Methods for identifying cognate pairs of ligands and receptors - Patents.com

Abstract

Provided herein is a method for identifying cognate pairs of ligand and receptor species. The method includes the steps of: (a) providing a set of ligand species, where each ligand species is displayed at least once; (b) providing a set of receptor species, where each receptor species is displayed at least once; (c) contacting the set of ligand species with the set of receptor species in a microreactor, where an enhanced signal is produced upon selective binding of the receptor species to the ligand species; (d) detecting the cognate pair of ligand and receptor species by production of the enhanced signal; and (a) identifying the cognate pair of ligand and receptor species.

Description

[Technical Field]

[0001] [Cross-reference to application] This application claims priority to U.S. Provisional Application No. 62 / 705,383, filed June 24, 2020, the disclosure of which is incorporated herein by reference in its entirety.

[0002] FIELD OF THE INVENTION The present invention relates to a method for identifying cognate pairs of ligands and receptors, in particular cognate pairs of T cell receptors and T cell antigens or cognate pairs of B cell receptors and B cell receptor antigens. [Background technology]

[0003] Immunotherapy has become an innovative and attractive therapeutic modality for cancer, offering potentially targeted therapy with fewer side effects compared to conventional treatments. One type of immunotherapy is checkpoint blockade therapy using humanized mAbs specific for CTL antigen 4 (CTLA-4), programmed cell death-1 (PD-1), or its ligand PD-L1. This therapy has induced significant and durable clinical responses in patients with melanoma, lung cancer, kidney cancer, and bladder cancer. However, only a small subset of patients (20–30%) responds to such immune checkpoint therapy, and only in patients with specific types of cancer.

[0004] Therefore, cancer immunotherapy using a vaccine approach may be relevant in patients who do not respond to such treatments.

[0005] However, very few tumor antigens are known that can induce effective and safe T cell-mediated antitumor immunity in cancer patients. In fact, such effective tumor antigens must be overexpressed in cancer tissues but not in normal tissues, and must be capable of inducing tumor antigen-specific T cell responses.

[0006] Therefore, reliable identification of T cell antigens addresses an unmet need in the field of cancer immunology.

[0007] Furthermore, immunotherapy approaches may potentially be applied to treat autoimmune, inflammatory and autoimmune, infectious and metabolic diseases, where again efficient and reliable identification of T cell antigens is of paramount importance.

[0008] Autoimmune diseases can also be treated by cell-based therapies or tolerization approaches, which also require reliable identification of the antigen of interest.

[0009] Unfortunately, despite this potential utility, the knowledge and characterization of T cell antigens has progressed very slowly, particularly due to both the large T cell repertoire and the large number of potential T cell antigens.

[0010] Current methods for identifying T cell epitopes generally involve isolating T cells, generating individual T cell clones, and screening a panel of tumor cell lines or expression libraries (fresh or derived from established cell lines) derived from autologous tumor cells (Boon et al. (1994) Annu Rev Immunol. 12:337-65). This process is labor-intensive and inefficient because both T cell clones and tumor cell lines must be established, which is time-consuming and not possible for all tumor types.

[0011] Recently, deep sequencing of tumor DNA along with RNA analysis has enabled the definition and ranking of candidate epitopes using peptide-binding prediction algorithms for specific MHC alleles without the need to establish tumor cell lines (Gubin et al. (2015) J. Clin. Invest. 125:3413-3421). However, prediction algorithms are less reliable for MHC class II-restricted epitopes recognized by CD4 T cells. Epitopes can also be identified by proteomic analysis of acid eluates derived from immunoprecipitation of MHC class I molecules obtained from tumor cells, further refining the predictive capabilities of the process. In both cases, MHC tetramers loaded with the most likely antigen candidates are synthesized and used to fluorescently label and isolate potentially reactive T cells (Yadav et al. (2014) Nature 515:572-576 and Andersen et al. (2012) Nat. Protoc. 7:891-902).

[0012] However, the generation of MHC class II tetramers remains challenging for many epitopes.

[0013] Alternatively, T cell clones or cell lines expressing cloned T cell receptors (TCRs) are functionally tested against antigen-presenting cells (APCs) loaded with synthetic peptides (expression libraries) (Gaugler et al. (1994) J. Exp. Med. 179:921-930) or transduced with mRNA encoding candidate epitopes (Holtkamp et al. (2006) Blood 108:4009-4017). DNA-tagged MHC oligomer technology (Bentzen et al. (2016) Nat. Biotechnol. 34:1037-1045) requires prior knowledge of candidate antigens and is currently only applicable to MHC-I-restricted epitopes.

[0014] However, each method has the following disadvantages: generating T cell clones is very labor-intensive because the resulting clones are screened for antigen specificity; identifying recurrent and / or tumor-reactive TCRs without cell expansion from mixed populations by deconvolution methods is applicable only when the frequency of some of the TCRs of interest is increased and sufficient cells are available; elution of peptides from tumor MHC molecules requires many tumor cells; and bioinformatic analysis of MHC epitopes from genomic data requires strong hypotheses about the nature of the epitopes.

[0015] Taken together, these methods are low-throughput and based on multi-step processes that often require the generation of specific reagents (clones, tumor cell lines, MHC tetramers, mRNA, peptides), and therefore are not applicable to the unbiased discovery of MHC class I and class II epitopes that can be produced by mechanisms other than mutation or overexpression (e.g., by post-translational modifications [phosphorylation, ubiquitination, sumoylation, among others], splicing, insertion / deletion).

[0016] Thus, there is a significant need for efficient methods for identifying cognate pairs of T cells and T cell antigens, particularly from subjects suffering from cancer, inflammatory and autoimmune diseases, infectious diseases, or metabolic diseases.

[0017] Furthermore, B cell repertoires (BCRs and antibodies) can be the source of therapeutic products and can be used for immune response monitoring. Such therapeutic products can be antibodies (and all their variants, including bispecific antibodies and nanobodies) and engineered cells (including CAR-T and NK cells, among others), which utilize the ability of antibodies to preferentially bind to specific antigens in their native format. Such antigens can be natural antigens potentially overexpressed in cancer cells or foreign antigens expressed in cancer cells or normal cells. Antigen libraries and / or B cell repertoires are typically screened to identify antigen / B cell repertoire pairs. Identification of antigens can lead to the identification of potential vaccines, biomarkers, and targets. Identification of B cell repertoires corresponding to specific antigens benefits from high-throughput methods to rapidly identify potent and specific therapeutics. Summary of the Invention [Problem to be solved by the invention]

[0018] Understanding T cell phenotypic responses can be used to monitor immune responses from patients before and / or after treatment. Therefore, defining T cells and / or B cells that respond to foreign antigens (e.g., T cell antigens, B cell antigens, viral antigens, bacterial antigens, parasitic antigens, etc.) can guide treatment of diseases caused by foreign antigens and lead to the identification of cognate vaccine epitopes. Furthermore, identifying foreign antigen-responsive T cell receptors (which may be comparable to antitumor T cell receptors) can guide the selection of T cell receptors with high affinity and specificity.

[0019] Linking T cell repertoires, antigens, and phenotypes over time is necessary to understand the diversity and heterogeneity of immunogenic antigens and responder T cells at both the phenotypic and transcriptomic levels over time. This will effectively help stratify patients based on their immunogenic responses at different stages and develop preventative and therapeutic vaccines.

[0020] Cell therapy (e.g., T cells, CAR T cells, Tregs, stem cells) is another promising treatment that demonstrates curative and beneficial clinical outcomes, requiring the identification of receptor sequences that respond to specific ligands and the characterization of this interaction and their downstream effects. Indeed, linking phenotype-genotype and ligand-receptor relationships can address these unmet needs and improve the selection of optimal responder cells that exhibit appropriate phenotypic and transcriptomic profiles. This linkage and matching is useful for fine-tuning the selection and selecting optimal cells (with the correct receptor, such as TCR) that exhibit the highest specific activity. [Means for solving the problem]

[0021] The present invention relies on the unexpected discovery that it is possible to rapidly and easily screen thousands of tumor antigens, including an unlimited set of antigens, for their ability to bind and activate T cells without any a priori selection, and to reliably identify such cognate pairs of T cell antigens and T cell receptors with a low error rate and without further confirmation of the identification. Furthermore, the designed identification method can be applied to any type of ligand-receptor binding pair, such as viral antigens and T cell receptors, bacterial antigens and T cell receptors, parasitic antigens and T cell receptors, and B cell antigens and B cell receptors.

[0022] Thus, provided herein is a method for identifying cognate pairs of ligand and receptor species, the method comprising the steps of: (a) providing a set of ligand species, wherein each ligand species is displayed at least once; (b) providing a set of receptor species, wherein each receptor species is displayed at least once; (c) contacting the set of ligand species with the set of receptor species in a microreactor, wherein an enhanced signal is produced upon selective binding of the receptor species to the ligand species; (d) detecting the cognate pair of ligand and receptor species by production of the enhanced signal; and (a) identifying the cognate pair of ligand and receptor species.

[0023] In certain embodiments, each ligand species comprises a barcode sequence. In certain embodiments, each receptor species comprises a barcode sequence. In certain embodiments, each ligand species and / or each receptor species comprises a barcode sequence.

[0024] In certain embodiments, each ligand species is expressed or presented on the surface of a cell or bead, or expressed or present in a cell-free extract or solution. The ligand species can be expressed or presented on the surface of, for example, an antigen-presenting cell. The antigen-presenting cell can be selected from, for example, macrophages, dendritic cells, Langerhans cells, B cells, monocyte-derived dendritic cells, or other cells that express MHC class I or II molecules.

[0025] In certain embodiments, each receptor species is expressed or displayed on the surface of a cell or bead, or is expressed or present within a cell-free extract or in solution.

[0026] In certain embodiments, the microreactor is selected from an aqueous droplet, a microcapsule, a microbead, a compartment of a microfluidic chip, or a well (e.g., a well of a tissue culture plate).

[0027] In certain embodiments, the signal is selected from the following: morphological changes in any one of cells, ligands, or receptors; fluorescent signal enhancement; modification of fluorescent signals using caged compounds or through quenching reactions; light absorption; modification / generation of visible structures; or a combination of these signals. The signal can be dynamic (on / off; versus on; versus off) and spatial-temporal changes. Modification of fluorescent signals through quenching reactions can include, for example, FRET, FLIP, FRAP, FLAP, BRET, or FLIM quenching reactions. The morphological change can be cell-cell interaction (e.g., immunological synapse-like), changes in cell size, changes in cell granularity, polarization of peptide / protein localization, or material transfer between cells (e.g., proteins, nucleic acids, lipids, and / or carbohydrates).

[0028] In certain embodiments, identifying the cognate pair of ligand and receptor species comprises amplifying the ligand and receptor species, and at least one of the amplified ligand and receptor species is sequenced for identification.

[0029] In certain embodiments, the set of ligand species may be selected from, for example, T cell antigens, B cell antigens, viral antigens, bacterial antigens, parasitic antigens, neoantigens, tumor-associated antigens (TAA), tumor-specific antigens, immune checkpoint molecules, cytokines, carbohydrates, members of the immunoglobulin superfamily, selectins, chemokines, hormones, growth factors, G protein-coupled receptor ligands, or enzyme substrates.

[0030] In certain embodiments, the set of receptor species may be selected from, for example, T cell receptors, B cell receptors, immune checkpoint receptors, cytokine receptors, selectins, integrins, members of the immunoglobulin superfamily, cadherins, chemokine receptors, hormone receptors, growth factor receptors, G protein-coupled receptors (GPCRs), or enzymes.

[0031] In certain embodiments, the ligand species is a T cell antigen and the receptor species is a T cell receptor, and upon selective binding of the T cell receptor to the T cell antigen, an enhanced signal is produced, wherein the enhanced signal produced is a result of T cell activation.

[0032] In certain embodiments, the ligand species is a viral antigen and the receptor species is a T cell receptor, and upon selective binding of the T cell receptor to the viral antigen, an enhanced signal is produced, wherein the enhanced signal produced is a result of T cell activation.

[0033] In certain embodiments, contacting the set of ligand species with the set of receptor species in the microreactor occurs for about 0.001 hours to about 8 hours, hi certain embodiments, contacting the set of ligand species with the set of receptor species in the microreactor occurs for at least about 8 hours, e.g., from about 8 hours to about 48 hours.

[0034] In certain embodiments, the contacting step occurs for about 0.1 hours to about 8 hours, and if the ligand and receptor species bind with high affinity, the enhanced signal produced is an early marker of T cell activation. In certain embodiments, the contacting step occurs for about 0.1 hours to about 8 hours, and if the ligand and receptor species bind with high affinity, the enhanced signal produced is a late marker of T cell activation.

[0035] In certain embodiments, when the contacting step occurs for at least about 8 hours and the ligand species and receptor species bind with high affinity, the enhanced signal produced can be an early or late marker of T cell activation.

[0036] In certain embodiments, the contacting step occurs for at least about 8 hours, and when the ligand and receptor species bind with low affinity, the enhanced signal produced is an early marker of T cell activation. Extending the contacting step for ligand and receptor species that bind with low affinity can ultimately result in an enhanced signal produced by a later marker of T cell activation.

[0037] In certain embodiments, the early marker of T cell activation may be, but is not limited to, CD69, CD107a, or transferrin receptor.

[0038] In certain embodiments, the late marker of T cell activation may be, but is not limited to, CD137, HLA-DR, VLA1, PTA1, CD71, CD27, PD-1, TIM3, LAG3, or CTLA4.

[0039] In certain embodiments, the signal is detected by an anti-CD69 antibody, an anti-CD107a antibody, an anti-transferrin receptor antibody, an anti-CD137 antibody, an anti-HLA-DR antibody, an anti-VLA1 antibody, an anti-PTA1 antibody, an anti-CD71 antibody, an anti-CD27 antibody, an anti-PD1 antibody, an anti-TIM3 antibody, an anti-LAG3 antibody, or an anti-CTLA4 antibody.

[0040] In certain embodiments, the ligand species is a B cell antigen and the receptor species is a B cell receptor, and an enhanced signal is produced upon selective binding of the B cell receptor to the B cell antigen. The enhanced signal produced can be the result of, for example, B cell activation, B cell (receptor)-specific antigen detection, or target cell activation.

[0041] In certain embodiments, the signal is detected by an anti-CD138 antibody, an anti-CD19 antibody, an anti-CD45R antibody, an anti-CD45 antibody, activation of fluorescent reporter expression, or inhibition of fluorescent reporter expression.

[0042] 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 is to be understood that the invention is not limited to the precise embodiments shown in the drawings. [Brief explanation of the drawings]

[0043] [Figure 1]Titration of anti-CD137 antibodies in droplets. Different concentrations of antibodies were co-flowed and incubated overnight with polyclonal pre-activated T cells in droplets. [Figure 2] Schematic of T cell activation by antigen-presenting cells (APCs) in a droplet workflow. Peptide-pulsed K562 APCs were resuspended in a co-flow containing anti-CD137 antibody and then co-flowed through a separate inlet with a second inlet containing a T cell clone specific for an EBV peptide. [Figure 3] T cell activation in droplets is shown. Peptide-pulsed K562 cells were resuspended in a parallel flow containing anti-CD137 antibody and then separately co-flowed with a T cell clone specific for an EBV peptide. [Figure 4] Images of antigen-presenting cell (APC, K562) and T cell interactions in droplets are shown. The top row shows micrographs of droplets co-encapsulating T cells and peptide-loaded K562 after overnight incubation at 37 °C. Others are associated fluorescent signals as indicated. These binding events result in a red fluorescent signal; the anti-CD137 antibody was concentrated on the T cells (yellow) rather than being uniformly distributed throughout the droplet volume or the antigen-presenting cells (purple) (indicated by arrows). [Figure 5] Representative fluorescence plots of IFN-γ secretion in droplets are shown. T cells were co-cultured overnight in droplets with mRNA-transfected antigen-presenting cells (APCs; K562 cells) in the presence of an anti-IFN-γ conjugated antibody (as shown, a PE fluorescent dye is linked to the antibody). IFN-γ release was detected in the droplets by an indirect ELISA assay using an anti-CD45 bispecific antibody preloaded on the T cells and a PE-linked anti-IFN-γ secondary antibody run in parallel. [Figure 6]A representative workflow for sequence recovery of activated T cells using APCs with the Cell-Cap system is shown. Enriched droplets (derived from sorted droplets containing cells with the desired phenotype) were collected into individual microwells (which may be confirmed under a microscope) and then fused with droplets containing hydrogel beads conjugated with cell barcodes and gene-specific primers. The gene-specific primers were designed to capture TCR, antigen information, and optionally additional gene sets. Sequences with the same cell barcode were then obtained from the same droplets corresponding to the appropriate T cell-APC pair, thus recovering sequences for the TCR and cognate antigen. [Figure 7] Linking TCR sequences to antigens: The same cell barcode and sequences (called TMGs) recovered from the same droplet display both TCR and antigen. As an example, a cell barcode consists of a series of four specific sets of 11-mer indices separated by a 4-mer linker. These sequences are blasted against public or private databases and matched with TCR alpha (SEQ ID NO: 1) and beta (SEQ ID NO: 2) sequences and the TMG sequence (SEQ ID NO: 3) (the tandem minigene corresponds to the antigen transfected into the APC). UMIs (unique molecular identifiers) are used to quantify the transcripts produced per cell, and are optional. DETAILED DESCRIPTION OF THE INVENTION

[0044] Various publications, articles, and patents are cited or described in the Background and throughout this specification; each of these references is incorporated herein by reference in its entirety. The disclosure of documents, acts, materials, devices, articles, and the like which has been included herein is for the purpose of providing a context for the present invention. Such discussion is not an admission that any or all of them form part of the prior art with respect to any invention(s) disclosed or claimed.

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains.Otherwise, certain terms used herein have the meanings defined in the specification.All patents, published patent applications, and publications cited herein are incorporated by reference as if set forth in their entirety herein.

[0046] Generally, the nomenclatures used 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.

[0047] 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.

[0048] It must be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0049] Unless otherwise specified, any numerical values, such as concentrations or concentration ranges, described herein should be understood to be modified in all instances by the term "about." Thus, numerical values ​​typically include ±10% of the recited value. For example, a concentration of 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Similarly, a concentration range of 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v). The use of numerical ranges herein explicitly includes all possible subranges, all individual numerical values ​​within that range, and includes integers and fractions of values ​​within such ranges, unless the context clearly indicates otherwise.

[0050] As used herein, the term "nucleic acid" generally refers to at least one molecule or strand of DNA or RNA, containing at least one nucleobase, e.g., 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).

[0051] "RNA," as used herein, refers to functional RNA such as mRNA, tRNA, ncRNA, lncRNA, miRNA, siRNA, piRNA, gRNA, telomerase RNA component, RNAi, CRISPR RNA, circular RNA, enhancer RNA, snoRNA, snRNA, and rRNA.

