoglobulinemia, fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, graft-versus-host disease, Hashimoto's thyroiditis , idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, juvenile arthritis, lichen planus, erythematous lupus, Meniere's disease, mixed connective tissue disease, multiple sclerosis mediated by IgG4-related disease, type 1 or immune-mediated diabetes mellitus , myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, inflammation of several cartilages, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary cholangiolytic cirrhosis of the liver, psoriatic arthritis, Raynaud's disease, Reiter's syndrome, rheumatoid arthritis, sa rcoidosis, scleroderma , Sjögren's syndrome, muscle stiffness syndrome, systemic lupus erythematous, Takayasu arteritis, temporal arteritis/giant cell arteritis, ulcerative colitis, uveitis, ANCA-associated vasculitis, other vasculitides such as dermatosus vasculitis herpetiformis, vitiligo, solid organ transplant rejection, graft reaction against the host", a decrease in the sensitivity of a group of reactive antibodies in kidney transplant recipients, islet cell transplantation and allogeneic hematopoietic stem cell transplantation, focal segmental glomerulosclerosis (FSGS), types of glomerulonephritis.

[0041] VIB4920, more specifically, can be used to treat RA, systemic lupus erythematous (SLE), myositis, antiphospholipid syndrome, autoimmune hepatitis, focal segmental glomerulosclerosis (FSGS), lupus nephritis, inflammatory myopathies, idiopathic thrombocytopenic purpura (ITP), systemic sclerosis, vasculitis, cutaneous lupus, autoimmune hemolytic anemia, myasthenia gravis, IgG4-related disease, or Sjögren's syndrome. In addition, VIB4920 can be used to treat graft-versus-host disease and/or to reduce or prevent organ or tissue graft rejection.

[0042] Treatment of an autoimmune disease or disorder can be in the form of suppression of the B cell or T cell mediated immune response, which may be a decrease in the level of class switched antibodies, a decrease in the level of circulating B cell subsets, a decrease in plasma activity, or reduction in plasma cell content and occurrence of the plasma cell gene signature. Treatment of an autoimmune disease or disorder may involve providing a reduction in inflammatory markers. Inflammatory markers may be one or more of autoantibody levels, plasma cell (PC) content or the occurrence of the PC gene signature (a signature characterized by expression of the IGHA1, IGJ, IGKC, IGKV4-1, and TNFRSF17 genes), circulating B cell subsets, and antibodies with switched class. Treatment of an autoimmune disease or disorder may involve a reduction in the severity of clinical signs and symptoms, such as clinical signs and symptoms measured by a global assessment by the patient or physician. Clinical signs and symptoms may include one or more of arthritis, pain, fatigue, fever, malaise, rash, weakness, or signs of organ dysfunction such as proteinuria or loss of kidney function.

[0043] If the method is a method of reducing the level of autoantibodies in a patient while being treated for an autoimmune disorder, the autoantibodies may be antinuclear antibodies, for example, in a patient being treated for SLE, Sjögren's syndrome, inflammatory myopathy, or systemic sclerosis. Antinuclear antibodies may be one or more of anti-SSA/Ro or SSB-La autoantibodies (SLE or Sjögren's syndrome), anti-dsDNA antibodies (SLE), anti-Smith antibodies (SLE), anti-topoisomerase antibodies (systemic sclerosis), or anti-histone antibodies (SLE). If the method is a method of reducing the level of autoantibodies in a patient while being treated for an autoimmune disorder, the autoantibodies may be antibodies to liver-kidney microsome type 1, for example, in a patient being treated for autoimmune hepatitis. If the method is a method of reducing the level of autoantibodies in a patient while being treated for an autoimmune disorder, the autoantibodies may be antibodies to the nicotinic acetylcholine receptor or muscle-specific kinase, for example, in a patient being treated for myasthenia gravis. If the method is a method of reducing the level of antibodies in a patient during transplantation treatment, the antibodies may be alloantibodies.

[0044] A reduction in the level of autoantibodies in a patient being treated for an autoimmune disorder may be a reduction in the percentage of autoantibodies to a level that is at least 20% less than the level before administration of VIB4920. It may be a reduction in the percentage of autoantibodies to a level of at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% relative to autoantibody levels before treatment with VIB4920. Reductions in autoantibody levels can be achieved within a month to three months from the start of VIB4920 administration.

[0045] If the autoimmune disease or disorder is RA, treatment of rheumatoid arthritis may involve reducing one or more of the following: anti-RF autoantibodies, anti-citrullinated peptide antibodies, Vectra DA biomarker score (Vectra DA biomarker score) by Vectra DA biomarker level, which is a composite score of expression levels of interleukin-6, tumor necrosis factor receptor type I, vascular cell adhesion molecule 1, epidermal growth factor, vascular endothelial growth factor A, YKL-40, matrix metalloproteinase 1, MMP -3, CRP, serum amyloid A, leptin and resistin), occurrence of plasma cell signature (PC), serum C-reactive protein (CRP), DAS28-CRP or Clinical Disease Activity Index (CDAI), or may represent a decrease number of painful joints, intensity of joint tenderness, number of swollen joints, or intensity of joint swelling. If the autoimmune disease or disorder is RA, treatment may represent achievement of ACR20, ACR50 or ACR70.

[0046] Treatment of an autoimmune disease or disorder may be characterized by a reduction in the severity of clinical symptoms of the disease or disorder by at least 20%, or a decrease in the severity of inflammation, or a decrease in the level of biomarkers of the disease or disorder relative to their levels before treatment with VIB4920. A reduction in the severity of any such symptoms or inflammation or biomarker levels may be a reduction in the severity of symptoms or inflammation or biomarker levels that is at least 50% relative to their levels prior to treatment with VIB4920. The decline may be such that the autoimmune disease or disorder is described as being in remission.

[0047] Additionally, if the autoimmune disease or disorder is rheumatoid arthritis, then treatment of the autoimmune disease or disorder may reduce the level of RF autoantibodies in the patient to levels that are about at least 20%, at least 30%, at least at least 40%, at least 45%, at least 50%, at least 60%, at least 75%, or at least 80% relative to RF autoantibody levels before treatment with VIB4920. If the autoimmune disease or disorder is rheumatoid arthritis, then the treatment for the autoimmune disease or disorder may be a decrease in DAS28-CRP, and the decrease in DAS28-CRP may be such that there is an adjusted mean difference of at least -1.2 or at least at least -1.5, or at least -2.0, or at least -2.2. Additionally, if the autoimmune disease or disorder is rheumatoid arthritis, then treatment of the autoimmune disease or disorder may be a reduction in the Vectra DA biomarker score, which reduction may be an adjusted mean difference of at least -10.3 or at least -10.5, or at least -10.8.

[0048] When VIB4920 is used in a method for reducing inflammation, the inflammation may be the result of an inflammatory disease or disorder, or may be due to injury or processes designed to prevent it, such as may be due to an organ or tissue transplantation procedure. When VIB4920 is used in a method of reducing inflammation in an inflammatory disease or disorder, the inflammatory disease or disorder may be inflammatory myopathy or lupus nephritis, cutaneous lupus, RA, SLE, ITP, myositis, Sjögren's syndrome, vasculitis, systemic sclerosis, autoimmune hemolytic anemia, myasthenia gravis or focal segmental glomerulosclerosis. When VIB4920 is used in a method to reduce inflammation, the inflammation may be due to injury or processes designed to prevent it, for example, may be due to an organ or tissue transplantation procedure.

[0049] When VIB4920 is used in a method of inducing immune tolerance to a replacement therapy in a patient, VIB4920 can induce the development of immune tolerance by reducing the level of production of neutralizing antibodies to the replacement therapy in the patient. If the patient has not been treated with a replacement therapy agent or has otherwise not developed neutralizing antibodies to the replacement therapy agent, then inducing immune tolerance may prevent the patient from developing neutralizing antibodies to the replacement therapy agent at the first encounter. However, if a patient develops neutralizing antibodies to the replacement therapy, then VIB4920 may induce the development of immune tolerance by reducing the levels of neutralizing antibodies to the replacement therapy that the patient produces. The patient's neutralizing antibody levels to the replacement therapy can be reduced by at least 50%, at least 55%, at least 60%, at least 65%, at least 70, at least 75% , at least 80%, at least 85%, at least 90%, at least 95% or to a level that is undetectable. The percentage reduction in the patient's levels of neutralizing antibodies to the replacement therapy may be a comparison or may be determined by comparing the first level of neutralizing antibodies produced in response to the replacement therapy before the first dose of VIB4920 with the second level of neutralizing antibodies produced in response to for replacement therapy after the first or second or third or fourth or fifth dose of VIB4920. Alternatively, the percentage reduction in a patient's levels of neutralizing antibody to the replacement therapy may be a comparison or may be determined by comparing the peak levels of neutralizing antibodies produced in response to the replacement therapy prior to the first dose of VIB4920 with the peak levels of neutralizing antibodies. produced in response to replacement therapy after administration of the first or second or third or fourth or fifth dose of VIB4920.

