CD40L antagonists and uses thereof

Abstract

This article provides a human CD40L-specific Tn3 molecule and its therapeutic use.

Description

Background Art

[0001] The CD40 / CD40L pathway plays a key role in driving humoral immune responses and has been implicated 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 non-hematopoietic cells.

[0002] Expression of the CD40 ligand, CD40L (also known as CD154), is highly regulated and is predominantly found on activated CD4+ T cells (2). The CD40 / CD40L interaction between B cells and activated T cells is crucial for the establishment of effective humoral responses to T-dependent antigens (3-5). The CD40 / CD40L axis drives B cell expansion, differentiation, and isotype switching in vitro (6-9). In vivo, CD40 signaling is essential for germinal center (GC) formation, somatic hypermutation, and the generation of memory B cells and long-lived plasma cells (10-13). CD40 or CD40L deficiency in humans results in X-linked hyperimmunoglobulinemia syndrome, a disease characterized by impaired isotype class switching, manifested by high levels of serum IgM accompanied by low to undetectable levels of IgG, IgA, or IgE and increased susceptibility to infection (14-16).

[0003] Clinical trials of compounds targeting CD40L have shown that targeting the CD40 pathway has potential benefits in autoimmune diseases. In a phase II trial, the humanized 5c8 anti-CD40L antibody BG9588 significantly reduced proteinuria and anti-dsDNA antibody titers in patients with proliferative lupus nephritis (17). Other studies have shown that anti-CD40L treatment can reduce circulating CD38 in patients with active SLE. hi Ig-secreting cells as well as peripheral GC B cells (18,19). Anti-CD40L monoclonal antibody (mAb) treatment has also been shown to induce profound responses in a subset of patients with immune thrombocytopenia (ITP) (20).

[0004] Although anti-CD40L mAb therapy has demonstrated potential in clinical trials, the program has been suspended due to adverse thromboembolic events. Although not precisely defined, one potential explanation for these unexpected safety concerns is that FcγRIIa (or CD32a) is expressed on human but not mouse platelets (21). CD40L is also highly expressed on activated platelets (22), where antibody-mediated simultaneous binding to CD40L and FcγRIIa on adjacent cells may lead to platelet aggregation. Mouse models support a role for FcγRIIa in anti-CD40L-induced thrombocytopenia. In transgenic mice for human FcγRIIa, anti-CD40L mAb caused shock and thrombocytopenia (23). This effect was not observed in wild-type mice or transgenic mice injected with an aglycosylated form of the antibody that cannot bind to FcγRs.

[0005] To target CD40L without the potential complications associated with mAbs, a CD40L-specific Tn3 scaffold protein was generated (24, 25). The Tn3 protein is derived from the third fibronectin type III domain of human tenascin C and can be engineered to confer target-specific binding properties (26, 27). Fusion of a bivalent CD40L-specific Tn3 protein with human serum albumin (HSA) resulted in a molecule, VIB4920, that binds to human CD40L and prevents its interaction with the CD40 receptor. Consistent with this disruption in the CD40L / CD40 interaction, VIB4920 was able to effectively inhibit the activation and differentiation of human B cells in vitro by blocking CD40 signaling events.

[0006] There is a need in the art for new therapeutic agents that can significantly affect the humoral immune response and treat autoimmune and / or inflammatory disorders. There is also a need in the art for inducing immune tolerance to alternative therapies in patients in need thereof.

[0007] It has been found that when administered to patients with autoimmune / inflammatory diseases or disorders, VIB4920 can alleviate clinical symptoms of the disease and reduce other markers. In particular, administration of VIB4920 at specific doses to rheumatoid arthritis (RA) subjects resulted in statistically significant reductions in rheumatoid factor (RF) autoantibody titers, Vectra DA biomarker scores, and disease activity measured by DAS28-CRP compared to placebo. Summary of the Invention

[0008] The present disclosure provides a method for inhibiting B cell and T cell-mediated immune responses in a subject. The method comprises the following steps: administering a dose of 500 mg to 3000 mg of VIB4920 to a subject in need thereof, and inhibiting B cell and T cell-mediated immune responses.

[0009] The present disclosure also provides a method for treating an autoimmune disease or disorder, comprising the steps of administering a dose of 500 mg to 3000 mg of VIB4920 to a subject in need thereof, thereby treating the autoimmune disease or disorder.

[0010] This specification further provides a method for reducing the measurement of RA disease activity in a patient receiving RA treatment. The method comprises the following steps: administering VIB4920 to the patient, and reducing the measurement of RA disease activity in the patient. The measurement of reduced RA disease activity can include one or more of DAS28-CRP, Clinical Disease Activity Index (CDAI), tender joint count, swollen joint count, patient's global assessment, or physician's global assessment. VIB4920 can be administered at a dosage of about 500 mg to 3000 mg.

[0011] The present disclosure also provides a method for reducing RF autoantibodies in a patient being treated for RA, comprising the steps of administering VIB4920 to the patient at a dose of about 500 mg to 3000 mg, and reducing the patient's RF autoantibodies.

[0012] This specification also provides a method for reducing a biomarker score in a patient being treated for RA. The method comprises the following steps: administering about 500 mg to 3000 mg of VIB4920 to the patient, and reducing the patient's biomarker score. In such a method, the biomarker score can be one or more of a plasma cell (PC) gene signature, a Vectra-DA score, or a serum C-reactive protein (CRP) level.

[0013] This specification also provides a method for reducing the PC gene signature in a patient in need thereof. The method comprises the following steps: administering VIB4920 to a patient in need thereof, and reducing the PC gene signature score in the patient. The patient in need thereof may be a patient currently receiving treatment for systemic lupus erythematosus, rheumatoid arthritis, myositis, antiphospholipid syndrome, autoimmune hepatitis, Sjögren's disease, or other autoimmune or inflammatory conditions, as well as transplantation and graft-versus-host disease. VIB4920 may be administered to a patient in need thereof at a dose of approximately 500 mg to 3000 mg.

[0014] This specification further provides a method for reducing autoantibodies in a patient being treated for an autoimmune disorder, or reducing alloantibodies in a patient being treated for a transplant. The method comprises the steps of administering VIB4920 to a patient in need thereof, and reducing the patient's autoantibodies or alloantibodies. In such a method, the patient is being treated for an autoimmune disease characterized by the presence of autoantibodies, or is being treated to prevent transplant rejection. VIB4920 is administered to the patient at a dose of approximately 500 mg to 3000 mg.

[0015] This specification also provides a method for reducing inflammation in a patient. The method comprises the steps of administering VIB4920 to a patient in need thereof, and reducing the patient's inflammation. The patient may be currently receiving treatment for an inflammatory disease or disorder, or may be currently receiving prophylactic treatment for anticipated inflammation in response to an organ or tissue transplant. VIB4920 may be administered at a dose of approximately 500 mg to 3000 mg.

[0016] This specification further provides a method for inducing immune tolerance to an alternative therapy in a patient. The method comprises the following steps: administering VIB4920 to a patient in need of an alternative therapy, and inducing immune tolerance to the alternative therapy in the patient. VIB4920 can be administered at a dose of about 1000 mg to 3000 mg. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figures 1A-1G Biochemical characterization of human CD40L-specific Tn3 clones, including that of VIB4920, a bivalent 342 clone fused to HSA, is provided. Figure 1A Shown is the ability of a panel of human CD40L-specific Tn3 clones to inhibit CD40-CD40L interaction as measured by Proteon. The percentage of inhibition shown is over a range of Tn3 concentrations. The average of two replicate wells is shown. Figure 1B The ability of anti-CD40L Tn3 proteins to inhibit CD40L-mediated signaling via NFkB is shown. HEK293 cells expressing CD40R and NFkB-luciferase-reporter genes were stimulated overnight with recombinant CD40L in the presence of anti-CD40L Tn3 proteins. Percent inhibition of luciferase activity is shown. Data represent the average of two replicate wells. Figure 1C Inhibition of CD86 upregulation by various concentrations of Tn3 constructs is shown. After preincubation with CD40L Tn3 protein, human PBMCs were stimulated with recombinant CD40L and CD86 expression assessed by flow cytometry. The average of two replicate wells is shown. Figure 1DThe indicated Tn3 molecules were tested for their ability to inhibit CD40-CD40L interaction in an ELISA assay. Data represent the mean of duplicate wells. Figure 1E Data are provided for screening of clone 342 for binding to a panel of related TNF family members, including Fas, TNFα, TNFβ, and OX40L. Clone 342 was found to selectively bind CD40L. Figure 1F The proposed structure of VIB4920 was based on the crystallization of 342Tn3 and the published crystal structure of HSA (1). Figure 1G Pictorial representation of the CD40 / CD40L and 342 / CD40L structures arranged by the common CD40L molecule. CD40L is shown in green, Tn3 in magenta, and the CD40 receptor in cyan.

[0018] Figures 2A-2D Structural characterization of the human CD40L-specific Tn3 clone 342 is provided. Figure 2A This is a pictorial illustration of the trimeric 342 / CD40L structure. The extracellular domain of CD40L is shown in green; 342 is shown in magenta. Figure 2B The interface between 342 and CD40L is shown. Fragments of CD40L and 342 are shown as green and magenta tubes, respectively. Amino acids involved in hydrogen bonds are represented by short lines. Hydrogen bonds are represented by α-terminal distances. The black dashed line shows the maximum bond distance. Figure 2C and Figure 2D Shown ( Figure 2C )CD40L and ( Figure 2D ) Electrostatic surface potential of the interaction surface of 342CD40L-specific Tn3. The molecule is rotated approximately 90 degrees to reveal the interface and is semi-transparent to allow visualization of amino acids involved in hydrogen bonding. Red indicates a negatively charged surface, and blue indicates a positive charge.

