Methods for identification of cognate pairs of ligands and receptors

Abstract

Provided herein are methods for high-throughput screening (HTS) for identifying cognate pairs of a ligand and receptor. The methods comprise (a) providing a library of tagged receptors; (b) providing a second library of nucleotide sequences that encode for ligands; (c) transmitting the nucleotide sequences of second library into cellular expression systems; (d) incubating cellular expression systems for expression of ligand molecules of second library on the surface of the plurality of cellular expression systems;(e) contacting the plurality of tagged receptors with expressed ligands on the surface of plurality of cellular expression systems for receptor target pairing; (f) encapsulating the plurality of cellular expression systems in a plurality of microreactors; (g) generating nucleotide sequences of the second library and the unique barcode sequence of tagged receptor of the first library; (h) analyzing sequences from step g. to identify cognate pairs of receptors of first library and ligands of second library.

Description

[0001] METHODS FOR IDENTIFICATION OF COGNATE PAIRS OF LIGANDS AND RECEPTORS

[0002] REFERENCE TO RELATED APPLICATION

[0003] The application claims priority to and the benefit of the filing date of U.S. Provisional Patent Application No. 63 / 624453, filed on January 24, 2025, the entire contents of which, including any drawings, are incorporated herein by reference.

[0004] FIELD OF THE INVENTION

[0005] The invention relates to methods for identifying cognate pairs of ligands and receptors.

[0006] BACKGROUND OF THE INVENTION

[0007] In phenotypic discovery, molecules with a desired effect on the phenotype of a cell are isolated, followed by identification of their targets. Target deconvolution can be achieved by multiple methods yet remains the bottleneck of the process due to its low efficiency. The two main existing deconvolution approaches include immunoprecipitation followed by mass spectrometry and protein library overexpression.

[0008] In immunoprecipitation followed by mass spectrometry, the method uses immobilized antibodies to immunoprecipitated antigens from a cell lysate or membrane extract that can be identified by mass spectrometry. This method is time and resource consuming and unreliable. There are multiple limiting factors such as abundance of cells expressing the antigen, as well as the abundance of the antigen on those cells, the stability of the complex formed by the antibody and the antigen as well as the risk of denaturing the conformational epitope.

[0009] In protein library overexpression, standard protein arrays involve generating proteins and spotting them onto a substrate. This method is relatively low-cost, but suffers from the risk of getting non appropriately folded proteins, and, as a consequence, of not presenting conformational epitopes for binding. Alternatively, the proteins can be presented by a membrane protein in a cellular context in situ; in this case, some receptors may be poorly expressed and interactions may be detectable. In addition, this method scales poorly with the number of antibodies.

[0010] Given that there is strong need to develop therapeutic antibodies against new targets, as the antibodies currently used in the clinic target a small number of antigens, there remains an unmet need in phenotypic discovery strategies where the target is unknown and discovered in the process, compared to target based approaches, requiring the identity of the target to discover drugs.

[0011] BRIEF SUMMARY OF THE INVENTION

[0012] Provided herein are high throughput screening (HTS) methods for identifying receptorligand cognate pairs in a microfluidics system. The methods comprise: a. providing a receptor library, wherein each receptor molecule of the library is tagged with a first nucleotide tag sequence resulting in tagged receptors, wherein the first nucleotide tag sequence comprises a unique barcode sequence and a universal primer sequence, b. providing a second library of nucleotide sequences, wherein the nucleotide sequences encode for ligands, c. transmitting the nucleotide sequences of second library into cellular expression systems by transfection or transduction, wherein each cellular expression system expresses one nucleotide sequence of the second library resulting in a plurality of cellular expression systems, d. incubating plurality of cellular expression systems for expression of ligand molecules of second library within the plurality of cellular expression systems, wherein the ligand molecules are presented on the surface of the plurality of cellular expression systems, e. contacting the plurality of tagged receptors with expressed ligands on the surface of plurality of cellular expression systems for receptor target pairing, f. encapsulating the plurality of cellular expression systems in a plurality of microreactors, wherein each microreactor comprises one cellular expression system expressing one ligand of the second library, g. generating combined nucleotide sequences by enzymatic reaction, wherein the combined nucleotide sequences comprise the nucleotide sequences of the second library and the unique barcode sequence of tagged receptor of the first library, h. sequencing of combined nucleotide sequences, i. analyzing sequences from step h. to identify cognate pairs of receptors of first library and ligands of second library.

[0013] In certain embodiments of the methods the nucleotide sequences of the second library comprises a sequence encoding a recombinant RNA which encodes the ligand, thus the second library encodes for a set of ligands. In certain embodiments of the methods the nucleotide tag sequence is a DNA sequence. In certain embodiments of the methods the recombinant RNA of the second library is linked to the nucleotide tag sequence through an enzymatic reaction. The enzymatic reaction can be performed with a polymerase such as Klenow-fragment without 5'-3' exonuclease activity - or BST polymerase or other polymerase or using a ligase enzyme. The skilled person in the field is well aware of a wide selection of available polymerase and knows how to select the appropriate enzyme. In certain embodiments of the methods the recombinant RNA of the second library further comprises a second nucleotide tag sequence, and wherein the second nucleotide tag sequence is a second unique barcode which identifies the recombinant RNA. In certain embodiments, each ligand of the set of ligands is displayed on the surface of a cell, wherein each cell expresses a single ligand.

[0014] In certain embodiments of the methods the tagged receptors of the first library are tagged antibodies. In certain embodiments of the methods, the method further comprises cell lysis of cellular expression system in the plurality of microreactors, wherein cell lysis releases the nucleotide sequences of the second library.

[0015] In certain embodiments, the first nucleotide tag is a DNA sequence. The first nucleotide tag can, for example, comprise at least one of a sequence complementary to a sequence of the second nucleotide tag attached to the recombinant RNA, the first nucleotide tag sequence may further comprise a barcode, and a sequence for amplification.

[0016] In certain embodiments, the recombinant RNA is linked to the second nucleotide tag through an enzymatic reaction. In certain embodiments, the second nucleotide tag is a DNA sequence In certain embodiments, identifying the receptor-ligand cognate pair comprises amplifying the recombinant RNA and sequencing the recombinant RNA sequence for ligand identification.

[0017] In certain embodiments, identifying the receptor-ligand cognate pair further comprises amplifying the first nucleotide tag of the tagged receptor before and sequencing the first nucleotide tag for receptor identification.

[0018] In certain embodiments, sequencing and identifying of the first nucleotide tag and the second nucleotide tag may be performed separately or simultaneously. In certain embodiments, the method occurs in a single reaction.

[0019] In certain embodiments, the microreactor is selected from an aqueous droplet, a microcapsule, a microbead, a compartment of a microfluidic chip, or a well.

[0020] In certain embodiments, the receptor is an antibody. The antibody can, for example, be isolated from an antibody-secreting B cell from a human subject. The antibody can, for example, be isolated from patient-derived xenograft mice or humanized mice. In certain embodiments, the antibody is identified using single-cell antibody sequencing. In certain embodiments, the subject has a disease or disorder.

[0021] BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. It should be understood that the invention is not limited to the precise embodiments shown in the drawings.

[0023] FIG. 1 shows a schematic representation of the microfluidic chip design used to encapsulate reagents with transfected cells labelled with antibodies.

[0024] FIG. 2 shows a schematic of the library preparation process from in droplet reverse transcription to next generation sequencing. Tag-ID corresponds to the DNA sequence of the first nucleotide tag identifying the tagged receptor (e.g. an antibody). PCRh is a DNA site for PCR amplification at PCR1. RD1, P5, P7 are DNA sites introduced for the sequencing of the library.

[0025] FIG. 3 shows a graph demonstrating the number of reads associated with each antibody tag.

[0026] FIGs. 4A-4B show graphs demonstrating the number of reads associated with each pool for a given antibody divided by the number of reads associated with the isotype control paired with the same pool.

[0027] FIG. 5 shows a schematic of recombinant RNA (Rec RNA) and antibody tags linked using a ligation reaction. The antibody DNA tag and the Rec RNA anneal adjacent to one another on a target DNA molecule.

[0028] FIG. 6 shows a schematic of DNA sequences enabling the binding of an antibody tag with Rec RNA in the case of a ligation.

[0029] DEFINITIONS

[0030] Various publications, articles and patents are cited or described in the background and throughout the specification; each of these references is herein incorporated by reference in its entirety. Discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is for the purpose of providing context for the invention. Such discussion is not an admission that any or all of these matters form part of the prior art with respect to any inventions disclosed or claimed.

[0031] Unless defined otherwise, all technical and scientific terms used herein have the same meaning commonly understood to one of ordinary skill in the art to which this invention pertains. Otherwise, certain terms used herein have the meanings as set in the specification. All patents, published patent applications, and publications cited herein are incorporated by reference as if set forth fully herein.

[0032] Generally, nomenclatures utilized in connection with, and techniques of, cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization described herein are those well-known and commonly used in the art. Standard techniques are used for recombinant DNA, oligonucleotide synthesis and tissue culture. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein.

[0033] It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise.

[0034] Unless otherwise stated, any numerical value, such as a concentration or a concentration range described herein, is to be understood as being modified in all instances by the term "about." Thus, a numerical value typically includes ± 10% of the recited value. For example, a concentration of 1 mg / ml includes 0.9 mg / ml to 1.1 mg / ml. Likewise, a concentration range of 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v). As used herein, the use of a numerical range expressly includes all possible subranges, all individual numerical values within that range, including integers within such ranges and fractions of the values unless the context clearly indicates otherwise.

[0035] As used herein, the term "high throughput" generally refers to a method for scientific experimentation in which researchers can test thousands or more variables in parallel to arrive at a result or results for the specific method being tested. High throughput screening methods can be relevant to the fields of biology, chemistry, and / or materials science and can utilize robotics, data processing / control software, liquid handling devices, other specialized hardware, and sensitive detectors to screen large numbers of samples simultaneously and arrive at a given result or results.

[0036] As used herein, the term "nucleic acid" generally refers to at least one molecule or strand of DNA or RNA, comprising at least one nucleobase, such as, for example, a naturally occurring purine or pyrimidine base found in DNA (e.g., adenine "A," guanine "G," thymine "T," and cytosine "C") or RNA (e.g., A, G, uracil "U," and C).

[0037] "RNA" refers herein to functional RNA, such as mRNA, tRNA, ncRNA, IncRNA, miRNA, siRNA, piRNA, gRNA, telomerase RNA component, RNAi, CRISPR RNA, circular RNA, enhancer RNA, snoRNA, snRNA and rRNA.

[0038] As it will be understood by those skilled in the art, the depiction of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid also encompasses the complementary strand of a depicted single strand. The term nucleic acid thus encompasses complementary DNA. As it will also be appreciated by those skilled in the art, many variants of a nucleic acid may be used for the same purpose as a given nucleic acid. Thus, a nucleic acid also encompasses substantially identical nucleic acids and complements thereof. As it will also be understood by those skilled in the art, a single strand nucleic acid, such as, a primer, may hybridize to the target sequence under hybridization conditions, preferably stringent hybridization conditions. Thus, a nucleic acid also encompasses a primer that hybridizes under hybridization conditions to a target sequence.

[0039] These definitions refer to at least one single-stranded molecule, but in some embodiments encompass also at least one additional strand that is partially, substantially or fully complementary to the at least one single-stranded molecule. Accordingly, in some embodiments said definitions refer to double stranded molecules.

[0040] Thus, in one embodiment, a nucleic acid refers to at least one double-stranded molecule that comprises one or more complementary strand(s) or "complement(s)" of a particular sequence comprising a strand of the molecule.

[0041] The "barcode sequence" or "barcode" herein refers to a unique nucleic acid sequence that can be distinguished by its sequence from another nucleic acid sequence, thus permitting to uniquely label a nucleic acid sequence so that it can be distinguished from another nucleic acid carrying another barcode sequence. In one embodiment, the barcode sequence uniquely identifies the nucleic acids contained in a particular microreactor from nucleic acids contained in other microreactors, for instance, even after the nucleic acids are pooled together. In some embodiments, the barcode sequence may be used to distinguish tens, hundreds, or even thousands of nucleic acids, e.g., arising from cells contained in different microreactors.

[0042] In one embodiment, the barcode sequence may be of any suitable length. The barcode sequence is preferably of a length sufficient to distinguish the barcode sequence from other barcode sequences. In one embodiment, a barcode sequence has a length of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 72, 74, 76, 78, 80, 85, 90 or more nucleotides, such as 50 to 85, 60 to 80, 70 to 80 nucleotides. In some embodiments, the barcode sequence uniquely identifies the identity of a receptor and / or a ligand present in a microreactor (e.g., a droplet).

[0043] In one embodiment, the barcode sequence consists of more than one barcode sequence, wherein the barcoded sequences are different. Such barcode sequence is called herein a "set of barcode sequences."

[0044] In a related embodiment, the different barcode sequences may be taken from a "pool" of potential barcode sequences. If the barcode sequence consists of more than one barcode sequence, the barcode sequences may be taken from the same, or different pools of potential barcode sequences. The pool of sequences may be selected using any suitable technique, e.g., randomly, or such that the sequences allow for error detection and / or correction, for example, by being separated by a certain distance (e.g., Hamming distance) such that errors in reading of the barcode sequence can be detected, and in some cases, corrected. The pool may have any number of potential barcode sequences, e.g., at least 100, at least 300, at least 500, at least 1,000, at least 3,000, at least 5,000, at least 10,000, at least 30,000, at least 50,000, at least 100,000, at least 300,000, at least 500,000, at least 1,000,000, at least 10,000,000, or at least 100,000,000 barcode sequences.

