Biosensors for detecting binding of bacteriophage and analytes binding to bacteriophage

Abstract

A biosensor for detecting the presence, amount, or binding kinetics of an analyte bound to an analyte binding molecule is provided. The biosensor includes a core component and a bacteriophage (such as an M13 bacteriophage) displaying the analyte binding molecule immobilized thereon. Also provided are methods for detecting or quantitating the presence or amount of an analyte bound to an analyte binding molecule, or binding kinetics, using the biosensor having an analyte binding molecule-displaying bacteriophage immobilized thereon. The biosensor can be a bio-layer interferometry (BLI) biosensor, and can detect the presence, amount, or binding kinetics of the analyte (e.g., antigen) binding to a plurality of phage-displayed analyte binding molecules (e.g., antibodies) efficiently and rapidly.

Description

Patent Application BA2404-WO S1163211080WO (00026) BIOSENSORS FOR DETECTING BINDING OF BACTERIOPHAGE AND ANALYTES BINDING TO BACTERIOPHAGE Background

[0001] The present disclosure relates to apparatus and methods for detecting and / or quantitating analyte binding to phage-displayed analyte binding molecules.

[0002] Phage display is a technique for discovering and engineering therapeutic proteins, including antibodies, antibody fragments, nanobodies, and peptides. In phage display, a gene encoding a protein of interest is inserted into a phage gene encoding a surface protein (e.g., a coat protein), causing a phage (also referred to as a bacteriophage) to display a protein of interest on its external surface. A diversity of proteins of interest can be displayed by a plurality of bacteriophages. The diversity of proteins that the phages display can then be screened for interaction with one or more target molecules (e.g., proteins, peptides, DNA molecules). Using phage display, large libraries of proteins can be screened for specific biological features by an in vitro selection.

[0003] Currently, there are no known biosensors that can assess binding amount or kinetics of the phage displayed proteins (e.g., antibodies, nanobodies, antibody fragments, enzymes, substrates, peptides, ligands, antigens) to the target molecules (e.g., antigens, DNA molecules, substrates, enzymes, receptors, antibodies). Accurate and rapid detection of binding of the phage displayed proteins to the target molecules would promote therapeutic drug discovery, engineering, and screening. 1 WBD (US) 4937-7073-4681v1Patent Application BA2404-WO S1163211080WO (00026) Summary

[0004] In view of the foregoing, there is a need for methods that efficiently, accurately, and rapidly detect the presence, amount, or binding kinetics of an analyte (e.g., antigen) binding to the phage-displayed analyte binding molecule (e.g., antibody) to facilitate discovery and engineering of therapeutic molecules (e.g., therapeutic antibodies). This disclosure is directed generally to systems and methods to address these shortcomings of the art and provide other additional or alternative advantages. The disclosure herein provides embodiments of biosensors, and methods using the bacteriophage-immobilized biosensors, for detecting the presence, amount, or binding kinetics of an analyte binding to an analyte binding molecule displayed by the bacteriophage. The biosensor for detecting the binding of the analyte to the analyte binding molecule contains a core component and a bacteriophage displaying the analyte binding molecule immobilized thereon. The biosensor can be a bio-layer interferometry (BLI) biosensor, and the signals generated by binding of the analyte to the biosensor can be indicative of spectral shift, which can be measured using BLI.

[0005] In certain aspects of the present disclosure, a method for detecting the presence, amount, or binding kinetics of an analyte bound to an analyte binding molecule is provided. The method includes contacting the analyte with a biosensor with a bacteriophage immobilized on the biosensor, such that the analyte binds to the analyte binding molecule of the biosensor, the bacteriophage displaying the analyte binding molecule, and detecting signals generated by binding the analyte to the biosensor over time to determine the presence, amount, or binding kinetics of the analyte bound to the analyte binding molecule.

[0006] In embodiments, the bacteriophage is an M13 bacteriophage. 2 WBD (US) 4937-7073-4681v1Patent Application BA2404-WO S1163211080WO (00026)

[0007] In certain embodiments, contacting the analyte with the biosensor includes contacting the analyte with a biosensor containing the bacteriophage bound to a first antibody bound to the biosensor. In specific embodiments, the first antibody is an anti-pVIII antibody (such as anti-pVIII bivalent (e.g., full length) antibody), an anti-pIII antibody (such as anti-pIII bivalent (e.g., full length) antibody), an anti-pVI antibody (such as anti-pVI bivalent (e.g., full length) antibody), an anti-pVII antibody (such as anti-pVII bivalent (e.g., full length) antibody), or an anti-pIX antibody (such as anti-pIX bivalent (e.g., full length) antibody). Contacting the analyte with the biosensor includes contacting the analyte with the biosensor containing the bacteriophage bound to: an anti- pVIII antibody via a pVIII molecule on the bacteriophage surface; an anti-pIII antibody via a pIII molecule on the bacteriophage surface; an anti-pVI antibody via a pVI molecule on the bacteriophage surface; an anti-pVII antibody via a pVII molecule on the bacteriophage surface; or an anti-pIX antibody via a pIX molecule on the bacteriophage surface, respectively. In further embodiments, contacting the analyte with the biosensor includes contacting the analyte with the biosensor containing the bacteriophage bound to a biotinylated antibody (e.g., a biotinylated anti- pVIII antibody, a biotinylated anti-pIII antibody, a biotinylated anti-pVI antibody, a biotinylated anti-pVII antibody, or a biotinylated anti-pIX antibody) bound to streptavidin bound to the biosensor.

[0008] In embodiments, contacting the analyte with the biosensor includes contacting an analyte attached to a detectable label with the biosensor. The signals are signals of the detectable label generated by binding of the analyte attached to the biosensor. In specific embodiments, the detectable label is a fluorescent label.

[0009] In some embodiments, the analyte is an antigen and the analyte binding molecule is a second antibody that binds the analyte. 3 WBD (US) 4937-7073-4681v1Patent Application BA2404-WO S1163211080WO (00026)

[0010] In further embodiments, the biosensor is a bio-layer interferometry (BLI) biosensor.

[0011] In certain embodiments, the signals are indicative of spectral shift measured using bio- layer interferometry (BLI). In embodiments, positive spectral shift indicates binding of the analyte to the biosensor.

[0012] In some embodiments, detecting the binding kinetics includes detecting an association rate constant (ka), a dissociation rate constant (kd), and / or an equilibrium dissociation constant (KD) of binding of the analyte binding molecule to the analyte.

[0013] In embodiments, the method further includes detecting the amount or binding kinetics of the analyte bound to the biosensor based on the signals generated by binding of the analyte to the biosensor over time and a standard curve of association between known amounts of the analyte bound to the biosensor and signals generated by each known amount of the analyte bound to the biosensor over time.

[0014] In certain embodiments, the presence or amount of the analyte bound to the biosensor is detected within 5 minutes of contacting the analyte with the biosensor.

[0015] In embodiments, the binding kinetics of the analyte binding molecule to the analyte is detected within 12 minutes of contacting the analyte with the biosensor.

[0016] In some embodiments, contacting the analyte with the biosensor includes contacting the analyte in a plurality of samples with a plurality of biosensors. Each sample is contacted with a biosensor of the plurality of biosensors. In embodiments, detecting the signals includes detecting the signals in the plurality of samples simultaneously. 4 WBD (US) 4937-7073-4681v1Patent Application BA2404-WO S1163211080WO (00026)

[0017] In specific embodiments, the signals are detected using a plate reader.

[0018] In embodiments, the plurality of biosensors include a plurality of heterogenous biosensors each comprising a bacteriophage displaying a heterogenous analyte binding molecule immobilized on each biosensor. In embodiments, the plurality of samples comprise the analyte in different concentrations.

