Method for screening target cell and cell surface receptor

Abstract

The present application relates to a method for screening a target cell, wherein the target cell expresses, on the surface thereof, a receptor capable of binding to a target ligand. The present application further relates to a target cell obtained by means of the method, and a receptor binding to a target ligand.

Description

A method for screening target cells and cell surface receptors TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to a method for screening target cells and cell surface receptors. BACKGROUND

[0002] The binding of cell surface receptors and ligands reflects two important dimensions: specificity and affinity. Examples of cell surface receptors with potential therapeutic applications for diseases are B cell surface receptors (BCR) and T cell surface receptors (TCR), whose ligands are antibodies and antigen-MHC complexes, respectively. Among them, monoclonal antibodies (mAbs) have the characteristics of strong specificity and the ability to enhance immune response, and have become an important drug for the treatment of cancer and immune diseases. Although technology is constantly updated, screening for high-affinity antibodies is still an expensive and laborious task, as it requires testing and validation from the expressed antibody pool. Over the past few decades, methods for therapeutic antibodies have mostly been based on hybridoma technology, in vitro display technology, and ex vivo single B cell detection, which require high technology or low affinity or insufficient throughput. Next-generation sequencing (NGS) has been applied to discover single B cell VH and VL sequences through microwell plates or droplets, which can achieve high throughput but cannot be used for antibody screening, such as 10X Genomics V(D)J sequencing. Combined with proteomics technology, LIBRA-seq (Linking B cell receptors to antigen specificity by sequencing) emerged, which added barcoded antigens and could read the labeled antigen abundance on a single B cell, with the characteristics of high throughput and applicability to many targets, but could not read other functional information of antibodies, especially the affinity to antigens. Most high-affinity functional antibody screening methods are fluorescence-based, such as the Beacon platform and CelliGO, which rely on expensive specialized instruments and cannot achieve high-throughput sequencing. Moreover, it is well known that high antigen binding rates on B cells can be due to high levels of BCR or high affinity of BCR, so when antibodies are sorted only according to the amount of bound antigen, the true antibody binding affinity cannot be accurately represented.

[0003] Therefore, a highly efficient and high-throughput method for discovering receptor-specific high-affinity ligands is crucial for the prevention and treatment of future diseases. SUMMARY

[0004] The present application provides a cell screening method for obtaining the affinity of cell surface receptors and their ligands based on high-throughput sequencing or low-throughput signal values. The method of the present application can significantly improve the screening efficiency of high-affinity ligands.

[0005] In one aspect, the present application provides a method for screening a target cell, the surface of which expresses a receptor capable of binding a target ligand, the method comprising the following steps: (1) providing a cell population expressing one or more cell surface receptors; (2) providing the target ligand with a first label; (3) providing a labeling protein with a second label, the labeling protein being capable of binding the receptor and the binding not being through the binding epitope of the receptor to the target ligand; (4) contacting the cell population in (1) with the target ligand in (2) and the labeling protein in (3); (5) obtaining a ligand-receptor affinity value of one or more cells in the cell population, wherein the ligand-receptor affinity value = signal value of the first label / signal value of the second label, (6) determining whether one or more cells in the cell population is the target cell according to the ligand-receptor affinity value of one or more cells obtained in (5). In other embodiments, the ligand-receptor affinity value = ln(signal value of the first label / signal value of the second label). It should be noted that the principle of the ligand-receptor affinity value of the present application is the ratio of two signal values, whether to take ln does not affect the judgment of multiple ratios, any method that can compare the ratio of two signal values can be used for the calculation method of the ligand-receptor affinity value of the present application.

[0006] In certain embodiments, the first label is a fluorescent label and / or a barcode.

[0007] In certain embodiments, the first label is a fluorescent label. In certain embodiments, the first label is a barcode.

[0008] In certain embodiments, the second label is a fluorescent label and / or a barcode. In certain embodiments, the second label is a fluorescent label. In certain embodiments, the second label is a barcode.

[0009] In certain embodiments, the fluorescent label is selected from the group consisting of Alexa Fluor series, FITC, 5-FAM, Cy5, Cy3, Cy2, TRITC, 5-TAMRA, HEX, Rhodamine B, and AMC.

[0010] In certain embodiments, the barcode comprises a DNA sequence or an RNA sequence. In certain embodiments, the barcode comprises a cell barcode. In certain embodiments, the barcode comprises a molecular barcode. In certain embodiments, the barcode comprises a unique molecular identifier (UMI). In certain embodiments, the barcode is delivered by a bead.

[0011] In certain embodiments, the first label is different from the second label. For example, the first and second labels are different sequences of DNA sequences or RNA sequences. For example, the first and second labels are different fluorescent labels.

[0012] In certain embodiments, the population of cells in (1) comprises a population of immunized B cells. For example, the population of immunized B cells can be immunized with an antigen that is SARS-Cov-2, OVA.

[0013] In certain embodiments, the antigen for immunization comprises an antigen from a pathogen or an animal. In certain embodiments, the antigen for immunization is ovalbumin OVA.

[0014] In certain embodiments, the antigen for immunization comprises an antigen from a virus. In certain embodiments, the antigen for immunization comprises an antigen from an influenza virus. In certain embodiments, the antigen for immunization comprises an antigen from a coronavirus. In certain embodiments, the antigen for immunization comprises an antigen from SARS-CoV-2.

[0015] In certain embodiments, the antigen for immunization comprises a tumor antigen. In certain embodiments, examples of the antigen for immunization can include, but are not limited to, selected from the group consisting of PD-1, HER2, CD20, CD3, CTLA4, PSMA, EGFR, EGFRviii, FAP, CD33, HER3, SIRPa, DLK1, PTK7, GPR20, CD71, CD16, CAIX, CAXII, CXCR3, CXCR5, CXCR4, GRPR, CD70, CD46, CD166, CD36, CD73, CD38, CD51, FGFR3, FcRH5, VEGF, VEGFR2, CD45, CCR4, CD25, ROR1, TROP-2, NECTIN4, cMET, CD19, CD22, CD30, CD33, CD123, BCMA, CD79b, AXL, RON, B7-H3, B7-H4, CD47, TNFa, IL17A, IL4Ra, IL23R, and EpCAM.

[0016] In certain embodiments, the target ligand with the first label is an antigen with a first fluorescent label and / or a first barcode.

[0017] In certain embodiments, the antigen comprises an antigen from a pathogen or an animal. For example, the antigen is ovalbumin OVA.

[0018] In certain embodiments, the antigen from a pathogen comprises an antigen from a virus. For example, the antigen from a virus comprises an antigen from an influenza virus. For example, the antigen from a virus comprises an antigen from a coronavirus. For example, the antigen from a virus comprises an antigen from SARS-CoV-2.

[0019] In certain embodiments, the antigen comprises a tumor antigen. Examples of tumor antigens can include, but are not limited to, selected from the group consisting of PD-1, HER2, CD20, CD3, CTLA4, PSMA, EGFR, EGFRviii, FAP, CD33, HER3, SIRPa, DLK1, PTK7, GPR20, CD71, CD16, CAIX, CAXII, CXCR3, CXCR5, CXCR4, GRPR, CD70, CD46, CD166, CD36, CD73, CD38, CD51, FGFR3, FcRH5, VEGF, VEGFR2, CD45, CCR4, CD25, ROR1, TROP-2, NECTIN4, cMET, CD19, CD22, CD30, CD33, CD123, BCMA, CD79b, AXL, RON, B7-H3, B7-H4, CD47, TNFa, IL17A, IL4Ra, IL23R, and EpCAM.

[0020] In certain embodiments, the labeled protein with the second label is a secondary antibody with a second fluorescent label and / or a second barcode.

[0021] In certain embodiments, after (4) and before (5), the method comprises performing cell sorting. Through cell sorting, one or more cells are isolated from the target cell population as single cells present in separate chambers.

[0022] In certain embodiments, wherein in (5), the signal value of the first label and / or the second signal value is a fluorescent intensity.

[0023] In certain embodiments, wherein (6) comprises comparing the ligand-receptor affinity value of the one or more cells to a reference ligand-receptor affinity value, and determining the one or more cells as the target cell if the ligand-receptor affinity value of the one or more cells is greater than the reference ligand-receptor affinity value.

[0024] In some embodiments, the reference ligand-receptor affinity value is derived from the one or more cell ligand-receptor affinity values. The main purpose of the reference ligand-receptor affinity value is to set a boundary for the range of ligand-receptor affinity values of the cells, so that ligands with relatively higher affinity can be selected. The reference ligand-receptor affinity value is a relative concept, which is adjusted according to the degree of affinity required in each experiment. The ligand-receptor affinity value is not a fixed value, but is derived from the one or more cell ligand-receptor affinity values in a specific embodiment, and the relative affinity required, and is used to set a boundary for selection. In a specific embodiment, after obtaining the one or more cell ligand-receptor affinity values, if it is required to screen ligands with relatively higher affinity, the reference ligand-receptor affinity value can be set to a certain value, so as to exclude ligands with lower affinity than the reference value. If it is required to screen ligands with higher affinity, the reference ligand-receptor affinity value can be set to a higher value, so as to exclude more ligands with lower affinity than the reference value.

[0025] In some embodiments, the target ligand has high affinity to the receptor.

