Bispecific anti-pseudomonas antibodies with modified fc regions and methods of use thereof

Abstract

The present invention relates to bispecific anti-Pseudomonas antibodies with modified Fc regions and methods of use thereof. The present disclosure relates to a bispecific antibody that specifically binds to Pseudomonas aeruginosa PcrV protein and Psl exopolysaccharide and comprises a modified Fc region. For example, such bispecific antibodies can have increased half-life and reduced aggregation in the manufacturing process without reduced anti-Pseudomonas efficacy compared to bispecific antibodies without the modified Fc region.

Description

[0001] This application is a divisional application of Chinese patent application 202480030566.0, filed on May 8, 2024, entitled "Bispecific anti-pseudomonas antibody with modified FC region and method of use thereof".

[0002] Cross-references to related applications This application claims priority to U.S. Provisional Application No. 63 / 501,036, filed May 9, 2023, which is incorporated herein by reference in its entirety.

[0003] Reference to the sequence listing submitted electronically The contents of the sequence list submitted electronically (name: PSEUD-110-WO-PCT_Seqlisting_ST26.xml; size: 58,580 bytes; and creation date: April 23, 2024) are incorporated herein by reference in their entirety. Technical Field

[0004] This disclosure relates to bispecific antibodies against Pseudomonas Psl and PcrV. Such antibodies can be used, for example, for the prevention and treatment of Pseudomonas infections. Advantageously, the bispecific antibodies against Pseudomonas Psl and PcrV exhibit reduced aggregation during manufacturing and have a clinically useful half-life. Furthermore, this disclosure provides compositions that can be used for such treatments. Background Technology

[0005] Pseudomonas aeruginosa (P. aeruginosa) is a Gram-negative opportunistic pathogen that can cause both acute and chronic infections in affected individuals (Ma et al., Journal of Bacteriology 189(22):8353-8356 (2007)). This is partly due to the bacteria's high innate resistance to clinically used antibiotics and partly due to the formation of highly antibiotic-resistant biofilms (Drenkard E., MicrobesInfect 5:1213-1219 (2003); Hancoke & Speert, Drug Resist Update 3:247-255 (2000)).

[0006] Pseudomonas aeruginosa is a common cause of hospital-acquired infections in the Western world. It is a common causative agent of bacteremia in burn patients and immunocompromised individuals (Lyczak et al., Microbes Infect 2:1051-1060 (2000)). Pseudomonas aeruginosa is also the most common cause of hospital-acquired Gram-negative pneumonia (Craven et al., SeminRespir Infect 11:32-53 (1996)), especially in mechanically ventilated patients, and is the most prevalent pathogen in the lungs of individuals with cystic fibrosis (Pier et al., ASM News 6:339-347 (1998)).

[0007] Furthermore, *Pseudomonas aeruginosa* is known to colonize the airways of patients with noncystic fibrotic bronchiectasis. Noncystic fibrotic bronchiectasis is a chronic disease characterized by abnormal and permanent dilation of the bronchi, leading to chronic cough, sputum production, and recurrent bacterial infections of the airways. Patients with bronchiectasis suffer from a high morbidity rate due to frequent exacerbations, which impair quality of life and promote antibiotic resistance, resulting in decreased lung function.

[0008] It has been reported that the extracellular polysaccharide of Pseudomonas aeruginosa is anchored to the surface of Pseudomonas aeruginosa and is considered important in promoting colonization of host tissues and in establishing / maintaining biofilm formation (Jackson, KD et al., JBacteriol 186, 4466-4475 (2004)). Its structure contains a repeating pentasaccharide rich in mannose (Byrd, MS et al., Mol Microbiol 73, 622-638 (2009)).

[0009] PcrV is a component of the type III secretory system. PcrV appears to be part of the transport mechanism of the type III secretory system that mediates the delivery of type III secretory toxins to target eukaryotic cells (Sawa T. et al., Nat. Med. 5, 392-398 (1999)). Active and passive immunization against PcrV improved acute lung injury and mortality in mice infected with cytotoxic Pseudomonas aeruginosa (Sawa et al. 2009). The primary effect of immunization against PcrV is due to blocking the transport of type III secretory toxins into eukaryotic cells.

[0010] Due to the increasing multidrug resistance, there is still a need in the field to develop improved agents for targeting Pseudomonas. Summary of the Invention

[0011] This article presents bispecific antibodies against Pseudomonas aeruginosa PcrV protein and Psl extracellular polysaccharide, which contain modifications to the Fc region that can lead to an increased half-life.

[0012] In some aspects provided herein, this disclosure relates to a bispecific antibody that specifically binds to Pseudomonas aeruginosa PcrV protein and Psl extracellular polysaccharide, wherein the antibody comprises a modified IgG Fc region containing amino acid substitutions at two or more positions relative to the wild-type IgG Fc region at positions 432 to 437 according to the Kabat EU number index; wherein (i) Positions 432 and 437 are each replaced by cysteine; (ii) Position 433 is histidine or is replaced by arginine, proline, threonine, lysine, serine, alanine, methionine or asparagine. (iii) Position 434 is asparagine or is replaced by arginine, tryptophan, histidine, phenylalanine, tyrosine, serine, methionine or threonine. (iv) Position 435 is histidine; and (v) Position 436 is tyrosine or phenylalanine, or is substituted by leucine, arginine, isoleucine, lysine, methionine, valine, histidine, serine, or threonine; and This antibody has an increased half-life compared to the corresponding antibody with the wild-type IgG Fc region.

[0013] In some respects, the modified IgG Fc region is a modified IgG1 Fc region. In other respects, the modified IgG Fc region is a modified human IgG Fc region (e.g., a modified human IgG1 Fc region).

[0014] In some respects, the bispecific antibody exhibits less aggregation in solution compared to antibodies comprising a heavy chain containing the amino acid sequence of SEQ ID NO:19 and a light chain containing the amino acid sequence of SEQ ID NO:20. In some respects, the bispecific antibody exhibits less aggregation in shake-plate overgrowth screening compared to antibodies comprising a heavy chain containing the amino acid sequence of SEQ ID NO:19 and a light chain containing the amino acid sequence of SEQ ID NO:20.

[0015] In some respects, bispecific antibodies promote opsonization and phagocytic activity of Pseudomonas aeruginosa, optionally wherein the bispecific antibody mediates in vitro opsonization and phagocytic activity of Pseudomonas aeruginosa similar to antibodies containing a heavy chain with the amino acid sequence of SEQ ID NO:19 and a light chain with the amino acid sequence of SEQ ID NO:20.

[0016] In some respects, the bispecific antibody also includes an amino acid insertion after position 437, optionally wherein the amino acid insertion is glutamic acid.

[0017] In some respects, the bispecific antibody exhibits a higher binding affinity for FcRn at pH 6.0 than its counterpart with the wild-type human IgG1 Fc region at pH 6. In other respects, the bispecific antibody exhibits a higher binding affinity for FcRn at pH 7.4 than its counterpart with the wild-type human IgG1 Fc region at pH 7.4. In some respects presented herein, the modified human IgG1 Fc region shows an increased pH-dependent binding affinity for FcRn compared to its counterpart with the wild-type human IgG1 Fc region.

[0018] In some aspects, the modified human IgG1 Fc region has amino acid substitutions at three of the following positions: 432, 433, 434, 435, 436, and 437. In some aspects, the modified human IgG1 Fc region has amino acid substitutions at four of the following positions: 432, 433, 434, 435, 436, and 437. In some aspects, the modified human IgG1 Fc region has amino acid substitutions at five of the following positions: 432, 433, 434, 435, 436, and 437. In some aspects, the modified human IgG1 Fc region has amino acid substitutions at six of the following positions: 432, 433, 434, 435, 436, and 437.

[0019] In some respects, the modified human IgG1 Fc region contains the amino acid sequence of SEQ ID NO:44 or the amino acid sequence of SEQ ID NO:33.

[0020] In some respects, bispecific antibodies are not HexaBody.

[0021] In some respects, bispecific antibodies competitively inhibit the binding of antibodies containing a heavy chain variable region (VH) with the amino acid sequence of SEQ ID NO:13 and a light chain variable region (VL) with the amino acid sequence of SEQ ID NO:14 to PcrV.

[0022] In some respects, the epitopes of PcrV bound by bispecific antibodies are the same as those of PcrV bound by antibodies containing the amino acid sequence of SEQ ID NO:13 (VH) and the amino acid sequence of SEQ ID NO:14 (VL).

[0023] In some respects, the bispecific antibody comprises an antigen-binding domain that binds to the Pseudomonas aeruginosa PcrV protein and comprises VH-CDR1 containing the amino acid sequence of SEQ ID NO:1, VH-CDR2 containing the amino acid sequence of SEQ ID NO:2, VH-CDR3 containing the amino acid sequence of SEQ ID NO:3, VL-CDR1 containing the amino acid sequence of SEQ ID NO:4, VL-CDR2 containing the amino acid sequence of SEQ ID NO:5, and VL-CDR3 containing the amino acid sequence of SEQ ID NO:6.

[0024] In some respects, the antigen-binding domain that binds to the Pseudomonas aeruginosa PcrV protein comprises a VH containing the amino acid sequence of SEQ ID NO:13 and / or a VL containing the amino acid sequence of SEQ ID NO:14.

[0025] In some respects, the antigen-binding domains that bind to Pseudomonas aeruginosa PcrV proteins contain heavy chain variable regions and light chain variable regions on separate polypeptides.

[0026] In some respects, the bispecific antibody competitively inhibits the binding of antibodies comprising VH containing the amino acid sequence of SEQ ID NO:15 and VL containing the amino acid sequence of SEQ ID NO:16 to Psl.

[0027] In some respects, the epitopes of Psl bound by bispecific antibodies are the same as those of Psl bound by antibodies containing the amino acid sequence of VH (SEQ ID NO: 15) and VL (SEQ ID NO: 16).

[0028] In some aspects provided herein, the antibody comprises an antigen-binding domain that binds to the extracellular polysaccharide of Pseudomonas aeruginosa Psl and comprises a heavy chain variable region VH-CDR1 containing the amino acid sequence of SEQ ID NO:7, VH-CDR2 containing the amino acid sequence of SEQ ID NO:8, VH-CDR3 containing the amino acid sequence of SEQ ID NO:9, a light chain variable region VL-CDR1 containing the amino acid sequence of SEQ ID NO:10, VL-CDR2 containing the amino acid sequence of SEQ ID NO:11, and VL-CDR3 containing the amino acid sequence of SEQ ID NO:12.

[0029] In some aspects provided herein, the antigen-binding domains binding to the Pseudomonas aeruginosa Psl extracellular polysaccharide include a VH containing the amino acid sequence of SEQ ID NO:15 and / or a VL containing the amino acid sequence of SEQ ID NO:16.

[0030] In some aspects, the antigen-binding domain bound to the Pseudomonas aeruginosa Psl extracellular polysaccharide comprises VH and VL on the same polypeptide. In some aspects, the antigen-binding domain bound to the Pseudomonas aeruginosa Psl extracellular polysaccharide includes a linker between VH and VL, optionally wherein the linker comprises the amino acid sequence of SEQ ID NO:18.

[0031] In some aspects, the antigen-binding domain binding to the Pseudomonas aeruginosa Psl extracellular polysaccharide comprises scFv. In some aspects, the scFv comprises a linker, optionally wherein the linker comprises the amino acid sequence of SEQ ID NO:18. In some aspects, the scFv is in a VH-linker-VL orientation. In some aspects, the scFv comprises the amino acid sequence of SEQ ID NO:17. In some aspects, the scFv is on the same polypeptide chain as the VH of the antigen-binding domain binding to the Pseudomonas aeruginosa PcrV protein. In some aspects, the scFv is the C-terminus of the VH of the antigen-binding domain binding to the Pseudomonas aeruginosa PcrV protein.

[0032] In some respects, the bispecific antibody comprises (i) a heavy chain of formula VH-CH1-H1-L1-S-L2-H2-CH2-CH3, wherein VH is a variable domain of the anti-Pseudomonas aeruginosa PcrV heavy chain; 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 a variable domain of the anti-Pseudomonas aeruginosa PcrV light chain, and CL is the antibody light chain κ constant region or the antibody light chain λ constant region.

[0033] In some aspects, CH1 contains the amino acid sequence of SEQ ID NO:21. In some aspects, H1 contains the amino acid sequence of SEQ ID NO:22. In some aspects, L1 contains the amino acid sequence of SEQ ID NO:28. In some aspects, L2 contains the amino acid sequence of SEQ ID NO:28. In some aspects, H2 contains the amino acid sequence of SEQ ID NO:23. In some aspects, CH2-CH3 contains the amino acid sequence of SEQ ID NO:30. In some aspects, CL is the κ constant region of the antibody light chain. In some aspects, CL contains the amino acid sequence of SEQ ID NO:24.

[0034] In some respects, bispecific antibodies comprise a heavy chain containing the amino acid sequence of SEQ ID NO:31 and / or a light chain containing the amino acid sequence of SEQ ID NO:20.

[0035] In some aspects provided herein, this disclosure relates to an isolated polynucleotide comprising a nucleic acid molecule encoding a heavy chain of the bispecific antibody described herein. In some aspects, the isolated polynucleotide also comprises a nucleic acid molecule encoding a light chain of the bispecific antibody described herein.

[0036] In some aspects provided herein, this disclosure relates to a vector comprising (i) a nucleic acid molecule encoding the heavy chain of a bispecific antibody, or (ii) a nucleic acid molecule encoding the heavy chain of the bispecific antibody described herein and a nucleic acid molecule encoding the light chain of the bispecific antibody described herein. In some aspects, this disclosure relates to a pair of vectors, wherein a first vector of the pair comprises a nucleic acid molecule encoding the heavy chain of the bispecific antibody described herein, and a second vector of the pair comprises a nucleic acid molecule encoding the light chain of the bispecific antibody described herein.

[0037] In some aspects provided herein, this disclosure relates to a host cell comprising (i) an isolated polynucleotide comprising a nucleic acid molecule encoding the heavy chain of the bispecific antibody described herein, (ii) a vector comprising a nucleic acid molecule encoding the heavy chain of the bispecific antibody described herein, or a nucleic acid molecule encoding both the heavy chain and a light chain of the bispecific antibody described herein, or (iii) a nucleic acid molecule encoding both the heavy chain and a light chain of the bispecific antibody described herein. In some aspects provided herein, this disclosure relates to a host cell comprising a pair of vectors, wherein a first vector of the pair of vectors comprises a nucleic acid molecule encoding the heavy chain of the bispecific antibody described herein, and a second vector of the pair of vectors comprises a nucleic acid molecule encoding the light chain of the bispecific antibody described herein.

[0038] In some aspects provided herein, this disclosure relates to a method for generating bispecific antibodies, the method comprising culturing the host cells described herein and optionally isolating the bispecific antibodies. In some aspects, this disclosure relates to bispecific antibodies generated by the methods described herein.

[0039] In some aspects provided herein, this disclosure relates to a composition comprising the bispecific antibody described herein and a pharmaceutically acceptable carrier.

[0040] In some aspects provided herein, this disclosure relates to a method of treating or preventing Pseudomonas infection in a subject of need, the method comprising administering to the subject a bispecific antibody described herein or a composition comprising a bispecific antibody described herein. In some aspects provided herein, this disclosure relates to the use of a bispecific antibody described herein or a composition comprising a bispecific antibody described herein in the preparation of a medicament for use in treating or preventing Pseudomonas infection in a subject of need. In some aspects provided herein, this disclosure relates to a bispecific antibody described herein or a composition comprising a bispecific antibody described herein for use in treating or preventing Pseudomonas infection in a subject of need.

[0041] In some respects, an infection is a lung infection, a respiratory infection, pneumonia, bacteremia, a bone infection, a joint infection, a skin infection, a burn infection, a wound infection, or any combination thereof.

[0042] In some aspects provided herein, this disclosure relates to a method of treating bronchiectasis in a subject of need, the method comprising administering the bispecific antibody described herein or a composition comprising the bispecific antibody described herein. In some aspects provided herein, this disclosure relates to the use of the bispecific antibody described herein or a composition comprising the bispecific antibody described herein in the preparation of a medicament for use in treating bronchiectasis in a subject of need. In some aspects provided herein, this disclosure relates to the bispecific antibody described herein or a composition comprising the bispecific antibody described herein for use in treating bronchiectasis in a subject of need.

[0043] In some aspects provided herein, this disclosure relates to a method for improving forced expiratory volume 1 (FEV1) before bronchodilator administration in a subject with bronchiectasis, the method comprising administering the bispecific antibody described herein or a composition comprising the bispecific antibody described herein. In some aspects provided herein, this disclosure relates to the use of the bispecific antibody described herein or a composition comprising the bispecific antibody described herein in the preparation of a medicament for use in improving forced expiratory volume 1 (FEV1) before bronchodilator administration in a subject with bronchiectasis. In some aspects provided herein, this disclosure relates to the bispecific antibody described herein or a composition comprising the bispecific antibody described herein for use in improving forced expiratory volume 1 (FEV1) before bronchodilator administration in a subject with bronchiectasis.

[0044] In some aspects provided herein, this disclosure relates to a method for reducing the Pseudomonas aeruginosa load in a subject suffering from bronchiectasis, the method comprising administering to the subject a bispecific antibody described herein or a composition comprising a bispecific antibody described herein. In some aspects provided herein, this disclosure relates to the use of a bispecific antibody described herein or a composition comprising a bispecific antibody described herein in the preparation of a medicament for use in reducing the Pseudomonas aeruginosa load in a subject suffering from bronchiectasis. In some aspects provided herein, this disclosure relates to a bispecific antibody described herein or a composition comprising a bispecific antibody described herein for use in reducing the Pseudomonas aeruginosa load in a subject suffering from bronchiectasis.

[0045] In some aspects provided herein, this disclosure relates to a method for reducing bronchiectasis exacerbation in a subject of need, the method comprising administering to the subject a bispecific antibody described herein or a composition comprising a bispecific antibody described herein. In some aspects provided herein, this disclosure relates to the use of a bispecific antibody described herein or a composition comprising a bispecific antibody described herein in the preparation of a medicament for use in reducing bronchiectasis exacerbation in a subject of need. In some aspects provided herein, this disclosure relates to a bispecific antibody described herein or a composition comprising a bispecific antibody described herein for use in reducing bronchiectasis exacerbation in a subject of need.

[0046] In some aspects provided herein, this disclosure relates to a method for reducing the need for intravenous antibiotics in a subject suffering from bronchiectasis, the method comprising administering to the subject a bispecific antibody described herein or a composition comprising a bispecific antibody described herein. In some aspects provided herein, this disclosure relates to the use of a bispecific antibody described herein or a composition comprising a bispecific antibody described herein in the preparation of a medicament for use in reducing the need for intravenous antibiotics in a subject suffering from bronchiectasis. In some aspects provided herein, this disclosure relates to a bispecific antibody described herein or a composition comprising a bispecific antibody described herein for use in reducing the need for intravenous antibiotics in a subject suffering from bronchiectasis.

[0047] In some aspects provided herein, this disclosure relates to a method for stabilizing lung function in a subject suffering from bronchiectasis, the method comprising administering to the subject a bispecific antibody described herein or a composition comprising a bispecific antibody described herein. In some aspects provided herein, this disclosure relates to the use of a bispecific antibody described herein or a composition comprising a bispecific antibody described herein in the preparation of a medicament for use in stabilizing lung function in a subject suffering from bronchiectasis. In some aspects provided herein, this disclosure relates to a bispecific antibody described herein or a composition comprising a bispecific antibody described herein for use in stabilizing lung function in a subject suffering from bronchiectasis.

[0048] In some respects, bronchiectasis is noncystic fibrotic bronchiectasis.

[0049] In some respects, the method or use, or the antibody or composition used, may also include the administration of antibiotics.

[0050] In some respects, the subjects were colonized by Pseudomonas aeruginosa, optionally in the respiratory tract of the subjects. Attached Figure Description

[0051] Figure 1A The opsonization phagocytosis (OPK) activity of AZD0292 and gremubamab (MEDI3902) in an opsonization phagocytosis assay is shown. Data indicate that AZD0292 exhibits opsonization phagocytosis activity comparable to that of gremubamab. (See Example 1.) Figure 1B The anticytotoxic killing activity of AZD0292 and glimepiride (MEDI3902) in an anticytotoxicity assay is shown. Data indicate that AZD0292 exhibits anticytotoxic activity comparable to glimepiride. (See Example 1.) Figure 2 The opsonization activities of AZD0292, garibumab (MEDI3902), fucosylated garibumab (garibumab-AFuc), garibumab-AFuc (garibumab-AFuc-YTE) containing a YTE half-life extension mutation in its Fc region, and the negative control IgG mAb are presented. The data indicate that AZD0292 containing the N3Y half-life extension mutation is more active in the opsonization assay compared to the garibumab YTE half-life extension derivative.

