Anti-PCRV and PSL Bispecific Antibody for the Treatment of Bronchiectasis

Abstract

The present disclosure relates to an anti-Pseudomonas Psl and PcrV bispecific antibody for use in the treatment of bronchiectasis.

Description

Technical Field

[0001] The present disclosure relates to the use of a bispecific antibody that specifically binds to Pseudomonas aeruginosa PcrV protein and Psl exopolysaccharide for treating bronchiectasis.

Background Art

[0002] Pseudomonas aeruginosa (P. aeruginosa) is a gram-negative opportunistic pathogen that causes both acute and chronic infections in susceptible individuals. This is due in part to the high natural resistance of the bacteria to clinically used antibiotics and in part to the formation of highly antibiotic-resistant biofilms. Furthermore, P. aeruginosa is known to colonize the airways in patients with non-cystic fibrosis bronchiectasis. Non-cystic fibrosis bronchiectasis is a chronic disease characterized by abnormal and persistent dilation of the bronchi, resulting in chronic cough, sputum production, and recurrent bacterial airway infections. Patients with bronchiectasis suffer from a severe medical condition due to impaired quality of life, promotion of resistance to antibiotics, and frequent exacerbations that lead to a decline in lung function.

[0003] The important medical need for the treatment of bronchiectasis is not met, and no approved therapies for reducing exacerbations are currently available. The 2017 European Respiratory Society (ERS) guidelines on the management of adult bronchiectasis suggest that there are no other recommended treatments in addition to antibiotics for treating acute exacerbations. Due to the increasing multi-drug resistance exhibited by bacteria, the development of new strategies for the identification of new Pseudomonas-specific prevention and treatment strategies is still needed in the art.

Summary of the Invention

[0004] Provided herein are anti-Pseudomonas Psl and PcrV bispecific antibodies for use in the treatment of bronchiectasis.

[0005] In some aspects provided herein, a method of treating bronchiectasis in a subject in need thereof comprises administering to the subject a bispecific antibody that specifically binds to Pseudomonas aeruginosa PcrV protein and Psl exopolysaccharide.

[0006] In some aspects provided herein, a method of improving forced expiratory volume 1 (FEV1) in a subject with bronchiectasis prior to administering a bronchodilator comprises administering to the subject a bispecific antibody that specifically binds to Pseudomonas aeruginosa PcrV protein and Psl exopolysaccharide.

[0007] In some aspects provided herein, a method of reducing Pseudomonas aeruginosa burden in a subject with bronchiectasis comprises administering to the subject a bispecific antibody that specifically binds to Pseudomonas aeruginosa PcrV protein and Psl exopolysaccharide.

[0008] In some aspects provided herein, a method of reducing exacerbation of bronchiectasis in a subject in need thereof comprises administering to the subject a bispecific antibody that specifically binds to Pseudomonas aeruginosa PcrV protein and Psl exopolysaccharide. In some aspects, the administration reduces exacerbations of bronchiectasis that require hospitalization. In some aspects, the administration reduces exacerbations of bronchiectasis that require antibiotics. In some aspects, the administration reduces exacerbations of bronchiectasis that require hospitalization and exacerbations of bronchiectasis that require antibiotics.

[0009] In some aspects provided herein, a method of reducing the need for intravenous antibiotics in a subject having bronchiectasis comprises administering to the subject a bispecific antibody that specifically binds to Pseudomonas aeruginosa PcrV protein and Psl exopolysaccharide.

[0010] In some aspects provided herein, a method of stabilizing lung function in a subject having bronchiectasis comprises administering to the subject a bispecific antibody that specifically binds to Pseudomonas aeruginosa PcrV protein and Psl exopolysaccharide.

[0011] In some aspects, the bronchiectasis is non-cystic fibrosis bronchiectasis. In some aspects, the non-cystic fibrosis bronchiectasis is confirmed by chest computed tomography (CT) showing bronchiectasis affecting one or more lobes.

[0012] In some aspects, the subject is colonized with Pseudomonas aeruginosa. In some aspects, the subject's airway is colonized with Pseudomonas aeruginosa. In some aspects, Pseudomonas aeruginosa colonization is detected by sputum culture.

[0013] In some aspects, the subject is chronically infected with Pseudomonas aeruginosa. In some aspects, the subject has an increased airway neutrophil count. In some aspects, the subject has an increased sputum neutrophil count. In some aspects, the subject has a history of at least two moderate to severe exacerbations of bronchiectasis per year that require antibiotics. In some aspects, the subject has a history of at least one exacerbation that requires hospital care. In some aspects, the subject is receiving long-term nebulized antibiotics. In some aspects, the subject has chronic obstructive pulmonary disease (COPD).

[0014] In some embodiments, bronchiectasis was caused by hypogammaglobulinemia. In some embodiments, bronchiectasis was caused by unclassifiable immunodeficiency. In some embodiments, bronchiectasis was caused by alpha-1-antitrypsin deficiency.

[0015] In some embodiments, administration reduces Pseudomonas aeruginosa in sputum cultures obtained from the subject. In some embodiments, the reduction occurs within 12 weeks of the first administration. In some embodiments, the reduction occurs within 8 weeks of the first administration. In some embodiments, the reduction occurs within 4 weeks of the first administration.

[0016] In some embodiments, administration reduces the use of antibiotics. In some embodiments, administration eradicates Pseudomonas aeruginosa in the subject.

[0017] In some embodiments, the bispecificity competitively inhibits the binding of an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14 to PcrV. In some embodiments, the bispecific antibody binds to the same PcrV epitope as an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14.

[0018] In some embodiments, the bispecific antibody comprises a PcrV binding domain comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 2, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 3, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the bispecific antibody comprises a PcrV binding domain comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13. In some embodiments, the bispecific antibody comprises a PcrV binding domain comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the bispecific antibody comprises a heavy chain variable domain having the amino acid sequence of SEQ ID NO: 13 and a light chain variable domain having the amino acid sequence of SEQ ID NO: 14.

[0019] In some embodiments, the bispecific antibody comprises a PcrV binding domain having a heavy chain variable region and a light chain variable region on separate polypeptides.

[0020] In some embodiments, the bispecific competitively inhibits the binding of an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16 to Psl. In some embodiments, the bispecific antibody binds to the same Psl epitope as an antibody comprising a heavy chain variable domain having the amino acid sequence of SEQ ID NO: 15 and a light chain variable domain having the amino acid sequence of SEQ ID NO: 16.

[0021] In some embodiments, the bispecific antibody comprises a Psl-binding domain comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 7, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 8, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 9, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 10, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the bispecific antibody comprises a Psl-binding domain comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15. In some embodiments, the bispecific antibody comprises a Psl-binding domain comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, the bispecific antibody comprises a Psl-binding domain comprising a heavy chain variable domain having the amino acid sequence of SEQ ID NO: 15 and a light chain variable domain having the amino acid sequence of SEQ ID NO: 16.

[0022] In some embodiments, the bispecific antibody comprises a Psl-binding domain having a heavy chain variable region and a light chain variable region on the same polypeptide. In some embodiments, the bispecific antibody comprises a Psl-binding domain that is a scFv. In some embodiments, the scFv comprises a linker. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, the scFv is in the VH-linker-VL orientation. In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 17.

[0023] In some embodiments, the bispecific antibody is an IgG antibody. In some embodiments, the IgG antibody is an IgG1 antibody.

[0024] In some embodiments, the bispecific antibody comprises: (i) a heavy chain of the formula VH-CH1-H1-L1-S-L2-H2-CH2-CH3, wherein VH is an anti-P. aeruginosa PcrV heavy chain variable domain, CH1 is a heavy chain constant region domain 1, H1 is a first heavy chain hinge region fragment, L1 is a first linker, S is an anti-P. aeruginosa Psl scFv molecule, L2 is a second linker, H2 is a second heavy chain hinge region fragment, CH2 is a heavy chain constant region domain-2, and CH3 is a heavy chain constant region domain-3; and (ii) a light chain of the formula VL-CL, wherein VL is an anti-P. aeruginosa PcrV light chain variable domain and CL is an antibody light chain kappa constant region or an antibody light chain lambda region. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 13. In some embodiments, the scFv comprises the amino acid sequences of SEQ ID NO: 15 and SEQ ID NO: 16. In some embodiments, VL comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 13 and the scFv comprises the amino acid sequences of SEQ ID NO: 15 and SEQ ID NO: 16. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 13 and VL comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the scFv comprises the amino acid sequences of SEQ ID NO: 15 and SEQ ID NO: 16 and VL comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 13, the scFv comprises the amino acid sequences of SEQ ID NO: 15 and SEQ ID NO: 16, and VL comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 13 and the scFv comprises the amino acid sequence of SEQ ID NO: 17. In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 17 and VL comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 13, the scFv comprises the amino acid sequence of SEQ ID NO: 17, and VL comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, CL is an antibody light chain kappa constant region. In some embodiments, the bispecific antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19.In some embodiments, the bispecific antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the bispecific antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 19 and a light chain having the amino acid sequence of SEQ ID NO: 20.

[0025] In some embodiments, the bispecific antibody neutralizes cytotoxicity. In some embodiments, the bispecific antibody targets Pseudomonas aeruginosa for killing by opsonophagocytosis. In some embodiments, the bispecific antibody prevents cell attachment. In some embodiments, the bispecific antibody disrupts biofilm formation. In some embodiments, the bispecific antibody inhibits primary colony formation.

[0026] In some embodiments, the subject is colonized with a strain of Pseudomonas aeruginosa having a genome comprising the Psl - operon. In some embodiments, the subject is colonized with a strain of Pseudomonas aeruginosa having a genome comprising the PcrV locus. In some embodiments, the subject is human.

[0027] In some embodiments, the bispecific antibody is administered intravenously. In some embodiments, the bispecific antibody is administered subcutaneously.

[0028] In some embodiments, the methods provided herein further comprise administering an antibiotic.

