Anti-Pcrv and Psl bispecific antibodies for treatment of bronchiectasia

Abstract

The present disclosure relates to anti-Pseudomonas Psl and PcrV bispecific antibodies for use in the treatment of bronchiectasia.

Description

Technical Field

[0001] The present disclosure relates to the use of bispecific antibodies that specifically bind 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 impaired individuals. This is partly due to the high innate resistance of bacteria to clinically used antibiotics, and partly due to the formation of a highly antibiotic-resistant biofilm. It is also known that Pseudomonas aeruginosa colonizes the airways of patients with non-cystic fibrosis bronchiectasis. Non-cystic fibrosis bronchiectasis is a chronic disease characterized by abnormal and permanent expansion of the bronchi, leading to chronic cough, sputum production and repeated bacterial infections of the airways. Patients with bronchiectasis suffer from high morbidity due to frequent exacerbations, which damage the quality of life and promote resistance to antibiotics, causing reduced lung function.

[0003] There is a significant unmet medical need for the treatment of bronchiectasis, and no approved therapies are currently available for reducing exacerbations. The European Respiratory Society (ERS) 2017 guidelines for the management of bronchiectasis in adults suggest that no other treatments can be recommended except antibiotics to treat acute exacerbations. Due to the increased multidrug resistance exhibited by bacteria, there remains a need in the art to develop new strategies for identifying new Pseudomonas-specific prevention and treatment strategies. Summary of the invention

[0004] Provided herein are anti-Pseudomonas Psl and PcrV bispecific antibodies for use in treating 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 a Pseudomonas aeruginosa PcrV protein and a Psl exopolysaccharide.

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

[0008] In some aspects provided herein, a method of reducing the 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 the exacerbation of bronchiectasis requiring hospitalization. In some aspects, the administration reduces the exacerbation of bronchiectasis requiring antibiotics. In some aspects, the administration reduces the exacerbation of bronchiectasis requiring hospitalization and the exacerbation of bronchiectasis requiring antibiotics.

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

[0010] In some aspects provided herein, a method of stabilizing lung function in a subject with bronchiectasis comprises administering to the subject a bispecific antibody that specifically binds to a Pseudomonas aeruginosa PcrV protein and a 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) demonstrating that the bronchiectasis affects 1 or more lobes.

[0012] In some aspects, the subject is colonized with Pseudomonas aeruginosa. In some aspects, the respiratory tract of the subject is colonized with Pseudomonas aeruginosa. In some aspects, Pseudomonas aeruginosa colonization has been detected by sputum culture.

[0013] In some aspects, the subject is chronically infected with Pseudomonas aeruginosa. In some aspects, the subject suffers from airway neutrophilia. In some aspects, the subject suffers from sputum neutrophilia. In some aspects, the subject has a moderate to severe bronchiectasis exacerbation history requiring antibiotics at least 2 times per year. In some aspects, the subject has a history of exacerbation requiring hospital care at least 1 time. In some aspects, the subject uses aerosolized antibiotics for a long time. In some aspects, the subject suffers from chronic obstructive pulmonary disease (COPD).

[0014] In some aspects, bronchiectasis is caused by hypogammaglobulinemia. In some aspects, bronchiectasis is caused by common variable immunodeficiency. In some aspects, bronchiectasis is caused by alpha-1-antitrypsin deficiency.

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

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

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

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

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

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

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

[0022] In some aspects, 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 aspects, the bispecific antibody comprises a Psl binding domain that is a scFv. In some aspects, the scFv comprises a linker. In some aspects, the linker comprises the amino acid sequence of SEQ ID NO: 18. In some aspects, the scFv is in the orientation of VH-linker-VL. In some aspects, the scFv comprises the amino acid sequence of SEQ ID NO: 17.

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

[0024] In some aspects, the bispecific antibody comprises a heavy chain of (i) formula VH-CH1-H1-L1-S-L2-H2-CH2-CH3, wherein VH is an anti-Pseudomonas 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-Pseudomonas 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 formula VL-CL, wherein VL is an anti-Pseudomonas 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 aspects, VH comprises the amino acid sequence of SEQ ID NO: 13. In some aspects, scFv comprises the amino acid sequence of SEQ ID NO: 15 and the amino acid sequence of SEQ ID NO: 16. In some aspects, the VL comprises the amino acid sequence of SEQ ID NO: 14. In some aspects, the VH comprises the amino acid sequence of SEQ ID NO: 13, and the scFv comprises the amino acid sequence of SEQ ID NO: 15 and the amino acid sequence of SEQ ID NO: 16. In some aspects, the VH comprises the amino acid sequence of SEQ ID NO: 13 and the VL comprises the amino acid sequence of SEQ ID NO: 14. In some aspects, the scFv comprises the amino acid sequence of SEQ ID NO: 15 and the amino acid sequence of SEQ ID NO: 16, and the VL comprises the amino acid sequence of SEQ ID NO: 14. In some aspects, the VH comprises the amino acid sequence of SEQ ID NO: 13, the scFv comprises the amino acid sequence of SEQ ID NO: 15 and the amino acid sequence of SEQ ID NO: 16, and the VL comprises the amino acid sequence of SEQ ID NO: 14. In some aspects, the 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 aspects, the scFv comprises the amino acid sequence of SEQ ID NO: 17 and the VL comprises the amino acid sequence of SEQ ID NO: 14. In some aspects, the VH comprises the amino acid sequence of SEQ ID NO: 13, the scFv comprises the amino acid sequence of SEQ ID NO: 17, and the VL comprises the amino acid sequence of SEQ ID NO: 14. In some aspects, CL is an antibody light chain kappa constant region. In some aspects, the bispecific antibody comprises a heavy chain having an amino acid sequence of SEQ ID NO: 19. In some aspects, the bispecific antibody comprises a light chain having an amino acid sequence of SEQ ID NO: 20. In some aspects, the bispecific antibody comprises a heavy chain having an amino acid sequence of SEQ ID NO: 19 and a light chain having an amino acid sequence of SEQ ID NO: 20.

[0025] In some aspects, bispecific antibodies neutralize cell poisoning. In some aspects, bispecific antibodies target Pseudomonas aeruginosa for opsonized phagocytosis and killing. In some aspects, bispecific antibodies prevent cell attachment. In some aspects, bispecific antibodies destroy biofilm formation. In some aspects, bispecific antibodies inhibit primary colony formation.

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

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

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

[0029] In some aspects, the present invention provides the use of bispecific antibodies. In some aspects, the present invention provides the use of bispecific antibodies that specifically bind to Pseudomonas aeruginosa PcrV protein and Psl exopolysaccharide in the preparation of a medicament for treating bronchiectasis in a subject in need thereof. Treating bronchiectasis may include any method as provided herein.

[0030] In some aspects, bispecific antibodies for use are provided herein. In some aspects, a bispecific antibody specifically binding to Pseudomonas aeruginosa PcrV protein and Psl exopolysaccharide is provided herein for use in treating bronchiectasis in a subject in need thereof. Treatment of bronchiectasis may include any method as provided herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Target conservation of Psl and PcrV in bronchiectasis (BE)-derived Pseudomonas aeruginosa isolates obtained by WGS (whole genome sequencing) is shown. White indicates the presence of pcrV or psl operon genes, and grey indicates the absence.

[0032] Figure 2 Shown are ELISA reactivity of NCFBE-derived P. aeruginosa strains 79 and 6 to Cam003 and MEDI3902, indicating expression of Psl in these strains.

[0033] Figure 3 Immunoblotting of NCFBE-derived P. aeruginosa strains using anti-PcrV antibody V2L2-MD and anti-PcrV polyclonal IgG is shown. (Strains 6077 and 6206 were used as positive controls).

[0034] Figure 4A The anti-cytotoxic activity of MEDI3902 and control IgG against representative ExoU-producing P. aeruginosa non-cystic fibrosis bronchial dilatation (NCFBE) isolates (NCFBE strains 84 and 55) is shown. The x-axis shows the logarithmic concentration (nM) of the mAb used.

[0035] Figure 4B Shown are in vitro opsonophagocytic (OPK) activities of MEDI3902 and control IgG against NCFBE-derived P. aeruginosa strain 6. The x-axis shows the logarithmic concentration (nM) of the mAb used.

[0036] Figure 5 The effect of MEDI3902 on peripheral blood neutrophil OPK against P. aeruginosa in patients with bronchiectasis and on peripheral blood neutrophil OPK against P. aeruginosa in neutrophils (from healthy controls and matched controls) is shown. Also shown is neutrophil activity after exposure to an IgG control.

[0037] Fig. 6A and Figure 6B Endogenous anti-Psl and anti-PcrV serum titers in bronchiectasis patients and matched controls are shown. Antibody titers were measured in serum samples from bronchiectasis patients divided into those with chronic P. aeruginosa airway infection (chronic PA) and those with no recent documented infection (unknown PA) as well as age-matched and sex-matched controls. Fig. 6A - Serum anti-Psl antibody titers were determined using ELISA plates coated with (left) wild-type (WT) PAO1 or (right) pslA-deficient (ΔpslA) PAO1. Sera were added in a dilution series of 1:200-1:437400 to determine endpoint titers, and PAO1 binding was detected using an anti-human HRP-conjugated antibody. Endpoint titers are those that show a difference in absorbance values ​​between two serial dilutions less than that of the blank, and if the titer is reduced in the wells coated with ΔPsl-PAO1 in the right panel, it is considered anti-Psl specific. Data points represent individuals identified with endogenous anti-Psl antibodies. Figure 6B - Anti-PcrV titer was determined using ELISA plates coated with recombinant human PcrV protein and as Fig. 6A Endpoint titers were selected in . Sera were added in a dilution series from 1:100 to 1:72900. Lines represent group medians.