[0052] As will be understood by those skilled in the art, the designation of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid also encompasses the complementary strand of the designated single strand. Thus, the term "nucleic acid" encompasses complementary DNA. As will also be recognized by those skilled in the art, many variants of nucleic acids can be used for the same purpose as a given nucleic acid. Thus, a nucleic acid also encompasses substantially identical nucleic acids and their complements. As will also be understood by those skilled in the art, a single-stranded nucleic acid, such as a primer, can hybridize to a target sequence under hybridization conditions, preferably stringent hybridization conditions. Thus, a nucleic acid also encompasses a primer that hybridizes to a target sequence under hybridization conditions.

[0053] 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. Specifically, a barcoded primer can be about 30 to about 150 nucleobases in length, about 40 to about 100 nucleobases in length, about 50 to about 90 nucleobases in length, or about 60 to about 80 or 70 nucleobases in length. More specifically, in the context of the present invention, a barcoded primer is an oligonucleotide comprising a barcode sequence or set of barcode sequences and a primer sequence, wherein each different primer sequence defines a different specificity of the barcoded primer. In one embodiment, the barcoded primer comprises, from 5' to 3', a universal primer sequence, a barcode sequence or set of barcode sequences, and a primer sequence.

[0054] These definitions refer to at least one single-stranded molecule, but in some embodiments also encompass at least one additional strand that is partially, substantially, or fully complementary to at least one single-stranded molecule. Thus, in some embodiments, the definitions refer to a double-stranded molecule.

[0055] Thus, in one embodiment, a nucleic acid refers to at least one double-stranded molecule comprising one or more complementary strands or "complements" of a particular sequence comprising a strand of the molecule.

[0056] As used herein, a "barcode sequence" refers to a unique nucleic acid sequence that can be distinguished from another nucleic acid sequence by its sequence, thus allowing the nucleic acid sequence to be uniquely labeled so that it can be distinguished from another nucleic acid bearing a different barcode sequence.

[0057] In one embodiment, the barcode sequence uniquely identifies the nucleic acid contained in a particular microreactor from the nucleic acid contained in other microreactors, even after the nucleic acids have been pooled together, for example.

[0058] In some embodiments, barcode sequences may be used to distinguish between tens, hundreds, or even thousands of nucleic acids, for example, resulting from cells contained within different microreactors.

[0059] In one embodiment, the barcode sequence can be of any suitable length. The barcode sequence is preferably long enough to distinguish it from other barcode sequences. In one embodiment, the 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, for example, 50-85, 60-80, 70-80 nucleotides.

[0060] In one embodiment, the barcode sequence consists of more than one barcode sequence, where the barcode sequences are different. Such barcode sequences are referred to herein as a "set of barcode sequences."

[0061] In a related embodiment, the different barcode sequences may be obtained from a "pool" of potential barcode sequences. Where a barcode sequence consists of more than one barcode sequence, the barcode sequences may be obtained from the same or different pools of potential barcode sequences. The pool of sequences may be selected using any suitable technique, for example randomly, or such that the sequences are separated by a particular distance (e.g., Hamming distance) to allow for error detection and / or correction, so that errors in the reading of the barcode sequences can be detected and potentially corrected. A pool can have any number of potential barcode sequences, for example, 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.

[0062] Methods for joining different barcode sequences obtained from one "pool" or more than one "pool" are known to those skilled in the art and include, but are not limited to, the use of ligases and / or annealing or primer extension methods.

[0063] In one embodiment, the barcode sequence is a double-stranded or single-stranded nucleic acid, or a partially single-stranded and double-stranded nucleic acid.

[0064] "Primer sequences" are typically short, single-stranded nucleic acids, 10 to 50 nucleotides in length, designed to perfectly or nearly perfectly match the nucleic acid of interest to be captured, and then amplified (e.g., by PCR) or reverse transcribed (e.g., by RT). Primer sequences are "specific" for the nucleic acid to which they hybridize; i.e., primer sequences hybridize preferably under stringent, and more preferably highly stringent, hybridization conditions, and are complementary or nearly complementary to the nucleic acid to which they hybridize (also called the target sequence).

[0065] Typically, a primer sequence serves as a starting point for nucleic acid synthesis, allowing a polymerase enzyme, such as a nucleic acid polymerase, to extend the primer sequence and replicate a complementary strand. The primer sequence may be complementary to and hybridize with a target nucleic acid. In some embodiments, the primer sequence is a synthetic primer sequence. In some embodiments, the primer sequence is a non-naturally occurring primer sequence. The primer sequence typically has a length of 10 to 50 nucleotides. For example, the 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, the primer sequence has a length of 18 to 24 nucleotides.

[0066] In one embodiment, the primer sequence is located 3' to the barcoded primer used in the context of the present invention (ie, the primer is 3' to the barcode sequence).

[0067] As used herein, "gene" can refer to a genomic gene containing transcriptional and / or transcriptional regulatory sequences and / or coding regions and / or non-translated sequences (e.g., introns, 5'- and 3'-untranslated sequences). The coding region of a gene can be a nucleotide sequence encoding an amino acid sequence or functional RNA, such as tRNA, rRNA, catalytic RNA, siRNA, miRNA, antisense RNA, lncRNA, and piRNA. A gene can also be an mRNA or cDNA corresponding to the coding region (e.g., exons and miRNA) and may include 5'- or 3'-untranslated sequences linked thereto. A gene can also be an in vitro-produced amplified nucleic acid molecule containing all or a portion of the coding region and / or 5'- or 3'-untranslated sequences linked thereto.

[0068] As used herein, the term "stringent conditions" or "high stringency conditions" refers to conditions that are suitable for forming binding pairs between nucleic acids with a determined level of complementarity, but are not suitable for forming binding pairs between nucleic acids exhibiting less than the determined level of complementarity. Stringent conditions are a combination of both hybridization and washing conditions and are sequence-dependent. These conditions can be modified according to methods known to those skilled in the art (Tijssen, 1993, Laboratory Techniques in Biochemistry and Molecular Biology - Hybridization with Nucleic Acid Probes, Part I, Chapter 2 "Overview of principles of hybridization and the strategy of nucleic acid probe assays", Elsevier, New York). Generally, high stringency conditions are selected to be about 5°C lower than the melting temperature (Tm), preferably a temperature close to the Tm of a perfectly base-paired duplex (Anderson, MLM (1999) Nucleic acid hybridization. New York: Bios Scientific Publisher p. 54). Hybridization procedures are well known in the art and are described, for example, in Ausubel, F M, Brent, R., Kingston, RE, Moore, D D, Seidman, J G, Smith, J A, Struhl, K. eds. (1998) Current protocols in molecular biology. V B Handa, series ed. New York: John Wiley & Sons.

[0069] High stringency conditions typically include hybridization at about 40°C to about 68°C, where the temperature is typically higher than the highest melting temperature, T, of the nucleic acid hybridized to the target sequence in, for example, 5xSSC / 5xDenhardt's solution / 1.0% SDS and a wash at about 60°C to about 68°C in 0.2xSSC / 0.1% SDS. M is equivalent to

[0070] As used herein, the term "tissue" refers to a group of cells, generally consisting of homogeneous cells that perform the same or similar functions. A tissue may be part of an organ or bone, or may be a loose association of cells, such as cells of the immune system. A tissue may be a healthy tissue or a diseased tissue. In particular, a tissue may be a cancerous tissue or a tumor-peripheral tissue.

[0071] As used herein, a "subject" is a mammal, such as a human, but may also be another animal, such as a dog, cat, cow, sheep, pig, horse, monkey, rat, mouse, rabbit, guinea pig, etc. Preferably, the subject is a human.

[0072] In a particular embodiment, the subject is suffering from a disease, in particular cancer, an inflammatory and autoimmune disease, an infectious disease or a metabolic disease.

[0073] "Cancer," as used herein, refers to a class of diseases involving abnormal proliferation, including both cancers associated with solid tumors and cancers that do not involve solid tumors (e.g., leukemia).

[0074] By "autoimmune disease" herein is meant a wide range of degenerative diseases caused by the immune system attacking one's own cells.

[0075] " Inflammatory and autoimmune diseases " as used herein refer to diseases that are initially caused by inflammatory processes, which begin with the activation of T cells by antigen-presenting cells, and then lead to the activation of other inflammatory cells, resulting in the release of pro-inflammatory cytokines, chemotactic substances and matrix-degrading enzymes.Examples of inflammatory and autoimmune diseases are well known to those skilled in the art, and include rheumatoid arthritis, osteoarthritis, osteoporosis, Crohn's disease, ulcerative colitis, multiple sclerosis, periodontitis, gingivitis, graft-versus-host reaction, psoriasis, scleroderma, alopecia areata, Sjogren's syndrome, polymyositis, pemphigus, uveitis, Addison's disease, atopic dermatitis, asthma, systemic lupus erythematosus (SLE), nephropathy and chronic obstructive pulmonary disease (COPD), diabetic retinopathy and age-related macular degeneration.

[0076] "Infectious disease" as used herein means a disease caused by infection with a microorganism. In the context of the present invention, the term "microorganism" refers equally to viruses, and in particular to lipid-enveloped viruses (e.g., influenza viruses), bacteria, parasites, and fungi.

[0077] "Metabolic disease" as used herein refers to any type of disorder in which metabolic errors and imbalances occur, causing metabolic processes to occur in a suboptimal manner. In a preferred embodiment, the metabolic disease is selected from the group consisting of hyperglycemia, diabetes, particularly type 2 diabetes, obesity, dyslipidemia, and hypercholesterolemia. In a particular embodiment, the metabolic disease is diabetes, more particularly type 2 diabetes.

[0078] In the context of the present invention, the term "cognate pair" of ligand and receptor refers to a pairing of a ligand species and a receptor species to which it selectively binds.

[0079] By "selectively bind" herein is meant that one member of a pair recognizes and binds to the other member of the pair with greater affinity than another member of the pair.

[0080] As used herein, "specifically bind" means that one member of a pair recognizes and binds to the other member of the pair and has no detectable binding activity for another member of the pair.

[0081] As used herein, the term "high affinity" refers to selective or specific binding of a ligand species to a receptor species, where the binding is at a level of 3 μM or less.

[0082] As used herein, the term "low affinity" refers to selective or specific binding of a ligand species to a receptor species where the binding is at a level of 3 μM or greater.

[0083] As used herein, the term "ligand species" refers to a member of a particular recognition pair that selectively binds, preferably specifically binds, to a second member of said particular recognition pair (or cognate pair).

[0084] As used herein, the term "receptor species" refers to a member of a specific recognition pair that is selectively bound, preferably specifically bound, by a second member of said specific recognition pair (or cognate pair).

[0085] Thus, since ligands and receptors are defined as binding partners, a molecule that is a ligand may also be a receptor, and conversely, a molecule that is a receptor may also be a ligand.

[0086] The term "set of ligands" as used herein refers to at least one ligand species, preferably a plurality of ligand species, in particular a plurality of ligand species, where at least two of the plurality of ligand species are part of distinct recognition pairs. Preferably, a set of ligands as used in the context of the present invention includes redundant ligand species, i.e., ligand species that are present in the set in multiple copies.

[0087] The term "receptor set" as used herein 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, a receptor set as used in the context of the present invention includes redundant receptor species, i.e., receptor species that are present in the set in multiple copies.

[0088] In certain embodiments, the set of receptors is expressed or displayed on the surface of a cell (or multiple cells), beads, particularly modified APC-like beads as disclosed in Neal et al. (2017) J. Immunol. Res. Ther. 2:68-79, or encoded in vitro (i.e., expressed or present in a cell-free extract or solution) as disclosed in Grubaugh et al. (2013) Vaccine 31:3805-3810. Preferably, the set of receptors is expressed or displayed on the surface of a cell (or multiple cells), a cell, or beads that express or display unique receptor species from the set of receptors.

[0089] In another specific embodiment, the set of ligands is expressed or displayed on the surface of a cell (or multiple cells), beads, particularly APC-like beads modified as disclosed in Neal et al. (2017) J. Immunol. Res. Ther. 2:68-79, or encoded in vitro (i.e., expressed or present in a cell-free extract or solution) as disclosed in Grubaugh et al. (2013) Vaccine 31:3805-3810. Preferably, the set of ligands is expressed or presented on the surface of a cell (or cells) that express or present one ligand species from the set of ligands or multiple distinct ligand species from the set of ligands, preferably 2-1000, 5-900, 10-800, 20-700, 30-600, 40-500, 50-400 distinct ligand species from the set of ligands, particularly 100-350, 150-300, or 200-250 distinct ligand species from the set of ligands.

[0090] In a particularly preferred embodiment, the set of receptors is expressed or presented on the surface of a cell (or multiple cells) and the set of ligands is expressed or presented on the surface of another cell (or multiple cells). Even more preferably, the set of receptors is expressed or presented on the surface of a cell (or multiple cells), each cell expressing or presenting a unique receptor species from the set of receptors, and the set of ligands is expressed or presented on the surface of another cell (or multiple cells), each cell expressing or presenting multiple distinct ligand species from the set of ligands.

[0091] In certain embodiments, the receptor may be, for example, a T cell receptor (TCR from a TCR / T cell antigen recognition pair, including a TCR from a TCR / viral antigen recognition pair), a B cell receptor (from a B cell receptor / B cell antigen recognition pair), a receptor for a stimulatory immune checkpoint molecule (e.g., OX40L from the OX40L / OX40 pair), a receptor for an inhibitory immune checkpoint molecule (e.g., PD-L1 from the PD-L1 / PD-1 pair), a cytokine receptor (from a cytokine / cytokine receptor pair), a selectin (from a selectin / carbohydrate pair), an integrin (from an integrin / immunoglobulin pair), or ... cytokine receptor (from a cytokine / cytokine receptor pair), a selectin (from a selectin / carbohydrate pair), an integrin (from an integrin / immunoglobulin pair), or a receptor for a stimulatory immune checkpoint molecule (e.g., OX40L from the OX40L / OX40 pair), a cytokine receptor (from a cytokine / cytokine receptor pair), a selectin (from a selectin / carbohydrate pair), an integrin (from an integrin / immunoglobulin pair), or a receptor for a stimulatory immune checkpoint molecule (e.g., OX40L from the OX40L / OX40 pair), a cytokine receptor (from a cytokine / The receptor may be a member of a selectin pair, a member of the immunoglobulin superfamily (from an immunoglobulin superfamily member / selectin pair, or from a pair containing two members of the immunoglobulin superfamily), a cadherin (from a pair containing two cadherins), a chemokine receptor (from a chemokine / chemokine receptor pair), a hormone receptor (from a hormone / hormone receptor pair), a growth factor receptor (from a growth factor / growth factor receptor pair), a G protein-coupled receptor (GPCR, from a GPCR / corresponding ligand pair), or an enzyme (from an enzyme / corresponding substrate pair).

[0092] In a preferred embodiment, the receptor pair is a set of T cell receptors.

[0093] In certain embodiments, the ligand may be, for example, a T cell antigen (from a TCR / T cell antigen recognition pair), a B cell antigen (from a B cell receptor / B cell antigen recognition pair), a viral antigen, a bacterial antigen, a parasitic antigen, a neoantigen (i.e., an antigen resulting from a genetic mutation or aberrant expression in tumor cells that is uniquely found in tumor cells), a tumor-associated antigen (TAA), a tumor-specific antigen, a stimulatory immune checkpoint molecule (e.g., OX40 from the OX40L / OX40 pair), an inhibitory immune checkpoint molecule (e.g., PD-1 from the PD-L1 / PD-1 pair), a cytokine (cytokinin), or a cytotoxic T cell antigen (cytotoxic T cell antigen). The set of ligands may be a set of T cell antigens (peptides, glycolipids, or small metabolites, e.g., 5-A-RU derivatives), preferably bound to a major histocompatibility complex (MHC molecule, which may be class I, class II, or MR1) or CD1a, b, c, or d molecule.

[0094] The foreign antigen, e.g., a viral antigen, a bacterial antigen, or a parasitic antigen, can include, for example, but is not limited to, a viral antigen, a bacterial antigen, or a 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), Escherichia 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), herpesvirus, Helicobacter pylori, human endogenous retrovirus K (HERV-K), human immunodeficiency virus (HIV), human T-cell leukemia virus (HTLV), influenza virus, Lassa virus, Plasmodium parasites (e.g., those causing malaria), mumps virus (e.g., Mumps orthorubulavirus), mycoplasma bacteria, norovirus, papillomavirus (HPV), parvovirus, rhinovirus, rotavirus, rubella virus, Salmonella bacteria (e.g., Salmonella Examples of foreign antigens include T. typhi, SARS coronavirus (SARS-CoV), toxoplasma parasites (e.g., Toxoplasma gondii), treponemal bacteria (e.g., Treponema pallidum, Treponema carateum), trypanosomatid parasites (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 can result in detection of a more enhanced signal in the methods disclosed herein. Foreign antigens are known to those skilled in the art and are described, for example, in Medical Microbiology, 4 thSee 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(1):e00245-10 (2011).

[0095] T cell antigen / T cell receptor In one particular embodiment, the set of receptors is a set of T cell receptors, preferably displayed on the surface of T cells, each T cell preferably having a unique T cell receptor, and the set of ligands is a set of T cell antigens, preferably bound to major histocompatibility complexes (MHC), typically displayed on the surface of antigen-presenting cells (APCs), each APC preferably presenting multiple antigen species.

[0096] "T cell antigen" as used herein refers to CD4 + T cell antigen or CD8 + It stands for T cell antigen. + T cell antigens are CD4 + Any antigen that can be recognized by T cells and trigger an immune response, e.g., CD4 through presentation of the antigen or a portion thereof bound to a class II major histocompatibility complex molecule (MHC) + Refers to the antigen specifically recognized by the T cell receptor on T cells. + T cell antigens are CD8 + Any antigen that can be recognized by T cells and trigger an immune response, e.g., CD8 through presentation of the antigen or a portion thereof bound to a class I major histocompatibility complex molecule (MHC) + Refers to an antigen that is specifically recognized by a T cell receptor on a T cell. T cell antigens are generally proteins or peptides, but can also be other molecules such as lipids and glycolipids, and any of their derivatives.

[0097] Tetramers, multimers, and their derivatives are also envisioned, in which antigen specificity is carried by barcode or gene. Tetramers, or any derivatives thereof, can be synthesized in droplets by in vitro transcription-translation (IVTT) containing antigen and corresponding vector. Such vectors can contain genes encoding the expression of soluble TCRs.

[0098] Preferably, the set of ligands is a set of T cell antigens bound to major histocompatibility complexes (MHC) presented on the surface of antigen-presenting cells (APCs).

[0099] In the context of the present invention, the term "antigen-presenting cell" or "APC" encompasses a heterogeneous group of immunocompetent cells that mediate cellular immune responses by processing and presenting antigens to T cells. Antigen-presenting cells include, but are not limited to, macrophages, dendritic cells, Langerhans cells, B cells, monocyte-derived dendritic cells, artificial APCs, modified APCs, or other cells that express MHC class I or MHC class II molecules.

[0100] The APC can be a B cell, and in particular an immortalized B cell, such as an Epstein-Barr virus (EBV)-immortalized B cell.

