Fusion proteins for use in treatment of thyroid eye disease

Abstract

The present disclosure relates to methods of treating thyroid eye disease (TED) by administering to a subject in need thereof a fusion protein, such as a fusion protein as defined herein. The present disclosure provides treatment of pathophysiological parameters of TED, including both systemic and local pathophysiological parameters, thereby effectively treating the thyroid eye disease.

Description

Technical Field

[0001] This disclosure relates to a method for treating thyroid eye disease (TED) by administering a fusion protein (such as a fusion protein as defined herein) to a subject in need. This disclosure provides treatment of TED pathophysiological parameters, including both systemic and local pathophysiological parameters, thereby effectively treating the disease. Background of the Invention

[0003] Autoimmune diseases encompass a wide range of clinical impairments defined by a pathological response to self-antigens or autoantigens, resulting in immune system-mediated damage. Typically, autoimmune diseases are characterized by the presence of abnormal lymphocyte (T and B cell) activation, in which non-lymphoid cells also play a significant role in disease pathogenesis, resulting from the disruption of immune tolerance and at least partially leading to dysregulated antibody production.

[0004] Autoimmune diseases can be systemic or tissue-specific in nature: systemic autoimmune disorders include systemic rheumatic diseases (such as systemic lupus erythematosus), while tissue / organ-specific diseases include endocrine disorders and neurological disorders (such as autoimmune thyroiditis and multiple sclerosis).

[0005] Thyroid eye disease (TED) is a painful, disabling, and disfiguring autoimmune condition commonly associated with Graves' disease (a form of autoimmune thyroiditis that causes hyperthyroidism) and can lead to visual impairments, including diplopia and vision loss (Joseph SS et al. 2015; Bartalena L. et al. 2020). TED can also be present in patients with other autoimmune disorders, including Hashimoto's thyroiditis (a form of autoimmune thyroiditis that causes hypothyroidism) (Patel A. et al. 2019). TED is characterized by the presence of bulging eyes (protruding eyeballs), which represents a disabling factor in patients. Moderate to severe disease can occur in up to one-third of patients and can be visually threatening.

[0006] The pathophysiology of TED is complex and comprises several parameters, including systemic pathophysiological components and localized inflammatory orbital pathology, as described in this article. Active phases of TED are characterized by orbital inflammation and infiltration of various immune cells, leading to extensive orbital remodeling. The triggering of TED may be due to loss of tolerance to the thyroid-stimulating hormone receptor (TSHR) and the production of stimulating autoantibodies. Orbital fibroblasts present in the retro-orbital tissue express TSHR, and binding of anti-TSHR antibodies to this receptor leads to cell activation. Subsequently, orbital fibroblasts differentiate into mature adipocytes and myofibroblasts, which increases the production of hyaluronic acid and pro-inflammatory cytokines. (Weiler DL. 2017; Jain AP. et al. 2021) Histopathological findings of the orbit include extensive deposition of hyaluronic acid between myofibrils, resulting in enlargement of the extraocular muscles and extensive inflammatory infiltration, as well as associated interstitial edema. (Joseph SS et al. 2015; Patel A. et al. 2019; Hwang CJ. et al. 2009) The local activation of these cells and the production of inflammatory molecules ultimately lead to the expansion and remodeling of the periorbital and orbital tissues, resulting in diplopia and proptosis.

[0007] Another cell population that plays a key role in TED is circulating fibroblasts. (Douglas RS. et al. 2014) Fibroblasts are mesenchymal cells derived from monocyte precursors. They can infiltrate damaged organs and possess the inflammatory characteristics of macrophages and the tissue remodeling properties of fibroblasts. Chronic inflammatory stimulation mediates the differentiation, transport, and accumulation of fibroblasts in autoimmune-related diseases. (Reilkoff RA. et al. 2011) Fibroblast precursors are present at an increased frequency in the peripheral blood of patients with TED and express both TSHR and insulin-like growth factor-1 receptor (IGF-1R), and can be directly activated by stimulating autoantibodies. Activated fibroblasts express CD40.

[0008] Cytokines (including pro-inflammatory and anti-inflammatory cytokines) are also important participants in the pathogenesis of TED and are involved in different processes, such as by promoting the initiation and spread of local autoimmune inflammation, which plays a key role in maintaining the autoimmune response within a specific tissue.

[0009] The binding of CD40 / CD40L has been proposed to play a role in the pathophysiology of TED. In orbital fibroblasts and infiltrating fibroblasts, the binding of CD40L to CD40 induces the upregulation and release of pro-inflammatory cytokines (such as IL-6 and IL-8). These pro-inflammatory cytokines induce genes encoding prostaglandin H synthase-2, hyaluronic acid synthase, and uridine diphosphate glucose dehydrogenase, directly leading to hyaluronic acid production, orbital inflammation, and ultimately, tissue remodeling. (Hwang CJ et al. 2009; Smith TJ et al. 2008)

[0010] IGF-1R has also been described in the pathophysiology of TED. IGF-1R and TSHR have been shown to form a complex of physical and functional interactions within orbital fibroblasts, and stimulation of IGF-1R similarly leads to activation of TSHR and promotes an inflammatory response. (Jain AP. et al. 2021)

[0011] IGF-1R inhibitors have been studied for the treatment of TED; however, although reductions in ptosis, for example, have been reported after IGF-1R inhibition, the relapse rate with IGF-1R inhibitors has been reported to be approximately 30%. (Couch, SM. 2022)

[0012] Other treatments for TED include the use of intravenous corticosteroids (± statins), mycophenolate mofetil (± steroids), CD20 inhibitors, IL-6 receptor antagonists, and orbital radiation. These treatments aim to reduce systemic and local inflammation or lower autoantibody titers. However, these treatments have been found to have only minor effects on proptosis and diplopia. Therefore, despite considerable efforts to identify effective treatment options for TED, challenges remain in determining the optimal treatment options. Consequently, there is an unmet need for new, effective treatments with prolonged efficacy and low relapse rates.

[0013] Many biologics targeting the CD40 / CD40L axis have been developed and have shown efficacy in inhibiting immune responses in preclinical models of autoimmune diseases.

[0014] The fusion protein disclosed herein was previously described in WO 2021 / 149015, which demonstrated its ability to block the interaction between CD40L in CD4+ T cells and CD40 in B cells, resulting in selective inhibition of B cell maturation and immunoglobulin class switching. Further description illustrates how the fusion protein provides reduced IgG antibody titers and decreased cellularity of germinal centers in lymph nodes following keyhole cyanobacterial (KLH) immunization in cynomolgus monkeys, demonstrating its effect on T cell-dependent antibody responses (TDAR).

[0015] Although the role of CD40 / CD40L binding has been proposed as part of the complex pathophysiology of TED, no potential therapeutic benefits have been reported from blocking the CD40 / CD40L interaction, such as reductions in fibroblast-induced systemic pathophysiology and / or localized inflammatory orbital disease. Therefore, as stated above, it remains unknown whether CD40L inhibition alone will have any effect on various disease parameters. Summary of the Invention

[0016] This article discloses methods for treating thyroid eye disease (TED) that involve administering a therapeutically effective amount of a fusion protein comprising the structure according to formula (I) to subjects in need: Formula (I), R1 and R2 are each an anti-CD40L single-chain variable fragment (scFv) linked to the N-terminus of the anti-serum albumin antigen-binding fragment (Fab), and Each of R1 and R2 is connected to either the heavy chain variable domain or the light chain variable domain of the antiserum albumin Fab.

[0017] This article also discloses methods for treating Graves' disease, which involve administering a therapeutically effective amount of a fusion protein comprising the structure according to formula (I) to subjects in need: Formula (I), R1 and R2 are each an anti-CD40L single-chain variable fragment (scFv) linked to the N-terminus of the anti-serum albumin antigen-binding fragment (Fab), and Each of R1 and R2 is connected to either the heavy chain variable domain or the light chain variable domain of the antiserum albumin Fab.

[0018] In some embodiments, R1 is connected to the heavy chain variable domain of antiserum albumin Fab, and R2 is connected to the light chain variable domain of antiserum albumin Fab.

[0019] In some embodiments, each of R1 and R2 is an anti-CD40L hu5c8 scFv. In some embodiments, R1 and R2 each comprise a complementarity-determining domain (CDR) region containing the following amino acid sequence: CDR VL1:RASQRVSSSTYSYMH (SEQ ID NO: 3); CDR VL2: YASNLES (SEQ ID NO: 4); CDR VL3: QHSWEIPPT (SEQ ID NO: 5); CDR VH1:SYYMY (SEQ ID NO: 6); CDR VH2: EINPSNGDTNFNEKFKS (SEQ ID NO: 7); and CDR VH3: SDGRNDMDS (SEQ ID NO: 8).

[0020] In some embodiments, R1 and R2 each comprise a heavy chain variable domain containing an amino acid sequence having at least 80% identity with the amino acid sequence of QVQLVQSGAEVVKPGASVKLSCKASGYIFTSYYMYWVKQAPGQGLEWIGEINPSNGDTNFNEKFKSKATLTVDKSASTAYMELSSLRSEDTAVYYCTRSDGRNDMDSWGQGTLVTVSS (SEQ ID NO: 10).

[0021] In some embodiments, R1 and R2 each comprise a light chain variable domain containing an amino acid sequence having at least 80% identity with the amino acid sequence of DIVLTQSPATLSVSPGERATISCRASQRVSSSTYSYMHWYQQKPGQPPKLLIKYASNLESGVPARFSGSGSGTDFTLTISSVEPEDFATYYCQHSWEIPPTFGGGTKLEIKR (SEQ ID NO: 9).

[0022] In some embodiments, R1 and R2 each comprise a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 10, and the light chain variable domain comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 9.

[0023] In some embodiments, each of R1 and R2 contains an amino acid sequence having at least 80% identity with SEQ ID NO: 11. In some embodiments, each of R1 and R2 contains the amino acid sequence of SEQ ID NO: 11. In some embodiments, each of R1 and R2 contains the amino acid sequence of either SEQ ID NO: 11 or SEQ ID NO: 12.

[0024] In some embodiments, each of R1 and R2 is linked to the antiserum albumin Fab via one or more adapters. In some embodiments, each adapter comprises 1 to 20 amino acids. In some embodiments, each adapter comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 23 or SEQ ID NO: 24. In some embodiments, each adapter comprises the amino acid sequence of SEQ ID NO: 23 or SEQ ID NO: 24. In some embodiments, the heavy chain variable domain and the light chain variable domain of R1 and R2 are linked via a (G4S)3 adapter comprising the amino acid sequence of SEQ ID NO: 23.

[0025] In some embodiments, anti-CD40L scFv is linked to the N-terminus of the heavy chain of anti-serum albumin Fab via a linker having the amino acid sequence of SEQ ID NO: 23. In some embodiments, anti-CD40L scFv is linked to the N-terminus of the light chain of anti-serum albumin Fab via a linker having the amino acid sequence of SEQ ID NO: 24.

[0026] In some embodiments, the first anti-CD40L scFv is linked to the N-terminus of the heavy chain of anti-serum albumin Fab via a linker having the amino acid sequence of SEQ ID NO: 23, and the second anti-CD40L scFv is linked to the N-terminus of the light chain of anti-serum albumin Fab via a linker having the amino acid sequence of SEQ ID NO: 24.

[0027] In some embodiments, antiserum albumin Fab comprises a complementarity-determining domain (CDR) region containing the following amino acid sequence: CDR VL1:RASQSVGSNLA (SEQ ID NO: 13); CDR VL2: GASTGAT (SEQ ID NO: 14); CDR VL3: QQYYSFLAKT (SEQ ID NO: 15); CDR VH1:AYSMN (SEQ ID NO: 16); CDR VH2: SISSSGRYIHYADSVKG (SEQ ID NO: 17); and CDR VH3:ETVMAGKALDY (SEQ ID NO: 18).

[0028] In some embodiments, the antiserum albumin Fab comprises a heavy chain variable domain containing an amino acid sequence having at least 80% identity with the amino acid sequence of QVQLVQSGGGPVKPGGSLRLSCAASGFMFRAYSMNWVRQAPGKGLEWVSSISSSGRYIHYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARETVMAGKALDYWGQGTLVTVSS (SEQ ID NO: 19).

[0029] In some embodiments, the antiserum albumin Fab comprises a light chain variable domain containing an amino acid sequence having at least 80% identity with the amino acid sequence of DIVLTQSPGTLSLSPGETATLSCRASQSVGSNLAWYQQKPGQAPRLLIYGASTGATGVPARFSGSRSGTDFTLTITSLQPEDFATYYCQQYYSFLAKTFGQGTQLEIKR (SEQ ID NO: 20).

[0030] In some embodiments, the antiserum albumin Fab comprises a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 19, and the light chain variable domain comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 20.

[0031] In some embodiments, the antiserum albumin Fab comprises a heavy chain domain containing an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 21 (VH-CH1 domain). In some embodiments, the antiserum albumin Fab comprises a light chain domain containing an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 22 (VL-CL domain). In some embodiments, the antiserum albumin Fab comprises a heavy chain domain and a light chain domain, the heavy chain domain containing an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 21 (VH-CH1 domain), and the light chain domain containing an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 22 (VL-CL domain).

[0032] In some embodiments, the fusion protein comprises a heavy chain containing an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 1. In some embodiments, the fusion protein comprises a light chain containing an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 2. In some embodiments, the fusion protein comprises a heavy chain and a light chain, the heavy chain containing an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 1, and the light chain containing an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 2.

[0033] In some embodiments, the fusion protein is administered as a pharmaceutical formulation. In some embodiments, the fusion protein is administered via transdermal, subcutaneous, intravenous, or intramuscular administration. In some embodiments, the fusion protein is administered at an amount of about 0.01 mg / kg to about 100 mg / kg of the subject's body weight.

[0034] In some embodiments, the treatments disclosed herein result in the treatment of orbital disease in patients with TED. In some embodiments, the treatment of orbital disease in patients with TED results in a reduction of inflammation in the periorbital space.

[0035] In some embodiments, the treatments disclosed herein result in systemic pathophysiological treatment of TED. In some embodiments, the systemic pathophysiological treatment of TED results in inactivation of PBMCs and decreased cytokine expression. In some embodiments, the systemic pathophysiological treatment of TED results in reduced infiltration of autoimmune cells in the periorbital space.

[0036] In some embodiments, the treatments disclosed herein result in treatment of the systemic pathophysiology of TED and treatment of orbital disease in TED.

[0037] In some embodiments, the treatment results in a reduction of eye bulging.

[0038] In some embodiments, the treatment resulted in a reduction in diplopia. Attached Figure Description

[0039] Figure 1 The percentage change in right ear thickness relative to day 7, day 8, and day 9 is shown in KLH-immunized mice in the untreated (medium-treated) or mice treated with m-Ab1 at doses of 6.8 or 13.6 mg / kg. A decrease in ear thickness over time was observed compared to the untreated (medium-treated) control.

[0040] Figures 2A-2C show the levels of pro-inflammatory cytokines TNF-α, IL-4, and IL-22 in the KLH immune site of the right ear of mice in untreated (medium-treated) or mice treated with mAb1 at doses of 6.8 or 13.6 mg / kg. Treatment with mAb1 significantly reduced TNF-α levels compared to the untreated (medium-treated) control (Figure 2A). Reduced levels of IL-4 (Figure 2B) and IL-22 (Figure 2C) were also observed after mAb1 treatment. Statistical significance: p < 0.05, p < 0.005.

[0041] Figures 3A-3D show the mRNA levels (relative expression 2) of CD40 (Figure 3A), IL-6 (Figure 3B), IL-8 (Figure 3C), and RANTES (Figure 3D) in PBMCs as monitored by real-time quantitative PCR (qPCR). -ΔΔCt These PBMCs were untreated (control), treated with IFN-γ alone (IFN-γ), or treated with IFN-γ and 15 nM megaCD40L alone (15 nM CD40L), or in the presence of 3.75 or 124 nM h-Ab2 (3.75 nM h-Ab2), (124 nM h-Ab2), or in the presence of control antibody (3.75 nM Ctr-Ab), (124 nM Ctr-Ab). For statistical analysis, one-way ANOVA was used along with Bonferroni correction. p < 0.05, p < 0.01, p < 0.0001.

[0042] Figures 4A-4D show the mRNA levels (relative expression 2) of CD40 (Figure 4A), IL-6 (Figure 4B), IL-8 (Figure 4C), and RANTES (Figure 4D) in fibroblasts as monitored by real-time quantitative PCR (qPCR). -ΔΔCtThese fibroblasts were untreated (control), treated with IFN-γ alone (IFN-γ), or treated with IFN-γ and 32 nM human recombinant soluble CD40L alone or in the presence of 5, 135, or 1215 nM h-Ab1 (5 nM h-Ab1), (135 nM h-Ab1), (1215 nM h-Ab1), or in the presence of control antibody (5 nM Ctr-Ab), (135 nM Ctr-Ab), (1215 nM Ctr-Ab). For statistical analysis, one-way ANOVA with Bonferroni correction was used. p < 0.05, p < 0.01, p < 0.0001.

[0043] Figures 5A-5C show the mRNA levels (relative expression 2) of CD40 (Figure 5A), IL-8 (Figure 5B), and RANTES (Figure 5C) in myoblasts as monitored by real-time quantitative PCR (qPCR). -ΔΔCt These myoblasts were untreated (control), treated with IFN-γ alone (IFN-γ), or treated with IFN-γ and 32 nM human recombinant soluble CD40L alone or in the presence of 5, 135, or 1215 nM h-Ab1 (5 nM h-Ab1), (135 nM h-Ab1), (1215 nM h-Ab1), or in the presence of control antibody (5 nM Ctr-Ab), (135 nM Ctr-Ab), (1215 nM Ctr-Ab). For statistical analysis, one-way ANOVA with Bonferroni correction was used. p < 0.05, p < 0.01, p < 0.0001.

[0044] Figures 6A-6C show the mRNA levels (relative expression 2) of IL-6 (Figure 6A), IL-8 (Figure 6B), and RANTES (Figure 6C) in fibroblasts as monitored by real-time quantitative PCR (qPCR). -ΔΔCtThese fibroblasts were treated with IFN-γ and 32 nM human recombinant soluble CD40L in the presence of increased concentrations of h-Ab1 or control antibody (0.56 nM to 3645 nM). Solid black circles: h-Ab1; gray triangles: Ctr-Ab. Data are presented as mean ± standard error of the mean (SEM) of N = 5 biological replicates, where n = 2 technical replicates / experiment.

