Bispecific binding proteins against alertin and uses thereof

Abstract

Provided herein are methods of treating allergic diseases such as moderate to severe asthma and atopic dermatitis, methods of reducing daily oral corticosteroid doses, methods of inhibiting congenital lymphocyte proliferation, methods of reconciling type 1 and type 2 immune responses using certain bispecific binding proteins (bsBp) capable of neutralizing two different alarm elements. Exemplary bsBp, features thereof, and methods of screening for additional therapeutic bsBp are also described herein.

Description

1. Technical field:

[0001] 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:

[0003] This application claims priority to U.S. Provisional Application No. 63 / 313,483, filed on February 24, 2022, the entire contents of which are incorporated herein by reference.

[0004] 3. Sequence Listing:

[0005] This application contains a sequence listing in accordance with WIPO Standard ST.26, named "SBL009PCTSL.xml", created on February 14, 2023, with a size of 93,089 bytes and is hereby incorporated by reference in its entirety. 4. Background technology:

[0006] Alarmins are endogenous, constitutively expressed chemotactic and immune-activating proteins / peptides released as a result of degranulation, cell damage or death, or in response to immune induction.

[0007] In particular, the "alarmins" thymic stromal lymphopoietin (TSLP), interleukin-33 (IL-33), and interleukin-25 (IL-25) are released from barrier tissues (such as airway epithelial cells) in response to allergens. These alarmins, by activating type 2 innate lymphocytes (ILC2) and Th2 cells, are upstream elements responsible for initiating Th2 immune responses, leading to a cascade of events such as the release of IL-4, IL-5, IL-13, and IgE, and resulting in the emergence of a variety of allergic reactions. Biologics targeting any of these alarmins and cytokines produce varying degrees of improvement in symptom scores for allergic reactions such as asthma and atopic dermatitis. Although the use of single anti-TSLP or anti-IL-33 antibodies to treat asthma has been clinically validated, the lack of complete efficacy may be because each therapy only targets some elements in the pathway that regulates type 2 inflammation, while other elements in the pathophysiology of the disease are not addressed. These alarmins should potentially interact with each other, leading to their own inflammatory responses. Using a chronic model of helminth infection and type 2 cytokine-driven lung 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]. Furthermore, in a model of chronic allergic lung inflammation induced by house dust mites, disruption of all three mediators resulted in reduced inflammation, mucus production, and lung remodeling, suggesting that these alarmins interact and enhance their individual effects in maintaining type 2 pathology. Therefore, there is a great need for new clinical approaches in which two or more of these pathogenic alarmins are inhibited to improve clinical outcomes for allergic diseases, such as reducing steroid use and maintaining long-term disease remission. The methods provided in the present disclosure meet this need and provide related advantages in attenuating disease progression. 5. Summary of the invention:

[0008] In view of the clear need in the art for new methods for treating allergic diseases and disorders, the object of the present invention is to provide bispecific antibodies (bsBp) or antibody-receptor fusion proteins directed against two different alarmins, characterized by one or more of the following activities: i) binding to purified human IL-17RB and IL-33 proteins, with a K D Less than 10 -8 M; ii) binds to purified human IL-17RB and TSLP proteins, and its K D Less than 10 -8M; iii) inhibiting the release of Th2-related cytokines to a greater extent than anti-IL17RB monoclonal antibodies; iv) inhibiting the proliferation and activation of ILC2s to a greater extent than anti-IL17RB monoclonal antibodies.

[0009] As shown herein, such bispecific antibodies (bsBp) or antibody-receptor fusion proteins have the potential to prevent, inhibit or / and delay the progression of allergic diseases such as asthma and atopic dermatitis (AD). Therefore, the object of the present invention is to provide a method for treating a disease or disorder associated with allergy in a subject in need thereof, the method comprising the step of administering to the subject a therapeutically effective amount of a bispecific binding protein directed against two different alarmins X and Y, wherein the bispecific binding protein consists of (a) anti-alarmin X receptor IgG and (b) anti-alarmin Y scFv and (c) a polypeptide linker.

[0010] In a preferred embodiment, the subject is a human suffering from clinical or preclinical asthma, atopic dermatitis, fibrotic diseases, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, chronic obstructive pulmonary disease, chronic sinusitis, chronic sinusitis with nasal polyps.

[0011] 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.

[0012] In other preferred embodiments, the anti-alarmin X IgG is selected from the group consisting of an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, and an IgG4 antibody.

[0013] Another object of the present invention is to provide a method for treating an allergy-related disease or disorder in a subject in need thereof, the method comprising the step of 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.

[0014] In a preferred embodiment, the subject (preferably a human) suffers from clinical or preclinical asthma, atopic dermatitis, fibrotic diseases, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, chronic obstructive pulmonary disease, chronic sinusitis, chronic sinusitis with nasal polyps.

[0015] In a particularly preferred embodiment, the bispecific binding protein is selected from anti-IL-17RB / anti-human TSLP bispecific antibody, anti-IL-17RB / anti-human IL-33 bispecific antibody, anti-IL-17RB / human ST2 antibody-receptor fusion protein and anti-IL-17RB / human TSLPR antibody-receptor fusion protein.

[0016] In other preferred embodiments, the bispecific binding protein comprises a light chain variable region (VL) and a heavy chain variable region (VH), the light chain variable region (VL) consisting of VL CDR1, VL CDR2 and VL CDR3 having amino acid sequences of SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, respectively, and the heavy chain variable region (VH) consisting of VH CDR1, VH CDR2 and VH CDR3 having amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

[0017] In a particularly preferred embodiment, 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 the light chain sequence of SEQ ID NO: 33 and a heavy chain sequence selected from any one of SEQ ID NOs: 60-75.

[0018] In a preferred embodiment, the bispecific antibody (bsBp) and / or antibody-receptor fusion protein is administered intravenously, intramuscularly, subcutaneously, intracranialy, 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.

[0019] These and other objects and features of the present invention will become more apparent when the following detailed description is read in conjunction with the accompanying drawings and examples. However, it should be understood that the above-mentioned content of the invention and the following detailed description are preferred embodiments of the present invention and do not limit the present invention or other optional embodiments of the present invention. In particular, although the present invention is described with reference to many specific embodiments, it should be understood that these descriptions are explanations of the present invention and do not constitute limitations of the present invention. Without departing from the spirit and scope of the present invention, as described in the appended claims, various modifications and applications can be imagined by those skilled in the art. 6. Description of the drawings:

[0020] Figure 1 Dose-dependency curves of SM17 binding to IL-17RB from different species are depicted.

[0021] Figure 2 The binding specificity of SM17 to members of the IL-17 receptor family is depicted.

[0022] Figure 3 Binding of SM17 to native human Il-17RB is depicted.

[0023] Figure 4 : Depicted is the dose-dependent inhibitory effect of SM17 on IL-5 in human PBMC cultures.

[0024] Figure 5 Depicted is the inhibition of IL-8 production by TK-10 cells by SM17.

[0025] Figure 6 The effect of SM-17 in suppressing airway hyperresponsiveness in OVA-induced experimental asthma in mice is depicted.

[0026] Figure 7 The effect of SM-17 in suppressing BALF IL-5 and IL-13 levels in OVA-induced murine experimental asthma is depicted.

[0027] Figure 8 The effect of SM-17 in suppressing BALF eosinophil counts in OVA-induced experimental asthma in mice is depicted.

[0028] Fig. 9 The IL25 / SM17 interaction pattern and possible disease indications are depicted.

[0029] Fig. 10A The design of bispecific binding proteins (bispecific antibodies and antibody-receptor fusion proteins) is depicted.

[0030] Fig. 10B Depicted is SDS-PAGE of SM17, SM17-anti-TSLP, and SM17-anti-IL-33bsBp purified from the ExpiCHO transient transfection system.

[0031] Fig.11 Antigen binding of anti-alarmin bispecific binding proteins is depicted.

[0032] Fig.12 Depicted are the induction of IFNγ, CCL8, CCL17, IL-5 and IL-13 by alarmins from human PBMCs and the inhibitory effect of SM17 on cytokine release.

[0033] Fig.13 The potency of different bispecific binding proteins to induce cytokine and chemokine release from human PBMCs is depicted.

[0034] Fig.14 Results depicting the pro-proliferative effects of alarmins on ILC2s.

[0035] Fig.15 The results of ILC2 and Th2 cell responses to steroid hormones and bsBp are depicted.

[0036] Fig.16 Depicted are the results of a dendritic cell potency assay. 7. Specific implementation methods:

[0037] Type II inflammatory diseases involve a large number of irritant alarmins and cytokines upon challenge with allergens. Atopic dermatitis (AD) and asthma are representatives of this class of diseases.

[0038] AD is a chronic inflammatory skin disease characterized by severe itching. It affects 15-30% of children and 2-10% of adults, with severe impairment of quality of life. Immune factors in the pathogenesis of AD include massive dysregulation of Th2 lymphocytes and the release of related cytokines such as IL-4, IL-5, and IL-13. These factors lead to increased production of IgE, which leads to increased skin inflammation and aggravates the skin barrier defects in AD patients.

[0039] Interleukin-4 (IL-4) and interleukin-13 (IL-13) are of particular interest: they are hallmark cytokines of AD type II inflammatory responses triggered by invading parasites or allergens. The receptors for IL-4 and IL-13 share a common receptor chain, IL-4Rα; the IL-4Rα / IL-2Rγc (γc) heterodimer constitutes the receptor for IL-4, while 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 with the γc receptor chain or the IL-13 / IL-4Rα complex with the IL-13Rα1 receptor chain, respectively. While the receptor chain IL-4Rα is ubiquitously expressed, it is present at low levels in some cell types, whereas the expression of the γc or IL-13Rα1 receptor chains is cell type restricted. For example, in non-hematopoietic cells, although IL-13Rα1 shows high expression, γc is expressed at low or no expression [Junttila, Ilkka S et al. The Journal of Experimental Medicine, vol. 205, 11 (2008): 2595-608]. Recent studies have portrayed that IL-4 plays a central role in the generation and regulation of humoral immunity involving IgE in T2 reactions within lymph nodes; while IL-13 plays a more prominent role in peripheral tissues. Both IL-4 and IL-13 significantly reduce the expression of key structural proteins such as filaggrin, filaggrin 2, loricrin, involucrin, keratin 1, keratin 10, hornerin, desmoglein and desmocollin 1, as well as the expression of lipid components important for normal skin barrier function, leading to an increase in transepidermal water loss (TEWL). TEWL is usually measured to reflect the severity of AD and even to predict the occurrence of AD. Furthermore, both IL-4 and IL-13, by inhibiting AMP production in keratinocytes, have been reported to contribute to the development of skin dysbiosis, which is typically characterized by intense colonization with Staphylococcus aureus; this has recently been shown to precede the appearance of AD lesions. To this end, two biologics targeting these pathways have been developed and approved for the treatment of moderate to severe AD. They are dupilumab (an anti-IL-4Ra antibody that inhibits IL-4 and IL-13 responses) and tralokinumab (an anti-IL-13 antibody). However, approximately 50% of AD patients do not respond to these antibodies after 1 year of 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].

[0040] 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 most patients with asthma can achieve disease control using standard control therapy, approximately 5-10% of patients suffer from severe asthma that is not adequately controlled despite adherence to standard therapy (high-dose inhaled corticosteroids (ICS) plus long-acting β-agonists (LABA)). For severe asthma that is uncontrolled by standard therapy, the Global Initiative for Asthma (GINA) guidelines recommend the use of oral corticosteroids (OCS) for maintenance therapy. However, adverse events associated with OCS, such as those affecting the cardiovascular, gastrointestinal and musculoskeletal systems and infections, are common and can sometimes be fatal. Therefore, patients with severe asthma are characterized by the most urgent unmet medical needs and may be eligible for additional biological therapy.

[0041] There are two main types of asthma: Th2 high and Th2 low. In Th2 high asthma, excessive production 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 respiratory problems [Kuruvilla, Merin E et al. Clinical Reviews in Allergy & Immunology, vol. 56, 2 (2019): 219-233]. Therefore, biological agents targeting IgE, IL-4, IL-5 and IL-13 have recently emerged as promising additional therapies for treating severe uncontrolled asthma with a Th2 high phenotype.

[0042] IL-4 and IL-13 are also thought to have some non-redundant functions in allergy and asthma. In particular, IL-4 is thought to play a role primarily in the early stages of asthma development through its role in regulating T cell proliferation and survival and IgE synthesis. In contrast, human T cells are unable to respond to IL-13 due to the lack of surface expression of IL13Ra1. Unlike IL-4, IL-13 is more primarily involved in the late stages of allergic reactions, such as airway remodeling and goblet cell mucus hypersecretion, fibrosis, smooth muscle changes, and increased airway hyperresponsiveness [Gour, N., & Wills-Karp, M. (2015). Cytokine, 75(1), 68–78].

[0043] However, the clinical results of antibodies targeting IL-4 (altrakincept, pascolizumab) or IL-13 (tralokinumab) alone for the treatment of asthma have been disappointing [Panettieri, Reynold AJr 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]. But when both IL-4 and IL-13 are targeted, as in the case of dupilumab, clinically proven to be effective in treating Th2-mediated asthma [Maspero, Jorge F et al. The Journal Of Allergy And Clinical Immunology. Inpractice, vol. 8, 2 (2020): 527-539.e9]. This result suggests that dual blockade of IL-4 and IL-13 is necessary in the treatment of Th2-mediated asthma.

[0044] IL-5 plays a core pathogenic role in the differentiation, recruitment, survival and degranulation of eosinophils [Pelaia, Corrado et al. Frontiers in Physiology, vol. 10 1514. December 17, 2019]. There are a large number of patients with severe asthma who express a Th2-high phenotype characterized by eosinophilic inflammation. More than half of asthma subjects may have airway eosinophilia, and high levels of eosinophils are associated with repeated exacerbations of asthma and severe bronchial obstruction. Th2-high asthma with eosinophilia usually responds to treatment with corticosteroids, which may be due to the removal of eosinophils through corticosteroid-induced apoptosis. However, severe eosinophilic asthma may be resistant to inhaled and systemic corticosteroids because of excessive amounts of IL-5 in the bronchi. Excessive amounts of IL-5 in the bronchi can overcome the pro-apoptotic effects of corticosteroids on eosinophils. Therefore, blocking IL-5 activity by anti-IL-5 antibodies may help maintain therapeutic responsiveness to corticosteroid therapy, and in fact, three antibodies that block the IL-5 pathway are being used in combination with corticosteroids for the treatment of Th2-high asthma, including Reslizumab (anti-IL-5 antibody), Mepolizumab (anti-IL-5 antibody), and Benralizumab (anti-IL-5Ra antibody).

[0045] Allergic asthma is a Th2-high asthma subtype characterized by the presence of IgE antibodies to one or more common environmental allergens, such as house dust mites. In patients with allergic asthma, anti-allergen IgE binds to IgE receptors (FcεRI) on the surface of mast cells. Exposure to allergen antigens results in cross-linking of FcεRI on the surface of mast cells; when the intensity and duration of this FcεRI cross-linking are sufficient, mast cells become activated, leading to the release of their own effective material mediators: histamine, prostaglandin (PG) D2, and leukotriene (LT) C4, ultimately leading to bronchoconstriction, mucus secretion, and mucosal edema. Activated mast cells can also synthesize and secrete a large number of proinflammatory cytokines (including IL-4, IL-5, and IL-13), which in turn can regulate the development of IgE synthesis and eosinophilic inflammation. The anti-IgE antibody omalizumab is the first and, for a long time, the only monoclonal antibody that can be used as an add-on treatment for severe allergic asthma. Omalizumab works by selectively preventing human IgE from binding to its receptors and thereby inhibiting mast cell activation.

[0046] Although great progress has been made in elucidating Th2-high inflammatory pathways and developing related biologics for the treatment of Th2-high asthma, effective approaches to address Th2-low asthma are still lacking. Th2-low asthma patients respond poorly to corticosteroids, which further exacerbates the severity of the problem. Therefore, there is an unmet medical need for effective treatments for Th2-low asthma.

[0047] Another problem with standard care for asthma is over-reliance on OCS. In addition to ICS, about 30% of adult patients with severe asthma rely on OCS therapy 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 Initiative for Asthma (GINA) group is committed to setting an "acceptable" OCS dose for maintenance therapy, and they recommend 7.5 mg per day as an acceptable level because it corresponds to the physiological level of steroid production. However, recent studies have shown that there is no benign OCS prescription because the risk of adverse events is cumulative and increases with each treatment. After only four short-term OCS courses, an increase in incidence is seen [Sullivan, Patrick W et al. The Journal of Allergy and Clinical Immunology, vol. 141, 1 (2018): 110-116.e7]. In terms of lifetime cumulative doses, most side effects occur after patients receive 1-2.5 g of corticosteroids, and the incidence of diabetes begins to increase in patients receiving only 0.5 g of corticosteroids [Price, David B et al. Journal of Asthma and Allergy, vol. 11 193-204. 29 Aug. 2018].

[0048] The OCS-sparing effects of dupilumab, mepolizumab, reslizumab, omalizumab, and benralizumab were clinically evaluated. Although treatment with these biologics can reduce the daily OCS dose by 50%, only 5%-20% of patients with severe asthma are able to stop OCS use. There is still a huge unmet medical need to develop new treatments or biologics for the treatment of asthma that can eliminate or further reduce dependence on OCS [Cataldo, Didier et al., The Journal of Asthma: Official Journal Of The Association For The CareOf Asthma, vol. 58, 4 (2021): 448-458].

[0049] Currently, FDA-approved biologics for the treatment of asthma and AD target pathways downstream of Th2 inflammation. However, there are still clinical deficiencies that need to be addressed in patients treated with these biologics, such as high relapse rates, limited efficacy against non-Th2 inflammation, and reliance on continued use of OCS (albeit at reduced levels). Recently, new biologics targeting alarmins, including IL-25, TSLP, and IL-33, are in development. They are upstream elements that affect the Th2 inflammatory pathway and have the potential to potentially lead to both Th2 and non-Th2 inflammatory responses. Therefore, anti-alarmin biologics may provide an additional option that may address current clinical deficiencies in patients with AD and Th2 high / low severe asthma.

[0050] IL-25 (also known as IL-17E) is a member of the IL-17 cytokine family, which covers IL-17A to IL-17F. IL-25 binds to its receptor 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 and can induce the expression of IL-4, IL-5 and IL-13 genes, and further amplify allergic inflammatory responses in the lungs and digestive tract. IL-25 is important in type 2 immune responses because it activates IL-17RA / IL-17RB complexes in multiple cell types (including epithelial cells, Th2 cells, and ILC2). It has been reported that IL-25 inhibits CD4+ T cell activation and differentiation into Th17 cells and plays an anti-inflammatory role in autoimmune and inflammatory diseases by downregulating Th1 and Th17 cell responses.

[0051] 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, acting on several immune cell types, including 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 key role in driving Th2-mediated inflammation by regulating antigen-presenting cells (such as dendritic cells) to amplify type 2 cytokines of T cells and innate lymphocytes [Ito, Tomoki et al. The Journal Of Experimental Medicine, vol. 202, 9 (2005): 1213-23].

[0052] IL-33 is a member of the IL-1 family and has recently been identified as a ligand for IL-1 receptor family member T1 / ST2 (ST2). IL-33 is a dual-function protein that acts as a proinflammatory cytokine and an intracellular nuclear factor with transcriptional regulatory properties. After cell stress or necrosis, IL-33 is released into the extracellular space and serves as an endogenous danger signal that warns the immune system of tissue damage during trauma or infection. IL-33 expands Th1 and Th2 type responses through its activity on human basophils, allergen-reactive Th2 cells, iNKT and NK cells. Although IL-33 triggers type 2 cytokine responses in a normative manner, this cytokine can also synergize with type 1 cytokines such as IL-12 to stimulate interferon gamma (IFNγ) [Komai-Koma, Mousa et al. Immunobiology, vol. 221, 3 (2016): 412-7]. Therefore, IL-33 is emerging as an important immune regulator, playing an important role in allergic, fibrotic, infectious and chronic inflammatory diseases.