[0038] Fig. 7A and Figure 7B Shown is the effect and impact of endogenous anti-Psl antibodies in bronchiectasis patients on the activity of MEDI3902. Fig. 7A-Isolated peripheral blood neutrophils were incubated with luminescent Pseudomonas aeruginosa (PAO1), either alone or with the indicated dilutions of serum. In the left panel, wild-type (WT) PAO1 was used to determine the effect on neutrophil-mediated bacterial killing, and in the right panel, Psl-deficient (ΔpslA) PAO1 was used to determine the specific contribution of anti-Psl antibodies. Figure 7B - To assess whether endogenous anti-Psl antibodies compete with MEDI3902, the OPK assay was repeated with the indicated concentrations of MEDI3902 alone or added to serum diluted 1:100 with WT PAO1 (left panel) or PAO1ΔpslA (right panel). OPK was measured 2 hours later by quantitative luminescence. The % increase in neutrophil OPK was calculated relative to the % increase in neutrophils incubated with PAO1 without added antibody. Data represent mean ± SD for MEDI3902 alone or mean of duplicate wells for serum experiments.

[0039] Fig. 8A and Figure 8B Shown is the effect of bronchiectasis patient sera containing endogenous anti-PcrV antibodies on P. aeruginosa cytotoxicity and MEDI3902 activity. Fig. 8A - Bronchiectasis patient sera containing endogenous anti-PcrV antibodies as tested by ELISA were added to A549 epithelial cells at the indicated dilutions together with a cytotoxic Pseudomonas aeruginosa strain (6077) to evaluate anti-PcrV functionality. Cell lysis was determined after 3 hours of incubation by measuring lactate dehydrogenase. Figure 8B -like Fig. 8A As indicated, MEDI3902 was used alone (n=3 separate experimental runs) or added to Fig. 8A The ability of serum samples used in the assay to eliminate the cytotoxic effects of Pseudomonas aeruginosa on epithelial cells was determined. Data represent mean ± SD of MEDI3902 alone or mean of replicate wells for serum experiments.

[0040] Fig. 9 A flow chart of a clinical trial demonstrating the efficacy of MEDI3902 in patients with bronchiectasis is provided. DETAILED DESCRIPTION

[0041] I. Definitions

[0042] The headings provided herein are not limitations of the various aspects or aspects of the disclosure, which can be obtained by reference to the specification as a whole. Therefore, the terms defined immediately below are more fully defined by reference to the entire content of the specification.

[0043] It should be noted that the term "a" or "an" entity refers to one or more of that entity; for example, "antibody" should be understood to mean one or more antibodies. Thus, the terms "a" (or "an"), "one or more" and "at least one" are used interchangeably herein.

[0044] In addition, "and / or" as used herein is considered to be a specific disclosure of each of the two specified features or components with or without the other. Therefore, the term "and / or" as used herein in phrases such as "A and / or B" 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).

[0045] As used herein, when used to modify a numerical value or numerical range, the terms "about" and "approximately" mean that deviations of up to 10% above and up to 10% below the numerical value or range are still within the intended meaning of the recited numerical value or range. It should be understood that wherever aspects are described herein using the language "about" or "approximately" numerical values ​​or ranges, other similar aspects involving specific numerical values ​​or ranges are also provided.

[0046] It should be understood that wherever aspects are described herein with the language "comprising", other similar aspects described with the terms "consisting of" and / or "consisting essentially of" are also provided. In the present disclosure, "comprises", "comprising", "containing", and "having" and the like may mean "includes", "including", and the like; "consisting essentially of" or "consists essentially of" are open ended, allowing for the presence of more than those listed, as long as the basic or novel features of those listed are not altered by the presence of more than those listed, but excluding prior art aspects.

[0047] Units, prefixes and symbols are expressed in the form accepted by the International System of Units (SI). Numerical ranges are inclusive of the values ​​defining the range.

[0048] Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxyl orientation.

[0049] Unless otherwise defined, the technical and scientific terms used herein have the same meanings as those commonly understood by those of ordinary skill in the art to which the present disclosure relates. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd edition, 2002, CRC Press; Dictionary of Cell and Molecular Biology, 3rd edition, 1999, Academic Press; and Oxford Dictionary of Biochemistry and Molecular Biology, Revised edition, 2000, Oxford University Press, provide a general dictionary of many of the terms used in the present disclosure for those of skill in the art.

[0050] 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) by at least one antigen recognition site in the variable region of an immunoglobulin molecule. The term "antibody" includes monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, bispecific antibodies, and any other immunoglobulin molecules, as long as the antibody exhibits the desired biological activity. The antibody can be any of the following five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or its subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), based on the identity of their heavy chain constant domains (referred to as α, δ, ε, γ, and μ, respectively). Different classes of antibodies have different and well-known subunit structures and three-dimensional configurations. For the structure and properties of different classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, CT, 1994, p. 71 and Chapter 6.

[0051] The term "antibody fragment" refers to a part of an antibody. An "antigen-binding fragment" of an antibody refers to a part of an antibody that binds to an antigen. An antigen-binding fragment of an antibody may include an antigen-determining region (e.g., a complementary determining region (CDR)) of an antibody. 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. An antigen-binding fragment of an antibody may be monovalent or multivalent (e.g., bivalent). An antigen-binding fragment of an antibody may be monospecific or multispecific (e.g., bispecific). An antigen-binding fragment of an antibody may be derived from any animal species, such as a rodent (e.g., a mouse, a rat or a hamster) and a human, or may be artificially produced.

[0052] An "antigen binding domain" or "antigen binding region" refers to a monovalent portion of an antibody that binds an antigen. An "antigen binding domain" may include an antigenic determining region (e.g., a complementarity determining region (CDR)) of an antibody. An antibody or antigen binding fragment thereof (including monospecific and multispecific (e.g., bispecific) antibodies or antigen binding fragments thereof) may include an antigen binding domain.

[0053] Antibody fragments (including single-chain antibodies) may include the variable region alone or in combination with all or part of the following: hinge region, CH1, CH2, and CH3 domains.

[0054] Also included are antigen-binding fragments that further comprise the variable region with any combination of the hinge region, CH1, CH2, and CH3 domains.

[0055] The antibodies or antigen-binding fragments thereof disclosed herein can be 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.

[0056] Light chains are classified as kappa or lambda (κ, λ). Each heavy chain category can be combined with a κ or λ light chain. Typically, the light chain and heavy chain are covalently bound to each other, and when the immunoglobulin is produced by a hybridoma, B cell, or genetically engineered host cell, the "tail" portions of the two heavy chains are bound to each other by covalent disulfide bonds or non-covalent bonds. In the heavy chain, the amino acid sequence extends from the N-terminus at the forked end of the Y configuration to the C-terminus at the bottom of each chain.

[0057] Both the light chain and the heavy chain are divided into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, it should be understood that the variable domains of both the light chain (VL) and the heavy chain (VH) portions determine antigen recognition and specificity. In contrast, the constant domains of the light chain (CL) and the heavy chain (CH1, CH2 or CH3) confer important biological properties, such as secretion, transplacental mobility, Fc receptor binding, complement fixation, etc. By convention, the numbering of the constant region domains increases as they become more distant 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 and CL domains include the carboxyl termini of the heavy and light chains, respectively.

[0058] As described above, the variable region allows the binding molecule to selectively recognize and specifically bind to an epitope on an antigen. That is, a subset of the VL domain and the VH domain or complementary determining region (CDR) of a binding molecule (e.g., an antibody) is combined to form a variable region that defines a three-dimensional antigen binding site. This quaternary binding molecule structure forms an antigen binding site present at each arm end of the Y. More specifically, the antigen binding site is defined by three CDRs on each VH and VL chain.

[0059] In naturally occurring antibodies, the six "complementarity determining regions" or "CDRs" present in each antigen binding domain are short, non-contiguous amino acid sequences that are specifically positioned to form an antigen binding domain when the antibody assumes its three-dimensional configuration in an aqueous environment. The remaining amino acids in the antigen binding domain (called "framework" regions) show less intermolecular variability. The framework regions primarily adopt a β-sheet conformation and form loops connecting the CDRs, and in some cases form part of the β-sheet structure. Thus, the framework regions serve to form a scaffold that provides for positioning the CDRs in the correct orientation through interchain, non-covalent interactions. The antigen binding domain formed by the positioned CDRs defines a surface that is complementary to an epitope on an immunoreactive antigen. This complementary surface promotes non-covalent binding of an antibody to its cognate epitope. For any given heavy or light chain variable region, one of ordinary skill in the art can readily identify the amino acids comprising the CDR and framework regions, respectively, since they have been 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), the entireties of which are incorporated herein by reference).

[0060] The term "Kabat numbering" and similar terms are generally recognized in the art and refer to a system in which amino acid residues in the variable region of the heavy chain and the variable region of the light chain of an antibody or its antigen-binding fragment are numbered. In some aspects, CDR can be determined according to the Kabat numbering system (see, for example, Kabat EA & Wu TT (1971), "Annual Report of the New York Academy of Sciences" (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 CDR in the antibody heavy chain molecule is usually present at amino acid positions 31 to 35, which can optionally include one or two additional amino acids after 35 (referred to as 35A and 35B in the Kabat numbering scheme) (CDR1), amino acid positions 50 to 65 (CDR2) and amino acid positions 95 to 102 (CDR3). Using the Kabat numbering system, the CDRs within an antibody light chain molecule are typically present at amino acid positions 24 to 34 (CDR1), amino acid positions 50 to 56 (CDR2), and amino acid positions 89 to 97 (CDR3). In some aspects, the CDRs of the antibodies described herein have been determined according to the Kabat numbering scheme.