[0045] Figures 7A-7B show the relative protein levels of IL6 (Figure 7A) and IL8 (Figure 7B) in fibroblasts, as quantified by MSD assay, after treatment with IFN-γ and 32 nM recombinant human soluble CD40L in the presence of increased concentrations of h-Ab1 or control antibody (0.56 nM to 3645 nM). 100% was set as the protein levels found against fibroblasts stimulated alone with IFN-γ + CD40L. Solid black circles: h-Ab1, gray triangles: Ctr-Ab. Data are presented as mean ± standard error of the mean (SEM) of N = 5 biological replicates, where n = 2 technical replicates / experiment.

[0046] Figures 8A-8D show the mRNA levels (relative expression 2) of CD40 (Figure 8A), IL-6 (Figure 8B), IL-8 (Figure 8C), and RANTES (Figure 8D) in myoblasts as monitored by real-time quantitative PCR (qPCR). -ΔΔCt These myoblasts were treated with IFN-γ followed by 32 nM recombinant human soluble CD40L in the presence of increased concentrations of h-Ab1 or control antibody (90 pM to 10 µM). Black solid circles: h-Ab1, gray triangles: Ctr-Ab. Data are presented as mean ± standard error of the mean (SEM) of N = 4–5 biological replicates. For statistical analysis, one-way ANOVA was used along with Dunnett's post-hoc test, with 32 nM CD40L as a reference. p < 0.0001.

[0047] Figures 9A-9B show the relative protein levels of IL-6 (Figure 9A) and IL-8 (Figure 9B) in myoblasts, as quantified by MSD assay, after treatment with IFN-γ followed by 32 nM recombinant human soluble CD40L in the presence of increased concentrations of h-Ab1 or control antibody (90 pM to 10 µM). 100% was defined as the protein level released by myoblasts stimulated alone with IFN-γ + CD40L. Data are presented as mean ± standard error of the mean (SEM) of N = 4–5 biological replicates. For statistical analysis, one-way ANOVA was used along with a post-hoc Dunnett test, with 32 nM CD40L as a reference. p < 0.0001.

[0048] Figures 10A-10B show (A) a PCA plot illustrating the different transcriptomic profiles of PBMCs based on their activation state and original source. PC1 effectively separates activated PBMCs from naïve PBMCs (regardless of their original source), while PC2 distinguishes between healthy individuals and those with TED, highlighting the differences in their transcriptomic profiles. (B) A volcano plot showing the DE genes between naïve TED PBMCs and healthy PBMCs. A total of 290 DE genes were identified, of which 105 were significantly downregulated and 185 were significantly upregulated in the TED samples. Upregulated genes associated with inflammatory cytokine and T cell regulation were tagged.

[0049] Figure 11A-E Histograms showing the percentage of different cell types after 24 hours of incubation of PBMCs with anti-CD3 / CD28 beads are presented. This incubation resulted in effective T cell activation in both healthy and TED samples, as demonstrated by increased expression of surface markers CD69 (C) and CD25 (A) and increased expression of CD40L (B and D) (characteristics of T effector daughter cells), as shown by FACS analysis. Additionally, a significant increase in CD40-expressing B cells was observed in TED samples, while no change was observed in healthy PBMCs (E).

[0050] Figure 12 A heatmap illustrating transcriptional analysis of TED PBMCs following anti-CD3 / CD28 stimulation is shown. The analysis confirmed extensive cellular activation, evidenced by increased expression of inflammation-related genes such as IFNG, TNF, IL2, IL4, IL6, and IL9.

[0051] Figure 13A-C shows (A) a violin plot illustrating downregulated CD40 expression in activated TED PBMCs after treatment with the CD40L inhibitor (h-Ab1), while CD40 expression remained unchanged after treatment with anti-TNP IgG (negative control). (B) From the same sample as in A), data on the release of chemokines CXCL9, CXCL10, and CXCL11 from dendritic cells after treatment with the CD40L inhibitor are shown. (C) A volcano plot illustrating DE genes in response to CD40L inhibitor treatment. Key inflammation-related genes, including CCL2, ACHE, and FCER2, are significantly downregulated. Detailed Implementation

[0052] As disclosed herein, the fusion protein described herein has been identified as having effects on both the systemic autoimmune component and the underlying etiology of the disease, as well as the local inflammatory orbital component of thyroid ophthalmopathy (TED). Both characterize the pathophysiology of TED, and treatment of both components can lead to effective treatment of TED, addressing both the symptoms of the local inflammatory orbital component of the disease and its underlying systemic etiology and progression.

[0053] As demonstrated by the data presented herein, the fusion protein described herein reduces both local and systemic inflammation. Specifically, the fusion protein disclosed herein was found to inhibit the production of pro-inflammatory cytokines by activated fibroblasts and myoblasts, as well as peripheral blood mononuclear cells (PBMCs) (a cell population involved in the pathogenesis of TED). Furthermore, the fusion protein disclosed herein was found to enter the local inflammatory environment, and administration of the fusion protein was demonstrated to lead to a reduction in the local inflammatory response associated with immune cell infiltration. Therefore, administration of the fusion protein disclosed herein may provide effective treatment for TED by acting on the underlying autoimmune causes of the disease and the resulting local inflammatory conditions (which contribute to disease symptoms).

[0054] Therefore, this document discloses methods for treating Graves' disease and / or thyroid eye disease (TED). In some embodiments, this disclosure relates to methods for treating TED.

[0055] Fusion protein

[0056] The treatment method disclosed herein involves administering a fusion protein comprising a structure according to formula (I) to a subject in need: Formula (I), R1 and R2 are each an anti-CD40L single-chain variable fragment (scFv) linked to the N-terminus of anti-serum albumin Fab, and Each of R1 and R2 is connected to either the heavy chain variable domain or the light chain variable domain of the antiserum albumin Fab.

[0057] In some embodiments, the fusion protein disclosed herein consists of a structure as defined herein according to formula (I).

[0058] The fusion protein disclosed herein is a recombinant bispecific (scFv)2-Fab fusion protein that targets human CD40L and simultaneously binds to human serum albumin (HSA). Binding to HSA serves to prolong the half-life of the fusion protein, as further described in the previous disclosure of WO 2021 / 149015. In some embodiments, the fusion protein is a CD40L inhibitor.

[0059] In some embodiments, R1 is connected to the heavy chain variable domain of antiserum albumin Fab, and R2 is connected to the light chain variable domain of antiserum albumin Fab.

[0060] In some embodiments, each of R1 and R2 is connected to antiserum albumin Fab via one or more adapters, for example, where each adapter contains 1 to 20 amino acids.

[0061] In some embodiments, each adapter comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 23 or SEQ ID NO: 24. In some embodiments, each adapter comprises an amino acid sequence of SEQ ID NO: 23 or SEQ ID NO: 24. In some embodiments, each adapter consists of an amino acid sequence of SEQ ID NO: 23 or SEQ ID NO: 24.

[0062] In some embodiments, the linker connecting the anti-CD40L scFv to the N-terminus of the heavy chain of anti-serum albumin Fab has the amino acid sequence of SEQ ID NO: 23. In some embodiments, the linker connecting the anti-CD40L scFv to the N-terminus of the light chain of anti-serum albumin Fab has the amino acid sequence of SEQ ID NO: 24.

[0063] In some embodiments, the first anti-CD40L scFv is linked to the N-terminus of the heavy chain of anti-serum albumin Fab via a linker having the amino acid sequence of SEQ ID NO: 23, and the second anti-CD40L scFv is linked to the N-terminus of the light chain of anti-serum albumin Fab via a linker having the amino acid sequence of SEQ ID NO: 24.

[0064] Structure of anti-CD40L scFv

[0065] The disclosed anti-CD40L scFv of the fusion protein provides binding of the fusion protein to CD40L, thereby inhibiting the binding of the CD40L target to CD40. As used herein, the term single-chain variable fragment (scFv) refers to an antibody fragment consisting of the variable regions of the heavy chain (VH) and light chain (VL) of an immunoglobulin linked by short linker peptides. The linker can connect the N-terminus of the VH to the C-terminus of the VL, or vice versa.

[0066] In some embodiments, each of R1 and R2 is an anti-CD40L hu5c8 scFv, wherein the anti-CD40L scFv contains the VH and VL fragments of hu5C8. The hu5C8 antibody is also known as ruplizumab (INN name) and has been assigned CAS number: 220651-94-5. In some embodiments, R1 and R2 are different anti-CD40L scFvs. In some embodiments, R1 and R2 are the same anti-CD40L scFv.

[0067] In some embodiments, R1 and R2 each comprise a complementarity-determining domain (CDR) region containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the following: CDR VL1:RASQRVSSSTYSYMH (SEQ ID NO: 3); CDR VL2: YASNLES (SEQ ID NO: 4); CDR VL3: QHSWEIPPT (SEQ ID NO: 5); CDR VH1:SYYMY (SEQ ID NO: 6); CDR VH2: EINPSNGDTNFNEKFKS (SEQ ID NO: 7); and CDR VH3: SDGRNDMDS (SEQ ID NO: 8).

[0068] In some embodiments, R1 and R2 each comprise a heavy chain complementarity-determining domain (CDR) containing the following amino acid sequence: CDR VH1:SYYMY (SEQ ID NO: 6); CDR VH2: EINPSNGDTNFNEKFKS (SEQ ID NO: 7); and CDR VH3: SDGRNDMDS (SEQ ID NO: 8).

[0069] In some embodiments, R1 and R2 each comprise a light chain complementarity-determining domain (CDR) region containing the following amino acid sequence: CDR VL1:RASQRVSSSTYSYMH (SEQ ID NO: 3); CDR VL2: YASNLES (SEQ ID NO: 4); and CDR VL3: QHSWEIPPT (SEQ ID NO: 5).

[0070] In some embodiments, R1 and R2 each comprise a complementarity-determining domain (CDR) region containing the following amino acid sequence: CDR VL1:RASQRVSSSTYSYMH (SEQ ID NO: 3); CDR VL2: YASNLES (SEQ ID NO: 4); CDR VL3: QHSWEIPPT (SEQ ID NO: 5); CDR VH1:SYYMY (SEQ ID NO: 6); CDR VH2: EINPSNGDTNFNEKFKS (SEQ ID NO: 7); and CDR VH3: SDGRNDMDS (SEQ ID NO: 8).

[0071] In some embodiments, R1 and R2 each comprise a complementarity-determining domain (CDR) region consisting of the following amino acid sequence: CDR VL1:RASQRVSSSTYSYMH (SEQ ID NO: 3); CDR VL2: YASNLES (SEQ ID NO: 4); CDR VL3: QHSWEIPPT (SEQ ID NO: 5); CDR VH1:SYYMY (SEQ ID NO: 6); CDR VH2: EINPSNGDTNFNEKFKS (SEQ ID NO: 7); and CDR VH3: SDGRNDMDS (SEQ ID NO: 8).

[0072] In some embodiments, R1 and R2 each comprise a heavy chain variable domain containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with QVQLVQSGAEVVKPGASVKLSCKASGYIFTSYYMYWVKQAPGQGLEWIGEINPSNGDTNFNEKFKSKATLTVDKSASTAYMELSSLRSEDTAVYYCTRSDGRNDMDSWGQGTLVTVSS (SEQ ID NO: 10). In some embodiments, R1 and R2 each comprise a heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 10. In some embodiments, R1 and R2 each comprise a heavy chain variable domain consisting of the amino acid sequence of SEQ ID NO: 10.

[0073] In some embodiments, R1 and R2 each comprise a light chain variable domain containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with DIVLTQSPATLSVSPGERATISCRASQRVSSSTYSYMHWYQQKPGQPPKLLIKYASNLESGVPARFSGSGSGTDFTLTISSVEPEDFATYYCQHSWEIPPTFGGGTKLEIKR (SEQ ID NO: 9). In some embodiments, R1 and R2 each comprise a light chain variable domain containing the amino acid sequence of SEQ ID NO: 9. In some embodiments, R1 and R2 each comprise a light chain variable domain consisting of the amino acid sequence of SEQ ID NO: 9.

[0074] In some embodiments, R1 and R2 each comprise a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 10, and the light chain variable domain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 9.

[0075] In some embodiments, R1 and R2 each comprise a heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 10 and a light chain variable domain containing the amino acid sequence of SEQ ID NO: 9.

[0076] In some embodiments, R1 and R2 each comprise a heavy chain variable domain consisting of the amino acid sequence of SEQ ID NO: 10 and a light chain variable domain consisting of the amino acid sequence of SEQ ID NO: 9.

[0077] In some embodiments, the heavy chain variable domains and light chain variable domains of R1 and R2 are connected by a linker, such as a G4S linker (e.g., a (G4S)3 linker comprising the amino acid sequence of SEQ ID NO: 23). In some embodiments, the heavy chain variable domains and light chain variable domains of R1 and R2 are connected by a (G4S)3 linker comprising the amino acid sequence of SEQ ID NO: 23.

[0078] In some embodiments, the connector connects the C end of VL to the N end of VH.

[0079] In some embodiments, each of R1 and R2 contains an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 11.

[0080] In some embodiments, each of R1 and R2 contains an amino acid sequence that is at least 80% identical to SEQ ID NO: 11.

[0081] In some embodiments, each of R1 and R2 comprises the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12. In some embodiments, each of R1 and R2 comprises the amino acid sequence of SEQ ID NO: 11. In some embodiments, each of R1 and R2 consists of the amino acid sequence of SEQ ID NO: 11.

[0082] Structure of antiserum albumin Fab

[0083] The anti-serum albumin Fab disclosed herein is a Fab fragment that provides binding of the fusion protein to serum albumin. Binding to serum albumin provides an extended half-life for the fusion protein.

[0084] As used herein, the term "Fab" refers to an antibody fragment, which is the fragment antigen-binding region (Fab region) of an antibody, the area on the antibody that binds to an antigen. It consists of a constant (C) domain and a variable (V) domain from each of the heavy and light chains, for example, VH-CH1 from the antibody heavy chain and VL-CL from the antibody light chain.

[0085] In some embodiments, antiserum albumin Fab comprises complementarity-determining domain (CDR) regions containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the following: CDR VL1:RASQSVGSNLA (SEQ ID NO: 13); CDR VL2: GASTGAT (SEQ ID NO: 14); CDR VL3: QQYYSFLAKT (SEQ ID NO: 15); CDR VH1:AYSMN (SEQ ID NO: 16); CDR VH2: SISSSGRYIHYADSVKG (SEQ ID NO: 17); and CDR VH3:ETVMAGKALDY (SEQ ID NO: 18).

[0086] In some embodiments, antiserum albumin Fab comprises a heavy chain complementarity-determining domain (CDR) containing the following amino acid sequence: CDR VH1:AYSMN (SEQ ID NO: 16); CDR VH2: SISSSGRYIHYADSVKG (SEQ ID NO: 17); and CDR VH3 is either ETMAGKALDY (SEQ ID NO: 18) or ETMAGKALDY (SEQ ID NO: 30).

[0087] In some embodiments, antiserum albumin Fab comprises a heavy chain complementarity-determining domain (CDR) containing the following amino acid sequence: CDR VH1:AYSMN (SEQ ID NO: 16); CDR VH2: SISSSGRYIHYADSVKG (SEQ ID NO: 17); and CDR VH3:ETVMAGKALDY (SEQ ID NO: 18).

[0088] In some embodiments, antiserum albumin Fab comprises a heavy chain complementarity-determining domain (CDR) region consisting of the following amino acid sequence: CDR VH1:AYSMN (SEQ ID NO: 16); CDR VH2: SISSSGRYIHYADSVKG (SEQ ID NO: 17); and CDR VH3 is either ETMAGKALDY (SEQ ID NO: 18) or ETMAGKALDY (SEQ ID NO: 30).

[0089] In some embodiments, antiserum albumin Fab comprises a heavy chain complementarity-determining domain (CDR) region consisting of the following amino acid sequence: CDR VH1:AYSMN (SEQ ID NO: 16); CDR VH2: SISSSGRYIHYADSVKG (SEQ ID NO: 17); and CDR VH3:ETVMAGKALDY (SEQ ID NO: 18).

[0090] In some embodiments, antiserum albumin Fab comprises a light chain complementarity-determining domain (CDR) containing the following amino acid sequence: CDR VL1:RASQSVGSNLA (SEQ ID NO: 13); CDR VL2: GASTGAT (SEQ ID NO: 14); and CDR VL3: QQYYSFLAKT (SEQ ID NO: 15).

[0091] In some embodiments, antiserum albumin Fab comprises a light chain complementarity-determining domain (CDR) region consisting of the following amino acid sequence: CDR VL1:RASQSVGSNLA (SEQ ID NO: 13); CDR VL2: GASTGAT (SEQ ID NO: 14); and CDR VL3: QQYYSFLAKT (SEQ ID NO: 15).

[0092] In some embodiments, antiserum albumin Fab comprises a complementarity-determining domain (CDR) region containing the following amino acid sequence: CDR VL1:RASQSVGSNLA (SEQ ID NO: 13); CDR VL2: GASTGAT (SEQ ID NO: 14); CDR VL3: QQYYSFLAKT (SEQ ID NO: 15); CDR VH1:AYSMN (SEQ ID NO: 16); CDR VH2: SISSSGRYIHYADSVKG (SEQ ID NO: 17); and CDR VH3 is either ETMAGKALDY (SEQ ID NO: 18) or ETMAGKALDY (SEQ ID NO: 30).

[0093] In some embodiments, antiserum albumin Fab comprises a complementarity-determining domain (CDR) region containing the following amino acid sequence: CDR VL1:RASQSVGSNLA (SEQ ID NO: 13); CDR VL2: GASTGAT (SEQ ID NO: 14); CDR VL3: QQYYSFLAKT (SEQ ID NO: 15); CDR VH1:AYSMN (SEQ ID NO: 16); CDR VH2: SISSSGRYIHYADSVKG (SEQ ID NO: 17); and CDR VH3:ETVMAGKALDY (SEQ ID NO: 18).