[0045] Methods to join different barcode sequences taken from one "pool" or more than one "pool" are known to a person skilled in the art, and include, but are not limited to, the use of ligases and / or using annealing or a primer extension method.

[0046] In one embodiment, the barcode sequence is a double stranded or single stranded nucleic acid, or a partially single and double stranded nucleic acid.

[0047] In certain embodiments, the barcode sequence is comprised within a barcoded primer. As used herein, the term "barcoded primer" refers to at least one molecule of about 20 to about 200 nucleobases in length that can function to prime nucleic acid synthesis. In particular, the barcoded primer may be of about 30 to about 150 nucleobases in length, of about 40 to about 100 nucleobases in length, of about 50 to about 90 nucleobases in length, of about 60 to about 80 or 70 nucleobases in length. More particularly, in the context of the invention, a barcoded primer is an oligonucleotide comprising a barcode sequence or barcode set of sequences and a primer sequence, wherein each different primer sequence defines a different specificity of barcoded primer. In one embodiment, the barcoded primer comprises from 5' to 3' a universal primer sequence, a barcode sequence or barcode set of sequences and a primer sequence.

[0048] A "primer sequence" or a "sequence for amplification" is typically a short singlestranded nucleic acid, of between 6 to 50 nucleotides in length, designed to perfectly or almost perfectly match a nucleic acid of interest, to be captured and then amplified (e.g., by PCR) or reverse transcribed (e.g., by RT). The primer sequences or sequences for amplification are "specific" to the nucleic acids they hybridize to, i.e., the primer sequences preferably hybridize under stringent hybridization conditions, more preferably under highly stringent hybridization conditions, and are complementary to or almost complementary to the nucleic acids they hybridize to, also called target sequence.

[0049] Typically, the primer sequence or the sequence for amplification serves as a starting point for nucleic acid synthesis, allowing polymerase enzymes such as nucleic acid polymerase to extend the primer sequence and replicate the complementary strand. A primer sequence or sequence for amplification may be complementary to and hybridize to a target nucleic acid. In some embodiments, a primer sequence is a synthetic primer sequence. In some embodiments, a primer sequence is a non-naturally-occurring primer sequence. A primer sequence typically has a length of 6 to 50 nucleotides. For example, a primer sequence may have a length of 10 to 40, 10 to 30, 10 to 20, 25 to 50, 15 to 40, 15 to 30, 20 to 50, 20 to 40, or 20 to 30 nucleotides. In some embodiments, a primer sequence has a length of 18 to 24 nucleotides.

[0050] In one embodiment, the primer sequence or sequence for amplification is located on the 3' side of the barcoded primer used in context with the invention (i.e., the primer is in a 3' position compared to the barcode sequence).

[0051] As used herein, a "subject" is a mammal, such as a human, but can also be another animal such as a dog, a cat, a cow, a sheep, a pig, a horse, a monkey, a rat, a mouse, a rabbit, a guinea pig etc. Preferably, the subject is a human.

[0052] In a particular embodiment, the subject suffers from a disease or disorder, in particular from cancer, inflammatory and autoimmune disease, infectious disease or metabolic disease. By "cancer" is meant herein a class of diseases involving neoplasia which include both cancers that involve a solid tumor and those that do not involve a solid tumor (e.g., leukemia).

[0053] By "autoimmune disease" is meant herein a wild range of degenerative diseases caused by the immune system attacking a person's own cells.

[0054] By "inflammatory and autoimmune disease" is meant herein a disease first induced by an inflammatory process, initiated by the activation of T cells by antigen-presenting cells, which subsequently leads to the activation of other inflammatory cells and in turn the release of pro-inflammatory cytokines, chemotactic agents and matrix degrading enzymes. Examples of inflammatory and autoimmune diseases are well-known from the person skilled in the art and include rheumatoid arthritis, osteoarthritis, osteoporosis, Crohn's disease, ulcerative colitis, multiple sclerosis, periodontitis, gingivitis, graft versus host reactions, psoriasis, scleroderma, allopeicia, Sjogren's syndrome, polymyosititis, pempligus, uveititis, Addison's disease, atopic dermatitis, asthma, systemic lupus erythematosus (SLE), nephropathy and chronic obstructive pulmonary disease (COPD), diabetic retinopathy, and age-related macular degeneration.

[0055] By "infectious disease" is meant herein a disease caused by the transmission of a microorganism. In the context of the invention, the term "microorganism" refers equally to viruses, in particular viruses which have a lipid envelope (e.g., an influenza virus), bacteria, parasites, and fungi.

[0056] By "metabolic disease" is meant herein any type of disorders in which metabolic errors and imbalances occur and in which the metabolic processes take place in a sub-optimal manner. In a preferred embodiment, the metabolic disease is selected from the group consisting of hyperglycemia, diabetes, in particular type 2 diabetes, obesity, dyslipidemia and hypercholesterolemia. In a particular embodiment, said metabolic disease is diabetes, more particularly type 2 diabetes.

[0057] In the context of the invention, the term "cognate pair" of ligands and receptors refers to the pair of a ligand species and the receptor species to which it selectively binds.

[0058] By "selectively binding" is meant herein that one member of the pair recognizes and binds to the other member of the pair with greater affinity than to a member of another pair.

[0059] By "specifically binding" is meant herein that one member of the pair recognizes and binds to the other member of the pair and has no detectable binding activity for a member of another pair.

[0060] As used herein, the term "ligand" refers to a member of a particular recognition pair, which selectively binds to, preferably specifically binds to, the second member of said particular recognition pair (or cognate pair).

[0061] As used herein, the term "receptor" refers to a member of a particular recognition pair, which is selectively bound by, preferably specifically bound to, the second member of said particular recognition pair (or cognate pair).

[0062] Accordingly, the cognate pair comprises two molecules which are selectively bound by, preferably specifically bound to each other. Within this pair the terms ligand and receptor maybe mutually interchangeable, i.e., a molecule that is a ligand can be a receptor and, conversely, a molecule that is a receptor can be a ligand since ligands and receptors are defined as binding partners.

[0063] As used herein, the term "set of ligands" refers to at least one ligand species, preferably a plurality of ligand species, in particular a plurality of ligand species wherein at least two of the plurality of ligands species are part of distinct recognition pairs. Preferably, the set of ligands used in the context of the invention comprises redundant ligand species, i.e. ligand species which are present in the set in multiple copies.

[0064] As used herein, the term "set of receptors" refers to at least one receptor species, preferably a plurality of receptor species, in particular a plurality of receptor species wherein at least two of the plurality of receptor species are part of distinct recognition pairs. Preferably, the set of receptors used in the context of the invention comprises redundant receptor species, i.e. receptor species that are present in the set in multiple copies.

[0065] DETAILED DESCRIPTION OF THE INVENTION

[0066] A first aspect of the disclosed in invention are methods for high-throughput screening (HTS) for identifying a receptor-ligand cognate pair in a microfluidics system. The methods comprise the following steps: a. providing a receptor library, wherein each receptor molecule of the library is tagged with a first nucleotide tag sequence resulting in tagged receptors, wherein the first nucleotide tag sequence comprises a unique barcode sequence and a universal primer sequence, b. providing a second library of nucleotide sequences, wherein the nucleotide sequences encode for ligands, c. transmitting the nucleotide sequences of second library into cellular expression systems by transfection or transduction, wherein each cellular expression system expresses one nucleotide sequence of the second library resulting in a plurality of cellular expression systems, d. incubating plurality of cellular expression systems for expression of ligand molecules of second library within the plurality of cellular expression systems, wherein the ligand molecules are presented on the surface of the plurality of cellular expression systems, e. contacting the plurality of tagged receptors with expressed ligands on the surface of plurality of cellular expression systems for receptor target pairing, f. encapsulating the plurality of cellular expression systems in a plurality of microreactors, wherein each microreactor comprises one cellular expression system expressing one ligand of the second library, g. generating combined nucleotide sequences by enzymatic reaction, wherein the combined nucleotide sequences comprise the nucleotide sequences of the second library and the unique barcode sequence of tagged receptor of the first library, h. sequencing of combined nucleotide sequences i. analyzing sequences from step h. to identify cognate pairs of receptors of first library and ligands of second library.

[0067] The methods of the present invention allow for significantly more efficient and faster identification of cognate pairs of receptors and ligands. By using the receptor of the first library to directly select and isolate the nucleotide sequence of the second library which reduces the identification of the cognate pair to a single sequencing step. In some embodiments of the methods of the invention, the set of ligands is expressed by, or displayed on the surface of a cell or cells after clonal expansion, a bead, in particular engineered APC-like beads as disclosed in Neal et al. (2017) J. Immunol. Res. Ther. 2:68-79, or in vitro encoded (i.e., expressed or present in a cell free extract or in solution), as disclosed in Grubaugh et al. (2013) Vaccine 31:3805-3810. Preferably, the set of ligands is expressed by, or displayed on the surface of a cell (or cells), one cell expressing or displaying one ligand species from the set of ligands

[0068] The ligand is thus presented on the surface of a cell or bead and can be accessed and recognized by the corresponding receptor. In the preferred embodiment of the method of the invention the cellular expression systems comprise a plurality of cells, which have been transfected to express a distinct ligand molecule of the second library and to present the ligand on the cell surface, or alternatively a plurality of clones of stably transfected cells, wherein each clone expresses a different ligand molecule of the second library, are contacted with a plurality of tagged receptor molecules of the first library. Within this mixture of a plurality of tagged receptor molecules and a plurality of transfected cells expressing ligand molecules specific / selective interaction of cognate pairs of receptors and ligands is achieved. Hence, formation of the cognate receptor and ligand pairs is performed under optimized conditions for the plurality of receptors. The skilled person will understand that the selection of receptors affects the selection of appropriate parameters. The parameters include salinity, osmolarity and pH of the solution, selection of buffers, temperature and selection of salts in the solution. In a preferred embodiment of the invention each individual ligand and / or receptor molecule is present in multiple copies in the mixture to form cognate pairs and the tested pools contain a plurality of ligands and a plurality of receptors.

[0069] After specific / selective interactions of receptors and ligands have been achieved any unbound receptors are washed from the suspension of cells. Subsequently, the cells including bound receptors can be separated into microreactors, e.g., by encapsulation, therein further analysis is performed to identify the individual cognate pairs of receptors and ligands. High through-put screening is achieved to a large extent by the simple combination of the plurality of ligands and the plurality of receptors. Thus, in a single step and recognition of the cognate pairs is achieved. In a preferred embodiment the microreactors each contain a single ligand, but multiple microreactors can contain the same ligand.

[0070] For identifying the formed cognate pairs of ligands and receptor individual cells which have been separated into microreactors are lysed to release cellular contents including cellular RNA as well as the nucleotide sequences of the second library which may be recombinant RNA molecules. Consequently, within the microreactor released nucleotide sequence of the second library can interact with the tagged receptor molecules. This enables contacting the nucleotide sequence of the second library, which encodes the ligand, with the first nucleotide tag, which is attached to the receptor of the first library. In some embodiments the first nucleotide tag may be released from its connection to the receptor to facilitate interaction with the second nucleotide tag and / or the nucleotide sequence of the second library. From this interaction the primer sequences of the first nucleotide tag can be used to achieve reverse transcription and if required amplification of the nucleotide sequence of the second library. As part of reverse transcription the unique barcode of the first nucleotide tag which identifies the receptor is attached to the nucleotide sequence of the second library and thus incorporated within the resulting cDNA. The obtained cDNA may be amplified if necessary using primer sequences within either or both the first or second nucleotide tags or using specific sequences contained in the nucleotide sequence of the second library. The obtained cDNAs are prepared to form a sequencing library according to commonly known and well-established techniques to facilitate subsequent sequencing for identification. The cognate pairs of receptors and ligands are finally identified by sequencing of the barcode sequence of the first nucleotide tag to identify the receptor and by sequencing of the nucleotide sequence of the second library, i.e., the sequence of the ligand to identify the ligand. If the recombinant RNA contains a second nucleotide tag which includes a unique barcode sequence, sequencing of the barcode is sufficient for identification of the ligand. Further, identification by sequencing may be achieved in separate reactions identifying each the receptor and the ligand independent of each other. Alternatively, identification of both ligand and receptor may be achieved in a single reaction if both barcodes have been combined into a single cDNA for sequencing. In alternative embodiments of the methods of the invention the connection of the first nucleotide tag and the second nucleotide tag and / or the nucleotide sequence of the second library are achieved by a reaction other than reverse transcription. Alternatively, enzymatic reactions or primer extension may be used which use polymerases or ligases. The enzymatic reaction or primer extension can be performed with a polymerase such as Klenow-fragment without 5'-3' exonuclease activity - or BST polymerase or other polymerase or using a ligase enzyme. The skilled person in the field is well aware of a wide selection of available polymerase and knows how to select the appropriate enzyme. DNA polymerases are appropriate for this application as the first and the second nucleotide tag which are connected are both DNA sequences. In a preferred embodiment the second nucleotide tag and / or second sequence of the second library are connected by a ligase reaction.

