Identification of T cell receptors

Abstract

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

Description

[Technical Field]

[0001] The present disclosure relates to the fields of molecular biology and immunology. [Background technology]

[0002] Adoptive T cell therapy (ACT) is a powerful approach to cancer treatment that uses cancer-specific T cells (Rosenberg and Restifo, Science (2015) 348 (6230): 62-68). The cells used in ACT are typically either naturally occurring cancer antigen-specific cells, or T cells engineered to express a TCR specific for cells expressing an MHC:peptide complex containing a peptide of the target antigen of interest (i.e., TCR-engineered T cells), or T cells engineered to express a chimeric antigen receptor (CAR) containing 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 an appropriate target antigen and target molecule (Leko and Rosenberg, Cancer Cell (2020) 38 (4): 454-472). TCR discovery remains a challenging endeavor (Joglekar and Li, Nat Methods (2021) 18(8):873-880). Rapid and accurate identification of neoantigens and evaluation of their immunogenicity are critical for the widespread adoption of ACT (Garcia-Garijo et al., Front Immunol. (2019) 10:1392).

[0003] Trogocytosis is a well-established process that occurs bidirectionally during immune interactions between T cells and cells expressing MHC:peptide complexes recognized by their TCRs (Miyake and Karasuyama, Cells (2021) 10(5):1255). Identification of the 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 remains a need in the art for techniques that provide high-throughput identification of TCR and MHC:peptide interaction partners. Summary of the Invention

[0005] In a first aspect, the present disclosure provides 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.

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

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

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

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

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

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

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

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

[0014] The present disclosure also provides a polypeptide comprising (i) the 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 a polypeptide according to the present disclosure.

[0016] The present disclosure also provides an MHC:peptide complex comprising 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 encoding a polypeptide according to the present disclosure.

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

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

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

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

[0022] The present disclosure also provides a method of producing cells comprising MHC molecules labeled with an identifier moiety, comprising: (1) introducing into a cell a nucleic acid or nucleic acids according to the present disclosure; and (2) contacting the cell with a labeling moiety that includes 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; The present invention provides a method comprising:

[0023] The present disclosure also provides a method for producing a cell containing an MHC:peptide complex comprising an MHC molecule labeled with a single-stranded DNA (ssDNA) moiety, the method comprising: (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) a HaloTag; (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 (3) contacting the cell with a HaloTag ligand comprising a ssDNA portion and a chloroalkane portion; The present invention provides a method comprising:

[0024] The present disclosure also provides a method for producing a cell containing an MHC:peptide complex comprising an MHC molecule labeled with a single-stranded DNA (ssDNA) moiety, the method comprising: (1) introducing into a cell a nucleic acid or nucleic acids encoding a polypeptide comprising (i) an amino acid sequence of β2 microglobulin, (ii) a sortase substrate motif, and (iii) a HaloTag; (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 (3) contacting the cell with a HaloTag ligand comprising a ssDNA portion and a chloroalkane portion; The present invention provides a method comprising:

[0025] The present disclosure also provides cells produced by the method of producing cells comprising an MHC:peptide complex according to the present disclosure.

[0026] The present disclosure also provides compositions comprising cells and T cells according to the present disclosure.

[0027] The present disclosure also provides a method for identifying a T cell receptor (TCR) that binds to an MHC:peptide complex, comprising: (1) contacting a cell comprising the MHC:peptide complex of 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 containing a TCR that binds to the MHC:peptide complex; and (3) subsequently analyzing the T cells to identify TCRs that bind to the MHC:peptide complex; The present invention provides a method comprising:

[0028] The present disclosure also provides a method for identifying a T cell receptor (TCR) that binds to an MHC:peptide complex, comprising: (1) contacting (a) cells containing 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 containing a TCR that binds to the MHC:peptide complex; and (3) subsequently analyzing the cells obtained after step (2) to identify TCRs that bind to the MHC:peptide complex; Including, The method includes the step of: providing an MHC:peptide complex comprising an MHC molecule, the MHC molecule comprising a polypeptide comprising (i) an amino acid sequence of an MHC polypeptide and (ii) an identifier portion, the identifier portion being covalently attached to the polypeptide via a bond formed by a self-labeling protein tag.

[0029] The present disclosure also provides a method for identifying a T cell receptor (TCR) that binds to an MHC:peptide complex, comprising: (1) contacting (a) cells comprising an MHC:peptide complex with (b) a population of cells comprising T cells comprising a polypeptide on the cell surface comprising a sortase acceptor motif in the presence of sortase; (2) incubating the cells obtained after step (1) under conditions suitable for interaction between the cells of (a) and the cell population of (b); and (3) subsequently analyzing the cells obtained after step (2) to identify T cells containing a TCR that binds to the MHC:peptide complex; Including, The method provides a method in which the MHC:peptide complex comprises an MHC molecule, the MHC molecule comprising a polypeptide comprising (i) an amino acid sequence of an MHC polypeptide, (ii) a sortase substrate motif, and (iii) an identifier moiety, the identifier moiety being covalently attached to the polypeptide via a bond formed by a self-labeling protein tag.

[0030] In some embodiments, the sortase is provided on the cell surface of a T cell comprising a polypeptide comprising a sortase acceptor motif.

[0031] In some embodiments according to various different aspects described herein for methods of identifying TCRs that bind to MHC:peptide complexes, the MHC polypeptide is β2 microglobulin.

[0032] In some embodiments according to various different aspects described herein for methods of identifying TCRs that bind to an MHC:peptide complex, the identifier portion is a nucleic acid portion. In some embodiments, the identifier portion comprises or consists of single-stranded DNA (ssDNA).

[0033] In some embodiments according to various different aspects described herein for methods of identifying TCRs that bind to MHC:peptide complexes, the self-labeling protein tag is or comprises a HaloTag.

[0034] In some embodiments according to various different aspects described herein for methods of identifying TCRs that bind to an MHC:peptide complex, the identifier moiety is covalently attached to the polypeptide via an ester bond formed by the dehalogenase activity of the HaloTag on a HaloTag ligand comprising the identifier moiety and a chloroalkane moiety. DETAILED DESCRIPTION OF THE INVENTION

[0035] explanation Polypeptides 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. Polypeptides generally include peptides containing 50 or fewer amino acids.

[0036] In some aspects and embodiments, a polypeptide of the present disclosure comprises the amino acid sequence of a major histocompatibility complex (MHC) polypeptide and a moiety that facilitates labeling of the polypeptide with an identifier moiety.

[0037] In some aspects and embodiments, a polypeptide of the present disclosure comprises an amino acid sequence of a major histocompatibility complex (MHC) polypeptide and an identifier portion, wherein the identifier portion is covalently attached to the polypeptide via a linkage formed by a self-labeling protein tag.

[0038] In some aspects and embodiments, a polypeptide of the present disclosure comprises a sortase substrate motif and a moiety that facilitates labeling of the polypeptide with an identifier moiety.

[0039] In some aspects and embodiments, a polypeptide of the present disclosure comprises a sortase substrate motif and an identifier moiety, wherein the identifier moiety is covalently attached to the polypeptide via a linkage formed by the self-labeling protein tag.

[0040] Proteins localized in the cell membrane In some aspects and embodiments, a polypeptide of the present disclosure comprises the amino acid sequence of a protein that is localized to a cell membrane. Such proteins are sometimes referred to as cell surface proteins.

[0041] A cell membrane-localized protein is a protein that, when expressed by a cell (e.g., a eukaryotic / mammalian cell), is detectable in or at the cell membrane. A cell membrane-localized protein can be detectable in or at the cell membrane, for example, by immunohistochemistry / cytochemistry or flow cytometry analysis, for example, using an antibody against the protein.

[0042] It will be understood that a polypeptide according to the present disclosure that comprises the amino acid sequence of a protein that localizes to the cell membrane will similarly localize to the cell membrane of a cell that contains / expresses the polypeptide.

[0043] In some embodiments, the protein localized in the cell membrane is a protein expressed by immune cells. In some embodiments, the immune cells can be cells of hematopoietic origin, such as neutrophils, eosinophils, basophils, dendritic cells, lymphocytes, or monocytes. The lymphocytes can be, for example, T cells, B cells, natural killer (NK) cells, NKT cells, or innate lymphoid cells (ILCs), or their precursors (e.g., thymocytes or pre-B cells).

[0044] In some embodiments, the immune cell is an antigen-presenting cell (APC). An APC is a cell that expresses an MHC molecule (e.g., an MHC class I and / or an MHC class II molecule) and can present an MHC:peptide complex.

[0045] The APCs according to the present disclosure may be professional APCs. Professional APCs are specialized for presenting antigens to T cells, are efficient in processing and presenting MHC-peptide complexes on the cell surface, and express high levels of costimulatory molecules. Professional APCs include dendritic cells (DCs), macrophages, and B cells. Non-professional APCs are other cells that can present MHC-peptide complexes to T cells, particularly MHC class I-peptide complexes to CD8+ T cells. Thus, in some embodiments, the protein localized on the cell membrane is a protein expressed by an APC (e.g., DC, macrophage, or B cell).

[0046] In some embodiments, the plasma membrane-localized protein is an interaction partner (e.g., a ligand) of a plasma membrane-localized protein of a T cell. A T cell 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 HIn some embodiments, the T cell is a cytotoxic T cell (e.g., a cytotoxic T lymphocyte (CTL)). In some embodiments, the protein localized to the cell membrane is an interaction partner (e.g., a ligand) of a protein selected from a CD3-TCR complex polypeptide (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.

[0047] In some embodiments, the protein that is localized to the cell membrane is selected from an MHC polypeptide (e.g., an MHC polypeptide as described herein below), PD-L1, PD-L2, CD80, CD86, HVEM, B7-H3, B7-H4, OX40L, 4-1BBL, ICOSL, CD40, B7RP1, CD70, and GAL9. In a preferred embodiment, the protein that is localized to the cell membrane is an MHC polypeptide.

[0048] Major histocompatibility complex (MHC) polypeptides In some aspects and embodiments, a polypeptide of the present disclosure comprises the amino acid sequence of a major histocompatibility complex (MHC) polypeptide.

[0049] As used herein, "MHC polypeptide" refers to a constituent polypeptide of an MHC molecule. An MHC molecule also refers to a polypeptide complex formed by non-covalent interactions between MHC polypeptides and capable of binding to and presenting peptides. Thus, an MHC polypeptide is a polypeptide that can interact with another MHC polypeptide to form an MHC molecule.

[0050] Polypeptide complexes according to the present disclosure can be characterized by non-covalent protein:protein interactions between the constituent polypeptides / peptides. In some embodiments, the association involves electrostatic interactions (e.g., ionic bonds, hydrogen bonds) and / or van der Waals forces.

[0051] MHC molecules are broadly divided into two classes: class I and class II. MHC class I molecules are noncovalent 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, designated α1, α2, and α3. The α1 and α2 domains together form a groove in which peptides presented by MHC class I molecules bind, forming an MHC class I:peptide complex. MHC class II molecules are noncovalent 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, designated α1 and α2, and the β chain contains two domains, designated β1 and β2. The α1 and β1 domains together form a groove in which peptides presented by MHC class II molecules bind, forming an MHC class II:peptide complex.

[0052] In humans, MHC class I and II polypeptides are encoded by polymorphic human leukocyte antigen (HLA) genes, which encode MHC complexes capable of binding and presenting different peptides. MHC class I α-chain polypeptides are encoded by HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G. MHC class II α-chain polypeptides are encoded by HLA-DPA1, HLA-DQA1, HLA-DQA2, and HLA-DRA. 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 MHC class I molecules is invariant and encoded by B2M.

[0053] In some embodiments, the MHC polypeptide can be selected from β2-microglobulin, an MHC class I α chain polypeptide, an MHC class II α chain polypeptide, or an 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.

[0054] In a preferred embodiment, the MHC polypeptide is an MHC class I molecule polypeptide. In a more preferred embodiment, the MHC polypeptide is β2 microglobulin. The use of β2 microglobulin in the polypeptide of the present disclosure is advantageous because it provides universal labeling of MHC class I molecules, i.e., it provides labeling of MHC class I molecules, including various MHC class I α chain polypeptides.

[0055] Reference herein to a given MHC polypeptide also encompasses isoforms, fragments, variants, or homologs of the associated polypeptide from any species. By way of example, a reference to β2 microglobulin includes human β2 microglobulin having the amino acid sequence set forth in SEQ ID NO: 1 or 3, and also includes isoforms, fragments, variants, or homologs of human β2 microglobulin.

[0056] As used herein, an isoform, fragment, variant, or homolog of a given reference polypeptide can be characterized as having at least 70% sequence identity with the amino acid sequence of the reference polypeptide, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity. 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 compared to the amino acid sequence of the reference protein, but retains a significant degree of sequence identity (e.g., at least 60%) with the amino acid sequence of the reference protein. An "isoform" generally refers to a variant of a reference protein that is expressed by the same species as the reference protein. A "homolog" generally refers to a variant of a reference protein that is produced by a different species compared to the species of the reference protein. Homologs include orthologs.

[0057] An isoform, fragment, variant or homologue of a given reference protein may optionally be characterized as 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 the immature or mature (i.e., after processing to remove the signal peptide) form of a particular isoform of the related polypeptide from a given species, e.g., human.

[0058] 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 to SEQ ID NO: 3 or 1.

[0059] Moieties that facilitate labeling of polypeptides with identifier moieties In aspects and embodiments of the present disclosure, the polypeptide further comprises a moiety that facilitates labeling of the polypeptide with an identifier moiety (eg, an identifier moiety as described below).

[0060] In some embodiments, the moiety that facilitates labeling of the polypeptide with the identifier moiety is or comprises an amino acid sequence that forms the peptide / polypeptide moiety.

[0061] In some embodiments, the moiety that facilitates labeling of the polypeptide with the identifier moiety is or includes a self-labeling protein tag, such as those described in Liss et al., Scientific Reports (2016) 5:17740 and Wilhelm et al., Biochemistry (2021) 60(33):2560-2575, both of which are incorporated herein by reference in their entireties.

[0062] Self-labeling protein tags contain or consist of a moiety with enzymatic activity that catalyzes the covalent attachment of a moiety of interest. For example, HaloTag is a haloalkane dehalogenase that irreversibly reacts with primary alkyl halides (e.g., haloalkanes, e.g., chloroalkanes). Nucleophilic attack causes the substitution of the halogen by an amino acid residue, resulting in the formation of a covalent alkyl-enzyme conjugate. Specifically, when the terminal halogen is substituted, the COO of Asp106 of HaloTag is substituted. - An ester bond is formed between the group and the terminal carbon of the alkane.

[0063] In some embodiments, the self-labeling protein tag is selected from a HaloTag, a SNAP tag, a CLIP tag, an ACP tag, or an MCP tag. In a preferred embodiment, the self-labeling protein tag is a HaloTag.

[0064] In some embodiments, a moiety that facilitates 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 to SEQ ID NO: 4 or 5, preferably one of ≧80%, ≧85%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99% or 100% amino acid sequence identity.

[0065] It will be understood that in some embodiments, labeling a polypeptide according to the present disclosure with an identifier moiety modifies that moiety to facilitate labeling of the polypeptide with the identifier moiety. In some embodiments, after labeling of the polypeptide with the identifier moiety, the moiety that facilitated labeling of the polypeptide via the moiety that facilitates labeling of the polypeptide with the identifier moiety is no longer suitable for facilitating (further) labeling of the polypeptide. Illustratively, in the example of a polypeptide comprising a HaloTag, after labeling the polypeptide using a labeling moiety comprising a HaloTag ligand comprising a chloroalkane moiety and an identifier moiety, the HaloTag is modified to modify the COO of Asp106. -Since the group is no longer available, it cannot facilitate (further) labelling of the polypeptide.

[0066] In some embodiments, a moiety that facilitates labeling of a polypeptide with an identifier moiety is N-terminal to the amino acid sequence of a major histocompatibility complex (MHC) polypeptide in the amino acid sequence of a polypeptide of the present disclosure, i.e., in some embodiments, the recombinant polypeptide comprises the following structure: N-terminus-[...]-[moiety that facilitates labeling of a polypeptide with an identifier moiety]-[amino acid sequence of an MHC polypeptide]-[...]-C-terminus.

[0067] When used in the representation of polypeptide structures herein, "[...]" indicates the optional presence of an additional amino acid sequence / protein domain. For example, in the structure in the last sentence of the previous paragraph, an additional sequence of amino acids / protein domains may optionally be present downstream of the amino acid sequence of the MHC polypeptide and before the C-terminus of the polypeptide. Furthermore, when used in the representation of polypeptide structures herein, "-" indicates an optional linker sequence. For example, in the structure in the last sentence of the previous paragraph, a linker sequence may optionally be provided between the moiety that facilitates labeling of the polypeptide with an identifier moiety and the amino acid sequence of the MHC polypeptide.

[0068] Sign part Aspects and embodiments of the present disclosure use labeling moieties, for example, to label polypeptides with identifier moieties.

[0069] It will be understood that the labeling moiety is selected for compatibility with a moiety that facilitates labeling of the polypeptide with an identifier moiety according to the present disclosure. A labeling moiety according to the present disclosure generally includes an identifier moiety according to the present disclosure and a moiety for covalently attaching the identifier moiety to the polypeptide via a moiety that facilitates labeling of the polypeptide with the identifier moiety.

[0070] In some embodiments, the labeling moiety according to the present disclosure is or includes a HaloTag ligand. The HaloTag ligand includes a moiety that can act as a substrate for HaloTag haloalkane dehalogenase to form a covalent alkyl-enzyme conjugate. For example, the HaloTag ligand can include a primary alkyl halide moiety, such as a haloalkane moiety (e.g., a chloroalkane moiety).

[0071] In some embodiments, a labeling moiety according to the present disclosure comprises an identifier moiety according to the present disclosure and a chloroalkane moiety.

[0072] an identifier moiety and a moiety that facilitates labeling of the polypeptide with the identifier moiety In aspects and embodiments of the present disclosure, the polypeptide comprises an identifier portion.

[0073] As used herein, an "identifier moiety" refers to a moiety that can serve as a moiety for identifying a polypeptide that includes the identifier moiety. The identifier moiety is preferably polymorphic, such that multiple polypeptides according to the present disclosure are labeled with non-identical identifier moieties. An "identifier moiety" according to the present disclosure can be any detectable moiety that can serve to distinguish a polypeptide labeled with an identifier moiety from a polypeptide that is not labeled with an identifier moiety (e.g., a polypeptide that is not labeled with an identifier moiety, or a polypeptide that is labeled with non-identical identifier moieties). An identifier moiety may also be referred to as a "barcode," and labeling polypeptides with non-identical identifier moieties may be referred to as "barcoding."

[0074] Identifier moieties contemplated in connection with the present disclosure include, for example, nucleic acid, fluorescent, phosphorescent, luminescent, immunodetectable (e.g., epitope tags), radioactive, chemical, and enzymatic labels. In preferred embodiments, the identifier moiety comprises or consists of a nucleic acid moiety. That is, in preferred embodiments, the identifier moiety is or comprises a nucleic acid identifier moiety.

[0075] Nucleic acids are particularly well suited for use as identifier moieties because they are very highly polymorphic (thus making it possible to produce a great variety of unique identifier moieties) and technologies for detecting them with high sensitivity and specificity (e.g., next-generation sequencing technologies) are widely available.

[0076] In some embodiments, a nucleic acid moiety according to the present disclosure may comprise or consist of DNA or RNA. In some embodiments, a nucleic acid moiety comprises or consists of a polynucleotide. A "polynucleotide" refers to a polymeric chain of multiple nucleotide monomers linked by bonds between the monomers, typically phosphodiester bonds (e.g., in the case of polynucleotides formed by naturally occurring nucleotide monomers). Polynucleotides generally include oligonucleotides containing 50 or fewer nucleotides. A polynucleotide may be single-stranded or double-stranded (i.e., may contain a duplex formed by hydrogen bonding between complementary nucleotides).

[0077] 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 comprises one of 5 to 200 nucleotides, e.g., 10 to 100, 20 to 80, or 30 to 75 nucleotides.

[0078] It will be understood that the nucleic acid portion of the present disclosure is used as an identifier portion. The nucleic acid portion can include a structure that allows for the detection of the polypeptide labeled with the nucleic acid portion by analyzing the nucleic acid portion. For example, if the nucleic acid portion comprises or consists of a polynucleotide, the nucleic acid portion can provide for detection by analyzing the nucleotide sequence of the polynucleotide.

[0079] Identifier moieties according to the present disclosure are preferably used to encode (i.e., as identifiers) a peptide of interest. That is, an identifier moiety is used in combination with a peptide of interest such that a given identifier moiety corresponds to a particular peptide. Identifier moieties are useful for easily identifying peptide:MHC complexes presenting the peptide of interest, for example, in T cells after internalization (e.g., by trogocytosis). Detection of a given identifier moiety in a T cell indicates that the T cell has an internalized peptide:MHC complex presenting the peptide it encodes.

[0080] The identifier moieties are also useful for readily identifying peptide:MHC complexes that have interacted with T cells as a result of sortase-mediated transfer through detection of the identifier moiety on the T cell. According to such embodiments, detection of a given identifier moiety in a T cell indicates that the T cell has interacted with a peptide:MHC complex that presents the peptide it encodes.

[0081] Sortase-mediated labeling Aspects and embodiments of the present disclosure relate to enzyme-catalyzed site-specific labeling of polypeptides with identifier moieties, articles used in such labeling reactions, and products thereof.

[0082] In particular, the present disclosure contemplates sortase-mediated transfer of identifier moieties between polypeptides. Site-specific labeling of polypeptides by 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 incorporated herein by reference in their entireties.

[0083] As used herein, "sortase" refers to an enzyme that recognizes and cleaves polypeptides containing a sortase substrate motif that fits 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 are useful for transferring amino acids 1-4 and N-terminal thereto of a sortase substrate motif into polypeptides containing an appropriate sortase acceptor motif via transpeptidation. Sortases and their functions are reviewed, for example, in Mazmanian et al. Science (1999) 285(5428):760-763 and Paterson and Mitchell, Trends Microbiol. (2004) 12(2):89-95, both of which are incorporated herein by reference in their entireties.