[0094] In some embodiments, antiserum albumin Fab comprises a complementarity-determining domain (CDR) region consisting of the following amino acid sequence: CDR VL1:RASQSVGSNLA (SEQ ID NO: 13); CDR VL2: GASTGAT (SEQ ID NO: 14); CDR VL3: QQYYSFLAKT (SEQ ID NO: 15); CDR VH1:AYSMN (SEQ ID NO: 16); CDR VH2: SISSSGRYIHYADSVKG (SEQ ID NO: 17); and CDR VH3 is either ETMAGKALDY (SEQ ID NO: 18) or ETMAGKALDY (SEQ ID NO: 30).

[0095] In some embodiments, antiserum albumin Fab comprises a complementarity-determining domain (CDR) region consisting of the following amino acid sequence: CDR VL1:RASQSVGSNLA (SEQ ID NO: 13); CDR VL2: GASTGAT (SEQ ID NO: 14); CDR VL3: QQYYSFLAKT (SEQ ID NO: 15); CDR VH1:AYSMN (SEQ ID NO: 16); CDR VH2: SISSSGRYIHYADSVKG (SEQ ID NO: 17); and CDR VH3:ETVMAGKALDY (SEQ ID NO: 18).

[0096] In some embodiments, the antiserum albumin Fab comprises a heavy chain variable domain containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with QVQLVQSGGGPVKPGGSLRLSCAASGFMFRAYSMNWVRQAPGKGLEWVSSISSSGRYIHYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARETVMAGKALDYWGQGTLVTVSS (SEQ ID NO: 19).

[0097] In some embodiments, antiserum albumin Fab comprises a heavy chain variable domain containing an amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 68.

[0098] In some embodiments, antiserum albumin Fab comprises a heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 19.

[0099] In some embodiments, antiserum albumin Fab comprises a heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 68.

[0100] In some embodiments, the antiserum albumin Fab comprises a heavy chain variable domain consisting of an amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 68.

[0101] In some embodiments, antiserum albumin Fab comprises a heavy chain variable domain consisting of the amino acid sequence of SEQ ID NO: 19.

[0102] In some embodiments, antiserum albumin Fab comprises a heavy chain variable domain consisting of the amino acid sequence of SEQ ID NO: 68.

[0103] In some embodiments, the antiserum albumin Fab comprises a light chain variable domain containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with DIVLTQSPGTLSLSPGETATLSCRASQSVGSNLAWYQQKPGQAPRLLIYGASTGATGVPARFSGSRSGTDFTLTITSLQPEDFATYYCQQYYSFLAKTFGQGTQLEIKR (SEQ ID NO: 20). In some embodiments, the antiserum albumin Fab comprises a light chain variable domain containing the amino acid sequence of SEQ ID NO: 20. In some embodiments, the antiserum albumin Fab comprises a light chain variable domain consisting of the amino acid sequence of SEQ ID NO: 20.

[0104] In some embodiments, the antiserum albumin Fab comprises a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 19, and the light chain variable domain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 20. In some embodiments, the antiserum albumin Fab comprises a heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 19 and a light chain variable domain containing the amino acid sequence of SEQ ID NO: 20. In some embodiments, the antiserum albumin Fab comprises a heavy chain variable domain consisting of the amino acid sequence of SEQ ID NO: 19 and a light chain variable domain consisting of the amino acid sequence of SEQ ID NO: 20.

[0105] In some embodiments, the antiserum albumin Fab comprises a heavy chain domain having an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 21 (VH-CH1 domain). In some embodiments, the antiserum albumin Fab comprises a heavy chain domain containing the amino acid sequence of SEQ ID NO: 21 (VH-CH1 domain). In some embodiments, the antiserum albumin Fab comprises a heavy chain domain containing the amino acid sequence of SEQ ID NO: 21 (VH-CH1 domain). In some embodiments, the antiserum albumin Fab comprises a heavy chain domain consisting of the amino acid sequence of SEQ ID NO: 69 (VH-CH1 domain). In some embodiments, the antiserum albumin Fab comprises a heavy chain domain consisting of the amino acid sequence of SEQ ID NO: 21 or SEQ ID NO: 69 (VH-CH1 domain). In some embodiments, the antiserum albumin Fab comprises a heavy chain domain consisting of the amino acid sequence of SEQ ID NO: 21 (VH-CH1 domain). In some embodiments, the antiserum albumin Fab comprises a heavy chain domain consisting of the amino acid sequence of SEQ ID NO: 69 (VH-CH1 domain).

[0106] In some embodiments, the antiserum albumin Fab comprises a light chain domain having an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the SEQ ID NO: 22 (VL-CL domain). In some embodiments, the antiserum albumin Fab comprises a light chain domain containing the amino acid sequence of SEQ ID NO: 22 (VL-CL domain). In some embodiments, the antiserum albumin Fab comprises a light chain domain consisting of the amino acid sequence of SEQ ID NO: 22 (VL-CL domain).

[0107] In some embodiments, the antiserum albumin Fab comprises a heavy chain domain and a light chain domain, the heavy chain domain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the SEQ ID NO: 21 (VH-CH1 domain), and the light chain domain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the SEQ ID NO: 22 (VL-CL domain). In some embodiments, the antiserum albumin Fab comprises a heavy chain domain containing the amino acid sequence of SEQ ID NO: 21 or SEQ ID NO: 69 (VH-CH1 domain) and a light chain domain containing the amino acid sequence of SEQ ID NO: 22 (VL-CL domain). In some embodiments, the antiserum albumin Fab comprises a heavy chain domain containing the amino acid sequence of SEQ ID NO: 21 (VH-CH1 domain) and a light chain domain containing the amino acid sequence of SEQ ID NO: 22 (VL-CL domain). In some embodiments, the antiserum albumin Fab comprises a heavy chain domain containing the amino acid sequence of SEQ ID NO: 69 (VH-CH1 domain) and a light chain domain containing the amino acid sequence of SEQ ID NO: 22 (VL-CL domain). In some embodiments, the antiserum albumin Fab comprises a heavy chain domain consisting of the amino acid sequence of SEQ ID NO: 21 or SEQ ID NO: 69 (VH-CH1 domain) and a light chain domain consisting of the amino acid sequence of SEQ ID NO: 22 (VL-CL domain). In some embodiments, the antiserum albumin Fab comprises a heavy chain domain consisting of the amino acid sequence of SEQ ID NO: 21 (VH-CH1 domain) and a light chain domain consisting of the amino acid sequence of SEQ ID NO: 22 (VL-CL domain). In some embodiments, the antiserum albumin Fab comprises a heavy chain domain consisting of the amino acid sequence of SEQ ID NO: 69 (VH-CH1 domain) and a light chain domain consisting of the amino acid sequence of SEQ ID NO: 22 (VL-CL domain).

[0108] In some embodiments, antiserum albumin Fab includes a heavy chain variable domain, which contains...

[0109] (a) Heavy chain complementarity-determining domain 1 (CDR1) containing the amino acid sequence SYGIS (SEQ ID NO: 47), heavy chain CDR2 containing the amino acid sequence WINT YSGGTKYAQKF QG (SEQ ID NO: 48), and heavy chain CDR3 containing the amino acid sequence LGHCQRGICSDALDT (SEQ ID NO: 49); (b) Heavy chain complementarity-determining domain 1 (CDR1) containing the amino acid sequence SYGIS (SEQ ID NO: 47), heavy chain CDR2 containing the amino acid sequence RINTYNGNTGYAQRLQG (SEQ ID NO: 50), and heavy chain CDR3 containing the amino acid sequence LGHCQRGICSDALDT (SEQ ID NO: 49); (c) Heavy chain complementarity-determining domain 1 (CDR1) containing the amino acid sequence NYGIH (SEQ ID NO: 51), heavy chain CDR2 containing the amino acid sequence SISYDGSNKYYADSVKG (SEQ ID NO: 52), and heavy chain CDR3 containing the amino acid sequence DVHYYGSGSYYNAFDI (SEQ ID NO: 53); (d) Heavy chain complementarity-determining domain 1 (CDR1) containing the amino acid sequence SYAMS (SEQ ID NO: 54), heavy chain CDR2 containing the amino acid sequence VISHDGGFQYYADSVKG (SEQ ID NO: 55), and heavy chain CDR3 containing the amino acid sequence AGWLRQYGMDV (SEQ ID NO: 56); (e) Heavy chain CDR1 containing the amino acid sequence AYWIA (SEQ ID NO: 57), heavy chain CDR2 containing the amino acid sequence MIWPPDADARYSPSFQG (SEQ ID NO: 58), and heavy chain CDR3 containing the amino acid sequence LYSGSYSP (SEQ ID NO: 59); or (f) Heavy chain complementarity-determining domain 1 (CDR1) containing the amino acid sequence AYSMN (SEQ ID NO: 16), heavy chain CDR2 containing the amino acid sequence SSISSSGRYIHYADSVKG (SEQ ID NO: 17), and heavy chain CDR3 containing the amino acid sequence ETVMAGKALDY (SEQ ID NO: 18).

[0110] In some embodiments, the antiserum albumin Fab includes a light chain variable domain, which contains...

[0111] (a) Light chain complementarity-determining domain 1 (CDR1) containing the amino acid sequence RASQSISRYLN (SEQ ID NO: 33), light chain CDR2 containing the amino acid sequence GASRLES (SEQ ID NO: 34), and light chain CDR3 containing the amino acid sequence QQSDSVPVT (SEQ ID NO: 35); (b) Light chain complementarity-determining domain 1 (CDR1) containing the amino acid sequence RASQSISSYLN (SEQ ID NO: 36), light chain CDR2 containing the amino acid sequence AASSLQS (SEQ ID NO: 37), and light chain CDR3 containing the amino acid sequence QQSYSTPPYT (SEQ ID NO: 38); (c) Light chain complementarity-determining domain 1 (CDR1) containing the amino acid sequence RASQSIFNYVA (SEQ ID NO: 39), light chain CDR2 containing the amino acid sequence DASNRAT (SEQ ID NO: 40), and light chain CDR3 containing the amino acid sequence QQRSKWPPTWT (SEQ ID NO: 41); (d) Light chain complementarity-determining domain 1 (CDR1) containing the amino acid sequence RASETVSSRQLA (SEQ ID NO: 42), light chain CDR2 containing the amino acid sequence GASSRAT (SEQ ID NO: 43), and light chain CDR3 containing the amino acid sequence QQYGSSPRT (SEQ ID NO: 44); (e) The light chain complementarity-determining domain 1 (CDR1) containing the amino acid sequence RASQSVSSSSLA (SEQ ID NO: 45), the light chain CDR2 containing the amino acid sequence GASSRAT (SEQ ID NO: 43), and the light chain CDR3 containing the amino acid sequence QKYSSYPLT (SEQ ID NO: 46); or (f) Light chain complementary determinant domain 1 (CDR1) containing the amino acid sequence RASQSVGSNLA (SEQ ID NO: 13), light chain CDR2 containing the amino acid sequence GASTGAT (SEQ ID NO: 14), and light chain CDR3 containing the amino acid sequence QQYYSFLAKT (SEQ ID NO: 15).

[0112] In some embodiments, antiserum albumin Fab comprises

[0113] (a) The light chain complementarity-determining domain 1 (CDR1) containing the amino acid sequence RASQSISRYLN (SEQ ID NO: 31), the light chain CDR2 containing the amino acid sequence GASRLES (SEQ ID NO: 32), and the light chain CDR3 containing the amino acid sequence QQSDSVPVT (SEQ ID NO: 33), and

[0114] The heavy chain complementary determinant domain 1 (CDR1) containing the amino acid sequence SYGIS (SEQ ID NO: 45), the heavy chain CDR2 containing the amino acid sequence WINT YSGGTKYAQKF QG (SEQ ID NO: 46), and the heavy chain CDR3 containing the amino acid sequence LGHCQRGICSDALDT (SEQ ID NO: 47). (b) Light chain complementarity-determining domain 1 (CDR1) containing the amino acid sequence RASQSISSYLN (SEQ ID NO: 34), light chain CDR2 containing the amino acid sequence AASSLQS (SEQ ID NO: 35), and light chain CDR3 containing the amino acid sequence QQSYSTPPYT (SEQ ID NO: 36), and The heavy chain complementary determinant domain 1 (CDR1) containing the amino acid sequence SYGIS (SEQ ID NO: 45), the heavy chain CDR2 containing the amino acid sequence RINTYNGNTGYAQRLQG (SEQ ID NO: 48), and the heavy chain CDR3 containing the amino acid sequence LGHCQRGICSDALDT (SEQ ID NO: 47). (c) The light chain complementarity-determining domain 1 (CDR1) containing the amino acid sequence RASQSIFNYVA (SEQ ID NO: 37), the light chain CDR2 containing the amino acid sequence DASNRAT (SEQ ID NO: 38), and the light chain CDR3 containing the amino acid sequence QQRSKWPPTWT (SEQ ID NO: 39), and The heavy chain complementary determinant domain 1 (CDR1) containing the amino acid sequence NYGIH (SEQ ID NO: 49), the heavy chain CDR2 containing the amino acid sequence SISYDGSNKYYADSVKG (SEQ ID NO: 50), and the heavy chain CDR3 containing the amino acid sequence DVHYYGSGSYYNAFDI (SEQ ID NO: 51); (d) The light chain complementarity-determining domain 1 (CDR1) containing the amino acid sequence RASETVSSRQLA (SEQ ID NO: 40), the light chain CDR2 containing the amino acid sequence GASSRAT (SEQ ID NO: 41), and the light chain CDR3 containing the amino acid sequence QQYGSSPRT (SEQ ID NO: 42), and The heavy chain complementary determinant domain 1 (CDR1) containing the amino acid sequence SYAMS (SEQ ID NO: 52), the heavy chain CDR2 containing the amino acid sequence VISHDGGFQYYADSVKG (SEQ ID NO: 53), and the heavy chain CDR3 containing the amino acid sequence AGWLRQYGMDV (SEQ ID NO: 54). (e) The light chain complementarity-determining domain 1 (CDR1) containing the amino acid sequence RASQSVSSSSLA (SEQ ID NO: 43), the light chain CDR2 containing the amino acid sequence GASSRAT (SEQ ID NO: 41), and the light chain CDR3 containing the amino acid sequence QKYSSYPLT (SEQ ID NO: 44), and Heavy chain complementarity-determining domain 1 (CDR1) containing the amino acid sequence AYWIA (SEQ ID NO: 55), heavy chain CDR2 containing the amino acid sequence MIWPPDADARYSPSFQG (SEQ ID NO: 56), and heavy chain CDR3 containing the amino acid sequence LYSGSYSP (SEQ ID NO: 57); or (f) The light chain complementarity-determining domain 1 (CDR1) containing the amino acid sequence RASQSVGSNLA (SEQ ID NO: 13), the light chain CDR2 containing the amino acid sequence GASTGAT (SEQ ID NO: 14), and the light chain CDR3 containing the amino acid sequence QQYYSFLAKT (SEQ ID NO: 15), and The heavy chain contains complementary determinant domain 1 (CDR1) of the amino acid sequence AYSMN (SEQ ID NO: 16), the heavy chain contains CDR2 of the amino acid sequence SSISSSGRYIHYADSVKG (SEQ ID NO: 17), and the heavy chain contains CDR3 of the amino acid sequence ETVMAGKALDY (SEQ ID NO: 18).

[0115] In some embodiments, the antiserum albumin Fab comprises a heavy chain variable domain containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 63, 64, 65, 66, 67, 68, or 19. In some embodiments, the antiserum albumin Fab comprises a light chain variable domain containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 58, 59, 60, 61, 62, or 20.

[0116] In some embodiments, the antiserum albumin Fab comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 63, 64, 65, 66, 67, 68, or 19, and the light chain variable domain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 58, 59, 60, 61, 62, or 20.

[0117] Structure of fusion proteins

[0118] In some embodiments, the fusion protein comprises a heavy chain having an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 1. In some embodiments, the fusion protein comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 29. In some embodiments, the fusion protein comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 1. In some embodiments, the fusion protein comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 29. In some embodiments, the fusion protein comprises a heavy chain consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 29. In some embodiments, the fusion protein comprises a heavy chain consisting of the amino acid sequence of SEQ ID NO: 1. In some embodiments, the fusion protein comprises a heavy chain consisting of the amino acid sequence of SEQ ID NO: 29.

[0119] In some embodiments, the fusion protein comprises a light chain having an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 2. In some embodiments, the fusion protein comprises a light chain containing the amino acid sequence of SEQ ID NO: 2. In some embodiments, the fusion protein comprises a light chain consisting of the amino acid sequence of SEQ ID NO: 2.

[0120] In some embodiments, the fusion protein comprises a heavy chain and a light chain, the heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 1, and the light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 2. In some embodiments, the fusion protein comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 29 and a light chain containing the amino acid sequence of SEQ ID NO: 2.

[0121] In some embodiments, the fusion protein comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 1 and a light chain containing the amino acid sequence of SEQ ID NO: 2. In some embodiments, the fusion protein comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 29 and a light chain containing the amino acid sequence of SEQ ID NO: 2. In some embodiments, the fusion protein comprises a heavy chain consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 29 and a light chain consisting of the amino acid sequence of SEQ ID NO: 2. In some embodiments, the fusion protein comprises a heavy chain consisting of the amino acid sequence of SEQ ID NO: 1 and a light chain consisting of the amino acid sequence of SEQ ID NO: 2. In some embodiments, the fusion protein comprises a heavy chain consisting of the amino acid sequence of SEQ ID NO: 29 and a light chain consisting of the amino acid sequence of SEQ ID NO: 2.

[0122] As used herein, the terms “variable region” and “variable domain” are used interchangeably and are common in the art. A variable region typically refers to a portion of an antibody, usually a portion of the light or heavy chain, typically about 110 to 120 amino acids from the amino terminus in the mature heavy chain and about 90 to 115 amino acids in the mature light chain. These vary considerably in sequence between antibodies and are responsible for the binding and specificity of a particular antibody to its specific antigen. Sequence variability is concentrated in those regions called complementarity-determining regions (CDRs), while more highly conserved regions within a variable domain are called framework regions (FRs). Without wishing to be bound to any particular mechanism or theory, it is believed that the CDRs of both the light and heavy chains are primarily responsible for antibody-antigen interactions and specificity.

[0123] The terms "VL" and "VL domain" are used interchangeably and refer to the variable region of the antibody's light chain. The terms "VH" and "VH domain" are used interchangeably and refer to the variable region of the antibody's heavy chain.

