WO2016187466A1

WO2016187466A1 - Methods, apparatus and compositions for expression and high throughput screening

Info

Publication number: WO2016187466A1
Application number: PCT/US2016/033348
Authority: WO
Inventors: Bryan Glaser; Ivar Meyvantsson; Roland Green; Kimberly KAUFMAN; Madison GREEN; Tye Gribb; John Painter; Bonnie HAMMER; Allyson Skoien
Original assignee: Invenra, Inc.
Priority date: 2015-05-20
Filing date: 2016-05-19
Publication date: 2016-11-24

Abstract

Methods and apparatus are provided for screening libraries of polypeptides linked to coding sequences for a biological activity. Libraries of nucleic acid molecules encoding candidate polypeptides, such as antibodies, can be amplified, expressed, and linked to beads. The library allows for efficient high-throughput screening to identify polypeptides having a desired biological activity. In some aspects, amplification, expression, and/or screening is performed in a high-density pico-, nano-, or micro-well plate format. Methods and compositions for efficient expression and screening of antibodies are likewise provided.

Description

DESCRIPTION

METHODS, APPARATUS AND COMPOSITIONS FOR EXPRESSION AND HIGH

THROUGHPUT SCREENING

[0001] This application claims the benefit of United States Provisional Patent Application Nos. 62/164,237 and 62/164,274, both of which were filed on May 20, 2015 and which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

[0002] The present invention relates generally to the fields of biochemistry and molecular biology. More particularly, it concerns methods and apparatus to express, isolate and identify biologically active polypeptides, such as antibodies.

2. Description of Related Art

[0003] Many of the most effective modern therapeutics are polypeptide molecules such as monoclonal antibodies. In the case of antibodies, the mammalian immune system provides a highly adapted system for development of antibody molecules that are specific for a given therapeutic target. Modern molecular biology techniques allow the sequences for these antibodies to be isolated, such that therapeutics based on the antibody sequences can be mass produced in fermentation systems. Unfortunately, the development of antibody therapeutics is limited in that the therapeutic target must be known, be antigenic, and be accessible on the surface of a target cell.

[0004] Accordingly, methods for identifying candidate biologically active polypeptides by using molecular libraries are being explored. However, any such system requires that the library have sufficient diversity to interrogate a vast range of candidate molecules. Moreover, any assay using such a library must provide a system for determining the coding sequence for polypeptides that are identified in a binding or biological activity screen. In some cases, the polypeptide sequence can be directly determined, such as by mass spectroscopy, but such a method requires a large amount of each given polypeptide. Alternatively, the polypeptide can be tethered to its nucleic acid coding sequence by some method. Such methods based on tethering are generally referred to as biological display (e.g., phage display). [0005] Phage display technology has been successful as providing a vehicle that allows for the selection of a displayed protein by providing an essential link between nucleic acid and the activity of the encoded polypeptide (for a review see, e.g., Clackson and Wells, 1994). In this case, filamentous phage particles act as genetic display and packages proteins on the outside of the particle and the genetic elements that encode them on the inside. However, phage display relies upon the creation of nucleic acid libraries in vivo in bacteria and this places a limitation on the library size that can be used. Additionally, all potentially useful candidate polypeptides are fused to phage sequences for display and such fusion may interfere with the polypeptide function. Thus, there remains no efficient system for screening and identification of biologically active polypeptide molecules.

SUMMARY OF THE INVENTION

[0006] In a first embodiment there is provided a method of identifying or isolating at least one nucleic acid molecule encoding a polypeptide having a desired biological activity, the method comprising the steps of: (i) obtaining a library of nucleic acid molecules, individual members of the library encoding polypeptides comprising different amino acid sequences and having desired properties; (ii) compartmentalizing the members of the library individually in the presence of a first bead comprising a first binding moiety; (iii) amplifying the individually compartmentalized members of the library in the presence of a tagged- primer, wherein the amplified individually compartmentalized members comprise a tag that is bound to the first binding moiety of the first bead, to generate an amplified DNA library (e.g., an amplified on-bead DNA library); (iv) expressing the amplified DNA library in the presence of a second bead comprising a second binding moiety to produce individually compartmentalized polypeptides, wherein the polypeptides are bound to the second binding moiety of the second bead, thereby producing an expressed on-bead polypeptide library; (v) testing the expressed on-bead polypeptide library for a desired activity on living target cells; and (vi) identifying tagged nucleic acid molecules encoding polypeptides with the desired activity.

[0007] In some aspects, a method may comprise selecting library for member polypeptides that have a desired property (e.g., bind to a target of interest), prior to testing on cells. For example, in some aspects, obtaining a library of nucleic acid molecules for use according to the methods comprises: (a) obtaining a first library of nucleic acid molecules, individual members of the first library comprising a sequence that encodes a polypeptide, wherein individual members of the first library have different amino acid sequences; (b) expressing the polypeptides encoded by the first library to produce polypeptides, wherein each polypeptide is associated with the member of the first library that encodes that polypeptide, thereby producing an expressed polypeptide library; (c) testing the expressed polypeptide library for a desired property; (d) identifying or isolating the members of the first library that encode polypeptides having the desired property, thereby producing an enriched library; and (e) making a library of nucleic acid molecules from the enriched library, individual members of the library comprising a sequence that encodes a polypeptide having the desired property, wherein individual members of the library encode polypeptides comprising different amino acid sequences. Thus, in some aspects, a method of the embodiments comprises the steps of: (a) obtaining a first library of nucleic acid molecules, individual members of the first library comprising a sequence that encodes a polypeptide, wherein individual members of the first library have different amino acid sequences; (b) expressing the polypeptides encoded by the first library to produce polypeptides, wherein each polypeptide is associated with the member of the first library that encodes that polypeptide, thereby producing an expressed polypeptide library; (c) testing the expressed polypeptide library for a desired property; (d) identifying or isolating the members of the first library that encode polypeptides having the desired property, thereby producing an enriched library; (e) making a second library of nucleic acid molecules, individual members of the second library comprising a sequence that encodes a polypeptide having the desired property, wherein individual members of the library encode polypeptides comprising different amino acid sequences; (f) compartmentalizing the members of the library individually in the presence of a first bead comprising a first binding moiety; (g) amplifying the individually compartmentalized members of the second library in the presence of a tagged-primer, wherein the amplified individually compartmentalized members comprise a tag that is bound to the first binding moiety of the first bead, to generate an amplified DNA library; (h) expressing the amplified DNA library in the presence of a second bead comprising a second binding moiety to produce individually compartmentalized polypeptides, wherein the polypeptides are bound to the second binding moiety of the second bead, thereby producing an expressed on-bead polypeptide library; (i) testing the expressed on-bead polypeptide library for a desired activity on living cells; and j) identifying tagged nucleic acid molecules encoding polypeptides with the desired activity. [0008] Certain aspects of the embodiments involve expressing an amplified DNA library. For example, in some aspects, expressing the amplified DNA library comprises expressing from a DNA library bound to a first bead. In some aspects, however, the amplified DNA library is transferred to a surface, such as the surface of a compartment (e.g., a well), prior to expressing. For example, in some cases, the surface is a hydrogel surface or a membrane such as a PDMS membrane.

[0009] In further aspects, (i) obtaining a library of nucleic acid molecules comprises providing a library of mutated sequences based on a known polypeptide having a desired property of activity. For example, the known polypeptide can be an enzyme or therapeutic polypeptides. In some instance the known polypeptide is an antibody and mutating the polypeptide may involve making changes to the variable domain, the constant domain or the CDRs.

[0010] In some aspects, (ii) compartmentalizing the members of the library can comprise depositing the members in a gel, an emulsion microcapsule or in a well. In certain aspects, the step of compartmentalizing comprises depositing members of the library into wells of a plate. For example, in some cases, members of the library are deposited individually into wells. In other cases the members are distributed such that the majority of wells comprise from 0 to 2 member(s) of the library per well. In a further aspect, the step of compartmentalizing the members of the library may comprise depositing at least one bead per well of a plate (e.g. , at least 2, 3, 4, 5, 10, 20 or more beads per well). In some aspects, the method may comprise sealing the wells of a plate, after depositing the members of the library and/or the beads. In a further aspect, the wells may be sealed, such as with permeable membrane material. For example, a permeable membrane material may allow for the diffusion of biological molecules, but prevent beads from passing out of the well. In one aspect, the plate may comprise about 10, 100, 1,000, or 10,000 to about 50,000, 100,000, 200,000 or 300,000 wells per mm². In some cases, a plate comprises about 10,000 wells per mm².

[0011] In some aspects, expressing an amplified on-bead library may comprise contacting the amplified on-bead library with cell-free transcription and translation system (e.g., a eukaryotic or prokaryotic cell-free transcription and translation system). In some aspects, expressing the amplified on-bead library may comprise expressing the members in the presence of a chaperone, such as a heat shock protein. In a further aspect, the chaperone may be a human Grp94, Grp78, Grpl70, ErDJ3, PDIAl, PDIA6, BIP, Cyclophillin B or a homolog thereof from another species, such as yeast. In some aspects, a mixture of chaperones can be used.

[0012] In a further aspect, testing the expressed on-bead library may comprise depositing the members of the expressed on-bead library into wells of a plate. In some cases, the member polypeptides are separated from the beads prior to contacting them with cells. For example, removing the polypeptides from the beads may comprise cleaving the polypeptides from the beads with a protease (e.g., TEV protease). In some specific aspects, the members of the expressed on-bead antibody library are disposed in wells of a plate and contacting the polypeptide members with a culture of living target cells comprises contacting the on-bead antibody library in the wells of the plate with a corresponding plate having wells comprising the living target cells. In some aspects, the wells comprising the expressed on- bead library are separated by the wells comprising the living target cells by a porous membrane, such as a membrane that allow diffusion of the polypeptide members of the on- bead library but prevents diffusion of the beads and cells between wells of the plates. In certain aspects, the wells of a plate may comprise between about 1, 3, 5, 10, 15 or 20 and about 50, 100, 500, 1,000 and 10,000 living cells per well. In a further aspect, a well comprising cells may be coated with an extracellular matrix component. In some aspects, depositing the members of the expressed on-bead library into wells may comprise depositing the expressed on-bead library such that the majority of wells comprise from 0 to 1 member of the expressed on-bead library per well. In a further aspect, testing the expressed on-bead library may comprise sealing the wells of a plate, after depositing the members of the expressed on-bead library. In some aspects, the plate may comprise about 5, 10, 30, 50, 100 or 200 to about 500, 1,000, 2,000 or 3,000 wells per mm². In some aspects, a plate comprises about 30 wells per mm².

[0013] In a further aspect, a step of identifying the tagged nucleic acid molecules associated with polypeptides that provide a biological activity on target cells may comprise binding the tagged nucleic acid molecules to a collection bead (e.g., a magnetic collection bead). In a further aspect, binding the nucleic acid molecules to a collection bead may comprise (i) binding the tagged nucleic acid to a collection bead; (ii) disassociating the tagged nucleic acid from the first bead; and (iii) identifying the tagged nucleic acid bound the collection bead. In yet a further aspect, a step of identifying the tagged nucleic acid molecules associated with polypeptides that provide a biological activity on target cells may comprise collecting the nucleic acid molecule from well of an array. Thus, in some aspects, identifying the tagged nucleic acid molecules of the embodiments comprises isolating the tagged nucleic acid molecules. In some aspects, the nucleic acid molecules are bound to a matrix (e.g., in a well) of an array and identifying the nucleic acid molecule comprises disassociating the nucleic acid molecules from the matrix. In further aspects, the nucleic acid molecules are immobilized in a gel distributed across an array.

[0014] In still a further aspect, all library beads may comprise a magnetic capture tag (e.g. , a magnetic nanoparticle bound to the end of the DNA) prior to testing for biological activity. In this case, a step of identifying the tagged nucleic acid molecules associated with polypeptides that provide a biological activity on target cells may comprise: (i) disassociating tagged nucleic acid from the first binding moiety of the bead; and (ii) isolating the disassociated nucleic acid via a magnetic collection system that can capture magnetically tagged DNA, but where the magnetic force on the capture tag is not strong enough to recover DNA that is still attached to the much larger library beads.

[0015] Certain aspects of the methods involve the use of cell-free transcription/translation systems. For example, the components for use according to the embodiments can comprise an RNA polymerase (e.g. , a T7 or SP6 RNA polymerase) and factors required for RNA polymerase activity. Furthermore, translation systems can comprise ribosomes (e.g. , eukaryotic or prokaryotic ribosomes) and translation factors required for protein synthesis. For example, an extract from a translation competent cell lysate, such as bacteria (e.g. , an E. coli bacterial lysate), yeast or mammalian cell lysate (e.g. , a rabbit reticulocyte lysate or wheat germ extract), can be used. Additional components that can be used include, without limitation, buffers (e.g. , HEPES), reducing agents, such as dithiothreitol (e.g. , to stabilize the T7 RNA polymerase), nucleotides, folinic acid, tRNAs (such as E. coli tRNAs), salts (e.g. , magnesium, potassium, ammonium), glucose, cyclic AMP, creatine phosphate, creatine kinase, protease inhibitors, RNase inhibitors, amino acids, and inhibitors of RNA polymerase (e.g. , rifampicin).

[0016] In certain aspects, polypeptides of a library comprise a protease cleavage site. Likewise, in some aspects, testing a library member for activity can comprise contacting the polypeptide with a protease (to free the polypeptide from the bead). For example, the protease can be the Tobacco Etch Virus (TEV) protease and the expressed polypeptide can have, in addition to a candidate polypeptide sequence and the peptide-tag described above, the recognition site for the TEV protease (i.e. , Glu-Asn-Leu-Tyr-Phe-Gln-[Gly/Ser]). In this aspect, the TEV protease can then cleave the polypeptide sequence and thus dissociate the polypeptide from the carrier such that the polypeptide diffuses freely in solution or across a memebrane.

[0017] In some aspects, a method of the embodiments further comprises testing for a biological activity on test cells. Testing cells can involve, for instance, detecting a change in the physical, optical or fluorescent properties of the test cells, such as by detecting uptake or exclusion of a fluorescent dye by the cells or by detecting the binding of a labeled reagent, by expression of a reporter protein (e.g. , a fluorescent protein), or by loss of expression of a protein (e.g. , a fluorescent protein). In certain cases, a biological activity is detected by use of a cell internalization indicator (e.g., a pH sensitive dye). In some aspects, binding of a labeled reagent can be detected by magnetic or affinity separation. Furthermore, the testing of the cells may, in some cases, involve the detection of a soluble factor secreted or released by the cells. For example, testing cells can comprise detecting the binding of an antibody, an aptamer, a lectin, a polypeptide, a receptor protein, a ligand or a carbohydrate to the test cells or a component thereof. Thus, in some cases, detection of such binding can comprise detecting binding of the further binding moiety associated with the nucleic acid molecules of the library. Alternatively, testing the cells can comprise detecting the product of an enzymatic reaction. Thus, in some cases, the biological activity may result in release or cell surface-presentation of an enzyme that can convert a substrate to a product, where the product is detectable by some method (e.g., fluorescence or luminescence). For example, a reporter cell line may, as a result of the biological activity, express a fluorescence protein or a luciferase enzyme that has a secretion tag. In this case, a luciferin substrate can be provided and any secreted enzyme can turn the substrate into a luminescent product, which can be detected. Testing the cells can be completed while the cells are in a microcapsule (e.g., within an emulsion) or micro-well or after the cells are removed from the micro-well or microcapsule. In a further aspect, testing for a biological activity may comprise contacting cells with an internalization indicator (e.g., a pH sensitive dye). For example, testing cells may comprise detecting cell death. [0018] In one aspect, testing may comprise using cells that express a fluorescent viability indicator. In one aspect, the fluorescent viability indicator may be a green fluorescent protein fused with a tag that increases degradation (e.g., a PEST).

[0019] A huge array of biological responses can be tested according to the methods of the embodiments. In some aspects, the biological response can be binding to a cell, cell internalization; a change in cell proliferation; a change in the expression in the cell; a change in the compartmentalization of a marker inside the cell; a change in cell phenotype; a change in cell function; permeability of a polypeptide through an epithelial layer; a change in the markers expressed on the cell surface; T-cell activation; a change in a response to a drug; differentiation; de-differentiation (i.e. , enhanced pluripotency); or cell death (e.g. , via necrosis or apoptosis). In the case of apoptosis, for instance, detecting a response can comprise detecting Annexin V binding to the test cell. Likewise, in the case of cell differentiation detecting a response can comprise detecting the expression of a differentiation marker. In another aspect, the library is a library of antibody sequences and the biological activity is antibody-dependent cellular cytotoxicity (ADCC). In some cases, a test cell can comprise a transgene such as a transgene for the expression of a reporter (e.g., a fluorescent protein) and detecting a biological response can comprise detecting expression of the reporter. In another aspect, the testing may comprise scanning of the plate to detect a signal indicating a biological activity. In a further aspect, the scanning may be automated. [0020] In a further specific embodiment, the present disclosure provides a method of identifying or isolating at least one nucleic acid molecule encoding an antibody having a desired biological activity, the method comprising the steps of: (i) obtaining a library of nucleic acid molecules, individual members of the library comprising a sequence that encodes an antibody having a desired binding specificity, wherein individual members of the library encode antibodies comprising different amino acid sequences; (ii) compartmentalizing the members of the library individually in the presence of a first bead comprising a first binding moiety; (iii) amplifying the individually compartmentalized members of the library in the presence of a tagged-primer, wherein the amplified individually compartmentalized members comprise a tag that is bound to the first binding moiety of the first bead, to generate an amplified on-bead DNA library; (iv) expressing the amplified on-bead DNA library in the presence of a second bead comprising a second binding moiety to produce individually compartmentalized antibodies, wherein the antibodies are bound to the second binding moiety of the second bead, thereby producing an expressed on-bead antibody library; (v) testing the expressed on-bead antibody library for a desired activity (e.g., a binding activity, an enzymatic activity or a biological activity); and (vi) identifying or isolating tagged nucleic acid molecules encoding antibodies with the desired activity. In further aspects, testing the expressed on-bead antibody library for a desired activity is performed on living test cells.

[0021] In still a further embodiment, a method is provided comprising identifying or isolating at least one nucleic acid molecule encoding an antibody having a desired biological activity, the method comprising the steps of: (a) obtaining a first library of nucleic acid molecules, individual members of the first library comprising a sequence that encode an antibody or a scFv fragment of an antibody, wherein individual members of the first library encode antibodies or scFvs having different amino acid sequences; (b) expressing the antibody or scFv polypeptides encoded by the first library to produce scFv polypeptides, wherein each scFv polypeptide is associated with the member of the first library that encodes that scFv, thereby producing an expressed scFv polypeptide library; (c) testing the expressed scFv polypeptide library for a desired property; (d) identifying or isolating the members of the first library that encode an scFv polypeptide having the desired property, thereby producing an enriched first library; optionally, repeating steps (a)-(d) one or more times; (e) making a second library of nucleic acid molecules, each member of the second library comprising a sequence that encodes an antibody polypeptide, wherein individual members of the second library encode antibody polypeptides comprising a CDR sequence from an scFv polypeptide of the enriched first library; (f) compartmentalizing the members of the second library individually in the presence of a first bead comprising a first binding moiety; (g) amplifying the individually compartmentalized members of the second library in the presence of a tagged-primer, wherein the amplified individually compartmentalized members comprise a tag that is bound to the first binding moiety of the first bead, to generate an amplified on- bead DNA library; (h) expressing the amplified on-bead DNA library in the presence of a second bead comprising a second binding moiety to produce individually compartmentalized antibody polypeptides, wherein the antibody polypeptides are bound to the second binding moiety of the second bead, thereby producing an expressed on-bead antibody library; (i) testing the expressed on-bead antibody library for a desired activity; and (j) identifying or isolating tagged nucleic acid molecules encoding antibody polypeptides with the desired activity. In certain aspects, a method according to the instant embodiment comprises a step for repeating steps (a)-(d) one or more times before step (e). For example, in some aspects, steps (a)-(d) are repeated 2, 3, 4, 5, 6, 7, 8 or more times. In some aspects, the second binding moiety of the second bead binds directly to antibodies. For example, in some aspects, the second binding moiety can be a protein A, ZZ or Fc receptor. In still further aspects, the second binding moiety cleavable, such that the antibody can be released from the second bead. For example, the second binding moiety can comprise a protease cleavage site (e.g., TEV protease cleavage site). This, in some aspects, the second bead is a His affinity bead and the second binding moiety comprises a His tagged and an antibodies binding polypeptide (e.g., protein A, ZZ or Fc receptor). In yet further aspects, the second binding moiety comprises (i) a His tag; (ii) a protease cleavage site and (iii) an antibodies binding polypeptide (e.g., a fusion protein of His-TEV-ZZ). Optionally, the antibodies binding polypeptide may comprise and additional amino acid sequence, such as a sequence encoding a reporter, a tag or a toxin.

[0022] In one aspect, individual members of the library may encode antibodies having different heavy chain CDR3 sequences or light chain CDR3 sequences or both. In one aspect, the antibody polypeptides may comprise a human or a humanized antibody sequence. In certain aspects, the antibody polypeptides may comprise a scFv sequence. In one aspect, the antibody polypeptides may comprise an IgA, IgG (e.g., IgG 1, IgG2, IgG3 or IgG4), or IgE antibody sequence.

[0023] Thus, in some aspects, a sequence that encodes the antibody polypeptide may comprise coding sequence for a full-length heavy and light chain. In one aspect, the sequence that encodes the antibody polypeptide may comprise coding sequence for a light chain and a heavy chain, and the heavy chain may comprise a protease cleavage sequence and a polypeptide tag fused to the c-terminus of the heavy chain, or alternatively to the c-terminus of the light chain. In a further aspect, the heavy and light chain sequence may be operably linked to different promoters. In another aspect, the sequence that encodes the antibody polypeptide may comprise, from 5' to 3', sequences encoding i) a first promoter and ribosome binding site (RBS); (ii) an antibody heavy chain coding sequence; (iii) protease cleavage site; (iv) a polypeptide tag; (v) a stop codon; (vi) a DNA spacer with a second promoter and RBS; (vii) an antibody light chain coding sequence; (viii) a stop codon; (ix) and transcription terminator. Optionally, the sequence may comprise a cloning site or a toxin coding sequence between the antibody heavy chain coding sequence and the protease cleavage site. In a further aspect, the sequence that encodes the antibody polypeptide may comprise, from 5' to 3', sequences encoding i) a first promoter and ribosome binding site (RBS); (ii) an antibody heavy chain coding sequence; (iii) a stop codon; (iv) a DNA spacer with a second promoter and RBS; (v) an antibody light chain coding sequence; (vi) a stop codon; (vii) and transcription terminator. [0024] In one aspect, identifying tagged nucleic acid molecules associated with antibody polypeptides that provide a desired activity may comprise (a) scanning the plate to detect a signal indicating a biological activity; (b) recording the plate locations of the signals; (c) dispensing a recovery solution containing a dissociation reagent to those locations; and (d) recovering only those nucleic acid molecules that have been dissociated from library beads. In some aspects, the recovery solution also contains magnetic capture tags (e.g. , nanoparticles). In some further aspects, the recovery solution is dispensed to the miniature wells using an inkjet system. In yet further aspects, the nucleic acid molecules are captured using a permanent magnet covered with a film (e.g., parafilm) and the film placed in a buffer to collect recovered nucleic acids. In still further aspects, identifying tagged nucleic acid molecules associated with antibody polypeptides can comprise collecting the nucleic acids from wells by suction or washing (e.g., with a syringe). In some aspects, nucleic acid molecules can be collected manually or the collecting can be automated.

