Antibodies binding interleukin 13 and TSLP or tslpr and methods of use thereof

Abstract

Described herein are novel bispecific antibodies that bind to interleukin 13 (IL-13) and TSLP or TSLPR and methods of use thereof. In certain aspects, described herein are methods of inhibiting the biological activity of IL-13 and TSLP or TSLPR. In certain aspects, described herein are pharmaceutical compositions comprising a bispecific antibody that binds to interleukin 13 (IL-13) and TSLP or TSLPR. In certain aspects, the antibodies and methods described herein are useful for the treatment of inflammatory diseases or disorders associated with elevated levels of IL-13 and / or IgE as well as TSLP or TSLPR.

Description

Cross-references to related applications

[0001] This application claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 522,631, filed June 22, 2023. background

[0002] Interleukin (IL)-13 is a T helper cell subclass 2 (Th2) cytokine, belonging to the type I cytokine family, and exhibits pleiotropic effects in multiple cellular pathways. IL-13 is involved in the differentiation of naive T cells into Th2 cells. When co-stimulated with CD40 / CD40L, IL-13 promotes B cell proliferation and induces immunoglobulin isotype conversion to IgG4 and IgE. It also upregulates FcεRI, thus contributing to IgE initiation in mast cells. In monocytes / macrophages, IL-13 upregulates the expression of CD23 and MHC class I and II antigens, downregulates CD14 expression, inhibits antibody-dependent cytotoxicity, and promotes eosinophil survival, activation, and recruitment. IL-13 also exhibits important functions in non-hematopoietic cells such as smooth muscle cells, epithelial cells, endothelial cells, and fibroblasts. IL-13 enhances smooth muscle proliferation and cholinergic-induced contraction. In epithelial cells, IL-13 is a potent inducer of chemokine production, alters mucosal ciliary differentiation, reduces the frequency of ciliary pulsation in ciliated epithelial cells, and leads to goblet cell metaplasia. In endothelial cells, IL-13 is a potent inducer of vascular cell adhesion molecule-1 (VCAM-1), which is important for the recruitment of eosinophils. In epithelial keratinocytes, IL-13 reduces the expression of barrier integrity molecules such as filaggrin and loricrin, while stimulating the secretion of CCL26 and CCL2, which are responsible for recruiting several myeloid inflammatory cells. In human dermal fibroblasts, IL-13 induces type I collagen synthesis.

[0003] Thymic stromal lymphopoietin (TSLP) is an epithelial cell-derived cytokine produced in response to pro-inflammatory stimuli. TSLP has been found to promote allergic inflammatory responses primarily through its activity on dendritic cells and mast cells. Human TSLP expression has been reported to increase in the airways of asthma in relation to disease severity. Furthermore, TSLP protein levels have been detected in concentrated bronchoalveolar lavage fluid (BAL) from asthmatic patients and other patients with allergic conditions. In addition, TSLP has been found to promote fibrosis.

[0004] TSLP binds to a heterodimer receptor composed of the TSLP receptor (TSLPR) and the IL-7 receptor α chain (IL-7Rα) in dendritic cells, thereby activating the cells. Upon activation, dendritic cells express inflammatory chemokines, such as thymic and activation-regulated chemokines (TARC (CCL17)) and macrophage-derived chemokines (MDC (CCL22)).

[0005] TSLP activates dendritic cells through the TSLP receptor and is associated with disease pathologies including allergic inflammatory diseases such as asthma and autoimmune diseases such as systemic sclerosis.

[0006] Therefore, there is a need in the art for antagonists of IL-13 and TSLP or TSLP receptor (TSLPR) for the prevention and treatment of diseases in which human IL-13, TSLP and human TSLP receptor are involved in the pathology of the disease, such as inflammatory and fibrotic conditions. Overview

[0007] In one aspect, this article provides a bispecific antibody comprising a first antigen-binding site and a second antigen-binding site, wherein the first antigen-binding site binds to interleukin-13 (IL-13) and comprises a first variable heavy (VH) chain sequence and a first variable light (VL) chain sequence, the first variable heavy (VH) chain sequence comprising three first heavy chain CDR sequences: first CDR-H1, first CDR-H2, and first CDR-H3, and the first variable light (VL) chain sequence comprising three first light chain CDR sequences: first CDR-L1, first CDR-L2, and first CDR-L3; wherein: first CDR-H1 comprises a sequence selected from the sequences shown in SEQ ID NO: 58-99 and 121; first CDR-H2 comprises a sequence selected from the sequences shown in SEQ ID NO: 100-111; first CDR-H3 comprises a sequence selected from the sequences shown in SEQ ID NO: 112-120 and 130-140; first CDR-L1 comprises a sequence selected from the sequences shown in SEQ ID NO: 100-111; and first CDR-H3 comprises a sequence selected from the sequences shown in SEQ ID NO: 112-120 and 130-140. The sequences shown in SEQ ID NO: 141-144 and 149-152; the first CDR-L2 comprises a sequence selected from the sequences shown in SEQ ID NO: 153-158 and LAS; and the first CDR-L3 comprises a sequence selected from the sequences shown in SEQ ID NO: 165-172; and wherein the second antigen binding site binds to TSLP or TSLPR and comprises a second variable heavy (VH) chain sequence and a second variable light (VL) chain sequence, the second variable heavy (VH) chain sequence comprising three second heavy chain CDR sequences: second CDR-H1, second CDR-H2 and second CDR-H3, the second variable light (VL) chain sequence comprising three second light chain CDR sequences: second CDR-L1, second CDR-L2 and second CDR-L3; wherein: second CDR-H1 comprises a sequence selected from the sequences shown in SEQ ID NO: 610-618; second CDR-H2 comprises a sequence selected from the sequences shown in SEQ ID NO: 610-618; The second CDR-H3 comprises a sequence selected from the sequences shown in SEQ ID NO: 628-636, the second CDR-L1 comprises a sequence selected from the sequences shown in SEQ ID NO: 637-645, the second CDR-L2 comprises a sequence selected from the sequences shown in SEQ ID NO: 646-651, and the second CDR-L3 comprises a sequence selected from the sequences shown in SEQ ID NO: 652-657.

[0008] In some embodiments, the first CDR-H1 comprises a sequence selected from the sequences shown in SEQ ID NO: 58, 68, and 85; the first CDR-H2 comprises a sequence selected from the sequences shown in SEQ ID NO: 100, 104, and 108; the first CDR-H3 comprises a sequence selected from the sequences shown in SEQ ID NO: 112 and 130; the first CDR-L1 comprises a sequence selected from the sequences shown in SEQ ID NO: 141 and 589; the first CDR-L2 comprises a sequence selected from the sequence shown in SEQ ID NO: 153 and a sequence of LAS; and the first CDR-L3 comprises a sequence shown in SEQ ID NO: 165; and the second CDR-H1 comprises a sequence selected from the sequences shown in SEQ ID NO: 610-618; the second CDR-H2 comprises a sequence selected from the sequences shown in SEQ ID NO: 619-627; the second CDR-H3 comprises a sequence selected from the sequences shown in SEQ ID NO: 628-636; the second CDR-L1 comprises a sequence selected from SEQ ID NO: 58, 68, and 85; the first CDR-H2 comprises a sequence selected from the sequences shown in SEQ ID NO: 100, 104, and 108; the first CDR-H3 comprises a sequence selected from the sequences shown in SEQ ID NO: 112 and 130; the second CDR-L1 comprises a sequence selected from the sequences shown in SEQ ID NO: 141 and 589; the first CDR-L2 comprises a sequence selected from the sequences shown in SEQ ID NO: 153 and a sequence of LAS; and the first CDR-L3 comprises a sequence selected from the sequences shown in SEQ ID NO: 165; the second CDR-H1 comprises a sequence selected from the sequences shown in SEQ ID NO: 610-618; the second CDR-H2 comprises a sequence The second CDR-L2 comprises a sequence selected from the sequences shown in SEQ ID NO: 637-645; the second CDR-L3 comprises a sequence selected from the sequences shown in SEQ ID NO: 646-651; and the second CDR-L3 comprises a sequence selected from the sequences shown in SEQ ID NO: 652-657.

[0009] In some embodiments, the antibody comprises a first VH sequence selected from SEQ ID NO: 1-32, 470 and 688 and a second VH sequence selected from SEQ ID NO: 659-661, 682, 684, 686 and 688.

[0010] In some embodiments, the antibody comprises a first VH sequence containing SEQ ID NO: 3 and a second VH sequence selected from SEQ ID NO: 659-661, 682, 684, 686 and 688.

[0011] In some embodiments, the antibody comprises a first VL sequence selected from SEQ ID NO: 33-57, 471 and 687 and a second VL sequence selected from SEQ ID NO: 662-664, 681, 683 and 685.

[0012] In some embodiments, the bispecific antibody comprises a first VL containing a first VL sequence of SEQ ID NO: 39 and a second VL sequence selected from SEQ ID NO: 662-664, 681, 683 and 685.

[0013] In some embodiments, the bispecific antibody comprises a first VH sequence selected from SEQ ID NO: 1-32, 470 and 688 and a second VH sequence selected from SEQ ID NO: 659-661, 682, 684, 686 and 688; and a first VL sequence selected from SEQ ID NO: 33-57, 471 and 687 and a second VL sequence selected from SEQ ID NO: 662-664, 681, 683 and 685.

[0014] In some embodiments, the bispecific antibody comprises a first VH sequence containing SEQ ID NO: 3 and a second VH sequence selected from SEQ ID NO: 659-661, 682, 684, 686 and 688; and a first VL sequence containing SEQ ID NO: 39 and a second VL sequence selected from SEQ ID NO: 662-664, 681, 683 and 685.

[0015] In some implementations, the bispecific antibody is a humanized, human, or chimeric antibody.

[0016] In some implementations, the bispecific antibody is a humanized antibody.

[0017] In some implementations, the bispecific antibody comprises a heavy chain human constant region selected from classes of IgG, IgA, IgD, IgE, and IgM.

[0018] In some implementations, the human Fc region includes the human heavy chain constant region of the IgG class and subclasses selected from IgG1, IgG2, IgG3 and IgG4.

[0019] In some implementations, the human Fc region contains human IgG1 Fc.

[0020] In some implementations, the human Fc region contains human IgG4 Fc.

[0021] In some implementations, the human Fc region contains human IgG2 Fc.

[0022] In some embodiments, the heavy chain comprises an Fc sequence selected from the sequences shown in SEQ ID NO: 425-468, 484-539, 670-680, 689-691 and 693-804.

[0023] In some implementations, the heavy chain comprises the constant heavy chain sequence shown in SEQ ID NO: 439.

[0024] In some embodiments, the bispecific antibody comprises a heavy chain / chain A from Table 11a or Table 11b, a light chain / chain B from Table 12a or Table 12b, and optionally an Fc sequence from Table 13 (SEQ ID NO: 425-468, 484-539, 670-680, 689-691, and 693-804).

[0025] In some embodiments, the Fc region contains one or more amino acid substitutions, wherein the one or more substitutions result in a change in antibody half-life, ADCC activity, ADCP activity, or CDC activity compared to antibodies that are otherwise equivalent to Fc regions without one or more substitutions.

[0026] In some implementations, the changes are (a) an increase in antibody half-life and (b) a decrease in ADCC activity, ADCP activity, or CDC activity compared to antibodies that are otherwise equivalent to those containing one or more substituted Fcs.

[0027] In some implementations, the substitution of one or more amino acids results in an increased antibody half-life compared to antibodies containing the wild-type Fc region.

[0028] In some implementations, any isolated bispecific antibody described herein is used to treat inflammatory conditions or diseases.

[0029] In one aspect, this document provides an isolated polynucleotide or a group of polynucleotides encoding a bispecific antibody according to any one of the preceding claims, its VH, its VL, its light chain, its heavy chain, or its antigen-binding portion thereof, and optionally, said polynucleotide or group of polynucleotides comprises cDNA.

[0030] In one aspect, this document provides a vector or a set of vectors containing the polynucleotides or a set of polynucleotides described herein.

[0031] In one aspect, this article provides a host cell comprising the polynucleotide or a set of polynucleotides or a vector or a set of vectors described herein.

[0032] In one aspect, this document provides a method for generating antibodies, the method comprising expressing a bispecific antibody using a host cell as described herein and isolating the expressed bispecific antibody.

[0033] In one aspect, this document provides a pharmaceutical composition comprising any of the bispecific antibodies described herein and a pharmaceutically acceptable excipient.

[0034] In one aspect, this document provides a kit containing any of the bispecific antibodies described herein or a pharmaceutical composition described herein, along with instructions for use.

[0035] In one aspect, this document provides a method for treating an inflammatory condition or disease in a mammalian subject with a corresponding need, the method comprising administering to the mammalian subject a therapeutically effective amount of any of the bispecific antibodies described herein or a pharmaceutical composition described herein.

[0036] In some embodiments, the isolated bispecific antibody is used to treat an inflammatory condition or disease. In some embodiments, the isolated bispecific antibody is used to treat atopic dermatitis. In some embodiments, the treatment reduces the severity of the subject's disease, and the severity of the disease is assessed using the Atopic Dermatitis Disease Severity Outcome Measure. In some embodiments, the isolated bispecific antibody is used to treat asthma. In some embodiments, the isolated bispecific antibody is used to treat idiopathic pulmonary fibrosis. In some embodiments, the isolated bispecific antibody is used to treat alopecia areata. In some embodiments, the isolated bispecific antibody is used to treat chronic sinusitis with nasal polyps. In some embodiments, the isolated bispecific antibody is used to treat chronic sinusitis without nasal polyps (CRSsNP). In some embodiments, the isolated bispecific antibody is used to treat eosinophilic esophagitis (EoE). In some embodiments, the isolated bispecific antibody is used to treat eosinophilic gastrointestinal disorders or diseases (ENIDs) selected from eosinophilic gastritis (EoG), eosinophilic enteritis (EoN), eosinophilic colitis (EoC), and eosinophilic gastroenteritis (EGE). In some embodiments, the isolated bispecific antibody is used to treat Churg-Strauss syndrome / eosinophilic granulomatosis with polyangiitis (EGPA). In some embodiments, the isolated bispecific antibody is used to treat nodular prurigo (PN). In some embodiments, the isolated bispecific antibody is used to treat chronic spontaneous urticaria (CSU). In some embodiments, the isolated bispecific antibody is used to treat chronic pruritus of unknown cause (CPUO). In some embodiments, the isolated bispecific antibody is used to treat bullous pemphigoid (BP). In some embodiments, the isolated bispecific antibody is used to treat cold-induced urticaria (ColdU). In some embodiments, the isolated bispecific antibody is used to treat allergic fungal sinusitis (AFRS). In some embodiments, the isolated bispecific antibody is used to treat allergic bronchopulmonary aspergillosis (ABPA). In some embodiments, the isolated bispecific antibody is used to treat chronic obstructive pulmonary disease (COPD). In some embodiments, the isolated bispecific antibody is used to treat inflammatory bowel disease, such as Crohn's disease or ulcerative colitis. In some embodiments, the isolated bispecific antibody is used to treat psoriasis. In some embodiments, the isolated bispecific antibody is used to treat lupus. In some embodiments, the isolated bispecific antibody is used to treat rheumatoid arthritis.

[0037] In some aspects, this document describes isolated polynucleotides or groups of polynucleotides encoding antibodies described herein, their VH, VL, light chains, heavy chains, or antigen-binding moieties, and optionally, said polynucleotides or groups of polynucleotides comprise cDNA. In some aspects, this document describes vectors or groups of vectors comprising polynucleotides or groups of polynucleotides. In some aspects, this document describes host cells comprising polynucleotides or groups of polynucleotides or vectors or groups of vectors.

[0038] In some respects, this document describes a method for generating antibodies, the method comprising expressing antibodies using host cells as described herein and isolating the expressed antibodies.

[0039] In some respects, this article describes a pharmaceutical composition comprising the bispecific antibody described herein and a pharmaceutically acceptable excipient.

[0040] In some respects, this document describes kits comprising the bispecific antibodies or pharmaceutical compositions described herein and instructions for use.

[0041] In some aspects, this document describes a method for treating an inflammatory condition or disease in a mammalian subject with a corresponding need, the method comprising administering to the mammalian subject a therapeutically effective amount of the bispecific antibody or pharmaceutical composition described herein. In some embodiments of the method described herein, the inflammatory condition or disease is atopic dermatitis. In some embodiments, the inflammatory condition or disease is asthma. In some embodiments, the inflammatory condition or disease is idiopathic pulmonary fibrosis. In some embodiments, the inflammatory condition or disease is alopecia areata. In some embodiments, the inflammatory condition or disease is chronic sinusitis with nasal polyps. In some embodiments, the inflammatory condition or disease is chronic sinusitis without nasal polyps (CRSsNP). In some embodiments, the inflammatory condition or disease is eosinophilic esophagitis (EoE). In some embodiments, the inflammatory condition or disease is an eosinophilic gastrointestinal condition or disease (ENID) selected from the group consisting of eosinophilic gastritis (EoG), eosinophilic enteritis (EoN), eosinophilic colitis (EoC), and eosinophilic gastroenteritis (EGE). In some embodiments, the inflammatory condition or disease is Chag-Strauss syndrome / eosinophilic granulomatosis with polyangiitis (EGPA). In some embodiments, the inflammatory condition or disease is nodular prurigo (PN). In some embodiments, the inflammatory condition or disease is chronic spontaneous urticaria (CSU). In some embodiments, the inflammatory condition or disease is chronic pruritus of unknown cause (CPUO). In some embodiments, the inflammatory condition or disease is bullous pemphigoid (BP). In some embodiments, the inflammatory condition or disease is cold-induced urticaria (ColdU). In some embodiments, the inflammatory condition or disease is allergic fungal sinusitis (AFRS). In some embodiments, the inflammatory condition or disease is allergic bronchopulmonary aspergillosis (ABPA). In some embodiments, the inflammatory condition or disease is chronic obstructive pulmonary disease (COPD). In some embodiments, the inflammatory condition or disease is inflammatory bowel disease, such as Crohn's disease or ulcerative colitis. In some embodiments, the inflammatory condition or disease is psoriasis. In some implementations, the inflammatory condition or disease is lupus. In some implementations, the inflammatory condition or disease is rheumatoid arthritis.