[0029] In some embodiments, the use of a bispecific antibody is provided herein. In some embodiments, the use of a bispecific antibody that specifically binds to the Pseudomonas aeruginosa PcrV protein and Psl exopolysaccharide in the preparation of a medicament for treating bronchiectasis in a subject in need of treating bronchiectasis is provided herein. Treating bronchiectasis can comprise any method provided herein.

[0030] In some embodiments, bispecific antibodies for use are provided herein. In some embodiments, bispecific antibodies that specifically bind to Pseudomonas aeruginosa PcrV protein and Psl exopolysaccharide for use in treating bronchiectasis in a subject in need thereof are provided herein. Treating bronchiectasis can include any method provided herein.

Brief Description of the Drawings

[0031]

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Mode for Carrying Out the Invention

[0032] I. Definitions The headings provided herein do not limit the various aspects or embodiments of the disclosure that can be obtained by reference to the entire specification. Thus, the terms defined immediately below are more fully defined by reference to the entire specification.

[0033] It should be noted that the term "a" or "an" entity refers to one or more of that entity. For example, "an antibody" is understood to represent one or more antibodies. Thus, the terms "a" (or "an"), "one or more", and "at least one" can be used interchangeably herein.

[0034] Furthermore, as used herein, "and / or" shall be construed as a specific disclosure of each of two designated features or components, with or without the other. Thus, the term "and / or" as used in phrases such as "A and / or B" herein is intended to include "A and B", "A or B", "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to include each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0035] As used herein, the terms "about" and "approximately", when used to modify a numerical value or numerical range, indicate that a deviation of up to 10% above and 10% below that value or range remains within the scope of the intended meaning of the recited value or range. It is understood that whenever an aspect is described herein using a numerical value or range with the words "about" or "approximately", other similar aspects that refer to the specified numerical value or range are also provided.

[0036] It is understood that whenever an aspect is described herein using the word "comprising", other similar aspects described in terms of "consisting of" and / or "consisting essentially of" are also provided. In the present disclosure, "comprises", "comprising", "containing", "having", etc. may mean "includes", "including", etc. "Consisting essentially of" or "consists essentially of" is open-ended and allows for more than what is described, as long as the basic or novel features of what is described are not changed by the presence of more than what is described, but excludes prior art aspects.

[0037] Units, prefixes, and symbols are presented in the form approved by the International System of Units (SI). Numerical ranges include the numbers defining the range.

[0038] Unless otherwise indicated, amino acid sequences are written left to right in an amino- to carboxy orientation.

[0039] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press, and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press provide many common dictionaries of terms used in the present disclosure to one of ordinary skill in the art.

[0040] As used herein, the terms "antibody" and "immunoglobulin" are used interchangeably and refer to antibody molecules that recognize and specifically bind to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination of the foregoing (e.g., glycoprotein), through at least one antigen recognition site within the variable region of the immunoglobulin molecule. The term "antibody" encompasses monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, bispecific antibodies, and any other immunoglobulin molecule as long as the antibody exhibits the desired biological activity. Antibodies can be of any of five major classes: IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), based on the identity of the heavy chain constant domain of the immunoglobulin, which are called alpha, delta, epsilon, gamma, and mu, respectively. Different classes of antibodies have different well-known subunit structures and three-dimensional configurations. For the structures and properties of different classes of antibodies, see, for example, Basic and Clinical Immunology, 8th Ed., Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, CT, 1994, page 71 and Chapter 6.

[0041] The term "antibody fragment" refers to a part of an antibody. An "antigen-binding fragment" of an antibody refers to the part of the antibody that binds to an antigen. The antigen-binding fragment of an antibody can include the antigen-determining region of the antibody (e.g., the complementarity determining region (CDR)). Examples of antigen-binding fragments of an antibody include, but are not limited to, Fab, Fab’, F(ab’)2, and Fv fragments, linear antibodies, and single-chain antibodies. The antigen-binding fragment of an antibody can be monovalent or multivalent (e.g., bivalent). The antigen-binding fragment of an antibody can be monospecific or multispecific (e.g., bispecific). The antigen-binding fragment of an antibody can be derived from any animal species, e.g., rodents (e.g., mice, rats, or hamsters) and humans, or can be produced artificially.

[0042] "Antigen-binding domain" or "antigen-binding region" refers to the monovalent part of an antibody that binds to an antigen. The "antigen-binding domain" can include the antigen-determining region of the antibody (e.g., the complementarity determining region (CDR)). An antibody or its antigen-binding fragment (including monospecific and multispecific (e.g., bispecific) antibodies or their antigen-binding fragments) can include an antigen-binding domain.

[0043] An antibody fragment containing a single-chain antibody can include the variable region alone or in combination with all or part of the following: the hinge region, CH1, CH2, and CH3 domains.

[0044] Also included are antigen-binding fragments that also include any combination of the variable region and the hinge region, CH1, CH2, and CH3 domains.

[0045] The antibodies or their antigen-binding fragments disclosed herein can be derived from any animal origin, including birds and mammals. The antibodies can be human, mouse, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies.

[0046] Light chains are classified into either kappa or lambda (κ, λ). Each heavy chain class can bind to either a kappa or a lambda light chain. Generally, the light and heavy chains are covalently bonded to each other, and the "tail" portions of the two heavy chains are joined to each other by either covalent disulfide bonds or non-covalent bonds when the immunoglobulin is produced by either a hybridoma, B cell, or genetically engineered host cell. In the heavy chain, the amino acid sequence extends from the N-terminus at the branched end of the Y configuration to the C-terminus at the bottom of each chain.

[0047] Both the light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, it will be understood that the variable domains of both the light chain (VL) and heavy chain (VH) portions determine antigen recognition and specificity. Conversely, the constant domains of the light chain (CL) and heavy chain (CH1, CH2, or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, etc. By convention, the numbering of the constant region domains increases as they become more distal from the antigen-binding site or amino terminus of the antibody. The N-terminal portion is the variable region, and the C-terminal portion is the constant region. The CH3 domain and the CL domain contain the carboxy termini of the heavy and light chains, respectively.

[0048] As indicated above, the variable region allows the binding molecule to selectively recognize and specifically bind to an epitope on the antigen. That is, a subset of the binding molecule, such as the VL and VH domains of an antibody, or the complementarity-determining regions (CDRs), combine to form a variable region that defines a three-dimensional antigen-binding site. This quaternary binding molecule structure forms the antigen-binding sites present at the ends of each arm of the Y. More specifically, the antigen-binding site is defined by three CDRs in each of the VH and VL chains.

[0049] In naturally occurring antibodies, the six "complementary determining regions" or "CDRs" present in each antigen-binding domain are short non-contiguous sequences of amino acids that are specifically arranged to form the antigen-binding domain when the antibody assumes its three-dimensional conformation in an aqueous environment. The remaining amino acids in the antigen-binding domain, called the "framework" regions, exhibit less intermolecular variability. The framework regions predominantly adopt a β-sheet conformation, and the CDRs form loops that connect the β-sheet structures and, in some cases, form part of them. Thus, the framework regions act to form a scaffold that provides for the proper orientation of the CDRs by interchain non-covalent interactions. The antigen-binding domain formed by the arranged CDRs defines a surface that is complementary to an epitope on an immunoreactive antigen. This complementary surface facilitates non-covalent binding of the antibody to its cognate epitope. The amino acids comprising the CDRs and framework regions, respectively, can be readily identified by one of ordinary skill in the art for any given heavy or light chain variable region because they are precisely defined (see "Sequences of Proteins of Immunological Interest", Kabat, E., et al., U.S. Department of Health and Human Services (1983), and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987), which are incorporated herein by reference in their entirety).

[0050] The term "Kabat numbering" and similar terms are recognized in the art and refer to a system for numbering amino acid residues in the heavy and light chain variable regions of an antibody or antigen-binding fragment thereof. In certain embodiments, the CDRs can be determined according to the Kabat numbering system (see, e.g., Kabat EA & Wu TT (1971) Ann NY Acad Sci 190:382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). Using the Kabat numbering system, the CDRs within an antibody heavy chain molecule are typically present at amino acid positions 31-35 (CDR1), amino acid positions 50-65 (CDR2), and amino acid positions 95-102 (CDR3), which can optionally include one or two additional amino acids (referred to as 35A and 35B in the Kabat numbering scheme) following position 35. Using the Kabat numbering system, the CDRs within an antibody light chain molecule are typically present at amino acid positions 24-34 (CDR1), amino acid positions 50-56 (CDR2), and amino acid positions 89-97 (CDR3). In some embodiments, the CDRs of the antibodies described herein are determined according to the Kabat numbering scheme.

[0051] Chothia, instead, refers to the position of structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The end of the Chothia CDR-H1 loop varies between H32 and H34 depending on the length of the loop when numbered using the Kabat numbering rules (this is because the Kabat numbering scheme places insertions at H35A and H35B, and when neither 35A nor 35B is present, the loop ends at 32, when only 35A is present, the loop ends at 33, and when both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and the Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software.

[0052]

Table 1

[0053] Single chain Fv (scFv) molecules are known in the art and are described, for example, in U.S. Patent No. 5,892,019. The immunoglobulin or antibody molecules encompassed by this disclosure can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules.

[0054] A "monoclonal" antibody or antigen-binding fragment thereof refers to a homogeneous population of antibodies or antigen-binding fragments that are involved in the highly specific recognition and binding of a single antigenic determinant or epitope. This is in contrast to polyclonal antibodies, which typically contain different antibodies against different antigenic determinants. The term "monoclonal" antibody or antigen-binding fragment thereof refers to both intact monoclonal antibodies and full-length monoclonal antibodies, as well as antibody fragments (e.g., Fab, Fab’, F(ab’)2, Fv), single-chain (scFv) variants, fusion proteins containing antibody moieties, and any other modified immunoglobulin molecule containing an antigen recognition site. Further, a "monoclonal" antibody or antigen-binding fragment thereof refers to such antibodies and antigen-binding fragments produced in any number of ways including, but not limited to, hybridomas, phage selection, recombinant expression, and transgenic animals.