[0025] As discussed above, in certain aspects, the nucleic acid molecules are associated with a bead. As used herein, a "bead" can be, for example, a microsphere, a bead, a nanoparticle, a macromolecule, a molecule, a microfabricated structure, or a nanostructure. In some aspects, a bead comprises first and/or second binding moiety that can be, without limitation, an antibody, an aptamer, a lectin, a polypeptide, a receptor protein, a ligand, a carbohydrate, or a metal-charged chelating group capable of binding a tagged protein (e.g. nickel-nitrilotriacetic acid capable of binding histidine-tagged proteins). In some aspects, a bead may comprise histidine binding moieties and a portion of the bead can be associated with a his-tagged protein that acts as a binding moiety. In some cases, a binding moiety as used here refers to one half of a binding pair (e.g. , the streptavadin or biotin of a streptavidin- biotin binding pair). The linkage between a binding moiety and the carrier can be, without limitation, thiol, amino, carboxylate, hydroxylate, histidine-tagging (e.g., hexa-histidine tagging), or biotin-streptavidin. For example the carrier can be a cross-linked agarose bead functionalized with a nickel-charged chelating group capable of binding histidine-tagged proteins. Alternatively, the carrier can be a silica bead functionalized with nickel- nitrilotriacetic acid, or a streptavidin-coated polystyrene or silica bead pre-loaded with nickel- charged biotin-nitrilotriacetic acid. Such a bead (agarose, polystyrene, or silica) can, for instance, can be incubated with histidine-tagged streptavidin molecules at a concentration where a fraction of the histidine-tag binding sites will be occupied by streptavidin molecules (e.g. , in this case the nucleic acid molecules can have a biotin-tag, which can bind the streptavidin molecules on the bead to provide a linkage between the nucleic acid and the carrier). Likewise, in some aspects, an expressed polypeptide can comprise a histidine-tag, such that the expressed polypeptide molecules can bind to a fraction of the remaining histidine-tag binding sites on the bead to provide a linkage between the polypeptide and the carrier (and the nucleic acid molecule(s)). In one aspect, the bead may be a magnetic bead.

[0026] In certain aspects, an antibody, such as a scFv, or candidate polypeptide may comprise a cytotoxic polypeptide. In further aspects, an antibody or candidate polypeptide may be bound to a molecule (e.g., an antibody-binding polypeptide such as protein A, ZZ or a Fc receptor) that is itself associated or fused with a cytotoxic polypeptide. In one aspect, the cytotoxic polypeptide may comprise gelonin or Pseudomonas enterotoxin. In one aspect, the cytotoxic polypeptide may comprise an enzyme. In a further aspect, each members of the library may comprise a sequence that encodes a cell penetrating peptide. In a further aspect, the method may comprise washing and chemically modifying the antibody or polypeptide library member deposited on the bead. In some aspects, the chemical modification comprises labeling lysines, cysteines, or other modalities of the polypeptide. In yet a further aspect, the polypeptide member would be chemically labeled with a fluorescent dye, a small molecule drug conjugate, a polypeptide toxin, or another functional modality.

[0027] In one aspect, the target cells may be mammalian cells. In a further aspect, the target cells may be human cells. In one aspect, the target cells may be cancer cells. In some aspects, the testing of cells may be performed in the wells of a plate. In this aspect, the well may be coated with an extracellular matrix component. In a further aspect, the testing may comprise scanning of the plate to detect a signal indicating a biological activity. In yet a further aspect, the scanning may be automated.

[0028] In some aspects, identifying tagged nucleic acid molecules associated with antibodies or polypeptides that provide a desired activity comprises (a) recording the plate location of signals indicating biological activity; (b) dispensing a recovery liquid to those locations, the liquid comprising a dissociation agent which releases the nucleic acids from the library bead; and (c) collecting nucleic acid molecules using a magnet covered with a capture film. In a further aspect, the recovery liquid also contains magnetic capture beads. In yet another aspect, the recovery liquid is dispensed using an inkjet dispenser.

[0029] In a further embodiment, the present disclosure provides a method of identifying or isolating a population of nucleic acid molecules encoding antibodies or polypeptides having a desired activity, the method comprising the steps of: (a) obtaining a library of nucleic acid molecules, each member of the library comprising a sequence that encodes an antibody polypeptide comprising an antibody, a protease cleavage sequence and a polypeptide tag, wherein individual members of the library encode antibodies having different CDR sequences and wherein the protease cleavage sequence is positioned between the antibody and the polypeptide tag; (b) individually compartmentalizing the members of the library in the presence of a bead comprising a first and second binding moiety; (c) amplifying the individually compartmentalized members of the library in the presence of tagged-primers, wherein the amplified individually compartmentalized members comprise a tag that is bound to the first binding moiety of the bead, to generate an amplified on-bead library; (d) expressing the amplified on-bead library to produce individually compartmentalized antibody polypeptides, wherein the polypeptide tag of the antibody polypeptides is bound to the second binding moiety of the bead, thereby producing an expressed on-bead library; (e) testing the expressed on-bead library for a desired activity; and (f) identifying or isolating tagged nucleic acid molecule associated with antibody polypeptides that provide a desired activity.

[0030] In one aspect, the desired activity may be high expression or correct folding. In a further aspect, testing the expressed on-bead library for a desired activity may comprise testing the on-bead library for binding to a protein that binds to an antibody constant domain. In yet a further aspect, the protein that binds to an antibody constant domain may be a mammalian Fc receptor such as FcyRIa, FcyRIIa, FcyRIIb, FcyRIIc, FcyRIIIa, FcyRIIIb, or FcaRI. In another aspect, identifying tagged nucleic acid molecules associated with antibody polypeptides that provide a desired activity may comprise binding the on-bead library to a column comprising a protein that binds to an antibody constant domain or interacts via biophysical properties such as hydrophobicity. In another aspect, identifying or isolating tagged nucleic acid molecules associated with antibody polypeptides that provide a desired activity may comprise binding the on-bead library to a column comprising human serum antibodies to select for antibodies with increased likelihood of aggregation. In yet a further aspect, the method may comprise amplifying the isolated tagged nucleic acid molecules to obtain an enriched library comprising sequences of nucleic acid molecules with a desired activity.

[0031] In a further aspect, the desired activity may be binding to a desired antigen. In a further aspect, testing the expressed on-bead library for a desired activity may comprise testing the on-bead library for binding to a desired antigen. In yet a further aspect, identifying or isolating tagged nucleic acid molecule associated with antibody polypeptides that provide a desired activity may comprise binding the on-bead library to a column comprising a desired antigen. In yet a further aspect, the method may comprise amplifying the isolated tagged nucleic acid molecules to obtain an enriched library comprising sequences of nucleic acid molecules with a desired activity.

[0032] Thus, in some embodiments, a method is provided of identifying or isolating a nucleic acid molecule encoding a biologically active polypeptide having a desired biological activity, the method comprising the steps of (a) obtaining a library of polypeptide molecules comprising at least 50,000 different molecules; (b) individually testing the different polypeptide molecules on live test cells for a biological response to the polypeptide molecules; and (c) identifying the sequences of nucleic acid molecules encoding the subset polypeptide molecules that are biologically active. For example, in some aspects, the library comprises at least 50,000, 100,000, 200,000, 500,000, 1 million, 10 million, 100 million, 1 billion, or 10 billion different molecules (e.g. , between about 50,000 and 2 million; 500,000 and 1.5 million; 1 million and 2 million; 5 million and 20 million; 50 million and 200 million; 200 million and 1 billion; or 1 billion and 10 billion different molecules). In certain aspects, a library of the embodiments encodes polypeptides having a wide range of net charge, such as from about -30 to +30, -20 to +20, -10 to +20 or -5 to +10 (e.g., between about -5 and +14). In still further aspects, a library of the embodiments encodes polypeptides having a diversity of hydrophobicity such as polypeptides comprising from about 1% to about 80% hydrophobic amino acid positions (e.g. , between about 5% and 70%, 5% and 60% or 10% and 50% hydrophobic residues).

[0033] In some aspects, individually testing the different polypeptide molecules comprises individually testing the different polypeptide molecules on single cells or on about 1-10, 5-500, 500-1,000, 1,000-5,000, 5,000-30,000, 30,000-50,000, 5-100, or 10-50 live cells. In further aspects, individually testing the different polypeptides can comprise testing the molecules on cells (or populations of cells) isolated in a gel, a well (e.g. , of a microtiter plate), a tube or in a microcapsule of an emulsion. In some aspects, to achieve individual testing the different polypeptides, each isolated cell or cell population is contacted with, on average, one of the different polypeptide molecules (e.g. , in a emulsion of microcapsules comprising on average one different polypeptide per microcapsule). In still further aspects, the testing of the embodiments is performed at concentration of at least 10,000 (e.g. , at least about 15,000, 150,000, 1,500,000, 15 million or 150 million) distinct polypeptide library members per 1 mL of test volume and wherein the distinct polypeptides are comprised in separate microcapsules of an emulsion. [0034] In one embodiment, the present disclosure provides an enriched library of nucleic acid molecules encoding candidate polypeptides, such as antibodies, with a desired activity obtained by a method of the present embodiments.

[0035] Thus, in a further embodiment a polypeptide library is provided comprising a plurality of carrier particles wherein each particle comprises (a) one or more copies of a distinct nucleic acid molecule associated with the particle by a first binding moiety; and (b) a plurality of polypeptide molecules encoded by the distinct nucleic acid molecule, wherein each of said plurality of polypeptides is associated with the particle by a second binding moiety. For example, in some aspects, a library comprises at least about 0.1, 1, 10, 100 million, 1 billion or 10 billion carrier particles. In certain aspects, each of the carrier particles comprises 10, 100, 1,000, 10,000 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 20 million, 50 million or more copies of the distinct nucleic acid molecule (e.g., between about 1,000-100,000, 0.5-50 million, 0.5-10 million or 0.5-5 million copies of the molecule). Thus, in some aspects, each of the carrier particles comprises a plurality of polypeptide molecules, such as between about 1,000-100,000, 100,000-1 million, 1-10 million, 20-500 million, 0.01- 1 billion, 0.05-0.5 billion, 10-50 billion, 1-20 billion or 1-10 billion polypeptide molecules (e.g., more than about 10 million copies of the polypeptide molecule). In still further aspects, a library of the embodiments can be further defined by its diversity, for instance, a library can comprise between about 50,000 and 500,000, 5,000,000 or 5,000,000,000 distinct nucleic acid molecules. In still further aspects, the carrier particles of the library are comprised in microcapsules, such as the microcapsules of an emulsion (e.g., an emulsion comprising on average one carrier particle and distinct nucleic acid molecule per microcapsule). In some aspects, the polypeptide molecules of the library comprise antibodies (e.g. , comprising both a heavy and light chain sequence).

[0036] In yet a further embodiment, a carrier bead is provided comprising a functionalized surface bound to 1-10 million nucleic acid molecules and 1-20 billion polypeptide molecules. Beads for use according to the embodiments include, for instance, magnetic beads, cross-linked agarose beads, polystyrene beads, silica beads, microparticles and microspheres. Beads can have, with limitation, an average diameter of about 1-100 or 5- 80 μηι. In some cases, a bead can comprise at least 5, 10 or 15 billion polypeptide molecules and/or at least 5, 10, or 15 million nucleic acid molecules. In certain aspects, the nucleic acid molecules are bound to the bead by a biotin-avidin interaction. In still further aspects, the polypeptide molecules are bound to the bead by the binding of charged Ni groups on the bead by His tag sequences of the polypeptide molecules. In some cases, the nucleic acid molecules and/or the polypeptide molecules on the bead all comprise essentially identical sequences. In still further aspects, the polypeptide molecules bound to the bead(s) are encoded by the nucleic acid molecules bound to the bead. Thus, in still a further embodiment, library is provided comprising a plurality of beads in accordance with the embodiments wherein each bead is bound to nucleic acid molecules (and polypeptide molecules) comprising a unique sequence relative to the other beads of the library. For example, the library can comprise about 50,000 to 15 million beads bound to different nucleic acid sequences (e.g., at least or at most about 15,000, 150,000, 1,500,000, 15,000,000, million or 150,000,000 million beads bound to different nucleic acid sequences). In further aspects, the library has a concentration of at least 10,000, 20,000, 30,000 40,000 or 50,000 distinct polypeptide library members per 1 mL of volume.

[0037] In yet a further embodiment, a carrier bead is provided comprising a functionalized surface bound to 1,000-100,000 nucleic acid molecules and 1-100 million polypeptide molecules. Beads can have, without limitation, an average diameter of about 0.5-100 or 1-5 μηι. In some cases, a bead can comprise at least 5, 10 or 15 million polypeptide molecules and/or at least 5, 50, or 500 thousand nucleic acid molecules.

[0038] In a specific aspect a library provided comprising a plurality of His-tag binding beads wherein each particle comprises (a) a plurality copies of a distinct nucleic acid molecule associated with the bead a biotin tag bound to a his-tagged streptavidin polypeptide (that is in turn bound to the bead); and (b) a plurality of His-tagged polypeptide molecules encoded by the distinct nucleic acid molecule, wherein each of said plurality of polypeptides is associated with the bead via the His tag and wherein the polypeptide molecules encode a protease cleavage site that allows for cleavage away from the bead. In certain preferred aspects, the polypeptide molecules comprises an antibody (e.g. , an antibody heavy chain associated with a light chain).

[0039] In one embodiment, the present disclosure provides a method for expression of an antibody comprising: (a) providing a DNA encoding from 5' to 3' sequences encoding (i) a first promoter; (ii) an antibody heavy chain coding sequence; (iii) a second promoter; and (iv) an antibody light chain coding sequence; (b) transcribing the DNA in vitro to provide transcribed RNA; and (c) expressing the transcribed RNA in a cell-free translation system to provide a full-length antibody.

[0040] In one embodiment, the present disclosure provides a method for identifying or isolating an antibody comprising: (a) providing a DNA encoding from 5' to 3' sequences encoding (i) a first promoter; (ii) an antibody heavy chain coding sequence; (iii) a second promoter; (iv) an antibody light chain coding sequence; (v) a protease cleavage sequence; and (vi) a polypeptide tag; (b) transcribing the DNA in vitro to provide transcribed RNA; (c) expressing the transcribed RNA in a cell-free translation system; (d) purifying the antibody by binding the antibody to a binding moiety that binds to the polypeptide tag. In one aspect, the method may comprise contacting the purified antibody with a protease that cleaves the protease cleavage sequence to release the antibody from the polypeptide tag.

[0041] In a further embodiment, the present disclosure provides a DNA molecule for expression of an antibody comprising, from 5' to 3', sequences encoding: (i) a first promoter; (ii) an antibody heavy chain coding sequence; (iii) a second promoter; and (iv) an antibody light chain coding sequence. In one aspect, the DNA molecule may comprise, 3' of the antibody heavy chain coding sequence, sequences encoding (v) a protease cleavage sequence; and (vi) a polypeptide tag. In a further embodiment a DNA molecule is provided for expression of an antibody- binding protein comprising, from 5' to 3', sequences encoding: (i) a first promoter; (ii) a tag sequence (e.g., a poly-His); (iii) optionally, a protease cleavage site (e.g., a TEV cleavage site); (iv) optionally a cytotoxic or reporter polypeptide and (iv) an antibody-binding polypeptide (e.g., ZZ). In a further aspect, there is provided a polypeptide produced by expression of one of the nucleic acid molecules detailed above. [0042] In yet a further embodiment there is provided an apparatus, such as an apparatus that may be used for expression and/or screening of polypeptide sequences, such as polypeptides from a library. Exemplary embodiments include an apparatus configured for dosing beads, the apparatus comprising: a block comprising a first surface with a serpentine channel, and an inlet and an outlet in fluid communication with the serpentine channel; a plate comprising a plurality of apertures containing beads; and a first membrane comprising a library array of nucleic acid molecules, where the plate is positioned between the block and the first membrane, and the serpentine channel is in fluid communication with the plurality of apertures. [0043] In certain embodiments, the first membrane is a polydimethylsiloxane

(PDMS) membrane comprising DNA molecules. Particular embodiments further comprise: a second membrane located between the first membrane and the plate; and a third membrane located between the first surface with the serpentine channel. In specific embodiments, the second and third membranes are track-etched membranes. In certain embodiments, the second and third membranes are formed from a microporous polycarbonate film material. In exemplary embodiments, the second and third membranes comprise pores between 5 and 50 microns. In particular embodiments, the first membrane comprises DNA molecules, and the second membrane comprises pores, where the pores are configured to allow the DNA molecules of the first membrane to pass through the second membrane, and the pores are configured to retain the beads in the plurality of apertures in the plate. In specific embodiments, the plate is a stainless steel plate.

[0044] Certain embodiments further comprise: a base; and a compression mechanism, where: the block, the plate, and the first, second and third membranes are positioned between the base and the compression mechanism; and the compression mechanism is configured to compress the block, the plate, and the first, second and third membranes. Particular embodiments further comprise a compression frame position between the compression mechanism and the block. Specific embodiments further comprise: a fluid supply coupled to the inlet of the block, and a vacuum source coupled to the outlet of the block.

[0045] In certain embodiments, the serpentine channel comprises a first plurality of rows; the plurality of apertures in the plate are arranged in a second plurality of rows; and the first plurality of rows and the second plurality of rows comprise equivalent spacing between the first plurality of rows and the second plurality of rows. [0046] Exemplary embodiments include an apparatus configured for imaging a dosing array and an analyte cell array, the apparatus comprising: a first plate comprising a plurality of apertures containing the dosing array; and a second plate comprising a plurality of wells containing analyte cells, where individual apertures in the plurality of apertures are aligned with individual wells in the plurality of wells. In particular embodiments, the wells are nano wells. In specific embodiments, the wells have a diameter between 100 microns and 800 microns and a volume between 5 nanoliters and 60 nanoliters, or more specifically a diameter between 200 microns and 700 microns and a volume between 10 nanoliters and 50 nanoliters, or more specifically a diameter between 300 microns and 600 microns and a volume between 20 nanoliters and 40 nanoliters, or more specifically a diameter between 400 microns and 500 microns and a volume between 25 nanoliters and 30 nanoliters.

[0047] Certain embodiments further comprise: a block comprising a first surface with a plurality of channels extending to a perimeter of the block, where the first plate is positioned between the second plate and the first surface of the block. Exemplary embodiments further comprise a sealing membrane positioned between the first surface of the block and the first plate. Particular embodiments further comprise: a support plate; and a translucent plate, where the translucent plate is positioned between the support plate and the second plate comprising the plurality of wells containing analyte cells.

[0048] In specific embodiments, the support plate is configured to be coupled to an imaging device. Certain embodiments further comprise: a retention plate; and a clamp. In particular embodiments, the retention plate is configured to couple to the support plate; the retention plate comprises a central opening configured to receive the first plate, the second plate, the sealing membrane and the block; and the clamp is configured to secure the first plate, the second plate, the sealing membrane and the block between the clamp and the support plate. In specific embodiments, the sealing membrane is an oxygen permeable membrane, and in certain embodiments the sealing membrane is a polydimethylsiloxane (PDMS) sheet.

[0049] Particular embodiments further comprise a second and a third membrane, where: the second membrane is positioned between the first plate and the second plate; and the first plate is positioned between the second membrane and the third membrane. In specific embodiments, the second and third membranes are track-etched membranes. In certain embodiments, the second and third membranes are formed from a microporous polycarbonate film material. In particular embodiments, the second and third membranes comprise pores between 5 and 50 microns, or more specifically between 10 and 40 microns, or more specifically between 20 and 30 microns.

[0050] Exemplary embodiments include an apparatus configured for loading beads into a dosing plate, the apparatus comprising: a base; a compression mechanism; a frame; a plate comprising a plurality of apertures; a membrane; and a gasket. In certain embodiments, the compression mechanism is coupled to the base; the frame, the plate, the membrane and the gasket are arranged in a stacked array; the compression mechanism is configured to compress the stacked array. In particular embodiments, the base comprises a reservoir containing a fluid, and the stacked array is inserted into the reservoir. Specific embodiments further comprise alignment pins configured to align the stacked array and the reservoir. In certain embodiments, the fluid fills the apertures when the compression mechanism is activated to compress the stacked array. In particular embodiments, a fluid mixture comprising beads is distributed across the plate and the fluid mixture is retained by the frame. In certain embodiments, the membrane is configured to allow fluid from the reservoir to pass through the membrane and configured to a retain beads in the fluid mixture in the apertures of the plate. In specific embodiments, the membrane is a track-etched membrane. In certain embodiments, the membrane is formed from a microporous polycarbonate film material. In particular embodiments, the membrane comprises pores between 5 and 50 microns, or more specifically between 10 and 40 microns, or more specifically between 20 and 30 microns.

[0051] Exemplary embodiments include a method for loading expression reagent, the method including: obtaining a block comprising a first surface with a serpentine channel, and an inlet and an outlet in fluid communication with the serpentine channel; obtaining a plate comprising a plurality of apertures containing beads; positioning the plate such that the plurality of apertures are in fluid communication with the serpentine channel; coupling the inlet to a fluid supply comprising expression reagent; and providing a vacuum at the outlet. Exemplary embodiments further comprise providing a first membrane comprising a library array of nucleic acid molecules, where the plate is positioned between the block and the first membrane. In particular embodiments, the first membrane is a polydimethylsiloxane (PDMS) membrane comprising DNA molecules. Specific embodiments further comprise: providing a second membrane located between the first membrane and the plate; and providing a third membrane located between the first surface with the serpentine channel. In certain embodiments, the second and third membranes are track-etched membranes, and in particular embodiments, the second and third membranes are formed from a microporous polycarbonate film material. In specific embodiments, the second and third membranes comprise pores between 5 and 50 microns, or more specifically between 10 and 40 microns, or more specifically between 20 and 30 microns.

[0052] In certain embodiments, the first membrane comprises DNA molecules; and the second membrane comprises pores, where: the pores are configured to allow the DNA molecules of the first membrane to pass through the second membrane; and the pores are configured to retain the beads in the plurality of apertures in the plate. Particular embodiments further comprise compressing the block, the plate, and the first, second and third membranes. In certain embodiments, the plate is a stainless steel plate. In particular embodiments, the serpentine channel comprises a first plurality of rows; the plurality of apertures in the plate are arranged in a second plurality of rows; and the first plurality of rows and the second plurality of rows comprise equivalent spacing between the first plurality of rows and the second plurality of rows.

[0053] In still a further embodiment there is provided a bacterial expression system comprising a bacterial supernatant, produced by centrifugation of a bacterial lysate at not more than 20,000 times gravity and exogenously added factors to support RNA transcription and translation in the system. In some aspects, the system comprises (i) an exogenously added folding chaperone and/or (ii) the bacterial supernatant used to make the system is from a recombinant bacteria that comprises an expressed transgene encoding a folding chaperone. In some aspects, the bacterial supernatant (used to produce the expression system) is produced by centrifugation of the bacterial lysate at between about 8,000 and 20,000 times gravity. In further aspects, the bacterial supernatant is produced by centrifugation of the bacterial lysate at about 8,000-18,000, 10,000-15,000 or 1 1 ,000-13,000 times gravity (e.g., at about 12,000 x G).

[0054] In some aspects, a bacterial supernatant of the embodiments may be produced from a bacterial lysate without dialysis. In particular aspects, the folding chaperone for inclusion in an expression system is a Hsp 90 family chaperone, Hsp70 family chaperone, Hsp70/Hspl l0 family chaperone, DnaJ/Hsp40 family chaperone, a disulfide isomerase, a prolyl isomerase or a mixture thereof. In certain specific aspects, the folding chaperone is Grp94, BiP, Grpl 70, ErDJ3, PDIA1, PDIA6, Cyclophillin B or a mixture thereof. In specific aspects, the system comprises an exogenously added folding chaperone. In certain aspects, the system comprises at least two different exogenously added folding chaperones (e.g., a disulfide isomerase and a prolyl isomerase).

[0055] In further aspects, the bacterial lysate is an E. coli lysate. The bacterial lysate may be an E. coli lysate having a BL21(DE3) genetic background. In some aspects, the bacterial lysate is produced by sheer-force lysis of the cells. In certain aspects, the bacterial supernatant is from a recombinant bacteria that comprises an expressed transgene encoding a RNA polymerase. In particular aspects, the RNA polymerase may be a T7 or SP6 polymerase. [0056] In certain aspects, the exogenously added factors to support RNA transcription and translation in the system comprise a RNA polymerase, a pH buffer, a reducing agent, a salt, nucleotides, an energy production system, tRNA, amino acids and/or PEG. In specific aspects, the exogenously added factors to support RNA transcription and translation in the system comprise HEPS-KOH, DTT, ATP, CTP, GTP, UTP, folinic acid, E. coli tRNA, potassium glutamate, ammonium acetate, magnesium acetate, glucose, cAMP, creatine phosphate, creatine kinase, amino acids and PEG. In some other aspects, the system further comprises DNA encoding a polypeptide.

