Compositions and methods for generating novel T cell receptors

Abstract

The present disclosure generally relates to the field of T cell receptors or T cell receptor mimics, and methods for obtaining novel T cell receptors or novel T cell receptor mimics.The compositions and methods described identify T cell receptor and antigen pairs.The compositions and methods allow high-throughput analysis, and many antigens can be paired with a large repertoire of T cell receptors.

Description

[Technical field]

[0001] Background of the Disclosure Immune cells are a heterogeneous group of cells involved in many important immune functions, including defense against pathogens, control of inflammation, and elimination of cancer cells. The major classes of immune cells include T cells, B cells, macrophages, neutrophils, natural killer (NK) cells, and dendritic cells. Although they differ in function and morphology, immune cells are all positively or negatively regulated by their surface receptors. [Background technology]

[0002] The T cell receptor (TCR) is a molecule found on the surface of T lymphocytes (i.e., T cells) that is responsible for the recognition of antigens bound to major histocompatibility complex (MHC) molecules. The TCR is a heterodimer composed of two different protein chains. In most T cells (about 95%), these two protein chains are called alpha and beta chains. However, in a small percentage of T cells (about 5%), these two protein chains are called gamma and delta (gamma / delta) chains.

[0003] Humans have billions of T cell receptors that provide a major component of the adaptive immune system, and T cell receptor responses have been demonstrated to provide an important contribution to protection from diseases including viral infections, cancer, and autoimmunity. Summary of the Invention [Problem to be solved by the invention]

[0004] There is great interest in the ability to discover T cell receptors for specific antigens. Such T cell receptors are useful as research tools and for diagnostic and therapeutic applications. However, it is difficult to identify such useful T cell receptors, and once identified, these T cell receptors often require extensive redesign before they are suitable for therapeutic applications in humans. [Means for solving the problem]

[0005] overview In one aspect, the present disclosure relates to compositions and methods for identifying receptors or receptor mimics (e.g., chimeric receptors) that bind to a particular ligand. The compositions and methods described herein may allow for high-throughput analysis. A high diversity repertoire of receptors (e.g., immune cell receptors) can be screened against a library of ligands to identify receptor and ligand pairs that interact with each other. Each nucleic acid encoding each receptor in the repertoire can be linked to a barcode that identifies the receptor encoded by the nucleic acid. Each nucleic acid encoding each ligand can also be linked to a barcode that identifies the ligand encoded by the nucleic acid.

[0006] In one aspect, the present disclosure relates to compositions and methods for identifying immune cell receptors or immune cell receptor mimics (e.g., chimeric antigen receptors) that bind to a particular ligand. The compositions and methods described herein may allow for high-throughput analysis. A high diversity repertoire of immune cell receptors (e.g., T cell receptors) can be screened against a library of ligands (e.g., antigens) to identify receptor and ligand pairs that interact with each other. Each nucleic acid encoding each receptor in the repertoire can be linked to a barcode that identifies the receptor encoded by the nucleic acid. Each nucleic acid encoding each ligand can also be linked to a barcode that identifies the ligand encoded by the nucleic acid.

[0007] A wide range of immune cells and / or immune cell receptors can be used with the methods disclosed herein. For T cells, applicable receptors include alpha beta T cell receptor, gamma delta T cell receptor, CD43, CD44, CD45, LFA1, CD4, CD8, CD3, LAT, CD27, CD96, CD28, TIGIT, ICOS, BTLA, HVEM, 4-1BB, OX40, DR3, GITR, CD30, SLAM, CD2, 2B4, TIM1, TIM2, TIM3, CD226, CD160, LAG3, LAIR1, CD112R, CTLA-4, PD-1, PD-L1, PD-L2. For NK cells, applicable receptors include TRAIL, CD16, NKp30ab, NKGC, NKG2D, 2B4, DNAM-1, NKG2A, KIR, CD137, OX40, CD27. For B cells, applicable receptors include Siglec-10, LILRB / PIR-B, CD31, FCγRIIIB, CD19, CD20, CD22, CD25, CD32, CD40, CD47, CD52, CD80, CD86, CD267, CD268, CD268, B cell receptor, antibodies, IgM, IgD, IgG, IgA, IgE. For dendritic cells, applicable receptors include DNGR-1, MICL, CLEC1, CLEC12B, LOX1, mannose receptor, DC-SIGN, L-SIGN, SIGN-R1, LSECtin, CIRE, Langerin, MGL, scavenger receptor (SR), DC-ASGPR, DC-STAMP, CD80 / 86, TLR, FIRE, FcR, DEC205, BDCA-2, Dectin-2, DCIR, and chemokine receptors. In macrophages, applicable receptors include CD300a, TREM2 / DAP12, Bal-1, TIM4, CR3, SCARF1, CD36, MARCO, scavenger receptor A1, RAGE, Axl, Mer, Tyro3, ​​CD93, Stabilin2, DNGR-1, SIRP. In neutrophils, applicable receptors include CXCR1, FCγRIIIB, FCγRII, CR3, CR1, C3aR, TNFR, TLR2 / 6, C5aR. Other receptors (e.g., G protein-coupled receptors, ion channel-linked (ionotropic) receptors, and / or enzyme-linked receptors) may also be used in the methods and compositions of the present disclosure.Any immune cell that may be used herein includes, for example, a T cell, a B cell, a natural killer cell, a dendritic cell, a macrophage, a monocyte, and / or a neutrophil.

[0008] In one embodiment, the above repertoire of receptors can be engineered into phagocytes, such as macrophages, dendritic cells, neutrophils, and / or THP-1 cells. The receptors of interest can be engineered by their endogenous intracellular activation domains or by phagocytosis-associated intracellular activation domains (e.g., Fc receptor intracellular domains and CD19 intracellular domains). The extracellular domains of the receptors can be wild-type or modified versions of the extracellular domains of the receptors of interest.

[0009] In one embodiment, a repertoire of receptors can be engineered into immune cells (e.g., T cells, natural killer cells, B cells, macrophages, neutrophils, dendritic cells, etc.). The repertoire of receptors is expressed on the surface of immune cells (e.g., T cells, natural killer cells, B cells, macrophages, neutrophils, dendritic cells, etc.) and uses trogocytosis to separate the receptor from its antigen / ligand. Most immune cells are capable of trogocytosis, including, for example, T cells, macrophages, B cells, neutrophils, natural killer (NK) cells, monocytes, and dendritic cells. Receptors of interest can be introduced by their intrinsic intracellular activation domains or by trogocytosis-associated intracellular activation domains (e.g., Fc receptor intracellular domains and TCR intracellular domains). The extracellular domain of the receptor can be a wild-type or modified version of the receptor of interest.

[0010] The antigen / ligand of the receptor of interest can be a peptide, an MHC-peptide complex, a fragment, a chimera, or a whole protein. The antigen / ligand can be a carbohydrate, a fatty acid or lipid, or a small molecule. The antigen / ligand can be associated with the antigen-presenting cell surface by covalent or non-covalent bonds. If the antigen / ligand is a polypeptide, they can be anchored to the antigen-presenting cell surface by their natural transmembrane domain or by a synthetic transmembrane domain. The synthetic transmembrane domain can be fused to the antigen / ligand extracellular domain by engineering the DNA sequence to operably link the synthetic transmembrane domain DNA sequence with the DNA sequence encoding the desired portion, part, or whole of the antigen / ligand. Exemplary synthetic transmembrane domains include CD2, CD3d, CD3g, CD3z, CD4, CD8A, CD8B, CD22, CD27, CD28, CD40, CD79a, CD79b, CD80, CD84, CD86, CD137, CD244, CRACC, CRTAM, CLTA-4, MHC-I, MHC-II, platelet-derived growth factor receptor, FCGR1A, FCGR2A, FCG2B, FCGR3A, FCRL1, FCRL2, FCRL3, FCRL4, FCRL5, F The antigen / ligand library may include wild-type or modified transmembrane domains from CRL6, KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL3, LAG3, GITR, OX40, PD-1, PD-L1, PD-L2, TLR, SLAMF, LILRB1, LILRB2, NKG2A, NKG2C, NKG2D, TIGIT, IgG, IgM, IgA, IgE, IgD, or immunoglobulin. Repertoires of antigens / ligands may be created and engineered into antigen / ligand presenting cells. In some cases, variations of the antigen / ligand library may be created by introducing genetic mutations (e.g., randomly or site-specifically) into desired positions of the antigen / ligand of interest. Genetic mutations may be substitutions, deletions, insertions, and rearrangements of DNA fragments. The specific genetic mutations of choice may be generated randomly, enriched in regions of interest (e.g., the interaction domain of a ligand), or recommended by computational algorithms.Examples of computational algorithms to recommend gene mutation strategies include protein structure analysis, computational docking, molecular dynamics simulation, regression, statistically-based classification, random forests, support vector machines, and neural networks.

[0011] The antigen / ligand binding portion of the immune cell receptor or immune cell receptor mimic can be fused to the intracellular domain of the Fc receptor. Immune cell receptor mimic refers to an antibody or antibody fragment that can bind and interact with the MHC::antigen complex (academic.oup.com / abt / article / 2 / 1 / 22 / 5290150). Optionally, these chimeric receptors can include the transmembrane domain of the Fc receptor, or the chimeric receptor can have a different transmembrane domain. These chimeric receptors have the binding specificity of the immune cell receptor and can activate macrophages to phagocytose targets that present the antigen bound by the immune cell receptor. Macrophage / monocyte cells with chimeric TCRs can also be engineered to express one or more of CD28, CD137, CD3, B7.1, CD19 intracellular activation domain, CD64 intracellular activation domain, CD32 intracellular activation domain, CD16 intracellular activation domain, CD23 intracellular activation domain, or Lck activation domain. These further modifications can be included in the chimeric immune receptor Fc fusion, for example as costimulatory domains, or can be expressed individually in monocyte / macrophage cells. When an engineered monocyte / macrophage phagocytose a cell presenting the appropriate antigen, it can produce a macrophage with an immune cell receptor bearing that barcode, and an antigen / ligand bearing that barcode. Sequencing of such individual macrophages can identify the immune cell receptor antigen / ligand pair.

[0012] The antigen-binding portion of the T cell receptor or T cell receptor mimic can be fused to the intracellular domain of the Fc receptor. The T cell receptor mimic refers to an antibody or antibody fragment that can bind and interact with the MHC::antigen complex (academic.oup.com / abt / article / 2 / 1 / 22 / 5290150). Optionally, these chimeric receptors can include the transmembrane domain of the Fc receptor, or the chimeric receptor can have a different transmembrane domain. These chimeric receptors have the binding specificity of the T cell receptor and can activate macrophages to phagocytose targets that present the antigen bound by the T cell receptor. Macrophages / monocyte cells with chimeric TCRs can also be engineered to express one or more of CD28, CD137, CD3, B7.1, CD19 intracellular activation domain, CD64 intracellular activation domain, CD32 intracellular activation domain, CD16 intracellular activation domain, CD23 intracellular activation domain, or Lck activation domain. These further modifications can be included in the chimeric TCR-Fc fusion, for example as a costimulatory domain, or can be expressed individually in monocyte / macrophage cells. When an engineered monocyte / macrophage phagocytose a cell that presents the appropriate antigen, it can produce a macrophage that has a T cell receptor with that barcode, and an antigen with that barcode. Sequencing of such individual macrophages can identify the T cell receptor-antigen pair.

[0013] The antigen / ligand library can be made in antigen-presenting cells (e.g., K562 cell line), where the antigen or ligand can be encoded by a nucleic acid encoding the antigen or ligand with a barcode, or an antigen / ligand polypeptide and a recombinant MHC protein fused to the barcode, if desired. If the antigen is a non-polypeptide antigen, the antigen-presenting cell can be engineered to make the antigen / ligand and present it on the surface of the antigen-presenting cell. Alternatively, the antigen / ligand can be made outside the antigen-presenting cell and then associated with the surface of the antigen-presenting cell. Polypeptide fragments from each antigen / ligand that can be presented can be identified by antigen-MHC binding experiments, mass spectrometry experiments, sliding windows of full-length protein sequences (8-25 per window), or fragments of known proteins predicted to be presentable by computer algorithms. These presentable peptide fragments from the antigen can be fused with the appropriate MHC molecule to create a library of antigens to be presented. The antigen-presenting cell can be a K562 cell line (a human immortalized myeloid leukemia cell line) or an engineered HEK293 cell line. MHC-antigen polypeptide fusions can be introduced into K562 cells to generate a library of antigenic peptides. Optionally, the K562 cells can be engineered with an optical reporter, such as GFP.

[0014] The immune cell receptor repertoire of macrophage cells (e.g., THP-1 cells, a human monocytic cell line derived from acute monocytic leukemia) can be mixed with the antigen library of K562 cells. Macrophage cells (e.g., THP-1 cells) with immune receptor chimeric receptors or immune cell receptor mimics that bind antigen peptide:MHC fusions (or antigen / ligands) can phagocytose K562 cells that present the appropriate peptide:MHC fusions (or antigen / ligands). Macrophage cells (e.g., THP-1 cells) with phagocytosed K562 cells can be isolated and individually sequenced to identify the barcodes associated with immune cell receptors and antigens / ligands. If the K562 cells contain a reporter, the reporter can be used to screen macrophage cells (e.g., THP-1 cells) that have phagocytosed K562 cells, for example, using flow cytometry. Alternatively, antigen:MHC fusions and immune receptor chimeric receptors can be sequenced directly to identify immune cell receptors and antigen / ligand pairs.

[0015] The T cell receptor repertoire of macrophage cells (e.g., THP-1 cells, a human monocytic cell line derived from acute monocytic leukemia) can be mixed with the antigen library of K562 cells. Macrophage cells (e.g., THP-1 cells) with TCR chimeric receptors or T cell receptor mimics that bind to antigen peptide:MHC fusions can phagocytose K562 cells that present the appropriate peptide:MHC fusions. Macrophage cells (e.g., THP-1 cells) with phagocytosed K562 cells can be isolated and sequenced individually to identify barcodes associated with T cell receptors and antigens. If the K562 cells contain a reporter, the reporter can be used to screen macrophage cells (e.g., THP-1 cells) that have phagocytosed K562 cells, for example, using flow cytometry. Alternatively, antigen:MHC fusions and TCR chimeric receptors can be sequenced directly to identify TCR and antigen pairs.

[0016] In one aspect, phagocytosis by macrophages / monocytes / dendritic cells / neutrophils with chimeric TCRs can be promoted by differentiation of macrophages / monocytes / dendritic cells / neutrophils (if not terminally differentiated cells), e.g., macrophage / monocyte differentiation, stimulating differentiation using T cell macrophage hybridomas, blocking the CD47 pathway, or blocking the CD24 pathway.

[0017] Alternatively, many immune cells, including T cells, B cells, natural killer cells, and macrophages, rapidly exchange cell contents and portions of membrane with target cells in a cell contact-dependent and activation-dependent manner (https: / / www.mdpi.com / 2073-4409 / 10 / 5 / 1255). This phenomenon is called "trogocytosis". In particular, T cells undergo trogocytosis when activated by antigen(s) presented by antigen-presenting presenting cells (https: / / pubmed.ncbi.nlm.nih.gov / 17406507 / , https: / / pubmed.ncbi.nlm.nih.gov / 10542149 / ). Cell membrane exchange by trogocytosis between two cells is bidirectional. This disclosure describes a method to transfer antigen / ligand or receptor (e.g., T cell receptor) information between a library of T cells and a library of antigen-presenting cells using trogocytosis and membrane-bound RNA / DNA barcodes. By tethering antigen / ligand barcode or receptor (e.g., TCR) barcode DNA or RNA to either antigen-presenting cells (APCs) or cell membranes, activation-triggered trogocytosis naturally transfers the barcode DNA or RNA between cells for downstream sequencing analysis.

[0018] Antigen / ligand libraries can be generated in antigen-presenting cells (e.g., K562 cell line) that contain nucleic acids encoding antigen polypeptides (or antigens / ligands) and recombinant MHC proteins fused to barcodes. Polypeptide fragments from each antigen that can be presented can be identified by antigen-MHC binding experiments, mass spectrometry experiments, sliding windows of full-length protein sequences (8-25 per window), or fragments of known proteins predicted to be presentable by computer algorithms. These presentable peptide fragments from the antigen can be fused with appropriate MHC molecules to generate a library of presented antigens. The antigen-presenting cells can be any antigen-presenting cell, including, for example, the K562 cell line (a human immortalized myeloid leukemia cell line) or an engineered HEK293 cell line. MHC-antigen polypeptide fusions can be introduced into K562 cells to generate a library of antigen peptides. To anchor antigen DNA or RNA barcode to cell membrane, K562 cells or similar APCs express antigen barcode DNA or RNA with affinity to membrane-bound DNA or RNA binding protein(s) and its corresponding membrane-bound DNA or RNA binding protein(s). DNA or RNA binding protein(s) can be fused to MHC protein or can be independent of MHC protein. Antigen barcode can be fused to MHC protein or can be independent of MHC protein. Optionally, K562 cells can be engineered with optical reporter, such as GFP, or stained with cell membrane dye, such as Dil.