[0071] In one embodiment of the invention ligases are used to connect the first and the second nucleotide tags. In this embodiment separate reverse transcription of the connected sequences is required prior to sequencing to achieve DNA sequences. Separate reverse transcription may be performed in bulk for all or at least some samples simultaneously.

[0072] For subsequent sequencing the obtained connected DNA sequences are prepared as a sequencing library according to common techniques as necessary and appropriate for the obtained samples. For the method of the invention library preparation involves attachment of the connected sequences to a flow cell by adapters. Attachment to the adapters is achieved by ligation. The adapters may comprise further indexing sequences if required for sequencing. Sequencing is then performed using the flow cell.

[0073] Provided herein are high throughput methods of identifying a receptor-ligand cognate pair as described above. The methods provided are sensitive and can detect weak interactions between ligand and receptor, the methods are high throughput, as a large number of receptor-ligand pairs can be screened in parallel, the methods are compatible with different architectural classes of receptor and preserve native receptor confirmation, and the methods are unbiased.

[0074] In certain alternative embodiments, the nucleotide sequence of the second library, for example, may be linked to the second nucleotide tag at a later stage of the method after cell lysis instead of before cell transfection. In this case the second nucleotide tag is used to facilitate interaction with the first nucleotide tag and may further contain primer sequences for reverse transcription, amplification and sequencing.

[0075] Independent of the timing within the method of the invention for attachment of the second nucleotide tag to the nucleotide sequence of the second library, attachment may be achieved through an enzymatic reaction. Examples of enzymatic reactions can include, but are not limited to: (a) pairing of the second nucleotide tag with the nucleotide sequence of the second library using a sequence present on the second nucleotide tag that is complementary to a sequence on the nucleotide sequence of the second library followed by extension of the sequence of the second nucleotide tag by reverse transcription (e.g., see, FIG. 2, wherein the sequence on the second nucleotide tag is P2ARev, which is complementary to the P2A sequence on the nucleotide sequence of the second library and the P2ARev sequence is extended by a reverse transcription reaction); (b) pairing of the second nucleotide tag with the nucleotide sequence of the second library using an oligonucleotide that contains one sequence A that is complementary to the second nucleotide tag, and one sequence B that is complementary to the nucleotide sequence of the second library, wherein the second nucleotide tag and the nucleotide sequence of the second library anneal adjacent to each other to sequence A and sequence B, respectively, on the oligonucleotide and are then ligated using a ligation assay; or (c) for reactions with beads coencapsulated with the receptor and ligand-displaying cells, the beads are functionalized with primers containing an amplification sequence, a barcode, and a capture sequence A that is complementary to a sequence present on the first nucleotide tag of the receptor and on the recombinant RNA, wherein the primers bind to both the first nucleotide tag of the receptor and the second nucleotide tag of the nucleotide sequence of the second library and are extended by reverse transcription. After library preparation, the barcode of the primers is used as a common sequence to pair the first nucleotide tag of the receptor and the second nucleotide tag of the nucleotide sequence of the second library.

[0076] In certain embodiments, the ligand of the receptor-ligand cognate pair is identified by amplifying the nucleotide sequence of the second library and subsequent sequencing of the nucleotide sequence of the second library for ligand identification. In certain embodiments, the ligand of the receptor-ligand cognate pair is identified by amplifying the second nucleotide tag attached to the nucleotide sequence of the second library and sequencing the second nucleotide tag for ligand identification.

[0077] In certain embodiments, the receptor of the receptor-ligand cognate pair is identified by amplifying the first nucleotide tag and sequencing the first nucleotide tag for receptor identification. In certain embodiments, the receptor is an antibody.

[0078] The person skilled in the art is well aware of different techniques and approaches for sequencing of nucleotide sequences and will be able to select the appropriate approach for each application. In a preferred embodiment of the methods of the invention next generation sequencing is used to identify the receptor and the ligand of the cognate pairs which are found by the methods of the invention.

[0079] In some embodiments of the methods of the invention the nucleotide sequences of the second library comprise recombinant RNA sequences which encode the set of ligands.

[0080] In some embodiments of the methods of the invention the nucleotide sequences of the second library comprise guide RNA molecules for CRISPR / Cas application.

[0081] In some embodiments of the methods of the invention the nucleotide sequences of the second library comprise expression plasmids which are suitable for expression in the transfected cells.

[0082] The skilled person in the art is well aware of different vectors which are suitable for expression of genes in cells after transfection. Expression vectors or expression plasmids have to be selected in dependence of the target cells which are transfected for expression of the gene of interest which is encoded by the expression vector. Certain expression vectors may achieve stable transfection of cells, i.e., the cell is permanently transfected and the transfection is maintained in subsequent cell divisions. Other expression vectors achieve temporary transfection, i.e., the transfected gene information and thus corresponding gene expression is lost over time and gene expression is not maintained after cell divisions. Depending on the application the skilled person will be able to select a suitable vector construct. Each transfection vector comprises at least the sequence of at least one gene for expression, further at least one promoter sequence controlling expression of the at least one gene, at least one sequence for replication of the vector, i.e., an origin of replication. The expression vector may further comprise suitable enhancer sequences and different tag sequences for the expressed RNA and / or protein sequences. The expression vector may further comprise signal sequences which may affect expression and translation of the RNA sequence, such as sequences which address the protein for incorporation into the cell membrane or secretion. The different elements of the expression vector have to be selected depending on the selected expression system, i.e, depending on the cell type which is selected for gene expression, as different sequences and elements are required for gene expression in bacterial cells or eukaryotic cells such as yeast cells, or plant or animal / human cells.

[0083] In some embodiments of the methods of the invention the recombinant RNA of the second library further comprises a second nucleotide tag sequence, and wherein the second nucleotide tag sequence is a second unique barcode which identifies the recombinant RNA.

[0084] In some embodiments of the methods of the invention different techniques of transfection or transduction of cells may be applied. Transfection techniques are selected from the group comprising electroporation, sonoporation, magnetofection, gene injection, gene gun, lipofection, transfection with polymers, transfection with nanoparticles, viral transfection and CRISPR / Cas gene editing. These techniques are well established in the art and the skilled person is aware to select an appropriate approach.

[0085] The skilled person in the field is aware of different techniques which may be used for transfecting a target cell with either DNA or RNA molecules. The transfected cargo can be selected from different DNA and RNA species, these include different vectors such as plasmids, cosmids, phages, artificial chromosomes and other vectors, recombinant RNA or siRNA. The use of recombinant RNA as is known to the skilled person in the field, represents the transfer on an artificially prepared mRNA molecule for translation into protein, wherein the recombinant mRNA is optimized for increased stability. Increased stability is achieved by using features selected form the list comprising 5'-cap, 5'- and 3'-untranslated regions, length of poly(A) tail, inclusion of these features protects the recombinant RNA from degradation. In addition modified nucleotides may be used to increase RNA stability, e.g., locked RNAs (LNAs) or RNA molecules which have modified backbones or modified nulceotides to reduce the rate of degradation of the recombinant RNA.

[0086] The transfected cargo can be transferred into the target cell by either viral, physical or chemical means. Viral transfection includes using viral delivery mechanisms of infecting cells, wherein the cargo DNA or RNA is encapsulated in a viral vector, which then utilizes viral proteins for transferring genetic material into a target cells. Chemical means of transfection involve encapsulating or complexing the cargo RNA or DNA with natural or artificial lipid molecules or polymers, which facilitate the uptake of the DNA or RNA into the target cells. Chemical transfection techniques commonly achieve transfection by fusing with the target cell and releasing the cargo into the target cell or alternatively facilitate active uptake of the particle by the cell and subsequent release of the cargo from the lysosome. Lastly, physical means of transfection involve either mechanical means of entering the target cells such as injections or gene guns. Alternatively, ultrasound or electrical fields may be used as in sonoporation or electroporation, respectively, to achieve porous and permeable cell membranes which allow the entry of RNA or DNA into the cells.

[0087] In alternative embodiments the nucleotide sequences of the second library comprise gRNA sequences which are delivered by transduction or transfection into suitable cellular expression systems. These gRNA sequences are subsequently used with suitable selected CRISPR / Cas-systems to achieve expression of the nucleotide sequences of the second libraries within the cellular expression systems. Within this context transduction of a cell refers to introduction of foreign genetic material into the cell by a viral vector. In a preferred embodiment of the methods of the invention CRISPR activation (CRISPRa) is used for targeted gene activation of gene of interest, wherein a deactivated enzyme (dCas) is used to reduce unspecific reactions.

[0088] In some embodiments of the methods of the invention the first and the second nucleotide tag sequences are DNA sequences. The first nucleotide tag is attached to the receptor of the first library and the second nucleotide tag if present is attached to the nucleotide sequence of the second library which encodes the ligand molecule.

[0089] In some embodiments of the methods of the invention the first nucleotide tag sequences comprise a universal primer sequence complementary to a sequence of the nucleotide sequence or the recombinant RNA of the second library, a barcode, and a binding site sequence for primers for amplification and / or sequencing. In another embodiment, the nucleotide sequence may also contain specific primer sequences which target the nucleotide sequence of the second library.

[0090] In some embodiments of the invention the second nucleotide tag may comprise a universal primer sequence complementary to a sequence of the nucleotide sequence or the recombinant RNA of the second library, a barcode, and a binding site sequence for primers for amplification and / or sequencing. In another embodiment, the nucleotide sequence may also contain specific primer sequences which target the nucleotide sequence of the second library.

[0091] In some embodiments of the methods of the invention the receptor of the first library is tagged with the first nucleotide tag sequence by different conjugation techniques selected from the group comprising biotin-streptavidin interaction, amine conjugation, wherein a N- terminal amino group or an amino group of a protein side chain is used for attachment of the first nucleotide tag, sulfhydryl conjugation using exposed reduced thiol groups of the antibodies for attachment, or carbohydrate conjugation using oxidized carbohydrate residues of the antibodies for attachment.

[0092] "Streptavidin" generally refers to a 52.8 kDa protein purified from the bacterium Streptomyces avidinii. Streptavidin homo-tetramers have an extraordinarily high affinity for biotin with a dissociation constant (Kd) on the order of =10“14mol / L, the binding of biotin to streptavidin is one of the strongest non-covalent interactions known in nature.

[0093] Streptavidin-Biotin bonds are known to the skilled in the art. The obtained bond is selective and strong non-covalent bond. This bond may be used to temporarily connect two molecules and if necessary subsequently specifically release the molecules in a controlled manner. In a preferred embodiment of the invention a Streptavidin-Biotin bond is used to connect the receptors of the first library with the first nucleotide tag.

[0094] In some embodiments of the methods of the invention the recombinant RNA of the second library is linked to the second nucleotide tag sequence through an enzymatic reaction. Independent of the type of bond used to link the barcoded primers to the particle, the barcoded primers may further comprise at least one linker sequence.

[0095] Accordingly, in a further embodiment, the barcoded primer further comprises at least one linker sequence, said linker sequence being preferably comprised at the 5' end. Accordingly, in one embodiment, the barcoded primer comprises from 5' to 3' a linker sequence, a barcode sequence, and a primer sequence.

[0096] In one embodiment, the "linker sequence" is a sequence with which the barcoded primer is optionally bonded to the particle.

[0097] "Optionally bonded" herein refers to the possibility that once the barcoded primers bonded to the particle are loaded into the microreactor or the plurality of microreactors, the barcoded primers might be released from the particle, so that the microreactor comprises the particle and the barcoded primers, said barcoded primers being separated from said particle.

[0098] Preferably, the linker sequence is a cleavable linker sequence, e.g., that can be cleaved upon application of a suitable stimulus, such as enzymatic and / or photocleavage.

[0099] "Cleavable linkers" are well known to the skilled in the art and are further described in Leriche et al. (2012) Bioorg. Med. Chem. 20:571-582. They may include, but are not limited to, TEV, trypsin, thrombin, cathepsin B, cathespin D, cathepsin K, caspase lumatrix metalloproteinase sequences, phosphodiester, phospholipid, ester, galactose, dialkyl dialkoxysilane, cyanoethyl group, sulfone, ethylene glycolyl disuccinate, 2-N-acyl nitrobenzenesulfonamide, a-thiophenylester, unsaturated vinyl sulfide, sulfonamide after activation, malondialdehyde (MDA)-indole derivative, levulinoyl ester, hydrazone, acylhydrazone, alkyl thioester, disulfide bridges, azo compounds, 2-Nitrobenzyl derivatives, phenacyl ester, 8-quinolinyl benzenesulfonate, coumarin, phosphotriester, bisarylhydrazone, bimane bi-thiopropionic acid derivative, paramethoxybenzyl derivative, tertbutylcarbamate analogue, dialkyl or diaryl dialkoxysilane, orthoester, acetal, aconityl, hydrazone, b-thiopropionate, phosphoramidate, imine, trityl, vinyl ether, polyketal, alkyl 2- (diphenylphosphino)benzoate derivatives, allyl ester, 8-hydroxyquinoline ester, picolinate ester, vicinal diols, and selenium compounds. Cleavage conditions and reagents include, but are not limited to, enzymes, nucleophilic / basic reagents, reducing agents, photo-irradiation, electrophilic / acidic reagents, organometallic and metal reagents, and oxidizing reagents.

[0100] In a preferred embodiment, the cleavable linker is a photocleavable moiety, for example a photolabile chemical group followed a chain of 1 to 30 carbon atoms, typically a chain of 6 to 10 carbon atoms.

