Method and system for screening using microcapillary array

Abstract

To provide a high-throughput method for screening a large population of mutant proteins.SOLUTION: The method uses a large-scale array of microcapillaries, each of which contains a solution containing a mutant protein, an immobilized target molecule, and a reporter element. The immobilized target molecules can include any molecules of interest, including proteins, nucleic acids, carbohydrates, and other biomolecules. The association of the mutant protein with the molecular target is evaluated by measuring a signal from the reporter element. The contents of the microcapillaries identified as containing the mutant protein of interest in the assay can be isolated, and the cells expressing the mutant protein of interest can be characterized. A system for carrying out the disclosed screening method is also provided.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 830,978, filed April 8, 2019, which is expressly incorporated by reference in its entirety for all purposes.

[0002] Technical Field The present disclosure provides methods and systems for screening with microcapillary arrays. [Background technology]

[0003] 2. Background of the Invention The analysis of biological samples, including the identification, characterization, and re-engineering of proteins, nucleic acids, carbohydrates, and other important biomolecules, has greatly benefited from the scaling up of sample numbers and the scaling down of sample sizes. For example, two-dimensional microarrays of biological materials, such as DNA microarrays, have enabled the development of high-throughput screening methods that involve multiplexed approaches for processing samples and detecting results.

[0004] The above approaches potentially benefit from combination with optical sensing techniques to identify specimens of interest using fluorescence or other corresponding specific and sensitive labeling approaches.

[0005] While such techniques provide analytical information about a particular sample, such as the presence and potentially the amount of a particular biomolecule in solution or the sequence of a particular nucleic acid or polypeptide, they do not typically allow for the recovery of the biological sample identified by the assay without inactivating or otherwise damaging the sample of interest.

[0006] Therefore, there is a continuing need to develop improved microscale screening and analysis methods and systems with high throughput capabilities, in particular methods and systems that allow for the recovery of samples identified in screening and analysis using additional assays using the claimed systems, including calcium dye assays, T cell activation assays, B cell assays, and GFP assays. Summary of the Invention

[0007] The present disclosure addresses these and other needs by providing, in one aspect, a method for screening a population of mutant proteins comprising the steps of: providing a microcapillary array comprising a plurality of microcapillaries, each microcapillary comprising a mutant protein, an immobilized target molecule, and a reporter element, wherein the mutant protein associates with the immobilized target molecule with a particular affinity; and Measuring a signal from at least one reporter element indicative of association of at least one mutant protein with at least one immobilized target molecule in a reporter assay selected from the group consisting of a calcium dye assay, a T cell activation assay, a B cell assay, and a GFP assay to identify at least one microcapillary of interest.

[0008] In some embodiments, the method further comprises isolating the contents of the microcapillary of interest.

[0009] In another aspect, a system is provided for screening a population of mutant proteins comprising: An array comprising a plurality of microcapillaries, each microcapillary comprising a mutant protein, an immobilized target molecule, and a reporter element, wherein the mutant protein associates with the immobilized target molecule with a particular affinity.

[0010] In some embodiments, the system further comprises a microscope.

[0011] In some embodiments, the system further comprises a light source and a detector.

[0012] In some embodiments, the system further comprises an extraction device.

[0013] In some embodiments, the system further comprises a two-stage sample collection element.

[0014] In some embodiments, the present invention provides a method of screening a population of mutant proteins comprising the steps of: providing a microcapillary array comprising a plurality of microcapillaries, each microcapillary comprising a mutant protein, an immobilized target molecule, and a reporter element, wherein the mutant protein associates with the immobilized target molecule with a particular affinity; and Measuring a signal from the at least one reporter element indicative of association of the at least one mutant protein with the at least one immobilized target molecule to identify at least one microcapillary of interest.

[0015] In some embodiments, the mutant protein is expressed by an expression system.

[0016] In some embodiments, the expression system is a cell-free expression system.

[0017] In some embodiments, the expression system is a cellular expression system.

[0018] In some embodiments, the cellular expression system is animal-based, avian-based, fungal-based, bacterial-based, insect-based, or plant-based.

[0019] In some embodiments, the cell expression system is an avian system.

[0020] In some embodiments, the avian expression system is a chicken system.

[0021] In some embodiments, the mutant protein is a soluble protein.

[0022] In some embodiments, the target molecule is a target antibody, a target protein or polypeptide, a target nucleic acid, a target carbohydrate, or a combination of each.

[0023] In some embodiments, the target molecule is immobilized on a surface.

[0024] In some embodiments, the surface is the surface of a cell.

[0025] In some embodiments, the target molecule is a naturally occurring protein.

[0026] In some embodiments, the surface is the surface of a bead.

[0027] In some embodiments, the surface is a surface of a microcapillary wall.

[0028] In some embodiments, the surface is a surface configured to settle into the microcapillary by gravitational settling.

[0029] In some embodiments, the reporter element is a labeled antibody or other binding molecule.

[0030] In some embodiments, the labeled antibody or other binding molecule is a fluorescently labeled antibody or other binding molecule.

[0031] In some embodiments, the labeled antibody is a primary antibody or a secondary antibody.

[0032] In some embodiments, the labeled antibody or other binding molecule is an enzyme-linked antibody or other binding molecule.

[0033] In some embodiments, the reporter element is activated within the cell and the target molecule is immobilized on the surface of the cell.

[0034] In some embodiments, the reporter element comprises a green fluorescent protein or a mutant.

[0035] In some embodiments, the signal is a fluorescent signal, an absorbance signal, a bright field signal, or a dark field signal.

[0036] In some embodiments, each microcapillary in the microcapillary array contains between 0 and 5 mutant proteins from the population of mutant proteins.

[0037] In some embodiments, the microcapillary array comprises at least 100,000, at least 300,000, at least 1,000,000, at least 3,000,000, or at least 10,000,000 microcapillaries.

[0038] In some embodiments, each microcapillary further comprises a substance to improve the viability of the cell expression system.

[0039] In some embodiments, the substance is methylcellulose, dextran Puluronic® F-68, polyethylene glycol, or polyvinyl alcohol.

[0040] In some embodiments, the substance is a growth medium.

[0041] In some embodiments, the signal is measured by an optical detector.

[0042] In some embodiments, the signal is measured by microscopy.

[0043] In some embodiments, the method further comprises isolating the contents of the microcapillary of interest.

[0044] In some embodiments, the contents of the microcapillary of interest are isolated by pulsing the microcapillary of interest with a laser.

[0045] In some embodiments, the laser is a diode-pumped Q-switched laser.

[0046] In some embodiments, a laser is directed at the water-glass interface between the microcapillary wall and the sample contained in the microcapillary.

[0047] In some embodiments, the contents of the microcapillary of interest are isolated using a two-stage sample collection element.

[0048] In some embodiments, the microcapillaries do not contain microparticles, magnetic microparticles, magnetic beads, or electromagnetic radiation absorbing materials capable of blocking the transmission of electromagnetic radiation.

[0049] The present invention also provides a system for screening a population of mutant proteins comprising: An array comprising a plurality of microcapillaries, each microcapillary comprising a mutant protein, an immobilized target molecule, and a reporter element, wherein the mutant protein associates with the immobilized target molecule with a particular affinity.

[0050] In some embodiments, the mutant protein is expressed by an expression system.

[0051] In some embodiments, the expression system is a cell-free expression system.

[0052] In some embodiments, the expression system is a cellular expression system.

[0053] In some embodiments, the cellular expression system is animal-based, avian-based, fungal-based, bacterial-based, insect-based, or plant-based.

[0054] In some embodiments, the cell expression system is an avian system.

[0055] In some embodiments, the avian expression system is a chicken system.

[0056] In some embodiments, the mutant protein is a soluble protein.

[0057] In some embodiments, the target molecule is a target protein or polypeptide, a target nucleic acid, a target carbohydrate, or a combination of each.

[0058] In some embodiments, the target molecule is immobilized on a surface.

[0059] In some embodiments, the surface is the surface of a cell.

[0060] In some embodiments, the target molecule is a naturally occurring protein.

[0061] In some embodiments, the surface is the surface of a bead.

[0062] In some embodiments, the surface is a surface of a microcapillary wall.

[0063] In some embodiments, the surface is a surface configured to settle into the microcapillary by gravitational settling.

[0064] In some embodiments, the reporter element is a labeled antibody or other binding molecule.

[0065] In some embodiments, the labeled antibody or other binding molecule is a fluorescently labeled antibody or other binding molecule.

[0066] In some embodiments, the labeled antibody is a primary antibody or a secondary antibody.

[0067] In some embodiments, the labeled antibody or other binding molecule is an enzyme-linked antibody or other binding molecule.

[0068] In some embodiments, the reporter element is activated within the cell and the target molecule is immobilized on the surface of the cell.

[0069] In some embodiments, the reporter element comprises a green fluorescent protein or a mutant.

[0070] In some embodiments, the signal is a fluorescent signal, an absorbance signal, a bright field signal, or a dark field signal.

[0071] In some embodiments, each microcapillary in the microcapillary array contains between 0 and 5 mutant proteins from the population of mutant proteins.

[0072] In some embodiments, the microcapillary array comprises at least 100,000, at least 300,000, at least 1,000,000, at least 3,000,000, or at least 10,000,000 microcapillaries.

[0073] In some embodiments, each microcapillary further comprises a substance to improve the viability of the cell expression system.

[0074] In some embodiments, the substance is methylcellulose, dextran Puluronic® F-68, polyethylene glycol, or polyvinyl alcohol.

[0075] In some embodiments, the substance is a growth medium.

[0076] In some embodiments, the system further comprises a light source and a detector.

[0077] In some embodiments, the system further comprises a microscope.

[0078] In some embodiments, the system further comprises an extraction device.

[0079] In some embodiments, the extraction device comprises a diode-pumped Q-switched laser.

[0080] In some embodiments, the system further comprises a two-stage sample collection element.

[0081] In some embodiments, the microcapillaries do not contain microparticles, magnetic microparticles, magnetic beads, or electromagnetic radiation absorbing materials capable of blocking the transmission of electromagnetic radiation.

[0082] The present invention also provides a method for screening a population of mutant proteins comprising the steps of: providing a microcapillary array comprising a plurality of microcapillaries, each microcapillary comprising a cellular expression system expressing a mutant protein, a target molecule immobilized on the surface of the cell, and a reporter element, wherein the mutant protein associates with the immobilized target molecule in the microcapillary with a specific affinity, and the cellular expression system is an avian system; and Measuring a signal from at least one reporter element indicative of association of the at least one mutant protein with the at least one immobilized target molecule in a reporter assay to identify at least one microcapillary of interest.

[0083] In some embodiments, the avian system is a chicken system.

[0084] In some embodiments, the target molecule is a target protein or polypeptide, a target nucleic acid, a target carbohydrate, a target antibody, or a combination of each.

[0085] In some embodiments, the targeting molecule is a targeting antibody.

[0086] In some embodiments, the reporter assay is selected from the group consisting of a calcium dye assay, a T cell activation assay, a B cell assay, and a GFP assay.

[0087] In some embodiments, the reporter element is a labeled antibody or other binding molecule that localizes to an epitope on the mutant protein.

[0088] In some embodiments, the labeled antibody or other binding molecule is a fluorescently labeled antibody or other binding molecule.

[0089] In some embodiments, the B cell assay comprises spleen-sourced B cells.

[0090] In some embodiments, a T cell activation assay is used to assess the ability of an antibody to induce cell signaling.

[0091] In some embodiments, T cell activation assays are used to measure the internal signaling or cell surface marker induction capacity of the antibody.

[0092] In some embodiments, a T cell activation assay is used to assess the ability of the antibody to induce cell proliferation.

[0093] In some embodiments, a T cell activation assay is used to assess the ability of the antibody to induce cytokine secretion.

[0094] In some embodiments, the T cell activation assay measures CD25 expression to determine the activation capacity of the antibody.

[0095] In some embodiments, CD25 expression is measured using a fluorescently labeled anti-CD25 antibody.

[0096] In some embodiments, the T cell activation assay measures calcium signaling to determine the activation capacity of an antibody.

[0097] In some embodiments, the T cell activation assay uses a calcium-sensitive fluorophore to measure calcium signaling.

[0098] In some embodiments, the calcium sensitive fluorophore is selected from the group consisting of Fluo-4 AM, Fura-2 AM, and Indo-1 AM.

[0099] In some embodiments, the T cell activation assay comprises a mixture of T cells and antibody secreting cells (ASCs).

[0100] In some embodiments, the mixture of T cells and ASCs varies from a ratio of 2:1 to 12:1.

[0101] In some embodiments, the mixture of T cells and ASCs is in a 5:1 ratio.

[0102] In some embodiments, the ASC is a B cell.

[0103] In some embodiments, the mixture of T cells and ASCs further comprises T cell activation beads and antibody capture beads.

[0104] In some embodiments, the T cells are purified from peripheral blood.

[0105] In some embodiments, the signal used to identify at least one microcapillary of interest is at least a 10% to 10,000% or greater increase over the signal compared to the baseline and / or control sample.

[0106] In some embodiments, the signal used to identify at least one microcapillary of interest is at least a 10% or greater increase over the signal compared to the baseline and / or control sample.