[0084] In aspects and embodiments according to the present disclosure, a sortase is used to catalyze the transfer of a region from (a) a polypeptide comprising a sortase substrate motif (e.g., a region comprising, e.g., covalently attached to, an identifier moiety) to (b) a polypeptide comprising a sortase acceptor motif. Sortase-mediated transfer indicates a physical interaction between polypeptides (a) and (b), as efficient transfer requires their physical proximity. Sortase-catalyzed labeling of cells in the developing immune synapse is described, for example, in Pasqual et al., Nature (2018) 553:496-500, incorporated herein by reference in its entirety.

[0085] 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).

[0086] Sortases include sortase A, which is identified by Enzyme Commission number 3.4.22.70. Sortase A includes sortase A from Staphylococcus aureus (also referred to as "Sa-SrtA"), which has the amino acid sequence set forth in SEQ ID NO: 19. The region of S. aureus sortase A that confers sortase activity is set forth in SEQ ID NO: 20. Sortase A also includes sortase A from Streptococcus pyogenes (also referred to as "Sp-SrtA"), which has the amino acid sequence set forth in SEQ ID NO: 23. The region of S. pyogenes sortase A that confers sortase activity is set forth in SEQ ID NO: 24.

[0087] The present disclosure also contemplates sortase variants, such as those described 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, all of which are incorporated herein by reference in their entireties.

[0088] In some embodiments according to various aspects of the disclosure, the sortase is selected from sortase A (EC 3.4.22.70), S. aureus sortase A or a variant thereof, and S. pyogenes sortase A or a variant thereof.

[0089] A variant of sortase A from Staphylococcus aureus (i.e., a variant of S. aureus sortase A) refers to a polypeptide having at least 70% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 19, for example, ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98% or ≥99% amino acid sequence identity. Similarly, a variant of sortase A from Streptococcus pyogenes (i.e., a variant of S. pyogenes sortase A) refers to a polypeptide having at least 70% amino acid sequence identity, such as one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98% or >99% amino acid sequence identity, to the amino acid sequence of SEQ ID NO: 23. It will be understood that variants of S. aureus sortase A and variants of S. pyogenes sortase A preferably retain sortase activity.

[0090] It will be understood that a sortase variant comprises one or more (e.g., one of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) modifications compared to the amino acid sequence of a reference sortase (e.g., S. aureus sortase A, S. pyogenes sortase A).

[0091] "Modification" refers to a difference relative to a reference amino acid sequence. The reference amino acid sequence may be the amino acid sequence encoded by the most common nucleotide sequence of a gene encoding a related protein. In embodiments herein (and more generally in the art), a "modification" may also be referred to as a "substitution" or a "mutation." A modification typically involves the replacement of an amino acid residue with a non-identical "replacement" amino acid residue. The replacement amino acid residue in the modification according to the present disclosure may be a naturally occurring amino acid residue (i.e., encoded by the genetic code) that is not identical to the amino acid residue at the relevant position in the amino acid sequence prior to modification, 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 replacement amino acid residue in the modification may be a non-naturally occurring amino acid residue, i.e., an amino acid residue other than those listed in the preceding sentence. Examples of unnatural 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.

[0092] In some embodiments, a sortase variant according to the disclosure comprises an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 19, 20, 23, or 24, and comprises one or more (e.g., one of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) modifications compared to SEQ ID NO: 19, 20, 23, or 24.

[0093] 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 compared 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 compared to SEQ ID NO: 19 or SEQ ID NO: 20. The modifications in the preceding sentence are numbered according to SEQ ID NO: 19, and it will be understood that where the relevant modification is provided in SEQ ID NO: 20 instead, the corresponding position in SEQ ID NO: 20 is modified instead. By way of example, when the modification "K196T" is provided in SEQ ID NO:20, the "K" at position 137 of SEQ ID NO:20 is replaced with a "T".

[0094] In some embodiments according to various aspects of the disclosure, the sortase comprises or consists of the amino acid sequence of SEQ ID NO: 19, 20, 21, 22, 23, or 24, or an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 19, 20, 21, 22, 23, or 24, such as one of ≧75%, ≧80%, ≧85%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, or ≧99% sequence identity. 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., ≧75%, ≧80%, ≧85%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, or ≧99% sequence identity, to 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., ≧75%, ≧80%, ≧85%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, or ≧99% sequence identity, to 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., ≧75%, ≧80%, ≧85%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, or ≧99% sequence identity, to 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., ≧75%, ≧80%, ≧85%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, or ≧99% sequence identity, to 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., ≧75%, ≧80%, ≧85%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, or ≧99% sequence identity, to 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., ≧75%, ≧80%, ≧85%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, or ≧99% sequence identity, to SEQ ID NO: 24.

[0095] In some embodiments according to various aspects of the disclosure, the sortase substrate motif comprises or consists of a substrate motif for a sortase selected from sortase A (EC 3.4.22.70), S. aureus sortase A or a variant thereof, and S. pyogenes sortase A or a variant thereof.

[0096] Sortase substrate motifs include those that fit the consensus Leu-Pro-Xaa-Thr-Gly (SEQ ID NO: 26), which serves as a substrate for S. aureus sortase A and S. pyogenes sortase A, and those that fit 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 S. pyogenes sortase A (see, e.g., Johnson et al., J Biol Chem. (2022) 298(10):102446).

[0097] As used herein, when a given sortase substrate motif is said to "serve as a substrate" for a given reference sortase, the given reference sortase is capable of catalyzing cleavage of the sortase substrate motif (i.e., at the peptide bond between positions 4 and 5 of SEQ ID NO:25).

[0098] Thus, in some embodiments, the sortase substrate motif comprises or consists of an amino acid sequence that matches the consensus sequence of one of SEQ ID NOs: 25, 26, 27, or 28. In some embodiments, the sortase substrate motif comprises or consists of an amino acid sequence that matches the consensus sequence of SEQ ID NO: 25. In some embodiments, the sortase substrate motif comprises or consists of an amino acid sequence that matches the consensus sequence of SEQ ID NO: 26. In some embodiments, the sortase substrate motif comprises or consists of an amino acid sequence that matches the consensus sequence of SEQ ID NO: 27. In some embodiments, the sortase substrate motif comprises or consists of an amino acid sequence that matches the consensus sequence of SEQ ID NO: 28.

[0099] In some embodiments, a sortase substrate motif may be provided at or proximal to the C-terminus of a polypeptide comprising a sortase substrate motif, i.e., in some embodiments, position 1 of a sequence matching the consensus sequence of SEQ ID NO:25 is provided within one of 50 amino acids, such as 40, 30, 25, 20, 15, 10 or 5 amino acids, of the C-terminus of the polypeptide.

[0100] Sortases catalyze the transfer of polypeptide / peptide moieties from proteins that contain a sortase substrate motif for the sortase to proteins that contain a sortase acceptor motif for the sortase. A sortase acceptor motif is typically an oligopeptide sequence of small, non-polar, hydrophobic amino acids (e.g., glycine or alanine) provided at the N-terminus of a polypeptide / peptide that comprises the oligopeptide. For example, the best-characterized sortase acceptor motif that serves as the acceptor motif for S. aureus sortase A is an N-terminal oligoglycine.

[0101] As used herein, when a given sortase acceptor motif is said to "serve as an acceptor motif" for a given reference sortase, the given sortase acceptor motif can serve as a nucleophile of an intermediate formed by cleavage of a polypeptide / peptide comprising the appropriate sortase substrate motif by the sortase, i.e., in the context of a transpeptidation reaction, of the given reference sortase.

[0102] In some embodiments, a sortase acceptor motif may comprise or consist of at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (e.g., 2-10, e.g., 4) amino acids provided at the N-terminus of a polypeptide / peptide comprising such a motif, wherein the amino acids are selected from glycine and alanine.

[0103] In some embodiments, the sortase acceptor motif is G n G provided at the N-terminus of a polypeptide / peptide containing n (i.e., "N-terminus-G n"), where "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 to 10. In some embodiments, "n" is 4. The sortase acceptor motif "N-terminus-G4" serves as the acceptor motif for S. aureus sortase A and S. pyogenes sortase A.

[0104] In some embodiments, the sortase acceptor motif is an A provided at the N-terminus. n (i.e., "N-terminus-A n "), where "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 to 10. In some embodiments, "n" is 4.

[0105] It will be understood that where an aspect or embodiment of the disclosure concerns (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 an aspect or embodiment concerns (i) a polypeptide comprising a sortase acceptor motif and (ii) a sortase, the sortase acceptor motif and the sortase are preferably selected to be compatible with each other. Similarly, where an aspect or embodiment concerns (i) a polypeptide comprising a sortase substrate motif, (ii) a polypeptide comprising a sortase acceptor motif, and (iii) a sortase, the sortase substrate motif, the sortase acceptor motif, and the sortase are preferably selected to be compatible with each other.

[0106] 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 positions 4 and 5 of SEQ ID NO: 25). By way of example, a sortase consisting of the amino acid sequence of SEQ ID NO: 22 will catalyze the cleavage of a polypeptide / peptide that contains a sortase substrate motif that fits the consensus of SEQ ID NO: 26; therefore, this combination of sortase substrate motif and sortase is compatible.

[0107] A given sortase acceptor motif and a given sortase are compatible if the sortase acceptor motif serves as a nucleophile of an intermediate formed by cleavage of a polypeptide / peptide containing a suitable sortase substrate motif for the sortase by the sortase. By way of example, the sortase acceptor motif "N-terminus-G4" serves as a nucleophile of an intermediate formed by cleavage of a polypeptide / peptide containing a suitable sortase substrate motif for a sortase consisting of the amino acid sequence of SEQ ID NO: 22, and therefore this combination of sortase acceptor motif and sortase is compatible.

[0108] A given sortase substrate motif, a given sortase acceptor motif, and a given sortase are compatible if the sortase catalyzes cleavage of the sortase substrate motif, and the sortase acceptor motif serves as a nucleophile of an intermediate formed by cleavage of the sortase substrate motif by the sortase. By way of example, the sortase acceptor motif "N-terminus-G4" serves as a nucleophile of an intermediate formed by cleavage of a polypeptide / peptide containing a sortase substrate motif that fits the consensus of SEQ ID NO:26 by a sortase consisting of the amino acid sequence of SEQ ID NO:22, and thus this combination of sortase acceptor motif, sortase substrate motif, and sortase is compatible.

[0109] In aspects and embodiments in which a polypeptide comprises a sortase substrate motif, it will be understood that the sortase substrate motif is positioned within the amino acid sequence of the polypeptide so as to allow for sortase-mediated transfer of the identifier moiety when the polypeptide comprises such a moiety (e.g., following covalent association of the identifier moiety with the polypeptide via a linkage formed by a self-labeling protein tag). That is, in embodiments in which a polypeptide comprises a sortase substrate motif and a moiety that facilitates labeling of the polypeptide with an identifier moiety (e.g., a self-labeling protein tag, e.g., HaloTag), the sortase substrate motif is provided in the amino acid sequence of the polypeptide downstream (i.e., C-terminal) of the moiety that facilitates labeling of the polypeptide with the identifier moiety. Similarly, in embodiments in which a polypeptide comprises a sortase substrate motif and an identifier moiety covalently attached to the polypeptide via a linkage formed by a self-labeling protein tag (e.g., HaloTag), the sortase substrate motif is provided downstream (i.e., C-terminal) of the self-labeling protein tag. That is, in some embodiments, the polypeptide comprises the following structure: N-terminus-[...]-[moiety that facilitates labeling of the polypeptide with an identifier moiety]-[sortase substrate motif]-[...]-C-terminus.

[0110] Similarly, in aspects and embodiments in which the polypeptide comprises a sortase acceptor motif, it will be understood that the sortase acceptor motif is positioned within the amino acid sequence of the polypeptide so as to allow sortase-mediated transfer of the identifier moiety into the polypeptide, i.e., when the polypeptide comprises a sortase acceptor motif, the sortase acceptor motif is provided at the N-terminus of the polypeptide.

[0111] Aspects and embodiments according to the present disclosure provide cells comprising a polypeptide, wherein the polypeptide comprises a sortase acceptor motif. In preferred embodiments, the cell is a T cell. A T cell 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 In some embodiments, the T cell is a cytotoxic T cell (e.g., a cytotoxic T lymphocyte (CTL)).

[0112] In some embodiments, the T cell comprises a polypeptide comprising a sortase acceptor motif on its cell surface, i.e., in some embodiments, a polypeptide comprising a sortase acceptor motif is present in or on the cell surface of the T cell.

[0113] The T cell may comprise a polypeptide comprising a sortase acceptor motif as a result of modifying the T cell to comprise the polypeptide, hi some embodiments, the T cell may comprise a polypeptide comprising a sortase acceptor motif as a result of modifying the T cell (e.g., using a suitable genetic engineering platform) to comprise a nucleic acid encoding a polypeptide comprising a sortase acceptor motif.

[0114] However, in a preferred embodiment, T cells are modified to contain a polypeptide comprising a sortase acceptor motif by non-genetic modification of the T cells. For example, the polypeptide can be non-genetically bioconjugated to the T cells. Non-genetic bioconjugation techniques are reviewed, for example, in Roy et al. Bioconjug Chem. (2020) 31(11):2465-2475, the entire contents of which are incorporated herein by reference. Advantageously, such techniques do not require genetic engineering to produce T cells comprising a polypeptide comprising a sortase acceptor motif.

[0115] In some embodiments, the T cells are non-genetically modified T cells. In some embodiments, the T cells according to the present disclosure do not comprise exogenous nucleic acids.

[0116] Non-genetic bioconjugate strategies typically involve attaching exogenous functional groups to cell membranes without altering the genetic makeup of the cells.

[0117] Polypeptides containing a sortase acceptor motif can be attached to the surface of T cells by conjugation to functional groups metabolically introduced into the T cell. Azide, alkyne, or ketone moieties can be introduced onto cell surface-displayed glycans by metabolic glycan tagging (MGL).

[0118] In some aspects and embodiments, the present disclosure provides a T cell comprising a polypeptide comprising a sortase acceptor 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 may be referred to as being or being "immobilized" on the T cell, i.e., on or at the cell surface of the T cell.

[0119] In some embodiments, a polypeptide comprising a sortase acceptor motif is provided at the cell surface of a T cell as a result of covalent binding of the polypeptide to a molecule in or at the cell membrane of the T cell, hi some embodiments, a polypeptide comprising a sortase acceptor motif is provided at the cell surface of a T cell as a result of a non-covalent interaction between the polypeptide and a molecule in or at the cell membrane of the T cell.

[0120] For example, sialic acid residues on cell surface glycans can be modified to contain azide moieties by culturing cells in cell culture medium containing N-azidoacetylmannosamine (ManNAz). Polypeptides containing a sortase acceptor motif and functionalized with a cycloalkyne moiety (e.g., dibenzocyclooctyl (DBCO) or azadibenzocyclooctyne (ADIBO) moiety) can then be conjugated to the azide-modified cell surface glycans via strain-promoted alkyne-azide cycloaddition (SPAAC).

[0121] Thus, in some embodiments, the polypeptide comprising a sortase acceptor motif further comprises a moiety suitable for conjugating the polypeptide to an azide moiety-labeled interaction partner via SPAAC. In some embodiments, the moiety suitable for conjugating the polypeptide to an azide moiety-labeled interaction partner via SPAAC is or comprises a cycloalkyne moiety (e.g., a DBCO moiety or an azadibenzocyclooctyne (ADIBO) moiety). In some embodiments, the cycloalkyne moiety is or comprises a DBCO moiety.

[0122] In some aspects and embodiments, the present disclosure provides a T cell comprising a polypeptide comprising a sortase acceptor motif, where the polypeptide 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.

[0123] In some aspects and embodiments, the T cell further comprises a sortase, such as a sortase according to the embodiments above. Accordingly, aspects and embodiments according to the present disclosure provide a T cell comprising (i) a polypeptide comprising a sortase acceptor motif and (ii) a sortase.

[0124] The T cells may comprise a sortase as a result of modifying the T cells to contain a sortase, hi some embodiments, the T cells may comprise a sortase as a result of modifying the T cells (e.g., using a suitable genetic engineering platform) to contain a nucleic acid encoding the sortase.

[0125] However, in preferred embodiments, T cells are engineered to contain sortase by non-genetic modification of the T cells. In some embodiments, T cells are engineered to contain sortase via a bioconjugate, for example, via metabolic glycan labeling of the T cells followed by a "click" chemistry-based conjugation of the sortase. The sortase may be referred to as being or being "immobilized" on the T cell, i.e., on or at the cell surface of the T cell.

[0126] In some embodiments, the sortase is provided to the cell surface of the T cell as a result of covalent binding of the sortase or a polypeptide comprising a sortase portion to a molecule in or at the cell membrane of the T cell. In some embodiments, the sortase, or a polypeptide comprising a sortase portion, is provided to the cell surface of the T cell as a result of a non-covalent interaction between the sortase / polypeptide comprising the sortase portion and a molecule in or at the cell membrane of the T cell. Such a non-covalent interaction may be a protein:protein interaction. In some embodiments, the interaction comprises electrostatic interactions (e.g., ionic bonds, hydrogen bonds) and / or van der Waals forces. Such a non-covalent interaction may be the type of non-covalent interaction observed in antibody:antigen interactions.

[0127] In some embodiments, T cells are engineered to contain a sortase via metabolic glycan labeling of the T cells, a "click" chemistry-based conjugate of a hapten, and application of an antigen-binding molecule that binds to the hapten, where the antigen-binding molecule comprises or is linked to the sortase. In some embodiments, the hapten may be selected from DOTAM, DOTA, digoxigenin, biotin, and fluorescein. In some embodiments, the hapten is DOTAM or DOTA.

[0128] For example, sialic acid residues on cell surface glycans can be modified to contain azide moieties by culturing cells in cell culture medium containing ManNAz. A molecule containing a cycloalkyne moiety (e.g., DBCO or ADIBO moiety) conjugated to a hapten (e.g., DOTAM or DOTA) can then be used to conjugate the hapten to the azide-modified cell surface glycans via SPAAC. An antigen-binding molecule that specifically binds to the hapten and contains a sortase (i.e., as a fusion polypeptide) can then be applied to the cells, thereby noncovalently modifying the cells to contain sortase on their surface.

[0129] In some aspects and embodiments, the present disclosure provides a T cell comprising a hapten (e.g., DOTAM or DOTA) conjugated to the cell surface of the T cell via a SPAAC reaction between a cycloalkyne moiety (e.g., DBCO or ADIBO moiety) and an azide-modified cell-surface glycan. In some aspects and embodiments, the present disclosure provides a T cell comprising a hapten (e.g., DOTAM or DOTA) conjugated to the cell surface of the T cell via a SPAAC reaction between a cycloalkyne moiety (e.g., DBCO or ADIBO moiety) and an azide-modified cell-surface glycan, the T cell further comprising an antigen-binding molecule specifically bound to the hapten, wherein the antigen-binding molecule comprises a sortase.

[0130] The present disclosure also provides compounds comprising a hapten (eg, DOTAM or DOTA) and a cycloalkyne moiety (eg, a DBCO moiety or an ADIBO moiety).

[0131] In some aspects and embodiments, the present disclosure provides compounds comprising a hapten (e.g., DOTAM or DOTA) conjugated to a cycloalkyne moiety (e.g., DBCO moiety) via a linkage. In some embodiments, the linkage is or comprises a thiourea moiety. Thus, conjugation of the hapten and the cycloalkyne moiety is achieved by forming a thiourea bond, for example, by reaction of an isothiocyanate (NCS) group with a primary amine group.

[0132] Accordingly, the present disclosure provides a compound of structure (I): ALB(I) wherein A is a hapten, L is a bond, and B is a cycloalkyne moiety. In some embodiments, linkage L is or includes a thiourea moiety.

[0133] In some embodiments, the hapten is or comprises DOTAM. In some embodiments, the DOTAM comprises a C-functionalized cyclen moiety. In some embodiments, the C-functionalized cyclen moiety comprises an NCS group.

[0134] Thus, in some embodiments, the hapten comprises or consists of structure (II). TIFF2025530126000001.tif69170

[0135] In some embodiments, the hapten comprises DOTA. In some embodiments, the DOTA comprises a C-functionalized cyclen moiety. In some embodiments, the C-functionalized cyclen moiety comprises an NCS group.

[0136] Thus, in certain embodiments, the hapten comprises structure III. TIFF2025530126000002.tif70170

[0137] In some aspects and embodiments, the cycloalkyne moiety comprises an amine group. In some embodiments, the cycloalkyne moiety comprises DBCO-amine. The DBCO-amine has structure IV. TIFF2025530126000003.tif53170

[0138] In preferred embodiments, the compound comprises DOTAM conjugated to DBCO via a thiourea bond. In some embodiments, the compound has structure V: TIFF2025530126000004.tif69170

[0139] In certain embodiments, the compound comprises DOTA conjugated to DBCO via a thiourea bond. In some embodiments, the compound has structure VI. TIFF2025530126000005.tif70170

[0140] It will be understood that references herein to compounds include the salts, hydrates and solvates thereof, where appropriate.

[0141] In aspects and embodiments of the present disclosure, the sortase is provided as a fusion protein with the polypeptide of the antigen-binding molecule, in such aspects and embodiments, the antigen-binding molecule may be described as comprising a sortase moiety.

[0142] Thus, the present disclosure also provides antigen-binding molecules comprising a sortase moiety. The present disclosure also provides a nucleic acid or nucleic acids encoding such antigen-binding molecules. An expression vector or expression vectors comprising a nucleic acid or nucleic acids encoding such antigen-binding molecules is also provided. Cells comprising such antigen-binding molecules, such nucleic acids or nucleic acids, or such expression vector or expression vectors are also provided. Compositions comprising such antigen-binding molecules, such nucleic acids or nucleic acids, such expression vector or expression vectors, or such cells are also provided.

[0143] The sortase portion may comprise or consist of a sortase or variant thereof according to any embodiment described herein.

[0144] As used herein, "antigen-binding molecule" refers to a molecule that binds to a given target antigen. Antigen-binding molecules include antibodies (i.e., immunoglobulins (Ig)), as well as antigen-binding fragments and derivatives thereof. In some embodiments, an antigen-binding molecule according to the present disclosure comprises or consists of a monoclonal antibody, a monospecific antibody, a multispecific (e.g., bispecific, trispecific, etc.) antibody, a variable fragment (Fv) molecule, a single-chain Fv (scFv) molecule, a fragment antigen-binding (Fab) molecule, a single-chain Fab molecule (scFab), a crossFab molecule, a Fab' molecule, a Fab'-SH molecule, a F(ab')2 molecule, a diabody molecule, a triabody molecule, a scFv-Fc molecule, a minibody molecule, a heavy-chain-only antibody (HCAb) molecule, or a single-domain antibody (dAb, VHH) molecule.

