Bispecific binding proteins for alarmins and uses thereof

Abstract

Provided herein are methods for treating allergic diseases, such as moderate to severe asthma and atopic dermatitis, reducing the daily dosage of oral corticosteroid use, inhibiting innate lymphocyte proliferation, and impairing type 1 and type 2 immune responses using specific bispecific binding proteins (bsBps) that can neutralize two different alarmins. Also described herein are exemplary bsBps, their characteristics, and methods for screening for additional therapeutic bsBps. TIFF2025508835000014.tif208103

Description

[Technical field]

[0001] 1. Field of the invention: The present invention relates to molecular biology and allergic diseases, and in particular to the identification and use of bispecific antibodies comprising an alarmin receptor-binding immunoglobulin G antibody (IgG) and an alarmin-binding protein for the treatment of various allergic diseases, such as moderate to severe asthma and atopic dermatitis.

[0002] 2. Priority: This application claims priority to U.S. Provisional Application No. 63 / 313,483, filed February 24, 2022, the entire contents of which are incorporated herein by reference.

[0003] 3. Sequence Listing: This application contains a sequence listing in accordance with WIPO Standard ST.26, the sequence listing is entitled "SBL009PCTSL.xml", was created on Feb. 14, 2023, is 93,089 bytes in size, and is incorporated by reference in its entirety. [Background technology]

[0004] 4. Background of the invention: Alarmins are endogenously constitutively expressed chemotactic and immunostimulatory proteins / peptides that are released as a result of degranulation, cell injury or death, or in response to immune induction.

[0005] In particular, the "alarmins" thymic stromal lymphopoietin (TSLP), interleukin-33 (IL-33) and interleukin-25 (IL-25) are released from barrier tissues (e.g., airway epithelium) in response to allergens. These alarmins serve as upstream elements responsible for initiating the Th2 immune response, where activation of type 2 innate lymphoid cells (ILC2) and Th2 cells results in a cascade of events, such as the release of IL-4, IL-5, IL-13 and IgE, resulting in the manifestation of numerous allergic reactions. Biologics targeting any one of these alarmins and cytokines have produced variable improvements in symptom scores of allergic reactions, such as asthma and atopic dermatitis. Single use of anti-TSLP or anti-IL-33 antibodies in the treatment of asthma has been clinically validated, although lacking complete efficacy, which may be because each therapy targets only some elements of the pathways regulating type 2 inflammation, leaving other elements of the disease pathophysiology untreated. These alarmins should potentially interact with each other and contribute to their respective inflammatory responses. Using a chronic-phase model of helminth infection and type 2 cytokine-driven pulmonary inflammation, it was demonstrated that targeting all three alarmins (TSLP, IL-25 and IL-33) appears to be more effective than blocking any single alarmin alone [Vannella, Kevin M et al. Science Translational Medicine vol.8,337(2016):337ra65. (Non-Patent Document 1)]. Furthermore, disruption of all three mediators in a model of chronic house dust mite-induced allergic pulmonary inflammation resulted in reduced inflammation, mucus production and pulmonary remodeling, suggesting that these alarmins interact with each other and enhance their individual effects in maintaining type 2 pathology. Thus, there is a great need for novel clinical methods to inhibit two or more of these disease-causing alarmins, thereby improving clinical outcomes for allergic diseases, for example reducing steroid use, and maintaining long-term disease remission. The methods provided in the present disclosure fulfill this need and provide associated benefits in attenuating disease progression. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Vannella, Kevin M et al.Science Translational Medicine vol.8,337(2016):337ra65. Summary of the Invention

[0007] 5. Summary of the invention: With the clear need in the art for new methods of treating allergic diseases and disorders in mind, it is an object of the present invention to provide a method for the treatment of allergic diseases and disorders comprising administering to a patient a therapeutically effective amount of the following: -8 K is smaller than M D ii) bind to purified human IL-17RB and TSLP proteins at 10 -8 K is smaller than M D The object of the present invention is to provide a bispecific antibody (bsBp) or antibody-receptor fusion protein against two different alarmins, characterized by one or more of the following: iii) binds to an IL17RB receptor; iv) inhibits the release of Th2-associated cytokines to a greater extent than an anti-IL17RB monoclonal antibody; and iv) inhibits the proliferation and activation of ILC2s to a greater extent than an anti-IL17RB monoclonal antibody.

[0008] As demonstrated herein, such bispecific antibodies (bsBp) or antibody-receptor fusion proteins have the potential to prevent, inhibit or / and slow the progression of allergic diseases, such as asthma and atopic dermatitis (AD). It is therefore an object of the present invention to provide a method of treating an allergy-related disease or disorder in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a bispecific binding protein against two different alarmins X and Y, said bispecific binding protein being composed of (a) an anti-alarmin X receptor IgG and (b) an anti-alarmin Y scFv and (c) a polypeptide linker.

[0009] In a preferred embodiment, the subject is a human suffering from clinical or preclinical asthma, atopic dermatitis, fibrotic disease, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, chronic obstructive pulmonary disease, chronic sinusitis, or chronic sinusitis with nasal polyps.

[0010] In particularly preferred embodiments, the alarmin X receptor is IL-17RB, the alarmin Y receptor is TSLP or IL-33, and the alarmin receptor is ST2 or TSLPR.

[0011] In another preferred embodiment, the anti-Alarmin X IgG is selected from among IgG1, IgG2, IgG3 and IgG4 antibodies.

[0012] Another object of the present invention is to provide a method of treating an allergy-related disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a bispecific binding protein capable of (a) blocking IL-25 and IL-33 signaling, and / or (b) blocking IL-25 and TSLP signaling.

[0013] In a preferred embodiment, the subject, preferably a human, suffers from post-symptomatic or pre-symptomatic asthma, atopic dermatitis, fibrotic disease, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, chronic obstructive pulmonary disease, chronic sinusitis, or chronic sinusitis with nasal polyps.

[0014] In particularly preferred embodiments, the bispecific binding protein is selected from among an anti-IL-17RB / anti-human TSLP bispecific antibody, an anti-IL-17RB / anti-human IL-33 bispecific antibody, an anti-IL-17RB / human ST2 antibody-receptor fusion protein, and an anti-IL-17RB / human TSLPR antibody-receptor fusion protein.

[0015] In another preferred embodiment, the bispecific binding protein comprises a light chain variable region (VL) composed of VL CDR1, VL CDR2 and VL CDR3 having the amino acid sequences of SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, respectively, and a heavy chain variable region (VH) composed of VH CDR1, VH CDR2 and VH CDR3 having the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively.

[0016] In a particularly preferred embodiment, the VL and VH of the anti-IL-17RB antibody have the amino acid sequences of SEQ ID NO:33 and SEQ ID NO:32, respectively, and / or have a light chain sequence of SEQ ID NO:33 and any one of the heavy chain sequences selected from SEQ ID NOs:60 to 75.

[0017] In preferred embodiments, bispecific antibodies (bsBps) and / or antibody-receptor fusion proteins are administered intravenously, intramuscularly, subcutaneously, intracranially, intrathecally, intracerebroventricularly, intraperitoneally, intranasally, parenterally, topically or intradermally, optionally in combination with a second therapeutic agent, examples of which include, but are not limited to, corticosteroids, DNA methyltransferase (DNMT) inhibitors, anti-IL17A antibodies, anti-IL12 / IL23 antibodies, anti-IL23 antibodies, anti-IL17RA antibodies and tyrosine kinase inhibitors.

[0018] These and other objects and features of the present invention will become more fully apparent from the following detailed description taken in conjunction with the accompanying figures and examples. However, it should be understood that both the foregoing summary of the invention and the following detailed description are preferred embodiments and are not intended to limit the present invention or other alternative embodiments of the present invention. In particular, while the present invention has been described herein with reference to certain specific embodiments, it will be recognized that the description is illustrative of the present invention and does not constitute a limitation of the present invention. Various modifications and applications may occur to those skilled in the art without departing from the spirit and scope of the present invention as set forth in the appended claims. [Brief description of the drawings]

[0019] 6. Brief description of the drawings: [Figure 1] 1 shows dose-response curves of SM17 binding to IL-17RB from different species. [Diagram 2] 1 shows the binding specificity of SM17 to members of the IL-17 receptor family. [Diagram 3] SM17 binding to native human Il-17RB is shown. [Figure 4] 1 shows dose-dependent inhibition of IL-5 by SM17 in human PBMC cultures. [Diagram 5] 1 shows that IL-8 production from TK-10 cells was inhibited by SM17. [Figure 6] 1 shows the effect of SM-17 in suppressing airway hyperresponsiveness in OVA-induced experimental asthma in mice. [Figure 7] 1 shows the effect of SM-17 in suppressing BALF IL-5 and IL-13 levels in OVA-induced experimental asthma in mice. [Figure 8] 1 shows the effect of SM-17 in suppressing eosinophil cell count in BALF in OVA-induced experimental asthma in mice. [Figure 9] 1 shows the mode of IL25 / SM17 interaction and possible disease indications. [Figure 10A] 1 shows the design of bispecific binding proteins (bispecific antibodies and antibody-receptor fusion proteins). [Figure 10B] Shows SDS-PAGE of SM17, SM17-anti-TSLP and SM17-anti-IL-33 bsBp purified from the ExpiCHO transient transfection system. [Figure 11] 1 shows antigen binding of anti-alarmin bispecific binding proteins. [Figure 12] 1 shows the inhibitory effect of SM17 on the induction of IFNγ, CCL8, CCL17, IL-5 and IL-13 by alarmins from human PBMCs and on cytokine release. [Figure 13] FIG. 1 shows the potency of different bispecific binding proteins on cytokine and chemotactic factor release from induced human PBMC. [Figure 14] 1 shows the results of the proliferation-promoting effect of alarmin on ILC2. [Figure 15] The results of ILC2 and Th2 cell responses to steroid hormones and bsBp are shown. [Figure 16] 1 shows the results of a dendritic cell potency assay. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] 7. Detailed Description of the Preferred Embodiments: Type II inflammatory diseases involve large amounts of stimulatory alarmins and cytokines upon antigenic stimulation with allergens, and atopic dermatitis (AD) and asthma are representative diseases of this category.

[0021] AD is a chronic inflammatory skin disease characterized by intense pruritus. It affects 15-30% of children and 2-10% of adults with severely impaired quality of life. Immunological factors in the pathogenesis of AD include the impairment of numerous Th2 lymphocytes and the release of related cytokines, such as IL-4, IL-5, and IL-13. These factors lead to high levels of IgE production, which leads to increased skin inflammation and exacerbates the skin barrier defect in AD patients.

[0022] Of particular importance are interleukin-4 (IL-4) and interleukin-13 (IL-13), which are signature cytokines of type II inflammatory responses in AD, induced either by invading parasites or allergens. Both IL-4 and IL-13 receptors share a common receptor chain, namely IL-4Rα; the IL-4Rα / IL-2Rγc (γc) heterodimer constitutes the receptor for IL-4, whereas the IL-4Rα / IL-13Rα1 heterodimer constitutes the receptor for IL-13. Binding of IL-4 or IL-13 to the IL-4Rα receptor chain allows further association of the IL-4 / IL-4Rα complex to the γc receptor chain or the IL-13 / IL-4Rα complex to the IL-13Rα1 receptor chain, respectively. The receptor chain IL-4Rα is widely expressed, albeit at low levels, in some cell types, whereas expression of the γc or IL-13Rα1 receptor chains is cell type restricted. For example, in non-hematopoietic cells, IL-13Rα1 shows rather high expression, whereas γc expression is low or absent [Junttila, Ilkka S et al. The Journal of Experimental Medicine,vol.205,11(2008):2595-608.]. Recent studies have demonstrated the central role of IL-4 in generating and regulating humoral immunity involving IgE for T2 responses in lymph nodes; whereas IL-13 plays a more prominent role in peripheral tissues. Both IL-4 and IL-13 significantly reduce the expression of basic structural proteins such as filaggrin, filaggrin 2, loricrin, involucrin, keratin 1, keratin 10, hornerin, desmoglein and desmocollin 1, as well as lipid compositions important for normal skin barrier function, resulting in an increase in transepidermal water loss (TEWL), which is typically measured to indicate severity and may even be used to predict the onset of AD.Moreover, by suppressing AMP production in keratinocytes, both IL-4 and IL-13 have been reported to be responsible for the development of skin dysbiosis, typically characterized by strong colonization by Staphylococcus aureus; this onset has recently been shown to precede the appearance of AD lesions. Towards this end, two biologics addressing these pathways have been developed and approved for the treatment of moderate to severe AD. Namely, these are dupilumab (an anti-IL-4Ra antibody that inhibits both IL-4 and IL-13 responses) and tralokinumab (an anti-IL-13 antibody). However, approximately 50% of AD patients are unresponsive to these antibodies at one year after treatment. [Bangert, Christine et al. Science Immunology, vol. 6, 55 (2021): eabe2749; Wollenberg. A et al., The British Journal of Dermatology vol. 184, 3 (2021): 437-449].

[0023] Asthma is a chronic inflammatory disorder of the airways characterized by bronchial hyperresponsiveness and variable airflow limitation. Asthma affects more than 300 million people worldwide [Braman, Sidney S. Chest, vol. 130, 1 Suppl (2006): 4S-12S.]. Although the majority of asthma patients achieve disease control with standard controller therapy, approximately 5-10% have severe asthma that remains poorly controlled despite adherence to standard treatment (high-dose inhaled corticosteroids (ICS) + long-acting beta-agonists (LABA)). In severe asthma uncontrolled with standard treatment, the Global Initiative for Asthma (GINA) guidelines recommend the use of oral corticosteroids (OCS) for maintenance therapy. However, OCS-related adverse events, such as those affecting the cardiovascular, gastrointestinal and musculoskeletal systems, as well as infections, are common and may be fatal in some cases. Therefore, patients with severe asthma are characterized as having the most urgent unmet medical need and may be candidates for add-on biologic therapies.

[0024] There are two main categories of asthma: Th2 high and Th2 low. In Th2 high asthma, overproduction of type 2 immune cytokines, such as IL-4, IL-5 and IL-13, can lead to pulmonary eosinophilia, elevated immunoglobulin (Ig)E-levels, increased mucus production and life-threatening problems in breathing [Kuruvilla, Merin E et al., Clinical Reviews in Allergy & Immunology, vol. 56, 2 (2019): 219-233]. Therefore, biologics targeting IgE, IL-4, IL-5 and IL-13 have recently emerged as promising add-on therapies for uncontrolled severe asthma with a Th2 high phenotype.

[0025] IL-4 and IL-13 are also thought to have some non-redundant functions in allergy and asthma. In particular, IL-4 appears to act primarily in the early stages of asthma development through its role in regulating T cell proliferation and survival and in the synthesis of IgE. In contrast, human T cells cannot respond to IL-13 due to the lack of surface expression of IL13Ra1. Unlike IL-4, IL-13 is more primarily involved in later allergic responses, such as airway remodeling by goblet cells and mucus hypersecretion, fibrosis, smooth muscle changes, and enhanced airway hyperresponsiveness [Gour, N., & Wills-Karp, M. (2015). Cytokine, 75(1), 68-78].

[0026] However, clinical outcomes for antibodies targeting only IL-4 (altrakincept, pascolizumab) or only IL-13 (tralokinumab) for the treatment of asthma have been disappointing [Panettieri, Reynold A Jr et al. The Lancet.Respiratory Medicine vol.6,7(2018):511-525;Bagnasco,Diego et al. International Archives of Allergy and Immunology,vol.170,2(2016):122-31.]. However, when targeting both IL-4 and IL-13, as in the case of dupilumab, effective treatment of Th2-mediated asthma has been clinically demonstrated [Maspero,Jorge F et al. The Journal Of Allergy And Clinical Immunology. In practice,vol.8,2(2020):527-539.e9.]. The results suggest that dual blockade of IL-4 and IL-13 is necessary for the treatment of Th2-mediated asthma.

[0027] IL-5 plays a central pathogenic role in eosinophil differentiation, recruitment, survival and degranulation [Pelaia,Corrado et al. Frontiers in Physiology,vol.10 1514.17 Dec.2019]. A significant number of severe asthma patients exhibit a Th2-high phenotype characterized by eosinophilic inflammation. Airway eosinophilia can occur in the majority of asthmatic subjects, and high eosinophil levels are associated with recurrent asthma exacerbations and severe bronchial obstruction. Th2-high asthma with eosinophilia is often therapeutically responsive to corticosteroids, possibly due to the clearance of eosinophils by corticosteroid-induced apoptosis. However, severe eosinophilic asthma may be resistant to both inhaled and systemic corticosteroids due to excessive bronchial IL-5 levels. Excessive bronchial IL-5 levels may overpower the proapoptotic effects of corticosteroids on eosinophils. Thus, blocking IL-5 activity with anti-IL-5 antibodies may help to prolong the therapeutic response to corticosteroid treatment, and indeed three antibodies that block the IL-5 pathway are used in conjunction with corticosteroids to treat Th2 high asthma; e.g., reslizumab (anti-IL-5 antibody), mepolizumab (anti-IL-5 antibody) and benralizumab (anti-IL-5Ra antibody).

[0028] Allergic asthma, a subtype of Th2-high asthma, is characterized by the presence of IgE antibodies against one or more common environmental allergens, such as house dust mites. In patients with allergic asthma, anti-allergen IgE binds to the IgE receptor (FcεRI) on the surface of mast cells. Exposure to allergen antigens can result in cross-linking of FcεRI on the surface of mast cells; if such FcεRI cross-linking is of sufficient strength and duration, mast cells become activated, leading to the release of autacoid mediators histamine, prostaglandin (PG) D2 and leukotriene (LT) C4, ultimately resulting in bronchoconstriction, mucus secretion and mucosal edema. A number of proinflammatory cytokines (e.g., IL-4, IL-5 and IL-13) can also be synthesized and secreted by activated mast cells, which can further regulate both the synthesis of IgE and the development of eosinophilic inflammation. The anti-IgE antibody omalizumab was the first monoclonal antibody for add-on treatment of severe allergic asthma and for a long time was the only monoclonal antibody available. Omalizumab works by selectively blocking the binding of human IgE to its receptor and thus suppressing mast cell activation.

[0029] Although there has been great progress in elucidating the Th2-high inflammatory pathway and developing related biologics to treat Th2-high asthma, there is still no effective approach to address Th2-low asthma. This problem is exacerbated by the fact that Th2-low asthma patients respond poorly to corticosteroids. Therefore, there is an unmet medical need for treatment modalities for the effective treatment of Th2-low asthma.

[0030] Another problem in the standard treatment of asthma is the over-reliance on OCS. Approximately 30% of adult patients with severe asthma rely on OCS therapy in addition to ICS to maintain an acceptable level of asthma control [Chung,Kian Fan et al. The European Respiratory Journal,vol.43,2(2014):343-73.]. The Global Indicators for Asthma Management (GINA) group has committed to establishing an "acceptable" OCS dose for maintenance therapy and has proposed 7.5 mg per day as an acceptable level because it corresponds to a physiological level of steroid production. However, recent studies have shown that no OCS regimen is harmless, as the risk of adverse events is cumulative and increases with each treatment. It has been found that there is an increased mortality rate after only four short courses of OCS [Sullivan,Patrick W et al. The Journal of Allergy and Clinical Immunology,vol.141,1(2018):110-116.e7.]. In terms of lifetime cumulative dose, most side effects appeared after patients were treated with 1-2.5 g of corticosteroids, and the incidence of diabetes began to rise in patients receiving as little as 0.5 g of corticosteroids [Price, David B et al. Journal of Asthma and Allergy,vol.11 193-204.29 Aug.2018,].

[0031] The OCS-sparing effects of dupilumab, mepolizumab, reslizumab, omalizumab and benralizumab have been clinically evaluated. Although treatment with these biologics allowed a reduction in daily OCS dose by 50%, only 5%-20% of severe asthma patients were able to discontinue OCS use. There remains a significant unmet medical need to develop new treatment modalities or biologics to treat asthma that may eliminate or even reduce dependence on OCS [Cataldo,Didier et al. The Journal of Asthma:Official Journal Of The Association For The Care Of Asthma,vol.58,4(2021):448-458.].

[0032] All current FDA approved biologics for treating asthma and AD target the downstream pathway of Th2 inflammation.However, there are clinical inadequacies that need to be addressed in patients treated with such biologics, such as high relapse rates, limited efficacy against non-Th2 inflammation, and low-level but continued dependency on OCS use.Recently, new biologics are being developed that target alarmins, such as IL-25, TSLP and IL-33.This is an upstream element that affects Th2 inflammatory pathways, and could potentially contribute to both Th2 and non-Th2 inflammatory responses.Therefore, anti-alarmin biologics could provide additional options that could address the current clinical inadequacies in patients with AD and severe Th2-high / low asthma.

[0033] IL-25 (also known as IL-17E) is a member of the IL-17 cytokine family, which includes IL-17A to IL-17F. IL-25 binds to its receptors, composed of IL-17 receptor A (IL-17RA) and IL-17 receptor B (IL-17RB), for signal transduction [Borowczyk, Julia et al. The Journal Of Allergy And Clinical Immunology, vol. 148, 1 (2021): 40-52.]. IL-25 is a type 2 cytokine produced by Th2 cells, which can induce gene expression of IL-4, IL-5 and IL-13, as well as amplify allergic inflammatory responses in the lungs and gastrointestinal tract. IL-25 is important in type 2 immune responses, as it activates the IL-17RA / IL-17RB complex in various cell types, such as epithelial cells, Th2 cells and ILC2s. IL-25 is a type 2 cytokine produced by CD4 + It has been reported to play an anti-inflammatory role by inhibiting T cell activation and differentiation into Th17 cells, as well as down-regulating Th1 and Th17 cellular responses in autoimmune and inflammatory diseases.

[0034] Thymic stromal lymphopoietin (TSLP) is a member of the IL-2 cytokine family and a distant paralog of IL-7. TSLP binds to a heterodimeric receptor formed by the TSLP-specific TSLPR subunit and the IL-7R signaling chain, and acts on several immune cell types, such as dendritic cells, ILC2, mast cells, basophils, and T cells. During allergic inflammation, the main producers of TSLP are epithelial cells, keratinocytes, and stromal cells. TSLP plays a pivotal role in driving Th2-mediated inflammation by modulating antigen-presenting cells (e.g., dendritic cells) to amplify type 2 cytokines by T cells and innate lymphoid cells [Ito, Tomoki et al. The Journal Of Experimental Medicine,vol.202,9(2005):1213-23].

[0035] IL-33 is a member of the IL-1 family and was recently identified as a ligand for T1 / ST2 (ST2), a member of the IL-1 receptor family. IL-33 is a dual-function protein that acts both as a proinflammatory cytokine and an intracellular nuclear factor with transcriptional regulatory properties. After cellular stress or necrosis, IL-33 is released into the extracellular space and serves as an endogenous danger signal that alerts the immune system to tissue damage during trauma or infection. IL-33 amplifies both Th1- and Th2-type responses through its activity on human basophils, allergen-reactive Th2 cells, iNKT and NK cells. Although IL-33 canonically induces type 2 cytokine responses, this cytokine can also synergize with type 1 cytokines such as IL-12 to induce interferon-gamma (IFNγ) [Komai-Koma,Mousa et al. Immunobiology,vol.221,3(2016):412-7]. IL-33 is thus emerging as a pivotal immune modulator with an important role in allergic, fibrotic, infectious and chronic inflammatory diseases.