[0019] In certain embodiments, the presence or amount of the analyte bound to the analyte binding molecule in 96 samples are detected within 1 hour. In some embodiments, the binding kinetics of 5 analyte binding molecules to the analyte is measured within 1 hour.

[0020] In some embodiments, the method further includes comparing the amount of the analyte bound to the analyte binding molecule or the KDwith a predetermined threshold, and when the amount of analyte bound to the analyte binding molecule is greater than the predetermined threshold, or when the KDis smaller than the predetermined threshold, selecting the analyte binding molecule as a candidate for a therapeutic molecule.

[0021] In further embodiments, the method further includes comparing the amount of the analyte bound to the analyte binding molecule with a predetermined threshold among different analyte binding molecules, and selecting a range of (best performing) analyte binding molecules having largest amounts of the analyte binding as candidates for a therapeutic molecule.

[0022] In further embodiments, the method further includes comparing the KD for binding of the analyte to different analyte binding molecules, and selecting a range of (best performing) analyte binding molecules having smallest KDvalues as candidates for a therapeutic molecule. 5 WBD (US) 4937-7073-4681v1Patent Application BA2404-WO S1163211080WO (00026)

[0023] In certain embodiments of the present disclosure, a biosensor for detecting the presence, amount, or binding kinetics of an analyte bound to an analyte binding molecule is provided. The biosensor contains a core component, and a bacteriophage displaying the analyte binding molecule immobilized on the biosensor. The biosensor is configured to detect the presence, amount, or binding kinetics of the analyte bound to the analyte binding molecule.

[0024] In some embodiments, the bacteriophage is an M13 bacteriophage.

[0025] In some embodiments, the bacteriophage is immobilized on the biosensor via an antibody bound to the biosensor and bound to the bacteriophage. In certain embodiments, the antibody is an anti-pVIII antibody, an anti-pIII antibody, an anti-pVI antibody, an anti-pVII antibody, or an anti-pIX antibody (such as anti-pVIII bivalent (or full length) antibody, anti-pIII bivalent (or full length) antibody, anti-pVI bivalent (or full length) antibody, anti-pVII bivalent (or full length) antibody, or anti-pIX bivalent (or full length) antibody), and the bacteriophage is immobilized on the biosensor via an anti-pVIII antibody bound to the biosensor and bound to a pVIII molecule; an anti-pIII antibody bound to the biosensor and bound to a pIII molecule; an anti- pVI antibody bound to the biosensor and bound to a pVI molecule; an anti-pVII antibody bound to the biosensor and bound to a pVII molecule; or an anti-pIX antibody bound to the biosensor and bound to a pIX molecule, respectively, on the surface of the bacteriophage. In certain embodiments, the antibody is biotinylated, and is bound to streptavidin bound to the biosensor.

[0026] In embodiments, the biosensor is a bio-layer interferometry (BLI) biosensor.

[0027] Further, to the extent consistent, any of the embodiments or aspects described herein may be used in conjunction with any or all of the other embodiments or aspects described herein. 6 WBD (US) 4937-7073-4681v1Patent Application BA2404-WO S1163211080WO (00026) Brief Description of the Drawings

[0028] Various aspects of the present disclosure are described herein below with reference to the drawings, which are incorporated in and constitute a part of this specification.

[0029] FIG.1 is a schematic representation of a process of detecting the presence, amount, or binding kinetics of an antigen bound to the phage-displayed antibody using a biosensor, where the bacteriophage displaying the antibody is bound to the streptavidin-bound biosensor via a biotinylated anti-pVIII antibody (or a biotinylated anti-pIII antibody, a biotinylated anti-pVI antibody, a biotinylated anti-pVII antibody, or a biotinylated anti-pIX antibody), according to embodiments of the present disclosure.

[0030] FIG. 2 is a flowchart with schematic representations of a process of detecting the presence, amount, or binding kinetics of an analyte (such as an antigen, DNA molecule, substrate, enzyme, receptor, antibody) bound to the phage-displayed protein (such as an antibody, antibody fragment, nanobody, enzyme, substrate, peptide, ligand, antigen) using a biosensor according to embodiments of the present disclosure. Detailed Description

[0031] The present disclosure will now be described more fully hereinafter with reference to example embodiments thereof and with reference to the drawings. These example embodiments are described so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art. Features from one embodiment or aspect can be combined with features from any other embodiment or aspect in any appropriate combination. For example, any individual or collective features of method aspects or embodiments can be applied to apparatus, product, or component aspects or embodiments and vice versa. The disclosure may 7 WBD (US) 4937-7073-4681v1Patent Application BA2404-WO S1163211080WO (00026) be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements.

[0032] As used herein, the term “biosensor” refers to a device that detects the presence, physical characteristics, or amount of a substance of interest (an analyte) using a biological molecule (such as an enzyme, an antibody, an aptamer, DNA), a living organism, or a cell or tissue thereof. In embodiments, the biosensor has a distal end and a proximal end. The distal end of the biosensor can have a surface coated with a thin layer of analyte binding molecules (such as antibodies, antibody fragments, or nanobodies displayed by bacteriophages). The proximal end of the biosensor can be connected to a transducer that converts bio-recognition events into a measurable signal and / or a display that displays the signals.

[0033] As used herein, the term “proximal” refers to the portion of the device or component thereof that is closer to the light source in the device, and the term “distal” refers to the portion of the device or component thereof that is farther from the light source in the device and closer to the subject sample.

[0034] An “analyte” in the context of biosensor detection refers to any molecule or ligand the presence, amount, or binding kinetics of which is detected (or measured) by a biosensor. Example analytes include, but are not limited to, peptides, antigens, antibodies, ligands, and receptors.

[0035] An “analyte binding” molecule refers to any molecule or ligand capable of participating in a specific binding reaction with an analyte. Example binding reactions include, but are not limited to, receptor-ligand binding reactions and antibody-antigen binding reactions. Example 8 WBD (US) 4937-7073-4681v1Patent Application BA2404-WO S1163211080WO (00026) analyte binding molecules include, but are not limited to, peptides, antibodies, antigens, receptors, and ligands.

[0036] As used herein, the term “bio-layer interferometry (BLI) biosensor” refers to a biosensor that uses BLI for detection of the presence, physical characteristics, or amount of a substance of interest (an analyte). BLI is an optical technique for measuring biomolecular interactions by analyzing interference patterns of light reflected from the surface of a biosensor tip. BLI allows for label-free optical analysis for real-time monitoring of biomolecular interactions.

[0037] An “antibody” refers to a peptide or polypeptide derived from, modeled after, or substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, capable of specifically binding an antigen or epitope. An antibody includes an immunoglobulin molecule having two heavy chains and two light chains prepared by any method known in the art or later developed. Antibodies can be produced by immunizing mice, rats, or rabbits or by genetic engineering methods such as cloning of native immunoglobulin genes (or humanized immunoglobulin genes) in mammalian plasmid vectors and then expressing them in mammalian cell lines. An antibody also refers to and includes an antibody fragment. An “antibody fragment” refers to a fragment of an antibody that retains capacity to bind an antigen, and includes an antigen binding site (e.g., a fragment, a subsequence, a complementarity determining region (CDR)); a Fab fragment, i.e., a monovalent fragment consisting of the VL, VH, CL and CHI domains; a F(ab’)2 fragment or a Fab2 fragment, i.e., a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CHI domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a dAb fragment, which consists of a VH domain; and an isolated complementarity determining 9 WBD (US) 4937-7073-4681v1Patent Application BA2404-WO S1163211080WO (00026) region (CDR). For example, a fragment of an antibody molecule, produced by chemical cleavage or genetic engineering techniques, is an antibody. Single chain variable fragments (SCFvs) such as those produced using combinatorial genetic libraries and phage display technologies are antibodies.