[0107] In some embodiments, the increase in the signal used to identify at least one microcapillary of interest represents a statistically significant increase when compared to a baseline and / or control. [Brief description of the drawings]

[0108] [Figure 1] Schematic diagram of the steps of an exemplary microcapillary screening assay. The left-hand view of each panel is a cross-sectional side view of a single microcapillary. The right-hand view of each panel is a bottom view of a small portion of a microcapillary array. The shading in each example is intended to indicate electromagnetic signals such as fluorescence. [Figure 2A] FIG. 2A shows a bottom view of a small portion of a microcapillary array illustrating hybridoma screening for mammalian cells, where cells are imaged using bright field (FIG. 2A). [Figure 2B] FIG. 1 shows a bottom view of a small portion of a microcapillary array illustrating hybridoma screening for mammalian cells, where cells are imaged using LiveGreen. [Figure 2C] A bottom view of a small portion of a microcapillary array illustrating hybridoma screening for mammalian cells is shown, where cells are imaged using a fluorescent anti-mouse secondary antibody (Figure 2C). [Diagram 3] Shown are images of microcapillaries containing both A431 target cells and hybridoma cells over a 4 hour incubation period. [Figure 4] Shown are images of a small section of a microcapillary array highlighting expressing and non-expressing yeast cells versus mammalian cells, where cells are imaged using either bright field (Figure 4A) or fluorescent antibodies (Figure 4B). [Diagram 5] 1 shows the growth of immortalized human cells in microcapillary arrays over a six day period. [Figure 6A] 1A-1D are different views of a microscope system designed to implement the screening method of the present disclosure. [Figure 6B] See legend to Figure 6A. [Figure 6C] See legend to Figure 6A. [Figure 6D] See legend to Figure 6A. [Figure 6E] See legend to Figure 6A. [Figure 7] 1 shows an exemplary embodiment of cellular (including mammalian or yeast) expression and binding to mammalian cells using the present invention, with each one of the four panels representing the microcavity of one microcapillary over time. [Figure 8A] 8A) Each one of the four panels represents the microcavity of one microcapillary over time. [Figure 8B] 8A) Exemplary embodiments of cellular (including mammalian or yeast) expression and binding to two or more mammalian cell types using the present invention are shown. 8B) Potential readouts from exemplary assay embodiments are provided. [Figure 9] 1 shows exemplary fluorescence and bright field data generated by the exemplary assay described in Example 6. [Figure 10] Illustrated are exemplary embodiments of cellular (including mammalian or yeast) expression and binding to immobilized targets on solid supports (such as beads) using the present invention. Each one of the four panels represents the microcavity of one microcapillary over time. [Figure 11]Shows an exemplary embodiment of cell (including mammalian or yeast) expression and functional reporter response using the present invention. Each of the four panels represents one microcavity of a microcapillary over time. Reporters can include any detectable reporter, including, for example, GFP, YFP, and / or RFP, as well as any fluorophore described herein or known in the art. [Figure 12A] Examples of IgG1, IgG2, IgG3, and IgG4 sequences are provided. [Figure 12B] This is a continuation of Figure 12A. [Figure 13] A) Provides image data of beads from the titration experiment of Example 7. B) Graph showing the optimal range of signal was approximately 1:500 to 1:5000 (the lower range of the manufacturer's recommended range). [Figure 14] Overview of screening for full-length soluble antibodies against cell surface targets. [Figure 15] Data from high-throughput morphological and fluorescent screens. [Figure 16] Three functional screens for use in the system of the invention. [Figure 17] Data showing that the integrated platform system described herein enables accurate identification of rare cells. [Figure 18] Data from antigen-specific B cell profiling of a single OmniChicken. OmniChickens express a fully human, highly diversified antibody repertoire. Genetic diversity from human and mouse allows for more diverse epitope coverage. Allows for deep immune profiling that may lead to a more diverse panel of functional antibodies. Exemplary assay shown: Profiling of antigen-specific antibody repertoires following immunization with Progranulin. [Figure 19] B cell assay. Top: assay conditions. Bottom: exemplary data / readout. [Figure 20] Binding analysis related to B cell assays. [Figure 21] Quantification and sorting are related to B cell assays. [Figure 22] Single cell VH / VK isolation and sequencing. Pairwise distance between linked HCDR3-LCDR3 of each cell. [Diagram 23] Deeper characterization identifies new clonal families. Screening identifies the majority of the clones identified by the GEM assay. Multiple new clonal families were identified. [Figure 24] Antigen-specific clones have high affinity and broad epitope coverage. Ligand expressed subset of discovered clones as scFv-Fc. Characterized by Carterra LSA (affinity and epitope binning data). Clusters map to distinct subdomains of Progranulin with broad coverage of 7 subdomains. Further validation with newly discovered clone clusters is ongoing. [Diagram 25] Representative B cell screening results. [Figure 26] Common xPloration T cell activation assay classes. [Figure 27] T cell activation by B cells (CD25 surface expression). [Figure 28] T cell activation by B cells (calcium signaling). Human T cell activation as measured via calcium signaling. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0109] Detailed Description of the Invention Microcapillary arrays have recently been used in approaches for high-throughput analysis and protein engineering with a large number of biological samples, for example in an approach termed "microcapillary single-cell analysis and laser extraction" or "μSCALE". See Chen et al. (2016) Nature Chem. Biol. 12:76-81; DOI:10.1038 / NCHEMBI0.1978. This approach relies on spatial separation of single cells within the microcapillary array, thus allowing repeated imaging, cell growth, and protein expression of separate samples within each microcapillary of the microcapillary array. Thus, this technique allows for massively parallel quantitative biochemical and biophysical measurements on millions of samples within the microcapillary array, for example in the analysis of millions of protein variants expressed from yeast, bacteria, or other suitable cells distributed across the array. Advantageously, this approach allowed for simultaneous time-resolved kinetic analysis of multiple samples and sorting of those cells based on targeted phenotypic features.

[0110] The development of the μSCALE method and device for quantitative biochemical and biophysical analysis of populations of biological variants has also been reported in US Patent Application Publication No. 2016 / 0244749 Al, which is incorporated herein by reference in its entirety. However, the extraction of the desired microcapillary contents by the μSCALE approach requires the inclusion of a radiation-absorbing material in each sample and the directing of electromagnetic radiation from a pulsed laser to this material, thus complicating the extraction method. In addition, to minimize the signal emitted from microcavities that lack the desired binding activity, early methods for screening biological variants in an array of microcavities relied on adding microparticles to the aligned samples to partially or completely block the transmission of electromagnetic radiation into and out of the sample. See US Patent Application Publication No. 2014 / 0011690 Al. In some aspects of the present disclosure, the screening method does not rely on these additional sample components or manipulations, thus simplifying and improving the efficiency of the screening technique.

[0111] In certain applications of these approaches, and as disclosed in more detail herein, the target molecule may be immobilized on a surface, such as a particle (e.g., a magnetic particle), a cell, or the surface of a microcapillary wall. The interaction of the mutant protein with the target molecule in these approaches may then be measured by a number of methods, including methods that utilize detectable antibodies and methods that measure detectable signals generated within the target cells. It will be appreciated that such methods may be used in high throughput screening to discover protein mutants that bind to target molecules, such as target molecules on cells or other surfaces.

[0112] Screening Methods Thus, in some aspects, the disclosure provides a method of screening a population of mutant proteins comprising the steps of: providing a microcapillary array comprising a plurality of microcapillaries, each microcapillary comprising a mutant protein, an immobilized target molecule, and a reporter element, wherein the mutant protein associates with the immobilized target molecule with a particular affinity; and Measuring a signal from the at least one reporter element indicative of association of the at least one mutant protein with the at least one immobilized target molecule to identify at least one microcapillary of interest.

[0113] In these methods, the microcapillary array preferably comprises a plurality of longitudinally fused capillaries, e.g., fused silica capillaries, although any other suitable material may be used for the array. See, e.g., PCT International Patent Publications WO2012 / 007537 and WO2014 / 008056, which are incorporated herein by reference in their entirety. Such arrays can be manufactured, for example, by bundling millions or billions of silica capillaries and fusing them through a thermal process, although other suitable manufacturing methods can also be used. The melting process may include, for example, i) heating the capillary single draw glass drawn under tension into a single clad fiber; ii) making capillary multi-draw single capillary from the single draw glass by bundling, heating, and drawing; iii) making capillary multi-multi-draw multi-capillary from the multi-draw single capillary by additional bundling, heating, and drawing; iv) making a block assembly of drawn glass from the multi-multi-draw multi-capillary by stacking into a pressing block; v) making a block pressing block from the block assembly by treating with heat and pressure; and vi) making block forming blocks by cutting the block pressing block at precise lengths (e.g., 1 mm).

[0114] In some embodiments, the method further comprises slicing the silica capillary, thereby forming a very high density glass microcapillary array. In some embodiments, the microcapillary array may be cut to a height of about 1 millimeter, although shorter microcapillary arrays are also contemplated, including arrays with a height of 10 μm or less. In some embodiments, longer microcapillary arrays are contemplated, including arrays with a height of 10 mm or more.

[0115] Such a process forms very high density microcapillary arrays suitable for use in the present method. In an exemplary array, each microcapillary has a diameter of about 5 μm and about 66% open space (i.e., representing the lumen of each microcapillary). In some arrays, the percentage of the array that is open ranges from about 50% to about 90%, for example about 60-75%, such as the microcapillary array provided by Hamamatsu, which has an open area of ​​about 67%. In one particular example, a 10×10 cm array with 5 μm diameter microcapillaries and about 66% open space has about 330 million microcapillaries.

[0116] In various embodiments, the inner diameter of each microcapillary in the array ranges from about 1 μm to 500 μm. In some arrays, each microcapillary can have an inner diameter ranging from about 1 μm to 300 μm, optionally from about 1 μm to 100 μm, further optionally from about 1 μm to 75 μm, further optionally from about 1 μm to 50 μm, and further optionally from about 5 μm to 50 μm.

[0117] In some microcapillary arrays, the open area of ​​the array accounts for up to 90% of the open area (OA), so that the number of microcapillaries per cm of array varies between about 4.6 million and 11 million or more when the pore diameter varies between 1 μm and 500 μm. In some microcapillary arrays, the open area of ​​the array accounts for about 67% of the open area, so that the number of microcapillaries per cm of array varies between about 340 and 800,000 or more when the pore diameter varies between 1 μm and 500 μm. In some embodiments, the pore size is 1 μm, 5 μm, 10 μm, 50 μm, 100 μm, 250 μm, 350 or 500 μm. In some embodiments, the pore size is 5 μm to 500 μm. In some embodiments, the pore size is 10 μm to 450 μm. In some embodiments, the pore size is 50 μm to 500 μm. In some embodiments, the pore size is 100 μm to 500 μm. In some embodiments, the pore size is 250 μm to 500 μm. In some embodiments, the pore size is 350 μm to 500 μm. In some embodiments, the pore size is 100 μm to 450 μm. In some embodiments, the pore size is 250 μm to 450 μm.

[0118] In some embodiments, the number of microcapillaries per square cm of the array is about 400, 500, 1000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 50,000, 100,000, 200,000, 300,000, 400,000, 500,000, 600, 000, 700,000, or 800,000. In some embodiments, the number of microcapillaries per square cm of the array varies between about 500 and 800,000. In some embodiments, the number of microcapillaries per square cm of the array varies between about 1000 and 700,000. In some embodiments, the number of microcapillaries per square cm of the array varies between about 2000 and 600,000. In some embodiments, the number of microcapillaries per square cm of the array varies between about 10,000 and 800,000. In some embodiments, the number of microcapillaries per square cm of the array varies between about 10,000 and 700,000. In some embodiments, the number of microcapillaries per square cm of the array varies between about 50,000 and 800,000. In some embodiments, the number of microcapillaries per square cm of the array varies between about 50,000 and 700,000. In some embodiments, the number of microcapillaries per square cm of the array varies between about 100,000 and 700,000. In some embodiments, the number of microcapillaries per square cm of the array varies between about 100,000 and 600,000. In some embodiments, the number of microcapillaries per square cm of the array varies between about 100,000 and 500,000. In some embodiments, the number of microcapillaries per square cm of the array varies between about 500,000 and 800,000.

[0119] In one particular embodiment, microcapillary arrays can be manufactured by joining billions of silica capillaries and then fusing them through a thermal process. Slices (0.5 mm or larger) are then cut to form glass microcapillary arrays with very high aspect ratios. Arrays are also commercially available from Hamamatsu Photonics KK (Japan), Incom, Inc. (Massachusetts), Photonis Technologies, SAS (France) Inc., etc. In some embodiments, the microcapillaries of the array are closed at one end with a solid substrate attached to the array.

[0120] The microcapillary array of the present screening methods can include any number of microcapillaries in the array, hi some embodiments, the microcapillary array includes at least 100,000, at least 300,000, at least 1,000,000, at least 3,000,000, at least 10,000,000, or even more microcapillaries. In some embodiments, the array comprises at least 100,000, at least 200,000, at least 300,000, at least 400,000, at least 500,000, at least 600,000, at least 700,000, at least 800,000, at least 1,000,000, at least 1,500,000, at least 2,000,000, at least 2,500,000, or at least 3,000,000 or more microcapillaries. The number of microcapillaries in the array is preferably selected taking into account the size of the mutant protein library to be screened.

[0121] As described above, each capillary in the microcapillary array used in the screening method comprises a mutant protein, an immobilized target molecule, and a reporter element, and the mutant protein is one of the mutant protein populations that are subjected to the screening method. The mutant protein population can be any protein population that can be appropriately distributed in the microcapillary array. Ideally, the mutant protein population is distributed in the microcapillary array so that each microcapillary contains a small number of different mutant proteins, preferably a single different mutant protein per microcapillary. What is important is that the mutant protein population is selected in combination with the immobilized target molecule such that at least some of the proteins in the population can associate with the immobilized target molecule with a specific affinity, such that the association can be detected by measuring the signal from the reporter element.

[0122] The term "protein" as used herein refers to both full-length proteins or polypeptide sequences, and fragments thereof. Such fragments may include fragments that retain functional activity, such as, for example, binding activity. The terms "protein" and "polypeptide" are used interchangeably throughout this disclosure and include a chain of amino acids covalently linked via peptide bonds, where each amino acid in a polypeptide may be referred to as an "amino acid residue." The use of the term "protein" or "polypeptide" should not be considered limited to any particular length of polypeptide, e.g., any particular number of amino acid residues. The subject proteins may include proteins having non-peptide modifications, such as post-translational modifications, including glycosylation, acetylation, phosphorylation, sulfation, and the like, or other chemical modifications, such as alkylation, acetylation, esterification, PEGylation, and the like. Further modifications, such as the inclusion of unnatural amino acids within the polypeptide sequence or non-peptide bonds between amino acid residues, should also be considered within the definition of the term "protein" or "polypeptide."

[0123] The population of mutant proteins is preferably a population of proteins with small mutations, for example, each protein has a slightly different amino acid sequence.Therefore, the screening assay can identify mutant protein sequences with desirable properties.The screening can be performed on a microscopic scale in very large quantities, so that a large number of mutant proteins can be assayed in a relatively short time.

[0124] The term "antibody" is used in the broadest sense, and includes, for example, intact immunoglobulins or antigen-binding portions. Antigen-binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Thus, the term antibody includes conventional tetrameric antibodies of two heavy chains and two light chains, as well as antigen-binding fragments such as Fv, Fab and scFv. In some cases, the present invention provides bispecific antibodies that include at least one antigen-binding domain, as outlined herein.