[0019] Figures 3A-3G It is shown how VIB4920 inhibits CD40 signaling and activation of human B cells in ex vivo studies, but does not induce platelet aggregation. Figure 3A Shown is the percent inhibition of NFkB luciferase signaling by VIB4920 in engineered HEK29 cells stimulated overnight with CD40L. Data represent the mean of duplicate wells. One of two independent studies is shown. Figure 3B Figure 4 shows how VIB4920 and anti-CD40L mAb can inhibit CD86 upregulation in stimulated human PBMCs. Human PBMCs were stimulated with recombinant human megaCD40L and the percentage of CD19+ / CD86+ cells was measured by flow cytometry at 24 hours. Data represent the mean of two replicate wells. Figure 3CHuman B cells were stimulated with IL-21 and megaCD40L in the presence of control or anti-CD40L (mAb or VIB4920Tn3). B cell expansion was quantified on day 3. The dotted line represents ATP levels in unstimulated cells. Data shown are the mean and SD of three replicate wells and are representative of two independent experiments. Figure 3D and Figure 3E The effect of anti-CD40L (mAb or VIB4920 Tn3) on human B cells that were unstimulated (nil) or stimulated with IL-21, anti-IgM, and megaCD40L is shown. PC numbers were quantified on day 7. Specifically, Figure 3D The effects of the indicated molecules on the percentage of IgD-CD38hi PCs on day 7 are shown. Figure 3E Shown are the effects of the indicated molecules at the indicated concentrations on PC number at day 7. Data shown are the mean and SD of three replicate wells and are representative of two independent experiments. **** = p < 0.0001 by two-tailed unpaired Student's t-test. Figure 3F and Figure 3G Shown are the effects of anti-CD40L (mAb or VIB4920 Tn3) molecules on washed human platelets incubated with preformed immune complexes; platelet aggregation, or lack thereof, was measured for 12-14 minutes. Figure 3F The percentage of aggregation is shown. Where indicated, platelets were pre-incubated with anti-CD32a antibody for 5 minutes before adding immune complexes. Adenosine diphosphate (ADP) was used as a positive control for aggregation. Figure 3G Presented are the percentages of aggregation following incubation of platelets with immune complexes using the indicated concentrations of VIB4920 (Tn3) or anti-CD40L mAb (5c8). Data are representative of two independent experiments.

[0020] Figure 4A and 4B Mouse surrogate CD40L-specific Tn3 displayed potent neutralizing activity in response to immunization in vivo. Figure 4A and Figure 4B In the present study, mice were immunized with sheep red blood cells (SRBC) on day 0 and administered control or anti-CD40L Tn3 (M31-MSA) daily from day 9 to day 13. Figure 4A The percentages of germinal center B cells in spleen and lymph nodes quantified by flow cytometry on day 14 are presented. Points represent individual animals, and data are representative of two independent studies. **** = p < 0.0001 by two-tailed unpaired Student's t-test. Figure 4B Presented are the production of anti-sheep erythrocyte IgG quantified from serum at day 14. Data represent the mean and SEM of four animals per group (n=1 study).

[0021] Figure 5A and 5B : Study design of a Phase 1a clinical study to evaluate the safety of VIB4920 in healthy volunteers. Figure 5A Study cohorts for the Phase 1a study are shown. Figure 5B The Phase 1a study dosing and immunization strategies are provided.

[0022] Figure 6A and 6B : In a Phase 1a study in healthy volunteers, VIB4920 showed a favorable PK profile. Figure 6A Circulating levels of VIB4920 as determined by ELISA at the indicated time points are shown. The dashed line represents the lower limit of sensitivity of the assay. Error bars represent the standard deviation of the mean, which was not calculated for the group with N=2 subjects. Figure 6B Soluble CD40L levels assessed by ELISA in all dose cohorts at the indicated time points are shown. The dotted line represents the lower limit of detection of the assay.

[0023] Figure 7 : In a Phase 1a study in healthy volunteers, VIB4920 inhibited anti-drug antibodies (ADA) at high doses. The presence of ADA was determined by ELISA. Each subject in each cohort is depicted by a separate line. Subjects with high ADA (>480 median titer) are represented by a magenta line; subjects with low ADA (<480 median titer) are represented by a dark blue line; and subjects with undetectable ADA are represented by a light blue line.

[0024] Figures 8A-8C VIB4920 inhibits B cell proliferation and TDAR in a dose-dependent manner in healthy human volunteers. Healthy volunteers were immunized with KLH 14 days before treatment with placebo or VIB4920 and re-challenged 15 days after dosing. Figure 8A Anti-KLH IgG titers in healthy volunteers at multiple time points and at different VIB4920 doses are presented. Figure 8B Anti-KLH IgM titers in healthy volunteers at multiple time points and at different doses of VIB4920 are provided. IgG and IgM titers were measured by ELISA. Figure 8C This is a dose-response model for inhibition of anti-KLH IgG at day 43.

[0025] Figures 9A-9C : VIB4920 inhibits B cell proliferation and plasma cell responses in reducing TDAR in healthy human subjects. Figure 9AProvided are the detected frequencies of circulating proliferating B cells (Ki67+CD19+) quantified by flow cytometry at various time points in volunteers who received placebo or high-dose VIB4920 in the TDAR test study. Figure 9B Provided are the detected frequencies of circulating class-switched memory B cells (Ki67+CD19+IgD-CD27+) quantified by flow cytometry at different time points in volunteers who received placebo or high-dose VIB4920 in the TDAR test study. Figure 9C PC signature scores in whole blood assessed by Taqman PCR are provided. Mean and standard error expression values ​​for placebo and high-dose VIB4920 groups are shown. * = P < 0.05, ** = P < 0.01 versus placebo by Mann-Whitney U test.

[0026] Figure 10 : Phase 1b study design to evaluate VIB4920 in patients with RA. Arrows indicate doses of VIB4920 or placebo.

[0027] Figure 11 : Cohort demographics and clinical characteristics of RA patients enrolled in the VIB4920 phase 1b clinical trial.

[0028] Figure 12 VIB4920 demonstrated an acceptable safety profile in RA patients. The most common TEAEs reported occurred in at least two subjects in the Phase 1b study of RA subjects.

[0029] Figures 13A-13C : VIB4920 demonstrated linear PK and dose-dependent reduction of ADA in a Phase 1b study in patients with RA. Figure 13A The concentration of circulating VIB4920 determined by ELISA at the indicated time points is provided. The dotted line represents the lower limit of sensitivity of the assay. The mean and standard error of the mean are shown. Figure 13B The percentage of subjects with positive ADA titers as determined by ELISA for each dose administered at any time point in the study is presented. Figure 13C ADA titers over time as determined by ELISA in subjects with detectable ADA are provided.

[0030] Figures 14A-14F : VIB4920 reduces disease index scores and autoantibodies in RA patients. Figure 14A Shown are changes from baseline in DAS28-CRP assessed at the indicated time points for the indicated doses of VIB4920 or placebo (means and standard errors are indicated). Figure 14BShown are changes from baseline in CDAI assessed at the indicated time points for the indicated doses of VIB4920 or placebo (means and standard errors are indicated). Figure 14C Shown are changes from baseline in patient global assessments assessed at the indicated time points for the indicated doses of VIB4920 or placebo (means and standard errors are indicated). Figure 14D Shown are changes from baseline in physician global assessment assessed at the indicated time points for the indicated doses of VIB4920 or placebo (means and standard errors are indicated). Figure 14E Shown are changes from baseline in Vectra DA scores assessed at the indicated time points for the indicated doses of VIB4920 or placebo (means and standard errors are indicated). Figure 14F Shown are measurements of the percent reduction in RF antibody titers measured by ELISA at the indicated time points for each indicated dose of VIB4920 or placebo.

[0031] Figures 15A-15B : VIB4920 dose-dependently reduces DAS28-CRP scores and RF autoantibodies in RA patients. Figure 15A Shown are the differences in DAS28-CRP scores between placebo and the indicated doses of VIB4920 at Day 85. A linear dose response was shown; this was identified as the best-fitting model for assessing the relationship between VIB4920 dose and reduction in disease activity. Figure 15B Shown are the percent reductions in RF autoantibodies relative to placebo at the indicated doses of VIB4920 on Day 85. The Emax model is shown; it was determined to be the best fit for assessing the relationship between VIB4920 dose and RF titer.

[0032] Figure 16 : VIB4920 improved the DAS28 category of treated RA patients. The DAS28 category at Day 85 is shown. 50% and 75% of RA patients treated in the 1000 mg and 1500 mg groups, respectively, had low disease activity or remission at Day 85.

[0033] Figures 17A-17C : Effects of VIB4920 on tender / swollen joint counts and CRP in a phase 1b study in RA subjects. Figure 17A Shown are changes from baseline in tender joint counts for RA subjects at the indicated doses and time points. Figure 17B Shown are the changes from baseline in swollen joint counts for RA subjects at the indicated doses and time points. Figure 17CShown are the changes in the ratio of CRP levels to baseline in RA subjects at the indicated doses and time points. The mean and standard error for each are shown.

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

[0035] Figures 19A-19B : Amino acid sequence of clone 342 CD40L-specific Tn3 molecule.

[0036] Figure 20 : Amino acid sequence of the bivalent clone 342 CD40L-specific Tn3 molecule.

[0037] Figure 21A and 21B : Amino acid sequence of clone 309 CD40L-specific Tn3 molecule.

[0038] Figures 22A-22F : VIB4920 reduced disease index scores and autoantibodies in RA patients during a 12-week VIB4920 dosing period and a 12-week observation period after the last VIB4920 dose. Figure 22A Shown are changes from baseline in DAS28-CRP assessed at the indicated time points for the indicated doses of VIB4920 or placebo (means and standard errors are indicated). Figure 22B Shown are changes from baseline in CDAI assessed at the indicated time points for the indicated doses of VIB4920 or placebo (means and standard errors are indicated). Figure 22C Shown are changes from baseline in patient global assessments assessed at the indicated time points for the indicated doses of VIB4920 or placebo (means and standard errors are indicated). Figure 22D Shown are changes from baseline in physician global assessment assessed at the indicated time points for the indicated doses of VIB4920 or placebo (means and standard errors are indicated). Figure 22E Shown are changes from baseline in Vectra DA scores assessed at the indicated time points for the indicated doses of VIB4920 or placebo (means and standard errors are indicated). Figure 22F Shown are measurements of the percent reduction in RF antibody titers measured by ELISA at the indicated time points for each indicated dose of VIB4920 or placebo.

