T-cell receptor identification

Abstract

The present disclosure relates to the field of molecular biology and immunology.

Description

FIELD OF THE TECHNOLOGY

[0001] The present disclosure relates to the fields of molecular biology and immunology. BACKGROUND OF THE DISCLOSURE

[0002] Adoptive T cell therapy (ACT) is a powerful cancer treatment method using cancer-specific T cells (Rosenberg and Restifo, Science (2015) 348(6230):62-68). The cells used in ACT are usually naturally occurring cancer antigen-specific cells; or T cells engineered to express a TCR (i.e., TCR-engineered T cells) that is specific for cells expressing an MHC:peptide complex presenting a peptide containing a target antigen of interest; or T cells engineered to express a chimeric antigen receptor (CAR) that contains an antigen-binding domain specific for the target antigen of interest (CAR-engineered T cells; Rosenberg and Restifo, Science (2015) 348(6230):62-68). Engineering such cells requires the identification of appropriate target antigens and targeting molecules (Leko and Rosenberg, Cancer Cell (2020) 38(4):454-472). TCR discovery remains a challenging task (Joglekar and Li, Nat Methods (2021) 18(8):873-880). Rapid and precise identification of neoantigens and assessment of their immunogenicity are important for the widespread adoption of ACT (Garcia-Garijo et al., Front Immunol. (2019) 10:1392).

[0003] Trogocytosis is a recognized process that occurs bidirectionally between T cells and cells expressing an MHC:peptide complex recognized by their expressed TCRs during immune interactions (Miyake and Karasuyama, Cells (2021) 10(5):1255). Identification of HLA:peptide targets of orphan TCRs has been achieved by assessing trogocytosis of membrane contents from T cells to HLA:peptide-presenting target cells (Li et al., Nat Methods. (2019) 16(2):183-190).

[0004] There is still a need in the art to provide techniques for high-throughput identification of TCR and MHC:peptide interaction partners. SUMMARY OF THE DISCLOSURE

[0005] In a first aspect, the present disclosure provides a polypeptide comprising (i) an amino acid sequence of a major histocompatibility complex (MHC) polypeptide, and (ii) a moiety that facilitates labeling of the polypeptide with an identifier moiety.

[0006] In some embodiments according to various different aspects of the polypeptide described herein, the MHC polypeptide is β2-microglobulin.

[0007] In some embodiments according to various different aspects of the polypeptide described herein, the identifier moiety is a nucleic acid moiety. In some embodiments, the identifier moiety comprises or consists of single-stranded DNA (ssDNA).

[0008] In some embodiments according to various different aspects of the polypeptide described herein, the moiety that facilitates labeling of the polypeptide with an identifier moiety is or comprises a self-labeling protein tag. In some embodiments, the moiety that facilitates labeling of the polypeptide with an identifier moiety is or comprises a HaloTag.

[0009] In some embodiments according to various different aspects of the polypeptide described herein, the polypeptide further comprises a sortase substrate motif.

[0010] In some embodiments according to various different aspects of the polypeptide described herein, the polypeptide further comprises a detectable moiety.

[0011] In some embodiments according to various different aspects of the polypeptide described herein, the detectable moiety is a fluorescent label.

[0012] In some embodiments according to various different aspects of the polypeptide described herein, the polypeptide comprises or consists of the following amino acid sequence having at least 70% amino acid sequence identity with SEQ ID NO:11, SEQ ID NO:10, SEQ ID NO:13 or SEQ ID NO:12.

[0013] The present disclosure also provides a polypeptide comprising (i) an amino acid sequence of β2-microglobulin and (ii) a HaloTag.

[0014] The present disclosure also provides a polypeptide comprising (i) an amino acid sequence of β2-microglobulin, (ii) a sortase substrate motif, and (iii) a HaloTag.

[0015] The present disclosure also provides an MHC molecule comprising the polypeptide according to the present disclosure.

[0016] The present disclosure also provides an MHC:peptide complex, which comprises an MHC molecule according to the present disclosure and a peptide presented by the MHC molecule.

[0017] The present disclosure also provides a nucleic acid or nucleic acids, which encode a polypeptide according to the present disclosure.

[0018] In some embodiments of the various different aspects related to nucleic acids described herein, the nucleic acid or nucleic acids further comprise a nucleic acid encoding a peptide presented by an MHC molecule, which MHC molecule comprises a polypeptide according to the present disclosure.

[0019] The present disclosure also provides an expression vector or expression vectors, which comprise a nucleic acid or nucleic acids according to the present disclosure.

[0020] The present disclosure also provides a cell, which comprises a polypeptide, an MHC molecule, an MHC:peptide complex, a nucleic acid or nucleic acids, or an expression vector or expression vectors according to the present disclosure.

[0021] In some embodiments of the various different aspects related to cells described herein, the cell is an antigen presenting cell (APC).

[0022] The present disclosure also provides a method for producing a cell comprising an MHC molecule labeled with an identifier moiety, the method comprising:

[0023] (1) introducing a nucleic acid or nucleic acids according to the present disclosure into a cell; and

[0024] (2) contacting the cell with a labeling moiety comprising an identifier moiety, wherein the labeling moiety is suitable for labeling a polypeptide encoded by the nucleic acid or nucleic acids of (1) with the identifier moiety.

[0025] The present disclosure also provides a method for producing a cell comprising an MHC:peptide complex, which MHC:peptide complex comprises an MHC molecule labeled with a single-stranded DNA (ssDNA) moiety, the method comprising:

[0026] (1) introducing a nucleic acid or nucleic acids encoding a polypeptide comprising: (i) the amino acid sequence of β2-microglobulin and (ii) a HaloTag into a cell;

[0027] (2) introducing into the cell: (i) a peptide presented by an MHC molecule comprising the polypeptide of (1), or (ii) a nucleic acid encoding a peptide presented by an MHC molecule comprising the polypeptide of (1); and

[0028] (3) contacting the cell with a HaloTag ligand comprising an ssDNA moiety and a chloroalkane moiety.

[0029] The present disclosure also provides a method for producing cells comprising an MHC:peptide complex, the MHC:peptide complex comprising an MHC molecule labeled with a single-stranded DNA (ssDNA) moiety, the method comprising:

[0030] (1) introducing into a cell a nucleic acid or nucleic acids encoding a polypeptide comprising: (i) the amino acid sequence of β2-microglobulin, (ii) a sortase substrate motif, and (iii) a HaloTag;

[0031] (2) introducing into the cell: (i) a peptide presented by an MHC molecule comprising the polypeptide of (1), or (ii) a nucleic acid encoding a peptide presented by an MHC molecule comprising the polypeptide of (1); and

[0032] (3) contacting the cell with a HaloTag ligand comprising an ssDNA moiety and a chloroalkane moiety.

[0033] The present disclosure also provides a cell produced by the method for producing a cell comprising an MHC:peptide complex according to the present disclosure.

[0034] The present disclosure also provides a composition comprising a cell according to the present disclosure and a T cell.

[0035] The present disclosure also provides a method for identifying a T cell receptor (TCR) that binds to an MHC:peptide complex, the method comprising:

[0036] (1) contacting a cell comprising the MHC:peptide complex described in the present disclosure with a population of T cells;

[0037] (2) incubating the cells under conditions suitable for trogocytosis of the MHC:peptide complex by T cells comprising a TCR that binds to the MHC:peptide complex; and

[0038] (3) subsequently analyzing the T cells to identify a TCR that binds to the MHC:peptide complex.

[0039] The present disclosure also provides a method for identifying a T cell receptor (TCR) that binds to an MHC:peptide complex, the method comprising:

[0040] (1) contacting (a) a cell comprising an MHC:peptide complex with (b) a population of T cells;

[0041] (2) incubating the cells obtained after step (1) under conditions suitable for trogocytosis of the MHC:peptide complex by T cells comprising a TCR that binds to the MHC:peptide complex;

[0042] and

[0043] (3) Subsequently analyze the cells obtained after step (2) to identify the TCRs that bind to the MHC:peptide complex;

[0044] wherein the MHC:peptide complex comprises an MHC molecule, and wherein the MHC molecule comprises a polypeptide that comprises (i) the amino acid sequence of the MHC polypeptide and (ii) an identifier portion, wherein the identifier portion is covalently associated with the polypeptide by a bond formed by a self-labeling protein tag.

[0045] The present disclosure also provides a method for identifying a T cell receptor (TCR) that binds to an MHC:peptide complex, the method comprising:

[0046] (1) Contacting (a) cells comprising the MHC:peptide complex with (b) a population of cells comprising T cells in the presence of sortase, wherein these T cells comprise a polypeptide containing a sortase receptor motif at the cell surface;

[0047] (2) Incubating the cells obtained after step (1) under conditions suitable for the interaction between the cells of (a) and the population of cells of (b); and

[0048] (3) Subsequently analyze the cells obtained after step (2) to identify T cells that comprise a TCR that binds to the MHC:peptide complex;

[0049] wherein the MHC:peptide complex comprises an MHC molecule, and wherein the MHC molecule comprises a polypeptide that comprises (i) the amino acid sequence of the MHC polypeptide, (ii) a sortase substrate motif, and (iii) an identifier portion, wherein the identifier portion is covalently associated with the polypeptide by a bond formed by a self-labeling protein tag.

[0050] In some embodiments, the sortase is provided at the cell surface of T cells comprising a polypeptide containing a sortase receptor motif.

[0051] In some embodiments according to various aspects of the methods for identifying TCRs that bind to MHC:peptide complexes described herein, the MHC polypeptide is β2-microglobulin.

[0052] In some embodiments according to various aspects of the methods for identifying TCRs that bind to MHC:peptide complexes described herein, the identifier portion is a nucleic acid portion. In some embodiments, the identifier portion comprises or consists of single-stranded DNA (ssDNA).

[0053] In some embodiments according to various aspects of the methods for identifying TCRs that bind to MHC:peptide complexes described herein, the self-labeling protein tag is or comprises a HaloTag.

[0054] In some embodiments according to various aspects of the methods for identifying TCRs that bind to MHC:peptide complexes as described herein, the identifier portion is covalently associated with the polypeptide through an ester bond formed by the dehalogenase activity of HaloTag on a HaloTag ligand comprising the identifier portion and a chloroalkane portion.

[0055] Specification

[0056] Polypeptide

[0057] Aspects and embodiments of the present disclosure relate to polypeptides. A "polypeptide" refers to a polymeric chain of multiple amino acid monomers linked by peptide bonds. A polypeptide includes peptides, which typically include ≤50 amino acids.

[0058] In some aspects and embodiments, the polypeptides of the present disclosure include the amino acid sequence of a major histocompatibility complex (MHC) polypeptide, and a portion that facilitates labeling of the polypeptide with an identifier portion.

[0059] In some aspects and embodiments, the polypeptides of the present disclosure include the amino acid sequence of a major histocompatibility complex (MHC) polypeptide, and an identifier portion, wherein the identifier portion is covalently associated with the polypeptide through a bond formed by a self-labeling protein tag.

[0060] In some aspects and embodiments, the polypeptides of the present disclosure include a sortase substrate motif, and a portion that facilitates labeling of the polypeptide with an identifier portion.

[0061] In some aspects and embodiments, the polypeptides of the present disclosure include a sortase substrate motif, and an identifier portion, wherein the identifier portion is covalently associated with the polypeptide through a bond formed by a self-labeling protein tag.

[0062] Protein localized to the cell membrane

[0063] In some aspects and embodiments, the polypeptides of the present disclosure include the amino acid sequence of a protein localized to the cell membrane. Such a protein may also be referred to as a cell surface protein.

[0064] A protein localized to the cell membrane is a protein that is detectable in or at the cell membrane when expressed by a cell (e.g., a eukaryotic / mammalian cell). A protein localized to the cell membrane can be detected in or at the cell membrane, for example, by analysis using immunohistochemistry / cytochemistry or flow cytometry techniques, such as using an antibody against the protein.

[0065] It should be understood that the polypeptides according to the present disclosure that include the amino acid sequence of a protein localized to the cell membrane are similarly localized to the cell membrane of the cell that contains / expresses the polypeptide.

[0066] In some embodiments, the protein localized to the cell membrane is a protein expressed by an immune cell. In some embodiments, the immune cell can be a cell of hematopoietic origin, such as a neutrophil, eosinophil, basophil, dendritic cell, lymphocyte, or monocyte. The lymphocyte can be, for example, a T cell, B cell, natural killer (NK) cell, NKT cell, or innate lymphoid cell (ILC) or its precursor (e.g., thymocyte or pre-B cell).

[0067] In some embodiments, the immune cell is an antigen-presenting cell (APC). An APC is a cell that expresses MHC molecules (e.g., MHC class I and / or MHC class II molecules) and is capable of presenting an MHC:peptide complex.

[0068] The APC according to the present disclosure can be a professional APC. Professional APCs are specialized in presenting antigens to T cells; they are capable of efficiently processing and presenting MHC peptide complexes on the cell surface and express high levels of co-stimulatory molecules. Professional APCs include dendritic cells (DCs), macrophages, and B cells. Non-professional APCs are other cells that are capable of presenting MHC peptide complexes to T cells, specifically MHC class I peptide complexes to CD8+ T cells. Thus, in some embodiments, the protein localized to the cell membrane is a protein expressed by an APC (e.g., a DC, macrophage, or B cell).

[0069] In some embodiments, the protein localized to the cell membrane is an interaction partner (e.g., a ligand) for a protein localized to the cell membrane of a T cell. The T cell according to the present disclosure can express a CD3-TCR complex. In some embodiments, the T cell is a CD3+, CD4+ T cell. In some embodiments, the T cell is a CD3+, CD8+ T cell. In some embodiments, the T cell is a T helper cell (T H cell). In some embodiments, the T cell is a cytotoxic T cell (e.g., cytotoxic T lymphocyte (CTL)). In some embodiments, the protein localized to the cell membrane is an interaction partner (e.g., a ligand) for a protein selected from: CD3-TCR complex polypeptides (e.g., TCRα, TCRβ, TCRγ, TCRδ, CD3ε, CD3δ, CD3γ, CD3ζ, or CD3η), CD3, CD8, CD4, CD28, PD-1, CTLA-4, LAG-3, TIM-3, VISTA, TIGIT, BTLA, OX40, 4-1BB, ICOS, and CD27.

[0070] In some embodiments, the protein localized to the cell membrane is selected from: MHC polypeptides (e.g., MHC polypeptides as described below), PD-L1, PD-L2, CD80, CD86, HVEM, B7-H3, B7-H4, OX40L, 4-1BBL, ICOS-L, CD40, B7RP1, CD70, and GAL9. In a preferred embodiment, the protein localized to the cell membrane is an MHC polypeptide.

[0071] Major histocompatibility complex (MHC) polypeptide

[0072] In some aspects and embodiments, the polypeptides of the present disclosure comprise the amino acid sequence of a major histocompatibility complex (MHC) polypeptide.

[0073] As used herein, an "MHC polypeptide" refers to the constituent polypeptides of an MHC molecule. An MHC molecule, in turn, refers to a polypeptide complex formed by non-covalent interactions between MHC polypeptides and that is capable of binding and presenting a peptide. Thus, an MHC polypeptide is a polypeptide capable of interacting with another MHC polypeptide to form an MHC molecule.

[0074] The polypeptide complexes according to the present disclosure may be characterized by non-covalent protein:protein interactions between the constituent polypeptides / peptides. In some embodiments, the association includes electrostatic interactions (e.g., ionic bonding, hydrogen bonding) and / or van der Waals forces.

[0075] MHC molecules are broadly divided into two classes: class I and class II. MHC class I molecules are non-covalent heterodimers of an MHC class I alpha (α) chain polypeptide and a beta (β)2 microglobulin polypeptide. The MHC class I α-chain polypeptide has three domains named α1, α2, and α3. The α1 and α2 domains together form a groove to which the peptide presented by the MHC class I molecule binds to form an MHC class I:peptide complex. MHC class II molecules are non-covalent heterodimers of an MHC class II alpha (α) chain polypeptide and an MHC class II beta (β) chain polypeptide. The α-chain contains two domains named α1 and α2, and the β-chain similarly contains two domains named β1 and β2. The α1 and β1 domains together form a groove to which the peptide presented by the MHC class II molecule binds to form an MHC class II:peptide complex.

[0076] In humans, MHC class I and class II polypeptides are encoded by polymorphic human leukocyte antigen (HLA) genes, thereby encoding MHC complexes capable of binding different peptides and presenting said different peptides. The MHC class I α-chain polypeptides are encoded by HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G. The MHC class II α-chain polypeptides are encoded by HLA-DPA1, HLA-DQA1, HLA-DQA2, and HLA-DRA. The MHC class II β-chain polypeptides are encoded by HLA-DPB1, HLA-DQB1, HLA-DQB2, HLA-DRB1, HLA-DRB3, HLA-DRB4, and HLA-DRB5. The β2-microglobulin component of the MHC class I molecule is invariant and is encoded by B2M.

[0077] In some embodiments, the MHC polypeptide is selected from: β2-microglobulin, MHC class I α-chain polypeptide, MHC class II α-chain polypeptide, or MHC class II β-chain polypeptide. The MHC class I α-chain polypeptide can be an HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G polypeptide (e.g., an HLA-A, HLA-B, or HLA-C polypeptide). The HLA-A polypeptide can be an HLA-A02, HLA-A01, HLA-A03, HLA-A11, or HLA-A24 polypeptide. The MHC class II α-chain polypeptide can be an HLA-DPA1, HLA-DQA1, HLA-DQA2, or HLA-DRA polypeptide. The MHC class II β-chain polypeptide can be an HLA-DPB1, HLA-DQB1, HLA-DQB2, HLA-DRB1, HLA-DRB3, HLA-DRB4, or HLA-DRB5 polypeptide.

[0078] In preferred embodiments, the MHC polypeptide is a MHC class I molecule polypeptide. In further preferred embodiments, the MHC polypeptide is β2-microglobulin. The use of β2-microglobulin in the polypeptides of the present disclosure is advantageous because it provides a common marker for MHC class I molecules. That is, it provides a marker for MHC class I molecules that includes a diversity of MHC class I α-chain polypeptides.

[0079] The reference herein to a given MHC polypeptide also encompasses isotypes, fragments, variants, or homologs of related polypeptides from any species. By way of illustration, the reference to β2-microglobulin includes human β2-microglobulin having the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:3, and also includes isotypes, fragments, variants, or homologs of human β2-microglobulin.

[0080] As used herein, an isotype, fragment, variant, or homolog of a given reference polypeptide can be characterized by having at least 70% sequence identity, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity with the amino acid sequence of the reference polypeptide. A "fragment" generally refers to a portion of a reference protein. A "variant" generally refers to a protein having an amino acid sequence that contains one or more amino acid substitutions, insertions, deletions, or other modifications relative to the amino acid sequence of a reference protein, but retains a substantial degree of sequence identity (e.g., at least 60%) with the amino acid sequence of the reference protein. An "isotype" generally refers to a variant of a reference protein expressed by the same species as the reference protein. A "homolog" generally refers to a variant of a reference protein produced by a different species compared to the species of the reference protein. Homologs include orthologs.

[0081] An isotype, fragment, variant, or homolog of a given reference protein can optionally be characterized by having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity with the amino acid sequence of a designated isotype in the immature or mature (i.e., after processing to remove the signal peptide) form of a related polypeptide from a given species (e.g., human).

[0082] In some aspects and embodiments according to the present disclosure, the polypeptide comprises the amino acid sequence of β2-microglobulin. In some embodiments, the amino acid sequence of β2-microglobulin comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity with SEQ ID NO:3 or SEQ ID NO:1.

[0083] Portion that facilitates labeling of a polypeptide with an identifier portion

[0084] In aspects and embodiments of the present disclosure, the polypeptide further comprises a portion that facilitates labeling of the polypeptide with an identifier portion (e.g., an identifier portion as described below).

[0085] In some embodiments, the portion that facilitates labeling of the polypeptide with an identifier portion is or comprises an amino acid sequence that forms a peptide / polypeptide portion.

[0086] In some embodiments, the moiety facilitating the labeling of a polypeptide with an identifier moiety is or comprises a self-labeling protein tag. Self-labeling protein tags are described, for example, in Liss et al., Scientific Reports (2016) 5:17740 and Wilhelm et al., Biochemistry (2021) 60(33):2560-2575, which are hereby incorporated by reference in their entireties.

[0087] The self-labeling protein tag comprises or consists of the following: a moiety with covalently attached enzymatic activity having a catalytic moiety. For example, HaloTag is a haloalkane dehalogenase that can undergo an irreversible reaction with a primary alkyl halide (e.g., a haloalkane, such as a chloroalkane). Nucleophilic attack causes the halogen to be replaced by an amino acid residue, thereby forming a covalent alkyl-enzyme conjugate. Specifically, after replacement of the terminal halogen, an ester bond is formed between the COO - group of Asp106 of HaloTag and the terminal carbon of the alkane.

[0088] In some embodiments, the self-labeling protein tag is selected from HaloTag, SNAP-tag, CLIP-tag, ACP tag, or MCP tag. In a preferred embodiment, the self-labeling protein tag is HaloTag.

[0089] In some embodiments, the moiety facilitating the labeling of a polypeptide with an identifier moiety according to the present disclosure comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity with SEQ ID NO:4 or SEQ ID NO:5.

[0090] It should be understood that in some embodiments, labeling a polypeptide according to the present disclosure with an identifier moiety modifies the moiety facilitating the labeling of the polypeptide with the identifier moiety. In some embodiments, after labeling the polypeptide with the identifier moiety, via the moiety facilitating the labeling of the polypeptide with the identifier moiety, the moiety facilitating the labeling of the polypeptide is no longer suitable for facilitating the (further) labeling of the polypeptide. By way of illustration, in the example of a polypeptide comprising HaloTag, after labeling the polypeptide with a labeling moiety comprising a HaloTag ligand (which comprises a chloroalkane moiety and an identifier moiety), HaloTag is no longer able to facilitate the (further) labeling of the polypeptide because the COO - group of Asp106 is no longer available.

[0091] In some embodiments, in the amino acid sequence of the polypeptide of the present disclosure, the portion that facilitates labeling of the polypeptide with an identifier portion is located at the N-terminus of the amino acid sequence of the major histocompatibility complex (MHC) polypeptide. That is, in some embodiments, the recombinant polypeptide has the following structure: N-terminus - [... ] - [portion that facilitates labeling of the polypeptide with an identifier portion] - [amino acid sequence of the MHC polypeptide] - [... ] - C-terminus.

[0092] As used in the representation of the polypeptide structure herein, "[... ]" indicates the optional presence of other amino acid sequences / protein domains. For example, in the structure of the last sentence of the previous paragraph, other sequences of amino acid / protein domains may optionally be present downstream of the amino acid sequence of the MHC polypeptide and before the C-terminus of the polypeptide. Further, as used in the representation of the polypeptide structure herein, "-" indicates an optional linker sequence. For example, in the structure of the last sentence of the previous paragraph, a linker sequence may optionally be provided between the portion that facilitates labeling of the polypeptide with an identifier portion and the amino acid sequence of the MHC polypeptide.

[0093] Labeling portion

[0094] Aspects and embodiments of the present disclosure employ a labeling portion, such as for labeling a polypeptide with an identifier portion.

[0095] It should be understood that the labeling portion is selected to be compatible with the portion that facilitates labeling of the polypeptide with an identifier portion according to the present disclosure. The labeling portion according to the present disclosure generally comprises the identifier portion according to the present disclosure, and a portion for covalently attaching the identifier portion to the polypeptide by the portion that facilitates labeling of the polypeptide with an identifier portion.

[0096] In some embodiments, the labeling portion according to the present disclosure is or comprises a HaloTag ligand. The HaloTag ligand comprises a portion that can serve as a substrate for the HaloTag haloalkane dehalogenase for forming a covalent alkyl-enzyme conjugate. For example, the HaloTag ligand may comprise a primary alkyl halide portion, such as a haloalkane portion (e.g., a chloroalkane portion).

[0097] In some embodiments, the labeling portion according to the present disclosure comprises the identifier portion according to the present disclosure, and a chloroalkane portion.

[0098] Identifier portion and portion that facilitates labeling of a polypeptide with an identifier portion

[0099] In aspects and embodiments of the present disclosure, the polypeptide comprises an identifier portion.

[0100] As used herein, "identifier portion" refers to the following portion: a portion that can be used to identify a polypeptide containing the identifier portion. The identifier portion is preferably polymorphic, thereby providing a variety of polypeptides according to the present disclosure labeled with different identifier portions. The "identifier portion" according to the present disclosure can be any detectable portion that can be used to distinguish a polypeptide labeled with an identifier portion from a polypeptide not labeled with an identifier portion (e.g., a polypeptide not labeled with an identifier portion, or a polypeptide labeled with a different identifier portion). The identifier portion can also be referred to as a "barcode", and labeling polypeptides with different identifier portions can be referred to as "barcoding".

[0101] Identifier portions contemplated in connection with the present disclosure include, for example, nucleic acid, fluorescent, phosphorescent, luminescent, immunodetectable (e.g., epitope tags), radioactive, chemical, and enzyme labels. In a preferred embodiment, the identifier portion comprises or consists of a nucleic acid portion. That is, in a preferred embodiment, the identifier portion is or comprises a nucleic acid identifier portion.

[0102] Nucleic acids are particularly suitable for use as identifier portions because they are highly polymorphic (and thus can generate unique identifier portions with a very large variety), and techniques for their detection with high sensitivity and specificity are widely used (e.g., next-generation sequencing techniques).

[0103] In some embodiments, the nucleic acid portion according to the present disclosure can comprise or consist of the following: DNA or RNA. In some embodiments, the nucleic acid portion comprises or consists of a polynucleotide. "Polynucleotide" refers to a polymeric chain of multiple nucleotide monomers linked by bonds between the monomers, which are typically phosphodiester bonds (e.g., in the case of a polynucleotide formed from naturally occurring nucleotide monomers). Polynucleotides include oligonucleotides, which typically contain ≤50 nucleotides. The polynucleotide can be single-stranded or can be double-stranded (i.e., can comprise a duplex formed by hydrogen bonds between complementary nucleotides).

[0104] In some embodiments, the nucleic acid portion comprises or consists of single-stranded DNA (ssDNA). That is, in some embodiments, the identifier portion is an ssDNA identifier portion. In some embodiments, the nucleic acid portion comprises or consists of an ssDNA polynucleotide. In some embodiments, the ssDNA polynucleotide contains from 5 to 200 nucleotides, such as one of 10 to 100, 20 to 80, or 30 to 75 nucleotides.

[0105] It should be understood that the nucleic acid portion of the present disclosure is employed as the identifier portion. The nucleic acid portion can comprise a structure that provides for the detection of a polypeptide labeled with the nucleic acid portion through analysis of the nucleic acid portion. For example, where the nucleic acid portion comprises or consists of a polynucleotide, the nucleic acid portion can provide for detection through analysis of the nucleotide sequence of the polynucleotide.

[0106] Preferably, the identifier portion according to the present disclosure is employed to encode a peptide of interest (i.e., as an identifier therefor). That is, the identifier portion and the peptide of interest are employed in combination such that a given identifier portion corresponds to a specific peptide. The identifier portion can be used to readily identify a peptide:MHC complex presenting the peptide of interest, for example, in an internalized T cell (e.g., by trogocytosis). Detection of a given identifier portion in a T cell indicates that the T cell has internalized a peptide:MHC complex that presents the peptide encoded thereby.

[0107] As a result of sortase-mediated transfer, the identifier portion can also be used to readily identify a peptide:MHC complex that has interacted with a T cell through detection of the identifier portion on the T cell. According to such embodiments, detection of a given identifier portion on a T cell indicates that the T cell has interacted with a peptide:MHC complex that presents the peptide encoded thereby.

[0108] Sortase-mediated labeling

[0109] Aspects and embodiments of the present disclosure relate to the enzyme-catalyzed, site-specific labeling of polypeptides with an identifier portion, articles for such labeling reactions, and products thereof.

[0110] In particular, the present disclosure contemplates sortase-mediated transfer of an identifier portion between polypeptides. For example, site-specific labeling of polypeptides via sortase-catalyzed transpeptidation is described, for example, in Antos et al., Curr Protoc Protein Sci. (2009) Chapter 15: Unit - 15.3; and Popp et al., Curr Protoc Protein Sci. (2009) 15:3.1 - 3.9, both of which are hereby incorporated by reference in their entireties.