[0053] Because IL-25, TSLP, and IL-33 (collectively referred to as anti-alarmins) exhibit broad functions beyond the Th2 immune response, in addition to the approved indications for biologics that interact with downstream targets of the Th2 pathway, biologics targeting these anti-alarmins are considered to be effective against additional allergic disease subtypes. 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-λ monoclonal antibody that specifically binds to TSLP and prevents TSLP from interacting with its receptor complex. In a Phase II randomized, double-blind, placebo-controlled study (ALLEVIAD; NCT02525094), patients with moderate to severe AD showed an improvement trend in all endpoints after receiving terzelumab plus topical corticosteroids (TCS), while the improvement of placebo plus TCS did not reach statistical significance, as assessed by EASI50 at week 12. Two independent Phase III trials (SOURCE and NAVIGATOR) were conducted to evaluate the clinical efficacy of terzelumab in the treatment of moderate to severe asthma. In the NAVIGATOR study, patients with moderate to severe asthma received terzelumab plus OCS or placebo plus OCS. Compared with the placebo group, the treatment group achieved the primary endpoint with statistical significance, indicating that the reduction in AAER (annual asthma exacerbation rate) was clinically significant [Menzies-Gow, Andrew et al., The New England Journal of Medicine, vol. 384, 19 (2021): 1800-1809]. In the NAVIGATOR study, terzelumab (used with OCS) was equally effective in suppressing severe asthma in both Th2-high and Th2-low patients. However, in the SOURCE study, terzelumab failed to meet the primary endpoint of reducing daily OCS without loss of asthma control compared with placebo – a statistically significant finding. This result suggests that terzelumab used as monotherapy may not be adequate for the treatment of moderate to severe asthma.

[0054] Etokimab (ABN020), an anti-IL-33 humanized IgG1 monoclonal antibody, is being clinically evaluated for the treatment of AD. In a Phase 1 trial, administration of ABN020 resulted in a significant reduction in the number of eosinophils in the blood. In a Phase IIa study, it was found that ABN020 significantly alleviated symptoms of house dust mite-induced dermatitis and reduced neutrophil skin infiltration (a sign of non-Th2 inflammation) [Chen, Yi-Ling et al. Science Translational Medicine, vol. 11, 515 (2019): eaax2945]. These results indicate that ABN020 can simultaneously inhibit Th2 and non-Th2 inflammation. However, in a subsequent Phase IIb study, ABN020 failed to reach the primary endpoint of treating moderate to severe AD. Another anti-IL-33 antibody, Itepekimab (REGN3500), is being clinically evaluated for the treatment of asthma. In a Phase II proof-of-concept (POC) study, patients who discontinued ICS and long-acting bronchodilator inhalers (LABA) received 12 weeks of treatment with either dupilumab or tekinumab [Wechsler, Michael E et al. The New England Journal Of Medicine, vol. 385, 18 (2021): 1656-1668]. The rate of asthma loss control in patients treated with tekinumab (22%) was significantly lower than that in patients treated with placebo (41%). These data suggest that tekinumab may be effective as a monotherapy without the use of ICS and LABA. However, the efficacy of tekinumab was slightly lower than that of the anti-IL4Ra antibody dupilumab (19%). Therefore, further clinical development of tekinumab for asthma was discontinued for strategic rather than scientific reasons. In any case, it was later found that eptekimab reduced the exacerbation rate and improved 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 studies of eptekimab for the treatment of COPD in former smokers are currently underway.

[0055] To date, the efficacy of truncating the IL-25 / IL-17RB pathway for immune diseases such as asthma and AD has not been clinically validated. The most advanced anti-IL25 antibody is XKH001 developed by Kanovabiopharma, which is currently being evaluated in a Phase I trial. LNR 125.38 is another anti-IL-25 antibody from Lanier Biotherapeutics, currently in the preclinical stage; LNR 125.38 significantly reduced the increase in type 2 cytokines and inflammatory cells in allergic mice and mice with rhinovirus-induced asthma exacerbation. SM17, developed by SinoMab BioScience Limited, is the first humanized anti-IL17RB monoclonal antibody to enter Phase I clinical trials. SM17 does not block the binding of IL-25 to IL-17RB, but rather inhibits signal transduction through the IL-25 / IL-17RB pathway. SM17 is humanized from its parental murine antibody D9.2, which has shown therapeutic potential in preclinical murine studies for the treatment of inflammatory bowel disease, idiopathic pulmonary fibrosis, asthma, and rhinovirus-induced asthma exacerbations.

[0056] Despite the demonstrated preclinical and clinical efficacy of these anti-alarmin antibodies (particularly anti-TSLP and anti-IL33 antibodies) in reducing both Th2 and non-Th2 inflammation, their therapeutic responses as add-on or monotherapy for the treatment of asthma and / or AD have been somewhat suboptimal. One 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 any single one of them is not sufficient to achieve optimal clinical response.

[0057] At the molecular level, all three alarmins can directly activate ILC2, a member of the innate lymphoid cell (ILC) family. 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 secrete IL-5 and IL-13 in response to these signals, thereby enhancing allergic responses. If ILC2s are dysregulated, they can lead to excessive secretion of Th2 inflammatory cytokines, contributing to the development of allergic asthma, AD, allergic rhinitis, ulcerative colitis, and many chronic fibroproliferative disorders. ILC2s are also closely associated with rapid relapses of AD disease and OCS dependence in severe asthma.

[0058] In patients with severe eosinophilic asthma, a course of OCS resulted in a decrease in circulating eosinophils, Th2 cells, and Tc2 cells, but not ILC2s. This suggests that ILC2s are more resistant to OCS than Th2 cells (Hynes, G., et al. 2018). Clinically, IL-13 + ILC2 has a stronger positive correlation with the patient's asthma control status and is more resistant to glucocorticoid-induced cell death and type 2 cytokine release inhibition [Jia, Yi et al. American Journal Of Respiratory Cell And Molecular Biology, vol. 55, 5 (2016): 675-683]. Recent studies have further shown that all three alarmins play an important role in the corticosteroid resistance of ILC2. It has been confirmed in a mouse model that IL-33 rapidly increases the number of ILC2s in the peribronchial / perivascular areas, and the lung accumulation of ILC2s depends on the CCL8-CCR8 signaling pathway. It is known that IL-33 treatment leads to the production of CCL8, which mainly comes from pulmonary airway macrophages. Signal transduction through the CCL8-CCR8 pathway in turn affects the production of ILC2 cytokines (IL-13 and IL-5) and IL-13 +Activating ILC2 motility plays a key role [Puttur, Franz et al. Science Immunology, vol.4, 36 (2019): eaav7638]. It was later discovered that a subpopulation of ILC2s called "inflammatory" ILC2s (iILC2s) was involved in the development of corticosteroid resistance. In patients with chronic rhinitis or asthma, a correlation between disease severity and resistance to corticosteroid therapy was established, especially when the number of circulating iILC2s and resident iILC2s in inflamed mucosal tissues increased. Interestingly, the development and migration of iILC2s 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]. TSLP treatment also reportedly increases steroid resistance of ILC2s. Bronchoalveolar lavage fluid (BALF) ILC2s collected from asthmatic patients with high TSLP levels are steroid-resistant. IL-7 and TSLP abolished the inhibitory effect of dexamethasone on the production of type 2 cytokines by blood ILC2s [Liu, Sucai et al. The Journal Of Allergy And Clinical Immunology, vol. 141, 1 (2018): 257-268.e6].

[0059] Alarmins also play a key role in Th2 memory cell-mediated relapse of AD. In patients treated with dupilumab for one year, persistent skin-resident, treatment-resistant immune Th2 memory in AD was identified. These memory Th2 cells are IL-13 + IL-17RB + 、ST2 + ,TSLPR + , indicating that they can respond to alarmins. Previous studies have reported that IL-33 can induce Th2 memory cells to produce IL-31, a cytokine that causes severe itching 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]. In another report, CRTH2 + CD4 +Th2 memory cell infiltration of AD skin lesions is associated with TSLP-activated DCs (TSLP-DCs) [Wang, Yui-I et al. Immunity, vol. 24, 6 (2006): 827-838]. In addition, IL-25 enhances the proliferation of TSLP-DC-stimulated Th2 memory cells [Wang, YIHsi et al. The Journal of Experimental Medicine, vol. 204, 8 (2007): 1837-47]. These studies suggest that the neutralization of IL-4 and IL-13 by dupilumab may not be sufficient to inhibit the activity of Th2 memory cells. This may help explain why a considerable proportion of AD patients experience partial and non-persistent responses when receiving dupilumab 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]. Unlike IL-4 and IL-13, alarmins regulate both short-term Th2 effector function and long-term Th2 memory in AD. Therefore, neutralizing alarmins serves as a novel approach for AD treatment.

[0060] Since the three alarmins play redundant and overlapping roles in type 2 immunity, blocking any one of the three alarmins is unlikely to be sufficient to completely suppress Th2 and ILC2 activity. This may explain why tezelumab and tekinumab, although they inhibit both Th2 and non-Th2 inflammatory responses, show limited clinical efficacy in asthma and AD.

[0061] Although redundant, these alarmins have non-overlapping functions in Th2 as well as non-Th2 responses. IL-33 acts synergistically with IL-12 to directly induce human NK cells to produce interferon gamma (IFNg), a well-known pathogenic factor in the pathogenesis of inflammatory bowel disease (IBD). Studies of patient biopsies have shown that IL-33 levels are elevated in patients with active IBD, especially ulcerative colitis (UC) [Kobori, Ayako et al. Journal of Gastroenterology, vol. 45, 10 (2010): 999-1007]. In addition, UC-associated IL-33 has been found in myofibroblasts, which are often located at the base of inflammatory ulcers in UC patients [Sponheim, Jon et al. The American Journal Of Pathology, vol. 177, 6 (2010): 2804-15]. Blocking the IL-33 / ST2 pathway showed that experimental colitis was improved by enhancing mucosal healing in mice, and active disease in humans was alleviated, indicating the pathogenic role of IL-33 in IBD [Sedhom, Mamdouh AK et al. Gut, vol. 62, 12 (2013): 1714-23]. In contrast, TSLP induces human DC to express OX40 ligand (OX40L), but does not express IL-12, which is a prerequisite for tregI naive CD4+T cells to produce IL-4, IL-5 and IL-13. In addition, TSLP-activated DCs produce chemokines such as CCL17 / TARC and CCL22 / MDC, which attract naive T cells. TSLP directly stimulates naive CD4+T cells by engaging OX40 on T cells through TSLPR or indirectly through OX40 ligand (induced by TSLP on DC), inducing specialized Th2 polarization.

[0062] Interestingly, human TSLP and TLR3 ligands promote the differentiation of Th17 cells with a central memory T cell phenotype through DC activation. Similarly, IL-25 from local mast cells in the skin stimulates dermal DC to produce IL-1β, thereby helping to activate Th17 cells, rather than Th2 cells, in the induction phase of contact dermatitis. In psoriatic skin, IL-25 stimulates keratinocyte proliferation by activating STAT3 transcription factors and induces the production of inflammatory cytokines and chemokines. IL-25 expression in keratinocytes also contributes to the expansion of psoriasis-like inflammation. In addition, IL-25 is more effective than IL-33 in inducing human peripheral blood mononuclear cells (PBMC) to secrete IL-5 and IL-13 [Bartemes, Kathleen R et al. The Journal Of Allergy And Clinical Immunology, vol. 134, 3 (2014): 671-678.e4].

[0063] Although redundant, the three alarmins can play different roles in regulating the activities and reactions of ILC2, Th1, Th2 and Th17. In fact, it is reported that they target different cell types of 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 (such as contact dermatitis, AD, Th2-high / low asthma) and autoimmune diseases (such as inflammatory bowel disease, psoriasis), blocking a single alarmin may only be effective for specific patient groups. For example, Th1 and Th2 mixed phenotypes are most common in European and American AD, while Th17 and Th2 mixed phenotypes are 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 leaves room for improvement in addressing these immune diseases and presents an unmet medical need for new approaches and therapies targeting different subtypes of immune diseases.

[0064] One approach to this alternative therapy may include co-administration of biologics (such as antibodies) directed against two or more alarmins to treat different aspects of allergic diseases (such as pediatric and adult AD). Co-administration requires injection of two separate products, or a single injection of a co-formulation of two different biologics. Although two injections allow flexibility in dosage and timing, it is not convenient for patient compliance. In addition, although co-formulations can provide some flexibility in dosage, due to the different molecular properties of the two or more anti-alarmin biologics, it is often quite challenging or impossible to find formulation conditions with acceptable viscosity (at relatively high concentrations) and promote chemical and physical stability. In addition, co-administration and co-formulation involve the additional cost of two or more different drug therapies, which may increase patient and / or payer costs. Therefore, there is still a need for alternative therapies to treat allergic diseases with disease modifications, and such alternative therapies preferably include bispecific or multispecific binding proteins directed against different alarmins.

[0065] For illustration purposes, the present invention provides a bispecific binding protein for 2 different alarmins; it can be in the form of a bispecific protein, one end of which is specific for IL-17RB and the other end of which is specific for soluble alarmins. Specifically, the bispecific protein can be an anti-IL17RB (IL-25 receptor) antibody, fused at its C-terminus with (a) a single-chain Fv (scFv) targeting IL-33 or TSLP alarmins; or (b) the extracellular domain of 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 also be extended to multispecific binding proteins targeting different alarmin receptors, alarmins and other downstream cytokines.

[0066] Before the present disclosure is further described, it is to be understood that the present disclosure is not limited to the particular embodiments described herein and that the terminology used herein is for the purpose of describing particular embodiments and is not intended to be limiting.

[0067] Unless otherwise defined herein, scientific and technical terms used in this disclosure shall have the meanings commonly understood by those of ordinary skill in the art. In addition, unless the context otherwise requires, singular terms shall include the plural, and plural terms shall include the singular. In general, the terms used in connection with cell and tissue culture, molecular biology, immunology, genetics, and protein and nucleic acid chemistry and hybridization described herein and these techniques are all commonly used terms and techniques well known in the art.

[0068] The term "a" or "an" entity refers to one entity; for example, "a vector" is understood to refer to one vector.

[0069] The term "and / or" as used herein should be understood to specifically disclose each of the two specified features or components, whether or not the other is included. Therefore, the term "and / or" used in phrases such as "A and / or B" herein is intended to include "A and B", "A or B", "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in phrases 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 (alone); B (alone); and C (alone).

[0070] Range: Throughout this disclosure, various aspects of the invention may be expressed in range format. It should be understood that description in range format is for convenience and brevity only and should not be construed as a rigid limitation on the scope of the invention. Therefore, description of a range should be considered to have specifically disclosed all possible subranges within that range as well as individual values. For example, description of a range of 1 to 6 should be considered to have specifically disclosed subranges of 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, and individual values ​​within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0071] Exemplary genes and polypeptides are described herein with reference to GenBank numbers, GI numbers and / or SEQ ID NOs. It should be understood that one skilled in the art can readily identify homologous sequences by reference to sequence sources, including but not limited to GenBank (ncbi.nlm.nih.gov / genbank / ) and EMBL (embl.org / ).

[0072] 7.1IL17RB protein

[0073] Human IL17RB is a 47.9 kDa transmembrane protein (462 aa) that belongs to the IL-17 receptor family. IL-17RB is expressed in various endocrine tissues and different organs, such as the kidney and liver and epithelial cells of mucosal tissues. Elevated expression of IL-17RB has also been found in lung tissue of asthmatic patients and skin lesions of AD patients. The expression of IL-17RB in human ILC2, natural killer T (NKT) cells and Th2 cells indicates a potential role in immune cells. IL-17RB is shared by two ligands: IL-17B and IL-25 (also known as IL-17E). IL-25 binds to the heterodimeric IL-17RA / IL-17RB complex, while IL-17B is reported to bind to both the heterodimeric receptor and the IL-17RB homodimeric receptor [Wu, Heng-Hsiung et al. Science Translational Medicine, vol. 13, 583(2021): eabc2823]. The binding affinity (K) of IL-17B to IL-17RB D ) has a binding affinity about 30-fold lower than that of IL-25 (IL-17E), and the association rate (K on ) are similar, but the dissociation rate (K off ) is much faster. Additional information about human IL-17RB, including its exemplary amino acid sequence, can be found in public databases such as GENEBANK (NCBI Ref.). Exemplary sequences are also provided below.

[0074] 1MSLVLLSLAA LCRSAVPREP TVQCGSETGPSPEWMLQHDL IPGDLRDLRV EPVTTSVATG

[0075] 61DYSILMNVSW VLRADASIRL LKATKICVTG KSNFQSYSCV RCNYTEAFQT QTRPSGGKWT

[0076] 121FSYIGFPVEL NTVYFIGAHN IPNANMNEDGPSMSVNFTSP GCLDHIMKYK KKCVKAGSLW

[0077] 181DPNITACKKN EETVEVNFTT TPLGNRYMAL IQHSTIIGFS QVFEPHQKKQ TRASVVIPVT

[0078] 241GDSEGATVQL TPYFPTCGSD CIRHKGTVVL CPQTGVPFPL DNNKSKPGGW LPLLLLSLLV

[0079] 301ATWVLVAGIYLMWRHERIKKTSFSTTTLLPPIKVLVVYPSEICFHHTICY FTEFLQNHCR

[0080] 361SEVILEKWQK KKIAEMGPVQ WLATQKKAAD KVVFLLSNDV NSVCDGTCGK SEGSPSENSQ

[0081] 421DLFPLAFNLF CSDLRSQIHL HKYVVVYFRE IDTKDDYNAL SVCPKYHLMK DATAFCAELL

[0082] 481HVKQQVSAGK RSQACHDGCC SL (SEQ ID NO: 88)

[0083] 7.2 Bispecific Binding Proteins

[0084] The present disclosure provides bispecific binding proteins that can specifically bind two antigens. The binding protein generally comprises a variable light chain and a variable heavy chain region or domain, and these regions or domains correspond to the variable light chain and the variable heavy chain region or domain of an immunoglobulin. At least one antigen binding portion of the binding protein is a single-chain form, referred to in the art as scFv. In some embodiments, another antigen binding portion comprises IgG. In other embodiments, other antigen binding portions comprise scFv.

[0085] In some embodiments, methods and uses of antibodies or antigen-binding fragments thereof are provided herein, which can specifically bind to alarmin receptors, alarmins, or both. As used herein, the term "antibody" and its grammatical equivalents refer to immunoglobulin molecules that recognize and specifically bind to targets (such as proteins, polypeptides, peptides, carbohydrates, polynucleotides, lipids, or combinations of any of the above substances) through at least one antigen binding site (wherein the antigen binding site is generally located in the variable region of the immunoglobulin molecule). As used herein, the term includes complete polyclonal antibodies, complete monoclonal antibodies, single domain antibodies (sdAb; for example, 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 (such as dual variable domain immunoglobulin molecules) containing antigen binding sites, as long as these antibodies exhibit the desired biological activity. Antibodies also include, but are not limited to, mouse antibodies, rabbit antibodies, camel antibodies, primate antibodies, chimeric antibodies, humanized antibodies, and human antibodies. Antibodies can be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes) (such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), which are called α, δ, ε, γ, and μ, respectively, based on the characteristics of their heavy chain constant domains. In some embodiments, an antibody may comprise four polypeptide chains, two heavy chains (H) and two light chains (L), which are interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. Each light chain consists of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. Unless otherwise expressly stated, as used herein, the term "antibody" includes an "antigen-binding fragment" of a complete antibody. As used herein, the term "antigen-binding fragment" refers to a portion or fragment of a complete antibody that is an antigen-determining variable region of a complete 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 attached 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 is a single polypeptide chain with VL and VH regions joined by recombinant means), heavy chain antibodies (HCAb), light chain antibodies (LCAb), disulfide bond-linked scFv (dsscFv), diantibodies (bivalent, bispecific antibodies), triantibodies, tetraantibodies, miniantibodies, dual variable domain antibodies (DVD), single variable domain antibodies (sdAb or dAb; for example: camelid antibodies, alpaca antibodies), single variable domains of heavy chain antibodies (VHH), and bispecific or multispecific antibodies formed by antibody fragments. "Bispecific" antibodies or binding proteins are artificial hybrid antibodies with two different antigen binding sites that recognize and specifically bind to two different target sites. Bispecific binding antibodies and proteins can be produced by a variety of methods, including fusion of hybridomas or connection of Fab' fragments. See, for example, 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.

[0086] When used to refer to antibodies, the term "heavy chain" refers to a polypeptide chain of about 50-70 kDa, wherein the amino terminal portion includes a variable region (VH) of about 120-130 or more amino acids and a carboxyl terminal portion including a constant region. In some embodiments, the heavy chain constant region consists of three domains, CH1, CH2 and CH3, and there is a short flexible hinge region connecting the CH1 and CH2 domains. Based on the amino acid sequence of the heavy chain constant region, the constant region can be one of five different types, respectively called alpha (a), delta (δ), epsilon (ε), gamma (γ) and mu (μ). Different heavy chains have different sizes: α, δ and γ contain about 450 amino acids, while μ and ε contain about 550 amino acids. When combined with a light chain, these different types of heavy chains produce five well-known classes of antibodies, IgA, IgD, IgE, IgG and IgM, including four subclasses of IgG, namely IgGl, IgG2, IgG3 and IgG4. The heavy chain can be a human heavy chain.