[0039] Figures 23A-23C VIB4920 affects tender / swollen joint counts and CRP in patients with RA. In a Phase 1b clinical trial in patients with RA, the effects of VIB4920 were detectable both during the dosing phase and during the 12-week post-dose observation period. Figure 23AShown are changes from baseline in tender joint counts for RA subjects at the indicated doses and time points. Figure 23B Shown are the changes from baseline in swollen joint counts for RA subjects at the indicated doses and time points. Figure 23C Shown are the changes in the ratio of CRP levels to baseline in RA subjects at the indicated doses and time points. The mean and standard error for each are shown. DETAILED DESCRIPTION

[0040] Described herein are VIB4920 and its use in methods for inhibiting a B cell-mediated immune response, in methods for treating an autoimmune disease or disorder, in methods for reducing inflammation, in methods for reducing autoantibodies in a patient, in methods for reducing a measure of RA disease activity in a patient, in methods for reducing RF autoantibodies in a patient, in methods for reducing a plasma cell gene signature score in a patient, and in methods for inducing immune tolerance to an alternative therapy in a patient.

[0041] 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, adrenal autoimmune disease, 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), corneal and other tissue transplants, and chronic Inflammation, sepsis, rheumatoid arthritis, peritonitis, Crohn's disease, reperfusion injury, septicemia, endotoxic shock, cystic fibrosis, endocarditis, psoriasis, arthritis (e.g., psoriatic arthritis), anaphylactic shock, organ ischemia, reperfusion injury, spinal cord injury and allograft rejection, autoimmune thrombocytopenia, Behçet's disease, bullous pemphigoid, cardiomyopathy, celiac disease-dermatitis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, allergic granulomatous vasculitis, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, discoid lupus, essential mixed 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, lupus erythematosus, Meniere's disease, mixed connective tissue disease, IgG4-mediated diseases multiple sclerosis, type 1 or immune-mediated diabetes mellitus, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary achromoproliferative disorder Hyperimmunoglobulinemia, primary biliary cirrhosis, psoriatic arthritis, Raynaud's phenomenon, Reiter's syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, stiff-person syndrome, systemic lupus erythematosus, Takayasu arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, ANCA-associated vasculitis, other vasculitis (e.g., dermatitis herpetiformis vasculitis), vitiligo, solid organ transplant rejection, graft-versus-host disease, colony-reactive antibody desensitization in renal transplant recipients, islet cell transplantation and allogeneic hematopoietic stem cell transplantation, focal segmental glomerulosclerosis (FSGS), glomerulonephritis.

[0042] More specifically, VIB4920 can be used to treat RA, systemic lupus erythematosus (SLE), myositis, antiphospholipid syndrome, autoimmune hepatitis, focal segmental glomerulosclerosis (FSGS), lupus nephritis, inflammatory myopathy, idiopathic thrombocytopenic purpura (ITP), systemic sclerosis, vasculitis, cutaneous lupus, autoimmune hemolytic anemia, myasthenia gravis, IgG4 related diseases or Sjögren's syndrome. In addition, VIB4920 can be used to treat graft-versus-host disease and / or alleviate or prevent the rejection of organ or tissue transplantation.

[0043] The treatment of autoimmune disease or disorder can take the form of suppressing B cell or T cell-mediated immune response, which may be the minimizing of class switching antibodies, the minimizing of circulating B cell subsets, the minimizing of plasma activity or the minimizing of plasma cells and plasma cell gene labels. The treatment of autoimmune disease or disorder can be the reduction of inflammatory markers. Inflammatory markers may be one or more of autoantibody levels, plasma cells (PC) or PC gene labels (characterized by the label of the expression of genes IGHA1, IGJ, IGKC, IGKV4-1 and TNFRSF17), circulating B cell subsets and class switching antibodies. The treatment of autoimmune disease or disorder can be the mitigation of clinical signs and symptoms, such as those measured by patient or physician's overall assessment and the mitigation of symptoms. Clinical signs and symptoms may include one or more of the signs (such as proteinuria or renal function loss) of arthritis, pain, fatigue, fever, discomfort, rash, weakness or organ dysfunction.

[0044] If the method is to reduce the one of the autoantibodies of the patient being treated for an autoimmune disorder, these autoantibodies can be, for example, the antinuclear antibodies of the patient being treated for SLE, Sjögren's syndrome, inflammatory myopathy or systemic sclerosis. Antinuclear antibodies can be one or more of anti-SSA / Ro or anti-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 to reduce the one of the autoantibodies of the patient being treated for an autoimmune disorder, these autoantibodies can be, for example, the liver and kidney microsomal type 1 antibodies of the patient being treated for autoimmune hepatitis. If the method is to reduce the one of the autoantibodies of the patient being treated for an autoimmune disorder, these autoantibodies can be, for example, anti-nicotinic acetylcholine receptor or anti-muscle specific kinase antibodies of the patient being treated for myasthenia gravis. If the method is to reduce the one of the antibodies of the patient being treated for a transplant, these antibodies can be alloantibodies.

[0045] Reducing autoantibodies in a patient being treated for an autoimmune disorder can be reducing the percentage of autoantibodies to a level that is at least 20% lower than before administration of VIB4920. The percentage of autoantibodies can be reduced to a level that is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% relative to the level of autoantibodies before treatment with VIB4920. Reduction in autoantibodies can be achieved within one to three months of starting VIB4920 administration.

[0046] If the autoimmune disease or disorder is RA, the rheumatoid arthritis treatment can be a reduction in one or more of RF autoantibodies, anti-citrullinated peptide antibodies, Vectra DA biomarker score (the Vectra DA biomarker score is a composite score of the 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), plasma cell (PC) signature, serum reactive protein C (CRP), DAS28-CRP, or clinical disease activity index (CDAI), or can be a reduction in tender joint count, joint tenderness intensity, swollen joint count, or joint swelling intensity. If the autoimmune disease or disorder is RA, the treatment can be achieving ACR20, ACR50, or ACR70.

[0047] Treatment of an autoimmune disease or disorder can be characterized by a reduction in clinical symptoms of the disease or disorder, or a reduction in inflammation, or a reduction in a biomarker for the disease or disorder by at least 20% relative to levels prior to treatment with VIB4920. A reduction in any of these symptoms, inflammation, or biomarkers can be a reduction in symptoms, inflammation, or biomarkers by at least 50% relative to levels prior to initiation of treatment with VIB4920. Such a reduction can result in the autoimmune disease or disorder being characterized as in remission.

[0048] Additionally, if the autoimmune disease or disorder is rheumatoid arthritis, treatment of the autoimmune disease or disorder can reduce the patient's RF autoantibodies to a level of at least 20%, at least 30%, at least 40%, at least 45%, at least 50%, at least 60%, at least 75%, or at least 80% relative to the RF autoantibody level before VIB4920 treatment. If the autoimmune disease or disorder is rheumatoid arthritis, treatment of the autoimmune disease or disorder can be a reduction in DAS28-CRP, and the reduction in DAS28-CRP can be an adjusted mean difference of at least -1.2, or at least -1.5, or at least -2.0, or at least -2.2. Additionally, if the autoimmune disease or disorder is rheumatoid arthritis, treatment of the autoimmune disease or disorder can be a reduction in the Vectra DA biomarker score, and the reduction can be an adjusted mean difference of at least -10.3, or at least -10.5, or at least -10.8.

[0049] If VIB4920 is used to reduce the method for inflammation, then the inflammation may be caused by inflammatory disease or obstacle, or may be due to or expected to be damage, such as due to organ or tissue transplantation.If VIB4920 is used to reduce the method for inflammation in inflammatory disease or obstacle, then the inflammatory disease or obstacle can 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.If VIB4920 is used to reduce the method for inflammation, then the inflammation may be due to or expected to be damage, such as due to organ or tissue transplantation.

[0050] If VIB4920 is used in a method of inducing immune tolerance to an alternative therapy in a patient, VIB4920 can induce immune tolerance by reducing the production of neutralizing antibodies against the alternative therapy in the patient. If the patient has not received an alternative therapy or has not yet produced neutralizing antibodies against the alternative therapy, inducing immune tolerance can, in the first case, prevent the patient from producing neutralizing antibodies against the alternative therapy. However, if the patient produces neutralizing antibodies against the alternative therapy, VIB4920 can induce immune tolerance by reducing the level of neutralizing antibodies against the alternative therapy produced by the patient. The level of neutralizing antibodies produced by the patient against the alternative 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 undetectable levels. The percentage reduction in the patient's production of neutralizing antibodies to an alternative therapy can be a comparison of, or can be determined by comparing, a first level of neutralizing antibodies produced in response to an alternative therapy before administration of the first dose of VIB4920 and a second level of neutralizing antibodies produced in response to the alternative therapy after administration of the first, second, third, fourth, or fifth dose of VIB4920. Alternatively, the percentage reduction in the patient's production of neutralizing antibodies to an alternative therapy can be a comparison of, or can be determined by comparing, a peak level of neutralizing antibodies produced in response to an alternative therapy before administration of the first dose of VIB4920 and a peak level of neutralizing antibodies produced in response to an alternative therapy after administration of the first, second, third, fourth, or fifth dose of VIB4920.

[0051] Alternatively or additionally, the patient's immune tolerance induction for alternative therapy can be a reduction in the T cell response to alternative therapy. If the patient has not received alternative therapy, or has received alternative therapy but has not yet produced a T cell immune response to alternative therapy, VIB4920 can reduce the patient's T cell response by preventing the formation of the initial T cell response for alternative therapy. However, if the patient already has a T cell response for alternative therapy, VIB4920 can induce immune tolerance by reducing the existing T cell response for alternative therapy. The T cell response for alternative 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 reach an undetectable level. The percentage reduction of the patient's T cell response to an alternative therapy can be a comparison or can be determined by comparing the first level of T cell response to an alternative therapy before administering the first dose of VIB4920 and the second level of T cell response to an alternative therapy after administering the first or second or third or fourth or fifth dose of VIB4920. Alternatively, the percentage reduction of the patient's T cell response to an alternative therapy can be a comparison or can be determined by comparing the peak T cell response level for an alternative therapy before administering the first dose of VIB4920 and the peak T cell response level for an alternative therapy after administering the first or second or third or fourth or fifth dose of VIB4920. The reduction in T cell response can be characterized as a reduction in the proliferation and / or stimulation of CD4+ T cells stimulated by the alternative therapy. The reduction in T cell response can be characterized as a reduction in the CD4-dependent CD8+ T cell response for an alternative therapy.