[0061] Chothia refers to the position of the structural loop (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). When numbered using the Kabat numbering convention, the end of the Chothia CDR-H1 loop varies between H32 and H34, depending on the length of the loop (this is because the Kabat numbering scheme places the insertion at H35A and H35B; if both 35A and 35B are absent, the loop ends at 32; if only 35A is present, the loop ends at 33; if 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 the AbM antibody modeling software of Oxford Molecular.

[0062]

[0063]

[0064] Single-chain Fvs (scFv) molecules are known in the art and are described, for example, in U.S. Pat. No. 5,892,019. The immunoglobulin or antibody molecules encompassed by the present disclosure may 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.

[0065] "Monoclonal" antibody or its antigen binding fragment refers to a homogeneous antibody or antigen binding fragment population that participates in the highly specific recognition and binding of a single antigenic determinant or epitope. This is in contrast to polyclonal antibodies, which typically include different antibodies directed directly to different antigenic determinants. The term "monoclonal" antibody or its antigen binding fragment includes both complete monoclonal antibodies and full-length monoclonal antibodies, as well as antibody fragments (such as Fab, Fab', F(ab')2, Fv), single-chain (scFv) mutants, fusion proteins including antibody portions, and any other modified immunoglobulin molecules including antigen recognition sites. In addition, "monoclonal" antibody or its antigen binding fragment refers to such antibodies and their antigen binding fragments prepared in many ways (including but not limited to by hybridoma, phage selection, recombinant expression, and transgenic animals).

[0066] As used herein, "human" antibodies include antibodies having the amino acid sequence of a human immunoglobulin, and include antibodies isolated from a human immunoglobulin library or 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. Pat. No. 5,939,598 to Kucherlapati et al.

[0067] "Specific binding" generally means that a binding molecule (e.g., a bispecific antibody or its fragment, variant or derivative) binds to an epitope via an antigen binding domain, and the binding requires some complementarity between the antigen binding domain and the epitope. As provided herein, a binding molecule may contain one, two, three, four or more binding domains, which may be identical or different, and may bind to the same epitope, or bind to two or more different epitopes. According to this definition, when a binding molecule binds to an epitope via its antigen binding domain more easily than a random, unrelated epitope, it is considered that the binding molecule "specifically binds" to the epitope. The term "specificity" is used herein to limit the relative affinity of certain binding molecules in conjunction with certain epitopes. For example, a binding molecule "A" may be considered to have a higher specificity than a binding molecule "B" to a given epitope, or a binding molecule "A" may be considered to bind to an epitope "C" with a higher specificity than its related epitope "D" than its related epitope.

[0068] An antibody that "binds to the same epitope" as a reference antibody is one that contacts the same amino acids 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.

[0069] An antibody is said to "competitively inhibit" binding of a reference antibody to a given epitope if the antibody preferentially binds to a given epitope or an overlapping epitope such that it blocks binding of the reference antibody to that epitope to some extent. Competitive inhibition can be determined by any method known in the art, such as a competition ELISA assay. An antibody can be said to competitively inhibit 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%.

[0070] 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 should be understood that in addition to the typical antibody structure, other molecules with two binding specificities can be constructed. It should also be understood that antigen binding by bispecific antibodies can be simultaneous or sequential. Triomas and hybridomas are two examples of cell lines that can secrete bispecific antibodies. Bispecific antibodies can also be constructed by recombinant methods. ( and Heiss, Future Oncol. 6:1387-94 (2010); Mabry and Snavely, ID Rugs. 13:543-9 (2010)).

[0071] 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.

[0072] As used herein, the term "engineered antibody" refers to an antibody in which the variable domains in the heavy chain and light chain or both are changed by at least partially replacing one or more CDRs from a known specific antibody, and if necessary, by partial framework region replacement and sequence alteration. Although CDRs may be derived from antibodies of the same class or even subclass as the antibody from which the framework region is derived, it is envisioned that CDRs will be derived from antibodies of different classes and preferably from antibodies of different species. Engineered antibodies in which one or more "donor" CDRs from non-human antibodies of known specificity are transplanted into human heavy or light chain framework regions are referred to herein as "humanized antibodies". It may not be necessary to replace all CDRs with complete CDRs from donor variable regions to transfer the antigen binding capacity of one variable domain to another variable domain. On the contrary, it may only be necessary to transfer those residues necessary for the activity of the target binding site. It is well within the capabilities of those skilled in the art to obtain functional engineered or humanized antibodies by performing routine experimentation or by trial and error as explained, for example, in US Pat. Nos. 5,585,089, 5,693,761, 5,693,762, and 6,180,370.

[0073] "Isolated" polypeptides, antibodies, polynucleotides, vectors, cells or compositions are polypeptides, antibodies, polynucleotides, vectors, cells or compositions that are 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 a form found in nature. In some aspects, the isolated antibodies, polynucleotides, vectors, cells or compositions are 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.

[0074] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may include modified amino acids, and it may be interrupted by non-amino acids. These terms also encompass amino acid polymers modified naturally or by intervention; the modification, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included in the definition are, for example, polypeptides containing one or more amino acid analogs (including, for example, non-natural amino acids, etc.), as well as other modifications known in the art. It should be understood that because the polypeptides of the present disclosure are antibody-based, in some aspects, the polypeptides may exist as single chains or associated chains.

[0075] Administration "in combination with" one or more other therapeutic agents includes simultaneous (concurrent) or consecutive administration in any order.

[0076] Terms such as "treating" or "treatment" or "to treat" or "alleviating" or "to alleviate" refer to therapeutic measures that cure, slow down, alleviate the symptoms of, and / or stop the progression of a diagnosed pathological condition or disorder. Thus, those in need of treatment include those already diagnosed with or suspected of having the disorder.

[0077] The term "airway neutrophilia" refers to the accumulation of neutrophils in the lung spaces.

[0078] The term "sputum neutrophilia" refers to the presence of neutrophils in the sputum of a subject. In some aspects, the neutrophils in the sputum of a subject in need of treatment (e.g., a subject with bronchiectasis) are increased relative to the neutrophils in the sputum of a healthy control.

[0079] "Subject" or "individual" or "animal" or "patient" or "mammal" means any subject, e.g., a mammalian subject, for whom diagnosis, prognosis, or treatment is desired. Mammalian subjects include humans, livestock, farm animals, and zoo, sport, or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, bears, and the like.

[0080] II. Methods and uses of bispecific antibodies in the treatment of bronchiectasis

[0081] As demonstrated herein, bispecific antibodies that specifically bind to Pseudomonas aeruginosa Psl and PcrV can be used to treat subjects with bronchiectasis. Thus, provided herein are methods of treatment using such bispecific antibodies, uses of such bispecific antibodies in the preparation of medicaments, and bispecific antibodies for treatment.

[0082] In some aspects, provided herein is a method for treating bronchiectasis (e.g., non-cystic fibrosis bronchiectasis). The method may include administering to a subject a bispecific antibody (e.g., MEDI3902) that specifically binds to Pseudomonas aeruginosa PcrV protein and Psl exopolysaccharide. The subject may be, for example, a subject colonized by Pseudomonas aeruginosa.

[0083] In some aspects, provided herein is a method for improving the forced expiratory volume before bronchodilator 1 (FEV1) in a subject with bronchiectasis (e.g., non-cystic fibrosis bronchiectasis). The method may 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 may be, for example, a subject colonized by Pseudomonas aeruginosa.

[0084] In some aspects, provided herein is a method for reducing the Pseudomonas aeruginosa load in a subject with bronchiectasis (e.g., non-cystic fibrosis bronchiectasis). The method may 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 may be, for example, a subject colonized by Pseudomonas aeruginosa.

[0085] In some aspects, provided herein is a method for reducing the exacerbation of bronchiectasis in a subject with bronchiectasis (e.g., non-cystic fibrosis bronchiectasis). The method can, for example, reduce the exacerbation of bronchiectasis requiring hospitalization and / or reduce the 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 by Pseudomonas aeruginosa.

[0086] In some aspects, provided herein is a method for reducing the demand for intravenous antibiotics in subjects with bronchiectasis (e.g., non-cystic fibrosis bronchiectasis). The method may include administering to the subject a bispecific antibody (e.g., MEDI3902) that specifically binds to Pseudomonas aeruginosa PcrV protein and Psl exopolysaccharide. The subject may be, for example, a subject colonized by Pseudomonas aeruginosa.

[0087] In some aspects, provided herein is a method for stabilizing lung function in a subject with bronchiectasis (e.g., non-cystic fibrosis bronchiectasis). The method may include administering to the subject a bispecific antibody (e.g., MEDI3902) that specifically binds to Pseudomonas aeruginosa PcrV protein and Psl exopolysaccharide. The subject may be, for example, a subject colonized by Pseudomonas aeruginosa.

[0088] In some aspects, provided herein is a method for improving the frequency and / or intensity of cough in a subject with bronchiectasis (e.g., non-cystic fibrosis bronchiectasis). The method may include administering to the subject a bispecific antibody (e.g., MEDI3902) that specifically binds to Pseudomonas aeruginosa PcrV protein and Psl exopolysaccharide. The subject may be, for example, a subject colonized by Pseudomonas aeruginosa.

[0089] In some aspects, provided herein is a method for reducing bronchiectasis symptoms in a subject with bronchiectasis (e.g., non-cystic fibrosis bronchiectasis). The method may include administering to the subject a bispecific antibody (e.g., MEDI3902) that specifically binds to Pseudomonas aeruginosa PcrV protein and Psl exopolysaccharide. The subject may be, for example, a subject colonized by Pseudomonas aeruginosa.

[0090] In some aspects, provided herein is a method for improving the quality of life of a subject suffering from bronchiectasis (e.g., non-cystic fibrosis bronchiectasis). The method may include administering to the subject a bispecific antibody (e.g., MEDI3902) that specifically binds to Pseudomonas aeruginosa PcrV protein and Psl exopolysaccharide. The subject may be, for example, a subject colonized by Pseudomonas aeruginosa.