[0101] In one specific embodiment, the APCs are autologous immortalized B cells from the subject of interest, as defined above, or xenogeneic immortalized B cells bearing the same MHC as the subject of interest, as defined above.

[0102] "Xenogeneic B cells bearing the same MHC as the target subject," as used herein, refers to B cells that are not derived from the target subject but that bear the same MHC as the target subject.

[0103] "MHC" or "major histocompatibility complex," as used herein, refers to the complex of genes (and molecules encoded thereby) that encode cell surface molecules necessary for antigen presentation to T cells and rapid graft rejection. In humans, the MHC complex is also known as the HLA complex. The proteins encoded by the MHC complex are known as "MHC molecules" and are classified into class I and class II MHC molecules. Class I MHC molecules comprise a membrane heterodimeric protein consisting of an α chain encoded in the MHC noncovalently bound to β2-microglobulin. Class I MHC molecules are expressed by nearly all nucleated cells and are expressed by CD8 + It has been shown to function in antigen presentation to T cells. In humans, class I molecules include HLA-A, -B, and -C. Class II MHC molecules also contain membrane heterodimeric proteins consisting of non-covalently linked α and β chains. Class II MHC is expressed by CD4 + It is known that MHCs interact with T cells, and in humans, they include HLA-DP, -DQ, and DR. The term "MHC restriction" refers to the characteristic of T cells that can only recognize antigens after the processed antigenic peptide is presented in association with either class I or class II MHC molecules. Methods for identifying and comparing MHCs are well known in the art and are described in Allen et al. (1994) Human Imm. 40:25-32; or Santamaria et al. (1993) Human Imm. 37:39-50.

[0104] In a particular embodiment, each APC is directed to at least one T cell antigen from the set of T cell antigens, preferably a plurality of distinct T cell antigens, preferably 2-1000, 5-900, 10-800, 20-700, 30-600, 40-500, 50-400 distinct T cell antigens from the set of T cell antigens, particularly 60, 70, 80, 90, 100, 110, 120, 140, 150, 160, 170, 180, 190, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 400, 410, 420, 430, 440, 450, 460, 470, 480, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910 In one specific embodiment, each APC expresses or presents 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 distinct T cell antigens. In one specific embodiment, each APC expresses or presents 10 to 1000, more preferably 300, distinct T cell antigen types.

[0105] In a particular embodiment, the set of APCs presenting at least one T cell antigen, in particular a plurality of distinct T cell antigens, is obtained by introducing a library of nucleic acids encoding T cell antigens obtained from a tissue of the subject as defined above into autologous APCs from the subject as defined above or into xenogeneic APCs bearing the same MHC as the subject.

[0106] In another particular embodiment, a set of APCs presenting at least one T cell antigen, in particular a plurality of distinct T cell antigens, is obtained by introducing into the APCs a library of synthetic mRNAs encoding the antigens (either as tandem genes or single genes), said mRNAs being identified by sequencing the tumor genome, exome, or transcriptome and produced by in vitro transcription.

[0107] In another particular embodiment, a set of APCs presenting at least one T cell antigen, in particular multiple distinct T cell antigens, is obtained by introducing into the APCs a library of synthetic mRNAs encoding the antigens (either as tandem genes or single genes), said mRNAs being identified by sequencing the tumor genome, exome, or transcriptome, and produced by splitting and pooling the individual mRNAs.

[0108] In another specific embodiment, a set of APCs presenting at least one T cell antigen, particularly multiple distinct T cell antigens, is obtained by introducing into the APCs a library of synthetic mRNAs encoding the antigens (either as tandem genes or single genes), said mRNAs being identified by sequencing the tumor genome, exome, or transcriptome and produced using permeability-based DNA delivery that allows mRNA translation.

[0109] In another particular embodiment, a set of APCs presenting at least one T cell antigen, in particular a plurality of distinct T cell antigens, is obtained by introducing into the APCs individual DNAs produced on beads and transcribed into mRNA, transfecting them in droplets (either as tandem genes or single genes encoding the antigens, said antigens being identified by sequencing the genome, exome or transcriptome of the tumor).

[0110] In another particular embodiment, a set of APCs presenting at least one T cell antigen, in particular a plurality of distinct T cell antigens, is obtained by introducing into the APCs individual DNAs produced on beads and transcribed into mRNA, and transfecting or transducing into the APCs (either as tandem genes or single genes encoding the antigens, said antigens being identified by sequencing the genome, exome, or transcriptome of the tumor).

[0111] In another specific embodiment, the set of APCs presenting at least one T cell antigen, in particular a plurality of distinct T cell antigens, is obtained by introducing known tagged T cell antigens and / or known tagged nucleic acids encoding the T cell antigens into the APCs.

[0112] "Tagged" as used herein means carrying a tag, such as a nucleic acid of known sequence, a fluorescent dye, or a label. Typically, the tag can be a barcode sequence as defined above.

[0113] In one specific embodiment, a library of nucleic acids encoding T cell antigens is chemically synthesized based on sequencing of patient or pathogen RNA or DNA.

[0114] In another specific embodiment, the library of nucleic acids encoding T cell antigens is obtained by amplification (eg, by PCR) of nucleic acids from tissue of interest as defined above.

[0115] In one specific embodiment, the library of nucleic acids encoding T cell antigens is a cDNA library, hi another embodiment, the library of nucleic acids encoding T cell antigens is an mRNA library.

[0116] A library of nucleic acids encoding T cell antigens obtained from a tissue of interest can be obtained by methods well known to those skilled in the art. In particular, cells, such as MHC-I or MHC-II-expressing cells or tumor cells, can be extracted from the tissue of interest using known techniques, such as DNase, proteases (e.g., collagenase), and mechanical digestion. RNA can be isolated from the cells using techniques well known to those skilled in the art, such as silica columns or acid phenol techniques. These RNAs can then be reverse transcribed using well-known techniques to obtain a cDNA library. Preferably, the cDNA library is obtained by reverse transcription using primers that hybridize to the RNA. Primers can prime against all mRNAs by hybridizing to the poly(A) tail (anchor oligo-dT primers), or they can be designed to prime only against a specific subset of RNAs, or they can be designed to prime randomly against any RNA.

[0117] In one particular embodiment, the library is normalized to reduce bias within the library due to differences in mRNA concentrations.

[0118] A range of normalization techniques are well known to those skilled in the art, such as double-strand-specific nuclease (DSN)-based cDNA library normalization (Bogdanov et al. (2010). Curr. Protoc. Mol. Biol. Chapter 5: Unit 5.12.1-27) and cDNA library normalization by mRNA-cDNA hybridization and subtraction (Chen (2003) In S.-Y. Ying (Ed.), Generation of cDNA Libraries: Methods and Protocols (pp. 33-40) Totowa, NJ: Humana Press).

[0119] In one particular embodiment, the library of nucleic acids encoding T cell antigens comprises universal sequences, as defined below, that allow for specific amplification and sequencing in subsequent steps of the identification method of the invention.

[0120] The term "universal sequence" as used herein refers to a sequence that can be attached to a nucleic acid sequence, particularly in a library of nucleic acid molecules, by, for example, ligation or any other suitable method (such as overlap extension PCR, PCR, primer extension, or direct DNA synthesis), thereby attaching the same sequence to multiple different nucleic acid molecules. Such universal sequences are particularly useful for simultaneously analyzing multiple samples. Examples of universal sequences are universal primers and universal priming sites. Universal priming sites include a "common priming site" to which an appropriate primer can bind and which can be used as a priming site for synthesizing a nucleic acid sequence complementary to the nucleic acid sequence attached to the universal primer.

[0121] Introduction of the library of nucleic acids encoding T cell antigens into APCs can be carried out by any method familiar to those skilled in the art, for example, by transduction with viral vectors, by electroporation, or by transfection (e.g., lipo-, nucleo-, nanoparticle-based, or using cell-penetrating peptides).

[0122] In a particular embodiment, the introduction of a library of nucleic acids encoding T cell antigens, in particular a cDNA library of nucleic acids encoding T cell antigens, into the APC is carried out by transducing the APC with a viral vector carrying the library.

[0123] As used herein, the term "viral vector" refers to a virus, or a recombinant thereof, that can encapsulate desired genetic material and transfect and integrate the desired genetic material into target cells, thereby enabling effective and targeted delivery of genetic material both ex vivo and in vivo. Examples of viral vectors include adenovirus vectors, adeno-associated virus vectors, herpes simplex virus vectors, retrovirus vectors, lentivirus vectors, Semliki Forest virus vectors, Sindbis virus vectors, vaccinia virus vectors, fowlpox virus vectors, baculovirus vectors, and Sendai virus vectors. Preferably, the viral vector is a lentivirus vector.

[0124] As will be understood by those skilled in the art, when nucleic acids encoding T cell antigens are introduced into APCs by transduction with a viral vector, the number of distinct T cell antigens expressed or presented by the APCs depends on the multiplicity of infection (MOI) of the viral vector used to transduce the APCs.

[0125] Thus, in a specific embodiment, the library of nucleic acids encoding T cell antigens is introduced into the APCs at a multiplicity of infection of 0.01 to 1000, 0.05 to 900, 0.1 to 800, 0.5 to 700, 1 to 600, 2 to 500, 3 to 450, 4 to 400, 5 to 350, 6 to 300, 7 to 250, 8 to 200, 9 to 150, or 10 to 100, specifically 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 , 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500, thereby generating APCs that express at least one antigen and up to multiple antigens.

[0126] In another embodiment, a library of nucleic acids encoding T cell antigens, particularly an RNA library of nucleic acids encoding T cell antigens, is introduced into the APCs by transfection of the APCs with the mRNA or corresponding DNA. Such transfection can typically be performed by lipofection, nucleofection, nanoparticle-based transfection, or using a cell-penetrating peptide covalently linked to the RNA (Radis-Baptista et al. (2017) Journal of Biotechnology 252:15-26), such as penetratin. The same principle applies to the expression of TCRs by autologous or heterologous T cells or T cell lines to screen for TCR specificity and / or affinity / binding to antigen / MHC complexes.

[0127] The term "T cell receptor" or "TCR," as used herein, refers to an antigen-recognition molecule present on the surface of a T cell (i.e., a T lymphocyte). This definition explicitly includes understanding of the term as known in the art, including, for example, receptors comprising or consisting of a disulfide-linked heterodimer of highly variable α or β chains expressed in the cell membrane as a complex with an invariant CD3 chain, or receptors comprising or consisting of variable γ and δ chains expressed in the cell membrane as a complex with CD3 on a subset of T cells. The antigen-recognition domain of a TCR typically consists of α and β chains, or γ and δ chains, encoded by separate genes.

[0128] T cell receptor genes undergo a unique mechanism of genetic recombination, called V(D)J recombination, which occurs only in developing lymphocytes during early T cell maturation, resulting in the highly diverse repertoire of T cell receptors (TCRs) found on T cells.

[0129] Preferably, the set of receptors is a set of TCRs displayed on the surface of a T cell.

[0130] The term "T cell" or "T lymphocyte" as generally used herein refers to a T cell, e.g., a CD4 + Helper T cells (e.g., TH1, TH2, TH9 and TH17 cells), CD8 + Cytotoxic T cells, antigen-experienced T cells, naive T cells, central T cells, effector T cells, CD4 + It may refer to regulatory / suppressive T cells (Treg cells), natural killer T cells, γδ T cells, and / or autologous invasive T cells (e.g., TH40 cells), mucosal-associated invariant T cells (MAIT), exhausted T cells, memory T cells, central memory T cells, effector memory T cells, tissue-resident T cells.

[0131] Preferably, in a set of TCR-presenting T cells, each T cell expresses or presents a unique T cell receptor (TCR).

[0132] In a particular embodiment, the T cells of the set of TCR-presenting T cells are derived from the same target as the library of nucleic acids encoding T cell antigens as defined above used to obtain the set of T cell antigen-presenting APCs. In particular, this embodiment facilitates the detection of private T cell antigens.

[0133] By "private antigen" herein is meant an antigenic specificity that is restricted to one or a few individuals.

[0134] In another specific embodiment, the T cells of the set of TCR-presenting T cells are not derived from the same target as the library of nucleic acids encoding T cell antigens as defined above used to obtain the set of T cell antigen-presenting APCs. In particular, this embodiment facilitates the detection of public T cell antigens.

[0135] By "public antigen" herein is meant an antigen that is present in more than 5%, more specifically more than 10% of the population.

[0136] In one specific embodiment, the T cells of the set of T cells presenting a TCR are activated to allow for proliferation after collection from the subject.

[0137] B cell receptor / B cell antigen In another specific embodiment, the set of receptors is a set of B cell receptors (BCRs or antibodies) and the set of ligands is a set of B cell antigens.

[0138] As used herein, the term "BCR" refers to a transmembrane receptor protein located on the outer surface of B cells. The binding portion of the receptor consists of a membrane-bound antibody with a unique and randomly determined antigen-binding site, like most antibodies (see V(D)J recombination). When a B cell is activated by first encountering an antigen that binds to its receptor (its "cognate antigen"), the cell proliferates and differentiates to produce a population of antibody-secreting plasma B cells and memory B cells.

[0139] The term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules containing an antigen-binding site that immunospecifically binds to an antigen. Thus, the term "antibody" encompasses not only whole antibody molecules but also antibody fragments and antibody variants, 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. Two types of light chains exist: lambda (λ) and kappa (κ). There are five major heavy chain classes (or isotypes) that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, IgE, and IgY. Each chain contains a distinct constant region sequence. The light chain contains two domains: a variable domain (VL) and a constant domain (CL). The heavy chain contains four domains: a variable domain (VH) and three constant domains (CH1, CH2, and CH3, collectively referred to as CH). The variable regions of both the light (VL) and heavy (VH) chains determine antigen binding recognition and specificity. The constant region domains of the light (CL) and heavy (CH) chains confer important biological properties, such as antibody chain association, secretion, placental transport, complement binding, and Fc receptor (FcR) binding. The Fv fragment is the N-terminal portion of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain. Antibody specificity resides in the structural complementarity between the antibody-combining site and antigenic determinants. The antibody-combining site is primarily composed of residues from hypervariable or complementarity-determining regions (CDRs). Occasionally, residues from non-hypervariable or framework regions (FRs) influence the overall domain structure and thus the binding site. The complementarity-determining regions (CDRs) together refer to the amino acid sequences that 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, and LCDR3, and HCDR1, HCDR2, and HCDR3, respectively. Thus, an antigen-binding site contains six CDRs, including the CDR sets from each of the heavy and light chain V regions.Framework region (FR) refers to the amino acid sequences located between the CDRs, i.e., the 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)).

[0140] The term "antibody" also refers to single chain antibodies, such as camelid antibodies, or nanobodies or V HH Represents.

[0141] Antibody genes generally undergo a unique mechanism of genetic recombination called V(D)J recombination, which occurs only in developing lymphocytes during early B-cell maturation. Antibody genes can also undergo 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.

[0142] In one particular embodiment, when the set of receptors is a set of B cell receptors, said B cell receptors are presented by B cells.

[0143] Microreactors and co-compartmentalization By "co-compartmentalizing" ligand and receptor species, we mean herein forming a plurality of microreactors, each microreactor separating a group of ligand and / or receptor species, preferably at least one ligand species and optionally at least one receptor species, from the remaining ligand and receptor species provided by the set of ligands and receptors.

[0144] In the context of the present invention, the co-compartmentalization / microreactor step may be performed by any suitable method, for example, by microfluidics, flow cytometry cell-based sorting, and / or limiting dilution.

[0145] In one particular embodiment, the microreactor is a well or a microfabricated well.

[0146] In another particular embodiment, the microreactor is an aqueous droplet, particularly in a continuous immiscible phase.

[0147] "Droplet" generally refers to a measure of volume and, in the context of the present invention, to a separated portion of a first fluid surrounded by a second fluid. It should be noted that droplets are not necessarily spherical, but may assume other shapes, for example depending on the external environment.

[0148] Preferably, each droplet has a volume at least equal to the volume of two mammalian cells.

[0149] In another specific embodiment, the microreactor is a microcapsule. Microcapsules can refer to a measure of volume, and in the context of the present invention, refer to a separate portion of a first coating material surrounded by a second material. It should be noted that microcapsules are not necessarily spherical, but other shapes can be assumed, for example, depending on the external environment. A "microcapsule" generally refers to a hollow microparticle consisting of a solid shell surrounding a space that forms a core that is accessible to the permanently or temporarily encapsulated substance. The substance can be a drug, pesticide, pigment, cell, combination thereof, or similar material. The solid shell can encapsulate a solid, liquid, or gas within its micrometer-sized walls, made, for example, of a hard or soft dissolvable film. Commonly used coating materials for coating include ethyl cellulose, polyvinyl alcohol, gelatin, and sodium alginate.

[0150] Preferably, each microcapsule has a volume at least equal to the volume of two mammalian cells.

[0151] In another specific embodiment, the microreactor is a microbead. Microbeads can refer to a measure of volume and further refer to a separated portion of a first semi-solid material surrounded by a fluid that may or may not be permeable to the semi-solid bead. It should be noted that microbeads are not necessarily spherical, but other shapes can be assumed, for example, depending on the external environment. "Microbead" generally refers to a semi-solid, porous or non-porous structure that occupies the entire volume available for a permanently or temporarily encapsulated substance. The substance can be a drug, pesticide, dye, cell, combination thereof, and similar materials. The semi-solid porous structure can encapsulate a solid, liquid, or gas within micrometer walls made, for example, of a hard or soft dissolvable film. Materials commonly used to form microbeads include polymers such as agarose, acrylamide, and sodium alginate.

[0152] Preferably, each microbead has a volume at least equal to the volume of two mammalian cells.

[0153] It will be appreciated that after encapsulating the cells within the microcapsules or microbeads, the cells can effect transformation of the droplets within the microcapsules or microbeads.

[0154] The average volume of a mammalian cell is well known to those skilled in the art and is typically about 0.002 nL.

[0155] In a particular embodiment, each droplet or microcapsule or microbead has a volume of less than 20 nL. In one embodiment, each droplet has a volume of less than 15 nL, less than 10 nL, less than 9 nL, less than 8 nL, less than 7 nL, less than 6 nL, less than 5 nL, less than 3 nL, less than 2.5 nL, less than 2 nL, less than 1.5 nL, less than 1 nL, less than 0.5 nL, less than 0.2 nL, less than 0.1 nL, less than 0.05 nL, for example, 20 pL to 3 nL, 30 pL to 1 nL, 40 pL to 5 nL, 50 pL to 6 nL, 60 pL to 8 nL, 70 pL to 9 nL, 80 pL to 1 nL, 90 pL to 1 nL, 100 pL to 1 nL, 120 pL to 1 nL, 140 pL to 1 nL, 150 pL to 1 nL, 160 pL to 1 nL, 170 pL to 1 nL, 180 pL to 1 nL, 190 pL to 2 nL, 20 pL to 3 nL, 20 pL to 1 nL, 250 pL to 2 nL, 260 pL to 1 nL, 270 pL to 2 nL, 280 pL to 3 nL, 290 pL to 3 nL, 30 pL to 1 nL, 30 pL to 1 nL, 40 pL to 5 nL, 450 pL to 5 nL, 50 pL to 1 nL, 550 pL to 1 nL, 550 pL to 1 The volume may range from 500 pL, 50 pL, 250 pL, 60 pL to 100 pL, or from 0.1 nL to 3 nL, 0.5 nL to 3 nL, or 1 nL to 3 nL, typically 0.1 nL, 0.5 nL, 1 nL, 1.2 nL, 1.4 nL, 1.6 nL, 1.8 nL, 2.0 nL, 2.2 nL, 2.4 nL, 2.6 nL, 2.8 nL, or 3 nL.