[0038] A “bivalent antibody” has two antigen binding domains (arms) and bind two copies of an antigen at the same time. For example, a natural, full length IgG antibody is a bivalent antibody that binds two copies of the same antigen at the same time. On the other hand, a Fab fragment is a “monovalent antibody” that binds one copy of an antigen.

[0039] A “monospecific” antibody is an antibody capable of binding one target (e.g., antigen, epitope, cell, tissue). An antibody (e.g., an engineered antibody) can have binding capability to more than one, e.g., two or three different targets (e.g., antigens, epitopes, cells, tissues) at the same time. Such antibodies are referred to as “multispecific” antibodies, e.g., “bispecific” or “trispecific” antibodies, respectively.

[0040] Antibodies include polyclonal antibodies and monoclonal antibodies. A “polyclonal antibody” refers to a heterologous mixture of immunoglobulins against an antigen, and can be produced by multiple cells by inoculating a mammal such as a goat, a mouse, and a rabbit with an immunogen. A “monoclonal antibody” refers to an antibody produced by identical immune cells which are clones of a single cell. A polyclonal antibody can bind to different epitopes of the same antigen, whereas a monoclonal antibody binds to one epitope of the antigen.

[0041] A “protein” as used herein refers to a molecule that contains one or more chains of amino acid residues. A linear chain of amino acid residues is referred to as a polypeptide. A “protein” includes amino acid molecules of varied sizes, including peptides, polypeptides, 10 WBD (US) 4937-7073-4681v1Patent Application BA2404-WO S1163211080WO (00026) antigens, ligands, enzymes, receptors, antibodies. Proteins differ from one another primarily in their sequence of amino acids, which is dictated by the nucleotide sequence of genes that encode the protein. Specific amino acid sequences usually result in proteins folding into specific 3D structures that determine the activity of the proteins.

[0042] A “subject sample” as used herein refers to a sample with unknown presence, amount, or binding kinetics of an analyte (e.g., for binding to a bacteriophage-displayed analyte binding molecule) to be detected. A “reference sample” as used herein refers to a sample with known presence, amount, or binding kinetics of an analyte (e.g., for binding to a bacteriophage-displayed analyte binding molecule).

[0043] As used herein, the singular forms “a,” “an,” “the,” and the like include plural referents unless the context clearly dictates otherwise. In addition, while reference may be made herein to quantitative measures, values, geometric relationships or the like, unless otherwise stated, any one or more if not all of these may be absolute or approximate to account for acceptable variations that may occur, such as those due to manufacturing or engineering tolerances or the like. Reference to a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se with in a range suitable in the context, for example ± 10%. For example, description referring to “X” includes description of “X” and extends to a suitable range.

[0044] A “bacteriophage,” also referred to interchangeably as a “phage,” is a virus that infects bacteria and archaea and replicates therein. Bacteriophages are composed of proteins that encapsulate a DNA or RNA genome. Phages replicate within the bacterium or archaeon following the injection of the phages’ genome into the cytoplasm of bacteria or archaea. In phage display, a library of phage particles expressing a wide diversity of proteins on their surface (phage library) 11 WBD (US) 4937-7073-4681v1Patent Application BA2404-WO S1163211080WO (00026) is used to select proteins that bind the desired target. Bacteriophages that can be used in phage display include M13 bacteriophage, fd bacteriophage, f1 bacteriophage, T4 bacteriophage, T7 bacteriophage, and λ bacteriophage. Several methods including recombinant techniques have been developed to prepare phage libraries displaying a variety of proteins and to increase the diversity of the libraries. Phage libraries can be screened for binding to synthetic or native targets. The rapid isolation of specific ligands by phage display is advantageous in many applications including epitope mapping; analysis of protein-protein interactions; site-directed mutagenesis and selection of peptides having mutations at the binding site to a known target and increased binding affinity and selectivity; selection of inhibitors for the active and allosteric sites of the enzymes, receptor agonists and antagonists, and G-protein binding modulatory peptides. The specific ligands isolated from phage libraries can be used in therapeutic target validation, drug design and vaccine development. Phage display can also be used in conjunction with other methods of protein characterization, selection, and development.

[0045] In certain aspects of the present disclosure, a biosensor for detecting the presence, amount, or binding kinetics of an analyte bound to an analyte binding molecule is provided. The biosensor contains a core component, and a bacteriophage displaying the analyte binding molecule immobilized on the biosensor. The biosensor is configured to detect the presence, amount, or binding kinetics of the analyte bound to the analyte binding molecule.

[0046] Any suitable material can be used for the core component. For example, the core component can comprise a silicon substrate, activated with silane group, such as aminopropylsilane (APS) and epoxypropylsilane (EPS). The core component (optionally with one or more additional components thereon) may be capable of binding biotin, collagen, or a surface protein of the bacteriophage, as further described below. 12 WBD (US) 4937-7073-4681v1Patent Application BA2404-WO S1163211080WO (00026)

[0047] The bacteriophage can be any phage suitable for display of proteins, and include but not limited to M13 bacteriophage, fd filamentous phage, T4 bacteriophage, T7 bacteriophage, and λ bacteriophage. In specific embodiments, the bacteriophage is an M13 bacteriophage. M13 bacteriophage has a filamentous structure that fits 6.4kb of single stranded DNA with its protein capsid, having a length of 880 nm and thickness of 6.6 nm. The entire phage is covered with 2700- 2800 copies of structural surface protein pVIII, also referred to as major coat protein or major capsid protein, accounting for 98% of the whole mass of M13 phage. A total of 2700 copies of pVIII protein are helically wrapped around phage DNA through electrostatic interaction between phage genomic DNA and the positively charged domain of pVIII protein, leaving the M13 surface with negative charge. M13 also has 4 other structural surface proteins pIII, pVI, pVII, and pIX, also referred to as minor coat proteins or minor capsid proteins. pIII and pVI are capped on one end of the bacteriophage, while pVII and pIX are capped on the other end, 3-5 copies of each minor protein contained in an M13 bacteriophage. Genes encoding proteins to be screened, such as antibody fragments (e.g., Fab, Fab2, nanobodies) and antigen peptides, can be inserted into genes encoding M13’s surface proteins, such as pVIII, pIII, pVI, pVII, and pIX, and can be displayed using copies of M13’s surface proteins.

[0048] The term “immobilize” or “immobilized” or “immobilizing” in the context of immobilizing a bacteriophage onto a biosensor or a structure thereof (such as a core component) refers to the process of attaching or binding the bacteriophage to the biosensor or the structure thereof (such as a core component). Bacteriophage may be immobilized (attached, bound) to specific zones of the biosensor either by conjugating directly to the biosensor surface, or by indirect binding. Immobilization of a bacteriophage on a biosensor can be done by any methods known in the art. For example, the bacteriophage can be immobilized on the biosensor via an antibody 13 WBD (US) 4937-7073-4681v1Patent Application BA2404-WO S1163211080WO (00026) bound to the biosensor and bound to the bacteriophage. The antibody that immobilizes the bacteriophage onto the biosensor can be any antibody that binds to the bacteriophage (e.g., targets a surface molecule of the bacteriophage) and also binds to the biosensor. For example, the antibody can target a major coat (capsid) protein pVIII (i.e., the antibody can be an anti-pVIII antibody that binds to a pVIII molecule on the surface of the bacteriophage) and also bind to the biosensor (e.g., core component of the biosensor). pVIII is the major capsid protein that forms the filamentous body of the filamentous phage (e.g., M13, fd, or f1 bacteriophage). Additionally or alternatively, the antibody can target a minor coat (capsid) protein such as pIII, pVI, pVII, and pIX (i.e., the antibody can be an anti-pIII antibody that binds to a pIII molecule; an anti-pVI antibody that binds to a pVI molecule; an anti-pVII antibody that binds to a pVII molecule; or an anti-pIX antibody that binds to a pIX molecule), and also bind to the biosensor (e.g., ore component of the biosensor). In specific embodiments, the antibody is a full antibody (bivalent antibody) that binds to two copies of a target molecule, such as an anti-pVIII bivalent antibody, an anti-pIII bivalent antibody, an anti-pVI bivalent antibody, an anti-pVII bivalent antibody, or an anti-pIX bivalent antibody. Binding of the antibody with the biosensor can be by biotin-streptavidin interaction. For example, the antibody can be biotinylated, and can be bound to streptavidin that is bound to the biosensor. Non-exhaustive examples of a biosensor having a streptavidin moiety for binding a biotinylated target molecule (e.g., biotinylated antibody) include Octet® SSA Biosensors.