[0019] The T cell library may contain T cells with a diverse set of immune cell receptors (e.g., T cell receptors). Similar to the antigen presenting cells described above, the T cells may express immune receptor (e.g., TCR) barcode DNA or RNA with affinity to membrane-bound DNA- or RNA-binding protein(s) and its corresponding membrane-bound DNA- or RNA-binding protein(s). The DNA- or RNA-binding protein(s) may be fused to the immune receptor protein (e.g., TCR) or may be immune receptor protein (e.g., TCR) independent. The antigen barcode may be fused to the immune receptor protein (e.g., TCR) or may be immune receptor protein (e.g., TCR) independent. Optionally, the T cell(s) may be engineered with an optical reporter, e.g., membrane-bound GFP, or stained with a cell membrane dye, e.g., Dil. When the engineered T cell interacts with a cell presenting the appropriate antigen, it may capture the barcode presented on the APC surface by biting off a piece of the cell membrane by trogocytosis. Activated T cells can be sorted or enriched by antigen-presenting cell membrane markers, including transfected / transduced fluorescent proteins, membrane-bound dyes, or APC surface markers, or by T cell activation markers, including CD11a, CD69, CD70, CD71, CD25, CD26, CD27, CD28, CD30, CD40L, CD86, CD134, CD154, PD1, IL-2, NKG2D, TNF-alpha, IFN-gamma, or HLA-DR. By trogocytosis, activated T cells contain DNA or RNA barcodes of the corresponding antigen. DNA or RNA sequencing of such T cells can identify immune cell receptor sequences (e.g., TCR), antigen sequences, and T cell receptor-antigen relationships.

[0020] The immune cell receptor repertoire (e.g., TCR repertoire) of engineered cell library cells (e.g., Jurkat) can be mixed with the antigen library of K562 cells. The receptor repertoire cells interact with K562 cells that present the appropriate antigen / ligand (e.g., peptide-MHC fusions), resulting in barcode transfer by trogocytosis. Cells activated by APCs (e.g., K562 cells) can be isolated and sequenced individually to identify the barcodes associated with the receptor (e.g., immune cell receptor such as TCR) and antigen. If the K562 cells contain a reporter, the reporter can be used to screen for receptor cells (e.g., Jurkat cells) that have interacted with the K562 cells using flow cytometry or magnetic bead enrichment. Since the sorted T cells contain both barcodes, the antigen barcode and the receptor (e.g., TCR) / receptor mimic (e.g., CAR) barcode can be sequenced directly to identify the receptor and antigen pair.

[0021] Similarly, receptor-antigen / ligand interaction pairs can also be extracted by sequencing the receptor cells after trogocytosis. The antigen / ligand cells can be engineered to have an optical reporter, such as membrane-bound GFP, or stained with a dye, such as Dil. The receptor cells (e.g., K562 cells) can undergo a trogocytosis event with the antigen / ligand cells and retain fragments of the antigen / ligand cell membrane with the optical reporter or dye (https: / / pubmed.ncbi.nlm.nih.gov / 30700903 / ). Such K562 cells can be sorted by flow cytometry or enriched by magnetic beads based on the membrane-bound optical reporter or dye. Since the sorted APC / K562 cells contain both barcodes, the antigen barcode and the receptor (e.g., TCR) / receptor mimic (e.g., CAR) barcode can be directly sequenced to identify the receptor (e.g., TCR) and antigen / ligand pairs.

[0022] In one embodiment, the antigen / ligand can be engineered into a retrovirus (e.g., lentivirus) with a nucleic acid encoding a mutant viral envelope glycoprotein that contains at least one mutation that reduces its native function, a pegRNA (prime editing guide RNA), and a non-viral membrane-associated protein with the structure: S-ETD-LGD-IRES-R (where S encodes a signal sequence, ETD encodes an extracellular targeting domain; LGD encodes the antigen / ligand, IRES encodes an internal ribosome entry site, and R encodes a reporter (e.g., a selectable marker such as a fluorescent protein or antibiotic resistance). IRES and R are optional, and the nucleic acid can encode S-ETD-LGD. The IRES sequence can be replaced by a 2A self-cleaving peptide sequence (T2A, P2A, F2A, and E2A), which also allows for co-expression of two genes by one promoter. Receptor-presenting cells to be paired with the retroviral library of antigens / ligands are engineered to contain an insertion site for pegRNA adjacent or near the nucleic acid encoding the receptor. The pegRNA (prime editing guide RNA) inserts a barcode corresponding to the antigen / ligand near or adjacent to the nucleic acid encoding the receptor. Sequencing of the retrovirally transduced cells will identify this barcode in the context of sequences identifying the receptor and ligand pair. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] Detailed Description Before various embodiments are described, it is to be understood that the teachings of the present disclosure are not limited to the particular embodiments described, which may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present teachings will be limited only by the appended claims.

[0024] 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 disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present teachings, some exemplary methods and materials are described herein.

[0025] It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional elements. Thus, this statement is intended to serve as a predicate for the use of exclusionary language such as "solely," "only," and the like in connection with the recitation of claim elements or the use of "negative" limitations. Numerical limitations given regarding concentrations or levels of a substance are intended to be approximate unless the context clearly dictates otherwise. Thus, if a concentration is stated to be (for example) 10 micrograms ("μg"), it is intended to be understood that the concentration is at least approximately or about 10 μg.

[0026] Recombinant techniques encompassed in this description include, for example, conventional techniques of molecular biology, protein biochemistry, cell biology, immunology, microbiology, and employ recombinant DNA. See, for example, Sambrook and Russell, eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd ed.; the series Ausubel et al., eds. (2007) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., NY 10221; 1999), all of which are incorporated by reference in their entirety for all purposes.;MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press);MacPherson et al. (1995) PCR 2: A Practical Approach;Harlow and Lane, eds. (1999) Antibodies, A Laboratory Manual;Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th ed.;Gait, ed. (1984) Oligonucleotide Synthesis;U.S. Patent No. 4,683,195;Hames and Higgins, eds. (1984) Nucleic Acid Hybridization;Anderson (1999) Nucleic Acid Hybridization;Hames and Higgins, eds. (1984) Transcription and Translation;Immobilized Cells and Enzymes (IRL Press (1986));Perbal (1984) A Practical Guide to Molecular Cloning;Miller and Calos, eds. (1987) Gene Transfer Vectors for See Mammalian Cells (Cold Spring Harbor Laboratory); Gene Transfer and Expression in Mammalian Cells, ed. Makrides (2003); Immunochemical Methods in Cell and Molecular Biology, eds. Mayer and Walker (1987) (Academic Press, London); and Weir's Handbook of Experimental Immunology, eds. Herzenberg et al. (1996).

[0027] As will be apparent to one of ordinary skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has distinct components and features which may be readily distinguished from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present teachings. Any recited method may be carried out in the order of events recited or in any other order which is logically possible.

[0028] definition As used herein, the term "antigen" refers to a substance that, under appropriate conditions, is capable of inducing a specific immune response and reacting with the products of that response, such as specific antibodies or specifically sensitized T lymphocytes, or both. Antigens may be soluble substances, such as toxins, foreign proteins, mutant proteins, self-proteins, or particles, such as bacteria and tissue cells, but only parts of protein or polysaccharide molecules, known as antigenic determinants (epitopes), bind to specific receptors on antibodies or lymphocytes.

[0029] As used herein, the term "binding specificity" of a TCR refers to the identity of the antigen to which the TCR binds, preferably the identity of the epitope to which the TCR binds and activates a T cell.

[0030] As used herein, "complementarity determining region" or "CDR" refers to the region of a TCR that is primarily responsible for binding to an epitope of an antigen or antigen:MHC complex. CDRs are also called hypervariable regions. The CDRs of each TCR are typically called CDR1, CDR2, and CDR3, are numbered sequentially starting from the N-terminus, and are typically also identified by the chain in which the particular CDR is located. TCRs with different specificities (i.e., different binding sites for different antigens) have different CDRs. Only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. These positions within the CDRs are called specificity determining residues (SDRs).

[0031] As used herein, the term "epitope" refers to a site on an antigen or hapten to which specific B cells and / or T cells respond. The term is also used interchangeably with "antigenic determinant" or "antigenic determinant site." An epitope includes a portion of an antigen or other macromolecule that can form a binding interaction that interacts with the variable region binding pocket of an antibody.

[0032] As used herein, the term "heterologous" when used in reference to a portion of a polynucleotide indicates that the nucleic acid comprises two or more subsequences that are not normally found in the same relationship to each other in nature. For example, the nucleic acid is typically produced recombinantly, e.g., has two or more sequences from unrelated genes arranged to create a new functional nucleic acid. Similarly, a "heterologous" polypeptide or protein refers to two or more subsequences that are not normally found in the same relationship to each other in nature.

[0033] As used herein, the term "host cell" refers to a prokaryotic or eukaryotic cell into which the vector of the present invention can be introduced, expressed and / or propagated. Microbial host cells are cells of prokaryotic or eukaryotic microorganisms, including bacteria, yeast, microscopic fungi, and the microscopic stages of the life cycle of fungi and slime molds. Typical prokaryotic host cells include various strains of Escherichia coli (E. coli). Typical eukaryotic host cells are yeast or filamentous fungi, or mammalian cells, such as Chinese hamster ovary cells, mouse NIH 3T3 fibroblast cells, human embryonic kidney 193 cells, or rodent myeloma or hybridoma cells.

[0034] As used herein, the term "humanized" form of a non-human protein (e.g., a mouse TCR) is a chimeric protein that contains minimal sequence from a non-human homolog of the protein. For the most part, a humanized protein is a human TCR in which the recipient variable region residues are replaced by variable region residues (e.g., CDRs) from a non-human species (donor TCR), such as a mouse, rat, rabbit, or non-human primate, having the desired specificity, affinity, and capacity.

[0035] As used herein, the term "immunological response" to a composition or vaccine is the development in a host of a cellular and / or antibody-mediated immune response to the composition or vaccine of interest. Typically, an "immunological response" includes, but is not limited to, one or more of the following effects: production of antibodies, B cells, helper T cells, and / or cytotoxic T cells that are specifically directed to the antigen(s) contained in the composition or vaccine of interest. Preferably, the host exhibits either a therapeutic or protective immunological response such that resistance to new infections is enhanced and / or the clinical severity of disease is reduced. Such protection is demonstrated by either a reduction or loss of symptoms normally exhibited by an infected host, a faster recovery time and / or a reduction in viral titers in the infected host.

[0036] As used herein, the term "isolated" refers to a nucleic acid or polypeptide that is separated from other nucleic acids or polypeptides present in the natural source of the nucleic acid or polypeptide, as well as from polypeptides, and preferably refers to a nucleic acid or polypeptide that is found in the presence of only solvents (if any), buffers, ions, or other components that are normally present in the same solution. The terms "isolated" and "purified" do not encompass nucleic acids or polypeptides present in their natural source.

[0037] As used herein, the term "mammal" refers to warm-blooded vertebrate animals, all of which have hair and which nurse their young.

[0038] As used herein, "MHC molecules", "MHC proteins" or "HLA proteins" are used interchangeably and should be understood to mean, in particular, proteins that arise from the proteolytic cleavage of protein antigens and are capable of binding peptides that display potential T cell epitopes and transporting them to the cell surface where they can be presented to specific cells, in particular naive T cells, cytotoxic T lymphocytes, regulatory T cells, or helper T cells. The major histocompatibility complex of the genome comprises gene regions whose gene products are expressed at the cell surface and are important for the binding and presentation of endogenous and / or foreign antigens and thus control immunological processes. The major histocompatibility complex is classified into two gene groups that code for different proteins: MHC class I and MHC class II molecules. The molecules of the two MHC classes are specialized for different antigen sources. The molecules of MHC class I typically present endogenously synthesized antigens, such as, but not limited to, viral proteins and tumor antigens. The molecules of MHC class II present protein antigens that arise from exogenous sources, such as bacterial products.

[0039] As used herein, the term "naturally occurring" means that the component is encoded by a single gene that has not been altered by recombinant means and is pre-existing in an organism, for example, an antibody library generated from naive cells or cells exposed to an antigen.

[0040] As used herein, the terms "protein", "peptide", "polypeptide" and "polypeptide fragment" are used interchangeably herein to refer to a polymer of amino acid residues of any length. The polymer may be linear or branched, may contain modified amino acids or amino acid analogs, and may be interrupted by chemical moieties other than amino acids. The terms also encompass amino acid polymers that are modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as labeling or conjugation with a biologically active component.

[0041] As used herein, the term "purified" means that the designated nucleic acid or polypeptide is present in the substantial absence of other biological macromolecules, e.g., polynucleotides, proteins, etc. In one embodiment, the polynucleotide or polypeptide is purified to comprise at least 95% by weight, more preferably at least 99.8% by weight, of the designated biological macromolecules present (although water, buffers, and other small molecules, particularly molecules having a molecular weight less than 1000 Daltons, may be present).

[0042] As used herein, the term "recombinant nucleic acid" refers to a nucleic acid in a form not normally found in nature. That is, a recombinant nucleic acid is flanked by nucleotide sequences that are not naturally adjacent to the nucleic acid or has sequences that are not normally found in nature. A recombinant nucleic acid may be initially formed in vitro by the manipulation of nucleic acid with restriction endonucleases or using techniques such as the polymerase chain reaction. It is understood that once a recombinant nucleic acid is made and reintroduced into a host cell or organism, it replicates non-recombinantly, i.e., using the host cell's in vivo cellular machinery rather than through in vitro manipulation, although such a nucleic acid, once produced recombinantly, is subsequently replicated non-recombinantly, but is still considered recombinant for the purposes of this disclosure.

[0043] As used herein, the term "recombinant polypeptide" refers to a polypeptide expressed from a recombinant nucleic acid or a polypeptide chemically synthesized in vitro.

[0044] As used herein, the term "repertoire" or "library" refers to a collection of antibodies, T cell receptors, or antibody fragments, such as Fab, scFv, Fd, LC, V, or VFv. H , or V LThe term "repertoire" refers to a library of genes encoding, for example, exchange cassettes, or subfragments of variable regions, derived from the natural ensemble, or "repertoire," of antibody genes present in a human donor, as well as derived primarily from cells of the peripheral blood and spleen. In some embodiments, the human donor is "non-immune," i.e., does not exhibit symptoms of infection. In the current invention, the library or repertoire often includes members that are exchange cassettes for a portion of a given V region.

[0045] As used herein, a "single chain TCR" is a single polypeptide chain having both the alpha and beta chains of the TCR, or both the gamma and delta chains of the TCR, arranged such that the two TCR chains can form a TCR. The term further includes, but is not limited to, covalently linked TCR alpha and TCR beta or TCR gamma and TCR delta variable chain fragments, with or without a linker. Single chain TCRs may optionally include a CD3 or CD3 zeta signaling domain, alone or in combination with a CD28 signaling domain.

[0046] As used herein, "T cells" are defined as hematopoietic cells that normally occur in the thymus. T cells include, but are not limited to, natural killer T cells, regulatory T cells, helper T cells, cytotoxic T cells, memory T cells, gamma delta T cells, and mucosal invariant T cells. T cells also include, but are not limited to, CD8+ T cells, CD4+ T cells, Th1 T cells, and Th2 T cells. T cells can be primary T cells or T cell lines.

[0047] As used herein, the terms "T cell receptor" or "TCR" are used interchangeably and refer to a heterodimeric cell surface protein of the immunoglobulin superfamily that associates with the invariant protein of the CD3 complex, which is involved in mediating signal transduction. The TCR is composed of two cognate protein chains: an alpha chain and a beta chain or a gamma chain and a delta chain.

[0048] As used herein, the term "TCR library" refers to a polyclonal collection of vectors encoding TCRs or cells containing those vectors. A "TCR library" may include a subset or selection of vectors encoding TCRs. A "TCR library" may refer to a collection of a repertoire of TCRs that may be found in a subject. A "TCR library" may also refer to a subset of the repertoire of TCRs found in a subject.

[0049] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly dictates otherwise. Numerical limitations given regarding concentrations or levels of a substance, such as an antigen, are intended to be approximate. Thus, if a concentration is indicated to be at least (for example) 200 μg, it is intended to be understood that the concentration is at least approximately "about" or "about" 200 μg.

[0050] Novel receptors A wide variety of immune cells and / or immune cell receptors may be used in the methods disclosed herein. For T cells, applicable receptors include chimeric antigen receptor, alpha beta T cell receptor, gamma delta T cell receptor, CD43, CD44, CD45, LFA1, CD4, CD8, CD3, LAT, CD27, CD96, CD28, TIGIT, ICOS, BTLA, HVEM, 4-1BB, OX40, DR3, GITR, CD30, SLAM, CD2, 2B4, TIM1, TIM2, TIM3, CD226, CD160, LAG3, LAIR1, CD112R, CTLA-4, PD-1, PD-L1, PD-L2. For NK cells, applicable receptors include TRAIL, CD16, NKp30ab, NKGC, NKG2D, 2B4, DNAM-1, NKG2A, KIR, CD137, OX40, CD27. For B cells, applicable receptors include Siglec-10, LILRB / PIR-B, CD31, FCγRIIIB, CD19, CD20, CD22, CD25, CD32, CD40, CD47, CD52, CD80, CD86, CD267, CD268, CD268, B cell receptor, antibodies, IgM, IgD, IgG, IgA, IgE. In dendritic cells, applicable receptors include DNGR-1, MICL, CLEC1, CLEC12B, LOX1, mannose receptor, DC-SIGN, L-SIGN, SIGN-R1, LSECtin, CIRE, Langerin, MGL, scavenger receptor (SR), DC-ASGPR, DC-STAMP, CD80 / 86, TLR, FIRE, FcR, DEC205, BDCA-2, Dectin-2, DCIR, and chemokine receptors. In macrophages, applicable receptors include CD300a, TREM2 / DAP12, Bal-1, TIM4, CR3, SCARF1, CD36, MARCO, scavenger receptor A1, RAGE, Axl, Mer, Tyro3, ​​CD93, Stabilin2, DNGR-1, SIRP. On neutrophils, applicable receptors include CXCR1, FCγRIIIB, FCγRII, CR3, CR1, C3aR, TNFR, TLR2 / 6, C5aR.Other receptors (e.g., G protein-coupled receptors, ion channel-linked (ionotropic) receptors, and / or enzyme-linked receptors) may also be used in the methods and compositions of the present disclosure. The ligands screened in the present disclosure may be known ligands of the target receptor (e.g., PD-1L for the PD-1 receptor), or whole proteins with unknown affinity, or peptides with unknown affinity.