[0057] In a further embodiment there is provided a method of expressing a polypeptide comprising, contacting a DNA encoding the polypeptide with an expression system in accordance with the embodiments.

[0058] In still a further embodiment, there is provided a recombinant bacterial cell comprising an expressed transgene encoding a folding chaperone. In certain aspects, the transgene encoding the folding chaperone comprises a sequence encoding the folding chaperone linked to a heterologous promoter. In particular aspects, the heterologous promoter is an inducible promoter. In further aspects, the folding chaperone is a Hsp 90 family chaperone, Hsp70 family chaperone, Hsp70/Hspl l 0 family chaperone, DnaJ/Hsp40 family chaperone, a disulfide isomerase, a prolyl isomerase or a mixture thereof. In specific aspects, the folding chaperone is Grp94, BiP, Grpl70, ErDJ3, PDIA1 , PDIA6, Cyclophillin B or a mixture thereof.

[0059] In some aspects, the bacterial cell comprises an expressed transgenes encoding at least two different folding chaperones. In certain aspects, the bacterial cell is an E. coli cell. The bacterial cell may be an E. coli cell having a BL21(DE3) genetic background. In further aspects, the bacterial cell additionally comprises an expressed transgene encoding a RNA polymerase. In particular aspects, the RNA polymerase may be a T7 or SP6 polymerase. [0060] In yet still a further embodiment the invention provides a bacterial cell lysate produced from a bacterial cell in accordance with the embodiments and aspects described above. In specific aspects, the bacterial cell lysate is a bacterial lysate supernatant produced by centrifugation at not more than 20,000 times gravity. In further aspects, the bacterial lysate supernatant is produced by centrifugation of the bacterial lysate at between about 8,000 and 20,000 times gravity. In certain aspects, the bacterial lysate supernatant is produced by centrifugation of the bacterial lysate at about 8,000-18,000, 10,000-15,000 or 1 1,000-13,000 times gravity. In a particular aspect, the bacterial lysate supernatant is produced by centrifugation of the bacterial lysate at about 12,000 times gravity. In still further aspects, the bacterial lysate additionally comprises exogenously added factors to support RNA transcription and translation in the lysate.

[0061] As used herein, "essentially free," in terms of a specified component, is used herein to mean that none of the specified component has been purposefully formulated into a composition and/or is present only as a contaminant or in trace amounts. The total amount of the specified component resulting from any unintended contamination of a composition is therefore well below 0.05%, preferably below 0.01%. Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods.

[0062] As used herein in the specification and claims, "a" or "an" may mean one or more. As used herein in the specification and claims, when used in conjunction with the word "comprising", the words "a" or "an" may mean one or more than one. As used herein, in the specification and claim, "another" or "a further" may mean at least a second or more.

[0063] As used herein in the specification and claims, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects. [0064] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating certain embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [0066] FIG. 1: Flowchart detailing an exemplary antibody screening system.

[0067] FIG. 2: An exploded view of an exemplary bead loading apparatus.

[0068] FIG. 3: An exploded view of an exemplary dosing array.

[0069] FIG. 4: An exploded view of an exemplary dosing apparatus.

[0070] FIG. 5: A perspective view of an exemplary serpentine channel loading block.

[0071] FIG. 6: An exploded view of an exemplary imaging apparatus.

[0072] FIG. 7: A perspective view of an exemplary channeled retention block.

[0073] FIG. 8: A section view of an exemplary assembly of components used for imaging. [0074] FIG. 9: Figure shows a reproduction of an SDS-PAGE gel analysis of antibody molecules produced by expression extracts according to the embodiments. Expression in each case was in an S 12 extract system, in the presence of a redox buffer and was compared when various folding chaperones were included in the extract. Lanes are as follows 1 = No Chaperones; 2 = PDIA1 ; 3 = PDIA1 and CypB; 4 = PDIA1, CypB and Bip; 5 = PDIA1, CypB, Bip and ErDJ3; 6 = PDIA1, Bip and Grp94; 7 = PDIA1, CypB, Bip, ErDJ3 and Grp94; 8 = PDIA6, CypB, Bip, ErDJ3 and Grp94. [0075] FIG. 10: Graph shows the amount of antibody production for a variety of different antibodies using an S12 expression system according to the embodiments and either a full compliments of folding chaperones (PDIA1, CypB, Bip, ErDJ3 and Grp94) or only PDIA and CypB. For most antibodies PDIA and CypB appear sufficient to provide robust expression.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0076] Detailed herein is a system that allows for the efficient screening of highly diverse libraries of polypeptide molecules. The molecules can be screened not only for a desired binding affinity, but also for a desired biological activity on living cells. Polypeptides that are identified to provide a desired biological activity can be correlated with their nucleic acid coding sequence, which allows for a rapid determination of their sequence. A library DNA molecule may be associated with a bead or a surface (such a well or hydrogel) by clonal amplification on the bead or association with bead via a tagged primer following amplification. In some aspects, a second bead may comprise a second binding moiety that can be used to attach polypeptides (encoded in the library) to the second bead. Nucleic acid molecules that are members of the library are individually amplified, such as in separate emulsion microcapsules (e.g. , using bead-based emulsion PCR) or in separate wells of a plate. Thus, following amplification, an amplified on-bead library is provided wherein each member (or essentially each member) comprises a bead that is coated with multiple copies of the member nucleic acid, by virtue of a first binding moiety. For example, the beads can be streptavidin-coated beads and the member nucleic acids can be amplified using a biotin- tagged primer. In still further aspects, the beads can comprise His-tag binding moieties (e.g., Ni resin), where a portion of the bead is bound to His-tagged streptavidin (which in turn can bind to biotin-labeled nucleic acid library members). In some aspects, once subjected to amplification, the amplified on-bead library is purified to remove the reagents used for amplification.

[0077] Clonal expression of an amplified on-bead library can be achieved, e.g., by separating individual members into separate wells of a plate. For example, an amplified on- bead library may be applied to the wells of a plate such that each well (or essentially each well) comprises a single library member. The amplified on-bead library is then contacted with a cell-free transcription and translation system, such as a prokaryotic transcription and translation system. Expressed polypeptides from each library member can, once translated, bind to a second binding moiety on the second bead (via binding the heavy chain of the encoded polypeptides). Thus, once formed, an expressed on-bead library can comprise the bead coated with a plurality of a candidate polypeptides (such that the polypeptides may be cleaved-away from the bead). In some cases, the expressed on-bead library can be further purified to remove components of the transcription/translation system.

[0078] Next, an expressed on-bead library can be tested for a particular activity. In some cases, such testing is completed relative to living cells. However, in other cases, candidate polypeptides are screened in a cell-free system. For example, the library can be screened for members having high expression, members with proper folding, and/or members that bind with a particular binding affinity for a target. For example, in the case of candidate polypeptides that are antibodies the library can be screened for members that can bind to Fc- binding components, such as mammalian Fc receptor. Likewise, candidate antibodies can be screened to identify members of a library that bind to an antigen of interest or that have a particular binding affinity for an antigen. In such examples, screening of a library may be accomplished by, for example, passing the library through a column with a bound antigen of interest. In some aspects, candidate polypeptides identified in cell-free screening are used to generate an enriched library, which can be tested for activity on living cells. For example, members of the expressed on-bead library obtained in a screen can be subject to amplification forming an enriched library that may be likewise screened using the methods detailed herein. [0079] In still further aspects, members of an expressed on-bead library are individually tested for activity on living cells. Testing of bioactivity can be accomplished by separately contacting the library members with cells. In some cases the members of an expressed on-bead library are distributed to test cells, such that most compartments isolate no more than one (in accordance with random distribution at limiting dilution) bead/polypeptide- nucleic acid complex with one or more test cells (e.g. , in a well, gel matrix, or microcapsule). For instance, in other aspects, test cells can be grown in the wells of a plate and the library applied to the plate such that each well (or essentially each well) includes no more than one expressed on-bead library member (or multiple beads each comprising the same member of the expressed library). In some aspects, a plate comprising wells with the individual members of an expressed on-bead library is contacted with a corresponding plate comprising wells with living cells used for testing. [0080] In some aspects, biological activity is detected by detecting an optical or fluorescence signal (or the quenching of such a signal or the loss of such a signal). In the case of a plate, the wells of the plate can be scanned, such as by using an automated plate reader, and members showing biological activity collected from the indicated well. A variety of methods can be used for collecting library members showing biological activity. For example, nucleic acid molecules in positive wells can be dissociated from the bead to which they are bound and bound to a collection bead (such as a magnetic bead) for collection. Alternatively all library beads can have magnetically tagged (e.g., by a nanoparticle) nucleic acids, and the collection system set up to only collect nucleic acids that have been disassociated from beads. In still another example, nucleic acid molecules that correlate to positive well positions can be collected by suction or washing, such as with a syringe.

[0081] Hence, a system of the embodiments offers many significant advantages relative to other potential screening systems. For example, because the library is generated in situ it can have a nearly limitless size and diversity of sequence. Importantly, the candidate polypeptides remain associated with their coding sequences, first by virtue of the beads and then the well plate system (or other method of compartmentalization), so active molecules can be identified by sequencing of the coding sequence. However, unlike a phage display system, candidate polypeptides need not be covalently tethered to superfluous sequences (e.g., phage protein sequences). This allows the candidate polypeptides to fold independently of such sequences, which may provide molecules with a higher activity than a sterically hindered fusion protein. Additionally, any biological activity of identified polypeptides is truly indicative of an activity of the candidate polypeptide rather than non-bioactive binding, or an artifact of a phage fusion protein. Furthermore, this system makes it possible to test biological activity in live cells; in other words, the system is not limited to binding assays as is generally the case for phage-display and other display approaches. Thus, the methods of the embodiments not only provide for screening of a vast diversity of sequences, but also provide a screen that can be far more effective than any previous technique in providing biologically active candidate molecules. Further aspects applicable to the methods of the embodiments are discussed in detail below.

I. DNA Libraries

[0082] The platform provided herein enables the screening of polypeptide libraries for effects on living cells, such as internalization of antibodies. This requires the preparation of a high diversity DNA library encoding polypeptides. In some aspects, the library may be a library of molecules that is first selected by a high-throughput method for a desired property (e.g., target binding, folding, and enzymatic activity). For example, the library may be the product of screening using a cell expression, ribosome display or phage display system. In further aspects, the library may be a library of mutated sequences based on a molecule having a known therapeutic or biological activity (e.g., a therapeutic monoclonal antibody).

[0083] Certain aspects of the embodiments concern a library of DNA sequence, at least a subset of which encode a translation open reading frame (ORF) and can thereby serve as a template for protein synthesis. Thus, as used herein the term "library" is used in reference to a collection of molecules (e.g. , nucleic acid or polypeptide molecules) or cells wherein a plurality of individual species comprising the library are distinct from other cells or molecules of the same library in at least one detectable characteristic. Examples of libraries of molecules include libraries of nucleic acids, peptides, polypeptides, proteins, fusion proteins, polynucleotides, or oligonucleotides. [0084] The library sequences themselves can be generated by a variety of methods that are well known to those of skill in the art. To touch on a few such methods, the ORF for the library can be completely or partially composed of a randomized set of sequences (either by chemically synthesizing random sequence or by using error-prone amplification of a known sequence). In other cases, the library ORF sequences can be segments of genomic or cDNA sequences from an organism. For example, the ORF can be composed of segments of human cDNA. As a further example, the ORF can be composed of segments of genomic DNA sequences, which have been re-arranged in software to mimic somatic recombination events, such as in the mammalian (e.g. , human) immune system, and then chemically synthesized and assembled. Regardless of how the ORF sequences are produced, the library template will preferably include an ATG translation initiation codon that is optimized for prokaryotic or eukaryotic translation initiation and a stop codon.

[0085] Other sequences can be included adjacent to the library ORF to optimize expression, such as an internal ribosome entry site (IRES) or a templated poly-A tail. Furthermore, in certain aspects, a peptide tag, protease cleavage site, a cell penetrating peptide (CPP) and/or a cytotoxic moiety is encoded by the assembled dsDNA library. In some cases, one ore more of these elements can be included on the forward or reverse primer segment, in or on the library template itself. It will be recognized by a skilled worker that, in certain aspects, the library is engineered such that the additional sequence (for fusion), when expressed, forms an amino- or carboxy-terminal fusion protein with the library ORF. In this case, it may be preferred to include a spacer coding sequence, such a sequence encoding a stretch of poly glycine residues between the additional element and the library ORF. [0086] In some aspects, these molecules can be purified following construction and prior to linkage with a bead, e.g. , by size exclusion chromatography or gel purification. Once constructed the DNA library can be immobilized such as on a bead. In general, the bead will include an affinity moiety that allows the bead to interact with a nucleic acid molecule. For instance, the bead may be a Ni-coated or streptavidin-coated bead and a nucleic acid molecule for immobilization on the bead can include a biotin moiety. In some cases, each DNA molecule can include two affinity moieties, such as biotin, to further stabilize the DNA. Beads can include additional features for use in immobilizing nucleic acids or that can be used in a downstream screening or selection processes. For example, the bead may include a binding moiety (e.g. , Annexin V), a fluorescent label, or a fluorescent quencher. In some cases, the bead can be magnetic.

[0087] In certain embodiments a DNA library of the embodiments comprises (i) an ORF, including a translation initiation site (e.g. , an ATG codon in a favorable Kozak consensus or a Shine-Dalgarno ribosome binding site (RBS)) and termination codon; (ii) a polymerase promoter sequence (e.g. , a T7 polymerase binding site); (iii) a polymerase terminator sequence; and (iv) primer sequences that flank the ORF. In some preferred aspects the nucleic acid molecules further comprise an affinity tag, such a His tag or a biotin tag. For example, a library may be composed of molecules comprising, in order from 5' to 3', a biotin tag - a forward primer binding sequence - a polymerase promoter sequence - an ORF - a polymerase terminator sequence - a reverse primer-binding sequence - a biotin tag (e.g. , 5'- biotin-primer-T7 promoter-ORF-T7 terminator-primer-biotin-3'). In further aspects the ORF sequence can be further flanked by additional or alternative primer binding sequences such as, in order from 5' to 3', a biotin tag - a forward primer binding sequence - a polymerase promoter sequence - an additional forward primer binding sequence - an ORF- an additional reverse primer binding sequence - a polymerase terminator sequence - a reverse primer- binding sequence - a biotin tag.

[0088] A DNA library of the embodiments may be composed of naturally occurring or artificially synthesized molecules. For example, in certain aspects, a library is composed of nucleic acid sequences that represent genomic DNA sequences or cDNA sequences (or portions thereof) from an organism, such as a human. In further aspects, a library may comprise an essentially random ORF coding sequence. ORF coding sequence in a library can also be chimeric sequences including segments of sequence from two different organisms or segments of sequence derived from cDNA and segments that are randomized. Likewise, DNA microarrays can be used as a template for construction of a DNA library of the embodiments. In some aspects, a DNA library represents the entire (or nearly the entire) proteome of an organism, such as a human. In some preferred aspects a library is composed of artificially synthesized nucleic acid sequences derived from cDNA with one or more site specific randomized variants. In some aspects a library is composed of artificially synthesized single chain antibody fragments (e.g., a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of an immunoglobulin), where specific sequence segments in the variable region are randomized. In further aspects, the library comprises VH and VL sequences that are associated with one another but not covalently linked. [0089] Furthermore, in certain aspects, library sequences can include segments of sequence that encode polypeptides having a known function, such as a cell-binding domain or cell penetrating peptide (CPP) in the ORF sequence along with sequence-derived from cDNA, genomic DNA, or randomized sequence (i.e., to generate an ORF encoding a fusion protein). Thus, in certain aspects, DNA molecules of the embodiments comprise an ORF that comprises a CPP coding sequence along with a segment of library sequence (such as randomized sequence), 5' of the CPP coding sequence 3' of the CPP coding sequence or both. As used herein the terms "cell penetrating peptide" and "membrane translocation domain" are used interchangeably and refer to segments of polypeptide sequence that allow a polypeptide to cross the cell membrane (e.g. , the plasma membrane in the case of a eukaryotic cell). Examples of CPP segments include, but are not limited to, segments derived from HIV Tat, herpes virus VP22, the Drosophila Antennapedia homeobox gene product, or protegrin I. In still further aspects, library sequences can include segments of sequence that encode polypeptides that facilitate intracellular localization of the library polypeptides, such as sequences that promote escape from endosomes, provide nuclear localization or mitochondrial localization.

[0090] Methods for generating and amplifying a nucleic acid library of the embodiments are well known in the art. In certain embodiments, it may be desired to employ one or more techniques for the manipulation, isolation or amplification of nucleic acids. Such techniques may include, for example, the preparation of vectors as well as methods for cloning selected nucleic acid segments from a cell (e.g. , cloning cDNA sequences or fragments thereof). [0091] Nucleic acids used as a template for amplification may be isolated from cells, tissues or other samples according to standard methodologies (Sambrook et al, 1989) or amplified from synthetic DNA, where the synthetic DNA is derived from linear strands, plasmids, or from a DNA microarray. In certain embodiments, nucleic acids may be amplified from whole cells or tissue homogenates or biological fluid samples (with or without substantial purification of the template nucleic acid). The nucleic acid may be genomic DNA or fractionated or whole cell RNA. Where RNA is used, it may be desired to first convert the RNA to a complementary DNA by use of a reverse transcriptase, as outlined below.

[0092] The term "primer," as used herein, is meant to encompass any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template-dependent process. Typically, primers are oligonucleotides from ten to twenty and/or thirty base pairs in length, but longer sequences can be employed. Primers may be provided in double-stranded and/or single-stranded form, although the single-stranded form is preferred.

[0093] Pairs of primers designed to selectively hybridize to nucleic acids corresponding to a selected nucleic acid sequence are contacted with the template nucleic acid under conditions that permit selective hybridization. Depending upon the desired application, high stringency hybridization conditions may be selected that will only allow hybridization to sequences that are completely complementary to the primers. In other embodiments, hybridization may occur under reduced stringency to allow for amplification of nucleic acids comprising one or more mismatches with the primer sequences. Once hybridized, the template-primer complex is contacted with one or more enzymes that facilitate template-dependent nucleic acid synthesis. Multiple rounds of amplification, also referred to as "cycles," are conducted until a sufficient amount of amplification product is produced. [0094] A number of template dependent processes are available to amplify the oligonucleotide sequences present in a given template sample. One of the best known amplification methods is the polymerase chain reaction (referred to as PCR) which is described in detail in U.S. Patents 4,683,195, 4,683,202 and 4,800,159, and in Innis et al , 1988, each of which is incorporated herein by reference in their entirety.

[0095] A reverse transcriptase PCR amplification procedure may be performed to generate cDNA sequence (or cDNA fragments). Methods of reverse transcribing RNA into cDNA are well known (see Sambrook et al , 1989). Alternative methods for reverse transcription utilize thermostable DNA polymerases. These methods are described in WO 90/07641. Polymerase chain reaction methodologies are well known in the art. Representative methods of RT-PCR are described in U.S. Patent 5,882,864. [0096] Another method for amplification is ligase chain reaction ("LCR"), disclosed in European Application 320 308, incorporated herein by reference in its entirety. U.S. Patent 4,883,750 describes a method similar to LCR for binding probe pairs to a target sequence. A method based on PCR and oligonucleotide ligase assay (OLA), disclosed in U.S. Patent 5,912,148, may also be used. [0097] Alternative methods for amplification of target nucleic acid sequences that may be used in the practice of the present invention are disclosed in U.S. Patents 5,843,650, 5,846,709, 5,846,783, 5,849,546, 5,849,497, 5,849,547, 5,858,652, 5,866,366, 5,916,776, 5,922,574, 5,928,905, 5,928,906, 5,932,451, 5,935,825, 5,939,291 and 5,942,391, GB Application No. 2 202 328, and in PCT Application No. PCT/US89/01025, each of which is incorporated herein by reference in its entirety.

[0098] Qbeta Replicase, described in PCT Application No. PCT/US87/00880, may also be used as an amplification method in the present invention. In this method, a replicative sequence of RNA that has a region complementary to that of a target is added to a sample in the presence of an RNA polymerase. The polymerase will copy the replicative sequence which may then be detected.

[0099] An isothermal amplification method, in which restriction endonucleases and ligases are used to achieve the amplification of target molecules that contain nucleotide 5'- [alpha-thio] -triphosphates in one strand of a restriction site may also be useful in the amplification of nucleic acids in the present invention (Walker et al, 1992). Strand Displacement Amplification (SDA), disclosed in U.S. Patent 5,916,779, is another method of carrying out isothermal amplification of nucleic acids, which involves multiple rounds of strand displacement and synthesis, i.e., nick translation.

[00100] Other nucleic acid amplification procedures include transcription- based amplification systems (TAS), including nucleic acid sequence based amplification (NASBA) and 3SR (Kwoh et al, 1989; Gingeras et al , PCT Application WO 88/10315, incorporated herein by reference in their entirety). European Application No. 329 822 discloses a nucleic acid amplification process involving cyclically synthesizing single- stranded RNA ("ssRNA"), ssDNA, and double-stranded DNA (dsDNA), which may be used in accordance with the present invention. [00101] PCT Application WO 89/06700 (incorporated herein by reference in its entirety) discloses a nucleic acid sequence amplification scheme based on the hybridization of a promoter region/primer sequence to a target single-stranded DNA ("ssDNA") followed by transcription of many RNA copies of the sequence. This scheme is not cyclic, i.e. , new templates are not produced from the resultant RNA transcripts. Other amplification methods include "race" and "one-sided PCR" (Frohman, 1990; Ohara et al, 1989).

[00102] As detailed herein, in certain aspects, a library of DNA molecules of the embodiment can be bound to a support such as bead. For example, in the case a library of DNA molecules that comprise a biotin moiety, the library can be bound to streptavidin-coated beads. In still further aspects, a bead for use in the embodiments can comprise one or more binding moieties (e.g., a polypeptide and a cell-binding moiety) and/or a moiety that aids in purification of the bead (e.g. , a bead may comprise a fluorescent marker or the beads can be magnetic).

[00103] As used herein a "cell-binding moiety" refers to a molecule that binds to a component of a test cell such as a cell surface protein or an intracellular protein. Such moieties can bind to cells generally or bind to specific cell populations (e.g., stem cells, cells of certain tissue type or cells that are apoptotic). For example, the cell-binding moiety can be an antibody (e.g. , a monoclonal antibody), an aptamer, a lectin, a proteoglycan, or a receptor or ligand polypeptide. In some specific aspects, the cell-binding moiety is Annexin V or an anti-CD34 antibody. In another example the cell-binding moiety is an anti-CD-63 antibody, which will bind to activated basophils. In this case, the assay could be used to screen polypeptides for induction of allergic reactions. Further examples of cell-binding moieties include anti-CD44+, anti-CD49fhi or CD133hi antibodies for binding to estrogen-negative breast cancer cells. In a further example, the cell-binding moiety can be a protein expressed by the cell as a transgene. For instance, an anti-microbial polypeptide that causes cell lysis of E. coli can be detected by (i) expressing maltose binding protein with a histidine-tag in the E. coli test cells, and (ii) using a nickel-charged chelating group as a binding moiety to capture the maltose binding protein that is released from E. coli cells that are lysed.

A. CDR region sequence preparation

[00104] In the case where the polypeptide of the library is an antibody, a library may comprise scFv coding sequence or partial or complete antibody coding sequences. Information regarding the general domain structure of an antibody can be found for example in Kabat (1991). Immunoglobulin G (IgG) antibodies consist of four polypeptide chains - two identical heavy chains and two identical light chains. The heavy and light chains are composed of constant and variable regions. In the final assembled and folded antibody macromolecules, the variable regions form a pocket of hypervariable sequence segments that is the antigen-binding site of the antibody. These variable regions are called complementarity-determining regions, or CDRs. The heavy and light chains each have three CDRs, which are responsible for the diversity and antigen specificity of the antibodies. The germline genomic DNA sequences that are expressed to form the CDRs, as well as the ways in which the human immune system introduces diversity, are well known. Three gene segments are involved in forming the heavy chain CDRs, known as the variable (V) segment, diversity (D) segment, and joining (J) segment. The light chain CDRs are formed from only V and J domains. Two types of light chains are possible in human IgG antibodies - called kappa and lambda. The human genome contains several different versions of each gene segment. As B-lymphocytes mature they each assemble their own antibody gene from these segments by somatic recombination.

