Hydrogel culture-based method for screening interacting TCR and pmhc

Abstract

A method for screening an interacting TCR and antigen-MHC complex (pMHC), a T cell population expressing an exogenous TCR and / or exogenous pMHC, and a use of the T cell population in screening an interacting TCR and pMHC.

Description

Method for screening interacting TCR and pMHC based on hydrogel culture TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and relates to a method for screening interacting TCR and pMHC. BACKGROUND

[0002] TCR (T cell receptor) is a specific T cell receptor on the surface of T cells, which is a molecule capable of recognizing endogenous / foreign antigens, and can realize killing and clearance of specific damaged cells, and can mediate immune response, and plays an important role in immune response and immune regulation. The receptor is also a molecular structure for specific recognition and binding of antigen peptide-MHC molecules by T cells, and usually exists in the form of a complex with CD3 molecules on the surface of T cells. In the process of T cell recognition of antigens, the receptor TCR and the pMHC (antigen molecule polypeptide or peptide-MHC) presented by the major histocompatibility complex (MHC) are closely combined, thereby activating T cells, i.e. after T cells are stimulated by antigens, they are differentiated, proliferated, and transformed into activated T cells, producing direct killing effect of antigens and synergistic killing effect of immune effectors of cytokines released by activated T cells. Tumor cell immunotherapy (TCR-T) is a treatment method for killing and clearing tumor cells by using T cells specifically combined with target tumor antigens.

[0003] TCR recognition of pMHC activates immune response, which is crucial for maintaining the health of the human body. In vitro evolved TCR with high affinity can improve the anti-tumor ability of modified T cells.

[0004] There is always a need in the art for high-throughput and large-scale bidirectional screening technology of TCR-pMHC. SUMMARY

[0005] The present application aims to provide a method for screening interacting TCR and antigen-MHC complex (pMHC), a cell population expressing exogenous TCR and / or expressing exogenous pMHC, and the use of the cell population in screening interacting TCR and pMHC.

[0006] In one aspect, the present application provides a method for screening interacting TCR and antigen-MHC complex (pMHC), comprising:

[0007] (a) expressing a TCR library and / or a pMHC library on the surface of a cell population to obtain a cell population expressing exogenous TCR and / or exogenous pMHC;

[0008] (b) culturing the cell population obtained in step (a) in a gel to obtain a plurality of cell colonies from single cells;

[0009] (c) sorting out the activated cells;

[0010] (d) determining the sequence of the TCR and the sequence of the pMHC on the surface of the cells in step (c);

[0011] to determine the interacting TCR and antigen-MHC complex.

[0012] In another aspect, the present application provides a T cell population expressing an exogenous TCR and / or an exogenous pMHC, preferably the cell population is a T cell population.

[0013] In another aspect, the present application provides a use of the cell population of the present disclosure in screening interacting TCR and pMHC.

[0014] In yet another aspect, the present application provides a use of a gel for culturing cells in screening interacting TCR and pMHC.

[0015] The inventors found that TCR and pMHC are co-expressed on the surface of T cells, and T cells are dispersed in a hydrogel for culture expansion, to obtain T cell colonies from the same cell. If the TCR and pMHC in the cell colony are interacting, the T cells in the cell colony are activated. By determining the TCR and pMHC on the surface of the activated T cells, the interacting TCR and pMHC can be screened, thereby realizing high-throughput screening. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1A: depicts the principle of the high-throughput screening of membrane receptor specific antigen method based on hydrogel 3D culture of the present application: first, pMHC and TCR are expressed in HLA-I knockout and TCR knockout Jurkat screening cell lines by lentiviral transduction, pMHC and TCR are randomly combined in cells, the cell population after transduction is mixed in a temperature-sensitive hydrogel, short-term 3D culture is carried out, a large number of cell colonies expanded from single cells are formed, cell colonies carrying interacting pMHC and TCR will activate the NFAT-zsGreen+ signal for cell recovery sequencing.

[0017] Figure IB: The antigen library screening process of one or a few TCRs: For a few TCRs to be screened, MHC and TCR are expressed in Jurkat screening cells by lentiviral transduction, and pep-SCD library is expressed in Jurkat screening cells by lentiviral transduction. The screened pep-SCD sequences are obtained by sorting NFAT-zsGreen positive cells, and the antigen sequence recognized by the TCR to be screened is obtained by high-throughput sequencing after 2-4 rounds of screening enrichment.

[0018] Figure 1C: Co-expression of pep-SCD and TCR using pBIPE structure.

[0019] Figure 2A: Obtain Jurkat cell lines with HLA-I (MHC-1) and TCR gene knockout, HLA-I (MHC-1) gene knockout lentivirus packaging cell 293T#7, and Jurkat screening cell line for expressing NFAT-zsGreen reporter system.

[0020] Figure 2B: Composition structure of SCT and SCD: SCT is a single-chain pMHC trimer molecule, and B2m signal\peptide\linkerl\B2m\linker2\MHC heavy chain sequence is expressed in sequence using GS-linker. SCD is a peptide-B2M dimer molecule, and the sequence structure is B2m signal\peptide\linkerl\B2m.

[0021] Figure 3: 3D culture commonly used material Matrigel cannot effectively wrap Jurkat cells to form regular monoclone cell colonies, and gel-X, i.e. pNIPaam-PEG thermosensitive hydrogel, can effectively wrap cells to form monoclone cell colonies.

[0022] Figure 4A: Specificity verification using antigen-TCR pairs with known specificity.

[0023] Figure 4B: Affinity verification using antigen-TCR pairs with known specificity.

[0024] Figure 5: Vector single copy activation efficiency verification using antigen-TCR pairs with known specificity and low cell infection multiplicity.

