Anti-il-33 and anti-tslp antibodies and mixtures thereof

Abstract

Described herein are anti-IL-33 and / or anti-TSLP antibodies, polynucleotides encoding anti-IL-33 and / or anti-TSLP antibodies, host cells containing such polynucleotides, methods of making and using such anti-IL-33 and / or anti-TSLP antibodies or polynucleotides, pharmaceutical compositions containing such anti-IL-33 and / or anti-TSLP antibodies or polynucleotides, and mixtures or bispecific antibodies comprising such anti-IL-33 and / or anti-TSLP antibodies or polynucleotides encoding such mixtures or bispecific antibodies.

Description

Technical Field

[0001] This invention belongs to the field of antibodies and their use in the treatment of various human diseases. Background Technology

[0002] Asthma is a chronic inflammatory respiratory disease typically characterized by variable airflow limitation and affects more than 300 million people worldwide. This disease results from a complex interplay between the environment, lung epithelial cells, and the ensuing immune response, leading to (allergic) airway inflammation. Within this pathobiological context, bronchial epithelial cells play a crucial role by producing innate cytokines called alarm factors, such as thymic stromal lymphopoietin (TSLP) and interleukin-33 (IL-33). These cytokines are secreted when airway epithelial cells are damaged by a variety of environmental triggers, such as allergens, respiratory viruses, bacteria, smoking, and air pollutants.

[0003] Following alarm release, a wide range of innate and adaptive immune cells—including dendritic cells, naïve and differentiated T cells, ILC2 cells, eosinophils, basophils, and mast cells—respond to TSLP and IL33 via their respective receptors. TSLP acts specifically through TSLPR (also known as CRLF2) and is signaled via a heterodimeric receptor complex consisting of TSLPR and the interleukin-7 receptor α chain. TSLP-mediated signaling leads to the activation of Janus kinase (JAK) and signal transducers with activating transcription factors (STATs), which in turn lead to the transcription of target genes and a subsequent tightly coordinated immune response.

[0004] IL-33 specifically functions via the ST2 receptor and is signaled via a receptor complex (IL1R-AcP) containing ST2 and the co-receptor IL-1R accessory protein. IL-33 binding leads to activation of the NF-κB signaling pathway via the classic MyD88 / IRAK / TRAF6 module, resulting in phosphorylation and activation of the ERK1 / 2, JNK, p38, and PI3K / AKT signaling modules, thereby leading to the production and release of pro-inflammatory cytokines.

[0005] Genome-wide association studies have shown a strong association between asthma and genetic polymorphisms of TSLP, IL33, and ST2. Furthermore, mouse studies also support the possibility of TSLP and IL33 as targets for asthma. In a chronic model of adaptive immune-mediated allergic airway inflammation, mice lacking the TSLPR or IL33 pathway showed significantly reduced airway inflammation and airway hyperresponsiveness (AHR). In addition, intranasal administration of IL33 or TSLP induced allergic airway inflammation in mice. Therefore, both TSLP and IL33 can activate multiple downstream inflammatory cascades, explaining why they are considered major disease drivers of asthma.

[0006] There is an unmet need for therapies that can treat or improve inflammatory diseases, such as chronic airway inflammatory diseases. This invention addresses this need. Invention Overview

[0007] This article describes anti-interleukin-33 (IL33) antibodies, anti-thymocyte stromal lymphopoietin (TSLP) antibodies, mixtures containing anti-IL-33 and anti-TSLP antibodies, bispecific antibodies containing anti-IL-33 and / or anti-TSLP antibodies, nucleic acids encoding these antibodies and mixtures, host cells containing these nucleic acids, pharmaceutical compositions comprising these antibodies, mixtures and nucleic acids, and treatment methods including administering these antibodies, mixtures, nucleic acids or pharmaceutical compositions to a patient.

[0008] For example, asthma and COPD (chronic obstructive pulmonary disease) are chronic inflammatory airway diseases characterized by obstructive airflow limitation. Both diseases impose a significant burden on patients and healthcare systems. While asthma affects 262 million people and causes 461,000 deaths globally [see Lancet 2020; 396:1204–1222], COPD has a greater disease burden and is the third leading cause of death worldwide, causing approximately 3.2 million deaths in 2019. These two diseases are heterogeneous in their clinical presentation and underlying inflammatory mechanisms; therefore, monotherapy with a single MOA (mechanism of action) may not be effective for all patients.

[0009] For both diseases, common triggers for acute exacerbations of airflow obstruction include viral or bacterial infections, cigarette smoke, allergens, and environmental factors such as air pollution. These triggers induce epithelial cells in the lungs to secrete IL-33 (interleukin-33), TSLP (thymic stromal lymphopoietin), and IL-25 (interleukin-25, or IL-17E), three alarming factors that drive type 2 inflammation. Particularly for asthma, genome-wide association studies have shown a strong association between the disease and genetic polymorphisms of TSLP, IL33, and ST2. Therefore, TSLP and IL-33 have become particularly attractive targets due to their strong genetic link to asthma and their broad impact on airway inflammation.

[0010] ILC2s are relatively newly discovered immune cells. They are tissue-resident cells, primarily distributed in mucosal tissues such as the lungs, small intestine, skin, and adipose tissue. Due to their location, they are believed to play a key role in the development of allergic diseases and type 2 inflammation. Upon binding to alarmins, ILC2s rapidly release cytokines such as IL-4, IL-5, and IL-13 to mediate responses from eosinophils, mast cells, basophils, dendritic cells (DCs), B cells, and Th2 cells.

[0011] The current standard treatment is a combination of inhaled beta-adrenergic agonists and inhaled corticosteroids (ICS), primarily providing symptom relief for disease control and often improving lung function and reducing the rate of acute exacerbations. However, asthma generally responds better than COPD, and patients with eosinophilic inflammation respond best to ICS. A subset of asthma and COPD patients are not sensitive to ICS. Common side effects in patients using steroids and beta-adrenergic agonists long-term include increased frequency with dose increases, muscle cramps, and muscle weakness.

[0012] Non-type 2 asthma patients assessed by low serum eosinophil count and low exhaled nitric oxide fraction are not suitable candidates for current biologic therapies. Furthermore, while azithromycin and the phosphodiesterase-4 inhibitor roflumilast can be used to reduce acute exacerbations of COPD, their use is limited due to side effects. For azithromycin, the main side effects are gastrointestinal reactions, with the potential for arrhythmias and antibiotic resistance; while for roflumilast, the main side effects are gastrointestinal, such as nausea.

[0013] Chronic obstructive pulmonary disease (COPD) is currently defined as a chronic disease state characterized by irreversible airway obstruction resulting from the progression of two major underlying diseases, including chronic bronchitis and emphysema. Chronic bronchitis is clinically defined as persistent cough, sputum production, and dyspnea, while emphysema is histopathologically defined as irreversible changes in the airway walls distal to the terminal bronchioles, clinically manifested as slowly progressive dyspnea. COPD is currently the fourth leading cause of death in the United States and Europe, and deaths in COPD patients are often due to complications of the disease, such as respiratory failure or infection (GOLD Symposium Summary, Am J Respir CritCare Med 2001; 163: 1256-1276).

[0014] In some patients with chronic asthma, irreversible airway obstruction can develop that is difficult to distinguish from COPD, and thus bronchial asthma can progress to COPD (Celli BR et al., Eur Respir J 2004; 23: 932-46). In a significant number of patients meeting the current diagnostic criteria for COPD, significant reversible improvement in airway obstruction has been demonstrated with short- or long-term inhaled bronchodilators and steroids (GOLD Symposium Summary, Am J Respir Crit CareMed 2001; 163: 1256-1276). Therefore, a significant number of patients meet the defining criteria for both bronchial asthma and COPD (Guerra S, Curr Opin Pulm Med 2005; 11: 17-13).

[0015] In humans, both TSLP and IL33 are associated with disease severity and have been shown to upregulate the expression levels of each other's receptors (Pebbles et al., Allergy, 2020), suggesting the existence of a regulatory feedback loop. Furthermore, TSLP and IL33 activate and recruit a variety of immune cells, many of which express both TSLP and IL33 receptors, and therefore these alarms have the potential to amplify downstream (adaptive) immune responses and airway inflammation.

[0016] Therefore, a significant unmet need remains for the treatment and / or prevention of chronic inflammatory diseases, including COPD and asthma. This paper anticipates that simultaneous blocking of both IL-33 and TSLP alarmins could improve the efficacy of treatments for chronic inflammatory diseases such as asthma and COPD, as blocking only one alarmin is insufficient. This paper anticipates that dual blocking of IL-33 and TSLP is more advantageous than targeting a single pathway; for example, anti-IL-5 therapy only improves type 2 inflammation. Dual blocking of IL-33 and TSLP will reduce the secretion of several downstream cytokines (i.e., IL-4, IL-5, and IL-13), which mediate inflammatory responses in eosinophils, B cells, mast cells, etc.

[0017] This invention anticipates that the anti-hIL33 and anti-hTSLP MabPair antibodies of this invention offer safety advantages over smaller molecules. The MabPair compositions of this invention are expected to avoid the side effects of long-term use of inhaled β-adrenergic agonists and inhaled corticosteroids. The MabPair compositions of this invention are expected to address unmet needs in the treatment of asthma, COPD, and other conditions.

[0018] Therefore, this article also provides methods for the prevention or treatment of inflammatory and / or inflammatory diseases (including asthma, COPD, etc.). In moderate to severe inflammatory and / or inflammatory diseases (such as asthma, COPD, etc.), simultaneous or concurrent inhibition of TSLP and IL-33 may achieve superior efficacy compared to single-agent therapy.

[0019] The following numbered items describe these compositions and methods. 1. An anti-human IL33 (anti-hIL33) antibody comprising a heavy chain variable domain (VH) and a light chain variable region (VL), wherein each of the VH and VL comprises a complementarity-determining region 1 (CDR1), a CDR2, and a CDR3. The anti-hIL33 antibody comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VLCDR3, each containing the following amino acid sequences: SEQ ID NO: 80, 81, 82, 77, 78, and 79. The anti-hIL33 antibody inhibits the interaction between human IL-33 and the ST2 / IL1AcP complex. 2. The anti-hIL33 antibody according to Item 1, wherein VH comprises an amino acid sequence with no more than four altered amino acid sequences relative to the group consisting of SEQ ID NO: 22 and 29, and / or VL comprises an amino acid sequence with no more than four altered amino acid sequences relative to the group consisting of SEQ ID NO: 20 and 25. 3. Based on the anti-hIL33 antibody described in Project 1-2, The anti-hIL33 antibody VH and VL each contain an amino acid sequence, and the amino acid sequences of VH and VL together comprise two sequences. One of the two sequences contains no more than four amino acid changes relative to one sequence in the VH / VL sequence pair, and the other of the two sequences contains no more than four amino acid changes relative to the other sequence in the VH / VL sequence pair. The VH / VL sequence pairs are selected from the group consisting of: SEQ ID NO: 29 (VH) and 25 (VL); and SEQ ID NO: 22 (VH) and 20 (VL). 4. The anti-hIL33 antibody as described in items 1-3, wherein... VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 contain the amino acid sequences of SEQ ID NO: 80, 81, 82, 77, 78, and 79, respectively. The VH contains no more than four altered amino acid sequences relative to the amino acid sequence of SEQ ID NO: 29, and The VL contains no more than four altered amino acid sequences relative to the amino acid sequence of SEQ ID NO: 25. 5. One or more polynucleotides encoding the anti-hIL33 antibody of item 1, said anti-hIL33 antibody comprising VH and VL, said VH and VL each comprising CDR1, CDR2 and CDR3. The anti-hIL33 antibody comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VLCDR3, each containing the following amino acid sequences: SEQ ID NO: 80, 81, 82, 77, 78, and 79; and The anti-hIL33 antibody inhibits the interaction between human IL-33 and the ST2 / IL1AcP complex. 6. A host cell comprising one or more polynucleotides encoding an anti-hIL33 antibody as described in item 5, said anti-hIL33 antibody comprising VH and VL, said VH and VL each comprising CDR1, CDR2 and CDR3. The anti-hIL33 antibody comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VLCDR3, which respectively contain the following sequences: SEQ ID NO: 80, 81, 82, 77, 78, and 79; and The anti-hIL33 antibody inhibits the interaction between human IL-33 and the ST2 / IL1AcP complex. 7. A method for treating a patient in need of treatment with inflammation, inflammatory disease, chronic inflammatory airway disease, asthma, COPD, and / or type 2 inflammation, said method comprising: (a) The anti-hIL33 antibody of Item 1 is administered to the patient, the antibody comprising VH and VL, each of VH and VL comprising CDR1, CDR2 and CDR3, wherein: (1) the anti-hIL33 antibody comprises VH CDR1, VH CDR2, VHCDR3, VL CDR1, VL CDR2 and VL CDR3, each comprising the following sequences: SEQ ID NO: 80, 81, 82, 77, 78 and 79; and (2) the anti-hIL33 antibody inhibits the interaction between human IL-33 and the ST2 / IL1AcP complex; or (b) Administer to the patient one or more polynucleotides encoding the anti-hIL33 antibody described in (a). 8. An anti-human TSLP (anti-hTSLP) antibody comprising VH and VL, wherein each of VH and VL comprises CDR1, CDR2, and CDR3. The anti-hTSLP antibody comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VLCDR3, each containing the following sequences: SEQ ID NO: 86, 87, 88, 83, 84, and 85; SEQ ID NO: 86, 87, 88, 83, 89, and 90; SEQ ID NO: 94, 95, 96, 91, 92, and 93. The anti-hTSLP antibody inhibits the binding of hTSLP to hTSLPR or the interaction of hTSLP with the hTSLPR / hIL7Rα complex. 9. The anti-hTSLP antibody as described in Project 8, The anti-hTSLP VH contains an amino acid sequence with no more than four altered amino acid sequences relative to the group consisting of SEQ ID NO: 43, 50, 65 and 70, and / or the anti-hTSLP VL contains an amino acid sequence with no more than four altered amino acid sequences relative to the group consisting of SEQ ID NO: 45, 47, 54, 67 and 74. 10. Based on the anti-hTSLP antibody described in items 9-10, The anti-hTSLP antibody VH and VL each contain an amino acid sequence, and the amino acid sequences of VH and VL together contain two sequences. One of the two sequences contains no more than four amino acid changes relative to one sequence in the VH / VL sequence pair, and the other of the two sequences contains no more than four amino acid changes relative to the other sequence in the VH / VL sequence pair. The VH / VL sequence pairs are selected from the group consisting of: SEQ ID NO: 43 (VH) and 45 (VL); SEQ ID NO: 43 (VH) and 47 (VL); SEQ ID NO: 50 (VH) and 54 (VL); SEQ ID NO: 65 (VH) and 67 (VL); and SEQ ID NO: 70 (VH) and 74 (VL). 11. The anti-hTSLP antibody as described in Projects 8-10, wherein... VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 contain the amino acid sequences of SEQ ID NO: 94, 95, 96, 91, 92, and 93, respectively. The VH contains no more than four altered amino acid sequences relative to the amino acid sequence of SEQ ID NO: 70, and The VL contains no more than four altered amino acid sequences relative to the amino acid sequence of SEQ ID NO: 74. 12. One or more polynucleotides encoding the anti-hTSLP antibody described in item 8, said anti-hTSLP antibody comprising VH and VL, said VH and VL each comprising CDR1, CDR2 and CDR3. The anti-hTSLP antibody comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VLCDR3, each containing the following sequences: SEQ ID NO: 86, 87, 88, 83, 84, and 85; SEQ ID NO: 86, 87, 88, 83, 89, and 90; SEQ ID NO: 94, 95, 96, 91, 92, and 93. The anti-hTSLP antibody inhibits the binding of hTSLP to hTSLPR or the interaction of hTSLP with the hTSLPR / hIL7Rα complex. 13. A host cell comprising one or more polynucleotides encoding an anti-hTSLP antibody as described in item 12, said anti-hTSLP antibody comprising VH and VL, said VH and VL each comprising CDR1, CDR2 and CDR3. The anti-hTSLP antibody comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VLCDR3, each containing the following sequences: SEQ ID NO: 86, 87, 88, 83, 84, and 85; SEQ ID NO: 86, 87, 88, 83, 89, and 90; SEQ ID NO: 94, 95, 96, 91, 92, and 93. The anti-hTSLP antibody inhibits the binding of hTSLP to hTSLPR or the interaction of hTSLP with the hTSLPR / hIL7Rα complex. 14. A method for treating a patient in need of treatment with inflammation, inflammatory disease, chronic inflammatory airway disease, asthma, COPD, and / or type 2 inflammation, the method comprising: (a) The anti-hTSLP antibody described in item 8 is administered to the patient, the antibody comprising VH and VL, each of VH and VL comprising CDR1, CDR2, and CDR3, wherein: (1) the anti-hTSLP antibody comprises VH CDR1, VH CDR2, VHCDR3, VL CDR1, VL CDR2, and VL CDR3, each comprising the following sequences: SEQ ID NO: 86, 87, 88, 83, 84, and 85; SEQ ID NO: 86, 87, 88, 83, 89, and 90; SEQ ID NO: 94, 95, 96, 91, 92, and 93; and (2) the anti-hTSLP antibody inhibits the binding of hTSLP to hTSLPR or the interaction of hTSLP with the hTSLPR / hIL7Rα complex; or (b) Administer to the patient one or more polynucleotides encoding the anti-hTSLP antibody described in (a). 15. A mixture comprising anti-hIL33 antibody and anti-hTSLP antibody, wherein: (a) (1) The anti-hIL33 antibody comprises VH and VL, each of which comprises CDR1, CDR2, and CDR3; (2) The anti-hIL33 antibody comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3, each comprising the following sequences: SEQ ID NO: 80, 81, 82, 77, 78, and 79; and (3) The anti-hIL33 antibody inhibits the interaction between human IL-33 and the ST2 / IL1AcP complex; and (b) (1) The anti-hTSLP antibody comprises VH and VL, each of which comprises CDR1, CDR2 and CDR3; (2) The anti-hTSLP antibody comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3, each comprising the following sequences: SEQ ID NO: 86, 87, 88, 83, 84 and 85; SEQ ID NO: 86, 87, 88, 83, 89 and 90; SEQ ID NO: 94, 95, 96, 91, 92 and 93; and (3) The anti-hTSLP antibody inhibits the binding of hTSLP to hTSLPR or the interaction of hTSLP with the hTSLPR / hIL7Rα complex. 16. The mixture according to item 15, wherein (a) (1) The VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VLCDR3 of the anti-hIL33 antibody respectively contain the amino acid sequences of SEQ ID NO: 80, 81, 82, 77, 78 and 79, (2) the VH of the anti-hIL33 antibody contains no more than four altered amino acid sequences relative to the amino acid sequence of SEQ ID NO: 29, and (3) the VL of the anti-hIL33 antibody contains no more than four altered amino acid sequences relative to the amino acid sequence of SEQ ID NO: 25, and (b) (1) The VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VLCDR3 of the anti-hTSLP antibody respectively contain the amino acid sequences of SEQ ID NO: 94, 95, 96, 91, 92 and 93, respectively; (2) the VH contains no more than four altered amino acid sequences relative to the amino acid sequence of SEQ ID NO: 70; and (3) the VL contains no more than four altered amino acid sequences relative to the amino acid sequence of SEQ ID NO: 74. 17. One or more polynucleotides encoding the mixture of anti-hIL33 antibody and anti-hTSLP antibody as described in item 15, wherein... (a) (1) The anti-hIL33 antibody comprises VH and VL, each of which comprises CDR1, CDR2, and CDR3; (2) The anti-hIL33 antibody comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3, each comprising the following sequences: SEQ ID NO: 80, 81, 82, 77, 78, and 79; and (3) The anti-hIL33 antibody inhibits the interaction between human IL-33 and the ST2 / IL1AcP complex; and (b) (1) The anti-hTSLP antibody comprises VH and VL, each of which comprises CDR1, CDR2 and CDR3; (2) The anti-hTSLP antibody comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3, each comprising the following sequences: SEQ ID NO: 86, 87, 88, 83, 84 and 85; SEQ ID NO: 86, 87, 88, 83, 89 and 90; SEQ ID NO: 94, 95, 96, 91, 92 and 93; and (3) The anti-hTSLP antibody inhibits the binding of hTSLP to hTSLPR or the interaction of hTSLP with the hTSLPR / hIL7Rα complex. 18. A host cell comprising one or more polynucleotides encoding a mixture as described in item 17, said mixture comprising an anti-hIL33 antibody and an anti-hTSLP antibody, wherein... (a) (1) The anti-hIL33 antibody comprises VH and VL, each of which comprises CDR1, CDR2, and CDR3; (2) The anti-hIL33 antibody comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3, each comprising the following sequences: SEQ ID NO: 80, 81, 82, 77, 78, and 79; and (3) The anti-hIL33 antibody inhibits the interaction between human IL-33 and the ST2 / IL1AcP complex; and (b) (1) The anti-hTSLP antibody comprises VH and VL, each of which comprises CDR1, CDR2 and CDR3; (2) The anti-hTSLP antibody comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3, each comprising the following sequences: SEQ ID NO: 86, 87, 88, 83, 84 and 85; SEQ ID NO: 86, 87, 88, 83, 89 and 90; SEQ ID NO: 94, 95, 96, 91, 92 and 93; and (3) The anti-hTSLP antibody inhibits the binding of hTSLP to hTSLPR or the interaction of hTSLP with the hTSLPR / hIL7Rα complex. 19. A method for treating a patient in need of treatment with inflammation, inflammatory disease, chronic inflammatory airway disease, asthma, COPD, and / or type 2 inflammation, the method comprising: (a) Administering the mixture described in item 15 to the patient, the mixture comprising anti-hIL33 antibody and anti-hTSLP antibody, wherein: (1) (A) The anti-hIL33 antibody comprises VH and VL, each of which comprises CDR1, CDR2, and CDR3; (B) The anti-hIL33 antibody comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3, each comprising the following sequences: SEQ ID NO: 80, 81, 82, 77, 78, and 79; and (C) The anti-hIL33 antibody inhibits the interaction between human IL-33 and the ST2 / IL1AcP complex; and (1) (B) The anti-hTSLP antibody comprises VH and VL, each of which comprises CDR1, CDR2, and CDR3; (B) The anti-hTSLP antibody comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3, each comprising the following sequences: SEQ ID NO: 86, 87, 88, 83, 84, and 85; SEQ ID NO: 86, 87, 88, 83, 89, and 90; SEQ ID NO: 94, 95, 96, 91, 92, and 93; and (C) The anti-hTSLP antibody inhibits the binding of hTSLP to hTSLPR or the interaction of hTSLP with the hTSLPR / hIL7Rα complex; or (b) Administer to the patient one or more polynucleotides encoding the mixture described in (a). 20. An antibody mixture comprising: (a) An anti-hIL33 antibody comprising a heavy chain (HC) and a light chain (LC), wherein (1) the HC of the anti-hIL33 antibody is encoded by a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 31, and (2) the LC of the anti-hIL33 antibody is encoded by a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 27; and (b) An anti-hTSLP antibody comprising HC and LC, wherein (1) the HC of the anti-hTSLP antibody is encoded by a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 72, and (2) the LC of the anti-hTSLP antibody is encoded by a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 76. 21. The mixture according to item 20, wherein: The amino acid sequences of the anti-hIL33 antibody HC and LC are encoded by the nucleic acid sequences of SEQ ID NO: 30 and 26, respectively; and The amino acid sequences of the anti-hTSLP antibody HC and LC are encoded by the nucleic acid sequences of SEQ ID NO: 71 and 75, respectively. 22. A pharmaceutical composition comprising the mixture described in any one of items 20 to 21. 23. One or more polynucleotides encoding any one of items 20 to 21. 24. The polynucleotide according to item 23, wherein the polynucleotide comprises the nucleic acid sequences of SEQ ID NO: 30, 26, 71 and 75. 25. One or more vectors comprising the polynucleotides described in item 23 or 24. 26. The vector described in Item 25 is a viral vector. 27. The vector described in Project 26 is an oncolytic virus vector. 28. The vector described in item 26 or 27 is a retrovirus, adenovirus, adeno-associated virus (AAV), vaccinia virus, modified Ankara vaccinia virus (MVA), herpesvirus, lentivirus, measles virus, Coxsackie virus, Newcastle disease virus, reovirus, or poxvirus vector. 29. A host cell comprising the polynucleotides described in item 23 or 24 and / or the vector described in item 5, wherein the host cell is capable of producing a mixture as described in any one of items 20 to 21. 30. The host cell as described in Item 29 is a CHO cell or a mouse myeloma cell. 31. A method for preparing an antibody mixture, comprising the following steps: Culture the host cells described in any one of items 29 to 30; and The antibody mixture is recovered from the culture supernatant or host cell clumps. 32. A method for treating a patient in need of treatment with inflammation, inflammatory disease, chronic inflammatory airway disease, asthma, COPD, and / or type 2 inflammation, the method comprising: (a) Administering to the patient a dose of any mixture of items 20 to 21 or the pharmaceutical composition of item 22, or (b) Administering to the patient a dose of the polynucleotide described in item 23 or 24 or the carrier described in any one of items 26 to 28. 33. An anti-hIL33 antibody, wherein HC contains an amino acid sequence relative to SEQ ID NO: 7, 11, 15, 19, 23, or 31 that includes no more than four, three, two, or one altered amino acid sequence, and LC contains an amino acid sequence relative to SEQ ID NO: 5, 9, 13, 17, 21 or 27 containing no more than four, three, two or one altered amino acid sequence. 34. The anti-hIL33 antibody according to item 33, wherein HC contains the amino acid sequence of SEQ ID NO: 31 and LC contains the amino acid sequence of SEQ ID NO: 27. 35. An anti-hTSLP antibody, wherein HC contains an amino acid sequence relative to SEQ ID NO: 44, 52, 62, 66, or 72 that includes no more than four, three, two, or one altered amino acid sequence, and LC contains an amino acid sequence relative to SEQ ID NO: 46, 48, 56, 64, 68 or 76 that contains no more than four, three, two or one altered amino acid sequence. 36. The anti-hTSLP antibody according to item 35, wherein HC comprises the amino acid sequence of SEQ ID NO: 72 and LC comprises the amino acid sequence of SEQ ID NO: 76. 37. A mixture of anti-hIL33 antibody and anti-hTSLP antibody, wherein The anti-hIL33 HC contains an amino acid sequence relative to SEQ ID NO: 7, 11, 15, 19, 23, or 31 that includes no more than four, three, two, or one altered amino acid sequence, and The anti-hIL33 LC contains an amino acid sequence with no more than four, three, two, or one altered amino acid sequence relative to SEQ ID NO: 5, 9, 13, 17, 21, or 27; and The anti-hTSLP HC comprises an amino acid sequence relative to SEQ ID NO: 44, 52, 62, 66, or 72 containing no more than four, three, two, or one altered amino acid sequence, and The anti-hTSLP LC contains an amino acid sequence that includes no more than four, three, two, or one altered amino acid sequence relative to SEQ ID NO: 46, 48, 56, 64, 68, or 76. Brief description of the attached figures

[0020] Figure 1 : Binding of hIL-33 to ST2-Fc and blocking of IL-33 / ST2 interaction by anti-hIL-33 antibodies. (A) Evaluation of the binding of different concentrations of active hIL-33 to ST2-Fc captured on a CM5 chip, with binding times set to 1 or 2 minutes and dissociation times maintained at 5 minutes. (B) Blocking analysis of different anti-hIL-33 antibodies. 10 nM of active hIL-33 was premixed with different concentrations of each antibody, and the mixture was then flowed through the surface of a CM5 chip containing ST2-Fc protein. (C) Blocking activity analysis of chimeric anti-hIL-33 antibody 10998, five humanized anti-hIL-33 antibody variants, and isotype control antibody 10825. Evaluation was performed using Biacore 2000 via SPR (surface plasmon resonance). The x-axis represents antibody concentration (nM), and the y-axis represents response units (RU) corrected for blank control buffer.

[0021] Figure 2 Mass spectrometry analysis to evaluate the deamidation sensitivity of humanized anti-hIL-33 antibodies QB11061 and QB11119. Anti-hIL-33 antibodies QB11061 (A and B) and QB11119 (C, D, and E) were denatured, reduced, alkylated, and digested with trypsin for accelerated deamidation. Pretreated antibody samples (A and C) and post-treated antibody samples (B and D) were digested with trypsin, and the eluted peptides were detected at UV 214 nm. Each peptide was analyzed by mass spectrometry to assess potential deamidation. Peaks marked with arrows and L3 (N→D) indicate peaks in VL-CDR2. 28 NG 29 Deamidinated peptides at motifs. Figure 1 E is Figure 1 A magnified view of D. The x-axis represents elution time, and the y-axis represents the response unit.

[0022] Figure 3 : Schematic diagram of the construction of a yeast display library for affinity maturation. (A) Four DNA fragments were mixed for electroporation of competent yeast cells, the four DNA fragments including PCR-amplified DNA fragments encoding the following: (1) a yeast signal peptide (SP1) and VL sequence; (2) an intermediate fragment containing human c-κ (Ck), a c-MYC tag, a furin-pep2A cleavage site (F2A), and yeast signal peptide 2 (SP2); (3) SP2, a VH sequence (VH), and a human CH1 sequence (CH1); (4) a pFab1.6 vector double-digested with Bgl II and NheI containing SP1, a hemagglutinin tag (HA), and the yeast Aga2 protein (Aga2). Homologous recombination in yeast cells assembled the DNA fragments into a full-length construct, which was transcribed under a galactose-inducible promoter. (B) After translation of the polypeptide, LC (VL+Ck) is cleaved by furin (after the c-MYC tag), and then LC is secreted and assembled with Fd (VH+CH1) to form the Fab fragment. The Aga2 protein in the introduced construct forms two disulfide bonds with the initial Aga1 protein expressed on yeast, thus anchoring (or displaying) the Fab fragment on the yeast surface.

[0023] Figure 4 Following the final round of FACS sorting, 94 individual yeast colonies were bound to 2 nM biotinylated hIL-33 antigen. Wells A1 and H12 contained parental QB11119 yeast clones as internal controls. Fifteen clones exhibiting strong antigen binding (boxed out) were selected for PCR amplification and DNA sequencing. The x-axis represents the binding strength of the FITC-conjugated anti-HA antibody, indicating the display level of the Fab fragment on the yeast surface. The y-axis measures the antigen-binding ability of the displayed Fab fragment by the binding of APC-conjugated streptavidin to biotinylated hIL-33.

[0024] Figure 5: Surface plasmon resonance sensing images of captured anti-hIL-33 antibody interacting with hIL-33 (A) and cyIL-33 (B), superimposed on a Biacore T200 using a 1:1 kinetic model. Polyclonal goat anti-human IgG (Fc-specific) antibody was immobilized on the CM4 chip surface using a standard amine conjugation method. Anti-hIL-33 antibodies QB11004, QB11061, QB11119, QB11421, QB11465 and reference-3 antibody QB11094 were captured and then serially diluted 1:3 at a rate of 50 μL / min with active hIL-33 (50 nM, 16.67 nM, 5.56 nM, 1.85 nM, 0.62 nM) or cyIL-33 (100 nM, 33.33 nM, 11.11 nM, 3.7 nM, 1.23 nM, 0.41 nM). Binding time was 3 minutes, and dissociation time was monitored for up to 1 hour, but only data from the first 5 minutes were evaluated. For evaluation of the hIL-33 antigen, cycling was performed at 37°C, and for the cyIL-33 antigen, cycling was performed at 25°C. After each cycle, the chip was regenerated with 10 mM glycine-HCl at pH 1.5. Results were analyzed using Biacore T200 evaluation software V3.2. Results were double-referenced and fitted to a 1:1 interaction model. Calculated parameter values ​​for the kinetic analysis are listed in Tables 9 and 10.

[0025] Figure 6Screening for antagonistic anti-hTSLP polyclonal rabbit antibodies using Biacore T200. (A) Comparison of purified rabbit anti-human TSLP polyclonal antibodies (from rabbits 7182, 7183, 7184, and 7186) with reference antibody 10985 and isotype control antibody 10987. Anti-His6 antibody was immobilized on the surface of a CM5 chip, and different concentrations of premixed His6-hTSLP antigen and 2 µg / ml purified rabbit antibody were injected. Finally, 100 nM hTSLPR-Fc was allowed to flow through the surface of the CM5 chip. (B) Binding sensor map of the interaction between the captured polyclonal antibody from rabbit 7186 and hTSLP. (C) Binding sensor map of the interaction between the captured polyclonal antibody from rabbit 7186 and cyTSLP. (D) Evidence of the direct interaction between hTSLP and hTSLPR-Fc. Anti-His6 antibody was immobilized on the surface of a CM5 chip, capturing 5 µg / ml of His6-hTSLP antigen. Blank buffer was injected for 1 minute, followed by a second injection of blank buffer, 100 nM hTSLPR-Fc, anti-hTSLP control blocking antibody 10985, or anti-hTSLP non-blocking antibody 10987, allowing it to flow through the CM5 chip surface for 3 minutes. (E) Polyclonal antibodies from rabbit 7186 can block the direct interaction between hTSLP and hTSLPR-Fc. Anti-His6 antibody was immobilized on the surface of a CM5 chip, capturing 5 µg / ml of His6-hTSLP antigen. 30 µg / ml of polyclonal antibody from rabbit 7186 was injected for 1 minute, followed by a second injection of blank buffer, 100 nM hTSLPR-Fc, anti-hTSLP control blocking antibody 10985, or anti-hTSLP non-blocking antibody 10987, allowing it to flow through the CM5 chip surface for 3 minutes. The difference in response between the test protein and the blank buffer was recorded on the y-axis, and the time (seconds) was recorded on the x-axis.

[0026] Figure 7 Antigen binding of single rabbit anti-hTSLP antibodies was confirmed by plate-based ELISA analysis. 2 µg / ml of hTSLP, cyTSLP, or unrelated antigen was immobilized in PBS in each well of a 96-well Maxisorp plate overnight. The plate was washed three times and blocked with 1xPBST containing 2% BSA. Supernatant from 177 rabbit hybridomas was added, and the plate was shaken at RT for 1 hour. HRP-conjugated goat anti-rabbit IgG (H+L) secondary antibody was added, the plate was shaken for 1 hour, washed, and then the substrate 3,3',5,5'-tetramethylbenzidine (TMB) was added for color development. The plate was read at 450 nm using a Perkin-Elmer microplate reader. The x-axis represents the clone name, and the y-axis represents the colorimetric value at 450 nm.

[0027] Figure 8Competitive ELISA assays were used to confirm the blocking activity of the first 10 rabbit anti-hTSLP mAbs. His6-hTSLP antigen was coated at 2 µg / ml into 96-well Maxisorp plates. Rabbit anti-hTSLP antibodies or rabbit isotype control antibodies were added at 5 µg / ml (in duplicate), followed by 2 µg / ml of biotinylated hTSLP, then streptavidin-conjugated HRP, and finally the substrate TMB for color development. The plate was shaken for 15 minutes, and the reaction was terminated by adding H2SO4 solution. The plate was read using a Perkin-Elmer microplate reader. The percentage of inhibition was plotted against antibody-free 1xPBST buffer.

[0028] Figure 9 The blocking activity of single rabbit anti-hTSLP mAbs was compared by screening using Biacore T200 (A) and CCL17 release assay (B). (A) hTSLPR-Fc protein was immobilized on the surface of a CM5 chip. 20 nM of hTSLP and different concentrations of anti-TSLP rabbit antibody were premixed and then flowed through the CM5 surface. After a 3-minute binding run, the binding level of hTSLP to the immobilized hTSLPR (as the response unit) was recorded on the y-axis, and the antibody concentration (nM) was plotted as logarithmic. 10 The scale is plotted on the x-axis. (B) Primary human mononuclear cells purified from PBMCs were seeded in 96-well plates and treated with a constant amount of hTSLP (concentration close to EC50). 50 The rabbit was treated with the preferred anti-hTSLP rabbit antibodies 60E10, 135F1, 139F11, and isotype control 11176 or 10861 for 24 hours using serially diluted values. CCL-17 chemokine levels (pg / ml) in the supernatant were determined using the R&D Systems Human CCL-17 ELISA Kit (catalog number DY364). Results were plotted using GraphPad Prism software. The IC50 values ​​of the first three clones #66 (clone 60E10), #70 (clone 135F1), and #72 (clone 139F11) were... 50 The values ​​are listed in the small table below the graph.

[0029] Figure 10Mechanism of action of anti-hTSLP antibodies QB11341 and QB11237 as determined by Biacore assay. (A) Direct ligand-receptor interaction between hTSLP and hTSLPR. hTSLPR-Fc protein was immobilized on the surface of a CM5 chip using a standard amine conjugation method. Monomeric hTSLP antigens of 0, 0.62, 1.85, 5.56, 16.7, and 50 nM were allowed to flow through the CM5 surface, with binding time for 3 minutes and dissociation time for 5 minutes. Kinetic sensor plots were analyzed using BIAevaluation 4.1.1 to measure the binding affinity between hTSLP and hTSLPR. (B) Blockade of the hTSLP-hTSLPR axis by antibody QB11341 and blockade of the hTSLP-hIL7Rα axis by antibody QB11237. hTSLPR-Fc protein was immobilized on the surface of a CM5 chip. The monomeric antigen hTSLP alone, or an hTSLP-antibody complex premixed with antibodies QB11341 or QB11237, was allowed to flow through the CM5 surface for 3 minutes. Finally, 50 nM hIL7Rα or blank run buffer was injected, with a binding time of 2 minutes and a dissociation time of 5 minutes. (C) Assessment of the inhibitory level of antibody QB11237 on the hTSLP-hIL7Rα interaction. hTSLPR-Fc was immobilized on a CM5 chip, and 20 nM of monomeric hTSLP was injected, binding for 2 minutes and dissociation for 100 seconds. Then, 20 nM of anti-hTSLP antibody QB11341 or QB11237 was allowed to flow through the CM5 surface, with a binding time of 3 minutes and a rapid dissociation time of 1 minute. Finally, 50 nM hIL7Rα or blank run buffer was injected, with a binding time of 2 minutes and a dissociation time of 2 minutes. The inhibitory level of antibody QB11237 on the hTSLP-hIL7Rα interaction was estimated as "100% minus the peak height difference (with and without antibody QB11237) divided by the percentage of the peak height without antibody QB11237".