[0049] A bacteriophage can also be immobilized on a biosensor by binding a human Fc tagged bacteriophage to Protein A bound to the core component of the biosensor; by passive adsorption of bacteriophage onto the core component of the biosensor, for example by incubating the biosensor with a solution containing the bacteriophage; or by using a crosslinker. Conventional immobilization chemistries can be used for chemically (such as covalently) attaching a layer of 14 WBD (US) 4937-7073-4681v1Patent Application BA2404-WO S1163211080WO (00026) the analyte binding molecule to the biosensor. For example, a variety of bifunctional reagents containing a siloxane group can be used for chemical attachment to SiO2, and a hydroxyl, amine, carboxyl or other reaction group can be used for attachment of biological molecules, such as proteins (such as antigens, antibodies). It is also well known to etch or otherwise treat glass or glass surfaces to increase the density of hydroxyl groups by which analyte binding molecules (such as antibodies) can be bound. Where the core component of the biosensor is formed of a polymer, such as polystyrene, a variety of methods are available for exposing available chemically active surface groups, such as amine, hydroxyl, and carboxyl groups, for binding with analyte binding molecules (such as antibodies).

[0050] In an example of indirect binding of a bacteriophage to the biosensor, a bacteriophage may be immobilized on particles or other solid supports, and the solid supports may be immobilized onto the biosensor surface. Solid supports that may be used to immobilize a bacteriophage include membrane filters, cellulose-based papers, beads (including polymeric, latex, and paramagnetic particles), silicon wafers, nanoparticles, gels, and multi-well plates.

[0051] FIG.1 schematically depicts an embodiment of a biosensor for detecting the presence, amount, or binding kinetics of an analyte bound to an analyte binding molecule. The biosensor 114 includes a core component (optionally with one or more additional components) 102 having a streptavidin moiety 104 and a biotinylated anti-pVIII antibody (or a biotinylated anti-pIII antibody, a biotinylated anti-pVI antibody, a biotinylated anti-pVII antibody, or a biotinylated anti- pIX antibody) 106 bound to the core component (optionally with one or more additional components) 102 via the streptavidin-biotin interaction. The biosensor further includes a bacteriophage 110 displaying an analyte binding molecule 112, such as an Fab2 or Fab, immobilized onto the biosensor by binding of the pVIII molecule (a protein moiety) 108 of the 15 WBD (US) 4937-7073-4681v1Patent Application BA2404-WO S1163211080WO (00026) bacteriophage 110 to the anti-pVIII antibody (or the anti-pIII antibody, the anti-pVI antibody, the anti-pVII antibody, or the anti-pIX antibody) 106 bound to the biosensor. The biosensor 114 is configured to detect the presence, amount, or binding kinetics of the analyte 116 bound to the displayed analyte binding molecule 112 of the biosensor.

[0052] The biosensor may be a BLI biosensor. The BLI biosensor uses BLI to measure the signal / wavelength / spectral shift (nm) over time generated by the biosensor and an analyte (such as bacteriophage), with a positive shift indicating binding of an analyte, such as bacteriophage (both in monomeric form and micellar form), to the biosensor.

[0053] Methods of the present disclosure for detecting the presence, amount, or binding kinetics of an analyte bound to an analyte binding molecule provided herein include contacting the analyte with a biosensor, and detecting signals generated by binding of the analyte to the biosensor over time and thus detecting the presence, amount, or binding kinetics of the analyte bound to the analyte binding molecule. The biosensor contains a bacteriophage displaying the analyte binding molecule. The bacteriophage is immobilized on the biosensor such that the analyte binds to the analyte binding molecule of the biosensor.

[0054] In addition to measuring the presence and amount of the analyte bound to the analyte binding molecule, the methods provided herein can measure the binding kinetics (e.g., KD, ka, kd) of the analyte (e.g., antigen) to the analyte binding molecule (e.g., antibody) displayed on the bacteriophage. In improving the binding of therapeutic molecules (e.g., antibodies) to the target molecule (e.g., antigen) in the process of affinity maturation, binding kinetics (as assessed by, e.g., KD, ka, kd) provides helpful information regarding whether the process of binding improvement (affinity maturation) is working during rounds of therapeutic molecule designing and panning. 16 WBD (US) 4937-7073-4681v1Patent Application BA2404-WO S1163211080WO (00026) The ability to measure binding kinetics provides an improvement and distinction over the existing methods of assessing binding of the analyte and the analyte binding molecule, such as ELISA, which can measure the presence and amount of analyte binding but does not measure the binding kinetics. In contrast, by using the methods provided herein, binding kinetics of an analyte to a bacteriophage-displayed analyte binding molecule can be measured, for example, by measuring the amount of binding over time of different concentrations of the analyte in samples to the bacteriophage-displayed analyte binding molecule on the biosensor, indicated by the biosensor signals.

[0055] As used herein, an association rate constant (also referred to as ka, kon, or on-rate) refers to the rate at which a first composition binds to a second composition to form a complex. kacan be expressed in per mole per second (M-1s-1). As used herein, a dissociation rate constant (also referred to as kd, koff, or off-rate) refers to the rate at which the binding of the first and second molecules (compositions) reverses. kdcan be expressed in per second (s-1). As used herein, an equilibrium dissociation constant KD refers to the tendency of the complex to break apart into the first and second compositions, and can be defined as KD= kd / ka. In a dissociation reaction represented as AxBy⇄  xA + yB, KDcan be also calculated as KD= [A]x[B]y / [AxBy] where [A], [B], and [AxBy]the equilibrium concentrations of the first composition A, the second composition B, and the fully formed complex AxBy, respectively.

[0056] The bacteriophage can be any phage suitable for display of proteins, and include but not limited to M13 bacteriophage, fd filamentous phage, T4 bacteriophage, T7 bacteriophage, and λ bacteriophage. In specific embodiments, the bacteriophage is an M13 bacteriophage. 17 WBD (US) 4937-7073-4681v1Patent Application BA2404-WO S1163211080WO (00026)

[0057] Contacting the analyte with the biosensor can include contacting the analyte with a biosensor containing the bacteriophage bound to an antibody bound to the biosensor. The antibody can be any antibody that binds (a surface protein of) the bacteriophage, such as an anti-pVIII antibody an anti-pIII antibody, an anti-pVI antibody, an anti-pVII antibody, or an anti-pIX antibody, that binds pVIII, pIII, pVI, pVII, or pIX, respectively, on the surface of the bacteriophage. The antibody can be a bivalent antibody, such as an anti-pVIII bivalent antibody, an anti-pIII bivalent antibody, an anti-pVI bivalent antibody, an anti-VII bivalent antibody, or an anti-pIX bivalent antibody. Binding of the antibody with the biosensor can be by biotin-streptavidin interaction. For example, the antibody can be biotinylated, and can be bound to streptavidin that is bound to the biosensor. Non-exhaustive examples of a biosensor having a streptavidin moiety for binding a biotinylated target molecule (e.g., biotinylated WGA) include Octet® SSA Biosensors.