[0124] The term "Kabat numbering" and similar terms are recognized in the art and refer to a system for numbering amino acid residues in the variable regions of the heavy and light chains of an antibody or its antigen-binding moiety. In some respects, the CDR of an antibody can be determined according to the Kabat numbering system (see, for example, Kabat EA and Wu TT (1971) Ann NY Acad Sci [Annals of the New York Academy of Sciences] 190: 382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, 5th Edition, US Department of Health and Human Services, NIH Publication No. 91-3242). In some embodiments, the CDR of the antibody described herein has been determined according to the Kabat numbering scheme.

[0125] In some embodiments, the position of one or more CDRs along the VH (e.g., CDR1, CDR2, or CDR3) and / or VL (e.g., CDR1, CDR2, or CDR3) regions of the antibody described herein may vary by one, two, three, four, five, or six amino acid positions, provided that immunospecific binding with the antigen is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). For example, the position defining the CDR of the antibody described herein can be varied by shifting the N-terminal and / or C-terminal boundary of the CDR relative to the CDR position of the multispecific antibody described herein by one, two, three, four, five, or six amino acids, provided that immunospecific binding with one or more antigens is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In other embodiments, the length of one or more CDRs along the VH (e.g., CDR1, CDR2, or CDR3) and / or VL (e.g., CDR1, CDR2, or CDR3) regions of the antibody described herein may vary (e.g., shorter or longer) by one, two, three, four, five, or more amino acids, provided that immune-specific binding to one or more antigens is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In some embodiments, the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and / or VH CDR3 described herein may be one, two, three, four, five, or more amino acids shorter than one or more of the CDRs described herein, provided that immune-specific binding to one or more antigens is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In other embodiments, the VL CDR1, VL CDR2, VL CDR3, VHCDR1, VH CDR2 and / or VH CDR3 described herein may be one, two, three, four, five or more amino acids longer than one or more of the CDRs described herein, provided that the immune-specific binding to one or more antigens is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%).In other embodiments, the amino terminus of VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and / or VH CDR3 described herein may be extended by one, two, three, four, five, or more amino acids compared to one or more of the CDRs described herein, provided that immunospecific binding to one or more antigens is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In other embodiments, the carboxyl terminus of VL CDR1, VLCDR2, VL CDR3, VH CDR1, VH CDR2, and / or VH CDR3 described herein may be extended by one, two, three, four, five, or more amino acids compared to one or more of the CDRs described herein, provided that immunospecific binding to one or more antigens is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In other embodiments, the amino terminus of VL CDR1, VL CDR2, VL CDR3, VH CDR1, VHCDR2, and / or VH CDR3 described herein may be shortened by one, two, three, four, five, or more amino acids compared to one or more of the CDRs described herein, provided that immunospecific binding to one or more antigens is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In some embodiments, the carboxyl terminus of VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and / or VH CDR3 described herein may be shortened by one, two, three, four, five, or more amino acids compared to one or more of the CDRs described herein, provided that immunospecific binding to one or more antigens is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). Any method known in the art can be used to determine whether immunospecific binding to one or more antigens is maintained.

[0126] The determination of the percentage of identity between two sequences (e.g., amino acid sequences or nucleic acid sequences) can also be accomplished using mathematical algorithms. A specific, non-limiting example of a mathematical algorithm used to compare two sequences is the algorithm of Karlin S and Altschul SF (1990) PNAS [Proceedings of the National Academy of Sciences] 87: 2264-2268, with modifications seen in Karlin S and Altschul SF (1993) PNAS [Proceedings of the National Academy of Sciences] 90: 5873-5877. This algorithm is incorporated into the NBLAST and XBLAST procedures of Altschul SF et al., (1990) J Mol Biol [Journal of Molecular Biology] 215: 403. BLAST nucleotide searches can be performed using the NBLAST nucleotide procedure parameter set (e.g., score = 100, word length = 12) to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed using the XBLAST procedure parameter set (e.g., score 50, word length = 3) to obtain amino acid sequences homologous to the protein molecules described herein. For obtaining vacancy alignments for comparative purposes, vacancy BLAST, as described in Altschul SF et al., (1997) Nuc Acids Res [Nucleic Acid Research] 25: 3389 3402, can be used. Alternatively, PSI BLAST can be used to perform iterative searches that detect long-distance relationships (Id) between molecules. When using BLAST, vacancy BLAST, and PSI BLAST procedures, the default parameters of the corresponding procedures (e.g., XBLAST and NBLAST) can be used (see, for example, the National Center for Biotechnology Information (NCBI) at ncbi.nlm.nih.gov). Another specific non-limiting example of a mathematical algorithm for sequence comparison is the algorithm of Myers and Miller, 1988, CABIOS [Computer Applications in the Biological Sciences] 4:11 17. This algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When comparing amino acid sequences using the ALIGN program, a PAM 120 weighted residue table, a 12-fold vacancy length penalty, and a 4-fold vacancy penalty can be used. The percentage of identity (%) between two sequences, with or without allowed vacancy, can be determined using techniques similar to those described above. When calculating the percentage of identity, only perfect matches are typically counted.

[0127] As used herein, "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having side chains have been defined in the art. These families include amino acids having the following side chains: basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In some embodiments, one or more amino acid residues within one or more CDRs or one or more frame regions of an antibody may be replaced by amino acid residues having similar side chains.

[0128] The fusion proteins disclosed herein can be produced by any method known in the art for synthesizing antibodies, such as by chemical synthesis or by recombinant expression techniques, for example by the method disclosed in WO 2021 / 149015 (which is incorporated herein by reference in its entirety). Suitable methods include molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and conventional techniques in related fields within the scope of this art.

[0129] In some embodiments, the fusion protein is administered as a pharmaceutical formulation (e.g., but not limited to, a liquid pharmaceutical formulation). The pharmaceutical composition may be formulated with pharmaceutically acceptable excipients according to conventional techniques, such as those disclosed in Remington, “The Science and Practice of Pharmacy”, 22nd edition (2012), edited by Allen, Loyd V., Jr. Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the doses and concentrations employed. The fusion protein may be administered via routes of administration known to a practicing physician, such as percutaneous, subcutaneous, intravenous, or intramuscular administration. In one embodiment, the fusion protein is administered via an intravenous route.

[0130] In some embodiments, the fusion protein is administered to the subject at a dose of about 0.01 to about 100 mg / kg of the subject's body weight. Typical daily doses may range from about 0.01 mg / kg to about 100 mg / kg or more, depending on several factors, such as the specific mammal being treated, the individual subject's clinical condition, the site of delivery of the drug, the method of administration, the schedule of administration, and other factors known to the medical practitioner.

[0131] Treatment

[0132] This document discloses methods for treating Graves' disease and / or thyroid eye disease (TED) in subjects in need. In some embodiments, this disclosure relates to methods for treating TED in subjects in need. TED may also be referred to as thyroid-associated eye disease (TAO) or Graves' eye disease (GO). In some embodiments, this disclosure relates to methods for treating Graves' disease in subjects in need.

[0133] In some embodiments, the method disclosed herein involves administering to a subject in need a fusion protein comprising a structure according to formula (I): Formula (I), R1 and R2 are each an anti-CD40L single-chain variable fragment (scFv) linked to the N-terminus of anti-serum albumin Fab, and Each of R1 and R2 is connected to either the heavy chain variable domain or the light chain variable domain of the antiserum albumin Fab.

[0134] In some embodiments, the method disclosed herein involves administering a fusion protein having formula (I) to a subject in need: Formula (I), R1 and R2 are each an anti-CD40L single-chain variable fragment (scFv) linked to the N-terminus of anti-serum albumin Fab, and Each of R1 and R2 is connected to either the heavy chain variable domain or the light chain variable domain of the antiserum albumin Fab.

[0135] In some embodiments, this disclosure relates to fusion proteins comprising a structure according to formula (I): Formula (I), R1 and R2 are each an anti-CD40L single-chain variable fragment (scFv) linked to the N-terminus of anti-serum albumin Fab, and Each of R1 and R2 is connected to either the heavy chain variable domain or the light chain variable domain of the antiserum albumin Fab. This fusion protein is intended for use in the treatment of TED.

[0136] In some embodiments, this disclosure relates to a fusion protein having formula (I): Formula (I), R1 and R2 are each an anti-CD40L single-chain variable fragment (scFv) linked to the N-terminus of anti-serum albumin Fab, and Each of R1 and R2 is connected to either the heavy chain variable domain or the light chain variable domain of the antiserum albumin Fab. This fusion protein is intended for use in the treatment of TED.

[0137] In some embodiments, this disclosure relates to the use of a fusion protein comprising the structure according to formula (I) for the manufacture of a medicament for the treatment of TED: Formula (I), R1 and R2 are each an anti-CD40L single-chain variable fragment (scFv) linked to the N-terminus of anti-serum albumin Fab, and Each of R1 and R2 is connected to either the heavy chain variable domain or the light chain variable domain of the antiserum albumin Fab.

[0138] In some embodiments, this disclosure relates to the use of a fusion protein having formula (I) in the manufacture of a medicament for the treatment of TED: Formula (I), R1 and R2 are each an anti-CD40L single-chain variable fragment (scFv) linked to the N-terminus of anti-serum albumin Fab, and Each of R1 and R2 is connected to either the heavy chain variable domain or the light chain variable domain of the antiserum albumin Fab.

[0139] In some embodiments, the subject has TED and Graves' disease. In some embodiments, the subject has TED and Hashimoto's thyroiditis. In some embodiments, the subject with TED is diagnosed with Graves' disease. In some embodiments, the subject with TED is diagnosed with Hashimoto's thyroiditis. In some embodiments, the methods disclosed herein relate to treating TED patients with comorbid Graves' disease disorder. In some embodiments, the methods disclosed herein relate to treating TED patients with comorbid Hashimoto's thyroiditis disorder.

[0140] In some embodiments, the subject has acute or chronic TED. In some embodiments, the subject has acute TED. A subject with acute TED is defined as a patient in whom disease progression evolves with increasing symptoms and severity, and has not yet reached a plateau according to the Rundle's curve of TED disease progression, such as, for example, a patient who had an onset of ophthalmic symptoms less than 6 months ago, at least 24 months ago, or less than 12 months ago. In some embodiments, acute TED refers to an onset of ophthalmic symptoms 0 to 18 months ago, 6 to 18 months ago, 6 to 15 months ago, 6 to 12 months ago, or 0 to 12 months ago. In some embodiments, the subject has chronic TED. Chronic TED refers to a disease state in which disease progression has reached a plateau according to the Rundle's curve of TED disease progression, and there has been no significant change in severity.

[0141] In some embodiments, the subject has active TED. TED activity can be assessed using methods known to the technician, such as by assessing different inflammatory parameters, such as spontaneous retrobulbar pain, gaze-induced orbital pain, eyelid redness, conjunctival redness, eyelid swelling, inflammation of the caruncle and / or folds, and / or conjunctival edema. The activity measure of TED can be presented as a Clinical Activity Score (CAS) based on the listed inflammatory parameters, where 1 point is assigned for each present parameter. In some embodiments, active TED can be represented by a CAS score equal to or greater than 3 on a 7-point CAS scale (e.g., a CAS score of 3 to 7) (Burch et al. 2022). In some embodiments, the subject has active TED and has a CAS score of 3 or higher, such as a CAS score of 4 or higher, such as a CAS score of 5 or higher, such as a CAS score of 6 or higher, such as a CAS score of 7. In some embodiments, the subject has inactive TED, such as having a CAS score equal to or less than 2 on a 7-point CAS scale.

[0142] In some embodiments, the subject has moderate to severe TED. The severity of TED can be assessed using methods known to the art, such as by assessing different severity indicators, such as palpebral fissure width, eyelid swelling, eyelid redness, conjunctival redness, conjunctival edema, inflammation of the caruncle or folds, proptosis, diplopia, extraocular muscle involvement, corneal involvement, and / or optic nerve involvement (Burch et al. 2022). In some embodiments, the subject has active, moderate to severe TED.

[0143] In some embodiments, the subjects being treated are thyroid function normal or have only mild hypothyroidism or hyperthyroidism, for example, free triiodothyronine (FT3) and free thyroxine (FT4) levels that are no more than + / - 50% from the normal limit.

[0144] This article discloses methods for treating TED, which include administering therapeutically effective amounts of the fusion protein as disclosed herein to subjects in need.

[0145] In some embodiments, treatment leads to the treatment of orbital lesions causing TED. In some embodiments, treatment of orbital lesions causing TED results in a reduction of inflammation in the periorbital space, such as, but not limited to, inactivation of orbital fibroblasts and / or myoblasts, leading to decreased cytokine expression.

[0146] In some embodiments, the treatment results in systemic pathophysiological effects of TED. In some embodiments, the systemic pathophysiological effects of TED result in PBMC inactivation and decreased cytokine expression, thereby leading to reduced infiltration of autoimmune cells in the periorbital space.

[0147] In some embodiments, treatments are used to address the systemic pathophysiology of TED and the orbital disease associated with TED.

[0148] In some embodiments, the treatment results in a reduction in eye protrusion, such as, but not limited to, a reduction in eye protrusion of at least 2 mm. In some embodiments, the treatment results in a reduction in eye protrusion of at least 2 mm, at least 2.5 mm, at least 3 mm, at least 4 mm, at least 5 mm, or any range thereof. In some embodiments, the treatment results in maximal reduction in eye protrusion, wherein the eye's positioning returns to its pre-TED positioning, such as a positioning corresponding to a standard positioning relative to sex and race. In some embodiments, the treatment results in a reduction in eye protrusion within the range of 2 mm to maximum reduction in eye protrusion, such as within the range of 3 mm to maximum reduction in eye protrusion, such as within the range of 4 mm to maximum reduction in eye protrusion, such as within the range of 5 mm to maximum reduction in eye protrusion. In some embodiments, reduction in eye protrusion is achieved after 6, 12, or 24 weeks of treatment. In some embodiments, the treatment results in a reduction in diplopia.

[0149] In some embodiments, the treatment results in a decrease in the CAS score, for example, a decrease of 1, 2, 3, 4, or 5 on a 7-point CAS scale. In some embodiments, the treatment results in a decrease of at least 1 on the 7-point CAS scale, for example, a decrease of at least 2, at least 3, at least 4, or at least 5 on the CAS score. In some embodiments, the treatment results in a reduction in eyelid retraction. In some embodiments, the treatment results in a reduction in the volume of the extraorbital muscles and / or the volume of the extraorbital adipose tissue.

[0150] In some embodiments, treatment results in a reduction of autoantibodies, such as a reduction of thyroid-stimulating hormone receptor autoantibody (TSH-R-Ab), anti-thyroid peroxidase antibody (TPO-Ab), and / or anti-thyroglobulin antibody (Tg-Ab). In some embodiments, treatment results in a reduction of thyroid dysfunction, such as a reduction of free triiodothyronine (FT3), free thyroxine (FT4), and / or thyroid-stimulating hormone (TSH).

[0151] In some embodiments, treatment results in an increase in Graves' ophthalmopathy quality of life (GO-QOL) scores, such as an increase in one or both of the following GO-QOL sub-scores: 1. measuring the consequences of diplopia and decreased visual acuity on visual function, and 2. measuring the psychosocial consequences of altered appearance (Terwee et al. 1998).

[0152] In some embodiments, administration of the fusion protein results in treatment of orbital TED. In some embodiments, administration of the fusion protein results in a reduction of orbital inflammation, such as a reduction in T cell-mediated local inflammation. In some embodiments, administration of the fusion protein results in a reduction of inflammatory responses in myoblasts and / or fibroblasts. In some embodiments, administration of the fusion protein results in a reduction of cytokine expression in the periorbital space, such as a reduction in the expression of TNF-α, IL-4, IL-22, IL-6, IL-8, and / or RANTES in the periorbital space. In some embodiments, administration of the fusion protein results in a reduction of cytokine expression in orbital fibroblasts and / or myoblasts, such as a reduction in the expression of IL-6, IL-8, and / or RANTES in orbital fibroblasts and / or myoblasts. In some embodiments, treatment results in a reduction in the production of hyaluronic acid and / or GAGs in the periorbital space, ultimately leading to a reduction or prevention of fibrosis. In some embodiments, treatment results in a reduction in the differentiation of orbital fibroblasts into mature adipocytes and myofibroblasts, ultimately leading to a reduction or prevention of the production of hyaluronic acid and pro-inflammatory cytokines.

[0153] In some embodiments, administration of the fusion protein results in a therapeutic effect on systemic pathophysiology. In some embodiments, administration of the fusion protein leads to reduced activation of PBMCs (e.g., B cells, monocytes, dendritic cells, and fibroblasts). In some embodiments, the treatment results in a reduced inflammatory response in the subject's PBMCs (e.g., B cells, monocytes, dendritic cells, and fibroblasts), such as a reduced inflammatory response in fibroblasts. In some embodiments, the treatment results in reduced PBMC activation and / or inflammatory response, such as decreased expression of IL-6, IL-8, and / or RANTES in PBMCs. In some embodiments, the treatment results in decreased CD40 expression in PBMCs. In one embodiment, the treatment results in decreased expression of CXCL9, CXCL10, and / or CXCL11 in dendritic cells. In one embodiment, the treatment results in downregulation of inflammation-related genes, such as downregulation of CCL2, ACHE, and / or FCER2 genes in PBMCs. The reduction in PBMC activation and inflammatory response can further lead to reduced infiltration of these activated PBMCs and / or cytokines derived from them into the orbital cavity, ultimately resulting in reduced activation of orbital fibroblasts and a reduction in resulting local inflammation. In some embodiments, administration of the fusion protein results in a reduction and / or prevention of autoimmune cell infiltration in the periorbital space. In some embodiments, administration of the fusion protein results in a reduction of anti-TSHR autoantibodies and / or anti-IGF1R autoantibodies.

[0154] In some embodiments, administration of the fusion protein results in treatment of systemic pathophysiological aspects of TED and orbital diseases, such as treatment of autoimmune components and treatment of inflammation in the periorbital tissues of TED. In some embodiments, administration of the fusion protein results in treatment of underlying TED-related autoimmune disorders in the subject, such as treatment of underlying Graves' disease or treatment of underlying Hashimoto's thyroiditis associated with TED.