[0087] When used to refer to antibodies, the term "light chain" refers to a polypeptide chain of about 25 kDa, wherein the amino terminal portion includes a variable region of about 100 to about 110 or more amino acids and a carboxyl terminal portion including a constant region. The light chain constant region consists of a domain CL. The approximate length of the light chain is 211 to 217 amino acids. Based on the amino acid sequence of the constant region, there are two different types, i.e. kappa (κ) and lambda (λ). The light chain amino acid sequence is well known in the art. The light chain can be a human light chain.

[0088] The term "variable domain" or "variable region" refers to a portion of an antibody light or heavy chain, usually located at the amino terminus of the light or heavy chain, and is about 120 to 130 amino acids in length in the heavy chain and about 100 to 110 amino acids in length in the light chain, and is used for the binding and specificity of each specific antibody to its specific antigen. The variable domains vary greatly in sequence between different antibodies. The variability of the sequence is mainly concentrated in the CDRs, while the less variable parts of the variable domain are called framework regions (FRs). The CDRs of the light and heavy chains are mainly responsible for the interaction between the antibody and the antigen. In some embodiments, each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The amino acid position numbering used herein is based on the EU index, such as Kabat et al. (1991) Sequences of proteins of immunological interest. (U.S. Department of Health and Human Services, Washington, DC) 5th edition.

[0089] CDR refers to one of the three hypervariable regions (H1, H2 or H3) in the non-framework region of the VHβ sheet framework of an immunoglobulin (Ig or antibody), or one of the three hypervariable regions (L1, L2 or L3) in the non-framework region of the VLβ sheet framework of an antibody. Therefore, CDR is a variable region sequence interspersed in the framework region sequence. CDR regions are well known to those skilled in the art and have been defined by a variety of methods / systems. These systems and / or definitions have been developed and improved for many years and include Kabat, Chothia, IMGT, ABM and Contact. For example, Kabat defines the region with the highest variability in the variable (V) domain of an antibody (Kabat, EA et al. The Journal Of Biological Chemistry, vol. 252, 19 (1977): 6609-16.; Kabat, EA Advances in protein chemistry vol. 32 (1978): 1-75.). The Chothia definition is based on the position of the structural loop regions, which defines the CDR region sequences as those residues that are not part of the conserved β-sheet framework and are therefore able to adapt to different conformations [Chothia, C, and AM Lesk. Journal of Molecular Biology, vol. 196, 4 (1987): 901-17]. Both terms are recognized in the art. In addition, the IMGT system is based on sequence variability and position in the variable region structure. The AbM definition is a compromise between Kabat and Chothia. The Contact definition is based on analysis of available antibody crystal structures. Software programs (such as abYsis) are available and known to those skilled in the art for analyzing antibody sequences and determining CDRs. The positions of CDRs in canonical antibody variable domains have been determined by comparing a large number of 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]. Since the number of residues in the hypervariable region varies among different antibodies, in the canonical variable domain numbering scheme, additional residues relative to the canonical position are usually numbered with a, b, c, etc. next to the residue number. This nomenclature is also well known to those skilled in the art.

[0090] For example, the table below lists CDRs defined according to Kabat (hypervariable) or Chothia (structural) nomenclature.

[0091] <![CDATA[Kabat 1 ]]> <![CDATA[Chothia 2 ]]> Ring position VHCDR 31-35 26-32 Connecting chains B and C VHCDR2 50-65 53-55 Connecting C' and C" chains VHCDR3 95-102 96-101 Connecting F and G strands VLCDR 24-34 26-32 Connecting chains B and C VLCDR2 50-56 50-52 Connecting C' and C" chains VLCDR3 89-97 91-96 Connecting F and G strands

[0092] 1 The residue numbering follows the nomenclature of Kabat et al., supra.

[0093] 2 The residue numbering follows the nomenclature of Chothia et al., supra.

[0094] One or more CDRs can also be incorporated into a molecule in a covalent or non-covalent manner to make it an immunoadhesin. An immunoadhesin can incorporate the CDR(s) as part of a larger polypeptide chain, can covalently link the CDR(s) to another polypeptide chain, or can incorporate the CDR(s) non-covalently. The CDRs allow the immunoadhesin to bind to a specific target antigen.

[0095] The terms "epitope" and "antigenic determinant" are used interchangeably herein and refer to the site where an antibody or antigen binding fragment binds to the surface of a target molecule, such as a localized region on the surface of an antigen. The target molecule may include a protein, peptide, nucleic acid, carbohydrate, or lipid. An epitope with immunogenic activity is a portion of a target molecule that causes 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 may be determined by any method known in the art (including, for example, by immunoassay). An antigenic epitope is not necessarily immunogenic. An epitope is typically composed of chemically active surface groups of molecules such as amino acids or sugar side chains, and has specific three-dimensional structural characteristics and specific charge characteristics. The term "epitope" includes linear epitopes and conformational epitopes. The region in a target molecule (e.g., a polypeptide) that acts on an epitope may be a continuous amino acid of a polypeptide, or an epitope may be composed of a combination of two or more non-continuous regions of a target molecule. An epitope may or may not be a three-dimensional surface feature of a target molecule. Epitopes formed by contiguous amino acids (also called linear epitopes) are usually retained when the protein is denatured, while epitopes formed by tertiary folding (also called conformational epitopes) are usually lost when the protein is denatured. Epitopes usually include at least 3, and more commonly, at least 5, 6, 7 or 8-10 amino acids in a unique spatial conformation.

[0096] As used herein, the term "specific binding" refers to the interaction of a polypeptide or molecule with an epitope, protein or target molecule that is more frequent, more rapid, longer lasting, stronger in affinity, or some combination thereof than with alternative substances (including related or unrelated proteins). A binding moiety (e.g., an antibody) that specifically binds to a target molecule (e.g., an antigen) can be identified by methods such as immunoassays, ELISA, biolayer interferometry ("BLI"), SPR (e.g., Biacore), or other techniques known to those skilled in the art. Typically, the specific response is at least twice the background signal or noise, and can be more than 10 times the background. For a discussion of antibody specificity, see Paul, ed., 1989. Fundamental Immunology Second Edition 2nd ed., Raven Press, New York, pp. 332-336. A binding moiety that specifically binds to a target molecule can bind to the target molecule with an affinity that is higher than its affinity for a different molecule. In some embodiments, a binding moiety that specifically binds to a target molecule can bind to the target molecule with an affinity that is at least 20 times higher, at least 30 times higher, at least 40 times higher, at least 50 times higher, at least 60 times higher, at least 70 times higher, at least 80 times higher, at least 90 times higher, or at least 100 times higher than its affinity for a different molecule. In some embodiments, a binding moiety that specifically binds to a particular target molecule binds to the different molecule with such low affinity that binding cannot be detected using an assay described herein or known in the art. In some embodiments, "specific binding" refers, for example, to a binding moiety that binds to a different molecule with a K of about 0.1 mM or less. D Bind to target molecule.

[0097] In some embodiments, "specific binding" refers to a polypeptide or molecule that binds with a K of about 10 μM or less or about 1 μM or less. D In some embodiments, "specific binding" refers to a polypeptide or molecule with a K of about 0.1 μM or less, about 0.01 μM or less, about 1 nM or less. DBinding target. Due to the sequence identity between homologous proteins of different species, specific binding may include polypeptides or molecules that recognize proteins or targets of more than one species. Similarly, due to the homology in certain regions of the polypeptide sequences of different proteins, specific binding may include polypeptides or molecules that recognize more than one protein or target. It is understood that, in some embodiments, the binding portion (such as an antibody) that specifically binds to the first target may or may not specifically bind to the second target. Therefore, "specific binding" does not necessarily require (although it may include) exclusive binding, i.e., binding to a single target. Therefore, in some embodiments, a binding portion (such as an antibody) can specifically bind to more than one target. For example, in some cases, an antibody may include two identical antigen binding sites, each of which can specifically bind to the same epitope on two or more proteins. In some alternative embodiments, an antibody may be bispecific and include at least two antigen binding sites with different specificities.

[0098] As used herein, the term "binding affinity" generally refers to the sum of the strengths of non-covalent interactions between a binding moiety and a target molecule (such as an antigen). The binding of a binding moiety to a target molecule is a reversible process, and the binding affinity is usually expressed in terms of the equilibrium dissociation constant (K D ) to report. D is the dissociation rate (k off or k d ) and the association rate (k on or k a ) ratio. D The lower the K, the higher the affinity. A is the equilibrium association constant, which is also the reciprocal of the equilibrium dissociation constant, i.e. = 1 / K D For antibody-antigen interactions, K D It can be calculated as the ratio of the product of the free antibody and free antigen concentrations to the antibody-antigen complex concentration, that is, [antigen] x [antibody] / [antigen-antibody].

[0099] There are many methods known in the art for measuring binding affinity, any of which may be used for the purposes of the present disclosure. Specific illustrative embodiments include the following. In some embodiments, "K D ” or “K D The "value" can be measured by assays known in the art, such as by binding assays. D Can 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 DK values ​​can also be measured by biolayer interferometry (BLI), for example using the Gator system (Probe Life) or the Octet-96 system (Sartorius, Gottingen, Germany). D or K D Values ​​can also be measured by surface plasmon resonance assay using a BIAcore system (eg, Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, New Jersey).

[0100] As used herein, the term "variant" in relation to a protein or polypeptide having a particular sequence feature ("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) amino acid substitutions, deletions, and / or additions compared to the reference protein or reference polypeptide. The change in the amino acid sequence may be an amino acid substitution. The change in the amino acid sequence may be a conservative amino acid substitution. A functional fragment or functional variant of a protein or polypeptide retains the basic structural and functional properties of the reference protein or polypeptide.

[0101] As used interchangeably herein, the terms "polypeptide", "peptide", "protein" and their grammatical equivalents refer to an amino acid polymer of any length, which may be linear or branched. It may include non-natural or modified amino acids, or may be interrupted by non-amino acids. The polypeptide, peptide or protein may also be modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification.

[0102] As used interchangeably herein, the terms "polynucleotide," "nucleic acid," and their grammatical equivalents refer to nucleotide polymers of any length, and include DNA and RNA. The nucleotides may be deoxynucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. Nucleic acid molecules may be single-stranded or double-stranded.

[0103] As used herein, the term "coding" and its grammatical equivalents refer to the inherent properties of a specific nucleotide sequence in a polynucleotide or nucleic acid (such as a gene, cDNA or mRNA), which serves as a template for synthesizing other polymers and macromolecules in a biological process, and these polymers and macromolecules have a determined nucleotide sequence (i.e., rRNA, tRNA and mRNA) or a determined amino acid sequence and the resulting biological properties. Therefore, if the transcription and translation of the mRNA corresponding to the gene produces a protein, the gene encodes a protein. Unless otherwise indicated, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate to each other and encode the same amino acid sequence. The nucleotide sequence encoding a protein and RNA may include introns.

[0104] An "isolated" polypeptide, peptide, protein, antibody, polynucleotide, vector, cell or composition refers to a polypeptide, peptide, protein, antibody, polynucleotide, vector, cell or composition in a form that does not exist in nature. An isolated polypeptide, peptide, protein, antibody, polynucleotide, vector, cell or composition includes those that have been purified to the point where they no longer exist in a form that occurs 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.

[0105] As used herein, the terms "identical (same)", "identity" percentage and grammatical equivalents thereof used when relating to two or more polynucleotides or polypeptides refer to two or more sequences or subsequences when comparing and aligning (if necessary, introducing room) to achieve maximum correspondence, without considering any conservative amino acid substitutions as part of sequence identity, the nucleotides or amino acid residues that are identical or have a specific percentage are identical. The identity percentage can be measured using sequence alignment software or algorithms or by visual estimation. Various algorithms and software that can be used to obtain amino acid or nucleotide sequence alignments are well known in the art. These algorithms and software include but are not limited to BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package and variants thereof. In some embodiments, two polynucleotides or polypeptides provided herein are substantially identical, which means that 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% nucleotide or amino acid residue identity when compared and aligned using a sequence comparison algorithm or by visual inspection to achieve maximum correspondence. In some embodiments, identity is present in a region of an amino acid sequence having a length of at least about 10 residues, at least about 20 residues, at least about 40-60 residues, at least about 60-80 residues, or any integer value therebetween. In some embodiments, identity is present in a region longer than 60-80 residues, such as at least about 80-100 residues, and in some embodiments, the sequence is substantially identical over the full length of the compared sequence (such as the coding region of a target protein or antibody). In some embodiments, the identity exists in 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, or any integer value therebetween. In some embodiments, the identity exists in a region that is longer than 60-80 bases, such as at least about 80-100 bases or more, and in some embodiments, the sequences are substantially identical over the full length of the compared sequences (such as the nucleotide sequence encoding the target protein).

[0106] As used herein, "conservative amino acid substitution" refers to an amino acid residue being replaced by another amino acid residue having a similar side chain. As used herein, "conservative similar" amino acids or residues refer to non-identical amino acid residues with similar side chains. Families of amino acid residues with similar side chains have been defined in the art, including 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), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0107] As used herein, the term "vector" and its grammatical equivalents refer to a vector for carrying genetic material (such as a polynucleotide sequence), which can be introduced into a host cell and replicated and / or expressed in the host cell. Suitable vectors include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which may include a selection sequence or a marker that can be operably stably integrated into the host cell chromosome. In addition, the vector may also include one or more selectable marker genes and appropriate expression control sequences. For example, the selectable marker gene that may be included provides resistance to antibiotics or toxins, supplements nutritional deficiency defects, or supplies key nutrients that are not present in the culture medium. The expression control sequence may include constitutive and inducible promoters, transcription enhancers, transcription terminators, etc., which are all well-known in the art. When two or more polynucleotides are to be co-expressed, the two polynucleotides may be inserted into, for example, a single expression vector or a separate expression vector. For single vector expression, the encoding polynucleotide may be operably connected to a common expression control sequence or connected to different expression control sequences, such as an inducible promoter and a constitutive promoter. The introduction of polynucleotides into a host cell may be confirmed using methods well-known in the art. Those skilled in the art understand that the polynucleotides are expressed in sufficient amounts to produce the desired product (e.g., IgG constituent bispecific binding protein), and further understand that expression levels can be optimized using methods well known in the art to obtain sufficient expression.

[0108] As used herein, the term "host cell" refers to a cell into which genetic material (such as a recombinant expression vector) may be or has been introduced. Host cells include not only subject cells into which exogenous genetic material has been introduced, but also the offspring of such cells. Due to mutations or environmental influences, offspring cells may undergo certain modifications, so these offspring may not be identical to parental cells.

[0109] As used herein and understood in the art, "EC" refers to the effective concentration of an agent (such as an antibody) and is commonly used in dose-response curves. The "effect" of a formulation can be a positive (activation) effect or a negative effect. The term "EC50" refers to the concentration at which an active agent (such as an antibody) produces a half-maximal response. Also as used herein and understood in the art, "IC" refers to the concentration of an agent with an inhibitory effect and is also commonly used in dose-response curves. The term "IC50" refers to the concentration of an agent (such as an antibody) at which the activity of its inhibition is reduced by half.

[0110] As described in the previous section, a bispecific binding protein can be composed of two polypeptides linked together by a disulfide bond, wherein each polypeptide comprises a first scFv region and a second scFv region at its N- and C-termini, respectively. The two scFv regions specifically bind to different antigens. Between the first and second scFv regions of each polypeptide is a sequence comprising the hinge, CH2, and CH3 domains of an immunoglobulin.

[0111] Alternatively, the bispecific binding protein may include an IgG with an scFv region attached to the C-terminus of each CH3 domain. In this case, the variable heavy and light chains in the Fab region of the IgG can specifically bind to one antigen, while the C-terminal scFv region includes different variable heavy and light chains that specifically bind to a second antigen.

[0112] As described above, a bispecific binding protein can bind to two different antigens through two domains, one domain at the N-terminus binds to antigen X and the other at the C-terminus binds to antigen Y. In some embodiments, the bispecific binding protein binds to the first domain of the alarmin receptor and the second domain of the alarmin. In a specific embodiment, the bispecific binding protein inhibits IL17RB activation by binding through the first domain and binds to TSLP through the second domain. In another embodiment, the bispecific binding protein inhibits IL17RB activation by binding through the first domain and binds to IL33 through the second domain.

[0113] In one embodiment, the bispecific binding protein comprises two polypeptides of the following formula:

[0114] X—H—Fc—L—scFv Y

[0115] Where X is scFv X or Fab region, wherein X specifically binds to a first antigen, and scFv Y It specifically binds to the second antigen. H is the hinge region. Fc includes the CH2 and CH3 regions of immunoglobulin. scFv X and scFv YEach independently is a single chain variable 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 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.

[0116] In an embodiment, the bispecific binding protein comprises two polypeptides of the following formula:

[0117] X—H—Fc—L—ECD Y

[0118] Where X is scFv X or Fab region, wherein X specifically binds to the first antigen, and ECD Y It is the extracellular domain of the alarmin receptor, H is the hinge region, Fc includes the CH2 and CH3 regions of the immunoglobulin, and scFv X is a single chain variable 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, then the first antigen is an immunomodulatory protein and the other is an alarmin. In some embodiments, ECD Y is the TSLP receptor extracellular domain. In some embodiments, ECD Y It is the extracellular domain of IL-33 receptor.

[0119] The VH and VL chains incorporated into the bispecific binding protein can be derived from a variety of sources, including pre-existing antibodies, newly generated antibodies, and VH and VL chain libraries. 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 immunomodulatory proteins (such as IL17RB or TSLPR), are provided in the Detailed Description section.

[0120] Nucleic acids comprising nucleotide sequences encoding the polypeptides of the present disclosure are provided herein. Methods of producing the polypeptides, culturing host cells, and recovering the polypeptides are also provided and are further discussed in the following detailed description.

[0121] In another aspect, the present disclosure provides compositions comprising the bispecific binding proteins described herein. The compositions generally include one or more bispecific binding proteins described herein and / or salts thereof, and one or more excipients, carriers or diluents.

[0122] Current anti-alarmin antibody therapies have limited efficacy in AD and moderate-to-severe asthma. Specifically, Phase II AD clinical trials of terzelumab (anti-TSLP antibody) and eteluzumab (anti-IL-33 antibody) failed to meet their respective primary endpoints. However, in the treatment of asthma, both terzelumab and entelostat met their primary endpoints in Phase II and Phase III trials, although their therapeutic efficacy was limited: terzelumab failed to reduce the daily dose of OCS use, while eteluzumab was relatively inferior to dupilumab. The bispecific proteins described herein specifically bind to two antigens, such as alarmin receptors and alarmins, and block two different alarmin-induced immune responses. This inhibition of alarmin-induced responses has the potential to reduce the frequency of dosing and prolong disease remission for allergic diseases or disorders.

[0123] In some embodiments, the present invention provides a bispecific binding protein thereof for the preparation of a drug for reducing the daily dose of OCS for treating Th2-high and / or Th2-low immune disorders such as asthma, wherein the bispecific binding protein specifically binds to an alarmin and / or an alarmin receptor. In the above-mentioned use, 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 to human IL-17RB and TSLP. In some embodiments, the bispecific binding protein specifically binds to human IL-17RB and IL-33.

[0124] Also provided herein is a method for treating an allergic disease or disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a bispecific binding protein thereof, the bispecific binding protein specifically binding to 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 thereof specifically binds to 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 preclinical AD and asthma.

[0125] As used herein, the term "treat" and its grammatical equivalents, when referring to a disease or condition or a subject suffering from a disease or condition, refers to the action of inhibiting, eliminating, alleviating and / or ameliorating the symptoms, symptom severity and / or symptom frequency associated with the disease or disorder being treated. For example, when used with reference to AD, the term "treat" and its grammatical equivalents refer to the action of reducing the severity of the disease, delaying or slowing the progression of the disease, which includes but is not limited to (a) reducing the frequency of dosing to achieve disease remission, or reducing the incidence of disease relapse, or (b) delaying, ameliorating or minimizing one or more symptoms associated with AD (quantified by the Eczema Area and Severity Index (EASI)), (c) reducing the amount and frequency of OCS use.