[0101] In a further preferred embodiment, the cleavable linker is a double-stranded DNA molecule containing a target site for a specific restriction endonuclease.

[0102] In a particular embodiment, the barcoded primers bound to a particle are released from the particle in the microreactor, in particular, prior to or after lysing the cells, as disclosed below.

[0103] The release of at least some of the barcoded primers may further occur after lysing the cells and before reverse transcribing the released nucleic acids hybridized to said barcoded primers or after lysing the cells and after reverse transcribing the released nucleic acids hybridized to said barcoded primers.

[0104] Those skilled in the art will understand that depending on the time point selected for releasing the barcoded primers, the term "at least some of the barcoded primers" might refer to, for example, at least some of the barcoded primers hybridized to the nucleic acids released by the cells or a DNA / RNA duplex.

[0105] In one embodiment, the at least some of the barcoded primers can be released using any means, such as enzymes, nucleophilic / basic reagents, reducing agents, photo-irradiation, electrophilic / acidic reagents, organometallic and metal reagents, and oxidizing reagents.

[0106] In one embodiment, the at least some of the barcoded primers can be released using enzymatic and / or photocleavage. For example, an endonuclease may be used to cleave a linker sequence or any other sequence to release the at least some of the barcoded primers from the particle.

[0107] In a further embodiment, releasing the barcoded primer refers to disrupting the bond, such as a streptavidin biotin. Methods to disrupt a streptavidin biotin bond are known to the skilled in the art and include enzymatic digestion of streptavidin and / or denaturation of streptavidin.

[0108] In one embodiment, the barcoded primer is released by enzymatic digestion of streptavidin.

[0109] Preferably, each particle carries a barcode sequence or barcode set of sequences distinguishable from barcode sequences or barcode sets of sequences carried by other beads. In other words, each particle carries a unique majority type of barcode sequence or barcode set of sequences, optionally comprised in several barcoded primers, preferably at least some being in association with different primer sequences, while two different particles preferably do not carry the same majority barcode sequence or barcode set of sequences.

[0110] In a preferred embodiment, each microreactor contains a single particle carrying barcoded primers or less than 10 particles, in particular, less than 9, 8, 7, 6, 5, 4, 3, or 2 particles carrying barcoded primers. In a particularly preferred embodiment, each microreactor carries a single particle carrying barcoded primers.

[0111] The "reverse transcriptase (RT)" in context of the present invention is an enzyme used to generate complementary DNA (cDNA) from an RNA template, in a process termed reverse transcription.

[0112] In one embodiment, the reverse transcriptase is selected from the group consisting of Superscriptase I, Superscriptase II, Superscriptase III, Superscriptase IV, Murine Leukemia RT, SmartScribe RT, Maxima H RT, or MultiScribe RT.

[0113] In one embodiment, the reverse transcriptase is at a concentration of 1 to 50 U / pL, preferably 5 to 25 U / pL, for example at 12.5 U / pL.

[0114] In the context of the present invention, the ligase is an enzyme capable of covalently linking a nucleic acid to another nucleic acid by forming a new chemical bond. By way of an example, a ligase can covalently link the recombinant RNA with the second nucleotide tag. In a preferred embodiment of the invention the ligase is selected from the group comprising E. coli DNA ligase, T4 DNA / RNA ligases, Ampligase DNA Ligase, DNA ligase I, DNA ligase III and DNA ligase IV. In an alternative embodiment of the methods of the invention barcode indexing of the tagged receptors of the first library and / or the nucleotide sequences of the second library may be achieved using hydrogel beads carrying barcoded primers. This approach for barcoding is described in PCT patent application WO2022195089, the method of barcoding of this application is incorporated by reference into the present application. Briefly, this method provides hydrogel beads which deliver barcoded primers into microreactors for indexing and labeling of target molecules and target sequences. The method further involves in situ amplification of a single barcode molecule based on polymerase / nicking cycles that allow generation of barcoded molecules in the same reactor wherein the cell is subjected to analysis. Accordingly, use of this method allows for in situ labeling of the nucleotide sequences of the second library.

[0115] In some embodiments of the methods of the invention the microreactor is selected from an aqueous droplet, a microcapsule, a microbead, a microfluidic droplet, a compartment of a microfluidic chip, or a well.

[0116] In a particular embodiment, the microreactors are wells or microfabricated wells.

[0117] In another particular embodiment, the microreactors are aqueous droplets, in particular in a continuous immiscible phase.

[0118] In embodiments of the invention, the methods occur in a single reaction. The single reaction can, for example, occur in a microreactor. In the context of the invention, the microreactor may be selected from any suitable method, such as by microfluidics, flow cytometry cell-based sorting, and / or limiting dilution.

[0119] In certain embodiments, each ligand of the set of ligands is displayed on the surface of a single cell. Each ligand can, for example, be encoded by a recombinant RNA within the single cell. In certain embodiments, the single cell is lysed within the microreactor.

[0120] A "droplet" generally refers to a measure of volume and further refers in context of the present invention, to an isolated portion of a first fluid that is surrounded by a second fluid. It is to be noted that a droplet is not necessarily spherical, but may assume other shapes as well, for example, depending on the external environment. Preferably, each droplet has a volume at least equal to the volume of one mammalian cells. Preferably, the droplet volume is less than 1 nl, more preferably less than 500 pl, more preferably less than 100 pl, more preferably less than 50 pl, even more preferably less than 10 pl and most preferably less than 5 pl.

[0121] In another particular embodiment, the microreactors are microcapsules. The microcapsules can refer to a measure of volume and further refer in context of the present invention, to an isolated portion of a first coating material that surround a second material. It is to be noted that a microcapsule is not necessarily spherical, but may assume other shapes as well, for example, depending on the external environment. A "microcapsule" generally refers to a hollow microparticle composed of a solid shell surrounding a core-forming space available to permanently or temporarily entrapped substances. The substances can be drugs, pesticides, dyes, cells, combinations thereof and similar materials. The solid shell can, for example, enclose solids, liquids, or gases inside a micrometric wall made of hard or soft soluble film. The coating materials generally used for coating are ethyl cellulose, polyvinyl alcohol, gelatin, sodium alginate.

[0122] Preferably, each microcapsule has a volume at least equal to the volume of one mammalian cells. Preferably, the droplet volume is less than 1 nl, more preferably less than 500 pl, more preferably less than 100 pl, more preferably less than 50 pl, even more preferably less than 10 pl and most preferably less than 5 pl.

[0123] In another particular embodiment, the microreactors are microbeads. The microbeads can refer to a measure of volume and further refers to an isolated portion of a first semi-solid material that is surrounded by a fluid, either permeant or not to the semi-solid bead. It is to be noted that a microbead is not necessarily spherical, but may assume other shapes as well, for example, depending on the external environment. A "microbead" generally refers to a semi-solid porous or not structure, occupying the whole volume available to permanently or temporarily entrapped substances. The substances can be drugs, pesticides, dyes, cells, combinations thereof and similar materials. The semi-solid porous structure can, for example, enclose solids, liquids, or gases inside a micrometric wall made of hard or soft soluble film. The materials generally used for forming microbeads include polymers like agarose, acrylamide, sodium alginate. Preferably, each microbead has a volume at least equal to the volume of one mammalian cells. Preferably, the droplet volume is less than 1 nl, more preferably less than 500 pl, more preferably less than 100 pl, more preferably less than 50 pl, even more preferably less than 10 pl and most preferably less than 5 pl.

[0124] It is understood that cells can be encapsulated in microcapsules or microbeads before the cells achieve their transformation of droplet into microcapsules or microbeads.

[0125] If the ligands are displayed on cells or particles, the distribution of cells or particles, and hence also the distribution of ligands, will typically follow a Poisson distribution. However, if the microreactors are droplets, a variety of microfluidic techniques, familiar to the skilled person, allow distributions other than Poisson distribution, in particular distributions in which a higher fraction of droplets contains single ce I Is / pa rticles. These techniques include methods consisting in ordering the particles / cells before compartmentalization into droplets using inertial forces and mediated by secondary flows such as Dean flow (see Edd et al. (2008) Lab Chip 8:1262-1264; Kemna et al. Lab Chip (2012) 12:2881-2887, Lagus and Edd (2013) RSC Advances 3:20512-20522, Schoeman et al. (2014) Electrophoresis 35:385-392, Schoeman et al. (2018) Scientific Reports 8:3714, US 2013 / 011210, US 2010 / 021984 and US 2011 / 0223314), methods consisting in the isolation / sorting of the droplet containing a single cel l / pa rticle or a pair of ce I Is / pa rticles to reduce the number of droplets to analyze / measure (see Hu et al. (2015) Lab Chip 15:3989-3993; Chung et al. (2017) Lab Chip 17:3664-3671 and Shembekar et al. (2018) Cell Reports 22:2206-2215), methods consisting in forcing the cells / particles to flow in a narrow bottleneck to reduce chances of encapsulating multiple cells / particles of the same sample (see Ramji et al. (2014) Biomicrofluidics 8:034104), methods consisting in the production of droplets on demand when a cel l / pa rticle is passing in front of the nozzle (see Schoendube et al. (2015) Biomicrofluidics 9:014117; Leibacher et al. (2015) Biomicrofluidics 9:024109 and Yusof et al. (2011) Lab. Chip 11:2447-2454).

[0126] Accordingly, in a particular embodiment, a plurality of receptors, in particular of antibodies comprised in an aqueous composition are co-compartmentalized with a plurality of ligands, in particular of target antigens, into a plurality of microreactors, in particular in a plurality of microfluidic droplets, and the number of receptor species, in particular of antibodies, co-compartmentalized into one microreactor, in particular co-compartmentalized in one droplet follows, depending on the parameters used, a probability distribution, in particular a Poisson distribution. The parameters can be adapted to obtain, for instance, most microreactors having either 1 or 0 receptor, in particular, an antibody, thus minimizing the number of compartments containing several receptors.

[0127] The parameters used to co-compartmentalize receptor species, in particular antibodies, with ligand species, in particular target antigen displaying cells, can be adapted to obtain at least some of the microreactors comprising a single ligand species in each microreactor. The single ligand species is then combined in each microreactor with a plurality of receptor species. Hence, to achieve high-throughput screening single ligand species are contacted by a plurality of receptor species. Subsequent washing steps remove all unbound or unspecifically bound receptors from the ligand presenting cells. Thus, only stably bound cognate ligand-receptor pairs remain. After cell lysis the first nucleotide tag of the tagged receptor may contact the second nucleotide tag of the recombinant RNA for subsequent amplification and sequencing for identifying the cognate ligand-receptor pairs. Wherein sequencing and identification may be performed separately for the receptor and the ligand or simultaneously in a single reaction.

[0128] The microreactors of the method of the invention may comprise less than 100 tagged receptors within each microreactor, preferably less than 90 tagged receptors within each microreactor, more preferably less than 80 tagged receptors within each microreactor, more preferably less than 70 tagged receptors within each microreactor, more preferably less than 60 tagged receptors within each microreactor, more preferably less than 50 tagged receptors within each microreactor, more preferably less than 40 tagged receptors within each microreactor, even more preferably less than 30 tagged receptors within each microreactor, even more preferably less than 20 tagged receptors within each microreactor, even more preferably less than 10 tagged receptors within each microreactor and most preferably less than 5 tagged receptors within each microreactor.

[0129] In another embodiment of the methods of the invention the microreactors of the method of the invention may comprise more than 5 tagged receptors within each microreactor, more preferably more than 10 tagged receptors within each microreactor, more preferably more than 20 tagged receptors within each microreactor, more preferably more than 30 tagged receptors within each microreactor, more preferably more than 40 tagged receptors within each microreactor, more preferably more than 50 tagged receptors within each microreactor, more preferably more than 60 tagged receptors within each microreactor, more preferably more than 70 tagged receptors within each microreactor, more preferably more than 80 tagged receptors within each microreactor, even more preferably more than 90 tagged receptors within each microreactor and most preferably more than 100 tagged receptors within each microreactor.

[0130] As will be understood by the skilled person, some microreactors may however be created which do not include any receptor species, do the statics of random distribution of events which in most cases can be accurately described by a Poisson distribution.

[0131] The set of microreactors, in particular the set of microfluidic droplets, may be obtained by any suitable technique. In particular, the set of microreactors may be formed by (a) providing a first fluid source, the first fluid comprising a suspension of a set of tagged receptors of the first library as defined above, (b) providing a second fluid source, the second fluid comprising a suspension of the cellular expression systems which present the set of ligands of the second library on their surface as defined above; (c) providing a carrier fluid, the carrier fluid being immiscible with the first fluid and the second fluid, (d) injecting the carrier fluid in a main channel of a chip, (e) generating a flow of microreactors, in particular droplets, in the carrier fluid by injecting the second fluid and the first fluid in at least a secondary channel of the chip, the secondary channel opening in the main channel, each generated microreactor, in particular droplet, comprising a mix of the first fluid and the second fluid, wherein the concentration of the receptors in the first fluid, the concentration of the ligands in the second fluid, the geometry of the main channel and the secondary channel, the injection parameters of the first fluid, of the second fluid and of the carrier fluid are adapted such that each microreactor, in particular droplet, comprises at least one receptor species and preferably one ligand species and preferably presents a volume of less than 20 nl, more preferably less than 15 nl and more preferably of less than 10 nl, more preferably less than 1 nl, more preferably less than 500 pl, more preferably less than 100 pl, more preferably less than 50 pl, even more preferably less than 10 pl and most preferably less than 5 pl. In one embodiment, the first and second fluid sources are organized in the form of a junction.