[0025] Figure 6: Comparison of the influence of cell colony-cell colony mutual contact generated during 3D culture.

[0026] Figure 7: depicts the results of screening the antigen recognition map of a single flu-TCR from the amino acid mutation library Flu-TCR-mut using the method of the present application.

[0027] Figure 8: depicts the results of screening individual DMF5-TCRs for antigen recognition profile from a random amino acid library A2-NNK using the method of the present application.

[0028] Figure 9: depicts the antigen recognition fingerprint of DMF5-TCR.

[0029] Figure 10: depicts that the antigen rare sequences of DMF5-TCR obtained from screening can be verified by peptide pulsing experiment.

[0030] Figure 11: depicts that the method of the present application can accomplish the recognition fingerprint screening of random library for 4 different TCRs simultaneously. DETAILED DESCRIPTION

[0031] The inventors found that the simultaneous expression of TCR and pMHC on the surface of T cells and the dispersion of T cells in hydrogel for culture can realize high-throughput TCR-pMHC bidirectional screening.

[0032] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The following definitions are provided for the following terms used herein for the purpose of the present application.

[0033] The following terms as used herein are explained below, but the present application is not limited by the following explanations.

[0034] As used herein, the term "about" or "approximately" means an amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length that varies by as much as 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% to a reference amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length. In one embodiment, the term "about" or "approximately" means a range of ± 15%, ± 10%, ± 9%, ± 8%, ± 7%, ± 6%, ± 5%, ± 4%, ± 3%, ± 2%, or ± 1% around a reference amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length.

[0035] As used herein, the terms "substantially" and "essentially" mean a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that is about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In one embodiment, the term "substantially the same" means a range of quantities, levels, values, numbers, frequencies, percentages, dimensions, sizes, amounts, weights or lengths that are about the same as a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0036] As used herein, the term "substantially free of, when used to describe a composition, such as a population of cells or a culture medium, means free of the specified material, for example, a composition that is 95% free, 96% free, 97% free, 98% free, 99% free of the specified material, or undetectable as measured by conventional means. A similar meaning can apply to the term "absence of, when referring to the absence of a particular material or component of a composition.

[0037] Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. In particular embodiments, the terms "comprise", "comprises", "comprising" and "have" are used synonymously.

[0038] "Consisting of means including, but limited to, any recited in the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that no other elements can be present.

[0039] "Consisting essentially of means including any elements recited in the phrase "consisting essentially of" and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. As used herein, the phrase "consisting essentially of" indicates that the listed elements are required or mandatory, but that other elements are optional and can or can not be present depending upon whether or not they affect the activity or action of the listed elements.

[0040] Throughout this specification, reference has been made to "one embodiment", "an embodiment", "a certain embodiment", "some embodiments", "another embodiment", "further embodiments" or "yet further embodiments" or similar phrases. It is emphasized that, in accordance with the teachings of the specification, the foregoing phrases are not necessarily all referring to the same embodiment. In particular, different embodiments can be combined in any suitable manner. Furthermore, specific features, structures, or characteristics can be combined in any suitable manner in one or more embodiments.

[0041] "Culturing" or "cell culturing" refers to the maintenance, growth and / or differentiation of cells in an in vitro environment. "Cell culture medium", "medium", "culture medium", "media", "supplement" and "culture supplement" refer to a nutritional composition for culturing a cell culture.

[0042] "Cultivate" refers to the maintenance, propagation (growth) and / or differentiation of cells outside of a tissue or organism, for example, in a sterile plastic (or coated plastic) cell culture dish or flask. Cultivation can utilize a medium as a source of nutrients, hormones and / or other factors that aid in the propagation and / or maintenance of cells.

[0043] "Propagation" refers to the property of one cell to divide into two substantially identical cells or an increase in the number of a population of cells (e.g., to replicate).

[0044] "Propagation" or "expansion" refers to the growth (e.g., replication via cell proliferation) of cells outside of a tissue or organism, for example, in a sterile container such as a plastic (or coated plastic) cell culture dish or flask.

[0045] The term "vector" or "expression vector" as used herein refers to a vehicle containing nucleic acid sequences that encode at least a portion of a gene product that is transcribed. In some cases, the RNA molecule is then translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, for example, in the production of antisense molecules or ribozymes. Expression vectors can contain a variety of control sequences, which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host organism. In addition to controlling transcription and translation, control sequences that are contained in vectors and expression vectors can have other functions and are described further infra.

[0046] The term "gene" as used herein is defined as a unit of coding for a functional protein, polypeptide, or peptide. It is understood that this functional term includes genomic sequences, cDNA sequences, and small engineered gene segments that express or are adapted to express proteins, polypeptides, domains, peptides, fusion proteins, and mutants.

[0047] The term "polynucleotide" or "nucleic acid" as used herein is defined as a chain of nucleotides. In addition, a nucleic acid is a polymer of nucleotides. Thus, as used herein, nucleic acid and polynucleotide are used interchangeably. A nucleic acid is a polynucleotide, which can be hydrolyzed into monomeric "nucleotides." The monomeric nucleotides and hydrolysis into nucleosides. The nucleotides in "polynucleotide" or "nucleic acid" as referred to herein can be modified or unmodified nucleotides.

[0048] The term "polypeptide" as used herein is defined as a chain of amino acid residues, which typically has a defined sequence. The term polypeptide as used herein can be used interchangeably with the term protein. The polypeptides and peptides of the present application can be modified by various amino acid deletions, insertions and / or substitutions. In particular embodiments, the modified polypeptides and / or peptides are capable of modulating an immune response in a subject. In some embodiments, a wild-type protein or peptide is used. In some embodiments, a modified protein or polypeptide is used to generate a pMHC.

