Screening methods

Abstract

The invention relates to screening methods. In particular, it relates to a method of identifying a functional TCR from a library of particles, which displays a plurality of different T cell receptors (TCRs). The method may comprise a) exposing the library of particles to a target antigen to identify TCRs that bind to the target antigen, b) transfecting a plurality of T cells with nucleic acid encoding TCRs identified in step (a), c) exposing the plurality of transfected cells to the target antigen, and d) selecting cells having TCR activity in the presence of the target antigen. Transfection of the T cells is such that each T cell comprises a nucleic acid encoding a single TCR from the library of particles, the nucleic acid is integrated into the genome of the T cell at a single identical pre-defined locus, and the TCR is in a single chain format and comprises an alpha chain variable domain, a beta cain variable domain and a constant domain. The T cells do not express endogenous TCR. A T-cell comprising a recombinase mediated landing pad, wherein the T cell constitutively expresses TCR constant domain and does not express endogenous TCR, is also claimed.

Description

[0001] SCREENING METHODS

[0002] Field of the invention

[0003] The invention relates to screening methods. In particular, it relates to methods of identifying functional TCRs from particle libraries.

[0004] Background of the invention

[0005] The selection of functional antigen binding molecules is critical for the development of therapeutic TCRs and antibodies. Molecule selection for discovery workflows typically relies on library-based methods, such as phage or yeast display, to screen large pools of variants (upwards of 108) for the best starting (or native) sequence for subsequent engineering. Conventional methods for screening large libraries rely on binding-based selection, wherein molecules are selected based on binding in solution to a target antigen. However, for molecules designed to target a peptide-MHC antigen, such as TCRs, emerging data is revealing that strong antigen binding in solution frequently does not correlate with the ability of the TCR to generate a T cell signalling response against antigen presenting cells (APCs) when the TCR is expressed on the cell surface, or when incorporated into a soluble bispecific molecule such as an ImmTAC®, or as a soluble TCR. Functional screening of TCRs in a cellular context (for example by viral transduction into primary T cells) may be used to select TCRs able to generate a signalling response. However, this is a low throughput approach and not amenable to screening of high diversity libraries. WO2021074249 discloses a method of identifying functional TCRs that uses a modified T cell line, but this method remains unsuitable for high-throughput screening of large, high diversity libraries. The methods disclosed in WO2021074249 are labour intensive and only suitable for small number of candidate sequences.

[0006] Summary of the invention

[0007] In one aspect of the invention, there is provided a method of identifying a functional T cell receptor (TCR) from a library of particles, the library displaying a plurality of different TCRs, the method comprising: a) exposing the library of particles to a target antigen to identify TCRs that bind to the target antigen, b) transfecting a plurality of T cells with nucleic acid encoding TCRs identified in step (a), such that each T cell comprises a nucleic acid encoding a single TCR from the library of particles, wherein the T cells do not express endogenous TCR, the nucleic acid is integrated into the genome of the T cell at a single identical pre-defined locus, and the TCR is in a single chain format and comprises an alpha chain variable domain, a beta cain variable domain and a constant domain, c) exposing the plurality of transfected cells to the target antigen, and d) selecting cells having TCR activity in the presence of the target antigen.

[0008] In a further aspect of the invention, there is provided a T cell comprising a recombinase mediated landing pad, wherein the T cell constitutively expresses exogenous TCR constant domain and does not express endogenous TCR.

[0009] In a further aspect of the invention, there is provided use of the cells described herein in the methods described herein.

[0010] In a further aspect of the invention, there is provided a mammalian display library comprising a plurality of T cells in accordance with the invention, wherein each of the plurality of cells has been transfected with nucleic acid encoding a single TCR that binds a target antigen, wherein the nucleic acid is integrated into the genome of the T cell at a single identical pre-defined locus, and wherein the TCR is in a single chain format and comprises an alpha chain variable domain, a beta chain variable domain and a constant domain.

[0011] In a further aspect of the invention, there is provided TCRs obtained or obtainable from the methods described herein.

[0012] The inventors have developed a novel high-throughput library-based approach to stably express single chain TCRs on the T cell surface and capture, with high fidelity, the high diversity output from a first selection step (of the order of 104TCRs), thereby capturing both strongly and more weakly binding TCRs for subsequent functional screening. The invention may rely on a modified TCR-null T cell line which incorporates a recombinase dependent landing pad to mediate single copy integration of the TCRs maintaining a tight phenotype- to-genotype linkage and ensuring transcriptional normalisation. Activated T cells may be sorted and deep sequenced to identify the most abundant, or enriched, clones. The invention provides for the first time a reliable and efficient discovery workflow for the selection of functional antigen binding TCRs that are suitable for development as therapeutics for the treatment of cancer, infectious disease and autoimmune disease.

[0013] An advantage of the present invention is that, in a typical discovery workflow, antigen binding TCRs are identified from large libraries displayed on phage, yeast or mammalian cells (such as CHO cells) using multiple rounds of selection; selected TCRs are subsequently cloned into a suitable T cell line and then are assessed for their ability to induce T cell signalling in the presence of antigen presenting cells (APCs). While this approach has been successful, it has two key drawbacks. Firstly, a cumbersome reformatting and cloning step is required to convert TCRs from the single chain format employed for phage and yeast display, to a full-length format capable of forming a TCR signalling complex on the T cell surface. Since this is a relatively low throughput approach, it means that in practice only a small proportion of the original library diversity can ever be tested for functional binding (typically tens up to several hundred). Secondly, binding-based selections are dominated by the strongest binders, which leads to a high attrition rate since a significant proportion of strong binders are not capable of T cell activation. Furthermore, functional TCRs with weaker binding may never make it through the selection process. The inventors of the present application have frequently found that TCRs which bind but are not functional are unable to be rescued, i.e. made functional, even when subjected to further engineering. These drawbacks lead to delays and inefficiencies in therapeutic development and can ultimately result in program failure. Therefore, there is a need for a high throughput approach for functional screening of TCRs, which can accommodate a high diversity input and be seamlessly integrated into a standard binding-based selection workflow.

[0014] The present invention provides a platform for identifying TCRs based on affinity and function, that relies on the initial selection of a high diversity pool of both strongly and weakly antigen binding TCRs followed by a second selection step based on antigen-induced TCR signalling. In addition, the invention uses a single chain TCR format, which allows for a seamless transition between existing display methods, to take advantage of both affinity and functional based selection.

[0015] An advantage of the method according to the invention compared to previous methods for the discovery of native T cell receptors is that the TCRs are in a single chain format. This allows for the direct transfer of the TCR sequences from a phage display system to a mammalian (T cell) display system. This in turn allows for high-throughput identification of TCRs that bind to specific target antigens and have the ability to activate T cells in vitro. In order to achieve this, the nucleic acids encoding the single chain TCRs are integrated into the T cell genome at a single identical pre-defined genomic locus. Due to the targeted single-copy integration of the nucleic acids, the integrated nucleic acids may be under the same promoter post integration. As a result, the transfected cells can all have normalised expression of the TCR at the transcriptional level, limiting variation at the protein level, and inaccurate results due to avidity issues. The method of the invention thereby allows for identification of antigen binding TCRs that activate T cells in vitro, in a high-throughput manner.

[0016] Detailed Description

[0017] As used herein, a “functional TCR” is a TCR that is capable of inducing T cell activation in vitro. A functional TCR is capable of recognizing antigens with high specificity and initiating appropriate immune responses, such as the activation of cytotoxic T cells, or helper T cells.

[0018] One method of determining whether a TCR is capable of inducing T cell activation in vitro is set out in Example 1 herein. TCR activity may be measured by any suitable means. TCR activity may be determined by expression of T cell activation markers. The activation markers may be CD69 and / or CD25. Activation may be measured by flow cytometry, cytokine release or gene expression levels. Gene expression may be measured by any suitable means, such as by polymerase chain reaction. In some embodiments, TCR activity may be determined by a reporter system. The reporter may be NFAT luciferase or a fluorescent protein. The reporter system may comprise a polynucleotide comprising a promoter region operably linked to a polynucleotide sequence encoding a detectable marker. The reporter system may provide an indication of one or more activities of the cell selected from the group consisting of nuclear factor of activated T-cells (NFAT) signalling, activator protein-1 (AP-1) signalling, NFKB / Rel signalling, and calcium flux.

[0019] The NFAT-inducible promoter may be any transcriptional promoter comprising an NFAT response element, i.e. a promoter that is active in the presence of dephosphorylated NFAT and essentially inactive in the absence of dephosphorylated NFAT. The promoter may be any promoter functional in mammalian cells, comprising one or several copies of an NFAT binding site.

