WO2022046997A1

WO2022046997A1 - Bama-binding agents and methods of use thereof

Info

Publication number: WO2022046997A1
Application number: PCT/US2021/047697
Authority: WO
Inventors: Jade BOJKOVIC; Jessica FERREYRA; Yan Wang
Original assignee: Ngm Biopharmaceuticals, Inc.
Priority date: 2020-08-27
Filing date: 2021-08-26
Publication date: 2022-03-03

Abstract

The present disclosure provides binding agents, such as VHHs, that specifically bind Acinetobacter BamA, as well as compositions comprising the binding agents, and methods of their use. The disclosure also provides related polynucleotides and vectors encoding the binding agents and cells comprising the binding agents.

Description

BAMA-BINDING AGENTS AND METHODS OF USE THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. Serial No. 63/070,996 filed August 27, 2020, which is incorporated herein by reference in its entirety.

SEQUENCE LISTING

The present specification is being filed with a computer readable form (CRF) copy of the Sequence Listing in ASCII text format submitted via EFS-Web. The CRF copy of the Sequence Listing, entitled 13370-115-228_SeqListing_ST25.txt, which was created on August 19, 2021 and is 49,058 bytes in size, is incorporated herein by reference in its entirety.

FIELD OF THE DISCLOSURE

[0001] The present disclosure generally relates to agents that bind bacterial outer membrane protein assembly factor BamA, particularly antibodies that bind Acinetobacter baumannii BamA, as well as compositions comprising the BamA-binding agents. Methods of making the binding agents and methods of using the agents and compositions are also disclosed.

BRIEF SUMMARY

[0002] The present disclosure provides agents that bind Acinetobacter BamA. The agents include, but are not limited to, polypeptides such as antibodies that specifically bind Acinetobacter BamA. The agents may be referred to herein as “BamA-binding agents”. The disclosure provides methods of making a BamA-binding agent. The disclosure provides methods of using a BamA-binding agent. In some embodiments, a BamA-binding agent inhibits Acinetobacter BamA activity. In some embodiments, a BamA-binding agent inhibits growth of Acinetobacter. In some embodiments, a BamA-binding agent is used to treat Acinetobacter infections. In some embodiments, a BamA-binding agent is used in a combination therapy. In some embodiments, a BamA-binding agent is used in combination with at least one additional therapeutic agent.

[0003] The disclosure also provides compositions comprising the BamA-binding agents described herein. In some embodiments, the disclosure provides pharmaceutical compositions comprising the BamA-binding agents described herein. Polynucleotides and/or vectors encoding the BamA-binding agents are provided. Cells comprising the polynucleotides and/or the vectors described herein are also provided. Cells comprising or producing the BamA-binding agents described herein are provided. Methods of making the BamA-binding agents described herein are also provided. [0004] In one aspect, the present disclosure provides agents that bind Acinetobacter BamA. In some embodiments, an agent binds A. baumcinnii BamA. In some embodiments, a BamA-binding agent binds the barrel domain of Acinetobacter BamA. In some embodiments, a BamA-binding agent binds the barrel domain of A. baumannii BamA. In some embodiments, a BamA-binding agent binds SEQ ID NO:2. In some embodiments, a BamA-binding agent binds one or more of the external outer membrane loops of the barrel domain of Acinetobacter BamA. In some embodiments, a BamA-binding agent binds loop 1, loop 2, loop 3, loop 4, loop 5, loop 6, loop 7, and/or loop 8 of the barrel domain of Acinetobacter BamA. In some embodiments, a BamA-binding agent is an antibody. In some embodiments, a BamA-binding agent is a VHH.

[0005] In some embodiments, a BamA-binding agent binds at last one of the external outer membrane loops of the barrel domain of A. baumannii BamA. In some embodiments, a BamA-binding agent binds within amino acids 438-442 of SEQ ID NO: 1. In some embodiments, a BamA-binding agent binds within amino acids 467-472 of SEQ ID NO: 1. In some embodiments, a BamA-binding agent binds within amino acids 496-514 of SEQ ID NO: 1. In some embodiments, a BamA-binding agent binds within amino acids 538-603 of SEQ ID NO: 1. In some embodiments, a BamA-binding agent binds within amino acids 635-643 of SEQ ID NO: 1. In some embodiments, a BamA-binding agent binds within amino acids 672-723 of SEQ ID NO: 1. In some embodiments, a BamA-binding agent binds within amino acids 754-799 of SEQ ID NO: 1. In some embodiments, a BamA-binding agent binds within amino acids 821-832 of SEQ ID NO: 1. In some embodiments, a BamA-binding agent binds a conformational epitope comprising one or more of the external outer membrane loops of BamA.

[0006] In another aspect, the present disclosure provides agents that have at least one or more of the following properties: (i) binds Acinetobacter baumannii; (ii) does not bind E. coli BamA; (iii) is an antagonist of A. baumannii BamA; (iv) inhibits growth of A. baumannii,' (v) inhibits BamA activity, and (vi) inhibits OmpT protease activity.

[0007] In another aspect, the present disclosure provide agents that bind BamA. In some embodiments, the BamA is an Acinetobacter BamA. In some embodiments, the BamA is an

Acinetobacter baumannii BamA. In some embodiments, a BamA-binding agent is an antibody. In some embodiments, a BamA-binding agent is a single domain antibody. In some embodiments, a BamA- binding agent is a single heavy chain variable domain antibody (VHH).

[0008] In some embodiments, a BamA-binding agent comprises a heavy chain variable region CDR1 comprising the amino acid sequence GLSFGLDAYAVA (SEQ ID NO: 11), a heavy chain variable region CDR2 comprising the amino acid sequence CISPTGSRVAYADSAKG (SEQ ID NO: 12), and a heavy chain variable region CDR3 comprising the amino acid sequence SNDKRCSDFGVDRVGY (SEQ ID NO: 13). In some embodiments, a BamA-binding agent comprises a heavy chain variable region CDR1 comprising the amino acid sequence GSTATRDTFSSHRMT (SEQ ID NO:21), a heavy chain variable region CDR2 comprising the amino acid sequence TITGDDITNYTGSVKG (SEQ ID NO:22), and a heavy chain variable region CDR3 comprising the amino acid sequence LERGIWAY (SEQ ID NO:23). In some embodiments, a BamA-binding agent comprises a heavy chain variable region CDR1 comprising the amino acid sequence GFSFRDYSMS (SEQ ID NO:31), a heavy chain variable region CDR2 comprising the amino acid sequence GIRSLGTTTYYADSVKG (SEQ ID NO:32), and a heavy chain variable region CDR3 comprising the amino acid sequence CLGKICDRFGIVDIYS (SEQ ID NO:33). In some embodiments, a BamA-binding agent binds one or more (1, 2, 3, 4, 5, 6, 7, or 8) of the external outer membrane loops of the barrel domain of Acinetobacter BamA.

[0009] In some embodiments, a BamA-binding agent comprises a heavy chain variable region CDR1 comprising the amino acid sequence GLSFGLDAYAVA (SEQ ID NO: 11), a heavy chain variable region CDR2 comprising the amino acid sequence CISPTGSRVAYADSAKG (SEQ ID NO: 12), and a heavy chain variable region CDR3 comprising the amino acid sequence SNDKRCSDFGVDRVGY (SEQ ID NO: 13). In some embodiments, a BamA-binding agent comprises a heavy chain variable region CDR1 comprising the amino acid sequence GLSFGLDAY (SEQ ID NO: 14), a heavy chain variable region CDR2 comprising the amino acid sequence SPTGSR (SEQ ID NO: 15), and a heavy chain variable region CDR3 comprising the amino acid sequence SNDKRCSDFGVDRVGY (SEQ ID NO: 13). In some embodiments, a BamA-binding agent comprises a heavy chain variable region CDR1 comprising the amino acid sequence GLSFGLDAYAVA (SEQ ID NO: 11), a heavy chain variable region CDR2 comprising the amino acid sequence CISPTGSRVA (SEQ ID NO: 16), and a heavy chain variable region CDR3 comprising the amino acid sequence SNDKRCSDFGVDRVGY (SEQ ID NO: 13). In some embodiments, a BamA-binding agent comprises a heavy chain variable region CDR1 comprising the amino acid sequence LDAYAVA (SEQ ID NO: 17), a heavy chain variable region CDR2 comprising the amino acid sequence CISPTGSRVAYADSAKG (SEQ ID NO: 12), and a heavy chain variable region CDR3 comprising the amino acid sequence SNDKRCSDFGVDRVGY (SEQ ID NO: 13). In some embodiments, a BamA-binding agent comprises a heavy chain variable region CDR1 comprising the amino acid sequence GLDAYAVA (SEQ ID NO: 18), a heavy chain variable region CDR2 comprising the amino acid sequence GVSCISPTGSRVA (SEQ ID NO: 19), and a heavy chain variable region CDR3 comprising the amino acid sequence ATSNDKRCSDFGVDRVG (SEQ ID NO:20).

[0010] In some embodiments, a BamA-binding agent comprises a heavy chain variable region having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:41. In some embodiments, a BamA-binding agent comprises a heavy chain variable region having at least 85% sequence identity to the amino acid sequence of SEQ ID NO:41. In some embodiments, a BamA-binding agent comprises a heavy chain variable region having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:41. In some embodiments, a BamA-binding agent comprises a heavy chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:41. In some embodiments, a BamA-binding agent comprises a heavy chain variable region having at least 96% sequence identity to the amino acid sequence of SEQ ID NO:41. In some embodiments, a BamA-binding agent comprises a heavy chain variable region having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:41. In some embodiments, a BamA-binding agent comprises a heavy chain variable region having at least 98% sequence identity to the amino acid sequence of SEQ ID NO:41. In some embodiments, a BamA-binding agent comprises a heavy chain variable region having at least 99% sequence identity to the amino acid sequence of SEQ ID NO:41. In some embodiments, a BamA-binding agent comprises a heavy chain variable region of amino acid sequence SEQ ID NO:41.

[0011] In some embodiments, a BamA-binding agent is VHH-29. In some embodiments, a BamA- binding agent is a humanized version of VHH-29. In some embodiments, a BamA-binding agent is a variant of VHH-29 or a variant of a humanized version of VHH-29.

[0012] In some embodiments, a BamA-binding agent comprises a heavy chain variable region CDR1 comprising the amino acid sequence GSTATRDTFSSHRMT (SEQ ID NO:21), a heavy chain variable region CDR2 comprising the amino acid sequence TITGDDITNYTGSSVKG (SEQ ID NO:22), and a heavy chain variable region CDR3 comprising the amino acid sequence LERGIWAY (SEQ ID NO:23). In some embodiments, a BamA-binding agent comprises a heavy chain variable region CDR1 comprising the amino acid sequence GSTATRDTFSSH (SEQ ID NO:24), a heavy chain variable region CDR2 comprising the amino acid sequence TGDDI (SEQ ID NO:25), and a heavy chain variable region CDR3 comprising the amino acid sequence LERGIWAY (SEQ ID NO:23). In some embodiments, a BamA- binding agent comprises a heavy chain variable region CDR1 comprising the amino acid sequence GSTATRDTFSSHRMT (SEQ ID NO:21), a heavy chain variable region CDR2 comprising the amino acid sequence TITGDDITN (SEQ ID NO:26), and a heavy chain variable region CDR3 comprising the amino acid sequence LERGIWAY (SEQ ID NO:23). In some embodiments, a BamA-binding agent comprises a heavy chain variable region CDR1 comprising the amino acid sequence RDTFSSHRMT (SEQ ID NO:27), a heavy chain variable region CDR2 comprising the amino acid sequence TITGDDITNYTGSSVKG (SEQ ID NO:22), and a heavy chain variable region CDR3 comprising the amino acid sequence LERGIWAY (SEQ ID NO:23). In some embodiments, a BamA-binding agent comprises a heavy chain variable region CDR1 comprising the amino acid sequence TRDTFSSHRMT (SEQ ID NO:28), a heavy chain variable region CDR2 comprising the amino acid sequence MVATITGDDITN (SEQ ID NO:29), and a heavy chain variable region CDR3 comprising the amino acid sequence HLLERGIWA (SEQ ID NO: 30). [0013] In some embodiments, a BamA-binding agent comprises a heavy chain variable region having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:42. In some embodiments, a BamA-binding agent comprises a heavy chain variable region having at least 85% sequence identity to the amino acid sequence of SEQ ID NO:42. In some embodiments, a BamA-binding agent comprises a heavy chain variable region having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:42. In some embodiments, a BamA-binding agent comprises a heavy chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:42. In some embodiments, a BamA-binding agent comprises a heavy chain variable region having at least 96% sequence identity to the amino acid sequence of SEQ ID NO:42. In some embodiments, a BamA-binding agent comprises a heavy chain variable region having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:42. In some embodiments, a BamA-binding agent comprises a heavy chain variable region having at least 98% sequence identity to the amino acid sequence of SEQ ID NO:42. In some embodiments, a BamA-binding agent comprises a heavy chain variable region having at least 99% sequence identity to the amino acid sequence of SEQ ID NO:42. In some embodiments, a BamA-binding agent comprises a heavy chain variable region of amino acid sequence SEQ ID NO:42.

[0014] In some embodiments, a BamA-binding agent is VHH-47. In some embodiments, a BamA- binding agent is a humanized version of VHH-47. In some embodiments, a BamA-binding agent is a variant of VHH-47 or a variant of a humanized version of VHH-47.

[0015] In some embodiments, a BamA-binding agent comprises a heavy chain variable region CDR1 comprising the amino acid sequence GFSFRDYSMS (SEQ ID NO:31), a heavy chain variable region CDR2 comprising the amino acid sequence GIRSLGTTTYYADSVKG (SEQ ID NO:32), and a heavy chain variable region CDR3 comprising the amino acid sequence CLGKICDRFGIVDIYS (SEQ ID NO:33). In some embodiments, a BamA-binding agent comprises a heavy chain variable region CDR1 comprising the amino acid sequence GFSFRDY (SEQ ID NO:34), a heavy chain variable region CDR2 comprising the amino acid sequence RSLGTT (SEQ ID NO:35), and a heavy chain variable region CDR3 comprising the amino acid sequence CLGKICDRFGIVDIYS (SEQ ID NO:33). In some embodiments, a BamA-binding agent comprises a heavy chain variable region CDR1 comprising the amino acid sequence GFSFRDYSMS (SEQ ID NO:31), a heavy chain variable region CDR2 comprising the amino acid sequence GIRSLGTTTY (SEQ ID NO:36), and a heavy chain variable region CDR3 comprising the amino acid sequence CLGKICDRFGIVDIYS (SEQ ID NO:33). In some embodiments, a BamA-binding agent comprises a heavy chain variable region CDR1 comprising the amino acid sequence DYSMS (SEQ ID NO:37), a heavy chain variable region CDR2 comprising the amino acid sequence GIRSLGTTTYYADSVKG (SEQ ID NO:32), and a heavy chain variable region CDR3 comprising the amino acid sequence CLGKICDRFGIVDIYS (SEQ ID NO:33). In some embodiments, a BamA-binding agent comprises a heavy chain variable region CDR1 comprising the amino acid sequence RDYSMS (SEQ ID NO:38), a heavy chain variable region CDR2 comprising the amino acid sequence WVSGIRSLGTTTY (SEQ ID NO:39), and a heavy chain variable region CDR3 comprising the amino acid sequence AKCLGKICDRFGIVDIY (SEQ ID NO: 40).

[0016] In some embodiments, a BamA-binding agent comprises a heavy chain variable region having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:43. In some embodiments, a BamA-binding agent comprises a heavy chain variable region having at least 85% sequence identity to the amino acid sequence of SEQ ID NO:43. In some embodiments, a BamA-binding agent comprises a heavy chain variable region having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:43. In some embodiments, a BamA-binding agent comprises a heavy chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:43. In some embodiments, a BamA-binding agent comprises a heavy chain variable region having at least 96% sequence identity to the amino acid sequence of SEQ ID NO:43. In some embodiments, a BamA-binding agent comprises a heavy chain variable region having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:43. In some embodiments, a BamA-binding agent comprises a heavy chain variable region having at least 98% sequence identity to the amino acid sequence of SEQ ID NO:43. In some embodiments, a BamA-binding agent comprises a heavy chain variable region having at least 99% sequence identity to the amino acid sequence of SEQ ID NO:43. In some embodiments, a BamA-binding agent comprises a heavy chain variable region of amino acid sequence SEQ ID NO:43.

[0017] In some embodiments, a BamA-binding agent is VHH-82. In some embodiments, a BamA- binding agent is a humanized version of VHH-82. In some embodiments, a BamA-binding agent is a variant of VHH-82 or a variant of a humanized version of VHH-82.

[0018] In another aspect of the disclosure, provided herein is a binding agent that competes for binding to Acinetobacter BamA with any of the BamA-binding agents described herein. In some embodiments, provided herein is an agent that competes for binding to Acinetobacter BamA with a reference VHH, wherein the reference VHH comprises a heavy chain variable region CDR1 comprising the amino acid sequence GFSFRDYSMS (SEQ ID NO:31), a heavy chain variable region CDR2 comprising the amino acid sequence GIRSLGTTTYYADSVKG (SEQ ID NO:32), and a heavy chain variable region CDR3 comprising the amino acid sequence CLGKICDRFGIVDIYS (SEQ ID NO:33).

[0019] In some embodiments of each of the aforementioned aspects and embodiments, as well as other aspects and embodiments described herein, a BamA-binding agent is an antibody. In some embodiments, the BamA-binding agent is a VHH. In some embodiments, the BamA-binding agent is a monoclonal antibody. In some embodiments, the BamA-binding agent is a humanized antibody. In some embodiments, the BamA-binding agent is a chimeric antibody. In some embodiments, the BamA-binding agent is a whole or intact antibody. In some embodiments, the BamA-binding agent is a bispecific antibody or a multispecific antibody. In some embodiments, the BamA-binding agent is an antibody fragment comprising at least one antigen-binding site. In some embodiments, the antibody fragment is a Fab, Fab’, F(ab’)2, Fv, scFv, (scFv)2, single chain antibody, dual variable region antibody, single variable region antibody, linear antibody, diabody, nanobody, or a V region antibody. In some embodiments, the antibody fragment is a Fab. In some embodiments, the antibody fragment is a Fab’. In some embodiments, the antibody fragment is a F(ab’)2. In some embodiments, the antibody fragment is a Fv. In some embodiments, the antibody fragment is a scFv. In some embodiments, the antibody fragment is a (SCFV)2. In some embodiments, the antibody fragment is a single chain antibody. In some embodiments, the antibody fragment is a dual variable region antibody. In some embodiments, the antibody fragment is a single variable region antibody. In some embodiments, the antibody fragment is a linear antibody. In some embodiments, the antibody fragment is a diabody. In some embodiments, the antibody fragment is a nanobody. In some embodiments, the antibody fragment is a V region antibody. In some embodiments, the BamA-binding agent is an IgG antibody. In some embodiments, the BamA-binding agent is an IgGl antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody.

[0020] In some embodiments of each of the aforementioned aspects and embodiments, as well as other aspects and embodiments described herein, a BamA-binding agent is attached (either directly or indirectly) to a half-life extending moiety. In some embodiments of each of the aforementioned aspects and embodiments, as well as other aspects and embodiments described herein, a BamA-binding agent is linked or conjugated to an antibacterial agent.

[0021] In some embodiments of each of the aforementioned aspects and embodiments, as well as other aspects and embodiments described herein, a BamA-binding agent comprises a polypeptide comprising at least one of the VHHs described herein. In some embodiments, a BamA-binding agent comprises a polypeptide comprising: a first VHH described herein and a second VHH described herein. In some embodiments, a BamA-binding agent comprises a polypeptide comprising: (a) a first VHH, wherein the first VHH comprises a CDR1 comprising the amino acid sequence GFSFRDYSMS (SEQ ID NO:31), a CDR2 comprising the amino acid sequence GIRSLGTTTYYADSVKG (SEQ ID NO:32), and a CDR3 comprising the amino acid sequence CLGKICDRFGIVDIYS (SEQ ID NO:33); and (b) a second VHH. In some embodiments, the polypeptide comprises a second VHH comprising a CDR1 comprising the amino acid sequence GLSFGLDAYAVA (SEQ ID NO: 11), a CDR2 comprising the amino acid sequence CISPTGSRVAYADSAKG (SEQ ID NO: 12), and a CDR3 comprising the amino acid sequence SNDKRCSDFGVDRVGY (SEQ ID NO: 13). In some embodiments, the polypeptide comprises a second VHH comprising a CDR1 comprising the amino acid sequence GSTATRDTFSSHRMT (SEQ ID NO:21), a CDR2 comprising the amino acid sequence TITGDDITNYTGSVKG (SEQ ID NO:22), and a CDR3 comprising the amino acid sequence LERGIWAY (SEQ ID NO:23).

[0022] In some embodiments of each of the aforementioned aspects and embodiments, as well as other aspects and embodiments described herein, a BamA -binding agent described herein is an antagonist of Acinetobacter . In some embodiments, a BamA -binding agent described herein is an antagonist of A. baumannii. In some embodiments, a BamA-binding agent inhibits Acinetobacter BamA activity. In some embodiments, the BamA-binding agent is an antagonist of Acinetobacter BamA.

[0023] In another aspect, the disclosure provides compositions comprising a BamA-binding agent described herein. In some embodiments, a composition comprises an anti-BamA antibody described herein. In some embodiments, a composition comprises an anti-BamA VHH described herein. In some embodiments, a composition comprises a monoclonal anti-BamA VHH described herein. In some embodiments, a composition comprises an anti-BamA VHH selected from the group consisting of: VHH- 29, VHH-47, and VHH-82.

[0024] In another aspect, the disclosure provides pharmaceutical compositions comprising a BamA- binding agent described herein and a pharmaceutically acceptable carrier. In some embodiments, a pharmaceutical composition comprises an anti-BamA antibody described herein and a pharmaceutically acceptable carrier. In some embodiments, a pharmaceutical composition comprises an anti-BamA VHH described herein and a pharmaceutically acceptable carrier. In some embodiments, a pharmaceutical composition comprises a monoclonal anti-BamA VHH described herein and a pharmaceutically acceptable carrier. In some embodiments, a pharmaceutical composition comprises an anti-BamA VHH selected from the group consisting of: VHH-29, VHH-47, and VHH-82 and a pharmaceutically acceptable carrier. In some embodiments, a pharmaceutical composition comprises the anti-BamA VHH- 82 and a pharmaceutically acceptable carrier. In some embodiments, a pharmaceutical composition comprises the anti-BamA VHH-29 and a pharmaceutically acceptable carrier. In some embodiments, a pharmaceutical composition comprises the anti-BamA VHH-47 and a pharmaceutically acceptable carrier.

[0025] In some embodiments of each of the aforementioned aspects, as well as other aspects and/or embodiments described elsewhere herein, the BamA-binding agent is isolated. In some embodiments, the BamA-binding agent is substantially pure.