[0026] In another aspect, the present application provides a system and / or device for screening target cells, the surface of the target cells expressing a receptor capable of binding to a target ligand, the method comprising the following steps: (1) providing a cell population expressing one or more cell surface receptors; (2) providing the target ligand with a first label; (3) providing a labeled protein with a second label, the labeled protein being capable of binding to the receptor, and the binding not being through the binding epitope of the receptor and the target ligand; (4) contacting the cell population in (1) with the target ligand in (2) and the labeled protein in (3); (5) obtaining a ligand-receptor affinity value of one or more cells in the cell population, wherein the ligand-receptor affinity value = the signal value of the first label / the signal value of the second label, (6) determining whether one or more cells in the cell population are the target cells according to the ligand-receptor affinity value of one or more cells obtained in (5).

[0027] In another aspect, the present application provides the target cells obtained by the method.

[0028] In another aspect, the present application provides a method for screening a target B cell, which expresses a B cell receptor capable of binding to an antigen on its surface: (1) providing a population of immunized B cells, (2) providing the antigen with a first label, (3) providing a labeled protein with a second label, which is capable of binding to the receptor and the binding is not through the binding epitope of the receptor to the target ligand, (4) contacting the population of B cells in (1) with the antigen in (2) and the labeled protein in (3), (5) obtaining a ligand-receptor affinity value of one or more B cells in the population of B cells, wherein the ligand-receptor affinity value = the signal value of the first label / the signal value of the second label, (6) determining whether one or more cells in the population of B cells is the target B cell according to the ligand-receptor affinity value of the one or more B cells obtained in (5).

[0029] In another aspect, the present application provides a system and / or device for screening a target B cell, which expresses a B cell receptor capable of binding to an antigen on its surface: (1) providing a population of immunized B cells, (2) providing the antigen with a first label, (3) providing a labeled protein with a second label, which is capable of binding to the receptor and the binding is not through the binding epitope of the receptor to the target ligand, (4) contacting the population of B cells in (1) with the antigen in (2) and the labeled protein in (3), (5) obtaining a ligand-receptor affinity value of one or more B cells in the population of B cells, wherein the ligand-receptor affinity value = the signal value of the first label / the signal value of the second label, (6) determining whether one or more cells in the population of B cells is the target B cell according to the ligand-receptor affinity value of the one or more B cells obtained in (5).

[0030] In another aspect, the present application provides the target B cell obtained by the method.

[0031] In another aspect, the present application provides a method for screening high affinity antibodies, comprising: (1) providing a population of immunized B cells, the B cell population comprising one or more B cells expressing a B cell receptor (BCR), (2) providing an antigen with a first label, (3) providing a label protein with a second label, the label protein being capable of binding to the BCR, and the binding not being through the binding epitope of the antigen by the BCR, (4) contacting the B cell population in (1) with the antigen in (2) and the label protein in (3), (5) obtaining a ligand-receptor affinity value of one or more B cells in the B cell population, wherein the ligand-receptor affinity value = signal value of the first label / signal value of the second label, (6) comparing the ligand-receptor affinity value of the one or more B cells with a reference ligand-receptor affinity value, and if the ligand-receptor affinity value of the one or more cells is greater than the reference ligand-receptor affinity value, determining the one or more cells as target B cells, (7) sequencing the BCR of the target B cells to obtain the high affinity antibodies.

[0032] In another aspect, the present application provides high affinity antibodies obtained by the method.

[0033] In another aspect, the present application provides a pharmaceutical composition comprising the high affinity antibodies.

[0034] In another aspect, the present application provides a method for screening target T cells, the surface of the T cells expressing a T cell receptor capable of binding to an antigen: (1) providing a population of immunized T cells, (2) providing the antigen with a first label, (3) providing a label protein with a second label, the label protein being capable of binding to the receptor, and the binding not being through the binding epitope of the target ligand by the receptor, (4) contacting the T cell population in (1) with the antigen in (2) and the label protein in (3), (5) obtaining a ligand-receptor affinity value of one or more T cells in the T cell population, wherein the ligand-receptor affinity value = signal value of the first label / signal value of the second label, (6) determining whether one or more cells in the T cell population are the target T cells according to the ligand-receptor affinity value of the one or more T cells obtained in (5).

[0035] In another aspect, the present application provides a system and a device for screening target T cells, the surface of which expresses T cell receptors capable of binding to antigens: (1) providing a population of immunized T cells, (2) providing the antigen with a first label, (3) providing a labeled protein with a second label, the labeled protein being capable of binding to the receptor, and the binding not being through the binding epitope of the receptor to the target ligand, (4) contacting the population of T cells in (1) with the antigen in (2) and the labeled protein in (3), (5) obtaining the ligand-receptor affinity value of one or more T cells in the population of T cells, wherein the ligand-receptor affinity value = the signal value of the first label / the signal value of the second label, (6) determining whether one or more cells in the population of T cells are the target T cells according to the ligand-receptor affinity value of one or more T cells obtained in (5).

[0036] In another aspect, the present application provides the target T cells obtained by the method.

[0037] One application of the method of the present application is to screen B cells by obtaining the affinity of B cell surface receptors (or antibodies) to antigens based on high-throughput sequencing and / or low-throughput fluorescent reading. In one embodiment, using OVA protein as an antigen, it is verified that the ligand-receptor affinity value obtained by the method of the present application is highly correlated with the ELISA result, indicating that the method of the present application can be used for efficient screening of antibodies, especially high-affinity antibodies. In another embodiment, using SARS-CoV-2 protein as an antigen, effective SARS-CoV-2 antibodies are identified, and 10 candidate antibodies obtained according to the method of the present application all show excellent biological activity in subsequent production and verification. However, the antibody screening method commonly used in the art usually needs to screen hundreds to thousands of candidate antibodies, and therefore, in the application of screening B cells or screening antibodies, the efficiency of the method of the present application is significantly improved compared with the screening method reported in the literature.

[0038] In the screening based on high-throughput sequencing, the method of the present application can use barcoded oligonucleotides to label cell surface receptors and their ligands, so that it can be used to simultaneously detect the binding of multiple cell surface receptors and ligands, i.e., the method of the present application can simultaneously screen receptors (e.g., antibodies) with high binding capacity for multiple ligands (e.g., antigens).

[0039] In the screening based on low-throughput methods (e.g., using fluorescent labeling), the method of the present application can not contain a high-throughput sequencing step, and the use of low-throughput signal values (e.g., fluorescence intensity values) can also accurately reflect the binding affinity of receptors and ligands, so as to screen target cells or their surface receptors in a convenient, fast and low-cost manner.

[0040] In addition to the B cell screening method as an example, the method of the present application can also be used to screen any target cells expressing surface receptors, or surface receptors with high affinity to specific ligands. For example, using labeled antigen peptides to contact T cells expressing TCR, T cells or TCRs capable of high affinity to antigen peptides can be obtained according to the method of the present application. For example, the method of the present application can also be used to screen antigen peptides or tumor neoantigens with high affinity to T cells or TCRs.

[0041] Other aspects and advantages of the present application will be readily appreciated by those skilled in the art from the detailed description that follows, when considered in connection with the accompanying drawings. The detailed description shows and describes only examples of the present application. As will be realized by those skilled in the art, the application is capable of modifications in the light of the above teachings. Accordingly, the drawings and descriptions are to be regarded as illustrative in nature and not restrictive. BRIEF DESCRIPTION OF DRAWINGS

[0042] The specific features of the application involved in the present application are shown in the appended claims. The features and advantages of the application involved can be better understood by referring to the detailed description of the exemplary embodiments and the accompanying drawings. A brief description of the drawings is as follows:

[0043] Figure 1 shows the detection schematic flow of the method described in the present application. B cells are isolated and enriched from immunized mice by density gradient centrifugation and MACS. Then stained with fluorescently labeled DNA barcode antigen and secondary antibody, followed by FACS sorting. Antigen positive B cells are collected and high-throughput single-cell V(D)J is performed using Feature Barcode technology. For this, bead-delivered oligonucleotides index BCR transcripts and antigen / secondary antibody barcodes during reverse transcription, enabling direct mapping of BCR sequences to antigen / secondary antibody information after sequencing. The number and position of oligonucleotides and fluorescent molecules on each antigen and secondary antibody can vary.

[0044] Figure 2 shows all B cells (dots) recovered from the AAB-seq experiment (n = 3,712) with paired heavy chain / light chain sequencing information and antigen / secondary antibody reactivity information, as well as the OVA score (x-axis) and ligand-receptor affinity (i.e., AAB-seq score) (y-axis) of LIBRA-seq. Dispersively selected antibodies for expression and validation (colored dots) are colored from lowest (purple) to highest (red) LG-AUC score.

[0045] Figure 3 shows sequence features and antigen specificity of the dispersed selected antibodies from OVA immunized mice. Data are presented as mean ± SD, n > 2. AAB-seq scores and lg(ELISA_AUC) of the selected antibodies are shown as a heatmap from minimum (light yellow) to maximum (purple). CDRH3 sequences of the antibodies are shown as SEQ ID NOs: 8-25, and CDRL3 sequences are shown as SEQ ID NOs: 26-41.

[0046] Figure 4 shows that OVA antigen-specific selected antibodies were verified as expected by ELISA. Data are presented as mean ± SD, n = 3.

[0047] Figure 5 shows that OVA antigen-specific selected high AAB score antibodies were verified as expected by ELISA. Data are presented as mean ± SD, n = 3.