[0132] The junction may be, for instance, a T-junction, a Y-junction, a channel- within-a- channel junction (e.g., in a coaxial arrangement, or comprising an inner channel and an outer channel surrounding at least a portion of the inner channel), a cross (or "X") junction, a flowfocusing junction, or any other suitable junction for creating droplets. See, for example, International Patent Application No. PCT / US2004 / 010903, filed April 9, 2004, entitled "Formation and Control of Fluidic Species," by Link, et ah, published as WO 2004 / 091763 on October 28, 2004, or International Patent Application No. PCT / US2003 / 020542, filed June 30, 2003, entitled "Method and Apparatus for Fluid Dispersion," by Stone, et ah, published as WO 2004 / 002627 on January 8, 2004.

[0133] In an alternative embodiment of the invention microfluidic droplets may be obtained using a vortexer as described by Clark et al. in Nature Biotechnology (2023), 41, 1557-1566, wherein emulsification is achieved by vortexing samples for between 30 s to 3 min at about 3,000 to 3,200 rpm.

[0134] In some embodiments, the junction may be configured and arranged to produce substantially monodisperse droplets.

[0135] The at least some microreactors may further comprise, in context of the present invention, a reverse transcriptase and barcoded primers, as further defined herein below.

[0136] In some embodiments of the methods of the invention the tagged receptors of the first library are tagged antibodies.

[0137] In certain embodiments, the ligand can, for example, be a target antigen for the antibody. The target antigen can, for example, be an antigen recognized by the antibody secreted from an antibody-secreting B cell from a subject. In certain embodiments, the ligands can, for example, be T cells antigens (from a TCR / T cell antigen recognition pair), B cell antigen (from a B cell receptor / B cell antigen recognition pair). As defined above the present invention identifies cognate pairs of ligands and receptors. Accordingly, ligands and receptors can be considered interchangeable. Hence, the ligand can also be a T cell receptor (TCR) or a B cell receptor. The ligand of the present invention may further refer to viral antigens, bacterial antigens, parasitic antigens, neoantigens (i.e., antigens which result from gene mutations or aberrant expression in tumor cells and whose expression is uniquely found in tumor cells), tumor associated antigens (TAAs), tumor specific antigens, stimulatory immune checkpoint molecules (e.g. 0X40 from an OX40L / OX40 pair), inhibitory immune checkpoint molecules (e.g. PD-1 from a PD-L1 / PD-1 pair), peptide-major histocompatibility complex (pMHC) multimers / monomers, cytokines and their respective receptors (from a cytokine / cytokine receptor pair), carbohydrates (from a selectin / carbohydrate pair), members of the immunoglobulin superfamily (from a pair comprising two members of the immunoglobulin superfamily), selectin (from a member of the immunoglobulin superfamily / selectin pair), chemokines and their respective receptors (from a chemokine / chemokine receptor pair), hormones and their respective receptors (from an hormone / hormone receptor pair), growth factors and their respective receptors (from a growth factor / growth factor receptor pair), ligands of GPCRs and the respective GPCRs (from a GPCR / corresponding ligand pair) or substrates and the respective enzymes (from an enzyme / corresponding substrate pair).

[0138] Foreign antigens, such as, viral antigens, bacterial antigens, or parasitic antigens can, for example, include, but are not limited to, a viral antigen, bacterial antigen, or parasitic antigen selected from at least one of the following organisms: Borrelia bacteria (e.g., Borrelia burgdorferi), Chikungunya Virus (CHIKV), Chlamydia bacteria (e.g., Chlamydia trachomatis), Cytomegalovirus (CMV), Dengue Virus (DENV), Ebola Virus (EVD), E. coli (e.g., Shiga-Like toxin), Epstein Barr Virus (EBV), Feline Leukemia Virus, Hantavirus, Hepatitis Virus (e.g., Hepatitis A, B, C, D, and / or E virus), Herpes Virus, Helicobacter pylori, Human endogenous retrovirus K (HERV-K), Human Immmunodeficiency Virus (HIV), Human T-cell Leukemia Virus (HTLV), Influenza Virus, Lassa Virus, Plasmodium parasites (e.g., which cause malaria), Mumps Virus (e.g., Mumps orthorubulavirus), Mycoplasma bacteria, Norovirus, Papillomavirus (HPV), Parvovirus, Rhinovirus, Rotavirus, Rubella virus, Salmonella bacteria (e.g., Salmonella typhi), SARS coronavirus (SARS-CoV), Toxoplasma parasite (e.g., Toxoplasma gondii), Treponema bacteria (e.g., Treponema pallidum, Treponema carateum), Trypanosoma parasite (e.g., Trypanosoma cruzi, which causes Chagas disease), Varicella Zoster Virus (VZV), Variola Virus, West Nile Virus (WNV), and / or Zika virus (ZIKV). Viral antigens are known to have stronger TCR affinity, see, e.g., Aleksic et al., Eur. J. Immunol. 42(12):3174-9 (2012), which could lead to higher enhanced signal detection in the methods disclosed herein. Foreign antigens are known to those skilled in art, see, e.g., Medical Microbiology, 4th edition, Chapter 6: Normal Flora; Baron S., editor; Galveston, TX; University of Texas Medical Branch at Galveston (1996); Laufer et al., "Microbial communities of the upper respiratory tract and otitis media in children," mBio 2(l):e00245-10 (2011).

[0139] In some embodiments of the methods of the invention the tagged antibody is isolated from an antibody-secreting B cell from a human subject.

[0140] In another particular embodiment, the receptor is an antibody. The antibody can, for example, be isolated from an antibody-secreting B cell from a subject, preferably a human subject, more preferably a human subject with a disease or disorder. In certain embodiments, the antibody can, for example, be isolated from a patient derived xenograft mouse or a humanized mouse. The antibody can, for example, be identified using single-cell antibody sequencing. In another particular embodiment, the ligand is a B cell antigen or a target antigen for the antibody.

[0141] In alternative embodiment the receptor may not be an antibody. As defined above the present invention identifies cognate pairs of ligands and receptors. Accordingly, ligands and receptors can be considered interchangeable. Hence, the receptor may refer to viral antigens, bacterial antigens, parasitic antigens, neoantigens (i.e., antigens which result from gene mutations or aberrant expression in tumor cells and whose expression is uniquely found in tumor cells), tumor associated antigens (TAAs), tumor specific antigens, stimulatory immune checkpoint molecules (e.g. 0X40 from an OX40L / OX40 pair), inhibitory immune checkpoint molecules (e.g. PD-1 from a PD-L1 / PD-1 pair), peptide-major histocompatibility complex (pMHC) multimers / monomers, cytokines and their respective receptors (from a cytokine / cytokine receptor pair), carbohydrates (from a selectin / carbohydrate pair), members of the immunoglobulin superfamily (from a pair comprising two members of the immunoglobulin superfamily), selectin (from a member of the immunoglobulin superfamily / selectin pair), chemokines and their respective receptors (from a chemokine / chemokine receptor pair), hormones and their respective receptors (from an hormone / hormone receptor pair), growth factors and their respective receptors (from a growth factor / growth factor receptor pair), ligands of GPCRs and the respective GPCRs (from a GPCR / corresponding ligand pair) or substrates and the respective enzymes (from an enzyme / corresponding substrate pair) .Vi ra I antigens, bacterial antigens, or parasitic antigens can, for example, include, but are not limited to, a viral antigen, bacterial antigen, or parasitic antigen selected from at least one of the following organisms: Borrelia bacteria (e.g., Borrelia burgdorferi), Chikungunya Virus (CHIKV), Chlamydia bacteria (e.g., Chlamydia trachomatis), Cytomegalovirus (CMV), Dengue Virus (DENV), Ebola Virus (EVD), E. coli (e.g., Shiga-Like toxin), Epstein Barr Virus (EBV), Feline Leukemia Virus, Hantavirus, Hepatitis Virus (e.g., Hepatitis A, B, C, D, and / or E virus), Herpes Virus, Helicobacter pylori, Human endogenous retrovirus K (HERV-K), Human Immmunodeficiency Virus (HIV), Human T-cell Leukemia Virus (HTLV), Influenza Virus, Lassa Virus, Plasmodium parasites (e.g., which cause malaria), Mumps Virus (e.g., Mumps orthorubulavirus), Mycoplasma bacteria, Norovirus, Papillomavirus (HPV), Parvovirus, Rhinovirus, Rotavirus, Rubella virus, Salmonella bacteria (e.g., Salmonella typhi), SARS coronavirus (SARS-CoV), Toxoplasma parasite (e.g., Toxoplasma gondii), Treponema bacteria (e.g., Treponema pallidum, Treponema carateum), Trypanosoma parasite (e.g., Trypanosoma cruzi, which causes Chagas disease), Varicella Zoster Virus (VZV), Variola Virus, West Nile Virus (WNV), and / or Zika virus (ZIKV).

[0142] The term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which immunospecifically binds an antigen. As such, the term antibody encompasses not only whole antibody molecules, but also antibody fragments as well as variants of antibodies, including derivatives such as humanized antibodies. In certain conventional antibodies, two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chain, lambda (A.) and kappa (K). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA, IgE and IgY. Each chain contains distinct constant region sequences. The light chain includes two domains, a variable domain (VL) and a constant domain (CL). The heavy chain includes four domains, a variable domain (VH) and three constant domains (CHI, CH2 and CH3, collectively referred to as CH). The variable regions of both light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen. The constant region domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, secretion, trans-placental mobility, complement binding, and binding to Fc receptors (FcR). The Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain. The specificity of the antibody resides in the structural complementarity between the antibody combining site and the antigenic determinant. Antibody combining sites are made up of residues that are primarily from the hypervariable or complementarity determining regions (CDRs). Occasionally, residues from nonhypervariable or framework regions (FR) influence the overall domain structure and hence the combining site. Complementarity determining regions (CDRs) refer to amino acid sequences which, together, define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding-site. The light and heavy chains of an immunoglobulin each have three CDRs, designated LCDR1, LCDR2, LCDR3, and HCDR1, HCDR2, HCDR3, respectively. Therefore, an antigen-binding site includes six CDRs, comprising the CDR set from each of a heavy and a light chain V region. Framework Regions (FRs) refer to amino acid sequences interposed between CDRs, i.e., to those portions of immunoglobulin light and heavy chain variable regions that are relatively conserved among different immunoglobulins in a single species, as defined by Kabat, et al. (Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md., 1991).

[0143] The term antibody further denotes single chain antibodies, for instance Camelidae antibodies, or nanobodies or VHH.

[0144] Antibody genes generally undergo a unique mechanism of genetic recombination, called V(D)J recombination, that occurs only in developing lymphocytes during the early stages of B cell maturation. The antibody genes may be further subjected to somatic hypermutation, and the combination of V(D)J recombination and somatic hypermutation results in the highly diverse repertoire of antibodies / immunoglobulins (Igs) found on B cells.

[0145] In some embodiments of the methods of the invention the tagged antibody is isolated from patient-derived xenograft (PDX) mice or humanized mice.

[0146] In some embodiments of the methods of the invention the method further comprises washing of the plurality of cellular expression systems to remove unbound tagged receptors and unspecifically bound tagged receptors.

[0147] In some embodiments of the methods of the invention the method further comprises adding reagents for reverse transcription to the plurality of microreactors within the microfluidic system, wherein the reagents for reverse transcription comprise a reverse transcriptase enzyme, mixture of dNTPs, and a suitable buffer.

[0148] In a particular embodiment, said microreactors include additional reagents.

[0149] Additional reagents typically include a reverse transcriptase (RT), a cell lysis buffer, deoxynucleotide triphosphates (dNTPs), ligase, and / or a plurality of barcoded primers specific for a nucleic acid sequence encoding the ligand or ligand candidate and of barcoded primers specific for a nucleic acid sequence encoding the receptor, as defined below.

[0150] When the ligand is a tagged ligand, as defined above, the tag may comprise at least one of a sequence complementary to a sequence of the recombinant RNA, a barcode, and / or a sequence for amplification. Similarly, when the receptor is a tagged receptor, as defined above, the tag may comprise at least one of a sequence complementary to a sequence of the recombinant RNA, a barcode, and / or a sequence for amplification.

[0151] Accordingly, in a particular embodiment, additional reagents are added to the microreactors, said additional reagents are selected from the group comprising at least a reverse transcriptase (RT), deoxynucleotide triphospates (dNTPs), ligase, an oligonucleotide partially complementary to the first nucleotide tag of the receptor and the second nucleotide tag of the ligand and / or optionally a cell lysis buffer.

[0152] The term "hybridization," as described herein, refers to a phenomenon in which the primer sequence present in the barcoded primer anneals to a complementary nucleic acid sequence of the released nucleic acids. Accordingly, as known by the skilled in the art, the temperature to use depends on the primer sequence and / or the polymerase enzyme used.