[0020] Library of particles

[0021] The library of particles used in the methods described herein displays a plurality of different T cell receptors (TCRs). The plurality of TCRs may consist essentially of TCRs comprising an alpha chain comprising an alpha chain variable domain from a natural repertoire and a beta chain comprising a beta chain variable domain from a natural repertoire. The plurality of TCRs may comprise TCRs comprising a gamma chain comprising a gamma chain variable domain from a natural repertoire and a delta chain comprising a delta chain variable domain from a natural repertoire. By "from a natural repertoire" it is meant that the TCR alpha and beta chain variable domains (or gamma and delta chain variable domains) are expressed from DNA sequences that have been obtained from human donors. In other words, the diversity of the variable domains of the TCRs of the library has been naturally generated during T cell development in vivo. Furthermore, this means that the sequences of all the alpha and beta chains (or gamma and delta chains) in the library will have been selected for during thymic selection. The random combination of these alpha and beta chains (or gamma and delta chains), which occurs during library creation, may result in an alternative repertoire of alpha beta chain (or gamma delta chain) combinations compared to that originally present in vivo (i.e. , in the donor(s)). The DNA sequences may be obtained indirectly e.g., by producing cDNA from donor mRNA. The cDNA sequences may then be used as templates to produce DNA sequences from which the plurality of different TCRs is produced.

[0022] The particles forming the library may be phage particles, ribosomes, yeast cells or mammalian cells. In some embodiments, the particles are phage particles. Phage display of TCRs is described in WO 2004 / 044004. Alternatively, the library may be a yeast display library, so the particles may be yeast cells. Alternatively, the library of particles may be a library of ribosomes. Ribosome display is known in the art. The particles may be complete ribosomal complexes or parts thereof. Any library of particles that is able to display single chain TCRs is encompassed. Alternatively, the library of particles may be a library of mammalian cells. The cells may be CHO cells.

[0023] The library of particles preferably comprises at least 1 x 108particles that display an op (or yb, gamma delta) TCR chain combination. TCR phage libraries can be used to isolate TCRs with novel antigen specificity in a discovery workflow. Such libraries are typically constructed with alpha and beta chain sequences corresponding to those found in a natural repertoire. However, the random combination of these alpha and beta chain sequences, which occurs during library creation, produces a repertoire of TCRs not present in nature (as described in WO2015 / 136072, WO 2017 / 046198, WO 2017 / 046201 , WO 2017 / 046202, WO 2017 / 046205, WO 2017 / 046207, WO 2017 / 046208, WO 2017 / 046211A1 and WO 2017 / 046212). The methods described herein may utilise the pooled output from several phage libraries.

[0024] The library of particles used herein may be non-naturally occurring as it may include TCR(s) that are not naturally occurring or those that would be considered “isolated” as that term is used herein. Accordingly, TCRs identified by the methods of the invention may likewise be patent-eligible subject matter as such TCRs are not naturally occurring or those that would be considered “isolated” as that term is used herein. Similarly, cells and particles described herein are patent-eligible subject matter because by displaying on its surface or expressing a TCR of the invention, the cell or particle is not naturally occurring or that which would be considered “isolated” as that term is used herein. Non-natural mutations may be introduced by any way known in the art. Non-natural mutations may be randomly generated, or specifically defined, or both. For example, randomly generated mutations may be incorporated at defined positions using site-saturation mutagenesis in which the native amino acid coding sequence is replaced by the coding sequence of all other naturally occurring amino acids; thereby, creating additional library diversity at a defined position. The method may involve replicating the DNA of interest using PCR amplification with degenerate synthetic oligonucleotides as primers. Alternatively, or additionally, defined mutations, including insertions and deletions, may be introduce at certain positions using, for example, commercially available kits, such as the Quik Change Site Directed Mutagenesis Kit from Stratagene. Preferably, non-natural mutations are incorporated into the CDR regions.

[0025] The library may display TCRs where 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the alpha chain variable domains or beta chain variable domains comprise a non-natural mutation.

[0026] The plurality of transfected T cells of the invention may be referred to herein as a “mammalian display library”. The mammalian display library preferably comprises T cells expressing exogenous TCRs, which were first displayed on particles in the library of particles.

[0027] Target antigen

[0028] TCRs specifically recognise peptide fragments that are derived from intracellular proteins and presented in complex with Major Histocompatibility Complex (MHC) molecules on the surface of antigen presenting cells (APCs). In humans, MHC molecules are known as human leukocyte antigens (HLA), and both terms are used synonymously herein. MHC molecules have a binding groove in which the peptide fragments bind.

[0029] As used herein, “exposing” to a target antigen means contacting the TCR with the target antigen or screening the library of particles, or mammalian display library with the target antigen. The target antigen may be used to screen the library of particles for a TCR to which it binds. As used herein, the term “binds” refers to both strong and weak binding, or refers to a measurable interaction between the target antigen and a TCR. In some embodiments, a binding affinity of 200 pM or stronger may be considered as binding. In some embodiments, the binding affinity between the target antigen and TCR in the library of particles is at least 200pM, at least 300pM, at least 400pM or at least 500pM. Binding may be measured by ELISA or SPR, typically binding is measured at room temperature for ELISA and 25°C for SPR.

[0030] The target antigen may also be used to screen the mammalian display library for a T cell expressing TCR which is activated in vitro in the presence of the target antigen.

[0031] The screening of the library of particles may include one or more steps as set out below: i) panning the library of particles using as a target the peptide antigen ii) repeating step i) one or more times iii) screening the particle clones identified in step i) or ii) iv) identifying a TCR that specifically binds the peptide antigen.

[0032] In accordance with step (ii), step (i) may be repeated once, twice, 3 times, 4 times, 5 times, or 6 times. It may be repeated up to 10 times. Preferably step (i) may be repeated once.

[0033] In some embodiments, the plurality of transfected cells (also called the mammalian display library) may be exposed to target antigen more than once. In the methods of the invention, the step of “exposing the plurality of T cells” may be repeated once, twice, 3 times, 4 times, 5 times, or 6 times. It may be repeated up to 10 times. The cells may be sorted or enriched for cells harboring TCRs that result in T cell activation in vitro after each exposure to target antigen.

[0034] By “panning”, it is meant that the particle clones are allowed to contact the target antigen and the bound particle clones may be separated from the non-bound particle clones. The terms “panning” and “selection” may be used herein synonymously. Target antigens may be provided in soluble form, or may be immobilised by attachment to a suitable solid support. Examples of solid supports include, but are not limited to, a bead, a membrane, sepharose, a magnetic bead, a plate, a tube, a column. Target antigens may be attached to an ELISA plate, a magnetic bead, or a surface plasmon resonance biosensor chip. Methods of attaching target antigen complexes to a solid support are known to the skilled person, and include, for example, using an affinity binding pair, e.g., biotin and streptavidin, or antibodies and antigens. An alternative method may include panning on intact cells. (The particles that do not bind (i.e., particles that do not display a TCR that binds to the target antigen) may be washed away. The bound particle clones may then be eluted by; enzymatic cleavage of a protease site, such as trypsin, between the TCR beta chain and gene III; extremes of pH; or competition with excess antigen. These particles clones may be taken through further rounds of panning, or on to screening experiments to identify clones with optimal binding characteristics. In the methods of the invention, the TCRs expressed on the bound particles may then be transfected into the plurality of T cells.

[0035] The method of identifying functional TCRs may further comprise a step of enriching for TCRs that bind target antigen, prior to transfecting the plurality of T cells. The enrichment may occur by separating the bound particles from the non-bound particles and / or panning the library multiple times, for example twice. In some embodiments, the library of particles is a phage display library, and the enrichment step comprises separating the bound phage clones from the non-bound phage clones.

[0036] The target antigen may be a peptide-MHC (pMHC) antigen. The target antigen may be a pH LA antigen. The target antigen may activate the TCR. The target antigen may be any known pMHC antigen. Novel peptide antigens may also be used, in order to identify specifically binding TCRs that may prove useful in therapeutic areas. In some embodiments, the target antigen may bind to an MHC molecule that is expressed on the surface of an antigen presenting cell (APC).

[0037] The target antigen may comprise a peptide derived from a tumour associated antigen (TAA). The target antigen may comprise a peptide derived from a viral antigen, or another disease associated antigen. The target antigen may comprise a peptide derived from an autoantigen. Examples of these antigens are known in the art. For example, known TAAs include cancer testis antigens, such as PRAME (Preferentially expressed Antigen in Melanoma), MAGEA (Melanoma-associated antigen) 4 (MAGEA4), MAGEA3, MAGEA1 , and other MAGE family antigens, and PIWIL1 (also known as piwi-like protein 1 or HIWI); overexpressed antigens include gp1OO (glycoprotein 100). Further examples of TAAs are provided in US20230346885. Known viral antigens may be from HIV (human immunodeficiency virus), HBV (Hepatitis B virus) or EBV (Epstein-Barr virus). Known antigens with a role in autoimmune diseases include PPI (pre-pro-insulin).