[0111] As used herein, "sortase" refers to an enzyme that recognizes and cleaves a polypeptide that contains a sortase substrate motif conforming to the consensus Leu-Pro / Ala-Xaa-Thr-Gly / Ala / Ser (SEQ ID NO: 25). Cleavage occurs between positions 4 and 5 of SEQ ID NO: 25 (i.e., at the peptide bond between Thr and Gly / Ala / Ser). Sortases can be used to transfer, via transpeptidation, positions 1 to 4 of the sortase substrate motif and the amino acids N-terminal thereto to a polypeptide that contains an appropriate sortase receptor motif. A review of sortases and their functions is provided, for example, in Mazmanian et al., Science (1999) 285(5428):760-763; and Paterson and Mitchell, Trends Microbiol. (2004) 12(2):89-95, which are hereby incorporated by reference in their entireties.

[0112] In aspects and embodiments according to the present disclosure, a sortase is employed to catalyze the transfer of (a) a region of a polypeptide that contains a sortase substrate motif (e.g., a region that contains an identifier portion, e.g., covalently linked thereto) to (b) a polypeptide that contains a sortase receptor motif. Sortase-mediated transfer indicates a physical interaction between polypeptides (a) and (b) because it requires their close physical proximity for efficient transfer to occur. Sortase-catalyzed labeling of cells during productive immunological synapses is described, for example, in Pasqual et al., Nature (2018) 553:496-500, which is hereby incorporated by reference in its entirety.

[0113] Based on sequence alignment and phylogenetic analysis of 61 sortases from Gram-positive bacterial genomes, sortases have been divided into four classes: sortase A, sortase B, sortase C, and sortase D (see Dramsi et al., Res Microbiol. (2005) 156(3):289-97).

[0114] Sortases include sortase A, which is identified by the International Enzyme Commission number 3.4.22.70. Sortase A encompasses sortase A from Staphylococcus aureus ( Also referred to as "Sa-SrtA"), which has the amino acid sequence shown in SEQ ID NO: 19. The region of Staphylococcus aureus sortase A that confers sortase activity is shown in SEQ ID NO: 20. Sortase A also encompasses sortase A from Streptococcus pyogenes ( Also referred to as“Sa-SrtA”), which has the amino acid sequence shown in SEQ ID NO: 23. The region of Streptococcus pyogenes sortase A that confers sortase activity is shown in SEQ ID NO: 24.

[0115] The present disclosure also contemplates sortase variants. Sortase variants are described, for example, in Dorr et al., PNAS US (2014) 111(37):13343-13348; Chen et al., PNAS USA (2011) 108(28):11399-11404; and Chen et al., Sci Rep. (2016) 6:31899, which are hereby incorporated by reference in their entireties.

[0116] In some embodiments according to various aspects of the present disclosure, the sortase is selected from: sortase A (EC 3.4.22.70), Staphylococcus aureus sortase A or a variant thereof, and Streptococcus pyogenes sortase A or a variant thereof.

[0117] A variant of sortase A from Staphylococcus aureus (i.e., a variant of Staphylococcus aureus sortase A) refers to a polypeptide having at least 70% amino acid sequence identity (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98% or ≥99% amino acid sequence identity) with the amino acid sequence of SEQ ID NO: 19. Similarly, a variant of sortase A from Streptococcus pyogenes (i.e., a variant of Streptococcus pyogenes sortase A) refers to a polypeptide having at least 70% amino acid sequence identity (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98% or ≥99% amino acid sequence identity) with the amino acid sequence of SEQ ID NO: 23. It should be understood that variants of Staphylococcus aureus sortase A and variants of Streptococcus pyogenes sortase A preferably retain sortase activity.

[0118] It should be understood that a sortase variant contains one or more (e.g., one of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) modifications (e.g., Staphylococcus aureus sortase A, Streptococcus pyogenes sortase A) relative to the amino acid sequence of a reference sortase.

[0119] "Modification" refers to a difference relative to a reference amino acid sequence. The reference amino acid sequence can be the amino acid sequence encoded by the most common nucleotide sequence of the gene encoding the relevant protein. In the examples herein (and more generally in the art), "modification" can also be referred to as "substitution" or "mutation". Modifications generally include substituting an amino acid residue with a "replacement" amino acid residue that is not the same. The replacement amino acid residue according to the modification of the present disclosure can be a naturally occurring amino acid residue (i.e., encoded by the genetic code), which is different from the amino acid residue at the relevant position of the amino acid sequence before modification, and is selected from: alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Val). In some embodiments, the modified replacement amino acid residue can be a non-naturally occurring amino acid residue - i.e., an amino acid residue other than those listed in the previous sentence. Examples of non-naturally occurring amino acid residues include norleucine, ornithine, norvaline, homoserine, aib, and other amino acid residue analogs, such as those described in Ellman et al., Meth. Enzym. 202 (1991) 301-336.

[0120] In some embodiments, the sortase variant according to the present disclosure comprises an amino acid sequence having at least 70% amino acid sequence identity with SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 23, or SEQ ID NO: 24, and comprises one or more (e.g., one of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) modifications relative to SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 23, or SEQ ID NO: 24.

[0121] In some embodiments, the sortase variant comprises one or more (e.g., one of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) of the following modifications relative to SEQ ID NO:19 or SEQ ID NO:20: K196T, P86L, P94S, P94R, N98S, A104T, E106G, A118T, F122S, F122Y, D124G, N127S, K134R, F154R, D160N, D165A, K173E, G174S, K177E, 1182V, and / or K190E. In some embodiments, the sortase variant comprises K196T relative to SEQ ID NO:19 or SEQ ID NO:20. It should be understood that the modifications in the previous sentence are numbered according to SEQ ID NO:19, and it should be understood that when the relevant modification is alternatively provided in SEQ ID NO:20, the corresponding position of SEQ ID NO:20 is alternatively modified. By way of illustration, when the modification "K196T" is provided in SEQ ID NO:20, the "K" at position 137 of SEQ ID NO:20 is replaced with "T".

[0122] In some embodiments according to various aspects of the present disclosure, the sortase comprises or consists of: an amino acid sequence having the amino acid sequence of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24, or an amino acid sequence having at least 70% amino acid sequence identity (e.g., one of ≥ 75%, ≥ 80%, ≥ 85%, ≥ 90%, ≥ 91%, ≥ 92%, ≥ 93%, ≥ 94%, ≥ 95%, ≥ 96%, ≥ 97%, ≥ 98%, or ≥ 99% sequence identity) with SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24. In some embodiments, the sortase comprises or consists of: an amino acid sequence having the amino acid sequence of SEQ ID NO: 19, or an amino acid sequence having at least 70% amino acid sequence identity (e.g., one of ≥ 75%, ≥ 80%, ≥ 85%, ≥ 90%, ≥ 91%, ≥ 92%, ≥ 93%, ≥ 94%, ≥ 95%, ≥ 96%, ≥ 97%, ≥ 98%, or ≥ 99% sequence identity) with SEQ ID NO: 19. In some embodiments, the sortase comprises or consists of: an amino acid sequence having the amino acid sequence of SEQ ID NO: 20, or an amino acid sequence having at least 70% amino acid sequence identity (e.g., one of ≥ 75%, ≥ 80%, ≥ 85%, ≥ 90%, ≥ 91%, ≥ 92%, ≥ 93%, ≥ 94%, ≥ 95%, ≥ 96%, ≥ 97%, ≥ 98%, or ≥ 99% sequence identity) with SEQ ID NO: 20. In some embodiments, the sortase comprises or consists of: an amino acid sequence having the amino acid sequence of SEQ ID NO: 21, or an amino acid sequence having at least 70% amino acid sequence identity (e.g., one of ≥ 75%, ≥ 80%, ≥ 85%, ≥ 90%, ≥ 91%, ≥ 92%, ≥ 93%, ≥ 94%, ≥ 95%, ≥ 96%, ≥ 97%, ≥ 98%, or ≥ 99% sequence identity) with SEQ ID NO: 21. In some embodiments, the sortase comprises or consists of: an amino acid sequence having the amino acid sequence of SEQ ID NO: 22, or an amino acid sequence having at least 70% amino acid sequence identity (e.g., one of ≥ 75%, ≥ 80%, ≥ 85%, ≥ 90%, ≥ 91%, ≥ 92%, ≥ 93%, ≥ 94%, ≥ 95%, ≥ 96%, ≥ 97%, ≥ 98%, or ≥ 99% sequence identity) with SEQ ID NO: 22.In some embodiments, the sortase comprises or consists of: an amino acid sequence having the amino acid sequence of SEQ ID NO:23, or an amino acid sequence having at least 70% amino acid sequence identity (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98% or ≥99% sequence identity) with SEQ ID NO:23. In some embodiments, the sortase comprises or consists of: an amino acid sequence having the amino acid sequence of SEQ ID NO:24, or an amino acid sequence having at least 70% amino acid sequence identity (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98% or ≥99% sequence identity) with SEQ ID NO:24.

[0123] In some embodiments according to various aspects of the present disclosure, the sortase substrate motif comprises or consists of: a substrate motif for a sortase selected from: sortase A (EC 3.4.22.70), Staphylococcus aureus sortase A or a variant thereof, and Streptococcus pyogenes sortase A or a variant thereof.

[0124] Sortase substrate motifs include those that conform to the consensus Leu - Pro - Xaa - Thr - Gly (SEQ ID NO:26), which serve as substrates for Staphylococcus aureus sortase A and Streptococcus pyogenes sortase A; and those that conform to the consensus Leu - Pro - Xaa - Thr - Ala (SEQ ID NO:27) or Leu - Pro - Xaa - Thr - Ser (SEQ ID NO:28), which serve as substrates for Streptococcus pyogenes sortase A (see, for example, Johnson et al., J Biol Chem. (2022) 298(10):102446).

[0125] As used herein, where a given sortase substrate motif is referred to as "serving as a substrate for a given reference sortase", the given reference sortase is capable of catalyzing the cleavage of the sortase substrate motif (i.e., at the peptide bond between positions 4 and 5 of SEQ ID NO:25).

[0126] Thus, in some embodiments, the sortase substrate motif comprises or consists of: an amino acid sequence conforming to the consensus sequence of any one of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27 or SEQ ID NO:28. In some embodiments, the sortase substrate motif comprises or consists of: an amino acid sequence conforming to the consensus sequence of SEQ ID NO:25. In some embodiments, the sortase substrate motif comprises or consists of: an amino acid sequence conforming to the consensus sequence of SEQ ID NO:26. In some embodiments, the sortase substrate motif comprises or consists of: an amino acid sequence conforming to the consensus sequence of SEQ ID NO:27. In some embodiments, the sortase substrate motif comprises or consists of: an amino acid sequence conforming to the consensus sequence of SEQ ID NO:28.

[0127] In some embodiments, the sortase substrate motif can be provided at or near the C-terminus of a polypeptide comprising the sortase substrate motif. That is, in some embodiments, position 1 of the sequence conforming to the consensus sequence of SEQ ID NO:25 can be provided within 50 amino acids, such as within one of 40, 30, 25, 20, 15, 10 or 5 amino acids of the C-terminus of the polypeptide.

[0128] Sortase catalyzes the transfer of a polypeptide / peptide moiety from a protein comprising a sortase substrate motif for the sortase to a protein comprising a sortase receptor motif for the sortase. The sortase receptor motif is typically an oligopeptide sequence of small, non-polar, hydrophobic amino acids (e.g., glycine or alanine), which is provided at the N-terminus of a polypeptide / peptide comprising the oligopeptide. The most characteristic sortase receptor motif is the N-terminal oligoglycine, which serves as a receptor motif for, for example, Staphylococcus aureus sortase A.

[0129] As used herein, where a given sortase receptor motif is referred to as "serving as a receptor motif for a given reference sortase", the given sortase receptor motif is capable of serving as a nucleophile for an intermediate formed by cleavage of a polypeptide / peptide by the sortase, the polypeptide / peptide comprising an appropriate sortase substrate motif for the given reference sortase, i.e., in the context of a transpeptidation reaction.

[0130] In some embodiments, the sortase receptor motif can comprise or consist of: at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (e.g., 2 to 10, such as 4) amino acids, which are provided at the N-terminus of a polypeptide / peptide comprising such a motif, wherein the amino acids are selected from glycine and alanine.

[0131] In some embodiments, the sortase receptor motif may comprise or consist of: providing in the presence of a G n G at the N-terminus of the polypeptide / peptide n (i.e., "N-terminal-G n ”), wherein “n” is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, “n” is at least 2. In some embodiments, “n” is 2 or 10. In some embodiments, “n” is 4. The sortase receptor motif “N-terminal-G4” is used as a receptor motif for Staphylococcus aureus sortase A and Streptococcus pyogenes sortase A.

[0132] In some embodiments, the sortase receptor motif may comprise or consist of: providing an A at the N-terminus n (i.e., "N-terminal-A n ”), wherein “n” is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, “n” is at least 2. In some embodiments, “n” is 2 or 10. In some embodiments, “n” is 4.

[0133] It should be understood that where aspects or embodiments of the present disclosure relate to (i) a polypeptide comprising a sortase substrate motif and (ii) a sortase, the sortase substrate motif and the sortase are preferably selected to be compatible with each other. Similarly, where aspects or embodiments relate to (i) a polypeptide comprising a sortase receptor motif and (ii) a sortase, the sortase receptor motif and the sortase are preferably selected to be compatible with each other. Similarly, where aspects or embodiments relate to (i) a polypeptide comprising a sortase substrate motif, (ii) a polypeptide comprising a sortase receptor motif and (iii) a sortase, the sortase substrate motif, the sortase receptor motif and the sortase are preferably selected to be compatible with each other.

[0134] A given sortase substrate motif and a given sortase are compatible if the sortase catalyzes cleavage of the sortase substrate motif (i.e., at the peptide bond between position 4 and position 5 of SEQ ID NO: 25). As an illustration, a sortase consisting of the amino acid sequence of SEQ ID NO: 22 catalyzes cleavage of a polypeptide / peptide comprising a sortase substrate motif that conforms to the consensus term of SEQ ID NO: 26, and thus this combination of sortase substrate motif and sortase is compatible.

[0135] A sortase receptor motif is compatible with a given sortase if the sortase receptor motif serves as a nucleophile for an intermediate formed by cleavage of a polypeptide / peptide by the sortase, where the polypeptide / peptide comprises an appropriate sortase substrate motif for the sortase. By way of illustration, the sortase receptor motif "N-terminal-G4" serves as a nucleophile for an intermediate formed by cleavage of a polypeptide / peptide that comprises an appropriate sortase substrate motif for a sortase consisting of the amino acid sequence of SEQ ID NO:22, and thus this combination of sortase receptor motif and sortase is compatible.

[0136] A given sortase substrate motif, a given sortase receptor motif, and a given sortase are compatible if the sortase catalyzes cleavage of the sortase substrate motif and the sortase receptor motif serves as a nucleophile for an intermediate formed by cleavage of the sortase substrate motif by the sortase. By way of illustration, the sortase receptor motif "N-terminal-G4" serves as a nucleophile for an intermediate formed by cleavage of a polypeptide / peptide by a sortase consisting of the amino acid sequence of SEQ ID NO:22, where the polypeptide / peptide comprises a sortase substrate motif conforming to the consensus of SEQ ID NO:26, and thus this combination of sortase receptor motif, sortase substrate motif, and sortase is compatible.

[0137] It should be understood that in aspects and embodiments in which a polypeptide comprises a sortase substrate motif, the sortase substrate motif is positioned within the amino acid sequence of the polypeptide such that, for example when the polypeptide comprises such a portion (e.g., after covalently associating an identifier portion with the polypeptide via a bond formed by a self-labeling protein tag), sortase-mediated transfer of the identifier portion is permitted. That is, in embodiments in which the polypeptide comprises a sortase substrate motif and a portion that facilitates labeling of the polypeptide with an identifier portion (e.g., a self-labeling protein tag such as HaloTag), in the amino acid sequence of the polypeptide, the sortase substrate motif is provided downstream (i.e., at its C-terminus) of the portion that facilitates labeling of the polypeptide with the identifier portion. Similarly, in embodiments in which the polypeptide comprises a sortase substrate motif and an identifier portion that is covalently associated with the polypeptide via a bond formed by a self-labeling protein tag (e.g., HaloTag), the sortase substrate motif is provided downstream (i.e., at its C-terminus) of the self-labeling protein tag. That is, in some embodiments, the polypeptide comprises the following structure: N-terminal - [... ] - [portion that facilitates labeling of the polypeptide with an identifier portion] - [sortase substrate motif] - [... ] - C-terminal.

[0138] Similarly, it should be understood that in aspects and embodiments in which the polypeptide comprises a sortase receptor motif, the sortase receptor motif is positioned within the amino acid sequence of the polypeptide, such as to permit sortase-mediated transfer of an identifier moiety to the polypeptide. That is, in the case where the polypeptide comprises a sortase receptor motif, the sortase receptor motif is provided at the N-terminus of the polypeptide.

[0139] Aspects and embodiments according to the present disclosure provide cells comprising a polypeptide, wherein the polypeptide comprises a sortase receptor motif. In preferred embodiments, the cell is a T cell. T cells according to the present disclosure may express a CD3-TCR complex. In some embodiments, the T cell is a CD3+, CD4+ T cell. In some embodiments, the T cell is a CD3+, CD8+ T cell. In some embodiments, the T cell is a T helper cell (T H cell). In some embodiments, the T cell is a cytotoxic T cell (e.g., cytotoxic T lymphocyte (CTL)).

[0140] In some embodiments, the T cell comprises, at its cell surface, a polypeptide comprising a sortase receptor motif. That is, in some embodiments, the polypeptide comprising a sortase receptor motif is present in or at the cell surface of the T cell.

[0141] As a result of modifying the T cell to comprise a polypeptide, the T cell may comprise a polypeptide comprising a sortase receptor motif. In some embodiments, as a result of modifying the T cell to comprise a nucleic acid encoding a polypeptide comprising a sortase receptor motif (e.g., using a suitable genetic engineering platform), the T cell may comprise a polypeptide comprising a sortase receptor motif.

[0142] However, in preferred embodiments, the T cell is modified to comprise a polypeptide comprising a sortase receptor motif by non-genetic modification of the T cell. For example, the polypeptide may be non-genetically bioconjugated to the T cell. Non-genetic bioconjugation techniques are reviewed in, for example, Roy et al., Bioconjug Chem. (2020) 31(11):2465-2475, which is hereby incorporated by reference in its entirety. Advantageously, such techniques do not require genetic engineering to produce T cells comprising a polypeptide comprising a sortase receptor motif.

[0143] In some embodiments, the T cell is a non-genetically modified T cell. In some embodiments, T cells according to the present disclosure do not comprise exogenous nucleic acids.

[0144] Non-genetic bioconjugation strategies generally involve attaching an exogenous functional group to the cell membrane without altering the genetic nature of the cell.

[0145] Polypeptides comprising sortase receptor motifs can be attached to the surface of T cells by binding to functional groups metabolically introduced into the T cells. An azide moiety, an alkyne moiety, or a ketone moiety can be introduced into the polysaccharides presented at the cell surface by metabolic glycan labeling (MGL).

[0146] In some aspects and embodiments, the present disclosure provides T cells comprising a polypeptide that comprises a sortase receptor motif, wherein the polypeptide is linked to the cell surface of the T cell via metabolic glycan labeling of the T cell, followed by click-chemistry-based conjugation of the polypeptide. The polypeptide can be said to be "anchored" or having been "anchored" on the T cell, i.e., on or at the cell surface of the T cell.

[0147] In some embodiments, the polypeptide comprising the sortase receptor motif is provided at the cell surface of the T cell because the polypeptide covalently associates with a molecule in or at the cell membrane of the T cell. In some embodiments, the polypeptide comprising the sortase receptor motif is provided at the cell surface of the T cell because of non-covalent interactions between the polypeptide and a molecule in or at the cell membrane of the T cell.

[0148] For example, by culturing cells in a cell culture medium containing N-azidoacetylmannosamine (ManNAz), the sialic acid residues of the cell surface glycans can be modified to contain an azide moiety. A polypeptide comprising a sortase receptor motif and functionalized with a cyclic alkyne moiety (e.g., a dibenzocyclooctyl (DBCO) moiety or an azidodibenzocyclooctyne (ADIBO) moiety) can then be conjugated to the azide-modified cell surface polysaccharides via strain-promoted alkyne-azide cycloaddition (SPAAC).

[0149] Thus, in some embodiments, the polypeptide comprising the sortase receptor motif further comprises a moiety suitable for conjugating the polypeptide to an interaction partner labeled with an azide moiety via SPAAC. In some embodiments, the moiety suitable for binding to an interaction partner conjugated to an azide moiety via SPAAC is or comprises a cyclic alkyne moiety (e.g., a DBCO moiety or an azidodibenzocyclooctyne (ADIBO) moiety). In some embodiments, the cyclic alkyne moiety is or comprises a DBCO moiety.

[0150] In some aspects and embodiments, the present disclosure provides T cells comprising a polypeptide that comprises a sortase receptor motif, wherein the polypeptide is conjugated to the cell surface of the T cell via a SPAAC reaction between a cyclic alkyne moiety (e.g., a DBCO moiety or an ADIBO moiety) and an azide-modified cell surface polysaccharide.

[0151] In some aspects and embodiments, the T cell further comprises a sortase, such as a sortase according to the embodiments described above. Thus, aspects and embodiments according to the present disclosure provide a T cell that comprises (i) a polypeptide comprising a sortase receptor motif and (ii) a sortase.

[0152] Since the T cell is modified to comprise a sortase, the T cell can comprise a sortase. In some embodiments, since the T cell is modified to comprise a nucleic acid encoding a sortase (e.g., using a suitable genetic engineering platform), the T cell can comprise a sortase.

[0153] However, in preferred embodiments, the T cell is modified to comprise a sortase by non-genetic modification of the T cell. In some embodiments, the T cell is modified to comprise a sortase via bioconjugation, such as via metabolic glycan labeling of the T cell, followed by click chemistry-based conjugation of the sortase. The sortase can be said to be "immobilized" on or at the cell surface of the T cell, i.e., on the cell surface of the T cell.

[0154] In some embodiments, the sortase is provided at the cell surface of the T cell because the sortase or a polypeptide comprising a sortase moiety is covalently associated with a molecule in or at the cell membrane of the T cell. In some embodiments, the sortase or a polypeptide comprising a sortase moiety is provided at the cell surface of the T cell because of non-covalent interactions between the sortase / comprising a sortase moiety polypeptide and a molecule in or at the cell membrane of the T cell. Such non-covalent interactions can be protein:protein interactions. In some embodiments, the interactions include electrostatic interactions (e.g., ionic bonding, hydrogen bonding) and / or van der Waals forces. Such non-covalent interactions can be of the type observed in antibody:antigen interactions.

[0155] In some embodiments, the T cell is modified to comprise a sortase via metabolic glycan labeling of the T cell, click chemistry-based conjugation of a hapten, and application of an antigen-binding molecule that binds to the hapten, wherein the antigen-binding molecule comprises a sortase or is linked to the sortase. In some embodiments, the hapten can be selected from DOTAM, DOTA, digoxin, biotin, and fluorescein. In some embodiments, the hapten is DOTAM or DOTA.

[0156] For example, cells can be cultured in a cell culture medium containing ManNAz to modify the sialic acid residues of cell surface polysaccharides to contain azide moieties. Subsequently, a molecule containing a cycloalkyne moiety (e.g., a DBCO moiety or an ADIBO moiety) conjugated to a hapten (e.g., DOTAM or DOTA) can be employed to conjugate the hapten to the azide-modified cell surface polysaccharide via SPAAC. Subsequently, an antigen-binding molecule that specifically binds to the hapten and contains sortase (i.e., as a fusion polypeptide) can be administered to the cells, thereby non-covalently modifying the cells to contain sortase at the cell surface.

[0157] In some aspects and embodiments, the present disclosure provides T cells containing a hapten (e.g., DOTAM or DOTA) that is conjugated to the cell surface of the T cell via a SPAAC reaction between a cycloalkyne moiety (e.g., a DBCO moiety or an ADIBO moiety) and an azide-modified cell surface polysaccharide. In some aspects and embodiments, the present disclosure provides T cells containing a hapten (e.g., DOTAM or DOTA) that is conjugated to the cell surface of the T cell via a SPAAC reaction between a cycloalkyne moiety (e.g., a DBCO moiety or an ADIBO moiety) and an azide-modified cell surface glycan, and the T cell further contains an antigen-binding molecule that specifically binds to the hapten, wherein the antigen-binding molecule contains sortase.

[0158] The present disclosure also provides a compound containing a hapten (e.g., DOTAM or DOTA) and a cycloalkyne moiety (e.g., a DBCO moiety or an ADIBO moiety).

[0159] In some aspects and embodiments, the present disclosure provides a compound containing a hapten (e.g., DOTAM or DOTA) that is conjugated to a cycloalkyne moiety (e.g., a DBCO moiety) via a bond. In some embodiments, the bond is or contains a thiourea moiety. Thus, the binding of the hapten to the cycloalkyne moiety is achieved by forming a thiourea bond, for example, by the reaction of an isothiocyanate (NCS) group with a primary amine group.

[0160] Accordingly, the present disclosure provides a compound of structure (I):

[0161] A-L-B (I)

[0162] wherein A is a hapten, L is a bond, and B is a cycloalkyne moiety. In some embodiments, the bond L is or contains a thiourea moiety.

[0163] In some embodiments, the hapten is or contains DOTAM. In some embodiments, DOTAM contains a C-functionalized cyclen moiety. In some embodiments, the C-functionalized cyclen moiety contains an NCS group.

[0164] Thus, in some embodiments, the hapten comprises or consists of structure (II):

[0165]

[0166] In some embodiments, the hapten comprises DOTA. In some embodiments, DOTA comprises a C-functionalized cyclen moiety. In some embodiments, the C-functionalized cyclen moiety comprises an NCS group.

[0167] Thus, in certain embodiments, the hapten comprises structure III:

[0168]

[0169] In some aspects and embodiments, the cycloalkyne moiety comprises an amine group. In some embodiments, the cycloalkyne moiety comprises DBCO-amine. DBCO-amine has structure IV:

[0170]

[0171] In preferred embodiments, the compound comprises DOTAM conjugated to DBCO via a thiourea bond. In some embodiments, the compound has structure V:

[0172]

[0173] In certain embodiments, the compound comprises DOTA conjugated to DBCO via a thiourea bond. In some embodiments, the compound has structure VI:

[0174]

[0175] It should be understood that the compounds recited herein include, where appropriate, their salts, hydrates, and solvates.

[0176] In aspects and embodiments of the present disclosure, sortase is provided as a fusion protein fused to a polypeptide of an antigen-binding molecule. In such aspects and embodiments, the antigen-binding molecule can be described as comprising a sortase moiety.

[0177] Accordingly, the present disclosure also provides an antigen-binding molecule comprising a sortase moiety. The present disclosure also provides a nucleic acid or nucleic acids encoding such an antigen-binding molecule. An expression vector or expression vectors comprising the nucleic acid or nucleic acids encoding such an antigen-binding molecule are also provided. A cell comprising such an antigen-binding molecule, such nucleic acid or nucleic acids, or such expression vector or expression vectors is also provided. A composition comprising such an antigen-binding molecule, such nucleic acid or nucleic acids, such expression vector or expression vectors, or such cell is also provided.

[0178] The sortase moiety may comprise or consist of: a sortase or variant thereof according to any of the embodiments described herein.

[0179] As used herein, an "antigen-binding molecule" refers to a molecule that binds to a given target antigen. Antigen-binding molecules include antibodies (i.e., immunoglobulins (Ig)) and antigen-binding fragments and derivatives thereof. In some embodiments, the antigen-binding molecule according to the present disclosure comprises or consists of: monoclonal antibodies, monospecific antibodies, multispecific (e.g., bispecific, trispecific, etc.) antibodies, variable fragment (Fv) molecules, single-chain Fv (scFv) molecules, fragment antigen-binding (Fab) molecules, single-chain Fab molecules (scFab), crossFab molecules, Fab' molecules, Fab'-SH molecules, F(ab')2 molecules, diabody molecules, triabody molecules, scFv-Fc molecules, microantibody molecules, heavy-chain-only antibody (HCAb) molecules, or single-domain antibody (dAb, VHH) molecules.

