Patent Description:
Previous methods for mining the antibody repertoire from human donors have helped identify therapeutically valuable antibodies, define novel targets, and offer insight into the immune response to a disease. The methods for isolating these antibodies generally fall under two categories: isolating antibodies directly from cells such as B-cells or selecting antibodies from combinatorial libraries such as phage display, yeast display or mammalian display. The two approaches have different strengths; for example, antibodies obtained directly from B-cells usually have better potency and manufacturing properties while the display platforms offer the ability for subsequent screening, deep mining and clonal stability (Burton, D et al. , (<NUM>), <NUM>: <NUM>-<NUM>). There is curently no high throughput technology that combines the benefits of both approaches.

Currently technologies are available to encapsulate large numbers of cells and subsequently sequence their VH and VL domains by next-generation sequencing technologies. The original pairing of variable domains are maintained by the use of barcoded primers on particles that are encapsulated along with the B-cells. These technologies enable phylogenetic analysis of the sequences without any information about the antigen specificity or other biological functions of the repertoire. These technologies do not enable high throughput translation and subsequent screening of the antibody sequences. This creates a serious limitation in the functional analysis of the immune repertoire (<NPL>; <NPL>; <NPL>; <NPL>;<NPL>; <NPL>; <NPL>). Moreover, validating antibody leads requires gene synthesis, cloning and expression which can create a severe bottleneck in the number of candidates that can be functionally assessed (<NPL>).

Generating recombinant antibody fragments, such as scFv and Fab, from single cells in microtiter plates has been described, although this approach is severely limited in throughput, in that it can handle at most a few thousands of cells at a time, with a maximum success rate of <NUM>-<NUM>% (<NPL>; <NPL>).

Both of these technologies suffer from low screening throughput that overwhelmingly under-samples the ~<NUM><NUM> B cells obtained from a typical blood draw.

Accordingly, there is a need in the art for a technology or approach that is able to rapidly isolate natively-paired antibody sequences from human donors at a high enough throughput to adequately cover the natural diversity and in a format that enables rapid screening for activity.

<NPL> reltaes to the persistence and evolution of allergen-specific IgE repertoires during subcutaneous specific immunotherapy. <NPL> relates to the pairing of T-cell receptor chains via emulsion PCR. <NPL> relates to high-throughput sequencing of the paired human immunoglobulin heavy and light chain repertoire.

The subject-matter of the invention is defined by the appended claims. Any further aspects of the disclosure that fall outside the claim-scope are provided for information only.

The present disclosure is concerned with a method for generating a library of natively-paired scFv amplicons that can be screened for antibody binding and function.

The present disclosure is also concerned with the use of microfluidics for encapsulating single cells in droplets.

The present invention will now be described in more detail with reference to the attached Figures, in which are shown:.

The present disclosure permits encapsulation of single cells isolated from patients, e.g. B-cells, in water-in-oil droplets, with reagents to amplify and link native pairings of heavy and light chain variable domain amplicons from single encapsulated cells, in order to create a recombinant library of scFv. Throughout the description reference is made to phage display, but it will be appreciated by the person skilled in the art that yeast display and mammalian display technologies are equally applicable, and the inventors have explicitly contemplated such alternative display systems.

The present disclosure involves cloning the variable domains (VH and VL) from single encapsulated cells and joining them to form an scFv. By physically separating each cell, native VH-VL pairing, which is critical to recovering antibody binding and function, is preserved. The resulting amplicon forms an expression-ready scFv. The library of scFv is a translatable scFv library that can either be directly screened for binding and function, enriched by phage-display panning, or deep-sequenced using next-generation sequencing.

The present disclosure further permits coupling of the expression-ready scFv library with the screening methods (e.g. phase-display) to enrich for antigen-specific clones. The present disclosure allows the high throughput identification of antigen-specific antibodies, in particular by mining the natural B-cell diversity to rapidly isolate antigen-specific antibodies from human patients. The present invention allows the identification of antibodies that are not found by existing technologies.

The present disclosure provides a method for producing encapsulated natively-paired scFv amplicons, the method comprising: encapsulating single B-cells in droplets, wherein the droplets further contain reagents for amplifying and linking native pairings of heavy and light chain variable domain amplicons from single encapsulated cells; lysing the single encapsulated cells and generating the encapsulated natively-paired scFv amplicons, wherein each scFv amplicon comprises a native pairing of heavy and light chain variable domain amplicons linked together by a linker, wherein the linker has a length of <NUM>-<NUM> amino acids.

Typically the scFv amplicon comprises the formula V1-L-V2. L is the linker. In one instance, V1 is the heavy chain variable domain and V2 is the light chain variable domain i.e. the scFv amplicon has the formula VH-L-VL. In another instance, V2 is the heavy chain variable domain and V1 is the light chain variable domain i.e. the scFv amplicon has the formula VL-L-VH.

In one instance, the reagents for amplifying and linking native pairings of heavy and light chain variable domain amplicons as defined anywhere herein comprise primers designed to human Ig sequences. In one instance, the reagents for amplifying and linking native pairings of heavy and light chain variable domain amplicons as defined anywhere herein comprise a primer pool comprising the primers as set out in Table <NUM>. In one instance, the reagents for amplifying and linking native pairings of heavy and light chain variable domain amplicons as defined anywhere herein comprise a primer pool comprising the primers as set out in Table <NUM>. The group of primers in Table <NUM> or Table <NUM> may be further subdivided if desired, for instance, appropriate sets of primers may be used where the cells are separated into kappa or lambda expressing cells or into cells expressing different Ig H isotypes. Primers may be designed with custom software known in the art. Heavy and light chain variable domain amplicons are initially formed from the native heavy and light chain variable domain sequences in the generation of a scFv amplicon.

In one instance, the generating step for generating the encapsulated scFv amplicons as defined anywhere herein comprises initially forming heavy and light chain variable domain amplicons from native heavy and light chain variable domain sequences and the reagents comprise a primer pool comprising first and second heavy chain variable domain primers; and first and second light chain variable domain primers, wherein the first heavy chain variable domain primer and the first light chain variable domain primer interact to join the heavy and light chain variable domain amplicons. In one instance, the primer pool comprises a lower concentration of the first primers than the second primers. Preferably, the concentration of the first primers is reduced by a factor of between two and eight, e.g. two, three, four, five, six, seven, eight, nine or ten, compared to the concentration of the second primers. Preferably, the concentration is reduced by a factor of eight. By providing a limiting amount of the nucleic acid primers that bind inside the variable domains, amplification of the full scFv is favoured over the individual VH and VL domains.

In one instance, the first heavy chain variable domain primer as defined anywhere herein is fused to a first overhang sequence and the first light chain variable domain primer as defined anywhere herein is fused to a second overhang sequence, wherein the overhang sequences interact to join the heavy and light chain variable domain amplicons. Preferably, the first and second overhang sequences are at least partially complementary. More preferably, the first and second overhang sequences are fully complementary. The two domain amplicons may be linked using overlap-extension PCR to generate a scFv amplicon.

In one instance, the RT-PCR is used in combination with overlapping-extension PCR.

In one instance, the first heavy chain variable domain primer as defined anywhere herein is the reverse primer which binds inside (typically at the <NUM>' terminus of FR4) the heavy chain variable domain of the native sequence/amplicon, and the second heavy chain variable domain primer as defined anywhere herein is the forward primer which binds outside the heavy chain variable domain of the native sequence/amplicon; and the first light chain variable domain primer as defined anywhere herein is the forward primer which binds inside (typically at the <NUM>' terminus of FR1) the light chain variable domain of the native sequence/amplicon, and the second light chain variable domain primer as defined anywhere herein is the reverse primer which binds outside the light chain variable domain of the native sequence/amplicon.

In another instance, the first heavy chain variable domain primer as defined anywhere herein is the forward primer which binds inside (typically at the <NUM>' terminus of FR1) the heavy chain variable domain of the native sequence/amplicon, and the second heavy chain variable domain primer as defined anywhere herein is the reverse primer which binds outside the heavy chain variable domain of the native sequence/amplicon; and the first light chain variable domain primer as defined anywhere herein is the reverse primer which binds inside (typically at the <NUM>' terminus of FR4) the light chain variable domain of the native sequence/amplicon, and the second light chain variable domain primer as defined anywhere herein is the forward primer which binds outside the light chain variable domain of the native sequence/amplicon.