[0052] Figure 3This study demonstrated increased serum exposure to AZD0292 in mice compared to glimepiride following intravenous (IV) administration of 10 mg / kg. The study was conducted in Tg32 human FcRn transgenic mice (n=4 at each time point). (See Example 2.) Figure 4A The opsonization and phagocytic activity of AZD0292 and glimepiride were shown compared to the control group when exposed to temperature variations (4°C and 45°C) and light. No differences were observed between AZD0292 and glimepiride under these stress conditions. (See Example 3.) Figure 4B The anticytotoxic activity of AZD0292 and glimepiride was shown compared to the control group when exposed to temperature variations (4°C and 45°C) and light. No differences were observed between AZD0292 and glimepiride under these stress conditions. (See Example 3.) Figure 5A The percentage of monomers in the AZD0292 (slanted column) and MEDI3902 (blank column) compositions is shown. (See Example 4.) Figure 5B The percentage of aggregates in the AZD0292 (slanted column) and MEDI3902 (blank column) compositions is shown. (See Example 4.) Figure 6 The aggregation percentages of AZD0292 and MEDI3902 for each test clone are shown. (See Example 4.) The Y-axis marked "AH" and the X-axis marked "1-12" indicate the grid positions in the 96-well plate, each position representing a different expression clone.

[0053] Figure 7 A scatter plot matrix showing the correlation between the percentage of high molecular weight substances, the average concentration of PhyTip protein A purified samples, and the titer from the fed-batch 96-well plate bioreactor on the last day is presented. (See Example 4.) Figures 8A-8B The results of the Anderson-Darling and Shapiro-Wilk tests for AZD0292 are shown. Figure 8A ) and MEDI3902 ( Figure 8B The results of the normality test for the aggregates. (See Example 4.) Figure 8C The results of a t-test comparing the average percentage of aggregation between clones expressing MEDI3902 and AZD0292 are shown. (See Example 4.) Figures 9A-9B The percentage of aggregation is shown. Figure 9A ) and the titer at the end of the culture ( Figure 9BAnd associated with each clone expressing AZD0292 (slashed bar) and MEDI3902 (blank bar). Clones are plotted in ascending order from left to right based on the aggregation percentage value. (See Example 4.) Figure 10 The percentage of aggregates stored at 40°C for several months is shown. Data points for MEDI3902 are shown as squares (■) and for AZD0292 as triangles (▲). (See Example 4.) Detailed Implementation

[0054] I. Definition The headings provided herein are not intended to limit any aspect or number of aspects of this disclosure, which can be obtained by referring to the specification as a whole. Therefore, the terms defined immediately below are more fully defined by reference to the entire specification.

[0055] It should be noted that the terms “a” or “an” entity refer to one or more of that entity; for example, “antibody” should be understood to mean one or more antibodies. Therefore, the terms “a” (or “an”), “one or more” and “at least one” are used interchangeably in this document.

[0056] Furthermore, the term “and / or” as used herein is considered to refer to each of two specified features or components, whether or not they are specifically disclosed with 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 cover each of the following: 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).

[0057] As used herein, when used to modify numerical values ​​or ranges, the terms “about” and “approximately” mean deviations of up to 10% above and below the stated value or range, respectively, still within the intended meaning of the enumerated value or range, and include the exact number modified by these terms. It should be understood that wherever the language “about” or “approximately” is used to describe a numerical value or range throughout this document, other similar aspects relating to specific numerical values ​​or ranges are also provided.

[0058] It should be understood that wherever the term "comprises" is used to describe an aspect herein, other similar aspects described by the terms "composed of" and / or "substantially composed of" are also provided. In this disclosure, "comprises," "comprising," "containing," and "having" may mean "includes," "including," etc.; "substantially composed of" or "substantially composed of" is open-ended and allows for the existence of more than those listed, provided that the essential or novel features of the listed ones are not altered by the existence of more than those listed, but excludes prior art aspects.

[0059] Units, prefixes, and symbols are represented in their internationally recognized (SI) form. Numerical ranges include the values ​​that define that range.

[0060] Unless otherwise stated, the amino acid sequence is written from left to right with the amino group to the carboxyl group orientation.

[0061] Unless otherwise defined, the technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd edition, 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd edition, 1999, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, revised edition, 2000, Oxford University Press provide a general dictionary for those skilled in the art of the use of many of the terms used in this disclosure.

[0062] As used herein, the terms “antibody” and “immunoglobulin” are used interchangeably and refer to an antibody molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination of the foregoing (e.g., glycoprotein), through at least one antigen recognition site within the variable region of an immunoglobulin molecule. The term “antibody” includes monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, bispecific antibodies, and any other immunoglobulin molecule that exhibits the desired biological activity. Antibodies can be any of the following five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their 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 conformations. For the structure and properties of different classes of antibodies, see, for example, Basic and Clinical Immunology, 8th ed., Daniel P. Stites, Abba I. Terr and Tristram G. Parslow, Appleton & Lange, Norwalk, CT, 1994, p. 71 and Chapter 6.

[0063] The term "antibody fragment" refers to a portion of an antibody. An "antigen-binding fragment" of an antibody refers to a portion of an antibody that binds to an antigen. An antigen-binding fragment of an antibody may contain an antigen-determining region (e.g., a complementarity-determining region (CDR)). Examples of antigen-binding fragments of antibodies 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 polyvalent (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 rodents (e.g., mice, rats, or hamsters) and humans, or may be artificially generated.

[0064] An "antigen-binding domain" or "antigen-binding region" refers to the monovalent portion of an antibody that binds to an antigen. An "antigen-binding domain" may include the antigen-determining region (e.g., a complementarity-determining region (CDR)) of the antibody. Antibodies or their antigen-binding fragments (including monospecific and multispecific (e.g., bispecific) antibodies or their antigen-binding fragments) may contain antigen-binding domains.

[0065] As used herein, the term "Fc region" (sometimes referred to as "Fc" or "Fc domain") refers to the portion of an IgG molecule associated with the crystallizable fragment obtained by digestion of the IgG molecule with papain. The Fc region consists of the C-terminal half of the two heavy chains of the IgG molecule linked by disulfide bonds. It is not antigen-binding active but contains a carbohydrate portion and binding sites for complement and Fc receptors (including FcRn receptors) (see below). The Fc region contains the entire second constant domain CH2 (residues 231-340 of human IgG1, according to the Kabat numbering system) and the third constant domain CH3 (residues 341-447). The amino acid residues of the IgG constant and variable domains mentioned herein are numbered according to the EU numbering index of Kabat et al. (Sequences of Proteins of Immunological Interest, 5th edition, 1991 NIH Publication No. 91-3242, which is incorporated herein by reference in its entirety) and include corresponding residues in other IgG constant domains identified by sequence alignment.

[0066] As used herein, the terms “hinge-Fc region,” “Fc-hinge region,” “hinge-Fc domain,” or “Fc-hinge domain” are used interchangeably and refer to a region of the IgG molecule consisting of the Fc region (residues 231-447) and the hinge region (residues 216-230) extending from the N-terminus of the Fc region.

[0067] Antibody fragments (including single-chain antibodies) may include a single variable region or a combination of variable regions with all or some of the following: hinge region, CH1, CH2 and CH3 domains.

[0068] It also includes antigen-binding fragments that contain any combination of variable regions and hinge regions, CH1, CH2 and CH3 domains.

[0069] The antibodies or antigen-binding fragments disclosed herein may be derived from any animal source, including birds and mammals. Antibodies may be human, mouse, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies.

[0070] Light chains are classified as kappa or lambda(λ). Each heavy chain class can bind to either a κ or λ light chain. Typically, light and heavy chains are covalently bonded to each other, and when immunoglobulins are generated by hybridomas, B cells, or genetically engineered host cells, the "tail" portions of the two heavy chains are linked together via covalent disulfide bonds or non-covalent bonds. In the heavy chain, the amino acid sequence extends from the N-terminus of the Y-configuration branch to the C-terminus at the bottom of each chain.

[0071] Both the light and heavy chains are divided into structurally and functionally homologous regions. The terms "constant" and "variable" are used functionally. The term "constant domain" refers to a portion of the immunoglobulin molecule that has a more conserved amino acid sequence compared to another portion of the immunoglobulin (the variable domain containing the antigen-binding site). The heavy chain constant domain contains CH1, CH2, and CH3 domains, while the light chain constant domain contains the CL domain. The variable domains of both the light chain (VL) and heavy chain (VH) portions determine antigen recognition and specificity. Conversely, the constant domains of the light chain (CL) and heavy chain (CH1, CH2, or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, etc. By convention, the numbering of constant domains increases as they become more distant from the antibody's antigen-binding site or N-terminus. The N-terminal portion is the variable region, and the C-terminal portion is the constant region; the CH3 and CL domains comprise the carboxyl termini of the heavy and light chains, respectively.

[0072] As described above, the variable region allows binding molecules to selectively recognize and specifically bind to epitopes on antigens. That is, subsets of the VL and VH domains, or complementarity-determining regions (CDRs), of a binding molecule (e.g., an antibody) are combined to form a variable region defining a three-dimensional antigen-binding site. This quaternary binding molecular structure forms an antigen-binding site present at the end of each arm of the Y-chain. More specifically, the antigen-binding site is defined by three CDRs on each VH and VL chain.

[0073] In naturally occurring antibodies, the six complementarity-determining regions (CDRs) present in each antigen-binding domain are short, discontinuous amino acid sequences that are specifically localized to form the antigen-binding domain when the antibody assumes its three-dimensional conformation in an aqueous environment. The remaining amino acids in the antigen-binding domain (called "framework" regions) exhibit less intermolecular variability. Framework regions primarily adopt a β-sheet conformation and form linked loops with the CDRs, and in some cases, form part of the β-sheet structure. Thus, the framework regions act as a scaffold that provides the correct orientation for the CDRs through interchain, non-covalent interactions. The antigen-binding domain formed by the localized CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface facilitates the non-covalent binding of the antibody to its homologous epitope. For any given heavy or light chain variable region, those skilled in the art can readily identify the amino acids comprising the CDR and framework regions, respectively, as they have been precisely defined (see, “Sequences of Proteins of Immunological Interest,” Kabat, E. et al., US Department of Health and Human Services, (1983); and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987), which are incorporated herein by reference in their entirety).

[0074] The terms “Kabat numbering,” “Kabat EU numbering index,” and similar terms are recognized in the art and refer to a system for numbering amino acid residues in the variable regions of the heavy and light chains of antibodies or their antigen-binding fragments. In some respects, CDRs can be determined according to the Kabat numbering system (see, for example, Kabat EA & Wu TT (1971) AnnNY Acad Sci 190: 382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, 5th ed., US Department of Health and Human Services, NIH Publication No. 91-3242). Using the Kabat numbering system, CDRs within the antibody heavy chain molecule are typically located at amino acid positions 31 to 35, which may optionally include one or two additional amino acids following 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 the antibody light chain molecule are typically located at amino acid positions 24 to 34 (CDR1), 50 to 56 (CDR2), and 89 to 97 (CDR3). In some respects, the CDRs of the antibodies described herein have been determined according to the Kabat numbering scheme.

[0075] Chothia refers to the location of the structural loop (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). When 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 neither 35A nor 35B is present, 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 region represents a compromise between the Kabat CDR and the Chothia structural loop and is used by Oxford Molecular Diagnostics' AbM antibody modeling software.

[0076] Single-chain Fvs (scFv) molecules are known in the art and described, for example, in U.S. Patent 5,892,019. The immunoglobulin or antibody molecules included in this disclosure can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules.

[0077] "Monoclonal" antibodies or their antigen-binding fragments refer to a homogeneous group of antibodies or antigen-binding fragments that participate in the highly specific recognition and binding of a single antigenic determinant or epitope. This contrasts with polyclonal antibodies, which typically consist of different antibodies targeting different antigenic determinants. The term "monoclonal" antibody or its antigen-binding fragment encompasses 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. Furthermore, "monoclonal" antibodies or their antigen-binding fragments refer to such antibodies and their antigen-binding fragments prepared in many ways, including but not limited to hybridoma, phage selection, recombinant expression, and transgenic animals.

[0078] As used herein, "human" antibodies include antibodies having the amino acid sequence of human immunoglobulins, and include antibodies isolated from human immunoglobulin libraries or from animals that are genetically modified with one or more human immunoglobulins and do not express endogenous immunoglobulins, as described below and, for example, in U.S. Patent No. 5,939,598 to Kucherlapati et al.

[0079] "Specific binding" generally means that a binding molecule (e.g., a bispecific antibody or a fragment, variant, or derivative thereof) binds to an epitope via an antigen-binding domain, and that this binding requires some complementarity between the antigen-binding domain and the epitope. Binding molecules, as described herein, may contain one, two, three, four, or more binding domains, which may be the same or different, and may bind the same epitope or two or more different epitopes. By this definition, a binding molecule is considered to "specifically bind" an epitope when it is easier for it to bind to an epitope via its antigen-binding domain than to bind to a random, unrelated epitope. The term "specific" is used herein to define the relative affinity of certain binding molecules for certain epitopes. For example, binding molecule "A" may be considered to have higher specificity for a given epitope than binding molecule "B," or binding molecule "A" may be considered to bind epitope "C" with higher specificity than it binds to related epitope "D."

[0080] An antibody that "binds to the same epitope" as the reference antibody is one that contacts the same amino acid and / or sugar residues as the reference antibody. The ability of an antibody to bind to the same epitope as the reference antibody can be determined using peptide scanning mutagenesis or high-throughput alanine scanning mutagenesis.

[0081] If an antibody preferentially binds to a given epitope or overlapping epitope, thereby partially blocking the binding of a reference antibody to that epitope, the antibody is considered to "competitively inhibit" the binding of the reference antibody to that epitope. Competitive inhibition can be determined by any method known in the art, such as a competitive ELISA assay. It can be considered that the antibody competitively inhibits the binding of the reference antibody to the given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

[0082] As used herein, the term "bispecific antibody" refers to an antibody that has binding domains within a single antibody molecule that are specific for two different antigens or epitopes. It should be understood that other molecules with two binding specificities can be constructed in addition to typical antibody structures. It should also be understood that antigen binding via bispecific antibodies can be simultaneous or sequential. Trisomy 11 and heterozygous hybridomas are two examples of cell lines that secrete bispecific antibodies. Bispecific antibodies can also be constructed via recombinant methods. (Ströhlein and Heiss, Future Oncol. 6:1387-94 (2010); Mabry and Snavely, IDrugs. 13:543-9 (2010)).

[0083] As used herein, the term “MEDI3902” or “gremubamab” refers to a bispecific antibody having a heavy chain containing the amino acid sequence of SEQ ID NO:19 and a light chain containing the amino acid sequence of SEQ ID NO:20. MEDI3902 is also known as gremubamab.

[0084] As used herein, the term "AZD0292" refers to a bispecific antibody having a heavy chain containing the amino acid sequence of SEQ ID NO:31 and a light chain containing the amino acid sequence of SEQ ID NO:20.

[0085] As used herein, the term "FcRn receptor" or "FcRn" refers to the Fc receptor ("n" indicating newborn) known to be involved in the transfer of maternal IgG from the human or primate placenta or yolk sac (rabbit) to the fetus and from colostrum to the newborn via the small intestine. FcRn is also known to participate in maintaining constant serum IgG levels by binding IgG molecules and recycling them into the serum. The binding of FcRn to naturally occurring IgG1, IgG2, and IgG4 molecules is strictly pH-dependent, with optimal binding at pH 6. IgG3 has a known variation at position 435 (i.e., human IgG has R435 instead of H435 found in human IgG1, IgG2, and IgG4), which may result in reduced binding at pH 6. FcRn comprises heterodimers of two polypeptides with molecular weights of approximately 50 kDa and 15 kDa, respectively. The extracellular domain of the 50 kDa peptide is associated with the major histocompatibility complex (MHC) class I α chain, and the 15 kDa peptide shows non-polymorphic β2-microglobulin (β2-m). Besides the placenta and neonatal intestine, FcRn is expressed in various tissues across species and in various types of endothelial cell lines. It is also expressed in adult human vascular endothelium, muscular vascular system, and hepatic sinusoids, suggesting that endothelial cells may be the primary cause of maintaining serum IgG levels in humans and mice. FcRn receptors include, for example, human and mouse FcRn proteins and their homologs with FcRn activity.

[0086] As used in this paper, the term “FcRn-binding fragment” of the IgG constant domain refers to a fragment of the IgG constant domain that binds to the FcRn receptor. The FcRn-binding fragment of the IgG constant domain may include an Fc region or a hinge Fc region; therefore, it may include the portion of the heavy chain CH2-CH3 region or the hinge-CH2-CH3 region involved in FcRn binding (see Roopenian et al., Nature Rev. Immunol. 7:715-725 (2007)).

[0087] As used in this article, "KD" (sometimes also called Kd, K) D or K d KD is the equilibrium dissociation constant of the binding interaction between two molecules (such as IgG and FcRn). KD can be determined by the observed association (k on ) and dissociation (k off The rate constant is calculated such that KD equals k. off / k on The ratio.

[0088] As used herein, the term "in vivo half-life" refers to the biological half-life of a particular type of IgG molecule or a fragment containing an FcRn binding site in circulation in a given animal, and is expressed as the time required for half the amount administered to the animal to be cleared from circulation and / or other tissues of the animal. When the clearance curve for a given IgG is constructed as a function of time, the curve is typically biphasic, consisting of a rapid α phase representing the equilibrium between the intravascular and extravascular spaces of the injected IgG molecule and determined in part by the size of the molecule, and a longer β phase representing the catabolic metabolism of the IgG molecule in the intravascular space. The term "in vivo half-life" actually corresponds to the half-life of the IgG molecule in the β phase.

[0089] As used herein, the term "engineered antibody" refers to an antibody in which a variable domain in the heavy and light chains, or both, is altered by at least partially replacing one or more CDRs from an antibody of known specificity, and, if necessary, by partial frame region substitution and sequence changes. While CDRs may originate from antibodies of the same class or even subclass from which frame regions are derived, it is envisioned that CDRs will originate from antibodies of different classes and preferably from antibodies of different species. Engineered antibodies in which one or more "donor" CDRs from a non-human antibody of known specificity are transplanted into a human heavy or light chain frame region are referred to herein as "humanized antibodies." It may not be necessary to replace all CDRs with intact CDRs from the donor variable region to transfer the antigen-binding capacity of one variable domain to another. Instead, it may only be necessary to transfer those residues necessary to maintain the activity of the target binding site. According to the interpretations described in, for example, U.S. Patents 5,585,089, 5,693,761, 5,693,762, and 6,180,370, obtaining functional engineered or humanized antibodies through routine experiments or through trial and error is entirely within the capabilities of those skilled in the art.

[0090] "Isolated" peptides, antibodies, polynucleotides, carriers, cells, or compositions are peptides, antibodies, polynucleotides, carriers, cells, or compositions in a form not found in nature. Isolated peptides, antibodies, polynucleotides, carriers, cells, or compositions include those that have been purified to the point that they no longer exist in a form found in nature. In some respects, isolated antibodies, polynucleotides, carriers, cells, or compositions are substantially pure. As used herein, "substantially pure" means material that is at least 50% pure (i.e., free from contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0091] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acids of any length. This polymer may be linear or branched, may include modified amino acids, and may be interrupted by non-amino acid components. These terms also cover polymers of naturally modified or intervened amino acids; such modifications include, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeled component. This definition also includes, for example, polypeptides containing one or more amino acid analogs (including, for example, non-natural amino acids) and other modifications known in the art. It should be understood that because the polypeptides of this disclosure are antibody-based, in some respects, the polypeptide may exist as a single chain or an associated chain.

[0092] Administering in combination with one or more other therapeutic agents (e.g., antibiotics) includes simultaneous (concurrent) or sequential administration in any order.

[0093] Terms such as “treating,” “treatment,” “to treat,” “alleviating,” or “to alleviate” refer to therapeutic measures that cure, slow down, reduce, alleviate the symptoms of a diagnosed pathological condition or symptom, and / or stop the progression of a diagnosed pathological condition or symptom. Therefore, those requiring treatment include those who have been diagnosed with or are suspected of having the condition.

[0094] The term "airway neutrophilia" refers to the accumulation of neutrophils in the lung space.

[0095] The term "sputum neutrophilia" refers to the presence of neutrophils in the sputum of a subject. In some respects, the number of neutrophils in the sputum of subjects requiring treatment (e.g., subjects with bronchiectasis) is increased compared to the number of neutrophils in the sputum of healthy controls.

[0096] The terms "subject," "individual," "animal," "patient," or "mammal" refer to any subject requiring diagnosis, prognosis, or treatment, such as a mammalian subject. Mammal subjects include humans, livestock, farm and zoo animals, sporting animals, or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, dairy cows, bears, etc.

[0097] II. Bispecific anti-pseudomonas antibodies with modified Fc regions This article presents bispecific antibodies that specifically bind to Pseudomonas aeruginosa Psl and PcrV and contain a modified Fc region. As presented herein, it was surprisingly found that modification of the Fc region of such bispecific antibodies can lead to an increased half-life, but also to a reduction in manufacturing-related aggregation without diminishing the efficacy against Pseudomonas aeruginosa.

[0098] Exemplary sequences that may be present in bispecific antibodies that specifically bind to Pseudomonas aeruginosa Psl and PcrV are presented in Table 1, defined according to Kabat nomenclature / numbering.