[0050] Inducing immune tolerance to the replacement therapy in a patient may alternatively or additionally be a decrease in the level of T cell response to the replacement therapy agent. If a patient has not been treated with a replacement therapy or has been treated with a replacement therapy but does not yet have a T-cell immune response to the replacement therapy, then VIB4920 may reduce the level of the patient's T-cell response by preventing the generation of an initial response T cells for replacement therapy. However, if the patient has an existing T cell response to the replacement therapy, then VIB4920 may induce the development of immune tolerance by reducing the level of the existing T cell response to the replacement therapy. The level of T cell response to the replacement therapy can be reduced by at least 50%, at least 55%, at least 60%, at least 65%, at least 70, at least 75%, at least at least 80%, at least 85%, at least 90%, at least 95% or to a level that is undetectable. The percentage reduction in T cell response to replacement therapy in a patient may be a comparison or may be determined by comparing the first level of T cell response to replacement therapy prior to the first dose of VIB4920 with the second level of T cell response to replacement therapy after the first or second or third or fourth or fifth dose of VIB4920. Alternatively, the percentage reduction in T-cell response to replacement therapy in a patient may be a comparison or may be determined by comparing the peak T-cell response to replacement therapy prior to the first dose of VIB4920 with the peak T-cell response for replacement therapy after the first or second or third or fourth or fifth dose of VIB4920. A decrease in the level of T cell response may be characterized by a decrease in the level of proliferation and/or stimulation of CD4+ T cells stimulated by the replacement therapy. Decreased T-cell responses may also be characterized by decreased CD4-dependent CD8+ T-cell responses to replacement therapy.

[0051] Replacement therapy for which the development of immune tolerance is induced may be peptide or protein replacement therapy. If the replacement therapy agent is a peptide or protein therapy agent, it may be a factor VIII or factor IX therapy agent and may be administered to treat a patient suffering from hemophilia. If the replacement therapy is a peptide or protein therapy, it may be an enzyme replacement therapy (ERT). If the replacement therapy is ERT, the replacement therapy agent may be agalsidase alfa or agalsidase beta, it can replace alpha-galactosidase A, and it can treat a patient suffering from Fabry disease. If the replacement therapy is ERT, the replacement therapy agent may be iaronidase, it may replace alpha-L-iduronidase and can treat a patient suffering from mucopolysaccharidosis (MPS) type 1 (also known as Hurler syndrome, Hurler-Scheie or Sheya depending on its severity). If the replacement therapy is ERT, the replacement therapy agent may be alglucosidase, it may replace alpha-glucosidase, and a patient suffering from Pompe disease may be treated with it. If the replacement therapy is ERT, the replacement therapy agent may be idursulfase, it may replace iduronate-2-sulfatase, and a patient suffering from MPS type II may be treated with it. If the replacement therapy is ERT, the replacement therapy agent may be imiglucerase or velaglucerase alfa, or taliglucerase alfa, it can replace beta-glucocerebrosidase, and it can treat a patient suffering from Gaucher disease. If the replacement therapy is ERT, the replacement therapy agent may be arylsulfatase B Naglazyme, it can replace N-acetylgalactosamine-4-sulfatase and can treat a patient suffering from MPS type VI. If the peptide or protein replacement therapy agent is a peptide or protein, inducing immune tolerance may reduce the level of neutralizing antibodies to the peptide or protein and/or may reduce the level of T cell response to the peptide or protein in the patient.

[0052] Additionally, the replacement therapy agent for which immune tolerance is induced may be a viral vector that contains a nucleic acid encoding a therapeutic peptide or protein. If the replacement therapy agent is a viral vector that contains a nucleic acid encoding a therapeutic peptide or protein, the viral vector may be an adenoviral vector, an adeno-associated virus vector, a retroviral vector, a poxvirus vector, an alphavirus, a herpes simplex virus, or any other a viral vector capable of delivering a nucleic acid encoding a therapeutic peptide or protein into a patient's cells. The viral vector can be modified, for example, by pseudotyping, and/or to remove its wild-type genes, and/or to incorporate nucleic acids encoding a therapeutic peptide or protein.

[0053] The therapeutic peptide or protein that is encoded by the nucleic acid from the viral vector may be a therapeutic peptide or protein that is factor VIII or factor IX, or it may be an ERT agent such as agalsidase alpha, agalsidase beta, idursulfase, yaronidase, alglucosidase alfa, imiglucerase, velaglucerase alfa, taliglucerase alfa or Naglazyme arylsulfatase B.

[0054] In addition, if the replacement therapy agent is a viral vector that contains a nucleic acid encoding a therapeutic peptide or protein, then VIB4920 can induce the development of immune tolerance by reducing the level of immune response to the viral vector or by reducing the level of immune response to the therapeutic peptide or a protein encoded by a viral vector, or both the first and second. VIB4920 may induce the development of immune tolerance to the viral vector by reducing neutralizing antibody levels and/or T cell responses to the viral vector, either the vector itself or cells infected with the viral vector. VIB4920 may additionally or alternatively induce the development of immune tolerance to a viral vector replacement therapy by reducing the level of neutralizing antibodies or T cell response to a therapeutic peptide or protein encoded by a viral vector nucleic acid.

[0055] VIB4920 for use in various methods may comprise the amino acid sequence shown in FIG. 18. VIB4920 may have the amino acid sequence shown in FIG. 18, or may be characterized by the presence of one or more amino acid residue substitutions relative to the amino acid sequence shown in FIG. 18. If VIB4920 is characterized by the presence of amino acid sequence changes relative to those shown in FIG. 18, the changes may be changes in one of the linkers. VIB4920 contains a Gly15 linker separating the two CD40L-specific monomers and a Gly10 linker separating the CD40L-specific monomer from the HSA sequence. Both or one of these linkers may be modified and may be replaced by an amino acid sequence (G_mX)_nwhere X is serine (S), alanine (A), glycine (G), Leu (L), isoleucine (I) or valine (V); m and n are integer values; m equals 1, 2, 3 or 4; and n is 1, 2, 3, 4, 5, 6, or 7. For example, one or both of the linkers may be modified to have an amino acid sequence that contains one of GGGGSGGGGS, GGGGSGGGGSGGGGS, GGGGGGGGGG, or GGGGGGGGGGGGGGG. If VIB4920 has an amino acid sequence related to the amino acid sequence shown in FIG. 18, this may be due to changes or changes in the amino acid sequence of HSA fused to two CD40L-specific monomers. An HSA fused to two CD40L-specific monomers may be changed to a related HSA fused to two CD40L-specific Tn3 monomers, except at least one amino acid substitution, numbered relative to a position in the full-length mature HSA, at a position selected from the group consisting of from 407, 415, 463, 500, 506, 508, 509, 511, 512, 515, 516, 521, 523, 524, 526, 535, 550, 557, 573, 574 and 580; wherein at least one amino acid change does not involve a change from lysine (K) to glutamic acid (E) at position 573. If VIB4920 is characterized by amino acid sequence changes relative to that shown in FIG. 18, changes may affect the amino acid sequence of one or both CD40L-specific Tn3 monomers, unless this results in an adverse effect on the effectiveness of VIB4920in vivo, for example, the amino acid sequence change is such that one or both of the CD40L-specific Tn3 monomers have the amino acid sequence shown in FIG. 19A.

[0056] The dose of VIB4920 administered in the methods may be a dose ranging from about 500 mg to about 3000 mg. The dosage may be from about 750 mg to about 3000 mg, or from about 1000 mg to about 3000 mg, or from about 1500 mg to about 3000 mg, or from about 500 mg to about 2000 mg, or from about 750 mg to about 2000 mg. mg, or from about 1000 mg to about 2000 mg, or from about 1000 mg to about 2500 mg, or from about 1000 mg to about 1500 mg. The dose may be 500 mg, 750 mg, 900 mg, 1000 mg, 1250 mg, 1500 mg, 1750 mg, 2000 mg, 2250 mg, 2500 mg or 3000 mg.

[0057] The dose of VIB4920 can be administered approximately every two weeks or can be administered twice a month. The dose of VIB4920 can also be administered approximately once per week or approximately once per month. VIB4920 can be dosed every 7 days, every 10 days, every 14 days, every 15 days, every 16 days, every 14-10 days, every 14-16 days, or every 30 days. The dose of VIB4920 can be administered by intravenous or subcutaneous injection.

[0058] If the administered dose of VIB4920 is a dose of 1000 mg, 1500 mg, or about 1000 mg to about 1500 mg, then the dose can be administered biweekly or it can be administered twice a month. If the dose of VIB4920 is 3000 mg, then the dose of VIB4920 can be administered once a month. If the dose of VIB4920 is 500 mg or 750 mg, then the dose of VIB4920 can be administered once every two weeks or alternatively administered twice a month. Any of these doses can be administered intravenously.