[0091] In some aspects, provided herein is a method for eradicating Pseudomonas aeruginosa in a subject with bronchiectasis (e.g., non-cystic fibrosis bronchiectasis). The method may include administering to the subject a bispecific antibody (e.g., MEDI3902) that specifically binds to Pseudomonas aeruginosa PcrV protein and Psl exopolysaccharide. The subject may be, for example, a subject colonized by Pseudomonas aeruginosa.

[0092] In some aspects, provided herein is a method for inducing sustained Pseudomonas aeruginosa inhibition in a subject with bronchiectasis (e.g., non-cystic fibrosis bronchiectasis). The method may 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 may be, for example, a subject colonized by Pseudomonas aeruginosa.

[0093] In some aspects, provided herein is a method for reducing the risk of bronchiectasis progression associated with Pseudomonas aeruginosa in a subject with bronchiectasis (e.g., non-cystic fibrosis bronchiectasis). The method may include administering to the subject a bispecific antibody (e.g., MEDI3902) that specifically binds to Pseudomonas aeruginosa PcrV protein and Psl exopolysaccharide. The subject may be, for example, a subject colonized by Pseudomonas aeruginosa.

[0094] Bronchiectasis suitable for treatment according to the methods and uses provided herein can be non-cystic fibrosis bronchiectasis. In some aspects, non-cystic fibrosis bronchiectasis is confirmed by chest computed tomography (CT) demonstrating that bronchiectasis affects 1 or more lobes of the subject.

[0095] Subjects suitable for treating bronchiectasis (e.g., non-cystic fibrosis bronchiectasis) according to the methods and uses provided herein can be subjects colonized by Pseudomonas aeruginosa. Subjects colonized with Pseudomonas aeruginosa can be identified, for example, using conventional sputum culture. In some aspects, subjects are colonized by Pseudomonas aeruginosa strains comprising a genome with a Psl-operon. In some aspects, subjects are colonized by Pseudomonas aeruginosa strains comprising a genome with a PcrV encoding locus.

[0096] In some aspects, the subject is chronically infected with Pseudomonas aeruginosa. As used herein, a "chronically infected" subject refers to a subject that is simultaneously or sequentially colonized with at least two (2) isolates of Pseudomonas aeruginosa while being clinically stable for more than one year.

[0097] A subject with bronchiectasis (eg, non-cystic fibrosis bronchiectasis) suitable for treatment according to the methods and uses provided herein can be a subject with airway neutrophilia and / or sputum neutrophilia.

[0098] A subject with bronchiectasis (eg, non-cystic fibrosis bronchiectasis) suitable for treatment according to the methods and uses provided herein can be a subject with a history of at least two (2) exacerbations of moderate to severe bronchiectasis requiring antibiotics per year.

[0099] A subject with bronchiectasis (eg, non-cystic fibrosis bronchiectasis) suitable for treatment according to the methods and uses provided herein can be a subject with a history of at least one (1) exacerbation requiring hospital care.

[0100] A subject with bronchiectasis (eg, non-cystic fibrosis bronchiectasis) suitable for treatment according to the methods and uses provided herein can be a subject who is chronically using nebulized antibiotics.

[0101] A subject with bronchiectasis (eg, non-cystic fibrosis bronchiectasis) suitable for treatment according to the methods and uses provided herein can be a subject with chronic obstructive pulmonary disease (COPD).

[0102] A subject having bronchiectasis (eg, non-cystic fibrosis bronchiectasis) suitable for treatment according to the methods and uses provided herein can be a human subject.

[0103] 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 aspects, bronchiectasis (e.g., non-cystic fibrosis bronchiectasis) is caused by hypogammaglobulinemia. In some aspects, bronchiectasis (e.g., non-cystic fibrosis bronchiectasis) is caused by common variable immunodeficiency. In some aspects, bronchiectasis (e.g., non-cystic fibrosis bronchiectasis) is caused by alpha-1-antitrypsin deficiency.

[0104] The methods and uses provided herein may include administering to a subject a bispecific antibody (e.g., MEDI3902) that specifically binds to Pseudomonas aeruginosa PcrV protein and Psl exopolysaccharide. Administration may be intravenous administration. Administration may be subcutaneous administration.

[0105] 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 Pseudomonas aeruginosa PcrV protein and Psl exopolysaccharide reduces Pseudomonas aeruginosa in sputum culture obtained from a subject, for example, compared to Pseudomonas aeruginosa in sputum culture obtained from a subject before administration. The reduction may 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 Pseudomonas aeruginosa PcrV protein and Psl exopolysaccharide reduces antibiotic use in a subject, for example, compared to use by the subject before administration. In some aspects of the methods and uses provided herein, administration of a bispecific antibody (eg, MEDI3902) that specifically binds to Pseudomonas aeruginosa PcrV protein and Psl exopolysaccharide eradicates Pseudomonas aeruginosa in a subject.

[0106] Method and purposes provided herein may include bispecific antibodies (e.g., MEDI3902) specifically binding Pseudomonas aeruginosa PcrV protein and Psl extracellular polysaccharides and antibiotic combinations are applied to subjects. In some aspects, methods and purposes provided herein include bispecific antibodies (e.g., MEDI3902) specifically binding Pseudomonas aeruginosa PcrV protein and Psl extracellular polysaccharides and aminoglycosides, ticarcillin, urea penicillin, ciprofloxacin, cefepime, gentamicin, amikacin, tobramycin, ceftazidime, aztreonam, cefotaxime, meropenem, polymyxin b or any combination thereof are applied to subjects.

[0107] III. Bispecific Antibodies

[0108] The methods and uses provided herein utilize bispecific antibodies that specifically bind to Pseudomonas aeruginosa Psl and PcrV. The bispecific antibodies include a Psl binding domain and a PcrV binding domain. Exemplary bispecific antibodies for the methods provided herein are disclosed in, for example, PCT Publication Nos. WO 2013 / 070615, WO 2014 / 074528, and WO 2015 / 171504, the disclosures of which are incorporated herein by reference in their entirety.

[0109] Exemplary sequences of bispecific antibodies for use in the methods provided herein are presented in Table 1.

[0110] Table 1: Bispecific antibody sequences

[0111]

[0112]

[0113] The bispecific antibodies for the methods and uses provided herein may include an antigen binding domain that specifically binds to Pseudomonas aeruginosa PcrV, and competitively inhibit the binding of an antibody comprising a heavy chain variable region having an amino acid sequence of SEQ ID NO: 13 and a light chain variable region having an amino acid sequence of SEQ ID NO: 14 to PcrV. In some aspects, the bispecific antibodies for the methods and uses provided herein include an antigen binding domain that specifically binds to Pseudomonas aeruginosa PcrV, and the epitope of the PcrV bound is the same as the epitope of the PcrV bound by the antibody comprising a heavy chain variable region having an amino acid sequence of SEQ ID NO: 13 and a light chain variable region having an amino acid sequence of SEQ ID NO: 14.

[0114] In some aspects, the bispecific antibodies for use in the methods and uses provided herein include an antigen binding domain that specifically binds to Pseudomonas aeruginosa PcrV and include (i) heavy chain CDR1, CDR2, and CDR3 having the amino acid sequence of the heavy chain CDR1, CDR2, and CDR3 sequence in SEQ ID NO:13 (e.g., Kabat-defined, AbM-defined, or Chothia-defined CDRs) and (ii) light chain CDR1, CDR2, and CDR3 having the amino acid sequence of the heavy chain CDR1, CDR2, and CDR3 sequence in SEQ ID NO:14 (e.g., Kabat-defined, AbM-defined, or Chothia-defined CDRs).

[0115] In some aspects, the bispecific antibodies for the methods and uses provided herein include an antigen binding domain that specifically binds to Pseudomonas aeruginosa PcrV, and include a heavy chain CDR1 having an amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 having an amino acid sequence of SEQ ID NO: 2, a heavy chain CDR3 having an amino acid sequence of SEQ ID NO: 3, a light chain CDR1 having an amino acid sequence of SEQ ID NO: 4, a light chain CDR2 having an amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 having an amino acid sequence of SEQ ID NO: 6. In some aspects, the bispecific antibodies for the methods and uses provided herein include an antigen binding domain that specifically binds to Pseudomonas aeruginosa PcrV, and include a heavy chain variable region having an amino acid sequence of SEQ ID NO: 13. In some aspects, the bispecific antibodies for the methods and uses provided herein include an antigen binding domain that specifically binds to Pseudomonas aeruginosa PcrV, and include a light chain variable region having an amino acid sequence of SEQ ID NO: 14. In some aspects, the bispecific antibodies for use in the methods and uses provided herein include an antigen binding domain that specifically binds to Pseudomonas aeruginosa PcrV and includes a heavy chain variable region having the amino acid sequence of SEQ ID NO:13 and a light chain variable region having the amino acid sequence of SEQ ID NO:14.

[0116] In some aspects, the bispecific antibodies for use in the methods and uses provided herein include a PcrV binding domain having a heavy chain variable region and a light chain variable region on separate polypeptides.

[0117] In some aspects, the bispecific antibodies for the methods and uses provided herein include an antigen binding domain that specifically binds to Pseudomonas aeruginosa Psl, and competitively inhibits the binding of an antibody comprising a heavy chain variable region having an amino acid sequence of SEQ ID NO: 15 and a light chain variable region having an amino acid sequence of SEQ ID NO: 16 to Psl. In some aspects, the bispecific antibodies for the methods and uses provided herein include an antigen binding domain that specifically binds to Pseudomonas aeruginosa Psl, and the epitope of Psl bound is the same as the epitope of Psl bound by an antibody comprising a heavy chain variable region having an amino acid sequence of SEQ ID NO: 15 and a light chain variable region having an amino acid sequence of SEQ ID NO: 16.