[0099] Table 1: Bispecific antibody sequences a. An antigen-binding domain of a bispecific anti-pseudomonas antibody with a modified Fc region. This article presents a bispecific antibody that specifically binds to the Pseudomonas aeruginosa Psl extracellular polysaccharide (Psl) and the type 3 secreted protein PcrV. Therefore, in some respects, the bispecific antibody comprises both a Psl-binding domain and a PcrV-binding domain.

[0100] The bispecific antibody provided herein may contain an antigen-binding domain that specifically binds to *Pseudomonas aeruginosa* PcrV and competitively inhibits the binding of an antibody containing a heavy chain variable region containing the amino acid sequence of SEQ ID NO:13 and a light chain variable region containing the amino acid sequence of SEQ ID NO:14 to PcrV. In some aspects, the bispecific antibody provided herein contains an antigen-binding domain that specifically binds to *Pseudomonas aeruginosa* PcrV, and the epitope of the bound PcrV is the same as the epitope of the PcrV bound by the antibody containing the heavy chain variable region containing the amino acid sequence of SEQ ID NO:13 and the light chain variable region containing the amino acid sequence of SEQ ID NO:14.

[0101] In some respects, the bispecific antibody provided herein comprises an antigen-binding domain that specifically binds to Pseudomonas aeruginosa PcrV and comprises (i) heavy chain CDR1, CDR2, and CDR3 containing the amino acid sequences of the heavy chain CDR1, CDR2, and CDR3 sequences in SEQ ID NO:13 (e.g., CDRs defined by Kabat, AbM, or Chothia) and (ii) light chain CDR1, CDR2, and CDR3 containing the amino acid sequences of the heavy chain CDR1, CDR2, and CDR3 sequences in SEQ ID NO:14 (e.g., CDRs defined by Kabat, AbM, or Chothia).

[0102] In some aspects, the bispecific antibody provided herein comprises an antigen-binding domain that specifically binds to *Pseudomonas aeruginosa* PcrV, and comprises a heavy chain CDR1 containing the amino acid sequence of SEQ ID NO:1, a heavy chain CDR2 containing the amino acid sequence of SEQ ID NO:2, a heavy chain CDR3 containing the amino acid sequence of SEQ ID NO:3, a light chain CDR1 containing the amino acid sequence of SEQ ID NO:4, a light chain CDR2 containing the amino acid sequence of SEQ ID NO:5, and a light chain CDR3 containing the amino acid sequence of SEQ ID NO:6. In some aspects, the bispecific antibody provided herein comprises an antigen-binding domain that specifically binds to *Pseudomonas aeruginosa* PcrV, and comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO:13. In some aspects, the bispecific antibody provided herein comprises an antigen-binding domain that specifically binds to *Pseudomonas aeruginosa* PcrV, and comprises a light chain variable region containing the amino acid sequence of SEQ ID NO:14. In some respects, the bispecific antibody provided herein comprises an antigen-binding domain that specifically binds to Pseudomonas aeruginosa PcrV, and comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO:13 and a light chain variable region containing the amino acid sequence of SEQ ID NO:14.

[0103] In some respects, the bispecific antibody presented in this article contains a PcrV binding domain, which has heavy chain variable regions and light chain variable regions on separate polypeptides.

[0104] In some aspects, the bispecific antibody provided herein comprises a PcrV-binding domain having a heavy chain variable region and a light chain variable region on the same polypeptide. In some aspects, the PcrV-binding domain having a heavy chain variable region and a light chain variable region on the same polypeptide comprises a linker. The linker may, for example, be located between the heavy chain variable region and the light chain variable region. The linker may be, for example, a glycine-rich linker or a glycine-serine linker. In some aspects, the linker comprises the amino acid sequence of SEQ ID NO: 18.

[0105] In some respects, the bispecific antibodies provided herein contain a PcrV-binding domain of scFv. In some respects, PcrV binds to scFv at a VH-VL orientation, such as a VH-linker-VL orientation. In some respects, PcrV binds to scFv at a VL-VH orientation, such as a VL-linker-VH orientation.

[0106] In some aspects, the bispecific antibody provided herein comprises 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 containing the amino acid sequence of SEQ ID NO:15 and a light chain variable region containing the amino acid sequence of SEQ ID NO:16 to Psl. In some aspects, the bispecific antibody provided herein comprises an antigen-binding domain that specifically binds to *Pseudomonas aeruginosa* Psl, and the epitope of the bound Psl is identical to the epitope of the Psl bound by the antibody comprising the heavy chain variable region containing the amino acid sequence of SEQ ID NO:15 and the light chain variable region containing the amino acid sequence of SEQ ID NO:16.

[0107] In some respects, the bispecific antibody provided herein comprises an antigen-binding domain that specifically binds to Pseudomonas aeruginosa Psl, and comprises (i) heavy chain CDR1, CDR2, and CDR3 containing the amino acid sequences of the heavy chain CDR1, CDR2, and CDR3 sequences in SEQ ID NO:15 (e.g., CDRs defined by Kabat, AbM, or Chothia) and (ii) light chain CDR1, CDR2, and CDR3 containing the amino acid sequences of the heavy chain CDR1, CDR2, and CDR3 sequences in SEQ ID NO:16 (e.g., CDRs defined by Kabat, AbM, or Chothia).

[0108] In some aspects, the bispecific antibody provided herein comprises an antigen-binding domain that specifically binds to *Pseudomonas aeruginosa* Psl, and comprises a heavy chain CDR1 containing the amino acid sequence of SEQ ID NO:7, a heavy chain CDR2 containing the amino acid sequence of SEQ ID NO:8, a heavy chain CDR3 containing the amino acid sequence of SEQ ID NO:9, a light chain CDR1 containing the amino acid sequence of SEQ ID NO:10, a light chain CDR2 containing the amino acid sequence of SEQ ID NO:11, and a light chain CDR3 containing the amino acid sequence of SEQ ID NO:12. In some aspects, the bispecific antibody provided herein comprises an antigen-binding domain that specifically binds to *Pseudomonas aeruginosa* Psl, and comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO:15. In some aspects, the bispecific antibody provided herein comprises an antigen-binding domain that specifically binds to *Pseudomonas aeruginosa* Psl, and comprises a light chain variable region containing the amino acid sequence of SEQ ID NO:16. In some respects, the bispecific antibody provided herein comprises an antigen-binding domain that specifically binds to Pseudomonas aeruginosa Psl, and comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO:15 and a light chain variable region containing the amino acid sequence of SEQ ID NO:16.

[0109] In some aspects, the bispecific antibody provided herein comprises 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 PcrV-binding domain having both heavy chain and light chain variable regions on the same polypeptide includes a linker. The linker may, for example, be located between the heavy chain and light chain variable regions. The linker may be, for example, a glycine-rich linker or a glycine-serine linker. In some aspects, the linker comprises the amino acid sequence of SEQ ID NO:18.

[0110] In some aspects, the bispecific antibody comprises a Psl-binding domain of the scFv. The scFv may contain a linker. The linker may be, for example, a glycine-rich linker or a glycine-serine linker. In some aspects, the linker comprises the amino acid sequence of SEQ ID NO:18. In some aspects, the scFv is in a VH-VL orientation, such as a VH-linker-VL orientation. In some aspects, the scFv is in a VL-VH orientation, such as a VL-linker-VH orientation. In some aspects, the scFv comprises the amino acid sequence of SEQ ID NO:17.

[0111] In some respects, the bispecific antibodies presented in this article contain a Psl binding domain, which has heavy chain variable regions and light chain variable regions on separate polypeptides.

[0112] b. Structure of a bispecific anti-pseudomonas antibody with a modified Fc region In some respects, the bispecific antibody described herein is an IgG antibody. An IgG antibody may be, for example, an IgG1 antibody. In some respects, the IgG1 antibody is a human IgG1 antibody. In some respects, the IgG1 antibody is a humanized IgG1 antibody.

[0113] IgG antibodies can be, for example, IgG2 antibodies. In some respects, IgG2 antibodies are human IgG2 antibodies. In other respects, IgG2 antibodies are humanized IgG1 antibodies.

[0114] IgG antibodies can be, for example, IgG3 antibodies. In some respects, IgG3 antibodies are human IgG3 antibodies. In other respects, IgG3 antibodies are humanized IgG3 antibodies.

[0115] IgG antibodies can be, for example, IgG4 antibodies. In some respects, IgG4 antibodies are human IgG4 antibodies. In other respects, IgG4 antibodies are humanized IgG4 antibodies.

[0116] In some respects, the bispecific antibodies disclosed herein have structures of BS1, BS2, BS3 or BS4, all of which are shown in Figure 17 of WO 2013 / 070615, which is incorporated herein by reference in its entirety.

[0117] In some respects, the bispecific antibodies disclosed herein have a BS4 structure, which is detailed in WO 2013 / 070615, which is incorporated herein by reference in its entirety. For example, this disclosure provides bispecific antibodies in which an anti-Psl scFv molecule is inserted into the hinge region of each heavy chain of an anti-PcrV antibody or a fragment thereof.

[0118] In some respects, the bispecific antibody provided herein comprises (i) a heavy chain of formula VH-CH1-H1-L1-S-L2-H2-CH2-CH3, wherein VH is a variable domain of the anti-Pseudomonas aeruginosa PcrV heavy chain; CH1 is 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 heavy chain constant region domain-2; and CH3 is 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 the antibody light chain κ constant region or antibody light chain λ region. In some respects, CL is the antibody light chain κ constant region. In some respects, 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, scFv contains the amino acid sequences of SEQ ID NO:15 and SEQ ID NO:16. In some aspects, VH contains the amino acid sequence of SEQ ID NO:13, VL contains the amino acid sequence of SEQ ID NO:14, and scFv contains the amino acid sequences of SEQ ID NO:15 and SEQ ID NO:16. In some aspects, scFv contains the amino acid sequence of SEQ ID NO:17. In some aspects, VH contains the amino acid sequence of SEQ ID NO:13, VL contains the amino acid sequence of SEQ ID NO:14, and scFv contains the amino acid sequence of SEQ ID NO:17. In some aspects, CH1 contains the amino acid sequence of SEQ ID NO:21. In some aspects, L1 and L2 may be the same or different, and may independently contain (a) [GGGGS]n, where n is 0, 1, 2, 3, 4, or 5 (SEQ ID NO:26), (b) [GGGG]n, where n is 0, 1, 2, 3, 4, or 5 (SEQ ID NO:27), or a combination of (a) and (b). In some aspects, H1 contains the amino acid sequence EPKSC (SEQ ID NO:22). In some aspects, L1 contains [GGGGS]n, where n is 2 (SEQ ID NO:28). In some aspects, L2 contains [GGGGS]n, where n is 2 (SEQ ID NO:28). In some aspects, H2 contains the amino acid sequence DKTHTCPPCP (SEQ ID NO:23). In some aspects, CH2-CH3 contains the amino acid sequence of SEQ ID NO:30. In some aspects, CL contains the amino acid sequence of SEQ ID NO:24.

[0119] In some aspects, the bispecific antibody provided herein comprises a polypeptide containing the amino acid sequence of SEQ ID NO:31. In some aspects, the bispecific antibody provided herein comprises a polypeptide containing the amino acid sequence of SEQ ID NO:20. In some aspects, the bispecific antibody provided herein comprises a polypeptide containing both the amino acid sequence of SEQ ID NO:31 and the amino acid sequence of SEQ ID NO:20.

[0120] In some aspects, the bispecific antibodies provided herein can be tandem single-chain (sc)Fv fragments containing two distinct scFv fragments covalently linked together by a linker (e.g., a peptide linker). (Ren-Heidenreich et al. Cancer 100:1095-1103 (2004); Korn et al. J Gene Med 6:642-651 (2004)). In some aspects, the linker can contain all or part of a heavy-chain polypeptide constant region (such as a CH1 domain). In some aspects, the two antibody fragments can be covalently linked together by a polyglycine-serine or polyserine-glycine linker, as described, for example, in U.S. Patent Nos. 7,112,324 and 5,525,491, respectively. Methods for generating bispecific tandem scFv antibodies are described, for example, in Maletz et al., Int J Cancer 93:409-416 (2001); and Honemann et al., Leukemia 18:636-644 (2004). Alternatively, the antibody may be a “linear antibody,” as described, for example, in Zapata et al., Protein Eng. 8:1057-1062 (1995). Briefly, these antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) forming a pair of antigen-binding regions.

[0121] This disclosure also covers variant forms of bispecific antibodies, such as the tetravalent bivariate domain immunoglobulin (DVD-Ig) molecule described in Wu et al. (2007) NatBiotechnol 25(11):1290-1297. The DVD-Ig molecule is engineered such that two distinct light chain variable domains (VLs) from two different parental antibodies are tandemly linked directly or via short linkers using recombinant DNA technology, followed by a light chain constant domain. For example, the DVD-Ig light chain polypeptide may contain, in tandem, a VL from the PcrV-binding domain; and (b) a VL from the Psl-binding domain. Similarly, the heavy chain comprises two distinct heavy chain variable domains (VHs) tandemly linked, followed by constant domains CH1 and Fc regions. For example, the DVD-Ig heavy chain polypeptide may contain, in tandem, a VH from the PcrV-binding domain; and (b) a VH from the Psl-binding domain. In this configuration, the expression of both chains in the cell produces a heterotetramer containing four antigen-binding sites, two of which specifically bind to PcrV and two of which specifically bind to Psl. Methods for generating DVD-Ig molecules from two parental antibodies are further described, for example, in PCT Publications WO 2008 / 024188 and WO 2007 / 024715, each of which is incorporated herein by reference in its entirety.

[0122] In some respects, the bispecific antibodies presented herein do not contain modifications that enhance hexamer formation. In some respects, the bispecific antibodies presented herein are not HexaBody. In some respects, the bispecific antibodies presented herein are not IgG-HexaBody. The HexaBody technique is discussed, for example, in de Jong RN et al., PLoS Biol. 2016 Jan 6; 14(1):e1002344. doi: 10.1371 / journal.pbio.1002344.PMID: 26736041; PMCID: PMC4703389, which is incorporated herein by reference in its entirety.

[0123] In some respects, the bispecific antibodies provided herein do not contain E345R substitution. In some respects, the bispecific antibodies provided herein do not contain E430G substitution. In some respects, the bispecific antibodies provided herein do not contain S440Y substitution. In some respects, the bispecific antibodies provided herein do not contain E345R, E430G, or S440Y substitution.

[0124] c. Modified Fc region in bispecific anti-pseudomonas antibodies As provided herein, a bispecific antibody with a modified Fc region may have one or more altered properties compared to its “corresponding” antibody without a modified Fc region (e.g., having a wild-type Fc region). A “corresponding” antibody to a reference antibody with a modified Fc region is an antibody containing the same amino acid sequence as the reference sequence, except for the specified modification in the Fc region. Thus, for example, ghrebamab (MEDI3902) is a “corresponding” antibody for AZD0292 because ghrebamab contains the same amino acid sequence as AZD0292, except for the N3Y modification in the Fc region of AZD0292.

[0125] The bispecific antibodies against Pseudomonas aeruginosa Psl and PcrV provided herein can utilize FcRn-mediated recycling to achieve serum half-lives that are similar to or different from those of endogenous IgG, depending on the desired properties. By further conferring improved pharmacokinetic properties on biotherapeutic and diagnostic agents, this disclosure offers the opportunity for more desirable dosages, reduced administration frequencies, or increased clearance rates while maintaining efficacy.

[0126] This disclosure provides bispecific antibodies against Pseudomonas aeruginosa Psl and PcrV, the in vivo half-life of which is altered (increased or decreased) by the presence of IgG constant domains or their FcRn binding fragments (e.g., Fc regions or hinge-Fc regions) (e.g., derived from human IgG, such as human IgG1), and these antibodies have modifications of one or more amino acid residues in at least the CH3 domain. Modifications may include amino acid substitutions, insertions, deletions, or any combination thereof. It should be understood that all references to amino acid residues in the IgG constant and variable domains appearing herein are numbered according to the EU number index of Kabat et al. (Sequences of Proteins of Immunological Interest, 5th edition, 1991 NIH Publication No. 91-3242, which is incorporated herein by reference in its entirety) and include corresponding residues in other IgG constant domains identified by sequence alignment.

[0127] More specifically, this disclosure provides bispecific antibodies against Pseudomonas aeruginosa Psl and PcrV, the in vivo half-life of which is altered (increased or decreased) by the presence of the IgG constant domain or its FcRn binding fragment (e.g., Fc region or hinge-Fc region (e.g., derived from human IgG, e.g., human IgG1)), the antibodies having modification with one or more of amino acid residues 432, 433, 434, 435, 436 or 437, and / or the antibodies having a single amino acid insertion between amino acids 437 and 438 in the His435 loop region of the CH3 domain, the insertion referred to herein as 437*, these amino acid substitutions and / or insertions alter (increase or decrease) the binding affinity of the IgG constant domain or its FcRn binding fragment to FcRn at a specific pH (e.g., pH 6.0 or pH 7.4). Such modifications (including insertions between residues 437 and 438) will generally be referred to as modifications within the His435 loop region, i.e., modifications at amino acid residues 432–437. In some respects, these modifications may exclude residue 435, such that the modified IgG constant domain or its FcRn binding portion (e.g., the Fc region or hinge-Fc region) contains His435, found in wild-type human IgG1, IgG2, and IgG4. In some respects, such as modifications to a similar His435 loop region in human IgG3, which includes arginine at position (R435) in the wild-type molecule instead of histidine (H435) found in IgG1, IgG2, and IgG4, and further, is a site of known allelic variation, these modifications involve replacing the wild-type non-histidine residue 435 with histidine to produce H435. In one respect, the modified IgG constant domain or its FcRn binding portion (e.g., Fc region or hinge-Fc region) is a human or humanized IgG constant domain or its FcRn binding portion, but it may be mouse. The human or humanized IgG constant domain may be a constant domain derived from the IgG1, IgG2, IgG3, or IgG4 domains or any of their subtypes.

[0128] As presented herein, amino acid modifications in the His435 loop region of the CH3 domain of the Fc fragment of human IgG can affect the binding affinity of bispecific antibodies to FcRn at one or more pH values. These modifications can lead to pH-dependent alterations in the binding of bispecific antibodies to FcRn. In some respects, amino acid modifications in the His435 loop region can result in higher binding affinity of bispecific antibodies to FcRn at pH 6, at pH 7.4, or at both pH 6.0 and 7.4 than that exhibited by corresponding antibodies possessing the constant domain of wild-type IgG. Additionally or alternatively, modifications can affect the in vivo half-life of the molecule.

[0129] The His435 loop region contains amino acid residues 432, 433, 434, 435, 436, and 437. The wild-type amino acid sequence of the His435 loop region (residues 432 to 437) of the CH3 domain of the Fc fragment of human IgG1, IgG2, and IgG4 is Leu-His-Asn-His-Tyr-Thr (SEQ ID NO:34), and the wild-type amino acid sequence of human IgG3 is Leu-His-Asn-Arg-Phe-Thr (SEQ ID NO:35). In some aspects, one or more amino acid modifications in the Fc region are made at or near one or more of residues 432, 433, 434, 435, 436, and 437, for example, in the constant domain of human IgG or its FcRn-binding domain (e.g., the Fc region or hinge-Fc region), or in similar residues of these residues as determined by amino acid sequence alignment in other IgGs. Such mutations include amino acid substitutions as well as deletions and insertions. The exemplary site for amino acid insertion is between residues 437 and 438, and this addition is referred to as 437* in this paper.

[0130] In one aspect of the modified IgG constant domain or its FcRn binding fragment (e.g., the Fc region or the hinge-Fc-Fc region), residue 435 remains histidine (His435) (as in wild-type human IgG1, IgG2, and IgG4) or is mutated to histidine (as in IgG3, which naturally contains R435, and is therefore mutated to R435H), while at least one of residues 432, 433, 434, 436, or 437 is substituted and / or inserted at position 437*. In another aspect, residues 435 (His435) and 433 (His 433) are not mutated (except for human IgG3, where residue 435 has the R435H mutation, and is therefore His435), while at least one of residues 432, 434, 436, or 437 is substituted and / or inserted at position 437*. In one aspect, the FcRn binding domain has substitutions at one, two, three, four, or all five residues of residues 432, 433, 434, 436, and 437, and / or has an insertion at position 437* in the His435 ring region. In another aspect, the FcRn binding domain has substitutions at three or more positions of residues 432, 433, 434, 435, 436, or 437. In yet another aspect, the FcRn binding domain has substitutions at four or more positions of residues 432, 433, 434, 435, 436, or 437.

[0131] In one aspect, at least one of positions 432 and 437 is substituted with a cysteine ​​residue, and residues 433, 434, 435, and 436 are each independently substituted or unsubstituted. In other aspects, both residues 432 and 437 are substituted with a cysteine ​​residue, and residues 433, 434, 435, and 436 are each independently substituted or unsubstituted.