[0055] As used herein, a "human" antibody includes an antibody having the amino acid sequence of a human immunoglobulin and includes antibodies isolated from a human immunoglobulin library or isolated from an animal that is transgenic for one or more human immunoglobulins and does not express endogenous immunoglobulins, as described below and, for example, in U.S. Patent No. 5,939,598 to Kucherlapati et al.

[0056] "Specifically binds" generally means that a binding molecule, such as a bispecific antibody or fragment, variant, or derivative thereof, binds to an epitope via an antigen-binding domain, and that the binding involves some complementarity between the antigen-binding domain and the epitope. The binding molecules provided herein can be the same or different and can contain one, two, three, four, or more binding domains that can bind to the same epitope or two or more different epitopes. According to this definition, a binding molecule is said to "specifically bind" to an epitope if it binds to the epitope via its antigen-binding domain more readily than it binds to a random, unrelated epitope. The term "specificity" is used herein to denote a relative affinity by which a particular binding molecule binds to a particular epitope. For example, binding molecule "A" may be considered to have a higher specificity for a given epitope than binding molecule "B", or binding molecule "A" may be said to bind to epitope "C" with a higher specificity than it has for related epitope "D".

[0057] An antibody that "binds to the same epitope" as a reference antibody refers to an antibody that contacts the same amino acid and / or sugar residues as the reference antibody. The ability of an antibody to bind to the same epitope as a reference antibody can be determined using peptide scanning mutagenesis or high-throughput alanine scanning mutagenesis.

[0058] An antibody is said to "competitively inhibit" the binding of a reference antibody to a given epitope, which is said when the antibody preferentially binds to that epitope or an overlapping epitope such that it blocks the binding of the reference antibody to the epitope to some extent. Competitive inhibition can be determined by any method known in the art, such as a competitive ELISA assay. An antibody may be said to competitively inhibit the binding of a reference antibody to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

[0059] As used herein, the term "bispecific antibody" refers to an antibody that has binding domains specific for two different antigens or epitopes within a single antibody molecule. It will be understood that, in addition to the standard antibody structure, other molecules having two binding specificities can be constructed. It will be further understood that antigen binding by a bispecific antibody can be simultaneous or sequential. Triomas and hybrid hybridomas are two examples of cell lines that can secrete bispecific antibodies. Bispecific antibodies can also be constructed by recombinant means. (Strohlein and Heiss, Future Oncol. 6:1387-94 (2010), Mabry and Snavely, IDrugs. 13:543-9 (2010)).

[0060] As used herein, the term "MEDI3902" refers to a bispecific antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 19 and a light chain having the amino acid sequence of SEQ ID NO: 20. MEDI3902 is also known as Gremubamab.

[0061] As used herein, the term "engineered antibody" refers to an antibody in which the variable domain in either or both of the heavy and light chains has been altered by at least partial substitution of one or more CDRs from an antibody of known specificity and, if necessary, by partial substitution of framework regions and sequence changes. The CDRs can be from an antibody of the same class or even subclass as the antibody from which the framework regions are derived, but it is contemplated that the CDRs can be from antibodies of different classes, preferably from antibodies of different species. An engineered antibody in which one or more "donor" CDRs from a non-human antibody of known specificity have been transplanted into a human heavy or light chain framework region is referred to herein as a "humanized antibody". It may not be necessary to replace all of the CDRs with complete CDRs from the donor variable region in order to transfer the antigen-binding ability of one variable domain to another. Rather, it may only be necessary to transfer the residues necessary to maintain the activity of the target binding site. For example, considering the descriptions set forth in U.S. Patent Nos. 5,585,089, 5,693,761, 5,693,762, and 6,180,370, obtaining a functional engineered antibody or humanized antibody is well within the ability of one of ordinary skill in the art by either routine experimentation or trial and error testing.

[0062] An "isolated" polypeptide, antibody, polynucleotide, vector, cell, or composition is a polypeptide, antibody, polynucleotide, vector, cell, or composition that is in a form not found in nature. Isolated polypeptides, antibodies, polynucleotides, vectors, cells or compositions include those that have been purified to the extent that they are no longer in their natural form. In some embodiments, an isolated antibody, polynucleotide, vector, cell, or composition is substantially pure. As used herein, "substantially pure" refers to a material that is at least 50% pure (i.e., free of contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0063] The terms "polypeptide", "peptide", and "protein" are used interchangeably herein and refer to a polymer of amino acids of any length. The polymer may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. These terms also include amino acid polymers that are naturally or artificially modified by any other operation or modification, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or conjugation with a labeling component. For example, polypeptides containing one or more analogs of amino acids (including, for example, non-natural amino acids) and other modifications known in the art are also included within this definition. Since the polypeptides of the present disclosure are antibody-based, it is understood that in some embodiments, the polypeptides can occur as single-chain or associated chains.

[0064] Administration "in combination with" one or more additional therapeutic agents includes simultaneous (parallel) administration or sequential administration in any order.

[0065] The terms "treating" or "treatment" or "treat" or "alleviating" or "alleviate" refer to a therapeutic means that cures, decelerates, reduces, and / or halts the progression of the symptoms of a diagnosed pathological condition or disorder. Thus, those in need of treatment include those already diagnosed with the disorder or those suspected of having the disorder.

[0066] The term "airway neutrophilia" refers to the accumulation of neutrophils in the airspaces of the lung.

[0067] The term "sputum neutrophilia" refers to the presence of neutrophils in the sputum of a subject. In some embodiments, the neutrophils in the sputum of a subject in need of treatment, such as a subject having bronchiectasis, are increased compared to the neutrophils in the sputum of a healthy control.

[0068] "Subject", "individual", "animal", "patient", or "mammal" means any subject for which diagnosis, prognosis, or therapy is desired, such as a mammalian subject. Mammalian subjects include humans, domestic animals, farm animals, and zoo, sports, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, beef cattle, dairy cows, bears, and the like.

[0069] II. Methods and Uses of Bispecific Antibodies in the Treatment of Bronchiectasis As shown herein, bispecific antibodies that specifically bind to Pseudomonas aeruginosa Psl and PcrV are useful in treating subjects having bronchiectasis. Accordingly, provided herein are methods of treatment using such bispecific antibodies, the use of such bispecific antibodies in the preparation of a medicament, and bispecific antibodies for use in treatment.

[0070] Some embodiments provide herein a method of treating bronchiectasis (e.g., non-cystic fibrosis bronchiectasis). The method can include administering to a subject a bispecific antibody (e.g., MEDI3902) that specifically binds to Pseudomonas aeruginosa PcrV protein and Psl exopolysaccharide. The subject can be, for example, a subject colonized with Pseudomonas aeruginosa.

[0071] Some embodiments provide herein a method of improving pre-bronchodilator forced expiratory volume in 1 second (FEV1) in a subject having bronchiectasis (e.g., non-cystic fibrosis bronchiectasis). The method can include administering to a subject a bispecific antibody (e.g., MEDI3902) that specifically binds to Pseudomonas aeruginosa PcrV protein and Psl exopolysaccharide. The subject can be, for example, a subject colonized with Pseudomonas aeruginosa.

[0072] In some embodiments, provided herein is a method for reducing Pseudomonas aeruginosa burden in a subject having bronchiectasis (e.g., non-cystic fibrosis bronchiectasis). The method can include administering to the subject a bispecific antibody (e.g., MEDI3902) that specifically binds to the Pseudomonas aeruginosa PcrV protein and the Psl exopolysaccharide. The subject can be, for example, a subject colonized with Pseudomonas aeruginosa.

[0073] In some embodiments, provided herein is a method for reducing exacerbation of bronchiectasis in a subject having bronchiectasis (e.g., non-cystic fibrosis bronchiectasis). The method can, for example, reduce exacerbation of bronchiectasis requiring hospitalization and / or reduce exacerbation of bronchiectasis requiring antibiotics. The method can include administering to the subject a bispecific antibody (e.g., MEDI3902) that specifically binds to the Pseudomonas aeruginosa PcrV protein and the Psl exopolysaccharide. The subject can be, for example, a subject colonized with Pseudomonas aeruginosa.

[0074] In some embodiments, provided herein is a method for reducing the need for intravenous antibiotics in a subject having bronchiectasis (e.g., non-cystic fibrosis bronchiectasis). The method can include administering to the subject a bispecific antibody (e.g., MEDI3902) that specifically binds to the Pseudomonas aeruginosa PcrV protein and the Psl exopolysaccharide. The subject can be, for example, a subject colonized with Pseudomonas aeruginosa.

[0075] In some embodiments, methods are provided herein for stabilizing lung function in a subject having bronchiectasis (e.g., non-cystic fibrosis bronchiectasis). The method can include administering to the subject a bispecific antibody that specifically binds to Pseudomonas aeruginosa PcrV protein and Psl exopolysaccharide (e.g., MEDI3902). The subject can be, for example, a subject colonized with Pseudomonas aeruginosa.

[0076] In some embodiments, methods are provided herein for improving the frequency and / or intensity of cough in a subject having bronchiectasis (e.g., non-cystic fibrosis bronchiectasis). The method can include administering to the subject a bispecific antibody that specifically binds to Pseudomonas aeruginosa PcrV protein and Psl exopolysaccharide (e.g., MEDI3902). The subject can be, for example, a subject colonized with Pseudomonas aeruginosa.

[0077] In some embodiments, methods are provided herein for reducing bronchiectasis symptoms in a subject having bronchiectasis (e.g., non-cystic fibrosis bronchiectasis). The method can include administering to the subject a bispecific antibody that specifically binds to Pseudomonas aeruginosa PcrV protein and Psl exopolysaccharide (e.g., MEDI3902). The subject can be, for example, a subject colonized with Pseudomonas aeruginosa.