[0156] Such droplets or microcapsules or microbeads may be prepared by any technique well known to those skilled in the art, in particular by microfluidic techniques.

[0157] As will be understood by those skilled in the art, and as further explained below, the number of ligands and receptors, specifically the number of APCs presenting T cell antigens and T cells presenting TCRs, that are co-compartmentalized within one microreactor, e.g., droplet, follows a probability distribution, e.g., a Poisson distribution, and depends, for example, on the concentration of the first type of cells in the first fluid, the concentration of the second type of cells in the second fluid, the geometry of the main and side channels, and the injection parameters of the first, second, and carrier fluids used.

[0158] When ligands and / or receptors are presented on cells or particles, the distribution of the cells or particles, and therefore also the distribution of the ligands and / or receptors, typically follows a Poisson distribution. However, when the microreactors are droplets, various microfluidic techniques familiar to those skilled in the art allow for distributions other than Poisson distributions, particularly distributions in which a larger fraction of the droplets contain single cells / particles. These techniques consist of arranging particles / cells using inertial forces before compartmentalization into droplets, mediated by secondary flows such as Dean flow (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, US2013 / 011210, US2010 / 021984, and US2011 / 0223314), methods consisting of isolating / sorting droplets containing single cells / particles or cell / particle pairs 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 of flowing cells / particles through narrow bottlenecks to reduce the possibility of trapping multiple cells / particles of the same sample (Ramji et al. (2014) Biomicrofluidics 8:034104), and methods consisting of the production of droplets on demand as cells / particles pass in front of a nozzle (see Schoendube et al. (2015) Biomicrofluidics 9:014117; Leibacher et al. (2015) Biomicrofluidics 9:024109 and Yusof et al. (2011) Lab.Chip11:2447-2454).

[0159] Thus, in a particular embodiment, multiple receptor species, specifically multiple receptor species of TCR-presenting T cells contained in an aqueous composition, are co-compartmentalized with multiple ligands, specifically multiple ligands of antigen-presenting APCs, in multiple microreactors, specifically multiple microfluidic droplets, and the number of receptor species, specifically the number of TCR-presenting T cells, co-compartmentalized in a single microreactor, specifically a single droplet, follows a probability distribution, specifically a Poisson distribution, depending on the parameters used. The parameters can, for example, be adapted so that most microreactors have one or zero receptors, specifically TCR-presenting T cells, thus minimizing the number of compartments containing few receptors.

[0160] The parameters used to co-compartmentalize a receptor species, particularly a TCR-presenting T cell, with a ligand species, particularly an antigen-presenting APC, can be adapted so that at least some microreactors contain a single receptor species, particularly a single TCR-presenting T cell.

[0161] Similarly, the parameters used to co-compartmentalize a receptor species, particularly a TCR-presenting T cell, with a ligand species, particularly an antigen-presenting APC, can be adapted so that at least some microreactors contain a single ligand species, particularly a single antigen-presenting APC.

[0162] In a particularly preferred embodiment, a set of microreactors is produced, each containing at least one ligand species, particularly at least one T cell antigen, more particularly at least one T cell antigen-presenting APC, and at least one receptor species, particularly at least one TCR, more particularly at least one TCR-presenting T cell.

[0163] However, as will be appreciated by those skilled in the art, some microreactors may be fabricated that do not contain any receptor species.

[0164] Preferably, a set of microreactors is produced, each containing at least one ligand species, particularly at least one T cell antigen, more particularly at least one T cell antigen-presenting APC, and one or a few receptor species, particularly one or a few TCRs, more particularly one or a few TCR-presenting T cells.

[0165] By "few" herein is meant less than 10, for example 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1.

[0166] In another preferred embodiment, a set of microreactors is produced, each containing one or a small number of ligand species, in particular one or a small number of T cell antigens, more in particular one or a small number of T cell antigen-presenting APCs, and at least one receptor species, in particular at least one TCR, more in particular at least one TCR-presenting T cell.

[0167] In a further preferred embodiment, a set of microreactors is produced, each containing one or a small number of ligand species, particularly one or a small number of T cell antigens, more particularly one or a small number of T cell antigen-presenting APCs, and one or a small number of receptor species, particularly one or a small number of TCRs, more particularly one or a small number of TCR-presenting T cells.

[0168] In a preferred embodiment, a set of microreactors is produced, each containing one ligand type, specifically one T cell antigen, more specifically one T cell antigen-presenting APC, and one receptor type, specifically one TCR, more specifically one TCR-presenting T cell.

[0169] In a particularly preferred embodiment, a set of microreactors is produced, each containing one APC presenting multiple ligand species, specifically multiple T cell antigens, more specifically multiple T cell antigens, or multiple APCs presenting a single (or multiple) T cell antigens, and one receptor species, specifically one TCR, more specifically one TCR-presenting T cell.

[0170] Thus, in one embodiment, the set of microreactors has a flow rate of 1 to 20,000 microreactors / second, e.g., 1 to 15,000 microreactors / second, 1 to 10,000 microreactors / second, 1 to 9000 microreactors / second, 1 to 8000 microreactors / second, 1 to 7000 microreactors / second, 1 to 6000 microreactors / second, 1 to 5000 microreactors / second, 1 to 4000 microreactors / second, 1 to 3000 microreactors / second, 1 to 2000 microreactors / second, 1 to 1000 microreactors / second, 1 to 800 microreactors / second, 1 to 700 microreactors / second, 1 to 600 microreactors / second, 1 to 500 microreactors / second, 1 to 400 microreactors / second, 1 to 5000 microreactors / second, 1 to 6000 microreactors / second, 1 to 700 microreactors / second, 1 to 800 microreactors / second, 1 to 700 microreactors / second, 1 to 600 microreactors / second, 1 to 500 microreactors / second, 1 to 400 microreactors / second, 1 to 500 microreactors / second, 1 to 600 microreactors / second, 1 to 700 microreactors / second, 1 to 800 microreactors / second, 1 to 800 microreactors / second, 1 to 900 microreactors / second, 1 to 1000 microreactors / second, 1 to 1000 microreactors / second, 1 to The receptor species, specifically antigen-presenting APCs, are formed by co-compartmentalization of a ligand species, specifically antigen-presenting APCs, and a receptor species, specifically TCR-presenting T cells, at a frequency of 1 to 300 microreactors / second, 1 to 200 microreactors / second, 1 to 100 microreactors / second, 1 to 80 microreactors / second, 1 to 70 microreactors / second, 1 to 50 microreactors / second, for example 10 to 300 microreactors / second, 50 to 300 microreactors / second, 100 to 300 microreactors / second, 150 to 300 microreactors / second, 150 to 250 microreactors / second, 175 to 250 microreactors / second, typically 1 to 1000 microreactors / second, preferably 175 to 250 microreactors / second.

[0171] The set of microreactors, specifically the set of microfluidic droplets, can be obtained by any suitable technique. Specifically, the set of microreactors can be obtained by the following steps: (a) providing a first fluid source, where the first fluid comprises a suspension of the set of receptors as defined above; (b) providing a second fluid source, where the second fluid comprises a suspension of the set of ligands as defined above; (c) providing a carrier fluid, where the carrier fluid is immiscible with the first and second fluids; (d) injecting the carrier fluid into a main channel of the chip; and (e) injecting the second fluid and the first fluid in at least a minor channel of the chip, thereby forming the microreactors, specifically the droplets, in the carrier fluid. The microreactors, specifically droplets, can be formed by a flow generating step, wherein the side channels open into the main channel, and each produced microreactor, specifically droplets, contains a mixture of a first fluid and a second fluid, wherein the concentration of the receptor in the first fluid, the concentration of the ligand in the second fluid, the geometry of the main channel and side channels, the injection parameters of the first fluid, the second fluid and the carrier fluid are adapted to provide a volume of preferably less than 20 nL, wherein each microreactor, specifically droplets, contains at least one receptor species (preferably only a single receptor species) and at least one ligand species.

[0172] Alternatively, a set of microreactors may be formed by (a) providing a first fluid source, where the first fluid comprises a suspension of a preformed set of receptors and ligands as defined above; (b) optionally providing a second fluid source, where the second fluid comprises reagents for detection as defined above; and (c) providing a carrier fluid, where the carrier fluid is immiscible with the first and second fluids.

[0173] In one embodiment, the first fluid source and the second fluid source are arranged in the form of a junction.

[0174] The junction can be, for example, a T-junction, a Y-junction, a channel-in-channel junction (e.g., a coaxial arrangement, or including an inner channel and an outer channel surrounding at least a portion of the inner channel), a cross (or "X") junction, a flow-focusing 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" (Link, et al., published October 28, 2004 as WO2004 / 091763), or International Patent Application No. PCT / US2003 / 020542, filed June 30, 2003, entitled "Method and Apparatus for Fluid Dispersion" (Stone, et al., published January 8, 2004 as WO2004 / 002627).

[0175] In some embodiments, the junction may be constructed and arranged to produce substantially monodisperse droplets.

[0176] The amount of receptor species / droplet can also be referred to as the loading rate. For example, the average loading rate can be less than about 1 receptor species / droplet, less than about 0.9 receptor species / droplet, less than about 0.8 receptor species / droplet, less than about 0.7 receptor species / droplet, less than about 0.6 receptor species / droplet, less than about 0.5 receptor species / droplet, less than about 0.4 receptor species / droplet, less than about 0.3 receptor species / droplet, less than about 0.2 receptor species / droplet, less than about 0.1 receptor species / droplet, less than about 0.05 receptor species / droplet, less than about 0.03 receptor species / droplet, less than about 0.02 receptor species / droplet, or less than about 0.01 receptor species / droplet. In some cases, a loading rate of fewer receptor species can be selected to minimize the possibility of producing droplets having more than one receptor species therein. Thus, for example, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% of the droplets may contain no receptor species or only one receptor species.

[0177] At least some of the microreactors, in the context of the present invention, may further comprise a reverse transcriptase and a barcoded primer as further defined herein below.

[0178] Recognition assays and classification "Recognition" as used herein preferably refers to binding between a ligand species and a receptor species, which induces a specific response by the ligand species and / or the receptor species.

[0179] As will be understood by those skilled in the art, the response induced by the recognition between a ligand species and a receptor species depends on the particular ligand and receptor considered. For example, the recognition between a T cell antigen and its corresponding TCR presented by a T cell induces the activation of the T cell. Similarly, the recognition between a B cell antigen and its corresponding B cell receptor presented by a B cell induces the activation of the B cell.

[0180] Thus, the assay used to determine recognition between a ligand and receptor species will depend on the particular ligand and receptor under consideration.

[0181] In one particular embodiment, when receptor species of a set of receptors are presented by cells, recognition between the ligand and receptor in each microreactor is assayed by determining whether a cellular response is induced in said microreactor; if an induced cellular response is determined in said microreactor, the microreactor is classified as positive (recognition between the ligand species and receptor species in the microreactor), and if no induced cellular response is determined in said microreactor, the microreactor is classified as negative (no recognition between the ligand species and receptor species in the microreactor).

[0182] More specifically, when the set of receptors are TCR-presenting T cells and the set of ligands are T cell antigen-presenting APCs, recognition between the ligands and receptors is assayed by determining whether T cell activation is induced in the microreactor; if induced T cell activation is determined in the microreactor, the microreactor is classified as positive (recognition between the ligand species and receptor species in the microreactor), and if induced T cell activation is not determined in the microreactor, the microreactor is classified as negative (no recognition between the ligand species and receptor species in the microreactor).

[0183] "T cell activation" as used herein refers to the regulated series of events induced by the recognition of an antigen, whatever its chemical nature, by a TCR, resulting in the activation, differentiation, proliferation, and acquisition of T cell immune functions by TCR-presenting T cells. As is well known to those skilled in the art, the signaling cascade initiated by TCR activation is the activation of the inositol triphosphate / Ca2+ receptor. 2+ These pathways include the diacylglycerol / protein kinase C, Ras / mitogen-activated protein kinase, and PI3-K pathways. Components of these pathways transmit signals to the nucleus to activate genes encoding various secreted factors such as IL-2, IL-4, IL-7, IL-9, IL-10, TNF-α, and interferon-γ, activate genes encoding various cell surface-expressed activation markers such as CD137, CD40L, and CD69, and induce the caspase 3 / 7 pathway, which is associated with the proliferation, maturation, and function of cellular components of the immune system.

[0184] In certain embodiments, the step of contacting the set of ligand species with the set of receptor species in the microreactor occurs over a short incubation time, e.g., from about 0.001 hours to about 8 hours. In certain embodiments, the contacting step may be from about 0.001 hours to about 8 hours, from about 0.001 hours to about 7 hours, from about 0.001 hours to about 6 hours, from about 0.001 hours to about 5 hours, from about 0.001 hours to about 4 hours, from about 0.001 hours to about 3 hours, from about 0.001 hours to about 2 hours, from about 0.001 hours to about 1 hour, from about 0.01 hours to about 8 hours, from about 0.01 hours to about 7 hours, from about 0.01 hours to about 6 hours, from about 0.01 hours to about 5 hours, from about 0.01 hours to about 4 hours, or from about 0.01 hours to about 1 hour. The reaction time may be from about 0.1 to about 3 hours, from about 0.01 to about 2 hours, from about 0.01 to about 1 hour, from about 0.1 to about 8 hours, from about 0.1 to about 7 hours, from about 0.1 to about 6 hours, from about 0.1 to about 5 hours, from about 0.1 to about 4 hours, from about 0.1 to about 3 hours, from about 0.1 to about 2 hours, from about 0.1 to about 1 hour, from about 1 to about 8 hours, from about 1 to about 7 hours, from about 1 to about 6 hours, from about 1 to about 5 hours, from about 1 to about 4 hours, from about 1 to about 3 hours, or from about 1 to about 2 hours.

[0185] In certain embodiments, the step of contacting the set of ligand species with the set of receptor species in the microreactor occurs over a long incubation time, e.g., at least about 8 hours, preferably about 8 hours to about 48 hours. In certain embodiments, the contacting occurs for about 8 hours to about 48 hours, about 8 hours to about 40 hours, about 8 hours to about 32 hours, about 8 hours to about 24 hours, about 8 hours to about 16 hours, about 16 hours to about 48 hours, about 16 hours to about 40 hours, about 16 hours to about 32 hours, about 16 hours to about 24 hours, about 24 hours to about 48 hours, about 24 hours to about 40 hours, about 24 hours to about 32 hours, about 32 hours to about 48 hours, about 32 hours to about 40 hours, or about 40 hours to about 48 hours.

[0186] In certain embodiments, when the contacting step involves a shorter incubation time (e.g., about 0.001 hour to about 8 hours) and the ligand and receptor species bind with high affinity, the enhanced signal produced is an early or late marker of T cell activation. When the ligand and receptor species have high affinity for each other, selective or specific binding can occur immediately, thereby resulting in immediate activation of the T cell (e.g., when the ligand species is a T cell antigen and the receptor species is a T cell receptor). Immediate activation of T cells can result in the immediate expression of early markers, which may include, but are not limited to, CD69, CD107a, and transferrin receptor. Immediate activation of T cells can also result in the immediate expression of continuously expressed markers, which may include, but are not limited to, IFNγ, CD25, CD154, TNF, IL-10, IL-2, and IL-1b. Immediate activation of T cells can also result in the expression of late markers during shorter incubation times, which may include, but are not limited to, CD137, HLA-DR, VLA1, PTA1, CD71, CD27, PD-1, TIM3, LAG3, or CTLA4.

[0187] In certain embodiments, when the contacting step involves a longer incubation time (e.g., at least about 8 hours, preferably about 8 to about 48 hours) and the ligand and receptor species bind with high affinity, the enhanced signal produced can be an early or late marker of T cell activation. Because of the longer incubation time, immediate activation of the T cells can result in the production of either the aforementioned early or late markers. Furthermore, because of the longer incubation time, continuously expressed markers can also be produced.

[0188] In certain embodiments, if the contacting step involves a longer incubation time (e.g., at least about 8 hours, preferably about 8 to about 48 hours) and the ligand and receptor species bind with low affinity, the enhanced signal produced is an early marker of T cell activation. With lower affinity, longer incubation times may be required to activate T cells, and thus, depending on the incubation time, either early or late markers of T cell activation may be observed. By extending the contacting step for ligand and receptor species with low affinity, the enhanced signal produced may ultimately be a late marker of T cell activation.

[0189] In certain embodiments, the early marker of T cell activation may be, but is not limited to, CD69, CD107a, or transferrin receptor.

[0190] In certain embodiments, the continuously expressed markers of T cell activation can be, but are not limited to, IFNγ, CD25, CD154, TNF, IL-10, IL-2, and IL-1b.

[0191] In certain embodiments, the late marker of T cell activation may be, but is not limited to, CD137, HLA-DR, VLA1, PTA1, CD71, CD27, PD-1, TIM3, LAG3, or CTLA4.

[0192] In certain embodiments, the signal is detected by an anti-CD69 antibody, an anti-CD107a antibody, an anti-transferrin receptor antibody, an anti-CD137 antibody, an anti-HLA-DR antibody, an anti-VLA1 antibody, an anti-PTA1 antibody, an anti-CD71 antibody, an anti-CD27 antibody, an anti-PD1 antibody, an anti-TIM3 antibody, an anti-LAG3 antibody, or an anti-CTLA4 antibody.

[0193] Assays for determining T cell activation are well known to those skilled in the art and include, inter alia, detection of upregulation of CD69, CD137, CD134 (OX40) or CD40L, detection of cytokine secretion, induction of the caspase 3 / 7 pathway, and secretion of perforin or granzymes, or detection of TNF-α, and interferon-γ.

[0194] The selective and / or specific binding of the ligand species to the receptor species may be in the range of about 1 nM to about 100 nM, about 1 nM to about 75 nM, about 1 nM to about 50 nM, about 1 nM to about 25 nM, about 10 nM to about 100 nM, about 10 nM to about 75 nM, about 10 nM to about 50 nM, about 10 nM to about 25 nM, about 20 nM to about 100 nM, about 20 nM to about 75 nM, about 20 nM to about 50 nM, about 30 nM to about 100 nM, about 30 nM to about 3 ... In a fluorescent assay using a fluorescent antibody, the antibody can be detected within the range of about 60 nM to about 100 nM, about 60 nM to about 75 nM, about 70 nM to about 100 nM, about 80 nM to about 100 nM, or about 90 nM to about 100 nM. The selective binding of the ligand species to the receptor species can be about 1 nM, about 5 nM, about 10 nM, about 15 nM, about 20 nM, about 25 nM, about 30 nM, about 35 nM, about 40 nM, about 45 nM, about 50 nM, about 55 nM, about 60 nM, about 65 nM, about 70 nM, about 75 nM, about 80 nM, about 85 nM, about 90 nM, about 95 nM, or about 100 nM.