[0049] In the context of TCR binding, the term "interacting" TCR and antigen-MHC complex (pMHC) refers to high avidity and / or high affinity binding of a TCR to a particular polypeptide (such as a particular antigen) (or more accurately, to a particular epitope of a particular polypeptide). The binding of a TCR to these epitopes is typically stronger than the binding of the same TCR to any other epitope or to any other polypeptide that does not contain the epitope.

[0050] The peptides or polypeptides of the antigen or antigen source that can be used in the present application can be any peptide or polypeptide of an antigen or antigen source of interest to the investigator, for example, can be a peptide or polypeptide of a tumor antigen.

[0051] The terms "transfection," "transduction," or "transformation" as used herein are used interchangeably, and refer to the introduction of foreign DNA into a host cell. Transfection (or transduction or transformation) can be achieved by any of a number of methods, including electroporation, microinjection, biolistic delivery, retroviral infection, lipofection, superfection, etc. Transfection (or transduction or transformation) can be either stably transfected (or transduced or transformed) or transiently transfected (or transduced or transformed). Stable transfection (or transduction or transformation) refers to the introduction of foreign DNA into a host cell wherein the DNA is incorporated into the chromosome of the cell. Transient transfection (or transduction or transformation) refers to the introduction of foreign DNA into a host cell wherein the DNA is not incorporated into the chromosome of the cell.

[0052] The term "determining sequence" as used herein refers to the process of obtaining the nucleotide sequence of a nucleic acid or deoxyribonucleic acid of a corresponding sample by routine techniques in the art. For example, the corresponding sequence can be detected by sequencing using a first generation sequencing method (e.g., Sanger sequencing), a second generation sequencing or a third generation sequencing method. In some embodiments, the sequence of the TCR on the surface of the T cell and the sequence of the pMHC are determined using a second generation sequencing or a third generation sequencing method. Preferably, in some embodiments, the sequence of the TCR on the surface of the T cell and the sequence of the pMHC are determined using a single cell sequencing method.

[0053] The term "library" or "pool" as used herein refers to a collection of a particular type of DNA, RNA or polypeptide, for example, a "TCR library" as used herein is a collection of one or more TCR sequences (e.g., DNA or RNA encoding a TCR, or polypeptide sequence of a TCR) that differ in sequence, for example, a "pMHC" pool as used herein is a collection of one or more complexes of an antigen and an MHC that differ in sequence.

[0054] The term "PCR amplification" as used herein refers to the use of polymerase chain reaction (PCR) to amplify a DNA sequence to obtain more copies of the DNA sequence.

[0055] The term "exogenous" as used herein refers to something that is derived from outside of an organism. For example, an exogenous TCR refers to a TCR that is not produced by the T cell expressing the TCR, but is introduced into the T cell by delivery (e.g., transformation or transfection) and expressed by the T cell. In some embodiments, exogenous materials (e.g., genes) are transduced into T cells using lentiviral transfection.

[0056] The term "endogenous" as used herein refers to something that is derived from an organism itself, i.e., a material that is naturally produced by the organism. For example, an endogenous TCR refers to a TCR that is produced by the T cell itself as encoded by the genes of the T cell.

[0057] The term "MHC" (major histocompatibility complex) as used herein is a family of genes found in the genomes of most vertebrates, consisting of a cluster of closely linked genes that are highly polymorphic, whose encoded products are antigen-presenting and T cell activating molecules that are intimately involved in the immune response and immunoregulation and are antigens that elicit rapid and strong rejection responses. The MHC in humans is known as HLA (human leukocyte antigen), i.e., human leukocyte antigen. MHCs are generally capable of binding to peptides or polypeptides that can serve as antigens to form a complex, thereby presenting the antigen to the cell surface. In the present invention, the complex of an antigen and MHC is referred to herein as "pMHC" or "antigen-MHC complex". In some embodiments, the MHC in the pMHC library of the present invention is MHC-1. In some embodiments, the MHC in the pMHC library of the present invention is MHC-2. In some embodiments, the MHC in the pMHC library of the present invention is a mixture of MHC-1 and MHC-2.

[0058] The T cell receptor (TCR) is a specific receptor on the surface of T cells that is responsible for recognizing antigens presented by major histocompatibility complex (MHC), unlike B cell receptors, TCRs cannot recognize free antigens. Generally, T cell receptors have low affinity for antigens, so the same antigen can be recognized by different T cell receptors, and a certain receptor can recognize many antigens.

[0059] The inventors have found, without being bound by any theory, that in the TCR-pMHC bi-directional screening process, a plurality of exogenous TCRs from a TCR library and a plurality of exogenous pMHCs from a pMHC library can be expressed on the surface of a T cell population to obtain a T cell population expressing exogenous TCRs and exogenous pMHCs. The T cell population is then dispersed in a gel for culture to obtain a plurality of cell colonies formed by proliferation of individual T cells. The cell colonies are then screened by a reporter system (such as a fluorescent reporter system) to screen for T cell colonies in which the surface pMHC and TCR interact. Through further testing (such as single-cell sequencing), the sequences of the pMHC and TCR that can interact are obtained.

[0060] An "activated cell" as described herein refers to a cell whose surface TCR and / or pMHC leads to activation of a downstream signaling pathway as a result of interaction with another cell's pMHC and / or TCR, thereby causing a change in the reporter status in the cell. For example, in some embodiments, a cell is activated as a result of interaction of its surface TCR with another cell's surface pMHC, resulting in increased expression of a fluorescent protein in the cell. One skilled in the art can sort activated cells based on the change in reporter status.

