Peptide markers for tracking genetically modified cells

Abstract

To enable management of injected genetically modified cells, it is useful for the genetically modified cells to express a marker that can be used to detect such cells in a pool of unmodified cells. Some embodiments relate to marker proteins that include a TCR constant domain and an exogenous amino acid variant present in the TCR constant domain that includes a sequence that allows for detection and identification. Other embodiments relate to antibody epitopes bound to TCR chains. Both marker proteins and antibody epitopes can be used to track genetically modified cells.

Description

[Technical Field]

[0001] Cross-reference of related applications All applications claiming foreign or domestic priority are identified in the application data sheet submitted with this application and are incorporated herein by reference in accordance with 37 CFR 1.57.

[0002] Sequence listing reference This application is filed together with an electronic sequence listing. The sequence listing was prepared on December 20, 2021, and is provided as a file titled "NTBV.016WO.xml" with a size of 57,943 bytes. The information contained in the electronic sequence listing is incorporated herein by reference in its entirety. [Background technology]

[0003] Cell therapy involves injecting, transplanting, or implanting living cells into a patient. For example, in immunotherapy, this involves transplanting T cells that fight cancer cells through cell-mediated immunity, or transplanting stem cells to regenerate diseased tissue, in order to achieve therapeutic effects. [Overview of the project]

[0004] Some aspects described herein relate to labeled proteins comprising a TCR constant domain and an exogenous amino acid variant present within the TCR constant domain that contains a detectable and identifiable sequence.

[0005] In some embodiments, labeled proteins can be used for the detection, isolation, or reduction of genetically modified cells. The labeled protein, or protein marker, can function as a marker; by finding this marker, any cell containing the labeled protein can be detected, isolated, or reduced. The labeled protein may contain an epitope peptide that can be identified by a suitable antibody.

[0006] Some aspects described herein relate to labeling proteins used for the detection, isolation, or reduction of genetically modified T cells altered by the introduction of therapeutic TCR genes, wherein the labeling proteins are derived from the mouse TCR Cβ domain and introduced into the human TCR Cβ2 domain by mutating amino acids present in the human TCR Cβ2 domain.

[0007] In some embodiments, a labeling protein is provided for detecting, isolating, or reducing cells modified by a novel TCR gene for cancer treatment, comprising any one of the labeling proteins provided herein.

[0008] Some embodiments relate to a kit comprising an antibody or conjugate that recognizes a labeled protein, used to detect, isolate, or reduce genetically modified cells having any one of the labeled proteins described above.

[0009] Some embodiments relate to labeled proteins used for targeted delivery of one or more loads to genetically modified cells expressing such labeled proteins, and comprising any one of the labeled proteins described herein.

[0010] Some embodiments relate to a method for targeted delivery of one or more loadings to genetically modified cells expressing a labeled protein. The method comprises a) obtaining a complex comprising one or more loadings and a binder that specifically binds to the labeled protein, and b) bringing the complex into contact with the genetically modified cell.

[0011] Several embodiments relate to antibody epitopes that can be inserted into or constitute part of the TCR chain. In some embodiments, the epitope can be used to detect genetically modified cells expressing such an antibody epitope, where the antibody epitope is bound to the TCR chain or a chimeric antigen receptor. In some embodiments, the antibody epitope comprises a 2A peptide sequence, an HA.11 epitope tag, a FLAG epitope tag, a Myc epitope tag, or a V5 epitope tag. In some embodiments, the antibody epitope is inserted into the constant domain of the TCR chain but not into the variable domain of the TCR. In some embodiments, the antibody epitope is introduced by mutating amino acids at one or more positions on the TCR chain but not by adding exogenous additional amino acids to the TCR chain.

[0012] Some embodiments relate to antibody epitopes used to detect genetically modified T cells altered by the introduction of a therapeutic TCR gene, comprising any one of the antibody epitopes described above.

[0013] Some embodiments relate to antibody epitopes used to detect cells modified by novel TCR or CAR genes for cancer treatment, comprising any one of the antibody epitopes described above.

[0014] Some embodiments relate to kits used to detect genetically modified cells expressing any one of the antibody epitopes described above.

[0015] Some embodiments relate to a gene construct comprising a nucleotide sequence capable of expressing a 2A peptide sequence or a sequence at least 90% identical thereto, wherein the construct is configured to express multiple proteins from a single reading frame, and wherein the nucleotide sequence does not increase the size of the gene construct beyond 25 amino acids. As used herein, "gene" in the context of a related peptide refers to the nucleic acid sequence encoding that peptide sequence and nothing more.

[0016] Some embodiments relate to genetically modified cells expressing a labeled protein of any one of the foregoing embodiments.

[0017] These and other features, aspects, and advantages of the present invention will be more deeply understood by reference to the following description and the appended claims.

Brief Description of the Drawings

[0018] [Figure 1] Schematic diagram of a TCR constant domain epitope. [Figure 2A] Shows a schematic diagram of an antibody epitope where the antibody epitope is bound to the C-terminus of the TCR chain. [Figure 2B] Shows a schematic diagram of an antibody epitope where the antibody epitope is bound to the N-terminus of the TCR chain. [Figure 2C] Shows a schematic diagram of an antibody epitope where the antibody epitope is inserted into the TCR chain. [Figure 2D] Shows a schematic diagram of an antibody epitope used to link the TCRα chain and the TCRβ chain. The positions of the TCRα chain and the TCRβ chain may be interchanged. [Figure 3]This paper presents FACS analysis of human primary T cells in which the NY-ESO-1 1G4 TCR was knocked into the endogenous TRAC locus using various repair templates. The data show that, when using the full-length mouse Trbc2 sequence, the exogenous poly(A) signaling pathway is effective in inducing TCR expression, and that the H57 antibody can recognize cells expressing the full-length mouse Trbc2 sequence. [Figure 4] This paper presents FACS analysis of human primary T cells in which the NY-ESO-1 1G4 TCR was knocked into the endogenous TRAC locus using various repair templates. The data indicate that it is sufficient to incorporate 10 amino acid residues derived from the A and B strands of mouse Trbc2, as well as an FG loop, into the human TRBC2 sequence to enable TCR expression and recognition by the H57 antibody. [Figure 5] This paper presents FACS analysis of human primary T cells in which the NY-ESO-1 1G4 TCR was knocked into the endogenous TRAC locus using various repair templates. The data indicate that six amino acid residues and an FG loop derived from the mouse Trbc2 A strand are required to enable recognition by the H57 antibody. [Figure 6] This paper presents FACS analysis of human primary T cells in which the NY-ESO-1 1G4 TCR was knocked into the endogenous TRAC locus using various repair templates. The data indicate that it is sufficient to incorporate only six amino acid residues and an FG loop derived from the mouse Trbc2 A strand into the human TRBC2 sequence to enable TCR expression and recognition by the H57 antibody. [Figure 7] This image shows FACS analysis of human primary T cells in which the NY-ESO-1 1G4 TCR was knocked into the endogenous TRAC locus using various repair templates. The data demonstrate that human primary T cells modified to express the NY-ESO-1 1G4 TCR containing the T2A peptide sequence can be detected by an anti-2A peptide antibody. [Figure 8]This paper presents FACS analysis of human primary T cells in which the NY-ESO-1 1G4 TCR was knocked into the endogenous TRAC locus using various repair templates. The data show that human primary T cells modified to express the NY-ESO-1 1G4 TCR containing the T2A peptide sequence can be detected by an anti-2A peptide antibody, and that there is a correlation between the staining of this 2A peptide and the staining of TCR Vβ13.1. [Figure 9] This shows FACS analysis of human primary T cells transduced via retrovirus to express the CD19 CAR construct. The data demonstrate that human primary T cells modified to express the CD19 CAR construct containing the 2A peptide sequence can be detected by an anti-2A peptide antibody, and that there is a correlation between the staining of this 2A peptide and the staining of a transduction marker protein (Ly6G) present in the same construct. [Figure 10] The sequence of the immunogen used to produce the 2A peptide antibody 3H4 is shown (SEQ ID NO: 1). [Figure 11] The T2A peptide sequence is shown (SEQ ID NO: 2). [Figure 12] The P2A peptide sequence is shown (SEQ ID NO: 3). [Figure 13] The sequence of the human TCR Cβ2 domain containing a mouse TCR epitope detectable by the H57 antibody is shown (SEQ ID NO: 4). [Figure 14] The sequence of the human TCR Cβ2 domain linked to the T2A peptide epitope detectable by the 3H4 antibody is shown (SEQ ID NO: 5). [Figure 15] This shows a fragment of a T2A or P2A peptide sequence (SEQ ID NO: 6). [Figure 16] This shows a fragment of an E2A or F2A peptide sequence (SEQ ID NO: 7). [Figure 17] The sequence of the human TCR Cβ2 domain is shown (SEQ ID NO: 8). [Figure 18]This figure shows histograms of FACS analysis of huTRBC2-Mur6 1G4 TCR jarcut cells stained with labeled H57 antibody after pre-incubation without antibody, or after adding H57, H57-IL2, or H57-IL2v. The decrease in staining in this figure reflects the binding of the antibody or fusion protein during pre-incubation. [Figure 19] The histograms and antagonist concentration curves of FACS analysis of huTRBC2-Mur6 1G4 TCR primary T cells stained with labeled H57 antibody after pre-incubation without antibody, or with H57, H57-IL2, or H57-IL2v. The decrease in staining in this figure reflects the binding of the antibody or fusion protein during pre-incubation. [Figure 20] The histogram and binding concentration curves of FACS analysis of huTRBC2-Mur6 1G4 TCR primary T cells stained with a labeled antibody that recognizes IL2 are shown. [Figure 21] The FACS plots of stimulus responses, measured by CD25 and CD69 expressed in response to IL2, H57-IL2, or H57-IL2v fusion proteins, are shown. [Figure 22] This study shows CD25 and CD69 expression in response to changes in the concentration of IL2, H57-IL2, or H57-IL2v fusion protein. [Figure 23] This is a histogram of FACS analysis showing increased proliferation as measured by cell trace violet in response to IL2, H57-IL2, or H57-IL2v fusion protein. [Figure 24] This shows increased proliferation as measured by cell trace violet in response to changes in the concentration of IL2, H57-IL2, or H57-IL2v fusion protein. [Figure 25] This shows FACS plots regarding the binding of H57 to antibodies that bind to the human TCRβ constant domain in cells expressing muTRBC2, huTRBC1-Mur6, and huTRBC2-Mur6 1G4. [Figure 26A] This shows increases in IFN-γ and IL2 in primary T cells expressing muTRBC2, huTRBC2, and huTRBC2-Mur6 1G4 TCR. It also shows changes in IFN-γ concentration in response to changes in the concentration of NY-ESO peptides applied to target cells. [Figure 26B] This shows increased IFN-γ and IL2 in primary T cells expressing muTRBC2, huTRBC2, and huTRBC2-Mur6 1G4 TCRs. A dot plot shows the percentage of cells expressing the marker (IFN-γ) in each experiment with the highest peptide load. [Figure 26C] This shows increases in IFN-γ and IL2 in primary T cells expressing muTRBC2, huTRBC2, and huTRBC2-Mur6 1G4 TCR. It also shows changes in IL2 concentration in response to changes in the concentration of NY-ESO peptides applied to target cells. [Figure 26D] This shows increased IFN-γ and IL2 in primary T cells expressing muTRBC2, huTRBC2, and huTRBC2-Mur6 1G4 TCRs. A dot plot shows the percentage of cells expressing the marker (IL2) in each experiment with the highest peptide load. [Figure 27A] This shows the time course of target cell numbers when co-cultured with T cells expressing muTRBC2, huTRBC2, and huTRBC2-Mur6 1G4 TCR. [Figure 27B] The dot plots show the number of remaining related cells in each experiment after the longest co-culture period (72 hours). [Modes for carrying out the invention]