[0155] Without being bound by theory, the systemic pathophysiology of TED and the treatment of orbital disease lead to improvements in the symptomatology and progression of TED, resulting in effective treatment with a low relapse rate. In some embodiments, the treatment results in no disease relapse in the treated subject after the last dose, for example, no relapse 28 weeks after the last dose.

[0156] In some embodiments, the treatment results in a reduction in eye protrusion compared to baseline protrusion before treatment, for example, but not limited to a reduction in eye protrusion of at least 2 mm. As used herein, the term eye protrusion (also known as bulging of the eyeball) refers to the protrusion of the eyeball. Eye protrusion can be evaluated using various methods known to those skilled in the art, such as measurements using a Hertel exophthalmometer or measurements using magnetic resonance imaging (MRI).

[0157] In some embodiments, treatment results in a reduction of diplopia. In some embodiments, treatment results in both eye protrusion and a reduction of diplopia. As used herein, the term diplopia refers to the double vision that results in a subject seeing two images due to misalignment of the eyes. Diplopia can be assessed by methods known to a technician, such as the Bahn-Gorman scale (grades 0-IV) or the Gorman score (grades 0-III). A reduction in diplopia as referred to herein may be a decrease of 1 or greater in the Bahn-Gorman scale or Gorman score, for example, a decrease of 1 or 2. In one embodiment, a reduction in diplopia results in diplopia of grade 0 (no diplopia) to grade II (non-constant diplopia) according to the Bahn-Gorman scale or Gorman score, such as grade II (non-constant diplopia) or lower according to the Bahn-Gorman scale or Gorman score, such as grade I (intermittent diplopia) or lower according to the Bahn-Gorman scale or Gorman score, such as grade 0 (no diplopia) according to the Bahn-Gorman scale or Gorman score.

[0158] In some embodiments, this disclosure relates to methods of treating Graves' disease, including administering a therapeutically effective amount of the fusion protein as disclosed herein to a subject in need. In some embodiments, this disclosure relates to the use of the fusion protein as disclosed herein in the treatment of Graves' disease. In some embodiments, this disclosure relates to the use of the fusion protein as disclosed herein in the manufacture of a medicament for the treatment of Graves' disease.

[0159] As used herein, the term "therapeuticly effective amount" of a compound means an amount sufficient to alleviate, block, partially block, eliminate, improve, or delay the clinical manifestations of a given disease and its complications in a therapeutic intervention, including the administration of the compound. An amount sufficient to achieve the above is defined as a "therapeuticly effective amount." The effective amount for each purpose will depend on the severity of the disease or lesion, as well as the subject's weight and general condition.

[0160] As used herein, the terms “treatment” or “treating” are intended to refer to the management and care of a subject for the purpose of alleviating, blocking, partially blocking, removing, reducing, lowering, improving, or delaying the progression of clinical manifestations of a disease or preventing such clinical manifestations, compared to a subject who has not received the treatment disclosed herein. Subjects to be treated may be mammals, particularly humans or non-human mammals.

[0161] As used herein, the terms "subject" and "patient" are used interchangeably. A subject can be an animal. In some embodiments, a subject is a mammal, such as a non-primate (e.g., a cow, pig, horse, cat, dog, rat, etc.) or a primate (e.g., a monkey or human), or a human. In some embodiments, a subject is a cynomolgus monkey or marmoset. In some embodiments, such a term refers to a non-human animal (e.g., a non-human animal such as a pig, horse, cow, cat, or dog). In some embodiments, such a term refers to a pet or farm animal. In particular embodiments, such a term refers to a human.

[0162] Fusion protein

[0163] This article discloses a fusion protein comprising a structure according to formula (I): Formula (I), R1 and R2 are each anti-CD40L single-chain variable fragments (scFv) linked to the N-terminus of anti-serum albumin Fab. Each of R1 and R2 is connected to either the heavy chain variable domain or the light chain variable domain of the antiserum albumin Fab, and R1 and R2 each contain the following complementarity-determining domain (CDR) regions: CDR VL1: RASQRVSSSTYSYMH (SEQ ID NO: 3); CDR VL2: YASNLES (SEQ ID NO: 4); CDR VL3: QHSWEIPPT (SEQ ID NO: 5); CDR VH1: SYYMY (SEQ ID NO: 6); CDR VH2: EINPSNGDTNFNEKFKS (SEQ ID NO: 7); and CDRVH3: SDGRNDMDS (SEQ ID NO: 8), and The antiserum albumin Fab contains the following complementarity-determining domains (CDRs): CDR VL1: RASQSVGSNLA (SEQ ID NO: 13); CDR VL2: GASTGAT (SEQ ID NO: 14); CDR VL3: QQYYSFLAKT (SEQ ID NO: 15); CDR VH1: AYSMN (SEQ ID NO: 16); CDR VH2: SISSGRYIHYADSVKG (SEQ ID NO: 17); and CDR VH3 ETVAAGKALDY (SEQ ID NO: 30).

[0164] Numbered Examples

[0165] E1. A method for treating thyroid eye disease (TED), the method comprising administering to a subject in need a therapeutically effective amount of a fusion protein comprising a structure according to formula (I): Formula (I), R1 and R2 are each an anti-CD40L single-chain variable fragment (scFv) linked to the N-terminus of anti-serum albumin Fab, and Each of R1 and R2 is connected to either the heavy chain variable domain or the light chain variable domain of the antiserum albumin Fab.

[0166] E2. The method according to Example E1, wherein R1 is connected to the heavy chain variable domain of the antiserum albumin Fab, and wherein R2 is connected to the light chain variable domain of the antiserum albumin Fab.

[0167] E3. The method according to any one of the foregoing embodiments, wherein each of R1 and R2 is anti-CD40L hu5c8 scFv.

[0168] E4. The method according to any one of the foregoing embodiments, wherein R1 and R2 each comprise a complementarity-determining domain (CDR) region containing the following amino acid sequence: CDR VL1:RASQRVSSSTYSYMH (SEQ ID NO: 3); CDR VL2: YASNLES (SEQ ID NO: 4); CDR VL3: QHSWEIPPT (SEQ ID NO: 5); CDR VH1:SYYMY (SEQ ID NO: 6); CDR VH2: EINPSNGDTNFNEKFKS (SEQ ID NO: 7); and CDR VH3: SDGRNDMDS (SEQ ID NO: 8).

[0169] E5. The method according to any one of the foregoing embodiments, wherein R1 and R2 each comprise a heavy chain variable domain, the heavy chain variable domain comprising an amino acid sequence having at least 80% identity with the amino acid sequence of QVQLVQSGAEVVKPGASVKLSCKASGYIFTSYYMYWVKQAPGQGLEWIGEINPSNGDTNFNEKFKSKATLTVDKSASTAYMELSSLRSEDTAVYYCTRSDGRNDMDSWGQGTLVTVSS (SEQ ID NO:10).

[0170] E6. The method according to any one of the foregoing embodiments, wherein R1 and R2 each comprise a light chain variable domain, the light chain variable domain comprising an amino acid sequence having at least 80% identity with the amino acid sequence of DIVLTQSPATLSVSPGERATISCRASQRVSSSTYSYMHWYQQKPGQPPKLLIKYASNLESGVPARFSGSGSGTDFTLTISSVEPEDFATYYCQHSWEIPPTFGGGTKLEIKR (SEQ ID NO: 9).

[0171] E7. The method according to any one of the foregoing embodiments, wherein R1 and R2 each comprise a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 10, and the light chain variable domain comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 9.

[0172] E8. The method according to any one of the foregoing embodiments, wherein the heavy chain variable domain and the light chain variable domain of R1 and R2 are connected by a (G4S)3 linker comprising the amino acid sequence of SEQ ID NO: 23.

[0173] E9. The method according to any one of the foregoing embodiments, wherein each of R1 and R2 comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 11.

[0174] E10. The method according to any one of the foregoing embodiments, wherein each of R1 and R2 comprises the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12.

[0175] E11. The method according to any one of the foregoing embodiments, wherein each of R1 and R2 comprises the amino acid sequence of SEQ ID NO: 11.

[0176] E12. The method according to any one of the foregoing embodiments, wherein each of R1 and R2 is connected to the antiserum albumin Fab via one or more connectors.

[0177] E13. The method according to Example E12, wherein each connector contains 1 to 20 amino acids.

[0178] E14. The method according to any one of Examples E12 and E13, wherein each adapter comprises an amino acid sequence having at least 90% identity with SEQ ID NO:23 or SEQ ID NO:24.

[0179] E15. The method according to any one of Examples E12 to E14, wherein each adapter comprises the amino acid sequence of SEQ ID NO:23 or SEQ ID NO:24.

[0180] E16. The method according to any one of the foregoing embodiments, wherein the anti-CD40L scFv is linked to the N-terminus of the heavy chain of the anti-serum albumin Fab via a linker having the amino acid sequence of SEQ ID NO: 23.

[0181] E17. The method according to any one of the foregoing embodiments, wherein the anti-CD40L scFv is linked to the N-terminus of the light chain of the anti-serum albumin Fab via a linker having the amino acid sequence of SEQ ID NO: 24.

[0182] E18. The method according to any one of the foregoing embodiments, wherein the first anti-CD40L scFv is connected to the N-terminus of the heavy chain of the anti-serum albumin Fab via a linker having the amino acid sequence of SEQ ID NO: 23, and the second anti-CD40L scFv is connected to the N-terminus of the light chain of the anti-serum albumin Fab via a linker having the amino acid sequence of SEQ ID NO: 24.

[0183] E19. The method according to any one of the foregoing embodiments, wherein the antiserum albumin Fab comprises a complementarity-determining domain (CDR) region containing the following amino acid sequence: CDR VL1:RASQSVGSNLA (SEQ ID NO: 13); CDR VL2: GASTGAT (SEQ ID NO: 14); CDR VL3: QQYYSFLAKT (SEQ ID NO: 15); CDR VH1:AYSMN (SEQ ID NO: 16); CDR VH2: SISSSGRYIHYADSVKG (SEQ ID NO: 17); and CDR VH3:ETVMAGKALDY (SEQ ID NO: 18).

[0184] E20. The method according to any one of the foregoing embodiments, wherein the antiserum albumin Fab comprises a heavy chain variable domain, the heavy chain variable domain comprising an amino acid sequence having at least 80% identity with the amino acid sequence of QVQLVQSGGGPVKPGGSLRLSCAASGFMFRAYSMNWVRQAPGKGLEWVSSISSSGRYIHYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARETVMAGKALDYWGQGTLVTVSS (SEQ ID NO: 19).

[0185] E21. The method according to any one of the foregoing embodiments, wherein the antiserum albumin Fab comprises a light chain variable domain, the light chain variable domain comprising an amino acid sequence having at least 80% identity with the amino acid sequence of DIVLTQSPGTLSLSPGETATLSCRASQSVGSNLAWYQQKPGQAPRLLIYGASTGATGVPARFSGSRSGTDFTLTITSLQPEDFATYYCQQYYSFLAKTFGQGTQLEIKR (SEQ ID NO:20).

[0186] E22. The method according to any one of the foregoing embodiments, wherein the antiserum albumin Fab comprises a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 19, and the light chain variable domain comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 20.

[0187] E23. The method according to any one of the foregoing embodiments, wherein the antiserum albumin Fab comprises a heavy chain domain comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 21 (VH-CH1 domain).

[0188] E24. The method according to any one of the foregoing embodiments, wherein the antiserum albumin Fab comprises a light chain domain comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 22 (VL-CL domain).

[0189] E25. The method according to any one of the foregoing embodiments, wherein the antiserum albumin Fab comprises a heavy chain domain and a light chain domain, the heavy chain domain comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 21 (VH-CH1 domain), and the light chain domain comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 22 (VL-CL domain).

[0190] E26. The method according to any one of the foregoing embodiments, wherein the fusion protein comprises a heavy chain containing an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 1.

[0191] E27. The method according to any one of the foregoing embodiments, wherein the fusion protein comprises a light chain containing an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 2.

[0192] E28. The method according to any one of the foregoing embodiments, wherein the fusion protein comprises a heavy chain and a light chain, the heavy chain comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 1, and the light chain comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 2.

[0193] E29. The method according to any one of the foregoing embodiments, wherein the fusion protein is administered as a pharmaceutical formulation.

[0194] E30. The method according to any one of the foregoing embodiments, wherein the fusion protein is administered via transdermal, subcutaneous, intravenous or intramuscular administration.

[0195] E31. The method according to any one of the foregoing embodiments, wherein the fusion protein is administered at an amount of about 0.01 mg / kg to about 100 mg / kg of the subject's body weight.

[0196] E32. The method according to any one of the foregoing embodiments, wherein the treatment results in the treatment of orbital disease of TED.

[0197] E33. The method according to embodiment E32, wherein the treatment of orbital disease of the TED results in a reduction of inflammation in the periorbital space.

[0198] E34. The method according to any one of the foregoing embodiments, wherein the treatment results in treatment of the systemic pathophysiology of TED.

[0199] E35. The method according to Example E34, wherein the systemic pathophysiological treatment of the TED results in PBMC inactivation and decreased cytokine expression.

[0200] E36. The method according to Example E34, wherein the systemic pathophysiological treatment of the TED results in a reduction of autoimmune cell infiltration in the periorbital space.

[0201] E37. The method according to any one of the foregoing embodiments, wherein the treatment results in treatment of the systemic pathophysiology of TED and treatment of orbital disease of TED.

[0202] E38. The method according to any one of the foregoing embodiments, wherein the treatment results in a reduction of eye protrusion.

[0203] E39. The method according to any one of the foregoing embodiments, wherein the treatment results in a reduction of diplopia.

[0204] Table 1. Sequence

[0205] References

[0206] 1. Bartalena L, Piantanida E, Gallo D, Lai A, Tanda ML. Epidemiology, Natural History, Risk Factors, and Prevention of Graves' Orbitopathy. Front Endocrinol (Lausanne). 2020; 11: 615993.

[0207] 2. Douglas RS, Mester T, Ginter A, Kim DS. Thyrotropin receptor and CD40 mediate interleukin-8 expression in fibrocytes: implications for thyroid-associated ophthalmopathy (an American Ophthalmological Society thesis). Trans Am Ophthalmol Soc. 2014; 112:26-37.

[0208] 3. Hwang CJ, Afifiyan N, Sand D, Naik V, Said J, Pollock SJ, et al. Orbital fibroblasts from patients with thyroid-associated ophthalmopathy overexpress CD40: CD154 hyperinduces IL-6, IL-8, and MCP-1. InvestOphthalmol Vis Sci. 2009; 50(5): 2262-2268.

[0209] 4. Jain AP, Jaru-Ampornpan P, Douglas RS. Thyroid eye disease: Redefining its management - A review. [Thyroid eye disease: Redefining its management - A review] Clin ExpOphthalmol. [Clinical and Experimental Ophthalmology] 2021; 49(2): 203-211.

[0210] 5. Joseph SS, Douglas RS. Thyroid Eye Disease: A Comprehensive Review. [Thyroid Eye Disease: A Comprehensive Review] in: Demirci H, ed. Orbital Inflammatory Diseases and Their Differential Diagnosis. [Orbital Inflammatory Diseases and Their Differential Diagnosis] Essentials in Ophthalmology Epub. [Essentials in Ophthalmology Epub] Springer-Verlag Berlin Heidelberg: Springer; 2015. pp. 73-88.

[0211] 6. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)), Methods, 2001, pp. 402-408.

[0212] 7. Patel A, Yang H, Douglas RS. A New Era in the Treatment of Thyroid Eye Disease. Am J Ophthalmol. 2019;208: 281-288.

[0213] 8. Reilkoff RA, Bucala R, Herzog EL. Fibrocytes: emerging effector cells in chronic inflammation. Nat RevImmunol. 2011; 11(6): 427-435.

[0214] 9. Smith TJ, Tsai CC, Shih MJ, Tsui S, Chen B, Han R, et al. Unique attributes of orbital fibroblasts and global alterations in IGF-1 receptor signaling could explain thyroid-associated ophthalmopathy. [Thyroid. 2008; 18(9): 983-988.]

[0215] 10. Weiler DL. Thyroid eye disease: a review. [Thyroid eye disease: a review] ClinExp Optom. [Clinical and Experimental Optics] 2017; 100(1): 20-25.

[0216] 11. Burch HB, Perros P, Bednarczuk T, Cooper DS, Dolman PJ, Leung AM, et al. Management of thyroid eye disease: a Consensus Statement by the American Thyroid Association and the European Thyroid Association. [European Thyroid Journal] 2022; 11(6).

[0217] 12. Terwee CB, Gerding MN, Dekker FW, Prummel MF, Wiersinga WM. Development of a disease-specific quality of life questionnaire for patients with Graves' ophthalmopathy: the GO-QOL. Br J Ophthalmol. [British Journal of Ophthalmology] 1998; 82(7): 773-779.

[0218] 13. Couch SM. Teprotumumab (Tepezza) for Thyroid Eye Disease. Mo Med. Molecular Medicine, 2022; 119(1), 36-41.

[0219] Example

[0220] Example 1 - Effect of CD40L inhibition on local inflammation in a mouse model of delayed-type hypersensitivity in the dermis

[0221] To understand the effect of CD40L inhibition on reducing T cell-mediated local inflammation, we tested the pharmacodynamic response of m-Ab1 using a mouse dermal delayed-type hypersensitivity (DTH) model. Molecular and cellular assessments of DTH responses are useful methods for evaluating the mechanism of action (MOA) of immunomodulators. Since h-Ab1 does not cross-react with rodent CD40L, mouse-specific m-Ab1 (SEQ ID NO: 25 and 26) was used in this study. m-Ab1 differs from h-Ab1 in that its anti-CD40L scFv is derived from an MR1 antibody and is therefore able to bind to mouse CD40L. Apart from this modification, m-Ab1 shares the same structure as h-Ab1 and shares similar affinity and potency.

[0222] In this study, we characterized the effect of CD40L inhibition on the DTH response induced by keyhole cyanin (KLH). Efficacy was determined by measurements of ear thickness, serum antigen-specific antibody concentrations, and cytokine levels in local immune regions.

[0223] Materials and Methods: Model induction: Female C57BL6 mice weighing 18–20 g (Charles River Laboratories) were used in the study. Animals were provided with Teklad 22 / 5 rodent diet (Envigo, catalog 8640) and water at will, were housed under standard conditions, and allowed to acclimatize for at least one week prior to use in the study.