[0061] As used herein, a reporter is generally a protein or gene that can be detected when expressed in a cell. In some embodiments, cells can be sorted based on the presence or absence of a reporter in a population or cell (e.g., using flow cytometry and / or fluorescence activated cell sorting). In some embodiments, the reporter is a fluorescent protein. The fluorescent protein can be a green fluorescent protein (GFP), a yellow fluorescent protein (YFP), a red fluorescent protein (RFP), zsGreen. The fluorescent protein can be as described in U.S. Patent No. 7,060,869 entitled "Fluorescent protein sensors for detection of analytes." In some embodiments, the reporter is an antibiotic resistance marker. In some embodiments, the antibiotic resistance marker is a protein or gene that confers a competitive advantage to a cell comprising the marker. In some embodiments, the antibiotic resistance marker comprises a hygromycin resistance protein or gene, a kanamycin resistance protein or gene, an ampicillin resistance protein or gene, a streptomycin resistance protein or gene, or a neomycin resistance protein or gene.

[0062] Screening methods

[0063] The present disclosure provides a method of screening for interacting TCR and antigen-MHC complex (pMHC), comprising:

[0064] (a) expressing a TCR library and / or a pMHC library on the surface of a population of cells, to obtain a population of cells expressing exogenous TCR and / or exogenous pMHC;

[0065] (b) culturing the population of cells obtained in step (a) in a gel to obtain a plurality of cell colonies from single cells;

[0066] (c) sorting for activated cells;

[0067] (d) determining the sequence of the TCR and the sequence of the pMHC on the surface of the cells in step (c);

[0068] thereby determining the interacting TCR and antigen-MHC complex.

[0069] Any TCR library or pMHC library in the art can be used in the present application. In some embodiments, the library is constructed using random mutagenic primers. In some embodiments, the pMHC library is a Flu-TCR-mut library. In some embodiments, the pMHC library is an A2-NNK library. In some embodiments, the pMHC library used is a minigene library. In some embodiments, the library is constructed using the methods of the examples of the present application. In some embodiments, the library is constructed using gene synthesis.

[0070] Any suitable technique in the art can be used to culture the cells of the present application. One of skill in the art is capable of selecting suitable culture conditions, such as temperature, time, etc. In some embodiments, the cells of the present application are cultured at 37°C.

[0071] One of skill in the art is capable of selecting suitable means to express the TCR library and / or the pMHC library on the surface of the cell population. In some embodiments, lentiviral transfection is used to express the TCR library and / or the pMHC library on the surface of the cell population. In some embodiments, retroviral transfection is used to express the TCR library and / or the pMHC library on the surface of the cell population.

[0072] In some embodiments, the cell population is a T cell population and the cells are T cells.

[0073] In some embodiments, step (b) comprises (1) resuspending the cell population in a hydrogel; (2) placing the hydrogel containing the cells in a culture medium for culturing.

[0074] Any means known in the art can be used to sort the activated cells. In some embodiments, step (c) is sorting the activated cells based on the presence or absence of a reporter. In some embodiments, the reporter is a fluorescent protein. In some embodiments, flow cytometry is used to sort the activated cells.

[0075] In some embodiments, the method of the present application further comprises (e) preparing the TCR sequences and the pMHC sequences identified in step (d) into a TCR library and a pMHC library; (f) repeating steps (a)-(f) one, two, three, four, five, six, seven, eight, nine, or ten times using the TCR library and the pMHC library prepared in (e).

[0076] In some embodiments, step (d) is determining the TCR and the pMHC by sequencing, preferably the sequencing is single cell sequencing. In some embodiments, the sequencing is next generation sequencing. In some embodiments, the sequencing is third generation sequencing.

[0077] Gel

[0078] Any known gel for cell culture in the art can be used in the present application.

[0079] Natural or synthetic gels can be used in the present application. Natural gels for cell culture are typically formed from proteins and extracellular matrix (ECM) components, as well as materials derived from other biological sources (e.g. chitosan, alginate, or silk fibres). As these gels are derived from natural sources, they have inherent biocompatibility and bioactivity. Synthetic hydrogels can be formed from purely non-natural molecules (e.g. polyethylene glycol (PEG), polyvinyl alcohol and poly(2-hydroxy methyl acrylate)). PEG hydrogels have been shown to be able to sustain the viability of encapsulated cells and allow ECM deposition during degradation. This inert gel has a high degree of reproducibility, is easy to tune the mechanical properties, and can be easily processed and manufactured.

[0080] Thermosensitive hydrogels are a class of functional hydrogels that can respond to environmental temperature rapidly and undergo reversible transformation. They are flowable liquids at lower temperature environment, and can be uniformly mixed with various seed cells, drugs and active molecules, etc. At physiological temperature environment (37°C), they rapidly gel to form three-dimensional hydrogels, thereby embedding cells and other substances in situ without the need for any other chemical reactions.

[0081] In some embodiments, the gel used in the present application can be a gel including, but not limited to, a gel including the following components: Matrigel rgf, BME1, BME1rgf, BME2, BME2rgf, BME3 (all Matrigel variants), Collagen I, Collagen IV, a mixture of Collagen I and IV, or a mixture of Collagen I and IV, and Collagen II and III, puramatix, hydrogel, Cell-Tak TM , Collagen I, Collagen IV, fibronectin, gelatin, laminin, osteopontin, polylysine (PDL, PLL), PDL / LM and PLO / LM, or vitronectin.

[0082] Preferably, in some embodiments, the gel used in the present application is a hydrogel. In some embodiments, the gel used in the present application is a thermosensitive hydrogel.

[0083] In some embodiments, the present application provides the use of a gel for culturing T cells in screening interacting TCRs and pMHCs.

[0084] Cell population

[0085] The present application also provides a cell population, which can be used in the screening of the present application. The cell population expresses an exogenous TCR and / or an exogenous pMHC.