[0118] In some aspects, the bispecific antibodies for use in the methods and uses provided herein include an antigen binding domain that specifically binds to Pseudomonas aeruginosa Psl and include (i) heavy chain CDR1, CDR2, and CDR3 having the amino acid sequence of the heavy chain CDR1, CDR2, and CDR3 sequence in SEQ ID NO: 15 (e.g., Kabat-defined, AbM-defined, or Chothia-defined CDRs) and (ii) light chain CDR1, CDR2, and CDR3 having the amino acid sequence of the heavy chain CDR1, CDR2, and CDR3 sequence in SEQ ID NO: 16 (e.g., Kabat-defined, AbM-defined, or Chothia-defined CDRs).

[0119] In some aspects, the bispecific antibodies for the methods and uses provided herein include an antigen binding domain that specifically binds to Pseudomonas aeruginosa Psl, and include a heavy chain CDR1 having an amino acid sequence of SEQ ID NO: 7, a heavy chain CDR2 having an amino acid sequence of SEQ ID NO: 8, a heavy chain CDR3 having an amino acid sequence of SEQ ID NO: 9, a light chain CDR1 having an amino acid sequence of SEQ ID NO: 10, a light chain CDR2 having an amino acid sequence of SEQ ID NO: 11, and a light chain CDR3 having an amino acid sequence of SEQ ID NO: 12. In some aspects, the bispecific antibodies for the methods and uses provided herein include an antigen binding domain that specifically binds to Pseudomonas aeruginosa Psl, and include a heavy chain variable region having an amino acid sequence of SEQ ID NO: 15. In some aspects, the bispecific antibodies for the methods and uses provided herein include an antigen binding domain that specifically binds to Pseudomonas aeruginosa Psl, and include a light chain variable region having an amino acid sequence of SEQ ID NO: 16. In some aspects, the bispecific antibodies for use in the methods and uses provided herein include an antigen binding domain that specifically binds to Pseudomonas aeruginosa Psl and includes a heavy chain variable region having the amino acid sequence of SEQ ID NO: 15 and a light chain variable region having the amino acid sequence of SEQ ID NO: 16.

[0120] In some aspects, the bispecific antibody for the methods and purposes provided herein includes an antigen binding domain that specifically binds to Pseudomonas aeruginosa Psl, and includes a heavy chain variable region and a light chain variable region on the same polypeptide. In some aspects, the bispecific antibody includes a Psl binding domain that is a scFv. The scFv may include a connexon. The connexon may be, for example, a glycine-rich connexon or a glycine-serine connexon. In some aspects, the connexon includes the amino acid sequence of SEQ ID NO:18. In some aspects, the scFv is in the orientation of VH-connexon-VL. In some aspects, the scFv is in the orientation of VL-connexon-VL. In some aspects, the scFv includes the amino acid sequence of SEQ ID NO:17.

[0121] In some aspects, the bispecific antibody used in the methods and uses provided herein is an IgG antibody. The IgG antibody can be, for example, an IgG1 antibody.

[0122] In some aspects, the bispecific antibodies for the methods and uses provided herein include (i) a heavy chain of formula VH-CH1-H1-L1-S-L2-H2-CH2-CH3, wherein VH is an anti-Pseudomonas 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-Pseudomonas 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 formula VL-CL, wherein VL is an anti-Pseudomonas 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 aspects, CL is an antibody light chain kappa constant region. In some aspects, 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 aspects, the scFv comprises the amino acid sequence of SEQ ID NO: 15 and the amino acid sequence of SEQ ID NO: 16. In some aspects, the VH comprises the amino acid sequence of SEQ ID NO: 13, the VL comprises the amino acid sequence of SEQ ID NO: 14, and the scFv comprises the amino acid sequence of SEQ ID NO: 15 and the amino acid sequence of SEQ ID NO: 16. In some aspects, the scFv comprises the amino acid sequence of SEQ ID NO: 17. In some aspects, the VH comprises the amino acid sequence of SEQ ID NO: 13, the 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 aspects, CH1 comprises the amino acid sequence of SEQ ID NO: 21. In some aspects, L1 and L2 can be the same or different and can independently include (a) [GGGGS]n, wherein n is 0, 1, 2, 3, 4 or 5 (SEQ ID NO: 26), (b) [GGGG]n, wherein n is 0, 1, 2, 3, 4 or 5 (SEQ ID NO: 27), or a combination of (a) and (b). In some aspects, H1 includes the amino acid sequence EPKSC (SEQ ID NO: 22). In some aspects, L1 includes [GGGGS]n, wherein n is 2 (SEQ ID NO: 28). In some aspects, L2 includes [GGGGS]n, wherein n is 2 (SEQ ID NO: 28) or n is 4 (SEQ ID NO: 29). In some aspects, H2 includes the amino acid sequence DKTHTCPPCP (SEQ ID NO: 23). In some aspects, CH2-CH3 includes the amino acid sequence of SEQ ID NO: 25. In some aspects, CL includes the amino acid sequence of SEQ ID NO: 24.

[0123] In some aspects, the bispecific antibodies for use in the methods and uses provided herein include a polypeptide having the amino acid sequence of SEQ ID NO: 19. In some aspects, the bispecific antibodies for use in the methods and uses provided herein include a polypeptide having the amino acid sequence of SEQ ID NO: 20. In some aspects, the bispecific antibodies for use in the methods and uses provided herein include a polypeptide having the amino acid sequence of SEQ ID NO: 19 and a polypeptide having the amino acid sequence of SEQ ID NO: 20.

[0124] In some aspects, the bispecific antibodies for the methods and purposes provided herein neutralize cell poisoning. In some aspects, the bispecific antibodies for the methods and purposes provided herein target Pseudomonas aeruginosa for opsonized phagocytosis and killing (OPK). The method for assessing the neutralization of cell poisoning and / or assessing OPK is known in the art and provided herein, for example, provided in Example 3. In some aspects, the bispecific antibodies for the methods and purposes provided herein prevent cell attachment, for example, prevent Pseudomonas aeruginosa from attaching to host cells. The method for assessing the prevention of attachment is known in the art, and is provided in, for example, WO 2013 / 070615, which is incorporated herein by reference in its entirety. In some aspects, the bispecific antibodies for the methods and purposes provided herein destroy biofilm formation. In some aspects, the bispecific antibodies for the methods and purposes provided herein inhibit primary colony formation.

[0125] The anti-Pseudomonas aeruginosa Psl and PcrV bispecific antibodies can be used to prepare a medicament for treating bronchiectasis (eg, non-cystic fibrosis bronchiectasis).

[0126] IV. Pharmaceutical Compositions

[0127] The pharmaceutical composition used in the present disclosure includes anti-Pseudomonas aeruginosa Psl and PcrV bispecific antibodies and pharmaceutically acceptable carriers well known to those of ordinary skill in the art. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.

[0128] The route of administration of the anti-Pseudomonas aeruginosa Psl and PcrV bispecific antibodies can be, for example, parenteral. The term parenteral as used herein includes, for example, intravenous injection and subcutaneous administration. Therefore, a pharmaceutical composition comprising the anti-Pseudomonas aeruginosa Psl and PcrV bispecific antibodies can be formulated for intravenous administration. In some aspects, a pharmaceutical composition comprising the anti-Pseudomonas aeruginosa Psl and PcrV bispecific antibodies can be formulated for subcutaneous administration. A suitable form of administration is a solution for injection.

[0129] Pharmaceutical compositions comprising anti-Pseudomonas aeruginosa Psl and PcrV bispecific antibodies can be administered and / or formulated, for example, for the treatment of bronchiectasis (eg, non-cystic fibrosis bronchiectasis).

[0130] 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 aspects, the bispecific antibodies are formulated for administration in combination with aminoglycosides, ticarcillin, urea penicillin, ciprofloxacin, cefepime, gentamicin, amikacin, tobramycin, ceftazidime, aztreonam, cefotaxime, meropenem, polymyxin b, or any combination thereof.

[0131] Example

[0132] Materials and Methods for Examples 1 to 4

[0133] patient

[0134] Patients with bronchiectasis were enrolled from the specialist bronchiectasis clinic at Nine Wells Hospital, Dundee, UK. Inclusion criteria for patients with bronchiectasis were age >18 years, bronchiectasis with respiratory symptoms confirmed by CT scan, and ability to give informed consent. Healthy control subjects and age-matched control subjects and sex-matched control subjects were also enrolled from Nine Wells Hospital; inclusion criteria were age >18 years and ability to give informed consent, and participants were excluded if they had active infection or inflammatory disease.

[0135] Pseudomonas aeruginosa isolates from patients with bronchiectasis

[0136] As previously described, Pseudomonas aeruginosa clinical isolates from sputum of 100 bronchiectasis patients were used for whole genome sequencing (WGS) (Tabor et al., The Journal of Infectious Diseases, 2018, 218: 1983-1994-DOI: 10.1093 / infdis / jiy438). For initial culture, frozen stored patient isolates were inoculated into lysate broth in 96-well Corning PP 1.2ml sterile cluster tubes (Sigma; CLS4411) and incubated at 37°C for approximately 24 hours under orbital shaking (200rpm). The plate was then centrifuged (5000g, 15 minutes) and the supernatant was removed. The plate containing the bacterial cell pellet was frozen at -80°C until thawed for DNA extraction.

[0137] Whole genome sequencing and genetic analysis

[0138] Whole genome sequencing and genetic analysis were performed as described in DOI: 10.1093 / infdis / jiy438. In brief, DNA was purified from bacterial culture, milled via beads, and then extracted using QIAamp DNA Mini kit (QIAGEN). Mechanical shearing by Covaris, followed by preparation of sequencing libraries by NEBNext Ultra DNA library preparation kit (New England Biolabs). Sequencing was performed via NovaSeq 2x250 sequencing run (Illumina).