[0036] Because IL-25, TSLP and IL-33 (collectively known as alarmins) exhibit broad functions beyond Th2 immune responses, biologics against these alarmins may be effective in additional allergic disease subtypes beyond the approved indications for biologics that interact with downstream targets in the Th2 pathway. Both anti-TSLP and anti-IL33 antibodies have been clinically evaluated in asthma and AD [Simpson, Eric L et al. Journal of the American Academy of Dermatology, vol.80,4(2019):1013-1021; Chen, Yi-Ling et al. Science Translational Medicine, vol.11,515(2019):eaax2945]. Tezepelumab (AMG 157 / MEDI9929) is a fully human immunoglobulin G2-lambda monoclonal antibody that specifically binds to TSLP and prevents TSLP from interacting with its receptor complex. A phase II randomized, double-blind, placebo-controlled trial (ALLEVIAD; NCT02525094) showed a trend towards improvement in all endpoints in patients with moderate-to-severe AD when treated with tezepelumab plus topical corticosteroid use (TCS) compared with placebo plus TCS, but such improvements failed to achieve statistical significance as estimated by EASI50 at week 12. Two separate phase III trials (SOURCE and NAVIGATOR) were conducted to evaluate the clinical efficacy of tezepelumab for treating moderate-to-severe asthma. In the NAVIGATOR trial, patients with moderate-to-severe asthma were treated with either tezepelumab plus OCS or placebo plus OCS. The primary endpoint met with statistical significance in the treatment group compared to the placebo group, demonstrating a clinically meaningful reduction in AAER (Annual Asthma Exacerbation Rate) [Menzies-Gow,Andrew et al. The New England Journal of Medicine,vol.384,19(2021):1800-1809].In the NAVIGATOR study, tezepelumab (combined with OCS) was equally effective in suppressing severe Th2-high and Th2-low asthma. However, in the SOURCE study, tezepelumab failed to meet the primary endpoint of a statistically significant reduction in daily OCS compared with placebo without poor asthma control. This result suggests that tezepelumab used as monotherapy may not be sufficient for the treatment of moderate-to-severe asthma.

[0037] Etoximab (ABN020), an anti-IL-33 humanized IgG1 monoclonal antibody, was clinically evaluated for the treatment of AD. In a phase 1 trial, administration of ABN020 led to a significant reduction in blood eosinophil counts. ABN020 was found to significantly alleviate symptoms of house dust mite-induced dermatitis and reduce neutrophil infiltration in the skin (a hallmark of non-Th2 inflammation) in a phase Iia trial [Chen, Yi-Ling et al. Science Translational Medicine,vol.11,515(2019):eaax2945]. These results suggested that ABN020 could suppress both Th2 and non-Th2 inflammation. However, ABN020 failed to meet its primary endpoint criteria in a subsequent phase Iib trial for the treatment of moderate to severe AD. Itepekimab (REGN3500), another anti-IL-33 antibody, was clinically evaluated for the treatment of asthma. In a phase II proof-of-concept (POC) study, patients who discontinued the use of ICS and inhaled long-acting bronchodilators (LABA) received treatment with either dupilumab or itepekimab for 12 weeks [Wechsler,Michael E et al. The New England Journal Of Medicine,vol.385,18(2021):1656-1668.]. The rate of poor asthma control in patients treated with itepekimab (22%) was significantly lower than in patients treated with placebo (41%). The data suggested that itepekimab may be effective as a monotherapy without ICS and LABA. However, the efficacy of itepekimab was slightly inferior to the anti-IL4Ra antibody dupilumab (19%). Further clinical development of itepekimab for asthma was therefore halted due to strategic rather than scientific considerations. Nevertheless, itepekimab was later found to reduce exacerbation rates and improve lung function in former smokers with chronic obstructive pulmonary disease (COPD), a disease characterized by neutrophilic inflammation [Rabe, Klaus F et al. The Lancet. Respiratory Medicine,vol.9,11(2021):1288-1298].Two phase III clinical trials are ongoing with itepekimab for the treatment of COPD in former smokers.

[0038] To date, the efficacy of blocking the IL-25 / IL-17RB pathway against immunological diseases, such as asthma and AD, has never been clinically verified. The latest anti-IL25 antibody is XKH001, developed by Kanovabiopharma and currently being evaluated in Phase I clinical trials. LNR 125.38 is another anti-IL-25 antibody developed by Lanier Biotherapeutics and currently in preclinical stage; LNR 125.38 significantly reduces the increase of type 2 cytokines and inflammatory cells in allergic mice and mice with rhinovirus-induced asthma exacerbation. SM17, developed by SinoMab BioScience Limited, is a first-in-class humanized anti-IL17RB monoclonal antibody in Phase I clinical trials. SM17 does not block IL-25 from binding to IL-17RB, but inhibits signaling through the IL-25 / IL-17RB pathway. SM17 is a humanized version of the parent murine antibody D9.2 that has demonstrated therapeutic potential in preclinical studies in mice for the treatment of inflammatory bowel disease, idiopathic pulmonary fibrosis, asthma and rhinovirus-induced asthma exacerbations.

[0039] Despite the demonstrated preclinical and clinical effectiveness of such anti-alarmin antibodies (especially anti-TSLP and anti-IL33 antibodies) in reducing both Th2 and non-Th2 inflammation, their therapeutic response is somewhat suboptimal when used as add-on or monotherapy for the treatment of asthma and / or AD. A possible explanation is that TSLP, IL-33, and possibly IL-25 play redundant and overlapping functions / roles in the pathogenesis of asthma and AD. Therefore, blocking only any one of them is insufficient to obtain optimal clinical response.

[0040] At the molecular level, all three alarmins directly activate ILC2, a family member of innate lymphoid cells (ILCs). ILC2s are tissue-resident sentinels that rapidly respond to their environment through soluble inflammatory mediators, neurotrophic factors and cell-cell interactions. ILC2s have been shown to express receptors for IL-25, IL-33 and TSLP and respond to these signals by secreting IL-5 and IL-13, which subsequently enhance allergic responses. When ILC2s are dysregulated, this can contribute to the overproduction of Th2-type inflammatory cytokines, leading to the development of allergic asthma, AD, allergic rhinitis, ulcerative colitis and many chronic fibroproliferative disorders. ILC2s are also closely associated with rapid disease relapse in AD and OCS dependency in severe asthma.

[0041] In patients with severe eosinophilic asthma, one course of OCS resulted in a decrease in circulating eosinophils, Th2 cells, and Tc2 cells, but not ILC2s, suggesting that ILC2s are more resistant to OCS than Th2 cells (Hynes, G., et al. 2018). +Compared with Th2 cells, ILC2 showed a stronger positive correlation with the asthma control status of patients and were more resistant to glucocorticoid-induced cell death and suppression of type 2 cytokine release [Jia, Yi et al. American Journal Of Respiratory Cell And Molecular Biology,vol.55,5(2016):675-683]. Recent studies further demonstrated that all three alarmins play an important role in the corticosteroid resistance of ILC2. IL-33 rapidly increases the number of ILC2 in the peribronchial / perivascular areas in a mouse model, but the accumulation of ILC2 in the lungs was dependent on the CCL8-CCR8 signaling pathway. It is known that IL-33 treatment mainly leads to the production of CCL8 from macrophages in the pulmonary airways. Signaling through the CCL8-CCR8 pathway further enhances ILC2 cytokine (IL-13 and IL-5) production as well as IL-13. +It played a pivotal role in activated ILC2 motility [Puttur,Franz et al. Science Immunology,vol.4,36(2019):eaav7638]. A subpopulation of ILC2s known as "inflammatory" ILC2s (iILC2s) was later found to be involved in the development of corticosteroid resistance. A correlation between disease severity and resistance to corticosteroid therapy was established in patients with chronic sinusitis or asthma, especially when the number of circulating iILC2s and resident iILC2s in inflamed mucosal tissues was increased. Interestingly, iILC2 development and migration is IL-25 dependent [Miller,Mindy M et al. Science Immunology,vol.5,43(2020):eaay3994;van der Ploeg,Esmee K et al. Science Immunology,vol.6,55(2021):eabd3489]. It has also been reported that TSLP treatment increases the steroid resistance of ILC2. Bronchoalveolar lavage fluid (BALF) ILC2 collected from asthma patients with high TSLP levels were steroid resistant. IL-7 and TSLP abolished the dexamethasone inhibition of type 2 cytokine production from blood ILC2 [Liu, Sucai et al. The Journal Of Allergy And Clinical Immunology,vol.141,1(2018):257-268.e6].

[0042] Alarmins also play a crucial role in AD relapse mediated by Th2 memory cells. Persistent skin-resident treatment-resistant immune memory Th2 in AD was identified in patients treated with dupilumab for one year. Such memory Th2 cells are involved in the upregulation of IL-13. + , IL-17RB + , ST2 + , TSLPR +It is suggested that they may be responsive to alarmins. Previous studies have reported that IL-33 may induce Th2 memory cells to produce IL-31, a cytokine that causes severe itch in AD [Maier,Elisabeth et al. Journal of Immunology(Baltimore,Md.:1950)vol.193,2(2014):645-54;Stott,Bryony et al. The Journal Of Allergy And Clinical Immunology,vol.132,2(2013):446-54.e5]. Another report showed that CRTH2 expression in AD skin lesions may be mediated by IL-33. + CD4 + Infiltration of Th2 memory cells was associated with TSLP-activated DCs (TSLP-DCs) [Wang,Yui-I et al. Immunity,vol.24,6(2006):827-838]. Furthermore, IL-25 enhanced the proliferation of Th2 memory cells stimulated by TSLP-DCs [Wang,YIHsi et al. The Journal of Experimental Medicine,vol.204,8(2007):1837-47]. These studies indicated that neutralization of IL-4 and IL-13 by dupilumab may be insufficient to suppress the activity of Th2 memory cells. This may help explain why a significant proportion of AD patients have partial and non-durable responses when treated with dupilumab [Bangert, Christine et al. Science Immunology, vol.6,55(2021):eabe2749][Wollenberg, A et al. The British Journal Of Dermatology, vol.184,3(2021):437-449]. Unlike IL-4 and IL-13, alarmins regulate both short-term Th2 effector function and long-lasting Th2 memory in AD. Thus, neutralization of alarmins is useful as a novel approach for AD treatment.

[0043] Because the three alarmins play redundant roles in type 2 immunity, blocking any one of these three alarmins is unlikely to be sufficient to fully suppress both Th2 and ILC2 activity, which may explain why tezepelumab and itepekimab have shown limited clinical efficacy in asthma and AD despite inhibiting both Th2 and non-Th2 inflammatory responses.

[0044] Although redundant, there are non-overlapping functions between these alarmins in Th2 as well as non-Th2 responses. IL-33 acts in concert with IL-12 to directly induce the production of interferon gamma (IFNg) from human NK cells, a well-known disease-causing factor in the pathogenesis of inflammatory bowel disease (IBD). Studies of patient biopsies have shown elevated levels of IL-33 in patients with active IBD, particularly ulcerative colitis (UC) [Kobori, Ayako et al. Journal of Gastroenterology, vol. 45, 10 (2010): 999-1007]. Furthermore, UC-associated IL-33 has been found in myofibroblasts that tend to localize at the base of inflammatory ulcerations in patients with UC [Sponheim, Jon et al. The American Journal Of Pathology, vol. 177, 6 (2010): 2804-15]. Blockade of the IL-33 / ST2 pathway was shown to ameliorate experimental colitis by improving mucosal healing in mice and active disease in humans, suggesting a pathogenic role for IL-33 in IBD [Sedhom, Mamdouh AK et al.Gut,vol.62,12(2013):1714-23]. In contrast, TSLP induces human DCs to express OX40 ligand (OX40L) but not IL-12, a prerequisite for tregI naive CD4+ T cells to produce IL-4, IL-5, and IL-13. Furthermore, TSLP-activated DCs produce chemokines, such as CCL17 / TARC and CCL22 / MDC, which attract naive T cells. TSLP stimulation of naive CD4+ T cells, either directly through TSLPR or indirectly through binding of OX40 ligand (induced by TSLP on DCs) to OX40 on T cells, induces specific Th2 polarization.

[0045] Interestingly, upon DC activation, human TSLP and TLR3 ligands promote the differentiation of Th17 cells with a central memory T cell phenotype. Similarly, IL-25 derived from skin-localized mast cells stimulates cutaneous DCs to produce IL-1β during the elicitation phase of contact dermatitis, thereby contributing to the activation of Th17 cells rather than Th2 cells. In psoriatic skin, IL-25 stimulates keratinocyte proliferation and induces the production of inflammatory cytokines and chemokines through activation of the STAT3 transcription factor. Also, IL-25 expression in keratinocytes contributes to the amplification of psoriasiform inflammation. Furthermore, IL-25 was more potent than IL-33 in inducing the secretion of IL-5 and IL-13 from human peripheral blood mononuclear cells (PBMCs) [Bartemes,Kathleen R et al. The Journal Of Allergy And Clinical Immunology,vol.134,3(2014):671-678.e4].

[0046] Although appearing redundant, the three alarmins may play different roles in regulating ILC2, Th1, Th2 and Th17 activity and responses. Indeed, they have been reported to target different cell types in the central and peripheral systems, including but not limited to basophils, macrophages, eosinophils, mast cells, fibroblasts and keratinocytes. Due to the heterogeneity of allergic diseases (e.g., contact dermatitis, AD, Th2-high / low asthma) and autoimmune diseases (e.g., inflammatory bowel disease, psoriasis), blocking a single alarmin may only be effective for certain patient groups. For example, a mixed Th1 and Th2 phenotype is most common in Western AD, whereas a mixed Th17 and Th2 phenotype is most common in Asian and pediatric AD [Renert-Yuval, Yael, and Emma Guttman-Yassky. Annals Of Allergy, Asthma & Immunology: Official Publication Of The American College Of Allergy, Asthma, & Immunology, vol. 124, 1 (2020): 28-35]. This indicates that there is still room for improvement in addressing these immunological diseases, presenting an unmet medical need for novel approaches and therapies tailored to different subtypes of immunological diseases.

[0047] One example of such an approach to alternative therapy may include co-administration of biologics (e.g., antibodies) against two or more alarmins to treat different modalities of allergic disease (e.g., pediatric AD and adult AD). Co-administration requires either injection of two separate products or a single injection of a co-formulation with two different biologics. Two injections allow flexibility in the amount and timing of administration, but are inconvenient for patients in terms of compliance. Furthermore, although co-formulation may provide some flexibility in the amount of administration, due to the different molecular characteristics of two or more anti-alarmin biologics, it is often quite difficult or impossible to find formulation conditions that have acceptable viscosity (at relatively high concentrations) and promote chemical and physical stability. Furthermore, co-administration and co-formulation entail the additional cost of two or more different drug therapies, which may result in high costs for patients and / or payers. Thus, there remains a need for alternative therapies for the treatment of allergic disease with disease modification, and preferably such alternative therapies include bispecific or multispecific binding proteins against different alarmins.

[0048] For illustrative purposes, the present invention provides a bispecific binding protein for two different alarmins; this can be in the form of a bispecific protein with specificity for IL-17RB on the one hand and for soluble alarmins on the other hand. Specifically, the bispecific protein can be an anti-IL17RB (IL-25 receptor) antibody fused to the C-terminus with either (a) a single chain Fv (scFv) targeting IL-33 or TSLP alarmins; or (b) the extracellular domain of the IL-33 receptor (ST2) or TSLP receptor (TSLPR). Although the present invention discloses the use of bispecific binding proteins for alarmin receptors and different alarmins, this approach can be subdivided into multispecific binding proteins targeting different alarmin receptors, alarmins, and other downstream cytokines.

[0049] Before the present disclosure is further described, it is to be understood that this disclosure is not limited to particular embodiments shown herein and that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0050] Unless otherwise defined herein, scientific and technical terms used in this disclosure shall have the meanings commonly understood by those skilled in the art.Furthermore, unless otherwise required by context, singular terms shall include the plural, and plural terms shall include the singular.In general, the nomenclature and techniques used in connection with cell and tissue culture, molecular biology, immunology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art.

[0051] The terms "a" or "an" entity refer to one entity; for example, "a vector" is understood to refer to one vector.

[0052] The term "and / or" as used herein should be interpreted as a specific disclosure of two specified features or components, each with or without the other. Thus, the term "and / or" when used in a phrase, such as "A and / or B" herein, is intended to include "A and B", "A or B", "A" (single) and "B" (single). Similarly, the term "and / or" when used in a phrase, such as "A, B and / or C", is intended to include each of the following aspects: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (single); B (single); and C (single).

[0053] Ranges: Throughout this disclosure, various aspects of the invention may be presented in a range format. It should be understood that the description in range format is merely abbreviated for convenience and should not be construed as an inflexible limitation on the scope of the invention. Thus, the description of a range should be considered to have all possible subranges specifically disclosed as well as individual numerical values ​​within the range. For example, the description of a range, such as 1-6, should be considered to have specifically disclosed subranges, such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as individual numerical values ​​within the range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0054] Exemplary genes and polypeptides are described herein by reference to GenBank numbers, GI numbers and / or SEQ ID NO. It will be appreciated that one of skill in the art can readily identify homologous sequences by reference to sequence sources such as, but not limited to, GenBank (ncbi.nlm.nih.gov / genbank / ) and EMBL (embl.org / ).

[0055] 7.1 IL17RB protein Human IL17RB is a 47.9 kDa transmembrane protein (462 aa) belonging to the IL-17 receptor family. IL-17RB is expressed in various endocrine tissues and in epithelial cells of different organs, such as kidney and liver, as well as mucosal tissues. High IL-17RB expression is also found in lung tissues of asthmatic patients and skin lesions of AD patients. IL-17RB expression in human ILC2, natural killer T (NKT) cells, and Th2 cells suggests a potential role in immune cells. IL-17RB is shared by two ligands, IL-17B and IL-25 (also known as IL-17E). It has been reported that IL-25 binds to the heterodimeric IL-17RA / IL-17RB complex, whereas IL-17B binds to both the heterodimeric and IL-17RB homodimeric receptors [Wu, Heng-Hsiung et al. Science Translational Medicine, vol. 13, 583 (2021): eabc2823]. The binding affinity of IL-17B to IL-17RB (K D ) is approximately 30-fold lower than that of IL-25 (IL-17E), and the binding rate (K on ) is similar, but the dissociation rate (K off ) is fairly fast. Further information regarding human IL-17RB, including an exemplary amino acid sequence thereof, may be found in public databases, such as GENEBANK (NCBI Ref. NP_061195.2). An exemplary sequence is also provided below. TIFF2025508835000002.tif37153

[0056] 7.2 Bispecific Binding Proteins The present disclosure provides a bispecific binding protein that can specifically bind to two antigens.The binding protein generally comprises a light chain variable region or domain and a heavy chain variable region or domain that correspond to the light chain variable region or domain and the heavy chain variable region or domain of an immunoglobulin.At least one antigen-binding portion of the binding protein is in a single-chain format known in the art as scFv.In some embodiments, the other antigen-binding portion comprises IgG.In other embodiments, the other antigen-binding portion comprises scFv.

[0057] In some embodiments, methods and uses of antibodies or antigen-binding fragments thereof that specifically bind to alarmin receptors, alarmins, or both are provided herein. The term "antibody" and its grammatical equivalents, as used herein, refers to an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, glycan, polynucleotide, lipid, or a combination of any of the above, through at least one antigen-binding site, where the antigen-binding site is usually located in the variable region of the immunoglobulin molecule. As used herein, the term encompasses intact polyclonal antibodies, intact monoclonal antibodies, single domain antibodies (sdAb; e.g., camelid antibodies, alpaca antibodies), single chain Fv (scFv) antibodies, heavy chain antibodies (HCAb), light chain antibodies (LCAb), multispecific antibodies, bispecific antibodies, monospecific antibodies, monovalent antibodies, and any other modified immunoglobulin molecules (e.g., dual variable domain immunoglobulin molecules) that contain antigen-binding sites, so long as the antibody exhibits the desired biological activity. Antibodies also include, but are not limited to, murine, rabbit, camelid, primate, chimeric, humanized, and human antibodies. Antibodies may be of any of the five major immunoglobulin classes, IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof based on the identity of the heavy chain constant domains, designated alpha, delta, epsilon, gamma, and mu, respectively (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). In some embodiments, an antibody may comprise four polypeptide chains, two heavy (H) chains and two light (L) chains, interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. Each light chain is composed of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. Unless otherwise indicated, the term "antibody" as used herein includes an "antigen-binding fragment" of an intact antibody. The term "antigen-binding fragment" as used herein refers to a portion or fragment of an intact antibody that is the antigen-determining variable region of the intact antibody.Examples of antigen-binding fragments include, but are not limited to, Fab (a monovalent fragment consisting of the VL, VH, CL and CH1 domains without the hinge region), Fab' (a monovalent fragment consisting of the VL, VH, CL and CH1 domains linked to the hinge region), F(ab')2 (a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region), Fd (a fragment consisting of the VH and CH1 domains), Fv (a fragment consisting of the VL and VH domains of a single arm of an antibody), linear antibodies, single-chain antibody molecules (e.g., scFv, which can be produced by recombinant techniques), and the like. These include antibodies that are a single polypeptide chain with VL and VH regions linked by a stagger, heavy chain antibodies (HCAbs), light chain antibodies (LCAbs), disulfide-linked scFvs (dsscFvs), diabodies (bivalent bispecific antibodies), tribodies, tetrabodies, minibodies, dual variable domain antibodies (DVDs), single variable domain antibodies (sdAbs or dAbs; e.g., camelid antibodies, alpaca antibodies), and single variable domains of heavy chain antibodies (VHHs), as well as bispecific or multispecific antibodies formed from antibody fragments. A "bispecific" antibody or binding protein is an artificial hybrid antibody that has two different antigen binding sites that recognize and specifically bind to two different targets. Bispecific binding antibodies and proteins can be produced in a variety of ways, such as by fusion of hybridomas or linking of Fab' fragments. See, e.g., Kostelny, SA et al. Journal of Immunology (Baltimore, Md.: 1950) vol. 148, 5 (1992): 1547-53; Songsivilai, S, and PJ Lachmann. Clinical And Experimental Immunology, vol. 79, 3 (1990): 315-21.

[0058] The term "heavy chain" when used in reference to an antibody refers to a polypeptide chain of about 50-70 kDa, with an amino-terminal portion containing a variable region (VH) of about 120-130 or more amino acids and a carboxy-terminal portion containing a constant region. In some embodiments, the heavy chain constant region is composed of three domains, CH1, CH2 and CH3, with a short flexible hinge region connecting the CH1 and CH2 domains. The constant region can be one of five different types, designated alpha (a), delta (δ), epsilon (ε), gamma (γ) and mu (μ), based on the amino acid sequence of the heavy chain constant region. The different heavy chains differ in size: α, δ and γ contain approximately 450 amino acids, while μ and ε contain approximately 550 amino acids. In combination with light chains, the different types of heavy chains give rise to the five well-known antibody classes: IgA, IgD, IgE, IgG, and IgM, with four subclasses of IgG: IgG1, IgG2, IgG3, and IgG4, respectively. The heavy chains can be human heavy chains.

[0059] The term "light chain" when used in reference to an antibody refers to a polypeptide chain of about 25 kDa, comprising an amino-terminal portion containing a variable region of about 100 to about 110 or more amino acids and a carboxy-terminal portion containing a constant region. The light chain constant region is composed of one domain, CL. The approximate length of a light chain is 211 to 217 amino acids. There are two distinct types, designated lambda (λ) and kappa (κ), based on the amino acid sequence of the constant domain. The amino acid sequences of light chains are well known in the art. The light chain may be a human light chain.

[0060] The term "variable domain" or "variable region" refers to a portion of an antibody light or heavy chain, generally located at the amino terminus of the light or heavy chain, having a length of about 120-130 amino acids in the heavy chain and about 100-110 amino acids in the light chain, that is used in the binding and specificity of each particular antibody to its particular antigen. Variable domains vary widely in sequence among different antibodies. The sequence variability is concentrated in the CDRs, while the less variable portions of the variable domains are referred to as framework regions (FRs). The CDRs of the light and heavy chains are primarily responsible for the interaction of the antibody with the antigen. In some embodiments, each VH and VL is composed of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The numbering of amino acid positions used herein is based on that of Kabat et al. (1991) Sequences of proteins of immunological interest. (USDepartment of Health and Human Services, Washington, DC) 5. th This is based on the EU Index as in ed.