[0153] The step of reverse transcription defined above refers to reverse transcribing the released nucleic acids hybridized to said barcoded primers using the primer sequence in at least some of the microreactors. Reverse transcription is performed using the reverse transcriptase (RT) comprised in at least some of the microreactors. "Reverse Transcription" or "RT reaction" is a process in which single-stranded RNA is reverse transcribed into a single-stranded complementary DNA (cDNA) by using total cellular RNA or poly(A) RNA, a reverse transcriptase enzyme, a primer, dNTPs and an RNase inhibitor. It will be understood by the skilled in the art, that the product of the reverse transcription is a RNA / DNA duplex comprising a single strand cDNA hybridized to its template RNA. As it will be further understood, said RNA / DNA duplex is further linked to the barcoded primer comprising the primer sequence used for the reverse transcription.

[0154] "Template switching" refers to a technology described originally in 2001, frequently referred to as "SMART" (switching mechanism at the 5' end of the RNA transcript) technology (Takara Bio USA, Inc). This technology has shown promise in generating full-length cDNA libraries, even from single-cell-derived RNA samples (Zhu et al. (2001) Biotechniques 30:892- 897). This strategy relies on the intrinsic properties of Moloney murine leukemia virus (MMLV) reverse transcriptase and the use of a unique template switching oligonucleotide (TS oligo, or TSO). During first-strand synthesis, upon reaching the 5' end of the RNA template, the terminal transferase activity of the MMLV reverse transcriptase adds a few additional nucleotides (mostly deoxycytidine) to the 3' end of the newly synthesized cDNA strand. These bases function as a TS oligo-anchoring site. Upon base pairing between the TS oligo and the appended deoxycytidine stretch, the reverse transcriptase "switches" template strands, from cellular RNA to the TS oligo, and continues replication to the 5' end of the TS oligo. By doing so, the resulting cDNA contains the complete 5' end of the transcript, and universal sequences of choice are added to the reverse transcription product. Along with tagging of the cDNA 3' end by oligo dT primers, this approach makes it possible to efficiently amplify the entire full- length transcript pool in a completely sequence-independent manner (Shapiro et al. (2013) Nat. Rev. Genet. 14:618-630).

[0155] Accordingly, it will be understood by the skilled in the art, that after reverse transcribing the nucleic acids, the microreactor further comprises cDNAs.

[0156] Accordingly, in one embodiment, at least some of the microreactors further comprise cDNAs produced by reverse transcription of nucleic acids from the cells contained in said microreactors. In one embodiment, said cDNA refers to a single-stranded complementary DNA.

[0157] In a further embodiment, said cDNA is comprised in a RNA / DNA duplex.

[0158] In one embodiment, the RNA / DNA duplex refers to the RNA that has been reverse transcribed and is hybridized to the primer sequence of at least one of the primers, which is optionally barcoded, contained in the microreactor.

[0159] As it will be understood by the skilled in the art, in one embodiment, the RNA / DNA duplex is linked to the primer, which is optionally barcoded, comprising the primer sequence to which the nucleic acid, preferably mRNA, was hybridized and which was used for reverse transcription.

[0160] In one example, hybridization and reverse transcription are performed by incubating the microreactors for example for 1 h or 2 h at 55°C or 50°C during typically mixing of the microreactors at for example 550 rpm.

[0161] In some embodiments of the methods of the invention the method further comprises adding reagents for cell lysis to the plurality of microreactors, wherein the reagents for cell lysis are selected from the group comprising hypotonic buffers, detergents and lysozyme.

[0162] In the context of the present invention, the "cell lysis buffer" is a composition enabling cell lysis, preferably without disruption of the microreactors, in particular, of the droplets.

[0163] Preferably, the cell lysis buffer is compatible with RT activity and / or with reagents used for the recognition assay.

[0164] In one embodiment, the lysis buffer comprises enzymes selected from the group consisting of lysozyme, lysostaphin, zymolase, mutanolysin, glycanases, proteases, and mannose.

[0165] In one preferred embodiment, the lysis buffer comprises magnesium chloride, a detergent, a buffered solution and an RNase inhibitor.

[0166] In one embodiment, the magnesium chloride is used at a concentration of between

[0167] 1 mM to 20 mM. In one embodiment, the detergent is selected from the group consisting of Triton-X- 100, NP-40, Nonidet P40, and Tween-20 and IGEPAL CA 630.

[0168] In one embodiment, the detergent is at a concentration of 0.1% to 10%.

[0169] Non-limiting examples of the buffered solution include Tris-HCI, Hepes-KOH, Pipes- NaOH, maleic acid, phosphoric acid, citric acid, malic acid, formic acid, lactic acid, succinic acid, acetic acid, pivalic (trimethylacetic) acid, pyridine, piperazine, picolinic acid, L-histidine, MES, Bis-tris, bis-tris propane, ADA, ACES, MOPSO, PIPES, imidazole, MOPS, BES, TES, HEPES, DIPSO, TAPSO, TEA (triethanolamine), N-Ethylmorpholine, POPSO, EPPS, HEPPS, HEPPSO, Tris, tricine, Glycylglycine, bicine, TAPS, morpholine, N-Methyldiethanolamine, AMPD (2-amino-2- methyl-l,3-propanediol), Diethanolamine, AMPSO, boric acid, CHES, glycine, CAPSO, ethanolamine, AMP (2-amino-2-methyl-l-propanol), piperazine, CAPS, 1, 3-Diaminopropane, CABS, or piperidine (see also, www.reachdevices.com / Protein / Biological Buffers.html).

[0170] Non-limiting examples of RNase inhibitors include RNase OUT, IN, SuperIN Rnase, and those inhibitors targeting a wide range of RNAse (e.g., A, B, C, 1 and Tl).

[0171] In one example the lysis buffer is typically 0.36% Igepal CA 630, 50 mM Tris-HCI pH 8.

[0172] In a particular embodiment, said additional reagents are added into the microreactor, in particular into the microfluidic droplet, by injection from a reservoir, for example using electrical forces (picoinjection) (Abate et al. (2010) Proc. Nat. Acad. Sci. USA 107:19163- 19166).

[0173] In another particular embodiment, said additional reagents are added into the microreactor, in particular into the microfluidic droplet, by coalescence with a second microreactor, in particular a second microfluidic droplet, comprising said additional reagents but not comprising any ligand or receptor. Droplets can be coalesced by a variety of methods known to the skilled person, including passive droplet coalescence (see Mazutis et al. (2009) Lab on a Chip, 9(18):2665-2672; Mazutis et al. (2012) Lab Chip, 12:1800-1806), droplet coalescence driven by local heating from a focused laser (Ba roud et al. (2007) Lab Chip 7:1029- 1033) or using electric forces (Chabert et al. (2005) Electrophoresis 26:3706-3715; Ahn et al. (2006) Appl. Phys. Lett., 88:264105; Link et al. (2006) Angew. Chem., Int. Ed., 45:2556-2560; Priest et al. (2006) Appl. Phys. Lett. 89:134101) or using magnetophoretic forces or using pneumatic controllers (see Xi et al. (2017) Lab Chip 17:751-771).

[0174] Said second microreactor, in particular said second microfluidic droplet, can be prepared by the same techniques as those disclosed above for the microreactors comprising the ligands and receptors.

[0175] By "coalescence" is meant herein the process by which two or more droplets or particles merge during contact to form a single daughter droplet or particle.

[0176] In a particular embodiment, in each microreactor in which the above additional reagents are added, barcoded cDNAs are prepared by (a) lysing the cells expressing or displaying receptors and the cells expressing or displaying ligands, to release mRNA from the cells, (b) hybridizing at least some of the released mRNA coding for the receptor (or for the receptor's tag) to the receptor (or the receptor's tag)-encoding nucleic acid sequence specific primer, being optionally barcoded, and at least some of the released mRNA coding for the ligand (or for the ligand's tag) to the ligand (or the ligand's tag)-encoding nucleic acid sequence specific barcoded primer, in at least some of the microreactors, and (c) reverse transcribing the released mRNA hybridized to the primers, being optionally barcoded thereby obtaining barcoded cDNAs.

[0177] As will be understood by the skilled person, when the ligand's tag (second nucleotide tag) or the receptor's tag (first nucleotide tag) is a barcode sequence, it is not necessary to prepare barcoded cDNAs as detailed above, since the nt sequences of the ligand or the receptor allow themselves their own identification. Under these conditions an enzymatic reaction may be used instead of reverse transcription to connect the tag sequences. The enzymatic reaction can be performed with a polymerase such as Klenow-fragment without 5'-3' exonuclease activity - or BST polymerase or other polymerase or using a ligase enzyme. The skilled person in the field is well aware of a wide selection of available polymerase and knows how to select the appropriate enzyme.

[0178] "Barcoding" herein refers to adding a genetic sequence, a so-called barcode sequence as further defined herein above, to a nucleic acid which allows to distinguish said barcoded nucleic acid from a nucleic acid having another added genetic sequence, i.e., another unique barcode sequence.

[0179] The term "cell lysis" in the context of the present invention may be accomplished by enzymatic, physical, and / or chemical means, or any combination thereof, in particular enzymatic, physical, and / or chemical means. Other cell disruption methods may also be used.

[0180] Accordingly, in one embodiment, the cells are lysed using enzymatic, physical, and / or chemical cell lysis.

[0181] "Enzymatic methods" to remove cell walls is well-established in the art. The enzymes are generally commercially available and, in most cases, were originally isolated from biological sources. Enzymes commonly used include lysozyme, lysostaphin, zymolase, mutanolysin, glycanases, proteases, and mannose.

[0182] As known by the skilled in the art "chemical cell lysis" is achieved using chemicals such as detergents, which disrupt the lipid barrier surrounding cells by disrupting lipid-lipid, lipid- protein, and protein-protein interactions. The ideal detergent for cell lysis depends on cell type and source. Nonionic and zwitterionic detergents are milder detergents. The Triton X series of nonionic detergents, the IGEPAL CA 630 nonionic detergent, and 3-[(3- Cholamidopropyl) dimethylammonio]-l-propanesulfonate (CHAPS), a zwitterionic detergent, are commonly used for these purposes. In contrast, ionic detergents are strong solubilizing agents and tend to denature proteins, thereby destroying protein activity and function. SDS, an ionic detergent that binds to and denatures proteins, is used extensively in the art to disrupt cells.

[0183] "Physical cell lysis" refers to the use of sonication, thermal shock (above 40°C, below 10°C), electroporation, or laser-induced cavitation.

[0184] In one example the cells are lysed on ice.

[0185] In one preferred embodiment, the cell lysis does not disrupt or destroy the microreactors, in particular, the droplets, in the context of the invention.

[0186] In some embodiments of the methods of the invention the method further comprises cell lysis of cellular expression systems in the plurality of microreactors, wherein cell lysis releases the nucleotide sequences of the second library.

[0187] In some embodiments of the methods of the invention the method further comprises contacting universal primer sequence of nucleotide tag sequence of tagged receptors to released nucleotide sequences of second library.

[0188] Recognition assay and classification

[0189] By "recognition" is meant herein a binding between a ligand species and a receptor species.

[0190] As will be understood by the skilled person, the reaction induced by a recognition between a ligand species and a receptor species will depend on the particular ligands and receptors considered. Accordingly, the assay used to determine the recognition between a ligand species and a receptor species will depend on the particular ligands and receptors considered.

[0191] In a particular embodiment, assay reagents are added to the microreactors. Preferably, said assay reagents are co-compartmentalized with said ligand species and said receptor species during the co-compartmentalization step.

[0192] For example, when the microreactors are microfluidic droplets, said assay reagents can be included in the fluid used for the formation of said droplets. Alternatively, said assay reagents may be provided through a third fluid.

[0193] Reagents can also be added to pre-formed droplets by a variety of methods known to the skilled person, including passive droplet coalescence (see Mazutis et al. (2009). Lab Chip, 9 (18), 2665-2672; Mazutis & Griffiths (2012) Lab Chip, 12:1800-1806), droplet coalescence driven by local heating from a focused laser (Baroud et al. (2007). Lab Chip 7:1029-1033) or using electric forces (Chabert et al. (2005) Electrophoresis, 26:3706-3715; Ahn et al. (2006) Appl. Phys. Lett., 88:264105; Link et al. (2006) Angew. Chem., Int. Ed., 45:2556-2560; Priest et al. (2006) Appl. Phys. Lett., 89:134101), or by injection of liquids into pre-formed droplets, for example using electrical forces (picoinjection) (Abate et al. (2010) Proc. Nat. Acad. Sci. USA, 107:19163-19166).

[0194] As will be understood by the skilled person, said assay reagents will depend on the particular recognition assay carried out.

[0195] Said separation can be carried out by any technique well-known from the skilled person, which will depend on the type of microreactors used. In particular, said separation may be carried out by sorting of the microreactors, in particular of the microfluidic droplets, for example, by detecting a reporter reagent. Said separation may also be carried out by sorting of the microreactors by flow cytometry.

[0196] In a preferred embodiment, when the microreactors are droplets, the droplets will be sorted in a microfluidic device by dielectrophoresis (Ahn et al. (2006) Appl. Phys. Lett. 88:024104) or using surface acoustic waves (Franke et al. (2009) Lab Chip 9:2625-2627), triggered, for example, by detecting a fluorescent signal in the droplets (Baret et al. (2009) Lab Chip, 9:1850-1858) or using magnetophoretic forces or using pneumatic controllers (see Xi et al. (2017) Lab Chip 17:751-771).