[0086] Preferably, in some embodiments, the cell population is a T cell population.

[0087] In some embodiments, no less than 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the T cells in the cell population described herein express only 1, 2, 3, 4, or 5 exogenous TCRs and only 1, 2, 3, 4, or 5 exogenous pMHCs.

[0088] In some embodiments, no less than 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the T cells in the cell population described herein express only 1, 2, 3, 4, or 5 exogenous TCRs.

[0089] In some embodiments, no less than 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the T cells in the cell population described herein express only 1, 2, 3, 4, or 5 exogenous pMHCs.

[0090] In some embodiments, the endogenous MHC-I, endogenous MHC-II, and / or endogenous TCR of the cell described herein is deleted.

[0091] In some embodiments, the cell described herein is a T cell, preferably a Jurkat T cell.

[0092] In some embodiments, the exogenous pMHC in the cell described herein comprises a mutated MHC. Preferably, in some embodiments, the MHC mutation is Y84C.

[0093] In some embodiments, the exogenous pMHC is in single-chain dimer (SCD) or single-chain trimer (SCT) form.

[0094] In another aspect, the present application also provides use of the cell population described herein in screening interacting TCRs and pMHCs.

[0095] Applications

[0096] The screening method of the present application can be used for high-throughput screening of interacting TCRs and pMHCs.

[0097] In some embodiments, the method of the present application can be used to obtain a TCR antigen recognition map for one determined TCR.

[0098] In some embodiments, the methods of the present application can be used to find antigens against clinical commonality or highly expanded TCRs, thereby for analyzing diseases, designing treatment strategies, such as designing TCR-T cell therapy.

[0099] In some embodiments, the data generated by the methods of the present application can be used for machine learning models. In some embodiments, the data generated by the methods of the present application can be used to train and learn models. This application can improve the prediction accuracy of machine learning models.

[0100] In some embodiments, the methods of the present application can be used to identify cross-reactivity against TCRs used in clinical practice.

[0101] Hereinafter, the present application will be illustrated by examples to show the beneficial effects of the present application. Those skilled in the art will recognize that these examples are illustrative rather than limiting. These examples will not limit the scope of the present application in any way. The experimental methods described in the following examples are conventional methods unless otherwise specified; reagents and materials are commercially available unless otherwise specified.

[0102] The experimental techniques and experimental methods used in this example are conventional techniques and methods unless otherwise specified, for example, the experimental methods not specified in the following examples are generally carried out under conventional conditions or under the conditions recommended by the manufacturer. The materials, reagents, etc. used in the examples can be obtained through regular commercial channels unless otherwise specified.

[0103] Example

[0104] Example 1, Construction of Starting SCD (peptide), TCR sequence library and lentivirus library packaging

[0105] 1. Use the oligo library synthesized by chip (or use NNK degenerate primer to construct random mutation library), and use NEBuilder HiFi DNA Assembly cloning method to construct SCD (pMHC) library into pHAGE-EF1A-SCD-IRES-ALNGFR (SEQ ID NO: 1) vector;

[0106] 2. Use NEBuilder HiFi DNA Assembly cloning method to construct gene-synthesized TCR sequence library in pHAGE-EF1A-TCR (SEQ ID NO: 2) vector.

[0107] The SCD (peptide) library is constructed as follows:

[0108] 1. Linearize the pBIPE-1-vector (SEQ ID NO: 25) using the primer pair (1), DNA polymerase and pBIPE-1-vector (SEQ ID NO: 25) for amplification;

[0109] 2. Use the synthesized pooled-library-A2 primer (SEQ ID NO: 3) and common-R primer (SEQ ID NO: 4) for amplification;

[0110] 3. NEBuilder HiFi DNA assembly: use NEBbuilder HiFi DNA Assembly Master Mix (E2621L) to connect the following system, 50°C for 1 hour:

[0111] 4. Use silica gel column to purify the DNA connection product;

[0112] 5. Electroporation: use electroporation competent cells for electroporation, and then culture the transformed library cells, and extract the plasmid library.

[0113] The pBIPE-final-library is constructed as follows:

[0114] Step 1

[0115] 1. Use the primer pair (1), DNA polymerase and pBIPE-1-vector (SEQ ID NO: 25) for amplification to obtain a linearized pBIPE-1-vector fragment;

[0116] 2. NEBuilder HiFi DNA Assembly recombination of the SCD-TCR library fragment and the pBIPE-1-vector fragment, and electroporation to obtain the transformed pBIPE-1-library intermediate plasmid library.

[0117] Step 2

[0118] 1. Use the pBIPE-1-library intermediate plasmid, and use Mlu1-HF (NEB) for single enzyme digestion to obtain a linearized SCD-TCR library 2;

[0119] 2. Use the primer pair (2), DNA polymerase and pBIPE-1-vector for amplification to obtain a linearized promoter-insert fragment;

[0120] 3. Linearized SCD-TCR library 2 and promoter-insert fragments were recombined by NEBuilder HiFi DNA Assembly, electrotransformation, and obtained transformed pBIPE-final-library plasmid library.

[0121] The lentivirus packaging method is as follows

[0122] The library plasmid, lentivirus packaging plasmid psPAX.2, and pMD2.G plasmid were used to transfect HEK-293T cells to produce a lentivirus library. After 24 hours of liquid change, the virus was collected at 48 hours and concentrated by ultracentrifugation.

[0123] Example 2, construction of Jurkat-NFAT-reporter cell line

[0124] Construction of HLA KO, TCR KO, overexpression of NFAT-zsGreen-humanCD8 reporter vector

[0125] 1. Knock out HLA-A\B\C and TCR of Jurkat-T cell line using CRISPR-Cas9 system (Figure 2A. ①②);

[0126] 2. After overexpression of NFAT-zsGreen-humanCD8 using lentivirus, monoclonal culture was used to obtain high-efficiency reporter cell line Jurkat T reporter cell line (Figure 2A. ③).

