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WO2025160372A1 - Procédés d'identification de paires apparentées de ligands et de récepteurs - Google Patents

Procédés d'identification de paires apparentées de ligands et de récepteurs

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Publication number
WO2025160372A1
WO2025160372A1 PCT/US2025/012918 US2025012918W WO2025160372A1 WO 2025160372 A1 WO2025160372 A1 WO 2025160372A1 US 2025012918 W US2025012918 W US 2025012918W WO 2025160372 A1 WO2025160372 A1 WO 2025160372A1
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WO
WIPO (PCT)
Prior art keywords
library
sequence
nucleotide
receptor
sequences
Prior art date
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Pending
Application number
PCT/US2025/012918
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English (en)
Inventor
Francisco Adrian
Pascaline Mary
Sophie JIN
Sami ELLOUZE
Liang SCHWEIZER
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Hifibio SAS
Original Assignee
Hifibio SAS
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Filing date
Publication date
Application filed by Hifibio SAS filed Critical Hifibio SAS
Publication of WO2025160372A1 publication Critical patent/WO2025160372A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502769—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements
    • B01L3/502784—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements specially adapted for droplet or plug flow, e.g. digital microfluidics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1065—Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1075—Isolating an individual clone by screening libraries by coupling phenotype to genotype, not provided for in other groups of this subclass
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0883—Serpentine channels
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B30/00—Methods of screening libraries
    • C40B30/04—Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses

Definitions

  • the invention relates to methods for identifying cognate pairs of ligands and receptors.
  • Target deconvolution 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.
  • 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.
  • 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.
  • 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.
  • HTS high throughput screening
  • each cellular expression system expresses one nucleotide sequence of the second library resulting in a plurality of cellular expression systems
  • 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.
  • 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.
  • the nucleotide tag sequence is a DNA sequence.
  • 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 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.
  • each ligand of the set of ligands is displayed on the surface of a cell, wherein each cell expresses a single ligand.
  • 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.
  • the recombinant RNA is linked to the second nucleotide tag through an enzymatic reaction.
  • 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.
  • 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.
  • the microreactor is selected from an aqueous droplet, a microcapsule, a microbead, a compartment of a microfluidic chip, or a well.
  • 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.
  • the antibody is identified using single-cell antibody sequencing.
  • the subject has a disease or disorder.
  • FIG. 1 shows a schematic representation of the microfluidic chip design used to encapsulate reagents with transfected cells labelled with antibodies.
  • 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.
  • FIG. 3 shows a graph demonstrating the number of reads associated with each antibody tag.
  • 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.
  • 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.
  • FIG. 6 shows a schematic of DNA sequences enabling the binding of an antibody tag with Rec RNA in the case of a ligation.
  • 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.”
  • a numerical value typically includes ⁇ 10% of the recited value.
  • a concentration of 1 mg/ml includes 0.9 mg/ml to 1.1 mg/ml.
  • a concentration range of 1% to 10% (w/v) includes 0.9% (w/v) to 11% (w/v).
  • 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.
  • 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.
  • 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).
  • a 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).
  • 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.
  • nucleic acid also encompasses the complementary strand of a depicted single strand.
  • the term nucleic acid thus encompasses complementary DNA.
  • nucleic acid also encompasses substantially identical nucleic acids and complements thereof.
  • a single strand nucleic acid such as, a primer, may hybridize to the target sequence under hybridization conditions, preferably stringent hybridization conditions.
  • a nucleic acid also encompasses a primer that hybridizes under hybridization conditions to a target sequence.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the barcode sequence uniquely identifies the identity of a receptor and/or a ligand present in a microreactor (e.g., a droplet).
  • 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.”
  • 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.
  • 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.
  • 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.
  • the barcode sequence is a double stranded or single stranded nucleic acid, or a partially single and double stranded nucleic acid.
  • the barcode sequence is comprised within a barcoded primer.
  • 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.
  • 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.
  • 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.
  • the barcoded primer comprises from 5' to 3' a universal primer sequence, a barcode sequence or barcode set of sequences and a primer sequence.