These reagents allow the cognate VH and VL domains to be amplified within droplets.

The present disclosure also permits encapsulation of single cells isolated from patients. In one instance, the encapsulating step as defined anywhere herein comprises using microfluidics. Microfluidics is able to capture millions of cells, potentially the entire repertoire, into picoliter-sized droplets for parallel amplification into a library and thus provides a high throughput approach. In one instance, the library is a scFv library (<FIG>).

In one instance, the microfluidics as defined anywhere herein comprises using a glass microfluidic chip with pressure pumps. In one instance the microfluidic chip as defined anywhere herein is a fluorophillic chip. The microfluidic chip is designed to merge two streams of aqueous fluids: one carrying a suspension of cells and the other containing reagents for one-step reverse transcription (RT) and overlap-extension PCR. Microfluidics is used to reliably generate evenly sized droplets at high rates.

Though it has been reported by several groups that cell-based RT-PCR is not feasible in volumes of less than <NUM> nL (<NPL>; <NPL>; <NPL>; <NPL>), the method of the disclosure is able to successfully amplify Ig transcripts directly from cells in picoliter-sized droplets e.g. droplets of 200pL in volume. In one instance, the droplets as defined anywhere herein are from about <NUM> pL to about <NUM> pL in volume. In one instance, the droplets are from about <NUM> pL to about <NUM> pL in volume. In one instance, the droplets are about <NUM> pL in volume.

In one instance, the encapsulating step as defined anywhere herein comprises combining an aqueous suspension with an oil to form an emulsion comprising the encapsulated single cells in water-oil droplets, wherein the aqueous suspension comprises the cells and the reagents for amplifying and linking native pairings of amplicons.

In one instance, the method further comprises a step prior to the encapsulating step, the step comprising providing the aqueous suspension comprising the cells and the reagents for amplifying and linking native pairings of amplicons. In one instance, the providing step comprises stimulating the cells in a first aqueous suspension comprising the cells and subsequently combining the cells with the reagents for amplifying and linking native pairings of amplicons to form the aqueous suspension comprising the cells and the reagents for amplifying and linking native pairings of amplicons. It is generally understood that the cells may be stimulated for about <NUM> hours, preferably at least <NUM> hours.

Titration of the cell suspension achieves approximately <NUM> cell for every <NUM> droplets which, based on Poisson statistics, results in single-cell encapsulation with ><NUM>% probability (<FIG>). In one instance, the suspension of cells as defined anywhere herein is at a density of about <NUM> to about <NUM> million cells/ml. In one instance, the suspension of cells as defined anywhere herein is at a density of about <NUM> to about <NUM> million cells/ml. In a more preferred instance, the suspension of cells as defined anywhere herein is at a density of about <NUM> million cells/ml. These densities are optimal for obtaining single-cell encapsulation into droplets (<FIG>). These densities also obtain single-cell encapsulation into droplets approximately <NUM> in diameter. Empty droplets may also be generated, although these do not contribute to the library since no template cells are present.

In one instance, the suspension of cells as defined anywhere herein comprises an anti-clumping agent. In another instance, prior to encapsulation, the suspension of cells as defined anywhere herein is stirred e.g. with a paramagnetic stir disk. Use of an anti-clumping excipient and/or stirring prevents suspended cells from settling prior to encapsulation. Since stimulated cells, e.g. B-cells or T-cells, have a tendency to aggregate over time, use of an anti-clumping excipient and/or stirring prevents changes in flow rates, as well as multiple cells being encapsulated together.

In one instance, the suspension of cells as defined anywhere herein comprises a stabilizing agent. Preferably the stabilizing agent is an amphipathic molecule. More preferably, the stabilizing agent is acetylated BSA. Acetylated BSA is an amphipathic molecule which can stabilize the water-oil interface and lower the interfacial tension (<NPL>). The present inventors have determined that use of acetylated BSA decreases droplet coalescence during the harsh conditions of PCR cycling. Further, the presence of acetylated BSA may protect enzymes such as reverse transcriptase from denaturation at the interface.

In one instance, the generating step for generating the encapsulated amplicons as defined anywhere herein comprises RT-PCR.

In one instance, the oil as defined anywhere herein is a low viscosity oil. In a preferred instance, the oil as defined anywhere herein is fluorinated oil. In a more preferred instance, the oil as defined anywhere herein is HFE-<NUM> fluorinated oil + <NUM>% w/v <NUM>-fluoro-surfactant (RAN Biotechnologies cat no <NUM>-FLUOROSURFACTANT-HFE7500. Due to the life span of cells used in the present disclosure, the throughput of the present method is limited by the time it takes to encapsulate the cells. The present inventors have determined that throughput can be improved by reducing the viscosity of the oil used to form the water-oil droplets for encapsulating the cells. Specifically, the present inventors have determined that a less viscous oil allows greater flow rates. In one instance, the microfluidic flow rate for the oil is between <NUM> and <NUM>µL/min , preferably <NUM>µL/min and the aqueous fluid is between <NUM> and <NUM>µL/min, preferably <NUM>µL/min. Reducing the viscosity of the oil is preferred over alternatives such as increasing cell density because it reduces the risk of more than one cell being encapsulated in a single droplet.

The methods of the invention as defined above, allow millions of cells to be encapsulated. Preferably, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM> or at least <NUM> million cells are encapsulated. The methods of the disclosure as defined above, allow one million cells to be encapsulated within about <NUM> minutes.

In one instance, the reagents for amplifying and linking native pairings of amplicons as defined anywhere herein comprise standard RT-PCR reagents. In one instance, the reagents as defined anywhere herein comprise Titan (Roche cat no <NUM>).

The above optimization of the aqueous components and microfluidic flow rates generates droplets that have improved homogeneity in size and improved integrity during RT-PCR. This allows improved reliability in the generating amplicons.

Prior to the cells being encapsulated in droplets, some cells die and lyse, releasing their genetic material (e.g. nucleic acid) into the surrounding media. The nucleic acids, e.g. those encoding VH or VL domains, may contaminate droplets and lead to non-natively paired products. It will be appreciated by the person skilled in the art that it is desirable to minimize the levels of contaminating nucleic acid present in the droplets. In particular, the free nucleic acid from dead or dying cells is RNA. In one instance, the method as defined anywhere herein further comprises preventing at least some free nucleic acid from dead or dying cells from being encapsulated in droplets. In one instance, the method as defined anywhere herein further comprises preventing substantially all free nucleic acid from dead or dying cells from being encapsulated in droplets. Preferably, the method as defined anywhere herein further comprises preventing free nucleic acid from dead or dying cells from being encapsulated in droplets. It will be appreciated by the person skilled in the art that any method for achieving this is within the scope of the present disclosure.

In one instance, the method as defined anywhere herein further comprises reducing the levels of free nucleic acid from dead or dying cells that are encapsulated in droplets. It will be understood that the levels of free nucleic acid from dead or dying cells are reduced as compared to the levels that would be encapsulated if this method step had not been carried out. It will be appreciated by the person skilled in the art that any method for achieving this is within the scope of the present disclosure. In one instance, the levels of free nucleic acid from dead or dying cells are reduced by <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%,or <NUM>%.

In one instance, the preventing comprises stimulating cells for less than <NUM> hours prior to encapsulating. In one instance, the reducing comprises stimulating cells in the first aqueous solution comprising cells for less than <NUM> hours.

In one instance, the preventing comprises selecting live cells prior to encapsulating. In one instance, the reducing comprises selecting live cells for combining with the reagents for amplifying and linking native pairings of amplicons in the aqueous suspension. In one instance, the selecting comprises Fluorescence-activated cell sorting (FACS). In one instance, the selecting comprises bead-based selection of cells. It will be appreciated by the person skilled in the art that the desired cells selected for encapsulating are the live cells and not the dead or dying cells.