[0224] KLH (MilliporeSigma, catalog number H7017, batch number 0000188788) was freshly prepared immediately prior to administration in Freund's complete adjuvant (FCA; MilliporeSigma, catalog number F5881, batch number 1003484857) fortified with Mycobacterium tuberculosis H37RA. KLH was administered subcutaneously (sc) at doses of 2 or 10 mg / mL on day 0 (sensitization) and day 7 (challenge), respectively, at a volume equal to 0.1 mL / mouse.

[0225] On day 0 of the study, ear thickness was measured in both ears using calipers, and KLH emulsion was injected subcutaneously. On day 7, ear thickness was measured in both ears using calipers. After caliper measurement, 10 µl of 10 mg / ml KLH emulsion was injected into the auricle of the animal's right ear. Ear thickness was measured using a spring-loaded micrometer caliper (Mitutuyo America Corporation, model 700-118). Baseline measurements of both ears were recorded before challenge on days 0 and 7. Measurements of both ears were taken on days 8 and 9, and the differences from baseline were calculated.

[0226] On day 9 (48 hours post-challenge), mice were euthanized with isoflurane, followed by exsanguination and cervical dislocation. The right ear was collected, and its weight was recorded. An 8-mm biopsy of the right ear was collected, weighed, placed in 10% neutral buffered formalin (NBF), and submitted for histopathological processing. The remaining right ear tissue not included in the 8-mm biopsy was weighed, rapidly frozen in liquid N2, and submitted for in vitro analysis. The left ear was collected, rapidly frozen in liquid N2, and submitted for in vitro analysis.

[0227] Whole blood (500 µL, yielding 3 x 20 µL and 2 x 45 µL of serum) was collected via retroorbital puncture at the time of euthanasia and processed to produce serum. The serum samples were immediately rapidly frozen in liquid N2.

[0228] Test sample and administration: m-Ab1 was prepared once in a mediator (20 mM L-histidine-HCl with 50 mg / mL sucrose) and maintained at 4°C. m-Ab1 was administered once intraperitoneally (ip) on day 7 at a dose of 3.4, 6.8, or 13.6 mg / kg and a volume equal to 5 mL / kg. The mediator was administered to the mediator control group starting from day 0.

[0229] In vitro analysis: Anti-KLH antibody enzyme-linked immunosorbent assaySerum samples were analyzed using an anti-KLH antibody enzyme-linked immunosorbent assay kit (ELISA; Life Diagnostics, catalog number KLHG-1, batch number KLHG1132223) based on the manufacturer’s instructions.

[0230] Luminex assay To measure the levels of pro-inflammatory cytokines at immune sites, supernatants from homogenized ear tissue samples were analyzed using a Luminex multiplex assay (mouse TH17 magnetic bead assay (Millibo Sigma, catalog number MTH17MAG-47K, lot number 3977881)) based on the manufacturer's instructions. Supernatants from homogenized ear tissue samples were also analyzed using a Pierce dicaprylic acid (BCA) protein assay (Thermo Fisher Scientific Inc., catalog number 23225, lot number XI351701) based on the manufacturer's instructions.

[0231] Statistical analysis: Data are presented as mean ± standard error of the mean (SEM). Statistical analysis of the data generated from this study was performed using GraphPad Prism 10 for Windows (GraphPad Software, Inc.). Statistical significance was set at p < 0.05. Only statistically significant observations are included in the results.

[0232] result: Response of m-Ab1 (CD40L inhibitor) dose / right ear thickness ( Figure 1 ): Compared with the mediator-treated control, m-Ab1 administered at 3.4 mg / kg showed no difference in right ear thickness.

[0233] Compared to the mordant-treated control, the percentage change in right ear thickness over time with m-Ab1 administration at 6.8 mg / kg (m-Ab1 day 8: 0.319 ± 0.007 mm; m-Ab1 day 9: 0.329 ± 0.007 mm; compared to mordant day 8: 0.383 ± 0.012 mm; mordant day 9: 0.393 ± 0.017 mm), from day 8 (m-Ab1: 39.41 ± 3.42% compared to mordant: 72.73 ± 5.57%) to day 9 (m-Ab1: 43.84 ± 3.83% compared to mordant: 77.15 ± 7.56%), and the area under the curve (AUC) from day 7 to day 9 (m-Ab1: 0.599 ± 0.001 compared to mordant: 0.691), were also observed. ± 0.021).

[0234] Compared to the mordant-treated control, the percentage change in right ear thickness over time with m-Ab1 administration at 13.6 mg / kg (m-Ab1 day 8: 0.293 ± 0.005 mm; m-Ab1 day 9: 0.305 ± 0.004 mm; compared to mordant day 8: 0.383 ± 0.012 mm; mordant day 9: 0.393 ± 0.017 mm), from day 8 (m-Ab1: 27.12 ± 1.57% compared to mordant: 72.73 ± 5.57%) to day 9 (m-Ab1: 32.64 ± 1.62% compared to mordant: 77.15 ± 7.56%), and the area under the curve (AUC) from day 7 to day 9 (m-Ab1: 0.560) were also considered. ±0.006 compared to the medium: 0.691 ± 0.021).

[0235] Treatment with mAb1 (a CD40L inhibitor) reduced the expression of local pro-inflammatory cytokines. Compared with the carcass-treated control, KLH mice treated with 3.4, 6.8, or 13.6 mg / kg m-Ab1 had significantly reduced tumor necrosis factor α (TNF-α) levels (m-Ab1 3.4 mg / kg: 8.22 ± 1.56 pg / mL, p = 0.028; 6.8 mg / kg: 6.43 ± 1.09 pg / mL, p = 0.005; 13.6 mg / kg: 5.99 ± 1.19 pg / mL, p = 0.003; compared with carcass: 16.10 ± 2.59 pg / mL) (Figure 2A).

[0236] Interleukin-4 (IL-4) levels were also significantly reduced by treatment with 3.4, 6.8, or 13.6 mg / kg m-Ab1 (m-Ab1 3.4 mg / kg: 1.72 ± 0.44 pg / mL, p = 0.088; 6.8 mg / kg: 1.95 ± 0.46 pg / mL, p = 0.112; 13.6 mg / kg: 1.70 ± 0.19 pg / mL, p = 0.086; compared to the mediator: 6.15 ± 2.04 pg / mL), but the reduction was not statistically significant (Figure 2B).

[0237] Similarly, IL-22 levels showed a non-statistically significant decrease after treatment (m-Ab1 3.4 mg / kg: 25.78 ± 5.42 pg / mL, p = 0.140; 6.8 mg / kg: 24.01 ± 6.21 pg / mL, p = 0.078; 13.6 mg / kg: 27.21 ± 5.57 pg / mL, p = 0.116; compared to the mediator: 63.81 ± 20.80 pg / mL) (Figure 2C).

[0238] in conclusion: Subcutaneous antigen sensitization followed by intradermal KLH challenge in the auricle resulted in significant skin inflammation, peaking at 48 h. Administration of the CD40L inhibitor m-Ab1 in mice reduced the inflammatory spectrum. m-Ab1 significantly reduced TNF-α levels. Decreased levels of cytokines IL-4 and IL-22 were also observed. The reduction in local inflammation led to a decrease in the thickness (i.e., swelling) of the challenged ear. These data support the entry of the disclosed fusion protein into the local inflammatory environment and demonstrate that inhibition of CD40L leads to a reduction in the local inflammatory response associated with immune cell infiltration. Since T cell and macrophage infiltration and local periorbital inflammation are characteristic of TED, the results of this study support that administration of the disclosed fusion protein and thereby inhibition of CD40L provides a treatment for orbital disease in TED.

[0239] Example 2 - Effects of CD40L inhibitors on inflammatory responses in PBMCs

[0240] In patients with TED, PBMCs have been described as being activated and playing a systemic role in the pathogenesis of TED. Activated T cells express CD40L, while other cell types (part of the PBMC pool) express CD40 (i.e., B cells, monocytes, dendritic cells, and fibroblasts). When activated, CD40+ PBMCs produce pro-inflammatory cytokines. Both activated cells (e.g., fibroblasts) and cytokines can infiltrate the orbital cavity, thereby activating resident fibroblasts and initiating the local inflammation involved in TED orbital disease.

[0241] The purpose of this example is to demonstrate the efficacy of CD40L inhibitors in suppressing the production of inflammatory cytokines by PMBCs by blocking the CD40 / CD40L interaction.

[0242] Materials and Methods: PBMC isolation and culture: PBMCs were isolated from whole blood obtained from healthy donors. 18 mL of blood was partitioned into a container containing 15 mL of Lymphoprep. TM The PBMCs were placed in 50 mL Leucosep tubes (Greiner) of density gradient medium (StemCell Technologies) and centrifuged at 1900 rpm for 15 min at room temperature with a deceleration rate of 1. The plasma layer was then discarded, and the PBMC fraction was collected in 50 mL Falcon tubes (ThermoFisher Scientific). The PBMCs were washed with 40 mL of DMEM (Gibco) and centrifuged at 1900 rpm for 10 min at 4°C. The supernatant was discarded, and two more washes were performed. The PBMCs were then counted using a Vi-Cell BLU cell counter (Beckman Coulter) at 5 x 10⁻⁶. 6 PBMCs were resuspended at 2.5 x 10⁻⁶ cells / ml. 6 cells / cm 2 The cells were plated in NUNC™ cell culture treated 24-well plates (Thermo Fisher Scientific) in DMEM with 10% FBS.

[0243] PBMC Stimulation and Treatment: After 10 days of culture, the medium was replaced with DMEM containing 1% FBS, and PBMCs were stimulated with 200 ng / ml IFN-γ (R&D Systems) for 72 hours. PBMCs were then treated with 15 nM megaCD40L (Enzo Lifesciences) alone or in combination with 5 nM or 135 nM h-Ab2 (a CD40L inhibitor with the same anti-CD40L scFv sequence as h-Ab1, but with a single-point mutation in anti-serum albumin Fab compared to h-Ab1). The same anti-CD40L scFv and the absence of serum albumin resulted in similar CD40L binding affinity and potency to h-Ab1 and the control antibody Ctr-Ab (which differs from h-Ab1 in having a TNP-specific scFv that does not bind to CD40L, while having the same anti-serum albumin Fab as h-Ab1) for 24 hours. MegaCD40L was premixed with the corresponding antibody at a specified concentration in culture medium for 5 min at room temperature, and then added to the cells. As a control, cells were either untreated or treated with IFN-γ only.

[0244] mRNA quantification via RT-PRC: CD40, IL-6 (Interleukin-6) IL-8 (Interleukin-8) and CCL5 (CC motif chemokine ligand 5, referred to as RANTES mRNA levels were monitored using real-time quantitative PCR (qPCR). After treatment, cells were lysed with 150 µl of Aurum total RNA lysis solution, and RNA was purified according to the manufacturer's instructions (Bio-Rad Laboratories). cDNA was synthesized using the TaqMan™ Reverse Transcription Kit (Thermo Fisher Scientific). Quantitative PCR was performed on a CFX96 thermal cycler (Bio-Rad Laboratories) using TaqMan™ Universal PCR Premix (Thermo Fisher Scientific) and pre-designed qPCR probes (Integrated DNA Technologies). GPI (Glucose-6-phosphate isomerase) was used as a housekeeping gene, and relative expression was calculated using the ΔΔCt method with 15 nM megaCD40L conditions as a reference (Livak et al. 2001).

[0245] Statistical analysis: Data are presented as mean ± standard error (SD) from n = 2–4 replicates of one experiment. For statistical analysis, one-way ANOVA with Bonferroni correction was performed using GraphPad Prism 10 for Windows (GraphPad Software, Inc.). p < 0.05 p < 0.01 p < 0,0001.

[0246] result: PBMC expression after IFN-γ stimulation CD40 .Although CD40 It is barely expressed in unstimulated PBMCs, but after 72 h of IFN-γ stimulation, CD40 Upregulated by 2.5-fold, and after IFN-γ stimulation, when PBMCs were additionally treated with 15 nM megaCD40L for 24 hours, CD40 Doubled further. Any antibody treatment against CD40 There was no significant effect on mRNA levels (Figure 3A).

[0247] PBMCs produce inflammatory cytokines as a response to megaCD40L. IL-6, IL-8 and RANTES Expression was almost absent in unstimulated PMBCs and showed little increase after IFN-γ stimulation; however, after stimulation with 15 nM megaCD40L, IL-6, IL-8 and RANTES The expression of cytokines was significantly increased (Figures 3B-3D).

[0248] Inflammatory cytokine expression can be blocked by treatment with CD40-L inhibitors. Pretreatment with h-Ab2 reduced megaCD40L in a dose-dependent manner. IL-6, IL-8 and RANTES Expression (Figures 3B-3D). When treated with 5 nM or 124 nM h-Ab2 respectively, IL-6 Relative expression reduced megaCD40L stimulation levels by 60% or 95%. Treatment with 5 nM or 124 nM h-Ab2, respectively, resulted in... IL-8 Relative expression reduced megaCD40L stimulation levels by 72% and 83%, respectively. Treatment with 5 nM or 124 nM h-Ab2 resulted in reductions in these levels. RANTESRelative expression reduced megaCD40L stimulation levels by 47% and 71%. This reduction in cytokine expression was not present when using the control antibody, indicating that the dose-dependent effect was mediated by direct blockade of CD40L with h-Ab2.

[0249] in conclusion: This example demonstrates the efficacy of h-Ab2 in reducing inflammatory cytokine production by PBMCs by blocking CD40:CD40L interaction. Activated PBMC cells (e.g., fibroblasts) and their derived cytokines participate in the pathogenesis of TED by activating orbital fibroblasts or orbital myoblasts and contributing to local inflammation; therefore, a positive effect on their inflammatory state is expected to have therapeutic implications for TED patients.

[0250] Example 3 - Effects of CD40L inhibitors on inflammatory responses in fibroblasts

[0251] Activation of orbital fibroblasts is a key feature of the pathogenesis of TED. Activation of orbital fibroblasts occurs when CD40L (expressed on activated T cells infiltrating the retro-orbital tissue in TED) binds to CD40 (a receptor localized on fibroblasts). This interaction triggers a signaling cascade that leads to the expression and secretion of various cytokines, such as IL-6, IL-8, and RANTES, which have pro-inflammatory and chemotherapeutic effects and thus contribute to the localized orbital inflammation observed in TED patients.

[0252] The purpose of this example is to demonstrate the efficacy of h-Ab1 in inhibiting the production of inflammatory cytokines by orbital fibroblasts by blocking the CD40:CD40L interaction.

[0253] Materials and Methods: Fibroblast Culture and Treatment: Human primary fibroblasts (Lonza) were seeded at 40,000 cells / cm² in an FGM™-2 Fibroblast Growth Medium-2 commercial culture system (Lonza). After 2 days of culture, the medium was replaced with DMEM containing 1% FBS, and the fibroblasts were stimulated with 200 ng / ml IFN-γ (R&D Systems) for 72 hours. Then, the fibroblasts were treated with 32 nM human recombinant soluble CD40L (Enzo Life Sciences) alone or in combination with 5 nM or 135 nM h-Ab1 or Ctr-Ab for 24 hours. Human recombinant soluble CD40L was premixed with the corresponding antibody at a specified concentration in the medium for 5 min at room temperature and then added to the cells. As a control, cells were untreated or treated with IFN-γ only.

[0254] mRNA quantification via RT-PRC: Real-time PCR monitoring CD40 , IL-6, IL-8 and RANTES mRNA levels. After treatment, cells were lysed with 100 µL homogenization buffer from the Maxwell® RSC simplyRNA Cell Kit, and RNA was purified using a Maxwell® RSC instrument (Promega) according to the manufacturer's instructions. cDNA was synthesized using the TaqMan™ Reverse Transcription Kit (Thermo Fisher Scientific). Quantitative PCR was performed on a CFX96 thermal cycler (Bio-Rayet Laboratories) using TaqMan™ Universal PCR Premix (Thermo Fisher Scientific) and pre-designed qPCR probes (Integrated DNA Technologies). GPI Used as a housekeeping gene, and relative expression was calculated using the ΔΔCt method with 32 nM human recombinant soluble CD40L conditions as a reference (Livak et al. 2001).

[0255] Statistical analysis: Data are presented as mean ± standard error (SD) from n = 2–4 replicates of one experiment. For statistical analysis, one-way ANOVA with Bonferroni correction was performed using GraphPad Prism 10 for Windows (GraphPad Software, Inc.). p < 0.05 p < 0.01 p < 0,0001.

[0256] result: Fibroblasts express CD40 after IFN-γ stimulation. Although CD40 expression is absent in unstimulated human primary fibroblasts, it is upregulated more than 15-fold after 72 h of IFN-γ stimulation. This increase in CD40 expression is further doubled when fibroblasts are treated with 32 nM recombinant human soluble CD40L for an additional 24 h following IFN-γ stimulation. Any antibody treatment has no effect on... CD40 mRNA levels were not significantly affected unless fibroblasts were treated with 1215 nM h-Ab1. CD40 mRNA levels decreased (Figure 4A).

[0257] Fibroblasts produce inflammatory cytokines in response to soluble CD40L. Treatment with 32 nM recombinant human soluble CD40L increased CD40 expression in fibroblasts. IL-6, IL-8 and RANTES The expression of CD40L, which was absent in control and IFN-γ-treated fibroblasts, indicates a direct effect of CD40L:CD40 interaction (Figs. 4B-4D). Human recombinant soluble CD40L exists in monomeric form, but treatment with it induces an inflammatory response in fibroblasts, likely due to the spontaneous formation of the trimer complex.

[0258] Inflammatory cytokine expression could be blocked by h-Ab1 treatment. When fibroblasts were treated with a combination of CD40L and h-Ab1, cytokine expression decreased in a dose-dependent manner (Figures 4B-4D). When fibroblasts were treated with 135 nM and 1215 nM h-Ab1, respectively, IL-6 expression decreased by 36% and 73%. When fibroblasts were treated with 135 nM or 1215 nM h-Ab1, respectively, IL-8 expression decreased by 46% and 93%. When 5 nM h-Ab1 or 5 nM or 135 nM Ctr-Ab was used, IL-6 and IL-8 This effect was not observed in other studies. When fibroblasts were treated with 5 nM, 135 nM, and 1215 nM h-Ab1, respectively, the relative expression of RANTES mRNA decreased by 30%, 72%, and 95%, respectively, and no decrease was observed when Ctr-Ab was used. In summary, these results demonstrate that the dose-dependent effect is mediated by direct blockade of CD40L with h-Ab1.