[0038] The MHC molecule may be MHC class I. The MHC class I molecule may be selected from HLA-A*02, HLA-A*01, HLA-A*03, HLA-A11 , HLA-A23, HLA-A24, HLA-B*07, HLA-B*08, HLA-B40, HLA-B44, HLA-B15, HLA-C*04, HU\*C*03 HLA-C*07. The MHC class I molecule may be HLA-A*02 or HLA-A24

[0039] Transfection As used herein “transfecting” means introducing (or integrating) nucleic acid into the genome of a target cell, such that the protein encoded by the nucleic acid is expressed. In the methods described herein, each of the plurality of cells is transfected with a nucleic acid encoding a single TCR, such that the TCR gene is integrated at single-copy genome locus. Thus, all TCRs identified as binding to the target antigen are transfected into the plurality of T cells, with each T cell expressing a single TCR.

[0040] Each nucleic acid may be stably integrated into the genome by any known method. Preferably the nucleic acid is stably integrated via site-specific integration. The integration may be a recombinase dependent integration, the recombinase dependent integration may be via a recombinase mediated landing pad, preferably a Bxb1 mediated landing pad. The nucleic acids encoding the exogenous TCRs may be integrated into the genome at any genomic location, provided the exogenous TCR is stably integrated and present at a single copy in the genome. Other method of transfecting the TCRs include CRISPR / cas9, or lentiviral transduction.

[0041] A "recombinase-mediated landing pad" is a genetic element engineered for precise genome editing. The term "landing pad" refers to a specific region within a genome where genetic material can be inserted with high precision. Recombinase enzymes, such as Bxb1 , Cre or Flp, are often used to facilitate the targeted integration or excision of DNA sequences at these landing pads. Alternative integrases / recombinases include serine integrases such as <t>C31 , <t>BT1 , <t>C1, 370, <t>K38, R4, TP901-1, W^ , RV, SPBc ,TG1 , MR11.A118, BL3 and tyrosine recombinases such as Cre, Flp and Dre recombinase

[0042] Use of a recombinase mediated landing pad allows for site specific integration of exogenous nucleic acids via recombinase-mediated cassette exchange (RCME). The nucleic acids may be inserted into the genome at a pre-defined locus, under the control of a promoter. In the methods of the invention, the nucleic acids encoding the single chain TCRs may be introduced as a cassette targeting vector (CTV), into a single copy, predefined genome locus flanked with recombinase integration sites. Each of the transfected plurality of T cells of the invention may therefore express the TCRs from the same genomic location, under the control of the same promoter. The plurality of transfected T cells of the invention may therefore express the same, or essentially the same, levels of TCRs at the transcriptional level. The plurality of transfected T cells of the invention may express similar levels of the exogenous TCR at RNA level. Expression of the TCRs may be normalised at RNA level. However, protein levels of the exogenous TCRs may vary. Bxb1 is a bacteriophage integrase site, which is a sequence recognized by the integrase enzyme derived from the bacteriophage. This integrase enzyme catalyses the insertion of DNA sequences into specific genomic locations. Herein, the Bxb1 landing pad serves as a target site for the integrase-mediated insertion of nucleic acids encoding a single chain TCR. The recombinase mediated landing pad may be introduced into the cell via any appropriate means. The skilled person is aware of appropriate methods. For example, the recombinase mediated landing pad may be introduced via lentiviral transduction. The landing pad may further comprise a fluorescent marker.

[0043] The method of the present invention relies on targeted genomic integration of single chain TCR sequences. This results in homogenous TCR expression from a single locus.

[0044] The plurality of T cells transfected in the methods described herein do not express endogenous TCRs. The T cells may have been modified or engineered to remove endogenous TCR expression. The removal of endogenous TCR expression can be achieved by any suitable means, for example, CRISPR, RNAi, antisense oligonucleotides (ASOs) or mutagenesis. In some embodiments, the endogenous expression of TCRs is removed by CRISPR-Cas9 methods. In some embodiments, the alpha chain constant domain and / or the beta chain constant domain are knocked out, thereby removing TCR expression.

[0045] As the T cells of the invention do not express endogenous TCRs, the surface expression of CD3 is reduced. Antibodies that are directed towards CD3 or extracellular parts of the TCR allow for discrimination of T cells that successfully integrate the single chain TCR from cells that either did not integrate the single chain TCR, or where integration resulted in an unproductive DNA sequence. Thus, the cells according to the invention have the advantage that CD3 is only expressed on the cell surface if the cells successfully integrate nucleic acid encoding an entire TCR.

[0046] The plurality of T cells used in the methods described herein constitutively express constant domain. Preferably the T cells constitutively express an exogenous full length TCR constant domain. The T cells may constitutively express exogenous alpha constant chain (Ca) or a beta constant chain (Cb). The constant domain may be human or murine. The constant domains are preferably murine. When the single chain format is Va-Vb-Cb (where Va is alpha chain variable domain and Vb is beta chain variable domain), the T cell may express a Ca. When the single chain format is Vb-Va-Ca, the T cell may express a Cb. The exogenous constant domain may be introduced into the genome by lentiviral transduction or any other suitable means.

[0047] The plurality of cells may be derived from a T cell that has been obtained from any organism. However, it is preferred that the cells of the present invention are derived from a human or murine T cell. The plurality of T cells may be derived from primary T cells. In a preferred embodiment, the T cells may be derived from an immortal T cell line. The person skilled in the art would understand which T cells would be appropriate for use in the methods described herein. The T cells may be mammalian, such as human or murine. In a preferred embodiment, the T cells are human.

[0048] The T cells may be derived from immortalised cell lines such as Jurkat cells, MOLT-3 cells, CEM cells, SLIP-T1 cells, T-AII-104 and MT-2 cells, another immortal T cell line includes SKW3 which was derived from a leukaemia patient. In a preferred embodiment, the T cells may be derived from a Jurkat cell. The Jurkat cell line is an immortalized T lymphocyte cell line that was originally obtained from the peripheral blood of a boy with T cell leukemia. The Jurkat cell line has most often been used as a prototypical T cell line to study multiple events in T cell biology, including T cell signalling and molecular events in the HIV infection life cycle. The Jurkat line may be derived from Jurkat E6.1. Other Jurkat derivates that may be used include J.RT3-T3.5-TIB-153 (known as J.RT3) and Jurkat 76.

[0049] The plurality of T cells that are transfected do not express endogenous TCRs but do constitutively express constant domain. The constant domain may be endogenous or exogenous. In a preferred embodiment, the cells constitutively express exogenous constant domain. Preferably the plurality of T cells include a recombinase mediated landing pad.

[0050] There are a number of known methods suitable for the transfection of T cells with DNA or RNA encoding the TCRs obtained from step (a) that bind the target antigen. The single chain TCRs may be transfected using any suitable means, such as chemical, physical, and viral-mediated transfection. In some embodiments, the TCR is transfected into plurality of T cells using electroporation, liposomes, microinjection, retroviruses, lentiviruses, adenoviruses, or adeno-associated viruses (AAVs). In one embodiment, the cells are transfected using electroporation. The single chain TCRs may be co-transfected with a Bxb1 recombinase expressing plasmid. The method may include selecting cells that have been successfully transfected. For example, the selection may be via antibiotic resistance, or via a fluorescent marker.

[0051] Transfecting the plurality of T cells with nucleic acid encoding TCRs identified in step (a) may be understood as the generation of a mammalian display library. In some embodiments, the mammalian display library comprises approximately 103cells, each expressing a specific TCR. In some embodiments, the mammalian display library comprises approximately 104, or 105variants.

[0052] The TCR sequence is a single chain format comprising an alpha chain variable domain sequence (Va), a beta chain variable domain sequence (VP) and an alpha constant chain (Ca) or a beta constant chain (CP) sequence. The single chain TCR format may comprise Va-Vb-Cb or Vb-Va-Ca. A “single chain TCR” is a TCR encoded by nucleic acids that encode each of the alpha and beta chain variable domains cloned into the same vector. The library of particles and the transfected T cells of the invention may both comprise single chain TCRs.

[0053] Single chain TCR

[0054] The TCR sequence is in a single chain format comprising an alpha chain variable domain sequence (Va), a beta chain variable domain sequence (Vb) and an alpha constant chain (Ca) or a beta constant chain (Cb) sequence. The single chain TCR format may comprise Va-Vb-Cb or Vb-Va-Ca. A “single chain TCR” may be a TCR encoded by nucleic acids that encode each of the alpha and beta chain variable domains cloned into the same vector. The library of particles and the transfected T cells of the invention may both comprise single chain TCRs.

[0055] Crucially for the invention, a single chain TCR is used, and integrated into a specific predefined genome location. This allows for direct transfer of the output of a library of particles to a mammalian library, without severance of the link between alpha and beta variable domain sequences, thereby allowing for affinity and function of the TCRs to be measured and selected for in a high-throughput method.