[0019] Specific features of the present invention have been referenced in the summary of the invention described above, the modes for carrying out the invention, and the claims described below. The disclosure of the present invention herein is understood to include all possible combinations of such specific features. For example, if a specific feature is disclosed in the context of a particular aspect or aspect of the present invention, or in the context of a particular claim, that feature may also be used, to the extent possible, in combination with other particular aspects and aspects of the present invention, and / or in the context of other particular aspects and aspects of the present invention, as well as in the present invention in general.

[0020] Cell therapy includes treatments that involve injecting or transplanting cells into a patient. Genetically modified T cells are one such material. To enable the management of injected genetically modified T cells, it is useful for genetically modified T cells to express markers that can be used to detect such T cells even in a pool of unmodified cells. Ideally, such markers can be used to isolate successfully modified T cells during the manufacturing process, or to track or reduce those T cells after administration to a patient. Furthermore, such markers must be non-immunogenic and constitute a natural part of the therapeutic gene construct. Such markers may be polypeptides, for example, that can form epitopes to which antibodies can bind.

[0021] Some embodiments described herein relate to labeled proteins that can be used for the detection, isolation, or reduction of genetically modified cells. The labeled protein, or protein marker, can function as a marker; by finding this marker, any cells containing the labeled protein can be detected, isolated, or reduced. In some embodiments, the labeled protein comprises a TCR constant domain and an exogenous amino acid variant present in the TCR constant domain containing a detectable and identifiable sequence.

[0022] Some embodiments relate to labeled proteins used for targeted delivery of one or more loads to genetically modified cells expressing such labeled proteins, and comprising any one of the labeled proteins described herein.

[0023] Some embodiments relate to antibody epitopes that can be inserted into or constitute part of a TCR chain. In some embodiments, the epitopes can be used to detect genetically modified cells expressing such antibody epitopes, where the antibody epitope is bound to a TCR chain or a chimeric antigen receptor. definition

[0024] Throughout this specification, the term “includes,” or variations such as “includes” or “contains,” is understood to mean including the element, integer, or step, or group of elements, integers, or steps being referred to, and not to exclude other elements, integers, or steps, or groups of elements, integers, or steps.

[0025] The following explanations of terms and methods are provided to better describe this disclosure and to guide those skilled in the art in implementing it. The singular forms “one,” “one kind,” and “this” refer to one or more unless the context makes it clear otherwise. For example, the term “contains a nucleic acid molecule” refers to one or more nucleic acid molecules and is considered equivalent to the phrase “contains at least one nucleic acid molecule.” The term “or” refers to one or more of the alternative elements described, or a combination of two or more elements, unless the context makes it clear otherwise. As used herein, “contains” means “includes.” Thus, “contains A or B” means “includes A, B, or A and B” without excluding any additional elements. Unless otherwise specified, if any of these definitions differ from any other definitions that may exist, the definitions provided herein shall prevail.

[0026] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure belongs. All identifiers (IDs) of the Human Gene Analysis Organization (HUGO Gene Nomenclature Committee (HGNC)) referred herein are incorporated in their entirety by reference. Similar or equivalent methods and materials may be used for the implementation or testing of this disclosure, but preferred methods and materials are listed below. The materials, methods, and examples are illustrative and not intended to limit the scope.

[0027] The term "T cell receptor" or "TCR" refers to a molecule present on the surface of T cells, or T lymphocytes, that recognizes antigens bound as peptides to major histocompatibility complex (MHC) molecules. The TCR is composed of two protein chains (i.e., a heterodimer). In humans, 95% of T cells have an alpha (α) chain and a beta (β) chain (encoded by TRA and TRB, respectively), while 5% of T cells have a gamma and delta (γ / δ) chain (encoded by TRG and TRD, respectively). This ratio changes during individual development and disease states (such as leukemia), and also varies by species. Each TCR chain consists of two extracellular domains: a variable (V) region and a constant (C) region. The constant region is close to the cell membrane and is followed by a transmembrane region and a short cytoplasmic tail, while the variable region binds to the peptide / MHC complex. The variable regions of both the TCRα and TCRβ chains contain three hypervariable complementarity-determining regions (CDRs) called CDR1, CDR2, and CDR3. In some embodiments, CDR3 is the primary antigen-recognition region. In some embodiments, the TCRα chain gene contains V and J, and the TCRβ chain gene contains V, D, and J gene segments that contribute to the diversity of the TCR. The constant domain of the TCR consists of short ligature sequences in which cysteine ​​residues form disulfide bonds, creating links between the two chains.

[0028] The terms “therapeutic TCR” or “therapeutic TCR gene” may refer to a specific combination of TCRα and TCRβ chains that mediates a desired function, such as enabling the host immune system to fight disease. Therapeutic TCR genes can be selected from in vitro mutant TCR chains expressed as recombinant TCR libraries by phages, yeast, or T cell-presenting systems. Therapeutic TCR genes may be autologous or allogeneic.

[0029] The term "cancer" refers to a malignant neoplasm exhibiting characteristic anaplasia with loss of differentiation ability, rapid growth rate, invasion into surrounding tissues, and metastatic potential. The term "cancer" includes diseases characterized by uncontrolled cell proliferation in the subject. In some embodiments, the terms "cancer" and "tumor" are used interchangeably. In some embodiments, the term "tumor" refers to benign, or non-malignant, growth.

[0030] As used herein, the term “nascent antigen” refers to an antigen derived from a tumor-specific genomic mutation. For example, a nascent antigen may arise from the expression of a mutant protein resulting from a non-synonymous single nucleotide mutation in a tumor sample, or from the expression of an alternate reading frame resulting from a mutagenic frameshift. Thus, nascent antigens may be associated with a pathological condition. In some embodiments, a “mutant protein” refers to a protein containing at least one amino acid different from the amino acid at the same position in the normal amino acid sequence. In some embodiments, a mutant protein includes the inclusion of amino acids resulting from insertions, deletions, substitutions, reading frame shifts, or any combination thereof, relative to the normal amino acid sequence.

[0031] An "antibody" is a polypeptide that contains at least one of the light or heavy chains of the immunoglobulin variable region and specifically recognizes and binds to an antigen epitope. In some embodiments, an antibody consists of a heavy chain and a light chain, and each chain has a variable weight (V H ) region and variable light (V L It has a variable region called the ) region.H Region and V L The regions together play a role in binding to the antigen recognized by the antibody. The term antibody includes intact immunoglobulins, as well as their variants and parts, such as Fab' fragments, F(ab)'2 fragments, and any other molecules derived from intact immunoglobulins.