[0030] Figure 11Chain loss assays were used to evaluate LC-HC pairing of anti-hIL-33 and anti-hTSLP antibodies before and after engineering. ExpiCHO cells were co-transfected with plasmid DNA (A and C) encoding LC1 and / or HC1 of anti-hTSLP QB11548 combined with LC2 and / or HC2 of anti-hIL33 QB11465, or plasmid DNA (B and D) encoding LC1 and / or HC1 of anti-hTSLP QB11237 combined with LC2 and / or HC2 of anti-hIL33 QB11465. ExpiCHO cells were incubated with shaking in a tissue incubator for 12 days. 10 µl of clear supernatant per lane was loaded onto a 4–15% Criterion™ TGX Stain-Free™ pre-prepared SDS-PAGE gel for antibody visualization using a CHEMIDOC™ XRS+ imager. Lanes carrying protein molecular weight standards (labeled M) ran on the far left of each gel, with molecular weights expressed in kilodaltons (kDa). (A) Lane 1 contained plasmid DNA encoding LC1 and HC1 of anti-hTSLP antibody QB11548; Lane 2 contained plasmid DNA encoding HC1 of anti-hTSLP antibody QB11548 and LC2 of anti-hIL-33 antibody QB11465; Lane 3 contained plasmid DNA encoding LC2 and HC2 of anti-hIL-33 antibody QB11465; Lane 4 contained plasmid DNA encoding LC1 of anti-hTSLP antibody QB11548 and HC2 of anti-hIL-33 antibody QB11465. (B) Lane 1 contains plasmid DNA encoding LC1 and HC1 of anti-hTSLP antibody QB11237; Lane 2 contains plasmid DNA encoding HC1 of anti-hTSLP antibody QB11237 and LC2 of anti-hIL-33 antibody QB11465; Lane 3 contains plasmid DNA encoding LC2 and HC2 of anti-hIL-33 antibody QB11465; Lane 4 contains plasmid DNA encoding LC1 of anti-hTSLP antibody QB11237 and HC2 of anti-hIL-33 antibody QB11465.(C) Lane 1 contains plasmid DNA encoding LC1 and HC1 of anti-hTSLP antibody QB11764; Lane 2 contains plasmid DNA encoding HC1 of anti-hTSLP antibody QB11764 and LC2 of anti-hIL-33 antibody QB11465; Lane 3 contains plasmid DNA encoding LC2 and HC2 of anti-hIL-33 antibody QB11465; Lane 4 contains plasmid DNA encoding LC1 of anti-hTSLP antibody QB11764 and HC2 of anti-hIL-33 antibody QB11465; Lane 5 contains four plasmid DNAs encoding LC1, HC1, LC2, and HC2 of both anti-hTSLP antibody QB11764 and anti-hIL-33 antibody QB11465; Lane 6 contains empty pSB01 plasmid DNA as a mimic transfection. (D) Lane 1 contains plasmid DNA encoding LC1 and HC1 of anti-hTSLP antibody QB11718; Lane 2 contains plasmid DNA encoding HC1 of anti-hTSLP antibody QB11718 and LC2 of anti-hIL-33 antibody QB11465; Lane 3 contains plasmid DNA encoding LC2 and HC2 of anti-hIL-33 antibody QB11465; Lane 4 contains plasmid DNA encoding LC1 of anti-hTSLP antibody QB11718 and HC2 of anti-hIL-33 antibody QB11465; Lane 5 contains four plasmid DNAs encoding LC1, HC1, LC2, and HC2 of both anti-hTSLP antibody QB11718 and anti-hIL-33 antibody QB11465; Lane 7 contains plasmid DNA encoding LC and HC of anti-HER2 trastuzumab to monitor transfection efficiency.

[0031] Figure 12Mass spectrometry analysis of MabPair antibody mixture QB11750, consisting of anti-hIL-33 IgG4 antibody QB11465 and anti-hTSLP IgG1-D265A antibody QB11718. Experimental procedures were as described in Example 10. The x-axis shows deconvolution mass, and the y-axis shows the count, reflecting the abundance of a given mass of protein. (A) UV detection of MabPair mixture QB11750 after PNGase F treatment for deglycosylation. (B) Mass of intact (unreduced) anti-hTSLP IgG1-D265A antibody QB11718 (top) and anti-hIL-33 IgG4 antibody QB11465 (bottom). (C) Reduction of MabPair mixture QB11750 after PNGase F treatment for deglycosylation, incubation at 55°C for 30 min in a buffer containing 4 M guanidine hydrochloride, 50 mM Tris pH 8.0, and 50 mM DTT. The reduced LC and HC were scanned using a TIC (total ion chromatogram), which was created by summing the intensities of all mass spectrometric peaks belonging to the same scan. (D) The mass of the deconvolution peak was matched from top to bottom with the LC of anti-hIL-33 antibody QB11465 (theoretical mass 24026.53 Da), the LC of anti-hTSLP antibody QB11718 (theoretical mass 23664.31 Da), the HC of anti-hTSLP antibody QB11718 (theoretical mass 49187.03 Da), and the HC of anti-hIL-33 antibody QB11465 (theoretical mass 48987.43 Da).

[0032] Figure 13 Mass spectrometry analysis of the Fab fragment of MabPair antibody mixture QB11750, consisting of anti-hIL-33 IgG4 antibody QB11465 and anti-hTSLP IgG1-D265A antibody QB11718. (A) 400 µg of MabPair antibody mixture QB11750 was digested overnight at 37°C with 100 units of IdeS, diluted 1:2 in reducing buffer containing 2-MEA, heated at 37°C for 1 hour, and the reaction was terminated with 10% formic acid. The treated sample was injected into an Agilent 6224 Precision Mass TOF mass spectrometer for detection and analysis under UV light. (B) Mass of all individual deconvolution peaks as shown in Figure (A). Main peaks 3, 6, 10, 11, 12, 13 and shoulder peak 14 are labeled in the respective subplots. The x-axis shows the deconvolution mass, and the y-axis shows the count, reflecting the abundance of protein of a given mass.

[0033] Figure 14Mass spectrometry analysis of MabPair antibody mixture QB11823, consisting of anti-hIL-33 IgG4 antibody QB11465 and anti-hTSLP IgG1-D265A antibody QB11764. The experimental procedures were as described in Example 11. (A) After deglycosylation with PNGase F, the unreduced MabPair mixture QB11823 was detected by UV. (B) After deglycosylation with PNGase F, the MabPair mixture QB11823 was digested overnight at 37°C with 100 units of IdeS to produce F(ab')2 and Fc / 2 fragments, followed by UV detection. (C) Mass of the four deconvolution peaks. Peak 1 matches the half-Fc (Fc / 2) of anti-hTSLP antibody QB11764, peak 2 matches the half-Fc (Fc / 2) of anti-hIL-33 antibody QB11465, peak 3 matches the F(ab')2 fragment of anti-hIL-33 antibody QB11465, and peak 4 matches the F(ab')2 fragment of anti-hTSLP antibody QB11764. The x-axis shows the deconvolution quality, and the y-axis shows the count, reflecting the abundance of a given protein quality.

[0034] Figure 15 Mass spectrometry analysis of the Fab fragment of MabPair antibody mixture QB11823, consisting of anti-hIL-33 IgG4 antibody QB11465 and anti-hTSLP IgG1-D265A antibody QB11764. (A) 400 µg of MabPair antibody mixture QB11823 was digested overnight at 37°C with 100 units of IdeS, diluted 1:2 in reducing buffer containing 2-MEA, heated at 37°C for 1 hour, and the reaction was terminated with 10% formic acid. The treated sample was injected into an Agilent 6224 Precision Mass-Time-of-Flight mass spectrometer for detection and analysis under UV light. (B) Mass of deconvolution peaks B and D as shown in Figure (A). Peaks 1 and 2 are the unreduced F(ab')2 of anti-hIL-33 antibody QB11465 and anti-hTSLP antibody QB11764, respectively. Peaks A and C are 2-MEA adduct species of the Fab fragments of anti-hIL-33 antibody QB11465 and anti-hTSLP antibody QB11764, respectively. The x-axis shows the deconvolution quality, and the y-axis shows the count, reflecting the abundance of a given protein quality.

[0035] Figure 16: Surface plasmon resonance sensing images of captured anti-hTSLP antibodies interacting with hTSLP (A) and cyTSLP (B) and superimposed with a 1:1 kinetic model fit in Biacore T200. (A) Polyclonal goat anti-human IgG (Fc specific) antibody was immobilized on the surface of a CM4 chip using a standard amine conjugation method. Anti-hTSLP antibodies QB10985 (reference antibody), QB11781, and QB11764 were captured and then injected at a rate of 50 uL / min with monomeric hTSLP antigens derived from E. coli (catalog number 1398-TS-010) (1.0, 0.5, 0.25, 0.13, 0.063, 0.031 nM), or internally prepared monomeric hTSLP antigens QB11630 (SEQ ID NO: 32) (10, 4, 1.6, 0.64, 0.256, 0.1024 nM), or internally prepared monomeric hTSLP antigens QB11631 (SEQ ID NO: 34) (8, 4, 2, 1, 0.5, 0.25, 0.13 nM). The binding time was 3–10 minutes, and the dissociation time was monitored for up to 12 minutes. The cycle was run at 25°C. After each cycle, the chip was regenerated with 10 mM glycine-HCl at pH 1.5. (B) The experimental procedure was the same as in (A), except that the internally prepared monomeric cyTSLP antigens QB11632 (SEQ ID NO: 35) (200, 66.7, 22.2, 7.4, 2.47, 0.823 nM) or QB11633 (SEQ ID NO: 37) (50, 16.7, 5.56, 1.85, 0.617, 0.206 nM) were used. The results were analyzed using Biacore T200 evaluation software V3.2. The results were double-referenced and fitted to a 1:1 Langmuir interaction model. The calculated parameter values ​​for the kinetic analysis are listed in Tables 30 and 31.

[0036] Figure 17 The blocking activity of anti-hIL-33 IgG4 antibody QB11465 was evaluated by cell-based assays. (A) Inhibition of IFNγ cytokine secretion by primary human NK cells by anti-hIL-33 antibody. Purified human NK cells were seeded at 30,000 cells / well in complete RPMI 1640 medium and injected with a constant amount of hIL-33 (concentration close to EC50). 50(a) Treatment with 1 ng / mL IL-12 in combination with serially diluted 1:3 anti-hIL-33 antibody for 24 hours. The level of IFNγ cytokines in the supernatant was measured (pg / ml) using the R&D Systems Human IFNγ ELISA kit, following the manufacturer's instructions. (B) Inhibition of IL-5 secretion by anti-hIL-33 antibody in primary human ILC2 cells. Purified ILC2 cells were cultured in RPMI 1640 complete medium containing 10 ng / ml each of human IL-2, IL-25, TSLP, and IL-33 cytokines for amplification. ILC2 cells were washed and then seeded at 30,000 cells / well with a certain amount of hIL-33 (concentration close to EC5). 90 (C) Inhibition of p38 MAPK phosphorylation in primary human ILC2 cells by anti-hIL-33 antibody in serially diluted 1:3 ratios or allotype control IgG4 antibody for 48 hours. The level of hIL-5 in the supernatant was measured (pg / ml) using a human IL-5 ELISA kit according to the manufacturer's instructions. ILC2 cells were seeded at 100,000 cells / well and treated with a certain amount of hIL-33 (concentration close to EC50). 90 The cells were treated with serially diluted anti-hIL-33 antibody at a 1:3 ratio for 15 minutes. p38 MAPK phosphorylation levels were determined using the AlphaLISA SureFire Ultra p-38 MAPK HV Detection Kit, following the manufacturer's instructions, and expressed as MFI (mean fluorescence intensity). (D) Inhibition of p38 MAPK phosphorylation in primary human ILC2 cells induced by hIL-33 or cyIL-33. The experimental procedure was the same as in (C), except that hIL-33 and cyIL-33 were used respectively to determine the cross-species blocking activity of antibody QB11465. IC50 50 The values ​​were calculated using GraphPadPrism software.

[0037] Figure 18 Anti-hTSLP antibody inhibited hTSLP-induced proliferation in BaF3 cells stably expressing hTSLPR / hIL7Rα. BaF3 cells stably expressing hTSLPR / hIL-7Rα were seeded at 10,000 cells / well and treated with a certain amount of hTSLP (concentration close to EC50). 50The cells were treated with serially diluted anti-hTSLP antibody or allotype control antibody for 72 hours. Proliferation (RLU) was measured using Promega's CellTiter-Glo chemiluminescent cell viability assay kit, according to the manufacturer's instructions. (A) Internally prepared hTSLP antigen QB11630 (SEQ ID NO: 32); (B) Internally prepared hTSLP antigen QB11631 (SEQ ID NO: 34); (C) Internally prepared cyTSLP antigen QB11632 (SEQ ID NO: 35); (D) Internally prepared cyTSLP QB11633 (SEQ ID NO: 37); (E) Initial hTSLP from healthy donor 1; (F) Initial hTSLP from healthy donor 2. Initial hTSLP was obtained from human small airway epithelial cells (SAEC) purchased from Lonza. The x-axis represents the antibody concentration (nM) used in the assay, and the y-axis represents the luminescence output in RLU (relative light units). IC50 50 The values ​​were calculated using GraphPad Prism software.

[0038] Figure 19 Inhibition of hTSLP-induced pSTAT5 in BaF3 cells stably expressing hTSLPR / hIL7Rα by anti-hTSLP antibody. BaF3 cells stably expressing hTSLPR / hIL-7Rα were seeded at 10,000 cells / well and treated with different forms of TSLP in combination with serially diluted anti-hTSLP antibody for 15 min. (A) Internally prepared hTSLP QB11630 (SEQ ID NO: 32); (B) Internally prepared hTSLP QB11631 (SEQ ID NO: 34); (C) Internally prepared cyTSLPQB11632 (SEQ ID NO: 35). The phosphorylation level of pSTAT5 was determined using the AlphaLISA SureFire Ultra p-STAT5 HV Detection Kit according to the manufacturer's instructions. The plate was read using a Perkin Elmer EnVision multi-label microplate reader. The x-axis represents the antibody concentration (nM) used in the assay, and the y-axis represents the phosphorylated pSTAT5 output in MFI (mean fluorescence intensity). 50 The values ​​were calculated using GraphPad Prism software.

[0039] Figure 20 Anti-hTSLP antibody inhibited hTSLP-induced CCL17 release. Purified human monocytes were seeded at 150,000 cells / well and treated with a constant amount of TSLP (concentration close to EC50). 50The supernatant was treated with serially diluted anti-TSLP antibody or allotype control antibody QB11571 for 24 hours. Then, the level of CCL17 chemokine in the supernatant was measured using the Human CCL17 / TARC DuoSet ELISA kit, following the manufacturer's instructions. (A) Internally prepared recombinant hTSLP QB11630, (B) Internally prepared hTSLP QB11631, (C) Internally prepared cyTSLP QB11632 were used for this assay. The x-axis represents the antibody concentration (nM) added in the assay, and the y-axis represents the CCL17 level (pg / ml) measured in the supernatant. IC50 50 The values ​​were calculated using GraphPadPrism software.

[0040] Figure 21 Anti-hTSLP antibody inhibited IL-5 secretion in hTSLP-induced human ILC2 cells. Purified human ILC2 cells were cultured in complete RPMI 1640 medium containing 10 ng / mL each of human IL-2, IL-25, TSLP, and IL-33 for expansion. ILC2 cells were washed and then seeded at 30,000 cells / well with antibiotics at concentrations close to EC5. 90 A fixed amount of internally prepared recombinant hTSLP QB11630 (A) or hTSLP QB11631 (B) was combined with serially diluted 1:4 anti-hTSLP antibody or allotype control antibody and treated for 48 hours. The level of hIL-5 in the supernatant was measured using the R&D Systems Human IL-5 DuoSet ELISA Kit (catalog number DY205) according to the manufacturer's instructions. The x-axis represents the antibody concentration added in the assay (nM), and the y-axis represents the IL-5 measured in the supernatant (pg / ml). IC50 50 The values ​​were calculated using GraphPad Prism software.

[0041] Figure 22 Inhibition of pSTAT5 in hTSLP-induced human ILC2 cells by anti-hTSLP antibody. Purified human ILC2 cells were cultured in complete RPMI 1640 medium containing 10 ng / mL each of human IL-2, IL-25, TSLP, and IL-33 for expansion. ILC2 cells were washed and then seeded at 30,000 cells / well with a fixed amount of internally prepared recombinant hTSLP QB11630 (A), hTSLP QB11631 (B), or cyTSLP QB11632 (C) (concentration close to EC50). 90The antibody was treated with serially diluted 1:4 anti-hTSLP antibody or allotype control antibody for 48 hours. The phosphorylation level of pSTAT5 was determined using the AlphaLISA SureFire Ultra p-STAT5HV assay kit, following the manufacturer's instructions. Plate readings were performed in a PerkinElmer EnVision multi-label microplate reader. The x-axis represents the antibody concentration (nM) used in the assay, and the y-axis represents the phosphorylated pSTAT5 output as MFI (mean fluorescence intensity). IC50 50 The values ​​were calculated using GraphPad Prism software.

[0042] Figure 23 The synergistic inhibition of IL-5 secretion by the combination of anti-hIL-33 IgG4 antibody QB11465 and anti-TSLP IgG1-D265A antibody QB11718 was investigated. Purified human ILC2 cells were cultured in complete RPMI 1640 medium containing 10 ng / mL each of human IL-2, IL-25, TSLP, and IL-33 for expansion. The expanded ILC2 cells were washed and then seeded at 30,000 cells / well, and treated with 5 ng / mL of recombinant hTSLP and hIL-33 in combination with 4x, 2x, 1x, 0.5x, or 0.25x IC50. 50 Treatment with different concentrations of anti-hTSLP antibody alone, anti-hIL-33 antibody alone, or a combination of anti-hTSLP antibody and anti-hIL-33 antibody for 48 hours. The level of hIL-5 in the supernatant was determined using the R&D Systems Human IL-5 DuoSet ELISA Kit (catalog number DY205) according to the manufacturer's instructions. (A) Synergistic effect of anti-hTSLP antibody QB11718 and anti-hIL-33 antibody QB11465. (B) Synergistic effect of anti-hTSLP reference antibody QB10985 and anti-hIL-33 antibody QB11465. (C) CI (combination index) calculated from (A) and (B).

[0043] Figure 24Lead anti-hIL-33 IgG4 antibody QB11465 significantly inhibited allergen-induced pulmonary eosinophilic inflammation and IL-4 production in hIL-33 knock-in mice. To induce airway inflammation, purified house dust mites (HDM) at a dose of 25 μg / 15 μL were administered intranasally to female C57BL / 6 hIL-33 knock-in mice under isoflurane anesthesia once daily for 5 days for a total of 6 weeks. Starting from day 22 of the study, mice in groups 3 and 4 were intraperitoneally injected twice weekly with lead anti-hIL-33 IgG4 antibody QB11465 (5 mg / kg) (group 3) or anti-hIL-33 reference-3 antibody QB11094 (5 mg / kg) (group 4) for 3 weeks. Group 1 did not receive HDM, and group 2 received HDM but did not receive anti-IL-33 antibody treatment. (A) Percentage of eosinophils in BALF in groups 1–4. (B) IL-4 levels (pg / ml) in BALF of mice in groups 1-4. A brief explanation of sequence listings Detailed description

[0044] This document provides antibodies that bind to human IL33 (hIL33), antibodies that bind to human TSLP (hTSLP), mixtures of these antibodies, polynucleotides encoding these antibodies and mixtures, host cells containing such polynucleotides, and therapeutic methods using these antibodies, mixtures, and polynucleotides. This document further describes a method for preparing said mixtures using a single host cell line that produces mixtures of these anti-hIL33 antibodies and anti-TSLP antibodies. The anti-hIL33 antibodies described herein bind to human and cynomolgus monkey IL33 and inhibit the interaction of hIL33 with hST2 and / or the ST2 / IL1AcP complex. The anti-hTSLP antibodies described herein inhibit the binding of hTSLP to hTSLPR (human thymic stromal lymphopoietin receptor) and / or block the interaction of hTSLP with hIL7Rα, and bind to human and cynomolgus monkey TSLP. Therefore, the invention of the anti-hTSLP antibodies provided herein can inhibit the formation of the hTSLP-hTSLPR complex or the hTSLP-hIL7Rα complex. Compared to either antibody alone, the mixture of anti-hIL33 and anti-hTSLP antibodies showed better efficacy in reducing the secretion of multiple downstream cytokines (i.e., IL-4, IL-5, and IL-13), which mediate inflammatory responses in eosinophils, B cells, mast cells, and others. Therefore, the invention of the mixture of anti-hIL33 and anti-hTSLP antibodies could treat or prevent inflammatory diseases such as asthma and COPD, while avoiding the side effects of long-term use of inhaled β-adrenergic agonists and inhaled corticosteroids. The invention of the mixture of anti-hIL33 and anti-hTSLP antibodies presented herein is also anticipated for the treatment of viral infections and cancer.

[0045] This article also provides methods for preparing the antibodies or antibody mixtures described herein using a single host cell line. This article further describes the polynucleotides encoding these antibodies and mixtures, the host cells containing such polynucleotides, and the therapeutic methods utilizing these antibodies, mixtures (including antibody mixtures), and polynucleotides. definition

[0046] As used herein, an "agonist" is a molecule that mimics or enhances the activity of a specific biologically active molecule or pathway. For example, when a cytokine binds to a protein expressed on the cell surface, that protein can mediate downstream effects of the molecule or pathway. When an agonist of that protein interacts with it, it can induce similar, larger, or smaller effects (compared to those induced by the cytokine), although the agonist may compete with or not compete with the cytokine for binding to the protein.

[0047] As used herein, a “change” refers to a alteration in an amino acid or nucleotide sequence. A change can be an insertion, deletion, or substitution. A “change” is the insertion, deletion, or substitution of a single amino acid or nucleotide. For example, if a deletion removes three amino acids or three nucleotides from an amino acid or nucleotide sequence, three changes occur (in this case, a deletion). An amino acid substitution change can be indicated by stating the amino acid present in the original sequence, then the position of that amino acid in the original sequence, and the amino acid that replaced the original amino acid. For example, G133M indicates that glycine, which was previously present at position 133 of the original sequence, has been replaced by methionine. Furthermore, 133M indicates that the amino acid at position 133 is methionine, but does not specify the identity of the original amino acid, which could be any amino acid, including methionine. Finally, G133 indicates that glycine is the amino acid at position 133 of the original sequence. Additionally, G133M / A indicates that glycine, which was previously present at position 133 of the original sequence, has been replaced by methionine or alanine.

[0048] As mentioned in this article, the "unfavorable factor for the alteration of heterodimers" is the third chain constant structural domain (C). H 3) Amino acid sequence (choose either human or primate C) H Substitution, insertion, or deletion of a single amino acid within a 3-amino acid sequence, wherein such substitution, insertion, or deletion, in the case of an antibody mixture, is detrimental to the formation of heterodimeric HC / HC pairs. An antibody may contain more than one detrimental alteration, and multiple detrimental alterations may occur at multiple sites in one or more antibodies within an antibody mixture. In some cases, a detrimental alteration alone may have little or no effect, but when one or more other detrimental alterations are present in the same antibody or in different antibodies within an antibody mixture, they can inhibit heterodimer formation. Alterations may include the substitution of residues present in the wild-type sequence with charged residues (which may be charged or uncharged). Alternatively, substitutions may create steric hindrance in the heterodimeric HC / HC pair, interfering with proper heavy chain / heavy chain (HC / HC) pairing, such as "protrusion" adjacent to another "protrusion" or "pore" adjacent to another "pore." Protrusions (or bumps) and pores are described in column 12, line 12 through column 13, line 2 of U.S. Patent 8,679,785, which is incorporated herein by reference. An example of a pair of alterations in the IgG heavy chain that can be detrimental to heterodimer formation is D399K / R and K409D / E.

[0049] As used herein, in the context of the antibodies of this invention, the phrase "inhibitory interaction" refers to an agent or "antagonist" that blocks or inhibits the activity or binding of a specific biologically active molecule. For example, a particular protein, when interacting with its binding partner, can activate a biological pathway with known downstream effects. The antibodies of this invention, as antagonists or inhibitors of such protein and / or its binding partner, reduce or eliminate those downstream effects, optionally by blocking or inhibiting the interaction between the protein and its binding partner. Exemplary antagonists are the anti-IL33 and anti-TSLP antibodies of this invention described herein.

[0050] As referred to in this article, "antibody" is an antibody containing at least one vitamin V. H or V L Proteins. Antibodies typically contain vitamin V. H and V L V H s and V L The term "antibody" is described in detail in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, Public Health Service, National Institutes of Health, NIH Publication No. 91-3242, 1991, pp. xvi-xix and 103-533, which are incorporated herein by reference. "Antibody" includes molecules in various forms, such as single-chain Fv (scFv) antibodies (which contain V antibodies linked by linkers). H and V L), Fab, F(ab')2, Fab' and scFv:Fc antibodies (as described in Carayannopoulos and Capra, FUNDAMENTAL IMMUNOLOGY, 3rd ed., edited by Paul, Raven Press, New York, 1993, Chapter 9, pp. 284-286, which are incorporated herein by reference), BiTE® antibodies, single-domain antibodies, bispecific antibodies, Fab-scFv, DVD-IgG, IgG(H)-scFv, nanobodies, nanobody-HSA, biantibodies, DART, TandAb, scDiabody, microantibodies, small antibodies, etc. (see, e.g., Spiess et al. (2015), Alternative molecular formats and therapeutic applications for bispecific antibodies, Molecular Immunology 67: 95-106), and IgG antibodies as defined below, as well as many other possible forms.

[0051] As used herein, a “bispecific antibody” is an antibody comprising at least one variable domain from a first antibody that binds to a first epitope or antigen and at least one variable domain from a second antibody that binds to a second epitope or antigen. In some cases, the first and second epitopes will be located on different molecules, optionally on different proteins. Thus, in some cases, the first and second antibodies will bind to different antigens. Bispecific antibodies can take many forms. For example, bispecific antibodies can be in many possible forms, such as bispecific T-cell adaptors (BiTE), dual-affinity heavy-targeting proteins (DART), biantibodies, tandem biantibodies (TandAb), or IgG antibodies. See, for example, Wang et al. (2019), Design and Production of Bispecific Antibodies, Antibodies 8, 43 (30 pages), which are incorporated herein by reference in their entirety; and Spiess et al. (2015), Alternative molecular formats and therapeutic applications for bispecific antibodies, Mol. Immunol. 67: 95-106, which are incorporated herein by reference in their entirety. Each of these exemplary forms contains V from two different antibodies that bind to different epitopes. H and V L Bispecific antibodies may contain components that promote V H s and VL Alterations in homologous pairing, referred to herein as couple-directed alterations, are discussed above and below. If a bispecific antibody is an IgG antibody composed of two heavy chains (each derived from two different antibodies) and two light chains (each derived from one of the two different antibodies), it may also contain alterations that promote heterodimeric HC / HC pairing formation. Many such alterations are known in the art.

[0052] As used herein, “cancer antigens” are molecules, optionally proteins, that are abundantly expressed on the surface of cancer cells. Cancer antigens are expressed at sufficiently high levels that they can be detected by typical immunohistochemistry (IHC). See, e.g., Parra et al. (2018), Appl. Immunohistochem. Mol. Morphol. 26(2): 83-93. Cancer antigens can be expressed at varying levels in different cancer cells and can also be expressed, at least to some extent, in normal cells. In some cases, cancer antigens are expressed only in cancer cells. For example, a rearranged form of the epidermal growth factor receptor (EGFR) called EGFRvIII is expressed in glioblastoma cells but not in normal cells. In another instance, carcinoembryonic antigen (CEA) is expressed in normal tissues during fetal development but not after birth. CEA is expressed in some cancer cells. Therefore, both EGFRvIII and CEA are cancer antigens as used herein. Other examples of cancer antigens include proteins encoded by genes including EGFR, V-ERB-B2 avian erythroblast leukemia virus oncogene homolog 2 (HER2), epithelial cell adhesion molecule (EpCAM), phosphatidylinositol proteoglycan 3 (GPC3), tumor necrosis factor receptor superfamily member 17 (TMFRSF17, referred to as BCMA in this paper), Claudin-18.2, CD20, and prostate-specific antigen (PSA).

[0053] As used herein, “charged” amino acids are acidic or basic amino acids that can carry a charge at near-physiological pH. These include the acidic amino acids glutamic acid (E) and aspartic acid (D), which are negatively charged at physiological pH, and the basic amino acids arginine (R) and lysine (K), which are positively charged at physiological pH. The weakly basic amino acid histidine can be partially charged at near-physiological pH and is not included in the definition of “charged” amino acids in this article. To avoid confusion, as used herein, a positive charge is considered to be “opposite” to a negative charge. Therefore, for example, the amino acids glutamic acid (E) and arginine (R) have opposite charges.

[0054] The "clearance" of antibodies in the body refers to the elimination of antibodies, which can be detected as the removal or reduction of antibody levels in the bloodstream or other tissues of mammals. Typically, to determine the clearance rate, antibodies are administered to mammals, followed by periodic sampling of the mammal's blood or tissues and quantitative testing for the presence of antibodies. From these tests, the in vivo half-life (T0) can be derived. 1 / 2 ) and / or the area under the curve (AUC) value. As indicated in this article, T 1 / 2 A decrease in AUC indicates increased clearance. An exemplary method for determining whether clearance of altered human IgG antibodies in mice is increased or decreased relative to unaltered antibodies includes the following steps: Unaltered and altered antibodies may be injected subcutaneously (e.g., subcutaneously in the shoulder area) into different mice. Approximately 0.1 mL of whole blood may be collected at each time point via retroorbital sinus puncture. The blood may be clotted and processed to obtain serum. The presence of human antibodies in the serum sample may be determined using an antibody specific to human Fc, such as a commercially available immunoassay system, such as one of those provided by Gyros US, Inc., Warren, NJ, USA. Blood samples may be collected at times such as 0, 0.5, 2, 8, 24, 72, 120, 168, 240, 312, 384, and 480 hours after injection. Pharmacokinetic parameters may be estimated from serum concentrations using software such as Phoenix.RTM. 6.3 (Pharsight, Sunnyvale, CA, USA).

[0055] Chemotherapy agents target dividing cells and interfere with processes associated with cell division, such as DNA replication, RNA synthesis, protein synthesis, the assembly, disassembly, or function of the mitotic spindle, and / or the synthesis or stability of molecules (such as nucleotides or amino acids) that play a role in these processes. Therefore, chemotherapeutic agents can kill cancer cells and other dividing cells. Chemotherapy agents are well known in the art. These include, for example, the following reagents: alkylating agents (e.g., busulfan, temozolomide, cyclophosphamide, lomustine (CCNU), streptozoline, methyllomustine, cis-diamminedichloroplatinum, thiotepa, and aziridinylbenzoquinone); inorganic ions (e.g., cisplatin and carboplatin); nitrogen mustards (e.g., melphalan hydrochloride, chlorambucil, ifosfamide, and nitrogen mustard hydrochloride); nitrosoureas (e.g., carmustine (BCNU)); antitumor antibiotics (e.g., doxorubicin, daunorubicin, styracin, doxorubicin, idarubicin, mitomycin C, and bleomycin); plant derivatives (e.g., vincristine, vinblastine, etc.). Diosmin, vinblastine, vinorelbine, paclitaxel, docetaxel, VP-16, and VM-26; antimetabolites (e.g., methotrexate (with or without leucovorin), 5-fluorouracil (with or without leucovorin), 5-fluorodeoxyuridine, 6-mercaptopurine, 6-thioguanine, gemcitabine, cytarabine, 5-azacytidine, hydroxyurea, deoxycofuran, and fludarabine); podophyllotoxins (e.g., etoposide, irinotecan, and topotecan); and actinomycin D, dacarbazine (DTIC), mAMSA, procarbazine, hexamethylmelamine, pentamethylmelamine, L-asparaginase, and mitoxantrone. See, for example, Cancer: Principles and Practice of Oncology, 4th ed., DeVita et al., eds., JB Lippincott Co., Philadelphia, Pa. (1993), relevant sections of which are incorporated herein by reference.

[0056] Other chemotherapeutic agents include those that act through the same general mechanisms as those listed above. For example, agents that act by alkylating DNA (such as alkylating agents and nitrogen mustards) are considered chemotherapeutic agents. Agents that interfere with nucleotide synthesis (such as methotrexate, cytarabine, 6-mercaptopurine, 5-fluorouracil, and gemcitabine) are considered chemotherapeutic agents. Mitotic spindle toxins are considered chemotherapeutic agents, such as paclitaxel and vincristine. Topoisomerase inhibitors (such as podophyllotoxins), which interfere with DNA replication, are considered chemotherapeutic agents. Antibiotics that interfere with DNA synthesis through multiple mechanisms (such as doxorubicin, bleomycin, and mitomycin) are considered chemotherapeutic agents. Agents that carbamylate amino acids (such as lomustine and carmustine) or deplete asparagine pools (such as L-asparaginase) are also considered chemotherapeutic agents. Merck Manual of Diagnosis and Therapy, 17th Edition, Part 11, Hematology and Oncology, 144. Principles of Cancer Therapy, Table 144-2 (1999). Chemotherapy agents specifically include those that directly affect the same cellular processes as those affected by the chemotherapeutic agents listed above.

[0057] In the context of antibody mixtures, a “homologous” HC, as used herein, is an HC to which a specific LC is known to pair to form a specific antigen-binding site. For example, if a full-length IgG antibody X is known to bind to antigen X, then antibody X HC is a homologous HC of antibody X LC, and vice versa. Furthermore, if the mixture also contains antibody Y that binds to antigen Y, then antibody Y HC is “non-homologous” relative to antibody X LC, and vice versa; and antibody Y LC is “non-homologous” relative to antibody X HC, and vice versa.

[0058] The "Complementarity Determinant Region" (CDR) is V H or V L The high-variability region within. Each V H and V L It contains three CDRs, called CDR1, CDR2, and CDR3. CDRs form a ring on the antibody surface and are primarily responsible for determining the antibody's binding specificity. CDRs are scattered among four more conserved framework regions (called FR1, FR2, FR3, and FR4), as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0059] The positions of the CDRs are indicated in Examples 2, 5, 6, and 7. VH CDRs are defined as follows: CDR1 is located at positions 31-35 (possible insertion numbers are 35a and 35b); CDR2 is located at positions 50-65 (possible insertion numbers are 52a-52c); and CDR3 is located at positions 95-102 (possible insertion numbers are 100A-100K). Kabat et al., ibid., page xvii, which is incorporated herein by reference. VL CDRs are defined as follows: CDR1 is located at positions 24-34 (possible insertion numbers are 27A-27F); CDR2 is located at positions 50-56; and CDR3 is located at positions 89-97 (possible insertion numbers are 95A-95F). These definitions of VH and VL CDRs are used herein.

[0060] Two treatments / medications are considered to be administered "in parallel" if they are administered within the same short period of time, such as on the same day, or within the same longer time frame. Such a longer time frame can include, for example, one treatment / medication administered weekly and the other every four days. Although the two treatments / medications may never or rarely be administered on the same day, they are administered continuously over a common period of weeks, months, or longer. Similarly, if one medication is administered annually and the other weekly, or if the weekly medication is administered before and / or for one year after the annual medication is administered, they are considered to be administered "in parallel." Therefore, as referred to herein, "in parallel" administration of two treatments / medications includes continuous treatment with two different treatments / medications over a common time frame.

[0061] As used herein, “conservative” amino acid substitution is the substitution of an amino acid with a different amino acid that has similar properties (such as similar polarity, hydrophobicity, or volume). Conservative substitution involves replacing an amino acid with another amino acid from the same group, which includes the following: (1) hydrophobic amino acids, including alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; (2) uncharged polar amino acids, including glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; (3) basic amino acids, including arginine, lysine, and histidine; and (4) acidic amino acids, including aspartic acid and glutamic acid. Conservative substitutions also include: (1) A replaced by V, L, or I; (2) R replaced by K, Q, or N; (3) N replaced by Q, H, K, or R; (4) D replaced by E; (5) C replaced by S or A; (6) Q replaced by N; (7) E replaced by D; (8) G replaced by P or A; (9) H replaced by N, Q, K, or R; (10) I replaced by L, V, M, A, or F; (11) L replaced by I, V, M, A, or F; (12) K replaced by R, Q, or N; (13) M replaced by L, F, or I; (14) F replaced by L, V, I, A, or Y; (15) P replaced by A; (16) S replaced by T, A, or G; (17) T replaced by S; (18) W replaced by Y or F; (19) Y replaced by W, F, T, or S; and (20) V is replaced by I, M, L, F, or A.

[0062] As referred to in this article, "cysteine ​​substitution" is an amino acid substitution in which cysteine ​​replaces another amino acid.

[0063] Two or more antibodies are "different" if the amino acid sequences of all polypeptide chains contained in an antibody are not "the same" as referred to herein.

[0064] Two amino acid sequences are considered "identical" as used herein if they can be encoded by the same DNA sequence. That is, amino acid sequences that differ only due to post-translational modifications (e.g., the removal of a carboxyl-terminal lysine or the cyclization of an N-terminal glutamate or glutamine residue) are considered "identical" as used herein.

[0065] Amino acid sequences are considered “different” as used herein if they have one or more amino acid substitutions, deletions, or insertions relative to each other. However, it should be noted that such “different” amino acid sequences are not considered different if the differences are caused solely by post-translational modifications, i.e., if the amino acid sequences can be encoded by the same DNA sequence.

[0066] As referred to herein, an amino acid sequence is “encoded by a nucleotide sequence,” which, given a known genetic code, can theoretically be encoded by that nucleotide sequence. As referred to herein, such a polypeptide chain does not need to be actually prepared from that nucleic acid to be “encoded” by that nucleotide sequence, and that nucleotide sequence does not need to contain all the auxiliary sequences required for transcription and / or translation termination and initiation to “encode” the amino acid sequence. As is known in the art, due to the degeneracy of the genetic code, a given amino acid sequence is “encoded” by a defined set of nucleic acid sequences. Furthermore, as stated above, an amino acid sequence “encoded” by a nucleotide sequence is still considered to be “encoded” by that nucleic acid sequence if its amino acid sequence is altered due to post-translational modifications (as referred to herein). Therefore, for example, if an amino acid sequence would be “encoded” by a nucleotide sequence, but only if the amino acids in that sequence are altered or deleted, and if such alteration or deletion can be proven to be due to post-translational modifications, then it is considered to be “encoded” by that nucleotide sequence. For example, recombinant humanized IgG antibodies produced from Chinese hamster ovary (CHO) cells often lack the C-terminal (C-terminal) lysine of the heavy chain, even if the nucleotide sequence encoding the antibody could encode a C-terminal lysine. This lysine is typically removed post-translational. This paper proposes that such antibodies are "encoded" by nucleotide sequences containing a C-terminal lysine.

[0067] As referred to in this article, the "Fc segment," "Fc region," or "Fc part" is basically composed of hinge structural domains (hinges) from the HC and second-chain constant structural domains (C). H 2) and C H It consists of 3 components, although in some isotypes (such as IgA or IgM) it may further include downstream regions of CH3.