[0056] To transfer the TCRs from the library of particles to the T cells of the invention a cassette targeting vector may be used, such as shown in Figure 3.

[0057] T cells In a further aspect of the invention, there is provided a T cell comprising a recombinase mediated landing pad, wherein the T cell constitutively expresses TCR constant domain and does not express endogenous TCR. The recombinase mediated landing pad may be a Bxb1 landing pad. The constitutive expression of TCR constant domain be exogenous or endogenous constant domain. In a preferred embodiment, the constitutively express constant domain is exogenous.

[0058] In the cell of the present invention, the TCR expression may have been modified such that surface expression of the TCR-CD3 complex is disrupted. The cell does not express endogenous TCRs, thereby removing interference from endogenous TCR expression and reducing TCR chain mispairing in the methods of the invention. The cells of the invention do not express endogenous functional TCRs. To express a functional TCR in the cells, it is required to introduce nucleic acid encoding a TCR alpha variable domain, a TCR beta variable domain and a TCR alpha constant domain or a TCR beta constant domain into the genome of the cells.

[0059] The T cell may be derived from an immortalised T cell line. The immortalised T cell line may be any T cell line, such as Jurkat cells, MOLT-3 cells, CEM cells, SLIP-T1 cells, T-AII- 104, SKW3, and MT-2 cells.

[0060] The constant domain may be a Ca or a Cb domain. The constant domain may be human or murine. In a preferred embodiment, the constant domain is murine.

[0061] Also encompassed herein is use of the cells described herein in the methods described herein.

[0062] In a particular embodiment, the invention relates to a method for producing a T cell expressing a recombinase mediated landing pad, and constitutive TCR constant domain, which does not express endogenous TCR. The recombinase mediated landing pad may be a Bxb1 landing pad. The TCR constant domain may be a Ca or a Cb domain. The constant domain may be human or murine. The T cell may be any suitable T cell. In some embodiments the T cell is primary T cell or an immortalised T cell. In a preferred embodiment, the cell is a Jurkat cell. The method may comprise the steps of:

[0063] (a) providing a T cell

[0064] (b) modifying the T cell to express a recombinase mediated landing pad, and a TCR constant domain; and

[0065] (c) modifying the cell to remove endogenous expression of TCR, thereby obtaining a cell expressing a recombinase mediated landing pad, and constitutive TCR constant domain, which does not express endogenous TCR.

[0066] The cell according to the invention may be used as a platform for the discovery of T cell receptors. The cells according to the invention may be distinguished from cells not according to the invention because the cells of the invention do not express exogenous TCR, express exogenous constant domain and express a recombinase mediated landing pad.

[0067] TCR activation

[0068] In the methods of the invention, the plurality of transfected cells is exposed to target antigen. The target antigen may be expressed on surface of an antigen presenting cell (APC). The APC may be selected from the group consisting of a T2 cell, disease specific cell line and primary cells. The APC may be a T2 cell that artificially presents high levels of target antigen. The target antigen may be a short peptide fragment bound to a MH C molecule. Alternatively artificial APCs may be used, such as a bead coated with pHLA and an anti- CD28 antibody.

[0069] Methods

[0070] The method of identifying a functional TCR may further comprise a step prior to step (b), where the library of particles is enriched for TCRs that bind target antigen. The enrichment may occur by separating particles that have bound target antigen from particles that have not bound target antigen. Following enrichment, only the TCRs that have bound to the target antigen are transfected into a plurality of T cells.

[0071] The method of identifying a functional TCR may further comprise sorting cells based on TCR activity. Cell sorting may be performed using Fluorescence-activated cell sorting (FACS), or any other suitable means.

[0072] The method of identifying a functional TCR may further comprise deep sequencing the TCR alpha and beta variable domains from the activated T-cells.

[0073] In some embodiments, exposing the plurality of T cells (or mammalian display library) of the invention to antigen may result in convergence of the TCR sequences and enrichment of the cells expressing the most potent TCR sequences. The level of enrichment can be used to rank the TCRs, with the most enriched cells expressing the most potent TCRs. In some embodiments, the cells may be sorted, or enriched, for cells expressing functional TCRs. The sorted cells may be exposed to antigen a second time and the sorting step repeated. In this way it may be possible to further enrich for the most potent TCRs. To determine the level of enrichment an enrichment score may be calculated by determining the ratio of activated-to-input TCR read count frequency, where 1 = no enrichment.

[0074] The method of identifying a functional TCR as described herein may further comprise engineering the identified TCRs. Methods to engineer TCRs for therapeutics are known in the art. For example, the TCR may be taken through further affinity maturation such that binding affinity and / or half-life is increased. The TCR may be soluble, i.e. it may be cleaved from the transmembrane domain. The TCR may contain a non-native disulphide bond.

[0075] Binding affinity (inversely proportional to the equilibrium constant KD) and binding half-life (expressed as T%) can be determined by any appropriate method. It will be appreciated that doubling the affinity of a TCR results in halving the KD. T1Z> is calculated as I n2 divided by the off-rate (kOff). So, doubling of T1Z> results in a halving in kOff. KD and kOff values for TCRs are usually measured for soluble forms of the TCR, i.e., those forms which are truncated to remove hydrophobic transmembrane domain residues. Therefore, it is to be understood that a given TCR meets the requirement that it has a binding affinity for, and / or a binding half-life for a peptide antigen if a soluble form of that TCR meets that requirement. Preferably the binding affinity or binding half-life of a given TCR is measured several times, at a defined temperature using the same assay protocol and an average of the results is taken. More preferable the binding affinity or binding half-life is measured by surface plasmon resonance at a temperature of 25°C.

[0076] The methods described herein may further comprise fusing the TCR to a second moiety. The second moiety may be a cytokine, antibody or fragment thereof, therapeutically active agent or chemical agent. A non-exhaustive list of exemplary second moieties is: immunomodulatory agents, radioactive compounds, enzymes, cytokines, cytotoxic agents, imaging compound and antibodies or fragments thereof. The TCR may be fused to detectable labels including, but not limited to, fluorescent labels, radiolabels, enzymes, nucleic acid probes and contrast reagents, or to therapeutic agents including, but not limited to, immunomodulators, radioactive compounds, enzymes (perforin for example) or chemotherapeutic agents (cis-platin for example). The TCR may subsequently be non- naturally expressed on the surface of cells, preferable mammalian cells, more preferably immune cells, even more preferable T cells. The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e. , molecules that contain an antigen binding site that specifically binds an antigen, whether natural or partly or wholly synthetically produced. The term "antibody" includes antibody fragments, derivatives, functional equivalents and homologues of antibodies, humanised antibodies, including any polypeptide comprising an immunoglobulin binding domain, whether natural or wholly or partially synthetic and any polypeptide or protein having a binding domain which is, or is homologous to, an antibody binding domain. Chimeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included.

[0077] Fragments of a whole antibody can perform the function of binding antigens. Examples of binding fragments are (i) the Fab fragment consisting of VL, VH, CL and CH1 domains; (ii) the Fd fragment consisting of the VH and CH1 domains; (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment which consists of a VH domain; (v) isolated CDR regions; (vi) F(ab')2 fragments, a bivalent fragment comprising two linked Fab fragments (vii) single chain Fv molecules (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site; (viii) bispecific single chain Fv dimers and (ix) "diabodies", multivalent or multispecific fragments constructed by gene fusion

[0078] The methods described herein may further comprise formulating the TCR into a pharmaceutical composition. The pharmaceutical composition may comprise a pharmaceutically acceptable carrier. This pharmaceutical composition may be in any suitable form, (depending upon the desired method of administering it to a patient). It may be provided in unit dosage form, will generally be provided in a sealed container and may be provided as part of a kit. Such a kit would normally (although not necessarily) include instructions for use. It may include a plurality of said unit dosage forms. Suitable compositions and methods of administration are known to those skilled in the art.

[0079] Additionally, T cells transfected with TCRs of the invention may be provided in pharmaceutical composition together with a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be a cream, emulsion, gel, liposome, nanoparticle or ointment.

[0080] The pharmaceutical composition may be adapted for administration by any appropriate route such as a parenteral (including subcutaneous, intramuscular, or intravenous), enteral (including oral or rectal), inhalation or intranasal routes. Such compositions may be prepared by any method known in the art of pharmacy, for example by mixing the active ingredient with the carrier(s) or excipient(s) under sterile conditions.

[0081] TCRs identified by the methods described herein may also be labelled with an imaging compound, for example a label that is suitable for diagnostic purposes. Such labelled TCRs are useful in a method for detecting a TCR ligand selected from CD1-antigen complexes, bacterial superantigens, and MHC-peptide / superantigen complexes which method comprises contacting the TCR ligand with a high affinity TCR (or a multimeric high affinity TCR complex) which is specific for the TCR ligand; and detecting binding to the TCR ligand. In tetrameric high affinity TCR complexes (formed, for example) using biotinylated heterodimers) fluorescent streptavidin (commercially available) can be used to provide a detectable label. A fluorescently-labelled tetramer is suitable for use in FACS analysis, for example to detect antigen presenting cells carrying the peptide for which the high affinity TCR is specific.