[00105] Sequence frameworks containing the variable regions of IgG heavy chains, and kappa light chains can be found for example in the databases provided to the scientific community by IMGT®, the international ImMunoGeneTics information system® (on the World Wide Web at imgt.org), VBASE2 (on the World Wide Web at vbase2.org/vbase2.php), or AbYsis (on the World Wide Web at bioinf.org.uk/abysis/). The amino acid sequences of the IgGl and kappa constant regions of the heavy and light chains can be obtained from the NCBI Protein database (on the World Wide Web at ncbi.nlm.nih.gov/protein). Such a framework, optimized for bacterial extract cell-free expression, and containing both primer sites and restriction enzyme cut sites flanking each CDR, may be used. The heavy and light chain frameworks may be designed into one plasmid where both genes have a transcription start site and ribosome binding site and one transcription termination site follows the last gene.

[00106] The amino acid sequences of all heavy chain CDRl and CDR2, and all kappa light chain CDRl and CDR2 regions observed in human B lymphocyte populations can be found, for example, in the IMGT® (on the World Wide Web at imgt.org), VBASE2 (on the World Wide Web at vbase2.org/vbase2.php), or AbYsis (on the World Wide Web at bioinf.org.uk/abysis/) databases.

[00107] A software program that mimics the somatic recombination events observed in the human immune system can be used to produce a large library of CDR3 sequences. The resulting sequences may contain all known IgG heavy chain V, D, and J domains, as well as IgG kappa light chain V and J domains. [00108] The heavy chain CDRl and CDR2, as well as the kappa light chain

CDRl and CDR2 amino acid sequences may be manually identified. A software program may be used to reverse translate the sequences into DNA sequences and add a specific set of primer and restriction enzyme cut sites to the sequences while keeping CDR sequences in the correct reading frame. [00109] A software program may be used to assemble heavy chain CDR3 regions. Data files containing DNA sequences of germline D and J regions may be prepared. In accordance with known patterns of B lymphocyte rearrangement of IgG genes, the program can be used to generate palindromic DNA sequences (P), randomly incorporated deletions and additions in a certain partem and at a certain pre-determined rate. For this, each output sequence has structure: [PNP>D<PNP>J], where P indicates a palindrome of length zero (80% probability), two (15%), or four (5%); N indicates zero (20%), one (20%), two (20%), three (20%), or four (20%) random nucleotides; the "D" and "J" sequences are read from the input files; the greater-than sign (>) indicates a downstream deletion of zero (80%), one (15%), or two (5%) bases; and the less-than sign (<) indicates an upstream deletion of zero (80%), one (15%), or two (5%) bases. Only sequences that contained no Cys, no Lys, no Asn, and no stop codon are recorded for use in the CDR library. Only sequences that are free from any of the restriction enzyme cut sites that are chosen to flank the CDRs or the variable regions are recorded. A sequence is only recorded if it contains the amino acid sequence WGXG. Each sequence is only recorded once. A specific set of primer and restriction enzyme cut site sequences that enable integration into the antibody framework are added to the CDR sequences, while keeping CDR sequences in the correct reading frame.

[00110] A software program may be used to assemble kappa light chain CDR3 regions. Data files containing DNA sequences of germline V and J regions can be prepared. In accordance with known patterns of B lymphocyte rearrangement of IgG genes, the program may randomly incorporated deletions and additions in a certain pattern and at a certain pre-determined rate. For this, each output sequence has the structure: [V< N>J], where N indicates zero (20%), one (20%), two (20%), three (20%), or four (20%) random nucleotides; the "V" and "J" sequences are read from the input files; the greater-than sign (>) indicates a downstream deletion of zero (80%), one (15%), or two (5%) bases; and the less- than sign (<) indicates an upstream deletion of zero (80%), one (15%), or two (5%) bases. The final sequence is determined by identifying the FGXG amino acid motif and an upstream Cys residue; the sequence starts after the Cys; if no FGXG motif was found, the sequence is rejected. Only sequences that contain no Cys, no Lys, no Asn, and no stop codon are recorded for use in the CDR library. Only sequences that are free from any of the restriction enzyme cut sites that are chosen to flank the CDRs are recorded. Each sequence is only recorded once. A specific set of primer and restriction enzyme cut site sequences that enable integration into the antibody framework are added to the CDR sequences, while keeping CDR sequences in the correct reading frame.

[00111] In order to prepare scFv and antibody sequences for use with the screening platform provided herein, isolated CDR sequences must be integrated into a scFv and/or an antibody framework at specific locations. This is achieved using a combination of overlap extension PCR, ligation, and circular amplification. Protocols for overlap extension PCR can be adapted and modified from Warrens et al. (1997).

B. Mutation and diversification of sequences

[00112] A wide range of methodologies are know for mutating a diversifying sequence for a library. In some cases random mutagenesis is used. In other aspects, a focused mutagenesis (e.g., of a particular region of a polypeptide) is employed. For example, protocols for high diversity DNA synthesis via mask-less photolithographic DNA microarrays can be found for example in Singh-Gasson (1999), incorporated herein by reference. The sequence data generated with the software programs may be used to generate exposure patterns for microarray synthesis, such that a library of 2 million DNA molecules, each with a different 105 base DNA sequence, may be synthesized. The process is expected to produce millions of variants of each DNA sequence. Free DNA in solution is derived from the microarray.

II. Amplification of individual library members

[00113] The screening platform provided herein provides the ability to go from a DNA library, in which each member contains open reading frames encoding a candidate polypeptide (e.g., light and heavy chains that form a scFv or an antibody), to information about which polypeptides have a desired property or activity. One aspect of this platform is the ability to compartmentalize library members through amplification (e.g. , PCR), transcription-translation (e.g. , cell-free expression), and screening. The inventors provide a method that is based on miniature wells ranging in density from 10 wells/mm² to 100,000 wells/mm².

[00114] One of the steps of the methods detailed herein involves amplification of individual library member (and association of the member with a bead). In some cases the individual member are deposited into the wells of a plate. In other aspects, the members are dispersed such that the majority of reactions comprise from 0 to 2 (or 0 to 1) member of the library separated from other library members. In some aspects the physical separation is provided by separate wells of a nano- or pico-well plate. In further aspects, microcapsules of an emulsion system may be used.

A. Plate and well systems

[00115] Micro-well plates (or nano-well or pico-well plates) are a well developed technology and can be constructed from a wide range of materials (plastics, rubber, metals, glass) and spanning sizes from 50 nm to around 1 mm. The filling of micro- wells can be accomplished, for example, by the liquid front associated with a rigid hydrophilic object (i.e., wiping). Similarly, patterned wetting was demonstrated using a thin glass probe. Loading of micro wells facilitated by a set array of venting holes has also been shown developed. Solution was first loaded into the space between the micro well plate at the bottom and the venting plate at the top; as the venting plate was moved to transiently overlap with the micro wells, air escaped from the wells as solution flowed from the interstitial space into the wells.

[00116] Most applications utilize some sort of functionalization of the well surface, and some use beads for one or more steps in a process. Nagai et al. (2001) found that the lower limit of volume for successful PCR in silicon micro wells was 86 pL using a quencher-based qPCR approach. Nanoliter quantitative PCR systems in through-hole arrays have been developed by Biotrove Inc. They use a stainless steel plate with through-holes, where the interior is coated with a PCR-compatible PEG hydrophilic layer, and the outer surface is coated with a fluoro-alkyl hydrophobic layer. The array of 3072 holes is split up into 48 regions of 64 holes. Each hole has a capacity of 33 nL. Each region of 64 holes is loaded with primer pairs simultaneously using pin tool transfer from a 384-well source-plate; the 64-hole regions are patterned with the same pitch as a 384-well plate. Miniaturized quantitative reverse-transcription PCR has been carried out in nanoliter wells. Wells were pre-loaded with primer pairs manually, and PCR mix loaded in a low-cell via vacuum, and sealed with PDMS.

[00117] There is a wide range of methods for construction of well/plate systems. For example, a hybrid elastomer-metal stencil was developed to enable precise patterning of protein microarrays. While the stencil is in contact with the substrate it forms nanoliter-scale wells. For digital biology applications, two micro well patterns were overlaid in a lattice arrangement inside a micro fluidic channel. The lattice arrangement enabled double area density of wells compared to a single layer. Filling without air bubbles and sealing with immiscible phase was also demonstrated.

B. Emulsion systems

[00118] A wide variety of microencapsulation procedures are available (see Benita, 1996) and may be used to create microcapsules used in accordance with the present embodiments. For example, in some aspects, an emulsion PCR system is used for amplifying library sequences. More than 200 microencapsulation methods have been identified in the literature (Finch, 1993). These include membrane enveloped aqueous vesicles such as lipid vesicles (liposomes; New, 1990) and non-ionic surfactant vesicles (van Hal et al , 1996). These are closed-membranous capsules of single or multiple bilayers of non-covalently assembled molecules, with each bilayer separated from its neighbor by an aqueous compartment. In the case of liposomes the membrane is composed of lipid molecules; these are usually phospholipids but sterols such as cholesterol may also be incorporated into the membranes (New, 1990). A variety of enzyme-catalyzed biochemical reactions, including RNA and DNA polymerization and RNA translation, can be performed within liposomes (Chakrabarti et al , 1994; Oberholzer et al, 1995a; Oberholzer et al , 1995b; Walde et al , 1994; Wick & Luisi, 1996). Enzyme-catalyzed biochemical reactions have also been demonstrated in microcapsules generated by a variety of other methods. Many enzymes are active in reverse micellar solutions (Bru & Walde, 1991 ; Bru & Walde, 1993; Creagh et al , 1993; Haber et al , 1993; Kumar et al. , 1989; Luisi and Steinmann-Hofmann, 1987; Mao & Walde, 1991 ; Mao et al, 1992; Perez-Gilabert et al , 1992; Walde et al , 1994; Walde et al , 1993; Walde et al, 1988) such as the AOT-isooctane-water system (Menger & Yamada,

1979) .

[00119] With a membrane-enveloped vesicle system much of the aqueous phase is outside the vesicles and is therefore non-compartmentalized. In some aspects, this continuous, aqueous phase is removed or the biological systems in it inhibited or destroyed (for example, by digestion of nucleic acids with DNase or RNase) in order that the reactions are limited to the microcapsules (Luisi and Steinmann-Hofmann, 1987).

[00120] Microcapsule droplets can also be generated by interfacial polymerization and interfacial complexation (Whateley, 1996). Microcapsules of this sort can have rigid, nonpermeable membranes, or semipermeable membranes. Semi-permeable microcapsules bordered by cellulose nitrate membranes, polyamide membranes and lipid- polyamide membranes can all support biochemical reactions, including multienzyme systems (Chang, 1987; Chang, 1992; Lim, 1984). Alginate/polylysine microcapsules (Lim & Sun,

1980) , which can be formed under very mild conditions, have also proven to be very biocompatible, providing, for example, an effective method of encapsulating living cells and tissues (Chang, 1992; Sun et al, 1992). Non-membranous microencapsulation systems based on phase partitioning of an aqueous environment in a colloidal system, such as an emulsion, may also be used.

[00121] Preferably, the microcapsule droplets of the present embodiments are formed from emulsions. The primary water-in-oil microcapsule droplets are formed from heterogeneous systems of two immiscible liquid phases with one of the phases dispersed in the other as droplets of microscopic or colloidal size (Becher, 1957; Sherman, 1968; Lissant, 1974; Lissant, 1984). Emulsions may be produced from any suitable combination of immiscible liquids. Preferably the emulsion of the present embodiments has water that contains the biochemical components, as the phase present in the form of finely divided microcapsules (the disperse, internal or discontinuous phase) and a hydrophobic, immiscible liquid (an "oil", such as mineral oil) as the matrix in which these microcapsules are suspended (the nondisperse, continuous or external phase). Such emulsions are termed "water-in-oil" (w/o). This has the advantage that the entire aqueous phase containing the biochemical components is compartmentalized in discreet microcapsules (the internal phase). The hydrophobic oil phase, generally contains none of the biochemical components and hence is inert. [00122] The primary emulsion may be stabilized by addition of one or more surface-active agents (surfactants). These surfactants are termed emulsifying agents and act at the water/oil interface to prevent (or at least delay) separation of the phases. Many oils and many emulsifiers can be used for the generation of water-in-oil emulsions; a recent compilation listed over 16,000 surfactants, many of which are used as emulsifying agents (Ash and Ash, 1993). Particularly suitable oils include light white mineral oil and non-ionic surfactants (Schick, 1966) such as sorbitan monooleate (Span™80; ICI), octyl phenol ethoxylate (Triton™ X-100) and polyoxyethylenesorbitan monooleate (Tween™80; ICI). Other emulsifying agents that may be used include, silicone-based emulsifier such as Bis- PEG/PPG-14/14 Dimethicone, Cyclopentasiloxane (ABIL EM 90). [00123] The use of anionic surfactants may also be beneficial. Suitable surfactants include sodium cholate and sodium taurocholate. Particularly preferred is sodium deoxycholate, at a concentration, such as 0.5% w/v, or less. Inclusion of such surfactants can, in some cases, increase the expression of the nucleic acids molecules and/or the activity of the encoded polypeptides. Addition of some anionic surfactants to a non-emulsified reaction system completely abolishes translation. During emulsification, however, the surfactant is transferred from the aqueous phase into the interface and activity is restored. Addition of an anionic surfactant to the mixtures to be emulsified ensures that reactions proceed only after compartmentalization.

[00124] Creation of an emulsion generally requires the application of mechanical energy to force the phases to mix together. There are a variety of ways of doing this, which utilize a variety of mechanical devices, including stirrers (such as magnetic stir- bars, propeller and turbine stirrers, vortexers, paddle devices and whisks), homogenizes (including rotor-stator homogenizes, high-pressure valve homogenizes and jet homogenizes), colloid mills, ultrasound and "membrane emulsification" devices (Becher, 1957; Dickinson, 1994).

[00125] Water-in-oil microcapsule emulsions of the present embodiments are generally stable with little if any exchange of contents (e.g. , nucleic acids) between the microcapsules. Additionally, biochemical reactions proceed in emulsion microcapsules. Moreover, complicated biochemical processes, notably gene transcription and translation are also active in emulsion microcapsules. The technology exists to create emulsions with volumes all the way up to industrial scales of thousands of liters (Becher, 1957; Sherman, 1968; Lissant, 1974; Lissant, 1984).

[00126] The preferred microcapsule size will vary depending upon the precise requirements of any individual selection process that is to be performed according to the present invention. In all cases, there will be an optimal balance between gene library size, the required enrichment and the required concentration of components in the individual microcapsules to achieve efficient expression and reactivity of the gene products.

[00127] For emulsion PCR, an emulsion PCR reaction is created by vigorously shaking or stirring a "water in oil" mix to generate a multitude of miniature aqueous compartments. The DNA library is mixed in a limiting dilution to generate compartments containing, on average, just one DNA molecule and bead (at the optimal dilution many compartments may be empty). To facilitate amplification efficiency, both an upstream (low concentration, matches primer sequence on bead) and downstream PCR primers (high concentration) are included in the reaction mix. Depending on the size of the aqueous compartments generated during the emulsification step, up to 3 X 10' individual PCR reactions per μΐ can be conducted simultaneously in the same tube. Essentially each little compartment in the emulsion forms a micro PCR reactor. The average size of a compartment in an emulsion ranges from sub-micron in diameter to over 100 microns, depending on the emulsification conditions.

III. Expression from an on-bead library

[00128] In preferred aspects, members of an on-bead library are individually expressed after being separately deposited in wells of a plate (as described above). In certain other aspects, an emulsion expression system is used to individually express member polypeptides of a library. For example, protocols are provided in Tawfik and Griffiths 1998; Ghadessy et al. 2001; Ghadessy and Hollinger 2004 and in U.S. Pat. Publns. 20070077572 and 20090197248, each of which is incorporated herein by reference in its entirety.

[00129] In general, expression involves providing the nucleic acid molecules in the presence of factors required for expression, which can be produced recombinantly, provided by cell lysates (or extracts thereof) or a combination of the two. In the case of nucleic acids molecules composed of RNA, only translation machinery needs to be provided. However, in preferred aspects the nucleic acid molecules are DNA and the expression system includes factors for RNA synthesis and protein synthesis (i.e. , transcription and translation). Reagents for such combined transcription and translation ("TnT") are commercially available and can be used in accordance with the embodiments (see e.g. , the TNT® systems available from Promega, Madison WI).

[00130] The processes of expression must occur within each individual well or microcapsule provided by the present embodiments. Both in vitro transcription and coupled transcription-translation become less efficient at sub-nanomolar DNA concentrations. Because of the requirement for only a limited number of DNA molecules to be present in each well or microcapsule. In some aspects a eukaryotic translation system (such as a mammalian cell lysate) is used in the expression system. In this case, the efficiency of protein synthesis may be significantly enhanced by providing a transcription system that includes reagents to mediate capping of the RNA transcripts and/or additional of a poly-A tail to the RNAs. In still further aspects, a stretch of poly-A residues may be template on the coding DNA molecules (e.g., following the ORF coding sequence).

[00131] The effective genetic element, namely, DNA or RNA, concentration in the microcapsules may be artificially increased by various methods that will be well known to those versed in the art. These include, for example, the addition of volume excluding chemicals such as polyethylene glycols (PEG) and a variety of gene amplification techniques, including transcription using RNA polymerases including those from bacteria such as E. coli (Roberts, 1969; Blattner and Dahlberg, 1972; Roberts et al, 1975; Rosenberg et al , 1975), eukaryotes e.g. (Weil et al , 1979; Manley et al , 1983) and bacteriophage such as T7, T3 and SP6 (Melton et al , 1984); the polymerase chain reaction (PCR) (Saiki et al , 1988); Q.beta. replicase amplification (Miele et al, 1983; Cahill et al , 1991 ; Chetverin and Spirin, 1995; Katanaev et al , 1995); the ligase chain reaction (LCR) (Landegren et al , 1988; Barany, 1991); and self-sustained sequence replication system (Fahy et al , 1991) and strand displacement amplification (Walker et al, 1992). Even gene amplification techniques requiring thermal cycling such as PCR and LCR could be used if the emulsions and the in vitro transcription or coupled transcription-translation systems are thermostable (for example, the coupled transcription-translation systems could be made from a thermostable organism such as Thermus aquaticus). Increasing the effective local nucleic acid concentration enables larger microcapsules to be used effectively.

[00132] The well or microcapsule size must be sufficiently large to accommodate all of the required components of the biochemical reactions that are needed to occur within the microcapsule. For example, in vitro, both transcription reactions and coupled transcription-translation reactions require a total nucleoside triphosphate concentration of about 2 mM. In the case of reactions involving translation, it is to be noted that the ribosomes necessary for the translation to occur are themselves approximately 20 nm in diameter. Hence, the preferred lower limit for microcapsules is a diameter of approximately 0.1 μιη (100 nm).

[00133] In some aspects, the platform provided herein enables the screening of an antibody library against live cells to identify those antibodies that exhibit a desired activity. A DNA library that encodes a diverse population of human antibodies can be expressed in vitro to form a library of full-size antibodies in a way that is suitable for the screening platform described. A modified cell-free expression system may include a mixture of protein chaperones (Table 1) that aid in the correct folding of full-length antibody. Such a procedure may be modified from examples found in Schwarz et al (2007) and S12 extract based on Kim et al. (2006).

Table 1. Chaperones to Improve Antibody Folding

[00134] The design of the plasmid encoding the heavy and light chains of the human antibody may influence the expression of a library of full-size antibodies in a way that is suitable for the screening platform described. Three DNA constructs have been designed for the use in full-length antibody library development and synthesized by DNA2.0 (Menlo Park, CA). Both designs contain the DNA sequence for the light chain (variable and constant) and the heavy chain (variable and constant, and optionally toxin cloning site, TEV protease site, and His tag). Library construct 1 is organized so that the heavy chain follows the primary T7 promoter and ribosome binding site (RBS) and possesses a start codon and ends with a stop codon to prevent translation read through. A spacer region comprising a secondary T7 promoter and RBS follow immediately downstream of the heavy chain and is responsible for initiating transcription/translation of the light chain. The light chain has a start and stop codon to enable production of the light chain independently of the heavy chain. A transcription termination site follows the light chain. A single transcription termination site enables production of mRNA that has both genes as well as a shorter mRNA for the second gene. Library construct 2 is similar to construct 1 but the gene order has been swapped. Library construct 3 is similar to construct 2 but the second T7 promoter upstream of the heavy chain gene was removed.

[00135] It is hypothesized that the gene order of construct 1 is more favorable than construct 2 because the light chain is present in both mRNA products enabling higher production of light chain than heavy chain. The presence of extra light chain aids in the correct folding of the heavy chain, which requires binding to the light chain. Construct 3 did not produce any full length antibody that could be purified by protein G. Most likely, the ratio of heavy to light chain protein was not optimal for correct assembly.

IV. Cell culture [00136] A wide range of cells can be compartmentalized such as in wells of plate or in microcapsules, such as the aqueous microcapsules of a water-in-oil emulsion (see, e.g. , Ghadessy, 2001). For micro-wells (or nano-wells), surfaces can be prepared in a similar fashion to conventional tissue culture; e.g., with tissue-culture treated plastic, or with extracellular matrix proteins (e.g. , collagen, fibronectin). As such, a wide variety of adherent cell types (e.g. , primary cells and cell lines) can be cultured in micro-wells under conditions similar to those in conventional macro-scale tissue culture. In the case of emulsions, cells that have been adapted for growth in suspension. For example, cells that overexpress MDM2 can be used, as can suspension adapted HeLa S3 cells a variety of leukemia cell lines (e.g. , Jurkat), and certain strains of 293T cells. In some other aspects, cells are not adapted for suspension growth. For instance such cells may be grown in the wells of a plate prior to testing. In these cases cells will be suspended for distribution into the wells of a plate. For example, cells isolated from a tissue being grown on a substrate can be disrupted by mechanical agitation and/or treatment with protease (e.g., trypsin) prior to distribution; in some cases such cells will grow in cluster or spheroids and exhibit desirable properties for bioactivity testing. In some aspects, cells are distributed in wells such that the majority of wells comprise between about 1 to 10, 5 to 20, 10 to 50, 10 to 100 or 50 to 500 cell per well. [00137] Microcapsules or wells comprising cells can further comprise components that will be used to assay for biological activity of the library polypeptides. For example, such components can include fluorescent dyes, buffers, ions (e.g. , Ca²⁺, or Mg²⁺), enzymes, antibodies, cofactors and the like. Likewise, nuclease inhibitors, protease inhibitors and/or non-specific blockers, to reduce non-specific or low affinity interactions between a binding moiety and its target, can be included. Non-specific blockers can be, for example, abundant serum proteins, such albumin (e.g. , bovine serum albumen (BSA)). In further aspects, any of the foregoing components can be added to the system just prior to performing an assay to identify cells that exhibit a biological response.

V. Assay for bioactive polypeptides [00138] In a further aspect, the embodiments provide systems and methods for screening or sorting test cells and/or microcapsules in a liquid (e.g., of a microcapsule), a well, a tube or a gel and assessing biological activity of polypeptides. For example, a characteristic of cells in a micro-well may be sensed and/or determined in some fashion (e.g. , as further described below), and the location of the well may be chosen for delivery of a reagent that makes it possible to specifically recover nucleic acid from that well. In further aspects that reagent delivered to micro-wells where cells have specific characteristics may disassociate nucleic acids from library beads. A magnetic collection system may then be applied to only collect nucleic acids that have been disassociated from library beads.

[00139] In some aspects, wide range of cellular assays can be employed in micro-well plates, such as uptake/exclusion of fluorescent dyes (e.g., nucleic acid dyes), fluorescent protein reporter systems, and cell viability assays. For example, cells expressing a fluorescent protein with a tag (e.g. , PEST) that causes high turnover of the protein will provide a good assay for any cellular effect that renders the cell unable to synthesize proteins.