[0132] In one aspect, at least one of positions 432 and 437 is substituted with an amino acid selected from the group consisting of glutamine, glutamic acid, aspartic acid, lysine, arginine, and histidine, and residues 433, 434, 435, and 436 are each independently substituted or unsubstituted. In other aspects, both positions 432 and 437 are substituted with an amino acid selected independently from the group consisting of glutamine, glutamic acid, aspartic acid, lysine, arginine, and histidine, and residues 433, 434, 435, and 436 are each independently substituted or unsubstituted.

[0133] In one aspect, the modified IgG constant domain or its FcRn binding fragment (e.g., the Fc region or hinge-Fc region) contains at least three mutations in the His435 loop region and has a histidine residue at position 435 (the histidine residue at position 435 can be a wild-type residue or a mutation). This disclosure covers any arrangement of various permutations of three or more mutations (excluding the mutation at 435, if present), including but not limited to mutations at the following sites: Position: 432, 433, and any of 434, 436, 437, and 437* Position: 432, 434, and any of 436, 437, and 437* Position: 432, 436, and any of 437 and 437* Locations: 432, 437, and 437* Position: 433, 434, and any of 436, 437, and 437* Position: 433, 436, and any of 437 and 437* Locations: 433, 437, and 437* Position: 434, 436, and any of 437 and 437* Locations: 434, 437, and 437* Locations: 436, 437 and 437* Position: Any of 432, 433, 434, 436, 437, and 437* Position: Any of 432, 433, 436, 437, and 437* Locations: 432, 433, 437 and 437* Position: Any of 432, 434, 436, 437, and 437* Locations: 432, 434, 437, and 437* Locations: 432, 436, 437 and 437* Position: Any of 433, 434, 436, 437, and 437* Locations: 433, 434, 437 and 437* Positions: 434, 436, 437 and 437* Position: Any of 432, 433, 434, 436, 437, and 437* Locations: 432, 433, 434, 437, and 437* Locations: 432, 433, 436, 437 and 437* Locations: 432, 434, 436, 437 and 437* Positions: 433, 434, 436, 437 and 437* Positions: 432, 433, 434, 436, 437 and 437* In some aspects, the modified IgG constant domain or its FcRn binding fragment (e.g., the Fc region or hinge-Fc region) contains mutations at positions 432, 433, 434, 436, and 437 in the His435 loop region, and has a histidine at position 435 (the histidine at position 435 can be a wild-type residue or a mutation). In one aspect, the modified IgG Fc region contains the amino acid sequence of SEQ ID NO:44. In one aspect, the modified IgG1 Fc region contains the amino acid sequence of SEQ ID NO:44. In one aspect, the modified human IgG Fc region contains the amino acid sequence of SEQ ID NO:44. In one aspect, the modified human IgG1 Fc region contains the amino acid sequence of SEQ ID NO:44. The amino acid sequence of SEQ ID NO:44 may be referred to as "N3Y" modification, "N3Y" mutation, etc.

[0134] In one aspect, the modified IgG Fc region comprises the amino acid sequence of SEQ ID NO:33. In one aspect, the modified IgG1 Fc region comprises the amino acid sequence of SEQ ID NO:33. In one aspect, the modified human IgG Fc region comprises the amino acid sequence of SEQ ID NO:33. In one aspect, the modified human IgG1 Fc region comprises the amino acid sequence of SEQ ID NO:33.

[0135] In one aspect of the modified IgG constant domain or its FcRn binding fragment (e.g., the Fc region or hinge-Fc region), the His435 loop region of the CH3 domain of the Fc fragment has the amino acid sequence CXXXXC (residues 432-437; SEQ ID NO:41) or CXXXXCE (residues 432 to 437*, where 437* is an insertion; SEQ ID NO:42). In one aspect, exemplary His435 loop amino acid sequences of various HB20.3IgG mutants can be generated by CXXXXCE (SEQ ID NO:42).

[0136] Unwilling to be bound by theory, the two cysteine ​​residues may play a stabilizing role in the ring region by forming a disulfide cysteine. The predicted distance between the two cysteine ​​residues is approximately 6.7 Å, which is within the range (4.6 Å–7 Å) compatible with the formation of cysteine. In some aspects based on the His435 ring motif CXXXXC (SEQ ID NO:41), the amino acid modification of the modified IgG constant domain or its FcRn binding fragment (e.g., the Fc region or hinge-Fc region) is a substitution at one or more positions 432–437, both of which are substituted with cysteine. Position 435 is histidine. Position 433 can be substituted with arginine, proline, threonine, lysine, serine, alanine, methionine, or asparagine; in one aspect, position 433 is serine. Position 434 can be substituted with arginine, tryptophan, histidine, phenylalanine, tyrosine, serine, methionine, or threonine; in one respect, position 434 is tyrosine. Position 436 can be substituted with leucine, arginine, isoleucine, lysine, methionine, valine, histidine, serine, or threonine; in one respect, position 436 is leucine. In some respects, the mutated His435 loop region contains a glutamate insertion at position 437*.

[0137] In one aspect, positions 432 and 437 are cysteine; position 433 is arginine, proline, threonine, lysine, serine, alanine, methionine, asparagine, or histidine; position 434 is arginine, tryptophan, histidine, phenylalanine, tyrosine, serine, methionine, threonine, or asparagine; position 435 is histidine; and position 436 is leucine, arginine, isoleucine, lysine, methionine, valine, histidine, serine, threonine, tyrosine, or phenylalanine. In another aspect, the His435 ring region is CXRHXC (SEQ ID NO:36), wherein position 433 is histidine or is substituted with arginine, proline, serine, or asparagine, and wherein position 436 is substituted with leucine, arginine, isoleucine, methionine, or serine. In one aspect, the His435 loop region is CRRHXC (SEQ ID NO:37), wherein position 436 is substituted with leucine, arginine, isoleucine, lysine, methionine, valine, histidine, serine, or threonine. In another aspect, the His435 loop region is CXRHRC (SEQ ID NO:38), wherein position 433 is arginine, proline, threonine, lysine, serine, alanine, methionine, or asparagine. In another aspect, the His435 loop region is CSWHLC (SEQ ID NO:39) or CSWHLE (SEQ ID NO:40). In some aspects, and in any of these aspects, the mutated His435 loop region contains a glutamate insertion at position 437*.

[0138] Amino acid modifications can be performed by any method known in the art, and many such methods are well-known and routine to those skilled in the art. For example, but not limited to, amino acid substitutions, deletions, and insertions can be performed using any well-known PCR-based technique. Amino acid substitutions can be performed by site-directed mutagenesis (see, for example, Zoller and Smith, Nucl. Acids Res. 10:6487-6500, 1982; Kunkel, Proc. Natl. Acad. Sci. USA 82:488, 1985, which are incorporated herein by reference in their entirety). Mutants resulting in increased affinity for FcRn and increased in vivo half-life can be readily screened using well-known and routine assays, such as those described herein. Amino acid substitutions can be introduced at one or more residues in the IgG constant domain or its FcRn-binding fragment (e.g., the Fc region or hinge-Fc region), and the mutated constant domain or fragment can be expressed on the surface of a phage, which can then be screened for the increased FcRn binding affinity.

[0139] Once generated, the mutated IgG constant domain or fragment thereof (e.g., an Fc region or hinge-Fc region) can be used to construct bispecific antibodies (e.g., by fusing with a variable portion of a bispecific antibody of interest) or Fc fusion molecules (e.g., by fusing / conjugating a heterologous portion). The modified IgG or Fc fusion molecules of this disclosure can be generated by methods well known to those skilled in the art. In short, such methods include, but are not limited to, combining a variable region having desired specificity (e.g., a variable region isolated from a phage display or expression library or derived from a human or non-human antibody) with a modified IgG constant region or its FcRn-binding fragment (e.g., an Fc region or hinge-Fc region) having a modified half-life as provided herein. Alternatively, those skilled in the art can generate the modified IgG or Fc fusion molecules of this disclosure by substituting at least one amino acid residue in the Fc region of an antibody or Fc fusion molecule.

[0140] In addition to affecting half-life, the amino acid modifications described herein can also alter (i.e., increase or decrease) the bioavailability of molecules (e.g., transport to mucosal surfaces or other target tissues), particularly alter (i.e., increase or decrease) the transport (or concentration or half-life) of molecules to (e.g., the mucosal surfaces of the lungs) or other parts of target tissues. In some aspects, amino acid modifications alter (e.g., increase or decrease) the transport, concentration, or half-life of molecules to the lungs. In other aspects, amino acid modifications alter (e.g., increase or decrease) the transport (or concentration or half-life) of molecules to the heart, pancreas, liver, kidneys, bladder, stomach, large or small intestine, respiratory tract, lymph nodes, nervous tissue (central and / or peripheral nervous tissue), muscle, epidermis, bone, cartilage, joints, blood vessels, bone marrow, prostate, ovary, uterus, tumor or cancerous tissue, etc.

[0141] In some respects, the amino acid modifications do not eliminate or alter the binding function of one or more other immune effectors or receptors in the constant domain, such as, but not limited to, complement binding, antibody-dependent cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), antibody-dependent phagocytosis (ADCP), and / or binding to one or more Fcγ receptors, such as FcγRI, FcγRII, and FcγRIII. The effector functions of the modified IgG and other molecules of this disclosure can be evaluated using methods well-known and conventional in the art.

[0142] Furthermore, this disclosure provides modified IgGs that are highly suitable for a variety of diagnostic and therapeutic purposes. This disclosure provides modified IgGs whose binding to FcRn exhibits varying levels of pH dependence. These varying levels of pH dependence can lead to or be associated with different pharmacokinetic properties, thereby resulting in modified IgGs that are more suitable for some purposes than others.

[0143] For example, some of the modified IgGs described herein exhibit high affinity binding to FcRn at pH 6.0, and show a high level of pH dependence in their binding to FcRn, and exhibit an observed increase in in vivo half-life. Modified IgGs of this aspect of the present disclosure are useful, for example, when used as therapeutic agents in applications where a longer in vivo half-life is desired. Optionally, the modified IgGs also exhibit other improved pharmacokinetic properties, such as retained or enhanced ability to interact with Fc ligands such as Fcγ receptors and C1q, potent opsonization-phagocytosis (OPK) activity, and the ability to mediate Fc effector functions (e.g., CDC, ADCC).

[0144] In contrast, some of the modified IgGs described herein exhibit high affinity binding to FcRn at pH 6.0, and show a lower level of pH dependence in their binding to FcRn (generally due to enhanced affinity for FcRn at pH 7.4), and exhibit an observed reduction in in vivo half-life. The modified IgGs of this aspect of the present disclosure are useful, for example, in applications where a shorter in vivo half-life is desired, such as when used as therapeutic agents in the treatment of certain autoimmune diseases. They are also well-suited for diagnostic applications, such as when used as biological contrast agents in cases requiring rapid removal from body fluids or tissues.

[0145] Furthermore, this disclosure relates to amino acid modifications (e.g., substitution, insertion, or deletion) in the constant domain of IgG or its FcRn-binding fragments (e.g., Fc regions or hinge-Fc regions), which have been found to increase the affinity of the constant domain of IgG or its fragments for FcRn at pH 6, and these amino acid modifications optionally alter the affinity of IgG or its fragments for FcRn at pH 7.4, thereby changing the pH dependence of the binding affinity of the constant domain of IgG or its fragments (e.g., Fc regions or hinge-Fc regions) for FcRn. In addition, these modifications can increase or decrease the in vivo half-life of the molecule.

[0146] In one aspect, this disclosure emphasizes the pharmaceutical importance of increasing the in vivo half-life of bispecific antibodies against Pseudomonas aeruginosa Psl and PcrV. To this end, this disclosure provides bispecific antibodies against Pseudomonas aeruginosa Psl and PcrV containing a modified IgG constant domain or its FcRn binding fragment (e.g., an Fc region or a hinge-Fc region (e.g., derived from human IgG, e.g., human IgG1)), which confer an increased in vivo half-life on immunoglobulins and other bioactive molecules. In this respect, this disclosure relates to bispecific antibodies against Pseudomonas aeruginosa Psl and PcrV with increased in vivo half-life due to the presence of a modified IgG constant domain or its FcRn-binding fragment (e.g., an Fc region or a hinge-Fc region (e.g., derived from human IgG, such as human IgG1)), wherein the IgG constant domain or its fragment (e.g., by amino acid substitution, deletion, or insertion) is modified to alter (increase or decrease) the binding affinity of the IgG constant domain or FcRn-binding fragment to FcRn at a specific pH (e.g., pH 6.0 or pH 7.4). In one aspect, the IgG constant domain or its FcRn-binding fragment is modified to increase the binding affinity to FcRn at pH 6.0 relative to the binding affinity to FcRn at pH 7.4. The in vivo half-life of the modified IgG of this disclosure can be conveniently assessed in human transgenic mouse models or cynomolgus monkey primate models, as described, for example, in Example 2 below.

[0147] Most of the modified antibodies disclosed herein, regardless of whether they exhibit an increased or decreased in vivo half-life compared to each other or their unmodified or wild-type counterparts, contain an IgG constant domain or its FcRn-binding fragment, which exhibits a higher affinity for FcRn binding at pH 6.0 than the wild-type IgG constant domain.

[0148] More generally, those skilled in the art will understand that the Fc variants of this disclosure, regardless of whether they exhibit an increased or decreased in vivo half-life compared to each other or their unmodified or wild-type counterparts, can possess altered FcRn binding properties. Examples of binding properties include, but are not limited to, binding specificity, equilibrium dissociation constant (KD), dissociation rate, and association rate (kN, KD, KN ... on and k off ), combining affinity and / or cohesive force. It is well known in the art that the equilibrium dissociation constant (KD) is defined as k off / k on It should be understood that higher affinity interactions will have lower KD, and conversely, lower affinity interactions will have higher KD. However, in some cases, the value of k... on or k offIt may be more relevant than the value of KD.

[0149] While the relationship between IgG binding affinity to FcRn, the pH dependence of such affinity, and in vivo half-life is complex, for constant domains of IgG that exhibit high affinity for FcRn at pH 6.0 (e.g., less than about 500 nM KD), as binding affinity to FcRn increases at pH 7.4 (generally reflecting a decrease in the pH dependence of FcRn binding), for example, if the KD at pH 7.4 decreases to below about 1 µM to the nanomolar range, in some cases, the result can be a shorter in vivo half-life for the modified IgG. Conversely, a decreased binding affinity for FcRn at pH 7.4 (e.g., above about 1 µM KD at pH 7.4) along with a high binding affinity at pH 6.0 (e.g., less than about 500 nM KD) generally reflects a greater pH dependence of FcRn binding, which in some cases can lead to a longer in vivo half-life.

[0150] In some aspects, the modified IgG and other molecules of this disclosure contain a modified IgG constant domain or its FcRn-binding fragment (e.g., an Fc region or hinge-Fc region) that exhibits a KD (kinetic density) of less than 100 nM, less than 200 nM, less than 300 nM, less than 400 nM, less than 500 nM, or less than 1000 nM for FcRn binding at pH 6.0. The modified IgG of this disclosure can be characterized, for example, by a KD value of 10 nM to 500 nM or 50 nM to 500 nM for FcRn binding at pH 6.0. In some aspects, the modified IgG of this disclosure exhibits at least a 10-fold, at least a 20-fold, or at least a 50-fold increase in binding affinity for FcRn at pH 6.0 compared to the wild-type IgG constant domain or its FcRn-binding fragment.

[0151] Additionally or alternatively, the modified IgG can exhibit binding affinity for FcRn between 10 nM and 50 µM at pH 7.4. To avoid being bound by theory, it was observed that a threshold may exist: at pH 7.4, a KD of about 1 µM or higher (e.g., 1 µM, 5 µM, 10 µM, 20 µM, 30 µM, 40 µM, 50 µM or higher; i.e., binding affinity in the micromolar or millimolecular range demonstrating lower binding affinity for FcRn) can be associated with modified IgG or other molecules with longer half-lives (slower clearance), while at pH 7.4, a KD of less than 1 µM (e.g., 50 nM, 100 nM, 200 nM, 500 nM, 800 nM to about 1000 nM; i.e., binding affinity in the nanomolar range demonstrating higher binding affinity for FcRn) can be associated with modified IgG or other molecules with shorter half-lives (faster clearance). The increased half-life of modified IgG or other molecules is generally, but not always, associated with pH-dependent binding to FcRn, characterized by a KD of 50 nM to 400 nM or 500 nM at pH 6 and a KD greater than 1 µM at pH 7.4.

[0152] The structure of the constant IgG domain outside the His435 loop region (or its FcRn binding fragment, such as the Fc region or hinge-Fc region) may herein be referred to as the IgG “base structure” or “background” of the molecule, and the two terms are used interchangeably. Therefore, this disclosure contemplates IgG modified with a mutation incorporated into the His435 loop region of a wild-type IgG base structure or a mutant IgG base structure. Any mutant IgG base structure may be utilized; exemplary but non-limiting mutant IgG base structures are described herein. In some aspects, the IgG base structure has the sequence according to SEQ ID NO: 25 or 19.

[0153] The modified immunoglobulin molecules disclosed herein include naturally occurring IgG molecules containing an FcRn-binding domain, as well as other non-IgG immunoglobulins (e.g., IgE, IgM, IgD, IgA, and IgY) or immunoglobulin fragments engineered to contain an FcRn-binding fragment (i.e., fusion proteins comprising a non-IgG immunoglobulin or a fragment thereof and an FcRn-binding domain, such as an Fc region or an Fc hinge region). In both cases, the FcRn-binding domain has one or more amino acid modifications that increase the affinity of the constant domain fragment for FcRn at pH 6.0 and optionally affect (increase or decrease) the pH dependence of FcRn binding.

[0154] Modified immunoglobulins include any immunoglobulin molecule that binds to (preferably, immune-specifically, i.e., competing out non-specific binding) an antigen as determined by an immunoassay well known in the art for determining specific antigen-antibody binding and contains an FcRn binding fragment. Such antibodies include, but are not limited to, polyclonal antibodies, monoclonal antibodies, bispecific antibodies, multispecific antibodies, human antibodies, humanized antibodies and chimeric antibodies, and single-chain antibodies.

[0155] Immunoglobulins (and other proteins used herein) can be derived from any animal source, including birds and mammals. Antibodies can be, for example, human, rodent (e.g., mouse and rat), donkey, sheep, rabbit, goat, guinea pig, camel, horse, or chicken. As used herein, "human" antibodies include antibodies having the amino acid sequence of human immunoglobulins and include antibodies isolated from human immunoglobulin libraries or from animals that are genetically modified with one or more human immunoglobulins and do not express endogenous immunoglobulins, as described below and, for example, in U.S. Patent No. 5,939,598 to Kucherlapati et al.

[0156] d. Additional properties of bispecific anti-pseudomonas antibodies with modified Fc regions In some aspects, the bispecific antibodies (e.g., AZD0292) provided herein mediate cytotoxic activity against Pseudomonas (e.g., Pseudomonas aeruginosa). In other aspects, the bispecific antibodies (e.g., AZD0292) provided herein target Pseudomonas (e.g., Pseudomonas aeruginosa) for opsonization phagocytic killing (OPK). Methods for evaluating cytotoxic activity and / or OPK are known in the art and are provided herein, for example, in Example 1.

[0157] In some respects, the bispecific antibody (e.g., AZD0292) provided herein exhibits cytotoxic activity against pseudomonads (e.g., *Pseudomonas aeruginosa*) similar to that of ghrebamazine (MEDI3902). In some respects, the bispecific antibody (e.g., AZD0292) provided herein exhibits OPK activity against pseudomonads (e.g., *Pseudomonas aeruginosa*) similar to that of ghrebamazine. In some respects, the bispecific antibody (e.g., AZD0292) provided herein exhibits both cytotoxic and OPK activity against pseudomonads (e.g., *Pseudomonas aeruginosa*) similar to that of ghrebamazine.

[0158] In some respects, the bispecific antibodies provided herein (e.g., AZD0292) prevent cell attachment, such as preventing Pseudomonas aeruginosa (e.g., Pseudomonas aeruginosa) from attaching to host cells. Methods for assessing attachment prevention are known in the art and are provided, for example, in WO 2013 / 070615, which is incorporated herein by reference in its entirety. In some respects, the bispecific antibodies provided herein (e.g., AZD0292) disrupt biofilm formation. In some respects, the bispecific antibodies provided herein (e.g., AZD0292) inhibit primary colony formation.

[0159] In some respects, the bispecific antibody provided herein (e.g., AZD0292) exhibits less aggregation in solution than garibalzine. In some respects, the bispecific antibody provided herein (e.g., AZD0292) exhibits less aggregation than garibalzine in shake-plate overgrowth screening (see, for example, Example 4).

[0160] III. Uses of bispecific anti-pseudomonas antibodies with modified Fc regions This document also provides methods for preparing and administering anti-pseudomonas Psl and / or PcrV binding molecules (e.g., antibodies, fragments, variants, or derivatives thereof) as disclosed herein to subjects in need. The route of administration of anti-pseudomonas Psl and / or PcrV binding molecules (e.g., antibodies, fragments, variants, or derivatives thereof) may be, for example, parenteral. As used herein, the term parenteral includes, for example, intravenous, intra-arterial, intraperitoneal, intramuscular, or subcutaneous administration. A suitable form of administration is a solution for injection, particularly a solution for intravenous or intra-arterial injection or infusion. However, in other methods compatible with the teachings herein, anti-pseudomonas Psl and / or PcrV binding molecules (e.g., antibodies, fragments, variants, or derivatives thereof) as disclosed herein may be delivered directly to sites of adverse cell populations (e.g., infection), thereby increasing the exposure of diseased tissue to the therapeutic agent. For example, anti-pseudomonas Psl and / or PcrV binding molecules may be directly administered to ocular tissue, burn tissue, or lung tissue.