[0195] T cell activation can also be detected by sequencing T cell mRNA using barcoded cDNA primers to detect specific mRNA expression patterns characteristic of activated T cells. These cDNA primers typically contain the same barcode sequences as the ligand-specific and receptor-specific cDNA barcoded primers in the same microreactor. Therefore, the cDNAs contain the same barcode sequences as those contained in the ligand and receptor cDNAs, allowing the ligand and receptor in positive droplets (containing activated T cells) to be identified by sequencing. The cDNA primers may also contain unique molecular identifiers (UMIs) to facilitate quantification and normalization of mRNA expression (Kivioja et al. (2012). Nat. Methods, 9, 72-74; Islam et al. (2014). Nat. Methods, 11, 163-166). The number of beads, or optionally the number of UMIs, is used to quantify and normalize mRNA expression.

[0196] Similarly, if the set of receptors are B cell receptor-presenting B cells and the set of ligands are B cell antigens, recognition between the ligands and receptors is assayed by determining whether B cell activation is induced in the microreactor; if induced B cell activation is determined in the microreactor, the microreactor is classified as positive (recognition between the ligand species and receptor species in the microreactor), and if induced B cell activation is not determined in the microreactor, the microreactor is classified as negative (no recognition between the ligand species and receptor species in the microreactor).

[0197] "B cell activation," as used herein, refers to the process or activity that causes a B cell to exhibit the phenotype of an activated B cell, and "activated B cells" refer to B cells that can exhibit some of the following phenotypes: antibody production (induction of >50-fold increased expression, up to 300-fold), surface expression versus secretion, antigen specificity, CD138 / CD38 expression, high endoplasmic reticulum network or abundance, antigen-mediated activation, T cell-dependent activation, T cell-independent activation (see Blood. 2003;102:592-600, Blood. 2002;99:2905-2912, and Blood. 2010;116(18):3445-3455), etc.

[0198] Assays for determining B cell activation are well known to those skilled in the art.

[0199] B cell activation can also be identified by sequencing B cell mRNA using barcoded cDNA primers to detect specific mRNA expression patterns characteristic of activated B cells. These cDNA primers typically contain the same barcode sequence as the ligand-specific and receptor-specific barcoded cDNA primers in the same microreactor. Thus, the cDNAs contain the same barcode sequences as those contained in the ligand and receptor cDNAs, allowing the ligand and receptor in positive droplets (containing activated B cells) to be identified by sequencing. The cDNA primers may also contain unique molecular identifiers (UMIs) to facilitate quantification of mRNA expression. The number of beads, or optionally the number of UMIs, is used to quantify mRNA expression.

[0200] In one particular embodiment, assay reagents are added to the microreactor, preferably said assay reagents are co-compartmentalized with said ligand species and said receptor species during a co-compartmentalization step.

[0201] For example, if the microreactor is a microfluidic droplet, the assay reagents may be included in the first and / or second fluids used to form the droplet, or alternatively, the assay reagents may be provided via a third fluid.

[0202] Reagents can also be coalesced by a variety of methods known to those skilled in the art, 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 localized heating with a focused laser (Baroud et al. (2007). Lab Chip 7:1029-1033), or using electrical 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. al. (2006) Appl. Phys. Lett., 89:134101), or by injecting liquid into preformed droplets using, for example, electrical forces (picoinjection) (Abate et al. (2010) Proc. Nat. Acad. Sci. USA, 107:19163-19166).

[0203] As will be appreciated by one of skill in the art, the assay reagents will depend on the particular recognition assay being performed.

[0204] In one particular embodiment, said assay reagents comprise a reporter reagent that allows direct sorting of positive microreactors by detection techniques as defined below.

[0205] Based on the results of the assays performed within each microreactor, each microreactor can be classified as positive or negative.

[0206] In one particular embodiment, the positive microreactors can be separated from the negative microreactors, thereby forming a group of positive microreactors.

[0207] The separation can be performed by any technique well known to those skilled in the art and depends on the type of microreactor used. In particular, the separation can be performed by sorting the microreactors, in particular the microfluidic droplets, for example by detecting a reporter reagent. The separation can also be performed by sorting the microreactors by flow cytometry.

[0208] In a preferred embodiment, when the microreactors are droplets, the droplets are sorted in a microfluidic device by dielectrophoresis (Ahn et al. (2006) Appl. Phys. Lett. 88:024104) or using triggered surface acoustic waves (Franke et al. (2009) Lab Chip 9:2625-2627), for example by detecting fluorescent signals within 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).

[0209] Alternatively, based on the classification of microreactors as positive or negative, a subset of positive microreactors can be demonstrated only intellectually without physically forming a group of positive microreactors.

[0210] Optional additional reagents and microreactor processing In one particular embodiment, the microreactor, in particular the positive microreactor, comprises an additional reagent.

[0211] The additional reagents typically include reverse transcriptase (RT), cell lysis buffer, deoxynucleotide triphosphates (dNTPs), and multiple barcoded primers specific for nucleic acid sequences encoding ligands or candidate ligands and multiple barcoded primers specific for nucleic acid sequences encoding receptors, as defined below.

[0212] When the ligand is a tagged ligand as defined above, the barcoded primer specific to a nucleic acid sequence encoding the ligand can be a barcoded primer specific to a nucleic acid sequence encoding a tag of said ligand. Similarly, when the receptor is a tagged receptor as defined above, the barcoded primer specific to a nucleic acid sequence encoding a receptor can be a barcoded primer specific to a nucleic acid sequence encoding a tag of said receptor.

[0213] Thus, in one particular embodiment, additional reagents are added to the microreactor, specifically to the positive microreactor, said additional reagents including at least a reverse transcriptase (RT), deoxynucleotide triphosphates (dNTPs), and a plurality of barcoded primers specific to a nucleic acid sequence encoding a ligand (or a tag of the ligand) and a plurality of barcoded primers specific to a nucleic acid sequence encoding a receptor (or a tag of the receptor), and may include a cell lysis buffer, wherein the barcoded primers specific to the nucleic acid sequence encoding the ligand (or a tag of the ligand) are a primer sequence specific to the nucleic acid sequence encoding the ligand (or a tag of the ligand) and a barcode sequence or a sequence encoding the barcode sequence. microreactors, wherein the barcoded primer specific to the nucleic acid sequence encoding the receptor (or receptor tag) comprises a primer sequence specific to the nucleic acid sequence encoding the receptor (or receptor tag) and a barcode sequence or set of barcode sequences, wherein the barcode sequence or set of barcode sequences contained within a microreactor is distinguishable from the barcode sequence or set of barcode sequences contained within other microreactors, but the barcoded primer specific to the nucleic acid sequence encoding the ligand (or ligand tag) and the nucleic acid sequence encoding the receptor (or receptor tag) contained within a given microreactor share a common barcode sequence or set of barcode sequences.

[0214] Thus, in one particular embodiment, additional reagents are added to the microreactor, specifically to the positive microreactor, and the additional reagents include at least a reverse transcriptase (RT), deoxynucleotide triphosphates (dNTPs), and a plurality of primers (which may be barcoded and serve as templates for a template switch reaction (described in US 5,962,272)), wherein either a free-floating specific primer for a nucleic acid sequence encoding a ligand (or a tag of the ligand) and a barcoded primer specific for a nucleic acid sequence encoding a receptor (or a tag of the receptor) may be added, and / or a polydT primer and / or a random primer may be added, and a cell lysis buffer may be added, wherein the primer (which may be barcoded) specific for the template switch reaction includes a primer sequence known to those skilled in the art to associate with a non-template nucleotide, typically a triple cytosine, generated during RT by the reverse transcriptase at the 3' end of the cDNA (Zajac et al. (2013) PLoS ONE 8:e85270), wherein a primer (which may be barcoded) specific for a non-template nucleotide (typically three cytosines) comprises a primer sequence specific for the non-template nucleotide and a barcode sequence or set of barcode sequences, wherein the barcode sequence or set of barcode sequences contained within a microreactor is distinguishable from the barcode sequence or set of barcode sequences contained within other microreactors, while the barcoded primers specific for the non-template nucleotide contained within a given microreactor share a common barcode sequence or set of barcode sequences.

[0215] When the receptor is a TCR, the nucleic acid sequence encoding the receptor is preferably a nucleic acid sequence encoding an αT cell receptor, βT cell receptor, γT cell receptor or δT cell receptor.

[0216] When the receptor is an antibody presenting B cell, the nucleic acid encoding the receptor is preferably a nucleic acid sequence encoding an antibody heavy chain variable domain or an antibody light chain variable domain.

[0217] If the receptor is composed of several polypeptides each encoded by a separate gene, several separate barcoded primers are preferably added, each specific to a nucleic acid sequence encoding one of the polypeptides. In other words, if the receptor is composed of n polypeptides each encoded by a separate gene, a first barcoded primer is added that comprises a primer sequence specific to a nucleic acid sequence encoding a first polypeptide of the receptor, a second barcoded primer is added that comprises a primer sequence specific to a nucleic acid sequence encoding a second polypeptide of the receptor, and so on. And an nth barcoded primer is added that comprises a primer sequence specific to a nucleic acid encoding the nth polypeptide of the receptor.

[0218] Similarly, if the ligand is composed of several polypeptides each encoded by a separate gene, several separate barcoded primers are preferably added, each specific to a nucleic acid sequence encoding one of the polypeptides. In other words, if the ligand is composed of a number n of polypeptides each encoded by a separate gene, a first barcoded primer is added that comprises a primer sequence specific to a nucleic acid sequence encoding a first polypeptide of the ligand, a second barcoded primer is added that comprises a primer sequence specific to a nucleic acid sequence encoding a second polypeptide of the ligand, and so on. Then an nth barcoded primer is added that comprises a primer sequence specific to a nucleic acid sequence encoding the nth polypeptide of the ligand.

[0219] In one particular embodiment, typically when a receptor is composed of two polypeptides, specifically two chains encoded by two separate genes, the barcoded primers specific to the nucleic acid sequence encoding the receptor are two different barcoded primers, each specific to the nucleic acid sequence encoding one of the two polypeptides, specifically the chains that make up the receptor.

[0220] Typically, when the receptor is a TCR, the barcoded primers specific to a nucleic acid sequence encoding the TCR are two different barcoded primers, where one barcoded primer is specific to a nucleic acid sequence encoding an alpha T cell receptor and the second barcoded primer is specific to a nucleic acid sequence encoding a beta T cell receptor.

[0221] Alternatively, if the receptor is a TCR, the barcoded primers specific to a nucleic acid sequence encoding a TCR are two different barcoded primers, where one barcoded primer is specific to a nucleic acid sequence encoding a gamma T cell receptor and the second barcoded primer is specific to a nucleic acid sequence encoding a delta T cell receptor.

[0222] Typically, when the receptor is a BCR and / or antibody (as defined above), the barcoded primers specific to a nucleic acid sequence encoding a BCR and / or antibody (as defined above) are two different barcoded primers, where one barcoded primer is specific to a nucleic acid sequence encoding a lambda or kappa chain of the BCR and / or antibody, and the second barcoded primer is specific to a nucleic acid sequence encoding a gamma, delta, epsilon, alpha, or mu chain of the BCR and / or antibody.

[0223] In the context of the present invention, a "nucleic acid sequence encoding" a ligand (or ligand tag) or receptor (or receptor tag) can be any type of nucleic acid as defined in the section "Definitions" above. In particular, it can be a DNA molecule or an RNA molecule. In one particular embodiment, said nucleic acid sequence encoding a ligand (or ligand tag) or receptor (or receptor tag) consists of or comprises an mRNA sequence encoding said ligand (or said ligand tag) or said receptor (or said receptor tag), a fragment thereof, or a complementary sequence thereof. In another particular embodiment, said nucleic acid sequence encoding a ligand (or ligand tag) or receptor (or receptor tag) consists of or comprises a cDNA sequence encoding said ligand (or said ligand tag) or said receptor (or said receptor tag), or a fragment thereof. In yet another embodiment, said nucleic acid sequence encoding a ligand (or ligand tag) or receptor (or receptor tag) consists of or comprises a gene encoding said ligand (or said ligand tag) or said receptor (or said receptor tag), or a fragment thereof.

[0224] As defined above, a barcode sequence or set of barcode sequences contained within a microreactor is distinguishable from barcode sequences or sets of barcode sequences contained within other microreactors, but the barcode primers specific to the nucleic acid sequence encoding the ligand (or ligand tag) and the nucleic acid sequence encoding the receptor (or receptor tag) contained within a given microreactor share a common barcode sequence or set of barcode sequences. In other words, each microreactor contains a unique type of barcode sequence or set of barcode sequences, and may contain several barcode primers, preferably associated with different primer sequences, but a particular barcode sequence or set of barcode sequences is preferably never contained within two different microreactors.

[0225] In some embodiments, the barcoded primers are delivered on particles. In a particularly preferred embodiment, the barcoded primers are initially attached to particles. Indeed, initially attaching the barcoded primers to particles facilitates the delivery of only one type of barcoded primer into each microreactor.

[0226] As used herein, the terms "particles" and "beads" are used interchangeably.

[0227] "Particle" in the context of the present invention refers to a fine particle.

[0228] In one embodiment, the particles are hydrogel particles, polymer particles or magnetic particles.

[0229] The particles may have an irregular or regular shape, for example, they may be spherical, ellipsoidal, or cubic.

[0230] "Hydrogel particles" are described, for example, in International Patent Application No. WO2008 / 109176, entitled "Assay and other reactions involving droplets." Examples of hydrogels include, but are not limited to, agarose, poly(ethylene glycol) diacrylate, or acrylamide-based gels, such as bis-acrylamide, polyacrylamide, streptavidin acrylamide, poly-N-isopropyl acrylamide, or poly-N-isopropyl polyacrylamide, or mixtures thereof. In one example, the hydrogel particles comprise acrylamide, bis-acrylamide, and streptavidin acrylamide.

[0231] For example, an aqueous solution of a monomer may be dispersed in a microreactor, e.g., a droplet, and then polymerized to form, e.g., a gel. Another example is a hydrogel, e.g., alginate, which can be gelled by the addition of calcium ions. In some cases, a gelling initiator (e.g., ammonium persulfate and TEMED for acrylamide, or Ca for alginate) is added. 2+ ) can be added to a microreactor, e.g., a droplet, by, for example, co-flow with an aqueous phase, diffusion and / or co-flow through an oil phase, or coalescence of two different droplets, as disclosed, for example, in U.S. Patent Application No. 11 / 360,845, filed February 23, 2006, entitled "Electronic Control of Fluidic Species" (Link, et al., published January 4, 2007 as U.S. Patent Application Publication No. 2007 / 000342; or U.S. Patent Application No. 11 / 698,298, filed January 24, 2007, entitled "Fluidic Droplet Coalescence" (Ahn et al.).

[0232] In another set of embodiments, the particles may comprise one or more polymers, and are therefore referred to herein as “polymer particles.” Exemplary polymers include, but are not limited to, polystyrene (PS), polycaprolactone (PCL), polyisoprene (PIP), poly(lactic acid), polyethylene, polypropylene, polyacrylonitrile, polyimide, polyamide, and / or mixtures and / or copolymers of these and / or other polymers.

[0233] Additionally, in some embodiments, the particles may be magnetic and thus referred to as "magnetic particles," allowing for magnetic manipulation of the particles. For example, the particles may comprise iron or other magnetic materials. The particles may also be functionalized so that other molecules, such as proteins, nucleic acids, or small molecules, can be attached.

[0234] In some embodiments, the particles may be fluorescent.

[0235] In some embodiments, the particles may be functionalized to facilitate their identification and / or their sorting, for example with histidine, Flag, HA, streptavidin, acrydite DNA or biotin, among others.

[0236] In one embodiment, the particles comprise streptavidin, which may be attached to the surface of the particles defined above or may be inside said particles.

[0237] In one embodiment, the hydrogel particles have a size of 1 pL to 1000 pL, e.g., 1 pL to 500 pL, 1 pL to 400 pL, 1 pL to 400 pL, 1 pL to 300 pL, e.g., 5 pL to 300 pL, 5 pL to 250 pL, 5 pL to 200 pL, 10 pL to 250 pL, 10 pL to 200 pL, 20 pL to 150 pL, 30 pL to 100 pL, 40 pL to 90 pL, 50 pL to 60 pL, preferably 60 pL to 100 pL.

[0238] It will be understood by those skilled in the art that temporarily binding barcoded primers to particles makes it possible to provide particles with a large number of barcoded primers. Furthermore, initially binding the barcoded primers to the particles facilitates the introduction of barcoded primers into each microreactor, specifically each droplet, where the barcoded primers have the same barcode sequence.

[0239] Thus, in one embodiment, the barcoded primers are covalently or non-covalently attached to the particles.

[0240] As used herein, "non-covalent binding" refers to, for example, streptavidin-biotin binding. Other non-covalent bindings, such as avidin-biotin binding or binding of His-tags to nickel, are known to those skilled in the art.

[0241] As used herein, "covalent bond" refers to, for example, an amino bond or an acrydite-phosphoramidite bond.

[0242] "Streptavidin" generally refers to a 52.8 kDa protein purified from the bacterium Streptomyces avidinii. The streptavidin homo-tetramer has a very high affinity for biotin (dissociation constant (Kd) of approximately 10 -14 mol / L), and the binding of biotin to streptavidin is one of the strongest non-covalent interactions known in nature.

[0243] In a preferred embodiment, the non-covalent bond is a streptavidin-biotin bond.

[0244] Streptavidin-biotin binding is known to those skilled in the art. Thus, in one embodiment, the particles defined herein contain streptavidin. Thus, in the same embodiment, the barcoded primers defined herein contain biotin. In other words, the barcoded primers are functionalized with biotin.

[0245] Independently of the type of bond used to link the barcoded primer to the particle, the barcoded primer may further comprise at least one linker sequence.

[0246] Thus, in a further embodiment, the barcoded primer further comprises at least one linker sequence, preferably at the 5' end. Thus, in one embodiment, the barcoded primer comprises, from 5' to 3', a linker sequence, a barcode sequence, and a primer sequence.

[0247] In one embodiment, a "linker sequence" is a sequence by which a barcoded primer is optionally attached to a particle.

[0248] As used herein, "optionally bound" refers to the possibility that once the barcoded primers bound to the particles are loaded into a microreactor or multiple microreactors, the barcoded primers may be released from the particles, such that the microreactor contains the particles and the barcoded primers and the barcoded primers are separated from the particles.

[0249] Preferably, the linker sequence is a cleavable linker sequence that can be cleaved upon application of an appropriate stimulus, such as, for example, enzyme and / or photocleavage.