[0059] The dosage and dosage schedule of VIB4920 may be such that any therapeutic effect achieved by administering VIB4920 to treat any autoimmune/inflammatory disease or disorder is e.g., decreased autoantibody levels, decreased Vectra DA score, decreased occurrence of plasma cell signature, decreased CRP levels, decreased DAS28-CRP, decreased number of swollen joints, decreased number of tender joints, decreased CDAI, improved patient global score, improved global score by a physician, achieving ACR20, achieving ACR50, or achieving ACR70 may be considered “continuous.” A "long-lasting" effect of VIB4920 in the treatment of an autoimmune/inflammatory disease or disorder is an effect in which the therapeutic effect achieved with VIB4920 is maintained (even though VIB4920 is no longer administered) for at least 4 weeks, at least 6 weeks, at least 8 weeks, at least 10 weeks, at least 12 weeks, at least 16 weeks, at least 20 weeks, or at least 24 weeks after the last dose of the course of VIB4920. A course of VIB4920 may be a dose of VIB4920 ranging from 500 mg to 3000 mg (e.g. 500 mg, 750 mg, 1000 mg, 1250 mg, 1500 mg, 1750 mg, 2000 mg, 2250 mg, 2500 mg, 2750 mg, or 3000 mg) over a period of about 8 to 24 weeks (e.g. 8 weeks, or 10 weeks, or 12 weeks, or 14 weeks, or 16 weeks, or 18 weeks, or 20 weeks, or 22 weeks, or 24 weeks, or 2 months, or 4 months, or 6 months) with an administration interval doses once every 7-31 days (eg, once every 7 days, once every 10 days, once every 14 days, once every 15 days, once every 16 days, once every 14-10 days, once every 14-16 days or once every 30 days).

[0060] Those skilled in the art will identify, or be able to determine, using no more than routine experimentation, a large number of equivalents to the specific embodiments described herein. Such equivalents are intended to be covered by the following claims.

[0061] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent or patent application had been specifically and individually indicated for inclusion herein via link.

EXAMPLES

Example 1: Isolation and Optimization of CD40L-Specific Tn3 Proteins

[0062] Tn3 is a small scaffold protein of approximately 90 amino acids in length that contains structures with immunoglobulin fold variations, including loops structurally similar to the complementarity-determining regions of an antibody that can be randomized to select for specific binding properties ( 24 ).

[0063] Human CD40L-specific Tn3 clones were isolated as detailed in WO2013/055745 (also see24, 27, 50). Briefly, selection of human CD40L-specific Tn3 involved five rounds of panning, alternating selection on recombinant human CD40L protein and on a CHO cell line expressing human CD40L. Mouse CD40L-specific Tn3 proteins were selected using only recombinant mouse CD40L protein. The Tn3 genes from the selection were co-cloned into an expression vector and individual His-tagged variants were assessed for CD40L binding by capture on Maxisorp plates coated with anti-His antibody (2 μg/ml in PBS). Biotinylated MegaCD40L (Enzo Biosciences, 0.5 μg/ml) was added and incubated for 1.5 h. After a single wash with PBS/Tween, the interaction between captured Tn3 variants and CD40L was monitored using SA-HRP (1:1000 dilution). After 20 minutes, plates were washed twice in PBS/Tween, allowed to develop with TMB substrate, and stopped with 2.5 M H3PO4. Absorbance was measured at 450 nm. Affinity maturation of CD40L-specific Tn3 proteins was performed by selecting improved candidates from phage display libraries in which random mutations were allowed to occur in CDR-like loops (24). This strategy resulted in clone 342 (FIG. 19), an improvement on human CD40L-specific protein clone 309 (FIG. 21A), and clone M31, an improvement on mouse CD40L-specific protein clone M13. Additional human CD40L-Tn3 clones were also obtained, e.g. clones 304, 311, 320, 310, 321 and 322. See WO 2013/055745, incorporated herein by reference.

[0064] This series of human CD40L-specific Tn3 clones were characterized for their ability to biochemically inhibit the binding of CD40L to its receptor (CD40). All seven elements in this series inhibited the binding of CD40L to CD40 with IC50 values below 1 μM (Fig. 1A). The two most potent inhibitors in the biochemical CD40L-CD40 inhibition assay were further evaluated for inhibition of CD40L-mediated signaling in a cell-based reporter gene assay. HEK-293 cells expressing human CD40 and luciferase as a reporter gene for NF-κB were stimulated with recombinant human CD40L protein. Human CD40L-specific Tn3 protein clones 309 and 311 dose-dependently inhibited CD40L-induced NF-κB reporter gene expression at micromolar concentrations (Fig. 1B), reflecting the ability of such proteins to functionally inhibit CD40/CD40L signaling.

[0065] Simultaneous binding to multiple targets, as occurs with divalent antibodies, can lead to a marked increase in avidity. To study the effect of the presence of two valencies on the activity of CD40L-specific Tn3 proteins, two copies of identical Tn3 modules (309-309; e.g. fig. 21B) was linked via a flexible spacer containing Gly₄Ser, to form a tandem divalent fusion protein. Primary human B cells upregulate expression of the activation marker CD86 in response to stimulation by CD40. Preincubation of human peripheral blood mononuclear cells (PBMCs) with monovalent CD40L-specific Tn3 clone 309 inhibited the upregulation of CD86 expression on human CD19+ B cells in a dose-dependent manner (Fig. 1C). Strikingly, compared to monovalent Tn3, there was an almost 1000-fold improvement in activity with the bivalent construct in this assay using primary cells ( Fig. 1C ). In addition, affinity maturation (by performing random mutagenesis in the variable CDR-like loop regions) of clone 309, resulting in clone 342, provided a significant improvement in CD40L binding affinity (309: 190 nm; 342: 1.4 nm) and showed ca. 300-fold improvement in CD40-CD40L interaction inhibitory activity (Figure 1D). Affinity matured clone 342 was also screened for binding to a panel of related TNF family members, including Fas, TNFα, TNFβ, and OX40L, and was found to selectively bind CD40L. See fig. 1E.

[0066] Finally, as with other alternative scaffold technologies and because of their small size, when administered systemically, fully purified Tn3 molecules are expected to exhibit very rapid clearance from the bloodstream. To improve the pharmacokinetic properties of the proteins, CD40-specific Tn3 proteins were fused to serum albumin ( 28 , 29 ). A murine substitute for the bivalent CD40L-specific Tn3 protein, M13-M13, had a half-life in mice when delivered systemically of <30 minutes. Fusion of murine serum albumin (MSA) with the Tn3 protein M13-M13 resulted in a 65-fold increase in serum half-life and a 345-fold decrease in clearance (Table 1).

Table 1. Fusion with serum albumin results in a strong improvement in the half-life of the Tn3 molecule M13-M13

Molecule M13-M13 M13-M13-MSA Half-life (days) 0.02 1.3 Cmax (µg/ml) 10.7 135.8 AUC (µg/day/ml) 0.4 142.1 CL (ml/day/kg) 24305.2 70.4 Vss (ml/kg) 346.5 126.8

CD-1 mice 5-7 weeks old received a single injection of the divalent CD40L-specific Tn3 molecule with or without MSA (n=12/group; 10 mg/kg, i.v.). Blood samples were collected from groups of n=3 mice/group at various time points ranging from 15 minutes to 72 hours and circulating Tn3 protein levels were determined by ELISA.

[0067] Based on such observations, the divalent human CD40L-specific Tn3 molecule, VIB4920, was composed of tandemly arranged clones of 342 CD40L-specific Tn3 proteins for optimal activity, fused to human serum albumin (HSA) to provide improved half-life (Figure 1F ; Fig. 18).

[0068] To better understand the molecular nature of the interaction between CD40L and VIB4920, crystallographic studies were performed. CD40L-specific Tn3 (342) and soluble CD40L proteins were expressed, purified, cocrystallized, and structure determined at 2.8 Å resolution. The molecular structure of trimeric soluble CD40L complexed with Tn3 is shown in FIG. 2A. The contact surface with CD40L is composed of amino acids, the majority of which belong to the second modified loop of Tn3, including eight of the ten hydrogen bonds formed between the molecules (within a distance of 3.5 Å, Fig. 2B). Initial characterization of the molecule revealed that Tn3 clone 342 was capable of blocking the interaction between CD40L and CD40. To visualize the details of this action, the structure of the CD40/CD40L complex was combined with the structure of the clone 342/CD40L complex (Fig. 1G). When combined, clone 342 and CD40 were demonstrated to share binding sites on CD40L. Therefore, VIB4920 competes with CD40 and prevents its association with CD40L.

Example 2: VIB4920 Blocks Human B Cell Activation and Differentiation

[0069] CD40-mediated signaling has been extensively characterized and involves the activation of a large number of different pathways and transcription factors, including NF-κB ( 30 ), which can promote B cell activation, proliferation, and differentiation ( 31 ). Therefore, the ability of VIB4920 to inhibit CD40L-mediated NF-kB activation was studied using a cell line that expresses human CD40 and luciferase as a reporter gene for NF-kB-. Stimulation of this cell line with recombinant human CD40L or with cells expressing CD40L induces NF-kB activation. VIB4920 was able to effectively block CD40 signaling in this cell line, as demonstrated by dose-dependent inhibition of NF-κB activation (IC50: 0.899 nM; Figure 3A).

[0070] Resting B cells constitutively express the co-stimulatory molecule CD86 at low levels, the expression level of which rapidly increases upon activation, including activation by CD40 ( 32 ). Primary human PBMCs were stimulated with recombinant human CD40L and CD86 expression was assessed on B cells after 16 hours by flow cytometry. VIB4920 completely prevented the CD40L-mediated increase in CD86 expression by primary human B cells (Fig. 3B).