In one instance, the preventing comprises sequestering the free nucleic acid from dead or dying cells using magnetic beads. In one instance, the reducing comprises sequestering the free nucleic acid from dead or dying cells from the first aqueous solution using magnetic beads. In one instance, the reducing comprises sequestering the free nucleic acid from dead or dying cells from the aqueous suspension comprising the cells and the reagents. Sequestering the free nucleic acid from dead or dying cells allows removal of this nucleic acid such that the levels of free nucleic acid from dead or dying cells that are encapsulated in droplets are reduced. In one instance, the magnetic beads are oligonucleotide-coated magnetic beads. In one instance, the magnetic beads are poly-dT beads. Magnetised beads with nucleic acid binding agents, in particular RNA binding agents, may be added to the cell suspension prior to encapsulation to 'mop up' free nucleic acid, in particular RNA, present in the surrounding media. The beads may be removed prior to encapsulation using a magnetic field, thereby removing the contaminating nucleic acid.

The present disclosure further provides a method for producing a library of natively-paired amplicons, the method comprising producing encapsulated natively-paired amplicons according to the method as defined above; and lysing the droplets to produce a library of natively-paired amplicons. Commercial kits are available for lysing the droplets (for example, the Micellula kit from EURx). The DNA from the droplets can be purified using a PCR purification kit from Qiagen or by other methods such as using isobutanol (<NPL>) and diethyl ether (<NPL>) have been described. A library of natively-paired amplicons produced according to the above method is within the scope of the present disclosure. A library of natively-paired amplicons having the features of a library of natively-paired amplicons produced according to this method is also within the scope of the present disclosure.

The present disclosure further provides a method for producing a library of natively-paired scFv amplicons for screening for antigen binding and/or function, the method comprising producing a library of natively-paired scFv amplicons according to the method as defined above; and producing a further library of natively-paired scFv amplicons, wherein the natively-paired scFv amplicons of the further library have the general formula R1-V1-L-V2-R2, wherein R1 and R2 are the same or different and each comprises a restriction enzyme site, V1 and V2 are the natively-paired heavy and light chain variable domain, wherein when V1 is the light chain variable domain, V2 is the heavy chain variable domain or when V1 is the heavy chain variable domain, V2 is the light chain variable domain, and L is a direct bond or linker. In one instance, the natively-paired scFv amplicons of the further library have the general formula R1-VH-L-VL-R2 i.e. V1 is VH and V2 is VL. In another instance, the natively-paired scFv amplicons of the further library have the general formula R1-VL-L-VH-R2 i.e. V1 is VL and V2 is VH.

In one instance, R1 and R2 as defined anywhere herein are different. L is a linker. Preferably, R1 and R2 are different and L is a linker.

In one instance, the producing a further library of natively-paired amplicons as defined anywhere herein comprises using Nested PCR. Nested PCR uses a second set of primers to amplify the full amplicon, which are different to those used to generate the amplicon. A subsequent run of nested PCR therefore amplifies the final expression-ready amplicon and reduces amplification of alternative PCR products formed due to non-specific primer binding. In one instance, the Nested PCR uses primers that bind to FR1 of V1 and FR4 of the V2. The full scFv amplicon product can therefore be amplified. In one instance, the Nested PCR as defined anywhere herein uses primers that are fused to overhang sequences that comprise a restriction enzyme site. Preferred restriction enzyme sites are Sfi1 and Not1. In one instance, R1 as defined anywhere herein comprises the Sfi1 restriction enzyme site and R2 as defined anywhere herein comprises the Not1 restriction enzyme site. The resulting amplicon forms an expression-ready amplicon that can easily be cloned into expression vectors for any number of expression systems for phage-display panning or direct screening.

A library of natively-paired scFv amplicons for screening for antigen binding and/or function produced by the method as defined above is within the scope of the present disclosure. A library of natively-paired scFv amplicons for screening for antigen binding and/or function having the features of a library of natively-paired scFv amplicons for screening for antigen binding and/or function produced by the method as defined above is also within the scope of the present disclosure.

The disclosure further provides a method for producing a natively-paired scFv library for screening for antigen binding and/or function, the method comprising producing a library of natively-paired scFv amplicons according to the method described herein; and expressing the natively-paired scFv. In one instance, this method comprises expressing the natively-paired scFv as scFv-Fc. In another instance, this method comprises reformatting the natively-paired scFv to IgG. Preferably, this reformatted IgG may be directly screened for binding and function, enriched by phage-display panning, or deep-sequenced using next-generation sequencing for repertoire characterisation. In a further instance, this method comprises expressing the natively-paired scFv as a scFv phage display library.

In one instance, each scFv of the scFv library as defined anywhere herein comprises the heavy and light chain variable domains of a native pairing of a single cell linked together by a linker. The linker must be of a length to allow pairing between the heavy and light chain variable domains. Preferably, the linker as defined anywhere herein is Glycine and/or Serine rich. More preferably the linker as defined anywhere herein is (Gly<NUM>Ser)<NUM> and encoded by the following sequence:
ggaggcggcggtagcggcggaggtggctcaggcggtggcggaagt (SEQ ID NO: <NUM>).

The linker as defined anywhere herein has a length of <NUM>-<NUM> amino acids. More preferably, the linker as defined anywhere herein has a length of <NUM>-<NUM> amino acids. Still more preferably, the linker as defined anywhere herein has a length of <NUM> amino acids. Suitable linkers will be well known to the person skilled in the art.

A natively-paired scFv library for screening for antigen binding and/or function produced by the method as defined above is within the scope of the present disclosure. A natively-paired scFv library for screening for antigen binding and/or function having the features of a natively-paired scFv library for screening for antigen binding and/or function produced by the method as defined above is also within the scope of the present disclosure. Advantageously, the scFv library is expression-ready.

The disclosure further provides a method for identifying an antigen-specific molecule, the method comprising producing a natively-paired scFv library according to the method described herein; interrogating the natively-paired scFv library with an antigen sample; and identifying an antigen-specific molecule.

This technology described herein is exemplified with B-cells and the platform is established for B-cell repertoire capture. Since T-cell receptors can also be converted into scFv format and retain activity (<NPL>), the technology can equally be applied to the capture of the T-cell receptor repertoire.

The present disclosure further provides a scFv library comprising natively-paired recombinant scFv for screening for antibody binding and/or function, wherein each scFv comprises the heavy and light chain variable domains of a native pairing of a single cell linked together as described herein. Instances as hereinbefore described in connection with the methods of the present invention may provide further information regarding features of the libraries. Advantageously, the libraries may be a translatable. The present libraries enable high throughput translation and subsequent screening of the sequences.

The present disclosure further provides a method for identifying an antigen-specific molecule, the method comprising interrogating a natively-paired scFv as defined anywhere herein with an antigen sample; and identifying an antigen-specific molecule. The present disclosure further provides a method for identifying an antigen-specific molecule, the method comprising interrogating a scFv library as defined anywhere herein with an antigen sample; and identifying an antigen-specific molecule.

In one instance, the antigen-specific molecule is an antibody. In one instance, the antibody as defined anywhere herein is a monoclonal antibody.

In one instance, the antigen sample as defined anywhere herein is tumour tissue. In one instance, the antigen sample as defined anywhere herein is whole bacteria. In one instance, the antigen sample as defined anywhere herein is viral particles. In one instance, the antigen sample as defined anywhere herein comprises hemagglutinin (HA) proteins.

It will be appreciated by the person skilled in the art that multiple rounds of interrogating the library can be carried out, preferably with different antigen samples, so as to allow the identification of cross-reactive antigen-specific molecules. In one instance, the antigen-specific molecule is cross reactive.

The present disclosure also provides the use of a natively-paired scFv library as defined anywhere herein for identifying an antigen-specific molecule. The present disclosure also provides the use of a scFv library as defined anywhere herein for identifying an antigen-specific molecule.

In one instance, the use as defined above comprises interrogating the scFv library with an antigen sample and identifying an antigen-specific molecule. All instances as hereinbefore described in connection with the methods of the present invention apply equally to the above defined use.