[0145] 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, finomers, atrimers, DARPins, affibodies, affilins, armadillo repeat proteins (ArmRPs), OBodys, and adnectins (reviewed, e.g., in Reverdatto et al., Curr Top Med Chem. 2015;15(12):1082-1101, which is incorporated herein by reference in its entirety (see also, e.g., 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, for example, in 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 about 300-700 daltons) organic compounds).

[0146] In some embodiments, an antigen-binding molecule according to the present disclosure is or comprises an antigen-binding peptide / polypeptide or an antigen-binding peptide / polypeptide complex. An antigen-binding molecule may comprise two or more peptides / polypeptides that together form the antigen-binding molecule. The peptides / polypeptides may be covalently or non-covalently associated. In some embodiments, the peptide / polypeptide forms part of a larger polypeptide that comprises the peptide / polypeptide (e.g., in the case of an scFv molecule comprising a VH domain and a VL domain, or in the case of an scFab molecule comprising a VH-CH1 and a VL-CL).

[0147] In some embodiments, the antigen-binding molecule comprises an antibody heavy chain variable (VH) region and an antibody light chain variable (VL) region of an antibody capable of binding to a given target antigen. In some embodiments, the antigen-binding molecule comprises or consists of an Fv molecule formed by the VH region and VL region of an antibody capable of binding to a given target antigen. In some embodiments, the VH region and VL region may be provided in the same polypeptide and connected 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.

[0148] The antigen-binding molecules of the present disclosure generally comprise six complementarity-determining region (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. The six CDRs together define the paratope of the antigen-binding molecule, which is the part of the molecule that binds to the target antigen. The VH and VL regions each comprise a framework region (FR) on either side of each CDR, which provides a scaffold for the CDR. From the N-terminus to the C-terminus, the VH region comprises the following structure: N-terminus-[HC-FR1]-[HC-CDR1]-[HC-FR2]-[HC-CDR2]-[HC-FR3]-[HC-CDR3]-[HC-FR4]-C-terminus; the VL region comprises the following structure: N-terminus-[LC-FR1]-[LC-CDR1]-[LC-FR2]-[LC-CDR2]-[LC-FR3]-[LC-CDR3]-[LC-FR4]-C-terminus.

[0149] There are several different conventions for defining antibody CDRs and FRs, such as (i) the Kabat system 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 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) using the IMGT V-DOMAIN numbering convention 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.

[0150] In some embodiments, the antigen-binding molecule of the present disclosure binds to a hapten, such as DOTAM or DOTA. In some embodiments, the antigen-binding molecule comprises the CDRs of an antigen-binding molecule that binds to a hapten (e.g., a hapten described herein, such as DOTAM or DOTA). In some embodiments, the antigen-binding molecule comprises the FRs of an antigen-binding molecule that binds to a hapten (e.g., a hapten described herein, such as DOTAM or DOTA). In some embodiments, the antigen-binding molecule comprises the CDRs and FRs of an antigen-binding molecule that binds to a hapten (e.g., a hapten described herein, such as DOTAM or DOTA). That is, in some embodiments, the antigen-binding molecule comprises the VH region and VL region of an antigen-binding molecule that binds to a hapten (e.g., a hapten described herein, such as DOTAM or DOTA).

[0151] In some embodiments, the antigen-binding molecule comprises the CDR, FR, and / or VH and / or VL regions of a DOTAM-binding antibody, or the CDR, FR, and / or VH and / or VL regions derived from those of a DOTAM-binding antibody. In some embodiments, the antigen-binding molecule comprises the CDR, FR, and / or VH and / or VL regions of a DOTAM-binding antibody described in WO 2019 / 202399, or the CDR, FR, and / or VH and / or VL regions derived from those of a DOTAM-binding antibody described in WO 2019 / 202399.

[0152] WO 2019 / 202399 describes DOTAM-binding antibodies, such as PRIT-213, whose antigen-binding region is formed from a VH region having the amino acid sequence of SEQ ID NO: 33 (SEQ ID NO: 7 in WO 2019 / 202399) and a VL region having the amino acid sequence of SEQ ID NO: 41 (SEQ ID NO: 8 in WO 2019 / 202399). In some embodiments, the antigen-binding molecule comprises the CDRs, FRs, and / or VH and / or VL regions of PRIT-213 or CDRs, FRs, and / or VH and / or VL regions derived from those of PRIT-213.

[0153] In some embodiments, the antigen binding molecule comprises: (1) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 34 HC-CDR2 having the amino acid sequence of SEQ ID NO: 35 HC-CDR3 having the amino acid sequence of SEQ ID NO: 36; or variants thereof in which one, two or three amino acids in HC-CDR1, and / or one, two or three amino acids in HC-CDR2, and / or one, two or three amino acids in HC-CDR3 are substituted with different amino acids.

[0154] In some embodiments, the antigen binding molecule comprises: (2) A VH region incorporating the following FR: HC-FR1 having the amino acid sequence of SEQ ID NO: 37 HC-FR2 having the amino acid sequence of SEQ ID NO: 38 HC-FR3 having the amino acid sequence of SEQ ID NO: 39 HC-FR4 having the amino acid sequence of SEQ ID NO: 40; or variants thereof in which one, two or three amino acids in HC-FR1, and / or one, two or three amino acids in HC-FR2, and / or one, two or three amino acids in HC-FR3, and / or one, two or three amino acids in HC-FR4 are substituted with other amino acids.

[0155] In some embodiments, the antigen-binding portion comprises a VH region comprising a CDR according to (1) and a FR according to (2).

[0156] In some embodiments, the antigen binding molecule comprises: (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 to the amino acid sequence of SEQ ID NO: 33.

[0157] In some embodiments, the antigen-binding moiety is (4) A VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 42 LC-CDR2 having the amino acid sequence of SEQ ID NO: 43 LC-CDR3 having the amino acid sequence of SEQ ID NO: 44; or variants thereof in which one, two or three amino acids in LC-CDR1, and / or one, two or three amino acids in LC-CDR2, and / or one, two or three amino acids in LC-CDR3 are substituted with another amino acid.

[0158] In some embodiments, the antigen-binding moiety is (5) A VL region incorporating the following FR: LC-FR1 having the amino acid sequence of SEQ ID NO: 45 LC-FR2 having the amino acid sequence of SEQ ID NO: 46 LC-FR3 having the amino acid sequence of SEQ ID NO: 47 LC-FR4 having the amino acid sequence of SEQ ID NO: 48; or variants thereof in which one, two or three amino acids in LC-FR1, and / or one, two or three amino acids in LC-FR2, and / or one, two or three amino acids in LC-FR3, and / or one, two or three amino acids in LC-FR4 are substituted with other amino acids.

[0159] In some embodiments, the antigen-binding portion comprises a VL region comprising a CDR according to (4) and a FR according to (5).

[0160] In some embodiments, the antigen-binding moiety is (6) The VL region comprises 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 to the amino acid sequence of SEQ ID NO: 41.

[0161] In some embodiments, the antigen-binding portion comprises a VH region described in any one of (1) to (3) above and a VL region described in any one of (4) to (6) above.

[0162] Amino acid substitutions according to the present disclosure can be biochemically conservative. In some embodiments, if the substituted amino acid is provided in one of rows 1-5 of the table below, the replacement amino acid for the substitution is another non-identical amino acid provided in the same row. TIFF2025530126000006.tif48170

[0163] By way of example, in some embodiments where the substitution is for a Met residue, the replacement amino acid may be selected from Ala, Val, Leu, Ile, Trp, Tyr, Phe, and norleucine.

[0164] In some embodiments, the replacement amino acid in the substitution may have the same side chain polarity as the amino acid residue it replaces. In some embodiments, the replacement amino acid in the substitution may have the same side chain charge (pH 7.4) as the amino acid residue it replaces. TIFF2025530126000007.tif153170

[0165] That is, in some embodiments, a non-polar amino acid is substituted with another non-identical non-polar amino acid; in some embodiments, a polar amino acid is substituted with another non-identical polar amino acid; in some embodiments, an acidic polar amino acid is substituted with another non-identical acidic polar amino acid; in some embodiments, a basic polar amino acid is substituted with another non-identical basic polar amino acid; in some embodiments, a neutral polar amino acid is substituted with another non-identical neutral polar amino acid; in some embodiments, a positive amino acid is substituted with another non-identical positive amino acid; in some embodiments, a negative amino acid is substituted with another non-identical negative amino acid.

[0166] In some embodiments, substitutions may be functionally conservative, i.e., in some embodiments, the substitution may not affect (or not substantially affect) one or more functional properties (e.g., target antigen binding) of the antigen-binding moiety comprising the substitution, compared to the equivalent unsubstituted molecule.

[0167] In some embodiments, the sortase moiety is provided as a fusion polypeptide with the constituent polypeptides of the antigen-binding molecule. The sortase moiety may be linked to the amino acid sequence of the antigen-binding molecule via a linker sequence.

[0168] 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 comprised in a polypeptide comprising a VH region (e.g., a VH region according to embodiments described hereinabove). In some embodiments, the sortase moiety is comprised in a polypeptide comprising a VL region (e.g., a VL region according to embodiments described hereinabove). In some embodiments, the sortase moiety is comprised in a polypeptide comprising a VH region and a VL region.

[0169] Immunoglobulins and their structures are described, for example, in Schroeder and Cavacini J Allergy Clin Immunol. (2010) 125(202):S41-S52, which is incorporated herein by reference in its entirety. G-type immunoglobulins (i.e., IgG) are glycoproteins of approximately 150 kDa comprising two heavy chains and two light chains. From the N-terminus to the C-terminus, immunoglobulin heavy chains comprise a VH followed by a heavy chain constant region comprising three constant domains (CH1, CH2, and CH3, with a CH1-CH2 hinge region between CH1 and CH2). Immunoglobulin light chains comprise a VL followed by a 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 chains can be kappa (κ) or lambda (λ).

[0170] In some embodiments, the antigen-binding molecule of the present disclosure comprises 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 is derived from the heavy chain constant region sequence of IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM, such as 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 is 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 molecule of the present disclosure comprises one or more domains of an immunoglobulin light chain constant region sequence. In some embodiments, the immunoglobulin light chain constant region sequence is human immunoglobulin kappa constant (IGKC; Cκ). In some embodiments, the immunoglobulin light chain constant region sequence is human immunoglobulin lambda constant (IGLC; Cλ), such as IGLC1, IGLC2, IGLC3, IGLC6, or IGLC7.

[0171] In some embodiments, the sortase moiety is comprised in a polypeptide comprising one or more domains of an immunoglobulin constant region: an immunoglobulin heavy chain constant region domain comprises a CH1 region, a CH1-CH2-hinge region, a CH2 region, and a CH3 region; an immunoglobulin light chain constant region domain comprises a CL region.

[0172] As used herein, "CH1 region" refers to the amino acid sequence corresponding to the CH1 region of an immunoglobulin. The CH1 region is the region of an Ig formed by positions 118-215 of the Ig constant region according to the EU numbering system described in Edelman et al., Proc Natl Acad Sci USA (1969) 63(1):78-85. The "CH1-CH2 hinge region" refers to the amino acid sequence corresponding to the CH1-CH2 hinge region of an immunoglobulin. The CH1-CH2 hinge region is the region of an Ig formed by positions 216-230 of the Ig constant region according to the EU numbering system described in Edelman et al., Proc Natl Acad Sci USA (1969) 63(1):78-85. The "CH2 region" refers to the amino acid sequence corresponding to the CH2 region of an immunoglobulin. The CH2 region is the region of an Ig formed by positions 231-340 of the Ig constant region according to the EU numbering system described in Edelman et al., Proc Natl Acad Sci USA (1969) 63(1):78-85. "CH3 region" refers to the amino acid sequence corresponding to the CH3 region of an immunoglobulin. The CH3 region is the region of an Ig formed by positions 341-447 of the Ig constant region according to the EU numbering system described in Edelman et al., Proc Natl Acad Sci USA (1969) 63(1):78-85. "CH2-CH3 region" refers to the amino acid sequence corresponding to the CH2 and CH3 regions of an immunoglobulin. The CH2-CH3 region is the region of an Ig formed by positions 231-447 of the Ig constant region according to the EU numbering system described in Edelman et al., Proc Natl Acad Sci USA (1969) 63(1):78-85.

[0173] In some embodiments, the antigen-binding molecule comprises a polypeptide comprising or consisting of one of the following structures: N-terminus - [VH domain] - [...] - [sortase domain] - C-terminus N-terminus-[VH region]-[CH1 region]-[...]-[sortase part]-C-terminus N-terminus-[VH region]-[CH1 region]-[CH1-CH2 hinge region]-[CH2 region]-[CH3 region]-[sortase portion]-C-terminus N-terminus - [VL domain] - [...] - [sortase domain] - C-terminus N-terminus - [VL domain] - [CL domain] - [...] - [sortase domain] - C-terminus

[0174] According to such an embodiment, the "VH region" may be a VH region as defined in one of (1) to (3) above, and the "VL region" may be a VL region as defined in one of (4) to (6) above.

[0175] In some embodiments, an antigen-binding molecule of the present disclosure comprises a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 29, or an amino acid sequence having at least 70% amino acid sequence identity, e.g., ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity, to SEQ ID NO: 29. In some embodiments, an antigen-binding molecule comprises a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 30, or an amino acid sequence having at least 70% amino acid sequence identity, e.g., ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity, to SEQ ID NO: 30. In some embodiments, the antigen-binding molecule comprises a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 31, or an amino acid sequence having at least 70% amino acid sequence identity, e.g., ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity, to SEQ ID NO: 31. In some embodiments, the antigen-binding molecule comprises a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 32, or an amino acid sequence having at least 70% amino acid sequence identity, e.g., ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity, to SEQ ID NO: 32.

[0176] In some embodiments, antigen-binding molecules of the present disclosure include (i) a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 29 or an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 29, e.g., ≧75%, ≧80%, ≧85%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, or ≧99% sequence identity, and (ii) a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 31 or an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 31, e.g., ≧75%, ≧80%, ≧85%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, or ≧99% sequence identity.

[0177] In some embodiments, antigen-binding molecules of the present disclosure include (i) polypeptides comprising or consisting of the amino acid sequence of SEQ ID NO: 30 or an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 30, e.g., ≧75%, ≧80%, ≧85%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, or ≧99% sequence identity, and (ii) polypeptides comprising or consisting of the amino acid sequence of SEQ ID NO: 32 or an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 32, e.g., ≧75%, ≧80%, ≧85%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, or ≧99% sequence identity.

[0178] Detectable Part In some aspects and embodiments, a polypeptide according to the present disclosure further comprises a detectable moiety. In some embodiments, the detectable moiety is provided at the N-terminus and / or C-terminus of the polypeptide.

[0179] It should be understood that according to such embodiments, the detectable moiety is not the same as (or is used in conjunction with) the identifier portion of the polypeptide. The detectable moiety is useful for identifying and / or selecting T cells that have internalized / internalized MHC molecules that comprise a polypeptide of the present disclosure.

[0180] In some embodiments, the detectable moiety is a fluorescent label, a phosphorescent label, a luminescent label, an immunodetectable label (e.g., an epitope tag), a radioactive label, a chemical label, a nucleic acid label, or an enzyme label. Polypeptides according to the present disclosure may be covalently or non-covalently labeled with a detectable moiety. In a preferred embodiment, the detectable moiety is a fluorescent label.

[0181] Fluorescent labels include, for example, fluorescein, rhodamine, allophycocyanin, eosin, and NDB, green fluorescent protein (GFP), enhanced GFP (eGFP), rare earth chelates such as europium (Eu), terbium (Tb), and samarium (Sm), tetramethylrhodamine, Texas Red, 4-methylumbelliferone, 7-amino-4-methylcoumarin, Cy3, and Cy5. Radioactive labels include 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 ,scandium47 , 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 Examples of luminescent labels include radioactive isotopes such as fluoroisotopes, ...

[0182] In some embodiments, the detectable moiety is an epitope tag, such as 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 a hapten (e.g., biotin, digoxigenin, dinitrophenol).

[0183] In some embodiments, the detectable moiety is a moiety having a detectable activity, such as an enzymatic moiety, including, for example, luciferase, glucose oxidase, galactosidase (e.g., beta-galactosidase), glucorinidase, phosphatase (e.g., alkaline phosphatase), peroxidase (e.g., horseradish peroxidase), and cholinesterase.

[0184] Additional MHC complex polypeptide sequences In some embodiments, the polypeptides of the present disclosure further comprise an amino acid sequence capable of associating with the amino acid sequence of an MHC polypeptide described above to form an MHC molecule.

[0185] That is, in some embodiments, the polypeptide is (i) the amino acid sequence of a major histocompatibility complex (MHC) polypeptide; (ii) an amino acid sequence capable of associating with the amino acid sequence of (i) to form an MHC molecule; and (iii) a moiety that facilitates labeling of the polypeptide with an identifier moiety Includes.

[0186] In some embodiments, the polypeptide is (i) the amino acid sequence of a major histocompatibility complex (MHC) polypeptide; (ii) an amino acid sequence capable of associating with the amino acid sequence of (i) to form an MHC molecule; and (iii) an identifier moiety covalently attached to the polypeptide via a linkage formed by the self-labeling protein tag; Includes.

[0187] In some embodiments, the polypeptide is (i) the amino acid sequence of a major histocompatibility complex (MHC) polypeptide; (ii) an amino acid sequence capable of associating with the amino acid sequence of (i) to form an MHC molecule; (iii) a moiety that facilitates labeling of the polypeptide with an identifier moiety, and (iv) Sortase substrate motif Includes.

[0188] In some embodiments, the polypeptide is (i) the amino acid sequence of a major histocompatibility complex (MHC) polypeptide; (ii) an amino acid sequence capable of associating with the amino acid sequence of (i) to form an MHC molecule; (iii) an identifier moiety covalently attached to the polypeptide via a linkage formed by the self-labeling protein tag; and (iv) Sortase substrate motif Includes.

[0189] For example, 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.

[0190] In embodiments in which the polypeptide of the present disclosure comprises the amino acid sequence (ii) above, it will 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 according to the identity of the amino acid sequence of (i) so that the amino acid sequences can associate with each other to form a competent MHC molecule. Illustratively, in embodiments in which 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 Iα 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).

[0191] In some embodiments, the polypeptide of the present disclosure comprises: (i) an amino acid sequence which is or is derived from an MHC polypeptide selected from column A of Table 1; (ii) an amino acid sequence that is or is derived from an MHC polypeptide selected from column B of Table 1; and (iii) a moiety that facilitates labeling of the polypeptide with an identifier moiety Includes.

[0192] In some embodiments, the polypeptide of the present disclosure comprises: (i) an amino acid sequence which is or is derived from an MHC polypeptide selected from column A of Table 1; (ii) an amino acid sequence that is or is derived from an MHC polypeptide selected from column B of Table 1; and (iii) an identifier moiety covalently attached to the polypeptide via a linkage formed by the self-labeling protein tag; wherein the MHC polypeptide selected from column A of Table 1 and the MHC polypeptide selected from column B of Table 1 are selected from the same row of Table 1.

[0193] In some embodiments, the polypeptide of the present disclosure comprises: (i) an amino acid sequence which is or is derived from an MHC polypeptide selected from column A of Table 1; (ii) an amino acid sequence which is or is derived from an MHC polypeptide selected from column B of Table 1; (iii) a moiety that facilitates labeling of the polypeptide with an identifier moiety, and (iv) Sortase substrate motif Includes.

[0194] In some embodiments, the polypeptide of the present disclosure comprises: (i) an amino acid sequence which is or is derived from an MHC polypeptide selected from column A of Table 1; (ii) an amino acid sequence which is or is derived from an MHC polypeptide selected from column B of Table 1; (iii) an identifier moiety covalently attached to the polypeptide via a linkage formed by the self-labeling protein tag; and (iv) Sortase substrate motif wherein the MHC polypeptide selected from column A of Table 1 and the MHC polypeptide selected from column B of Table 1 are selected from the same row of Table 1. TIFF2025530126000008.tif144170

[0195] Linkers, Labels and Conjugates Polypeptides of the present disclosure may further comprise additional amino acids or sequences of amino acids.

[0196] A polypeptide may include one or more linker sequences between sequences of amino acids. For example, a linker sequence may be provided between different domains of a polypeptide (e.g., between the amino acid sequence of an MHC polypeptide and a moiety that facilitates labeling of the polypeptide with an identifier moiety).

[0197] Linker sequences are known to those skilled in the art, and are described, for example, in Chen et al., Adv Drug Deliv Rev (2013) 65 (10): 1357-1369, the entire contents of which are incorporated herein by reference. In some embodiments, the linker sequence can be a flexible linker sequence. A flexible linker sequence allows the relative movement of the amino acid sequences connected by the linker sequence. Flexible linkers are known to those skilled in the art, and some are identified in Chen et al., Adv Drug Deliv Rev (2013) 65 (10): 1357-1369. Flexible linker sequences often contain a high proportion of glycine and / or serine residues.

[0198] 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 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 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.

[0199] In some embodiments, the linker sequence comprises one or more copies of the amino acid sequence according to SEQ ID NO: 7 or 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.

[0200] 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.

[0201] The polypeptides of the present disclosure may comprise an amino acid sequence to facilitate expression, folding, transport, processing, purification, or detection of the antigen-binding molecule / polypeptide. For example, the polypeptides of the present disclosure may further comprise a sequence of amino acids that form a detectable moiety, for example, as described hereinabove.

[0202] A polypeptide may further contain a signal peptide (also known 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 alpha helix. Secreted proteins and proteins expressed on the cell surface often 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 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).

[0203] A signal peptide may be present at the N-terminus of a polypeptide or in a newly synthesized polypeptide. The signal peptide provides efficient transport of the polypeptide. The signal peptide is often removed by cleavage and therefore is not included in the mature polypeptide.

[0204] 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 using amino acid sequence analysis tools such as, for example, SignalP (Petersen et al., 2011 Nature Methods 8:785-786) or Signal-BLAST (Frank and Sippl, 2008 Bioinformatics 24:2172-2176).