[0052] The alternative therapy for inducing immune tolerance can be a peptide or protein alternative therapy. If alternative therapy is peptide therapy or protein therapy, it can be factor VIII or factor IX therapy, and can be applied to treat the patient with hemophilia. If alternative therapy is peptide therapy or protein therapy, it can be enzyme replacement therapy (ERT). If alternative therapy is ERT, the alternative therapy can be agalsidase (agalsidase) α or agalsidase β, which can replace α-galactosidase A, and can treat the patient with Fabry disease. If alternative therapy is ERT, the alternative therapy can be laronidase (iaronidase), which can replace α-L-iduronidase, and the alternative therapy can treat the patient with mucopolysaccharidosis (MPS) type 1 (also referred to as Hurler syndrome, Hurler's Schein syndrome or Schein syndrome, depending on its severity). If alternative therapy is ERT, the alternative therapy can be alglucosidase (alglucosidase), which can replace α-glucosidase, and the alternative therapy can treat the patient with Pompe disease. If the alternative therapy is ERT, the alternative therapy can be idursulfase, which can replace iduronate-2-sulfatase, and the alternative therapy can treat patients with type II MPS. If the alternative therapy is ERT, the alternative therapy can be imiglucerase or velaglucerase α or talicidinase α, which can replace β-glucocerebrosidase, and the alternative therapy can treat patients with Gaucher disease. If the alternative therapy is ERT, the alternative therapy can be Naglazyme arylsulfatase B, which can replace N-acetylgalactosamine-4-sulfatase, and the alternative therapy can treat patients with MPS VI. If the peptide or protein alternative therapy is a peptide or protein, immune tolerance induction can reduce the generation of neutralizing antibodies against the peptide or protein in the patient and / or can reduce the T cell response against the peptide or protein.

[0053] In addition, the alternative therapy for inducing immune tolerance can be a viral vector comprising a nucleic acid encoding a therapeutic peptide or protein. If the alternative therapy is a viral vector comprising a nucleic acid encoding a therapeutic peptide or protein, the viral vector can be an adenoviral vector, an adeno-associated viral vector, a retroviral vector, a poxvirus, an alphavirus, a herpes simplex virus vector, or any other viral vector capable of delivering a nucleic acid encoding a therapeutic peptide or protein to a patient's cells. The viral vector can be modified, for example, by pseudotyping and / or deleting its wild-type gene and / or including a nucleic acid encoding a therapeutic peptide or protein.

[0054] The therapeutic peptide or protein encoded by the nucleic acid of the viral vector can be a therapeutic peptide or protein Factor VIII or Factor IX, or can be an ERT, such as agalsidase alfa, agalsidase beta, idursulfatase, laronidase, alglucosidase alfa, imiglucerase, velaglucerase alfa, talicidinase alfa, or Naglazyme arylsulfatase B.

[0055] In addition, if the alternative therapy is a viral vector comprising a nucleic acid encoding a therapeutic peptide or protein, VIB4920 can induce immune tolerance by reducing the immune response to the viral vector, or by reducing the immune response to the therapeutic peptide or protein encoded by the viral vector, or both. VIB4920 can induce immune tolerance to the viral vector by reducing neutralizing antibodies and / or T cell responses to the viral vector (the vector itself or cells infected by the viral vector). Additionally or alternatively, VIB4920 can induce immune tolerance to alternative therapies comprising viral vectors by reducing neutralizing antibodies or T cell responses to therapeutic peptides or proteins encoded by the nucleic acid of the viral vector.

[0056] VIB4920 for various methods may include Figure 18 The amino acid sequence shown in FIG. VIB4920 may have Figure 18 The amino acid sequence shown, or may have relative Figure 18 One or more amino acid residues in the amino acid sequence shown are changed. Figure 18 If the amino acid sequence of the VIB4920 is altered from that shown, the changes may be to one of the linkers. VIB4920 contains a Gly15 linker that separates the two CD40L-specific monomers and a Gly10 linker that separates the CD40L-specific monomer from the HSA sequence. Two or one of these linkers may be altered and may be replaced by (G m X) n wherein X is serine (S), alanine (A), glycine (G), Leu (L), isoleucine (I), or valine (V); m and n are integer values; m is 1, 2, 3, or 4; and n is 1, 2, 3, 4, 5, 6, or 7. For example, one or both linkers can be altered to have an amino acid sequence comprising one of GGGGSGGGGS, GGGGSGGGGSGGGGS, GGGGGGGGGGG, or GGGGGGGGGGGGGGGG. If VIB4920 has an amino acid sequence relative to Figure 18The amino acid sequence of the HSA fused to the two CD40L-specific monomers may be altered relative to the HSA fused to the two CD40L-specific Tn3 monomers except for at least one amino acid substitution relative to the position numbered in the full-length mature HSA at a position selected from the group consisting of: 407, 415, 463, 500, 506, 508, 509, 511, 512, 515, 516, 521, 523, 524, 526, 535, 550, 557, 573, 574, and 580; wherein the at least one amino acid substitution does not comprise a substitution of lysine (K) to glutamic acid (E) at position 573. If VIB4920 has a relative Figure 18 These changes may be directed to the amino acid sequence of one or two of the CD40L-specific Tn3 monomers, as long as it does not adversely affect the in vivo efficacy of VIB4920. For example, the amino acid sequence changes may result in one or two CD40L-specific Tn3 monomers having Figure 19A The amino acid sequence shown.

[0057] The dose of VIB4920 administered in the methods can be a dose between about 500 mg and about 3000 mg. The dose can be between about 750 mg and about 3000 mg, or between about 1000 mg and about 3000 mg, or between about 1500 mg and about 3000 mg, or between about 500 mg and about 2000 mg, or between about 750 mg and about 2000 mg, or between about 1000 mg and about 2000 mg, or between about 1000 mg and about 2500 mg, or between about 1000 mg and about 1500 mg. The dose can be 500 mg, 750 mg, 900 mg, 1000 mg, 1250 mg, 1500 mg, 1750 mg, 2000 mg, 2250 mg, 2500 mg, or 3000 mg.

[0058] A dose of VIB4920 may be administered about every other week, or twice a month. A dose of VIB4920 may also be administered about once a week, or about once a month. A dose of VIB4920 may be administered 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. A dose of VIB4920 may be administered by intravenous or subcutaneous injection.

[0059] If the dose of VIB4920 administered is 1000 mg, 1500 mg, or one of about 1000 mg to about 1500 mg, the dose can be administered every other week, or twice a month. If the dose of VIB4920 is 3000 mg, the dose can be administered once a month. If the dose of VIB4920 is 500 mg or 750 mg, the dose can be administered once every other week, or alternatively, twice a month. Any of these doses can be administered intravenously.

[0060] The dosage and dosing regimen of VIB4920 can be such that administration of VIB4920 achieves any therapeutic effect for the treatment of any autoimmune / inflammatory disease or disorder, e.g., reduction in autoantibodies, reduction in Vectra DA score, reduction in plasma cell signature, reduction in CRP, reduction in DAS28-CRP, reduction in swollen joint count, reduction in tender joint count, reduction in CDAI, improvement in patient global assessment, improvement in physician global assessment, achievement of ACR20, achievement of ACR50, or achievement of ACR70, which can be considered "durable." A "durable" effect of VIB4920 in the treatment of an autoimmune / inflammatory disease or disorder is that the therapeutic effect achieved by VIB4920 is maintained (despite no further administration of VIB4920) 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 a course of VIB4920. A course of treatment with VIB4920 can be administering a dose of 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) of VIB4920 for 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) at an interval of every 7 to 31 days (e.g., 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).

[0061] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. Such equivalents are intended to be encompassed by the following claims.

[0062] 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 was specifically and individually indicated to be incorporated herein by reference.

[0063] Examples

[0064] Example 1 - Isolation and Optimization of CD40L-Specific Tn3 Protein

[0065] Tn3 is a small protein scaffold, approximately 90 amino acids in length, with an immunoglobulin-like fold, including loops structurally similar to the complementarity-determining regions of antibodies, which can be randomized to select for specific binding properties (24).

[0066] Human CD40L-specific Tn3 clones were isolated as described in detail in WO 2013 / 055745 (see also 24, 27, 50). Briefly, selection for human CD40L-specific Tn3s involved five rounds of panning, alternating between selection for recombinant human CD40L protein and selection for a CHO cell line expressing human CD40L. Mouse CD40L-specific Tn3 proteins were selected using only recombinant mouse CD40L protein. Tn3 genes from the selection output were collectively cloned into expression vectors, and CD40L binding of each His-tagged variant was assessed 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 hours. After washing once with PBS / Tween, the interaction between the captured Tn3 variants and CD40L was monitored using SA-HRP (1:1000 dilution). After 20 minutes, the plates were washed twice in PBS / Tween, developed with TMB substrate, and then stopped with 2.5 M H 3 PO 4 . The absorbance was measured at 450 nm. Affinity maturation of the CD40L-specific Tn3 protein was performed by selecting improved candidates from a phage-displayed library in which the CDR-like loops were randomly mutated ( 24 ). This strategy resulted in clone 342 ( FIG. 19 ), an improved variant of human CD40L-specific 309 ( Figure 21A )) and clone M31 (an improved variant of murine CD40L-specific M13). Additional human CD40L-Tn3 clones have also been prepared, such as clones 304, 311, 320, 310, 321, and 322. See, WO 2013 / 055745, which is incorporated herein by reference.

[0067] This panel of human CD40L-specific Tn3 clones was characterized for their ability to biochemically inhibit the binding of CD40L to its receptor (CD40). All seven in the panel inhibited the binding of CD40L to CD40 with IC50 values ​​below 1 μM ( Figure 1AThe inhibition of CD40L-mediated signaling by the two most potent inhibitors in the biochemical CD40L-CD40 inhibition assay was further evaluated in a cell-based reporter gene assay. HEK-293 cells expressing human CD40 and NF-κB-luciferase reporter genes were stimulated with recombinant human CD40L protein. Human CD40L-specific Tn3 proteins 309 and 311 inhibited CD40L-induced NF-κB reporter gene expression in a dose-dependent manner at micromolar concentrations ( Figure 1B ), highlighting the ability of these proteins to functionally inhibit CD40 / CD40L signaling.