While the methods defined herein enable isolation of antibodies from human B cells, it can readily be extended to isolate antibodies from any species for which V-gene sequence information is available. This can be particularly useful for expanding the breadth and depth of the hybridoma technology, where low fusion efficiencies (less than <NUM>% (<NPL>)) lead to significant loss of repertoire. The methods defined herein may also be applied for generating monoclonal antibodies from organisms for which myeloma fusion partners are not available. T cell receptor (TCR) repertoires (consisting of paired α/β or δ/γ chains) could also be captured in a similar recombinant format and single-chain TCR has been shown to be amenable to selection by phage and yeast display (<NPL>; <NPL>).

The methods enable the rapid capture of the native repertoire from millions of primary human B cells into a powerful and sensitive screening platform, with significant implications for therapeutic antibody development, immune repertoire characterization and rational vaccine design. For example, linking the variable domains into a translatable scFv format allows the combination of the strengths of multiple technologies: using the immense screening power of display platforms to mine the full richness of a naturally evolved antibody response.

The present disclosure provides a fast method for lead antibody generation from natural repertoires - a single researcher can rapidly progress from millions of primary B cells to specific monoclonal antibodies within <NUM> weeks. This could be especially valuable for combating emerging infectious diseases, e.g. an Ebola outbreak.

The libraries of the present disclosure constitute a renewable resource that can be expanded as new donors are added, panned repeatedly against a multitude of targets (including whole bacteria or tumor tissue), or archived indefinitely for future use.

Large-scale efforts that use next generation sequencing to predict antibody function could particularly benefit, such as the recently launched Human Immunome Program (<NPL>). This project aims to sequence the expressed antibody repertoires from <NUM> individuals and infer vaccine reactivity based on sequencing information alone. An exciting addition to this project could be to use the method outlined here to build pooled display libraries from these individuals, such that one could directly measure the reactivity of the human repertoire to any number of vaccine candidates.

Primers were designed using custom software written in Perl for maximal coverage of all human Ig sequences (Table <NUM> and Table <NUM>). Nucleotide sequences for leader, variable and constant regions were downloaded from IMGT (<NPL>) for the human heavy, lambda and kappa genes (excluding pseudogenes and truncated transcripts) and four sets of primers were designed for each gene family. Outside primers ("out" subscript in the primer names) were designed by calling Primer3 and design primers to span the splice junction of the leader sequence ("out_5" primers) or bind within the first <NUM> bases of the constant domain ("out_3" primers). For VK_out_3, a manually designed primer was created to span the variable domain-constant domain splice junction. All primers were designed to anneal with a minimal melting temperature of <NUM>. Inside primers ("in" subscript in the primer names) were designed by fixing the <NUM>' end of the primer at the start ("in_5") or end ("in_3") of the V coding sequence and extended until the Tm reached <NUM> using the OligoTm module from Primer3. FR4 specific primers were also manually extended for increased specificity. Where possible, primers within each set were consolidated to have at most <NUM> degenerate bases. Where possible, primers within each set were fused to overhangs to enable linker formation (VH_in_3 and VK/L_in_5). Where possible, primers within each set were fused to overhangs to enable restriction digestion by Notl or Sfil. Where possible, primers within each set were barcoded for MiSeq deep sequencing (Table <NUM>).

This method described below provides a platform to capture the antibody repertoire from pools of primary B-cells into a screenable format while maintaining the cognate heavy and light chain pairing (<FIG>). To achieve this, each B-cell is encapsulated into a water-in-oil droplet containing reagents for RT-PCR amplification of the heavy and light variable domain gene transcripts and their pairing by overlap-extension PCR to generate a scFv amplicon.

B-cells were isolated from healthy human blood samples using the RoboSep Human B-cell Enrichment Kit (StemCell Technologies, 19054RF). Cells were centrifuged at 500xg for <NUM> minutes and re-suspended in RPMI1640 (Invitrogen, A10491-<NUM>), supplemented with insulin-transferrin-selenium (Invitrogen, <NUM>-<NUM>), <NUM>% fetal bovine serum (Invitrogen, <NUM>-<NUM>), <NUM>. 5µg/ml megaCD40L (Enzo, ALX-<NUM>-<NUM>-C010), 33ng/ml IL-<NUM> (internally produced) and penicillin-streptomycin-glutamine (Invitrogen, <NUM>-<NUM>) and grown at <NUM> and <NUM>% CO<NUM> for <NUM> hours. Prior to encapsulation, cells were washed in PBS (<NUM> minutes at 700xg) before re-suspending in hypoosmolar electrofusion buffer (Eppendorf, <NUM>) containing <NUM>:<NUM>,<NUM> dilution of Anti-Clumping Agent (Invitrogen, 0010057DG) and <NUM>/ml acetylated BSA (EURx, E4020-<NUM>).

Stimulated B-cells have a tendency to aggregate over time and this can cause changes in flow rates, as well as multiple cells being encapsulated together. Use of an anti-clumping excipient and a paramagnetic stir disk were found to keep cells from settling prior to encapsulation.

Acetylated BSA is an amphipathic molecule which can stabilize the water-oil interface and lower the interfacial tension (<NPL>). Droplet coalescence during the harsh conditions of PCR cycling was optimized. A combination of lower denaturation temperatures and the use of acetylated BSA decreased droplet coalescence and improved droplet stability (<FIG>). Preferably, the denaturation temperatures are within the range of about <NUM> and about <NUM>. More preferably, the denaturation temperatures are within the range of about <NUM> and about <NUM>. For instance, the denaturation temperature may be about <NUM>. Further, the presence of acetylated BSA may protect enzymes such as reverse transcriptase from denaturation at the interface.

As reagents within the droplets cannot be added or subtracted once the droplet has formed, a reaction mixture was optimized to perform all steps in a single reaction mix. Cells were encapsulated at a <NUM>:<NUM> ratio with 2xRT-PCR master mix. Stock primers were mixed at <NUM> in equal amounts to create pools that were added to the RT-PCR mix.

A typical master mix of 300µl was composed of <NUM>. 86µl VH-out-F-T7, <NUM>. 86µl VL-out-R-T3, 2µl VH-in-R and 2µl VL-in-F (Table <NUM>), 120ul 5x OneStep RT-PCR buffer, 24µl OneStep RT-PCR enzyme mix, <NUM>. 1µl Q solution (Qiagen, <NUM>), 12µl <NUM> dNTP (Invitrogen, <NUM>-<NUM>) and 30µl RNaseOUT (Invitrogen, <NUM>-<NUM>). Reagents were left to incubate on ice before centrifuging through a <NUM> spin filter (Corning, <NUM>).

The organic phase of the emulsion was made using the Micellula emulsion PCR kit (EURx, <NUM>-<NUM>) using <NUM>% component <NUM>, <NUM>% component <NUM> and <NUM>% component <NUM>. Reagents were mixed and vortexed for <NUM> seconds at maximum speed and incubated at room temperature for at least <NUM> minutes. Organic phase was filtered through a <NUM> filter prior to encapsulation.

Encapsulation was performed on a <NUM>-reagent droplet generation chip (Dolomite, <NUM>) with fluids pumped using an OB1 flow controller (Elvesys). Aqueous liquids of cells and RT-PCR mix were each pumped at 57mbar while the oil liquid was pumped at 101mbar. The resulting emulsion was collected in <NUM>-minute fractions (about 40µl emulsion per fraction) in PCR strip tubes. As a control, an open PCR reaction was made by combining equal volumes of cell and RT-PCR mixes and divided in PCR strip tubes in 40µl aliquots.

It was found that by reducing the relative amount of 'inside' relative to 'outside' primers (<FIG>) by a factor of <NUM>, it was possible to selectively deplete the inside primers during the early PCR cycles and favour production of the full linked product over individual variable domain amplicons (data not shown).