[0058] The biosensor can be a BLI biosensor. In some embodiments, the signals are indicative of signal / wavelength / spectral (nm) shift, which can be measured using BLI. The signal / wavelength / spectral (nm) shift over time can indicate binding of an analyte to the BLI biosensor. In some embodiments, a positive signal / wavelength / spectral shift indicates binding of the analyte to the biosensor, which can be measured using BLI.

[0059] Signals generated by binding of the bacteriophage to the biosensor may be amplified by using a targeting molecule and / or a detectable label. For example, the analyte can be attached to a detectable label. Contacting the analyte with the biosensor can include contacting an analyte attached to a detectable label with the biosensor. In this example, the signals are signals of the detectable label generated by binding of the analyte attached to the biosensor. Any detectable label that can generate signals indicative of the presence, amount, or binding kinetics of the detectable 18 WBD (US) 4937-7073-4681v1Patent Application BA2404-WO S1163211080WO (00026) label and the molecules it is bound can be used in the methods provided herein. In specific embodiments, the detectable label is a fluorescent label, and contacting the analyte with the biosensor includes contacting an analyte attached to a fluorescent label as the detectable label. The fluorescent signals of fluorescence labeled analyte bound to the biosensor is measured to detect the presence, amount, or binding kinetics of analyte bound to the biosensor. Fluorescence can be measured using any methods known in the art, such as using a fluorometer. In other embodiments, the detectable label is an HRP label, the method further includes contacting the HRP-labeled analyte bound to the biosensor with an HRP substrate, thereby generating the signals of the detectable label. Catalysis of the substrate by HRP generates a chromogenic signal at 428 nm, which may be measured to detect the presence, amount, or binding kinetics of the analyte in the sample bound to the biosensor. The signal can be enhanced in the presence of an enhancer by methods well known in the art, and resulting enhanced chemiluminescence may be measured, to detect the presence, amount, or binding kinetics of the analyte in the sample. Chromogenic signals or enhanced chemiluminescence can be measured using any methods known in the art, such as using a luminometer.

[0060] Additionally or alternatively, the method can further include, after contacting the analyte with the biosensor, contacting the biosensor-bound analyte with a targeting molecule attached to a detectable label such that the targeting molecule binds to the biosensor-bound analyte; and detecting the signals of the detectable label generated by binding of the targeting molecule to the biosensor-bound analyte. The detectable label can be, for example, a fluorescent label or a horseradish peroxidase (HRP) label. Where the detectable label is an HRP label, the method can further include contacting the HRP-labeled targeting molecule bound to the biosensor-bound analyte with an HRP substrate, thereby generating the signals of the detectable label. The 19 WBD (US) 4937-7073-4681v1Patent Application BA2404-WO S1163211080WO (00026) chromogenic signals generated by the enzymatic reaction and / or enhanced chemiluminescence using an enhancer may be measured to detect the presence, amount, or binding kinetics of the analyte bound to the biosensor, as described above, for example using a luminometer.

[0061] The methods of detecting binding of the analyte to the biosensor using a detectable label (such as a fluorescent label or an HRP label) optionally with a targeting molecule targeting the analyte may be used independently from, or in conjunction with the label free methods of detecting analyte binding to the analyte binding molecule based on the signals generated by binding of the analyte to the biosensor (such as by using BLI).

[0062] The biosensors, systems, and methods provided herein can be used, for example, to screen for therapeutic molecules for affinity and specificity of binding to a target molecule. The analyte binding molecules (e.g., therapeutic molecule candidates to be screened) that are displayed by the bacteriophage and screened for binding to the analyte can be, for example and without limitation, peptides (e.g., for improved binding to a receptor (analyte)), ligands (e.g., for improved binding to a receptor (analyte)), antigens (e.g., for improved binding to an antigen (analyte)), receptors (e.g., for improved binding to a receptor (analyte)), enzymes (e.g., for improved binding to a substrate (analyte)), or antibodies (e.g., for improved binding to an antigen (analyte)). In specific embodiments, the analyte binding molecule (e.g., therapeutic molecule candidate to be screened) that is displayed by the bacteriophage is an antibody, and the analyte (e.g., a target molecule) is an antigen. Thus, contacting the analyte with the biosensor can include contacting an antigen as the analyte with the biosensor comprising a bacteriophage displaying an antibody as the analyte binding molecule. 20 WBD (US) 4937-7073-4681v1Patent Application BA2404-WO S1163211080WO (00026)

[0063] Contacting the analyte with the biosensor may include contacting the analyte in a plurality of samples with a plurality of heterogenous biosensors. The plurality of heterogenous biosensors can each include a bacteriophage displaying a heterogenous analyte binding molecule, e.g., with varied sequences or mutations at specific sites of interest in the analyte binding molecule, immobilized on the biosensor. Each sample is contacted with a biosensor of the plurality of heterogenous biosensors. The signals in the plurality of samples can be detected simultaneously and / or consecutively.

[0064] The signals in a plurality of samples can be detected simultaneously, for example using BLI and / or using a detectable label attached to the analyte or a targeting molecule that binds to the analyte. To facilitate simultaneous or high throughput detection, the plurality of samples can be each placed in a plurality of wells in a plate (such as wells in a 96 well plate or a 384 well plate), and the signals in the plurality of wells can be detected in plate reader format. Any other sample placement format may be used to facilitate simultaneous or high throughput detection. In particular, the method provided herein may be used in high throughput detection of the presence, amount, or binding kinetics of the analyte bound to the analyte binding molecule (and thereby to the biosensor) in multiple samples, such as multiple samples containing different biosensors containing bacteriophages displaying different analyte binding molecules to be screened for the binding to the analyte of interest. Thus, the methods provided herein can streamline screening of therapeutic molecules (e.g., proteins, peptides, antibodies, antigens, ligands, receptors) in drug discovery and biologics manufacturing.

[0065] In some embodiments, the method further includes quantitating the amount of the analyte bound to the analyte binding molecule (and thereby to the biosensor). The amount of the analyte bound to the biosensor in a test sample (subject sample) can be quantitated based on the 21 WBD (US) 4937-7073-4681v1Patent Application BA2404-WO S1163211080WO (00026) signals generated by binding of the analyte in the test sample (subject sample) to the biosensor and a standard curve (or a calibration curve). The standard curve can represent an association between known amounts of the analyte bound to the biosensor and signals generated by each known amount of analyte bound to the biosensor, obtained for example by using reference samples.

[0066] The method provided herein can provide fast and accurate detection of the presence, amount, or binding kinetics of the analyte bound to the analyte binding molecule in a sample or in multiple samples. For example, the presence, amount, or binding kinetics of the analyte bound to the analyte binding molecule can be detected within 1 hour, 30 minutes, 20 minutes, 10 minutes, or 5 minutes of contacting the analyte with the biosensor. The method provided herein can detect the presence, amount, or binding kinetics of the analyte bound to the analyte binding molecule in multiple samples, such as 2, 3, 4, 5, 6, 7, 8, 9, 10. 11, 12, 13, 14, 15, 16, or more samples simultaneously, and thus may be capable of high throughout detection or screening. For example, the method can detect the presence, amount, or binding kinetics of the analyte bound to the analyte binding molecule in multiple samples in a plate reader format.

[0067] In certain embodiments, the method can detect the presence, amount, or binding kinetics of an analyte in multiple samples in a 96 well, where a standard curve representing an association between known amounts of the analyte bound to the biosensor and signals generated by each known amount of the analyte bound to the analyte binding molecule of the biosensor is recorded from first column containing 8 reference samples within 5 minutes. The presence or amount of the bacteriophage bound to the biosensor in the rest of the 88 samples (columns 2-12) can be measured within 55 minutes (5 minutes per column). Thus, the presence or amount of bacteriophage in 88 subject samples in a 96 well plate can be measured within 1 hour, which 22 WBD (US) 4937-7073-4681v1Patent Application BA2404-WO S1163211080WO (00026) indicates the capability to detect the presence or amount of bacteriophage in approximately 1.5 samples per minute.