[0205] 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 to SEQ ID NO:2.

[0206] Certain Exemplary Polypeptides, MHC Molecules, and MHC:Peptide Complexes In some embodiments, a polypeptide according to the present disclosure comprises or consists of one of the following structures: N-terminus-[signal peptide]-[a portion that facilitates tagging of the polypeptide with an identifier portion]-[amino acid sequence of the MHC polypeptide]-C-terminus N-terminus-[a moiety that facilitates labeling of the polypeptide with an identifier moiety]-[amino acid sequence of the MHC polypeptide]-C-terminus. N-terminus - [signal peptide] - [HaloTag] - [mature β2 microglobulin sequence] - C-terminus N-terminus-[HaloTag]-[mature β2 microglobulin sequence]-C-terminus N-terminus-[signal peptide]-[a portion that facilitates labeling of the polypeptide with an identifier portion]-[amino acid sequence of an MHC polypeptide]-[an amino acid sequence that can associate with the amino acid sequence of an MHC polypeptide to form an MHC molecule]-C-terminus N-terminus-[a moiety that facilitates labeling of the polypeptide with an identifier moiety]-[amino acid sequence of an MHC polypeptide]-[an amino acid sequence that can associate with the amino acid sequence of an MHC polypeptide to form an MHC molecule]-C-terminus N-terminus - [signal peptide] - [HaloTag] - [mature β2 microglobulin sequence] - [mature HLA-A02 sequence] - C-terminus N-terminus - [signal peptide] - [HaloTag] - [mature β2 microglobulin sequence] - [mature HLA class I α polypeptide sequence] - C-terminus N-terminus - [HaloTag] - [mature β2 microglobulin sequence] - [mature HLA class I α polypeptide sequence] - C-terminus N-terminus - [signal peptide] - [a portion that facilitates tagging of the polypeptide with an identifier] - [sortase substrate motif] - [amino acid sequence of a protein localized in the cell membrane] - C-terminus N-terminus - [a portion that facilitates labeling of the polypeptide with an identifier] - [sortase substrate motif] - [amino acid sequence of a protein localized in the cell membrane] - C-terminus N-terminus - [signal peptide] - [a portion that facilitates tagging of the polypeptide with an identifier portion] - [sortase substrate motif] - [amino acid sequence of the MHC polypeptide] - C-terminus N-terminus-[a moiety that facilitates labeling of the polypeptide with an identifier moiety]-[sortase substrate motif]-[amino acid sequence of the MHC polypeptide]-C-terminus N-terminus - [signal peptide] - [HaloTag] - [sortase substrate motif] - [mature β2-microglobulin sequence] - C-terminus N-terminus - [HaloTag] - [sortase substrate motif] - [mature β2-microglobulin sequence] - C-terminus N-terminus-[signal peptide]-[moiety that facilitates labeling of the polypeptide with the identifier moiety]-[sortase substrate motif]-[amino acid sequence of an MHC polypeptide]-[amino acid sequence that can associate with the amino acid sequence of an MHC polypeptide to form an MHC molecule]-C-terminus N-terminus-[a moiety that facilitates labeling of a polypeptide with an identifier moiety]-[sortase substrate motif]-[amino acid sequence of an MHC polypeptide]-[an amino acid sequence that can associate with an amino acid sequence of an MHC polypeptide to form an MHC molecule]-C-terminus N-terminus - [signal peptide] - [HaloTag] - [sortase substrate motif] - [mature β2 microglobulin sequence] - [mature HLA-A02 sequence] - C-terminus N-terminus - [signal peptide] - [HaloTag] - [sortase substrate motif] - [mature β2-microglobulin sequence] - [mature HLA class I α polypeptide sequence] - C-terminus N-terminus - [HaloTag] - [sortase substrate motif] - [mature β2-microglobulin sequence] - [mature HLA class I α polypeptide sequence] - C-terminus

[0207] The present disclosure further includes: (1) A polypeptide comprising or consisting of (e.g., from N-terminus to C-terminus): (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 to SEQ ID NO: 2; (ii) an amino acid sequence encoding a moiety that facilitates labeling of a polypeptide with an identifier moiety (e.g., a self-labeling protein tag, e.g., a 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 (iii) An amino acid sequence encoding the amino acid sequence of 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.

[0208] (2) A polypeptide comprising or consisting of (e.g., from N-terminus to C-terminus): (i) an amino acid sequence encoding a moiety that facilitates labeling of a polypeptide with an identifier moiety (e.g., a self-labeling protein tag, e.g., a 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 (ii) An amino acid sequence encoding the amino acid sequence of 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.

[0209] (3) A polypeptide comprising or consisting of (e.g., from N-terminus to C-terminus): (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 to SEQ ID NO: 2; (ii) an amino acid sequence encoding a moiety that facilitates labeling of a polypeptide with an identifier moiety (e.g., a self-labeling protein tag, e.g., a 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; (iii) 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 (iv) An amino acid sequence encoding an amino acid sequence of an HLA class Iα 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.

[0210] (4) A polypeptide comprising or consisting of (e.g., from N-terminus to C-terminus): (i) an amino acid sequence encoding a moiety that facilitates labeling of a polypeptide with an identifier moiety (e.g., a self-labeling protein tag, e.g., a 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; (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 (iii) An amino acid sequence encoding an amino acid sequence of an HLA class Iα 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.

[0211] (5) A 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 to SEQ ID NO:10.

[0212] (6) A 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 to SEQ ID NO:11.

[0213] (7) A 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 to SEQ ID NO:12.

[0214] (8) A 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 to SEQ ID NO: 13.

[0215] (9) A polypeptide comprising or consisting of (e.g., from N-terminus to C-terminus): (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 to SEQ ID NO: 2; (ii) an amino acid sequence encoding a moiety that facilitates labeling of a polypeptide with an identifier moiety (e.g., a self-labeling protein tag, e.g., a 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; (iii) an amino acid sequence that matches the consensus of SEQ ID NO: 25, and (iv) An amino acid sequence encoding the amino acid sequence of 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.

[0216] (10) A polypeptide comprising or consisting of (e.g., from N-terminus to C-terminus): (i) an amino acid sequence encoding a moiety that facilitates labeling of a polypeptide with an identifier moiety (e.g., a self-labeling protein tag, e.g., a 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; (ii) an amino acid sequence that matches the consensus of SEQ ID NO: 25; (iii) An amino acid sequence encoding the amino acid sequence of 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.

[0217] (11) A polypeptide comprising or consisting of (e.g., from N-terminus to C-terminus): (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 to SEQ ID NO: 2; (ii) an amino acid sequence that matches the consensus of SEQ ID NO: 25; (iii) an amino acid sequence encoding a moiety that facilitates labeling of a polypeptide with an identifier moiety (e.g., a self-labeling protein tag, e.g., a 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; (iv) 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 (v) An amino acid sequence encoding an amino acid sequence of an HLA class Iα 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.

[0218] (12) A polypeptide comprising or consisting of (e.g., from N-terminus to C-terminus): (i) an amino acid sequence encoding a moiety that facilitates labeling of a polypeptide with an identifier moiety (e.g., a self-labeling protein tag, e.g., a 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; (ii) an amino acid sequence that matches the consensus of SEQ ID NO: 25; (iii) 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 (iv) An amino acid sequence encoding an amino acid sequence of an HLA class Iα 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.

[0219] The present disclosure further includes: (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 Iα polypeptide. (B) An MHC molecule comprising or consisting of the polypeptide described in (4) or (8) above. (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 Iα polypeptide. (D) An MHC molecule comprising or consisting of the polypeptide described in (12) above.

[0220] The present disclosure further includes: (I) An MHC:peptide complex comprising the MHC molecule described in (A) or (B) above and a peptide presented by the MHC molecule. (II) An MHC:peptide complex comprising: (i) an MHC molecule described in (A) or (B) above, (ii) a peptide presented by the MHC molecule, and (ii) an identifier portion (e.g., a nucleic acid identifier portion, e.g., an ssDNA identifier portion). (III) An MHC:peptide complex comprising the MHC molecule described in (C) or (D) above and a peptide presented by the MHC molecule. (IV) An MHC:peptide complex comprising: (i) an MHC molecule described in (C) or (D) above, (ii) a peptide presented by the MHC molecule, and (ii) an identifier portion (e.g., a nucleic acid identifier portion, e.g., an ssDNA identifier portion).

[0221] Nucleic acids and vectors The present disclosure provides a nucleic acid or multiple nucleic acids encoding 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.

[0222] Polypeptides according to the present disclosure can be produced intracellularly by translation of RNA encoding the polypeptide. Polypeptides 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.

[0223] In some embodiments, the nucleic acid may be or may be contained in a vector or vectors. As used herein, a "vector" is a nucleic acid molecule used as a vehicle to transfer exogenous nucleic acid into a cell.

[0224] Therefore, the present disclosure also provides a vector or vectors containing a nucleic acid or nucleic acids according to the present disclosure. The vector can facilitate the delivery of a nucleic acid encoding a polypeptide according to the present disclosure to a cell. The vector can be an expression vector containing elements necessary for the expression of a polypeptide according to the present disclosure. The vector can also contain elements that facilitate the integration of the nucleic acid into the genomic DNA of a cell into which the vector is introduced.

[0225] The nucleic acids and vectors according to the present disclosure can be provided in purified or isolated form, i.e., from other nucleic acids or naturally occurring biological materials.

[0226] The vector may be a vector for expressing a nucleic acid in a cell (i.e., an expression vector). Such a vector may include a promoter sequence 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' in the nucleotide sequence of the vector to the nucleotide sequence encoding the polypeptide) and an expression enhancer. Any suitable vector, promoter, enhancer, and stop codon known in the art may be used to express a peptide or polypeptide from a vector according to the present disclosure.

[0227] The term "operably linked" can include a situation in which a nucleic acid encoding a polypeptide according to the present disclosure and a regulatory nucleic acid sequence (e.g., a promoter and / or enhancer) are covalently linked in such a manner that expression of the nucleic acid encoding the polypeptide is under the influence or control of the regulatory nucleic acid sequence (thereby forming an expression cassette). Thus, a regulatory sequence is operably linked to a selected nucleic acid sequence if the regulatory sequence is capable of effecting transcription of the nucleic acid sequence. The resulting transcript can then be translated into the desired polypeptide.

[0228] Vectors contemplated in connection 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, vaccinia viral vectors, and herpes viral vectors), transposon-based vectors, and artificial chromosomes (e.g., yeast artificial chromosomes), as described, for example, in Maus et al., Annu Rev Immunol (2014) 32:189-225 and Morgan and Boyerinas, Biomedicines (2016) 4:9, both of which are incorporated herein by reference in their entireties. In some embodiments, a vector according to the present disclosure is a lentiviral vector.

[0229] In some embodiments, the vector may be a eukaryotic vector, i.e., a vector that contains elements necessary for expression of a protein from the vector in a eukaryotic cell, hi some embodiments, the vector may be a mammalian vector that includes, for example, a cytomegalovirus (CMV) or SV40 promoter driving protein expression.

[0230] peptide The articles and methods of the present disclosure are used to identify T cell receptors (TCRs) that can bind to MHC:peptide complexes. In particular, it is contemplated that the articles and methods of the present disclosure are used to identify TCRs that can bind to MHC molecules that present a peptide of interest.

[0231] A peptide of the present disclosure can be a peptide of a polypeptide of interest. A peptide of a reference polypeptide of interest can be any fragment of the polypeptide.

[0232] The polypeptide of interest can be any polypeptide. For example, the polypeptide of interest can be a polypeptide for which it is desirable to direct an immune response (e.g., a cell-mediated immune response), or a polypeptide of an infectious agent or cell. The polypeptide of interest can be a disease-associated antigen.

[0233] A "disease-associated antigen" refers to an antigen whose presence indicates a given disease / disease state, or whose elevated levels positively correlate with a given disease / disease state. A disease-associated antigen may be an antigen whose expression is associated with the onset, progression, or severity of symptoms of a given disease. A disease-associated antigen may be associated with the cause or pathology of a disease, or may be abnormally expressed as a result of a disease. A disease-associated antigen may be an antigen of an infectious agent or pathogen, a cancer-associated antigen, or an autoimmune disease-associated antigen.

[0234] In some embodiments, the disease-associated antigen is an antigen of a pathogen, which can be a prokaryote (bacterium), a eukaryote (e.g., protozoa, helminths, fungi), a virus, or a prion.

[0235] In some embodiments, pathogen is intracellular pathogen.In some embodiments, pathogen is virus.Virus can be dsDNA virus (for example, adenovirus, herpesvirus, poxvirus), ssRNA virus (for example, parvovirus), dsRNA virus (for example, reovirus), (+)ssRNA virus (for example, picornavirus, togavirus), (-)ssRNA virus (for example, orthomyxovirus, rhabdovirus), ssRNA-RT virus (for example, retrovirus) or dsDNA-RT virus (for example, hepadnavirus). In particular, viruses of the Adenoviridae, Herpesviridae, Papillomaviridae, Polyomaviridae, Poxviridae, Hepadnaviridae, Parvoviridae, Astroviridae, Caliciviridae, Picornaviridae, Coronaviridae, Flaviviridae, Togaviridae, Hepeviridae, Retroviridae, Orthomyxoviridae, Arenaviridae, Bunyaviridae, Filoviridae, Paramyxoviridae, Rhabdoviridae, and Reoviridae families are contemplated. In some embodiments, the virus is Epstein-Barr virus, adenovirus, herpes simplex 1 virus, herpes simplex 2 virus, 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 The virus is selected from 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 virus, Hantaan virus, Ebola virus, Marburg virus, measles virus, mumps virus, parainfluenza virus, picornavirus, respiratory syncytial virus, rabies virus, hepatitis D virus, rotavirus, orbivirus, coltivirus, and bannavirus.In some embodiments, the pathogen is a bacterium. The bacterium can be gram-positive or gram-negative. In particular, bacteria of the genera Bacillus, Bartonella, Bordetella, Borrelia, Brucella, Campylobacter, Chlamydia, Chlamydophila, Clostridium, Corynebacterium, Enterococcus, Escherichia, Francisella, Haemophilus, Helicobacter, Legionella, Leptospira, Listeria, Mycobacteria, Mycoplasma, Neisseria, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Treponema, Ureaplasma, Vibrio, and Yersinia are contemplated. In some embodiments, the pathogen is a protozoan. In particular, protozoa of the genera Entamoeba, Plasmodium, Giardia, Trypanosoma, Leishmania, Besnoitia, and Toxoplasma are contemplated. In some embodiments, the pathogen is a fungus. In particular, fungi of the genera Candida, Aspergillus, Blastomyces, Coccidioides, Sporothrix, Cryptococcus, Histoplasma, Pneumocystis, Stachybotrys, Rhizopus, Mucor, Kuningamella, Apophysomyces, Trichophyton, Microsporum, Epidermophyton, Fusarium, and Lictheimia are contemplated.

[0236] In some embodiments, the polypeptide of interest is a polypeptide whose expression / activity, or its upregulated expression / activity, is positively associated 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.

[0237] 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, glycan, glycolipid, lipid, or fragment thereof. The expression of a cancer cell antigen can be associated with cancer. A cancer cell antigen can be aberrantly expressed by cancer cells (e.g., the cancer cell antigen can be expressed with abnormal localization) or can be expressed in an abnormal structure by cancer cells. A cancer cell antigen can induce an immune response. A cancer cell antigen can be a cancer-associated antigen. In some embodiments, a cancer cell antigen is an antigen whose expression is associated with the onset, progression, or severity of cancer symptoms. A cancer-associated antigen can be associated with the cause or pathology of cancer, or can be aberrantly expressed as a result of cancer. In some embodiments, a cancer cell antigen is an antigen whose expression is upregulated (e.g., at the RNA and / or protein level) by cancer cells, e.g., compared to expression levels by comparable non-cancerous cells (e.g., non-cancerous cells derived from the same tissue / cell type). In some embodiments, a cancer-associated antigen may be preferentially expressed by cancerous cells and not expressed by comparable non-cancerous cells (e.g., non-cancerous cells derived from the same tissue / cell type). In some embodiments, a cancer-associated antigen may 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, a cancer-associated antigen may be an overexpressed cellular protein, a cancer antigen produced by an oncogenic virus, a carcinoembryonic antigen, or the product of a cell surface glycolipid or glycoprotein.

[0238] It will be understood that in preferred embodiments, a peptide according to the present disclosure is a peptide that is capable of participating in an MHC:peptide complex, i.e., 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.

[0239] In some embodiments, the peptide has a length of 5 to 30, for example 10 to 25 or 8 to 11 amino acids.

[0240] MHC molecules and MHC:peptide complexes The present disclosure provides an MHC molecule comprising a polypeptide according to the present disclosure. The MHC molecule can be an MHC class I molecule or an MHC class II molecule. In a preferred embodiment, the MHC molecule is an MHC class I molecule.

[0241] An MHC molecule is formed from a polypeptide complex formed by association between a polypeptide according to the present disclosure and another MHC polypeptide. It will be understood that the constituent polypeptides of the MHC molecule are selected to form a competent MHC molecule. By way of example, in an embodiment in which the polypeptide of the present disclosure comprises the amino acid sequence of β2-microglobulin, the other MHC polypeptide of the MHC molecule comprising the polypeptide of the present disclosure is an MHC class I α chain polypeptide.

[0242] The MHC molecule is capable of presenting the peptides of the present disclosure (ie, in the form of an MHC:peptide complex) to T cells expressing a TCR that binds to the MHC:peptide complex.

[0243] Accordingly, the present disclosure also provides an MHC:peptide complex comprising an MHC molecule according to the present disclosure (ie, comprising a polypeptide of the present disclosure) and a peptide presented by the MHC molecule.

[0244] Cells containing / expressing the polypeptides and nucleic acids / vectors of the disclosure The present disclosure also provides cells comprising a polypeptide, MHC molecule, MHC:peptide complex, antigen-binding molecule, nucleic acid(s) or vector(s) according to the present disclosure.

[0245] It will be understood that where a cell is referred to herein in the singular (ie, "a / the cell"), a plurality / population of such cells is also contemplated.

[0246] The present disclosure further provides a plurality of cells comprising a polypeptide according to the present disclosure, wherein the polypeptides are not identical. In particular, the present disclosure provides a plurality of cells, wherein different cells of the plurality of cells comprise polypeptides comprising identifier portions that are not identical. That is, the present disclosure provides a plurality of cells (which may be referred to as a "library"), wherein different cells of the plurality of cells comprise different polypeptides according to the present disclosure that comprise identifier portions that are not identical.

[0247] The present disclosure also provides a plurality of cells comprising non-identical MHC molecules according to the present disclosure, particularly wherein the plurality of cells comprises MHC molecules comprising polypeptides with non-identical identifier portions. That is, the present disclosure provides a plurality of cells ("libraries") wherein a plurality of different cells comprise non-identical MHC molecules according to the present disclosure, wherein the non-identical MHC molecules comprise polypeptides of the present disclosure with non-identical identifier portions.

[0248] The present disclosure also provides a plurality of cells comprising non-identical MHC:peptide complexes according to the present disclosure, and in particular, the plurality of cells comprises MHC:peptide complexes comprising MHC molecules comprising polypeptides with non-identical identifier portions, and / or the plurality of cells comprises MHC:peptide complexes comprising non-identical peptides. That is, the present disclosure provides a plurality of cells ("libraries") wherein a plurality of different cells comprise non-identical MHC:peptide complexes according to the present disclosure, wherein the non-identical MHC:peptide complexes comprise MHC molecules comprising polypeptides with non-identical identifier portions, and / or the non-identical MHC:peptide complexes comprise non-identical peptides.

[0249] The cell may be a eukaryotic cell, for example, a mammalian cell. The mammal may 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 animal of the order Rodents), cat, dog, pig, sheep, goat, cattle (including cattle, e.g., dairy cows, or any animal of the order Bovidae), horse (including any animal of the order Equidae), donkey, and non-human primate). In a preferred embodiment, the cell is a human cell.

[0250] In some embodiments, the cell is or is derived from a cell type commonly used for the expression of polypeptides for therapeutic use in humans. 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 cell is or is derived from a CHO cell.

[0251] The present disclosure also provides methods for producing cells according to the present disclosure, and cells obtained or obtainable by such methods. Methods for producing cells containing / expressing a polypeptide / polypeptide complex of interest are well known to those skilled in the art and generally involve introducing a nucleic acid / vector encoding the polypeptide of interest into a cell.

[0252] Such methods may include nucleic acid transfer for permanent (i.e., stable) or transient expression of the transferred nucleic acid. In some embodiments, after introduction into a cell, the nucleic acid encoding the polypeptide of interest may be integrated into or form part of the genomic DNA of the cell. In some embodiments, after introduction into a cell, the nucleic acid encoding the polypeptide of interest may be maintained extrachromosomally.

[0253] Any suitable genetic engineering platform can be used, including gammaretroviral vectors, lentiviral vectors, adenoviral vectors, DNA transfection, transposon-based gene delivery and RNA transfection, as described, for example, in Maus et al., Annu Rev Immunol (2014) 32:189-225, which is incorporated herein by reference in its entirety. Methods also include, for example, those described in Wang and Riviere Mol Ther Oncolytics. (2016) 3:16015, which is incorporated herein by reference in its entirety. Suitable methods for introducing nucleic acids / vectors into cells include transduction, transfection and electroporation.

[0254] Aspects and embodiments of the present disclosure relate particularly to antigen-presenting cells (APCs). APCs are cells that express MHC molecules (e.g., MHC class I and / or MHC class II molecules) and can present MHC:peptide complexes. It will be understood that APCs according to the present disclosure can present MHC:peptide complexes according to the present disclosure.

[0255] The APC according to the present disclosure can be a professional APC. Professional APCs are specialized to present antigens to T cells; they are efficient in processing and presenting MHC-peptide complexes on the cell surface and express high levels of costimulatory molecules. Professional APCs include dendritic cells (DCs), macrophages, and B cells. Non-professional APCs are other cells that can present MHC-peptide complexes to T cells, particularly MHC class I-peptide complexes to CD8+ T cells.