[0068] As referred to in this article, "heavy chain (HC)" contains at least V. H C H 1. Hinges, C H 2 and C H 3. An HC containing all of these domains may also be referred to as a "full-length HC," or in some implementations as an "IgG HC." Certain isotypes such as IgA or IgM may contain additional sequences, such as, for example, IgM C. H 4. Structural Domains. The numbering system of Kabat et al. (ibid.) is used for V. H (see Figure 1 ), and the EU system (Edelman et al. (1969), Proc. Natl. Acad. Sci. USA 63: 78-85, which is incorporated herein by reference in its entirety) is used for C H 1. Hinges, C H 2 and C H3. The use of these well-known numbering systems can lead to discrepancies between the actual amino acid positions in the sequences disclosed herein and the numbers assigned to those positions using the Kabat or Edelman numbering systems. However, those skilled in the art can assign Kabat or Edelman numbers to any particular position in the disclosed antibody sequences by referring to their knowledge in the art and the tables disclosed below (which show how to assign Kabat or Edelman numbers with reference to conserved features of the antibody sequence that can be found in the disclosed sequences). Tables 1 and 2 below illustrate such numbering on universal HC sequences. Table 1: Person V H s common sequence

[0069] Table 1: This table shows the number of people V based on Kabat et al. (ibid.). H The amino acid sequence (subgroups I-III) is "invariant" (according to Kabat et al., ibid.). Numbering is based on Kabat et al. (ibid.). Site numbers within the CDR are written in bold italics. Positions followed by letters, such as 100A, may or may not be filled with amino acids, except for 82A-82C, due to the variable length of the CDR. Positions 82A-82C in the framework region are almost always filled with amino acids from subtypes I-III. H The amino acids occupy a specific position in the body. A single bolded amino acid at a specific position indicates a position in human V as described by Kabat et al. (ibid.). H The "invariant" amino acids in all three subtypes I-III of s. Unlabeled amino acid positions do not conform to this standard.

[0070] Table 1 shows that many conserved amino acids have conserved spacing, making any V... H The sequences can be visually compared with the conserved amino acids spaced as shown above. Alternatively, new sequences can be compared using alignment software with known V... H Sequence alignment, for example, the International ImMunoGeneTics (IMGT) Information System ®Alignment software available online (e.g., IMGT / DomainGapAlign, which is available at http: / / www.imgt.org or CLUSTAL Omega (Sievers et al., (2011), Fast, scalable generation of high-quality protein multiple sequencealignments using Clustal Omega, Molecular Systems Biology 7(1): 539).

[0071] Table 2 below shows the alignment of human IgG Fc regions for four human IgG subclasses: IgG1, IgG2, IgG3, and IgG4. This alignment demonstrates the differences between these subclasses and the high degree of sequence conservation. Table 2: Amino acid sequence of the Fc region of human IgG

[0072] "Human" nucleotide or amino acid sequences, proteins, or antibodies are those naturally occurring in the human body, or sequences, proteins, or antibodies that are equivalent to such sequences or proteins except for minor mutations or alterations as described below. Many human nucleotide and amino acid sequences are reported, for example, by Kabat et al. (ibid.), demonstrating the use of the term "human" in this art. As referred to herein, a "human" amino acid sequence or antibody may contain one or more insertions, deletions, or substitutions relative to a naturally occurring sequence, provided that the "human" amino acid sequence contains no more than 10 single amino acid insertions, deletions, and / or substitutions per 100 amino acids. Similarly, a human nucleotide sequence contains no more than 30 single nucleotide insertions, deletions, and / or substitutions per 300 nucleotides. In V H or V L In specific cases of amino acid sequences (or nucleotide sequences encoding such amino acid sequences), CDRs are expected to be highly variable, and in order to determine a specific V... H or V L Whether an amino acid sequence (or the nucleotide sequence that encodes it) is a "human" sequence, CDRs (or the nucleotides that encode them) are not considered part of the sequence.

[0073] As referred to in this article, a "heterodimer" is a protein dimer in which the two proteins in the dimer have different amino acid sequences. In the specific case of IgG antibodies containing heterodimeric HC / HC pairs, the two different HCs in the heterodimer pair have V proteins with different amino acid sequences. H Structural domain.

[0074] As referred to in this article, “humanized” antibodies are derived from non-human sources but engineered to be as human as possible, thus potentially reducing immunogenicity in humans while maintaining antibody stability and functional properties such as binding. Typically, this means that most or all of the frame regions of the constant and variable domains are human or near-human sequences, while the CDR is derived from a different organism. However, simply transplanting the CDR of a mouse antibody into a human frame does not produce antibodies with the desired properties and may require further modification. In recent years, various methods have been developed to simplify and improve humanization outcomes. See, for example, Choi et al. (2015), mAbs 7(6): 1045-1057 and the references cited therein. However, the results of modifications made to improve one or more properties of an antibody are not entirely predictable, primarily due to the high flexibility of the CDR 3 loop. See, for example, dos Santos et al. (2018), Advances and challenges in therapeutic monoclonal antibodies drug development. Braz. J. Pharm. Sci. 54(Special):e01007.

[0075] As referred to in this article, “IgG antibody” contains: (1) two HCs, each containing V H C H 1. Hinge structural domain, C H 2 and C H 3; and (2) two light chains (LC), each containing V L and LC constant structure domain (C L The heavy chain constant domains of IgG antibodies belong to IgG isotypes, such as IgG1, IgG2, IgG3, or IgG4 subclasses. These domains are described in, for example, Kabat et al. (ibid.), pp. xv-xix and 647-699, which are incorporated herein by reference. The numbering system used by Kabat et al. (ibid.) is for V H s and V L s (see Tables 1 and 2 in this paper). The EU system (Edelman et al. (1969), Proc. Natl. Acad. Sci. USA 63: 78-85, which is incorporated herein by reference in its entirety) is used for C L C H 1. Hinges, C H 2 and C H3. In some embodiments, certain portions of the constant domain of an IgG antibody may belong to one subclass, such as IgG4, while another portion of the same antibody may belong to another subclass, such as IgG1. Such antibodies are still IgG antibodies as referred to herein. Furthermore, as indicated herein, the amino acid sequence of the constant domain of an IgG antibody may deviate to a limited extent from the naturally occurring sequence without altering the antibody to a substance other than an IgG antibody. For example, an IgG antibody may contain C14 amino acid substitutions, deletions, or insertions relative to the naturally occurring IgG amino acid sequence, comprising no more than 24, 20, 16, 14, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions, deletions, or insertions. H 1 to C H 3 fragments. However, the IgG subclass of IgG antibodies can be found in C... H 1 to C H The length of the 3 fragments may vary or remain unchanged, such that, for example, a portion of the amino acid sequence of the fragment can be compared with, for example, an IgG1 antibody sequence, while another portion of the amino acid sequence can be compared with, for example, an IgG4 antibody sequence, to determine whether the antibody is the IgG antibody referred to herein.

[0076] As indicated herein, the "inhibition" of antibody against the interaction or binding of hIL33 with hST2 and / or the ST2 / IL1AcP complex can be achieved, as described in Example 13, using IFNγ cytokine secreted by primary human NK cells. Figure 17 A) IL-5 secretion in primary human ILC2 cells ( Figure 17 B) and the phosphorylation level of p38 MAPK in primary human ILC2 cells ( Figure 17 The measurements of C and 17D are used to determine this.

[0077] Similarly, the “inhibition” of the interaction or binding of hTSLP to hTSLPR (human thymic stromal lymphopoietin receptor), the interaction of hTSLP to hIL7Rα, and / or the interaction of hTSLP to the hTSLPR / hIL7Rα complex can be determined by the Biacore assay described in Example 8 and the cell-based assays described in Examples 14-18 herein.

[0078] As referred to in this article, "light chain (LC)" includes V L and C L C L It could be κ(C) L κ) or λ(C L λ) domains. These domains, including their exemplary amino acid sequences, are described in, for example, Kabat et al. (ibid.), pp. xiii-lix, 103-309 and 647-660, which are incorporated herein by reference. This article is for V LThe numbering system is the one described by Kabat et al. (ibid.) and is used for C L The EU numbering system is the one described in Edelman et al. (ibid.). Tables 3 and 4 below illustrate the application of these systems to various light chain sequences. Those skilled in the art can use this information to assign Kabat or Edelman numbers to specific positions in the sequences disclosed herein. Table 3: Person V L Common sequence of s

[0079] Table 3: Numbering is based on Kabat et al. (ibid.). Bold italic numbers indicate the positions of CDRs. The position number is followed by a letter, such as 27A. Since the length of a CDR is variable, it may or may not be filled with amino acids. All invariant residues of the human light chain in Kabat et al. (ibid.) are shown in bold letters, indicating the amino acid found at that position. At selected sites, one or two amino acids commonly found at that site are indicated in normal font. Furthermore, many other amino acids are present at κ or λ V. L Within certain subgroups, it is invariant or highly conserved, which can help classify specific amino acid sequences as V. L Select the site to be altered in PCT / US2018 / 089293 or PCT / US2017 / 030676. Bold underline Type representation. Table 4: C L Common sequence and number

[0080] Table 4: Numbering is based on Edelman et al. (ibid.), which, along with Kabat et al. (ibid.), is used for C. L The numbers are the same. The amino acids shown in bold below the numbers are based on κ and λ C from multiple species, according to Kabat et al. (ibid.). L The “unchanged” residues in the alignment. Conserved amino acids in the 10 human κ chains (above) and 28 human λ chains (below) reported by Kabat et al. (ibid.) are shown in normal font, as indicated by the selected sites (131, 160, 162, 174, 176, and 178). In cases where either of two different amino acids is found at one of these sites, the more common amino acid is shown before the less common amino acid, such as A / M. Bold underline The numbers represent altered sites reported in PCT / US2018 / 089293 or PCT / US2017 / 030676. In addition, many other amino acids are altered at C... L κ or C LCertain subgroups of the λ domain are invariant or highly conserved, which can help classify specific amino acid sequences as C. L The position of an unspecified amino acid is not fixed.

[0081] As used herein, “MabPair” or “MabPair mixture” refers to a pair of antibodies, i.e., two antibodies, produced in a culture of a single host cell line in which DNA encoding antibodies has been introduced. The host cell produces only two major antibody species. For a further description of how MabPairs are produced, refer to the description in US 11,130,808, Examples 1, 2, 3, 4, 5, 6, and 7, and the accompanying drawings described therein, all of which are incorporated herein by reference in their entirety for all purposes.

[0082] In the context of antibody mixtures, the “major species” antibody, as referred to herein, is a specific antibody that constitutes at least 10% of the total antibody count in the mixture. To determine the quantity of the major species in an antibody mixture, methods as described in Example 5 and as per US 11,130,808 can be employed. Figure 14 The low-pH cation exchange (CEX) chromatography method is shown in (part of US 11,130,808, which is incorporated herein by reference). This method is described by Chen et al. (2010), Protein Science, 19:1191-1204, which is incorporated herein by reference in its entirety. Briefly, the method utilizes a Waters Alliance 2695 high-performance liquid chromatography (HPLC) system with a Thermo ProPac™ WCX-10 weak CEX column (4 x 250 mm), previously protected by a 50 mm guard column (ProPac™ WCX-10G). Chromatography can be run over 30 minutes with a linear gradient from 100% buffer A (20 mM sodium acetate, pH 5.2) to 100% buffer B (20 mM sodium acetate with 250 mM sodium chloride, pH 5.2). The column can be washed with high salt (1 M sodium chloride) and reequilibrated to the starting conditions of buffer A. Antibodies in column effluent can be detected by absorbance at 214 nm. The relative amount of the detection peak can be determined using EMPOWER™ software (Waters Corp., Milford, MA, USA). Low pH CEX can distinguish different full-length antibody species and can be used to quantify the relative amount of a specific antibody species in a mixture.

[0083] As referred to in this article, “minor species” antibodies in an antibody mixture constitute less than 10% of the total antibody content in the mixture. This can be determined by low-pH CEX chromatography as described in the definition of “major species”.

[0084] The terms “nucleic acid” and “polynucleotide” are used interchangeably in this article, as are “nucleic acid sequence”, “nucleotide sequence”, or “polynucleotide sequence”.

[0085] As referred to in this article, "spousal-oriented change" is V H C H 1. V L Or C L The substitution, insertion, or deletion of a single amino acid at the HC / LC interface in an amino acid sequence, optionally replacing a naturally occurring amino acid with a charged amino acid or cysteine, leads to a stronger binding between HC and LC (optionally human and / or primate HC and LC). More specifically, "HC-pairing directional alteration" is V L Or C L The changes in V sometimes only occur in V. H Or C H When "LC pairing orientation alteration" is present at the "contact" residue in 1, it leads to stronger binding between HC and LC. Similarly, "LC pairing orientation alteration" is V H Or C H The changes in 1, which sometimes only occur in V L Or C L When a "HC-LC pairing orientation change" is present at the "contact" residue, it results in a stronger binding of HC and LC. In some embodiments, the HC-LC pairing orientation change of the contact pair can be a substitution of charged amino acids with opposite charges. In other embodiments, a charged amino acid is already present at one of the contact sites of HC or LC, so only one chain needs to be modified to create a pair of oppositely charged residues, i.e., a charge pair, at the contact site in the homologous HC / LC pair. In other embodiments, a cysteine ​​residue can be introduced at the contact site to form a disulfide bridge in the homologous HC / LC pair. In a further embodiment, the HC-LC pairing orientation change can be a substitution or pre-existing amino acid that creates a mortise (or protrusion and cavity) at the contact residue, as described in U.S. Patent 8,679,785, the relevant portion of which is incorporated herein by reference. HC can be an isoform of IgG, IgA, IgD, IgM, or IgE, optionally IgG1, IgG2, IgG3, or IgG4. The HC-LC pairing orientation change occurs at the contact amino acid site that forms part of the HC / LC interface. L and C H The interface residues in 1 include those within 4.5 Å, as described in U.S. Patent 8,592,562, Tables 4 and 5, and the texts appended in columns 10 and 11, all of which are incorporated herein by reference. H 1 and C L These positions are listed in Table 5 below. Table 5: C H 1 and C LContact residues between

[0086] In V H and V L In specific cases of contact residues at the interface between two residues, the following criteria can be used to select suitable residue pairs for modification, one in V H In the middle, one in V L In this context, the following residues are considered: (1) buried or partially buried, meaning they are difficult to access within the tertiary structure of the full-length antibody; (2) spatially close, meaning that, according to known structural models, the Cα (Cα being the central carbon atom of the amino acid to which the amino, carboxyl, and side chains are attached) of two amino acids are within approximately 12 Å, or the distance between a side chain heavy atom (any atom other than hydrogen) of one amino acid and any heavy atom of another amino acid is at most 5.5 Å; (3) highly conserved residues, although they do not necessarily have to be completely unchanged; and (4) residues not within the CDR or not interacting with the CDR. Examples of such contacting residues include, but are not limited to, the following: position 44 (V H ) and the 100th (V L ); 39th (V) H ) and the 38th (V L ); and the 105th (V) H ) and the 43rd (V L ).

[0087] Roughly speaking, changes in HC / LC binding strength due to HC and / or LC mating orientation can be determined by the "chain loss" experiment described in Example 11 of US 11,124,470 and the accompanying drawings mentioned therein, and Example 3 of US 11,130,808 and the accompanying drawings mentioned therein, all of which are incorporated herein by reference for all purposes.

[0088] To confirm, or in some cases clarify, the results of chain loss experiments can be obtained through methods such as those described in Example 12 of this document. Figure 24 Thompson et al. (2014), mAbs 6:1, 197-203 (which is incorporated herein in its entirety) and US 11,130,808 Figure 15The size of the Fab fragment generated in transfections containing DNA encoding HC and LC of the first antibody (Mab1) and HC and LC of the second antibody (Mab2) was determined by mass spectrometry as described in Example 5 (which is incorporated herein by reference). In most cases, such techniques can be used to distinguish homologous and non-homologous pairs by mass. If non-homologous pairs are the predominant species in cells transfected with DNA encoding unaltered Mab1 HC and LC and unaltered Mab2 HC and LC, and not the predominant species in cells transfected with DNA encoding Mab1 HC and LC and Mab2 HC and LC (where at least one of the said antibodies contains a mating-oriented alteration), then at least one alteration is considered a favorable mating-oriented alteration.

[0089] Examples of mate orientation changes include partial or complete changes to any of the following charge pairs: 44D / E (V H ) and 100R / K (V L ); 44R / K(V H ) and 100D / E (V L ); 105R / K(V H ) and 43D / E(V L ); 105D / E(V H ) and 43R / K (V L ); 147D / E(C H 1) and 131R / K(C L ); 147R / K(C H 1) and 131D / E(C L ); 168D / E(C H 1) and 174R / K(C L ); 168R / K(C H 1) and 174D / E(C L ); 181R / K(C H 1) and 178E / D(C L ); and 181E / D(C H 1) and 178R / K(C L Furthermore, mate-directed alterations include replacing one amino acid with a cysteine ​​residue, resulting in a cysteine ​​pair substitution in both antibody HC and LC, such as any of the following pairs: 126C(C H 1) and 121C(C L ); 126C(C H 1) and 124C (C L ); 127C(C H 1) and 121C(C L ); 128C(C H 1) and 118C(CL ); 133C(C H 1) and 117C(C L ); 133C(C H 1) and 209C(C L ); 123C(C H 1) and 116C(C L ); 141C(C H 1) and 116C(C L ); 168C(C H 1) and 174C(C L ); 170C (C H 1) and 162C(C L ); 183C(C H 1) and 176C(C L ); 173C(C H 1) and 160C (C L ); 170C (C H 1) and 176C(C L ); and 173C(C H 1) and 162C(C L ).

[0090] "Primate" nucleotide or amino acid sequences or proteins are sequences or proteins that are naturally occurring in nucleic acids or proteins found in primates, or sequences or proteins that are equivalent to such sequences or proteins except for minor alterations described below. Primates include animals from many families, including but not limited to the suborder Prosimians (including lemurs), New World monkeys, chimpanzees, humans, gorillas, orangutans, gibbons, and Old World monkeys. Specific primate species include, but are not limited to, Homo sapiens, rhesus macaques (Macaca mulata), cynomolgus monkeys (Macaca fascicularis), and chimpanzees (Pan troglodytes). Many primate nucleotide and amino acid sequences are known in the art, such as those reported by Kabat et al. (ibid.). Generally, a "primate" amino acid sequence as referred to herein may contain one or more insertions, deletions, or substitutions relative to naturally occurring primate sequences, provided that the "primate" amino acid sequence contains no more than 10 single amino acid insertions, deletions, and / or substitutions per 100 amino acids. Similarly, primate nucleotide sequences contain no more than 30 single nucleotide insertions, deletions, and / or substitutions per 300 nucleotides relative to naturally occurring primate sequences. In V H or V L In specific cases of sequences, CDR is expected to be highly variable, and in order to determine a specific V H or V LWhether an amino acid sequence (or the nucleotide sequence that encodes it) is a "primate" sequence, the CDR (or the nucleotide that encodes it) is not considered part of the sequence.

[0091] As referred to in this article, a "signal peptide" is an amino acid sequence on a protein, often the N-terminal sequence, that, together with signal recognition particles, targets the protein to the endoplasmic reticulum (and possibly the cell surface) in eukaryotes or the plasma membrane in prokaryotes. See, for example, Hegde and Bernstein (2006), Trends in Biochemica. L Sciences 31(6): 563-571. Although the primary sequence of a signal peptide is somewhat variable, a variety of signal peptides are known in the art. N-terminal signal peptides are typically cleaved and removed during the maturation of the protein.

[0092] As used herein, a “targeted biologic” is a protein that can affect a particular aspect of the biological state of a cell through its interaction with another specific molecule, which may be a protein. For example, a “targeted biologic” can affect a cell’s survival, proliferation, ability to produce specific cytokines or proteins, etc. For example, the anti-hIL33 and anti-TSLP antibodies described herein are “targeted biologics” because they interact with hIL33 and hTSLP, which causes many of the biological effects described in the examples herein.

[0093] Similarly, as referred to in this article, a "targeted inhibitor" is a small molecule that can affect a specific aspect of cellular biological state through its interaction with a particular cellular molecule (which may be a protein). For example, a "tyrosine kinase inhibitor" is a small molecule that affects the activity of tyrosine kinases (which can affect a variety of cellular functions) through its interaction with tyrosine kinases.

[0094] As referred to herein, “treatment” for a specific disease or condition refers to a series of actions that may include the administration of one or more antibodies, polynucleotides encoding one or more antibodies, and / or one or more other molecules that result in the reduction or interruption of one or more symptoms of the disease or condition in a human patient, an animal model system believed to reflect the disease or condition, or an in vitro cell-based assay believed to reflect the disease or condition. This can be determined by objectively measuring the symptoms in a human or animal, or by measuring various parameters in a cell-based assay, such as the production of one or more cytokines (e.g., IFNγ), cell proliferation, cell death, etc. For example, “treatment” for cancer may result in a reduction in tumor volume, the absence of expected tumor metastasis in a human or animal model system, increased survival time, or increased progression-free or disease-free survival in a human or animal with cancer. Cancer treatment may also result in an increase in indicators of activation of certain aspects of the immune system in cell-based assays, such as macrophage phagocytosis of cancer cells, T cell proliferation, and / or an increase in cytokines (e.g., type I IFN, IFNγ, and / or IL-2) produced by one or more cell types that function in the immune system. Anti-IL33 antibody

[0095] On the one hand, this document provides variable domains of anti-IL-33 monoclonal antibodies (Mabs) with unique amino acid sequences, including those listed in the sequence listing. As illustrated in the examples below, these monoclonal antibodies (Mabs) can bind to the antigen encoded by the human and cynomolgus monkey IL-33 allele, namely the IL-33 protein, and can inhibit the interaction of human IL-33 with the ST2 / IL1AcP complex. Interleukin 33 (IL-33) is a member of the IL-1 cytokine family. IL-33 binding to the IL-33 receptor (composed of ST2 and IL-1RAcP) promotes a pro-inflammatory response. IL-33 is secreted in a full-length 270-amino-acid precursor form, which can be cleaved by proteases secreted by mast cells. In specific embodiments, the anti-IL-33 antibodies provided herein inhibit the interaction of IL-33 with ST2 and / or block the binding of IL-33 to ST2.

[0096] On the one hand, these antibodies can be, for example, human, humanized, or primate IgG antibodies, which can be IgG1, IgG2, IgG3, or IgG4 antibodies. On the other hand, the antibody is a human or humanized IgG1 antibody.

[0097] On one hand, the VH of the anti-hIL33 antibody contains VH CDR1, VH CDR2, and VH CDR3, which contain the amino acid sequences SEQ ID NO:80 (CDR1), SEQ ID NO:81 (CDR2), and SEQ ID NO:82 (CDR3). Antibodies containing VH (which includes any one of these CDR sequence groups) can inhibit the interaction between human IL-33 and the ST2 / IL1AcP complex.

[0098] Furthermore, the VH of the anti-hIL33 antibody may contain an amino acid sequence of any one of SEQ ID NO: 29, 22, 6, 10, 14, or 18; or may contain slightly modified versions of these sequences. For example, the VH may contain one or more pairwise directional alterations, which may be amino acid substitutions relative to any one of SEQ ID NO: 29, 22, 6, 10, 14, and / or 18. In some embodiments, the VH may contain no more than 6, 5, 4, 3, 2, or 1 amino acid alterations relative to any one of SEQ ID NO: 29, 22, 6, 10, 14, or 18. These amino acid alterations may be substitutions and / or pairwise directional alterations. In a further embodiment, such alterations occur only in the frame region and not in the CDR. A VH containing such altered sequences may be part of an antibody capable of inhibiting the interaction of human IL-33 with the ST2 / IL1AcP complex.

[0099] Similarly, the VL of an anti-hIL33 antibody may comprise VL CDR1, VL CDR2, and VL CDR3, which contain the amino acid sequences of SEQ ID NO:77 (CDR1), SEQ ID NO:78 (CDR2), and SEQ ID NO:79 (CDR3). Antibodies containing a VH (which comprises any one of these CDR sequence groups) can inhibit the interaction between human IL-33 and the ST2 / IL1AcP complex.

[0100] Furthermore, the VL of the anti-hIL33 antibody may contain the amino acid sequence of any one of SEQ ID NO:25, 20, 4, 8, 12, and 16, or may contain slightly modified versions of these sequences. For example, the VL may contain one or more pairwise directional alterations, which may be amino acid substitutions relative to any one of SEQ ID NO:25, 20, 4, 8, 12, and / or 16. In some embodiments, the VL may contain no more than 6, 5, 4, 3, 2, or 1 amino acid alterations relative to any one of SEQ ID NO:25, 20, 4, 8, 12, and 16. These amino acid alterations may be substitutions and / or pairwise directional alterations as described in U.S. Patent No. 11,124,570, etc. A VL containing such altered sequences may be part of an antibody capable of inhibiting the interaction of human IL-33 with the ST2 / IL1AcP complex.

[0101] On the other hand, anti-hIL33 antibodies may comprise VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences SEQ ID NO: 80, 81, 82, 77, 78, and 79. Antibodies containing such CDR sequence sets can inhibit the interaction between human IL-33 and the ST2 / IL1AcP complex.

[0102] On the other hand, anti-hIL33 antibodies VH and VL may each comprise an amino acid sequence from any of the following two sets of amino acid sequences: SEQ ID NO: 29 (VH) and 25 (VL); SEQ ID NO: 22 (VH) and 20 (VL); SEQ ID NO: 6 (VH) and 4 (VL); SEQ ID NO: 10 (VH) and 8 (VL); SEQ ID NO: 14 (VH) and 12 (VL); and SEQ ID NO: 18 (VH) and 16 (VL). In some embodiments, VH and VL may comprise slightly modified versions of one of these two sets of sequences. For example, VH may comprise one or more pairwise directional changes, which may be amino acid substitutions relative to one sequence in one of the two sets of sequences, and VL may comprise one or more pairwise directional changes, which may be amino acid substitutions relative to the other sequence in the same set of two sequences. The VH and VL that form an antibody or a portion thereof may each contain an amino acid sequence in which the first (VH) and second (VL) amino acid sequences relative to the two sequences contain no more than 6, 5, 4, 3, 2, or 1 amino acid alterations (optionally substitutions). These alterations may be couple-directed alterations. VH and VL containing such altered sequences may be part of an antibody capable of inhibiting the interaction of human IL-33 with the ST2 / IL1AcP complex.

[0103] In one specific embodiment, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VLCDR2, and VL CDR3 of the anti-hIL33 antibody respectively comprise the amino acid sequences of SEQ ID NO: 80, 81, 82, 77, 78, and 79; VH comprises no more than four altered amino acid sequences relative to the amino acid sequence of SEQ ID NO: 29, and VL comprises no more than four altered amino acid sequences relative to the amino acid sequence of SEQ ID NO: 25. In a more specific embodiment of the anti-hIL33 antibody of the present invention, HC comprises no more than four, three, two, or one altered amino acid sequences relative to the amino acid sequences of SEQ ID NO: 7, 11, 15, 19, 23, or 31, and LC comprises no more than four, three, two, or one altered amino acid sequences relative to the amino acid sequences of SEQ ID NO: 5, 9, 13, 17, 21, or 27. In yet another more specific embodiment of the anti-hIL33 antibody of the present invention, HC comprises the amino acid sequence of SEQ ID NO:31, and LC comprises the amino acid sequence of SEQ ID NO:27.

[0104] In other respects, the anti-hIL33 antibody may be part of a bispecific antibody that binds hIL33 and another antigen. It is also contemplated that the anti-hIL33 antibody of the present invention may be in different forms, such as scFv, scFv-Fc, BiTE, etc. ® Single-domain antibodies, bispecific antibodies, Fab-scFv, DVD-IgG, IgG(H)-scFv, nanobodies, nanobody-HAS, biantibodies, DART, TandAb, scDiabody, microantibodies, small antibodies, etc. See, for example, Spiess et al. (2015), Molecular Immunology 67:95-106. Anti-TSLP antibody

[0105] On the one hand, this article provides variable domains of anti-TSLP monoclonal antibodies (Mabs) with unique amino acid sequences, including those sequences shown in the sequence listing. As illustrated in the examples below, these monoclonal antibodies (Mabs) can bind to antigens encoded by the human and cynomolgus monkey TSLP alleles, namely the TSLP protein, and can inhibit the interaction (e.g., binding) of hTSLP with hTSLPR or hTSLP with hIL7Rα; and / or the interaction of hTSLP with the hTSLPR / hIL7Rα complex. Mature human TSLP (NCBI accession number: NP_149024.1) is a polypeptide composed of 131 amino acids. It is well known that 36NNT38 and 91NAT93 are N-glycosylated when the antigen is produced from mammalian cells.

[0106] On the one hand, these antibodies can be, for example, human, humanized, or primate IgG antibodies, which can be IgG1, IgG2, IgG3, or IgG4 antibodies. On the other hand, the antibody is a human or humanized IgG1 antibody.

[0107] On one hand, the VH of the anti-TSLP antibody contains VH CDR1, VH CDR2, and VH CDR3, which contain the amino acid sequences SEQ ID NO: 86 or 94 (CDR1), SEQ ID NO: 87 or 95 (CDR2), and SEQ ID NO: 88 or 96 (CDR3). Therefore, the anti-hTSLP antibody VH CDR1, VL CDR2, and VL CDR3 may contain the amino acid sequences SEQ ID NO: 86, 87, and 88; or SEQ ID NO: 94, 95, and 96, respectively. Antibodies containing VH (which contains any of these CDR sequence groups) can inhibit the interaction between hTSLP and hTSLPR, or the interaction between hTSLP and hIL-7Rα; and / or inhibit the interaction between hTSLP and the hTSLPR / hIL-7Rα complex.

[0108] Furthermore, the VH of the anti-hTSLP antibody may contain an amino acid sequence of any one of SEQ ID NO: 70, 65, 50, 43, or 39; or may contain slightly modified versions of these sequences. For example, the VH may contain one or more coupler-directed alterations, which may be amino acid substitutions relative to any one of SEQ ID NO: 70, 65, 50, 43, and / or 39. In some embodiments, the VH may contain no more than 6, 5, 4, 3, 2, or 1 amino acid alterations relative to any one of SEQ ID NO: 70, 65, 50, 43, or 39. These amino acid alterations may be substitutions and / or coupler-directed alterations. In further embodiments, such alterations occur only in the frame region and not in the CDRs. The VH containing such altered sequences may be part of an antibody that inhibits the interaction of hTSLP with hTSLPR or hTSLP with hIL7Rα; and / or the interaction of hTSLP with the hTSLPR / hIL7Rα complex.

[0109] Similarly, the VL of an anti-hTSLP antibody may comprise VL CDR1, VL CDR2, and VL CDR3, which contain the amino acid sequences of SEQ ID NO: 83 or 91 (CDR1), SEQ ID NO: 84, 89, or 92 (CDR2), and SEQ ID NO: 85, 90, or 93 (CDR3). Therefore, the anti-hTSLP antibody VL CDR1, VL CDR2, and VL CDR3 may contain the amino acid sequences of SEQ ID NO: 83, 84, and 85; SEQ ID NO: 83, 89, and 90; or SEQ ID NO: 91, 92, and 93, respectively. An antibody containing a VH (which comprises any of these CDR sequence groups) can inhibit the interaction of hTSLP with hTSLPR or hTSLP with hIL7Rα; and / or inhibit the interaction of hTSLP with the hTSLPR / hIL7Rα complex.

[0110] Furthermore, the VL of the anti-hTSLP antibody may contain the amino acid sequence of any one of SEQ ID NO: 74, 67, 54, 47, 45, and 41, or may contain slightly modified versions of these sequences. For example, the VL may contain one or more pairwise directional alterations, which may be amino acid substitutions relative to any one of SEQ ID NO: 74, 67, 54, 47, 45, and / or 41. In some embodiments, the VL may contain no more than 6, 5, 4, 3, 2, or 1 amino acid alteration relative to any one of SEQ ID NO: 74, 67, 54, 47, 45, and 41. These amino acid alterations may be substitutions and / or pairwise directional alterations as described in U.S. Patent No. 11,124,570, etc. A VL containing such altered sequences may be part of an antibody capable of inhibiting the interaction of human TSLP with the TSLPR / IL7Rα complex.

[0111] On the other hand, anti-hTSLP antibodies may comprise VH CDR1, VHCDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NO: 86, 87, 88, 83, 84, and 85; SEQ ID NO: 86, 87, 88, 83, 89, and 90; or SEQ ID NO: 94, 95, 96, 91, 92, and 93. Antibodies containing such CDR sequence sets can inhibit the interaction of hTSLP with hTSLPR or hTSLP with hIL7Rα; and / or the interaction of hTSLP with the hTSLPR / hIL7Rα complex.

[0112] On the other hand, anti-hTSLP antibodies VH and VL may each comprise an amino acid sequence from any of the following two sets of amino acid sequences: SEQ ID NO: 70 (VH) and 74 (VL); SEQ ID NO: 65 (VH) and 67 (VL); SEQ ID NO: 50 (VH) and 54 (VL); SEQ ID NO: 43 (VH) and 47 (VL); SEQ ID NO: 43 (VH) and 45 (VL); and SEQ ID NO: 39 (VH) and 41 (VL). In some embodiments, VH and VL may comprise slightly modified versions of one of these two sets of sequences. For example, VH may comprise one or more pairwise directional changes, which may be amino acid substitutions relative to one sequence in one set of the two sets of sequences, and VL may comprise one or more pairwise directional changes, which may be amino acid substitutions relative to the other sequence in the same set of two sequences. The VH and VL forming an antibody or a portion thereof may each contain an amino acid sequence in which the first (VH) and second (VL) amino acid sequences relative to the two sequences contain no more than 6, 5, 4, 3, 2, or 1 amino acid alterations (optionally substitutions). These alterations may be couple-directed alterations. VH and VL containing such altered sequences may be part of an antibody capable of inhibiting the interaction of hTSLP with hTSLPR or hTSLP with hIL7Rα; and / or capable of inhibiting the interaction of hTSLP with the hTSLPR / hIL7Rα complex.

[0113] In one specific embodiment, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VLCDR2, and VL CDR3 of the anti-hTSLP antibody respectively comprise the amino acid sequences of SEQ ID NO: 94, 95, 96, 91, 92, and 93; VH comprises no more than four altered amino acid sequences relative to the amino acid sequence of SEQ ID NO: 70, and VL comprises no more than four altered amino acid sequences relative to the amino acid sequence of SEQ ID NO: 74. In a more specific embodiment of the anti-hTSLP antibody of the present invention, HC comprises no more than four, three, two, or one altered amino acid sequences relative to the amino acid sequence of SEQ ID NO: 44, 52, 58, 62, 66, or 72, and LC comprises no more than four, three, two, or one altered amino acid sequences relative to the amino acid sequence of SEQ ID NO: 46, 48, 56, 60, 64, 68, or 76. In yet another more specific embodiment of the anti-hTSLP antibody of the present invention, HC comprises the amino acid sequence of SEQ ID NO:72, and LC comprises the amino acid sequence of SEQ ID NO:76.

[0114] In other respects, the anti-hTSLP antibody may be part of a bispecific antibody that binds hTSLP and another antigen. It is also contemplated that the anti-hTSLP antibody of the present invention may be in different forms, such as scFv, scFv-Fc, BiTE. ® Single-domain antibodies, bispecific antibodies, Fab-scFv, DVD-IgG, IgG(H)-scFv, nanobodies, nanobody-HAS, biantibodies, DART, TandAb, scDiabody, microantibodies, small antibodies, etc. See, for example, Spiess et al. (2015), Molecular Immunology 67:95-106. Other aspects of anti-hIL33 and / or anti-hTSLP antibodies

[0115] The anti-hIL33 and anti-hTSLP antibodies described herein can be human, humanized, or primate antibodies and / or IgG antibodies, such as IgG1, IgG2, IgG3, or IgG4 antibodies. Such IgG antibodies may contain mating orientation alterations, such as HC and / or LC mating orientation alterations. Furthermore, such IgG antibodies may contain one or more alterations that are unfavorable to heterodimerization. Such alterations can increase the likelihood that a single host cell line incorporating DNA encoding at least two different IgG antibodies will produce no more than two or three different major antibody classes.

[0116] Pair orientation alterations can form part of a charge pair or contact cysteine ​​pair within IgG anti-hIL33 and / or anti-hTSLP antibodies (optionally human or primate IgG antibodies) as described herein. For example, pairwise orientation changes include changes that produce, in whole or in part, any of the following charge pairs (including amino acid substitutions): 44D / E (VH) and 100R / K (VL); 44R / K (VH) and 100D / E (VL); 105R / K (VH) and 43D / E (VL); 105D / E (VH) and 43R / K (VL); 147D / E (CH1) and 131R / K (CL); 147R / K (CH1) and 131D / E (CL); 168D / E (CH1) and 174R / K (CL); 168R / K (CH1) and 174D / E (CL); 181R / K (CH1) and 178E / D (CL); and 181E / D (CH1) and 178R / K (CL). If a charged amino acid is already present at one of these sites, only one pairing orientation change is required to generate a charge pair. In other cases, two pairing orientation changes (one in HC and one in LC) will be required to generate a charge pair. Furthermore, pairing orientation changes include substitutions in which a cysteine ​​replaces another amino acid to generate a contact cysteine ​​pair, which can form a disulfide bridge. In human IgG1 antibodies, these may include the following pairs: 126 (CH1) and 121 (CL), 170C (CH1) and 162C (CL), 170 (CH1) and 176 (CL), 173 (CH1) and 160 (CL), and 183 (CH1) and 176 (CL). In human IgG4 antibodies, these may include the following pairs: 170C (CH1) and 162C (CL), 173C (CH1) and 162C (CL), and 183 (CH1) and 176 (CL). In an antibody mixture, the contact cysteine ​​pair in the homologous CH1 / CL pair of one antibody may be located at a different position than that of the other antibody in the mixture, which may increase the selectivity for the formation of homologous HC / LC pairs. US11,130,808, including those portions describing the alteration of pair orientation, including Examples 1-3 and 5 and their referenced figures, is incorporated herein by reference for all purposes.

[0117] DNA encoding HC and / or LC containing mating-directed alterations can be prepared using known methods. Such methods are described in US 11,130,808 and include, for example, the artificial synthesis of DNA sequences (e.g., from commercial suppliers such as Integrated DNA Technologies, Coralville, Iowa, USA, or Genewiz, South Plainfield, NJ, USA) and the ligation of DNA fragments via a Gibson reaction (i.e., overlap PCR), as described in the Gibson Assembly® Master Mix Instruction Manual, New England Biolabs Inc. (NEB), Version 3.3, NEB Catalog No. #E2611S / L, NEB Inc., Ipswich, MA, USA. The design, preparation, and testing of mating-directed alterations are described in detail in US 11,130,808. Examples 1-3 and 5 of US 11,130,808 and... Figure 4-7 Articles 12-15 are incorporated herein by reference. Once the DNA encoding the antibody has been prepared, the antibody can be prepared by transfecting host cells as described in the examples herein.