[0068] In further embodiments, binding kinetics (e.g., KD, ka, kd) of an analyte (e.g., antigen) to a bacteriophage-displayed analyte binding molecule (e.g., antibody) can be measured by measuring the amount of binding over time of different concentrations of the analyte in samples to the bacteriophage-displayed analyte binding molecule on the biosensor in a plate reader format. For example, binding kinetics can be measured by measuring the amount of binding over time of 8 different concentrations of the analyte to the bacteriophage-displayed analyte binding molecule, and the binding kinetics of each analyte binding molecule to the analyte can be measured in about 12 minutes. In this embodiment, binding kinetics of 5 analyte-binding molecules to the analyte can be measured per hour. Currently no other methods can provide the binding kinetics of the analyte and the analyte binding molecule, e.g., for therapeutic molecule screening, efficiently. For example, ELISA can measure the amount of the analyte bound to the analyte binding molecule, but cannot measure the binding kinetics. The capability to detect binding kinetics of the analyte and the analyte binding molecule, short time until detection of the presence, amount, or binding kinetics of the analyte bound to the analyte binding molecule, and the capability of high throughput detection (e.g., to screen multiple therapeutic candidates (phage-displayed analyte binding molecules) for binding to the target molecule (analyte)) of the methods provided herein may offer advantages over currently available methods of detecting binding of therapeutic molecule candidates (e.g., phage-displayed therapeutic candidates) to a target molecule, which are low throughput, inconsistent, time-consuming, and / or expensive, and unable to detect binding kinetics.

[0069] Once the presence, amount, or binding kinetics of the analyte bound to the biosensor is detected (for example by using the biosensor provided herein having a bacteriophage displaying 23 WBD (US) 4937-7073-4681v1Patent Application BA2404-WO S1163211080WO (00026) the analyte binding molecule immobilized thereon), a skilled artisan (such as a scientist, an immunologist) can select a procedure that is compatible with the detection results. For example, the methods provided herein can further include comparing the amount of the analyte bound to the analyte binding molecule or KD of binding between the analyte and the analyte binding molecule with a predetermined threshold, and when the amount of analyte bound to the analyte binding molecule is greater than the predetermined threshold, or when the KD is smaller than the predetermined threshold (which indicates greater affinity of the analyte and the analyte binding molecule), selecting the analyte binding molecule as a candidate for a therapeutic molecule. Additionally or alternatively, the method can further include comparing the amount of the analyte bound to the analyte binding molecule with a predetermined threshold among different analyte binding molecules, and selecting a range of (best performing) analyte binding molecules having largest amounts of the analyte binding as candidates for a therapeutic molecule. The method can also include comparing the KDfor binding of the analyte to different analyte binding molecules, and selecting a range of (best performing) analyte binding molecules having smallest KD values (having largest affinity to the analyte) as candidates for a therapeutic molecule. A skilled artisan can select an appropriate threshold or range. The detection results of the present methods can be used to facilitate discovery, development, and screening of therapeutic molecules (e.g., antibodies, enzymes, receptors, antigens, substrates, and ligands).

[0070] FIG.2 schematically represents an embodiment of a process of detecting the presence, amount of an analyte bound to an analyte binding molecule. Process 200, for detecting the presence, amount, or binding kinetics of an analyte bound to an analyte binding molecule, begins with a step 202 of immobilizing the bacteriophage onto the biosensor core component (optionally with one or more additional components). Any suitable method of immobilization may be used. 24 WBD (US) 4937-7073-4681v1Patent Application BA2404-WO S1163211080WO (00026) For example, the bacteriophage can be immobilized on the biosensor via an antibody bound to the biosensor and bound to the bacteriophage. The antibody that immobilizes the bacteriophage onto the biosensor can be any antibody that binds to the bacteriophage (e.g., targets a surface molecule of the bacteriophage) and also binds to the biosensor. For example, the antibody can be an anti- pVIII antibody that binds to a pVIII molecule (e.g., an anti-pVIII bivalent antibody that binds to two copies of a pVIII molecule); an anti-pIII antibody that binds to a pIII molecule (e.g., an anti- pIII bivalent antibody that binds to two copies of a pIII molecule); an anti-pVI antibody that binds to a pVI molecule (e.g., an anti-pVI bivalent antibody that binds to two copies of a pVI molecule); an anti-pVII antibody that binds to a pVII molecule (e.g., an anti-pVII bivalent antibody that binds to two copies of a pVII molecule); or an anti-pIX antibody that binds to a pIX molecule (e.g., an anti-pIX bivalent antibody that binds to two copies of a pIX molecule) on the surface of the bacteriophage and also binds to the biosensor (e.g., core component of the biosensor). Binding of the antibody with the biosensor can be by biotin-streptavidin interaction. For example, the antibody can be biotinylated, and can be bound to streptavidin that is bound to the biosensor. Non- exhaustive examples of a biosensor having a streptavidin moiety for binding a biotinylated target molecule (e.g., biotinylated antibody) include Octet® SSA Biosensors.

[0071] A bacteriophage can also be immobilized on the core component of the biosensor by binding a human Fc tagged bacteriophage to Protein A bound to the core component of the biosensor; by passive adsorption of bacteriophage onto the core component of the biosensor, for example by incubating the biosensor with a solution containing the bacteriophage; or by using a crosslinker.

[0072] Process 200 continues to step 204, to provide the biosensor having a bacteriophage displaying analyte binding molecule immobilized thereon. Process 200 continues to step 206, to 25 WBD (US) 4937-7073-4681v1Patent Application BA2404-WO S1163211080WO (00026) contact the analyte with the biosensor. Contacting the analyte with an analyte binding molecule bound biosensor allows for binding of the analyte to the biosensor.

[0073] Process 200 continues to step 208, to detect signals generated by binding of the analyte to the biosensor, thereby detecting the presence, amount, or binding kinetics of the analyte bound to the analyte binding molecule of the biosensor. In embodiments, the biosensor is a BLI biosensor. In some embodiments, the signals are generated by binding of the analyte to the BLI biosensor over time, and are indicative of signal / wavelength / spectral (nm) shift, which can be measured using BLI. In some embodiments, a positive signal / wavelength / spectral shift may indicate binding of the analyte to the biosensor, which can be measured using BLI. The binding kinetics (e.g., KD, ka, kd) can be measured at least in part by measuring the amount of binding of multiple different concentrations of the analyte in samples to the analyte binding molecule of the biosensor (e.g., displayed by a bacteriophage). In this step, use of a standard curve (or calibration curve) may facilitate accurate quantitation of the amount or binding kinetics of the analyte bound to the biosensor. The standard curve (or calibration curve) may represent an association between known amounts of the analyte bound to the biosensor and signals generated by each known amount of the analyte bound to the biosensor over time obtained for example by using reference samples. The presence, amount, or binding kinetics of bacteriophage in each subject sample can be determined based on the signals generated by binding of the analyte to the biosensor over time, and the standard curve.