[0161] As disclosed herein, anti-Pseudomonas Psl and / or PcrV binding molecules (e.g., antibodies or fragments, variants, or derivatives thereof) can be administered in pharmaceutically effective amounts for in vivo treatment of Pseudomonas infections. In this regard, it should be understood that the disclosed bispecific antibodies will be formulated to facilitate the administration of the active agent and promote its stability. For the purposes of this application, a pharmaceutically effective amount refers to an amount sufficient to achieve effective binding to the target and to provide a benefit (e.g., treatment, improvement, mitigation, clearance, or prevention of Pseudomonas infection).

[0162] Some aspects relate to a method for preventing or treating a Pseudomonas infection in a subject of need, the method comprising administering to the subject an effective amount of a binding molecule or a fragment thereof, an antibody or a fragment thereof. In other aspects, the Pseudomonas infection is a Pseudomonas aeruginosa infection. In some aspects, the subject is a human being. In some aspects, the infection is an eye infection, a lung infection, a burn infection, a wound infection, a skin infection, a blood infection, a bone infection, or a combination of two or more of said infections. In other aspects, the subject has acute pneumonia, burns, a corneal infection, cystic fibrosis, or a combination thereof.

[0163] Some aspects relate to a method for blocking or preventing the adhesion of Pseudomonas aeruginosa to epithelial cells, the method comprising contacting a mixture of epithelial cells and Pseudomonas aeruginosa with a binding molecule or fragment thereof, an antibody or fragment thereof, as described herein.

[0164] A method for enhancing OPK in Pseudomonas aeruginosa is also disclosed, comprising contacting a mixture of phagocytes and Pseudomonas aeruginosa with a binding molecule or fragment thereof, antibody or fragment thereof, composition, polynucleotide, carrier, or host cell as described herein. In another aspect, the phagocytes are differentiated HL-60 cells or human polymorphonuclear leukocytes (PMNs).

[0165] a. Methods and applications of bispecific anti-pseudomonas antibodies with modified Fc regions in the treatment of bronchiectasis. As demonstrated in this article, bispecific antibodies that specifically bind to Pseudomonas aeruginosa Psl and PcrV and have a modified Fc region (e.g., AZD0292) can be used to treat subjects with bronchiectasis. Therefore, this article provides treatment methods using such bispecific antibodies, their use in pharmaceutical preparation, and bispecific antibodies for treatment.

[0166] In some respects, this article provides methods for treating bronchiectasis (e.g., noncystic fibrotic bronchiectasis). These methods may include administering to a subject a bispecific antibody (e.g., AZD0292) that specifically binds to the Pseudomonas aeruginosa PcrV protein and Psl extracellular polysaccharide and contains a modified Fc region. The subject may be, for example, a subject colonized by Pseudomonas aeruginosa.

[0167] In some respects, this article provides methods for improving forced expiratory volume 1 (FEV1) before bronchodilator administration in subjects suffering from bronchiectasis (e.g., noncystic fibrotic bronchiectasis). These methods may include administering to the subject a bispecific antibody (e.g., AZD0292) that specifically binds to the Pseudomonas aeruginosa PcrV protein and Psl extracellular polysaccharide and contains a modified Fc region. The subject may be, for example, a subject colonized by Pseudomonas aeruginosa.

[0168] In some respects, this article provides methods for reducing the Pseudomonas aeruginosa burden in subjects suffering from bronchiectasis (e.g., noncystic fibrotic bronchiectasis). These methods may include administering to the subject a bispecific antibody (e.g., AZD0292) that specifically binds to the P. aeruginosa PcrV protein and Psl extracellular polysaccharide and contains a modified Fc region. The subject may be, for example, a subject colonized by P. aeruginosa.

[0169] In some respects, this article provides methods for reducing bronchiectasis exacerbations in subjects with bronchiectasis (e.g., noncystic fibrotic bronchiectasis). These methods can, for example, reduce bronchiectasis exacerbations requiring hospitalization and / or reduce bronchiectasis exacerbations requiring antibiotics. These methods may include administering to the subject a bispecific antibody (e.g., AZD0292) that specifically binds to the Pseudomonas aeruginosa PcrV protein and Psl extracellular polysaccharide and contains a modified Fc region. The subject may be, for example, a subject colonized by Pseudomonas aeruginosa.

[0170] In some respects, this article provides methods for reducing the need for intravenous antibiotics in subjects suffering from bronchiectasis (e.g., noncystic fibrotic bronchiectasis). These methods may include administering to the subject a bispecific antibody (e.g., AZD0292) that specifically binds to the Pseudomonas aeruginosa PcrV protein and Psl extracellular polysaccharide and contains a modified Fc region. The subject may be, for example, a subject colonized by Pseudomonas aeruginosa.

[0171] In some respects, this article provides methods for stabilizing lung function in subjects with bronchiectasis (e.g., noncystic fibrotic bronchiectasis). These methods may include administering to the subject a bispecific antibody (e.g., AZD0292) that specifically binds to the Pseudomonas aeruginosa PcrV protein and Psl extracellular polysaccharide and contains a modified Fc region. The subject may be, for example, a subject colonized by Pseudomonas aeruginosa.

[0172] In some respects, this article provides methods for improving cough frequency and / or intensity in subjects suffering from bronchiectasis (e.g., noncystic fibrotic bronchiectasis). These methods may include administering to the subject a bispecific antibody (e.g., AZD0292) that specifically binds to the Pseudomonas aeruginosa PcrV protein and Psl extracellular polysaccharide and contains a modified Fc region. The subject may be, for example, a subject colonized by Pseudomonas aeruginosa.

[0173] In some respects, this article provides methods for reducing bronchiectasis symptoms in subjects suffering from bronchiectasis (e.g., noncystic fibrotic bronchiectasis). These methods may include administering to the subject a bispecific antibody (e.g., AZD0292) that specifically binds to the Pseudomonas aeruginosa PcrV protein and Psl extracellular polysaccharide and contains a modified Fc region. The subject may be, for example, a subject colonized by Pseudomonas aeruginosa.

[0174] In some respects, this article provides methods for improving the quality of life of subjects suffering from bronchiectasis (e.g., noncystic fibrotic bronchiectasis). These methods may include administering to the subject a bispecific antibody (e.g., AZD0292) that specifically binds to the Pseudomonas aeruginosa PcrV protein and Psl extracellular polysaccharide and contains a modified Fc region. The subject may be, for example, a subject colonized by Pseudomonas aeruginosa.

[0175] In some respects, this article provides methods for eradicating *Pseudomonas aeruginosa* in subjects suffering from bronchiectasis (e.g., noncystic fibrotic bronchiectasis). These methods may include administering to the subject a bispecific antibody (e.g., AZD0292) that specifically binds to the *P. aeruginosa* PcrV protein and Psl extracellular polysaccharide. The subject may be, for example, a subject colonized by *P. aeruginosa*.

[0176] In some respects, this article provides methods for inducing sustained Pseudomonas aeruginosa inhibition in subjects suffering from bronchiectasis (e.g., noncystic fibrotic bronchiectasis). These methods may include administering to the subject a bispecific antibody (e.g., AZD0292) that specifically binds to the P. aeruginosa PcrV protein and Psl extracellular polysaccharide and contains a modified Fc region. The subject may be, for example, a subject colonized by P. aeruginosa.

[0177] In some respects, this article provides methods for reducing the risk of Pseudomonas aeruginosa-associated bronchiectasis progression in subjects with bronchiectasis (e.g., noncystic fibrotic bronchiectasis). These methods may include administering to the subject a bispecific antibody (e.g., AZD0292) that specifically binds to the P. aeruginosa PcrV protein and Psl extracellular polysaccharide and contains a modified Fc region. The subject may be, for example, a subject colonized by P. aeruginosa.

[0178] The bronchiectasis suitable for treatment according to the methods and uses provided herein can be noncystic fibrotic bronchiectasis. In some respects, noncystic fibrotic bronchiectasis is confirmed by chest computed tomography (CT) scans, demonstrating that the bronchiectasis affects one or more lobes of the subject.

[0179] Subjects with bronchiectasis (e.g., noncystic fibrotic bronchiectasis) suitable for treatment according to the methods and uses provided herein may be subjects colonized by *Pseudomonas aeruginosa*. Subjects colonized with *P. aeruginosa* can be identified, for example, using routine sputum culture. In some aspects, the subject is colonized by a *P. aeruginosa* strain containing a genome containing the Psl-operon. In some aspects, the subject is colonized by a *P. aeruginosa* strain containing a genome containing the PcrV coding locus.

[0180] In some respects, subjects were chronically infected with Pseudomonas aeruginosa. As used herein, "chronic infection" refers to subjects who were simultaneously or sequentially colonized by at least two isolates of Pseudomonas aeruginosa and who were clinically stable for more than one year.

[0181] Subjects with bronchiectasis (e.g., noncystic fibrotic bronchiectasis) who are eligible for treatment according to the methods and uses provided herein may be subjects with airway neutrophilia and / or sputum neutrophilia.

[0182] Subjects with bronchiectasis (e.g., noncystic fibrotic bronchiectasis) who are eligible for treatment according to the methods and uses provided herein may be subjects with a history of moderate to severe bronchiectasis exacerbations requiring antibiotics at least twice a year.

[0183] Subjects with bronchiectasis (e.g., noncystic fibrotic bronchiectasis) who are eligible for treatment according to the methods and uses provided herein may be subjects with at least one history of exacerbation requiring hospital care.

[0184] Subjects with bronchiectasis (e.g., noncystic fibrotic bronchiectasis) who are eligible for treatment according to the methods and uses provided herein may be subjects who have been on long-term nebulized antibiotics.

[0185] Subjects with bronchiectasis (e.g., noncystic fibrotic bronchiectasis) who are eligible for treatment according to the methods and uses provided herein may be subjects with chronic obstructive pulmonary disease (COPD).

[0186] Subjects with bronchiectasis (e.g., noncystic fibrotic bronchiectasis) who are suitable for treatment according to the methods and uses provided herein may be human subjects.

[0187] As provided herein, bronchiectasis of any cause (e.g., noncystic fibrotic bronchiectasis) can be treated according to the methods and uses provided herein. For example, in some respects, bronchiectasis (e.g., noncystic fibrotic bronchiectasis) is caused by hypogammaglobulinemia. In some respects, bronchiectasis (e.g., noncystic fibrotic bronchiectasis) is caused by common variable immunodeficiency. In some respects, bronchiectasis (e.g., noncystic fibrotic bronchiectasis) is caused by α-1-antitrypsin deficiency.

[0188] The methods and uses described herein may include administering to a subject a bispecific antibody (e.g., AZD0292) that specifically binds to the Pseudomonas aeruginosa PcrV protein and Psl extracellular polysaccharide. Administration may be intravenous or subcutaneous.

[0189] The methods and uses described herein are effective in treating bronchiectasis (e.g., noncystic fibrotic bronchiectasis). In some aspects of the methods and uses described herein, administration of a bispecific antibody (e.g., AZD0292) that specifically binds to the Pseudomonas aeruginosa PcrV protein and Psl extracellular polysaccharide and contains a modified Fc region reduces Pseudomonas aeruginosa in sputum cultures obtained from the subject, for example, compared to Pseudomonas aeruginosa in sputum cultures obtained from the subject prior to administration. This reduction can occur, for example, within 12 weeks of the first administration of the bispecific antibody, within 8 weeks of the first administration of the bispecific antibody, or within 4 weeks of the first administration of the bispecific antibody. In some aspects of the methods and uses described herein, administration of a bispecific antibody (e.g., AZD0292) that specifically binds to the Pseudomonas aeruginosa PcrV protein and Psl extracellular polysaccharide and contains a modified Fc region reduces antibiotic use in the subject, for example, compared to the subject's use prior to administration. In some aspects of the methods and uses provided herein, the administration of a bispecific antibody (e.g., AZD0292) that specifically binds to the Pseudomonas aeruginosa PcrV protein and Psl extracellular polysaccharide eradicates Pseudomonas aeruginosa in a subject.

[0190] The methods and uses provided herein may include administering a bispecific antibody (e.g., AZD0292) that specifically binds to the Pseudomonas aeruginosa PcrV protein and Psl extracellular polysaccharide and contains a modified Fc region to a subject in combination with an antibiotic. In some aspects, the methods and uses provided herein include administering a bispecific antibody (e.g., AZD0292) that specifically binds to the Pseudomonas aeruginosa PcrV protein and Psl extracellular polysaccharide and contains a modified Fc region to a subject in combination with: aminoglycosides, ticarcillin, ureapeptide, ciprofloxacin, cefepime, gentamicin, amikacin, tobramycin, ceftazidime, aztreonam, cefotaxime, meropenem, polymyxin B, or any combination thereof.

[0191] b. Methods and uses of bispecific anti-pseudomonas antibodies with modified Fc regions in the prevention or treatment of hospital-acquired infections. This disclosure provides a method for preventing hospital-acquired infections in susceptible human subjects, wherein the method includes administering to the subject a bispecific antibody (e.g., AZD0292) that specifically binds to the Pseudomonas aeruginosa PcrV protein and Psl extracellular polysaccharide. Susceptible human subjects are individuals at risk of contracting a hospital-acquired infection but who are not infected or do not show symptoms of infection at the time of administration; or individuals who have already contracted a hospital-acquired infection requiring intervention or mitigation. The method also includes monitoring the subject for symptoms following administration of the bispecific antibody, for example, for 1, 3, 5, 7, 10, 15, 21, 28, or 30 days. In one aspect, the method includes monitoring the subject for symptoms for at least about 21 days or longer from the date of administration. "Hospital-acquired infection" is defined elsewhere herein and includes, for example, pneumonia, bacteremia, bone infection, joint infection, skin infection, burn infection, wound infection, peritonitis, sepsis, and / or abscess. Symptoms associated with hospital-acquired infections (e.g., pneumonia) are known in the art. In some respects, hospital-acquired infections are caused by or exacerbated by *Pseudomonas aeruginosa*. According to this method, a human subject is successfully treated if, for example, at 1, 3, 5, 8, 10, 15, 21, 28, or 30 days after administration, the subject remains asymptomatic (if the subject was asymptomatic at the time of administration) or exhibits symptoms less severe than expected without treatment with a bispecific anti-pseudomonas antibody containing a modified Fc region (e.g., AZD0292). In another respect, a human subject is successfully treated if, at 21 days after administration, the subject remains asymptomatic (if the subject was asymptomatic at the time of administration) or exhibits symptoms less severe than expected without treatment with a bispecific anti-pseudomonas antibody containing a modified Fc region (e.g., AZD0292). On the other hand, if a human subject remains asymptomatic (if the subject was asymptomatic at the time of administration) or exhibits symptoms less severe than expected if treated with a bispecific anti-pseudomonas antibody containing a modified Fc region (e.g., AZD0292) 28 or 30 days after administration, the human subject is considered successfully treated.

[0192] In another aspect, this disclosure provides a method for preventing or treating pneumonia (e.g., hospital-acquired or non-hospital-acquired pneumonia) in a susceptible human subject, wherein the method comprises administering to the subject a bispecific antibody (e.g., AZD0292) that specifically binds to the Pseudomonas aeruginosa PcrV protein and Psl extracellular polysaccharide and comprises a modified Fc region. In some aspects, the pneumonia is hospital-acquired or iatrogenic. The susceptible human subject is a person at risk of contracting pneumonia but without pneumonia symptoms at the time of administration; or a person already infected with pneumonia requiring intervention or relief. The method also includes monitoring the subject for pneumonia symptoms after administration of the bispecific antibody, for example, for durations of 1 day, 3 days, 5 days, 7 days, 10 days, 15 days, 21 days, 28 days, or 30 days. In one aspect, the method includes monitoring the subject's symptoms for at least about 21 days from the date of administration. Symptoms associated with pneumonia are known in the art. In some aspects, the pneumonia is caused by or exacerbated by Pseudomonas aeruginosa. According to this method, a human subject is successfully treated if, for example, at 1, 3, 5, 8, 10, 15, 21, 28, or 30 days after administration, the subject remains asymptomatic (if the subject was asymptomatic at the time of administration) or exhibits symptoms of less severity than expected without treatment with a bispecific anti-pseudomonas antibody containing a modified Fc region (e.g., AZD0292). In one aspect, a human subject is successfully treated if, at 7 days after administration, the subject remains asymptomatic (if the subject was asymptomatic at the time of administration) or exhibits symptoms of less severity than expected without treatment with a bispecific anti-pseudomonas antibody containing a modified Fc region (e.g., AZD0292). In another aspect, a human subject is successfully treated if, at 21 days after administration, the subject remains asymptomatic (if the subject was asymptomatic at the time of administration) or exhibits symptoms of less severity than expected without treatment with a bispecific anti-pseudomonas antibody containing a modified Fc region (e.g., AZD0292). On the other hand, if a human subject remains asymptomatic (if the subject was asymptomatic at the time of administration) or exhibits symptoms less severe than expected if treated with a bispecific anti-pseudomonas antibody containing a modified Fc region (e.g., AZD0292) 28 or 30 days after administration, the human subject is considered successfully treated.

[0193] On the other hand, this disclosure provides a method for preventing or treating diseases caused by *Pseudomonas aeruginosa* (e.g., pneumonia, tracheobronchitis, bacteremia, endocarditis, meningitis, otitis media, bacterial keratitis, endophthalmitis, osteomyelitis, gastrointestinal disease, skin infection, sepsis, or any combination thereof) in susceptible human subjects. In some aspects, the diseases caused by *Pseudomonas aeruginosa* are hospital-acquired or iatrogenic. Susceptible human subjects are those at risk of infection with a disease that can be treated or prevented by the methods provided herein but who do not have symptoms of the disease at the time of administration; or those who have already been infected with a disease caused by *Pseudomonas aeruginosa* requiring treatment, intervention, or remission.

[0194] The methods described herein are applicable to susceptible human subjects as described elsewhere herein. Examples include subjects who are about to be hospitalized, currently hospitalized, recently hospitalized, and who are about to, currently, or recently using a mechanical ventilator or a combination thereof. In some cases, hospitalization may be in the intensive care unit (ICU). If necessary, mechanical ventilation may be performed via intubation, such as endotracheal or nasotracheal intubation, or via tracheostomy. Patients who are about to, currently, or recently receiving mechanical ventilation may have an increased risk of contracting respiratory infections, such as pneumonia, such as Pseudomonas aeruginosa pneumonia. In those cases where mechanical ventilation is indicated, administration of a bispecific antibody provided by the disclosed methods may reduce the risk of contracting pneumonia, for example, while currently receiving mechanical ventilation, after mechanical ventilation is no longer required, or a combination thereof.

[0195] In some respects, the subject's respiratory tract (e.g., lower respiratory tract) is colonized by *Pseudomonas aeruginosa* upon administration of the bispecific antibody. In some respects, the subject's respiratory tract is colonized by *P. aeruginosa* one, two, three, or four days prior to administration of the bispecific antibody. Colonization can be measured, for example, by detecting *P. aeruginosa* in tracheal aspirates 6, 12, 24, 48, 72, or 96 hours prior to administration of the bispecific antibody. In some respects of the methods provided herein, the subject has not received an antibiotic believed to be active against the *P. aeruginosa* strains that have colonized the subject prior to administration of the bispecific antibody. In some respects, the subject's respiratory tract may additionally be colonized by *Staphylococcus aureus* upon administration of a bispecific anti-pseudomonas antibody containing a modified Fc region (e.g., AZD0292).

[0196] In some respects, subjects did not experience pneumonia symptoms when administered the bispecific antibody. Symptoms can be assessed using the Clinical Pulmonary Infection Score (CPIS). For example, if a subject had a CPIS score of less than 6 24 hours prior to administration of the bispecific antibody, asymptomatic individuals could be presumed.

[0197] The methods described herein include monitoring a subject for disease symptoms, such as pneumonia symptoms, after administration of bispecific antibodies. In some respects, pneumonia can be monitored in a subject by chest X-ray, observation of respiratory signs or symptoms of pneumonia, microbiological confirmation of pneumonia, or any combination thereof. For example, a subject may be diagnosed with pneumonia when new or worsening infiltrates consistent with pneumonia are observed on a chest X-ray, when the subject displays at least two minor or at least one major respiratory sign or symptom of pneumonia, when a sample obtained from the subject is positive for Pseudomonas aeruginosa by culture, or any combination thereof. In some respects, the sample is the subject's respiratory secretions. Respiratory secretions can be obtained by endotracheal aspiration, bronchoscopy using bronchoalveolar lavage, sampling using a contamination-resistant sample brush in intubated subjects, or any combination thereof from coughed-up sputum.