[0250] "Cleavable linkers" are well known to those of skill in the art and are further described in Leriche et al. (2012) Bioorg. Med. Chem. 20:571-582. These include, but are not limited to, TEV, trypsin, thrombin, cathepsin B, cathespin D, cathepsin K, caspase, lumatrix metalloproteinase sequences, phosphodiesters, phospholipids, esters, galactose, dialkyldialkoxysilanes, cyanoethyl groups, sulfones, ethylene glycolyl disuccinate, 2-N-acylnitrobenzenesulfonamides, α-thiophenyl esters, unsaturated vinyl sulfides, activated sulfonamides, malondialdehyde (MDA) indole derivatives, levulinoyl esters, hydrazones, acylhydrazones, alkylthioesters, disulfide bridges, azo compounds, 2-nitrobenzyl derivatives, phenacyl esters, 8-quinolinylbenzenesulfonic acid, coumarins, phosphotriesters, bis-arylhydrazones, bimane bithiopropionic acid Cleavage conditions and reagents include, but are not limited to, enzymes, nucleophilic / basic reagents, reducing agents, light irradiation, electrophilic / acidic reagents, organometallic and metallic reagents, and oxidizing reagents.

[0251] In a preferred embodiment, the cleavable linker is a photocleavable moiety, for example a photolabile chemical group followed by a chain of 1 to 30 carbon atoms, typically a chain of 6 to 10 carbon atoms.

[0252] In a further preferred embodiment, the cleavable linker is a double-stranded DNA molecule that contains a target site for a specific restriction endonuclease.

[0253] In one particular embodiment, the barcoded primers bound to the particles are released from the particles in the microreactor, particularly before or after lysing the cells as disclosed below.

[0254] The release of at least some of the barcoded primers can further occur after lysing the cells and before reverse transcribing the released nucleic acids hybridized to the barcoded primers, or after lysing the cells and reverse transcribing the released nucleic acids hybridized to the barcoded primers.

[0255] Those skilled in the art will understand that depending on the time point selected for release of the barcoded primers, the term "at least some barcoded primers" can refer, for example, to at least a portion of the barcoded primers hybridized to nucleic acids or DNA / RNA duplexes released by the cells.

[0256] In one embodiment, at least a portion of the barcoded primers can be released using any means, such as enzymes, nucleophilic / basic reagents, reducing agents, light irradiation, electrophilic / acidic reagents, organometallic and metallic reagents, and oxidizing reagents.

[0257] In one embodiment, at least a portion of the barcoded primers can be released using enzymes and / or photocleavage, for example, an endonuclease can be used to cleave the linker sequence or any other sequence to release at least a portion of the barcoded primers from the particle.

[0258] In a further embodiment, release of the barcoded primer refers to disruption of a streptavidin-biotin bond, etc. Methods for disrupting a streptavidin-biotin bond are known to those skilled in the art and include enzymatic digestion of streptavidin and / or denaturation of streptavidin.

[0259] In one embodiment, the barcoded primers are released by enzymatic digestion of streptavidin.

[0260] Preferably, each particle carries a barcode sequence or set of barcode sequences that is distinguishable from barcode sequences or sets of barcode sequences carried by other beads. In other words, each particle carries a unique majority type of barcode sequence or set of barcode sequences, and may include several barcoded primers, preferably at least some of which are associated with different primer sequences, but two different particles preferably do not carry the same majority barcode sequence or set of barcode sequences.

[0261] In a preferred embodiment, each microreactor contains a single particle carrying a barcoded primer or fewer than 10 particles carrying a barcoded primer, specifically fewer than 9, 8, 7, 6, 5, 4, 3, or 2 particles. In a particularly preferred embodiment, each microreactor contains a single particle carrying a barcoded primer.

[0262] "Reverse transcriptase (RT)", in the context of the present invention, is an enzyme used to produce complementary DNA (cDNA) from an RNA template in a process called reverse transcription.

[0263] 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.

[0264] In one embodiment, the reverse transcriptase is at a concentration of 1 to 50 U / μL, preferably 5 to 25 U / μL, for example, 12.5 U / μL.

[0265] In the context of the present invention, a "cell lysis buffer" is a composition that allows cell lysis, preferably without disrupting the microreactors (specifically the droplets).

[0266] Preferably, the cell lysis buffer is compatible with the reagents used in the RT activity and / or recognition assay.

[0267] In one embodiment, the lysis buffer comprises an enzyme selected from the group consisting of lysozyme, lysostaphin, zymolase, mutanolysin, glycanase, protease, and mannose.

[0268] In one preferred embodiment, the lysis buffer comprises magnesium chloride, a detergent, a buffered solution and an RNase inhibitor.

[0269] In one embodiment, magnesium chloride is used at a concentration of 1 mM to 20 mM.

[0270] 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.

[0271] In one embodiment, the detergent is at a concentration of 0.1% to 10%.

[0272] Non-limiting examples of buffered solutions include Tris-HCl, Hepes-KOH, Pipes-NaOH, maleic acid, phosphoric acid, citric acid, malic acid, formic acid, lactic acid, succinic acid, acetic acid, pivalic acid (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-ethylmorpholinium, POPSO, EPPS, HEPPS, HEPPSO , Tris, Tricine, glycylglycine, bicine, TAPS, morpholine, N-methyldiethanolamine, AMPD (2-amino-2-methyl-1,3-propanediol), diethanolamine, AMPSO, boric acid, CHES, glycine, CAPSO, ethanolamine, AMP (2-amino-2-methyl-1-propanol), piperazine, CAPS, 1,3-diaminopropane, CABS, or piperidine (see also www.reachdevices.com / Protein / BiologicalBuffers.html).

[0273] Non-limiting examples of RNase inhibitors include inhibitors that target RNase OUT, IN, SuperIN Rnases, and broad-spectrum RNAses (eg, A, B, C, 1 and T1).

[0274] In one example, the lysis buffer is typically 0.36% Igepal CA 630, 50 mM Tris-HCl (pH 8).

[0275] In one particular embodiment, the additional reagent is added into the microreactor, in particular into the microfluidic droplet, by injection from a reservoir, e.g., using electrical forces (picoinjection) (Abate et al. (2010) Proc. Nat. Acad. Sci. USA 107:19163-19166).

[0276] In another particular embodiment, the additional reagent is added into the microreactor, particularly into a microfluidic droplet, by combining it with a second microreactor, particularly a second microfluidic droplet, that contains the additional reagent but does not contain any ligand or receptor. Droplets can be coalesced by a variety of methods known to those skilled in the art, 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 with a focused laser (Baroud et al. (2007) Lab Chip 7:1029-1033), or using electrical 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 ... al. (2006) Appl. Phys. Lett. 89:134101) or magnetophoretic forces, or using a pneumatic controller (see Xi et al. (2017) Lab Chip 17:751-771).

[0277] The second microreactor, specifically the second microfluidic droplet, can be prepared by the same techniques as those disclosed above for the microreactor containing the ligand and receptor.

[0278] "Coalescence," as used herein, refers to the process by which two or more droplets or particles merge during contact to form a single daughter droplet or particle.

[0279] Preferably, said additional reagent is selectively added to the positive microreactor, in particular after a step of separating the positive microreactor from the negative microreactor.

[0280] In a particular embodiment, in each microreactor, and particularly in each positive microreactor to which the above-mentioned additional reagents are added, the barcoded cDNA is prepared by (a) lysing cells expressing or presenting the receptor and cells expressing or presenting the ligand to release mRNA from the cells, (b) in at least some microreactors, hybridizing at least a portion of the released mRNA encoding the receptor (or a tag for the receptor) to a nucleic acid sequence-specific primer (which may be barcoded) encoding the receptor (or a tag for the receptor) and hybridizing at least a portion of the released mRNA encoding the ligand (or a tag for the ligand) to a nucleic acid sequence-specific barcoded primer encoding the ligand (or a tag for the ligand), and (c) reverse transcribing the released mRNA hybridized to the primer (which may be barcoded), thereby obtaining barcoded cDNA.

[0281] As will be appreciated by those skilled in the art, if the ligand tag or receptor tag is a barcode sequence, there is no need to prepare barcoded cDNA as detailed above, since the nt sequence of the ligand or receptor allows for their own identification.

[0282] "Barcoding" as used herein refers to the addition of a genetic sequence (a so-called barcode sequence, as further defined herein above) to a nucleic acid, thereby allowing said barcoded nucleic acid to be distinguished from a nucleic acid having another added genetic sequence, i.e., another unique barcode sequence.

[0283] The term "cell lysis" in the context of the present invention may be achieved by enzymatic, physical, and / or chemical means, or any combination thereof (e.g., enzymatic, physical, and / or chemical means). Other cell disruption methods may also be used.

[0284] Thus, in one embodiment, cells are lysed using enzymatic, physical, and / or chemical cell lysis.

[0285] "Enzymatic methods" for removing cell walls are well documented in the art. Enzymes are commonly available commercially and are in most cases originally isolated from biological sources. Commonly used enzymes include lysozyme, lysostaphin, zymolase, mutanolysin, glycanase, protease, and mannose.

[0286] As known by those skilled in the art, "chemical cell lysis" is achieved using chemicals, such as detergents, that disrupt the lipid barrier surrounding cells by disrupting lipid-lipid, lipid-protein, and protein-protein interactions. The ideal detergent for cell lysis depends on the cell type and origin. Nonionic and zwitterionic detergents are milder detergents. The Triton X series of nonionic detergents, IGEPAL CA 630 nonionic detergent, and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), a zwitterionic detergent, are commonly used for these purposes. In contrast, ionic detergents are strong solubilizers and tend to denature proteins, thereby disrupting their activity and function. SDS, an ionic detergent that binds to and denatures proteins, is widely used in the art to disrupt cells.

[0287] "Physical cell lysis" refers to the use of sonication, temperature shock (above 40°C, below 10°C), electroporation, or laser-induced cavitation.

[0288] In one example, the cells are lysed on ice.

[0289] In one preferred embodiment, cell lysis does not disrupt or break the microreactor, specifically the droplet in the context of the present invention.

[0290] The term "hybridization" as used herein refers to the phenomenon in which the primer sequence present in the barcoded primer anneals to the complementary nucleic acid sequence of the released nucleic acid. Therefore, as known by those skilled in the art, the temperature used depends on the primer sequence and / or polymerase enzyme used.

[0291] The step of reverse transcription as defined above refers to reverse transcribing, in at least some of the microreactors, the released nucleic acids hybridized to the barcoded primers using a primer sequence. The reverse transcription is carried out using a reverse transcriptase (RT) enzyme contained in at least some of the microreactors.

[0292] "Reverse transcription" or "RT reaction" is a process in which single-stranded RNA is reverse-transcribed into single-stranded complementary DNA (cDNA) using total cellular RNA or poly(A) RNA, reverse transcriptase enzyme, primers, dNTPs, and an RNase inhibitor. It will be understood by those skilled in the art that the product of reverse transcription is an RNA / DNA duplex containing a single-stranded cDNA hybridized to the template RNA. It will also be understood that the RNA / DNA duplex is further linked to a barcoded primer containing the primer sequence used for reverse transcription.

[0293] "Template switching" refers to a technology originally described in 2001 and is often referred to as "SMART" (Switch Mechanism at the 5' of RNA Transcripts) technology (Takara Bio USA, Inc.). This technology has shown promise for 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 endogenous properties of Moloney murine leukemia virus (MMLV) reverse transcriptase and the use of a unique template switch oligonucleotide (TS oligo, or TSO). During first-strand synthesis, upon reaching the 5' end of the RNA template, the terminal transferase activity of MMLV reverse transcriptase adds several additional nucleotides (mostly deoxycytidine) to the 3' end of the newly synthesized cDNA strand. These bases act as TS oligo-anchor sites. Upon base pairing between the TS oligo and the added stretch of deoxycytidine, reverse transcriptase "switches" the template strand from the cellular RNA to the TS oligo and continues replicating up to the 5' end of the TS oligo. The resulting cDNA contains the complete 5' end of the transcript, and a universal sequence of choice is added to the reverse transcript. Combined with tagging the 3' end of the cDNA with an oligo-dT primer, this approach allows for efficient amplification of the entire full-length transcript pool in a completely sequence-independent manner (Shapiro et al. (2013) Nat. Rev. Genet. 14:618-630).

[0294] Thus, it will be understood by those skilled in the art that after reverse transcribing the nucleic acid, the microreactor further contains cDNA.

[0295] Thus, in one embodiment, at least some of the microreactors further comprise cDNA produced by reverse transcription of nucleic acid from cells contained within said microreactors.

[0296] In one embodiment, the cDNA refers to a single-stranded complementary DNA.

[0297] In a further embodiment, the cDNA is comprised within an RNA / DNA duplex.

[0298] In one embodiment, the RNA / DNA duplex refers to reverse-transcribed RNA hybridized to the primer sequence of at least one primer, which may be barcoded, contained within the microreactor.

[0299] As will be appreciated by those skilled in the art, in one embodiment, the RNA / DNA duplex is bound to a primer, which may be barcoded, that contains the primer sequence used for reverse transcription, to which the nucleic acid (preferably mRNA) hybridizes.

[0300] In one example, hybridization and reverse transcription is carried out by incubating the microreactor for, for example, 1 or 2 hours at 55° C. or 50° C., typically while mixing the microreactor at, for example, 550 rpm.

[0301] Identification of ligand and receptor species The identification of the ligand and receptor species contained in each microreactor, in particular each positive microreactor, can be performed by any technique well known to those skilled in the art. In particular, the identification of the ligand and receptor species contained in each microreactor, in particular each positive microreactor, can be performed by sequencing, in particular by sequencing the DNA, the barcoded cDNA obtained as detailed above, or the tags.

[0302] In one embodiment, the barcoded cDNA produced by reverse transcription as defined above is recovered and typically further used for subsequent amplification and identification by sequencing of the library preparation.

[0303] Thus, in one embodiment, the method of the invention further comprises the step of recovering the cellular cDNA produced by reverse transcription in at least some of the microreactors, preferably in the positive microreactors.

[0304] As used herein, the term "recovering" refers to isolating barcoded cDNA produced by reverse transcription in at least some microreactors from the plurality of microreactors.

[0305] In one embodiment, the term "recovering" as used herein refers to a step of collecting a microreactor containing barcoded cDNA produced by reverse transcription, or a step of collecting an aqueous composition contained in the microreactor containing the barcoded cDNA and separating the barcoded cDNA contained in the aqueous composition.

[0306] In one particular embodiment, "recovering" as used herein refers to collecting microfluidic droplets containing barcoded cDNA produced by reverse transcription, disrupting the microfluidic droplets, and separating the barcoded cDNA contained within an aqueous composition from the oil phase of the microfluidic droplets.

[0307] Methods for isolating nucleic acids, specifically cDNA, from microfluidic droplets are known to those skilled in the art and include, for example, collecting the microfluidic droplets and breaking the emulsion, for example, by applying an electric field (electrocoalescence) or by adding a chemical emulsion breaker (e.g., perfluoro-octanol in the case of droplets in a fluorinated carrier oil). In one example, the broken emulsion is typically centrifuged, for example, at 10,000 g for 10 minutes at 4°C, and the supernatant containing the barcoded cDNA in the aqueous phase is collected.

[0308] In one embodiment, the method further comprises 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 is performed after the step of recovering the barcoded cDNA produced by reverse transcription as defined herein above.

[0309] Preferably, the step of removing unincorporated barcoded primers precedes the amplification and / or sequencing steps as defined herein below.

[0310] In one embodiment, removing unincorporated barcoded primers comprises contacting the aqueous composition of 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 and may form a column. In a further example, unincorporated barcoded primers are removed by size selection, for example, using an acrylamide or agarose gel.

[0311] In one embodiment, removing unincorporated barcoded primers comprises contacting the aqueous composition of at least some of the microreactors with an exonuclease, such as exonuclease Exo1, to degrade unincorporated barcoded primers in the aqueous composition of at least some of the microreactors.

[0312] In certain embodiments of this step, the exonuclease degrades single-stranded nucleic acid sequences from the aqueous composition containing the cDNA.

[0313] It will be understood by those skilled in the art that the barcoded cDNA obtained after reverse transcription is typically in the form of an RNA / DNA complex and is therefore protected from said exonucleases.

[0314] In one embodiment, the barcoded cDNA comprises one or more modified nucleotides or nucleotide analogs, e.g., to facilitate purification of the barcoded cDNA sequence or molecule.

[0315] For example, nucleotides can be used as phosphorothioate derivatives (substitution of a non-bridging phosphoryl oxygen atom with a sulfur atom) which increase resistance to nuclease digestion. 2'-Methoxyethyl (MOE) modifications (e.g., modified backbones marketed by ISIS Pharmaceuticals) are also effective.

[0316] Other examples of modified nucleotides include derivatives of nucleotides with substitutions at the 2' position of the sugar, specifically the following chemical modifications: O-methyl (2'-O-Me), 2-methoxyethyl (2'-O-MOE), fluoro (2'-fluoro), chloro (2'-Cl), bromo (2'-Br), cyanide (2'-CN), trifluoromethyl (2'-CF), OCF (2'-OCF), OCN (2'-OCN), O-alkyl (2'-O-a) substituted, S-alkyl (2'-S-alkyl), N-alkyl (2'-N-alkyl), O-alkenyl (2'-O-alkenyl), S-alkenyl (2'-S-alkenyl), N-alkenyl (2'-N-alkenyl), SOCH3 (2'-SOCH3), SO2CH3 (2'-SO2CH3), ONO2 (2'-ONO2), NO2 (2'-NO2), N3 (2'-N3), and / or NH2 (2'-NH2) groups. Other examples of modified nucleotides include biotin-labeled nucleotides.

[0317] Other examples of modified nucleotides include nucleotides in which the ribose moiety is used to produce locked nucleic acids (LNAs) locked in a 3'-end configuration by forming a covalent bridge between the 2' oxygen and the 4' carbon of the ribose.

[0318] Another example of a nucleotide analog includes deoxyinosine.

[0319] Other examples of nucleotide analogs include biotinylated, fluorescently labeled nucleotides. For example, biotin-11-dCTP can be used as a substrate for reverse transcriptase to incorporate biotin into cDNA during polymerization, allowing affinity purification with streptavidin or avidin.

[0320] In one embodiment, the barcoded cDNA is further treated with RNAse A and / or RNAse H.

[0321] "RNAse A" is an endoribonuclease that specifically degrades single-stranded RNA at C and U residues. In one embodiment, RNAse A is at a concentration of 10 to 1000 μg / μL, preferably 50 to 200 μg / μL, for example, 100 μg / μL.

[0322] "RNAse H" is a family of nonspecific endonucleases that catalyze the cleavage of RNA via a hydrolytic mechanism. RNase H ribonuclease activity cleaves the 3'-OP bond of RNA in DNA / RNA duplex substrates to produce 3'-hydroxyl and 5'-phosphate terminated products. In one embodiment, RNAse H is at a concentration of 10-1000 μg / μL, preferably 50-200 μg / μL, e.g., 100 μg / μL.