[0061] CDR refers to one of three hypervariable regions (H1, H2 or H3) in the non-framework region of the VH β-sheet framework part of an immunoglobulin (Ig or antibody) or one of three hypervariable regions (L1, L2 or L3) in the non-framework region of the VL β-sheet framework part of an antibody. Thus, CDR is a variable region sequence interspersed within the framework region sequence. CDR regions are well known to those skilled in the art and have been defined by various methods / systems. Such systems and / or definitions have been developed and modified over the years and include Kabat, Chothia, IMGT, AbM and Contact. For example, Kabat defines the most hypervariable regions within the variable (V) domain of an antibody (Kabat, EA et al. The Journal Of Biological Chemistry, vol. 252, 19 (1977): 6609-16.; Kabat, E A. Advances in protein chemistry vol. 32 (1978): 1-75.). The Chothia definition is based on the location of structural loop regions, and the CDR region sequences are defined as residues that are not part of the conserved β-sheet framework regions and therefore can adopt different conformations [Chothia, C, and AM Lesk. Journal of Molecular Biology, vol. 196, 4 (1987): 901-17]. Both terminologies are well recognized in the art. Furthermore, the IMGT system is based on the variability of the sequence and the location of the variable regions in the structure. The AbM definition is a compromise between Kabat and Chothia. The Contact definition is based on the analysis of available antibody crystal structures. Software programs (e.g., abYsis) are available for analyzing antibody sequences and determining CDRs and are known to those skilled in the art.The positions of the CDRs within canonical antibody variable domains have been determined by comparison of numerous structures [Al-Lazikani, B et al. Journal Of Molecular Biology, vol. 273, 4 (1997): 927-48] [Morea, V et al. Methods (San Diego, Calif.) vol. 20, 3 (2000): 267-79]. Because the number of residues in the hypervariable regions varies between antibodies, the additional residues relative to the canonical positions are conventionally numbered a, b, c, etc. next to the residue number in the canonical variable domain numbering scheme. Such nomenclature is similarly well known to those of skill in the art.

[0062] For example, CDRs defined according to either the Kabat (hypervariable criteria) or Chothia (structural criteria) designations are shown in the tables below. TIFF2025508835000003.tif49160 1 Residue numbering follows the nomenclature of Kabat et al., supra. 2 Residue numbering follows the nomenclature of Chothia et al., supra.

[0063] One or more CDRs may also be incorporated into a molecule, either covalently or non-covalently, to form an immunoadhesin. In an immunoadhesin, the CDRs may be incorporated as part of a larger polypeptide chain, to which another polypeptide chain may be covalently linked, or the CDRs may be incorporated non-covalently. The CDRs enable the immunoadhesin to bind to a particular antigen of interest.

[0064] The terms "epitope" and "antigenic determinant" are used interchangeably herein and refer to the site on the surface of a target molecule to which an antibody or antigen-binding fragment binds, e.g., a localized area on the surface of an antigen. Target molecules can include proteins, peptides, nucleic acids, sugar chains, or lipids. An epitope with immunogenic activity is a portion of a target molecule that elicits an immune response in an animal. An epitope of a target molecule with antigenic activity is a portion of a target molecule to which an antibody binds, and is determined by any method known in the art, such as by immunoassay. An epitope of an antigen is not necessarily immunogenic. Epitopes often consist of chemically active surface groups of molecules, e.g., amino acids or sugar side chains, and have specific three-dimensional structural characteristics as well as specific charge characteristics. The term "epitope" includes linear epitopes and structural epitopes. The region of a target molecule (e.g., a polypeptide) that contributes to an epitope can be contiguous amino acids of the polypeptide, or an epitope can be two or more non-contiguous regions of the target molecule combined. An epitope may or may not be a three-dimensional surface feature of the target molecule. Epitopes formed by contiguous amino acids (also called linear epitopes) are typically retained when a protein is denatured, whereas epitopes formed by folding into a three-dimensional structure (also called structural epitopes) are typically lost when a protein is denatured. An epitope typically includes at least 3, more usually at least 5, 6, 7 or 8-10 amino acids in a unique spatial conformation.

[0065] The term "specifically binds" as used herein means that a polypeptide or molecule interacts with an epitope, protein or target molecule more frequently, more rapidly, with a longer duration, with greater affinity, or with any combination of the above, than with another substance, such as related and unrelated proteins. Binding moieties (e.g., antibodies) that specifically bind to a target molecule (e.g., an antigen) can be identified, for example, by immunoassays, ELISA, Bio-Layer Interferometry ("BLI"), SPR (e.g., Biacore) or other techniques known to those of skill in the art. Typically, a specific response is at least twice the background signal or noise, and may be more than 10 times the background. See, for example, Paul, ed., 1989, for a discussion of antibody specificity, FUNDAMENTAL IMMUNOLOGY SECOND EDITION , Raven Press, New York, pp. 332-336. A binding moiety that specifically binds to a target molecule may bind to the target molecule with a higher affinity than the affinity for a different molecule. In some embodiments, a binding moiety that specifically binds to a target molecule may bind to the target molecule with an affinity that is at least 20 times greater, at least 30 times greater, at least 40 times greater, at least 50 times greater, at least 60 times greater, at least 70 times greater, at least 80 times greater, at least 90 times greater, or at least 100 times greater than the affinity for a different molecule. In some embodiments, a binding moiety that specifically binds to a particular target molecule binds to a different molecule with such low affinity that binding cannot be detected using the assays described herein or known in the art. In some embodiments, "specifically binds" refers to, for example, a binding moiety that binds to a molecular target with a K of about 0.1 mM or less. D This means that they are joined by

[0066] In some embodiments, "specifically binds" means that a polypeptide or molecule binds to a target with a K DIn some embodiments, "specifically binds" means that a polypeptide or molecule binds to a target with a K of about 0.1 μM or less, about 0.01 μM or less, or about 1 nM or less. D Specific binding means binding at a specific target. Due to sequence identity between homologous proteins in different species, specific binding may include a polypeptide or molecule recognizing a protein or target in more than one species. Similarly, due to homology in certain regions of the polypeptide sequences of different proteins, specific binding may include a polypeptide or molecule recognizing more than one protein or target. It will be understood that in some embodiments, a binding moiety (e.g., an antibody) that specifically binds to a first target may or may not specifically bind to a second target. Thus, "specific binding" does not necessarily require (but may include) exclusive binding, i.e., binding to a single target. Thus, a binding moiety (e.g., an antibody) may in some embodiments specifically bind to more than one target. For example, an antibody may in some particular cases contain two identical antigen binding sites that each specifically bind to the same epitope on two or more proteins. In some particular alternative embodiments, an antibody may be bispecific, containing at least two antigen binding sites with different specificities.

[0067] The term "binding affinity," as used herein, generally refers to the strength of the overall result of non-covalent interactions between a binding moiety and a target molecule (e.g., an antigen). Binding between a binding moiety and a target molecule is a reversible process, and the affinity of binding is typically measured by the equilibrium dissociation constant (K D ) is reported as K D is the binding rate (k on or k a ) to dissociation rate (k off or k d ) of the bond pair. D The smaller the K, the higher the affinity. A is the equilibrium binding constant, which is also the reciprocal of the equilibrium dissociation constant, i.e., = 1 / K D For antibody-antigen interactions, KD can be calculated as the ratio of the product of the concentrations of free antibody and free antigen to the concentration of the antibody-antigen complex, ie, [antigen] x [antibody] / [antigen-antibody].

[0068] A variety of methods for measuring binding affinity are known in the art, any of which may be used for purposes of the present disclosure. Specific exemplary embodiments include the following. In some embodiments, the "K D " or "K D The "K value" can be measured by assays known in the art, for example by binding assays. D may be measured in a radiolabeled antigen binding assay (RIA) (Chen, Y et al. Journal of Molecular Biology, vol. 293, 4 (1999): 865-81). D or K D The value of K can also be measured by using biolayer interferometry (BLI), for example using the Gator system (Probe Life) or the Octet-96 system (Sartorius, Göttingen, Germany). D or K D The value of can also be measured by using a surface plasmon resonance assay by using a BIAcore system (e.g., Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ).

[0069] The term "variant" as used herein in the context of a protein or polypeptide having particular sequence characteristics ("reference protein" or "reference polypeptide") refers to a different protein or polypeptide having one or more (e.g., about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5, etc.) amino acid substitutions, deletions, and / or additions compared to the reference protein or polypeptide. The amino acid sequence changes may be amino acid substitutions. The amino acid sequence changes may be conservative amino acid substitutions. A functional fragment or functional variant of a protein or polypeptide maintains the essential structural and functional properties of the reference protein or polypeptide.

[0070] The terms "polypeptide", "peptide", "protein" and grammatical equivalents are used interchangeably herein to refer to polymers of amino acids of any length, which may be linear or branched, which may include unnatural or modified amino acids, and which may be interrupted by non-amino acids. A polypeptide, peptide or protein may also be modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification.

[0071] The terms "polynucleotide", "nucleic acid" and grammatical equivalents are used interchangeably herein to refer to a polymer of nucleotides of any length, including DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and / or their analogs, or any substance that can be synthesized into a polymer by DNA polymerase or RNA polymerase. Nucleic acid molecules can be single-stranded or double-stranded.

[0072] As used herein, the term "encoding" and its grammatical equivalents refer to the inherent property that a particular sequence of nucleotides within a polynucleotide or nucleic acid, such as a gene, cDNA or mRNA, serves as a template for the synthesis of other polymers and macromolecules of biological processes that have either a defined nucleotide sequence (i.e., rRNA, tRNA and mRNA) or a defined amino acid sequence and biological properties derived therefrom. Thus, a gene encodes a protein when the protein is produced by transcription and translation of the mRNA corresponding to the gene. Unless otherwise stated, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. Protein and RNA encoding nucleotide sequences may include introns.

[0073] An "isolated" polypeptide, peptide, protein, antibody, polynucleotide, vector, cell or composition is a polypeptide, peptide, protein, antibody, polynucleotide, vector, cell or composition that is in a form not found in nature. Isolated polypeptides, peptides, proteins, antibodies, polynucleotides, vectors, cells or compositions include those that have been purified to a degree that they are not in a form found in nature. In some embodiments, an isolated polypeptide, peptide, protein, antibody, polynucleotide, vector, cell or composition is substantially pure. In some embodiments, an isolated polypeptide, peptide, protein, antibody, polynucleotide, vector, cell or composition is substantially free of other cellular material and / or chemicals.

[0074] The terms "identical", "percent identity" and grammatical equivalents herein, when used in the context of two or more polynucleotides or polypeptides, indicate that two or more sequences or subsequences are the same or have a specified percentage of the same nucleotide or amino acid residues when compared and aligned (introducing gaps, if necessary) to maximize correspondence, without considering any conservative amino acid substitutions as part of the sequence identity. Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to obtain amino acid or nucleotide sequence alignment are well known in the art. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package and its variants. In some embodiments, two polynucleotides or polypeptides provided herein are substantially identical, meaning that when compared and aligned for maximum correspondence, they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity of nucleotide or amino acid residues as measured using a sequence comparison algorithm or by visual inspection. In some embodiments, the identity exists over a region of the amino acid sequence at least about 10 residues, at least about 20 residues, at least about 40-60 residues, at least about 60-80 residues in length, or any integer value of the number of residues therebetween. In some embodiments, the identity exists over a region longer than 60-80 residues, e.g., at least about 80-100 residues, and in some embodiments, the sequences are substantially identical over the entire length of the sequences being compared, e.g., the coding regions of the target proteins or antibodies.In some embodiments, the identity exists over a region of the nucleotide sequence that is at least about 10 bases, at least about 20 bases, at least about 40-60 bases, at least about 60-80 bases in length, or any integer number of residues in between. In some embodiments, the identity exists over a region longer than 60-80 bases, e.g., at least about 80-1000 bases or more, and in some embodiments, the sequences are substantially identical over the entire length of the sequences being compared, e.g., nucleotide sequences encoding a protein of interest.

[0075] "Conservative amino acid substitution" as used herein refers to the replacement of an amino acid residue with another amino acid residue that has a similar side chain. "Conservatively similar" amino acids or residues as used herein refer to non-identical amino acid residues that have similar side chains. Families of amino acid residues with similar side chains have been defined in the art, such as 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), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0076] The term "vector" and its grammatical equivalents, as used herein, refers to a vehicle used to carry genetic material (e.g., a polynucleotide sequence) that can be introduced into a host cell and replicated and / or expressed therein. Vectors applicable for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which may include a selection sequence or marker that is functional for stable integration into the host cell chromosome. In addition, the vector may include one or more selectable marker genes and appropriate expression control sequences. The selectable marker genes that may be included provide, for example, resistance to antibiotics or toxins, complement auxotrophic deficiencies, or provide important nutrients that are not in the culture medium. Expression control sequences may include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like, which are well known in the art. When two or more polynucleotides are co-expressed, both polynucleotides may be inserted, for example, into a single expression vector or into separate expression vectors. In the expression in a single vector, the coding polynucleotides may be operatively linked to a common expression control sequence or may be linked to different expression control sequences, for example, one inducible promoter and one constitutive promoter. The introduction of the polynucleotide into the host cell may be confirmed using methods well known in the art. It will be understood by those skilled in the art that the polynucleotide is expressed in an amount sufficient to produce the desired product (e.g., an IgG-consisting bispecific binding protein), and further understood that the expression level may be optimized to obtain sufficient expression using methods well known in the art.

[0077] As used herein, the term "host cell" refers to a cell into which genetic material, such as a recombinant expression vector, can be or has been introduced. Host cells include not only the cell into which exogenous genetic material is introduced, but also the progeny of such cells. Such progeny may not be identical to the parent cell, since certain modifications may occur in successive generations, either due to mutation or environmental influences.

[0078] As used herein and as understood in the art, "EC" refers to the effective concentration of an agent (e.g., an antibody) and is commonly used in dose-response curves. The "effect" of an agent can be a positive (stimulatory) effect or a negative effect. The term "EC50" refers to the concentration of an active agent (e.g., an antibody) that produces a half-maximal effect. Also, as used herein and as understood in the art, "IC" refers to the concentration of an agent that has an inhibitory effect and is also commonly used in dose-response curves. The term "IC50" refers to the concentration of an agent (e.g., an antibody) that reduces the inhibitory activity by half.

[0079] As described in the above section, a bispecific binding protein can comprise two polypeptides linked together by disulfide bonds, each polypeptide comprising a first scFv region and a second scFv region at its respective N-terminus and C-terminus. The two scFv regions specifically bind to different antigens. Between the first and second scFv regions of each polypeptide are sequences comprising the hinge, CH2, and CH3 domains of an immunoglobulin.

[0080] Alternatively, the bispecific binding protein can comprise an IgG with an scFv region linked to the C-terminus of each CH3 domain, where the heavy and light chain variable regions of the Fab region of the IgG specifically bind one antigen, and the C-terminal scFv region contains different heavy and light chain variable regions that specifically bind a second antigen.

[0081] As described above, a bispecific binding protein can bind to two different antigens through two domains, one at the N-terminus and binding to antigen X, and the other at the C-terminus and binding to antigen Y. In some embodiments, the bispecific binding protein binds to a first domain of an alarmin receptor and a second domain of an alarmin. In one particular embodiment, the bispecific binding protein inhibits activation of IL17RB by binding through the first domain and binds to TSLP through the second domain. In another embodiment, the bispecific binding protein inhibits activation of IL17RB by binding through the first domain and binds to IL33 through the second domain.

[0082] In one embodiment, the bispecific binding protein has the formula: XH-Fc-L-scFv Y wherein X is an scFv X or a Fab region, where X specifically binds to a first antigen, and Y specifically binds to a second antigen, H is a hinge region, Fc includes the CH2 and CH3 regions of an immunoglobulin, and scFv X and scFv Y are each independently a single chain variable region fragment and L is a polypeptide linker. In some embodiments, one of the two antigens is an immunomodulatory protein and the other is an alarmin. In some embodiments, when X is a Fab region, the first antigen is an immunomodulatory protein and the other is an alarmin. In some embodiments, when X is a Fab region, the first antigen is an alarmin and the other is an immunomodulatory protein.

[0083] In one embodiment, the bispecific binding protein has the formula: XH-Fc-L-ECD Y wherein X is an scFv X or a Fab region, where X specifically binds to a first antigen and Yis the extracellular domain of an alarmin receptor, H is the hinge region, Fc contains the CH2 and CH3 regions of an immunoglobulin, and scFv X is a single chain variable region fragment and L is a polypeptide linker. In some embodiments, one of the two antigens is an immunomodulatory protein and the other is an extracellular domain of an alarmin receptor. In some embodiments, when X is a Fab region, the first antigen is an immunomodulatory protein and the other is an alarmin. In some embodiments, the ECD Y is the extracellular domain of the TSLP receptor. In some embodiments, the ECD Y is the extracellular domain of the IL-33 receptor.

[0084] The VH and VL chains incorporated into the bispecific binding proteins can be derived from many sources, including existing antibodies, newly created antibodies, and libraries of VH and VL chains. Specific exemplary embodiments of VH and VL chains that can be incorporated into bispecific binding proteins, as well as specific exemplary embodiments of bispecific binding proteins that compete for binding to immune-modulating proteins, such as IL17RB or TSLPR, are provided in the detailed description section.

[0085] Nucleic acids comprising nucleotide sequences encoding the polypeptides of the present disclosure are provided herein, and methods for making the polypeptides, culturing host cells, and recovering the polypeptides are also provided and further discussed in the detailed description below.

[0086] In another aspect, the disclosure provides compositions comprising the bispecific binding proteins described herein. The compositions generally comprise one or more bispecific binding proteins described herein, and / or salts thereof, and one or more excipients, carriers, or diluents.

[0087] Currently used anti-alarmin antibody therapies have limited efficacy in AD and moderate to severe asthma. Specifically, the anti-TSLP antibody tezepelumab and the anti-IL-33 antibody etokimab failed to meet the criteria for their respective primary endpoints in Phase II clinical trials for AD. However, in the treatment of asthma, both tezepelumab and itepekimab met the criteria for their primary endpoints in both Phase II and Phase III clinical trials, despite their limited therapeutic efficacy (tezepelumab was unable to reduce the daily dose of OCS use, and itepekimab was inferior compared to dupilumab). The bispecific proteins described herein specifically bind to two antigens, e.g., an alarmin receptor and an alarmin, and block the immune response induced by two different alarmins. Such inhibition of alarmin-induced responses potentially allows for reduced dosing frequency and long-lasting disease remission in allergic diseases or disorders.

[0088] In some embodiments, provided herein is a use of a bispecific binding protein that specifically binds to an alarmin and / or an alarmin receptor for the preparation of a medicament for reducing the daily dose of OCS in the treatment of Th2-high and / or Th2-low immunological disorders, such as asthma. In the above uses, the bispecific binding protein (a) blocks both IL-25 signaling and TSLP signaling, and / or (b) blocks both IL-25 signaling and IL-33 signaling. In some embodiments, the bispecific binding protein specifically binds human IL-17RB and TSLP. In some embodiments, the bispecific binding protein specifically binds human IL-17RB and IL-33.

[0089] Also provided herein is a method of treating an allergic disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a bispecific binding protein thereof that specifically binds IL-17RB and TSLP or IL-17RB and IL-33, wherein the bispecific binding protein (a) blocks both IL-25 signaling and TSLP signaling, and / or (b) blocks both IL-25 signaling and IL-33 signaling. In some embodiments, the bispecific binding protein specifically binds human IL-17RB and TSLP or human IL-17RB and IL-33. In some embodiments, the allergic disease or disorder is AD and asthma. In some embodiments, the allergic disease or disorder is pre-clinical AD and asthma.

[0090] As used herein, the term "treat" and its grammatical equivalents in the context of a disease or condition or a subject having a disease or condition refer to the act of suppressing, eliminating, reducing, and / or relieving the symptoms, severity of symptoms, and / or frequency of symptoms associated with the disease or disorder being treated.For example, when used in the context of AD, the term "treat" and its grammatical equivalents refer to the act of reducing the severity of the disease or slowing or slowing the progression of the disease, including, but not limited to, (a) reducing the frequency of dosing to achieve disease remission or reduce the occurrence of disease relapse, or (b) delaying, relieving, or minimizing one or more symptoms associated with AD as determined by the Eczema Area and Severity Index (EASI), and (c) reducing the amount and frequency of OCS use.

[0091] The term "block" and grammatical equivalents refer to the act of reducing in some way the biological function of an alarmin, such as, but not limited to, (a) directly competing for the alarmin's binding site on the corresponding receptor, or (b) inhibiting heterodimerization of the corresponding receptor, thereby reducing the biological effect induced by alarmin occupancy.

[0092] As used herein, the term "administer" and its grammatical equivalents refer to the act of delivering or causing the delivery of a therapeutic or pharmaceutical composition to the body of a subject by the methods described herein or known in the art. The therapeutic substance may be a compound, a polypeptide, or a cell. Administering a therapeutic or pharmaceutical composition includes formulating the therapeutic or pharmaceutical composition to be delivered to the body of a subject. Exemplary administration forms include oral dosage forms, such as tablets, capsules, syrups, suspensions; injectable dosage forms, such as intravenous (IV), intramuscular (IM) or intraperitoneal (IP); subcutaneous (SC), transdermal dosage forms, such as creams, jellies, powders or patches; buccal dosage forms; inhalation powders, sprays, suspensions, and rectal suppositories.

[0093] As used herein, the terms "effective amount", "therapeutically effective amount" and grammatical equivalents refer to an amount that, when administered to a subject, either alone or as part of a pharmaceutical composition, and either in a single dose or as part of a series of doses, can have any detectable positive effect on any symptoms, aspects or characteristics of a disease, disorder or condition. A therapeutically effective amount can be confirmed by measuring the relevant physiological effect. The exact amount required will vary from subject to subject, depending on the age, weight and general health of the subject, the severity of the condition being treated, the judgment of the clinician, and the like. A therapeutically effective amount is also an amount in which any toxic or adverse effects of the therapeutic agent do not outweigh the therapeutically beneficial effects. The appropriate "effective amount" in any individual case may vary depending on factors such as the disease state, age, sex, and weight of the individual, and can be determined by one of ordinary skill in the art using routine experimental procedures. A "prophylactically effective amount" refers to an amount that is effective, at the dosage and for the period required, to obtain the desired prophylactic outcome, e.g., delay or suppression of the onset of a disease or disorder. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will generally be less than the therapeutically effective amount.

[0094] The term "subject," as used herein, refers to any animal (e.g., mammal), including but not limited to, humans, non-human primates, dogs, cats, rodents, etc., that will be the recipient of a particular treatment.

[0095] In some embodiments, the bispecific binding proteins disclosed herein (a) inhibit IL5 / CCL17 / IL13 / CCL8 / IFNγ secretion from human PBMCs and / or (b) inhibit ILC2 migration and proliferation. Thus, the bispecific binding proteins disclosed herein exhibit long-lasting efficacy by directly suppressing ILC2 activity. Furthermore, the methods disclosed herein have the additional therapeutic benefit of reducing Th2 and ILC2 cell migration. In some embodiments, provided herein are methods of treating OCS-resistant related diseases or disorders using the anti-IL17RB / anti-TSLP or anti-IL17RB / anti-IL33 bispecific binding proteins disclosed herein.

[0096] The methods provided herein can treat allergic diseases or disorders. The allergic disease or disorder can be post-symptomatic or pre-symptomatic allergic asthma, allergic sinusitis, allergic conjunctivitis, or AD.

[0097] While the above indications are preferred, other diseases, disorders or conditions may be amenable to treatment or may be prevented by administration to a subject of a bispecific antigen binding protein as disclosed herein. Such diseases, disorders and conditions include, but are not limited to, inflammation, autoimmune diseases, inflammation of cartilage, fibrotic diseases and / or bone aging, arthritis, rheumatoid arthritis, juvenile arthritis, juvenile rheumatoid arthritis, oligoarticular juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, systemic onset juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteropathic arthritis, juvenile reactive arthritis, juvenile Reter's syndrome, S. EA syndrome (seronegativity, enthesopathy, arthropathy syndrome), juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, oligoarticular rheumatoid arthritis, polyarticular rheumatoid arthritis, systemic onset rheumatoid arthritis, ankylosing spondylitis, enteropathic arthritis, reactive arthritis, Reter Syndrome, SEA syndrome (seronegative, enthesopathy, arthropathy syndrome), dermatomyositis, psoriatic arthritis, scleroderma, systemic lupus erythematosus, vasculitis, myelitis, polymyositis, dermatomyositis, osteoarthritis, polyarteritis nodosa, Wegener's granulomatosis, arteritis, polymyalgia rheumatica, sarcoidosis, scleroderma, sclerosis, primary biliary sclerosis, sclerosing cholangitis, Sjogren's syndrome, psoriasis, plaque psoriasis, guttate psoriasis , inverse psoriasis, pustular psoriasis, erythrodermic psoriasis, dermatitis, AD, atherosclerosis, lupus, Still's disease, systemic lupus erythematosus (SLE), myasthenia gravis, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, celiac disease, multiple sclerosis (MS), asthma, COPD, Guillain-Barre disease, diabetes mellitus type I, Graves' disease, Addison's disease, Raynaud's phenomenon, autoimmune hepatitis, GVHD, and the like. In a specific embodiment, a pharmaceutical composition is provided comprising a therapeutically effective amount of an anti-IL17RB / anti-TSLP bispecific binding protein. In another specific embodiment, a pharmaceutical composition is provided comprising a therapeutically effective amount of an anti-IL17RB / anti-IL33 bispecific binding protein.