[0127] Construction of MHC, TCR overexpression cell line

[0128] 1. Specific MHC (including Y84C mutation) such as HLA-A2 / HLA-B35 / HLA-B27, etc. was expressed in Jurkat T reporter cell line using MHC overexpression lentivirus (Figure 2B);

[0129] 2. TCR library was expressed in the above cell line using TCR lentivirus library;

[0130] 3. Flow cytometry was used to sort cells to obtain cells with high expression of MHC and TCR.

[0131] Example 3, single cell cloning culture of temperature-sensitive hydrogel

[0132] 1. Jurkat cells were completely resuspended in 1 / 10 gel volume of complete culture medium, passed through a 70 μm cell filter, and obtained a single dispersed cell suspension in a 50 mL centrifuge tube, and placed at 4°C;

[0133] 2. Add 10 times cell suspension volume of pNIPaam-PEG hydrogel (Mebiol Gel), pipette on ice to mix;

[0134] 3. According to the pNIPaam-PEG instruction, add cell-hydrogel mixture to cold 6-well cell culture plate, 1 mL / well, place in 37℃ incubator for 5 min to solidify;

[0135] 4. Add 4 mL of 37℃ preheated complete medium to each well of the solidified hydrogel, place in 37℃ incubator for culture, replace with preheated complete medium every 24 h.

[0136] Example 4. Identification of interacting TCR and pMHC based on hydrogel culture

[0137] Using Jurkat-T-reporter cell line overexpressing HLA-A2, overexpress 1G4, DMF5, Flu TCR using lentivirus, overexpress NYESO-1, MART-1 SCD using lentivirus, after infection, place cells in hydrogel for single cell clonal culture, experimental results show that Jurkat T reporter cells only activate NFAT-GFP reporter signal when carrying specific peptide-TCR (such as known specific recognition of 1G4-TCR / NYESO and DMF5 / MART-1) (Figure 4A).

[0138] High-throughput identification of interacting TCR and pMHC using the following method

[0139] DNA extraction and sequencing library construction

[0140] 1. Use cell sorting to obtain single cell population cell suspension carrying specific SCD-TCR sequence pairs, after centrifugation

[0141] 2. Resuspend in 23.3% percoll-sucrose solution to form aqueous phase 1, cell density not more than 1 million cells per mL;

[0142] 3. Configure amplification reaction solution: use DNA polymerase, PCR buffer, primer pair (3) to form aqueous phase 2;

[0143] 4. Use biorad ddPCR droplet generator oil as oil phase 1.

[0144] Example 5. Double cell or clone-to-clone influence of temperature-sensitive hydrogel

[0145] The activation efficiency of peptide-carrying cells, specific TCR\MHC cells, and cells carrying the same TCR\MHC but not carrying the peptide, labeled with CellTrace Violet and CellTrace Far Red respectively, at different cell culture densities, shows that the activation efficiency of peptide-carrying cells is independent of cell density, and the activation degree of non-peptide-carrying cells affected by peptide-carrying cells increases with cell density, and is less than 5% at a cell inoculation density of 5M / mL, showing good cell wrapping and isolation effect of the method (Figure 6).

[0146] Example 6, Screening of DMF5 TCR Specific Antigens

[0147] Cell library infection and culture

[0148] 1. Use A2-NNK library to package initial A2-NNK library virus according to the above steps, and infect the Jurkat-T-reporter cell strain expressing DMF5 TCR constructed in the above steps;

[0149] 2. Resuspend the cells completely in 1 / 10 gel volume of complete culture medium, pass through a 70 μm cell filter, and obtain a single dispersed cell suspension in a 50 mL centrifuge tube, and stand at 4°C;

[0150] 3. Add 10 times the volume of the cell suspension of pNIPaam-PEG hydrogel (Mebiol Gel), and mix well on ice using a pre-cooled pipette;

[0151] 4. Add the cell-hydrogel mixture to a cold 6-well cell culture plate, 1 mL per well, and place in a 37°C incubator for 5 min to solidify;

[0152] 5. Add 4 mL of 37°C preheated complete culture medium to each well of the solidified hydrogel, and place in a 37°C incubator for culture, and replace the preheated complete culture medium every 24 h.

[0153] Cell recovery and sorting

[0154] 1. After 48-72 h of culture according to the above steps, remove the complete culture medium on top of the hydrogel, add 4°C pre-cooled PBSD solution, and cool the gel on ice, constantly shaking until the cell gel and PBSD form a uniform solution; centrifuge to recover the cells and use a flow cytometer to sort zsGreen+ positive cells.

[0155] Cell genome extraction and PCR amplification

[0156] 1. Take the zsGreen positive cells obtained by the above step, centrifuge and resuspend in PBS, use Qiagen genomic extraction kit (Ref 69504) to extract genomic DNA,

[0157] 2. Use primer pair (5) to perform fragment amplification with DNA polymerase,

[0158] 3. Use 2% DNA agarose gel to perform gel recovery and purification.

[0159] PCR amplification product adds sequencing adapter and subcloning

[0160] 1. Take the DNA purified by the above gel recovery, use Nextra primer index primer pair to add library sequencing adapter and index, use 2% DNA agarose gel to perform gel recovery and purification;

[0161] 2. Take the DNA purified by the above gel recovery, use primer pair (6) to perform library subcloning, use 2% DNA agarose gel to perform gel recovery and purification to obtain subcloning PCR product;

[0162] 3. Take the pHAGE-EF1A-SCD-IRES-ALNGFR linearized vector digested by BsmB1, the above subcloning PCR product to perform NEBuilder HiFi DNA Assembly recombination, electrotransformation, and obtain the transformed Phage-EF1A-SCD-IRES-library-round2 plasmid library.