[00140] Alternatively, a characteristic of a cell or microcapsule may be sensed and/or determined in some fashion (e.g., as further described below), then the microcapsule or cell may be selected or directed towards a particular region of the device, for example, for sorting or screening purposes. In further aspects, cells or microcapsules can be purified based on a detectable bioactivity of a polypeptide. For example, in the case an activity that changes the composition at the cell surface, a moiety, such as an antibody that detects this change can be used to purify the cell. [00141] In some aspects, the micro-wells may be opened before an assay to detect or select cells that exhibit a biological response is performed. Accordingly, reagents for use in selection or screening can be added to the wells just after removing the seal separating the well compartments. For example such components can include fluorescent dyes, buffers, ions (e.g., Ca²⁺, or Mg²⁺), enzymes, antibodies, cofactors and the like. Likewise, non-specific blockers, such as serum proteins (e.g., BSA) can be added. In further aspects, nuclease inhibitors and/or excess amounts of irrelevant nucleic acid can be added to aid in preserving the nucleic acid molecules that constitute the library.

[00142] In some cases, a specific blocker can be added, such as an excess amount of a soluble component recognized by the binding moiety associated with the nucleic acid library. In the case of a binding moiety that is an antibody, a peptide containing the antibody-recognized epitope can be added. Such blockers will block the binding moieties on the majority of unbound antibodies (i.e. , from droplets that were negative for a biological activity) and thereby prevent them from binding to positive cells or cell components after the emulsion is broken (when the aqueous phases become mixed). For instance, the DNA coated beads will, in many cases, have multiple copies of the binding moiety and multiple binding events per bead will greatly increase the strength of the binding. However, once the aqueous phases are mixed, beads from all of the microcapsules could potentially bind to cells that exhibit a biological response. The use of such specific blockers at this step reduces these interactions and thereby decreases the number of false positives that could be identified. This step can also be performed with a large dilution and/or at a low temperature to slow the binding kinetics and reduce binding of false positives. [00143] In some aspects, biologically active polypeptides may be detecting an enzymatic activity or a fluorescence signal. For example, in some aspects, a test cell may be a transgenic cell that comprises an enzyme, such that a desirable biological activity results in a detectable enzymatic catalysis. For instance, a test cell may express luciferase such that if cell lysis releases the enzyme (in the presence of a substrate) a detectable luminesce signal is produced indicating cell lysis. In another example, a test cell may have a promoter responsive to a desired biological activity that controls expression of a reporter gene (such as GFP). In this case activation of the promoter would result in detectable expression of the gene indicative of the biological activity of the polypeptide. [00144] One example of a biological response that can be screened or selected in accordance with the methods of the embodiments is cell death or lysis. For example, lysis of bacterial cells that have been incubated with the products of the in vitro transcription/translation reactions in water-in-oil emulsions can be detected using antibodies to intracellular targets such as sigma 70 family proteins, housekeeping proteins or RNA polymerase subunits. Alternatively, the intracellular target detected can be a protein expressed by the cell as a transgene. Similar methods can likewise be employed to measure the lysis of eukaryotic cells using antibodies specific for intracellular targets such as housekeeping proteins or RNA polymerase subunits GAPDH or actin. In either case, beads including the DNA library can be conjugated to a primary antibody. The beads can then be used for emulsion transcription/translation reactions in water-in-oil emulsion and fused with the bacterial (or eukaryotic) cells and incubated for a period of time (protease inhibitors can be added to the emulsions when necessary to protect the integrity of the target protein). The water-in-oil emulsions are then broken using previously described methods and the aqueous phase is passed over a resin coupled to the secondary binding moiety (such as an antibody that binds to a different epitope on the same target as the primary antibody). Beads that do not contain the protein of interest bound to the primary antibody are washed from the resin and collected. Beads containing the protein of interest bound to the primary antibody are eluted from the column using standard methods and as detailed below and the isolated nucleic acids (e.g., isolated from the eluted beads) are sequenced. [00145] In some aspects, detecting a biological response can involve detecting a characteristic such as fluorescence of a cell or microcapsule may be determined, and an electric field may be applied or removed from the cell or microcapsule to direct it to a particular channel. In some cases, high sorting speeds may be achievable using certain systems and methods of the invention. Thus, in one embodiment of the invention, fluorescence activated cell sorting (FACS) screening or other automated flow cytometric techniques may be used for the efficient isolation of test cells or microcapsules (and associated nucleic acid molecules) that exhibit a response to a candidate polypeptide. Instruments for carrying out flow cytometry are known to those of skill in the art and are commercially available. Examples of such instruments include FACS Star Plus, FACScan and FACSort instruments from Becton Dickinson (Foster City, Calif.) Epics C from Coulter Epics Division (Hialeah, Fla.) and MOFLO™ from Cytomation (Colorado Springs, Co). [00146] Flow cytometric techniques in general involve the separation of cells, emulsion microcapsules or other particles in a liquid sample. Typically, the purpose of flow cytometry is to analyze the separated cells or particles for one or more characteristics thereof, for example, presence of a labeled ligand or other molecule. The basic steps of flow cytometry involve the direction of a fluid sample through an apparatus such that a liquid stream passes through a sensing region. The particles should pass one at a time by the sensor and are categorized based on size, refraction, light scattering, opacity, roughness, shape, fluorescence, etc.

[00147] Rapid quantitative analysis of cells can thus be achieved with FACS.

The system permits quantitative multiparameter analysis of cellular properties at rates of several thousand cells per second. These instruments provide also the ability to differentiate among cell types, for example, in an assay to identify cell differentiation promoting molecules. Importantly, cells or particles that display a desired parameter (e.g. , fluoresce) can be channeled into a separate flow stream, thereby isolating the cell and/or particle. Thus, not only is cell analysis performed by flow cytometry, but so too is sorting of cells. In U.S. Patent 3,826,364, an apparatus is disclosed which physically separates particles, such as functionally different cell types. In this machine, a laser provides illumination, which is focused on the stream of particles by a suitable lens or lens system so that there is highly localized scatter from the particles therein. In addition, high intensity source illumination is directed onto the stream of particles for the excitation of fluorescent particles in the stream. Certain particles in the stream may be selectively charged and then separated by deflecting them into designated receptacles. A classic form of this separation is via fluorescent-tagged antibodies, which are used to mark one or more cell types for separation. [00148] Other examples of methods for flow cytometry that could include, but are not limited to, those described in U.S. Patent Nos. 4,284,412; 4,989,977; 4,498,766; 5,478,722; 4,857,451 ; 4,774,189; 4,767,206; 4,714,682; 5,160,974; and 4,661,913, each of the disclosures of which are specifically incorporated herein by reference. [00149] For the present invention, another advantage known to those of skill in the art is that nonviable cells can be recovered using flow cytometry. Since flow cytometry is essentially a particle sorting technology, the ability of a cell to grow or propagate is not necessary. Thus, FACS can be used to screen for polypeptides that induce cell death, such as apoptosis. Techniques for the recovery of nucleic acids from such non-viable cells are well known in the art and may include, for example, use of template-dependent amplification techniques including PCR.

[00150] While various embodiments contemplate the use of microfluidic methods for screening a biological activity it is also contemplated that cells may be screened while compartmentalized or immobilized, such as in gel, a well or on a slide. For example, the test cells can comprise an array with each compartment or isolated zone comprising test cells and (on average) one member of a library for testing. Methods for assessing activity may be employed as outlined above (e.g. , enzymatic activity, fluorescence, luminescence, etc.) and positive hits can be selected from each of the isolated cell populations. As with flow cytometry methods, methods using plates or arrays of cell populations are highly amenable to automation, as would be preferable for high-throughput screening. Furthermore, methods involving the use of immobilized cells can also employ antibodies or other binding moieties to detect a biological activity in cells (e.g. , as in a modified ELISA assay).

[00151] Once a cell and associated nucleic acid have been isolated the nucleic acid can be sequenced to provide the structure of the polypeptide having the desired biological activity. For instance, primer binding sequences comprised in the nucleic acid molecules can be used to rapidly amplify and/or sequence the molecules. In some cases, a coding sequence with an identified biological activity is used as the basis for a new library in a screening method such as that detailed here. For example, the identified coding sequence can be partially randomized and subjected to one or more additional screening steps to identify coding sequences that have enhanced biological activity or to determine which portions of a coding sequence are required for a biological activity. [00152] Protocols for the fabrication, preparation and handling of miniature well plates, as well as the use of such plates for cell culture, can be found for example in Guldevall et al. (2010), incorporated herein by reference.

[00153] Protocols for the preparation of antibodies for cell internalization studies can be found for example in GE Healthcare Application note 28-9042-47 AA, "Practical aspects of live CypHer5E assays using the IN Cell Analyzer 1000," (on the World Wide Web at gelifesciences.com/gehcls_images/GELS/Related%20Content/Files/1314750913712/litdoc28 904247_20110831034447.pdf), incorporated herein by reference. [00154] The internalization of an antibody into cells is dependent on the specific interaction of the antibody with the receptor on the surface of the cell. Upon binding of the receptor and stimulation with the receptor's ligand, the antibody is internalized into the cell. Antibodies labeled with the pH sensitive dye, CypHer5E, will become fluorescent when internalized into the acidic lysosome. VI. Examples

[00155] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques and apparatus disclosed in the examples which follow represent techniques and apparatus discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1 - Antibody screening [00156] This example describes a screening platform that enables the identification of antibodies that have functional effects on live cells. In particular this describes a specific example where this platform is utilized to synthesize a library of human antibodies and, in the presence of TNF-a ligand, screen that library against a cell line that has an NF-κΒ reporter construct, such that specific antibodies that inhibit TNF-a from triggering NF-κΒ signaling in those cells can be detected and the sequence encoding those antibodies retrieved. The process is illustrated in the flowchart shown in FIG. 1.

Library Preparation [00157] Synthetic biology provides the ability to synthesize DNA molecules with a user-defined sequence that can include one or more genes along with additional sequences necessary to regulate transcription and translation. A variety of methods have been reported that enable variability to be added to the synthetic DNA molecules such that a library can be created. Such methods include error-prone PCR, degenerate randomization of specific bases, and random ligation of DNA fragments.

[00158] Here, synthetic biology (e.g. Gen9, Inc.; DNA2.0, Inc.) is used to establish a framework for the antibody library that encodes both the heavy and the light chain of a human IgG antibody in a single-chain variable fragment (scFv) construct. The variable regions of this construct are initially created as sequence data in a software process that mimics the human immune system. Germline sequence data is used as the input into software programs that combines gene segments in a way comparable that of naive human B-cells. In the human body, the antibody genes of maturing B-cells are assembled via somatic recombination from these same germline sequences. A large portion of the antibody sequence is constant among all antibodies. The variability (and specificity) is mostly due to six specific segments of sequence - the complementarity determining regions (CDRs) - which are derived from a diverse set of germline gene segments that in vivo are combined by somatic recombination as antibody expressing cells (i.e. B-lymphocytes) mature.

[00159] The scFv framework DNA sequence has a placeholder for each CDR flanked by specific primer sequences. The CDR sequences that the inventors' software generates can be synthesized by a variety of methods as described above. A preferred process by which the CDR sequences can be synthesized is DNA microarrays (e.g. , Roche Nimblegen, Inc.), where the sequences are flanked by the same primers as the placeholders in the scFv framework (protocols for high diversity DNA synthesis via mask-less photolithographic DNA microarrays can be found for example in Singh-Gasson, 1999). The intrinsic error rate of the microarray synthesis provides millions of variations of each CDR sequence. This is a library of CDR regions. For each of the six CDR regions, all library members specific to that region can be amplified specifically using the primer sequences flanking that CDR. Then, a combination of polymerase chain reaction (PCR) and ligation steps are used to integrate the library of CDR regions into the scFv framework to create a DNA library that encodes a scFv with highly diverse CDRs that are analogous to those of naive human B-lymphocytes. Likewise, full length antibody libraries are commercially available from companies such as DNA2.0, Inc.

Ribosome display and conversion from scFv to full antibody

[00160] The scFv library is screened for binding using ribosome display in order to enrich for antibodies that bind the desired target (see for example Zahnd et al. Nature Methods 4:269, 2007). It may also be of interest to pre-screen for other properties, such as proper folding and assembly of the heavy and light chains of the antibody. This can be detected, for example, by the extent to which hydrophobic dyes stain the antibody (properly folded proteins tend to have fewer hydrophobic regions on their outside surface). Furthermore, the library could be enriched for manufacturability (e.g. expression yield), heat stability (e.g. judged by retention of proper folding), or freeze-thaw stability. Pre-screens can be run in series to create a single, tightly focused, sub-library or in parallel to create several sub-libraries that can be screened separately. After pre-screening with ribosome display, the enriched population of DNA sequences can be reconstituted using suitable primers and backfilling to incorporate the CDRs from the hits in a full antibody construct with individual heavy and light chain sequences in a construct suitable for cell-free expression with bacterial extracts.

Bead amplification of DNA library encoding antibodies

[00161] In order to amplify each library member individually the DNA library is compartmentalized in emulsion droplets with all the reagents necessary to run polymerase chain reaction (PCR) as described in Williams et al. 2006, incorporated herein by reference. For compartmentalization in the emulsion droplets, the DNA library is diluted such that the majority of wells have only a single library member, many have no DNA, and some have two or more DNA molecules. In addition to the DNA, beads that can bind biotin-tagged molecules are distributed in the wells such that droplets generally have only a single bead. Only the forward PCR primer is biotinylated so that only one end of the amplified DNA can bind the DNA library bead. Once bead amplification is completed, the DNA library is now on beads, where most beads contain many copies (approximately 1-10 million copies) of one library member, bound to the bead via a biotin-streptavidin complex, and where most of the original library members are represented on a bead.

Bead-to-bead expression of an antibody library

[00162] A second bead population is prepared, that is capable of binding human antibodies; these are antibody dosing beads. Each library member from the previous step is expressed individually so that the antibody encoded on a certain DNA library bead is represented as a polypeptide on a specific antibody dosing bead. In a preferred embodiment the antibody dosing beads are coated with a fusion polypeptide of a zz-tag (binds human IgG heavy chain constant region), a protease cleavage site (e.g. Tobacco Etch Virus protease), and a His tag or a sequence that can be biotinylated (e.g. AviTag); and bound to the antibody dosing beads via a His-Nickel interaction or biotin-streptavidin complex (as appropriate). The DNA library beads are distributed into the wells of a miniature well plate (see for example: Morrison et al. "Nanoliter high throughput quantitative PCR," Nucleic Acids Research, vol. 34, no. 18, p. el23, 2006), and the antibody dosing beads are distributed into the wells of a second miniature well plate. In both cases each well generally has a one or more identical single bead(s) and the well plates are designed to mate such that each well in the DNA library plate connects with a specific well in the antibody dosing plate, but isolated from other wells of the plates. The DNA library well plate has a solid bottom, whereas the antibody library plate has through-wells that are sealed with a permeable membrane on both sides. All wells in both plates are loaded with cell-free expression reagents. These reagents enable the transcription of the DNA bound to the DNA library beads, and the translation of both the heavy chain and the light chain encoded in that DNA. The cell-free expression reagents furthermore provide the appropriate environment for the antibody to fold into a functional conformation. The antibody dosing beads can bind the heavy chain of the expressed antibodies. Once bead expression is completed, the antibody library is now on the antibody dosing beads, which are sitting in an antibody dosing plate. Each antibody dosing bead contains many copies (approximately 1-20 billion) of the antibody polypeptide macromolecules.

Screening of antibody libraries against live cells [00163] Cultured cells are prepared in a third plate - a cell plate. Solid-bottom miniature well plates are tissue-culture treated (e.g. oxygen plasma) and optionally coated with collagen using established protocols (e.g., Ostrovidov et al, Biomedical Microdevices 6:279, 2004). Then cells are seeded in the plate and allowed to attach for 24 hours without sealing the plate. The seeding density ranges from 100 to 1000 cells per mm² of the wells; in one embodiment this amounts to 2-20 cells per well. The cell plate is designed to mate with the antibody dosing plate such that each well in the antibody dosing plate connects with a specific well in the cell plate, but are isolated from other wells of the plates. A variety of assays have been described that enable the detection of cellular responses to treatments, such as antibodies. In the case of screening for inhibition of TNF-a signaling, the preferred method is a transgenic reporter cell line, which expresses a luciferase reporter when exposed to active TNF-a in a way that triggers intra-cellular signaling. The assay is run using regular cell culture medium (e.g. Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum) supplemented with TNF-a as well as the protease (e.g. Tobacco Etch Virus protease) that can cleave the antibodies from the antibody dosing beads and release them into the cell culture medium. The mated cell and antibody dosing plates are sealed to compartmentalize each bead of the antibody dosing plate with a specific population of cells in the cell plate. The sandwiched antibody dosing and cell plates are clamped together and placed in a cell culture incubator for the duration of the assay; e.g. 24 hours.

Hit identification and recovery

[00164] In some aspects, a reagent solution may be introduced in to the (third) cell plate that facilitates hit identification (for example a solution comprising luciferin and appropriate buffer). In some case the a reagent mixture may be added through a common channel in fluid communication with all wells of the cell plate (through permeable membranes). In still other cases, a fourth plate - reagent plate - is prepared that, like the antibody dosing plate, has through-wells and is sealed with a permeable membrane on both sides. After incubation of the antibody dosing and cell plate sandwich, a reagent plate containing luciferase readout reagents (i.e. luciferin and appropriate buffer) is added on top of the sandwich of antibody dosing plate and cell plate, such that a specific set of wells - one in each of the three plates - is connected.

[00165] Regardless of the method by which the reagents are delivered to the cell plate the distribution of the resulting luminescent signal in the plate is recorded. This can be done using a variety of instruments, such as plate readers, laser scanning cytometers, high content screening instruments, microarray scanners, or epifluorescence microscope with a motorized stage. Our preferred method is a high resolution, high sensitivity, digital camera (e.g. Nikon D800) that can capture the entire array in a single image. The distribution of fluorescence signal in the miniaturized well plates is recorded. Using image processing software (e.g. Genepix, Image/J, Cellprofiler, or Python Imaging Library), a luminescent intensity value can be assigned to each well in the array, and that signal linked on a well-by- well basis back to the DNA library plate. Those wells where the luminescent signal is substantially absent are considered hits. The absence of luminenscence indicates that the antibody in that position inhibited the TNF-a signaling.

[00166] Identification of positive hits from the cell plate can then be correlated by well position to the corresponding well from the library plate (comprising the DNA library sequences). Once identified, the DNAs corresponding to the positive hits are collected and amplified or sequenced. For example, positive hit DNAs can be collected by puncture of the sealing membrane with a syringe and wash and/or suction to remove the DNA.

[00167] In an alternative strategy, in order to link specific DNA sequences (i.e. library members) to the cell responses, well-specific DNA sequences - or zip codes - are delivered to the DNA library plate. In some embodiments the sequences are in the form of primers that amplify the CDRs from the DNA library beads and also have a well-specific tag; each well will receive two (or more) sets of primers: one for the heavy chain CDR3 and one for the light chain CDR3. In some cases additional sequences might be recovered from the library, such as the antibody framework or CDR1 or CDR2 sequences. The zip code sequences will be prepared on a DNA microarray that is designed to mate with the DNA library plate, such that each spot (or set of spots) on the array is connected with a specific well in the DNA library plate. In a preferred embodiment, the DNA microarray is prepared with DNA spots that are not covalently linked to the substrate, so that the DNA molecules can spontaneously dissociate from the substrate upon contact with an appropriate buffer. Alternatively, before the zip code microarray and DNA library plate are joined, the DNA library plate is loaded with a restriction enzyme that can cut the zip code sequences free from the microarray. Before the microarray is connected with the DNA library plate, the wells are loaded with PCR mix that will allow the CDR sequences to be amplified from the DNA library beads and at the same time have the well-specific tags incorporated in the amplicons. Amplicons are then pooled from the entire plate in a tube for further processing. [00168] Once the hits sequences are identified and collected by one of the methods above, the hits are then sequenced using next-generation sequencing (e.g. Illumina MiSeq). There will be two amplicons for each hit - one for the heavy chain CDR3 and one for the light chain CDR3; in some cases additional amplicons will be generated. The sequencing results will be analyzed to identify clusters of sequences with similar properties. This will ensure that the maximum diversity possible will be brought forward into hit validation. In some cases multiple rounds of screening will be performed and sequences ranked in terms of enrichment through successive rounds.

[00169] The hit sequences can also be used to build new more focused libraries for additional screens; for example some of the CDRs can be kept constant and the others randomized using the CDR library described above, or the entire sequence can be subjected to random mutagenesis. Once hit identification is completed, a list of DNA sequences that encode the light and heavy chains of antibodies that are internalized into the test cells is known. Hit validation

[00170] Ultimately a certain set of hit sequences is used to synthesize larger quantities of antibodies for hit validation. The matched light and heavy chain CDR sequences for each hit are re-integrated into the antibody framework and cloned into a vector suitable for mammalian cell expression. Antibodies are then purified using conventional methods [see for example: Burgess and Deutscher, "Guide to Protein Purification, Second Edition", Academic Press, 2009] and used in bench-scale assays to validate binding kinetics and cellular effects.

[00171] The purified antibodies can be used to identify and/or verify the cellular target recognized by the antibody, as well as to further characterize and compare drug-related properties, such as dose response and kinetics. For example, target characterization assays can include enzyme-linked immunosorbent assays (ELISAs), reporter assays for different signaling pathways or cellular backgrounds. The antibodies can be further optimized for manufacturability, heat stability, freeze-thaw stability, and related properties that will be important if the antibodies were to become part of a therapeutic. Example 2 - EGFR antibody screening

[00172] This example describes a screening platform that enables the identification of antibodies that have functional effects on live cells. In particular this example describes a process by which a library of human antibodies is synthesized and screened against a cell line that expresses the epidermal growth factor receptor 1 (EGFR1), such that sequences that encode antibodies that are internalized into those cells can be detected.

Library preparation

[00173] Synthetic biology provides the ability to synthesize DNA molecules with a user-defined sequence that can include one or more genes along with additional sequences necessary to regulate transcription and translation. A variety of methods have been reported that enable variability to be added to the synthetic DNA molecules such that a library can be created. Such methods include error-prone PCR, degenerate randomization of specific bases, and random ligation of DNA fragments.

[00174] Here, synthetic biology (e.g. , Gen9, Inc.; DNA2.0, Inc.) is used to establish a framework for the antibody library that encodes both the heavy and the light chain of a human IgG antibody in a single DNA construct. Then, in a process that mimics the human immune system, germline sequence data is used as the input into software programs that combines gene segments in a way comparable to that of naive human B cells; in the human body, the antibody genes of maturing B cells are assembled via somatic recombination from these same germline sequences. A large portion of the antibody sequence is constant among all antibodies. The variability (and specificity) is due to six specific segments of sequence - the complementarity determining regions (CDRs) - which are derived from a diverse set of germline gene segments that in vivo are combined by somatic recombination as antibody expressing cells (i.e. , B lymphocytes) mature.

[00175] The synthetic antibody framework DNA sequence has a placeholder for each CDR flanked by specific primer sequences and restriction enzyme cut sites. The CDR sequences that the inventors' software generates are synthesized on DNA microarrays (e.g., Roche Nimbi egen, Inc.) flanked by the same primers and restriction enzyme cut sites as the placeholders in the antibody framework. The intrinsic error rate of the microarray synthesis provides millions of variations of each CDR sequence. This is a library of CDR regions. For each of the six CDR regions, all library members specific to that region can be amplified specifically using the primer sequences flanking that CDR. Then, a combination of polymerase chain reaction (PCR) and ligation steps are used to integrate the library of CDR regions into the antibody framework to create a DNA library that encodes both the heavy and the light chain of human IgG antibodies, with highly diverse CDRs that is analogous to those of naive human B-lymphocytes. In addition to the antibody, the DNA will encode a toxin (e.g. , Pseudomonas Enterotoxin), separated from the Fc end of the heavy chain by a spacer, as well as a highly specific protease cleavage site (e.g., Tobacco Etch Virus protease) separated from the toxin by a spacer, and a histidine tag following the protease cleavage site. Bead amplification of DNA library encoding antibodies

[00176] In order to amplify each library member individually the DNA library is compartmentalized in a miniature well array with all the reagents necessary to run polymerase chain reaction (PCR). Miniature well arrays with well densities ranging from 10 wells/mm² to 1 million wells/mm² can be readily fabricated using previously described methods (see, for example, Hatch et al , 2011, incorporated herein by reference). For compartmentalization in the wells, the DNA library is diluted such that many of the wells have only a single library member, many have no DNA, and some have two or more DNA molecules. In addition to the DNA, bi-functional beads, which can bind both histidine-tagged molecules and biotin-tagged molecules, are distributed in the wells such that the majority of wells have at least one bead. The dual functionality is preferably achieved by using a bead that binds histidine-tagged proteins, and covering a small fraction of the binding sites with histidine-tagged streptavidin. Both PCR primers are biotinylated so that one end of the amplified DNA can bind the bi-functional bead, and the other end is available on the other end of the DNA for downstream manipulation. The beads are pre-incubated with the forward primer to minimize "bridging," where both ends of a given DNA molecule are bound the bead; thus the vast majority of DNA molecules should only have the forward primer bound to the bead (this has been verified experimentally). The PCR protocol is optimized in the context of cell-free expression (see Example 2). Once bead amplification is completed, the library is now on beads, where most beads contain many copies (approximately 1,000- 100,000 or 1-10 million copies) of one library member, bound to the bead via a biotin- streptavidin complex, and where most of the original library members are represented on a bead. Bead expression of an antibody library

[00177] Each library member from the previous step is expressed individually.