[0118] In a further embodiment, the anti-hIL33 and / or anti-hTSLP antibodies as described herein may include one or more modifications that are detrimental to HC / HC heterodimer formation. In a further embodiment, the anti-hIL33 and / or anti-hTSLP antibodies as described herein may include one or more modifications that reduce one or more aspects of antibody effector function. Examples of such alterations include: (1) D265A or D265X in IgG antibody HC (where X is any amino acid other than D); (2) E318X, K320X and / or K322X in IgG2 antibody (where X is any amino acid other than the original amino acid); (3) D270X, K322X, P329X and / or P331X in IgG1 antibody (where X is any amino acid other than the original amino acid); (4) P329A; (5) L234A, L235A and / or P329A in IgG1 antibody; and / or (6) L234A, L235E and G237A in the constant region of IgG1. A mixture of anti-HIL33 and anti-HTSLP antibodies

[0119] This document provides antibody mixtures comprising the anti-hIL33 and anti-hTSLP antibodies described herein. In some embodiments, such antibody mixtures are prepared in a single host cell line having been introduced with DNA encoding one or more of the two antibodies. This method for preparing antibody pairs is described in detail in WO 2017 / 205014 (corresponding to US 11,130,808), as in Examples 1-12, pages 68-103, and the accompanying drawings mentioned therein. WO 2017 / 205014 and US 11,130,808 are incorporated herein by reference in their entirety for all purposes. Mixtures of the two antibodies prepared using these methods are referred to herein as MabPairs.

[0120] More specifically, these mixtures may contain any of the anti-hTSLP antibodies described above. In some embodiments, the anti-hTSLP antibody contains D399R and K409E, as well as other modifications, in its HC. For example, as described in Example 9, a set of substitutions K147D, V173C, C220G, D265A, D399R, and K409E were introduced into the HC of anti-hTSLP antibody QB11237; while substitutions S131K, S162C, and C214S were introduced into the LC of anti-hTSLP antibody QB11237, and the new anti-hTSLP antibody with these substitutions was renamed clone QB11718. SEQ ID NO: 72 and 71 show the amino acid sequence of anti-hTSLP QB11718 HC and the nucleic acid sequence encoding it, respectively. SEQ ID NO: 76 and 75 show the amino acid sequence of anti-hTSLP QB11718 LC and the nucleic acid sequence encoding it, respectively. In a particular embodiment, the mixture may further comprise any anti-hIL33 antibody described herein, such as QB11465, which is converted from anti-hIL-33 IgG1 antibody QB11421 to IgG4 antibody (SEQ ID NO: 25 and 27), for co-expression with a lead anti-TSLP IgG1 antibody as MabPair.

[0121] Examples of the preparation of other MabPair mixtures are described in Example 9, and include modified versions of the anti-hTSLP antibody and the unchanged anti-hIL-33 antibody prepared as follows. Substitutions K147D, V173C, and C220G were introduced into the HC of anti-hTSLP antibody QB11548 to enhance homologous chain pairing in CH1; substitution D265A was introduced to weaken effector functions (ADCC, ADCP, CDC) in CH2; and substitutions D399R and K409E were introduced to prevent the formation of heterodimer HC in CH3. Therefore, substitutions S131K, S162C, and C214S were introduced into the LC of anti-hTSLP antibody QB11548, and the modified anti-hTSLP antibody was renamed clone QB11764. SEQ ID NO: 52 and 51 show the amino acid sequence of anti-hTSLP QB11764 HC and the nucleic acid sequence encoding it, respectively. SEQ ID NO:56 and 55 show the amino acid sequence of anti-hTSLP QB11764 LC and the nucleic acid sequence encoding it, respectively.

[0122] Other MabPairs containing anti-hIL33 and anti-hTSLP antibodies with other alterations to the HC and LC sequences described herein (as described in US 11,130,808) are also included in the antibody mixtures provided herein. Furthermore, in some embodiments, the anti-hTSLP IgG1 antibody may contain 147D, 170C, 173C, 220G, 399R, and 409E in its HC and 131K, 160C, 162C, and 214S in its LC, while the anti-hIL33 IgG4 antibody has a native 409R in its HC.

[0123] Table 6 below lists exemplary mate orientation changes, where one or more changes may be included in the anti-hIL33 and / or anti-hTSLP antibodies in the antibody mixture. Table 6: Exemplary Spouse Orientation Changes *Antibody 1 and Antibody 2 are different antibodies. For the purposes of this table, they are interchangeable. #Changes to the heavy and light chains (e.g., HC1 and LC1) of a single primary antibody listed in the same row may be present simultaneously as listed in the table. However, the secondary antibody in the mixture may or may not include changes to antibody 2 listed in the same row. In some embodiments, the antibody may include changes listed in two or more rows, such as 105R / K and 147R / K in the heavy chain, and 43E / D and 131E / D in the light chain. @ Not all changes apply to all IgG subtypes.

[0124] When anti-hIL33 and / or anti-hTSLP antibodies are part of an antibody mixture, the anti-hIL33 and / or anti-hTSLP antibodies may contain modifications unfavorable to heterodimerization, provided that both antibodies are IgG antibodies. In one embodiment, one antibody may be an IgG4 antibody (which has a naturally occurring arginine at position 409) or an IgG1 antibody that has been modified to have an arginine at position 409, i.e., having a K409R modification, while the other antibody has amino acid 399K / R and 409D / E.

[0125] In some embodiments, anti-hIL33 antibodies and anti-hTSLP antibodies, including the anti-hIL33 VH and VL and anti-hTSLP VH and VL described herein, may be part of a chimeric antigen receptor (CAR), which may also contain a portion of a T-cell receptor and be used in CAR-T cell therapy. CAR-T cell therapy is explained in references such as Yu et al. (2019), Molecular Cancer 18: 125 (: / / doi.org / 10.1186 / s12943-019-1057-4); and Lemal and Tournilhac (2019), J. ImmunoTher. Cancer 7: 202 (…). https: / / doi.org / 10.1186 / s40425-019-0686-x )middle. Polynucleotides, vectors and host cells

[0126] Polynucleotides, such as DNA or other nucleic acids, encoding the antibodies and antibody mixtures described herein are provided. Using the guidance provided herein, those skilled in the art can combine known or novel nucleic acid sequences encoding antibodies and modify them by known methods to produce polynucleotides encoding the antibodies and antibody mixtures described herein, which contain the VH and VL amino acid sequences described herein. Such nucleotide sequences encoding VH, VL, HC, or LC, or portions of such sequences, are disclosed as in, such as SEQ ID NO: 24, 26, 28, 30, 49, 51, 53, 55, 69, 71, 73, and 75, and throughout the specification. In some embodiments, the polynucleotide may encode HC and / or LC containing alterations (such as mating orientation alterations) relative to the amino acid sequences disclosed herein. Such alterations may be amino acid substitutions. Furthermore, such polynucleotides may encode HC and / or LC containing one or more mating orientation alterations outside the variable domain and / or one or more alterations unfavorable to heterodimerization. Many nucleic acid sequences encoding constant domains (e.g., CL, CH1, hinge, CH2, and CH3) of human, mammalian, and primate immunoglobulins are known in the art. See also, e.g., Kabat et al., ibid. Optionally, the polynucleotide sequence encoding the variable domain described herein may be combined with a polynucleotide sequence encoding such a constant domain to produce any form of antibody, such as IgG, IgM, IgD, IgE, IgA, bispecific forms, scFv, scFv-Fc, Fabs, BiTE (scFc-linker-scFv), Fab-scFv, IgG-scFv. In some embodiments, the polynucleotide sequence may encode an HC in which the hinge or hinge portion may be derived from an isotype or isotype subclass different from one or more other constant domains, and / or the hinge domain may have an amino acid sequence altered relative to naturally occurring hinge domains. In some embodiments, these antibodies may contain coupler orientation alterations and / or alterations detrimental to heterodimerization. In a further embodiment, the polynucleotide sequence may encode HC that has been modified to enhance effector functions such as complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, these antibodies may be mammalian antibodies, optionally human, humanized, or primate antibodies.

[0127] Methods for modifying polynucleotides are well known in the art. The most straightforward method for creating modified polynucleotides may be the synthesis of polynucleotides with the desired sequence. Many companies, such as DNA 2.0 (Menlo Park, Calif., USA), BlueHeron (Bothell, Washington), Genewiz (South Plainfield, New Jersey), Gen9 (Cambridge, Massachusetts), and Integrated DNA Technologies (Coralville, Iowa), offer this service. Other known methods for introducing mutations, such as site-directed mutagenesis using polymerase chain reaction (PCR), are also employed. See, for example, Zoller (1991), Curr. Opin. Biotechnol. 2(4): 526-531; Reikofski and Tao (1992), Biotechnol. Adv. 10(4): 535-547.

[0128] The vector containing a polynucleotide (optionally DNA) encoding the antibody and mixtures thereof described herein can be any vector suitable for expressing the antibody in the selected host cell. The vector may include selection markers for selecting the host cell containing the vector and / or for maintaining and / or amplifying the vector in the host cell. Such markers include, for example, (1) genes conferring resistance to antibiotics or other toxins (such as ampicillin, tetracycline, or kanamycin) to prokaryotic host cells; (2) genes compensating for cellular nutrient deficiencies; or (3) genes whose operation provides key nutrients unavailable in complex or limited culture media. Specific selection markers may be kanamycin resistance genes, ampicillin resistance genes, and tetracycline resistance genes. Bleomycin resistance or neomycin resistance genes may also be used for selection in prokaryotic and eukaryotic host cells. Dihydrofolate reductase (DHFR) genes and / or promoterless thymidine kinase genes may be used in mammalian cells, as known in the art. See, eg, Kingston et al. 2002, AMPLIFICATION USING CHO CELL EXPRESSION VECTORS, Current Protocols in Molecular Biology, Ch. 16, Unit 16.23, Wiley 2002.

[0129] In addition, the vector may contain one or more other sequence elements required for maintaining the vector and / or expressing the sequence encoding the antibody or antibody mixture described herein. Such elements include, for example, origin of replication, promoter, one or more enhancers, transcription terminators, ribosome binding sites, polyadenylation sites, multiple adapter insertion sites for foreign sequences (such as DNA encoding antibodies or antibody mixtures described herein), and intercalation sequences between two insert sequences, such as DNA encoding HC and LC. These sequence elements can be selected to function in desired host cells to facilitate vector replication and / or amplification and expression of heterologous sequences in the insert vector. Such sequence elements are well known in the art and are available from a wide range of commercially available vectors.

[0130] In some embodiments, the polynucleotide encoding an antibody or antibody mixture may be carried on one or more viral vectors, optionally oncolytic viral vectors. Examples of such viral vectors include adenovirus, adeno-associated virus (AAV), retrovirus, vaccinia virus, modified Ankara vaccinia virus (MVA), herpesvirus, lentivirus, Newcastle disease virus, measles virus, Coxsackie virus, reovirus, and poxvirus vectors. In such embodiments, these viral vectors containing polynucleotides encoding the antibodies or antibody mixtures described herein can be administered to patients to treat disease. For example, in cancer patients, such viral vectors containing polynucleotides encoding antibodies or antibody mixtures can be directly administered to the primary site of tumors or cancer cells in the patient by means of, for example, injection, inhalation (for lung cancer), topical application (for skin cancer), and / or application to mucous membranes (nucleic acids can be absorbed through mucous membranes). Alternatively, such viral vectors can be administered systemically, for example, orally, topically, through mucous membranes, or via subcutaneous, intravenous, intra-arterial, intramuscular, or intraperitoneal injection as described herein. Similarly, the polynucleotides encoding the antibody mixture described herein can be encapsulated in liposomes and administered to patients with the disease.

[0131] The polynucleotides and / or vectors described herein can be introduced into host cells, for example, for the purpose of producing one or more antibodies. The host cell containing one or more polynucleotides and / or vectors encoding one or more antibodies can be any of a variety of cells suitable for expressing recombinant proteins. These include, for example, Gram-negative or Gram-positive prokaryotes, such as bacteria like *Escherichia coli*, *Bacillus subtilis*, or *Salmonella typhimurium*. In other embodiments, the host cell can be a eukaryotic cell, including such species as *Saccharomyces cerevisiae*, *Schizosaccharomyces cerevisiae*, or eukaryotera species, or any cell capable of expressing a heterologous polypeptide. In further embodiments, the host cell can be a mammalian cell. Many mammalian cell lines suitable for expressing heterologous polypeptides are known in the art and are available from a variety of suppliers, including the American Type Culture Collection (ATCC). Suitable mammalian host cell lines include, for example, the COS-7 line (ATCC CRL 1651) (Gluzman et al., 1981, Cell 23:175), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells or their derivatives such as VeggieCHO ​​and related cell lines (which are grown in serum-free medium (Rasmussen et al., 1998, Cytotechnology 28: 31)), CHO-K1 and CHO pro-3 cell lines and their derivatives such as DUKX-X11 and DG44 cell lines (which lack dihydrofolate reductase (DHFR) activity), HeLa cells, young hamster kidney (BHK) cells (such as ATCC CRL 10), and CVI / EBNA cell lines derived from the African green monkey kidney cell line CVI (ATCC CCL 70) (such as McMahan et al., 1991, EMBO J). (As described in 10:2821), human embryonic kidney (HEK) cells such as 293, 293 EBNA or MSR 293, human epidermal A431 cells, human Colo205 cells, HL-60 cells, U937 cells, HaK cells, Jurkat cells, HepG2 / 3B cells, KB cells, NIH 3T3 cells, S49 cells, and mouse myeloma cells, including NSO and Sp2 / 0 cells. Other prokaryotic, eukaryotic or mammalian cell types capable of expressing heterologous peptides may also be used. Methods for preparing antibodies and antibody mixtures

[0132] Typically, the individual anti-IL33 and / or anti-hTSLP monoclonal antibodies and mixtures thereof described herein can be produced by introducing DNA encoding the antibody into a host cell, culturing the host cell under conditions suitable for antibody production in the host cell, and recovering the antibody from cell clumps or cell supernatant. For example, any suitable method can be used to introduce DNA encoding one or more antibodies into a host cell, as described above, including, for example, transfection, transduction, lipid transfection, transformation, microparticle bombardment, microinjection, or electroporation. In some embodiments, DNA encoding two full-length antibodies can be introduced into the host cell. Such methods are known in the art and described, for example, by Kaestner et al. (2015), Bioorg. Med. Chem. Lett. 25:1171-1176, which is incorporated herein by reference.

[0133] Host cells with DNA encoding one or more antibodies can be cultured, and antibodies can be recovered from cell culture supernatants or cell clumps. Antibodies can undergo further purification steps, such as various centrifugal precipitation, precipitation, dialysis, and / or column chromatography, including affinity chromatography such as protein A chromatography, anion exchange chromatography, cation exchange chromatography, reversed-phase chromatography, hydrophobic interaction chromatography, and size exclusion chromatography, among many other possible purification steps.

[0134] Antibodies produced individually using the methods described above can be mixed to create a mixture. Alternatively, antibody mixtures can be produced in a similar manner, except that DNA encoding two different antibodies can be introduced into the host cell simultaneously or sequentially. Host cells containing DNA encoding two different IgG antibodies (i.e., two different heavy and light chains) can potentially produce up to ten different types of IgG antibodies due to mixed HC / HC and HC / LC pairings. See, for example, US 11,130,808. Figure 4 To limit the number of this type of antibody, antibodies may contain HC and LC pairing modifications and / or modifications that are unfavorable to heterodimers. Such modifications can limit the number of major antibody types produced by the host cell. Such mixtures can be purified as described above. Similar problems can arise when producing bispecific IgG antibodies in a single cell line. In this case, pairing modifications can be used to ensure that only homologous HC / LC pairing is used, and modifications that favor heterodimer HC / HC pairing can also be used. Such modifications are described in, for example, U.S. Patent 8,592,562. Examples 1 and 2 of U.S. Patent 8,592,562 and the accompanying drawings mentioned herein are incorporated herein by reference.

[0135] Those skilled in the art will understand that producing antibody mixtures in a single host cell line, rather than in two, represents a significant improvement in ease of production and efficiency compared to developing and operating two commercial production processes. Developing a commercial production process for any antibody requires optimizing numerous factors, including the expression system, the host cell line (if a cell line is used for expression), the cell culture process (including physical variables such as the use of stirred tanks vs. perfusion vs. many other culture methods, and the culture medium and feed strategy used to culture the host cell line), and antibody purification and formulation. Furthermore, once a process is developed, it must be characterized and validated and transferred to a production facility that complies with current good manufacturing practices (cGMP). See, for example, Li et al. (2010), Cell culture processes for monoclonal antibody production, mAbs 2(5): 466-477. Therefore, it is clear that producing antibody mixtures in a single process represents a significant improvement in ease of production and efficiency compared to producing them in two processes, not to mention a significant reduction in cost. Pharmaceutical Compositions and Administration Methods

[0136] The anti-hIL33 and / or anti-hTSLP antibodies, mixtures of anti-hIL33 and anti-hTSLP antibodies, bispecific antibodies, polynucleotides, and / or carriers described herein can be administered in pharmaceutically acceptable formulations. Regarding mixtures of anti-hIL33 and anti-hTSLP antibodies, each antibody may be formulated and administered individually or as part of a MabPair, as described in Examples 9-11 herein. Many pharmaceutical formulations are known in the art. Many such formulations are described in Remington: The Science and Practice of Pharmacy, 21. st ed., Lippincott Williams & Wilkins, Philadelphia, PA, 2005, the relevant portion of which is incorporated herein by reference.

[0137] Polynucleotides and proteins, such as antibodies, are typically administered parenterally rather than orally. Depending on the formulation, oral administration exposes the protein or polynucleotide to the acidic environment of the stomach, which can inactivate it. In some embodiments, specific formulations may allow for the oral administration of specific proteins or polynucleotides that are insensitive to gastric acid or are adequately protected from exposure to an acidic environment. Formulations may also be administered via mucosal administration. In some embodiments, formulations may also be administered topically. Typically, antibodies and polynucleotides are administered via injectable liquid formulations. Treatment

[0138] This article provides a method for treating patients in need of treatment with inflammation (e.g., type 2 inflammation), inflammatory diseases, and chronic inflammatory airway diseases such as asthma and COPD, the method comprising administering to the patient a mixture comprising: (a) an anti-hIL33 antibody and / or an anti-hTSLP antibody; or administering to the patient one or more polynucleotides encoding (a). For example, asthma and COPD (chronic obstructive pulmonary disease) are chronic inflammatory airway diseases characterized by obstructive airflow limitation. Both diseases are a significant burden on patients and the healthcare system. While asthma affects 262 million people and causes 461,000 deaths globally [see Lancet 2020; 396:1204–1222], COPD has a greater disease burden and is the third leading cause of death worldwide, causing approximately 3.2 million deaths in 2019. Both diseases are heterogeneous in their clinical presentation and underlying inflammatory mechanisms; therefore, monotherapy with a single MOA (mechanism of action) may not be sufficient to effectively treat all patients.

[0139] For both diseases, common triggers for acute exacerbations of airflow obstruction include viral or bacterial infections, cigarette smoke, allergens, and environmental factors such as air pollution. These triggers induce epithelial cells in the lungs to secrete IL-33 (interleukin-33), TSLP (thymic stromal lymphopoietin), and IL-25 (interleukin-25, or IL-17E), three alarming factors that drive type 2 inflammation. Particularly for asthma, genome-wide association studies have shown a strong association between the disease and genetic polymorphisms of TSLP, IL33, and ST2. Therefore, both TSLP and IL-33 have become particularly attractive targets due to their strong genetic link to asthma and their broad impact on airway inflammation.

[0140] ILC2s are relatively newly discovered immune cells. They are tissue-resident cells, primarily distributed in mucosal tissues such as the lungs, small intestine, skin, and adipose tissue. Due to their location, they are believed to play a key role in the development of allergic diseases and type 2 inflammation. Upon binding to alarmins, ILC2s rapidly release cytokines such as IL-4, IL-5, and IL-13 to mediate responses from eosinophils, mast cells, basophils, dendritic cells (DCs), B cells, and Th2 cells.

[0141] The current standard treatment is a combination of inhaled beta-adrenergic agonists and inhaled corticosteroids (ICS), primarily providing symptom relief for disease control and often improving lung function and reducing the rate of acute exacerbations. However, asthma generally responds better than COPD, and patients with eosinophilic inflammation respond best to ICS. A subset of asthma and COPD patients are not sensitive to ICS. Common side effects in patients using steroids and beta-adrenergic agonists long-term include increased frequency with dose increases, muscle cramps, and muscle weakness.

[0142] Non-type 2 asthma patients assessed by low serum eosinophil count and low exhaled nitric oxide fraction are not suitable candidates for current biologic therapies. Furthermore, while azithromycin and the phosphodiesterase-4 inhibitor roflumilast can be used to reduce acute exacerbations of COPD, their use is limited due to side effects. For azithromycin, the main side effects are gastrointestinal reactions, with the potential for arrhythmias and antibiotic resistance; while for roflumilast, the main side effects are gastrointestinal, such as nausea.

[0143] Chronic obstructive pulmonary disease (COPD) is currently defined as a chronic disease state characterized by irreversible airway obstruction resulting from the progression of two major underlying diseases, including chronic bronchitis and emphysema. Chronic bronchitis is clinically defined as persistent cough, sputum production, and dyspnea, while emphysema is histopathologically defined as irreversible changes in the airway walls distal to the terminal bronchioles, clinically manifested as slowly progressive dyspnea. COPD is currently the fourth leading cause of death in the United States and Europe, and deaths in COPD patients are often due to complications of the disease, such as respiratory failure or infection (GOLD Symposium Summary, Am J Respir CritCare Med 2001; 163: 1256-1276).

[0144] In some patients with chronic asthma, irreversible airway obstruction can develop that is difficult to distinguish from COPD, and thus bronchial asthma can progress to COPD (Celli BR et al., Eur Respir J 2004; 23: 932-46). In a significant number of patients meeting the current diagnostic criteria for COPD, significant reversible improvement in airway obstruction has been demonstrated with short- or long-term inhaled bronchodilators and steroids (GOLD Symposium Summary, Am J Respir Crit CareMed 2001; 163: 1256-1276). Therefore, a significant number of patients meet the defining criteria for both bronchial asthma and COPD (Guerra S, Curr Opin Pulm Med 2005; 11: 17-13).

[0145] Therefore, a significant unmet need remains for the treatment and / or prevention of disease maintenance and exacerbation reduction in chronic inflammatory diseases, including COPD and asthma. This paper anticipates that simultaneous blocking of both IL-33 and TSLP alarmins could improve the efficacy of treatments for chronic inflammatory diseases such as asthma and COPD, as blocking only one alarmin is insufficient. This paper anticipates that dual blocking of IL-33 and TSLP is more advantageous than targeting a single pathway; for example, anti-IL-5 therapy only improves type 2 inflammation. Dual blocking of IL-33 and TSLP will reduce the secretion of several downstream cytokines (i.e., IL-4, IL-5, and IL-13), which mediate inflammatory responses in eosinophils, B cells, mast cells, etc.

[0146] This invention anticipates that the anti-hIL33 and anti-hTSLP MabPair antibodies of this invention offer a safety advantage over smaller molecules. The MabPair compositions of this invention are expected to avoid the side effects of long-term use of inhaled β-adrenergic agonists and inhaled corticosteroids. The MabPair compositions of this invention are expected to address an unmet need for the treatment of COPD, as no biologics are currently approved by the FDA for this indication.

[0147] In addition to asthma and COPD, other inflammatory diseases that this study anticipates treating include, but are not limited to, arthritis, dermatitis, psoriasis, cystic fibrosis, late post-transplant and chronic solid organ rejection, multiple sclerosis, systemic lupus erythematosus, inflammatory bowel disease, autoimmune diabetes, diabetic retinopathy, diabetic nephropathy, diabetic vascular disease, ocular inflammation, uveitis, rhinitis, ischemia-reperfusion injury, restenosis after angioplasty, glomerulonephritis, Graves' disease, gastrointestinal irritation, conjunctivitis, atherosclerosis, coronary artery disease, angina pectoris, and small artery disease.

[0148] This article also anticipates that the anti-hIL33 and anti-hTSLP MabPair antibodies of the present invention may have the potential to treat viral infections and cancer, see Stanbery AG et al., J Allergy Clin Immunol. 2022;150(6):1302-1313.

[0149] In a particular embodiment of the treatment method provided herein, (1)(A) the anti-hIL33 antibody comprises VH and VL, each of which comprises CDR1, CDR2, and CDR3; (B) the anti-hIL33 antibody comprises VH CDR1, VH CDR2, VHCDR3, VL CDR1, VL CDR2, and VL CDR3, each comprising the following sequences: SEQ ID NO: 80, 81, 82, 77, 78, and 79; and (C) the anti-hIL33 antibody inhibits the interaction between human IL-33 and the ST2 / IL1AcP complex; and (1)(B) the anti-hTSLP antibody comprises VH and VL, each of which comprises CDR1, CDR2, and CDR3; (B) the anti-hTSLP antibody comprises VHCDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3, each comprising the following sequences: SEQ ID NO: 80, 81, 82, 77, 78, and 79. NO: 86, 87, 88, 83, 84 and 85; SEQ ID NO: 86, 87, 88, 83, 89 and 90; SEQ ID NO: 94, 95, 96, 91, 92 and 93; and (C) anti-hTSLP antibody inhibits the binding of hTSLP to the hTSLPR / hIL7Rα complex. In another embodiment, the antibody mixture comprises: (a) an anti-hIL33 antibody comprising a heavy chain (HC) and a light chain (LC), wherein (1) the HC of the anti-hIL33 antibody is encoded by a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:31, and (2) the LC of the anti-hIL33 antibody is encoded by a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:27; and (b) an anti-hTSLP antibody comprising HC and LC, wherein (1) the HC of the anti-hTSLP antibody is encoded by a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:72, and (2) the LC of the anti-hTSLP antibody is encoded by a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:76. In a particular embodiment, the anti-hIL33 and anti-hTSLP antibody mixture used in the method of the present invention corresponds to MabPairsQB11750 and QB11823 described in Examples 10 and 11. MabPair QB11750 contains anti-IL-33 IgG4 antibody QB11465 and anti-TSLP IgG1-D265A antibody QB11718; and MabPair QB11823 contains anti-IL-33 IgG4 antibody QB11465 and anti-TSLP IgG1-D265A antibody QB11764.

[0150] Anti-hIL33 and / or anti-hTSLP mixtures containing anti-hIL33 and / or anti-hTSLP antibodies can be administered alone or as a mixture (such as as MabPairs QB11750 and QB11823, etc.; as described in Examples 10 and 11). Bispecific antibodies containing anti-hIL33 and / or anti-hTSLP and a second antibody, or polynucleotides or vectors encoding such antibodies or combinations, can be administered with additional therapies before, after, and / or in parallel with the antibodies, combinations, or polynucleotides or vectors.

[0151] Regarding antibodies or mixtures thereof, they may be administered to patients at therapeutically effective doses at appropriate intervals. Therapeuticly effective doses can be determined by methods known in the art, including in vitro assays, rodent and / or primate model systems, and / or clinical trials. Doses of antibodies, antibody mixtures, or the polynucleotides encoding them may be administered once or twice over a period of time, or at intervals over a period of time. In some cases, administration may be stopped and restarted. In some embodiments, a mixture comprising anti-hIL33 and anti-hTSLP antibodies may be administered, such that both antibodies can be administered simultaneously. After one or more doses of the mixture, one antibody may be administered alone. In some embodiments, administration of the antibody may be sustained for a period of time. In some embodiments, administration of the antibody or antibody mixture may be stopped and resumed once or multiple times.

[0152] In cases where one or more polynucleotides or vectors are encoded in the antibody or antibody mixture described herein, the dosage may be, for example, about 5 x 10⁻⁶ per kilogram of body weight. 9 Copy (copies / kg) the polynucleotide or vector to about 10 15 copies / kg, approximately 10 10 Copy / kg to approximately 10 14 Copies / kg, or approximately 5 x 10 10 Copy / kg to approximately 5 x 10 13 Copies / kg. Alternatively, the dosage can be approximately 10. 10 10 11 10 12 10 13 5 x 10 13 10 14 2 x 10 14 3 x 10 14 4 x 10 14 5 x 10 14 6 x 10 14 7 x 10 14 8 x 10 14 9 x 10 14 , or 1015 The polynucleotide or vector is copied. The dosing frequency can be adjusted as needed and can be as described above or, for example, daily, every other day, twice a week, once a week, once every ten days, once every two weeks, once every three weeks, once a month, or once every two, three, four, five, six, seven, eight, nine, ten, eleven or twelve months.

[0153] The present invention has been generally described above. The specific embodiments described below are intended to illustrate the invention and not to limit its scope. It should be understood that various changes and modifications can be made to the invention, which are consistent with the spirit of the invention as described herein and will be apparent to those skilled in the art. Such changes and modifications are within the scope of the invention and are included in the appended claims. Example Example 1: Humanization and Engineering of Mouse Anti-human IL-33 Monoclonal Antibody

[0154] Interleukin-33 (IL-33) is a member of the IL-1 cytokine family. Binding of hIL-33 to the hIL-33 receptor, composed of ST2 and IL-1RAcP, promotes a pro-inflammatory response. Our aim is to develop an anti-hIL-33 antibody that can block the binding of hIL-33 to ST2.

[0155] Full-length hIL-33 is secreted in a 270-amino acid precursor form, which can be cleaved by serine proteases (such as trypsin and chymotrypsin secreted by mast cells) to produce a more efficient cleavage form of the cytokine. See LeFrancais. E. et al. Proc NatL Acad Sci USA 2014; 111(43):15502-15507.

[0156] Recombinant hIL-33 antigen was prepared internally via transient transfection of Expi293 cells. This antigen, an isoform A of the protein (SEQ ID NO: 1), begins at Ser112 and ends at Thr270, carrying N-terminal tags including Avitag, c-MYC, and His6, for biotinylation, detection, and purification, respectively. Briefly, the DNA fragment encoding the hIL-33 antigen (SEQ ID NO: 1) was ordered from Integrated DNA Technologies Inc., Coralville, IA, USA. The DNA fragment was assembled into the pSB01 expression vector using a Gibson assembly reaction (see Gibson G. Methods Enzymol. 2011;498: 349–361). Insertion was confirmed by Sanger sequencing at Genewiz Inc. (219 Terry Ave N, Floor 2, Seattle, WA98109). Using the ExpiFectamine™ 293 Transfection Kit (ThermoFisherScientific, catalog number A14525), plasmid DNA encoding the recombinant hIL-33 antigen was transiently transfected into Expi293 cells according to the manufacturer's instructions. After 5 days of incubation with shaking at 37°C, 120 rpm, and 5% CO2, the supernatant was harvested and the recombinant hIL-33 antigen was purified using standard techniques on an AKTA Avant chromatography system (Cytiva Inc., Marlborough, MA) using a HisTrap Excel column (Cytiva Inc., catalog number 17371205). Human IL-33 can be oxidized, resulting in the formation of two intracellular disulfide bonds. This oxidation alters the conformation of hIL-33, leading to loss of binding to the receptor ST2; see Verstraete K et al., Nat. Commun. 2017; 8:14937. To maintain constant activity of the recombinant hIL-33 antigen in its reduced form, a mutant hIL-33 protein was designed by introducing two substitutions, C208S and C232S, into QB11935 (SEQ ID NO: 2), see Cohen E. et al. Nat. Comm. 2015; 6:8327. This antioxidant hIL-33 was tagged with His6 at the N-terminus for purification. The antioxidant hIL-33 antigen was produced via transient transfection of Expi293 cells as described above.

[0157] The cynomolgus macaque IL-33 homologue (cyIL-33) is secreted as a full-length protein of 270 amino acids. Engineered anti-hIL-33 antibodies were also designed to block binding along the cyIL-33 / ST2 axis. To assess the cross-species binding of the anti-hIL-33 antibody and affinity maturation as described in Example 3, a recombinant cyIL-33 antigen was produced. This recombinant cyIL-33 antigen was an isoform X1 version of the protein (NCBI accession number XP_005581824), beginning at Ser112 and ending at ILe270. N-terminal tags, including Avitag, FLAG, and His6, were added for biotinylation, detection, and purification, respectively. The cyIL-33 protein (SEQ ID NO: 3) was produced via transient transfection of Expi293 cells as described above.

[0158] Mouse hybridomas were prepared using reduced active hIL-33 antigen as an immunogen, followed by DNA immunization boosting as described by Liu S et al., Hum Gene Ther. 2018; 29(9): 997–1003. The blocking antibody clone 18G9, which blocks the interaction between hIL-33 and human ST2, was identified by ELISA. cDNA sequences encoding VL and VH were obtained using standard molecular biology techniques (Meyer L et al., PLoSONE 2019;14(6):e0218717). The mouse VH region was combined with the human IgG4 constant region, and the mouse VL region was combined with the human κ constant region to express chimeric antibodies, which were confirmed to bind to hIL-33 and cyIL-33 antigens using surface plasmon resonance (SPR) technology in Cytiva's Biacore T200, as described below.

[0159] The receptor hST2-Fc protein from R&D Systems (catalog number 523-ST-100) was directly immobilized on the CM5 surface in flow cells 2, 3, and 4 via amine coupling, while hST2-Fc was not immobilized in flow cell 1. Running buffer was passed through flow cell 1, so background binding from flow cell 1 was used as a reference. The reference was subtracted from the results of flow cells 2, 3, or 4 to obtain the true binding. Active hIL-33 monomer (reduced to 2 mM DTT) was injected onto the CM5 surface at 0, 0.62, 1.85, 5.56, 16.7, and 50 nM, with binding times set to 1 or 2 minutes and dissociation times to 5 minutes. Binding capacity was recorded in response units (RU). Figure 1 As shown in Figure A, the hIL-33 ligand binds to immobilized hST2-Fc in a dose-dependent manner in a 1:1 ratio, binding EC for 1 minute. 50 10.82 nM, EC combined in 2 minutes 50 It is 5.175 nM.

[0160] The direct interaction of hIL-33-hST2 on the CM5 surface was confirmed. Figure 1 A) Subsequently, the gradient-premixed hIL-33-antibody complex was injected into the same CM5 chip immobilized with hST2-Fc: hIL-33 was combined with isotype control antibody 10825, anti-hIL-33 reference antibody 1 10997, anti-hIL-33 reference antibody 2 10996, or anti-hIL-33 chimeric antibody 10998 (SEQ ID NO: 5 and 7), respectively. The binding time was set to 2 minutes, and the dissociation time was set to 5 minutes. Figure 1 As shown in Figure B, when the isotype control antibody 10825 at concentrations of 100 nM or 33.3 nM was premixed with 10 nM hIL-33, the binding capacity was equivalent, approximately 60 RUs. Both anti-hIL-33 reference antibody 1 and reference antibody 2 showed additional binding upon premixing with the hIL-33 ligand, indicating that neither reference antibody blocked the direct interaction between hIL-33 and hST2. However, the anti-hIL-33 chimeric antibody 10998 strongly inhibited the interaction between hIL-33 and hST2 in a dose-dependent manner, with an IC50 value of [missing value]. 50 It is 1.5 nM.

[0161] Humanization of the anti-hIL-33 antibody 10998 was achieved by transplanting the CDR into five different combinations of human VH and VL germline lines. Appropriate reversion mutations were introduced into the human germline sequences to maintain the correct folding of the antibody. Humanization of mouse antibodies has been well-described by many researchers; see the review paper by Safdari Y et al., Biotechnol Genet Eng Rev. 2013; 29:175–186. Five initially humanized anti-hIL-33 antibodies were expressed via transient transfection into ExpiCHO-S cells (ThermoFisher Scientific, Waltham MA, A29127) using Expifectamine CHO transfection reagent (ThermoFisher Scientific, A29192) and a 12-day culture protocol. The recombinant antibodies were purified from the clear supernatant by protein A affinity chromatography on an AKTA Avant HPLC system using HiTrap MabSelect Sure resin (Cytiva, Marlborough MA, 29049104). Purified isotype control antibody 10825, anti-hIL-33 chimeric antibody 10998, or individually humanized anti-hIL-33 antibodies (10999, 11000, 11001, 11002, 11004) were serially diluted 1:3 and premixed with 10 nM hIL-33, and then injected onto the hST2-Fc-fixed CM5 surface as described above. Figure 1 As shown in Figure C, the isotype control antibody 10825 did not inhibit the interaction between hIL-33 and hST2 even at 100 nM, while the chimeric antibody 10998 and four humanized anti-hIL-33 antibodies (10999, 11001, 11002, and 11004) inhibited the hIL-33 / hST2 interaction considerably in a dose-dependent manner. However, the humanized antibody 11000 had lower potency, with an IC50 value of [missing value]. 50 It is 2.269 nM.

[0162] Humanized anti-hIL-33 antibody 11004 (SEQ ID NO: 9 and 11) was selected for further engineering and affinity maturation because it exhibits robust production, minimal reversion mutations, and good blocking activity (IC50). 50= 0.9271 nM). However, humanized antibody 11004 showed an 89.8% main peak and a 10.2% HMW (high molecular weight) peak in SEC analysis, indicating that the antibody could aggregate during production and purification processes. To improve purity by mitigating the aggregation problem, another round of engineering was carried out, mutating several framework residues to the typical residues of the VH3 / VK2 lineage in antibody 11004. Antibody variant QB11061 (SEQ ID NO: 13 and 15), with a Q41P substitution in VH-FW2, showed a 97.4% main peak and a 2.6% HMW peak.

[0163] Computer analysis of antibody QB11061 identified readily deamide-compatible sequence motifs in VH and VL. VL-CDR2 is located in the sequence motif... 28 NG 29 and 30 NT 31 It contains two potential deamidation tendencies. VH-CDR2 contains 56 NT 57Potential deamidation susceptibility. Accelerated deamidation experiments were performed to assess the veracity of the deamidation susceptibility. The anti-hIL-33 antibody QB11061 was denatured, reduced, and alkylated prior to trypsin digestion. Briefly, 1 mg of antibody was concentrated to approximately 10 mg / mL and then exchanged three times with 0.1 M triethylammonium bicarbonate buffer (pH approximately 8.5) at 14,000 × g and 15°C for 5 minutes each time using a 10 kDa molecular weight cutoff (MWCO) microcentrifuge tube. 350 µg (~35 µL) of protein was added to a 1.5 mL screw-cap microcentrifuge tube, followed by 315 µL of denaturing buffer containing 7.5 M guanidine hydrochloride and 50 mM Tris pH 8.0. Then, 8.5 µL of 0.1 M dithiothreitol (DTT) in 50 mM Tris pH 8.0 solution was added, the sample was briefly vortexed, and incubated at 37°C for 30 minutes. The reduced antibody was cooled to room temperature, and then 25.5 µL of 0.1 M iodoacetamide (IAM) aqueous solution was added. The sample was briefly vortexed and incubated at room temperature in the dark for 30 minutes. 300 µL of the resulting reduced and alkylated sample was loaded onto a NAP5 desalting column, which had been pre-equilibrated with 10 x 1 mL of freshly prepared digestion buffer containing 1 M urea and 50 mM Tris at pH 8.0. After loading, the column was washed with 200 µL of digestion buffer, and then eluted with 600 µL of digestion buffer into 1.5 mL screw-cap microcentrifuge tubes. TEAB (triethylamine bicarbonate) was added to half of the eluted sample to a final concentration of 0.1 M, while no TEAB was added to the other half. Both samples were incubated at 45°C for 36 hours. Sequencing-grade modified trypsin (Promega, catalog number V5113) was added to the sample and digested at 37°C for 4 hours. Finally, 20 µL of 10% (v / v) formic acid was added to completely quench the reaction. Mass spectrometry analysis was performed on untreated and treated antibody QB11061 to assess actual deamidation at susceptible residues.