[0032] Genetically modified cells are cells whose genetic structure has been altered using biotechnology. Such alterations include the transfer of genes within and across species to create improved or novel organisms. New DNA is obtained by isolating and copying the desired genetic material using recombinant DNA methods, or by artificially synthesizing DNA.

[0033] "Genetically modified T cells" are T cells whose genetic structure has been altered using biotechnology.

[0034] In this specification, the terms epitope peptide, epitope protein, and antibody epitope are used interchangeably. For individually named epitopes, the name may be replaced with the word epitope or a phrase containing the word epitope. For example, an epitope named "mur6" may refer to either huTRBC1-Mur6 or huTRBC2-Mur6, where appropriate in the context. Various aspects of labeled proteins

[0035] Some embodiments described herein relate to labeled proteins that can be used to detect, isolate, or reduce genetically modified cells. The labeled protein, or protein marker, can function as a marker; by finding this marker, any cells containing the labeled protein can be detected, isolated, or reduced. The labeled protein may contain an epitope peptide that can be identified by a suitable antibody. In some embodiments, the labeled protein can be used to stimulate cells. In some embodiments, the labeled protein is introduced into T cells to enable detection, isolation, or reduction of genetically modified T cells. In some embodiments, the labeled protein is introduced into T cells so that modified T cells can be stimulated. In some embodiments, the labeled protein is introduced into T cells to create a “handle” for delivering components including, but not limited to, cytokines, nucleic acids, and small molecules. In some embodiments, the labeled protein is not used for stimulation.

[0036] Schematic diagrams of several embodiments are shown in Figure 1. In some embodiments, the TCR constant domain shown in Figure 1 may be the constant domain of TCRα or TCRβ. The entire TCR constant domain or only a portion of the TCR constant domain may be used.

[0037] In some embodiments, the labeled protein comprises a TCR constant domain and an exogenous amino acid variant present in the TCR constant domain containing a detectable and identifiable sequence. In some embodiments, the amino acid variant is introduced into the TCR constant domain rather than the TCR variable domain to preserve the specificity and sensitivity of the TCR. In some embodiments, the antibody epitope is inserted into the constant domain of the TCR chain but not into the TCR variable domain. In some embodiments, the antibody epitope is introduced by mutating amino acids at one or more positions on the TCR chain, but not by adding exogenous additional amino acids to the TCR chain.

[0038] To be detectable and identifiable means that it can be discovered or identified by molecular biological techniques or other related techniques. For example, a labeled protein can be detected and identified if it binds to a specific antibody and can then be detected and identified by standard molecular biological techniques, including but not limited to flow cytometry, IHC, immunofluorescence microscopy, Western blotting, and ELISA. In some embodiments, labeled proteins are expressed on the cell surface and can therefore be used for extracellular antibody binding.

[0039] An exogenous amino acid variant refers to an amino acid sequence of the TCR constant domain that is not naturally found in the species. The exogenous amino acid variant may be an amino acid sequence derived from another species or an artificial sequence. Furthermore, the exogenous amino acid variant may be a continuous or discontinuous amino acid sequence. For example, in some embodiments, the TCR constant domain is derived from one species, and the exogenous amino acid variant is derived from another species. In some embodiments, the TCR constant domain is derived from humans, and the exogenous amino acid variant is derived from a non-human species. In some embodiments, the non-human species is the mouse.

[0040] Exogenous amino acid variants can be created by mutating specific amino acids in the TCR constant domain. For example, by mutating specific amino acids in the human TCR constant domain, a portion of the amino acid sequence of the human TCR constant domain can be altered to match the sequence of the mouse TCR. This method allows for the introduction of a mouse epitope into the human TCR constant domain to obtain a labeled protein while maintaining the total number of amino acids constituting the labeled protein to be the same as that of the natural human TCR constant protein. Therefore, one advantage of this method is that the size of the labeled protein does not become larger than that of the natural human TCR constant domain. In addition, because the labeled protein is incorporated into the TCRβ constant domain, it has the same stability and expression as the TCR itself. Furthermore, by introducing a selection of amino acids from the mouse TCR constant domain rather than using the full-length mouse TCR constant domain, it may be possible to reduce the immunogenicity of the labeled protein. In some embodiments, the mutation includes 10 amino acid mutations. In some embodiments, the mutation includes 6 amino acid mutations. In some embodiments, the mutation is introduced into only one of the two TCR constant domains.

[0041] Exogenous amino acid variants can also be introduced into the TCR constant domain by other methods. For example, a labeled protein can be created by inserting a portion of the amino acid sequence of the TCR constant domain of the first species into the TCR constant domain of the second species, which has the same number of amino acids, thereby substituting the sequence. Even with this method, the size of the labeled protein will not be larger than that of the natural TCR constant domain of the second species.

[0042] In some embodiments, the TCR constant domain includes a sequence encoded by the human T cell receptor β constant 2 (TRBC2) gene. In some embodiments, the exogenous amino acid variant includes a sequence of the mouse TCR β chain constant region (TCR Cβ domain). In some embodiments, the exogenous amino acid variant includes one or more of the following 10 amino acid mutations: K4R, F7T, Y37F, N106E, E108K, T110P, Q111E, D112G, R113S, A114P. In some embodiments, the exogenous amino acid variant includes two, three, four, five, six, seven, eight, nine, or ten of these mutations.

[0043] To rapidly identify genetically modified cells from unmodified cells at the single-cell level, antibody reagents that detect protein markers specific to modified cells are useful. Previously published studies have shown that hybrid mouse TCRs, in which the human TCR variable domain is conjugated to the mouse TCR constant domain, are functional and detectable by the anti-mouse TCR Cβ antibody H57 (Cohen et al., Journal of Cancer Research, 2006). As used herein, "H57" may refer to any antibody or its antigen-binding fragment that retains the selective binding characteristics described herein and has at least the H57 variable region. Since this antibody does not bind to the human TCR Cβ domain, it can identify only modified T cells expressing the mouse TCR. Disadvantages of mouse-forming such full-length TCR constant domains include the potential for increased immunogenicity of the introduced TCR gene (Davis et al., Clin Cancer Res, 2010), and the inability to in-frame knock-in therapeutic TCR genes into exons of the constant loci of human TCRα or TCRβ. To overcome these limitations, in some embodiments, a "minimized" mouse epitope-containing TCR Cβ domain is generated by swapping 10 amino acids in the sequence of the human TRBC2 gene (K4R, F7T, Y37F, N106E, E108K, T110P, Q111E, D112G, R113S, A114P, where the numbering follows that of the TRBC2 gene (ENSG00000211772) in the Ensembl genome browser). In some embodiments, the numbering of these residues refers to the numbering sequence in Sequence ID No. 8. Therefore, the reference "as numbered in Sequence ID No. 8" refers not to the sequence itself, but to the method of numbering the amino acid positions within Sequence ID No. 8. Of these 10 amino acids, K4 and F7 are part of the A strand of TCR Cβ, Y37 is part of the B strand of TCR Cβ, and N106-A114 are part of the FG loop of TCR Cβ (Sasada et al., Journal of Experimental Medicine (J Exp Med), 2002).In several embodiments, a “minimized” mouse epitope-containing TCR Cβ domain was generated by substituting six amino acids in the human TRBC2 gene sequence (K4R, E108K, T110P, Q111E, D112G, R113S) (“huTRBC2-Mur6”). These mouse epitope-containing TCRβ chains efficiently pair with full-length human TCRα chains and are detected on the cell surface by the H57 antibody, enabling the detection, isolation, and reduction of TCR-modified T cells. Furthermore, these mouse epitope-containing TCRβ chains are also suitable for in-frame knock-in of therapeutic TCR genes into exons of human TCRα or TCRβ constant loci.

[0044] In some embodiments, exogenous amino acid variants can be detected and identified by antibodies, nanobodies, Fab fragments, or Darpin. Nanobodies, also known as single-domain antibodies, are antibody fragments consisting of a single monomeric variable antibody domain that, like the whole antibody, can selectively bind to specific antigens. Darpin (an acronym for designed ankyrin repeat proteins) is a genetically modified antibody-mimetic protein that typically exhibits high specificity and high affinity for binding to labeled proteins; Darpin is derived from naturally occurring ankyrin proteins, a group of proteins that mediate high-affinity protein-protein interactions in nature. In some embodiments, exogenous amino acid variants contain antibody epitopes that can bind to certain antibodies and be detected and identified by such antibodies. Epitopes may be continuous or discontinuous sequences. In some embodiments, the 4th and 108-113th positions of TRBC2 are mouse-derived. Detection of TCR-modified T cells by labeled proteins in some embodiments is based on antibody staining detectable by flow cytometry.