[0082] A high affinity TCR (or multivalent complex thereof) identified by the present invention may alternatively or additionally be associated with (e.g. covalently or otherwise linked to) a therapeutic agent which may be, for example, a toxic moiety for use in cell killing, or an immunostimulating agent such as an interleukin or a cytokine. A multivalent high affinity TCR complex of the present invention may have enhanced binding capability for a TCR ligand compared to a non-multimeric wild-type or high affinity T cell receptor heterodimer. Thus, the multivalent high affinity TCR complexes according to the invention are particularly useful for tracking or targeting cells presenting particular antigens in vitro or in vivo, and are also useful as intermediates for the production of further multivalent high affinity TCR complexes having such uses. The high affinity TCR or multivalent high affinity TCR complex may therefore be provided in a pharmaceutically acceptable formulation for use in vivo.

[0083] High affinity TCRs of the invention may be used in the production of soluble bi-specific reagents. In a preferred embodiment, these are ImmTAC® , ImmTAAl ® or ImmTAV ® reagents. ImmTAC® and ImmTAV ® reagents comprise a soluble TCR, fused via a linker to an anti-CD3 specific antibody fragment. ImmTAAl ® reagents comprise a soluble TCR, fused via a linker to an immune suppressor (for example a PD-1 agonist). Further details including how to produce such reagents are described in WO10 / 133828 and WO2019219709. The TCRs identified by the methods described herein may be used in adoptive therapy. The TCRs may be glycosylated when expressed by the transfected T cells. As is well known, the glycosylation pattern of transfected TCRs may be modified by mutations of the transfected gene.

[0084] The TCRs identified by the methods described herein may be provided in pharmaceutical composition together with a pharmaceutically acceptable carrier. The pharmaceutical composition may be adapted for administration by any appropriate route such as a parenteral (including subcutaneous, intramuscular, intravenous, or intraperitoneal), inhalation or oral route. Such compositions may be prepared by any method known in the art of pharmacy, for example by mixing the active ingredient with the carrier(s) or excipient(s) under sterile conditions.

[0085] Mammalian display library

[0086] In a further aspect of the invention, there is provided a mammalian display library comprising a plurality of T cells as described herein, wherein each of the plurality of T cells has been transfected with a nucleic acid encoding a single TCR that binds a target antigen, wherein each nucleic acid is integrated into the genome of the T cell at a single identical pre-defined locus, and wherein the TCR is in a single chain format and comprises an alpha chain variable domain, a beta chain variable domain and a constant domain.

[0087] The mammalian display library may comprise approximately 103cells, each expressing a specific TCR. In some embodiments, the mammalian display library may comprise approximately 104, or 105variants.

[0088] The mammalian display library may comprise T cells derived from immortalised cell lines such as Jurkat cells, MOLT-3 cells, CEM cells, SLIP-T1 cells, T-AII-104, MT-2 cells and SKW3 cells.

[0089] In a further aspect of the invention, there is provided TCRs and functional fragments thereof, obtained / obtainable from, or identified by the methods described herein. Also specifically envisaged are polynucleotides encoding the TCRs or fragments thereof, obtained from the methods described herein. The TCRs identified by the methods described herein may be affinity matured to increase binding to target antigen. The TCRs identified by the methods described herein may be fused to a second moiety.

[0090] Definitions

[0091] It is also noted that in this disclosure and particularly in the claims and / or paragraphs, terms such as “comprises”, “comprised”, “comprising” and the like can have the usual meaning attributed to it; e.g., they can mean “includes”, “included”, “including”, and the like; and that terms such as “consisting essentially of’ and “consists essentially of” have the meaning generally ascribed to them e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention.

[0092] The terms "T cell receptor" and "TCR", as used herein, are used interchangeably. As used herein, the term "TCR variable domain" is understood to encompass amino acids of a given TCR which are not included within the non-variable or constant domain as encoded by the TRAC gene for TCR alpha chains and either the TRBC1 or TRBC2 genes for TCR beta chains. As used herein, the term "constant domain" with respect to a TCR refers to the extracellular portion of a TCR that is encoded by the TRAC gene for TCR a chains and either the TRBC1 or TRBC2 genes for TCR beta chains. The term constant domain does not include a TCR variable domain encoded by a TRAV gene or a TRAJ gene for a TCR alpha chain or a TRBV gene, a TRBD gene, or a TRBJ gene for a TCR beta chain.

[0093] Within the present invention, an endogenous gene is a naturally-occurring gene that can be found in a given cell at a known position in the chromosome. A recombinant gene or exogenous gene, is a gene that has been introduced into a cell by methods of genetic engineering. An endogenous gene and a recombinant gene may have identical DNA sequences and may only differ in their position in the chromosome. As used herein, “endogenous” relates to genetic material originating from within the same organism or cell line that is being studied or manipulated. For example, endogenous genes are those naturally present in the genome of the target cells. As used herein, “exogenous” relates to genetic material originates from an external source outside the organism or cell line of interest. Exogenous transfection involves introducing foreign genetic material, such as plasmid DNA, RNA, or viral vectors, into the cells. Exogenous genetic material may come from the same species (homologous) or from a different species (heterologous). As used herein, the term "promoter" refers to a regulatory region of DNA that is in operable linkage with a nucleotide sequence, e.g., a protein coding sequence. The promoter contains specific DNA sequences and response elements that are recognized by proteins known as transcription factors. These factors bind to and recruit RNA polymerase II to the promoter thereby facilitating transcription of the downstream nucleotide sequence. The transcribed mRNA comprising a protein coding sequence is then translated to produce the expressed protein.

[0094] As used herein, the term "operable linkage" refers to the configuration between a promoter sequence and a protein coding sequence that allows for the transcription of the protein coding sequence in a host cell.

[0095] The term "encoding" or "encoded" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an RNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e. , rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.

[0096] Preferred or optional features of each aspect of the invention are as for each of the other aspects mutatis mutandis. Accordingly, although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined in the appended claims. The documents referred to herein are incorporated by reference to the fullest extent permitted by law.

[0097] In the event of any discrepancy between a nucleic acid sequence or amino acid sequence provided in the description, claims and / or drawings and the corresponding sequence in the sequence listing, the sequence in the description, claims and / or drawings prevails.

[0098] The present invention will be further illustrated in the following figures and Examples which are given for illustration purposes only and are not intended to limit the invention in any way.

[0099] List of Figures

[0100] FIG 1. Generation of TCR-null NFAT Luciferase Jurkat cell line A. Flow cytometry assessment of endogenous Jurkat TCRa&p constant domain knockout through CRISPR / Cas9 NHEJ. The selected clone is CD3 and TRBC1 negative B. NFAT signalling pathway is functional in TCR-null Jurkat cells C. TCR-null Jurkats cannot induce TCR-mediated T cell activation confirming the absence of functional TCR / CD3 complex on the surface of the cells.

[0101] FIG 2. Development of Jurkat based mammalian display system for functional screening of TCRs A. Schematic representation of the engineering steps carried out to generate TCR-null Jurkat landing pad cells B. Flow cytometry assessment of endogenous Jurkat TCR knockout through CRISPR / Cas9 NHEJ followed by introduction of single copy Bxb1 landing pad through lenti-viral mediated gene transduction.

[0102] FIG 3. RMCE enables the efficient and directed insertion of a DNA fragment, encoding TCR alpha and beta chains, via a cassette targeting vector (CTV) into a single copy, predefined genome locus flanked with Bxb1 integration sites for Bxb1 integrase recognition. The CTV accommodates the insertion of single chain TCRs comprising Va and Vp with a short GS linker between the two domains. The murine constant domain (muTRBC) is downstream of p. SP indicates signal peptide.

[0103] FIG 4. Jurkat landing pad system enables stable and homogenous expression of TCRs A. Schematic representation of targeted replacement of mCherry coding gene for a transgenic full length TCR a / p cassette via RMCE B. Flow cytometry assessment of CD3 restoration in TCR-null Jurkat landing pad cells after targeted TCR reconstitution via Bxb1 mediated cassette exchange and puromycin selection.

[0104] FIG 5. Representative flow cytometry dot plots showing surface TCR display of ten PIWIL1 specific TCRs 10 days post electroporation and puromycin selection.

[0105] FIG 6. Representative flow cytometry dot plots showing surface upregulation of T cell activation markers, CD25 and CD69, in Jurkat landing pad cells displaying 10 different PIWIL1 specific TCRs individually after overnight coculture with T2 cells pulsed with irrelevant peptide (control) or cognate peptide (PIWIL1)

[0106] FIG 7. Schematic representation of the workflow for high throughput functional TCR library screening in Jurkat landing pad cells

[0107] FIG 8. FACS plots showing generation of PIWIL1 and target2 TCR libraries. Flow cytometry assessment of CD3 restoration in TCR-null Jurkat landing pad cells after targeted TCR reconstitution via Bxb1 mediated cassette exchange and puromycin selection.