[0068] As occurs in the case of bivalent antibodies, simultaneous binding to multiple targets can lead to a significant increase in avidity. To explore the effect of bivalency on the potency of CD40L-specific Tn3 proteins, two copies of the same Tn3 module (309-309; e.g., Figure 21B ) are linked via a flexible Gly4Ser containing spacer to form a tandem bivalent fusion protein. Human primary B cells upregulate the activation marker CD86 in response to stimulation by CD40. Preincubation of human peripheral blood mononuclear cells (PBMCs) with monovalent CD40L-specific Tn3 309 inhibits the upregulation of CD86 on human CD19+ B cells in a dose-dependent manner ( Figure 1C Surprisingly, the potency was increased nearly 1000-fold in this primary cell assay using the bivalent construct compared to monovalent Tn3 ( Figure 1C Furthermore, affinity maturation of clone 309 (by random mutagenesis in the variable CDR-like loop regions) resulted in clone 342, which significantly improved binding affinity to CD40L (309: 190 nM; 342: 1.4 nM) and showed approximately 300-fold greater potency in inhibiting the CD40-CD40L interaction ( 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. Figure 1E .

[0069] Finally, as with other alternative scaffold technologies and due to its small size, naked Tn3 molecules are expected to exhibit very rapid clearance from the circulation upon systemic administration. To improve the pharmacokinetic properties of the protein, CD40-specific Tn3 proteins were fused to serum albumin (28, 29). The bivalent mouse surrogate CD40L-specific Tn3 protein, M13-M13, had a half-life of <30 minutes in mice when delivered systemically. Fusion of mouse serum albumin (MSA) to the M13-M13 Tn3 protein resulted in a 65-fold increase in serum half-life and a 345-fold decrease in clearance (Table 1).

[0070] Table 1: Fusion to serum albumin significantly increases the half-life of the M13-M13 Tn3 molecule

[0071] molecular 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

[0072] 5-7 week old CD-1 mice received a single injection of bivalent CD40L-specific Tn3 molecules with or without MSA (n=12 / group; 10 mg / kg, iv). At various time points between 15 minutes and 72 hours, blood was drawn from n=3 mice / group and circulating levels of Tn3 protein were determined by ELISA.

[0073] Based on these observations, the bivalent human CD40L-specific Tn3 molecule VIB4920 consists of 342 CD40L-specific Tn3 proteins in series, which are fused to human serum albumin (HSA) for optimal potency, thereby improving half-life ( Figure 1F ; Figure 18 ).

[0074] To better understand the molecular nature of the interaction between CD40L and VIB4920, crystallographic studies were performed. CD40L-specific Tn3 (342) and soluble CD40L protein were expressed, purified, and co-crystallized. The molecular structure of trimeric soluble CD40L in complex with Tn3 is shown in Figure 2A The interface with CD40L is composed largely of amino acids from the second modified loop of Tn3, including eight of the ten hydrogen bonds formed between the molecules ( Within the distance, Figure 2B Initial characterization of the molecule indicated that 342Tn3 was able to block the interaction between CD40L and CD40. To visualize the details of this interaction, the structure of the CD40 / CD40L complex was superimposed on the structure of 342 / CD40L ( Figure 1G The overlap shows that 342 and CD40 share a common binding site on CD40L. Therefore, VIB4920 competes with CD40 and prevents its binding to CD40L.

[0075] Example 2 - VIB4920 blocks activation and differentiation of human B cells

[0076] CD40 signaling has been extensively characterized and involves the activation of multiple different pathways and transcription factors, including NF-κB (30), which promotes B cell activation, proliferation, and differentiation (31). Therefore, the ability of VIB4920 to inhibit CD40L-mediated NF-κB activation was investigated using a cell line expressing human CD40 and an NF-κB luciferase reporter gene. NF-κB activation was induced by stimulating this cell line with recombinant human CD40L or with cells expressing CD40L. VIB4920 was able to effectively block CD40 signaling using this cell line, as demonstrated by dose-dependent inhibition of NF-κB activation (IC50: 0.899 nM; Figure 3A ).

[0077] Resting B cells constitutively express low levels of the co-stimulatory molecule CD86, which is rapidly upregulated upon activation, including activation by CD40 (32). Primary human PBMCs were stimulated with recombinant human CD40L, and CD86 expression on B cells was assessed by flow cytometry 16 hours later. VIB4920 completely blocked CD40L-mediated upregulation of CD86 by primary human B cells ( Figure 3B ).

[0078] Example 3 - VIB4920 does not induce platelet aggregation in vitro

[0079] Anti-CD40L directed mAbs have failed in clinical trials due to safety concerns, largely due to thromboembolic complications associated with cross-linked CD40L on the platelet cell surface. To confirm that VIB4920, which lacks the Fc domain, does not induce platelet aggregation, we evaluated its effects on washed human platelets in vitro. As previously described, anti-CD40L mAb (human IgG1) showed a significant ability to induce platelet aggregation when pre-complexed with sCD40L ( Figure 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 the important role of Fc receptors in this response ( Figure 3F In contrast, VIB4920 showed no tendency to induce platelet aggregation in this assay at several concentrations tested ( Figure 3G). These data suggest that the absence of the Fc region in the Tn3 construct can reduce the risk of platelet aggregation and thromboembolic events observed in the clinic with therapeutic anti-CD40L antibodies. To confirm this result, data from long-term (7-month) studies in non-human primates dosed up to 300 mg / kg of VIB4920 (not shown) did not identify adverse findings in platelet function, e.g., no adverse findings in D-dimer (to monitor blood clots), PFA100 (to assess platelet function), or TAT complex (thrombin antithrombin complex) tests were identified.

[0080] Example 4 - CD40L-specific Tn3 protein modulates immune responses in vivo

[0081] The central role of CD40L in promoting T-dependent immune responses has been well characterized (9, 35). Therefore, a T-dependent immune model was used to evaluate the ability of the Tn3-MSA fusion protein to block humoral immune responses in vivo. Due to insufficient sequence homology between human and murine CD40L, the CD40L-specific mouse surrogate, Tn3 M31, was used in these studies.

[0082] To test whether the Tn3-MSA fusion protein could block the immune response in vivo, mice were vaccinated with sheep red blood cells (SRBC) and then treated daily with anti-CD40 LTn3 protein for 9-13 days after vaccination. On day 14, the immune response in the treated animals was assessed by flow cytometry quantification of spleen and lymph node germinal center B cells. As expected, immunization with SRBC resulted in a significant expansion of germinal center frequency in control-treated mice ( Figure 4A A dose-dependent decrease in germinal center B cell frequency was observed in mice treated with the CD40L-specific Tn3-MSA fusion protein ( Figure 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 near absence of germinal center B cells in the spleen and lymph nodes), comparable to control unimmunized mice. Drug administration did not interfere with other cell subsets, including specific T cell populations, ensuring that the observed effects were not secondary to T cell exhaustion (data not shown). In addition, anti-SRBC IgG levels reflected the level of germinal B cell response, with SRBC-specific Ig titers significantly reduced at higher doses of anti-CD40LTn3 ( Figure 4B Example 5 - VIB4920 was well tolerated by healthy volunteers

[0083] The safety profile of VIB4920 was evaluated in a Phase 1a (Ph1a) study in healthy adults aged 18-49 years. Subjects were enrolled in seven single-dose escalation cohorts, with doses of VIB4920 up to 3000 mg, and randomized to VIB4920 or placebo ( Figure 5A and 5B The primary endpoint, safety and tolerability, was measured as the incidence of treatment-emergent adverse events (TEAEs) and treatment-emergent serious adverse events (TESAEs). Across all dose cohorts, TEAEs were generally clinically insignificant, with the most common events including nasopharyngitis (common cold) and headache (Table 2).

[0084] Table 2: VIB4920 shows a favorable safety profile in humans

[0085]

[0086] The most common TEAEs occurring in at least 2 subjects in the Ph1a study in healthy volunteers

[0087] Importantly, the overall percentage of subjects with one or more study product-related TEAEs was comparable between the total VIB4920 group (40.9%) and placebo (33.3%). In addition, no infusion-related reactions, serious infections, or deaths occurred, and only one TESAE was reported: a tibial fracture in the placebo group. Notably, no clinically significant coagulation or platelet function abnormalities were observed following treatment with VIB4920 in this Ph1a study.

[0088] Example 6 - VIB4920 shows a favorable PK / PD profile in healthy volunteers.

[0089] In addition to evaluating the safety profile of VIB4920, the Ph1a study also evaluated pharmacokinetic (PK) and pharmacodynamic (PD) endpoints. Following a single intravenous dose of 3-3000 mg, the PK profile of VIB4920 was linear and increased in a dose-proportional manner ( Figure 6A The molecule had a mean terminal half-life of 8 days, with the highest dose reaching a half-life of 10.1 + / - 1.87 days.

[0090] CD40L is a transmembrane protein; however, it can be cleaved and shed by activated T cells and platelets. Soluble CD40L (sCD40L) is an 18-kDa trimer that is detected at low levels in healthy donors and is increased in the circulation of patients with autoimmune diseases (36, 37). Measurement of sCD40L levels after administration of VIB4920 represents a potential measure of target engagement because sCD40L bound to VIB4920 can be retained and accumulated in the circulation. As expected, there was a dose-dependent increase in total sCD40L in plasma after administration of VIB4920 ( Figure 6B When the dose was increased from 3 mg to 3000 mg, the time to reach maximum total sCD40L in plasma increased from 11.5 days to 84 days, indicating that target engagement was maintained longer in the highest dose group.