[0078] In some embodiments, methods are provided herein for improving the quality of life in a subject having bronchiectasis (e.g., non-cystic fibrosis bronchiectasis). The method can include administering to the subject a bispecific antibody that specifically binds to Pseudomonas aeruginosa PcrV protein and Psl exopolysaccharide (e.g., MEDI3902). The subject can be, for example, a subject colonized with Pseudomonas aeruginosa.

[0079] In some embodiments, provided herein are methods for eradicating Pseudomonas aeruginosa in a subject having bronchiectasis (e.g., non-cystic fibrosis bronchiectasis). The methods can include administering to the subject a bispecific antibody (e.g., MEDI3902) that specifically binds to the Pseudomonas aeruginosa PcrV protein and the Psl exopolysaccharide. The subject can be, for example, a subject colonized with Pseudomonas aeruginosa.

[0080] In some embodiments, provided herein are methods for inducing persistent suppression of Pseudomonas aeruginosa in a subject having bronchiectasis (e.g., non-cystic fibrosis bronchiectasis). The methods can include administering to the subject a bispecific antibody (e.g., MEDI3902) that specifically binds to the Pseudomonas aeruginosa PcrV protein and the Psl exopolysaccharide. The subject can be, for example, a subject colonized with Pseudomonas aeruginosa.

[0081] In some embodiments, provided herein are methods for reducing the risk of bronchiectasis progression associated with Pseudomonas aeruginosa in a subject having bronchiectasis (e.g., non-cystic fibrosis bronchiectasis). The methods can include administering to the subject a bispecific antibody (e.g., MEDI3902) that specifically binds to the Pseudomonas aeruginosa PcrV protein and the Psl exopolysaccharide. The subject can be, for example, a subject colonized with Pseudomonas aeruginosa.

[0082] Bronchiectasis suitable for treatment by the methods and uses provided herein can be non-cystic fibrosis bronchiectasis. In some embodiments, non-cystic fibrosis bronchiectasis is confirmed by chest computed tomography (CT) showing bronchiectasis affecting one or more lobes in the subject.

[0083] Subjects having bronchiectasis (e.g., non-cystic fibrosis bronchiectasis) suitable for treatment by the methods and uses provided herein can be subjects colonized with Pseudomonas aeruginosa. Subjects having Pseudomonas aeruginosa colonization can be identified, for example, using a routine sputum culture. In some embodiments, the subject is colonized with a strain of Pseudomonas aeruginosa that contains a genome that includes the Psl-operon. In some embodiments, the subject is colonized with a strain of Pseudomonas aeruginosa that contains a genome that includes the PcrV coding locus.

[0084] In some embodiments, the subject is chronically infected with Pseudomonas aeruginosa. As used herein, a "chronically infected" subject refers to a subject who is clinically stable over a period of one year while being colonized simultaneously or sequentially with at least two isolates of Pseudomonas aeruginosa.

[0085] Subjects having bronchiectasis (e.g., non-cystic fibrosis bronchiectasis) suitable for treatment by the methods and uses provided herein can be subjects having an increased airway neutrophil count and / or an increased sputum neutrophil count.

[0086] Subjects having bronchiectasis (e.g., non-cystic fibrosis bronchiectasis) suitable for treatment by the methods and uses provided herein can be subjects having a history of at least two moderate to severe exacerbations of bronchiectasis per year that require antibiotics.

[0087] Subjects having bronchiectasis (e.g., non-cystic fibrosis bronchiectasis) suitable for treatment by the methods and uses provided herein can be subjects having a history of at least one exacerbation that requires hospital care.

[0088] Subjects having bronchiectasis (e.g., non-cystic fibrosis bronchiectasis) suitable for treatment by the methods and uses provided herein can be subjects receiving long-term aerosolized antibiotics.

[0089] Subjects having bronchiectasis (e.g., non-cystic fibrosis bronchiectasis) suitable for treatment by the methods and uses provided herein can be subjects having chronic obstructive pulmonary disease (COPD).

[0090] Subjects having bronchiectasis (e.g., non-cystic fibrosis bronchiectasis) suitable for treatment by the methods and uses provided herein can be human subjects.

[0091] As provided herein, bronchiectasis of any cause (e.g., non-cystic fibrosis bronchiectasis) can be treated according to the methods and uses provided herein. For example, in some embodiments, bronchiectasis (e.g., non-cystic fibrosis bronchiectasis) is caused by hypogammaglobulinemia. In some embodiments, bronchiectasis (e.g., non-cystic fibrosis bronchiectasis) is caused by unclassifiable immunodeficiency. In some embodiments, bronchiectasis (e.g., non-cystic fibrosis bronchiectasis) is caused by alpha-1-antitrypsin deficiency.

[0092] The methods and uses provided herein can include administering to a subject a bispecific antibody (e.g., MEDI3902) that specifically binds to the Pseudomonas aeruginosa PcrV protein and the Psl exopolysaccharide. Administration can be intravenous administration. Administration can be subcutaneous administration.

[0093] The methods and uses provided herein are effective in treating bronchiectasis (e.g., non-cystic fibrosis bronchiectasis). In some aspects of the methods and uses provided herein, administration of a bispecific antibody (e.g., MEDI3902) that specifically binds to the Pseudomonas aeruginosa PcrV protein and the Psl exopolysaccharide reduces Pseudomonas aeruginosa in a sputum culture obtained from a subject, for example, as compared to Pseudomonas aeruginosa in a sputum culture obtained from the subject prior to administration. The reduction can occur, for example, within 12 weeks of the first administration of the bispecific antibody, within 8 weeks of the first administration of the bispecific antibody, or within 4 weeks of the first administration of the bispecific antibody. In some aspects of the methods and uses provided herein, administration of a bispecific antibody (e.g., MEDI3902) that specifically binds to the Pseudomonas aeruginosa PcrV protein and the Psl exopolysaccharide decreases antibiotic use by the subject, for example, as compared to antibiotic use by the subject prior to administration. In some aspects of the methods and uses provided herein, administration of a bispecific antibody (e.g., MEDI3902) that specifically binds to the Pseudomonas aeruginosa PcrV protein and the Psl exopolysaccharide eradicates Pseudomonas aeruginosa in the subject.

[0094] The methods and uses provided herein can include administering to a subject a bispecific antibody (e.g., MEDI3902) that specifically binds to the Pseudomonas aeruginosa PcrV protein and the Psl exopolysaccharide, in combination with an antibiotic. In some embodiments, the methods and uses provided herein include administering to a subject a bispecific antibody (e.g., MEDI3902) that specifically binds to the Pseudomonas aeruginosa PcrV protein and the Psl exopolysaccharide, in combination with an aminoglycoside, ticarcillin, ureidopenicillin, ciprofloxacin, cefepime, gentamicin, amikacin, tobramycin, ceftazidime, aztreonam, cefotaxime, meropenem, polymyxin b, or any combination thereof.

[0095] III. Bispecific Antibodies The methods and uses provided herein utilize a bispecific antibody that specifically binds to Pseudomonas aeruginosa Psl and PcrV. The bispecific antibody includes a Psl binding domain and a PcrV binding domain. Exemplary bispecific antibodies for use in the methods provided herein are disclosed, for example, in PCT Publications Nos. 2013 / 070615, 2014 / 074528, and 2015 / 171504, the disclosures of which are incorporated herein by reference in their entireties.

[0096] Exemplary sequences of bispecific antibodies for use in the methods provided herein are shown in Table 1.

[0097] [Table 2-1]

[0098] [Table 2-2]

[0099] The bispecific antibodies for the methods and uses provided herein can include an antigen-binding domain that specifically binds to P. aeruginosa PcrV and competitively inhibits the binding of an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14 to PcrV. In some embodiments, the bispecific antibodies for the methods and uses provided herein specifically bind to P. aeruginosa PcrV and include an antigen-binding domain that binds to the same epitope of PcrV as an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14.

[0100] In some embodiments, the bispecific antibodies for the methods and uses provided herein specifically bind to P. aeruginosa PcrV and include an antigen-binding domain comprising: (i) heavy chain CDR1, CDR2, and CDR3 (e.g., CDRs defined by Kabat, AbM, or Chothia) comprising the amino acid sequences of the heavy chain CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 13; and (ii) light chain CDR1, CDR2, and CDR3 (e.g., CDRs defined by Kabat, AbM, or Chothia) comprising the amino acid sequences of the light chain CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 14.

[0101] In some embodiments, the bispecific antibodies for the methods and uses provided herein specifically bind to P. aeruginosa PcrV and comprise an antigen-binding domain that includes a heavy-chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a heavy-chain CDR2 comprising the amino acid sequence of SEQ ID NO: 2, a heavy-chain CDR3 comprising the amino acid sequence of SEQ ID NO: 3, a light-chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a light-chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a light-chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the bispecific antibodies for the methods and uses provided herein specifically bind to P. aeruginosa PcrV and comprise an antigen-binding domain that includes a heavy-chain variable region comprising the amino acid sequence of SEQ ID NO: 13. In some embodiments, the bispecific antibodies for the methods and uses provided herein specifically bind to P. aeruginosa PcrV and comprise an antigen-binding domain that includes a light-chain variable region comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the bispecific antibodies for the methods and uses provided herein specifically bind to P. aeruginosa PcrV and comprise an antigen-binding domain that includes a heavy-chain variable region comprising the amino acid sequence of SEQ ID NO: 13 and a light-chain variable region comprising the amino acid sequence of SEQ ID NO: 14.

[0102] In some embodiments, the bispecific antibodies for the methods and uses provided herein comprise a PcrV-binding domain having a heavy-chain variable region and a light-chain variable region on separate polypeptides.