[0108] FIG 9. T cell activation profiling of PIWIL1 and target 2 TCR libraries via fluorescence- activated cell sorting. Jurkat landing pad cells expressing PIWIL1 and target 2 libraries were co-cultured with T2 cells pulsed with irrelevant (control) or cognate peptide (PIWIL1) The percentages of CD69 and CD25 double positive population were determined by flow cytometry in comparison to the control sample. Sorted cells were subjected to Sanger or deep sequencing to identify enriched TCRs in each population.

[0109] FIG 10. Graph showing enrichment score correlation with TCR potency. TCR activationbased enrichment score is calculated as the ratio of activated to input TCR read count frequency. Each circle on the graph represents a unique TCR. Higher the activation score indices higher probability of a given TCR to be potent.

[0110] Examples

[0111] Example 1 : Methods

[0112] Cell lines and cell culture

[0113] The Jurkat NFAT luciferase T cell line was purchased from Promega. Engineered Jurkat landing pad cells were maintained in RPMI-1640 supplemented with 10% heat- inactivated FBS, 2 mM L-glutamine, 0.1 mM MEM nonessential amino acids, 1 mM sodium pyruvate, pen / strep (50 U / rnL penicillin and 50 pg / mL strep), 200 pg / mL hygromycin B and 2 mg / ml G418 (Jurkat growth media). Once electroporated, correct integrants were selected with Jurkat growth media containing 250 ng / ml puromycin. T2 antigen presenting cells (174xCEM.T2) were obtained from American Type Culture Collection (ATCC) and were cultured in RP I media supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine and pen / strep (50 U / rnL penicillin and 50 pg / mL strep) (R10 media). HEK-293T cells were purchased from ATCC and maintained in DM EM (4.5 g / L glucose) supplemented with 10% heat-inactivated FBS and 2 mM L-glutamine. Detachment of HEK-293T adherent cell lines was performed using the 0.25% Trypsin-EDTA solution (Thermo Fisher Scientific, # 25200056). All standard media (RPMI-1640, DMEM), FBS, glutamine, MEM nonessential amino acids, sodium pyruvate, pen / strep, PHA, and PBS were purchased from Thermo Fisher Scientific. Hygromycin B was purchased from Invitrogen. G418 and puromycin were purchased from InvivoGen. All cell lines were cultured at 37°C, 5% CO2 humidified tissue culture incubator. Cloning and Polymerase Chain Reaction (PCR)

[0114] PCRs for cloning into lentiviral expression vectors were done with Phusion High Fidelity DNA Polymerase (NEB, #M0530S) with custom designed primers and using the following cycling conditions: 98°C for 30 sec; 30 cycles of 98°C for 10 sec, 58°C for 1 min, 72°C for 2 min; final extension 72°C for 10 min. PCR products were column purified using Machery- Nagel NucleoSpin Gel and PCR Clean-up kit (Fisher Scientific, #740609.50) and cloned into desired plasmid backbones via Gibson assembly using NEBuilder HiFi DNA assembly master mix (NEB, #E2621) using manufacturer's instructions.

[0115] PCRs for genotyping of bacterial colonies after transformation were performed using Taq 2X Master Mix (NEB, #M0270L) with custom designed primers and using the following cycling conditions: 95°C for 5 min; 30 cycles of 95°C for 30 sec, 56°C for 30 sec, 72°C for 2min 20 sec; final extension 70°C for 2 min.

[0116] Flow Cytometry and cell sorting

[0117] Flow cytometric analysis was performed on BD LSRFortessa X-20 Cell Analyzer (BD Biosciences). FACS sorting was performed using BD FACSAria Fusion (BD Biosciences). Data analysis was performed using FlowJo. Surface staining for flow cytometry was performed pelleting cells at 400 g for 5 min and resuspending in cells in flow cytometry buffer (2% FBS and 2 mM EDTA in DPBS) with antibodies and viability dye for 30 minutes at 4 °C in the dark. Cells were washed once in flow cytometry buffer before resuspension. The following antibodies were diluted in the flow cytometry buffer: BB515 anti-human CD3 (BD Biosciences, clone SK7, 2 pg ml’1, #564560), PerCP / Cyanine5.5 anti-human HLA-A2 (BioLegend, clone BB7.2, 4 pg ml’1, #343316), APC anti-human CD69 (BioLegend, clone FN50, 1 pg ml’1, #310910), PE anti-human CD25 (BioLegend, clone BC96, 0.5 pg ml’1, #302606), R-PE anti-human TRBC1 (LSBio, 1:100 dilution, clone JOVI-1,) and fixable viability dye eFluor 780 (Thermo Fisher Scientific, 1:1000 dilution, #65086514) Blocking of Fc receptors was performed prior to staining using the Human TruStain FcX (BioLegend, #422302) Single cell sorts were collected in 96-well U-bottom plates and clones were cultured 2-3 weeks prior to characterisation.

[0118] CRISPR-Cas9 genome editing

[0119] Cas9 ribonucleoprotein complexes were generated by mixing 240 ng synthetic gRNAs (sgRNA) (Thermo Fisher Scientific) with 1250 ng TrueCut Cas9 protein v2 (Thermo Fisher Scientific, #A36498) in Resuspension buffer R by incubating 10-15 minutes at room temperature. For optimal editing, 2x105Jurkat cells were used per electroporation using Neon Transfection System 10 pl Kit (Thermo Fisher Scientific, #MPK1025) according to manufacturer's instructions. Prior to electroporation, cells were transferred to a sterile tube and centrifuged at 400 x g for 5 minutes. Cells were washed once with 1 ml PBS without Ca2+and Mg2+. Upon centrifugation, the supernatant carefully aspirated to remove all the PBS. Cells were resuspended in 5pl Resuspension Buffer R. Cells were then mixed with 5 pl of Cas9 RNP complex and electroporated using Program #5 (1700V, 20ms, 1 pulse). Cells were transferred into a 6-well dish containing 5ml R10 media without any antibiotics for 72 hrs post electroporation for recovery. Cells were screened for the absence of surface CD3 and TRBC1 expression by flow cytometry. TCR null cell population were single cell sorted to achieve monoclonality. Absence of wild type sequences and the presence of indels were confirmed by sanger sequencing followed by sequence trace decomposition using CRISP-ID (http: / / crispid.gbiomed.kuleuven.be / ) and TIDE (https: / / tide.nki.nl / ). sgRNA sequences targeting TRAC (5'- AGAGTCTCTCAGCTGGTACA-3' (SEQ ID NO: 1)) and TRBC1 (5'- GGAGAATGACGAGTGGACCC-3' (SEQ ID NO: 2)) were purchased from Thermo Fisher Scientific.

[0120] Lentiviral transduction of TCR-null Jurkat cells

[0121] 15 million HEK-293T cells were transfected with 10 pg Bxb1 mCherry landing pad or full length murine TRAC lenti expression constructs under the control of human elongation factor-1 alpha (EF-1a) promotor, 12 pg pRSV.REV, 12 pg pMDLg and 12 pg pMDg.2 using PEI-Max (Polysciences) following manufacturer's protocol. Lentiviral supernatant was collected at 72 h after transfection, spun down at 500 g for 10 min and clarified through a 0.45-pm syringe filter. Cleared supernatant was precipitated with Lenti-X concentrator (Takara-Clontech) according to manufacturer's instructions and incubated overnight at 4°C. Samples were centrifuged next day at 1500 g for 3 h at 4°C. The pellet containing lenti-viral particles was gently resuspended in complete RPMI medium and added to Jurkat cells in RPMI medium containing 10% FBS and 12 pg / ml polybrene (Merck) Samples were centrifuged at 700 g for 2 h at 32°C. Three days after viral transduction, cells were analysed by flow cytometry to measure transduction efficiencies. To enrich mCherry+ landing pad cells post transduction, 2-5 million cells were stained with viability dye (1 / 1000) (eFluor780). Cells were then sorted on a BD FACSAria Fusion platform for live mCherry+ cells. The sorted Jurkat cells were recovered and expanded in RPMI medium with 10% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 1X MEM non-essential amino acid solution and 200 mg / ml hygromycin. Jurkat landing pad cells that were transduced with lenti construct expressing full length murine TRAC were selected with 2 mg / ml G418. Co-culture Assays

[0122] Peptides were supplied as dried pellets with 2 mg per peptide, reconstituted in 500 pl of DMSO and stored at -80°C for long term storage. All peptides were purchased from Peptide Protein Research Ltd. For peptide pulsing, peptides were diluted to 10 pM in R10 media, and the solution was used to resuspend T2 cells at 1x106cells ml’1. Cells were incubated for 2 hours at 37°C, 5% CO2 humidified tissue culture incubator and added to the coculture wells (see below). Jurkat landing pad cells expressing TCR of interest were harvested, pelleted by centrifugation and resuspended in fresh R10 media at 1x106cells ml’1. Peptide-pulsed T2 cells were combined with Jurkat cells at a 1 :1 ratio in a final volume of 200 pl and incubated overnight (16 hours) at 37°C, 5% CO2 humidified tissue culture incubator. The next day, flow cytometry was used to assess TCR responses through detecting upregulation of CD69 and CD25 expression. Jurkat and T2 cells were identified using surface staining for CD3 and HLA-A2, respectively. Viable cells were identified by exclusion of viability dye.