[0098] Based on the data presented herein, it is expected that treating a subject with an allergic disease or disorder with the bispecific binding proteins described herein will provide therapeutic benefit.

[0099] 7.2.1 Exemplary Anti-IL17RB / Anti-Alarmin Bispecific Binding Proteins The present invention provides a bispecific binding protein comprising an Immunoglobulin G antibody (IgG) with specificity for an alarmin receptor, wherein the Immunoglobulin G antibody (IgG) is fused at each C-terminus of the immunoglobulin chain to either (a) a single chain variable fragment (scFv) specific for a particular alarmin, or (b) the extracellular domain of an alarmin receptor.

[0100] In some embodiments, the present invention provides a bispecific antibody comprising an IgG and two scFvs, wherein (a) the IgG comprises two heavy chains (HC) and two light chains (LC), each HC comprising a heavy chain variable region (HCVR1) comprising heavy chain CDRs (HCDRs) 1 to 3, each light chain comprising a light chain variable region (LCVR1) comprising light chain CDRs (LCDRs) 1 to 3, the amino acid sequence of HCDR1 is SEQ ID NO:1, the amino acid sequence of HCDR2 is SEQ ID NO:2, the amino acid sequence of HCDR3 is SEQ ID NO:3, the amino acid sequence of LCDR1 is SEQ ID NO:4, the amino acid sequence of LCDR2 is SEQ ID NO:5, and the amino acid sequence of LCDR3 is SEQ ID NO:6. (b) each scFv comprises a heavy chain variable region (HCVR2) and a light chain variable region (LCVR2), HCVR2 comprises HCDRs 4 to 6, and LCVR2 comprises LCDRs 4 to 6, the amino acid sequence of HCDR4 is SEQ ID NO:7, the amino acid sequence of HCDR5 is SEQ ID NO:8, the amino acid sequence of HCDR6 is SEQ ID NO:9, the amino acid sequence of LCDR4 is SEQ ID NO:10, the amino acid sequence of LCDR5 is SEQ ID NO:11, and the amino acid sequence of LCDR6 is SEQ ID NO:12, and each scFv comprises an N-terminus of HCVR2 of each scFv, and The bispecific antibodies include a HCVR2 of each scFv linked at the C-terminus of the HC to said IgG antibody via a polypeptide linker (L1), and the HCVR2 of each scFv is linked at the C-terminus of the HCVR2 to the LCVR2 of the same scFv at the N-terminus of the LCVR2 of the same scFv via a second polypeptide linker (L2). In some embodiments, the HCDR variants have up to about 3, about 5, about 8, about 10, about 12 or about 15 amino acid substitutions, additions and / or deletions in SEQ ID NOs: 1-3. In some embodiments, the LCDR variants have up to about 3, about 5, about 8, about 10, about 12 or about 15 amino acid substitutions, additions and / or deletions in SEQ ID NOs: 4-6.

[0101] In some embodiments, the present invention provides a bispecific antibody comprising an IgG and two scFvs, wherein (a) the IgG comprises two heavy chains (HC) and two light chains (LC), each HC comprising a heavy chain variable region (HCVR1) comprising heavy chain CDRs (HCDRs) 1 to 3, each light chain comprising a light chain variable region (LCVR1) comprising light chain CDRs (LCDRs) 1 to 3, the amino acid sequence of HCDR1 is SEQ ID NO:1, the amino acid sequence of HCDR2 is SEQ ID NO:2, the amino acid sequence of HCDR3 is SEQ ID NO:3, the amino acid sequence of LCDR1 is SEQ ID NO:4, the amino acid sequence of LCDR2 is SEQ ID NO:5, and the amino acid sequence of LCDR3 is SEQ ID NO:6. (b) each scFv comprises a heavy chain variable region (HCVR2) and a light chain variable region (LCVR2), HCVR2 comprises HCDRs 4 to 6, and LCVR2 comprises LCDRs 4 to 6, the amino acid sequence of HCDR4 is SEQ ID NO:36, the amino acid sequence of HCDR5 is SEQ ID NO:37, the amino acid sequence of HCDR6 is SEQ ID NO:38, the amino acid sequence of LCDR4 is SEQ ID NO:39, the amino acid sequence of LCDR5 is SEQ ID NO:40, and the amino acid sequence of LCDR6 is SEQ ID NO:41, and each scFv comprises an IgG The bispecific antibodies include a HCVR2 of each scFv linked at the C-terminus of the HC to said IgG antibody via a polypeptide linker (L1), and the HCVR2 of each scFv is linked at the C-terminus of the HCVR2 to the LCVR2 of the same scFv at the N-terminus of the LCVR2 of the same scFv via a second polypeptide linker (L2). In some embodiments, the HCDR variants have up to about 3, about 5, about 8, about 10, about 12 or about 15 amino acid substitutions, additions and / or deletions in SEQ ID NOs: 1-3. In some embodiments, the LCDR variants have up to about 3, about 5, about 8, about 10, about 12 or about 15 amino acid substitutions, additions and / or deletions in SEQ ID NOs: 4-6.

[0102] In some embodiments, the present invention provides a bispecific antibody comprising an IgG and two scFvs, wherein (a) the IgG comprises two heavy chains (HC) and two light chains (LC), each HC comprising a heavy chain variable region (HCVR1) comprising heavy chain CDRs (HCDRs) 1 to 3, each light chain comprising a light chain variable region (LCVR1) comprising light chain CDRs (LCDRs) 1 to 3, the amino acid sequence of HCDR1 is SEQ ID NO:1, the amino acid sequence of HCDR2 is SEQ ID NO:2, the amino acid sequence of HCDR3 is SEQ ID NO:3, the amino acid sequence of LCDR1 is SEQ ID NO:4, the amino acid sequence of LCDR2 is SEQ ID NO:5, and the amino acid sequence of LCDR3 is SEQ ID NO:6. (b) each scFv comprises a heavy chain variable region (HCVR2) and a light chain variable region (LCVR2), wherein HCVR2 comprises HCDRs 4 to 6 and LCVR2 comprises LCDRs 4 to 6, the amino acid sequence of HCDR4 is SEQ ID NO:42, the amino acid sequence of HCDR5 is SEQ ID NO:43, the amino acid sequence of HCDR6 is SEQ ID NO:44, the amino acid sequence of LCDR4 is SEQ ID NO:45, the amino acid sequence of LCDR5 is SEQ ID NO:46, and the amino acid sequence of LCDR6 is SEQ ID NO:47, and each scFv comprises an IgG The bispecific antibodies include a HCVR2 of each scFv linked at the C-terminus of the HC to said IgG antibody via a polypeptide linker (L1), and the HCVR2 of each scFv is linked at the C-terminus of the HCVR2 to the LCVR2 of the same scFv at the N-terminus of the LCVR2 of the same scFv via a second polypeptide linker (L2). In some embodiments, the HCDR variants have up to about 3, about 5, about 8, about 10, about 12 or about 15 amino acid substitutions, additions and / or deletions in SEQ ID NOs: 1-3. In some embodiments, the LCDR variants have up to about 3, about 5, about 8, about 10, about 12 or about 15 amino acid substitutions, additions and / or deletions in SEQ ID NOs: 4-6.

[0103] In some embodiments, the present invention provides a bispecific antibody comprising an IgG and two scFvs, wherein (a) the IgG comprises two heavy chains (HC) and two light chains (LC), each HC comprising a heavy chain variable region (HCVR1) comprising heavy chain CDRs (HCDRs) 1 to 3, each light chain comprising a light chain variable region (LCVR1) comprising light chain CDRs (LCDRs) 1 to 3, the amino acid sequence of HCDR1 is SEQ ID NO:1, the amino acid sequence of HCDR2 is SEQ ID NO:2, the amino acid sequence of HCDR3 is SEQ ID NO:3, the amino acid sequence of LCDR1 is SEQ ID NO:4, the amino acid sequence of LCDR2 is SEQ ID NO:5, and the amino acid sequence of LCDR3 is SEQ ID NO:6. (b) each scFv comprises a heavy chain variable region (HCVR2) and a light chain variable region (LCVR2), HCVR2 comprises HCDRs 4 to 6, and LCVR2 comprises LCDRs 4 to 6, the amino acid sequence of HCDR4 is SEQ ID NO:13, the amino acid sequence of HCDR5 is SEQ ID NO:14, the amino acid sequence of HCDR6 is SEQ ID NO:15, the amino acid sequence of LCDR4 is SEQ ID NO:16, the amino acid sequence of LCDR5 is SEQ ID NO:17, and the amino acid sequence of LCDR6 is SEQ ID NO:18, and each scFv comprises an N-terminus of HCVR2 of each scFv, and The bispecific antibodies include a HCVR2 of each scFv linked at the C-terminus of the HC to said IgG antibody via a polypeptide linker (L1), and the HCVR2 of each scFv is linked at the C-terminus of the HCVR2 to the LCVR2 of the same scFv at the N-terminus of the LCVR2 of the same scFv via a second polypeptide linker (L2). In some embodiments, the HCDR variants have up to about 3, about 5, about 8, about 10, about 12 or about 15 amino acid substitutions, additions and / or deletions in SEQ ID NOs: 1-3. In some embodiments, the LCDR variants have up to about 3, about 5, about 8, about 10, about 12 or about 15 amino acid substitutions, additions and / or deletions in SEQ ID NOs: 4-6.

[0104] In some embodiments, the present invention provides a bispecific antibody comprising an IgG and two scFvs, wherein (a) the IgG comprises two heavy chains (HC) and two light chains (LC), each HC comprising a heavy chain variable region (HCVR1) comprising heavy chain CDRs (HCDRs) 1 to 3, each light chain comprising a light chain variable region (LCVR1) comprising light chain CDRs (LCDRs) 1 to 3, the amino acid sequence of HCDR1 is SEQ ID NO:1, the amino acid sequence of HCDR2 is SEQ ID NO:2, the amino acid sequence of HCDR3 is SEQ ID NO:3, the amino acid sequence of LCDR1 is SEQ ID NO:4, the amino acid sequence of LCDR2 is SEQ ID NO:5, and the amino acid sequence of LCDR3 is SEQ ID NO:6. (b) each scFv comprises a heavy chain variable region (HCVR2) and a light chain variable region (LCVR2), wherein HCVR2 comprises HCDRs 4 to 6 and LCVR2 comprises LCDRs 4 to 6, the amino acid sequence of HCDR4 is SEQ ID NO:48, the amino acid sequence of HCDR5 is SEQ ID NO:49, the amino acid sequence of HCDR6 is SEQ ID NO:50, the amino acid sequence of LCDR4 is SEQ ID NO:51, the amino acid sequence of LCDR5 is SEQ ID NO:52, and the amino acid sequence of LCDR6 is SEQ ID NO:53, and each scFv comprises an IgG The bispecific antibodies include a HCVR2 of each scFv linked at the C-terminus of the HC to said IgG antibody via a polypeptide linker (L1), and the HCVR2 of each scFv is linked at the C-terminus of the HCVR2 to the LCVR2 of the same scFv at the N-terminus of the LCVR2 of the same scFv via a second polypeptide linker (L2). In some embodiments, the HCDR variants have up to about 3, about 5, about 8, about 10, about 12 or about 15 amino acid substitutions, additions and / or deletions in SEQ ID NOs: 1-3. In some embodiments, the LCDR variants have up to about 3, about 5, about 8, about 10, about 12 or about 15 amino acid substitutions, additions and / or deletions in SEQ ID NOs: 4-6.

[0105] In some embodiments, the present invention provides a bispecific antibody comprising an IgG and two scFvs, wherein (a) the IgG comprises two heavy chains (HC) and two light chains (LC), each HC comprising a heavy chain variable region (HCVR1) comprising heavy chain CDRs (HCDRs) 1 to 3, each light chain comprising a light chain variable region (LCVR1) comprising light chain CDRs (LCDRs) 1 to 3, the amino acid sequence of HCDR1 is SEQ ID NO:1, the amino acid sequence of HCDR2 is SEQ ID NO:2, the amino acid sequence of HCDR3 is SEQ ID NO:3, the amino acid sequence of LCDR1 is SEQ ID NO:4, the amino acid sequence of LCDR2 is SEQ ID NO:5, and the amino acid sequence of LCDR3 is SEQ ID NO:6. (b) each scFv comprises a heavy chain variable region (HCVR2) and a light chain variable region (LCVR2), wherein HCVR2 comprises HCDRs 4 to 6 and LCVR2 comprises LCDRs 4 to 6, the amino acid sequence of HCDR4 is SEQ ID NO:54, the amino acid sequence of HCDR5 is SEQ ID NO:55, the amino acid sequence of HCDR6 is SEQ ID NO:56, the amino acid sequence of LCDR4 is SEQ ID NO:57, the amino acid sequence of LCDR5 is SEQ ID NO:58 and the amino acid sequence of LCDR6 is SEQ ID NO:59, and each scFv comprises an IgG The bispecific antibodies include a HCVR2 of each scFv linked at the C-terminus of the HC to said IgG antibody via a polypeptide linker (L1), and the HCVR2 of each scFv is linked at the C-terminus of the HCVR2 to the LCVR2 of the same scFv at the N-terminus of the LCVR2 of the same scFv via a second polypeptide linker (L2). In some embodiments, the HCDR variants have up to about 3, about 5, about 8, about 10, about 12 or about 15 amino acid substitutions, additions and / or deletions in SEQ ID NOs: 1-3. In some embodiments, the LCDR variants have up to about 3, about 5, about 8, about 10, about 12 or about 15 amino acid substitutions, additions and / or deletions in SEQ ID NOs: 4-6.

[0106] In some embodiments, the present invention provides a bispecific antibody comprising an IgG and an extracellular domain of an alarmin receptor, wherein (a) the IgG comprises two heavy chains (HC) and two light chains (LC), each HC comprising a heavy chain variable region (HCVR1) comprising heavy chain CDRs (HCDRs) 1-3, each light chain comprising a light chain variable region (LCVR1) comprising light chain CDRs (LCDRs) 1-3, the amino acid sequence of HCDR1 is SEQ ID NO:1, the amino acid sequence of HCDR2 is SEQ ID NO:2, the amino acid sequence of HCDR3 is SEQ ID NO:3, the amino acid sequence of LCDR1 is SEQ ID NO:4, the amino acid sequence of LCDR2 is SEQ ID NO:5, and the amino acid sequence of LCDR3 is SEQ ID NO:6; (b) the extracellular domain of the alarmin receptor comprises the amino acid sequence of SEQ ID NO:19 or SEQ ID NO:34, and each of the alarmin extracellular domains is linked at the C-terminus of each domain's amino acid sequence to the IgG antibody at the C-terminus of each IgG HC via a polypeptide linker (L1). In some embodiments, the HCDR variants have up to about 3, about 5, about 8, about 10, about 12 or about 15 amino acid substitutions, additions and / or deletions in SEQ ID NOs:1-3.

[0107] In some embodiments, the present invention provides a bispecific antibody comprising an IgG and an extracellular domain of an alarmin receptor, wherein (a) the IgG comprises two heavy chains (HC) and two light chains (LC), each HC comprising a heavy chain variable region (HCVR1) comprising heavy chain CDRs (HCDRs) 1-3, each light chain comprising a light chain variable region (LCVR1) comprising light chain CDRs (LCDRs) 1-3, the amino acid sequence of HCDR1 is SEQ ID NO:1, the amino acid sequence of HCDR2 is SEQ ID NO:2, the amino acid sequence of HCDR3 is SEQ ID NO:3, the amino acid sequence of LCDR1 is SEQ ID NO:4, the amino acid sequence of LCDR2 is SEQ ID NO:5, and the amino acid sequence of LCDR3 is SEQ ID NO:6; (b) the extracellular domain of the alarmin receptor comprises the amino acid sequence of SEQ ID NO:20 or SEQ ID and each of the alarmin extracellular domains is linked at the C-terminus of the amino acid sequence of each domain to said IgG antibody at the C-terminus of each IgG HC via a polypeptide linker (L1). In some embodiments, the HCDR variants have up to about 3, about 5, about 8, about 10, about 12 or about 15 amino acid substitutions, additions and / or deletions in SEQ ID NOs:1-3.

[0108] In some embodiments, a bispecific binding protein of the disclosure comprises a polypeptide linker (L1 and / or L2) having a sequence corresponding to a sequence selected from one of the sequences in the table below. TIFF2025508835000004.tif63128

[0109] In a further embodiment of the bispecific binding protein of the invention, the amino acid sequence of each HC is SEQ ID NO: 32 and the amino acid sequence of each LC is SEQ ID NO: 33. In yet a further embodiment of the bispecific binding protein of the invention, the polypeptide linker L1 has the sequence of SEQ ID NO: 30.

[0110] In some embodiments of the bispecific binding proteins of the invention, the amino acid sequence of each LC is SEQ ID NO: 33. In a further embodiment, the amino acid sequence of each HC can be any one selected from SEQ ID NOs: 60-75.

[0111] In some embodiments, the anti-IL17RB IgG-containing bispecific binding protein that may be used in the methods disclosed herein is an IgA, IgD, IgE, IgG, or IgM antibody. In some embodiments, the antibody is an IgA antibody. In some embodiments, the antibody is an IgD antibody. In some embodiments, the antibody is an IgE antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgM antibody. In some embodiments, the antibody provided herein can be an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody. In some embodiments, the antibody is an IgG1 antibody. In some embodiments, the antibody is an IgG2 antibody. In some embodiments, the antibody is an IgG3 antibody. In some embodiments, the antibody is an IgG4 antibody. In certain embodiments, the antibody comprises a heavy chain constant region, e.g., an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, or a constant region of any of the above having a glycosylation site, and / or a modified glycoform at the glycosylation site.

[0112] In some embodiments, the anti-IL17RB binding portion of the bispecific binding protein used in the methods disclosed herein is in the form of a single domain antibody (sdAb), a heavy chain antibody (HCAb), an Fv, a single chain variable region fragment (scFv) or (scFv)2 fused with the constant region of an IgA, IgD, IgE, IgG or IgM antibody. In some embodiments, the antibody is an IgA antibody. In some embodiments, the antibody is an IgD antibody. In some embodiments, the antibody is an IgE antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgM antibody. In some embodiments, the antibody provided herein can be an IgG1 antibody, an IgG2 antibody, an IgG3 antibody or an IgG4 antibody. In some embodiments, the antibody is an IgG1 antibody. In some embodiments, the antibody is an IgG2 antibody. In some embodiments, the antibody is an IgG3 antibody. In some embodiments, the antibody is an IgG4 antibody. In certain embodiments, the antibody comprises a heavy chain constant region, e.g., an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, or a constant region of any of the above having a glycosylation site, and / or a modified glycoform at the glycosylation site.

[0113] In some embodiments, the antigen-binding fragment of an anti-alarmin antibody is used in the methods disclosed herein. In some embodiments, the antigen-binding fragment provided herein can be a single domain antibody (sdAb), a heavy chain antibody (HCAb), a Fab, a Fab', a F(ab')2, an Fv, a single chain variable region fragment (scFv) or a (scFv)2. In some embodiments, the antigen-binding fragment of an anti-alarmin antibody is a single domain antibody (sdAb). In some embodiments, the antigen-binding fragment of an anti-alarmin antibody is a heavy chain antibody (HCAb). In some embodiments, the antigen-binding fragment of an anti-alarmin antibody is a Fab. In some embodiments, the antigen-binding fragment of an anti-alarmin antibody is a Fab'. In some embodiments, the antigen-binding fragment of an anti-alarmin antibody is a F(ab')2. In some embodiments, the antigen-binding fragment of an anti-alarmin antibody is an Fv. In some embodiments, the antigen-binding fragment of an anti-alarmin antibody is a scFv. In some embodiments, the antigen-binding fragment of the anti-alarmin antibody is a disulfide-linked scFv [(scFv)2]. In some embodiments, the antigen-binding fragment of the anti-alarmin antibody is a diabody (dAb). In a further embodiment, the anti-alarmin antigen-binding fragment neutralizes the activity of alarmins, such as IL-25, IL-33 and TSLP.

[0114] The term "activity" includes, for example, properties such as the ability to bind with specificity to a target protein, the affinity of an antibody or binding protein for a protein, the ability to neutralize the biological activity of a target protein, and the ability to inhibit the interaction of a target protein with its natural receptor or natural ligand.

[0115] In some embodiments, the method disclosed herein uses a recombinant anti-IL17RB / anti-TSLP bispecific antibody. In some embodiments, the method disclosed herein uses a recombinant anti-IL17RB / anti-IL33 bispecific antibody. In some embodiments, the method disclosed herein uses a recombinant anti-IL17RB / TSLP receptor bispecific binding protein. In some embodiments, the method disclosed herein uses a recombinant anti-IL17RB / IL33 receptor bispecific binding protein.

[0116] In some embodiments, the IgG-containing bispecific binding protein provided herein is a chimeric antibody. In some embodiments, the IgG-containing bispecific binding protein provided herein is a humanized antibody. In some embodiments, the IgG-containing bispecific binding protein provided herein is a human antibody. In some embodiments, the scFv-containing bispecific binding protein provided herein is a chimeric scFv. In some embodiments, the scFv-containing bispecific binding protein provided herein is a humanized scFv. In some embodiments, the scFv-containing bispecific binding protein provided herein is a human scFv. In some embodiments, the bispecific binding protein used in the methods provided herein is isolated. In some embodiments, the bispecific binding protein used in the methods provided herein is substantially pure.

[0117] Various methods are known in the art for making humanized antibodies and scFvs. Methods are known in the art for obtaining high affinity binding using humanized antibodies. A non-limiting example of such a method is hypermutation of the variable region and selection of cells expressing such high affinity antibodies (affinity maturation). In addition to using display libraries, a designated antigen (e.g., recombinant IL17RB or an epitope thereof) can be used to immunize non-human animals, such as rodents. In some specific embodiments, rodent antigen-binding fragments (e.g., mouse antigen-binding fragments) can be made and isolated using methods known in the art and / or disclosed herein. In some embodiments, mice can be immunized with an antigen (e.g., recombinant IL17RB or an epitope thereof).

[0118] Human antibodies and scFvs can be prepared using a variety of techniques known in the art. In some embodiments, human antibodies are generated from immortalized human B lymphocytes immunized in vitro. In some embodiments, human antibodies are generated from lymphocytes isolated from immunized individuals. In any case, cells producing antibodies against the target antigen can be generated and isolated. In some embodiments, human antibodies are selected from phage libraries expressing human antibodies. Alternatively, phage display technology can be used to generate human antibodies and antibody fragments in vitro from immunoglobulin variable region gene repertoires from non-immunized donors. Techniques for generating and using antibody phage libraries are well known in the art. Once antibodies are identified, higher affinity human antibodies can be generated using affinity maturation strategies known in the art, including but not limited to chain shuffling and site-directed mutagenesis. In some embodiments, human antibodies are generated in transgenic mice containing human immunoglobulin loci. Upon immunization, such mice are capable of producing a full human antibody repertoire in the absence of endogenous immunoglobulin production.

[0119] In some embodiments, the IgG-containing bispecific binding protein used in the methods provided herein comprises a monovalent antigen-binding site. In some embodiments, the IgG-containing bispecific binding protein comprises a monospecific binding site. In some embodiments, the IgG-containing bispecific binding protein comprises a bivalent binding site. In some embodiments, the scFv-containing bispecific binding protein used in the methods provided herein comprises a monovalent antigen-binding site. In some embodiments, the scFv-containing bispecific binding protein comprises a monospecific binding site. In some embodiments, the scFv-containing bispecific binding protein comprises a bivalent binding site.

[0120] In some embodiments, the IgG-containing bispecific binding protein used in the methods provided herein is SM17. The term "SM17" refers to a humanized antibody against human IL17RB (hIL17RB). The sequence characteristics of SM17 are shown in the table below. Further description of the structural and functional characteristics of SM17 can be found in WO2020115319A1, which is incorporated by reference in its entirety. The HC sequence of SM17 is SEQ ID NO:32. The LC sequence of SM17 is SEQ ID NO:32. The amino acid sequence of SM17 HCDR1 is SEQ ID NO:1, the amino acid sequence of SM17 HCDR2 is SEQ ID NO:2, the amino acid sequence of SM17 HCDR3 is SEQ ID NO:3, the amino acid sequence of SM17 LCDR1 is SEQ ID NO:4, the amino acid sequence of SM17 LCDR2 is SEQ ID NO:5, and the amino acid sequence of SM17 LCDR3 is SEQ ID NO:6.