[0125] The mutant proteins and / or mutant polypeptides may include, but are not limited to, secreted proteins. In some embodiments, the secreted proteins are derived from a recombinant protein and / or polypeptide library. In some embodiments, the secreted proteins are derived from a recombinant protein and / or polypeptide library. In some embodiments, the secreted proteins are derived from a recombinant protein and / or polypeptide library from a mammalian cell line. In some embodiments, the recombinant protein and / or polypeptide library comprises full-length mammalian antibodies. In some embodiments, the recombinant protein and / or polypeptide library comprises full-length mammalian antibodies, including IgG1, IgG2, and IgG4 antibodies and variants thereof. In some embodiments, the recombinant protein and / or polypeptide library comprises full-length human antibodies. In some embodiments, the recombinant protein and / or polypeptide library comprises full-length human antibodies, including IgG1, IgG2, and IgG4 antibodies. In some embodiments, the recombinant protein and / or polypeptide library comprises full-length mouse antibodies. In some embodiments, the recombinant protein and / or polypeptide library comprises full-length mouse antibodies, including IgG1, IgG2, and IgG4 antibodies. In some embodiments, the recombinant protein and / or polypeptide library comprises full-length rat antibodies. In some embodiments, the recombinant protein and / or polypeptide library comprises full length rat antibodies, including IgG1, IgG2, and IgG4 antibodies. In some embodiments, the recombinant protein and / or polypeptide library comprises antibody fragments (Fab). In some embodiments, the recombinant protein and / or polypeptide library comprises single chain variable fragments (scFv). In some embodiments, the recombinant protein and / or polypeptide library comprises natural protein ligands. In some embodiments, the recombinant protein and / or polypeptide library comprises natural protein ligands for defined target proteins and / or polypeptides.In some embodiments, the recombinant protein and / or polypeptide library comprises a target antibody and / or fragment thereof. In some embodiments, the recombinant protein and / or polypeptide library comprises a target antibody heavy chain and / or fragment thereof, such as a variable heavy chain. In some embodiments, the recombinant protein and / or polypeptide library comprises a target antibody light chain and / or fragment thereof, such as a variable light chain. In some embodiments, the system of the present invention allows for precise pairing of VH / VL (variable heavy chain and variable light chain).

[0126] In some embodiments, each microcapillary in the microcapillary array contains 0-5 different mutant proteins from the population of mutant proteins. In certain embodiments, each microcapillary in the microcapillary array contains 0-4, 0-3, 0-2, or even 0-1 different mutant proteins from the population of mutant proteins. It is understood that the different mutant proteins in the population of mutant proteins differ in their molecular structure, whether or not there are differences in their amino acid sequence or any other chemical modification of the protein.

[0127] It should be understood that each microcapillary typically contains many multiple copies of the same mutant protein, depending on the origin and expression level of the particular mutant protein (see below). In some embodiments, each microcapillary contains thousands, tens of thousands, hundreds of thousands, millions, billions, or even more molecules of a particular mutant protein, depending on how the mutant protein is delivered to or expressed in the microcapillary. In some embodiments, the mutant protein can bind to one, two, three, or four or more target molecules. In some embodiments, the mutant protein can bind to one target molecule. In some embodiments, the mutant protein can bind to two target molecules. In some embodiments, the mutant protein can bind to three target molecules. In some embodiments, the mutant protein can bind to four target molecules. In some embodiments, the mutant protein can bind to more than four target molecules. In some embodiments, this assay can alternatively be used to screen for antibodies that bind to both the mouse and human (or other animal combination) variants of the target protein, i.e., to discover "cross-reactive" antibodies. For example, the presence of stained cells and the presence of stained beads in the microcapillary indicates the presence of an antibody that binds to the "target protein" (e.g., mouse target) and also binds to the "target protein analog" (e.g., human target) to identify antibodies that bind to both mouse and human targets. For example, the presence of stained cells and the presence of stained beads in the microcapillary indicates the presence of an antibody that binds to the "target protein" (e.g., cynomolgus target) and also binds to the "target protein analog" (e.g., human target) to identify antibodies that bind to both cynomolgus and human targets.

[0128] A population of mutant proteins is typically produced using a genetic library in a biological expression system, such as an in vitro (i.e., cell-free) expression system or an in vivo or cellular expression system. Exemplary cellular expression systems include, for example, an animal system (e.g., a mammalian system), a fungal system (e.g., a yeast system), a bacterial system, an insect system, or a plant system. In certain embodiments, the expression system is a mammalian system or a yeast system. In certain embodiments, the expression system is an avian system (e.g., a chicken system). An expression system, whether cellular or acellular, typically comprises a library of genetic material that encodes a population of mutant proteins. A cellular expression system offers the advantage of being able to grow and propagate cells with a desired phenotype, for example, cells expressing a particular mutant protein of interest, such as a mutant protein that can associate with an immobilized target molecule with high affinity, thus simplifying the identification and characterization of the protein of interest expressed by the cell. In some embodiments, the biological expression system comprises a mammalian cell line. In some embodiments, the mammalian cell line is selected from the group consisting of CHO-K1, CHO-S, HEK293T, and / or any derivative of these cell types. In some embodiments, the mammalian cell line is CHO-K1. In some embodiments, the mammalian cell line is CHO-S. In some embodiments, the mammalian cell line is HEK293T. In some embodiments, the mammalian cell line is selected from the group consisting of human, mouse, and / or rat hybridoma cell lines. In some embodiments, the mammalian cell line is a human hybridoma cell line. In some embodiments, the mammalian cell line is a mouse hybridoma cell line. In some embodiments, the mammalian cell line is a rat hybridoma cell line.

[0129] Gene libraries encoding large populations of mutant proteins are well known in the field of biotechnology. Such libraries are often utilized in systems that rely on the process of directed evolution to identify proteins with advantageous properties, such as high affinity binding to a target molecule, stability, high expression, or specific spectroscopic activity, e.g., fluorescence or enzymatic activity. Often, the library contains genetic fusions with sequences from the host expression system, e.g., fragments of proteins that direct subcellular localization, and the expression population of mutant fusion proteins is directed by the targeting fragment to a specific location in a cell or virus particle for activity screening of the mutant protein population. As is well known in the art, routine biotechnology techniques can be used to generate large numbers of mutant proteins (e.g., up to 10 6 Mutant, 10 8 Mutant, 10 10 Mutant, 10 12 A library of variants (variants, or even more variants) can be generated. Such libraries can include any of the variant proteins described herein, including antibodies, antibody fragments, single chain variable fragments, or natural protein ligands. In some embodiments, the system of the invention allows for precise pairing of VH / VL (variable heavy and variable light chains).

[0130] Thus, in some embodiments, the mutant protein is a soluble protein, e.g., a soluble protein secreted by a cell expression system. Exemplary soluble mutant proteins include antibodies and antibody fragments, alternative protein scaffolds such as disulfide-bonded peptide scaffolds, extracellular domains of cell surface receptor proteins, receptor ligands such as G protein-coupled receptor ligands, other peptide hormones, lectins, and the like. Advantageously, the mutant protein with the desired binding activity and the cell expressing it are co-localized in the same microcapillary throughout the assay, so that the mutant proteins screened for binding activity in the present method do not need to be covalently linked to the cell or virus expressing them in order to be identified after the screening assay. Isolation of the contents of the desired microcapillary, followed by growth of the cell or virus clone responsible for the expression of the desired mutant protein, thereby allows the protein to be identified and characterized. Unlike screening assays in which the mutant protein of interest is presented by fusion of the protein to a molecule on the surface of a cell or virus particle, the mutant proteins identified in the present screening method do not need to be modified in any way after their identification. Thus, the activities of the mutant proteins observed in the screen are more likely to represent the actual activities of those proteins in their subsequent applications.

[0131] However, in other embodiments, it may be desirable for the mutant protein to be a membrane-bound protein, for example, a protein that remains bound to the surface of a cell or a viral particle in an expression system. Screening for cell-bound mutant proteins may be desirable when the mutant protein and its target molecule mediate the interaction between two cells in a living tissue. The ability to screen for cell-bound mutant proteins may also be desirable when screening for interactions with traditionally "undruggable" protein targets, such as, for example, G-protein-coupled receptors or ion channels.

[0132] In addition to mutant proteins, each microcapillary in the microcapillary array of the screening method also contains an immobilized target molecule. The immobilized target molecule serves as a potential binding partner for the mutant protein in the screening assay. Unlike a population of mutant proteins, where each microcapillary ideally contains mutant proteins with slightly different sequences, the immobilized target molecule ideally has the same molecular structure in each microcapillary of the array. In some embodiments, there is no binding or other interaction between the mutant protein and another agent or molecule (e.g., a target molecule) before the addition of the mutant protein to the microcapillary. In some embodiments, the interaction between the mutant protein and the target molecule occurs within the microcapillary and / or microcavity.

[0133] In some embodiments, the target molecule is a target protein or polypeptide, a target nucleic acid, a target carbohydrate, a target lipid, or a combination of two or more of these target molecules. For example, in some embodiments, the target molecule can be a lipid-modified protein or a glycosylated protein. In some embodiments, the target molecule is immobilized on a surface. In more specific embodiments, the target molecule is immobilized on the surface of a cell, such as a target cell, on the surface of a bead, on the surface of a microcapillary wall, or on another suitable surface. In other more specific embodiments, the target molecule is a natural protein, such as a natural protein immobilized on the surface of a cell. In yet other more specific embodiments, the target molecule is immobilized on a surface configured to settle in a microcapillary by gravitational sedimentation. In some embodiments, one, two, three, four, or more target molecules are used to identify mutants that bind to one, two, three, four, or more target molecules. In some embodiments, the target molecules are separately contained in separate, different microcapillaries. In some embodiments, the target molecules are separately contained in separate, different microcapillaries within a single array. In some embodiments, the target molecules are contained separately in separate, distinct microcapillaries in one or more arrays. In some embodiments, the target molecules are contained together in a single microcapillary. In some embodiments, the target molecules are contained together in a single microcapillary in a single array. In some embodiments, the one, two, three, or four or more target molecules to which the variant binds are derivatives or variants of the original target molecule, including chemical modifications, secondary post-translational modifications, or sequence identity variants (e.g., variants having 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to the original nucleic acid or amino acid target sequence).

[0134] As mentioned above, in the disclosed method, the mutant protein associates with the immobilized target molecule with a specific affinity in the microcapillary. Importantly, such affinity must be strong enough for the mutant protein of interest such that the association can be measured by a signal from the reporter element. As will be appreciated by those skilled in the art, binding affinity is typically measured using a dissociation constant (K d ), with the lower the dissociation constant the higher the affinity. In some embodiments, the association of the mutant protein of interest with the immobilized target molecule exhibits a dissociation constant in the millimolar to micromolar range. In certain embodiments, the association is in the micromolar to high nanomolar (i.e., 10 -6 M~10 -8 In more specific embodiments, the association exhibits a dissociation constant of from low nanomolar to high picomolar (i.e., 10 -8 M~10 -10 In even more specific embodiments, the association is in the picomolar range (i.e., 10 -10 M~10 -12 M), or even lower dissociation constant. In some embodiments, the first cell expresses and secretes the mutant protein or polypeptide and the second cell contains the target, such that the first cell binds to the second cell. In some embodiments, the second cell expresses the target. In some embodiments, the second cell is labeled with the target. In some embodiments, the first cell binds to the second cell in a microcapillary. In some embodiments, the first cell binds to the second cell in a microcapillary and / or microcavity.

[0135] In addition to the mutant protein and the immobilized target molecule, each microcapillary in the microcapillary array of the present screening method also contains a reporter element. Importantly, the reporter element provides a measurable signal indicative of the association of the mutant protein with the immobilized target molecule, and thus serves to identify the microcapillary that contains the mutant protein of interest.

[0136] In some embodiments, the reporter element is a labeled antibody or other molecule capable of binding to each mutant protein in the population of mutant proteins. More specifically, the reporter element is a fluorescently labeled antibody or other binding molecule.

[0137] In some embodiments, the labeled antibody is a labeled primary antibody or a labeled secondary antibody. For the purposes of this disclosure, a primary antibody is typically considered to be an antibody that directly binds to an antigen of interest, and a secondary antibody is typically considered to be an antibody that binds to a constant region on a primary antibody for the purpose of labeling the primary antibody. Thus, secondary antibodies are frequently labeled with a fluorophore or other detectable label, or with an enzyme that can generate a detectable signal. They are generally specific to a primary antibody from a different species. For example, as will be understood by those skilled in the art, goats or other animal species can be used to make secondary antibodies against almost any primary antibody other than those from mice, chickens, rabbits, or that animal species. In certain embodiments, the labeled antibody is a fluorescent antibody or an enzyme-linked antibody.

[0138] In some of the embodiments of the present methods, for example in the screening methods shown in Figures 1A-1C, the mutant protein mediates the association of the reporter element with a target molecule, in this example a target molecule on the surface of a target cell. As shown in Figure 1B, if the mutant protein (referred to here as a "secreted protein") has sufficient affinity for its target molecule on the target cell, the mutant protein will associate with the target cell under the conditions of the microcapillary solution. The reporter element (referred to here as a "fluorescent detection antibody") ideally binds to the mutant protein at an epitope that does not affect the affinity of the mutant protein for the target molecule, as shown in Figure 1C.

[0139] As will be appreciated by those skilled in the art, when soluble reporter elements such as fluorescent antibodies are used in the present screening methods, the signal emitted by any excess reporter elements that remain free in solution in the microcapillary (i.e., not bound to mutant proteins or bound to mutant proteins that are not bound to target molecules) should not be so high as to overwhelm the signal of reporter elements associated with target molecules via mutant proteins (see, for example, the unassociated fluorescent detection antibody shown in FIG. 1C). However, such background signals can be minimized by limiting the concentration of labeled antibodies or other reporter elements in the microcapillary solution. Furthermore, when signals from the screening methods are measured using a fluorescent microscope, background signals from reporter elements not associated with target molecules can be minimized by configuring the microscope to image a relatively narrow depth of field surrounding the location of the target molecules (e.g., the bottom of the microcapillary if the target cells have settled there by gravity).

[0140] In other embodiments, the reporter element is an intracellular reporter element that generates a detectable signal in association with a binding event, such as, for example, the association of a mutant protein with an immobilized target molecule, such as a receptor or other molecular target on the surface of a cell. In these embodiments, the reporter element may include an entire cellular pathway, such as, for example, an intracellular signaling pathway. Such pathways should contain, or be engineered to contain, a detectable signal as a downstream readout of the pathway. In contrast to the assays shown in Figures 1A-1C, in which the detectable signal is bound to the outer surface of the target cell, the detectable signal in these embodiments will typically occur inside the target cell.