[0051] The compositions and methods described herein can be used to find novel pairs of antigens and T cell receptors or T cell receptor mimics. A T cell receptor mimic refers to an antibody or antibody fragment that can bind to an MHC-presented antigen. A T cell receptor repertoire is generated in monocytes (e.g., macrophages) or other phagocyte cell types. The antigen-binding portion of the T cell receptor is fused to all or a portion of an Fc receptor, such that when this chimeric receptor binds to an antigen, the macrophage phagocytose the antigen. The nucleic acid encoding the TCR receptor or T cell receptor mimic includes a barcode that identifies the TCR and coactivation domains (e.g., Fc domain). The TCR chimeric receptor or T cell receptor mimic library can be expressed in appropriate monocyte cells (e.g., macrophages) or other phagocyte cell types (e.g., neutrophils, dendritic cells, mast cells, etc.).

[0052] The compositions and methods described herein can be used to find new pairs of other receptors and ligands (e.g., antigens). In addition to T cell receptors and T cell receptor mimics, other receptors may be used. For example, ion channel-linked (ionotropic) receptors, G protein-coupled (metabotropic) receptors, and enzyme-linked receptors can be associated with transgene expression. One class of receptors that can be used is immune receptors, such as T cell receptors, B cell receptors (also known as antigen receptors or immunoglobulin receptors), and innate immune receptors.

[0053] T cell receptors are heterodimers of two different polypeptide chains. In humans, most T cells have a T cell receptor made of gamma and delta (γ / δ) chains (encoded by TRG and TRD, respectively) with the T cell receptor made of alpha (α) and beta (β) chains. Techniques and primers for amplifying nucleic acids encoding T cell receptor chains from lymphocytes are well known in the art and are described, for example, in the SMARTer Human TCR a / b Profiling Kits marketed by Clontech, Boria et al., BMC Immunol. 9:50-58 (2008); Moonka et al., J. Immunol. Methods 169:41-51 (1994); Kim et al., PLoS ONE 7:e37338 (2012); Seitz et al., Proc. Natl Acad. Sci. 103:12057-62 (2006), all of which are incorporated by reference in their entirety for all purposes. TCR repertoires can be used as separate chains to form antigen binding domains. TCR repertoires can be converted into single chain antigen binding domains. Single chain TCRs can be made from nucleic acids encoding human alpha and beta chains using techniques well known in the art, including, for example, those described in U.S. Patent Application Publication No. 2012 / 0252742; Schodin et al., Mol. Immunol. 33:819-829 (1996); Aggen et al., "Engineering Human Single-Chain T Cell Receptors," PhD dissertation, University of Illinois at Urbana-Champaign (2010), all of which are incorporated by reference in their entireties for all purposes (a copy of which can be found at ideals.illinois.edu / bitstream / handle / 2142 / 18585 / Aggen_David.pdf?sequence=1).

[0054] B cell receptors include immunoglobulins, signaling moieties, CD79, and ITAMs, which are membrane-bound. Techniques and primers for amplifying nucleic acids encoding human antibody light and heavy chains are well known in the art and are described, for example, in ProGen's Human IgG and IgM Library Primer Set, Catalog No. F2000; Andris-Widhopf et al., "Generation of Human Fab Antibody Libraries: PCR Amplification and Assembly of Light and Heavy Chain Coding Sequences," Cold Spring Harb.Protoc.2011; Lim et al., Nat.Biotechnol.31:108-117 (2010); Sun et al., World J.Microbiol.Biotechnol.28:381-386 (2012); Coronella et al., Nucl.Acids.Res.28:e85 (2000), all of which are incorporated by reference in their entirety for all purposes. Techniques and primers for amplifying nucleic acids encoding mouse antibody light and heavy chains are well known in the art and are described, for example, in U.S. Pat. No. 8,143,007; Wang et al., BMC Bioinform. Vol. 7 (Suppl.): S9 (2006), both of which are incorporated by reference in their entirety for all purposes. Antibody repertoires may be used as separate chains in the antigen-binding domain or converted to single-chain antigen-binding domains. Single-chain antibodies can be made from nucleic acids encoding human light and heavy chains using techniques well known in the art, including, for example, those described in Pansri et al., BMC Biotechnol. Vol. 9: 6 (2009); Peraldi-Roux, Methods Molc. Biol. Vol. 907: 73-83 (2012), both of which are incorporated by reference in their entirety for all purposes.Single chain antibodies can be made from nucleic acids encoding murine light and heavy chains using techniques well known in the art, including, for example, those described in Imai et al., Biol. Pharm. Bull. 29:1325-1330 (2006); Cheng et al., PLoS ONE 6:e27406 (2011), both of which are incorporated by reference in their entireties for all purposes.

[0055] Innate immune receptors include, for example, the CD94 / NKG2 receptor family (e.g., NKG2A, NKG2B, NKG2C, NKG2D, NKG2E, NKG2F, NKG2H), 2B4 receptors, NKp30, NKp44, NKp46, and NKp80 receptors, Toll-like receptors (e.g., TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, RP105).

[0056] G protein-linked receptors, also known as seven transmembrane domain receptors, are a large family of receptors that couple receptor binding of ligand to cellular responses through G proteins. These G proteins are trimers of α, β, and γ subunits (known as Gα, Gβ, and Gγ, respectively) that are active when bound to GTP and inactive when bound to GDP. When the receptor binds to a ligand, it undergoes a conformational change that allosterically activates the G protein to exchange GTP for bound GDP. After GTP binding, the G protein dissociates from the receptor to yield Gα-GTP monomers and Gβγ dimers. G protein-linked receptors have been grouped together into classes that include, for example, rhodopsin-like receptors, selectin receptors, metabotropic glutamate / pheromone receptors, fungal mating pheromone receptors, cyclic AMP receptors, and frizzled / smoothened receptors. G protein receptors are used in a wide variety of physiological processes, including the detection of electromagnetic radiation, taste (gustation), the sense of smell, neurotransmission, immune system regulation, proliferation, cell density sensing, and more.

[0057] Enzyme-linked receptors, also known as catalytic receptors, are transmembrane receptors in which the binding of extracellular ligands causes enzymatic activity inside the cell. Enzyme-linked receptors have two domains joined together by a transmembrane portion (or domain) of a polypeptide. The two terminal domains are the extracellular ligand-binding domain and the intracellular domain with catalytic function. There are numerous families of enzyme-linked receptors, including, for example, the Erb receptor family, the glial cell line-derived neurotrophic factor receptor family, the natriuretic peptide receptor family, the trk neurotrophin receptor family, and the toll-like receptor family.

[0058] Ion channel-linked receptors, also known as ligand-gated ion channels, are receptors that transport ions, such as Na, into the blood in response to binding of a ligand to the receptor. + , K + , Ca 2+ and Cl - These are receptors that allow molecules to cross membranes. For example, there are numerous families of ligand-gated ion channels, including cationic cys-loop receptors, anionic cys-loop receptors, ionotropic glutamate receptors (AMPA receptors, NMDA receptors), GABA receptors, 5-HT receptors, ATP-gated channels, and PIP2-gated channels.

[0059] T cell receptor repertoires may be generated from engineered cell lines (e.g., Jurkat cells) from donors / patients with appropriate HLA alleles (e.g., HLA-A02:01) or synthesized from known sequences (e.g., GenBank). Diversity may be introduced into engineered cells via plasmids that introduce specific alpha and beta chain TCRs spanning the V and J genes. Combinations of CDR3, alpha, and beta genes may be put together in any of the following ways: (1) optionally, sampling of known unpaired (e.g., beta or alpha) TCRs known to interact with the antigen target; (2) optionally, sampling of paired TCRs known to interact with the antigen target; (3) starting with a known paired or unpaired TCR known to interact with the antigen target and mutating its CDR3 region in either areas known to be important for binding or known not to be important for binding.

[0060] Other receptor repertoires may also be generated from donors / patients with appropriate HLA alleles (e.g., HLA-A02:01), or harvested from appropriate cell lines, or synthesized from known sequences (e.g., sequences found in GenBank). Diversity can be introduced into the receptor repertoire using methods known in the art (e.g., engineering mutations into the receptor, chain swapping, creating receptor chain chimeras, swapping chains of receptors made from two or more polypeptides, etc.).

[0061] DNA encoding the receptors of interest may be synthesized individually or in a pooled library format. Variation (e.g., diversity) may be introduced into the receptor library or repertoire using genetic mutations introduced at any position of the receptor of interest. Alternatively, variation may be introduced by strand swapping techniques, or by mixing segments of nucleic acid encoding the receptor (strand(s)), as is done, for example, in some guided evolution methods. Genetic mutations may be substitutions, deletions, insertions, and rearrangements of DNA fragments. The specific genetic mutations of choice may be randomly generated, enriched in regions of interest (e.g., receptor interaction domains), or recommended by computational algorithms. Examples of computational algorithms to recommended genetic mutation strategies include protein structure analysis, computational docking, molecular dynamics simulations, regression, statistically-based classification, random forests, support vector machines, and neural networks.

[0062] The antigen / ligand of the receptor of interest can be a peptide, an MHC-peptide complex, a fragment, a chimera, or a whole protein. The antigen / ligand can be a carbohydrate, a fatty acid or lipid, or a small molecule. The antigen / ligand can be associated with the antigen-presenting cell surface by covalent or non-covalent bonds. If the antigen / ligand is a polypeptide, they can be anchored to the antigen-presenting cell surface by their natural transmembrane domain or by a synthetic transmembrane domain. The synthetic transmembrane domain can be fused to the antigen / ligand extracellular domain by engineering the DNA sequence to operably link the synthetic transmembrane domain DNA sequence with the DNA sequence encoding the desired portion, part, or whole of the antigen / ligand. Exemplary synthetic transmembrane domains include CD2, CD3d, CD3g, CD3z, CD4, CD8A, CD8B, CD22, CD27, CD28, CD40, CD79a, CD79b, CD80, CD84, CD86, CD137, CD244, CRACC, CRTAM, CLTA-4, MHC-I, MHC-II, platelet-derived growth factor receptor, FCGR1A, FCGR2A, FCG2B, FCGR3A, FCRL1, FCRL2, FCRL3, FCRL4, FCRL5, F The antigen / ligand library may include wild-type or modified transmembrane domains from CRL6, KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL3, LAG3, GITR, OX40, PD-1, PD-L1, PD-L2, TLR, SLAMF, LILRB1, LILRB2, NKG2A, NKG2C, NKG2D, TIGIT, IgG, IgM, IgA, IgE, IgD, or immunoglobulin. Repertoires of antigens / ligands may be created and engineered into antigen / ligand presenting cells. In some cases, variations of the antigen / ligand library may be created by introducing genetic mutations (e.g., randomly or site-specifically) into desired positions of the antigen / ligand of interest. Genetic mutations may be substitutions, deletions, insertions, and rearrangements of DNA fragments. The specific genetic mutations of choice may be generated randomly, enriched in regions of interest (e.g., interaction domains of a ligand), or recommended by computational algorithms.Examples of computational algorithms to recommend gene mutation strategies include protein structure analysis, computational docking, molecular dynamics simulation, regression, statistically-based classification, random forests, support vector machines, and neural networks.

[0063] Antigen libraries can be created as fusions of antigen polypeptides with appropriate MHC molecules or two separate molecules, peptide and MHC. Algorithms can be used to identify presentable antigens, including, for example, those disclosed in Chen et al., Nature Biotechnology 37:1332-43 (2019), Jurtz et al., J. Immunol. 199:3360-68 (2017), and Abelin et al., Immunity 51:766-79 (2019), all of which are incorporated by reference in their entirety for all purposes. The nucleic acid encoding the antigen-MHC fusions can optionally include a barcode that identifies the antigen polypeptide of the fusion. The library of antigen-MHC molecule fusions is placed into an appropriate host cell (e.g., a cell line such as K562) that displays the fused or unfused antigen-MHC molecule complexes as well as the barcode on its surface. The barcode may be DNA or RNA and may be attached directly to the cell membrane, to an MHC molecule, or to another protein (e.g., GFP) that, when expressed, transports the barcode to the cell surface. Host cells for antigen libraries may also contain a reporter (e.g., cells may be engineered to express membrane GFP).

[0064] A repertoire of TCR-Fc fusions on monocytic cells can be mixed with a library of antigen::MHC fusion proteins. Macrophage cells with TCR antigen binding domains capable of binding to antigen polypeptides presented on MHC can phagocytose cells presenting antigen-MHC fusions that bind to the TCR of the TCR-Fc chimeric receptor. If the cells presenting antigen-MHC fusions contain a reporter, the signal from the receptor can be used to separate TCR-Fc cells that have phagocytosed the antigen-MHC fusion presenting cells. For example, if the reporter is an optical reporter, the optical reporter can be used to separate TCR-Fc cells that have phagocytosed the antigen-MHC using FACs sorting. Optionally, the phagocytic cell type with the chimeric TCR repertoire can contain a reporter (e.g., an optical reporter different from the antigen presenting cell reporter). If both the cells bearing the chimeric TCR repertoire and the cells bearing the antigen library have different optical reporters, FACs sorting can be used to select cells that have both optical signals (e.g., by first selecting cells that have one of the optical signals and then selecting cells that have the other optical signal).

[0065] Alternatively, a repertoire of engineered receptor cells (e.g., monocytes with TCR) can be mixed with a library of ligands (e.g., antigen-MHC fusion proteins). The receptor cells interact with and activate against cells presenting a ligand (e.g., fused or unfused antigen-MHC complexes) that binds to the receptor (e.g., TCR) of the engineered cells (e.g., monocytes or macrophages). The receptor cells capture fragments of the membrane of the ligand cells, including the cell surface DNA or RNA barcode (that identifies the ligand), by trogocytosis. If the cells presenting the ligand (e.g., antigen-MHC fusions) contain a membrane-bound reporter or dye, the signal from the membrane-bound reporter can be used to separate the engineered receptor cells that have interacted with the ligand-presenting cells (e.g., antigen-MHC fusion-presenting cells) from other cells. For example, if the reporter is an optical reporter, the optical reporter can be used to separate the receptor cells that have been activated against the ligand (e.g., antigen-MHC) using FACs sorting. Optionally, the trogocytic cell type may contain a reporter (e.g., an optical reporter different from the antigen presenting cell reporter). If both the engineered receptor cells and the cells with the ligand library have different optical reporters, FACs sorting can be used to select cells with both optical signals (e.g., by first selecting cells with one of the optical signals and then selecting cells with the other optical signal).

[0066] The engineered receptor cell(s) activated against the ligand (e.g., antigen-MHC) can be separated for bulk or single cell sequencing to identify the antigen polypeptide and receptor (e.g., TCR variable region) that formed the receptor-ligand pair. The barcodes of the receptor and ligand can be sequenced to identify the receptor and ligand, or the nucleic acids encoding the receptor (e.g., TCR variable region) and receptor (e.g., antigen) can be directly sequenced to identify the receptor and ligand.

[0067] Libraries of engineered receptors (e.g., T cell receptors or T cell receptor mimics) can be generated with up to 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , or 10 10 A library of ligands (e.g., antigen-MHC fusions) may represent up to 10 different receptors (e.g., T cell receptors). 3 or 10 4 or 10 5 or 10 6 or 10 7 or 10 8 or 10 9 or 10 10 The antigen-MHC fusion may represent a number of different ligands (e.g., antigen-MHC fusions).

[0068] Barcode receptor repertoire Receptor repertoires can be obtained starting with nucleic acids encoding the receptor of interest from an appropriate cell type (e.g., the disclosed receptors for T cells, B cells, NK cells, macrophages and / or neutrophils above). For example, T cell receptor repertoires can be obtained from one or more of CD8+ T cells, CD4+ T cells, regulatory T cells, memory T cells, helper T cells, or cytotoxic T cells. T cells may be naive or include effector and / or memory T cells that have been exposed to an antigen of interest or other antigens. Memory T cells can be stem cell memory T cells (T SCM ), central memory cells (T CM ), transitional memory cells (T TM ), and / or effector memory cells (T EM The T cells may be Th1, Th2, Th9, Th 17 , Th 22 , T reg , T fhand cytotoxic T lymphocytes (CTLs). T cell receptors can be obtained from either (or both) of genomic DNA of T cells (or subpopulations of T cells) and / or mRNA of T cells (or subpopulations of T cells). Repertoires of T cell receptors are well known in the art and can be obtained using techniques and primers described, for example, in SMARTer Human TCR a / b Profiling Kits marketed by Clontech, Boria et al., BMC Immunol. 9:50-58 (2008); Moonka et al., J. Immunol. Methods 169:41-51 (1994); Kim et al., PLoS ONE 7:e37338 (2012); Seitz et al., Proc. Natl Acad. Sci. 103:12057-62 (2006), all of which are incorporated by reference in their entirety for all purposes. T cell receptor polypeptides can be used as separate chains to form a TCR. Alternatively, the T cell receptor polypeptide chains can be converted into a single chain TCR. Single chain T cell receptors can be made from nucleic acids encoding human alpha and beta chains using techniques well known in the art, including, for example, those described in U.S. Patent Application Publication No. 2012 / 0252742; Schodin et al., Mol. Immunol. 33:819-829 (1996); Aggen et al., "Engineering Human Single-Chain T Cell Receptors," PhD dissertation, University of Illinois at Urbana-Champaign (2010), a copy of which can be found at ideals.illinois.edu / bitstream / handle / 2142 / 18585 / Aggen_David.pdf?sequence=1, all of which are incorporated by reference in their entirety for all purposes.