[0048] Figure 6 shows that all B cells (dots) recovered (n = 3,712) are shown as in (A). Selected high AAB score antibodies (colored dots) are colored by LG-AUC score, scale consistent with (B).

[0049] Figure 7 shows sequence features and antigen specificity of the selected high AAB score antibodies from OVA immunized mice. Data are presented as mean ± SD, n > 2. AAB-seq scores and lg(ELISA_AUC) of the selected antibodies are shown as a heatmap from minimum (light yellow) to maximum (purple). CDRH3 sequences of the antibodies are shown as SEQ ID NOs: 42-51, and CDRL3 sequences are shown as SEQ ID NOs: 29, 36, and 52-58.

[0050] Figure 8 shows that OVA antigen-specific selected high AAB score antibodies were verified by ELISA. Data are presented as mean ± SD, n = 3.

[0051] Figure 9 shows that all B cells (dots) recovered from the AAB-seq experiment of SARS-COV-2 RBD antibodies (n = 3,712) with paired heavy / light chain sequencing information and antigen / secondary antibody reactivity information are shown in the plot, and CLR (RBD_UMI) (x-axis) and AAB score (y-axis). Dispersed selected antibodies for expression and verification (colored dots) are colored by LG (ELISA_AUC) score from lowest (purple) to highest (red).

[0052] Figure 10 shows sequence features and antigen specificity of selected antibodies in SARS-COV-2 RBD immunized mice. Data are presented as mean ± SD, n > 2. AAB-seq scores and lg(ELISA_AUC) of selected antibodies are shown as a heatmap from minimum (light yellow) to maximum (purple). CDRH3 sequences of the antibodies are shown as SEQ ID NOs: 59-65, and CDRL3 sequences are shown as SEQ ID NOs: 66-71.

[0053] Figure 11 shows the predicted SARS-CoV-2 RBD antigen-specific antibodies were verified by ELISA. Data are presented as mean ± SD, n = 3.

[0054] Figure 12 shows BLI measurements to determine the affinity of selected antibodies to RBD.

[0055] Figure 13 shows neutralization assays using pseudoviruses displaying the S protein of SARS-CoV-2 WT shown. Values are mean ± SD, n = 3.

[0056] Figure 14 shows SARS-CoV-2 WT pseudovirus neutralization assay data (%). Data are presented as mean ± SD, n = 3.

[0057] Figure 15 shows antibodies were tested for antibody-dependent cellular phagocytosis activity (ADCP) against SARS-CoV-2 RBD compared to the positive control MM17T. The AUC of phagocytosis score is shown in the graph, which is calculated from the data in Figure S4B. Data are presented as mean ± SD, n = 3.

[0058] Figure 16 shows antibodies were tested for antibody-dependent complement deposition (ADCD) activity against SARS-CoV-2 RBD compared to the positive control MM17T. The AUC of C3b deposition score is shown, which is calculated from the data in Figure S4D. Data are presented as mean ± SD, n = 3.

[0059] Figure 17 shows antibodies were tested for antibody-dependent cellular phagocytosis activity against SARS-CoV-2 RBD compared to the positive control MM17T. Phagocytosis score (see Methods) is shown on the y-axis, and antibody concentration is shown on the x-axis. Data are presented as mean ± SD, n = 3.

[0060] Figure 18 shows phagocytosis was confirmed by confocal microscopy images. Nuclei are blue, beads are red, and membranes are green with concanavalin A (FITC-ConA).

[0061] Figure 19 shows a schematic flow of the detection method described herein.

[0062] Figure 20 shows the Pearson correlation of Fluo_Ratio (x-axis) and oligo ratio (y-axis). DETAILED DESCRIPTION

[0063] The following examples illustrate the practice of the application in specific embodiments, and it will be apparent to those skilled in the art that many more embodiments can be practiced based on the disclosure herein.

[0064] In the present application, the terms "isolated," "purified," and their grammatical equivalents, as used herein, refer to a reduction in the amount of at least one contaminant (such as a protein and / or nucleic acid sequence) from a sample or source (e.g., a cell) from which the material is isolated. Thus, purification results in "enrichment," e.g., an increase in the amount of a desired protein and / or nucleic acid sequence in a sample.

[0065] In the present application, "high affinity" or "high affinity" is generally used to describe a molecule (such as an antibody, ligand, receptor, probe, etc.) that has a strong binding ability with its target. In one embodiment, "high affinity" in the present application refers to the binding between the ligand and its target with a dissociation constant (Kd) less than 1 x 10 -9 M, preferably less than 1 x 10 -10 M, which can be determined by Surface Plasmon Resonance (SPR) technology, for example, using a Biacore TM instrument at 25°C in PBS buffer at neutral pH. In one embodiment, "high affinity" in the present application refers to the ability of an antibody to achieve specific binding and produce a detectable signal at a concentration of less than 1 μg / mL, for example, in ELISA, FACS, immunoblotting.

[0066] In the present application, the term "comprising" generally indicates a range or elements that includes other elements or features. This is used to define the scope of a particular technology more flexibly and inclusively. When the word "comprising" is used, it means that the scheme includes not only the explicitly listed elements, but also other related elements or features that can achieve similar functions.

[0067] In the present application, the term "and / or" is generally used to indicate a selection range, indicating an inclusive relationship between two or more elements or conditions. When "and / or" is used, it allows one element or condition, or both, to exist to achieve the same technical effect. For example, if the description contains expressions such as "A and / or B", it means that it can include A or B, or both A and B.

[0068] DETAILED DESCRIPTION

[0069] Method for screening target cells

[0070] In one aspect, the present application provides a method for screening target cells, the surface of which expresses a receptor capable of binding a target ligand, the method comprising the steps of:

[0071] (1) providing a cell population expressing one or more cell surface receptors;

[0072] (2) providing the target ligand with a first label;

[0073] (3) providing a labeled protein with a second label, the labeled protein being capable of binding the receptor, and the binding not being through the binding epitope of the receptor to the target ligand;

[0074] (4) contacting the cell population in (1) with the target ligand in (2) and the labeled protein in (3);

[0075] (5) obtaining a ligand-receptor affinity value for one or more cells in the cell population, wherein the ligand-receptor affinity value = signal value of the first label / signal value of the second label, and

[0076] (6) determining whether one or more cells in the cell population are the target cells based on the ligand-receptor affinity value(s) obtained for the one or more cells in (5).

[0077] For example, in (1), the cell population can be a population of immunized B cells.

[0078] Thus, in another aspect, the present application provides a method for screening target B cells, the surface of which expresses a B cell receptor capable of binding an antigen:

[0079] (1-1) providing a population of immunized B cells,

[0080] (2-1) providing the antigen with a first label,

[0081] (3-1) providing a labeled protein with a second label, the labeled protein being capable of binding the receptor, and the binding not being through the binding epitope of the receptor to the target ligand

[0082] (4-1) contacting the population of B cells in (1-1) with the antigen in (2-1) and the labeled protein in (3-1),

[0083] (5-1) obtaining a ligand-receptor affinity value for one or more B cells in the population of B cells, wherein the ligand-receptor affinity value = signal value of the first label / signal value of the second label, and

[0084] (6-1) determining whether one or more cells in the B cell population are the target B cell based on the ligand-receptor affinity value(s) of the one or more B cells obtained in (5-1).

[0085] For example, the B cell population can be a B cell population comprising B cells or a peripheral blood mononuclear cell (PBMC) population comprising B cells.

[0086] For example, the B cell includes a memory B cell, a plasma cell, a naive B cell, an activated B cell, or a B cell line.

[0087] For example, the method can comprise immunizing an animal with one or more antigens and then isolating the immunized B cell population from the animal. For example, the antigen in the present application can be from a pathogen or an animal. For example, the antigen in the present application can be a tumor antigen. For example, the antigen in the present application can be from a human.

[0088] For example, in (1), the cell population can be a T cell population expressing a T cell surface receptor (TCR).

[0089] Accordingly, in another aspect, the present application provides a method of screening for a target T cell whose surface expresses a T cell receptor capable of binding to an antigen:

[0090] (1-2) providing an immunized T cell population,

[0091] (2-2) providing the antigen with a first label,

[0092] (3-2) providing a labeled protein with a second label, the labeled protein being capable of binding to the receptor and the binding not being through the binding epitope of the receptor to the target ligand,

[0093] (4-2) contacting the T cell population in (1-2) with the antigen in (2-2) and the labeled protein in (3-2),

[0094] (5-2) obtaining a ligand-receptor affinity value of one or more T cells in the T cell population, wherein the ligand-receptor affinity value = signal value of the first label / signal value of the second label, and

[0095] (6-2) determining whether one or more cells in the T cell population are the target T cell based on the ligand-receptor affinity value(s) of the one or more T cells obtained in (5-2).

[0096] In certain embodiments, the cell surface receptor is a receptor protein that is naturally expressed on the surface of a cell. In certain embodiments, the cell surface receptor is a receptor protein that is expressed on the surface of a cell by transgenic means. In certain embodiments, the cell surface receptor is a receptor protein that is expressed on the surface of a cell by transgenic means. In certain embodiments, the cell surface receptor is expressed directly on the surface of a cell. In certain embodiments, the cell surface receptor is indirectly linked to the surface of a cell, for example, by a linker. In certain embodiments, the cell surface receptor is a cell specific receptor.