[0323] In one embodiment, the barcoded cDNA is further treated with proteinase K. "Proteinase K" is a widely used serine protease that digests proteins preferentially after hydrophobic amino acids. In one embodiment, proteinase K is at a concentration of 0.1-5 mg / mL, preferably 0.1-1 mg / mL, e.g., 0.8 mg / mL.

[0324] In one embodiment, the barcoded cDNA obtained after reverse transcription is sequenced to allow identification of receptors and ligands contained within the same microreactor.

[0325] In one embodiment, sequencing the barcoded cDNA may include performing a next-generation sequencing (NGS) protocol on the sequencing library. Any type of NGS protocol can be used, such as MiSeq Systems (Illumina®), HiSeq Systems (Illumina®), NextSeq Systems (Illumina®), NovaSeq Systems (Illumina®), IonTorrent Systems (Thermo Fisher), IonProton Systems (Thermo Fisher), or sequencing systems produced by Pacific Biosciences or Nanopore.

[0326] In certain embodiments, the NGS protocol includes loading an amount of sequencing library per flow cell of the reagent kit between 1 pM and 20 pM, specifically between 1.5 pM and 20 pM.

[0327] In one embodiment, the NGS sequencing protocol further comprises adding 5-60% PhiX to the volume of the sequencing library or to the flow cell of the reagent kit.

[0328] In one embodiment, the barcoded cDNA is further amplified prior to sequencing.

[0329] In one embodiment, the amplification step is performed by polymerase chain reaction (PCR) and / or linear amplification.

[0330] In one embodiment, a linear amplification precedes the PCR reaction.

[0331] In one embodiment, the linear amplification is in vitro transcription.

[0332] In one embodiment, the linear amplification is an isothermal amplification.

[0333] In one embodiment, the amplification step is performed after removing unincorporated barcoded primers. In one embodiment, the amplification step is performed before the sequencing step defined herein above.

[0334] In one embodiment, the barcoded cDNA produced after reverse transcription is quantified using qPCR.

[0335] In one embodiment, specific sequences required for sequencing are added during amplification or by adapter ligation, thereby generating a sequencing library.

[0336] As will be appreciated by those skilled in the art, because the barcoded cDNA from a particular microreactor will possess the same specific majority barcode sequence or set of barcode sequences that is different from the majority barcode sequence or set of barcode sequences contained in other microreactors, it is possible to determine which identified ligand species was contained in the same microreactor as a particular identified ligand receptor, specifically a positive microreactor.

[0337] [Embodiment] The present invention also provides the following non-limiting embodiments.

[0338] Embodiment 1 is a method for identifying cognate pairs of a ligand and receptor species, the method comprising: a. providing a set of ligand species, wherein each ligand species is represented at least once; b. providing a set of receptor species, wherein each receptor species is represented at least once; c. contacting a set of ligand species with a set of receptor species in a microreactor, wherein an enhanced signal is produced upon selective binding of the receptor species to the ligand species; d. detecting the cognate pair of ligand and receptor species by production of a signal; and e. Identifying cognate pairs of ligand and receptor species.

[0339] Embodiment 2 is the method of embodiment 1, wherein each ligand species comprises a barcode sequence.

[0340] Embodiment 3 is the method of embodiment 1 or 2, wherein each receptor species comprises a barcode sequence.

[0341] Embodiment 4 is the method of any one of Embodiments 1 to 3, wherein each ligand species is expressed or presented on the surface of a cell or bead, or expressed or present in a cell-free extract or in solution.

[0342] Embodiment 5 is the method of embodiment 4, wherein the ligand species is expressed or presented on the surface of the antigen-presenting cell.

[0343] Embodiment 6 is the method of embodiment 5, wherein the antigen-presenting cell is selected from a macrophage, a dendritic cell, a Langerhans cell, a B cell, a monocyte-derived dendritic cell, or another cell that expresses an MHC class I or II molecule.

[0344] Embodiment 7 is the method of any one of Embodiments 1 to 6, wherein each receptor species is expressed or displayed on the surface of a cell or bead, or expressed or present in a cell-free extract or in solution.

[0345] Example 8 is the method of any one of Examples 1 to 7, wherein the microreactor is selected from an aqueous droplet, a microcapsule, a microbead, a compartment of a microfluidic chip, or a well.

[0346] Embodiment 9 is the method of any one of embodiments 1 to 8, wherein the signal is selected from a morphological change in one of the cell, the ligand, or the receptor; fluorescent signal enhancement; modification of the fluorescent signal using a caged compound or by a quenching reaction; light absorption; modification / generation of a visible structure; or a combination of these signals.

[0347] Embodiment 10 is the method of any one of embodiments 2 to 9, wherein identifying the cognate pair of ligand and receptor species comprises amplifying the ligand and / or receptor species, and at least one of the amplified ligand and receptor species is sequenced for identification.

[0348] Embodiment 11 is the method of any one of Embodiments 1 to 10, wherein the set of ligand species is selected from a T cell antigen, a B cell antigen, a viral antigen, a bacterial antigen, a parasitic antigen, a neoantigen, a tumor-associated antigen (TAA), a tumor-specific antigen, an immune checkpoint molecule, a cytokine, a carbohydrate, a member of the immunoglobulin superfamily, a selectin, a chemokine, a hormone, a growth factor, a G protein-coupled receptor ligand, or an enzyme substrate.

[0349] Embodiment 12 is the method of any one of Embodiments 1 to 11, wherein the set of receptor species is selected from T cell receptors, B cell receptors, immune checkpoint receptors, cytokine receptors, selectins, integrins, members of the immunoglobulin superfamily, cadherins, chemokine receptors, hormone receptors, growth factor receptors, G protein-coupled receptors (GPCRs), or enzymes.

[0350] Embodiment 13 is any one of the methods of Embodiments 1 to 12, wherein the ligand species is a T cell antigen, the receptor species is a T cell receptor, an enhancement signal is produced upon selective binding of the T cell receptor to the T cell antigen, and the produced enhancement signal is a result of T cell activation.

[0351] Embodiment 14 is the method of any one of Embodiments 1 to 12, wherein the ligand species is a viral antigen, the receptor species is a T cell receptor, an enhancement signal is produced upon selective binding of the T cell receptor to the viral antigen, and the produced enhancement signal is a result of T cell activation.

[0352] Embodiment 15 is the method of embodiment 13 or 14, wherein the step of contacting the set of ligand species with the set of receptor species in the microreactor occurs for about 0.001 hours to about 8 hours.

[0353] Embodiment 16 is the method of embodiment 13 or 14, wherein the step of contacting the set of ligand species with the set of receptor species in the microreactor occurs for at least about 8 hours.

[0354] Embodiment 17 is the method of embodiment 15 or 16, wherein the ligand species and receptor species bind with high affinity and the enhanced signal produced is an early or late marker of T cell activation.

[0355] Embodiment 18 is the method of embodiment 16, wherein the ligand species and receptor species bind with low affinity and the enhanced signal produced is an early or late marker of T cell activation.

[0356] Embodiment 19 is the method of embodiment 17 or 18, wherein the early marker of T cell activation is selected from CD69, CD107a, or transferrin receptor.

[0357] Embodiment 20 is the method of embodiment 17 or 18, wherein the late marker of T cell activation is selected from CD137, HLA-DR, VLA1, PTAl, CD71, CD27, PD-1, TIM3, LAG3, or CTLA4.

[0358] Embodiment 21 is the method of Embodiment 19 or 20, wherein the enhanced signal is detected by an anti-CD69 antibody, an anti-CD107a antibody, an anti-transferrin receptor antibody, an anti-CD137 antibody, an anti-HLA-DR antibody, an anti-VLA1 antibody, an anti-PTA1 antibody, an anti-CD71 antibody, an anti-CD27 antibody, an anti-PD1 antibody, an anti-TIM3 antibody, an anti-LAG3 antibody, or an anti-CTLA4 antibody.

[0359] Embodiment 22 is the method of any one of Embodiments 1 to 12, wherein the ligand species is a B cell antigen, the receptor species is a B cell receptor, and an enhanced signal is produced upon selective binding of the B cell receptor to the B cell antigen.

[0360] Embodiment 23 is the method of embodiment 22, wherein the signal is detected by an anti-CD138 antibody, an anti-CD19 antibody, an anti-CD45R antibody, an anti-CD45 antibody, activation of fluorescent reporter expression, or inhibition of fluorescent reporter expression. [Example]

[0361] The following examples of the present invention are intended to further illustrate the nature of the present invention. It should be understood that the following examples do not limit the present invention, and that the scope of the present invention should be determined by the appended claims.

[0362] [material and method] Cell preparation:T cells were cultured x vivo with 5% human serum supplemented with 1% penicillin, streptomycin, 1% sodium pyruvate, and non-essential amino acids (GIBCO). Cells were counted, washed with PBS, and spun down at 450g for 5 minutes at 4°C. Cell pellets were resuspended at 2µM / ml in PBS and stained for 20 minutes at 37°C with cell traces previously dissolved in DMSO at 5µM (except for cell traces (red) which were at 1µM). The reaction was stopped by adding protein-containing buffer (complete medium) at least five more times and incubating at room temperature for 5 minutes. Cells were spun down at 450g for 3 minutes at 4°C and resuspended in MACS buffer (phosphate-buffered saline (PBS), pH 7.2, 0.5% bovine serum albumin (BSA), and 2 mM EDTA (by diluting MACS® BSA stock solution (#130-091-376) 1:20 with auto MACSR rinse solution (#130-091-222)).

[0363] T cell purification T cells were purified using negative selection as recommended by the manufacturer (Miltenyi; Bergisch Gladbach, Germany). PBMCs were resuspended at 250 μg / ml in MACs buffer. Antibody cocktails targeting different subsets were added at a 1 / 5 dilution and incubated for 5 minutes at 4°C. The cell concentration was adjusted to 125 μg / ml with MACs buffer, and a microbead cocktail was added at a 1 / 5 dilution. T cells were extracted using an LS column in the magnetic field of a MACS separator.

[0364] Peptide loading of K562 K562 cells were counted, washed with PBS, and spun down at 450g for 5 minutes. The cell pellet was resuspended in PBS at 2µg / ml and stained with Cell Trace at the recommended concentration. The staining reaction was stopped with X vivo 5% human serum for 5 minutes at room temperature. The cells were then washed and resuspended at 1µg / ml in X vivo containing 5% human serum. Peptides were then added to the cell suspension at 10µg / ml and incubated at 37°C, 5% CO2 for 90 minutes to allow peptide presentation by HLA0201.

[0365] mRNA transfection The medium for K562 cells was changed every 2–3 days. For mRNA transfection, K562 cells must be in the growth phase and subcultured less than 10 times (subcultured cells after reaching 1 M:ml). K562 cells were cultured at 1 × 10 5 Cells were seeded at 3 x 10 cells / ml and incubated at 3 x 10 for 2 days prior to Nucleofection™. 5 Cells were cultured at a density of 1000 cells / ml. After uploading the experimental parameter file, the appropriate Nucleofector™ Program (FF-120) was selected. Cell culture plates were prepared by filling the appropriate number of wells with the desired volume of recommended culture medium and pre-incubated / equilibrated in a humidified 37°C, 5% CO2 incubator. K562 cells were counted and the required cell number was centrifuged at 90g for 10 minutes at room temperature. The supernatant was completely removed, and the cells were resuspended in 4D Nucleofector solution at the appropriate concentration (1000 M / ml). 1 M cells were mixed with the required amount of mRNA substrate (10 μg) and transferred to a Nucleocuvette™ vessel, the lid of which was placed in the retainer of the 4D-Nucleofector™ X unit. After pulse transfection, the Nucleocuvette™ vessel was carefully removed from the retainer and incubated at room temperature for 10 minutes. The cells were then resuspended in pre-warmed medium (500 μl per 1 M cells), gently mixed by pipetting up and down 2-3 times, and plated. The plated cells were then incubated in a humidified 37°C, 5% CO2 incubator until analysis.

[0366] Cell preparation for IFN-γ in dropletsFor effector cells, T cells were labeled with Cell Trace (yellow) and then with a bispecific antibody targeting CD45 and IFNγ (1 / 10 dilution in chilled medium, 5 min at 4°C, 10 µM / ml cell concentration). T cells were then washed and resuspended in a parallel flow containing X vivo medium containing 5% human serum supplemented with Pluronic F68 (0.1%), 23.6% Nycodenz, and 6% DNA marker (Nucgreen).

[0367] For peptide loading, K562 cells were labeled at 5 μM using the indicated cell traces, and then loaded with 10 μM or no peptide. The cells were then washed by adding cold buffer, spun down for 10 minutes at 4°C, and resuspended in parallel flow as described above. For mRNA-transfected K562 cells, the cells were not labeled because the transfected mRNA encodes a fluorescent reporter.

[0368] Detection antibodies were added to the cell suspension. Finally, these cells were separately co-flowed into a microfluidic device, thus generating 100 picoliter droplets, which were then incubated overnight at 37°C and 5% CO2.

[0369] Droplet production Aqueous Phase I: Preparing cells for compartmentalization in droplets Cell suspensions were prepared at 4°C as described for compartmentalization in droplets, and cells were resuspended in X vivo supplemented with 0.1% Pluronic F68 (LifeTechnologies; Carlsbad, CA), 25 mM HEPES (pH 7.4) (LifeTechnologies), 1% Pen / Strep (ThermoFisher; Waltham, MA), 23.6% Nycodenz (Fisher Scientifique), and 6% DNA marker (Nucgreen-Thermofisher) to achieve a λ (average number of cells per droplet) of approximately 0.9 and 0.45 for K562 and T cells, respectively, in the droplet T cell activation assay.

[0370] Aqueous Phase II: Preparation of bioassay reagents and reporter cells for compartmentalization in droplets K562 cells stained with Cell Trace and loaded with peptide for their cognate antigen were resuspended in working buffer containing Fc block (1 / 5 dilution) (Miltenyi) and either anti-CD137 antibody at 2x the indicated concentration (yielding final droplet concentrations as indicated) or IFN-γ at 1 / 25 dilution. K562 cells displaying peptides on their cell surface were pre-labeled with the indicated Cell Trace and resuspended as defined above in X Vivo medium supplemented with 23.6% (vol / vol) Nycodenz, containing 5% human serum, to achieve a λ (average number of cells per droplet) of approximately 0.9 for reporter cells in the cell-based assay. T cells pre-labeled with the indicated Cell Trace were resuspended in the same medium described above with the required concentration of Drop Code DY754 at a λ of approximately 0.45.

[0371] Microfluidic ChipSeparate microfluidic chips were used. Device 1 was used to compartmentalize bioassay reagents and single cells into droplets, or antigen-presenting K562 cells and T cells and bioassay reagents into droplets; Device 2 was used to sort droplets by fluorescence-activated dielectrophoresis; Device 3 produced hydrogel beads, and Device 4 (CellCap) compartmentalized single hydrogel beads and single sorted cells. All chips were fabricated by soft lithography in polydimethylsiloxane (PDMS) (Sylgard). Masters were fabricated using one or two layers of SU-8 photoresist, depending on the design (MicroChem; Bear, DE). The list depth of the photoresist layer for Devices 1 and 2 was 40 μm ± 1 μm, and for Device 3 it was 55 μm ± 1 μm. For device 4, the first layer (70–75 μm deep) was used for the inlet for the hydrogel beads, and the second layer (130–145 μm deep) was used for the inlet for the cells, the inlet for the reverse transcriptase enzyme, and the outlet. The electrodes were prepared by melting 51In 32.5Bi 16.5Sn alloy (Indium Corporation of America) into the electrode channel.

[0372] Droplet production, collection, and incubation Aqueous phases I and II were flowed in parallel and partitioned into droplets using hydrodynamic flow focusing in dripping mode on a microfluidic chip equipped with a 15 μm wide, 40 μm deep, and 10 μm long nozzle. The continuous phase consisted of 2-3% (wt / wt) 008-FluoroSurfactant (RAN Biotechnologies; Beverly, MA) in Novec HFE7500 fluorinated oil (3M; Saint Paul, MN). The flow rate was adjusted to produce monodisperse droplets of 80 μl ± 8 μl (for cell-based assays using membrane-bound antigens). Immediately after production, droplets were collected in a 5 ml hemolysis tube filled with 5 ml of Novec HFE7500 fluorinated oil containing 0.1% (wt / wt) 008-FluoroSurfactant.

[0373] Depending on the readout used in the droplet assay, at least two emulsions were produced containing either the indicated concentration (20 nM) of anti-CD137 antibody or 1 / 50 of the anti-IFNγ detection reagent in the droplets. These emulsions were then differentiated using Dy754 (Dyomics) to optically code droplets in positive and negative control emulsions to screen conditions. T cells were co-compartmentalized in the droplets with peptide-presenting K562 (pulsed or transfected). In certain circumstances, T cells encapsulated in the positive emulsion may be labeled with a different color than T cell labels in the negative emulsion. This is primarily used to distinguish positive emulsions from negative emulsions containing cells during flow cytometry post-QC for specific cell enrichment.

[0374] Microfluidic Platform Droplet fluorescence analysis and sorting were performed on a dedicated droplet microfluidic station equipped with a fixed-focus laser line (solid-state laser with wavelengths of 405 nm, 488 nm, 561 nm, or 635 nm, Omicron) oriented parallel to the bead line for fluorescence analysis, using photomultiplier bandpass filters of 440 / 40-25 nm, 525 / 40-25 nm, 593 / 46-25 nm, and 708 / 75-25 nm (Hamamatsu; Shizuoka, Japan).

[0375] Gating strategies for droplet sortingDroplets were first gated to remove coalesced droplets and retain only droplets of the desired size. Optical droplet barcoding was used to detect negative control droplets, positive control droplets, and droplets containing test cells. Fluorescence relocalization to T cells was measured by plotting the maximum peak fluorescence signal (Fp) within the droplet against the integrated fluorescence signal (Fi) from the droplet. Fluorescence relocalization to T cells results in an increase in the ratio Fp / Fi. Non-coalesced droplets containing T cells and K562 cells were sorted if they met all of the following criteria: 1) the droplet contained K562 cells, 2) it contained T cells, 3) relocalization of readout markers (IFN-γ and CD137) on cells within the droplet, and 4) colocalization of marker fluorescence peaks on T cells. Therefore, the colocalization value (c) was bounded between 0 and 1, with 1 being perfect colocalization of the two peaks. Droplets were sorted if c > 0.95. For all antigens, the colocalization parameter (c) was calculated from the time interval between the peak in the fluorescence of the activation marker and the T cell fluorescence (tp) and the time interval from the beginning to the end of the droplet (td): c = 1 - (tp / td). The value of c is therefore bounded between 0 and 1, with 1 being perfect colocalization of the two peaks.