[0103] In some embodiments, the bispecific antibodies for the methods and uses provided herein specifically bind to P. aeruginosa Psl and comprise an antigen-binding domain that competitively inhibits the binding of an antibody comprising a heavy-chain variable region comprising the amino acid sequence of SEQ ID NO: 15 and a light-chain variable region comprising the amino acid sequence of SEQ ID NO: 16 to Psl. In some embodiments, the bispecific antibodies for the methods and uses provided herein specifically bind to P. aeruginosa Psl and comprise an antigen-binding domain that binds to the same epitope of Psl as an antibody comprising a heavy-chain variable region comprising the amino acid sequence of SEQ ID NO: 15 and a light-chain variable region comprising the amino acid sequence of SEQ ID NO: 16.

[0104] In some embodiments, the bispecific antibodies for the methods and uses provided herein specifically bind to P. aeruginosa Psl and comprise an antigen-binding domain comprising (i) heavy-chain CDR1, CDR2, and CDR3 comprising the amino acid sequences of the heavy-chain CDR1, CDR2, and CDR3 sequences at SEQ ID NO: 15 (e.g., CDRs defined by Kabat, AbM, or Chothia definitions), and (ii) light-chain CDR1, CDR2, and CDR3 comprising the amino acid sequences of the heavy-chain CDR1, CDR2, and CDR3 sequences at SEQ ID NO: 16 (e.g., CDRs defined by Kabat, AbM, or Chothia definitions).

[0105] In some embodiments, the bispecific antibodies for the methods and uses provided herein specifically bind to P. aeruginosa Psl and comprise an antigen-binding domain that includes a heavy-chain CDR1 comprising the amino acid sequence of SEQ ID NO: 7, a heavy-chain CDR2 comprising the amino acid sequence of SEQ ID NO: 8, a heavy-chain CDR3 comprising the amino acid sequence of SEQ ID NO: 9, a light-chain CDR1 comprising the amino acid sequence of SEQ ID NO: 10, a light-chain CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a light-chain CDR3 comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the bispecific antibodies for the methods and uses provided herein specifically bind to P. aeruginosa Psl and comprise an antigen-binding domain that includes a heavy-chain variable region comprising the amino acid sequence of SEQ ID NO: 15. In some embodiments, the bispecific antibodies for the methods and uses provided herein specifically bind to P. aeruginosa Psl and comprise an antigen-binding domain that includes a light-chain variable region comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, the bispecific antibodies for the methods and uses provided herein specifically bind to P. aeruginosa Psl and comprise an antigen-binding domain that includes a heavy-chain variable region comprising the amino acid sequence of SEQ ID NO: 15 and a light-chain variable region comprising the amino acid sequence of SEQ ID NO: 16.

[0106] In some embodiments, the bispecific antibodies for the methods and uses provided herein specifically bind to P. aeruginosa Psl and comprise an antigen-binding domain that includes a heavy-chain variable region and a light-chain variable region on the same polypeptide. In some embodiments, the bispecific antibody comprises a Psl-binding domain that is a scFv. The scFv can include a linker. The linker can be, for example, a glycine-rich linker or a glycine-serine linker. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, the scFv is in the VH-linker-VL orientation. In some embodiments, the scFv is in the VL-linker-VL orientation. In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 17.

[0107] In some embodiments, the bispecific antibodies for the methods and uses provided herein are IgG antibodies. The IgG antibodies can be, for example, IgG1 antibodies.

[0108] In some embodiments, the bispecific antibodies for the methods and uses provided herein comprise: (i) a heavy chain of the formula VH-CH1-H1-L1-S-L2-H2-CH2-CH3, wherein VH is an anti-P. aeruginosa PcrV heavy chain variable domain, CH1 is a heavy chain constant region domain 1, H1 is a first heavy chain hinge region fragment, L1 is a first linker, S is an anti-P. aeruginosa Psl scFv molecule, L2 is a second linker, H2 is a second heavy chain hinge region fragment, CH2 is a heavy chain constant region domain-2, and CH3 is a heavy chain constant region domain-3; and (ii) a light chain of the formula VL-CL, wherein VL is an anti-P. aeruginosa PcrV light chain variable domain and CL is an antibody light chain kappa constant region or an antibody light chain lambda region. In some embodiments, CL is an antibody light chain kappa constant region. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 13, and VL comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the scFv comprises the amino acid sequences of SEQ ID NO: 15 and SEQ ID NO: 16. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 13, VL comprises the amino acid sequence of SEQ ID NO: 14, and the scFv comprises the amino acid sequences of SEQ ID NO: 15 and SEQ ID NO: 16. In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 17. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 13, VL comprises the amino acid sequence of SEQ ID NO: 14, and the scFv comprises the amino acid sequence of SEQ ID NO: 17. In some embodiments, CH1 comprises the amino acid sequence of SEQ ID NO: 21. In some embodiments, L1 and L2 may be the same or different and independently may comprise (a) [GGGGS]n, where n is 0, 1, 2, 3, 4, or 5 (SEQ ID NO: 26), (b) [GGGG]n, where n is 0, 1, 2, 3, 4, or 5 (SEQ ID NO: 27), or a combination of (a) and (b). In some embodiments, H1 comprises the amino acid sequence EPKSC (SEQ ID NO: 22). In some embodiments, L1 comprises [GGGGS]n, where n is 2 (SEQ ID NO: 28).In some embodiments, L2 comprises [GGGGS]n, wherein n is 2 (SEQ ID NO: 28) or n is 4 (SEQ ID NO: 29). In some embodiments, H2 comprises the amino acid sequence DKTHTCPPCP (SEQ ID NO: 23). In some embodiments, CH2-CH3 comprises the amino acid sequence of SEQ ID NO: 25. In some embodiments, CL comprises the amino acid sequence of SEQ ID NO: 24.

[0109] In some embodiments, the bispecific antibodies for the methods and uses provided herein comprise a polypeptide comprising the amino acid sequence of SEQ ID NO: 19. In some embodiments, the bispecific antibodies for the methods and uses provided herein comprise a polypeptide comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the bispecific antibodies for the methods and uses provided herein comprise a polypeptide comprising the amino acid sequence of SEQ ID NO: 19 and a polypeptide comprising the amino acid sequence of SEQ ID NO: 20.

[0110] In some embodiments, the bispecific antibodies for the methods and uses provided herein neutralize cytotoxicity. In some embodiments, the bispecific antibodies for the methods and uses provided herein target Pseudomonas aeruginosa for opsonophagocytic killing (OPK). Methods for evaluating cytotoxicity neutralization and / or methods for evaluating OPK are known in the art and are provided herein, for example, in Example 3. In some embodiments, the bispecific antibodies for the methods and uses provided herein prevent cell attachment, for example, prevent the attachment of Pseudomonas aeruginosa to host cells. Methods for evaluating prevention of attachment are known in the art and are provided, for example, in International Publication No. WO 2013 / 070615, which is incorporated herein by reference in its entirety. In some embodiments, the bispecific antibodies for the methods and uses provided herein disrupt biofilm formation. In some embodiments, the bispecific antibodies for the methods and uses provided herein inhibit primary colony formation.

[0111] The anti-Pseudomonas aeruginosa Psl and PcrV bispecific antibody can be used in the preparation of a medicament for treating bronchiectasis (for example, non-cystic fibrosis bronchiectasis).

[0112] IV. Pharmaceutical composition The pharmaceutical composition used in the present disclosure includes an anti-Pseudomonas aeruginosa Psl and PcrV bispecific antibody and a pharmaceutically acceptable carrier well known to those skilled in the art. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.

[0113] The administration route of the anti-Pseudomonas aeruginosa Psl and PcrV bispecific antibody can be, for example, parenteral. The term parenteral as used herein includes, for example, intravenous and subcutaneous administration. Thus, the pharmaceutical composition containing the anti-Pseudomonas aeruginosa Psl and PcrV bispecific antibody can be formulated for intravenous administration. In some embodiments, the pharmaceutical composition containing the anti-Pseudomonas aeruginosa Psl and PcrV bispecific antibody can be formulated for subcutaneous administration. A suitable form for administration would be an injectable solution.

[0114] The pharmaceutical composition containing the anti-Pseudomonas aeruginosa Psl and PcrV bispecific antibody can be administered and / or formulated, for example, for the treatment of bronchiectasis (for example, non-cystic fibrosis bronchiectasis).

[0115] As provided herein, pharmaceutical compositions comprising anti-Pseudomonas aeruginosa Psl and PcrV bispecific antibodies can be formulated for administration in combination with antibiotics. In some embodiments, the bispecific antibodies are formulated for administration in combination with an aminoglycoside, ticarcillin, ureidopenicillin, ciprofloxacin, cefepime, gentamicin, amikacin, tobramycin, ceftazidime, aztreonam, cefotaxime, meropenem, polymyxin b, or any combination thereof.

Example

[0116] Materials and Methods for Examples 1-4 Patients Patients with bronchiectasis were enrolled from the Bronchiectasis Specialist Clinic at Ninewells Hospital in Dundee, UK. Inclusion criteria for bronchiectasis patients were being 18 years of age or older, having bronchiectasis confirmed by CT scan, having associated respiratory symptoms, and being able to give informed consent. Healthy age- and sex-matched control subjects were also enrolled from Ninewells Hospital. Inclusion criteria were being 18 years of age or older and being able to give informed consent, and participants were excluded if they had an ongoing infection or inflammatory disease.

[0117] Isolates from P. aeruginosa bronchiectasis patients One hundred clinical isolates of P. aeruginosa from sputum of patients with bronchiectasis were used for whole genome sequence (WGS) as previously described (Tabor et al., the Journal of Infectious Diseases, 2018, 218:1983 - 1994 - DOI:10.1093 / infdis / jiy438). For the initial culture, cryopreserved patient isolates were inoculated into lysogeny broth in 96 - well Corning PP 1.2 ml sterile cluster tubes (Sigma; CLS4411) and incubated at 37 °C for approximately 24 hours with circular shaking (200 rpm). Subsequently, the plates were centrifuged (5000 g, 15 minutes) and the supernatant was removed. The plates containing the bacterial cell pellet were frozen at - 80 °C until thawed for DNA extraction.