[0198] Secondary respiratory signs or symptoms of pneumonia include, but are not limited to, a body temperature greater than approximately 38°C, a core body temperature less than approximately 35°C, and a respiratory count greater than approximately 10,000 cells / mm³. 3 White blood cell count less than approximately 4,500 cells / mm² 3 A white blood cell count greater than approximately 15% band neutrophil count, production of new purulent tracheal secretions or sputum, new auscultatory findings, dull percussion, new onset of cough, dyspnea, tachypnea, hypoxemia, or any combination thereof. Major respiratory signs or symptoms of pneumonia may include, but are not limited to, acute changes in a ventilatory support system used to enhance oxygenation (including a PaO2 / FiO2 ratio less than approximately 240 mm Hg maintained for at least four hours, a decrease in a PaO2 / FiO2 ratio greater than approximately 50 mm Hg maintained for at least four hours), the need to initiate or restart mechanical ventilation in non-mechanically ventilated subjects, or any combination thereof. Microbiological confirmation of pneumonia may include, but is not limited to, cultures of respiratory samples positive for Pseudomonas aeruginosa, blood cultures positive for Pseudomonas aeruginosa, pleural fluid aspirates or lung tissue cultures positive for Pseudomonas aeruginosa, or any combination thereof.

[0199] In some aspects, the methods provided by this disclosure may also include administering an antibiotic to the subject before, concurrently with, and / or after the administration of the bispecific antibody. Suitable antibiotics may include, but are not limited to, aminoglycosides, ticarcillin, ureapeptides, ciprofloxacin, cefepime, gentamicin, amikacin, tobramycin, ceftazidime, aztreonam, cefotaxime, or any combination thereof. Suitable dosage and treatment duration may be readily determined by the healthcare provider. In some aspects, the subject is susceptible to the antibiotic selected for administration by the Pseudomonas aeruginosa strain colonized by him / her. However, in other aspects, the subject is resistant or partially resistant to one or more of the available antibiotics selected for administration by the Pseudomonas aeruginosa strain colonized by him / her.

[0200] IV. Methods for generating bispecific anti-pseudomonas antibodies The bispecific anti-pseudomonas antibody (e.g., AZD0292) containing the modified Fc region disclosed herein can be generated by any method known in the art for synthesizing antibodies, particularly by chemical synthesis or by recombinant expression technology.

[0201] This disclosure also provides a polynucleotide comprising a nucleic acid sequence encoding a bispecific antibody against Pseudomonas aeruginosa Psl and PcrV, the bispecific antibody comprising the modified IgG constant domain of this disclosure or its FcRn binding fragment (e.g., an Fc region or a hinge-Fc region); and a vector comprising said polynucleotide. In a particular aspect, this disclosure provides an isolated polynucleotide comprising a nucleic acid molecule encoding the heavy chain of the bispecific antibody described herein. In some aspects, the isolated polynucleotide also comprises a nucleic acid molecule encoding the light chain of the bispecific antibody described herein.

[0202] The nucleotide sequence of the anti-Pseudomonas aeruginosa Psl and PcrV bispecific antibody containing the modified IgG constant domain and the polynucleotide encoding the sequence can be obtained by any method known in the art, including general DNA sequencing methods such as dideoxy chain termination (Sanger sequencing) and oligonucleotide priming in combination with PCR.

[0203] The nucleotide sequence encoding the antibody can be obtained from any information available to those skilled in the art (i.e., from Genbank, literature, or through conventional cloning). If a clone containing a nucleic acid encoding a specific antibody or its epitope-binding fragment is unavailable, but the sequence of the antibody molecule or its epitope-binding fragment is known, the nucleic acid encoding the immunoglobulin can be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from any tissue or cell expressing the antibody, such as hybridoma cells selected to express the antibody, or nucleic acids isolated therefrom, such as poly A+ RNA). This is achieved by PCR amplification using synthetic primers that hybridize to the 3' and 5' ends of the sequence, or by cloning using oligonucleotide probes specific to a specific gene sequence, to identify, for example, a cDNA clone encoding the antibody from the cDNA library. The amplified nucleic acid produced by PCR can then be cloned into a reproducible cloning vector using any method well known in the art.

[0204] Once the nucleotide sequence of the antibody is determined, methods well-known in the art for manipulating nucleotide sequences, such as recombinant DNA techniques, site-directed mutagenesis, and PCR, can be used to manipulate the antibody's nucleotide sequence (see, for example, techniques described in Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d, ColdSpring Harbor Laboratory, Cold Spring Harbor, NY; and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY, all of which are incorporated herein by reference in their entirety) to produce antibodies with different amino acid sequences by, for example, introducing amino acid substitutions, deletions, and / or insertions into the epitope-binding domain region of the antibody, such as into the hinge-Fc region of an antibody that interacts with FcRn. Antibodies with one or more modifications at amino acid residues 432-437 or other positions can be produced.

[0205] In a specific embodiment, the nucleic acid molecule encoding the heavy chain of the bispecific antibody has the sequence according to SEQ ID NO:45. In a specific embodiment, the nucleic acid molecule encoding the light chain of the bispecific antibody has the sequence according to SEQ ID NO:46.

[0206] Recombinant expression of antibodies requires the construction of an expression vector containing a nucleotide sequence encoding an antibody. Once the nucleotide sequence encoding an antibody molecule or a portion thereof (optionally, but not necessarily, containing a variable region of the heavy chain or a variable region of the light chain) is obtained, a vector for generating the antibody molecule can be produced using recombinant DNA techniques known in the art. Therefore, methods for preparing proteins by expressing polynucleotides containing antibody-encoding nucleotide sequences are described herein. Expression vectors containing antibody-encoding sequences and appropriate transcriptional and translational control signals can be constructed using methods known to those skilled in the art. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Therefore, this disclosure provides a reproducible vector containing a nucleotide sequence encoding a constant region of an antibody molecule, wherein the constant region of the antibody molecule has one or more modifications in amino acid residues involved in interaction with FcRn (see, for example, PCT Publication WO 86 / 05807; PCT Publication WO 89 / 01036; and U.S. Patent No. 5,122,464). Nucleotide sequences encoding antibody heavy chain variable regions, light chain variable regions, both heavy chain variable regions and light chain variable regions, epitope-binding fragments of heavy chain variable regions and / or light chain variable regions, or one or more complementarity-determining regions (CDRs) can be cloned into such vectors for expression.

[0207] Therefore, in some aspects provided herein, this disclosure relates to a vector comprising (i) a nucleic acid molecule encoding the heavy chain of a bispecific antibody, or (ii) a nucleic acid molecule encoding the heavy chain of the bispecific antibody described herein and a nucleic acid molecule encoding the light chain of the bispecific antibody described herein. In some aspects, this disclosure relates to a pair of vectors, wherein a first vector of the pair comprises a nucleic acid molecule encoding the heavy chain of the bispecific antibody described herein, and a second vector of the pair comprises a nucleic acid molecule encoding the light chain of the bispecific antibody described herein. In a specific embodiment, the nucleic acid molecule encoding the heavy chain of the bispecific antibody has the sequence according to SEQ ID NO: 45. In a specific embodiment, the nucleic acid molecule encoding the light chain of the bispecific antibody has the sequence according to SEQ ID NO: 46.

[0208] Expression vectors are transferred into host cells using conventional techniques, and the transfected cells are then cultured using conventional techniques to produce antibodies with increased affinity for FcRn and increased in vivo half-life. Therefore, this disclosure includes host cells containing a polynucleotide encoding an antibody, its constant domain, or an FcRn-binding fragment having one or more modifications at amino acid residues 432-437 or other positions, optionally operably linked to a heterologous promoter.

[0209] Various host-expression vector systems can be used to express the antibody molecules disclosed herein. Such host-expression systems represent media by which the coding sequences of interest can be generated and subsequently purified, but also represent cells that can express the antibody molecules disclosed herein in situ when transformed or transfected with appropriate nucleotide coding sequences. These include, but are not limited to, microorganisms such as bacteria (e.g., *Escherichia coli* and *Bacillus subtilis*) transformed with recombinant phage DNA, plasmid DNA, or coliform DNA expression vectors containing antibody coding sequences. Yeast transformed with recombinant yeast expression vectors containing antibody-encoding sequences (e.g., *Saccharomyces* and *Pichia*); insect cell systems infected with recombinant viral expression vectors containing antibody-encoding sequences (e.g., baculoviruses); plant cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus, CaMV; and tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors containing antibody-encoding sequences (e.g., Ti plasmids); and mammalian cell systems carrying recombinant expression constructs (e.g., COS, CHO, BHK, 293, 3T3, and NSO cells) containing promoters derived from mammalian cell genomes (e.g., metallothionein promoters) or from mammalian viruses (e.g., adenovirus late promoters; vaccinia virus 7.5K promoters). Bacterial cells such as *Escherichia coli* and eukaryotic cells are well-suited for expressing complete recombinant antibody molecules. For example, mammalian cells such as Chinese hamster ovary cells (CHO) together with vectors such as major intermediate early gene promoter elements from human cytomegalovirus are efficient antibody expression systems (Foecking et al., Gene, 45:101, 1986, and Cockett et al., Bio / Technology, 8:2, 1990).

[0210] In bacterial systems, a variety of expression vectors can be advantageously selected based on the intended use of the expressed antibody molecule. For example, when producing large quantities of such a protein, a vector that directs the expression of a high-level fusion protein product that is easily purified may be needed to generate a pharmaceutical composition of the antibody molecule. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al., EMBO, 12:1791, 1983), in which the antibody-coding sequence can be individually linked to a frame containing the lacZ coding region in the vector to produce a fusion protein; and the pIN vector (Inouye & Inouye, NucleicAcids Res., 13:3101-3109, 1985 and Van Heeke & Schuster, J. Biol.Chem., 24:5503-5509, 1989).

[0211] In insect systems, the alfalfa silver-striped armyworm nucleopolyhedrovirus (AcNPV) is used as a vector for expressing exogenous genes. This virus grows in Spodoptera frugiperda cells. Antibody-coding sequences can be cloned separately into non-essential regions of the virus (e.g., polyhedromic protein genes) and placed under the control of AcNPV promoters (e.g., polyhedromic protein promoters).

[0212] In mammalian host cells, numerous virus-based expression systems can be used to express the antibody molecules disclosed herein. When adenovirus is used as the expression vector, the antibody-coding sequence of interest can be linked to an adenoviral transcription / translation control complex, such as a late promoter and a triple leader sequence. This chimeric gene can then be inserted into the adenoviral genome via in vitro or in vivo recombination. Insertion into non-essential regions of the viral genome (e.g., regions E1 or E3) will yield a recombinant virus that is viable and capable of expressing the antibody molecule in an infected host (e.g., see Logan & Shenk, Proc. Natl. Acad. Sci. USA, 81:355-359, 1984). Effective translation of the inserted antibody-coding sequence may also require specific initiation signals. These signals include the ATG start codon and adjacent sequences. Furthermore, the start codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire inserted sequence. These exogenous translation control signals and start codons can be of various origins, i.e., natural and synthetic. Expression efficiency can be enhanced by including appropriate transcriptional enhancer elements, transcription terminators, etc. (see, for example, Bitter et al., Methods in Enzymol., 153:516-544, 1987).

[0213] Furthermore, host cell lines can be selected to regulate antibody sequence expression or modify and process antibodies in a desired specific manner. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can be crucial for antibody function. Different host cells possess characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be selected to ensure proper modification and processing of expressed antibodies. For this purpose, eukaryotic host cells with cellular machinery for the proper processing, glycosylation, and phosphorylation of primary transcripts of gene products can be used. Such mammalian host cells include, but are not limited to, CHO, VERY, BHK, HeLa, COS, MDCK, 293, 3T3, W138, and particularly myeloma cells (such as NSO cells) and related cell lines, see, for example, Morrison et al., U.S. Patent No. 5,807,715, which is incorporated herein by reference in its entirety.

[0214] For long-term, high-yield production of recombinant antibodies, stable expression is preferred. For example, cell lines stably expressing antibody molecules can be engineered. Instead of using expression vectors containing viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) and selectivity markers. After introducing exogenous DNA, engineered cells can be grown in enriched medium for 1–2 days, then switched to selective medium. Selectivity markers in the recombinant plasmid confer resistance to selection and enable cells to stably integrate the plasmid into their chromosome and grow to form focal clusters (foci), which can then be cloned and amplified into cell lines. This method can be advantageously used for the engineering of cell lines expressing antibody molecules. Such engineered cell lines can be particularly used for screening and evaluating compositions that interact directly or indirectly with antibody molecules.

[0215] Many selection systems can be used, including but not limited to herpes simplex virus thymidine kinase (Wigler et al., Cell, 11:223, 1977), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA, 48:202, 1992), and adenine phosphoribosyltransferase (Lowy et al., Cell, 22:8-17, 1980) genes, which can be used for tk-cells, hgprt-cells, or aprt-cells, respectively. Furthermore, antimetabolite resistance can be used as a basis for selecting the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Natl. Acad. Sci. USA, 77:357, 1980 and O'Hare et al., Proc. Natl. Acad. Sci. USA, 78:1527, 1981); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA, 78:2072, 1981); neo, which confers resistance to aminoglycoside G-418 (Wu and Wu, Biotherapy, 3:87-95, 1991; Tolstoshev, Ann. Rev. Pharmacol. Toxicol., 32:573-596, 1993; Mulligan, Science, 260:926-932, 1993; and Morgan and Anderson, Ann. Rev. Biochem., 62: 191-217, 1993; and May, TIB TECH, 11(5):155-215, 1993); and hygro, which confers resistance to hygromycin (Santerre et al., Gene, 30:147, 1984).Commonly known methods in the field of recombinant DNA technology can be routinely applied to select desired recombinant clones, and such methods are described in, for example, Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY, 1993; Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY, 1990; in Chapters 12 and 13; Dracopoli et al. (eds.), Current Protocols in Human Genetics, John Wiley & Sons, NY, 1994; and Colberre-Garapin et al., J. Mol. Biol., 150:1, 1981, which are incorporated herein by reference in their entirety.

[0216] The expression level of antibody molecules can be increased by vector amplification (see Bebbington and Hentschel, 1987, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3, Academic Press, New York). When the marker in the antibody-expressing vector system is amplifiable, the increased level of inhibitors present in the host cell culture will increase the copy number of the marker gene. Because the amplified region associates with the antibody gene, antibody production will also increase (Crouse et al., Mol., Cell. Biol., 3:257, 1983).

[0217] Host cells can be co-transfected using the two expression vectors disclosed herein: a first vector encoding a heavy-chain-derived polypeptide and a second vector encoding a light-chain-derived polypeptide. Both vectors may contain the same selective markers enabling equal expression of the heavy-chain and light-chain polypeptides, or different selective markers to ensure the maintenance of both plasmids. Alternatively, a single vector encoding and capable of expressing both the heavy-chain and light-chain polypeptides may be used. In such cases, the light chain should be placed before the heavy chain to avoid excessive amounts of toxic free heavy chain (Proudfoot, Nature, 322:52, 1986; and Kohler, Proc. Natl. Acad. Sci. USA, 77:2 197, 1980). The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.

[0218] Once the bispecific anti-pseudomonas antibody (e.g., AZD0292) containing the modified Fc region of this disclosure is generated through recombinant expression, it can be purified using any method known in the art for purifying immunoglobulin molecules, such as chromatography (e.g., ion exchange chromatography, affinity chromatography (particularly after protein A purification by affinity for a specific antigen), and size sieving column chromatography), centrifugation, differential solubility, or any other standard technique for purifying proteins. Furthermore, the antibody or fragment thereof of this disclosure can be fused with heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification.

[0219] V. Pharmaceutical Composition Pharmaceutical compositions used in this disclosure may comprise a bispecific antibody against Pseudomonas aeruginosa Psl and PcrV containing a modified Fc region (e.g., AZD0292) and a pharmaceutically acceptable carrier well known to those skilled in the art. Formulations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.

[0220] The route of administration for bispecific anti-Pseudomonas aeruginosa Psl and PcrV antibodies with modified Fc regions can be, for example, parenteral. As used herein, parenteral administration includes, for example, intravenous and subcutaneous administration. Therefore, pharmaceutical compositions comprising bispecific anti-Pseudomonas aeruginosa Psl and PcrV antibodies (e.g., AZD0292) with modified Fc regions can be formulated for intravenous administration. In some aspects, pharmaceutical compositions comprising bispecific anti-Pseudomonas aeruginosa Psl and PcrV antibodies (e.g., AZD0292) with modified Fc regions can be formulated for subcutaneous administration. A suitable form of administration is a solution for injection.

[0221] Pharmaceutical compositions comprising a bispecific antibody against Pseudomonas aeruginosa Psl and PcrV containing a modified Fc region (e.g., AZD0292) may be applied and / or formulated for, for example, to treat the diseases disclosed herein.

[0222] Bispecific antibodies against Pseudomonas aeruginosa Psl and PcrV containing modified Fc regions (e.g., AZD0292) can be administered in pharmaceutically effective amounts for in vivo treatment or prophylaxis of Pseudomonas aeruginosa infections.

[0223] As provided herein, pharmaceutical compositions comprising a bispecific antibody against Pseudomonas aeruginosa Psl and PcrV containing a modified Fc region (e.g., AZD0292) can be formulated for use in combination with antibiotics. In some aspects, the bispecific antibody is formulated for use in combination with aminoglycosides, ticarcillin, ureapeptide, ciprofloxacin, cefepime, gentamicin, amikacin, tobramycin, ceftazidime, aztreonam, cefotaxime, meropenem, polymyxin B, or any combination thereof.

[0224] VI. Kits containing bispecific anti-pseudomonas antibodies with modified Fc regions This disclosure also provides a pharmaceutical package or kit comprising one or more containers filled with one or more ingredients of a pharmaceutical composition of this disclosure. Optionally, associated with such containers may be a label in the form prescribed by a government agency regulating the production, use, or sale of pharmaceutical or biological products, reflecting that agency’s approval for the production, use, or sale of the pharmaceutical or biological product for human administration.

[0225] This disclosure provides kits that can be used in the methods described above. In one aspect, the kit contains, optionally in purified form, a bispecific antibody (e.g., AZD0292) of this disclosure containing a modified Fc region against *Pseudomonas aeruginosa* Psl and PcrV, in one or more containers. In a specific aspect, the kit of this disclosure contains substantially isolated antigens or combinations of antigens (e.g., PcrV and Psl) as controls. Optionally, the kit of this disclosure also contains control antibodies, fusion proteins, or conjugate molecules that do not react with the antigens contained in the kit. In another specific aspect, the kit of this disclosure contains tools for detecting the binding of the bispecific antibody (e.g., AZD0292) of this disclosure containing a modified Fc region against *Pseudomonas aeruginosa* Psl and PcrV to the antigen (e.g., the bispecific antibody (e.g., AZD0292) containing a modified Fc region against *Pseudomonas aeruginosa* Psl and PcrV may be conjugated to a detectable substrate such as a fluorescent compound, enzyme substrate, radioactive compound, or luminescent compound, or a second antibody recognizing the first antibody may be conjugated to a detectable substrate). Specifically, the kit may contain recombinant or chemically synthesized antigens or combinations of antigens. The antigens provided in the kit may also be attached to a solid carrier. More specifically, the detection tool of the aforementioned kit includes a solid carrier to which the antigen or combination of antigens is attached. Such kits may also include anti-human antibodies without attached reporter molecular markers. In this respect, the binding of the antibody to the antigen can be detected by the binding of the antibody to the reporter molecular marker.

[0226] VII. Immunological assay The immunospecific binding of the disclosed bispecific antibody against Pseudomonas Psl and PcrV (e.g., AZD0292) containing the modified Fc region can be determined by any method known in the art. Immunoassays that can be used include, but are not limited to, competitive and non-competitive assay systems using techniques such as: Western blotting, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), sandwich immunoassay, immunoprecipitation assay, precipitin reaction, gel diffusion precipitin reaction, immunodiffusion assay, agglutination assay, complement fixation assay, immunoradioassay, fluorescence immunoassay, protein A immunoassay, to name just a few. Such assays are routine and well known in the art (see, for example, Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, Vol. 1 (1994), which is incorporated herein by reference in its entirety). Exemplary immunoassays are briefly described below (but are not intended to be limiting).

[0227] Several methods exist for measuring the affinity of antibody-antigen interactions, but relatively few are available for determining the rate constant. Most methods rely on labeled antibodies or antigens, which inevitably complicates routine measurements and introduces uncertainty into the measured quantity. Antibody affinity can be measured using various methods, including OCTET. ® BIACORE ® ELISA and FACS.

[0228] OCTET ® The system uses a biosensor in the form of a 96-well plate to report kinetic analyses. Protein binding and dissociation events can be monitored by measuring the binding of one protein in solution to a second protein immobilized on the FortéBio biosensor. This is achieved through immobilization onto OCTET. ® At the tip, the binding of antibodies in solution was then analyzed to measure the binding of Fc-modified anti-Pseudomonas Psl and PcrV bispecific antibodies. The association and dissociation of the antibodies with the immobilized Fc-modified anti-Pseudomonas Psl and PcrV bispecific antibodies were then detected by an instrument sensor. Data were then collected and output to a GraphPad Prism for affinity curve fitting.