[0164] Figure 2 The spectra of trypsin-digested peptides from untreated anti-hIL-33 antibody QB11061 (A) and treated anti-hIL-33 antibody QB11061 (B) are shown under UV 215 nm scanning; as are the spectra of trypsin-digested peptides from untreated anti-hIL-33 antibody QB11119 (C) and treated anti-hIL-33 antibody QB11119. (Representative) 28 NG 29 The peak of deamidation (L3N → D) at the L3N → D site increased significantly after accelerated deamidation treatment, compared to... Figure 2 A and Figure 2 B indicates 28 NG 29 The motif is readily deamided.

[0165] To alleviate the effects of anti-hIL-33 antibody QB11061 VL-CDR2 28 NG 29 Deamidation motifs were performed by replacing the N28 residue with glutamine, glutamate, histidine, lysine, or arginine. Additionally, N28-retained residues were simultaneously prepared. 28 NA 29 However, antibody variants containing G29A substitutions, such as QB11119, were evaluated. Each LC variant was co-expressed with HC of QB11061 to generate hIgG1 with L234A + L235A + P329A substitutions, which eliminated Fc effector function (see Wang X. et al. Protein Cell. 2018; 9(1): 63–73). These antibody variants were produced via transient transfection of ExpiCHO cells and purified by protein A affinity chromatography. SPR analysis showed that the anti-hIL-33 antibodies QB11119 (SEQ ID NO: 17 and 19) containing G29A substitutions in VL-CDR2 maintained affinity for hIL-33 compared to the humanized antibodies QB11004 and QB11061 (see Figure 5 and Table 7 below). 28 NG 29 motif change to 28 NA 29 Subsequently, the anti-hIL-33 antibody QB11119 was subjected to accelerated deamidation as described above. The peak representing the deamidated peptide L3 (N → D) is observed in the untreated ( Figure 2 C) or processed ( Figure 2 D) The anti-hIL-33 antibody QB11119 showed a significant reduction, indicating that the deamidation problem at N28 was alleviated when the G29A mutation was introduced. Spectroscopically magnified images of the peptide showed... Figure 2 In E, the peak representing the deamidated peptide is very small. No peak corresponding to VL-CDR2 was observed in untreated or treated samples. 30 NT 31 In deamidation or VH-CDR2 56 NT 57 The deamidated modification products (data not shown) indicated that neither of the two sequence motifs exhibited significant deamidation susceptibility. Therefore, after mitigating deamidation, the engineered anti-hIL-33 antibody variant QB11119 was selected for further affinity maturation via yeast display. Example 2: Affinity maturation of humanized anti-hIL-33 antibody QB11119 via yeast display

[0166] Typically, antagonistic antibodies with high affinity exhibit high biological activity. To achieve high biological activity, a Fab library of the anti-hIL-33 antibody QB11119 for yeast display was generated to identify beneficial variants that result in high binding affinity. The procedure for constructing the Fab library for yeast display is briefly described in our patent US11,124,570.

[0167] The VL amino acid sequence (SEQ ID NO: 16) of antibody QB11119 was used via the Kabat numbering scheme. www.bioinf.org.uk / abs / abnum / The positions of all residues were determined, and the results are shown below. Five CDR residues (K27, M51, M89, L92, and E93) in VL were randomized using the NNK codon. Table 7. Kabat numbers of amino acid residues in the VL region of humanized anti-hIL-33 antibody QB11119 The CDR1 (positions 24-34), CDR2 (positions 50-56), and CDR3 (positions 89-97) regions are highlighted in gray. The bold residues at positions 27, 51, 89, 92, and 93 are randomized using the NNK codon to further enhance binding affinity through yeast display. The mutant residue A29, which mitigates deamidation, is indicated in bold and underline.

[0168] Six independent oligonucleotides covering five randomized residues were synthesized by Integrated DNA Technologies, Inc. (Coralville IA, USA). The six overlapping oligonucleotides were added to the same tube with short 5' forward and 3' reverse primers, and the entire VL region was assembled and amplified by PCR. PCR bands of the desired size were excised from a 1.5% agarose gel and purified using the QIAquick Gel Extraction Kit (Qiagen, catalog number 28704). The purified PCR products were used for a second round of PCR amplification to obtain a large quantity of material for library construction. The intermediate fragment containing the κ constant region, c-MYC-R6-pep2A sequence, and signal peptide 2 (SP2), as well as the VH fragment of the anti-hIL-33 antibody QB11119, were amplified by PCR to obtain a large quantity of material.

[0169] To display a library of randomized Fab fragments in yeast, linearized pFab1.6 was used to electroporate Saccharomyces cerevisiae cells. This vector has a sequence encoding a signal peptide (SP1) at one end (preceded by a galactose-inducible promoter) and a sequence encoding a CH1 domain at the other end (followed by a lectin-encoding sequence). This was combined with the three purified PCR fragments described above, encoding (1) a randomized SP1-VL-Ck at 5 residues, (2) an intermediate fragment Ck-MYC-R6-Pep2A-SP2, and (3) the wild-type SP2-VH-CH1. PCR fragment (1) overlapped with the SP1 end of the vector and the Ck end of PCR fragment 2. The SP2 end of the signal peptide of PCR fragment (2) overlapped with the SP2 end of PCR fragment (3), and the CH1 end of PCR fragment 3 overlapped with the CH1 end of the vector. Since all these overlaps are within the range of 30 to 60 base pairs, homologous recombination in yeast allows fragments to assemble into a single vector containing an insert with the following sequence element order: SP1-VL-Ck-MYC-R6-Pep2A-SP2-VH-CH1-HA-lectin, where R6 encodes six consecutive arginine residues to serve as a furin cleavage site, and HA is the HA tag, a small peptide derived from human influenza hemagglutinin, which has been widely used as a protein tag. See [link to relevant documentation]. Figure 3 A. Expression of these sequences was driven by a galactose-inducible promoter upstream of SP1. Transformants were grown on selective agar plates prepared in a yeast medium containing glucose and lacking uracil. The vector contained genes that compensated for the host yeast strain's inability to synthesize uracil. The library size (i.e., the total number of transformants) was approximately 1.36 x 10⁻⁶. 8 Within the scope of the transformant, the computational complexity is more than 10 times that of the library. The estimated complexity of the library is less than approximately 10. 7 This means that each library contains fewer than approximately 10 7 Different combinations of nucleotide sequences encoding Fab fragments. These estimates are based on the number of possible combinations under randomization of the variable sites.

[0170] To assess the actual diversity and quality of the library, DNA segments encoding Fab fragments from 50 randomly selected yeast clones were examined. VL fragments from these clones were amplified by yeast colony PCR and sequenced by Genewiz Inc. DNA sequence analysis showed that 60% of the colonies encoded in-frame VL and CL regions, as well as in-frame VH and CH1 regions; therefore, the translated peptides could be processed to be displayed as Fab on the yeast surface. See [link to relevant documentation]. Figure 3B. The VH-CH1-Aga2 polypeptide is anchored to the yeast surface by forming two disulfide bonds with the Aga1 protein, while processed VL-CL is captured due to the strong interaction at the VH-VL and CH1-CL interfaces (see Mei M. et al. Microbio. Res. 2017; 196:118-128). The DNA encoding the VL region contains the expected amino acid variation at the target location.

[0171] Five CDR residues (S32, G98, R99, R100, and D100b) in antibody QB11119 VH were randomized using the NNK codon, as shown in Table 8 below. Table 8. Kabat numbers of amino acid residues in the VH region of humanized anti-hIL-33 antibody QB11119 The CDR1 (positions 31-35), CDR2 (positions 50-65), and CDR3 (positions 100a-102) regions are highlighted in gray. The bold residues at positions 32, 98, 99, 100, and 100b are randomized using the NNK codon for use in yeast display technology to further improve binding affinity.

[0172] The VH library for yeast display was constructed similarly to the one described above, except that it used wild-type VL fragments and randomized VH fragments amplified by PCR. The quality of the VH library was similarly assessed by sequencing the DNA of 50 randomly selected yeast colonies.

[0173] Biotinylated hIL-33 QB10975 (SEQ ID NO: 1) and cyIL-33 QB10976 (SEQ ID NO: 3) proteins were reduced at 50°C for 30 minutes using 10 mM DTT (Milwaukee WI, catalog number 3483-12-3) from Sigma Aldrich to prepare active antigens. The reduced hIL-33 or cyIL-33 antigens were used alternately in screening to enrich antibody variants capable of binding to both hIL-33 and cyIL-33 antigens across species.

[0174] Yeast cells containing VL and VH libraries corresponding to >10 times the library size were subjected to 0.1 OD. 600 Inoculated into URA-free CM glucose medium (Teknova Inc., Hollister CA, catalog number C18140), and grown overnight at 30°C to OD. 600 The value is 3~5. 100 OD was collected by centrifugation. 600A certain amount of yeast cells were introduced and then resuspended in 200 mL of 90% CM galactose + 10% URA-free glucose medium at 20°C for 2 days to induce Fab expression on the yeast surface. As described by Chao et al., Nature Protocols, 2006 doi:10.1038 / nprot.2006.94, an inducible library of antibody variants binding to biotinylated active antigens was enriched by streptavidin-coupled magnetic bead sorting (MACS). The first round of MACS was performed using 500 nM biotinylated hIL-33 antigen (SEQ ID NO: 1), with an input yeast amount of 3.0 x 10⁻⁶. 9 The second round of MACS used 100 nM biotinylated cyIL-33 antigen (SEQ ID NO: 3).

[0175] The MACS-enriched Fab yeast pools were further sorted by FACS to identify clones with very high binding affinity for hIL-33. The yeast VL pools were incubated with 125 nM bn-hIL-33, then stained with FITC-labeled anti-HA antibody (Waltham MA, catalog number A-21287) from Thermo Fisher Scientific and APC-labeled streptavidin (catalog number S868) from Thermo Fisher Scientific, followed by FACS sorting. The enriched anti-hIL-33 VL pools were further sequentially sorted with 15 nM bn-cyIL-33 antigen. Similarly, the VH pools obtained after MACS enrichment were sequentially sorted with biotinylated 30 nM hIL-33 and 50 nM cyIL-33 antigen. After final FACS sorting, yeast cells were plated on uridine-free CM glucose agar plates (Teknova Inc., catalog number C3080) and cultured at 30°C for 3 days.

[0176] A new combinatorial library containing beneficial VL and VH variants was constructed to identify Fab conjugates with very high affinity. For each pool, an OD representing >10 times the pool size was used. 600Plasmid DNA was extracted from yeast. Variable regions were amplified from the VL or VH pools via PCR using specific primers. The gel-purified VL and VH PCR fragments were amplified by a second round of PCR to obtain a large quantity of material. The amplified VL, intermediate, and VH fragments were combined with the Bgl II / Nhe I-digested pFab1.6 vector for electroporation into competent yeast cells. Electroporated yeast cells were plated on URA-free CM glucose agar plates, grown for 3 days, and harvested. A combined library was induced as described above, and three rounds of FACS sorting were performed consecutively using biotinylated 20 nM hIL-33, 4 nM cyIL-33, and 1 nM hIL-33 antigens.

[0177] Ninety-six yeast colonies were individually selected and induced, and their binding to the hIL-33 antigen was ranked by the % Q2 double-positive signal determined by FACS analysis. (See [link to FACS analysis]). Figure 4 Fifteen colonies exhibiting the strongest binding to the hIL-33 antigen were selected for further analysis. Plasmid DNA was extracted from single yeast colonies using the Zymoprep Yeast Plasmid Miniprep II Kit (Zymo Research, Irvine CA, catalog number D2004). The VL and VH regions were amplified by PCR using specific primers. DNA encoding the VL and VH variable regions was revealed by Sanger DNA sequencing from Genewiz, Inc.

[0178] DNA gblocks encoding unique VL and VH sequences of a yeast clone with the highest affinity for the hIL-33 antigen were synthesized by IDT Inc. The VL gblocks were assembled with the κ constant region into the pSB01 expression vector via a Gibson reaction; the VH gblocks were assembled with the IgG1 constant region into the pSB01 expression vector via a Gibson reaction. The IgG1 HC plasmid contained L234A, L235A, and P329A to eliminate antibody effector functions such as ADCC, CDC, and ADCP. After DNA sequencing confirmation, the LC and HC plasmids were co-transfected into ExpiCHO cells, and the secreted recombinant antibodies in the supernatant were purified by protein A affinity chromatography as described above. SEC analysis showed that the main peak purity of all antibodies was >98%, and mass spectrometry analysis showed that all antibodies had the expected LC and HC quality. Preliminary data from the Octet analysis indicate that antibody variants QB11421 (SEQ ID NO: 21 and 23) have the highest binding affinity for hIL-33 and cyIL-33 antigens (Figure 5). Example 3: Biacore measurement of the preferred anti-IL-33 antibody QB11421 after affinity maturation via yeast display.

[0179] Kinetic binding data were obtained using SPR on a Biacore T200 device. Anti-hIL-33 antibodies were captured separately onto CM4 chips immobilized with polyclonal goat anti-human Fc-specific antibodies. Analytes hIL-33 and cyIL-33 were serially diluted 1:3 in run buffer containing HBS-EP + 0.05% BSA and injected into flow cells (fc) 1 through 4, where fc2, fc3, and fc4 contained different captured anti-hIL-33 antibodies, while fc1 was injected with blank run buffer as a reference. True antigen binding for each antibody was obtained by subtracting the binding from fc2, fc3, and fc4. Analytes were injected at a flow rate of 50 µL / min, with detection temperatures of 37°C for hIL-33 antigen and 25°C for cyIL-33 antigen. Complex binding was allowed for 3 minutes, with dissociation up to 1 hour. The CM4 chip was regenerated with 10 mM glycine at pH 1.5 at the end of each cycle. The data were aligned and double-referenced using T200 evaluation software V3.2. The results were fitted to a 1:1 combined model using the software's global data analysis option. Table 9. Summary of anti-hIL-33 antibody binding to hIL-33 antigen on Biacore T200 device. Table 10. Summary of anti-hIL-33 antibody binding to cyIL-33 antigen on Biacore T200 device.

[0180] As shown in Table 9 above, SPR analysis of the preferred anti-IL-33 IgG1 antibody QB11421 after yeast affinity maturation showed that its affinity for hIL-33 increased by approximately 2-fold compared to the parental antibody QB11119. This increase was primarily due to faster binding (Ka), while dissociation (Kd) remained almost unchanged. SPR analysis also showed that the binding affinity of QB11421 for hIL-33 was very comparable to that of the control anti-IL-33 antibody QB11094, which had slower binding (Ka) and slower dissociation (Kd). After converting the anti-hIL-33 IgG1 antibody QB11421 into IgG4 antibodies QB11465 (SEQ ID NO: 25 and 27) using methods well-known in the art for co-expression with the preferred anti-TSLP IgG1 antibody as MabPair, the binding affinity was very similar because the VH and VL variable regions remained unchanged. The antibodies described herein are intended to bind cyIL-33. SPR analysis was performed using cyIL-33 protein as an analyte, as described above (see above). Figure 10The results showed that the preferred clones after yeast display, IgG1 (QB11421) and IgG4 (QB11465), exhibited similar binding affinity to the cyIL-33 antigen, with both showing approximately a 3-fold increase in binding affinity compared to the parental antibody QB11119. However, the binding of the anti-hIL-33 reference-3 antibody QB11094 to the cyIL-33 antigen derived from mammalian cells was negligible. Example 4: Preparation of anti-TSLP antibodies and identification of blocking antibodies via rabbit hybridoma

[0181] Three forms of recombinant hTSLP protein with Avitag, c-MYC, and His6 tags (used for biotinylation, detection, and purification, respectively) at the N-terminus were prepared by transient transfection of Expi293 cells and purified by standard nickel column chromatography: (1) wild-type TSLP, SEQ ID NO: 32, but produced in the presence of furin protease inhibitor II (Sigma catalog number SCP0148); (2) TSLP with five consecutive residues deleted. 126 KRRKR 130 (2) The furin cleavage motif SEQ ID NO: 33; (3) Two substitutions (R127A + R130S) were introduced to eliminate the furin cleavage site, SEQ ID NO: 34. Similarly, three forms of recombinant cyTSLP protein with Avitag, FLAG, and His6 tags at the N-terminus were prepared by transient transfection of Expi293 cells and purified by standard nickel column chromatography: SEQ ID NO: 35, wild-type TSLP produced in the presence of furin inhibitor II; SEQ ID NO: 36, lacking the furin cleavage motif; SEQ ID NO: 37, mutant protein with R127A+R130S. The internally prepared antigens were used for Biacore measurements, yeast display, and cell-based functional assays as described below.

[0182] Rabbit monoclonal antibodies can recognize a variety of epitopes, including those with poor immunogenicity in mice and humans. Because rabbits have a more diverse natural antibody library than mice, rabbit antibodies exhibit higher affinity for antigens than mouse antibodies. Rabbits have larger spleens, greater blood volume, and more bone marrow tissue than mice, facilitating manipulation. In Genscript (Piscataway, NJ), using… 126 KRRKR 130Four New Zealand white rabbits were immunized with the missing hTSLP QB11033 antigen. Rabbit serum was collected 8-10 weeks post-immunization, and polyclonal antibodies in the serum were purified on a small scale using a protein A column. The purified polyclonal antibodies were normalized and used in a competitive Biacore assay. Different concentrations of His6-tagged hTSLP antigen were captured onto a CM5 chip immobilized with anti-His6 antibody, and 100 nM hTSLPR-Fc (SEQ ID NO: 38) was allowed to flow through the surface of the CM5 chip. Due to the interaction between hTSLP and hTSLPR, the binding signal of the RU (response unit) was detected using a Biacore T200. If an antibody blocking the interaction between hTSLP and hTSLPR is present, the binding signal will be reduced in this competitive Biacore assay.

[0183] Anti-hTSLP reference blocking antibody 10985 (hollow circle) showed very strong inhibition, while the non-blocking anti-hTSLP antibody 10987 (hollow square) showed enhanced binding because 10987 binds to a different epitope outside the hTSLP / hTSLPR interface. Polyclonal antibodies from rabbit 7184 and 7186 showed strong inhibition, while polyclonal antibodies from rabbit 7183 showed moderate levels of inhibition, and antibodies from rabbit 7182 showed low levels of inhibition. See [link to relevant documentation]. Figure 6 A. The binding of polyclonal antibodies from rabbit 7184 and 7186 to hTSLP and cyTSLP was tested using Biacore. The polyclonal antibody from rabbit 7186 showed similar RU binding levels to hTSLP and cyTSLP, while the polyclonal antibody from rabbit 7184 showed half the binding level to cyTSLP compared to hTSLP (data not shown).

[0184] Capture 5 µg / mL of His6-tagged hTSLP antigen onto a CM5 chip immobilized with anti-His6 antibody at a rate of 20 µL / min. Inject blank buffer for 1 minute, then inject a second batch of blank buffer, 100 nM hTSLPPR-Fc, anti-hTSLP control blocking antibody 10985, or anti-hTSLP non-blocking antibody 10987, allowing it to flow over the CM5 chip surface for 3 minutes. Figure 6As shown in Figure D, after the His6-tagged hTSLP antigen was captured onto the CM5 chip, the first injection of blank buffer did not interrupt the binding of hTSLP ligand, anti-hTSLP non-blocking antibody 10987, or anti-hTSLP control blocking antibody 10985, because they could all bind to hTSLP. However, no additional binding occurred when the blank buffer was injected a second time across the CM5 surface. These results indicate that interactions between the hTSLP ligand and the hTSLP ligand receptor, and between the hTSLP antigen and the anti-hTSLP antibody, were observed using the Biacore assay.

[0185] After the His6-tagged hTSLP antigen is captured onto the CM5 chip, a first injection of purified rabbit polyclonal antibody from rabbit 7186 is passed through the CM5 surface at 30 µg / mL. If any blocking antibody is present in the rabbit polyclonal antibody that binds to hTSLP and inhibits the interaction between hTSLP and hTSLPR, the binding signal of the second injection of hTSLPR should be reduced. Figure 6 As shown in Figure E, the second injection of blank buffer did not produce any additional binding signal; instead, the binding signal gradually drifted and decreased because there was dissociation between the captured hTSLP and the flowing anti-hTSLP antibody present in the rabbit 7186 polyclonal antibody. The second injection of hTSLPR did not produce any additional binding; instead, the binding signal between hTSLP and the purified polyclonal antibody from rabbit 7186 decreased, indicating the presence of some blocking antibody in the rabbit 7186 polyclonal antibody to prevent interaction between hTSLP and hTSLPR. Both the non-blocking antibody 10987 and the anti-TSLP control blocking antibody 10985 showed additional binding signals as expected within 60–180 seconds, because only a small amount of blocking antibody was present in the rabbit 7186 polyclonal antibody. Based on all the above data, rabbit 7186 spleen cells were fused with the rabbit plasmacytoma cell line 240E-1 and cultured in a medium containing HAT (hypoxanthine, aminopterin, and thymidine) to produce rabbit anti-hTSLP monoclonal antibody.

[0186] ELISA was used to screen supernatants from 14,100 wells in 150 96-well plates (one well used as a positive control and one as a negative control) for binding to hTSLP, cyTSLP, or unrelated antigens. A total of 177 hTSLP conjugates with a signal-to-noise ratio >3 were selected for a second round of ELISA assays for confirmation. Sixteen clones showed strong binding to both hTSLP and cyTSLP but not to unrelated antigens: clones 8F7, 13A8, 37E4, 40H8, 45E7, 60E10, 110D9, 120D6, 121B5, 124A2, 124G7, 132A7, 133F7, 135F1, 139B10, and 139F11. Two clones showed much stronger binding to hTSLP but weaker binding to cyTSLP: clones 82C8 and 128D9. Two clones showed strong binding to hTSLP but no binding to cyTSLP at all; these were clones 23G3 and 130E10. See also Figure 7 .

[0187] Sixteen anti-hTSLP antibodies were produced by transiently transfecting ExpiCHO cells with plasmid DNAs encoding HC and LC for each antibody, followed by purification by protein A affinity chromatography as described above. 2 µg / mL of His6-tagged hTSLP antigen was coated in 1x PBS buffer and incubated overnight at 4°C. The plates were washed three times with 1x PBST, blocked with 1x PBST containing 2% BSA, and shaken at 200 rpm for 1 hour at room temperature. Single anti-hTSLP rabbit antibodies or rabbit isotype control antibodies were repeatedly added to a final concentration of 5 µg / mL, followed by a final concentration of 2 µg / mL biotinylated hTSLP. The plates were shaken at 200 rpm for 1 hour at room temperature and washed three times with 1x PBST. HRP-conjugated streptavidin was added to each well, the plates were washed three times, HRP substrate was added to each well for color development, and the reaction was terminated by adding H2SO4 solution after shaking the plates for 15 min. Read the plate using a Perkin-Elmer microplate reader. Plot the percentage of inhibition relative to antibody-free blank 1x PBST buffer.

[0188] like Figure 8As shown, the rabbit isotype control antibody had a baseline inhibition level of 5%; however, in a competitive ELISA assay, 10 out of 16 clones strongly inhibited the binding of hTSLP to hTSLPR (≥ 70%), namely clones 8F7, 37E4, 40H8, 60E10, 110D9, 120D6, 121B5, 135F1, 139B10, and 139F11. The inhibition rates of the other 6 clones (13A8, 45E7, 124A2, 124G7, 132A7, and 133F7) were <70% (data not shown). These top 10 purified rabbit IgGs were further screened using a competitive Biacore assay as described above. Figure 9 As shown in Figure A, the clonal 135F1 rabbit IgG antibody exhibited the highest activity in inhibiting the hTSLP-hTSLPR interaction, followed by 139F11, reference antibody 10985, 139B10, 37E4, 110D9, and 121B5. The preferred clone 135F1 completely blocked the hTSLP-hTSLPR interaction at 5.5 nM. Isotype control antibodies 11239, 8F7, 40H8, and 120D6 did not show inhibition in this assay.

[0189] Primary human monocytes were purified from PBMCs using Stemcell Technologies' EasySep™ Human Monocyte Isolation Kit (catalog number 19359). On the day of assay, monocytes were seeded in 96-well plates and treated with a constant amount of hTSLP (concentration close to EC50). 50 The rabbit was treated with preferred anti-TSLP rabbit antibodies 135F1, 139F11, or 139B10 at serial dilutions for 24 hours. The level of CCL-17 chemokine in the supernatant was determined using a human CCL-17 ELISA kit (R&D catalog number DY364). Figure 9 As shown in Figure B, compared with the blank buffer, human isotype control antibody 10861 or rabbit isotype control antibody 11176 did not inhibit CCL17 release at the highest concentration of 100 nM. However, all three rabbit monoclonal antibodies, 135F1 (#70), 139F11 (#72), and 139B10 (#66), inhibited CCL17 release, with an IC50 value of [missing value]. 50 The concentrations were 4.238 nM, 5.275 nM, and 7.500 nM, respectively. Clearly, clone 135F1 exhibited the highest potency in this cell-based biological assay. In conclusion, the lead rabbit antibody clone 135F1 (SEQ ID NO: 39-42) was selected for humanization via CDR transplantation and yeast display. Example 5: Humanization of rabbit anti-hTSLP antibody clone 135F1 by CDR transplantation and yeast display

[0190] To successfully develop rabbit monoclonal antibodies into therapeutic agents, the rabbit antibodies should be humanized to reduce the likelihood of patients developing neutralizing antibodies against non-human molecules. The humanization of the rabbit antibody is based on the pioneering work of Zhang Y et al., mAbs 2017; 9(3):419–429. The VL amino acid sequence (SEQ ID NO: 41) of the lead rabbit antibody clone 135F1 was obtained using the Kabat numbering scheme (…). www.bioinf.org.uk / abs / abnum / The location of all residues was determined, and the results are shown below. Table 11. Kabat numbers of amino acid residues in the VL region of rabbit anti-human TSLP antibody clone 135F1 The regions CDR1 (24-34), CDR2 (50-56), and CDR3 (89-97) are highlighted in gray, and the additional Cys residue at position 80 in FW3 is shown in bold.

[0191] The VH amino acid sequence (SEQ ID NO: 39) of the lead rabbit antibody 135F1 was also used via the Kabat numbering scheme. www.bioinf.org.uk / abs / abnum / The location of all residues was determined, and the results are shown below. Table 12. Kabat numbers of amino acid residues in the VH region of rabbit anti-human TSLP antibody clone 135F1 The regions CDR1 (positions 31-35), CDR2 (positions 50-65), and CDR3 (positions 95-102) are highlighted in gray, and the additional Cys residue at position 50 in VH-CDR2 is shown in bold.

[0192] In all rabbit antibodies, the Cys residue at position 80 of VL-FW3 forms a covalent disulfide bond with the Cys residue at position 50 of VH-CDR2. However, this additional disulfide bond is absent in all human antibodies. To begin humanization of clone 135F1, the Cys residue at position 80 of VL-FW3 was changed to Pro because this Cys80 residue is located at the corner of the VL structure; the Cys residue at position 50 of VH-CDR2 was changed to Ser because Cys residues and Ser residues have similar sizes, see Zhang Y et al. mAbs 2017; 9(3):419–429. Using the new VL (C80P) and VH (C50S) sequences derived from clone 135F1, the humanization was performed by running the IgBlast program ( www.ncbi.nlm.nih.gov / igblast / Search for homologous human VL and VH sequences respectively. IGKV1-13*02 The germline shared the highest identity (67.8%) with the VL sequence of 135F1, and humans... IGHV3-74*01 The germline showed the highest sequence identity (60.4%) with the VH sequence of 135F1. The Kabat CDRs of the cloned 135F1 VL and VH were transplanted into human homologous sequences. IGKV1-13*02 and IGHV3-74*01 In the phylogenetic family.

[0193] Two β-sheets formed by the β-chain ↓C''↑C'↓C↑F↓G from the VL and VH regions stack together to form a barrel-like structure, aligning the connected CDR loops to confer antigen binding sites. Therefore, the β-chain ↓C''↑C'↓C↑F↓G between VL and VH is crucial for the correct folding and stability of the antibody; see Chiu M. L et al. Antibodies (Basel) 2019; 8(4):55. Using CLUSTALW software (… www.genome.jp / tools-bin / clustalw The amino acid sequence of the original rabbit VL clone 135F1 was compared with that of the human clone. IGKV1-13*02 The VL amino acid sequence was obtained by CDR transplantation in germline. Similarly, the original rabbit VH clone 135F1 and human-based clones were transplanted using CLUSTALW software. IGHV3-74*01 The amino acid sequences of VH transplanted from the CDR line were compared. Significant differences were found between the interface residues at positions 38, 42, 43, and 83 of the Kabat line in rabbit VL and human IGKV1-13*02, as well as at position 70 (Q / D), although this position is located in loop 4 of VL and may facilitate antigen binding (see Zhang Y et al. mAbs 2017; 9(3):419–429). Similarly, significant differences were found between the interface residues at positions 44, 45, 89, and 91 of the Kabat line in rabbit VH and human IGHV3-74*01. VH-CDR2... 55 DS 56 Motifs are potential hotspots for isomerization, which can lead to some instabilities. It is preferable to eliminate these potential instabilities in the early stages. Table 13. Degenerate codons used to construct the display library of humanized yeast clonal 135F1 against hTSLP. Key residues at the VL-VH interface were alternated between rabbit and human strains using degenerate codons, and the VH-CDR2 isomerization motif was paired with NNK codons. 55 DS 56 Potential hotspots are completely randomized. Due to the degeneracy of codons, additional amino acid residues can be introduced at certain positions. For example, the degenerate codon (A / G) (G / T) at position 43 of VL encodes Arg(R), Ile(I), Gly(G), and Val(V), not just the alternating R and V residues between rabbit 135F1 VL and human IGKV1-13*02.

[0194] To rapidly humanize the rabbit 135F1 antibody, six long oligonucleotides, approximately 100 nt in length, covering five degenerate codons in VL (as shown in Table 13 above), were used to assemble the full-length VL via PCR with short forward and reverse oligonucleotides. Similarly, six long oligonucleotides, approximately 100 nt in length, covering four degenerate codons and two randomized codons in VH, were used to assemble the full-length VH via PCR with another short forward and another reverse oligonucleotide. Fab libraries for yeast display were constructed as described in Example 2 above to select humanized Fab variants using MACS and FACS methods.

[0195] The induced yeast library was subjected to a first round of MACS with 500 nM biotinylated hTSLP (bn-hTSLP) and a second round of MACS with 100 nM biotinylated cyTSLP (bn-cyTSLP) to enrich positive conjugates. The collected yeast pools were further sorted by FACS in three rounds, with the antigen concentration decreasing sequentially: 10 nM bn-hTSLP antigen; 3 nM bn-cyTSLP antigen; 0.5 nM bn-hTSLP antigen. After final FACS sorting, 94 individual yeast colonies were randomly selected; well A01 contained unrelated yeast colonies, and well B01 contained positive yeast colonies. Yeast cells were induced using a streptavidin-APC complex with bn-hTSLP (or bn-cyTSLP) and ALEXA FLUOR. ® Labeled antibodies specific to the HA tag were analyzed by FACS. Of the 94 single colonies, 10 showed strong antigen binding (Q2 > 30%), 17 showed moderate binding (Q2 = 20–30%), and 21 showed weak binding (Q2 = 10–20%). The remaining yeast colonies showed negligible binding. All 48 conjugates were subjected to PCR to amplify the VL and VH fragments, followed by DNA sequencing. After sequence alignment, 6 unique VHs and 6 unique VLs constituted the combinations of all 48 conjugates. Redundancy existed; for example, 20 conjugates shared the same VL, while the other 28 conjugates shared 5 other distinct VLs.

[0196] DNA gBlocks encoding the six VH and six VL molecules were synthesized and converted into human IgG1, as described in Example 2. The purified antibody was subjected to Biacore analysis as described above. Table 14 below shows that the preferred clone QB1341 (VH and HC are SEQ ID NO: 43 and 44, respectively; VL and LC are SEQ ID NO: 45 and 46, respectively) has a binding affinity of 1.76 nM for the hTSLP (R127A+R130S) antigen (SEQ ID NO: 34), which is about 10 times lower than the 0.184 nM binding affinity of the reference antibody QB10985. Table 14. Biacore kinetics of the lead humanized anti-hTSLP antibody QB11341

[0197] Human monocytes were purified from healthy donors using Stemcell Technologies' EasySep™ Human Monocyte Isolation Kit (catalog number 19059). On the day of assay, monocytes were seeded in 96-well plates and treated with constant amounts of different forms of hTSLP (concentration close to EC50). 50 The cells were treated with serially diluted anti-hTSLP antibody for 24 hours. The levels of hTSLP- or cyTSLP-induced CCL-17 chemokines in the supernatant were measured using a human CCL-17 ELISA kit (R&D Systems, catalog number DY364). Table 15. Inhibition of CCL-17 secretion in human PBMCs by anti-hTSLP antibody (NA means "unavailable").

[0198] The results in Table 15 show that, in hTSLP-induced CCL-17 secretion, the preferred anti-hTSLP clone QB11341 was 30 to 50 times less potent than the reference analog QB10985, while the antibody isotype control showed no inhibitory effect. Table 16 Inhibition of CCL-17 secretion in human PBMCs by anti-hTSLP antibody NA means "unavailable".

[0199] The results in Table 16 show that, in cyTSLP-induced CCL-17 secretion, the potency of the preferred anti-hTSLP clone QB11341 was 15 to 40 times lower than that of the reference analog QB10985, while the antibody isotype control showed no inhibitory effect. Example 6: Affinity maturation of the preferred anti-hTSLP antibody QB11341 by yeast display to improve its activity.

[0200] The preferred humanized anti-hTSLP clone QB11341 exhibits a binding affinity of 1.76 nM for the hTSLP (R127A+R130S) antigen, approximately 10-fold lower than that of the anti-hTSLP reference analog QB10985 (see Table 14). Generally, higher binding affinity of antagonistic antibodies leads to higher blocking activity. Further engineering of the VL region of clone QB11341 using yeast display technology can improve binding affinity for both hTSLP and cyTSLP antigens, potentially enhancing blocking efficacy.

[0201] The degenerate codon NNK was used to completely randomize five residues in the designed oligonucleotides: VL-CDR1 (position 34), VL-CDR2 (position 53), and VL-CDR3 (positions 91, 92, and 93), as shown in Table 17 below. Table 17. Kabat numbers of amino acid residues in the VL region of humanized anti-hTSLP clone QB11341 The CDR1 (positions 24-34), CDR2 (positions 50-56), and CDR3 (positions 89-97) regions are highlighted in gray. The bold residues at positions 34, 53, 91, 92, and 93 are randomized using the NNK codon for yeast display technology to further enhance binding affinity. The additional Cys residue at position 80 of VL-FW3, indicated by bold and underline, is changed to Pro.

[0202] A novel VL library was constructed and linked to the VH region of the parental clone QB11341 to display Fab fragments on the yeast surface. Two rounds of MACS selection and three rounds of FACS sorting were performed as described in Example 2 to screen for beneficial VL variants that would result in higher binding affinity. To identify Fab fragments with cross-species binding to human and cynomolgus monkey TSLP antigens, bn-hTSLP and bn-cyTSLP antigens were alternately applied during yeast display. After screening 94 single colonies by FACS analysis, the VL fragments of the top 20 conjugates were amplified by PCR and their sequences were revealed by DNA sequencing. The results showed that there were 8 different VLs among the top 20 conjugates. gBlocks of DNA sequences were designed using the amino acid sequences of all 8 VL variants and converted to full-length LCs as described in Example 2. Finally, 8 novel recombinant antibodies were prepared by transient transfection of ExpiCHO cells, purified using a protein A column, and subjected to Biacore analysis. The results showed that the novel antibody QB11548 (SEQ ID NO: 43, 44, 47, 48) exhibited higher binding affinity for both hTSLP (R127A+R130S) and cyTSLP (R127A+R130S) antigens than its parental clone QB11341. QB11548 incorporated the K53R substitution in VL-CDR2 and the F92R substitution in VL-CDR3 to enhance binding affinity. The inhibition of CCL17 secretion from TSLP-induced PBMCs also indicated that clone QB11548 possessed stronger blocking activity than its parental clone QB11341. Table 18. Inhibition of CCL-17 secretion in human PBMCs by anti-hTSLP antibody NA means "unavailable".

[0203] The results in Table 18 show that, in hTSLP-induced CCL-17 secretion, the preferred anti-hTSLP clone QB11548 was 5 to 10 times less potent than the reference analog QB10985, while the antibody isotype control showed no inhibitory effect. Table 19 Inhibition of CCL-17 secretion in human PBMCs by anti-hTSLP antibody NA means "unavailable".

[0204] The results in Table 19 show that, in cyTSLP-induced CCL-17 secretion, the preferred anti-hTSLP clone QB11548 was 7-12 times less potent than the reference analog QB10985, while the antibody isotype control showed no inhibitory effect. Example 7: Humanization of rat anti-hTSLP antibody and affinity maturation via yeast display

[0205] A rat anti-hTSLP antibody clone 23B12 derived from a hybridoma has been deposited at the American Center for Type Culture Collection (10801 University Blvd., Manassas, VA 20110-2209, USA) under patent accession designation "PTA-7951". This hybridoma was deposited on October 26, 2006, under the conditions of the Budapest Treaty, and received accession number PTA-79-51. The VH and VL amino acid sequences of clone 23B12 were extracted from patent US8,232,372B2, SEQ NO: 57 and 59. The CDR in the VH of clone 23B12 was transplanted into a human lineage. IGHV1-69*06 and IGHJ1*01 In China, the CDR from clone 23B12 VL was transplanted into a human lineage. IGKV3-20*01 and IGKJ4*01 In this study, appropriate reversion mutations were introduced into the humanized antibodies to maintain correct folding and structure. Four humanized antibody variants were prepared by transient transfection of ExpiCHO cells and purified by protein A chromatography as described in Example 2 above. One variant, hz-3C QB10990 (SEQ NO: 61-64), showed good yield and comparable binding to hTSLP and cyTSLP antigens; however, this antibody exhibited only a 90.2% main peak and a 9.2% HMW peak in SEC analysis. To mitigate the aggregation problem, a new round of antibody engineering was conducted by testing the effects of different germline residues in the original rat antibody 23B12. An antibody variant, hz-3C-V6 (QB11060, SEQ NO: 63-66), exhibited high yields with D9P and S108L reversion mutations in FW1 and FW4 of the VH region, respectively; high SEC characteristics (97.9% main peak); rapid Kon and high RU (response units) as analyzed by Biacore; and a binding difference of <10-fold compared to the cyTSLP antigen. However, the binding affinity to hTSLP (R127A+R130S) was approximately 1.62 nM, which was 8.8-fold lower than that of the reference QB10985 (see Table 20 below). Table 20 Biacore kinetics of humanized anti-hTSLP antibody clone QB11060 (hz-3C-V6) and reference antibody QB10985.