[0026] In another aspect, the disclosure provides polynucleotides comprising a polynucleotide that encodes a BamA-binding agent described herein. In some embodiments, a polynucleotide encodes an anti-BamA VHH described herein. In some embodiments, the polynucleotide is isolated. In some embodiments, a vector comprises a polynucleotide that encodes a BamA-binding agent described herein. In some embodiments, an isolated cell comprises a polynucleotide that encodes a BamA-binding agent described herein. In some embodiments, an isolated cell comprises a vector comprising a polynucleotide that encodes a BamA-binding agent described herein. In some embodiments, a cell comprises a BamA- binding agent described herein. In some embodiments, a cell produces a BamA-binding agent described herein. In some embodiments, a cell produces an anti-BamA VHH described herein. In some embodiments, a cell is a monoclonal cell line.

[0027] In another aspect, the disclosure provides methods of using the BamA-binding agents described herein. In some embodiments, a method comprises using a composition comprising a BamA-binding agent described herein. In some embodiments, a method comprises using a pharmaceutical composition comprising a BamA-binding agent described herein.

[0028] In some embodiments, a method of inhibiting the growth of Acinetobacter comprises contacting Acinetobacter with an effective amount of a BamA-binding agent described herein. In some embodiments, a method of inhibiting an Acinetobacter infection in a subject, comprises administering to the subject a therapeutically effective amount of a BamA-binding agent described herein. In some embodiments, a method of treating an Acinetobacter infection in a subject comprises administering to the subject a therapeutically effective amount of a BamA-binding agent described herein.

[0029] In some embodiments of the methods described herein, the Acinetobacter is A. baumannii. In some embodiments of the methods described herein, the BamA-binding agent comprises VHH-29. In some embodiments of the methods described herein, the BamA-binding agent is VHH-29. In some embodiments of the methods described herein, the BamA-binding agent comprises VHH-47. In some embodiments of the methods described herein, the BamA-binding agent is VHH-47. In some embodiments of the methods described herein, the BamA-binding agent comprises VHH-82. In some embodiments of the methods described herein, the BamA-binding agent is VHH-82.

[0030] In some embodiments of any of the methods described herein, a BamA-binding agent or VHH described herein is administered with at least one additional therapeutic agent.

[0031] In some embodiments of each of the aforementioned aspects and embodiments, as well as other aspects and embodiments described herein, the subject is human.

[0032] Where aspects or embodiments of the disclosure are described in terms of a Markush group or other grouping of alternatives, the present disclosure encompasses not only the entire group listed as a whole, but also each member of the group individually and all possible subgroups of the main group, and also the main group absent one or more of the group members. The present disclosure also envisages the explicit exclusion of one or more of any of the group members in the claimed disclosure.

BRIEF DESCRIPTION OF THE FIGURES

[0033] Figure 1. Inhibition of Acinetobacter growth with sera from llamas immunized with A. baumannii BamA. Sera from pre-immunization bleed, intermediate bleed (week 6), and the final bleed (week 30) were diluted in PBS and added to wells containing A. baumannii ATCC 19606 or E. coli ATCC 25922. Ciprofloxacin was used as a control. Bacterial growth was determined by measuring the absorbance at 600 nm.

[0034] Figure 2. FACS screening and enrichment of VHHs that bound A. baumannii BamA. A VHH- expressing yeast library was panned with biotinylated A. baumannii BamA. Cells binding to BamA were selected and enriched through 3 rounds of screening.

[0035] Figure 3. Growth inhibitory screening assay. 52 VHHs were prepared in a 3 -fold serial dilution in PBS and data was taken from the highest concentration within a range of 4-20 pM (55-300 pg/ml) in PBS and added to wells containing A. baumannii ATCC 19606 in the presence or absence of polymyxin B nonapeptide (PMBN). Ciprofloxacin and tetracycline were used as positive controls and an anti -A. coli BamA VHH was used as a negative control. After 18-20 hours, bacterial growth was determined by measuring the absorbance at 600 nM.

[0036] Figure 4. Antimicrobial minimum inhibitory concentration (MIC) assay. Exemplary VHH-82 was prepared in a 3-fold serial dilution in PBS and added to wells containing A. baumannii in the presence of PMB or PMBN. Ciprofloxacin was used as a positive control and an anti -A. coli BamA VHH was used as a negative control. After 18-20 hours, bacterial growth was determined by measuring the absorbance at 600 nM.

[0037] Figure 5. Growth inhibitory screening assay. 52 VHHs were prepared in a 3 -fold serial dilution in PBS and data was taken from the highest concentration within a range of 4-20 pM (55-300 pg/ml) in PBS and added to wells containing LPS-deficient strain A. baumannii ATCC 19606-AlpxC. Ciprofloxacin and tetracycline were used as positive controls. After 18-20 hours, bacterial growth was determined by measuring the absorbance at 600 nM.

[0038] Figure 6. Antimicrobial MIC assay. Exemplary VHH-82 was prepared at 10000-0.50 nM in a 3-fold serial dilution in PBS and added to wells containing A. baumannii-

or wild-type A. baumannii in the presence of 4 pg/mL CHIR-090. Ciprofloxacin was used as a positive control. After 18-20 hours, bacterial growth was determined by measuring the absorbance at 600 nM.

[0039] Figure 7. Binding competition assay. Purified VHH-82 or VHH-29 was immobilized on a CM5 chip surface using amine coupling chemistry. VHH-29/BamA, VHH-47/BamA, VHH-67/BamA, and VHH-82/BamA mixtures were prepared (antibody concentration was titrated from 0.05-1000 nM; Bam A concentration held constant at 50 nM) in a 96-well microplate. The mixtures were injected over the coated chips. The normalized signal was plotted against the VHH concentration.

[0040] Figure 8. Epitope binding ELISA. Peptides representing the predicted outer membrane loops of A. baumannii BamA protein were synthesized by custom peptide synthesis (Anaspec). The peptides were synthesized with a biotin molecule attached at the N-terminus and peptides for loop 4 and loop 7 were prepared in both linearized and cyclized forms. The biotinylated peptides were added to a streptavidin-coated plate at 2 pg/ml and VHH-47-Fc was added to the plate at 20 pg/ml. An HRP -labeled secondary anti-Fc antibody (Jackson Immuno Research Laboratories Inc.) was added to the plate, followed by a chemiluminescent detection reagent.

[0041] Figure 9. Acinetobacter OmpT assay. A. baumannii cells containing a vector encoding an inducible OmpT were grown overnight in LB media containing 30 pg/ml carbenicillin. Anti -A. baumannii BamA VHH-82 and VHH-47 and control anti-/:', coli BamA VHH-1A2 were added at concentrations ranging from 2 to 0.003 pM (3-fold dilutions) prior to bacterial addition. For induction of OmpT, 1 pM IPTG was added to the wells and plates were incubated at 37° C for 2 hours. 100 pl from each well was transferred to a black clear bottom 96-well plate and peptide Abz-ARRA(NO2Y)-amide substrate was added at 500 pM. OmpT activity was measured at 2.5 hours post-IPTG induction in a fluorimeter using excitation and emission wavelengths of 325 and 430 nm, respectively.

[0042] Figure 10. Time kill assay. A. baumannii ATCC 19606 was cultured in the presence of 25% human serum and VHH-82 (2, 4, 7, and 15 pM). Bacterial growth was quantified after 0, 2, 5, and 24 hour incubations at 37° C by plating 10-fold dilutions on Mueller Hinton II agar plates.

[0043] Figure 11. Time kill assay. A. baumannii ATCC 19606 was cultured in the presence of 21 pM PMBN and VHH-82 (0.6, 1, and 2 pM). Bacterial growth was quantified after 0, 2, 5, and 24 hour incubations at 37° C by plating 10-fold dilutions on Mueller Hinton II agar plates.

DETAILED DESCRIPTION

[0044] The present disclosure provides novel agents, including but not limited to, polypeptides such as antibodies, that bind bacterial outer membrane protein assembly factor BamA (referred to herein as BamA). The BamA-binding agents include, but are not limited to, polypeptides, antibodies (including antigen-binding fragments thereof), scaffold proteins, and heterodimeric molecules. BamA-binding agents include, but are not limited to, agents that inhibit the growth of gram-negative bacteria, particularly Acinetobacter species. Related polypeptides, polynucleotides, vectors, compositions comprising the agents, cells comprising the related polynucleotides or vectors, and methods of making the agents are provided. Methods of using the novel BamA-binding agents are also provided.

I. Definitions

[0045] Unless otherwise defined herein, technical and scientific terms used in the present description have the meanings that are commonly understood by those of ordinary skill in the art. Whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any description of a term set forth conflicts with any document incorporated herein by reference, the description of the term set forth below shall control. [0046] The term “binding agent” as used herein refers to a molecule that binds a specific antigen or target (e.g., BamA). A binding agent may comprise a protein, peptide, nucleic acid, carbohydrate, lipid, or small molecular weight compound. In some embodiments, a binding agent comprises a full-length antibody. In some embodiments, a binding agent is an antigen-binding fragment of an antibody. In some embodiments, a binding agent comprises an alternative protein scaffold or artificial scaffold (e.g, a nonimmunoglobulin backbone). In some embodiments, a binding agent is a fusion protein comprising an antigen-binding site. In some embodiments, a binding agent is a bispecific or multispecific molecule comprising at least one antigen-binding site.

[0047] The term “antibody” is used herein in the broadest sense and encompasses various antibody structures, including but not limited to, an immunoglobulin molecule that recognizes and binds a target through at least one antigen-binding site, polyclonal antibodies, recombinant antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, bispecific antibodies, multispecific antibodies, diabodies, tribodies, tetrabodies, single chain Fv (scFv) antibodies, single variable domain antibodies (e.g., VHHs), and antibody fragments as long as they exhibit the desired antigen-binding activity. Thus, the term “antibody” includes both intact antibody and antibody fragment. The term “intact antibody” or “full-length antibody” refers to an antibody having a structure substantially similar to a native antibody structure. This includes, for example, an antibody comprising two light chains each comprising a variable region and a light chain constant region (CL) and two heavy chains each comprising a variable region and at least heavy chain constant regions CHI, CH2, and CH3.

Depending on the isotype of antibody, an intact antibody may include a hinge region (or a portion thereof) between the CHI and CH2 regions. The term “antibody fragment” as used herein refers to a molecule other than an intact antibody that comprises a portion of an antibody and generally an antigen-binding site. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, single chain antibody molecules, scFv, sc(Fv)2, disulfide-linked scFv (dsscFv), single domain antibodies, diabodies, tribodies, tetrabodies, minibodies, and multispecific antibodies formed from antigen-binding antibody fragments.

[0048] The term “VHH” as used herein refers to a single variable domain antibody comprising a heavy chain only devoid of a light chain. VHH includes, but not limited to a single variable domain antibody derived from a heavy chain antibody raised in a camelid animal, such as a llama, alpaca, or camel. Other terms for VHHs used by those skilled in the art include, but are not limited to, single domain antibodies (sdAbs), single variable domain antibodies, heavy chain variable domain antibodies, and Nanobodies®. A VHH as defined herein comprises one antigen-binding site.

[0049] The term “monoclonal antibody” as used herein refers to a substantially homogenous antibody population involved in the highly specific recognition and binding of a single antigenic determinant or epitope. The term “monoclonal antibody” encompasses intact and full-length antibodies, as well as antibody fragments (e.g., Fab, Fab', F(ab')2, Fv), VHHs, single chain antibodies, scFv, fusion proteins comprising an antigen-binding antibody fragment, and any other modified immunoglobulin molecule comprising at least one antigen-binding site. Furthermore, “monoclonal antibody” refers to such antibodies made by any number of techniques, including but not limited to, hybridoma production, phage display libraries, yeast display libraries, recombinant expression, and transgenic animals.

[0050] The term “chimeric antibody” refers to an antibody in which a portion of the heavy and/or light chain is derived from a first source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.

[0051] The term “humanized antibody” as used herein refers to an antibody that includes sequences from human immunoglobulins (e.g., recipient antibody) in which the native CDR residues are replaced by residues from the corresponding CDR of a nonhuman species (e.g., donor antibody) such as camelid, mouse, rat, rabbit, or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, one or more FR region residues of the human immunoglobulin sequences are replaced by corresponding nonhuman residues. Furthermore, humanized antibodies can comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. A humanized antibody heavy or light chain can comprise substantially all of at least one or more variable regions, in which all or substantially all of the CDRs correspond to those of a nonhuman immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. In certain embodiments, the humanized antibody will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see, Jones et al., Nature 321 :522-25 (1986); Riechmann et al., Nature 332:323-29 (1988); Presta, Curr. Op. Struct. Biol. 2:593-96 (1992); Carter et al., Proc. Natl. Acad. Sci. USA 89:4285-89 (1992); U.S. Pat. Nos: 6,800,738; 6,719,971; 6,639,055; 6,407,213; and 6,054,297. [0052] The terms “epitope” and “antigenic determinant” are used interchangeably herein and refer to that portion of an antigen or target capable of being recognized and bound by a particular antibody. When the antigen or target is a polypeptide, epitopes can be formed both from contiguous amino acids and noncontiguous amino acids juxtaposed by tertiary folding of the protein. Epitopes formed from contiguous amino acids (also referred to as linear epitopes) are typically retained upon protein denaturing, whereas epitopes formed by tertiary folding (also referred to as conformational epitopes) are typically lost upon protein denaturing. An epitope typically includes at least 3, and more usually, at least 5, 6, 7, or 8- 10 amino acids in a unique spatial conformation. Epitopes can be predicted using any one of a large number of publicly available bioinformatic software tools. X-ray crystallography can be used to characterize an epitope on a target protein by analyzing the amino acid residue interactions of an antigen/antibody complex.

[0053] The term “specifically binds” as used herein refers to an agent that interacts more frequently, more rapidly, with greater duration, with greater affinity, or with some combination of the above to a particular antigen, epitope, protein, or target molecule than with alternative substances. A binding agent that specifically binds an antigen can be identified, for example, by immunoassays, ELISAs, surface plasmon resonance (SPR), or other techniques known to those of skill in the art. Generally, a binding agent that specifically binds an antigen will bind the target antigen at a higher affinity than its affinity for a different antigen. The different antigen can be a related antigen. In some embodiments, a binding agent that specifically binds an antigen can bind the target antigen with an affinity that is at least 20 times greater, at least 30 times greater, at least 40 times greater, at least 50 times greater, at least 60 times greater, at least 70 times greater, at least 80 times greater, at least 90 times greater, or at least 100 times greater, than its affinity for a different antigen. In some embodiments, a binding agent that specifically binds a particular antigen binds a different antigen at such a low affinity that binding cannot be detected using an assay described herein or otherwise known in the art. In some embodiments, affinity is measured using SPR technology in a Biacore system as described herein or as known to those of skill in the art.

[0054] The terms “polypeptide” and “peptide” and “protein” are used interchangeably herein and refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid, including but not limited to, unnatural amino acids, as well as other modifications known in the art. It is understood that, because the polypeptides of this disclosure may be based upon antibodies, the term “polypeptide” encompasses polypeptides as a single chain and polypeptides of two or more associated chains.

[0055] The terms “polynucleotide” and “nucleic acid” and “nucleic acid molecule” are used interchangeably herein and refer to polymers of nucleotides of any length, and include DNA and RNA. The nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that may be incorporated into a polymer by DNA or RNA polymerase. [0056] The terms “identical” or percent “identity” in the context of two or more nucleic acids or polypeptides, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity. The percent identity may be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that may be used to obtain alignments of amino acid or nucleotide sequences are well-known in the art. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and variants thereof. In some embodiments, two nucleic acids or polypeptides of the disclosure are substantially identical, meaning they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments at least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection. In some embodiments, identity exists over a region of the sequences that is at least about 10, at least about 20, at least about 20-40, at least about 40-60, at least about 60-80 nucleotides or amino acids in length, or any integral value there between. In some embodiments, identity exists over a longer region than 60-80 nucleotides or amino acids, such as at least about 80-100 nucleotides or amino acids, and in some embodiments the sequences are substantially identical over the full length of the sequences being compared, for example, (i) the coding region of a nucleotide sequence or (ii) an amino acid sequence.

[0057] The phrase “conservative amino acid substitution” as used herein refers to a substitution in which one amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been generally defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, substitution of an alanine for a valine is considered to be a conservative substitution. Methods of identifying nucleotide and amino acid conservative substitutions that do not eliminate binding are well-known in the art.

[0058] The term “vector” as used herein means a construct that is capable of delivering, and usually expressing, one or more gene(s) or sequence(s) of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid, or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, and DNA or RNA expression vectors encapsulated in liposomes.

[0059] The term “isolated” as used herein refers to a polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition that is in a form not found in nature. An “isolated” antibody is substantially free of material from the cellular source from which it is derived. In some embodiments, isolated polypeptides, soluble proteins, antibodies, polynucleotides, vectors, cells, or compositions are those that have been purified to a degree that they are no longer in a form in which they are found in nature. In some embodiments, a polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition that is isolated is substantially pure. A polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition may be isolated from a natural source (e.g. , tissue) or from a source such as an engineered cell line.

[0060] The term “substantially pure” as used herein refers to 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. [0061] The term “subject” refers to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, canines, felines, rabbits, rodents, and the like.

[0062] The term “pharmaceutically acceptable” as used herein refers to a substance approved or approvable by a regulatory agency or listed in the U.S. Pharmacopeia, European Pharmacopeia, or other generally recognized pharmacopeia for use in animals, including humans.

[0063] The terms “pharmaceutically acceptable excipient, carrier, or adjuvant” or “acceptable pharmaceutical carrier” as used herein refer to an excipient, carrier, or adjuvant that may be administered to a subject, together with at least one therapeutic agent, and that is generally safe, non-toxic, and has no effect on the pharmacological activity of the therapeutic agent. In general, those of skill in the art and government agencies consider a pharmaceutically acceptable excipient, carrier, or adjuvant to be an inactive ingredient of any formulation or any pharmaceutical composition.

[0064] The term “pharmaceutical formulation” or “pharmaceutical composition” as used herein refers to a preparation that is in such form as to permit the biological activity of the agent to be effective. A pharmaceutical formulation or composition generally comprises additional components, such as a pharmaceutically acceptable excipient, carrier, adjuvant, buffers, etc.

[0065] The term “effective amount” or “therapeutically effective amount” as used herein refers to the amount of an agent that is sufficient to reduce and/or ameliorate the severity and/or duration of (i) a disease, disorder or condition in a subject, and/or (ii) a symptom in a subject. The term also encompasses an amount of an agent necessary for the (i) reduction or amelioration of the advancement or progression of a given disease, disorder, or condition, (ii) reduction or amelioration of the recurrence, development, or onset of a given disease, disorder, or condition, and/or (iii) the improvement or enhancement of the prophylactic or therapeutic effect(s) of another agent or therapy (e.g., an agent other than the binding agents provided herein).

[0066] The term “therapeutic effect” as used herein refers to the effect and/or ability of an agent to reduce and/or ameliorate the severity and/or duration of (i) a disease, disorder, or condition in a subject, and/or (ii) a symptom in a subject. The term also encompasses the ability of an agent to (i) reduce or ameliorate the advancement or progression of a given disease, disorder, or condition, (ii) reduce or ameliorate the recurrence, development, or onset of a given disease, disorder, or condition, and/or (iii) to improve or enhance the prophylactic or therapeutic effect(s) of another agent or therapy (e.g., an agent other than the binding agents provided herein).

[0067] The term “treat” or “treatment” or “treating” or “to treat” or “alleviate” or alleviation” or “alleviating” or “to alleviate” as used herein refers to both (i) therapeutic measures that aim to cure, slow down, lessen symptoms of, and/or halt progression of a pathologic condition or disorder and (ii) prophylactic or preventative measures that aim to prevent or slow the development of a targeted pathologic condition or disorder. Thus, those in need of treatment include those already with the disorder, those at risk of having/deve loping the disorder, and those in whom the disorder is to be prevented.

[0068] The term “prevent” or “prevention” or “preventing” as used herein refers to the partial or total inhibition of the development, recurrence, onset, or spread of a disease, disorder, or condition, or a symptom thereof in a subject.

[0069] The term “inhibit” as used herein refers to reduction or decrease, e.g., of an activity or effect. The term includes completely, substantially, or partially blocking the activity or effect. For example, the term “inhibit,” when used in the context of inhibiting an Acinetobacter infection, refers to reducing the Acinetobacter infection, and as a result the infection is completely removed or decreased to a less degree.

[0070] As used herein, reference to “about” or “approximately” a value or parameter includes (and describes) embodiments that are directed to that value or parameter. For example, a description referring to “about X” includes description of “X”.

[0071] As used in the present disclosure and claims, the singular forms “a”, “an” and “the” include plural forms unless the context clearly dictates otherwise.

[0072] It is understood that wherever embodiments are described herein with the term “comprising” otherwise analogous embodiments described in terms of “consisting of’ and/or “consisting essentially of’ are also provided. It is also understood that wherever embodiments are described herein with the phrase “consisting essentially of’ otherwise analogous embodiments described in terms of “consisting of’ are also provided.

[0073] The term “and/or” as used in a phrase such as “A and/or B” herein is intended to include both A and B; A or B; A (alone); and B (alone). Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following embodiments: 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). II. Bacterial BamA-binding agents

[0074] BamA is an outer membrane protein (OMP) that is an essential and central component of the [3- barrel assembly machine (BAM) in Gram-negative bacteria. OMPs are a class of unique integral membrane proteins anchored in the outer membrane, whose [3-barrel structure are formed by 8 to 26 strands. There are large extended loops between the strands on the extracellular side and short loops on the periplasmic side. BamA can automatically insert into the outer membrane, but is necessary and responsible for the assembly of other OMPs. OMPs are crucial for multiple cellular function including construction of the outer membrane itself, nutrient acquisition and antibiotic efflux. Because depletion of the BAM complex is detrimental to bacterial viability, the BAM complex and particularly BamA is a potential target for new and novel antibacterial agents.

[0075] An exemplary amino acid (aa) sequence for A. baumannii BamA ATCC 19606 (UniProtKB No. D0C6H3) is provided herein as SEQ ID NO: 1. As used herein, reference to amino acid positions of BamA refer to the numbering of amino acid sequences including the signal sequence.

[0076] BamA consists of an N-terminal periplasmic domain that contains five polypeptide transport associated (POTRA) domains and a C-terminal 16-stranded [3-barrel domain and about 94 kDa. A. baumannii BamA is a protein of 841 amino acids (aa) - the signal sequence is aa 1-24 and the predicted barrel domain is aa 427-841. Within the barrel domain there are 8 external outer membrane loops: loop 1 is aa 438-442, loop 2 is aa 467-472, loop 3 is aa 496-514, loop 4 is aa 538-603, loop 5 is aa 635-643, loop 6 is aa 672-723, loop 7 is aa 754-799, and loop 8 is 821-832.