[0069] Immune cells (e.g., T cells) can be obtained from several sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, spleen tissue, embryos, and tumors. Immune cells (e.g., T cells) can be obtained from a unit of blood collected from a subject using a number of techniques known to those skilled in the art, such as Ficoll separation. Immune cells (e.g., T cells) from an individual's circulating blood can be obtained by apheresis or leukapheresis. Genomic DNA or mRNA / cDNA can be used to obtain nucleic acids encoding receptors (e.g., T cell receptors).

[0070] Tumor-infiltrated T cells can be used as a source of T cell repertoire. T cells can be removed during surgery. T cells can be isolated after removal of tumor tissue by biopsy. T cells can be isolated from the bulk population of T cells from tumor samples by any suitable method known in the art. For example, the bulk population of T cells can be obtained from tumor samples by dissociating the tumor samples into cell suspensions from which specific cell populations can be selected. Suitable methods for obtaining the bulk population of T cells can include, but are not limited to, any one or more of mechanically dissociating the tumor (e.g., mincing), enzymatically dissociating the tumor (e.g., digesting), and aspiration (e.g., as by injection).

[0071] Enrichment of T cell population by negative selection can be achieved by a combination of antibodies against surface markers unique to negatively selected cells.A preferred method is cell sorting and / or selection via negative magnetic immunoadherence or flow cytometry, using a cocktail of monoclonal antibodies against cell surface markers present on negatively selected cells.For example, to enrich CD4+ cells by negative selection, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8.

[0072] T cells can also be antigen-specific T cells. For example, tumor-specific T cells can be used. Antigen-specific T cells can be isolated from a patient of interest, such as a patient suffering from cancer or infectious disease. Antigen-specific T cells can be induced by vaccination of a subject with a specific antigen, alone or in combination with an adjuvant, or pulsed with dendritic cells. Antigen-specific cells for use in expansion can also be generated in vitro using a number of methods known to those skilled in the art.

[0073] Each T cell receptor is made up of two protein chains: an alpha chain and a beta chain, or a gamma chain and a delta chain. Each chain has two extracellular domains: a variable (V) region and a constant (C) region. The constant region is proximal to the cell membrane and is followed by a transmembrane region and a short cytoplasmic tail, while the variable region binds to the antigen:MHC complex. The variable domains of both the TCR alpha and beta chains each have three hypervariable regions or complementarity determining regions (CDRs). There is also an additional area of ​​hypervariability in the beta chain (HV4) that does not normally contact the antigen and is therefore not considered a CDR. CDR3 is the main CDR responsible for recognition of processed antigens, and CDR1 of the alpha chain has also been shown to interact with the N-terminal portion of antigenic peptides, whereas CDR1 of the beta chain interacts with the C-terminal portion of the peptide. CDR2 recognizes the MHC. CDR4 of the beta chain is not thought to participate in antigen recognition, but has been shown to interact with superantigens. The constant domain of the TCR consists of a short connective sequence in which cysteine ​​residues form disulfide bonds, forming the link between the two chains.

[0074] Forms of TCR mimics include chimeric T cell receptors or chimeric antigen receptors (CARs). Chimeric T cell receptors are made by fusing a suitable cytoplasmic region with one chain of a single chain TCR or T cell receptor. Single chain T cell receptors can be made according to Zhang et al., Cancer Gene Therapy vol. 11:487-496 (2004), which is incorporated by reference in its entirety for all purposes. Suitable cytoplasmic regions include one or more of the following: Fc cytoplasmic domain, CD3zeta, CD28, B7.1, CD137, CD19 intracellular activation domain, CD64 intracellular activation domain, CD32 intracellular activation domain, CD16 intracellular activation domain, CD23 intracellular activation domain, or Lck activation domain. Costimulatory domains that may be used in chimeric TCRs include, for example, CD3zeta, CD28, B7.1, CD137, CD19 intracellular activation domain, CD64 intracellular activation domain, CD32 intracellular activation domain, CD16 intracellular activation domain, CD23 intracellular activation domain, Lck activation domain, CD27, OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, lymphocyte function associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, MyD88, etc. Chimeric T cell receptors fused with an Fc signaling domain, and optionally one or more costimulatory domains, can trigger phagocytosis of monocytes / macrophages in response to ligand binding to the TCR. Alternatively, a chimeric T cell receptor or a chimeric antigen receptor having a signaling domain and, optionally, one or more costimulatory domains, can trigger trogocytosis in response to an antigen that interacts with the receptor.

[0075] In some cases, T cells contain TCR barcodes bound to their cell membranes via membrane-bound DNA or RNA binding proteins. The antigen barcodes of antigen-presenting cells can be introduced simultaneously or sequentially with antigen coding cDNA or mRNA. Membrane-bound nucleic acid (DNA or RNA) binding proteins can be engineered from zinc finger nucleases, transcription factor binding proteins, replication protein A, CRISPR Cas9, Cas13, Cpf1, phage RNA binding proteins, bacterial single-stranded binding proteins, or engineered restriction digestion enzymes. Nucleic acid binding proteins have binding motifs, which are described for an exemplary set of proteins in Table 1.

[0076] [Table 1]

[0077] Membrane-bound nucleic acid binding proteins can be engineered from engineered nucleic acid binding proteins with (if necessary) synthetic transmembrane domains. Exemplary synthetic transmembrane domains include CD2, CD3d, CD3g, CD3z, CD4, CD8A, CD8B, CD22, CD27, CD28, CD40, CD79a, CD79b, CD80, CD84, CD86, CD137, CD244, CRACC, CRTAM, CLTA-4, MHC-I, MHC-II, platelet-derived growth factor receptor, FCGR1A, FCGR2A, FCG2B, FCGR3A, FCRL1, FCRL2, FCRL3, FCRL4, FCRL5, F The nucleic acid binding domain may be engineered to be present on the cytoplasmic side of the membrane using engineering techniques known in the art. For example, many synthetic transmembrane domains have the c-terminus of the polypeptide chain on the cytoplasmic side of the membrane. Thus, the nucleic acid binding protein may be engineered to be present on the c-terminus of the synthetic transmembrane domain, and thus the nucleic acid binding protein portion is present on the cytoplasmic side of the membrane. The barcode may be a unique sequence of the CDR of the TCR or the antigen, or the barcode may be introduced into the nucleic acid encoding the receptor or antigen or ligand so that the expressed mRNA contains the barcode. The nucleic acid encoding the receptor / antigen / ligand may also be engineered to contain a binding motif of a membrane-bound nucleic acid binding protein so that the expressed mRNA contains the binding motif. This results in the mRNA of the receptor or antigen being bound to the membrane-bound nucleic acid binding protein. When trogocytosis occurs between the receptor cell and the ligand-presenting cell, the membrane-bound nucleic acid binding protein with the barcode is also transferred, resulting in both cells having membrane-bound barcodes of the receptor (e.g., TCR or CAR) and the ligand (e.g., antigen).

[0078] The repertoire of chimeric TCRs is 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , or 10 10 The repertoire of chimeric TCRs may have different TCRs. The repertoire of chimeric TCRs may represent the population of TCRs found in the genomic DNA of the subject, or of naive T cells, or of T cells from the subject that initiates an immune response to an antigen of interest or from any of the above populations of T cells. The preferred cell type of the repertoire of chimeric TCRs is, for example, monocytes of macrophages, including THP-1 cells.

[0079] Barcoded antigen / ligand repertoire A ligand library is created from the desired ligands (e.g., antigens). The HLA genes and antigen sequences can be fused to a single peptide or encoded as separate peptides. The HLA alpha and beta chains can be fused or encoded as separate peptides. Because HLA can only bind and present fragments of full-length proteins, the antigen sequences are 8-25 amino acids long. Computer algorithms may be used to select peptides for use as presentable antigens, and suitable methods for identifying presentable peptide antigens are described in Chen et al., Nature Biotechnol. 37:1332-43 (2019); Jurtz et al., J. Immunol. 199:3360-68 (2017); Abelin et al., J. Thero. Biol. 389:214-224 (2016), all of which are incorporated by reference in their entirety for all purposes.

[0080] T cell receptors can recognize antigens bound to major histocompatibility complex (MHC) molecules. MHC molecules include class I, class II, and class III. Both class I and class II MHC molecules play a role in immune responses. Class I MHC molecules consist of heavy and light chains and can bind peptides of about 8-11 amino acids, but usually 9 or 10 amino acids, and present them to naive and cytotoxic T lymphocytes. The peptides bound by class I MHC molecules can arise from endogenous protein antigens. The alpha chain of class I MHC molecules can be HLA-A, HLA-B, or HLA-C monomers, and the beta chain can be beta 2 microglobulin.

[0081] MHC class II molecules consist of alpha and beta chains and can bind peptides of about 8-24 amino acids, which they present to T cells. The peptides bound by MHC class II molecules can originate from extracellular or exogenous protein antigens. The alpha and beta chains can be HLA-DR, HLA-DQ and HLA-DP monomers. MHC class II can be expressed by all cell types, but usually occurs in professional antigen-presenting cells (APCs): macrophages, B cells, and especially dendritic cells. APCs take up antigenic proteins, perform antigen processing, and bring back molecular fractions of them - fractions called epitopes - and present them on the surface of the APC coupled within MHC class II molecules (antigen presentation). At the surface of the cell, the epitopes can be recognized by immune structures such as T cell receptors (TCRs).

[0082] In some cases, antigen-presenting cells contain antigen barcodes bound to their cell membranes via membrane-bound DNA or RNA-binding proteins. The antigen barcodes of antigen-presenting cells can be introduced simultaneously or sequentially with antigen-encoding cDNA or mRNA. The membrane-bound DNA or RNA-binding proteins can be engineered by zinc finger nucleases, transcription factor binding proteins, replication protein A, CRSIPR Cas9, Cas13, Cpf1, phage RNA-binding proteins, bacterial single-stranded binding proteins, or engineered restriction digestion enzymes. The membrane-bound antigen barcodes can transfer information between APCs and T cells during trogocytosis.

[0083] MHC-antigen complexes can be linked together in one or any combination of the following ways to create an antigen library: genetically linked MHC alpha chain, MHC beta chain, and antigen coding sequences with flexible linkers (e.g., GCGGSGGGSGGGGGS), genetically linked MHC genes with self-cleaving peptide sequences (e.g., T2A, P2A, or F2A), or overexpression of antigen peptides in cell lines with the desired MHC alleles. Antigen DNA or RNA can be recovered by bulk or single cell sequencing of the APCs of the library, serving as a natural barcode. Antigen DNA / RNA barcodes (antigen sequences) can be amplified using PCR and primers targeting antigen-adjacent regions. After phagocytosis, where the APCs are consumed by macrophages, paired antigen and TCR data can be recovered from one cell (the macrophage that "ate" the APC) via DNA or RNA sequencing. Alternatively, after T cell activation and trogocytosis, where APCs and T cells exchange membrane pieces, the barcode (either DNA or RNA) allows paired antigen and TCR data to be retrieved from one cell (APC or T cell containing both antigen and TCR barcodes) via DNA or RNA sequencing. Methods for generating single chain MHC molecules with antigenic peptides are described, for example, in Mottez et al., Cells expressing a major histocompatibility complex class I molecule with a single covalently bound peptide are highly immunogenic, J. Exp. Med. 181:493-502 (1995); Ignatowicz et al., Cell surface expression of class II MHC proteins bound by a single peptide, J. Immunol. 1999, all of which are incorporated by reference in their entirety for all purposes.Volume 154: pages 3852 - 3862 (1995); Uger et al., Creating CTL targets with epitope-linked B2 microglobulin constructs, J. Immunol. Volume 160: pages 1598 - 1605 (1998); Yu et al., Cutting edge: single-chain trimers of MHC class I molecules form stable structures that potently stimulate antigen-specific T cells and B cells, J. Immunol. Volume 168: pages 3145 - 3149 (2002); Hansen et al., Translational and Basic applications of peptide-MHC I single chain trimers, Trends Immunol 31: pages 363 - 369 (2010); Kotsiou et al., Properties and applications of single-chain major histocompatibility complex class I molecules, Antioxidants Redox Signaling 15: pages 645 - 655 (2011); Zhao et al., Use of single chain MHC technology to investigate co-agonism in human CD8+ T cell activation, J. Vis. Exp.144:e59126 (2019). Alternatively, MHC chains (class I or II) are produced recombinantly separately and assembled in recombinant cells to form MHC molecules. Antigens can be co-expressed in cells as peptides for combination with MHC molecules or as whole antigen proteins that are processed for degradation into peptides that can be presented to MHC molecules. Methods for expressing antigen::MHC complexes in this manner are described, for example, in Stevens et al., Efficient generation of major histocompatibility complex class I peptide complexes using synthetic peptide libraries, J. Bio. Chem. vol. 273: pp. 2874-2884 (1998); Braendstrup et al., MHC class II tetramers from isolated recombinant alpha and beta chains refolded with affinity tagged peptides, PLoS ONE vol. 8: E73648 (2013); Saini et al., Empty peptide receptive MHC class I molecules for efficient detection of antigen specific T cells, Sci. Immunol. vol. 4: eaau9039 (2019).

[0084] Infectious disease antigens that may be used in the antigen library include antigens derived from infectious pathogens, which may be bacterial or viral pathogens. Bacterial pathogens include, for example, Staphylococci, Streptococcus, Escherichia coli, Pseudomonas, Salmonella, Staphylococcus aureus, Neisseria gonorrhoeae, Streptococcus pyogenes, Group A Streptococcus, Group B Streptococcus (Streptococcus agalactiae), Streptococcus pneumoniae, Clostridium tetani, Helicobacter pyloris, Legionella pneumophila, and the like. pneumophilia strains, Mycobacteria sps. (e.g., M. tuberculosis, M. avium, M. intracellulare, M. kansaii, or M. gordonea), Neisseria meningitides, Listeria monocytogenes, R. rickettsia, Salmonella spp., Brucella spp., Shigella spp. strains, certain strains of Escherichia coli or other bacteria that have acquired genes with invasiveness factors, and any of the foregoing or other bacteria that have acquired antibiotic resistance factors.

[0085] Viral pathogens include, for example, Ebola, Zika, RSV, Retroviridae (e.g., human immunodeficiency viruses such as HIV-1 and HIV-LP), Picornaviridae (e.g., poliovirus, hepatitis A virus, enterovirus, human cossackievirus, rhinovirus, and echovirus), rubella virus, coronavirus, vesicular stomatitis virus, rabies virus, Ebola virus, parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus, and the like. Somatic viruses include influenza viruses, Hepatitis B virus, Parvovirus, AdenoViridae, Herpesyiridae (e.g., Herpes simplex virus types 1 and 2 (HSV), Varicella zoster virus, Cytomegalovirus (CMV), and Herpes virus), Poxviridae (e.g., Smallpox virus, Vaccinia virus, and Pox virus), Hepatitis C virus, Epstein-Barr virus, or Kaposi's sarcoma virus.