[0180] Antigen-binding molecules according to the present disclosure also include additional target antigen-binding peptides / polypeptides, such as peptide aptamers, thioredoxins, anticalins, Kunitz domains, Avimers, knottins, fynomers, atrimers, DARPins, affibodies, affilins, ArmRPs, O-Bodies, and adnectins (reviewed in, for example, Reverdatto et al., Curr Top Med Chem. 2015;15(12):1082–1101, which is hereby incorporated by reference in its entirety (see also, for example, Boersma et al., J Biol Chem (2011) 286:41273-85 and Emanuel et al., Mabs (2011) 3:38-48)). Antigen-binding molecules according to the present disclosure also include target antigen-binding nucleic acids, such as nucleic acid aptamers (reviewed in, for example, Zhou and Rossi Nat Rev Drug Discov. 2017 16(3):181-202). Antigen-binding molecules according to the present disclosure also include target antigen-binding small molecules (e.g., low molecular weight (<1000 daltons, typically between about 300 daltons and 700 daltons) organic compounds).

[0181] In some embodiments, the antigen-binding molecule according to the present disclosure is or comprises an antigen-binding peptide / polypeptide or an antigen-binding peptide / polypeptide complex. The antigen-binding molecule can comprise more than one peptide / polypeptide, and the more than one peptide / polypeptide together form the antigen-binding molecule. The peptide / polypeptide can be covalently or non-covalently associated. In some embodiments, the peptide / polypeptide forms part of a larger polypeptide comprising the peptide / polypeptide (e.g., in the case of an scFv molecule comprising a VH region and a VL region, or in the case of an scFab molecule comprising a VH-CH1 and a VL-CL region).

[0182] In some embodiments, the antigen-binding molecule comprises the variable heavy (VH) region of an antibody and the variable light (VL) region of an antibody that are capable of binding to a given target antigen. In some embodiments, the antigen-binding molecule comprises or consists of an Fv molecule: the Fv molecule is formed by the VH region and the VL region of an antibody that are capable of binding to a given target antigen. In some embodiments, the VH region and the VL region can be provided in the same polypeptide and are linked by a linker sequence. In some embodiments, the antigen-binding molecule comprises or consists of: an scFv molecule that binds to a given target antigen.

[0183] Antigen-binding molecules of the present disclosure generally comprise six complementarity-determining regions (CDRs); three in the heavy-chain variable (VH) region: HC-CDR1, HC-CDR2, and HC-CDR3, and three in the light-chain variable (VL) region: LC-CDR1, LC-CDR2, and LC-CDR3. Together, these six CDRs define the paratope of the antigen-binding molecule, which is the portion of the molecule that binds to the target antigen. The VH and VL regions contain framework regions (FRs) flanking each CDR, which provide a scaffold for the CDRs. From the N-terminus to the C-terminus, the VH region has the following structure: N-terminus - [HC-FR1] - [HC-CDR1] - [HC-FR2] - [HC-CDR2] - [HC-FR3] - [HC-CDR3] - [HC-FR4] - C-terminus; and the VL region has the following structure: N-terminus - [LC-FR1] - [LC-CDR1] - [LC-FR2] - [LC-CDR2] - [LC-FR3] - [LC-CDR3] - [LC-FR4] - C-terminus.

[0184] There are several different conventions for defining antibody CDRs and FRs, such as (i) the Kabat system, which is described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991); (ii) the Chothia system, which is described in Chothia et al., J. Mol. Biol. 196:901-917 (1987); and (iii) the international IMGT (ImMunoGeneTics) information system (LeFranc et al., Nucleic Acids Res. (2015) 43 (Database issue): D413-22), which uses the IMGT V-DOMAIN numbering rules as described in Lefranc et al., Dev. Comp. Immunol. (2003) 27:55-77. In some embodiments, the CDRs and FRs of the VH and VL regions of the antigen-binding molecules described herein are defined according to the Kabat system, the Chothia system, or the IMGT information system.

[0185] In some embodiments, the antigen-binding molecules of the present disclosure bind to a hapten (e.g., DOTAM or DOTA). In some embodiments, the antigen-binding molecule comprises the CDRs of the antigen-binding molecule that binds to a hapten (e.g., the haptens as described herein, e.g., DOTAM or DOTA). In some embodiments, the antigen-binding molecule comprises the FRs of the antigen-binding molecule that binds to a hapten (e.g., the haptens as described herein, e.g., DOTAM or DOTA). In some embodiments, the antigen-binding molecule comprises the CDRs and FRs of the antigen-binding molecule that binds to a hapten (e.g., the haptens as described herein, e.g., DOTAM or DOTA). That is, in some embodiments, the antigen-binding molecule comprises the VH and VL regions of the antigen-binding molecule that binds to a hapten (e.g., the haptens as described herein, e.g., DOTAM or DOTA).

[0186] In some embodiments, the antigen-binding molecule comprises the CDRs, FRs, and / or VH and / or VL regions of a DOTAM-binding antibody, or the CDRs, FRs, and / or VH and / or VL regions of those DOTAM-binding antibodies derived from a DOTAM-binding antibody. In some embodiments, the antigen-binding molecule comprises the CDRs, FRs, and / or VH and / or VL regions of the DOTAM-binding antibody described in WO 2019 / 202399 A1, or the CDRs, FRs, and / or VH and / or VL regions of those DOTAM-binding antibodies derived from the DOTAM-binding antibody described in WO 2019 / 202399 A1.

[0187] WO 2019 / 202399 A1 describes a DOTAM-binding antibody such as PRIT-213, the antigen-binding region of which is formed by a VH region having the amino acid sequence of SEQ ID NO:33 (SEQ ID NO:7 of WO 2019 / 202399 A1) and a VL region having the amino acid sequence of SEQ ID NO:41 (SEQ ID NO:8 of WO 2019 / 202399 A1). In some embodiments, the antigen-binding molecule comprises the CDRs, FRs, and / or VH and / or VL regions of PRIT-213, or the CDRs, FRs, and / or VH and / or VL regions of those CDRs, FRs, and / or VH and / or VL regions derived from PRIT-213.

[0188] In some embodiments, the antigen-binding molecule comprises:

[0189] (1) A VH region incorporating the following CDRs:

[0190] HC-CDR1 having the amino acid sequence of SEQ ID NO:34

[0191] HC-CDR2 having the amino acid sequence of SEQ ID NO:35

[0192] HC-CDR3 having the amino acid sequence of SEQ ID NO:36,

[0193] or a variant thereof, wherein one or two or three amino acids in HC-CDR1, and / or one or two or three amino acids in HC-CDR2, and / or one or two or three amino acids in HC-CDR3 are replaced by another amino acid.

[0194] In some embodiments, the antigen-binding molecule comprises:

[0195] (2) a VH region incorporating the following FRs:

[0196] HC-FR1 having the amino acid sequence of SEQ ID NO:37

[0197] HC-FR2 having the amino acid sequence of SEQ ID NO:38

[0198] HC-FR3 having the amino acid sequence of SEQ ID NO:39

[0199] HC-FR4 having the amino acid sequence of SEQ ID NO:40,

[0200] or a variant thereof, wherein one or two or three amino acids in HC-FR1, and / or one or two or three amino acids in HC-FR2, and / or one or two or three amino acids in HC-FR3, and / or one or two or three amino acids in HC-FR4 are replaced by another amino acid.

[0201] In some embodiments, the antigen-binding portion comprises a VH region comprising the CDRs according to (1) and the FRs according to (2).

[0202] In some embodiments, the antigen-binding molecule comprises:

[0203] (3) a VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least ≥75%, ≥80%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity with the amino acid sequence of SEQ ID NO:33.

[0204] In some embodiments, the antigen-binding portion comprises:

[0205] (4) The VL region incorporates the following CDRs:

[0206] LC-CDR1, which has the amino acid sequence of SEQ ID NO:42

[0207] LC-CDR2, which has the amino acid sequence of SEQ ID NO:43

[0208] LC-CDR3, which has the amino acid sequence of SEQ ID NO:44,

[0209] or a variant thereof, wherein one or two or three amino acids in LC-CDR1, and / or one or two or three amino acids in LC-CDR2, and / or one or two or three amino acids in LC-CDR3 are replaced by another amino acid.

[0210] In some embodiments, the antigen-binding portion comprises:

[0211] (5) The VL region incorporates the following FRs:

[0212] LC-FR1, which has the amino acid sequence of SEQ ID NO:45

[0213] LC-FR2, which has the amino acid sequence of SEQ ID NO:46

[0214] LC-FR3, which has the amino acid sequence of SEQ ID NO:47

[0215] LC-FR4, which has the amino acid sequence of SEQ ID NO:48,

[0216] or a variant thereof, wherein one or two or three amino acids in LC-FR1, and / or one or two or three amino acids in LC-FR2, and / or one

[0217] or two or three amino acids in LC-FR3, and / or one or two or three amino acids in LC-FR4 are replaced by another amino acid.

[0218] In some embodiments, the antigen-binding portion comprises a VL region, and the VH region comprises the CDRs according to (4) and the FRs according to (5).

[0219] In some embodiments, the antigen-binding portion comprises:

[0220] (6) The VL region, which contains an amino acid sequence having at least 70% sequence identity, more preferably at least ≥75%, ≥80%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 41.

[0221] In some embodiments, the antigen-binding portion comprises a VH region according to any one of (1) to (3) above, and a VL region according to any one of (4) to (6) above.

[0222] Substitutions of the amino acids according to the present disclosure may be biochemically conservative. In some embodiments, when the amino acid to be substituted is provided in one of the first to fifth rows of the following table, the alternative amino acid for the substitution is another different amino acid provided in the same row:

[0223]

[0224] By way of illustration, in some embodiments where the substitution has a Met residue, the alternative amino acid may be selected from Ala, Val, Leu, Ile, Trp, Tyr, Phe, and norleucine.

[0225] In some embodiments, the alternative amino acid in the substitution may have the same side chain polarity as the amino acid residue it replaces. In some embodiments, the alternative amino acid in the substitution may have the same side chain charge (at pH 7.4) as the amino acid residue it replaces:

[0226]

[0227]

[0228] That is, in some embodiments, a non-polar amino acid is replaced by another different non-polar amino acid. In some embodiments, a polar amino acid is replaced by another different polar amino acid. In some embodiments, an acidic polar amino acid is replaced by another different acidic polar amino acid. In some embodiments, a basic polar amino acid is replaced by another different basic polar amino acid. In some embodiments, a neutral amino acid is replaced by another different neutral amino acid. In some embodiments, a positive amino acid is replaced by another different positive amino acid. In some embodiments, a negative amino acid is replaced by another different negative amino acid.

[0229] In some embodiments, the substitution can be functionally conservative. That is, in some embodiments, the substitution may not affect (or may substantially not affect) one or more functional properties (e.g., target antigen binding) of the antigen-binding portion containing the substitution as compared to an equivalent unsubstituted molecule.

[0230] In some embodiments, the sortase moiety is provided as a fusion polypeptide fused to a constituent polypeptide of an antigen-binding molecule. The sortase moiety can be linked to the amino acid sequence of the antigen-binding molecule via a linker sequence.

[0231] In some embodiments, the sortase moiety is fused to the N-terminus or C-terminus of a constituent polypeptide of the antigen-binding molecule. In some embodiments, the sortase moiety is included in a polypeptide comprising a VH region (e.g., a VH region according to the embodiments described above). In some embodiments, the sortase moiety is included in a polypeptide comprising a VL region (e.g., a VL region according to the embodiments described above). In some embodiments, the sortase moiety is included in a polypeptide comprising a VH region and a VL region.

[0232] Immunoglobulins and their structures are described, for example, in Schroeder and Cavacini J Allergy Clin Immunol. (2010) 125(202):S41-S52, which is hereby incorporated by reference in its entirety. The G-type immunoglobulin (i.e., IgG) is a glycoprotein of approximately 150 kDa and comprises two heavy chains and two light chains. From the N-terminus to the C-terminus, the immunoglobulin heavy chain comprises VH, followed by a heavy chain constant region that comprises three constant domains (CH1, CH2, and CH3, where the CH1-CH2 hinge region provides between CH1 and CH2). The immunoglobulin light chain comprises VL, followed by CL. Depending on the heavy chain, immunoglobulins can be classified as IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM. The light chain can be kappa (κ) or lambda (λ).

[0233] In some embodiments, the antigen-binding molecules of the present disclosure comprise one or more domains of an immunoglobulin heavy chain constant region sequence (e.g., CH1, CH1-CH2 hinge, CH2, CH3, etc.). In some embodiments, the immunoglobulin heavy chain constant region sequence is or derived from IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM, such as the heavy chain constant region sequence of human IgG (e.g., hIgG1, hIgG2, hIgG3, hIgG4), hIgA (e.g., hIgA1, hIgA2), hIgD, hIgE, or hIgM. In some embodiments, the immunoglobulin heavy chain constant region sequence is or derived from the heavy chain constant region sequence of a human IgG1 allotype (e.g., G1m1, G1m2, G1m3, or G1m17). In some embodiments, the antigen-binding molecules of the present disclosure comprise one or more domains of an immunoglobulin light chain constant region sequence. In some embodiments, the immunoglobulin light chain constant region sequence is the human immunoglobulin kappa constant region (IGKC; Cκ). In some embodiments, the immunoglobulin light chain constant region sequence is the human immunoglobulin lambda constant region (IGLC; Cλ), such as IGLC1, IGLC2, IGLC3, IGLC6, or IGLC7.

[0234] In some embodiments, the sortase moiety is included in a polypeptide that comprises one or more domains of an immunoglobulin constant region. The immunoglobulin heavy chain constant region comprises a CH1 domain, a CH1-CH2 hinge region, a CH2 domain, and a CH3 domain. The immunoglobulin light chain constant region comprises a CL domain.

[0235] In this text, the "CH1 region" refers to the amino acid sequence corresponding to the CH1 region of an immunoglobulin. According to the EU numbering system described by Edelman et al., Proc Natl Acad Sci USA (1969) 63(1):78-85, the CH1 region is the region of the Ig formed by positions 118 to 215 of the Ig constant region. The "CH1-CH2 hinge region" refers to the amino acid sequence corresponding to the CH1-CH2 hinge region of an immunoglobulin. According to the EU numbering system described by Edelman et al., Proc Natl Acad Sci USA (1969) 63(1):78-85, the CH1-CH2 hinge region is the region of the Ig formed by positions 216 to 230 of the Ig constant region. The "CH2 region" refers to the amino acid sequence corresponding to the CH2 region of an immunoglobulin. According to the EU numbering system described by Edelman et al., Proc Natl Acad Sci USA (1969) 63(1):78-85, the CH2 region is the region of the Ig formed by positions 231 to 340 of the Ig constant region. The "CH3 region" refers to the amino acid sequence corresponding to the CH3 region of an immunoglobulin. According to the EU numbering system described by Edelman et al., Proc Natl Acad Sci USA (1969) 63(1):78-85, the CH3 region is the region of the Ig formed by positions 341 to 447 of the Ig constant region. The "CH2-CH3 region" refers to the amino acid sequence corresponding to the CH2 and CH3 regions of an immunoglobulin. According to the EU numbering system described by Edelman et al., Proc Natl Acad Sci USA (1969) 63(1):78-85, the CH2-CH3 region is the region of the Ig formed by positions 231 to 447 of the Ig constant region.

[0236] In some embodiments, the antigen-binding molecule comprises a polypeptide that comprises or consists of one of the following structures:

[0237] N-terminus - [VH region] - [... ] - [sortase moiety] - C-terminus

[0238] N-terminus - [VH region] - [CH1 region] - [... ] - [sortase moiety] - C-terminus

[0239] N-terminus - [VH region] - [CH1 region] - [CH1-CH2 hinge region] - [CH2 region] - [CH3 region] -

[0240] [sortase moiety] - C-terminus

[0241] N-terminus - [VL region] - [... ] - [sortase moiety] - C-terminus

[0242] N-terminus - [VL region] - [CL region] - […]- [sortase moiety] - C-terminus

[0243] According to such embodiments, the "VH region" can be a VH region as defined in one of (1) to (3) above, and the "VL region" can be a VL region as defined in one of (4) to (6) above.

[0244] In some embodiments, the antigen-binding molecule of the present disclosure comprises a polypeptide that comprises or consists of: an amino acid sequence having the amino acid sequence of SEQ ID NO:29, or an amino acid sequence having at least 70% amino acid sequence identity (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98% or ≥99% sequence identity) with SEQ ID NO:29. In some embodiments, the antigen-binding molecule comprises a polypeptide that comprises or consists of: an amino acid sequence having the amino acid sequence of SEQ ID NO:30, or an amino acid sequence having at least 70% amino acid sequence identity (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98% or ≥99% sequence identity) with SEQ ID NO:30. In some embodiments, the antigen-binding molecule comprises a polypeptide that comprises or consists of: an amino acid sequence having the amino acid sequence of SEQ ID NO:31, or an amino acid sequence having at least 70% amino acid sequence identity (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98% or ≥99% sequence identity) with SEQ ID NO:31. In some embodiments, the antigen-binding molecule comprises a polypeptide that comprises or consists of: an amino acid sequence having the amino acid sequence of SEQ ID NO:32, or an amino acid sequence having at least 70% amino acid sequence identity (e.g., one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98% or ≥99% sequence identity) with SEQ ID NO:32.

[0245] In some embodiments, the antigen-binding molecules of the present disclosure comprise: (i) a polypeptide comprising or consisting of an amino acid sequence having the amino acid sequence of SEQ ID NO: 29, or an amino acid sequence having at least 70% amino acid sequence identity (e.g., one of ≥ 75%, ≥ 80%, ≥ 85%, ≥ 90%, ≥ 91%, ≥ 92%, ≥ 93%, ≥ 94%, ≥ 95%, ≥ 96%, ≥ 97%, ≥ 98% or ≥ 99% sequence identity) with SEQ ID NO: 29; and (ii) a polypeptide comprising or consisting of an amino acid sequence having the amino acid sequence of SEQ ID NO: 31, or an amino acid sequence having at least 70% amino acid sequence identity (e.g., one of ≥ 75%, ≥ 80%, ≥ 85%, ≥ 90%, ≥ 91%, ≥ 92%, ≥ 93%, ≥ 94%, ≥ 95%, ≥ 96%, ≥ 97%, ≥ 98% or ≥ 99% sequence identity) with SEQ ID NO: 31.

[0246] In some embodiments, the antigen-binding molecules of the present disclosure comprise: (i) a polypeptide comprising or consisting of an amino acid sequence having the amino acid sequence of SEQ ID NO: 30, or an amino acid sequence having at least 70% amino acid sequence identity (e.g., one of ≥ 75%, ≥ 80%, ≥ 85%, ≥ 90%, ≥ 91%, ≥ 92%, ≥ 93%, ≥ 94%, ≥ 95%, ≥ 96%, ≥ 97%, ≥ 98% or ≥ 99% sequence identity) with SEQ ID NO: 30; and (ii) a polypeptide comprising or consisting of an amino acid sequence having the amino acid sequence of SEQ ID NO: 32, or an amino acid sequence having at least 70% amino acid sequence identity (e.g., one of ≥ 75%, ≥ 80%, ≥ 85%, ≥ 90%, ≥ 91%, ≥ 92%, ≥ 93%, ≥ 94%, ≥ 95%, ≥ 96%, ≥ 97%, ≥ 98% or ≥ 99% sequence identity) with SEQ ID NO: 32.

[0247] Detectable portion

[0248] In some aspects and embodiments, the polypeptides according to the present disclosure further comprise a detectable moiety. In some embodiments, the detectable moiety is provided at the N-terminus and / or C-terminus of the polypeptide.

[0249] In accordance with such embodiments, it should be understood that the detectable moiety is not identical to (or taken with) the identifier portion of the polypeptide. The detectable moiety can be used to identify and / or select T cells that have absorbed / internalized MHC molecules that comprise the polypeptides of the present disclosure.

[0250] In some embodiments, the detectable moiety is a fluorescent label, a phosphorescent label, a luminescent label, an immuno-detectable label (e.g., an epitope tag), a radioactive label, a chemical label, a nucleic acid label, or an enzyme label. The polypeptides according to the present disclosure can be labeled covalently or non-covalently with a detectable moiety. In a preferred embodiment, the detectable moiety is a fluorescent label.

[0251] Fluorescent labels include, for example, fluorescein, eosin, allophycocyanin, eosin and NDB, green fluorescent protein (GFP), enhanced GFP (eGFP), chelates of rare earths such as europium (Eu), terbium (Tb), and samarium (Sm), tetramethylrhodamine, Texas Red, 4-methylumbelliferone, 7-amino-4-methylcoumarin, Cy3, and Cy5. Radioactive labels include radioisotopes such as hydrogen 3 , sulfur 35 , carbon 14 , phosphorus 32 , iodine 123 , iodine 125 , iodine 126 , iodine 131 , iodine 133 , bromine 77 , technetium 99m , indium 111 , indium 113m , gallium 67 , gallium 68 , ruthenium 95 , ruthenium 97 , ruthenium 103 , ruthenium 105 , mercury 207 , mercury 203 , rhenium 99m , rhenium 101 , rhenium 105 , scandium 47 , tellurium 121m , tellurium 122m , tellurium 125m , thulium 165 , thulium 167 , thulium 168 , copper 67 , fluorine 18 , yttrium 90 , palladium 100 , bismuth 217 and antimony 211 . Luminescent labels include radioluminescence, chemiluminescence (e.g., acridinium esters, luminol, isoluminol), and bioluminescent labels. Immuno-detectable labels include haptens, peptides / polypeptides, antibodies, receptors, and ligands such as biotin, avidin, streptavidin, or digoxin. Nucleic acid labels include aptamers.

[0252] In some embodiments, the detectable moiety is an epitope tag, for example, His (e.g., 6XHis), FLAG, c-Myc, StrepTag, hemagglutinin, E, calmodulin-binding protein (CBP), glutathione-S-transferase (GST), maltose-binding protein (MBP), thioredoxin, S-peptide, T7 peptide, SH2 domain, avidin, streptavidin, or hapten (e.g., biotin, digoxin, dinitrophenol).

[0253] In some embodiments, the detectable moiety is a moiety having detectable activity, for example, an enzyme moiety. Enzyme moieties include, for example, luciferase, glucose oxidase, galactosidase (e.g., β-galactosidase), glucuronidase, phosphatase (e.g., alkaline phosphatase), peroxidase (e.g., horseradish peroxidase), and cholinesterase.

[0254] Additional MHC complex polypeptide sequences

[0255] In some embodiments, the polypeptide of the present disclosure further comprises an amino acid sequence that is capable of associating with the amino acid sequence of an MHC polypeptide as described above for the formation of an MHC molecule.

[0256] That is, in some embodiments, the polypeptide comprises:

[0257] (i) the amino acid sequence of a major histocompatibility complex (MHC) polypeptide;

[0258] (ii) an amino acid sequence that is capable of associating with the amino acid sequence of (i) to form an MHC molecule; and

[0259] (iii) a moiety that facilitates labeling of the polypeptide with an identifier moiety.

[0260] In some embodiments, the polypeptide comprises:

[0261] (i) the amino acid sequence of a major histocompatibility complex (MHC) polypeptide;

[0262] (ii) an amino acid sequence that is capable of associating with the amino acid sequence of (i) to form an MHC molecule

[0263] sequence; and

[0264] (iii) an identifier moiety, wherein the identifier moiety is covalently associated with the polypeptide via a bond formed by a self-labeling protein tag.

[0265] In some embodiments, the polypeptide comprises:

[0266] (i) the amino acid sequence of a major histocompatibility complex (MHC) polypeptide;

[0267] (ii) an amino acid sequence capable of associating with the amino acid sequence of (i) to form an MHC molecule;

[0268] (iii) a moiety that facilitates labeling of the polypeptide with an identifier moiety; and

[0269] (iv) a sortase substrate motif.

[0270] In some embodiments, the polypeptide comprises:

[0271] (i) an amino acid sequence of a major histocompatibility complex (MHC) polypeptide;

[0272] (ii) an amino acid sequence capable of associating with the amino acid sequence of (i) to form an MHC molecule;

[0273] (iii) an identifier moiety, wherein the identifier moiety is covalently associated with the polypeptide via a bond formed by a self-labeling protein tag; and

[0274] (iv) a sortase substrate motif.

[0275] By way of illustration, the polypeptide of SEQ ID NO:13 herein comprises the mature sequence of β2-microglobulin and further comprises the amino acid sequence of mature HLA-A02 (linked via a flexible GS linker). The B2M and HLA-A02 sequences of the polypeptide associate to form an MHC molecule.

[0276] In embodiments where the polypeptide of the present disclosure comprises the amino acid sequence according to (ii) above, it should be understood that the amino acid sequence capable of associating with the amino acid sequence of the MHC polypeptide to form an MHC molecule is selected based on the identity of the amino acid sequence according to (i), such that the amino acid sequences can associate with each other to form a competent MHC molecule. By way of illustration, in embodiments where the amino acid sequence of (i) is or is derived from the amino acid sequence of β2-microglobulin, the amino acid sequence of (ii) is or is derived from the amino acid sequence of an HLA class I α polypeptide (e.g., an HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G polypeptide; e.g., an HLA-A, HLA-B, or HLA-C polypeptide).

[0277] In some embodiments, the polypeptide of the present disclosure comprises:

[0278] (i) an amino acid sequence that is or is derived from: an MHC polypeptide selected from column A of Table 1;

[0279] (ii) an amino acid sequence that is or is derived from: an MHC polypeptide selected from column B of Table 1; and

[0280] (iii) a moiety that facilitates labeling of the polypeptide with an identifier moiety.

[0281] In some embodiments, the polypeptides of the present disclosure comprise:

[0282] (i) an amino acid sequence that is or is derived from: an MHC polypeptide selected from column A of Table 1;

[0283] (ii) an amino acid sequence that is or is derived from: an MHC polypeptide selected from column B of Table 1; and

[0284] (iii) an identifier portion, wherein the identifier portion is covalently associated with the polypeptide by a bond formed by a self-labeling protein tag;

[0285] wherein the MHC polypeptide selected from column A of Table 1 and the MHC polypeptide selected from column B of Table 1 are from the same row of Table 1.

[0286] In some embodiments, the polypeptides of the present disclosure comprise:

[0287] (i) an amino acid sequence that is or is derived from: an MHC polypeptide selected from column A of Table 1;

[0288] (ii) an amino acid sequence that is or is derived from: an MHC polypeptide selected from column B of Table 1;

[0289] (iii) a portion that facilitates labeling of the polypeptide with the identifier portion; and

[0290] (iv) a sortase substrate motif.

[0291] In some embodiments, the polypeptides of the present disclosure comprise:

[0292] (i) an amino acid sequence that is or is derived from: an MHC polypeptide selected from column A of Table 1;

[0293] (ii) an amino acid sequence that is or is derived from: an MHC polypeptide selected from column B of Table 1;

[0294] (iii) an identifier portion, wherein the identifier portion is covalently associated with the polypeptide by a bond formed by a self-labeling protein tag; and

[0295] (iv) a sortase substrate motif;

[0296] wherein the MHC polypeptide selected from column A of Table 1 and the MHC polypeptide selected from column B of Table 1 are from the same row of Table 1.

[0297] Table 1

[0298]

[0299]

[0300] Linkers, labels, and conjugates

[0301] The polypeptides of the present disclosure may additionally comprise additional amino acids or sequences of amino acids.

[0302] The polypeptide may have one or more linker sequences between sequences of amino acids. For example, the linker sequence may be provided between different domains of the polypeptide (e.g., between the amino acid sequence of an MHC polypeptide and the portion that facilitates labeling of the polypeptide with an identifier moiety).

[0303] Linker sequences are known to the person skilled in the art and are described, for example, in Chen et al., Adv Drug Deliv Rev (2013) 65(10):1357 - 1369, which is hereby incorporated by reference in its entirety. In some embodiments, the linker sequence may be a flexible linker sequence. A flexible linker sequence allows relative movement of the amino acid sequences joined by the linker sequence. Flexible linkers are known to the person skilled in the art and several flexible linkers are identified in Chen et al., Adv Drug Deliv Rev (2013) 65(10):1357 - 1369. Flexible linker sequences typically contain a high proportion of glycine residues and / or serine residues.