[0077] The present disclosure provides agents that bind Gram-negative bacteria BamA. In some embodiments, a BamA-binding agent binds Acinetobacter BamA. In some embodiments, a BamA- binding agent binds one of the external outer membrane loops of Acinetobacter BamA. In some embodiments, a BamA-binding agent binds loop 1 of Acinetobacter BamA. In some embodiments, a BamA-binding agent binds loop 2 of Acinetobacter BamA. In some embodiments, a BamA-binding agent binds loop 3 of Acinetobacter BamA. In some embodiments, a BamA-binding agent binds loop 4 of Acinetobacter BamA. In some embodiments, a BamA-binding agent binds loop 5 of Acinetobacter BamA. In some embodiments, a BamA-binding agent binds loop 6 of Acinetobacter BamA. In some embodiments, a BamA-binding agent binds loop 7 of Acinetobacter BamA. In some embodiments, a BamA-binding agent binds loop 8 of Acinetobacter BamA. In some embodiments, a BamA-binding agent binds more than one loop of Acinetobacter BamA. In some embodiments, a BamA-binding agent binds an epitope comprising one or more amino acids in more than one loop of Acinetobacter BamA. [0078] In some embodiments, a BamA-binding agent binds within amino acids 438-442 of SEQ ID NO: 1. In some embodiments, a BamA-binding agent binds within amino acids 467-472 of SEQ ID NO: 1. In some embodiments, a BamA-binding agent binds within amino acids 496-514 of SEQ ID NO: 1. In some embodiments, a BamA-binding agent binds within amino acids 538-603 of SEQ ID NO: 1. In some embodiments, a BamA-binding agent binds within amino acids 635-643 of SEQ ID NO: 1. In some embodiments, a BamA-binding agent binds within amino acids 672-723 of SEQ ID NO: 1. In some embodiments, a BamA-binding agent binds within amino acids 754-799 of SEQ ID NO: 1. In some embodiments, a BamA-binding agent binds within amino acids 821-832 of SEQ ID NO: 1. It is understood that the barrel domain and/or external outer membrane loops of BamA may be defined differently by those of skill in the art, therefore the N-terminal amino acids and the C-terminal amino acids of the barrel domain of BamA or any of the loops may vary by 1, 2, 3, 4, 5, or more amino acid residues.

[0079] In some embodiments, a BamA-binding agent binds within SEQ ID NO:3. In some embodiments, a BamA-binding agent binds within SEQ ID NO:4. In some embodiments, a BamA- binding agent binds within SEQ ID NO:5. In some embodiments, a BamA-binding agent binds within SEQ ID NO:6. In some embodiments, a BamA-binding agent binds within SEQ ID NO:7. In some embodiments, a BamA-binding agent binds within SEQ ID NO:8. In some embodiments, a BamA- binding agent binds within SEQ ID NO:9. In some embodiments, a BamA-binding agent binds within SEQ ID NO: 10.

[0080] In some embodiments, a BamA-binding agent is an antibody. In some embodiments, the antibody is a recombinant antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody.

[0081] In some embodiments, the antibody is an antibody fragment comprising an antigen-binding site. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, single chain antibody molecules, scFv, disulfide-linked scFv (dsscFv), diabodies, tribodies, tetrabodies, minibodies, dual variable domain antibodies (DVD), and single variable domain antibodies. In some embodiments, the antibody fragment is a Fab. In some embodiments, the antibody fragment is a Fab’. In some embodiments, the antibody fragment is a F(ab’)2. In some embodiments, the antibody fragment is a Fv. In some embodiments, the antibody fragment is a scFv. In some embodiments, the antibody fragment is a (SCFV)2. In some embodiments, the antibody fragment is a disulfide-linked scFv (dsscFv). In some embodiments, the antibody fragment is a single chain antibody. In some embodiments, the antibody fragment is a dual variable domain (DVD) antibody. In some embodiments, the antibody fragment is a single variable domain antibody. In some embodiments, the antibody fragment is a linear antibody. In some embodiments, the antibody fragment is a diabody. In some embodiments, the antibody fragment is a tribody. In some embodiments, the antibody fragment is a tetrabody. In some embodiments, the antibody fragment is a minibody. In some embodiments, the antibody fragment is a nanobody. In some embodiments, the antibody fragment is a V region antibody.

[0082] In some embodiments, the antibody is a heavy chain only antibody (e.g., came lid antibodies). In some embodiments, the antibody is a single variable domain antibody. In some embodiments, the antibody is a single variable domain of a heavy chain (VHH) antibody. VHHs are also referred to as nanobodies®.

[0083] In some embodiments, the antibody is a monospecific antibody. In some embodiments, the antibody is a bispecific antibody. In some embodiments, the antibody is a multispecific antibody. In some embodiments, the antibody is a monovalent antibody. In some embodiments, the antibody is a bivalent antibody. In some embodiments, the antibody is a tetravalent antibody.

[0084] In some embodiments, the antibody is isolated. In some embodiments, the antibody is substantially pure.

[0085] In some embodiments, a BamA-binding agent is a polyclonal antibody. Polyclonal antibodies may be prepared by any method known to those of skill in the art. In some embodiments, polyclonal antibodies are produced by immunizing an animal (e.g., a rabbit, rat, mouse, goat, donkey, llama) with an antigen of interest (e.g., a purified peptide fragment, a recombinant protein, or a fusion protein) using multiple subcutaneous or intraperitoneal injections. In some embodiments, the antigen is conjugated to a carrier such as keyhole limpet hemocyanin (KLH), serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor. The antigen (with or without a carrier protein) is diluted in sterile saline and usually combined with an adjuvant (e.g., Complete or Incomplete Freund's Adjuvant) to form a stable emulsion. After a period of time, polyclonal antibodies are recovered from the immunized animal (e.g., from blood or ascites). In some embodiments, the polyclonal antibodies are purified from serum or ascites according to standard methods in the art including, but not limited to, affinity chromatography, ion-exchange chromatography, gel electrophoresis, and/or dialysis.

[0086] In some embodiments, a BamA-binding agent is a monoclonal antibody. Monoclonal antibodies may be prepared by any method known to those of skill in the art. In some embodiments, monoclonal antibodies are prepared using hybridoma methods known to one of skill in the art. For example, using a hybridoma method, a mouse, rat, rabbit, hamster, llama, or other appropriate host animal, is immunized as described above. In some embodiments, lymphocytes are immunized in vitro. In some embodiments, the immunizing antigen is a human protein or a fragment thereof. In some embodiments, the immunizing antigen is a bacterial protein or a fragment thereof. In some embodiments, the immunizing antigen is a combination of two or more (e.g., 2, 3, 4) related proteins or fragments thereof. [0087] Following immunization, lymphocytes are isolated and fused with a suitable myeloma cell line using, for example, polyethylene glycol or electrofusion. The hybridoma cells are selected using specialized media as known in the art and unfused lymphocytes and myeloma cells do not survive the selection process. Hybridomas that produce monoclonal antibodies directed specifically against a chosen antigen may be identified by a variety of methods including, but not limited to, immunoprecipitation, immunoblotting, and in vitro binding assays (e.g., flow cytometry, FACS, ELISA, SPR (e.g., Biacore), and radioimmunoassay). Once hybridoma cells that produce antibodies of the desired specificity, affinity, and/or activity are identified, the clones may be subcloned by limiting dilution techniques. In some embodiments, high-throughput methods are used to distribute single cell hybridoma cells into plates. In some embodiments, high-throughput methods are used to directly distribute single cells from original fusion into plates. The hybridomas may be propagated either in in vitro culture using standard methods or in vivo as ascites tumors in an animal. The monoclonal antibodies may be purified from the culture medium or ascites fluid according to standard methods in the art including, but not limited to, affinity chromatography, ion-exchange chromatography, gel electrophoresis, and dialysis.

[0088] In some embodiments, monoclonal antibodies are made using recombinant DNA techniques as known to one skilled in the art. For example, the polynucleotides encoding an antibody are isolated from mature B-cells or hybridoma cells, such as by RT-PCR using oligonucleotide primers that specifically amplify the genes encoding the heavy and light chains of the antibody, and their sequence is determined using standard techniques. The isolated polynucleotides encoding the heavy and light chains are then cloned into suitable expression vectors that produce the monoclonal antibodies when transfected into host cells such as E. coli, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin proteins.

[0089] In some embodiments, monoclonal antibodies are isolated from phage display libraries expressing variable domains or CDRs of a desired species. Screening of phage libraries can be accomplished by various techniques known in the art. In some embodiments, monoclonal antibodies are isolated from yeast display libraries expressing variable domains or CDRs of a desired species. Screening of yeast libraries can be accomplished by various techniques known in the art. A major advantage of cellsurface display is the compatibility of these methods with analysis and sorting by flow cytometry. Each individual cell of the library can be investigated for the binding characteristics of the binding entity expressed on the cell surface. High-throughput cell sorting allows for the selection and isolation of separate cell populations based on well-controlled parameters. In some embodiments, a library of VHHs is displayed on the surface of yeast cells; yeast cells are mixed with labeled-target protein (e.g., BamA) or labeled bacterial cells; and cells expressing VHHs bound to target are sorted and isolated. [0090] In some embodiments, a monoclonal antibody is modified by using recombinant DNA technology to generate alternative antibodies. In some embodiments, the constant domains of the light chain and heavy chain of a mouse monoclonal antibody are substituted for constant regions of a human antibody to generate a chimeric antibody. In some embodiments, the constant regions are truncated or removed to generate a desired antibody fragment of a monoclonal antibody. In some embodiments, site- directed or high-density mutagenesis of a variable region is used to optimize specificity and affinity of a monoclonal antibody.

[0091] In some embodiments, a BamA-binding agent is a humanized antibody. Various methods for generating humanized antibodies are known in the art. In some embodiments, a humanized antibody comprises one or more amino acid residues that have been introduced into it from a source that is nonhuman. In some embodiments, humanization is performed by replacing all the CDR sequences in a human antibody with corresponding CDR sequences of a non-human antibody, for example by replacing the CDRs of a human heavy chain variable domain with the CDRs of a camelid VHH domain antibody. [0092] The choice of which human heavy chain variable region and/or light chain variable region to use for generating humanized antibodies can be made based on a variety of factors and by a variety of methods known in the art. In some embodiments, the “best-fit” method is used where the sequence of the variable region of a non-human antibody is screened against the entire library of known human variable region sequences. The human sequence that is most similar to that of the non-human sequence is selected as the human variable region framework for the humanized antibody. In some embodiments, a particular variable region framework derived from a consensus sequence of all human antibodies of a particular subgroup of light or heavy chains is selected as the variable region framework. In some embodiments, the variable region framework sequence is derived from the consensus sequences of the most abundant human subclasses. In some embodiments, human germline genes are used as the source of the variable region framework sequences.

[0093] Other methods for humanization include, but are not limited to, a method called “superhumanization” which is described as the direct transfer of CDRs to a human germline framework, a method termed Human String Content (HSC) which is based on a metric of “antibody humanness”, methods based on generation of large libraries of humanized variants (including phage, ribosomal, and yeast display libraries), and methods based on framework region shuffling.

[0094] In some embodiments, a humanized VHH comprises one or more amino acid residues that have been introduced into it from a human heavy chain variable region. In some embodiments, humanization is performed by substituting one or more amino acids within the framework sequences of the camelid VHH with amino acids corresponding to framework sequences of a human heavy chain variable region. In some embodiments, the humanized VHHs are constructed by substituting amino acids within one of the framework regions of the VHH with amino acids corresponding to framework regions of a human heavy chain variable region. In some embodiments, the humanized VHHs are constructed by substituting amino acids within two of the framework regions of the VHH with amino acids corresponding to framework regions of a human heavy chain variable region. In some embodiments, the humanized VHHs are constructed by substituting amino acids within three four of the framework regions of the VHH with amino acids corresponding to framework regions of a human heavy chain variable region. In some embodiments, the humanized VHHs are constructed by substituting amino acids within four of the framework regions of the VHH with amino acids corresponding to framework regions of a human heavy chain variable region.

[0095] In some embodiments, a BamA-binding agent is a bispecific antibody. Bispecific antibodies are capable of recognizing and binding at least two different antigens or epitopes. The different epitopes can either be within the same molecule (e.g., two epitopes on BamA) or on different molecules (e.g., one epitope on BamA and one epitope on a different target). In some embodiments, a bispecific antibody has enhanced potency as compared to an individual antibody or to a combination of more than one antibody. In some embodiments, a bispecific antibody has reduced toxicity as compared to an individual antibody or to a combination of more than one antibody. It is known to those of skill in the art that any therapeutic agent may have unique pharmacokinetics (PK) (e.g., circulating half-life). In some embodiments, a bispecific antibody has the ability to synchronize the PK of two active binding agents wherein the two individual binding agents have different PK profdes. In some embodiments, a bispecific antibody has the ability to concentrate the actions of two agents in a common area (e.g. , tissue) in a subject. In some embodiments, a bispecific antibody has the ability to concentrate the actions of two agents to a common target (e.g., a specific cell type). In some embodiments, a bispecific antibody has the ability to target the actions of two agents to more than one biological pathway or function. In some embodiments, a bispecific antibody has the ability to target two different cells and bring them closer together.

[0096] In some embodiments, a bispecific antibody has decreased toxicity and/or side effects. In some embodiments, a bispecific antibody has decreased toxicity and/or side effects as compared to a mixture of the two individual antibodies or the antibodies as single agents. In some embodiments, a bispecific antibody has an increased therapeutic index. In some embodiments, a bispecific antibody has an increased therapeutic index as compared to a mixture of the two individual antibodies or the antibodies as single agents.

[0097] Many techniques for making bispecific antibodies are known to those skilled in the art. In some embodiments, a bispecific antibody comprises heavy chain constant regions with modifications in the amino acids that are part of the interface between the two heavy chains. These modifications are made to enhance heterodimer formation and generally reduce or eliminate homodimer formation. In some embodiments, the bispecific antibody is generated using a knobs-into-holes (KIH) strategy. In some embodiments, the bispecific antibody comprises variant hinge regions incapable of forming disulfide linkages between identical heavy chains (e.g., reduce homodimer formation). In some embodiments, the bispecific antibody comprises heavy chains with changes in amino acids that result in altered electrostatic interactions. In some embodiments, the bispecific antibodies comprise heavy chains with changes in amino acids that result in altered hydrophobic/hydrophilic interactions.

[0098] Bispecific antibodies can be intact antibodies or antibody fragments comprising antigenbinding sites. In some embodiments, the bispecific antibodies comprise antibody fragments comprising antigen binding sites.

[0099] In some embodiments, a BamA-binding agent is a single domain antibody (e.g., a VHH) that binds BamA. In some embodiments, an anti-BamA antibody or anti-BamA VHH binds A. baumannii BamA. In some embodiments, an anti-BamA antibody or anti-BamA VHH binds A. baumannii BamA and does not bind E. coli BamA. In some embodiments, an anti-BamA antibody or anti-BamA VHH binds a BamA epitope. In some embodiments, an anti-BamA antibody or anti-BamA VHH binds a BamA epitope within at least one of the external loops of the barrel domain of BamA. In some embodiments, an anti-BamA antibody or anti-BamA VHH binds a BamA epitope within loop 4 of the barrel domain of BamA. In some embodiments, an anti-BamA antibody or anti-BamA VHH binds a BamA epitope within loop 6 of the barrel domain of BamA. In some embodiments, an anti-BamA antibody or anti-BamA VHH binds a BamA epitope within loop 7 of the barrel domain of BamA. [00100] In some embodiments, an anti-BamA antibody or anti-BamA VHH binds an epitope comprising at least one amino acid (e.g. , 1, 2, 3, 4, 5, 6, 7, 8, 9) within amino acids 496-514 of SEQ ID NO: 1. In some embodiments, an anti-BamA antibody or anti-BamA VHH binds an epitope comprising at least one amino acid within amino acids 538-603 of SEQ ID NO: 1. In some embodiments, an anti-BamA antibody or anti-BamA VHH binds an epitope comprising at least one amino acid within amino acids 672-723 of SEQ ID NO: 1. In some embodiments, an anti-BamA antibody or anti-BamA VHH binds an epitope comprising at least one amino acid within amino acids 754-799 of SEQ ID NO: 1. In some embodiments, an anti-BamA antibody or anti-BamA VHH binds an epitope comprising amino acids within SEQ ID NO:5. In some embodiments, an anti-BamA antibody or anti-BamA VHH binds an epitope comprising amino acids within SEQ ID NO:6. In some embodiments, an anti-BamA antibody or anti-BamA VHH binds an epitope comprising amino acids within SEQ ID NO:8. In some embodiments, an anti-BamA antibody or anti-BamA VHH binds an epitope comprising amino acids within SEQ ID NOV.

[00101] In some embodiments, the epitope is a conformational epitope. In some embodiments, the epitope is a linear epitope. [00102] In some embodiments, an anti-BamA antibody or anti-BamA VHH competes with a second agent for binding within at least one of the external outer membrane loops of A. baumannii BamA. In some embodiments, an anti-BamA antibody or anti-BamA VHH competes with a second agent for binding within amino acids 496-514 of SEQ ID NO: 1. In some embodiments, an anti-BamA antibody or anti-BamA VHH competes with a second agent for binding within amino acids 538-603 of SEQ ID NO: 1. In some embodiments, an anti-BamA antibody or anti-BamA VHH competes with a second agent for binding within amino acids 672-723 of SEQ ID NO: 1. In some embodiments, an anti-BamA antibody or anti-BamA VHH competes with a second agent for binding within amino acids 754-799 of SEQ ID NO: 1. In some embodiments, an anti-BamA antibody or anti-BamA VHH competes with a second agent for binding within amino acid sequence SEQ ID NO:5. In some embodiments, an anti-BamA antibody or anti-BamA VHH competes with a second agent for binding within amino acid sequence SEQ ID NO:6. In some embodiments, an anti-BamA antibody or anti-BamA VHH competes with a second agent for binding within amino acid sequence SEQ ID NO:8. In some embodiments, an anti-BamA antibody or anti-BamA VHH competes with a second agent for binding within amino acid sequence SEQ ID NOV. [00103] In some embodiments, a BamA-binding agent comprises an anti-BamA VHH described herein. In some embodiments, the BamA-binding agent comprises a variant of an anti-BamA VHH described herein. In some embodiments, a variant of an anti-BamA VHH comprises one to thirty amino acid substitutions. In some embodiments, a variant of the anti-BamA VHH comprises one to twenty-five amino acid substitutions. In some embodiments, a variant of the anti-BamA VHH comprises one to twenty amino acid substitutions. In some embodiments, a variant of the anti-BamA VHH comprises one to fifteen amino acid substitutions. In some embodiments, a variant of the anti-BamA VHH comprises one to ten amino acid substitutions. In some embodiments, a variant of the anti-BamA VHH comprises one to five amino acid substitutions. In some embodiments, the variant of the anti-BamA VHH comprises one to three amino acid substitutions. In some embodiments, the amino acid substitution(s) is in a CDR of the VHH. In some embodiments, the amino acid substitution(s) is not in a CDR of the VHH. In some embodiments, the amino acid substitution(s) is in a framework region of the VHH. In some embodiments, the amino acid substitution(s) is a conservative amino acid substitution.

[00104] CDRs of an antibody are defined using a variety of methods/systems by those skilled in the art. These systems and/or definitions have been developed and refined over a number of years and include Kabat, Chothia, IMGT, AbM, and Contact. The Kabat definition is based on sequence variability and is commonly used. The Chothia definition is based on the location of the structural loop regions. The IMGT system is based on sequence variability and location within the structure of the variable domain. The AbM definition is a compromise between Kabat and Chothia. The Contact definition is based on analyses of the available antibody crystal structures. An Exemplary system is a combination of Kabat and Chothia. Software programs (e.g., abYsis) are available and known to those of skill in the art for analysis of antibody sequence and determination of CDRs.

[00105] The specific CDR sequences defined herein are generally based on a combination of Kabat and Chothia definitions (Exemplary definition). However, it will be understood that reference to a VHH CDR of a specific antibody will encompass all CDR definitions as known to those of skill in the art.

[00106] In some embodiments, a BamA-binding agent described herein comprises the three CDRs of VHH-29 based on the Kabat definition. In some embodiments, a BamA-binding agent described herein comprises the three CDRs of VHH-29 based on the Chothia definition. In some embodiments, a BamA- binding agent described herein comprises the three CDRs of VHH-29 based on the AbM definition. In some embodiments, a BamA-binding agent described herein comprises the three CDRs of VHH-29 based on the IMGT definition. In some embodiments, a BamA-binding agent described herein comprises the three CDRs of VHH-29 based on the Contact definition. In some embodiments, a BamA-binding agent described herein comprises the three CDRs of VHH-29 based on the Exemplary definition.

[00107] In some embodiments, a BamA-binding agent described herein comprises the three CDRs of VHH-47 based on the Kabat definition. In some embodiments, a BamA-binding agent described herein comprises the three CDRs of VHH-47 based on the Chothia definition. In some embodiments, a BamA- binding agent described herein comprises the three CDRs of VHH-47 based on the AbM definition. In some embodiments, a BamA-binding agent described herein comprises the three CDRs of VHH-47 based on the IMGT definition. In some embodiments, a BamA-binding agent described herein comprises the three CDRs of VHH-47 based on the Contact definition. In some embodiments, a BamA-binding agent described herein comprises the three CDRs of VHH-47 based on the Exemplary definition.

[00108] In some embodiments, a BamA-binding agent described herein comprises the three CDRs of VHH-82 based on the Kabat definition. In some embodiments, a BamA-binding agent described herein comprises the three CDRs of VHH-82 based on the Chothia definition. In some embodiments, a BamA- binding agent described herein comprises the three CDRs of VHH-82 based on the AbM definition. In some embodiments, a BamA-binding agent described herein comprises the three CDRs of VHH-82 based on the IMGT definition. In some embodiments, a BamA-binding agent described herein comprises the three CDRs of VHH-82 based on the Contact definition. In some embodiments, a BamA-binding agent described herein comprises the three CDRs of VHH-82 based on the Exemplary definition.

[00109] In some embodiments, a BamA-binding agent comprises one, two, and/or three CDRs of any one of the VHHs described herein. In some embodiments, a BamA binding agent comprises one, two, and/or three heavy chain variable region CDRs from Table 1. In some embodiments, a BamA binding agent comprises one, two, and/or three heavy chain variable region CDRs from Table 2. In some embodiments, a BamA binding agent comprises one, two, and/or three heavy chain variable region CDRs from Table 3.

[00112] In some embodiments, a BamA-binding agent is a variant of a BamA-binding agent described herein. In some embodiments, a BamA-binding agent variant comprises amino acid substitutions in the heavy chain variable region CDR1, CDR2, and/or CDR3 as compared to a BamA-binding agent described herein. In some embodiments, a BamA-binding agent comprises one or more (e.g., 1, 2, 3, 4, etc.) amino acid substitutions in a CDR of an VHH described herein. In some embodiments, the amino acid substitutions are conservative substitutions. In some embodiments, a CDR comprises one amino acid substitution. In some embodiments, a CDR comprises two amino acid substitutions. In some embodiments, a CDR comprises three amino acid substitutions. In some embodiments, a CDR comprises four amino acid substitutions. In some embodiments, the CDR is a heavy chain variable region CDR1. In some embodiments, the CDR is a heavy chain variable region CDR2. In some embodiment, the CDR is a heavy chain variable region CDR3. In some embodiments, the substitutions are made as part of a humanization process. In some embodiments, the substitutions are made as part of a germline humanization process. In some embodiments, the substitutions are made as part of an affinity maturation process. In some embodiments, the substitutions are made as part of an optimization process.