[0163] Multiple rounds of screening and sequencing

[0164] 1. According to the above steps, use the subcloned pHAGE-EF1A-SCD-IRES-library-round2 and subsequent round subcloned plasmid library to package two rounds, three rounds, four rounds of virus, infect the Jurkat cell strain constructed in the above step to express a single or several TCRs, and perform cell library infection, culture, recovery, sorting, genomic extraction and PCR amplification, PCR amplification product subcloning, until the screening ends when the proportion of cells with more than 1% zsGreen positive ratio appears in the BD Aria III flow sorting;

[0165] 2. Use sequencing fragments with different index sequences to perform NGS sequencing to obtain peptide sequence information obtained by screening.

[0166] Experimental results

[0167] For single TCR screening, pMHC library or genome predicted library E6 was constructed using E8-E9 NNK random primers, first TCR was overexpressed in Jurkat reporter cells, then pMHC random library was introduced. After sorting GFP+ cells, genomic sequence was recovered, and the screened sequence was used to construct the next round of pMHC library. (Figure 1A) (Figure 1B) After three rounds of screening, Flu TCR antigen fingerprint was obtained from the mutant library (Figure 7), and DMF5 TCR antigen fingerprint was obtained from the NNK library (Figure 8, 9), and the rare sequence was verified by peptide pulsing experiment (Figure 10).

[0168] For 4-6 TCRs, TCR expression cell lines were constructed respectively and mixed, the same library and mixed screening strategy was used, and after 3-4 rounds, the cells were wrapped separately, and sorting could complete 1G4 DMF5 Flu EBV 4 TCR synchronous mixed screening. (Figure 11)

[0169] Example 7, Screening of 100 TCR Specific Antigens Specific to B27 Ankylosing Spondylitis

[0170] Cell library infection and culture

[0171] 1. Using the initial NNK-B27 library virus packaged according to the above steps, infect the Jurkat cell strain expressing the 100 TCR library, stand for 2 hours at 37°C, centrifuge at 1700 rpm for 5 min, then discard the supernatant;

[0172] 2. Resuspend the cells completely in 1 / 10 gel volume of complete medium, pass through a 70 μm cell filter to obtain a single dispersed cell suspension in a 50 mL centrifuge tube, and stand at 4°C;

[0173] 3. Add 10 times the volume of the cell suspension to the pNIPaam-PEG hydrogel, and use a pre-cooled pipette to mix on ice;

[0174] 4. According to the instructions for use of pNIPaam-PEG, add the cell-hydrogel mixture to a cold 6-well cell culture plate, 1 mL / well, and place in a 37°C incubator for 5 min to solidify;

[0175] 5. Add 4 mL of 37°C preheated complete medium to each well of the solidified hydrogel, and place in a 37°C incubator for culture, and replace the preheated complete medium every 24 h

[0176] Cell recovery and sorting

[0177] 1. After 48h to 72h of culture, remove the supernatant of the hydrogel and add the PBSD solution pre-cooled at 4°C. Shake the gel on ice until the cell gel and PBSD form a uniform solution. Centrifuge the cells and use the flow cytometer to sort the zsGreen+positive cells.

[0178] After centrifugation, use the single-cell suspension carrying the specific SCD-TCR sequence pair obtained by cell sorting

[0179] 1. Resuspend in 23.3% percoll-sucrose solution to form aqueous phase 1, with a cell density of no more than 1 million cells per mL.

[0180] 2. Prepare the amplification reaction solution: use DNA polymerase, PCR buffer, and primer pair (3) to form aqueous phase 2.

[0181] 3. Use biorad ddPCR droplet generator oil as oil phase 1.

[0182] Use the double-channel water-in-oil chip PDMS-SCE-50 (Zhiheng)

[0183] 1. Connect the chip clamp.

[0184] 2. Pre-rinse the aqueous phase 1 / 2 and oil phase 1.

[0185] 3. Replace the aqueous phase 1 with the cell suspension, adjust the air pump pressure and flow rate to 3ul / min for the aqueous phase 1 and aqueous phase 2, and adjust the input pressure of the oil phase 1 to form stable 50um diameter droplets.

[0186] 4. Collect the droplets in a 1.5mL centrifuge tube and transfer them to a PCR eight-tube tube for single-cell droplet PCR capture of SCD-TCR sequences. The single-cell droplet PCR reaction conditions are: 98°C for 1min (98°C for 10s, 55°C for 10s, 68°C for 2min30s) for 30 cycles, 68°C for 7min, and 4°C storage.

[0187] 5. Concentrate the droplet PCR reaction solution in a 1.5mL EP tube, wash the PCR tube with HFE7500 (Novec 3M) to collect all the droplets, transfer them to a 1.5mL EP tube, and stand for 1min.

[0188] 6. Use a low-adhesion tip to carefully remove the lower oil phase in the EP tube, add 1x droplet volume of TE solution, and 1x droplet volume of 20% PFO / HFE7500 (CF3(CF2)5CH2CH2OH CAS: 647-42-7) for demulsification treatment. After centrifugation at 3000g for 5min, remove the oil phase.

[0189] 7. Add 1x droplet volume of 20% PFO / HFE7500 (CF3(CF2)5CH2CH2OH CAS: 647-42-7) for demulsification, centrifuge at 3000g for 5 min and carefully pipette as much of the aqueous phase as possible.

[0190] Single cell SCD-TCR paired PCR product recovery, nested PCR, vector cloning

[0191] 1. Remove the aqueous phase from the above step and purify the DNA fragment using a silica gel column purification method to obtain a pure DNA fragment.