[0256] In some embodiments, the APC is an APC that can cross-present an antigen internalized by the APC on MHC class I (e.g., taken up by endocytosis / phagocytosis). Cross-presentation of an internalized antigen to CD8+ T cells on MHC class I is described, for example, in Alloatti et al., Immunological Reviews (2016), 272(1):97-108, which is incorporated herein by reference in its entirety. APCs capable of cross-presentation include, for example, dendritic cells (DCs), macrophages, B cells, and sinusoidal endothelial cells.

[0257] In some embodiments, the APC expresses / comprises a peptide presented by an MHC molecule comprising a polypeptide according to the present disclosure.

[0258] In some embodiments, the APC expresses / contains a peptide presented by an MHC molecule according to the present disclosure as a result of contact with the peptide or contact with and internalization of a polypeptide comprising the peptide. When 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 produce the peptide.

[0259] In some embodiments, the APCs may be "pulsed" with the polypeptide / peptide, which may involve culturing the APCs in vitro in the presence of the polypeptide / peptide for a period of time sufficient for the APCs to internalize the polypeptide / peptide.

[0260] In some embodiments, the APC expresses / comprises a peptide presented by an MHC molecule according to the present disclosure as a result of expression in the cell of a nucleic acid encoding the peptide or a nucleic acid encoding a polypeptide comprising the peptide. In some embodiments, the APC comprises a peptide presented by an MHC molecule, a nucleic acid encoding a polypeptide according to the present disclosure, or a nucleic acid encoding a polypeptide comprising the peptide. The APC may comprise such a nucleic acid as a result of a nucleic acid encoding the peptide / polypeptide (e.g., in the form of a vector comprising such a nucleic acid) being introduced into the cell by, for example, transfection, transduction, electroporation, etc.

[0261] In some embodiments, the APCs are cells of a cell line (eg, an immortalized cell line).

[0262] In some embodiments, the APC comprises a modification to reduce / prevent expression of one or more MHC polypeptides (i.e., compared to the expression level of the relevant MHC polypeptide by an equivalent unmodified cell). In some embodiments, the APC comprises a modification to reduce expression of one or more endogenous MHC polypeptides, i.e., MHC polypeptides encoded by the genome of an equivalent unmodified cell. In some embodiments, the APC comprises a modification to reduce 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 comprises a modification to reduce expression of one or more MHC class I α chain polypeptides.

[0263] In particular, modifications to reduce / prevent expression of one or more MHC polypeptides are contemplated when the APC is allogeneic with respect to the subject from which the T cells to be screened according to the methods for identifying a TCR according to the present disclosure are isolated / obtained.

[0264] When a subject is referred to herein as being "allogeneic" or "non-autologous" to a reference subject, the subjects are not the same. An allogeneic subject may not be genetically identical to the reference subject. An allogeneic subject may contain MHC / HLA genes encoding MHC / HLA molecules (e.g., MHC class Iα and / or MHC class II molecules) that are not identical to the MHC / HLA molecules (e.g., MHC class Iα and / or MHC class II molecules) encoded by the reference subject.

[0265] Conversely, when a subject is referred to herein as "autologous" or "self," the subject may be genetically identical. An autologous subject may contain MHC / HLA genes encoding identical MHC / HLA molecules (e.g., MHC class Iα and / or MHC class II molecules). An autologous subject may be the same subject.

[0266] 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 target nucleic acid editing using site-specific nucleases (SSNs).

[0267] Suitable methods may use targeting by homologous recombination, as outlined in, for example, Mortensen Curr Protoc 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 outlined 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 the target nucleic acid sequence of interest. DSBs can be repaired either by error-prone non-homologous end joining (NHEJ), in which the two ends of the break are rejoined, often with the insertion or deletion of nucleotides. Alternatively, DSBs can be repaired by homology-directed repair (HDR), a high-fidelity mechanism in which a DNA template with ends homologous to the break is provided and introduced at the site of the DSB.

[0268] 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. ZFN systems are described, for example, in Umov et al., Nat Rev Genet. (2010) 11(9):636-46, the entire contents of which are incorporated herein by reference. ZFNs contain a programmable zinc finger DNA binding domain and a DNA cleavage domain (e.g., a FokI endonuclease domain). DNA binding domains can be identified by screening zinc finger arrays that can bind to target nucleic acid sequences. The TALEN system is generally described, for example, in Mahfouz et al., Plant Biotechnol J. (2014) 12(8):1006-14, which is incorporated herein by reference in its entirety. TALENs contain a programmable DNA-binding TALE domain and a DNA-cleavage domain (e.g., a FokI endonuclease domain). TALEs contain a repeat domain consisting of 33-39 amino acid repeats that are identical except for two residues at positions 12 and 13 of each repeat, the repeat variable di-residues (RVDs). Each RVD determines the binding of the repeat to a nucleotide in the target DNA sequence according to the following relationship: "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 incorporated herein by reference in its entirety.These systems include an endonuclease (e.g., Cas9, Cpf1, etc.) and a single guide RNA (sgRNA) molecule. The sgRNA can be engineered to target the endonuclease activity to a nucleic acid sequence of interest.

[0269] In some embodiments, particularly in embodiments in which the APC comprises a modification to reduce / prevent expression of one or more endogenous MHC polypeptides, the APC further comprises a modification to comprise / express a nucleic acid encoding one or more MHC polypeptides. In such embodiments, the APC is preferably engineered to express an MHC polypeptide corresponding to one in which the APC comprises a modification to reduce / prevent endogenous expression (e.g., if the APC comprises a modification to reduce / prevent expression of an endogenous MHC class I α chain polypeptide, the APC is engineered to express the relevant MHC class I α 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 which T cells to be screened according to the methods for identifying TCRs disclosed herein are isolated / obtained. In some embodiments, the APC is engineered to express one or more MHC polypeptides encoded by the subject comprising a disease-associated antigen according to the present disclosure. In some embodiments, the APC is engineered to express one or more MHC polypeptides encoded by the subject comprising a cancer-associated neoantigen (i.e., a subject comprising cells comprising / expressing a cancer-associated neoantigen).

[0270] The preceding paragraph particularly relates to embodiments in which the articles and methods of the present disclosure are used to identify TCRs that bind to a particular MHC:complex of interest. Modifying APCs to reduce / prevent expression of endogenous MHC polypeptide(s) and engineering APCs to express MHC polypeptides encoded by the genome of the subject of interest provides for antigen presentation to occur in the subject.

[0271] In some embodiments, APCs are primary cells, e.g., cells isolated / obtained directly from a living subject / tissue (e.g., via biopsy). APCs can be isolated / obtained or derived from cells isolated / obtained from a subject of interest, e.g., a patient (e.g., a subject / patient comprising a disease-associated antigen according to the present disclosure, e.g., a subject / patient comprising a cancer-associated neoantigen (i.e., a subject / patient comprising cells comprising / expressing a cancer-associated neoantigen). In such embodiments, APCs can be from an autologous subject to the subject from which T cells to be screened according to the methods for identifying TCRs according to the present disclosure are isolated / obtained. In such embodiments, APCs already express appropriate MHC molecules for presentation of a peptide of interest when the peptide of interest occurs in the subject, and therefore it is generally not intended that the APCs be modified to reduce / prevent expression of endogenous MHC polypeptides.

[0272] The present disclosure provides methods for producing cells comprising MHC molecules 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 the identifier moiety (e.g., HaloTag); and (2) contacting the cell with a labeling moiety comprising the identifier moiety, where 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).

[0273] The present disclosure also provides methods for producing cells containing MHC:peptide complexes comprising MHC molecules labeled with identifier moieties, the methods comprising: (1) introducing into the cells (e.g., APCs) 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 cells a nucleic acid encoding (i) a peptide presented by an MHC molecule comprising the polypeptide encoded by the nucleic acid or nucleic acids of (1), or (ii) a peptide presented by an MHC molecule comprising the polypeptide encoded by the nucleic acid or nucleic acids of (1); and (3) contacting the cells 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 cells with a HaloTag ligand comprising an ssDNA moiety and a chloroalkane moiety).

[0274] In accordance with the previous two paragraphs, in some embodiments, the cell is an APC, e.g., an APC described herein. In some embodiments, the method further includes method steps for producing an APC described herein, e.g., by modification to reduce / prevent expression of one or more endogenous MHC polypeptides (e.g., in the case of APCs allogeneic to the subject from which the T cells screened according to the methods of the present disclosure are isolated / obtained), and / or modification to express one or more non-endogenous MHC polypeptides (e.g., one or more MHC polypeptides encoded by the genome of the subject from which the T cells screened according to the methods of the present disclosure are isolated / obtained).

[0275] composition The present disclosure provides compositions comprising the polypeptides, antigen-binding molecules, nucleic acids, expression vectors, and cells of the present disclosure.

[0276] The present disclosure also provides a composition comprising a cell (eg, an antigen-presenting cell) according to the present disclosure and a T cell.

[0277] The present disclosure also provides (a) a cell (e.g., an antigen-presenting cell) comprising, on its cell surface, a polypeptide according to the present disclosure (e.g., a polypeptide comprising a moiety that facilitates labeling of the polypeptide with an identifier moiety, or a polypeptide comprising an identifier moiety); and (b) a cell (e.g., a T cell) comprising a polypeptide on its cell surface that comprises a sortase acceptor motif; and (c) Sortase Also provided is a composition comprising:

[0278] In some embodiments, the composition comprises: (a) a cell (e.g., an antigen-presenting cell) comprising, on its cell surface, a polypeptide according to the present disclosure (e.g., a polypeptide comprising a moiety that facilitates labeling of the polypeptide with an identifier moiety, or a polypeptide comprising an identifier moiety); and (b) a cell (e.g., a T cell) comprising, on its cell surface, (i) a polypeptide comprising a sortase acceptor motif, and (ii) a sortase Includes.

[0279] It will be understood that cells according to the present disclosure are contemplated for use in methods in which a T cell according to the present disclosure is contacted with a cell according to the present disclosure (e.g., an APC) for the purpose of identifying a T cell that expresses a TCR that binds to an MHC:peptide complex presented by the cell.

[0280] It will of course be understood that a composition according to the present disclosure may include a plurality of cells and a plurality of T cells according to the present disclosure.

[0281] In some embodiments, the composition comprises a plurality of non-identical T cells. In some embodiments, the composition comprises a plurality of T cells encoding / comprising non-identical TCRs. It will be appreciated 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 encoding / comprising diverse TCRs for ultimate identification of TCRs capable of binding to MHC:peptide complexes presented by the antigen-presenting cell.

[0282] In some embodiments, a composition according to the present disclosure comprises a plurality of non-identical cells according to the present disclosure. In some embodiments, a composition comprises a plurality of antigen-presenting cells that contain / present non-identical MHC:peptide complexes. It will 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 non-identical peptides for presentation by the MHC:peptide complexes and are contacted with a population of T cells encoding / containing diverse TCRs for ultimate identification of TCRs that can bind to a variety of different MHC:peptide complexes presented by the antigen-presenting cells.

[0283] Methods for identifying T cell receptor-bound MHC:peptide complexes and for identifying cells that have interacted with each other Aspects and embodiments of the present disclosure relate, in particular, to identifying T cell receptors (TCRs) that bind to MHC:peptide complexes. In particular, 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 containing a peptide of interest.

[0284] Accordingly, the present disclosure provides a method for identifying T cell receptors (TCRs) that bind to an MHC:peptide complex, comprising: (1) contacting (a) cells comprising the 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 TCRs that bind to the MHC:peptide complex, wherein the MHC:peptide complex comprises an MHC molecule, and 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 attached to the polypeptide via a bond formed by a self-labeling protein tag.

[0285] The disclosure also provides a method for identifying a T cell receptor (TCR) that binds to an MHC:peptide complex, comprising: (1) contacting (a) cells comprising the MHC:peptide complex with (b) a cell population comprising T cells that comprise a polypeptide on the cell surface comprising a sortase acceptor motif in the presence of a sortase; (2) incubating the cells obtained after step (1) under conditions suitable for interaction between the cells of (a) and the cell population of (b); and (3) subsequently analyzing the cells obtained after step (2) to identify T cells that comprise a TCR that binds to the MHC:peptide complex, wherein the MHC:peptide complex comprises an MHC molecule, and the MHC molecule comprises a polypeptide comprising (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 attached to the polypeptide via a bond formed by a self-labeling protein tag. In some embodiments, incubating the cells under conditions suitable for interaction between the cells of (a) and the cell population of (b) (i.e., co-culturing the cells) comprises culturing under conditions suitable for sortase-mediated transfer of an identifier moiety (i.e., from a polypeptide comprising (i) an amino acid sequence of an MHC polypeptide, (ii) a sortase substrate motif, and (iii) an identifier moiety) into a polypeptide comprising a sortase acceptor motif.

[0286] The cell comprising the MHC:peptide complex comprising an MHC molecule comprising a polypeptide according to the present disclosure can be a cell described herein, e.g., an APC. The step of "contacting" such a cell with a T cell can include contacting the cells with each other in a co-culture.

[0287] Incubating cells under conditions suitable for trogocytosis of the MHC:peptide complex by T cells comprising a TCR that binds to the MHC:peptide complex (i.e., co-culturing the cells) may include maintaining the cells at 37°C in a humidified atmosphere containing 5% CO2. Similarly, incubating cells under conditions suitable for interaction between the cells of (a) and the cell population of (b) (i.e., co-culturing the cells) may include maintaining the cells at 37°C in a humidified atmosphere containing 5% CO2. Similarly, incubating cells under conditions suitable for interaction between the cells of (a) and the cell population of (b) (i.e., co-culturing the cells) may include culturing under conditions suitable for sortase-mediated transfer of an identifier moiety into a polypeptide comprising a sortase acceptor motif, and may include maintaining the cells at 37°C in a humidified atmosphere containing 5% CO2. Suitable culture conditions can be readily determined by one of skill in the art.

[0288] Trogocytosis refers to the internalization of the cell membrane by one cell and / or cell membrane-bound material of another cell. Trogocytosis is described, for example, in Zhao et al., Front Immunol. (2022) 13:791006, the entire contents of which are incorporated herein by reference. The present disclosure is particularly interested in trogocytosis by T cells of MHC:peptide complexes presented by APCs.

[0289] In some embodiments, the sortase is provided on the cell surface of a T cell comprising a polypeptide comprising a sortase acceptor motif. The sortase may 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 linked to 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., via a protein:protein interaction, e.g., via an antibody:antigen interaction).

[0290] In some aspects and embodiments, the methods of the present disclosure further comprise analyzing the T cells to identify TCRs that bind to the MHC:peptide complex.

[0291] In some embodiments, the method further comprises determining a peptide of the MHC:peptide complex that was internalized by the T cell. In preferred embodiments, analyzing the T cell comprises analyzing the T cell to determine the identity of the identifier moiety (i.e., the identifier moiety of the MHC:peptide complex or the identifier moiety transferred to the polypeptide comprising a sortase acceptor motif). As explained hereinabove, a given identifier moiety is preferably used with and encodes a particular peptide, and thus determining the identity of the identifier moiety provides a determination of the peptide of the MHC:peptide complex with which the T cell interacted (e.g., which may have been internalized by the T cell).

[0292] The analysis used to determine the identity of the identifier portion will, of course, be selected according to 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.

[0293] The nucleotide sequence of a polynucleotide / nucleic acid can be determined by any suitable technique well known to those skilled in the art, including, for example, Sanger sequencing. In a preferred embodiment, next generation sequencing (NGS) techniques can be used.

[0294] In preferred embodiments, analyzing the T cells further comprises analyzing the T cells to determine the identity of the TCR encoded by the T cells. Determining the identity of the TCR encoded by the T cells may comprise determining the nucleotide sequence of a nucleic acid encoding a 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 encoding nucleic acid sequence). In some embodiments, the method comprises determining the nucleotide sequence of a gene region encoding the variable region of the TCR alpha chain (e.g., the Valpha and / or Jalpha regions) and / or determining the nucleotide sequence of a gene region encoding the variable region of the TCR beta chain (e.g., the Vbeta, Dbeta and / or Jbeta regions).

[0295] It will be appreciated that in some embodiments, the method comprises determining the peptide of the MHC:peptide complex that was internalized by the T cell (e.g., via determining the identity of the identifier portion) and determining the identity of the TCR encoded by the T cell. Similarly, it will be appreciated that in some embodiments, the method comprises determining the peptide of the MHC:peptide complex that interacted with the T cell (e.g., via determining the identity of the identifier portion transferred to the T cell) and determining the identity of the TCR encoded by the T cell. In this manner, the method provides for easy identification of a specific TCR that recognizes an MHC:peptide complex that presents a particular peptide of interest. In preferred embodiments, the identifier portion comprises or consists of a nucleic acid portion (e.g., a polynucleotide), and the method comprises analyzing the nucleotide sequences of the nucleic acid encoding the identifier portion and the variable region of the TCR using next-generation sequencing (NGS) technology.

[0296] It will be understood that the analysis is performed on single cells that have internalized an MHC:peptide complex according to the present disclosure or that have an identifier moiety transferred thereto via sortase-mediated transfer. Single-cell sequencing methods are reviewed, for example, in Tang et al., Cell & Bioscience (2019) 9:53, which is incorporated herein by reference in its entirety.

[0297] The use of multiple identifier moieties and corresponding peptides and diverse populations of T cells encoding different TCRs provides for the simultaneous identification of multiple TCR-MHC:peptide complex interaction pairs in a single experiment.

[0298] In some embodiments of the disclosed methods, APCs containing MHC molecules comprising a polypeptide according to the present disclosure, comprising (i) the amino acid sequence of the MHC polypeptide and (ii) a moiety that facilitates labeling of the polypeptide with an identifier moiety, may be modified to contain a peptide according to the present disclosure (e.g., by pulsing the APCs with a peptide containing the relevant peptide or by introducing a nucleic acid encoding the peptide into the APCs), and the APCs may also be labeled with an identifier moiety corresponding to the peptide (e.g., using a labeling moiety). APCs may be labeled with different peptide and identifier pairs, for example, in wells of a polypropylene plate. Various peptide-labeled and identifier-labeled APCs may then be pooled, contacted with a population of T cells, and incubated under conditions suitable for trogocytosis of the MHC:peptide complex by T cells containing a TCR that binds to the MHC:peptide complex. The T cells of the population may then be analyzed to determine the identity of the TCR encoded by the T cells and the identity of the peptide in the MHC:peptide complex internalized by the T cells, i.e., via determining the identity of the identifier moiety.

[0299] In some embodiments of the methods of the present disclosure, APCs comprising MHC molecules comprising a polypeptide according to the present disclosure comprising (i) the amino acid sequence of the MHC polypeptide, (ii) a sortase substrate motif, and (iii) a moiety that facilitates labeling of the polypeptide with the identifier moiety may be modified to contain a peptide according to the present disclosure (e.g., by pulsing the APCs with a peptide comprising the relevant peptide or by introducing into the APCs a nucleic acid encoding the peptide), and the APCs may also be labeled with an identifier moiety corresponding to the peptide (e.g., using a labeling moiety). APCs may be labeled with different peptide and identifier pairs, for example, in the wells of a polypropylene plate. The various peptide-labeled and identifier-labeled APCs may then be pooled and contacted with a population of T cells comprising a polypeptide comprising a sortase acceptor motif on their cell surface in the presence of sortase and incubated under conditions suitable for sortase-mediated transfer of the identifier moiety into the polypeptide comprising the sortase acceptor motif. The T cells of the population can then be analyzed to determine the identity of the TCR encoded by the T cells and the identity of the peptide in the MHC:peptide complex that interacted with the T cells (e.g., via determining the identity of the identifier moiety transferred to the T cells).

[0300] It should be understood that in some embodiments, it is possible that a T cell expressing a given single TCR may recognize, and therefore interact with and / or internalize, multiple non-identical peptide:MHC complexes. In such embodiments, multiple different identifier moieties may be detected within or on a given T cell.

[0301] In some embodiments, the method may further comprise a step of isolating / selecting T cells that were or are likely to have been involved in the formation of a TCR-MHC:peptide complex (i.e., prior to analysis of the T cells to determine the identity of the T cells and the identifier moiety). According to such embodiments, the cells are selected / isolated for subsequent analysis (i.e., from other cells, e.g., T cells that are not / are unlikely to have been involved in the formation of such a complex). This method step focuses downstream analysis of T cells that have interacted with and / or internalized the MHC:peptide complex of interest.

[0302] In some embodiments, T cells are isolated / selected based on characterization of markers of correlation of CD3-TCR complex-mediated signaling, e.g., 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.

[0303] In some embodiments, particularly those in which a polypeptide of the present disclosure comprises a detectable moiety as described herein, T cells can be isolated / selected for subsequent analysis based on detection of a detectable marker in or on the T cells. By way of example, in embodiments in which a polypeptide of the present disclosure comprises a fluorescent label, T cells with internalized MHC:peptide complexes comprising a polypeptide of the present disclosure can be detected and sorted (e.g., by fluorescence-activated cell sorting (FACS)) based on detection of T cells containing the fluorescent label. The T cells containing the fluorescent label can then be further analyzed as described above to determine the identity of the TCR and the identity of the peptide in the peptide:MHC complex they internalized.

[0304] It will be understood that in some embodiments, the method for identifying T cell receptor-bound MHC:peptide complexes further comprises method steps for producing APCs comprising MHC molecules according to the present disclosure and / or for producing APCs comprising MHC:peptide complexes according to the present disclosure, as described herein above.

[0305] In further aspects and embodiments, the disclosure more generally provides methods for identifying cells that have interacted with a cell comprising a polypeptide comprising (i) a sortase substrate motif and (ii) an identifier moiety at its cell surface, the method comprising: (1) (a) contacting cells comprising (i) a sortase substrate motif and (ii) a polypeptide comprising (i) a sortase substrate motif and (ii) an identifier moiety at its cell surface with a cell population comprising cells comprising a polypeptide comprising a sortase acceptor motif at their cell surface in the presence of (b) a sortase; and (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 (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 the identifier moiety, thereby identifying cells that have interacted with a cell comprising (i) a sortase substrate motif and (ii) an identifier moiety. In some embodiments, incubating the cells under conditions suitable for interaction between the cells of (a) and the cell population of (b) (i.e., co-culturing the cells) comprises culturing under conditions suitable for sortase-mediated transfer of an identifier moiety (i.e., from a polypeptide comprising (i) a sortase substrate motif, and (ii) an identifier moiety) into a polypeptide comprising a sortase acceptor motif.