[0304] In some embodiments, the linker sequence comprises at least one glycine residue and / or at least one serine residue. In some embodiments, the linker sequence comprises or consists of: glycine residues and serine residues. In some embodiments, the linker sequence has the following structure: (GxS)n or (GxS)nGm; where G = glycine, S = serine, x = 3 or 4, n = 2, 3, 4, 5 or 6, and m = 0, 1, 2 or 3. In some embodiments, the linker sequence comprises one or more (e.g., 1, 2, 3, 4, 5 or 6) copies (e.g., in tandem) of the sequence motif G4S. In some embodiments, the linker sequence comprises or consists of: (G4S)3 or (G4S)4. In some embodiments, the linker sequence has a length of from 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 10, 1 to 15, 1 to 20, 1 to 25 or 1 to 30 amino acids.

[0305] In some embodiments, the linker sequence comprises one or more copies of the amino acid sequence according to SEQ ID NO:7 or SEQ ID NO:8. In some embodiments, the linker sequence comprises at least 1, 2, 3 or 4 copies of the amino acid sequence according to SEQ ID NO:6.

[0306] In some embodiments, the linker sequence comprises or consists of: an amino acid sequence having at least 60%, preferably ≥70%, ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity to SEQ ID NO:7 or 8.

[0307] The polypeptides of the present disclosure may comprise amino acid sequences to facilitate the expression, folding, transport, processing, purification or detection of antigen-binding molecules / polypeptides. For example, the polypeptides of the present disclosure may additionally comprise sequences of amino acids that form a detectable moiety such as those described above.

[0308] The polypeptide may additionally comprise a signal peptide (also referred to as a leader sequence or signal sequence). Signal peptides typically consist of a sequence of 5 to 30 hydrophobic amino acids that form a single α helix. Secreted proteins as well as proteins expressed at the cell surface typically contain signal peptides. Signal peptides are known for many proteins and are recorded in databases such as GenBank, UniProt, and Ensembl, and / or can be identified / predicted, for example, using amino acid sequence analysis tools such as SignalP (Petersen et al., 2011 Nature Methods 8:785-786) or Signal-BLAST (Frank and Sippl, 2008 Bioinformatics 24:2172-2176).

[0309] The signal peptide may be present at the N-terminus of the polypeptide and may be present in the newly synthesized polypeptide. The signal peptide provides efficient transport of the polypeptide. The signal peptide is typically removed by cleavage and is thus not included in the mature polypeptide.

[0310] Signal peptides are known for many proteins and are recorded in databases such as GenBank, UniProt, Swiss-Prot, TrEMBL, Protein Information Resource, Protein Data Bank, Ensembl, and InterPro, and / or can be identified / predicted, for example, using amino acid sequence analysis tools such as SignalP (Petersen et al., 2011 Nature Methods 8:785-786) or Signal-BLAST (Frank and Sippl, 2008 Bioinformatics 24:2172-2176).

[0311] In some embodiments, the signal peptide comprises or consists of: an amino acid sequence having at least 60%, preferably ≥70%, ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity with SEQ ID NO:2.

[0312] Specific exemplary polypeptides, MHC molecules, and MHC:peptide complexes

[0313] In some embodiments, the polypeptide according to the present disclosure comprises or consists of one of the following structures:

[0314] N-terminus - [signal peptide] - [portion for facilitating labeling of the polypeptide with an identifier] - [amino acid sequence of the MHC polypeptide] - C-terminus

[0315] N-terminus - [portion for facilitating labeling of the polypeptide with an identifier] - [amino acid sequence of the MHC polypeptide] - C-terminus

[0316] N-terminus - [signal peptide] - [HaloTag] - [mature β2-microglobulin sequence] - C-terminus

[0317] N-terminus - [HaloTag] - [mature β2-microglobulin sequence] - C-terminus

[0318] N-terminus - [signal peptide] - [portion for facilitating labeling of the polypeptide with an identifier] - [amino acid sequence of the MHC polypeptide] - [amino acid sequence capable of associating with the amino acid sequence of the MHC polypeptide to form an MHC molecule] - C-terminus

[0319] N-terminus - [portion for facilitating labeling of the polypeptide with an identifier] - [amino acid sequence of the MHC polypeptide] - [amino acid sequence capable of associating with the amino acid sequence of the MHC polypeptide to form an MHC molecule] - C-terminus

[0320] N-terminus - [signal peptide] - [HaloTag] - [mature β2-microglobulin sequence] - [mature HLA-A02 sequence] - C-terminus

[0321] N-terminus - [signal peptide] - [HaloTag] - [mature β2-microglobulin sequence] - [mature HLA class I α polypeptide sequence] - C-terminus

[0322] N-terminus - [HaloTag] - [mature β2-microglobulin sequence] - [mature HLA class I α polypeptide sequence] - C-terminus

[0323] N-terminus - [signal peptide] - [portion facilitating labeling of the polypeptide with an identifier moiety] - [sortase substrate motif] - [amino acid sequence of a protein localized to the cell membrane] - C-terminus

[0324] N-terminus - [portion facilitating labeling of the polypeptide with an identifier moiety] - [sortase substrate motif] - [amino acid sequence of a protein localized to the cell membrane] - C-terminus

[0325] N-terminus - [signal peptide] - [portion facilitating labeling of the polypeptide with an identifier moiety] - [sortase substrate motif] - [amino acid sequence of an MHC polypeptide] - C-terminus

[0326] N-terminus - [portion facilitating labeling of the polypeptide with an identifier moiety] - [sortase substrate motif] - [amino acid sequence of an MHC polypeptide] - C-terminus

[0327] N-terminus - [signal peptide] - [HaloTag] - [sortase substrate motif] - [mature β2-microglobulin sequence] - C-terminus

[0328] N-terminus - [HaloTag] - [sortase substrate motif] - [mature β2-microglobulin sequence] - C-terminus

[0329] N-terminus - [signal peptide] - [portion facilitating labeling of the polypeptide with an identifier moiety] - [sortase substrate motif] - [amino acid sequence of an MHC polypeptide] - [amino acid sequence capable of associating with the amino acid sequence of the MHC polypeptide to form an MHC molecule] - C-terminus

[0330] N-terminus - [portion facilitating labeling of the polypeptide with an identifier moiety] - [sortase substrate motif] - [amino acid sequence of an MHC polypeptide] - [amino acid sequence capable of associating with the amino acid sequence of the MHC polypeptide to form an MHC molecule] - C-terminus

[0331] N-terminus - [signal peptide] - [HaloTag] - [sortase substrate motif] - [mature β2-microglobulin sequence] - [mature HLA-A02 sequence] - C-terminus

[0332] N-terminus - [signal peptide] - [HaloTag] - [sortase substrate motif] - [mature β2-microglobulin sequence] - [mature HLA class I α polypeptide sequence] - C-terminus

[0333] N-terminus - [HaloTag] - [sortase substrate motif] - [mature β2-microglobulin sequence] - [mature HLA class I α polypeptide sequence] - C-terminus

[0334] The present disclosure further provides:

[0335] (1) A polypeptide comprising or consisting of, for example, from the N-terminus to the C-terminus:

[0336] (i) an amino acid sequence encoding a signal peptide, for example an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity to SEQ ID NO:2;

[0337] (ii) an amino acid sequence encoding a part that promotes the labeling of the polypeptide with an identifier moiety (e.g., a self-labeling protein tag such as HaloTag); for example an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity to SEQ ID NO:4 or 5; and

[0338] (iii) an amino acid sequence encoding an MHC polypeptide (e.g., β2-microglobulin); for example an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity to SEQ ID NO:3.

[0339] (2) A polypeptide comprising or consisting of, for example, from the N-terminus to the C-terminus:

[0340] (i) an amino acid sequence encoding a part that promotes the labeling of the polypeptide with an identifier moiety (e.g., a self-labeling protein tag such as HaloTag); for example an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity to SEQ ID NO:4 or 5; and

[0341] (ii) an amino acid sequence encoding an MHC polypeptide (e.g., β2-microglobulin); for example an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity to SEQ ID NO:3.

[0342] (3) A polypeptide comprising or consisting of the following (e.g., from the N-terminus to the C-terminus):

[0343] (i) An amino acid sequence encoding a signal peptide, e.g., an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity with SEQ ID NO:2;

[0344] (ii) An amino acid sequence encoding a moiety that facilitates labeling of the polypeptide with an identifier moiety (e.g., a self-labeling protein tag, e.g., HaloTag); e.g., an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity with SEQ ID NO:4 or 5;

[0345] (iii) An amino acid sequence encoding an amino acid sequence of β2-microglobulin; e.g., an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity with SEQ ID NO:3; and

[0346] (iv) An amino acid sequence encoding an amino acid sequence of an HLA class I α polypeptide; e.g., an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity with SEQ ID NO:9.

[0347] (4) A polypeptide comprising or consisting of the following (e.g., from the N-terminus to the C-terminus):

[0348] (i) An amino acid sequence encoding a moiety that facilitates labeling of the polypeptide with an identifier moiety (e.g., a self-labeling protein tag, e.g., HaloTag); e.g., an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity with SEQ ID NO:4 or 5;

[0349] (ii) An amino acid sequence encoding the amino acid sequence of β2-microglobulin; for example, an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity with SEQ ID NO:3; and

[0350] (iii) An amino acid sequence encoding the amino acid sequence of HLA class I α polypeptide; for example, an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity with SEQ ID NO:9.

[0351] (5) The polypeptide according to the present disclosure comprises or consists of: an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity with SEQ ID NO:10.

[0352] (6) The polypeptide according to the present disclosure comprises or consists of: an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity with SEQ ID NO:11.

[0353] (7) The polypeptide according to the present disclosure comprises or consists of: an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity with SEQ ID NO:12.

[0354] (8) The polypeptide according to the present disclosure comprises or consists of: an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity with SEQ ID NO:13.

[0355] (9) A polypeptide comprising or consisting of (for example, from the N-terminus to the C-terminus):

[0356] (i) An amino acid sequence encoding a signal peptide, e.g., an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity with SEQ ID NO:2;

[0357] (ii) An amino acid sequence encoding a moiety that facilitates the labeling of a polypeptide with an identifier moiety (e.g., a self-labeling protein tag, such as HaloTag); e.g., an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity with SEQ ID NO:4 or 5;

[0358] (iii) An amino acid sequence conforming to the consensus item of SEQ ID NO:25; and

[0359] (iv) An amino acid sequence encoding an MHC polypeptide (e.g., β2-microglobulin); e.g., an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity with SEQ ID NO:3.

[0360] (10) A polypeptide comprising or consisting of (e.g., from the N-terminus to the C-terminus):

[0361] (i) An amino acid sequence encoding a moiety that facilitates the labeling of a polypeptide with an identifier moiety (e.g., a self-labeling protein tag, such as HaloTag); e.g., an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity with SEQ ID NO:4 or 5;

[0362] (ii) An amino acid sequence conforming to the consensus item of SEQ ID NO:25;

[0363] (iii) An amino acid sequence encoding an MHC polypeptide (e.g., β2-microglobulin); for example, an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity with SEQ ID NO:3.

[0364] (11) A polypeptide comprising or consisting of (e.g., from the N-terminus to the C-terminus):

[0365] (i) An amino acid sequence encoding a signal peptide, for example, an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity with SEQ ID NO:2.

[0366] (ii) An amino acid sequence conforming to the consensus of SEQ ID NO:25.

[0367] (iii) An amino acid sequence encoding a moiety that facilitates labeling of the polypeptide with an identifier moiety (e.g., a self-labeling protein tag, such as HaloTag); for example, an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity with SEQ ID NO:4 or 5.

[0368] (iv) An amino acid sequence encoding an amino acid sequence of β2-microglobulin; for example, an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity with SEQ ID NO:3; and

[0369] (v) An amino acid sequence encoding an amino acid sequence of an HLA class I α polypeptide; for example, an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity with SEQ ID NO:9.

[0370] (12) A polypeptide comprising or consisting of (e.g., from the N-terminus to the C-terminus):

[0371] (i) An amino acid sequence encoding a portion that facilitates tagging a polypeptide with an identifier portion (e.g., a self - labeling protein tag, such as HaloTag); for example, an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity to SEQ ID NO:4 or 5;

[0372] (ii) An amino acid sequence conforming to the consensus item of SEQ ID NO:25;

[0373] (iii) An amino acid sequence encoding an amino acid sequence of β2 - microglobulin; for example, an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity to SEQ ID NO:3; and

[0374] (iv) An amino acid sequence encoding an amino acid sequence of an HLA class I α polypeptide; for example, an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity to SEQ ID NO:9.

[0375] The present disclosure further provides:

[0376] (A) An MHC molecule comprising or consisting of: (i) a polypeptide according to (2) or (6) above and (ii) an MHC polypeptide capable of associating with the polypeptide of (i) to form an MHC molecule; for example, an HLA class I α polypeptide.

[0377] (B) An MHC molecule comprising or consisting of a polypeptide according to (4) or (8) above.

[0378] (C) An MHC molecule comprising or consisting of: (i) a polypeptide according to (10) above and (ii) an MHC polypeptide capable of associating with the polypeptide of (i) to form an MHC molecule; for example, an HLA class I α polypeptide.

[0379] (D) An MHC molecule comprising or consisting of a polypeptide according to (12) above.

[0380] The present disclosure further provides:

[0381] (I) An MHC:peptide complex comprising an MHC molecule according to (A) or (B) above and a peptide presented by the MHC molecule.

[0382] (II) An MHC:peptide complex comprising: (i) an MHC molecule according to (A) or (B) above, (ii) a peptide presented by the MHC molecule, and (ii) an identifier moiety (e.g., a nucleic acid identifier moiety, e.g., an ssDNA identifier moiety).

[0383] (III) An MHC:peptide complex comprising an MHC molecule according to (C) or (D) above and a peptide presented by the MHC molecule.

[0384] (IV) An MHC:peptide complex comprising: (i) an MHC molecule according to (C) or (D) above, (ii) a peptide presented by the MHC molecule, and (ii) an identifier moiety (e.g., a nucleic acid identifier moiety, e.g., an ssDNA identifier moiety).

[0385] Nucleic acids and vectors

[0386] The present disclosure provides a nucleic acid or nucleic acids that encode a polypeptide according to the present disclosure (e.g., a polypeptide or antigen-binding molecule according to any aspect or embodiment described herein). In some embodiments, the nucleic acid comprises or consists of: DNA and / or RNA.

[0387] A polypeptide according to the present disclosure can be produced intracellularly by translation of RNA encoding the polypeptide. A polypeptide according to the present disclosure can be produced intracellularly by transcription from a nucleic acid encoding the polypeptide and subsequent translation of the transcribed RNA.

[0388] In some embodiments, the nucleic acid can be a vector or vectors, or can be comprised / included in a vector or vectors. As used herein, a "vector" is a nucleic acid molecule that serves as a vehicle for transferring an exogenous nucleic acid into a cell.

[0389] Accordingly, the present disclosure also provides a vector or vectors that comprise a nucleic acid or nucleic acids according to the present disclosure. The vector can facilitate delivery of the nucleic acid encoding the polypeptide according to the present disclosure to a cell. The vector can be an expression vector that contains elements required for expression of the polypeptide according to the present disclosure. The vector can contain elements that facilitate integration of the nucleic acid into the genomic DNA of the cell into which the vector is introduced.

[0390] The nucleic acids and vectors according to the present disclosure can be provided in a purified or isolated form, i.e., away from other nucleic acids or naturally-occurring biological materials.

[0391] The vector can be a vector for expressing nucleic acids in cells (i.e., an expression vector). Such vectors may contain a promoter sequence that is operably linked to a nucleotide sequence encoding a polypeptide according to the present disclosure. The vector may also include a stop codon (i.e., 3' to the nucleotide sequence encoding the polypeptide in the nucleotide sequence of the vector) and an expression enhancer. Any suitable vector, promoter, enhancer, and stop codon known in the art can be used to express a peptide or polypeptide from the vector according to the present disclosure.

[0392] The term "operably linked" can include the situation where the nucleic acid encoding the polypeptide according to the present disclosure and a regulatory nucleic acid sequence (e.g., a promoter and / or an enhancer) are covalently linked in such a way that the expression of the nucleic acid encoding the polypeptide is placed under the influence or control of the regulatory nucleic acid sequence (thus forming an expression cassette). Therefore, if a regulatory sequence can affect the transcription of a nucleic acid sequence, the regulatory sequence is operably linked to the selected nucleic acid sequence. The resulting transcript can then be translated into the desired polypeptide.

[0393] Vectors contemplated in combination with the present disclosure include DNA vectors, RNA vectors, plasmids (e.g., conjugative plasmids (e.g., F plasmids), non-conjugative plasmids, R plasmids, col plasmids, episomes), viral vectors (e.g., retroviral vectors, e.g., gamma-retroviral vectors (e.g., murine leukemia virus (MLV)-derived vectors, e.g., SFG vectors), lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, poxviral vectors, and herpesviral vectors), transposon-based vectors, and artificial chromosomes (e.g., yeast artificial chromosomes), e.g., as described in Maus et al., Annu Rev Immunol (2014) 32:189-225 and Morgan and Boyerinas, Biomedicines (2016) 4:9, which are hereby incorporated by reference in their entireties. In some embodiments, the vector according to the present disclosure is a lentiviral vector.

[0394] In some embodiments, the vector can be a eukaryotic vector, i.e., a vector that contains the components necessary for expressing a protein from the vector in a eukaryotic cell. In some embodiments, the vector can be a mammalian vector, e.g., which contains a cytomegalovirus (CMV) or SV40 promoter to drive protein expression.

[0395] Peptide

[0396] The compositions and methods of the present disclosure can be used to identify T cell receptors (TCRs) that can bind to MHC:peptide complexes. Specifically, it is contemplated to use the compositions and methods of the present disclosure to identify TCRs that can bind to MHC molecules presenting a peptide of interest.

[0397] The peptides of the present disclosure can be peptides of a target polypeptide. The peptides of the target reference polypeptide can be any fragment of the polypeptide.

[0398] The target polypeptide can be any polypeptide. The target polypeptide can be, for example, a polypeptide for which an immune response (e.g., a cell-mediated immune response) is desired to be elicited, or a polypeptide of an infective agent or cell. The target polypeptide can be a disease-related antigen.

[0399] "Disease-related antigen" refers to an antigen whose presence indicates a given disease / disease state, or an antigen for which an elevated antigen level is positively correlated with a given disease / disease state. A disease-related antigen can be an antigen whose expression is related to the development, progression, or severity of the symptoms of a given disease. A disease-related antigen can be associated with the etiology or pathology of a disease, or can be abnormally expressed due to a disease. A disease-related antigen can be an antigen of an infective agent or pathogen, a cancer-related antigen, or an autoimmune disease-related antigen.

[0400] In some embodiments, the disease-related antigen is an antigen of a pathogen. The pathogen can be a prokaryote (bacteria), a eukaryote (e.g., protozoa, worms, fungi), a virus, or a prion.

[0401] In some embodiments, the pathogen is an intracellular pathogen. In some embodiments, the pathogen is a virus. The virus can be a dsDNA virus (e.g., adenovirus, herpesvirus, poxvirus), an ssRNA virus (e.g., parvovirus), a dsRNA virus (e.g., reovirus), a (+)ssRNA virus (e.g., picornavirus, togavirus), a (-)ssRNA virus (e.g., orthomyxovirus, rhabdovirus), an ssRNA-RT virus (e.g., retrovirus) or a dsDNA-RT virus (e.g., hepadnavirus). Specifically, the families Adenoviridae, Herpesviridae, Papillomaviridae, Polyomaviridae, Poxviridae, Hepadnaviridae, Parvoviridae, Astroviridae, Caliciviridae, Picornaviridae, Coronaviridae, Flaviviridae, Togaviridae, Hepadnaviridae, Retroviridae, Orthomyxoviridae, Arenaviridae, Bunyaviridae, Filoviridae, Paramyxoviridae, Rhabdoviridae and Reoviridae are contemplated. In some embodiments, the virus is selected from Epstein-Barr virus, adenovirus, herpes simplex virus type 1, herpes simplex virus type 2, varicella-zoster virus, cytomegalovirus, human herpesvirus 8, human papillomavirus, BK virus, JC virus, smallpox, hepatitis B virus, parvovirus B19, human astrovirus, lymphocytic choriomeningitis virus, norwalk virus, coxsackievirus, hepatitis A virus, poliovirus, rhinovirus, severe acute respiratory syndrome virus, hepatitis C virus, yellow fever virus, dengue virus, West Nile virus, TBE virus, rubella virus, hepatitis E virus, human immunodeficiency virus, influenza virus, Lassa virus, Crimean-Congo hemorrhagic fever virus, Hantaan virus, Ebola virus, Marburg virus, measles virus, mumps virus, parainfluenza virus, picornavirus, respiratory syncytial virus, rabies virus, hepatitis D virus, rotavirus, orbivirus, tick-borne encephalitis virus and Banna virus. In some embodiments, the pathogen is a bacterium. The bacterium can be Gram-positive or Gram-negative. Specifically, bacteria of the genera Bacillus, Bartonella, Bordetella, Borrelia, Brucella, Campylobacter, Chlamydia, Chlamydophila, Clostridium, Corynebacterium, Enterococcus, Escherichia, Francisella, Haemophilus, Helicobacter, Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Treponema, Ureaplasma, Vibrio and Yersinia are contemplated. In some embodiments, the pathogen is a protozoan. Specifically, protozoa of the genera Entamoeba, Plasmodium, Giardia, Trypanosoma, Leishmania, Besnoitia and Toxoplasma are contemplated. In some embodiments, the pathogen is a fungus.Specifically, fungi of the genera Candida, Aspergillus, Blastomyces, Coccidioides, Sporothrix, Cryptococcus, Histoplasma, Pneumocystis, Stachybotrys, Rhizopus, Mucor, Cunninghamella, Lepidomyces, Trichophyton, Microsporum, Epidermophyton, Fusarium, and Mortierella are contemplated.

[0402] In some embodiments, the polypeptide of interest is a polypeptide whose expression / activity or upregulated expression / activity is positively correlated with a disease or disorder (e.g., cancer, infectious disease, or autoimmune disease). In some embodiments, the polypeptide of interest is associated with cancer, infectious disease, or autoimmune disease.

[0403] In some embodiments, the disease-associated antigen is a cancer cell antigen. A cancer cell antigen is an antigen expressed or overexpressed by cancer cells. A cancer cell antigen can be any peptide / polypeptide, glycoprotein, lipoprotein, polysaccharide, glycolipid, lipid, or fragment thereof. The expression of a cancer cell antigen can be associated with cancer. A cancer cell antigen can be abnormally expressed by cancer cells (e.g., a cancer cell antigen can be expressed with abnormal localization), or can be expressed by cancer cells with an abnormal structure. A cancer cell antigen can be capable of eliciting an immune response. A cancer cell antigen can be a cancer-associated antigen. In some embodiments, the cancer cell antigen is an antigen whose expression is associated with the development, progression, or severity of cancer symptoms. A cancer-associated antigen can be associated with the etiology or pathology of cancer, or can be abnormally expressed due to cancer. In some embodiments, the cancer cell antigen is an antigen whose expression is upregulated by cancer cells (e.g., at the RNA and / or protein level), e.g., compared to the expression level expressed by comparable non-cancerous cells (e.g., non-cancerous cells derived from the same tissue / cell type). In some embodiments, the cancer-associated antigen can be preferentially expressed by cancerous cells and not by comparable non-cancerous cells (e.g., non-cancerous cells derived from the same tissue / cell type). In some embodiments, the cancer-associated antigen can be the product of a mutated oncogene or a mutated tumor suppressor gene. In some embodiments, the polypeptide of interest is a cancer-associated neoantigen. In some embodiments, the cancer-associated antigen can be the product of: an overexpressed cellular protein, a cancer antigen produced by an oncogenic virus, a carcinoembryonic antigen, or a cell surface glycolipid or glycoprotein.

[0404] It should be understood that in preferred embodiments, the peptides according to the present disclosure are peptides capable of participating in an MHC:peptide complex. That is, the peptide is capable of associating with an MHC molecule according to the present disclosure (i.e., an MHC molecule comprising a polypeptide according to the present disclosure) to form an MHC:peptide complex.

[0405] In some embodiments, the peptide has a length of 5 to 30 (e.g., 10 to 25 or 8 to 11) amino acids.

[0406] MHC molecules and MHC:peptide complexes

[0407] The present disclosure provides MHC molecules, which comprise a polypeptide according to the present disclosure. The MHC molecules can be MHC class I molecules or MHC class II molecules. In a preferred embodiment, the MHC molecule is an MHC class I molecule.

[0408] The MHC molecule is formed by a polypeptide complex, which is formed by the association between a polypeptide according to the present disclosure and another MHC polypeptide. It should be understood that the constituent polypeptides of the MHC molecule are selected to form a competent MHC molecule. By way of illustration, in an embodiment where the polypeptide according to the present disclosure comprises the amino acid sequence of β2-microglobulin, the other MHC polypeptide of the MHC molecule comprising the polypeptide according to the present disclosure is an MHC class I α-chain polypeptide.

[0409] The MHC molecule is capable of presenting the peptide according to the present disclosure (i.e., in the form of an MHC:peptide complex) to a T cell expressing a TCR that binds to the MHC:peptide complex.

[0410] Accordingly, the present disclosure also provides an MHC:peptide complex, which comprises an MHC molecule according to the present disclosure (i.e., comprising a polypeptide according to the present disclosure) and a peptide presented by the MHC molecule.

[0411] Cells comprising / expressing the polypeptides and nucleic acids / vectors of the present disclosure

[0412] The present disclosure also provides a cell, which comprises a polypeptide, an MHC molecule, an MHC:peptide complex, an antigen-binding molecule, a nucleic acid / nucleic acids or a vector / vectors according to the present disclosure.

[0413] It should be understood that when a cell is referred to in the singular herein (i.e., "a / the cell"), populations of such cells are also contemplated.

[0414] The present disclosure further provides a plurality of cells comprising a polypeptide according to the present disclosure, wherein the polypeptides are different. Specifically, the present disclosure provides a plurality of cells, wherein different cells among the plurality of cells comprise polypeptides containing different identifier portions. That is, the present disclosure provides a plurality of cells (which may be referred to as a "library"), wherein different cells among the plurality of cells comprise different polypeptides according to the present disclosure, and the different polypeptides comprise different identifier portions.

[0415] The present disclosure similarly provides a plurality of cells comprising different MHC molecules according to the present disclosure, particularly wherein the plurality of cells comprise MHC molecules, and the MHC molecules comprise polypeptides having different identifier portions. That is, the present disclosure provides a plurality of cells (“library”), wherein different cells among the plurality of cells comprise different MHC molecules according to the present disclosure, and the different MHC molecules comprise polypeptides of the present disclosure having different identifier portions.

[0416] The present disclosure similarly provides a plurality of cells, the plurality of cells comprising different MHC:peptide complexes according to the present disclosure, particularly wherein the plurality of cells comprise MHC:peptide complexes containing MHC molecules, and the MHC molecules comprise polypeptides having different identifier portions, and / or wherein the plurality of cells comprise MHC:peptide complexes that comprise different peptides. That is, the present disclosure provides a plurality of cells (“library”), wherein different cells among the plurality of cells comprise different MHC:peptide complexes according to the present disclosure, and the different MHC:peptide complexes comprise MHC molecules, and the MHC molecules comprise polypeptides having different identifier portions, and / or wherein the different MHC:peptide complexes comprise different peptides.

[0417] The cells can be eukaryotic cells, such as mammalian cells. The mammal can be a primate (rhesus monkey, cynomolgus monkey, non-human primate or human) or a non-human mammal (e.g., rabbit, guinea pig, rat, mouse or other rodent (including any rodentia), cat, dog, pig, sheep, goat, cow (including cattle, such as dairy cows, or any bovidae), horse (including any equidae), donkey and non-human primate). In a preferred embodiment, the cells are human cells.