  • 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).
  • 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.
  • the subject is a human.
  • the subject suffers from a disease or disorder, in particular from cancer, inflammatory and autoimmune disease, infectious disease or metabolic disease.
  • 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).
  • infectious disease is meant herein a disease caused by the transmission of a microorganism.
  • microorganism refers equally to viruses, in particular viruses which have a lipid envelope (e.g., an influenza virus), bacteria, parasites, and fungi.
  • 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.
  • the metabolic disease is selected from the group consisting of hyperglycemia, diabetes, in particular type 2 diabetes, obesity, dyslipidemia and hypercholesterolemia.
  • said metabolic disease is diabetes, more particularly type 2 diabetes.
  • cognate pair of ligands and receptors refers to the pair of a ligand species and the receptor species to which it selectively binds.
  • selective 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.
  • 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.
  • 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).
  • 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).
  • the cognate pair comprises two molecules which are selectively bound by, preferably specifically bound to each other.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the microreactors each contain a single ligand, but multiple microreactors can contain the same ligand.
  • nucleotide sequence 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a polymerase such as Klenow-fragment without 5'-3' exonuclease activity - or BST polymerase or other polymerase or using a ligase enzyme.
  • DNA polymerases are appropriate for this application as the first and the second nucleotide tag which are connected are both DNA sequences.
  • the second nucleotide tag and/or second sequence of the second library are connected by a ligase reaction.
  • ligases are used to connect the first and the second nucleotide tags.
  • 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.
  • the obtained connected DNA sequences are prepared as a sequencing library according to common techniques as necessary and appropriate for the obtained samples.
  • 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.
  • 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.
  • the nucleotide sequence of the second library may be linked to the second nucleotide tag at a later stage of the method after cell lysis instead of before cell transfection.
  • 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.
  • attachment may be achieved through an enzymatic reaction.
  • 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.
  • 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
  • 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.
  • the receptor is an antibody.
  • nucleotide sequences of the second library comprise recombinant RNA sequences which encode the set of ligands.
  • nucleotide sequences of the second library comprise guide RNA molecules for CRISPR/Cas application.
  • 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.
  • RNA or DNA DNA into the cells.
  • mechanical means of entering the target cells such as injections or gene guns.
  • 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.
  • 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.
  • the nucleotide sequence may also contain specific primer sequences which target the nucleotide sequence of the second library.
  • 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.
  • the nucleotide sequence may also contain specific primer sequences which target the nucleotide sequence of the second library.
  • 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.
  • 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.
  • Kd dissociation constant
  • 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.
  • a Streptavidin-Biotin bond is used to connect the receptors of the first library with the first nucleotide tag.
  • the recombinant RNA of the second library is linked to the second nucleotide tag sequence through an enzymatic reaction.
  • the barcoded primers may further comprise at least one linker sequence.
  • 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.
  • 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.
  • the cleavable linker is a double-stranded DNA molecule containing a target site for a specific restriction endonuclease.
  • 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.
  • 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.
  • 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.
  • 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.
  • the at least some of the barcoded primers can be released using enzymatic and/or photocleavage.
  • 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.
  • 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.
  • the barcoded primer is released by enzymatic digestion of streptavidin.
  • each particle carries a barcode sequence or barcode set of sequences distinguishable from barcode sequences or barcode sets of sequences carried by other beads.
  • 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.
  • 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.
  • RT reverse transcriptase
  • cDNA complementary DNA
  • 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.
  • 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.
  • the ligase is an enzyme capable of covalently linking a nucleic acid to another nucleic acid by forming a new chemical bond.
  • a ligase can covalently link the recombinant RNA with the second nucleotide tag.
  • 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.
  • 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.
  • the microreactor is selected from an aqueous droplet, a microcapsule, a microbead, a microfluidic droplet, a compartment of a microfluidic chip, or a well.
  • the microreactors are wells or microfabricated wells.
  • the microreactors are aqueous droplets, in particular in a continuous immiscible phase.
  • the methods occur in a single reaction.