[0141] Many intracellular signaling pathways have been developed for use in high-throughput screening assays, particularly in drug discovery screening, and can be adapted for use in the present assay. See, for example, Michelini et al. (2010) Anal. Bioanal. Chem. 398:227-38. In particular, any cellular assay in which a binding event with a target molecule on the cell surface results in the generation of a measurable signal, particularly a fluorescent signal, can be used as a reporter element in the present assay. Preferably, cells can be engineered to express a target molecule of interest on their surface, such that binding of a particular mutant protein to the target molecule and the resulting activation of an intracellular signaling pathway results in the generation of a detectable signal from the reporter element, thus allowing the identification of the microcapillary as a positive hit. Expression of green fluorescent protein (GFP), or any of a wide variety of mutant fluorescent proteins, is often used as a readout in such cellular assays and can serve as the endpoint of the reporter element in the present method. Alternatively, as will be well understood by those skilled in the art, a readout of signaling can be provided by luciferase or other related enzymes that generate a bioluminescent signal. See, for example, Kelkar et al. (2012) Curr. Opin. Pharmacol. 12:592-600. Reporter elements can also include RFP (red fluorescent protein) and YFP (yellow fluorescent protein), as well as variants thereof. Other well-known enzyme reporters from bacterial and plant systems include β-galactosidase, chloramphenicol acetyltransferase, β-glucuronidase (GUS), etc., which can be adapted for use in the present screening assays using appropriate chromogenic substrates. Transcriptional reporters using firefly luciferase and GFP have been widely used to study the function and regulation of transcription factors. They can be similarly adapted for use in the present screening assays.Exemplary intracellular signaling systems are commercially available, for example, Qiagen's Cignal™ Reporter Assay kits, available with either luciferase or GFP readouts (see, for example, www.sabiosciences.com / reporterassays.php). Such systems can be appropriately re-engineered for use in the present screening methods.

[0142] It should be understood that the mutant protein expression system, particularly when the expression system is a cellular expression system, may be combined with immobilized target molecules and reporter elements (or suitable components, such as cellular components involved in the creation of the immobilized target molecules and / or reporter elements) prior to expression of the mutant protein and / or prior to delivery of the assay mixture to the array of microcapillaries. Such an approach advantageously allows flexibility and control in the timing of interactions between components, compared to prior art microcapillary screening systems in which all components of the screening assay are typically mixed in a static format and loaded into the microcapillary. In contrast, the present method allows some or all of the components of the binding assay to be created in situ within the microcapillary by allowing proliferation of cellular components, expression of genetic components, or both. In some embodiments, cell culture medium and / or growth agents are used to maintain the health of the cells during the assay process. In some embodiments, components that promote the metabolic health of the cells are included.

[0143] It should also be understood that the concentration of each component of the screening assay in the microcapillary, including the concentration of the mutant protein, the concentration of the immobilized target molecule, and the concentration of the reporter element, can be adjusted as desired in the assay to obtain optimal results. In particular, it may be desirable to adjust the concentration of the mutant protein and / or the immobilized target molecule to achieve a desired level of association between these components. The level of association also depends on the specific affinity between these components, with higher affinity resulting in a higher level of association for a given concentration of the components, and lower affinity resulting in a lower level of association of the components for a given concentration. As will be appreciated by those skilled in the art, the concentration of the reporter element can be similarly adjusted to achieve an optimal level of signal output. In some embodiments, the reporter element used includes a secondary antibody, including those commercially available. In some embodiments, the dilution range is 1:200 to 1:2000. In some embodiments, the dilution range is 1:300 to 1:2000. In some embodiments, the dilution range is 1:300 to 1:1500. In some embodiments, the dilution range is 1:400 to 1:1500. In some embodiments, the dilution range is 1:500 to 1:1500. In some embodiments, the dilution range is 1:200 to 1:1000. In some embodiments, the dilution range is 1:500 to 1:1000. In some embodiments, the dilution range is 1:1000 to 1:2000. In some embodiments, the dilution range is 1:1500 to 1:2000. In some embodiments, the dilution is 1:200, 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, 1:1000, 1:1500, or 1:2000. In some embodiments, the fluorophore is AlexaFluor 3, AlexaFluor 5, AlexaFluor 350, AlexaFluor 405, AlexaFluor 430, AlexaFluor 488, AlexaFluor 500, AlexaFluor 514, AlexaFluor 532, AlexaFluor 546, AlexaFluor 555, AlexaFluor 568, AlexaFluor594, AlexaFluor 610, AlexaFluor 633, AlexaFluor 647, AlexaFluor 660, AlexaFluor 680, AlexaFluor 700, and AlexaFluor 750 (Molecular Probes AlexaFluor dyes available from Life Technologies, Inc. (USA)). In some embodiments, the fluorophore may include, but is not limited to, a Cy dye, including, but not limited to, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, and Cy7 (available from GE Life Sciences or Lumiprobes). In some embodiments, fluorophores may include, but are not limited to, DyLight 350, DyLight 405, Dy Light 488, DyLight 550, DyLight 594, DyLight 633, DyLight 650, DyLight 680, DyLight 750 and DyLight 800 (available from Thermo Scientific (USA)). In some embodiments, the fluorophore is selected from the group consisting of FluoProbes 390, FluoProbes 488, FluoProbes 532, FluoProbes 547H, FluoProbes 594, FluoProbes 647H, FluoProbes 682, FluoProbes 752 and FluoProbes 782, AMCA, DEAC (7-diethylaminocoumarin-3-carboxylic acid); 7-hydroxy-4-methylcoumarin-3; 7-hydroxycoumarin-3; MCA (7-methoxycoumarin-4-acetic acid); 7-methoxycoumarin-3; AMF (4'-(aminomethyl)fluorescein); 5-DTAF (5-(4,6-dichlorotriazinyl)aminofluorescein); 6-DTAF (6-(4,6-dichlorotriazinyl)aminofluorescein); 6-FAM. (6-carboxyfluorescein), 5(6)-FAM cadaverine; 5-FAM cadaverine; 5(6)-FAM ethylenediamine; 5-FAM ethylenediamine; 5-FITC (FITC isomer I;Fluorescein-5-isothiocyanate; 5-FITC cadaverine; Fluorescein-5-maleimide; 5-IAF (5-iodoacetamidofluorescein); 6-JOE (6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein); 5-CRl l0 (5-carboxyrhodamine 110); 6-CRl l0 (6-carboxyrhodamine 110); 5-CR6G (5-carboxyrhodamine 6G); 6-CR6G (6-carboxyrhodamine 6G); 5(6)-Carboxyrhodamine 6G cadaverine; 5(6)-Carboxyrhodamine 6G ethylenediamine; 5-ROX (5-carboxy-X-rhodamine); 6-ROX (6-carboxy-X-rhodamine); 5-TAMRA (5-carboxytetramethylrhodamine); 6-TAMRA (6-carboxytetramethylrhodamine); 5-TAMRA cadaverine; 6-TAMRA cadaverine; 5-TAMRA ethylenediamine; 6-TAMRA ethylenediamine; 5-TMR C6 maleimide; 6-TMR C6 maleimide; TR C2 maleimide; TR cadaverine; 5-TRITC; G isomer (tetramethylrhodamine-5-isothiocyanate); 6-TRITC; R isomer (tetramethylrhodamine-6-isothiocyanate); dansylcadaverine (5-dimethylaminonaphthalene-l-(N-(5-aminopentyl))sulfonamide); EDANS C2 maleimide; fluorescamine; NBD; and pyrromethene, and derivatives thereof, but are not limited thereto. In some embodiments, the reporter element used may be a donkey anti-goat IgG secondary antibody labeled with AlexaFluor 633.

[0144] In some embodiments, each microcapillary in the microcapillary array of the screening method further comprises an agent for improving the viability of the cell expression system. Specifically, the agent is included to prevent cell damage during the step of isolating the contents of the microcapillary of interest, for example, by a laser pulse (see below). In preferred embodiments, the agent is methylcellulose (e.g., 0.001-10% by weight), dextran (e.g., 0.5-10% by weight), Pluronic® F-68 (e.g., 0.01-10% by weight), polyethylene glycol ("PEG") (e.g., 0.01-10% by weight), polyvinyl alcohol ("PVA") (e.g., 0.01-10% by weight), or the like. Alternatively, or in addition, each microcapillary in the microcapillary array of the screening method may further comprise a growth additive, such as, for example, 50% conditioned growth medium, 25% standard growth medium, or 25% serum. In some embodiments, the conditioned growth medium is conditioned for 24 hours. In some embodiments, the added agent is insulin, transferrin, ethanolamine, selenium, insulin-like growth factor, or a combination of these agents, or a combination of any of the above agents.

[0145] The screening method of the present disclosure further comprises measuring the signal from at least one reporter element that indicates the association of at least one mutant protein with at least one immobilized target molecule to identify at least one microcapillary of interest.In some embodiments, the signal that is measured is a fluorescent signal, an absorbance signal, a bright-field signal, or a dark-field signal, a phase contrast signal, etc.Therefore, the measuring step can be carried out by a suitable detector device, for example, a device that can detect electromagnetic radiation or any other suitable signal.In certain embodiments, the measuring step is carried out by a microscope, such as a fluorescent microscope or other microscope that is configured to detect the above-mentioned signal.

[0146] It should be understood that in a preferred embodiment, the microcapillaries utilized in the present screening method do not contain microparticles capable of suppressing the transmission of electromagnetic radiation. In other words, the microcapillaries are preferably completely transparent to the electromagnetic radiation incident on the microcapillary array, particularly along the longitudinal axis of the microcapillary. In another preferred embodiment, the microcapillaries of the present screening method do not contain magnetic microparticles or beads. In yet another preferred embodiment, the microcapillaries of the present screening method do not contain microparticles, magnetic microparticles, or magnetic beads capable of suppressing the transmission of electromagnetic radiation.

[0147] In other preferred embodiments, the microcapillaries utilized in the present screening methods do not contain electromagnetic radiation absorbing materials, however, it should be understood that components of reporter elements involved in generating a measurable signal in the screening methods, such as fluorophores on fluorescent antibodies, are not to be considered electromagnetic radiation absorbing materials for purposes of this aspect of the invention.

[0148] In some embodiments, the screening method further comprises isolating the contents of the microcapillary of interest. In certain embodiments, the contents of the microcapillary of interest are isolated by pulsing the microcapillary of interest with a laser. More specifically, the laser can be a diode laser or a diode-pumped Q-switched Nd:YLF laser. In some embodiments, the laser can be directed at the water-glass interface between the microcapillary wall and the sample contained in the microcapillary. Without intending to be bound by theory, it is believed that firing a UV laser at this interface can break the meniscus / water surface tension that normally holds the sample in the microcapillary, thus allowing the sample to fall from the array by the force of gravity. In other embodiments, the contents of the microcapillary of interest are isolated by laser-induced expansion by steam force. In some embodiments, the contents of the microcapillary are isolated by breaking the glass of the microcapillary itself.

[0149] In some embodiments, the microcapillary screening method of the present invention allows for screening of reactions and / or interactions (including binding interactions) between mutant proteins and target molecules that occur within minutes of adding the components to the microcapillary. In some embodiments, the reaction and / or interaction between the mutant protein and the target molecule occurs and / or is detectable within about 1 minute to about 10 minutes. In some embodiments, the reaction and / or interaction between the mutant protein and the target molecule occurs and / or is detectable within about 1 hour to about 6 hours. In some embodiments, the reaction and / or interaction occurs within about 1 hour, about 2 hours, about 4 hours, about 6, about 10 hours, about 12 hours, about 16 hours, about 24 hours, about 36 hours, or about 48 hours. hours minutes to about 10 minutes. In some embodiments, the reaction and / or interaction between the mutant protein and the target molecule occurs and / or is detectable within a time period such that the cells in the microcapillary are viable and healthy. In some embodiments, the reaction and / or interaction between the mutant protein and the target molecule occurs and / or is detectable within a period of time such that the cells in the microcapillary are viable. In some embodiments, the cells can be grown after removal from the microcapillary and / or microcavity. In some embodiments, the cells are viable after removal from the microcapillary and / or microcavity. In some embodiments, the reaction and / or interaction between the mutant protein and the target molecule occurs within the microcapillary.

[0150] Systems for screening According to another aspect of the present invention, there is provided a system for screening a population of mutant proteins comprising: An array comprising a plurality of microcapillaries, each microcapillary comprising a mutant protein, an immobilized target molecule, and a reporter element, wherein the mutant protein associates with the immobilized target molecule with a specific affinity. These components of the screening device are described in detail above, and any of them can be incorporated into the system for screening.

[0151] In some embodiments, the screening system further comprises a light source and a detector. The light source and the detector are selected according to the specific reporter element used in the screening system. For example, if the reporter element generates a fluorescent signal, the light source provides an excitation light of a suitable wavelength to excite the fluorescent probe. Similarly, the detector is selected to be sensitive to the wavelength of light emitted by the fluorescent probe. As will be understood by those skilled in the art, the light source and the detector can be components of a microscope, such as a fluorescent microscope, or they can be separate devices.

[0152] In some embodiments, the screening system further comprises an extraction device, such as a diode laser, a diode-pumped Q-switched laser, or other suitable components for isolating the contents of the microcapillary of interest.

[0153] An exemplary microscope for screening a population of mutant proteins according to the present methods is shown in the drawings in Figures 6A-6E. Figure 6A is a top perspective view of the microscope, showing both the array holding stage and the sample collection stage. Figure 6B shows a front view of the device. Figure 6C shows a view from the right side. A close-up view of the right side of the device is provided in Figure 6D, detailing the relationship between the array holding stage and the sample collection stage. Figure 6E provides an exploded view of the various components of a multi-stage sample collection microscope suitable for use in the present screening system.

[0154] It will be readily apparent to those skilled in the relevant art that other suitable modifications and adaptations may be made to the methods and applications described herein without departing from the scope of the present invention or any of its aspects. Having described the present invention in detail, the present invention will be more clearly understood by reference to the following examples, which are not intended to limit the present invention but are for illustrative purposes only. In some embodiments, any aspect disclosed in the method section above can also be readily applied to the system of the present invention. The system of the present invention can be used with any of the methods described herein.

[0155] Microcavity Array In these methods, the microcavity array includes any array that includes individual chambers and allows light to pass through the array to a detector. In some embodiments, the array is a microcapillary array. In some embodiments, the microcapillary array includes a plurality of longitudinally fused capillaries, such as fused silica capillaries, although any other suitable material may be used in the array. See, for example, the arrays described in U.S. Patent Application No. 62 / 433,210, filed December 12, 2016, U.S. Patent Application No. 15 / 376,588, filed December 12, 2016, PCT International Patent Publication Nos. WO 2012 / 007537 and WO 2014 / 008056, the disclosures of which are all incorporated herein by reference in their entirety.

[0156] Such arrays can be fabricated, for example, by bundling millions or billions of silica capillaries and fusing them through a thermal process, although other suitable fabrication methods can be used. The fusing process can include, for example, i) heating capillary single-draw glass drawn into a single-clad fiber under tension, ii) creating capillary multi-draw single capillaries from the single-draw glass by bundling, heating, and drawing, iii) creating multi-draw multi-capillaries from the multi-draw single capillaries by additional bundling, heating, and drawing, iv) creating a block assembly of drawn glass from the multi-multi-draw multi-capillaries by stacking them into a pressing block, v) creating a block pressed block from the block assembly by treating with heat and pressure, and vi) creating block-forming blocks by cutting the block pressed block at precise lengths (e.g., 1 mm).