[0418] In some embodiments, the cells are or are derived from cell types commonly used for the expression of polypeptides for use in human therapy. Exemplary cells are described, for example, in Kunert and Reinhart, Appl Microbiol Biotechnol. (2016) 100:3451–3461 (incorporated herein by reference in its entirety), and include, for example, CHO, HEK 293, PER.C6, NS0 and BHK cells. In a preferred embodiment, the cells are or are derived from CHO cells.

[0419] The present disclosure also provides methods for producing the cells according to the present disclosure, and cells obtained or obtainable by such methods. Methods for producing cells comprising / expressing a polypeptide / polypeptide complex of interest are well known to those skilled in the art and generally include introducing a nucleic acid / carrier encoding the polypeptide of interest into the cells.

[0420] Such methods can include nucleic acid transfer for permanent (i.e., stable) or transient expression of the transferred nucleic acid. In some embodiments, after introduction into the cell, the nucleic acid encoding the polypeptide of interest can integrate into the genomic DNA of the cell or form part of the genomic DNA of the cell. In some embodiments, after introduction into the cell, the nucleic acid encoding the polypeptide of interest can be maintained episomally.

[0421] Any suitable genetic engineering platform can be used, and the genetic engineering platform includes gamma-retroviral vectors, lentiviral vectors, adenoviral vectors, DNA transfection, transposon-based gene delivery, and RNA transfection, such as described in Maus et al., Annu Rev Immunol (2014) 32:189-225, which is hereby incorporated by reference in its entirety. Methods also include those described in, for example, Wang and Rivière Mol Ther Oncolytics. (2016) 3:16015, which is hereby incorporated by reference in its entirety. Suitable methods for introducing the nucleic acid / vector into the cell include transduction, transfection, and electroporation.

[0422] Aspects and embodiments of the present disclosure particularly relate to antigen-presenting cells (APCs). An APC is a cell that expresses MHC molecules (e.g., MHC class I and / or MHC class II molecules) and is capable of presenting an MHC:peptide complex. It should be understood that an APC according to the present disclosure is capable of presenting an MHC:peptide complex according to the present disclosure.

[0423] An APC according to the present disclosure can be a professional APC. Professional APCs are specialized for presenting antigens to T cells; they are capable of efficiently processing and presenting MHC peptide complexes on the cell surface and express high levels of co-stimulatory molecules. Professional APCs include dendritic cells (DCs), macrophages, and B cells. Non-professional APCs are other cells that are capable of presenting MHC peptide complexes to T cells, specifically MHC class I peptide complexes to CD8+ T cells.

[0424] In some embodiments, the APC is an APC capable of cross-presenting on an antigen of MHC class I internalized by the APC (e.g., absorbed by endocytosis / phagocytosis). Cross-presentation to CD8+ T cells on internalized antigens of MHC class I is described in, for example, Alloatti et al., Immunological Reviews (2016), 272(1):97-108, which is hereby incorporated by reference in its entirety. APCs capable of cross-presentation include, for example, dendritic cells (DCs), macrophages, B cells, and liver sinusoidal endothelial cells.

[0425] In some embodiments, the APC expresses / contains a peptide presented by an MHC molecule comprising a polypeptide according to the present disclosure.

[0426] In some embodiments, the APC expresses / contains a peptide presented by an MHC molecule according to the present disclosure because it has been in contact with a peptide, or has been in contact with a polypeptide comprising a peptide and has internalized it. In cases where the peptide is provided to the APC in the form of a polypeptide comprising the peptide, the polypeptide can be processed by the cell to generate the peptide.

[0427] In some embodiments, the APC can have been "pulsed" with a polypeptide / peptide, which can involve culturing the APC in vitro in the presence of the polypeptide / peptide for a time sufficient for the APC to internalize the polypeptide / peptide.

[0428] In some embodiments, the APC expresses / contains a peptide presented by an MHC molecule according to the present disclosure due to the expression of a nucleic acid encoding the peptide intracellularly or the expression of a nucleic acid encoding a polypeptide comprising the peptide. In some embodiments, the APC contains a nucleic acid encoding a peptide presented by an MHC molecule, a polypeptide according to the present disclosure, or a nucleic acid encoding a polypeptide comprising the peptide. The APC can contain such a nucleic acid because a nucleic acid encoding the peptide / polypeptide (e.g., in the form of a vector comprising such a nucleic acid) has been introduced into the cell (e.g., via transfection, transduction, electroporation, etc.).

[0429] In some embodiments, the APC is a cell of a cell line (e.g., an immortalized cell line).

[0430] In some embodiments, the APC contains a modification for reducing / preventing the expression of one or more MHC polypeptides (i.e., compared to the expression level of the relevant MHC polypeptides expressed by equivalent unmodified cells). In some embodiments, the APC contains a modification for reducing the expression of one or more endogenous MHC polypeptides (i.e., MHC polypeptides encoded by the genome of equivalent unmodified cells). In some embodiments, the APC contains a modification for reducing the expression of one or more endogenous MHC polypeptides that are capable of associating with a polypeptide according to the present disclosure to form a functional MHC molecule (i.e., capable of antigen presentation). In some embodiments, the APC contains a modification for reducing the expression of one or more MHC class I α-chain polypeptides.

[0431] Modifications for reducing / preventing the expression of one or more MHC polypeptides are contemplated, particularly in cases where the APC is allogeneic with respect to a subject (from which T cells to be screened according to a method for identifying TCRs according to the present disclosure are isolated / obtained).

[0432] When a subject is referred to herein as "allogeneic" or "non - autologous" relative to a reference subject, the subject is different. An allogeneic subject can be genetically different from the reference subject. An allogeneic subject can contain MHC / HLA genes encoding MHC / HLA molecules (e.g., MHC class I α and / or MHC class II molecules) that are different from the MHC / HLA molecules (e.g., MHC class I α and / or MHC class II molecules) encoded by the reference subject.

[0433] In contrast, when a subject is referred to herein as "autogeneic" or "autologous", the subject can be genetically identical. An autologous subject can contain MHC / HLA genes encoding the same MHC / HLA molecules (e.g., MHC class I α and / or MHC class II molecules). An autologous subject can be the same subject.

[0434] Modification of a given target nucleic acid can be achieved in a variety of ways known to those skilled in the art, including modifying the target nucleic acid by homologous recombination and editing the target nucleic acid using site - specific nucleases (SSNs).

[0435] Suitable methods can employ targeting by homologous recombination, which is reviewed in, for example, Mortensen CurrProtoc Neurosci. (2007) Chapter 4: Unit 4.29 and Vasquez et al., PNAS 2001, 98(15):8403 - 8410, both of which are incorporated herein by reference in their entirety. Targeting by homologous recombination involves the exchange of nucleic acid sequences through crossover events guided by homologous sequences. Other suitable techniques include nucleic acid editing using SSNs. Gene editing using SSNs is reviewed in, for example, Eid and Mahfouz, Exp Mol Med. 2016 Oct;48(10):e265, which is incorporated herein by reference in its entirety. Enzymes capable of generating site - specific double - strand breaks (DSBs) can be engineered to introduce DSBs into a target nucleic acid sequence of interest. DSBs can be repaired by error - prone non - homologous end joining (NHEJ), in which the two ends of the break are rejoined, typically by nucleotide insertion or deletion. Alternatively, DSBs can be repaired by homologous directed repair (HDR), a high - fidelity mechanism in which a DNA template with ends homologous to the break site is provided and introduced at the site of the DSB.

[0436] SSNs that can be engineered to generate target nucleic acid sequence-specific DSBs include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats / CRISPR-associated 9 (CRISPR / Cas9) systems. The ZFN system is reviewed, for example, in Umov et al., Nat Rev Genet. (2010) 11(9):636-46, which is hereby incorporated by reference in its entirety. ZFNs contain programmable zinc finger DNA-binding domains and DNA cleavage domains (e.g., the FokI endonuclease domain). The DNA-binding domains can be identified by screening zinc finger arrays capable of binding to the target nucleic acid sequence. The TALEN system is reviewed, for example, in Mahfouz et al., Plant Biotechnol J. (2014) 12(8):1006-14, which is hereby incorporated by reference in its entirety. TALENs contain programmable DNA-binding TALE domains and DNA cleavage domains (e.g., the FokI endonuclease domain). TALEs contain a repeat domain consisting of repeats of 33 to 39 amino acids that are identical except for two residues at positions 12 and 13 of each repeat, which are the repeat variable diresidues (RVDs). Each RVD determines the binding of the repeat to a nucleotide in the target DNA sequence according to the following relationships: "HD" binds to C, "NI" binds to A, "NG" binds to T, and "NN" or "NK" binds to G (Moscou and Bogdanove, Science (2009) 326(5959):1501). CRISPR / Cas9 and related systems, such as CRISPR / Cpf1, CRISPR / C2c1, CRISPR / C2c2, and CRISPR / C2c3, are reviewed, for example, in Nakade et al., Bioengineered (2017) 8(3):265-273, which is hereby incorporated by reference in its entirety. These systems contain endonucleases (e.g., Cas9, Cpf1, etc.) and single guide RNA (sgRNA) molecules. The sgRNA can be engineered to target endonuclease activity to the nucleic acid sequence of interest.

[0437] In some embodiments, and particularly in embodiments where the APC comprises modifications for reducing / preventing the expression of one or more endogenous MHC polypeptides, the APC further comprises modifications for comprising / expressing a nucleic acid encoding one or more MHC polypeptides. In such embodiments, the APC is preferably engineered to express MHC polypeptides corresponding to those MHC polypeptides, and the APC comprises modifications for reducing / preventing the endogenous expression of those MHC polypeptides (e.g., if the APC comprises modifications for reducing / preventing the expression of an endogenous MHC class I alpha chain polypeptide, the APC is engineered to express the relevant MHC class I alpha chain polypeptide). The APC can be engineered to express one or more MHC polypeptides encoded by the genome of a subject of interest (e.g., a patient). The subject of interest can be the same subject from whom the T cells to be screened according to the TCRs of the present disclosure are isolated / obtained. In some embodiments, the APC is engineered to express one or more MHC polypeptides encoded by a subject comprising a disease-related antigen according to the present disclosure. In some embodiments, the APC is engineered to express one or more MHC polypeptides encoded by a subject comprising a cancer-related neoantigen (i.e., a subject comprising cells containing / expressing a cancer-related neoantigen).

[0438] The foregoing paragraphs particularly relate to embodiments where the articles and methods of the present disclosure are used to identify TCRs that bind to MHC:complexes of particular interest. The modifications of the APC for reducing / preventing the expression of endogenous MHC polypeptides, and the engineering of the APC for expressing MHC polypeptides encoded by the genome of a subject of interest, provide for antigen presentation that would occur in the subject.

[0439] In some embodiments, the APC is a primary cell, such as a cell directly isolated / obtained from a living subject / tissue (e.g., via biopsy). The APC can have been isolated / obtained from or be derived from a cell that is isolated / obtained from a subject of interest (e.g., a patient (e.g., a subject / patient comprising a disease-related antigen according to the present disclosure, e.g., a subject / patient comprising a cancer-related neoantigen (i.e., a subject / patient comprising cells containing / expressing a cancer-related neoantigen)). In such embodiments, the APC can range from an autologous subject to a subject from whom the T cells to be screened according to the method for identifying TCRs of the present disclosure are isolated / obtained. In such embodiments, it is generally not contemplated to modify the APC to reduce / prevent the expression of endogenous MHC polypeptides, since the APC already expresses the appropriate MHC molecules for the presentation of the target peptides as they occur in the subject of interest.

[0440] The present disclosure provides a method for producing a cell comprising an MHC molecule labeled with an identifier moiety, the method comprising: (1) introducing into a cell (e.g., an APC) a nucleic acid or nucleic acids encoding a polypeptide comprising: (i) the amino acid sequence of an MHC polypeptide (e.g., β2-microglobulin) and (ii) a moiety that facilitates labeling of the polypeptide with an identifier moiety (e.g., HaloTag); and (2) contacting the cell with a labeling moiety comprising the identifier moiety, wherein the labeling moiety is suitable for labeling the polypeptide encoded by the nucleic acid or nucleic acids of (1) with the identifier moiety (e.g., contacting the cell with a HaloTag ligand comprising an ssDNA moiety and a chloroalkane moiety).

[0441] The present disclosure also provides a method for producing a cell comprising an MHC:peptide complex comprising an MHC molecule labeled with an identifier moiety, the method comprising: (1) introducing into a cell (e.g., an APC) a nucleic acid or nucleic acids encoding a polypeptide comprising: (i) the amino acid sequence of an MHC polypeptide (e.g., β2-microglobulin) and (ii) a moiety that facilitates labeling of the polypeptide with an identifier moiety (e.g., HaloTag); (2) introducing into the cell: (i) a peptide presented by an MHC molecule comprising the polypeptide encoded by the nucleic acid or nucleic acids of (1), or (ii) a nucleic acid encoding a peptide presented by an MHC molecule comprising the polypeptide encoded by the nucleic acid or nucleic acids of (1); and (3) contacting the cell with a labeling moiety comprising the identifier moiety, wherein the labeling moiety is suitable for labeling the polypeptide encoded by the nucleic acid or nucleic acids of (1) with the identifier moiety (e.g., contacting the cell with a HaloTag ligand comprising an ssDNA moiety and a chloroalkane moiety).

[0442] According to the foregoing two paragraphs, in some embodiments, the cell is an APC, e.g., an APC as described herein. In some embodiments, the method further comprises method steps for generating an APC as described herein, e.g., by modification for reducing / preventing the expression of one or more endogenous MHC polypeptides (e.g., in the case of an APC that is allogeneic to a subject, isolating / obtaining T cells to be screened according to the method of the present disclosure from the subject), and / or by modification for expressing one or more non-endogenous MHC polypeptides (e.g., one or more MHC polypeptides encoded by the genome of the subject, isolating / obtaining T cells to be screened according to the method of the present disclosure from the subject).

[0443] Compositions

[0444] The present disclosure provides compositions comprising the polypeptides, antigen-binding molecules, nucleic acids, expression vectors, and cells of the present disclosure.

[0445] The present disclosure also provides a composition comprising a cell (e.g., an antigen-presenting cell) according to the present disclosure and a T cell.

[0446] The present disclosure also provides a composition comprising:

[0447] (a) a cell (e.g., an antigen-presenting cell) that comprises, at its cell surface, a polypeptide according to the present disclosure (e.g., a polypeptide that comprises a portion that facilitates labeling of the polypeptide with an identifier moiety, or a polypeptide that comprises an identifier moiety); and

[0448] (b) a cell (e.g., a T cell) that comprises, at its cell surface, a polypeptide that comprises a sortase receptor motif; and

[0449] (c) a sortase.

[0450] In some embodiments, the composition comprises:

[0451] (a) a cell (e.g., an antigen-presenting cell) that comprises, at its cell surface, a polypeptide according to the present disclosure (e.g., a polypeptide that comprises a portion that facilitates labeling of the polypeptide with an identifier moiety, or a polypeptide that comprises an identifier moiety); and

[0452] (b) a cell (e.g., a T cell) that comprises, at its cell surface: (i) a polypeptide that comprises a sortase receptor motif and (ii) a sortase.

[0453] It should be understood that, for the purpose of identifying T cells that express a TCR that binds to an MHC:peptide complex presented by a cell, it is contemplated that the cells according to the present disclosure are used in a method in which a T cell according to the present disclosure is contacted with a cell according to the present disclosure (e.g., an APC).

[0454] Of course, it should be understood that the composition according to the present disclosure may comprise multiple cells according to the present disclosure and multiple T cells.

[0455] In some embodiments, the composition comprises multiple different T cells. In some embodiments, the composition comprises multiple T cells that encode / contain different TCRs. It should be understood that, in embodiments of the methods of the present disclosure, an antigen-presenting cell according to the present disclosure is contacted with a population of T cells that encode / contain diverse TCRs for the ultimate identification of TCRs that are capable of binding to an MHC:peptide complex presented by the antigen-presenting cell.

[0456] In some embodiments, a composition according to the present disclosure comprises a plurality of different cells according to the present disclosure. In some embodiments, the composition comprises a plurality of antigen-presenting cells, the plurality of antigen-presenting cells comprising / presenting different MHC:peptide complexes. It should be understood that in embodiments of the methods of the present disclosure, a plurality of antigen-presenting cells according to the present disclosure are engineered to express / contain different peptides for presentation by MHC:peptide complexes and contacted with a population of T cells encoding / containing diverse TCRs for eventual identification of TCRs capable of binding to the various different MHC:peptide complexes presented by the antigen-presenting cells.

[0457] Methods for identifying T cell receptor binding to MHC:peptide complexes, and methods for identifying cells that have interacted with each other Methods

[0458] Aspects and embodiments of the present disclosure specifically relate to the identification of T cell receptors (TCRs) that bind to MHC:peptide complexes. Specifically, it is contemplated to use the articles described herein (e.g., polypeptides, MHC molecules, MHC:peptide complexes, antigen-binding molecules, and cells according to the present disclosure) in methods for identifying TCRs that bind to MHC:peptide complexes comprising a peptide of interest.

[0459] Accordingly, the present disclosure provides a method for identifying a T cell receptor (TCR) that binds to an MHC:peptide complex, the method comprising: (1) contacting (a) a cell comprising an MHC:peptide complex with (b) a population of T cells; (2) incubating the cells obtained after step (1) under conditions suitable for trogocytosis of the MHC:peptide complex by T cells comprising a TCR that binds to the MHC:peptide complex; and (3) subsequently analyzing the cells obtained after step (2) to identify the TCR that binds to the MHC:peptide complex; wherein the MHC:peptide complex comprises an MHC molecule, and wherein the MHC molecule comprises a polypeptide comprising (i) the amino acid sequence of an MHC polypeptide and (ii) an identifier portion, wherein the identifier portion is covalently associated with the polypeptide by a bond formed by a self-labeling protein tag.

[0460] The present disclosure also provides a method for identifying a T cell receptor (TCR) that binds to an MHC:peptide complex, the method comprising: (1) contacting (a) cells comprising the MHC:peptide complex with (b) a population of cells comprising T cells in the presence of sortase, wherein the T cells comprise a polypeptide containing a sortase receptor motif at the cell surface; (2) incubating the cells obtained after step (1) under conditions suitable for interaction between the cells of (a) and the population of cells of (b); and (3) subsequently analyzing the cells obtained after step (2) to identify T cells comprising a TCR that binds to the MHC:peptide complex, wherein the MHC:peptide complex comprises an MHC molecule, and wherein the MHC molecule comprises a polypeptide that comprises (i) the amino acid sequence of an MHC polypeptide, (ii) a sortase substrate motif, and (iii) an identifier portion, wherein the identifier portion is covalently associated with the polypeptide by a bond formed from a self-labeling protein tag. In some embodiments, incubating the cells (i.e., the co-culture of cells) under conditions suitable for interaction between the cells of (a) and the population of cells of (b) comprises culturing under conditions suitable for sortase-mediated transfer of the identifier portion (i.e., from the polypeptide comprising (i) the amino acid sequence of an MHC polypeptide, (ii) a sortase substrate motif, and (iii) an identifier portion) to the polypeptide comprising the sortase receptor motif.

[0461] The cells comprising the MHC:peptide complex can be the cells as described herein, such as APCs, and the MHC:peptide complex comprises an MHC molecule containing a polypeptide according to the present disclosure. The step of "contacting" such cells with the T cells can include bringing the cells into contact with each other in a co-culture.

[0462] Incubating the cells (i.e., the co-culture of cells) under conditions suitable for trogocytosis of the MHC:peptide complex by T cells comprising a TCR that binds to the MHC:peptide complex can include maintaining the cells in a humidified environment at 37°C with 5% CO2. Similarly, incubating the cells (i.e., the co-culture of cells) under conditions suitable for interaction between the cells of (a) and the population of cells of (b) can include maintaining the cells in a humidified environment at 37°C with 5% CO2. Similarly, incubating the cells (i.e., the co-culture of cells) under conditions suitable for interaction between the cells of (a) and the population of cells of (b) includes culturing under conditions suitable for sortase-mediated transfer of the identifier portion to the polypeptide comprising the sortase receptor motif, and such culturing can include maintaining the cells in a humidified environment at 37°C with 5% CO2. Appropriate culture conditions can be readily determined by one of ordinary skill in the art.

[0463] Trognocytosis refers to the internalization of the cell membrane and / or cell membrane-bound materials of one cell by another cell. Trognocytosis is described, for example, in Zhao et al., Front Immunol. (2022) 13:791006, which is hereby incorporated by reference in its entirety. The present disclosure particularly focuses on trognocytosis of MHC:peptide complexes presented by APCs by T cells.

[0464] In some embodiments, the sortase is provided at the cell surface of a T cell comprising a polypeptide containing a sortase receptor motif. The sortase can be immobilized on the T cell, i.e., on or at the cell surface of the T cell. In some embodiments, the sortase is covalently associated with a molecule in or at the cell membrane of the T cell. In some embodiments, the sortase is non-covalently associated with a molecule in or at the cell membrane of the T cell (e.g., by protein:protein interactions, such as by antibody:antigen interactions).

[0465] In some aspects and embodiments, the methods of the present disclosure further comprise analyzing the T cell to identify a TCR that binds to an MHC:peptide complex.

[0466] In some embodiments, the method further comprises determining the peptide of the MHC:peptide complex internalized by the T cell. In preferred embodiments, analyzing the T cell comprises analyzing the T cell to determine the characteristics of an identifier portion (i.e., the identifier portion of the MHC:peptide complex, or the identifier portion transferred to the polypeptide containing the sortase receptor motif). As explained above, a given identifier portion is preferably employed with a specific peptide and encodes that specific peptide, and thus determination of the characteristics of the identifier portion provides determination of the peptide of the MHC:peptide complex that has interacted with the T cell (and which may, for example, have been internalized by the T cell).

[0467] Of course, the analysis for determining the characteristics of the identifier portion is selected according to the nature of the identifier portion. In embodiments in which the identifier portion comprises or consists of a nucleic acid portion, the analysis can include determining the structure of the nucleic acid portion. In some embodiments, the analysis includes determining the nucleotide sequence of an identifier portion comprising or consisting of a polynucleotide.

[0468] The nucleotide sequence of a polynucleotide / nucleic acid can be determined by any suitable technique that is well known to those skilled in the art and includes, for example, Sanger sequencing. In preferred embodiments, next-generation sequencing (NGS) techniques can be employed.

[0469] In a preferred embodiment, analyzing the T cell further comprises analyzing the T cell to determine the characteristics of the TCR encoded by the T cell. Determining the characteristics of the TCR encoded by the T cell may include: determining the nucleotide sequence of the nucleic acid encoding the variable region of the TCR, and / or determining the amino acid sequence of the variable region of the TCR (e.g., via in silico translation of the encoded nucleic acid sequence). In some embodiments, the method comprises: determining the nucleotide sequence of the gene region encoding the variable region of the TCR α chain (e.g., Vα and / or Jα regions), and / or determining the nucleotide sequence of the gene region encoding the variable region of the TCR β chain (e.g., Vβ, Dβ, and / or Jβ regions).

[0470] It should be understood that in some embodiments, the method comprises: determining the peptide of the MHC:peptide complex internalized by the T cell (e.g., via determination of the identity of the identifier portion) and determining the characteristics of the TCR encoded by the T cell. Similarly, it should be understood that in some embodiments, the method comprises: determining the peptide of the MHC:peptide complex that has interacted with the T cell (e.g., via determination of the characteristics of the identifier portion transferred to the T cell) and determining the characteristics of the TCR encoded by the T cell. In this way, the method provides facile discrimination of a specific TCR that identifies the MHC:peptide complex presenting the peptide of particular interest. In a preferred embodiment, the identifier portion comprises or consists of a nucleic acid portion (e.g., polynucleotide), and the method comprises using next-generation sequencing (NGS) technology to analyze the identifier portion and the nucleotide sequence of the nucleic acid encoding the variable region of the TCR.

[0471] It should be understood that the analysis is performed on a single cell that has internalized the MHC:peptide complex according to the present disclosure, or on a single cell that has had an identifier portion transferred to it via sortase-mediated transfer. Single-cell sequencing methods are reviewed in, for example, Tang et al., Cell & Bioscience (2019) 9:53, which is hereby incorporated by reference in its entirety.

[0472] The use of multiple identifier portions and corresponding peptides and diverse populations of T cells encoding different TCRs provides simultaneous discrimination of multiple TCR-MHC:peptide complex interaction pairs in a single experiment.

[0473] In some embodiments of the methods of the present disclosure, an APC comprising an MHC molecule containing a polypeptide that comprises (i) the amino acid sequence of an MHC polypeptide and (ii) a portion that facilitates labeling of the polypeptide with an identifier portion can be modified to comprise a peptide according to the present disclosure (e.g., by pulsing the APC with a peptide comprising the relevant peptide, or by introducing a nucleic acid encoding the peptide into the APC), and the APC can also be labeled with an identifier portion corresponding to the peptide (e.g., using a labeling portion). The APCs can be labeled with different peptide and identifier pairs, e.g., in the wells of a polypropylene plate. Then, the various peptide-labeled and identifier-labeled APCs can be pooled and contacted with a population of T cells, and incubated under conditions suitable for trogocytosis of the MHC:peptide complex by T cells comprising a TCR that binds to the MHC:peptide complex. The T cell population can then be analyzed to determine the characteristics of the TCR encoded by the T cells, and the characteristics of the peptide of the MHC:peptide complex internalized by the T cells, i.e., via determination of the characteristics of the identifier portion.

[0474] In some embodiments of the methods of the present disclosure, an APC comprising an MHC molecule containing a polypeptide that comprises (i) the amino acid sequence of an MHC polypeptide, (ii) a sortase substrate motif, and (iii) a portion that facilitates labeling of the polypeptide with an identifier portion can be modified to comprise a peptide according to the present disclosure (e.g., by pulsing the APC with a peptide comprising the relevant peptide, or by introducing a nucleic acid encoding the peptide into the APC), and the APC can also be labeled with an identifier portion corresponding to the peptide (e.g., using a labeling portion). The APCs can be labeled with different peptide and identifier pairs, e.g., in the wells of a polypropylene plate. Then, the various peptide-labeled and identifier-labeled APCs can be pooled and contacted with a population of T cells that comprise a polypeptide containing a sortase receptor motif at the cell surface, in the presence of sortase, and incubated under conditions suitable for sortase-mediated transfer of the identifier portion to the polypeptide containing the sortase receptor motif. The T cell population can then be analyzed to determine the characteristics of the TCR encoded by the T cells, and the characteristics of the peptide of the MHC:peptide complex that has interacted with the T cells (e.g., via determination of the characteristics of the identifier portion transferred to the T cells).

[0475] It should be understood that in some embodiments, it is possible that T cells expressing a given single TCR can recognize multiple distinct peptide:MHC complexes and can thus interact with and / or internalize them. In such embodiments, multiple different identifier portions can be detected in or on a given T cell.

[0476] In some embodiments, the method may further include the step of isolating / selecting T cells that have participated or may have participated in the formation of the TCR-MHC:peptide complex (i.e., before analyzing the T cells to determine the characteristics of the T cells and the identifier moiety). According to such embodiments, the cells are selected / isolated (i.e., selected / isolated from other cells, such as T cells that have not participated / are unlikely to have participated in the formation of such complexes) for subsequent analysis. This method step focuses downstream analysis on those T cells that have interacted with and / or internalized the MHC:peptide complex of interest.

[0477] In some embodiments, the T cells are isolated / selected based on the characterization of correlates of CD3-TCR complex-mediated signaling, such as markers of proliferation / population expansion, growth factor (e.g., IL-2) expression, IFNγ expression, CD107a expression, TNFα expression, GM-CSF expression, perforin expression, granzyme expression, granulysin expression, and / or FAS ligand (FASL) expression.

[0478] In some embodiments, particularly those in which the polypeptides of the present disclosure comprise a detectable moiety as described herein, T cells may be isolated / selected for subsequent analysis based on the detection of a detectable marker in or on the T cell. By way of illustration, in embodiments in which the polypeptides of the present disclosure comprise a fluorescent label, T cells having internalized MHC:peptide complexes comprising the polypeptides of the present disclosure may be detected and sorted based on the detection of the T cells comprising the fluorescent label (e.g., by fluorescence-activated cell sorting (FACS)). The T cells comprising the fluorescent label may then be further analyzed as described above to determine the characteristics of the TCR and the characteristics of the peptides of the peptide:MHC complexes that they have internalized.