[0126] The term "blocking" and its grammatical equivalents refer to the action of reducing the biological function of the alarmin in ways including, but not limited to, (a) directly competing for the binding site of the alarmin on its corresponding receptor, or (b) preventing heterodimerization of the corresponding receptor, reducing the biological effect induced by the alarmin occupancy.

[0127] As used herein, the term "administering" and its grammatical equivalents refer to the act of delivering or causing a therapeutic agent or pharmaceutical composition to be delivered to a subject's body by methods described herein or known in the art. The therapeutic agent can be a compound, a polypeptide, or a cell. Administering a therapeutic agent or pharmaceutical composition includes prescribing a prescription for delivering the therapeutic agent or pharmaceutical composition to a subject's body. 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, including creams, gels, powders, or patches; oral dosage forms; inhalation powders, sprays, suspensions, and rectal suppositories.

[0128] As used herein, the terms "effective amount", "therapeutically effective amount" and their grammatical equivalents refer to an amount of an agent that, when administered to a subject alone or as part of a pharmaceutical composition, in a single dose or as part of a series of doses, has any detectable positive effect on any symptom, aspect or feature of a disease, disorder or condition. The therapeutically effective amount can be determined by measuring the relevant physiological effects. The exact amount required varies from subject to subject, depending on the subject's age, weight and general condition, the severity of the disease being treated, the judgment of the clinician, etc. The therapeutically effective amount also refers to the amount in which the toxic or harmful effects of the therapeutic agent are exceeded by the beneficial effects of the treatment. In any individual case, an appropriate "effective amount" may vary due to factors such as the disease state, age, sex and weight of the individual, and can be determined by a person of ordinary skill in the art using routine experiments. A "prophylactic effective amount" refers to an amount effective at the dose and time period required to achieve the desired prophylactic effect (e.g., delaying or preventing the occurrence of a disease or disorder). Typically, since a prophylactic dose is used for a subject before or in the early stages of a disease, the prophylactic effective amount is usually less than the therapeutically effective amount.

[0129] As used herein, the term "subject" refers to any animal (eg, mammal) that is to be the recipient of a particular treatment, including but not limited to humans, non-human primates, canines, felines, rodents, and the like.

[0130] In some embodiments, the bispecific binding protein disclosed herein (a) inhibits IL5 / CCL17 / IL13 / CCL8 / IFNγ secretion of human PBMC, and / or (b) inhibits ILC2 migration and proliferation. Therefore, the bispecific binding protein disclosed herein shows a lasting therapeutic effect by directly inhibiting the activity of ILC2. In addition, the method disclosed herein has the additional therapeutic benefit of reducing Th2 and ILC2 cell migration. In some embodiments, provided herein is a method for treating OCS resistance-related diseases or disorders using the anti-IL17RB / anti-TSLP or anti-IL17RB / anti-IL33 bispecific binding proteins disclosed herein.

[0131] The methods provided herein can treat allergic diseases or disorders. The allergic disease or disorder can be clinical or preclinical allergic asthma, allergic rhinitis, allergic conjunctivitis or AD.

[0132] While the above indications are preferred, administration of a bispecific antigen binding protein as disclosed herein to a subject may also treat or prevent other diseases, disorders or conditions. These diseases, disorders and conditions include, but are not limited to, inflammation, autoimmune diseases, cartilage inflammation, fibrotic diseases and / or bone degeneration, arthritis, rheumatoid arthritis, juvenile arthritis, juvenile rheumatoid arthritis, oligoarticular juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, systemic juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteropathic arthritis, juvenile reactive arthritis, juvenile Rett syndrome, SEA syndrome (seronegative, enthesopathy, arthropathy syndrome), juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, oligoarticular rheumatoid arthritis, polyarticular rheumatoid arthritis, systemic rheumatoid arthritis, ankylosing spondylitis, enteropathic arthritis, reactive arthritis, ... end disease, arthropathy syndrome), dermatomyositis, psoriatic arthritis, scleroderma, systemic lupus erythematosus, vasculitis, myositis, polymyositis, dermatomyositis (dermatomyolitis), osteoarthritis, polyarteritis nodosa, Wegener's granulomatosis, arteritis, polymyalgia rheumatica, sarcoidosis, scleroderma, sclerosis, primary biliary sclerosis, sclerosing cholangitis, Sjögren'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, type I diabetes, Graves' disease, Addison's disease, Raynaud's phenomenon, autoimmune hepatitis, GVHD, etc. In a specific embodiment, a pharmaceutical composition comprising a therapeutically effective amount of an anti-IL17RB / anti-TSLP bispecific binding protein is provided. In another specific embodiment, a pharmaceutical composition comprising a therapeutically effective amount of an anti-IL17RB / anti-IL33 bispecific binding protein is provided.

[0133] Based on the data provided herein, treatment of subjects with an allergic disease or disorder using the bispecific binding proteins described herein is expected to provide therapeutic benefit.

[0134] 7.2.1 Exemplary Anti-IL17RB / Anti-Alarmin Bispecific Binding Proteins

[0135] The present invention provides a bispecific binding protein comprising an immunoglobulin G antibody (IgG) specific for an alarmin receptor, which is fused at the C-terminus of the immunoglobulin chain to (a) a single-chain variable fragment (scFv) specific for a specific alarmin or (b) the extracellular domain of the alarmin receptor, respectively.

[0136] 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), the heavy chain variable region comprising heavy chain CDRs (HCDRs) 1-3, and each light chain comprising a light chain variable region (LCVR1), the light chain variable region comprising light chain CDRs (LCDRs) 1-3, wherein 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; and (b) each scFv comprises a heavy chain variable region (HCVR2) and a light chain variable region (LCVR2), the HCVR2 comprising HCDRs 4-6, and the LCVR2 comprising LCDRs 4-6, wherein 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, wherein each scFv is linked to the IgG antibody at the C-terminus of each IgG HC via a polypeptide linker (L1) at the N-terminus of the HCVR2 of each scFv, and wherein the HCVR2 of each scFv is linked to the LCVR2 of the same scFv at the N-terminus of the LCVR2 of the same scFv via a second polypeptide linker (L2) at the C-terminus of the HCVR2. In some embodiments, the HCDR variants thereof 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 thereof 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.

[0137] 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), the heavy chain variable region comprising heavy chain CDRs (HCDRs) 1-3, and each light chain comprising a light chain variable region (LCVR1), the light chain variable region comprising light chain CDRs (LCDRs) 1-3, wherein 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; and (b) each scFv comprises a heavy chain variable region (HCVR2) and a light chain variable region (LCVR2), the HCVR2 comprising HCDRs 4-6, and the LCVR2 comprising LCDRs 4-6, wherein 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, wherein each scFv is linked to the IgG antibody at the C-terminus of each IgG HC via a polypeptide linker (L1) at the N-terminus of the HCVR2 of each scFv, and wherein the HCVR2 of each scFv is linked to the LCVR2 of the same scFv at the N-terminus of the LCVR2 of the same scFv via a second polypeptide linker (L2) at the C-terminus of the HCVR2. In some embodiments, the HCDR variants thereof 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 thereof 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.

[0138] 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), the heavy chain variable region comprising heavy chain CDRs (HCDRs) 1-3, and each light chain comprising a light chain variable region (LCVR1), the light chain variable region comprising light chain CDRs (LCDRs) 1-3, wherein 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; and (b) each scFv comprises a heavy chain variable region (HCVR2) and a light chain variable region (LCVR2), the HCVR2 comprising HCDRs 4-6, and the LCVR2 comprising LCDRs 4-6, wherein 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, wherein each scFv is linked to the IgG antibody at the C-terminus of each IgG HC via a polypeptide linker (L1) at the N-terminus of the HCVR2 of each scFv, and wherein the HCVR2 of each scFv is linked to the LCVR2 of the same scFv at the N-terminus of the LCVR2 of the same scFv via a second polypeptide linker (L2) at the C-terminus of the HCVR2. In some embodiments, the HCDR variants thereof 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 thereof 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.

[0139] 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), the heavy chain variable region comprising heavy chain CDRs (HCDRs) 1-3, and each light chain comprising a light chain variable region (LCVR1), the light chain variable region comprising light chain CDRs (LCDRs) 1-3, wherein 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; and (b) each scFv comprises a heavy chain variable region (HCVR2) and a light chain variable region (LCVR2), the HCVR2 comprising HCDRs 4-6, and the LCVR2 comprising LCDRs 4-6, wherein 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, wherein each scFv is linked to the IgG antibody at the C-terminus of each IgG HC via a polypeptide linker (L1) at the N-terminus of the HCVR2 of each scFv, and wherein the HCVR2 of each scFv is linked to the LCVR2 of the same scFv at the N-terminus of the LCVR2 of the same scFv via a second polypeptide linker (L2) at the C-terminus of the HCVR2. In some embodiments, the HCDR variants thereof 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 thereof 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.

[0140] 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), the heavy chain variable region comprising heavy chain CDRs (HCDRs) 1-3, and each light chain comprising a light chain variable region (LCVR1), the light chain variable region comprising light chain CDRs (LCDRs) 1-3, wherein 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; and (b) each scFv comprises a heavy chain variable region (HCVR2) and a light chain variable region (LCVR2), the HCVR2 comprising HCDRs 4-6, and the LCVR2 comprising LCDRs 4-6, wherein 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, wherein each scFv is linked to the IgG antibody at the C-terminus of each IgG HC via a polypeptide linker (L1) at the N-terminus of the HCVR2 of each scFv, and wherein the HCVR2 of each scFv is linked to the LCVR2 of the same scFv at the N-terminus of the LCVR2 of the same scFv via a second polypeptide linker (L2) at the C-terminus of the HCVR2. In some embodiments, the HCDR variants thereof 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 thereof 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.

[0141] 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), the heavy chain variable region comprising heavy chain CDRs (HCDRs) 1-3, and each light chain comprising a light chain variable region (LCVR1), the light chain variable region comprising light chain CDRs (LCDRs) 1-3, wherein 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; and (b) each scFv comprises a heavy chain variable region (HCVR2) and a light chain variable region (LCVR2), the HCVR2 comprising HCDRs 4-6, and the LCVR2 comprising LCDRs 4-6, wherein 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, wherein each scFv is linked to the IgG antibody at the C-terminus of each IgG HC via a polypeptide linker (L1) at the N-terminus of the HCVR2 of each scFv, and wherein the HCVR2 of each scFv is linked to the LCVR2 of the same scFv at the N-terminus of the LCVR2 of the same scFv via a second polypeptide linker (L2) at the C-terminus of the HCVR2. In some embodiments, the HCDR variants thereof 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 thereof 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.

[0142] In some embodiments, the present invention provides a bispecific antibody comprising an IgG and an extracellular domain of an alarmin receptor, (a) the IgG comprises two heavy chains (HC) and two light chains (LC), each HC comprising a heavy chain variable region (HCVR1), the heavy chain variable region comprising heavy chain CDRs (HCDRs) 1-3, and each light chain comprising a light chain variable region (LCVR1), the light chain variable region comprising light chain CDRs (LCDRs) 1-3, wherein 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; and (b) the extracellular domain of the alarmin receptor, wherein the amino acid sequence is SEQ ID NO: 19 or SEQ ID NO: 20. NO: 34, wherein each alarmin extracellular domain is linked to the IgG antibody at the C-terminus of each IgGHC via a polypeptide linker (L1) at the C-terminus of the amino acid sequence of each domain. In some embodiments, the HCDR variants thereof 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 NO: 1-3.

[0143] In some embodiments, the present invention provides a bispecific antibody comprising an IgG and an extracellular domain of an alarmin receptor, (a) the IgG comprises two heavy chains (HC) and two light chains (LC), each HC comprising a heavy chain variable region (HCVR1), the heavy chain variable region comprising heavy chain CDRs (HCDRs) 1-3, and each light chain comprising a light chain variable region (LCVR1), the light chain variable region comprising light chain CDRs (LCDRs) 1-3, wherein 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; and (b) the extracellular domain of the alarmin receptor, wherein the amino acid sequence is SEQ ID NO: 20 or SEQ ID NO: 35, wherein each alarmin extracellular domain is linked to the IgG antibody at the C-terminus of each IgGHC via a polypeptide linker (L1) at the C-terminus of the amino acid sequence of each domain. In some embodiments, the HCDR variants thereof 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 NO: 1-3.

[0144] In some embodiments, the bispecific binding protein of the present disclosure comprises a polypeptide linker (L1 and / or L2) having a sequence corresponding to one of the sequences selected from the following table:

[0145]

[0146]

[0147] 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 a still further embodiment of the bispecific binding protein of the invention, the polypeptide linker L1 has the sequence of SEQ ID NO:30.

[0148] In some embodiments of the bispecific binding protein of the invention, the amino acid sequence of each LC is SEQ ID NO: 33. In another embodiment, the amino acid sequence of each HC can be any one selected from SEQ ID NO: 60-75.

[0149] In some embodiments, the anti-IL17RB IgG bispecific binding protein that can 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 includes a heavy chain constant region, such as an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, or any of the above constant regions, whose glycosylation sites and / or glycoforms on glycosylation sites are modified.

[0150] 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), heavy chain antibody (HCAb), Fv, single chain variable fragment (scFv) or (scFv)2 fused to 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 may 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, such as an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, or any of the above constant regions, whose glycosylation sites and / or glycoforms at the glycosylation sites are modified.

[0151] In some embodiments, an antigen-binding fragment of an anti-alarmone 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), Fab, Fab', F(ab')2, Fv, a single chain variable fragment (scFv) or (scFv)2. In some embodiments, the antigen-binding fragment of an anti-alarmone antibody is a single domain antibody (sdAb). In some embodiments, the antigen-binding fragment of an anti-alarmone antibody is a heavy chain antibody (HCAb). In some embodiments, the antigen-binding fragment of an anti-alarmone antibody is Fab. In some embodiments, the antigen-binding fragment of an anti-alarmone antibody is Fab'. In some embodiments, the antigen-binding fragment of an anti-alarmone antibody is F(ab')2. In some embodiments, the antigen-binding fragment of an anti-alarmone antibody is Fv. In some embodiments, the antigen-binding fragment of an anti-alarmone antibody is scFv. In some embodiments, the antigen-binding fragment of an anti-alarmone antibody is a disulfide-linked scFv [(scFv)2]. In some embodiments, the antigen-binding fragment of an anti-alarmin antibody is a diabody (dAb). In further embodiments, the anti-alarmin antigen-binding fragment neutralizes the activity of alarmins, including IL-25, IL-33, and TSLP.

[0152] The term "activity" includes properties such as the ability to specifically bind to a target protein, the affinity of an antibody or binding protein for a protein, the ability to neutralize a biological activity of a target protein, the ability to inhibit the interaction of a target protein with its natural receptor or natural ligand.

[0153] In some embodiments, the methods disclosed herein use recombinant anti-IL17RB / anti-TSLP bispecific antibodies. In some embodiments, the methods disclosed herein use recombinant anti-IL17RB / anti-IL33 bispecific antibodies. In some embodiments, the methods disclosed herein use recombinant anti-IL17RB / anti-TSLP receptor bispecific binding proteins. In some embodiments, the methods disclosed herein use recombinant anti-IL17RB / anti-IL33 receptor bispecific binding proteins.

[0154] In some embodiments, the IgG provided herein constitutes a bispecific binding protein and is a chimeric antibody. In some embodiments, the IgG provided herein constitutes a bispecific binding protein and is a humanized antibody. In some embodiments, the IgG provided herein constitutes a bispecific binding protein and is a human antibody. In some embodiments, the scFv provided herein constitutes a bispecific binding protein and is a chimeric scFv. In some embodiments, the scFv provided herein constitutes a bispecific binding protein and is a humanized scFv. In some embodiments, the scFv provided herein constitutes a bispecific binding protein and is a human scFv. In some embodiments, the bispecific binding protein used in the method provided herein is separated. In some embodiments, the bispecific binding protein used in the method provided herein is substantially pure.

[0155] Various methods for generating humanized antibodies and scFv are known in the art. Methods for achieving high affinity binding to humanized antibodies are known in the art. A non-limiting example of this method is hypermutation of the variable region and selection of cells expressing such high affinity antibodies (affinity maturation). In addition to using display libraries, designated antigens (such as recombinant IL17RB or an epitope thereof) can also be used to immunize non-human animals, such as rodents. In certain embodiments, rodent antigen binding fragments (such as mouse antigen binding fragments) can be generated and separated using methods known in the art and / or methods disclosed herein. In some embodiments, mice can be immunized with antigens (such as recombinant IL17RB or an epitope thereof).

[0156] Human antibodies and scFv can be prepared using various techniques known in the art. In some embodiments, human antibodies are generated by immortalized human B lymphocytes immunized in vitro. In some embodiments, human antibodies are generated by lymphocytes separated from immune individuals. In any case, cells that produce antibodies against target antigens can be generated and separated. In some embodiments, human antibodies are selected from phage libraries, wherein phage libraries express human antibodies. Alternatively, phage display technology can also be used to produce human antibodies and antibody fragments in vitro from immunoglobulin variable region gene libraries of unimmunized donors. The generation and use of antibody phage libraries are well known in the art. Once the antibody is determined, affinity maturation strategies known in the art (including but not limited to chain shuffling and site-directed mutagenesis) can be used to generate human antibodies with higher affinity. In some embodiments, human antibodies are produced in transgenic mice containing human immunoglobulin loci. After immunization, these mice can produce a complete human antibody library in the absence of endogenous immunoglobulin production.

[0157] In some embodiments, the IgG used in the method provided herein forms a bispecific binding protein comprising a monovalent antigen binding site. In some embodiments, the IgG forms a bispecific binding protein comprising a monospecific binding site. In some embodiments, the IgG forms a bispecific binding protein comprising a bivalent binding site. In some embodiments, the scFv used in the method provided herein forms a bispecific binding protein comprising a monovalent antigen binding site. In some embodiments, the scFv forms a bispecific binding protein comprising a monospecific binding site. In some embodiments, the scFv forms a bispecific binding protein comprising a monospecific binding site. In some embodiments, the scFv forms a bispecific binding protein comprising a bivalent binding site.

[0158] In some embodiments, the IgG composition 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 provided in the table below. Additional descriptions of the structural and functional characteristics of SM17 can be found in WO2020115319A1, which is incorporated herein 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.

[0159] In some embodiments, the IgG composition bispecific binding protein that can be used for the methods provided herein comprises one, two, three, four, five and / or six CDRs of SM17. In some embodiments, the IgG composition bispecific binding protein comprises a VL, and the VL comprises one, two and / or three VL CDRs of SM17. In some embodiments, the IgG composition bispecific binding protein provided herein comprises a VH, and the VH comprises one, two and / or three VH CDRs of SM17. In some embodiments, the IgG composition bispecific binding protein provided herein comprises one, two and / or three VLCDRs and one, two and / or three VH CDRs of SM17.

[0160] It is well known in the art that the VH CDR3 and VL CDR3 domains play an important role in the binding specificity / affinity of antibodies to antigens. Therefore, in some embodiments, the IgG-composition bispecific binding proteins that can be used in the methods disclosed herein can have appropriate association / dissociation kinetics with human IL17RB and have VH CDR3 and VL CDR3 structures that are identical or related to the VH CDR3 and VLCDR3 of SM17. The consensus motif of SM17 VL CDR3 comprising the amino acid sequence of SEQ ID NO: 6 can be modified by replacing one or more amino acids to adjust the antibody affinity without changing its binding specificity, or alternatively, by replacing the VLCDR3 of an unrelated human antibody that exhibits sufficient similarity to SM17 VL CDR3, using the standards described in Chinese Patent No. ZL200880024788.2, which is incorporated herein by reference. Similarly, the consensus motif of SM17VH CDR3 comprising the amino acid sequence of SEQ ID NO: 3 can be modified by substituting one or more amino acids to adjust the antibody affinity without changing its binding specificity, or alternatively by substituting by a VHCDR3 of an unrelated human antibody that exhibits sufficient similarity to SM17VH CDR3, using the standards described in Chinese Patent No. ZL200880024788.2, which is incorporated herein by reference.