[0074] From step 206, process 200 may alternatively or additionally continue to step 210, to detect the presence, amount, or binding kinetics of the analyte bound to analyte binding molecule based on signals of detectable labels. A detectable label may be attached to the analyte, and the presence, amount, or binding kinetics of the analyte bound to the biosensor may be detected based 26 WBD (US) 4937-7073-4681v1Patent Application BA2404-WO S1163211080WO (00026) on signals of the detectable label generated by binding of the analyte attached to the detectable label to the biosensor. Additionally or alternatively, a detectable label may be attached to a targeting molecule. Step 210 may further include contacting the biosensor-bound analyte with a targeting molecule attached to a detectable label such that the targeting molecule binds to the biosensor-bound analyte, and the presence, amount, or binding kinetics of the analyte bound to the biosensor may be detected based on signals of the detectable label generated by binding of the targeting molecule attached to the detectable label to the analyte bound to the biosensor. The binding kinetics (e.g., KD, ka, kd) can be measured at least in part by measuring the amount of binding of multiple different concentrations of the analyte in samples to the analyte binding molecule of the biosensor (e.g., displayed by a bacteriophage) based on signals of the detectable label.

[0075] The detectable label may be a fluorescent label or a HRP label. To generate the HRP signals, the HRP-labeled targeting molecule bound to the biosensor-bound bacteriophage is contacted with an HRP substrate, and optionally with a chemiluminescence enhancer according to the procedures well known in the art. Signals of the detectable label can be measured by standard methods, such as using a fluorometer for detecting signals of a fluorescent label, and using a luminometer for detecting chromogenic signals or enhanced chemiluminescence. Step 210 for detecting presence, amount, or binding kinetics of the analyte bound to the analyte binding molecule of the biosensor using a detectable label (e.g., attached to the analyte or attached to a targeting molecule that binds the analyte) may be used independently from, or in conjunction with, step 208 for detecting presence, amount, or binding kinetics of the analyte bound to the analyte binding molecule based on the signals generated by binding of the analyte to the biosensor without using labels, for example by using BLI. 27 WBD (US) 4937-7073-4681v1Patent Application BA2404-WO S1163211080WO (00026)

[0076] In some embodiments, the biosensor provided herein contains an optical fiber having a proximal end portion and a distal end portion, the proximal end portion configured to receive light from a light source and configured to deliver reflected light to a detector. The distal end portion configured to have analytes bind thereto such that light reflected from the distal end portion is phase shifted based on a thickness of analytes bound to the distal end portion. In some embodiments, the biosensor further comprises an optical resonator at a distal end portion of the optical fiber, the optical resonator including a first reflective surface and a second reflective surface, the first reflective surface configured to reflect light with a first phase and the second reflective surface configured to reflect light with a second phase which is phase shifted based on a thickness of analytes bound to the optical resonator.

[0077] In some embodiments of the methods provided herein, the signals such as those generated by binding of bacteriophage to the biosensor, or binding of a targeting molecule (such as an anti-LPS antibody) are measured by a detector. The signals can be detected based on any label-free technique for detecting a change in a property of a sensor surface, such as BLI, Surface Plasmon Resonance (SPR), Surface Acoustic Wave (SAW), Quartz Crystal Microbalance (QCM), and Reflectometric Interference Spectroscopy (RIfS). Additionally or alternatively, the signals of the detectable label, such as fluorescent label, can be detected by a detector. In specific embodiments, the signals are measured by an interferometer. The interferometer can comprise the biosensor, and can constitute a BLI sensor. In some embodiments, the interferometer further comprises: a first optical waveguide configured to receive light from a light source; a second optical waveguide configured to deliver reflected light to a detector; and an optical coupler spatially separates a distal portion of the first optical waveguide from a distal portion of the second optical waveguide, wherein the biosensor is attached to the optical coupler. In some embodiments, 28 WBD (US) 4937-7073-4681v1Patent Application BA2404-WO S1163211080WO (00026) the interferometer further comprises a light source that is in optical communication with the first optical waveguide and configured to provide light to the first optical waveguide. In some embodiments, the interferometer further comprises a detector configured to receive light from the second optical waveguide. In some embodiments, the first optical waveguide and the second optical waveguide are disposed in a fiber optic bundle.

[0078] For example, an interferometer can include a light source, an optical assembly, and a detector unit. The BLI sensor or optical assembly functions as a sensing element or detector tip to detect analytes attached to an end thereof. The detector unit detects interference signals produced by interfering light waves reflected from the optical assembly. The light source directs light into the optical assembly, which is reflected back to the detector unit through an optical coupling assembly. The coupling assembly includes a first optical waveguide or fiber that extends from the light source to the optical assembly, a second optical waveguide or fiber which carry reflected light from the optical assembly to the detector, and an optical coupler which optically couples the first optical waveguide and the second optical waveguide. In some embodiments, the coupling assembly includes a lens system constructed to focus a light beam on an upper surface of the optical assembly and to direct reflected interfering light from the optical assembly to the detector.

[0079] The light source can be a white light source, such as a light emitting diode (LED), that produces light over a broad spectrum, e.g., 400 nm or less to 700 nm or greater, typically over a spectral range of at least 100 nm. In some embodiments, the light source can be a plurality of sources each having a different characteristic wavelength, such as LEDs designed for light emission at different selected wavelengths in the visible light range. The same function can be achieved by a single light source, such as, white light source, with suitable filters for directing light with different selected wavelengths onto the optical assembly. 29 WBD (US) 4937-7073-4681v1Patent Application BA2404-WO S1163211080WO (00026)

[0080] The detector may be a spectrometer, such as charge-coupled device (CCD), capable of recording the spectrum of the reflected interfering light from the optical assembly. In some embodiments, where the light source operates to direct different selected wavelengths onto the optical assembly, the detector may be a simple photodetector for recording light intensity at each of the different irradiating wavelengths. In certain embodiments, the detector may include one or more filters which allows detection of light intensity, for instance from a white-light source, at each of a plurality of selected wavelengths of the interference reflectance wave.

[0081] The first optical waveguide and / or the second optical waveguide may be in the form of a fiber optic bundle (FOB). As shown, the first optical waveguide includes several fiber optic elements surrounding a single fiber optic element of the second optical waveguide. This arrangement separates delivery of light from the light source from delivery of the reflected light from the optical assembly to the detector. It will be appreciated that other arrangements of the first optical waveguide and the second optical waveguide may allow for spatial separation of the light from the light source and the reflected light from the optical assembly. The separation of the light from the light source and the reflected light from the optical assembly may improve a signal to noise ratio (SNR) of the apparatus. In some embodiments, the first optical waveguide is a single fiber and the second fiber optical waveguide is formed of a plurality of fibers.

[0082] The distal tip of the fiber optic bundle can be aligned with a proximal end portion of the optical fiber when the BLI sensor is attached to the optical coupler. The BLI sensor may be fixedly attached to the optical coupler to align and maintain a position of the proximal end portion with respect to the tip. 30 WBD (US) 4937-7073-4681v1Patent Application BA2404-WO S1163211080WO (00026)

[0083] The BLI sensor can include the optical fiber having a proximal end and a distal end. The proximal end and / or the distal end of the optical fiber may be polished ends. The BLI sensor can have an optical resonator having a first reflecting surface and a second reflecting surface distal of the first reflecting surface. The optical fiber is substantially transparent between the proximal end and distal end thereof. The optical resonator may be transparent between the first and second reflecting surfaces. The distance between the first and second reflecting surfaces defines a thickness of the optical resonator. The thickness of the optical resonator may be in a range of 50 nm to 5,000 nm, such as between 400 nm and 1,000 nm.

[0084] The second reflecting surface is formed of a layer of analyte binding molecules which are effective to bind analyte molecules specifically and with high affinity. That is, the analyte and anti-analyte molecules are opposite members of a binding pair which can include, without limitations, antigen-antibody pairs, complementary nucleic acids, and receptor-binding agent pairs. In specific embodiments, the analyte molecule is an antigen, and the anti-analyte molecule is an antibody (e.g., Fab2) displayed by a bacteriophage as a candidate for binding to the antigen.