[0045] In some embodiments, exogenous amino acid variants can be detected and identified by the anti-mouse TCR Cβ antibody H57. H57 specifically binds to the mouse TCR Cβ domain but not to the human TCR Cβ domain. By introducing a mouse epitope detectable by H57 into the human TCR Cβ domain, the resulting labeled protein can be distinguished from proteins with the native human TCR Cβ domain. Any antibody epitope domain present in the constant domains of other species of TCRα or TCRβ (TRAC and TRBC, respectively), such as an antibody epitope detectable by the mouse anti-rat TCRαβ antibody R73, may be used in a similar manner.

[0046] In some embodiments, an artificial antibody epitope may be created using targeted amino acid mutations in human TRAC or TRBC, and an antibody against it may be produced.

[0047] In some embodiments, labeled proteins are provided. These labeled proteins can be used to detect, isolate, or reduce genetically modified cells expressing such labeled proteins, and include any of the labeled proteins described above. In some embodiments, genetically modified cells include genetically modified T cells. In some embodiments, genetically modified T cells include T cells modified by the introduction of a therapeutic TCR gene. The term “therapeutic TCR gene” may refer to a specific combination of TCRα and TCRβ chains that mediates a desired function, such as enabling the host immune system to fight disease.

[0048] Currently available techniques for detecting TCR-modified T cells have various drawbacks. Typically, the expression of TCR proteins in TCR-modified T cells is detected by antibodies specific to MHC multimers or the TCR V domain (Altman et al., Science, 2006; Faint et al., Journal of Immunological Methods, 1999). In addition, TCR Cβ domain-specific antibodies have been used to detect TCRs possessing the human TRBC1 domain (clone IP26; Schober et al., Nat Biomed Eng, 2019), or the mouse Trbc1 / 2 domain (clone H57; Mall et al., Journal of the American Cancer Society, 2016), as pan-αβ TCR antibodies. However, MHC multimers must be specifically generated for the specificity of each TCR and cannot be used for specific TCRs due to HLA allele constraints; TCR V domain-specific antibodies can only be used for specific TCR V domains; and TCR V domain-specific and TCR Cβ domain-specific antibodies cannot be used when TCR-modified T cells are present in a pool of other T cells that utilize the same TCR V domain or TCR Cβ domain. In addition, the use of the full-length mouse TCR constant domain is undesirable because a large mouse protein sequence may increase immunogenicity; furthermore, therapeutic TCR gene constructs based on the full-length mouse TCR constant domain cannot be integrated in-frame into the human TCRα or TCRβ constant gene loci, and therefore need to be co-delivered with an exogenous polyA signal for efficient expression. As a result, the size of the gene template required for the full-length mouse TCR constant domain increases, which may negatively affect the gene transfer efficiency when using site-directed knock-in approaches.

[0049] Similarly, currently available techniques for isolating TCR-modified T cells also have various drawbacks. Such techniques utilize isolation methods that employ some kind of antibody (e.g., flow cytometry or magnetic bead isolation). Most commonly, TCR-modified T cells are isolated using MHC multimer-based reagents (Knabel et al., Nature Medicine, 2002). As mentioned earlier, MHC multimers need to be specifically generated for the specificity of each TCR and cannot be used for specific TCRs due to HLA allele constraints. Other isolation methods include: - Utilization of antibody epitopes positioned within the TCR antigen-binding domain (for example, described by Kieback et al., Proceedings of the National Academy of Sciences, 2008). Amino acid addition or modification may alter the structure of the binding domain, potentially interfering with the fine specificity of the TCR antigen and the sensitivity of the TCR. Therefore, this concept requires research to be implemented for each individual TCR, and it is highly likely that it will affect antigen binding for some TCRs. - Use of antibodies specific to the TCR V domain or human TRBC1. As mentioned above, such reagents can only be used for specific TCR V domains and cannot be used when TCR-modified T cells are present in a pool of other T cells that utilize the same TCR V domain or human TRBC1. - The use of cell surface marker proteins expressed in conjunction with therapeutic TCR genes, such as co-expression of cleaved LNGFR or EGFR proteins. However, including such cell surface markers increases the size of the transgene, which may impair the efficiency of gene modification and subsequent transgene expression, as well as potentially increase the immunogenicity of the modified T cells.

[0050] In addition, methods for reducing TCR-modified T cells based on currently available technologies also have various drawbacks. Numerous "safety switches" for adoptive T cell therapies, including TCR therapy and CAR therapy, have been reported, and some of the reported systems include: - Utilization of antibody epitopes positioned within the TCR antigen-binding domain (see, for example, Keeback et al., Proceedings of the National Academy of Sciences, 2008). This concept needs to be tested for each individual TCR, and for some TCRs, it is likely to affect antigen binding. - The use of further transgenes that can be selectively activated in vivo, such as herpes simplex virus TK (Bonini et al., Science, 1997; Ciceri et al., Lancet Oncology, 2009) or inducible caspase 9 (Straathof et al., Blood, 2005; DiStasi et al., New England Journal of Medicine, 2011). However, including such markers increases the size of the transgene that needs to be introduced into the cell, which may impair the efficiency of gene modification and subsequent transgene expression.

[0051] Compared to currently available technologies, some embodiments provided herein can offer one or more of the following advantages: 1. Low immunogenicity: In some embodiments, a limited number of amino acid mutations are introduced into the human TRBC domain. As described by Cohen et al., Journal of the American Cancer Society, 2006, the full-length mouse Trbc2 gene contains 33 different amino acids compared to the human TRBC2 gene. On the other hand, as described in some embodiments, the mouse epitope-containing TRBC2 gene contains only 10 or fewer mouse-derived amino acids that are different from those of human, for example, 9, 8, 7, 6, 5, 4, 3, or 2. 2. Broad and universal utility for all human TCRs: Unlike alternative technologies such as TCR V domain-specific antibodies, MHC polymers, or modifications of the TCR framework, some embodiments described herein can be used for all TCRs, regardless of the use of the TCR V domain, MHC restriction, and antigen specificity. 3. Detection with high specificity: In some embodiments, TCR-modified T cells can be detected with high specificity without genetically knocking out the endogenous TCR chain. Alternative technologies, such as TCR V domain-specific antibodies or human TRBC1-specific antibodies, react specifically with a large portion of the endogenous TCR chain, which can hinder the accurate detection, specific isolation, or reduction of TCR-modified T cells. 4. Therapeutic TCR genes can be knocked in-frame into the exons of the human TCR locus: For example, the knock-in of a therapeutic TCR construct using the full-length mouse-derived Trbc gene into the TCRα locus, as described by Schober et al. (Nature Biomedical Engineering, 2019), requires co-delivery of the full-length mouse-derived Trac gene and the poly(A) sequence of bovine growth hormone as an exogenous transcription termination factor. In contrast, TCRβ chains utilizing the mouse epitope-containing TRBC2 gene, as described in some embodiments, can efficiently pair with full-length human TCRα chains, thus enabling in-frame knock-in into the TCRα locus exon and compatibility with endogenous transcription termination factors. This means that the size of the therapeutic gene construct can be reduced by approximately 500 base pairs, thereby increasing the efficiency of the gene modification process. 5. Avoiding a substantial increase in the size of the therapeutic TCR gene cassette: Some embodiments are based on mutating several amino acids in a selected human TCR constant domain to their mouse counterparts. Alternative techniques used for the detection, isolation, and / or reduction of TCR-modified T cells include the use of additional proteins (e.g., cleaved EGFR, cleaved LNGFR, HSV-TK, and inducible caspase 9). This can increase the size of the delivered transgene, potentially impairing the efficiency of the gene modification and subsequent transgene expression. Furthermore, including additional proteins expressed on the cell surface in the transgene may enhance the immunogenicity of the modified T cells.

[0052] In some embodiments, T cells modified by the introduction of therapeutic TCR genes are used to treat cancer. In some embodiments, the cancer is a solid tumor. The therapeutic TCR gene can be selected from TCR chains that have been mutated in vitro and expressed as recombinant TCR libraries by phages, yeast, or T cell presentation systems. The therapeutic TCR gene may also refer to a newly generated antigen-specific TCR gene derived from an individual patient-specific cancer biopsy. After identification, such a newly generated antigen TCR gene is introduced into the patient's T cells by genetic engineering, thereby directing the T cells' antigen specificity to the tumor-generated antigen. Finally, the genetically modified T cells are returned to the patient by intravenous infusion to treat the cancer. These embodiments can be applied to all cancers suitable for modified adoptive T cell therapy.

[0053] In some embodiments, a mouse epitope is introduced into the human TCR Cβ domain to incorporate a labeled protein into a therapeutic TCR gene construct, allowing the TCR introduced into genetically modified T cells to be detected by the anti-mouse TCR Cβ antibody H57. H57 is an antibody first reported by Kubo et al. in the Journal of Immunology (J Immunol), 1989. In some embodiments, H57-597 is used. H57-597 is a hamster mAb that targets the C-region epitope of the TCRβ chain. The H57-597 antibody does not cross-react with T cells having γ / δ TCRs. Immobilized or soluble H57-597 antibodies can activate T cells having α / β TCRs. Some aspects described herein relate to labeling proteins used to detect, isolate, or reduce genetically modified T cells that have been altered by the introduction of therapeutic TCR genes, wherein the labeling proteins are derived from the mouse TCR Cβ domain and introduced into the human TCR Cβ2 domain by mutating amino acids present in the human TCR Cβ2 domain.