[0042] In some respects, this document describes a method for treating a pathology associated with elevated IL-13 levels in a mammalian subject with a corresponding need, the method comprising administering to the mammalian subject a therapeutically effective amount of the bispecific antibody or pharmaceutical composition described herein.

[0043] In some respects, this document describes a method for reducing the biological activity of IL-13 in mammalian subjects with appropriate need, the method comprising administering to the mammalian subject a therapeutically effective amount of the bispecific antibody or pharmaceutical composition described herein.

[0044] In some respects, this document describes a method for suppressing TH2-type hypersensitivity reactions in mammalian subjects with appropriate need, the method comprising administering to the mammalian subject a therapeutically effective amount of the bispecific antibody or pharmaceutical composition described herein.

[0045] In some respects, this article describes a method for reducing thymus and activated regulatory chemokine (TARC) / CCL17 levels in mammalian subjects with corresponding needs, the method comprising administering to the mammalian subject a therapeutically effective amount of the bispecific antibody or pharmaceutical composition described herein.

[0046] In some respects, this document describes a method for preventing inflammatory conditions or diseases in mammalian subjects with appropriate need, the method comprising administering to the mammalian subject a therapeutically effective amount of the bispecific antibody or pharmaceutical composition described herein. Brief description of the attached diagram

[0047] These and other features, aspects, and advantages of the invention will become better understood in light of the following description and accompanying drawings, wherein: Figure 1 An exemplary IgG-scFv bispecific antibody form is described.

[0048] Figure 2 An exemplary DVD-Ig bispecific antibody form is described.

[0049] Figure 3 An exemplary form of the CrossMab bispecific antibody is described. Detailed Explanation

[0050] This disclosure relates in part to antibodies (e.g., bispecific antibodies) that bind (1) IL-13 and (2) TSLP or TSLP receptor (TSLPR).

[0051] definition Unless otherwise defined, all technical terms, symbols, and other scientific terms used herein are intended to have the meanings commonly understood by those skilled in the art. In some instances, terms with commonly understood meanings are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not necessarily be construed as indicating a difference from what is commonly understood in the art. The techniques and procedures described or referenced herein are generally well understood by those skilled in the art and typically employ conventional methods, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, and are widely used molecular cloning methods. Unless otherwise stated, procedures involving the use of commercially available kits and reagents are generally performed according to the manufacturer's prescribed protocols and conditions, where appropriate.

[0052] As used herein, unless otherwise stated, the singular forms “a”, “an” and “the” include plural referents.

[0053] It will be understood that the aspects and embodiments of the invention described herein include “comprising the aspect and embodiments,” “consisting of the aspect and embodiments,” and “mainly consisting of the aspect and embodiments.”

[0054] For all compositions described herein and all methods of using the compositions described herein, the compositions may comprise the listed components or steps, or may be “substantially composed of the listed components or steps.” When a composition is described as “substantially composed of the listed components,” the composition comprises the listed components and may comprise other components that do not substantially affect the treated condition, but does not comprise any other components that substantially affect the treated condition besides those explicitly listed; or, if the composition does contain additional components that substantially affect the treated condition besides those listed, the composition does not contain an additional concentration or amount of the additional components that substantially affect the treated condition. When a method is described as “substantially composed of the listed steps,” the method comprises the listed steps and may comprise other steps that do not substantially affect the treated condition, but does not comprise any other steps that substantially affect the treated condition besides those explicitly listed. As a non-limiting specific example, when a composition is described as “substantially composed of components,” the composition may additionally comprise any amount of pharmaceutically acceptable carriers, mediators, or diluents and other such components that do not substantially affect the treated condition.

[0055] As used herein, the term "vector" refers to a nucleic acid molecule capable of proliferating another nucleic acid linked to it. This term includes vectors as self-replicating nucleic acid structures as well as vectors incorporated into the genome of a host cell that has been introduced therein. Some vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0056] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells in which exogenous nucleic acids have been introduced, as well as the progeny of such cells. Host cells include “transformers” (or “transformed cells”) and “transfectants” (or “transfected cells”), each comprising primary transformed or transfected cells and their derived progeny. Such progeny may not be identical to the parent cells in terms of nucleic acid contents and may contain mutations. “Recombinant host cell” or “host cell” refers to a cell containing exogenous polynucleotides, regardless of the method used for insertion, such as direct uptake, transduction, f-crossing, or other methods known in the art for producing recombinant host cells.

[0057] As used in this article, the term "eukaryote" refers to organisms belonging to the phylogenetic domain Eucarya, such as animals (including but not limited to mammals, insects, reptiles, birds, etc.), ciliates, plants (including but not limited to monocots, dicots, algae, etc.), fungi, yeasts, flagellates, microsporidia, protozoa, etc.

[0058] As used herein, the term "prokaryote" refers to a prokaryotic organism. For example, non-eukaryotic organisms can belong to the phylogenetic domain Eubacteria (including, but not limited to, *Escherichia coli*). Escherichia coli Thermostats ( ) Thermus thermophilus ), thermophilic steatobacterium ( Bacillus stearothermophilus ), Fluorescent Pseudomonas ( Pseudomonas fluorescens ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), Pseudomonas putida ( Pseudomonas putida (etc.), or the phylogenetic archaea domain (including but not limited to methanococcus janthii) Methanococcus jannaschii Thermotrophic methanobacterium ( Methanobacterium thermoautotrophicum ), halophilic bacteria such as Halophilus warwick ( Haloferax volcanii ) and species of the genus *Halobacter* NRC-1 ( Halobacterium species NRC-1 Archaeocystis scintillans ( ) Archaeoglobus fulgidus ), Vibrio parahaemolyticus ( Pyrococcus furiosus ), Horikoshi fireball ( Pyrococcus horikoshii ), Agile Fire Bacteria ( Aeropyrum pernix )wait).

[0059] As used herein, "effective dose" or "therapeutic effective dose" refers to the amount of a therapeutic compound (e.g., an anti-IL-13 antibody) administered to an individual as a single dose or as part of a series of doses, which, alone or in combination with another mode of treatment, effectively produces or contributes to the desired therapeutic effect. Examples of desired therapeutic effects include enhanced immune response, slowing or delaying tumor development; disease stabilization; and improvement of one or more symptoms. An effective dose may be administered in one or more doses.

[0060] The term "treating" (and its variations, such as "treat" or "treatment") refers to a clinical intervention that attempts to alter the natural course of a disease or condition in a subject with a corresponding need. Treatment can be performed during the clinicopathological process. The desired effects of treatment include preventing disease recurrence, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing disease progression, improving or mitigating the disease state, and alleviating or improving prognosis.

[0061] The term "sufficient amount" refers to an amount sufficient to produce the desired effect, such as an amount sufficient to modulate the immune response of a subject.

[0062] As used herein, the terms "subject" or "individual" refer to a mammalian subject. Exemplary subjects include humans, monkeys, dogs, cats, mice, rats, cattle, horses, camels, goats, rabbits, and sheep. In some embodiments, the subject is a human. In some embodiments, the subject suffers from a disease or condition that can be treated with the antibodies provided herein. In some embodiments, the disease or condition is cancer. In some embodiments, the disease or condition is a viral infection.

[0063] The term "in vitro" refers to a process that occurs in living cells that grow separately from a living organism, such as a process that grows in a tissue culture.

[0064] The term "in vivo" refers to processes that occur within a living organism.

[0065] The term "packaging insert" is used to refer to the instruction leaflet typically included in the commercial packaging of therapeutic or diagnostic products (e.g., test kits), which contains information concerning indications, usage, dosage, administration, combination therapy, contraindications, and / or warnings related to the use of such therapeutic or diagnostic products.

[0066] The term "pharmaceutical composition" refers to an article whose form allows the biological activity of the active ingredient contained therein to be effective in treating a subject, and which does not contain any additional components that would have unacceptable toxicity to the subject in the amount provided in the pharmaceutical composition.

[0067] The terms "co-administered," "combined," and "in combination with" include the simultaneous, parallel, or sequential administration of two or more therapeutic agents without a specific time limit. In one embodiment, the agents are simultaneously present in cells or within the subject's body or exert their biological or therapeutic effects simultaneously. In one embodiment, the therapeutic agents are contained in the same composition or unit dosage form. In other embodiments, the therapeutic agents are contained in separate compositions or unit dosage forms. In some embodiments, the first agent may be administered prior to the administration of the second therapeutic agent.

[0068] The terms “modulate” and “modulation” refer to reducing or suppressing, or activating or increasing, the stated variable.

[0069] The terms “increase” and “activation” refer to an increase of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 2 times, 3 times, 4 times, 5 times, 10 times, 20 times, 50 times, 100 times or more in the stated variable.

[0070] The terms “reduction” and “suppression” refer to a reduction of the stated variable by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 2 times, 3 times, 4 times, 5 times, 10 times, 20 times, 50 times, 100 times or more.

[0071] The term "about" indicates and covers an indicated value as well as a range above and below that value. In some embodiments, the term "about" means ±10%, ±5%, or ±1% of the specified value. In some embodiments, where applicable, the term "about" means the specified value ± one standard deviation of that value.

[0072] The term "agonist" refers to the activation of receptor signaling to induce a biological response associated with receptor activation. An "agonist" is an entity that binds to and activates a receptor.

[0073] The term "antagonist" refers to the inhibition of receptor signaling to suppress biological responses associated with receptor activation. An "antagonist" is an entity that binds to and antagonizes a receptor.

[0074] For any structural and functional features described herein, the methods for determining these features are known in the art.

[0075] When used sequentially, the term "optionally" means to include all listed combinations from one to all, and to cover all sub-combinations.

[0076] The term "amino acid" refers to twenty common, naturally occurring amino acids. These include alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0077] The term "affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen or epitope). Unless otherwise indicated, as used herein, "affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen or epitope).

[0078] As used herein, the term "kd" (sec-1) refers to the dissociation rate constant of a specific antibody-antigen interaction. This value is also known as the koff value.

[0079] As used herein, the term "ka" (M⁻¹×sec⁻¹) refers to the association rate constant of a specific antibody-antigen interaction. This value is also known as the kon value.

[0080] As used herein, the term "KD" (M) refers to the dissociation equilibrium constant of a particular antibody-antigen interaction. KD = kd / ka. In some embodiments, the affinity of an antibody is described based on the KD of the interaction between such an antibody and its antigen. For clarity, as is known in the art, a smaller KD value indicates a higher affinity interaction, while a larger KD value indicates a lower affinity interaction.

[0081] As used herein, the term "KA" (M-1) refers to the association equilibrium constant of a specific antibody-antigen interaction. KA = ka / kd.

[0082] The term "antibody" is used in its broadest sense herein and includes certain types of immunoglobulin molecules that contain one or more antigen-binding domains that specifically bind to antigens or epitopes. Antibodies specifically include intact antibodies (e.g., intact immunoglobulins), antibody fragments, and multispecific antibodies.

[0083] "Anti-IL-13 antibody", "IL-13 antibody" or "IL-13 specific antibody" is an antibody that specifically binds to the antigen IL-13, as described herein.

[0084] The term "epitope" refers to the part of an antigen that specifically binds to an antibody.

[0085] As used herein, the term “hypervariant region” or “HVR” refers to each region of an antibody variable domain that is sequence hypervariable and / or forms a structurally defined loop (“hypervariant loop”).

[0086] The term "antigen-binding domain" refers to the portion of an antibody that can specifically bind to an antigen or epitope.

[0087] The term "chimeric antibody" refers to an antibody in which a portion of the heavy chain and / or light chain originates from a specific source or species, while the remaining portion of the heavy chain and / or light chain originates from a different source or species.

[0088] The term "human antibody" refers to an antibody having an amino acid sequence corresponding to that of antibodies produced by humans or human cells, or an antibody derived from a non-human source (e.g., obtained from a human source or designed de novo) using a human antibody library or human antibody encoding sequence. Human antibodies specifically exclude humanized antibodies.

[0089] The term "humanized antibody" refers to a protein having a sequence that differs from that of an antibody derived from a non-human species by one or more amino acid substitutions, deletions, and / or additions, such that when administered to human subjects, the humanized antibody is less likely to induce an immune response and / or induce a less severe immune response compared to antibodies from non-human species.

[0090] The term "multispecific antibody" refers to an antibody that contains two or more different antigen-binding domains that commonly and specifically bind to two or more different epitopes.

[0091] "Bispecific antibodies" are antibodies that contain two different antigen-binding domains, each binding to a different epitope.

[0092] A "monospecific antibody" is an antibody that contains one or more binding sites that specifically bind to a single epitope. An example of a monospecific antibody is the naturally occurring IgG molecule, which, although bivalent (i.e., having two antigen-binding domains), recognizes the same epitope at each of the two antigen-binding domains. Binding specificity can exist in any suitable valence state.

[0093] The term "monoclonal antibody" refers to an antibody derived from a substantially homogeneous group of antibodies. Aside from the variants that normally occur during the production of monoclonal antibodies, a substantially homogeneous group of antibodies contains antibodies that are substantially similar and bind to the same epitopes. Such variants are typically present only in small quantities. Monoclonal antibodies are generally obtained by a process involving the selection of a single antibody from more than one. For example, the selection process may involve choosing a unique clone from a pool of multiple clones such as hybridoma clones, phage clones, yeast clones, bacterial clones, or other recombinant DNA clones. The selected antibody can be further modified, for example, to increase its affinity for the target ("affinity maturation"), to humanize the antibody, to improve its production in cell cultures, and / or to reduce its immunogenicity in subjects.

[0094] The term "single-chain" refers to a molecule comprising amino acid monomers linearly linked by peptide bonds. In a particular such embodiment, in a single-chain Fab molecule, the C-terminus of the Fab light chain is attached to the N-terminus of the Fab heavy chain. As described in more detail herein, scFv has a light chain variable domain (VL) that is attached from its C-terminus to the N-terminus of the heavy chain variable domain (VH) via a polypeptide chain. Optionally, scFv comprises a polypeptide chain in which the C-terminus of the VH is attached to the N-terminus of the VL via the polypeptide chain.

[0095] The “Fab fragment” (also known as the antigen-binding fragment) contains a constant domain (CL) of the light chain and a first constant domain (CH1) of the heavy chain, as well as variable domains VL and VH on the light and heavy chains, respectively. The variable domains contain a complementarity-determining loop (CDR, also known as a hypervariable region) involved in antigen binding. The Fab' fragment differs from the Fab fragment in that it has residues added to the carboxyl terminus of the CH1 domain of the heavy chain, including one or more cysteine ​​residues from the antibody hinge region.

[0096] The “F(ab’)2” fragment contains two Fab’ fragments linked by disulfide bonds near the hinge region. The F(ab’)2 fragment can be generated, for example, by recombinant methods or by digesting an intact antibody with pepsin. The F(ab’) fragment can be dissociated, for example, by treatment with β-mercaptoethanol.

[0097] The “Fv” fragment is a non-covalently linked dimer of a heavy chain variable domain and a light chain variable domain.

[0098] A “single-chain Fv” or “sFv” or “scFv” comprises the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. In one embodiment, the Fv polypeptide also includes a polypeptide linker between the VH and VL domains, which allows the scFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun, Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Rosenburg and Moore, Springer-Verlag, New York, pp. 269–315 (1994). HER2 antibody scFv fragments are described in WO93 / 16185; U.S. Patent No. 5,571,894; and U.S. Patent No. 5,587,458.

[0099] The “scFv-Fc” fragment contains an scFv attached to an Fc domain. For example, the Fc domain may be attached to the C-terminus of the scFv. The Fc domain may be following VH or VL, depending on the orientation of the variable domain in the scFv (i.e., VH-VL or VL-VH). Any suitable Fc domain known in the art or described herein may be used. In some cases, the Fc domain contains an IgG4 Fc domain.

[0100] The term "sdAb" refers to a molecule in which one variable domain of the antibody specifically binds to an antigen while the other variable domain is absent. Single-domain antibodies and their fragments are described in Arabi Ghahroudi et al. (1998). FEBS Letters 414:521-526 and Muyldermans et al. (2001) Trends in Biochem. Sci. 26:230-245, each incorporated herein by reference in its entirety. Single-domain antibodies are also known as sdAbs or nanobodies. sdAbs are quite stable and readily expressed as fusion-paired forms with the antibody Fc chain (Harmsen MM, De Haard HJ (2007) “Properties, production, and applications of camelid single-domain antibody fragments”). Appl. Microbiol Biotechnol . 77(1): 13-22).

[0101] The terms “full-length antibody,” “intact antibody,” and “total antibody” are used interchangeably in this document and refer to an antibody that has a structure substantially similar to that of naturally occurring antibodies and has a heavy chain containing an Fc region. For example, when used to refer to an IgG molecule, a “full-length antibody” is an antibody that consists of two heavy chains and two light chains.

[0102] The term "antibody fragment" refers to an antibody that contains a portion of a complete antibody, such as the antigen-binding region or variable region of a complete antibody. Antibody fragments include, for example, Fv fragments, Fab fragments, F(ab')2 fragments, Fab' fragments, scFv (sFv) fragments, and scFv-Fc fragments.

[0103] The term "Fc domain" or "Fc region" in this article is used to define the C-terminal region of the immunoglobulin heavy chain that contains at least a portion of the constant region. This term includes both native sequence Fc regions and variant Fc regions.

[0104] The term “substantially purified” refers to the constructs described herein or variants thereof, which may be substantially or substantially free of components that are typically associated with or interact with proteins, such as those found in their natural environment, i.e., native cells, or in the case of recombinant antibodies, and in some embodiments substantially free of cellular material, including protein articles having less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% (by dry weight) of contaminating proteins.