[0259] in conclusion: This example demonstrates the efficacy of h-Ab1 in reducing the production of inflammatory cytokines by activated fibroblasts by blocking CD40:CD40L interactions. Activated fibroblasts are a key driver of TED pathogenesis in the orbital cavity and are largely responsible for local inflammation; therefore, a positive effect on their inflammatory state is expected to have significant therapeutic implications for TED patients.

[0260] Example 4 - Effects of h-Ab1 on inflammatory responses in myoblasts

[0261] Resident myoblasts in the orbital cavity express CD40 on their surface and can produce inflammatory cytokines in response to CD40L stimulation. The production of cytokines by myoblasts following CD40:CD40L interaction is associated with TED, in which muscle cells contribute to local inflammation in the orbital cavity.

[0262] The purpose of this example is to demonstrate the efficacy of h-Ab1 in inhibiting the production of inflammatory cytokines by orbital myoblasts by blocking the CD40:CD40L interaction.

[0263] Materials and Methods: Myoblast culture and treatment: Human primary myoblasts (Thermo Fisher Scientific) were seeded at a density of 50,000 cells / cm² in a solution supplemented with 10% FBS (Gibco) and 1 ng / ml. TGF-β1 (R&D Systems) was cultured in DMEM (Gibco). After 2 days of culture, the medium was replaced with DMEM containing 1% FBS, and myoblasts were stimulated with 200 ng / ml IFN-γ (R&D Systems) for 72 hours. Myoblasts were then treated for 24 hours with 32 nM human recombinant soluble CD40L (Enzo Life Sciences) alone or in combination with 5 nM or 135 nM h-Ab1 (a CD40L inhibitor) and the control antibody Ctr-Ab. Human recombinant soluble CD40L and the corresponding antibody were premixed and incubated for 5 minutes before being added to the cells. As a control, one group of myoblasts received no stimulation or treatment at all, while another group received only IFN-γ.

[0264] mRNA quantification via RT-PRC: Real-time PCR monitoring CD40 IL-6, IL-8, and RANTES mRNA levels were measured. Following treatment, cells were lysed with 100 µL homogenization buffer from the Maxwell® RSC Simply RNA Cell Kit, and RNA was purified using a Maxwell® RSC instrument (Promega) according to the manufacturer's instructions (Promega). cDNA was synthesized using the TaqMan™ Reverse Transcription Kit (Thermo Fisher Scientific). Quantitative PCR was performed on a CFX96 thermal cycler (Bio-Rayet Laboratories) using TaqMan™ Universal PCR Premix (Thermo Fisher Scientific) and pre-designed qPCR probes (Integrated DNA Technologies). GPI Used as a housekeeping gene, and relative expression was calculated using the ΔΔCt method with 32 nM human recombinant soluble CD40L conditions as a reference (Livak et al. 2001).

[0265] Statistical analysis: Data are presented as mean ± standard error (SD) from n = 2–4 replicates of one experiment. For statistical analysis, one-way ANOVA with Bonferroni correction was performed using GraphPad Prism 10 for Windows (GraphPad Software, Inc.). p < 0.05 p < 0.01 p < 0.001 and p < 0,0001.

[0266] result: Myoblasts begin to express CD40 after IFN-γ stimulation. Although CD40 It is hardly expressed in unstimulated myoblasts, but after 72 h of IFN-γ stimulation, CD40 Upregulated 13-fold, and after IFN-γ stimulation, when myoblasts were further treated with 32 nM recombinant human soluble CD40L for 24 hours, CD40 Doubled further. Any antibody treatment against CD40 mRNA levels were not significantly affected (Figure 5A).

[0267] Myoblasts produce inflammatory cytokines in response to soluble CD40L. Treatment with 32 nM recombinant human soluble CD40L increased the number of CD40-expressing myoblasts. IL-8 and RANTES The expression of CD40L:CD40 was absent in control and IFN-γ-treated myoblasts alone, indicating a direct effect of CD40L:CD40 interaction (Figs. 5B-5C). However, it was not detected in either stimulated or unstimulated myoblasts. IL-6 The transcripts indicate that human primary myoblasts cannot produce this cytokine under in vitro conditions. Human recombinant soluble CD40L exists in monomeric form, but treatment with it induces an inflammatory response in myoblasts, likely due to the spontaneous formation of the trimer complex.

[0268] Inflammatory cytokine expression can be blocked by treatment with h-Ab1. When myoblasts were treated with a combination of CD40L and h-Ab1, cytokine expression decreased in a dose-dependent manner (Figures 5B-5C). When myoblasts were treated with 5 nM, 135 nM, or 1215 nM h-Ab1, respectively, IL-8 Relative expression decreased by 37%, 85%, and 100%. When myoblasts were treated with 5 nM or 135 nM h-Ab1... RANTES mRNA relative expression decreased by 50%, and when myoblasts were treated with 1215 nM h-Ab1, RANTES The relative expression of mRNA decreased by 80%. No decrease was observed when Ctr-Ab was used. These results demonstrate that the dose-dependent effect is mediated by direct blockade of CD40L with h-Ab1.

[0269] in conclusion: This example demonstrates the efficacy of h-Ab1 in reducing the production of inflammatory cytokines by activated myoblasts by blocking the CD40:CD40L interaction. Activated myoblasts are involved in the pathogenesis of TED in the orbital cavity and contribute to local inflammation; therefore, a positive effect on their inflammatory state is expected to have therapeutic implications for TED patients.

[0270] Example 5 - Dose-dependent effects of CD40L inhibitors on inflammatory responses in fibroblasts

[0271] The purpose of this example is to demonstrate the efficacy of h-Ab1 in inhibiting the production of inflammatory cytokines by fibroblasts by blocking the CD40:CD40L interaction.

[0272] Materials and Methods: Fibroblast Culture and Treatment: Human primary fibroblasts (Lonza) were seeded at 40,000 cells / cm² in an FGM™-2 Fibroblast Growth Medium-2 commercial culture system (Lonza). After 2 days of culture, the medium was replaced with DMEM containing 1% FBS, and the fibroblasts were stimulated with 200 ng / ml IFN-γ (R&D Systems) for 72 hours. Then, the fibroblasts were treated for 24 hours with 32 nM human recombinant soluble CD40L (Enzo Life Sciences) alone or in combination with 0.56 nM to 3645 nM h-Ab1 or Ctr-Ab increased 3-fold. Human recombinant soluble CD40L was premixed with the corresponding antibody at a specified concentration in the medium for 5 min at room temperature and then added to the cells. As a control, cells were either untreated or treated with IFN-γ only.

[0273] mRNA quantification via RT-PRC: Real-time PCR monitoring CD40 IL-6, IL-8, and RANTES mRNA levels were measured. Following treatment, cells were lysed with 100 µL homogenization buffer from the Maxwell® RSC simplyRNA Cell Kit, and RNA was purified using a Maxwell® RSC instrument (Promega) according to the manufacturer's instructions (Promega). cDNA was synthesized using the TaqMan™ Reverse Transcription Kit (Thermo Fisher Scientific). Quantitative PCR was performed on a CFX96 thermal cycler (Bio-Ray Laboratories) using TaqMan™ Universal PCR Premix (Thermo Fisher Scientific) and pre-designed qPCR probes (Integrated DNA Technologies). GPIUsed as housekeeping genes, and with 32 nM human recombinant soluble CD40L as a reference, relative expression was calculated using the ΔΔCt method (Livak et al. 2001). Based on the concentration range of h-Ab1 or Ctr-Ab, compared to a fixed concentration of human recombinant soluble CD40L, a four-parameter logistic (4PL) regression curve could be fitted, and the IC50 (half-maximum inhibitory concentration) value for antibody inhibition of cytokine-related gene expression could be determined.

[0274] Protein quantification was performed using MSD assays: Supernatants were collected before and after treatment with CD40L and antibodies, and IL6 and IL8 protein production was quantified using a custom-designed U-PLEX biomarker (hu) according to the manufacturer's instructions (MSD). Relative cytokine production was calculated using 32 nM human recombinant soluble CD40L conditions as a reference (100%). Four-parameter logistic (4PL) regression curves were fitted based on the concentration range of h-Ab1 or Ctr-Ab, compared to a fixed concentration of human recombinant soluble CD40L, to determine the IC50 (half-maximum inhibitory concentration) value for antibody inhibition of cytokine protein production.

[0275] Statistical analysis: Data are presented as mean ± standard error of the mean (SEM) of N = 5 biological replicates, where n = 2 technical replicates / experiment. IC50 values ​​are presented as the geometric mean and 95% confidence interval of the IC50 calculated for each experiment.

[0276] result: Inflammation-related gene expression can be blocked in a dose-dependent manner with h-Ab1 treatment. When fibroblasts are treated with a combination of CD40L and h-Ab1, the expression of inflammation-related genes (…) IL6, IL 8 and RANTES The relative expression of ) was significantly blocked, showing a maximum efficacy of 100% ( Figure 6A-C Cytokine expression decreased in a dose-dependent manner, among which those targeting... IL6 , IL8 and RANTES The IC50 values ​​were 157.1 nM (56.1–439.8 nM), 94.2 nM (30.1–295.1 nM), and 29.6 nM (3.7–238.5 nM), respectively. No decrease was observed when using Ctr-Ab. In summary, these results demonstrate that the dose-dependent effect is mediated by direct blockade of CD40L with h-Ab1.

[0277] Inflammatory cytokine protein production can be blocked in a dose-dependent manner with h-Ab1 treatment. When fibroblasts are treated with a combination of CD40L and h-Ab1, cytokine protein production is significantly blocked. IL6 and IL8 The maximum efficacy is shown as 100%. Figure 7A-B Cytokine expression decreased in a dose-dependent manner, with IC50 values ​​specifically targeting [specific cytokine expression levels]. IL6 The value is 139.9 nM (50.9-383.9 nM) and is targeted at IL8 The value was 106.6 nM (34.1–333.4 nM). No decrease was observed when using Ctr-Ab.

[0278] in conclusion: This example demonstrates the efficacy of h-Ab1 in reducing the production of inflammatory cytokines by activated fibroblasts by binding to recombinant human soluble CD40L and thus blocking CD40:CD40L interaction. It blocks gene expression and protein production in a dose-dependent manner, with a maximum efficacy of 100%. Activated fibroblasts are a key driver of TED pathogenesis in the orbital cavity and are largely responsible for local inflammation; therefore, a positive effect on their inflammatory state is expected to have significant therapeutic implications for TED patients.

[0279] Example 6 - Concentration-dependent effects of CD40L inhibitors on inflammatory responses in myoblasts

[0280] The aim of this study is to determine the efficacy of h-Ab1 in inhibiting the gene expression and secretion of pro-inflammatory cytokines in primary myoblasts by suppressing the interaction between CD40L and its receptor.

[0281] Materials and Methods: Myoblast culture and treatment: Human primary myoblasts (Lonza Biosciences) were cultured at a concentration of 50,000 cells / cm³. 2 Cells were plated at a density in complete SkGM-2 medium (Lonza). After 3 days of culture, the cell culture medium was replaced with one supplemented with 1% FBS (Gibco) and 1 ng / mL. TGF-β1 (R&D Systems) in DMEM (Gibco) was used to prevent cell differentiation into myotubes. Myoblasts were stimulated with 200 ng / ml IFN-γ (R&D Systems) for 72 hours. Cells not treated with IFN-γ served as a control.

[0282] Myoblasts were then treated for 4 hours with 32 nM recombinant human soluble CD40L (Enzo Life Sciences) alone or in the presence of a CD40L inhibitor h-Ab1 (90 pM–10 µM) or a control molecule Ctr-Ab (90 pM–10 µM). The recombinant human soluble CD40L was briefly (approximately 5 minutes) premixed with the test compound and then added to the cells. Cells pretreated with IFN-γ but not treated with CD40L served as an additional control.

[0283] mRNA quantification by RT-PCR: determined by real-time PCR. CD40, IL-6, IL-8 and RANTES mRNA levels. After treatment, cells were lysed with 100 μL homogenization solution (Promega), and RNA was purified using the MaxWell HT SimpleRNA Kit according to the manufacturer's instructions (Promega). Quantitative PCR was performed on a CFX96 thermal cycler (Bio-Ray Laboratories) using PrimeTime One-Step Wide-Range Premix and pre-designed qPCR probes (Integrated DNA Technologies). GPI Used as a housekeeping gene, and relative expression was calculated using the ΔΔCt method with IFN-γ treatment alone as a reference (Livak et al. 2001).

[0284] Protein quantification was performed using the MSD assay: Cell supernatant was collected before and after treatment with soluble CD40L. IL-6 and IL-8 proteins present in the supernatant were quantified using a custom-designed U-Plex biomarker (hu) assay, following the manufacturer's instructions (Meso Scale Discovery).

[0285] Data processing and statistical analysis: All data are presented as mean ± standard error of the mean (SEM) of N = 5 (IL-6, IL-8) or N = 4 (RANTES) independent experiments, with each experiment performed in triplicate wells for each condition. For normalization of cytokine mRNA expression data, expression relative to the 32 nM CD40L treatment condition was set to 100%, while cytokine expression in the control treated with IFN-γ only was set to 0%. Similarly, for normalization of cytokine protein data, cytokines present in the supernatant of cells treated with 32 nM CD40L were set to 100%, while cytokines present in the supernatant of cells treated with IFN-γ only were set to 0%. To compare CD40 mRNA expression, data were normalized for expression in cells treated with IFN-γ for 72 hours and those treated with 32 nM CD40L for 4 hours.

[0286] In GraphPad Prism version 10 (GraphPad Software, Inc.), data is fitted to a four-parameter logic (4PL) equation to generate a regression curve. The regression curve is then used to estimate the IC50 of h-Ab1. 50 (Half-maximal inhibitory concentration) values. Statistical differences between relative CD40 expression levels were determined using one-way ANOVA along with a post-hoc Dennehy test, employing GraphPad Prism version 10 (GraphPad Software, Inc.).

[0287] result: CD40 expression was upregulated in myoblasts following IFN-γ stimulation. CD40 mRNA expression was significantly upregulated (on average 10-fold) in myoblasts treated with IFN-γ for 72 hours compared to untreated control levels (P < 0.0001) (Fig. 8A). No significant changes in CD40 expression were observed after treatment with 32 nM CD40L alone or in the presence of any tested concentration of h-Ab1 or Ctr-Ab (Fig. 8A) (the highest and lowest concentrations of h-Ab1 and Ctr-Ab tested only are shown in the figure).

[0288] Effects of h-Ab1 treatment on gene expression of pro-inflammatory cytokines. Pretreatment of myoblasts with IFN-γ for 72 h followed by treatment with 32 nM soluble CD40L resulted in upregulation of mRNA expression of pro-inflammatory cytokines IL-6 and IL-8, as well as the chemokine RANTES (Fig. 8B-D). h-Ab1 inhibited the upregulation of IL-6 (Fig. 8B), IL-8 (Fig. 8C), and RANTES (Fig. 8D) mRNA in a concentration-dependent manner. pIC of h-Ab1 inhibition of mRNA expression 50 The estimated values ​​are: 6.55 ± 0.11 (mean ± sem N = 5) for IL-6, 6.45 ± 0.37 (N = 5) for IL-8, and 9.14 ± 0.21 (N = 4) for RANTES. This corresponds to the IC50 of h-Ab1. 50 The values ​​were: 280.3 nM (142.0–553.5 nM) for IL-6 inhibition, 353.5 nM (32.4–3855 nM) for IL-8 inhibition, and 0.72 nM (0.15–3.49 nM) for RANTES inhibition. (Geometric mean (95% confidence interval)). No decrease in IL-6, IL-8, or RANTES mRNA expression was observed in the control Ctr-Ab (Figures 8B–D).

[0289] Effect of h-Ab1 treatment on the release of pro-inflammatory cytokines. Pretreatment of myoblasts with IFN-γ for 72 hours followed by treatment with 32 nM soluble CD40L resulted in the release of pro-inflammatory cytokines IL-6 and IL-8. Figure 9A-B The release of both IL-6 and IL-8 could be significantly inhibited by h-Ab1 in a concentration-dependent manner, with a maximum efficacy of 100% inhibition. In the presence of 90 pM, 90 nM, or 10 µM h-Ab1, the release of IL-6 was 88.2 ± 8.3% (mean ± sem, N = 4), 50.5 ± 6.3% (N = 5, p < 0.001), and -3.8 ± 2.3% (N = 4, p < 0.001), respectively, compared to the release in response to 32 nM soluble CD40L alone (Figure 9A). Similarly, in the presence of 90 pM, 90 nM, or 10 µM h-Ab1, compared to responses to 32 nM soluble CD40L alone, IL-8 release was 106.1 ± 9.9% (mean ± sem, N = 4), 46.9 ± 5.2% (N = 5, p < 0.001), and -8.1 ± 1.1% (N = 4, p < 0.001) (Figure 9B). No reduction in IL-6 or IL-8 release was observed in the presence of the control Ctr-Ab. Figure 9A-B ).

[0290] in conclusion: This example demonstrates the efficacy of h-Ab1 in reducing the increased expression and secretion of inflammatory cytokines IL-6 and IL-8, as well as the chemokine RANTES, induced by soluble CD40L in myoblasts. By binding to soluble CD40L, h-Ab1 prevents the interaction between CD40L and its receptor. Activated myoblasts are involved in the pathogenesis of TED in the orbital cavity and contribute to local inflammation. Therefore, a reduction in the inflammatory potential of myoblasts is expected to have a positive therapeutic impact on patients with TED.

[0291] Example 7 - CD40L inhibitors block the innate immune response in activated PBMCs from TED patients.