[0086] The antigen library may include any of the tumor-associated antigens, tumor neo-antigens, or other antigens that are preferentially found on cancer cells. Tumor-associated antigens that may be used in the antigen library include any gene or protein that is over- or aberrantly expressed in tumor cells, such as mesothelin, disialoganglioside (GD2), Her-2, MUC1, GPC3, EGFRVIII, CEA, CD19, EGFR, PSMA, GPC2, folate receptor beta, IgG Fc receptor, PSCA, PD-L1, EPCAM, Lewis Y antigen, L1CAM, FOLR, CD30, CD20, EPHA2, PD-1, C-MET, ROR1, CLDN18.2, NKG2D, CD133, TSHR, CD70, ERBB, AXL, Death receptor 5, VEGFR-2, CD123, CD80, CD86, TSHR, ROR2, CD147, kappa IGG, IL-13, MUC16, IL-13R, NY-ESO-1, IL13RA2, DLL3, FAP, LMP1, TSHR, BCMA, NECTIN-4, MG7, AFP (alpha-fetoprotein), GP100, B7-H3, Nectin-4, MAGE-A1, MAGE-A4, MART-1, HBV, MAGE-A3, TAA, GP100, Thyroglobulin, EBV, HPV E6, PRAME, HERV-E, WT1, GRAS G12V, p53, TRAIL, MAGE-A10, HPV-E7, KRAS G12D, MAGE-A6, CD19, BCMA, CD22, CD123, CD20, CD30, CD33, CD138, CD38, CD7, SLAMF7, IGG FC, MUC1, LewisY antigen, CD133, ROR1, FLT3, NKG2D, kappa light chain, CD34, CLL-1, TSLP, CD10, PD-L1, CD44V6, EBV, CD5, GPC3, CD56, integrin B7, CD70, MUCL, CKIT, CLDN18.2, TRBC1, TAC1, CD56, CD4, CD2, CD18, CD27, CD37, CD72, CD79A, CD79B, CD83, CD117, CD172 , ERBB3, ERBB4, DR5, HER2, CS1, IL-1RAP, ITGB7, SLC2A14, SLC4A1, SLC6A11, SLC7A3, SLC13A5, SLC19A1, SLC22A12, SLC34A1, slc45A3, SLC46A2, Fra, IL-13Ra2, ULBP3, ULBP1, CLD18, NANOG, CEACAM8, TSPAN16, GLRB, DYRK4, SV2C, SI GLEC8, RBMXL3, HIST1HIT, CCR8, CCNB3, ALPPL2, ZP2, OTUB2, LILRA4, GRM2, PGG1, NBIF3, GYPA, ALPP, SPATA19, FCRL I, FCRLA, CACNG3, UPK3B, 12UMO4, MUC12, HEPACAM, BPI, ATP6V0A4, HMMR, UPK1A, ADGRV1, HERC5, C3AR1, FASLG, NGB, These include CELSR3, CD3G, CEACAM3, TNFRSFBC, MS4AB, S1PR5, EDNRB, SCN3A, ABCC8, ABCB1, ANO1, KCND2, HTR4, CACNB4, HTR4, CNR2, 26LRB, EXOC1, ENTPP1, ICAM3, ABCGB, SCN4B, SPN, CD68, ITGAL, ITGAM, SCTR, CYYR1, CLCN2, SLARA3 and JAG3. Tumor neo-antigens that can be used in the antigen library include any mutant gene or protein that is unique or enriched in tumor cells, such as ACVR2A K435fs, AKT1 E17K, AR Q58L, ASXL1 G610, ATR K771fs, BRAF V600E, CHEK2 K416E, CRIPAK R154fs, CTNNB1 S26F, CTNNB1 S30C, CTNNB1 D25N, CTNNB1 G27R, CTNNB1 S26C, CTNNB1 S30F, DNMT3AR882H、DNMT3A R882C、EGFR A244V、EGFR G553V、EGFR A244T、EGFR R177C、EGFR L813R、EP300 D1399N、FBXW7 R465H、FBXW7 R505G、FBXW7 R505C、FBXW7 R465C、FGFR2 N550K、FGFR2 S252W、FGFR3 S249C、FLT3 D835Y、GATA3 P409fs、IDH1 R132C、IDH1 R132H、IDH2 R88Q、KRAS G12V、KRAS G13D、KRAS G12D、KRAS G12C、KRAS A146T、KRAS G12A、KRAS Q61L、MTOR S2215Y、NFE2L2 R18G、NFE2L2 E63Q、NFE2L2 D13H、NPM1 W288fs、NRAS G13D、NRAS Q61R、NRAS G12D、NRAS Q61K、NRAS Q61L、PIK3CA E453K、PIK3CA E545A、PIK3CA M1043I、PIK3CA M1043V、PIK3CA R108H、PIK3CA H1047R、PIK3CA Q546K、PIK3CA G118D、PIK3CA N345K、PIK3CA H1047L、PIK3CA R88Q、PIK3CA K111E、PIK3CA Q546R、PIK3CA Q546P、PIK3CA E726K、PIK3CA E542K、PIK3CA E81K、PIK3CA E545K、PIK3CA C420R、PIK3CA R93Q、PPP2R1A P99R、PPP2R1A R103W、PTEN A328fs、PTEN R130Q、PTEN R130G、PTEN T319fs、RPL22 K15fs、SF3B1 K700E、SMAD4 R361H、TGFBR2 E150fs、TP53 R249M、TP53 E285K、TP53 R249S、TP53 G266V、TP53 R158H、TP53 C141Y、TP53 Y236C、TP53 V173M、TP53 S241F、TP53 V173L、TP53 E271K、TP53 E286K、TP53 C275Y、TP53 R110L、TP53 R273L、TP53 H179Y、TP53 V216M、TP53 V157F、TP53H193R, TP53 T125, TP53 I195T, TP53 H179R, TP53 G245S, TP53 R282W, TP53 R273C, TP53 Y220C, TP53 R248W, TP53 R248Q, TP53 R273H, TP53 R175H, TP53 Y205C, TP53 M237I, TP53 C238F, TP53 V272M, TP53 C242F, TP53 L194R, TP53 A159V, TP53 G245D, TP53 H193L, TP53 Y163C, TP53 R158L, TP53 G108fs, TP53 G245V, TP53 C238Y, TP53 C176F, TP53 Tumor neoantigens include Y234C, TP53 C176Y, TP53 K132N, U2AF1 S34F, VHL L89H, VHL S111N, VHL W117fs. Tumor neoantigens can be generated by single nucleotide mutations, frameshift mutations, insertions, deletions, alternative splicing, or extrachromosomal circular DNA.

[0087] Suitable antigens for the library include KRAS variants, a tumor neo-antigen often found in pancreatic, lung, colon, and bile duct cancers. Table 2 below shows common KRAS mutations associated with certain cancers.

[0088] [Table 2]

[0089] These KRAS mutant alleles can be used in antigen libraries to screen for TCRs that can bind to the mutant alleles but not the wild-type allele. Such discriminatory TCRs can be used to target immunotherapy to cancers that harbor these KRAS mutations.

[0090] A suitable cell type for the antigen library is an antigen-presenting cell. The APC may lack MHC molecules on its surface. Suitable antigen-presenting cells include, for example, K562 cells.

[0091] Also described herein is a retrovirus-based system that repurposes viral tropism as a method of selecting molecular interactions, for example by encoding these antigen / ligand variants in the corresponding transfer plasmid used to generate the virus, replacing the binding function of the wild-type viral surface protein with that of the antigen / ligand of interest, thereby ensuring that the resulting virus displays the antigen / ligand variant on its surface and packages the corresponding genetic sequence. Thus, when the virus enters a target cell (e.g., bearing a receptor that binds to the extracellular targeting domain of the displayed antigen / ligand variant), cell entry results in the integration of the genetic sequence of the displayed protein into the genome of the target cell at the desired location.

[0092] The retrovirus may have a nucleic acid encoding the structure: S-ETD-LGD-IRES-R (where S encodes a signal sequence, ETD encodes an extracellular targeting domain; LGD encodes an antigen / ligand, IRES encodes an internal ribosome entry site, and R encodes a reporter (e.g., a selectable marker such as a fluorescent protein or antibiotic resistance). IRES and R are optional, and the nucleic acid may encode S-ETD-LGD. The retrovirus also encodes a mutant viral envelope protein that contains at least one mutation that reduces its native entry function. The IRES sequence may be replaced by a 2A self-cleaving peptide sequence (T2A, P2A, F2A, and E2A), which also allows for co-expression of two genes by one promoter.

[0093] Described herein are retroviruses that include a viral envelope protein with at least one mutation that reduces its native function; a non-viral membrane-bound protein that includes a membrane-binding domain, an extracellular antigen / ligand domain, and a nucleic acid that encodes a reporter. The retroviruses disclosed herein can have one or more elements from a suitable species of retroviral genome (naturally occurring or modified). Retroviruses include seven families: alpharetroviruses (avian leukemia viruses), betaretroviruses (mouse mammary tumor viruses), gammaretroviruses (mouse leukemia viruses), deltaretroviruses (bovine leukemia viruses), epsilonretroviruses (walleye cutaneous sarcoma viruses), lentiviruses (human immunodeficiency virus 1), and spumaviruses (human spumaviruses). Other examples of retroviruses are provided in U.S. Patent No. 7,901,671, which is incorporated by reference in its entirety for all purposes.

[0094] Modified lentiviral genome can be useful as a viral vector for delivery of nucleic acid to host cells.Host cells can be transfected with lentiviral vector and optionally with additional vectors for expressing lentiviral packaging proteins (e.g., VSV-G, Rev, and Gag / Pol) to produce lentiviral particles in culture medium.Non-limiting examples of retroviral constructs useful herein include, for example, lentiviral vectors, human immunodeficiency virus (HIV) vectors, avian leukemia virus (ALV) vectors, murine leukemia virus (MLV) vectors, murine mammary tumor virus (MMTV) vectors, murine stem cell virus, and human T-cell leukemia virus (HTLV) vectors.These retroviral constructs contain proviral sequences from corresponding retroviruses.

[0095] The retroviral vectors described herein may further comprise additional functional elements known in the art that address safety concerns and / or improve vector function, such as packaging efficiency and / or viral titer. Further information can be found in US20150316511, WO2015 / 117027, and WO2019 / 056015, each of which is incorporated by reference in its entirety for all purposes.

[0096] The viral envelope protein may be any viral envelope protein of any retrovirus (e.g., lentivirus). The viral envelope protein may be a VSV-G envelope protein, a measles virus envelope protein, a Nipah virus envelope protein, or a coccus virus G protein. The native function that is reduced by the mutation of the viral envelope protein may be viral tropism (e.g., the ability to infect cells, bind to cells, etc.). For example, the mutant VSV-G envelope protein may be mutated at amino acids H8, K47, Y209, and / or R354. The mutant VSV-G envelope protein may have one or more of the following changes: H8A, K47A, K47Q, Y209A, R354A, and / or R354Q. Exemplary mutant VSV-G envelope proteins are described in Nikolic et al., "Structural basis for the recognition of LDL-receptor family members by VSV glycoprotein." Nature Comm., 2018, 9:1029, which is incorporated by reference in its entirety for all purposes. The mutant measles virus envelope protein may have a mutation(s) at Y481, R533, S548, and / or F549. The mutant measles virus envelope protein may have one or more of Y481A, R533A, S548L, and / or F549S. The mutant Nipah virus envelope protein may have a mutation(s) at E501, W504, Q530, and / or E533. The mutant measles virus envelope protein may have one or more of E501A, W504A, Q530A, and / or E533A. The mutant coccal viral G protein may have a mutation(s) at K64 and / or R371. The mutant coccal viral G protein may have one or more of K64Q, and / or R371A.

[0097] A membrane-binding domain is a protein or peptide having an amino acid sequence that allows the protein or peptide to be fully or partially embedded or associated with the membrane (e.g., envelope) of a retrovirus. The membrane-binding domain may allow the presentation and delivery of an extracellular antigen / ligand domain to the extracellular environment. The membrane-binding domain may have an intracellular domain, a transmembrane domain, and an extracellular domain. The membrane-binding domain may be a major histocompatibility complex (MHC) protein or a fragment thereof. The MHC protein may be a class I or class II MHC protein.

[0098] The retroviruses present in the retroviral library may collectively comprise a library of different antigens / ligands fused to a membrane-binding domain. The extracellular antigen / ligand can bind to a target cell. The extracellular antigen / ligand domain can bind to a protein or receptor ligand (e.g., a T cell receptor) present on the cell surface of a cell or a subset of a population of cells. The ligand-extracellular antigen / ligand domain binds to a receptor present on the cell surface of a T cell or a subset of a population of T cells. This binding interaction between the extracellular antigen / ligand domain of the retrovirus and the cell's receptor, protein or ligand can allow the retrovirus to enter a cell (e.g., an antigen-specific cell, e.g., a T cell).

[0099] The non-viral membrane-associated protein may include a linker located between the membrane-associated domain and the extracellular antigen / ligand domain. The linker may be an amino acid linker, a rigid linker, a flexible linker, or an oligomerization linker. A rigid linker may be an amino acid sequence that lacks flexibility (e.g., may contain at least one proline). For example, a rigid linker may be derived from the platelet-derived growth factor receptor (PDGFR) stalk or the CD8-alpha stalk. A flexible linker is an amino acid sequence that has many degrees of freedom (e.g., may contain multiple amino acids with small side chains, such as glycine or alanine). An oligomerization linker is an amino acid that can oligomerize to another related amino acid. An oligomerization linker may be an amino acid sequence that can form a dimer, trimer, or tetramer. For example, an oligomerization linker may be made from an IgG4 hinge domain.

[0100] The present specification also relates to a library of retroviruses, the library comprises a plurality of unique retroviruses, each unique retrovirus comprises a viral envelope protein that comprises at least one mutation that reduces its native function, a non-viral membrane-associated protein that comprises a membrane-associated domain and an extracellular targeting domain, and optionally a nucleic acid that encodes a reporter, each unique retrovirus comprises a different and unique extracellular targeting domain.The present specification also describes a library of cells that comprises retroviruses, the library comprises a plurality of unique cells, each unique cell comprises a unique retrovirus.

[0101] The library may contain multiple retroviruses with nucleic acids encoding different MHC-peptide fusions for use in screening populations of T cells. In such libraries, the MHC-peptide fusions displayed on the viral surface allow for T cell infection in a TCR-specific manner. Infected T cells can be collected and sequenced, allowing for the identification of MHC-peptide ligands capable of infecting a subset of the T cell population of interest, as well as the ability to simultaneously track the TCR sequence and the reactive MHC-peptide ligand. MHC-peptide retroviral libraries may contain randomized transfer vectors containing randomized MHC-peptide targeting elements. Randomly generated libraries may be generated using degenerate oligonucleotide primers. Targeted libraries may be specific to a unique set of antigens (e.g., all possible viral or bacterial antigens for a particular target of interest - human immunodeficiency virus, tuberculosis TB, etc.; or all possible neoantigens of a particular subject). Antigen / ligand sequences may be selected to maximize their chances of interacting with a receptor or binding protein. Variations of antigen / ligand libraries can be created by introducing genetic mutations (e.g., randomly or site-specifically) into desired positions of the antigen / ligand of interest. Genetic mutations can be substitutions, deletions, insertions, and rearrangements of DNA fragments. Specific genetic mutations of choice can be generated randomly, enriched in regions of interest (e.g., interaction domains of ligands), or recommended by computational algorithms. Examples of computational algorithms for genetic mutation strategies include protein structure analysis, computational docking, molecular dynamics simulation, regression, statistically-based classification, random forests, support vector machines, and neural networks.

[0102] The barcode sequence can be a randomly generated nucleotide sequence of any length greater than 4. For example, a DNA sequence of 15 nucleotides can be 4 15 =1e 9The barcode sequence may provide a diversity of marker sequences. The barcode sequence may be an amino acid that codes for a marker sequence or may function purely as a marker sequence. For example, one design of retroviral plasmid contains two cloning sites: one for the antigen-encoding sequence and one for the barcode sequence.

[0103] After inserting both the barcode and the ligand sequence into a viral plasmid and amplifying the viral plasmid by either PCR or E. coli amplification, a portion of the plasmid can be sequenced by bulk sequencing. Because the barcode and ligand sequences are close to each other, the bulk sequencing data can establish a one-to-one mapping between the barcode sequence and the ligand sequence.

[0104] host cell The nucleic acid encoding the polypeptide (e.g., TCR or antigen) described herein is cloned into a suitable expression vector for expression of the polypeptide in a host cell. The host cell may be a mammalian cell, such as a mouse, rat, rabbit, hamster, pig, cow, cat, or dog. The mammalian cell may also be a primate cell, including but not limited to a monkey, chimpanzee, gorilla, and human. The mammalian cell may be a mouse cell, since mice typically serve as a model for other mammals, most specifically humans (see, e.g., Hanna, J. et al., Science 318:1920-23, 2007; Holtzman, DM et al., J Clin Invest. 103(6):R15-R21, 1999; Warren, RS et al., J Clin Invest. 95:1789-1797, 1995; each publication is incorporated by reference in its entirety for all purposes). Host cells include, for example, fibroblasts, epithelial cells (e.g., kidney, breast, prostate, lung), keratinocytes, hepatocytes, adipocytes, endothelial cells, and hematopoietic cells (e.g., macrophages, other monocytes, antigen presenting cells, B cells, neutrophils, mast cells, dendritic cells, or other phagocyte cell types). Host cells may be mature cells (e.g., terminally differentiated, dividing or non-dividing) or embryonic cells (e.g., blastocyst cells, etc.) or stem cells. Host cells may be cell lines from animals or other sources.

[0105] The mammalian cell may be a cell found in the circulatory system of a mammal, including a human. Exemplary circulatory cells include, among others, red blood cells, platelets, plasma cells, T cells, natural killer cells, B cells, macrophages, neutrophils, etc., and their precursor cells. As a group, these cells are defined as circulating eukaryotic cells. Such circulating cells may be primary cells or derived from primary cells. Circulating cells may be autologous, syngeneic, or allogeneic. Mammalian cells may be derived from any of these circulating eukaryotic cells. Circulating cells or cells derived from circulating cells may be used. The mammalian cell may be a macrophage or a precursor or progenitor cell to a macrophage. The monocyte or macrophage cell may be a raw264.7 cell, a K562 cell, or a THP-1 cell. The mammalian cell may be a T cell or a T cell precursor or progenitor cell. The mammalian cell may be a helper T cell, a cytotoxic T cell, a memory T cell, a regulatory T cell, a natural killer T cell, a mucosal-associated invariant T cell, a gamma delta T cell, or a precursor or progenitor to the aforementioned cells. The mammalian cell may be a natural killer cell, or a precursor or progenitor to a natural killer cell. The mammalian cell may be a B cell, or a plasma cell, or a B cell precursor or progenitor. The mammalian cell may be a neutrophil or a neutrophil precursor or progenitor. The mammalian cell may be a megakaryocyte or a precursor or progenitor to a megakaryocyte. The mammalian cell may be an antigen presenting cell, such as a monocyte, macrophage, dendritic cell, epithelial cell, etc. The APC may be engineered to lack MHC molecules on its surface. Such an APC will only present MHC molecules from the MHC:antigen fusion polypeptides of the antigen library.