[0105] In the context of two or more nucleic acid or polypeptide sequences, the term "percentage of identity" refers to the percentage of identical nucleotide or amino acid residues in two or more sequences or subsequences when compared and aligned for maximum correspondence, measured either by using one of the following sequence comparison algorithms (e.g., using publicly available computer software such as BLAST, BLASTP, BLASTN, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, CLUSTAL OMEGA, or MUSCLE software, or other algorithms available to a technician) or by visual inspection. Software used for BLAST analysis (Altschul et al. (1990)) J. Mol. Biol(215:403-410) is publicly available from the National Center for Biotechnology Information (ncbi.nlm.nih.gov). Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithm required to achieve maximum alignment across the full length of the compared sequences. Depending on the application, the "identity" percentage may exist within regions of the compared sequences, such as within functional domains, or, alternatively, across the full length of both compared sequences.

[0106] For sequence comparisons, a reference sequence is typically used, and the test sequence is compared to it. When using a sequence comparison algorithm, the test and reference sequences are input into the computer, and the coordinates of the subsequences are specified if necessary, along with the sequence algorithm program parameters. The sequence comparison algorithm then calculates the percentage of sequence identity between the test sequence and the reference sequence based on the specified program parameters.

[0107] The optimal alignment of sequences for comparison can be performed, for example, by Smith & Waterman (1981). Adv. Appl. Math 2:482 Local homology algorithm, Needleman & Wunsch (1970) J. Mol. Biol. The homology alignment algorithm for 48:443, Pearson & Lipman (1988). Proc. Nat ' l. Acad. Sci. Similarity search methods as described in USA 85:2444, computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics software package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al., below).

[0108] The ranges stated herein should be understood as abbreviations of all values ​​within that range, including the stated endpoints. For example, the range 1 to 50 is understood to include any number, combination of numbers, or subrange of numbers that comes from the following groups: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50.

[0109] It must be noted that, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” as used in the specification and appended claims include plural indicators.

[0110] Bispecific anti-IL-13 / TSLP antibody Basic antibody structure Recognized immunoglobulin (antibody) genes include κ, λ, α, γ, δ, ε, and μ constant region genes, as well as numerous immunoglobulin variable region genes. Light chains are classified as κ or λ. The "class" of an antibody or immunoglobulin refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The constant domains of the heavy chain corresponding to different classes of immunoglobulins are respectively called α, δ, ε, γ, and µ.

[0111] An exemplary immunoglobulin structural unit comprises two pairs of polypeptide chains, each pair having a “light” chain (approximately 25 kD) and a “heavy” chain (approximately 50-70 kD). The N-terminal domain of each chain defines a variable region of approximately 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chain domains, respectively. The IgG1 heavy chain comprises VH, CH1, CH2, and CH3 domains from its N to C-terminus, respectively. The light chain comprises VL and CL domains from its N to C-terminus. The IgG1 heavy chain comprises a hinge between the CH1 and CH2 domains. In some embodiments, the immunoglobulin construct comprises at least one immunoglobulin domain from IgG, IgM, IgA, IgD, or IgE linked to a therapeutic polypeptide. In some embodiments, the immunoglobulin domains found in the antibodies provided herein are derived from or derived from immunoglobulin-based constructs, such as biantibodies or nanobodies. In some embodiments, the immunoglobulin constructs described herein comprise at least one immunoglobulin domain from a heavy chain antibody such as a camel antibody. In some embodiments, the immunoglobulin constructs provided herein comprise at least one immunoglobulin domain from a mammalian antibody such as a bovine antibody, human antibody, camel antibody, mouse antibody, or any chimeric antibody.

[0112] In some embodiments, the antibodies provided herein comprise a heavy chain. In one embodiment, the heavy chain is IgA. In one embodiment, the heavy chain is IgD. In one embodiment, the heavy chain is IgE. In one embodiment, the heavy chain is IgG. In one embodiment, the heavy chain is IgM. In one embodiment, the heavy chain is IgG1. In one embodiment, the heavy chain is IgG2. In one embodiment, the heavy chain is IgG3. In one embodiment, the heavy chain is IgG4. In one embodiment, the heavy chain is IgA1. In one embodiment, the heavy chain is IgA2.

[0113] In some embodiments, the antibody is an IgG1 antibody. In some embodiments, the antibody is an IgG3 antibody. In some embodiments, the antibody is an IgG2 antibody. In some embodiments, the antibody is an IgG4 antibody.

[0114] Typically, natural four-chain antibodies contain six hypervariable regions (HVRs); three in the variable ring (VH) (H1, H2, and H3) and three in the variable chain (VL) (L1, L2, and L3). The HVRs usually contain amino acid residues from the hypervariable loop and / or from the complementarity-determining region (CDR), which has the highest sequence variability and / or is involved in antigen recognition. Except for CDR1 in the VH, the CDR usually contains amino acid residues that form the hypervariable loop. HVRs are also referred to as CDRs, and these terms are used interchangeably throughout this text, referring to the portion of the variable region that forms the antigen-binding region. (References: Kabat et al. (1983), US Dept. of Health and Human Services, Sequences of Proteins of Immunological Interest; Chothia et al. (1987)) J Mol Biol 196:901-917 has already described this specific region, where the definition includes overlap or subsets of amino acid residues when compared with each other. However, the application of either definition to refer to the CDR of an antibody or its variants is intended to fall within the scope of the terminology defined and used herein. The exact number of residues containing a particular CDR will vary depending on the sequence and size of the CDR. Given the amino acid sequence of the variable region of an antibody, those skilled in the art can routinely determine which residues contain a particular CDR.

[0115] The amino acid sequence boundaries of a CDR can be determined by those skilled in the art using any of many known numbering schemes, including Kabat et al., above (“Kabat” numbering scheme); Al-Lazikani et al. (1997). J. Mol. Biol., 273:927-948 (“Chothia” numbering scheme); MacCallum et al. (1996). J. Mol. Biol. 262:732-745 (“Contact” numbering scheme); Lefranc et al. (2003). Dev. Comp. Immunol. 27:55-77 (“IMGT” numbering scheme); and Honegge and Plückthun (2001). J. Mol. Biol. Those described in 309:657-70 (“AHo” numbering scheme); each of them is incorporated herein by reference in its entirety.

[0116] Table 1 provides the locations of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 identified using the Kabat and Chothia schemes. For CDR-H1, residue numbers were provided using both the Kabat and Chothia numbering schemes.

[0117] CDRs can be assigned, for example, using antibody numbering software such as Abnum, which is available at www.bioinf.org.uk / abs / abnum / and described in Abhinandan and Martin (2008). Immunology In , 45:3832-3839, it is incorporated in its entirety by reference.

[0118] Table 1. Residues in the CDR according to the Kabat and Chothia numbering scheme.

[0119] *When using the Kabat numbering convention, the C-terminus of CDR-H1 varies between H32 and H34 depending on the length of the CDR.

[0120] When referring to residues in the constant region of the antibody heavy chain, the “EU numbering scheme” is generally used (e.g., as reported by Kabat et al., ibid.). Unless otherwise stated, the EU numbering scheme is used to refer to residues in the constant region of the antibody heavy chain as described herein.

[0121] One example of an antigen-binding domain is an antigen-binding domain formed by the VH-VL dimer of an antibody. Another example of an antigen-binding domain is an antigen-binding domain formed by the diversification of certain loops in the tenth fibronectin type III domain of Adnectin. An antigen-binding domain may sequentially contain CDRs 1, 2, and 3 from the heavy chain; and sequentially contain CDRs 1, 2, and 3 from the light chain.

[0122] Epitopes typically consist of surface-accessible amino acid residues and / or sugar side chains, and can possess specific three-dimensional structural features and specific charge characteristics. The difference between conformational and non-conformational epitopes is that binding to the former, but not the latter, can be lost in the presence of denaturing solvents. Epitopes can contain amino acid residues that directly participate in binding and other amino acid residues that do not directly participate in binding. Epitopes that bind to antibodies can be determined using known techniques for epitope identification, such as testing the binding of antibodies to IL-13 variants with different point mutations or to chimeric IL-13 variants.

[0123] To screen for antibodies that bind to epitopes on target antigens (e.g., IL-13, TSLP, or TSLPR) to which the antibody of interest binds, routine cross-blocking assays can be performed, as described in Antibodies, A Laboratory Manual, ColdSpring Harbor Laboratory, Ed Harlow, and David Lane (1988). Alternatively or additionally, epitope mapping can be performed using methods known in the art.

[0124] Chimeric antibodies are antibodies in which a portion of the heavy chain and / or light chain originates from a specific source or species, while the remainder of the heavy chain and / or light chain originates from different sources or species.

[0125] Human antibodies are antibodies having an amino acid sequence that corresponds to the amino acid sequence of antibodies produced by humans or human cells, or antibodies derived from non-human sources (e.g., obtained from human sources or designed de novo) using human antibody libraries or human antibody encoding sequences. Human antibodies specifically exclude humanized antibodies.

[0126] Humanized antibodies have sequences that differ from those of antibodies derived from non-human species by substitution, deletion, and / or addition of one or more amino acids, such that when administered to human subjects, humanized antibodies are less likely to induce an immune response and / or induce a less severe immune response compared to antibodies from non-human species. In one embodiment, certain amino acids in the framework and constant domains of the heavy and / or light chains of a non-human species antibody are mutated to produce a humanized antibody. In another embodiment, a constant domain from a human antibody is fused to a variable domain from a non-human species. In yet another embodiment, one or more amino acid residues in one or more CDR sequences of a non-human antibody are altered to reduce its potential immunogenicity when the non-human antibody is administered to a human subject, wherein the altered amino acid residues are not important for the immune-specific binding of the antibody to its antigen, or the alteration to the amino acid sequence is a conserved alteration such that the binding of the humanized antibody to the antigen is not significantly worse than the binding of the non-human antibody to the antigen. Examples of how to generate humanized antibodies can be found in U.S. Patents 6,054,297, 5,886,152, and 5,877,293. For further details, see Jones et al. (1986). Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-329; and Presta (1992) Curr. Op. Struct. Biol. 2:593-596, each of which is incorporated in its entirety by reference.

[0127] Two or more distinct epitopes can be epitopes on the same antigen (e.g., a single IL-13) or epitopes on different antigens (e.g., different IL-13 molecules, or IL-13 molecules and non-IL-13 molecules). In some embodiments, the multispecific antibody binds to two distinct epitopes (i.e., a "bispecific antibody"). In some embodiments, the multispecific antibody binds to three distinct epitopes (i.e., a "trispecific antibody").

[0128] Anti-IL-13 / TSLP or TSLPR antibodies may include those described herein, such as the clones shown in the figures and / or tables. In some embodiments, the bispecific antibody comprises a replacement scaffold. In some embodiments, the bispecific antibody consists of a replacement scaffold. In some embodiments, the bispecific antibody consists essentially of a replacement scaffold. In some embodiments, the bispecific antibody comprises antibody fragments. In some embodiments, the bispecific antibody consists essentially of antibody fragments.

[0129] In some implementations, the bispecific antibody is a monoclonal antibody.

[0130] In some implementations, the bispecific antibody is a polyclonal antibody.

[0131] In some embodiments, the bispecific antibody is generated by a hybridoma. In other embodiments, the bispecific antibody is generated by recombinant cells engineered to express the desired variable and constant domains.

[0132] In some implementations, the bispecific antibody may be a single-chain antibody or other antibody derivative or variant thereof that retains antigen specificity and the lower hinge region.

[0133] In some embodiments, the bispecific antibody may be a multifunctional antibody, a recombinant antibody, a human antibody, a humanized antibody, a fragment thereof, or a variant thereof. In a particular embodiment, the antibody fragment or a derivative thereof is selected from Fab fragments, Fab'2 fragments, CDRs, and scFvs.

[0134] In some implementations, bispecific antibodies can form immune complexes. For example, immune complexes can be tumor cells covered by bispecific antibodies.

[0135] For sequence comparisons, a reference sequence is typically used, and the test sequence is compared to it. When using a sequence comparison algorithm, the test and reference sequences are input into the computer, and the coordinates of the subsequences are specified if necessary, along with the sequence algorithm program parameters. The sequence comparison algorithm then calculates the percentage of sequence identity between the test sequence and the reference sequence based on the specified program parameters.

[0136] Bispecific antibody structure This application provides antibodies and compositions comprising bispecific antibodies binding to IL-13 and TSLP or TSLPR. The bispecific antibodies disclosed herein may have any structure known in the art.

[0137] Bispecific antibodies can take any of the forms shown in the examples below, but are not limited to. One form is a bispecific antibody comprising a first immunoglobulin heavy chain, a second immunoglobulin heavy chain, and an immunoglobulin light chain. The first immunoglobulin heavy chain comprises a first Fc (hinge-CH2-CH3) domain, a first variable heavy chain domain, and optionally a first CH1 heavy chain domain. The immunoglobulin light chain comprises a variable light chain domain and a constant light chain domain; the immunoglobulin light chain, together with the first immunoglobulin heavy chain, forms an antigen-binding site that binds to IL-13. The second immunoglobulin heavy chain comprises a second Fc (hinge-CH2-CH3) domain, a second variable heavy chain domain, and a second CH1 heavy chain domain, which can pair with the same immunoglobulin light chain that pairs with the first immunoglobulin heavy chain, except that when the immunoglobulin light chain pairs with the second immunoglobulin heavy chain, the resulting antigen-binding site binds to TSLP or TSLPR.

[0138] like Figure 1 Another exemplary form shown relates to a bispecific antibody (IgG-scFv form) comprising two pairs of immunoglobulin light chains and immunoglobulin heavy chains. The first light chain / heavy chain pair comprises a first immunoglobulin heavy chain, an immunoglobulin light chain, and an scFv. The first immunoglobulin heavy chain includes, from its N-terminus to its C-terminus, a first VH1 domain, a first CH1 domain, a first Fc (hinge-CH2-CH3) domain, and a single-chain Fv (scFv) fused to the first Fc (hinge-CH2-CH3) domain via a linker or antibody hinge. Various linkers can be used to attach the scFv to the first Fc domain (linker H2) or between VL2 and VH2 of the scFv itself (see [linker H2]). Figure 1 The black line connecting VH2 and VL2 of scFv in the illustration is referred to as linker H3 in this document. The immunoglobulin light chain contains a first variable light chain (VL1) domain and a first constant light chain (CL) domain from the N-terminus to the C-terminus. The first light chain / heavy chain pair binds to a second light chain / heavy chain pair to form two Fab regions and an Fc domain containing a first Fc domain and a second Fc domain from the second heavy chain. The C-terminus of each of the first and second Fc domains is attached to scFv. The Fab region can bind IL-13 and the scFv region can bind TSLP or TSLPR, or vice versa.

[0139] In some implementations, the bispecific antibody is in the form of a dual variable domain immunoglobulin (DVD-Ig™), such as... Figure 2As shown, DVD-Ig™ combines the target-binding domains of two monoclonal antibodies via a flexible, naturally occurring linker, producing a tetravalent IgG-like molecule. The DVD-Ig form comprises two pairs of immunoglobulin light chains and immunoglobulin heavy chains. The first heavy chain, from its N-terminus to its C-terminus, includes a first heavy chain variable domain (VH1), a second heavy chain variable domain (VH'), and a first heavy chain constant domain (CH1-CH2-CH3). In some embodiments, the VH1 and VH' domains are linked by a linker. The first light chain, from its N-terminus to its C-terminus, includes a first light chain variable domain (VL1), a second light chain variable domain (VL'), and a constant light chain domain (CL). In some embodiments, VL' is fused to VL2 via a linker.

[0140] In some implementations, the bispecific antibody is in the form of a CrossMab, such as Figure 3 As shown, the CrossMab form is a bispecific antibody comprising two pairs of immunoglobulin light chains and immunoglobulin heavy chains. The first light chain / heavy chain pair comprises a first immunoglobulin heavy chain (HC1) and an immunoglobulin light chain (LC1) having an Fc domain. The first immunoglobulin heavy chain contains a first VH domain (VH1) and a first constant heavy chain domain (CH1-CH2-CH3) from the N-terminus to the C-terminus, and the first immunoglobulin light chain contains a first VL domain and a first constant light chain (CL) domain from the N-terminus to the C-terminus. The second immunoglobulin heavy chain (HC2) contains a second VH domain (VH2) and a second constant heavy chain domain (CL'-CH2-CH3) from the N-terminus to the C-terminus, and the second immunoglobulin light chain (LC2) contains a second VL domain (VL2) and a second constant heavy chain (CH1') domain. In some implementations, the CH3 domains of HC1 and HC2 may contain mutations that promote heterodimerization (e.g., knot and hole mutations).

[0141] In some implementations, the bispecific antibody is in the form of Triomab, a trifunctional, bispecific antibody that maintains an IgG-like shape. This chimera consists of two hemiantibodies derived from two parental antibodies, each hemiantibody having one light chain and one heavy chain.

[0142] In some implementations, the bispecific antibody is in the form of a KiH common light chain (LC), which involves knots-into-holes (KIH) technology. KIH involves engineered C H The three domains facilitate heterodimerization by creating "nodes" or "pores" in each heavy chain. The concept behind the "node-in-pore (KiH)" Fc technology is to introduce "nodes" (e.g., T366W in EU designation) within a CH3 domain (CH3A) by replacing small residues with large residues.CH3A To accommodate the "node," a complementary "pore" surface is created on another CH3 domain (CH3B) by replacing the nearest adjacent residue with a smaller residue (e.g., T366S / L368A / Y407V). CH3B ) Optimization of "pore" mutations through structure-guided phage library screening (Atwell et al. (1997) "Stable heterodimers from remodeling the domain interface of ahomodimer using a phage display library," J. Mol. Biol. 270(1):26–35). X-ray crystal structure of KiH Fc variant (Elliott et al. (2014) "Antiparallel conformation of knoband hole aglycosylated half-antibody homodimers is mediated by a CH2-CH3hydrophobic interaction," J. Mol. Biol. 426(9):1947–57; Mimoto et al. (2014) “Crystal structure of a novel asymmetrically engineered Fc variant with improved affinity for FcgammaRs,” Mol. Immunol. 58(1):132–8) proved that heterodimerization is thermodynamically supported by hydrophobic interactions driven by spatial complementarity at the core interface between CH3 structural domains, while the segment-to-segment and pore-to-pore interfaces do not support homodimerization due to steric hindrance and disruption of favorable interactions, respectively.