[0206] To obtain higher blocking activity, as described in our patent US11,124,570 and Example 2 above, five amino acid residues in the VL of the NNK codon-resistant-hTSLP clone QB11060 were completely randomized for yeast display to rapidly identify beneficial variants that confer higher binding affinity and potentially higher biological activity, see Table 21 below. Table 21. Kabat numbers of amino acid residues in the VL region of humanized anti-hTSLP antibody clone QB11060 The regions CDR1 (positions 24-34), CDR2 (positions 50-56), and CDR3 (positions 89-97) are highlighted in gray. The bold residues at positions 28, 31, 91, 92, and 94 are randomized using the NNK codon and used in yeast display technology to further improve binding affinity.

[0207] During the selection process, bn-hTSLP and bn-cyTSLP antigens were used alternately for two rounds of MACS selection and three rounds of FACS sorting to enrich high-affinity binders and thus identify cross-species binding clones. The VL fragments of the top 20 binders were amplified by PCR and sequenced. Six unique clones with novel VL sequences were then converted into human IgG4 produced in ExpiCHO cells. Characterization, including Biacore, SEC analysis, and cell-based CCL17 release assays, was performed to identify the lead clone QB11237 IgG4, which, compared to the original clone 23B12, contained five novel amino acid residues in its VL-CDR: P28H, I31R, T91S, F92Y, and L94F. Example 8: Analysis of the inhibitory mechanisms of anti-hTSLP antibodies QB11341 and QB11237 using Biacore

[0208] To explore how anti-hTSLP antibodies QB11341 and QB11237 inhibit hTSLP-induced biological effects, a Biacore assay was performed to assess (1) the ligand-receptor interaction between hTSLP and hTSLPR; (2) the direct blocking of the hTSLP-hTSLPR axis by antibody QB11341 and the blocking of the hTSLP-hIL7Rα interaction by antibody QB11237; and (3) the inhibitory level of the hTSLP-hIL7Rα interaction by antibody QB11237.

[0209] R&D Systems' hTSLPR-Fc dimer (catalog number 981-TR) was directly immobilized on the CM5 surface via amine coupling. Flow cells 2, 3, and 4 were set to high, medium, and low densities, respectively, while flow cell 1 was left unimmobilized with hTSLPR-Fc. Running buffer was passed through flow cell 1, thus the background binding in flow cell 1 was used as a reference. The reference binding was subtracted from the results of flow cells 2, 3, or 4 to obtain the true binding. The hTSLP monomer antigen ligand was injected onto the CM5 surface at concentrations of 0, 0.62, 1.85, 5.56, 16.7, and 50 nM, with binding times of 3 minutes and dissociation times of 5 minutes. Kinetic sensor plots were analyzed using BIAevaluation 4.1.1. Figure 10 As shown in Figure A, the hTSLP ligand binds to the fixed hTSLPR-Fc in a dose-dependent manner in a 1:1 ratio, and the estimated binding affinity between hTSLP and hTSLPR is approximately 6 nM.

[0210] After demonstrating direct hTSLP-hTSLPR interaction on the CM5 surface, CM5 chips immobilized with the same hTSLPR-Fc were injected with individual hTSLP or hTSLP-antibody complexes premixed with antibodies QB11341 or QB11237 for 3 minutes. Finally, 50 nM hIL7Rα (R&D Systems, catalog number 206-IR) or blank buffer was injected onto the CM5 surface, with a binding time of 2 minutes and a dissociation time of 5 minutes. Figure 10 As shown in section B, when only hTSLP was implanted, a peak of 0-300 RU was observed at time points 20-200 seconds. Subsequently, when hIL7Rα was implanted consecutively, an additional peak of 300-900 RU was observed. When no hIL7Rα was implanted, the peak gradually shifted downwards due to the slow dissociation of the hTSLP-hTSLPR complex. These results reflect that hTSLP can bind to hTSLPR on the CM5 chip surface, and that hIL7Rα further binds to hTSLP-hTSLPR to form a three-molecule complex. Figure 10 As shown above (B), a peak of 0-1200 RU was observed when the mixed hTSLP-QB11237 was injected, indicating that hTSLP in the hTSLP-QB11237 complex can still bind to hTSLPR on the CM5 surface. From time point 200-450 seconds, no additional peaks were observed regardless of whether hIL7Rα was injected; instead, the sensing plot decreased from 200 seconds onwards. The results indicate that hIL7Rα can no longer bind to the hTSLPR-hTSLP-QB11237 complex. Figure 10As shown below, when the premixed hTSLP-QB11341 complex was first injected into the CM5 surface immobilized with hTSLPR-Fc, no additional sensing peaks were observed, indicating that QB11341 completely prevented the binding of hTSLP to hTSLPR-Fc.

[0211] The hTSLPR-Fc-immobilized CM5 chip was injected with 20 nM hTSLP, with a binding time of 2 minutes and a dissociation time of 100 seconds. Subsequently, 20 nM anti-hTSLP antibody QB11341 or QB11237 was injected onto the CM5 surface, with a binding time of 3 minutes and a rapid dissociation time of 1 minute. Finally, 50 nM hIL7Rα or blank running buffer was injected, with a binding time of 2 minutes and a dissociation time of 2 minutes. Figure 10 As shown in Figure C, a peak of 0-300 RU was observed from time point 20-140 seconds, indicating that the ligand hTSLP binds to the hTSLPR immobilized on the CM5 surface. From time point 140-240 seconds, the peak gradually decreased, indicating that the ligand hTSLP began to dissociate from the hTSLP-hTSLPR complex. At time point 240 seconds, when anti-hTSLP antibody QB11237 or QB11341 was injected, a sharp peak of approximately 1000 RU was observed for QB11237, while the sensor map for antibody QB11341 continued to shift downwards. The results indicate that antibody QB11237 can still bind to the hTSLP-hTSLPR complex, but once hTSLP forms a complex with hTSLPR, antibody QB11341 cannot bind to hTSLP. This means that QB11237 is not a direct blocking antibody for hTSLP, while QB11341 is a direct blocking agent for hTSLP. At time 440 seconds, either hIL7Rα or blank buffer was injected. A peak of 100 RU to 200 RU was observed for antibody QB11341, indicating that hIL7Rα can bind to the formed hTSLPR-hTSLP-QB11341 triploidal complex, as hTSLP still has an interface available for hIL7Rα binding. However, no significant peak was observed for antibody QB11237 regardless of the injection of hIL7Rα. By calculating the peak height difference with and without QB11237 antibody, the inhibition was estimated to be approximately 93%. The results indicate that antibody QB11237 blocks the interaction between hTSLP and hIL7Rα.

[0212] In summary, two types of hTSLP inhibitors were identified. Anti-hTSLP antibody QB11341 (and its derivatives QB11548 and QB11764 described in Examples 9-12 below) is a direct blocker of hTSLP, preventing the interaction between hTSLP and hTSLPR. Anti-hTSLP antibody QB11237 (and its derivative QB11718 described in Examples 9-12 below) does not directly block the hTSLP-hTSLPR interaction; instead, antibody QB11237 prevents the binding of hTSLP to hIL7Rα. As described in the background section, the hTSLP receptor consists of two subunits, hTSLPR and hIL7Rα. Both receptor units have intracellular domains (ICDs) that can be phosphorylated for signal transduction. Therefore, inhibiting the formation of either the hTSLP-hTSLPR complex or the hTSLP-hIL7Rα complex can prevent hTSLP-mediated biological effects. Example 9: Generation of MabPair composed of anti-IL-33 IgG4 and different anti-TSLP IgG1-D265A antibodies

[0213] As described in our patent application WO2021041678A1, chain loss experiments were performed to evaluate the natural pairing ability of matched and mismatched HC and LC. Qiagen was used. ® The Midi-prep kit (Qiagen NV, the Netherlands) was used to purify HC and LC plasmid DNA encoding anti-hIL-33 antibody QB11465, anti-hTSLP antibody QB11548, and anti-hTSLP antibody QB11237, respectively. The resulting DNA was diluted in water and purified using EPPENDORF TUBES. ® Mixed DNA. A set of 4 tubes of mixed DNA was transiently transfected into EXPICHO. ™ In cells, to assess whether non-homologous HC / LC pairing occurs spontaneously. Tube 1 contains DNA encoding anti-hTSLP antibody HC (HC1) and its homologous LC (LC1). Tube 2 contains DNA encoding a non-homologous HC / LC pair consisting of anti-hTSLP antibody HC1 and anti-hIL-33 antibody LC2. Tube 3 contains DNA encoding a homologous HC / LC pair consisting of anti-hIL-33 antibody HC2 and LC2. Tube 4 contains DNA encoding anti-hIL-33 non-homologous HC2 and anti-hTSLP antibody LC1.

[0214] More details, using LIPOFECTAMINE ® In 2000, EXPICHO was transfected with plasmid DNA encoding the test antibody in a 24-well deep-well blocking plate. ™Cells. Cells were continuously shaken at 150 rpm for 12 days at 37°C. The supernatant was harvested by centrifuging at 1200 rpm for 15 minutes to precipitate the cells. For all samples (none reduced), 5 μL of supernatant and 5 μL of 2X Laemmli sample buffer (65.8 mM Tris-HCl, pH 6.8; 2.1% sodium dodecyl sulfate (SDS); 26.3% (w / v) glycerol; 0.01% bromophenol blue) were heated at 70°C for 10 minutes. The treated samples were loaded into 4–15% CRITERION. ™ TGX STAIN-FREE ™ Fill pre-prepared SDS-PAGE gels (Bio-Rad Laboratories, Inc., Hercules, CA, catalog number 567-8085) into the wells. Electrophoresis was run at 200 V for 45 minutes. CHEMIDOC from Bio-Rad Laboratories, Inc. was used. ™ XRS+ imager visualization.

[0215] like Figure 11 As shown in Figure A, anti-hTSLP QB11548 (lane 1) and anti-hIL-33 QB11465 (lane 3) antibodies were well expressed due to the strong intensity of the full-length antibody band at approximately 150 kDa. The expression level of the antibody generated from the homologous HC1 / LC1 pair of anti-hTSLP QB11548 (lane 1) was slightly higher than that generated from the non-homologous pair of HC1 (from anti-hTSLP) and LC2 (from anti-hIL-33) in lane 2. This indicates that the expression of HC1 of anti-hTSLP QB11548 together with its own LC1 is superior to that together with the non-homologous LC2 from the anti-hIL-33 antibody QB11465. Interestingly, the expression levels of the non-homologous LC1 (from anti-hTSLP) and HC2 (from anti-hIL-33) pairs were slightly higher than those of the homologous HC2 / LC2 pair, see [reference needed]. Figure 11 A. Compare lanes 3 and 4. These results indicate that HC2 expression of anti-hIL-33 QB11465 is superior to that of anti-hTSLP QB11548 when expressed together with its non-homologous LC1. Therefore, a key issue to address in engineering these antibodies to prepare MabPair is the mismatch between HC2 expression of anti-hIL-33 QB11465 and LC1 expression of anti-hTSLP antibody QB11548, which is a direct blocker of the hTSLP-hTSLPR axis.

[0216] Chain deletion experiments were performed on anti-hTSLP antibody QB11237 and anti-IL-33 antibody QB11465, similar to those described above. Figure 11 As shown in B, the full-length antibody produced by co-expression of matched HC1 and LC1 of anti-hTSLP QB11237 (lane 1) was more abundant than the full-length antibody produced by co-expression of a mismatch between HC1 of anti-hTSLP antibody QB11237 and LC2 of anti-IL-33 antibody QB11465 (lane 2). However, the full-length antibody produced by matched HC2 and LC2 of anti-IL-33 antibody QB11237 (lane 3) was less abundant than the full-length antibody produced by a mismatch between HC2 of anti-hIL-33 antibody QB11237 and LC1 of anti-hTSLP antibody QB11237 (lane 4). This indicates that the main problem in preparing MabPair for these two antibodies in a single cell line is resolving the mismatch between HC2 of anti-hIL-33 antibody QB11465 and LC1 of anti-hTSLP antibody QB11237.

[0217] In the following experiments, the antibody was modified to enhance homologous HC / LC pairs, prevent non-homologous HC / LC pairs, and prevent HC / HC heterodimers. MabPair antibodies, consisting of a modified version of the anti-hTSLP antibody and an unchanged anti-hIL-33 antibody, were prepared as described in Examples 2 and 3 of US11,130,808 (which is incorporated herein by reference). Substitutions K147D, V173C, and C220G were introduced into the HC of the anti-hTSLP antibody QB11548 to enhance homologous strand pairing in CH1; substitution D265A was introduced to weaken effector functions (ADCC, ADCP, CDC) in CH2; and substitutions D399R and K409E were introduced to prevent the formation of heterodimer HCs in CH3. The DNA fragment encoding the modified HC of the anti-hTSLP antibody QB11548 was synthesized by IDT and subsequently subjected to a Gibson reaction with the expression vector pSB01 to assemble the DNA encoding the full-length HC. Accordingly, substitutions for S131K, S162C, and C214S were introduced into the LC of anti-hTSLP antibody QB11548. The modified anti-hTSLP antibody was renamed clone QB11764. SEQ ID NO: 52 and 51 show the amino acid sequence of anti-hTSLP QB11764 HC and the nucleic acid sequence encoding it, respectively. SEQ ID NO: 56 and 55 show the amino acid sequence of anti-hTSLP QB11764 LC and the nucleic acid sequence encoding it, respectively.

[0218] Because the main problem in preparing MabPairs for anti-hIL-33 QB11465 and anti-hTSLP QB11548 or QB11237 is to prevent mismatch between the HC2 of anti-hIL-33 QB11465 and the LC1 of anti-hTSLP antibody QB11548 or QB11237, the same set of substitutions K147D, V173C, C220G, D265A, D399R and K409E were introduced into the HC of anti-hTSLP antibody QB11237; and S131K, S162C and C214S were introduced into the LC of anti-hTSLP antibody QB11237. The new anti-hTSLP antibody with these substitutions was renamed clone QB11718. SEQ ID NO: 72 and 71 show the amino acid sequence of anti-hTSLP QB11718 HC and the nucleic acid sequence encoding it, respectively. SEQ ID NO: 76 and 75 show the amino acid sequence of anti-hTSLP QB11718 LC and the nucleic acid sequence encoding it, respectively.

[0219] Plasmid DNA encoding the HC and LC of one or two different antibodies was placed in a series of EPPENDORF tubes. The tubes contained DNA encoding the following antibodies: Tube 1 contained DNA encoding anti-hTSLP antibody QB11764 HC (HC1) and its homologous LC (LC1). Tube 2 contained DNA encoding a non-homologous HC / LC pair consisting of HC1 of anti-hTSLP QB11764 and LC2 of anti-hIL-33 antibody QB11465. Tube 3 contained DNA encoding a homologous HC2 / LC2 pair consisting of HC2 and LC2 of anti-hIL-33 antibody QB11465. Tube 4 contained DNA encoding a non-homologous HC2 of anti-hIL-33 QB11465 and LC1 of anti-hTSLP antibody QB11764. Tube 5 contained all four DNA sequences encoding anti-hTSLP QB11764 (HC1 and LC1) and anti-hIL-33 antibody QB11465 (HC2 and LC2). Tube 6 contained no plasmid DNA as a dummy control. Tube 7 contained DNA encoding anti-HER2 trastuzumab IgG1 (HC and LC) to assess transfection efficiency. The mixed plasmid DNA was used to transfect 3 mL of EXPICHO in a 24-well plate. ™ Cells. The cells containing transfected EXPICHO... ™ The cell plates were shaken at 37°C and 10% CO2 for 12 days. As described above, 10 µl of the harvested supernatant was loaded onto 4–15% CRITERION. ™ TGX STAIN-FREE ™ In each lane of the pre-prepared SDS-PAGE gel.

[0220] like Figure 11 As shown in C, full-length antibodies of 150 kDa were observed for homologous HC1 and LC1 (lane 1) of anti-hTSLP antibody QB11764 and homologous HC2 and LC2 (lane 3) of anti-hIL-33 antibody QB11465. However, no obvious band of 150 kDa was observed for non-homologous HC1 of anti-hTSLP antibody QB11764 and LC2 (lane 2) of anti-hIL-33 antibody QB11465; or for HC2 of anti-hIL-33 antibody QB11465 and LC1 of anti-hTSLP antibody QB11764. A mixture of all four chains of anti-hTSLP QB11764 and anti-hIL-33 antibody QB11465 produced an antibody mixture of approximately 150 kDa (lane 5), while simulated transfection in lane 6 did not produce any antibody. The results showed that when MabPair was prepared in the same host cell, only matched HC and LC could produce full-length antibodies, while mismatched HC and LC were prevented from producing full-length antibodies.

[0221] Similar results were obtained for the preparation of MabPair, composed of anti-hTSLP antibody QB11718 and anti-hIL-33 antibody QB11465. Figure 11 As shown in lane D, only the matched HC and LC pairs of anti-hTSLP antibody QB11718 produced full-length antibodies (lane 1); the matched HC and LC pairs of anti-hIL-33 antibody QB11465 produced full-length antibodies (lane 3). Mismatched HC1 and LC2 in lane 2, or mismatched HC2 and LC1 in lane 4, did not produce any full-length antibodies. A mixture of all four strands of anti-hTSLP QB11764 and anti-hIL-33 antibody QB11465 produced an antibody mixture of approximately 150 kDa (lane 5), and the HC and LC pairs of anti-HER2 trastuzumab produced full-length antibodies (lane 7), indicating good transfection efficiency in the experiment. The results suggest that after introducing an appropriate mutation into anti-hTSLP antibody QB11718, only matched HC and LC pairs can produce full-length antibodies when preparing MabPair products, while mismatched HC and LC pairs are prevented from producing full-length antibodies. Example 10: Mass spectrometry confirmation of MabPair QB11750, composed of anti-IL-33 IgG4 antibody QB11465 and anti-TSLP IgG1-D265A antibody QB11718.

[0222] EXPICHO was transiently transfected with four plasmid DNAs encoding the corresponding HC and LC. ™Cells were used to produce recombinant monoclonal anti-hIL33 IgG4 antibody QB11465 and anti-hTSLP IgG1-D265A antibody QB11718. After incubation with shaking at 37°C and 10% CO2 for 12 days, the supernatant was harvested and purified by protein A column according to standard protocol to obtain MabPair product QB11750. Mass spectrometry analysis was performed to confirm the absence of any mismatch between HC and LC. The mass spectrometry method used is described by Thompson et al. (2014), mAbs6:1: 197-203, which is incorporated herein by reference in its entirety in WO 2017 / 205014, page 92, line 31 to page 94, line 10. Figure 18 As described in the document, these sections of WO 2017 / 205014 are incorporated herein by reference.

[0223] For this analysis, 20 µg of purified MabPair QB11750 (composed of anti-hIL-33 IgG4 antibody QB11465 and anti-hTSLP IgG1-D265A antibody QB11718) was incubated at 37 °C with 2 µl of PNGase F endopeptidase (NewEngland Biolabs, catalog number P0704S) in 20 µl of 50 mM Tris at pH 7.5 for 16 h. PNGase F is the most efficient enzyme for removing almost all N-linked oligosaccharides from glycoproteins. PNGase F is an amidase that cleaves between the innermost GlcNAc and asparagine residues of high-mannose, heterozygous, and complex oligosaccharides. After deglycosylation, half of the sample was reduced by incubation at 55 °C for 30 min in a buffer containing 4 M guanidine hydrochloride, 50 mM Tris at pH 8.0, and 50 mM DTT. Non-reduced and reduced samples were analyzed by HPLC-MS using an Agilent 6224 Precision Mass Time-of-Flight mass spectrometer equipped with an ESI source and coupled to an Agilent 1200 HPLC. An Agilent Pursuit diphenyl column (2.0 × 150 mm, 3 μm) was used at 80 °C and a flow rate of 0.4 µL / min. Mobile phase A consisted of water containing 0.1% trifluoroacetic acid (TFA), and mobile phase B consisted of isopropanol (IPA):acetonitrile (ACN):water (70:30:10) containing 0.9% TFA. Mobile phase B was initially maintained at 10%, then increased to 32% B over 5 minutes, and then to 42% B over 35 minutes. The solvent was then changed to 90% B and held for 4 minutes to clean the column. Finally, the solvent was restored to 10% B and held for 4 minutes to reequilibrate the column. The MS instrument parameters were as follows: drying gas temperature, drying gas flow rate, and nebulizer were set to 300ºC, 12 L / min, and 40 psig, respectively. Capillary, fragmentation, skimmer 1, and Oct RF Vpp were set to 4500V, 250V, 60V, and 750V, respectively. The instrument was calibrated at a high resolution of 4 GHz in the m / z range of 100 to 3000. HPLC-MS data were analyzed using Agilent MassHunter Qualitative and BioConfirm software. The theoretical sizes of all deglycosylated antibody species that may form in cells containing DNA encoding anti-hIL-33 IgG4 antibody QB11465 and anti-hTSLP IgG1-D265A antibody QB11718 are shown in Table 22 below. Table 22: Theoretical mass of deglycosylated antibodies in MabPair QB11750

[0224] Figure 12 Subfigure A shows the two main peaks of deglycosylated antibodies produced by host cells containing DNA encoding MabPair QB11750 (composed of anti-hIL-33 IgG4 antibody QB11465 and anti-hTSLP IgG1-D265A antibody QB11718) under UV detection. Subfigure B shows the complete mass of anti-hTSLP IgG1-D265A antibody QB11718 (top) and anti-hIL-33 IgG4 antibody QB11465 (bottom). As shown, the observed mass of the anti-hTSLP IgG1-D265A antibody QB11718 main peak is 145678.44 Daltons (Da), which differs from the theoretical mass of 145670.02 Da by 8.42 Da, a deviation of 57.8 ppm. The minor peak at 145839.43 Da was glycosylated anti-hTSLP IgG1-D265A antibody QB11718, as the observed difference was 160.99 Da (145839.43 – 145678.44), with the 162 Da mass increase representing glycosylated lysine, see Wei B. et al. MABS 2017; 9(4):586–594 and Lapolla A et al. J Am Soc Mass Spectrom 2004;15:496–509. Another minor peak was disaccharidated, as the observed difference between the two minor peaks (146001.51 – 145839.43) was 162.08 Da. Similarly, the main peak of the anti-hIL-33 IgG4 antibody QB11465 showed an observed mass of 146002.30 Da, a difference of 45.48 ppm (6.64 Da) from the theoretical mass of 145995.66 Da. The minor peak at 146163.42 Da is the glycosylated version, and the minor peak at 146325.70 Da is the disaccharidated version of the anti-hIL-33 IgG4 antibody QB11465. Since the mass deviations from the theoretical masses are all less than 100 ppm, these experimentally determined masses indicate that the main peaks of both antibodies can originate from antibodies with homologous HC / LC pairs.

[0225] Under reduction conditions, the MabPair QB11750 exhibits four distinct peaks under UV detection. Figure 12 C), anti-hIL-33 LC ( Figure 12 The mass of (D, above) is 24026.88 Da, which differs from the theoretical mass of 24026.53 Da by 14.6 ppm; anti-hTSLP LC ( Figure 12The mass of D (second from the top) is 23664.36 Da, which differs from the theoretical mass of 23664.31 Da by 2.1 ppm; anti-hTSLP HC ( Figure 12 C, the third one from the top) has a mass of 49188.91 Da, which differs from the theoretical mass of 49187.03 Da by 38.2 ppm; anti-hIL-33 HC ( Figure 12 The mass of (D, below) was 48989.36 Da, differing from the theoretical mass of 48987.43 Da by 39.4 ppm. All experimental errors were well below the allowable error of 100 ppm. These results clearly demonstrate that MabPair QB11750, composed of anti-hIL-33 IgG4 antibody QB11465 and anti-hTSLP IgG1-D265A antibody QB11718, contains four different chains with expected masses.

[0226] MabPair product QB11750 was digested with the IdeS protease to generate the F(ab')2 fragment, as the IdeS protease preferentially cleaves sequences below the hinge regions of IgG1 (CPPCPAPELLG ↓ GPSVFLFPP) and IgG4 (CPPCPAPELG ↓ GPSVFLFPP). Partial reduction was then performed using 2-MEA to generate the Fab fragment, as 2-MEA preferentially reduces interchain disulfide bonds in the hinge region while preserving intrachain disulfide bonds in VH and VL. Therefore, the Fab fragment in MabPair can be easily analyzed by mass spectrometry to assess the presence of chain mismatches in MabPair QB11750.

[0227] 400 µg of sample buffer was centrifuged three times at 15°C and exchanged three times with 14,000 xg to 50 mM sodium phosphate (NaP) and 150 mM NaCl at pH 6.6. The sample was digested overnight at 37°C with 100 units of IdeS. For the reduced sample, the non-reduced digest was diluted 1:2 in 100 mM NaP, 150 mM NaCl, 5 mM EDTA at pH 7.3, and 2.2 mM 2-MEA. The sample was thoroughly mixed and heated at 37°C for up to 1 hour, then quenched with 1 / 10 volume of 10% (v / v) formic acid. The non-reduced and reduced samples were injected separately into an Agilent 6224 Precision Mass Time-of-Flight mass spectrometer for analysis as described above.

[0228] like Figure 13As shown in Figure A, MabPair QB11750 treated with IdeS and 2-MEA exhibited 14 peaks under UV detection, with peaks 3, 6, 10, 11, and 13 being the main peaks. The expected theoretical masses of all possible combinations of F(ab')2 and Fab fragments are listed in Tables 23 and 24 below. Main peak 3 at 25230.94 Da is the half-Fc of anti-hTSLP antibody QB11718, differing from the theoretical mass of 25230.55 ppm by 15 ppm. Main peak 6 at 25216.28 Da is the half-Fc of anti-hIL-33 antibody QB11465, differing from the theoretical mass of 25216.76 Da by 19 ppm. Main peak 10 at 49071.14 Da is the Fab fragment of anti-hTSLP antibody QB11718, with an experimental error of 1 ppm compared to the theoretical mass of 49071.05 Da. Shoulder peak 9 at 49087.87 Da is the oxidized Fab fragment of anti-hTSLP antibody QB11718, as the mass difference between shoulder peak 9 and main peak 10 is 16.73 Da. Main peak 11 at 49247.96 Da is the Fab fragment of anti-hIL-33 antibody QB11465, with an experimental error of 0.8 ppm compared to the theoretical mass of 49248.00 Da. Shoulder peak 12 at 98140.84 Da is the F(ab')2 of anti-hTSLP antibody QB11718, and main peak 13 at 98492.51 Da is the F(ab')2 of anti-hIL-33 antibody QB11465, with experimental errors of 27.9 ppm and 5.2 ppm, respectively. Minor peak 14 at 98493.90 Da could be the F(ab')2 of anti-hIL-33 with reduced disulfide bonds, as the mass difference between main peak 13 and minor peak 14 is 1.39 Da. The presence of the F(ab')2 fragment indicates that 2-MEA did not completely reduce all F(ab')2 fragments to Fab fragments. Other minor peaks (1, 2, 4, 5, 7, 8) around the main Fc / 2 peaks 3 and 6 are modified Fc / 2 or LC of anti-hTSLP antibody or anti-hIL-33 antibody. All observed masses were well below the permissible experimental error of 100 ppm; for F(ab')2, there were no peaks with masses close to the eight non-homologous fragments; for Fab, there were no peaks with masses close to the two non-homologous fragments (see Tables 23 and 24 below). All these results clearly demonstrate that all HC-HC and LC-HC pairs were correctly paired to produce the designed MabPair antibody mixture. Table 23: Theoretical quality of the F(ab')2 fragment in MabPair QB11750 Table 24: Theoretical quality of the Fab fragment in MabPair QB11750 Example 11: Mass spectrometry confirmation of MabPair QB11823, composed of anti-IL-33 IgG4 antibody QB11465 and anti-TSLP IgG1-D265A antibody QB11764.

[0229] MabPair QB11823, composed of anti-hIL33 IgG4 antibody QB11465 and anti-hTSLP IgG1-D265A antibody QB11764, was produced via transient transfection and analyzed by mass spectrometry in the same manner as described in Example 10. Anti-hTSLP antibody QB11718 blocks the interaction between hTSLP and hIL7Rα, while anti-hTSLP antibody QB11764 directly blocks the interaction between hTSLP and hTSLPR (see Example 8 above). Figure 14 Subfigure A shows the two main peaks under UV detection after deglycosylation with PNGase F treatment. As indicated, the observed quality of the main peak at 144,022.65 Da is that of intact anti-hTSLP IgG1-D265A antibody QB11764, differing from the theoretical quality of 144,018.08 Da by 4.66 Da (32.36 ppm). The minor peak at 144,184.95 Da is that of glycosylated anti-hTSLP IgG1-D265A antibody QB11764, as the observed difference is 162.3 Da. Similarly, the main peak of anti-hIL-33 IgG4 antibody QB11465 shows an observed quality of 146,000.75 Da, differing from the theoretical quality of 145,995.66 Da by 35.28 ppm (5.15 Da). The minor peak at 146163.7 Da is the glycosylated version of the anti-hIL-33 IgG4 antibody QB11465, with a difference of 162.95 Da. Since the mass deviation from the theoretical mass is less than 100 ppm and no other antibody species originating from the mismatched HC / LC pair were observed (see Table 25 below), the masses determined by these experiments indicate that the major peaks of each antibody can originate solely from antibodies with homologous HC / LC pairs. Table 25: Theoretical mass of deglycosylated antibodies in MabPair QB11823

[0230] like Figure 14 As shown in Figure B, MabPair QB11823 processed by IdeS exhibits four main peaks under UV detection. Figure 14As shown in Figure C, peak 1 at 25230.30 Da represents the half-Fc (Fc / 2) of anti-hTSLP antibody QB11764, differing from the theoretical mass of 25230.55 Da by 9.9 ppm. Peak 2 at 25216.15 Da represents the half-Fc of anti-hIL-33 antibody QB11465, differing from the theoretical mass of 25216.76 Da by 24.2 ppm. Peak 3 at 98492.43 Da represents the F(ab')2 fragment of anti-hIL-33 antibody QB11465, with an experimental error of 17.4 ppm compared to the theoretical mass of 98490.72 Da. Peak 4 at 98486.25 Da represents the F(ab')2 fragment of anti-hTSLP antibody QB11764, with an experimental error of 14.6 ppm compared to the theoretical mass of 98484.81 Da. All observations were well below the allowable experimental error of 100 ppm; there were no peaks with masses close to the eight non-homologous F(ab')2 fragments, see Table 26 below. Table 26: Theoretical quality of the F(ab')2 fragment in MabPair QB11823

[0231] like Figure 15 As shown in Figure A, MabPair QB11823 treated with IdeS and 2-MEA exhibited four main peaks under UV detection. Main peaks 1 and 2 represent the F(ab')2 fragments of anti-hTSLP antibody QB11764 and anti-hIL-33 antibody QB11465, respectively. The presence of the F(ab')2 fragment indicates that 2-MEA did not completely reduce all F(ab')2 fragments to Fab fragments. Main peak B, with a mass of 49247.72 Da, is the Fab fragment of anti-hIL-33 antibody QB11465, with an experimental error of 6.9 ppm compared to the theoretical mass of 49247.38 Da. Main peak D, with a mass of 48246.10 Da, is the Fab fragment of anti-hTSLP antibody QB11764, with an experimental error of 34.8 ppm compared to the theoretical mass of 48244.42 Da. Other minor peaks A and C are Fab fragments added by 2-MEA. All observed masses were well below the permissible experimental error of 100 ppm; no peaks with masses close to the two non-homologous Fab fragments (48950.00 Da and 48541.80 Da) were observed (see Table 27 below). All of the above results clearly demonstrate that all HC-HC and LC-HC pairings were correctly matched to produce the designed MabPair antibody mixture. Table 27: Theoretical quality of the Fab fragment in MabPair QB11823 Example 12: Evaluation of the binding characteristics of anti-TSLP IgG1-D265A antibody by Biacore analysis

[0232] To comprehensively characterize the binding properties of lead anti-hTSLP antibodies QB11764 and QB11718, which have different mechanisms of inhibiting hTSLP-induced biological effects (Examples 4 and 6), a variety of commercial antigens were purchased and in-house prepared antigens were purified to provide a comprehensive binding profile. Table 28: TSLP antigens from different sources used for Biacore measurements

[0233] Goat anti-human IgG capture antibody was immobilized on the flow cells of a CM4 sensor chip using an amine conjugation kit and HBS-EP as the run buffer. Goat anti-human IgG reagent was prepared at 30 µg / mL in acetate 5.5 buffer for immobilization. Approximately 8000 RU were immobilized on each flow cell surface. Table 29. Biacore Measurement Conditions for Evaluating the Binding Properties of Anti-hTSLP Antibodies

[0234] To collect kinetic binding data, the analytes listed in Table 29 were diluted in running buffer (composed of HBS-EP + 0.05% BSA) and injected into flow cells (fc) 1 through 4, where fc2 captured QB11718, fc3 captured QB11764, and fc4 captured QB10985, all at approximately 100 RU. Fc1 was used as the reference flow cell. Analytes were injected at the concentrations listed in Table 29 at a flow rate of 30 µL / min and a detection temperature of 25 °C. The complexes were allowed to bind and dissociate according to the times specified in Table 29. At the end of each cycle, the surface was regenerated for 20 seconds with 10 mM glycine-HCl (pH 1.5). One analyte concentration for each analyte was repeatedly injected, and buffer blanks were flowed through the reference and captured ligand surfaces. Data alignment and dual-reference were performed using T200Evaluation software. Using the global data analysis option in the software, the data is fitted to a simple 1:1 Langmuir interaction model. Table 30: Summary of the binding characteristics of anti-hTSLP antibodies to monomeric hTSLP antigens Table 31: Summary of the binding characteristics of anti-hTSLP antibodies to monomeric cyTSLP antigen

[00235] When Biacore was measured using hTSLP antigen produced by *E. coli* (R&D Systems, 1398-TS-010), the lead anti-hTSLP antibodies QB11718 and QB11764 exhibited very comparable binding properties, with similar Ka, Kd, ​​and single-digit pM binding affinity (KD), see Table 30. When Biacore was measured using commercially available hTSLP antigen produced by mammalian cells (Acro, TSP-H52Hb; Sino 16135-H08H), the lead anti-hTSLP antibody QB11718 showed very comparable binding affinity to the reference antibody QB10985, while no significant binding was observed with the other lead anti-hTSLP antibody QB11764. When Biacore was performed using the internally prepared hTSLP natural antigen QB11630 generated in the presence of furin inhibitor, the lead anti-hTSLP antibody QB11718 showed a binding affinity very similar to the reference antibody QB10985, while another lead anti-hTSLP antibody QB11764 showed a binding affinity (KD = 1.54 x 10⁻⁶). -10 M) compared to QB10985 (KD = 5.07 x 10) -11 The affinity for cyTSLP was approximately 3-fold lower than that for reference QB10985; very similar results were obtained when the internally prepared hTSLP antigen QB11631, with two substituted R127A+R130S, was applied to Biacore measurements. These results indicate that antigens from different sources (prokaryotes and eukaryotes) can have different binding characteristics due to different post-translational modifications that produce antigens; the lead anti-hTSLP antibody QB11718 showed binding characteristics very comparable to the control QB10985 on different antigens, while another lead anti-hTSLP antibody QB11764 showed reduced or negligible binding when using different antigens, possibly due to the different epitopes (the epitopes bound by QB11764) being affected by post-translational modifications. As summarized in Table 31, the affinity of the lead anti-hTSLP antibody QB11718 for cyTSLP was slightly lower than that of the reference QB10985 and approximately 3-fold higher than that of the other lead anti-hTSLP antibody QB11764. Example 13: Evaluation of the blocking activity of anti-hIL-33 IgG4 antibody QB11465 by cell-based assay

[0235] Several cell-based assays were performed, including measuring IFNγ cytokine secretion from primary human NK cells. Figure 17 A) IL-5 secretion in primary human ILC2 cells ( Figure 17 B) and the phosphorylation level of p38 MAPK in primary human ILC2 cells ( Figure 17(C, 17D) to evaluate the biological activity of the lead anti-hIL-33 IgG4 blocking antibody QB11465.

[0236] Using Stemcell Technologies' EasySep ™ Human NK cell isolation kit (catalog number 17955) was used to purify primary human NK cells. The purified human NK cells were cultured at 30,000 cells / well in RPMI 1640 medium containing 2 mM L-glutamine, 10% heat-inactivated fetal bovine serum, and 100 units / mL penicillin-streptomycin. A constant amount of hIL-33 (concentration close to EC50) was added. 50 The supernatant was treated with 1 ng / mL IL-12 and serially diluted 1:3 anti-hIL-33 antibody for 24 hours. IFNγ cytokine levels in the supernatant were determined using the R&D Systems Human IFNγ ELISA Kit (catalog number DY285B) according to the manufacturer's instructions.

[0237] like Figure 17 As shown in Figure A, the isotype control antibody QB11827 failed to inhibit IFNγ secretion from primary human NK cells at a concentration of 1,000 nM, with IFNγ levels measured at approximately 1,200 pg / mL. However, the reference antibody QB11094 (anti-hIL-33 IgG4) effectively inhibited IFNγ secretion, with an IC50 concentration of [missing value]. 50 = 0.05 nM. The lead anti-hIL-33 IgG4 antibody QB11465 strongly inhibits IFNγ secretion, IC50... 50 = 1.0 nM, with a potency 20 times lower than the reference -3 antibody QB11094.

[0238] Using Stemcell Technologies' EasySep ™The Human ILC2 Enrichment Kit (catalog number 17972) was used to purify primary human type 2 innate lymphoid cells (ILC2) from PBMCs. The purified ILC2 cells were cultured in RPMI 1640 assay medium containing 2 mM L-glutamine, 10% heat-inactivated fetal bovine serum, 1 mM sodium pyruvate, 0.075% sodium bicarbonate, 100 mM HEPES, and 100 units / mL penicillin-streptomycin. Supplementation was performed every two weeks with 10 ng / mL each of human cytokines IL-2, IL-25, TSLP, and IL-33 to amplify for at least 3–4 weeks (approximately 40-fold amplification) or up to 6–8 weeks (>100-fold amplification). Prior to assay, ILC2 cells were washed three times with assay medium and then incubated overnight in a tissue culture incubator to reduce background signal. On the day of the assay, ILC2 cells were seeded at 30,000 cells / well and treated with a certain amount of hIL-33 (Biolegend, catalog number 581804) (concentration close to EC50). 90 The supernatant was treated with serially diluted anti-hIL-33 antibody at a 1:3 ratio for 48 hours, with isotype control IgG4 antibody added at the highest concentration of 1,000 nM. The hIL-5 level in the supernatant was determined using a human IL-5 ELISA kit (R&D Systems, catalog number DY205) according to the manufacturer's instructions.