[0123] Genotyping of cell lines and integrants

[0124] Genomic DNA was extracted from 1x106cells by using Monarch Genomic DNA Purification Kit (NEB, #T3010L) according to manufacturer's instructions. Genomic DNA concentration was measured using NanoDrop 2000. 50 ng / pl genomic DNA was then used as a template for 50pl PCR reactions using Phusion High Fidelity DNA Polymerase (NEB, #M0530S) with custom designed primers and using the following cycling conditions: 98°C for 30 sec; 30 cycles of 98°C for 10 sec, 59°C for 15 sec, 72°C for 1 min; final extension 72°C for 2 min.

[0125] Cloning of TCR cassettes

[0126] PCRs for cloning phage TCR outputs into the cassette targeting vector (CTV) for the generation of Jurkat TCR libraries were performed using KOD Hot Start DNA Polymerase (Merck, # 71086) with custom designed primers and using the following cycling conditions: 95°C for 2 min; 20 cycles of 95°C for 20 sec, 59°C for 10 sec, 70°C for 10 sec per kb; final extension 70°C for 1 min per kb. TCR amplicons were then cloned into cassette targeting vector between Nhel and Hindlll sites using NEBuilder HIFI DNA Assembly Master Mix (NEB, #E2621S)

[0127] TCR reconstitution via Bxb1 mediated cassette exchange

[0128] Jurkat landing pad cells were sub-cultured 2-3 days before electroporation to achieve a final density of 1x106 / ml on the day of electroporation. Cell transfections were performed using electroporation (Neon Transfection System, Thermo Fisher Scientific, #MPK5000) using 100 l Kit (Thermo Fisher Scientific, #MPK10096) 1-5pg of cassette targeting vector (CTV) containing TCR of interest and 2-10 pg Bxb1 expression plasmid were mixed and used for electroporating 1x106cells for each transfection. Following electroporation, cells were recovered in in R10 media without any antibiotics. At three days postelectroporation, Bxb1 integration efficiency was analysed by calculating the % of cells with restored surface TCR / CD3 via flow cytometry. Cells were then selected with Jurkat growth media containing 250 ng / ml puromycin to select the integrants. Finally, Jurkat landing pad cells expressing individual TCRs were characterised by flow cytometry, PCR and Sanger sequencing. pH LA library screening and functional selections

[0129] CTVs containing PIWIL1 pan2 phage outputs (5 pg / pl) and the Bxb1 expression plasmid (1 Opg / pl) were used to electroporate 10x106Jurkat landing pad cells using Neon Transfection System 100pl Kit (Thermo Fisher Scientific, #MPK10096). 72 hrs post electroporation cells were transferred into 250 ng / ml puromycin containing media. To enrich correct integrants cells were grown for 7-10 days until all cells die off in the mock electroporation control. Jurkat cells with restored surface CD3 expression were bulk sorted and expanded for 10 days prior to overnight co-culture with T2 cells pulsed with cognate peptide (PIWIL1) or irrelevant peptide (TAX). After co-culture, CD69-and-CD25- double-positive cells for cognate peptide and negative for irrelevant control peptide were bulk sorted for genomic DNA extraction, Sanger and deep sequencing.

[0130] Deep sequencing of recombinant, chromosomally integrated TCRs

[0131] One microgram of extracted genomic DNA, equivalent to ~1.5 x105cells, was used in a UMI-tagging reaction using a single primer consisting of a universal PCR amplification handle, patterned N12 UMI and TRAV-specific annealing sequence. Single-primer extension was performed with KAPA HiFi HotStart ReadyMix (Roche, #07958935001) and 0.2 pM of primer, with following programme: 3 min at 95°C; 1 cycle of 20 sec at 98°C, 15 sec at 60°C and 1 min at 72°C. ExoProStar 1-STEP Kit (I llustra, #US77705) was used to remove excess UMI oligos, according to manufacturer’s instructions. Reaction was additionally purified and concentrated by AMPure XP beads (Beckman Coulter, #A63881) clean-up in 0.7:1 beads-to-reaction ratio. Full volume of UMI-tagged template was subjected to PCR amplification using two pools (For and Rev) of primers with degenerate spacer nucleotides (N2-8) designed to introduce diversity at the start of Illumina reads, while adding i5 and i7 binding sites. PCR was performed with KAPA HiFi HotStart ReadyMix and 0.2 pM of each primer pool, with following programme: 3 min at 95°C; 15 cycles of 20 sec at 98°C and 1 min at 72°C; 5 min at 72°C. PCR products were bead- purified in 1:1 beads-to-reaction ratio. Target amplicons were indexed using NEBNext Multiplex Oligos for Illumina (New England Biolabs, #E7600S) and NEBNext Ultra II Q5 Master Mix (New England Biolabs, #M0544S), with following programme: 30 sec at 98°C; 12-15 cycles of 10 sec at 98°C and 75 sec at 65°C; 5 min at 65°C. Final libraries were bead-purified in 0.7:1 beads-to-reaction ratio, quantified with the Qubit 1X dsDNA High Sensitivity Assay kit (Thermo Fisher Scientific, #Q33231) and fragment-analysed on a Bioanalyzer (Agilent, #5067-4626). Libraries were pooled and sequenced using MiSeq 600 cycle v3 kit (Illumina, #MS-102-3003) in paired-end 2x 300 cycles mode.

[0132] Sequencing analysis

[0133] First, using MIGEC v1.2.9, UMI sequences were extracted from reads and adapter regions were trimmed. Primer sequences are provided below Reads processed by MiGEC were aligned to human reference using MiXCR v3.0.13, separately for TRA and TRB.

[0134] Following clone assembly with default parameters, and clone and alignments export, reads were paired using a custom R script. Final paired clonotypes were defined and quantified as unique combinations of CDR3 amino acid sequence, V and J gene for both alpha and beta variable chains.

[0135] *N2-8 indicates a pool of primers comprising 2 to 8 random nucleotides to form diversity spacers

[0136] Calculation of binding affinity

[0137] SPR measurements were carried out on a BIAcore 8K, BIAcore 3000 or BIAcore T200 instrument. Briefly, biotinylated class I HLA-A*02 molecules were refolded with the peptide of interest and purified. Biotinylated peptide-HLA monomers were immobilized on to streptavidin-coupled CM-5 sensor chips. Equilibrium binding constants were determined using serial dilutions of soluble TCR. KD values were obtained by non-linear curve fitting using Prism software and the 20 Langmuir binding isotherm, bound = C*Max / (C + KD), where “bound” is the equilibrium binding in response units at injected TCR concentration C and Max is the maximum binding. Measurements were performed at 25°C, unless otherwise indicated, in Dulbecco’s PBS buffer, supplemented with 0.005% P20. Example 2: Generation of a modified signalling competent Jurkat T cell line

[0138] Disruption of endogenous TCR genes

[0139] To prevent mispairing between the introduced TCR a and p chain and the endogenous TCR a and chain, endogenous TCR genes were inactivated using CRISPR-Cas9 genome editing to knock out both alpha and beta TCR constant chains. NFAT-Luciferase Jurkat cells were sequentially electroporated with Cas9 protein along with synthetic guide RNAs (sgRNA) targeting the 5' end of the first exon of TRAC and TRBC1. Cells were screened for the absence of surface CD3 and TRBC1 expression by flow cytometry and monoclonal cell populations created by single cell sorting (Figure 1A). Endogenous TCR disruption was confirmed to have no detectable effect on Jurkat cell growth or NFAT- reporter function (Figure 1B). No TCR-mediated T cell activation was observed, confirming the absence of surface TCR (Figure 1C).

[0140] Integration of Bxb1 landing pad for recombinase-mediated cassette exchange (ROME) A Bxb1 recombinase dependent landing pad was introduced into the TCR-null Jurkat cells (Figure 2A). The system was designed for stable, single-copy integration of TCR molecules with high efficiency into a defined genetic locus, resulting in a strict phenotype- to-genotype linkage and transcriptional normalisation. Bxb1 integrase is a serine recombinase derived from phage and is known to be highly effective in mammalian cells. Bxb1 catalyses irreversible unidirectional recombination between attP (attachment site for phage) and attB (attachment site for bacteria) sites, resulting in hybrid product sites attL (attachment left) and attR (attachment right). A fluorescent marker (mCherry) was included to allow enrichment of landing pad containing cells via FACS sorting (Figure 2B). mCherry fluorescent protein was flanked with Bxb1 attP and mut attP recognition sites with expression driven by an upstream promoter.