[0097] In the specific example of a secreted antibody, a linker (e.g., for an antibody or other protein) can be attached to the surface of a B cell that links the secreted antibody such that the secreted antibody is indirectly linked to the surface of a cell.

[0098] In other examples, the linker can be a small molecule linker, a polypeptide, or a nucleic acid.

[0099] The cell surface receptor can be a secreted receptor, a transmembrane receptor, or a non-transmembrane receptor. In addition to the BCR or TCR mentioned above, examples of cell surface receptors can include, but are not limited to, cytokine receptors and growth factor receptors, such as ion channel receptors, G protein-coupled receptors, or enzyme-linked receptors.

[0100] Thus, the methods of the application can be used for screening of any receptor-ligand of interest, as long as the receptor of interest can be linked to the surface of a cell directly or indirectly by artificial means.

[0101] In one specific embodiment, the target cell is a B cell. In certain embodiments, the target cell is a B cell and the receptor is a B cell receptor, or a secreted antibody, for example, the secreted antibody can be linked to the surface of a B cell directly or indirectly.

[0102] In one specific embodiment, the target cell is a T cell. In certain embodiments, the target cell is a T cell and the receptor is a T cell receptor, and the ligand is an antigenic peptide that can bind to the T cell receptor in the context of MHC.

[0103] In one specific embodiment, the target cell is a yeast cell. In certain embodiments, the target cell is a yeast cell and the receptor is an antibody displayed on the surface of a yeast cell, and the ligand is an antigen.

[0104] In the present application, the term "antigen" generally refers to a substance capable of eliciting an immune system to produce antibodies. In some embodiments, an antigen stimulates an immune response, for example by producing antibodies specific to the antigen. Antigens of the present application can be antigens from human immunodeficiency virus, antigens from influenza virus, antigens from respiratory syncytial virus (RSV). Antigens of the present application can also be, for example, human antigens (e.g., oncogene-encoded proteins).

[0105] In certain embodiments, the antigen comprises an antigen from a pathogen or an animal.

[0106] In certain embodiments, the antigen from a pathogen comprises an antigen from a virus. In some embodiments, the antigen from a virus comprises an antigen from human immunodeficiency virus (HIV), an antigen from influenza virus, or an antigen from respiratory syncytial virus (RSV).

[0107] In certain embodiments, the antigen comprises a tumor antigen. In some embodiments, the antigen from an animal comprises an antigen from a human. In some embodiments, the antigen from a human comprises an oncogene-encoded protein, including, for example, HER-2 / neu, RAS, MYC, SRC, telomerase, BCL-2, EGFR, p53, BRCA, Rb, APC, CDKN2A, PTEN, VHL, or WRN.

[0108] In the present application, the term "antibody" is used in a broad sense and includes polyclonal and monoclonal antibodies. In addition to intact immunoglobulin molecules, the term "antibody" includes fragments or aggregates of these immunoglobulin molecules, conjugates, and human or humanized versions of immunoglobulin molecules or fragments thereof, as long as they are selected to be capable of interacting specifically with an antigen. The desired activity of the antibodies can be tested using the in vitro assays described herein or by analogous methods, and then their in vivo therapeutic and / or prophylactic activity can be tested according to known clinical testing methods. The antibodies include the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and their subtypes, e.g., IgGl, IgG2, IgG3, and IgG4, IgAl and IgA2. The term also includes immunoglobulins of different classes of heavy chain constant domains (a, d, e, g, and m, respectively), and immunoglobulins of light chain constant regions, kappa and lambda. "Antibody or antigen-binding fragment thereof" or "antibody or fragment thereof" refers to antibody fragments that retain the ability to bind their specific antigen, including chimeric antibodies and hybrid antibodies with dual or multiple antigen or epitope specificity, as well as fragments, such as F(ab')2, Fab', Fab, Fv, sFv, scFv, etc. or their hybrid fragments. The meaning of "antibody or antigen-binding fragment thereof" also includes conjugates of antibody fragments and antigen-binding proteins (single-chain antibodies). The meaning of "antibody or antigen-binding fragment thereof" also includes immunoglobulin single variable domains, such as nanobodies. Antibodies can include insertions, deletions, substitutions, or other selected modifications of particular regions or particular amino acid residues, as long as the activity of the antibody or fragment is not significantly altered or impaired compared to before.

[0109] First label and second label

[0110] In the methods of the present application, a ligand for a cell surface receptor can be labeled. The appropriate ligand is selected according to the desired binding properties of the surface receptor of the target cell. For example, if B cell surface receptors (or antibodies in their secreted form) capable of binding to SARS-Cov-2 proteins are desired, the SARS-Cov-2 proteins can be labeled. For example, with a first label. The labeled ligand (e.g., antigen) is then contacted with the target cell population to be screened. After incubation under suitable conditions, if the target cell population to be screened contains one or more surface receptors expressing receptors against that particular ligand, the labeled ligand binds to the cell surface via the receptor. The labeled ligand can bind to a specific epitope of the corresponding cell surface receptor.

[0111] The methods of the present application include detecting the signal value of the first label of one or more cells in the target cell population, which reflects the number or level of ligand bound to the surface of each cell.

[0112] For example, the first label can be a fluorescent label and / or a barcode. For example, the first label can be a fluorescent label. For example, the first label can be a barcode.

[0113] In the method of the present application, a labeling protein of a cell surface receptor can be labeled. The labeling protein can bind to one or more cell surface receptors in a target cell population, whether the one or more cell surface receptors can specifically bind to the ligand or not. Thus, the binding of the labeling protein to the cell surface receptor is independent of the binding epitope of the cell surface receptor to its corresponding ligand. Alternatively, the binding of the labeling protein to the cell surface receptor does not affect the binding of the cell surface receptor to its corresponding ligand. For example, the labeling protein is labeled with a second label. The labeled labeling protein is then contacted with a target cell population to be screened.

[0114] In one specific example, the labeling protein is a secondary antibody. The secondary antibody can bind to the BCR. For example, the secondary antibody can bind to the light chain of the BCR. For example, the secondary antibody can bind to the Fc of the BCR. For example, the secondary antibody can be a goat anti-mouse Ig light chain antibody.

[0115] The method of the present application comprises detecting a signal value of the second label of one or more cells in the target cell population, which signal value represents the number or level of receptors expressed on the surface of each cell.

[0116] For example, the second label can be a fluorescent label and / or a barcode. For example, the second label can be a fluorescent label. For example, the second label can be a barcode.

[0117] The first label or the second label of the present application can comprise a fluorescent label.

[0118] The fluorescent labeling techniques in the art can be used in the method of the present application. A fluorescent substance can be used as a fluorescent label. A fluorescent substance refers to a compound having a conjugated double bond system chemical structure, which can be excited to an excited state when irradiated with ultraviolet light or blue-violet light, and emit fluorescence when returning to the ground state from the excited state. The fluorescent labeling technique generally refers to using a fluorescent substance covalently bound or physically adsorbed on a certain group of a molecule to be studied, and using its fluorescent properties to provide information of the object to be studied.

[0119] Common fluorescent labels can include, but are not limited to, a substance selected from the group consisting of Alexa Fluor series, Cyanine series (e.g., Cy5, Cy3, Cy2), Rhodamine series (e.g., Rhodamine 6G, Rhodamine B), DyLight series (e.g., DyLight 488, DyLight 550, DyLight 650) FITC, 5-FAM, TRITC, 5-TAMRA, HEX, and AMC.

[0120] The first label or the second label of the present application can include a barcode label. The barcode of the present application can include a DNA sequence or an RNA sequence or an RNA-DNA hybrid comprising a barcode (also referred to as, barcoded). The barcode can also be single-stranded or double-stranded.

[0121] The barcode of the first label or the second label can comprise a primer sequence (e.g., a universal primer sequence, a target primer sequence, a random primer sequence, a sequencing primer sequence, or an amplification primer sequence) recognition region, a primer annealing sequence, an attachment sequence, and the like. For example, a sequencing primer sequence, the resulting amplified target sequence will comprise such a primer and is readily transferred into a sequencing system. For example, when an Illumina sequencing system is employed to sequence the amplified target, the sequencing primer sequence can comprise a read length primer sequence (Read).

[0122] The barcode of the first label or the second label can comprise a capture sequence, which can be used to link with a cell barcode delivered by a bead or a particle.

[0123] For example, the first label can comprise a barcode and a fluorescent label. For example, the second label can comprise a barcode and a fluorescent label.

[0124] For example, the first label can comprise a fluorescent label. For example, the second label can comprise a fluorescent label.

[0125] For example, the first label can comprise a barcode. For example, the second label can comprise a barcode.

[0126] For example, the first label and the second label can be different. For example, the first label and the second label can both be fluorescent labels. For example, the first label and the second label are different fluorescent labels.

[0127] For example, the first label and the second label can both be DNA sequences or RNA sequences. For example, the first label and the second label are different sequences of DNA sequences or RNA sequences.

[0128] The methods of the application can further comprise providing an auxiliary substance that facilitates the recognition or binding of the ligand and the receptor. The types of such auxiliary substances and the contexts in which they are used are known to those skilled in the art, and the appropriate auxiliary substance can be selected based on the mechanism of the ligand-receptor recognition or binding.