[0106] Any immune cell can be used as host cell herein, including, for example, T cells, B cells, natural killer cells, dendritic cells, macrophages, monocytes, and / or neutrophils.Host cell can be receptor-specific immune cell or cell line genetically modified to express target receptor.Host cell includes human primary immune cell, mouse primary immune cell, rat primary immune cell, and immortalized mammalian cell line. Examples of immortalized mammalian cell lines that can be used are Jurkat, K562, Tall-104, Raji, HEK293, HEK293T, 3T6, A559, A9, AtT-20, 3T3, BHK-21, BHL-100, BT, Caco-2, Chang, CHO-K1, COS-1, COS -3, COS-7, Daudi, H9, HeLa, Hep-2, HL-60, HT-1080, HT-29, HUVEC, I-10, IM-9, JEG-2, MDA-MB-231, L2, KB, KG-1, MCF7, WI-38, WISH, XC, Y1, Jeko, NK-92, NK-92 Including MI, J76, CCRF-CEM, DND-41, HPB-ALL, MOL-4, RPMI-8402, GRANTA, MINO, REC-1, U-2940, BJAB, VAL, THP-1, MUTZ-3, U-937, ME-1, MOLM13, U-937, and SEM.

[0107] The host cells can be obtained from a subject. The subject can be any living organism. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. Macrophages, T cells, antigen-presenting cells, etc. can be obtained from several sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, splenic tissue, and tumors. Any number of macrophage cell lines, antigen-presenting cell lines, or T cell lines are available in the art and can be used. Macrophages, antigen-presenting cells, or T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to those skilled in the art, such as Ficoll separation. Host cells from an individual's circulating blood can be obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. The cells collected by apheresis may be washed to remove the plasma fraction and place the cells in a suitable buffer or medium for subsequent processing steps. The cells may be washed with phosphate buffered saline (PBS). In alternative embodiments, the washing solution may be calcium-deficient, magnesium-deficient, or may be deficient in many, if not all, divalent cations. An initial activation step in the absence of calcium may result in escalation of activation.

[0108] In one embodiment, the genome of the host cell is engineered with a first nucleic acid that has a unique site for insertion of the insert nucleic acid by a targeting system such as CRISPR. The host cell can optionally be engineered with the enzymes of the targeting system, including, for example, Cas9, Cas12a (aka Cpf1), suitable zinc finger nucleases (ZENs), or transcription activator-like nucleases (TALENs). The first nucleic acid can be engineered near or adjacent to a nucleic acid encoding a receptor or binding protein of the host cell. This allows for directional insertion of the nucleic acid into this site near or adjacent to the nucleic acid encoding the receptor. If the antigen / ligand interacts with the receptor / binding protein of the host cell, it can introduce the nucleic acid into the host cell. If this nucleic acid is accompanied by a suitable targeting nucleic acid (e.g., guide RNA), the nucleic acid showing the antigen / ligand can be integrated near or adjacent to the nucleic acid encoding the receptor. For example, a pegRNA as described herein can be inserted into this site. Appropriate primers will amplify and / or sequence both the receptor nucleic acid (or barcode) and the antigen / ligand nucleic acid (or barcode).

[0109] nucleic acid The nucleic acid may at least partially encode the individual peptides, polypeptides, proteins and barcodes described herein. The nucleic acid may be natural, synthetic or a combination thereof. The nucleic acid may be RNA, mRNA, DNA or cDNA.

[0110] Nucleic acid also includes expression vectors, such as plasmids, or viral vectors, or linear vectors, or vectors that integrate into chromosomal DNA. Expression vectors can contain nucleic acid sequences that allow vectors to replicate in one or more selected host cells. Such sequences are well known for various cells. The origin of replication from plasmid pBR322 is suitable for most gram-negative bacteria. In eukaryotic host cells, such as mammalian cells, expression vectors can be integrated into the host cell chromosome and then replicated by the host chromosome. Similarly, vectors can be integrated into the prokaryotic cell chromosome.

[0111] Expression vectors also usually contain a selection gene, also called a selectable marker. Selectable markers are well known in the art for prokaryotic and eukaryotic cells, including the host cells of the present invention. Usually, a selection gene encodes a protein necessary for the survival or growth of a transformed host cell grown in a selective culture medium. Host cells not transformed by a vector containing a selection gene will not survive in the culture medium. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply vital nutrients not available from complex media, such as the gene encoding D-alanine racemase in Bacillus. An exemplary selection scheme can utilize a drug that stops the growth of the host cell. Those cells successfully transformed with a heterologous gene produce a protein that confers drug resistance and thus survive the selection regime. Other selectable markers for use in bacterial or eukaryotic (including mammalian) systems are well known in the art.

[0112] An example of a promoter capable of expressing a transgene encoding a TCR or antigen described herein in a mammalian host cell is the EF1a promoter. The native EF1a promoter drives expression of the alpha subunit of the elongation factor 1 complex, which is responsible for the enzymatic delivery of aminoacyl-tRNA to the ribosome. The EF1a promoter is widely used in mammalian expression plasmids and has been shown to be effective in driving expression from transgenes cloned into lentiviral vectors. See, for example, Milone et al., Mol. Ther. 17(8):1453-1464 (2009), which is incorporated by reference in its entirety for all purposes. Another example of a promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operably linked to it. Other constitutive promoter sequences may also be used, including, but not limited to, Simian Virus 40 (SV40) early promoter, mouse mammary tumor virus promoter (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, phosphoglycerate kinase (PGK) promoter, MND promoter (a synthetic promoter containing the U3 region of a modified MoMuLV LTR with a myeloproliferative sarcoma virus enhancer, see, for example, Li et al., J. Neurosci. Methods vol. 189, pp. 56-64 (2010), which is incorporated by reference in its entirety for all purposes), avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, including, but not limited to, actin promoter, myosin promoter, elongation factor-1a promoter, hemoglobin promoter, and creatine kinase promoter. Furthermore, the expression vectors herein are not limited to the use of constitutive promoters.

[0113] Inducible promoters are also contemplated for use in the expression vector. Examples of inducible promoters include, but are not limited to, metallothionein promoter, glucocorticoid promoter, progesterone promoter, tetracycline promoter, c-fos promoter, ThermoFisher's T-REx system that drives expression from the human cytomegalovirus immediate early promoter under the control of tetracycline operator(s), and Intrexon's RheoSwitch promoter. See, e.g., Karzenowski, D. et al., BioTechiques 39:191-196 (2005); Dai, X. et al., Protein Expr. Purif 42:236-245 (2005); Palli, SR et al., Eur. J. Biochem. 270:1308-1515 (2003); Dhadialla, TS et al., Annual Rev. Entomol. 43:545-569 (1998); Kumar, MB, et al., J. Biol. Chem. 279:27211-27218 (2004); Verhaegent, M. et al., Annal. Chem. 74:4378-4385 (2002); Katalam, AK, et al., Molecular Therapy 13:S103 (2006); and Karzenowski, D. et al., Molecular Therapy 13:S194 (2006), U.S. Patent Nos. 8,895,306, 8,822,754, 8,748,125, and 8,536,354.

[0114] Expression vectors typically have promoter elements, e.g., enhancers, to control the frequency of transcription initiation. Typically, these are located in a region 30-110 bp upstream of the start site, although some promoters have been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements is often flexible, so that promoter function is preserved when elements are inverted or moved with respect to each other. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decrease. Depending on the promoter, individual elements appear to be able to function either cooperatively or independently to activate transcription.

[0115] It may be desirable to modify the polypeptides described herein. Those skilled in the art will recognize many ways to make changes to a given nucleic acid construct to generate variant polypeptides. Such well-known methods include site-directed mutagenesis, PCR amplification using degenerated oligonucleotides, exposing cells containing the nucleic acid to mutagens or radiation, chemical synthesis of the desired oligonucleotides (e.g., in conjunction with ligation and / or cloning to generate large nucleic acids) and other well-known techniques (see, e.g., Gillam and Smith, Gene 8:81-97, 1979; Roberts et al., Nature 328:731-734, 1987, which are incorporated by reference in their entirety for all purposes). Recombinant nucleic acids encoding the polypeptides described herein can be modified to provide preferred codons that enhance translation of the nucleic acid in a selected organism.

[0116] The polynucleotides of the present invention also include polynucleotides that contain substantially the same nucleotide sequence as the polynucleotides described herein. The polynucleotides may have at least about 80%, more typically at least about 90%, and even more typically at least about 95% sequence identity to the polynucleotides encoding the polypeptide of interest. The polynucleotides include the complements of the polynucleotides that contain nucleotide sequences that have at least about 80%, more typically at least about 90%, and even more typically at least about 95% sequence identity to the polynucleotides encoding the polypeptides described above. The polynucleotides may be DNA (genomic, cDNA, amplified, or synthetic) or RNA. Methods and algorithms for obtaining such polynucleotides are well known to those skilled in the art and may include, for example, methods for determining hybridization conditions that can usually isolate polynucleotides of desired sequence identity.

[0117] Nucleic acids encoding protein analogs or variants (i.e., designed to have one or more amino acids different from wild-type polypeptides) can be produced using site-directed mutagenesis or PCR amplification, with the primer(s) carrying the desired point mutation. For detailed descriptions of suitable mutagenesis techniques, see Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989) and / or Current Protocols in Molecular Biology, Ausubel et al., eds., Green Publishers Inc. and Wiley and Sons, NY (1994), each of which is incorporated by reference in its entirety for all purposes. Chemical synthesis using methods well known in the art, for example, as described by Engels et al., Angew Chem Intl ed. vol. 28:716-34, 1989 (incorporated by reference in its entirety for all purposes), can also be used to prepare such nucleic acids.

[0118] The amino acid "substitution" to generate a variant may be the result of replacing one amino acid with another amino acid having similar structure and / or chemical properties, i.e., conservative amino acid substitution. Amino acid substitutions may be made based on the similarity of polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathicity of the residues involved. For example, non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

[0119] The nucleic acid can be linked to another nucleic acid so that it is expressed under the control of a suitable promoter.The nucleic acid can also be linked to the nucleic acid that contains promoter or other regulatory elements that cooperate with the transcription initiation site, such as enhancer sequence, polyA site, or termination sequence, to achieve efficient transcription of the nucleic acid.In addition to the nucleic acid, a gene that can be a marker for confirming the expression of the nucleic acid (e.g., a drug resistance gene, a gene that codes for a reporter enzyme, or a gene that codes for a fluorescent protein) can be incorporated.

[0120] When nucleic acid is introduced into cells ex vivo, nucleic acid may be combined with a substance that promotes the transfer of nucleic acid into cells, for example, in addition to the above-mentioned excipients, a reagent for introducing nucleic acid, such as liposome or cationic lipid. Alternatively, a vector carrying nucleic acid is also useful. In particular, a composition in a form suitable for administration to a living body that contains nucleic acid carried by a suitable vector is suitable for in vivo gene therapy.

[0121] Introduction of polynucleotides into host cells Nucleic acids can be introduced by transfection (e.g., Gorman et al. Proc. Natl. Acad. Sci. 79:22 (1982):6777-6781, which is incorporated by reference in its entirety for all purposes), transduction (e.g., Cepko and Pear (2001) Current Protocols in Molecular Biology unit 9.9; DOI:10.1002 / 0471142727.mb0909s36, which is incorporated by reference in its entirety for all purposes), calcium phosphate transformation (e.g., Kingston, Chen and Okayama (2001) Current Protocols in Molecular Biology Appendix 1C; DOI:10.1002 / 0471142301.nsa01cs01, which is incorporated by reference in its entirety for all purposes), calcium chloride and polyethylene glycol (PEG)-based methods for introducing recombinant DNA into microalgae cells (Chlorella ellipsoidea), and the like. Other examples of this method include those reported for use in transforming protoplasts of Escherichia coli (such as Escherichia coli), which are incorporated by reference in their entireties for all purposes, using techniques such as the use of peptides (see, e.g., Kim et al. (2002) Mar. Biotechnol. 4:63-73, which is incorporated by reference in its entirety for all purposes), cell penetrating peptides (e.g., Copolovici, Langel, Eriste, and Langel (2014) ACS Nano 2014 8(3), 1972-1994; DOI: 10.1021 / nn4057269, which are incorporated by reference in their entireties for all purposes), electroporation (see, e.g., Potter (2001) Current Protocols in Molecular Biology unit 10.15; DOI: 10.1002 / 0471142735.im1015s03 and Kim et al. (2014) Genome pp. 1012-19.doi:10.1101 / gr.171322.113, Kim et al. 2014 describe an optimized electroporation system, the Amaza Nucleofector), microinjection (e.g., McNeil (2001) Current Protocols in Cell Biology unit 20.1; DOI: 10.1002 / 0471143030.cb2001s18, which is incorporated by reference in its entirety for all purposes), liposomes or cell fusion (e.g., Hawley-Nelson and Ciccarone (2001) Current Protocols in Neuroscience Appendix 1F; DOI: 10.1002 / 0471142301.nsa01fs10, which is incorporated by reference in its entirety for all purposes), mechanical manipulation (e.g., Sharon et al. (2013) PNAS 2013, which is incorporated by reference in its entirety for all purposes). 110(6); DOI:10.1073 / pnas.1218705110), gene gun methods (see, e.g., Sanford, Trends in Biotech. (1988) 6:299-302; U.S. Pat. No. 4,945,050, incorporated by reference in its entirety for all purposes), lithium acetate / PEG transformation (Gietz and Woods (2006) Methods Mol. Biol. 313, 107-120) and modifications thereof, or other well-known techniques for delivery of nucleic acids to host cells. Once introduced, the nucleic acids of the invention can be expressed episomally or can be synthesized using well-known techniques, such as recombination (e.g., Lisby and Rothstein (2015) Cold Spring Harb Perspect Biol. Mar 2;7(3). pii:a016535. doi:10.1101 / cshperspect.a016535, incorporated by reference in its entirety for all purposes), non-homologous integration (e.g., Deyle and Russell (2009) Curr Opin Mol Ther.2009 Aug;11(4):442-7) or transposition (as described above for mobile genetic elements) can be used to integrate into the genome of the host cell. The efficiency of homologous and non-homologous recombination can be enhanced by genome editing techniques that introduce targeted single- or double-strand breaks (DSBs). Examples of DSB generating techniques include CRISPR / Cas9, TALEN, zinc finger nucleases, or equivalent systems (e.g., Cong et al. Science 339(6121)(2013):819-823; Li et al. Nucl. Acids Res (2011):gkr188; Gaj et al. Trends in Biotechnology 31(7)(2013):397-405, all of which are incorporated by reference in their entirety for all purposes), transposons, e.g., Sleeping Beauty (e.g., Singh et al. (2014) Immunol Rev. 2014 Jan;257(1):181-90. doi:10.1111 / imr.12137, all of which are incorporated by reference in their entirety for all purposes), targeted recombination using, for example, FLP recombinase (e.g., O'Gorman, Fox and Wahl, 2013, incorporated by reference in their entirety for all purposes), and the like. Science (1991) 15:251(4999):1351-1355), CRE-LOX (e.g., Sauer and Henderson PNAS (1988):85;5166-5170), or equivalent systems, or other techniques known in the art for integrating the nucleic acids of the invention into the genome of eukaryotic cells.

[0122] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microparticles, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is liposomes (e.g., artificial membrane vesicles). Other methods of state-of-the-art targeted delivery of nucleic acids are available, such as delivery of polynucleotides by targeted nanoparticles or other suitable submicron-sized delivery systems.

[0123] Novel receptors The T cell receptor and corresponding antigen / ligand are found by mixing the TCR repertoire described herein with the antigen library described herein and isolating individual cells with chimeric TCRs that have phagocytosed cells with a particular antigen-MHC fusion protein. Alternatively, the T cell receptor and corresponding antigen / ligand are found by mixing the TCR repertoire described herein with the antigen library described herein and isolating individual cells with trogocytosis markers and antigen / TCR barcodes. For example, the cells with the antigen library can be labeled with an optical probe, and optionally the cells with the TCR repertoire can be labeled with a different optical probe. FACs sorting can then be used to isolate cells from the TCR repertoire that obtain the antigen barcode from the antigen library. The separated cells are then sequenced to identify the TCR and antigen pair. Sequencing can be performed to identify the TCR and antigen. For example, sequencing can identify the barcode associated with each TCR and each antigen, or the TCR and antigen fusion can be sequenced in whole or in part. The high throughput of these systems and methods allows for the identification of TCRs and the antigens they bind, including many novel TCRs not previously known to interact with the antigen of interest.

[0124] The methods and compositions described herein can be used to find new TCRs useful in the treatment of cancer, infectious diseases, autoimmune diseases, and the like. Target antigens associated with these diseases are known and can be used to find new TCRs that interact with the antigens. The TCRs identified by these methods can be used to produce immunotherapies to treat these diseases. For example, many tumor-associated antigens (TAAs) are known. The methods and compositions described herein can produce antigen libraries from TAAs that can be used to find TCRs that bind to the TAAs. These TCRs can be used to create chimeric TCRs and / or chimeric antigen receptors that target cytotoxic T cells and natural killer cells to cancer cells. Similarly, many antigens are known for various infectious diseases. The methods and compositions described herein can identify TCRs that can be used to treat these infectious diseases. For example, chimeric TCRs that combine an Fc signaling portion (and optionally a coactivator) with a TCR ligand binding portion can be used to create macrophages or other phagocytes that phagocytose microbial agents that cause infectious diseases. Alternatively, chimeric TCRs or chimeric antigen receptors induce T cell activation, trogocytosis and antigen / TCR barcode exchange. TCRs can also be used to create chimeric antigen receptors that can activate helper T cells to lyse tumor cells, activate the tumor microenvironment, or assist B cells in producing neutralizing antibodies.