[0306] The step of "contacting" the cells of (a) with the cells of (b) may include contacting the cells with each other in a co-culture.

[0307] Incubating the cells under conditions suitable for interaction between the cells of (a) and the cell population of (b) (i.e., co-culturing the cells) includes culturing under conditions suitable for sortase-mediated transfer of an identifier moiety into a polypeptide comprising a sortase acceptor motif, which may include maintaining the cells at 37° C. in a humidified atmosphere containing 5% CO2. Suitable culture conditions can be readily determined by one of skill in the art.

[0308] In some embodiments, the sortase is provided to the cell surface of a cell that comprises a polypeptide comprising a sortase acceptor motif. The sortase may be immobilized on the cell, i.e., on or at the cell surface of the cell. In some embodiments, the sortase is covalently bound to 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 cell (e.g., via a protein:protein interaction, e.g., via an antibody:antigen interaction).

[0309] The analysis used to identify cells within the cell population of (b) that have received sortase-mediated transfer of the identifier moiety will, of course, be selected according to the nature of the identifier moiety. In embodiments in which the identifier moiety comprises or consists of a nucleic acid moiety, the analysis may include determining the structure of the nucleic acid moiety. In some embodiments, the analysis includes determining the nucleotide sequence of the identifier moiety that comprises or consists of a polynucleotide. The nucleotide sequence of the polynucleotide / nucleic acid may be determined by any suitable technique, such as those described hereinabove.

[0310] It will be understood that the analysis is performed on a single cell that has an identifier moiety transferred thereto via sortase-mediated transfer. Single-cell sequencing methods are reviewed, for example, in Tang et al., Cell & Bioscience (2019) 9:53, the entire contents of which are incorporated herein by reference. The use of multiple identifier moieties and diverse cell populations provides for the simultaneous identification of multiple cell-cell interaction pairs in a single experiment.

[0311] In some embodiments, cells comprising a polypeptide comprising (i) a sortase substrate motif and (ii) a moiety that facilitates labeling of the polypeptide with the identifier moiety may be labeled with different identifier moieties, for example in the wells of a polypropylene plate. The cells may then be pooled and contacted with a population of cells comprising a polypeptide comprising a sortase acceptor motif on its cell surface in the presence of sortase, and incubated under conditions suitable for sortase-mediated transfer of the identifier moiety to the polypeptide comprising the sortase acceptor motif.

[0312] In some embodiments, the method may further comprise a step of isolating / selecting cells that have been or are likely to have been involved in a cell-cell interaction (i.e., prior to analyzing the cells to determine the identity of the identifier moiety). According to such embodiments, the cells are selected / isolated for subsequent analysis (i.e., from other cells, e.g., cells that are not / are unlikely to have been involved in such an interaction). This method step focuses downstream analysis of cells that were involved in the cell-cell interaction of interest.

[0313] In some embodiments, particularly those in which the polypeptides of the present disclosure comprise a detectable moiety as described herein, cells may be isolated / selected for subsequent analysis based on detection of a detectable marker in or on the cells.

[0314] It will be appreciated that in some embodiments the method further comprises method steps for producing cells comprising a polypeptide comprising (i) a sortase substrate motif and (ii) an identifier moiety at their cell surface, as described hereinabove.

[0315] kit The present disclosure also provides kits of parts. 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 carrying out methods according to the present disclosure.

[0316] 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, the labeling moiety being suitable for labeling 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 by the identifier moiety.

[0317] In some embodiments, a kit according to the present disclosure includes: (1) a nucleic acid or multiple nucleic acids encoding a polypeptide comprising (i) the amino acid sequence of β2 microglobulin and (ii) a HaloTag; and (2) a HaloTag ligand comprising an ssDNA portion and a chloroalkane portion.

[0318] In some aspects, a kit according to the present disclosure comprises: (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) a HaloTag; and (2) a HaloTag ligand comprising a ssDNA portion and a chloroalkane portion. In some embodiments, the kit further comprises a sortase and reagents for modifying cells to comprise a polypeptide comprising a sortase acceptor motif on the cell surface. In some embodiments, the kit comprises reagents for modifying cells to comprise a sortase on the cell surface.

[0319] In some embodiments, the kit includes (3) a peptide presented by an MHC molecule comprising a polypeptide encoded by the nucleic acid or nucleic acids described in (1), or a nucleic acid encoding a peptide presented by an MHC molecule comprising a polypeptide encoded by the nucleic acid or nucleic acids described in (1).

[0320] In some embodiments, a kit according to the present disclosure includes (1) a cell comprising an MHC molecule according to the present disclosure, or a plurality of such cells, and (2) a peptide presented by the MHC molecule of the cell described in (1), or a nucleic acid encoding a peptide presented by the MHC molecule of the cell described in (1).

[0321] In some embodiments, a kit according to the present disclosure comprises a cell, or a plurality of such cells, comprising an MHC:peptide complex comprising an MHC molecule comprising a polypeptide according to the present disclosure and a peptide presented by the MHC molecule.

[0322] In some embodiments, the cells of the plurality of cells according to the previous two paragraphs are not identical. In some embodiments, the plurality of cells of a kit according to the present disclosure comprises cells comprising an MHC molecule, and the MHC molecules expressed by the plurality of different cells comprise a polypeptide of the present disclosure labeled with non-identical identifier moieties. In some embodiments, the plurality of cells of a kit according to the present disclosure comprises cells comprising non-identical peptides.

[0323] A kit-of-parts according to the present disclosure may include predetermined amounts of the items described in the previous six paragraphs. In some embodiments, the relevant items are provided in a container (e.g., in a vial or bottle). The kit may provide the relevant items along with instructions (e.g., protocols) on how to use them in accordance with the methods described herein.

[0324] In some embodiments, the kit of parts includes materials for producing a polypeptide according to the present disclosure. In some embodiments, the kit of parts includes materials for producing an MHC molecule according to the present disclosure. In some embodiments, the kit of parts includes materials for producing an MHC:peptide complex according to the present disclosure. In some embodiments, the kit of parts includes materials for producing a cell according to the present disclosure. In some embodiments, the kit of parts includes materials for producing a composition according to the present disclosure.

[0325] In some embodiments, the kit-of-parts may include a nucleic acid(s) or expression vector(s) according to the present disclosure, and, optionally, materials for introducing the nucleic acid(s) or expression vector(s) into a cell.

[0326] In some embodiments, reagents for modifying cells to contain polypeptides comprising a sortase acceptor motif on their cell surface comprise nucleic acid(s) encoding the polypeptides comprising a sortase acceptor motif, or expression vector(s) comprising such nucleic acids, and, optionally, materials for introducing the nucleic acid(s) or expression vector(s) into the cell. However, in preferred embodiments, reagents for modifying cells to contain polypeptides comprising a sortase acceptor motif on their cell surface are reagents that provide a non-genetic bioconjugate of the polypeptide to the cell. In some embodiments, the reagent comprises ManNAz. In some embodiments, the reagent comprises a reagent for functionalizing a polypeptide comprising a sortase acceptor motif with a cycloalkyne moiety (e.g., a dibenzocyclooctyl (DBCO) moiety or an azadibenzocyclooctyne (ADIBO) moiety).

[0327] In some embodiments, a reagent for modifying a cell to contain a sortase on its cell surface comprises a nucleic acid(s) encoding a polypeptide comprising a sortase acceptor motif, or an expression vector(s) comprising such nucleic acids, and, optionally, materials for introducing the nucleic acid(s) or expression vector(s) into the cell. In some embodiments, a reagent for modifying a cell to contain a sortase on its cell surface is a reagent that provides a non-genetic bioconjugate of a polypeptide to the cell. In some embodiments, the reagent comprises ManNAz. In some embodiments, the reagent comprises a reagent that provides a non-genetic bioconjugate 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).

[0328] 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 that comprises a sortase moiety (e.g., DOTAM or DOTA).

[0329] The manufacture of a kit-of-parts according to the present disclosure preferably follows standard procedures known to those skilled in the art.

[0330] Sequence identity As used herein, "sequence identity" refers to the percentage of nucleotides / amino acid residues in a subject sequence that are identical to the nucleotides / amino acid residues in a reference sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity between the sequences. Pairwise and multiple sequence alignment for determining the percent sequence identity between two or more amino acid or nucleic acid sequences can be achieved by various methods known to those skilled in the art, for example, using publicly available computer software such as ClustalOmega (Soding, 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 preferable to use default parameters, such as gap penalty and extension penalty. TIFF2025530126000009.tif239170TIFF2025530126000010.tif242170TIFF2025530126 000011.tif240170TIFF2025530126000012.tif239170TIFF2025530126000013.tif96170

[0331] Numbered paragraphs The following numbered paragraphs (para) provide further descriptions of features and combinations of features contemplated in connection with the present disclosure.

[0332] 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.

[0333] 2A. The polypeptide of paragraph 1A, wherein the MHC polypeptide is β2 microglobulin.

[0334] 3A. The polypeptide of para. 1A or para. 2A, wherein the identifier moiety is a nucleic acid moiety.

[0335] 4A. The polypeptide of any one of paragraphs 1A to 3A, wherein the identifier portion comprises or consists of single-stranded DNA (ssDNA).

[0336] 5A. The polypeptide of any one of paragraphs 1A to 4A, wherein the moiety that facilitates labeling of the polypeptide with the identifier moiety is or includes a self-labeling protein tag.

[0337] 6A. The polypeptide of any one of paragraphs 1A to 5A, wherein the moiety that facilitates labeling of the polypeptide with an identifier moiety is or includes a HaloTag.

[0338] 7A. A polypeptide comprising (i) the amino acid sequence of β2 microglobulin and (ii) a HaloTag.

[0339] 8A. The polypeptide of any one of paragraphs 1A to 7A, further comprising a detectable moiety.

[0340] 9A. The polypeptide of para. 8A, wherein the detectable moiety is a fluorescent label.

[0341] 10A. A polypeptide according to any one of paragraphs 1A to 9A, comprising or consisting of an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 11, 10, 13 or 12.

[0342] 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 attached to the polypeptide via a linkage formed by a self-labeling protein tag.

[0343] 12A. The polypeptide of paragraph 11A, wherein the MHC polypeptide is β2 microglobulin.

[0344] 13A. The polypeptide of paragraph 11A or paragraph 12A, wherein the identifier moiety is a nucleic acid moiety.

[0345] 14A. The polypeptide of any one of paragraphs 11A to 13A, wherein the identifier portion comprises or consists of single-stranded DNA (ssDNA).

[0346] 15A. The polypeptide of any one of paragraphs 11A to 14A, wherein the self-labeling protein tag is or comprises a HaloTag.

[0347] 16A. The polypeptide of paragraph 15A, wherein the identifier moiety is covalently attached to 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.

[0348] 17A. A polypeptide comprising (i) an amino acid sequence of β2 microglobulin, (ii) a HaloTag, and (iii) a single-stranded DNA (ssDNA) portion linked to the polypeptide via an ester bond formed by the dehalogenase activity of the HaloTag on a HaloTag ligand comprising an ssDNA portion and a chloroalkane portion.

[0349] 18A. The polypeptide of any one of paragraphs 11A to 17A, further comprising a detectable moiety.

[0350] 19A. The polypeptide of para. 18A, wherein the detectable moiety is a fluorescent label.

[0351] 20A. An MHC molecule comprising a polypeptide according to any one of paragraphs 1A to 19A.

[0352] 21A. An MHC:peptide complex comprising an MHC molecule according to paragraph 20A and a peptide presented by the MHC molecule.

[0353] 22A. A nucleic acid or nucleic acids encoding a polypeptide according to any one of paragraphs 1A to 10A.

[0354] 23A. The nucleic acid or nucleic acids of para. 11A or para. 12A, further comprising a nucleic acid encoding a peptide presented by an MHC molecule comprising a polypeptide of any one of paras. 1A-10A.

[0355] 24A. An expression vector or vectors comprising a nucleic acid or nucleic acids according to paragraph 22A or paragraph 23A.

[0356] 25A. A cell comprising a polypeptide described in any one of paragraphs 1A to 19A, an MHC molecule described in para. 20A, an MHC:peptide complex described in para. 21A, a nucleic acid or nucleic acids described in any one of paras 21A to 23A, or an expression vector or expression vectors described in para. 24A.

[0357] 26A. The cell described in para. 15A, which is an antigen-presenting cell (APC).

[0358] 27A. A method for producing cells comprising MHC molecules labeled with an identifier moiety, comprising: (1) introducing into a cell a nucleic acid or nucleic acids described in paragraph 22A; and (2) contacting the cell with a labeling moiety that includes 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; A method comprising:

[0359] 28A. A method for producing cells containing MHC molecules labeled with a single-stranded DNA (ssDNA) moiety, comprising: (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) a HaloTag; and (2) contacting the cell with a HaloTag ligand comprising a ssDNA portion and a chloroalkane portion; A method comprising:

[0360] 29A. A method for producing cells containing MHC:peptide complexes comprising MHC molecules labeled with an identifier moiety, comprising: (1) introducing into a cell a nucleic acid or nucleic acids described in paragraph 22A; (2) introducing 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); and (3) 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 via a moiety that facilitates labeling of the polypeptide by the identifier moiety; A method comprising:

[0361] 30A. A method for producing cells containing MHC:peptide complexes comprising MHC molecules labeled with a single-stranded DNA (ssDNA) moiety, comprising: (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) a HaloTag; (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 (3) contacting the cell with a HaloTag ligand comprising a ssDNA portion and a chloroalkane portion; A method comprising:

[0362] 31A. Cells produced by the method described in any one of paragraphs 27A to 30A.

[0363] 32A. A composition comprising a cell according to any one of paragraphs 25A, 26A or 31A and a T cell.

[0364] 33A. A method for identifying T cell receptors (TCRs) that bind to MHC:peptide complexes, comprising: (1) contacting a cell containing an MHC:peptide complex described in paragraph 21A with a population of T cells; (2) incubating the cells under conditions suitable for trogocytosis of the MHC:peptide complex by T cells containing a TCR that binds to the MHC:peptide complex; and (3) subsequently analyzing the T cells to identify TCRs that bind to the MHC:peptide complex; A method comprising:

[0365] 34A. A kit comprising: (1) a nucleic acid or nucleic acids described in paragraph 22A, and (2) a labeling moiety comprising an identifier moiety, the labeling moiety being suitable for labeling 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 by the identifier moiety; Kit including:

[0366] 35A. A kit comprising: (1) a nucleic acid or nucleic acids encoding a polypeptide comprising (i) an amino acid sequence of β2 microglobulin and (ii) a HaloTag; and (2) HaloTag ligands containing a ssDNA moiety and a chloroalkane moiety; Kit including:

[0367] 36A. A kit according to paragraph 35A or paragraph 36A, (3) A kit comprising a peptide presented by an MHC molecule comprising a polypeptide encoded by the nucleic acid or nucleic acids described in (1), or a nucleic acid encoding a peptide presented by an MHC molecule comprising a polypeptide encoded by the nucleic acid or nucleic acids described in (1).

[0368] 37A. A kit comprising: (1) a cell or a plurality of such cells containing an MHC molecule described in paragraph 20A; and (2) A kit comprising a peptide presented by an MHC molecule of the cell according to (1) or a nucleic acid encoding a peptide presented by an MHC molecule of the cell according to (1).

[0369] 38A. A kit comprising a cell or a plurality of such cells comprising an MHC:peptide complex as described in para. 21A.

[0370] 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.

[0371] 2B. The polypeptide of paragraph 1B, wherein the MHC polypeptide is β2 microglobulin.

[0372] 3B. The polypeptide of paragraph 1B or paragraph 2B, wherein the identifier moiety is a nucleic acid moiety.

[0373] 4B. The polypeptide of any one of paragraphs 1B to 3B, wherein the identifier portion comprises or consists of single-stranded DNA (ssDNA).

[0374] 5B. The polypeptide of any one of paragraphs 1B to 4B, wherein the moiety that facilitates labeling of the polypeptide with the identifier moiety is or includes a self-labeling protein tag.

[0375] 6B. The polypeptide of any one of paragraphs 1B to 5B, wherein the moiety that facilitates labeling of the polypeptide with an identifier moiety is or includes a HaloTag.

[0376] 7B. The polypeptide of any one of paragraphs 1B to 6B, further comprising a sortase substrate motif.

[0377] 8B. A polypeptide comprising (i) the amino acid sequence of β2 microglobulin and (ii) a HaloTag.

[0378] 9B. A polypeptide comprising (i) the amino acid sequence of β2 microglobulin, (ii) a sortase substrate motif, and (iii) a HaloTag.

[0379] 10B. The polypeptide of any one of paragraphs 1B to 9B, further comprising a detectable moiety.

[0380] 11B. The polypeptide of paragraph 10B, wherein the detectable moiety is a fluorescent label.

[0381] 12B. A polypeptide according to any one of paragraphs 1B to 11B, comprising or consisting of an amino acid sequence having at least 70B% amino acid sequence identity to SEQ ID NO: 11, 10, 13 or 12.

[0382] 13B. 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 attached to the polypeptide via a linkage formed by a self-labeling protein tag.

[0383] 14B. The polypeptide of paragraph 13B, wherein the MHC polypeptide is β2 microglobulin.

[0384] 15B. The polypeptide of paragraph 13B or paragraph 14B, wherein the identifier moiety is a nucleic acid moiety.

[0385] 16B. The polypeptide of any one of paragraphs 13B to 15B, wherein the identifier portion comprises or consists of single-stranded DNA (ssDNA).

[0386] 17B. The polypeptide of any one of paragraphs 13B to 16B, wherein the self-labeling protein tag is or comprises a HaloTag.

[0387] 18B. The polypeptide of paragraph 17B, wherein the identifier moiety is covalently attached to 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.

[0388] 19B. The polypeptide of any one of paragraphs 13B to 18B, further comprising a sortase substrate motif.

[0389] 20B. A polypeptide comprising (i) an amino acid sequence of β2 microglobulin, (ii) a HaloTag, and (iii) a single-stranded DNA (ssDNA) portion linked to the polypeptide via an ester bond formed by the dehalogenase activity of the HaloTag on a HaloTag ligand comprising an ssDNA portion and a chloroalkane portion.

[0390] 21B. A polypeptide comprising (i) an amino acid sequence of β2-microglobulin, (ii) a sortase substrate motif, (iii) a HaloTag, and (iv) a single-stranded DNA (ssDNA) portion linked to the polypeptide via an ester bond formed by the dehalogenase activity of the HaloTag on a HaloTag ligand comprising an ssDNA portion and a chloroalkane portion.

[0391] 22B. The polypeptide of any one of paragraphs 12B to 21B, further comprising a detectable moiety.

[0392] 23B. The polypeptide of para. 22B, wherein the detectable moiety is a fluorescent label.

[0393] 24B. An MHC molecule comprising a polypeptide according to any one of paragraphs 1B to 23B.

[0394] 25B. An MHC:peptide complex comprising an MHC molecule according to para. 24B and a peptide presented by the MHC molecule.

[0395] 26B. A nucleic acid or nucleic acids encoding a polypeptide according to any one of paragraphs 1B to 12B.

[0396] 27B. The nucleic acid or nucleic acids of Paragraph 26B, further comprising a nucleic acid encoding a peptide presented by an MHC molecule comprising a polypeptide of any one of Paragraphs 1B-12B.

[0397] 28B. An expression vector or vectors comprising a nucleic acid or nucleic acids according to para. 26B or para. 27B.

[0398] 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, a nucleic acid or nucleic acids according to paragraph 26B or para 27B, or an expression vector or expression vectors according to para 28B.

[0399] 30B. The cell of para. 29B, which is an antigen-presenting cell (APC).

[0400] 31B. A method for producing cells comprising MHC molecules labeled with an identifier moiety, comprising: (1) introducing into a cell a nucleic acid or nucleic acids described in paragraph 26B; and (2) contacting the cell with a labeling moiety that includes 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; A method comprising:

[0401] 32B. A method for producing cells containing MHC molecules labeled with a single-stranded DNA (ssDNA) moiety, comprising: (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) a HaloTag; and (2) contacting the cell with a HaloTag ligand comprising a ssDNA portion and a chloroalkane portion; A method comprising:

[0402] 33B. A method for producing cells containing MHC molecules labeled with a single-stranded DNA (ssDNA) moiety, comprising: (1) introducing into a cell a nucleic acid or nucleic acids encoding a polypeptide comprising (i) an amino acid sequence of β2 microglobulin, (ii) a sortase substrate motif, and (iii) a HaloTag; and (2) contacting the cell with a HaloTag ligand comprising a ssDNA portion and a chloroalkane portion; A method comprising:

[0403] 34B. A method for producing cells containing MHC:peptide complexes comprising MHC molecules labeled with an identifier moiety, comprising: (1) introducing into a cell a nucleic acid or nucleic acids described in paragraph 26B; (2) introducing 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); and (3) 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 via a moiety that facilitates labeling of the polypeptide by the identifier moiety; A method comprising:

[0404] 35B. A method for producing cells containing MHC:peptide complexes comprising MHC molecules labeled with a single-stranded DNA (ssDNA) moiety, comprising: (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) a HaloTag; (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 (3) contacting the cell with a HaloTag ligand comprising a ssDNA portion and a chloroalkane portion; A method comprising:

[0405] 36B. A method for producing cells containing MHC:peptide complexes comprising MHC molecules labeled with a single-stranded DNA (ssDNA) moiety, comprising: (1) introducing into a cell a nucleic acid or nucleic acids encoding a polypeptide comprising (i) an amino acid sequence of β2 microglobulin, (ii) a sortase substrate motif, and (iii) a HaloTag; (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 (3) contacting the cell with a HaloTag ligand comprising a ssDNA portion and a chloroalkane portion; A method comprising:

[0406] 37B. Cells produced by the method described in any one of paragraphs 31B to 36B.

[0407] 38B. A composition comprising the cells of any one of paragraphs 29B, 30B, or 37B and T cells.

[0408] 39B. A method for identifying a T cell receptor (TCR) that binds to an MHC:peptide complex, comprising: (1) contacting (a) cells containing 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 containing a TCR that binds to the MHC:peptide complex; and (3) subsequently analyzing the cells obtained after step (2) to identify TCRs that bind to the MHC:peptide complex; Including, A method wherein the MHC:peptide complex comprises an MHC molecule, the MHC molecule comprising a polypeptide comprising (i) an amino acid sequence of an MHC polypeptide and (ii) an identifier portion, the identifier portion being covalently attached to the polypeptide via a bond formed by a self-labeling protein tag.