[0129] For example, in the context of T cell receptors, it is known to those skilled in the art that antigens typically need to be bound to an auxiliary protein (major histocompatibility complex, MHC) before they can bind to T cells. Thus, in embodiments in which T cell receptors or T cells of interest are screened, the methods further comprise, when contacting the population of T cells with the antigen and the labeled protein, adding the auxiliary protein (e.g., MHC) such that. For another example, when the antigen is provided with the first label, the antigen is presented by the MHC. In this context, the antigen can be displayed by a self-major histocompatibility complex (MHC) molecule, i.e., the MHC-antigen complex is the receptor. For example, using MHC tetramers, the antigen peptide is incubated to bind as a water-soluble MHC-antigen complex, wherein the antigen or MHC complex is labeled with a DNA barcode and a fluorescent molecule. Similarly, an antibody against TCR is prepared as a DNA barcode complex and a fluorescent molecule. The T cells are incubated with both, and the same FACS sorting and analysis is performed.

[0130] Single cell sorting

[0131] After obtaining the target ligand with the first label and the labeled protein with the second label, the labeled target ligand and the labeled protein are contacted with the population of cells to be screened. After contacting under suitable incubation conditions, the population of cells is subjected to cell sorting, such that one or more single cells in the target cell population are isolated as single cells. For example, the sorting is performed using flow cytometry sorting techniques.

[0132] In some embodiments, the target cell population is isolated into single cells by cell emulsions (droplet fluids). In some embodiments, the target cell population is isolated into single cells in a microwell or plate-based assay.

[0133] In the present application, the methods can comprise introducing a unique cell barcode-labeled bead or particle into each single cell emulsion. The cell barcode- containing oligonucleotide can amplify the cell mRNA transcript via a template switch oligo, which is part of the cell barcode-containing oligonucleotide. The cell barcode- containing oligonucleotide can directly anneal to the oligonucleotide from the molecule.

[0134] For example, the cell barcode can comprise a unique molecular identifier (UMI).

[0135] When the first label and / or second label is a barcode, the methods and systems of the application further comprise using a primer that is capable of interacting with the barcode. In certain embodiments, the primer is used to support a DNA amplification reaction. For example, the primer is capable of extending in a sequence-specific manner. Extension of a primer in a sequence-specific manner includes any method that makes the sequence and / or composition of a nucleic acid molecule to which the primer hybridizes or otherwise associates direct or influence the composition or sequence of the product of primer extension. Thus, extension of a primer in a sequence-specific manner includes, but is not limited to, PCR, DNA sequencing, DNA extension, DNA polymerization, RNA transcription, or reverse transcription. Preferred techniques and conditions for amplifying a primer in a sequence-specific manner. In certain embodiments, the primer is used in a DNA amplification reaction, such as PCR or direct sequencing. In certain embodiments, the primer can also be extended using non-enzymatic techniques, for example, the nucleotides or oligonucleotides used to extend the primer can be modified or optimized so that they are capable of chemically reacting in a sequence-specific manner to extend the primer. The primer of the application hybridizes to a nucleic acid or nucleic acid region of the application, or to a complementary region of a nucleic acid or nucleic acid region.

[0136] For example, the cell barcode can be delivered by a bead or a particle. The particle or bead size can be optimized to bind the cell in a single cell emulsion and optimized for subsequent PCR reactions. As used herein, "bead" or "particle" is not limited to a particular type of bead or particle. Suitable beads or particles are known to one of ordinary skill in the art and can be selected depending on the desired end use.

[0137] In some embodiments, combining a single cell with a unique cell barcode-labeled bead is performed by a cell emulsion (droplet fluid). In some embodiments, combining a single B cell with a unique cell barcode-labeled bead is performed by co-encapsulating a single B cell with a unique cell barcode-labeled bead.

[0138] The methods of the application further comprise, after sorting into single cells, obtaining a signal value of the first label for the single cell. The signal value of the first label can be a fluorescence intensity from which the number or level of the first label carried by the single cell can be derived. The signal value of the first label can be a number or level of unique molecular identifier (UMI) from which the number or level of the first label carried by the single cell can be derived.

[0139] The method of the present application further comprises obtaining a signal value of a second marker of the single cell after sorting into single cells. The signal value of the second marker can be fluorescence intensity, from which the number or level of the second marker carried by the single cell can be obtained. The signal value of the second marker can be the number or level of unique molecular identifier (UMI), from which the number or level of the second marker carried by the single cell can be obtained.

[0140] The method of the present application further comprises obtaining a ligand-receptor affinity score, wherein the ligand-receptor affinity is the ratio of the signal value of the first marker of a single cell to the signal value of the second marker of the single cell. The ligand-receptor affinity value of the present application reflects the ratio of the number or level of the target ligand bound on the cell surface receptor of a cell to the number or level of the cell surface receptor of the cell, which can reflect the affinity of the cell surface receptor and the target ligand. The higher the ratio, the higher the affinity of the cell surface receptor and the target ligand; the lower the ratio, the lower the affinity of the cell surface receptor and the target ligand.

[0141] The method of the present application can further comprise comparing the ligand-receptor affinity value of the one or more cells with a reference ligand-receptor affinity value, and determining that the one or more cells are high-affinity target cells for surface receptors if the ligand-receptor affinity value of the one or more cells is greater than the reference ligand-receptor affinity value, and determining that the one or more cells are low-affinity target cells for surface receptors if the ligand-receptor affinity value of the one or more cells is lower than the reference ligand-receptor affinity value.

[0142] In another aspect, the present application provides a target cell obtained by the method.

[0143] In another aspect, the present application provides a target B cell obtained by the method.

[0144] In another aspect, the present application provides a method for screening high affinity antibodies, comprising: (1) providing an immunized B cell population, the B cell population comprising one or more B cells expressing B cell receptor (BCR), (2) providing an antigen with a first label, (3) providing a marker protein with a second label, the marker protein being capable of binding to the BCR, and the binding not being through the BCR to the binding epitope of the antigen, (4) contacting the B cell population in (1) with the antigen in (2) and the marker protein in (3), (5) obtaining a ligand-receptor affinity value of one or more B cells in the B cell population, wherein the ligand-receptor affinity value = signal value of the first label / signal value of the second label, (6) comparing the ligand-receptor affinity value of the one or more B cells with a reference ligand-receptor affinity value, if the ligand-receptor affinity value of the one or more cells is greater than the reference ligand-receptor affinity value, determining the one or more cells as target B cells, (7) sequencing the BCR of the target B cells to obtain the high affinity antibodies.

[0145] In another aspect, the present application provides a method for screening high affinity antibodies, comprising: labeling an antigen with an antigen barcode; labeling a secondary antibody with an antibody barcode; providing the labeled antigen and the labeled secondary antibody to a B cell population; allowing the barcode-labeled antigen and the barcode-labeled secondary antibody to bind to the B cell population; washing unbound antigen and secondary antibody from the B cell population; separating the B cells into single cell emulsions; introducing a unique cell barcode-labeled bead into each single cell emulsion; preparing a single cell cDNA library from the single cell emulsions; performing a PCR amplification reaction to produce a plurality of amplicons, wherein the amplicons comprise: a cell barcode, an antibody barcode, and / or an antigen barcode; sequencing the plurality of amplicons.

[0146] In another aspect, the present application provides high affinity antibodies obtained by the method.

[0147] For example, the antibody comprises heavy chain complementarity determining region 3 (CDRH3), heavy chain complementarity determining region 2 (CDRH3), and heavy chain complementarity determining region 1 (CDRH1), light chain complementarity determining region 3 (CDRL3), light chain complementarity determining region 2 (CDRL3), and light chain complementarity determining region 1 (CDRL1), wherein the CDRH3 comprises an amino acid sequence as shown in any one of SEQ ID NOs: 8-25, the CDRL3 comprises an amino acid sequence as shown in any one of SEQ ID NOs: 26-41.

[0148] For example, the antibody comprises a heavy chain complementarity determining region 3 (CDRH3), a heavy chain complementarity determining region 2 (CDRH3), and a heavy chain complementarity determining region 1 (CDRH1), a light chain complementarity determining region 3 (CDRL3), a light chain complementarity determining region 2 (CDRL3), and a light chain complementarity determining region 1 (CDRL1), wherein the CDRH3 comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 42-51, the CDRL3 comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 29, 36, and 52-58.

[0149] For example, the antibody comprises a heavy chain complementarity determining region 3 (CDRH3), a heavy chain complementarity determining region 2 (CDRH3), and a heavy chain complementarity determining region 1 (CDRH1), a light chain complementarity determining region 3 (CDRL3), a light chain complementarity determining region 2 (CDRL3), and a light chain complementarity determining region 1 (CDRL1), wherein the CDRH3 comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 59-65, the CDRL3 comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 66-71.

[0150] In another aspect, the present application provides a pharmaceutical composition comprising the high affinity antibody.

[0151] In another aspect, the present application provides a target T cell obtained by the method.

[0152] In another aspect, the present application provides a system and / or device for screening a target cell, the surface of which expresses a receptor capable of binding a target ligand, the system comprising (1) a cell population expressing one or more cell surface receptors, (2) the target ligand with a first label, (3) a marker protein with a second label, the marker protein being capable of binding the receptor, and the binding not being through the receptor to a binding epitope of the target ligand.

[0153] In another aspect, the present application provides a system and / or device for screening a target B cell, the system comprising (1) an immunized B cell population, the B cell population comprising one or more B cells expressing a B cell receptor (BCR), (2) an antigen with a first label, (3) a marker protein with a second label, the marker protein being capable of binding the BCR, and the binding not being through the BCR to a binding epitope of the antigen.