[0121] In some embodiments, IgG-containing bispecific binding proteins that can be used in the methods provided herein comprise one, two, three, four, five, and / or six CDRs of SM17. In some embodiments, the IgG-containing bispecific binding proteins comprise a VL that comprises one, two, and / or three VL CDRs of SM17. In some embodiments, the IgG-containing bispecific binding proteins provided herein comprise a VH that comprises one, two, and / or three VH CDRs of SM17. In some embodiments, the IgG-containing bispecific binding proteins provided herein comprise one, two, and / or three VL CDRs and one, two, and / or three VH CDRs of SM17.

[0122] It is well known in the art that the VH and VL CDR3 domains play an important role in the binding specificity / affinity of an antibody to an antigen. Thus, in some embodiments, the IgG-containing bispecific binding protein that may be used in the methods disclosed herein may have suitable binding / dissociation kinetics with human IL17RB and may have VH and VL CDR3s that are structurally identical or related to those of SM17. The consensus motif of SM17 VL CDR3 comprising the amino acid sequence SEQ ID NO:6 may be modified by substituting one or more amino acids so that the antibody affinity is adjusted without changing the binding specificity, or alternatively, may be replaced with the VL CDR3 of an unrelated human antibody that shows sufficient similarity to SM17 VL CDR3 using criteria such as those described in Chinese Patent No. ZL200880024788.2, which is incorporated herein by reference. Similarly, the consensus motif of SM17 VH CDR3 comprising the amino acid sequence SEQ ID NO:3 may be modified by substituting one or more amino acids to adjust the antibody affinity without altering the binding specificity, or alternatively, may be replaced with the VH CDR3 of an unrelated human antibody that shows sufficient similarity to SM17 VH CDR3 using criteria such as those set forth in Chinese Patent No. ZL200880024788.2, which is incorporated herein by reference.

[0123] One of skill in the art will recognize that certain substitutions of amino acids within the CDR3 domain, particularly conservative amino acids, do not alter the epitope specificity of the antibody. Thus, in some embodiments, the CDR3 of an antibody or antigen-binding fragment provided herein (e.g., SM17 or ) comprises: (1) the same number of residues and 50% or more sequence identity with SM17 CDR3; (2) contains at least one, and preferably more, aromatic residues that are identical or conservatively related to the residues at the corresponding positions in SM17 CDR3; (3) contains at least one, and preferably more, charged residues that are identical or conservatively related to the residues at the corresponding positions in SM17 CDR3; and / or (4) A human or primate antibody that contains at least one, and preferably more, amino acid residues that are identical or conservatively related to the residues at the corresponding positions in SM17 CDR3. or a CDR3 from an unrelated primate or human antibody containing no more than one to five conservatively related residues is used to replace the VL and / or VH CDR3 of SM17. In some embodiments, no more than one to three conservative amino acid substitutions are made in the SM17 VL and / or VH CDR3 domains, or a VL and / or VH CDR3 from an unrelated primate or human antibody containing no more than one to three conservatively related residues is used to replace the VL and / or VH CDR3 of SM17.

[0124] In some embodiments, an IgG-containing bispecific binding protein used in the methods provided herein specifically binds to IL17RB comprising a VL having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:33. In some embodiments, an IgG-containing bispecific binding protein used in the methods provided herein specifically binds IL17RB comprising a VH having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:32. In some embodiments, an IgG-containing bispecific binding protein used in the methods provided herein specifically binds to IL17RB comprising: (a) a VL having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:33; and (b) a VH having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:32.

[0125] In some embodiments, an IgG-containing bispecific binding protein used in the methods provided herein specifically binds to IL17RB comprising a VL having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:33. In some embodiments, an IgG-containing bispecific binding protein used in the methods provided herein has a VL having at least 85% sequence identity to SEQ ID NO:33. In some embodiments, an IgG-containing bispecific binding protein used in the methods provided herein has a VL having at least 90% sequence identity to SEQ ID NO:33. In some embodiments, an IgG-containing bispecific binding protein used in the methods provided herein has a VL having at least 95% sequence identity to SEQ ID NO:33. In some embodiments, an IgG-containing bispecific binding protein used in the methods provided herein has a VL with at least 98% sequence identity to SEQ ID NO: 33. In some embodiments, an IgG-containing bispecific binding protein used in the methods provided herein that specifically binds to IL17RB comprises a VL having the amino acid sequence of SEQ ID NO:33.

[0126] In some embodiments, an IgG-containing bispecific binding protein used in the methods provided herein specifically binds to IL17RB comprising a VH having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:32. In some embodiments, an IgG-containing bispecific binding protein used in the methods provided herein has a VH having at least 85% sequence identity to SEQ ID NO:32. In some embodiments, an IgG-containing bispecific binding protein used in the methods provided herein has a VH having at least 90% sequence identity to SEQ ID NO:32. In some embodiments, an IgG-containing bispecific binding protein used in the methods provided herein has a VH having at least 95% sequence identity to SEQ ID NO:32. In some embodiments, an IgG-containing bispecific binding protein used in the methods provided herein has a VH with at least 98% sequence identity to SEQ ID NO: 32. In some embodiments, an IgG-containing bispecific binding protein used in the methods provided herein that specifically binds to IL17RB comprises a VH having the amino acid sequence of SEQ ID NO:32.

[0127] In some embodiments, the IgG-containing bispecific binding protein that specifically binds to IL17RB comprises a VL and a VH, the VL and VH having the amino acid sequences of SEQ ID NOs: 32 and 33, respectively. The IgG-containing bispecific binding protein that specifically binds to IL17RB can comprise any combination of VL disclosed herein with any VH disclosed herein.

[0128] In some embodiments, an IgG-containing bispecific binding protein that specifically binds to IL17RB comprising: (a) a VL comprising VL CDR1, 2 and 3 derived from a VL having the amino acid sequence of SEQ ID NO:33; and / or (b) a VH comprising VH CDR1, 2 and 3 derived from a VH having the amino acid sequence of SEQ ID NO:32.

[0129] In some embodiments, an IgG-containing bispecific binding protein that specifically binds IL17RB comprises a VL, said VL comprising VL CDR1, 2 and 3 derived from a VL having the amino acid sequence of SEQ ID NO:33.

[0130] In some embodiments, an IgG-containing bispecific binding protein that specifically binds IL17RB comprises a VH, the VH comprising VH CDR1, 2 and 3 derived from a VH having the amino acid sequence of SEQ ID NO:32.

[0131] In some embodiments, an IgG-containing bispecific binding protein that specifically binds IL17RB comprises a VL and a VH, wherein the VL comprises a VL CDR1, CDR2 and CDR3 derived from a VL having the amino acid sequence of SEQ ID NO:33, and the VH comprises a VH CDR1, CDR2 and CDR3 derived from a VH having the amino acid sequence of SEQ ID NO:32.

[0132] In some embodiments, the IgG-containing bispecific binding proteins provided herein are variants of SM17. The SM17 variants may have a VL that is a variant of the VL of SM17 with up to about 5 amino acid substitutions, additions, and / or deletions in SEQ ID NO:33. The SM17 variants may have a VH that is a variant of the VH of SM17 with up to about 5 amino acid substitutions, additions, and / or deletions in SEQ ID NO:32. The amino acid substitutions, additions, and / or deletions may be in the CDRs of the VH or the CDRs of the VL. In some embodiments, the amino acid substitutions, additions, and / or deletions are not in the CDRs. In some embodiments, the SM17 variants have up to about 5 conservative amino acid substitutions. In some embodiments, the SM17 variants have up to 3 conservative amino acid substitutions.

[0133] In some embodiments, the IgG-containing bispecific binding proteins that can be used in the methods disclosed herein comprise a VH or VL having at least one framework (FR) region. In some embodiments, the FR1 region of the VL is κ ID human germline family, and the FR2 region of the VL is V κ 1 human germline family, and the FR3 region of the VL is V κ 1 may be derived from a human germline family, and the FR4 region of the VL may be V κ In some embodiments, the FR1, FR2, FR3, and FR4 of the VL may have the amino acid sequences of SEQ ID NOs: 36, 37, 38, and 39, respectively (framework sequences set forth in WO2020115319A1, which is incorporated herein by reference). In some embodiments, the framework 1 (FR1) region of the VH may have the amino acid sequences of SEQ ID NOs: 36, 37, 38, and 39, respectively (framework sequences set forth in WO2020115319A1, which is incorporated herein by reference). H 3 human germline family; the framework 2 (FR2) region of VH is V H 3 human germline family; the framework 3 (FR3) region of VH is V H3 human germline family; the framework 4 (FR4) region of VH is V H It may be derived from the J5 human germline family. In some embodiments, FR1, FR2, FR3 and FR4 of the VH may have the amino acid sequences of SEQ ID NOs: 40, 41, 42 and 43, respectively (framework sequences shown in WO2020115319A1).

[0134] The present disclosure further contemplates further variants and equivalents that are substantially homologous to the recombinant, monoclonal, chimeric, humanized and human antibodies or antibody fragments described herein.In some embodiments, it is desirable to improve the binding affinity of the antibody.In some embodiments, it is desirable to adjust the biological properties of the antibody, including but not limited to specificity, thermostability, expression level, effector function, glycosylation, immunogenicity and / or solubility.Those skilled in the art will recognize that amino acid changes, such as changing the number or position of glycosylation sites or altering membrane anchoring characteristics, may alter the post-translational processing of the antibody.

[0135] A variation can be a substitution, deletion or insertion of one or more nucleotides encoding the antibody or polypeptide that results in a change in the amino acid sequence when compared to the native antibody or polypeptide sequence. In some embodiments, the amino acid substitution is the result of replacing one amino acid with another amino acid having similar structural and / or chemical properties, e.g., replacing a leucine with a serine, e.g., a conservative replacement of amino acids. The insertion or deletion can range from about 1-5 amino acids. In some embodiments, the substitution, deletion or insertion can include less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions compared to the parent molecule. In some embodiments, biologically useful and / or relevant amino acid sequence variations can be determined by systematically making insertions, deletions or substitutions in the sequence and testing the resulting variant proteins for activity compared to the parent protein.

[0136] It is known in the art that the constant region of an antibody mediates several effector functions, and such effector functions may vary depending on the antibody isotype. For example, the complement system is activated when the C1 component of complement binds to the Fc region of an IgG or IgM antibody (which is bound to an antigen). Complement activation is important in the opsonization and lysis of cellular pathogens. Complement activation may also stimulate inflammatory responses and be involved in autoimmune hypersensitivity. The Fc region of an antibody may also bind to cells expressing Fc receptors (FcR). There are several specific Fc receptors for different antibody classes, such as IgG (gamma receptors), IgE (epsilon receptors), IgA (alpha receptors) and IgM (mu receptors). Binding of antibodies to Fc receptors on cell surfaces elicits many important and diverse biological responses, such as engulfment and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (termed antibody-dependent cellular cytotoxicity or ADCC), release of inflammatory mediators, placental transfer, and control of immunoglobulin production. In some embodiments, the bispecific binding proteins described herein comprise at least one constant region of a human IgA antibody. In some embodiments, the bispecific binding proteins described herein comprise at least one constant region of a human IgD antibody. In some embodiments, the bispecific binding proteins described herein comprise at least one constant region of a human IgE antibody. In some embodiments, the bispecific binding proteins described herein comprise at least one constant region of a human IgG antibody. In some embodiments, the bispecific binding proteins described herein comprise at least one constant region of a human IgM antibody. In some embodiments, the bispecific binding proteins described herein comprise at least one constant region of a human IgG1 antibody. In some embodiments, the bispecific binding proteins described herein comprise at least one constant region of a human IgG2 antibody. In some embodiments, the bispecific binding proteins described herein comprise at least one constant region of a human IgG3 antibody. In some embodiments, the bispecific binding proteins described herein comprise at least one constant region of a human IgG4 antibody.

[0137] In some embodiments, at least one or more constant regions are modified or deleted in the bispecific binding proteins described herein. In some embodiments, the bispecific binding proteins include modifications to one or more of the three heavy chain constant regions (CH1, CH2, or CH3) and / or the light chain constant region (CL). In some embodiments, the heavy chain constant region of the modified bispecific binding protein includes at least one human constant region. In some embodiments, the heavy chain constant region of the modified bispecific binding protein includes more than one human constant region. In some embodiments, the modifications to the constant region include the addition, deletion, or substitution of one or more amino acids in one or more regions.

[0138] In some embodiments, one or more regions are partially or completely deleted from the constant region of the modified bispecific binding protein. In some embodiments, the entire CH2 domain is removed from the bispecific binding protein (ΔCH2 construct). In some embodiments, the deleted constant region is replaced with a short amino acid spacer that provides some of the molecular flexibility typically provided by the absent constant region. In some embodiments, the modified bispecific binding protein comprises a CH3 domain that is fused directly to the hinge region of the bispecific binding protein. In some embodiments, the modified bispecific binding protein comprises a peptide spacer inserted between the hinge region and the modified CH2 and / or CH3 domain.

[0139] In some embodiments, the bispecific binding protein comprises an Fc region. In some embodiments, the Fc region is fused via a hinge. The hinge can be an IgG1 hinge, an IgG2 hinge, or an IgG3 hinge. The amino acid sequences of the Fc regions of human IgG1, IgG2, IgG3, and IgG4 are known to those skilled in the art. In some cases, Fc regions with amino acid variations have been identified in native antibodies. In some embodiments, the modified bispecific binding protein (e.g., the modified Fc region) results in altered effector function, which in turn affects the biological profile of the bispecific binding protein. For example, in some embodiments, the deletion or inactivation (by point mutation or other means) of the constant region reduces Fc receptor binding of the modified bispecific binding protein in circulation. In some embodiments, the modification of the constant region reduces the immunogenicity of the bispecific binding protein. In some embodiments, the modification of the constant region increases the serum half-life of the bispecific binding protein. In some embodiments, the modification of the constant region decreases the serum half-life of the bispecific binding protein. In some embodiments, constant region modifications reduce or eliminate ADCC and / or complement dependent cytotoxicity (CDC) of the bispecific binding protein. In some embodiments, specific amino acid substitutions in the human IgG1 Fc region with corresponding IgG2 or IgG4 residues reduce the effector function (e.g., ADCC and CDC) of the modified bispecific binding protein. In some embodiments, the bispecific binding protein does not have one or more effector functions (e.g., an "effectorless" antibody). In some embodiments, the bispecific binding protein does not have ADCC activity and / or does not have CDC activity. In some embodiments, the bispecific binding protein does not bind to Fc receptors and / or complement factors. In some embodiments, the bispecific binding protein does not have effector functions. In some embodiments, constant region modifications increase or improve ADCC and / or CDC of the bispecific binding protein. In some embodiments, the constant region is modified to eliminate disulfide bonds or oligosaccharide moieties.In some embodiments, the constant region is modified to add / substitute one or more amino acids to provide one or more cytotoxin, oligosaccharide or glycan binding sites. In some embodiments, the bispecific binding protein comprises a variant Fc region that has been engineered with substitutions at specific amino acid positions compared to the native Fc region. In some embodiments, the bispecific binding protein described herein comprises an IgG1 heavy chain constant region comprising one or more amino acid substitutions selected from the group consisting of K214R, L234A, L235E, G237A, D356E and L358M according to EU numbering. In some embodiments, the IgG1 heavy chain constant region comprises one or more amino acid substitutions selected from the group consisting of K214R, L234A, L235E, G237A, A330S, P331S, D356E and L358M according to EU numbering. In some embodiments, the IgG1 heavy chain constant region comprises one or more amino acid substitutions selected from the group consisting of K214R, C226S, C229S and P238S according to EU numbering. In some embodiments, the IgG1 heavy chain constant region comprises one or more amino acid substitutions selected from the group consisting of K214R, D356E and L358M according to EU numbering. In some embodiments, the IgG1 heavy chain constant region comprises one or more amino acid substitutions selected from the group consisting of S131C, K133R, G137E, G138S, Q196K, I199T, N203D, K214R, C226S, C229S and P238S according to EU numbering. In some embodiments, the bispecific binding proteins described herein comprise an IgG2 heavy chain constant region comprising one or more amino acid substitutions selected from the group consisting of V234A, G237A, P238S, H268A, V309L, A330S and P331S. In some embodiments, the bispecific binding proteins described herein comprise an IgG4 heavy chain constant region comprising one or more amino acid substitutions selected from the group consisting of S228P, L234A and L235A.

[0140] In some embodiments, variants may include the addition of amino acid residues at the amino and / or carboxyl termini of the antibody or polypeptide. The length of the added amino acid residues may range from 1 residue to 100 or more residues. In some embodiments, variants include an N-terminal methionyl residue. In some embodiments, variants include an additional polypeptide / protein (e.g., an Fc region) to create a fusion protein. In some embodiments, variants are engineered to be detectable and may include a detectable label and / or protein (e.g., a fluorescent tag or an enzyme).

[0141] The variant antibodies or antigen-binding fragments described herein can be generated using methods known in the art, including but not limited to site-directed mutagenesis, alanine scanning mutagenesis, and PCR mutagenesis.

[0142] In some embodiments, variants of IgG-containing bispecific binding proteins disclosed herein can retain the ability to bind to IL17RB to a similar, equal, or greater extent than the parent antibody or antigen-binding fragment. In some embodiments, the variants can be at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more identical in amino acid sequence to the parent antibody or antigen-binding fragment. In some specific embodiments, variants of IgG-containing bispecific binding proteins comprise the amino acid sequence of the parent IgG-containing bispecific binding protein with one or more conservative amino acid substitutions. Conservative amino acid substitutions are known in the art and include amino acid substitutions in which one amino acid with certain physical and / or chemical properties is replaced with another amino acid with the same or similar chemical or physical properties.

[0143] In some embodiments, a variant of a bispecific binding protein comprises an amino acid sequence of a parent antibody or antigen-binding fragment with one or more non-conservative amino acid substitutions. In some embodiments, a variant of a bispecific binding protein comprises an amino acid sequence of a parent binding antibody or antigen-binding fragment with one or more non-conservative amino acid substitutions, which do not interfere with or inhibit one or more biological activities (e.g., IL17RB binding) of the variant. In some specific embodiments, the one or more conservative amino acid substitutions and / or the one or more non-conservative amino acid substitutions may improve the biological activity of the variant, such that the biological activity of the functional variant is increased when compared to the parent binding moiety.

[0144] In some embodiments, the bispecific binding proteins described herein are chemically modified, either naturally or by intervention. In some embodiments, the bispecific binding proteins are chemically modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization, by known protecting / blocking groups, proteolytic cleavage, and / or linkage with cellular ligands or other proteins. Any of a number of chemical modifications can be made by known techniques. The bispecific binding proteins can include one or more amino acid analogs (e.g., non-natural amino acids, etc.), as well as other modifications known in the art.

[0145] The bispecific binding proteins of the present disclosure may be analyzed for their physical, chemical and / or biological properties by various methods known in the art. In some embodiments, the bispecific binding proteins are tested for their ability to bind to two different alarmins (e.g., human IL17RB and TSLP). Binding assays include, but are not limited to, surface plasmon resonance (e.g., BIAcore), ELISA and FACS. In some embodiments, the dissociation constant of the binding agent (e.g., antibody) to the alarmin is determined by surface plasmon resonance (e.g., BIAcore). Antibodies may also be evaluated for solubility, stability, thermostability, viscosity, expression level, expression quality and / or purification efficiency.

[0146] SM17 binds human IL17RB with a dissociation constant (K D In some embodiments, the bispecific binding proteins used in the methods disclosed herein bind to IL17RB (e.g., human IL17RB) with a dissociation constant (K) of about 100 nM or less, about 40 nM or less, about 20 nM or less, about 10 nM or less, about 1 nM or less, about 0.1 nM or less, 50 pM or less, 10 pM or less, or 1 pM or less. D In some embodiments, K D In some embodiments, the K D In some embodiments, the K D In some embodiments, the K D In some embodiments, the K D In some embodiments, the K D In some embodiments, K D In some embodiments, K D In some embodiments, K D In some embodiments, the K D is about 10 pM or less.

[0147] In some embodiments, the bispecific binding protein binds IL17RB (e.g., human IL17RB) with a K in the range of 0.1-1 nM, 0.5-5 nM, 1-10 nM, 1-5 nM, 5-50 nM, 10-100 nM, or 50-500 nM. D In some embodiments, K D In some embodiments, K is in the range of 0.1 to 1 nM. D In some embodiments, K is in the range of 0.5 to 5 nM. D In some embodiments, K is in the range of 1 to 10 nM. D In some embodiments, K is in the range of 1 to 5 nM. D In some embodiments, K is in the range of 5 to 50 nM. D In some embodiments, K is in the range of 10 to 100 nM. D is in the range of 50 to 500 nM.

[0148] In some embodiments, the bispecific binding protein used in the methods disclosed herein binds IL17RB (e.g., human IL17RB) at a concentration of about 0.8×10 9 M -1 The binding constant (K A In some embodiments, the bispecific binding protein used in the methods disclosed herein binds to IL17RB (e.g., human IL17RB) at about 1×10 6 M -1 More than that, about 1×10 7 M -1 More than that, about 1×10 8 M -1 That's about 5 x 10 8 M -1 That's about 8 x 10 8 M -1 More than that, about 1×10 9 M -1 That's about 5 x 10 9 M -1 More than that, about 1×10 10 M -1 That's about 5 x 10 10 M -1 More than that, about 1×10 11 M -1 That's about 5 x 1011 M -1 or more or about 1×10 12 M -1 More than K A In some embodiments, K A is about 1×10 7 M -1 That is all. In some embodiments, K A is about 5×10 7 M -1 That is all. In some embodiments, K A is about 1×10 8 M -1 That is all. In some embodiments, K A is about 5×10 8 M -1 That is all. In some embodiments, K A is about 8 x 10 8 M -1 That is all. In some embodiments, K A is about 1×10 9 M -1 That is all. In some embodiments, K A is about 5×10 9 M -1 That is all. In some embodiments, K A is about 1×10 10 M -1 That's all.

[0149] In some embodiments, the bispecific binding protein used in the methods disclosed herein binds IL17RB (e.g., human IL17RB) at about 1×10 6 ~1×10 7 M -1 , 5×10 6 ~5×10 7 M -1 , 1×10 7 ~1×10 8 M -1 , 5×10 7 ~5×10 8 M -1 , 1×10 8 ~5×10 8 M -1 , 1×10 8 ~1×10 9 M -1 , 5×108 ~1×10 9 M -1 , 5×10 8 ~5×10 9 M -1 , 1×10 9 ~1×10 10 M -1 , 5×10 9 ~5×10 10 M -1 , 1×10 10 ~1×10 11 M -1 , 5×10 10 ~5×10 11 M -1 , 1×10 11 ~1×10 12 M -1 or 5×10 11 ~5×10 12 M -1 K in the range A In some embodiments, K A is about 1×10 6 ~1×10 7 M -1 In some embodiments, K A is about 1×10 7 ~1×10 8 M -1 In some embodiments, K A is about 1×10 8 ~1×10 9 M -1 In some embodiments, K A is about 5×10 8 ~1×10 9 M -1 In some embodiments, K A is about 5×10 8 ~5×10 9 M -1 In some embodiments, K A is about 1×10 9 ~1×10 10 M -1 is within the range.

[0150] In some embodiments, the bispecific binding protein used in the methods disclosed herein binds to human IL17RB in a concentration of 1.38×10 -7 s -1 In some embodiments, the bispecific binding protein used in the methods disclosed herein dissociates from human IL17RB with a kd of about 5×10 -4 s -1 Below, approximately 1×10 -4 s -1 Below, approximately 2×10 -5 s -1 Below, approximately 4×10 -6 s -1 Below, approximately 8×10 -7 s -1 Below, approximately 2×10 -7 s -1 Below, approximately 4×10 -8 s -1 In some embodiments, the bispecific binding protein used in the methods disclosed herein dissociates from human IL17RB with a kd of about 5×10 -4 s -1 In some embodiments, the kd is about 1×10 -4 s -1 In some embodiments, the kd is about 2×10 -5 s -1 In some embodiments, the kd is about 4×10 -6 s -1 In some embodiments, the kd is about 8×10 -7 s -1 In some embodiments, the kd is about 2×10 -7 s -1 In some embodiments, the kd is about 4×10 -8 s -1 The following is the result.