[0139] Sequencing data were quality trimmed by BBDuk (Bushnell B., http: / / sourceforge.net / projects / bbmap / ), de novo assembled by 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). Multilocus sequence typing (MLST) and Psl operon gene content assessment were performed using SRST2 (Inouye et al., Genome Medicine, 2014, 6:90-DOI:10.1186 / s13073-014-0090-6), with a minimum cutoff of 20-fold coverage for gene presence detection. The PcrV gene sequence was assembled de novo (Tovchigrechko et al., DOI:10.5281 / zenodo.3619527. Accessed on January 21, 2020), translated into protein sequence, and amino acid substitutions were identified using PAO1 NC_002516pcrV as a reference sequence. Core genome multiple alignments were performed using Parsnp and PAO1 with reference to NC_002516 (Treangen et al., Genome Biology, 2014, 15:524-DOI:10.1186 / s13059-014-0524-x). Phylogenetic trees were visualized and annotated using ITOL, version 3 (Letunic and Bork, Nucleic Acids Research, 2016, 44:W242-W245-DOI:10.1093 / nar / gkw290).

[0140] Endogenous anti-Psl and anti-PcrV antibody levels in serum

[0141] Endpoint antibody titers were determined by enzyme-linked immunosorbent assay (ELISA) as described (Thaden et al., The Journal of Infectious Diseases, 2016, 213:640-648 - DOI: 10.1093 / infdis / jiv436).

[0142] Human neutrophil isolation and opsonophagocytic killing (OPK) assay

[0143] Venous blood (20 ml) containing EDTA anticoagulant was used for primary neutrophil isolation within 2 hours of venipuncture. TM Peripheral blood neutrophils were isolated using the EasySep Direct Human Neutrophil Isolation Kit (StemCell, catalog number 19666) according to the manufacturer's instructions. Briefly, 50 μl of antibody selection cocktail and 50 μl of RapidSpheres were added per ml of whole blood. TM Easy50 magnets were 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 precipitated at 300g for 6 min. Neutrophils were resuspended in OPK buffer (RPMI 1640 [Gibco, catalog number 10363083] without phenol red, containing 1% BSA) at 20×106 cells / ml for later use.

[0144] Neutrophil OPK of P. aeruginosa was measured as previously described (DOI: 10.1093 / infdis / jiv436), with modifications as described herein. To test the effect of MEDI3902 on primary neutrophil OPK activity, an antibody dilution series from 40 μg / ml to 0.04 μg / ml was prepared in OPK buffer. Nonspecific activity was evaluated using an isotype control antibody with a 3-fold dilution series from 30 μg / ml to 3.33 μg / ml. To evaluate the functionality of serum anti-Psl antibodies, heat-inactivated (56°C, 30 min) serum samples were prepared with a 10-fold dilution series from 1:100 to 1:1,000,000. In all assays, OPK was measured 2 hours after the addition of P. aeruginosa.

[0145] Cytotoxicity assay

[0146] The cytotoxicity of P. aeruginosa strain 6077 and NCFBE clinical isolates in A549 epithelial cells and the effects of MEDI3902 or endogenous anti-PcrV were investigated as previously described (DOI:10.1093 / infdis / jiv436) with some modifications. To determine endogenous anti-PcrV antibody functionality, a 3-fold dilution series of heat-inactivated sera from 1:100 to 1:24,300 was utilized. Competition of endogenous anti-PcrV antibodies with MEDI3902 was assessed using sera at 1:100 and a 5-fold dilution series of MEDI3902 from 300 μg / ml to 0.096 μg / ml. CytoTox-ONE was used according to the manufacturer's instructions. TM The A549 cell death assay was performed using a 400 μg / mL PCR product.

[0147] Detection of Psl Expression in NCFBE Clinical Isolates

[0148] Psl expression in NCFBE clinical isolates was measured by indirect enzyme-linked immunosorbent assay (ELISA) as previously described (DiGiandomenico et al., Sci Transl Med, 2014, 12:262 - DOI: 10.1126 / scitranslmed.3009655) using anti-Psl mAb Cam003 (DiGiandomenico et al., J Exp Med, 2012, 209:1273-1287 - DOI: 10.1084 / jem.20120033) or MEDI3902.

[0149] Detection of PcrV expression from clinical isolates

[0150] PcrV expression was assessed by Western immunoblot analysis from whole cell lysates after induction of the type III secretion system (T3S) of P. aeruginosa in vitro as described elsewhere (Warrener et al., Antimicrobial Agents and Chemotherapy, 2014, 58:8 - DOI: 10.1128 / AAC.02643-14; DOI: 10.1093 / infdis / jiv436).

[0151] Example 1: Target Conservation of Psl and PcrV in BE-Derived Pseudomonas aeruginosa Isolates

[0152] Psl operon genes in NCFBE isolates

[0153] Of the isolates sequenced, 72 strains contained a complete complement of the psl operon genes (pslA to pslO). In contrast, 17 strains lacked the entire operon, while 11 strains lacked one or more genes ( Figure 1 Representative isolates were tested in a whole cell Psl ELISA with anti-Psl mAb Cam003 to identify which psl operon genes are essential for Psl synthesis. Isolates that were completely devoid of the operon, lacked pslA, or lacked pslF-pslO genes were negative for Psl expression ( Figure 1 ). It has been previously shown that pslM-O is not required for Psl expression (DOI:10.1093 / infdis / jiy438). Strain 79 lacks pslK but shows binding to Cam003 and MEDI3902, but not to control IgG ( Figure 2 Strain 6 is an isolate containing a fully intact psl locus and served as a positive control for this assay ( Figure 2 ). A total of 73 of the 100 isolates contained the genetic elements necessary for the expression of Psl ( Figure 1 ).

[0154] PcrV genes are highly prevalent in NCFBE P. aeruginosa isolates

[0155] The full-length pcrV gene was present in 99 of the 100 sequenced isolates, demonstrating a high prevalence in NCFBE-derived P. aeruginosa strains ( Figure 1 ). In a global surveillance study of P. aeruginosa isolates from acute infection sources, 46 PcrV variant sequences have been previously isolated using PAO1 PcrV as the amino acid sequence reference strain (DOI: 10.1093 / infdis / jiy438). PcrV amino acid sequence analysis of NCFBE clinical isolates revealed substitutions at 18 positions, corresponding to 20 different PcrV full-length protein isoforms. Although most PcrV variants from this strain collection have been previously reported (DOI: 10.1093 / infdis / jiy438), 6 new variant sequences (shown in bold in Table 2) were identified in NCFBE-derived P. aeruginosa isolates (Table 2).

[0156] Table 2

[0157] Cluster ID Cluster Count 1 Same as NC_002516 (PAO1 PcrV) 50 2 L6F:S21P:S225K 10 3 L6F:A9G:S21P:S225R 7 4 S225R 6 5 L6F:A9G:S21P:S225K 4 6 A9G:S21P 3 7 L6F:S21P:S225R 3 8 S21P:K118E:D119A:S146G:V172L:D188G:D199G:D209N:S225R 3 9 S21P 2 10 V99A 1 11 S285I 1 12 S225G 1 13 E76D 1 14 L6F:A9G:S21P 1 15 A9G:S21P:S225R:V237I 1 16 L6F:A9S:S21P:S225R 1 17 L6F:A9G:S21P:D133E:S225R 1 18 S21P:K118E:D119A:S146G:V172L:D188G:D209N:S225R 1 19 L6F:A9G:S21P:L33M:S225R 1 20 L6F:A9G:S21P:E30K:S225R 1 sum: 99

[0158] Example 2: Binding of NCFBE PcrV subtypes by the anti-PcrV MEDI3902 binding domain

[0159] Although none of the newly identified amino acid substitutions were contact points with MEDI3902 anti-PcrV, the ability of the anti-PcrV mAb V2L2MD (which contains the complementary determining regions, VH, and VL contained in MEDI3902) to bind to PcrV expressed in whole cell lysates of subtype-specific strains cultured under type 3 secretion inducing conditions was evaluated. The reactivity of the lysates against purified polyclonal IgG derived from rabbits immunized with recombinant PcrV was evaluated to assess whether potential differences in protein reactivity were due to anti-PcrV mAb binding or to in vitro PcrV expression levels. Immunoblot analysis indicated that anti-PcrV mAb binding was comparable to that of anti-PcrV polyclonal IgG against all newly identified PcrV variants that could express PcrV under in vitro conditions ( Figure 3 ). In summary, the data indicate that V2L2-MD binds to all variant PcrV sequences expressed in vitro.

[0160] Example 3: MEDI3902 maintains functional activity against NCFBE-derived P. aeruginosa isolates

[0161] ExoU is a protein delivered by the type III secretion system of P. aeruginosa and is an important cytotoxin for this pathogen. The anti-cytotoxic activity of MEDI3902 was evaluated against representative ExoU-producing P. aeruginosa NCFBE isolates (NCFBE strain 84 and strain 55), which were cytotoxic against an epithelial cell line (A549). MEDI3902 inhibited cytotoxicity of both strains, whereas control IgG had no protective activity ( Figure 4A In addition, an in vitro OPK assay was performed with a representative P. aeruginosa isolate that has a fully intact psl locus (NCFBE strain 6) and was shown to be bound by the anti-Psl mAbs Cam003 or MEDI3902. MEDI3902 and Cam003, but not control IgG, exhibited OPK activity against NCFBE isolate 6 ( Figure 4B ).