[0161] Those skilled in the art will appreciate that certain substitutions of amino acids in the CDR3 domain will not alter the epitope specificity of the antibody, particularly substitutions with conservative amino acids. Thus, in some embodiments, the CDR3 of an antibody or antigen-binding fragment (e.g., SM17 or) provided herein can be replaced with a CDR3 from a human or primate antibody that: (1) has the same number of residues as SM17 CDR3 and exhibits 50% or greater sequence homology; (2) contains at least one, preferably multiple, aromatic residues that are identical or conservatively similar to the residues at the corresponding positions in SM17 CDR3; (3) contains at least one, preferably multiple, charged residues that are identical or conservatively similar to the residues at the corresponding positions in SM17 CDR3; and / or (4) contains at least one, preferably multiple, amino acid residues that are identical or conservatively similar to the residues at the corresponding positions in SM17 CDR3, which positions are known to be important for maintaining the binding site structure / contact of the anti-IL17RB antibody, as determined by crystal structure and / or computer database analysis. In some embodiments, no more than one to five conservative amino acid substitutions are made to the SM17 VL and / or VH CDR3 domains, or the VL and / or VH CDR3 of an unrelated primate or human antibody containing no more than one to five conservative similar 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 within the SM17 VL and / or VH CDR3 domains, or the VL and / or VH CDR3 of an unrelated primate or human antibody containing no more than one to three conservative similar residues is used to replace the VL and / or VH CDR3 of SM17.

[0162] In some embodiments, the IgG composition bispecific binding protein used in the methods provided herein specifically binds to IL17RB, which IL17RB comprises 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, the IgG composition bispecific binding protein used in the methods provided herein specifically binds to IL17RB, which IL17RB comprises 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, the IgG composition bispecific binding protein used in the methods provided herein specifically binds to IL17RB, which IL17RB comprises: (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.

[0163] In some embodiments, the IgG composition bispecific binding protein used in the methods provided herein specifically binds to IL17RB comprising a VL, wherein the VL has 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, the IgG composition 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, the IgG composition 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, the IgG composition 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, the IgG composition bispecific binding protein used in the methods provided herein has a VL having at least 98% sequence identity to SEQ ID NO: 33. In some embodiments, the IgG composition bispecific binding protein used in the methods provided herein specifically binds to IL17RB, which IL17RB comprises a VL having an amino acid sequence of SEQ ID NO: 33.

[0164] In some embodiments, the IgG constituent bispecific binding proteins used in the methods provided herein specifically bind to IL17RB comprising a VH, wherein the VH has 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, the IgG constituent bispecific binding proteins used in the methods provided herein have a VH having at least 85% sequence identity to SEQ ID NO: 32. In some embodiments, the IgG constituent bispecific binding proteins used in the methods provided herein have a VH having at least 90% sequence identity to SEQ ID NO: 32. In some embodiments, the IgG constituent bispecific binding proteins used in the methods provided herein have a VH having at least 95% sequence identity to SEQ ID NO: 32. In some embodiments, the IgG composition bispecific binding protein used in the methods provided herein has a VH having at least 98% sequence identity to SEQ ID NO: 32. In some embodiments, the IgG composition bispecific binding protein used in the methods provided herein specifically binds to IL17RB, which IL17RB comprises a VH having the amino acid sequence of SEQ ID NO: 32.

[0165] In some embodiments, the IgG component bispecific binding protein thereof specifically binds to IL17RB comprising VL and VH, wherein VL and VH have the amino acid sequences of SEQ ID NOs: 32 and 33, respectively. The IgG component bispecific binding protein thereof that specifically binds to IL17RB may comprise a combination of any VL disclosed herein and any VH disclosed herein.

[0166] In some embodiments, the IgG composition bispecific binding protein specifically binds to IL17RB, which IL17RB comprises: (a) a VL, which VL comprises VL CDR1, 2 and 3 from a VL having an amino acid sequence of SEQ ID NO: 33; and / or (b) a VH, which VH comprises VH CDR1, 2 and 3 from a VH having an amino acid sequence of SEQ ID NO: 32.

[0167] In some embodiments, the IgG component bispecific binding protein specifically binds IL17RB, which IL17RB comprises a VL, wherein the VL comprises VL CDRs 1, 2, and 3 from a VL having the amino acid sequence of SEQ ID NO:33.

[0168] In some embodiments, the IgG component bispecific binding protein specifically binds to IL17RB, which IL17RB comprises a VH, wherein the VH comprises VH CDRs 1, 2, and 3 from a VH having the amino acid sequence of SEQ ID NO:32.

[0169] In some embodiments, the IgG composition bispecific binding protein specifically binds to IL17RB, which IL17RB comprises VL and VH, wherein VL comprises VL CDR1, CDR2 and CDR3 from VL having the amino acid sequence of SEQ ID NO: 33, and VH comprises VH CDR1, CDR2 and CDR3 from VH having the amino acid sequence of SEQ ID NO: 32.

[0170] In some embodiments, the IgG composition bispecific binding protein provided herein is a variant of SM17. The SM17 variant may have a VL, which 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 variant may have a VH, which 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 VH CDR or VLCDR. In some embodiments, the amino acid substitutions, additions and / or deletions are not in CDR. In some embodiments, the variant of SM17 has up to about 5 conservative amino acid substitutions. In some embodiments, the variant of SM17 has up to 3 conservative amino acid substitutions.

[0171] In some embodiments, the IgG bispecific binding proteins that can be used in the methods disclosed herein include VH or VL with at least one framework (FR) region. In some embodiments, the FR one (FR1) region of VL may be from the VκID human germline family, the FR two (FR2) region of VL may be from the Vκ1 human germline family, the FR three (FR3) region of VL may be from the Vκ1 human germline family, and the FR four (FR4) region of VL may be from the VκJ1 human germline family. In some embodiments, the FR1, FR2, FR3 and FR4 of VL may have the amino acid sequences of SEQ ID NOs: 36, 37, 38 and 39, respectively (the framework sequences shown in WO2020115319A1 are incorporated herein by reference). In some embodiments, the framework one (FR1) region of VH may be from V H 3 human germline families; the framework 2 (FR2) region of VH can be derived from V H 3 human germline families; the framework three (FR3) region of VH can be derived from V H3 human germline families; and the framework four (FR4) region of VH can be derived from V H J5 human germline family. In some embodiments, FR1, FR2, FR3, and FR4 of VH may have amino acid sequences of SEQ ID NOs: 40, 41, 42, and 43, respectively (framework sequences shown in WO2020115319A1).

[0172] The present disclosure further contemplates additional variants and equivalents that are substantially homologous to the recombinant antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, and human antibodies or antibody fragments thereof described herein. In some embodiments, it is desirable to improve the binding affinity of the antibody. In some embodiments, it is desirable to modulate the biological properties of the antibody, including but not limited to specificity, thermostability, expression level, effector function (one or more), glycosylation, immunogenicity, and / or solubility. It will be appreciated by those skilled in the art that amino acid changes may alter post-translational processes of the antibody, such as changing the number or location of glycosylation sites, or changing membrane anchoring properties.

[0173] Variation can be the replacement, deletion or insertion of one or more nucleotides encoding antibodies or polypeptides, which causes the amino acid sequence to change compared to the native antibody or polypeptide sequence. In some embodiments, amino acid substitution is the result of replacing an amino acid with another amino acid having similar structure and / or chemical properties, for example, replacing leucine with serine, such as conservative amino acid substitution. Insertion or deletion can be within the range of about 1 to 5 amino acids. In some embodiments, relative to the parent molecule, substitution, deletion or insertion includes 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. In some embodiments, biologically useful and / or relevant variations in the amino acid sequence can be determined by systematically inserting, deleting or replacing in the sequence, and testing the activity of the resulting variant protein compared to the parent protein.

[0174] As known in the art, the constant region of an antibody mediates a variety of effector functions, and these effector functions may vary depending on the isotype of the antibody. For example, the C1 component of complement binds to the Fc region of an IgG or IgM antibody (binding to an antigen) to activate the complement system. Complement activation is important for the opsonization and lysis of cellular pathogens. The activation of complement also stimulates inflammatory responses and may participate in autoimmune hypersensitivity reactions. In addition, the Fc region of an antibody may bind to cells expressing Fc receptors (FcRs). There are several Fc receptors that are specific for different classes of antibodies, including IgG (gamma receptors), IgE (epsilon receptors), IgA (alpha receptors), and IgM (mu receptors). Antibodies bind to Fc receptors on the cell surface and trigger many important and diverse biological responses, including phagocytosis and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (called antibody-dependent cellular toxicity or ADCC), release of inflammatory mediators, placental transfer, and control of immunoglobulin production. In some embodiments, the bispecific binding protein described herein comprises at least one constant region of a human IgA antibody. In some embodiments, the bispecific binding proteins described herein include at least one constant region of a human IgD antibody. In some embodiments, the bispecific binding proteins described herein include at least one constant region of a human IgE antibody. In some embodiments, the bispecific binding proteins described herein include at least one constant region of a human IgG antibody. In some embodiments, the bispecific binding proteins described herein include at least one constant region of a human IgM antibody. In some embodiments, the bispecific binding proteins described herein include at least one constant region of a human IgG1 antibody. In some embodiments, the bispecific binding proteins described herein include at least one constant region of a human IgG2 antibody. In some embodiments, the bispecific binding proteins described herein include at least one constant region of a human IgG3 antibody. In some embodiments, the bispecific binding proteins described herein include at least one constant region of a human IgG4 antibody.

[0175] In some embodiments, in the bispecific binding protein described herein, at least one or more constant regions have been modified or deleted. In some embodiments, the bispecific binding protein includes one or more of the three heavy chain constant regions (CH1, CH2 or CH3) and / or modification of 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 modification of the constant region includes the addition, deletion or substitution of one or more amino acids in one or more regions.

[0176] 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 has been removed from the bispecific binding protein (ΔCH2 construct). In some embodiments, the deleted constant region is replaced by a short amino acid spacer, which provides some molecular flexibility usually conferred by the non-existent constant region. In some embodiments, the modified bispecific binding protein includes a CH3 domain fused directly to the hinge region of the bispecific binding protein. In some embodiments, the modified bispecific binding protein includes a peptide spacer inserted between the hinge region and the modified CH2 and / or CH3 domains.

[0177] In some embodiments, the bispecific binding protein comprises an Fc region. In some embodiments, the Fc region is fused by a hinge. The hinge can be an IgG1 hinge, an IgG2 hinge, or an IgG3 hinge. The amino acid sequence of the Fc region of human IgG1, IgG2, IgG3, and IgG4 is known to those of ordinary skill in the art. In some cases, Fc regions with amino acid variations have been identified in native antibodies. In some embodiments, modified bispecific binding proteins (such as modified Fc regions) provide altered effector functions, which in turn affect the biological signature spectrum of the bispecific binding protein. For example, in some embodiments, the deletion or inactivation of the constant region (by point mutation or other methods) reduces the Fc receptor binding of the modified bispecific binding protein during its circulation. In some embodiments, constant region modifications reduce the immunogenicity of the bispecific binding protein. In some embodiments, constant region modifications increase the serum half-life of the bispecific binding protein. In some embodiments, constant region modifications reduce the serum half-life of the bispecific binding protein. In some embodiments, constant region modifications reduce or remove the ADCC and / or complement dependent cytotoxicity (CDC) of the bispecific binding protein. In some embodiments, replacing specific amino acids in the human IgG1 Fc region with corresponding IgG2 or IgG4 residues reduces the effector functions (such as ADCC and CDC) of the modified bispecific binding protein. In some embodiments, the bispecific binding protein does not have one or more effector functions (such as "effectless" antibodies). In some embodiments, the bispecific binding protein has no ADCC activity and / or 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 has no effector function (one or more). In some embodiments, constant region modifications increase or enhance 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 / replace one or more amino acids to provide one or more cytotoxins, oligosaccharides or carbohydrate attachment sites. In some embodiments, the bispecific binding protein comprises a variant Fc region, which is engineered to be substituted at a specific amino acid position compared to the native Fc region. In some embodiments, the bispecific binding proteins described herein comprise 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 protein described herein comprises 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 S228P, L234A, and L235A.

[0178] In some embodiments, variants may include adding amino acid residues at the amino and / or carboxyl terminal of an antibody or polypeptide. The length of the extra amino acid residues may range from one residue to one hundred or more residues. In some embodiments, variants include N-terminal methionyl residues. In some embodiments, variants include extra polypeptides / proteins (such as Fc regions) to produce fusion proteins. In some embodiments, variants are engineered to detect, and may include detectable markers and / or proteins (such as fluorescent tags or enzymes).

[0179] The variant antibodies or antigen-binding fragments described herein can be produced using methods known in the art, including, but not limited to, site-directed mutagenesis, alanine scanning mutagenesis, and PCR mutagenesis.

[0180] In some embodiments, the variants of the IgG bispecific binding proteins disclosed herein may retain the ability to bind to IL17RB to a similar, identical or higher degree as the parent antibody or antigen binding fragment. In some embodiments, the amino acid sequence of the variant may 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 to the parent antibody or antigen binding fragment. In certain embodiments, the variants of the IgG bispecific binding proteins include the amino acid sequence of the parent of the IgG 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 an amino acid having certain physical and / or chemical properties is replaced with another amino acid having the same or similar chemical or physical properties.

[0181] In some embodiments, the variant of the bispecific binding protein includes the amino acid sequence of the parent antibody or antigen-binding fragment with one or more non-conservative amino acid substitutions. In some embodiments, the variant of the bispecific binding protein includes the amino acid sequence of the parent binding antibody or antigen-binding fragment with one or more non-conservative amino acid substitutions, wherein the one or more non-conservative amino acid substitutions do not interfere with or inhibit one or more biological activities (e.g., IL17RB binding) of the variant. In certain embodiments, one or more conservative amino acid substitutions and / or one or more non-conservative amino acid substitutions can enhance the biological activity of the variant, thereby improving the biological activity of the functional variant compared to the parent binding portion.

[0182] In some embodiments, the bispecific binding proteins described herein are chemically modified naturally or by intervention. In some embodiments, the bispecific binding proteins have been chemically modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protection / blocking groups, proteolytic cleavage and / or connection with cell ligands or other proteins. Any of numerous chemical modifications can be performed by known techniques. The bispecific binding proteins may include one or more amino acid analogs (including, for example, non-natural amino acids), as well as other modifications known in the art.

[0183] The bispecific binding proteins disclosed herein can be analyzed for their physical, chemical and / or biological properties by various methods known in the art. In some embodiments, the ability of the bispecific binding protein to bind to two different alarmins (such as human IL17RB and TSLP) is detected. Binding assays include but are not limited to surface plasmon resonance (such as BIAcore), ELISA and FACS. In some embodiments, the dissociation constant of a binding agent (such as an antibody) and an alarmin is a dissociation constant determined by surface plasmon resonance (such as BIAcore). In addition, the solubility, stability, thermal stability, viscosity, expression level, expression quality and / or purification efficiency of the antibody can also be evaluated.

[0184] SM17 binds to human IL17RB with a dissociation constant (K) of approximately 1 pM. D In some embodiments, the bispecific binding protein used in the methods disclosed herein binds to IL17RB (such as human IL17RB) with a dissociation constant (K D ) is 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. In some embodiments, K D is about 20 nM or less. In some embodiments, K D is about 10 nM or less. In some embodiments, K D is about 5 nM or less. In some embodiments, K D is about 2 nM or less. In some embodiments, K D is about 1.5 nM or less. In some embodiments, K D is about 1 nM or less. In some embodiments, K D is about 0.5 nM or less. In some embodiments, K D is about 0.1 nM or less. In some embodiments, K D is about 50 pM or less. In some embodiments, K D About 10 pM or less.

[0185] In some embodiments, the K of the bispecific binding protein to IL17RB (eg, human IL17RB) is D 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. In some embodiments, K D In the range of 0.1-1 nM. In some embodiments, K D In the range of 0.5-5 nM. In some embodiments, KD In the range of 1-10 nM. In some embodiments, K D In the range of 1-5 nM. In some embodiments, K D In the range of 5-50 nM. In some embodiments, K D In the range of 10-100 nM. In some embodiments, K D In the range of 50-500nM.

[0186] In some embodiments, the bispecific binding proteins used in the methods disclosed herein bind to IL17RB (e.g., human IL17RB) with an association constant (K A ) is about 0.8x10 9 M -1 In some embodiments, the bispecific binding protein used in the methods disclosed herein binds to IL17RB (eg, human IL17RB) with an association constant (K A ) is about 1×10 6 M -1 or higher, about 1×10 7 M -1 or higher, about 1×10 8 M -1 or higher, about 5×10 8 M -1 or higher, about 8×10 8 M -1 or higher, about 1×10 9 M -1 or higher, about 5×10 9 M -1 or higher, about 1×10 10 M -1 or higher, about 5×10 10 M -1 or higher, about 1×10 11 M -1 or higher, about 5×10 11 M -1 or higher, or about 1×10 12 M -1 Or higher. In some embodiments, K A About 1×10 7 M -1 Or higher. In some embodiments, K A About 5×10 7 M -1 Or higher. In some embodiments, K A About 1×10 8 M -1 Or higher. In some embodiments, KA About 5×10 8 M -1 Or higher. In some embodiments, K A About 8×10 8 M -1 Or higher. In some embodiments, K A About 1×10 9 M -1 Or higher. In some embodiments, K A About 5×10 9 M -1 Or higher. In some embodiments, K A About 1×10 10 M -1 or higher.

[0187] In some embodiments, the bispecific binding proteins used in the methods disclosed herein bind to IL17RB (such as human IL17RB) and their K A In 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 -1×1012 M -1 or 5×10 11 -5×10 12 M -1 In some embodiments, K A In about 1×10 6 -1×10 7 M -1 In some embodiments, K A In about 1×10 7 -1×10 8 M -1 In some embodiments, K A In about 1×10 8 -1×10 9 M -1 In some embodiments, K A In about 5×10 8 -1×10 9 M -1 In some embodiments, K A In about 5×10 8 -5×10 9 M -1 In some embodiments, K A In about 1×10 9 -1×10 10 M -1 within the range.

[0188] In some embodiments, the bispecific binding proteins used in the methods disclosed herein dissociate from human IL17RB with a kd of 1.38×10 -7 s -1 or less, as determined by Octet (e.g., biolayer interferometry). In some embodiments, the bispecific binding proteins used in the methods disclosed herein dissociate from human IL17RB with a kd of about 5×10 -4 s -1 or less, about 1×10 -4 s -1 or less, about 2×10 -5 s -1 or less, about 4×10 -6 s -1 or less, about 8×10 -7 s -1 or less, about 2×10 -7 s -1 or less, about 4×10 -8 s -1In 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, kd is about 1×10 -4 s -1 In some embodiments, kd is about 2×10 -5 s -1 or less. In some embodiments, kd is about 4×10 -6 s -1 or less. In some embodiments, kd is about 8×10 -7 s -1 In some embodiments, kd is about 2×10 -7 s -1 or less. In some embodiments, kd is about 4×10 -8 s -1 or less.

[0189] Epitope mapping is a method for identifying binding sites, regions or epitopes on a target protein to which a bispecific binding protein binds. There are various methods for mapping epitopes on target proteins known in the art. These methods include, but are not limited to, shotgun mutagenesis, site-directed mutagenesis and alanine scanning; domain or fragment scanning; peptide scanning (such as Pepscan technology); display methods (such as phage display, microbial display and ribosome / mRNA display); methods involving protein hydrolysis and mass spectrometry; and structural determination (such as X-ray crystallography and NMR). In some embodiments, the bispecific binding proteins described herein are characterized by assays including, but not limited to, N-terminal sequencing, amino acid analysis, HPLC, mass spectrometry, ion exchange chromatography, and papain digestion.

[0190] In some embodiments, the bispecific binding proteins useful in the methods disclosed herein can bind to the conformational epitope, whose K A About 0.8x10 9 M -1 In some embodiments, the bispecific binding protein used in the methods disclosed herein can bind to the conformational epitope, whose K A About 1×10 7 M -1 or higher, about 1×10 8 M -1 or higher, about 5×10 8 M -1 or higher, about 1×10 9 M -1 or higher, about 5×10 9 M -1 or higher, about 1×1010 M -1 or higher, about 5×10 10 M -1 or higher, about 1×10 11 M -1 or higher, about 5×10 11 M -1 or higher, or about 1×10 12 M -1 Or higher. In some embodiments, K A About 5×10 7 M -1 Or higher. In some embodiments, K A About 1×10 8 M -1 Or higher. In some embodiments, K A About 5×10 8 M -1 Or higher. In some embodiments, K A About 8×10 8 M -1 Or higher. In some embodiments, K A About 1×10 9 M -1 Or higher. In some embodiments, K A About 5×10 9 M -1 In some embodiments, the bispecific binding protein used in the methods disclosed herein binds to the conformational epitope and its K A In 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 , 1×109 -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 In some embodiments, K A In about 1×10 6 -1×10 7 M -1 In some embodiments, K A In about 1×10 6 -1×10 7 M -1 In some embodiments, K A In about 1×10 7 -1×10 8 M -1 In some embodiments, K A In about 1×10 8 -1×10 9 M -1 In some embodiments, K A In about 5×10 8 -1×10 9 M -1 In some embodiments, K A In about 5×10 8 -5×10 9 M -1 In some embodiments, K A In about 1×10 9 -1×10 10 M -1 within the range.