Encapsulated and open reverse-transcription PCR reactions were performed with a reverse transcription step of <NUM> minutes at <NUM> followed by heat-inactivation of RT/activation of Taq polymerase of <NUM> minutes at <NUM>. This was followed by <NUM> cycles of PCR (<NUM> for <NUM> seconds, <NUM> for <NUM> seconds, <NUM> for <NUM> minute) and a final extension step of <NUM> minutes at <NUM>. Excess oil above the droplets was manually removed and the droplets were lysed by adding 5x excess of Buffer PB from the QiaQuick PCR purification kit (Qiagen, <NUM>) and PCR product was purified according to the manufacturer's instructions. The products were size-selected on <NUM>% agarose to between <NUM>-1200bp using the QiaQuick gel-extraction kit (Qiagen, <NUM>) and eluted in 40µl EB buffer.

Nested PCR amplification was performed in 15µl reactions using mixtures of VH-in-F and either VK-in-R or VL-in-R (at <NUM>:<NUM> dilution, Table <NUM>), consisting of 1µl purified RT-PCR product as template, 3µl diluted primer mix, <NUM>. 5µl 10x Hifi Platinum PCR buffer, <NUM>. 3µl <NUM> dNTP, <NUM>. 6µl <NUM> MgSO<NUM> and <NUM>. 06µl Hifi Platinum Taq (Invitrogen, <NUM>-<NUM>). Cycling conditions consisted of an initial denaturation step of <NUM> minutes at <NUM> followed by <NUM> cycles of PCR (<NUM> for <NUM> sec, <NUM> for <NUM> sec, <NUM> for <NUM> sec) and a final extension step of <NUM> minutes at <NUM>. Products were purified using the QiaQuick PCR purification kit and eluted in 40µl EB buffer.

In order to demonstrate single-cell encapsulation, two aliquots of <NUM> million mouse hybridoma cells were stained with red or green fluorescence using CellTracker dyes (Invitrogen, C34552 and C7025) according to the manufacturer's instructions. Stained cells were resuspended in PBS and encapsulated using the conditions described above, substituting the RT-PCR mix with PBS. Droplets were collected in <NUM>-well dishes and imaged at 200x magnification using the Evos FL Auto Cell Imaging System (Invitrogen).

A density of <NUM> million cells per ml was found to be optimal for obtaining mostly single-cell encapsulation into droplets of approximately <NUM> in diameter (<FIG>). Although a number of empty droplets were generated using this process, these did not contribute to the scFv library as no template cells are present.

In order to measure improvements in droplet stability, droplets were generated with mouse hybridoma cells and RT-PCR buffer using two methods. The first method used two syringe pumps (Razel R-<NUM>) to deliver aqueous and oil fluids, respectively, to the microfluidic chip. Aqueous fluids were loaded into <NUM> syringes and dispensed simultaneously from a single pump at <NUM>. 5µl/min, whereas the oil:surfactant solution was loaded into a <NUM> syringe and dispensed from a separate pump at <NUM>. The second method was as described above using a pulseless pressure pump. 5µl emulsion was transferred to <NUM> well microtiter plates and imaged at 25x magnification to inspect for coalescence and droplet homogeneity. The emulsions were then subjected to <NUM> cycles of RT-PCR (as described above) and imaged once again.

Optimized PCR conditions and excipients contributed to increased stability, as did generating monodisperse droplets (<FIG>).

To test the optimization at achieving single-cell encapsulation and droplet stability during RT-PCR, a mixture of primary human and mouse B-cells were used and primer sets were designed to amplify and link the CH1 and Cκ domains.

In more detail, primary human B-cells from healthy donors were processed and stimulated as described above. Primary mouse B-cells were isolated from splenocytes using the Mouse B-cell Isolation Kit (StemCell Technologies, <NUM>) according to the manufacturer's instructions. These cells were stimulated in identical conditions as human B-cells, substituting megaCD40L with mouse CD40L (Enzo, ALX-<NUM>-<NUM>-C010) and mouse IL21 (internally produced). <NUM>-hour stimulated cells were combined in a <NUM>:<NUM> ratio and <NUM>,<NUM> cells were encapsulated as described above. A parallel "open" reaction was performed by combining <NUM>,<NUM> cells directly in RT-PCR mix without encapsulation. ScFv-like amplicons were generated using RT-PCR and nested PCR as described above, with primer sets designed to amplify and pair the CH1 and Cκ domains.

Using the constant instead of the variable domains greatly reduced the complexity of expected outputs to just <NUM> possibilities: two amplicons with paired fragments (hCH1-hCκ and mCH1-mCκ) and two amplicons with scrambled fragments (hCH1-mCκ and mCH1-hCκ) which were easily detectable by nested PCR with specific primers and confirmed by Sanger sequencing. As expected, all possible products were identified using an open reaction but strikingly the Immune Replica technology only generated correctly paired amplicons (<FIG>). This provides evidence that chain pairing is maintained within droplets.

Serum from two healthy donors (<NUM> and <NUM>) was isolated by centrifugation of whole blood at 500xg for <NUM> minutes, then diluted in ELISA blocking buffer (<NUM>% nonfat milk - Bio-Rad, 106404XTU + <NUM>% Tween-<NUM> - BDH,BDH4210 in PBS). albicans mannan and Influenza hemagglutinin (South Dakota) antigens were produced in-house and coated on <NUM>-well High Binding plates (Corning, <NUM>) at 4µg/ml and incubated overnight at <NUM>. Plates were blocked in ELISA blocking buffer for <NUM> hours before being washed <NUM> times with ELISA washing buffer (PBS + <NUM>% Tween-<NUM>) and incubated with serial dilutions of the sera for <NUM> hour. Plates were washed <NUM> times and bound IgG was detected with a <NUM>:<NUM>,<NUM> dilution of anti-human Fc-gamma-HRP (Jackson labs, <NUM>-<NUM>-<NUM>), with TMB development over <NUM> minutes (KPL, <NUM>-<NUM>-<NUM>). The reaction was stopped by adding an equal volume of <NUM> hydrochloric acid before colorimetric analysis was performed by measuring absorbance at <NUM>.

Both donors had moderate serum titers against two common therapeutic targets: Influenza hemagglutinin (H1, South-Dakota variant) and C. albicans mannan (<FIG>).

In order to validate the Immune Replica technology, a head-to-head comparison of libraries generated from primary human B-cells with and without single-cell encapsulation was conducted.

For each of the two healthy donors (<NUM> and <NUM>), approximately <NUM>,<NUM> primary B-cells were encapsulated ("em") with the full set of primers for human variable gene amplification and chain pairing as described above. In parallel, reference libraries were also generated for each donor using <NUM>,<NUM> primary B-cells that were not encapsulated (open ("op") reaction), where variable gene pairings are expected to be scrambled.

Following RT-PCR and nested PCR amplification, scFv amplicon bands were subcloned into a scFv-Fc expression vector with NotI/SfiI (New England Biolabs cat no R0189S and R0123S) for high-throughput screening by ELISA (<FIG>).

Transformants from the <NUM> libraries (<NUM>-em, <NUM>-op, <NUM>-em and <NUM>-op) were plated on Qtrays containing 2xYT agar+ 100µg/ml carbenicillin + <NUM>% glucose (Teknova, Y6260) and <NUM>,<NUM> colonies were picked for each into <NUM>-well plates containing 60ul LB + 100µg/ml carbenicillin (Invitrogen, <NUM>-<NUM>) + <NUM>% glucose (Teknova, G0535) and grown overnight at <NUM>. 5µl of the overnight culture was used to inoculate <NUM> deep-well plates containing 250µl reconstituted MagicMedia (Invitrogen, K6803) + 100µg/ml carbenicillin and grown over <NUM> days at <NUM>. After <NUM> days the cultures were treated with <NUM>:<NUM> dilution of PopCulture reagent (Novagen, <NUM>) + <NUM>:<NUM>,<NUM> dilution of DNAse I (Invitrogen, <NUM>-<NUM>) and debris was cleared by centrifugation at 4000xg for <NUM> minutes. Antigen binding was performed by ELISA, as described above, except that the blocking step used <NUM>% BSA (Sigma, A7030) + <NUM>% Tween-<NUM> (BDH, BDH4210) in PBS. Antibodies were deemed specific to the antigen where the signal was greater than the mean background signal plus <NUM>-times the background standard deviation. Hits were verified by re-expressing the antibody from glycerol stocks and repeating the ELISA.