[0125] Exemplary tumor-associated antigens are KRAS variant alleles associated with certain cancers.90% of pancreatic cancers and more than 30% of lung, colon and bile duct cancers have one of these KRAS variant alleles.KRAS variant alleles include, for example, G12D, G12V, G12R, G12H, G12S, G12L, Q61H, Q61K, Q61R, A11T, G13P, G13D, and double mutations G12D and Q61H.Antigen libraries are made by some or all of these mutant KRAS variant alleles, concentrating on peptides that contain mutations for presentation by antigen libraries.Many different T cell receptor repertoires can be combined with KRAS antigen libraries to find TCRs that bind to different KRAS variant alleles. The T cell receptor repertoire used may originate from a TCR or genomic library of TCRs from cancer patients who have survived the cancer of interest (e.g., pancreatic, or lung, or colon, or cholangiocarcinoma). The TCR repertoire may be generated from patients who have undergone and survived (or showed a partial response to) immunotherapy, indicating that the patient's immune system was able to fight the cancer.

[0126] Anti-KRAS variant TCRs can be evaluated for their ability to distinguish between different KRAS variant alleles and wild-type KRAS. The affinity of anti-KRAS variant TCRs for KRAS variants and wild-type can also be evaluated. Anti-KRAS variant T cell receptors can be stratified into groups based on these characteristics, and anti-KRAS variant T cell receptors can be used to create chimeric antigen receptors for immunotherapy.

[0127] Other tumor-associated antigens include, for example, mesothelin, disialoganglioside (GD2), Her-2, MUC1, GPC3, EGFRVIII, CEA, CD19, EGFR, PSMA, GPC2, folate receptor beta, IgG Fc receptor, PSCA, PD-L1, EPCAM, Lewis Y antigen, L1CAM, FOLR, CD30, CD20, EPHA2, PD-1, C-MET, ROR1, CLDN18.2, NKG2D, CD133, TSHR, CD70, ERBB, AXL, Death receptor 5, VEGFR-2, CD123, CD80, CD86, TSHR, ROR2, CD147, kappa IGG, IL-13, MUC16, IL-13R, NY-ESO-1, IL13RA2, DLL3, FAP, LMP1, TSHR, BCMA, NECTIN-4, MG7, AFP (alpha-fetoprotein), GP100, B7-H3, Nectin-4, MAGE-A1, MAGE-A4, MART-1, HBV, MAGE-A3, TAA, GP100, Thyroglobulin, EBV, HPV E6, PRAME, HERV-E, WT1, GRAS G12V, p53, TRAIL, MAGE-A10, HPV-E7, KRAS G12D, MAGE-A6, CD19, BCMA, CD22, CD123, CD20, CD30, CD33, CD138, CD38, CD7, SLAMF7, IGG FC, MUC1, Lewis Y antigen, CD133, ROR1, FLT3, NKG2D, kappa light chain, CD34, CLL-1, TSLP, CD10, PD-L1, CD44V6, EBV, CD5, GPC3, CD56, integrin B7, CD70, MUCL, CKIT, CLDN18.2、TRBC1、TAC1、CD56、CD4、CD2、CD18、CD27、CD37、CD72、CD79A、CD79B、CD83、CD117、CD172、ERBB3 、ERBB4、DR5、HER2、CS1、IL-1RAP、ITGB7、SLC2A14、SLC4A1、SLC6A11、SLC7A3、SLC13A5、SLC19A1、S LC22A12、SLC34A1、slc45A3、SLC46A2、Fra、IL-13Ra2、ULBP3、ULBP1、CLD18、NANOG、CEACAM8、TSPAN16、GLRB、DYRK4、SV2C、SIGLEC8、RBMXL3、HIST1HIT、CCR8、CCNB3、ALPPL2、ZP2、OTUB2、LILRA4、GR M2、PGG1、NBIF3、GYPA、ALPP、SPATA19、FCRLI、FCRLA、CACNG3、UPK3B、12UMO4、MUC12、HEPACAM、BPI、ATP6V0A4、HMMR、UPK1A、ADGRV1、HERC5、C3AR1、FASLG、NGB、CELSR3、CD3G、CEACAM3、TNFRSFBC、M Including S4AB、S1PR5、EDNRB、SCN3A、ABCC8、ABCB1、ANO1、KCND2、HTR4、CACNB4、HTR4、CNR2、26LRB、EXOC1、ENTPP1、ICAM3、ABCGB、SCN4B、SPN、CD68、ITGAL、ITGAM、SCTR、CYYR1、CLCN2、SLARA3、JAG3.

[0128] Other tumor-associated antigens include, for example, complement factor H (e.g., lung cancer, breast cancer, other solid tumors), delta opioid receptor (e.g., small cell lung cancer), c-Met (e.g., NSCLC), gpNMB (e.g., melanoma, breast cancer, other solid tumors), TRAP-2 (e.g., epithelial tumors and other solid tumors), CEACAM5 (e.g., colorectal cancer), CD56 (e.g., SCLC), CD25 (e.g., hematological cancers), guanyl cyclase C (e.g., pancreatic cancer), CAG (e.g., solid tumors), LIV-1 (e.g., breast cancer), PTK7 (e.g., lung cancer, colorectal cancer, breast cancer, and ovarian cancer), LAMP-1 (e.g., For example, colorectal cancer, melanoma, laryngeal cancer), P-cadherin 3 (e.g., epithelial tumors), HER-3 (e.g., breast cancer), CD133 (e.g., hepatocellular carcinoma, pancreatic cancer, colorectal cancer, cholangiocarcinoma), GPRC5D (e.g., multiple myeloma), BCMA (e.g., multiple myeloma), CD138 (e.g., multiple myeloma), Ig kappa light chain (e.g., leukemia, lymphoma, NHL, and multiple myeloma), CD30 (e.g., NHL, HD), IL13Ra2 (e.g., glioblastoma), and ligands for NKG2D (e.g., using the NKG2D receptor as a binding domain, such as AML, MDS, and MM).

[0129] Alternatively, the retroviruses described above can be used with host cells that present a receptor or other binding molecule on their surface. The retrovirus (presenting the antigen / ligand) and a population of host cells are combined (e.g., physically combined or contacted). The population of host cells can be sorted based on the presence or absence of a reporter (encoded in the nucleic acid carried by the retrovirus). A subset of the population of host cells that contains the reporter (e.g., expresses the reporter) can be sorted from the remaining subset of the population of host cells that does not contain the reporter. Sorting of the population of cells can be performed using flow cytometry (e.g., fluorescence-activated cell sorting), magnetic enrichment, or antibiotic selection. This FACs or other sorting separates host cells infected by the retrovirus from uninfected host cells (which do not have retroviral nucleic acid).

[0130] Lentiviruses or retroviruses can carry several genetic components that are expressed after infection of host cells: 1) the gene sequence of the antigen / ligand, 2) a prime editing guide RNA (pegRNA) that contains a barcode in the template region, 3) a reporter gene (e.g., a reporter such as GFP) that indicates the presence of viral transduction, and 4) other helper proteins (e.g., MHC). These genetic components are expressed in host cells (e.g., T cells) that have a receptor or binding protein. Retroviruses (e.g., lentiviruses) can display potential antigens / ligands and helper proteins necessary to bind to the host cell's receptor or binding protein. The reporter gene (e.g., GFP) is expressed by host cells infected by retroviruses (e.g., lentiviruses) and allows screening and sorting (e.g., by FACs) of infected host cells from uninfected host cells. Retroviral (e.g., lentiviral) vectors deliver pegRNA that serve two functions: 1) targeting the CRISPR editing complex to a specific insertion site based on sequence homology with a unique site integrated near or adjacent to the nucleic acid encoding the receptor or binding protein, and 2) inserting the retroviral (e.g., lentiviral) nucleic acid (e.g., a barcode sequence) into the specific insertion site by homology-based correction.

[0131] The construct packaged into retrovirus (e.g., lentivirus) may have a constant region and two variable regions. One variable region encodes a barcode that is inserted by the pegRNA scaffold into the target site of the host cell. The second variable region encodes the variable region of an antigen / ligand to interact with a receptor / binding protein, for example, S-ETD-LGD-IRES-R (where S encodes a signal sequence, ETD encodes an extracellular targeting domain; LGD encodes an antigen / ligand, IRES encodes an internal ribosome entry site, and R encodes a reporter (e.g., a fluorescent protein or antibiotic resistance). IRES and M are optional, and the nucleic acid may encode S-ETD-LGD. Nucleotide sequences may be inserted into these two variable regions simultaneously or sequentially by type I restriction digestion-based assembly, Golden Gate assembly, Gateway assembly, or Gibson assembly. When using type I restriction digestion-based assembly, two different digestion enzymes are used for each variable region. When using Golden Gate assembly, four type II restriction digestion sites can be introduced to allow for simultaneous insertion of barcode and antigen nucleotide sequences.

[0132] Fusogens for viral constructs are engineered to allow membrane fusion between cells only upon antigen / ligand-receptor (or binding protein) interaction. One example of such a fusogen is a mutant vesicular stomatitis virus G protein (VSV-G). VSV-G is a viral envelope protein that has been widely used to pseudotype lentiviruses. Examples of mutations are K47Q and R354A, which interfere with its direct recognition and interaction with the host cell's LDLR (Nikolic et al., 2018 CITE, incorporated by reference in its entirety for all purposes). Here, VSV-G can mediate cell entry based on a user-defined ligand-receptor interaction.

[0133] During budding, lentiviruses and / or retroviruses can use the host cell membrane as their own viral membrane, and therefore any protein present in the producer cell membrane can be presented by the viral particle.Producer cells can present full-length antigen / ligand proteins on their cell membranes, as long as the lentivirus and / or retrovirus construct contains the antigen / ligand protein sequence and the antigen / ligand protein is membrane-bound.Many ligands are naturally membrane proteins (e.g., PD-L1 and MHC-antigen complexes).Antigens / ligands that are not naturally membrane-bound can be anchored to the cell membrane by fusing them to a transmembrane domain DNA sequence, such as a viral coat protein. Exemplary transmembrane domains include CD2, CD3d, CD3g, CD3z, CD4, CD8A, CD8B, CD22, CD27, CD28, CD40, CD79a, CD79b, CD80, CD84, CD86, CD137, CD244, CRACC, CRTAM, CLTA-4, MHC-I, MHC-II, platelet-derived growth factor receptor, FCGR1A, FCGR2A, FCG2B, FCGR3A, FCRL1, FCRL2, FCRL3, FCRL4, FCRL5, FC and wild-type or modified transmembrane domains derived from RL6, KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL3, LAG3, GITR, OX40, PD-1, PD-L1, PD-L2, TLR, SLAMF, LILRB1, LILRB2, NKG2A, NKG2C, NKG2D, TIGIT, IgG, IgM, IgA, IgE, IgD, or immunoglobulins.

[0134] MHC-antigen complexes can be presented in retroviruses (e.g., lentiviruses) using single-chain trimers (Mottez et al., Cells expressing a major histocompatibility complex class I molecule with a single covalently bound peptide are highly immunogenic, J. Exp. Med. vol. 181:493-502 (1995), incorporated by reference in its entirety for all purposes). The single-chain trimers can have in tandem a signal peptide, an antigen peptide, a linker, an MHC beta chain, a second linker, and an MHC alpha chain. For example, DNA encoding the human growth hormone signal peptide through beta 2 microglobulin (MHC beta chain) can be synthesized and inserted into a lentivirus construct along with the MHC alpha chain. Co-transfecting the single-chain trimer DNA (part of our lentivirus and retrovirus plasmids) into a packaging cell line (293T) will produce membrane-bound MHC-antigen complexes. When lentiviruses and retroviruses use the packaging cell membrane as their viral membrane, MHC-antigen complexes can be presented on the viral membrane.

[0135] Reporters (e.g., a reporter such as GFP) can be introduced into host cells in two ways to indicate viral entry: 1) lentiviruses or retroviruses carry pre-translated reporter gene products (proteins), and 2) lentiviruses or retroviruses deliver and integrate reporter genes into recipient cell genomes. An exemplary method of carrying reporter gene products by viruses is to fuse GFP with viral structural proteins (e.g., viral envelope proteins or M proteins). Lentiviruses and retroviruses naturally deliver reporter-bearing constructs into host cell genomes.

[0136] After lentivirus or retrovirus is produced by packaging cells, the virus suspension can be mixed with the receptor host library. The lentivirus or retrovirus will infect the receptor host cells if there is a correct antigen / ligand-receptor / binding protein pair between them. The lentivirus or retrovirus will deliver viral RNA and associated proteins (reverse transcriptase and / or reporter protein) to the receptor cells. The viral RNA or viral DNA integrated by reverse transcriptase can produce reporter gene products (e.g., GFP) and pegRNA (barcode sequence).

[0137] The pegRNA can introduce a barcode into a specific site of the receptor host cell genome (e.g., a region near or adjacent to the receptor gene) by interacting with Cas9 or Cas12a or other gene editing enzymes. The CRISPR editing enzyme can be produced by the host cell, lentivirus viral DNA, or introduced by electroporation. The barcode can then be sequenced by the receptor (and / or its barcode) to identify antigen / ligand and receptor / binding protein pairs.

[0138] A pegRNA may have three components: 1) a scaffold region (also known as the constant region), 2) a sequence targeting region, and 3) a barcode region. The sequence targeting region is specific to the insertion site of the host cell. Sequence targeting regions of 1e0 to 1e5 can be used to target multiple regions of the recipient host cell, but each individual pegRNA can only target one region. The barcode region determines the specific sequence that is inserted at the insertion site. Different barcodes of 1e0 to 1e10 can be used. The sequence targeting region and barcode region are close to each other on the viral plasmid, therefore, they can use the same cloning site or separate cloning sites.

[0139] Infected cells can be isolated based on reporter gene product (GFP), for example, using flow cytometry FACS or magnetic bead pulldown. Magnetic beads can be conjugated to proteins or antibodies that can bind to surface receptor proteins (e.g., MYC-tag or FLAG-tag). Cells that bind to magnetic beads are enriched for infected cells.

[0140] These enriched host cells are bulk and can be sequenced high-throughput to identify the receptor / binding protein and antigen / ligand. This sequencing identifies the pegRNA barcode to identify the antigen / ligand that binds to the receptor. Because the pegRNA is inserted near or adjacent to the receptor gene, either conventional sequencing (100-600 nt in length) or long-read sequencing (600-30,000 nt) can be performed to capture the relationship between the antigen / ligand barcode and the receptor / binding protein. The barcode-ligand relationship can be established ahead of time by bulk sequencing of the construct, so that the binding pairing can be computationally inferred.

[0141] The invention disclosed herein will be better understood through the experiments detailed below. However, those skilled in the art will readily recognize that the specific methods and results discussed are merely illustrative of the invention, as more fully described in the claims that follow. Unless otherwise indicated, the disclosure is not limited to specific procedures, materials, etc., which may vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to be limiting.

[0142] Example 1: Creating TCR mimic-like chimeric TCRs or chimeric antigen receptors (CARs) Single chain T cell receptors are made according to Zhang et al., Cancer Gene Therapy 11:487-496 (2004), which is incorporated by reference in its entirety for all purposes. Appropriate cytoplasmic regions are fused to the single chain TCR to make chimeric T cell receptors. The cytoplasmic regions include one or more of the following: Fc cytoplasmic domain, CD3zeta, CD28, B7.1, CD137, CD19 intracellular activation domain, CD64 intracellular activation domain, CD32 intracellular activation domain, CD16 intracellular activation domain, CD23 intracellular activation domain, or Lck activation domain.

[0143] Single chain or natural dual chain TCRs or natural dual chain TCRs are generated from the T cell receptors Flu-TCR1, Flu-TCR2, DMF-5, 1G4, CSS-944-8, CSS-930-1a, CSS-930-1b, PMEL-1, PMEL-2 and PMEL-3. Certain sequences of the alpha and beta TCR chains of each of these TCRs are given in Tables 3 and 4 below:

[0144] [Table 3]

[0145] [Table 4]

[0146] Some of the TCRs and their antigens are described in Spindler et al., Nature Biotechnol. 38:609-619 (2020).

[0147] Multiple chimeric antigen receptors are generated for each single-chain TCR. The TCR CDR3 region and its adjacent regions (approximately 150 nt) serve as barcodes for TCR identification.

[0148] The TCR repertoire may also include TCR sequences identified in human donor tissues (including blood), modified native human TCR sequences, or TCR sequences generated by computer algorithms. To increase the success rate of screening, TCR sequences can be selected using one of the following approaches: selecting TCRs from donors with the desired HLA alleles, selecting donors with the desired condition (e.g., using HLA predicting CMV status or cancer status, or algorithms based on the TCR sequences.

[0149] Example 2: TCR repertoire in THP-1 cells The repertoire of chimeric TCRs prepared in Example 1 is engineered into THP-1 cells. A construct encoding the chimeric TCR is introduced so that each THP-1 cell has one construct and expresses one chimeric TCR. The population of THP-1 cells carrying the repertoire of chimeric TCRs can be used in the subsequent steps.

[0150] Example 3: TCR repertoire in Jurkat cells The repertoire of chimeric TCRs prepared in Example 1 is engineered into Jurkat cells. A construct encoding the chimeric TCR is introduced so that each Jurkat cell has one construct and expresses one chimeric TCR. The population of Jurkat cells carrying the repertoire of chimeric TCRs can be used in the subsequent steps.