[0157] In some embodiments, the method further comprises slicing the silica capillary, thereby forming a very high density glass microcapillary array. In some embodiments, the microcapillary array may be cut to a height of about 1 millimeter, although shorter microcapillary arrays are also contemplated, including arrays with a height of 10 μm or less. In some embodiments, longer microcapillary arrays are contemplated, including arrays with a height of 10 mm or more.

[0158] Such a process forms a very high density microcapillary array suitable for use in the present method. In an exemplary array, each microcapillary has a diameter of about 5 μm and about 66% open space (i.e., representing the lumen of each microcapillary). In some arrays, the percentage of the array that is open ranges from about 50% to about 90%, for example, about 60% to about 75%, such as the microcapillary array provided by Hamamatsu, which has an open area of ​​about 67%. In one particular example, a 10×10 cm array with 5 μm diameter microcapillaries and about 66% open space has about 330 million microcapillaries.

[0159] In various embodiments, the inner diameter of each microcapillary in the array ranges from about 1 μm to 500 μm. In some arrays, each microcapillary can have an inner diameter ranging from about 1 μm to 300 μm, optionally from about 1 μm to 100 μm, further optionally from about 1 μm to 75 μm, further optionally from about 1 μm to 50 μm, and further optionally from about 5 μm to 50 μm.

[0160] In some microcapillary arrays, the open area of ​​the array represents up to 90% of the open area (OA), so the number of microcapillaries per cm of array varies from about 4.6 to over 11 million when pore diameters vary from 1 μm to 500 μm. In some microcapillary arrays, the open area of ​​the array represents about 67% of the open area, so the number of microcapillaries per cm of array varies from about 3.4 to over 800,000 when pore diameters vary from 1 μm to 500 μm. In some embodiments, the number of microcapillaries per square cm of the array is about 400, 800, 1000, 2000, 4000, 5000, 10,0000, 25,000, 50,000, 75,000, 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1,000,000 or more.

[0161] In one particular embodiment, microcapillary arrays can be manufactured by joining billions of silica capillaries and then fusing them through a thermal process. Slices (0.5 mm or larger) are then cut to form glass microcapillary arrays with very high aspect ratios. Arrays are also commercially available from Hamamatsu Photonics KK (Japan), Incom, Inc. (Massachusetts), Photonis Technologies, SAS (France) Inc., etc. In some embodiments, the microcapillaries of the array are closed at one end with a solid substrate attached to the array.

[0162] The microcapillary array of the present screening method can include any number of microcapillaries in the array. In some embodiments, the microcapillary array includes at least 100,000, at least 300,000, at least 1,000,000, at least 3,000,000, at least 10,000,000, or even more microcapillaries. In some embodiments, the microcapillary array includes at least 100,000. In some embodiments, the microcapillary array includes at least 200,000 microcapillaries. In some embodiments, the microcapillary array includes at least 300,000 microcapillaries. In some embodiments, the microcapillary array includes at least 400,000 microcapillaries. In some embodiments, the microcapillary array includes at least 500,000 microcapillaries. In some embodiments, the microcapillary array includes at least 600,000 microcapillaries. In some embodiments, the microcapillary array comprises at least 700,000 microcapillaries. In some embodiments, the microcapillary array comprises at least 800,000 microcapillaries. In some embodiments, the microcapillary array comprises at least 900,000 microcapillaries. In some embodiments, the microcapillary array comprises at least 1,000,000 microcapillaries. In some embodiments, the microcapillary array comprises at least 2,000,000 microcapillaries. In some embodiments, the microcapillary array comprises at least 3,000,000 microcapillaries. In some embodiments, the microcapillary array comprises at least 4,000,000 microcapillaries. In some embodiments, the microcapillary array comprises at least 5,000,000 microcapillaries.In some embodiments, the microcapillary array comprises at least 10,000,000 microcapillaries. In some embodiments, the microcapillary array comprises at least 15,000,000 microcapillaries. In some embodiments, the microcapillary array comprises at least 20,000,000 microcapillaries. The number of microcapillaries in the array is preferably selected taking into account the size of the mutant protein library to be screened.

[0163] The microcavity array is about 0.2 mm (200 μm) to about 1 mm thick and about 50 μm to about 200 μm in diameter. In some embodiments, the microcavity array is about 1.5 mm thick and about 150 μm in diameter. In some embodiments, the microcavity array is about 2 mm thick and about 200 μm in diameter. In some embodiments, the microcavity array is about 1 mm thick and about 100 μm in diameter. In some embodiments, the microcavity array is about 1 mm thick and about 10 μm in diameter. In some embodiments, the microcavity array is about 1 μm, 5 μm, and / or 10 μm in diameter. In some embodiments, the microcavity array is about 10 μm in diameter.

[0164] A variety of microcavity arrays may find use in the methods of the invention. Exemplary microcavity array sizes are provided herein. In some embodiments, the microcavities in the array are about 50 μm to about 200 μm in diameter. In some embodiments, the microcavities in the array are about 75 μm to about 150 μm in diameter. In some embodiments, the microcavities in the array are about 75 μm to about 125 μm in diameter. In some embodiments, the microcavities in the array are about 75 μm to about 110 μm in diameter. In some embodiments, the microcavities in the array are about 80 μm to about 110 μm in diameter. In some embodiments, the microcavities in the array are about 75 μm to about 150 μm in diameter. In some embodiments, the microcavities in the array are about 80 μm, about 90 μm, about 100, or about 110 μm in diameter. In some embodiments, the microcavities in the array are about 100 μm in diameter.

[0165] A variety of microcavity arrays may find use in the methods of the invention. Exemplary sample volumes are provided herein. In some embodiments, the sample volume in each microcavity is less than about 500 nL. In some embodiments, the sample volume in each microcavity is about 5 nL to about 500 nL. In some embodiments, the volume in each microcavity is about 5 nL to about 400 nL. In some embodiments, the volume in each microcavity is about 5 nL to about 300 nL. In some embodiments, the volume in each microcavity is about 5 nL to about 200 nL. In some embodiments, the volume in each microcavity is about 5 nL to about 100 nL. In some embodiments, the volume in each microcavity is about 5 nL to about 90 nL. In some embodiments, the volume in each microcavity is about 5 nL to about 80 nL. In some embodiments, the volume in each microcavity is about 5 nL to about 70 nL. In some embodiments, the volume in each microcavity is about 5 nL to about 60 nL. In some embodiments, the volume in each microcavity is about 5 nL to about 50 nL. In some embodiments, the volume in each microcavity is about 5 nL to about 40 nL. In some embodiments, the volume in each microcavity is about 5 nL to about 30 nL. In some embodiments, the volume in each microcavity is about 5 nL to about 20 nL. In some embodiments, the volume in each microcavity is about 5 nL to about 10 nL. In some embodiments, the volume in each microcavity is about 5 nL to about 8 nL. In some embodiments, the volume in each microcavity is about 7 nL to about 8 nL. In some embodiments, the volume in each microcavity is about 7.8 nL. In some embodiments, the volume in each microcavity is about 70 pL to about 100 pL. In some embodiments, the volume in each microcavity is about 70 pL to about 90 pL. In some embodiments, the volume in each microcavity is about 70 pL to about 80 pL.In some embodiments, the volume in each microcavity is about 78.5 pL. In some embodiments, the volume in each microcavity is about 150 fL to about 1000 fL. In some embodiments, the volume in each microcavity is about 200 fL to about 1000 fL. In some embodiments, the volume in each microcavity is about 300 fL to about 1000 fL. In some embodiments, the volume in each microcavity is about 400 fL to about 900 fL. In some embodiments, the volume in each microcavity is about 500 fL to about 800 fL. In some embodiments, the volume in each microcavity is about 150 fL to 200 fL. In some embodiments, the volume in each microcavity is about 157 fL.

[0166] In some embodiments, each microcavity in the microcapillary array of the present screening method further comprises an agent for improving the viability of the cell expression system when a cell expression assay is used. Specifically, the agent is included to prevent cell damage during the step of isolating the contents of the microcapillary of interest, for example, by a laser pulse (see below). In preferred embodiments, the agent is methylcellulose (e.g., 0.001% to 10% by weight), dextran (e.g., 0.5% to 10% by weight), Pluronic® F-68 (e.g., 0.01% to 10% by weight), polyethylene glycol ("PEG") (e.g., 0.01% to 10% by weight), polyvinyl alcohol ("PVA") (e.g., 0.01% to 10% by weight), or the like. Alternatively, or in addition, each microcapillary in the microcapillary array of the present screening method may further comprise a growth additive, such as, for example, 50% conditioned growth medium, 25% standard growth medium, or 25% serum. In some embodiments, the conditioned growth medium is conditioned for 24 hours. In some embodiments, the agent added is insulin, transferrin, ethanolamine, selenium, insulin-like growth factor, or a combination of these agents, or any combination of the above agents. It should also be understood that the concentration of each component of the screening assay in the microcavity can be adjusted as desired in the assay to achieve optimal results. In particular, it may be desirable to adjust the concentration of proteins, polypeptides, nucleic acids, small molecules, and / or cells to achieve a desired level of association between these components. The level of association also depends on the specific affinity between these components, with higher affinity resulting in a higher level of association for a given concentration of the components, and lower affinity resulting in a lower level of association of the components for a given concentration. As will be understood by those skilled in the art, the concentrations of various components can be adjusted in a similar manner to achieve an optimal level of signal output.

[0167] Sample and / or library components Libraries that can be screened by the methods of the present invention include any library that includes a plurality of molecules and mixtures and / or combinations thereof. In some embodiments, the library includes a sample that includes a biological material. In some embodiments, the library includes a sample that includes a plurality of one or more molecules and / or cells and mixtures and / or combinations thereof. In some embodiments, the library includes a sample that includes a plurality of one or more proteins (including antibodies), polypeptides, nucleic acids, small molecules, dyes, and / or cells and mixtures and / or combinations thereof. In some embodiments, the molecule includes any molecule. In some embodiments, the molecule includes, but is not limited to, proteins, polypeptides, nucleic acids, small molecules, and / or dyes and mixtures and / or combinations thereof. In some embodiments, the library includes a sample that includes a biological material that includes polypeptides, nucleic acids, small molecules, and / or cells and mixtures and / or combinations thereof. In some embodiments, the library includes a sample. In some embodiments, the sample includes, but is not limited to, biological material that includes polypeptides, nucleic acids, small molecules, dyes, and / or cells and mixtures and / or combinations thereof. In some embodiments, the sample includes at least one molecule and / or cell to be screened. In some embodiments, the sample comprises at least 1 to 10 molecules and / or cells to be screened, as well as mixtures and / or combinations thereof. In some embodiments, the sample comprises a plurality of molecules and / or cells to be screened, as well as mixtures and / or combinations thereof. In some embodiments, the molecules to be screened are referred to as target molecules. In some embodiments, the cells to be screened are referred to as target cells.

[0168] The arrays provided herein allow for the screening of libraries composed of proteins, polypeptides, nucleic acids, small molecules, dyes, and / or cells, as well as mixtures and / or combinations thereof. In some embodiments, the target molecules to be screened are proteins, polypeptides, nucleic acids, small molecules, dyes, carbohydrates, lipids, or combinations of two or more of these target molecules. In some embodiments, the proteins and / or polypeptides are selected from the group consisting of enzymes, ligands, and receptors. For example, in some embodiments, the target molecules can be lipid-modified or glycosylated proteins. In some embodiments, the target molecules are natural proteins.

[0169] As described above, each capillary in the microcavity array used in the present screening method contains a different sample component. Such sample components may include, but are not limited to, proteins, polypeptides, nucleic acids, small molecules, dyes, and / or cells (i.e., target molecules and / or target cells), as well as mixtures and / or combinations thereof. In some embodiments, the library for screening contains mutant proteins, mutant polypeptides, mutant nucleic acids, mutant small molecules, mutant dyes, and / or mutant cells that exhibit distinctive characteristics. In some embodiments, the mutant proteins, mutant polypeptides, mutant nucleic acids, mutant small molecules, mutant dyes, and / or mutant cells exhibit distinctive characteristics such that each microcavity contains a sample that contains a target molecule and / or target cell that is different from the sample found in each of the other microcavities in the array. In some embodiments, one or more microcavities in the array contain a sample that contains the same target molecule and / or target cell (e.g., in duplicate for comparison) as the sample found in at least one other microcavity in the array.

[0170] In some embodiments, the proteins and / or polypeptides in the library screened in the microcavity array can be mutant proteins and / or polypeptides. Mutant proteins include proteins and polypeptides that can be distinguished from each other based on at least one feature or characteristic. In some embodiments, the mutant proteins and / or polypeptides exhibit different amino acid sequences, exhibit different amino acid sequence lengths, are produced / created by different methods, exhibit different activities, exhibit different chemical modifications, and / or exhibit different post-translational modifications. In some embodiments, the mutant proteins and / or polypeptides exhibit different amino acid sequences. In some embodiments, the mutant proteins and / or polypeptides exhibit different amino acid sequence lengths. In some embodiments, the mutant proteins and / or polypeptides are produced / created by different methods. In some embodiments, the mutant proteins and / or polypeptides exhibit different activities. In some embodiments, the mutant proteins and / or polypeptides exhibit different chemical modifications. In some embodiments, the mutant proteins and / or polypeptides exhibit different post-translational modifications. In some embodiments, the mutant proteins are one of a population of mutant proteins and / or polypeptides that have been subjected to screening methods and analyzed using the microcavity array disclosed herein. The population of variant proteins and / or polypeptides can be any population of proteins that can be suitably distributed within a microcapillary array.

[0171] In some embodiments, the nucleic acids in the library screened in the microcavity array can be mutant nucleic acids. The mutant nucleic acids include nucleic acids that can be distinguished from each other based on at least one feature or characteristic. In some embodiments, the mutant nucleic acids have different nucleotide sequences, have different nucleotide sequence lengths, are produced / created by different methods, have different methylation patterns, have different chemical modifications, and / or exhibit other distinctive modifications. In some embodiments, the mutant nucleic acids have different nucleotide sequences. In some embodiments, the mutant nucleic acids have different nucleotide sequence lengths. In some embodiments, the mutant nucleic acids are produced / created by different methods. In some embodiments, the mutant nucleic acids have different methylation patterns. In some embodiments, the mutant nucleic acids have different chemical modifications. In some embodiments, the mutant nucleic acids exhibit other distinctive modifications. In some embodiments, the nucleic acid is one of a population of mutant nucleic acids that has been subjected to a screening method and analyzed using the microcavity array disclosed herein. The population of mutant nucleic acids can be any nucleic acid population that can be appropriately distributed in a microcapillary array.