[0197] Identification of ligand species and receptor species

[0198] Identification of the ligand species as defined by the second library and the receptor species as defined by the first library contained in each microreactor can be carried out by any technique well-known from the skilled person. In particular, identification of the ligand species and the receptor species contained in each microreactor can be carried out by sequencing, in particular, by sequencing DNA, the barcoded cDNAs obtained as detailed above, or the first and second nucleotide tags.

[0199] In one embodiment, the barcoded cDNAs produced by the reverse transcription as defined above are recovered and further used for identification, typically, by subsequent amplification and sequencing library preparation.

[0200] Accordingly, in one embodiment, the method of the invention further comprises recovering cell cDNAs produced by reverse transcription in at least some of the microreactors, preferably in the positive microreactors.

[0201] "Recovering" herein refers to isolating the barcoded cDNAs produced by reverse transcription in at least some of the microreactors from said plurality of microreactors.

[0202] In one embodiment, recovering herein refers to collecting the microreactors comprising barcoded cDNA produced by reverse transcription or collecting the aqueous composition contained in said microreactors comprising said barcoded cDNA, and separating the barcoded cDNA comprised in the aqueous composition.

[0203] In one particular embodiment, recovering herein refers to collecting the microfluidic droplets comprising barcoded cDNA produced by reverse transcription, breaking the microfluidic droplets and separating the barcoded cDNA comprised in the aqueous composition from the oil phase of said microfluidic droplets.

[0204] Methods to isolate nucleic acids, in particular cDNA from microfluidic droplets are known to the skilled in the art and comprise for example, collecting the microfluidic droplets and breaking the emulsion by, for example, applying an electrical field (electrocoalescence) or by adding a chemical emulsion breaking agent, such as perfluoro-octanol in the case of droplets in fluorinated carrier oils. In one example, the broken emulsion is typically centrifuged for, for example, 10 minutes at 10,000 g at 4°C and the supernatant comprising the barcoded cDNA in the aqueous phase is recovered.

[0205] In one embodiment, the method further comprises the step of removing unincorporated barcoded primers from the aqueous composition of the microreactors. In one preferred embodiment, the step of removing unincorporated barcoded primers from the aqueous composition of at least some of the microreactors takes place after the step of recovering the barcoded cDNA produced by reverse transcription as defined herein above.

[0206] Preferably, the step of removing unincorporated barcoded primers precedes the amplification step and / or the sequencing step defined herein below.

[0207] In one embodiment, removing unincorporated barcoded primers comprises contacting the aqueous composition of the at least some of the microreactors with a purification substrate wherein the purification substrate removes unincorporated barcoded primers. In one embodiment, the purification substrate comprises beads or particles, which, optionally, form a column. In a further example, unincorporated barcoded primers are removed by size selection using for example an acrylamide or an agarose gel.

[0208] In one embodiment, the step of removing unincorporated barcoded primers comprises contacting the aqueous composition of the at least some of the microreactors with an exonuclease, such as the exonuclease Exol, to degrade the unincorporated barcoded primers within the aqueous composition of the at least some of the microreactors.

[0209] In certain embodiments of this step, the exonuclease degrades single stranded nucleic acid sequences from the aqueous compositions comprising the cDNA.

[0210] It will be understood by the skilled in the art, that the barcoded cDNA obtained after reverse transcription is typically present in the form of a RNA / DNA complex and thus protected from said exonucleases.

[0211] In one embodiment, the barcoded cDNA comprises one or more modified nucleotides or nucleotide analogs, for example for facilitating purification of the barcoded cDNA sequences or molecules.

[0212] For example, the nucleotides may be employed as phosphorothioate derivatives (replacement of a non-bridging phosphoryl oxygen atom with a sulfur atom) which have increased resistance to nuclease digestion. 2'-methoxyethyl (MOE) modification (such as the modified backbone commercialized by ISIS Pharmaceuticals) is also effective.

[0213] Other examples of modified nucleotides include derivatives of nucleotides with substitutions at the 2' position of the sugar, in particular with the following chemical modifications: O-methyl group (2'-0-Me) substitution, 2-methoxyethyl group (2'-0-M0E) substitution, fluoro group (2'-fluoro) substitution, chloro group (2'-CI) substitution, bromo group (2'-Br) substitution, cyanide group (2'-CN) substitution, trifluoromethyl group (2'-CF3) substitution, OCF3 group (2'-OCF3) substitution, OCN group (2'-OCN) substitution, O-alkyl group (2'-O-a Ikyl) substitution, S-alkyl group (2'-S-a I kyl) substitution, N-alkyl group (2'-N-a kyl) substitution, O-alkenyl group (2'-O-alkenyl) substitution, S-alkenyl group (2'-S-alkenyl) substitution, N-alkenyl group (2'-N-alkenyl) substitution, SOCH3 group (2'-SOCH3) substitution, SO2CH3 group (2'-SO2CH3) substitution, ONO2 group (2'-ONO2) substitution, NO2 group (2'-NO2) substitution, N3 group (2'-N3) substitution and / or NH2 group (2'-NH2) substitution. Other examples of modified nucleotides include biotin labeled nucleotides.

[0214] Other examples of modified nucleotides include nucleotides wherein the ribose moiety is used to produce locked nucleic acid (LNA), in which a covalent bridge is formed between the 2' oxygen and the 4' carbon of the ribose, fixing it in the 3'-endo configuration.

[0215] Other examples of nucleotide analogs include deoxyinosine.

[0216] Other examples of nucleotide analogs include Biotinylated, fluorescently labelled nucleotide. For example, Biotin-ll-dCTP can be used as a substrate for the reverse transcriptase to incorporate biotins into the cDNA during polymerization, allowing affinity purification using streptavidin or avidin.

[0217] In one embodiment, the barcoded cDNA is further treated with RNAse A and / or RNAse H.

[0218] "RNAse A" is an endoribonuclease that specifically degrades single-stranded RNA at C and U residues. In one embodiment, the RNAse A is at a concentration of 10 to 1000 pg / pL, preferably 50 to 200 pg / pL, for example at 100 pg / pL.

[0219] "RNAse H" is a family of non-specific endonucleases that catalyze the cleavage of RNA via a hydrolytic mechanism. RNase H ribonuclease activity cleaves the 3'-O-P bond of RNA in a DNA / RNA duplex substrate to produce 3'-hydroxyl and 5'-phosphate terminated products. In one embodiment, the RNAse H is at a concentration of 10 to 1000 pg / pL, preferably 50 to 200 pg / pL, for example at 100 pg / pL.

[0220] In one embodiment, the barcoded cDNA is further treated with Proteinase K. "Proteinase K" is a broad-spectrum serine protease and digests proteins, preferentially after hydrophobic amino acids. In one embodiment, the Proteinase K is at a concentration of 0.1 to 5 mg / mL, preferably 0.1 to 1 mg / mL, for example at 0.8 mg / mL.

[0221] In one embodiment, the barcoded cDNAs obtained after reverse transcription are sequenced to allow identification of receptors and ligands contained in the same microreactor.

[0222] In one embodiment, the step of sequencing the barcoded cDNA may comprise performing a next generation sequencing (NGS) protocol on a sequencing library. Any type of NGS protocol can be used such as the MiSeq Systems (illumina®), the HiSeq Systems (illumina®), the NextSeq System (illumina®), the NovaSeq Systems (illumina®), the lonTorrent system (ThermoFisher), the lonProton system (ThermoFisher), or the sequencing systems produced by Pacific Biosciences or by Nanopore.

[0223] In certain embodiments, the NGS protocol comprises loading an amount of the sequencing library between 1 pM and 20 pM, in particular between 1.5 pM and 20 pM, per flow cell of a reagent kit.

[0224] In one embodiment, the NGS sequencing protocol further comprises the step of adding 5-60% PhiX to the amount of the sequencing library or to the flow cell of the reagent kit.

[0225] In one embodiment, prior to sequencing, the barcoded cDNAs are further amplified.

[0226] In one embodiment, the amplification step is performed by a polymerase chain reaction (PCR), and / or a linear amplification.

[0227] In one embodiment, the linear amplification precedes the PCR reaction.

[0228] In one embodiment, the linear amplification is an in vitro transcription.

[0229] In one embodiment, the linear amplification is an isothermal amplification.

[0230] In one embodiment, said amplification step is performed after removing unincorporated barcoded primers. In one embodiment, said amplification step is performed prior to the sequencing step defined herein above.

[0231] In one embodiment, the barcoded cDNA produced after reverse transcription is quantified using qPCR.

[0232] In one embodiment, specific sequences necessary for sequencing are added during amplification or by ligation of adaptors, thereby generating a sequencing library.

[0233] As will be understood by the skilled person, since the barcoded cDNAs from a particular microreactor carry a same specific majority barcode sequence or barcode set of sequences which is different from the majority barcode sequences or barcode sets of sequences included in other microreactors, it is possible to determine which identified ligand species were contained in the same microreactor, in particular in positive microreactors, as a particular identified ligand receptor.

[0234] EMBODIMENTS

[0235] The invention also provides the following non-limiting embodiments.

[0236] Embodiment 1 is a method for high-throughput screening (HTS) for identifying a receptor-ligand cognate pair in a microfluidics system, the method comprising: a. providing a receptor library, wherein each receptor molecule of the library is tagged with a first nucleotide tag sequence resulting in tagged receptors, wherein the first nucleotide tag sequence comprises a unique barcode sequence and a universal primer sequence, b. providing a second library of nucleotide sequences, wherein the nucleotide sequences encode for ligands, c. transmitting the nucleotide sequences of second library into cellular expression systems by transfection or transduction, wherein each cellular expression system expresses one nucleotide sequence of the second library resulting in a plurality of cellular expression systems, d. incubating plurality of cellular expression systems for expression of ligand molecules of second library within the plurality of cellular expression systems, wherein the ligand molecules are presented on the surface of the plurality of cellular expression systems, e. contacting the plurality of tagged receptors with expressed ligands on the surface of plurality of cellular expression systems for receptor target pairing, f. encapsulating the plurality of cellular expression systems in a plurality of microreactors, wherein each microreactor comprises one cellular expression system expressing one ligand of the second library, g. generating combined nucleotide sequences by enzymatic reaction, wherein the combined nucleotide sequences comprise the nucleotide sequences of the second library and the unique barcode sequence of tagged receptor of the first library, h. sequencing of combined nucleotide sequences i. analyzing sequences from step h. to identify cognate pairs of receptors of first library and ligands of second library. Embodiment 2 is the method of embodiment 1, wherein the nucleotide sequences of the second library comprise recombinant RNA molecules which encode the set of ligands.

[0237] Embodiment 3 is the method of any one of embodiments 1 and 2, wherein the nucleotide sequences of the second library comprise expression plasmids which are suitable for expression in the cellular expression system.

[0238] Embodiment 4 is the method of any one of embodiments 1 to 3, wherein the recombinant RNA of the second library further comprises a second nucleotide tag sequence, and wherein the second nucleotide tag sequence is a second unique barcode which identifies the recombinant RNA.

[0239] Embodiment 5 is the method of any one of embodiments 1 to 4, wherein technique for transfection or transduction of cells is selected from the group comprising electroporation, sonoporation, magnetofection, gene injection, gene gun, lipofection, transfection with polymers, transfection with nanoparticles, viral transfection and CRISPR / Cas gene editing.

[0240] Embodiment 6 is the method of any one of embodiments 1 and 5, wherein the first and the second nucleotide tag sequences are DNA sequences.

[0241] Embodiment 7 is the method of any one of embodiments 1 to 6, wherein the first nucleotide tag sequences comprise a universal primer sequence complementary to a sequence of the recombinant RNA of the second library, a barcode, and a sequence for amplification.

[0242] Embodiment 8 is the method of any one of embodiments 1 to 7, wherein the receptor of the first library is tagged with the first nucleotide tag sequence by a techniques selected from the group comprising biotin-streptavidin interaction, amine conjugation, sulfhydryl conjugation and carbohydrate conjugation.

[0243] Embodiment 9 is the method of any one of embodiments 1 to 6, wherein the recombinant RNA of the second library is linked to the second nucleotide tag sequence through an enzymatic reaction.

[0244] Embodiment 10 is the method of any one of embodiments 1 to 6, wherein the nucleotide sequences of the second library comprise guide RNA molecules.

[0245] Embodiment 11 is the method of any one of embodiments 1 to 10, wherein the microreactor is selected from an aqueous droplet, a microcapsule, a microbead, a microfluidic droplet, a compartment of a microfluidic chip, or a well.

[0246] Embodiment 12 is the method of any one of embodiments 1 to 11, wherein the tagged receptors of the first library are tagged antibodies.

[0247] Embodiment 13 is the method of embodiment 12, wherein the tagged antibody is isolated from an antibody-secreting B cell from a human subject.

[0248] Embodiment 14 is the method of embodiment 12, wherein the tagged antibody is isolated from patient-derived xenograft (PDX) mice or humanized mice.

[0249] Embodiment 15 is the method of any one of embodiments 1 to 14, wherein the method further comprises washing of the plurality of cellular expression systems to remove unbound tagged receptors and unspecifically bound tagged receptors.

[0250] Embodiment 16 is the method of any one of embodiments 1 to 15, wherein the method further comprises adding reagents for reverse transcription to the plurality of microreactors within the microfluidic system, wherein the reagents for reverse transcription comprise a reverse transcriptase enzyme, mixture of dNTPs, and a suitable buffer.