Example 3 VIB4920 does not induce platelet aggregation in vitro

[0071] mAbs targeting CD40L have not been approved in clinical trials due to safety concerns, largely due to thromboembolic complications associated with cross-linking of CD40L on the surface of platelet cells. To confirm that VIB4920, which lacks the Fc domain, does not induce platelet aggregation, we evaluated its effect on washed human plateletsin vitro. As previously described, when preformed as a complex with sCD40L, the anti-CD40L (human IgG1) mAb exhibited a significant ability to induce platelet aggregation (Fig. 3F). The response was rapid, with mAb-sCD40L immune complexes inducing 80% platelet aggregation within 8 minutes. Importantly, preincubation of platelets with an antibody that blocks FcγRIIa (mAb IV.3) prevented mAb-immune complex-mediated aggregation, consistent with an essential role of Fc receptors in this response (Fig. 3F). In contrast, at several concentrations tested, VIB4920 did not show a propensity to induce platelet aggregation in this assay (Fig. 3G). These data indicate that the absence of the Fc region in Tn3-based constructs may reduce the risk of platelet aggregation and thromboembolic events that have been observed with the use of therapeutic anti-CD40L antibodies in clinical practice. In support of this result, data (not shown) from a long-term (seven-month) study in non-human primates administered a dose of VIB4920 not exceeding 300 mg/kg showed no adverse effects on platelet function, e.g. showed no adverse results in tests for D-dimers (to monitor the presence of blood clots), PFA100 (to assess platelet function), or the TAT complex (thrombin-antithrombin complex).

Example 4: CD40L-Specific Tn3 Proteins Modulate Immune Responses in Vivo

[0072] The central role of CD40L in promoting T cell-dependent immune responses has been well characterized ( 9, 35 ). Therefore, a T-dependent immunization model was used to evaluate the ability of the Tn3-MSA fusion protein to block humoral immune responses in vivo . Due to the lack of sequence homology between human and mouse CD40L, a murine CD40L-specific Tn3 surrogate, M31, was used for such studies.

[0073] To test whether the Tn3-MSA fusion protein was able to block immune responses in vivo , mice were injected with sheep red blood cells (SRBC) and then treated daily on days 9-13 post-administration with the CD40L-specific binding protein Tn3 . The immune response in treated animals was assessed on day 14 by quantifying germinal center B cells in the spleen and lymph nodes by flow cytometry. As expected, immunization with SRBC in control-treated mice resulted in a significant increase in germinal center frequency (Fig. 4A). A dose-dependent reduction in the frequency of germinal center B cells was observed in mice treated with the CD40L-specific Tn3-MSA fusion protein (Fig. 4A). At a dose of 30 mg/kg, the CD40L-specific Tn3-MSA fusion protein induced complete inhibition of germinal center formation, as assessed by the almost complete absence of germinal center B cells in the spleen and lymph nodes, equivalent to control non-immunized mice. Other cell subsets were not disrupted by drug administration, including specific T cell populations, ensuring that the observed effects were not secondary to T cell depletion (data not shown). In addition, SRBC IgG levels reflected the level of germline B cell response. however, a significant decrease in SRBC-specific Ig titers occurred at higher doses of Tn3, which specifically binds CD40L (Fig. 4B).

Example 5 VIB4920 is well tolerated in healthy volunteers

[0074] The safety properties of VIB4920 were assessed in humans in a Phase 1a (Ph1a) study conducted in healthy adults aged 18-49 years. Subjects were enrolled in seven single-dose escalation cohorts, with doses of VIB4920 no more than 3000 mg, and were randomized to VIB4920 or placebo (Figures 5A and 5B). The primary endpoint of safety and tolerability was measured by the incidence of treatment-emergent adverse events (TEAEs) and treatment-emergent serious adverse events (TESAEs). Across all dosing cohorts, TEAEs were generally of low clinical significance, with the most common events being nasopharyngitis (the common cold) and headache (Table 2).

Table 2. VIB4920 demonstrates a good safety profile in humans

Most common TEAE occurring in at least 2 subjects in the Ph1a Healthy Volunteer Study.

[0075] Importantly, the overall percentage of subjects with one or more TEAEs associated with the study product was comparable for the overall VIB4920 group (40.9%) and the placebo group (33.3%). Additionally, there were no infusion-related reactions, severe infections, or deaths, and only one TESAE, tibial fracture, was reported in the placebo group. Notably, in this Ph1a study, no clinically significant clotting or platelet function abnormalities were observed following treatment with VIB4920.

Example 6: VIB4920 Demonstrates a Preferred PK/PD Profile in Healthy Volunteers

[0076] In addition to evaluating the safety profile of VIB4920, the Ph1a study also evaluated pharmacokinetic (PK) and pharmacodynamic (PD) endpoints. The PK profile of VIB4920 following a single intravenous dose of 3-3000 mg was linear, with exposure increasing proportionally to dose (Figure 6A). The mean terminal half-life of the molecule was 8 days, with the half-life at the highest dose being no more than 10.1+/-1.87 days.

[0077] CD40L is a transmembrane protein; however, it can be cleaved off and shed by both activated T cells and platelets. Soluble CD40L (sCD40L) is an 18-kDa trimer that is detected at low levels in healthy donors and at elevated levels in the circulation of patients with autoimmune disease ( 36, 37 ). Measuring sCD40L levels after administration of VIB4920 represents a potential indicator of the level of target binding, since sCD40L bound to VIB4920 may persist and accumulate in the bloodstream. As expected, a dose-dependent increase in total sCD40L levels was observed in plasma following administration of VIB4920 (Fig. 6B), indicating target binding. The time to reach maximum plasma total sCD40L levels increased from 11.5 to 84 days when the dose was increased from 3 mg to 3000 mg, indicating that target binding was maintained for a longer period in the highest dose group.

Example 7 Reduced levels of ADA variants were observed in healthy subjects receiving higher doses of VIB4920

[0078] Biological drugs are by nature highly specific/selective; however, they are complex molecules capable of inducing an immune response. Anti-drug antibody (ADA) variants provide an indicator of the immunogenicity of a therapeutic. In healthy volunteers, ADA variants were detected in the vast majority of patients receiving low doses of VIB4920 (Fig. 7). More specifically, 18 of 20 subjects dosed in the 3-100 mg range had detectable levels of ADA variants, with 10 of these subjects demonstrating high ADA titers (titer value greater than the median value of 480). In contrast, the incidence of ADA variants was significantly reduced at higher levels of VIB4920 (Fig. 7), and only 1 of 8 subjects in the 3000 mg dose group developed detectable antibody titers to the drug. The reduction in ADA incidence observed at high doses of VIB4920 confirms the immunomodulatory properties of the molecule. Additionally, lower percentages and titers of ADA variants may indicate a better tolerated, more effective therapeutic agent.

Example 8 VIB4920 Inhibits T Cell-Dependent Antibody Response in Healthy Volunteers

[0079] VIB4920 was further evaluated for its ability to influence humoral immune responses in healthy subjects. This evaluation was carried out by determining the effect of VIB4920 on T cell-dependent antibody response (TDAR) induced by immunization with hemocyanin from Megathura crenulata (KLH). For healthy subjects in all treatment groups, two immunization sessions were administered by subcutaneous administration of KLH: ( first ) 14 days before dosing with either VIB4920 or placebo, and ( second ) 15 days after dosing (Fig. 5B). Both IgM and IgG antibodies raised to KLH were monitored until day 113.

[0080] TDAR occurred as expected in subjects treated with placebo, i.e., a pattern that included a sharp rise in KLH IgG titers on day 22 (one week after booster), peak IgG levels observed on day 29, and then a drop in KLH-specific IgG antibodies by the end of the monitoring period (Fig. 8A ). Additionally, and consistent with previous publications, the secondary response to KLH in the placebo-treated group was dominated by IgG, with an overall much more modest increase in KLH-specific IgM detected after rechallenge (Figure 8B) (38-41).

[0081] As expected, healthy volunteers treated with VIB4920 at low doses had anti-KLH antibody titers similar to those of the placebo-treated group. In contrast, healthy volunteers treated with VIB4920 at high doses showed a significantly reduced level of secondary response to KLH, such that at day 43 there was a statistically significant decrease in KLH IgG levels starting at the 300 mg dose (p=0.035 ), and increased at doses of 1000 mg (p=0.002) and 3000 mg (p<0.001). Of note, anti-KLH IgG levels were reduced by 78% and 86% compared with placebo on day 43 in the 1000 mg and 3000 mg cohorts, respectively (Figure 8C). In the highest dose group, 7 of 8 subjects had undetectable anti-KLH IgG titers at day 43, suggesting near complete suppression of the humoral immune response by VIB4920.

Example 9 Immunosuppression by VIB4920 is Mediated by Inhibition of B Cell Proliferation and Plasma Cell Responses

[0082] The mechanism by which VIB4920 suppresses secondary immune response types was better defined by collecting peripheral blood from subjects before and after immunization and characterizing circulating lymphocyte subsets by flow cytometry. In healthy subjects treated with placebo, booster immunization induced B cell proliferation, which was detected by detecting an increase in the frequency of Ki67+ CD19+ B cells in the circulation at the day 22 visit, i.e. e. 7 days after re-administration (Fig. 9A).