[0151] Epitope mapping is a method to identify the binding site, region or epitope on target protein that bispecific binding protein binds.Various methods for mapping epitopes on target protein are known in the art.Such methods include, but are not limited to, shotgun mutagenesis, site-directed mutagenesis and alanine scanning; domain or fragment scanning; peptide scanning (e.g., Pepscan technology); display methods (e.g., phage display, microbial display and ribosome / mRNA display); methods involving proteolysis and mass spectrometry; and structure determination (e.g., X-ray crystallography and NMR).In some embodiments, the bispecific binding protein described herein is characterized by assays such as, but not limited to, N-terminal sequencing, amino acid analysis, HPLC, mass spectrometry, ion exchange chromatography and papain digestion.

[0152] In some embodiments, a bispecific binding protein that may be used in the methods disclosed herein binds to this structural epitope at a concentration of about 0.8×10 9 M -1 K A In some embodiments, the bispecific binding proteins used in the methods disclosed herein can bind to this structural epitope at about 1×10 7 M -1 More than that, about 1×10 8 M -1 That's about 5 x 10 8 M -1 More than that, about 1×10 9 M -1 That's about 5 x 10 9 M -1 More than that, about 1×10 10 M -1 That's about 5 x 10 10 M -1 More than that, about 1×10 11 M -1 That's about 5 x 10 11 M -1 or more or about 1×10 12 M -1 More than K AIn some embodiments, K A is about 1×10 7 M -1 That is all. In some embodiments, K A is about 5×10 7 M -1 That is all. In some embodiments, K A is about 1×10 8 M -1 That is all. In some embodiments, K A is about 5×10 8 M -1 That is all. In some embodiments, K A is about 8 x 10 8 M -1 That is all. In some embodiments, K A is about 1×10 9 M -1 That is all. In some embodiments, K A is about 5×10 9 M -1 In some embodiments, the bispecific binding protein used in the methods disclosed herein binds to this structural epitope at about 1×10 6 ~1×10 7 M-1, 5×10 6 ~5×10 7 M-1, 1×10 7 ~1×10 8 M-1, 5×10 7 ~5×10 8 M-1, 1×10 8 ~5×10 8 M-1, 1×10 8 ~1×10 9 M-1, 5×10 8 ~1×10 9 M-1, 5×10 8 ~5×10 9 M-1, 1×10 9 ~1×10 10 M-1, 5×10 9 ~5×10 10 M-1, 1×10 10 ~1×10 11 M-1, 5×10 10 ~5×10 11 M-1, 1×10 11 ~1×10 12M-1 or 5 × 10 11 ~5×10 12 K within M-1 range A In some embodiments, K A is about 1×10 6 ~1×10 7 In some embodiments, K is in the range of M-1. A is about 1×10 6 ~1×10 7 In some embodiments, K is in the range of M-1. A is about 1×10 7 ~1×10 8 In some embodiments, the K is in the range of about 1×10 to 1×10 M. In some embodiments, the K A is about 5×10 8 ~1×10 9 In some embodiments, K is in the range of M-1. A is about 5×10 8 ~5×10 9 In some embodiments, K is in the range of M-1. A is about 1×10 9 ~1×10 10 It is within the range of M-1.

[0153] In some embodiments, the present invention provides a bispecific binding protein that competes with another bispecific binding protein (e.g., human IL17RB) for binding to alarmin. A bispecific binding protein that "competes with another antibody for binding to a target" refers to a bispecific binding protein that inhibits (partially or completely) the binding of the other bispecific binding protein to the same target. Whether or not they compete with each other for binding to a target, i.e., whether and to what extent a bispecific binding protein inhibits the binding of another bispecific binding protein to a target, can be determined using known competition experiments, such as Bio-layer Interferometry Kinetic Analysis. In some embodiments, the bispecific binding protein that contains the bispecific binding protein competes with the binding of another bispecific binding protein to alarmin (e.g., human IL17RB) and inhibits it by at least 50%, 60%, 70%, 80%, 90% or 100%. Competitive assays can be carried out, for example, as described by Ed Harlow and David Lane, Cold Spring Harb Protoc; 2006; doi:10.H0l / pdb.prot4277 or Ed Harlow and David Lane, " Using Antibodies ", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA 1999, Chapter 11.

[0154] In some embodiments, the bispecific binding protein used in the methods disclosed herein binds a radiolabeled IL17RB to native IL17RB on renal cancer cells (e.g., TK-10 cells). 125 - SM17 binding and an EC of about 0.1 μg / ml or less, about 0.2 μg / ml or less, about 0.5 μg / ml or less, about 0.8 μg / ml or less, about 1 μg / ml or less, about 2 μg / ml or less, about 5.0 μg / ml or less, about 8 μg / ml or less, about 10 μg / ml or less, or about 50 μg / ml or less 50 In some embodiments, the EC 50 In some embodiments, the EC 50In some embodiments, the EC 50 In some embodiments, the EC 50 In some embodiments, the EC 50 In some embodiments, the EC 50 In some embodiments, the EC 50 In some embodiments, the bispecific binding protein used in the methods disclosed herein binds to a radiolabeled IL17RB antibody against native IL17RB on renal cancer cells (e.g., TK-10 cells). 125 - SM17 binding with an EC within the range of about 0.1-50 μg / ml, about 0.1-10 μg / ml, about 0.1-5 μg / ml, about 0.5-10 μg / ml, about 0.5-5 μg / ml, about 1-10 μg / ml or about 1-5 μg / ml 50 In some embodiments, the EC 50 In some embodiments, the EC 50 In some embodiments, the EC 50 In some embodiments, the EC 50 In some embodiments, the EC 50 In some embodiments, the EC 50 In some embodiments, the EC 50 is in the range of approximately 1 to 5 μg / ml.

[0155] In some embodiments, the bispecific binding proteins provided herein can be derivatized or linked to another functional molecule (e.g., another peptide or protein) and used in the methods disclosed herein. Thus, in some embodiments, the antibodies and antigen-binding fragments used in the methods disclosed herein include derivatized and otherwise modified forms of the human anti-IL17RB antibodies described herein, such as immune adhesion molecules. For example, the antibodies and antigen-binding fragments can be functionally linked (by chemical coupling, genetic fusion, non-covalent binding or introduction of artificial amino acids / functional groups suitable for site-specific conjugation) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or diabody), a detectable agent, a cytotoxic agent, a pharmaceutical agent, and / or a protein or peptide that can mediate the binding of the antibody or antigen-binding fragment to another molecule (e.g., a streptavidin core region or a polyhistidine tag).

[0156] In some embodiments, the bispecific binding proteins described herein are conjugated to detectable substances or molecules that allow the agents to be used for diagnosis and / or detection. Detectable substances can also include, but are not limited to, enzymes, such as horseradish peroxidase, alkaline phosphatase, glucose oxidase, beta-galactosidase, and acetylcholinesterase; prosthetic groups, such as biotin and flavins; fluorescent substances, such as umbelliferone, fluorescein, fluorescein isothiocyanate (FITC), rhodamine, tetramethylrhodamine isothiocyanate (TRITC), dichlorotriazinylamine fluorescein, dansyl chloride, cyanine (Cy3), 5-dimethylamine-1-naphthalenesulfonyl chloride, and phycoerythrin; bioluminescent substances, such as luciferase; radioactive substances, such as 212 Bi, 14 C. 57 Co, 51 Cr, 67 Cu, 18 F, 68 Ga,67 Ga, 153 Gd, 159 Gd, 68 Ge, 3 H, 166 Ho, 131 I, 125 I, 123 I, 121 I, 115 In, 113 In, 112 In, 111 In, 140 La, 177 Lu, 54 Mn, 99 Mo, 32 P, 103 Pd, 149 Pm, 142 Pr, 186 Re, 188 Re, 105 Rh, 97 Ru, 35 S, 47 Sc, 75 Se, 153 Sm, 113 Sn, 117 Sn, 85 Sr, 99m Tc, 201 Ti, 133 Xe, 90 Y, 69 Yb, 175 Yb, 65 Zn; positron emitting metals; and magnetic metal ions Positron emitting metals; and magnetic metal ions may also be mentioned.

[0157] The bispecific binding proteins described herein may be attached to a solid support. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene. In some embodiments, the immobilized bispecific binding proteins are used in immunoassays. In some embodiments, the immobilized IgG-containing bispecific binding proteins are used in the purification of target antigens (e.g., human IL17RB).

[0158] 7.3 Preparation method Bispecific binding proteins and antibodies thereof that may be used in the methods disclosed herein, including but not limited to bispecific antibodies, antibody-alarmin receptor fusion proteins, monoclonal antibodies, chimeric antibodies, human antibodies, and humanized antibodies, may be prepared by any method disclosed herein or known in the art. Methods for making antibodies are well known in the art. See, for example, Harlow et al., ANTIBODIES:A LABORATORY MANUAL ,(Cold Spring Harbor Labora to Hammerling et al.: Monoclonal Antibodies and T-Cell Hybridomas 563, 681 (Elsevier, NY, 1981), each of which is incorporated by reference in its entirety.

[0159] In some embodiments, the bispecific binding proteins that can be used in the methods provided herein are recombinant, i.e., prepared, expressed, produced or isolated by recombinant means. In some embodiments, the bispecific binding proteins disclosed herein can be prepared, for example, by introducing a recombinant expression vector into a host cell, a recombinant combinatorial human antibody library, an antibody isolated from an animal (e.g., a mouse) transgenic for human immunoglobulin genes [Taylor, LD et al. Nucleic Acids Research, vol. 20, 23 (1992): 6287-95], or an antibody prepared, expressed, produced or isolated by any other means involving splicing of human immunoglobulin gene sequences into other DNA sequences.

[0160] In some embodiments, bispecific binding proteins can be prepared by recombinant expression of immunoglobulin light and heavy chain genes in a host cell. To express a bispecific binding protein, a host cell is introduced with one or more recombinant expression vectors carrying DNA fragments encoding the immunoglobulin light and heavy chains of the bispecific binding protein such that the light and heavy chains are expressed in the host cell and preferably secreted into the medium in which the host cell is cultured, from which the bispecific binding protein can be recovered. Standard recombinant DNA methodologies, such as those described in Sambrook, Fritsch and Maniais (eds), Bispecific Binding Protein Heavy and Light Chain Genes, Incorporating These Genes into a Recombinant Expression Vector and Introducing the Vector into a Host Cell, can be used. MOLECULAR CLONING:A LABORATORY MANUAL ,Second Edition, Cold Spring Harbor, NY,(1989),Ausubel et al.(eds.) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY , Greene Publishing Associates, (1989) and U.S. Pat. No. 4,816,397 are used.

[0161] To express a recombinant bispecific binding protein, such as a SM17-related bispecific binding protein, first obtain DNA fragments encoding the light and heavy chain variable regions. These DNAs can be obtained by hybridoma amplification and modification of the light and heavy chain variable sequences of a mouse antibody using polymerase chain reaction (PCR), or by oligo synthesis using standard methods known to those skilled in the art based on the amino acid sequences encoding the designed light and heavy chain variable sequences. The coding DNA sequences can be further optimized to facilitate mammalian expression of the resulting antibody.

[0162] Once the VH and VL fragments of the mouse antibody are obtained, these sequences can be mutated to encode framework-patched versions, as described in WO2020115319A1, which is incorporated herein by reference in its entirety.

[0163] Once DNA fragments encoding the VH and VL segments of the bispecific binding protein are obtained (e.g., by amplifying and mutating the original mouse VH and VL genes as described above), these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example to convert the variable region genes into full-length antibody chain genes, Fab fragment genes or scFv genes. In such manipulations, the VL-encoding DNA fragment or the VH-encoding DNA fragment is operably linked to another DNA fragment encoding another protein, for example an antibody constant region or a flexible linker. The term "operably linked" as used in this context is intended to mean that the two DNA fragments are joined in such a way that the amino acid sequences encoded by the two DNA fragments remain in frame.

[0164] The isolated VH region-encoding DNA can be converted to a full-length heavy chain gene by operably linking the VH-encoding DNA to another DNA molecule encoding a heavy chain constant region (CH1, CH2 and CH3). The sequences of human heavy chain constant region genes are known in the art (see, for example, Kabat, EA, et al (1991)). SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST , Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242), and a DNA fragment containing this region can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, Ig4, IgA, IgE, IgM or IgD constant region, but is most preferably an IgG1 or IgG4 constant region. In the Fab fragment heavy chain gene, the VH-encoding DNA can be operably linked to another DNA molecule encoding only the CH1 constant region of the heavy chain.

[0165] The isolated VL region-encoding DNA can be converted to a full-length light chain gene (as well as a Fab light chain gene) by operably linking the VL-encoding DNA to another DNA molecule encoding the light chain constant region, CL. The sequences of human light chain constant region genes are known in the art (see, for example, Kabat, EA, et al (1991)). SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST (See, e.g., Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242), and a DNA fragment containing this region can be obtained by standard PCR amplification. The light chain constant region can be a kappa constant region or a lambda constant region, but is most preferably a kappa constant region.

[0166] To prepare an scFv gene, the VH-encoding DNA fragment and the VL-encoding DNA fragment are operably linked to another fragment encoding a flexible linker, such as another fragment encoding the amino acid sequence (Gly4-Ser)3, such that the VH and VL sequences can be expressed as a continuous single-chain protein in which the VL and VH regions are linked by the flexible linker [Bird, RE et al. Science, (New York, NY) vol. 242, 4877 (1988): 423-6; Huston, JS et al. Proceedings of the National Academy of Sciences of the United States of America, vol. 85, 16 (1988): 5879-83; McCafferty, J et al. Nature, vol. 348, 6301 (1990): 552-4].

[0167] To express the bispecific binding protein that can be used in the methods disclosed herein, DNA encoding the light and heavy chains, either partial or full length, obtained as described above, is inserted into an expression vector such that the genes are operably linked to transcriptional and translational control sequences. In this context, the term "operably linked" is intended to mean that the antibody gene is ligated into a vector such that the transcriptional and translational control sequences in the vector perform their intended function of regulating the transcription and translation of the antibody gene. The expression vector and expression control sequences are selected to be compatible with the expression host cell used. The antibody light chain gene and the bispecific binding protein heavy chain gene can be inserted into separate vectors, or more typically, both genes are inserted into the same expression vector. The antibody gene is inserted into the expression vector by standard methods (e.g., ligation of the vector with complementary restriction sites on the antibody gene fragment, or blunt end ligation if no restriction sites are present). In some embodiments, prior to insertion of the SM17-related bispecific binding protein light or heavy chain sequences, the expression vector already carries the bispecific binding protein constant region sequences. For example, one approach for converting the SM17-related bispecific binding protein VH and VL sequences into a full-length bispecific binding protein gene is to insert them into an expression vector already encoding the heavy and light chain constant regions, respectively, such that the VH segment is operably linked to a CH segment in the vector and the VL segment is operably linked to a CL segment in the vector. Additionally or alternatively, the recombinant expression vector may encode a signal peptide that facilitates secretion of the antibody chain from the host cell. The antibody chain gene may be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

[0168] In addition to the bispecific binding protein chain genes, the recombinant expression vectors provided herein may carry regulatory sequences that control the expression of the bispecific binding protein chain genes in a host cell. The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the bispecific binding protein chain genes. Such regulatory sequences are described, for example, in Goeddel; GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Those skilled in the art will recognize that the design of an expression vector, such as the selection of regulatory sequences, depends on factors such as the choice of the host cell to be transformed and the level of expression of protein desired. Preferred regulatory sequences for expression in mammalian host cells include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and / or enhancers derived from the immunoglobulin heavy chain (IgH) enhancer (Gillies, SD et al. Cell, vol. 33, 3 (1983): 717-28. doi: 10.1016 / 0092-8674 (83) 90014-4), metallothionein (MT), cytomegalovirus (CMV) (e.g., the CMV promoter / enhancer), simian virus 40 (SV40) (e.g., the SV40 promoter / enhancer), adenovirus (e.g., the adenovirus major late promoter (AdMLP)), and polyoma. For further description of viral regulatory elements and sequences thereof, see, e.g., U.S. Patent Nos. 5,665,578; 5,168,062; 4,510,245; and 4,968,615.

[0169] In addition to the gene and regulatory sequence of the bispecific binding protein chain, the recombinant expression vector provided herein may carry additional sequences, such as sequences that regulate the replication of the vector in host cells (e.g., origin of replication) and selectable marker genes. The selectable marker gene facilitates the selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and 5,179,017). For example, typically, the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, to the host cells into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr- host cells using methotrexate selection / amplification), the glutamate synthase (GS) gene and the neo gene (for G418 selection).

[0170] For expression of the light and heavy chains, expression vectors encoding the heavy and light chains are transfected into a host cell by standard techniques. The term "transfection" in its various forms is intended to encompass a wide variety of techniques commonly used for the introduction of foreign DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, lipofection, protoplast fusion, etc. Although bispecific binding proteins can be produced in either prokaryotic or eukaryotic host cells, expression of bispecific binding proteins in eukaryotic cells, particularly mammalian host cells, is preferred, since such host cells are more amenable to synthesis and secretion of properly folded, immunologically active bispecific binding proteins than prokaryotic cells.

[0171] Preferred mammalian host cells for expressing recombinant bispecific binding proteins for use in the methods described herein include SP2 / 0 myeloma cells, NSO myeloma cells, COS cells and Chinese hamster ovary (CHO) cells (e.g., dfhr-CHO cells as described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4200 used with a DHFR selectable marker as described in RJ Kaufman and PA Sharp (1982) J. Mol. Biol. 159:601-621). Once the recombinant protein-encoding expression vector has been introduced into the mammalian host cell, the bispecific binding protein is produced by culturing the host cell for a period of time sufficient to allow expression of the bispecific binding protein within the host cell or, more preferably, for a period of time sufficient to allow secretion of the bispecific binding protein into the culture medium in which the host cell is cultured. The bispecific binding protein may be recovered from the culture medium using standard protein purification methods.

[0172] Host cells may also be used to produce portions of intact antibodies, such as Fab fragments or scFv molecules. Variations of the above procedures are clearly contemplated herein. For example, it may be desirable to transfect host cells with DNA encoding either the light or heavy chain (but not both) of the antibody used in the methods disclosed herein. Recombinant DNA techniques may also be used to remove some or all of the DNA encoding either or both of the light and heavy chains that are not necessary for binding to alarmins. Molecules expressed from such truncated DNA molecules are also encompassed by the antibodies provided herein. Bispecific binding proteins, in which one heavy chain and one light chain are specific for human IL17RB and the other heavy and light chains are specific for an antigen other than IL17RB, may also be made by crosslinking the antibody of the invention with a second antibody by standard chemical crosslinking methods.

[0173] In some embodiments of the recombinant expression system of the bispecific binding proteins that can be used in the methods disclosed herein, a recombinant expression vector encoding both the heavy chain and the antibody light chain is introduced into SP2 / 0 cells by electroporation. In some embodiments of the expression system, a recombinant expression vector encoding both the heavy chain and the antibody light chain is introduced into CHO cells by standard techniques, such as lipofection.

[0174] In the recombinant expression vector, the heavy and light chain genes are each operably linked to mouse or human immunoglobulin heavy chain (IgH), CMV enhancer, metallothionein or AdMLP promoter regulatory elements to drive high levels of transcription of the genes. The recombinant expression vector also carries a DHFR gene that allows SP2 / 0 cells transfected with the vector to be selected using methotrexate selection / amplification. Alternatively, a recombinant expression vector containing the heavy and light chain genes operably linked to mouse or human IgH, CMV enhancer / AdMLP / metallothionein promoter regulatory elements and a DHFR gene can be used to transfect SP2 / 0 or CHO cells that are dhfr-. SP2 / 0 or CHO cells transfected with the vector can be selected, and the gene expression level in the vector is amplified by increasing the methotrexate level in the culture medium. The selected transformant host cells are cultured to allow expression of the heavy and light chains, and the intact bispecific binding protein is recovered from the culture medium. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells, and recover the bispecific binding protein from the culture medium.

[0175] 7.4 Pharmaceutical Compositions Also provided herein is a pharmaceutical composition comprising the bispecific binding protein that can be used in the method disclosed herein.In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the bispecific binding protein disclosed herein and a pharma- ceutical acceptable carrier.In some embodiments, the pharmaceutical composition is useful for treating AD and asthma.In some embodiments, the pharmaceutical composition is useful for preventing the progression of AD and asthma in a subject (e.g., a human patient).

[0176] The amount of therapeutic bispecific binding protein that may be combined with a carrier material in the pharmaceutical compositions disclosed herein may vary. In some embodiments, the amount of bispecific binding protein present in the pharmaceutical composition is an amount that provides a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 0.01 percent to about 99 percent of active ingredient, from about 0.1 percent to about 70 percent of active ingredient, or from about 1 percent to about 30 percent, in combination with a pharma- ceutically acceptable carrier.

[0177] The pharmaceutical compositions provided herein comprise a bispecific binding protein provided herein, such as a SM17-related bispecific binding protein. The bispecific binding protein may be present in various concentrations. In some embodiments, the pharmaceutical compositions provided herein comprise 1-1000 mg / ml of a soluble bispecific binding protein provided herein. In some embodiments, the pharmaceutical compositions comprise 10-500 mg / ml, 10-400 mg / ml, 10-300 mg / ml, 10-200 mg / ml, 10-100 mg / ml, 20-100 mg / ml, or 50-100 mg / ml of a soluble bispecific binding protein provided herein. In some embodiments, the pharmaceutical compositions provided herein comprise about 10 mg / ml, about 20 mg / ml, about 30 mg / ml, about 40 mg / ml, about 50 mg / ml, about 60 mg / ml, about 70 mg / ml, about 80 mg / ml, about 90 mg / ml, about 100 mg / ml, about 120 mg / ml, about 150 mg / ml, about 180 mg / ml, about 200 mg / ml, about 300 mg / ml, about 500 mg / ml, about 800 mg / ml, or about 1000 mg / ml of a bispecific binding protein provided herein. Dosages can be readily adjusted by one of skill in the art; for example, lower purity requires higher dosages.

[0178] The pharmaceutical compositions provided herein can be in various forms.These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injection and infusion solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories.Preferred forms depend on intended mode of administration and therapeutic application.Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions or formulations described herein include water, ethanol, polyol (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil and injectable organic esters such as ethyl oleate.

[0179] Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. In some embodiments, the pharmaceutical compositions provided herein are in the form of a liquid for injection or infusion. In some embodiments, the pharmaceutical compositions are aqueous formulations. Such formulations are typically solutions or suspensions, but may also include colloids, dispersions, emulsions, and multi-phase materials. The term "aqueous formulation" is defined as a formulation that contains at least 50% w / w water. Similarly, the term "aqueous liquid" is defined as a liquid that contains at least 50% w / w water, and the term "aqueous suspension" is defined as a suspension that contains at least 50% w / w water. The compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentration.

[0180] In some embodiments, the pharmaceutical compositions disclosed herein are in a freeze-dried form to which the physician or the patient adds solvents and / or diluents prior to use.

[0181] The pharmaceutical compositions provided herein can include pharmaceutically acceptable carriers.Pharmaceutically acceptable carriers include any physiologically compatible solvent, dispersion medium, coating, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. Examples include water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, etc., and combinations thereof.In some embodiments, pharmaceutically acceptable carriers include isotonic agents in the composition, such as sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride.

[0182] In some embodiments, the pharma- ceutically acceptable carrier further comprises minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antibody or antigen-binding fragment. In some embodiments, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active ingredient (i.e., the bispecific binding protein) may be coated with a material to protect the active ingredient from the action of acids and other natural conditions that may inactivate the active ingredient.

[0183] Also provided herein is a kit for preparing a pharmaceutical composition having a bispecific binding protein disclosed herein, such as a SM17-related bispecific binding protein. In some embodiments, the kit comprises a bispecific binding protein disclosed herein and a pharma- ceutically acceptable carrier in one or more containers. In another embodiment, the kit can comprise a binding protein disclosed herein for administration to a subject. In a specific embodiment, the kit includes instructions for preparing and / or administering the bispecific binding protein.