[00113] In some embodiments, a BamA-binding agent comprises one or more heavy chain variable region CDRs that have been modified to reduce deamidation within the CDR sequence. Deamidation is a chemical reaction in which an amide functional group in the side chain of the amino acids asparagine (Asn or N) or glutamine (Gin or Q) is removed or converted to another functional group. Generally, asparagine is converted to aspartic acid or isoaspartic acid and glutamine is converted to glutamic acid or polyglutamic acid. In some situations, deamidation may change the structure, function, and/or stability of a polypeptide, potentially resulting in decreased biological activity. In some embodiments, the heavy chain variable region CDR1, CDR2, and/or CDR3 of a VHH described herein is modified to reduce deamidation.

[00114] In some embodiments, a BamA-binding agent comprises one or more heavy chain variable region CDRs that have been modified to reduce isomerization. Isomerization is a chemical process by which a compound is transformed into any of its isomeric forms, i.e., forms with the same chemical composition but with different structure or configuration and, potentially with different physical and chemical properties. Studies have shown that aspartate (Asp or D) isomerization within a CDR can impact antibody binding and/or stability. In some embodiments, the heavy chain variable region CDR1, CDR2, and/or CDR3 of a VHH described herein is modified to reduce isomerization.

[00115] In some embodiments, a BamA-binding agent comprises one or more heavy chain variable region CDRs that have been modified to reduce oxidation. Oxidation is a chemical process by which an oxygen is added to an atom, for example, methionine is converted to methionine sulfoxide by addition of an oxygen to the sulfur atom. Oxidation of one or more amino acids can potentially affect the physical and chemical properties of a protein. Studies have shown that oxidation of methionine (Met or M) within a CDR has the potential to impact antibody binding and/or stability. In some embodiments, the heavy chain variable region CDR1, CDR2, and/or CDR3 of a VHH described herein is modified to reduce oxidation.

[00116] In some embodiments, a BamA-binding agent comprises a heavy chain variable region that comprises a modification within the amino acid sequence wherein the modification eliminates a glycosylation site. In some embodiments, a BamA-binding agent comprises one or more heavy chain variable region CDRs that have been modified to eliminate a glycosylation site. The consensus glycosylation site for N-linked glycans is N-X-S/T, wherein X can be any amino acid except proline. Generally, a glycosylation site within a variable region and/or within a CDR will impact antibody structure, binding, and/or stability.

[00117] In some embodiments, a BamA-binding agent comprises a heavy chain variable region CDR1, CDR2, and CDR3 from VHH-29, a humanized version thereof, or variants thereof. In some embodiments, a BamA-binding agent comprises VHH-29. In some embodiments, a BamA-binding agent comprises a humanized version of VHH-29. In some embodiments, a BamA-binding agent comprises a variant of VHH-29 or a variant of humanized VHH-29.

[00118] In some embodiments, a BamA-binding agent comprises a heavy chain variable region CDR1 comprising the amino acid sequence GLSFGLDAYAVA (SEQ ID NO: 11), a heavy chain variable region CDR2 comprising the amino acid sequence CISPTGSRVAYADSAKG (SEQ ID NO: 12), and a heavy chain variable region CDR3 comprising the amino acid sequence SNDKRCSDFGVDRVGY (SEQ ID NO: 13). In some embodiments, a BamA-binding agent comprises a heavy chain variable region CDR1 comprising the amino acid sequence GLSFGLDAY (SEQ ID NO: 14), a heavy chain variable region CDR2 comprising the amino acid sequence SPTGSR (SEQ ID NO: 15), and a heavy chain variable region CDR3 comprising the amino acid sequence SNDKRCSDFGVDRVGY (SEQ ID NO: 13). In some embodiments, a BamA-binding agent comprises a heavy chain variable region CDR1 comprising the amino acid sequence GLSFGLDAYAVA (SEQ ID NO: 11), a heavy chain variable region CDR2 comprising the amino acid sequence CISPTGSRVA (SEQ ID NO: 16), and a heavy chain variable region CDR3 comprising the amino acid sequence SNDKRCSDFGVDRVGY (SEQ ID NO: 13). In some embodiments, a BamA-binding agent comprises a heavy chain variable region CDR1 comprising the amino acid sequence LDAYAVA (SEQ ID NO: 17), a heavy chain variable region CDR2 comprising the amino acid sequence CISPTGSRVAYADSAKG (SEQ ID NO: 12), and a heavy chain variable region CDR3 comprising the amino acid sequence SNDKRCSDFGVDRVGY (SEQ ID NO: 13). In some embodiments, a BamA-binding agent comprises a heavy chain variable region CDR1 comprising the amino acid sequence GLDAYAVA (SEQ ID NO: 18), a heavy chain variable region CDR2 comprising the amino acid sequence GVSCISPTGSRVA (SEQ ID NO: 19), and a heavy chain variable region CDR3 comprising the amino acid sequence ATSNDKRCSDFGVDRVG (SEQ ID NO:20).

[00119] In some embodiments, a BamA-binding agent comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising the amino acid sequence GLSFGLDAYAVA (SEQ ID NO: 11), a heavy chain variable region CDR2 comprising the amino acid sequence CISPTGSRVAYADSAKG (SEQ ID NO: 12), and a heavy chain variable region CDR3 comprising the amino acid sequence SNDKRCSDFGVDRVGY (SEQ ID NO: 13). In some embodiments, a BamA- binding agent comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising the amino acid sequence GLSFGLDAY (SEQ ID NO: 14), a heavy chain variable region CDR2 comprising the amino acid sequence SPTGSR (SEQ ID NO: 15), and a heavy chain variable region CDR3 comprising the amino acid sequence SNDKRCSDFGVDRVGY (SEQ ID NO: 13). In some embodiments, a BamA-binding agent comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising the amino acid sequence GLSFGLDAYAVA (SEQ ID NO: 11), a heavy chain variable region CDR2 comprising the amino acid sequence CISPTGSRVA (SEQ ID NO: 16), and a heavy chain variable region CDR3 comprising the amino acid sequence SNDKRCSDFGVDRVGY (SEQ ID NO: 13). In some embodiments, a BamA-binding agent comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising the amino acid sequence LDAYAVA (SEQ ID NO: 17), a heavy chain variable region CDR2 comprising the amino acid sequence CISPTGSRVAYADSAKG (SEQ ID NO: 12), and a heavy chain variable region CDR3 comprising the amino acid sequence SNDKRCSDFGVDRVGY (SEQ ID NO: 13). In some embodiments, a BamA- binding agent comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising the amino acid sequence GLDAYAVA (SEQ ID NO: 18), a heavy chain variable region CDR2 comprising the amino acid sequence GVSCISPTGSRVA (SEQ ID NO: 19), and a heavy chain variable region CDR3 comprising the amino acid sequence ATSNDKRCSDFGVDRVG (SEQ ID NO:20).

[00120] In some embodiments, a BamA-binding agent comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising the amino acid sequence GLSFGLDAYAVA (SEQ ID NO: 11), a heavy chain variable region CDR2 comprising the amino acid sequence CISPTGSRVAYADSAKG (SEQ ID NO: 12), and a heavy chain variable region CDR3 comprising the amino acid sequence SNDKRCSDFGVDRVGY (SEQ ID NO: 13).

[00121] In some embodiments, a BamA-binding agent comprises a heavy chain variable region CDR1 comprising the amino acid sequence GLSFGLDAYAVA (SEQ ID NO: 11), or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions, a heavy chain variable region CDR2 comprising the amino acid sequence CISPTGSRVAYADSAKG (SEQ ID NO: 12), or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions, and a heavy chain variable region CDR3 comprising the amino acid sequence SNDKRCSDFGVDRVGY (SEQ ID NO: 13), or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions.

[00122] In some embodiments, a BamA-binding agent comprises a heavy chain variable region having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:41. In some embodiments, a BamA- binding agent comprises a VHH having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:41. In some embodiments, a BamA-binding agent comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO:41. In some embodiments, a BamA-binding agent comprises a VHH comprising an amino acid sequence of SEQ ID NO:41.

[00123] In some embodiments, a BamA-binding agent comprises a VHH having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:41. In some embodiments, a BamA-binding agent comprises a VHH having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:41. In some embodiments, a BamA-binding agent comprises a VHH having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:41. In some embodiments, a BamA-binding agent comprises a VHH comprising the amino acid sequence of SEQ ID NO:41.

[00124] In some embodiments, a BamA-binding agent comprises a heavy chain variable region CDR1, CDR2, and CDR3 from VHH-47, a humanized version thereof, or variants thereof. In some embodiments, a BamA-binding agent comprises VHH-47. In some embodiments, a BamA-binding agent comprises a humanized version of VHH-47. In some embodiments, a BamA-binding agent comprises a variant of VHH-47 or a variant of humanized VHH-47.

[00125] In some embodiments, a BamA-binding agent comprises a heavy chain variable region CDR1 comprising the amino acid sequence GSTATRDTFSSHRMT (SEQ ID NO:21), a heavy chain variable region CDR2 comprising the amino acid sequence TITGDDITNYTGSSVKG (SEQ ID NO:22), and a heavy chain variable region CDR3 comprising the amino acid sequence LERGIWAY (SEQ ID NO:23). In some embodiments, a BamA-binding agent comprises a heavy chain variable region CDR1 comprising the amino acid sequence GSTATRDTFSSH (SEQ ID NO:24), a heavy chain variable region CDR2 comprising the amino acid sequence TGDDI (SEQ ID NO:25), and a heavy chain variable region CDR3 comprising the amino acid sequence LERGIWAY (SEQ ID NO:23). In some embodiments, a BamA- binding agent comprises a heavy chain variable region CDR1 comprising the amino acid sequence GSTATRDTFSSHRMT (SEQ ID NO:21), a heavy chain variable region CDR2 comprising the amino acid sequence TITGDDITN (SEQ ID NO:26), and a heavy chain variable region CDR3 comprising the amino acid sequence LERGIWAY (SEQ ID NO:23). In some embodiments, a BamA-binding agent comprises a heavy chain variable region CDR1 comprising the amino acid sequence RDTFSSHRMT (SEQ ID NO:27), a heavy chain variable region CDR2 comprising the amino acid sequence TITGDDITNYTGSSVKG (SEQ ID NO:22), and a heavy chain variable region CDR3 comprising the amino acid sequence LERGIWAY (SEQ ID NO:23). In some embodiments, a BamA-binding agent comprises a heavy chain variable region CDR1 comprising the amino acid sequence TRDTFSSHRMT (SEQ ID NO:28), a heavy chain variable region CDR2 comprising the amino acid sequence MVATITGDDITN (SEQ ID NO:29), and a heavy chain variable region CDR3 comprising the amino acid sequence HLLERGIWA (SEQ ID NO: 30).

[00126] In some embodiments, a BamA-binding agent comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising the amino acid sequence GSTATRDTFSSHRMT (SEQ ID NO:21), a heavy chain variable region CDR2 comprising the amino acid sequence TITGDDITNYTGSSVKG (SEQ ID NO:22), and a heavy chain variable region CDR3 comprising the amino acid sequence LERGIWAY (SEQ ID NO:23). In some embodiments, a BamA- binding agent comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising the amino acid sequence GSTATRDTFSSH (SEQ ID NO:24), a heavy chain variable region CDR2 comprising the amino acid sequence TGDDI (SEQ ID NO:25), and a heavy chain variable region CDR3 comprising the amino acid sequence LERGIWAY (SEQ ID NO:23). In some embodiments, a BamA-binding agent comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising the amino acid sequence GSTATRDTFSSHRMT (SEQ ID NO:21), a heavy chain variable region CDR2 comprising the amino acid sequence TITGDDITN (SEQ ID NO:26), and a heavy chain variable region CDR3 comprising the amino acid sequence LERGIWAY (SEQ ID NO:23). In some embodiments, a BamA-binding agent comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising the amino acid sequence RDTFSSHRMT (SEQ ID NO:27), a heavy chain variable region CDR2 comprising the amino acid sequence TITGDDITNYTGSSVKG (SEQ ID NO:22), and a heavy chain variable region CDR3 comprising the amino acid sequence LERGIWAY (SEQ ID NO:23). In some embodiments, a BamA-binding agent comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising the amino acid sequence TRDTFSSHRMT (SEQ ID NO:28), a heavy chain variable region CDR2 comprising the amino acid sequence MVATITGDDITN (SEQ ID NO:29), and a heavy chain variable region CDR3 comprising the amino acid sequence HLLERGIWA (SEQ ID NO: 30).

[00127] In some embodiments, a BamA-binding agent comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising the amino acid sequence GSTATRDTFSSHRMT (SEQ ID NO:21), a heavy chain variable region CDR2 comprising the amino acid sequence TITGDDITNYTGSSVKG (SEQ ID NO:22), and a heavy chain variable region CDR3 comprising the amino acid sequence LERGIWAY (SEQ ID NO:23).

[00128] In some embodiments, a BamA-binding agent comprises a heavy chain variable region CDR1 comprising the amino acid sequence GSTATRDTFSSHRMT (SEQ ID NO:21), or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions, a heavy chain variable region CDR2 comprising the amino acid sequence TITGDDITNYTGSSVKG (SEQ ID NO:22), or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions, and a heavy chain variable region CDR3 comprising the amino acid sequence LERGIWAY (SEQ ID NO:23), or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions.

[00129] In some embodiments, a BamA-binding agent comprises a heavy chain variable region having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:42. In some embodiments, a BamA- binding agent comprises a VHH having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:42. In some embodiments, a BamA-binding agent comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO:42. In some embodiments, a BamA-binding agent comprises a VHH comprising an amino acid sequence of SEQ ID NO:42.

[00130] In some embodiments, a BamA-binding agent comprises a VHH having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:42. In some embodiments, a BamA-binding agent comprises a VHH having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:42. In some embodiments, a BamA-binding agent comprises a VHH having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:42. In some embodiments, a BamA-binding agent comprises a VHH comprising the amino acid sequence of SEQ ID NO:42.

[00131] In some embodiments, a BamA-binding agent comprises a heavy chain variable region CDR1, CDR2, and CDR3 from VHH-82, a humanized version thereof, or variants thereof. In some embodiments, a BamA-binding agent comprises VHH-82. In some embodiments, a BamA-binding agent comprises a humanized version of VHH-82. In some embodiments, a BamA-binding agent comprises a variant of VHH-82 or a variant of humanized VHH-82.

[00132] In some embodiments, a BamA-binding agent comprises a heavy chain variable region CDR1 comprising the amino acid sequence GFSFRDYSMS (SEQ ID NO:31), a heavy chain variable region CDR2 comprising the amino acid sequence GIRSLGTTTYYADSVKG (SEQ ID NO:32), and a heavy chain variable region CDR3 comprising the amino acid sequence CLGKICDRFGIVDIYS (SEQ ID NO:33). In some embodiments, a BamA-binding agent comprises a heavy chain variable region CDR1 comprising the amino acid sequence GFSFRDY (SEQ ID NO:34), a heavy chain variable region CDR2 comprising the amino acid sequence RSLGTT (SEQ ID NO:35), and a heavy chain variable region CDR3 comprising the amino acid sequence CLGKICDRFGIVDIYS (SEQ ID NO:33). In some embodiments, a BamA-binding agent comprises a heavy chain variable region CDR1 comprising the amino acid sequence GFSFRDYSMS (SEQ ID NO:31), a heavy chain variable region CDR2 comprising the amino acid sequence GIRSLGTTTY (SEQ ID NO:36), and a heavy chain variable region CDR3 comprising the amino acid sequence CLGKICDRFGIVDIYS (SEQ ID NO:33). In some embodiments, a BamA-binding agent comprises a heavy chain variable region CDR1 comprising the amino acid sequence DYSMS (SEQ ID NO:37), a heavy chain variable region CDR2 comprising the amino acid sequence GIRSLGTTTYYADSVKG (SEQ ID NO:32), and a heavy chain variable region CDR3 comprising the amino acid sequence CLGKICDRFGIVDIYS (SEQ ID NO:33). In some embodiments, a BamA-binding agent comprises a heavy chain variable region CDR1 comprising the amino acid sequence RDYSMS (SEQ ID NO:38), a heavy chain variable region CDR2 comprising the amino acid sequence WVSGIRSLGTTTY (SEQ ID NO:39), and a heavy chain variable region CDR3 comprising the amino acid sequence AKCLGKICDRFGIVDIY (SEQ ID NO: 40).

[00133] In some embodiments, a BamA-binding agent comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising the amino acid sequence GFSFRDYSMS (SEQ ID NO:31), a heavy chain variable region CDR2 comprising the amino acid sequence GIRSLGTTTYYADSVKG (SEQ ID NO:32), and a heavy chain variable region CDR3 comprising the amino acid sequence CLGKICDRFGIVDIYS (SEQ ID NO:33). In some embodiments, a BamA-binding agent comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising the amino acid sequence GFSFRDY (SEQ ID NO:34), a heavy chain variable region CDR2 comprising the amino acid sequence RSLGTT (SEQ ID NO:35), and a heavy chain variable region CDR3 comprising the amino acid sequence CLGKICDRFGIVDIYS (SEQ ID NO:33). In some embodiments, a BamA-binding agent comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising the amino acid sequence GFSFRDYSMS (SEQ ID NO:31), a heavy chain variable region CDR2 comprising the amino acid sequence GIRSLGTTTY (SEQ ID NO:36), and a heavy chain variable region CDR3 comprising the amino acid sequence CLGKICDRFGIVDIYS (SEQ ID NO:33). In some embodiments, a BamA-binding agent comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising the amino acid sequence DYSMS (SEQ ID NO:37), a heavy chain variable region CDR2 comprising the amino acid sequence GIRSLGTTTYYADSVKG (SEQ ID NO:32), and a heavy chain variable region CDR3 comprising the amino acid sequence CLGKICDRFGIVDIYS (SEQ ID NO:33). In some embodiments, a BamA-binding agent comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising the amino acid sequence RDYSMS (SEQ ID NO:38), a heavy chain variable region CDR2 comprising the amino acid sequence WVSGIRSLGTTTY (SEQ ID NO:39), and a heavy chain variable region CDR3 comprising the amino acid sequence AKCLGKICDRFGIVDIY (SEQ ID NO:40).

[00134] In some embodiments, a BamA-binding agent comprises a heavy chain variable region comprising a heavy chain variable region comprising a heavy chain variable region CDR1 comprising the amino acid sequence GFSFRDYSMS (SEQ ID NO:31), a heavy chain variable region CDR2 comprising the amino acid sequence GIRSLGTTTYYADSVKG (SEQ ID NO:32), and a heavy chain variable region CDR3 comprising the amino acid sequence CLGKICDRFGIVDIYS (SEQ ID NO:33).

[00135] In some embodiments, a BamA-binding agent comprises a heavy chain variable region CDR1 comprising the amino acid sequence GFSFRDYSMS (SEQ ID NO:31), or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions, a heavy chain variable region CDR2 comprising the amino acid sequence GIRSLGTTTYYADSVKG (SEQ ID NO:32), or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions, and a heavy chain variable region CDR3 comprising the amino acid sequence CLGKICDRFGIVDIYS (SEQ ID NO:33), or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions.

[00136] In some embodiments, a BamA-binding agent comprises a heavy chain variable region having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:43. In some embodiments, a BamA- binding agent comprises a VHH having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:43. In some embodiments, a BamA-binding agent comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO:43. In some embodiments, a BamA-binding agent comprises a VHH comprising an amino acid sequence of SEQ ID NO:43.

[00137] In some embodiments, a BamA-binding agent comprises a VHH having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:43. In some embodiments, a BamA-binding agent comprises a VHH having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:43. In some embodiments, a BamA-binding agent comprises a VHH having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:43. In some embodiments, a BamA-binding agent comprises a VHH comprising the amino acid sequence of SEQ ID NO:43.

[00138] Provided herein are agents that compete with one or more of the binding agents described herein for binding to Acinetobacter BamA. In some embodiments, an agent competes with one or more of the binding agents described herein for binding to A. baumannii BamA. In some embodiments, an agent that competes with one or more of the binding agents described herein is an antibody. In some embodiments, an agent that competes with one or more of the binding agents described herein is a VHH. In some embodiments, an agent binds the same epitope as one of the BamA-binding agents described herein. In some embodiments, an agent binds an epitope overlapping with an epitope bound by one of the BamA-binding agents described herein. Antibodies and antigen-binding fragments (e.g., VHHs) that compete with or bind the same epitope as the BamA-binding agents described herein are expected to show similar functional properties.

[00139] In some embodiments, a BamA-binding agent comprises a VHH described herein and at least one heavy chain constant regions (e.g., CHI, CH2, CH3, and/or CH4). In some embodiments, one or more of the constant regions of the binding agent has been modified. In some embodiments, the one or more of the constant regions of a modified agent comprises at least one human constant region. In some embodiments, the one or more of the constant regions of the modified agent comprises more than one human constant region. In some embodiments, modifications to a constant region comprise additions, deletions, or substitutions of one or more amino acids in one or more regions. In some embodiments, a constant region is replaced by a short amino acid spacer that provides some of the molecular flexibility typically imparted by a constant region.

[00140] It is known in the art that the constant region(s) of an antibody mediates several effector functions and these effector functions can vary depending on the isotype of the antibody. For example, binding of the C Iq component of complement to the Fc region of IgG or IgM antibodies when the antibodies are bound to antigen activates the complement system. Activation of complement is important in the opsonization and lysis of cell pathogens. The activation of complement also stimulates the inflammatory immune response and can be involved in autoimmune hypersensitivity. In addition, the Fc region of an antibody can bind a cell expressing a Fc receptor (FcR). There are a number of Fc receptors that are specific for different classes of antibody, including IgG (gamma receptors), IgE (epsilon receptors), IgA (alpha receptors) and IgM (mu receptors). Binding of antibody to Fc receptors on cell surfaces triggers a number of important and diverse biological responses including, but not limited to, engulfment and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody -coated target cells by killer cells (i.e., antibody-dependent cell cytotoxicity or ADCC), release of inflammatory mediators, placental transfer, and control of immunoglobulin production.