[0083] In subjects who received a high dose of VIB4920, and before rechallenge, the baseline frequency of proliferating B cells was reduced compared to the placebo-treated group. This is consistent with the proposed mechanism of action of the molecule. In addition, in cohorts receiving high dose VIB4920, the proliferative B cell response following immunization was largely impaired, as demonstrated by the lack of increase in Ki67+ B cells. See 3000 mg cohort, one week post-administration (Figure 9A). Additional phenotyping revealed that the greatest effect of VIB4920 on proliferating B cells was within the isotype-switched IgD-CD27+ memory cell population (Figure 9B). These data are consistent with the TDAR results, which demonstrate that VIB4920 has an inhibitory effect on IgG production in response to secondary administration.

[0084] Monitoring of changes in gene expression levels was also performed in the peripheral blood of subjects treated with placebo or VIB4920 before and after booster immunization with KLH. Specifically, the plasma cell (PC) gene signature, which is an accurate and reliable signature that can detect even subtle changes in the frequency of circulating PCs ( 42 ), was used to confirm the presence of specific changes in gene expression levels. Consistent with the results of the TDAR study, immunization induced a significant increase in whole blood PC gene signature score in placebo-treated subjects in the first week after booster administration, which returned to baseline within two weeks (Figure 9C ). In the cohort with the highest dose of VIB4920 (3000 mg), the peripheral blood PC gene signature score was significantly reduced compared with placebo-treated subjects before KLH reintroduction (Fig. 9C). Importantly, volunteers receiving high dose VIB4920 did not have an increase in PC gene signature score after booster immunization. These data depict the mechanism of action of VIB4920 and demonstrate its potent ability to suppress B cell and PC response types.

Example 10 Multiple doses of VIB4920 in patients with RA are safe and well tolerated

[0085] After establishing that VIB4920 had an acceptable safety profile and demonstrated evidence of mechanism of action in healthy volunteers, the Ph1b clinical proof-of-concept trial was conducted using multiple escalating doses in adult patients with moderate to severe RA. Patients with RA were treated with VIB4920 (75 mg, n=8; 500 mg, n=10; 1000 mg, n=12; or 1500 mg, n=12) or placebo (n=15) administered by intravenous ( i. v.) infusions every two weeks for 12 weeks (Fig. 10). Patients were then followed for an additional 12 weeks after treatment. Key endpoints measured at week 12 included safety, tolerability, PK parameters, ADA variants, and change in disease activity (DAS28-CRP), as well as additional biomarkers such as RF autoantibody levels, serum C-reactive protein (CRP) levels blood and Vectra-DA score. Fifty-three patients completed 12 weeks of treatment; two patients (one in the VIB4920 75 mg group and one in the VIB4920 1500 mg group) discontinued treatment due to adverse events, one patient in the placebo group withdrew informed consent, and one patient in the VIB4920 75 mg group was lost to follow-up. Basic demographic and clinical characteristics of the study population at baseline are presented in Fig. eleven.

[0086] Overall, VIB4920 was generally safe and well tolerated with a balanced distribution of observed TEAEs between the placebo group and the four dose groups. The most common TEAEs reported were diarrhea, hyperhidrosis, upper respiratory tract infection, and urinary tract infection, each occurring in 3 patients (7.1%). See fig. 12. No thrombotic adverse events or clinically significant coagulation disorders were noted. One adverse event (preferred term encephalitis) that was serious and life-threatening was reported in the 1500 mg dose group after 6 doses of study drug. No etiologic infectious agent was identified, and several months after stopping VIB4920, similar symptoms returned and the patient was subsequently diagnosed with metastatic melanoma of the brain.

Example 11 VIB4920 exhibited a linear PK profile and dose-dependently reduced levels of ADA variants in patients with RA

[0087] ADA variants similar to those in healthy volunteers from the Ph1a study treated with low dose VIB4920 were observed in RA patients receiving low dose VIB4920. Three of 8 (37.5%) RA patients receiving 75 mg VIB4920 and 3 of 10 (30%) RA patients receiving 500 mg VIB4920 developed ADA variants (Figure 13B). In the VIB4920 75 mg dose group, 2 of 8 subjects developed detectable levels of ADA variants during the treatment phase; All 3 subjects in the 500 mg VIB4920 treatment group developed detectable levels of ADA variants after treatment (Figure 13C). No ADA variants were identified during the treatment period in the 1000 mg dose group; one subject had detectable ADA levels after the treatment phase. No ADA variants were detected in the 1500 mg dose group (Figure 13B), suggesting that VIB4920 effectively suppresses the ADA response at higher doses.

Example 12: VIB4920 Reduces Disease Activity in Patients with RA

[0088] DAS-28/CRP scores were determined to understand whether VIB4920 reduced disease activity in patients with RA during a phase 1b clinical trial. The DAS-28/CRP score is a composite RA Clinical Disease Activity Score that includes the following: the number of swollen joints, the number of tender joints, CRP levels, and the patient's overall health status. VIB4920 significantly reduced disease activity, as quantified by DAS28-CRP score, in RA patients at higher doses (Figure 14A and Figure 22A). At week 12 post-treatment, the adjusted mean change from baseline in DAS28-CRP (SE) was: -2.3 (0.3) in the VIB4920 1500 mg group, -2.2 (0.3) in the VIB4920 1000 mg group, -1.2 (0.3) in the VIB4920 500 mg group, 0.1 (0.4) in the VIB4920 75 mg group, and -1.0 (0.3) in the placebo group (Figure 14A). Surprisingly, this observed reduction in DAS28-CRP score in patients with RA was maintained for at least an additional 12 weeks after the last dose of VIB4920. (See Figure 22A, particularly visit days 113, 141 and 169). The effect of VIB4920 on DAS28-CRP was rapid, with the reduction in score becoming noticeable by day 15, which occurred after only one dose of the drug.

[0089] Moreover, the reduction in disease activity at the two highest doses of VIB4920 when compared with placebo was both clinically and statistically significant; the adjusted mean difference at week 12 (SE) for the VIB4920 1500 mg group was -1.4 (0.4) and for the VIB4920 1000 mg group was -1.2 (0.4), p-value was 0.002 and 0.006, respectively. Using a linear dose-response model, a statistically significant dose-response relationship was demonstrated for DAS28CRP (p<0.001). The significant result was mainly achieved by the 1000 mg and 1500 mg treatment groups; 500 mg and 75 mg showed virtually no benefit over placebo (Figure 15A). In terms of clinical response in an individual patient, 75% of patients in the 1500 mg group and 50% of patients in the 1000 mg dose group achieved a DAS28-CRP score of 3.2 or less at week 12, indicating that they had there was low disease activity or clinical remission at the primary endpoint. See fig. 16.

Example 13: VIB4920 Provides Reduction in Immunologic and Inflammatory Biomarkers in Patients with RA

[0090] The effect of VIB4920 on immunological and inflammatory biomarkers was determined using the Vectra DA blood test. The Vectra DA test is a commercially available and validated test that measures 12 biomarkers (adhesion molecules, growth factors, cytokines, matrix metalloproteinases, skeletal proteins, hormones and acute phase proteins) of disease activity and combines them into a single score to assess key mechanisms and pathways that mediate disease activity of RA. At doses of 1500 mg and 1000 mg, VIB4920 significantly reduced the Vectra DA biomarker score during both the 12-week period during which VIB4920 was administered biweekly (Figure 14E) and an observation period of 12 weeks, during which VIB4920 was no longer administered (Fig. 22E). The adjusted mean difference for VIB4920 at 1500 mg compared with placebo (week 12) was 14.4 (-21.5, -7.2), p=0.001, and the adjusted mean difference for VIB4920 at 1000 mg compared with placebo (week 12) was -10.3 (-17.4, -3.3), p=0.018 (Figure 14E).

[0091] The efficacy results were in good agreement with other endpoints assessed in this trial (including Clinical Disease Activity Index - CDAI, number of tender and swollen joints, patient and physician global assessment, and serum CRP levels), confirming that 1000 and 1500 mg represent clinically effective doses in this study (FIGS. 14B-14D and FIGS. 17A-17C; see also FIGS. 22B-22D and FIGS. 23A-23C).

Example 14 VIB4920 Provides Significant Reduction in Rheumatoid Factor Autoantibodies in Subjects with RA

[0092] Rheumatoid factor (RF) autoantibodies are a family of autoantibodies produced against the Fc portion of IgG. Their levels are elevated in RA and are associated with a poor prognosis. Given the mechanism of action of VIB4920, its effect on autoantibody titers in subjects with RA was assessed. Notably, VIB4920 significantly reduced RF titers at dose levels of 500, 1000 and 1500 mg (Figure 14F) over a 12-week treatment period with biweekly dosing. Additionally and unexpectedly, the reduced RF titers at the 1000 and 1500 mg dose levels were maintained over a 12-week follow-up period after the last dose of VIB4920 (Figure 22F). The reduction in RF titers in response to VIB4920 from baseline was noticeable as early as day 29, with high doses of VIB4920 reducing RF titers by approximately 50% by day 85. Using the Emax model, VIB4920 demonstrated a statistically significant dose-response relationship for the reduction in RF titers from baseline (p<0.001) (Fig. 15B).