The beads are again compartmentalized in a miniature well plate, but now with cell-free expression reagents. These reagents enable the transcription of the DNA bound to the bead, and the translation of both the heavy chain and the light chain encoded by that DNA. The cell-free expression reagents furthermore provide the appropriate redox environment and chaperones for the antibody to fold into a functional conformation. The heavy chain of the antibody is expressed with a histidine-tag and a high-specificity protease site in between the histidine-tag and the antibody. Once bead expression is completed, the antibody library is now on the beads. Each bead contains many copies (approximately 1,000-100,000 or 1-10 million) of the DNA encoding one specific library member, and many copies (approximately 1-100 million or 1-20 billion) of the antibody polypeptide macromolecule, which are bound to the bead via a histidine tag.

Pre-screening of antibody libraries [00178] Before the antibody libraries are screened against cells, the libraries can optionally be pre-screened for a variety of desirable properties. If targeting of a specific marker is desired, the library could be enriched for antibodies that bind that target. Such pre- screening could be performed either in solution or against cells over-expressing that target. For example, if the desired target is EGFRl, then the library could be incubated with fluorescently-labeled extracellular fragments of the EGFRl protein. Beads carrying antibodies that bind the EGFRl extracellular protein fragment would become fluorescent and could be separated out by fluorescence-activated cell sorting (FACS). Alternatively, the beads could be distributed in an agarose gel at a density of approximately 1 million per cm² and individual hits could be collected using a fixed tip liquid handler (e.g. , Gilson 223 sample changer); this approach would collect approximately 10,000 beads in each sample; this however, would enrich the library significantly for binding of the desired target. Other properties are also of interest, such as proper folding and assembly of the heavy and light chains of the antibody. This can be detected by binding for protein A or protein G, or by the extent to which hydrophobic dyes stain the antibody (properly folded proteins tend to have fewer hydrophobic regions on their outside surface) or interactions with specific chromatography resins (hydrophobic interaction chromatography resin or antibody cross- interaction chromatography (CIC) resin). Furthermore, the library could be enriched for manufacturability (e.g. , expression yield), heat stability (e.g. , judged by retention of proper folding), or freeze-thaw stability. Pre-screens can be run in series to create a single, tightly focused, sub-library or in parallel to create several sub-libraries that can be screened separately. After pre-screening the resulting DNA sequences can be reconstituted using suitable primers and back-filling to re-create the original DNA sequence that can be amplified on beads as described above.

Screening of antibody libraries against live cells

[00179] Once a library has been pre-screened for the desired parameters (either target-focused or non-target-focused), it can be screened against cells; in some cases it may be desirable to screen the full diversity of the original DNA library against cells. A variety of assays have been described to detect internalization, such as pH-sensitive dyes that are minimally fluorescent in the cell culture medium, but become significantly brighter when they are in the cell endosome as a result of internalization. Viability assays can be used if a toxin is linked to the antibodies. As described above, the preferred approach here is to express a polypeptide toxin fused with the antibody. The cells used for the assay will be transfected with a constitutively expressed fluorescent protein, which is augmented with a PEST tag (i.e. , a peptide sequence enriched with proline, glutamic acid, serine, and threonine), which greatly increases degradation of an otherwise relatively stable fluorescent protein. Thus, healthy cells will be positive for fluorescent protein. When these cells internalize a toxin, especially if the toxin is a ribosome inhibitor, they will cease to produce the fluorescent protein and that already expressed will be degraded rapidly (e.g. , in less than 4 hours).

[00180] For the activity screen, miniature well plates are first coated (e.g., with collagen) using established coating protocols. Then cells are seeded in the plate and allowed to attach for 24 hours without sealing the plate. The seeding density ranges from 100 to 1000 cells per mm²; in one embodiment this amounts to 2-20 cells per well. The library beads are distributed to the wells such that most wells have one bead, many have no beads, and some have two or more. The assay will be run using regular cell culture medium (e.g., Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum) supplemented with the protease (e.g. , Tobacco Etch Virus protease) that can cleave the antibodies with the toxin from the bead and release them into the cell culture medium as well as EGF, the natural ligand for EGFR, which enhances receptor internalization. The plates are sealed to compartmentalize each library bead with a population of cells. The miniature well plates are placed in a cell culture incubator for 24 hours.

Hit identification and recovery

[00181] Immediately after sealing and before incubation the distribution of fluorescent signal in the plate is recorded; this is the time zero image. This can be done using a variety of instruments, such as plate readers, laser scanning cytometers, high content screening instruments, microarray scanners, or high resolution digital cameras. The inventors' preferred method is an inverted epifluorescence microscope (e.g. , Nikon TE-2000) with a motorized stage (e.g. , Prior Scientific). The miniaturized well plates will be fitted with marks that will enable the accurate determination of the location of wells with that fit a given set of criteria. The distribution of fluorescence signal in the miniaturized well plates is recorded again after incubation; this is the end-point image. Using image processing software (e.g. , Image/J, Cellprofiler, or Python Imaging Library), a fluorescence intensity value can be assigned to each well in the array, and that signal compared on a well-by-well basis between the time zero and end-point images. Those wells where the fluorescent signal was substantially eliminated are considered hits. The location of these wells relative to specific alignment marks is recorded. Once this information has been gathered, the plates are opened and dried.

[00182] The hits will be collected by collection with a syringe or by delivering elution and recovery reagents specifically to the wells that were identified as hits. The reagents will consist of a 500 mM solution of imidazole, which will elute the histidine-tagged streptavidin, which links the DNA to the library beads, such that the DNA that encodes that hit is released into solution, as well as small, streptavidin-coated, magnetic particles (e.g. , 1 micron beads) that can bind the free biotin at the end of the DNA that was away from the bead. Thus, wells that receive the elution/recovery mix should have magnetically tagged DNA in solution. In addition the reagent mix can contain viscosity modifiers (e.g. , glycerol, ethylene glycol, propylene glycol), surfactants (e.g. , Tween 20, Triton X-100), and humectants (e.g. , poly (ethylenegly col), ethylene glycol, propylene glycol).

[00183] Using the list of hit locations from the fluorescence image processing, a piezo-electric inkjet system is programmed to deliver in the range of 10 pL to 10 nL of the elution/recovery mix to each of the wells identified as a hit. Such inkjet systems can have accuracy down to 5 microns given appropriate automation and mechanical design. Once the elution/recovery mix has been delivered to the hit wells, those are the only wells that are wet and contain magnetic tags. The DNA from the hits is recovered using a permanent magnet covered with a collection film (e.g. , non-woven sheet wet with PCR buffer, parafilm). The film-covered magnet is slowly translated across the top surface of the miniature well plate with only a minimal gap (e.g. , 0.1 - 0.5 mm) between the top surface of the plate and the film. The film is then removed from the magnet and placed in a centrifuge tube with PCR buffer, where the DNA is released from the film and the film removed from the tube. The DNA is then concentrated using magnetic collection. In order to facilitate hit identification (see below), the volume density of magnetic beads will be optimized so that a relatively small number of magnetic beads are isolated for each hit (e.g., 1-10 magnetic beads). After hit recovery, the centrifuge tube contains a mixture of DNA molecules representing all the antibodies that were identified as having been internalized into cells; many identical DNA molecules are bound to small magnetic beads, and approximately 1-10 magnetic beads represent each hit.

[00184] The hits are then sequenced. If a large number of hits are collected, they can be sequenced using next-generation sequencing (e.g. , Illumina MiSeq). However, this approach requires some DNA processing such that the available read-length can cover at least two of the CDRs; the heavy chain CDR3 and light chain CDR3 are known to be most influential in the specificity of the antibody; knowing the sequence of those two would support follow-up screening where those are kept constant and CDRl and CDR2 regions of the heavy and light chain are varied two at a time to optimize binding. An example of DNA manipulation to enable next generation sequencing of two CDRs is described for example in DeKosky et al. (2013). The preferred approach here is to collect a relatively small number of hits (e.g. , 100 hits) and perform a limiting dilution such that individual magnetic beads could be isolated in the wells of a 384-well PCR plate. This plate can then be used to produce two amplicon samples for each hit - one for the heavy chain CDRs (approximately 300 bp) and one for the light chain CDRs (approximately 260 bp). The amplicon samples are then sequenced by Sanger sequencing on an individual basis. The hit sequences can be used to build new more focused libraries for additional screens; for example, some of the CDRs can be kept constant and the others randomized using the CDR library described above, or the entire sequence can be subjected to random mutagenesis. Once hit identification is completed, a list of DNA sequences that encode the light and heavy chains of antibodies that are internalized into the test cells is known.

Hit validation

[00185] The hit sequences are used to synthesize larger quantities of antibodies for hit validation. The matched light and heavy chain CDR sequences for each hit are reintegrated into the antibody framework and cloned into a vector suitable for cell-free expression using the same reagents as the screening library expression before. A 10 mL cell- free expression reaction run in tubes with gas permeable seals on an incubated shaker can each yield approximately 0.5 mg of functional antibody, which can be purified using conventional methods (see, for example, Burgess and Deutscher, 2009).

[00186] The purified antibodies can be used to identify and/or verify the cellular target recognized by the antibody, as well as to further characterize and compare internalization properties, such as dose response and kinetics. For example, target characterization assays can include enzyme-linked immunosorbent assays (ELISAs). Internalization assays include pH-sensitive dyes, cell death, and reporter assays. Cell death assays could be run using a variety of cell types and with a variety of conjugated drug molecules, either fused polypeptides (as described above) or chemically conjugated small molecules. The antibodies can be further optimized for manufacturability, heat stability, freeze-thaw stability, and related properties that will be important if the antibodies were to become part of a therapeutic.

Example 3 - Apparatus

[00187] This example describes features and operational aspects of apparatus configured to perform methods disclosed herein. For purposes of clarity, not all features are labeled with reference numbers in each figure. Referring initially to FIG. 2, an exploded view is provided of an apparatus 10 configured to load beads into a dosing plate 50. Apparatus 10 comprises a base 20 with a reservoir 25, and a compression mechanism 30 coupled to base 20. In addition, apparatus 10 comprises a gasket 35 configured to form a seal between reservoir 25 and a track-etched membrane 51 and a dosing plate 50. Apparatus 10 also comprises a compression frame 40 with apertures 45. Alignment pins 55 serve to align base 20 and the stacked array of components inserted into reservoir 25 (e.g. gasket 35, membrane 51, dosing plate 50 and compression frame 40). [00188] In the embodiment shown, dosing plate 50 is configured as a thin steel plate comprising a plurality of apertures 54 configured to receive antibody beads and other matter as desired (e.g. expression reagent). Membrane 51 is semi-permeable and serves to help retain matter in apertures 54 of plate 50 in this embodiment. In certain embodiments, membrane 51 is a polycarbonate track etched (PCTE) membrane made from a thin, microporous polycarbonate film material and has a pore size of 5-50 microns, or more particularly, 10-40 microns, or more particularly 15-25 microns, or more particularly 20 microns.

[00189] During operation of apparatus 10, gasket 35 is inserted into reservoir 25 and located by alignment pins 55. A fluid 26 (e.g. ethanol) can be added to reservoir 25 and dosing plate 50 (with membrane 51) lowered into reservoir onto gasket 35. Compression frame can then be placed on top of dosing plate 50, and compression mechanism 30 then activated to compress compression frame 40, dosing plate 50, membrane 51, and gasket 35 toward reservoir 25 in base 20. In the embodiment shown, compression mechanism 30 includes a cam device 31 activated by a lever 32 to compress the assembled components. Compression mechanism 30 can also comprise an adjustment knob 33 to adjust the amount of compression placed on compression frame 40 (and the associated components between it and reservoir 25). Compression of the assembly helps to purge any air bubbles from apertures 54 and ensure that apertures 54 are filled with fluid 26 from reservoir 25. [00190] Distilled water can then be added (e.g. via apertures 45 in compression frame 40) to the top of dosing plate 50 to further lubricate apertures 54. A mixture of beads (as described elsewhere in this disclosure) and distilled water can then be evenly distributed across the top of dosing plate 50 (again via apertures 45) and retained by frame 40. Apparatus 10 may then be manipulated (e.g. tilted or rotated) to ensure the beads are distributed across dosing plate 50 and settled into apertures 54.

[00191] The excess liquid can then be aspirated from dosing plate 50 and lever 32 manipulated to release the compressive force provided by compression mechanism 30. Compression frame 40 can then be removed from dosing plate 50. Dosing plate 50 and membrane 51 can then be lifted off of gasket 35. [00192] Referring now to FIG. 3, a second membrane 52 can be placed on top of dosing plate 50, such that dosing plate 50 is located between membranes 51 and 52 to form a dosing array 57. In certain embodiments, membrane 52 is equivalent to membrane 51. While FIG. 3 is shown in an exploded view, it is understood that during operation membranes 51 and 52 will be in contact with the lower and upper surfaces, respectively, of dosing plate 50. In the embodiment shown, apertures 54 are arranges in a pattern of rows with spacing SI between the rows. Membranes 51 and 52 can be configured with a pore size small enough to retain beads in apertures 54, but large enough to allow biological materials (e.g. nucleic acid molecules) to pass through the membranes to apertures 54 in dosing plate 50. After beads have been loaded into apertures 54, dosing array 57 can be allowed to dry before proceeding to the next processing step. [00193] FIG. 4 provides an exploded view of an apparatus 60 configured to add additional material (e.g. nucleic acids) to apertures 54 of dosing array 57. Apparatus 60 comprises a base 70 with alignment pins 77. In addition, apparatus 60 comprises compression mechanism 30 (previously described in the discussion of FIG. 2 and apparatus 10) and a compression frame 47 with apertures 49. Apparatus 60 further comprises a loading block 80 and a library array 90 comprising biologic molecules. In particular embodiments, library array 90 may comprise DNA molecules arranged in a particular array on a polydimethylsiloxane (PDMS) sheet.

[00194] During operation, library array 90 can be placed on based 70 and within alignment pins 77. Dosing array 57 can then be placed on top of library array 90, and loading block 80 placed on top of dosing array 57. Compression frame 47 can then be placed on top of loading block 80. Compression mechanism 30 can be manipulated to compress the components in a manner similar to that of apparatus 10 described in FIG. 2.

[00195] Referring now to FIGS. 4 and 5, loading block 80 comprises an inlet port 81, an outlet port 82 and a serpentine channel 85 on a surface 83 that is in contact with dosing array 57 when apparatus 60 is assembled. In the assembled position, serpentine channel 85 is in fluid communication with apertures 54 of dosing array 57. FIG. 5 is a view of loading block 80 showing serpentine channel 85, which is not visible in FIG. 4. During operation, a vacuum is pulled on outlet port 82 of loading block 80 with inlet port 81 closed. Inlet port 81 can be coupled to a fluid supply (not shown) including for example, a fluid supply comprising expression reagent. Compression mechanism 30 provides sufficient compressive force to seal loading block to dosing array 57 and library array 90. Accordingly, when inlet port 81 is opened with outlet port 82 still under vacuum, fluid (e.g. expression reagent) from the fluid supply will be drawn into inlet 81, through serpentine channel 85, and exit through outlet 82. Serpentine channel 85 is configured such that fluid will be directed sequentially to apertures 54. For example, the spacing S2 between rows in serpentine channel 85 is equivalent to the spacing SI between rows of apertures 54 in dosing array 57 (shown in FIG. 3). This configuration and loading procedure can help to ensure that each aperture 54 is filled with the desired fluid. Membrane 52 is semi-permeable such that fluid from serpentine channel 85 can pass through membrane 52 and into apertures 54. In addition, membrane 51 is semi-permeable such that material from library array 90 can pass through membrane 51 and into apertures 54. Serpentine channel 85 can then be cleared by de-coupling inlet 81 from the fluid supply and providing pressurized air to inlet 81 (or applying vacuum to outlet 82). Dosing array 57 and library array 90 can then be placed in an incubator and allowed to express.

[00196] Referring now to FIG. 6, an exploded view of an apparatus 100 is shown that is configured to allow for imaging of material in dosing array 57 and an analyte cell array 110. Apparatus 100 further comprises (from bottom to top in FIG. 6), a support plate 103, a plate 105 that is translucent (e.g. permits light to pass through), analyte cell array 110, dosing array 57, a sealing membrane or membrane 120, a retention plate 130, a retention block 140 and a clamp 150.

[00197] During operation, plate 105 can be inserted into a recess 104 in support plate 103. Analyte cell array 110 can then be placed on top of plate 105, and retention plate 130 can be secured to support plate 103. Threaded fasteners 131 can be inserted through apertures 132 in retention plate 130 and apertures 112 in analyte cell array 110 and into threaded holes 106 into support plate 103.

[00198] Retention plate 130 comprises a central opening 137 into which dosing array 57 and sealing membrane 120 can be inserted. Retention plate 130 also comprises tapered surfaces 135 configured to align dosing array 57 and sealing membrane 120 with analyte cell array 110. In exemplary embodiments, analyte cell array 110 is configured as a plate comprising wells 115 that are arranged and spaced in an array that is equivalent to that of apertures 54 in dosing array 57. Accordingly, when dosing array 57 is aligned with analyte cell array 110, individual wells 115 will be aligned with individual apertures 54. In specific embodiments, wells 115 can be configured as nano wells, including for example, nanowells having a diameter of approximately 420 microns and a capacity of approximately 35 nanoliters. In certain embodiments, wells 115 may have a diameter between 100 and 800 microns, or between 200 and 600 microns or between 300 and 500 microns, and have a volume between 5 and 60 nanoliters or between 15 and 50 nanoliters or between 25 and 40 nanoliters. [00199] Retention block 140 can then be placed into central opening 137 and onto sealing membrane 120 and secured with clamp 150. Threaded fasteners 151 can be inserted through apertures 152 in clamp 150 and into apertures 108 of support plate 103.

[00200] Referring now to FIG. 7, a view of retention block 140 shows a more detailed view of a surface 142 that engages sealing membrane 120 when apparatus 100 is assembled. As show in this view, surface 142 of block 140 comprises a series of intersecting reliefs or channels 145 extending to the perimeter of block 140.

[00201] Referring now to FIG. 8, a section view is shown of an assembly 160 comprising block 140, sealing membrane 120, dosing array 57 (e.g. dosing plate 50 and membranes 51 and 52), and analyte cell array 110. As previously mentioned, when dosing array 57 is aligned with analyte cell array 110, individual apertures 54 and wells 115 are also aligned. Channels 145 in block 140 can provide a pathway for atmospheric air (including oxygen) to reach material in apertures 54 and wells 115. In certain embodiments, sealing membrane 120 can be formed from an oxygen permeable material, including for example, PDMS. In addition, membranes 51 and 52 can be formed from oxygen permeable material. Accordingly, oxygen from atmospheric air may diffuse through channels 145 (which extend to the perimeter of block 140), through sealing membrane 120 and membrane 52 into apertures 54. Oxygen can further diffuse through membrane 51 and into wells 115. This can promote cell viability of cells in wells 115 and increase the time available for analysis of the interaction between cells in wells 115 and material (e.g. DNA) in apertures 54. [00202] In exemplary embodiments apparatus 100 can be coupled to an imaging device (e.g. a microscope or digital camera) to observe the interaction of the contents of apertures 54 and wells 115 (explained in more detail elsewhere in this disclosure). In specific embodiments, support plate 103 can be configured to engage a motorized stage of a microscope. Such interactions may include for example, fluorescence or luminescence, which can be directly observed via apparatus 100 and an imaging device. Example 4 - PD1 antibody screening

[00203] This example describes apparatus configured to perform the methods disclosed herein, including a screening platform that enables the identification of antibodies that have functional effects on live cells. In particular, this example describes a process by which a library of human antibodies is synthesized and screened against a cell line that expresses the programmed cell death 1 (PD-1), such that sequences that encode antibodies that prevent cell activation can be detected.

Library preparation

[00204] Synthetic biology provides the ability to synthesize DNA molecules with a user-defined sequence that can include one or more genes along with additional sequences necessary to regulate transcription and translation. A variety of methods have been reported that enable variability to be added to the synthetic DNA molecules such that a library can be created. Such methods include error-prone PCR, degenerate randomization of specific bases, and random ligation of DNA fragments.

[00205] Here, synthetic biology (e.g. , Gen9, Inc.; DNA2.0, Inc.) is used to establish a framework for the antibody library that encodes both the heavy and the light chain of a human IgG antibody in a single DNA construct. Then, in a process that mimics the human immune system, germline sequence data is used to combine gene segments in a way comparable to that of naive human B cells; in the human body, the antibody genes of maturing B cells are assembled via somatic recombination from these same germline sequences. A large portion of the antibody sequence is constant among all antibodies. The variability (and specificity) is due to six specific segments of sequence - the complementarity determining regions (CDRs) - which are derived from a diverse set of germline gene segments that in vivo are combined by somatic recombination as antibody expressing cells (i.e. , B lymphocytes) mature.

In this embodiment, a fully human library is created using a combinatorial synthesis process to mimic the V-D-J recombination of the human immune system. The heavy chain is composed of the human V-D-J and the light chain is composed of the V and J segments. To simplify the construction of the library, two heavy V segments (VH1-46 and VH3-23) and two light V segments (VK1-39 and VK3-15) are chosen. These V segments provide the CDR1 and CDR2 sequences for the library. The heavy CDR3 is created through the combinatorial construction of the V segments with 36 D segments and 4 J segments. Degenerate codons based on the Germline codon on the flanking segments are used to increase amino acid diversity in the joining regions. The addition of 0 to 3 amino acids to the flanking ends of the D segment is used to add additional length variation to the CDR3. The light CDR3 is created through the combinatorial construction of the V segments 4 J segments. Degenerate codons based on the Germline codon on the flanking segments are used to increase amino acid diversity in the joining regions. The addition of 0 to 3 amino acids to the flanking ends of the D segment is used to add additional length variation to the CDR3. The V-D-J recombination mimic plus the degeneracy and length diversity results in ~9xl0⁷ HCDR3 and ~ 1200 LCDR3 for a total diversity of - lxlO¹¹ members.

The antibody library is also constructed with select restriction enzyme recognition sequences before the heavy and light chain and in the interior of the two constant regions. The restriction enzyme sites enable the rapid conversion from a full-length IgG library to an scFv and the removal/addition of stop codons for a library that is suitable for ribosome display.

Ribosome display and conversion from scFv to full antibody

[00206] The scFv library is screened for binding using ribosome display in order to enrich for antibodies that bind the desired target (see for example Zahnd et al. Nature Methods 4:269, 2007). Pre-screens can be run in series to remove members with non-specific interactions or cross-reactivities to create a single, tightly focused, sub-library or in parallel to create several sub-libraries that can be screened separately. Screening rounds are performed in which the library is contacted with the antigen of focus and specific binders are identified. Techniques such as limiting the amount of antigen in the screen or off-rate selection can be used on later screening rounds if high affinity interactions are sought. Upon completion of ribosome display the enriched pool of any round is then converted to full-length IgG. The RNA message from the ribosome display is converted to cDNA by RT-PCR and cloned into a vector using the upstream Ndel and downstream Hindlll restriction enzyme sites to add a stop codon after the light chain constant region. Two methods are used to remove the scFv liker region and insert the heavy chain constant region, stop codon, and spacer with the regulatory elements needed for the expression of the light chain fragment. In the first embodiment, the Sacl and Ncol are used to remove the scFV spacer from the cloned vector and replace it with a DNA block containing the heavy constant region and regulatory elements using standard DNA cloning techniques. In the preferred embodiment, PCR primers are designed and used to amplify around the plasmid to remove the spacer region. The new DNA block is then cloned into the plasmid using traditional methods.