  • the single reaction can, for example, occur in a microreactor.
  • the microreactor may be selected from any suitable method, such as by microfluidics, flow cytometry cell-based sorting, and/or limiting dilution.
  • 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.
  • the single cell is lysed within the microreactor.
  • 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.
  • each droplet has a volume at least equal to the volume of one mammalian cells.
  • 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.
  • 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.
  • each microcapsule has a volume at least equal to the volume of one mammalian cells.
  • 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.
  • 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.
  • each microbead has a volume at least equal to the volume of one mammalian cells.
  • 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.
  • cells can be encapsulated in microcapsules or microbeads before the cells achieve their transformation of droplet into microcapsules or microbeads.
  • 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.
  • 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.
  • 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.
  • 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.
  • the junction may be configured and arranged to produce substantially monodisperse droplets.
  • 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.
  • the tagged receptors of the first library are tagged antibodies.
  • 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.
  • 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).
  • TCR T cell receptor
  • B cell receptor from a B cell receptor/B cell antigen recognition pair
  • the present invention identifies cognate pairs of ligands and receptors. Accordingly, ligands and receptors can be considered interchangeable.
  • 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).
  • pMHC peptide-major histocompatibility complex
  • 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.
  • Borrelia bacteria e.g., Borrelia burgdorferi
  • Chikungunya Virus CHIKV
  • Chlamydia bacteria e.g., Chlamydia trachomatis
  • CMV Cytomegalovirus
  • DEV Dengue Virus
  • Ebola Virus Ebola Virus
  • 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., To
  • 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).
  • 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.
  • 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.
  • the ligand is a B cell antigen or a target antigen for the antibody.
  • the receptor may not be an antibody.
  • the present invention identifies cognate pairs of ligands and receptors. Accordingly, ligands and receptors can be considered interchangeable.
  • 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
  • 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., To
  • 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.
  • 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.
  • 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).
  • 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).
  • VL variable domain
  • VH variable domain
  • CH constant domain
  • 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 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 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).
  • antibody further denotes single chain antibodies, for instance Camelidae antibodies, or nanobodies or VHH.
  • 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.
  • the tagged antibody is isolated from patient-derived xenograft (PDX) mice or humanized mice.
  • PDX patient-derived xenograft
  • the method further comprises washing of the plurality of cellular expression systems to remove unbound tagged receptors and unspecifically bound tagged receptors.
  • 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.
  • said microreactors include additional reagents.
  • 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.
  • RT reverse transcriptase
  • dNTPs deoxynucleotide triphosphates
  • ligase e
  • 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.
  • 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.
  • 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.
  • 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.
  • RT reverse transcriptase
  • dNTPs deoxynucleotide triphospates
  • 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.
  • hybridization 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.
  • 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.
  • RT reverse transcriptase
  • "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.
  • the product of the reverse transcription is a RNA/DNA duplex comprising a single strand cDNA hybridized to its template RNA.
  • said RNA/DNA duplex is further linked to the barcoded primer comprising the primer sequence used for the reverse transcription.
  • Temporal 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).
  • MMLV Moloney murine leukemia virus
  • 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.
  • the reverse transcriptase 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.
  • the resulting cDNA contains the complete 5' end of the transcript, and universal sequences of choice are added to the reverse transcription product.
  • 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).
  • the microreactor further comprises cDNAs.
  • the microreactors further comprise cDNAs produced by reverse transcription of nucleic acids from the cells contained in said microreactors.
  • said cDNA refers to a single-stranded complementary DNA.
  • said cDNA is comprised in a RNA/DNA duplex.
  • 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.
  • 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.
  • 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.
  • 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.
  • the "cell lysis buffer” is a composition enabling cell lysis, preferably without disruption of the microreactors, in particular, of the droplets.
  • the cell lysis buffer is compatible with RT activity and/or with reagents used for the recognition assay.
  • the lysis buffer comprises enzymes selected from the group consisting of lysozyme, lysostaphin, zymolase, mutanolysin, glycanases, proteases, and mannose.