[0184] In some embodiments, the pharmaceutical composition or formulation disclosed herein comprises: (a) a bispecific binding protein disclosed herein; (b) a buffer; (c) a stabilizer; (d) a salt; (e) a bulking agent; and / or (f) a surfactant. In some embodiments, the pharmaceutical composition or formulation is stable for at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 1 year, at least 2 years, at least 3 years, at least 5 years or longer. In some embodiments, the pharmaceutical composition or formulation is stable when stored at 4°C, 25°C or 40°C. In some embodiments, also provided herein are pharmaceutical compositions or formulations that improve the stability of the bispecific binding protein to allow for its long-term storage. The pharmaceutical composition disclosed herein may further comprise one or more of a preservative, an isotonicity agent, a chelating agent, a stabilizer and / or a surfactant, as well as various combinations thereof. The use of preservatives, isotonicity agents, chelating agents, stabilizers and surfactants in pharmaceutical compositions is well known to those skilled in the art. See Remington: The Science and Practice of Pharmacy, 19th edition, 1995.

[0185] Buffers useful in the pharmaceutical compositions or formulations disclosed herein can be weak acids or weak bases used to maintain the acidity (pH) of a solution near a selected value after the addition of another acid or base. A suitable buffer can be one that maximizes the stability of a pharmaceutical formulation by maintaining pH control of the formulation. A suitable buffer can also be one that ensures physiological compatibility or optimizes solubility. Rheology, viscosity, and other properties can also depend on the pH of the formulation. Common buffers include, but are not limited to, histidine, citrate, succinate, acetate, and phosphate. In some embodiments, the buffer includes histidine (e.g., L-histidine) along with an isotonicity agent and potential pH adjustment with an acid or base known in the art. In some specific embodiments, the buffer is L-histidine. In some specific embodiments, the pH of the formulation is maintained at about 2 to about 10 or about 4 to about 8.

[0186] Stabilizing agents are added to pharmaceutical products to stabilize them. Such agents can stabilize proteins in various ways. Common stabilizers include, but are not limited to, amino acids such as glycine, alanine, lysine, arginine or threonine, carbohydrates such as glucose, sucrose, trehalose, raffinose or maltose, polyols such as glycerol, mannitol, sorbitol, cyclodextrin or dextran of any type and molecular weight or PEG. In some embodiments, the stabilizer is selected to maximize the stability of FIX polypeptide in lyophilized preparation. In some specific embodiments, the stabilizer is sucrose and / or arginine.

[0187] Fillers may be added to pharmaceutical compositions or formulations to add volume and mass to the product, thereby facilitating its accurate measurement and handling. Common fillers include, but are not limited to, lactose, sucrose, glucose, mannitol, sorbitol, calcium carbonate, or magnesium stearate.

[0188] Surfactants are amphiphilic substances that have lyophilic and lyophobic groups.Surfactants can be anionic, cationic, zwitterionic or nonionic.Examples of nonionic surfactants include, but are not limited to, alkyl ethoxylates, nonylphenol ethoxylates, amine ethoxylates, polyethylene oxide, polypropylene oxide, fatty alcohols such as cetyl alcohol or oleyl alcohol, cocamide MEA, cocamide DEA, polysorbate or dodecyl dimethylamine oxide.In some embodiments, the surfactant is polysorbate 20 or polysorbate 80.

[0189] Pharmaceutical compositions disclosed herein may also contain pharmaceutically acceptable antioxidants. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butyl hydroxyanisole (BHA), dibutyl hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, etc.; and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0190] Such compositions may also contain adjuvants, such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the presence of microorganisms can be ensured both by the above-mentioned sterilization procedures and by including various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol sorbic acid, etc. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, etc., in the compositions. Prolonged absorption of injectable pharmaceutical forms can also be brought about by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0191] Pharmaceutical compositions or preparations must typically be sterile and stable under the conditions of manufacture and storage. Pharmaceutically acceptable carriers include sterile aqueous solutions or suspensions and sterile powders for extemporaneous preparation of sterile injectable solutions or suspensions. Sterile injectable solutions can be prepared by incorporating the required amount of therapeutic antibody or antigen-binding fragment in a suitable solvent, with one or a combination of the above-listed components as required, followed by sterile filtration. The use of such media and agents for pharmaceutically active substances is known in the art. In general, dispersions are prepared by incorporating active compounds into a sterile vehicle that contains a base dispersion medium and the other required components listed above. In the case of sterile powders for preparation of sterile injectable solutions, one example of the preparation method is vacuum drying and freeze-drying (lyophilization) to obtain a powder of active ingredient plus any additional desired components from its solution that has been previously sterile-filtered.

[0192] The pharmaceutical compositions disclosed herein can be prepared with carriers that protect active ingredients against rapid release, and can be, for example, controlled release formulations, such as implants, transdermal patches and microencapsulated delivery systems.Biodegradable biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid.Many methods for preparing such formulations are patented or generally known to those skilled in the art.See, for example, SUSTAINED AND CONTROLLED RELEASE DRUG DELIVERY SYSTEMS, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0193] In some embodiments, the bispecific binding proteins described herein can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) ​​excludes many highly hydrophilic compounds. To facilitate the passage of the therapeutic antibodies described herein through the BBB, they can be formulated, for example, in liposomes. For methods of making liposomes, see, for example, U.S. Patents 4,522,811; 5,374,548; and 5,399,331. Liposomes may contain one or more moieties that selectively transport into specific cells or organs, thus improving targeted drug delivery (see, for example, VV Ranade (1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties include folate or biotin [see, e.g., U.S. Patent 5,416,016 to Low et al.] mannosides [Umezawa, F, and Y Eto. Biochemical And Biophysical Research Communications vol. 153, 3 (1988): 1038-44.]; antibodies [Bloemen, PG et al. FEBS letters vol. 357, 2 (1995): 140-4.] [Owais, M et al. Antimicrobial Agents And Chemotherapy vol. 39, 1 (1995): 180-4.]; surfactant protein A receptor [Briscoe, P et al. The American Journal Of Physiology vol. 268, 3 Pt 1 (1995): L374-80]; pl20 [Schreier, H et al. The Journal Of Biological Chemistry vol.269,12(1994):9090-8][Keinanen,K,and ML Laukkanen.FEBS letters vol.346,1(1994):123-6][Killion,JJ,and IJ Fidler.ImmunoMethods vol.4,3(1994):273-9].

[0194] 7.5 Treatment Method As described in the above section, herein is provided a medical use of a bispecific binding protein (e.g., SM17-related bispecific binding protein) in the treatment of allergic diseases or disorders. Any bispecific binding protein disclosed herein may be used in the methods disclosed herein. In some embodiments, the method disclosed herein is used with a recombinant anti-IL17RB / anti-TSLP bispecific antibody. In some embodiments, the method disclosed herein is used with a recombinant anti-IL17RB / anti-IL33 bispecific antibody. In some embodiments, the method disclosed herein is used with a recombinant anti-IL17RB / TSLP receptor bispecific binding protein. In some embodiments, the method disclosed herein is used with a recombinant anti-IL17RB / IL33 receptor bispecific binding protein.

[0195] In some embodiments, provided herein is a method of reducing pulmonary ILC2 proliferation in a subject in need thereof. In some embodiments, provided herein is a method of treating a disease or disorder associated with allergy in a subject in need thereof. In some embodiments, provided herein is a method of reducing eosinophilic inflammation in a subject in need thereof. In some embodiments, provided herein is a method of treating a disease or disorder associated with neutrophilic inflammation in a subject in need thereof. In some embodiments, provided herein is a method of treating a disease or disorder by reducing the daily dose of OCS by at least 20%, at least 50%, or at least 75% in a subject in need thereof. In some embodiments, the subject is a human.

[0196] The methods of reducing pulmonary ILC2 proliferation, treating a disease or disorder associated with neutrophilic inflammation, treating a disease or disorder associated with eosinophilic inflammation, and treating a disease or disorder by reducing the daily dose of OCS comprise administering to a subject a therapeutically effective amount of a bispecific binding protein that specifically binds (a) IL17RB and TSLP, and / or (b) IL17RB and IL-33.

[0197] Suitable subjects for this method include human patients in whom blocking the activity of alarmins may be desirable. In some embodiments, the subject to be treated with the methods disclosed herein has been diagnosed with allergy-related disease or disorder, which may be post-symptomatic or pre-symptomatic asthma, AD, fibrotic disease, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, chronic obstructive pulmonary disease, chronic sinusitis, chronic sinusitis with nasal polyps. In some embodiments, the subject may be a mammal. In some embodiments, the subject is a human. In some embodiments, the subject to be treated with the methods disclosed herein continues OCS. In some embodiments, the subject has not been previously treated.

[0198] A bispecific binding protein (e.g., a SM17-related bispecific binding protein) or pharmaceutical composition provided herein can be administered to a subject by any method known in the art, including, but not limited to, intravenous, subcutaneous, intramuscular, intracranial, intrathecal, intraventricular, intraperitoneal, spinal, intranasal, intrapleural, topical, or intradermal administration.

[0199] In some embodiments, a bispecific binding protein (e.g., SM17-related bispecific binding protein) or pharmaceutical composition provided herein may be administered to a subject using parenteral administration. The phrase "parenteral administration," as used herein, refers to a mode of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion. In some embodiments, the bispecific binding protein is administered by intravenous infusion or injection. In some embodiments, the bispecific binding protein is administered by intramuscular injection. In some embodiments, the bispecific binding protein is administered by subcutaneous injection.

[0200] The bispecific binding protein (e.g., SM17-related bispecific binding protein) or pharmaceutical composition provided herein can be administered using medical devices known in the art.For example, in some embodiments, needleless hypodermic injection devices can be used, such as the devices disclosed in U.S. Patent Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556. Examples of well-known implants and modules for use herein include U.S. Patent No. 4,487,603, which discloses an implantable infusion micropump for dispensing a dose at a controlled rate; U.S. Patent No. 4,486,194, which discloses a therapeutic device for administering medicine through the skin; U.S. Patent No. 4,447,233, which discloses a dose infusion pump for delivering a dose at a precise infusion rate; U.S. Patent No. 4,447,224, which discloses an implantable variable flow rate infusion device for continuous drug delivery; U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system with multiple chamber compartments; and U.S. Patent No. 4,475,196, which discloses an osmotic drug delivery system. These patents are incorporated herein by reference. Many other such implants, delivery systems and modules are known to those skilled in the art.

[0201] In some aspects, the bispecific binding proteins disclosed herein may be administered orally, for example, with an inert diluent or an absorbable edible carrier. The therapeutic bispecific binding proteins may also be enclosed in gelatin shell hard or soft capsules, compressed into tablets, or directly incorporated into the subject's diet. For oral therapeutic administration, the bispecific binding proteins may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, and the like.

[0202] The methods provided herein include administering a therapeutically effective amount of a bispecific binding protein (e.g., a SM17-related bispecific binding protein) described herein. The actual dosage level of the therapeutic antibody can be varied to obtain an amount effective for a particular patient to obtain the desired therapeutic response without causing toxicity to the patient. The dosage level selected will depend on various pharmacokinetic factors, such as the activity of the particular composition described herein, the route of administration, the duration of administration, the rate of excretion, the duration of treatment, other drugs, compounds and / or substances used in combination with the particular composition used, the age, sex, weight, medical condition, general health and medical history of the patient being treated, and other factors well known in the medical arts.

[0203] In general, dosages can range, for example, from about 0.1 to 100 mg / kg of host body weight for a single dose. In some embodiments, the bispecific binding protein (e.g., SM17-related bispecific binding protein) is administered at about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, about 10 mg / kg, about 20 mg / kg, about 30 mg / kg, about 40 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg. In some embodiments, the bispecific binding protein is administered at about 1 mg / kg. In some embodiments, the bispecific binding protein is administered at about 5 mg / kg. In some embodiments, the bispecific binding protein is administered at about 10 mg / kg. In some embodiments, the bispecific binding protein is administered at about 20 mg / kg. In some embodiments, the bispecific binding protein is administered at about 40 mg / kg. In some embodiments, the bispecific binding protein is administered at about 60 mg / kg. In some embodiments, the bispecific binding protein is administered at about 100 mg / kg.

[0204] In some embodiments, the bispecific binding protein (e.g., SM17-related bispecific binding protein) is administered at a dose in the range of about 1-5 mg / kg, about 1-10 mg / kg, about 1-20 mg / kg, about 1-50 mg / kg, about 1-100 mg / kg, about 5-10 mg / kg, about 5-20 mg / kg, about 5-50 mg / kg, about 5-100 mg / kg, about 10-50 mg / kg, or about 10-100 mg / kg. In some embodiments, the bispecific binding protein is administered at a dose in the range of about 1-5 mg / kg. In some embodiments, the bispecific binding protein is administered at a dose in the range of about 1-10 mg / kg. In some embodiments, the bispecific binding protein is administered at a dose in the range of about 1-50 mg / kg. In some embodiments, the bispecific binding protein is administered at a dose in the range of about 10-50 mg / kg. In some embodiments, the bispecific binding protein is administered at a dose in the range of about 10-100 mg / kg.

[0205] In some embodiments, the methods provided herein include administering a bispecific binding protein (e.g., a SM17-related bispecific binding protein) at a dose of about 10 to 2000 mg. In some embodiments, the dose is about 10 mg, about 50 mg, about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, about 1400 mg, about 1500 mg, about 1600 mg, about 1700 mg, about 1800 mg, about 1900 mg, or about 2000 mg. In some embodiments, the antibody is administered at a dose of 100 mg. In some embodiments, the antibody is administered at a dose of 300 mg. In some embodiments, the antibody is administered at a dose of 600 mg. In some embodiments, the antibody is administered in a dose of 900 mg. In some embodiments, the antibody is administered in a dose of 1200 mg.

[0206] In some embodiments, the methods provided herein include administering an IgG-containing bispecific binding protein (e.g., an SM17-related bispecific binding protein) at a dose in the range of about 10-50 mg, 10-100 mg, 10-200 mg, 100-300 mg, 100-500 mg, 300-600 mg, 300-900 mg, 300-1200 mg, 600-1200 mg, 600-1800 mg, or 1000-2000 mg. In some embodiments, the antibody is administered at a dose in the range of 100-500 mg. In some embodiments, the antibody is administered at a dose in the range of 300-600 mg. In some embodiments, the antibody is administered at a dose in the range of 300-900 mg. In some embodiments, the antibody is administered at a dose in the range of 600-1200 mg.

[0207] During treatment, it is common to start with a low dose and then linearly titrate the dose up to a target dose. For illustrative purposes, in some embodiments, the methods provided herein comprise administering a bispecific binding protein at a dose of about 100 mg, and gradually linearly titrating the dose up to a target dose of about 600 mg.

[0208] Such doses may be administered to a subject daily, every other day, weekly, biweekly, monthly, or according to any other schedule determined by empirical analysis. Exemplary treatments include administration in repeated dosages over an extended period of time, for example, at least 6 months. In some embodiments, the methods provided herein include administering the bispecific binding protein weekly. In some embodiments, the methods include biweekly administration. In some embodiments, the methods include monthly administration. In some embodiments, the bispecific binding protein (e.g., SM17-related bispecific binding protein) is administered subcutaneously every week, every other week, or every month. In some embodiments, the IgG-containing bispecific binding protein (e.g., SM17-related bispecific binding protein) is administered intravenously every week, every other week, or every month.

[0209] Bispecific binding proteins (e.g., SM17-related bispecific binding proteins) may be administered for up to 3 months, 6 months, 9 months, 12 months, 18 months, 24 months, 30 months, or 36 months, as appropriate, as needed. In some embodiments, treatment is sustained for at least 3 months. In some embodiments, treatment is sustained for at least 6 months. In some embodiments, treatment is sustained for at least 12 months. In some embodiments, treatment is sustained for at least 24 months.

[0210] Any permutation and combination of the various aspects, such as number of administrations, dosage amounts, frequency of treatment, and length of treatment, are expressly contemplated herein and may be employed in the therapeutic methods disclosed herein.

[0211] By way of example, the following treatment regimens may be employed in the methods disclosed herein that involve administering an anti-IL17RB antibody or antigen-binding fragment, either as disclosed herein (e.g., an SM17-related bispecific binding protein) or as identified in the methods disclosed herein.

[0212] In some embodiments, the therapeutic antibody is administered intravenously or subcutaneously at a dose of about 10 mg / kg at least 21 days apart every 4 weeks. Some embodiments include the following titration schedule: infusions 1-2: 1 mg / kg IV; infusions 3-4: 3 mg / kg IV; infusions 5-6: 6 mg / kg IV; infusions 7 and beyond: 10 mg / kg IV.

[0213] In some embodiments, the therapeutic antibody is administered intravenously or subcutaneously at a single dose of 10, 20 or 40 mg / kg, a second dose of 10 mg / kg every other week for 24 weeks, and a third dose of 10 or 20 mg / kg monthly for 16 months.

[0214] In some embodiments, therapeutic antibodies are administered intravenously or subcutaneously at a dose of about 250 mg weekly or 500 mg every other week for up to two years, hi some embodiments, treatment begins with a monthly injection of about 120 mg.

[0215] Dosage regimen can be adjusted to bring about the desired optimal response (e.g., therapeutic or prophylactic response). For example, a single bolus can be administered, or several divided doses can be administered over time, or the dose can be proportionally reduced or increased as the exigencies of the therapeutic situation dictate. It is particularly convenient to formulate parenteral compositions into unit dosage form for easy administration and uniform dosage. Unit dosage form, as used herein, refers to a physically separate unit suitable as a single dosage for the mammalian subject to be treated; each unit contains a predetermined amount of therapeutic antibody calculated to bring about the desired therapeutic effect, together with the required pharmaceutical carrier. It should be noted that the appropriate dosage varies according to the type and severity of the condition to be alleviated. It is further understood that for any particular subject, specific dosage regimens will be adjusted over time according to the individual needs and the professional judgment of the person administering or supervising the administration of the compositions, and that the dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions.

[0216] In the treatment of allergy-related diseases or disorders, in some cases the disease or disorder may be cured by the methods provided herein, but any clinical improvement constitutes a benefit. In some embodiments, the methods provided herein reduce daily OCS use by an average of about 2.5 mg / day, about 5 mg / day, about 10 mg / day, about 20 mg / day, about 40 mg / day, about 95 CL. In some embodiments, the methods provided herein reduce daily OCS dose by an average of 25 percent. In some embodiments, the methods provided herein reduce daily OCS dose by an average of 50 percent. In some embodiments, the methods provided herein reduce daily OCS dose by an average of 75 percent. In some embodiments, the methods provided herein reduce daily OCS dose by an average of 100 percent. In some embodiments, the methods provided herein reduce daily OCS dose by an average of 25-50 percent. In some embodiments, the methods provided herein reduce the daily OCS dose by an average of 50-75 percent. In some embodiments, the methods provided herein reduce the daily OCS dose by an average of 75-100 percent.

[0217] In some embodiments, the methods provided herein reduce ILC2 proliferation in the lung. In some embodiments, the methods provided herein reduce ILC2 migration to the lung. In some embodiments, the methods provided herein reduce IL-5 levels in bronchoalveolar fluid. In some embodiments, the methods provided herein reduce IL-13 levels in bronchoalveolar fluid. In some embodiments, the methods provided herein reduce eosinophilic inflammation in the lung. In some embodiments, the methods provided herein reduce neutrophilic inflammation in the lung. In some embodiments, the methods provided herein reduce annual asthma exacerbation rates. In some embodiments, the methods provided herein reduce exhaled nitric oxide concentrations. In some embodiments, the methods provided herein reduce eosinophil counts in the blood. In some embodiments, the methods provided herein reduce transepidermal water loss.

[0218] In some embodiments, the method provided herein prevents the onset of AD or delays or stops the progression of AD.In some embodiments, the method provided herein alleviates AD symptoms.In some embodiments, the method provided herein prevents the onset of asthma or delays or stops the progression of asthma.In some embodiments, the method provided herein alleviates asthma symptoms.

[0219] The bispecific binding proteins disclosed herein can be administered by various methods known in the art. As will be appreciated by those skilled in the art, the route and / or mode of administration will vary depending on the desired results. In some embodiments, the bispecific binding proteins can be prepared with a carrier that protects against rapid release, such as a controlled release formulation, such as an implant, a transdermal patch, and a microencapsulated delivery system. Biodegradable biocompatible polymers, such as ethylene vinyl acetate, polyethylene glycol (PEG), polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, can be used. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, for example, SUSTAINED AND CONTROLLED RELEASE DRUG DELIVERY SYSTEMS, JR Robinson ed., Marcel Dekker, Inc., New York, 1978. In therapeutic applications, in some cases, relatively high dosages are required for a relatively short period of time until the progression of the disease is reduced or terminated, and until the patient shows partial or complete remission of the symptoms of the disease.

[0220] Combination therapy using agents with different mechanisms of action may result in additive or synergistic effects. Combination therapy may allow for lower doses of each agent than those used in monotherapy, thereby reducing toxic side effects and / or increasing the therapeutic index of the agents disclosed herein. Combination therapy may reduce the likelihood that drug resistance may develop. In some embodiments, the additional therapy results in an increased therapeutic index of the bispecific binding protein or pharmaceutical composition described herein. In some embodiments, the additional therapy results in a reduced toxicity and / or side effects of the bispecific binding protein or pharmaceutical composition described herein. In some embodiments, the bispecific binding protein or pharmaceutical composition described herein may be administered in combination with an additional therapy.

[0221] In some embodiments, the second therapeutic agent is a corticosteroid, a DNA methyltransferase (DNMT) inhibitor, an anti-IL4 antibody, an anti-IL5 antibody, an anti-IL4Ra antibody, an anti-IL13 antibody, an anti-IgE antibody, an anti-IL17A antibody, an anti-IL12 / IL23 antibody, an anti-IL23 antibody, an anti-IL17RA antibody, a tyrosine kinase inhibitor. In some embodiments, the second therapeutic agent can be a second antibody that suppresses the release of proinflammatory cytokines.

[0222] The second therapeutic agent can be administered before, in parallel with, or after the administration of the bispecific binding protein or pharmaceutical composition described herein.Combined administration can include co-administration, either using a single pharmaceutical formulation or separate formulations, or sequential administration, in any order, but generally within a period of time such that all active agents can simultaneously exert their biological activity.Those skilled in the art can easily determine the appropriate regimen for administering the pharmaceutical composition described herein in combination with additional therapy, such as the timing and dosing of the additional agent used in the combination therapy, based on the needs of the subject being treated.

[0223] All articles, publications and patents cited herein are incorporated by reference as if each article, publication or patent was specifically and individually set forth and incorporated by reference, and are incorporated by reference herein to disclose and describe the methods and / or materials related to which the publications are cited. However, any mention of references, journal articles, publications, patents, patent publications and patent applications cited herein is not, and should not be construed as, an admission or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

[0224] Unless otherwise indicated in the text, it is specifically contemplated that the various features described herein can be used in any combination.

[0225] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. EXAMPLES

[0226] 7.6 Experimental Examples Example 1: SM17 binds to exogenous mouse, monkey and human IL-17RB proteins A standard enzyme-linked immunosorbent assay (ELISA) method was used to measure the species specificity of SM17 against IL-17RB protein. Briefly, IL-17RB protein from human, mouse, cynomolgus monkey or rhesus monkey (R&D systems, Minneapolis, MN) was diluted to 2.5 μg / ml in PBS, and 50 μl of IL-17RB protein was added to each well of the ELISA strip. The strip was sealed with parafilm and incubated overnight at 4°C for coating. The next day, the strips were washed three times with washing buffer (0.05% Tween 20 in PBS) and blocked with 100 μl per well of blocking buffer (3% BSA in PBS) for 2 hours at room temperature, followed by the addition of serially diluted SM17 (at final concentrations of 10, 2, 0.4, 0.08, 0.016, 0.0032 and 0.00064 μg / ml, respectively). After a 2 hour incubation period at room temperature, the amount of bound SM17 was visualized by the addition of peroxidase-conjugated goat anti-human F(ab')2 specific antibody (Jackson ImmunoResearch) and TMB material (Sigma-Aldrich, St. Louis, MO) at OD 450 nM according to standard ELISA protocols known to those skilled in the art. The results show that SM17 binds to IL-17RB proteins from mouse, human, cynomolgus and rhesus monkeys with comparable affinity and in a dose-responsive manner (see Table 1 and Figure 1).