[0251] Embodiment 17 is the method of any one of embodiments 1 to 16, wherein the method further comprises adding reagents for cell lysis to the plurality of microreactors, wherein the reagents for cell lysis are selected from the group comprising hypotonic buffers, detergents and lysozyme.

[0252] Embodiment 18 is the method of any one of embodiments 1 to 17, wherein the method further comprises cell lysis of cellular expression systems in the plurality of microreactors, wherein cell lysis releases the nucleotide sequences of the second library.

[0253] Embodiment 19 is the method of any one of embodiments 1 to 18, wherein the method further comprises contacting universal primer sequence of first nucleotide tag sequence of tagged receptors to released second nucleotide tag sequences of second library. EXAMPLES

[0254] The following examples of the invention are to further illustrate the nature of the invention. It should be understood that the following examples do not limit the invention and that the scope of the invention is to be determined by the appended claims.

[0255] Example 1: Target deconvolution of antibodies

[0256] The objective of this example was to perform target convolution of 8 antibodies (7 antibodies specific to a target and one isotype control) against 18 antigens (including the specific targets of the selected antibodies) to confirm that the 7 target specific antibodies were associated with the corresponding antigen.

[0257] Materials and Methods

[0258] Antibody Tagging

[0259] Eight (8) commercially available antibodies were used as described in Table 1. The applied antibodies are examples and do not limit the invention.

[0260] Table 1: Antibodies used in deconvolution example

[0261] Antibodies were tagged with DNA oligonucleotides (Table 2) using a biotin-streptavidin interaction as follow: antibodies were conjugated with streptavidin using LYNX Rapid Streptavidin Antibody Conjugation Kit 100 pg (Biorad, # LNK161STR; Hercules, CA) following manufacturer instructions and incubated overnight with DNA oligonucleotides (Integrated DNATechnologies, HPLC purification; Coralville, IA) conjugated with Biotin added at the 5'end of the DNA molecule. The nucleotide tags listed in Table 2 are only examples to illustrate the invention and do not limit the invention. The tagged antibody was size purified using a 50 kDa Amicon Ultra Column followed by six washes in PBS. After size purification, left over unconjugated unbound DNA was removed by using magnetic streptavidin beads (BioAdem beads Streptavidin plus, Ademtech, #323; Pessac, France). Table 2: Tag sequences conjugated to each antibody used for target deconvolution example

[0262] Antigen library preparation

[0263] Three pools of cells were transfected with 6 antigens each (see Error! Reference source not found, for the list of antigens). Transfection was performed as followed: ExpiCHO-S Cells (ThermoFisher Scientific, #A29127; Waltham, MA), at a density of 3 to 4 x 106cells / ml, were grown overnight in ExpiCHO Expression Medium (ThermoFisher Scientific, #A2910001) for 24 hours. The cells were transfected with plasmids using ExpiFectamine (ThermoFisher Scientific, #A29129). Briefly, cells at a density of 7 to 10 x 106cells / ml and 95% minimal viability were washed and suspended in fresh ExpiCHO Expression Medium at a final density of 6 x 106cells / ml. Cells were distributed in 3 wells of a 6 well-plate (2.5 ml per well) and put at

[0264] 37 °C, 8% CO2, 120 rpm orbital shaking, until transfection. For each 2.5 ml well; 2.5 pg total of DNA plasmid (3 pools of 6 plasmids at equimolarity) were diluted in 102 pL final of OptiPRO SFM (ThermoFisher Scientific, #12309050) and mixed by swirling. 8 pL of ExpiFectamine CHO reagent were diluted in 100 pL final volume of OptiPRO SFM and mixed by inversion. Within 5 minutes maximum, diluted ExpiFectamine was added to diluted plasmid DNA. The final solution was mixed by pipetting 2-3 times, incubated for 1 to 5 minutes, then transferred to the cells in each well (drop by drop). The 6 well plate with transfected cells was incubated for ~24 h in New Brunswick Galaxy 170S incubator (Eppendorf; Hamburg, Germany) at 37 °C, 8% CO2, 85-95% humidity, with a shake speed of 120 rpm.

[0265] Table 3: List of transfected antigens into each pool. The antigens highlighted in bold correspond to the targets of the tested antibodies.

[0266] Plasmid

[0267] The open reading frames (ORF) of interest (CDS sequence, human surface protein) were cloned in the pcDNA3.4 vector (ThermoFisher Scientific), downstream of a P7-Kozak sequence, and upstream of an eGFP reporter gene. The ORF and eGFP sequences were separated by a nucleotide sequence encoding a P2A peptide sequence.

[0268] Target deconvolution in droplets

[0269] Cell library incubation with barcoded antibodies

[0270] Cells were incubated in labelling and washing buffer (LBW) (pH 7.2 with 1% BSA) completed with 1% (wt / v) final dextran sulfate sodium salt (MP Biomedicals, #101516; Irvine, CA) with approximately 5 pg / ml of each labelled antibody at 4 °C for 30 minutes and then washed 3 times in LBW, followed by one wash in PBS by centrifugation.

[0271] Cell encapsulation in droplets and in droplets RT

[0272] Cells were co-encapsulated with reverse transcription and lysis reagents to reach final concentration in drop of lx First Strand Buffer (Invitrogen, #18090200; Waltham, MA), 0.27% Igepal CA_630 (Sigma-Aldrich, #I88956-5OML; St. Louis, MO), 0.5 mM dNTPs (ThermoFisher Scientific #R0181), 5 mM DTT (Invitrogen, #18090200), 1 U / pl SUPERase IN (Invitrogen #AM2696), 10 U / pl SuperScript IV (Invitrogen, #18090200) , 1.2 pM DYE754 (Dyomics, #754- 00) and 10% (w / v) Nycodenz (Serva, #31000.01). 100 pl droplets were generated using a microfluidic device (Microfactory, #HFBDM) with a flow-focusing junction composed of a 15 pm wide, 40 pm deep and 10 pm long nozzle. The microfluidic device was manufactured by soft-lithography in poly-dimethylsiloxane (FIG. 1). The droplets were generated in a continuous phase composed of 2% (wt / wt) 008-FluoroSurfactant (RAN Biotechnologies; Beverly, MA) in Novec HFE 7500 fluorinated oil (3M). The final concentration of ~0.3 cells per droplet was to ensure that most droplets have 1 or less cells. The design of microfluidic device used is represented in FIG. 1. Emulsion was collected in collection tube pre-filled with 0.5% (wt / wt) 008-FluoroSurfactant in Novec HFE 7500 fluorinated oil and incubated for 1 hour 30 minutes at 55 °C followed by 20 minutes at 70 °C. After incubation, the emulsion was broken using lH,lH,2H,2H-perfluoro-l-octanol (Sigma, #370533-25G) mixed with HFE 7500 at a 1:5 ratio.

[0273] The cDNA sample was then first enzymatically purified using Exol (NEB, #M0293S; Ipswich, MA) followed by a purification step using paramagnetic beads (AMPure XP beads, Beckman Coulter, #A63881). Two rounds of PCR with Kapa High Fidelity polymerase (Roche, #7958935001; Indianapolis, IN) incorporate the Illumina adapter and index sequences: after each PCR the amplicons were purified using paramagnetic AMPure XP beads. After PCRs, the quality of the library was assessed using Tapestation and sequenced using a MiSeq system, MiSeq v3 flow cell Single-read sequencing 150 cycles, no Index, 20% PhiX (Illumina; San Diego, CA). The library preparation process is shown in more details in FIG. 2.

[0274] Data Analysis

[0275] Raw reads were aligned against a fasta reference containing the 18 ORF reverse sequences (from first ATG to P2Arev sequence) using Bowtie 2 (version 2.3.5.1) and the following argument: bowtie2 -local -x [bowtie_index] [sample-name.fastq] -S [output-name. sam] -no-hd

[0276] For each read, the sample index (UDP), antibody tag ID and readJD were retrieved, then merged with RNA alignment output based on readsJD. Then, the number of reads associated with each antibody tag paired with an identified gene was counted, in each sample. Finally, all reads from antigens coming from the same antigen pool were combined, ending up with a table containing the number of reads per antigen pool (1 to 3) for each antibody tag, in each sample.

[0277] Results

[0278] The number of reads per antibody tag was measured for each antibody present in the study and a minimal threshold defined by the number of reads associated with the isotype control was set, below which an antibody-target pair could not be confidently identified. All of the antibodies had a number of reads above threshold except for anti-IL3RA.

[0279] In a second step, for each antibody, Rn[poolx] was measured, which corresponded to the number of reads R[poolx] associated with antigen poolxdivided by the number of reads associated with the isotype control paired with the corresponding pool, Riso[poolx],

[0280] / ?rpooixi

[0281] Rn[poolx] = - - - —,with x = 1, 2 or 3 xJRiso [poolx]

[0282] For the 6 antibodies, the largest number of normalized reads corresponded to the pool expressing their respective specific targets (FIG. 4).

[0283] While the invention has been described in detail, and with reference to specific embodiments thereof, it will be apparent to one of ordinary skill in the art that various changes and modifications can be made therein without departing from the spirit and scope of the invention.

Claims

CLAIMS1. A method for high-throughput screening (HTS) for identifying a receptor-ligand cognate pair in a microfluidics system, the method comprising: a. providing a receptor library, wherein each receptor molecule of the library is tagged with a first nucleotide tag sequence resulting in tagged receptors, wherein the first nucleotide tag sequence comprises a unique barcode sequence and a universal primer sequence, b. providing a second library of nucleotide sequences, wherein the nucleotide sequences encode for ligands, c. transmitting the nucleotide sequences of second library into cellular expression systems by transfection or transduction, wherein each cellular expression system expresses one nucleotide sequence of the second library resulting in a plurality of cellular expression systems, d. incubating plurality of cellular expression systems for expression of ligand molecules of second library within the plurality of cellular expression systems, wherein the ligand molecules are presented on the surface of the plurality of cellular expression systems, e. contacting the plurality of tagged receptors with expressed ligands on the surface of plurality of cellular expression systems for receptor target pairing, f. encapsulating the plurality of cellular expression systems in a plurality of microreactors, wherein each microreactor comprises one cellular expression system expressing one ligand of the second library, g. generating combined nucleotide sequences by enzymatic reaction, wherein the combined nucleotide sequences comprise the nucleotide sequences of the second library and the unique barcode sequence of tagged receptor of the first library, h. sequencing of combined nucleotide sequences, i. analyzing sequences from step h. to identify cognate pairs of receptors of first library and ligands of second library.

2. The method of claim 1, wherein the nucleotide sequences of the second library comprise recombinant RNA molecules which encode the set of ligands or guide RNA molecules.

3. The method of any one of claims 1 and 2, wherein the nucleotide sequences of the second library comprise expression plasmids which are suitable for expression in the cellular expression system.

4. The method of any one of claims 1 to 3, wherein the recombinant RNA of the second library further comprises a second nucleotide tag sequence, and wherein the second nucleotide tag sequence is a second unique barcode which identifies the recombinant RNA.

5. The method of any one of claims 1 to 4, wherein technique for transfection or transduction of cells is selected from the group comprising electroporation, sonoporation, magnetofection, gene injection, gene gun, lipofection, transfection with polymers, transfection with nanoparticles, viral transfection CRISPR-mediated transcriptional activation and CRISPR / Cas gene editing.

6. The method of any one of claims 1 and 5, wherein the first and the second nucleotide tag sequences are DNA sequences.

7. The method of any one of claims 1 to 6, wherein the first nucleotide tag sequences comprise a universal primer sequence complementary to a sequence of the recombinant RNA of the second library, a barcode, and a sequence for amplification.

8. The method of any one of claims 1 to 7, wherein the receptor of the first library is tagged with the first nucleotide tag sequence by a techniques selected from the group comprising biotin-streptavidin interaction, amine conjugation, sulfhydryl conjugation and carbohydrate conjugation.

9. The method of any one of claims 1 to 6, wherein the recombinant RNA of the second library is linked to the second nucleotide tag sequence through an enzymatic reaction.

10. The method of any one of claims 1 to 9, wherein the microreactor is selected from an aqueous droplet, a microcapsule, a microbead, a microfluidic droplet, a compartment of a microfluidic chip, or a well.

11. The method of any one of claims 1 to 10, wherein the tagged receptors of the first library are tagged antibodies, preferably wherein the tagged antibody is isolated from an antibody-secreting B cell from a human subject or wherein the tagged antibody is isolatedfrom patient-derived xenograft (PDX) mice or humanized mice.

12. The method of any one of claims 1 to 11, wherein the method further comprises washing of the plurality of cellular expression systems to remove unbound tagged receptors and unspecifically bound tagged receptors.

13. The method of any one of claims 1 to 12, wherein the method further comprises adding reagents for reverse transcription to the plurality of microreactors within the microfluidic system, wherein the reagents for reverse transcription comprise a reverse transcriptase enzyme, mixture of dNTPs, and a suitable buffer and / or wherein the method further comprises adding reagents for cell lysis to the plurality of microreactors, wherein the reagents for cell lysis are selected from the group comprising hypotonic buffers, detergents and lysozyme.

14. The method of any one of claims 1 to 13, wherein the method further comprises cell lysis of cellular expression system in the plurality of microreactors, wherein cell lysis releases the nucleotide sequences of the second library.

15. The method of any one of claims 1 to 14, wherein the method further comprises contacting universal primer sequence of first nucleotide tag sequence of tagged receptors to released second nucleotide tag sequences of second library.

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