[0192] 2. Take 1 / 5 of the DNA fragment obtained in the above step, add the nested PCR primer pair (4), and use DNA polymerase to amplify to obtain (SCD-TCR library).

[0193] 3. Clone according to the method described in step one, and construct the SCD-TCR library into the pBIPE-1-vector vector to become pBIPE-final-library

[0194] Use pBIPE-final-library for multiple rounds of screening and sequencing

[0195] Cell genomic extraction and PCR amplification

[0196] 1. Take the zsGreen positive cells obtained in the above step, centrifuge and resuspend in PBS, and use the Qiagen genomic extraction kit (Ref 69504) to extract genomic DNA.

[0197] 2. Use primer pair (5), DNA polymerase to amplify the fragment,

[0198] 3. Use 0.5% DNA agarose gel for gel recovery and purification.

[0199] PCR amplification product adds sequencing adapter and subcloning

[0200] 1. Take the DNA purified by the above gel recovery, use primer pair (8) for library subcloning, use 1% DNA agarose gel for gel recovery and purification to obtain subcloning PCR product;

[0201] 2. Take the pBIPE-1-vector linearized by primer pair (9) PCR, the above subcloning PCR product for Gibson assembly recombination, electrotransformation, to obtain the transformed pBIPE-final-library-round2 plasmid library.

[0202] Multiple rounds of screening and sequencing

[0203] 1. According to the above steps, using subcloned pBIPE-final-library-round2 and subsequent round subcloned plasmid library to package round 2, 3, 4 virus, infect the Jurkat cell strain expressing single or several MHC constructed in the above steps, after cell library infection, culture, recovery, sorting, genome extraction and PCR amplification, subclone the PCR amplification product, until the end of the screening when more than 1% of the zsGreen positive proportion of cells appears in the BD Aria III flow sorting

[0204] 2. Perform pacbio sequencing of the sequence of the last round of PCR product to obtain the peptide and TCR sequence information obtained by screening.

[0205] Experimental results: After screening, 16 TCR recognition spectra were obtained from 100 TCRs, such as TCR#16, which is one of the TCR sequences obtained by recognizing the antigen recognition spectrum, recognizing xRxxFxMxx.

Claims

A method of screening for interacting TCRs and antigen-MHC complexes (pMHCs) comprising: (a) expressing a TCR library and / or a pMHC library on the surface of a population of cells to obtain a population of cells expressing exogenous TCRs and / or exogenous pMHCs; (b) culturing the population of cells obtained in step (a) in a gel to obtain a plurality of cell pellets from single cells; (c) sorting for activated cells; (d) determining the sequences of TCRs and pMHCs on the surface of the cells in step (c); thereby determining interacting TCRs and antigen-MHC complexes; optionally, the population of cells is a population of T cells; and the cells are T cells. The method of claim 1, further comprising: (e) preparing the TCR sequences and pMHC sequences determined in step (d) into a TCR library and a pMHC library; (f) repeating steps (a)-(f) one, two, three, four, five, six, seven, eight, nine, or ten times using the TCR library and pMHC library prepared in (e). The method of the preceding claims, wherein the gel is selected from the group consisting of Matrigel or a hydrogel; preferably, the gel is a hydrogel. The method of claim 3, wherein the hydrogel is a temperature-sensitive hydrogel, preferably a PEG-based temperature-sensitive hydrogel, more preferably a poly-N- isopropylacrylamide (PNIPAM) PEG temperature-sensitive hydrogel. The method of the preceding claims, wherein step (b) comprises (1) resuspending the population of cells in a hydrogel; (2) placing the hydrogel containing the cells in a culture medium for culturing. The method of the preceding claims, wherein step (a) is expressing the TCR library and the pMHC library on the surface of the population of cells using lentiviral transfection. The method of the preceding claims, wherein step (d) is determining the TCRs and pMHCs by sequencing, preferably the sequencing is single-cell sequencing. The method of the preceding claims, wherein step (c) is sorting for activated cells based on the presence or absence of a reporter. The method of claim 8, wherein the reporter is a fluorescent protein, preferably the fluorescent protein is zsGreen. The method of the preceding claims, wherein no less than 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the T cells in the population of T cells obtained in step (a) express only 1, 2, 3, 4, or 5 exogenous TCRs and only 1, 2, 3, 4, or 5 exogenous pMHCs. A population of cells expressing exogenous TCRs and / or exogenous pMHCs, preferably the population of cells is a population of T cells. The cell population of claim 11, wherein not less than 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the T cells express only 1, 2, 3, 4, or 5 exogenous TCRs and only 1, 2, 3, 4, or 5 exogenous pMHCs. The method or cell population of the preceding claim, wherein the endogenous MHC-I, endogenous MHC-II, and / or endogenous TCR of the cell is deleted. The method or cell population of the preceding claim, wherein the MHC in the exogenous pMHC is a wild-type MHC or is a MHC comprising a mutation, preferably the mutation is Y84C. The method or cell population of the preceding claim, wherein the cell is a Jurkat T cell. The method or cell population of the preceding claim, wherein the exogenous pMHC is in single chain dimer (SCD) or single chain trimer (SCT) form. Use of the cell population of the preceding claim in screening interacting TCRs and pMHCs. Use of a gel for culturing cells in screening interacting TCRs and pMHCs, preferably the gel is a hydrogel, preferably the cells are T cells. The use of claim 18, wherein the hydrogel is a thermoresponsive hydrogel, preferably a PEG-based thermoresponsive hydrogel, more preferably a poly-N-isopropylacrylamide (PNIPAM) PEG thermoresponsive hydrogel.

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