[0292] In patients with TED, PBMCs have been identified as active contributors to the systemic pathogenesis of the disease. In particular, activated T cells expressing CD40L play a crucial role in immune signaling. Other cell types within the PBMC pool (e.g., B cells, monocytes, and dendritic cells) express CD40 and can be activated by CD40L to release pro-inflammatory cytokines that significantly affect surrounding tissues. Activated cells and their cytokine products can infiltrate the orbital cavity, where they interact with resident fibroblasts to trigger a series of local inflammatory responses, thereby exacerbating the symptoms associated with TED orbital disease. The aim of this example is to demonstrate the efficacy of CD40L inhibitors in reducing the production of inflammatory cytokines by PBMCs.

[0293] Materials and Methods: PBMC Culture: Cryopreserved PBMCs from 3 TED patients and 3 healthy volunteers were obtained from Cureline. The PBMCs were thawed by placing the frozen vials in a 37°C water bath and gently agitating until most of the contents were thawed. The cells were then transferred to tubes containing 10 mL of pre-warmed DMEM with 10% FBS and centrifuged at 300 x G for 5 min. The supernatant was removed, and the PBMCs were cultured at 1 x 10⁻⁶ cells / mL. 6 Resuspend PBMCs at 250,000 cells / ml in DMEM with 10% FBS in Nunc™ cell culture treated 24-well plates.

[0294] PBMC Stimulation and Treatment: Six hours later, PBMCs were stimulated with aCD3 / CD28-coated beads (Thermo Fisher Scientific) at a 1:1 cell-to-bead ratio to induce polyclonal T cell activation. Cells were stimulated for 24 hours with aCD3 / CD28 beads alone or in the presence of 100 nM h-Ab1 (a CD40L inhibitor) or anti-TNP IgG (negative control). As controls, cells were either untreated (initial) or treated with beads only (activated).

[0295] Flow cytometry analysis: PBMCs were harvested, beads were removed by magnetic separation, and cells were washed with PBS. After centrifugation at 2600 RPM for 2 min, 1 μL / million cells of LIVE / DEAD™ near-IR staining agent (except for FMO LD, which contained PBS) was added and incubated at room temperature for 10 min (protected from light). Next, BD Fc Block™ was added to the cells and incubated for 10 min. The samples were then washed with PBS, centrifuged at 2600 RPM for 2 min, and BD OptiBuild™ mouse anti-human antibody (Table 2) was added and incubated at 4°C for 20 min (protected from light). The samples were then washed twice with PBS, and 100 μL of BDCytofix™ fixation buffer 100 was added to the wells, mixed by pipetting, and incubated at room temperature for 20 min. The samples were then washed twice with PBS and analyzed in a NovoCyte Advanteon flow cytometer to assess their immunophenotype.

[0296] Table 2. Antibodies used for PBMC immunophenotyping by flow cytometry.

[0297]

[0298] RNA Extraction: Following treatment, PBMCs were harvested, beads were removed by magnetic separation, and cells were lysed with 100 μL of homogenization buffer from the Maxwell® RSC simply RNA Cell Kit. RNA was purified using a Maxwell® RSC instrument (Promega) according to the manufacturer's instructions (Promega). RNA quality was assessed using an Agilent 2100 Bioanalyzer system (Agilent Technologies). All samples had RIN values ​​greater than 9. RNA sequencing was performed by Eurofins to evaluate transcriptional changes.

[0299] RNA sequencing analysis: Eurofins Genomics provided the RNA sequencing files as FASTQ files. FastQC software was used to assess data quality; all samples passed QC and were therefore included for downstream analysis. The STAR alignment tool was used to perform each step of genome indexing, mapping, and read counting. Reads were mapped to the Homo sapiens reference genome (Ensembl version-101). Differential gene expression analysis was calculated using DESeq2 with default parameters, filtered for Log2 fold changes > ±1 and p-adjustment <= 0.05. Variance-stabilized transformation (VST) was applied to some downstream analyses and read count visualizations using the DESeq2 package. Gene set enrichment analysis was performed using the R package gProfiler2, with genes centrally expressed in the dataset as background. All plots and downstream analyses were performed in R (v4.0.2).

[0300] result: Gene expression analysis revealed immune activation in nascent TED PBMCs compared to healthy donors: RNA-seq analysis revealed distinct transcriptomic profiles among PBMCs, demonstrating significant differences in gene expression based on the naive and activated states of PBMCs. The PCA plot (Figure 10A) illustrates these findings, where the second principal component (PC2) distinguishes between healthy individuals and those with TED. Furthermore, the first principal component (PC1) effectively separates activated PBMCs from naive PBMCs, regardless of their naive origin. This separation highlights unique transcriptomic features associated with cell activation. Examining specific transcriptomic features, naive TED PBMCs exhibited 290 differentially expressed (DE) genes when compared to healthy PBMCs, including several upregulated inflammatory cytokine genes (e.g., IL6, IL1A, IL1B, CXCR3, and CCL20), as well as T cell regulation-related genes (e.g., CD274, PRF1, and ADGRG1) (Figure 10B). Consistently, gene set enrichment analysis (GSEA) revealed that nascent TED PBMCs exhibited enhanced immune system activation and upregulation of various inflammation-related pathways, including a gene ontology pathway named "Overview of proinflammatory and profibrotic mediators," which is associated with TED pathophysiology (Table 3). Table 3 depicts the significant pathways identified by GSEA associated with the DE gene in TED PBMCs. The analysis revealed increased immune system activation and upregulation of various inflammation-related pathways, particularly the gene ontology pathway "Overview of proinflammatory and profibrotic mediators," which is associated with TED pathophysiology.

[0301] Table 3. Top enrichment pathways of genes upregulated in TED-initiated PBMCs compared to healthy-initiated PBMCs

[0302] Incubation of PBMCs with anti-CD3 / CD28 beads resulted in effective T cell activation, leading to cytokine expression. Incubation of PBMCs with anti-CD3 / CD28 beads for 24 h resulted in effective T cell activation in both naïve and TED samples. This activation was demonstrated by increased expression of surface markers such as CD69 and CD25 and CD40L (a characteristic of T effector daughter cells), as shown by FACS analysis. Additionally, activation resulted in a significant increase in CD40-expressing B cells in TED samples, a change not observed in healthy PBMCs. Figure 11A-E Transcriptional analysis confirmed widespread cellular activation following anti-CD3 / CD28 stimulation (increased expression levels of IFNG, TNF, IL2, IL4, IL6, IL9, etc. were observed). Figure 12 ).

[0303] Treatment with CD40L inhibitors blocks the innate immune response in activated PBMCs from TED patients.

[0304] Although, as expected, the transcriptional upregulation of CD40L following aCD3 / CD28 activation was not blocked after treatment with the CD40L inhibitor (h-Ab1) (data not shown), CD40 expression was significantly downregulated. Figure 13A This potentially alters the interaction between T cells and antigen-presenting cells. According to the study, treatment with a CD40L inhibitor resulted in a significant reduction in the release of cytokines from dendritic cells. Specifically, the levels of chemokines CXCL9, CXCL10, and CXCL11 were significantly decreased. Figure 13B This reduction suggests a potential therapeutic effect in preventing the migration of activated T cells into the orbital cavity, a migration particularly relevant to the pathophysiology of TED, as infiltrating activated T cells are associated with characteristic symptoms such as swelling, proptosis, and extraocular muscle involvement. Notably, the expression of cytokines released by T cells (e.g., IL9 and IL2) was not affected by CD40L inhibitor treatment (data not shown), indicating that while CD40L inhibitors effectively modulate dendritic cell activity and cytokine release, they do not affect the intrinsic cytokine production capacity of activated T cells. Treatment with anti-TNP IgG (negative control) did not alter any gene expression patterns.

[0305] DE gene analysis showed that CD40L inhibitor treatment also significantly downregulated other related inflammation-related genes, including CCL2 (a cytokine involved in transporting activated T cells to inflammatory sites), ACHE (a gene associated with B cell growth, differentiation, and IgE regulation), and FCER2 (which regulates IgE production, B cell differentiation, and antigen uptake). Figure 13C To explore pathways associated with these DE genes, GSEA showed that treatment of activated TED PBMCs with CD40L inhibitors led to downregulation of immune system activation and inflammatory pathways (Table 4). GSEA revealed significant downregulation of pathways that play key roles in immune responses and inflammation, such as T cell regulation and complement system pathways.

[0306] Table 4. Top enrichment pathways of downregulated genes in TED PBMCs activated after CD40L inhibitor treatment.

[0307]

[0308] in conclusion : These findings collectively highlight the aberrant immune phenotype of TED PBMCs and reveal the potential of CD40L inhibition to modulate the immune response by selectively downregulating CD40 expression and reducing the release of pro-inflammatory cytokines from dendritic cells, thereby potentially limiting the pathological transport of T cells into the orbital cavity.

[0309] It should be understood that the embodiments described herein are to be considered in a descriptive sense only and not for limiting purposes. The description of features or aspects within each embodiment should generally be considered applicable to other similar features or aspects in other embodiments. While one or more embodiments have been described with reference to the accompanying drawings, those skilled in the art will understand that various changes in form and detail may be made therein without departing from the spirit and scope of this disclosure as defined by the following claims. All the various aspects, embodiments, and options described herein can be combined in any and all variations.

[0310] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the same extent that each individual publication, patent or patent application is expressly and individually indicated to be incorporated herein by reference.

Claims

1. A fusion protein comprising the structure according to formula (I): Formula (I), R1 and R2 are each an anti-CD40L single-chain variable fragment (scFv) linked to the N-terminus of anti-serum albumin Fab, and Each of R1 and R2 is linked to either the heavy chain variable domain or the light chain variable domain of the antiserum albumin Fab, and this fusion protein is used to treat thyroid eye disease (TED).

2. The fusion protein for the use of claim 1, wherein the fusion protein comprises a structure according to formula (I).

3. A fusion protein for use according to any one of the preceding claims, wherein R1 is linked to the heavy chain variable domain of the antiserum albumin Fab, and wherein R2 is linked to the light chain variable domain of the antiserum albumin Fab.

4. The fusion protein for use according to any one of the preceding claims, wherein each of R1 and R2 is an anti-CD40L hu5c8 scFv.

5. A fusion protein for the use according to any one of the preceding claims, wherein R1 and R2 each comprise a complementarity-determining domain (CDR) region containing the following amino acid sequence: CDR VL1:RASQRVSSSTYSYMH (SEQ ID NO: 3); CDR VL2: YASNLES (SEQ ID NO: 4); CDR VL3: QHSWEIPPT (SEQ ID NO: 5); CDR VH1:SYYMY (SEQ ID NO: 6); CDR VH2: EINPSNGDTNFNEKFKS (SEQ ID NO: 7); and CDR VH3: SDGRNDMDS (SEQ ID NO: 8).

6. A fusion protein for use according to any one of the preceding claims, wherein R1 and R2 each comprise a heavy chain variable domain comprising an amino acid sequence having at least 80% identity with the amino acid sequence of QVQLVQSGAEVVKPGASVKLSCKASGYIFTSYYMYWVKQAPGQGLEWIGEINPSNGDTNFNEKFKSKATLTVDKSASTAYMELSSLRSEDTAVYYCTRSDGRNDMDSWGQGTLVTVSS (SEQ ID NO: 10).

7. A fusion protein for use according to any one of the preceding claims, wherein R1 and R2 each comprise a light chain variable domain comprising an amino acid sequence having at least 80% identity with the amino acid sequence of DIVLTQSPATLSVSPGERATISCRASQRVSSSTYSYMHWYQQKPGQPPKLLIKYASNLESGVPARFSGSGSGTDFTLTISSVEPEDFATYYCQHSWEIPPTFGGGTKLEIKR (SEQ ID NO: 9).

8. A fusion protein for use according to any one of the preceding claims, wherein R1 and R2 each comprise a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 10, and the light chain variable domain comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO:

9.

9. A fusion protein for use according to any one of the preceding claims, wherein the heavy chain variable domain and the light chain variable domain of R1 and R2 are linked by a (G4S)3 linker comprising the amino acid sequence of SEQ ID NO:

23.

10. A fusion protein for use according to any one of the preceding claims, wherein each of R1 and R2 comprises an amino acid sequence having at least 80% identity with SEQ ID NO:

11.

11. A fusion protein for use according to any one of the preceding claims, wherein each of R1 and R2 comprises the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO:

12.

12. The fusion protein for use according to any one of the preceding claims, wherein each of R1 and R2 comprises the amino acid sequence of SEQ ID NO:

11.

13. A fusion protein for use according to any one of the preceding claims, wherein each of R1 and R2 is linked to the antiserum albumin Fab via one or more adapters.

14. The fusion protein for the use of claim 13, wherein each linker comprises 1 to 20 amino acids.

15. The fusion protein for use according to any one of claims 13 and 14, wherein each linker comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 23 or SEQ ID NO:

24.

16. The fusion protein for use according to any one of claims 13 to 15, wherein each linker comprises the amino acid sequence of SEQ ID NO: 23 or SEQ ID NO:

24.

17. A fusion protein for use according to any one of the preceding claims, wherein the anti-CD40L scFv is linked to the N-terminus of the heavy chain of the anti-serum albumin Fab via a linker having the amino acid sequence of SEQ ID NO:

23.

18. A fusion protein for use according to any one of the preceding claims, wherein the anti-CD40L scFv is linked to the N-terminus of the light chain of the anti-serum albumin Fab via a linker having the amino acid sequence of SEQ ID NO:

24.

19. A fusion protein for use according to any one of the preceding claims, wherein a first anti-CD40L scFv is linked to the N-terminus of the heavy chain of the anti-serum albumin Fab via a linker having the amino acid sequence of SEQ ID NO: 23, and a second anti-CD40L scFv is linked to the N-terminus of the light chain of the anti-serum albumin Fab via a linker having the amino acid sequence of SEQ ID NO:

24.

20. A fusion protein for the use according to any one of the preceding claims, wherein the antiserum albumin Fab comprises a complementarity-determining domain (CDR) region containing the following amino acid sequence: CDR VL1:RASQSVGSNLA (SEQ ID NO: 13); CDR VL2: GASTGAT (SEQ ID NO: 14); CDR VL3: QQYYSFLAKT (SEQ ID NO: 15); CDR VH1:AYSMN (SEQ ID NO: 16); CDR VH2: SISSSGRYIHYADSVKG (SEQ ID NO: 17); and CDR VH3:ETVMAGKALDY (SEQ ID NO: 18).

21. A fusion protein for use according to any one of the preceding claims, wherein the antiserum albumin Fab comprises a heavy chain variable domain comprising an amino acid sequence having at least 80% identity with the amino acid sequence of QVQLVQSGGGPVKPGGSLRLSCAASGFMFRAYSMNWVRQAPGKGLEWVSSISSSGRYIHYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARETVMAGKALDYWGQGTLVTVSS (SEQ ID NO: 19).

22. The fusion protein for use according to any one of the preceding claims, wherein the antiserum albumin Fab comprises a light chain variable domain comprising an amino acid sequence having at least 80% identity with the amino acid sequence of DIVLTQSPGTLSLSPGETATLSCRASQSVGSNLAWYQQKPGQAPRLLIYGASTGATGVPARFSGSRSGTDFTLTITSLQPEDFATYYCQQYYSFLAKTFGQGTQLEIKR (SEQ ID NO: 20).

23. The fusion protein for the use according to any one of the preceding claims, wherein the antiserum albumin Fab comprises a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 19, and the light chain variable domain comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO:

20.

24. The fusion protein for use according to any one of the preceding claims, wherein the antiserum albumin Fab comprises a heavy chain domain comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 21 (VH-CH1 domain).

25. The fusion protein for use according to any one of the preceding claims, wherein the antiserum albumin Fab comprises a light chain domain comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 22 (VL-CL domain).

26. The fusion protein for the use according to any one of the preceding claims, wherein the antiserum albumin Fab comprises a heavy chain domain and a light chain domain, the heavy chain domain comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 21 (VH-CH1 domain), and the light chain domain comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 22 (VL-CL domain).

27. A fusion protein for use according to any one of the preceding claims, wherein the fusion protein comprises a heavy chain comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO:

1.

28. A fusion protein for use according to any one of the preceding claims, wherein the fusion protein comprises a light chain comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO:

2.

29. A fusion protein for use according to any one of the preceding claims, wherein the fusion protein comprises a heavy chain and a light chain, the heavy chain comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 1, and the light chain comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO:

2.

30. A fusion protein for use according to any one of the preceding claims, wherein the fusion protein comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 1 and a light chain containing the amino acid sequence of SEQ ID NO:

2.

31. A fusion protein for use according to any one of the preceding claims, wherein the fusion protein is administered as a pharmaceutical formulation.

32. A fusion protein for use according to any one of the preceding claims, wherein the fusion protein is administered via transdermal, subcutaneous, intravenous or intramuscular administration.

33. The fusion protein for use according to any one of the preceding claims, wherein the fusion protein is administered in an amount of about 0.01 mg / kg to about 100 mg / kg of the subject's body weight.

34. A fusion protein for use according to any one of the preceding claims, wherein the treatment results in the treatment of orbital disease of TED.

35. The fusion protein for the use of claim 34, wherein the treatment of orbital disease with this TED results in a reduction of inflammation in the periorbital space.

36. A fusion protein for use according to any one of the preceding claims, wherein the treatment results in a systemic pathophysiological treatment of TED.

37. The fusion protein for the use of claim 36, wherein treatment of the systemic pathophysiology of the TED results in PBMC inactivation and decreased cytokine expression.

38. A fusion protein for use according to any one of the preceding claims, wherein the treatment results in the blocking of the innate immune response in PBMCs.

39. The fusion protein for the use of claim 38, wherein the treatment results in a decrease in CD40 expression in these PBMCs.

40. The fusion protein for use according to claim 38, wherein the treatment results in a decrease in the expression of CXCL9, CXCL10 and / or CXCL11 in dendritic cells.

41. The fusion protein for use according to claim 38, wherein the treatment results in the downregulation of inflammation-related genes in PBMCs, such as CCL2, ACHE, and / or FCER2 genes.

42. The fusion protein for any one of claims 36 to 41, wherein the systemic pathophysiological treatment of the TED results in a reduction of autoimmune cell infiltration in the periorbital space.

43. A fusion protein for use according to any one of the preceding claims, wherein the treatment results in treatment of the systemic pathophysiology of TED and treatment of orbital disease of TED.

44. The fusion protein for use according to any one of the preceding claims, wherein the treatment results in a reduction of eye protrusion.

45. The fusion protein for use according to any one of the preceding claims, wherein the treatment results in a reduction of diplopia.

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