[0239] like Figure 17 As shown in Figure B, the isotype control antibody QB11827 failed to inhibit IFNγ secretion from primary human NK cells at a concentration of 1,000 nM, with hIL-5 levels measured at approximately 300 pg / mL. However, the reference-3 anti-hIL-33 IgG4 antibody QB11094 effectively inhibited hIL-5 secretion, with an IC50 value of [missing value]. 50 = 0.228 nM. The lead anti-hIL-33 IgG4 antibody QB11465 strongly inhibits hIL-5 secretion, IC50... 50 = 2.39 nM, its potency is about 10 times lower than that of the reference -3 antibody QB11094.

[0240] Prior to the assay, isolated ILC2 cells were washed three times with assay medium and then incubated overnight in a tissue culture incubator to reduce background signal. On the day of assay, ILC2 cells were seeded at 100,000 cells / well and treated with a specific amount of hIL-33 or cyIL-33 (concentration close to EC50). 90Potency was determined by treating the wells with serially diluted anti-IL33 antibody at a 1:3 ratio for 15 minutes. p38 MAPK phosphorylation levels were measured using the Perkin Elmer AlphaLISA SureFire Ultra p-38 MAPK HV Detection Kit (Shelton, CT) according to the manufacturer's instructions. Signals in each well were read using a microplate reader with α-signal detection capability.

[0241] like Figure 17 As shown in Figure C, the isotype control antibody QB11827 did not inhibit p38 MAPK phosphorylation of ILC2. However, the reference-3 anti-hIL-33 IgG4 antibody QB11094 strongly inhibited p38 MAPK phosphorylation, with an IC50 score of 1.5%. 50 = 0.87 nM. The lead anti-hIL-33 IgG4 antibody QB11465 effectively inhibited p38 MAPK phosphorylation, IC50... 50 = 0.33 nM, its potency is approximately 2.5 times higher than the reference -3 antibody QB11094. To test whether the anti-hIL-33 antibody can effectively block cyIL-33-induced p38 MAPK phosphorylation in ILC2, hIL-33 and cyIL-33 ( Figure 17 D). The isotype control antibody QB11827 did not inhibit hIL-33 or cyIL-33-induced p38 MAPK phosphorylation at the highest concentration of 2,000 nM. The lead anti-hIL-33 IgG4 antibody QB11465 effectively inhibited p38 MAPK phosphorylation induced by hIL-33, with an IC50 value of [missing value]. 50 = 0.82 nM; When using cyIL-33 to induce p38 MAPK phosphorylation, IC50 = 0.82 nM; 50 = 4.4 nM.

[0242] In summary, our lead anti-hIL-33 IgG4 antibody QB11465 strongly inhibits hIL-33-induced biological functions, and cross-species binding to cyIL-33 leads to inhibition of cyIL-33-induced biological activity. The anti-hIL-33 reference-3 antibody QB11094 is generally more effective than our lead antibody QB11465 in long-term cell-based assays (24 h or 48 h), most likely due to its slower dissociation rate (Kd), but less potent than our lead antibody QB11465 in short-term p38 MAPK cell-based assays (15 min). Example 14: Anti-TSLP IgG1-D265A antibodies QB11718 and QB11764 strongly inhibited hTSLP-induced proliferation of BaF3 cells stably expressing hTSLPR / hIL7Rα.

[0243] BaF3 cells stably expressing hTSLPR / hIL-7Rα were seeded at 10,000 cells / well and treated with a certain amount of hTSLP (concentration close to EC50). 50 The efficacy was determined by treating cells with serially diluted anti-hTSLP antibody for 72 hours. Proliferation was measured using the Promega (Madison, WI) CellTiter-Glo luminescent cell viability assay kit according to the manufacturer's instructions. Multiple forms of TSLP were used in this assay. Figure 18 hTSLP QB11630 (SEQ ID NO: 32) in A; Figure 18 hTSLP QB11631 (SEQ ID NO: 34) in B; Figure 18 cyTSLP QB11632 (SEQ ID NO: 35) in C; Figure 18 cyTSLP QB11633 (SEQ ID NO: 37) in D; Figure 18 E contains natural hTSLP derived from healthy donor 1; natural hTSLP derived from healthy donor 2. Natural hTSLP was derived from human small airway epithelial cells (SAECs) purchased from Lonza (catalog number CC-2547). SAECs were cultured to subconfluence according to the manufacturer's instructions, and then treated in growth medium at 37°C for 48 hours with 25 ng / mL human TNFα, 10 ng / mL IL-1α, and 10 ng / mL IL-1β. After treatment, the supernatant was collected; half was kept as a stock solution and frozen at -80°C, while the other half was concentrated 20-fold by centrifugation and then frozen at -80°C.

[0244] In summary, the isotype control antibody QB11827 did not inhibit the proliferation of BaF3 cells stably expressing hTSLPR / hIL-7Rα at all. The lead anti-hTSLP antibody QB11718 was the most effective inhibitor, inhibiting the proliferation of BaF3 cells stably expressing hTSLPR / hIL-7Rα with a potency 3-10 times higher than the anti-hTSLP reference antibody QB10985. Another lead anti-hTSLP antibody, QB11764, could inhibit the proliferation of BaF3 cells induced by different forms of hTSLP and cyTSLP, but its potency was generally lower (>10-fold) than the anti-hTSLP reference antibody QB10985. Example 15: Anti-TSLP IgG1-D265A antibody QB11718 strongly inhibits hTSLP-induced pSTAT5 phosphorylation in BaF3 cells stably expressing hTSLPPR / hIL-7Rα.

[0245] TSLP mediates signal transduction by establishing a heteromeric complex involving TSLPR and IL-7Rα. In the absence of the ligand TSLP, the interaction between TSLPR and IL-7Rα is very weak, with an affinity of 20 µM, and this weak interaction cannot trigger activation of downstream pathways. Once TSLP binds to TSLPR, a conformational change allows the TSLP / TSLPR complex to rapidly bind IL-7Rα, making this binary assembly a prerequisite for efficient signal transduction. Following TSLP binding, dimerization of the two receptor chains leads to activation of Janus kinase (JAK), signal transducers, and activators of transcription (STATs), resulting in transcription of target genes and subsequent tightly coordinated immune responses. STAT5 is the primary substrate phosphorylated shortly after the formation of the tertiary TSLP / TSLPR / IL-7Rα complex.

[0246] BaF3 cells stably expressing hTSLPR / hIL-7Rα were seeded at 10,000 cells / well and treated for 15 minutes with different forms of TSLP stimulants in combination with serially diluted antibodies. The TSLP stimulants included... Figure 19 hTSLP QB11630 (SEQ ID NO: 32) in A; Figure 19 hTSLP QB11631 (SEQ ID NO: 34) in B; Figure 19 The cyTSLP QB11632 (SEQ ID NO: 35) in C was used. pSTAT5 phosphorylation levels were determined using the Perkin Elmer AlphaLISA SureFire Ultra p-STAT5 HV Detection Kit (catalog number ALSU-PST5-B-HV) according to the manufacturer's instructions. The plate was read in a microplate reader with α-signal detection capability, and the IC50 was calculated using GraphPad Prism software. 50 value.

[0247] In summary, the isotype control antibody QB11827 failed to inhibit pSTAT5 phosphorylation in BaF3 cells stably expressing hTSLPR / hIL-7Rα. The lead antibody QB11718 was a highly effective inhibitor, showing comparable efficacy to the anti-hTSLP reference antibody QB10985. Figure 19 A) or slightly higher ( Figure 19 (B and 19C) validity. Example 16: Anti-TSLP IgG1-D265A antibody QB11718 strongly inhibits TSLP-induced CCL17 secretion in human monocytes.

[0248] Once TSLP binds to TSLPR and forms a tertiary complex with IL7Rα, myeloid dendritic cells (mDCs) upregulate the production of CCL17 and CCL22, which are chemokines of the CCR4 receptor on Th2 cells. OX40L is also induced, thereby promoting Th2-type immune responses, such as the secretion of IL-4, IL-5, and IL-13. See Gu C. et al. Front Immunol. 2021;12: 678036.

[0249] Using Stemcell Technologies' EasySep ™ The Human Monocyte Isolation Kit (catalog number 19359) was used to purify primary human monocytes from PBMCs. The purified human monocytes were seeded at 150,000 cells / well and treated with a constant amount of TSLP (concentration close to EC50). 50 The supernatant was treated with serially diluted anti-TSLP antibody for 24 hours. CCL17 chemokine levels in the supernatant were determined using the R&D Systems Human CCL17 / TARC DuoSet ELISA Kit (catalog number DY364) according to the manufacturer's instructions. Multiple forms of recombinant TSLP were used in this assay, including hTSLP QB11630 (SEQ ID NO:32) generated in the presence of furin inhibitor. Figure 20 A) hTSLP QB11631 with two substitutions (R127A + R130S) at the furin cleavage site. Figure 20 B), and cyTSLP QB11632 (SEQ ID NO:35) generated in the presence of furin inhibitor. Figure 20 C). Calculate IC using GraphPad Prism software. 50 value.

[0250] In summary, the isotype control antibody QB11827 did not inhibit the secretion of CCL17 in human monocytes, while the lead anti-hTSLP antibody QB11718 was a very effective blocker, with a potency 2 to 3 times higher than that of the anti-hTSLP reference antibody QB10985. Example 17: Anti-TSLP IgG1-D265A antibody QB11718 strongly inhibits TSLP-induced secretion of IL-5 by human ILC2.

[0251] Type 2 innate lymphoid cells (ILC2) belong to an expanding family of innate lymphocytes and provide a potent source of immune effector cytokines at the initiation of immune responses. Human ILC2s have been reported to be present in human lung parenchyma and bronchoalveolar lavage (BAL) fluid, and are lineage-negative cells expressing the ST2 receptors TSLPR, IL-7Rα, and IL-33 (see Monticelli L.A. et al. Nat Immunol. 2011; 12:1045–1054). Under the control of transcription factors RORα and GATA3, ILC2s originate from lymphoprogenitor cells in the bone marrow to secrete type 2 cytokines, including IL-5 and IL-13.

[0252] Human ILCs were isolated from PBMCs as described in Example 13. The purified ILC2 cells were cultured in RPMI 1640 assay medium containing 2 mM L-glutamine, 10% heat-inactivated fetal bovine serum, 1 mM sodium pyruvate, 0.075% sodium bicarbonate, 100 mM HEPES, and 100 units / mL of penicillin-streptomycin. The medium was supplemented every two weeks with 10 ng / mL each of human cytokines IL-2, IL-25, TSLP, and IL-33 to amplify for at least 3-4 weeks (approximately 40-fold amplification) or up to 6-8 weeks (>100-fold amplification). Prior to the assay, ILC2 cells were washed three times with the assay medium and then incubated overnight in a tissue culture incubator to reduce background signal. On the day of the assay, ILC2 cells were seeded at 30,000 cells / well and treated with a specific amount of hTSLP QB11630 (SEQ ID NO:32). Figure 21 A) and hTSLP QB11631 (SEQ ID NO: 34, Figure 21 B) Concentration close to EC 90 The antibody was treated with a 1:4 serial dilution for 48 hours. The hIL-5 level in the supernatant was determined using a Human IL-5 ELISA kit (R&D Systems, catalog number DY205) according to the manufacturer's instructions. The IC50 was calculated using GraphPad Prism software. 50 value.

[0253] like Figure 21 As shown, hTSLP-induced IL-5 secretion was strongly inhibited by the anti-TSLP antibody, but not by the isotype control antibody QB11571. The lead anti-hTSLP antibody QB11718 showed IC50 values ​​of 0.10 nM and 0.35 nM, respectively, when used with natural hTSLP QB11630 and hTSLP (R127A+R130S) QB11631. 50The anti-hTSLP reference antibody QB10985 has low potency. When using natural hTSLP QB11630 and hTSLP (R127A+R130S) QB11631, the IC50 is low. 50 The values ​​are 0.26 nM and 0.61 nM, respectively. Example 18: Anti-TSLP IgG1-D265A antibody QB11718 strongly inhibits hTSLP-induced pSTAT5 phosphorylation in ILC2.

[0254] Although the lead anti-hTSLP antibody QB11718 inhibited TSLP-induced pSTAT5 phosphorylation in BaF3 cells stably expressing TSLPR / IL-7Rα (see Example 15 above), it is unclear whether this antibody also inhibits TSLP-induced pSTAT5 phosphorylation in human primary cells, given that BaF3 / TSLPR / IL-7Rα cells were artificially created.

[0255] Human ILC2s were isolated and cultured in the same manner as described in Example 17 above. A concentration close to EC was used. 90 The value of hTSLP QB11630 (SEQ ID NO: 32) Figure 22 A), hTSLP QB11631 (SEQ ID NO: 34, Figure 22 B) or cyTSLP QB11632 (SEQ ID NO: 35, Figure 22 C) Treat ILC2 with a 1:4 serially diluted antibody. Measure pSTAT5 phosphorylation levels using the Perkin Elmer AlphaLISA SureFire Ultra p-STAT5 HV assay kit (catalog number ALSU-PST5-B-HV) according to the manufacturer's instructions. Read the plate using a microplate reader with α-signal detection capability and calculate the IC50 using GraphPad Prism software. 50 value.

[0256] like Figure 22 As shown, the isotype control antibody QB11571 did not inhibit pSTAT5 phosphorylation in cultured human ILC2. However, the lead anti-hTSLP antibody QB11718 and the anti-hTSLP reference antibody QB10985 considerably inhibited pSTAT5 phosphorylation in cultured human ILC2, with an IC50 score of [missing value]. 50 The value is a sub-single digit or a low single digit nM. Example 19: The combined use of lead anti-hIL-33 IgG4 antibody QB11465 and anti-TSLP IgG1-D265A antibody QB11718 synergistically inhibits hTSLP-induced human ILC2 secretion of IL-5.

[0257] Human ILC2s express IL-7Rα (CD127), IL-33 receptor (ST2), IL-2 receptor (CD25), induced T cell co-stimulatory molecules (ICOS), thymocyte markers (CD90), hematopoietic progenitor cell marker c-kit (CD117), stem cell antigen 1 (Sca-1; Ly6A / E), and hematopoietic marker (CD45). Receptors for IL-2, IL-7, IL-25, and IL-33 are all important for ILC2 development and activation. See Moro K. et al., Nature 2010; 463:540-544, Neill DR et al., Nature 2010; 464:1367-1370, and Walker JA et al., Nat Rev Immunol. 2013; 13:75-87. Although the lead anti-hIL-33 IgG4 antibody inhibited hIL-33-induced IL-5 secretion in ILC2 (Example 13), and the lead anti-hTSLP IgG1-D265A antibody QB11718 also inhibited hTSLP-induced IL-5 secretion in ILC2 (Example 17), there is no public information regarding the combined effect of anti-hTSLP and anti-hIL-33 blocking antibodies.

[0258] Human ILC2 cells were isolated and cultured in the same manner as described in Example 17 above. Prior to the assay, ILC2 cells were washed three times with assay medium and then incubated overnight in a tissue incubator to reduce background signal. On the assay day, ILC2 cells were seeded at 30,000 cells / well and treated with 5 ng / mL of internally prepared hTSLP QB11630 and BioLegend's hIL-33 (catalog number 581814) in combination with 4x, 2x, 1x, 0.5x, or 0.25x IC50. 50The concentrations of (1) single lead anti-hTSLP IgG1-D265A antibody QB11718; (2) single anti-hTSLP reference antibody QB10985; (3) single lead anti-hIL-33 IgG4 antibody QB11465; (4) single anti-hIL-33 reference-3 antibody QB11094; (5) combination of lead anti-hTSLP IgG1-D265A antibody QB11718 and lead anti-hIL-33 IgG4 antibody QB11465; (6) combination of lead anti-hTSLP IgG1-D265A antibody QB11718 and anti-hIL-33 reference-3 antibody QB11094; (7) combination of anti-hTSLP reference antibody QB10985 and lead anti-hIL-33 IgG4 antibody QB11465 were treated for 48 hours. The level of hIL-5 cytokine in the supernatant was measured using the R&D Systems Human IL-5 DuoSet ELISA Kit (catalog number DY205). Results were processed using GraphPad Prism software. The percentage response of the inhibitory reaction relative to the untreated antibody was expressed as an IC50 value. 50 Multiples plotted. The combination index (CI) was calculated according to the Chou-Talalay method, see Chou TC Pharmacol Rev. 2006; 58(3): 621-681 and Cancer Res.2010; 70(2):440-446.

[0259] The Chou-Talalay drug combination method is based on the intermediate-effect equation, derived from the law of mass action. This unified theory provides common connections between single-entity and multi-entity drug combinations, as well as between first-order and higher-order kinetics. This universal equation encompasses the Michaelis-Menten, Hill, Henderson-Hasselbalch, and Scatchard equations in biochemistry and biophysics. The resulting Chou-Talalay combination index (CI) theorem provides quantitative definitions for additive effects (CI=1), synergistic effects (CI<1), and antagonistic effects (CI>1) in drug combinations. This theory also provides algorithms for automated computer simulations of synergistic and / or antagonistic effects at any effect and dose level, as illustrated in the CI plot and isoelectric line plot, respectively.

[0260] like Figure 23As shown in A, the lead anti-hTSLP IgG1-D265A antibody QB11718 effectively inhibited ILC2 secretion of hIL-5. Anti-hIL-33 antibody IgG4 antibody QB11465 and anti-hIL-33 reference antibody QB11094 moderately inhibited hIL-5 secretion, but neither was as effective as anti-hTSLP antibody QB11718. However, the combination of (5) lead anti-hTSLP IgG1-D265A antibody QB11718 and lead anti-hIL-33 IgG4 antibody QB11465, and (6) lead anti-hTSLP IgG1-D265A antibody QB11718 and anti-hIL-33 reference antibody QB11094 showed even greater inhibition of hIL-5 secretion, as the curve shifted to the left. The calculated CI values ​​were all below 1.0 (CI<1), see [link to relevant documentation]. Figure 23 The table in C shows that the combination of anti-hTSLP and anti-hIL-33 antibodies is more effective than the sum of the individual antibodies, and that the anti-hTSLP / anti-hIL-33 MabPair product has the potential to achieve a synergistic effect in the treatment of patients with asthma and COPD. Similar results were obtained when the lead anti-hTSLP IgG1-D265A antibody QB11718 was replaced with the anti-hIL-33 IgG4 antibody QB11465 as a reference antibody for anti-hTSLP QB10985. To our knowledge, this is the first time that a combination of anti-hTSLP blocking antibodies and anti-hIL-33 blocking antibodies has achieved a synergistic biological effect in human primary cells. Example 20: Anti-hIL-33 IgG4 antibody QB11465 significantly inhibited allergen-induced eosinophilic inflammation of the lungs and IL-4 production.

[0261] As shown in Example 13 above, the lead anti-hIL-33 IgG4 antibody QB11465 inhibited the secretion of IFNγ cytokines from primary human NK cells. Figure 17 A) IL-5 secretion in primary human ILC2 ( Figure 17 B) and the phosphorylation level of p38 MAPK in primary human ILC2 ( Figure 17 It exhibits strong biological activity in C and 17D. Testing whether this antibody can demonstrate biological efficacy in a mouse asthma model is crucial.

[0262] Five- to seven-week-old female C57BL / 6 knock-in mice (C57BL / 6-IL33tm1(hIL33) / Bcgen) with hIL-33 were housed in a controlled environment at Biocytogen Inc. (Beijing, China). The mice had free access to autoclaved pellet food and tap water.

[0263] Purified house dust mite (HDM) extract, purchased from Greer Laboratories (Lenoir, NC), was resuspended in PBS to a final concentration of 1.42 mg / ml. Under isoflurane anesthesia, female C57BL / 6 hIL-33 knock-in mice were intranasally administered a purified HDM extract containing 25 μg of protein (17.5 µl / mouse / day) once daily for 10 days to induce airway inflammation. Five mice were in each group: (1) no antibody treatment (HDM only); (2) lead anti-hIL-33 IgG4 antibody QB11465, 5 mg / kg; (3) anti-hIL-33 reference-3 antibody QB11094, 5 mg / kg, administered intraperitoneally twice weekly for 3 weeks (days 22, 25, 29, 32, 36, and 39). Five untreated hIL-33 knock-in mice served as controls. Animals were sacrificed on day 48 after HDM administration. At the study endpoint, each animal was anesthetized by isoflurane inhalation, and bronchoalveolar lavage fluid (BALF) was collected, as described in Hoecke LV et al. J Vis Exp. 2017; (123): 55398. Mouse cytokines IL-4, IL-5, IL-13, TSLP, and IL-33 in BALF were measured using the LEGENDplex™ 5-PlexPanel kit (Biolegend), and human IL-33 in BALF was measured using the human IL-33 pre-coated ELISA kit (Biolegend). All cytokines were measured repeatedly on the day of BALF collection. Immune cells were collected from BALF, centrifuged at 800 x g for 5 min, and washed once with 200 µL DPBS. Cells were incubated at RT for 20 min with 100 µL DPBS containing the immobilizable viability dye Zombie Aqua. Immune cells were washed again with DPBS and resuspended in 100 µL eBioscience. TMImmunocytes were stained with flow cytometry staining buffer (Thermo Fisher, Cat # 00-4222-26). Immunocytes were blocked with Mouse TruStain FcX™ Plus at 4°C for 15 min, then stained with the fluorescent antibodies shown in Table 32 at 4°C in the dark for 45 min, and washed twice with flow cytometry staining buffer. Immunocytes were resuspended in 100 µL DPBS + 100 µL IC fixation solution (Thermo Fisher, Cat # 00-8222-49) and analyzed using a BD® LSR II flow cytometer. After excluding erythrocytes, debris, and diploids, eosinophils were identified as CD45+, SiglecF+, CD3-, CD19-, and NKp46-, and reported as the percentage of viable cells. Data were analyzed using FlowJo software, and statistical analysis was performed using one-way ANOVA. Table 32: Fluorescently labeled antibodies used for flow cytometry analysis of different cell types in BAL solution

[0264] like Figure 24 As shown in Figure A, the baseline eosinophil level in hIL-33 knock-in C57BL / 6 mice (labeled "control") was approximately 6%, and HDM stimulation increased eosinophils to approximately 27%, indicating that HDM stimulation induced a lung inflammatory response. The lead anti-hIL-33 IgG4 antibody QB11465 reduced eosinophil levels from approximately 27% to approximately 15%, indicating that anti-hIL-33 antibody QB11465 significantly inhibited HDM-induced airway inflammation (p = 0.0002). The anti-hIL-33 reference-3 antibody QB11094 reduced eosinophils to approximately 17%, indicating that this reference antibody also significantly inhibited HDM-induced airway inflammation (p = 0.0024). Mouse cytokines IL-5, IL-13, TSLP, and human IL-33 in BALF were all below the detection limit; however, mouse IL-4 production increased after HDM stimulation. Treatment with the lead anti-hIL-33 IgG4 antibody QB11465 and the anti-hIL-33 reference-3 antibody QB11094 significantly reduced IL-4 production in mice (p = 0.0462 and p = 0.0046, respectively). Since IL-4 is a major TH2 cytokine, the reduction in IL-4 production indicates suppression of lung inflammation.