[0085] The index of refraction of the optical fiber may be similar to that of the second reflecting surface so that light reflected from the second reflecting surface occurs predominantly from the layer formed by the analyte binding molecules, rather than from the interface between the optical fiber and the analyte binding molecules. Similarly, as analyte molecules bind to distal end portion of the optical assembly, light reflected from the distal end portion of the assembly occurs predominantly from the layer formed by the analyte binding molecules and bound analyte, rather than from the interface region. 31 WBD (US) 4937-7073-4681v1Patent Application BA2404-WO S1163211080WO (00026)

[0086] The first reflecting surface of the optical assembly is formed as a layer of transparent material having an index of refraction that is substantially different from that of the optical fiber such that this layer functions to reflect a portion of the light directed onto the optical assembly.

[0087] The thickness of an analyte-binding layer disposed in the distal end portion of the optical element may be designed to optimize the overall sensitivity based on specific hardware and optical components. Conventional immobilization chemistries are used in chemically, such as covalently, attaching a layer of analyte binding molecules to the lower surface of the optical element. For example, a variety of bifunctional reagents containing a siloxane group for chemical attachment to SiO2, and a hydroxyl, amine, carboxyl or other reaction group for attachment of biological molecules, such as proteins (such as antigens, antibodies), or nucleic acids. It is also well known to etch or otherwise treat glass or glass surfaces to increase the density of hydroxyl groups by which analyte binding molecules can be bound. Where the optical fiber is formed of a polymer, such as polystyrene, a variety of methods are available for exposing available chemically active surface groups, such as amine, hydroxyl, and carboxyl groups.

[0088] In certain embodiments, the analyte-binding layer is formed under conditions in which a distal end surface of the optical fiber is densely coated, so that binding of analyte molecules to the layer forces a change in the thickness of the layer, rather than filling in the layer. The analyte- binding layer can be either a monolayer or a multi-layer matrix.

[0089] The measurement of the presence, amount, or binding kinetics of analyte (such as antigen) to the optical assembly is enabled by the interference of reflected light beams from the two reflecting surfaces in the optical assembly. Specifically, as analyte molecules attach to or detach from the surface, the average thickness of the second reflecting surfaces changes 32 WBD (US) 4937-7073-4681v1Patent Application BA2404-WO S1163211080WO (00026) accordingly. Because the thickness of all other layers remains the same, the interference wave formed by the light waves reflected from the two surfaces is phase shifted in accordance with this thickness change.

[0090] Assuming that there are two reflected beams, the first beam is reflected from the first reflecting surface and the second beam is reflected from the analyte binding molecules and bound analyte and the surrounding medium at the second reflecting surface. The conversion of the phase shifting to a thickness change of the bound analytes is well known in the art.

[0091] While several embodiments of the disclosure have been shown in the drawings, it is not intended that the disclosure be limited thereto, as it is intended that the disclosure be as broad in scope as the art will allow and that the specification be read likewise. Any combination of the above embodiments is also envisioned and is within the scope of the appended claims. Therefore, the above description should not be construed as limiting, but merely as exemplifications of particular embodiments. Those skilled in the art will envision other modifications within the scope of the claims appended hereto. 33 WBD (US) 4937-7073-4681v1

Claims

Patent Application BA2404-WO S1163211080WO (00026) What is Claimed:

1. A method for detecting the presence, amount, or binding kinetics of an analyte bound to an analyte binding molecule, the method comprising: contacting the analyte with a biosensor having a bacteriophage immobilized on the biosensor such that the analyte binds to the analyte binding molecule of the biosensor, wherein the bacteriophage displays the analyte binding molecule; and detecting signals generated by binding the analyte to the biosensor over time to determine the presence, amount, or binding kinetics of the analyte bound to the analyte binding molecule.

2. The method of claim 1, further comprising, prior to contacting the analyte with the biosensor, binding the bacteriophage to a first antibody bound to the biosensor.

3. The method of claim 2, wherein the first antibody is an anti-pVIII antibody, an anti-pIII antibody, an anti-pVI antibody, an anti-pVII antibody, or an anti-pIX antibody.

4. The method of claim 1, wherein contacting the analyte with the biosensor includes contacting an analyte attached to a detectable label with the biosensor, wherein the signals are signals of the detectable label generated by binding of the analyte attached to the biosensor.

5. The method of claim 4, wherein to the detectable label is a fluorescent label.

6. The method of claim 1, wherein the analyte binding molecule comprises a second antibody that binds the analyte. 34 WBD (US) 4937-7073-4681v1Patent Application BA2404-WO S1163211080WO (00026) 7. The method of claim 1, wherein the biosensor is a bio-layer interferometry (BLI) biosensor .

8. The method of claim 1, wherein the signals are indicative of spectral shift measured using bio-layer interferometry (BLI), wherein a positive spectral shift indicates binding of the analyte to the biosensor.

9. The method of claim 1, wherein detecting the binding kinetics comprises detecting an association rate constant (ka), a dissociation rate constant (kd), and / or an equilibrium dissociation constant (KD) of binding of the analyte binding molecule to the analyte.

10. The method of claim 1, wherein the method further comprises: detecting the amount or binding kinetics of the analyte bound to the biosensor based on the signals generated by binding of the analyte to the biosensor over time and a standard curve of association between known amounts of the analyte bound to the biosensor and signals generated by each known amount of the analyte bound to the biosensor over time.

11. The method of claim 1, wherein the presence or amount of the analyte bound to the analyte binding molecule is detected within 5 minutes of contacting the analyte with the biosensor.

12. The method of claim 1, wherein the binding kinetics of the analyte binding molecule to the analyte is detected within 12 minutes of contacting the analyte with the biosensor. 35 WBD (US) 4937-7073-4681v1Patent Application BA2404-WO S1163211080WO (00026) 13. The method of claim 1: wherein contacting the analyte with the biosensor includes contacting the analyte in a plurality of samples with a plurality of biosensors, wherein each sample is contacted with one of the plurality of biosensors; and wherein detecting the signals includes detecting the signals in the plurality of samples simultaneously.

14. The method of claim 13, the signals are detected using a plate reader.

15. The method of claim 13, wherein the plurality of biosensors comprise a plurality of heterogenous biosensors each comprising a bacteriophage displaying a heterogenous analyte binding molecule immobilized on each biosensor, or wherein the plurality of samples comprise the analyte in different concentrations.

16. The method of claim 13, wherein the presence or amount of the analyte bound to the analyte binding molecule in 96 samples are detected within 1 hour.

17. The method of claim 13, wherein the binding kinetics of 5 analyte binding molecules to the analyte is measured within 1 hour.

18. The method of claim 1, further comprising: comparing the amount of the analyte bound to the analyte binding molecule among different analyte binding molecules; and 36 WBD (US) 4937-7073-4681v1Patent Application BA2404-WO S1163211080WO (00026) selecting a range of analyte binding molecules having largest amounts of the analyte binding as candidates for a therapeutic molecule.

19. A biosensor for detecting the presence, amount, or binding kinetics of an analyte bound to an analyte binding molecule, the biosensor comprising: a core component; and a bacteriophage displaying the analyte binding molecule immobilized thereon, the biosensor being configured to detect the presence, amount, or binding kinetics of the analyte bound to the analyte binding molecule.

20. The biosensor of claim 19, wherein the bacteriophage is immobilized on the biosensor via an antibody bound to the biosensor and bound to the bacteriophage.

21. The biosensor of claim 20, wherein the antibody is an anti-pVIII antibody, an anti-pIII antibody, an anti-pVI antibody, an anti-pVII antibody, or an anti-pIX antibody.

22. The biosensor of claim 19, wherein the bacteriophage is an M13 bacteriophage.

23. The biosensor of claim 19, wherein the biosensor is a bio-layer interferometry (BLI) biosensor. 37 WBD (US) 4937-7073-4681v1

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