[0229] Compared to conventional methods for measuring antibody-antigen interaction affinity, in BIACORE ® Surface plasmon resonance (SPR) on cells offers numerous advantages: (i) no labeling of antibodies or antigens is required; (ii) antibodies do not require pre-purification and can be used directly from cell culture supernatant; (iii) real-time measurements allowing for rapid semi-quantitative comparisons of interactions between different monoclonal antibodies are feasible and sufficient for many assessment purposes; (iv) biologically specific surfaces can be regenerated, making it easy to compare a range of different monoclonal antibodies under identical conditions; and (v) the analytical procedure is fully automated and allows for extensive series measurements without user intervention. (BIA Applications Handbook, Edition AB (1998 reprint), BIACORE) ® Code number BR-1001-86; BIAtechnology Handbook, version AB (1998 reprint), BIACORE ® Code number BR-1001-84.

[0230] SPR-based binding studies require immobilizing one member of a binding pair onto the sensor surface. The immobilized binding pair is called a ligand. The binding pair in solution is called the analyte. In some cases, the ligand is indirectly attached to the surface by binding to another immobilized molecule, which is called a trapping molecule. The SPR response reflects the change in mass concentration at the detector surface when the analyte binds or dissociates.

[0231] Based on SPR, real-time BIACORE ® The measurement directly monitors the interaction as it occurs. This technique is well-suited for determining kinetic parameters. Comparing affinity rankings is extremely simple to perform, and both the kinetic and affinity constants can be derived from the sensor data.

[0232] When an analyte is injected into the ligand surface in discrete pulses, the resulting sensor map can be divided into three basic phases: (i) association of the analyte with the ligand during sample injection; (ii) equilibrium or steady state during sample injection, where the analyte binding rate is balanced by dissociation from the complex; and (iii) dissociation of the analyte from the surface during buffer flow.

[0233] The association and dissociation phases provide information about the kinetics of analyte-ligand interactions (k... a and k d The rate of complex formation and dissociation, k d / k a =K D The equilibrium phase provides information about the affinity (K) of analyte-ligand interactions. D ).

[0234] BIAevaluation software provides comprehensive tools for curve fitting using both numerical integration and global fitting algorithms. Through appropriate data analysis, it is possible to perform curve fitting from simple BIACORE. ® The study yielded individual rates of interaction and affinity constants. The range of affinities measurable using this technique is very wide, from mM to pM.

[0235] Epitope specificity is an important characteristic of monoclonal antibodies. Compared with conventional techniques using radioimmunoassay, ELISA, or other surface adsorption methods, using BIACORE... ® Epitope mapping does not require labeling or purifying antibodies and allows for multi-site specificity assays using sequences from several monoclonal antibodies. Furthermore, it can automate large-scale analysis.

[0236] Pair binding assays test the ability of two MAbs to simultaneously bind to the same antigen. MAbs targeting different epitopes will bind independently, while MAbs targeting the same or closely related epitopes will interfere with each other's binding. These assays use BIACORE. ® The combination experiment is easy to conduct.

[0237] For example, this experiment can use a capture molecule to bind to a first Mab, followed by the sequential addition of an antigen and a second Mab. The sensor map will reveal: 1. how much antigen binds to the first Mab, 2. the extent to which the second Mab binds to the surface-attached antigen, and 3. whether reversing the order of the pairwise tests changes the results if the second Mab does not bind.

[0238] Peptide inhibition is another technique used for epitope mapping. This method complements paired antibody binding studies and, when the primary sequence of the antigen is known, can correlate functional epitopes with structural features. The inhibition of binding of peptides or antigen fragments to immobilized antigens by different MAbs is tested. Peptides that interfere with the binding of a given MAb are considered structurally associated with the epitope defined by that MAb.

[0239] VIII. Diagnostic Uses In some aspects provided herein, bispecific antibodies against *Pseudomonas aeruginosa* Psl and PcrV with modified Fc regions can be used to detect the presence of *Pseudomonas aeruginosa* Psl and / or PcrV in a sample or individual. As used herein, the term "detection" encompasses both quantitative and qualitative detection. This document provides methods for using the antibodies of this disclosure for diagnostic purposes, such as detecting *Pseudomonas aeruginosa* Psl and / or PcrV in an individual or tissue samples derived from an individual. In some aspects, the individual is a human. Detection methods may involve the quantification of antigen-binding antibodies. Antibody detection in biological samples can be performed using any method known in the art, including immunofluorescence microscopy, immunocytochemistry, immunohistochemistry, ELISA, FACS analysis, immunoprecipitation, or micropositron emission tomography. In some aspects, antibodies are radiolabeled, for example, with 18F, and subsequently detected using micropositron emission tomography analysis. Antibody binding can also be quantified in patients using non-invasive techniques such as positron emission tomography (PET), X-ray computed tomography, single-photon emission computed tomography (SPECT), computed tomography (CT), and computed axial computed tomography (CAT).

[0240] The present invention also relates to the following embodiments: 1. A bispecific antibody that specifically binds to Pseudomonas aeruginosa PcrV protein and Psl extracellular polysaccharide, wherein the antibody comprises a modified IgG Fc region, the modified IgG Fc region containing amino acid substitutions at two or more positions relative to the wild-type IgG Fc region at positions 432 to 437 according to the Kabat EU number index; wherein (i) Positions 432 and 437 are each replaced by cysteine; (ii) Position 433 is histidine or is replaced by arginine, proline, threonine, lysine, serine, alanine, methionine or asparagine. (iii) Position 434 is asparagine or is replaced by arginine, tryptophan, histidine, phenylalanine, tyrosine, serine, methionine or threonine. (iv) Position 435 is histidine; and (v) Position 436 is tyrosine or phenylalanine, or is substituted by leucine, arginine, isoleucine, lysine, methionine, valine, histidine, serine, or threonine; and The antibody has an increased half-life compared to the half-life of the corresponding antibody having the wild-type IgG Fc region.

[0241] 2. The bispecific antibody according to embodiment 1, wherein the modified IgG Fc region is a modified IgG1 Fc region.

[0242] 3. The bispecific antibody according to embodiment 1 or 2, wherein the modified IgG Fc region is a modified human IgG Fc region.

[0243] 4. The bispecific antibody according to any one of embodiments 1 to 3, wherein the bispecific antibody exhibits less aggregation in solution compared to antibodies comprising a heavy chain containing the amino acid sequence of SEQ ID NO:19 and a light chain containing the amino acid sequence of SEQ ID NO:20.

[0244] 5. A bispecific antibody according to any one of embodiments 1 to 4, wherein the bispecific antibody promotes opsonization and phagocytic activity of Pseudomonas aeruginosa, optionally wherein the bispecific antibody mediates in vitro opsonization and phagocytic activity of Pseudomonas aeruginosa similar to antibodies comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 19 and a light chain containing the amino acid sequence of SEQ ID NO: 20.

[0245] 6. The bispecific antibody according to any one of embodiments 1 to 5, wherein the bispecific antibody further comprises an amino acid insertion after position 437, optionally wherein the amino acid insertion is glutamic acid.

[0246] 7. The bispecific antibody according to any one of embodiments 1 to 6, wherein the bispecific antibody has a higher binding affinity for FcRn at pH 6.0 than the corresponding antibody having the wild-type human IgG1 Fc region has a higher binding affinity for FcRn at pH 6.

[0247] 8. The bispecific antibody according to any one of embodiments 1 to 7, wherein the bispecific antibody has a higher binding affinity for FcRn at pH 7.4 than the corresponding antibody having the wild-type human IgG1 Fc region has a higher binding affinity for FcRn at pH 7.4.

[0248] 9. A bispecific antibody according to any one of embodiments 1 to 8, wherein the modified human IgG1 Fc region exhibits increased pH-dependent binding affinity for FcRn compared to a corresponding antibody having the wild-type human IgG1 Fc region.

[0249] 10. The bispecific antibody according to any one of embodiments 1 to 9, wherein the modified human IgG1 Fc region has amino acid substitutions at three of the positions 432, 433, 434, 435, 436 and 437.

[0250] 11. The bispecific antibody according to any one of embodiments 1 to 9, wherein the modified human IgG1 Fc region has amino acid substitutions at four positions 432, 433, 434, 435, 436 and 437.

[0251] 12. The bispecific antibody according to any one of embodiments 1 to 9, wherein the modified human IgG1 Fc region has amino acid substitutions at five positions 432, 433, 434, 435, 436 and 437.

[0252] 13. The bispecific antibody according to any one of embodiments 1 to 9, wherein the modified human IgG1 Fc region has amino acid substitutions at six positions 432, 433, 434, 435, 436 and 437.

[0253] 14. The bispecific antibody according to any one of embodiments 1 to 9, wherein the modified human IgG1 Fc region comprises the amino acid sequence of SEQ ID NO:44 or the amino acid sequence of SEQ ID NO:33.

[0254] 15. The bispecific antibody according to any one of embodiments 1 to 14, wherein the bispecific antibody is not HexaBody.

[0255] 16. The bispecific antibody according to any one of embodiments 1 to 15, wherein the bispecific antibody competitively inhibits the binding of an antibody comprising a heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO:13 and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO:14 to PcrV.

[0256] 17. The bispecific antibody according to any one of embodiments 1 to 16, wherein the epitope of PcrV bound by the bispecific antibody is the same as the epitope of PcrV bound by an antibody comprising VH containing the amino acid sequence of SEQ ID NO: 13 and VL containing the amino acid sequence of SEQ ID NO: 14.

[0257] 18. The bispecific antibody according to any one of embodiments 1 to 17, wherein the bispecific antibody comprises an antigen-binding domain that binds to the Pseudomonas aeruginosa PcrV protein and comprises VH-CDR1 containing the amino acid sequence of SEQ ID NO:1, VH-CDR2 containing the amino acid sequence of SEQ ID NO:2, VH-CDR3 containing the amino acid sequence of SEQ ID NO:3, VL-CDR1 containing the amino acid sequence of SEQ ID NO:4, VL-CDR2 containing the amino acid sequence of SEQ ID NO:5, and VL-CDR3 containing the amino acid sequence of SEQ ID NO:6.

[0258] 19. The bispecific antibody according to embodiment 18, wherein the antigen-binding domain binding to the Pseudomonas aeruginosa PcrV protein comprises VH containing the amino acid sequence of SEQ ID NO:13 and / or VL containing the amino acid sequence of SEQ ID NO:14.

[0259] 20. The bispecific antibody according to embodiment 18 or embodiment 19, wherein the antigen-binding domain binding to the Pseudomonas aeruginosa PcrV protein comprises a heavy chain variable region and a light chain variable region on separate polypeptides.

[0260] 21. The bispecific antibody according to any one of embodiments 1 to 20, wherein the bispecific antibody competitively inhibits the binding of an antibody comprising VH containing the amino acid sequence of SEQ ID NO:15 and VL containing the amino acid sequence of SEQ ID NO:16 to Psl.

[0261] 22. The bispecific antibody according to any one of embodiments 1 to 21, wherein the epitope of Psl bound by the bispecific antibody is the same as the epitope of Psl bound by an antibody comprising VH containing the amino acid sequence of SEQ ID NO:15 and VL containing the amino acid sequence of SEQ ID NO:16.

[0262] 23. The bispecific antibody according to any one of embodiments 1 to 22, wherein the antibody comprises an antigen-binding domain that binds to the extracellular polysaccharide of Pseudomonas aeruginosa Psl and comprises a heavy chain variable region VH-CDR1 containing the amino acid sequence of SEQ ID NO:7, a VH-CDR2 containing the amino acid sequence of SEQ ID NO:8, a VH-CDR3 containing the amino acid sequence of SEQ ID NO:9, a light chain variable region VL-CDR1 containing the amino acid sequence of SEQ ID NO:10, a VL-CDR2 containing the amino acid sequence of SEQ ID NO:11, and a VL-CDR3 containing the amino acid sequence of SEQ ID NO:12.

[0263] 24. The bispecific antibody according to embodiment 23, wherein the antigen-binding domain binding to the Pseudomonas aeruginosa Psl extracellular polysaccharide comprises a VH containing the amino acid sequence of SEQ ID NO:15 and / or a VL containing the amino acid sequence of SEQ ID NO:16.

[0264] 25. The bispecific antibody according to embodiment 23 or 24, wherein the antigen-binding domain binding to the Pseudomonas aeruginosa Psl extracellular polysaccharide comprises VH and VL on the same polypeptide.

[0265] 26. The bispecific antibody according to any one of embodiments 23 to 25, wherein the antigen-binding domain bound to the Pseudomonas aeruginosa Psl extracellular polysaccharide includes a linker between the VH and the VL, optionally wherein the linker includes the amino acid sequence of SEQ ID NO:18.

[0266] 27. The bispecific antibody according to any one of embodiments 23 to 26, wherein the antigen-binding domain binding to the Pseudomonas aeruginosa Psl extracellular polysaccharide comprises scFv.

[0267] 28. The bispecific antibody according to embodiment 27, wherein the scFv comprises a linker, optionally wherein the linker comprises the amino acid sequence of SEQ ID NO:18.

[0268] 29. The bispecific antibody according to embodiment 27 or embodiment 28, wherein the scFv is oriented at VH-linker-VL.

[0269] 30. The bispecific antibody according to any one of embodiments 27 to 29, wherein the scFv comprises the amino acid sequence of SEQ ID NO:17.

[0270] 31. The bispecific antibody according to any one of embodiments 27 to 30, wherein the scFv and the VH of the antigen-binding domain of the Pseudomonas aeruginosa PcrV protein are on the same polypeptide chain.

[0271] 32. The bispecific antibody according to any one of embodiments 27 to 31, wherein the scFv is the C-terminus of the VH of the antigen-binding domain of the Pseudomonas aeruginosa PcrV protein.

[0272] 33. A bispecific antibody according to any one of embodiments 1 to 32, wherein the bispecific antibody comprises (i) a heavy chain of formula VH-CH1-H1-L1-S-L2-H2-CH2-CH3, wherein VH is a variable domain of the anti-Pseudomonas aeruginosa PcrV heavy chain; 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 constant region.

[0273] 34. The bispecific antibody according to embodiment 33, wherein CH1 contains the amino acid sequence of SEQ ID NO:21.

[0274] 35. The bispecific antibody according to embodiment 33 or embodiment 34, wherein H1 comprises the amino acid sequence of SEQ ID NO:22.

[0275] 36. The bispecific antibody according to any one of embodiments 33 to 35, wherein L1 comprises the amino acid sequence of SEQ ID NO:28.

[0276] 37. The bispecific antibody according to any one of embodiments 33 to 36, wherein L2 comprises the amino acid sequence of SEQ ID NO:28.

[0277] 38. The bispecific antibody according to any one of embodiments 33 to 37, wherein H2 comprises the amino acid sequence of SEQ ID NO:23.

[0278] 39. The bispecific antibody according to any one of embodiments 33 to 38, wherein CH2-CH3 comprises the amino acid sequence of SEQ ID NO:30.

[0279] 40. The bispecific antibody according to any one of embodiments 33 to 39, wherein CL is the constant region of the antibody light chain κ.

[0280] 41. The bispecific antibody according to any one of embodiments 33 to 40, wherein CL comprises the amino acid sequence of SEQ ID NO:24.

[0281] 42. The bispecific antibody according to any one of embodiments 1 to 41, wherein the bispecific antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NO:31 and / or a light chain containing the amino acid sequence of SEQ ID NO:20.

[0282] 43. An isolated polynucleotide comprising a nucleic acid molecule encoding the heavy chain of a bispecific antibody according to any one of embodiments 1 to 42.

[0283] 44. The isolated polynucleotide according to embodiment 43, wherein the isolated polynucleotide further comprises a nucleic acid molecule encoding the light chain of the bispecific antibody according to any one of embodiments 1 to 42.

[0284] 45. A vector comprising a polynucleotide according to embodiment 43 or 44.

[0285] 46. ​​A host cell comprising (i) a polynucleotide according to embodiment 43 or 44, or (ii) a vector according to embodiment 45.

[0286] 47. A method for generating a bispecific antibody, the method comprising culturing a host cell according to embodiment 46, and optionally isolating the bispecific antibody.

[0287] 48. A bispecific antibody, said bispecific antibody being generated by the method according to embodiment 47.

[0288] 49. A composition comprising a bispecific antibody according to any one of embodiments 1 to 42 or 48 and a pharmaceutically acceptable carrier.

[0289] 50. A method for treating or preventing a Pseudomonas infection in a subject of need, the method comprising administering to the subject a bispecific antibody according to any one of embodiments 1 to 42 or 48 or a composition according to embodiment 49.

[0290] 51. The method according to embodiment 50, wherein the infection is a lung infection, respiratory tract infection, pneumonia, bacteremia, bone infection, joint infection, skin infection, burn infection, wound infection, or any combination thereof.

[0291] 52. A method of treating bronchiectasis in a subject in need, the method comprising administering to the subject a bispecific antibody according to any one of embodiments 1 to 42 or 48 or a composition according to embodiment 49.

[0292] 53. A method for improving the forced expiratory volume 1 (FEV1) before bronchodilator in a subject suffering from bronchiectasis, the method comprising administering to the subject a bispecific antibody according to any one of embodiments 1 to 42 or 48 or a composition according to embodiment 49.

[0293] 54. A method for reducing the Pseudomonas aeruginosa load in a subject suffering from bronchiectasis, the method comprising administering to the subject a bispecific antibody according to any one of embodiments 1 to 42 or 48 or a composition according to embodiment 49.

[0294] 55. A method for reducing bronchiectasis exacerbation in a subject in need, the method comprising administering to the subject a bispecific antibody according to any one of embodiments 1 to 42 or 48 or a composition according to embodiment 49.

[0295] 56. A method for reducing the need for intravenous antibiotics in a subject suffering from bronchiectasis, the method comprising administering to the subject a bispecific antibody according to any one of embodiments 1 to 42 or 48 or a composition according to embodiment 49.

[0296] 57. A method for stabilizing lung function in a subject suffering from bronchiectasis, the method comprising administering to the subject a bispecific antibody according to any one of embodiments 1 to 42 or 48 or a composition according to embodiment 49.

[0297] 58. The method according to any one of embodiments 52 to 57, wherein the bronchiectasis is noncystic fibrotic bronchiectasis.

[0298] 59. The method according to any one of embodiments 50 to 58, wherein the method further comprises administering antibiotics.

[0299] 60. The method according to any one of embodiments 50 to 59, wherein the subject is colonized by Pseudomonas aeruginosa, optionally wherein the subject's respiratory tract is colonized by Pseudomonas aeruginosa.

[0300] 61. Use of the bispecific antibody of any one of embodiments 1 to 42 or 48, or the composition of embodiment 49, in the preparation of a medicament for use in any one of embodiments 50 to 60.

[0301] 62. The bispecific antibody according to any one of embodiments 1 to 42 or 48, or the composition according to embodiment 49, for use in the method according to any one of embodiments 50 to 60.

[0302] *** Unless otherwise stated, the practice of this disclosure will employ conventional techniques within the scope of the art, including those of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology. These techniques are well explained in the literature. See, for example, *Molecular Cloning: A Laboratory Manual*, 2nd edition, edited by Sambrook et al., Cold Spring Harbor Laboratory Press: (1989); *Molecular Cloning: A Laboratory Manual*, edited by Sambrook et al., Cold Springs Harbor Laboratory, New York (1992); *DNA Cloning*, edited by DN Glover, Volumes I and II (1985); *Oligonucleotide Synthesis*, edited by MJ Gait, (1984); U.S. Patent No. 4,683,195 to Mullis et al.; *Nucleic Acid Hybridization*, edited by BD Hames & SJ Higgins (1984); *Transcription And Translation*, edited by BD Hames & SJ Higgins (1984); *Culture Of Animal Cells*, RI Freshney, Alan R. Liss, Inc., (1987); *Immobilized Cells And Enzymes*, IRL Press, (1986); B. Perbal, A Practical Guide To MolecularCloning (1984); Paper, Methods In Enzymology, Academic Press, Inc., NY; GeneTransfer Vectors For Mammalian Cells, JH Miller and MPCalos (ed.), Cold Spring Harbor Laboratory (1987); Methods in Enzymology, Volumes 154 and 155 (eds. Wu et al.); Immunochemical Methods in Cell and Molecular Biology, edited by Mayer and Walker, Academic Press, London (1987); Handbook of Experimental Immunology, Volumes I-IV, edited by D.M. Weir and C.C. Blackwell (1986); Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1986); and Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1989).

[0303] The general principles of antibody engineering are described in *Antibody Engineering*, 2nd ed., edited by CAKBorrebaeck, Oxford University Press (1995). The general principles of protein engineering are described in *Protein Engineering*, *A Practical Approach*, edited by Rickwood, D. et al., IRL Press at Oxford University Press, Oxford, Eng. (1995). The general principles of antibody and antibody-hapten binding are described in the following references: Nisonoff, A., *Molecular Immunology*, 2nd ed., Sinauer Associates, Sunderland, MA (1984); and Steward, MW, *Antibodies, Their Structure and Function*, Chapman and Hall, New York, NY (1984). In addition, standard immunological methods known in the art but not specifically described generally follow Current Protocols in Immunology, John Wiley & Sons, New York; Stites et al. (eds.), Basic and Clinical - Immunology (8th edition), Appleton & Lange, Norwalk, CT (1994); and Mishell and Shiigi (eds.), Selected Methods in Cellular Immunology, WHFreeman and Co., New York (1980).