[0143] In some implementations, the bispecific antibody is presented as an orthogonal Fab interface. In the orthogonal Fab IgG method (Lewis et al. (2014) "Generation of bispecific IgG antibodies by structure-based design of an orthogonal Fab interface," Nat. Biotechnol. 32(2):191–8), structure-based regional design in only one Fab in LC and HC VH-CH1 A complementary mutation is introduced at the interface without making any changes to the other Fab.

[0144] In some embodiments, the bispecific antibody is in a 2-in-1 Ig form. In some embodiments, the bispecific antibody is in an ES form, which is a heterodimeric construct containing two different Fabs fused to an Fc to bind to target 1 and target 2. Heterodimerization is ensured by electrostatically directed mutations in the Fc. In some embodiments, the bispecific antibody is in a Kλ-monomer form, which is a heterodimeric construct having two different Fabs fused to an Fc stable by a heterodimerization mutation: Fab 1 targeting antigen 1 contains κLC, while the second Fab targeting antigen 2 contains λLC.

[0145] In some embodiments, bispecific antibodies are in the form of Fab arm exchange (antibodies that exchange Fab arms by exchanging the heavy chain and the attached light chain (half-molecule) with a heavy-light chain pair from another molecule, thus generating bispecific antibodies). In some embodiments, bispecific antibodies are in the form of SEED bodies. The chain exchange engineered domain (SEED) platform is designed to generate asymmetric and bispecific antibody-like molecules, a capability that expands the therapeutic applications of natural antibodies. This protein engineering platform is based on exchanging structurally relevant sequences of immunoglobulins within a conserved CH3 domain. The SEED design allows for the efficient generation of AG / GA heterodimers while inhibiting homodimerization of the AG and GA SEED CH3 domains. (Muda M. et al. (2011)) Protein Eng. Des. Sel. 24(5):447-54). In some embodiments, the bispecific antibody is in the form of LuZ-Y, where the leucine zipper is used to induce heterodimerization of two different HCs. (Wranik et al. (2012)) J. Biol. Chem. 287:43331-9).

[0146] In some implementations, the bispecific antibody is in the form of a Cov-X body. In the bispecific Cov-X body, two distinct peptides are linked together using a branched-chain azacyclic butane ketone linker and fused to the scaffold antibody in a site-specific manner under mild conditions. Although the pharmacophores are responsible for functional activity, the antibody scaffold confers a long half-life and Ig-like partitioning. The pharmacophores can be chemically optimized or replaced with other pharmacophores to produce optimized or unique bispecific antibodies. (Doppalapudi et al. (2010)) PNAS 107(52): 22611-22616).

[0147] In some implementations, the bispecific antibody is in the form of an Oasc-Fab heterodimer, comprising a Fab that binds to target 1 and an scFab fused to the Fc that binds to target 2. Heterodimerization is ensured by mutation in the Fc.

[0148] In some implementations, the bispecific antibody is in the form of DuetMab, a heterodimeric construct containing two different Fabs that bind antigens 1 and 2 and an Fc stabilized by heterodimerization mutation. Fabs 1 and 2 contain different SS bridges, which ensure proper LC and HC pairing.

[0149] In some embodiments, the bispecific antibody is in the form of a CrossmAb, which is a heterodimeric construct having two different Fabs of binding targets 1 and 2 fused with an Fc stable by heterodimerization. The CL and CH1 domains, as well as the VH and VL domains, are substituted, for example, CH1 is fused directly (in-line) with VL, while CL is fused directly with VH.

[0150] In some embodiments, the bispecific antibody is in the form of Fit-Ig, which is a homodimeric construct in which the N-terminus of the Fab binding antigen 2 is fused to the HC of the Fab binding antigen 1. The construct contains wild-type Fc.

[0151] Table 2 lists peptide sequences that, when combined, can bind the heavy chain variable domain and light chain variable domain of IL13. Table 1a lists peptide sequences that, when combined, can bind the heavy chain variable domain and light chain variable domain of TSLP or TSLPR.

[0152] Sequence of bispecific anti-IL-13 / TSLP or TSLPR antibodies VH domain In some embodiments, the bispecific antibody provided herein comprises a first VH sequence selected from SEQ ID NO: 1-32, 470, and 688 and a second VH sequence selected from SEQ ID NO: 659-661, 682, 684, 686, and 688. In some embodiments, the bispecific antibody provided herein comprises a first VH sequence containing SEQ ID NO: 3 and a second VH sequence selected from SEQ ID NO: 659-661, 682, 684, 686, and 688.

[0153] In some embodiments, the bispecific antibody provided herein comprises a first VH sequence having at least about 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with a VH sequence selected from SEQ ID NO: 1-32, 470, and 688, and a second VH sequence selected from SEQ ID NO: 659-661, 682, 684, 686, and 688. In some embodiments, the bispecific antibody provided herein comprises a first VH sequence having at least about 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with a VH sequence comprising SEQ ID NO: 3, and a second VH sequence selected from SEQ ID NO: 659-661, 682, 684, 686, and 688. In some embodiments, the bispecific antibody provided herein comprises a first VH sequence selected from SEQ ID NO: 1-32, 470, and 688 and a second VH sequence selected from SEQ ID NO: 659-661, 682, 684, 686, and 688, having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions. In some embodiments, the bispecific antibody provided herein comprises a first VH sequence containing SEQ ID NO: 3 and a second VH sequence selected from SEQ ID NO: 659-661, 682, 684, 686, and 688, having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions. In some embodiments, the amino acid substitutions are conserved amino acid substitutions. In some embodiments, the bispecific antibody described in this paragraph is referred to herein as a "variant". In some embodiments, such variants are derived from the sequences provided herein, for example by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, but can be de novo isolated, for example, according to the methods provided herein for obtaining bispecific antibodies.

[0154] VL domain In some embodiments, the bispecific antibody provided herein comprises a first VL sequence selected from SEQ ID NO: 33-57, 471, and 687 and a second VL sequence selected from SEQ ID NO: 662-664, 681, 683, and 685. In some embodiments, the bispecific antibody provided herein comprises a first VL sequence containing SEQ ID NO: 39 and a second VL sequence selected from SEQ ID NO: 662-664, 681, 683, and 685.

[0155] In some embodiments, the bispecific antibody provided herein comprises a first VL sequence having at least about 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with a VL sequence selected from SEQ ID NO: 33-57, 471, and 687, and a second VL sequence selected from SEQ ID NO: 662-664, 681, 683, and 685. In some embodiments, the bispecific antibody provided herein comprises a first VL sequence having at least about 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with a VL sequence comprising SEQ ID NO: 39, and a second VL sequence selected from SEQ ID NO: 662-664, 681, 683, and 685. In some embodiments, the bispecific antibody provided herein comprises a first VL sequence selected from SEQ ID NO: 33-57, 471, and 687 and a second VL sequence selected from SEQ ID NO: 662-664, 681, 683, and 685, having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions. In some embodiments, the bispecific antibody provided herein comprises a first VL sequence containing SEQ ID NO: 39 and a second VL sequence selected from SEQ ID NO: 662-664, 681, 683, and 685, having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions.

[0156] Table 1a. Sequences of TSLP or TSLPR binding to heavy chain variable region (VH) and light chain variable region (VL) binding regions

[0157] VH-VL combination In some embodiments, the bispecific antibody provided herein comprises a first VH sequence selected from SEQ ID NO: 1-32, 470, and 688 and a second VH sequence selected from SEQ ID NO: 659-661, 682, 684, 686, and 688; and a first VL sequence selected from SEQ ID NO: 33-57, 471, and 687 and a second VL sequence selected from SEQ ID NO: 662-664, 681, 683, and 685. In some embodiments, the bispecific antibody provided herein comprises a first VH sequence containing SEQ ID NO: 3 and a second VH sequence selected from SEQ ID NO: 659-661, 682, 684, 686, and 688; and a first VL sequence containing SEQ ID NO: 39 and a second VL sequence selected from SEQ ID NO: 662-664, 681, 683, and 685.

[0158] In some respects, any one of SEQ ID NO: 1-32, 470 and 688 can be combined with any one of SEQ ID NO: 33-57, 471 and 687, and any one of SEQ ID NO: 659-661, 682, 684, 686 and 688 can be combined with any one of SEQ ID NO: 662-664, 681, 683 and 685.

[0159] In some embodiments, the bispecific antibody provided herein comprises a first VH sequence having at least about 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the VH sequences provided in SEQ ID NO: 1-32, 470, and 688, and a second VH sequence selected from SEQ ID NO: 659-661, 682, 684, 686, and 688; and a first VL sequence having at least about 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the VL sequences provided in SEQ ID NO: 33-57, 471, and 687, and a second VL sequence selected from SEQ ID NO: 662-664, 681, 683, and 685. In some embodiments, the bispecific antibody provided herein comprises a first VH sequence having at least about 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the VH sequence comprising SEQ ID NO: 3 and a second VH sequence selected from SEQ ID NO: 659-661, 682, 684, 686, and 688; and a first VL sequence having at least about 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the VL sequence comprising SEQ ID NO: 39 and a second VL sequence selected from SEQ ID NO: 662-664, 681, 683, and 685. In some embodiments, the bispecific antibodies provided herein comprise a first VH sequence provided in SEQ ID NO: 1-32, 470 and 688 and a second VH sequence selected from SEQ ID NO: 659-661, 682, 684, 686 and 688, having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid substitutions; and a first VL sequence provided in SEQ ID NO: 33-57, 471 and 687 and a second VL sequence selected from SEQ ID NO: 662-664, 681, 683 and 685, having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid substitutions.In some embodiments, the bispecific antibody provided herein comprises a first VH sequence of SEQ ID NO: 3 and a second VH sequence selected from SEQ ID NO: 659-661, 682, 684, 686, and 688, having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions; and a first VL sequence of SEQ ID NO: 39 and a second VL sequence selected from SEQ ID NO: 662-664, 681, 683, and 685, having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions. In some embodiments, the amino acid substitutions are conserved amino acid substitutions. In some embodiments, the antibody described in this paragraph is referred to herein as a "variant". In some embodiments, such variants are derived from the sequences provided herein, for example by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, but can be de novo isolated, for example, according to the methods provided herein for obtaining antibodies.

[0160] In some embodiments, the isolated bispecific antibody comprises a heavy chain variable domain comprising a frame region sequence selected from the sequences shown in SEQ ID NO: 198-229, 255-256, 258-259, 261-285, 311-315, 317-342, 368-369, 371-399, and 540-580. In some embodiments, the isolated bispecific antibody comprises a heavy chain variable domain comprising one, two, three, or four frame region sequences selected from the sequences shown in SEQ ID NO: 198-229, 255-256, 258-259, 261-285, 311-315, 317-342, 368-369, 371-399, and 540-580.

[0161] In some embodiments, the isolated bispecific antibody comprises a light chain variable domain comprising a frame region sequence selected from the sequences shown in SEQ ID NO: 230-231, 233-235, 239, 241-254, 286, 288, 290-291, 293, 296-310, 343-345, 347, 400-424, and 581-609. In some embodiments, the isolated bispecific antibody comprises a light chain variable domain comprising one, two, three, or four frame region sequences selected from those shown in SEQ ID NO: 230-231, 233-235, 239, 241-254, 286, 288, 290-291, 293, 296-310, 343-345, 347, 400-424, and 581-609.

[0162] In some embodiments, the isolated bispecific antibody comprises a heavy chain variable domain comprising one, two, three, or four frame region sequences selected from those shown in SEQ ID NO: 198-229, 255-256, 258-259, 261-285, 311-315, 317-342, 368-369, 371-399, and 540-580, and a light chain variable domain comprising one, two, three, or four frame region sequences selected from those shown in SEQ ID NO: 230-231, 233-235, 239, 241-254, 286, 288, 290-291, 293, 296-310, 343-345, 347, 400-424, and 581-609.

[0163] Table 2. VH-VL sequence of anti-interleukin (IL)-13 antibody

[0164] *The names correspond to the names in the informal sequence list. In some implementations, such IgG4-SP HC constant domains have the following sequence: ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:427).

[0165] In some implementations, such hIgG1-LALA-YTE HC constant domains have the following sequence: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:439).

[0166] In some implementations, such hIgG1-LAGA YTE HC constant domains have the following sequence: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELAGAPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:440).

[0167] In some implementations, such hIgG1-LALA-LS HC constant domains have the following sequence: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPG (SEQ ID NO:446).

[0168] In some implementations, such IgG4-YTE HC constant domains have the following sequence: ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:457).

[0169] In some implementations, such IgG4-LS HC constant domains have the following sequence: ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHSYTQKSLSLSLGK (SEQ ID NO:460).

[0170] In some implementations, such human κLC constant domains have a sequence: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:469).

[0171] CDR In some embodiments, the bispecific antibody provided herein comprises a first antigen-binding site and a second antigen-binding site, the first antigen-binding site comprising three CDRs of a first VH domain selected from SEQ ID NO: 1-32, 470, and 688, such as any CDRs listed in Tables 3, 4, or 5 below, and the second antigen-binding site comprising three CDRs of a second VH domain selected from SEQ ID NO: 659-661, 682, 684, 686, and 688, such as any CDRs listed in Table 3a. In some embodiments, the CDRs are exemplary CDRs. In some embodiments, the CDR is the Kabat CDR. In some embodiments, the CDR is the Chothia CDR. In some embodiments, the CDR is the IMGT CDR. In some embodiments, the CDR is the AbM CDR. In some embodiments, the CDR is the Contact CDR.

[0172] In some embodiments, the first antigen-binding site comprises a CDR having at least about 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with CDR-H1, CDR-H2, or CDR-H3 of SEQ ID NO: 58-140, and the second antigen-binding site comprises a CDR having at least about 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with CDR-H1, CDR-H2, or CDR-H3 of SEQ ID NO: 610-636. In some embodiments, the amino acid substitutions are conserved amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as “variants.” In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some implementations, such variants are not derived from the sequences provided herein, but can be isolated de novo, for example, according to the methods provided herein for obtaining antibodies.

[0173] In some embodiments, the bispecific antibody provided herein comprises a first antigen-binding site and a second antigen-binding site, wherein the first antigen-binding site comprises one to three CDRs of the VL domain of SEQ ID NO: 33-57, 471, and 687, such as any CDRs listed in Tables 6, 7, or 8, and the second antigen-binding site comprises one to three CDRs of the VL domain of SEQ ID NO: 662-664, 681, 683, and 685, such as any CDRs listed in Table 3a below. In some embodiments, the CDRs are exemplary CDRs. In some implementations, the CDR is the Kabat CDR. In some implementations, the CDR is the Chothia CDR. In some implementations, the CDR is the IMGT CDR. In some implementations, the CDR is the AbM CDR. In some implementations, the CDR is the Contact CDR.

[0174] In some embodiments, the first antigen-binding site comprises a CDR having at least about 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with CDR-L1, CDR-L2, or CDR-L3 of SEQ ID NO: 141-188, and the second antigen-binding site comprises a CDR having at least about 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with CDR-L1, CDR-L2, or CDR-L3 of SEQ ID NO: 637-657. In some embodiments, the amino acid substitutions are conserved amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as “variants.” In some embodiments, such variants are derived from the sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some implementations, such variants are not derived from the sequences provided herein, but can be isolated de novo, for example, according to the methods provided herein for obtaining antibodies.

[0175] In some embodiments, the bispecific antibody provided herein includes a first antigen-binding site and a second antigen-binding site. The first antigen-binding site includes three CDRs of the VH domain selected from SEQ ID NO: 1-32, 470, and 688 and three CDRs of the VL domain selected from SEQ ID NO: 33-57, 471, and 687. The second antigen-binding site includes three CDRs of the VH domain selected from SEQ ID NO: 659-661, 682, 684, 686, and 688 and three CDRs of the VL domain selected from SEQ ID NO: 662-664, 681, 683, and 685. In some embodiments, the bispecific antibody provided herein comprises a first antigen-binding site and a second antigen-binding site. The first antigen-binding site comprises three CDRs containing the VH domain of SEQ ID NO: 3 and three CDRs containing the VL domain of SEQ ID NO: 39. The second antigen-binding site comprises three CDRs containing the VH domain selected from SEQ ID NO: 659-661, 682, 684, 686, and 688, and three CDRs containing the VL domain of SEQ ID NO: 662-664, 681, 683, and 685. In some embodiments, the CDRs are exemplary CDRs. In some embodiments, the CDR is the Kabat CDR. In some embodiments, the CDR is the Chothia CDR. In some embodiments, the CDR is the IMGT CDR. In some embodiments, the CDR is the AbM CDR. In some embodiments, the CDR is the Contact CDR.

[0176] In some embodiments, the bispecific antibody provided herein comprises a first antigen-binding site and a second antigen-binding site. The first antigen-binding site comprises CDR-H3 selected from SEQ ID NO: 112-120 and 130-140, CDR-H2 selected from SEQ ID NO: 100-111, CDR-H1 selected from SEQ ID NO: 58-99 and 121, CDR-L3 selected from SEQ ID NO: 165-172, CDR-L2 selected from SEQ ID NO: 153-158 and LAS, and CDR-L1 selected from SEQ ID NO: 141-144 and 149-152. The second antigen-binding site comprises CDR-H3 selected from SEQ ID NO: 628-636, CDR-H2 selected from SEQ ID NO: 619-627, CDR-H1 selected from SEQ ID NO: 610-618, and CDR-L1 selected from SEQ ID NO: 610-618. CDR-L3 of SEQ ID NO: 652-657, CDR-L2 of SEQ ID NO: 646-651, and CDR-L1 of SEQ ID NO: 637-645. In some embodiments, the bispecific antibody provided herein comprises a first antigen-binding site and a second antigen-binding site. The first antigen-binding site comprises CDR-H3 selected from SEQ ID NO: 112 and 130, CDR-H2 selected from SEQ ID NO: 100, 104, and 108, CDR-H1 selected from SEQ ID NO: 58, 68, and 85, CDR-L3 selected from SEQ ID NO: 165, CDR-L2 selected from SEQ ID NO: 153 and LAS, and CDR-L1 selected from SEQ ID NO: 141 and 589. The second antigen-binding site comprises CDR-H3 selected from SEQ ID NO: 628-636, CDR-H2 selected from SEQ ID NO: 619-627, CDR-H1 selected from SEQ ID NO: 610-618, CDR-L3 selected from SEQ ID NO: 652-657, and CDR-L3 selected from SEQ ID NO: 141 and 589. CDR-L2 of SEQ ID NO: 646-651, and CDR-L1 selected from SEQ ID NO: 637-645.