[0479] It should be understood that in some embodiments, the method for identifying T cell receptor binding to MHC:peptide complexes additionally includes method steps for generating APCs comprising MHC molecules according to the present disclosure and / or generating APCs comprising MHC:peptide complexes according to the present disclosure, as described above.

[0480] In other aspects and embodiments, the present disclosure more generally provides methods for identifying cells that have interacted with cells that comprise a polypeptide at their cell surface, the polypeptide comprising (i) a sortase substrate motif and (ii) an identifier portion, the method comprising: (1) contacting (a) cells that comprise a polypeptide at their cell surface, the polypeptide comprising (i) a sortase substrate motif and (ii) an identifier portion, with (b) a population of cells that comprise a polypeptide that comprises a sortase receptor motif at their cell surface, in the presence of sortase; and (2) incubating the population of cells obtained after step (1) under conditions suitable for interaction between the cells of (a) and the population of cells of (b); and (3) subsequently analyzing the cells obtained after step (2) to identify cells within the population of cells of (b) that have undergone sortase-mediated transfer of the identifier portion, thereby identifying cells that have interacted with cells that comprise (i) a sortase substrate motif and (ii) an identifier portion. In some embodiments, incubating the cells (i.e., a co-culture of cells) under conditions suitable for interaction between the cells of (a) and the population of cells of (b) comprises incubating under conditions suitable for sortase-mediated transfer of the identifier portion (i.e., from the polypeptide that comprises (i) a sortase substrate motif and (ii) an identifier portion) to the polypeptide that comprises a sortase receptor motif.

[0481] The step of "contacting" the cells of (a) with the cells of (b) may comprise bringing the cells into contact with one another in a co-culture.

[0482] Incubating the cells (i.e., a co-culture of cells) under conditions suitable for interaction between the cells of (a) and the population of cells of (b) comprises culturing under conditions suitable for sortase-mediated transfer of the identifier portion to the polypeptide that comprises a sortase receptor motif, which culturing may comprise maintaining the cells at 37 °C in a humidified atmosphere containing 5% CO2. Suitable culturing conditions can be readily determined by one of ordinary skill in the art.

[0483] In some embodiments, the sortase is provided at the cell surface of the cells that comprise a polypeptide that comprises a sortase receptor motif. The sortase can be immobilized on the cells, i.e., on or at the cell surface of the cells. In some embodiments, the sortase is covalently associated with a molecule in or at the cell membrane of a T cell. In some embodiments, the sortase is non-covalently associated with a molecule in or at the cell membrane of the cells (e.g., by a protein:protein interaction, such as by an antibody:antigen interaction).

[0484] Of course, the analysis for identifying cells within the cell population for (b) that have undergone sortase-mediated transfer of the identifier portion is selected based on the nature of the identifier portion. In embodiments where the identifier portion comprises or consists of a nucleic acid portion, the analysis may include determining the structure of the nucleic acid portion. In some embodiments, the analysis includes determining the nucleotide sequence of an identifier portion that comprises or consists of a polynucleotide. The nucleotide sequence of the polynucleotide / nucleic acid can be determined by any suitable technique, as described above.

[0485] It should be understood that the analysis is performed on single cells that already have an identifier portion transferred to them via sortase-mediated transfer. Single cell sequencing methods are reviewed, for example, in Tang et al., Cell & Bioscience (2019) 9:53, which is hereby incorporated by reference in its entirety. The use of multiple identifier portions and diverse cell populations provides for the simultaneous identification of multiple cell-cell interaction pairs in a single experiment.

[0486] In some embodiments, cells comprising a polypeptide can be labeled with different identifier portions, for example, in the wells of a polypropylene plate, the polypeptide comprising (i) a sortase substrate motif and (ii) a portion that facilitates labeling of the polypeptide with the identifier portion. The cells can then be pooled and contacted with a cell population comprising a polypeptide containing a sortase receptor motif at the cell surface in the presence of sortase and incubated under conditions suitable for sortase-mediated transfer of the identifier portion to the polypeptide containing the sortase receptor motif.

[0487] In some embodiments, the method may further comprise the step of isolating / selecting cells that have participated or may have participated in a cell-cell interaction (i.e., prior to analyzing the cells to determine the characteristics of the identifier portion). According to such embodiments, the cells are selected / isolated (i.e., selected / isolated from other cells, such as cells that have not participated / are unlikely to have participated in such interactions) for subsequent analysis. This method step focuses the downstream analysis on those cells that have participated in the cell-cell interaction of interest.

[0488] In some embodiments, particularly those in which the polypeptides of the present disclosure comprise a detectable portion as described herein, cells can be isolated / selected for subsequent analysis based on the detection of a detectable marker in or on the cell.

[0489] It should be understood that in some embodiments, the method additionally includes method steps for producing cells that comprise, at their cell surface, a polypeptide that comprises (i) a sortase substrate motif and (ii) an identifier portion, as described above.

[0490] Kits

[0491] The present disclosure also provides multi-component kits. Aspects and embodiments of the present disclosure relate to kits for producing cells (e.g., antigen-presenting cells) according to the present disclosure. Aspects and embodiments of the present disclosure relate to kits for performing methods according to the present disclosure.

[0492] In some aspects, a kit according to the present disclosure includes: (1) a nucleic acid or nucleic acids encoding a polypeptide according to the present disclosure; and (2) a labeling moiety comprising an identifier moiety, wherein the labeling moiety is adapted to label a polypeptide encoded by the nucleic acid or nucleic acids of (1) with the identifier moiety via a moiety that facilitates labeling of the polypeptide with the identifier moiety.

[0493] In some aspects, a kit according to the present disclosure includes: (1) a nucleic acid or nucleic acids encoding a polypeptide comprising: (i) the amino acid sequence of β2-microglobulin and (ii) HaloTag; and (2) a HaloTag ligand comprising an ssDNA moiety and a chloroalkane moiety.

[0494] In some aspects, a kit according to the present disclosure includes: (1) a nucleic acid or nucleic acids encoding a polypeptide comprising: (i) the amino acid sequence of β2-microglobulin, (ii) a sortase substrate motif, and (iii) HaloTag; and (2) a HaloTag ligand comprising an ssDNA moiety and a chloroalkane moiety. In some embodiments, the kit further includes sortase, and a reagent for modifying a cell to comprise a polypeptide containing a sortase receptor motif at the cell surface. In some embodiments, the kit includes a reagent for modifying a cell to comprise sortase at the cell surface.

[0495] In some embodiments, the kit further comprises: (3) a peptide presented by an MHC molecule comprising the polypeptide, the polypeptide being encoded by the nucleic acid or nucleic acids of (1), or a nucleic acid encoding a peptide presented by an MHC molecule comprising the polypeptide, the polypeptide being encoded by the nucleic acid or nucleic acids of (1).

[0496] In some aspects, a kit according to the present disclosure includes: (1) a cell or cells comprising an MHC molecule according to the present disclosure, and (2) a peptide presented by the MHC molecule of the cell of (1), or a nucleic acid encoding a peptide presented by the MHC molecule of the cell of (1).

[0497] In some aspects, a kit according to the present disclosure includes a cell or cells comprising an MHC:peptide complex, the MHC:peptide complex comprising an MHC molecule comprising a polypeptide according to the present disclosure and a peptide presented by the MHC molecule.

[0498] In some embodiments, the cells in the various cell populations according to the foregoing two paragraphs are different. In some embodiments, the various cell populations of the kit according to the present disclosure include cells containing MHC molecules, wherein the MHC molecules expressed by different cells in the various cell populations include the polypeptides of the present disclosure labeled with different identifier moieties. In some embodiments, the various cell populations of the kit according to the present disclosure include cells containing different peptides.

[0499] The multi-component kit according to the present disclosure may include a predetermined number of the articles described in the foregoing two paragraphs. In some embodiments, the related articles are provided in a container (e.g., in a vial or bottle). The kit may provide the related articles and instructions (e.g., protocols) on how to use them according to the methods described herein.

[0500] In some embodiments, the multi-component kit includes materials for generating the polypeptides according to the present disclosure. In some embodiments, the multi-component kit includes materials for generating the MHC molecules according to the present disclosure. In some embodiments, the multi-component kit includes materials for generating the MHC:peptide complexes according to the present disclosure. In some embodiments, the multi-component kit includes materials for generating the cells according to the present disclosure. In some embodiments, the multi-component kit includes materials for generating the compositions according to the present disclosure.

[0501] In some embodiments, the multi-component kit may include the nucleic acid / multiple nucleic acids or expression vector / multiple expression vectors according to the present disclosure, and optionally, materials for introducing the nucleic acid / multiple nucleic acids or expression vector / multiple expression vectors into cells.

[0502] In some embodiments, the reagent for modifying cells to contain a polypeptide containing a sortase receptor motif at the cell surface comprises: a nucleic acid / multiple nucleic acids encoding a polypeptide containing a sortase receptor motif, or an expression vector / multiple expression vectors containing such nucleic acids, and optionally, materials for introducing the nucleic acid / multiple nucleic acids or expression vector / multiple expression vectors into cells. However, in a preferred embodiment, the reagent for modifying cells to contain a polypeptide containing a sortase receptor motif at the cell surface is a reagent that provides a non-genetic bioconjugation of the polypeptide to the cell. In some embodiments, the reagent includes ManNAz. In some embodiments, the reagent includes a reagent for functionalizing a polypeptide containing a sortase receptor motif with a cycloalkyne moiety (e.g., a dibenzocyclooctyl (DBCO) moiety or an azadibenzocyclooctyne (ADIBO) moiety).

[0503] In some embodiments, the reagent for modifying a cell to comprise sortase at the cell surface comprises: a nucleic acid / nucleic acids encoding a polypeptide comprising a sortase receptor motif, or an expression vector / expression vectors comprising such nucleic acid, and optionally, a material for introducing the nucleic acid / nucleic acids or expression vector / expression vectors into the cell. In some embodiments, the reagent for modifying a cell to comprise sortase at the cell surface is a reagent that provides non-genetic bioconjugation of a polypeptide to the cell. In some embodiments, the reagent comprises ManNAz. In some embodiments, the reagent comprises a reagent that provides non-genetic bioconjugation of a hapten (e.g., DOTAM or DOTA) to the cell. In some embodiments, the reagent comprises a compound comprising a cycloalkyne moiety (e.g., a DBCO moiety or an ADIBO moiety) and a hapten (e.g., DOTAM or DOTA).

[0504] In some embodiments, the reagent comprises an antigen-binding molecule according to the present disclosure, i.e., an antigen-binding molecule that binds to a hapten (e.g., DOTAM or DOTA), the antigen-binding molecule comprising a sortase moiety.

[0505] The manufacture of the multi-component kit according to the present disclosure preferably follows standard procedures known to those skilled in the art.

[0506] Sequence identity

[0507] As used herein, "sequence identity" refers to the percentage of nucleotide / amino acid residues in a subject sequence that are identical to the nucleotide / amino acid residues in a reference sequence, after aligning the sequences and introducing gaps (if necessary) to achieve the maximum percentage of sequence identity between the sequences. Pairwise and multiple sequence alignments for the purpose of determining the percentage of sequence identity between two or more amino acid or nucleic acid sequences can be achieved in a variety of ways known to those skilled in the art, e.g., using publicly available computer software such as ClustalOmega( J. 2005, Bioinformatics 21, 951-960), T-coffee (Notredame et al. 2000, J. Mol. Biol. (2000) 302, 205-217), Kalign (Lassmann and Sonnhammer 2005, BMC Bioinformatics, 6(298)) and MAFFT (Katoh and Standley 2013, Molecular Biology and Evolution, 30(4) 772–780) software. When using such software, it is preferred to use default parameters, e.g., for gap penalties and extension penalties.

[0508] Sequence

[0509]

[0510]

[0511]

[0512]

[0513]

[0514] Numbered paragraphs

[0515] The following numbered paragraphs provide further statements of features and combinations of features contemplated in connection with the present disclosure:

[0516] 1A. A polypeptide comprising (i) the amino acid sequence of a major histocompatibility complex (MHC) polypeptide and (ii) a moiety that facilitates labeling of the polypeptide with an identifier moiety.

[0517] 2A. The polypeptide according to paragraph 1A, wherein the MHC polypeptide is β2-microglobulin.

[0518] 3A. The polypeptide according to paragraph 1A or paragraph 2A, wherein the identifier moiety is a nucleic acid moiety.

[0519] 4A. The polypeptide according to any one of paragraphs 1A to 3A, wherein the identifier moiety comprises or consists of single-stranded DNA (ssDNA).

[0520] 5A. The polypeptide according to any one of paragraphs 1A to 4A, wherein the moiety that facilitates labeling of the polypeptide with an identifier moiety is or comprises a self-labeling protein tag.

[0521] 6A. The polypeptide according to any one of paragraphs 1A to 5A, wherein the moiety that facilitates labeling of the polypeptide with an identifier moiety is or comprises a HaloTag.

[0522] 7A. A polypeptide comprising (i) the amino acid sequence of β2-microglobulin and (ii) a HaloTag.

[0523] 8A. The polypeptide according to any one of paragraphs 1A to 7A, further comprising a detectable moiety.

[0524] 9A. The polypeptide according to paragraph 8A, wherein the detectable moiety is a fluorescent label.

[0525] 10A. A polypeptide according to any one of paragraphs 1A to 9A, wherein the polypeptide comprises or consists of the following: an amino acid sequence having at least 70% amino acid sequence identity with SEQ ID NO: 11, SEQ ID NO: 10, SEQ ID NO: 13 or SEQ ID NO: 12.

[0526] 11A. A polypeptide comprising (i) an amino acid sequence of a major histocompatibility complex (MHC) polypeptide and (ii) an identifier portion, wherein the identifier portion is covalently associated with the polypeptide via a bond formed by a self-labeling protein tag.

[0527] 12A. The polypeptide according to paragraph 11A, wherein the MHC polypeptide is β2-microglobulin.

[0528] 13A. The polypeptide according to paragraph 11A or paragraph 12A, wherein the identifier portion is a nucleic acid portion.

[0529] 14A. The polypeptide according to any one of paragraphs 11A to 13A, wherein the identifier portion comprises or consists of single-stranded DNA (ssDNA).

[0530] 15A. The polypeptide according to any one of paragraphs 11A to 14A, wherein the self-labeling protein tag is or comprises a HaloTag.

[0531] 16A. The polypeptide according to paragraph 15A, wherein the identifier portion is covalently associated with the polypeptide via an ester bond formed by the dehalogenase activity of HaloTag on a HaloTag ligand comprising the identifier portion and a chloroalkane portion.

[0532] 17A. A polypeptide comprising (i) an amino acid sequence of β2-microglobulin, (ii) a HaloTag, and (iii) a single-stranded DNA (ssDNA) portion, wherein the ssDNA portion is linked to the polypeptide via an ester bond formed by the dehalogenase activity of HaloTag on a HaloTag ligand comprising the ssDNA portion and a chloroalkane portion.

[0533] 18A. The polypeptide according to any one of paragraphs 11A to 17A, further comprising a detectable portion.

[0534] 19A. The polypeptide according to paragraph 18A, wherein the detectable portion is a fluorescent label.

[0535] 20A. An MHC molecule comprising a polypeptide according to any one of paragraphs 1A to 19A.

[0536] 21A. An MHC:peptide complex comprising an MHC molecule according to paragraph 20A and a peptide presented by the MHC molecule.

[0537] 22A. A nucleic acid or nucleic acids encoding a polypeptide according to any one of paragraphs 1A to 10A.

[0538] 23A. The nucleic acid or nucleic acids according to paragraph 11A or paragraph 12A, further comprising a nucleic acid encoding a peptide presented by an MHC molecule, the MHC molecule comprising a polypeptide according to any one of paragraphs 1A to 10A.

[0539] 24A. An expression vector or expression vectors comprising the nucleic acid or nucleic acids according to paragraph 22A or paragraph 23A.

[0540] 25A. A cell comprising a polypeptide according to any one of paragraphs 1A to 19A, an MHC molecule according to paragraph 20A, an MHC:peptide complex according to paragraph 21A, the nucleic acid or nucleic acids according to any one of paragraphs 21A to 23A, or an expression vector or expression vectors according to paragraph 24A.

[0541] 26A. The cell according to paragraph 15A, wherein the cell is an antigen presenting cell (APC).

[0542] 27A. A method for producing a cell comprising an MHC molecule labeled with an identifier moiety, the method comprising:

[0543] (1) introducing the nucleic acid or nucleic acids according to paragraph 22A into a cell; and

[0544] (2) contacting the cell with a labeling moiety comprising an identifier moiety, wherein the labeling moiety is adapted to label a polypeptide encoded by the nucleic acid or nucleic acids of (1) with the identifier moiety.

[0545] 28A. A method for producing a cell comprising an MHC molecule labeled with a single-stranded DNA (ssDNA) moiety, the method comprising:

[0546] (1) introducing into a cell a nucleic acid or nucleic acids encoding a polypeptide comprising (i) the amino acid sequence of β2-microglobulin and (ii) HaloTag; and

[0547] (2) contacting the cell with a HaloTag ligand comprising an ssDNA moiety and a chloroalkane moiety.

[0548] 29A. A method for producing a cell comprising an MHC:peptide complex comprising an MHC molecule labeled with an identifier moiety, the method comprising:

[0549] (1) Introduce the nucleic acid or nucleic acids as described in paragraph 22A into a cell;

[0550] (2) Introduce into the cell: (i) a peptide presented by an MHC molecule comprising a polypeptide encoded by the nucleic acid or nucleic acids of (1), or (ii) a nucleic acid encoding a peptide presented by an MHC molecule comprising a polypeptide encoded by the nucleic acid or nucleic acids of (1);

[0551] and

[0552] (3) Contact the cell with a labeling moiety comprising an identifier moiety, wherein the labeling moiety is adapted to label the polypeptide encoded by the nucleic acid or nucleic acids of (1) with the identifier moiety via a moiety that facilitates labeling of the polypeptide with the identifier moiety.

[0553] 30A. A method for producing a cell comprising an MHC:peptide complex, the MHC:peptide complex comprising an MHC molecule labeled with a single-stranded DNA (ssDNA) moiety, the method comprising:

[0554] (1) Introduce into a cell a nucleic acid or nucleic acids encoding a polypeptide comprising: (i) the amino acid sequence of β2-microglobulin and (ii) HaloTag;

[0555] (2) Introduce into the cell: (i) a peptide presented by an MHC molecule comprising the polypeptide of (1), or (ii) a nucleic acid encoding a peptide presented by an MHC molecule comprising the polypeptide of (1); and

[0556] (3) Contact the cell with a HaloTag ligand comprising an ssDNA moiety and a chloroalkane moiety.

[0557] 31A. A cell produced by the method according to any one of paragraphs 27A to 30A.

[0558] 32A. A composition comprising a cell according to any one of paragraphs 25A, 26A or 31A and a T cell.

[0559] 33A. A method for identifying a T cell receptor (TCR) that binds to an MHC:peptide complex, the method comprising:

[0560] (1) Contact a cell comprising an MHC:peptide complex as described in paragraph 21A with a population of T cells;

[0561] (2) Incubate the cells under conditions suitable for trogocytosis of the MHC:peptide complex by T cells comprising a TCR that binds to the MHC:peptide complex; and

[0562] (3) Subsequently, T cells are analyzed to identify the TCRs that bind to the MHC:peptide complex.

[0563] 34A. A kit comprising:

[0564] (1) The nucleic acid or nucleic acids according to paragraph 22A; and

[0565] (2) A labeling moiety comprising an identifier moiety, wherein the labeling moiety is suitable for labeling the polypeptide encoded by the nucleic acid or nucleic acids according to (1) with the identifier moiety via a moiety that facilitates labeling of the polypeptide with the identifier moiety.

[0566] 35A. A kit comprising:

[0567] (1) A nucleic acid or nucleic acids encoding a polypeptide comprising: (i) the amino acid sequence of β2-microglobulin and (ii) HaloTag; and

[0568] (2) A HaloTag ligand comprising an ssDNA moiety and a chloroalkane moiety.

[0569] 36A. The kit according to paragraph 35A or paragraph 36A, further comprising:

[0570] (3) A peptide presented by an MHC molecule comprising the polypeptide encoded by the nucleic acid or nucleic acids according to (1), or a nucleic acid encoding a peptide presented by an MHC molecule comprising the polypeptide encoded by the nucleic acid or nucleic acids according to (1).

[0571] 37A. A kit comprising:

[0572] (1) A cell or cells comprising the MHC molecule according to paragraph 20A, and

[0573] (2) A peptide presented by the MHC molecule of the cell according to (1), or a nucleic acid encoding a peptide presented by the MHC molecule of the cell according to (1).

[0574] 38A. A kit comprising a cell or cells containing the MHC:peptide complex according to paragraph 21A.

[0575] 1B. A polypeptide comprising (i) the amino acid sequence of a major histocompatibility complex (MHC) polypeptide and (ii) a moiety that facilitates labeling of the polypeptide with an identifier moiety.

[0576] 2B. The polypeptide according to paragraph 1B, wherein the MHC polypeptide is β2-microglobulin.

[0577] 3B. A polypeptide according to paragraph 1B or paragraph 2B, wherein the identifier portion is a nucleic acid portion.

[0578] 4B. A polypeptide according to any one of paragraphs 1B to 3B, wherein the identifier portion comprises or consists of single-stranded DNA (ssDNA).

[0579] 5B. A polypeptide according to any one of paragraphs 1B to 4B, wherein the portion that facilitates labeling of the polypeptide with the identifier portion is or comprises a self-labeling protein tag.

[0580] 6B. A polypeptide according to any one of paragraphs 1B to 5B, wherein the portion that facilitates labeling of the polypeptide with the identifier portion is or comprises a HaloTag.

[0581] 7B. A polypeptide according to any one of paragraphs 1B to 6B, wherein the polypeptide further comprises a sortase substrate motif.

[0582] 8B. A polypeptide comprising (i) the amino acid sequence of β2-microglobulin and (ii) a HaloTag.

[0583] 9B. A polypeptide comprising (i) the amino acid sequence of β2-microglobulin, (ii) a sortase substrate motif, and (iii) a HaloTag.

[0584] 10B. A polypeptide according to any one of paragraphs 1B to 9B, wherein the polypeptide further comprises a detectable moiety.

[0585] 11B. A polypeptide according to paragraph 10B, wherein the detectable moiety is a fluorescent label.

[0586] 12B. A polypeptide according to any one of paragraphs 1B to 11B, wherein the polypeptide comprises or consists of: an amino acid sequence having at least 70% amino acid sequence identity with SEQ ID NO:11, SEQ ID NO:10, SEQ ID NO:13, or SEQ ID NO:12.

[0587] 13B. A polypeptide comprising (i) the amino acid sequence of a major histocompatibility complex (MHC) polypeptide and (ii) an identifier portion, wherein the identifier portion is covalently associated with the polypeptide through a bond formed by a self-labeling protein tag.

[0588] 14B. A polypeptide according to paragraph 13B, wherein the MHC polypeptide is β2-microglobulin.

[0589] 15B. A polypeptide according to paragraph 13B or paragraph 14B, wherein the identifier portion is a nucleic acid portion.

[0590] 16B. A polypeptide according to any one of paragraphs 13B to 15B, wherein the identifier portion comprises or consists of single-stranded DNA (ssDNA).

[0591] 17B. A polypeptide according to any one of paragraphs 13B to 16B, wherein the self-labeling protein tag is or comprises a HaloTag.

[0592] 18B. A polypeptide according to paragraph 17B, wherein the identifier portion is covalently associated with the polypeptide via an ester bond formed by the dehalogenase activity of the HaloTag on a HaloTag ligand comprising the identifier portion and an alkyl chloride portion.

[0593] 19B. A polypeptide according to any one of paragraphs 13B to 18B, wherein the polypeptide further comprises a sortase substrate motif.

[0594] 20B. A polypeptide comprising (i) the amino acid sequence of β2-microglobulin, (ii) a HaloTag, and (iii) a single-stranded DNA (ssDNA) portion, wherein the ssDNA portion is linked to the polypeptide via an ester bond formed by the dehalogenase activity of the HaloTag on a HaloTag ligand comprising the ssDNA portion and an alkyl chloride portion.

[0595] 21B. A polypeptide comprising (i) the amino acid sequence of β2-microglobulin, (ii) a sortase substrate motif, (iii) a HaloTag, and (iv) a single-stranded DNA (ssDNA) portion, wherein the ssDNA portion is linked to the polypeptide via an ester bond formed by the dehalogenase activity of the HaloTag on a HaloTag ligand comprising the ssDNA portion and an alkyl chloride portion.

[0596] 22B. A polypeptide according to any one of paragraphs 12B to 21B, wherein the polypeptide further comprises a detectable moiety.

[0597] 23B. A polypeptide according to paragraph 22B, wherein the detectable moiety is a fluorescent label.

[0598] 24B. An MHC molecule comprising a polypeptide according to any one of paragraphs 1B to 23B.

[0599] 25B. An MHC:peptide complex comprising the MHC molecule according to paragraph 24B and a peptide presented by the MHC molecule.

[0600] 26B. A nucleic acid or nucleic acids encoding a polypeptide according to any one of paragraphs 1B to 12B.

[0601] 27B. The nucleic acid or nucleic acids according to paragraph 26B, wherein the nucleic acid or nucleic acids further comprise a nucleic acid encoding a peptide presented by an MHC molecule, and the MHC molecule comprises a polypeptide according to any one of paragraphs 1B to 12B.

[0602] 28B. An expression vector or expression vectors comprising the nucleic acid or nucleic acids according to paragraph 26B or paragraph 27B.

[0603] 29B. A cell comprising a polypeptide according to any one of paragraphs 1B to 23B, an MHC molecule according to paragraph 24B, an MHC:peptide complex according to paragraph 25B, the nucleic acid or nucleic acids according to paragraph 26B or paragraph 27B, or the expression vector or expression vectors according to paragraph 28B.

[0604] 30B. The cell according to paragraph 29B, wherein the cell is an antigen presenting cell (APC).

[0605] 31B. A method for producing a cell comprising an MHC molecule labeled with an identifier moiety, the method comprising:

[0606] (1) introducing the nucleic acid or nucleic acids according to paragraph 26B into a cell; and

[0607] (2) contacting the cell with a labeling moiety comprising an identifier moiety, wherein the labeling moiety is suitable for labeling a polypeptide encoded by the nucleic acid or nucleic acids of (1) with the identifier moiety.

[0608] 32B. A method for producing a cell comprising an MHC molecule labeled with a single-stranded DNA (ssDNA) moiety, the method comprising:

[0609] (1) introducing a nucleic acid or nucleic acids encoding a polypeptide comprising: (i) the amino acid sequence of β2-microglobulin and (ii) HaloTag into a cell; and

[0610] (2) contacting the cell with a HaloTag ligand comprising an ssDNA moiety and a chloroalkane moiety.

[0611] 33B. A method for producing a cell comprising an MHC molecule labeled with a single-stranded DNA (ssDNA) moiety, the method comprising:

[0612] (1) introducing a nucleic acid or nucleic acids encoding a polypeptide comprising: (i) the amino acid sequence of β2-microglobulin, (ii) a sortase substrate motif, and (iii) HaloTag into a cell; and

[0613] (2) Contact the cell with a HaloTag ligand comprising an ssDNA moiety and a chloroalkane moiety.

[0614] 34B. A method for producing a cell comprising an MHC:peptide complex, the MHC:peptide complex comprising an MHC molecule labeled with an identifier moiety, the method comprising:

[0615] (1) Introduce into the cell the nucleic acid or nucleic acids according to paragraph 26B;

[0616] (2) Introduce into the cell: (i) a peptide presented by an MHC molecule comprising a polypeptide encoded by the nucleic acid or nucleic acids of (1), or (ii) a nucleic acid encoding a peptide presented by an MHC molecule comprising a polypeptide encoded by the nucleic acid or nucleic acids of (1);

[0617] and

[0618] (3) Contact the cell with a labeling moiety comprising an identifier moiety, wherein the labeling moiety is adapted to label, via a moiety facilitating the labeling of a polypeptide with the identifier moiety, the polypeptide encoded by the nucleic acid or nucleic acids of (1).