[0054] In some embodiments, a labeling protein is provided for detecting, isolating, or reducing cells modified with a novel TCR gene for cancer treatment, comprising any one of the labeling proteins provided herein.

[0055] Several embodiments relate to a kit comprising an antibody or conjugate that recognizes a labeled protein, used to detect, isolate, or reduce genetically modified cells having any one of the labeled proteins described above. In some embodiments, the kit comprises magnetic beads conjugated to an H57 antibody, which can be used to isolate or reduce T cells expressing a mouse epitope-containing TCRβ chain. In some embodiments, the kit comprises a stain for detecting genetically modified cells in tissue. In some embodiments, the genetically modified cells include T cells. In some embodiments, the T cells are modified by the introduction of a therapeutic TCR gene. In some embodiments, the T cells modified by the introduction of a therapeutic TCR gene are used to treat cancer. Various aspects of labeled proteins used for targeted delivery

[0056] Some embodiments relate to labeled proteins used for targeted delivery of one or more loads to genetically modified cells expressing such labeled proteins, and comprising any one of the labeled proteins described herein.

[0057] In some embodiments, delivery of one or more loaders is achieved by conjugation to an antibody, antibody-mimicking protein, or any other antigen-binding scaffold. In some embodiments, delivery of one or more loaders is achieved by conjugation to an H57 antibody.

[0058] In some embodiments, the delivered payload is a protein, small molecule, nucleic acid, liposome, or nanoparticle. In some embodiments, the payload is a bispecific or trispecific antibody.

[0059] In some embodiments, the delivered cargo is a cytokine. In some embodiments, the delivered cargo is selected from the group consisting of IL-1β, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, IL-23, IL-27, IFN-α, IFN-β, IFN-γ, and TNF-α. In some embodiments, the cytokine is modified so that its sequence modifies its interaction with its native receptor molecule.

[0060] In some embodiments, the loader is an agonist or antagonist of a receptor molecule expressed by T cells. In some embodiments, the agonist binds to CD27, CD28, CD137, or CD278. In some embodiments, the antagonist is the TGF-β receptor, PD-1, CTLA-4, Vista, steroid receptor, or A1-, A 2A -, A 2B -or it binds to the A3-adenosine receptor.

[0061] In some embodiments, the loaded molecules are small molecules that regulate T cell activation, differentiation, proliferation, survival, or effector function. In some embodiments, the small molecules are TGF-β receptors, PD-1, CTLA-4, VISTA, steroid receptors, or A1-, A 2A -, A 2B - Or inhibits either A3-adenosine receptor signaling.

[0062] In some embodiments, the load-bearings are nucleic acids that regulate the activation, differentiation, proliferation, survival, or effector function of T cells. In some embodiments, the nucleic acids include miRNA, shRNA, or siRNA.

[0063] Some embodiments relate to a method for targeted delivery of one or more loadings to genetically modified cells expressing a labeled protein. The method comprises a) obtaining a complex comprising one or more loadings and a binder that specifically binds to the labeled protein, and b) bringing the complex into contact with the genetically modified cell.

[0064] In some embodiments, the binding agent is an antibody, an antibody-mimicking protein, or any other antigen-binding scaffold.

[0065] In some embodiments, one or more loads are proteins, small molecules, nucleic acids, liposomes, or nanoparticles.

[0066] As recognized herein, there are various drawbacks to the techniques for targeted delivery of payloads to modified T cells based on currently available technologies. Most techniques target either mouse T cells or unmodified human T cells using antibodies that bind to endogenous markers present on the cell surface (e.g., CD3e or PD-1). Some studies have reported targeting fluorescent liposomes conjugated with anti-Thy1.1F(ab')2 fragments to adoptively transferred mouse T cells by conjugating anti-Thy1.1F(ab')2 fragments to pegylated liposomes labeled with the lipophilic tracer fluorescent dye DiD (Zheng et al., Journal of Controlled Release, 2013). Another study reported targeting TGFβ receptor inhibition to adoptively transferred mouse T cells by conjugating anti-Thy1.1F(ab')2 fragments to pegylated liposomes loaded with the TGFβR1 inhibitor SB525334 (Zheng et al., ACS Nano, 2017). In addition, there is a report of targeting TGFβ receptor inhibition to mouse T cells using nanoparticles based on PLGA that are pegylated, loaded with the TGFβR1 inhibitor SD-208, and conjugated with anti-PD-1F(ab')2 fragments (Schmid et al., Nature Communications, 2017). There is also a description of targeting a CAR DNA construct to mouse T cells using nanoparticles loaded with two DNA plasmids encoding anti-CD3εF(ab')2 fragments, a plasmid encoding a CD19 CAR gene adjacent to a piggyBac inverted repeat, and a plasmid encoding a hyperactive piggyBac iPB7 transposase gene (Smith et al., Nature Nanotechnology, 2017). In addition, Foxo1 3AThere are reports of targeting the mRNA encoding a mutant transcription factor to human T cells by conjugating an anti-CD3ε antibody onto nanoparticles (Moffett et al., Nature Communications, 2017). In addition, there is a report of targeting TGFβ receptor inhibition to mouse T cells by conjugating anti-CD8α VHH nanobodies onto amphiphilic gold nanoparticles loaded with the TGFβR1 inhibitor SB525334 (Yang et al., Biomaterials Science, 2019). However, these technologies do not provide a solution for targeting and delivering loaders to modified human T cells used in adoptive T cell therapy.

[0067] There is also a technique that enables the targeting of IL-2 cytokine activity to human T cells and NK cells using an anti-PD-1 antibody fused to a mutant IL-2 polypeptide (IL2v) that has been modified to bind to IL-2Rβγ but not to IL-2Rα (WO / 2018 / 184964, 2018). Other studies describe the targeting of the cytokine type I interferon activity to mouse T cells and cDC1 DCs by fusing an anti-CD8α antibody with a mutant human IFN-α that has been modified to have approximately 1 / 100th the activity on mouse cells compared to wild-type mouse IFN-α (Huyghe et al., EMBO Mol Med, 2020). In addition, there have been reports of targeting the activity of the cytokine IL-21 to human T cells by fusing an anti-PD-1 antibody with a mutant IL-21 polypeptide modified to have less than 1 / 1000th the activity of wild-type free IL-21 (Shen et al., Frontiers in Immunology, 2020). However, these technologies do not provide a solution for targeting and delivering cytokine activity to modified human T cells used in adoptive T cell therapy.

[0068] Several studies have targeted modified human CAR T cells using Darpin, which binds to introduced truncated HER2 marker genes. This involves fusing Neo-2 / 15 and IL-2 and IL-15 mimics, modified to bind to IL-2Rβγ but not IL-2Rα, with anti-HER2 Darpin G3 to target the cytokines IL-2 and IL-15 to human CAR T cells expressing truncated HER2 as transduction marker genes (Leung et al., AACR2020, abstract 2222, 2020). However, since modified human T cells used in adoptive T cell therapy do not co-introduce additional marker genes in addition to the therapeutic TCR or CAR gene, this method does not provide a solution for targeted delivery of cytokine activity to modified human T cells used in adoptive T cell therapy.

[0069] Some embodiments described herein can overcome one or more of the aforementioned drawbacks. Various embodiments described herein target modified human T cells while maximizing gene editing efficiency without requiring the introduction of further marker genes, and thus without increasing the size of the therapeutic gene cassette.

[0070] In some embodiments, the TCR Cβ domain mouse epitope configuration differs from the aforementioned techniques in that it does not require the introduction of further marker genes, and therefore can target modified human T cells while assisting gene editing efficiency without increasing the size of the therapeutic gene cassette. Various aspects of 2A peptide epitopes

[0071] Some embodiments relate to antibody epitopes that can be inserted into or constitute part of the TCR chain. In some embodiments, the epitopes can be used to detect genetically modified cells expressing such antibody epitopes, where the antibody epitope is bound to the TCR chain or a chimeric antigen receptor. In some embodiments, the genetically modified cells include a nucleotide construct encoding a peptide containing the antibody epitope. In some embodiments, the detection of the genetically modified cells is based on antibody staining detectable by flow cytometry. The antibody can specifically bind to and recognize the antibody epitope.

[0072] As shown in Figures 2A-2D, in some embodiments, the antibody epitope is bound to the C-terminus or N-terminus of the TCR chain, where the TCR chain may be either a constant or variable chain. In other embodiments, the antibody epitope is inserted into the TCR chain. In some embodiments, the antibody epitope is used to link the TCRα and TCRβ chains. In some embodiments, the arrangement of the antibody epitope depends on the order in which the TCR chains are expressed. In some embodiments, genes other than TCRs may be included that encode proteins having the antibody epitope sequence. An example is a chimeric antigen receptor (CAR) transgene that is fused to a second transgene to regulate T cell function. In some embodiments, two or more antibody epitopes may be included. In some embodiments, there may be two, three, four, five, six, seven, eight, nine, or ten antibody epitopes, where each antibody epitope may be the same or different. In these embodiments, any antibody epitope can be used, as long as an antibody capable of specifically detecting the epitope exists. The antibodies used in some embodiments are suitable for use in flow cytometry. In some embodiments, the antibody epitope is localized intracellularly because it binds to the C-terminus of the TCR constant domain. This reduces immunogenicity because the antibody epitope cannot utilize the antibody-mediated immune response. In some embodiments, the antibody epitope is bound to the TCR constant domain to avoid interference with the specificity and sensitivity of the TCR.