[0191] In some embodiments, provided herein is a bispecific binding protein that can compete with another bispecific binding protein (such as human IL17RB) for binding to an alarmin. A bispecific binding protein that "competes with another antibody for binding to a target" refers to a bispecific binding protein that (partially or completely) inhibits the binding of another bispecific binding protein to the same target. Whether they compete with each other for binding to a target, that is, 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 biolayer interferometry kinetic analysis. In some embodiments, the constituent bispecific binding proteins compete and inhibit the binding of another bispecific binding protein to an alarmin (such as human IL17RB) by at least 50%, 60%, 70%, 80%, 90% or 100%. Competition assays can be performed, for example, according to Ed Harlow and David Lane, Cold Spring Harb Protoc; 2006; doi:l0.H0l / pdb.prot4277 or Ed Harlow and David Lane's " Using Antibodies ” Chapter 11, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA 1999.

[0192] In some embodiments, the bispecific binding proteins used in the methods disclosed herein bind to radiolabeled I that binds to native IL17RB on renal cancer cells (e.g., TK-10 cells). 125 -SM17 Competition, EC 50 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. In some embodiments, EC 50 is about 0.1 μg / ml or less. In some embodiments, EC 50 is about 0.2 μg / ml or less. In some embodiments, EC 50 is about 0.5 μg / ml or less. In some embodiments, EC 50 is about 1 μg / ml or less. In some embodiments, EC 50 is about 2 μg / ml or less. In some embodiments, EC 50 is about 5 μg / ml or less. In some embodiments, EC 50In some embodiments, the bispecific binding protein used in the methods disclosed herein binds to a radiolabeled I that binds to native IL17RB on a renal cancer cell (e.g., TK-10 cell). 125 -SM17 Competition, EC 50 In 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. In some embodiments, EC 50 In the range of about 0.1-50 μg / ml. In some embodiments, EC 50 In the range of about 0.1-10 μg / ml. In some embodiments, EC 50 In the range of about 0.1-5 μg / ml. In some embodiments, EC 50 In the range of about 0.5-10 μg / ml. In some embodiments, EC 50 In the range of about 0.5-5 μg / ml. In some embodiments, EC 50 In the range of about 1-10 μg / ml. In some embodiments, EC 50 In the range of about 1-5 μg / ml.

[0193] In some embodiments, the bispecific binding proteins provided herein can be derivatized or connected to another functional molecule (such as another peptide or protein) and used in the methods disclosed herein. Therefore, in some embodiments, the antibodies and antigen-binding fragments used in the methods disclosed herein include the derivatized and otherwise modified forms of the human anti-IL17RB antibodies described herein, including immunoadhesion molecules. For example, antibodies and antigen-binding fragments can be functionally connected (by chemical coupling, gene fusion, non-covalent association or introduction of artificial amino acids / functional groups suitable for site-specific conjugation) with one or more other molecular entities, such as another antibody (such as a bispecific antibody or a second antibody), a detectable agent, a cytotoxic agent, a pharmaceutical agent and / or a protein or peptide that can mediate the association of an antibody or antigen-binding fragment with another molecule (such as a streptavidin core region or a polyhistidine tag).

[0194] In some embodiments, the bispecific binding proteins described herein are conjugated to detectable substances or molecules, allowing the reagents to be used for diagnosis and / or detection. Detectable substances may also include, but are not limited to, enzymes such as horseradish peroxidase, alkaline phosphatase, glucose oxidase, β-galactosidase, and acetylcholinesterase; prosthetic groups such as biotin and flavin (one or more); fluorescent materials 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 materials such as luciferase; radioactive materials 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 Such as 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.

[0195] The bispecific binding proteins described herein can 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 protein is used for immunoassays. In some embodiments, the immobilized IgG composition bispecific binding protein is used for purification of the target antigen (such as human IL17RB).

[0196] Generation method

[0197] Bispecific binding proteins and antibodies thereof that can be used in the methods disclosed herein include, but are not limited to, bispecific antibodies, antibody-alarmin receptor fusion proteins, monoclonal antibodies, chimeric antibodies, human antibodies, and humanized antibodies, which can be prepared by any method disclosed herein or otherwise known in the art. Methods for antibody production are well known in the art. For example, see Harlow et al., Antibodies:A Laboratory Manual ,(Cold Spring HarborLabora to ry Press, 2nd ed. 1988); Hammerling et al., In: Monoclonal Antibodies and T-Cell Hybridomas 563, 681 (Elsevier, NY, 1981), each of which is incorporated herein by reference in its entirety.

[0198] 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 by, for example, introducing a recombinant expression vector into a host cell, a recombinant, a combinatorial human antibody library, antibodies isolated from animals (such as mice) with transgenic human immunoglobulin genes [Taylor, LD et al. Nucleic Acids Research, vol. 20, 23 (1992): 6287-95] or by any other means involving splicing human immunoglobulin gene sequences to other DNA sequences, preparing, expressing, producing, or isolating antibodies.

[0199] In some embodiments, the bispecific binding protein can be prepared by recombinantly expressing immunoglobulin light and heavy chain genes in a host cell. To express the bispecific binding protein, one or more recombinant expression vectors are introduced into the host cell, which carry DNA fragments of the immunoglobulin light and heavy chains encoding the bispecific binding protein, so that the light and heavy chains are expressed in the host cell and, preferably, secreted into the culture medium in which the host cells are cultured, from which the bispecific binding protein can be recovered. Standard recombinant DNA methods are used to obtain the bispecific binding protein heavy and light chain genes, incorporate these genes into recombinant expression vectors, and introduce the vectors into the host cell, as described in Sambrook, Fritsch and Maniais (eds), Molecular Cloning:A Laboratory Manual , 2nd ed., Cold Spring Harbor, NY, (1989), Ausubel et al. (eds.) Current Protocols in Molecular Biology , Greene Publishing Associates, (1989) and described in U.S. Patent No. 4,816,397.

[0200] To express a recombinant bispecific binding protein, such as an SM17-related bispecific binding protein, first obtain DNA fragments encoding the light and heavy chain variable regions. These DNAs can be obtained by using polymerase chain reaction (PCR) to amplify and modify hybridomas of murine antibody light and heavy chain variable sequences, or by oligosynthesis based on the designed amino acid sequences encoding the light and heavy chain variable sequences using standard methods known to those skilled in the art. The encoded DNA sequence can be further optimized to facilitate mammalian expression of the resulting antibody.

[0201] Once the VH and VL fragments of the murine antibodies are obtained, these sequences can be mutated to encode framework-patched versions, the methods of which have been described in WO2020115319A1, the entire text of which is incorporated herein by reference.

[0202] Once the DNA fragments encoding the VH and VL fragments of the bispecific binding protein are obtained (e.g., by amplification and mutagenesis of the original mouse VH and VL genes, as described above), these DNA fragments can be further manipulated by standard DNA recombination techniques, such as converting the variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these manipulations, the DNA fragment encoding VL or VH is operably linked to another DNA fragment encoding another protein (such as an antibody constant region or a flexible linker). As used in this context, the term "operably linked" is intended to refer to the joining of two DNA fragments so that the amino acid sequences encoded by the two DNA fragments remain in frame.

[0203] The isolated DNA encoding the VH region is converted to a full-length heavy chain gene by operably linking the VH encoding DNA to another DNA molecule encoding the heavy chain constant region (CH1, CH2 and CH3). The sequences of human heavy chain constant region genes are known in the art (e.g., see Kabat, EA, et al. (1991)). SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST , Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be IgG1, IgG2, IgG3, Ig4, IgA, IgE, IgM or IgD constant region, but most preferably IgG1 or IgG4 constant region. For the Fab fragment heavy chain gene, the VH encoding DNA can be operably linked to another DNA molecule encoding only the heavy chain CH1 constant region.

[0204] The isolated DNA encoding the VL region is converted into 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 (e.g., see Kabat, EA, et al. (1991) SEQUENCE OF PROTEINS OF IMMUNOLOGICAL INTEREST , Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments containing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region, but the kappa constant region is most preferred.

[0205] To prepare scFv genes, the VH and VL encoding DNA fragments are operably linked to another fragment encoding a flexible linker (such as encoding the amino acid sequence (Gly4-Ser)3) so that the VH and VL sequences can be expressed as a continuous single-chain protein, in which the VL and VH regions are joined by a 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].

[0206] In order to express the bispecific binding protein that can be used in the disclosed method herein, the DNA of the coding part or full-length light chain and heavy chain obtained as described above can be inserted into an expression vector so that the gene is operably connected to the transcription and translation control sequence. In this context, the term "operably connected" is intended to refer to the antibody gene being connected to the vector so that the transcription and translation control sequence in the vector can play the expected function of regulating the transcription and translation of the antibody gene. Expression vector and expression control sequence are selected to be compatible with the expression host cell used. Antibody light chain gene and bispecific binding protein heavy chain gene can be inserted into a separate vector respectively, and more typically, two genes are inserted into the same expression vector. Antibody gene is inserted into expression vector (for example, connection of complementary restriction sites on antibody gene fragment and vector, or blunt end connection in the absence of restriction sites) by standard methods. In some embodiments, before inserting the relevant bispecific binding protein light chain or heavy chain sequence of SM17, the expression vector has carried the bispecific binding protein constant region sequence. For example, one method of converting the SM17-related bispecific binding protein VH and VL sequences into full-length bispecific binding protein genes is to insert them into expression vectors that have encoded heavy chain constant regions and light chain constant regions, respectively, so that the VH segment is operably connected to the CH segment (one or more) in the vector, and the VL segment is operably connected to the CL segment in the vector. Additionally or alternatively, the recombinant expression vector can encode a signal peptide that promotes secretion of the antibody chain from the host cell. The antibody chain gene can be cloned into a vector so that the signal peptide is connected to the amino terminal frame of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin).

[0207] In addition to the bispecific binding protein chain gene, the recombinant expression vector provided herein may also carry a regulatory sequence that controls the expression of the bispecific binding protein chain gene in the host cell. The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (such as polyadenylation signals) that control the transcription or translation of the bispecific binding protein chain gene. For example, in Goeddel; GENEEXPRESSIONTECHNOLOGY:METHODSINENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990), describes such regulatory sequences. As will be appreciated by those skilled in the art, the design of the expression vector, including the selection of regulatory sequences, depends on the choice of the host cell to be transformed, the level of expression of the desired protein, and other factors. Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high-level 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): doi: 10.1016 / 0092-8674 (83) 90014-4), metallothionein (MT), cytomegalovirus (CMV) (e.g., CMV promoter / enhancer), simian virus 40 (SV40) (e.g., SV40 promoter / enhancer), adenovirus (e.g., adenovirus major late promoter (AdMLP)), and polyoma virus. 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.

[0208] In addition to the bispecific binding protein chain gene and regulatory sequence, the recombinant expression vector provided herein may also carry additional sequences, such as sequences (such as replication origins) and selectable marker genes that regulate the replication of the vector in the host cell. The selectable marker gene helps to select the host cell into which the vector has been introduced (for example, see U.S. Patent Nos. 4,399,216, 4,634,665 and 5,179,017). For example, the selectable marker gene can usually confer resistance to drugs such as G418, hygromycin or methotrexate on the host cell into which the vector has been introduced. Preferred selectable marker genes include dihydrofolate reductase (DHFR) gene (for dhfr-host cells selected / amplified by methotrexate), glutamate synthetase (GS) gene and neo gene (for G418 selection).

[0209] In order to express the light chain and the heavy chain, the expression vectors encoding the heavy chain and the light chain can be transfected into the host cell by standard techniques. The various forms of the term "transfection" are intended to include various techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, lipofection, protoplast fusion, etc. Although the bispecific binding protein can be produced in prokaryotic or eukaryotic host cells, it is preferred to express the bispecific binding protein in eukaryotic cells, particularly mammalian host cells, because such host cells are more likely to assemble and secrete properly folded and immunologically active bispecific binding proteins than prokaryotic cells.

[0210] Preferred mammalian host cells for expressing the recombinant bispecific binding proteins used in the methods described herein include SP2 / 0 myeloma cells, NSO myeloma cells, COS cells, and Chinese hamster ovary (CHO cells) (including dfhr-CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4200, using a DHFR selectable marker, e.g., as described in RJ Kaufman and PA Sharp (1982) J. Mol. Biol. 159:601-621). When the recombinant protein encoding expression vector is introduced into a 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 in the host cell, or more preferably, secretion of the bispecific binding protein into the culture medium in which the host cells are grown. The bispecific binding protein can be recovered from the culture medium using standard protein purification methods.

[0211] Host cells can also be used to produce portions of intact antibodies, such as Fab fragments or scFv molecules. Variations of the above procedures are expressly contemplated herein. For example, it may be desirable to transfect host cells with DNA encoding the light chain or heavy chain (but not both) of the antibodies used in the methods disclosed herein. Recombinant DNA technology can also be used to remove some or all of the DNA encoding one or both of the light and heavy chains, which are not necessary for binding to alarmins. Molecules expressed by such truncated DNA molecules are also included in the antibodies provided herein. In addition, by cross-linking the antibodies of the present invention with a second antibody by standard chemical cross-linking methods, a bispecific binding protein can be produced, in which one heavy chain and one light chain are antibodies that specifically bind to human IL17RB, and the other heavy chain and light chain are specific for antigens other than IL17RB.

[0212] In some embodiments of the bispecific binding protein recombinant expression system that can be used for the methods disclosed herein, the 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, the recombinant expression vector encoding both the heavy chain and the antibody light chain is introduced into CHO cells by standard techniques such as lipofection.

[0213] In the recombinant expression vector, the heavy chain and light chain genes are operably connected to mouse or human immunoglobulin heavy chain (IgH), CMV enhancer, metallothionein or AdMLP promoter regulatory elements to drive high-level transcription of the gene. The recombinant expression vector also carries the DHFR gene, which allows the use of methotrexate selection / amplification to select the SP2 / 0 cells that have been transfected with the vector. Alternatively, the recombinant expression vector containing the heavy chain and light chain genes operably connected to mouse or human IgH, CMV enhancer / AdMLP / metallothionein promoter regulatory elements and the DHFR gene can be used to transfect dhfr-SP2 / 0 or CHO cells. SP2 / 0 or CHO cells transfected with the vector can be selected, and the gene expression level in the vector is expanded by increasing the level of methotrexate in the culture. Cultivate the selected transformant host cell to allow it to express heavy and light chains, and reclaim the complete bispecific binding protein 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.

[0214] Pharmaceutical composition

[0215] Also provided herein is a pharmaceutical composition comprising a bispecific binding protein that can be used for the methods disclosed herein. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of a bispecific binding protein disclosed herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition can be used to treat AD and asthma. In some embodiments, the pharmaceutical composition can be used to inhibit the progression of AD and asthma in a subject (e.g., a human patient).

[0216] In the pharmaceutical compositions disclosed herein, the amount of therapeutic bispecific binding protein that can be combined with a carrier material can vary. In some embodiments, the amount of bispecific binding protein present in the pharmaceutical composition is an amount that produces a therapeutic effect. Generally, in combination with a pharmaceutically acceptable carrier, this amount ranges from about 0.01% to about 99% of the active ingredient, about 0.1% to about 70% of the active ingredient, or about 1% to about 30% of the active ingredient in one hundred percent.

[0217] The pharmaceutical composition provided herein comprises the bispecific binding protein provided herein, such as the bispecific binding protein associated with SM17. The bispecific binding protein can be present in various concentrations. In some embodiments, the pharmaceutical composition provided herein comprises the soluble bispecific binding protein provided herein of 1-1000mg / ml. In some embodiments, the pharmaceutical composition comprises the soluble bispecific binding protein provided herein of 10-500mg / ml, 10-400mg / ml, 10-300mg / ml, 10-200mg / ml, 10-100mg / ml, 20-100mg / ml or 50-100mg / ml. In some embodiments, the pharmaceutical compositions provided herein include 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 the bispecific binding proteins provided herein. Those skilled in the art can easily adjust the dosage; for example, a reduction in purity requires an increase in dosage.

[0218] The pharmaceutical compositions provided herein can be in various forms. These forms include, for example, liquid, semisolid and solid dosage forms, such as liquid solutions (such as injectable solutions and infusible solutions), dispersants or suspensions, tablets, pills, powders, liposomes and suppositories. The preferred form depends on the expected mode of administration and therapeutic application. Examples of suitable aqueous and non-aqueous carriers that can be used for pharmaceutical compositions or preparations described herein include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters (such as ethyl oleate).

[0219] For example, appropriate fluidity can be maintained by using a coating material such as lecithin, by maintaining the desired particle size in the case of a dispersant, and by using a surfactant. In some embodiments, the pharmaceutical compositions provided herein are in the form of injectable or infusible solutions. In some embodiments, the pharmaceutical composition is an aqueous preparation. Such preparations are typically solutions or suspensions, but may also include colloids, dispersants, emulsions, and multiphase materials. The term "aqueous preparation" is defined as a preparation comprising at least 50% w / w water. Similarly, the term "aqueous solution" is defined as a solution comprising at least 50% w / w water, and the term "aqueous suspension" is defined as a suspension comprising at least 50% w / w water. The composition can be formulated into a solution, microemulsion, liposome, or other ordered structure suitable for high drug concentrations.

[0220] In some embodiments, the pharmaceutical compositions disclosed herein are freeze-dried, to which a physician or patient adds solvents and / or diluents prior to use.

[0221] The pharmaceutical compositions provided herein may include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents, etc. Examples include one or more of water, saline, phosphate buffered saline, glucose, glycerol, ethanol, etc., and combinations thereof. In some embodiments, pharmaceutically acceptable carriers include isotonic agents, such as sugars, polyols such as mannitol, sorbitol, or sodium chloride in the composition.

[0222] In some embodiments, the pharmaceutically acceptable carrier further comprises a small amount of auxiliary substances, such as a wetting agent or emulsifier, a preservative or a buffer, which improves 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 (such as injection or infusion). Depending on the route of administration, the active ingredient (i.e., the bispecific binding protein) can be coated in a material to protect the active ingredient from the effects of acids and other natural conditions that can inactivate the active ingredient.

[0223] Also provided herein are kits for preparing pharmaceutical compositions having bispecific binding proteins disclosed herein (e.g., bispecific binding proteins associated with SM17). In some embodiments, the kit comprises bispecific binding proteins disclosed herein and a pharmaceutically acceptable carrier in one or more containers. In another embodiment, the kit may comprise binding proteins disclosed herein for administration to a subject. In a specific embodiment, the kit comprises instructions for preparing and / or administering bispecific binding proteins.

[0224] In some embodiments, the pharmaceutical compositions or formulations disclosed herein include: (a) a bispecific binding protein disclosed herein; (b) a buffer; (c) a stabilizer; (d) a salt; (e) a filler; 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, the present invention also provides a pharmaceutical composition or formulation that improves the stability of the bispecific binding protein to allow for its long-term storage. The pharmaceutical compositions disclosed herein may further include one or more preservatives, tonicity agents, chelating agents, stabilizers and / or surfactants, and various combinations thereof. The use of preservatives, isotonic agents, chelating agents, stabilizers and surfactants in pharmaceutical compositions is well known to those skilled in the art. Reference may be made to Remington: The Science and Practice of Pharmacy, 19th edition, 1995.

[0225] The buffer that can be used for the pharmaceutical composition or formulation disclosed herein can be a weak acid or a weak base, which is used to keep the acidity (pH) of the solution near a selected value after adding another acid or base. Suitable buffers can maximize the stability of the pharmaceutical formulation by maintaining the pH control of the formulation. Suitable buffers can also ensure physiological compatibility or optimize solubility. Rheology, viscosity and other characteristics also depend on the pH value of the formulation. Common buffers include but are not limited to histidine, citrate, succinate, acetate and phosphate. In some embodiments, the buffer includes histidine (such as L-histidine) and an isotonic agent, and may be adjusted to pH with an acid or base known in the art. In certain embodiments, the buffer is L-histidine. In certain embodiments, the pH of the formulation is maintained between about 2 and about 10, or between about 4 and about 8.