Of the <NUM>,<NUM> colonies that were screened for binding to immobilized antigen, <NUM> binders to hemagglutinin were identified and <NUM> binder to mannan (Table <NUM>). The majority of hits (<NUM>/<NUM>) came from the droplet libraries, suggesting that by preserving native chain pairing the identification of clones that may be lost during construction of a combinatorial library is facilitated.

In parallel, the repertoire of the encapsulated and open libraries was characterized by paired-end Illumina MiSeq deep sequencing.

As scFv amplicons are too large for MiSeq deep sequencing, separate VH and VK/L fragments were amplified using specific primer sets (Table <NUM>). Nested PCR amplification was performed in 15µl reactions using mixtures of iVH-in-F/iVH-in-R, iVK-in-F/iVK-in-R and iVL-in-F/iVL-in-R (at <NUM>:<NUM> dilution, Table <NUM>), consisting of 1µl template, 3µl <NUM>:<NUM> dilution of primer mix (VH-in-F + VL-in-R), <NUM>. 5µl 10x Hifi Platinum PCR buffer, <NUM>. 3µl <NUM> dNTP, <NUM>. 6µl <NUM> MgSO<NUM> and <NUM>. 06µl Hifi Platinum Taq (Invitrogen, <NUM>-<NUM>). Cycling conditions consisted of an initial denaturation step of <NUM> minutes at <NUM> followed by <NUM> cycles of PCR (<NUM> for <NUM> sec, <NUM> for <NUM> sec, <NUM> for <NUM> sec) and a final extension step of <NUM> minutes at <NUM>. Products were submitted for MiSeq 2x250bp paired-end sequencing (SeqMatic). Raw reads were quality filtered based on the reported quality score in the FASTQ files. Unique reads were mapped to IMGT V and J gene germline sequences to determine any biases that may have been introduced as a result of amplification within droplets.

All gene families that were detected in the open library were also identified within the encapsulated one at very similar proportions, suggesting that droplet amplification does not bias the repertoire (<FIG>).

In order to increase the throughput of the Immune Replica technology to <NUM><NUM> cells, a less viscous oil carrier (fluorinated oil) was used to enable the use of greater flow rates. <NUM><NUM> cells counted by ViCell were successfully encapsulated within <NUM>-<NUM> minutes.

RT-PCR components, cell encapsulation buffers and microfluidic flow rates were further optimized to generate droplets from <NUM><NUM> B cells that had improved stability during RT-PCR and were homogenous in size.

The method described below shows the capture of two million primary B cells into natively-paired expressible libraries that can be directly enriched and screened for function, while still maintaining the ability to profile the paired repertoire by next-generation sequencing (<FIG>). This is achieved by encapsulating B cells into picoliter-sized droplets (approximately <NUM> pL in volume), in which their cognate V genes are fused in-frame to form an scFv cassette. Glass microfluidic chips were used with pressure pumps to reliably generate evenly sized droplets at high rates, such that one million B cells could be encapsulated within <NUM> minutes.

The power of this approach is demonstrated by constructing natively-paired phage-display libraries from the peripheral blood cells of two healthy donors, which allowed selection to be driven towards antibodies cross-reactive to multiple influenza hemagglutinin (HA) subtypes.

Progression from whole blood isolation to <NUM> unique anti-HA monoclonal antibodies was achieved within four weeks. Six of these antibodies were cross-reactive to multiple HA subtypes, including one that showed cross-reactivity to <NUM> different subtypes from influenza A (Group <NUM> and <NUM>) and B lineages. The vast majority of these antibody sequences were not detected by next-generation sequencing of the paired repertoire, illustrating how this method can isolate extremely rare leads not likely found by existing technologies.

To capture the paired immunoglobulin repertoire into an expressible format, primer sets for multiplex amplification of all known human V and J genes were computationally designed from IMGT consensus sequences as described in Example <NUM>. In total, <NUM> primers were designed to amplify the <NUM> functional human V and J alleles with the appropriate overhangs for scFv generation (Table <NUM>).

Total B cells from healthy donors were isolated and stimulated as described in the examples above. For memory B cell isolation, the Human Switched Memory B Cell Isolation Kit (Miltenyi Biotec) is further used.

Two million B cells were separately isolated from the blood of two healthy donors: total B cells from Donor <NUM> and IgG+/IgA+ switched memory B cells from Donor <NUM>.

For each donor, the cells were washed in PBS (<NUM> minutes at <NUM>) and split into two halves.

One million cells were encapsulated with the optimized RT-PCR mix to generate natively-paired amplicon libraries ("emulsion library"). Specifically, one million cells were re-suspended in encapsulation buffer: hypo-osmolar electrofusion buffer (Eppendorf, <NUM>) containing <NUM>:<NUM>,<NUM> dilution of Anti-Clumping Agent (Invitrogen, <NUM>-0057AE) and <NUM>% OptiPrep Density Gradient medium (Sigma, D1556).

Cells were encapsulated at a <NUM>:<NUM> ratio with 2xRT-PCR master mix. The primers within each set were mixed in equal amounts and optimised concentrations of each set were added to the RT-PCR mix. A typical 2x master mix was composed of <NUM> VH-out-F, <NUM> VL-out-R, <NUM> VH-in-R and <NUM> VL-in-F (Table <NUM>), 2x One Tube RT-PCR reaction buffer (Roche cat no <NUM>), <NUM>% Titan One Tube RT-PCR enzyme mix (Roche, <NUM>), <NUM>% Q solution (Qiagen, <NUM>), <NUM> dNTP (Invitrogen, <NUM>), <NUM> DTT (Roche, <NUM>) and <NUM> units RNaseOUT (Invitrogen, <NUM>).

Encapsulation was performed on a <NUM>-reagent droplet generation fluorophilic chip (Dolomite, <NUM>) with fluids pumped using an OB1 flow controller (Elveflow, MKII). Aqueous liquids of cells and RT-PCR mix were each pumped at 30mbar while HFE-<NUM> fluorinated oil + <NUM>% w/v <NUM>-fluoro-surfactant (RAN Biotechnologies, <NUM>-FLUOROSURFACTANT-HFE7500) was pumped at 67mbar, with pressures fine-tuned to obtain a <NUM>:<NUM> mix of aqueous fluids. The resulting emulsion was collected in fractions (about 40µl emulsion per fraction) in PCR strip tubes and overlaid with mineral oil. Excess fluorinated oil was removed to maintain the overall volume at 100µL.

It was found that, using this method, it was possible to encapsulate one million B cells within <NUM> minutes. This method allows reliable generation of evenly sized droplets at high rates.

The remaining million cells were used to build a combinatorial scFv library ("combinatorial library"). Specifically, one million cells were processed for total RNA using the RNEasy RNA isolation kit (Qiagen) according to the manufacturer's instructions. 250ng total RNA was used for RT-PCR using the same master mix as with emulsions, except that the VH and VL sequences were amplified separately and then paired by overlap extension PCR (using the same primer sets).

Encapsulated and combinatorial libraries were created by reverse transcription for <NUM> minutes at <NUM> followed by heat-inactivation of RT/activation of Taq polymerase of <NUM> minutes at <NUM>. This was followed by <NUM> (emulsion) or <NUM> (combinatorial) cycles of PCR (<NUM> for <NUM> seconds, <NUM> for <NUM> seconds, <NUM> for <NUM> seconds) and a final extension step of <NUM> minutes at <NUM>. Excess oil below the droplets was manually removed and the droplets chemically coalesced using an equal volume of Pico-Break <NUM> (Dolomite). Amplified DNA was size-selected on <NUM>% agarose using the QIAquick gel-extraction kit (Qiagen).

Though it has been reported by several groups that cell-based RT-PCR is not feasible in volumes of less than <NUM> nL (<NPL>; <NPL>; <NPL>; <NPL>), it was found that this method is able to successfully amplify Ig transcripts directly from cells in droplets of approximately <NUM> pL in volume.