[0073] In some embodiments, genetically modified cells include T cells. In some embodiments, T cells are modified by the introduction of therapeutic TCR or CAR genes. In some embodiments, T cells modified by the introduction of therapeutic TCR or CAR genes are used to treat cancer. The term “therapeutic TCR gene” may refer to a specific combination of TCRα and TCRβ chains that mediates a desired function, such as enabling the host immune system to fight disease.

[0074] In some embodiments, the antibody epitope comprises a 2A peptide sequence, an HA.11 epitope tag, a FLAG epitope tag, a Myc epitope tag, or a V5 epitope tag. These epitope tags can be specifically detected using flow cytometry-compatible antibodies. In some embodiments, the antibody epitope comprises a peptide containing up to the same number of amino acids as the 2A peptide. Those skilled in the art will understand that any epitope tag may be used to construct an antibody epitope, as long as it contains up to the same number of amino acids as the 2A peptide.

[0075] 2A peptides, or 2A self-cleaving peptides, are peptides derived from viruses that are 18 to 22 amino acids long. In life science research, four members of the 2A peptide family are often used: P2A, T2A, E2A, or F2A. P2A is derived from porcine teschovirus-1 2A; T2A is derived from thosea asigna virus 2A; E2A is derived from equine rhinitis A virus; and F2A is derived from foot-and-mouth disease virus 18.

[0076] In some embodiments, the 2A peptide may be P2A, T2A, E2A, or F2A. In some embodiments, the 2A peptide serves two purposes: firstly, to enable the linking of the expression of two protein sequences; and secondly, to enable the detection of at least one of those two protein sequences. As used herein, “gene” means the nucleotide sequence encoding one of the peptides, such as the 2A peptide, that peptide (e.g., the 2A peptide). This is not intended to refer to the sequence of a naturally occurring gene, but rather to an abbreviated representation of the nucleic acid sequence encoding the relevant peptide.

[0077] In some embodiments, the antibody epitope comprises a 2A peptide sequence fragment or a sequence that is at least 90% identical thereto, where the 2A peptide sequence is not a full-length 2A sequence.

[0078] In some embodiments, the peptide sequence includes the sequence of SEQ ID NO: 1 (CGDVEENPG). In some embodiments, the peptide sequence includes the sequence of SEQ ID NO: 6 (GDVEENPG). In some embodiments, the peptide sequence includes the sequence of SEQ ID NO: 7 (GDVESNPG). In some embodiments, the peptide sequence includes a sequence that is at least 75% identical to SEQ ID NO: 1.

[0079] In some embodiments, antibody epitopes can be identified by the monoclonal anti-2A peptide antibody 3H4. 3H4 is an antibody recently developed by Novus Biologicals and can be used in Western blotting (Yu et al., Viruses, 2020), immunoprecipitation, and immunohistochemistry / immunofluorescence.

[0080] Some embodiments relate to antibody epitopes used for detecting genetically modified T cells altered by the introduction of a therapeutic TCR gene, wherein the antibody epitope comprises one of the antibody epitopes described above.

[0081] Some embodiments relate to antibody epitopes for detecting cells modified with novel TCR or CAR genes for cancer treatment, comprising any one of the antibody epitopes described above.

[0082] Some embodiments relate to kits used to detect genetically modified cells expressing any one of the antibody epitopes described above. In some embodiments, the genetically modified cells include T cells. In some embodiments, the T cells are modified by introducing a therapeutic TCR gene. In some embodiments, the T cells modified by introducing a therapeutic TCR gene are used to treat cancer. In some embodiments, the kit includes an antibody or conjugate that specifically binds to the antibody epitope. In some embodiments, the antibody or conjugate may include a fluorescent or detectable marker.

[0083] Some embodiments relate to a gene construct comprising a gene capable of expressing a 2A peptide sequence or a sequence at least 90% identical thereto, wherein the construct is configured to express multiple proteins from a single read frame, and the gene does not exceed 25 amino acids in size of the gene construct.

[0084] In some embodiments, the 2A peptide sequence comprises a 2A peptide sequence fragment or a sequence that is at least 90% identical thereto. This 2A peptide sequence is not a full-length 2A sequence. In some embodiments, the 2A peptide sequence comprises the sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6, or a sequence that is at least 75% identical to SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 6 (SEQ ID NO: 2=EGRGSLLTCGDVEENPGP; SEQ ID NO: 3=ATNFSLLKQAGDVEENPGP; SEQ ID NO: 6=GDVEENPG).

[0085] In cell therapy using genetically modified T cells, it is necessary to redirect the antigen specificity of these T cells. To redirect the antigen specificity of T cells, it is necessary to introduce both exogenous TCRα and TCRβ chain sequences. Therefore, to maximize the efficiency of gene modification, the therapeutic TCR gene construct must be able to easily express both TCR genes simultaneously. One way to achieve this is to use a 2A self-cleaving peptide sequence that allows the co-expression of two proteins in equimolar ratios from a single read frame (Ryan and Drew, EMBO J, 1994). Notably, TCR transgenes based on 2A peptides show higher expression and functionality than TCR transgenes based on IRES (Leisegang et al., Journal of Molecular Medicine, 2008), which is thought to be due to the higher pairing ability of the introduced TCR chains.

[0086] In some embodiments, the TCRα and TCRβ chains are co-expressed in therapeutic TCR gene constructs using an intervening epitope (e.g., marker or label) sequence, such as a 2A peptide sequence, covalently bound to the TCR Cα or TCR Cβ domain (depending on the order of expression). Since the 2A peptide sequence is not normally present in mammalian cells, such peptides can also function as protein markers for tracking genetically modified T cells. To distinguish modified T cells from unmodified T cells, antibody reagents capable of detecting modified T cells at the single-cell level can be used. Flow cytometry-based staining procedures have been developed that enable the detection of therapeutic TCR genes using the monoclonal anti-2A peptide antibody 3H4. Because the 2A peptide marker is covalently bound to the intracellular domain of the TCRβ chain, it is important to perform this staining procedure in membrane-permeable cells. These 2A peptide-bound TCRβ chains enable efficient detection of TCR-modified T cells using the 3H4 antibody, while also significantly reducing background signaling from wild-type T cells. Furthermore, these 2A peptide-linked TCRβ chains are also suitable for in-frame knock-in of therapeutic TCR genes into exons of human TCR loci.

[0087] Compared to currently available technologies, the antibody epitope-based protein markers described herein in various embodiments for detecting, isolating, or reducing TCR-modified T cells have the following advantages: (a) the size of the therapeutic gene construct does not exceed 25 amino acids; (b) immunogenicity is minimized; (c) they can be used with all therapeutic TCR genes without the need for optimization according to the antigen specificity of the TCR; and (d) they are compatible with the use of the full-length human TCR constant domain. Because they are localized within cells, these markers are suitable for detection but not for isolating or reducing T cells modified to express therapeutic TCRs.

[0088] More specifically, advantages over currently available technologies for antibody epitopes containing 2A peptide epitopes may, in some embodiments, include one or more of the following: 1. Low immunogenicity: The invention described herein introduces only a limited number of amino acid mutations compared to alternative techniques, such as the full-length mouse TCR constant domain. Notably, although the 2A peptide sequence is derived from a virus, it does not appear to induce an immune response in immunocompetent individuals (Arber et al., Gene Ther, 2013). 2. Broad and versatile utility for all human TCRs: It can be used for all therapeutic TCR genes without the need for optimization according to the antigen specificity of the TCR. 3. Detection with high specificity: Details can be seen in Examples 3 and 4. 4. Therapeutic TCR genes can be knocked in-frame into the exons of the human TCR gene locus. 5. Avoid a substantial increase in the size of therapeutic TCR gene cassettes: Because 2A peptides are small, adding them does not substantially change the size of the transgene. This is important because using larger transgenes usually reduces integration efficiency and increases DNA toxicity during gene delivery.

[0089] Some embodiments relate to genetically modified cells expressing any one of the labeled proteins described above. Some embodiments relate to genetically modified cells expressing any one of the antibody epitopes described above. Some embodiments relate to genetically modified cells containing any one of the gene constructs described above.