[00141] In some embodiments, a BamA-binding agent comprises a VHH and a variant constant region or Fc region. The amino acid sequences of the constant region or Fc region of human IgGl, IgG2, IgG3, and IgG4 are known to those of ordinary skill in the art (e.g., a representative human IgGl constant region is SEQ ID NO:44). In some cases, constant regions or Fc regions with amino acid variations have been identified in native antibodies. In some embodiments, a variant constant region or Fc region is engineered with substitutions at specific amino acid positions as compared to a native constant region or Fc region. Variant constant region or Fc regions are known in the art and include, but are not limited to, SEQ ID NOs:45-51. [00142] In some embodiments, a modified antibody provides for altered effector functions that, in turn, affect the biological profile of the antibody. For example, in some embodiments, the deletion or inactivation (through point mutations or other means) of a constant region reduces binding of a modified antibody to a Fc receptor. In some embodiments, constant region modifications increase the serum halflife of an antibody. In some embodiments, constant region modifications reduce the serum half-life of an antibody. In some embodiments, constant region modifications decrease or remove ADCC and/or complement-dependent cytotoxicity (CDC) of an antibody. In some embodiments, a human IgGl Fc region with specific amino acid substitutions corresponding to IgG2 or IgG4 residues reduce effector functions (e.g., ADCC and CDC) in a modified antibody. In some embodiments, a modified antibody does not have one or more effector functions. In some embodiments, a modified antibody has no ADCC activity and/or no CDC activity. In some embodiments, a modified antibody does not bind an Fc receptor and/or complement factors. In some embodiments, a modified antibody does not have any detectable effector functions (e.g., an “effectorless” antibody). In some embodiments, constant region modifications increase or enhance ADCC and/or CDC of an antibody. In some embodiments, the constant region is modified to eliminate disulfide linkages or oligosaccharide moieties. In some embodiments, the constant region is modified to add/substitute one or more amino acids to provide one or more cytotoxin, oligosaccharide, or carbohydrate attachment sites.

[00143] Modifications to a constant region of a binding agent described herein may be made using well- known biochemical or molecular engineering techniques. In some embodiments, antibody variants are prepared by introducing appropriate nucleotide changes into the encoding DNA, and/or by synthesis of the desired antibody or polypeptide. Using these engineering techniques to modify an antibody it may be possible to disrupt the activity or effector function provided by a specific sequence or region while substantially maintaining the structure, binding activity, and other desired characteristics of the modified antibody.

[00144] The present disclosure provides fusion proteins comprising a BamA-binding agent described herein. In some embodiments, a BamA-binding agent comprises a polypeptide comprising a VHH described herein. In some embodiments, a BamA-binding agent comprises a polypeptide comprising at least one of the VHHs described herein. In some embodiments, a BamA-binding agent comprises a polypeptide comprising: a first VHH described herein and a second VHH described herein. In some embodiments, a BamA-binding agent comprises a polypeptide comprising: (a) a first VHH comprising a CDR1 comprising the amino acid sequence GFSFRDYSMS (SEQ ID NO:31), a CDR2 comprising the amino acid sequence GIRSLGTTTYYADSVKG (SEQ ID NO:32), and a CDR3 comprising the amino acid sequence CLGKICDRFGIVDIYS (SEQ ID NO:33); and (b) a second VHH. In some embodiments, a BamA-binding agent comprises a polypeptide comprising: (a) a first VHH comprising a CDR1 comprising the amino acid sequence GFSFRDYSMS (SEQ ID NO:31), a CDR2 comprising the amino acid sequence GIRSLGTTTYYADSVKG (SEQ ID NO:32), and a CDR3 comprising the amino acid sequence CLGKICDRFGIVDIYS (SEQ ID NO:33); and (b) a second VHH comprising a CDR1 comprising the amino acid sequence GLSFGLDAYAVA (SEQ ID NO: 11), a CDR2 comprising the amino acid sequence CISPTGSRVAYADSAKG (SEQ ID NO: 12), and a CDR3 comprising the amino acid sequence SNDKRCSDFGVDRVGY (SEQ ID NO: 13). In some embodiments, a BamA-binding agent comprises a polypeptide comprising: (a) a first VHH comprising a CDR1 comprising the amino acid sequence GFSFRDYSMS (SEQ ID NO:31), a CDR2 comprising the amino acid sequence GIRSLGTTTYYADSVKG (SEQ ID NO:32), and a CDR3 comprising the amino acid sequence CLGKICDRFGIVDIYS (SEQ ID NO:33); and (b) a second VHH comprising a CDR1 comprising the amino acid sequence GSTATRDTFSSHRMT (SEQ ID NO:21), a CDR2 comprising the amino acid sequence TITGDDITNYTGSVKG (SEQ ID NO:22), and a CDR3 comprising the amino acid sequence LERGIWAY (SEQ ID NO:23).

[00145] In some embodiments, a BamA-binding agent comprises VHH-82 linked to VHH-29. In some embodiments, a BamA-binding agent comprises VHH-82 linked to VHH-47. In some embodiments, a BamA-binding agent comprises VHH-29 linked to VHH-47. In some embodiments, the first VHH is directly linked to the second VHH. In some embodiments, the first VHH is indirectly linked to the second VHH via a linker.

[00146] The present disclosure further embraces additional variants and equivalents that are substantially homologous to the recombinant, monoclonal, chimeric, humanized, human antibodies, antibody fragments thereof, or VHHs described herein. In some embodiments, it is desirable to improve the binding affinity of the antibody (e.g., VHH). In some embodiments, it is desirable to modulate biological properties of the antibody (e.g., VHH), including but not limited to, specificity, thermostability, expression level, effector fimction(s), glycosylation, immunogenicity, and/or solubility. Those skilled in the art will appreciate that amino acid changes may alter post-translational processes of an antibody (e.g., VHH), such as changing the number or position of glycosylation sites or altering membrane anchoring characteristics.

[00147] Variations may be a substitution, deletion, or insertion of one or more nucleotides encoding the binding agent or polypeptide that results in a change in the amino acid sequence as compared with the parent molecule or polypeptide sequence. In some embodiments, amino acid substitutions are the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine (i.e., conservative amino acid replacements). In some embodiments, the substitution, deletion, or insertion includes less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions relative to the parent molecule. In some embodiments, variations in the amino acid sequence that are biologically useful and/or relevant are determined by systematically making insertions, deletions, or substitutions in the sequence and testing the resulting variant proteins for activity as compared to the parental molecule.

[00148] In some embodiments, variants may include addition of amino acid residues at the amino- and/or carboxyl-terminal end of the binding agent or polypeptide. The length of additional amino acids residues may range from one residue to a hundred or more residues. In some embodiments, a variant comprises an N-terminal methionyl residue. In some embodiments, the variant comprises an additional polypeptide/protein to create a fusion protein. In some embodiments, a variant is engineered to be detectable and may comprise a detectable label and/or protein (e.g. , a fluorescent tag, a fluorescent protein, or an enzyme).

[00149] In some embodiments, a cysteine residue not involved in maintaining the proper conformation of an antibody or binding agent is substituted or deleted to modulate the protein’s characteristics, for example, to improve oxidative stability and/or prevent aberrant disulfide crosslinking. Conversely, in some embodiments, one or more cysteine residues are added to create disulfide bond(s) to improve stability.

[00150] In some embodiments, an antibody (e.g., VHH) of the present disclosure is “deimmunized”. The deimmunization of antibodies generally consists of introducing specific amino acid mutations (e.g., substitutions, deletions, additions) that result in removal of T-cell epitopes (known or predicted) without significantly reducing the binding affinity or other desired activities of the antibody.

[00151] The variant antibodies, VHHs, or polypeptides described herein may be generated using methods known in the art, including but not limited to, site-directed mutagenesis, alanine scanning mutagenesis, and PCR mutagenesis.

[00152] In some embodiments, a BamA-binding agent described herein is chemically modified. In some embodiments, a BamA-binding agent is an anti-BamA VHH that is chemically modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, and/or linkage to a cellular ligand or other protein. Any of numerous chemical modifications can be carried out by known techniques. In some embodiments, a BamA-binding agent is attached (either directly or indirectly) to a half-life extending moiety including, but not limited to, polyethylene glycol (PEG), a PEG mimetic, XTEN®, serum albumin, polysialic acid, N-(2-hydroxypropyl)methacrylamide, or dextran. In some embodiments, a BamA-binding agent is a VHH, wherein the VHH is attached (either directly or indirectly) to a half-life extending moiety including, but not limited to, polyethylene glycol (PEG), a PEG mimetic, XTEN®, serum albumin, polysialic acid, N-(2-hydroxypropyl)methacrylamide, or dextran.

[00153] In some embodiments, a composition comprises a BamA-binding agent described herein. In some embodiments, a composition comprises an anti-BamA VHH described herein. In some embodiments, a composition comprises a monoclonal anti-BamA VHH described herein. In some embodiments, a composition comprises a VHH selected from the group consisting of: VHH-29, VHH-47, VHH-82, and humanized versions thereof. In some embodiments, a composition comprises VHH-82. [00154] In some embodiments, a pharmaceutical composition comprises a BamA-binding agent described herein and a pharmaceutically acceptable carrier. In some embodiments, a pharmaceutical composition comprises an anti-BamA VHH described herein and a pharmaceutically acceptable carrier. In some embodiments, a pharmaceutical composition comprises a monoclonal anti-BamA VHH described herein and a pharmaceutically acceptable carrier. In some embodiments, a pharmaceutical composition comprises a VHH selected from the group consisting of: VHH-29, VHH-47, VHH-82, and humanized versions thereof and a pharmaceutically acceptable carrier. In some embodiments, a pharmaceutical composition comprises VHH-82 and a pharmaceutically acceptable carrier.

[00155] In some embodiments, a BamA-binding agent is isolated. In some embodiments, a BamA- binding agent is substantially pure.

[00156] Generally speaking, antigen-antibody interactions are non-covalent and reversible, formed by a combination of hydrogen bonds, hydrophobic interactions, electrostatic and van der Waals forces. When describing the strength of an antigen-antibody complex, the terms affinity and/or avidity are commonly used. The binding of an antibody to its antigen is a reversible process, and the affinity of the binding is typically reported as an equilibrium dissociation constant (KD). KD is the ratio of an antibody dissociation rate (k_Off) (how quickly it dissociates from its antigen) to the antibody association rate (k_on) (how quickly it binds to its antigen). In some embodiments, KD values are determined by measuring the k_on and k_Off rates of a specific antibody/antigen interaction and then using a ratio of these values to calculate the KD value. KD values may be used to evaluate and rank order the strength of individual antibody/antigen interactions. The lower the KD of an antibody, the higher the affinity of the antibody for its target. In some embodiments, affinity is measured using SPR technology in a Biacore system. Avidity gives a measure of the overall strength of an antibody-antigen complex. It is dependent on three major parameters: (i) affinity of the antibody for the target, (ii) valency of both the antibody and antigen, and (iii) structural arrangement of the parts that interact.

[00157] In some embodiments, a BamA-binding agent binds BamA with a dissociation constant (K_D) of 1 pM or less, 100 nM or less, 40 nM or less, 20 nM or less, 10 nM or less, 1 nM or less, 0. 1 nM or less, 50 pM or less, 10 pM or less, or 1 pM or less. In some embodiments, a BamA-binding agent binds BamA with a KD of about 20 nM or less. In some embodiments, a BamA-binding agent binds BamA with a KD of 10 nM or less. In some embodiments, a BamA-binding agent binds BamA with a KD of 5 nM or less. In some embodiments, a BamA-binding agent binds BamA with a KD of 3 nM or less. In some embodiments, a BamA-binding agent binds BamA with a KD of 2 nM or less. In some embodiments, a BamA-binding agent binds BamA with a KD of 1 nM or less. In some embodiments, a BamA-binding agent binds BamA with a KD of 0.5 nM or less. In some embodiments, a BamA-binding agent binds BamA with a KD of 0.1 nM or less. In some embodiments, a BamA-binding agent binds BamA with a KD of 50 pM or less. In some embodiments, a BamA-binding agent binds BamA with a KD of 25 pM or less. In some embodiments, a BamA-binding agent binds BamA with a KD of 10 pM or less. In some embodiments, a BamA-binding agent binds BamA with a KD of 1 pM or less. In some embodiments, a BamA-binding agent binds BamA with a KD of 0.01 nM to 2.5 nM. In some embodiments, a BamA- binding agent binds BamA with a KD of 0.1 nM to 5 nM. In some embodiments, a BamA-binding agent binds BamA with a KD of 1 nM to 5 nM. In some embodiments, the dissociation constant of the binding agent for BamA is the dissociation constant determined using a BamA protein immobilized on a Biacore chip and the binding agent flowed over the chip. In some embodiments, the dissociation constant of the binding agent for BamA is the dissociation constant determined using the binding agent captured on a Biacore chip and soluble BamA flowed over the chip.

[00158] In some embodiments, a BamA-binding agent binds BamA with a half-maximal effective concentration (EC50) of 1 pM or less, 100 nM or less, 40 nM or less, 20 nM or less, 10 nM or less, 1 nM or less, or 0.1 nM or less. In some embodiments, a BamA-binding agent binds BamA with a half- maximal effective concentration (EC50) of 1 pM or less. In some embodiments, a BamA-binding agent binds BamA with a half-maximal effective concentration (EC50) of 100 nM or less. In some embodiments, a BamA-binding agent binds BamA with a half-maximal effective concentration (EC50) of 40 nM or less. In some embodiments, a BamA-binding agent binds BamA with a half-maximal effective concentration (EC50) of 20 nM or less. In some embodiments, a BamA-binding agent binds BamA with a half-maximal effective concentration (EC50) of 10 nM or less. In some embodiments, a BamA-binding agent binds BamA with a half-maximal effective concentration (EC50) of 1 nM or less. In some embodiments, a BamA-binding agent binds BamA with a half-maximal effective concentration (EC50) of 0.1 nM or less. In some embodiments, a BamA-binding agent binds BamA with an EC50 of 1 pM or less, 100 nM or less, 40 nM or less, 20 nM or less, 10 nM or less, 1 nM or less, or 0. 1 nM or less. In some embodiments, a BamA-binding agent binds BamA with an EC50 of 1 pM or less. In some embodiments, a BamA-binding agent binds BamA with an EC50 of 100 nM or less. In some embodiments, a BamA- binding agent binds BamA with an EC50 of 40 nM or less. In some embodiments, a BamA-binding agent binds BamA with an EC50 of 20 nM or less. In some embodiments, a BamA-binding agent binds BamA with an EC50 of 10 nM or less. In some embodiments, a BamA-binding agent binds BamA with an EC50 of 1 nM or less. In some embodiments, a BamA-binding agent binds BamA with an EC50 of 0.1 nM or less. In some embodiments, a BamA-binding agent binds BamA with an EC50 of 0. 1 nM to about 3 nM. In some embodiments, a BamA-binding agent binds BamA with an EC50 of 0. 1 nM to 2 nM. In some embodiments, a BamA-binding agent binds BamA with an EC50 of 0.1 nM to 1 nM. In some embodiments, a BamA-binding agent binds BamA with an EC50 of 0.5 nM to 3 nM. In some embodiments, a BamA-binding agent binds BamA with an EC50 of 0.5 nM to 2 nM. In some embodiments, a BamA-binding agent binds BamA with an EC50 of 0.5 nM to 1 nM.

[00159] In some embodiments, a BamA-binding agent has at least one or more of the following properties: (i) binds Acinetobacter baumannii; (ii) does not bind E. coli BamA; (iii) is an antagonist of A. baumannii BamA; (iv) inhibits growth of A. baumannii,' (v) inhibits Bam activity, and (vi) inhibits OmpT protease activity.

[00160] The BamA-binding agents described herein can be produced by any suitable method known in the art. Such methods range from direct protein synthesis methods to constructing a DNA sequence encoding polypeptide sequences and expressing those sequences in a suitable host. In some embodiments, a DNA sequence is constructed using recombinant technology by isolating or synthesizing a DNA sequence encoding a wild-type protein of interest. Optionally, the sequence can be mutagenized by site-specific mutagenesis to provide functional variants thereof. In some embodiments, a DNA sequence encoding a polypeptide of interest is constructed by chemical synthesis using an oligonucleotide synthesizer. Oligonucleotides can be designed based on the amino acid sequence of the desired polypeptide and selecting those codons that are favored in the host cell in which the recombinant polypeptide of interest will be produced. Standard methods can be applied to synthesize a polynucleotide sequence encoding an isolated polypeptide of interest. For example, a complete amino acid sequence can be used to construct a back-translated gene. Further, a DNA oligomer containing a nucleotide sequence coding for the particular isolated polypeptide can be synthesized. For example, several oligonucleotides coding for portions of the desired polypeptide can be synthesized and then ligated. The individual oligonucleotides typically contain 5' or 3' overhangs for complementary assembly.

[00161] Once assembled (by synthesis, site-directed mutagenesis, or another method), the polynucleotide sequences encoding a particular polypeptide of interest can be inserted into an expression vector and operatively linked to an expression control sequence appropriate for expression of the protein in a desired host. Proper assembly can be confirmed by nucleotide sequencing, restriction enzyme mapping, and/or expression of a biologically active polypeptide in a suitable host.

[00162] In some embodiments, recombinant expression vectors are used to amplify and express DNA encoding the BamA-binding agents described herein. For example, recombinant expression vectors can be replicable DNA constructs that have synthetic or cDNA-derived DNA fragments encoding a polypeptide chain of a BamA-binding agent, such as an anti-BamA antibody (e.g., VHH) operatively linked to suitable transcriptional and/or translational regulatory elements derived from mammalian, microbial, viral or insect genes. A transcriptional unit generally comprises an assembly of (i) a genetic element or elements having a regulatory role in gene expression, for example, transcriptional promoters or enhancers, (ii) a structural or coding sequence that is transcribed into mRNA and translated into protein, and (iii) appropriate transcription and translation initiation and termination sequences. Regulatory elements can include an operator sequence to control transcription. The ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants can additionally be incorporated. DNA regions are “operatively linked” when they are functionally related to each other. For example, DNA for a signal peptide (secretory leader) is operatively linked to DNA for a polypeptide if it is expressed as a precursor that participates in the secretion of the polypeptide; a promoter is operatively linked to a coding sequence if it controls the transcription of the sequence; or a ribosome binding site is operatively linked to a coding sequence if it is positioned so as to permit translation. In some embodiments, structural elements intended for use in yeast expression systems include a leader sequence enabling extracellular secretion of translated protein by a host cell. In some embodiments, in situations where recombinant protein is expressed without a leader or transport sequence, a polypeptide may include an N-terminal methionine residue. This residue can optionally be subsequently cleaved from the expressed recombinant protein to provide a final product.

[00163] The choice of an expression control sequence and an expression vector generally depends upon the choice of host. A wide variety of expression host/vector combinations can be employed. Useful expression vectors for eukaryotic hosts include, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus, and cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from E. coli, including pCRl, pBR322, pMB9 and their derivatives, and wider host range plasmids, such as M13 and other filamentous single-stranded DNA phages.

[00164] Suitable host cells for expression of a BamA-binding agent or a BamA protein or fragment thereof to use as an antigen or immunogen include prokaryotes, yeast cells, insect cells, or higher eukaryotic cells under the control of appropriate promoters. Prokaryotes include gram -negative or grampositive organisms, for example E. coli or Bacillus. Higher eukaryotic cells include established cell lines of mammalian origin as described herein. Cell-free translation systems may also be employed. Appropriate cloning vectors and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts, as well as methods of protein production, including antibody production are well known in the art. [00165] Various mammalian culture systems can be used to express recombinant polypeptides. Expression of recombinant proteins in mammalian cells may be desirable because these proteins are generally correctly folded, appropriately modified, and biologically functional. Examples of suitable mammalian host cell lines include, but are not limited to, COS-7 (monkey kidney-derived), L-929 (murine fibroblast-derived), C127 (murine mammary tumor-derived), 3T3 (murine fibroblast-derived), CHO (Chinese hamster ovary-derived), HeLa (human cervical cancer-derived), BHK (hamster kidney fibroblast-derived), HEK-293 (human embryonic kidney-derived) cell lines and variants thereof. Mammalian expression vectors can comprise non-transcribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5' or 3' flanking nontranscribed sequences, and 5' or 3' non-translated sequences, such as necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, and transcriptional termination sequences.

[00166] Expression of recombinant proteins in insect cell culture systems (e.g., baculovirus) also offers a robust method for producing correctly folded and biologically functional proteins. Baculovirus systems for production of heterologous proteins in insect cells are well-known to those of skill in the art.

[00167] Proteins produced by a host cell can be purified according to any suitable method. Standard methods include chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for protein purification. Affinity tags such as hexahistidine (His6; SEQ ID NO:52), maltose binding domain, influenza coat sequence, and glutathione-S-transferase can be attached to the protein to allow easy purification by passage over an appropriate affinity column. Affinity chromatography methods used for purifying immunoglobulins can include, but are not limited to, Protein A, Protein G, and Protein L chromatography. Isolated proteins can be physically characterized using techniques that include, but are not limited to, proteolysis, size exclusion chromatography (SEC), mass spectrometry (MS), nuclear magnetic resonance (NMR), isoelectric focusing (IEF), high performance liquid chromatography (HPLC), and x-ray crystallography. The purity of isolated proteins can be determined using techniques known to those of skill in the art, including but not limited to, SDS-PAGE, SEC, capillary gel electrophoresis, IEF, and capillary isoelectric focusing (cIEF).

[00168] In some embodiments, supernatants from expression systems that secrete recombinant protein into culture media are first concentrated using a commercially available protein concentration filter, for example, an Amicon® or Millipore Pellicon® ultrafiltration unit. Following the concentration step, the concentrate can be applied to a suitable purification matrix. In some embodiments, an anion exchange resin is employed, for example, a matrix or substrate having pendant diethylaminoethyl (DEAE) groups. The matrices can be acrylamide, agarose, dextran, cellulose, or other types commonly employed in protein purification. In some embodiments, a cation exchange step is employed. Suitable cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups. In some embodiments, a hydroxyapatite media is employed, including but not limited to, ceramic hydroxyapatite (CHT). In some embodiments, one or more reverse-phase HPLC steps employing hydrophobic RP- HPLC media, e.g., silica gel having pendant methyl or other aliphatic groups, are employed to further purify a recombinant protein. In some embodiments, hydrophobic interaction chromatography (HIC) is used to separate recombinant proteins based on their hydrophobicity. HIC is a useful separation technique for purifying proteins while maintaining biological activity due to the use of conditions and matrices that operate under less denaturing conditions than some other techniques. Some or all of the foregoing purification steps, in various combinations, can be employed to provide a homogeneous recombinant protein.

[00169] BamA-binding agents of the present disclosure may be analyzed for their physical/chemical properties and/or biological activities by various assays known in the art. In some embodiments, a BamA-binding agent is tested for its ability to bind BamA. Binding assays include, but are not limited to, SPR (e.g., Biacore), ELISA, and FACS. In some embodiments, a BamA-binding agent is tested for its ability to inhibit growth of Acinetobacter cells. In some embodiments, a BamA-binding agent is tested for its ability to inhibit growth of A. baumannii cells. In addition, binding agents may be evaluated for solubility, stability, thermostability, viscosity, expression levels, expression quality, and/or purification efficiency.