[0226] Stabilizers are added to drug products to stabilize the product. These agents can stabilize proteins in different 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), cyclodextrins, or dextran or PEG of any type and molecular weight. In some embodiments, stabilizers are selected to maximize the stability of the FIX polypeptide in the lyophilized formulation. In certain embodiments, the stabilizer is sucrose and / or arginine.

[0227] Fillers can be added to pharmaceutical compositions or formulations to increase the volume and mass of the product, thereby facilitating its accurate metering and handling. Common fillers include, but are not limited to, lactose, sucrose, glucose, mannitol, sorbitol, calcium carbonate, or magnesium stearate.

[0228] Surfactant is an amphoteric substance with lyophilic and lyophobic groups. Surfactant 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 dimethyl amine oxide. In some embodiments, surfactant is polysorbate 20 or polysorbate 80.

[0229] The pharmaceutical composition disclosed herein may also include a pharmaceutically acceptable antioxidant. 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, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelators, such as citric acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, etc.; (3) metal chelators, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0230] These compositions can also contain adjuvants, such as preservatives, wetting agents, emulsifiers and dispersants. The presence of microorganisms can be ensured by the sterilization procedures described above and by including various antibacterial and antifungal agents (such as parabens, chlorobutanol, phenol sorbic acid, etc.). It is expected that isotonic agents, such as sugars, sodium chloride, etc., can also be included in the composition. In addition, the absorption of injectable drug forms can be extended by including agents that delay absorption such as aluminum monostearate and gelatin.

[0231] Pharmaceutical compositions or formulations must generally be sterile and stable under the conditions of manufacture and storage. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for immediate preparation of sterile injectable solutions or dispersions. Sterile injectable solutions can be prepared by the following method: the desired amount of therapeutic antibodies or antigen-binding fragments are incorporated into an appropriate solvent with one or a combination of the above-mentioned ingredients as required, and then filtered and sterilized. The use of such media and formulations for pharmaceutically active substances is known in the art. In general, dispersions are prepared by the following method: the active compound is incorporated into a sterile carrier containing a basic dispersion medium and the required other ingredients mentioned above. In the case of sterile powders for the preparation of sterile injectable solutions, some preparation methods are vacuum drying and freeze drying (lyophilization), which obtain active ingredient powders and any additional required ingredients from their previously sterile filtered solutions.

[0232] The pharmaceutical compositions disclosed herein can be prepared with carriers that protect the active ingredients from rapid release, such as controlled release formulations, including implants, transdermal patches, and microcapsule delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Many methods for preparing such formulations have been patented or are well 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.

[0233] 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 help the therapeutic antibodies described herein cross the BBB, they can be formulated, for example, in liposomes. For methods of making liposomes, see U.S. Patents 4,522,811; 5,374,548; and 5,399,331. Liposomes may include one or more parts that are selectively transported to specific cells or organs, thereby enhancing targeted drug delivery (see, e.g., VVRanade (1989) J. Clin. Pharmacol. 29: 685). Exemplary targeting moieties include folic acid or biotin [see U.S. Pat. No. 5,416,016 by 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]; p120 [Schreier, H et al. The Journal Of Biological Chemistry vol. 269, 12 (1994): 9090-8] [ K and ML Laukkanen. FEBS letters vol. 346, 1 (1994): 123-6] [Killion, JJ and IJ Fidler. ImmunoMethods vol. 4, 3 (1994): 273-9].

[0234] Treatment

[0235] As described in the above sections, the present invention provides medical uses of bispecific binding proteins (such as SM17-related bispecific binding proteins) in the treatment of allergic diseases or disorders. Any bispecific binding protein disclosed herein can be used in the methods disclosed herein. In some embodiments, recombinant anti-IL17RB / anti-TSLP bispecific antibodies are used in the methods disclosed herein. In some embodiments, recombinant anti-IL17RB / anti-IL33 bispecific antibodies are used in the methods disclosed herein. In some embodiments, recombinant anti-IL17RB / anti-TSLP receptor bispecific binding proteins are used in the methods disclosed herein. In some embodiments, recombinant anti-IL17RB / anti-IL33 receptor bispecific binding proteins are used in the methods disclosed herein.

[0236] In some embodiments, provided herein is a method for reducing the proliferation of lung ILC2 in a subject in need. In some embodiments, provided herein is a method for treating a disease or disorder associated with allergies in a subject in need. In some embodiments, provided herein is a method for reducing eosinophilic inflammation in a subject in need. In some embodiments, provided herein is a method for treating a disease or disorder associated with neutrophilic inflammation in a subject in need. In some embodiments, provided herein is a method for treating a disease or disorder associated with neutrophilic inflammation in a subject in need. In some embodiments, provided herein is a method for treating a disease or disorder by reducing the daily dose of OCS in a subject in need by at least 20%, at least 50%, or at least more than 75%. In some embodiments, the subject is human.

[0237] Methods for reducing lung ILC2 proliferation, treating diseases or disorders associated with neutrophilic inflammation, treating diseases or disorders associated with eosinophilic inflammation, and treating diseases or disorders by reducing the daily dose of OCS include administering to a subject a therapeutically effective amount of a bispecific binding protein that specifically binds to (a) IL17RB and TSLP and / or (b) IL17RB and IL-33.

[0238] Subjects suitable for the present methods include human patients in whom it is desired to block the activity of alarmins. In some embodiments, the subject treated with the methods disclosed herein is diagnosed with a disease or disorder related to allergies, which can be clinical or preclinical asthma, AD, fibrotic diseases, 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 can be a mammal. In some embodiments, the subject is human. In some embodiments, the subject treated with the methods disclosed herein already has OCS. In some embodiments, the subject has not been previously treated.

[0239] The bispecific binding proteins (such as SM17-related bispecific binding proteins) or pharmaceutical compositions provided herein can be administered to a subject by any method known in the art, including but not limited to intravenous administration, subcutaneous administration, intramuscular administration, intracranial administration, intrathecal administration, intracerebroventricular administration, intraperitoneal administration, spinal administration, intranasal administration, intrathoracic administration, topical administration, or intradermal administration.

[0240] In some embodiments, the bispecific binding proteins (such as SM17 related bispecific binding proteins) or pharmaceutical compositions provided herein can be administered to a subject using parenteral administration. As used herein, the term "parenteral administration" refers to the mode of administration beyond enteral and topical administration, usually by injection, and includes but is not limited to intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and stem injection and infusion. In some embodiments, the bispecific binding proteins are administered by intravenous infusion or injection. In some embodiments, the bispecific binding proteins are administered by intramuscular injection. In some embodiments, the bispecific binding proteins are administered by subcutaneous injection.

[0241] The bispecific binding proteins (such as SM17-related bispecific binding proteins) or pharmaceutical compositions provided herein can be administered using medical devices known in the art. For example, in some embodiments, a needle-free subcutaneous injection device can be used, such as the device disclosed in U.S. Pat. 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 known implants and module uses described herein include: U.S. Pat. No. 4,487,603, which discloses an implantable microinfusion pump that dispenses drugs at a controlled rate; U.S. Pat. No. 4,486,194, which discloses a therapeutic device for administering drugs through the skin; U.S. Pat. No. 4,447,233, which discloses a drug infusion pump that delivers drugs at a precise infusion rate; U.S. Pat. No. 4,447,224, which discloses a variable flow implantable infusion device for continuous drug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drug delivery system with a multi-chamber compartment; and U.S. Pat. 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.

[0242] In some embodiments, the bispecific binding proteins disclosed herein can be administered orally, for example, with an inert diluent or an assimilable edible carrier. Therapeutic bispecific binding proteins can also be encapsulated in hard or soft shell gelatin capsules, pressed into tablets, or directly incorporated into the diet of the subject. For oral therapeutic administration, the bispecific binding protein can be combined with an excipient and used in the form of an ingestible tablet, buccal tablet, lozenge, capsule, elixir, suspension, syrup, wafer, etc.

[0243] The methods provided herein include administering a therapeutically effective amount of a bispecific binding protein as described herein (such as a SM17-related bispecific binding protein). The actual dosage level of the therapeutic antibody can be varied so as to obtain a dosage that is effective for achieving the desired therapeutic response for a particular patient without being toxic to the patient. The selected dosage level will depend on various pharmacokinetic factors, including the activity of the specific compositions described herein, the route of administration, the time of administration, the excretion rate, the duration of treatment, other drugs, compounds and / or materials used in combination with the specific compositions used, the age, sex, weight, condition, general health and previous medical history of the patient being treated, and similar factors well known in the medical field.

[0244] In general, the dosage range of a single dose can be, for example, about 0.1 to 100 mg / kg of host body weight. In some embodiments, bispecific binding protein (e.g., SM17 related bispecific binding protein) is used in the following amounts: 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, bispecific binding protein is used at about 1 mg / kg. In some embodiments, bispecific binding protein is used at about 5 mg / kg. In some embodiments, bispecific binding protein is used at about 10 mg / kg. In some embodiments, bispecific binding protein is used 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.

[0245] In some embodiments, the bispecific binding protein (e.g., SM17-related bispecific binding protein) is administered at a dosage in the range of about 1 to 5 mg / kg, about 1 to 10 mg / kg, about 1 to 20 mg / kg, about 1 to 50 mg / kg, about 1 to 100 mg / kg, about 5 to 10 mg / kg, about 5 to 20 mg / kg, about 5 to 50 mg / kg, about 5 to 100 mg / kg, about 10 to 50 mg / kg, or about 10 to 100 mg / kg. In some embodiments, the bispecific binding protein is administered at a dosage in the range of about 1 to 5 mg / kg. In some embodiments, the bispecific binding protein is administered at a dosage in the range of about 1 to 10 mg / kg. In some embodiments, the bispecific binding protein is administered at a dosage in the range of about 1 to 50 mg / kg. In some embodiments, the bispecific binding protein is administered at a dosage in the range of about 10 to 50 mg / kg. In some embodiments, the bispecific binding protein is administered at a dose in the range of about 10 to 100 mg / kg.

[0246] In some embodiments, the methods provided herein include administering a bispecific binding protein (e.g., SM17-related bispecific binding protein) at a dosage of about 10-2000 mg. In some embodiments, the dosage 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 dosage of 100 mg. In some embodiments, the antibody is administered at a dosage of 300 mg. In some embodiments, the antibody is administered at a dosage of 600 mg. In some embodiments, the antibody is administered at a dosage of 900 mg. In some embodiments, the antibody is administered at a dose of 1200 mg.

[0247] In some embodiments, the methods provided herein include administering an IgG composition bispecific binding protein (e.g., SM17 related bispecific binding protein) at a dosage within 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 dosage within the range of 100-500 mg. In some embodiments, the antibody is administered at a dosage within the range of 300-600 mg. In some embodiments, the antibody is administered at a dosage within the range of 300-900 mg. In some embodiments, the antibody is administered at a dosage within the range of 600-1200 mg.

[0248] During treatment, it is usually started at a lower dose and then gradually increased to the target dose. For illustration, in some embodiments, the methods provided herein include administering a bispecific binding protein at a dose of about 100 mg and then gradually increasing to a target dose of about 600 mg.

[0249] Subjects can be administered with such dosages every day, every other day, every week, every two weeks, every month, or any other schedule determined according to empirical analysis. Exemplary treatments require administration with multiple dosages over an extended period (e.g., at least six months). In some embodiments, methods provided herein include weekly administration of bispecific binding proteins. In some embodiments, methods include biweekly administration. In some embodiments, the methods include monthly administration. In some embodiments, bispecific binding proteins (such as SM17-related bispecific binding proteins) are administered subcutaneously every week, every two weeks, or every month. In some embodiments, IgG-composition bispecific binding proteins (such as SM17-related bispecific binding proteins) are administered intravenously every week, every two weeks, or every month.

[0250] As needed and appropriate, the bispecific binding protein (such as SM17-related bispecific binding protein) can be administered for up to 3 months, 6 months, 9 months, 12 months, 18 months, 24 months, 30 months, or 36 months. In some embodiments, the treatment lasts for at least 3 months. In some embodiments, the treatment lasts for at least 6 months. In some embodiments, the treatment lasts for at least 12 months. In some embodiments, the treatment lasts for at least 24 months.

[0251] All permutations and combinations of the various embodiments, such as administration cycles, dosages, treatment frequencies, and treatment durations, are expressly contemplated herein and may be employed in the treatment methods disclosed herein.

[0252] For illustration, the following treatment regimens can be employed in the methods disclosed herein, which include administering an anti-IL17RB antibody or antigen-binding fragment (such as a SM17-related bispecific binding protein) disclosed herein or an anti-IL17RB antibody or antigen-binding fragment identified in the methods disclosed herein:

[0253] In some embodiments, the intravenous or subcutaneous dose of the therapeutic antibody is about 10 mg / kg, once every 4 weeks, at least 21 days apart. In some embodiments, the following infusion schedule is included: 1-2 infusions: 1 mg / kg IV; 3-4 infusions: 3 mg / kg IV; 5-6 infusions: 6 mg / kg IV; 7 and above: 10 mg / kg IV.

[0254] In some embodiments, the therapeutic antibody is administered intravenously or subcutaneously as 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.

[0255] In some embodiments, the therapeutic antibody is administered intravenously or subcutaneously at a dose of about 250 mg per week, or 500 mg every two weeks, for up to 2 years. In some embodiments, treatment begins with monthly shots of about 120 mg.

[0256] The dosage regimen may be adjusted to provide the best expected response (such as a therapeutic or preventive response). For example, a single bolus may be administered, several separate doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the urgency of the treatment situation. It is particularly advantageous to prepare parenteral compositions in dosage unit form for ease of administration and uniform dosage. As used herein, dosage unit form refers to physically discrete units suitable for use as unit doses for mammalian subjects receiving treatment; each unit contains a predetermined amount of therapeutic antibodies, which is calculated to produce the desired therapeutic effect in combination with the desired drug carrier. It should be noted that the appropriate dosage varies with the type and severity of the condition to be alleviated. It should be further understood that for any particular subject, the specific dosage regimen should be adjusted over time according to personal needs and the professional judgment of the person administering or supervising the administration of the composition, and the dosage ranges described herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.

[0257] In the treatment of diseases or disorders associated with allergies, sometimes the methods provided herein can cure the disease or disorder, but any clinical improvement constitutes a benefit. In some embodiments, the methods provided herein can 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 can reduce the daily dosage of OCS by an average of 25%. In some embodiments, the methods provided herein can reduce the daily dosage of OCS by an average of 50%. In some embodiments, the methods provided herein can reduce the daily dosage of OCS by an average of 75%. In some embodiments, the methods provided herein can reduce the daily dosage of OCS by an average of 100%. In some embodiments, the methods provided herein can reduce the daily dosage of OCS by an average of 25-50%. In some embodiments, the methods provided herein can reduce the daily dosage of OCS by an average of 50-75%. In some embodiments, the methods provided herein can reduce the daily dosage of OCS by an average of 75-100%.

[0258] In some embodiments, the methods provided herein reduce ILC2 proliferation in the lungs. In some embodiments, the methods provided herein reduce ILC2 migration to the lungs. 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 pulmonary eosinophilic inflammation. In some embodiments, the methods provided herein reduce pulmonary neutrophilic inflammation. In some embodiments, the methods provided herein reduce the annual exacerbation rate of asthma. In some embodiments, the methods provided herein reduce the ratio of exhaled nitric oxide. In some embodiments, the methods provided herein reduce blood eosinophil counts. In some embodiments, the methods provided herein reduce transepidermal water loss.

[0259] In some embodiments, the methods provided herein prevent the occurrence of AD, or delay or prevent the progression of AD. In some embodiments, the methods provided herein relieve the symptoms of AD. In some embodiments, the methods provided herein prevent the occurrence of asthma, or delay or prevent the progression of asthma. In some embodiments, the methods provided herein relieve the symptoms of asthma.

[0260] 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 of administration and / or mode vary according to the desired results. In some embodiments, the bispecific binding proteins can be prepared together with a carrier that protects them from being rapidly released, such as a controlled release formulation, which includes an implant, a transdermal patch, and a microcapsule 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 preparing such preparations have been patented or are well known to those skilled in the art. For example, see SUSTAINED AND CONTROLLED RELEASE DRUG DELIVERY SYSTEMS, JR Robinson ed., Marcel Dekker, Inc., New York, 1978. In therapeutic applications, relatively high doses are sometimes required within relatively short time intervals until the progression of the disease is reduced or terminated, until the patient's disease symptoms are partially or completely relieved.

[0261] Combination therapy using agents with different mechanisms of action can result in additional or synergistic effects. Combination therapy can reduce the dosage of each agent compared to monotherapy, thereby reducing toxic side effects and / or improving the therapeutic index of agents disclosed herein. Combination therapy can reduce the possibility of developing drug resistance. In some embodiments, additional therapy results in an increase in the therapeutic index of the bispecific binding protein or pharmaceutical composition described herein. In some embodiments, additional therapy results in a reduction in 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 can be administered in combination with additional therapy.

[0262] 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, and 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 may be a second antibody that inhibits the release of proinflammatory cytokines.

[0263] The second therapeutic agent can be administered before, simultaneously with, or after the administration of the bispecific binding protein or pharmaceutical composition described herein. The combined administration may include co-administration with a single pharmaceutical formulation or using separate formulations, or continuous administration in any order, but generally over a period of time so that all active agents can simultaneously exert their biological activities. Those skilled in the art can easily determine the appropriate regimen for the combined administration of the pharmaceutical composition described herein and the additional therapy, including the timing and administration of the additional formulation used in the combined therapy, based on the needs of the subject being treated.

[0264] All papers, publications and patents cited in this specification are incorporated into this specification by reference, just as each individual paper, publication or patent is specifically and individually indicated to be incorporated by reference herein, and incorporated by reference herein to disclose and describe the methods and / or materials related to the cited publications. However, any references, articles, publications, patents, patent publications and patent applications cited herein do not and should not be regarded as admitting or indicating in any form that they constitute valid prior art or form part of the general knowledge in any country in the world.

[0265] Unless the context dictates otherwise, the various features described herein may be used in any combination.

[0266] Unless defined otherwise, 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.

[0267] Experimental Examples

[0268] Example 1: SM17 binds to exogenous mouse, monkey and human IL-17RB proteins

[0269] The species specificity of SM17 for IL-17RB protein was determined using a standard enzyme-linked immunosorbent assay (ELISA) method. Briefly, IL-17RB protein from humans, mice, cynomolgus monkeys, or rhesus monkeys (R&D systems, Minneapolis, MN) was diluted to 2.5 μg / ml in PBS, and then 50 μl of IL-17RB protein was added to each well of the ELISA test strip. The test strip was sealed with plastic wrap and incubated overnight at 4°C for coating.

[0270] The next day, the cells were washed three times with washing buffer (0.05% Tween 20 in PBS), blocked with 100 μl of blocking buffer (3% BSA in PBS) for 2 hours (RT) per well, and then serially diluted SM17 was added (final concentrations were 10, 2, 0.4, 0.08, 0.016, 0.0032 and 0.00064 μg / ml, respectively). After incubation for 2 hours at RT, the amount of bound SM17 was revealed by adding peroxidase-conjugated goat anti-human F(ab')2 specific antibody (Jackson ImmunoResearch) and TMB substrate (Sigma-Aldrich, St. Louis, MO) at OD450 nM according to standard ELISA protocols known to those skilled in the art.

[0271] The results showed that SM17 bound to IL-17RB proteins from mice, humans, cynomolgus monkeys, and rhesus monkeys in a dose-response manner with comparable affinities (see Tables 1 and Figure 1 ).

[0272] Table 1: Optical density of IL-17RB antigen binding to different species at 450 nm

[0273]

[0274] Example 2: SM17 does not cross-react with other IL17 receptor subunits

[0275] The specificity of SM17 for other human IL17 receptor subunits was determined using a standard ELISA method. Similarly, SM17 was added to the wells of an ELISA plate coated with human IL-17 receptor subunits A, C, D, and E (IL17RA, IL17RC, IL17RD, and IL17RE) (R&D systems) at different concentrations (final concentrations of 0.4, 0.08, 0.016, 0.0032, and 0.00064 μg / ml, respectively). No significant binding of SM17 to other known human IL-17 receptor subunits (IL17RA, IL17RC, IL17RD, and IL17RE) was detected (see Tables 2 and Figure 2 ).