[0227] Table 1. Optical density at 450 nm of antigen binding to IL-17RB from different species. TIFF2025508835000005.tif62160

[0228] Example 2: SM17 does not cross-react with other IL17 receptor subunits Standard ELISA methods were used to measure the specificity of SM17 for other human IL17 receptor subunits. Similarly, serially diluted SM17 at different concentrations (final concentrations of 0.4, 0.08, 0.016, 0.0032 and 0.00064 μg / ml, respectively) were added to wells of ELISA plates coated with either human IL-17 receptor subunits A, C, D and E (IL17RA, IL17RC, IL17RD and IL17RE) (R&D systems). No obvious binding of SM17 to other known human IL-17 receptor subunits (IL17RA, IL17RC, IL17RD and IL17RE) was detected (see Table 2 and Figure 2).

[0229] Table 2. Optical density at 450 nm of antigen binding to IL-17 receptor family members TIFF2025508835000006.tif62160

[0230] Example 3: SM17 binds to native IL-17RB from human and rhesus monkey expressed on the surface of HEK293 cells HEK293 cells (ATCC, Manassas, VA) transfected with either full-length human or rhesus IL-17RB (Sino Biological, Beijing, China) were examined for binding to SM17 according to standard protocols for flow cytometry. Wild-type HEK293 stained with IgG4 isotype control (Sino Biological) was used as a negative control and for gating purposes. Briefly, 3 × 10 5HEK293 cells were seeded into each well of a 6-well plate at a density of 1000 x 1000 cells. The next day, full-length cDNAs of human or rhesus IL-17RB were first cloned into pCMV3-untagged expression plasmids by standard molecular cloning techniques, and then HEK293 cells were transfected with the expression plasmids of human or rhesus IL-17RB by lipofection (Lipofectamine 3000 Reagent; Thermo Fisher Scientific). Transfected cells were trypsinized, harvested, and fixed with 4% paraformaldehyde (5 min). SM17 at 1 μg / ml in wash buffer (3% BSA in PBS) or human IgG4 control was added to the suspension cells for 30 min incubation at room temperature, followed by Alexa Fluor® 647-conjugated goat anti-human IgG specific antibody (1:2000 dilution in wash buffer) (Jackson ImmunoResearch) for flow cytometry analysis using a BD FACSVerse cell analyzer (Becton Dickenson, Franklin Lakes, NJ). Enhanced binding of SM17 to HEK293 transfected with human IL17RB (hIL17RB-HEK293: 46%) and rhesus IL17RB (RhIL17RB-HEK293: 39%) compared to wild-type HEK293 (approximately 7%) demonstrated that SM17 binds to native human or rhesus IL17RB expressed on HEK293 cells (Figure 3).

[0231] Example 4: SM17 inhibits IL-5 release from human PBMCs co-cultured with IL-2 and IL-25 PBMCs (Ixcells, San Diego, CA) co-cultured with IL-2 and IL-25 (PeproTech, Cranbury, NJ) can result in the release of IL-5, a cytokine known to exacerbate asthma pathology. Therefore, the inhibitory effect of SM17 on IL-5 release from IL-2 / IL-25-treated human PBMCs was evaluated. Briefly, cryopreserved human PBMCs were thawed and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (Thermo Fisher Scientific). Serial dilutions of SM17 at concentrations ranging from 0.16 ng / mL to 5 mg / mL were added to 4 × 10 5 Human PBMCs were added in the presence of 10 units / mL IL-2 and 10 μg / mL IL-25. Non-specific human IgG4 (SinoBiological) was used as a control antibody. Treated cells were incubated at 37°C for 72 hours, and the levels of IL-5 released in the culture supernatant were measured by standard ELISA assay (R&D systems). The results showed that SM17 inhibited IL-2- and IL-25-induced IL-5 release from human PBMCs in a dose-dependent manner (Figure 4). Results are presented as mean ± SEM. One-way ANOVA, Dunnett's multiple comparison test, * :P<0.05; ** :P<0.01.

[0232] Example 5: Inhibition of IL-8 release by SM17 in renal carcinoma cell line TK-10 Human renal carcinoma cell line TK-10 (NCI-60, NIH, Bethesda, MD) treated with human IL-25 (PeproTech) and TNFa (R&D systems) can result in the release of IL-8, also known as neutrophil chemotactic factor, which is a key mediator of immune responses in the response of the innate immune system. Therefore, the inhibitory effect of SM17 and its murine counterpart (D9.2) on IL-8 release from IL-25 / TNFa-treated TK-10 cells was evaluated. Briefly, SM17 (human IgG4 isotype, SinoMab BioScience Limited, Hong Kong, China), SM17-IgG1 (human IgG1 isotype, SinoMab BioScience Limited) and its murine counterpart (D9.2, SinoMab BioScience Limited) at a concentration of 1 mg / mL were added to 2 × 10 4 TK-10 cells were added in the presence of 100 ng / mL IL-25 and 10 ng / mL TNFa. Non-specific human IgG1 (SM03, anti-CD22 chimeric antibody, SinoMab BioScience Limited) was used as a control antibody. Treated cells were cultured in OptiMEM (Thermo Fisher Scientific) for 24 or 48 hours at 37°C, and the level of IL-8 release in the culture supernatant was measured by standard ELISA assay using a commercial kit (R&D systems). The results showed that at 1 mg / mL, either SM17, SM17-IgG1 or D9.2 could efficiently suppress the release of IL-8 by IL-25 / TNFa-induced TK-10 cells at both time points (Figure 5).

[0233] Example 6: Measurement of antigen (IL17RB) binding kinetics of SM17 Bio-Layer Interferometry analysis (Octet ReD96 system, Sartorius) was used to measure the binding affinity of SM17 to human and cynomolgus IL17RB proteins (R&D systems). Briefly, SM17 (20 μg / mL) was immobilized on a biosensor by interaction with anti-human Fab CH1; serially diluted Cyno-IL17RB and Human-IL17RB (at concentrations of 158.7 nM, 79.4 nM and 39.7 nM, respectively) were added sequentially according to the standard operating protocol of the Octet ReD96 system to plot the association and dissociation curves. An irrelevant antibody (SM03, anti-human CD22 chimeric IgG1 antibody, SinoMab BioScience Limited) was used as a control reference. The K a , K dis and K. D The values ​​of are summarized in Table 3 below. It is noteworthy that K D is in the picomolar to single digit nanomolar range.

[0234] Table 3. Binding kinetics of SM17 to human and cynomolgus IL-17RB. TIFF2025508835000007.tif39160

[0235] Example 7: Therapeutic effect of SM17 on ovalbumin-induced asthma in mice Female BALB / c mice were induced with ovalbumin to elicit asthma-like symptoms in mice. Briefly, mice were sensitized on days 0 and 12 by intraperitoneal (IP) injection with 10 mg ovalbumin (OVA) emulsified in a total volume of 200 mL containing 1 mg aluminum hydroxide, respectively. Mice were divided into six groups of eight mice per group. Sensitized mice were exposed to aerosolized OVA 5% in sterile water for 20 min / day for six consecutive days (days 19, 20, 21, 22, 23, and 24). Naive mice inhaled atomized water for 20 min / day for six consecutive days. Mice were then iv injected with different concentrations of SM17, PBS (control) or dexamethasone (Dex) for 4 h, followed by aerosol challenge once a day (days 19-24). On day 25 (24 hours after the last OVA challenge), mice were anesthetized with Telazol® (20-40 mg / kg ip) and connected to a computer-controlled ventilator via a tracheal cannula. Inhalation / exhalation times and respiratory rates were preset to 1.5:1 and 90 breaths / min, respectively. After a steady baseline was established, pulmonary resistance (RL) was recorded to assess the response of mice to methacholine chloride gradients (0.025 and 0.05 mg / kg body weight); methacholine chloride was injected by fine needle at 5-minute intervals into the external jugular vein. Airway hyperresponsiveness was prevented in mice treated with 5 mg / kg SM17 or 1 mg / kg Dex. Administration of 1 mg / kg or 3 mg / kg SM17 before each OVA aerosol application resulted in a mild but non-significant abatement in AHR after methacholine challenge. As the results shown in Figure 6 indicate, the therapeutic effect of SM17 is dose-dependent. BALF was collected from mice sacrificed on day 25, and the number of inflammatory cells and the concentration of inflammatory cytokines in the BALF were measured. In addition, SM17 administered at 1, 3, and 5 mg / kg significantly reduced IL-5 levels in BALF. SM17 administered at 5 mg / kg significantly reduced IL-13 levels and eosinophil counts in BALF (Figure 7). SM17 and Dex at 5 mg / kg significantly reduced the number of lung-infiltrating eosinophils. Results are shown as mean ± SEM. One-way ANOVA, Dunnett's multiple comparison test, * :P<0.05; ** : P<0.01. Dex = dexamethasone. Figure 9 shows the proposed mechanism of action of SM17 to treat various indications.

[0236] Example 8: Construction and expression of anti-alarmin bispecific binding protein (bsBp) The light chain of SM17 (SEQ ID NO:33) is cloned into the pcDNA3.3 expression vector by TA cloning (Thermo Fisher Scientific). The cDNA encoding the heavy chain of SM17 operatively linked to the sequence of a specific alarmin-binding protein (e.g., an alarmin-binding receptor or an alarmin-specific antibody scFv) is gene synthesized (Genscript Biotech Corp., Piscataway, NJ) and cloned into the NheI / NotI cloning sites of the pEGFP-N1 expression vector (Clontech Laboratories, Mountain View, CA) (Figure 10a). The expression vector containing the heavy chain of a specific SM17-alarmin-binding protein and the expression vector of the SM17 light chain are co-transfected into expiCHO-S cells according to the manufacturer's specifications (Thermo Fisher Scientific). The specific bispecific binding protein (bsBp) containing the SM17 antibody linked to a specific alarmin-binding moiety is harvested 12 days after transfection and purified by Protein A affinity chromatography. A reducing SDS-PAGE of purified bsBp is shown in Figure 10b indicating that most of the bispecific antibody is intact in its native state. The heavy and light chain sequences of bsBp are summarized in Table 4 below.

[0237] Table 4: Amino acid sequences of bsBp and monoclonal antibodies TIFF2025508835000008.tif104133

[0238] Example 9: Specificity of anti-alarmin bsBp The binding specificity of the bsBp was evaluated by standard ELISA assay. Briefly, ELISA strips were coated with the respective target antigens including IL17RB, IL-33 or TSLP (R&D systems) at a final concentration of 2 μg / mL. Bispecific antibodies were added at 33.5 nM to the ELISA strips coated with the desired antigen. After 2 hours of incubation at room temperature, the ELISA strips were washed 5 times with PBS. Binding was visualized according to standard procedures by adding a goat anti-human F(ab')2 specific horseradish peroxidase (HRP) conjugated secondary antibody (1:5000 dilution, Jackson ImmunoResearch) followed by a solution of TMB material (Sigma-Aldrich-) (Figure 11). The antigen binding specificity of the bsBp is summarized in Figure 11 and Table 5 below. The results show specificity for IL17RB and the designed alarmins as originally designed. The results for SM17-human TSLPR are not shown as the yield was too low to be detected.

[0239] Table 5. Antigen-binding specificity of bsBp in ELISA TIFF2025508835000009.tif62154bsBp specific antigen binding affinity strength in a semi-quantitative assessment scale of + / +++ (-=no binding, +, ++, +++=weak, moderate, strong binding), N / A indicates no binding affinity.

[0240] Example 10: Alarmin binding kinetics of bispecific binding proteins The binding affinity of bsBp to the targeted alarmins was measured using bio-Layer Interferometry analysis (Octet ReD96 system, Sartorius). bsBp or SM17 (20 μg / mL) was immobilized on the biosensor by binding to the anti-human Fab CH1 region; serial dilutions of human IL33 and TSLP (158.7 nM, 79.4 nM, 39.7 nM) (R&D systems) were added according to the manufacturer's specifications, and association and dissociation curves were plotted. A biosensor immobilized with an irrelevant antibody (SM03, anti-human CD22 chimeric IgG1 antibody, SinoMab BioScience Limited, 20 μg / mL) was used as a control reference. The predicted Ka, Kdis and KD values ​​of each bispecific binding protein are summarized in Table 6 below.

[0241] Table 6. Binding kinetics of bsBp to human TSLP and IL-33 TIFF2025508835000010.tif103128 The binding affinity of SM17-human TSLPR is not shown due to the low yields.

[0242] Example 11: Induction of release of IFNγ, CCL8, CCL17, IL-5 and IL-13 from human PBMCs by alarmins and the inhibitory effect of SM17 on cytokine release Human PBMCs (4 × 10 5PBMCs (10 units / group, Ixcells) were incubated with IL-2 (10 units / mL, PeproTech) and various alarmins / alarmin combinations to mimic proinflammatory cytokine release during allergic disease. For IL-5 and IL-13 release, PBMCs were incubated with IL-2 and the three alarmins (all at 10 ng / ml) for 3 days. For CCL17 release, PBMCs were incubated with TSLP (10 ng / ml) for 1 day. For CCL8 and IFNγ release, PBMCs were incubated with IL-12 (10 ng / ml, Sino Biological) and IL-33 (10 ng / ml) for 1 day. Cells were pretreated with SM17 (5 μg / ml) or IgG4 isotype control (5 μg / ml) for 1 hour before cytokine addition. Supernatants were collected and cytokine concentrations were measured by ELISA kits (R&D systems). Using such established assays, the potency of SM17 and bsBp was tested. SM17 could effectively suppress the secretion of IL-5 and IL-13 in whole alarmin-stimulated PBMC cultures. However, the release assays of CCL17, CCL8 and IFNγ showed no inhibitory effect of SM17 treatment compared to the IgG4 isotype control (see FIG. 12). Results were expressed in absolute amounts in pg / ml.

[0243] Example 12: Efficacy of different bispecific binding proteins in inhibiting cytokine and chemotactic factor release from induced human PBMC SM17, three different SM17-anti-IL33 bsBps and three different SM17-anti-TSLP bsBps (all at 5 μg / ml) were added to human PBMC cultures under conditions that should induce the release of IFNγ, CCL8, CCL17 and IL-5 as described in Example 11 above. The levels of different cytokines and chemotactic factors in the supernatants obtained in induced PBMCs were evaluated using standard ELISA methods. The results show that the different pairs of SM17 / anti-TSLP and SM17 / anti-IL33 bsBps show improved or / and synergistic effects in suppressing the release of IFNγ, CCL8, CCL17, IL-5 and IL-13 when compared to that observed with SM17 alone (see FIG. 13). Specifically, in the IL-5 release assay, SM17-anti-IL-33 3# showed the strongest inhibitory effect when compared to SM17 treatment. In the CCL8 release assay, both SM17-anti-IL-33 1# and SM17-anti-IL-33 3# showed stronger inhibitory effects when compared to SM17. In the IFNγ release assay, all three SM17-anti-IL-33 bsBps showed stronger inhibitory effects when compared to SM17. In the CCL17 release assay, both SM17-anti-TSLP 1# and SM17-anti-TSLP 3# bsBps showed stronger inhibitory effects when compared to SM17 treatment. The potency of the bsBps in the different cytokine release assays is summarized in Figure 13 and Table 7.

[0244] Table 7. Potency of bsBp in cytokine release assay TIFF2025508835000011.tif55160bsBp specific cytokine inhibition intensity in the + / +++ semiquantitative scale (-=no inhibition, +, ++, +++=weak, moderate, strong inhibition). Nil: not investigated.

[0245] Example 13: Growth-promoting effect of alarmins on ILC2 Isolated human ILC2 cells can proliferate in response to alarmins. Human ILC2s were enriched from fresh human PBMCs using the EasySep™ Human ILC2 Isolation Kit (STEMCELL Technologies Inc. Cambridge, MA). The enriched ILC2s were then cultured in RPMI1640 medium (Thermo Fisher Scientific) containing 10% human AB serum (Sigma). After adding IL-2 (10 units / mL) together with IL-25 (10 ng / mL) or TSLP (10 ng / mL) or IL-33 (10 ng / mL) or a combination of these three alarmins to ILC2 cultures for 7 days, changes in ILC2 cell populations were measured by flow cytometry (BD FACSVerse) using a lineage-specific antibody panel against ILC2s (CRTH2+, IL-7Ra+; BioLegend, San Diego, CA). The results show that the major contributor to ILC2 proliferation is IL-33, and the combination of all alarmins further enhances ILC2 expansion (see FIG. 14).

[0246] Example 14: Suppression of ILC2 and Th2 cell populations in human PBMCs by dexamethasone and bsBp Alarmins are known to act in concert to enhance, e.g., increase, the function of ILC2 and Th2 cells in cell populations, while steroids, e.g., dexamethasone (Dex), can counteract such effects, resulting in mitigation of type 2 immune responses [Jia, Yi et al. American Journal Of Respiratory Cell And Molecular Biology, vol. 55, 5 (2016): 675-683]. ILC2 and Th2 cell populations can be measured by flow cytometry (BD FACSVerse) according to standard procedures known to those skilled in the art. Herein, ILC2 and Th2 cell populations were experimentally enhanced by a combination of IL-2 (10 units / mL) and three alarmins (10 ng / ml) for 5 days. The results show that Dex can significantly reduce the Th2 cell population (CD4+GATA3+ population, gated by anti-CD4 PE antibody (BioLegend) and anti-GATA3 APC antibody (Abcam, Cambridge, UK)) in human PBMC cultures, but not the ILC2 population (lin-IL-7Ra+CRTH2+, gated by anti-IL-7Ra percp / Cyanine5.5 antibody and anti-CRTH2 FITC antibody, BioLegend) (see FIG. 15). Furthermore, the addition of Bsbp significantly reduced the ILC2 cell population, and the efficacy of bsBp is summarized in FIG. 15 and Table 8 below.

[0247] Table 8. Potency of bsBp in ILC2 and Th2 proliferation assays TIFF2025508835000012.tif41160bsBp cell proliferation inhibition in semi-quantitative assessment steps of + / +++++ (-=no inhibition, +, ++, +++=weak, moderate, strong inhibition)

[0248] Example 15: Dendritic Cell Potency Assay TSLP derived from inflamed epithelium promotes dendritic cell (DC) maturation and primes Th2 responses via CCL17, which induces CD4+ T cell chemotaxis to mediate inflammation (Kitajima and Ziegler. 2013. “Cutting Edge: Identification Of The Thymic Stromal Lymphopoietin-Responsive Dendritic Cell Subset Critical For Initiation Of Type 2 Contact Hypersensitivity”. J Immunol 191:4903-4907; Bleck et al. 2015. “Co-Expression Of Type 2 Immune Targets In Sputum-Derived Epithelial And Dendritic Cells From Asthmatic Subjects”. J Allergy Clin Immunol 136:619-627.e5). Normal human DCs were obtained from Lonza (Bend, OR). The DCs were cultured in LGM-3 medium (Lonza). TSLP (10 ng / mL) is added to DC cultures for 5 days in the presence of 5 μg / mL SM17, IgG4 isotype control (Sino Biological), SM17-TSLPR bsBp (SM17 fused to the extracellular domain of the TSLP receptor) or SM17-anti-TSLP bsBp 2#. Supernatants are collected and CCL17 levels are measured by ELISA according to the manufacturer's specifications (R&D systems). Results show that the bispecific SM17-TSLPR and SM17-anti-TSLP bsBp 2# are efficient in suppressing TSLP-induced CCL17 production in DCs (Figure 16). The potencies of SM17, IgG4 and bsBp are summarized in Table 9 below.

[0249] Table 9. Potency of bsBp in CCL17 release assay in dendritic cells TIFF2025508835000013.tif35128bsInhibition of CCL17 release in semi-quantitative grades of + / +++ for Bp, SM17 and IgG4 isotypes (-=no inhibition, +, ++, ++=weak, moderate, strong inhibition)

[0250] Although the present invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to those skilled in the art that various changes and modifications can be made in the present invention without departing from the spirit and scope of the invention.

Claims

1. A pharmaceutical composition for use in a method of treating an allergy-related disease or disorder in a subject in need thereof, comprising a bispecific binding protein to two different alarmins X and Y, wherein the bispecific binding protein consists of (a) an anti-alarmin X receptor IgG, and (b) an anti-alarmin Y scFv, and (c) a polypeptide linker, and wherein the pharmaceutical composition is used to deliver a therapeutically effective amount of the bispecific binding protein to a cell or tissue of the subject.

2. A pharmaceutical composition for use in a method of treating an allergy-related disease or disorder in a subject in need thereof, comprising a bispecific binding protein to two different alarmins X and Y, wherein the bispecific binding protein consists of (a) anti-alarmin X receptor IgG, and (b) alarmin Y receptor, and (c) a polypeptide linker, and wherein the pharmaceutical composition is used to deliver a therapeutically effective amount of the bispecific binding protein to a cell or tissue of the subject.

3. 3. The pharmaceutical composition of claim 1 or 2, wherein the alarmin X receptor is IL-17RB.

4. 2. The pharmaceutical composition of claim 1, wherein the alarmin Y is TSLP.

5. 2. The pharmaceutical composition of claim 1, wherein the alarmin Y is IL-33.

6. 3. The pharmaceutical composition of claim 2, wherein the alarmin Y receptor is ST2.

7. 3. The pharmaceutical composition of claim 2, wherein the alarmin Y receptor is TSLPR.

8. 3. The pharmaceutical composition of claim 1, wherein the anti-alarmin X receptor IgG is an antibody selected from the group consisting of an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, and an IgG4 antibody.

9. 3. The pharmaceutical composition of claim 1 or 2, wherein the subject has clinical or preclinical asthma, atopic dermatitis, fibrotic disease, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, chronic obstructive pulmonary disease, chronic sinusitis, or chronic sinusitis with nasal polyps.

10. 1. A pharmaceutical composition for use in a method of treating an allergy-related disease or disorder in a subject in need thereof, comprising a bispecific binding protein that blocks (a) IL-25 and IL-33 signaling and / or (b) IL-25 and TSLP signaling, wherein the pharmaceutical composition is used to deliver a therapeutically effective amount of the bispecific binding protein to a cell or tissue of the subject.

11. 11. The pharmaceutical composition of claim 10, wherein the subject has symptomatic or pre-symptomatic asthma, atopic dermatitis, fibrotic disease, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, chronic obstructive pulmonary disease, chronic sinusitis, or chronic sinusitis with nasal polyps.

12. 11. The pharmaceutical composition of claim 10, wherein the bispecific binding protein is an anti-IL-17RB / anti-human TSLP bispecific antibody.

13. 11. The pharmaceutical composition of claim 10, wherein the bispecific binding protein is an anti-IL-17RB / anti-human IL-33 bispecific antibody.

14. 11. The pharmaceutical composition of claim 10, wherein the bispecific binding protein is an anti-IL-17RB / human ST2 antibody-receptor fusion protein.

15. 11. The pharmaceutical composition of claim 10, wherein the bispecific binding protein is an anti-IL-17RB / human TSLPR antibody-receptor fusion protein.

16. 16. The pharmaceutical composition of any one of claims 10 to 15, wherein the bispecific binding protein comprises a light chain variable region (VL) comprising VL CDR1, VL CDR2 and VL CDR3 having the amino acid sequences of SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively; and a heavy chain variable region (VH) comprising VH CDR1, VH CDR2 and VH CDR3 having the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively.

17. 17. The pharmaceutical composition of claim 16, wherein the VL and VH of the anti-IL-17RB antibody have the amino acid sequences of SEQ ID NO: 33 and SEQ ID NO: 32, respectively.

18. 16. The pharmaceutical composition of claims 10-15, wherein the bispecific binding protein comprises a light chain sequence of SEQ ID NO:33 and a heavy chain sequence selected from the group consisting of SEQ ID NOs:60-74.

19. 11. The pharmaceutical composition of claim 1, 2, or 10, wherein the bispecific binding protein is delivered to said cells or tissue intravenously, intramuscularly, subcutaneously, intracranially, intrathecally, intracerebroventricularly, intraperitoneally, intranasally, parenterally, topically, or intradermally.

20. 11. The pharmaceutical composition of claim 1, 2, or 10, wherein the bispecific binding protein is delivered in combination with a second therapeutic agent.

21. 21. The pharmaceutical composition of claim 20, wherein the second therapeutic agent is selected from the group consisting of a corticosteroid, a DNA methyltransferase (DNMT) inhibitor, an anti-IL17A antibody, an anti-IL12 / IL23 antibody, an anti-IL23 antibody, an anti-IL17RA antibody, and a tyrosine kinase inhibitor.

22. 11. The pharmaceutical composition of claim 1, 2, or 10, wherein the subject is a human subject and the cells or tissues of the subject are immune cells harvested and isolated from the subject.

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