[00170] In some embodiments, monoclonal antibodies or VHHs generated against BamA are grouped based upon the epitope each individual antibody (e.g., VHH) recognizes, a process known as “epitope binning”. Generally, antibodies or VHHs are tested in a pairwise combinatorial manner and antibodies or VHHs that compete with each other are grouped together into bins. For example, in a premix binning assay, a first antibody is immobilized on a surface and a premixed solution of a second antibody and antigen is flowed over the immobilized first antibody. In tandem, the antigen is immobilized on a surface and the two antibodies are flowed over the immobilized antigen and compete to bind. Using these techniques, antibodies that block one another can be identified. A competitive blocking profile is created for each antibody relative to the other antibodies. The blocking results determine which bin each antibody is placed in. High-throughput methods of epitope binning are known in the art and allow for screening and characterization of large numbers of antibodies within a short period of time. Antibodies that bind similar epitopes often share similar functions and/or capabilities. Conversely, antibodies that bind different epitopes may have different functional activities.

[00171] In some embodiments, an epitope bin comprises at least one VHH from the group consisting of: VHH-29, VHH-47, and VHH-82. In some embodiments, an epitope bin comprises VHH-29. In some embodiments, an epitope bin comprises VHH-47. In some embodiments, an epitope bin comprises VHH- 82. In some embodiments, an epitope bin comprises VHH-29, VHH-47, and VHH-82.

[00172] Epitope mapping is the process of identifying the binding site, or epitope on a target protein/antigen where an antibody (e.g., VHH) binds. A variety of methods are known in the art for mapping epitopes on target proteins. These methods include (i) mutagenesis, including but not limited to, shotgun mutagenesis, site-directed mutagenesis, and alanine scanning; (ii) domain or fragment scanning; (iii) peptide scanning (e.g., Pepscan technology); (iv) display methods, including but not limited to, phage display, microbial display, and ribosome/mRNA display; (v) methods involving proteolysis and mass spectroscopy; (vi) methods involving amide hydrogen/deuterium exchange; and (vii) structural determination, including but not limited to, x-ray crystallography and NMR.

[00173] In some embodiments, purified anti-BamA antibodies are characterized by assays including, but not limited to, N-terminal sequencing, amino acid analysis, HPLC, mass spectrometry, differential scanning fluorimetry (DSF), nanoDSF, capillary isoelectric focusing (cIEF), ion exchange chromatography, and papain digestion.

[00174] In some embodiments, assays are provided for identifying BamA-binding agents that inhibit growth of A. baumannii. In some embodiments, assays are provided for identifying an anti-BamA VHH that inhibits growth of A. baumannii. These assays may include, but are not limited to, antimicrobial minimum inhibitory concentration assays, growth inhibitory assays, and time-kill assays.

[00175] In some embodiments, the terms “inhibiting”, “reducing”, “blocking”, “antagonizing”, “suppressing”, and “interfering” are relative to levels and/or activity in the absence of treatment with the BamA-binding agent. In some embodiments, the terms “inhibiting”, “reducing”, “blocking”, “antagonizing”, “suppressing”, and “interfering” are relative to levels and/or activity prior to treatment with the BamA-binding agent.

[00176] The present disclosure also provides conjugates comprising a BamA-binding agent described herein. In some embodiments, a conjugate comprises an anti-BamA VHH described herein. In some embodiments, the binding agent or VHH is attached to a second molecule. In some embodiments, the binding agent or VHH is conjugated to a cytotoxic agent or moiety. In some embodiments, the binding agent or VHH is conjugated to a cytotoxic agent to form an antibody-drug conjugate (ADC). In some embodiments, the cytotoxic agent is a chemotherapeutic agent including, but not limited to, methotrexate, adriamycin/doxorubicin, melphalan, mitomycin C, chlorambucil, duocarmycin, daunorubicin, pyrrolobenzodiazepines (PBDs), or other intercalating agents. In some embodiments, the cytotoxic agent is a microtubule inhibitor including, but not limited to, auristatins, maytansinoids (e.g., DM1 and DM4), and tubulysins. In some embodiments, the cytotoxic agent is an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof, including, but not limited to, diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americcinci proteins (PAPI, PAPII, and PAP-S), Momordica charcintici inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. In some embodiments, an antibody is conjugated to one or more small molecule toxins, such as calicheamicins, maytansinoids, trichothenes, and CC1065. A derivative of any one of these toxins may be used as long as the derivative retains the cytotoxic activity of the parent molecule.

[00177] Conjugates comprising a BamA-binding agent described herein may be made using any suitable method known in the art. In some embodiments, conjugates are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyidithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HC1), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)- ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro-2,4-dinitrobenzene).

[00178] In some embodiments, a BamA-binding agent described herein is conjugated to a detectable substance or molecule that allows the agent to be used for diagnosis and/or detection. In some embodiments, an anti-BamA VHH described herein is conjugated to a detectable substance or molecule that allows the VHH to be used for diagnosis and/or detection. In some embodiments, a labeled anti- BamA is used to monitor growth of an A. baumannii culture. In some embodiments, a labeled anti-BamA is used to monitor growth of an A. baumannii infection. A detectable substance can include but is not limited to, enzymes, such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase, and acetylcholinesterase; prosthetic groups, such as biotin and flavine(s); fluorescent materials, such as, umbelliferone, fluorescein, fluorescein isothiocyanate (FITC), rhodamine, tetramethylrhodamine isothiocyanate (TRITC), dichlorotriazinylamine fluorescein, dansyl chloride, cyanine (Cy3), and phycoerythrin; bioluminescent materials, such as luciferase; radioactive materials, such as ²¹²Bi, ¹⁴C, ⁵⁷Co, ⁵¹Cr, ⁶⁷Cu, ¹⁸F, ⁶⁸Ga, ⁶⁷Ga, ¹⁵³Gd, ¹⁵⁹Gd, ⁶⁸Ge, ³H, ¹⁶⁶Ho, ¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I, ¹¹⁵In, ¹¹³In, ¹¹²In, ¹¹¹In, ¹⁴⁰La, ¹⁷⁷LU, ⁵⁴Mn, ⁹⁹Mo, ³²P, ¹⁰³Pd, ¹⁴⁹Pm, ¹⁴²Pr, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁰⁵Rh, ⁹⁷Ru, ³⁵S, ⁴⁷Sc, ⁷⁵Se, ¹⁵³Sm, ¹¹³Sn, ¹¹⁷Sn, ⁸⁵Sr, "^mTc, ²⁰¹Ti, ¹³³Xe, ⁹⁰Y, ⁶⁹Yb, ¹⁷⁵Yb, ⁶⁵Zn; positron emitting metals; and magnetic metal ions.

[00179] In some embodiments, an anti-BamA VHH described herein is conjugated to a second antibody to form an antibody heteroconjugate.

[00180] A BamA-binding agent described herein can be attached to a solid support. In some embodiments, an anti-BamA VHH described herein is attached to a solid support. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene. In some embodiments, an immobilized anti-BamA VHH is used in an immunoassay. In some embodiments, an immobilized anti-BamA VHH is used in purification of the target antigen.

[00181] In some embodiments, an anti-BamA VHH described herein is used in an immunoassay. Immunoassays are known to those of skill in the art and include, but are not limited to, ELISA, SPR (e.g., Biacore), FACS, and immunohistochemistry (IHC). In some embodiments, an anti-BamA VHH described herein is used on a tissue sample. In some embodiments, an anti-BamA VHH described herein is used on a bacterial culture sample.

III. Polynucleotides, Vectors, and Cells

[00182] In some embodiments, the disclosure encompasses polynucleotides comprising polynucleotides that encode a polypeptide (e.g., a BamA-binding agent) described herein. The term “polynucleotides that encode a polypeptide” encompasses a polynucleotide that includes only coding sequences for the polypeptide as well as a polynucleotide that includes additional coding and/or non-coding sequences. The polynucleotides of the disclosure can be in the form of RNA or in the form of DNA. DNA includes cDNA, genomic DNA, and synthetic DNA; and can be double -stranded or single -stranded, and if single stranded can be the coding strand or non-coding (anti-sense) strand.

[00183] In some embodiments, a polynucleotide comprises a polynucleotide encoding a heavy chain variable region of a BamA-binding agent described herein. In some embodiments, a polynucleotide comprises a polynucleotide encoding an anti-BamA VHH described herein.

[00184] In some embodiments, the polynucleotide comprises a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NO:41, SEQ ID NO:42, and SEQ ID NO:43. In some embodiments, the polynucleotide encodes an amino acid sequence of SEQ ID NO: 41. In some embodiments, the polynucleotide encodes an amino acid sequence of SEQ ID NO: 42. In some embodiments, the polynucleotide encodes an amino acid sequence of SEQ ID NO: 43.

[00185] In some embodiments, the polynucleotide comprises a polynucleotide encoding a polypeptide comprising more than one amino acid sequence selected from the group consisting of: SEQ ID NO:41, SEQ ID NO:42, and SEQ ID NO:43. In some embodiments, the polynucleotide comprises a polynucleotide encoding a polypeptide comprising an amino acid sequence of SEQ ID NO:41. In some embodiments, the polynucleotide comprises a polynucleotide encoding a polypeptide comprising an amino acid sequence of SEQ ID NO:42. In some embodiments, the polynucleotide comprises a polynucleotide encoding a polypeptide comprising an amino acid sequence of SEQ ID NO:43.

[00186] The present disclosure also provides variants of the polynucleotides described herein, wherein the variant encodes, for example, fragments, analogs, and/or derivatives of a polypeptide. In some embodiments, the present disclosure provides a polynucleotide comprising a polynucleotide having a nucleotide sequence at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, and in some embodiments, at least 96%, at least 97%, at least 98%, or at least 99% identical to a polynucleotide encoding a polypeptide described herein.

[00187] In some embodiments, a polynucleotide comprises a polynucleotide having a nucleotide sequence at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, and in some embodiments, at least 96%, at least 97%, at least 98%, or at least 99% identical to a polynucleotide encoding an amino acid sequence of: SEQ ID NO:41. In some embodiments, the polynucleotide comprises a nucleotide sequence at least 80% identical to a polynucleotide encoding an amino acid sequence of SEQ ID NO: 41. In some embodiments, the polynucleotide comprises a nucleotide sequence at least 85% identical to a polynucleotide encoding an amino acid sequence of SEQ ID NO: 41. In some embodiments, the polynucleotide comprises a nucleotide sequence at least 90% identical to a polynucleotide encoding an amino acid sequence of SEQ ID NO: 41. In some embodiments, the polynucleotide comprises a nucleotide sequence at least 95% identical to a polynucleotide encoding an amino acid sequence of SEQ ID NO: 41. In some embodiments, the polynucleotide comprises a nucleotide sequence at least 96% identical to a polynucleotide encoding an amino acid sequence of SEQ ID NO: 41. In some embodiments, the polynucleotide comprises a nucleotide sequence at least 97% identical to a polynucleotide encoding an amino acid sequence of SEQ ID NO: 41. In some embodiments, the polynucleotide comprises a nucleotide sequence at least 98% identical to a polynucleotide encoding an amino acid sequence of SEQ ID NO: 41. In some embodiments, the polynucleotide comprises a nucleotide sequence at least 99% identical to a polynucleotide encoding an amino acid sequence of SEQ ID NO: 41.

[00188] In some embodiments, a polynucleotide comprises a polynucleotide having a nucleotide sequence at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, and in some embodiments, at least 96%, at least 97%, at least 98%, or at least 99% identical to a polynucleotide encoding an amino acid sequence of: SEQ ID NO:42. In some embodiments, the polynucleotide comprises a nucleotide sequence at least 80% identical to a polynucleotide encoding an amino acid sequence of SEQ ID NO: 42. In some embodiments, the polynucleotide comprises a nucleotide sequence at least 85% identical to a polynucleotide encoding an amino acid sequence of SEQ ID NO: 42. In some embodiments, the polynucleotide comprises a nucleotide sequence at least 90% identical to a polynucleotide encoding an amino acid sequence of SEQ ID NO: 42. In some embodiments, the polynucleotide comprises a nucleotide sequence at least 95% identical to a polynucleotide encoding an amino acid sequence of SEQ ID NO: 42. In some embodiments, the polynucleotide comprises a nucleotide sequence at least 96% identical to a polynucleotide encoding an amino acid sequence of SEQ ID NO: 42. In some embodiments, the polynucleotide comprises a nucleotide sequence at least 97% identical to a polynucleotide encoding an amino acid sequence of SEQ ID NO: 42. In some embodiments, the polynucleotide comprises a nucleotide sequence at least 98% identical to a polynucleotide encoding an amino acid sequence of SEQ ID NO: 42. In some embodiments, the polynucleotide comprises a nucleotide sequence at least 99% identical to a polynucleotide encoding an amino acid sequence of SEQ ID NO: 42.

[00189] In some embodiments, a polynucleotide comprises a polynucleotide having a nucleotide sequence at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, and in some embodiments, at least 96%, at least 97%, at least 98%, or at least 99% identical to a polynucleotide encoding an amino acid sequence of: SEQ ID NO:43. In some embodiments, the polynucleotide comprises a nucleotide sequence at least 80% identical to a polynucleotide encoding an amino acid sequence of SEQ ID NO: 43. In some embodiments, the polynucleotide comprises a nucleotide sequence at least 85% identical to a polynucleotide encoding an amino acid sequence of SEQ ID NO: 43. In some embodiments, the polynucleotide comprises a nucleotide sequence at least 90% identical to a polynucleotide encoding an amino acid sequence of SEQ ID NO: 43. In some embodiments, the polynucleotide comprises a nucleotide sequence at least 95% identical to a polynucleotide encoding an amino acid sequence of SEQ ID NO: 43. In some embodiments, the polynucleotide comprises a nucleotide sequence at least 96% identical to a polynucleotide encoding an amino acid sequence of SEQ ID NO: 43. In some embodiments, the polynucleotide comprises a nucleotide sequence at least 97% identical to a polynucleotide encoding an amino acid sequence of SEQ ID NO: 43. In some embodiments, the polynucleotide comprises a nucleotide sequence at least 98% identical to a polynucleotide encoding an amino acid sequence of SEQ ID NO: 43. In some embodiments, the polynucleotide comprises a nucleotide sequence at least 99% identical to a polynucleotide encoding an amino acid sequence of SEQ ID NO: 43.

[00190] Also provided is a polynucleotide that comprises a polynucleotide that hybridizes to a polynucleotide encoding an amino acid sequence selected from the group consisting of: SEQ ID NO:41, SEQ ID NO:42, and SEQ ID NO:43. In some embodiments, the amino acid sequence is SEQ ID NO: 41. In other embodiments, the amino acid sequence is SEQ ID NO: 42. In yet other embodiments, the amino acid sequence is SEQ ID NO: 43. In some embodiments, the hybridization is under conditions of high stringency as is known to those skilled in the art.

[00191] As used herein, the phrase “a polynucleotide having a nucleotide sequence at least 95% identical to a polynucleotide sequence” is intended to mean that the nucleotide sequence of the polynucleotide is identical to a reference sequence except that the polynucleotide sequence can include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence can be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence can be inserted into the reference sequence. It is understood by those of skill in the art that an appropriate calculation would be made for other “% identical” statements, for example, 90% identical or 85% identical. Mutations of the reference sequence can occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.

[00192] The polynucleotide variants can contain alterations in the coding regions, non-coding regions, or both. In some embodiments, a polynucleotide variant contains alterations that produce silent substitutions, additions, or deletions, but does not alter the properties or activities of the encoded polypeptide. In some embodiments, a polynucleotide variant comprises silent substitutions that results in no change to the amino acid sequence of the polypeptide (due to the degeneracy of the genetic code). In some embodiments, a polynucleotide variant comprises one or more mutated codons comprising one or more (e.g., 1, 2, or 3) substitutions to the codon that change the amino acid encoded by that codon. Methods for introducing one or more substitutions into a codon are known in the art, including but not limited to, PCR mutagenesis and site-directed mutagenesis. Polynucleotide variants can be produced for a variety of reasons, for example, to optimize codon expression for a particular host (e.g., change codons in the human mRNA to those preferred by a bacterial host such as E. coll). In some embodiments, a polynucleotide variant comprises at least one silent mutation in a non-coding or a coding region of the sequence.

[00193] In some embodiments, a polynucleotide variant is produced to modulate or alter expression (or expression levels) of the encoded polypeptide. In some embodiments, a polynucleotide variant is produced to increase expression of the encoded polypeptide. In some embodiments, a polynucleotide variant is produced to decrease expression of the encoded polypeptide. In some embodiments, a polynucleotide variant has increased expression of the encoded polypeptide as compared to a parental polynucleotide sequence. In some embodiments, a polynucleotide variant has decreased expression of the encoded polypeptide as compared to a parental polynucleotide sequence.

[00194] In some embodiments, a polynucleotide comprises the coding sequence for a polypeptide fused in the same reading frame to a polynucleotide that aids in expression and secretion of a polypeptide from a host cell. In some embodiments, the polynucleotide that aids in expression and secretion is a leader sequence that functions as a secretory sequence for controlling transport of a polypeptide. In some embodiments, the polypeptide has a leader sequence cleaved by the host cell to form a “mature” form of the polypeptide.

[00195] In some embodiments, a polynucleotide comprises the coding sequence for a polypeptide fused in the same reading frame to a marker or tag sequence. For example, in some embodiments, a marker sequence is a hexa-histidine tag (SEQ ID NO:52) that allows for efficient purification of the polypeptide fused to the marker. In some embodiments, a marker sequence is a hemagglutinin (HA) tag derived from the influenza hemagglutinin protein when a mammalian host is used. In some embodiments, the marker sequence is a FLAG™ tag. In some embodiments, a marker is used in conjunction with other markers or tags.

[00196] In some embodiments, a polynucleotide is isolated. In some embodiments, a polynucleotide is substantially pure.

[00197] Vectors comprising any of the polynucleotides described herein are also provided. In some embodiments, a vector comprises a polynucleotide encoding a BamA-binding agent described herein. In some embodiments, a vector comprises a polynucleotide encoding a polypeptide that is part of a BamA- binding agent described herein. In some embodiments, a vector comprises a polynucleotide encoding an anti-BamA VHH described herein.

[00198] Cells comprising any of the polynucleotides described herein are provided. In some embodiments, a cell comprises a polynucleotide encoding a BamA-binding agent described herein. In some embodiments, a cell comprises a polynucleotide encoding a polypeptide that is part of a BamA- binding agent described herein. In some embodiments, a cell comprises a polynucleotide encoding an anti-BamA VHH described herein.

[00199] In addition, cells comprising the vectors described herein are provided. In some embodiments, a cell comprises a vector expressing a BamA-binding agent described herein. In some embodiments, a cell comprises a vector expressing a polypeptide that is part of a BamA-binding agent described herein. In some embodiments, a cell comprises a vector expressing an anti-BamA VHH described herein. In some embodiments, a cell comprises a vector encoding a BamA-binding agent described herein.

[00200] In some embodiments, a cell produces a BamA-binding agent described herein. In some embodiments, a cell produces an anti-BamA VHH described herein. In some embodiments, a cell produces a VHH selected from the group consisting of: VHH-29, VHH-47, and VHH-82. In some embodiments, the VHH is VHH-29. In some embodiments, the VHH is VHH-47. In some embodiments, the VHH is VHH-82.

[00201] In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is isolated. In some embodiments, the cell is a hybridoma. IV. Methods of Making Binding Agents

[00202] The disclosure provides methods for making the BamA-binding agents described herein. In some embodiments, a method comprises providing a cell comprising a BamA-binding agent described herein, culturing the cell under conditions that permit the expression of the binding agent, and isolating the binding agent. In some embodiments, a method comprises providing a cell comprising a polypeptide encoding a BamA-binding agent described herein, culturing the cell under conditions that permit the expression of the binding agent, and isolating the binding agent. In some embodiments, a method comprises providing a cell comprising an anti-BamA VHH described herein, culturing the cell under conditions that permit the expression of the VHH, and isolating the VHH. In some embodiments, a method further comprises purifying the binding agent or VHH. In some embodiments, a method further comprises formulating the binding agent or VHH as a pharmaceutical composition.

[00203] In some embodiments, a polynucleotide encoding a BamA-binding agent described herein is transiently transfected into a cell. In some embodiments, a polynucleotide encoding a BamA-binding agent described herein is stably transfected into a cell. In some embodiments, a vector comprising a polynucleotide encoding a BamA-binding agent described herein is transiently transfected into a cell. In some embodiments, a vector comprising a polynucleotide encoding a BamA-binding agent described herein is stably transfected into a cell.

[00204] In some embodiments, the cell used to make a BamA-binding agent is a bacterial cell. In some embodiments, the cell used to make a BamA-binding agent is a yeast cell. In some embodiments, the cell used to make a BamA-binding agent is a mammalian cell. In some embodiments, the cell used to make a BamA-binding agent is a CHO cell. In some embodiments, the cell used to make a BamA-binding agent is a HEK-293 cell.

V. Methods of use and pharmaceutical compositions

[00205] The BamA-binding agents of the disclosure are useful in a variety of applications including, but not limited to, therapeutic treatment methods, such as treatment of A. baumannii infections. The methods of use may be in vitro, ex vivo, or in vivo methods.

[00206] The present disclosure provides methods for inhibiting the growth of Acinetobacter using a BamA-binding agent described herein. In some embodiments, a method of inhibiting growth of Acinetobacter comprises contacting Acinetobacter with a BamA-binding agent described herein. In some embodiments, a method of inhibiting growth of Acinetobacter comprises contacting Acinetobacter with a BamA-1 -binding agent in vitro. In some embodiments, a method of inhibiting growth of Acinetobacter comprises contacting Acinetobacter with a BamA-binding agent in a growth inhibition assay. In some embodiments, a method of inhibiting growth of Acinetobacter comprises contacting Acinetobacter with a BamA-binding agent in a time-kill assay. In some embodiments, a method of inhibiting growth of Acinetobacter comprises contacting Acinetobacter a BamA-binding agent described herein in combination with at least one additional In some embodiments, a BamA-binding agent has a bactericidal effect on Acinetobacter. In some embodiments, a BamA-binding agent has a bacteriostatic effect on Acinetobacter. In some embodiments, of the methods described herein the Acinetobacter is A. baumannii.

[00207] In some embodiments, the BamA-binding agent is an anti-A. baumannii BamA VHH described herein. In some embodiments, the BamA binding agent is VHH-29, VHH-47, or VHH-82. In some embodiments, the BamA-binding agent is VHH-82.

[00208] In some embodiments, an in vitro method of inhibiting growth of Acinetobacter comprises contacting Acinetobacter with a BamA-binding agent described herein in combination with at least one additional agent that enhances and/or potentiates the activity of the BamA-binding agent.

[00209] In some embodiments, a method of inhibiting growth of Acinetobacter comprises contacting Acinetobacter with a BamA-binding agent described herein in vivo. In some embodiments, contacting Acinetobacter with a BamA-binding agent is undertaken in an animal model (e.g., a mouse model). In some embodiments, an agent is administered to mice that have been infected with Acinetobacter. In some embodiments, the agent is a BamA binding agent that binds A. baumannii. In some embodiments, a BamA-binding agent is administered at the same time or shortly before introduction of Acinetobacter into the animal to prevent infection (“preventative model”). In some embodiments, a BamA-binding agent is administered after an Acinetobacter infection has been established (“therapeutic model”). In some embodiments, a BamA-binding agent is administered to a transgenic animal (e.g. , a transgenic mouse). [00210] The present disclosure provides methods of treating Acinetobacter infections. In some embodiments, a method of inhibiting an Acinetobacter infection in a subject comprises administering to the subject a therapeutically effective amount of a BamA-binding agent described herein. In some embodiments, a method of treating an Acinetobacter infection in a subject comprises administering to the subject a therapeutically effective amount of a BamA-binding agent described herein.