Example 15. Methods

[0093] NF-kB reporter assay.

[0094] HEK293 cells expressing luciferase as a reporter for NF-kB-(Panomics) were engineered to stably express full-length human CD40R. Cells were seeded at a density of 5 x 10 ⁴ cells/well in 96-well poly-D-lysine-coated plates (BD Biosciences) and stimulated with recombinant megaCD40L protein (1.5 μg/ml, Enzo Biosciences) or Jurkat subclone D1.1 cells overexpressing CD40L (ATCC) for 16-24 hours in the presence or absence of control or CD40L-specific Tn3 at the indicated concentrations. Luminescence was detected using the Bright-Glo Luciferase Assay System (Promega) on a SpectraMax M5 plate reader (Molecular Devices).

[0095] CD86 upregulation assay

[0096] Human blood was collected from healthy donors after obtaining informed consent approved by the MedImmune Institutional Review Board. Peripheral blood mononuclear cells were isolated from CPT tubes (BD Biosciences) after centrifugation. PBMC (2.5-5.0 x 10 ⁵ cells/well) were stimulated in a 96-well round bottom plate with recombinant megaCD40L (100 ng/ml, Enzo Biosciences) for 16-18 hours in the presence of CD40L-specific Tn3 or mAb (clone 5c8) as stated. Flow cytometry was used to assess the expression level of CD86 on CD19+ B cells. The following antibodies were used: anti-CD86 (clone 2331, BD Pharmingen), anti-CD19 (clone HIB19, BD Pharmingen).

[0097] Human B cell assay

[0098] PBMC were isolated. Total B cells were negatively selected using MACS cell separation technology (Miltenyi Biotec), which typically provides purity levels greater than 95%. Purified peripheral blood B cells were cultured at a density of 0.5 to 1.0 x 10 ⁵ B cells per well in 96-well round bottom plates in a final volume of complete medium of 150 µL. Culture medium for B cell experiments was RPMI 1640 (Invitrogen) supplemented with 10% FCS, penicillin-streptomycin (100 U/ml penicillin, 100 μg/ml streptomycin), 2-mercaptoethanol (55 μM), L-glutamine (2 mM) and HEPES (5 mM). During the initial culture phase, B cells were stimulated with a combination of IL-21 (33 ng/ml, PeproTech Inc.) and megaCD40L (1.5 nM, Enzo Biosciences) with or without F(ab') ₂ to IgM (5.0 µg/ml, Jackson ImmunoResearch Laboratories). The level of B cell expansion was quantified by measuring ATP levels on day 3 or day 4 of culture using the Cell Titer-Glo luminescence assay (Promega) according to the manufacturer's instructions. PC differentiation was quantified at day 7 by flow cytometry. Cells were harvested for a fixed period of time and PC was defined as CD19 ⁺ IgD ^- CD38 ^high. cells.

[0099] Mouse model of immunization with SRBC

[00100] Balb/c mice (Jackson Laboratories) were immunized on day 0 with 0.2 ml SRBC (Colorado Serum Company) by intraperitoneal injection after removal directly from the bottle. Control (30 mg/kg) or CD40L-specific Tn3 (no more than 30 mg/kg as indicated) was administered daily on days 9–13 (intravenous). The frequency of germinal center B cells in the spleen was quantified at day 14 by flow cytometry. GC B cells were defined as CD19 ⁺ B220 ⁺ Fas ⁺ PNA ⁺ B cells.

[00101] Platelet Aggregation Assay

[00102] Human blood was collected from healthy donors into tubes containing ACD Solution B containing citric acid, dextrose and sodium. After centrifugation, two-thirds of the platelet-rich plasma was transferred to a polypropylene tube and incubated for 10 minutes with apyrase (2 U/ml) to prevent platelet activation during processing. Platelets were pelleted and resuspended in modified Tyrode buffer (137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 5.6 mM dextrose, 3.3 mM NaH2PO4, 20 mM HEPES, 0.1% BSA and pH 7.4) .

[00103] An immune complex (IC) was prepared by mixing mAb (h5c8 or negative control antibody) or Tn3 that specifically binds CD40L with hCD40L (293 cell source) for 5 minutes at room temperature. In some experiments, platelets were preincubated with anti-CD32a antibody (IV.3) for five minutes before adding IC. The platelet aggregation assay was performed according to the manufacturer's instructions with stirring at 37°C in a four-channel optical platelet aggregometer (model 700, Chrono-Log, Havertown, PA). Transmission monitoring was carried out for 12-20 minutes after mixing washed platelets with agonists.

[00104] Subjects and study design for Ph1a

[00105] A randomized, blinded, placebo-controlled Phase I study was conducted in healthy adults aged 18-49 years, including women not capable of childbearing (NCT02151110). Subjects were randomized into seven dose cohorts (3, 10, 30, 100, 300, 1000, or 3000 mg) and dosed sequentially based on the study protocol and recommendations from the dose escalation committee (DEC) that reviewed the safety data. and tolerability data obtained for the current dosing cohort, as well as accumulated data from previous dosing cohorts. Subjects were induced to TDAR by performing two separate immunization procedures with 1 mg KLH administered subcutaneously. The first KLH immunization session was performed during the screening period, 14 days before dosing with either VIB4920 or placebo, and the second KLH immunization session was performed on day 15 after dosing with either VIB4920 or placebo. Three interim analyzes were performed per protocol when all subjects in Cohort 5 (300 mg), Cohort 6 (1000 mg), and Cohort 7 (3000 mg) completed day 43, respectively.

[00106] Analysis of PK VIB4920

[00107] VIB4920 in K2EDTA human plasma was measured using a validated sandwich ELISA method in which each incubation step was followed by a wash step with 1 x PBS/0.1% Tween 20 (PBST) to remove unbound components. Briefly, Nunc microtiter plates were coated with mouse monoclonal antibody to VIB4920 (MedImmune) at a concentration of 1 μg/ml overnight at 2–8°C. Standards, quality controls (QC), and samples containing VIB4920 were diluted to the method's minimum required dilution (MRD) of 1:50 in 0.5% bovine serum albumin (BSA)/PBST before adding to the plate. After a 2-hour incubation, 1 μg/ml biotin-labeled rat anti-VIB4920 antibody (MedImmune) was added to the plate and incubated for 1 hour. The binding complex was visualized by successive incubation steps with streptavidin-linked horseradish peroxidase (HRP, GE Healthcare) and the peroxidase substrate SureBlue™ tetramethylbenzidine (TMB) (KPL, Inc.). Color development was stopped with 0.2 M sulfuric acid before analysis at 450 nm in a microplate reader. The quantitative range was from 0.05 to 1.60 μg/ml; Samples above the quantitative range were diluted with K2EDTA pooled blood plasma to bring the concentration within the range measured by the method.

[00108] Quantification of sCD40L

[00109] Plasma samples were collected to measure sCD40L concentrations during the screening period and on days 1, 2, 3, 5, 8, 15, 22, 29, 43, 57, 85, and 113. Total soluble CD40L (free sCD40L and sCD40L bound to VIB4920) in K2EDTA human plasma was measured using the Platinum Human sCD40L ELISA kit (eBioscience), which was modified to meet program requirements and qualified to ensure its accuracy and precision. Briefly, standards, QCs, and samples containing sCD40L were diluted to the method MRD of 1:50 in assay diluent containing 0.5% BSA/PBST and VIB4920 to ensure comparability and consistency of results. Wash steps with PBST followed each incubation step to remove unbound components. Diluted samples were added to a plate precoated with anti-sCD40L antibody and incubated for 1.5 hours. HRP-conjugated anti-human sCD40L antibodies were then added to ensure binding to sCD40L captured by the coating antibody. The binding complex was visualized by sequential addition of the peroxidase substrate TMB and stop solution (phosphoric acid) before analysis at 450 and 540 nm on a microplate reader. The quantitative range was from 6.25 to 400.00 ng/ml; Samples above the quantitative range were diluted with K2EDTA pooled blood plasma to bring the concentration within the range measured by the method.

[00110] ADA level measurement

[00111] The presence of ADA variants to VIB4920 in K2EDTA human plasma was determined using a validated sandwich ELISA method in which wash steps with PBST followed each incubation step to remove unbound components. Briefly, QC and samples were diluted to the method MRD of 1:60 in assay diluent containing 0.5% BSA/PBST, added to a washed Pierce™ Protein G coated plate (ThermoFisher) and incubated for 2 hours. Specific detection of ADA to VIB4920 was performed by overnight incubation with 1 μg/ml biotin-labeled VIB4920 prepared in assay diluent. The binding complex was visualized by successive incubation steps with streptavidin-linked HRP (GE Healthcare) and the TMB peroxidase substrate SureBlue™ (KPL, Inc.). Color development was stopped with 0.2 M sulfuric acid before analysis at 450 nm in a microplate reader. Each sample was subjected to a three-tier process in which the sample's response was first compared to a statistically determined OD cutoff value: if the value for the sample was at or above this value, the sample was considered potentially positive, and if below, the sample was determined to be ADA negative. Potentially positive samples were subjected to a second, competitive assessment in the presence of excess VIB4920; samples with percent inhibition at or above the statistically determined confirmation cutoff point were defined as confirmed positive and used for titer assessment. Samples with values below the confirmation cut-off point were considered negative for ADA to VIB4920. Titrated samples were serially diluted in pooled human plasma with K2EDTA to a level below the screening cut-off value, and the titer result was reported as the reciprocal of the highest dilution at which the sample was measured as positive before it was measured as negative. .