[0177] In some embodiments, at the first antigen binding site, CDR-H3 has at least about 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with CDR-H3 selected from SEQ ID NO: 112 and 130; CDR-H2 has at least about 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with CDR-H2 selected from SEQ ID NO: 100, 104, and 108; CDR-H1 has at least about 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with CDR-H1 selected from SEQ ID NO: 58, 68, and 85; and CDR-L3 has at least about 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with CDR-H1 selected from SEQ ID NO: 58, 68, and 85. CDR-L3 of SEQ ID NO: 165 has at least about 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with CDR-L2 selected from SEQ ID NO: 153 and LAS, and CDR-L1 has at least about 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with CDR-L2 selected from SEQ ID NO: 141 and 589. Furthermore, at the second antigen binding site, CDR-H3 is identical to CDR-L2 selected from SEQ ID NO: 141 and 589. CDR-H3 of SEQ ID NOs 628-636 has at least about 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with CDR-H2 of SEQ ID NOs 619-627; CDR-H1 has at least about 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with CDR-H2 of SEQ ID NOs 610-618; CDR-L3 has at least about 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with CDR-H1 of SEQ ID NOs 610-618; and CDR-L3 has at least about 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with CDR-H2 of SEQ ID NOs 619-627. The CDR-L3 of SEQ ID NO: 652-657 has at least about 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the CDR-L2 selected from SEQ ID NO: 646-651.Furthermore, CDR-L1 shares at least approximately 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with CDR-L1 selected from SEQ ID NO: 637-645.

[0178] In some embodiments, the amino acid substitutions are conserved amino acid substitutions. In some embodiments, the antibodies described in this disclosure are referred to herein as “variants” or “clones.” In some embodiments, such variants or clones are derived from the sequences provided herein, for example by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants or clones are not derived from the sequences provided herein, but can be de novo isolated, for example, according to the methods provided herein for obtaining antibodies.

[0179] In some respects, the antibodies disclosed herein do not include those disclosed in U.S. Patent No. 9,067,994.

[0180]

[0181]

[0182] In some embodiments, the bispecific antibodies disclosed herein are in any of the bispecific antibody forms described herein. In some embodiments, the bispecific antibody forms disclosed herein are selected from the group consisting of: (a) single-chain Fv (scFv), (b) tandem scFv forms of bispecific T-cell adaptors (BiTE), (c) disulfide-linked bispecific antibody forms of dual affinity retargeting (DART) bsAbs, (d) tandem bispecific antibodies (TandAbs), (e) conventional immunoglobulin G (IgG), (f) IgG with additional binding units such as scFv, (g) dual variable domain immunoglobulin (DVD-Ig), (h) quadromab bsAbs, (i) knobs-into-holes (KiH) bsAbs with a common light chain, (j) KiH-CrossMabCH1-CL, and (k) bsAbs via controlled Fab arm exchange (cFAE). (See Shim et al. (2020)) Biomolecules 26;10(3):360.) In some embodiments, the bispecific antibody disclosed herein is selected from the group consisting of IgG-scFv and DVD-Ig. In some embodiments, the bispecific antibody disclosed herein is in the form of IgG-scFv. In some embodiments, the bispecific antibody disclosed herein is in the form of DVD-Ig. In some embodiments, the bispecific antibody disclosed herein is in the form of kiH-CrossMabCH1-CL.

[0183] In some implementations, the heavy chain / chain A construct with an alias and construct number in Table 11a or Table 11b is combined with the light chain / chain B construct with the same alias and construct number in Table 12a or Table 12b.

[0184] The abbreviations used in the following paragraphs to describe the first VH sequence, heavy chain constant region, first VL sequence, second VH sequence, joint, and light chain constant structural domain correspond to the abbreviations used in the column headings of Tables 11a, 11b, 12a, and 12b.

[0185] In some embodiments, the bispecific antibody disclosed herein is in the form of IgG-scFv. In some embodiments, the bispecific form comprises a first VH sequence (VH1) containing SEQ ID NO: 3, a heavy chain constant region (CH(1-3)) containing SEQ ID NO: 439, a linker H2 containing SEQ ID NO: 665, a first VL sequence (VL1) containing SEQ ID NO: 662, a linker H3 containing SEQ ID NO: 666, a second VH (VH2) sequence containing SEQ ID NO: 659, a second VL sequence (VL2) containing SEQ ID NO: 39, and a light chain constant domain (CL) containing SEQ ID NO: 469.

[0186] In some embodiments, the bispecific form comprises a first VH sequence (VH1) containing SEQ ID NO: 3, a heavy chain constant region (CH(1-3)) containing SEQ ID NO: 439, a linker H2 containing SEQ ID NO: 665, a first VL sequence (VL2) containing SEQ ID NO: 663, a linker H3 containing SEQ ID NO: 666, a second VH (VH2) sequence containing SEQ ID NO: 660, a second VL sequence (VL1) containing SEQ ID NO: 39, and a light chain constant domain (CL) containing SEQ ID NO: 469.

[0187] In some embodiments, the bispecific form comprises a first VH sequence (VH1) containing SEQ ID NO: 3, a heavy chain constant region (CH(1-3)) containing SEQ ID NO: 439, a linker H2 containing SEQ ID NO: 665, a first VL sequence (VL2) containing SEQ ID NO: 664, a linker H3 containing SEQ ID NO: 666, a second VH (VH2) sequence containing SEQ ID NO: 661, a second VL sequence (VL1) containing SEQ ID NO: 39, and a light chain constant domain (CL) containing SEQ ID NO: 469.

[0188] In some embodiments, the bispecific form comprises a first VH sequence (VH1) containing SEQ ID NO: 659, a heavy chain constant region (CH(1-3)) containing SEQ ID NO: 439, a linker H2 containing SEQ ID NO: 665, a first VL sequence (VL2) containing SEQ ID NO: 39, a linker H3 containing SEQ ID NO: 666, a second VH (VH2) sequence containing SEQ ID NO: 3, a second VL sequence (VL1) containing SEQ ID NO: 662, and a light chain constant domain (CL) containing SEQ ID NO: 669.

[0189] In some embodiments, the bispecific form comprises a first VH sequence (VH1) containing SEQ ID NO: 660, a heavy chain constant region (CH(1-3)) containing SEQ ID NO: 439, a linker H2 containing SEQ ID NO: 665, a first VL sequence (VL2) containing SEQ ID NO: 39, a linker H3 containing SEQ ID NO: 666, a second VH (VH2) sequence containing SEQ ID NO: 3, a second VL sequence (VL1) containing SEQ ID NO: 663, and a light chain constant domain (CL) containing SEQ ID NO: 469.

[0190] In some embodiments, the bispecific form comprises a first VH sequence (VH1) containing SEQ ID NO: 661, a heavy chain constant region (CH(1-3)) containing SEQ ID NO: 439, a linker H2 containing SEQ ID NO: 665, a first VL sequence (VL2) containing SEQ ID NO: 39, a linker H3 containing SEQ ID NO: 666, a second VH (VH2) sequence containing SEQ ID NO: 3, a second VL sequence (VL1) containing SEQ ID NO: 664, and a light chain constant domain (CL) containing SEQ ID NO: 469.

[0191] In some embodiments, the bispecific form comprises a first VH sequence (VL') containing SEQ ID NO: 3, a adapter containing SEQ ID NO: 667, a second VH sequence (VL1) containing LAS, a heavy chain constant region (CH(1-3)) containing SEQ ID NO: 439, a first VL sequence (VL') containing SEQ ID NO: 39, a adapter containing SEQ ID NO: 668, a second VL (VL1) sequence containing SEQ ID NO: 662, and a light chain constant domain (CL) containing SEQ ID NO: 469.

[0192] In some embodiments, the bispecific form comprises a first VH sequence (VL') containing SEQ ID NO: 3, a adapter containing SEQ ID NO: 667, a second VH sequence (VL1) containing SEQ ID NO: 660, and a heavy chain constant region (CH(1-3)) containing SEQ ID NO: 439, and a first VL sequence (VL') containing SEQ ID NO: 39, a adapter containing SEQ ID NO: 668, a second VL (VL1) sequence containing SEQ ID NO: 663, and a light chain constant domain (CL) containing SEQ ID NO: 469.

[0193] In some embodiments, the bispecific form comprises a first VH sequence (VL') containing SEQ ID NO: 3, a adapter containing SEQ ID NO: 667, a second VH sequence (VL1) containing SEQ ID NO: 661, a heavy chain constant region (CH(1-3)) containing SEQ ID NO: 439, a first VL sequence (VL') containing SEQ ID NO: 39, a adapter containing SEQ ID NO: 668, a second VL (VL1) sequence containing SEQ ID NO: 664, and a light chain constant domain (CL) containing SEQ ID NO: 469.

[0194] In some embodiments, the bispecific form comprises a first VH sequence (VL') containing SEQ ID NO: 659, a linker containing SEQ ID NO: 667, a second VH sequence (VL1) containing SEQ ID NO: 3, a heavy chain constant region (CH(1-3)) containing SEQ ID NO: 439, a first VL sequence (VL') containing SEQ ID NO: 662, a linker containing SEQ ID NO: 668, a second VL (VL1) sequence containing SEQ ID NO: 39, and a light chain constant domain (CL) containing SEQ ID NO: 469.

[0195] In some embodiments, the bispecific form comprises a first VH sequence (VL') containing SEQ ID NO: 660, a linker containing SEQ ID NO: 667, a second VH sequence (VL1) containing SEQ ID NO: 3, a heavy chain constant region (CH(1-3)) containing SEQ ID NO: 439, and a first VL sequence (VL') containing SEQ ID NO: 663, a linker containing SEQ ID NO: 668, a second VL (VL1) sequence containing SEQ ID NO: 39, and a light chain constant domain (CL) containing SEQ ID NO: 469.

[0196] In some embodiments, the bispecific form comprises a first VH sequence (VL') containing SEQ ID NO: 661, a linker containing SEQ ID NO: 667, a second VH sequence (VL1) containing SEQ ID NO: 3, a heavy chain constant region (CH(1-3)) containing SEQ ID NO: 439, and a first VL sequence (VL') containing SEQ ID NO: 664, a linker containing SEQ ID NO: 668, a second VL (VL1) sequence containing SEQ ID NO: 39, and a light chain constant domain (CL) containing SEQ ID NO: 469.

[0197] In some embodiments, the bispecific antibody disclosed herein is in the form of IgG-scFv. In some embodiments, the bispecific form comprises a first VH sequence (VH1) containing SEQ ID NO: 3, a heavy chain constant region (CH(1-3)) containing SEQ ID NO: 439, a linker H2 containing SEQ ID NO: 665, a first VL sequence (VL2) containing SEQ ID NO: 681, a linker H3 containing SEQ ID NO: 666, a second VH (VH2) sequence containing SEQ ID NO: 682, a second VL sequence (VL1) containing SEQ ID NO: 39, and a light chain constant domain (CL) containing SEQ ID NO: 469.

[0198] In some embodiments, the bispecific form comprises a first VH sequence (VH1) containing SEQ ID NO: 3, a heavy chain constant region (CH(1-3)) containing SEQ ID NO: 439, a linker H2 containing SEQ ID NO: 665, a first VL sequence (VL2) containing SEQ ID NO: 683, a linker H3 containing SEQ ID NO: 666, a second VH (VH2) sequence containing SEQ ID NO: 684, a second VL sequence (VL1) containing SEQ ID NO: 39, and a light chain constant domain (CL) containing SEQ ID NO: 469.

[0199] In some embodiments, the bispecific form comprises a first VH sequence (VH1) containing SEQ ID NO: 3, a heavy chain constant region (CH(1-3)) containing SEQ ID NO: 439, a linker H2 containing SEQ ID NO: 665, a first VL sequence (VL2) containing SEQ ID NO: 685, a linker H3 containing SEQ ID NO: 666, a second VH (VH2) sequence containing SEQ ID NO: 686, a second VL sequence (VL1) containing SEQ ID NO: 39, and a light chain constant domain (CL) containing SEQ ID NO: 469.

[0200] In some embodiments, the bispecific form comprises a first VH sequence (VH1) containing SEQ ID NO: 659, a heavy chain constant region (CH(1-3)) containing SEQ ID NO: 439, a linker H2 containing SEQ ID NO: 665, a first VL sequence (VL2) containing SEQ ID NO: 687, a linker H3 containing SEQ ID NO: 666, a second VH (VH2) sequence containing SEQ ID NO: 688, a second VL sequence (VL1) containing SEQ ID NO: 662, and a light chain constant domain (CL) containing SEQ ID NO: 669.

[0201] In some embodiments, the bispecific form comprises a first VH sequence (VH1) containing SEQ ID NO: 660, a heavy chain constant region (CH(1-3)) containing SEQ ID NO: 439, a linker H2 containing SEQ ID NO: 665, a first VL sequence (VL2) containing SEQ ID NO: 687, a linker H3 containing SEQ ID NO: 666, a second VH (VH2) sequence containing SEQ ID NO: 688, a second VL sequence (VL1) containing SEQ ID NO: 663, and a light chain constant domain (CL) containing SEQ ID NO: 469.

[0202] In some embodiments, the bispecific form comprises a first VH sequence (VH1) containing SEQ ID NO: 661, a heavy chain constant region (CH(1-3)) containing SEQ ID NO: 439, a linker H2 containing SEQ ID NO: 665, a first VL sequence (VL2) containing SEQ ID NO: 687, a linker H3 containing SEQ ID NO: 666, a second VH (VH2) sequence containing SEQ ID NO: 688, a second VL sequence (VL1) containing SEQ ID NO: 664, and a light chain constant domain (CL) containing SEQ ID NO: 469.

[0203] In some embodiments, the bispecific form comprises a first VH sequence (VH1) containing SEQ ID NO: 3, a heavy chain constant region (CH(1-3)) containing SEQ ID NO: 689, a first VL sequence (VL1) containing SEQ ID NO: 39 and a light chain constant domain (CL) containing SEQ ID NO: 469, a second VH sequence (VH2) containing SEQ ID NO: 659, a heavy chain constant domain (CH2) containing SEQ ID NO: 690, a second VL sequence (VL2) containing SEQ ID NO: 662 and a second light chain constant domain containing SEQ ID NO: 692.

[0204] In some embodiments, the bispecific form comprises a first VH sequence (VH1) containing SEQ ID NO: 3, a heavy chain constant region (CH(1-3)) containing SEQ ID NO: 689, a first VL sequence (VL1) containing SEQ ID NO: 39 and a light chain constant domain (CL) containing SEQ ID NO: 469, a second VH sequence (VH2) containing SEQ ID NO: 660, a heavy chain constant domain (CH2) containing SEQ ID NO: 691, a second VL sequence (VL2) containing SEQ ID NO: 663 and a second light chain constant domain containing SEQ ID NO: 692.

[0205] In some embodiments, the bispecific form comprises a first VH sequence (VH1) containing SEQ ID NO: 659, a heavy chain constant region (CH(1-3)) containing SEQ ID NO: 689, a first VL sequence (VL1) containing SEQ ID NO: 662 and a light chain constant domain (CL) containing SEQ ID NO: 669, a second VH sequence (VH2) containing SEQ ID NO: 3, a heavy chain constant domain (CH2) containing SEQ ID NO: 691, a second VL sequence (VL2) containing SEQ ID NO: 39 and a second light chain constant domain containing SEQ ID NO: 692.

[0206] In some embodiments, the bispecific form comprises a first VH sequence (VH1) containing SEQ ID NO: 660, a heavy chain constant region (CH(1-3)) containing SEQ ID NO: 689, a first VL sequence (VL1) containing SEQ ID NO: 663 and a light chain constant domain (CL) containing SEQ ID NO: 469, a second VH sequence (VH2) containing SEQ ID NO: 3, a heavy chain constant domain (CH2) containing SEQ ID NO: 691, a second VL sequence (VL2) containing SEQ ID NO: 39 and a second light chain constant domain containing SEQ ID NO: 692.

[0207]

[0208] Fc modification This article describes bispecific antibodies containing a modified Fc region that bind (1) IL-13 and (2) TSLP or TSLPR. Unless otherwise stated herein, the amino acid residues in the Fc region or constant region are numbered according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991.

[0209] In some embodiments, the bispecific antibody comprises a modified Fc containing one or more modifications. In some embodiments, one or more modifications are located in the Fc of IgG1 (e.g., human IgG1 (hIgG1)). In some embodiments, one or more modifications are located in the Fc of IgG4 (e.g., human IgG4 (hIgG4)). In some embodiments, one or more modifications are located in the Fc of IgG2. In some embodiments, one or more modifications promote selective binding to the Fc-γ receptor. In any embodiment, the constant heavy chain region may include a C-terminal lysine residue.

[0210] Table 13 provides an example amino acid sequence for the Fc region.

[0211]

[0212]

[0213]

[0214]

[0215] In some embodiments, any bispecific antibody described herein may comprise an Fc, said Fc comprising one or more modifications having any of the Fc modifications described herein. In some embodiments, any bispecific antibody described herein may comprise any of the Fc sequences in Table 13 (SEQ ID NO: 425-468, 484-539, 670-680, 689-691, and 693-804). In some embodiments, the CH(1-3) domain of the bispecific antibody in Table 11a or Table 11b is replaced by any of the Fc sequences in Table 13 (SEQ ID NO: 425-468, 484-539, 670-680, 689-691, and 693-804).