  • the lysis buffer comprises magnesium chloride, a detergent, a buffered solution and an RNase inhibitor.
  • the magnesium chloride is used at a concentration of between
  • the detergent is selected from the group consisting of Triton-X- 100, NP-40, Nonidet P40, and Tween-20 and IGEPAL CA 630.
  • the detergent is at a concentration of 0.1% to 10%.
  • 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, bo
  • 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).
  • 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).
  • 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.
  • 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.
  • 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.
  • the ligand's tag (second nucleotide tag) or the receptor's tag (first nucleotide tag) is a barcode sequence
  • 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.
  • a polymerase such as Klenow-fragment without 5'-3' exonuclease activity - or BST polymerase or other polymerase or using a ligase enzyme.
  • 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.
  • 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.
  • the cells are lysed using enzymatic, physical, and/or chemical cell lysis.
  • 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.
  • 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.
  • 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.
  • Physical cell lysis refers to the use of sonication, thermal shock (above 40°C, below 10°C), electroporation, or laser-induced cavitation.
  • the cells are lysed on ice.
  • the cell lysis does not disrupt or destroy the microreactors, in particular, the droplets, in the context 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.
  • the method further comprises contacting universal primer sequence of nucleotide tag sequence of tagged receptors to released nucleotide sequences of second library.
  • recognition is meant herein a binding between a ligand species and a receptor species.
  • 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.
  • assay reagents are added to the microreactors.
  • said assay reagents are co-compartmentalized with said ligand species and said receptor species during the co-compartmentalization step.
  • said assay reagents can be included in the fluid used for the formation of said droplets.
  • said assay reagents may be provided through a third fluid.
  • 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.
  • said assay reagents will depend on the particular recognition assay carried out.
  • Said separation can be carried out by any technique well-known from the skilled person, which will depend on the type of microreactors used.
  • 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.
  • the droplets 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • Recovering herein refers to isolating the barcoded cDNAs produced by reverse transcription in at least some of the microreactors from said plurality of microreactors.
  • 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.
  • 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.
  • Methods to isolate nucleic acids, in particular cDNA from microfluidic droplets 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.
  • 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.
  • the method further comprises the step of removing unincorporated barcoded primers from the aqueous composition of the microreactors.
  • 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.
  • the step of removing unincorporated barcoded primers precedes the amplification step and/or the sequencing step defined herein below.
  • 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.
  • the purification substrate comprises beads or particles, which, optionally, form a column.
  • unincorporated barcoded primers are removed by size selection using for example an acrylamide or an agarose gel.
  • 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.
  • an exonuclease such as the exonuclease Exol
  • the exonuclease degrades single stranded nucleic acid sequences from the aqueous compositions comprising the cDNA.
  • the barcoded cDNA obtained after reverse transcription is typically present in the form of a RNA/DNA complex and thus protected from said exonucleases.
  • the barcoded cDNA comprises one or more modified nucleotides or nucleotide analogs, for example for facilitating purification of the barcoded cDNA sequences or molecules.
  • 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.
  • 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'-alken
  • 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.
  • LNA locked nucleic acid
  • nucleotide analogs include deoxyinosine.
  • nucleotide analogs include Biotinylated, fluorescently labelled nucleotide.
  • 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.
  • the barcoded cDNA is further treated with RNAse A and/or RNAse H.
  • RNAse A is an endoribonuclease that specifically degrades single-stranded RNA at C and U residues.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the barcoded cDNAs obtained after reverse transcription are sequenced to allow identification of receptors and ligands contained in the same microreactor.
  • the step of sequencing the barcoded cDNA may comprise performing a next generation sequencing (NGS) protocol on a sequencing library.
  • NGS next generation sequencing
  • 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.
  • 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.
  • 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.
  • the barcoded cDNAs are further amplified.
  • the amplification step is performed by a polymerase chain reaction (PCR), and/or a linear amplification.
  • PCR polymerase chain reaction
  • the linear amplification precedes the PCR reaction.
  • the linear amplification is an in vitro transcription.
  • the linear amplification is an isothermal amplification.