[0162] Example 4: MEDI3902 enhances the activity of peripheral blood neutrophils in patients with bronchiectasis against Pseudomonas aeruginosa Killing

[0163] Neutrophil-mediated bacterial killing has been previously reported to be impaired in bronchiectasis. The ability of neutrophils to kill P. aeruginosa in the presence of increasing concentrations of MEDI3902 or IgG control antibody was investigated and compared with neutrophils from healthy controls and age-matched controls and sex-matched controls. Killing of P. aeruginosa was similar in all three participant groups ( Figure 5). The highest concentration of MEDI3902 (200 nM) produced an average increase of 34.6% ± 8.1% (mean ± SD) in neutrophil-mediated killing of P. aeruginosa; the value was 36% ± 8.6% in healthy control neutrophils and 30.1% ± 7.6% in matched controls. At equimolar concentrations (200 nM), the addition of control IgG antibodies did not produce a change in killing in any group (1.1% ± 6.6%, -0.9% ± 3.6%, 2.1% ± 1.8%, respectively). Enhanced bacterial killing was observed with MEDI3902 at as low as the lowest concentration used (0.2 nM).

[0164] Example 5: Endogenous anti-Psl antibodies are absent in most bronchiectasis serum samples, whereas most patients Those showing detectable anti-PcrV

[0165] It has been previously reported that endogenous anti-Psl and PcrV serum IgG levels are variable in patients with acute P. aeruginosa infection and are indistinguishable from those in non-Pseudomonas infection samples. In the present study, titers were determined in sera from bronchiectasis patients with chronic P. aeruginosa airway infection or without recent documented infection, as well as from age-matched controls and sex-matched controls. Three of thirty-one (31) bronchiectasis patients had detectable anti-Psl titers; all of these participants were part of the chronic infection group, while one (1) participant in the matched control group also had measurable anti-Psl ( Fig. 6A In conclusion, the bronchiectasis patient group appeared to display higher serum anti-Pseudomonas antibody levels than the matched control group. Different levels of anti-PcrV titers were detected in all bronchiectasis samples, with the chronic infection group displaying the highest titers ( Figure 6B ).

[0166] Example 6: Endogenous anti-Psl antibody functional activity is variable in sera of bronchiectasis patients but is not associated with MEDI3902 Competition

[0167] Three patients with bronchiectasis demonstrated detectable serum anti-Psl antibody titers 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 patient sera at a 1:100 dilution resulted in increased killing in 2 of 3 samples (14.7% and 21.3% increases in serum 1 and serum 2, respectively; Fig. 7A (left panel)). Further serum dilutions showed no functional effects. The specific contribution of anti-Psl to these observed effects was determined in the same assay using Psl-deficient (ΔpslA) Pseudomonas aeruginosa, where the increased killing activity of serum 2 was abolished, while serum 1 retained its activity ( Fig. 7A(Right panel) Only 1 of the 3 samples (serum 2) showed functional anti-Psl antibodies, while the effect on neutrophil-mediated killing in the other samples may be due to other factors.

[0168] The ability of endogenous anti-Psl to compete for ligand binding sites and inhibit the effects of MEDI3902 was then investigated by spiking patient sera at 1:100 with the indicated concentrations of MEDI3902. All concentrations of MEDI3902 alone, as well as MEDI3902 with each serum sample, demonstrated increased killing of WT P. aeruginosa ( Figure 7B These effects were abolished in Psl-deficient bacteria when MEDI3902 was used alone or together with serum 2 ( Figure 7B (Right panel). The increase in killing was maintained in serum 1 and serum 3, which did not present functional anti-Psl. This data indicates that endogenous anti-Psl antibodies (including potentially functional anti-Psl antibodies) in the sera of bronchiectasis patients do not appear to interfere with or compete with MEDI3902.

[0169] Example 7: Serum from bronchiectasis patients with endogenous anti-PcrV has anti-PcrV activity against Pseudomonas aeruginosa cells The protective effect of MEDI3902 on cytotoxicity was not impaired.

[0170] Since a high proportion of bronchiectasis patient serum samples contained detectable levels of anti-PcrV antibodies, the potential functionality of endogenous anti-PcrV was tested to inhibit the effects of the cytotoxic ExoU+6077 P. aeruginosa strain on A549 epithelial cell lysis as an output. All five serum samples tested demonstrated a protective effect, with an average inhibition of LDH release and thus cell death, with 6077 P. aeruginosa being 71.9% ± 18.6% ( Fig. 8A ). Except for one of these samples, protective effects were observed up to the highest serum dilution of 1:24,300 (lysis inhibition rate of 53.2%±30.1%).

[0171] To determine whether endogenous anti-PcrV competes with MEDI3902 for binding and thus alters activity, the experiment was repeated with serum spiked with the indicated concentrations of MEDI3902 at a 1:100 dilution or with MEDI3902 alone ( Figure 8B). In the presence of patient serum, no reduction in MEDI3902 activity was observed at any concentration used, and for most serum samples, the anti-cytotoxic activity appeared to be additive when combined. In particular, for the lowest MEDI3902 concentration (0.48 nM), MEDI3902 alone produced 60.4 ± 4.5% protection, while MEDI3902 plus serum showed an average of 84.2% ± 6.4% inhibition of cell lysis. The data indicate that endogenous anti-PcrV antibodies (including potentially functional anti-PcrV antibodies) in the serum of bronchiectasis patients do not interfere with or compete with MEDI3902. In addition, the data indicate that adding MEDI3902 to serum has an additive anti-cytotoxic effect.

[0172] Example 8: MEDI3902 in patients with bronchiectasis and chronic Pseudomonas aeruginosa

[0173] A randomized, double-blind, phase 2 trial of MEDI3902 versus placebo was conducted in participants with bronchiectasis and chronic Pseudomonas aeruginosa infection. The flow chart of the trial is shown in Fig. 9 middle.

[0174] patient

[0175] Ninety (90) participants who met the following inclusion criteria were randomly assigned:

[0176] ●Age 18 to 85;

[0177] ●Clinical diagnosis of bronchiectasis;

[0178] ● Previous chest CT scan demonstrating bronchiectasis in 1 or more lobes;

[0179] ●Pseudomonas aeruginosa detected in sputum, bronchoalveolar lavage fluid, or another airway specimen at least once within 24 months prior to screening*

[0180] ●Sputum culture positive for P. aeruginosa at the screening visit and within 35 days after randomization*

[0181] * Participants whose previous samples have not tested positive for P. aeruginosa may submit two samples during the 35-day screening period, with at least 21 days between the two samples. If both of these samples are positive for P. aeruginosa, the last two inclusion criteria will be considered met and the patient may be enrolled

[0182] All participants were randomized to receive MEDI3902 1500 mg (IV, once every 4 weeks for 12 weeks), MEDI3902 500 mg (IV, once every 4 weeks for 12 weeks), or placebo (IV, once every 4 weeks for 12 weeks) in addition to standard care. The randomization was 2:1:1, MEDI3902 1500 mg:MEDI3902 500 mg:placebo.

[0183] Application

[0184] MEDI3902 (1500 mg or 500 mg) or placebo (liquid buffer in saline) was administered by intravenous infusion (total volume 250 mL) every 4 weeks for 12 weeks.

[0185] Evaluate

[0186] Participants were asked to bring spontaneous morning sputum samples to the visit. Sputum color was assessed using the Murray sputum color chart. P. aeruginosa bacterial load in sputum was assessed on days 7, 14, 28, 56, and 84.

[0187] Postbronchodilator spirometry at the visit was performed using American Thoracic Society / European Respiratory Society guidelines.

[0188] The forced expiratory volume in 1 second (FEV1), forced vital capacity (FVC), and forced expiratory flow 25% to 75% (FEF25-75) were measured.

[0189] The severity of bronchiectasis was assessed using the Bronchiectasis Severity Index and the MRC Dyspnea Score. Exacerbations were assessed using the EMBARC definition of exacerbations. (Hill AT et al. Eur Respir J 2017;49(6). doi:10.1183 / 13993003.00051-2017.)

[0190] The quality of life bronchiectasis questionnaire (QOL-B) (Quittner AL et al., Quality of Life Questionnaire-Bronchiectasis: Final psychometric analysis and determination of minimally 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(2I):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) assesses quality of life. The SGRQ has a 3-month recall period and is therefore administered at baseline and 3 months. The QOL-B and BIM questionnaires have shorter recall periods and are administered monthly.

[0191] result

[0192] Evaluation of P. aeruginosa bacterial load in sputum at Week 12 (Day 84) demonstrated the efficacy of MEDI3902 in treating bronchiectasis in patients with chronic P. aeruginosa infection.

[0193] P. aeruginosa eradication was also assessed, defined as a negative sputum culture for P. aeruginosa at the end of treatment.

[0194] Changes from baseline in the QOL-B and BIM questionnaires (Days 28, 56, 84, and 168) and SGRQ (Days 84 and 168) were evaluated to demonstrate the efficacy of MEDI3902 in improving the quality of life of patients with bronchiectasis.

[0195] The incidence of exacerbations (according to the EMBARC definition of exacerbation; Visit 1 to Day 84) was evaluated to demonstrate the effect of MEDI3902 on the time to first exacerbation.

[0196] Changes from baseline in FEV1 (Days 28, 56, and 84) were evaluated to demonstrate the effect of MEDI3902 on lung function.

[0197] It should be understood that the Detailed Description section (rather than the Summary and Abstract sections) is intended to be used to interpret the claims. The Summary and Abstract sections may set forth one or more but not all exemplary aspects of the present invention as contemplated by the inventors, and thus are not intended to limit the present invention and the appended claims in any way.

[0198] The breadth and scope of the present invention should not be limited by any of the above-described exemplary aspects, but should be defined only in accordance with the following claims and their equivalents.

Claims

1. A method for treating bronchiectasis in a subject in need thereof, the method comprising administering to the subject a bispecific antibody that specifically binds to Pseudomonas aeruginosa PcrV protein and Psl exopolysaccharide.

2. A method for improving pre-bronchodilator forced expiratory volume 1 (FEV1) in a subject with bronchiectasis, the method comprising administering to the subject a bispecific antibody that specifically binds to Pseudomonas aeruginosa PcrV protein and Psl exopolysaccharide.