[0151] Example 3: Antigen library in K562 cells Antigen libraries are generated from appropriate influenza HLA, human MART-1, pMHC dextramer of epitopes NY-ESO-1 (SLLMWITQC), LMP2 (CLG), A2 / MEL (KTW), gp100, as well as for T cell receptors Flu-TCR1, Flu-TCR2, DMF-5, 1G4, CSS-944-8, CSS-930-1a, CSS-930-1b, PMEL-1, PMEL-2 and PMEL-3. HLA genes and antigen sequences can be fused into a single peptide or coded as separate peptides. HLA alpha and beta chains can be fused or coded as separate peptides. Antigen sequences are 8-25 amino acids long, as HLA can only bind and present fragments of full-length proteins. A computer algorithm selects peptides for use as presentable antigens, and other epitopes have been previously reported in IEDB (iedb.org), for example, Vita et al., The Immune Epitope Database (IEDB): 2018 Revision, which is incorporated by reference in its entirety for all purposes. The antigen barcode of the antigen-presenting cell can be introduced simultaneously or sequentially with the DNA or RNA encoding the antigen. The antigen barcode made of either DNA or RNA can be bound to the cell membrane via DNA or RNA binding proteins, which can be engineered zinc finger nucleases, transcription factor binding proteins, replication protein A, CRSIPR Cas9, Cpf1, Cas13, phage RNA binding proteins, bacterial single-stranded binding proteins, or engineered restriction digestion enzymes.

[0152] For full data generation experiments, a repertoire of antigen sequences (>100) is introduced into K562 cells. Antigen sequences can be peptide sequences identified in in vitro binding experiments, mass spectrometry experiments, sliding windows of protein sequences (8-25 per window), or fragments of known proteins predicted to be presentable by a computer algorithm. The computer algorithm is trained on either the in vitro binding data, mass spectrometry data, or gene expression data to identify sequences likely to be presented by the MHC / HLA complex.

[0153] Example 4: Screening of chimeric T cell receptor repertoires against an antigen library THP-1 cells with a TCR repertoire and K562 cells with an antigen library are also labeled with different fluorescent proteins or live cell dyes restricted to their nuclei, respectively. THP-1 cells with a chimeric TCR repertoire are mixed with K562 cells with an antigen library, and the THP-1 cells can phagocytose the K562 cells that present the MHC:antigen fusion recognized by the chimeric TCR. After incubation, all cells are analyzed by flow cytometry and cell sorting to isolate cells with both fluorescent signals (indicating that THP-1 cells and K562 cells are together). Cells with two different fluorescent signals are THP-1 cells that have phagocytosed K562 cells. This cell population is sequenced by either bulk or single-cell DNA sequencing. Primers are used to amplify the antigen coding region (antigen barcode) and the CDR3 region (TCR barcode). From bulk sequencing, binding pairs of antigen and TCR sequences are identified.

[0154] From single cell sequencing, paired antigen and TCR sequences involved in activation events can be identified. The frequency of antigen or TCR reads can be used to identify pairs most likely to interact.

[0155] Example 5: Screening of T cell receptor repertoires against antigen libraries Jurkat cells carrying the TCR repertoire and K562 cells carrying the antigen library are also labeled with different membrane-bound fluorescent proteins or live cell dyes, respectively. In addition, the TCR repertoire and the antigen library are engineered with appropriate binding motifs for nucleic acid-binding proteins. Jurkat cells and K562 cells are engineered with fusion proteins of nucleic acid-binding proteins and synthetic transmembrane domains so that the nucleic acid-binding domain is present on the cytoplasmic side of the membrane. Jurkat cells carrying the chimeric TCR repertoire are mixed with K562 cells carrying the antigen library, and Jurkat cells can trogocytose membrane pieces from K562 cells that present MHC:antigen fusions recognized by the chimeric TCR (K562 cells can also trogocytose Kurkat cell membrane pieces). After incubation, all cells are analyzed by flow cytometry and cell sorting to isolate cells with both fluorescent signals (indicating that Jurkat cells have trogocytosed K562 cell membranes). The cells with two different fluorescent signals are Jurkat cells activated by K562 cells. The portions of the nucleic acid encoding the antigen and TCR are sequenced by either bulk or single-cell DNA sequencing. Primers are used to amplify the antigen coding region (and / or antigen barcode) and the CDR3 region (and / or TCR barcode). From the bulk sequencing, the binding pairs of antigen and TCR sequences are identified.

[0156] From single cell sequencing, paired antigen and TCR sequences involved in activation events can be identified. The frequency of antigen or TCR reads can be used to identify pairs most likely to interact.

[0157] Example 6: Screening of T cell receptor repertoires against a retroviral antigen library Jurkat cells with the above TCR repertoire are used in this example. These Jurkat cells are engineered to express Cas12a. The lentiviral construct is engineered with pegRNA and antigen-MHC complexes fused with GFP. The pegRNA targets the lentiviral construct to sequence near the TCR gene in the Jurkat cell genome. The pegRNA contains a barcode sequence that can be a random sequence of 15 nt in length (theoretical diversity of 1e9). The pegRNA sequence is expressed by a U6 promoter and terminated by a polyT sequence. The lentiviral construct expresses a library of antigen-MHC complexes that are introduced as single-chain trimers fused with GFP. The lentiviral particles present the antigen-MHC complex library on different viral particles. The expression of the MHC single-chain trimers is controlled by a mammalian gene promoter (e.g., EF1a promoter). The antigen diversity is 1e5 and can be adjusted based on the application. The MHC allele can be any human MHC allele or any human MHC allele with a mutation. Both the barcode and ligand sequence regions contain PaqCI cloning sites (hence four PaqCI cleavage sites with different cleavage site sequences). The barcode and ligand sequences can be introduced into the lentiviral construct library by Golden Gate assembly. After plasmid amplification, 1% of the construct material has the pegRNA-antigen region sequenced to establish the barcode-antigen pairing.

[0158] The lentiviral constructs are packaged by HEK293T packaging cells engineered with packaging helper plasmids (e.g., Gag-pol plasmid and VSV-G plasmid). The VSV-G plasmid contains a mutation in the coding region of VSV-G that precludes direct interaction of VSV-G with the LDLR on T cells, but still allows virus entry when antigen-TCR interaction brings the virus into the vicinity of the cell. The virus suspension is collected 48 and 72 hours after transfection of the packaging cell line. The membrane-bound GFP signal is checked to confirm the MHC single-chain trimer fused with GFP that is normally displayed on the cell and viral surfaces.

[0159] A library of lentiviral particles carrying the MHC-antigen library is used to transfect a population of Jurkat cells carrying the TCR repertoire. The pegRNA of the lentiviral construct targets the construct for insertion into the Jurkat genome adjacent to human TRBC1 (TCR beta constant region 1), TRBC2 (TCR beta constant region 2) and / or TRAC (TCR alpha constant region). The transduced Jurkat cells are identified and sorted by detecting the GFP signal in a flow cytometry FACS device. A live cell dye can be used to remove dead cells to reduce false positive signals of GFP. Jurkat cells sorted by GFP expression are pooled, lysed and DNA is extracted. The DNA is sequenced using amplification primers for the TCR and lentiviral barcodes. The purified PCR products are sequenced in bulk sequencing format by long-read sequencing (e.g., PacBio HiFi long-read sequencing). Pairwise relationships between TCR sequences and antigen barcodes can be established by ranking the frequency of common TCR-antigen pairs compared to a background distribution, which can be established by computational modeling or by sequencing negative control samples.

[0160] Optionally, the target antigen identified by sequencing can be verified as follows: The antigen can be introduced into an MHC allele-matched antigen-presenting cell line (APC, e.g., primary B cells, T2 cells, or K562 cells) by DNA plasmid, mRNA, peptide, or whole protein. A T cell line with a TCR of interest can be mixed with an antigen-presenting APC, and T cell activation markers can be measured by imaging, ELISA, or flow cytometry. T cell activation markers include IL-2, TNF-alpha, IFN-gamma, NFAT-mediated transcriptional activity, AP-1-mediated transcriptional activity, NF-kB-mediated transcriptional activity, CD69, CD107a, CD137 (4-1BB), HLA-DR, CD38, trogocytosis, PD-1, CD25, CD71, and morphological changes. The correct target antigen of the TCR activates the T cell as measured by one or more of the above markers after co-incubation of the T cell and the APC.

[0161] All publications and patents cited in this specification are incorporated by reference herein to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference, and are incorporated by reference herein to disclose and describe the methods and / or materials for which the publications are cited.

[0162] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the following claims.

Claims

1. A method for obtaining a T cell receptor or a T cell receptor mimic, comprising mixing a plurality of engineered immune cells containing a first plurality of nucleic acids encoding a repertoire of chimeric T cell receptors or T cell receptor mimics with a mutant viral envelope protein containing at least one mutation that reduces its natural function, and a plurality of nucleic acids encoding a ligand library, and a plurality of lentiviral particles containing the PegRNA, the PegRNA containing a barcode and a second nucleic acid sequence that interacts with the CRISPR editing system and directs the insertion of the PegRNA at a site within the genome of the immune cell, wherein a ligand from the ligand library is present on the surface of each lentiviral particle; transducing the engineered immune cells with the lentiviral particles, wherein the ligand of the lentiviral particles binds to the T cell receptor or the T cell receptor mimic of the immune cells; expressing a nucleic acid encoding a reporter in the engineered immune cells; isolating the immune cells expressing the reporter; and sequencing the nucleic acid encoding the T cell receptor or the T cell receptor mimic and the barcode.

2. The method according to claim 1, wherein the ligand is an antigen.

3. The method according to claim 2, wherein the antigen is fused to an MHC molecule or presented by an MHC molecule.

4. The method according to any one of claims 1 to 3, wherein the immune cells are T cells, natural killer cells, neutrophils, macrophages or monocytes.

5. The method according to any one of claims 1 to 3, wherein the TCR mimic comprises a single-chain antigen interaction receptor.

6. The method according to claim 5, wherein the TCR mimetic receptor further comprises a co-activation domain, and the co-activation domain is selected from the group consisting of CD3ζ, CD28, B7.1, CD137, CD19 intracellular activation domain, CD64 intracellular activation domain, CD32 intracellular activation domain, CD16 intracellular activation domain, CD23 intracellular activation domain, or Lck activation domain. **Claim 7** The method according to any one of claims 1 to 3, wherein the repertoire of the T cell receptor or TCR mimetic is derived from genomic DNA of a plurality of T cells from a subject or a plurality of cDNAs obtained from a plurality of T cells from the subject. **Claim 8** The method according to any one of claims 1 to 3, wherein the repertoire of the T cell receptor or TCR mimetic is obtained from a subject naive to an antigen library or a subject that has initiated an immune response to one or more antigens of the antigen library. **Claim 9** A method for obtaining a T cell receptor or TCR mimetic, comprising mixing a plurality of macrophage cells comprising a first plurality of nucleic acids encoding a repertoire of chimeric T cell receptors or TCR mimetics with a plurality of antigen-presenting cells comprising an optical reporter and a second plurality of nucleic acids encoding an antigen library, wherein the antigens of the antigen library are fusions of peptides from the antigen and MHC molecules; expressing the first plurality of nucleic acids encoding the repertoire of chimeric T cell receptors in the plurality of macrophage cells; expressing the second plurality of nucleic acids encoding the antigen library in the plurality of antigen-presenting cells; isolating macrophage cells that have phagocytosed the antigen-presenting cells; and sequencing the first plurality of nucleic acids and the second plurality of nucleic acids. **Claim 10** A method for obtaining a T cell receptor or a T cell receptor mimic, comprising mixing a plurality of engineered immune cells comprising a first plurality of nucleic acids encoding a repertoire of chimeric T cell receptors or T cell receptor mimics with a plurality of antigen-presenting cells comprising a membrane conjugated to an optical reporter and a second plurality of nucleic acids encoding an antigen library, wherein the antigens of the antigen library are fused or presented by MHC molecules, the membrane of the engineered immune cells is associated with a set of nucleic acid barcodes, and the membrane of the antigen-presenting cells is associated with a second set of nucleic acid barcodes; expressing the first plurality of nucleic acids encoding the repertoire of chimeric T cell receptors in the plurality of T cells; expressing the second plurality of nucleic acids encoding the antigen library in the plurality of antigen-presenting cells; isolating the antigen-presenting cells whose membranes have been trogocytosed by the immune cells or isolating the immune cells whose membranes have been trogocytosed by the antigen-presenting cells; and sequencing the first barcode and the second barcode from the isolated immune cells or the isolated antigen-presenting cells.

11. The method according to claim 9 or 10, wherein the first plurality of nucleic acids further comprises a plurality of different nucleic acid barcodes, and each T cell receptor or T cell receptor mimic is associated with a different barcode.

12. The method according to claim 9 or 10, wherein the second plurality of nucleic acids further comprises a plurality of different nucleic acid barcodes, and each antigen is associated with a different barcode.

13. The method according to claim 9 or 10, wherein sequencing the first plurality of nucleic acids sequences the barcode.

14. The method according to claim 11, wherein sequencing the second plurality of nucleic acids sequences the barcode.

15. The method according to claim 9 or 10, wherein the repertoire of T cell receptors is derived from genomic DNA of a plurality of T cells from a subject or a plurality of cDNAs obtained from a plurality of T cells from a subject. **Claim 16** The method according to claim 9 or 10, wherein the antigen library is a plurality of cancer-related antigens. **Claim 17** The method according to claim 16, wherein the cancer-related antigen is a KRAS variant allele. **Claim 18** The method according to claim 17, wherein the KRAS variant allele is G12D, G12V, G12R, G12H, G12S, G12L, Q61H, Q61K, Q61R, A11T, G13P, G13D, or the double mutation G12D and Q61H. **Claim 19** The plurality of cancer-related antigens are mesothelin, disialoganglioside (GD2), Her-2, MUC1, GPC3, EGFRVIII, CEA, CD19, EGFR, PSMA, GPC2, folate receptor β, IgG Fc receptor, PSCA, PD-L1, EpCAM, Lewis Y antigen, L1CAM, FOLR, CD30, CD20, EphA2, PD-1, c-Met, ROR1, CLDN18.2, NKG2D, CD133, TSHR, CD70, ERBB, AXL, cell death receptor 5, VEGFR-2, CD123, CD80, CD86, TSHR, ROR2, CD147, kappa IgG, IL-13, MUC16, IL-13R, NY-ESO-1, IL13RA2, DLL3, FAP, LMP1, TSHR, BCMA, nectin-4, MG7, AFP (alpha-fetoprotein), GP100, B7-H3, nectin-4, MAGE-A1, MAGE-A4, MART-1, HBV, MAGE-A3, TAA, GP100, thyroglobulin, EBV, HPV E6, PRAME, HERV-E, WT1, GRAS G12V, p53, TRAIL, MAGE-A10, HPV-E7, KRAS G12D, MAGE-A6, CD19, BCMA, CD22, CD123, CD20, CD30, CD33, CD138, CD38, CD7, SLAMF7, IgG FC, MUC1, LewisY antigen, CD133, ROR1, FLT3, NKG2D, kappa light chain, CD34, CLL-1, TSLP, CD10, PD-L1, CD44V6, EBV, CD5, GPC3, CD56, integrin B7, CD70, MUC-L, C-KIT, CLDN18.2, TRBC1, TAC1, CD56, CD4, CD2, CD18, CD27, CD37, CD72, CD79A, CD79B, CD83, CD117, CD172, ERBB3, ERBB4, DR5, HER2, CS1, IL-1RAP, ITGB7, SLC2A14, SLC4A1, SLC6A11, SLC7A3, SLC13A5, SLC19A1, SLC22A12, SLC34A1, slc45A3, SLC46A2, Fra, IL-13Ra2, ULBP3, ULBP1, CLD18, NANOG, CEACAM8, TSPAN16, GLRB, DYRK4, SV2C, SIGLEC8, RBMXL3, HIST1HIT, CCR8, CCNB3, ALPPL2, ZP2, OTUB2, LILRA4, GRM2, PGG1, NBIF3, GYPA, ALPP, SPATA19, FCRLI, FCRLA, CACNG3, UPK3B, 12UMO4, MUC12, HEPACAM, BPI, ATP6V0A4, HMMR, UPK1A, ADGRV1, HERC5, C3AR1, FASLG, NGB, CELSR3, CD3G, CEACAM3, TNFRSFBc, MS4AB, S1PR5, EDNRB, SCN3A, ABCC8, ABCB1, ANO1, KCND2, HTR4, CACNB4, HTR4, CNR2, 26LRB, EXOC1, ENTPP1, ICAM3, ABCGB, SCN4B, SPN, CD68, ITGAL, ITGAM, SCTR, CYYR1, CLCN2, SLARA3, or JAG3, the method according to claim 16.

20. The method according to claim 16, wherein the cancer-related antigen is a tumor neoantigen.

21. The method according to claim 9 or 10, wherein the antigen library is a plurality of infectious disease-related antigens.

22. The method according to claim 21, wherein the infectious disease is caused by a bacterial pathogen.

23. The method according to claim 21, wherein the infectious disease is caused by a viral pathogen.

24. The method according to claim 9 or 10, wherein the immune cell is a T cell, neutrophil, macrophage or monocyte.

25. The method according to claim 9 or 10, wherein the TCR mimic comprises a single-chain antigen interaction receptor.