[0091] Example 7 - ADA reduction observed in healthy subjects receiving higher doses of VIB4920

[0092] Biologic drugs are inherently highly specific / selective; however, they are complex molecules capable of eliciting an immune response. Anti-drug antibodies (ADA) are a measure of the immunogenicity of a therapeutic agent. In healthy volunteers, ADA were detected in the vast majority of patients receiving low-dose VIB4920 ( Figure 7 More specifically, 18 of the 20 subjects in the 3-100 mg dose range had detectable ADA, 10 of whom exhibited high ADA titers (greater than a median titer of 480). In contrast, at higher dose levels of VIB4920, the frequency of ADA was significantly reduced ( Figure 7 ), where only one of eight subjects in the 3000 mg dose group developed a detectable anti-drug titer. The reduced ADA frequency observed at high doses of VIB4920 supports the immunomodulatory potential of this molecule. Additionally, low percentages and low titers of ADA may translate into a more effective therapeutic agent with better tolerability.

[0093] Example 8 - VIB4920 inhibits T cell-dependent antibody responses in healthy volunteers

[0094] The ability of VIB4920 to affect the humoral immune response in healthy subjects was further evaluated. This evaluation was performed by measuring the effect of VIB4920 on the T cell-dependent antibody response (TDAR) induced by immunization with keyhole limpet hemocyanin (KLH). Healthy subjects in all treatment groups received two subcutaneous KLH immunizations: (first) 14 days before dosing with VIB4920 or placebo, and (second) 15 days after dosing ( Figure 5BThe production of IgM and IgG antibodies against KLH was monitored until day 113.

[0095] The TDAR followed the expected trend in placebo-treated subjects, i.e., the trend included a sharp increase in anti-KLH IgG titers on day 22 (one week after the second immunization), a peak IgG level observed on day 29, and then a decline in KLH-specific IgG antibodies until the end of the monitoring period ( Figure 8A Furthermore, consistent with previous reports, secondary anti-KLH responses in the placebo-treated group were predominantly IgG, with an overall more modest increase in KLH-specific IgM detected after rechallenge ( Figure 8B )(38-41).

[0096] As expected, anti-KLH titers in healthy volunteers treated with low-dose VIB4920 approached those in the placebo-treated group. In contrast, healthy volunteers treated with higher doses of VIB4920 demonstrated a marked reduction in secondary responses to KLH, such that on day 43, anti-KLH IgG levels were statistically significantly reduced starting with the 300 mg dose (p = 0.035) and increasing with the 1,000 mg (p = 0.002) and 3,000 mg (p < 0.001) doses. Notably, on day 43, IgG to KLH was reduced by 78% and 86% in the 1,000 mg and 3,000 mg cohorts, respectively, compared to placebo. Figure 8C In the highest-dose group, seven of eight subjects had undetectable anti-KLH-IgG titers at day 43, indicating that VIB4920 almost completely suppressed the humoral immune response.

[0097] Example 9 - VIB4920 mediates immunosuppression by inhibiting B cell proliferation and plasma cell responses

[0098] The mechanism by which VIB4920 inhibits the secondary immune response can be better defined by collecting peripheral blood from subjects before and after immunization and characterizing circulating lymphocyte subsets by flow cytometry. In healthy placebo-treated subjects, secondary immunization induced B cell proliferation, as demonstrated by an increase in the frequency of circulating Ki67+CD19+ B cells detected at visit day 22 (i.e., 7 days after rechallenge). Figure 9A ).

[0099] In subjects who received high-dose VIB4920, the baseline frequency of proliferating B cells prior to rechallenge was reduced compared to the placebo-treated group. This is consistent with the proposed molecular mechanism of action. In addition, the B cell proliferative response following immunization was significantly impaired in the cohort that received high-dose VIB4920, as indicated by the lack of an increase in Ki67+ B cells. See the 3000 mg cohort one week after challenge ( Figure 9A Further phenotypic analysis revealed that the greatest effect of VIB4920 on proliferating B cells was found in the IgD-CD27+ isotype-switched memory population ( Figure 9B These data are consistent with the TDAR results showing that VIB4920 has an inhibitory effect on IgG production in response to a secondary challenge.

[0100] Gene expression changes in the peripheral blood of subjects treated with placebo or VIB4920 were also monitored before and after secondary immunization with KLH. Specifically, the plasma cell (PC) gene signature, an accurate and robust signature capable of detecting even subtle changes in circulating PC frequency (42), was used to determine certain changes in gene expression. Consistent with the TDAR results, immunization induced a significant increase in the PC gene signature score in the whole blood of placebo-treated subjects one week after rechallenge, which returned to baseline at two weeks ( Figure 9C In the highest VIB4920 dose cohort (3000 mg), PC gene signature scores in peripheral blood were significantly reduced compared to placebo-treated subjects before re-challenge with KLH ( Figure 9C Importantly, volunteers who received a high dose of VIB4920 did not experience an increase in PC gene signature scores after re-immunization. These data highlight the mechanism of action of VIB4920 and demonstrate its potent ability to suppress B cell and PC responses.

[0101] Example 10 - Multiple-dose administration of VIB4920 is safe and well tolerated in RA patients

[0102] VIB4920 has an established acceptable safety profile and demonstrated proof of mechanism in healthy volunteers. The multiple ascending dose, proof-of-concept Ph1b clinical study was conducted in adult patients with moderately to severely active RA. RA patients 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 (iv) infusion every other week for 12 weeks ( Figure 10). Patients were then observed for an additional 12 weeks after treatment. Key endpoints measured at week 12 included safety, tolerability, PK parameters, ADA and changes in disease activity (DAS28-CRP), as well as additional biomarkers such as RF autoantibodies, serum C-reactive protein (CRP), and Vectra-DA score. 53 patients completed 12 weeks of treatment; 2 patients (1 in the VIB4920 75 mg group and 1 in the VIB4920 1500 mg group) discontinued treatment due to adverse events, 1 patient in the placebo group withdrew informed consent, and 1 patient in the VIB4920 75 mg group was lost to follow-up. The main demographic and clinical characteristics of the study population at baseline were Figure 11 Given in.

[0103] Overall, VIB4920 was generally safe and well tolerated, with a balanced distribution of TEAEs observed between placebo and the four active dose groups. The most common TEAEs reported were diarrhea, hyperhidrosis, upper respiratory tract infection, and urinary tract infection, each occurring in three patients (7.1%). Figure 12 No thrombotic adverse events or clinically significant coagulation abnormalities were observed. One adverse event (preferred term: "encephalitis") was reported as severe and life-threatening in the 1500 mg dose group after six doses of study drug. No causative infectious agent was identified; similar symptoms recurred several months after discontinuation of VIB4920, and the patient was subsequently diagnosed with brain metastatic melanoma.

[0104] Example 11 - VIB4920 shows a linear PK profile and dose-dependent reduction in ADA in RA patients

[0105] ADA were observed in RA patients receiving low-dose VIB4920, similar to healthy Ph1a study volunteers who received low-dose VIB4920. Three of eight RA patients (37.5%) receiving 75 mg VIB4920 and three of ten RA patients (30%) receiving 500 mg VIB4920 developed ADA ( Figure 13B In the 75 mg VIB4920 dose group, 2 of 8 subjects developed detectable ADA during the treatment period; all 3 subjects in the 500 mg VIB4920 treatment group developed detectable ADA after treatment ( Figure 13C During the treatment period, no ADA was detected in the 1000 mg dose group; one subject had detectable ADA after the treatment period. No ADA was detected in the 1500 mg dose group ( Figure 13B ), indicating that VIB4920 effectively inhibited ADA responses at higher doses.

[0106] Example 12 - VIB4920 reduces disease activity in RA patients

[0107] The DAS-28 / CRP score was measured to determine whether VIB4920 reduced disease activity in RA patients in the Phase 1b clinical trial. The DAS-28 / CRP score is a composite clinical disease activity score for RA that takes into account: the number of swollen joints, the number of tender joints, CRP levels, and the patient's overall health assessment. At the higher dose, VIB4920 significantly reduced disease activity quantified by the DAS28-CRP score in RA patients ( Figure 14A and Figure 22A At week 12 after the start of treatment, the adjusted mean changes from baseline in DAS28-CRP(SE) were -2.3 (0.3) for the VIB4920 1500 mg group, -2.2 (0.3) for the VIB4920 1000 mg group, -1.2 (0.3) for the VIB4920 500 mg group, 0.1 (0.4) for the VIB4920 75 mg group, and -1.0 (0.3) for the placebo group ( Figure 14A Surprisingly, the reduction in DAS28-CRP scores observed in RA patients persisted for at least another 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 scores significantly reduced by day 15 (after only a single dose of drug).

[0108] In addition, the reduction in disease activity at the two highest doses of VIB4920 was clinically meaningful and statistically significant compared to placebo; the adjusted mean difference (SE) at week 12 was -1.4 (0.4) for the VIB4920 1500 mg group and -1.2 (0.4) for the VIB4920 1000 mg group, with p-values ​​of 0.002 and 0.006, respectively. Using a linear dose-response model, a statistically significant dose response was shown for DAS28CRP (p<0.001). The significant results were primarily from the 1000 mg and 1500 mg treatment groups; the 500 mg and 75 mg groups had little or no benefit compared to placebo ( Figure 15A In terms of individual clinical responses, 75% of patients in the 1500 mg group and 50% of patients in the 1000 mg dose group had a DAS28-CRP score of 3.2 or less at Week 12, indicating that they were in low disease activity or clinical remission, the primary endpoint. Figure 16 .

[0109] Example 13 - VIB4920 reduces immune and inflammatory biomarkers in RA patients

[0110] The Vectra DA blood test was used to determine the effects of VIB4920 on immune and inflammatory biomarkers. The Vectra DA test is a commercially available and validated test that measures 12 biomarkers of disease activity (adhesion molecules, growth factors, cytokines, matrix metalloproteinases, skeletal muscle proteins, hormones, and acute phase proteins) and combines them into a single score to assess the key mechanisms and pathways driving RA disease activity. During a 12-week period ( Figure 14E ) and during the 12-week observation period without further VIB4920 administration ( Figure 22E ), both 1500 mg and 1000 mg of VIB4920 significantly reduced the Vectra DA multi-biomarker score. The adjusted mean difference for the 1500 mg dose of VIB4920 relative to placebo (week 12) was -14.4 (-21.5, -7.2), p = 0.001, and the adjusted mean difference for the 1000 mg dose of VIB4920 relative to placebo (week 12) was -10.3 (-17.4, -3.3), p = 0.018 ( Figure 14E ).