[0154] In another aspect, the present application provides a system and / or device for screening a B cell expressing a B cell receptor on the surface thereof capable of binding to an antigen, the system comprising (1) a population of B cells, (2) the antigen with a first label, (3) a label protein with a second label, the label protein being capable of binding to the BCR, and the binding not being through the binding epitope of the antigen to the BCR.

[0155] In another aspect, the present application provides a system and / or device for screening a T cell expressing a T cell receptor on the surface thereof capable of binding to an antigen, the system comprising (1) a population of T cells, (2) the antigen with a first label, (3) a label protein with a second label, the label protein being capable of binding to the receptor, and the binding not being through the binding epitope of the antigen to the receptor.

[0156] In one embodiment, the ligand secreted by the cell is captured on the surface of the cell or on a microsphere / matrix coupled / bound to the cell, for example, a complex of one or more proteins can be used, one part of which can specifically (to a cell surface antigen) or non-specifically (cell membrane, etc.) bind to the surface of the secretory cell, and the other part can specifically bind to the secreted ligand. Through incubation on the surface of the cell, the cell can specifically capture the ligand secreted by the cell and bind to the surface, and the ligand-receptor affinity value can be obtained by the method of the present application, so as to obtain a high affinity ligand.

[0157] For example, in a secretory B cell, biotinylated antibodies against secretory B cell surface antigens are used, first incubated with secretory B cells, then the cells are incubated with streptavidin, and finally the cells are incubated with biotinylated anti-IgG antibodies, so that the antibodies on the surface of the secretory B cells can capture IgG proteins, and then IgG is captured on the surface of the corresponding plasma cells. Detection of secondary antibodies (oligo-coupled goat anti-mouse IgG light chain antibodies) and detection of antigens (fluorescent and oligo-labeled antigens) are used for labeling. Subsequently, flow sorting is used, 10X scV(D)J library is constructed and sequenced, and data analysis is performed. The results show that the antibody with a higher ligand-receptor affinity value has a higher affinity.

[0158] In another embodiment, the target cell of the present application can be a yeast cell. The method of the present application can be used to screen yeast expressing high affinity antibodies in a yeast display library to obtain the sequence of the high affinity antibody.

[0159] Without wishing to be bound by any theory, the examples below are merely intended to illustrate the fusion proteins, preparation methods and uses of the present application, and are not intended to limit the scope of the present application.

[0160] Examples

[0161] Method

[0162] Binding of oligonucleotide barcode to protein

[0163] Using oligonucleotides with 15 base pair protein barcodes, the sequence is able to anneal to the template switch oligonucleotide, which is part of the 10x bead delivery oligonucleotide, and contains a truncated TruSeq small RNA read 1 sequence, with the structure as follows: 5'-CGGAGATGTGTATAAGAGACAG (SEQ ID NO: 1) - 9 or 10 base random nucleotides 1 (i.e. UMI, unique molecular identifier) - protein barcode of about 15 amino acid length - 9 or 10 base random nucleotides 2 (i.e. UMI, unique molecular identifier) - CCCATATAAGA*A*A (SEQ ID NO: 2, where * indicates a phosphorothioate modification in the middle of the backbone to the previous nucleotide when synthesizing the oligonucleotide sequence, making the oligonucleotide sequence more stable) - 3'. Among them, the protein barcode includes the barcodes of OVA (Sigma-Aldrich A5503), SARS-COV-2 RBD (2019-nCoV Spike (S) Protein RBD, His-Tag), LC (AffiniPure Goat Anti-Mouse IgG, Light Chain Specific, Jackson ImmunoResearch, 115-005-174). Among them, the barcodes of the exemplary OVA are: GACAAGTGATCTGCA (SEQ ID NO: 3) and TCTGCACCTTGCACG (SEQ ID NO: 4), the barcodes of the exemplary SARS-COV-2 RBD are CATTGTCGACCGCGA (SEQ ID NO: 5), and the barcodes of the exemplary LC are TCATTTCCTCCGATT (SEQ ID NO: 6) and GCACAGTCGCAATC (SEQ ID NO: 7). The oligonucleotide is ordered from Sangon Biotech and IDT, and is subjected to 5' amino modification and HPLC purification.

[0164] Fluorescent labeling of protein

[0165] After binding, the barcoded antigen is mixed with Alexa Fluor TM 647 NHS ester (Thermo Fisher Scientific, A37573), and the barcoded antibody is mixed with Alexa Fluor TM488 NHS ester (Thermo Fisher Scientific, A20000) was mixed and the reaction mixture was incubated at room temperature for 2 hours. 1 M glycine pH 8.5 (final concentration ~20 mM) was added and incubated at room temperature for 5 minutes to quench residual NHS groups. Excess oligonucleotides were removed from the protein-oligonucleotide conjugate using AKTA FPLC. The concentration of the antigen-oligonucleotide conjugate was determined by NanoDrop ONE (Thermo Scientific).

[0166] Mouse immunization and cell sorting

[0167] 6-8 month old female BALB / c mice (BALB / c) were immunized with 70 pg OVA (Sigma-Aldrich, A5503) or SARS-COV2-RBD (BD, Franklin Lakes, NJ) mixed with complete Freund’s adjuvant. The second and subsequent immunizations used 35 ug of protein antigen mixed with incomplete Freund’s adjuvant and cells were extracted from the spleen 2 days after the last immunization. Cells were then stained with a mixture of PE fluorescent dye conjugated anti-mouse B220 antibody (BD Biosciences, San Jose, CA), AF488 labeled anti-mouse IgG light chain antibody and AF647 labeled antigen (OVA or SARS-COV-RBD) for sorting of antigen positive B cells on a Beckman Coulter CytoFLEX SRT cell sorter.

[0168] Sample preparation, library preparation and sequencing

[0169] After FACS sorting into tubes, cells were centrifuged, resuspended in D-PBS (Sigma-Aldrich) and cell mass control was performed using a TC20 automated cell counter (Bio-rad, 145010). Cell suspension was loaded onto a Chromium Controller microfluidic device (10x Genomics) and processed according to manufacturer’s recommendations using the B cell single cell V(D)J solution to capture 10,000 B cells, amplify and purify the antigen barcode library. Gel bead emulsion (GEM) was formed in the channels of the chip and then collected for GEM reverse transcription (GEM-RT) reaction. cDNA was amplified and primers were added to increase the yield of antigen derived transcription products. After cDNA amplification, antigen derived transcription products were size separated from mRNA derived cDNA products using SPRI selection and further purification. After purification, antigen derived transcription sequencing library was prepared using a PCR reaction and purified using SPRI purification. Antigen and VDJ libraries were then analyzed, quantified and sequenced using the Illumina NovaSeq platform.

[0170] Sequence processing and bioinformatics analysis

[0171] Sequencing data was mapped to the GRCm38 reference genome using Cell Ranger multi (10x Genomics, v6.0.2) and a barcode-UMI, barcode-antigen matrix was generated for each sample from the paired-end FASTQ files of the oligonucleotide library as input. The protein barcode library was also processed using Cell Ranger (10X Genomics). BCR data was processed using cellranger multi and the same version of the reference genome. Overlapping barcodes were kept for downstream analysis. Integrated analysis of cell barcode-UMI-antigen matrix and BCR sequences was performed using the R package Seurat (v4.1.1). Cells were discarded if they met any of the following criteria: mitochondrial count percentage > 25%; number of unique features < 50; number of unique features > 3000. Cell barcodes with only non-functional heavy chain sequences and cells with multiple functional heavy chain sequences and / or multiple functional light chain sequences were removed, deducing that these were likely multiplets. The antigen count for each cell was clr transformed using the combinations package (v2.0-1).

[0172] Determination of ligand-receptor affinity values

[0173] After obtaining the surface protein UMI count matrix, noise with UMI < 4 was removed. Then, unique and complete heavy and light chain variable domain pairs were filtered. Given that UMI is a parameter of the actual number of cell-bound proteins, the ligand-receptor affinity value was defined as AAB-score = ln(x1 / x2), where x represents the surface protein UMI count, x1 and x2 correspond to the antigen and AAB antibody, respectively. Although the UMI count of each protein can differ in scale, which can be due to the difference in oligonucleotide loading during binding, the distribution peak of a specific protein in a cell population remains consistent under the same conditions. Therefore, the UMI ratio can accurately reflect the relative abundance of two proteins bound to the surface of a single cell. The final AAB-score (i.e., ligand-receptor affinity) is derived by transforming, scaling, and correcting these values using the natural logarithm (LN) function.

[0174] Antibody expression and purification

[0175] For each antibody, the variable genes were inserted into plasmids encoding the constant regions of the heavy chain (pFUSEss-CHIghG1, Invivogen) and light chain (pFUSE2ss-CLIg-hl2, Invivogen and pFUSE2ss-CLIg-hk Invivogen) and synthesized by GenScript. mAbs were expressed in CHO cells. After transfection, cells were cultured, centrifuged, eluted, and antibodies were purified.