[0172] In some embodiments, the small molecules in the library screened in the microcavity array can be mutant and / or different small molecules. Mutant small molecules include small molecules that can be distinguished from each other based on at least one feature or characteristic. In some embodiments, mutant small molecules have different structures, are produced / created by different methods, have different chemical modifications, and / or exhibit other distinctively different properties. In some embodiments, mutant small molecules have different structures. In some embodiments, mutant small molecules are produced / created by different methods. In some embodiments, mutant small molecules have different chemical modifications. In some embodiments, mutant small molecules exhibit other distinctively different properties. In some embodiments, the small molecules are derivatives of each other. In some embodiments, the small molecule is one of a population of small molecules that is subjected to a screening method and analyzed using the microcavity array disclosed herein. The population of small molecules can be any small molecule population that can be appropriately distributed in a microcapillary array.

[0173] In some embodiments, the cells in the library screened in the microcavity array can be mutant cells and / or cells of various types. Mutant cells include cells that are distinguishable from one another based on at least one feature or characteristic. In some embodiments, the cells are from different samples, from different patients, from different diseases, have different chemical modifications, and / or are genetically modified. The cells can include eukaryotic and prokaryotic cells. In some embodiments, the cells are from different samples. In some embodiments, the cells are from different patients. In some embodiments, the cells are from different diseases. In some embodiments, the cells have different chemical modifications. In some embodiments, the cells are genetically modified. In some embodiments, the cells can include human cells, mammalian cells, bacterial cells, avian cells, including chicken cells, and fungal cells, including yeast cells. In some embodiments, the cells can include human cells. In some embodiments, the cells can include mammalian cells. In some embodiments, the cells can include avian cells. In some embodiments, the cells can include chicken cells. In some embodiments, the cells can include bacterial cells. In some embodiments, the cells can include fungal cells. In some embodiments, the cell can comprise yeast cells. In some embodiments, the cell can comprise chicken cells. In some embodiments, the cell is one of the cell populations that are subjected to screening method and analyzed using the microcavity array disclosed herein. The cell population can be any cell population that can be appropriately distributed in the microcapillary array.

[0174] In some embodiments, the population of proteins, polypeptides, nucleic acids, and / or cells are distributed in a microcavity array such that each microcavity contains a small number of different mutant proteins, mutant polypeptides, mutant nucleic acids, and / or cells. In some embodiments, each microcavity contains a single different mutant protein, mutant polypeptide, mutant nucleic acid, and / or cell per microcavity. In some embodiments, each microcavity contains a single different mutant protein per microcavity. In some embodiments, each microcavity contains a single different mutant polypeptide per microcavity. In some embodiments, each microcavity contains a single different mutant nucleic acid per microcavity. In some embodiments, each microcavity contains a single different cell per microcavity. The population of mutant proteins, mutant polypeptides, mutant nucleic acids, and / or cells are selected in combination with other components in the composition.

[0175] In some embodiments, each microcavity in the microcavity array contains 0-5 different mutant proteins, mutant polypeptides, mutant nucleic acids, and / or cells from a population of mutant proteins. In some embodiments, each microcavity in the microcavity array contains 0-4, 0-3, 0-2, or even 0-1 different mutant proteins from a population of mutant proteins, mutant polypeptides, mutant nucleic acids, and / or cells. Thus, in some embodiments, the mutant proteins are soluble proteins, e.g., soluble proteins secreted by a cellular expression system. Exemplary soluble mutant proteins include antibodies and antibody fragments, alternative protein scaffolds such as disulfide-bonded peptide scaffolds, extracellular domains of cell surface receptor proteins, receptor ligands such as G protein-coupled receptor ligands, other peptide hormones, lectins, and the like. In some embodiments, mutant proteins screened using the present methods do not need to be covalently attached to the cells or viruses expressing them to be identified following a screening assay. Isolation of the desired microcapillary contents, followed by growth of the cell or virus clone responsible for the expression of the desired mutant protein, thereby allows the identification and characterization of the mutant protein. Unlike screening assays in which the mutant protein of interest is presented by fusion of the protein to a molecule on the surface of a cell or virus particle, the mutant proteins identified in this screening method do not need to be modified at all before or after their identification. Therefore, the activity of the mutant proteins observed in screening is more likely to represent the actual activity of the proteins in their subsequent applications. Not needing to modify mutant proteins or polypeptides before screening also allows for more efficient screening, saving the cost and time of library preparation.

[0176] In some embodiments, the mutant protein to be screened is a membrane-bound protein, for example, a protein that typically binds to the surface of a cell or a viral particle in an expression system. Screening for cell-bound mutant proteins may be desirable when the mutant protein and its target molecule mediate the interaction between two cells in a living tissue. The ability to screen for cell-bound mutant proteins may also be desirable when screening for interactions with traditionally "undruggable" protein targets, such as, for example, G-protein-coupled receptors or ion channels. Again, not needing to modify mutant proteins or polypeptides prior to screening also allows for more efficient screening, saving costs and time for library preparation.

[0177] In some embodiments, the mutant nucleic acids to be screened include any nucleic acid or polynucleotide, including those that bind to or interact with proteins. Again, not having to modify the nucleic acid or polynucleotide prior to screening can also allow for more efficient screening, saving costs and time for library preparation.

[0178] In some embodiments, the protein to be screened is an antibody, an antibody fragment such as an Fc, or an antibody fusion, including, for example, an Fc fusion. In some embodiments, the antibody or antibody fragment can be labeled.

[0179] In some embodiments, the method employs the use of an antibody to bind to the target molecule being screened. In some embodiments, the antibody is a labeled primary antibody or a labeled secondary antibody that is used to bind to the target molecule. A primary antibody is usually considered to be an antibody that directly binds to the antigen of interest, while a secondary antibody is usually considered to be an antibody that binds to the constant region of the primary antibody in order to label the primary antibody. Thus, secondary antibodies are often labeled with a fluorophore or other detectable label, or with an enzyme that can generate a detectable signal. They are generally specific to a primary antibody from a different species. As will be appreciated by those skilled in the art, for example, goats or other animal species can be used to create secondary antibodies for almost any primary antibody other than those from mice, chickens, rabbits, or that animal species. In some embodiments, the labeled antibody is a primary or secondary antibody. In some embodiments, the labeled antibody is a fluorescent antibody or an enzyme-linked antibody.

[0180] As will be appreciated by those skilled in the art, for example, when fluorescent antibodies are used in the present screening methods, the signal emitted by any excess reporter elements that remain free in solution in the microcavity (i.e., not bound to mutant proteins or bound to mutant proteins that are not bound to target molecules) should not be so high as to overwhelm the signal of reporter elements associated with target molecules via mutant proteins (see, e.g., unassociated fluorescent antibodies). However, such background signals can be minimized by limiting the concentration of labeled antibodies or other reporter elements in the microcapillary solution. Furthermore, when signals from the screening methods are measured using a fluorescent microscope, background signals from reporter elements that are not associated with target molecules can be minimized by configuring the microscope to image a relatively narrow depth of field that surrounds the location of the target molecules (e.g., the bottom of the microcapillary if the target cells have settled there by sedimentation gravity).

[0181] In some embodiments, the reporter element is part of a reporter assay. In some embodiments, the reporter assay may include, but is not limited to, a calcium dye assay, a T cell activation assay, a B cell assay, and a GFP assay. In some embodiments, the reporter assay is selected from the group consisting of a calcium dye assay, a T cell activation assay, a B cell assay, and a GFP assay. In some embodiments, the reporter assay is a calcium dye assay. In some embodiments, the reporter assay is a T cell activation assay. In some embodiments, the reporter assay is a B cell assay. In some embodiments, the reporter assay is a GFP assay.

[0182] In some embodiments, purified or enriched cell populations can be used in reporter assays. In some embodiments, the source of cells can be organs, tissues, tumors, or fluids from animals, including but not limited to humans. The cell source can be from various sources with different animal preferences. In some embodiments, the cells are immune cells. In some embodiments, the cells are T cells. In some embodiments, the cells are B cells. In some embodiments, the cells are subtypes of T cells. In some embodiments, the cells are subtypes of B cells. In some embodiments, the cells are NK cells. In some embodiments, the cells are phagocytes. In some embodiments, the cells are macrophages. In some embodiments, the cells are neutrophils. In some embodiments, the cells are mast cells. In some embodiments, the cells are monocytes. In some embodiments, the cells are subtypes of any of the cell types described herein. Purification or enrichment of cell populations can be performed using a variety of methods, including but not limited to density gradient separation, immunodensity cell isolation, immunomagnetic cell separation, fluorescence activated cell sorting (FACS), and microfluidic cell sorting. Other forms of purification or enrichment may involve treating a heterogeneous cell population with factors that alter cell growth or proliferation of one cell type relative to another. In some embodiments, for example, in isolating cells from a tissue or tumor, a step prior to the tissue being disaggregated, homogenized, and / or filtered may be necessary to aid in purification.

[0183] For example, in one embodiment, B cells are immunolabeled using a commercially available kit. + The cells are enriched from mouse splenocytes by isolating them. The splenocytes are incubated with magnetic microbeads coated with CD138. In some embodiments, the mixture is loaded onto a magnetic column and unbound cells are washed away. The magnetically labeled cells are then eluted from the column and washed with warmed PBMC medium. The washed CD138 +The cells were immediately used for binding screening. In some embodiments, a pan B cell kit can be used (commercially available from Miltenyi Biotec).

[0184] In some embodiments, the reporter assay is a B cell assay. In some embodiments, the source of B cells can be organ, tissue, tumor or liquid from animals, including but not limited to humans. The B cell source used to provide cells for B cell assay can be from various sources, with different animal preferences. In some embodiments, the source that provides B cells for B cell assay is spleen. Representative hits from an example of B cell assay are found in Figure 25.

[0185] In some embodiments, the reporter assay is a T cell activation assay. In some embodiments, the source of T cells can be organs, tissues, tumors, or fluids from animals, including but not limited to humans. The T cell source used to provide cells for T cell activation assays can be from various sources with different animal preferences. In some embodiments, the source of T cells for T cell assays is peripheral blood. In some embodiments, the source of T cells can include, but is not limited to, spleen, tonsils, bone marrow or expanded lymphoid progenitor cells, and T lymphocytes derived from induced pluripotent stem cells (iPSCs). In some embodiments, the source of T cells is selected from the group consisting of spleen, tonsils, bone marrow or expanded lymphoid progenitor cells, and T lymphocytes derived from induced pluripotent stem cells (iPSCs). In some embodiments, the source of T cells is spleen. In some embodiments, the source of T cells is tonsils. In some embodiments, the source of T cells is bone marrow. In some embodiments, the source of T cells is expanded lymphoid progenitor cells. In some embodiments, the source providing the T cells is T lymphocytes derived from induced pluripotent stem cells (iPSCs). Some embodiments of the T cell activation assay are found in Figure 26, Figure 27, and Figure 28. In some embodiments, the T cell activation assay is used to evaluate the cell signaling induction ability of the antibody. In some embodiments, the ability is determined by examining the antibody-induced response compared to a baseline. In some embodiments, the ability is determined by examining the antibody-induced response compared to a control. In some embodiments, the baseline is determined using a negative control. In some embodiments, the negative control is a control without stimulation. In some embodiments, the negative control is a non-functional antibody. In some embodiments, the antibody-induced response is about 10% or more increase. In some embodiments, the antibody-induced response is 10% or more increase. In some embodiments, the antibody-induced response is 10% or more increase compared to the baseline and / or the control.In some embodiments, the antibody-induced response is an increase of 20% or more compared to baseline and / or control. In some embodiments, the antibody-induced response is an increase of 30% or more compared to baseline and / or control. In some embodiments, the antibody-induced response is an increase of 40% or more compared to baseline and / or control. In some embodiments, the antibody-induced response is an increase of 50% or more compared to baseline and / or control. In some embodiments, the antibody-induced response is an increase of 60% or more compared to baseline and / or control. In some embodiments, the antibody-induced response is an increase of 70% or more compared to baseline and / or control. In some embodiments, the antibody-induced response is an increase of 80% or more compared to baseline and / or control. In some embodiments, the antibody-induced response is an increase of 90% or more compared to baseline and / or control. In some embodiments, the antibody-induced response is an increase of 100% or more compared to baseline and / or control. In some embodiments, the antibody-induced response is an increase of 200% or more compared to baseline and / or control. In some embodiments, the antibody-induced response is an increase of 300% or more compared to baseline and / or control. In some embodiments, the antibody-induced response is an increase of 400% or more compared to baseline and / or control. In some embodiments, the antibody-induced response is an increase of 500% or more compared to baseline and / or control. In some embodiments, the antibody-induced response is an increase of 600% or more compared to baseline and / or control. In some embodiments, the antibody-induced response is an increase of 700% or more compared to baseline and / or control. In some embodiments, the antibody-induced response is an increase of 800% or more compared to baseline and / or control. In some embodiments, the antibody-induced response is an increase of 900% or more compared to baseline and / or control. In some embodiments, the antibody-induced response is an increase of 1000% or more compared to baseline and / or control.In some embodiments, the antibody-induced response is an increase of 2,000% or more compared to baseline and / or control. In some embodiments, the antibody-induced response is an increase of 3,000% or more compared to baseline and / or control. In some embodiments, the antibody-induced response is an increase of 4,000% or more compared to baseline and / or control. In some embodiments, the antibody-induced response is an increase of 5,000% or more compared to baseline and / or control. In some embodiments, the antibody-induced response is an increase of 6,000% or more compared to baseline and / or control. In some embodiments, the antibody-induced response is an increase of 7,000% or more compared to baseline and / or control. In some embodiments, the antibody-induced response is an increase of 8,000% or more compared to baseline and / or control. In some embodiments, the antibody-induced response is an increase of 9,000% or more compared to baseline and / or control. In some embodiments, the antibody-induced response is an increase of 10,000% or more compared to baseline and / or control. In some embodiments, the antibody-induced response is an increase of 10% to 10,000% when compared to baseline and / or control. In some embodiments, the antibody-induced response is an increase of 100% to 1,000% when compared to baseline and / or control. In some embodiments, the antibody-induced response is an increase of 10% to 100% when compared to baseline and / or control. In some embodiments, the antibody-induced response is an increase of 100% to 5,000% when compared to baseline and / or control. In some embodiments, the antibody-induced response is an increase of 10% to 1000%. In some embodiments, the increase in the signal used to identify at least one microcapillary of interest is a statistically significant increase when compared to baseline and / or control. In some embodiments, the response is a statistically significant response when measured, for example, using one-way analysis of variance or T-test or other standard statistical parameters, and when compared to baseline and / or control.In some embodiments, the T cell activation assay is used to measure the antibody's ability to induce internal signaling or cell surface markers. In some embodiments, the T cell activation assay is used to evaluate the antibody's ability to induce cell expansion. In some embodiments, the T cell activation assay is used to evaluate the antibody's ability to induce cytokine secretion. In some embodiments, the T cell activation assay measures CD25 expression to determine activation. In some embodiments, the T cell activation assay measures CD25 expression using a fluorescently labeled anti-CD25 antibody. In some embodiments, the T cell activation assay measures calcium signaling to determine the antibody's activation ability. In some embodiments, the T cell activation assay measures calcium signaling using a calcium sensitive fluorophore. Some examples of calcium sensitive fluorophores include Fluo-4 AM, Fura-2 AM, and Indo-1 AM. In some embodiments, the calcium dye assay has a dynamic range of 1:20000. In some embodiments, the calcium dye assay has a dynamic range of 1:5000. In some embodiments, the calcium dye assay has a dynamic range of 1:10000. In some embodiments, the calcium dye assay has a dynamic range of 1:15000. In some embodiments, the T cell activation assay comprises a mixture of T cells and antibody secreting cells (ASCs). In some embodiments, the mixture of T cells and ASCs varies from a ratio of 2:1 to 12:1. In some embodiments, the mixture of T cells and ASCs is at a ratio of 5:1. In some embodiments, the T cells are purified from peripheral blood. In some embodiments, the ASCs are B cells. In some embodiments, the mixture of T cells and ASCs further comprises T cell activation beads and antibody capture beads.