[0141] Integration of transgenic single chain TCRs Transgenic TCRs were integrated using a promoter-less cassette targeting vector (CTV) which was transcriptionally linked to a puromycin resistance gene for positive selection of TCR expressing cells, along with a Bxb1 recombinase expression vector (Figures 3 and 4A). The CTV was designed to accommodate the insertion of single chain TCRs comprising Va and p with a short GS linker between the two domains. The murine Cp domain was included downstream of p. Hybrid TCRs with murine constant regions and human variable regions are known in the art to have higher surface density and display higher biological activity towards APCs than fully human molecules. Integration of transgenic TCRs was assessed by restoration of surface CD3 expression and loss of mCherry expression (Figure 4B). Correct integration of TCR cassettes in Jurkat landing pad cells was confirmed by genomic DNA PCR and Sanger sequencing.

[0142] Constitutive expression of Ca

[0143] To allow functional expression of single chain TCRs Jurkat cells were further modified to constitutively express murine Co.

[0144] Example 3: Assessment of TCR functionality

[0145] To confirm that integrated TCRs could generate a functional response, co-culture assays were performed using the modified Jurkat cells expressing a defined TCR and peptide- pulsed T2 cells. In this example ten native TCRs that recognise a HLA-A2 restricted peptide from PIWIL1 (SLSNRLYYL (SEQ ID NO: 6)) were introduced as full length alpha and beta chains into the modified Jurkat cells and cultured in the presence of T2 cells pulsed with PIWIL1 peptide. Each of the TCRs were shown to bind to the cognate peptide-HLA complex in solution using Biacore (Table 1). TCR activation was assessed by measuring the upregulation of T cell activation markers CD25 and CD69 by flow cytometry. As controls, TCR expressing modified Jurkat cells were co-cultured with unpulsed T2 cells, or T2 cells pulsed with an irrelevant peptide.

[0146] Results

[0147] Surface display of PIWIL TCRs on the modified Jurkat cells was confirmed 10 days post electroporation (Figure 5). In the presence of PIWIL peptide-HLA complex strong T cell activation was observed for two of 10 different TCRs tested (#659 and #489), weak activation for one (#444), and no activation for the remaining samples even though all these TCRs showed specific binding to the cognate peptide-HLA complex in solution (Figure 6 and Table 1).

[0148] Table 1. Table showing list of 10 PIWIL1 specific TCRs along with their CDR3a / p sequences and binding affinity to PI WIL peptide-HLA complex. These data demonstrate that TCR functionality can be assessed in the modified Jurkat cells. Furthermore, the data show that in vitro solution binding of a TCR to its cognate peptide-HLA is not a reliable indicator of TCR signalling capacity and therefore functional screening is critical to ensure TCRs that are unable to signal in the presence of APCs are eliminated from further development.

[0149] Example 4: A high throughput workflow for functional selection of target specific TCRs from large libraries

[0150] High throughput screening of functional TCRs from high diversity libraries is challenging. Therefore, to combine the power of binding-based selection with functional screening of large libraries the output from a second round of selection from a TCR phage library (~104TCRs) was seamlessly integrated into the modified Jurkat cells (Figure 7).

[0151] Phage library selection A phage library comprising approximately 109native TCRs displayed in single chain format was subjected to two rounds of panning with either PIWIL peptide-HLA complex or an alternative pHLA complex (termed target2 herein).

[0152] Transfer of phage library output to modified Jurkat cells

[0153] TCR alpha and beta variable domains from the selected phage were cloned into the cassette targeting vector (CTV) as single chain constructs and electroporated into the modified Jurkat cells as described in the methods. Deep sequencing showed a greater than 70% overlap between phagemid and CTV encoded TCRs, indicating a high-fidelity cloning process that effectively mirrored the input diversity.

[0154] Selection of functional TCRs from Jurkat library

[0155] Jurkat cells with integrated TCRs were first isolated by FACS based on rescued CD3 expression (Figure 8). Cells were subsequently expanded, co-cultured with cognate- peptide loaded T2 cells and monitored for upregulation of CD69 and CD25 to identify cells displaying target reactive TCRs (Figure 9). Deep and Sanger sequencing was then used to identify TCRap clonotypes that were enriched in each population. For PIWIL 22 unique TCR sequences were obtained (18 in the top 0.5% of activated cells). For target2 15 unique TCR sequence were obtained (7 from the top 1% of activated cells). A second round of activation and was performed on the sorted PIWIL clones to identify the most enriched clones.

[0156] To further assess the utility of the approach as an indicator of TCR potency, and thus TCR developability, an enrichment score was calculated (ratio of activated-to-input TCR read count frequency, where 1 = no enrichment) and used to rank each of the TCRs (Figure 10). Among the most enriched clones in the PIWIL selection, one TCR had been previously identified from the same phage library and has been successfully further developed as a clinical candidate, thereby demonstrating the utility of this approach to rapidly identify functional antigen binding TCRs with therapeutic potential.

Claims

Claims1. A method of identifying a functional TCR from a library of particles, the library displaying a plurality of different T cell receptors (TCRs), the method comprising: a) exposing the library of particles to a target antigen to identify TCRs that bind to the target antigen, b) transfecting a plurality of T cells with nucleic acid encoding TCRs identified in step (a), such that each T cell comprises a nucleic acid encoding a single TCR from the library of particles, wherein the T cells do not express endogenous TCR, the nucleic acid is integrated into the genome of the T cell at a single identical pre-defined locus, and the TCR is in a single chain format and comprises an alpha chain variable domain, a beta cain variable domain and a constant domain, c) exposing the plurality of transfected cells to the target antigen, and d) selecting cells having TCR activity in the presence of the target antigen.

2. The method of claim 1 , wherein the single chain TCR format comprises Va-Vb-Cb or Vb-Va-Ca.

3. The method of claim 1 or claim 2, wherein the T cells constitutively express Ca or Cb.

4. The method of any preceding claim, wherein when the single chain format is Va- Vb-Cb, the T cell expresses a Ca.

5. The method of any of claims 1 to 3, wherein when the single chain format is Vb- Va-Ca, the T cell expresses a Cb.

6. The method of any preceding claim, wherein the target antigen is expressed on surface of an antigen presenting cell (APC).

7. The method of any preceding claim, wherein the T cells are derived from an immortal T cell line.

8. The method of any preceding claim, wherein the T cells are Jurkat cells.

9. The method of any preceding claim, wherein the nucleic acid is integrated into the genome by site-specific integration.

10. The method of claim 9, wherein the integration is a recombinase dependent integration.

11. The method of claim 10, wherein recombinase dependent integration is a Bxb1 mediated landing pad.

12. The method of any preceding claim, wherein prior to step (c) the plurality of T cells is enriched for cells expressing TCR that bind to the target antigen.

13. The method of any preceding claim, wherein the method further comprises sequencing the TCR alpha and beta variable domains from the activated cells.

14. The method of any preceding claim, wherein the method further comprises affinity maturing the TCRs identified by the method.

15. The method of any preceding claim, wherein the method further comprises fusing the TCR to a second moiety.

16. The method of any preceding claim, wherein the library of particles are phage particles, ribosomes, mammalian cells or yeast cells.

17. The method of any preceding claim, wherein the library of particles are phage particles.

18. The method of any preceding claim, wherein the library of particles comprises at least 105variants.

19. A T cell comprising a recombinase mediated landing pad, wherein the T cell constitutively expresses TCR constant domain and does not express endogenous TCR.

20. The cell of claim 19, wherein the constant domain is a Ca or a Cb domain.

21. The cell of claim 19 or 20, wherein the constant domain is an exogenous constant domain.

22. The cell of any of claims 19 to 21, wherein the cell is a Jurkat cell.

23. The cell of any of claims 19 to 22, wherein the recombinase mediated landing pad is a BxB1 landing pad.

24. Use of the cell of any of claims 19 to 23 in the method of any of claims 1 to 18.

25. A mammalian display library comprising a plurality of cells according to any of claims 19 to 23, wherein each of the plurality of cells has been transfected with nucleic acid encoding a single TCR that binds a target antigen, wherein each nucleic acid is integrated into the genome at a single identical pre-defined genomic locus, and wherein the TCR is in a single chain format and comprises an alpha chain variable domain a beta chain variable domain and a constant domain.

26. A TCR obtained from method of any of claims 1 to 18.

27. The TCR of claim 26, wherein the TCR is affinity matured to increase binding to target antigen28. The TCR of claim 26 or 27, wherein the TCR is fused to a second moiety.

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