[0118] Whole - genome sequencing and genetic analysis Whole - genome sequencing and genetic analysis were performed as described in DOI:10.1093 / infdis / jiy438. Briefly, DNA was purified from bacterial cultures via bead beating followed by extraction using the QIAamp DNA Mini Kit (QIAGEN). Sequencing libraries were prepared by Covaris mechanical shearing followed by the NEBNext Ultra DNA Library Prep Kit (New England BioLabs). Sequencing was performed via NovaSeq 2×250 sequencing run (Illumina).

[0119] The array data was quality - trimmed via BBDuk (Bushnell B. http: / / sourceforge.net / projects / bbmap / ), de novo assembled via SPAdes (Bankevich et al., J Comput Biol., 2012, 19:455 - 477 - DOI:10.1089 / cmb.2012.0021), and annotated using Prokka (Seeman, Bioinformatics, 2014, 30:2068 - 2069 - DOI:10.1093 / bioinformatics / btu153). SRST2 (Inouye et al., Genome Medicine, 2014, 6:90 - DOI:10.1186 / s13073 - 014 - 0090 - 6) was used to perform multilocus sequence typing (MLST) and Psl operon gene content assessment with a minimum cutoff of 20 - fold coverage for gene presence calling. The PcrV gene sequence was de novo assembled (Tovchigrechko et al. DOI:10.5281 / zenodo.3619527. Accessed 2020 / 01 / 21, 2020), translated into a protein sequence, and amino acid substitutions were identified using the PAO1 NC_002516 pcrV as a reference sequence. Core - genome multiple alignment was performed using Parsnp and the PAO1 reference NC_002516 (Treangen et al., Genome Biology, 2014, 15:524 - DOI:10.1186 / s13059 - 014 - 0524 - x). The phylogenetic tree was visualized and annotated using ITOL, version 3 (Letunic and Bork, Nucleic Acids Research, 2016, 44:W242 - W245 - DOI:10.1093 / nar / gkw290).

[0120] Endogenous anti - Psl and anti - PcrV antibody levels in serum The endpoint antibody titer was determined by enzyme-linked immunosorbent assay (ELISA) as described in (Thaden et al., The Journal of Infectious Diseases, 2016, 213:640 - 648 - DOI:10.1093 / infdis / jiv436).

[0121] Assay for human neutrophil isolation and killing by opsonophagocytosis (OPK) Venous blood (20 ml) containing EDTA anticoagulant was used for primary neutrophil isolation within 2 hours of venipuncture. EasySep™ Direct Human Neutrophil Isolation Kit (StemCell, catalog number 19666) was used according to the manufacturer's instructions to isolate peripheral blood neutrophils. Briefly, 50 μl of antibody selection cocktail and 50 μl of RapidSpheres were added per 1 ml of whole blood. The EasySep™ Easy50 magnet was used for immunomagnetic separation. After incubation, RapidSpheres were added again and the blood was incubated for two more separation cycles. The isolated cells were quantified using a hemocytometer, washed in DPBS, and pelleted at 300 g for 6 minutes. Neutrophils were resuspended in OPK buffer (phenol red - free RPMI 1640 with 1% BSA [Gibco, catalog number 10363083]) at 20×106 cells / ml for use.

[0122] The neutrophil OPK of P. aeruginosa was measured as previously described (DOI: 10.1093 / infdis / jiv436) with the modifications described herein. To test the effect of MEDI3902 on primary neutrophil OPK activity, an antibody dilution series from 40 to 0.04 μg / ml was prepared in OPK buffer. An isotype control antibody was used to evaluate non-specific activity using a three-fold dilution series from 30 to 3.33 μg / ml. To evaluate the functionality of serum anti-Psl antibodies, heat-inactivated (56 °C, 30 min) serum samples were prepared in a ten-fold dilution series from 1:100 to 1:1,000,000. OPK was determined 2 hours after P. aeruginosa addition in all assays.

[0123] Cytotoxicity assay The cytotoxicity of P. aeruginosa strains 6077 and NCFBE clinical isolates, and the effect of MEDI3902 or endogenous anti-PcrV in A549 epithelial cells were investigated with some modifications as previously described (DOI: 10.1093 / infdis / jiv436). To determine the functionality of the endogenous anti-PcrV antibody, a three-fold dilution series of heat-inactivated serum from 1:100 to 1:24300 was utilized. The competition between the endogenous anti-PcrV antibody and MEDI3902 was evaluated using a 1:100 serum with a five-fold MEDI3902 dilution series from 300 to 0.096 μg / ml. CytoTox-ONE™ kit was used to determine A549 cell death according to the manufacturer's instructions.

[0124] Detection of Psl expression in NCFBE clinical isolates The expression of Psl in NCFBE clinical isolates was measured by indirect enzyme-linked immunosorbent assay (ELISA) using anti-Psl mAb Cam003 (DiGiandomenico et al., J Exp Med., 2012, 209:1273-1287 - DOI:10.1084 / jem.20120033) or MEDI3902 as previously described (DiGiandomenico et al., Sci Transl Med., 2014, 12:262 - DOI:10.1126 / scitranslmed.3009655).

[0125] Detection of PcrV expression from clinical isolates PcrV expression was evaluated from whole cell lysates by Western immunoblot analysis after in vitro induction of the type III secretion system (T3S) of Pseudomonas aeruginosa as described in other literature (Warrener et al., Antimicrobial Agents and Chemotherapy, 2014, 58:8 - DOI:10.1128 / AAC.02643-14; DOI:10.1093 / infdis / jiv436).

[0126] Example 1: Target conservation of Psl and PcrV in Pseudomonas aeruginosa isolates from BE Psl operon genes in the NCFBE isolates In the sequenced isolates, 72 strains contained the complete complement of the psl operon genes (pslA through pslO). In contrast, 17 strains lacked the complete operon and 11 strains lacked one or more genes (Figure 1). Representative isolates were tested in whole-cell Psl ELISA using anti-Psl mAb Cam003 to identify which psl operon genes were required for Psl synthesis. Isolates that were completely operon-null, lacked pslA, or lacked the genes pslF through pslO were negative for Psl expression (Figure 1). pslM through O have previously been shown not to be required for Psl expression (DOI:10.1093 / infdis / jiy438). Strain 79 was null for pslK but was shown to be bound by Cam003 and MEDI3902 but not by control IgG (Figure 2). Strain 6, an isolate containing a completely intact psl locus, functioned as a positive control for the assay (Figure 2). In total, 73 out of 100 isolates contained the genetic elements necessary to express Psl (Figure 1).

[0127] The PcrV gene is highly prevalent in NCFBE P. aeruginosa isolates The full-length pcrV gene was present in 99 out of 100 sequenced isolates and showed a high incidence among P. aeruginosa isolates from NCFBE (Figure 1). In a global surveillance study of P. aeruginosa isolates from acute infections, 46 PcrV variant sequences had been previously isolated using PAO1 PcrV as the amino acid reference strain (DOI:10.1093 / infdis / jiy438). Analysis of the PcrV amino acid sequences of NCFBE clinical isolates revealed substitutions at 18 positions corresponding to 20 different PcrV full-length protein subtypes. Most of the PcrV variants from this strain collection had been previously reported (DOI:10.1093 / infdis / jiy438), but six new variant sequences (shown in bold in Table 2) were identified in P. aeruginosa isolates from NCFBE (Table 2).

[0128]

Table 3

[0129] Example 2: The NCFBE PcrV subtype is bound by the anti-PcrV MEDI3902 binding domain None of the newly identified amino acid substitutions were due to the MEDI3902 anti-PcrV contact points, but the anti-PcrV mAb V2L2MD (containing the complementarity-determining regions, VH, and VL contained within MEDI3902) was evaluated for its ability to bind to PcrV expressed from whole cell lysates of subtype-specific strains cultured under type III secretion-inducing conditions. The reactivity of the lysates against purified polyclonal IgG from rabbits immunized with recombinant PcrV was performed to evaluate whether potential differences in protein reactivity were due to anti-PcrV mAb binding or in vitro PcrV expression levels. Immunoblot analysis showed that anti-PcrV mAb binding was comparable to anti-PcrV polyclonal IgG binding for all newly identified PcrV variants that were able to express PcrV under in vitro conditions (Figure 3). Overall, the data indicate that V2L2-MD bound to all mutant PcrV sequences expressed in vitro.

[0130] Example 3: MEDI3902 maintains functional activity against P. aeruginosa isolates from NCFBE ExoU is a protein transported by the type III secretion system of Pseudomonas aeruginosa and is an important cytotoxin for this pathogen. MEDI3902 was evaluated for its anti-cytotoxic activity against representative ExoU-producing P. aeruginosa NCFBE isolates (NCFBE strains 84 and 55) that exhibited cytotoxicity against an epithelial cell line (A549). MEDI3902 inhibited cytotoxicity in both strains, while control IgG had no protective activity (Figure 4A). In addition, when an in vitro OPK assay was performed using a representative P. aeruginosa isolate (NCFBE strain 6) that harbored a completely intact locus, this was shown to be bound by the anti-Psl mAb Cam003 or MEDI3902. MEDI3902 and Cam003 showed OPK activity against NCFBE isolate 6, while control IgG did not (Figure 4B).

[0131] Example 4: MEDI3902 enhances the killing of P. aeruginosa by peripheral blood neutrophils from patients with bronchiectasis Neutrophil-mediated bactericidal activity has previously been reported to be impaired in bronchiectasis. The ability of neutrophils to kill P. aeruginosa in the presence of increasing concentrations of MEDI3902 or an IgG control antibody was investigated and compared to the ability in neutrophils from healthy controls and age- and sex-matched controls. Killing of P. aeruginosa was similar in all three participant groups (Figure 5). MEDI3902 at the highest concentration used (200 nM) resulted in an average increase in neutrophil-mediated P. aeruginosa killing of 34.6 ± 8.1% (mean ± SD), which was 36 ± 8.6% in healthy control neutrophils and 30.1 ± 7.6% in the matched controls. At an equimolar concentration (200 nM), addition of the control IgG antibody did not result in a change in killing in any of the groups (1.1 ± 6.6%, -0.9 ± 3.6%, 2.1 ± 1.8%, respectively). Enhancement of bactericidal activity by MEDI3902 was observed at the lowest concentration used (0.2 nM).