[0186] Exemplary embodiments: The present application provides a method for screening a population of mutant proteins comprising the steps of: providing a microcapillary array comprising a plurality of microcapillaries, each microcapillary comprising a mutant protein, an immobilized target molecule, and a reporter element, wherein the mutant protein associates with the immobilized target molecule with a particular affinity; and Measuring a signal from the at least one reporter element indicative of association of the at least one mutant protein with the at least one immobilized target molecule to identify at least one microcapillary of interest.

[0187] In some embodiments, the mutant protein is expressed by an expression system.

[0188] In some embodiments, the expression system is a cell-free expression system.

[0189] In some embodiments, the expression system is a cellular expression system.

[0190] In some embodiments, the cellular expression system is animal-based, avian-based, fungal-based, bacterial-based, insect-based, or plant-based.

[0191] In some embodiments, the cell expression system is an avian system.

[0192] In some embodiments, the cell expression system is a chicken system.

[0193] In some embodiments, the mutant protein is a soluble protein.

[0194] In some embodiments, the target molecule is a target protein or polypeptide, a target nucleic acid, a target carbohydrate, or a combination of each.

[0195] In some embodiments, the target molecule is immobilized on a surface.

[0196] In some embodiments, the surface is the surface of a cell.

[0197] In some embodiments, the target molecule is a naturally occurring protein.

[0198] In some embodiments, the surface is the surface of a bead.

[0199] In some embodiments, the surface is a surface of a microcapillary wall.

[0200] In some embodiments, the surface is a surface configured to settle into the microcapillary by gravitational settling.

[0201] In some embodiments, the reporter element is a labeled antibody or other binding molecule.

[0202] In some embodiments, the labeled antibody or other binding molecule is a fluorescently labeled antibody or other binding molecule.

[0203] In some embodiments, the labeled antibody is a primary antibody or a secondary antibody.

[0204] In some embodiments, the labeled antibody or other binding molecule is an enzyme-linked antibody or other binding molecule.

[0205] In some embodiments, the reporter element is activated within the cell and the target molecule is immobilized on the surface of the cell.

[0206] In some embodiments, the reporter element comprises a green fluorescent protein or a mutant.

[0207] In some embodiments, the signal is a fluorescent signal, an absorbance signal, a bright field signal, or a dark field signal.

[0208] In some embodiments, each microcapillary in the microcapillary array contains between 0 and 5 mutant proteins from the population of mutant proteins.

[0209] In some embodiments, the microcapillary array comprises at least 100,000, at least 300,000, at least 1,000,000, at least 3,000,000, or at least 10,000,000 microcapillaries.

[0210] In some embodiments, each microcapillary further comprises a substance to improve the viability of the cell expression system.

[0211] In some embodiments, the substance is methylcellulose, dextran Puluronic® F-68, polyethylene glycol, or polyvinyl alcohol.

[0212] In some embodiments, the substance is a growth medium.

[0213] In some embodiments, the signal is measured by an optical detector.

[0214] In some embodiments, the signal is measured by microscopy.

[0215] In some embodiments, the method further comprises isolating the contents of the microcapillary of interest.

[0216] In some embodiments, the contents of the microcapillary of interest are isolated by pulsing the microcapillary of interest with a laser. EXAMPLES

[0217] Example 1. Screening for secreted EGFR-binding proteins 1A-1C show an exemplary method for screening soluble proteins, in this case immobilized target proteins, that can associate with a cell surface protein (e.g., epidermal growth factor receptor ("EGFR")) as an immobilized target molecule. FIG. 1A (left panel) shows a target cell expressing EGFR on its surface. Also shown are "library-expressing cells" expressing a population of mutant proteins, and a number of "fluorescent detection antibodies" in the microcapillary solution. A bottom view of the microcapillary array is shown in the right panel.

[0218] Each microcapillary component of this screening assay: 1. Cells secreting a mutant protein of interest ("library-expressing cells") The mutant protein of interest is preferably a member of a population of mutant proteins, i.e. a protein library. 2. A target protein immobilized on the surface of a "target cell." In this example, the target protein is a natural cell surface receptor (i.e., EGFR). Alternatively, however, the target protein may be immobilized on another surface, such as the surface of a bead or the surface of the microcapillary itself. 3. Reporter Elements In this example, the reporter element corresponds to a fluorescently labeled antibody (i.e., a "fluorescent detection antibody") specific for the secreted protein. The antibody specifically localizes to an epitope on the secreted protein, but ideally does not interfere with binding of the secreted protein to the target protein on the target cell. Alternatively, the reporter element may be a signaling pathway in cells expressing the target protein. If the secreted mutant protein binds to the target protein on the cell surface and activates a signaling pathway in the target cell, the binding interaction will generate a fluorescent signal within the cell (not shown). 4. Reaction buffer: a. Can be a medium for library expressing cells or target cells (e.g., such a medium can be a hybridoma medium, including, for example, those commercially available from ThermoFisher; thermofisher.com / order / catalog / product / 11279023; see World Wide Web at CD Hybridoma Medium). b. It can be a mammalian imaging solution (eg, such an imaging solution can be an optically clear physiological solution buffered with HEPES at pH 7.4).

[0219] How to explain: Step 1: All components are added to a microcapillary (see FIG. 1A). Step 2: Specific "secreted proteins" are expressed in the microcapillary by the library-expressing cells. Secreted protein variants capable of binding to the target protein are localized to the target cell surface as shown (see Figure 1B). Step 3: The fluorescent detection antibody associated with the bound secreted protein variant is observed associated with the target cell in the specific microcapillary (see Figure 1C).

[0220] Detailed explanation and sample data: To demonstrate this method, a yeast vector library was generated that expressed proteins designed to bind to EGFR on human cancer cells. In this library, some yeast mutants were able to express the proteins, while others were unable to express the proteins. Yeast cells, cancer cells, and fluorescent antibodies against the expressed proteins were added to the microcapillary. After 18 hours, the microcapillary array was imaged. Further details and results of the screening are provided in Example 3 below.

[0221] Example 2. Hybridoma screening for mammalian cells General Background Current methods for screening binding interactions between proteins or other target molecules typically rely on the use of "display" methods, such as phage display, bacterial display, yeast display, mammalian display, or viral display. In display methods, libraries of genes encoding protein variants are expressed on the surface of cells or phages. The protein variants are incubated with soluble forms of the target molecule to identify protein variants capable of binding to the target. The library can be screened by panning or fluorescence-activated cell sorting ("FACS"). Such assays have two main constraints: 1) the engineered protein is typically tethered to a display platform, and 2) it is usually advantageous for a soluble form of the target molecule to be present. Thus, it can be difficult to develop reliable assays for mutant proteins that bind to many target molecules, especially membrane proteins such as G protein-coupled receptors and other such receptors.

[0222] Hybridoma screening for mammalian cells To identify antibody variants that specifically bind to a target molecule, the hybridomas (which secrete the antibody variants) were added to a cancer cell line that expressed high levels of EGFR as a target molecule. A labeled antibody specific for the secreted antibody was then added.

[0223] material: cell: Mouse hybridoma A431 target cells (a human cancer cell line expressing high levels of EGFR) Detection antibody: Anti-mouse secondary antibody conjugated with Alexa488 (fluorophore) Cell Culture Media: DMEM-10% fetal bovine serum DMEM-10% horse serum

[0224] Cell line growth and preparation Mouse hybridoma cells were cultured in complete medium (Dulbecco's modified Eagle medium with 10% horse serum). Hybridoma cells were washed twice with PBSA and suspended in complete medium at 600 cells / μL. A431 cells were cultured in complete medium (Dulbecco's modified Eagle medium with 10% fetal bovine serum). A431 cells were washed twice with PBSA and stained with LiveGreen fluorescent signal. A431 cells were then suspended in complete medium with hybridoma at a final concentration of 1800 cells / μL.

[0225] Assay setup After mixing the two cell types, a detection antibody was added to the reaction mixture: secondary (anti-mouse Alexa488) at a 1:100 dilution. This reaction mixture was then loaded onto a corona-treated microcapillary array (40 μm diameter, 1 mm thick) that had been sterilized with ethanol. A 2 mm thick slab of 1% weight / volume agarose was placed over the array to help prevent evaporation. After each time period, samples were imaged under fluorescence and brightfield microscopy.

[0226] Sample data: Figures 2A-2C show images of subsections of the microcapillary array showing either all cells (Figure 2A, brightfield signal), A431 target cells (Figure 2B, LiveGreen signal), or cells labeled with a fluorescent anti-mouse secondary antibody (Figure 2C, Ab-a555 signal). Microcapillaries containing hybridoma cells expressing an antibody specific for EGFR are indicated by two arrows in each image.

[0227] Figure 3 shows images of a microcapillary containing both A431 target cells and hybridoma cells over a 4-hour incubation, where the antibody binding signal to the A431 target cells increased over the time course of the assay as mouse antibodies specific for EGFR were produced (center panel). LiveGreen staining of the A431 target cells decreased over the same period (right panel).

[0228] Example 3. Yeast library screening against mammalian cells To determine the best secretory yeast plasmid vector, a yeast vector library was generated expressing a scaffold protein designed to bind to EGFR on the surface of cancer cells. The library contained yeast cells with various soluble expression levels of the scaffold protein. Using the described assay, the mutant expression library was screened to recover a plasmid vector that highly expressed the desired scaffold protein. In this experiment, the secreted scaffold had a c-Myc tag, which can be labeled with a fluorescently labeled antibody.

[0229] material: cell: Yeast secreted library of scaffold proteins A431 cells (a human cancer cell line that expresses high levels of EGFR) Detection antibody: Chicken anti-c-Myc Alexa488-labeled anti-chicken secondary antibody Cell Culture Media: DMEM-10% FBS SD-CAA Minimal Yeast Medium Reaction buffer: SD-CAA Minimal Yeast Medium

[0230] method: Cell line growth and preparation Yeast libraries were grown in SD-CAA minimal yeast medium (20 g dextrose, 6.7 g Difco yeast nitrogen base medium, 5 g Bacto casamino acids, 5.4 g Na2HPO4, 8.56 g NaH2PO4·H2O; dissolved in deionized H2O to a volume of 1 liter). After growth, yeast cells were washed twice with PBSA (phosphate buffered saline + 1 mg / ml BSA) and suspended in SD-CAA to a final concentration of 2,400 cells / μL.

[0231] A431 cells were cultured in complete medium (Dulbecco's modified Eagle's medium containing 10% fetal bovine serum). A431 cells were washed twice with PBSA and suspended in SD-CAA containing yeast cells at a final concentration of 600 cells / μL.

[0232] Assay setup After mixing the two cell types, two antibodies were added to the reaction mixture: an unlabeled primary antibody (chicken anti-c-Myc) at a dilution of 1:250 and a labeled secondary antibody (anti-chicken Alexa488) at a dilution of 1:200. This reaction mixture was then loaded onto a corona-treated microcapillary array (40 μm diameter, 1 mm thickness) that had been sterilized with ethanol. A 2 mm thick slab of 1% weight / volume agarose was placed over the array to help prevent evaporation. After 18 hours of growth, samples were imaged under fluorescence and brightfield microscopy.

[0233] Microcapillary array extraction A Triton UV laser was used to extract the desired capillary contents. The laser operates for 18 ± 2 ms (n = 5 measurements) delivering a train of 2.5 kHz pulses with a total energy of approximately 100 μJ. The microcapillary contents were extracted onto a glass coverslip, which was then placed into yeast growth medium (liquid medium or agar plates) to grow the extracted cells.

[0234] Sample data: 4A and 4B show images of a subsection of a microcapillary array using bright field imaging (FIG. 4A) and fluorescent imaging (FIG. 4B) to identify microcapillaries with expressing and non-expressing cells.

[0235] Example 4. Growth of cultured human cells in microcapillary arrays Figures 5A-5G show the growth of K562 cells (a human immortalized myeloid leukemia cell line) in growth medium over a 6-day period in an array of microcapillaries. Brightfield images of the same portion of the array were taken every 24 h. Figure 5A: day 0, Figure 5B: day 1, Figure 5C: day 2, Figure 5D: day 3, Figure 5E: day 4, Figure 5F: day 5, and Figure 5G: day 6. A scale bar of 40 µm is indicated for each image.

[0236] Example 5. Hybridoma screening for mammalian reporter cells To identify antibody variants that activate specific signaling pathways, hybridomas secreting different antibody variants were added to a microcapillary array along with reporter cells. For example, reporter cells can be obtained from Qiagen (see http: / / www.sabiosciences.com / reporter_assay_product / HTML / CCS-013L.html). When a protein variant binds to the reporter cell and activates a signaling pathway, the reporter cell expresses a fluorescent protein. The signal fluorescence of the activated cells is observed in the microcapillaries containing the desired protein variant and used to isolate the contents of those microcapillaries.

[0237] Example 6. Sample Data for Multiple Target Binding Research purpose: Hybridomas secreting antibodies that specifically bind to the target protein but not to proteins of similar structure are identified. See, for example, Figure 9. This method allows for the screening of antibody libraries and does so on an ongoing basis.

[0238] material: Hybridoma Library

[0239] The target protein is displayed on the cancer cell type. See, e.g., FIG.

[0240] The target protein analogue is immobilized on the beads, see for example Figure 9. protocol: 1. Cells displaying target protein A on their surface were cultured. 2. Protein Dynabeads Biotin Binder were labeled with target protein B according to the manufacture's instructions. 3. A rat hybridoma library was cultured. 4. The cells, beads and hybridomas were diluted so that there was an average of 1 hybridoma, approximately 2 cells of target protein A cells, and approximately 10 beads coated with target protein B per microcapillary. 5. Anti-rat secondary antibody (reporter element) was added to the prepared cell sample. 6. The samples were loaded onto the array. 7. Incubate for 1 hour before imaging. 8. Cells that bind target protein A but not target protein B and beads were collected.