[0304] Standard references explaining the general principles of immunology include: Current Protocols in Immunology, John Wiley & Sons, New York; Klein, J., Immunology: The Science of Self-Nonself Discrimination, John Wiley & Sons, New York (1982); Kennett, R. et al., eds., Monoclonal Antibodies, Hybridoma: A New Dimension in Biological Analyses, Plenum Press, New York (1980); Campbell, A., “Monoclonal Antibody Technology” in Burden, R. et al., eds., Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 13, Elsevere, Amsterdam (1984); Kuby Immunnology, 4th edition, Richard A. Goldsby, Thomas J. Kindt and Barbara A. Osborne, H., eds., Freemand & Co. (2000); Roitt, I., Brostoff, J. and Male D., Immunology 6th edition London: Mosby (2001); Abbas A., Abul, A. and Lichtman, A., Cellular and Molecular Immunology 5th edition, Elsevier Health Sciences Division (2005); Kontermann and Dubel, AntibodyEngineering, Springer Verlan (2001); Sambrook and Russell, Molecular Cloning: ALLaboratory Manual.Cold Spring Harbor Press (2001); Lewin, Genes VIII, PrenticeHall (2003); Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988); Dieffenbach and Dveksler, PCR Primer Cold Spring Harbor Press (2003). .

[0305] Example This disclosure is illustrated by the following examples. It should be understood that the specific embodiments, materials, quantities, and procedures are to be interpreted broadly within the scope and spirit of this disclosure set forth herein.

[0306] Example 1: Construction of AZD0292 and modulation of phagocytic killing and anticytotoxic activities Grebamazepine antibody (also known as MEDI3902) is a bispecific antibody containing an anti-Psl antigen-binding domain and an anti-PcrV antigen-binding domain. The sequence of grebamazepine antibody is provided in Table 1 above. A novel antibody called AZD0292 was constructed using the same anti-Psl and anti-PcrV antigen-binding domains, but with an N3Y half-life extension modification within the CH3 domain. The AZD0292 antibody was constructed as described in DiGiandomenico et al., 2014 (Sci. Trans. Med.), except that the wild-type sequence MHEALHNHYTQKSLSLS (SEQ ID NO: 32) was replaced by a sequence (underlined) containing the modified Fc subsequence with the N3Y mutation: MHEA CSY H LC QKSLSLS (SEQ ID NO: 33).

[0307] Monoclonal antibodies targeting Psl and PcrV are known to mediate opsonization-phagocytosis (OPK) and anticytotoxic activity against *Pseudomonas aeruginosa*, respectively (see DiGiandomenico et al., 2014 Sci. Trans. Med.). Therefore, the ability of AZD0292 to mediate complement-dependent opsonization-phagocytosis activity in *P. aeruginosa* strains was evaluated compared to gravidarumab, the anti-Psl monoclonal antibody (mAb) Psl0096, the anti-PcrV mAb V2L2-MD, and control IgG (DiGiandomenico et al., 2014 Sci. Trans. Med.). The assay was performed in 96-well plates, using 0.025 mL of each component: *P. aeruginosa* strain PAO1 (DiGiandomenico et al., 2012 J. Exp. Med.), diluted baby rabbit serum (Cedar Lane), differentiated HL-60 cells, and the monoclonal antibody. Data were acquired using the Tecan Spark multimodal microplate reader (Tecan) and then plotted as percentage kill compared to antibody-deficient controls at different temperatures. Results were... Figure 1A As shown in the figure, AZD0292, gravidamen, and anti-Psl mAb Psl0096 mediate similar OPK activity against Pseudomonas aeruginosa. As expected, no opsonization phagocytic activity was observed with anti-PcrV mAb V2L2-MD or control IgG antibody.

[0308] The anticytotoxic activity of AZD0292 was also evaluated. This assay was performed as described in DiGiandomenico et al., 2014, Sci. Trans. Med. The antibody to be tested was first added to A549 cells (2 × 10⁻⁶ cells). 4 Cells were seeded in white 96-well plates (provided by Nunc Nunclon Delta) on Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Log-phase *Pseudomonas aeruginosa* strain 6077 expressing extracellular enzyme U was then added at a desired multiplicity of infection (MOI) of 10 and incubated at 37°C, 5% CO2 for 2 hours. Lactate dehydrogenase release from lysed cells was then measured. Data were obtained using the Tecan Spark multimodal microplate reader (Tecan). Results are presented in […]. Figure 1B As shown in the figure. In this assay, AZD0292, gravidamen, and anti-PcrVmAb V2L2-MD prevented cell death with similar efficacy, while control IgG and anti-Psl mAb Psl0096 did not have the expected protective activity.

[0309] These results indicate that AZD0292 and grabamab have equivalent effects in the assays of anti-Psl and anti-PcrV functional activity.

[0310] Next, to test the effects of different half-life extension techniques, the ability of AZD0292 to mediate complement-dependent opsonization phagocytic activity in *Pseudomonas aeruginosa* strains was evaluated compared to ghrebamazine, an afucosylated form of ghrebamazine with a YTE half-life extension (SEQ ID NO:43) but without the YTE half-life extension (M252Y / S254T / T256E substitution in the Fc region, according to Kabat numbering) and control IgG (DiGiandomenico et al., 2014 Sci. Trans. Med). Afucosylated IgG was generated using a transient expression system from Chinese hamster ovaries (CHO). Several methods for generating recombinant afucosylated proteins have now been established. For example, the titration of fucose into cell culture medium (Louie et al., Biotechnol Bioeng. Mar 2017; 114(3):632-644), co-expression of GDP-6-deoxy-D-lythreo-4-hexylose reductase (von Horsten et al., Glycobiology. Dec 2010; 20(12):1607-18), co-expression of anti-FUT8 intracellular antibody (Joubert et al., Biotechnol Bioeng. Aug 2022; 119(8):2206-2220), and FUT8 knockout cell lines (Yamane-Ohnuki et al., Biotechnol Bioeng. Sep 5 2004; 87(5):614-22; Malphettes et al., Biotechnol Bioeng. Aug 1 2010; 106(5):774-83) and other strategies (Pereira et al., MAbs. July 2018; 10(5):693-711).

[0311] The assay was performed in 96-well plates, with 0.025 mL of each component used; *Pseudomonas aeruginosa* strain PAO1 (DiGiandomenico et al., 2012 J. Exp. Med), diluted young rabbit serum (Cedar Lane), differentiated HL-60 cells, and monoclonal antibody. Data were obtained using a Tecan Spark multimodal microplate reader (Tecan) and then plotted as percentage of kill compared to an antibody-deficient control at different temperatures. Results were... Figure 2As shown in the figure, AZD0292, grubab, and grubab-Afuc mediate similar OPK activity against Pseudomonas aeruginosa. However, grubab-Afuc-YTE exhibited reduced activity compared to AZD0292, grubab, and grubab-Afu. No opsonization phagocytic activity was observed with the control IgG antibody.

[0312] These results surprisingly demonstrate that different half-life extension techniques for modifying the Fc region can affect the opsonization and phagocytic activity of antibodies. Although the YTE half-life extension modification resulted in a significant decrease in activity (note that the activity of fucosylated glimebumab was similar to that of glimebumab), the N3Y half-life extension modification did not affect the opsonization and phagocytic activity.

[0313] Example 2: AZD0292 has increased serum exposure compared to glimepiride. The N3Y modification of AZD0292 increases its binding affinity to the neonatal Fc receptor (FcRn), which functions as a recirculating receptor responsible for maintaining circulating IgG. To confirm that AZD0292 exhibits increased exposure relative to glimepiride, the pharmacokinetics of each molecule were compared in a Tg32 human FcRn transgenic mouse model.

[0314] In pharmacokinetic analysis, both AZD0292 and grabamab (10 mg / kg) were intravenously delivered to 7-week-old mice. Blood samples were collected at 1, 4, 12, 54, 72, and 102 hours and at 7, 9, 11, 14, 17, 21, 24, 28, 35, and 42 days after antibody administration. Blood was collected in BD microcapsule blood collection tubes, and serum was then processed by centrifugation at 500 × g for approximately 10 minutes. The processed serum was stored at -80°C until antibody quantification.

[0315] Quantification of ghrebamab and AZD0292 in mouse serum was performed using an antigen-specific ELISA. NuncMaxiSorp plates (Thermo Fisher Scientific) were coated overnight at 4°C with anti-idiotype antibodies targeting the anti-PcrV fraction of ghrebamab and AZD0292. After washing with PBS containing 0.1% Tween 20 (wash buffer), the plates were blocked at room temperature (RT) with PBS + 5% BSA for 1 hour. After washing three times with wash buffer, the plates were incubated with mouse serum diluted in PBS. AZD0292 or ghrebamab was then used as a standard.

[0316] After 1.5 hours of incubation, the plate was washed and incubated for 30 minutes with 0.05 mL of anti-idiotypic antibody targeting the anti-Psl arm of gravidamen / MEDI3902 and AZD0292 by shaking (200 rpm) at room temperature (RT). After washing three times with washing buffer, 0.05 mL of horseradish peroxidase (HRP)-conjugated goat anti-human IgG (1:10,000; Jackson Laboratories) was added, and the plate was incubated for 30 minutes at room temperature. After washing, 0.05 mL of 3,3',5,5'-tetramethylbenzidine (TMB) substrate (KPL) was added, and the reaction was stopped with 0.05 mL of 0.2 M H2SO4 after approximately 10 minutes. The optical density (OD) at 450 nm was measured using a spectrophotometer (Molecular Devices). 450 ).

[0317] The result is Figure 3 As shown in the figure, after administration of 10 mg / kg IV AZD0292 and glibenclamide, the clearance rate of AZD0292 was estimated to be 50% lower than that of glibenclamide.

[0318] Example 3: AZD0292 under photo-stress and heat-stress retains anti-Psl and anti-PcrV functional activities. To determine whether AZD0292 retains its functional activity after exposure to light and heat stress conditions, its activity was assessed by comparison with similarly treated glimepiride in OPK and anticytotoxic assays.

[0319] Accelerated photo- and heat-stress stability assays were performed as described (Dippel et al., 2023 MABS). Briefly, in the heat-stress assay, the antibody was diluted to 1 mg / mL in PBS (pH 7.2) and incubated at 4°C or 45°C for 2 weeks. In the photo-stress assay, the antibody was prepared at 2.5 mg / mL in PBS (pH 7.2), filled into 1 cc Schott glass vials, stoppered / sealed, and placed in an ICH-compliant photostable chamber (Caron model 6545-2). The samples were exposed to cold white light at 3000 lux for 1 week, for a total exposure of approximately 500,000 lux hours.

[0320] In OPK and anticytotoxicity assays, AZD0292 and glimepiride antibodies under photo- and heat-stressed conditions showed similar performance to those under non-stressed conditions. See also Figure 4A and Figure 4B The percentage changes in monomers, aggregates, and fragments compared to non-stressed materials are shown in Table 2.

[0321] Table 2. This data indicates that there is no difference in the functional activity of AZD0292 or glibenclamide between stressed and non-stressed conditions.

[0322] Example 4: AZD0292 surprisingly exhibits reduced aggregation Studies were conducted to assess whether there were any differences in product quality between clones expressing AZD0292 and MEDI3902 (specifically, assessing aggregate levels, monomer levels, protein concentration, and expression titers).

[0323] To assess the impact of the N3Y mutation in AZD0292 on affinity product aggregate levels, shaker overgrowth screening (SPOG) was performed. In this study, 384 clones were screened, and the top 96 clones were selected based on their expression titers. Small-scale protein A capture was used, followed by aggregation analysis of the top 96 clones using high-throughput size exclusion chromatography (HTSEC).

[0324] For shaker overgrowth screening, in short, during clonal cell line expansion, cell cultures from each stable clone are cultured at 0.7 × 10⁻⁶. 5 Cells / ml were seeded in 96-well plates with a total volume of 350 μl, and fed-batch culture was performed for 10 days using cell growth medium, supplemental nutrients, and glucose. On day 10 of this fed-batch experiment, cell cultures from each evaluated stable clone were harvested. Cell viability and viability were measured for each clone. The cell culture medium was then clarified by centrifugation, and the supernatant samples were sent for rProtein titer analysis (quantified by comparing the peak size from each sample to a calibration curve using high-performance liquid chromatography (HPLC) on an Agilent HP1100 or HP1200 (Agilent Technologies, Santa Clara, CA). HTP aggregation analysis is described in more detail below.

[0325] Used with Freedom EVOware ® Version 2.7 (TECAN Group Ltd.) of Tecan FreedomEVO ® The 200 robotic liquid handling platform (TECAN Group Ltd.) operates a liquid containing 20µL ProPlus (MabSelect SuRe) liquid. ™ Protein purification was performed from 96-well cell cultures using a PhyTip 200µL volume column made of Biotage GB Limited (Hengoed, United Kingdom).

[0326] Phosphate-buffered saline was used as the buffer for column equilibration and the first wash step (wash 1), while the second wash step (wash 2) buffer consisted of 25 mM sodium acetate and 120 mM sodium chloride at pH 5.5. Elution was performed using 100 mM glycine buffer at pH 2.6 or 25 mM sodium acetate buffer at pH 3.6. After purification, the sample was neutralized using 1 M Tris buffer at pH 7.5.

[0327] Aggregation analysis was performed using high-throughput size exclusion chromatography (HTSEC) with UV absorbance at 280 nm. The equipment and software used for chromatographic analysis were purchased from Agilent Technologies Inc., and included the Agilent 1260 Infinity II UHPLC system with a degasser, quaternary pump, isothermal multiplexer, diode array detector (DAD), and multi-column compartments with column selection valves. All UHPLC components were connected via 1.6 mm OD, 0.12 µm ID stainless steel capillaries with stainless steel fittings. System control and data analysis were performed using Agilent OpenLAB CDS ChemStation Edition (version C.01.07). HTSEC analysis was performed using an Acquity UPLC Protein BEH SEC 200 Å, 1.7 µm (Waters) column with a 2.1 × 150 mm (ID × length) ID and a mobile phase (pH 7.0) consisting of 50 mM sodium phosphate and 450 mM arginine. Data visualization and statistical analysis were performed using the JMP Pro16 (SAS) software package.

[0328] Figure 5A and Figure 5B The percentage of monomers from AZD0292 and MEDI3902 is shown. Figure 5A ) and percentage of higher molecular weight aggregates ( Figure 5B The data obtained from this study are represented by a bar chart. "Monomer percentage" refers to the expected monoclonal antibody (with 2 heavy chains and 2 light chains). Data from A5, A6, A9, B9, and E6 are missing due to insufficient sample availability for analysis. The data obtained from this study surprisingly show that AZD0292 has significantly less high molecular weight aggregate material than MEDI3902. This evidence across multiple expression clones suggests that this observation is not merely an artifact of a single clone, but a fundamental aspect of the N3Y mutation in AZD0292 compared to MEDI3902.

[0329] From Figure 5B Another representation of this data is in Figure 6 As shown in the image. Figure 6 The percentage of aggregates (high molecular weight substance) for each clone is shown. Light (fewer aggregates) to dark (more aggregates) coloring is proportional to the percentage of aggregates measured by HTSEC in each sample. This visual representation clearly shows the surprising reduction in the percentage of aggregates of AZD0292 compared to MEDI3902.

[0330] Plot the data Figure 7 The study aimed to assess the presence of any correlation between the percentage of high molecular weight substances (e.g., aggregates), the average concentration of PhyTip protein A purified samples, and the titer from the last day of the fed-batch 96-well plate bioreactor. However, no correlation was observed in the scatter plot matrix. Figure 7 These data indicate that aggregation was not induced by the sample concentration levels within the assessed range, and that aggregation was not induced by the final titer on the feed batch day.

[0331] To further compare the aggregate levels between clones expressing MEDI3902 and AZD0292, the data were tested by comparing them to the normality assumption required using the Student's t-test statistic. The Anderson-Darling and Shapiro-Wilk tests were used for both (AZD0292 and MEDI3902 samples, respectively). Figure 8A and Figure 8B The data were confirmed to be normally distributed (p > 0.05). The t-test used, based on the two-tailed F-test to assess the assumption of equal variance, yielded a p > 0.05. The t-test results showed that the mean aggregation levels observed in clones expressing MEDI3902 and AZD0292 were different, which was statistically significant (p < 0.05 based on 95% confidence interval). Figure 8C On average, the observed difference was that sample AZD0292 had 2.99% fewer aggregates compared to sample MEDI3902. Figure 8C ).

[0332] Figure 9A The cluster percentage was shown, and Figure 9B The antibody titer levels of clones expressing AZD0292 and MEDI3902, measured in all samples, are shown and sorted from low to high aggregate levels (in Figure 9A From left to right, the corresponding final titer levels for the same clone on the feed batch day are shown in the bottom row. Figure 9B middle). Figure 9A The data show reduced aggregation in the AZD0292 sample compared to the MEDI3902 sample. The data indicate that the reduced aggregation in AZD0292 is not correlated with a decrease in titer level.

[0333] Figure 10The aggregate percentage data for AZD0292 and MEDI3902 over three months at 40°C are shown. The increase in aggregate percentage of AZD0292 and MEDI3902 over time is comparable, indicating that the two molecules (MEDI3902 and AZD0292) have similar degradation rates.

[0334] Overall, these results indicate that clones expressing AZD0292 had significantly fewer aggregates than clones expressing MEDI3902. Although the mean expression titer of clones expressing MEDI3902 was slightly higher than that of clones expressing AZD0292, the difference was not significant. Data showed that the relative aggregate levels in AZD0292 ranged from 3.9% to 18.0%, while the aggregate levels in MEDI3902 ranged from 7.0% to 22.2%. Figure 9A and Figure 9B However, clones expressing AZD0292 reported 3% fewer aggregates than those expressing MEDI3902. This percentage difference in aggregate levels was greater than the analytical method error (0.1% area difference) and therefore a significant difference. Thus, clones expressing AZD0292 reported significantly fewer aggregates than those expressing MEDI3902. The observed aggregate levels showed no correlation with expression titer, confluence, viable cell density, or cell viability. These data surprisingly suggest that the N3Y mutation in AZD0292 reduces aggregate levels relative to MEDI3902.

[0335] All patents, patent applications (including provisional patent applications), publications (including patent publications and non-patent publications) cited herein, including their full disclosures and electronically available materials (including nucleotide sequence submissions in, for example, GenBank and RefSeq, and amino acid sequence submissions in, for example, SwissProt, PIR, PRF, PDB, and translations of annotated coding regions from GenBank and RefSeq), are incorporated herein by reference. The specific embodiments and examples described above are given for clarity only. Unnecessary limitations should not be construed as such. The invention is not limited to the exact details shown and described, as variations that will be apparent to those skilled in the art will be included within the scope of the invention as defined by the claims.

Claims

1. A bispecific antibody that specifically binds to Pseudomonas aeruginosa PcrV protein and Psl extracellular polysaccharide, wherein the antibody comprises a modified IgG Fc region, the modified IgG Fc region containing amino acid substitutions at two or more positions relative to the wild-type IgG Fc region at positions 432 to 437 according to the Kabat EU number index; wherein (i) Positions 432 and 437 are each replaced by cysteine; (ii) Position 433 is histidine or is replaced by arginine, proline, threonine, lysine, serine, alanine, methionine or asparagine. (iii) Position 434 is asparagine or is replaced by arginine, tryptophan, histidine, phenylalanine, tyrosine, serine, methionine or threonine. (iv) Position 435 is histidine; and (v) Position 436 is tyrosine or phenylalanine, or is substituted by leucine, arginine, isoleucine, lysine, methionine, valine, histidine, serine, or threonine; and The antibody has an increased half-life compared to the half-life of the corresponding antibody having the wild-type IgG Fc region.

2. The bispecific antibody according to claim 1, wherein the bispecific antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NO:31 and / or a light chain containing the amino acid sequence of SEQ ID NO:

20.

3. An isolated polynucleotide comprising a nucleic acid molecule encoding the heavy chain of the bispecific antibody according to claim 1.

4. A vector comprising the polynucleotide according to claim 3.

5. A host cell comprising (i) the polynucleotide of claim 3, or (ii) the vector of claim 4.

6. A method for generating a bispecific antibody, the method comprising culturing a host cell according to claim 5, and optionally isolating the bispecific antibody.

7. A bispecific antibody, said bispecific antibody being generated by the method according to claim 6.

8. A composition comprising a bispecific antibody according to any one of claims 1 to 2 or 7 and a pharmaceutically acceptable carrier.

9. A method for treating or preventing Pseudomonas infection in a subject of need, the method comprising administering to the subject a bispecific antibody according to any one of claims 1 to 2 or 7 or a composition according to claim 8.

10. A method of treating bronchiectasis in a subject in need, the method comprising administering to the subject a bispecific antibody according to any one of claims 1 to 2 or 7 or a composition according to claim 8.

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