[0216] In some implementations, one or more of the modifications in Fc are selected from the group consisting of: S298A, E333A, K334A, K326A, F243L, R292P, Y300L, V305I, P396L, F243L, R292P, Y300L, L235V, P396L, F243L, S239D, I332E, A330L, S267E, L328F, D265S, S239E, K326A, A327H, G237F, K326E, G236A, D270L, H268D, S324T, L234F, N325L, V266L, and S267D. In some implementations, one or more of the modifications of Fc are selected from the group consisting of: S228P, M252Y, S254T, T256E, T256D, T250Q, H285D, T307A, T307Q, T307R, T307W, L309D, Q411H, Q311V, A378V, E380A, M428L, N434A, N434S, N297A, D265A, L234A, L235A, and N434W.

[0217] In some embodiments, the modified Fc comprises a specific combination of amino acid substitutions selected from the group consisting of: L234A / L235A; V234A / G237A; L235A / G237A / E318A; S228P / L236E; H268Q / V309L / A330S / A331S; C220S / C226S / C229S / P238S; C226S / C229S / E3233P / L235V / L235A; L234F / L235E / P331S; C226S / P230S; L234A / G237A; L234A / L235A / G237A; and L234A / L235A / P329G.

[0218] In some embodiments, the modified Fc comprises a specific combination of amino acid substitutions selected from the group consisting of: M428L / N434S (LS); M252Y / S254T / T256E (YTE); T250Q / M428L; T307A / E380A / N434A; T256D / T307Q (DQ); T256D / T307W (DW); M252Y / T256D (YD); T307Q / Q311V / A378V (QVV); T256D / H285D / T307R / Q311V / A378V (DDRVV); L309D / Q311H / N434S (DHS); S228P / L235E (SPLE); L234A / L235A (LALA); M428L / N434A (LA); L234A / G237A (LAGA); L234A / L235A / G237A (LALAGA); L234A / L235A / P329G (LALAPG); N297A / YTE; D265A / YTE; LALA / YTE; LAGA / YTE; LALAGA / YTE; LALAPG / YTE; N297A / LS; D265A / LS; LALA / LS; LAGA / LS; LALAGA / LS; LALAPG / LS; N297A / DHS; D265A / DHS; LALA / DHS; LAGA / DH S;LALAGA / DHS;LALAPG / DHS;SP / YTE;SPLE / YTE;SP / LS;SPLE / LS;SP / DHS;SPLE / DHS;N297A / LA;D 265A / LA; LALA / LA; LAGA / LA; LALAGA / LA; LALAPG / LA; N297A / N434A; D265A / N434A; LALA / N434A; L AGA / N434A; LALAGA / N434A; LALAPG / N434A; N297A / N434W; D265A / N434W; LALA / N434W; LAGA / N43 4W; LALAGA / N434W; LALAPG / N434W; N297A / DQ; D265A / DQ; LALA / DQ; LAGA / DQ; LALAGA / DQ; LALAPG / DQ; N297A / DW; D265A / DW; LALA / DW; LAGA / DW; LALAGA / DW; LALAPG / DW; N297A / YD; D265A / YD; LALA / YD;LAGA / YD;LALAGA / YD;LALAPG / YD;N297A / QVV;D265A / QVV;LALA / QVV;LAGA / QVV;LALAGA / QVV;LALAPG / QVV; N297A / DDRVV; D265A / DDRVV; LALA / DDRVV; LAGA / DDRVV; LALAGA / DDRVV; and LALAPG / DDRVV. In some embodiments, the modified Fc comprises a specific combination of amino acid substitutions selected from the group consisting of M428L / N434S (LS) and M252Y / S254T / T256E (YTE). In some embodiments, the modified Fc comprises M428L / N434S (LS) (e.g., SEQ ID NO: 93, SEQ ID NO: 110, SEQ ID NO: 117). In some embodiments, the modified Fc comprises M252Y / S254T / T256E (YTE) modification.

[0219] In some embodiments, the bispecific antibodies described herein include modifications to enhance their ability to mediate effector function. Such modifications are known in the art and include defucosylation or modification of Fc affinity for activating receptors (primarily FCGR3a for antibody-dependent cytotoxicity (ADCC)) and for C1q for complement-dependent cytotoxicity (CDC).

[0220] In some respects, the bispecific antibodies presented herein comprise an Fc domain (e.g., IgG1) with a reduced fucose content at position Asn 297 (EU number) compared to the naturally occurring Fc domain. Such Fc domains are known to have improved ADCC. In some respects, such antibodies do not contain any fucose at position Asn 297.

[0221] In some embodiments, the bispecific antibody described herein comprises an Fc region having one or more amino acid substitutions that improve ADCC, such as substitutions at positions 298, 333, and 334 of the Fc region. In some embodiments, the bispecific antibody provided herein comprises an Fc region having one or more amino acid substitutions at positions 239, 332, and 330.

[0222] In some embodiments, Fc comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence according to any SEQ ID NO in Table 13. In some embodiments, Fc comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence according to any SEQ ID NO in Table 13. In some embodiments, Fc comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence according to any SEQ ID NO in Table 13. In some embodiments, Fc comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence according to any SEQ ID NO in Table 13. In some embodiments, Fc comprises an amino acid sequence having at least 96% sequence identity with the amino acid sequence according to any SEQ ID NO in Table 13. In some embodiments, Fc comprises an amino acid sequence having at least 97% sequence identity with the amino acid sequence according to any SEQ ID NO in Table 13. In some embodiments, Fc comprises an amino acid sequence having at least 98% sequence identity with the amino acid sequence according to any SEQ ID NO in Table 13. In some embodiments, Fc comprises an amino acid sequence having at least 99% sequence identity with the amino acid sequence according to any SEQ ID NO in Table 13.

[0223] In some embodiments, the bispecific antibody described herein comprises an Fc region in which at least one galactose residue of the oligosaccharide is attached. Such variants may have improved CDC function.

[0224] In some implementations, the bispecific antibodies described herein contain one or more alterations that improve or reduce C1q binding and / or CDC.

[0225] In some embodiments, the Fc region contains one or more amino acid substitutions, wherein the one or more substitutions result in an increase in one or more of the antibody half-life, ADCC activity, ADCP activity, or CDC activity compared to an Fc region without said one or more substitutions. In some embodiments, the one or more amino acid substitutions result in an increased antibody half-life at pH 6.0 compared to an antibody containing a wild-type Fc region. In some embodiments, the bispecific antibody has a half-life that is approximately 10,000, 1,000, 500, 100, 50, 20, 10, 9, 8, 7, 6, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.95, 1.9, 1.85, 1.8, 1.75, 1.7, 1.65, 1.6, 1.55, 1.50, 1.45, 1.4, 1.35, 1.3, 1.25, 1.2, 1.15, 1.1, or 1.05 times longer than that of an antibody containing the wild-type Fc region.

[0226] In some embodiments, the Fc region contains one or more amino acid substitutions, wherein the one or more substitutions result in a reduction of one or more of ADCC activity, ADCP activity, or CDC activity compared to an Fc without said one or more substitutions.

[0227] In some embodiments, the Fc region binds to an Fcγ receptor selected from the group consisting of: FcγRI, FcγRIIa, FcγRIIb, FcγRIIC, FcγRIIIa, and FcγRIIIb. In some embodiments, the Fc region binds to the Fcγ receptor with higher affinity at pH 6.0 compared to antibodies containing a wild-type Fc region.

[0228] In some embodiments, the bispecific antibody described herein comprises an extended half-life (i.e., serum half-life). In some embodiments, the bispecific antibody described herein comprises a half-life of at least about 14, 28, 42, 56, 70, 84, 96, or greater than 96 weeks. In some embodiments, the TSLP or TSLPR binding protein described herein comprises a half-life in the range of about 14 days to about 96 days, about 14 days to about 84 days, about 14 days to about 70 days, about 14 days to about 56 days, about 14 days to about 42 days, about 14 days to about 28 days, about 28 days to about 96 days, about 28 days to about 84 days, about 28 days to about 70 days, about 28 days to about 56 days, about 28 days to about 42 days, about 42 days to about 96 days, about 42 days to about 84 days, about 42 days to about 70 days, or about 42 days to about 56 days. In some embodiments, the bispecific antibody described herein comprises a half-life in the range of about 42 days to about 56 days. In some embodiments, the bispecific antibody described herein has a half-life of at least about 50 days. Methods for measuring the half-life are known in the art. In some embodiments, the half-life is measured in non-human primates. In some embodiments, the half-life is measured in humans. In some embodiments, the half-life is measured after intravenous administration. In some embodiments, the half-life is measured after subcutaneous administration.

[0229] In some embodiments, the bispecific antibody described herein has a half-life at least 20% longer than that of the comparative antibody. In some embodiments, the comparative antibody comprises the same complementarity-determining region and variable region but different Fc regions. In some embodiments, the half-life of the bispecific antibody described herein is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% longer than that of the comparative antibody. In some embodiments, the half-life of the bispecific antibody described herein is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 times longer than that of the comparative antibody.

[0230] Combination The affinity of molecule X for its partner Y can be expressed by the dissociation equilibrium constant (K). D The following describes in more detail the kinetic components that contribute to the dissociation equilibrium constant. Affinity can be measured by methods commonly known in the art, including those described herein, such as surface plasmon resonance (SPR) techniques (e.g., BIACORE®) or biolayer interferometry (e.g., FORTEBIO®).

[0231] Regarding antibody binding to target molecules, the terms "binding," "specific binding," "specifically bound to," "specific for," "selectively binding," and "selective for" refer to binding to a specific antigen (e.g., a peptide target) or an epitope on a specific antigen that is measurably different from nonspecific or nonselective interactions (e.g., with non-target molecules). Specific binding can be measured, for example, by measuring binding to a target molecule (i.e., IL-13, TSLP, or TSLPR) and comparing it to binding to a non-target molecule. Specific binding can also be determined by competition with a control molecule that mimics an epitope recognized on a target molecule. In this case, if the binding of the antibody to the target molecule is competitively inhibited by the control molecule, it indicates specific binding. In some embodiments, the bispecific antibody disclosed herein has an affinity for non-target molecules of less than about 50% of its affinity for IL-13, TSLP, or TSLPR. In some embodiments, the bispecific antibody disclosed herein has an affinity for non-target molecules of less than about 40% of its affinity for IL-13, TSLP, or TSLPR. In some embodiments, the bispecific antibodies disclosed herein have an affinity for non-target molecules of less than about 40% for IL-13, TSLP, or TSLPR. In some embodiments, the bispecific antibodies disclosed herein have an affinity for non-target molecules of less than about 20% for IL-13, TSLP, or TSLPR. In some embodiments, the bispecific antibodies disclosed herein have an affinity for non-target molecules of less than about 10% for IL-13, TSLP, or TSLPR. In some embodiments, the bispecific antibodies disclosed herein have an affinity for non-target molecules of less than about 1% for IL-13, TSLP, or TSLPR. In some embodiments, the bispecific antibodies disclosed herein have an affinity for non-target molecules of less than about 0.1% for IL-13, TSLP, or TSLPR.

[0232] When used herein in the context of two or more antibodies, the terms “competing with” or “cross-competing with” mean that two or more antibodies compete to bind to an antigen (e.g., IL-13). In one exemplary assay, IL-13 is coated on a surface and contacted with a first anti-IL-13 antibody, followed by the addition of a second anti-IL-13 antibody. In another exemplary assay, a first anti-IL-13 antibody is coated on a surface and contacted with IL-13, followed by the addition of a second anti-IL-13 antibody. If the presence of the first anti-IL-13 antibody in either assay reduces the binding of the second anti-IL-13 antibody, these antibodies compete with each other. The term “competing with” also includes combinations of antibodies where one antibody reduces the binding of another antibody, but where no competition is observed when the antibodies are added in reverse order. However, in some embodiments, the first and second antibodies inhibit each other’s binding regardless of the order in which they are added. In some implementations, one antibody reduces the binding of another antibody to its antigen by at least 25%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%, as measured in a competitive binding assay. A technician can select the concentration of the antibody used in a competitive assay based on the antibody's affinity for IL-13 and the antibody's valence state. The assays described in this definition are illustrative, and a technician can use any suitable assay to determine whether antibodies compete with each other. Suitable assays are described, for example, in Cox et al., “Immunoassay Methods,” in the Assay Guidance Manual [Internet], Updated December 24, 2014 (ncbi.nlm.nih.gov / books / NBK92434 / ; accessed September 29, 2015); Silman et al., Cytometry , 2001, 44:30-37; and Finco et al., J. Pharm. Biomed. Anal., 2011, 54:351-358; each of the references is incorporated in its entirety by reference.

[0233] If an excess of the test antibody (e.g., at least 2x, 5x, 10x, 20x, or 100x) inhibits or blocks the binding of the reference antibody by, for example, at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, as measured in a competitive binding assay, then the test antibody competes with the reference antibody. Antibodies identified by the competitive assay (competitive antibodies) include antibodies that bind to the same epitope as the reference antibody and antibodies that bind to a neighboring epitope sufficiently close to the epitope bound by the reference antibody due to steric hindrance. For example, a second competitive antibody can be identified that competes with the first antibody described herein for binding to IL-13. In some cases, the second antibody can block or inhibit the binding of the first antibody by, for example, at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, as measured in a competitive binding assay. In some cases, the second antibody can displace the first antibody by greater than 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%.

[0234] In some implementations, the antibody binds to human IL-13 and TSLP or TSLPR.

[0235] In some implementations, the bispecific antibody binds to the IL-13 sequence shown in SEQ ID NO: 472-475.

[0236] In some implementations, the bispecific antibody cross-reacts with cynomolgus monkey IL-13.

[0237] In some implementations, the bispecific antibody is in a concentration of less than or equal to about 1, 2, 3, 4, 5, 6, 7, 8, or 9 x 10⁻⁶. -9 M of K D Combined with the IL-13 sequence shown in SEQ ID NO: 472-475, as measured by SPR. In some embodiments, the bispecific antibody is in a dose of less than or equal to about 1 x 10⁻⁶. -10 M of K D Combined with the IL-13 sequence shown in SEQ ID NO: 472-475, as measured by SPR. In some embodiments, the bispecific antibody is in a dose of less than or equal to about 1 x 10⁻⁶. -9 M of K D Combined with human IL-13, as measured by SPR.

[0238] In some implementations, the bispecific antibodies provided herein are in quantities less than or equal to about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 1.95, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9 or 10 x 10 -8 M of K D Combined with IL-13, as measured by ELISA or any other suitable method known in the art. In some embodiments, the antibodies provided herein are in doses less than or equal to about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 1.95, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9 or 10 x 10 -9 M of K D Combined with IL-13, as measured by ELISA or any other suitable method known in the art.

[0239] In some implementations, the antibodies provided herein bind to IL-13, TSLP, or TSLPR K. D In approximately 0.001-0.01, 0.01-0.1, 0.01-0.05, 0.05-0.1, 0.1-0.5, 0.5-1, 0.25-0.75, 0.25-0.5, 0.5-0.75, 0.75-1, 0.75-2, 1.1-1.2, 1.2-1.3, 1.3-1.4, 1.4-1.5, 1.5-1.6, 1.6-1.7, 1.7-1.8, 1.8-1.9, 1.9-2, 1-2, 1-5, 2-7, 3-8, 3-5, 4-6, 5-7, 6-8, 7-9, 7-10 or 5-10 x 10 -8 The values ​​between M and M are measured, such as by ELISA or any other suitable method known in the art. In some embodiments, the antibodies provided herein are in quantities less than or equal to about 1 x 10⁻⁶. -8 M, or less than or equal to 1 x 10 -9 M of K D Combined with IL-13, TSLP, or TSLPR, as measured by ELISA or any other suitable method known in the art.

[0240] In some implementations, the antibodies provided herein are in quantities less than or equal to about 10, 9, 8, 7, 6, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.98, 1.95, 1.9, 1.85, 1.8, 1.75, 1.7, 1.65, 1.6, 1.55, 1.50, 1.45, 1.4, 1.3, 1.2, 1.1, 1, 0.9, 0.85, 0.8, 0.75, 0.7, 0.65, 0.6, 0.55, 0.5, 0.45, 0.4, 0.35, 0.3, 0.25, 0.2, 0.15, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, or 0.0001 x 10⁻⁶. -8 M or smaller K D Combined with IL-13, TSLP, or TSLPR, as measured by ELISA or any other suitable method known in the art. In some embodiments, the antibodies provided herein are in the form of 5-3, 4-2, 3-1, 1.9-1.8, 1.8-1.7, 1.7-1.6, 1.6-1.5, 1.9-1.5, 1.5-1, 1-0.8, 1-0.5, 0.9-0.6, 0.7-0.4, 0.6-0.2, 0.5-0.3, 0.3-0.2, 0.2-0.1, 0.1-0.01, 0.01-0.001, or 0.001-0.0001 x 10 -8 K between M D Combined with IL-13, TSLP, or TSLPR, as measured by ELISA or any other suitable method known in the art.

[0241] Function "Effective functions" refer to those biological activities mediated by the Fc region of an antibody, which can vary depending on the antibody isotype. Examples of antibody effector functions include receptor-ligand blocking, agonism or antagonism, C1q binding to activate complement-dependent cytotoxicity (CDC), Fc receptor binding to activate antibody-dependent cytotoxicity (ADCC), and antibody-dependent phagocytosis (ADCP). In some embodiments, the effector functions of the bispecific anti-IL-13 / TSLP or TSLPR antibodies described herein are to antagonize and block the binding of the IL-13 receptor to IL-13 and / or to block the binding of TSLP to TSLPR.

[0242] Pharmaceutical Composition This application provides compositions comprising bispecific antibodies, including pharmaceutical compositions comprising any one or more bispecific antibodies described herein and one or more pharmaceutically acceptable excipients. In some embodiments, the composition is sterile. The pharmaceutical composition typically contains an effective amount of bispecific antibody.

[0243] In addition to one or more bispecific antibodies disclosed herein, these compositions may also contain pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The exact nature of the carrier or other material may depend on the route of administration, such as oral, intravenous, skin or subcutaneous, nasal, intramuscular, or intraperitoneal routes.