[0276] Table 2: Optical density of IL-17 receptor family member antigen binding at 450 nm

[0277]

[0278] Example 3: SM17 binds to native human and rhesus monkey IL-17RB expressed on the surface of HEK293 cells

[0279] HEK293 cells (ATCC, Manassas, VA) transfected with full-length human or rhesus macaque IL-17RB (SinoBiological, Beijing, China) were examined for binding to SM17 following standard flow cytometry protocols. Wild-type HEK293 cells stained with IgG4 isotype control (Sino Biological) were used as negative controls and for gating purposes. Briefly, cells were plated at a density of 3 × 10 5 HEK293 cells with 10 cells were seeded into each well of a 6-well plate. The next day, the full-length cDNA of human or rhesus macaque IL-17RB was first cloned into the pCMV3-untagged expression plasmid by standard molecular cloning techniques, and then the expression plasmid of human or rhesus macaque IL-17RB was transfected into HEK293 cells by lipofection (Lipofectamine 3000 reagent; Thermo Fisher Scientific). The transfected cells were trypsinized, harvested, and fixed with 4% paraformaldehyde (5 minutes). SM17 or human IgG4 control at 1 μg / ml in wash buffer (3% BSA in PBS) was added to the resuspended cells and incubated for 30 minutes at room temperature before adding Alexa Fluor. 647-conjugated goat anti-human IgG specific antibody (1:2000 dilution in wash buffer) (Jackson ImmunoResearch), and flow cytometric analysis was performed using a BD FACSVerse cell analyzer (Becton Dickenson, Franklin Lakes, NJ).

[0280] Compared with wild-type HEK293 (approximately 7%), SM17 binding to HEK293 transfected with human IL17RB (hIL17RB-HEK293: 46%) and rhesus IL17RB (RhIL17RB-HEK293: 39%) was enhanced, indicating that SM17 binds to native human or rhesus IL17RB expressed on HEK293 cells ( Figure 3 ).

[0281] Example 4: SM17 inhibits the release of IL-5 from human PBMC co-cultured with IL-2 and IL-25

[0282] Co-culture of PBMCs (Ixcells, San Diego, CA) with IL-2 and IL-25 (PeproTech, Cranbury, NJ) results in the release of IL-5, a cytokine known to exacerbate asthma symptoms. 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). Serially diluted SM17 ranging from 0.16 ng / mL to 5 ng / mL was added to 4 × 10 5 Nonspecific human IgG4 (SinoBiological) was used as a control antibody. The treated cells were incubated at 37°C for 72 hours, and the IL-5 release level in the culture supernatant was measured by a standard ELISA assay (R&D systems). The results showed that SM17 inhibited IL-5 release from human PBMCs induced by IL-2 and IL-25 in a dose-dependent manner ( Figure 4 ). The results are expressed as mean ± SEM. One-way ANOVA, Dunnett's multiple comparison test, *: P < 0.05; **: P < 0.01.

[0283] Example 5: SM17 inhibits IL-8 release in the renal cancer cell line TK-10

[0284] Treatment of the human renal cancer cell line TK-10 (NCI-60, NIH, Bethesda, MD) with human IL-25 (PeproTech) and TNFa (R&D systems) leads to the release of IL-8, also known as neutrophil chemotactic factor, an important mediator of immune response in the innate immune system response. Therefore, the inhibitory effect of SM17 and its mouse counterpart (D9.2) on the release of IL-8 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 their mouse counterpart (D9.2, SinoMab BioScience Limited) at a concentration of 1 mg / mL were added to 2×10 cells in the presence of 100 ng / mL IL-25 and 10 ng / mL TNFa. 4in TK-10 cells. Nonspecific human IgG1 (SM03, anti-CD22 chimeric antibody, SinoMab BioScience Limited) was used as a control antibody. The treated cells were cultured in OptiMEM (Thermo Fisher Scientific) at 37°C for 24 or 48 hours, and the IL-8 release level 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, SM17, SM17-IgG1 or D9.2 could effectively inhibit IL-25 / TNFa-induced TK-10 cell release of IL-8 ( Figure 5 ).

[0285] Example 6: Determination of Antigen (IL17RB) Binding Kinetics of SM17

[0286] Biolayer interferometry analysis (Octet ReD96 system, Sartorius) was used to determine the binding affinity of SM17 to human and cynomolgus monkey IL17RB proteins (R&D systems). Briefly, SM17 (20 μg / mL) was immobilized on the biosensor through interaction with anti-human Fab CH1; serial dilutions of Cyno-IL17RB and human IL17RB (concentrations of 158.7 nM, 79.4 nM, and 39.7 nM, respectively) were then added according to the standard operating protocol of the Octet ReD96 system to plot 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 values ​​of the respective antibodies were 0.04 and 0.06, respectively. a , K dis and K D The values ​​are summarized in Table 3 below. It is worth noting that K D In the picomolar to single-digit nanomolar range.

[0287] Table 3: Binding kinetics of SM17 to human and cynomolgus monkey IL-17RB

[0288]

[0289] Example 7: Therapeutic effect of SM17 on ovalbumin-induced asthma in mice

[0290] Female BALB / c mice were induced with ovalbumin to induce asthma-like symptoms in mice. Briefly, mice were sensitized by intraperitoneal (IP) injection of 10 mg ovalbumin (OVA) (emulsified in 1 mg aluminum hydroxide) in a total of 200 ml on days 0 and 12. The mice were divided into 6 groups of 8 mice each. Sensitized mice were exposed to aerosolized 5% OVA in sterile water for 20 minutes / day for six consecutive days (days 19, 20, 21, 22, 23 and 24). Naive mice inhaled aerosolized water for 20 minutes / day for six consecutive days. Then mice were injected iv with different concentrations of SM17, PBS (control) or dexamethasone (Dex) 4 hours before aerosol attack, once a day (days 19 to 24).

[0291] On day 25 (24 hours after the last OVA challenge), Mice were anesthetized (20-40 mg / kg ip) and connected to a computer-controlled ventilator via endotracheal intubation. Expiratory / inspiratory time and respiratory rate were preset to 1.5:1 and 90 / min, respectively. After establishing a stable baseline, pulmonary resistance (RL) was recorded to assess the response of mice to a methacholine chloride gradient (0.025 and 0.05 mg / kg body weight); methacholine chloride was injected into the external jugular vein via a fine needle at 5-min intervals. Treatment of mice with 5 mg / kg SM17 or 1 mg / kg Dex prevented airway hyperresponsiveness. Administration of 1 mg / kg or 3 mg / kg SM17 before each OVA nebulization resulted in a slight but insignificant abolition of the AHR after methacholine challenge. As Figure 6 The results showed that the therapeutic effect of SM17 was dose-dependent. BALF of mice sacrificed on day 25 was retrieved to determine the number of inflammatory cells and the concentration of inflammatory cytokines in BALF.

[0292] 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 ). It was found that 5 mg / kg of SM17 and Dex significantly reduced the number of lung infiltrating eosinophils. The results are expressed as mean ± SEM. One-way ANOVA, Dunnett's multiple comparison test, *: P < 0.05; **: P < 0.01. Dex = dexamethasone. Fig. 9 The proposed mechanism of action of SM17 for the treatment of various indications is shown.

[0293] Example 8: Construction and expression of anti-alarmin bispecific binding protein (bsBp)

[0294] The light chain of SM17 (SEQ ID NO: 33) was cloned into the pcDNA3.3 expression vector by TA cloning (Thermo Fisher Scientific). The cDNA encoding the SM17 heavy chain was gene synthesized (Genscript Biotech Corp., Piscataway, NJ) and cloned into the NheI / NotI cloning site of the pEGFP-N1 expression vector (Clontech Laboratories, Mountain View, CA) (Figure 10a), which was operably linked to the sequence of a specific alarmin binding protein (e.g., an alarmin protein binding receptor or an scFv of an alarmin-specific antibody). The expression vector containing the specific SM17-alarmin binding protein heavy chain and the expression vector for the SM17 light chain were co-transfected into expiCHO-S cells according to the manufacturer's instructions (Thermo Fisher Scientific). The specific bispecific binding protein (bsBp) containing the SM17 antibody linked to the specific alarmin binding moiety was harvested on day 12 after transfection and purified by protein A affinity chromatography. A reducing SDS-PAGE of the purified bsBp is shown in Figure 10b, indicating that most of the bispecific antibodies are intact in nature. Table 4 below summarizes the heavy and light chain sequences of bsBp.

[0295] Table 4: Amino acid sequences of bsBp and monoclonal antibodies

[0296] Constructs HC Sequence ID LC Sequence ID SM17-anti-TSLP 1#bsBp 60 33 SM17-anti-TSLP 2#bsBp 61 33 SM17-anti-TSLP 3#bsBp 62 33 SM17-anti-IL-33 1#bsBp 63 33 SM17-anti-IL-33 2#bsBp 64 33 SM17-anti-IL-33 3#bsBp 65 33 Anti-TSLP Antibody 1# 75 76 Anti-TSLP Antibody 2# 77 78 Anti-TSLP Antibody 3# 79 80 Anti-IL-33 Antibody 1# 81 82 Anti-IL-33 Antibody 2# 83 84 Anti-IL-33 Antibody 3# 85 86 SM17-hTSLPR 75 33 SM17-hST2 74 33

[0297] Example 9: Specificity of anti-alarmin bsBp

[0298] The binding specificity of 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. 33.5 nM of the bispecific antibody was added to the ELISA strips coated with the desired antigen. After incubation for 2 hours at room temperature, the ELISA strips were washed 5 times with PBS. According to standard procedures, binding was revealed by adding goat anti-human F(ab')2-specific horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5000 dilution, Jackson ImmunoResearch) and then adding TMB substrate solution (Sigma-Aldrich-). Fig.11 ). the following Fig.11The antigen binding specificity of the bsBp is summarized in Table 5. The results indicate specificity for IL17RB and alarmin, as originally designed. Results for SM17-human TSLPR are not presented because the yield was too low to detect.

[0299] Table 5: Antigen binding specificity of bsBp in ELISA

[0300]

[0301] The strength of the specific antigen binding affinity of bsBp is semi-quantitatively measured with + / +++ (- = no binding, +, ++, +++ = weak, medium, strong binding), and N / A means no binding affinity.

[0302] Example 10: Alarmin Binding Kinetics of Bispecific Binding Proteins

[0303] The binding affinity of bsBp to the targeted alarmin was determined using biolayer interferometry (Octet RED96 system, Sartorius). bsBp or SM17 (20 μg / mL) was immobilized on the biosensor by binding to the anti-human Fab CH1 region; serially diluted human IL33 and TSLP (158.7 nM, 79.4 nM, 39.7 nM) (R&D systems) were added, and association and dissociation curves were drawn according to the manufacturer's instructions. Biosensors immobilized with an irrelevant antibody (SM03, anti-human CD22 chimeric IgG1 antibody, SinoMab BioScience Limited, 20 μg / mL) were used as control references. Table 6 below summarizes the expected K values ​​of the respective bispecific binding proteins a , K dis and KD value.

[0304] Table 6: Binding kinetics of bsBp to human TSLP and IL-33

[0305] TSLP <![CDATA[K a (1 / Ms)]]> <![CDATA[K dis (1 / s)]]> <![CDATA[K D (nM)]]> SM17-anti-TSLP 1#bsBp 4.72×10 2.41×10 0.511 SM17-anti-TSLP 2#bsBp 3.74×10 <1×10 <0.001 SM17-anti-TSLP 3#bsBp 4.81×10 3.17×10 0.0658 SM17-TSLPR N / A N / A N / A SM17 1×10 1×10 100 IL33 <![CDATA[K a (1 / Ms)]]> <![CDATA[K dis (1 / s)]]> <![CDATA[K D (nM)]]> SM17-anti-IL-331#bsBp 9.81×10 2.83×10 28.9 SM17-anti-IL-332#bsBp 3.73×10 <1×10 0.0259 SM17-anti-IL-333#bsBp 2.27×10 <1×10 0.0425 SM17-ST2 5.56×10 2.26×10 0.407 SM17 2.72×10 1.79x10 659000

[0306] The binding affinity of SM17-human TSLPR is not presented due to low yield.

[0307] Example 11: Alarmins induce the release of IFNγ, CCL8, CCL17, IL-5 and IL-13 from human PBMCs and the inhibitory effect of SM17 on cytokine release

[0308] Human PBMC (4×10 5, Ixcells) were incubated with IL-2 (10 units / mL, PeproTech) and various alarmins / alarmin combinations to simulate proinflammatory cytokine release during allergic diseases. For IL-5 and IL-13 release, PBMCs were incubated with IL-2 and three anti-allergics (all at 10 ng / ml) for three 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 one hour before adding cytokines.

[0309] Supernatants were collected and cytokine concentrations were measured by ELISA kits (R&D systems). These established assays were used to test the efficacy of SM17 and bsBp. SM17 was able to effectively inhibit the secretion of IL-5 and IL-13 in total alarmin-stimulated PBMC cultures. However, no inhibitory effect of SM17 treatment was indicated in CCL17, CCL8, and IFNγ release assays compared to IgG4 isotype controls (see Fig.12 ). The results are expressed as absolute amounts of pg / ml.

[0310] Example 12: Efficacy of different bispecific binding proteins in inhibiting the release of cytokines and chemokines induced by human PBMC

[0311] SM17, three different SM17-anti-IL33 bsBp and three different SM17-anti-TSLP bsBp (all at a concentration of 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 chemokines in the supernatants obtained from the induced PBMCs were evaluated using standard ELISA methods. The results showed that different pairs of SM17 / anti-TSLP and SM17 / anti-IL33 bsBp showed enhanced or / and synergistic effects compared to what was observed with SM17 alone in inhibiting the release of IFNγ, CCL8, CCL17, IL-5 and IL-13 (see Fig.13). Specifically, in the IL-5 release assay, SM17-anti-IL-33 3# showed the strongest inhibitory effect compared to SM17 treatment. In the CCL8 release assay, SM17-anti-IL-33 1# and SM17-anti-IL-33 3# both showed stronger inhibitory effects compared to SM17. In the IFNγ release assay, all three SM17-anti-IL-33bsBp showed stronger inhibitory effects compared to SM17. In the CCL17 release assay, SM17-anti-TSLP 1# and SM17-anti-TSLP 3#bsBp both showed stronger inhibitory effects compared to SM17 treatment. Fig.13 and Table 7 summarize the potency of bsBp in different cytokine release assays.

[0312] Table 7: Potency of bsBp in cytokine release assay

[0313]

[0314] The specific cytokine inhibition strength of bsBp was semi-quantitatively measured with + / +++ (- = no inhibition, +, ++, +++ = weak, moderate, strong inhibition). Nil: not investigated.

[0315] Example 13: Proliferation-promoting effect of alarmins on ILC2

[0316] Isolated human ILC2 cells can proliferate in response to alarmins. TM Human ILC2 isolation kit (STEMCELL Technologies Inc. Cambridge, MA) was used to enrich human ILC2 from fresh human PMBC. The concentrated ILC2 was then cultured in RPMI1640 medium (Thermo Fisher Scientific) containing 10% human AB serum (Sigma). 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 3 alarmins was added to the ILC2 culture for 7 days, and then the changes in the ILC2 cell population were measured by flow cytometry (BD FACSVerse) using a panel of lineage-specific antibodies against ILC2 (CRTH2+, IL-7Ra+; BioLegend, San Diego, CA). The results showed that the major contributor to ILC2 proliferation was IL-33, and the combination of all alarmins further enhanced ILC2 expansion (see Fig.14 ).

[0317] Example 14: Dexamethasone and bsBp inhibit ILC2 and Th2 cell populations in human PBMCs

[0318] Although alarmins are known to synergistically enhance ILC2 and Th2 cell functions, such as increasing cell populations, steroids (such as dexamethasone (Dex)) can offset these effects, leading to a reduction in 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, a combination of IL-2 (10 units / ml) and three alarmins (10 ng / ml) was continued for 5 days, which enhanced the cell populations of ILC2 and Th2 cells in the experiment. The results showed that Dex could significantly reduce the Th2 cell population (CD4+GATA3+ population, gated by anti-CD4 PE antibody (BioLegend) and anti-GATA3 APC antibody (Abcam, Cambridge, UK)) but not the ILC2 cell population (lin-IL-7Ra+CRTH2+, gated by anti-IL-7Ra percp / cyanine 5.5 antibody and anti-CRTH2 FITC antibody, BioLegend) in human PBMC cultures (see Fig.15 ). In addition, the addition of Bsbp significantly reduced the ILC2 cell population, and the efficacy of bsbp is summarized below Fig.15 and Table 8.

[0319] Table 8: Efficacy of bsBp in ILC2 and Th2 proliferation assays

[0320]

[0321] The cell proliferation inhibitory effect of bsBp was measured using + / +++ as a semi-quantitative standard (- = no inhibitory effect, +, ++, +++ = weak, moderate, and strong inhibitory effects).

[0322] Example 15: Dendritic cell potency assay

[0323] TSLP from inflammatory epithelial cells promotes dendritic cell (DC) maturation to stimulate Th2 response through CCL17, and CCL17 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 DC was obtained from Lonza (Bend, OR). DC was cultured in LGM-3 medium (Lonza). TSLP (10 ng / mL) was 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 TSLP receptor extracellular domain), or SM17-anti-TSLP bsBp 2#. Supernatants were collected and CCL17 levels were measured by ELISA according to the manufacturer's instructions (R&D systems). The results showed that the bispecific SM17-TSLPR and SM17-anti-TSLP bsBp 2# effectively inhibited TSLP-induced CCL17 production in DCs ( Fig.16 ). The potency of SM17, IgG4 and bsBp is summarized in Table 9 below.

[0324] Table 9: Efficacy of bsBp in CCL17 release assay on dendritic cells

[0325]

[0326] Inhibition of CCL17 release by bsBp, SM17 and IgG4 isotypes, with + / +++ as a semi-quantitative standard (- = no inhibition, +, ++, +++ = weak, moderate, strong inhibition)

[0327] While the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope of the invention.

Claims

1. A method for treating an allergy-related disease or disorder in a subject in need thereof, the method comprising the step of delivering to cells or tissues of the subject a therapeutically effective amount of a bispecific binding protein directed against two different alarmins X and Y, wherein the bispecific binding protein consists of (a) anti-alarmin X receptor IgG and (b) anti-alarmin Y scFv and (c) a polypeptide linker.

2. A method for treating an allergy-related disease or disorder in a subject in need thereof, the method comprising the step of delivering to cells or tissues of the subject a therapeutically effective amount of a bispecific binding protein directed against 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.

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

4. The method of claim 1, wherein the alarmin Y is TSLP.

5. The method of claim 1, wherein the alarmin Y is IL-33.

6. The method of claim 2, wherein the alarmin Y receptor is ST2.

7. The method of claim 2, wherein the alarmin Y receptor is TSLPR.

8. The method according to claim 1 or 2, 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. The method of claim 1 or 2, wherein the subject suffers from clinical or preclinical asthma, atopic dermatitis, fibrotic diseases, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, chronic obstructive pulmonary disease, chronic sinusitis, chronic sinusitis with nasal polyps.

10. A method for treating an allergy-related disease or disorder in a subject in need thereof, the method comprising the step of delivering to cells or tissues of the subject a therapeutically effective amount of a bispecific binding protein that blocks (a) IL-25 and IL-33 signaling and / or (b) IL-25 and TSLP signaling.

11. The method of claim 10, wherein the subject suffers from clinical or preclinical asthma, atopic dermatitis, fibrotic disease, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, chronic obstructive pulmonary disease, chronic sinusitis, chronic sinusitis with nasal polyps.

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

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

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

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

16. The method 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 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) comprising VH CDR1, VH CDR2 and VH CDR3 having amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

17. The method of claim 16, wherein 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. The method of claims 10 to 15, wherein the bispecific binding protein comprises a light chain sequence of SEQ ID NO: 33 and a heavy chain sequence selected from SEQ ID NOs: 60-75.

19. The method according to any one of claims 1 to 17, wherein the bispecific binding protein is delivered to the cell or tissue intravenously, intramuscularly, subcutaneously, intracranially, intrathecally, intracerebroventricularly, intraperitoneally, intranasally, parenterally, topically or intradermally.

20. The method according to any one of claims 1 to 19, wherein the bispecific binding protein is delivered in conjunction with a second therapeutic agent.

21. The method of claim 20, wherein the second therapeutic agent is selected from the group consisting of corticosteroids, DNA methyltransferase (DNMT) inhibitors, anti-IL17A antibodies, anti-IL12 / IL23 antibodies, anti-IL23 antibodies, anti-IL17RA antibodies, and tyrosine kinase inhibitors.

22. The method according to any one of claims 1 to 21, wherein the subject is a human subject, and the subject's cells or tissues are immune cells obtained and isolated from the subject.

Images