[0132] Example 5: Endogenous anti-Psl antibodies were not present in most of the bronchiectasis serum samples, although most patients showed detectable anti-PcrV. Previously, it has been reported that endogenous anti-Psl and PcrV serum IgG levels varied among patients with acute P. aeruginosa infections and were not different from those in non-Pseudomonas infection samples. In this study, titers were determined in sera from bronchiectasis patients with either chronic P. aeruginosa airway infections or no recently documented infections, as well as age- and sex-matched controls. Three out of 31 bronchiectasis patients had detectable anti-Psl titers, and all of these participants were part of the chronic infection group, although one participant from the matched control group also had measurable anti-Psl (Figure 6A). Overall, the bronchiectasis patient group appeared to have higher serum anti-Pseudomonas antibody levels than the matched control group. Anti-PcrV titers were detectable at varying levels in all bronchiectasis samples, with the chronic infection group showing the highest titers (Figure 6B).

[0133] Example 6: Endogenous anti-Psl antibody functional activity varied in bronchiectasis patient sera but did not compete with MEDI3902. Three patients with bronchiectasis showed serum anti-Psl antibody titers detectable by ELISA. The functionality of endogenous anti-Psl antibodies was tested in these samples for their ability to increase neutrophil-mediated killing of P. aeruginosa. Incubation of human neutrophils and wild-type (WT) P. aeruginosa with 1:100 diluted patient sera resulted in increased killing for two out of the three samples (14.7% and 21.3% increases in sera 1 and 2, respectively, Figure 7A (left panel)). Further serum dilutions showed no functional effect. When Psl-deficient (ΔpslA) P. aeruginosa was utilized in the same assay to determine the specific contribution of anti-Psl to these observed effects, the increase in killing activity by serum 2 was abolished, while serum 1 retained its activity (Figure 7A (right panel)). Only one out of the three samples (serum 2) showed functional anti-Psl antibodies, while the effects on neutrophil-mediated killing in the other samples may be due to other factors.

[0134] Subsequently, the ability of endogenous anti-Psl to compete for the ligand-binding site and the potential to inhibit the effect of MEDI3902 were investigated by spiking patient sera 1:100 with MEDI3902 at the indicated concentrations. All concentrations of MEDI3902 alone and MEDI3902 with each serum sample showed increased killing of WT P. aeruginosa (Figure 7B (left panel)). These effects were abolished in Psl-deficient bacteria when MEDI3902 was used alone or with serum 2 (Figure 7B (right panel)). The increase in killing was maintained for sera 1 and 3, which did not exhibit functional anti-Psl. This data indicates that endogenous anti-Psl antibodies in bronchiectasis patient sera, including potentially functional anti-Psl antibodies, do not interfere with or compete with MEDI3902.

[0135] Example 7: Bronchiectasis patient sera with endogenous anti-PcrV had a protective effect against P. aeruginosa cytotoxicity and did not impair MEDI3902 activity. Since a high percentage of bronchiectasis patient serum samples contained detectable levels of anti-PcrV antibody, the potential functionality of endogenous anti-PcrV was tested, and the ability to inhibit the action on A549 epithelial cell lysis by the cytotoxic ExoU+ 6077 P. aeruginosa strain was obtained as a result. All five serum samples tested showed a protective effect and, on average, when added at a 1:100 dilution, inhibited LDH release and thus cell death by 6077 P. aeruginosa by 71.9 ± 18.6% (Figure 8A). The protective effect was seen up to the highest serum dilution of 1:24300 (inhibition of lysis of 53.2 ± 30.1%) for all but one of these samples.

[0136] To determine whether endogenous anti-PcrV competes with MEDI3902 for binding and thus alters activity, experiments were repeated with 1:100 diluted serum spiked with the indicated concentrations of MEDI3902, or MEDI3902 alone (Figure 8B). In the presence of patient serum, no reduction in MEDI3902 activity was observed at any of the concentrations used, and for most serum samples, the anti-cytotoxic activity appeared to be additive when combined. In particular, at the lowest MEDI3902 concentration (0.48 nM), 60.4 ± 4.5% protection was obtained with MEDI3902 alone, while MEDI3902 + serum showed an average of 84.2 ± 6.4% inhibition of cell lysis. This data indicates that endogenous anti-PcrV antibodies in bronchiectasis patient serum, including potentially functional anti-PcrV titers, do not interfere with or compete with MEDI3902. In addition, this data indicates that the addition of MEDI3902 to serum has an additive anti-cytotoxic effect.

[0137] Example 8: MEDI3902 in Patients with Bronchiectasis and Chronic Pseudomonas aeruginosa A randomized, double-blind, phase 2 trial of MEDI3902 compared to placebo is conducted in participants with bronchiectasis and chronic Pseudomonas aeruginosa infection. The flowchart of the trial is shown in Figure 9.

[0138] Patient Randomize 90 participants who meet the following inclusion criteria. · 18 to 85 years old, · Clinical diagnosis of bronchiectasis, · Previous chest CT scan showing bronchiectasis in one or more lobes, · P. aeruginosa in sputum, bronchoalveolar lavage, or another airway sample at least once in the 24 months prior to screening * 、 · Sputum samples that are culture positive for P. aeruginosa sent at the screening visit and within 35 days of randomization * 、 * Participants without a previous positive sample for P. aeruginosa may submit two samples separated by at least 21 days during the 35-day screening period. If both of these samples are positive for P. aeruginosa, the last two inclusion criteria are considered met and the patient may be enrolled.

[0139] Participants are randomly assigned to receive either MEDI3902 1500 mg (IV, 4 times weekly for 12 weeks), MEDI3902 500 mg (IV, 4 times weekly for 12 weeks), or placebo (IV, 4 times weekly for 12 weeks), all in addition to standard therapy. Randomization is 2:1:1, MEDI3902 1500 mg:MEDI3902 500 mg:placebo.

[0140] Administration Administer MEDI3902 (1500 mg or 500 mg) or placebo (liquid buffer in saline) by intravenous infusion (total volume 250 mL) 4 times weekly for 12 weeks.

[0141] Evaluation Participants are required to bring a spontaneous early morning sputum sample to the visit. Evaluate the color of the sputum using the Murray Sputum Color Chart. Evaluate the P. aeruginosa bacterial load in the sputum on days 7, 14, 28, 56, and 84.

[0142] Using the guidelines of the American Thoracic Society / European Respiratory Society, perform post - bronchodilator spirometry at the time of visit.

[0143] Measure the forced expiratory volume in 1 second (FEV1), forced vital capacity (FVC), and forced expiratory flow at 25 - 75% (FEF25 - 75).

[0144] The severity of bronchiectasis is evaluated using the bronchiectasis severity index and the MRC dyspnea score. The exacerbation assessment uses the EMBARC definition of exacerbation. (Hill AT, et al. Eur Respir J. 2017;49(6). doi:10.1183 / 13993003.00051 - 2017.)

[0145] Quality of life is evaluated using the Quality of Life Bronchiectasis Questionnaire (QOL-B) (Quittner AL et al. Quality of Life Questionnaire-Bronchiectasis: final psychometric analyses and determination of minimal important difference scores. Thorax. 2015;70(1):12-20. doi:10.1136 / thoraxjnl-2014-205918), the St. George's Respiratory Questionnaire (SGRQ) (Wilson CB, et al., Am J Respir Crit Care Med. 1997;156(2 I):536-541. doi:10.1097 / 00008483-199803000-00011), and the Bronchiectasis Impact Measure (BIM) questionnaire (Crichton ML, et al. Eur Respir J. 2021;57(5). doi:10.1183 / 13993003.03156-2020). The SGRQ has a 3-month follow-up period and is thus performed at baseline and at 3 months. The QOL-B and BIM questionnaires have a shorter follow-up period and are performed monthly.

[0146] Results Evaluation of the P. aeruginosa bacterial load in sputum at week 12 (day 84) demonstrates the effect of MEDI3902 in the treatment of bronchiectasis in patients with chronic Pseudomonas aeruginosa infection.

[0147] Eradication of P. aeruginosa, defined by a negative sputum culture for P. aeruginosa at the end of treatment, is also evaluated.

[0148] Evaluate the changes from baseline in QOL-B and BIM questions (at days 28, 56, 84, and 168) and SGRQ (at days 84 and 168) to show the effectiveness of MEDI3902 in improving quality of life in patients with bronchiectasis.

[0149] Evaluate the occurrence of exacerbation (according to the EMBARC definition of exacerbation; days 1 to 84 of visit) to show the effect of MEDI3902 on the time to first exacerbation.

[0150] Evaluate the change from baseline in FEV1 (at 28, 56, and 84) to show the effect of MEDI3902 on lung function.

[0151] It should be understood that it is intended to interpret the claims using the section "Detailed Description of the Invention" rather than the sections "Summary" and "Abstract". The sections "Summary" and "Abstract" can explain one or more, but not all, of the exemplary aspects of the invention contemplated by the inventor, and thus are never intended to limit the invention and the appended claims.

[0152] The breadth and scope of the present invention should not be limited by any of the above exemplary aspects, but should be defined only in accordance with the following claims and their equivalents.

Claims

1. A pharmaceutical composition for use in the treatment of bronchiectasis in subjects requiring treatment of bronchiectasis, comprising a bispecific antibody that specifically binds to Pseudomonas aeruginosa PcrV protein and Psl extracellular polysaccharide.