[0244] Pharmaceutical compositions intended for oral administration may be in tablet, capsule, powder, or liquid form. Tablets may include solid carriers such as gelatin or excipients. Liquid pharmaceutical compositions typically include liquid carriers such as water, petroleum, animal or vegetable oils, mineral oils, or synthetic oils. They may include physiological saline solutions, dextran or other sugar solutions, or glycols such as ethylene glycol, propylene glycol, or polyethylene glycol.

[0245] For intravenous, skin, or subcutaneous injection, or injection at the lesion site, the active ingredient will be a parenteral acceptable aqueous solution that is pyrogen-free and has suitable pH, isotonicity, and stability. Those skilled in the art can readily use, for example, isotonic media such as sodium chloride injection, Ringer's solution, or lactated Ringer's solution to prepare suitable solutions. Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included as needed.

[0246] Bispecific anti-IL-13 / TSLP or TSLPR antibodies administered to an individual are preferably given at a “therapeutic effective dose” or a “prophylactic effective dose” (whichever is appropriate, although prophylaxis may be considered treatment), which is sufficient to demonstrate benefit to the individual. The actual dose administered, as well as the rate and timing of administration, will depend on the nature and severity of the protein aggregation disorder being treated. Treatment prescriptions, such as decisions regarding dosage, are within the responsibility of general practitioners and other physicians, and generally take into account the condition to be treated, the individual patient’s condition, the site of delivery, the method of administration, and other factors known to the practitioner. Examples of the above techniques and protocols can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed.), 1980.

[0247] Depending on the condition to be treated, the composition can be administered alone or in combination with other treatments (simultaneous or sequential administration).

[0248] method Preparation method Methods for preparing bispecific antibodies are known in the art. See Milstein and Cuello (1983) NATURE 305:537, International (PCT) Publication WO93 / 08829, and Traunecker et al. (1991) EMBOJ., 10:3655. For further details on the production of bispecific antibodies, see, for example, Suresh et al. (1986) METHODS ENZYMOL. 121:210. Bispecific antibodies include cross-linked or “heteroconjugated” or “heterodimeric” antibodies. For example, one antibody in a heterodimer may be conjugated to antibiotin and the other to biotin. Heterodimeric antibodies can be prepared using any convenient cross-linking method. Suitable cross-linking agents are well known in the art and are disclosed, along with some cross-linking techniques, in U.S. Patent No. 4,676,980.

[0249] For example, the bispecific antibodies described herein can be generated using recombinant methods and compositions, such as those described in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid encoding the bispecific antibody described herein is provided. Such a nucleic acid may encode an amino acid sequence containing a VL sequence and / or an amino acid sequence containing a VH sequence (e.g., the light chain and / or heavy chain of the antibody). In another embodiment, one or more vectors (e.g., expression vectors) containing such a nucleic acid are provided. In one embodiment, the nucleic acid is provided in a polycistronic vector. In another embodiment, a host cell containing such a nucleic acid is provided. In one such embodiment, the host cell comprises (e.g., having been transformed with): (1) a vector containing a nucleic acid encoding an amino acid sequence containing a VL sequence of an antibody and an amino acid sequence containing a VH sequence of an antigen-binding polypeptide construct, or (2) a first vector containing a nucleic acid encoding an amino acid sequence containing a VL sequence of an antigen-binding polypeptide construct and a second vector containing a nucleic acid encoding an amino acid sequence containing a VH sequence of an antigen-binding polypeptide construct. In one embodiment, the host cell is a eukaryotic cell, such as Chinese hamster ovary (CHO) cells or human embryonic kidney (HEK) cells or lymphocytes (e.g., Y0, NSO, Sp20 cells). In one embodiment, a method for preparing a bispecific antibody is provided, wherein the method includes culturing a host cell containing nucleic acid encoding a bispecific antibody as provided above under conditions suitable for antibody expression, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0250] To recombinantly generate antibodies, nucleic acids encoding antibodies such as those described above are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be readily isolated and sequenced using routine procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody).

[0251] When antibodies or their variants are recombinantly produced from host cells, in some embodiments, the protein is present in the culture medium at about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% or less of the cell dry weight. When antibodies or their variants are recombinantly produced from host cells, in some embodiments, the protein is present in the culture medium at about 5 g / L, about 4 g / L, about 3 g / L, about 2 g / L, about 1 g / L, about 750 mg / L, about 500 mg / L, about 250 mg / L, about 100 mg / L, about 50 mg / L, about 10 mg / L, or about 1 mg / L or less of the cell dry weight. In some embodiments, the "substantially purified" antibody produced by the methods described herein has a purity level of at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, specifically, at least about 75%, 80%, 85%, more specifically, at least about 90%, at least about 95%, at least about 99% or higher, as determined by appropriate methods such as SDS / PAGE analysis, RP-HPLC, SEC, and capillary electrophoresis.

[0252] Suitable host cells for cloning or expressing vectors encoding antibodies include prokaryotic or eukaryotic cells as described herein.

[0253] A recombinant host cell, or host cell, is a cell containing exogenous polynucleotides, regardless of the method used for insertion, such as direct uptake, transduction, f-crossing, or other methods known in the art for generating recombinant host cells. The exogenous polynucleotides may remain as non-integrating vectors, such as plasmids, or optionally may be integrated into the host genome. Host cells may include CHO, CHO derivatives, NSO, Sp20, CV-1, VERO-76, HeLa, HepG2, Per.C6, or BHK.

[0254] For example, antibodies can be generated in bacteria, especially where glycosylation and Fc effector function are not required. For information on the expression of antibody fragments and peptides in bacteria, see, for example, U.S. Patents 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in *Escherichia coli*.) After expression, the antibody can be separated from the bacterial cell paste in soluble fractions and can be further purified.

[0255] Besides prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains whose glycosylation pathways have been "humanized," resulting in antibodies with partial or complete human glycosylation patterns. See Gerngross. Nat. Biotech. 22:1409-1414 (2004), and Li et al. Nat. Biotech. 24:210-215 (2006).

[0256] Suitable host cells for expressing glycosylated antibodies also originate from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains have been identified that can be used in combination with insect cells, particularly for transfecting fall armyworm (Spodoptera frugiperda) cells.

[0257] Plant cell cultures can also be used as hosts. See, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe PLATNIBODIES™ technology for generating antibodies in transgenic plants).

[0258] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for suspension growth can be useful. Other examples of useful mammalian host cell lines are the monkey kidney CV1 line transformed from SV40 (COS-7); human embryonic kidney lines (293 or 293 cells, such as Graham et al.), J. Gen Virol. (described in 36:59 (1977)); young hamster kidney cells (BHK); mouse supporting cells (TM4 cells, such as Mather, etc.). Biol. Reprod.23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumors (MMT 060562); TRI cells, as described, for example, in Mather et al., Annals NY Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980) and myeloma cell lines such as Y0, NSO, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0259] In one embodiment, the antibody described herein is generated in stable mammalian cells by a method comprising the steps of: transfecting at least one mammalian cell with a nucleic acid encoding the antibody at a predetermined ratio; and expressing the nucleic acid in said at least one mammalian cell. In some embodiments, the predetermined ratio of nucleic acid is determined in a transient transfection experiment to determine the relative ratio of the input nucleic acid that results in the highest percentage of antibody in the expression product.

[0260] In some embodiments, a method for generating antibodies in stable mammalian cells as described herein is provided, wherein the expression product of at least one stable mammalian cell contains a larger percentage of the desired glycosylated antibody compared to a single heavy chain or light chain polypeptide or other antibody.

[0261] In some embodiments, a method is provided for generating glycosylated antibodies in stable mammalian cells as described herein, the method comprising identifying and purifying the desired glycosylated antibodies. In some embodiments, the identification is performed by one or both of liquid chromatography and mass spectrometry.

[0262] If desired, antibodies can be purified or isolated after expression. Proteins can be isolated or purified in a variety of ways known to those skilled in the art. Standard purification methods include chromatographic techniques, including ion exchange chromatography, hydrophobic interaction chromatography, affinity chromatography, size chromatography, or gel filtration chromatography, as well as reversed-phase chromatography, performed at atmospheric or high pressure using systems such as FPLC and HPLC. Purification methods also include electrophoresis, immunology, precipitation, dialysis, and chromatography focusing techniques. Ultrafiltration and percolation techniques combined with protein concentration are also useful. As is known in the art, many natural proteins bind to Fc and antibodies, and these proteins can be used in this invention to purify antibodies. For example, bacterial proteins A and G bind to Fc regions. Similarly, bacterial protein L binds to the Fab regions of some antibodies. Purification can often be achieved using specific fusion couplers. For example, if a GST fusion is used, the antibody can be purified using glutathione resin; if a His tag is used, Ni... +2 Affinity chromatography can be used to purify antibodies. If flag tags are used, fixed anti-flag antibodies can be employed. For general guidance on suitable purification techniques, see, for example, Protein Purification: Principles and Practice, 3. rd Ed., Scopes, Springer-Verlag, NY, 1994, incorporated in its entirety by reference. The required level of purification will vary depending on the intended use of the antibody. In some cases, purification is not required.

[0263] In some embodiments, anion exchange chromatography is used to purify antibodies, including but not limited to chromatography on Q-sepharose, DEAE sepharose, poros HQ, poros DEAE, Toyopearl Q, ToyopearlQAE, Toyopearl DEAE, Resource / Source Q and DEAE, and Fractogel Q and DEAE columns.

[0264] In a specific implementation, the proteins described herein are purified using cation exchange chromatography, including but not limited to SP-sepharose, CM sepharose, poros HS, poros CM, Toyopearl SP, Toyopearl CM, Resource / Source S and CM, Fractogel S and CM columns, and their equivalents and comparables.

[0265] Furthermore, the antibodies described herein can be chemically synthesized using techniques known in the art (see, for example, Creighton, 1983, Proteins: Structures and Molecular Principles, WH Freeman & Co., NY and Hunkapiller et al.). Nature , 310:105-111 (1984)). For example, peptides corresponding to polypeptide fragments can be synthesized using a peptide synthesizer. Furthermore, if desired, non-classical amino acids or chemical amino acid analogs can be introduced into the polypeptide sequence as substitutions or additions. Non-classical amino acids typically include, but are not limited to, D-isomers of common amino acids, 2,4-diaminobutyric acid, α-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, g-Abu, e-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, leucine, valine, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteine, tert-butylglycine, tert-butylalanine, phenylglycine, cyclohexylalanine, alanine, fluoroamino acids, designed amino acids such as methyl amino acids, C-methyl amino acids, N-methyl amino acids, and amino acid analogs. Furthermore, amino acids can be D-(dextral) or L-(levorotatory) amino acids.

[0266] In some embodiments, the aggregation temperature of the antibodies described herein is greater than about 69°C, greater than about 70°C, greater than about 71°C, greater than about 72°C, greater than about 73°C, greater than about 74°C, greater than about 75°C, or greater than about 76°C, for example, between about 69°C and about 77°C, between about 70°C and about 76°C, or between about 71°C and about 75°C. In some embodiments, the aggregation temperature is measured using DSF.

[0267] In some embodiments, the antibody described herein exhibits reduced hydrophobicity compared to lebrikizumab, as measured by hydrophobic interaction chromatography (HIC). In some embodiments, the antibody demonstrates a HIC retention time of less than about 15.2 minutes. In some embodiments, the antibody demonstrates a HIC retention time between about 13 minutes and about 15 minutes.

[0268] How to use In one aspect, this application provides a method for contacting IL-13 with the bispecific antibody described herein, which results in inhibition of the binding of IL-13 to the IL-13 receptor expressed on cells and / or inhibition of the binding of TSLP to TSLPR on cells.

[0269] In one aspect, this application provides a method for treating a subject with a condition or disease using the bispecific antibody described herein. In other aspects, this application describes a method for treating a subject with a corresponding need using a bispecific antibody, the method comprising administering a therapeutically effective amount of the bispecific antibody described herein or a pharmaceutical composition comprising a bispecific antibody to a mammalian subject. In some embodiments, this application provides a method for treating a subject with a condition or disease associated with elevated levels of IL-13 and / or IgE and / or TSLP.

[0270] In some respects, this article describes methods for treating pathologies associated with IL-13 and / or TSLP activity, the methods comprising administering to a mammalian subject a therapeutically effective amount of the bispecific antibody described herein or a pharmaceutical composition containing a bispecific antibody.

[0271] In some aspects, this document describes a method for treating an inflammatory condition or disease in a mammalian subject with a corresponding need, the method comprising administering to the mammalian subject a therapeutically effective amount of the antibody or pharmaceutical composition described herein. In some embodiments of the method described herein, the inflammatory condition or disease is atopic dermatitis. In some embodiments, the inflammatory condition or disease is asthma. In some embodiments, the inflammatory condition or disease is idiopathic pulmonary fibrosis. In some embodiments of the method described herein, the inflammatory condition or disease is alopecia areata. In some embodiments, the inflammatory condition or disease is chronic sinusitis with nasal polyps. In some embodiments, the inflammatory condition or disease is chronic sinusitis without nasal polyps (CRSsNP). In some embodiments, the inflammatory condition or disease is eosinophilic esophagitis (EoE). In some embodiments, the inflammatory condition or disease is an eosinophilic gastrointestinal condition or disease (ENID) selected from the group consisting of eosinophilic gastritis (EoG), eosinophilic enteritis (EoN), eosinophilic colitis (EoC), and eosinophilic gastroenteritis (EGE). In some embodiments, the inflammatory condition or disease is Chag-Strauss syndrome / eosinophilic granulomatosis with polyangiitis (EGPA). In some embodiments, the inflammatory condition or disease is nodular prurigo (PN). In some embodiments, the inflammatory condition or disease is chronic spontaneous urticaria (CSU). In some embodiments, the inflammatory condition or disease is chronic pruritus of unknown cause (CPUO). In some embodiments, the inflammatory condition or disease is bullous pemphigoid (BP). In some embodiments, the inflammatory condition or disease is cold-induced urticaria (ColdU). In some embodiments, the inflammatory condition or disease is allergic fungal sinusitis (AFRS). In some embodiments, the inflammatory condition or disease is allergic bronchopulmonary aspergillosis (ABPA). In some embodiments, the inflammatory condition or disease is chronic obstructive pulmonary disease (COPD). In some embodiments, the inflammatory condition or disease is inflammatory bowel disease, such as Crohn's disease or ulcerative colitis. In some embodiments, the inflammatory condition or disease is psoriasis. In some implementations, the inflammatory condition or disease is lupus. In some implementations, the inflammatory condition or disease is rheumatoid arthritis.

[0272] In some respects, this article describes a method for treating pathology associated with elevated IL-13 levels in mammalian subjects with corresponding needs, the method comprising administering to the mammalian subject a therapeutically effective amount of the antibody or pharmaceutical composition described herein.

[0273] In some respects, this document describes a method for reducing the biological activity of IL-13 and / or TSLP in mammalian subjects with appropriate need, the method comprising administering to the mammalian subject a therapeutically effective amount of the antibody or pharmaceutical composition described herein.

[0274] In some respects, this document describes a method for suppressing TH2-type hypersensitivity reactions in mammalian subjects with a corresponding need, the method comprising administering to the mammalian subject a therapeutically effective amount of the bispecific antibody or pharmaceutical composition described herein.

[0275] In some respects, this article describes a method for inhibiting IL-13-induced STAT6 phosphorylation in cells, the method comprising contacting cells with the antibody described herein.

[0276] In some respects, this article describes a method for inhibiting IL-13-induced CD23 expression in cells, the method comprising contacting cells with the bispecific antibody described herein.

[0277] In some respects, this article describes a method for inhibiting IL-13-induced secretion of CCL2 and CCL26 from cells, the method comprising contacting the cells with the bispecific antibody described herein.

[0278] In some respects, this article describes a method for inhibiting IL-13-induced NTRK1 expression in cells, the method comprising contacting cells with the bispecific antibody described herein.

[0279] In some respects, this article describes a method for reducing thymus and activated regulatory chemokine (TARC) / CCL17 levels in mammalian subjects with corresponding needs, the method comprising administering to the mammalian subject a therapeutically effective amount of the bispecific antibody or pharmaceutical composition described herein.

[0280] In some respects, this document describes a method for preventing inflammatory conditions or diseases in mammalian subjects with appropriate need, the method comprising administering to the mammalian subject a therapeutically effective amount of the bispecific antibody or pharmaceutical composition described herein.

[0281] Application method In some embodiments, the methods provided herein can be used to treat an individual's disease or condition. In one embodiment, the individual is a human and the antibody is the anti-IL-13 antibody described herein.

[0282] In some implementations, antibodies are administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, via implantation, inhalation, intrathecally, intraventricularly, or intranasally. An effective amount of bispecific antibody can be administered to treat a disease or condition. The appropriate dose of the bispecific antibody can be determined based on the type of disease or condition to be treated, the type of bispecific antibody, the severity and duration of the disease or condition, the individual's clinical condition, the individual's clinical history and response to treatment, and the attending physician's judgment.

[0283] In some embodiments, the antibodies provided herein are administered in combination with at least one additional therapeutic agent. Any suitable additional therapeutic agent or immunotherapeutic agent may be administered in combination with the antibodies provided herein. Additional therapeutic agents include those for the treatment or prevention of diseases or conditions, such as, but not limited to, those for inflammatory diseases or conditions associated with elevated levels of IL-13 and / or IgE and / or TSLP.

[0284] Additional therapeutic agents may be administered in any suitable manner. In some embodiments, the antibodies provided herein and additional therapeutic agents are contained in the same pharmaceutical composition. In some embodiments, the antibodies provided herein and additional therapeutic agents are contained in different pharmaceutical compositions.