[00211] In some embodiments, the disclosure provides use of a BamA-binding agent described herein in the manufacture or preparation of a medicament for treating an Acinetobacter infection.

[00212] In some embodiments of the methods described herein, a method is an in vitro method comprising contacting a cell with a BamA-binding agent described herein. In some embodiments of the methods described herein, a method is an in vivo method comprising administering a therapeutically effective amount of a BamA-binding agent described herein to a subject. [00213] In some embodiments of the methods described herein, the BamA-binding agent is an anti-A. baumannii BamA VHH described herein. In some embodiments, the BamA binding agent is VHH-29, VHH-47, or VHH-82. In some embodiments, the BamA-binding agent is VHH-82.

[00214] In some embodiments of the methods described herein, the BamA-binding agent comprises a heavy chain variable region CDR1, CDR2, and CDR3 of VHH-29. In some embodiments of the methods described herein, the BamA-binding agent comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising the amino acid sequence GLSFGLDAYAVA (SEQ ID NO: 11), a heavy chain variable region CDR2 comprising the amino acid sequence CISPTGSRVAYADSAKG (SEQ ID NO: 12), and a heavy chain variable region CDR3 comprising the amino acid sequence SNDKRCSDFGVDRVGY (SEQ ID NO: 13).

[00215] In some embodiments of the methods described herein, the BamA-binding agent comprises the amino acid sequence SEQ ID NO:41. In some embodiments of the methods described herein, the BamA- binding agent comprises a heavy chain variable region of amino acid sequence SEQ ID NO:41. In some embodiments of the methods described herein, the BamA-binding agent comprises VHH-29. In some embodiments of the methods described herein, the BamA-binding agent is VHH-29.

[00216] In some embodiments of the methods described herein, the BamA-binding agent comprises a heavy chain variable region CDR1, CDR2, and CDR3 of VHH-47. In some embodiments of the methods described herein, the BamA-binding agent comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising the amino acid sequence GSTATRDTFSSHRMT (SEQ ID NO:21), a heavy chain variable region CDR2 comprising the amino acid sequence TITGDDITNYTGSVKG (SEQ ID NO:22), and a heavy chain variable region CDR3 comprising the amino acid sequence LERGIWAY (SEQ ID NO:23).

[00217] In some embodiments of the methods described herein, the BamA-binding agent comprises the amino acid sequence SEQ ID NO:42. In some embodiments of the methods described herein, the BamA- binding agent comprises a heavy chain variable region of amino acid sequence SEQ ID NO:42. In some embodiments of the methods described herein, the BamA-binding agent comprises VHH-47. In some embodiments of the methods described herein, the BamA-binding agent is VHH-47.

[00218] In some embodiments of the methods described herein, the BamA-binding agent comprises a heavy chain variable region CDR1, CDR2, and CDR3 of VHH-82. In some embodiments of the methods described herein, the BamA-binding agent comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising the amino acid sequence GFSFRDYSMS (SEQ ID NO:31), a heavy chain variable region CDR2 comprising the amino acid sequence GIRSLGTTTYYADSVKG (SEQ ID NO:32), and a heavy chain variable region CDR3 comprising the amino acid sequence CLGKICDRFGIVDIYS (SEQ ID NO: 33). [00219] In some embodiments of the methods described herein, the BamA-binding agent comprises the amino acid sequence SEQ ID NO:43. In some embodiments of the methods described herein, the BamA- binding agent comprises a heavy chain variable region of amino acid sequence SEQ ID NO:43. In some embodiments of the methods described herein, the BamA-binding agent comprises VHH-82. In some embodiments of the methods described herein, the BamA-binding agent is VHH-82.

[00220] In some embodiments of the methods described herein, the subject is human.

[00221] For the treatment of an infection and/or disease, the appropriate dosage of a BamA-binding agent of the present disclosure depends on the infection or disease to be treated, the severity and course of the infection or disease, the responsiveness of the infection or disease, whether the agent is administered for therapeutic or preventative purposes, previous therapy, the patient's clinical history, and so on. A BamA-binding agent can be administered one time or over a series of treatments lasting from several days to several months, or until a cure is effected or a diminution of the infection and/or disease state is achieved.

[00222] In some embodiments, dosage of a BamA-binding agent is from 0.01 pg/kg to 100 mg/kg of body weight, from 0. 1 pg/kg to 100 mg/kg of body weight, or from 1 pg/kg to 100 mg/kg of body weight. In some embodiments, a BamA-binding agent is dosed once or more multiple times during a doing period.

[00223] In some embodiments of the methods described herein, a method comprises administering a BamA-binding agent described herein in combination with at least one additional therapeutic agent or therapeutic therapy. Treatment with two or more therapeutic agents often uses agents that work by different mechanisms of action, although this is not required. Combination therapy using agents with different mechanisms of action may result in additive or synergetic effects. Combination therapy may allow for a lower dose of each agent than is used in monotherapy, thereby reducing toxic side effects and/or increasing the therapeutic index of the agent(s). Combination therapy may decrease the likelihood that resistance to an agent will develop.

[00224] In some embodiments of the methods described, the combination of a BamA-binding agent described herein and at least one additional therapeutic agent results in additive or synergistic results. In some embodiments, the combination therapy results in an increase in the therapeutic index of the BamA- binding agent. In some embodiments, the combination therapy results in an increase in the therapeutic index of the additional therapeutic agent(s). In some embodiments, the combination therapy results in a decrease in the toxicity and/or side effects of the BamA-binding agent. In some embodiments, the combination therapy results in a decrease in the toxicity and/or side effects of the additional therapeutic agent(s). [00225] In some embodiments of the methods described herein, a combination treatment comprises one additional therapeutic agent. In some embodiments of the methods described herein, a combination treatment comprises two or more additional therapeutic agents.

[00226] In some embodiments, an in vitro method of inhibiting growth of Acinetobacter comprises contacting Acinetobacter with a BamA-binding agent described herein in combination with at least one additional antibacterial agent. In some embodiments, a method of inhibiting growth of Acinetobacter in a subject comprises administering a therapeutically effective amount of a BamA-binding agent described herein to the subject in combination with at least one additional antibacterial agent.

[00227] In some embodiments, an in vitro method of inhibiting growth of Acinetobacter comprises contacting Acinetobacter with a BamA-binding agent described herein in combination with at least one additional therapeutic agent, wherein the additional therapeutic agent enhances and/or potentiates the antibacterial activity of a BamA-binding agent described herein. In some embodiments, a method of inhibiting growth of Acinetobacter in a subject comprises administering a therapeutically effective amount of a BamA-binding agent described herein to the subject in combination with at least one additional therapeutic agent, wherein the additional therapeutic agent enhances and/or potentiates the antibacterial activity of a BamA-binding agent described herein. In some embodiments, the additional therapeutic agent that enhances and/or potentiates the antibacterial activity of a BamA-binding agent described herein is polymyxin B nonapeptide (PMBN), other polymyxin derivatives, phenylalanyl arginine b-naphthylamide (PAbN), an efflux inhibitor, or a LPS biosynthesis inhibitor such as CHIR-090. [00228] It will be appreciated that the combination of a BamA-binding agent described herein and at least one additional therapeutic agent can be administered in any order or concurrently. In some embodiments, a BamA-binding agent is administered to subjects that have previously undergone treatment with a therapeutic agent. In some embodiments, a BamA-binding agent and a second therapeutic agent are administered substantially simultaneously or concurrently. For example, a subject may be given a BamA-binding agent while undergoing a course of treatment with a second therapeutic agent (e.g., an antibacterial agent). In some embodiments, a BamA-binding agent is administered within 1 year of the treatment with a second therapeutic agent. In some embodiments, a BamA-binding agent is administered within 10, 8, 6, 4, or 2 months of any treatment with a second therapeutic agent. In some embodiments, a BamA-binding agent is administered within 4, 3, 2, or 1 weeks of any treatment with a second therapeutic agent. In some embodiments, a BamA-binding agent is administered within 5, 4, 3, 2, or 1 days of any treatment with a second therapeutic agent. It will further be appreciated that the two (or more) agents or treatments can be administered to the subject within a matter of hours or minutes (i. e. , substantially simultaneously). [00229] In some embodiments, treatment with a BamA-binding agent can occur prior to, concurrently with, or subsequent to administration of the additional therapeutic agents. In some embodiments, combined administration includes co-administration, either in a single pharmaceutical formulation or using separate formulations, or consecutive administration in either order but generally within a time period such that all active agents can exert their biological activities. In some embodiments, preparation of agents and/or dosing schedules for additional therapeutic agents are according to manufacturers' instructions or as determined empirically by the skilled practitioner.

[00230] The present disclosure provides compositions comprising a BamA-binding agent described herein. In some embodiments, a composition comprises a VHH selected from the group consisting of: VHH-29, VHH-47, and VHH-82. In some embodiments, a composition comprises VHH-82.

[00231] The present disclosure provides pharmaceutical compositions comprising a BamA-binding agent described herein and a pharmaceutically acceptable vehicle. In some embodiments, a pharmaceutical composition comprises a VHH selected from the group consisting of: VHH-29, VHH-47, and VHH-82 and a pharmaceutically acceptable vehicle. In some embodiments, a pharmaceutical composition comprises VHH-82 and a pharmaceutically acceptable vehicle.

[00232] Formulations are prepared for storage and use by combining a binding agent of the present disclosure with a pharmaceutically acceptable vehicle (e.g., a carrier or excipient). Those of skill in the art generally consider pharmaceutically acceptable carriers, excipients, and/or stabilizers to be inactive ingredients of a formulation or pharmaceutical composition.

[00233] Suitable pharmaceutically acceptable vehicles include, but are not limited to, nontoxic buffers; salts; antioxidants; preservatives; hydrophilic polymers; carbohydrates; chelating agents; sugars; saltforming counter-ions; metal complexes; and non-ionic surfactants. In some embodiments, the formulation is in the form of an aqueous solution. In some embodiments, the formulation is stored in a lyophilized or in an alternative dried form.

[00234] The binding agents of the present disclosure can be formulated in any suitable form for delivery to a target cell/tissue. In some embodiments, a BamA-binding agent is formulated as a liposome, microparticle, microcapsule, albumin microsphere, microemulsion, nanoparticle, nanocapsule, or macroemulsion.

[00235] In some embodiments, a BamA-binding agent is formulated with liposomes. Methods to produce liposomes are known to those of skill in the art. For example, some liposomes can be generated by reverse phase evaporation with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE).

[00236] In some embodiments, a BamA-binding agent is formulated as a sustained-release preparation. Suitable examples of sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers containing an agent, where the matrices are in the form of shaped articles (e.g., fdms or microcapsules). Sustained-release matrices include but are not limited to polyesters, hydrogels such as poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol), polylactides, copolymers of L- glutamic acid and 7 ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid- glycolic acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3- hydroxybutyric acid.

[00237] The pharmaceutical compositions or formulations of the present disclosure can be administered in any number of ways for either local or systemic treatment. Administration can be topical by epidermal or transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders; pulmonary by inhalation or insufflation of powders or aerosols, including by nebulizer, intratracheal, and intranasal; oral; or parenteral including intravenous, intraarterial, intratumoral, subcutaneous, intraperitoneal, intramuscular (e.g., injection or infusion), or intracranial (e.g., intrathecal or intraventricular).

VI. Kits and Articles of Manufacture

[00238] Further provided are kits, unit dosages, and articles of manufacture comprising any of the BamA-binding agents (e.g., the BamA-binding VHHs) described herein. In some embodiments, a kit is provided which contains any one of the pharmaceutical compositions described herein and preferably provides instructions for its use. The kits of the present application are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Kits may optionally provide additional components such as buffers and interpretative information. The present application thus also provides articles of manufacture, which include vials (such as sealed vials), bottles, jars, flexible packaging, and the like.

[00239] In some embodiments, a BamA-binding agent in the kits provided herein is conjugated to a detectable substance or molecule that allows the agent to be used for diagnosis and/or detection. In some embodiments, an anti-BamA VHH described herein is conjugated to a detectable substance or molecule that allows the VHH to be used for diagnosis and/or detection. In some embodiments, a labeled anti- BamA is used to monitor growth of an A. baumannii culture. In some embodiments, a labeled anti-BamA is used to monitor growth of an A. baumannii infection. A detectable substance can include but is not limited to, enzymes, such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase, and acetylcholinesterase; prosthetic groups, such as biotin and flavine(s); fluorescent materials, such as, umbelliferone, fluorescein, fluorescein isothiocyanate (FITC), rhodamine, tetramethylrhodamine isothiocyanate (TRITC), dichlorotriazinylamine fluorescein, dansyl chloride, cyanine (Cy3), and phycoerythrin; bioluminescent materials, such as luciferase; radioactive materials, such as ²¹²Bi, ¹⁴C, ⁵⁷Co, ⁵¹Cr, ⁶⁷Cu, ¹⁸F, ⁶⁸Ga, ⁶⁷Ga, ¹⁵³Gd, ¹⁵⁹Gd, ⁶⁸Ge, ³H, ¹⁶⁶Ho, ¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I, ¹¹⁵In, ¹¹³In, ¹¹²In, ¹¹¹ In, ¹⁴⁰La, ¹⁷⁷LU, ⁵⁴Mn, ⁹⁹MO, ³²P, ¹⁰³Pd, ¹⁴⁹Pm, ¹⁴²Pr, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁰⁵Rh, ⁹⁷Ru, ³⁵S, ⁴⁷Sc, ⁷⁵Se, ¹⁵³Sm, ¹¹³Sn, ¹¹⁷Sn, ⁸⁵Sr, "^mTc, ²⁰¹Ti, ¹³³Xe, ⁹⁰Y, ⁶⁹Yb, ¹⁷⁵Yb, ⁶⁵Zn; positron emitting metals; and magnetic metal ions.

[00240] In other embodiments, the kits provided herein further comprise an additional agent capable of detecting the VHH. In some embodiments, the additional agent is an antibody capable of binding the VHH provided herein. In some embodiments, the antibody capable of binding to the VHH provided herein is conjugated to a detectable moiety.

[00241] A BamA-binding agent described herein can be attached to a solid support. In some embodiments, an anti-BamA VHH described herein is attached to a solid support. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

[00242] The article of manufacture can comprise a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The containers may be formed from a variety of materials such as glass or plastic. Generally, the container holds a composition which is effective for treating a disease or disorder (such as cancer) described herein, and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The label or package insert indicates that the composition is used for treating the particular condition in an individual. The label or package insert will further comprise instructions for administering the composition to the individual. The label may indicate directions for reconstitution and/or use. The container holding the pharmaceutical composition may be a multi-use vial, which allows for repeat administrations (e.g. from 2-6 administrations) of the reconstituted formulation. Package insert refers to instructions customarily included in commercial packages of therapeutic products that contain information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products. Additionally, the article of manufacture may further comprise a second container comprising a pharmaceutically -acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, fdters, needles, and syringes.

[00243] The kits or article of manufacture may include multiple unit doses of the pharmaceutical composition and instructions for use, packaged in quantities sufficient for storage and use in pharmacies, for example, hospital pharmacies and compounding pharmacies. EXAMPLES Example 1 Generation of anti-Acinelohacler BamA VHH molecules [00244] Anti-A. baumcinnii BamA antibodies were generated using amino acids 427-841 of SEQ ID NO: 1. This polypeptide corresponds to the predicted barrel domain of A. baumcinnii BamA. Four llamas were immunized with the purified A. baumannii BamA protein solubilized in amphipol. The animals were injected with the immunogen once a week for four weeks, followed by a two month rest period, and two additional immunizations.

[00245] The antimicrobial activity of llama serum was determined by first preparing serum samples from pre-bleed, intermediate bleed (week 6), and final bleed (week 20) in dilutions of 1: 1, 1:2, 1:3, 1:9, and 1:27 in IX phosphate buffered solution (PBS) in 96-well polystyrene plates (Coming), with wells designated as growth and sterility controls. Ciprofloxacin hydrochloride (Alfa Aesar) was used as a quality control. Bacterial cell suspensions from A. baumannii ATCC 19606 and E.coli ATCC 25922 were prepared using the BBL™ Prompt™ Inoculation System (BD) as per the manufacturer’s guidelines, to achieve a pre-incubation density of 4 x 10⁷ CFU/ml with 60 pL per well in MHB II media. Bacterial suspensions were added to the wells containing the llama sera dilutions and the plates were incubated for 1 hour at 37° C. Samples were further diluted 1 : 100 into 160 pL of MHB II media to achieve a final density of 4 x 10⁵ CFU/ml. The plates were then incubated for 18-20 hours at 37° C and bacterial growth was determined by measuring the absorbance at 600 nm.

[00246] As shown in Figure 1, sera from llamas immunized with the A. baumannii BamA protein generated an immune response that specifically inhibited the growth of A. baumannii in vitro. In contrast, the sera did not inhibit the growth of E. coli, demonstrating the specificity of the immune response.

[00247] B-cells were isolated from blood samples using a Ficoll® column, and total DNA was isolated using standard methods. VHH regions were amplified using universal VHH PCR primers and cloned into a yeast vector pYD5. EBY 100 yeast cells were transfected with the vector to generate a yeast VHH display library.

[00248] The library was screened/panned using biotinylated A. baumannii BamA protein. Yeast cells from the library were mixed with biotinylated BamA protein and incubated for 1 hour before being selected/sorted either through a MACS® column or FACS using standard techniques. The amount of biotinylated BamA was decreased from 50 nM to 1 nM through multiple rounds of selection/panning. Positive BamA binding yeast populations were selected and positive single clones were moved forward for further binding and sequence analysis (Figure 2).

Example 2 VHH cloning, expression, and purification

[00249] DNA was isolated from cloned cells expressing VHHs that had been identified in the binding assays described above. DNA was inserted into a pTT5 vector and expressed in Expi293F cells using a transient expression system kit (ThermoFisher Scientific) following the manufacturer’s instructions. Media containing the secreted VHH molecules were clarified by centrifugation and using a 0.2 pm filter. VHHs (labeled with a His-Tag) were adsorbed to a HisTrap excel column (Cytiva) and following a stringent column wash were eluted using a 250 mM imidazole -containing buffer. VHHs were further purified either by size exclusion chromatography (Superdex 75; Cytiva), or alternatively, cation exchange chromatography (HiTrap™ SP HP, Cytiva). VHHs were formulated in a buffer containing 25 mM HEPES, 150 mM NaCl, pH 7.5. Protein purity was assessed by SDS-PAGE and analytical size exclusion chromatography. VHH protein identity was confirmed by mass spectrometry.

Example 3 Antimicrobial minimum inhibitory concentration assay (MIC) and growth inhibitory screening assays [00250] The antimicrobial activity of 52 VHHs was determined by the Minimum Inhibitory Concentration (MIC) broth microdilution method (see, Wiegand et al., 2008, Nature Protoc., 3: 163-175). Briefly, VHH samples were prepared using three-fold serial dilutions (304 to 0.02 pg/mL) in PBS and dispersed into 96-well polystyrene plates (Coming). Ciprofloxacin hydrochloride (Alfa Aesar) and tetracycline were used as positive controls; an anti-/'.'. coli BamA VHH was used as a negative control. A. baumannii ATCC 19606 or A. baumannii ATCC 19606-AlpxC suspensions were prepared using the BBL™ Prompt™ Inoculation System (BD) following the manufacturer’s guidelines. Bacterial cells were added to the plates at 5 x 10⁵ CFU/mL in MHB II media. The plates were incubated for 18-20 hours at 37° C and bacterial growth was determined by measuring the absorbance at 600 nm using a SpectraMax M3 plate reader and SoftMax Pro 5.4 software (Molecular Devices).

[00251] A screen was performed using wild-type A. baumannii in the absence or presence of polymyxin B nonapeptide (PMBN) to identify growth inhibitory anti -BamA VHHs. PMBN is an outer membrane permeabilizer that it does not lyse bacterial cells at subinhibitory concentrations.

[00252] As shown in Figure 3, several VHHs (VHH-29, VHH-40, VHH-47, VHH-67, and VHH-82) were identified that inhibited bacterial growth at the highest concentration tested by at least 50% and two, VHH-29 and VHH-82, inhibited bacterial growth to a level similar to the antibiotic controls.

[00253] These results demonstrated that several anti-A. baumannii BamA VHHs inhibited bacterial growth at levels similar to standard-of-care antibiotics. A representative set of these VHHs was sequenced and further characterized. [00254] The antimicrobial activity of VHH-82 was determined by the MIC broth microdilution method, as described above. Briefly, VHH solutions were prepared by a three-fold serial dilution in IX PBS in 96-well polystyrene plates (Coming), with wells designated as growth and sterility controls. VHH-82 were incubated in the presence of 21 pM of PMBN or 0. 16 pM of PMB . VHH-51 (anti -E . coli BamA control) was incubated in the presence of 21 pM of PMBN. Ciprofloxacin hydrochloride (Alfa Aesar) was used as a positive control. A. baumannii bacterial cell suspensions were prepared using the BBL Prompt™ Inoculation system (BD) as per the manufacturer’s guidelines, to achieve a final density of 5 x 10⁵ CFU/ml in MHB II media. [WHAT WAS ADDED TO PLATES?] The plates were then incubated for 18-20 hours at 37° C and bacterial growth was determined by measuring the absorbance at 600nm. [00255] As shown in Figure 4, VHH-82 inhibited growth of A. baumannii in a dose dependent manner. In contrast, anti-/? coli BamA VHH-51 did not inhibit growth of A. baumannii demonstrating that the activity was specific to VHH-82. VHH-82 in the presence of polymyxin B (PMB) as well as PMBN resulted in significant growth inhibition and an IC50 of 0.15 and 0.22 pM, respectively. These results were similar to ciprofloxacin (0.35 pM (0.116 pg/ml)).

[00256] Anti-A. baumannii BamA VHHs were screened for the ability to inhibit the growth of an LPS- deficient mutant, A. baumannii ATCC 19606-AlpxC. Lipopolysaccharide (LPS), is the primary lipid on the surface of Gram-negative bacteria, and is believed to act as a permeability barrier. The barrier acts to make the outer membrane relatively impermeable to a variety of molecules, such as hydrophobic antibiotics, detergents, and host proteins. A. baumannii ATCC 19606-AlpxC lacks the LPS barrier and allows for a more inclusive screening assay for antibacterial agents. Ciprofloxacin hydrochloride (Alfa Aesar) was used as a positive control.

[00257] As shown in Figure 5, several VHHs were identified that inhibited growth of A. baumannii to a similar level as antibiotic controls. These VHHs included VHH-29 and VHH-82.