[0241] Sample results: Microcapillaries that had appropriately stained cells but no stained beads were identified. The presence of stained cells and the absence of stained beads in the microcapillary indicated the presence of an antibody that binds to the target protein but not to the target protein analogue.

[0242] In some embodiments, the assay tested in this example can be adapted to screen for antibodies that bind to both mouse and human (or other animal combination) variants of a target protein, i.e., to discover "cross-reactive" antibodies. For example, the presence of stained cells and the presence of stained beads in the microcapillary indicates the presence of an antibody that binds to the "target protein" (e.g., mouse target) and also binds to the "target protein analog" (e.g., human target) to identify antibodies that bind to both mouse and human targets. For example, the presence of stained cells and the presence of stained beads in the microcapillary indicates the presence of an antibody that binds to the "target protein" (e.g., cynomolgus target) and also binds to the "target protein analog" (e.g., human target) to identify antibodies that bind to both cynomolgus and human targets.

[0243] Example 7. Titration of reporter elements for optimal signal output A frequently used reporter element is a fluorescently labeled secondary antibody. In this assay format, a secondary antibody is added at the start of the assay and, over time, this antibody binds to the secreted antibody (mutant protein) that is bound to the target protein.

[0244] An important consideration was the amount of secondary antibody used. Too much secondary antibody will result in too high background noise. Using too little secondary antibody will result in too low a signal. In this experiment, beads were labeled with a primary antibody and then titrated with various levels of secondary antibody to determine the optimal signal to noise ratio.

[0245] The reporter element used was a donkey anti-goat IgG secondary antibody labeled with Alexa Fluor633.

[0246] Manufacturer recommended usage range: 1:200 to 1:2000 dilution range.

[0247] The dilution series tested was 1:100, 1:200, 1:500, 1:1000, 1:2000, 1:5000. Images of beads from the experiment are shown in FIG.

[0248] Example 8. Reporter cell assay Calcium dye assay Jurkat cells were loaded with 10 μM Fluo-4 AM in IMDM with 10% FBS for 25 min at 37°C and washed twice with imaging buffer (PBS with 1 mM MgCl2, 1 mM CaCl2, 1 mM HEPES, and 0.1% BSA). Cells were resuspended at 5×106 / mL in imaging buffer. Dye-loaded cells were activated by adding α-CD28 (28.2) and α-CD3 (OKT3), both of which were mouse IgG antibodies cross-linked by preincubation with goat anti-mouse IgG. The final concentrations of antibodies were 5 μg / mL, 2.5 μg / mL, and 5 μg / mL, respectively. Activated cells were immediately loaded onto 40 μm μ-pore arrays, and samples were overlaid with PBS 1% agarose gel for imaging of Ca2+ signaling.

[0249] T cell activation assay Human T cells were isolated from peripheral blood mononuclear cells (PBMCS) via MACS (Pan T Cell Isolation Kit, human, Miltenyi Biotec). Cells were mixed with beads coated with agonistic antibodies. In this case, we used Dynabead Humans T-Activator beads, which are coated with anti-CD3 / CD28. The mixture of beads and cells was loaded onto a 40 μm μ-pore array and the sample was overlaid with a 1% agarose gel (containing culture medium). T cells are activated by the antibodies and express activation markers on their surface. After 24-72 hours, cells are stained with fluorescently labeled antibodies against the activation markers and imaged by a flow cell system.

[0250] GFP reporter cells GLP-1 receptor expressing cells were transfected with a cAMP response element (CRE) reporter that expresses GFP when the CRE signaling pathway is activated. These GLP-1 reporter cells were placed in a 40 μm μ-pore array with 10 uM of its cognate ligand GLP-1. After 24 hours, the cells were imaged for fluorescence. For the actual screening, we co-incubated antibody-secreting cells with GLP-1 CRE reporter cells in the μ-pore array. The antibody-secreting cells secrete antibodies that bind to and activate the GLP-1 reporter cells, activating GFP or other fluorescent protein variant signals.

[0251] Optionally, chicken cells can be used in the reporter cell assay.

[0252] Example 9. Chicken cell assay OmniChicken expresses a fully human, highly diversified antibody repertoire. Genetic diversity from humans and mice allows for more diverse epitope coverage. Deep immune profiling can lead to a more diverse panel of functional antibodies. It can also be referred to as a B cell assay.

[0253] Test Case: Profiling of antigen-specific antibody repertoires following immunization with Progranulin. See Figures 18-19.

[0254] Assay conditions - see FIG.

[0255] protocol: Combine mAb-secreting cells and target beads. Load cells + detection antibody onto array (homogeneous assay). Incubate for 3 hours. Image and quantitate data.

[0256] Example 10. T cell activation by B cells (CD25 surface expression) Objective: to find antibodies that can activate T cells as measured by induction of CD25 expression on the T cell surface. See Figure 27.

[0257] protocol: 1. Isolate CD3+ T cells from human peripheral blood mononuclear cells (PBMCs) using the MACS Pan T Cell Isolation Kit according to the supplier's procedures. 2. Label antibody-secreting cells (ASCs) with CellTrace Far Red according to the supplier's protocol. 3. Prepare a 1:1 mixture of CD3 T cell activation beads and antibody capture beads and wash the mixture with PBMC medium. 4. Wash Dynabeads MyOne Silane beads with PBMC medium. 5. Combine the beads and resuspend them in PBMC medium with 1:200 anti-human CD25 Alexa Fluor 488 detection antibody. 6. Mix T cells and ASCs at a ratio of 5:1 (can be from 2:1 to 12:1). 7. Wash the cell mixture with PBMC medium. Pellet the cells and resuspend in the bead mixture. 8. Load the assay mixture onto a 40 μm μpore array and overlay the samples with a 1% RPMI agarose gel and incubate at 37° C. in a humid environment for 2 days. 9. After 2 days of incubation, image the chip on xPloration and screen for activated T cells labeled with αCD25.

[0258] B Cell Isolation Protocol: B cells were enriched from mouse splenocytes by isolating CD138+ cells using a commercial kit. Splenocytes were incubated with CD138-coated magnetic microbeads. The mixture was loaded onto a magnetic column, and unbound cells were washed away. The magnetically labeled cells were then eluted from the column and washed with warmed PBMC medium. The washed CD138+ cells were immediately used for binding screening.

[0259] B cells can also be obtained using Pan B Cell Beads, for example using the Pan B Cell Isolation Kit from Miltenyi Biotec.

[0260] Example 11. T cell activation by B cells (calcium signaling) Objective: to find antibodies capable of activating T cells as measured by induction of calcium signaling. See Figure 28.

[0261] protocol: 1. Isolate CD3+ T cells from human peripheral blood mononuclear cells (PBMCs) using the MACS Pan T Cell Isolation Kit according to the supplier's procedures. 2. Label antibody-secreting cells (ASCs) with CellTrace Far Red according to the supplier's protocol. 3. Stain T cells with 10 μM calcium-sensitive fluorophore, such as Fluo-4 AM, Fura-2 AM, Indo-1 AM, in AIM V medium with 10% FBS for 25 minutes at 37° C. 4. Wash T cells twice with imaging buffer (PBS with 1 mM MgCl2, 1 mM CaCl2, 1 mM HEPES, and 0.1% BSA). 5. Mix T cells and ASCs at a ratio of 5:1 (can be from 2:1 to 12:1). 6. Wash the cell mixture with PBMC medium. Pellet the cells and resuspend in the bead mixture. 7. Load the assay mixture onto a 40 μm μpore array, overlay the samples with a 1% RPMI agarose gel, and incubate at 37°C for 0-6 hours. 8. Image the chip with xPloration and screen for activated T cells via calcium signaling.

[0262] All patents, patent publications, and other published references mentioned herein are incorporated by reference in their entirety herein, as if each was individually and specifically incorporated by reference.

[0263] Although specific examples have been provided, the above description is illustrative and not restrictive. Any one or more of the features of the above-mentioned aspects can be combined in any manner with one or more features of any other aspect of the present invention. Furthermore, upon review of this specification, many variations of the present invention will become apparent to those skilled in the art. Therefore, the scope of the present invention should be determined by reference to the appended claims, along with their full scope of equivalents.

[0264] The above examples are provided to give those skilled in the art a complete disclosure and description of how to make and use the compositions, systems, and method aspects of the present invention, and are not intended to limit the scope of what the inventors regard as their invention. Modifications of the above modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the appended claims. All patents and publications mentioned in this specification are indicative of the level of skill of those skilled in the art to which the invention pertains. All references cited in this disclosure are incorporated by reference to the same extent as if each reference was individually incorporated by reference in its entirety.

[0265] All heading and section designations are used for clarity and reference purposes only and should not be considered limiting in any way For example, one of ordinary skill in the art will recognize the utility of combining various aspects from different headings and sections as appropriate in accordance with the spirit and scope of the invention described herein.

[0266] All references cited in this specification are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[0267] It will be apparent to those skilled in the art that many modifications and variations of this application can be made without departing from the spirit and scope of the present application. The specific aspects and examples described herein are offered by way of example only and should be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.

[0268] Sequence information SEQUENCE LISTING <110> xCella Biosciences, Inc. <120> METHODS AND SYSTEMS FOR SCREENING USING MICROCAPILLARY ARRAYS <150> US 62 / 830,978 <151> 2019-04-08 <160> 4 <170> FastSEQ for Windows Version 4.0 <210> 1 <211> 330 <212> PRT <213> Homo sapiens <400> 1 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 2 <211> 326 <212> PRT <213> Homo sapiens <400> 2 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro 100 105 110 Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 130 135 140 Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly 145 150 155 160 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 165 170 175 Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp 180 185 190 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 195 200 205 Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu 210 215 220 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 225 230 235 240 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 245 250 255 Ser Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270 Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 275 280 285 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 290 295 300 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 305 310 315 320 Ser Leu Ser Pro Gly Lys 325 <210> 3 <211> 377 <212> PRT <213> Homo sapiens <400> 3 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro 100 105 110 Arg Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg 115 120 125 Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys 130 135 140 Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro 145 150 155 160 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 165 170 175 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 180 185 190 Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Lys Trp Tyr 195 200 205 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 210 215 220 Gln Tyr Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Leu His 225 230 235 240 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 245 250 255 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln 260 265 270 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 275 280 285 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 290 295 300 Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn 305 310 315 320 Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu 325 330 335 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile 340 345 350 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln 355 360 365 Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375 <210> 4 <211> 327 <212> PRT <213> Homo sapiens <400> 4 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro 100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325

Claims

1. 1. A method for screening a population of mutant proteins, comprising the steps of: providing a microcapillary array comprising a plurality of microcapillaries, each of the plurality of microcapillaries comprising a mutant protein, an immobilized target molecule, and a reporter element, wherein the mutant protein associates with the immobilized target molecule with a particular affinity; and and identifying at least one microcapillary of interest by measuring a signal from at least one reporter element indicative of association of at least one mutant protein with at least one immobilized target molecule in a reporter assay, the reporter assay being a B cell assay.

2. 2. The method of claim 1, wherein the target molecule is a target protein or polypeptide, a target nucleic acid, a target carbohydrate, or a combination of each.

3. 10. The method of claim 1, wherein the target molecule is immobilized on a surface.

4. 4. The method of claim 3, wherein the surface is the surface of a cell.

5. The method of claim 3, wherein the surface is the surface of a B cell.

6. 10. The method of claim 1, wherein the signal is measured by an optical detector.

7. 10. The method of claim 1, further comprising isolating the contents of the microcapillary of interest.

8. 10. The method of claim 1, wherein the microcapillary does not contain microparticles, magnetic microparticles, magnetic beads, or electromagnetic radiation absorbing materials capable of blocking the transmission of electromagnetic radiation.

9. The method of claim 1, wherein each of the plurality of microcapillaries contains B cells.

10. 1. A system for screening a population of mutant proteins, comprising: an array comprising a plurality of microcapillaries, each of the plurality of microcapillaries comprising a mutant protein, an immobilized target molecule, and a reporter element, wherein the mutant protein associates with the immobilized target molecule with a particular affinity; and and at least one reporter element of a reporter assay, which is a B-cell assay, configured to indicate association of at least one mutant protein with at least one immobilized target molecule to identify at least one microcapillary of interest.

11. 11. The screening system of claim 10, wherein the target molecule is a target protein or polypeptide, a target nucleic acid, a target carbohydrate, or a combination of each.

12. 11. The screening system of claim 10, wherein the target molecule is immobilized on a surface.

13. 13. The screening system of claim 12, wherein the surface is the surface of a cell.

14. 13. The screening system of claim 12, wherein the surface is the surface of a B cell.

15. 11. The screening system of claim 10, wherein the reporter element is a labeled antibody or other binding molecule.

16. 11. The screening system of claim 10, wherein the reporter element is activated within the cell and the target molecule is immobilized on the surface of the cell.

17. 11. The screening system of claim 10, wherein each microcapillary further comprises a substance for improving the viability of the cell expression system.

18. 11. The screening system of claim 10, wherein the microcapillary does not contain microparticles, magnetic microparticles, magnetic beads, or electromagnetic radiation absorbing materials capable of blocking the transmission of electromagnetic radiation.

19. The screening system of claim 10, wherein each of the plurality of microcapillaries contains B cells.

20. 1. A method for screening a population of mutant proteins comprising the steps of: providing a microcapillary array comprising a plurality of microcapillaries, each microcapillary comprising a cellular expression system expressing a mutant protein, a target molecule immobilized on the surface of the cell, and a reporter element, wherein the mutant protein associates with the immobilized target molecule in the microcapillary with a specific affinity, and the cellular expression system is an avian system; and and identifying at least one microcapillary of interest by measuring a signal from at least one reporter element indicative of association of at least one mutant protein with at least one immobilized target molecule in a reporter assay, the reporter assay being a B cell assay.

21. 10. The method of claim 1, wherein the signal used to identify the at least one microcapillary of interest is at least a 10% to 10,000% or more increase over the signal compared to a baseline and / or control sample.

22. The method of claim 1, wherein in each microcapillary, the reporter element is diluted at a factor ranging from 1:500 to 1:5000.

23. The method of claim 22, wherein the reporter element is diluted at a factor ranging from 1:2000 to 1:5000.

24. The screening system of claim 10, wherein in each microcapillary, the reporter element is diluted at a ratio ranging from 1:500 to 1:5000.

25. The screening system of claim 24, wherein the reporter element is diluted at a ratio ranging from 1:2000 to 1:5000.

26. The method of claim 20, wherein in each microcapillary, the reporter element is diluted at a factor ranging from 1:500 to 1:5000.

27. The method of claim 26, wherein the reporter element is diluted at a factor ranging from 1:2000 to 1:5000.

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