2. A pharmaceutical composition for use in improving pre-bronchodilator forced expiratory capacity 1 (FEV 1) in subjects with bronchiectasis, comprising a bispecific antibody that specifically binds to Pseudomonas aeruginosa PcrV protein and Psl extracellular polysaccharide.

3. A pharmaceutical composition for use in reducing Pseudomonas aeruginosa load in subjects with bronchiectasis, comprising a bispecific antibody that specifically binds to Pseudomonas aeruginosa PcrV protein and Psl extracellular polysaccharide.

4. A pharmaceutical composition for use in reducing exacerbations of bronchiectasis in subjects who need to reduce exacerbations of bronchiectasis, comprising a bispecific antibody that specifically binds to Pseudomonas aeruginosa PcrV protein and Psl extracellular polysaccharide.

5. The pharmaceutical composition for use according to claim 4, wherein the pharmaceutical composition reduces exacerbations of bronchiectasis requiring hospitalization.

6. The pharmaceutical composition for use according to claim 4 or 5, wherein the pharmaceutical composition reduces the exacerbation of bronchiectasis requiring antibiotics.

7. A pharmaceutical composition for use in reducing the need for intravenous antibiotics in subjects with bronchiectasis, comprising a bispecific antibody that specifically binds to Pseudomonas aeruginosa PcrV protein and Psl extracellular polysaccharide.

8. A pharmaceutical composition for use in stabilizing lung function in subjects with bronchiectasis, comprising a bispecific antibody that specifically binds to Pseudomonas aeruginosa PcrV protein and Psl extracellular polysaccharide.

9. The pharmaceutical composition for use according to any one of claims 1 to 5, 7, or 8, wherein the bronchiectasis is non-cystic fibrotic bronchiectasis.

10. The pharmaceutical composition for use according to claim 9, wherein the non-cystic fibrotic bronchiectasis is confirmed by computed tomography (CT) of the chest showing bronchiectasis affecting one or more lobes.

11. A pharmaceutical composition for use according to any one of claims 1 to 5, 7, or 8, wherein the subject is colonized with Pseudomonas aeruginosa.

12. The pharmaceutical composition for use according to claim 11, wherein the formation of Pseudomonas aeruginosa colonies is detected by sputum culture.

13. A pharmaceutical composition for use according to any one of claims 1 to 5, 7, or 8, wherein the subject is chronically infected with Pseudomonas aeruginosa.

14. A pharmaceutical composition for use according to any one of claims 1 to 5, 7, or 8, wherein the subject has an increase in airway neutrophils.

15. A pharmaceutical composition for use according to any one of claims 1 to 5, 7, or 8, wherein the subject has sputum neutrophilia.

16. A pharmaceutical composition for use according to any one of claims 1 to 5, 7, or 8, wherein the subject has a history of moderate to severe exacerbations of bronchiectasis occurring at least twice a year, requiring antibiotics.

17. A pharmaceutical composition for use according to any one of claims 1 to 5, 7, or 8, wherein the subject has a history of at least one exacerbation requiring hospital nursing.

18. A pharmaceutical composition for use according to any one of claims 1 to 5, 7, or 8, wherein the subject is receiving long-term spray antibiotic treatment.

19. A pharmaceutical composition for use according to any one of claims 1 to 5, 7, or 8, wherein the subject has chronic obstructive pulmonary disease (COPD).

20. A pharmaceutical composition for use according to any one of claims 1 to 5, 7, or 8, wherein the bronchiectasis is caused by hypogammaglobulinemia, unclassifiable immunodeficiency, or alpha-1-antitrypsin deficiency.

21. The pharmaceutical composition for use according to any one of claims 1 to 5, 7, or 8, wherein the pharmaceutical composition reduces Pseudomonas aeruginosa in a sputum culture obtained from the subject.

22. The pharmaceutical composition for use according to any one of claims 1 to 5, 7, or 8, wherein the pharmaceutical composition reduces the use of antibiotics.

23. A pharmaceutical composition for use according to any one of claims 1 to 5, 7, or 8, wherein the pharmaceutical composition eradicates Pseudomonas aeruginosa in the subject.

24. A pharmaceutical composition for use according to any one of claims 1 to 5, 7, or 8, wherein the bispecific antibody competitively inhibits the binding of the antibody, which includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 13 and / or a light chain variable region containing the amino acid sequence of SEQ ID NO: 14, to PcrV.

25. A pharmaceutical composition for use according to any one of claims 1 to 5, 7, or 8, wherein the bispecific antibody binds to the same PcrV epitope as the antibody comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 13 and / or a light chain variable region containing the amino acid sequence of SEQ ID NO:

14.

26. A pharmaceutical composition for use according to any one of claims 1 to 5, 7, or 8, wherein the bispecific antibody comprises a PcrV binding domain comprising a heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 2, a heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 3, a light chain CDR1 containing the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 containing the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 containing the amino acid sequence of SEQ ID NO:

6.

27. A pharmaceutical composition for use according to any one of claims 1 to 5, 7, or 8, wherein the bispecific antibody comprises a PcrV binding domain comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13 and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:

14.

28. The pharmaceutical composition for use according to any one of claims 1 to 5, 7, or 8, wherein the bispecific antibody comprises a PcrV-binding domain having a heavy chain variable region and a light chain variable region on a separate polypeptide.

29. The pharmaceutical composition for use according to any one of claims 1 to 5, 7, or 8, wherein the bispecific antibody competitively inhibits the binding of the antibody, which includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 15 and / or a light chain variable region containing the amino acid sequence of SEQ ID NO: 16, to Psl.

30. A pharmaceutical composition for use according to any one of claims 1 to 5, 7, or 8, wherein the bispecific antibody binds to the same Psl epitope as the antibody comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 15 and / or a light chain variable region containing the amino acid sequence of SEQ ID NO:

16.

31. A pharmaceutical composition for use according to any one of claims 1 to 5, 7, or 8, wherein the bispecific antibody comprises a Psl-binding domain comprising a heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 7, a heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 8, a heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 9, a light chain CDR1 containing the amino acid sequence of SEQ ID NO: 10, a light chain CDR2 containing the amino acid sequence of SEQ ID NO: 11, and a light chain CDR3 containing the amino acid sequence of SEQ ID NO:

12.

32. A pharmaceutical composition for use according to any one of claims 1 to 5, 7, or 8, wherein the bispecific antibody comprises a Psl-binding domain comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15 and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:

16.

33. The pharmaceutical composition for use according to any one of claims 1 to 5, 7, or 8, wherein the bispecific antibody comprises a Psl-binding domain having a heavy chain variable region and a light chain variable region on the same polypeptide.

34. A pharmaceutical composition for use according to any one of claims 1 to 5, 7, or 8, wherein the bispecific antibody comprises a Psl-binding domain which is scFv.

35. The pharmaceutical composition for use according to claim 34, wherein the scFv comprises a linker.

36. The pharmaceutical composition for use according to claim 35, wherein the linker comprises the amino acid sequence of SEQ ID NO:

18.

37. The pharmaceutical composition for use according to claim 36, wherein the scFv is in the VH-linker-VL orientation.

38. The pharmaceutical composition for use according to claim 34, wherein the scFv comprises the amino acid sequence of SEQ ID NO:

17.

39. The pharmaceutical composition for use according to any one of claims 1 to 5, 7, or 8, wherein the bispecific antibody is an IgG antibody.

40. The bispecific antibody comprises (i) a heavy chain of formula VH-CH1-H1-L1-S-L2-H2-CH2-CH3, where VH is the anti-P. aeruginosa PcrV heavy chain variable domain, CH1 is the heavy chain constant region domain 1, H1 is the first heavy chain hinge region fragment, L1 is the first linker, S is the anti-P. aeruginosa Psl scFv molecule, L2 is the second linker, H2 is the second heavy chain hinge region fragment, CH2 is the heavy chain constant region domain-2, and CH3 is the heavy chain constant region domain-3, and (ii) a light chain of formula VL-CL, where VL is the anti-P. A pharmaceutical composition for use according to any one of claims 1 to 5, 7, or 8, comprising a light chain which is an aeruginosa PcrV light chain variable domain, wherein CL is the antibody light chain kappa constant region or the antibody light chain lambda region.

41. The pharmaceutical composition for use according to claim 40, wherein VH comprises the amino acid sequence of SEQ ID NO: 13, scFv comprises the amino acid sequence of SEQ ID NO: 15 and the amino acid sequence of SEQ ID NO: 16, and / or VL comprises the amino acid sequence of SEQ ID NO:

14.

42. The pharmaceutical composition for use according to claim 40, wherein VH comprises the amino acid sequence of SEQ ID NO: 13, scFv comprises the amino acid sequence of SEQ ID NO: 17, and / or VL comprises the amino acid sequence of SEQ ID NO:

14.

43. The pharmaceutical composition for use according to claim 40, wherein CL is the constant region of the antibody light chain kappa.

44. The pharmaceutical composition for use according to any one of claims 1 to 5, 7, or 8, wherein the bispecific antibody neutralizes cytotoxicity.

45. A pharmaceutical composition for use according to any one of claims 1 to 5, 7, or 8, wherein the bispecific antibody targets Pseudomonas aeruginosa for killing by opsonin phagocytosis.

46. The pharmaceutical composition for use according to any one of claims 1 to 5, 7, or 8, wherein the bispecific antibody prevents cell adhesion.

47. The pharmaceutical composition for use according to any one of claims 1 to 5, 7, or 8, wherein the bispecific antibody disrupts biofilm formation.

48. A pharmaceutical composition for use according to any one of claims 1 to 5, 7, or 8, wherein the subject is colonized with a strain of Pseudomonas aeruginosa comprising a genome containing a Psl operon and / or a PcrV coding locus.

49. A pharmaceutical composition for use according to any one of claims 1 to 5, 7, or 8, wherein the bispecific antibody is administered intravenously.

50. A pharmaceutical composition for use according to any one of claims 1 to 5, 7, or 8, wherein the subject is a human.

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