[00258] As shown in Figure 6, to validate the activity of anti-A. baumannii BamA VHH-82 on the LPS- deficient A. baumannii mutant, a range of concentrations 10000-0.50 nM in 3-fold dilutions of VHH-82 was tested on A. baumannii ATCC 19606-AlpxC cells in a MIC assay as described herein. Ciprofloxacin hydrochloride (Alfa Aesar) was used as a positive control and an anti-/'.'. coli BamA VHH-51 was used as a negative control. A similar experiment was performed with wild-type A. baumannii cells in the presence of 4 pg/mL of CHIR-090, which is a small molecule inhibitor of LpxC. LpxC is an essential enzyme in the lipid A biosynthetic pathway and inhibition of this enzyme should result in depleted LPS levels on bacterial cells that are equivalent to the IpxC -deficient mutants.

[00259] As shown in Figure 6, VHH-82 inhibited growth of the LPS-deficient A. baumannii mutant in a dose dependent manner. VHH-82 had strong single agent activity with an IC₅₀ of 9 nM. These results were mirrored in the follow-up experiment with wild-type A. baumannii cells in the presence of CHIR- 090. VHH-82 had strong single agent activity with an IC50 of 41 nM.

Example 4

Binding Affinity of anti -A. baumannii Bam A VHHs

[00260] The affinities of VHHs that bound to A. baumannii BamA were measured using a Biacore system (GE Healthcare Life Sciences). Briefly, purified VHH-29-Fc, VHH-47-Fc, and VHH-82-Fc fusion proteins were captured (100-150 RUs) on flow cell 2 of a Protein-A chip (using flow cell 1 as a reference). Concentrations ranging from 0.4 to 200 nM (2-fold dilutions) of A. baumannii BamA were injected at a flow rate of 30 pL/min at 25° C. Kinetic data were collected over time and fit to a 1 : 1 Langmuir binding model using BIAevaluation™ software to calculate binding parameters, including KD. [00261] Binding data for the three representative VHHs are shown in Table 4.

[00262] Biacore experiments were undertaken to evaluate whether the representative VHHs competed with each other for binding to A. baumannii BamA. VHH-29, VHH-47, VHH-67, and VHH-82 were shown to inhibit bacterial growth (see Example 3 herein). Purified VHH-82 or VHH-29 were immobilized on a CM5 chip surface using amine coupling chemistry. VHH-29/BamA, VHH-47/BamA, VHH-67/BamA, and VHH-82/BamA mixtures were prepared (antibody concentration was titrated from 0.05-1000 nM; Bam A concentration held constant at 50 nM) in a 96-well microplate. The mixtures were injected over the coated chips. The normalized signal was plotted against the VHH concentration. If a VHH in solution blocked Bam A interaction with immobilized VHH-82 or VHH-29, then a decrease in RU was observed as the concentration of VHH in solution was increased (i.e. competition for the binding site on Bam A was observed).

[00263] As shown in Figure 7, VHH-29, VHH-47, and VHH-67 all competed for binding to BamA with VHH-82. Similarly, VHH-47, VHH-67, and VHH-82 all competed for binding to BamA with VHH-29. These results suggest that these exemplary VHHs (i.e., VHH-29, VHH-47, VHH-69, and VHH-82) that inhibited growth of A baumannii all bound the same epitope or a similar epitope on BamA. Example 5

Characterization of binding epitope on BamA

[00264] Peptides representing the predicted eight (8) outer membrane loops of A. baumannii BamA protein were synthesized by custom peptide synthesis (Anaspec; Table 5). The peptides were synthesized with a biotin molecule attached at the N-terminus and peptides for loop 4 and loop 7 were prepared in both linearized and cyclized forms.

[00265] The biotinylated peptides were added to a streptavidin-coated plate at 2 pg/ml and VHH-47-Fc was added to the plate at 20 pg/ml. An HRP-labeled secondary anti-Fc antibody (Jackson Immuno Research Laboratories) was added to the plates, followed by a chemiluminescent detection reagent.

[00266] As shown in Figure 6, VHH-47 bound to loop 4 of BamA.

Example 6

Acinetobacter OmpT assay

[00267] In order to assess whether the VHHs described herein had functional inhibitory activity on BamA, a cell-based assay was developed for A. baumannii based upon an E. coli OmpT enzymatic activity assay (Kramer et al., 2000, Eur. J. Biochem., 267:885-893). Outer membrane protein T (OmpT) is a protease present in the outer membrane of gram negative bacteria. The assembly of OmpT (and other Omps) is dependent on BamA. The assay measures the protease activity that occurs when OmpT is correctly folded and assembled into the membrane by BamA. To develop the assay for A. baumannii, several putative A. baumannii homologs of the E. coli OmpT gene were cloned and expressed using IPTG induction. The protein expressed from Acinetobacter spp. Rootl280 OmpT gene 5 was observed to have protease activity when recombinantly expressed in A. baumannii ATCC 19606. Using A. baumannii cells expressing this OmpT protein, VHH-47 and VHH-82 were assayed for their ability to inhibit BamA and subsequent OmpT protease activity.

[00268] For the fluorimetric assay, A.baumannii cells containing a vector encoding an inducible OmpT were grown overnight in LB media plus 30 pg/ml carbenicillin. The following day, the bacteria culture was diluted 1/20 in LB media and grown to an OD600 of 0.5. The culture was further diluted in PBS to a final bacterial inoculum of 5 x 10⁷ CFU/mL and 100 pl/well were dispersed into a 96-well microplate. Anti-A. baumannii BamA VHH-82 and VHH-47 and control anti -A. coli BamA VHH-1A2 were added at concentrations ranging from 2 to 0.003 pM (3-fold dilutions) prior to bacterial addition. For induction of OmpT, 1 pM IPTG was added to the wells and plates were incubated at 37°C for 2 hours. 100 pl from each well was transferred to a black clear bottom 96-well plate (Coming) and peptide Abz- ARRA(NO2Y)-amide substrate (New England Peptide) was added at 500 pM. OmpT activity was measured at 2.5 hours post-IPTG induction in a fluorimeter using excitation and emission wavelengths of 325 and 430 nm, respectively.

[00269] As shown in Figure 9, VHH-82 significantly inhibited (2.9-fold, ***p<0.001) the activity of OmpT protease in the presence of PMBN, as compared to the negative control.

[00270] These results suggest that exemplary anti-A. baumannii BamA VHHs inhibit and/or block BamA function in cells leading to an impairment of OmpT assembly in the outer membrane and a reduction of OmpT protease activity in A. baumannii cells.

Example 7 Time-kill assays

[00271] Time-kill assays are used to study the activity of an antimicrobial agent against a bacterial strain and can be used to determine the bactericidal or bacteriostatic activity of an agent overtime. Antimicrobials are defined as bactericidal when there is a greater than three log₁₀-fold decrease in CFU/mL when compared to initial inocula. Thus, to assess the kinetics of VHH-82 activity on A. baumannii cells, several time-kill analyses were performed. Briefly, A. baumannii ATCC 19606 was cultured in Mueller Hinton II broth in the presence of 25% human serum and VHH-82 (2, 4, 7, and 15 pM). In a second assay, A. baumannii ATCC 19606 was cultured in Mueller Hinton II broth in the presence of 21 μM PMBN and VHH-82 (0.6, 1, and 2 pM). Bacterial growth was quantified after 0, 2, 5, and 24 hour incubation at 37° C by plating 10-fold dilutions on Mueller Hinton II agar plates.

[00272] As shown in Figure 10, VHH-82 significantly inhibited A. baumannii growth in the presence of serum starting at concentrations as low as 4 pM and in a dose-dependent manner. At 24 hours bacteriostatic activity and prevention of regrowth was achieved with 15 pM VHH-82 in the presence of human sera.

[00273] As shown in Figure 11, VHH-82 significantly inhibited A. baumannii growth in the presence of PMBN starting at concentrations as low as 0.6 pM and in a dose-dependent manner. At 24 hours bactericidal activity was achieved at 2 pM VHH-82 in combination with PMBN.

[00274] These data demonstrate that exemplary anti-BamA VHH-82 robustly inhibits the growth of A. baumannii in the presence of human serum and in combination with PMBN.

[00275] Although the foregoing present disclosure has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the present disclosure. The embodiments of the present disclosure described herein are intended to be merely exemplary, and those skilled in the art will recognize numerous equivalents to the specific procedures described herein. All such equivalents are considered to be within the scope of the present disclosure and are covered by the embodiments.

[00276] All publications,, patents, patent applications, internet sites, and accession numbers/database sequences including both polynucleotide and polypeptide sequences cited herein are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, internet site, or accession number/database sequence were specifically and individually indicated to be so incorporated by reference.

[00277] Following are sequences disclosed in the application. CDR sequences are listed in Tables 1-3 as SEQ ID NOs: 11-40.

Acinetobacter baumannii BamA(ATCC 19606) (SEQ ID NO: 1)

MRHTHFLMPLALVSAMAAVQQAYAADDFVVRDIRVNGLVRLTPANVYTMLPINSGDRVNE PMIAEAIRTLYATGLFDDIKASKENDTLVFNVIERPIISKLEFKGNKLIPKEALEQGLKK MGIAEGEVFKKSALQTIETELEQQYTQQGRYDADVTVDTVARPNNRVELKINFNEGTPAK VFDINVIGNTVFKDSEIKQAFAVKESGWASVVTRNDRYAREKMAASLEALRAMYLNKGYI NFNINNSQLNISEDKKHI FIEVAVDEGSQFKFGQTKFLGDALYKPEELQALKIYKDGDTY SQEKVNAVKQLLLRKYGNAGYYFADVNIVPQINNETGVVDLNYYVNPGQQVTVRRINFTG NSKTSDEVLRREMRQMEGALASNEKIDLSKVRLERTGFFKTVDIKPARIPNSPDQVDLNV NVEEQHSGTTTLAVGYSQSGGITFQAGLSQTNFMGTGNRVAIDLSRSETQDYYNLSVTDP YFTIDGVSRGYNVYYRKTKLNDDYNVNNYVTDSFGGSLSFGYPIDENQSLSASVGVDNTK VTTGPYVSTYVRDYLLANGGKATSKGTYCPTDANGDSQYDTEKGECKVPEETYDNAFEGE FFTYNLNLGWSYNTLNRPIFPTSGMSHRVGLEIGLPGSDVDYQKVTYDTQAFFPIGSTGF VLRGYGKLGYGNDLPFYKNFYAGGYGSVRGYDNSTLGPKYPSVNLQETKQNDSSPEEVGG

NALVQFGTELVLPMPFKGDWTRQVRPVLFAEGGQVFDTKCNIDNSVYGNKGMKINGQTIT

DVRKYCEDNYGFDLGNLRYSVGVGVTWITMIGPLSLSYAFPLNDKPGDETKEIQFEIGRT F

Acinetobacter baumannii (ATCC 19606) Predicted barrel domain (aa 427-841) (SEQ ID NO:2)

SGTTTLAVGYSQSGGITFQAGLSQTNFMGTGNRVAIDLSRSETQDYYNLSVTDPYFTIDG

VSRGYNVYYRKTKLNDDYNVNNYVTDSFGGSLSFGYPIDENQSLSASVGVDNTKVTTGPY

VSTYVRDYLLANGGKATSKGTYCPTDANGDSQYDTEKGECKVPEETYDNAFEGEFFTYNL

NLGWSYNTLNRPIFPTSGMSHRVGLEIGLPGSDVDYQKVTYDTQAFFPIGSTGFVLRGYG

KLGYGNDLPFYKNFYAGGYGSVRGYDNSTLGPKYPSVNLQETKQNDSSPEEVGGNALVQF

GTELVLPMPFKGDWTRQVRPVLFAEGGQVFDTKCNIDNSVYGNKGMKINGQTITDVRKYC EDNYGFDLGNLRYSVGVGVTWITMIGPLSLSYAFPLNDKPGDETKEIQFEIGRTF

Acinetobacter baumannii (ATCC 19606) Predicted barrel domain loop 1 (aa 438-442) (SEQ ID NO:3)

QSGGI

Acinetobacter baumannii (ATCC 19606) Predicted barrel domain loop 2 (aa 467-472) (SEQ ID NO:4)

SETQDY

Acinetobacter baumannii (ATCC 19606) Predicted barrel domain loop 3 (aa 496-514) (SEQ ID NO:5)

RKTKLNDDYNVNNYVTDSF

Acinetobacter baumannii (ATCC 19606) Predicted barrel domain loop 4 (aa 538-603) (SEQ ID NO:6)

NTKVTTGPYVSTYVRDYLLANGGKATSKGTYCPTDANGDSQYDTEKGECKVPEETYDNAF EGEFFT

Acinetobacter baumannii (ATCC 19606) Predicted barrel domain loop 5 (aa 635-643) (SEQ ID NO:7)

LPGSDVDYQ

Acinetobacter baumannii (ATCC 19606) Predicted barrel domain loop 6 (aa 672-723) (SEQ ID NO:8)

NDLPFYKNFYAGGYGSVRGYDNSTLGPKYPSVNLQETKQNDSSPEEVGGNAL

Acinetobacter baumannii (ATCC 19606) Predicted barrel domain loop 7 (aa 754-799) (SEQ ID NO:9)

QVFDTKCNIDNSVYGNKGMKINGQTITDVRKYCEDNYGFDLGNLRY

Acinetobacter baumannii (ATCC 19606) Predicted barrel domain loop 8 (aa 821-832) (SEQ ID NO: 10)

PLNDKPGDETKE

VHH-29 (SEQ ID NO:41)

QVQLVESGGGLVQPGGSLRLSCTASGLSFGLDAYAVAWFRQVPGKGREGVSCISPTGSRV

AYADSAKGRFTISRDNVQKTVALQIHTLKPEDTAKYFCATSNDKRCSDFGVDRVGYWGQG

TQVTVSS

VHH-47 (SEQ ID NO:42)

QVQLVESGGGLVQAGGSLRLSCTASGSTATRDTFSSHRMTWYRQAPGKQREMVATITGDD ITNYTGSVKGRFTISRDSPKKTMYLQMNNLRPEDTAVYYCHLLERGIWAYWGQGTQVTVSS

VHH-82 (SEQ ID NO:43)

QVQLVESGGGLVQPGGSLRLSCTASGFSFRDYSMSWVRQAPGKGLEWVSGIRSLGTTTYY

ADSVKGRFTISRDNARKTLYLQMNNLKPDDTATYYCAKCLGKICDRFGIVDIYSRGQGTL VTVSS

Human IgGl constant region - CHI, hinge, CH2, and CH3 (SEQ ID NO:44)

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREE

MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Human IgGl constant region E233A/L235A (SEQ ID NO:45)

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPALAGG PSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREE

Human IgGl constant region L234A/L235A (SEQ ID NO:46)

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGG PSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREE

Human IgGl constant region L234A/L235A/P329G (SEQ ID NO:47)

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGG PSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKT ISKAKGQPREPQVYTLPPSREE

Human IgGl constant region N297G (SEQ ID NO:48)

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYG STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREE

Human IgGl constant region N297G/H310A (SEQ ID NO:49)

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYG STYRVVSVLTVLAQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREE

Human IgGl constant region L234F/L235E/P331G (SEQ ID NO:50) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAGIEKTISKAKGQPREPQVYTLPPSREE

MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Human IgGl constant region L234A/L235A/P331G (SEQ ID NO:51)

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAGIEKTISKAKGQPREPQVYTLPPSREE

Hexahistidine peptide tag (SEQ ID NO:52)

HHHHHH

Claims

WHAT IS CLAIMED:

1. A VHH that specifically binds Acinetobacter BamA, wherein the VHH comprises:

(a) a CDR1 comprising the amino acid sequence GLSFGLDAYAVA (SEQ ID NO: 11), a CDR2 comprising the amino acid sequence CISPTGSRVAYADSAKG (SEQ ID NO: 12), and a CDR3 comprising the amino acid sequence SNDKRCSDFGVDRVGY (SEQ ID NO: 13);

(b) a CDR1 comprising the amino acid sequence GSTATRDTFSSHRMT (SEQ ID NO:21), a CDR2 comprising the amino acid sequence TITGDDITNYTGSVKG (SEQ ID NO:22), and a CDR3 comprising the amino acid sequence LERGIWAY (SEQ ID NO:23); or

(c) a CDR1 comprising the amino acid sequence GFSFRDYSMS (SEQ ID NO:31), a CDR2 comprising the amino acid sequence GIRSLGTTTYYADSVKG (SEQ ID NO:32), and a CDR3 comprising the amino acid sequence CLGKICDRFGIVDIYS (SEQ ID NO:33).

2. The VHH of claim 1, which binds one or more of the external outer membrane loops of the barrel domain of Acinetobacter BamA.

3. The VHH of claim 1 or claim 2, which comprises a CDR1 comprising the amino acid sequence GLSFGLDAYAVA (SEQ ID NO: 11), a CDR2 comprising the amino acid sequence CISPTGSRVAYADSAKG (SEQ ID NO: 12), and a CDR3 comprising the amino acid sequence SNDKRCSDFGVDRVGY (SEQ ID NO: 13).

4. The VHH of claim 3, which has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:41.

5. The VHH of claim 3, which has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:41.

6. The VHH of claim 3, which comprises the amino acid sequence of SEQ ID NO:41.

7. The VHH of claim 1 or claim 2, which comprises a CDR1 comprising the amino acid sequence GSTATRDTFSSHRMT (SEQ ID NO:21), a CDR2 comprising the amino acid sequence TITGDDITNYTGSVKG (SEQ ID NO:22), and a CDR3 comprising the amino acid sequence LERGIWAY (SEQ ID NO: 23). The VHH of claim 7, which has at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 42. The VHH of claim 7, which has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 42. The VHH of claim 7, which comprises the amino acid sequence of SEQ ID NO:42. The VHH of claim 1 or claim 2, which comprises a CDR1 comprising the amino acid sequence GFSFRDYSMS (SEQ ID NO:31), a CDR2 comprising the amino acid sequence GIRSLGTTTYYADSVKG (SEQ ID NO:32), and a CDR3 comprising the amino acid sequence CLGKICDRFGIVDIYS (SEQ ID NO:33). The VHH of claim 11, which has at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 43. The VHH of claim 11, which has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 43. The VHH of claim 11, which comprises the amino acid sequence of SEQ ID NO:43. The VHH of any one of claims 1-5, 7-9, or 11-13, which is a humanized antibody. The VHH of any one of claims 1-15, which is a monoclonal antibody. The VHH of any one of claims 1-16, which is linked to at least one heavy chain constant region. The VHH of claim 17, wherein the at least one heavy chain constant region is from an IgGl antibody, an IgG2 antibody, or an IgG4 antibody. The VHH of any one of claims 1-16, which is linked to a Fc region. The VHH of claim 19, wherein the Fc region is from an IgGl antibody, an IgG2 antibody, or an IgG4 antibody. The VHH of any one of claims 1-20, which antagonizes A. baumannii BamA. The VHH of any one of claims 1-21, which inhibits the insertion of one or more outer membrane proteins (OMPs) into the outer membrane of A. baumannii. The VHH of any one of claims 1-20, which is attached to a half-life extending moiety. The VHH of any one of claims 1-20, which is linked or conjugated to an antibacterial agent. An antibody that competes with the VHH of any one of claims 1-20 for binding to A. baumannii BamA. A polypeptide comprising:

(a) a first VHH that specifically binds Acinetobacter BamA, wherein the first VHH comprises a CDR1 comprising the amino acid sequence GFSFRDYSMS (SEQ ID NO:31), a CDR2 comprising the amino acid sequence GIRSLGTTTYYADSVKG (SEQ ID NO:32), and a CDR3 comprising the amino acid sequence CLGKICDRFGIVDIYS (SEQ ID NO:33).; and

(b) a second VHH that specifically binds Acinetobacter BamA. The polypeptide of claim 26, wherein the second VHH comprises a CDR1 comprising the amino acid sequence GLSFGLDAYAVA (SEQ ID NO: 11), a CDR2 comprising the amino acid sequence CISPTGSRVAYADSAKG (SEQ ID NO: 12), and a CDR3 comprising the amino acid sequence SNDKRCSDFGVDRVGY (SEQ ID NO: 13). The polypeptide of claim 26, wherein the second VHH comprises a CDR1 comprising the amino acid sequence GSTATRDTFSSHRMT (SEQ ID NO:21), a CDR2 comprising the amino acid sequence TITGDDITNYTGSVKG (SEQ ID NO:22), and a CDR3 comprising the amino acid sequence LERGIWAY (SEQ ID NO:23). A pharmaceutical composition that comprises the VHH of any one of claims 1-24 or the polypeptide of any one of claims 26-28 and a pharmaceutically acceptable carrier. An isolated polynucleotide encoding the VHH of any one of claims 1-20 or the polypeptide of any one of claims 26-28. A vector comprising the polynucleotide of claim 30. An isolated cell comprising the polynucleotide of claim 30. An isolated cell comprising the vector of claim 31. An isolated cell producing the VHH of any one of claims 1-20 or the polypeptide of any one of claims 26-28. A method of inhibiting the growth of Acinetobacter, comprising contacting Acinetobacter with an effective amount of the VHH of any one of claims 1-24, the polypeptide of any one of claims 26- 28, or the pharmaceutical composition of claim 29. The method of claim 35, wherein the Acinetobacter is A. baumannii. The method of claim 35 or claim 36, further comprising contacting Acinetobacter with at least one additional therapeutic agent. The method of claim 37, wherein the additional therapeutic agent is an antibacterial agent. A method of inhibiting an Acinetobacter infection in a subject, comprising administering to the subject a therapeutically effective amount of the VHH of any one of claims 1-24, the polypeptide of any one of claims 26-28, or the pharmaceutical composition of claim 29. A method of treating an Acinetobacter infection in a subject, comprising administering to the subject a therapeutically effective amount of the VHH of any one of claims 1-24, the polypeptide of any one of claims 26-28, or the pharmaceutical composition of claim 29. The method of claim 39 or claim 40, wherein the Acinetobacter is A. baumannii. The method of any one of claims 39-41, further comprising the administration of at least one additional therapeutic agent. The method of claim 42, wherein the additional therapeutic agent is an antibacterial agent. The method of any one of claims 39-43, wherein the subject is a human. A method of making the VHH of any one of claims 1-20 or the polypeptide of any one of claims 26-28, comprising:

(a) culturing a cell expressing the VHH or the polypeptide, and

(b) isolating the VHH or polypeptide. The method of claim 45, further comprising purifying the VHH or polypeptide. The VHH of any one of claims 1-20, further comprising a detectable moiety. The VHH of claim 47, wherein the detectable moiety is a fluorescent label, a biolumine scent label, a chemiluminescent label, an enzyme, a small molecule, a radioisotope, or colloidal gold. A method of detecting Acinetobacter in a biological sample comprising:

(a) contacting the biological sample with the VHH of claim 47 or claim 48; and

(b) detecting the binding of the VHH to Acinetobacter in the sample. The method of claim 49, which comprises using flow cytometry, immunohistochemistry (IHC), western blot analysis, ELISA, or mass spectrometry. A kit for detecting Acinetobacter in a biological sample comprising an agent comprising the VHH of any one of claims 1 to 20. The kit of claim 51, wherein the agent further comprises a detectable moiety. The kit of claim 51, wherein the kit further comprises an agent for detecting the VHH.