[0265] Sequence list (using the Kabat numbering system): SEQ ID NO:1 Amino acid sequence of hIL-33 (Ser112 – Thr270) tagged with Avitag_c-MYC_His6, QB10975 GLNDIFEAQKIEWHEGGGGSEQKLISEEDLGSSHHHHHHSSGSITGISPITEYLASLSTYNDQSITFALEDESYEIYVEDLKKDEKKDKVLLSYYESQHPSNESGDGVDGKMLMVTLSPTKDFWLHANNKEHSVELHKCEKPLPDQAFFVLHNMHSNCVSFECKTDPGVFIGVKDNHLALIKVDSSENLCTENILFKLSET SEQ ID NO:2 His6-tagged hIL-33 (Ser112 – Thr270) (with C208S + C232S) amino acid sequence, QB11921 HHHHHHSSGSITGISPITEYLASLSTYNDQSITFALEDESYEIYVEDLKKDEKKDKVLLSYYESQHPSNESGDGVDGKMLMVTLSPTKDFWLHANNKEHSVELHKSEKPLPDQAFFVLHNMHSNCVSFESKTDPGVFIGVKDNHLALIKVDSSENLCTENILFKLSET SEQ ID NO:3 The amino acid sequence of cyIL-33 (Ser112 – Ile270) tagged with His6, QB10976 GLNDIFEAQKIEWHEGGGGSDYKDDDDKGSSHHHHHHSGSITGISPITESLASLSTYNDQSITFALEDESYEIYVEDLKKDKKKDKVLLSYYESQHPSSESGDGVDGKMLMVTLSPTKDFWLQANNKEHSVELHKCEKPLPDQAFFVLHNRSFNCVSFECKTDPGVFIGVKDNHLALIKVDYSENLGSENILFKLSEI Amino acid sequence of SEQ ID NO:4 mouse anti-hIL-33 QB10998 VL DIVMTQAAPSIPVTPGESVSISCKSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLENPYTFGGGTKLELN Amino acid sequence of SEQ ID NO:5 chimeric anti-hIL-33 QB10998 LC DIVMTQAAPSIPVTPGESVSISCKSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLENPYTFGGGTKLELNRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:6 Amino acid sequence of murine anti-hIL-33 QB10998 VH: EVMLVESGGGLVKPGGSLKLSCAASGFTFYSSAMSWVRQTPEKRLEWVATISSGGSNTYYPDSVKGRFTISRDNAKNTLYLQMSSLGSEDTAMYYCASAYYGRRYDAMDYWGQGTSVTVSS SEQ ID NO:7 Amino acid sequence of chimeric anti-hIL-33 QB10998 HC EVMLVESGGGLVKPGGSLKLSCAASGFTFYSSAMSWVRQTPEKRLEWVATISSGGSNTYYPDSVKGRFTISRDNAKNTLYLQMSSLGSEDTAMYYCASAYYGRRYDAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:8 Amino acid sequence of anti-hIL-33 QB11004 VL DVVMTQSPPSLPVTLGQSASISCKSSKSLLHSNGNTYLYWFQQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLENPYTFGQGTKVEIK SEQ ID NO:9 Amino acid sequence of anti-hIL-33 QB11004 LC DVVMTQSPPSLPVTLGQSASISCKSSKSLLHSNGNTYLYWFQQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLENPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:10 Amino acid sequence of anti-hIL-33 QB11004 VH EVQLLESGGGLVQPGGSLRLSCAASGFTFYSSAMSWVRQTQGKGLEWVSTISSGGSNTYYPDSVKGRFTISRDNSKNTLYLQMNSLGAEDTAVYYCASAYYGRRYDAMDYWGQGTLVTVSS SEQ ID NO:11 Amino acid sequence of anti-hIL-33 QB11004 HC EVQLLESGGGLVQPGGSLRLSCAASGFTFYSSAMSWVRQTQGKGLEWVSTISSGGSNTYYPDSVKGRFTISRDNSKNTLYLQMNSLGAEDATVYYCASAYYGRRYDAMDYW GQGTLVTSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:12 Amino acid sequence of anti-hIL-33 QB11061 VL DVVMTQSPPSLPVTLGQSASISCKSSKSLLHSNGNTYLYWFQQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLENPYTFGQGTKVEIK SEQ ID NO:13 Amino acid sequence of anti-hIL-33 QB11061 LC DVVMTQSPPSLPVTLGQSASISCKSSKSLLHSNGNTYLYWFQQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLENPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:14 Amino acid sequence of anti-hIL-33 QB11061 VH EVQLLESGGGLVQPGGSLRLSCAASGFTFYSSAMSWVRQTPGKGLEWVSTISSGGSNTYYPDSVKGRFTISRDNSKNTLYLQMNSLGAEDTAVYYCASAYYGRRYDAMDYWGQGTLVTVSS SEQ ID NO:15 Amino acid sequence of anti-hIL-33 QB11061 HC EVQLLESGGGLVQPGGSLRLSCAASGFTFYSSAMSWVRQTPGKGLEWVSTISSGGSNTYYPDSVKGRFTISRDNSKNTLYLQMNSLGAEDATVYYCASAYYGRRYDAMDYWG QGTLVTSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:16 Amino acid sequence of anti-hIL-33 QB11119 VL DVVMTQSPPSLPVTLGQSASISCKSSKSLLHSNANTYLYWFQQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLENPYTFGQGTKVEIK SEQ ID NO:17 Amino acid sequence of anti-hIL-33 QB11119 LC DVVMTQSPPSLPVTLGQSASISCKSSKSLLHSNANTYLYWFQQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLENPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:18 Amino acid sequence of anti-hIL-33 QB11119 VH EVQLLESGGGLVQPGGSLRLSCAASGFTFYSSAMSWVRQTQGKGLEWVSTISSGGSNTYYPDSVKGRFTISRDNSKNTLYLQMNSLGAEDTAVYYCASAYYGRRYDAMDYWGQGTLVTVSS SEQ ID NO:19 Amino acid sequence of anti-hIL-33 QB11119 HC EVQLLESGGGLVQPGGSLRLSCAASGFTFYSSAMSWVRQTQGKGLEWVSTISSGGSNTYYPDSVKGRFTISRDNSKNTLYLQMNSLGAEDATVYYCASAYYGRRYDAMDYWG QGTLVTSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPPAEAPEAGGPSVFLFPPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:20 Amino acid sequence of anti-hIL-33 QB11421 VL DVVMTQSPPSLPVTLGQSASISCKSSESLLHSNANTYLYWFQQRPGQSPQLLIYRGSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLRNPYTFGQGTKVEIK SEQ ID NO:21 Amino acid sequence of anti-hIL-33 QB11421 LC DVVMTQSPPSLPVTLGQSASISCKSSESLLHSNANTYLYWFQQRPGQSPQLLIYRGSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLRNPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:22 Amino acid sequence of anti-hIL-33 QB11421 VH EVQLLESGGGLVQPGGSLRLSCAASGFTFYSSAMSWFRQTQGKGLEWVSTISSGGSNTYYPDSVKGRFTISRDNSKNTLYLQMNSLGAEDTAVYYCASAYYGRRYDAMDYWGQGTLVTVSS SEQ ID NO:23 Amino acid sequence of anti-hIL-33 QB11421 HC EVQLLESGGGLVQPGGSLRLSCAASGFTFYSSAMSWFRQTQGKGLEWVSTISSGGSNTYYPDSVKGRFTISRDNSKNTLYLQMNSLGAEDATVYYCASAYYGRRYDAMDYWG QGTLVTSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPPAEAPEAGGPSVFLFPPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:24 Nucleotide sequence encoding anti-hIL-33 QB11465 VL GACGTAGTGATGACTCAGAGTCCTCCAAGCCTCCCTGTTACTCTCGGTCAGTCCGCCAGCATAAGCTGTAAGTCCTCCGAATCACTTCTGCATTCAAATGCTAACACTTATCTCTACTGGTTTCAGCAAAGACCTGGCCAATCACCCCAGTTGCTCATTTATAGGGGGAGCAATTTGGCTAGTGGGGTTCCAGATCGCTTTTCAGGAAGCGGCTCTGGTACCGACTTTACCCTCAAAATCAGTCGAGTAGAAGCTGAGGACGTTGGAGTTTACTATTGTATGCAGCACCTCCGAAATCCATACACTTTTGGGCAGGGGACAAAGGTCGAAATAAAG SEQ ID NO:25 Amino acid sequence of anti-hIL-33 QB11465 VL DVVMTQSPPSLPVTLGQSASISCKSSESLLHSNANTYLYWFQQRPGQSPQLLIYRGSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLRNPYTFGQGTKVEIK SEQ ID NO:26 Nucleotide sequence encoding anti-hIL-33 QB11465 LC GACGTAGTGATGACTCAGAGTCCTCCAAGCCTCCCTGTTACTCTCGGTCAGTCCGCCAGCATAAGCTGTAAGTCCTCCGAATCACTTCTGCATTCAAATGCTAACACTTATCTCTACTGGTTTCAGCAAAGACCTGGCCAATCACCCCAGTTGCTCATTTATAGGGGGAGCAATTTGGCTAGTGGGGTTCCAGATCGCTTTTCAGGAAGCGGCTCTGGTACCGACTTTACCCTCAAAATCAGTCGAGTAGAAGCTGAGGACGTTGGAGTTTACTATTGTATGCAGCACCTCCGAAATCCATACACTTTTGGGCAGGGGACAAAGGTCGAAA TAAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTGATAA SEQ ID NO:27 Amino acid sequence of anti-hIL-33 QB11465 LC DVVMTQSPPSLPVTLGQSASISCKSSESLLHSNANTYLYWFQQRPGQSPQLLIYRGSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLRNPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:28 Nucleotide sequence encoding anti-hIL-33 QB11465 VH GAGGTTCAACTTTTGGAGTCTGGCGGTGGTCTGGTGCAACCCGGTGGGAGTTTGCGACTTAGCTGTGCAGCCAGTGGATTCACTTTTTATAGCTCTGCCATGTCATGGTTTCGGCAGACTCAAGGAAAGGGACTGGAATGGGTTTCCACCATATCCTCAGGGGGCTCAAATACATACTACCCAGACTCCGTGAAAGGCCGGTTTACCATTTCACGAGACAACTCAAAGAACACACTTTACCTGCAAATGAATAGTCTGGGCGCTGAAGATACAGCCGTATATTATTGTGCATCAGCATACTACGGACGCCGATACGACGCAATGGATTATTGGGGACAGGGCACCCTGGTCACTGTCAGCTCT SEQ ID NO:z9 Amino acid sequence of anti-hIL-33 QB11465 VH EVQLLESGGGLVQPGGSLRLSCAASGFTFYSSAMSWFRQTQGKGLEWVSTISSGGSNTYYPDSVKGRFTISRDNSKNTLYLQMNSLGAEDTAVYYCASAYYGRRYDAMDYWGQGTLVTVSS SEQ ID NO:30 Nucleotide sequence encoding anti-hIL-33 QB11465 IgG4 HC It should be noted that there is a possible error in your original text where "SEQ ID NO:z9" in the translation of should probably be "SEQ ID NO:29".GAGGTTCAACTTTTGGAGTCTGGCGGTGGTCTGGTGCAACCCGGTGGGAGTTTGCGACTTAGCTGTGCAGCCAGTGGATTCACTTTTTATAGCTCTGCCATGTCATGGTTTCGGCAGACTCAAGGAAAGGGACTGGAATGGGTTTCCACCATATCCTCAGGGGGCTCAAATACATACTACCCAGACTCCGTGAAAGGCCGGTTTACCATTTCACGAGACAACTCAAAGAACACACTTTACCTGCAAATGAATAGTCTGGGCGCTGAAGATACAGCCGTATATTATTGTGCATCAGCATACTACGGACGCCGATACGACGCAATGGATTATTGGGGACAGGGCACCCTGGTCACTGTCAGCTCTGCTAGCACCAAGGGGCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGCCCCCCATGCCCACCATGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGAC CACGCCTCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAATGATAA Amino acid sequence of SEQ ID NO:31 anti-hIL-33 QB11465 IgG4 HC EVQLLESGGGLVQPGGSLRLSCAASGFTFYSSAMSWFRQTQGKGLEWVSTISSGGSNTYYPDSVKGRFTISRDNSKNTLYLQMNSLGAEDATVYYCASAYYGRRYDAMDYW GQGTLVTSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG PPCPPPAPEFLGGPSVFLFPPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK The amino acid sequence of SEQ ID NO:32 Avitag_c-MYC_His6_hTSLP(wt) QB11630, which was generated from wild-type hTSLP in the presence of 25 µM furin inhibitor II (Sigma, catalog number SCP0148). GLNDIFEAQKIEWHEGGGSEQKLISEEDLGSSHHHHHHSSGLVPRGSHMYDFTNCDFEKIKAAYLSTISKDLITYMSGTKSTEFNNTVSCSNRPHCLTEIQSLTFNPTAGCASLAKEMFAMKTKAALAIWCPGYSETQINATQAMKKRRKRKVTTNKCLEQVSQLQGLWRRFNRPLLKQQ SEQ ID NO:33 Avitag_c-MYC_His6_hTSLP QB11033 (missing) 126 KRRKR 130 amino acid sequence GLNDIFEAQKIEWHEGGGSEQKLISEEDLGSSHHHHHHSSGLVPRGSHMYDFTNCDFEKIKAAYLSTISKDLITYMSGTKSTEFNNTVSCSNRPHCLTEIQSLTFNPTAGCASLAKEMFAMKTKAALAIWCPGYSETQINATQAMKKVTTNKCLEQVSQLQGLWRRFNRPLLKQQ The amino acid sequence of SEQ ID NO:34 Avitag_c-MYC_His6_hTSLP QB11631 (with R127A+R130S mutation) GLNDIFEAQKIEWHEGGGSEQKLISEEDLGSSHHHHHHSSGLVPRGSHMYDFTNCDFEKIKAAYLSTISKDLITYMSGTKSTEFNNTVSCSNRPHCLTEIQSLTFNPTAGCASLAKEMFAMKTKAALAIWCPGYSETQINATQAMKKARKSKVTTNKCLEQVSQLQGLWRRFNRPLLKQQ The amino acid sequence of SEQ ID NO:35 Avitag_FLAG_His6_cyTSLP QB11632 is wild-type cyTSLP produced in the presence of 25 µM furin inhibitor II (Sigma, catalog number SCP0148). GLNDIFEAQKIEWHEGGGGSDYKDDDDKGSSHHHHHHSSGLVPRGSHMYDFTNCDFQKIEADYLRTISKDLITYMSGTKSTDFNNTVSCSNRPHCLTEIQSLTFNPTPRCASLAKEMFARKTKATLALWCPGYSETQINATQAMKKRRKRKVTTNKCLEQVSQLLGLWRRFIRTLLKKQ SEQ ID NO:36 Amino acid sequence of Avitag_FLAG_His6_cyTSLP QB10974 (missing 126 KRRKR 130 ) GLNDIFEAQKIEWHEGGGGSDYKDDDDKGSSHHHHHHSSGLVPRGSHMYDFTNCDFQKIEADYLRTISKDLITYMSGTKSTDFNNTVSCSNRPHCLTEIQSLTFNPTPRCASLAKEMFARKTKATLALWCPGYSETQINATQAMKKVTTNKCLEQVSQLLGLWRRFIRTLLKKQ SEQ ID NO:37 Amino acid sequence of Avitag_FLAG_His6_cyTSLP QB11633 (with R127A + R130S mutations) GLNDIFEAQKIEWHEGGGGSDYKDDDDKGSSHHHHHHSSGLVPRGSHMYDFTNCDFQKIEADYLRTISKDLITYMSGTKSTDFNNTVSCSNRPHCLTEIQSLTFNPTPRCASLAKEMFARKTKATLALWCPGYSETQINATQAMKKARKSKVTTNKCLEQVSQLLGLWRRFIRTLLKKQ SEQ ID NO:38 Amino acid sequence of hTSLPR-Fc fusion protein QB11034 GAAEGVQIQIIYFNLETVQVTWNASKYSRTNLTFHYRFNGDEAYDQCTNYLLQEGHTSGCLLDAEQRDDILYFSIRNGTHPVFTASRWMVYYLKPSSPKHVRFSWHQDAVTVTCSDLSYGDLLYEVQYRSPFDTEWQSKQENTCNVTIEGLDAEKCYSFWVRVKAMEDVYGPDTYPSDWSEVTCWQRGEIRDACAETPTPPKPKLSKAAAEPKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:39 Amino acid sequence of anti-hTSLP VH of rabbit hybridoma clone 135F1 QEQLEESGGDLVKPEGSLTLTCKASGFDFSSYWISWVRQAPGKRPEWIACIDSDIDSADYASWAKGRFTMSRTSSTTVTLQMTSLTAADTATYFCVRNLGLWGPGTLVTVSS SEQ ID NO:40 Amino acid sequence of anti-hTSLP HC of rabbit hybridoma clone 135F1 QEQLEESGGDLVKPEGSLTLTCKASGFDFSSYWISWVRQAPGKRPEWIACIDSDIDSADYASWAKGRFTMSRTSSTTVTLQMTSLTAADTATYFCVRNLGLWGPGTLV TVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPMC PPPELLGGPSVFIFPPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISK ARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPTVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK Amino acid sequence of rabbit hybridoma clone 135F1 against hTSLP VL (SEQ ID NO:41) AQVLTQTASPVSAAVGGTVTINCQSSQNVYDKDALAWYQHKPGQRPKLLIYEASKLASGVPSRFSGSGAGTQFTLTISGVQCDDAATYYCAGTFIDNIYTFGGGTEVVVK Amino acid sequence of rabbit hybridoma clone 135F1 against hTSLP LC (SEQ ID NO:42) AQVLTQTASPVSAAVGGTVTINCQSSQNVYDKDALAYQHKPGQRPKLLIYEASKLASGVPSRFSGSGAGTQFTLTISGVQCDDAATYYCAGTFIDNIIYTFGGGTEV VVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC Amino acid sequences of anti-hTSLP QB11341 and QB11548 VH of humanized clone 135F1 (SEQ ID NO:43) EVQLVESGGGLVQPGGSLRLSCAASGFDFSSYWISWVRQAPGKGPEWIASIDIDIDIADYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVRNLGLWGPGTLVTVSS Amino acid sequences of anti-hTSLP QB11341 and QB11548 HC from humanized clone 135F1 (SEQ ID NO:44) EVQLVESGGGLVQPGGSLRLSCAASGFDFSSYWISWVRQAPGKGPEWIASIDIDIDIADYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVRNLGLWGPGTLV TVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPC PPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLAAPIEKTIS KTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Amino acid sequence of anti-hTSLP QB11341 VL of humanized clone 135F1 (SEQ ID NO:45) DIQMTQSPSSSLSASVGDRVTITCQSSQNVYDKDALAWYQHKPGKRPKLLIYEASKLASGVPSRFSGSGSGTHFTLTISSLQPEDAATYYCAGTFIDNIYTFGGGTKVEIK Amino acid sequence of humanized clone 135F1 against hTSLP QB11341 LC (SEQ ID NO:46) DIQMTQSPSSSLSASVGDRVTITCQSSQNVYDKDALAWYQHKPGKRPKLLIYEASKLASGVPSRFSGSGSGTHFTLTISSLQPEDAATYYCAGTFIDNIYTFGGGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC The amino acid sequence of the humanized yeast clone 135F1 with anti-hTSLP QB11548 VL, SEQ ID NO:47. DIQMTQSPSSSLSASVGDRVTITCQSSQNVYDKDALAWYQHKPGKRPKLLIYEASRLASGVPSRFSGSGSGTHFTLTISSLQPEDAATYYCAGTRIDNIYTFGGGTKVEIK SEQ ID NO:48 Amino acid sequence of yeast-displayed humanized clone 135F1 anti-hTSLP QB11548 LC DIQMTQSPSSSLSASVGDRVTITCQSSQNVYDKDALAWYQHKPGKRPKLLIYEASRLASGVPSRFSGSGSGTHFTLTISSLQPEDAATYYCAGTRIDNIYTFGGGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:49 encodes the nucleotide sequence of the final cloned anti-hTSLP QB11764 VH. GAGGTGCAATTGGTGGAGTCTGGTGGGGGACTTGTGCAACCCGGCGGATCACTGCGGCTTAGCTGCGCAGCATCAGGATTTGATTTTAGTAGCTATTGGATCAGTTGGGTTAGGCAGGCACCAGGGAAACGCCCCGAGTGGATAGCAAGTATTGACATCGATATTGATA TTGCCGATTATGCTTCCTGGGCCAAAGGCCGATTTACAAATAAGTCGCGATAACTCAAAAAACACCCTTTATCTTCAAATGAATTCCCTCCGAGCAGAAGATACCGCTGTTTACTACTGTGTCCGAAATCTGGGATTGTGGGGCCCAGGTACCCTTGTTACAGTTTCTTCC SEQ ID NO:50 The amino acid sequence of the finally cloned anti-hTSLP QB11764 VH EVQLVESGGGLVQPGGSLRLSCAASGFDFSSYWISWVRQAPGKRPEWIASIDIDIDIADYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVRNLGLWGPGTLVTVSS SEQ ID NO:51 encodes the nucleotide sequence of the final clone of anti-hTSLP QB11764 IgG1-D265A for MabPair production. GAGGTGCAATTGGTGGAGTCTGGTGGGGGACTTGTGCAACCCGGCGGATCACTGCGGCTTAGCTGCGCAGCATCAGGATTTGATTTTAGTAGCTATTGGATCAGTTGGGTTAGGCAGGCACCAGGGAAACGCCCCGAGTGGATAGCAAGTATTGACATCGATATTGATATTGCCGATTATGCTTCCTGGGCCAAAGGCCGATTTACAATAAGTCGCGATAACTCAAAAAACACCCTTTATCTTCAAATGAATTCCCTCCGAGCAGAAGATACCGCTGTTTACTACTGTGTCCGAAATCTGGGATTGTGGGGCCCAGGTACCCTTGTTACAGTT TCTTCCGCCTCAACTAAGGGACCAAGCGTCTTCCCCCTTGCTCCCTCAAGCAAGTCCACAAGCGGGGGGACCGCTGCTTTGGGCTGCCTCGTTGATGACTACTTTCCCGAACCTGTCACCGTGTCATGGAATTCCGGAGCCTTGACTTCTGGGGTACATACCTGCCCTGCTGTATTGCAGAGTTCTGGACTGTATTCTCTGAGTAGTGTAGTGACTGTTCCATCTAGCTCCCTGGGTACTCAGACCTACATTTGTAATGTCAATCACAAGCCTAGTAACACCAAAGTGGATAAGAAAGTGGAGCCTAAGTCTGGTGATAAGACACACACATGCCCTCCCTGTCCAGCACCAGAGCTCCTTGGGGGACCTTCCGTCTTTCTTTTCCCTCCCAAACCCAAGGATACACTTATGATTAGCCGGACACCAGAAGTTACTTGCGTCGTTCGTTGCAGTGAGCCATGAGGACCCAGAAGTTAAGTTCAATTGGTACGTGGATGGCGTCGAGGTACATAATGCCAAGACCAAGCCACGTGAGGAGCAGTACAACAGTACATATAGGGTCGTGTCCGTACTTACAGTGCTCCACCAAGATTGGCTGAATGGTAAGGAATATAAGTGTAAGGTTAGTAATAAAGCACTGCCCGCCCCTATCGAAAAGACCATATCT AAAGCCAAAGGCCAGCCCCGTGAACCCCAGGTATATACACTTCCACCATCCCGTGAGGAAATGACTAAAAATCAGGTATCTCTTACCTGCCTCGTAAAAGGTTTCTACCCATCCGATATAGCAGTAGAGTGGGAAAGCAATGGCCAACCCGAGAACAATTACAAAACCACCCCCCCTGTGCTGCGTAGCGATGGTTCTTTTTTTCTTTACTCCGAACTTACAGTGGATAAGTCCCGTTGGCAGCAAGGAAACGTATTCTCTTGTTCTGTAATGCATGAAGCACTTCATAATCATTATACTCAAAAGTCCCTGTCTCTCTCCCCCGGCAAGTGA SEQ ID NO:52 Amino acid sequence of anti-hTSLP QB11764 IgG1-D265A, the final clone for MabPair production EVQLVESGGGLVQPGGSLRLSCAASGFDFSSYWISWVRQAPGKRPEWIASIDIDIDIDIADYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVRNLGLWGPGTLV TSSASTKGPSVFPLAPSSKSTSGGTAALGCLVDDYFPEPVTVSWNSGALTSGVHTCPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSGDKTHT CPPCPAPELLGGPSVFLFPPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLRSDGSFFLYSELTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:53 Nucleotide sequence encoding the ultimately cloned anti-hTSLP QB11764 VL[[ID=]3] GACATACAGATGACCCAGTCCCCCAGCTCTTTGTCAGCATCCGTAGGAGATCGGGTCACCATCACCTGTCAATCATCTCAAAACGTTTATGATAAAGACGCATTGGCCTGGTACCAGCATAAGCCAGGTAAAAGGCCAAAATTGCTCATCTACGAAGCTAGTCGGCTGGCATCAGGGGTGCCATCTAGGTTTTCCGGTAGCGGAAGTGGTACACATTTTACACTTACCATTTCCTCTCTTCAACCAGAAGATGCCGCCACCTATTATTGTGCCGGTACAAGAATCGACAATATATACACTTTCGGAGATGGGACAAAGGTAGAAATAAAG SEQ ID NO:54 Amino acid sequence of the ultimately cloned anti-hTSLP QB11764 VL DIQMTQSPSSLSASVGDRVTITCQSSQNVYDKDALAWYQHKPGKRPKLLIYEASRLASGVPSRFSGSGSGTHFTLTISSLQPEDAATYYCAGTRIDNIYTFGDGTKVEIK SEQ ID NO:55 Nucleotide sequence encoding anti-hTSLP QB11764 LC, the final clone for MabPair production GACATACAGATGACCCAGTCCCCCAGCTCTTTGTCAGCATCCGTAGGAGATCGGGTCACCATCACCTGTCAATCATCTCAAAACGTTTATGATAAAGACGCATTGGCCTGGTACCAGCATAAGCCAGGTAAAAGGCCAAAATTGCTCATCTACGAAGCTAGTCGGCTGGCATCAGGGGTGCCATCTAGGTTTTCCGGTAGCGGAAGTGGTACACATTTTACACTTACCATTTCCTCTCTTCAACCAGAAGATGCCGCCACCTATTATTGTGCCGGTACAAGAATCGACAATATATACACTTTCGGAGATGGGACAAAGGTAGAAATA AAGCGTACGGTGGCCGCTCCTAGTGTATTCATCTTTCCCCCTTCTGATGAACAATTGAAGTCTGGGACTGCAAAGGTTGTATGTCTCCTTAATAACTTCTACCCACGAGAAGCCAAAGTACAGTGGAAGGTGGATAATGCCCTCCAAAGCGGAAACAGTCAGGAATGCGTAACTGAACAAGACAGCAAGGACTCAACCTACTCTCTTTCCTCCACACTCACACTGTCAAAGGCAGATTACGAGAAGCACAAAGTCTACGCTTGCGAGGTCACTCATCAGGGCCTCAGCTCACCCGTCACTAAGTCTTTTAACAGAGGCGAATCTTGA SEQ ID NO:56 Amino acid sequence of anti-hTSLP QB11764 LC, the final clone for MabPair production DIQMTQSPSSSLSASVGDRVTITCQSSQNVYDKDALAWYQHKPGKRPKLLIYEASRLASGVPSRFSGSGSGTHFTLTISSLQPEDAATYYCAGTRIDNIYTFGDGTKVE IKRTVAAPSVFIFPPSDEQLKSGTAKVVCLLNNFYPREAKVQWKVDNALQSGNSQECVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES SEQ ID NO:57 Anti-hTSLP QB10987 VH amino acid sequence of chimeric antibody clone 23B12 EEKLQQSGDDLVRPGAAVKMSCKASGYIFTDYAMHWVKQRPGQGLEWIGTFIPLLDTSDYNQNFKGRATLTADKSSNTAYMELSRLTSEDSAVYYCARMGVTHSYVMDAWGQGASVTVSS Amino acid sequence of anti-hTSLP QB10987 HC of chimeric antibody clone 23B12 (SEQ ID NO:58) EEKLQQSGDDLVRPGAAVKMSCKASGYIFTDYAMHWVKQRPGQGLEWIGTFIPLLDTSDYNQNFKGRATLATADKSSNTAYMELSRLTSEDSAVYYCARMGVTHSYVMDA WGQGASVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES KYGPPCPPPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLAPIEKTI SKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Amino acid sequence of anti-hTSLP QB10987 VL of chimeric antibody clone 23B12 (SEQ ID NO:59) DIVLTQSPATLSVTPGESVLSCRASQPISISVHWFQQKSNESPRLLIKFASQSISGIPSRFSGSGSGTDFTLNINRVESEDFSVYYCQQTFSLPYTFGTGTKLELK Amino acid sequence of anti-hTSLP QB10987 LC of chimeric antibody clone 23B12 (SEQ ID NO: 60) DIVLTQSPATLSVTPGESVLSCRASQPISISVHWFQQKSNESPRLIKFASQSISGIPSRFSGSGSGTDFTLNINRVESEDFSVYYCQQTFSLPYTFGTGTGTKLEL KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Amino acid sequence of anti-hTSLP QB10990 VH of humanized antibody clone hz-3C (SEQ ID NO:61) QVQLVQSGDEVKKPGSSVKVSCKASGYIFTDYAMHWVRQRPGQGLEWMGTFIPLLDTSDYNQNFKGRATITADKSTSTAYMELSSLRSEDTAVYYCARMGVTHSYVMDAWGQGTSVTVSS Amino acid sequence of anti-hTSLP QB10990 HC of humanized antibody clone hz-3C (SEQ ID NO:62) QVQLVQSGDEVKKPGSSVKVSCKASGYIFTDYAMHWVRQRPGQGLEWMGTFIPLLDTSDYNQNFKGRATITADKSTSTAYMELSSLRSEDTAVYYCARMGVTHSYVMDAW GQGTSVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVSKYGPPCPPPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLAAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:63 Amino acid sequence of the anti-hTSLP QB10990 VL of the humanized antibody clone hz-3C, equivalent to QB11060 VL EIVLTQSPGTLSLSPGERATLSCRASQPISISVHWYQQKPGEAPRLLIYFASQSISGIPDRFSGSGSGTDFTLTISRLESEDFAVYYCQQTFSLPYTFGTGTKVEIK SEQ ID NO:64 Amino acid sequence of the anti-hTSLP QB10990 LC of the humanized antibody clone hz-3C, equivalent to QB11060 LC EIVLTQSPGTLSLSPGERATLSCRASQPISISVHWYQQKPGEAPRLLIYFASQSISGIPDRFSGSGSGTDFTLTISRLESEDFAVYYCQQTFSLPYTFGTGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC The amino acid sequence of the humanized antibody clone hz-3C anti-hTSLP QB11060 VH (with D9P+S108L substitution), SEQ ID NO:65, is equivalent to QB11237 VH. QVQLVQSGPEVKKPGSSVKVSCKASGYIFTDYAMHWVRQRPGQGLEWMGTFIPLLDTSDYNQNFKGRATITADKSTSTAYMELSSLRSEDTAVYYCARMGVTHSYVMDAWGQGTLVTVSS SEQ ID NO:66 The amino acid sequence of the humanized antibody clone hz-3C anti-hTSLP QB11060 HC (with D9P+S108L substitution) is equivalent to QB11237 HC. QVQLVQSGPEVKKPGSSVKVSCKASGYIFTDYAMHWVRQRPGQGLEWMGTFIPLLDTSDYNQNFKGRATITADKSTSTAYMELSSLRSEDTAVYYCARMGVTHSYVMDAWG QGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG PPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLAAPIEKT ISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Amino acid sequence of anti-hTSLP QB11237 VL of yeast-displayed humanized antibody clone hz-3C. (SEQ ID NO:67) EIVLTQSPGTLSLSPGERATLSCRASQHISRSVHWYQQKPGEAPRLLIYFASQSISGIPDRFSGSGSGTDFTLTISRLESEDFAVYYCQQSYSFPYTFGTGTKVEIK SEQ ID NO:68 Amino acid sequence of the humanized antibody clone hz-3C against hTSLP QB11237 LC after yeast display EIVLTQSPGTLSLSPGERATLSCRASQHISRSVHWYQQKPGEAPRLLIYFASQSISGIPDRFSGSGSGTDFTLTISRLESEDFAVYYCQQSYSFPYTFGTGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:69 encodes the nucleotide sequence of the final cloned anti-hTSLP QB11718 VH. CAGGTCCAACTTGTTCAGTCAGGCCCAGAAGTCAAAAAACCTGGTAGTTCCGTCAAGGTATCCTGCAAGGCAAGTGGATACATCTTCACAGACTATGCTATGCACTGGGTGCGTCAGAGACCAGGTCAGGACCTCGAATGGATGGGCACTTTCATTCCCCTTCTGGATACCTCAGACTAC AATCAGAATTTCAAGGGAAGAGCAACCATTACTGCTGATAAGTCAACAAGTACTGCTTATATGGAACTGAGCTCCTTGCGCAGCGAAGACACAGCCGTTTATTATTGCGCCCGGATGGGTGTTACACATAGTTACGTGATGGACGCATGGGGACAGGGAACACTCGTAACAGTGTCTTCA SEQ ID NO:70 The amino acid sequence of the finally cloned anti-hTSLP QB11718 VH QVQLVQSGPEVKKPGSSVKVSCKASGYIFTDYAMHWVRQRPGQDLEWMGTFIPLLDTSDYNQNFKGRATITADKSTSTAYMELSSLRSEDTAVYYCARMGVTHSYVMDAWGQGTLVTVSS SEQ ID NO:71 Nucleotide sequence of the final clone of anti-hTSLP QB11718 IgG1-D265A HC for MabPair production CAGGTCCAACTTGTTCAGTCAGGCCCAGAAGTCAAAAAACCTGGTAGTTCCGTCAAGGTATCCTGCAAGGCAAGTGGATACATCTTCACAGACTATGCTATGCACTGGGTGCGTCAGAGACCAGGTCAGGACCTCGAATGGATGGGCACTTTCATTCCCCTTCTGGATACCTCAGACTACAATCAGAATTTCAAGGGAAGAGCAACCATTACTGCTGATAAGTCAACAAGTACTGCTTATATGGAACTGAGCTCCTTGCGCAGCGAAGACACAGCCGTTTATTATTGCGCCCGGATGGGTGTTACACATAGTTACGTGATGGACGCATGGGGACAGGGAACACTCGTAACAGTGTCTTCAGCTAGCACTAAAGGCCCTAGTGTGTTCCCACTCGCTCCAAGCTCCAAGTCAACCTCCGGCGGCACCGCTGCCCTGGGTTGTCTCGTCGATGATTATTTTCCCGAACCAGTGACTGTGAGCTGGAATAGCGGAGCTCTCACAAGTGGCGTGCACACTTGTCCTGCCGTATTGCAATCCTCCGGCCTGTATTCACTCAGTTCTGTCGTTACCGTCCCCTCATCATCCCTCGGGACACAAACATATATATGCAATGTCAATCACAAGCCTAGTAACACAAAAGTAGATAAAAAAGTCGAGCCAAAGTCCGGCGATAAAACCCATACTTGCCCACCCTGTCCAGCTCCCGAGCTTCTGGGGGGACCCAGTGTATTTCTCTTTCCACCAAAGCCTAAAGACACACTGATGATTAGCAGAACCCCCGAAGTTACCTGTGTCGTGGTAGCAGTTTCCCATGAAGATCCTGAAGTCAAGTTCAATTGGTACGTAGATGGGGTGGAGGTCCATAACGCAAAAACAAAACCCCGCGAAGAGCAGTATAACTCTACTTACCGGGTTGTGTCCGTGCTCACCGTGCTGCATCAAGATTGGCTGAATGGAAAGGAATACAAATGTAAGGTCAGCAATAAGGCCCTTCCCGCACCTATTGAAAAAACAATCTCTAAAGCTAAGGGCCAACCACGAGAGCCTCAAGTTTATACCCTCCCTCCATCTAGAGAAGAGATGACTAAAAACCAGGTGTCCCTCACTTGCCTCGTTAAGGGCTTTTACCCAAGTGACATTGCTGTGGAGTGGGAATCAAATGGACAACCTGAAAACAATTACAAAACTACACCCCCAGTCTTGAGGAGCGACGGCTCATTTTTTTTTGTACTCTGAGCTTACCGTTGACAAAAGCCGCTGGCAGCAAGGAAACGTTTTTTCATGCAGTGTAATGCATGAGGCACTTCACAATCATTACACACAGAAGTCATTGAGCCTCAGCCCTGGGAAGTGA SEQ ID NO:72 Amino acid sequence of the final clone anti-hTSLP QB11718 IgG1-D265A HC for MabPair production QVQLVQSGPEVKKPGSSVKVSCKASGYIFTDYAMHWVRQRPGQDLEWMGTFIPLLDTSDYNQNFKGRATITADKSTSTAYMELSSLRSEDTAVYYCARMGVTHSYVMDAWGQ GTLVTSSASTKGPSVFPLAPSSKSTSGGTAALGCLVDDYFPEPVTVSWNSGALTSGVHTCPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSGDK THTCPPCPAPELLGGPSVFLFPPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLRSDGSFFLYSELTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:73 Nucleotide sequence encoding the final clone anti-hTSLP QB11718 VL GAAATAGTCCTCACACAGTCCCCAGGTACCCTCTCACTGTCACCCGGCGAAAGGGCCACTCTGTCCTGTCGTGCAAGCCAGCACATCAGTCGCTCTGTCCATTGGTATCAACAGAAGCCAGGTGAGGCCCCACGTCTTCTGATTTACTTCGCTAGTCAAT CTATTCTCCGGCATTCCTGACCGATTTAGCGGCAGCGGAAGCGGAACCGACTTCACTCTCACAATATCACGGCTTGAATCAGAGGATTTCGCCGTCTATTACTGCCAGCAGAGTTACAGCTTCCCCTATACTTTCGGGCGCGGAACTAAGGTAGAAATTAAG SEQ ID NO:74 The amino acid sequence of the finally cloned anti-hTSLP QB11718 VL EIVLTQSPGTLSLSPGERATLSCRASQHISRSVHWYQQKPGEAPRLLIYFASQSISGIPDRFSGSGSGTDFTLTISRLESEDFAVYYCQQSYSFPYTFGRGTKVEIK SEQ ID NO:75 encodes the nucleotide sequence of the final clone of anti-hTSLP QB11718 LC used in MabPair production. GAAATAGTCCTCACACAGTCCCCAGGTACCCTCTCACTGTCACCCGGCGAAAGGGCCACTCTGTCCTGTCGTGCAAGCCAGCACATCAGTCGCTCTGTCCATTGGTATCAACAGAAGCCAGGTGAGGCCCCACGTCTTCTGATTTACTTCGCTAGTCAATCTATCTCCGGCATTCCTGACCGATTTAGCGGCAGCGGAAGCGGAACCGACTTCACTCTCACAATATCACGGCTTGAATCAGAGGATTTCGCCGTCTATTACTGCCAGCAGAGTTACAGCTTCCCCTATACTTTCGGGCGCGGAACTAAGGTAGAAATTAAGC GTACGGTCGCCGCCCCCAGCGTATTCATATTTCCTCCTAGCGATGAGCAGCTTAAATCAGGAACAGCAAAGGTTGTCTGTTTGTTGAACAATTTCTATCCTCGGGAAGCTAAGGTCCAATGGAAAGTGGACAACGCCTTGCAGAGCGGTAATTCACAAGAATGCGTGACAGAGCAAGATAGCAAAGACTCAACCTACTCACTGAGCTCTACTCTGACACTTTCTAAAGCTGATTATGAGAAACACAAGGTATATGCATGTGAAGTGACTCACCAGGGGCTCAGCTCACCTGTGACTAAATCCTTTAACAGGGGCGAATCCTGA SEQ ID NO:76 Amino acid sequence of the anti-hTSLP QB11718 LC, the final clone for MabPair production EIVLTQSPGTLSLSPGERATLSCRASQHISRSVHWYQQKPGEAPRLLIYFASQSISGIPDRFSGSGSGTDFTLTISRLESEDFAVYYCQQSYSFPYTFGRGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAKVVCLLNNFYPREAKVQWKVDNALQSGNSQECVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES SEQ ID NO:77 Amino acid sequence of the VL CDR1 region of anti-hIL-33 antibodies QB11421 and QB11465 KSSESLLHSNANTYLY SEQ ID NO:78 Amino acid sequence of the VL CDR2 region of anti-hIL-33 antibodies QB11421 and QB11465 RGSNLAS SEQ ID NO:79 Amino acid sequences of the VL CDR3 region of anti-hIL-33 antibodies QB11421 and QB11465 MQHLRNPYT SEQ ID NO:80 Amino acid sequence of the VH CDR1 region of anti-hIL-33 antibodies QB11421 and QB11465 SSAMS SEQ ID NO:81 Amino acid sequence of the VH CDR2 region of anti-hIL-33 antibodies QB11421 and QB11465 TISSGGSNTYYPDSVKG SEQ ID NO:82 Amino acid sequences of the VH CDR3 region of anti-hIL-33 antibodies QB11421 and QB11465 AYYGRRYDAMDY SEQ ID NO:83 Amino acid sequence of VL CDR1 region of anti-hTSLP antibodies QB11341, QB11548, and QB11764 QSSQNVYDKDALA SEQ ID NO:84 Amino acid sequence of the VL CDR2 region of anti-hTSLP antibody QB11341 EASKLAS SEQ ID NO:85 Amino acid sequence of the VL CDR3 region of anti-hTSLP antibody QB11341 AGTFIDNIYT SEQ ID NO:86 Amino acid sequences of the VH CDR1 region of anti-hTSLP antibodies QB11341, QB11548, and QB11764 SYWIS SEQ ID NO:87 Amino acid sequences of the VH CDR2 region of anti-hTSLP antibodies QB11341, QB11548, and QB11764 SIDIDIDADYASWAKG SEQ ID NO:88 Amino acid sequence of VH CDR3 region of anti-hTSLP antibodies QB11341, QB11548, and QB11764 NLGL SEQ ID NO:89 Amino acid sequence of the VL CDR2 region of anti-hTSLP antibodies QB11548 and QB11764 EASRLAS SEQ ID NO:90 Amino acid sequence of VL CDR3 region of anti-hTSLP antibodies QB11548 and QB11764 AGTRIDNIYT SEQ ID NO:91 Amino acid sequence of the VL CDR1 region of anti-hTSLP antibodies QB11237 and QB11718 RASQHISRSVH SEQ ID NO:92 Amino acid sequence of the VL CDR2 region of anti-hTSLP antibodies QB11237 and QB11718 FASQSIS SEQ ID NO:93 Amino acid sequence of VL CDR3 region of anti-hTSLP antibodies QB11237 and QB11718 QQSYSFPYT SEQ ID NO:94 Amino acid sequence of the VH CDR1 region of anti-hTSLP antibodies QB11237 and QB11718 DYAMH SEQ ID NO:95 Amino acid sequence of the VH CDR2 region of anti-hTSLP antibodies QB11237 and QB11718 TFIPLLDTSDYNQNFKG SEQ ID NO:96 Amino acid sequence of VH CDR3 region of anti-hTSLP antibodies QB11237 and QB11718 MGVTHSYVMDA

Claims

1. An anti-human IL33 (anti-hIL33) antibody comprising a heavy chain variable domain (VH) and a light chain variable region (VL), wherein each of VH and VL comprises complementarity-determining regions 1 (CDR1), 2, and 3. The anti-hIL33 antibody comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3, which respectively contain the following amino acid sequences: SEQ ID NO: 80, 81, 82, 77, 78, and 79. The anti-hIL33 antibody inhibits the interaction between human IL-33 and the ST2 / IL1AcP complex.

2. The anti-hIL33 antibody according to claim 1, wherein VH comprises an amino acid sequence with no more than four altered amino acid sequences relative to the group consisting of SEQ ID NO: 22 and 29, and / or VL comprises an amino acid sequence with no more than four altered amino acid sequences relative to the group consisting of SEQ ID NO: 20 and 25.

3. The anti-hIL33 antibody according to claims 1-2, The anti-hIL33 antibody VH and VL each contain an amino acid sequence, and the amino acid sequences of VH and VL together contain two sequences. One of the two sequences contains no more than four amino acid changes relative to one sequence in the VH / VL sequence pair, and the other of the two sequences contains no more than four amino acid changes relative to the other sequence in the VH / VL sequence pair. The VH / VL sequence pairs are selected from the group consisting of: SEQ ID NO: 29 (VH) and 25 (VL); and SEQ ID NO: 22 (VH) and 20 (VL).

4. The anti-hIL33 antibody according to claims 1-3, wherein... VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 contain the amino acid sequences of SEQ ID NO: 80, 81, 82, 77, 78, and 79, respectively. VH contains an amino acid sequence relative to SEQ ID NO:29 that includes no more than four altered amino acid sequences, and VL contains an amino acid sequence relative to SEQ ID NO:25 that includes no more than four altered amino acid sequences.

5. One or more polynucleotides encoding the anti-hIL33 antibody of claim 1.

6. A host cell comprising one or more polynucleotides as described in claim 5.

7. A method for treating a patient in need of treatment with inflammation, inflammatory disease, chronic inflammatory airway disease, asthma, COPD, and / or type 2 inflammation, said method comprising: (a) Administering the anti-hIL33 antibody of claim 1 to the patient; or (b) Administer to the patient one or more polynucleotides encoding the anti-hIL33 antibody described in (a).

8. An anti-human TSLP (anti-hTSLP) antibody comprising VH and VL, wherein each of VH and VL comprises CDR1, CDR2, and CDR3. The anti-hTSLP antibody comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3, which respectively contain the following sequences: SEQ ID NO: 86, 87, 88, 83, 84, and 85; SEQ ID NO: 86, 87, 88, 83, 89, and 90; SEQ ID NO: 94, 95, 96, 91, 92, and 93, and The anti-hTSLP antibody inhibits the binding of hTSLP to hTSLPR or the interaction of hTSLP with the hTSLPR / hIL7Rα complex.

9. The anti-hTSLP antibody according to claim 8, The anti-hTSLP VH contains an amino acid sequence with no more than four altered amino acid sequences relative to the group consisting of SEQ ID NO:43, 50, 65 and 70, and / or the anti-hTSLP VL contains an amino acid sequence with no more than four altered amino acid sequences relative to the group consisting of SEQ ID NO:45, 47, 54, 67 and 74.

10. The anti-hTSLP antibody according to claims 8-9, The anti-hTSLP antibody VH and VL each contain an amino acid sequence, and the amino acid sequences of VH and VL together contain two sequences. One of the two sequences contains no more than four amino acid changes relative to one sequence in the VH / VL sequence pair, and the other of the two sequences contains no more than four amino acid changes relative to the other sequence in the VH / VL sequence pair. The VH / VL sequence pairs are selected from the group consisting of: SEQ ID NO:43(VH) and 45; SEQ ID NO:43(VH) and 47(VL); SEQ ID NO:50(VH) and 54(VL); SEQ ID NO:65(VH) and 67(VL); and SEQ ID NO:70(VH) and 74(VL).

11. The anti-hTSLP antibody according to claims 8-10, wherein... VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 contain the amino acid sequences of SEQ ID NO: 94, 95, 96, 91, 92, and 93, respectively. VH contains an amino acid sequence that is modified by no more than four amino acid sequences relative to SEQ ID NO:70, and VL contains an amino acid sequence that is modified by no more than four amino acid sequences relative to SEQ ID NO:

74.

12. One or more polynucleotides encoding the anti-hTSLP antibody of claim 8.

13. A host cell comprising one or more polynucleotides as described in claim 12.

14. A method for treating a patient in need of treatment with inflammation, inflammatory disease, chronic inflammatory airway disease, asthma, COPD, and / or type 2 inflammation, the method comprising: (A) Administering the anti-hTSLP antibody of claim 8 to the patient; or (B) Administer to the patient one or more polynucleotides encoding the anti-hTSLP antibody described in (a).

15. A mixture comprising anti-hIL33 antibody and anti-hTSLP antibody, wherein: (a) (1) The anti-hIL33 antibody comprises VH and VL, each of which comprises CDR1, CDR2, and CDR3; (2) The anti-hIL33 antibody comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3, each comprising the following sequences: SEQ ID NO: 80, 81, 82, 77, 78, and 79; and (3) The anti-hIL33 antibody inhibits the interaction between human IL-33 and the ST2 / IL1AcP complex; and (b) (1) The anti-hTSLP antibody comprises VH and VL, each of which comprises CDR1, CDR2 and CDR3; (2) The anti-hTSLP antibody comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3, each comprising the following sequences: SEQ ID NO: 86, 87, 88, 83, 84 and 85; SEQ ID NO: 86, 87, 88, 83, 89 and 90; SEQ ID NO: 94, 95, 96, 91, 92 and 93; and (3) The anti-hTSLP antibody inhibits the binding of hTSLP to hTSLPR or the interaction of hTSLP with the hTSLPR / hIL7Rα complex.

16. The mixture according to claim 15, wherein (a) (1) VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 of the anti-hIL33 antibody contain the amino acid sequences of SEQ ID NO:80, 81, 82, 77, 78, and 79, respectively; (2) VH of the anti-hIL33 antibody contains no more than four altered amino acid sequences relative to the amino acid sequence of SEQ ID NO:29; and (3) VL of the anti-hIL33 antibody contains no more than four altered amino acid sequences relative to the amino acid sequence of SEQ ID NO:

25. (b) (1) The VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 of the anti-hTSLP antibody contain the amino acid sequences of SEQ ID NO:94, 95, 96, 91, 92 and 93, respectively; (2) VH contains no more than four altered amino acid sequences relative to the amino acid sequence of SEQ ID NO:70; and (3) the VL contains no more than four altered amino acid sequences relative to the amino acid sequence of SEQ ID NO:

74.

17. One or more polynucleotides encoding the mixture of claim 15.

18. A host cell comprising one or more polynucleotides as described in claim 17.

19. A method for treating a patient in need of treatment with inflammation, inflammatory disease, chronic inflammatory airway disease, asthma, COPD, and / or type 2 inflammation, the method comprising: (a) Administering the mixture of claim 15 to the patient; or (b) Administer to the patient one or more polynucleotides encoding the mixture described in (a).

20. An antibody mixture comprising: (a) An anti-hIL33 antibody comprising a heavy chain (HC) and a light chain (LC), wherein (1) the HC of the anti-hIL33 antibody is encoded by a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:31, and (2) the LC of the anti-hIL33 antibody is encoded by a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:27; and (b) An anti-hTSLP antibody comprising HC and LC, wherein (1) the HC of the anti-hTSLP antibody is encoded by a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:72, and (2) the LC of the anti-hTSLP antibody is encoded by a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:

76.

21. The mixture according to claim 20, wherein The amino acid sequences of the anti-hIL33 antibody HC and LC are encoded by the nucleic acid sequences of SEQ ID NO:30 and 26, respectively; and The amino acid sequences of the anti-hTSLP antibody HC and LC are encoded by the nucleic acid sequences of SEQ ID NO:71 and 75, respectively.

22. A pharmaceutical composition comprising the mixture of any one of claims 20 to 21.

23. One or more polynucleotides encoding the mixture of any one of claims 20 to 21.

24. The polynucleotide of claim 23, wherein the polynucleotide comprises the nucleic acid sequences of SEQ ID NO: 30, 26, 71 and 75.

25. A vector comprising the polynucleotide of claim 23 or 24.

26. The vector according to claim 25, wherein it is a viral vector.

27. The vector according to claim 26 is an oncolytic virus vector.

28. The vector according to claim 26 or 27 is a retrovirus, adenovirus, adeno-associated virus, vaccinia virus, modified Ankara vaccinia virus, herpesvirus, lentivirus, measles virus, Coxsackie virus, Newcastle disease virus, reovirus or vaccinia virus vector.

29. A host cell comprising the polynucleotide of claim 23 or 24 and / or the vector of claim 5, wherein the host cell is capable of producing the mixture of any one of claims 20 to 21.

30. The host cell according to claim 29, wherein it is a CHO cell or a mouse myeloma cell.

31. A method for preparing an antibody mixture, comprising the following steps: Culture the host cells according to any one of claims 29 to 30; and The antibody mixture was recovered from the culture supernatant or host cell clumps.

32. A method for treating a patient in need of treatment with inflammation, inflammatory disease, chronic inflammatory airway disease, asthma, COPD, and / or type 2 inflammation, the method comprising: (a) Administering to the patient a dose of the mixture of any one of claims 20 to 21 or the pharmaceutical composition of claim 22, or (b) Administering to the patient a dose of the polynucleotide of claim 23 or 24 or the carrier of any one of claims 26 to 28.

33. An anti-hIL33 antibody, wherein HC contains an amino acid sequence relative to SEQ ID NO:7, 11, 15, 19, 23, or 31 that includes no more than four, three, two, or one altered amino acid sequence, and LC contains an amino acid sequence relative to SEQ ID NO:5, 9, 13, 17, 21 or 27 that includes no more than four, three, two or one altered amino acid sequence.

34. The anti-hIL33 antibody according to claim 33, wherein HC comprises the amino acid sequence of SEQ ID NO:31, and LC comprises the amino acid sequence of SEQ ID NO:

27.

35. An anti-hTSLP antibody, wherein HC contains amino acid sequences relative to SEQ ID NO:44, 52, 62, 66, or 72 that include no more than four, three, two, or one altered amino acid sequence, and LC contains an amino acid sequence relative to SEQ ID NO:46, 48, 56, 64, 68 or 76 that contains no more than four, three, two or one altered amino acid sequence.

36. The anti-hTSLP antibody according to claim 35, wherein HC comprises the amino acid sequence of SEQ ID NO:72, and LC comprises the amino acid sequence of SEQ ID NO:

76.

37. A mixture of anti-hIL33 antibody and anti-hTSLP antibody, wherein The anti-hIL33 HC contains an amino acid sequence relative to SEQ ID NO:7, 11, 15, 19, 23, or 31 that includes no more than four, three, two, or one altered amino acid sequence, and The anti-hIL33 LC contains an amino acid sequence relative to SEQ ID NO:5, 9, 13, 17, 21, or 27 that includes no more than four, three, two, or one altered amino acid sequence; and The anti-hTSLP HC contains an amino acid sequence relative to SEQ ID NO:44, 52, 62, 66, or 72 that includes no more than four, three, two, or one altered amino acid sequence. The anti-hTSLP LC contains an amino acid sequence relative to SEQ ID NO:46, 48, 56, 64, 68 or 76 that includes no more than four, three, two or one altered amino acid sequence.

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