[00112] Evaluation of antibodies to KLH

[00113] Megathura crenulata (KLH) hemocyanin IgG antibodies in human serum were measured using a validated sandwich ELISA method in which wash steps with PBST followed each incubation step to remove unbound components; a volume of 100 μl/well was used for all steps. Briefly, nunc microtiter plates were coated with 3 μg/ml KLH (Immucothel, biosyn Arzneimittel GmbH) prepared in 1x PBS, pH 7.2, overnight at 2–8°C. Standards and QCs consisting of a mixture of nine anti-KLH IgG monoclonal antibodies of varying isotype and affinity (AstraZeneca) and samples containing anti-KLH antibodies were diluted to the MRD method of 1:250 in 0.5% BSA/PBST before adding to tablet. After a 2-hour incubation, HRP-conjugated mouse anti-human IgG antibodies (Invitrogen) were added to the plate and incubated for 1 hour to specifically detect anti-KLH IgG antibodies. The binding complex was visualized by successive steps of adding the peroxidase substrate TMB and stop solution (0.2 M sulfuric acid) before analysis at 450 on a microplate reader. The quantitative range was from 163.30 to 10000.00 ng/ml; samples with values above the quantitative range were diluted with serum to bring the concentration within the range measured by the method.

[00114] Flow cytometry in Ph1a

[00115] Blood was collected into Cytochex BCT tubes (Streck), sent to Covance Central Laboratory Services (Indianapolis, Indiana) and tested by flow cytometry using a validated method. Briefly, cells were stained with fluorochrome-labeled antibodies to CD45 (clone HI30), CD19 (clone HIB19), IgD (clone IA6-2), CD27 (clone M-T271), and CD38 (clone HIT2, all BD) to identify B cell populations . Cells were then treated with FACSPerm2 (Becton Dickenson) and stained for intracellular expression of Ki67 (clone KI67, Biolegend) to measure the number of proliferating cells.

[00116] PC signature

[00117] The PC gene signature was determined as previously described (Streicher 2014). Briefly, total RNA was isolated from PAXgene blood tubes using the PAXgene Blood RNA kit (Qiagen). For TaqMan qPCR, cDNA was prepared using the SuperScript III First-Strand Synthesis SuperMix kit (Life Technologies) and random primers. Samples were prepared using the TaqMan Pre-Amp Master Mix kit and analyzed using the BioMark Real-Time PCR System. We calculated ΔΔCt values using the average of 2 reference genes (β-actin and GAPDH) and the baseline expression level of each patient as a control. Fold change values were determined by calculating 2-ΔΔCt.

[00118] Patients and study design for Ph1b

[00119] A randomized, blinded, placebo-controlled Phase 1b study was conducted in patients aged 18-70 years who had been diagnosed with RA according to EULAR/ACR criteria (Aletaha et al. 2010) for at least 6 months prior to enrollment. study. Subjects had moderate to severe activity, defined as a DAS28-CRP score of at least 3.2 at screening, and at least 4 swollen and 4 tender joints at screening and randomization. At the time of screening, patients were positive for either rheumatoid factor (RF-IgM ≥ 14 units/mL) or anti-citrullinated peptide antibodies (ACPA). Patients received methotrexate (MTX) at a dose of 7.5-25 mg per week, or, if MTX intolerant, another conventional DMARD for at least 12 weeks and at a stable dose for at least 6 weeks before screening. Previous treatment with biologic agents (except rituximab or other B-cell depleting agents) prescribed for RA was acceptable provided that adequate washout was performed before randomization in the present study. Patients were treated with placebo (n=15) or VIB4920 (75 mg, n=8; 500 mg, n=10; 1,000 mg, n=12; or 1,500 mg, n=12) administered by route i. v. infusions every two weeks for 12 weeks, followed by post-treatment observation for 12 weeks. VECTRA-DA score measurements were performed at Crescendo Bioscience (San Francisco, CA), and RF autoantibody measurements were performed at Covance Central Laboratory Services (Princeton, NJ).

[00120] Statistical analysis

[00121] A two-tailed unpaired Student's t test was used to evaluate the effect of treatment on primary human B cell expansion, plasma cell differentiation, and GC B cell response in the SRBC model. The Mann-Whitney U test was used to compare the gene signature occurrence score across multiple time points between VIB4920 and placebo (Figure 9C). Statistical tests and plotting were performed using Graphpad Prism software. Dose-response relationships for changes in DAS28-CRP and RF from baseline at day 85 were analyzed using the MCP-Mod approach, including the corresponding baseline level as a covariate, with three pre-specified candidate models for the dose-response relationship (linear , Emax and Hill's Emax model). Dose-response signal testing was adjusted for multiplicity with an error correction for the effect of multiple comparisons set at 0.10. The final model was selected from those identified as significant based on Akaike's information criteria. Changes from baseline in DAS28-CRP, RF, Vectra DA, CDAI, number of tender joints, number of swollen joints, patient and physician global assessment, and serum CRP were analyzed using a mixed model for repeated measures analysis (MMRM) including the corresponding outcome at baseline as a covariate.

References cited in this disclosure

1. S. Sugio, A. Kashima, S. Mochizuki, M. Noda, K. Kobayashi, Crystal structure of human serum albumin at 2.5 A resolution. Protein Eng 12, 439-446 (1999)

2. S. Lederman et al. , Identification of a novel surface protein on activated CD4+ T cells that induces contact-dependent B cell differentiation (help). The Journal of experimental medicine 175, 1091-1101 (1992).

3. M. Croft, R. M. Siegel, Beyond TNF: TNF superfamily cytokines as targets for the treatment of rheumatic diseases. Nature reviews. Rheumatology 13, 217-233 (2017).

4. TM Foy, FH Durie, RJ Noelle, The expansive role of CD40 and its ligand, gp39, in immunity. Seminars in immunology 6, 259-266 (1994).

5. JB Splawski, PE Lipsky, CD40-mediated regulation of human B-cell responses. Research in immunology 145, 226-234; discussion 244-229 (1994).

6. RJ Armitage et al. , Molecular and biological characterization of a murine ligand for CD40. Nature 357, 80-82 (1992).

7. P. Garside et al. , Visualization of specific B and T lymphocyte interactions in the lymph node. Science 28 1 , 96-99 (1998).

8. D. Hollenbaugh et al. , The human T cell antigen gp39, a member of the TNF gene family, is a ligand for the CD40 receptor: expression of a soluble form of gp39 with B cell co-stimulatory activity. The EMBO journal 11, 4313-4321 (1992).

9. RJ Noelle et al. , A 39-kDa protein on activated helper T cells binds CD40 and transduces the signal for cognate activation of B cells. Proceedings of the National Academy of Sciences of the United States of America 89, 6550-6554 (1992).

10. TM Foy et al. ,gp39-CD40 interactions are essential for germinal center formation and the development of B cell memory. The Journal of experimental medicine 180, 157-163 (1994).

11. TM Foy et al. , In vivo CD40-gp39 interactions are essential for thymus-dependent humoral immunity. II. Prolonged suppression of the humoral immune response by an antibody to the ligand for CD40, gp39. The Journal of experimental medicine 178, 1567-1575 (1993).

12. S. Han et al. , Cellular interaction in germinal centers. Roles of CD40 ligand and B7-2 in established germinal centers. Journal of immunology 155, 556-567 (1995).

13. T. Kawabe et al. , The immune responses in CD40-deficient mice: impaired immunoglobulin class switching and germinal center formation. Immunity 1, 167-178 (1994).

14. RC Allen et al. , CD40 ligand gene defects responsible for X-linked hyper-IgM syndrome. Science 259, 990-993 (1993).

15. A. Aruffo et al. , The CD40 ligand, gp39, is defective in activated T cells from patients with X-linked hyper-IgM syndrome. Cell 72, 291-300 (1993).

16. JP DiSanto, JY Bonnefoy, JF Gauchat, A. Fischer, G. de Saint Basile, CD40 ligand mutations in x-linked immunodeficiency with hyper-IgM. Nature 361, 541-543 (1993).

17. D. T. Boumpas et al. , A short course of BG9588 (anti-CD40 ligand antibody) improves serologic activity and decreases hematuria in patients with proliferative lupus glomerulonephritis. Arthritis and rheumatism 48, 719-727 (2003).

18. AC Grammer et al. , Abnormal germinal center reactions in systemic lupus erythematosus demonstrated by blockade of CD154-CD40 interactions. The Journal of clinical investigation 112, 1506-1520 (2003).