Both cases resulted in a linked product consisting of (from <NUM>' to <NUM>') part of the VH leader sequence, VH, (Gly<NUM>-Ser)<NUM> linker, VL, N-terminus of CL. This product was then used as template for nested PCR with VH FR1 and VL FR4 specific primer sets to generate full-length scFv amplicon libraries (<FIG>).

To obtain an in-depth assessment of the captured repertoire, the nested PCR primers contained barcoded overhangs that enable next-generation sequencing on the Illumina MiSeq (<FIG>).

Nested PCR amplification consisted of <NUM>% purified RT-PCR product, <NUM> VH-in-F and VL-in-R primer pools (Table <NUM>), 1x Hifi Platinum PCR buffer, <NUM> dNTP, <NUM> MgSO<NUM> and <NUM> units Hifi Platinum Taq (Invitrogen). Cycling conditions consisted of an initial denaturation step of <NUM> minutes at <NUM> followed by <NUM> cycles of PCR (<NUM> for <NUM> sec, <NUM> for <NUM> sec, <NUM> for <NUM> sec) and a final extension step of <NUM> minutes at <NUM>. Products were again size-selected as above.

A final scale-up PCR was performed using common forward (Illu scaleup_F) and barcoded reverse primers (Illu_R_N50X) to enable library construction and Illumina sequencing (Table <NUM>). We used the Q5 polymerase (NEB) according to manufacturer's instructions with the following thermocycling program: <NUM> for <NUM> minutes, <NUM>-<NUM> cycles of <NUM> for <NUM> seconds and <NUM> for <NUM> seconds, <NUM> for <NUM> minutes.

Each barcoded library was size-selected to 850bp, combined in equal amounts and subjected to <NUM>×300bp MiSeq sequencing using a custom priming approach (SeqMatic). The custom priming strategy was designed to obtain paired 300bp reads of the <NUM>' ends of VH and VL, consisting of FR4, CDR3 and FR3 (<FIG>). The R1 and R2 primers (Table <NUM>) were used to generate VL and VH sequences, respectively, whereas the standard Illumina P5 primer was used for the index read. Whereas the VL read was obtained using a priming site introduced at the <NUM>' end of the construct, the VH read required an internal primer annealing to the (G<NUM>S)<NUM> linker sequence (Table <NUM>).

Following demultiplexing, raw Fastq reads were quality-filtered using FastQC, paired by the Illumina Fastq ID, aligned to IMGT V and J genes and annotated according to Kabat definition (<NPL>) to extract CDR3 sequences. Subsequently, CDRH3 and CDRL3 sequences were concatenated and clustered where the amino acid identity was greater than <NUM>%.

Unique CDRH3 and CDRL3 sequences were counted and the numbers of unique VL sequences pairing with each unique VH were calculated as a measure of pairing efficiency. For CDRH3 sequences paired with multiple CDRL3, the top-pair weight is determined as the ratio of counts between the most abundant CDRL3 and all CDRL3 sequences (<NPL>). A total of <NUM>,<NUM> and <NUM>,<NUM>,<NUM> unique CDRH3:CDRL3 clusters were recovered for the two emulsion and combinatorial libraries, respectively (Table <NUM>).

The clustering parameters were validated using the error-corrected asymptotic Chao richness estimator (<NPL>) and it was found that the computed diversity of the amplicon library adjusted for sequencing artifacts is very close to the observed number of clusters. This provides evidence that the clustering parameters reliably corrected for sequencing errors while minimising the loss of truly unique sequences.

To validate this approach at achieving single-cell encapsulation and cognate chain pairing, primary human and mouse B cells were isolated and stimulated as described in Example <NUM>. Equal amounts of primary stimulated mouse and human B cells were mixed and <NUM>,<NUM> cells from this mixture were encapsulated as described in Example <NUM>.

A parallel "combinatorial" reaction was performed by combining <NUM>,<NUM> cells directly in RT-PCR mix without encapsulation. RT-PCR and nested PCR conditions were as described in Example <NUM> with primer sets designed to amplify and pair the CH1 and Cκ domains (Table <NUM>).

As expected, the combinatorial format produced all possible products but, strikingly, only natively-paired amplicons were generated with encapsulation (<FIG>).

One million total B cells isolated from the blood of a healthy donor were mixed with <NUM>,<NUM> IM-<NUM> cells (<NUM>%) before being encapsulated with the optimised RT-PCR mix to generate a natively-paired amplicon library, consisting of (from <NUM>' to <NUM>') part of the VH leader sequence, VH, (Gly<NUM>-Ser)<NUM> linker, VL, N-terminus of CL. This product was then used as template for nested PCR with VH FR1 and VL FR4 specific primer sets to generate a full-length scFv amplicon library (<FIG>).

As a further validation of correct chain pairing, a primer specific to the IM-<NUM> CDRH3 sequence (RRGVTDIDPFDI; IM9-CDRH3-Fwd) was used with a generic reverse primer (R1, Table <NUM>) to amplify all VL sequences that paired with the IM-<NUM> heavy chain. The resulting amplicon was cloned and analysed by Sanger sequencing, which showed correct pairing with the known IM-<NUM> VL (QHYNRPWT) in <NUM>/<NUM> colonies (<NUM>% pairing accuracy). This confirms that even with an overwhelming abundance of competing B cells; the present method maintains correct chain pairing.

As a yet further validation that the Immune Replica system preserves chain pairing, the number of unique CDRL3 sequences that paired with each CDRH3 sequence was determined.

As expected, the combinatorial library displayed promiscuous pairing, with each CDRH3 sequence paired with a median of <NUM>-<NUM> unique CDRL3 sequences (<FIG>, Table <NUM>). Given that the sequencing depth (<NUM><NUM>) vastly under-samples the theoretical sequence diversity of the combinatorial libraries (<NUM><NUM>), the true rate of combinatorial pairing would likely be considerably higher. This was in stark contrast to the emulsion libraries, where a median of <NUM>:<NUM> CDRH3/CDRL3 pairing with narrow distribution was observed. In cases where multiple pairings were detected, top-pair analysis determined a <NUM>%-<NUM>% accuracy in VH-VL pairing (<FIG>). The top-pair method has been used to validate cognate chain pairing in a previously published method that generates amplicons only suitable for sequencing, but not for screening (<NPL>). However, using similar sequencing depth and starting cell numbers the pairing efficiency was found to be significantly better with the present method (p < <NUM>, <FIG> and Table <NUM>).

B cells were stained using CellTracker Red CMTPX or CellTracker Green CMFDA dyes (Life Technologies) according to the manufacturer's instructions. Stained cells were re-suspended in PBS and encapsulated using the conditions described above, substituting the RT-PCR mix with PBS. Cell lysis was imaged in two ways: (<NUM>) stained cells were re-suspended in encapsulation buffer, encapsulated with RT-PCR mix and heated to <NUM> for <NUM> minutes; (<NUM>) unstained cells were encapsulated with RT-PCR mix containing 2x SYBR-Green (Invitrogen) and heated to <NUM> for <NUM> minutes. Droplets were collected in µ-Slide<NUM> channel slides (lbidi) and imaged at 200x magnification using the Evos FL Auto Cell Imaging System (Invitrogen).

Robust cell lysis caused by addition of RT-PCR buffer and incubation at <NUM>, was observed by Trypan Blue staining (<FIG>), and detection of release of cytosolic dyes and nuclear material by SYBR-Green (<FIG>).

As proof of principle, the scFv libraries were used to isolate antibodies against influenza hemagglutinin, an antigen to which humans are commonly exposed.

The emulsion and combinatorial libraries were bulk subcloned into a phagemid vector (<NPL>) to construct phage-display libraries of over <NUM>×<NUM><NUM> transformants.