[0285] In embodiments in which the bispecific antibody and additional therapeutic agent provided herein are included in different pharmaceutical compositions, administration of the bispecific antibody may be performed before, simultaneously with, and / or after administration of the additional therapeutic agent. In some embodiments, administration of the antibody and additional therapeutic agent provided herein occurs within approximately one month of each other. In some embodiments, administration of the bispecific antibody and additional therapeutic agent provided herein occurs within approximately one week of each other. In some embodiments, administration of the bispecific antibody and additional therapeutic agent provided herein occurs within approximately one day of each other. In some embodiments, administration of the bispecific antibody and additional therapeutic agent provided herein occurs within approximately 12 hours of each other. In some embodiments, administration of the bispecific antibody and additional therapeutic agent provided herein occurs within approximately 1 hour of each other.

[0286] reagent kits and products This application provides kits comprising any one or more bispecific antibodies, compositions, and instructions for use described herein. In some embodiments, the kits further comprise a selection of any secondary antibody, reagents for immunohistochemical analysis, pharmaceutically acceptable excipients, and components as specified in the instructions, and any combination thereof. In one specific embodiment, the kit comprises a pharmaceutical composition comprising any one or more bispecific antibodies, compositions, and one or more pharmaceutically acceptable excipients described herein.

[0287] This application also provides articles comprising any of the bispecific antibodies, compositions, or kits described herein. Examples of articles include vials (including sealed vials).

[0288] Example This document provides embodiments for carrying out specific implementations of the invention. These embodiments are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way. Efforts have been made to ensure the accuracy of the figures used (e.g., quantities, temperatures, etc.), but some experimental error and deviation should certainly be allowed.

[0289] Unless otherwise stated, the practice of this invention will be carried out using conventional methods of protein chemistry, biochemistry, recombinant DNA techniques, and pharmacology, which are within the scope of the art. Such techniques are well explained in the literature. See, for example, TECreighton, Proteins: Structures and Molecular Properties (WH Freeman and Company, 1993); AL Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook et al., Molecular Cloning: A Laboratory Manual (2 nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18 th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3 rd Ed. (Plenum Press) Vols A and B(1992).

[0290] method Gene synthesis and plasmid construction The coding sequences for bispecific antibodies HC and LC were generated through DNA synthesis and PCR, and then subcloned into plasmids for expression in mammalian cell systems. The gene sequences in the expression vector were confirmed by DNA sequencing.

[0291] Expression of antibody constructs Transient expression of bispecific antibodies was achieved by co-transfecting paired HC and LC constructs into CHO cells using the PEI method. In short, approximately 5.5 × 10⁻⁶ cells were used in shake flasks.6 CHO cells were used as the host at / mL. Transfection was initiated by adding a mixture of 1 mg / L DNA and 7 mg / L PEI in OptiMEM™ medium (Invitrogen) to the cells and then gently mixing. The cells were then cultured for 9 days in an incubator shaker at 120 rpm, 37°C, and 8% CO2. Peptone and glucose were fed every 2–3 days after 24 hours and thereafter, depending on cell density and viability. Cell culture was terminated on day 9, at which point cell viability decreased to <80%. The conditioned culture was harvested for protein purification.

[0292] Purification of antibody constructs Protein purification was performed using an AKTA Pure instrument (GE Lifesciences) via affinity chromatography and ion exchange chromatography. Conditioned cultures expressing the target bispecific antibody were harvested by centrifugation at 4000 rpm for 50 min and filtered through a 0.22 μm filter. The harvested supernatant was loaded onto a Mabselect™ SuRe™ (GE Healthcare) column. After washing the column with buffer A (PBS, pH 7.4), proteins were eluted with buffer B (1 M glycine, pH 2.7) and immediately neutralized with 1 / 10 volume of buffer D (1 M sodium citrate, pH 6.0). The affinity-purified antibody buffer was then exchanged with 20 mM sodium acetate at pH 5.5.

[0293] SEC-HPLC analysis of antibody constructs Analytical SEC-HPLC was performed using a Shimadzu LC-10 HPLC instrument (Shimadzu Corp.). 20 µL of 1 mg / mL sample was added to a Superdex® 200 Increase 5 / 150GL column (GE Lifesciences). The mobile phase was 2*PBS, the flow rate was 0.3 mL / min, and the induction time was 15 min.

[0294] Antibody-IL13 binding kinetics were measured using surface plasmon resonance. Binding kinetics and affinity constants were determined at 25 °C and in a run buffer of HBS-EP+ (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20) using a Biacore 8KSPR system (GE HealthCare) equipped with an S-series sensor chip protein G (Cytiva, catalog number 29179315). After stabilization in the run buffer, bispecific constructs (diluted to 1 μg / mL) were captured onto flow cell 2 (active) at a flow rate of 10 μL / min for 60 seconds. Recombinant human IL13, TSLP, and / or TSLPR proteins with a His tag were prepared at concentrations of 0, 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, and 0 nM and injected into flow cells 1 (reference) and 2 (active) at a flow rate of 30 μL / min for 180 seconds. Recombinant cynomolgus monkey IL13, TSLP, and / or TSLPR proteins, and His tags were prepared at concentrations of 0, 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25, and 0 nM and injected into flow cells 1 (reference) and 2 (active) at a flow rate of 30 μL / min for 180 seconds. The capture surface was regenerated by injecting glycine (pH 1.5) at a flow rate of 30 μL / min for 30 seconds, and samples were injected onto freshly captured bispecific antibodies in a multi-cycle manner. Data were processed and analyzed using Biacore Insight assessment software version 2.0.15.12933 (GE Healthcare) as follows: The response from flow cell 1 (reference) was subtracted from the response from flow cell 2 (active). Then, the responses from two buffer blank injections were subtracted from the data subtracted from the reference (2-1) to produce dual-reference data, which were fitted to a 1:1 binding model to determine the apparent association (ka) and dissociation rate constant (kd). Their ratio provides the apparent equilibrium dissociation constant or affinity constant (KD = kd / ka).

[0295] Assessing blockade using cell line-based assays Multiple assays were used to assess the blocking of the intact signaling complex of IL-13 / IL4a and / or TSLP / TSLPR and the inhibition of downstream signaling. Briefly, HEK293 cells previously transduced to stably express both hIL-13 / IL4a and / or hTSLP / TSLPR were cultured and harvested. Cells were seeded at 200,000 cells per well at 100 μL / well. Cells were washed and the supernatant was discarded. A mixture of 100 μL of biotinylated hIL-13 and / or hTSLP with purified bispecific antibody (1:1 v / v) was prepared and incubated for 1 h. The incubated mixture was added to resuspend the cells, resulting in a final concentration of 0.05 μg / mL of hIL-13 and / or hTSLP and 0–100 nM of purified bispecific antibody. Cells were stained in this mixture at 4°C for 1 h. Cells were then washed and stained with 100 μL of Alexa Fluor 488-conjugated streptavidin at a 1:1000 dilution to detect binding of biotinylated hIL-13 on the cell surface. Cells were incubated at 4°C in the dark for 1 hour. Cells were then washed again, and the median fluorescence intensity (MFI) of cells in each well was recorded via FACS using BDFACSCanto II. Subsequent data were analyzed using GraphPad Prism. IC50 50 The value was determined to be the concentration of bispecific antibody required to inhibit 50% of the maximum MFI on the surface of biotinylated hIL-13 or hTSLP, the concentration being detected by incubation of 0.05 μg / mL of hIL-13 or hTSLP alone.

[0296] Assessing blockade using ELISA A 96-well plate (Costar #9018) was coated overnight at 4°C with TSLP R in 2 μg / ml PBS pH 7.4, and then blocked at 37°C with PBST + 1% BSA for 2 h. Serially diluted test samples (in duplicate, 1 / 5 dilution from 10 nM, 7 doses + blank) were mixed with 5 ng / ml biotin-TSLP at room temperature for 30 min. The test antibody (TA)-TSLP mixture was then added to the blocked plate and incubated at 37°C for 1 h. After washing, the plate was incubated with streptavidin-HRP at 37°C for 1 h. After washing, TMB was added to each well and incubated at room temperature until color development (approximately 10 min). The reaction was terminated by adding 1N HCl, and the optical density (OD) was read at 450 nm. Inhibition % was calculated as 1 - (OD450 of sample / OD450 of 'ligand only'). The IC50 of TA was calculated by nonlinear regression.

[0297] Inhibit the binding of IL-13 or TSLP to cells overexpressing IL-13 / IL4a or TSLP / TSLPR The functional blockade of the antibody against this binding interaction was assessed using the binding of IL-13 and / or TSLP to cells overexpressing IL-13 / IL4RA and / or TSLP / TSLPR. Cell line-based assays are expected to show that the bispecific antibody will exhibit low IC50. 50 It also inhibits the binding of IL-13 and / or TSLP in IL-13 / IL4Rα or TSLP / TSLPR overexpressing cell lines.

[0298] Example 1: Engineered bispecific antibodies exhibited improved affinity and potency in blocking IL-13 / IL4a and / or TSLP / TSLPR. result Determination of antibody affinity for IL-13 and / or TSLP or TSLPR Using the methods described above, surface plasmon resonance (SPR) was used to evaluate the affinity of bispecific antibodies for IL-13 and / or TSLP / TSLPR and their binding kinetics.

[0299] Bispecific antibodies bound human IL-13 and / or TSLP with sub-nanomolar affinity, as measured by SPR. Results are summarized in Table 14.

[0300] Informal sequence list 。

Claims

1. A bispecific antibody, wherein the bispecific antibody comprises a first antigen-binding site and a second antigen-binding site, wherein a) The first antigen-binding site binds to interleukin-13 (IL-13) and comprises a first variable heavy (VH) chain sequence and a first variable light (VL) chain sequence, wherein the first variable heavy (VH) chain sequence comprises three first heavy chain CDR sequences: first CDR-H1, first CDR-H2, and first CDR-H3, and the first variable light (VH) chain sequence comprises three first light chain CDR sequences: first CDR-L1, first CDR-L2, and first CDR-L3; wherein: i. The first CDR-H1 comprises a sequence selected from the sequences shown in SEQ ID NO: 58-99 and 121; ii. The first CDR-H2 comprises a sequence selected from the sequences shown in SEQ ID NO: 100-111; iii. The first CDR-H3 comprises a sequence selected from the sequences shown in SEQ ID NO: 112-120 and 130-140. iv. The first CDR-L1 comprises a sequence selected from the sequences shown in SEQ ID NO: 141-144 and 149-152. v. The first CDR-L2 comprises a sequence selected from the sequences shown in SEQ ID NO: 153-158 and a sequence from LAS; and vi. The first CDR-L3 comprises a sequence selected from the sequences shown in SEQ ID NO: 165-172; and b) wherein the second antigen binding site binds to TSLP or TSLPR and comprises a second variable heavy (VH) chain sequence and a second variable light (VL) chain sequence, the second variable heavy (VH) chain sequence comprising three second heavy chain CDR sequences: second CDR-H1, second CDR-H2, and second CDR-H3, and the second variable light (VL) chain sequence comprising three second light chain CDR sequences: second CDR-L1, second CDR-L2, and second CDR-L3; wherein: vii. The second CDR-H1 comprises a sequence selected from the sequences shown in SEQ ID NO: 610-618; viii. The second CDR-H2 comprises a sequence selected from the sequences shown in SEQ ID NO: 619-627; ix. The second CDR-H3 comprises a sequence selected from the sequences shown in SEQ ID NO: 628-636. x. The second CDR-L1 comprises a sequence selected from the sequences shown in SEQ ID NO: 637-645. xi. The second CDR-L2 comprises a sequence selected from the sequences shown in SEQ ID NO: 646-651; and xii. The second CDR-L3 comprises a sequence selected from the sequences shown in SEQ ID NO: 652-657.

2. The isolated bispecific antibody according to claim 1, wherein... i. The first CDR-H1 comprises a sequence selected from the sequences shown in SEQ ID NO: 58, 68 and 85; ii. The first CDR-H2 comprises a sequence selected from the sequences shown in SEQ ID NO: 100, 104 and 108; iii. The first CDR-H3 comprises a sequence selected from the sequences shown in SEQ ID NO: 112 and 130; iv. The first CDR-L1 comprises a sequence selected from the sequences shown in SEQ ID NO: 141 and 589; v. The first CDR-L2 comprises a sequence selected from the sequence shown in SEQ ID NO: 153 and a sequence from LAS; and vi. The first CDR-L3 contains the sequence shown in SEQ ID NO: 165; and vii. The second CDR-H1 comprises a sequence selected from the sequences shown in SEQ ID NO: 610-618; viii. The second CDR-H2 comprises a sequence selected from the sequences shown in SEQ ID NO: 619-627; ix. The second CDR-H3 comprises a sequence selected from the sequences shown in SEQ ID NO: 628-636; x. The second CDR-L1 comprises a sequence selected from the sequences shown in SEQ ID NO: 637-645; xi. The second CDR-L2 comprises a sequence selected from the sequences shown in SEQ ID NO: 646-651; and xii. The second CDR-L3 comprises a sequence selected from the sequences shown in SEQ ID NO: 652-657.

3. The isolated bispecific antibody according to claim 1 or 2, wherein the antibody comprises a first VH sequence selected from SEQ ID NO: 1-32, 470 and 688 and a second VH sequence selected from SEQ ID NO: 659-661, 682, 684, 686 and 688.

4. The isolated bispecific antibody according to any one of claims 1-3, wherein the antibody comprises a first VH sequence containing SEQ ID NO: 3 and a second VH sequence selected from SEQ ID NO: 659-661, 682, 684, 686 and 688.

5. The isolated bispecific antibody according to any one of claims 1-4, wherein the antibody comprises a first VL sequence selected from SEQ ID NO: 33-57, 471 and 687 and a second VL sequence selected from SEQ ID NO: 662-664, 681, 683 and 685.

6. The isolated bispecific antibody according to any one of claims 1-5, wherein the bispecific antibody comprises a first VL sequence containing SEQ ID NO: 39 and a second VL sequence selected from SEQ ID NO: 662-664, 681, 683 and 685.

7. The isolated bispecific antibody according to any one of claims 1-3, wherein the bispecific antibody comprises a first VH sequence selected from SEQ ID NO: 1-32, 470 and 688 and a second VH sequence selected from SEQ ID NO: 659-661, 682, 684, 686 and 688; and a first VL sequence selected from SEQ ID NO: 33-57, 471 and 687 and a second VL sequence selected from SEQ ID NO: 662-664, 681, 683 and 685.

8. The isolated bispecific antibody according to any one of claims 1-3 or 7, wherein the bispecific antibody comprises a first VH sequence containing SEQ ID NO: 3 and a second VH sequence selected from SEQ ID NO: 659-661, 682, 684, 686 and 688; and a first VL sequence containing SEQ ID NO: 39 and a second VL sequence selected from SEQ ID NO: 662-664, 681, 683 and 685.

9. The isolated bispecific antibody according to any one of claims 1-8, wherein the bispecific antibody is a humanized antibody, a human antibody, or a chimeric antibody.

10. The isolated bispecific antibody according to any one of claims 1-9, wherein the bispecific antibody is a humanized antibody.

11. The isolated bispecific antibody according to any one of claims 1-10, wherein the bispecific antibody comprises a heavy chain human constant region selected from the classes IgG, IgA, IgD, IgE and IgM.

12. The isolated bispecific antibody according to claim 11, wherein the human Fc region comprises a human heavy chain constant region of IgG class and subclasses selected from IgG1, IgG2, IgG3 and IgG4.

13. The isolated bispecific antibody according to claim 12, wherein the human Fc region comprises human IgG1 Fc.

14. The isolated bispecific antibody according to claim 12, wherein the human Fc region comprises human IgG4 Fc.

15. The isolated bispecific antibody according to claim 12, wherein the human Fc region comprises human IgG2Fc.

16. The isolated bispecific antibody according to any one of claims 11-15, wherein the Fc region comprises a sequence selected from any one of the sequences shown in SEQ ID NO: 425-468, 484-539, 670-680, 689-691 and 693-804.

17. The isolated bispecific antibody according to any one of claims 11-16, wherein the heavy chain comprises the constant heavy chain sequence shown in SEQ ID NO:

439.

18. The isolated bispecific antibody according to any one of claims 1-17, wherein the bispecific antibody comprises a heavy chain / chain A from Table 11a or Table 11b, a light chain / chain B from Table 12a or Table 12b, and optionally an Fc sequence from Table 13.

19. The isolated antibody according to any one of claims 11-18, wherein the Fc region comprises one or more amino acid substitutions, wherein the one or more substitutions result in a change in antibody half-life, ADCC activity, ADCP activity, or CDC activity compared to an antibody that is otherwise equivalent to an Fc region without the one or more substitutions.

20. The isolated bispecific antibody according to claim 19, wherein the change is (a) an increase in antibody half-life and (b) a decrease in ADCC activity, ADCP activity or CDC activity compared to an antibody equivalent in other aspects containing one or more of the substituted Fc.

21. The isolated bispecific antibody according to claim 19, wherein the one or more amino acid substitutions result in an increased antibody half-life compared to an antibody containing a wild-type Fc region.

22. The isolated bispecific antibody according to any one of claims 1-21, for the treatment of inflammatory conditions or diseases.

23. An isolated polynucleotide or a group of polynucleotides encoding a bispecific antibody, its VH, its VL, its light chain, its heavy chain or its antigen-binding portion, according to any one of claims 1-21, and optionally, wherein said polynucleotide or group of polynucleotides comprises cDNA.

24. A vector or a group of vectors comprising the polynucleotide or a group of polynucleotides of claim 23.

25. A host cell comprising the polynucleotide or a group of polynucleotides of claim 23 or the vector or a group of vectors of claim 24.

26. A method for generating an antibody, the method comprising expressing the bispecific antibody in a host cell of claim 25 and isolating the expressed bispecific antibody.

27. A pharmaceutical composition comprising a bispecific antibody of any one of claims 1-21 and a pharmaceutically acceptable excipient.

28. A kit comprising a bispecific antibody of any one of claims 1-21 or a pharmaceutical composition of claim 27 and instructions for use.

29. A method for treating an inflammatory condition or disease in a mammalian subject with a corresponding need, the method comprising administering to the mammalian subject a therapeutically effective amount of the bispecific antibody of any one of claims 1-21 or the pharmaceutical composition of claim 27.

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