Bead amplification of DNA library encoding antibodies [00207] In order to amplify each library member individually, the DNA library is compartmentalized in emulsion droplets with all the reagents necessary to run polymerase chain reaction (PCR) as described in Williams et al. 2006, known as emulsion PCR or EmPCR, incorporated herein by reference. The biotinylated DNA library is diluted such that the majority of streptavidin beads have only a single library member, many have no DNA, and some have two or more DNA molecules. Biotinylated forward primer is also attached to the streptavidin on bead. The beads are heated to denature any streptavidin that is not bound to biotin, as it is less stable. Amplification of the clonal DNA on bead is accomplished with biotinylated reverse primer in solution. Once bead amplification is completed, the DNA library is now on beads, where most beads contain many copies (approximately 1-10 million copies) of one library member, bound to the bead via a biotin-streptavidin complex and with a biotin on the end of the DNA in solution, and where most of the original library members are represented on a bead.

DNA Array Construction from EmPCR Beads

[00208] A DNA array containing the library DNA amplified during the EmPCR reaction is prepared by first creating a hydrogel array on a silicone rubber substrate such as PDMS. The hydrogel array is prepared following the protocol (described in Lofas and Hohnsson J. Chem. Soc, Chem. Commun:. 1526-1528, 1990), with the following modification. The silicone rubber is first exposed to oxygen plasma while contacting a patterning foil that creates active and inactive regions on the substrate. The active regions will be the attachment points for the hydrogels. After plasma treatment and while still contacting the patterning foil, the substrate is silanized with an amine functional silane such as APTES before following the Lofas and Hohnsson protocol. After the hydrogel array is created, neutravadin or another coupling reagent is coupled to the hydrogels following the protocol described in Johnsson et al. Anal. Biochem:. 198, 268, 1991. [00209] The neutravidin hydrogel array is now ready for transfer of DNA from the EmPCR beads. The neutravidin hydrogel array is placed face up in the bead loading jig. In one embodiment, a patterning foil similar to dosing plate 50 is placed on top of the array to limit the beads to the hydrogels and to create wells for the beads to settle into. Loading block 80 is placed on top of the foil and the assembly is pressed together with compression frame 47. Inlet port 81 is closed, the EmPCR beads are suspended in PBS-Tween 20 (0.05% v/v) and placed into the loading chamber fluid supply attached to port 81. Vacuum is pulled on outlet port 82 and inlet port 81 is opened filling the patterning wells with beads. The compression frame 47 is removed along with loading block 80 and a dialysis membrane with a cutoff small enough to retain DNA is applied on top of the patterning foil. Loading block 80 is placed on top of the dialysis membrane and the assembly is pressed together with the compression frame 47. Inlet port 81 is closed, vacuum is applied to outlet port 82, the elution buffer is applied to the loading chamber fluid supply, inlet port 81 is opened and the assembly is filled with the elution buffer. The elution buffer passes through the dialysis membrane and elutes the DNA off of the EmPCR beads whereupon it binds to the capture agent on the hydrogel, typically neutravidin. After elution and binding, the assembly is disassembled and the DNA library array 90 is washed with water.

Antibody Capture Array Construction

[00210] The Antibody Capture Arrays consist of a core stainless steel foil, dosing plate 50, that has apertures 54 and track etched polycarbonate membrane 51 or other suitable material on both sides of the foil to retain the Antibody Capture Beads. This membrane allows reagents to flow through the holes but retains the beads creating a miniature purification column. To create the Antibody Capture Array dosing plate 50 and membranes 51 and 52 are treated with oxygen plasma and coated with a suitable silane or silane blend for bonding polycarbonate to steel. In a preferred embodiment a blend of an amine functional silane and a dipodal silane are used to bond the bottom membrane 51 to the bottom of foil dosing plate 50. After drying, Antibody Capture Beads are added to the array by placing fluid 26, 1 mL of 100% ethanol, in reservoir 25 inside gasket 35 of the bead loading apparatus 10. Then the dosing plate 50 is placed, with the membrane 51 side down, on top of gasket 35. The metal compression frame 40 is then placed on top of the gasket 35 and the compression mechanism 30 is slowly pushed down. Then 4 mL of dH20 is placed on dosing plate 50 and then the bead solution is distributed via pipetting across the surface of dosing plate 50. After letting the beads settle into the apertures 54 in dosing plate 50, the liquid is removed with a pipette. After drying, excess beads on the surface of dosing plate 50 are removed with a pressure sensitive adhesive tape that is applied and then removed from the foil. Membrane 52 is then applied to the top of the Antibody Capture Array using the same protocol used for applying membrane 51. After membrane 52 is applied and dry, the antibody capture array, (e.g. dosing array 57), is blocked with a suitable blocking solution such as Denhardts for at least 4 hours. After blocking, the arrays are washed in dH20, dried and stored at RT.

In situ synthesis of the antibodies

[00211] DNA library array 90 is placed face up in the Cell Free Expression Jig, apparatus 60. Antibody capture/dosing array 57 is placed on top of the DNA library array 90. Loading block 80 is placed on top of the antibody capture/dosing array 57 andcompression frame 47 is placed on top of the serpentine loading block 80. Alignment pins 77 on base 70 ensure the beads in the antibody capture/dosingarray 57 are aligned with the hydrogels containing the DNA on DNA library array 90. The assembly is compressed with compression mechanism 30, inlet port 81 is closed, vacuum is pulled on outlet port 82, the expression mix is added to the loading chamber fluid supply, inlet port 81 is opened and the antibody capture/dosing array 57 is filled and the expression mix contacts the DNA on the DNA library array 90 initiating the in situ synthesis of the antibodies . As the antibodies are synthesized, they are captured on the caged beads sitting above the DNA. After synthesis is complete, the assembly of apparatus 60 is taken apart and antibody capture/dosing array 57 is washed to remove any traces of the expression mix. The result is a set of caged beads, each cage containing beads binding antibodies expressed from the clonal DNA from the hydrogel array directly underneath and contacting the antibody capture/dosing array 57.

Screening of antibody libraries against live cells

[00212] Cultured cells are prepared in a third plate - a cell plate. In certain embodiments, solid-bottom miniature well plates, e.g. analyte cell array 110, are tissue- culture treated (e.g. oxygen plasma) and optionally coated with collagen using established protocols (e.g. Ostrovidov et al. Biomedical Microdevices 6:279, 2004). Then cells are seeded in analyte cell array 110 and allowed to attach for 24 hours without sealing the plate. The seeding density ranges from 100 to 1000 cells per mm² of the wells; in one embodiment this amounts to 2-20 cells per well. Analyte cell array l l Ois designed to mate with the antibody dosing array 57 such that each well in antibody dosing array 57 connects with a specific well in analyte cell array 110, but are isolated from other wells of the plates. A variety of assays have been described that enable the detection of cellular responses to treatments, such as antibodies. In the case of screening for inhibition of PD-1 signaling, the preferred method is a transgenic reporter cell line, which expresses a GFP reporter when active. Active cells are culture in the presence of either cells expressing PD-L1 or recombinant PD-L1 to inhibit the activation and production of GFP. Library members that inhibit the interaction of PD-1 with PD-L1 will block the inhibition and result in GFP production. The assay is run using regular cell culture medium (e.g. Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum) supplemented with the protease (e.g. Tobacco Etch Virus protease) that can cleave the antibodies from the antibody dosing array 57 and release them into the cell culture medium. The mated cell and antibody dosing plates (e.g. analyte cell array 110 and antibody dosing array 57) are sealed to compartmentalize each bead of the antibody dosing plate with a specific population of cells in analyte cell array 110. The sandwiched antibody dosing and cell plates (e.g. analyte cell array 110 and antibody dosing array 57) are clamped together using retention plate 130, retention block 140 and clamp 150 and placed in a cell culture incubator for the duration of the assay; e.g. 24 hours.

Hit identification and recovery

[00213] Immediately after sealing and before incubation the distribution of fluorescent signal in the plate is recorded; this is the time zero image. This can be done using a variety of instruments, such as plate readers, laser scanning cytometers, high content screening instruments, microarray scanners, or high resolution digital cameras. An exemplary method utilizes an inverted epifluorescence microscope (e.g. , Nikon TE-2000) with a motorized stage (e.g., Prior Scientific). The distribution of fluorescence signal in the miniaturized well plates in analyte cell array 110 is recorded again after incubation; this is the end-point image. Using image processing software (e.g. , GenePix, or Python Imaging Library), a fluorescence intensity value can be assigned to each well in analyte cell array 110, and that signal compared on a well-by-well basis between the time zero and end-point images. Those wells where the fluorescent signal was substantially increased are considered hits. The location of these wells relative to specific alignment marks is recorded. Once this information has been gathered, the plates are opened and dried. [00214] The hits will be collected from the DNA array wells of interest using a syringe puncture using a microscope system for visualization of the array spots. The PDMS plugs are placed into PCR strip tubes. The heavy and light chains along with the spacer region are amplified using standard PCR techniques. Overlap extension PCR is used to combine the heavy chain to the spacer region and the spacer region to the light chain. The resulting PCR fragment is digested with Ndel and Hindlll so as to be cloned into an expression vector containing the proper regulatory elements for cell-free expression.

[00215] The hits are then sequenced. If a large number of hits are collected, they can be sequenced using next-generation sequencing (e.g. , Illumina MiSeq). However, this approach requires some DNA processing such that the available read-length can cover at least two of the CDRs; the heavy chain CDR3 and light chain CDR3 are known to be most influential in the specificity of the antibody; knowing the sequence of those two would support follow-up screening where those are kept constant and CDRl and CDR2 regions of the heavy and light chain are varied two at a time to optimize binding. An example of DNA manipulation to enable next generation sequencing of two CDRs is described for example in DeKosky et al. (2013). The preferred approach here is to collect a relatively small number of hits (e.g. , 100 hits) and perform a limiting dilution such that individual magnetic beads could be isolated in the wells of a 384-well PCR plate. This plate can then be used to produce two amplicon samples for each hit - one for the heavy chain CDRs (approximately 300 bp) and one for the light chain CDRs (approximately 260 bp). The amplicon samples are then sequenced by Sanger sequencing on an individual basis. The hit sequences can be used to build new more focused libraries for additional screens; for example, some of the CDRs can be kept constant and the others randomized using the CDR library described above, or the entire sequence can be subjected to random mutagenesis. Once hit identification is completed, a list of DNA sequences that encode the light and heavy chains of antibodies that block the PD-1 interaction with PD-L1 into the test cells is known.

Hit validation

[00216] The rebuilt hit DNA is used to synthesize larger quantities of antibodies for hit validation. A 10 mL cell-free expression reaction run in tubes with gas permeable seals on an incubated shaker can each yield approximately 0.5 to 5 mg of functional antibody, which can be purified using conventional methods (see, for example, Burgess and Deutscher, 2009). [00217] The purified antibodies can be used to identify and/or verify the cellular target recognized by the antibody, as well as to further characterize and compare cell- based activities, such as dose response and kinetics. For example, target characterization assays can include enzyme-linked immunosorbent assays (ELISAs). The antibodies can be further optimized for manufacturability, heat stability, freeze-thaw stability, and related properties that will be important if the antibodies were to become part of a therapeutic.

Example 5 - Cell-Free Expression

[00218] This example describes cell free expression systems that may be used according to the embodiments, with systems for expression of full-length IgG or IgG-like proteins as an example. In general, expression involves providing the nucleic acid molecules in the presence of factors required for expression, which can be produced recombinantly, provided by cell lysates (or extracts thereof) or a combination of the two. In the case of nucleic acids molecules composed of RNA, only translation machinery needs to be provided. However, in preferred aspects the nucleic acid molecules are DNA and the expression system includes factors for RNA synthesis and protein synthesis (i.e. , transcription and translation). Reagents for such combined transcription and translation ("TnT") are commercially available and can be used in accordance with the embodiments (see e.g. , the TNT® systems available from Promega, Madison WI).

Typical cell-free expression reagents utilize a S30 cellular extract. These extracts are centrifuged as 30,000 xg and dialyzed to remove both small molecule and large molecule components from the original extract. The S30 extract is then supplemented with expression factors and energy sources to reconstitute the active system. An alternative approach to the S30 extract systems is the S12 extract-based system. The S 12 extract is only spun at 12,000 xg and does not utilize a dialysis procedure to remove endogenous elements from the bacterial lysis. The use of an S12 extract removes some control over the exact composition of the reaction but is more easily made and less expensive than the S30-based systems.

[00219] The processes of expression must occur within each individual well or microcapsule provided by the present embodiments. Both in vitro transcription and coupled transcription-translation become less efficient at sub-nanomolar DNA concentrations. In some aspects a eukaryotic translation system (such as a mammalian cell lysate) is used in the expression system. In this case, the efficiency of protein synthesis may be significantly enhanced by providing a transcription system that includes reagents to mediate capping of the RNA transcripts and/or addition of a poly-A tail to the RNAs. In still further aspects, a stretch of poly-A residues may be template on the coding DNA molecules (e.g., following the ORF coding sequence).

[00220] The effective genetic element, namely, DNA or RNA, concentration in the microcapsules may be artificially increased by various methods that will be well known to those versed in the art. These include, for example, the addition of volume excluding chemicals such as polyethylene glycols (PEG) and a variety of gene amplification techniques, including transcription using RNA polymerases including those from bacteria such as E. coli (Roberts, 1969; Blattner and Dahlberg, 1972; Roberts et al, 1975; Rosenberg et al, 1975), eukaryotes e.g. (Weil et al , 1979; Manley et al , 1983) and bacteriophage such as T7, T3 and SP6 (Melton et al , 1984); the polymerase chain reaction (PCR) (Saiki et al , 1988); Q.beta. replicase amplification (Miele et al, 1983; Cahill et al , 1991 ; Chetverin and Spirin, 1995; Katanaev et al , 1995); the ligase chain reaction (LCR) (Landegren et al , 1988; Barany, 1991); and self-sustained sequence replication system (Fahy et al , 1991) and strand displacement amplification (Walker et al, 1992). Even gene amplification techniques requiring thermal cycling such as PCR and LCR could be used if the emulsions and the in vitro transcription or coupled transcription-translation systems are thermostable (for example, the coupled transcription-translation systems could be made from a thermostable organism such as Thermus aquaticus). Increasing the effective local nucleic acid concentration enables larger microcapsules to be used effectively.

[00221] The well or microcapsule size must be sufficiently large to accommodate all of the required components of the biochemical reactions that are needed to occur within the microcapsule. For example, in vitro, both transcription reactions and coupled transcription-translation reactions require a total nucleoside triphosphate concentration of about 2 mM. In the case of reactions involving translation, it is to be noted that the ribosomes necessary for the translation to occur are themselves approximately 20 nm in diameter. Hence, the preferred lower limit for microcapsules is a diameter of approximately 0.1 μιη (100 nm).

[00222] In some aspects, the platform provided herein enables the screening of an antibody library against live cells to identify those antibodies that exhibit a desired activity. A DNA library that encodes a diverse population of human antibodies can be expressed in vitro to form a library of full-size antibodies in a way that is suitable for the screening platform described. A modified cell-free expression system may include a mixture of protein chaperones (see, e.g., Table 1, above) that aid in the correct folding of full-length antibody. Such a procedure may be modified from examples found in Schwarz et al. (2007) and S12 extract based on Kim et al. (2006) or from US 20140315245 Al, incorporated herein by reference.

Creation of an SI 2 E. coli lysate for cell-free expression

[00223] The following reagents are required for the production of the overexpressing strains that are used to create the S 12 extract:

1. YTPG medium (2x): For 10 liters aqueous solution: 29.9 g KH2PO4 , 91.3 g K2HPO4, 100 g yeast extract, 160 g bactotryptone, 50 g NaCl, 198 g glucose. Sterilize phosphate buffer separately from the other dissolved components by autoclaving. Sterilize glucose by filtering.

2. S12 A buffer: 10 mM Tris-acetate (pH 8.2), 14 mM Mg2+ acetate, 60 mM potassium glutamate, and 1 mM DTT containing 0.05% (v/v) 2-mercaptoethanol. Sterilize by filtering.

3. S12 B buffer: 10 mM Tris-acetate (pH 8.2), 14 mM Mg2+ acetate, 60 mM potassium glutamate, and 1 mM DTT. Sterilize by filtering.

[00224] The described S12-based cell-free expression system utilizes multiple bacterial expression strains to provide the necessary chaperones and T7 polymerase. In certain embodiments, the expressed chaperones and polymerase can be purified and added to the final mix at known concentrations. In other embodiments, the S 12 lysate can be created from bacterial strains that are each over expressing a unique chaperone. In this embodiment, the resulting lysates can be blended to achieve the desired ratio of chaperones that are needed for antibody expression.

[00225] To begin, 75 ml of fresh overnight culture of BL21(DE3) - Tuner + chaperone (Table 2) in LB + Antibiotic is initiated by scraping from the desired glycerol stock with sterile pipette tip and injecting into sterile LB in 250 mL flask. (The resulting cultures are grown overnight at 37 °C. Table 2- Cha erone ex ression strains

[00226] For each required chaperone, 80 mL of 1M Glucose, 80 mL of 10X

Phosphate Buffer, and 800 uL of 1000X antibiotic is added to 750 mL of 2X YT Media. The flasks are inoculated with 12 mL of the pre-culture and incubated with vigorous stirring to ensure good aeration (220 rpm) at 37 deg. C. . The cells are grown until an Οϋβοο of approximately 0.5 and 0.5 mL of 1 M IPTG is added to induce T7 polymerase and chaperone expression. After induction, the cells are grown until an Οϋβοο of approximately 2.5.

[00227] When the cells are ready for harvest they are chilled rapidly to below 12 deg. C by adding 1 L of ice-cold diWater to each flask. The cells are harvested by centrifugation at 2,000 rpm for 10 min at 4 deg. C. and the supernatant is discarded. The cell pellets are resuspended in a total volume of 500 ml of 4 deg. C cold S12 A buffer and pelleted again at 2,000g for 10 min at 4 deg. C. This step is then repeated with a final centrifugation step for 15 min.

[00228] The cell pellet is weighed the pellet and is re-suspend in 1.27 mL of ice-cold Buffer B per gram of wet weight cells. The cells are lysed using the French press at 20,000 PSI and the cell lysate is collected on ice. The lysate is then centrifuge at 12000 xg for 10 min at 4 deg. C. to pellet insoluble material and the non-turbid supernatant fraction is collected. Finally, the cleared lysate is incubated 10 minutes at 37 deg. C. with shaking at 230 rpm. The cell lysate is then aliquoted in appropriate volumes and rapidly frozen in liquid nitrogen. Creation of the S-12 Reaction Mix

[00229] The S12 cell-free expression mix requires the following reaction mix to provide the needed small molecules for correct expression:

[00230] The S12 reaction also requires the following amino acid mix complete the reaction:

[00231] The S 12 reaction mix is used to express IgG or IgG-like molecules from a DNA source. In some embodiments, the chaperones needed for efficient expression of IgG are purified and added to the reaction mix. In other embodiments, the chaperones are provided through a blend of lysates, each of which provides a unique chaperone. The appropriate redox environment (GSG to GSSG ratio) and disulfide isomerase (PDIA1 or PDIA6) are important factors for cell-free expression of IgG (see, e.g., FIG. 9). In some embodiments, it may be beneficial to add additional chaperones for the expression of IgG. For example, as shown in FIGs 9-10, PDIA1 and CypB in the expression extract had the most profound beneficial effects for antibody expression. Cell-free expression using the described S12 systems is performed using the following general expression reaction:

[00232] The design of the plasmid encoding the heavy and light chains of the human antibody may influence the expression of a library of full-size antibodies in a way that is suitable for the screening platform described. Three DNA constructs have been designed for the use in full-length antibody library development and synthesized by DNA2.0 (Menlo Park, CA). Both designs contain the DNA sequence for the light chain (variable and constant) and the heavy chain (variable and constant, and optionally toxin cloning site, TEV protease site, and His tag). Library construct 1 is organized so that the heavy chain follows the primary T7 promoter and ribosome binding site (RBS) and possesses a start codon and ends with a stop codon to prevent translation read through. A spacer region comprising a secondary T7 promoter and RBS follow immediately downstream of the heavy chain and is responsible for initiating transcription/translation of the light chain. The light chain has a start and stop codon to enable production of the light chain independently of the heavy chain. A transcription termination site follows the light chain. A single transcription termination site enables production of mRNA that has both genes as well as a shorter mRNA for the second gene. Library construct 2 is similar to construct 1 but the gene order has been swapped. Library construct 3 is similar to construct 2 but the second T7 promoter upstream of the heavy chain gene was removed.

[00233] It is hypothesized that the gene order of construct 1 is more favorable than construct 2 because the light chain is present in both mRNA products enabling higher production of light chain than heavy chain. The presence of extra light chain aids in the correct folding of the heavy chain, which requires binding to the light chain. Construct 3 did not produce any full-length antibody that could be purified by protein G. Most likely, the ratio of heavy to light chain protein was not optimal for correct assembly.

* * *

[00234] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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Claims

WHAT IS CLAIMED IS;

1. A method of identifying at least one nucleic acid molecule encoding a polypeptide having a desired activity, the method comprising the steps of:

(i) obtaining a library of nucleic acid molecules, individual members of the library encoding polypeptides comprising different amino acid sequences and having desired property;

(ii) compartmentalizing the members of the library individually in the presence of a first bead comprising a first binding moiety;

(iii) amplifying the individually compartmentalized members of the library in the presence of a tagged-primer, wherein the amplified individually compartmentalized members comprise a tag that is bound to the first binding moiety of the first bead, to generate an amplified DNA library;

(iv) expressing the amplified DNA library in the presence of a second bead comprising a second binding moiety to produce individually compartmentalized polypeptides, wherein the polypeptides are bound to the second binding moiety of the second bead, thereby producing an expressed on-bead polypeptide library;

(v) testing the expressed on-bead polypeptide library for a desired activity; and

(vi) identifying tagged nucleic acid molecules encoding polypeptides with the desired activity.

2. The method of claim 1, wherein (iv) expressing the amplified DNA library comprises expressing the amplified DNA library is bound to said first bead.

3. The method of claim 1 , wherein the amplified DNA library is transferred to a surface of a compartment prior to said expressing.

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

5. The method of claim 1 , wherein identifying tagged nucleic acid molecules comprises isolating the tagged nucleic acid molecules.

6. The method of claim 1 , wherein the obtaining the library of nucleic acid molecules comprises: (a) obtaining a first library of nucleic acid molecules, individual members of the first library comprising a sequence that encodes a polypeptide, wherein individual members of the first library have different amino acid sequences;

(b) expressing the polypeptides encoded by the first library to produce polypeptides, wherein each polypeptide is associated with the member of the first library that encodes that polypeptide, thereby producing an expressed polypeptide library;

(c) testing the expressed polypeptide library for a desired property;

(d) identifying the members of the first library that encode polypeptides having the desired property, thereby producing an enriched library; and

(e) making a library of nucleic acid molecules from the enriched library, individual members of the library comprising a sequence that encodes a polypeptide having the desired property, wherein individual members of the library encode polypeptides comprising different amino acid sequences.

7. The method of claim 1, further comprising the steps of:

(a) obtaining a first library of nucleic acid molecules, individual members of the first library comprising a sequence that encodes a polypeptide, wherein individual members of the first library have different amino acid sequences;

(c) testing the expressed polypeptide library for a desired property;

(d) identifying or isolating the members of the first library that encode polypeptides having the desired property and, optionally repeating steps (a)-(d) one or more times, thereby producing an enriched library;

(e) making a second library of nucleic acid molecules, individual members of the second library comprising a sequence that encodes a polypeptide having the desired property, wherein individual members of the library encode polypeptides comprising different amino acid sequences;

(f) compartmentalizing the members of the library individually in the presence of a first bead comprising a first binding moiety;

(g) amplifying the individually compartmentalized members of the second library in the presence of a tagged-primer, wherein the amplified individually compartmentalized members comprise a tag that is bound to the first binding moiety of the first bead, to generate an amplified DNA library;

(h) expressing the amplified DNA library in the presence of a second bead comprising a second binding moiety to produce individually compartmentalized polypeptides, wherein the polypeptides are bound to the second binding moiety of the second bead, thereby producing an expressed on-bead polypeptide library;

(i) testing the expressed on-bead polypeptide library for a desired activity; and j) identifying tagged nucleic acid molecules encoding polypeptides with the desired activity.