[0111] Efficacy results were highly consistent across other endpoints evaluated in the trial (including Clinical Disease Activity Index (CDAI), tender and swollen joint counts, patient and physician global assessments, and serum CRP levels), supporting 1000 and 1500 mg as clinically effective doses in this study ( Figures 14B-14D and Figures 17A-17C ; See also Figures 22B-22D and Figures 23A-23C ).

[0112] Example 14 - VIB4920 significantly reduces rheumatoid factor autoantibodies in RA subjects

[0113] Rheumatoid factor autoantibodies (RF) are a family of autoantibodies produced against the Fc portion of IgG. They are elevated in RA and are associated with a poor prognosis. Given the mechanism of action of VIB4920, its effect on autoantibody titers in RA subjects was evaluated. Notably, VIB4920 significantly reduced RF titers at the 500, 1000, and 1500 mg dose levels during the 12-week, every-other-week treatment period ( Figure 14F Furthermore, surprisingly, the reduced RF titers were maintained at both the 1000 and 1500 mg dose levels throughout the 12-week observation period following the last dose of VIB4920 ( Figure 22F). As early as day 29, a reduction in RF titers relative to baseline was evident in response to VIB4920; by day 85, high-dose VIB4920 reduced RF titers by approximately 50%. Using the Emax model, VIB4920 showed a statistically significant dose response (p<0.001) in reducing RF titers relative to baseline ( Figure 15B ).

[0114] Example 15 - Method

[0115] NF-kB reporter gene assay.

[0116] HEK293 cells (Panomics) expressing the NF-κB luciferase reporter gene were engineered to stably express full-length human CD40R. The cells were cultured at 5×10 4 Cells were seeded at a density of 10 cells / well in 96-well poly-D-lysine-coated plates (BD Biosciences) and stimulated for 16-24 hours with megaCD40L recombinant protein (1.5 μg / ml, Enzo Biotech) or D1.1 Jurkat subclone (ATCC) cells overexpressing CD40L 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 microplate reader (Molecular Devices).

[0117] CD86 upregulation assay

[0118] Human blood was collected from healthy donors after obtaining informed consent approved by MedImmune's Institutional Review Board. Peripheral blood mononuclear cells were isolated from CPT tubes (BD Biosciences) after centrifugation. PBMCs (2.5–5.0 × 10 β-cells) were stimulated with recombinant megaCD40L (100 ng / ml, Enzo Biosciences) in 96-well round-bottom plates in the presence of the indicated CD40L-specific Tn3 or mAb (clone 5c8). 5 Cells / well) for 16-18 hours. Flow cytometry was used to assess CD86 expression on CD19+ B cells. The following antibodies were used: CD86 (clone 2331, BD Pharmingen) and CD19 (clone HIB19, BD Pharmingen).

[0119] Human B cell assay

[0120] PBMCs were isolated. Total B cells were negatively selected using MACS cell separation technology (Miltenyi Biotec), which typically yielded a purity greater than 95%. Purified peripheral blood B cells were plated at 0.5 to 1.0 × 10 cells per well. 5 The density of B cells was cultured in a final volume of 150 μl complete medium in a 96-well round-bottom plate. The culture medium for B cell experiments was RPMI 1640 (Invitrogen), supplemented with 10% FCS, penicillin-streptomycin (100 units / ml penicillin, 100 μg / ml streptomycin), 2-mercaptoethanol (55 μM), L-glutamine (2 mM) and HEPES (5 mM). At the beginning of the culture, in the presence or absence of anti-IgM F (ab') 2 (5.0 μg / ml, Jackson ImmunoResearch Laboratories), B cells were stimulated with a combination of IL-21 (33 ng / ml, PeproTech Inc.) and megaCD40L (1.5 nM, Enzo Biotech). B cell expansion was quantified by measuring ATP on day 3 or 4 of culture using the Cell Titer-Glo luminescence assay (Promega) according to the manufacturer's instructions. PC differentiation was quantified by flow cytometry on day 7. Cells were obtained for a fixed amount of time, and PCs were defined as CD19 + IgD - CD38 hi cell.

[0121] Mouse SRBC immunization model

[0122] On day 0, balb / c mice (Jackson Laboratories) were immunized with 0.2 ml of SRBC (Colorado Serum Company) injected intraperitoneally directly from the bottle. From day 9 to day 13, control (30 mg / kg) or CD40L-specific Tn3 (up to 30 mg / kg as indicated) were administered daily (intravenously). The frequency of germinal center B cells in the spleen was quantified by flow cytometry on day 14. GC B cells were defined as CD19 + B220 + Fas + PNA + B cells.

[0123] Platelet aggregation assay

[0124] Human blood from healthy donors was collected into ACD solution B tubes containing citric acid, dextrose, and sodium. After centrifugation, two-thirds of the platelet-rich plasma was transferred to a polypropylene tube and incubated with apyrase (2 U / ml) for 10 minutes to prevent platelet activation during treatment. Platelets were pelleted and resuspended in modified Tyrode's 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).

[0125] Immune complexes (ICs) were generated by mixing mAb (h5c8 or negative control antibody) or anti-CD40L Tn3 with hCD40L (293 cell-derived) for 5 minutes at room temperature. In some experiments, platelets were preincubated with anti-CD32a antibody (IV.3) for 5 minutes before adding the IC. Platelet aggregation was measured with stirring at 37°C in a four-channel optical aggregometer (Model 700, Chrono-Log, Havertown, PA) according to the manufacturer's instructions. After mixing washed platelets with the agonist, light transmission was monitored for 12-20 minutes.

[0126] Ph1a subjects and study design

[0127] A phase I, randomized, blinded, placebo-controlled study (NCT 02151110) was conducted in healthy adults aged 18-49 years (including women of non-fertile potential). Subjects were randomly divided into 7 dose cohorts (3, 10, 30, 100, 300, 1000 or 3000 mg) and administered sequentially according to the study protocol and the recommendations of the dose escalation committee (DEC), which reviewed the safety and tolerability data from the current dose cohort and the cumulative data from the previous dose cohort. TDAR was induced in subjects by subcutaneous administration of two separate 1 mg KLH immunizations. The first KLH immunization was administered during the screening period, 14 days before administration of VIB4920 or placebo; the second KLH immunization was administered on the 15th day after administration of VIB4920 or placebo. When all subjects in the 5th cohort (300 mg), the 6th cohort (1000 mg) and the 7th cohort (3000 mg) were completed on day 43, three interim analyses were performed according to the protocol.

[0128] PK assay of VIB4920

[0129] The VIB4920 in human K2EDTA plasma was measured using a sandwich ELSA method of validation, wherein a washing step was performed with 1x PBS / 0.1% Tween 20 (PBST) after each incubation to remove unbound components. In brief, Nunc microtiter plates were coated overnight with 1 μg / mL anti-VIB4920 mouse monoclonal antibodies (MedImmune Co., Ltd. (MedImmune)) at 2°C-8°C. Before being added to the plate, standards, quality controls (QC), and samples containing VIB4920 were diluted to a method minimum required dilution (MRD) of 1:50 in 0.5% bovine serum albumin (BSA) / PBST. After incubation for 2 hours, 1 μg / mL biotin-labeled anti-VIB4920 rat antibodies (MedImmune Co., Ltd.) were added to the plate and incubated for 1 hour. Streptavidin-linked horseradish peroxidase (HRP, GE Healthcare) and SureBlue TM Continuous incubation with tetramethylbenzidine (TMB) peroxidase substrate (KPL, Inc.) visualized the bound complex. Color development was stopped with 0.2 M sulfuric acid, and analysis was performed on a microplate reader at 450 nm. The quantitation range was 0.05 to 1.60 μg / mL; samples outside the quantitation range were diluted with pooled K2EDTA plasma to bring the concentration within the measurable range of the method.

[0130] Quantification of sCD40L

[0131] Plasma samples were collected during screening and on days 1, 2, 3, 5, 8, 15, 22, 29, 43, 57, 85, and 113 to measure sCD40L concentrations. Total soluble CD40L (free sCD40L and sCD40L bound to VIB4920) was measured in human K2EDTA plasma using a human sCD40L Platinum ELISA kit (eBioscience), which was modified to meet procedural requirements and qualified to ensure accuracy and precision. Briefly, standards, QCs, and sCD40L-containing samples were diluted to the method MRD of 1:50 in assay diluent containing 0.5% BSA / PBST and VIB4920 to ensure comparable and consistent results. Each incubation was followed by washing with PBST to remove unbound components. The diluted samples were added to plates pre-coated with anti-sCD40L antibody and incubated for 1.5 hours. HRP-conjugated anti-human sCD40L was then added to bind to the sCD40L captured by the coated antibody. The bound complex was visualized by sequential addition of TMB peroxidase substrate and stop solution (phosphoric acid) and analyzed on a microplate reader at 450 and 540 nm. The quantitation range was 6.25 to 400.00 ng / mL; samples outside the quantitation range were diluted with pooled K2EDTA plasma to bring the concentration within the measurable range of the method.

[0132] Measurement of ADA

[0133] The presence of ADA against VIB4920 was determined in human K2EDTA plasma using a validated sandwich ELISA method with PBST washes after each incubation to remove unbound components. Briefly, QCs and samples were diluted to the method MRD of 1:60 in assay diluent containing 0.5% BSA / PBST and added to washed Pierce TM Protein G-coated plates (ThermoFisher) were incubated for 2 hours. ADA against VIB4920 was specifically detected by incubating 1 μg / mL biotinylated VIB4920 prepared in assay diluent overnight. TMContinuous incubation of TMB peroxidase substrate (KPL company) visualizes the binding complex. The color development was stopped with 0.2M sulfuric acid and then analyzed at 450nm on a microplate reader. Each sample was processed in three layers, in which the sample response was first compared with a statistically determined cutoff OD value. When the sample was equal to or higher than the value, the sample was considered to be potentially positive, and when the sample was lower than the value, the sample was determined to be ADA negative. In the presence of excel VIB4920, a second competitive assessment was performed on the potential positive samples; samples with an inhibition percentage equal to or higher than the statistically determined confirmation cutoff point were defined as confirmed positive and titer assessment was performed. Samples below the confirmation cutoff point were considered to be negative for ADA against VIB4920. The titrated samples were serially diluted in pooled human K2EDTA plasma to below the screening cutoff value, and the titer results were reported as the reciprocal of the highest dilution at which the sample was measured to be positive before measuring negative.