[0090] Labeled proteins or antibody epitopes can be introduced into any suitable cells. Suitable cells include, but are not limited to, mammalian cells, insect cells, yeast, and bacteria. In some embodiments, suitable carriers include viruses, yeast, bacteria, and phages. Throughout this disclosure, for simplicity, the term “cell” is used, but all such disclosures of “cell” are intended to be applicable not only to various forms of T cells (e.g., immortalized T cells), yeast, and bacteria, but more broadly to any carrier, including viruses and phages. Accordingly, disclosures of “cell” as used herein (related to cells into which a combinatorial library may be introduced) may include eukaryotic cells, prokaryotic cells, and also refer to the option of using viruses and phages as carriers. Cells may be cell lines, immortalized cells, or primary cells. In some embodiments, cells are human cells or derived from human cells. In some embodiments, the population of cells includes immortalized T cells or primary T cells. In some embodiments, cells are modified, e.g., genetically modified, to suppress or eliminate the intrinsic or background expression of their functional characteristics. In some embodiments, cells are modified, e.g., genetically modified, to enhance their ability to exhibit their functional characteristics when a labeled protein or antibody epitope is introduced. In some embodiments, cells are modified, e.g., genetically modified, to promote the growth and / or maintenance of a population in a culture medium. In some embodiments, a cell population does not contain an intrinsic polypeptide that confers at least one functional characteristic to the cells. In some embodiments, cells are genetically modified to introduce, enhance, eliminate, or suppress the expression of one or more of CD4, CD8, and CD28. In some embodiments, the genetically modified cells are T cells.

[0091] Figures 3-9 show some data supporting the various embodiments described herein. The sequences listed in Table 1 will be useful in these and other embodiments. Figures 10-17 also show various sequences from the following table.

[0092] Table 1-1 Table 1-2 Table 1-3 Table 1-4 Table 1-5 Table 1-6 Table 1-7 Table 1-8 Table 1-9 Table 1-10 Table 1-11

[0093] Figure 3 shows FACS analysis of human primary T cells in which the NY-ESO-1 1G4 TCR was knocked into the endogenous TRAC locus using various repair templates. huTRBC1 represents the full-length human TRBC1 sequence; muTrbc2 represents the full-length mouse Trbc2 sequence with the poly(A) signal removed; muTrbc2-BGHpA represents the full-length mouse Trbc2 sequence with the BGH poly(A) signal added; and huTRBC1-muFG represents the human TRBC1 sequence incorporating the mouse Trbc2 FG loop (presumably a H57-binding epitope; Wang et al., Embo Journal, 1998). Human primary CD3 + T cells were selected, activated with anti-CD3 / CD28 beads for 2 days, and then electroporated with a repair template leading to TRAC RNP and TCR knock-in. Cells were harvested 5 days after electroporation and analyzed by co-staining with APC anti-mouse TCR Cβ chain antibody (clone H57-597, catalog number 109212, BioLegend) and PE-Cy7 anti-human TCR Vβ13.1 antibody (clone H131, catalog number 362406, BioLegend). This data indicates that poly(A) signaling is useful for TCR expression when using the full-length mouse Trbc2 sequence, and that cells expressing the full-length mouse Trbc2 sequence can be recognized by the H57 antibody. However, while incorporating the mouse Trbc2 FG loop into the human TRBC1 sequence could induce TCR expression, it was not sufficient for recognition by the H57 antibody.

[0094] Figure 4 shows FACS analysis of human primary T cells in which the NY-ESO-1 1G4 TCR was knocked into the endogenous TRAC locus using various repair templates. huTRBC1 represents the full-length human TRBC1 sequence; muTrbc2-BGHpA represents the full-length mouse Trbc2 sequence with added BGH polyA signaling; and huTRBC2-muABFG represents the human TRBC2 sequence incorporating 10 amino acid residues derived from mouse Trbc2 A and B strands and an FG loop (presumably a H57-binding epitope; Wang et al., Embo Journal, 1998 and Sasada et al., Journal of Experimental Medicine, 2002). Human primary CD3 +T cells were selected, activated with anti-CD3 / CD28 beads for 2 days, and then electroporated with a repair template leading to TRAC RNP and TCR knock-in. Cells were harvested 5 days after electroporation and analyzed by co-staining with APC anti-mouse TCR Cβ chain antibody (clone H57-597, catalog number 109212, BioLegend) and PE-Cy7 anti-human TCR Vβ13.1 antibody (clone H131, catalog number 362406, BioLegend). This data shows that TCR expression and recognition by H57 antibody are possible simply by incorporating 10 amino acid residues and an FG loop derived from the A and B strands of mouse Trbc2 into the human TRBC2 sequence (amino acid mutations are K4R, F7T, Y37F, N106E, E108K, T110P, Q111E, D112G, R113S, A114P).

[0095] Figure 5 shows FACS analysis of human primary T cells in which the NY-ESO-1 1G4 TCR was knocked into the endogenous TRAC locus using various repair templates. huTRBC2 is the full-length human TRBC2 sequence; huTRBC2-muABFG is the human TRBC2 sequence incorporating 10 amino acid residues from the A and B strands of mouse Trbc2 and an FG loop (a putative H57-binding epitope); muABFG R4K is the human TRBC2 sequence incorporating 9 amino acid residues from the A and B strands of mouse Trbc2 and an FG loop, with one amino acid residue replaced by the human counterpart to position the minimum H57-binding epitope (R4K); muABFG T7F is the human TRBC2 sequence incorporating 9 amino acid residues from the A and B strands of mouse Trbc2 and an FG loop, with one amino acid residue replaced by the human counterpart to position the minimum H57-binding epitope (T7F); muABFG F37Y is a human TRBC2 sequence (F37Y) in which nine amino acid residues derived from the A and B strands of mouse Trbc2 and an FG loop are incorporated, and one amino acid residue is replaced with that of the human counterpart to position the minimum H57-binding epitope; muABFG. E106N is a human TRBC2 sequence (E106N) in which nine amino acid residues derived from the A and B strands of mouse Trbc2 and an FG loop are incorporated, and one amino acid residue is replaced with that of the human counterpart to position the minimum H57-binding epitope; muABFG. K108E is a human TRBC2 sequence (K108E) in which nine amino acid residues derived from the A and B strands of mouse Trbc2 and an FG loop are incorporated, and one amino acid residue is replaced with that of the human counterpart to position the minimum H57-binding epitope; muABFG. P110T incorporates nine amino acid residues and an FG loop derived from the A and B strands of mouse TRBC2, and replaces one amino acid residue with that of the human counterpart to position the minimum H57-binding epitope (P110T) human TRBC2 sequence;muABFG E111Q is a human TRBC2 sequence (E111Q) incorporating nine amino acid residues and an FG loop derived from the A and B strands of mouse Trbc2, with one amino acid residue replaced by that of the human counterpart to position the minimum H57-binding epitope; muABFG G112D is a human TRBC2 sequence (G112D) incorporating nine amino acid residues and an FG loop derived from the A and B strands of mouse Trbc2, with one amino acid residue replaced by that of the human counterpart to position the minimum H57-binding epitope; muABFG S113R is a human TRBC2 sequence (S113R) incorporating nine amino acid residues and an FG loop derived from the A and B strands of mouse Trbc2, with one amino acid residue replaced by that of the human counterpart to position the minimum H57-binding epitope; muABFG P114A shows a human TRBC2 sequence (P114A) incorporating nine amino acid residues and an FG loop derived from the A and B strands of mouse TRBC2, with one amino acid residue replaced by that of the human counterpart to position the minimum H57-binding epitope. Human primary CD3; + T cells were selected, activated with anti-CD3 / CD28 beads for 2 days, and then electroporated with a repair template leading to TRAC RNP and TCR knock-in. Cells were harvested 5 days after electroporation and analyzed by co-staining with APC anti-mouse TCR Cβ chain antibody (clone H57-597, catalog number 109212, BioLegend) and PE-Cy7 anti-human TCR Vβ13.1 antibody (clone H131, catalog number 362406, BioLegend). The data indicate that six amino acid residues and an FG loop derived from the A strand of mouse Trbc2 are required to enable recognition by the H57 antibody (the amino acids are R4, K108, P110, E111, G112, and S113).

[0096] Figure 6 shows FACS analysis of human primary T cells in which the NY-ESO-1 1G4 TCR was knocked into the endogenous TRAC locus using various repair templates. huTRBC2 represents the full-length human TRBC2 sequence; huTRBC2-mu10 represents the human TRBC2 sequence incorporating 10 amino acid residues and an FG loop (presumably an H57-binding epitope) derived from the A and B strands of mouse Trbc2; huTRBC2-mu6 represents the human TRBC2 sequence incorporating 6 amino acid residues and an FG loop (the smallest identified H57-binding epitope) derived from the A strand of mouse Trbc2; and huTRBC2-mu7 represents the human TRBC2 sequence incorporating 7 amino acid residues and an FG loop (the smallest identified H57-binding epitope + T7) derived from the A strand of mouse Trbc2. Human primary CD3 + T cells were selected, activated with anti-CD3 / CD28 beads for 2 days, and then electroporated with a repair template leading to TRAC RNP and TCR knock-in. Cells were harvested 6 days after electroporation and analyzed by co-staining with APC anti-mouse TCR Cβ chain antibody (clone H57-597, catalog number 109212, BioLegend) and PE-Cy7 anti-human TCR Vβ13.1 antibody (clone H131, catalog number 362406, BioLegend). The data show that TCR expression and recognition by the H57 antibody are possible simply by incorporating six amino acid residues and an FG loop derived from the A strand of mouse Trbc2 into the human TRBC2 sequence (amino acid mutations are K4R, E108K, T110P, Q111E, D112G, R113S).