[0619] 35B. A method for producing a cell comprising an MHC:peptide complex, the MHC:peptide complex comprising an MHC molecule labeled with a single-stranded DNA (ssDNA) moiety, the method comprising:

[0620] (1) Introduce into the cell a nucleic acid or nucleic acids encoding a polypeptide comprising: (i) the amino acid sequence of β2-microglobulin and (ii) HaloTag;

[0621] (2) Introduce into the cell: (i) a peptide presented by an MHC molecule comprising the polypeptide of (1), or (ii) a nucleic acid encoding a peptide presented by an MHC molecule comprising the polypeptide of (1); and

[0622] (3) Contact the cell with a HaloTag ligand comprising an ssDNA moiety and a chloroalkane moiety.

[0623] 36B. A method for producing a cell comprising an MHC:peptide complex, the MHC:peptide complex comprising an MHC molecule labeled with a single-stranded DNA (ssDNA) moiety, the method comprising:

[0624] (1) Introduce into the cell a nucleic acid or nucleic acids encoding a polypeptide comprising: (i) the amino acid sequence of β2-microglobulin, (ii) a sortase substrate motif, and (iii) HaloTag;

[0625] (2) Introduce the following into the cell: (i) a peptide presented by an MHC molecule comprising the polypeptide of (1), or (ii) a nucleic acid encoding a peptide presented by an MHC molecule comprising the polypeptide of (1); and

[0626] (3) Contact the cell with a HaloTag ligand comprising an ssDNA moiety and a chloroalkane moiety.

[0627] 37B. A cell produced by the method according to any one of paragraphs 31B to 36B.

[0628] 38B. A composition comprising a cell according to any one of paragraphs 29B, 30B or 37B and a T cell.

[0629] 39B. A method for identifying a T cell receptor (TCR) that binds to an MHC:peptide complex, the method comprising:

[0630] (1) Contacting (a) a cell comprising an MHC:peptide complex with (b) a population of T cells;

[0631] (2) Incubating the cells obtained after step (1) under conditions suitable for trogocytosis of the MHC:peptide complex by T cells comprising a TCR that binds to the MHC:peptide complex;

[0632] and

[0633] (3) Subsequently analyzing the cells obtained after step (2) to identify the TCR that binds to the MHC:peptide complex;

[0634] wherein the MHC:peptide complex comprises an MHC molecule, and wherein the MHC molecule comprises a polypeptide comprising (i) the amino acid sequence of an MHC polypeptide and (ii) an identifier portion, wherein the identifier portion is covalently associated with the polypeptide by a bond formed by a self-labeling protein tag.

[0635] 40B. A method for identifying a T cell receptor (TCR) that binds to an MHC:peptide complex, the method comprising:

[0636] (1) Contacting (a) a cell comprising an MHC:peptide complex with (b) a population of cells comprising T cells in the presence of sortase, wherein the T cells comprise a polypeptide containing a sortase receptor motif at the cell surface;

[0637] (2) Incubating the cells obtained after step (1) under conditions suitable for the interaction between the cells of (a) and the population of cells of (b); and

[0638] (3) Subsequently, the cells obtained after step (2) are analyzed to identify T cells comprising a TCR that binds to the MHC:peptide complex;

[0639] wherein the MHC:peptide complex comprises an MHC molecule, and wherein the MHC molecule comprises a polypeptide that comprises (i) the amino acid sequence of an MHC polypeptide, (ii) a sortase substrate motif, and (iii) an identifier portion, wherein the identifier portion is covalently associated with the polypeptide by a bond formed by a self-labeling protein tag.

[0640] 41B. The method according to paragraph 40B, wherein the sortase is provided at the cell surface of T cells comprising a polypeptide containing a sortase receptor motif.

[0641] 42B. The method according to any one of paragraphs 39B to 41B, wherein the MHC polypeptide is β2-microglobulin.

[0642] 43B. The method according to any one of paragraphs 39B to 42B, wherein the identifier portion is a nucleic acid portion.

[0643] 44B. The method according to any one of paragraphs 39B to 43B, wherein the identifier portion comprises or consists of single-stranded DNA (ssDNA).

[0644] 45B. The method according to any one of paragraphs 39B to 44B, wherein the self-labeling protein tag is or comprises a HaloTag.

[0645] 46B. The method according to paragraph 45B, wherein the identifier portion is covalently associated with the polypeptide by an ester bond formed by the dehalogenase activity of the HaloTag on a HaloTag ligand comprising the identifier portion and a chloroalkane portion.

[0646] 47B. The method according to any one of paragraphs 39B to 46B, wherein the polypeptide comprising: the amino acid sequence of an MHC polypeptide, and the identifier portion, further comprises a detectable portion.

[0647] 48B. The method according to paragraph 47B, wherein the detectable portion is a fluorescent label.

[0648] 49B. A kit comprising:

[0649] (1) The nucleic acid or nucleic acids according to paragraph 26B; and

[0650] (2) A labeled portion comprising an identifier portion, wherein the labeled portion is suitable for labeling a polypeptide encoded by the nucleic acid or nucleic acids according to (1) with the identifier portion via a moiety that facilitates labeling of the polypeptide with the identifier portion.

[0651] 50B. A kit, comprising:

[0652] (1) A nucleic acid or nucleic acids that encode a polypeptide comprising: (i) the amino acid sequence of β2-microglobulin and (ii) HaloTag; and

[0653] (2) A HaloTag ligand comprising an ssDNA moiety and a chloroalkane moiety.

[0654] 51B. A kit, comprising:

[0655] (1) A nucleic acid or nucleic acids that encode a polypeptide comprising: (i) the amino acid sequence of β2-microglobulin, (ii) a sortase substrate motif, and (iii) HaloTag; and

[0656] (2) A HaloTag ligand comprising an ssDNA moiety and a chloroalkane moiety.

[0657] 52B. The kit according to paragraph 51B, wherein the kit further comprises:

[0658] Sortase, and a reagent for modifying a cell to comprise a polypeptide containing a sortase receptor motif at the cell surface.

[0659] 53B. The kit according to paragraph 51B or paragraph 52B, wherein the kit comprises a reagent for modifying a cell to comprise sortase at the cell surface.

[0660] 54B. The kit according to any one of paragraphs 49B to 53B, wherein the kit further comprises:

[0661] A peptide presented by an MHC molecule comprising a polypeptide, the polypeptide being encoded by the nucleic acid or nucleic acids according to (1), or a nucleic acid encoding a peptide presented by an MHC molecule comprising a polypeptide, the polypeptide being encoded by the nucleic acid or nucleic acids according to (1).

[0662] 55B. A kit, comprising:

[0663] (1) A cell or cells comprising the MHC molecule according to paragraph 24B, and

[0664] (2) A peptide presented by the MHC molecule of the cell according to (1), or a nucleic acid encoding a peptide presented by the MHC molecule of the cell according to (1).

[0665] 56B. A kit, comprising a cell or cells containing the MHC:peptide complex according to paragraph 25B.

[0666] 57B. A polypeptide comprising (i) a sortase substrate motif and (ii) a moiety that facilitates labeling of the polypeptide with an identifier moiety.

[0667] 58B. The polypeptide according to paragraph 57B, wherein the polypeptide further comprises the amino acid sequence of a protein localized to the cell membrane.

[0668] 59B. The polypeptide according to any one of paragraphs 57B to 58B, wherein the moiety that facilitates labeling of the polypeptide with an identifier moiety is or comprises a self-labeling protein tag.

[0669] 60B. The polypeptide according to any one of paragraphs 57B to 59B, wherein the moiety that facilitates labeling of the polypeptide with an identifier moiety is or comprises a HaloTag.

[0670] 61B. The polypeptide according to any one of paragraphs 57B to 60B, wherein the identifier moiety is a nucleic acid moiety.

[0671] 62B. The polypeptide according to any one of paragraphs 57B to 61B, wherein the identifier moiety comprises or consists of single-stranded DNA (ssDNA).

[0672] 63B. A polypeptide comprising (i) the amino acid sequence of a protein localized to the cell membrane, (ii) a sortase substrate motif, and (iii) a HaloTag.

[0673] 64B. The polypeptide according to any one of paragraphs 57B to 63B, wherein the polypeptide further comprises a detectable moiety.

[0674] 65B. The polypeptide according to paragraph 64B, wherein the detectable moiety is a fluorescent label.

[0675] 66B. A polypeptide comprising (i) a sortase substrate motif and (ii) an identifier moiety, wherein the identifier moiety is covalently associated with the polypeptide via a bond formed by a self-labeling protein tag.

[0676] 67B. The polypeptide according to any one of paragraphs 63B to 66B, wherein the self-labeling protein tag is or comprises a HaloTag.

[0677] 68B. The polypeptide according to paragraph 67B, wherein the identifier moiety is covalently associated with the polypeptide via an ester bond formed by the dehalogenase activity of HaloTag on a HaloTag ligand comprising the identifier moiety and a chloroalkane moiety.

[0678] 69B. The polypeptide according to any one of paragraphs 63B to 68B, wherein the identifier moiety is a nucleic acid moiety.

[0679] 70B. A polypeptide according to any one of paragraphs 63B to 68B, wherein the identifier portion comprises or consists of single-stranded DNA (ssDNA).

[0680] 71B. A polypeptide comprising (i) the amino acid sequence of a protein localized to the cell membrane, (ii) a sortase substrate motif, (iii) a HaloTag, and (iv) a single-stranded DNA (ssDNA) moiety, the ssDNA moiety being linked to the polypeptide via an ester bond formed by the dehalogenase activity of the HaloTag on a HaloTag ligand, the HaloTag ligand comprising the ssDNA moiety and a chloroalkane moiety.

[0681] 72B. A polypeptide according to any one of paragraphs 63B to 71B, wherein the polypeptide further comprises a detectable moiety.

[0682] 73B. A polypeptide according to paragraph 72B, wherein the detectable moiety is a fluorescent label.

[0683] 74B. A nucleic acid or nucleic acids encoding a polypeptide according to any one of paragraphs 57B to 65B.

[0684] 75B. An expression vector or expression vectors comprising the nucleic acid or nucleic acids according to paragraph 74B.

[0685] 76B. A cell comprising a polypeptide according to any one of paragraphs 57B to 71B, a nucleic acid or nucleic acids according to paragraph 74B, or an expression vector or expression vectors according to paragraph 75B.

[0686] 77B. A cell according to paragraph 76B, wherein the cell is an antigen-presenting cell (APC).

[0687] 78B. A method for producing a cell comprising a polypeptide labeled with an identifier portion, the method comprising:

[0688] (1) introducing the nucleic acid or nucleic acids according to paragraph 74B into a cell; and

[0689] (2) contacting the cell with a labeling moiety comprising the identifier portion, wherein the labeling moiety is adapted to label the polypeptide encoded by the nucleic acid or nucleic acids of (1) with the identifier portion.

[0690] 79B. A method according to paragraph 78B, wherein the identifier portion is a nucleic acid moiety.

[0691] 80B. A method according to paragraph 78B or paragraph 79B, wherein the identifier portion comprises or consists of single-stranded DNA (ssDNA).

[0692] 81B. A method according to any one of paragraphs 78B to 80B, wherein the moiety that facilitates labeling of the polypeptide with the identifier moiety is or comprises a HaloTag, and wherein the labeling moiety is a HaloTag ligand comprising the identifier moiety and a chloroalkane moiety.

[0693] 82B. A method for producing a cell comprising a polypeptide labeled with a single-stranded DNA (ssDNA) moiety, the method comprising:

[0694] (1) introducing into the cell a nucleic acid or nucleic acids encoding a polypeptide comprising: (i) the amino acid sequence of a protein localized to the cell membrane, (ii) a sortase substrate motif, and (iii)

[0695] a HaloTag; and

[0696] (2) contacting the cell with a HaloTag ligand comprising an ssDNA moiety and a chloroalkane moiety.

[0697] 83B. A cell produced by a method according to any one of paragraphs 78B to 82B.

[0698] 84B. A composition comprising:

[0699] (a) a cell comprising on its cell surface a polypeptide according to any one of paragraphs 57B to 73B;

[0700] (b) a cell comprising on its cell surface a polypeptide comprising a sortase receptor motif; and

[0701] (c) a sortase.

[0702] (d) a sortase.

[0703] (e) a sortase.

[0704] 85B. The composition according to paragraph 84B, wherein the composition comprises:

[0705] (a) a cell comprising on its cell surface a polypeptide according to any one of paragraphs 57B to 73B; and

[0706] (b) a cell comprising on its cell surface: (i) a polypeptide comprising a sortase receptor motif and (ii) a sortase.

[0707] (c) a cell comprising on its cell surface: (i) a polypeptide comprising a sortase receptor motif and (ii) a sortase.

[0708] 86B. The composition according to paragraph 84B or paragraph 85B, wherein the cell according to (a) is an antigen-presenting cell and / or wherein the cell according to (b) is a T cell.

[0709] 87B. A method for identifying cells that have interacted with cells comprising a polypeptide according to any one of paragraphs 66B to 73B on their cell surface, the method comprising:

[0710] (1) contacting (a) cells comprising a polypeptide according to any one of paragraphs 66B to 73B on their cell surface with (b) a cell population comprising cells that comprise a polypeptide comprising a sortase receptor motif on the cell surface, in the presence of a sortase; and

[0711] (2) incubating the cell population obtained after step (1) under conditions suitable for interaction between the cells of (a) and the cell population of (b); and

[0712] (3) subsequently analyzing the cells obtained after step (2) to identify cells within the cell population of (b) that have undergone sortase-mediated transfer of an identifier moiety, thereby identifying cells that have interacted with cells comprising a polypeptide according to any one of paragraphs 66B to 73B on their cell surface.

[0713] 88B. The method according to paragraph 87B, wherein the sortase is provided on the cell surface of cells comprising a polypeptide comprising a sortase receptor motif.

[0714] 89B. A kit comprising:

[0715] (1) a nucleic acid or nucleic acids according to paragraph 74B; and

[0716] (2) a labeling moiety comprising an identifier moiety, wherein the labeling moiety is suitable for labeling a polypeptide encoded by the nucleic acid or nucleic acids according to (1) with the identifier moiety via a moiety that facilitates labeling of the polypeptide with the identifier moiety.

[0717] 90B. A kit comprising:

[0718] (1) a nucleic acid or nucleic acids encoding a polypeptide comprising: (i) the amino acid sequence of a protein localized to the cell membrane, (ii) a sortase substrate motif, and (iii) a HaloTag; and

[0719] (2) a HaloTag ligand comprising an ssDNA moiety and a chloroalkane moiety.

[0720] 91B. The kit according to paragraph 89B or paragraph 90B, wherein the kit further comprises:

[0721] a sortase, and a reagent for modifying cells to comprise a polypeptide comprising a sortase receptor motif on the cell surface.

[0722] The kit according to any one of paragraphs 89B to 91B, wherein the kit comprises a reagent for modifying a cell to comprise sortase at the cell surface.

[0723] ***

[0724] The present disclosure includes combinations of the described aspects and preferred features, unless such combinations are clearly impermissible or expressly avoided.

[0725] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0726] Aspects and embodiments of the invention will now be illustrated by way of example with reference to the accompanying drawings. Other aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

[0727] Throughout the specification and the following claims, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" shall be understood to imply the inclusion of the stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0728] As used herein, "peptide" refers to a chain of two or more amino acid monomers linked by peptide bonds. Peptides typically have a length of about 2 to 50 amino acids in a region. "Polypeptide" is a polymer chain of two or more peptides. Polypeptides typically have a length greater than about 50 amino acids. References herein to peptides, polypeptides, and proteins also include glycopeptides / glycopolypeptides / glycoproteins, lipopeptides / lipopolypeptides / lipoproteins, nucleopeptides / nucleopolypeptides / nucleoproteins, etc.

[0729] As used herein, an amino acid sequence or a region of a polypeptide that "corresponds" to a specified reference amino acid sequence or polypeptide has at least 60% (e.g., at least one of ≥65%, ≥70%, ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) sequence identity with the amino acid sequence / polypeptide / amino acid sequence of the amino acid sequence / polypeptide / region. The amino acid sequence / region / position of a polypeptide / amino acid sequence that "corresponds" to a specified reference polypeptide / amino acid sequence can be identified by sequence alignment of the subject sequence with the reference sequence, e.g., using sequence alignment software such as ClustalOmega( J. 2005, Bioinformatics 21, 951 - 960).

[0730] As used herein, an amino acid sequence (e.g., the amino acid sequence of a peptide / polypeptide / domain / region) "derived from" a reference amino acid sequence (e.g., the amino acid sequence of a reference peptide / polypeptide / domain / region) comprises or consists of the following: an amino acid sequence having at least 60% (e.g., at least one of 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the reference amino acid sequence.

[0731] It must be noted that, unless the context clearly dictates otherwise, as used in this specification and the appended claims, the singular forms "a", "an", "the" and "said" include plural referents. Ranges may be expressed herein as from "about" one particular value and / or to "about" another particular value. When expressing such a range, another embodiment includes from one particular value and / or to another particular value. Similarly, when a value is expressed as an approximation by use of the antecedent "about", it should be understood that the particular value forms another embodiment.

[0732] Where nucleic acid sequences are disclosed herein, their reverse complements are also specifically contemplated.

[0733] The methods described herein may preferably be carried out in vitro. The term "in vitro" is intended to encompass procedures carried out with cells in culture, while the term "in vivo" is intended to encompass procedures carried out with / on a whole multicellular organism.

[0734] Brief Description of the Drawings

[0735] Embodiments and experiments demonstrating the principles of the present disclosure will now be discussed with reference to the drawings.

[0736] Figure 1 : 293T cells KO HLA were transfected to express HaloTag-β2M-HLA-A*02:01. Cells derived from a single clone generated by antibiotic selection and cells from the parental cell line were labeled with the HaloTag ligand tetramethylrhodamine (HTL TMR). HaloTag ligand (HTL) staining reaches saturation at a concentration of about 1 μM, and the same concentration only results in a slight increase in the background signal of fluorescence on the parental cell line.

[0737] Figure 2A and Figure 2B: HaloTag-β2M-HLA-A*02:01 293T cells were labeled with ssDNA-HTL and then tracked with HTL AF488 (500 nM). The ssDNA-HTL reacts specifically with the HaloTag (HT) and is thus attached to the cell surface, as evident from the increased fluorescence for the DNA-binding dye and the decreased tracking label. At approximately 10 μM ssDNA-HTL, the labeling reached 60% to 70% integrity.

[0738] Figure 3 : HaloTag-β2M-HLA-A*02:01 293T cells were either unlabeled or labeled with HTL AF660 or ssDNA-HTL Atto655. After pulsing the cells with a dilution series of the NY-ESO-1 peptide, these cells were used as target cells in the Jurkat activation assay. The Jurkats were modified to convert the intensity of TCR signaling into relative luminescence in this assay. The curves for the three conditions were close to each other and thus conjugation with HTL did not interfere with immune activation.

[0739] Figure 4: HaloTag-β2M-HLA-A*02:01 293T cells were labeled with HTL TMR. After pulsing the cells with the WT-1 peptide in medium at 37 °C for one hour, they were co-cultured with donor T cells and a bispecific antibody. The WT-1 bispecific antibody induces an artificial synapse by bridging CD3 on the T cell and WT-1pHLA on the target cell. DP47 is a bispecific antibody that has the exact same CD3 binder and the same layout but does not recognize the target in this assay. The co-cultures were set up with different target cell:effector cell ratios in the range of 5:1 to 1:5. After 2 hours, the extent of trogocytosis in those co-cultures was analyzed by flow cytometry. Target cells and effector cells were distinguished by CD3 staining in the live single cell population. In the co-culture treated with the WT-1 bispecific antibody (which had an E:T ratio of 1:5), the median fluorescence intensity of the TMR increased to over 10,000. Lower E:T ratios (which thus resulted in fewer target cells) led to lower median fluorescence intensities, but the percentage of positive cells remained high. The background noise observed in the DP47 control was very low.

[0740] Figure 5: HaloTag-β2M-HLA-A*02:01 293T cells were labeled with HTL TMR. After pulsing the cells with WT-1 peptide in medium at 37 °C for one hour, they were co-cultured with donor T cells and a bispecific antibody. The WT-1 bispecific antibody induces an artificial synapse by bridging CD3 on the T cell and WT-1pHLA on the target cell. DP47 is a bispecific antibody that has an identical CD3 binder and the same layout but does not recognize the target in this assay. The co-cultures were set up to have different target cell:effector cell ratios in the range of 5:1 to 1:5. After 24 hours, the degree of trogocytosis in those co-cultures was analyzed by flow cytometry. Target cells and effector cells were distinguished by CD3 staining in live single cell populations. In the E:T 1:5 co-culture treated with the WT-1 bispecific antibody, the median fluorescence intensity of TMR increased to over 10,000. Lower E:T ratios (which thus resulted in fewer target cells) led to lower median fluorescence intensities, but the percentage of positive cells remained high. Compared to the 2-hour experiment, the percentage of positive cells also increased from 40% to nearly 80% in the co-culture with an E:T ratio of 5:1. The background noise over-observed in the DP47 control was low but slightly higher compared to the 2-hour experiment.

[0741] Figure 6 : HaloTag-β2M-HLA-A*02:01 293T cells were labeled with HTL TMR. After pulsing the cells with MART-1 peptide in medium at 37 °C for one hour, they were co-cultured with a pool of T cells specific for MART-1. The E:T was set to 1:1. After 1 hour, 2 hours, 3 hours, and 24 hours, the degree of trogocytosis in those co-cultures was analyzed by flow cytometry. Target cells and effector cells were distinguished by CD3 staining in live single cell populations.

[0742] Figure 7 : Additionally, the activation of T cells in the co-cultures was analyzed by looking at the upregulation of CD69. Antibody staining showed an increase in CD69 expression on T cells in the pulsed co-cultures. In the unpulsed co-cultures, the signal remained at a high level throughout the 24-hour time window with a low signal background.

[0743] Figure 8 : Additionally, the activation of T cells in the co-cultures was analyzed by looking at the upregulation of CD137. Antibody staining showed an increase in CD137 expression on T cells in the pulsed co-cultures at the 24-hour time point. The signal in the unpulsed co-cultures remained at a low level throughout the 24-hour time window.

[0744] Figure 9:In addition, degranulation of T cells in the co-cultures was analyzed by looking at the upregulation of CD107a. Antibody staining showed that the CD107a expression on T cells in the pulsed co-cultures started at the one-hour time point and increased. The signal decreased over time and approached the background signal of T cells in the unpulsed co-cultures at the 24-hour time point.

[0745] Figure 10: HaloTag-β2M-HLA-A*02:01 293T cells were labeled with HTL AF660. After pulsing the cells with NLV peptide in medium at 37 °C for one hour, they were co-cultured with donor T cells (where approximately 1% were specific for the CMV-derived epitope NLV). The E:T was set to 1:1. After 3 hours, the degree of trogocytosis in those co-cultures was analyzed by flow cytometry. Target cells and effector cells were distinguished by CD3 staining in the live single-cell population. T cells specific for the NLV pHLA complex were shown by Dextramer staining ( Figure 10A ). In all co-cultures ( Figure 10B ), a certain percentage of T cells became positive for the HaloTagged protein by trogocytosis. However, in the pulsed co-cultures, all T cells specific for the NLV epitope were also positive for HTL.

[0746] Figure 11 : HaloTag-β2M-HLA-A*02:01 293T cells were labeled with HTL-46bp-Atto647N. After pulsing the cells with MART-1 peptide in medium at 37 °C for one hour, they were co-cultured with donor T cells (where approximately 1% were specific for the tumor antigen MART-1). The E:T was set to 1:1. After 14 hours, the degree of trogocytosis in those co-cultures was analyzed by flow cytometry. Target cells and effector cells were distinguished by CD3 staining in the live single-cell population. T cells specific for the MART-1 pHLA complex were shown by Dextramer staining. The MFI of barcode-HTL-derived on Dextramer-positive T cells in the pulsed was higher compared to the unpulsed co-cultures.

[0747] Figure 12 : T cells obtained from healthy donor subjects were fed with azide-modified sugars to install azide groups on their cell surfaces for subsequent strain-promoted alkyne-azide cycloaddition. After 48 hours, the cells were incubated with a fluorescently labeled peptide-DBCO conjugate for 120 minutes. Flow cytometric analysis of single live cells showed that, within the concentration range studied, the peptide specifically attached to cells fed with azide-modified sugars but not to control T cells.

[0748] Figure 13A and Figure 13B : Transduced Jurkat cells and T cells obtained from healthy donor subjects were fed with azide-modified sugars to install azide groups on their cell surfaces for subsequent strain-promoted alkyne-azide cycloaddition. After 48 hours, the cells were incubated with different ratios of fluorescently labeled peptide-DBCO conjugates and DOTAM-DBCO for 120 minutes. Flow cytometric analysis of single live cells showed that for both T cells (A) and Jurkat cells (B), for a peptide:DOTAM ratio of 10:1, both components were highly attached.

[0749] Figure 14A and Figure 14B : Transduced Jurkat cells were fed with azide-modified sugars to install azide groups on their cell surfaces for subsequent strain-promoted alkyne-azide cycloaddition. After 48 hours, the cells were incubated with sortase A receptor peptide-DBCO conjugate and DOTAM_DBCO for 120 minutes. An antibody-enzyme conjugate against a hapten was used to tether sortase A to effector cells. 293T HaloTag-β2M-HLA-A*02:01 cells were labeled with a biotinylated sortase donor peptide-HTL conjugate and pulsed to present the NY-ESO-1 antigen. Co-cultures of these cells were found to result in peptide transfer after three hours (A) and 16 hours (B), as determined by an increase in biotin staining on single live effector cells.

[0750] Figure 15 : Transduced Jurkat cells were fed with azide-modified sugars to install azide groups on their cell surfaces for subsequent strain-promoted alkyne-azide cycloaddition. After 48 hours, the cells were incubated with sortase A receptor peptide-DBCO conjugate and DOTAM_DBCO for 120 minutes. An antibody-enzyme conjugate against a hapten was used to tether sortase A to effector cells. 293T HaloTag-β2M-HLA-A*02:01 cells were labeled with a biotinylated ssDNA peptide-HTL conjugate and pulsed to present the NY-ESO-1 antigen. Co-cultures of these cells were found to result in peptide transfer after 16 hours, as determined by an increase in biotin staining on single live effector cells.

[0751] Examples

[0752] Example 1: Materials and Methods

[0753] 1.1 Recombinant DNA techniques

[0754] Standard methods were used to manipulate DNA as described in Sambrook et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Molecular biology reagents were used according to the manufacturer's instructions.

[0755] 1.2 DNA sequencing

[0756] DNA sequences were determined by double-stranded Sanger sequencing.

[0757] 1.3 Gene synthesis

[0758] The desired gene fragments were synthesized by automated gene synthesis from synthetic oligonucleotides and PCR products by Genscript Biotech (New Jersey, US). Gene segments flanked with single restriction endonuclease cleavage sites were cloned into standard cloning / sequencing vectors. Plasmid DNA was purified from transformed bacteria and its concentration was determined by UV spectroscopy. The DNA sequence of the subcloned gene fragment was confirmed by DNA sequencing. Gene segments with appropriate restriction sites were designed to allow subcloning into the corresponding expression vectors.

[0759] 1.4 Generation of Halo-tagged cell lines

[0760] To produce cell lines expressing HLA-A*02:01 containing a HaloTag fusion at the cell surface, a gene fusion of the HaloTag enzyme with human β2M (which was linked to HLA-A*02:01 via a linker) was generated by standard cloning techniques and subcloned into a suitable expression vector. The amino acid sequences of the nascent and mature HaloTag-B2M-HLA-A02 fusion proteins are shown in SEQ ID NO:12 and SEQ ID NO:13.