[0409] 40B. A method for identifying a T cell receptor (TCR) that binds to an MHC:peptide complex, comprising: (1) contacting (a) cells comprising an MHC:peptide complex with (b) a population of cells comprising T cells comprising a polypeptide on the cell surface comprising a sortase acceptor motif in the presence of sortase; (2) incubating the cells obtained after step (1) under conditions suitable for interaction between the cells of (a) and the cell population of (b); and (3) subsequently analyzing the cells obtained after step (2) to identify T cells containing a TCR that binds to the MHC:peptide complex; Including, A method wherein the MHC:peptide complex comprises an MHC molecule, the MHC molecule comprising a polypeptide comprising (i) an amino acid sequence of an MHC polypeptide, (ii) a sortase substrate motif, and (iii) an identifier moiety, the identifier moiety being covalently attached to the polypeptide via a bond formed by a self-labeling protein tag.

[0410] 41B. The method of paragraph 40B, wherein the sortase is provided to the cell surface of the T cell comprising a polypeptide comprising a sortase acceptor motif.

[0411] 42B. The method of any one of paragraphs 39B to 41B, wherein the MHC polypeptide is β2 microglobulin.

[0412] 43B. The method of any one of paragraphs 39B to 42B, wherein the identifier moiety is a nucleic acid moiety.

[0413] 44B. The method of any one of paragraphs 39B to 43B, wherein the identifier portion comprises or consists of single-stranded DNA (ssDNA).

[0414] 45B. The method of any one of paragraphs 39B to 44B, wherein the self-labeling protein tag is or includes a HaloTag.

[0415] 46B. The method of paragraph 45B, wherein the identifier moiety is covalently attached to 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.

[0416] 47B. The method of any one of paragraphs 39B to 46B, wherein the polypeptide comprising the amino acid sequence of the MHC polypeptide and the identifier portion further comprises a detectable moiety.

[0417] 48B. The method of paragraph 47B, wherein the detectable moiety is a fluorescent label.

[0418] 49B. A kit comprising: (1) a nucleic acid or nucleic acids described in paragraph 26B, and (2) a labeling moiety comprising an identifier moiety, the labeling moiety being suitable for labeling 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 by the identifier moiety; Kit including:

[0419] 50B. A kit comprising: (1) a nucleic acid or nucleic acids encoding a polypeptide comprising (i) an amino acid sequence of β2 microglobulin and (ii) a HaloTag; and (2) HaloTag ligands containing a ssDNA moiety and a chloroalkane moiety; Kit including:

[0420] 51B. A kit comprising: (1) a nucleic acid or nucleic acids encoding a polypeptide comprising (i) an amino acid sequence of β2 microglobulin, (ii) a sortase substrate motif, and (iii) a HaloTag; and (2) HaloTag ligands containing a ssDNA moiety and a chloroalkane moiety; Kit including:

[0421] 52B. The kit of paragraph 51B, further comprising a sortase and reagents for modifying cells to contain a polypeptide comprising a sortase acceptor motif on the cell surface.

[0422] 53B. A kit according to paragraph 51B or paragraph 52B, comprising a reagent for modifying cells to contain a sortase on the cell surface.

[0423] 54B. A kit according to any one of paragraphs 49B to 53B, A kit comprising a peptide presented by an MHC molecule comprising a polypeptide encoded by the nucleic acid or nucleic acids described in (1), or a nucleic acid encoding a peptide presented by an MHC molecule comprising a polypeptide encoded by the nucleic acid or nucleic acids described in (1).

[0424] 55B. A kit comprising: (1) a cell or a plurality of such cells containing an MHC molecule described in paragraph 24B; and (2) A peptide presented by an MHC molecule of the cell according to (1) or a nucleic acid encoding a peptide presented by an MHC molecule of the cell according to (1), Kit including:

[0425] 56B. A kit comprising a cell or a plurality of such cells comprising an MHC:peptide complex as described in para. 25B.

[0426] 57B. A polypeptide comprising (i) a sortase substrate motif, and (ii) a moiety that facilitates labeling of the polypeptide with an identifier moiety.

[0427] 58B. The polypeptide of paragraph 57B, further comprising the amino acid sequence of a protein that localizes to a cell membrane.

[0428] 59B. The polypeptide of any one of paragraphs 57B to 58B, wherein the moiety that facilitates labeling of the polypeptide with the identifier moiety is or includes a self-labeling protein tag.

[0429] 60B. The polypeptide of any one of paragraphs 57B to 59B, wherein the moiety that facilitates labeling of the polypeptide with an identifier moiety is or includes a HaloTag.

[0430] 61B. The polypeptide of any one of paragraphs 57B to 60B, wherein the identifier moiety is a nucleic acid moiety.

[0431] 62B. The polypeptide of any one of paragraphs 57B to 61B, wherein the identifier portion comprises or consists of single-stranded DNA (ssDNA).

[0432] 63B. A polypeptide comprising (i) an amino acid sequence of a protein localized to a cell membrane, (ii) a sortase substrate motif, and (iii) a HaloTag.

[0433] 64B. The polypeptide of any one of paragraphs 57B to 63B, further comprising a detectable moiety.

[0434] 65B. The polypeptide of para. 64B, wherein the detectable moiety is a fluorescent label.

[0435] 66B. A polypeptide comprising (i) a sortase substrate motif and (ii) an identifier moiety, wherein the identifier moiety is covalently attached to the polypeptide via a linkage formed by a self-labeling protein tag.

[0436] 67B. The polypeptide of any one of paragraphs 63B to 66B, wherein the self-labeling protein tag is or comprises a HaloTag.

[0437] 68B. The polypeptide of para. 67B, wherein the identifier moiety is covalently attached to 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.

[0438] 69B. The polypeptide of any one of paragraphs 63B to 68B, wherein the identifier moiety is a nucleic acid moiety.

[0439] 70B. The polypeptide of any one of paragraphs 63B to 68B, wherein the identifier portion comprises or consists of single-stranded DNA (ssDNA).

[0440] 71B. A polypeptide comprising: (i) an amino acid sequence of a protein localized to a cell membrane; (ii) a sortase substrate motif; (iii) a HaloTag; and (iv) a single-stranded DNA (ssDNA) portion linked to the polypeptide via an ester bond formed by the dehalogenase activity of the HaloTag on a HaloTag ligand comprising an ssDNA portion and a chloroalkane portion.

[0441] 72B. The polypeptide of any one of paragraphs 63B to 71B, further comprising a detectable moiety.

[0442] 73B. The polypeptide of para. 72B, wherein the detectable moiety is a fluorescent label.

[0443] 74B. A nucleic acid or nucleic acids encoding a polypeptide according to any one of paragraphs 57B to 65B.

[0444] 75B. An expression vector or vectors comprising a nucleic acid or nucleic acids according to para. 74B.

[0445] 76B. A cell comprising a polypeptide according to any one of paragraphs 57B to 71B, a nucleic acid or nucleic acids according to para. 74B, or an expression vector or vectors according to para. 75B.

[0446] 77B. The cell of para. 76B, which is an antigen-presenting cell (APC).

[0447] 78B. A method for producing a cell comprising a polypeptide labeled with an identifier moiety, comprising: (1) introducing into a cell a nucleic acid or nucleic acids described in paragraph 74B; and (2) contacting the cell with a labeling moiety that includes 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; A method comprising:

[0448] 79B. The method of paragraph 78B, wherein the identifier moiety is a nucleic acid moiety.

[0449] 80B. The method of paragraph 78B or paragraph 79B, wherein the identifier portion comprises or consists of single-stranded DNA (ssDNA).

[0450] 81B. The method of any one of paragraphs 78B to 80B, wherein the moiety that facilitates labeling of the polypeptide with an identifier moiety is or comprises a HaloTag, and the labeling moiety is a HaloTag ligand comprising an identifier moiety and a chloroalkane moiety.

[0451] 82B. A method for producing a cell comprising a polypeptide labeled with a single-stranded DNA (ssDNA) moiety, comprising: (1) introducing into a cell a nucleic acid or nucleic acids encoding a polypeptide comprising (i) an amino acid sequence of a protein localized to the cell membrane, (ii) a sortase substrate motif, and (iii) a HaloTag; and (2) contacting the cell with a HaloTag ligand comprising a ssDNA portion and a chloroalkane portion; A method comprising:

[0452] 83B. A cell produced by the method described in any one of paragraphs 78B to 82B.

[0453] 84B. A composition comprising: (a) a cell comprising on its cell surface a polypeptide according to any one of paragraphs 57B to 73B; (b) a cell comprising a polypeptide comprising a sortase acceptor motif on its cell surface; and (c) Sortase, A composition comprising:

[0454] 85B. A composition according to paragraph 84B, (a) a cell comprising on its cell surface a polypeptide according to any one of paragraphs 57B to 73B; and (b) a cell comprising, on its cell surface, (i) a polypeptide comprising a sortase acceptor motif, and (ii) a sortase; A composition comprising:

[0455] 86B. The composition of paragraph 84B or paragraph 85B, wherein the cell of (a) is an antigen-presenting cell and / or the cell of (b) is a T cell.

[0456] 87B. A method for identifying a cell that has interacted with a cell containing a polypeptide according to any one of paragraphs 66B to 73B on its cell surface, comprising: (1) (a) contacting a cell comprising, on its cell surface, a polypeptide according to any one of paragraphs 66B to 73B; and (b) contacting, in the presence of a sortase, a cell population comprising cells comprising, on their cell surface, a polypeptide comprising a sortase acceptor motif; (2) incubating the cell population obtained after step (1) under conditions suitable for the interaction between the cells of (a) and the cell population of (b); and (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 the identifier moiety, thereby identifying cells that have interacted with a cell comprising a polypeptide described in any one of paragraphs 66B to 73B on its cell surface; A method comprising:

[0457] 88B. The method of para. 87B, wherein the sortase is provided to the cell surface of a cell comprising a polypeptide comprising a sortase acceptor motif.

[0458] 89B. A kit comprising: (1) a nucleic acid or nucleic acids described in paragraph 74B, and (2) a labeling moiety comprising an identifier moiety, the labeling moiety being suitable for labeling 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 by the identifier moiety; Kit including:

[0459] 90B. A kit comprising: (1) a nucleic acid or nucleic acids encoding a polypeptide comprising (i) an amino acid sequence of a protein that localizes to a cell membrane, (ii) a sortase substrate motif, and (iii) a HaloTag; and (2) HaloTag ligands containing a ssDNA moiety and a chloroalkane moiety; Kit including:

[0460] 91B. The kit of paragraph 89B or paragraph 90B, further comprising a sortase and reagents for modifying cells to contain a polypeptide comprising a sortase acceptor motif on the cell surface.

[0461] 92B. A kit according to any one of paragraphs 89B to 91B, comprising a reagent for modifying cells to contain a sortase on the cell surface.

[0462] The present disclosure includes combinations of the described aspects and preferred features unless such combinations are expressly disallowed or explicitly avoided.

[0463] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0464] Aspects and embodiments of the present disclosure will now be described, by way of example, with reference to the accompanying drawings. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned herein are incorporated by reference.

[0465] Throughout this specification, including the claims which follow, unless the context requires otherwise, the word "comprise," and variations such as "comprises" and "comprising," will be understood to imply the inclusion of stated integers or steps, or groups of integers or steps, but not the exclusion of any other integers or steps, or groups of integers or steps.

[0466] As used herein, "peptide" refers to a chain of two or more amino acid monomers linked by peptide bonds. Peptides typically have a length in the region of about 2 to 50 amino acids. A "polypeptide" is a polymeric 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, and the like.

[0467] As used herein, an amino acid sequence or region of a polypeptide that "corresponds" to a specific reference amino acid sequence or region of a polypeptide has at least 60%, for example, at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the 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 specific reference amino acid sequence / region / position of a polypeptide / amino acid sequence can be identified by sequence alignment of the subject sequence to the reference sequence, for example, using sequence alignment software such as ClustalOmega (Soding, J. 2005, Bioinformatics 21, 951-960).

[0468] As used herein, an amino acid sequence (e.g., the amino acid sequence of a peptide / polypeptide / domain / region) that is "derived" from a reference amino acid sequence (e.g., the amino acid sequence of a reference peptide / polypeptide / domain / region) consists of, or consists of, an amino acid sequence that has at least 60%, e.g., at least 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.

[0469] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment.

[0470] When a nucleic acid sequence is disclosed herein, the reverse complement thereof is also expressly contemplated.

[0471] The methods described herein may preferably be performed in vitro. The term "in vitro" is intended to encompass procedures performed with cells in culture, while the term "in vivo" is intended to encompass procedures with / on intact multicellular organisms.

[0472] Embodiments and experiments illustrating the principles of the present disclosure will now be described with reference to the accompanying drawings. [Brief explanation of the drawings]

[0473] [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 from the parental cell line were labeled with the HaloTag ligand tetramethylrhodamine (HTL TMR). Saturation in HaloTag ligand (HTL) staining was achieved at concentrations near 1 μM; the same concentration resulted in only a slight background signal increase in fluorescence in the parental cell line. [Figure 2] Figure 2A and Figure 2B: HaloTag-β2M-HLA-A*02:01 293T cells were labeled with ssDNA-HTL and subsequently chased with HTL AF488 (500 nM). The ssDNA-HTL reacted specifically with HaloTag (HT) and thus attached to the cell surface, as evidenced by an increase in fluorescence of the DNA-conjugated dye and a decrease in the chase label. At around 10 µM ssDNA-HTL, labeling achieved 60-70% completeness. [Figure 3] HaloTag-β2M-HLA-A*02:01 293T cells were either left unlabeled or labeled with HTL AF660 or ssDNA-HTL Atto655. After pulsing the cells with a dilution series of NY-ESO-1 peptide, the cells served as target cells in a Jurkat activation assay. Jurkat is modified to measure the intensity of TCR signaling in this assay as relative luminescence. The curves for the three conditions were close to each other, indicating that conjugation with HTL did not interfere with immune activation. [Figure 4]HaloTag-β2M-HLA-A*02:01 293T cells were labeled with HTL TMR. After pulsing the cells with WT-1 peptide in culture medium for 1 hour at 37°C, they were used in coculture with donor T cells and bispecific antibodies. The WT-1 bispecific antibody induces an artificial synapse by crosslinking CD3 on T cells with WT-1 pHLA on target cells. DP47 is a bispecific antibody with the same CD3 binding agent and layout but does not recognize the target in this assay. Cocultures were set up at different target-to-effector cell ratios ranging from 5:1 to 1:5. After 2 hours, the extent of trogocytosis in these cocultures was analyzed by flow cytometry. Target and effector cells were distinguished by CD3 staining in live single-cell populations. The median fluorescence intensity of the TMR increased to over 10,000 in WT-1 bispecific antibody-treated cocultures at an E:T ratio of 1:5. Lower E:T ratios, and therefore fewer target cells, resulted in lower median fluorescence intensities, but still a high percentage of positive cells. The background noise observed in the DP47 control was very low. [Figure 5]HaloTag-β2M-HLA-A*02:01 293T cells were labeled with HTL TMR. The cells were pulsed with WT-1 peptide in culture medium for 1 hour at 37°C and then cocultured with donor T cells and a bispecific antibody. The WT-1 bispecific antibody induces an artificial synapse by crosslinking CD3 on T cells with WT-1 pHLA on target cells. DP47 is a bispecific antibody with the same CD3 binding agent and layout but does not recognize the target in this assay. Cocultures were set up at different target-to-effector cell ratios ranging from 5:1 to 1:5. After 24 hours, the extent of trogocytosis in these cocultures was analyzed by flow cytometry. Target and effector cells were distinguished by CD3 staining in live single-cell populations. The median fluorescence intensity of the TMR increased to over 10,000 in WT-1 bispecific antibody-treated E:T 1:5 cocultures. Lower E:T ratios, and therefore fewer target cells, resulted in lower median fluorescence intensities, but still a higher percentage of positive cells. Compared to the 2-hour experiment, the percentage of positive cells also increased from 40% to nearly 80% in the 5:1 E:T co-cultures. The background noise observed in the DP47 control was low, but slightly higher compared to the 2-hour experiment. [Figure 6] HaloTag-β2M-HLA-A*02:01 293T cells were labeled with HTL TMR. They were pulsed with MART-1 peptide in culture medium for 1 h at 37°C and then cocultured with a pool of T cells specific for MART-1 at a 1:1 E:T ratio. After 1, 2, 3, and 24 h, the extent of trogocytosis in the cocultures was analyzed by flow cytometry. Target and effector cells were distinguished by CD3 staining in live single-cell populations. [Figure 7]T cells from the cocultures were further analyzed for activation by examining the upregulation of CD69. Antibody staining revealed increased CD69 expression on T cells in the pulsed cocultures. The signal remained elevated throughout the 24-hour window, while the signal background was low in the non-pulsed cocultures. [Figure 8] T cells from the cocultures were further analyzed for activation by examining the upregulation of CD137. Antibody staining revealed increased CD137 expression on T cells in the pulsed cocultures at 24 h. The signal in the non-pulsed cocultures remained low throughout the 24 h window. [Figure 9] T cells from the cocultures were further analyzed for degranulation by examining upregulation of CD107a. Antibody staining revealed increased CD107a expression on T cells in the pulsed cocultures, starting at 1 hour. The signal decreased over time and approached background signal of T cells in the non-pulsed cocultures at 24 hours. [Figure 10] HaloTag-β2M-HLA-A*02:01 293T cells were labeled with HTL AF660. After pulsing with NLV peptide in medium at 37°C for 1 hour, they were cocultured with donor T cells, of which approximately 1% had specificity for the CMV-derived epitope NLV. The E:T ratio was set at 1:1. After 3 hours, the extent of trogocytosis in these cocultures was analyzed by flow cytometry. Target and effector cells were distinguished by CD3 staining in live single-cell populations. T cells with specificity for the NLV-pHLA complex were revealed by dextramer staining (Figure 10A). In all cocultures (Figure 10B), a percentage of T cells became positive for the HaloTag-tagged protein upon trogocytosis. However, in pulsed cocultures, all T cells with specificity for the NLV epitope were also positive for HTL. [Figure 11]HaloTag-β2M-HLA-A*02:01 293T cells were labeled with HTL-46bp-Atto647N. After pulsing with MART-1 peptide in medium for 1 hour at 37°C, they were cocultured with donor T cells, of which approximately 1% had specificity for the tumor antigen MART-1. The E:T ratio was set at 1:1. After 14 hours, the extent of trogocytosis in these cocultures was analyzed by flow cytometry. Target and effector cells were distinguished by CD3 staining in live single-cell populations. T cells with specificity for the MART-1 pHLA complex were revealed by dextramer staining. The MFI derived from barcode-HTL was higher in pulsed dextramer-positive T cells compared with unpulsed cocultures. [Figure 12] T cells obtained from healthy donor subjects were supplied with azide-modified sugars to install azide groups on their cell surface, followed by strain-promoted alkyne-azide cycloaddition. After 48 hours, the cells were incubated with fluorescently labeled peptide-DBCO conjugates for 120 minutes. Flow cytometry analysis of single live cells demonstrated specific attachment of the peptide to azide-modified sugar-supplemented cells, but not to control T cells, across the concentration range tested. [Figure 13] Figures 13A and 13B: Transduced Jurkat cells and T cells obtained from healthy donor subjects were supplied with azide-modified sugars to install azide groups on their cell surface, which were then used in strain-promoted alkyne-azide cycloaddition. After 48 hours, cells were incubated with fluorescently labeled peptide-DBCO conjugates and DOTAM-DBCO at different ratios for 120 minutes. Flow cytometry analysis of single live cells showed high adhesion of both components at a 10:1 peptide:DOTAM ratio for T cells (A) and Jurkat cells (B). [Figure 14]Figures 14A and 14B: Transduced Jurkat cells were supplied with azide-modified sugars to install azide groups on their cell surface, which were then used in strain-promoted alkyne-azide cycloaddition. After 48 hours, cells were incubated with sortase A acceptor peptide-DBCO conjugate and DOTAM-DBCO for 120 minutes. Sortase A was tethered to effector cells using an antibody-enzyme conjugate against the hapten. 293T HaloTag-β2M-HLA-A*02:01 cells were labeled with biotin-labeled sortase donor peptide-HTL conjugate and pulsed to present the NY-ESO-1 antigen. Co-culture of cells was found to result in peptide import after 3 hours (A) and 16 hours (B), as determined by increased biotin staining on single, live effector cells. [Figure 15] Transduced Jurkat cells were supplied with azide-modified sugars to install azide groups on their cell surface, followed by strain-promoted alkyne-azide cycloaddition. After 48 hours, cells were incubated with sortase A acceptor peptide-DBCO conjugates and DOTAM-DBCO for 120 minutes. Sortase A was tethered to effector cells using an antibody-enzyme conjugate against the hapten. 293T HaloTag-β2M-HLA-A*02:01 cells were labeled with a biotin-labeled ssDNA peptide-HTL conjugate and pulsed to present the NY-ESO-1 antigen. Co-culture of these cells was found to result in peptide import after 16 hours, as determined by increased biotin staining on single live effector cells. [Example]

[0474] Example 1: Materials and Methods 1.1 Recombinant DNA Technology Standard methods were used to manipulate DNA as described by Sambrook et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Molecular biological reagents were used according to the manufacturer's instructions.

[0475] 1.2 DNA sequencing The DNA sequence was determined by double-stranded Sanger sequencing.

[0476] 1.3 Gene synthesis Desired gene segments were synthesized by Genscript Biotech (New Jersey, USA) from synthetic oligonucleotides and PCR products by automated gene synthesis, as needed. Gene segments flanked by single restriction endonuclease cleavage sites were cloned into standard cloning / sequencing vectors. Plasmid DNA was purified from transformed bacteria, and the concentration was measured by UV spectroscopy. The DNA sequences of the subcloned gene fragments were confirmed by DNA sequencing. Gene segments were designed with appropriate restriction sites to allow subcloning into the respective expression vectors.