[0176] Enzyme-linked immunosorbent assay (ELISA)

[0177] For antibody binding assessment, soluble OVA protein was seeded at 2 pg / mL overnight at 4°C. The next day, plates were washed three times with PBS with 0.05% Tween-20 (PBS-T) and coated with 5% BSA (Sigma-Aldrich, V900933) in PBS-T. Plates were incubated for 1 hour at 37°C and washed three times with PBS-T. Primary antibodies were diluted in 0.1% BSA in PBS-T starting at 10 pg / mL with serial dilutions of 1:3 added to the plates. Plates were incubated for 1 hour at 37°C and washed three times in PBS-T. Secondary antibodies (goat anti-mouse IgG heavy and light chain antibodies coupled to HRP) were added to 0.1% BSA in PBS-T at a dilution of 1:20,000 and plates were incubated for 1 hour at 37°C. Plates were washed four times with PBS-T and developed by adding TMB substrate (Biolegend, 421101) to each well. Plates were incubated for 15 minutes at room temperature and then 2N sulfuric acid was added to stop the reaction. Plates were read at 450 nm. Data is presented as the mean ± s.e.m. of one ELISA experiment. ELISAs were repeated two or more times. AUC was calculated using Origin 2017.

[0178] Bio-layer interferometry (BLI) analysis

[0179] Tested antibodies were diluted to a concentration of 5 pg / mL with PBS containing 0.02% Tween20 (Solarbio Life Sciences) and 0.1% (w / v) BSA and then immobilized on an anti-mIgG Fc Capture (AMC) biosensor (Sartorius). After a 60-second wash with PBST, the biosensor tip was dipped into a well containing SARS-CoV-2 RBD protein (Sino Biological) diluted to a concentration of 200 nM and allowed to bind for 300 seconds followed by a 600-second dissociation step. KD was calculated using a 1:1 binding model in the data analysis software 11 (ForteBio).

[0180] Pseudovirus neutralization assay

[0181] Pseudovirus neutralization assays were performed using 293T cells stably expressing ACE2. Cells (30,000 cells per well in DMEM with 10% fetal bovine serum) were seeded in 96-well plates overnight. Different concentrations of mAbs (starting at 200 pg / mL with double serial dilutions, 50 pL aliquots in duplicate) were mixed with the same volume of SARS-CoV-2 pseudovirus in 96-well plates. The mixtures were incubated at 37°C with 5% CO2for 1 hour. Virus-free control wells were provided with 100 pL of DMEM [10% (v / v) FBS]. Virus-only control wells contained 50 pL of media and 50 pL of pseudovirus. After 1 hour, the media was removed from the 293T cells, and 100 pL of the pseudovirus and antibody mixture was incubated with the cells at 37°C, 5% CO2for 1 hour. An additional 100 pL of DMEM was added to each well and incubated with the cells at 37°C, 5% CO2for 48 hours. After incubation, the supernatant was removed, and 100 pL of Nano-Glo luciferase detection reagent (Promega) diluted 1 : 1 in PBS was added to each well and incubated for 5 minutes. Luminescence was measured using Centro LB 963 microplate (Berthold Technologies). Relative luciferase units were calculated by normalizing the luminescence signal to the virus-only control group. IC50values were determined using GraphPad Prism 9.0 (GraphPad Software Inc.) by four-parameter nonlinear regression. 50 .

[0182] Antibody-dependent cellular phagocytosis (ADCP)

[0183] Beads were incubated with antibodies at a starting concentration of 50 mg / mL for two hours and titrated five-fold. MM17T (Sino Biological, Cat: 40592-MM17T) was used as a positive control. Antibodies and beads were incubated with THP-1 cells overnight, fixed and detected on a Beckman Coulter CytoFLEX SRT. Phagocytosis scores were calculated as the percentage of THP-1 cells phagocytosing fluorescent beads multiplied by the geometric mean fluorescence intensity of the population in the FITC channel. To facilitate display, these scores were subsequently divided by 10,000.

[0184] Antibody-dependent complement deposition (ADCD)

[0185] Biotinylated SARS-Cov-2 RRBD protein was coated on fluorescent neutravidin beads at a 1:1 ratio for 2 hours at 37 degrees. These beads were incubated with 100 ug / ml antibody for 1 hour and with guinea pig complement diluted 1:50 with gelatin / veronal buffer for 15 minutes at 37 degrees. Beads were washed twice in PBS at 2000g and stained with anti-guinea pig C3b-FITC, fixed and detected on a Beckman Coulter CytoFLEX SRT. Complement deposition score was calculated as the percentage of C3b-FITC positive beads multiplied by the geometric mean fluorescence intensity of FITC in that population. To facilitate presentation, these scores were divided by 1000.

[0186] Example 1 Simultaneous acquisition of antigen-antibody affinity and paired BCR sequences using AAB-seq for rapid and efficient screening of functional antibodies

[0187] Mice were immunized with chicken ovalbumin (OVA). B cells were stained with Alexa Fluor 647 and oligonucleotide barcode labeled OVA, oligonucleotide barcode labeled goat anti-mouse Ig light chain secondary antibody (AAB-ab). Approximately 10 7 B cells were enriched from the spleen of OVA immunized mice by density gradient centrifugation and MACS. B220 PE + / DAPI - / AF647 + cells were then stained and sorted using FACS technology for 10x high throughput single cell V(D)J with Feature Barcode to capture 10,000 B cells. The schematic workflow is shown in Figure 1.

[0188] After bioinformatics processing, paired heavy / light chain sequences and antigen reactivity information were obtained for 3,712 cells (Figure 2). The results showed that the ADT level of the antigen distribution group was consistent with the gold standard flow cytometry. For each cell, the AAB-score was calculated as an important indicator of antibody affinity, with a value between -2.73 and 3.68.

[0189] To validate the correlation between AAB-score and affinity, 16 BCRs were selected for expression and protein assay (Figure 3). The predicted antigen specificity and binding capacity were confirmed by enzyme-linked immunosorbent assay (ELISA) (Figure 4), indicating that antibodies with high AAB-score exhibited greater affinity to the antigen, and vice versa. Among all the tested antibodies, the correlation between AAB-score and the area under the curve (AUC) value derived from ELISA [(lg(ELISA_AUC)] was strong (Pearson correlation R = 0.8597, P = 9.72E-06) (Figure 5). These findings suggest that the binding affinity of BCR to antigen can be reliably inferred by measuring the relative ratio of antigen bound to BCR on the surface of B cells using high-throughput cell surface protein sequencing, thereby directly reading the antigen affinity in a single B cell sequencing experiment.

[0190] The BCR dataset was then utilized to identify high-affinity OVA-specific antibodies. B cells with high AAB-score determined from the high-throughput experiment described above were selected for subsequent recombinant antibody expression and characterization (Figure 6). Notably, these selected B cells exhibited OVA antigen positive characterization. The expression sequences of 10 selected antibodies were analyzed, and their antigen specificity was tested using ELISA. The results showed that all antibodies with high AAB-score bound to OVA and exhibited high antigen affinity (Figure 6, Figure 7, Figure 8). This validated that functional antibodies can be effectively screened in a targeted manner directly from sequencing data, thereby saving the resources normally required for expression and purification of large antibody pools for ELISA testing.

[0191] Example 2 AAB-seq screening of SARC-COV2-RBD antibodies

[0192] To illustrate the utility of AAB-seq in functional antibody screening, the antibody repertoire of mice immunized with SARS-CoV-2 receptor binding domain (RBD) was analyzed. After sequencing and subsequent bioinformatics analysis, paired VH:VL antibody sequences and corresponding AAB-scores of 4,143 cells were recovered (Figure 9). From this dataset, 10 antibodies were selected for recombinant antibody production (Figure 10). The reactivity of purified mAbs against SARS-CoV-2 RBD was tested using ELISA and biolayer interferometry (BLI), and it was found that 8 mAbs with higher AAB-scores (1743-3, 1743-6, 1743-8, 1743-9, 1743-10, 1743-11, 1743-12, 1743-13) bound to RBD and exhibited strong antigen-binding capacity (Figure 11), with Kd values ranging from 0.211 to 7.80 nM (Figure 12) as shown by BLI results, with a 100% success rate for antibody screening. Similarly, 2 mAbs with lower AAB-scores did not bind to the antigen. Subsequently, these antibodies were tested for neutralizing activity against wild-type SARS-CoV-2 pseudovirus. The results showed that antibodies 1743-3 and 1743-13 effectively neutralized the SARS-CoV-2 WT strain with IC 50 values of 2.25 pg / mL and 18.58 pg / mL, respectively (Figures 13, 14). Next, these antibodies were evaluated for antibody-dependent cellular phagocytosis (ADCP) function against SARS-CoV-2 RBD protein in vitro. Antibodies 1743-8, 1743-9, 1743-10, 1743-11, 1743-12, and 1743-13 exhibited good ADCP activity (Figures 15-16, 17, 18). Finally, antibodies 1743-3 and 1743-13 also exhibited effective antibody-dependent complement deposition (ADCD) (Figures 15, 17, 18). These results reflect the different characteristics of F(ab) and Fc effector functions of antibodies. These results highlight the effectiveness of AAB-seq in identifying high-affinity antibodies, thereby demonstrating its practicality in screening functional antibodies.