19. W. Huang et al. , The effect of anti-CD40 ligand antibody on B cells in human systemic lupus erythematosus. Arthritis and rheumatism 46, 1554-1562 (2002).

20. V. L. Patel, J. Schwartz, J. B. Bussel, The effect of anti-CD40 ligand in immune thrombocytopenic purpura. British journal of haematology 141, 545-548 (2008).

21. SE McKenzie et al. , The role of the human Fc receptor Fc gamma RIIA in the immune clearance of platelets: a transgenic mouse model. Journal of immunology 162, 4311-4318 (1999).

22. JE Freedman, CD40-CD40L and platelet function: beyond hemostasis. Circulation research 92, 944-946 (2003).

23. L. Robles-Carrillo et al. , Anti-CD40L immune complexes potently activate platelets in vitro and cause thrombosis in FCGR2A transgenic mice. Journal of immunology 185, 1577-1583 (2010).

24. JS Swers et al. , Multivalent scaffold proteins as superagonists of TRAIL receptor 2-induced apoptosis. Molecular cancer therapeutics 12, 1235-1244 (2013).

25. R. Vazquez-Lombardi et al. , Challenges and opportunities for non-antibody scaffold drugs. Drug discovery today 20, 1271-1283 (2015).

26. R. N. Gilbreth, B. M. Chacko, L. Grinberg, J. S. Swers, M. Baca, Stabilization of the third fibronectin type III domain of human tenascin-C through minimal mutation and rational design. Protein engineering, design & selection: PEDS 27, 411-418 (2014).

27. V. Oganesyan et al. , Fibronectin type III domains engineered to bind CD40L: cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of two complexes. Acta crystallographica. Section F, Structural biology and crystallization communications 69, 1045-1048 (2013).

28. D. Muller et al. , Improved pharmacokinetics of recombinant bispecific antibody molecules by fusion to human serum albumin. The Journal of biological chemistry 282, 12650-12660 (2007).

29. BJ Smith et al. , Prolonged in vivo residence times of antibody fragments associated with albumin. Bioconjugate chemistry 12, 750-756 (2001).

30. I. Berberich, G. L. Shu, E. A. Clark, Cross-linking CD40 on B cells rapidly activates nuclear factor-kappa B. Journal of immunology 153, 4357-4366 (1994).

31. M. Kaileh, R. Sen, NF-kappaB function in B lymphocytes. Immunological reviews 246, 254-271 (2012).

32. M. Roy et al. , Studies on the interdependence of gp39 and B7 expression and function during antigen-specific immune responses. European journal of immunology 25, 596-603 (1995).

33. R. Ettinger et al. , IL-21 induces differentiation of human naive and memory B cells into antibody-secreting plasma cells. Journal of immunology 175, 7867-7879 (2005).

34. JL Karnell et al. , CD19 and CD32b differentially regulate human B cell responsiveness. Journal of immunology 192, 1480-1490 (2014).

35. BR Renshaw et al. , Humoral immune responses in CD40 ligand-deficient mice. The Journal of experimental medicine 180, 1889-1900 (1994).

36. K. Kato et al. , The soluble CD40 ligand sCD154 in systemic lupus erythematosus. The Journal of clinical investigation 104, 947-955 (1999).

37. RK Vakkalanka et al. , Elevated levels and functional capacity of soluble CD40 ligand in systemic lupus erythematosus sera. Arthritis and rheumatism 4 2 , 871-881 (1999).

38. P. Bird, J. E. Calvert, P. L. Amlot, Distinctive development of IgG4 subclass antibodies in the primary and secondary responses to keyhole limpet haemocyanin in man. Immunology 69, 355-360 (1990).

39. M. E. Devey, K. M. Bleasdale-Barr, P. Bird, P. L. Amlot, Antibodies of different human IgG subclasses show distinct patterns of affinity maturation after immunization with keyhole limpet haemocyanin. Immunology 70, 168-174 (1990).

40. J. Ferbas et al. , A novel assay to measure B cell responses to keyhole limpet haemocyanin vaccination in healthy volunteers and subjects with systemic lupus erythematosus. British journal of clinical pharmacology 76, 188-202 (2013).

41. JS Miller et al. , Diminished neo-antigen response to keyhole limpet hemocyanin (KLH) vaccines in patients after treatment with chemotherapy or hematopoietic cell transplantation. Clinical immunology 117, 144-151 (2005).

42. K. Streicher et al. , The plasma cell signature in autoimmune disease. Arthritis & rheumatology 66, 173-184 (2014).

43. FJ Dumont, IDEC-131. IDEC/Eisai. Current opinion in investigational drugs 3, 725-734 (2002).

44. W. Schuler et al. , Efficacy and safety of ABI793, a novel human anti-human CD154 monoclonal antibody, in cynomolgus monkey renal allotransplantation. Transplantation 77, 717-726 (2004).

45. F. Langer et al. , The role of CD40 in CD40L- and antibody-mediated platelet activation. Thrombosis and haemostasis 93, 1137-1146 (2005).

46. C. Heeschen et al. , Soluble CD40 ligand in acute coronary syndromes. The New England journal of medicine 348, 1104-1111 (2003).

47. F. Mach, U. Schonbeck, G. K. Sukhova, E. Atkinson, P. Libby, Reduction of atherosclerosis in mice by inhibition of CD40 signaling. Nature 394, 200-203 (1998).

48. T. Oura et al. , Long-term hepatic allograft acceptance based on CD40 blockade by ASKP1240 in nonhuman primates. American journal of transplantation: official journal of the American Society of Transplantation and the American Society of Transplant Surgeons 12, 1740-1754 (2012).

49. M. Watanabe et al. , ASKP1240, a fully human anti-CD40 monoclonal antibody, prolongs pancreatic islet allograft survival in nonhuman primates. American journal of transplantation: official journal of the American Society of Transplantation and the American Society of Transplant Surgeons 13, 1976-1988 (2013).

50. D. Spencer et al. , O-xylosylation in a recombinant protein is directed at a common motif on glycine-serine linkers. J Pharm Sci 102, 3920-3924 (2013).

Claims (17)

1. A method of treating or preventing an autoimmune disease or disorder, comprising administering a CD40L-specific Tn3 scaffold protein at a dose of 500 mg to 3000 mg to a subject in need thereof, wherein the CD40L-specific Tn3 scaffold protein contains: two monomeric subunit sequences connected by a first linker; And a human serum albumin sequence, wherein the human serum albumin sequence is linked to one of the two monomeric subunit sequences by a second linker, wherein each monomeric subunit sequence comprises the polypeptide sequence of SEQ ID NO:3, and wherein the autoimmune disease or disorder is one of focal segmental glomerulosclerosis, systemic sclerosis, myositis, antiphospholipid syndrome, autoimmune hepatitis, lupus nephritis, idiopathic thrombocytopenic purpura, vasculitis, cutaneous lupus, autoimmune hemolytic anemia, Sjogren's syndrome, IgG4-related disease, or systemic erythematous lupus. 2. A method of treating or preventing an autoimmune disease or disorder, comprising administering a CD40L-specific Tn3 scaffold protein at a dose of 500 mg to 3000 mg to a patient in need thereof, wherein the CD40L-specific Tn3 scaffold protein comprises SEQ ID NO: 1, wherein the autoimmune disease or disorder is one of focal segmental glomerulosclerosis , systemic sclerosis, myositis, antiphospholipid syndrome, autoimmune hepatitis, lupus nephritis, idiopathic thrombocytopenic purpura, vasculitis, cutaneous lupus, autoimmune hemolytic anemia, Sjogren's syndrome, IgG4-related disease, or systemic erythematous lupus. 3. Method according to any one of paragraphs. 1-2, where the dose is from 1000 mg to 1500 mg. 4. Method according to claim 3, where the dose is 1000 mg. 5. Method according to any one of paragraphs. 1-2, where the dose is 1500 mg to 3000 mg. 6. Method according to any one of paragraphs. 1-5, where the dose is administered once every 14 days or administered twice a month. 7. Method according to any one of paragraphs. 1-6, where the administration is intravenous. 8. Method according to any one of paragraphs. 1-7, wherein administration is effective in reducing the level of an inflammatory marker in a subject in need thereof. 9. The method of claim 8, wherein the inflammatory marker includes one or more of an autoantibody level, a plasma cell signature (PC) occurrence, a circulating B cell count, and an antibody class switch level. 10. The method of claim 9, wherein the inflammatory marker comprises reducing the occurrence of the PC signature. 11. The method according to claim 10, where the decrease in the occurrence of the PC signature is characterized by a decrease in the level of expression of a gene selected from the group consisting of immunoglobulin heavy constant alpha 1 (IGHA1), connecting chains of multimeric IgA and IgM (IGJ), kappa constant domain of immunoglobulin ( IGKC), immunoglobulin kappa variable domain 4-1 (IGKV4-1) and TNF receptor superfamily member 17 (TNFRSF17). 12. The method of claim 1 or 2, wherein the autoimmune disease or disorder is Sjögren's syndrome. 13. The method of claim 1 or 2, wherein the autoimmune disease or disorder is focal segmental glomerulosclerosis. 14. The method according to claim 1 or 2, wherein the inflammatory disease or disorder is lupus nephritis.

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