[0134] Assessment of anti-KLH antibodies

[0135] Anti-keyhole limpet hemocyanin (KLH) IgG antibodies in human serum were measured using a validated sandwich ELISA method, with PBST washing after each incubation to remove unbound components; 100 μL volume / well was used for all steps. Briefly, Nunc microtiter plates were coated overnight at 2°C-8°C with 3 μg / mL KLH (Immucothel, biosyn Arzneimittel GmbH) prepared in 1x PBS (pH 7.2). Standards and QCs (consisting of a mixture of nine monoclonal anti-KLH IgG antibodies (AstraZeneca) of varying isotypes and affinities) and samples containing anti-KLH antibodies were diluted to the method MRD of 1:250 in 0.5% BSA / PBST and then added to the plates. After 2 hours of incubation, HRP-conjugated mouse anti-human IgG (Invitrogen) was added to the plate and incubated for 1 hour to specifically detect anti-KLH IgG antibodies. The bound complex was visualized by sequential addition of TMB peroxidase substrate and stop solution (0.2 M sulfuric acid) and then analyzed on a microplate reader at 450 rpm. The quantitative range was 163.30 to 10,000.00 ng / mL; samples outside the quantitative range were diluted with serum to bring the concentration within the measurable range of the method.

[0136] Flow cytometry in Ph1a

[0137] Blood is collected in Cytochex BCT tubes (Streck (Streck)), transported to Covance Central Laboratory Services (Covance Central Laboratories Services) (Indianapolis, Indiana), and tested by flow cytometry using the method for verification. In brief, cells are stained with fluorescent dye-labeled antibodies for CD45 (clone HI30), CD19 (clone HIB19), IgD (clone IA6-2), CD27 (clone M-T271) and CD38 (clone HIT2, all BD) to identify B cell colonies. Subsequently, cells are processed with FACSPerm2 (BD company (BectonDickenson)), and stained to measure proliferating cells for intracellular Ki67 (clone KI67, 100 companies (Biolegend)) expression.

[0138] PC Tags

[0139] The PC gene signature was determined as previously described (Streicher 2014). Briefly, total RNA was extracted from PAXgene blood tubes using the PAXgene Blood RNA Kit (Qiagen). For TaqMan qPCR, cDNA was generated 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. ΔΔCt values ​​were calculated using the mean of two reference genes (β-actin and GAPDH) and each patient's baseline expression level as a control. Fold change values ​​were determined by calculating 2-ΔΔCt.

[0140] Ph1b patients and study design

[0141] A phase Ib, randomized, blinded, placebo-controlled study was conducted in patients aged 18-70 years diagnosed with RA according to the EULAR / ACR criteria (Aletaha et al. 2010) at least 6 months prior to study entry. Subjects had moderate to severe activity, as defined by a DAS28-CRP score of at least 3.2 at screening and at least 4 swollen joints and 4 tender joints at screening and randomization. At screening, patients were positive for 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 another conventional DMARD if MTX was intolerant, starting at least 12 weeks prior to screening and continuing at a stable dose for at least 6 weeks. Prior treatment with biological agents for RA (except rituximab or other B cell depleting agents) was acceptable as long as appropriate washout was performed before randomization in our study. Patients were treated with placebo (n=15) or VIB4920 (75 mg, n=8; 500 mg n=10; 1000 mg n=12; or 1500 mg n=12) administered by IV infusion every other week for 12 weeks, followed by a 12-week post-treatment observation period. VECTRA-DA scores were measured by Crescendo Bioscience (San Francisco, CA), while RF autoantibody measurements were performed by Covance Central Laboratory Services (Princeton, NJ).

[0142] Statistical analysis

[0143] In the SRBC model, the effects of treatment on primary human B cell expansion, plasma differentiation, and GC B cell responses were assessed using two-tailed unpaired Student's t-tests. The gene signature scores for VIB4920 and placebo were compared at multiple time points using the Mann-Whitney U test. Figure 9C). Statistical tests and plotting were performed using Graphpad Prism software. The dose response of the changes in DAS28-CRP and RF relative to baseline on day 85 was analyzed using the MCP-Mod method, including the corresponding baseline as a covariate, and using three pre-specified candidate models for dose response (linear, Emax, and Hill-Emax models). The test of the dose-response signal was adjusted for multiplicity to control the overall error rate (family-wise error rate) at the 0.10 level. The final model was selected from those models indicated as significant based on the Akaike information criterion. Using a mixed model analysis of repeated measures (MMRM), the changes in DAS28-CRP, RF, Vectra DA, CDAI, tender joint counts, swollen joint counts, patient and physician overall assessments, and serum CRP relative to baseline were analyzed, including the corresponding baseline results as covariates.

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Claims

1. Use of a CD40L-specific Tn3 scaffold protein in the preparation of a medicament for use in a method for inhibiting a B cell-mediated immune response in a subject, the method comprising: Administering a dose between 500 mg and 3000 mg of CD40L-specific Tn3 scaffold protein to subjects in need; and Inhibit the B cell-mediated immune response, wherein the subject has an autoimmune disease or disorder, and the autoimmune disease or disorder is Sjögren's syndrome, and wherein the CD40L-specific Tn3 scaffold protein comprises two monomer sequences and a human serum albumin sequence, wherein each monomer sequence is represented by the polypeptide sequence of SEQ ID NO:

3.

2. The use according to claim 1, wherein the dose is between 1000 mg and 1500 mg of CD40L-specific Tn3 scaffold protein.

3. The use according to claim 2, wherein the dose is 1000 mg of CD40L-specific Tn3 scaffold protein.

4. The use according to claim 2, wherein the dose is 1500 mg of CD40L-specific Tn3 scaffold protein.

5. The use according to any one of claims 1 to 4, wherein the dose is administered intravenously.

6. Use of a CD40L-specific Tn3 scaffold protein in the preparation of a medicament for use in a method for treating an autoimmune disease or disorder, the method comprising: Administer a dose between 500 mg and 3000 mg of CD40L-specific Tn3 scaffold protein to patients in need; and treating the autoimmune disease or disorder, wherein the autoimmune disease or disorder is Sjögren's syndrome, and wherein the CD40L-specific Tn3 scaffold protein comprises two monomer sequences and a human serum albumin sequence, wherein each monomer sequence is represented by the polypeptide sequence of SEQ ID NO:

3.

7. The use according to claim 6, wherein the dose is between 1000 mg and 1500 mg of CD40L-specific Tn3 scaffold protein.

8. The use according to claim 7, wherein the dose is 1000 mg of CD40L-specific Tn3 scaffold protein.

9. The use according to claim 8, wherein the dose is 1500 mg of CD40L-specific Tn3 scaffold protein.

10. The use according to any one of claims 6 to 9, wherein the dose is administered intravenously.

11. Use of a CD40L-specific Tn3 scaffold protein in the preparation of a medicament for use in a method for reducing a plasma cell (PC) gene signature in a patient in need thereof, the method comprising: administering a CD40L-specific Tn3 scaffold protein to a patient in need thereof, The patient has Sjögren's syndrome and is being treated for Sjögren's syndrome, and wherein the CD40L-specific Tn3 scaffold protein is administered at a dose of 500 mg to 3000 mg; and Lower the patient's PC gene signature score, and The CD40L-specific Tn3 scaffold protein comprises two monomer sequences and a human serum albumin sequence, wherein each monomer sequence is shown in the polypeptide sequence of SEQ ID NO:

3.

12. The use according to claim 11, wherein the dose is between 1000 mg and 2000 mg.

13. The use according to claim 12, wherein the dose is between 1000 mg and 1500 mg.

14. The use according to claim 13, wherein the dose is 1000 mg.

15. The use of claim 14, wherein the dose is 1500 mg.

16. The use of any one of claims 11 to 15, wherein the dose is administered intravenously.

17. Use of a CD40L-specific Tn3 scaffold protein in the preparation of a medicament for use in a method for reducing autoantibodies in a patient being treated for an autoimmune disorder, the method comprising: administering a CD40L-specific Tn3 scaffold protein to a patient in need thereof, wherein the patient has Sjögren's syndrome; and wherein the CD40L-specific Tn3 scaffold protein is administered at a dose of 500 mg to 3000 mg; and Reduce the patient's autoantibodies, The CD40L-specific Tn3 scaffold protein comprises two monomer sequences and a human serum albumin sequence, wherein each monomer sequence is shown in the polypeptide sequence of SEQ ID NO:

3.

18. The use according to claim 17, wherein the dose is between 1000 mg and 2000 mg.

19. The use according to claim 18, wherein the dose is between 1000 mg and 1500 mg.

20. The use of claim 19, wherein the dose is 1000 mg.

21. The use of claim 19, wherein the dose is 1500 mg.

22. The use of any one of claims 17-21, wherein the dose is administered intravenously.

23. Use of a CD40L-specific Tn3 scaffold protein in the preparation of a medicament for use in a method for reducing inflammation in a patient, the method comprising: administering a CD40L-specific Tn3 scaffold protein to a patient in need thereof, wherein the patient has Sjögren's syndrome; and wherein the CD40L-specific Tn3 scaffold protein is administered at a dose of 1000 mg to 3000 mg; and Reduce the patient's inflammation, The inflammation is Sjögren's syndrome, and the CD40L-specific Tn3 scaffold protein comprises two monomer sequences and a human serum albumin sequence, wherein each monomer sequence is represented by the polypeptide sequence of SEQ ID NO:

3.

24. The use according to claim 23, wherein the dose is between 1000 mg and 2000 mg.

25. The use of claim 24, wherein the dose is between 1000 mg and 1500 mg.

26. The use of claim 23, wherein the dose is 1000 mg.

27. The use of claim 23, wherein the dose is 1500 mg.

28. The use of claim 23, wherein the dose is 3000 mg.

29. The use of any one of claims 23-28, wherein the dose is administered intravenously.

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