[0477] 1.4 Generation of Halo-tagged cell lines To generate cell lines expressing HLA-A*02:01 containing HaloTag fusions on the cell surface, gene fusions of the HaloTag enzyme to human β2M linked to HLA-A*02:01 by a linker were generated by standard cloning techniques and subcloned into an appropriate expression vector. The amino acid sequences of the nascent and mature HaloTag-B2M-HLA-A02 fusion proteins are shown in SEQ ID NOs: 12 and 13.

[0478] A plasmid encoding the HaloTag-B2M-HLA-A02 fusion protein was introduced by lipofection into 293T cells modified by CRIPSR to knock out the endogenous HLA locus. Transfected clones were selected by standard antibiotic selection. The resulting clones were further selected based on positive antibody staining for HLA-A*02:01 and β2M, as determined by flow cytometry analysis. Selected positive clones were finally exposed to a series of HaloTag ligands (HTLs) containing the fluorophore tetramethylrhodamine (TMR; HTL TMR). Briefly, cells were detached, washed with PBS, and incubated with 1 x 10 cells in PBS with HTL TMR. 6 The cells were incubated at a density of 1000 cells / ml for 30 minutes. Cells were then washed with PBS and stained with a standard live / dead stain before analysis by flow cytometry. Events were gated based on forward and side scatter, followed by exclusion of doublets and dead cells. The median fluorescence intensity (MFI) of the cells was plotted against the concentration of HTL. Complete labeling was achieved for the final clones starting at 1 μM (Figure 1). Low background labeling of the parental cell line was observed at 1 μM.

[0479] 1.5 Attachment of DNA barcodes to cells via the HaloTag system The DNA oligonucleotide barcodes used in the 293T HaloTag-β2M-HLA-A*02:01 cell line were prepared as follows: A 69-bp oligo with a nucleotide sequence matching the consensus shown in SEQ ID NO: 18 was modified at the 3' end with a fluorophore (Atto655 or Cy3b) and conjugated at the 5' end to an HTL. The modified oligo was obtained from Biomers GmbH and is hereafter referred to as "DNA-HTL."

[0480] 293T HaloTag-β2M-HLA-A*02:01 cells were detached, washed with PBS, and then cultured at 1 × 10 6Cells were incubated with a dilution series of DNA-HTL in modified DPBS (Gibco, #14287080) at a cell density of 1000 cells / ml for 120 min. Cells were then washed with PBS and stained with a standard live / dead stain. Additionally, HTL AF488 chase was included in the PBS solution at 500 nM for 5 min. Cells were then analyzed by flow cytometry. Events were gated based on forward and side scatter, followed by exclusion of doublets and dead cells. The median fluorescence intensity (MFI) of DNA-HTL and chase was plotted against the concentration of DNA-HTL. Starting at a concentration of 10 μM, approximately 60-70% labeling was achieved (Figure 2).

[0481] 1.6 Preparation of virus-like particles (VLPs) Lipofectamine LTX™-based transfection was performed using approximately 70% confluent Lenti-X™ 293T cells (Takara, #632180) with a 2:1:2 molar ratio of the transfer vector-encoding construct and the packaging vectors pCAG-VSVG and psPAX2 (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 all experiments, mock virus-like particles (VLPs) were produced using only the packaging vector and no transfer vector. After 48 hours, the supernatant was collected and remaining cells were removed by centrifugation. VLPs were either used directly or stored at -80°C.

[0482] 1.7 Transduction of Jurkat cells 1 x 10 per well 6Jurkat cells were seeded into wells of a 24-well plate. VLPs were used fresh or thawed at 37°C. 300 μl was added to the 24-well plate along with 8 μg / ml polybrene (Sigma-Aldrich) and Lentiboost P (1:100) (Sirion Biotech, #SB-P-LV-101-12) and spin-transfected at 1100 × g for 99 minutes at 31°C. Cells were incubated at 37°C, 5% CO2 for at least 72 hours, after which transduction was assessed by flow cytometry.

[0483] 1.8 Isolation of primary T cells from blood Donor blood was provided by Blutspende Zurich (Rutistrasse 19, 8952 Schlieren). LeucoSEP tubes (Fisher Scientific, #10349081) containing 15 mL of room-temperature Histopaque density gradient medium (Sigma-Aldrich, #10771) were prepared and centrifuged at 400 × g for 5 minutes until the Histopaque passed through the filter. The blood was diluted with an equal volume of PBS. 30 mL of the blood / buffer mixture was added to the LeucoSEP tube. The tube was broken and centrifuged at 1200 × g for 20 minutes. The band containing peripheral blood mononuclear cells (PBMCs) was carefully pipetted into a new 50 mL Falcon tube and topped up to 50 mL with DPBS. The cells were washed three times with DPBS and finally resuspended in DPBS and counted. Pan T cell isolation was performed by negative selection using a Pan T cell isolation kit (Miltenyi, #130-096-535) according to the manufacturer's instructions. Cells were frozen or used immediately after isolation. Cells were cultured in modified RPMI (Gibco, #11530446), 10% FBS (Sigma, #F4135-500ML), 1% Glutamax (Gibco, #35050-038), 50 IU / ml Proleukin (Novartis), 25 ng / ml IL-7 (Miltenyi, #130-095-364), and 50 ng / ml IL-15 (Miltenyi, #130-095-766).

[0484] 1.9 Cellular Metabolic Glycoengineering and Strain-Promoted Alkyne-Azide Cycloaddition of Peptides and Small Molecules Cells were cultured for 24–48 hours in standard cell culture medium containing 50 μM N-azidoacetylmannosamine tetraacylated (ManNAz) to allow the click handles to be attached to the cell surface. Functionalized cells were then incubated with DOTAM-DBCO and / or peptide conjugates with the following structures: [6-FAM]-GGGGG-[CYS(DBCO-MAL)] or GGGGG-[CYS(DBCO-MAL)] in PBS at different concentrations and ratios for 2 hours.

[0485] 1.10 Production of IgG-like proteins in Expi293F cells Antibodies and bispecific antibodies were produced by transient transfection of Expi293F cells. Cells were grown in Expi293 medium (Gibco, 1435101) at a density of 2.5 x 10 6 Cells were seeded at 1000kJ / ml. The expression vector and ExpiFectamine (Gibco, ExpiFectamine Transfection Kit, #13385544) were mixed separately in OptiMEM (Gibco, #11520386). After 5 minutes, both solutions were combined, mixed by pipetting, and incubated at room temperature for 25 minutes. Cells were added to the vector / ExpiFectamine solution and incubated for 24 hours at 37°C in a shaking incubator with a 5% CO2 atmosphere. One day after transfection, supplements (Enhancer 1+2, ExpiFectamine Transfection Kit) were added. After 4-5 days, the cell supernatant was harvested by centrifugation and subsequent filtration (0.2 μm filter). Protein was purified from the harvested supernatant using standard methods as described below.

[0486] 1.11 Purification of IgG-like proteins Proteins were purified from filtered cell culture supernatants. Briefly, proteins were purified from cell culture supernatants by protein A or kappa 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). Elution was achieved at pH 3.0, followed immediately by neutralization of the sample pH. Proteins were concentrated by centrifugation (Millipore Amicon® ULTRA-15, #UFC903096), and aggregated proteins were separated from monomeric proteins by size exclusion chromatography in 20 mM histidine, 140 mM sodium chloride, pH 6.0.

[0487] 1.12 Analysis of IgG-like proteins The concentration of purified protein was determined by measuring absorbance at 280 nm using the mass extinction coefficient calculated based on the amino acid sequence according to Pace et al., Protein Science (1995) 4:2411-1423. Protein purity and molecular weight were analyzed by CE-SDS in the presence and absence of reducing agents using a LabChip GXII or LabChip GX Touch (Perkin Elmer). Aggregate content was determined by HPLC chromatography at 25°C using an analytical size-exclusion column (TSKgel G3000 SW XL or UP-SW3000) equilibrated in running buffer (200 mM KH2PO4, 250 mM KCl pH 6.2, 0.02% NaN3).

[0488] 1.13 Coupling of IgG with fluorescent dyes Purified IgG was labeled with fluorophores for subsequent use in flow cytometry analysis. Briefly, an appropriate amount of protein was labeled using a commercially available kit such as Alexa Fluor™ 647 Antibody Labeling Kit #A20186 (ThermoFisher). The resulting conjugate was analyzed to determine the degree of labeling, and the ideal amount of antibody used in the staining procedure was individually determined in a titration experiment.

[0489] 1.14 Peptide barcodes used in the HaloTag system A peptide with the structure "[biotin-Ahx]-SELPETGK" was conjugated to HTL via an esterification reaction. The resulting conjugate is hereafter referred to as "peptide HTL."

[0490] 293T HaloTag-β2M-HLA-A*02:01 cells were detached, washed with PBS, and then cultured at 3 × 10 6 The cells were incubated with 100 μM of peptide HTL in PBS at a cell density of 1000 cells / ml for 120 min. The cells were then washed with PBS and used in co-culture.

[0491] 1.15 DNA-peptide barcodes for use with the HaloTag system The DNA oligonucleotide peptide barcodes used in the 293T HaloTag-β2M-HLA-A*02:01 cell line were prepared as follows: A 69-bp oligo with a nucleotide sequence matching the consensus shown in SEQ ID NO: 18 was modified at the 3' end with "biotin-TEG" and at the 5' end with "DBCO-TEG." The modified oligo was obtained from IDT as an HPLC-purified product. A peptide with the structure "[Lys(N3)]-SELPETGK" was conjugated to HTL via an esterification reaction and then conjugated to the oligo via strain-promoted alkyne-azide cycloaddition. The resulting conjugate is hereafter referred to as "DNA-peptide HTL" and has the following structure: biotin-DNA-[DBCO / azide]-peptide-[amine / ester]-HTL (where "[DBCO / azide]" denotes the bond formed by strain-promoted alkyne-azide cycloaddition reaction between the DBCO and azide moieties, and "[amine / ester]" denotes the ester bond formed by the dehalogenase activity of HaloTag on the HaloTag ligand containing the chloroalkane moiety).

[0492] 293T HaloTag-β2M-HLA-A*02:01 cells were detached, washed with PBS, and then cultured at 3 × 10 6 The cells were incubated with 100 μM of DNA-peptide HTL in modified DPBS (Gibco, #14287080) at a cell density of 100 cells / ml for 120 minutes. Cells were then washed with modified PBS and used in co-culture.

[0493] Example 2: Conjugates of HaloTag Ligand Do Not Interfere with Immune Cell Activation We investigated whether the proximity of HTL conjugated to the 293T HaloTag-β2M-HLA-A*02:01 cell line to the TCR of immune cells interferes with proper synapse formation and activation. Potential steric and charge-based interference was assessed using a Jurkat activation assay. A Jurkat parent cell line engineered to express luciferase under the control of a TCR signaling-inducible promoter, TCR deleted, endogenously expressing CD4, and also engineered to express CD8a was obtained from Promega (#GA1162). The cells were further engineered to present the 1G4 TCR specific for NY-ESO-1 by lentiviral transduction. The assay was performed according to the provided protocol. Briefly, 293T HaloTag-β2M-HLA cells were detached, washed with PBS, and labeled with HTL AF660 and HTL-ssDNA-(69bp)-Atto655 under the optimized conditions 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 for 1 hour at 37°C. The labeled and pulsed cells were then co-cultured with Jurkat cell lines at a 1:1 ratio and 1 x 10 5 Cells / well were assayed. Assays were performed in 96-well white F-bottom chimney plates. After 7 hours, the provided substrate (Promega, #J3081) was added, and the resulting luminescence was read on a Tecan Spark multimode microplate reader. Plotting peptide concentration versus luminescence and fitting concentration-response curves using GraphPad Prism version 8 revealed no significant differences in fluorophore- or DNA-conjugated cells compared to cells bearing an empty HaloTag (Figure 3).

[0494] Example 3: Trogocytosis of HaloTagged HLA induced by synthetic immunization 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. At the artificial synapse, materials are transferred from the engaged target cell to the CD3-TCR complex-expressing cell by trogocytosis. A bispecific antibody recognizing WT-1 (Augsberger et al., Blood (2021) 138(25):2655-2669) was used to evaluate whether HaloTag-tagged HLA molecules could be transferred in this manner.

[0495] 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 antigen peptide "RMFPNAPYL" (SEQ ID NO: 15), which forms the epitope of the WT-1-binding arm of the bispecific antibody (described above). Next, cocultures of effector and WT-1 peptide-pulsed target cells with the bispecific antibody were set up in technical duplicates. The bispecific antibody concentration was fixed at 10 μg / ml, but the effector-to-target cell ratio was varied at 5:1, 1:1, and 1:5, with 5 × 10 cells per well in 100 μl of medium. 5 The total number of cells was constant. The resulting trogocytosis was evaluated by flow cytometry after 2 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 TMR-positive cells among all effector cells were exported and analyzed further. The WT-1-targeting bispecific antibody induced trogocytosis of HaloTag-β2M-HLA-A*02:01 from target to effector cells already after 2 hours (Figure 4) and again after 24 hours (Figure 5). Very low background staining was observed with the non-targeting control bispecific antibody DP47 after 2 hours (Figure 4) and 24 hours (Figure 5).

[0496] Example 4: Trogocytosis of HaloTagged HLA induced by TCR interactions in pools with a given specificity To test whether HaloTag-tagged HLA could also be transferred by TCR-driven trogocytosis, we used a polyclonal pool of T cells with specificity for the tumor-associated target MART-1. 293T HaloTag-β2M-HLA-A*02:01 cells were labeled with HTL TMR (as described above) and pulsed with the MART-1 antigen peptide "ELAGIGILTV" (SEQ ID NO: 16). Effector and target cells were then transferred at a 1:1 ratio and 5 × 10 cells per well. 5 Co-cultures were established with T cells. The resulting trogocytosis was analyzed by flow cytometry after 1, 2, 3, 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 TMR-positive cells among all effector cells were exported and further analyzed, along with the median fluorescence intensity of CD69, CD107a, and CD137. T cells indeed became positive for HaloTag-tagged molecules as early as 1 hour after the initiation of co-culture (Figure 6). MFI was consistently higher compared to unpulsed control cells over the entire period. Furthermore, T cells were activated, as evidenced by early CD69 upregulation (Figure 7) and CD137 upregulation after 24 hours (Figure 8). Finally, T cells exhibited degranulation, as evidenced by the appearance of CD107a on the cell surface (Figure 9).

[0497] Example 5: Trogocytosis of HaloTagged HLA induced by TCR interaction with donor T cells with more than one specificity To test whether HaloTag-tagged HLA could be transferred to T cells with a given specificity that are present at low frequency within a pool of T cells with diverse 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 coculture. 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). Effector and target cells were then cultured at a 1:1 ratio and 3 × 10 cells per well. 5 Co-cultures with 1000 T cells were established. The resulting trogocytosis was analyzed by flow cytometry after 3 hours. Target 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). While a specific fraction of T cells became positive for HaloTag regardless of peptide pulsing, in peptide-pulsed co-cultures, all NLV-reactive cells were HaloTag positive (Figure 10).

[0498] Example 6: Trogocytosis of ssDNA barcodes attached to HaloTag-tagged HLA induced by TCR interaction with donor T cells with more than one specificity To assess whether DNA conjugated to HaloTag-β2M-HLA-A*02:01 molecules could be transferred to low-frequency T cells within a pool of diverse specificities, donor-derived T cells were screened for the presence of HLA-A*02:01-restricted tumor-associated T cell epitopes, such as MART-1, by dextramer staining (Immudex, Denmark). These donor T cells were used for coculture. 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). Effector and target cells were then cultured at a 1:1 ratio and 3 × 10 cells per well.5 Co-cultures with 1000 cells were established. The resulting trogocytosis was analyzed by flow cytometry after 14 hours. Target 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). Pulsed cultures had higher MFI in the Atto647N channel of the MART-1-positive population compared to non-pulsed controls (Figure 11).

[0499] Example 7: Functionalization of glycoengineered cells to attach peptides to their surface Healthy donor T cells were functionalized with peptides attached to glycoproteins as follows. Cells were cultured for 48 hours in the presence of azide-modified sugars. As a result, the azide moiety was presented on the cell surface and thus available for strain-promoted alkyne-azide cycloaddition. Cells were incubated with different concentrations of fluorescently labeled peptide-DBCO conjugates for 2 hours. Washed cells were then analyzed by flow cytometry. Analysis of single live cells revealed specific and concentration-dependent attachment of the peptide compared to unmanipulated control cells (Figure 12).

[0500] Example 8: Functionalization of glycoengineered cells to attach small molecules and peptides to their surface Transduced Jurkat cells and healthy donor T cells were functionalized with peptides and small molecules attached to glycoproteins as follows. Cells were cultured for 48 hours in the presence of azide-modified sugars. As a result, the azide moiety was displayed on the cell surface and therefore available for strain-promoted alkyne-azide cycloaddition. Cells were incubated with different ratios of fluorescently labeled peptide-DBCO conjugates and / or DOTAM-DBCO for 2 hours, with the total concentration fixed at 10 μg / ml. DOTAM conjugates were detected using a fluorescently labeled DOTAM-specific antibody (an antibody formed from polypeptides having the amino acid sequences of SEQ ID NOs: 29 and 31, labeled with AF647). Washed cells were then analyzed by flow cytometry. Analysis of single live cells revealed specific and ratio-dependent attachment of peptides and haptens (Figure 13).

[0501] Example 9: Cell-to-cell peptide transfer by tethered sortase A in co-cultures Transduced Jurkat cells were functionalized with the peptide, and DOTAM was attached to the glycoprotein as described in Example 8. A modified version of sortase A was then tethered to the cells using an anti-DOTAM antibody-sortase A conjugate (formed from a polypeptide having the amino acid sequences of SEQ ID NOs: 30 and 32).

[0502] 293T HaloTag-β2M-HLA-A*02:01 cells were labeled with peptide HTL (described in Example 1.14) and pulsed with the NY-ESO-1 antigen peptide "SLLMWITQC" (SEQ ID NO: 14). Effector and target cells were then incubated at a 1:1 ratio and 3 x 10 cells per well. 5 Co-cultures were established with 1000 cells. The resulting peptide transfer was analyzed by flow cytometry after 3 and 16 hours. Within the population of live single cells, target and effector cells were distinguished by CD45 staining. A specific increase in peptide HTL on effector cells was detected by anti-biotin antibody staining and observed after 3 hours (Figure 14A) and 16 hours (Figure 14B) of co-culture.

[0503] Example 10: Transfer of barcode constructs between cells by tethered sortase A in co-cultures Transduced Jurkat cells were functionalized with the peptide, and DOTAM was attached to the glycoprotein as described in Example 8. A modified version of sortase A was then tethered to the cells using an anti-DOTAM antibody-sortase A conjugate (formed from a polypeptide having the amino acid sequences of SEQ ID NOs: 30 and 32).

[0504] 293T HaloTag-β2M-HLA-A*02:01 cells were labeled with DNA-peptide HTL (described in Example 1.15) and pulsed with the NY-ESO-1 antigen peptide "SLLMWITQC" (SEQ ID NO: 14). Effector and target cells were then incubated at a 1:1 ratio and 3 x 10 cells per well. 5 Co-cultures with cells were established. The resulting peptide transfer was analyzed 16 hours later by flow cytometry. Within the population of live single cells, target and effector cells were distinguished by CD45 staining. The specific increase in DNA-peptide HTL on effector cells was detected by anti-biotin antibody staining (Figure 15).

Claims

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

2. The polypeptide of claim 1, wherein the MHC polypeptide is β2 microglobulin.

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

4. 4. The polypeptide of claim 1, wherein the moiety that facilitates labeling of the polypeptide with an identifier moiety is or comprises a self-labeling protein tag.

5. The polypeptide of 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 of any one of claims 1 to 5, further comprising a sortase substrate motif.

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

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

9. 9. The polypeptide of any one of claims 1 to 8, comprising or consisting of an amino acid sequence having at least 70% amino acid sequence identity with SEQ ID NO: 11, 10, 13 or 12.

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

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

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

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

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

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

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

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

18. The cell of claim 17, which is an antigen-presenting cell (APC).

19. 1. A method of producing cells comprising MHC molecules labeled with an identifier moiety, comprising: (1) introducing into a cell the nucleic acid or nucleic acids according to claim 15; and (2) contacting the cell with 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 of (1) with an identifier moiety; A method comprising:

20. 1. A method for producing cells containing MHC:peptide complexes comprising MHC molecules labeled with a single-stranded DNA (ssDNA) moiety, the method comprising: (1) introducing into a cell a nucleic acid or nucleic acids encoding a polypeptide comprising (i) an amino acid sequence of β2 microglobulin and (ii) a HaloTag; (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 (3) contacting the cell with a HaloTag ligand comprising the ssDNA portion and a chloroalkane portion; A method comprising:

21. 1. A method for producing cells containing MHC:peptide complexes comprising MHC molecules labeled with a single-stranded DNA (ssDNA) moiety, the method comprising: (1) introducing into a cell a nucleic acid or nucleic acids encoding a polypeptide comprising (i) an amino acid sequence of β2 microglobulin, (ii) a sortase substrate motif, and (iii) a HaloTag; (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 (3) contacting the cell with a HaloTag ligand comprising the ssDNA portion and a chloroalkane portion; A method comprising:

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

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

24. 1. A method for identifying a T cell receptor (TCR) that binds to an MHC:peptide complex, comprising: (1) contacting a cell comprising the MHC:peptide complex of 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 TCRs that bind to the MHC:peptide complex.

25. 1. A method for identifying a T cell receptor (TCR) that binds to an MHC:peptide complex, comprising: (1) contacting (a) cells containing MHC:peptide complexes with (b) a population of T cells; (2) incubating the cells obtained after step (1) under conditions suitable for trogocytosis of the MHC:peptide complexes by T cells containing a TCR that binds to the MHC:peptide complexes; and (3) subsequently analyzing the cells obtained after step (2) to identify TCRs that bind to the MHC:peptide complex; Including, the MHC:peptide complex comprises an MHC molecule, the MHC molecule comprising a polypeptide comprising (i) an amino acid sequence of an MHC polypeptide and (ii) an identifier portion, the identifier portion being covalently attached to the polypeptide via a bond formed by a self-labeling protein tag.

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

27. 27. The method of claim 26, wherein the sortase is provided on the cell surface of a T cell comprising a polypeptide comprising a sortase acceptor motif.

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

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

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

31. 31. The method of claim 30, wherein the identifier moiety is covalently attached to the polypeptide via 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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