8. The method of claim 1, wherein the individual members of the library comprise a sequence that encodes an antibody having a desired binding specificity.

9. The method of claim 8, wherein the individual members of the library comprise a sequence that encodes a scFv.

10. The method of claim 8, wherein the individual members of the library comprise a sequence that encodes an antibody VH and VL chain.

11. The method of claim 8, wherein the individual members of the library comprise a sequence that encodes an IgG VH and VL chain.

12. The method of claim 11, wherein the individual members of the library comprise a sequence that encodes full-length IgG VH and VL chains.

13. The method of claim 7, wherein (b) expressing the polypeptides encoded by the first library to produce polypeptides comprises ribosome display, phage display or cell surface display.

14. The method of claim 1, wherein the desired property is high expression, heat stability, binding specificity or correct folding.

15. The method of claim 7, wherein (c) testing the expressed polypeptide library for a desired property comprises testing the expressed polypeptide library for binding to a desired target.

16. The method of claim 7, wherein the individual members of the library comprise a sequence that encodes an antibody and wherein (c) testing the expressed polypeptide library for a desired property comprises testing the expressed polypeptide library for binding to a protein that binds to an antibody constant domain.

17. The method of claim 16, wherein the protein that binds to an antibody constant domain is a mammalian Fey receptor.

18. The method of claim 7, further comprising amplifying the isolated members of the first library to obtain an enriched first library comprising nucleic acid molecules that encode polypeptides having a desired property.

19. The method of claim 1, wherein the desired activity is a binding activity.

20. The method of claim 1, wherein the desired activity is an enzymatic activity.

21. The method of claim 18, wherein (i) obtaining a library of nucleic acid molecules comprises providing a library of mutated sequences based on a known therapeutic polypeptide.

22. The method of claim 21, wherein the known therapeutic polypeptide is an antibody.

23. The method of claim 22, wherein the mutations are in the antibody constant domain.

24. The method of claim 1, wherein (ii) compartmentalizing the members of the library individually comprises individually compartmentalizing the members in a gel, a well of a plate or a microcapsule of an emulsion.

25. The method of claim 24, wherein (ii) compartmentalizing the members of the library individually comprises depositing members of the library into wells.

26. The method of claims 25, further comprising sealing the wells of a plate after depositing the members of the library and/or the beads.

27. The method of claims 25, wherein the plate comprises 1 to 300,000 wells per mm².

28. The method of claim 1, wherein (iv) expressing the amplified DNA library comprises contacting the members with a cell-free transcription and translation system.

29. The method of claim 28, wherein the members amplified DNA library are bound to a surface in individual compartments.

30. The method of claim 28, wherein (iv) expressing the amplified DNA library comprises contacting the members with a eukaryotic cell-free transcription and translation system.

31. The method of claim 28, wherein (iv) expressing the amplified DNA library comprises contacting the members with a prokaryotic cell-free transcription and translation system.

32. The method of claim 1, wherein (iv) expressing the amplified DNA library comprises expressing the members in the presence of a chaperone.

33. The method of claim 32, wherein the chaperone is Grp94, Grp78, Grpl 70, ErDJ3, PDIA1, PDIA6, Cyclophillin B, BIP or a mixture thereof.

34. The method of claim 1, wherein the desired activity is a biological activity and testing the expressed on-bead polypeptide library for a desired activity comprises testing on living target cells.

35. The method of claim 34, wherein the biological activity comprises cell binding, cell penetration, increased cell proliferation, pathway activation, apoptosis, pathway inhibition, receptor antagonism or cytotoxicity.

36. The method of claim 34, wherein the biological activity is T-cell activation.

37. The method of claim 34, wherein the library is a library of antibody sequences and the biological activity is antibody-dependent cellular cytotoxicity (ADCC).

38. The method of claim 34, wherein (v) testing the expressed on-bead library comprises contacting the polypeptide members of the expressed on-bead library with a culture of living target cells in wells of a plate.

39. The method of claim 38, wherein the wells of a plate comprise between 1 and 10,000 living cells per well.

40. The method of claim 38, wherein contacting the polypeptide members of the expressed on-bead antibody library with a culture of living target cells comprises removing the polypeptides from the beads.

41. The method of claim 40, wherein removing the polypeptides from the beads comprises cleaving the polypeptides from the beads with a protease.

42. The method of claim 38, wherein the members of the expressed on-bead antibody library are disposed in wells of a plate and wherein contacting the polypeptide members with a culture of living target cells comprises contacting the on-bead antibody library in the wells of the plate with a corresponding plate having wells comprising the living target cells.

43. The method of claim 42, wherein the wells comprising the expressed on-bead antibody library are separated from the wells comprising the living target cells by a porous membrane.

44. The method of claim 43, wherein the porous membrane comprises pores that allow diffusion of the polypeptide members of the on-bead antibody library.

45. The method of claim 43, wherein the porous membrane comprises pores that prevent diffusion of the beads of the on-bead antibody library.

46. The method of claim 38, further comprising sealing the wells of a plate comprising the living cells after said contacting.

47. The method of claim 46, wherein the wells are sealed with a permeable membrane.

48. The method of claim 38, wherein the wells are coated with an extracellular matrix component.

49. The method of claim 38, wherein testing comprises scanning of the plate to detect a signal indicating a biological activity.

50. The method of claim 49, wherein the scanning is automated.

51. The method of claim 38, wherein (v) testing further comprises contacting the living target cells with an internalization indicator.

52. The method of claim 51, wherein the internalization indicator is a pH sensitive dye.

53. The method of claim 38, wherein (v) testing further comprises contacting cells in the presence of a fluorescent viability marker or using living target cells that express a fluorescent viability indicator.

54. The method of claim 38, wherein the target cells are mammalian cells.

55. The method of claim 54, wherein the target cells are human cells.

56. The method of claim 38, wherein the target cells are cancer cells.

57. The method of claim 8, wherein testing the expressed on-bead antibody library for a desired activity comprises testing the on-bead antibody library for binding to a desired antigen.

58. The method of claim 1, wherein (vi) identifying the tagged nucleic acid molecules encoding polypeptides with a desired activity comprises collecting the nucleic acid molecules from well and sequencing them.

59. The method of claim 1, wherein (vi) identifying the tagged nucleic acid molecules encoding polypeptides with a desired activity comprises binding the tagged nucleic acid molecules to a collection bead.

60. The method of claim 59, wherein binding the tagged nucleic acid molecules to a collection bead comprises (i) disassociating the tagged nucleic acid from the first binding moiety of the first bead; (ii) binding the tagged nucleic acid to a collection bead; and (iii) identifying or isolating the tagged nucleic acid bound to the collection bead.

61. The method of claim 58, wherein the collection bead is magnetic.

62. The method of claim 1, wherein (vi) identifying tagged nucleic acid molecules encoding antibody polypeptides with a desired activity comprises: (i) recording the plate location of signals indicating the desired activity; (ii) dispensing a recovery liquid to those locations, the liquid comprising a dissociation agent that releases the tagged nucleic acids from the first library bead; and (iii) collecting the tagged nucleic acid molecules using a magnet covered with a capture film.

The method of claim 62, wherein the recovery liquid further comprises magnetic beads.

64. The method of claim 63, wherein the recovery liquid is dispensed using an inkjet dispenser.

65. The method of claim 1, wherein the polypeptide members of the library are bound to a cytotoxic agent.

66. The method of claim 1, wherein the polypeptide members of the library further comprise a cytotoxic polypeptide.

67. The method of claim 66, wherein the cytotoxic polypeptide comprises gelonin or Pseudomonas enterotoxin.

68. The method of claim 66, wherein the cytotoxic polypeptide comprises an enzyme.

69. The method of claim 1, wherein the individual members of the library encode antibody polypeptides having different heavy chain CDR3 sequences or light chain CDR3 sequences or both.

70. The method of claim 1, wherein the individual members of the library encode human or humanized antibody sequences.

71. The method of claim 1, wherein the individual members of the library encode IgA, IgG, or IgE antibody sequences.

72. The method of claim 1, wherein the individual members of the library encode IgGl, IgG2 or IgG4 antibody sequences.

73. The method of claim 70, wherein the sequence that encodes the antibody polypeptide comprises coding sequence for the heavy and light chain.

74. The method of claim 73, wherein the heavy and light chain sequences are operably linked to different promoters.

75. The method of claim 73, wherein the heavy and light chain sequences are operably linked to the same promoter.

76. The method of claim 1, wherein the first and/or second bead is a magnetic bead.

77. The method of claim 1, wherein the first and/or second bead is not magnetic.

78. The method of claim 1, wherein the second bead is coated with a polypeptide comprising a region that binds IgG heavy chain constant region, a protease cleavage site, and a polypeptide tag, wherein the protease cleavage site is positioned between the region that binds IgG heavy chain constant region and the polypeptide tag.

79. The method of claim 78, wherein the polypeptide tag is a His tag, a Myc tag, a FLAG tag or a sequence that can be biotinylated.

80. The method of claim 79, wherein the polypeptide is bound to the bead by a biotin- streptavidin complex.

81. The method of claim 78, wherein the protease cleavage site is a Tobacco Etch Virus protease cleavage site.

82. The method of claim 78, wherein the region that binds IgG heavy chain constant region is a zz-tag, protein A or protein G.

83. The method of claim 1, further comprising amplifying the isolated tagged nucleic acid molecules to obtain an enriched library comprising nucleic acid molecules with a desired activity.

84. An enriched library of nucleic acid molecules encoding antibody polypeptides with a desired activity obtained by a method of claim 83.

85. A method of identifying at least one nucleic acid molecule encoding an antibody having a desired biological activity, the method comprising the steps of:

(i) obtaining a library of nucleic acid molecules, individual members of the library comprising a sequence that encodes an antibody having a desired binding specificity, wherein individual members of the library encode antibodies comprising different amino acid sequences;

(iii) amplifying the individually compartmentalized members of the library in the presence of a tagged-primer, wherein the amplified individually compartmentalized members comprise a tag that is bound to the first binding moiety of the first bead, to generate an amplified DNA library; (iv) expressing the amplified DNA library in the presence of a second bead comprising a second binding moiety to produce individually compartmentalized antibodies, wherein the antibodies are bound to the second binding moiety of the second bead, thereby producing an expressed on-bead antibody library;

(v) testing the expressed on-bead antibody library for a desired activity on living cells; and

(vi) identifying tagged nucleic acid molecules encoding antibodies with the desired activity.

86. A method of identifying at least one nucleic acid molecule encoding an antibody having a desired biological activity, the method comprising the steps of:

(a) obtaining a first library of nucleic acid molecules, individual members of the first library comprising a sequence that encode an antibody, wherein individual members of the first library encode antibodies having different amino acid sequences;

(b) expressing the antibody polypeptides encoded by the first library to produce scFv polypeptides, wherein each scFv polypeptide is associated with the member of the first library that encodes that scFv, thereby producing an expressed scFv polypeptide library;

(c) testing the expressed scFv polypeptide library for a desired property;

(d) identifying or isolating the members of the first library that encode an scFv polypeptide having the desired property, thereby producing an enriched first library;

(e) making a second library of nucleic acid molecules, each member of the second library comprising a sequence that encodes an antibody polypeptide, wherein individual members of the second library encode antibody polypeptides comprising a CDR sequence from an scFv polypeptide of the enriched first library;

(f) compartmentalizing the members of the second library individually in the presence of a first bead comprising a first binding moiety;

(h) expressing the amplified DNA library in the presence of a second bead comprising a second binding moiety to produce individually compartmentalized antibody polypeptides, wherein the antibody polypeptides are bound to the second binding moiety of the second bead, thereby producing an expressed on-bead antibody library; (i) testing the expressed on-bead antibody library for a desired activity; and j) identifying tagged nucleic acid molecules encoding antibody polypeptides with the desired activity.

87. The method of claim 85, wherein identifying tagged nucleic acid molecules comprises isolating the tagged nucleic acid molecules.

88. The method of claim 85, wherein (iv) expressing the amplified DNA library comprises expressing the amplified DNA library is bound to said first bead.

89. The method of claim 85, wherein the amplified DNA library is transferred to a membrane of a compartment prior to said expressing.

90. The method of claim 89, wherein the membrane is a hydrogel membrane.

91. An apparatus configured for dosing beads, the apparatus comprising:

a block comprising:

a first surface with a serpentine channel; and

an inlet and an outlet in fluid communication with the serpentine channel;

a plate comprising a plurality of apertures containing beads; and

a first membrane comprising a library array of nucleic acid molecules, wherein:

the plate is positioned between the block and the first membrane; and

the serpentine channel is in fluid communication with the plurality of apertures.

92. The apparatus of claim 91 wherein the first membrane is a polydimethylsiloxane (PDMS) membrane comprising DNA molecules.

93. The apparatus of claim 91 further comprising:

a second membrane located between the first membrane and the plate; and

a third membrane located between the first surface with the serpentine channel.

94. The apparatus of claim 93 wherein the second and third membranes are track-etched membranes.

95. The apparatus of claim 93 wherein the second and third membranes are formed from a microporous polycarbonate film material.

96. The apparatus of claim 93 wherein the second and third membranes comprise pores between 5 and 50 microns.

97. The apparatus of claim 93 wherein:

the first membrane comprises DNA molecules; and

the second membrane comprises pores, wherein:

the pores are configured to allow the DNA molecules of the first membrane to pass through the second membrane; and

the pores are configured to retain the beads in the plurality of apertures in the plate.

98. The apparatus of claim 97 wherein the plate is a stainless steel plate.

99. The apparatus of claim 93 further comprising:

a base; and

a compression mechanism, wherein:

the block, the plate, and the first, second and third membranes are positioned between the base and the compression mechanism; and

the compression mechanism is configured to compress the block, the plate, and the first, second and third membranes.

100. The apparatus of claim 99 further comprising a compression frame position between the compression mechanism and the block.

101. The apparatus of claim 91 further comprising:

a fluid supply coupled to the inlet of the block; and

a vacuum source coupled to the outlet of the block.

102. The apparatus of claim 91 wherein:

the serpentine channel comprises a first plurality of rows;

the plurality of apertures in the plate are arranged in a second plurality of rows; and the first plurality of rows and the second plurality of rows comprise equivalent spacing between the first plurality of rows and the second plurality of rows.

103. An apparatus configured for imaging a dosing array and an analyte cell array, the apparatus comprising:

a first plate comprising a plurality of apertures containing the dosing array; and a second plate comprising a plurality of wells containing analyte cells, wherein:

individual apertures in the plurality of apertures are aligned with individual wells in the plurality of wells.

104. The apparatus of claim 103 wherein the wells are nano wells.

105. The apparatus of claim 103 wherein the wells as nanowells having a diameter between 100 microns and 800 microns and a volume between 5 nanoliters and 60 nanoliters.

106. The apparatus of claim 103 further comprising:

a block comprising a first surface with a plurality of channels extending to a perimeter of the block, wherein:

the first plate is positioned between the second plate and the first surface of the block.

107. The apparatus of claim 106 further comprising a sealing membrane positioned between the first surface of the block and the first plate.

108. The apparatus of claim 107 further comprising:

a support plate; and

a translucent plate, wherein:

the translucent plate is positioned between the support plate and the second plate comprising the plurality of wells containing analyte cells.

109. The apparatus of claim 108 wherein the support plate is configured to be coupled to an imaging device.

110. The apparatus of claim 108 further comprising:

a retention plate; and

a clamp, wherein:

the retention plate is configured to couple to the support plate;

the retention plate comprises a central opening configured to receive the first plate, the second plate, the sealing membrane and the block; and

the clamp is configured to secure the first plate, the second plate, the sealing membrane and the block between the clamp and the support plate.

1 11. The apparatus of claim 107 wherein the sealing membrane is an oxygen permeable membrane.

1 12. The apparatus of claim 107 wherein the sealing membrane is a polydimethylsiloxane (PDMS) sheet.

1 13. The apparatus of claim 106 further comprising a second and a third membrane, wherein:

the second membrane is positioned between the first plate and the second plate; and the first plate is positioned between the second membrane and the third membrane.

114. The apparatus of claim 113 wherein the second and third membranes are track-etched membranes.

1 15. The apparatus of claim 1 13 wherein the second and third membranes are formed from a microporous polycarbonate film material.

116. The apparatus of claim 113 wherein the second and third membranes comprise pores between 5 and 50 microns.

1 17. An apparatus configured for loading beads into a dosing plate, the apparatus comprising:

a base;

a compression mechanism;

a frame;

a plate comprising a plurality of apertures;

a membrane; and

gasket, wherein:

the compression mechanism is coupled to the base;

the frame, the plate, the membrane and the gasket are arranged in a stacked the compression mechanism is configured to compress the stacked array.

1 18. The apparatus of claim 117 wherein:

the base comprises a reservoir containing a fluid;

the stacked array is inserted into the reservoir.

1 19. The apparatus of claim 118 further comprising alignment pins configured to align the stacked array and the reservoir.

120. The apparatus of claim 118 wherein the fluid fills the apertures when the compression mechanism is activated to compress the stacked array.

121. The apparatus of claim 120 wherein a fluid mixture comprising beads is distributed across the plate and the fluid mixture is retained by the frame.

122. The apparatus of claim 121 wherein the membrane is configured to allow fluid from the reservoir to pass through the membrane and configured to a retain beads in the fluid mixture in the apertures of the plate.

123. The apparatus of claim 117 wherein the membrane is a track-etched membrane.

124. The apparatus of claim 117 wherein the membrane is formed from a microporous polycarbonate film material.

125. The apparatus of claim 117 wherein the membrane comprises pores between 5 and 50 microns.

126. A method for loading expression reagent, the method comprising:

obtaining a block comprising:

a first surface with a serpentine channel; and

an inlet and an outlet in fluid communication with the serpentine channel;

obtaining a plate comprising a plurality of apertures containing beads; and

positioning the plate such that the plurality of apertures are in fluid communication with the serpentine channel;

coupling the inlet to a fluid supply comprising expression reagent; and

providing a vacuum at the outlet.

127. The method of claim 126 further comprising:

providing a first membrane comprising a library array of nucleic acid molecules, wherein the plate is positioned between the block and the first membrane.

128. The method of claim 127 wherein the first membrane is a polydimethylsiloxane (PDMS) membrane comprising DNA molecules.

129. The method of claim 127 further comprising:

providing a second membrane located between the first membrane and the plate; and providing a third membrane located between the first surface with the serpentine channel.

130. The method of claim 129 wherein the second and third membranes are track-etched membranes.

131. The method of claim 129 wherein the second and third membranes are formed from a microporous polycarbonate film material.

132. The method of claim 129 wherein the second and third membranes comprise pores between 5 and 50 microns.

133. The method of claim 129 wherein:

the first membrane comprises DNA molecules; and

the second membrane comprises pores, wherein:

134. The method of claim 129 further comprising compressing the block, the plate, and the first, second and third membranes.

135. The method of claim 126 wherein the plate is a stainless steel plate.

136. The method of claim 126 wherein:

the serpentine channel comprises a first plurality of rows;

137. A bacterial expression system comprising a bacterial supernatant, produced by centrifugation of a bacterial lysate at not more than 20,000 times gravity and exogenously added factors to support RNA transcription and translation in the system, wherein:

(i) the system comprises an exogenously added folding chaperone; or

(ii) the bacterial supernatant is from a recombinant bacteria that comprises an expressed transgene encoding a folding chaperone.

138. The system of claim 137, wherein the bacterial supernatant is produced by centrifugation of the bacterial lysate at between about 8,000 and 20,000 times gravity.

139. The system of claim 138, wherein the bacterial supernatant is produced by centrifugation of the bacterial lysate at about 8,000-18,000, 10,000-15,000 or 11 ,000-13,000 times gravity.

140. The system of claim 139, wherein the bacterial supernatant is produced by centrifugation of the bacterial lysate at about 12,000 times gravity.

141. The system of claim 137, wherein the bacterial supernatant is produced from the bacterial lysate without dialysis.

142. The system of claim 142, wherein the folding chaperone is a Hsp 90 family chaperone, Hsp70 family chaperone, Hsp70/Hspl l 0 family chaperone, DnaJ/Hsp40 family chaperone, a disulfide isomerase, a prolyl isomerase or a mixture thereof.

143. The system of claim 142, wherein the folding chaperone is Grp94, BiP, Grpl70, ErDJ3, PDIA1 , PDIA6, Cyclophillin B or a mixture thereof.

144. The system of claim 142, wherein the system comprises an exogenously added folding chaperone.

145. The system of claim 144, wherein the system comprises at least two different exogenously added folding chaperones.

146. The system of claim 145, wherein the system comprises an exogenously added disulfide isomerase and an exogenously added prolyl isomerase.

147. The system of claim 137, wherein the bacterial lysate is an E. coli lysate.

148. The system of claim 147, wherein the bacterial lysate is an E. coli lysate having a BL21(DE3) genetic background.

149. The system of claim 137, wherein the bacterial lysate is produced by sheer-force lysis of the cells.

150. The system of claim 137, wherein the bacterial supernatant is from a recombinant bacteria that comprises an expressed transgene encoding a RNA polymerase.

151. The system of claim 150, wherein the RNA polymerase is a T7 or SP6 polymerase.

152. The system of claim 137, wherein the exogenously added factors to support RNA transcription and translation in the system comprise a RNA polymerase, a pH buffer, a reducing agent, a salt, nucleotides, an energy production system, tRNA, amino acids and/or PEG.

153. The system of claim 152, wherein the exogenously added factors to support RNA transcription and translation in the system comprise HEPS-KOH, DTT, ATP, CTP, GTP, UTP, folinic acid, E. coli tRNA, potassium glutamate, ammonium acetate, magnesium acetate, glucose, cAMP, creatine phosphate, creatine kinase, amino acids and PEG.

154. The system of claim 137, further comprising DNA encoding a polypeptide.

155. A method of expressing a polypeptide comprising, contacting a DNA encoding the polypeptide with a system in accordance with anyone claims 137-153.

156. A recombinant bacterial cell comprising an expressed transgene encoding a folding chaperone.

157. The cell of claim 156, wherein the transgene encoding the folding chaperone comprises a sequence encoding the folding chaperone linked to a heterologous promoter.

158. The cell of claim 157, wherein the heterologous promoter is an inducible promoter.

159. The cell of claim 156, wherein the folding chaperone is a Hsp 90 family chaperone, Hsp70 family chaperone, Hsp70/Hspl l 0 family chaperone, DnaJ/Hsp40 family chaperone, a disulfide isomerase, a prolyl isomerase or a mixture thereof.

160. The cell of claim 159, wherein the folding chaperone is Grp94, BiP, Grpl 70, ErDJ3, PDIA1, PDIA6, Cyclophillin B or a mixture thereof.

161. The cell of claim 156, wherein the cell comprises an expressed transgenes encoding at least two different folding chaperones.

162. The cell of claim 156, wherein the bacterial cell is an E. coli cell.

163. The cell of claim 162, wherein the bacterial cell is an E. coli cell having a BL21 (DE3) genetic background.

164. The cell of claim 156, wherein the bacterial cell further comprises an expressed transgene encoding a RNA polymerase.

165. The cell of claim 162, wherein the RNA polymerase is a T7 or SP6 polymerase.

166. A bacterial cell lysate produced from a bacterial cell in accordance with anyone of claims 156-165.

167. The bacterial lysate of claim 166, is a bacterial lysate supernatant produced by centrifugation at not more than 20,000 times gravity.

168. The bacterial lysate of claim 167, wherein the bacterial lysate supernatant is produced by centrifugation of the bacterial lysate at between about 8,000 and 20,000 times gravity.

169. The bacterial lysate of claim 168, wherein the bacterial lysate supernatant is produced by centrifugation of the bacterial lysate at about 8,000-18,000, 10,000-15,000 or 1 1 ,000- 13,000 times gravity.

170. The bacterial lysate of claim 169, wherein the bacterial lysate supernatant is produced by centrifugation of the bacterial lysate at about 12,000 times gravity.

171. The bacterial lysate of claim 166, further comprising exogenously added factors to support RNA transcription and translation in the lysate.