[0193] Example 3 Application of AAB-seq in a fluorescent low-throughput experiment

[0194] This example uses FACS to directly enrich antibodies with high antigen / BCR ratio, followed by single B cell sequencing and direct cloning (Figure 19). Therefore, using DNA barcoded oligos to detect antigen / BCR ratio, whether the values are consistent with the fluorescence values detected at the single cell level by flow cytometry. Specifically, B cells are incubated with AAB-ab labeled with fluorescein and barcodes, and antigen labeled with fluorescein and barcodes, and then single B cells are sorted into 96-well plates with indexed functions to record the fluorescence values of each sorted cell. Then the cell surface protein oligos are converted into sequencing libraries. For each cell sorted by FACS, similar AAB-seq relative ratios are observed by both methods (Pearson R = 0.95185, P = 1.63815E-48) (Figure 20). This means that the fluorescence values can be used instead of the number of molecules (ADT UMIs).

[0195] Since the fluorescence ratio and ADT ratio show a high correlation, it is believed that the AAB-seq process can be simplified by only measuring the fluorescence ratio, and single B cells can be screened for direct BCR cloning. This not only skips the droplet scRNA-seq and surface protein sequencing process, but also directly clones the BCR gene for antibody expression without the need to synthesize the antibody gene, greatly simplifying the method steps and saving costs.

[0196] Example 4 Application of the method of the present application in screening high affinity secreted antibodies in secreted B cells

[0197] The ligands secreted by the cells can be captured on the cell surface or on microspheres / matrices / coupled / combined with the cells, etc., for example, a complex of one or more proteins can be used, part of which can be specifically (to cell surface antigens) or non-specifically (cell membrane, etc.) bound to the surface of the secreted cells, and the other part can specifically bind to the secreted ligand. By incubating the cell surface, the cells can specifically capture the ligands they secrete and bind to the surface, and the subsequent experiments use the same strategy as described above.

[0198] In secreted B cells, biotinylated antibodies against secreted B cell surface antigens are first incubated with secreted B cells, then the cells are incubated with streptavidin, and finally the cells are incubated with biotinylated anti-IgG antibodies, so that the antibodies on the surface of the secreted B cells can capture IgG proteins, and then capture IgG to the corresponding plasma cell surface.

[0199] The secondary antibody (oligo-conjugated goat anti-mouse IgG light chain antibody) and the detection antigen (fluorescently and oligo-labeled antigen) are used to label the antibody. Subsequently, the same as the previous step, after flow sorting, 10X scV(D)J library is constructed and sequenced, and the data analysis method is consistent with the previous embodiment. The higher the ligand-receptor affinity value, the higher the affinity. The results show that high-affinity receptors are screened.

[0200] Example 5 Application of the method of the present application in T cell receptor / ligand binding specificity and affinity detection

[0201] In addition to B cells, this embodiment verifies that the method of the present application can also be used to screen high-affinity T cells from T cells against antigens to obtain TCR sequences.

[0202] The design of this experiment is consistent with the antibody screening described in the previous embodiment, and the antigen is displayed by the major histocompatibility complex (MHC) molecule, specifically, using MHC tetramer, and the antigen peptide segment is incubated to form a water-soluble MHC-antigen complex, wherein the antigen or MHC complex carries a DNA barcode and a fluorescent molecule. Similarly, the anti-TCR antibody is prepared into a DNA barcode complex and a fluorescent molecule. Then incubate the T cells with both of them, and perform the same FACS sorting and analysis. The results show that high-affinity receptors are screened by ligand-receptor affinity value.

[0203] Example 6 Application of the method of the present application in yeast display library

[0204] The method of the present application can be used to screen yeast expressing high-affinity antibodies in a yeast display library to obtain the sequence of high-affinity antibodies.

[0205] The screening method is consistent with the antibody screening described in the above embodiment, except that the screening object is replaced by the yeast library displaying antibodies. Specifically, first, the gene sequences of a plurality of foreign proteins (e.g., antibodies) are inserted into yeast by genetic engineering techniques to form a yeast library expressing a plurality of antibodies on the outer surface.

[0206] In the yeast library, the yeast surface has antibody proteins, and then the yeast can be labeled with antigens with fluorescent AF647 and secondary antibodies (goat anti-mouse IgG light chain antibody) with AF488, and then the yeast with high AF488 / AF647 fluorescence ratio is sorted by flow sorting, which is considered to be yeast carrying high-affinity antibodies. After amplification culture, it is labeled and sorted again, and preferably screened several times to increase its accuracy. After several rounds of screening of the obtained yeast, the antibody affinity information and the sequence of high-affinity antibodies can be obtained by sequencing.

Claims

1. A method of screening for a target cell, the surface of the target cell expressing a receptor capable of binding a target ligand: (1) providing a cell population expressing one or more cell surface receptors, (2) providing the target ligand with a first label, (3) providing a labeled protein with a second label, the labeled protein being capable of binding the receptor and the binding not being through the receptor to the binding epitope of the target ligand, (4) contacting the cell population in (1) with the target ligand in (2) and the labeled protein in (3), (5) determining the signal value of the first label and the signal value of the second label, the ligand-receptor affinity value = the signal value of the first label / the signal value of the second label, (6) determining whether one or more cells in the cell population are the target cell based on the ligand-receptor affinity value of the one or more cells obtained in (5).

2. The method of claim 1, wherein the first label is a fluorescent label and / or a barcode.

3. The method of claims 1-2, wherein the second label is a fluorescent label and / or a barcode.

4. The method of claim 2 or 3, wherein the barcode comprises a DNA sequence or an RNA sequence.

5. The method of claim 2 or 3, wherein the first label is different from the second label. (5) obtaining a ligand-receptor affinity value for one or more cells in the cell population, wherein, 6. The method of any one of claims 1-3, wherein the cell population in (1) comprises a population of immunized B cells.

7. The method of any one of claims 1-5, wherein the target ligand with the first label is an antigen with a first fluorescent label and / or a first barcode.

8. The method of claim 7, wherein the antigen comprises an antigen from a pathogen or an animal.

9. The method of claim 8, wherein the antigen from a pathogen comprises an antigen from a virus.

10. The method of any one of claims 1-6, wherein the labeled protein with the second label is a secondary antibody with a second fluorescent label and / or a second barcode.

11. The method of any one of claims 1-10, after (4) and before (5), the method comprising performing cell sorting.

12. The method of any one of claims 1-11, wherein in (5), the signal value of the first label and / or the second signal value is a fluorescent intensity.

13. The method of any one of claims 1-12, wherein in (5), the signal value of the first label and / or the second signal value is a unique molecular identifier (UMI) count.

14. The method of any one of claims 1-13, wherein (6) comprises comparing the ligand-receptor affinity value of the one or more cells to a reference affinity value, and determining that the one or more cells are the target cell if the ligand-receptor affinity value of the one or more cells is greater than the reference affinity value.

15. The method of any one of claims 1-14, wherein the target ligand is high affinity to the receptor.

16. A target cell obtained by the method of any one of claims 1-15. ​ ​ ​ ​ ​ ​ 17. A method for screening a target B cell, the surface of which expresses a B cell receptor capable of binding an antigen, comprising: (1) providing a population of immunized B cells, (2) providing the antigen with a first label, (3) providing a labeled protein with a second label, the labeled protein being capable of binding the receptor and the binding not being through the binding epitope of the receptor to the target ligand, (4) contacting the population of B cells in (1) with the antigen in (2) and the labeled protein in (3), (5) obtaining a ligand-receptor affinity value for one or more B cells in the population, wherein the ligand-receptor affinity value = the signal value of the first label / the signal value of the second label, and (6) determining whether one or more cells in the population of B cells is the target B cell based on the ligand-receptor affinity value of the one or more B cells obtained in (5).

18. A B cell obtained by the method of claim 17.

19. A method for screening a high affinity antibody, the method comprising: (1) providing a population of immunized B cells, the population of B cells comprising one or more B cells expressing a B cell receptor (BCR), (2) providing an antigen with a first label, (3) providing a labeled protein with a second label, the labeled protein being capable of binding the BCR and the binding not being through the binding epitope of the BCR to the antigen, (4) contacting the population of B cells in (1) with the antigen in (2) and the labeled protein in (3), (5) obtaining a ligand-receptor affinity value for one or more B cells in the population, wherein the ligand-receptor affinity value = the signal value of the first label / the signal value of the second label, (6) comparing the ligand-receptor affinity value of the one or more B cells with a reference ligand-receptor affinity value, and determining that the one or more cells is a target B cell if the ligand-receptor affinity value of the one or more cells is greater than the reference ligand-receptor affinity value, and (7) sequencing the BCR of the target B cell to obtain the high affinity antibody.

20. An antibody obtained by the method of claim 20.

21. A method for screening a target T cell, the surface of which expresses a T cell receptor capable of binding an antigen, comprising: (1) providing a population of T cells, (2) providing the antigen with a first label, (3) providing a labeled protein with a second label, the labeled protein being capable of binding the receptor and the binding not being through the binding epitope of the receptor to the antigen, (4) contacting the population of T cells in (1) with the antigen in (2) and the labeled protein in (3), (5) obtaining a ligand-receptor affinity value for one or more T cells in the population, wherein the ligand-receptor affinity value = the signal value of the first label / the signal value of the second label, and (6) determining whether one or more cells in the population of T cells is the target T cell based on the ligand-receptor affinity value of the one or more T cells obtained in (5). (5) obtaining a ligand-receptor affinity value for one or more B cells in the B cell population, wherein, 22. A T cell obtained by the method of claim 21. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ (5) obtaining a ligand-receptor affinity value for one or more T cells in the population of T cells, wherein, ​ ​ ​

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