  • 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.
  • the barcoded cDNA produced after reverse transcription is quantified using qPCR.
  • specific sequences necessary for sequencing are added during amplification or by ligation of adaptors, thereby generating a sequencing library.
  • 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.
  • the invention also provides the following non-limiting embodiments.
  • 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.
  • HTS high-throughput screening
  • each cellular expression system expresses one nucleotide sequence of the second library resulting in a plurality of cellular expression systems
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Embodiment 12 is the method of any one of embodiments 1 to 11, wherein the tagged receptors of the first library are tagged antibodies.
  • Embodiment 13 is the method of embodiment 12, wherein the tagged antibody is isolated from an antibody-secreting B cell from a human subject.
  • Embodiment 14 is the method of embodiment 12, wherein the tagged antibody is isolated from patient-derived xenograft (PDX) mice or humanized mice.
  • PDX patient-derived xenograft
  • 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.
  • 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.
  • the reagents for reverse transcription comprise a reverse transcriptase enzyme, mixture of dNTPs, and a suitable buffer.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • ExpiCHO-S Cells (ThermoFisher Scientific, #A29127; Waltham, MA), at a density of 3 to 4 x 10 6 cells/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).
  • 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.
  • Table 3 List of transfected antigens into each pool. The antigens highlighted in bold correspond to the targets of the tested antibodies.
  • ORF open reading frames of interest
  • CDS sequence human surface protein
  • pcDNA3.4 vector ThermoFisher Scientific
  • P7-Kozak sequence downstream of a P7-Kozak sequence
  • eGFP reporter gene upstream of an eGFP reporter gene.
  • the ORF and eGFP sequences were separated by a nucleotide sequence encoding a P2A peptide sequence.
  • LBW labelling and washing buffer
  • MP Biomedicals, #101516; Irvine, CA dextran sulfate sodium salt
  • 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.
  • 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).
  • AMPure XP beads paramagnetic beads
  • 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.
  • sample index UDP
  • antibody tag ID 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.
  • 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.

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Abstract

L'invention concerne des procédés de criblage à haut rendement (HTS) pour identifier des paires apparentées d'un ligand et d'un récepteur. Les procédés comprennent les étapes suivantes : (a) la fourniture d'une bibliothèque de récepteurs marqués ; (b) la fourniture d'une seconde bibliothèque de séquences nucléotidiques qui codent pour des ligands ; (c) la transmission des séquences nucléotidiques de la seconde bibliothèque dans des systèmes d'expression cellulaire ; (d) l'incubation de systèmes d'expression cellulaire pour l'expression de molécules de ligand d'une seconde bibliothèque sur la surface de la pluralité de systèmes d'expression cellulaire ; (e) la mise en contact de la pluralité de récepteurs marqués avec des ligands exprimés sur la surface d'une pluralité de systèmes d'expression cellulaire pour un appariement de cible de récepteur ; (f) l'encapsulation de la pluralité de systèmes d'expression cellulaire dans une pluralité de microréacteurs ; (g) la génération de séquences nucléotidiques de la seconde bibliothèque et de la séquence de code à barres unique du récepteur marqué de la première bibliothèque ; (h) l'analyse de séquences de l'étape g afin d'identifier des paires apparentées de récepteurs de la première bibliothèque et des ligands de la seconde bibliothèque.
PCT/US2025/012918 2024-01-24 2025-01-24 Procédés d'identification de paires apparentées de ligands et de récepteurs Pending WO2025160372A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210146366A1 (en) * 2018-04-18 2021-05-20 Hifibio Sas Microfluidic method for single cell analysis
US20230071148A1 (en) * 2021-07-15 2023-03-09 Vcreate, Inc. Compositions and Methods for Making Novel T-Cell Receptors

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210146366A1 (en) * 2018-04-18 2021-05-20 Hifibio Sas Microfluidic method for single cell analysis
US20230071148A1 (en) * 2021-07-15 2023-03-09 Vcreate, Inc. Compositions and Methods for Making Novel T-Cell Receptors

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