3. A method for reducing the Pseudomonas aeruginosa load in a subject with bronchiectasis, the method comprising administering to the subject a bispecific antibody that specifically binds to the Pseudomonas aeruginosa PcrV protein and Psl exopolysaccharide.

4. A method for reducing exacerbation of bronchiectasis in a subject in need thereof, the method comprising administering to the subject a bispecific antibody that specifically binds to Pseudomonas aeruginosa PcrV protein and Psl exopolysaccharide.

5. The method of claim 4, wherein the administration reduces bronchiectasis exacerbations requiring hospitalization.

6. The method of claim 4 or 5, wherein the administration reduces exacerbations of bronchiectasis requiring antibiotics.

7. A method for reducing the need for intravenous antibiotics in a subject with bronchiectasis, the method comprising administering to the subject a bispecific antibody that specifically binds to Pseudomonas aeruginosa PcrV protein and Psl exopolysaccharide.

8. A method for stabilizing lung function in a subject with bronchiectasis, the method comprising administering to the subject a bispecific antibody that specifically binds to Pseudomonas aeruginosa PcrV protein and Psl exopolysaccharide.

9. The method of any one of claims 1 to 8, wherein the bronchiectasis is non-cystic fibrosis bronchiectasis.

10. The method of claim 9, wherein the non-cystic fibrosis bronchiectasis is confirmed by chest computed tomography (CT) demonstrating bronchiectasis affecting one or more lobes.

11. The method according to any one of claims 1 to 10, wherein the subject is colonized with Pseudomonas aeruginosa, optionally wherein the respiratory tract of the subject is colonized with Pseudomonas aeruginosa.

12. The method of claim 11, wherein the P. aeruginosa colonization has been detected by sputum culture.

13. The method according to any one of claims 1 to 12, wherein the subject is chronically infected with Pseudomonas aeruginosa.

14. The method of any one of claims 1 to 13, wherein the subject has airway neutrophilia.

15. The method of any one of claims 1 to 14, wherein the subject has sputum neutrophilia.

16. The method of any one of claims 1 to 15, wherein the subject has a history of at least 2 moderate to severe bronchiectasis exacerbations per year requiring antibiotics.

17. The method of any one of claims 1 to 16, wherein the subject has a history of at least 1 exacerbation requiring hospital care.

18. The method of any one of claims 1 to 17, wherein the subject is on chronic use of aerosolized antibiotics.

19. The method according to any one of claims 1 to 18, wherein the subject suffers from chronic obstructive pulmonary disease (COPD).

20. The method of any one of claims 1 to 19, wherein the bronchiectasis is caused by hypogammaglobulinemia.

21. The method of any one of claims 1 to 19, wherein the bronchiectasis is caused by common variable immunodeficiency.

22. The method of any one of claims 1 to 19, wherein the bronchiectasis is caused by alpha-1 -antitrypsin deficiency.

23. The method of any one of claims 1 to 22, wherein the administration reduces Pseudomonas aeruginosa in a sputum culture obtained from the subject, optionally wherein the reduction occurs within 12 weeks of the first administration, within 8 weeks of the first administration, or within 4 weeks of the first administration.

24. The method of any one of claims 1 to 23, wherein the administration reduces antibiotic use.

25. The method of any one of claims 1 to 24, wherein the administration eradicates Pseudomonas aeruginosa from the subject.

26. The method according to any one of claims 1 to 25, wherein the bispecific competitive inhibition comprises binding of an antibody having a heavy chain variable region having the amino acid sequence of SEQ ID NO: 13 and / or a light chain variable region having the amino acid sequence of SEQ ID NO: 14 to PcrV.

27. A method according to any one of claims 1 to 26, wherein the epitope of PcrV bound by the bispecific antibody is the same as the epitope of PcrV bound by an antibody comprising a heavy chain variable region having an amino acid sequence of SEQ ID NO: 13 and / or a light chain variable region having an amino acid sequence of SEQ ID NO:

14.

28. The method according to any one of claims 1 to 27, wherein the bispecific antibody comprises a PcrV binding domain, wherein the PcrV binding domain comprises a heavy chain CDR1 having an amino acid sequence of SEQ ID NO:1, a heavy chain CDR2 having an amino acid sequence of SEQ ID NO:2, a heavy chain CDR3 having an amino acid sequence of SEQ ID NO:3, a light chain CDR1 having an amino acid sequence of SEQ ID NO:4, a light chain CDR2 having an amino acid sequence of SEQ ID NO:5, and a light chain CDR3 having an amino acid sequence of SEQ ID NO:

6.

29. The method according to any one of claims 1 to 28, wherein the bispecific antibody comprises a PcrV binding domain, wherein the PcrV binding domain comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO: 13 and / or a light chain variable region having an amino acid sequence of SEQ ID NO:

14.

30. The method of any one of claims 1 to 29, wherein the bispecific antibody comprises a PcrV binding domain having a heavy chain variable region and a light chain variable region on separate polypeptides.

31. The method according to any one of claims 1 to 30, wherein the bispecific competitive inhibition comprises binding of an antibody having a heavy chain variable region having the amino acid sequence of SEQ ID NO: 15 and / or a light chain variable region having the amino acid sequence of SEQ ID NO: 16 to Psl.

32. The method according to any one of claims 1 to 31, wherein the epitope of Psl bound by the bispecific antibody is the same as the epitope of Psl bound by an antibody comprising a heavy chain variable region having an amino acid sequence of SEQ ID NO: 15 and / or a light chain variable region having an amino acid sequence of SEQ ID NO:

16.

33. The method of any one of claims 1 to 32, wherein the bispecific antibody comprises a Psl binding domain comprising a heavy chain CDR1 having an amino acid sequence of SEQ ID NO:7, a heavy chain CDR2 having an amino acid sequence of SEQ ID NO:8, a heavy chain CDR3 having an amino acid sequence of SEQ ID NO:9, a light chain CDR1 having an amino acid sequence of SEQ ID NO:10, a light chain CDR2 having an amino acid sequence of SEQ ID NO:11, and a light chain CDR3 having an amino acid sequence of SEQ ID NO:

12.

34. The method of any one of claims 1 to 33, wherein the bispecific antibody comprises a Psl binding domain comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 15 and / or a light chain variable region having the amino acid sequence of SEQ ID NO:

16.

35. The method of any one of claims 1 to 34, 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.

36. The method of any one of claims 1 to 35, wherein the bispecific antibody comprises a Psl binding domain that is a scFv.

37. The method of claim 36, wherein the scFv comprises a linker.

38. The method of claim 37, wherein the linker comprises the amino acid sequence of SEQ ID NO:

18.

39. The method of claim 37 or 38, wherein the scFv is in the orientation of VH-linker-VL.

40. The method of any one of claims 36 to 39, wherein the scFv comprises the amino acid sequence of SEQ ID NO:

17.

41. The method of any one of claims 1 to 40, wherein the bispecific antibody is an IgG antibody, optionally wherein the IgG antibody is an IgG1 antibody.

42. according to the method described in any one of claims 1 to 41, wherein the bispecific antibody comprises (i) a heavy chain of formula VH-CH1-H1-L1-S-L2-H2-CH2-CH3, wherein VH is an anti-Pseudomonas 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-Pseudomonas 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 formula VL-CL, wherein VL is an anti-Pseudomonas aeruginosa PcrV light chain variable domain, and CL is an antibody light chain κ constant region or an antibody light chain λ region.

43. The method of claim 42, wherein the VH comprises the amino acid sequence of SEQ ID NO: 13, the scFv comprises the amino acid sequence of SEQ ID NO: 15 and the amino acid sequence of SEQ ID NO: 16, and / or the VL comprises the amino acid sequence of SEQ ID NO:

14.

44. The method of claim 42, wherein the VH comprises the amino acid sequence of SEQ ID NO: 13, the scFv comprises the amino acid sequence of SEQ ID NO: 17, and / or the VL comprises the amino acid sequence of SEQ ID NO:

14.

45. The method of any one of claims 42 to 44, wherein the CL is an antibody light chain kappa constant region.

46. ​​The method of any one of claims 1 to 45, wherein the bispecific antibody neutralizes cell intoxication.

47. The method of any one of claims 1 to 46, wherein the bispecific antibody targets Pseudomonas aeruginosa for opsonophagocytic killing.

48. The method of any one of claims 1 to 47, wherein the bispecific antibody prevents cell attachment.

49. The method of any one of claims 1 to 48, wherein the bispecific antibody disrupts biofilm formation.

50. The method of any one of claims 1 to 49, wherein the subject is colonized with a Pseudomonas aeruginosa strain comprising a genome having a Psl-operon.

51. A method according to any one of claims 1 to 50, wherein the subject is colonized with a Pseudomonas aeruginosa strain comprising a genome having a PcrV encoding locus.

52. The method of any one of claims 1 to 51, wherein the bispecific antibody is administered intravenously.

53. The method of any one of claims 1 to 51, wherein the bispecific antibody is administered subcutaneously.

54. The method of any one of claims 1 to 53, further comprising administering an antibiotic.

55. The method of any one of claims 1 to 54, wherein the subject is a human.

56. Use of a bispecific antibody that specifically binds to Pseudomonas aeruginosa PcrV protein and Psl exopolysaccharide in the preparation of a medicament for treating bronchiectasis in a subject in need thereof, optionally wherein the treatment of bronchiectasis comprises a method according to any one of claims 1 to 55.

57. A bispecific antibody that specifically binds to Pseudomonas aeruginosa PcrV protein and Psl exopolysaccharide for use in treating bronchiectasis in a subject in need thereof, optionally wherein the treatment of bronchiectasis comprises a method according to any one of claims 1 to 55.

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