[0761] Plasmids encoding the HaloTag-B2M-HLA-A02 fusion protein were introduced into CRIPSR-modified 293T cells by lipofection to knockout the endogenous HLA locus. Transfected clones were selected by standard antibiotic selection. The resulting clones were further selected based on positive antibody staining against HLA-A*02:01 and β2M as determined by analysis performed by flow cytometry techniques. The selected positive clones were finally exposed to a dilution series of the HaloTag ligand (HTL) with the fluorophore tetramethylrhodamine (TMR) (HTL TMR). Briefly, the cells were detached, washed with PBS, and incubated with HTL TMR in PBS at 1x 106 Incubate together at a density of 6 cells / ml for 30 minutes. Then, before analysis by flow cytometry, wash the cells with PBS and stain with a standard live / dead stain. Events are gated based on forward scatter and side scatter, and then doublets and dead cells are excluded. Plot the median fluorescence intensity (MFI) of the cells against the concentration of HTL. Starting from a concentration of 1 μM, the final clone achieved complete labeling ( Figure 1 ). Low background labeling was observed on the parental cell line at 1 μM.

[0762] 1.5 Attachment of DNA barcodes to cells via the HaloTag system

[0763] Prepare the DNA oligonucleotide barcode to be used with the 293T HaloTag-β2M-HLA-A*02:01 cell line as follows. Modify the 3' end of a 69 bp oligonucleotide with a nucleotide sequence conforming to the consensus shown in SEQ ID NO:18 with a fluorophore (Atto655 or Cy3b), and bind the 5' end to HTL. The modified oligonucleotide is obtained from Biomers GmbH and is hereinafter referred to as "DNA-HTL".

[0764] Isolate the 293T HaloTag-β2M-HLA-A*02:01 cells, wash them with PBS, and incubate them with a dilution series of DNA-HTL in modified DPBS (Gibco, #14287080) at a cell density of 1x 10 6 cells / ml for 120 minutes. Then wash the cells with PBS and stain with a standard live / dead stain. Additionally, include HTL AF488 in PBS at 500 nM for 5 minutes. Subsequently, analyze the cells by flow cytometry. Events are gated based on forward scatter and side scatter, and then doublets and dead cells are excluded. Plot the median fluorescence intensity (MFI) of DNA-HTL and the tracer against the concentration of DNA-HTL. Starting from a concentration of 10 μM, achieve approximately 60% to 70% labeling (Figure 2).

[0765] 1.6 Preparation of virus-like particles (VLPs)

[0766] Use approximately 70% confluent Lenti-X TM 293T cells (Takara, #632180) and the chimeric receptor-encoding transfer vector and the packaging plasmids pCAG-VSVG and psPAX2 in a molar ratio of 2:1:2 for Lipofectamine LTX-based TMTransfection (Giry-Laterriere M et al. Methods Mol Biol. 2011;737:183-209; Myburgh R et al. Mol Ther Nucleic Acids. 2014). As a control for each experiment, mock virus-like particles (VLPs) were produced using only the packaging vector without the transfer vector. After 48 hours, the supernatant was collected and the remaining cells were removed by centrifugation. The VLPs were used directly or stored at -80 °C.

[0767] 1.7 Transduction of Jurkat cells

[0768] 1 x 10 6 Jurkat cells were seeded into the wells of a 24-well plate. Fresh or thawed at 37 °C VLPs were used, and 300 μl of VLPs were added to the 24-well plate together with 8 μg / ml polybrene (Sigma Aldrich) and Lentiboost P (1:100) (Sirion Biotech, #SB-P-LV-101-12) for infection by rotation at 1100 x g and at 31 °C for 99 min. The cells were incubated at 37 °C, 5% CO2 for at least 72 hours before transduction was evaluated by flow cytometry.

[0769] 1.8 Isolation of primary T cells from blood

[0770] The donor blood is from Blutspende Zürich (Rütistrasse 19, 8952 Schlieren). Prepare a LeucoSEP tube (Fisher Scientific, #10349081) with 15 mL of Histopaque density gradient medium (Sigma-Aldrich, #10771) at room temperature and centrifuge at 400 x g for 5 minutes until the Histopaque has passed through the filter. Dilute the blood with an equal volume of PBS. Add 30 ml of the blood / buffer mixture to the LeucoSEP tube. Centrifuge the tube at 1200 x g for 20 min and decant. Carefully transfer the band containing peripheral blood mononuclear cells (PBMCs) to a fresh 50 ml falcon tube and supplement to 50 ml with DPBS. Wash the cells 3 times with DPBS and finally resuspend in DPBS and count. Pan T cell isolation is performed by negative selection using a Pan T cell isolation kit (Miltenyi, #130-096-535) according to the manufacturer's instructions. After isolation, the cells are either frozen or used directly. The cells are cultured in Advanced RPMI (Gibco, #11530446), 10% FBS (Sigma, #F4135-500ML), 1% Glutamax (Gibco, #35050-038), 50 IU / Proleukin (Novartis), 25 ng / ml IL-7 (Miltenyi, #130-095-364) and 50 ng / ml IL-15 (Miltenyi, #130-095-766).

[0771] 1.9 Metabolic glycosylation engineering of cells and strain-promoted alkyne-azide cycloaddition of peptides and small molecules Addition

[0772] The cells are cultured in standard cell medium containing 50 μM tetra-acylated N-azidoacetylmannosamine (ManNAz) for 24 to 48 hours to install click handles on the surface of the cells. The functionalized cells are incubated with DOTAM-DBCO and / or peptide conjugates having the following structures: "[6-FAM]-GGGGG-[CYS(DBCO-MAL)]" or "GGGGG-[CYS(DBCO-MAL)]", and the incubation is carried out in PBS for two hours with different concentrations and ratios of components.

[0773] 1.10 Production of IgG-like proteins in Expi293F cells

[0774] Antibodies and bispecific antibodies are produced by transient transfection of Expi293F cells. The cells are at 2.5x10 6Cells were inoculated at a density of / ml in Expi293 medium (Gibco, #1435101). The expression vector and ExpiFectamine (Gibco, ExpiFectamine transfection kit, #13385544) were separately mixed in OptiMEM (Gibco, #11520386). After 5 min, the two solutions were combined, mixed by pipetting and incubated at room temperature for 25 min. The cells were added to the vector / ExpiFectamine solution and cultured in an orbital incubator at 37 °C and 5% CO2 atmosphere for 24 h. One day after transfection, supplements (Enhancer 1 + 2, ExpiFectamine transfection kit) were added. Four to five days later, the cell supernatant was harvested by centrifugation and subsequent filtration (0.2 μm filter), and the protein was purified from the harvested supernatant by the standard method shown below.

[0775] 1.11 Purification of IgG-like proteins

[0776] Purify the protein from the filtered cell culture supernatant. Briefly, purify the protein from the cell culture supernatant by Protein A or κ-selective affinity chromatography (equilibration buffer: 20 mM sodium citrate, 20 mM sodium phosphate, pH 7.5; elution buffer: 20 mM sodium citrate, pH 3.0) to purify the Fc-containing protein from the filtered cell culture supernatant. Elution was achieved at pH 3.0, and the pH of the sample was immediately neutralized thereafter. Concentrate the protein by centrifugation (Millipore ULTRA-15, #UFC903096), and separate the aggregated protein from the monomeric protein by size exclusion chromatography in 20 mM histidine, 140 mM sodium chloride (pH 6.0).

[0777] 1.12 Analysis of IgG-like proteins

[0778] According to Pace et al., Protein Science (1995) 4:2411 - 1423, the concentration of purified protein was determined by measuring the absorbance at 280 nm using the mass extinction coefficient calculated based on the amino acid sequence. In the presence and absence of reducing agent, the purity and molecular weight of the protein were analyzed by CE - SDS using LabChip GXII or LabChip GX Touch (Perkin Elmer). The aggregate content was determined by HPLC chromatography using an analytical size - exclusion column (TSKgel G3000 SW XL or UP - SW3000, Tosoh Bioscience) equilibrated in running buffer (200 mM KH2PO4, 250 mM KCl pH 6.2, 0.02% NaN3) at 25 °C.

[0779] 1.13 Conjugation of IgG with fluorescent dyes

[0780] The purified IgG was labeled with a fluorophore for subsequent analysis by flow cytometry. Briefly, an appropriate amount of the protein was labeled using a commercially available kit such as Alexa Fluor TM 647 Antibody Labeling Kit #A20186 (ThermoFisher). The resulting conjugate was analyzed to determine the degree of labeling, and the ideal amount of antibody for the staining procedure was determined individually in titration experiments.

[0781] 1.14 Peptide barcodes for use with the HaloTag system

[0782] The peptide with the structure "[biotin - Ahx] - SELPETGK" was conjugated to HTL via an ester reaction. The resulting conjugate is hereinafter referred to as "peptide HTL".

[0783] 293T HaloTag - β2M - HLA - A*02:01 cells were isolated, washed with PBS, and incubated with 100 μM peptide HTL in PBS at a cell density of 3 x 10 6 cells / ml for 120 minutes. The cells were then washed with PBS and used in co - cultures.

[0784] 1.15 DNA-peptide barcodes for use with the HaloTag system

[0785] Prepare the DNA oligonucleotide peptide barcodes to be used with the 293T HaloTag-β2M-HLA-A*02:01 cell line as follows. The 3' end of a 69 bp oligonucleotide having a nucleotide sequence conforming to the consensus shown in SEQ ID NO:18 was modified with "biotin-TEG" and the 5' end was modified with "DBCO-TEG". The modified oligonucleotide was obtained from IDT as an HPLC-purified product. A peptide having the structure "[Lys(N3)]-SELPETGK" was conjugated to HTL via an ester reaction and bound to the oligonucleotide via strain-promoted alkyne-azide cycloaddition. The resulting conjugate is hereinafter referred to as "DNA-peptide HTL" and has the following structure: biotin-DNA-[DBCO / azide]-peptide-[amine / ester]-HTL, where "[DBCO / azide]" indicates the linkage formed by the strain-promoted alkyne-azide cycloaddition reaction between the DBCO and azide moieties, and where "[amine / ester]" indicates the ester bond formed on the HaloTag ligand by the dehalogenase activity of HaloTag, the HaloTag ligand comprising a chloroalkane moiety.

[0786] Isolate 293T HaloTag-β2M-HLA-A*02:01 cells, wash them with PBS, and incubate them with 100 μM of DNA-peptide HTL in modified DPBS (Gibco, #14287080) at a cell density of 3×10 6 cells / ml for 120 minutes. Then wash the cells with modified DPBS and use them in co-cultures.

[0787] Example 2: Conjugation of HaloTag ligands does not interfere with activation of immune cells

[0788] The present inventors investigated whether the proximity of the bound HTL to the TCR of immune cells in the 293T HaloTag-β2M-HLA-A*02:01 cell line interferes with proper synapse formation and activation of immune cells. Possible steric as well as charge-based interference was evaluated in a Jurkat activation assay. Jurkat parental cell lines modified to express luciferase under the control of a TCR signaling-inducible promoter were obtained from Promega (#GA1162), which have a deleted TCR, endogenously express CD4, and have also been engineered to express CD8a. The cells were additionally modified by lentiviral transduction to present the 1G4 TCR specific for NY-ESO-1. The assay was performed according to the provided protocol. Briefly, 293T HaloTag-β2M-HLA cells were isolated, washed with PBS, and labeled with HTL AF660 and HTL-ssDNA-(69bp)-Atto655 under optimal conditions as described above. The labeled cells were pulsed with a dilution series of the NY-ESO-1 antigen peptide “SLLMWITQC” (SEQ ID NO:14) in growth medium at 37 °C for one hour. Thereafter, the labeled and pulsed cells were co-cultured with the Jurkat cell line at a ratio of 1:1 and at 1×10 5 cells / well. The assay was performed in a 96-well white F-bottom chimney plate. After 7 hours, the provided substrate (Promega, #J3081) was added, and the resulting luminescence was read at a Tecan Spark multimode microplate reader. GraphPad Prism version 8 was used to plot the peptide concentration versus luminescence and fit a concentration-response curve, which did not show significant differences between fluorophore- or DNA-binding cells compared to cells with an empty HaloTag ( Figure 3 ).

[0789] Example 3: Trogocytosis of HaloTagged HLA induced by synthetic immunity

[0790] Bispecific antibodies that engage CD3 on T cells and target antigens on tumor cells can be used to engage and activate T cells and direct their effector activity against cells expressing the target antigen. In an artificial synapse, material is transferred from the engaged target cell to the cell expressing the CD3-TCR complex by trogocytosis. The ability of HaloTagged-HLA molecules to be transferred in this manner was evaluated using a bispecific antibody that recognizes WT-1 (Augsberger et al., Blood (2021) 138(25):2655-2669).

[0791] Human T cells were isolated from donor blood by standard procedures. 293T HaloTag-β2M-HLA-A*02:01 cells were labeled with HTL TMR (as described above) and pulsed with the WT-1 antigenic peptide "RMFPNAPYL" (SEQ ID NO:15), which forms the epitope of the WT-1 binding arm of the bispecific antibody (as described above). Next, co-cultures of effector cells and WT-1 peptide-pulsed target cells with the bispecific antibody were set up in technical replicates. The concentration of the bispecific antibody was fixed at 10 μg / ml, while the effector cell to target cell ratio was varied from 5:1, 1:1, and 1:5, with the total cell count kept constant at 5×10 5 cells / well in 100 μl of medium. The resulting trogocytosis was evaluated by flow cytometry after 2 hours and 24 hours. Target and effector cells were distinguished from the live single cell population by CD3 staining. The median fluorescence intensity of effector cells in the TMR channel and the percentage of cells positive for TMR among total effector cells were exported and further analyzed. After two hours, the WT-1-targeted bispecific antibody had induced trogocytosis of HaloTag-β2M-HLA-A*02:01 from target cells to effector cells (Figure 4), and this was also observed after 24 hours (Figure 5). Very low background staining was observed with the non-target control bispecific antibody DP47 after two hours (Figure 4) and 24 hours (Figure 5).

[0792] Example 4: Trogocytosis of HaloTagged HLA induced by TCR interaction in a pool with a given specificity

[0793] To test whether HaloTagged HLA was also capable of being transferred by TCR-driven trogocytosis, a polyclonal pool of T cells specific for the tumor-associated target MART-1 was used. 293T HaloTag-β2M-HLA-A*02:01 cells were labeled with HTL TMR (as described above) and pulsed with the MART-1 antigenic peptide "ELAGIGILTV" (SEQ ID NO:16). Next, co-cultures of effector cells and target cells were set up at a 1:1 ratio and 5×10 5 cells / well. The resulting trogocytosis was analyzed by flow cytometry after 1 hour, 2 hours, 3 hours, and 24 hours. Target and effector cells were distinguished from the live single cell population by CD3 staining. The median fluorescence intensity of effector cells in the TMR channel and the percentage of cells positive for TMR among total effector cells, as well as the median fluorescence intensity of CD69, CD107a, and CD137, were exported and further analyzed. As early as 1 hour after the start of co-culture, the T cells did become positive for the HaloTagged molecule ( Figure 6)。Over the entire period, the MFI was consistently higher compared to non-pulsed control cells. Additionally, the T cells were activated, as seen from the early upregulation of CD69 ( Figure 7 ) and CD137 after 24 hours ( Figure 8 ). Finally, the T cells showed degranulation, as evident from CD107a that appeared on the cell surface ( Figure 9 ).

[0794] Example 5: Trogocytosis of HaloTagged HLA induced by TCR interaction using donor T cells with more than one specificity Trogocytosis of HaloTagged HLA

[0795] To test whether HaloTagged HLA could also be transferred to T cells with a given specificity that are present at low frequency within a T cell pool with different specificities, donor-derived T cells were screened for the presence of HLA-A*02:01-restricted antiviral T cells by Dextramer staining (Immudex, Denmark). These donor T cells were used for co-culture. 293T HaloTag-β2M-HLA-A*02:01 cells were labeled with HTL AF660 (as described above) and pulsed with the CMV antigen peptide "NLVPMVATV" (SEQ ID NO:17). Next, co-cultures of effector cells and target cells were set up at a 1:1 ratio and 3×10 5 cells / well. The resulting cytolysis was analyzed by flow cytometry after three hours. Target cells and effector cells were distinguished from the live single cell population by CD3 staining. Cells with NLV specificity were identified by Dextramer staining (Immudex, Denmark). Although a fraction of the T cells became positive for HaloTag independent of peptide pulsing, all NLV-reactive cells were HaloTag positive in the peptide-pulsed co-cultures (Figure 10).

[0796] Example 6: Trogocytosis of ssDNA barcodes attached to HaloTagged HLA induced by TCR interaction using donor T cells with more than one specificity Trogocytosis of HaloTagged HLA

[0797] To evaluate whether DNA bound to the HaloTag-β2M-HLA-A*02:01 molecule can also be transferred into a T cell pool with different specificities and exist at low frequencies, the presence of HLA-A*02:01-restricted tumor-associated T cell epitopes such as MART-1 was screened for donor-derived T cells by Dextramer staining (Immudex, Denmark). These donor T cells were used for co-culture. 293T HaloTag-β2M-HLA-A*02:01 cells were labeled with HTL 46bp Atto647N (as described above) and pulsed with MART-1 peptide (SEQ ID NO:16). Next, co-culture of effector cells and target cells was set up at a 1:1 ratio and 3×10 5 cells / well. The resulting trogocytosis was analyzed by flow cytometry after 14 hours. Target cells and effector cells were distinguished from the live single-cell population by CD3 staining. Cells with MART-1 specificity were identified by Dextramer staining (Immudex, Denmark). Compared to the unpulsed control, the pulsed cultures had a higher MFI ( Figure 11 ) in the Atto647N channel in the MART-1 positive population.

[0798] Example 7: Functionalization of glycosylation-engineered cells for attachment of peptides to the cell surface

[0799] Healthy donor T cells were functionalized with peptides attached to glycoproteins as described below. Cells were cultured for 48 hours in the presence of azide-modified sugars. Thus, azide moieties were presented on the cell surface and could therefore be used for strain-promoted alkyne-azide cycloaddition. Cells were incubated with different concentrations of fluorescently labeled peptide-DBCO conjugates for two hours. Subsequently, the washed cells were analyzed by flow cytometry. Analysis of single live cells revealed specific and concentration-dependent attachment of the peptides ( Figure 12 ) compared to unengineered control cells.

[0800] Example 8: Functionalization of glycosylation-engineered cells for attachment of small molecules and peptides to the cell surface

[0801] Transduced Jurkat cells and healthy donor T cells were functionalized with peptides and small molecules attached to glycoproteins as described below. The cells were cultured for 48 hours in the presence of azide-modified sugars. Thus, the azide moiety was presented on the surface of the cells and could therefore be used for strain-promoted alkyne-azide cycloaddition. The cells were incubated for two hours with different ratios of fluorescently labeled peptide-DBCO conjugates and / or DOTAM-DBCO, with the total concentration fixed at 10 μg / ml. DOTAM binding was detected using a fluorescently labeled DOTAM-specific antibody (an antibody formed by a polypeptide having the amino acid sequences of SEQ ID NO:29 and SEQ ID NO:31, labeled with AF647). The washed cells were then analyzed by flow cytometry. Analysis of single live cells revealed specific and ratio-dependent attachment of the peptide and hapten (Figure 13).

[0802] Example 9: Transfer of peptides between cells by tethered sortase A in co-cultures

[0803] Transduced Jurkat cells were functionalized with peptides, and DOTAM was attached to glycoproteins as described in Example 8. The modified version of sortase A was then tethered to the cells by using an anti-DOTAM antibody-sortase A conjugate (formed by a polypeptide having the amino acid sequences of SEQ ID NO:30 and SEQ ID NO:32).

[0804] 293T HaloTag-β2M-HLA-A*02:01 cells were labeled with peptide HTL (as described in Example 1.14) and pulsed with the NY-ESO-1 antigen peptide "SLLMWITQC" (SEQ ID NO:14). Next, co-culture of effector cells and target cells was set up at a 1:1 ratio and 3×10 5 cells / well. The resulting peptide transfer was analyzed by flow cytometry after three hours and sixteen hours. Within the live single cell population, target cells and effector cells were distinguished by CD45 staining. Specific increase of peptide HTL on effector cells was detected by anti-biotin antibody staining and was observed after three hours ( Figure 14A ) and sixteen hours ( Figure 14B ) of co-culture.

[0805] Example 10: Transfer of barcode constructs between cells by tethered sortase A in co-cultures

[0806] Transduced Jurkat cells were functionalized with peptides, and DOTAM was attached to glycoproteins as described in Example 8. The modified version of sortase A was then tethered to the cells by using an anti-DOTAM antibody-sortase A conjugate (formed by a polypeptide having the amino acid sequences of SEQ ID NO:30 and SEQ ID NO:32).

[0807] The 293T HaloTag-β2M-HLA-A*02:01 cells were labeled with DNA-peptide HTL (as described in Example 1.15) and pulsed with the NY-ESO-1 antigen peptide "SLLMWITQC" (SEQ ID NO:14). Next, co-cultures of effector cells and target cells were set up at a 1:1 ratio and 3×10 5 cells / well. The resulting peptide transfer was analyzed by flow cytometry 16 hours later. Within the live single cell population, target cells and effector cells were distinguished by CD45 staining. The increase in specificity of DNA-peptide HTL on effector cells was detected by anti-biotin antibody staining ( Figure 15 ).

Claims

1. A polypeptide comprising (i) an amino acid sequence of a major histocompatibility complex (MHC) polypeptide and (ii) a moiety that facilitates labeling of the polypeptide with an identifier moiety.

2. The polypeptide according to claim 1, wherein the MHC polypeptide is β2-microglobulin.

3. The polypeptide according to claim 1 or claim 2, wherein the identifier moiety is a nucleic acid moiety, optionally wherein the identifier moiety comprises or consists of single-stranded DNA (ssDNA).

4. The polypeptide according to any one of claims 1 to 3, wherein the moiety that facilitates labeling of the polypeptide with an identifier moiety is or comprises a self-labeling protein tag.

5. The polypeptide according to any one of claims 1 to 4, wherein the moiety that facilitates labeling of the polypeptide with an identifier moiety is or comprises a HaloTag.

6. The polypeptide according to any one of claims 1 to 5, wherein the polypeptide further comprises a sortase substrate motif.

7. The polypeptide according to any one of claims 1 to 6, which further comprises a detectable moiety.

8. The polypeptide according to claim 7, wherein the detectable moiety is a fluorescent label.

9. The polypeptide according to any one of claims 1 to 8, wherein the polypeptide comprises or consists of the following: An amino acid sequence having at least 70% amino acid sequence identity with SEQ ID NO:11, SEQ ID NO:10, SEQ ID NO:13 or SEQ ID NO:

12.

10. A polypeptide comprising (i) an amino acid sequence of β2-microglobulin and (ii) a HaloTag.

11. A polypeptide comprising (i) an amino acid sequence of β2-microglobulin, (ii) a sortase substrate motif and (iii) a HaloTag.

12. An MHC molecule comprising the polypeptide according to any one of claims 1 to 11.

13. An MHC:peptide complex comprising the MHC molecule according to claim 12 and a peptide presented by the MHC molecule.

14. A nucleic acid or nucleic acids encoding the polypeptide according to any one of claims 1 to 13.

15. The nucleic acid or nucleic acids according to claim 14, which further comprises a nucleic acid encoding a peptide presented by an MHC molecule, the MHC molecule comprising the polypeptide according to any one of claims 1 to 11.

16. An expression vector or expression vectors comprising the nucleic acid or nucleic acids according to claim 14 or 15.

17. A cell comprising the polypeptide according to any one of claims 1 to 11, the MHC molecule according to claim 12, the MHC:peptide complex according to claim 13, the nucleic acid or nucleic acids according to claim 14 or 15, or the expression vector or expression vectors according to claim 16.

18. The cell according to claim 17, wherein the cell is an antigen-presenting cell (APC).

19. A method for producing a cell comprising an MHC molecule labeled with an identifier moiety, the method comprising: (1) introducing the nucleic acid or nucleic acids according to claim 15 into a cell; and (2)Contact the cells with a labeling moiety comprising an identifier moiety, wherein the labeling moiety is adapted to label the polypeptide encoded by the nucleic acid or nucleic acids of (1) with the identifier moiety.

20. A method for producing cells comprising an MHC:peptide complex, the MHC:peptide complex comprising an MHC molecule labeled with a single-stranded DNA (ssDNA) moiety, the method comprising: (1) Introducing into the cells a nucleic acid or nucleic acids encoding a polypeptide comprising: (i) the amino acid sequence of β2-microglobulin and (ii) a HaloTag; (2) Introducing into the cells: (i) a peptide presented by an MHC molecule comprising the polypeptide of (1), or (ii) a nucleic acid encoding a peptide presented by an MHC molecule comprising the polypeptide of (1); and (3) Contacting the cells with a HaloTag ligand comprising the ssDNA moiety and a chloroalkane moiety.

21. A method for producing cells comprising an MHC:peptide complex, the MHC:peptide complex comprising an MHC molecule labeled with a single-stranded DNA (ssDNA) moiety, the method comprising: (1) Introducing into the cells a nucleic acid or nucleic acids encoding a polypeptide comprising: (i) the amino acid sequence of β2-microglobulin, (ii) a sortase substrate motif, and (iii) a HaloTag; (2) Introducing into the cells: (i) a peptide presented by an MHC molecule comprising the polypeptide of (1), or (ii) a nucleic acid encoding a peptide presented by an MHC molecule comprising the polypeptide of (1); and (3) Contacting the cells with a HaloTag ligand comprising the ssDNA moiety and a chloroalkane moiety.

22. A cell produced by the method according to any one of claims 19 to 21.

23. A composition comprising the cell according to any one of claims 17, 18 or 22 and T cells.

24. A method for identifying a T cell receptor (TCR) that binds to an MHC:peptide complex, the method comprising: (1) Contacting cells comprising the MHC:peptide complex according to claim 13 with a population of T cells; (2) Incubating the cells under conditions suitable for trogocytosis of the MHC:peptide complex by T cells comprising a TCR that binds to the MHC:peptide complex; and (3) Subsequently analyzing the T cells to identify a TCR that binds to the MHC:peptide complex.

25. A method for identifying a T cell receptor (TCR) that binds to an MHC:peptide complex, the method comprising: (1) Contacting (a) cells comprising an MHC:peptide complex with (b) a population of T cells; (2) Incubating the cells obtained after step (1) under conditions suitable for trogocytosis of the MHC:peptide complex by T cells comprising a TCR that binds to the MHC:peptide complex; and (3) Subsequently analyzing the cells obtained after step (2) to identify a TCR that binds to the MHC:peptide complex; wherein the MHC:peptide complex comprises an MHC molecule, and wherein the MHC molecule comprises a polypeptide that comprises (i) the amino acid sequence of an MHC polypeptide and (ii) an identifier moiety, wherein the identifier moiety is covalently associated with the polypeptide by a bond formed by a self-labeling protein tag.

26. A method for identifying a T cell receptor (TCR) that binds to an MHC:peptide complex, the method comprising: (1) contacting (a) a cell comprising an MHC:peptide complex with (b) a population of cells comprising T cells in the presence of sortase, wherein the T cells comprise a polypeptide containing a sortase receptor motif at the cell surface; (2) incubating the cells obtained after step (1) under conditions suitable for interaction between the cells of (a) and the population of cells of (b); and (3) subsequently analyzing the cells obtained after step (2) to identify T cells comprising a TCR that binds to the MHC:peptide complex; wherein the MHC:peptide complex comprises an MHC molecule, and wherein the MHC molecule comprises a polypeptide that comprises (i) the amino acid sequence of an MHC polypeptide, (ii) a sortase substrate motif, and (iii) an identifier moiety, wherein the identifier moiety is covalently associated with the polypeptide by a bond formed by a self-labeling protein tag.

27. The method according to claim 26, wherein the sortase is provided at the cell surface of T cells comprising a polypeptide containing a sortase receptor motif.

28. The method according to any one of claims 25 to 27, wherein the MHC polypeptide is β2-microglobulin.

29. The method according to any one of claims 25 to 28, wherein the identifier moiety is a nucleic acid moiety, optionally wherein the identifier moiety comprises or consists of single-stranded DNA (ssDNA).

30. The method according to any one of claims 25 to 29, wherein the self-labeling protein tag is or comprises a HaloTag.

31. The method according to claim 30, wherein the identifier moiety is covalently associated with the polypeptide by an ester bond formed by the dehalogenase activity of the HaloTag on a HaloTag ligand comprising the identifier moiety and a chloroalkane moiety.

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