[0376] Droplet sorting and cell recovery The droplets were sorted by a SAW sorter or by dielectrophoresis. This device can sort droplets into up to two bins by activating electrodes above or below the channel, but only one bin was used in this study. The inlet flow (Qem for emulsion and Qoil for oil) was adjusted for 600 s -1 The droplets were sorted at 1000 kJ / min; typical parameters for sorting were Q = 50 μl h -1 (180mbar), Qoil=50μlh -1The pressure was (180 mbar), F (the frequency of the sorting pulse) = 1.5 kHz, τsort (the duration of the sorting pulse) = 2,000 μs, and Usort (the peak-to-peak voltage applied across the electrodes) = 400 kV p-p. Sorted droplets were collected in 1.5 ml tubes cooled to 4 °C. Cells were harvested by adding 100 μl of X vivo supplemented with 5% human serum followed by 100 μl of 1H,1H,2H,2H-perfluoro-1-octanol (Sigma, 370533; Sigma, St. Louis, MO); the cells were then gently mixed and centrifuged at 300 g for 10 min at 4 °C to ensure complete phase separation. The cells were then washed in 400 μl of 0.1% Pluronic F-68 non-ionic surfactant (Thermo Fisher Scientific, 24040032), 25 mM Hepes, 5% (vol / vol) human serum (X vivo), centrifuged at 400 g for 5 min at 4°C, and then resuspended in 21.82% (vol / vol) Optiprep density gradient solution (Sigma) and 0.01 mg ml -1 Resuspended in 50 μl of 1×PBS containing BSA.

[0377] Production of barcoded hydrogel beads 60 μm diameter polyacrylamide hydrogel beads were produced by polymerization in droplets generated by a microfluidic device. However, barcoded primers were then added to the beads by split-and-pool synthesis using ligation rather than primer extension. One million beads—approximately 10 each with a 5' overhang (complementary to the first index 5' overhang sequence), a photocleavable site, and the T7-SBS12 sequence—were generated. 9100 copies of double-stranded DNA oligonucleotides were distributed into 96 wells of a microtiter plate. Each well contained 10 μl of 5 μM double-stranded DNA with a different first index (index A), a complementary 5' overhang to the first DNA at one end, and a different 5' overhang at the other end. These were ligated for 15 minutes at 23°C using T7 DNA ligase (New England Biolabs) according to the manufacturer's instructions. The hydrogel beads were then pooled, washed as described, and redistributed as described above into the wells of a second microtiter plate. Each well of the second microtiter plate contained double-stranded DNA with a different second index (index B), a complementary 5' overhang to index A at one end, and a different 5' overhang at the other end, which was ligated to index A. This splitting and pooling process was repeated 3–4 times (adding three indexes) to obtain 96 wells. 3 This results in a combination of approximately 10 6 After the addition of the final index, the beads were pooled, and a mixture of double-stranded DNA molecules containing gene-specific primer regions complementary to the regions encoding the minigenes TCR α and β and 20 selected genes, with complementary 5' overhangs to index C, was ligated to the barcodes on the beads. The second strand of the primer was then removed by incubation with 300 mM NaOH for 2 minutes at 22°C. After the process was completed, each hydrogel bead contained a total of approximately 10 identical bead-specific barcodes. 9 The primers are:

[0378] Barcoded complementary DNA synthesis in single cells Individual sorted cells were co-compartmentalized into droplets with individual barcoded hydrogel beads and lysis and reverse transcription reagents using a microfluidic device. Droplets with a volume of approximately 1 nL were then incubated for 250 s. -1Droplets were collected in 1.5 ml tubes containing the solvent HFE-7500 (Fluorochem; Derbyshire, United Kingdom) and 0.1% surfactant, photocleaved with ultraviolet light (OmniCure, AC475; 365 nm) for 90 seconds, and then incubated at 50°C for cell lysis and cDNA synthesis.

[0379] Sequencing library preparation The emulsion containing the barcoded cDNA was broken by adding 1 volume of 1H,1H,2H,2H-perfluoro-1-octanol. The pooled barcoded cDNA was further purified with RNA CleanXP beads (Beckman, A63987; Beckman Coulter; Brea, CA) in a 1:1 ratio (vol / vol) and eluted in 40 μl of DNase- and RNase-free HO. Sequencing libraries were produced by two-step nested PCR using GoTaq polymerase (Promega), outer (PCR1) and inner (PCR2) reverse primers (SBS12 primer followed by Illumina TruSeq indexing primer-P7), and outer (PCR1) and inner (PCR2) forward primers (specific for TCRα and β, TMG, and selected genes).

[0380] Sequencing : The final product was sequenced on an Illumina (MiSeq / Nextseq) which allows sequencing of the entire CDR3 domains of the α and β TCRs, the antigens of the TMG and 20 genes, as well as the barcode sequences.

[0381] Example 1: Titration of anti-CD137 antibodies in droplets To evaluate the signal detection sensitivity and dynamic range of immune responses based on CD137 activation marker expression in droplets, anti-CD137 antibody titration was performed in droplets. T cells were activated with Transact (1 / 100) for 48 hours at 37°C and 5% CO2, then stained with 5 μM cell trace (yellow) in PBS. Cells were then spun down at 450 g for 5 minutes and resuspended in a parallel flow containing complete X vivo medium with 5% human serum supplemented with Pluronic F68 (0.1%), 23.6% Nycodenz, and 6% DNA marker (Nucgreen).

[0382] The second parallel flow contained each of the different antibody concentrations tested in several emulsions assigned to different droplet code concentrations of DY754 (see Gerard et al., "High-throughput single-cell activity-based screening and sequencing of antibodies using droplet microfluidics," Nat. Biotechnology 38:715-21 (2020)). The anti-CD137 BV421 antibody was added at twice the concentration of the parallel flow to reach the indicated concentrations (e.g., 5 nM, 10 nM, and 20 nM) in the produced droplets. Cells and anti-CD137 antibody were injected separately in two parallel flows from two different inlets. The results of the experiment are shown in Figure 1.

[0383] As exemplified by the change in maximum peak detection across the UserInt signal, a minimum anti-CD137 antibody concentration of 20 nM in a 100 pL droplet was required to efficiently detect antibody relocalization on the surface of T cells. Therefore, the sensitivity of the assay was calculated as the minimum number of molecules expressed on the surface of T cells that would lead to sufficient antibody relocalization and detection of a fluorescent signal. The calculated number of molecules in this assay was 120,460 molecules of CD137 per activated T cell, leading to the detection of 80% of activated T cells. In such an assay format, lower CD137 expression would preclude detection of activated T cells.

[0384] Example 2: T cell activation in droplets To assess the efficiency of T cell activation in droplets and the sensitivity of signal detection based on CD137 activation marker expression in droplets, T cells were stimulated with antigen-presenting cells and activation with anti-CD137 antibodies was monitored under conditions conducive to efficient CD137 detection (defined above).

[0385] K562 cells were stained with 1 μM cell trace (red) and then loaded with 10 μM of a viral peptide (Epstein-Barr virus antigen BMLF1). (In other experiments, the peptide can be any T cell antigen, including viral, bacterial, or parasitic antigens.) These cells were resuspended in a parallel flow containing complete X vivo medium with 5% human serum supplemented with Pluronic F68 (0.1%), 23.6% Nycodenz, and 6% DNA marker (Nucgreen). Meanwhile, a T cell clone specific for the BMLF1 antigen was labeled with 5 μM cell trace (yellow) and suspended in the same parallel flow composition as described above. Anti-CD137 BV421 antibody was added to the K562 cell suspension at 40 nM, resulting in a droplet concentration of 20 nM after the two cell suspensions were parallel flowed separately in two different inlets. Therefore, 100 picoliters (pl) of droplets were generated using 2% HFE surfactant. These droplets were sorted based on expression of markers of interest and their localization in / on T cells after incubation at 37°C and 5% CO. Figure 2 shows an overview of T cell activation in droplets.

[0386] Figure 3 shows data collected using the schematic approach described in Figure 2. Briefly, 100 picoliter (pl) droplets were produced to simultaneously encapsulate two cell types (i.e., K562 cells and T cells). These droplets were collected in a 5 ml tube containing 0.1% HFE surfactant. The tube was incubated overnight at 37 °C. These droplets were then injected into a microfluidic device, and the droplets were examined for CD137 expression in droplets simultaneously containing viable K562 and T cells labeled with different colors. User integration, a feature in the microfluidic device, allowed for estimation of the area under the peak, which was used to remove any potential background and nonspecific signal (Figure 3).

[0387] Efficient (approximately 94%) T cell activation with minimal nonspecific detection (0%-0.1%) was confirmed by detecting 20 nM of CD137 antibody in droplets using high-affinity antigen and T cell clones.

[0388] Example 3: Antigen-presenting cell (APC)-T cell interactions using different readouts To confirm that the assay is specific to the association / interaction of T cells and APC cells in a droplet format at the single-cell level, T cell stimulation was performed with antigen-presenting cells in droplets, and cell-cell interactions were monitored in bright field. Activation with anti-CD137 antibody, T cell killing activity (using the NucGreen readout), and controlled specific localization were monitored for each readout.

[0389] For this experiment, cells were stained with specific fluorometric constructs and checked under a fluorescence microscope. K562 cells were labeled with 5 μM cell trace (purple) in PBS and then loaded with 10 μM peptide, while T cells were labeled with 5 μM cell trace (yellow) in PBS. Both cells were resuspended in parallel flows containing X vivo medium with 5% human serum supplemented with Pluronic F68 (0.1%), 23.6% Nycodenz, and 6% DNA marker (Nucgreen). Before droplet production began, 40 nM of antibody CD137 APC was added to the parallel flow of K562 cells, while Droplet Code was added at the desired concentration to the parallel flow of T cells. After overnight incubation at 37 °C and 5% CO2, the droplets were checked under a fluorescence microscope. These images showed clear localization of the red signal (CD137) on viable T cells (yellow) but not on viable T cells (purple) (Figure 4). Nucgreen is a viability marker that can be used to control the coincidence (and kinetics) of T cell killing activity (or not) by co-expressing activation markers.

[0390] It was confirmed that T-APC cells associate and interact within the droplets, that activated T cells produce / express detectable CD137 protein as visualized by relocalization of CD137 antibody on the surface of the T cells, and that no killing activity was detected during the course of the experiment, confirming that relocalization of CD137 antibody is not due to dead / dying T cells.

[0391] Example 4: Use of anti-IFN-γ antibodies for the detection of activated cells To assess the efficiency of T cell activation in droplets under physiologically relevant conditions by T cells and APCs (APCs presenting CMV pp65 viral antigen) and the sensitivity of signal detection based on cytokine secretion, IFNγ activation marker expression was used as a readout. T cell stimulation was performed by antigen-presenting cells, and activation was monitored using an anti-IFNγ antibody under conditions that resulted in efficient IFNγ detection (not shown).

[0392] K562 cells were labeled with 5 μM Cell Trace (purple) and then loaded with 10 μM peptide (or not). The K562 cells were then washed by adding chilled buffer and spun down at 4°C for 10 minutes. The cells were then resuspended in a parallel flow of X vivo medium containing 5% human serum supplemented with Pluronic F68 (0.1%), 23.6% Nycodenz, and 6% DNA marker (Nucgreen). An anti-IFN-γ detection antibody was added to the cell suspension at a 1 / 25 dilution. For effector cells, T cells were labeled with Cell Trace (yellow) and then with a bispecific antibody targeting CD45 and IFN-γ (1 / 10 dilution in chilled medium, 5 minutes at 4°C, 10 μM / ml cell concentration). The T cells were then washed and resuspended in a parallel flow containing X Vivo medium with 5% human serum supplemented with Pluronic F68 (0.1%), 23.6% Nycodenz, 6% DNA marker (Nucgreen), and the appropriate concentration of dropcode. Finally, these cells were separately parallel flowed into a microfluidic device, thus producing 100 μl droplets, which were then incubated overnight at 37 °C and 5% CO2. A similar experiment was performed using K562 cells transfected with 10 μg of mRNA encoding a fluorescent reporter and the viral peptide CMV pp65. The results of the experiment are shown in Figure 5.

[0393] Using cytokine secretion (e.g., IFNg) as a readout in droplets using high affinity antigen and T cell clones, specific T cell activation was detected with minimal nonspecific detection.

[0394] Example 5: Recovery of TCR and antigen linkage sequences from enriched cells using the CellCap device To retrieve antigens and / or TCR sequences, cells with the desired phenotype were sorted into a microfluidic chip called CellCap. After visual inspection of the phenotype, information on TCR and antigen pairs was retrieved by barcoding single cells using barcoded beads in droplets containing enriched droplets of antigen-expressing APCs and T cells. Alternative droplet and sequence recovery methods were possible.

[0395] During cell sorting into the cell cap, the prepared barcoded bead library was washed five times in 5 ml of 1x BW buffer (20 mM Tris-HCl (pH 8.0), 50 mM NaCl, 0.1% Tween 20). The hydrogel beads were then spun down at 3200 g for 2 minutes at 4°C, followed by denaturation with 1 ml of denaturing solution (970 μl HO + 30 μl 10 M NaOH, 300 mM final) for 2 minutes at room temperature (RT). The barcoded beads were then washed three times with 5 ml of BW buffer, after which the barcoded beads were labeled with biotinylated FITC (5 μM final concentration) for 10 minutes at room temperature. After one wash, the barcoded beads were resuspended in 1 ml of library buffer (Tris (pH 8), 10 mM, EDTA 0.1 mM, Tween 20 0.1%), heated to 70°C for 2 minutes, and spun down at 3200g for 2 minutes at 4°C. The pellet was resuspended in a buffer containing first strand buffer, Igepal CA_630, sulforhodamine B, and nuclease-free water, and run separately in parallel with the reverse transcription (RT) mix in a separate inlet.

[0396] Once the CellCap was filled with 100 μl droplets containing cells, the CellCap reservoir was inverted and the air was removed from the chip using a syringe (without a chamber) connected to the inlet. HFE was flushed at 2000 μl / hr to completely remove any air bubbles from the system. The flow was then reduced to 150 μl / hr, and the tubing containing the barcoded bead emulsion was replaced until each well was filled with 1 nl droplets, flushing all remaining and floating droplets out of the chip. The droplets were then flushed to 5 μl / hr. * After fusion for 5 s and clamping both ends (inlet and outlet), reverse transcription (RT) was initiated using a thermomixer, and the droplets were incubated at 50 °C for 2 h and then maintained at 4 °C after which RT was inactivated and the library preparation was processed for sequencing. Figure 6 shows an overview of a representative workflow.

[0397] Figure 7 shows a representative diagram of predicted sequencing data recovered from TCR α and β and antigen to identify cognate pairs of receptor and ligand.

[0398] While the present invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to those skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope of the invention.

Claims

1. 1. A method for identifying cognate pairs of a ligand and receptor species, comprising: a. providing a set of ligand species; wherein each ligand species is displayed at least once within a microfluidic droplet, microcapsule, or microbead; b. Providing a set of receptor species; wherein each receptor species is displayed at least once; c. contacting said set of ligand species with said set of receptor species in a microreactor; wherein an enhanced signal is produced upon selective binding of the receptor species to the ligand species; d. detecting a cognate pair of ligand and receptor species by production of said signal within said microfluidic droplet, microcapsule, or microbead under flow conditions; and e. Identifying said cognate pairs of ligand and receptor species. Including, method.

2. 10. The method of claim 1, each ligand species and / or each receptor species comprises a barcode sequence; method.

3. 3. The method of claim 1 or 2, Each ligand species and / or each receptor species is expressed or presented on the surface of a cell or bead, or is expressed or present in a cell-free extract or in solution; method.

4. 4. The method of claim 3, the cell is an antigen-presenting cell selected from a macrophage, a dendritic cell, a Langerhans cell, a B cell, a monocyte-derived dendritic cell, or another cell that expresses an MHC class I or II molecule; method.

5. 3. The method of claim 1 or 2, the microreactor is selected from an aqueous droplet, a microcapsule, a microbead, a compartment of a microfluidic chip, or a well; and / or the signal is selected from a morphological change of any one of a cell, a ligand, or a receptor; a fluorescent signal enhancement; a modification of a fluorescent signal using a caged compound or by a quenching reaction; light absorption; a modification / generation of a visible structure; or a combination of these signals. method.

6. 3. The method of claim 2, wherein identifying cognate pairs of ligand and receptor species comprises amplifying said ligand and / or receptor species, and at least one of the amplified ligand and receptor species is sequenced for identification; method.

7. 3. The method of claim 1 or 2, (a) the set of ligand species is selected from T cell antigens, B cell antigens, viral antigens, bacterial antigens, parasitic antigens, neoantigens, tumor-associated antigens (TAA), tumor-specific antigens, immune checkpoint molecules, cytokines, carbohydrates, members of the immunoglobulin superfamily, selectins, chemokines, hormones, growth factors, G protein-coupled receptor ligands, or enzyme substrates; and / or (b) the set of receptor species is selected from T cell receptors, B cell receptors, immune checkpoint receptors, cytokine receptors, selectins, integrins, members of the immunoglobulin superfamily, cadherins, chemokine receptors, hormone receptors, growth factor receptors, G protein-coupled receptors (GPCRs), or enzymes. method.

8. 3. The method of claim 1 or 2, a) the ligand species is a T cell antigen and the receptor species is a T cell receptor, and the enhanced signal is produced upon selective binding of the T cell receptor to the T cell antigen, and the produced enhanced signal is a result of T cell activation; b) the ligand species is a viral antigen and the receptor species is a T cell receptor, and the enhanced signal is produced upon selective binding of the T cell receptor to the viral antigen, and the produced enhanced signal is the result of T cell activation; or c) the ligand species is a B cell antigen and the receptor species is a B cell receptor, and the enhanced signal is produced upon selective binding of the B cell receptor to the B cell antigen. method.

9. 9. The method of claim 8, contacting the set of ligand species with the set of receptor species in a microreactor; a) from about 0.001 hours to about 8 hours; or b) occurs over at least about 8 hours; method.

10. 10. The method of claim 9, the ligand species and the receptor species bind with high affinity; the enhanced signal produced is an early or late marker of T cell activation; method.

11. 10. The method of claim 9, the ligand species and the receptor species bind with low affinity; the enhanced signal produced is an early or late marker of T cell activation; method.

12. 12. The method of claim 10 or 11, (a) the early marker of T cell activation is selected from CD69, CD107a, or transferrin receptor; and / or (b) the late marker of T cell activation is selected from CD137, HLA-DR, VLA1, PTA1, CD71, CD27, PD-1, TIM3, LAG3, or CTLA4. method.

13. 13. The method of claim 12, the enhanced signal is detected by an anti-CD69 antibody, an anti-CD107a antibody, an anti-transferrin receptor antibody, an anti-CD137 antibody, an anti-HLA-DR antibody, an anti-VLA1 antibody, an anti-PTA1 antibody, an anti-CD71 antibody, an anti-CD27 antibody, an anti-PD1 antibody, an anti-TIM3 antibody, an anti-LAG3 antibody, or an anti-CTLA4 antibody; method.

14. 14. The method of claim 13, The signal is detected by an anti-CD138 antibody, an anti-CD19 antibody, an anti-CD45R antibody, an anti-CD45 antibody, activation of fluorescent reporter expression, or inhibition of fluorescent reporter expression. method.

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