Specifically, the emulsion and combinatorial amplicon libraries were subcloned into a phagemid vector (pCANTAB6) using Not1/Sfi1 restriction enzymes (NEB) and phage display libraries were generated as described in <NPL>. <NUM> colonies from each of the <NUM> libraries were cultured to mid-log phase and infected with M13-K07 (Invitrogen) to initiate overnight monoclonal phage production. Antibody display was determined by ELISA. 1µg/ml anti-myc antibody (Invitrogen) was immobilised overnight on <NUM> well MAXISORP plates (Nunc) and blocked for <NUM> hours with <NUM>% BSA (Sigma) and <NUM>% Tween-<NUM> (BDH). Following washing with PBST (PBS pH7. <NUM> (Invitrogen) + <NUM>% Tween-<NUM>), diluted phage supernatant was bound and detected using an anti-M13-HRP antibody (<NUM>:<NUM>, GE Healthcare) and visualised with TMB (KPL). Monoclonal phage ELISA against the myc tag fused to the scFv indicated that the libraries mostly displayed scFv well, with positive display seen for <NUM>-<NUM>% of clones (Table <NUM>).

Recombinant hemagglutinin proteins were expressed and purified as described in <NPL>. HA proteins used are as follows: H1 CA/<NUM>, A/California/<NUM>/<NUM> H1N1; H1 SD/<NUM>, A/South Dakota/<NUM>/<NUM> H1N1; H2 MO/<NUM>, A/Swine/Missouri/<NUM> H2N3; H5 VN/<NUM>, A/Vietnam/<NUM>/<NUM> H5N1; H6 HK/<NUM>, A/teal/Hong Kong/W312/<NUM> H6N1; H9 HK/<NUM>, A/chicken/Hong Kong/G9/<NUM> H9N2; H3 PE/<NUM>, A/Perth/<NUM>/<NUM> H3N2; H7 NL/<NUM>, A/Netherlands/<NUM>/<NUM> H7N7; B FL/<NUM>, B/Florida/<NUM>/<NUM> Yamagata lineage; B BR/<NUM>, B/Brisbane/<NUM>/<NUM> Victoria lineage).

The four libraries were subjected to two rounds of enrichment using used <NUM> biotinylated hemagglutinin H1 (A/California/<NUM>/<NUM> H1N1) as described in <NPL>.

Amplified phage outputs were profiled by polyclonal ELISA, using immobilised NeutrAvidin (Thermo Fisher Scientific) to capture specific biotinylated antigen prior to incubation with phage. Polyclonal phage ELISA confirmed robust enrichment for specific hemagglutinin H1 binders regardless of the B cell source (<FIG>, <FIG>). Of note, while the combinatorial libraries showed an overall stronger specific enrichment, monoclonal sequencing of the enriched clones revealed a strong bias (<NUM>%) for IGHV1-<NUM> germline sequences as compared to the corresponding emulsion library (<NUM>%, <FIG>).

Since it has previously been shown that IGHV1-<NUM> containing antibodies can contact group <NUM> hemagglutinin subtypes through heavy chain interactions alone (<NPL>), it is suggested that enrichment of combinatorial libraries was driven by selecting for VL partners to IGHV1-<NUM> that expressed or folded well in bacteria. This highlights a key bias with combinatorial libraries.

To specifically enrich for cross-reactive antibodies, the first round output was panned from the emulsion libraries on a non-circulating group <NUM> subtype, influenza A hemagglutinin H5 (<NUM>, A/Vietnam/<NUM>/<NUM>).

Enriched libraries were bulk subcloned into an scFv-Fc expression vector (<NPL>) using Not1/Sfi1 restriction enzymes (NEB) and transformed into chemically competent Top10 cells (Invitrogen). Single clones were grown overnight in LB containing 100µg/ml carbenicillin (Invitrogen) and <NUM>% Glucose (TekNova) before being diluted <NUM>:<NUM> in reconstituted MagicMedia (Invitrogen) containing 100µg/ml carbenicillin (Invitrogen). Cells were induced over <NUM> hours at <NUM> and pelleted by centrifugation. Diluted supernatants were used to determine antigen reactivity by ELISA as described above, using an anti-Fc-gamma-HRP secondary antibody (Jackson ImmunoResearch). Following sequencing, unique clones were expressed in HEK-<NUM> Freestyle cells for <NUM> days and supernatants were used to confirm binding by ELISA.

Of the <NUM>,<NUM> clones screened, <NUM> clones showed specific binding to H1, consisting of <NUM> unique sequences. This included six unique antibodies that showed cross-reactivity to both antigens used in the panning (<FIG>). Surprisingly, one of these antibodies (IGHV1-<NUM>/IGLV1-<NUM>) displayed specific binding to all <NUM> HA subtypes tested, including subtypes from the A (Group <NUM> and <NUM>) and both lineages of influenza B (Yamagata and Victoria), with relatively similar EC<NUM> values ranging between <NUM> to <NUM> (<FIG>). Such universal anti-influenza antibodies are thought to be extremely rare, having long been sought out, yet only identified once by combinatorial phage-display (<NPL>). Whereas the existence of such an antibody within the healthy donor sample used was unexpected, the ability to isolate it through deep mining of the repertoire illustrates the power of the present method.

To ascertain the relative frequency of the hits within the captured B cell repertoire, their respective CDRH3:CDRL3 pairs were searched within the next generation sequencing dataset, allowing for up to <NUM> amino acid mismatches to account for possible sequencing-induced mutations. Only one of the <NUM> antigen-specific sequences was observed among the <NUM>,<NUM> unique paired sequence clusters, implying that the remaining hits were too rare to be detected by next generation sequencing. This sequence (0089EA-C02) accounted for <NUM> out of <NUM>,<NUM>,<NUM> mapped reads (Table <NUM>). Following selection, it was found this clone repeated in <NUM> out of the <NUM>,<NUM> clones screened, an enrichment of <NUM>,<NUM> fold.

It will be appreciated by the person skilled in the art that other platforms for displaying native human antibodies are equally applicable, and the inventors have explicitly contemplated such alternative display systems. For example, yeast display systems might allow the identification of further antigen-specific sequences which might have existed in the repertoire, but were not selected because of differences in expression and folding of human antibodies in bacteria.

As this platform depends on successful PCR from gene-specific primers, it is possible that antibody genes mutated within the primer binding sites may be excluded from the resulting library. Ancestral antibodies of equal activity yet having fewer mutations (<NPL>) could still be captured.

Nevertheless, this particular set of leads could not have been predicted from sequencing information alone. To the extent that the scarcity of these leads determined by next generation sequencing represents that within the original B cell pool, this is suggestive that these leads could not be found through standard methods of culturing and screening individual B cells.

It was thought that encapsulation of free RNA coming from dead or dying cells before they have entrained into droplets could interfere with the isolation of natively-paired libraries. In particular, the RNA encoding VH or VL domains might contaminate droplets and lead to non-natively paired products. In order to determine whether cell lysis prior to encapsulation can contaminate droplets with RNA, <NUM> stimulated cells were incubated for the same duration (<NUM> minutes) as a typical encapsulation and then bulk RT-PCR of VH domains was performed from either the supernatant or the cell pellet (positive control). A water control was included as a negative control.

Results indicate that a significant amount of RNA is released from the cells (<FIG>).

Methods to mitigate this issue include reduced stimulation time, more stringent selection of live cells (e.g. FACS, bead-based), and sequestering RNA using oligo-coated magnetic beads.

Theoretically, the number of cells encapsulated in a single droplet follows a Poisson distribution with λ = <NUM>, given that the ratio between cells and droplets is <NUM>:<NUM>.

Single-cell encapsulation percentage is defined as the percentage of the droplets with single cell out of droplets with ≥<NUM> cell, therefore, the probability could be calculated as: <MAT>.

Claim 1:
A method for producing encapsulated natively-paired scFv amplicons, the method comprising:
a. encapsulating single B-cells in droplets, wherein the droplets further contain reagents for amplifying and linking native pairings of heavy and light chain variable domain amplicons from single encapsulated B-cells;
b. lysing the single encapsulated B-cells; and
c. generating the encapsulated natively-paired scFv amplicons, wherein each scFv amplicon comprises a native pairing of heavy and light chain variable domain amplicons linked together by a linker, wherein the linker has a length of <NUM>-<NUM> amino acids.