[0097] Figure 7 shows FACS analysis of human primary T cells in which the NY-ESO-1 1G4 TCR was knocked into the endogenous TRAC locus using various repair templates. "Simulated electroporation" indicates cells that were not electroporated; "TRAC RNP" indicates cells in which only TRAC RNP was electroporated; "TRAC RNP + circular repair template" indicates cells in which both TRAC RNP and the circular repair template were electroporated; and "TRAC RNP + linear repair template" indicates cells in which both TRAC RNP and the linear repair template were electroporated. Human primary CD3 + T cells were selected, activated with anti-CD3 / CD28 beads for 2 days, and then electroporated with a repair template leading to TRAC RNP and TCR knock-in. Ten days after electroporation, cells were harvested, permeabilized, and then FACS analysis was performed by staining with unconjugated mouse anti-2A peptide antibody (clone 3H4, catalog number NBP2-59627, Novus Biologicals) and BV421 rat anti-mouse IgG1 antibody (clone A85-1, catalog number 562580, BD Biosciences). BD Perm / Wash buffer (1x) was used for all staining. The data demonstrate that human primary T cells modified to express the NY-ESO-1 1G4 TCR containing the T2A peptide sequence can be detected with the anti-2A peptide antibody.

[0098] Figure 8 shows FACS analysis of human primary T cells in which the NY-ESO-1 1G4 TCR was knocked into the endogenous TRAC locus using various repair templates. Simulated electroporation indicates cells that were not electroporated; TRAC RNP indicates cells in which only TRAC RNP was electroporated; TRAC RNP + circular repair template indicates cells in which both TRAC RNP and the circular repair template were electroporated; and TRAC RNP + linear repair template indicates cells in which both TRAC RNP and the linear repair template were electroporated. Human primary CD3 +T cells were selected, activated with anti-CD3 / CD28 beads for 2 days, and then electroporated with a repair template leading to TRAC RNP and TCR knock-in. Cells were harvested 11 days after electroporation, permeabilized, and then subjected to FACS analysis by staining with unconjugated mouse anti-2A peptide antibody (clone 3H4, catalog number NBP2-59627, Novus Biologicals), BV421 rat anti-mouse IgG1 antibody (clone A85-1, catalog number 562580, BD Biosciences), and PE-Cy7 anti-human TCR Vβ13.1 antibody (clone H131, catalog number 362406, BioLegend). BD permeabilization / wash buffer (1x) was used for all staining. The data demonstrate that human primary T cells modified to express the NY-ESO-1 1G4 TCR containing the T2A peptide sequence can be detected with an anti-2A peptide antibody, and that there is a correlation between the staining of this 2A peptide and the staining of TCR Vβ13.1.

[0099] Figure 9 shows FACS analysis of human primary T cells transduced via retrovirus to express the CD19 CAR construct. Mock transduction refers to untransformed cells; Ly6G-Puro refers to cells transformed with retrovirus containing the Ly6G-P2A-Puro construct;1 st Gen CAR-Ly6G-Puro is 1 st Cells transformed with a retrovirus containing the Gen CD19 CAR-T2A-Ly6G-P2A-Puro construct;2 nd Gen CAR-Ly6G-Puro is 2 nd This shows cells transformed with a retrovirus containing the Gen CD19 CAR-T2A-Ly6G-P2A-Puro construct. +T cells were selected, activated with anti-CD3 / CD28 beads for 2 days, and then infected with retrovirus as described above. Cells were harvested 14 days after transduction, permeabilized, and then subjected to FACS analysis by staining with unconjugated mouse anti-2A peptide antibody (clone 3H4, catalog number NBP2-59627, Novus Biologicals), BV421 rat anti-mouse IgG1 antibody (clone A85-1, catalog number 562580, BD Biosciences), and PE / Dazzle 594 anti-Ly6G antibody (clone 1A8, catalog number 127647, BioLegend). BD permeabilization / wash buffer (1x) was used for all staining. The data demonstrate that human primary T cells modified to express a CD19 CAR construct containing the 2A peptide sequence can be detected by the anti-2A peptide antibody, and that there is a correlation between the staining of this 2A peptide and the staining of the transduction marker protein (Ly6G) contained in the same construct.

[0100] Where this specification refers to a method comprising two or more defined steps, these defined steps may be performed in any order or simultaneously (unless the context excludes such possibility). The method may also include one or more other steps performed before any of the defined steps, between two defined steps, or after all of the defined steps (unless the context excludes such possibility).

[0101] In some cases, one of the following arrangements is conceivable: 1. A labeled protein, TCR constant domain; and A labeled protein containing exogenous amino acid variants present in the TCR constant domain that include detectable and identifiable sequences. 2. A labeled protein of arrangement 1, wherein the TCR constant domain includes a TCRα or TCRβ constant domain. 3. A labeled protein of arrangement 1, wherein the exogenous amino acid variant contains a mutation in the sequence of the TCR constant domain, and the labeled protein optionally contains huTRBC1-mur6 or huTRBC2-mur6. 4. A labeled protein of arrangement 1, wherein the constant TCR domain is derived from one species and the exogenous amino acid variant is derived from another species. 5. A labeled protein of arrangement 4, wherein the constant TCR domain is derived from humans and the exogenous amino acid variant is derived from a non-human species. 6. A labeled protein of arrangement 5, wherein the non-human species is mouse. 7. A labeled protein of arrangement 3, wherein the TCR constant domain contains the sequence encoded by the human TRBC2 gene. 8. A labeled protein of arrangement 3, wherein the exogenous amino acid variant contains the sequence of the mouse TCR Cβ domain. 9. A labeled protein of arrangement 3, wherein the mutation contains 10 amino acid mutations. 10. A labeled protein of arrangement 9 in which the mutation forms a discontinuous sequence. The 11.10 amino acid mutations are numbered as K4R, F7T, Y37F, N106E, E108K, T110P, Q111E, D112G, R113S, and A114P according to the numbering method of Sequence ID No. 8, and are labeled proteins of arrangement 9. 12. A labeled protein of arrangement 3, wherein the mutation contains 6 amino acid mutations. The 13.6 amino acid mutations are K4R, E108K, T110P, Q111E, D112G, and R113S, and the labeled protein of arrangement 12 optionally includes SEQ ID NO: 27. 14. A labeled protein of arrangement 1 capable of detecting and identifying the exogenous amino acid variant by antibody, nanobody, Fab fragment, or DARPin. 15. A labeled protein of arrangement 1 in which the exogenous amino acid variant can be detected and identified with the anti-mouse TCR Cβ antibody H57-597. 16. A labeled protein used for detection, isolation, or reduction of genetically modified cells expressing such labeled protein, comprising the labeled protein described in any one of arrangements 1 to 15. 17. A labeled protein of arrangement 16, wherein the genetically modified cells include genetically modified T cells. 18. A labeled protein of arrangement 17, wherein the genetically modified T cells include T cells modified by the introduction of a therapeutic TCR gene. 19. A labeled protein of arrangement 18, wherein T cells modified by the introduction of a therapeutic TCR gene are used for the treatment of cancer. 20. A labeling protein used for detection, isolation, or reduction of genetically modified T cells altered by the introduction of a therapeutic TCR gene, wherein the labeling protein is derived from the mouse TCR Cβ domain and introduced into the human TCR Cβ2 domain by mutating an amino acid present in the human TCR Cβ2 domain. 21. A labeled protein used for the detection, isolation, or reduction of cells modified with the novel TCR gene for cancer treatment, comprising the labeled protein described in any one of arrangements 1 to 20. 22. A kit for detecting, isolating, or reducing genetically modified cells having a labeled protein described in any one of arrangements 1 to 21, comprising an antibody that recognizes the labeled protein. 23. A kit of arrangement 22 in which the genetically modified cells include T cells. 24. A kit for arrangement 23, in which the T cells are modified by introducing a therapeutic TCR gene. 25. A kit for arrangement 24 in which T cells modified by the introduction of a therapeutic TCR gene are used for the treatment of cancer. 26. A labeled protein used for targeted delivery of one or more packages to genetically modified cells expressing such labeled proteins, comprising a labeled protein described in any one of arrangements 1 to 15. 27. A labeled protein of arrangement 26, wherein the delivery of one or more loads is achieved by conjugation to an antibody, antibody-mimetic protein, or any other antigen-binding scaffold. 28. A labeled protein of arrangement 27, wherein the antibody is the anti-mouse TCR Cβ antibody H57-597. 29. A labeled protein of arrangement 26, wherein one or more of the loaded elements are proteins, small molecules, nucleic acids, liposomes, or nanoparticles. 30. A labeled protein of arrangement 26, wherein the delivered load is a cytokine. 31. A labeled protein of arrangement 30, wherein the cytokine is selected from the group consisting of IL-1β, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, IL-23, IL-27, IFN-α, IFN-β, IFN-γ, and TNF-α. 32. A labeled protein of arrangement 30, wherein the sequence of the cytokine is modified to regulate its interaction with its native receptor molecule. 33. A labeled protein of arrangement 30, wherein the cytokine is an agonist or antagonist of a receptor molecule expressed by T cells. 34. A labeled protein of arrangement 26, wherein the packaged substance is an agonist or antagonist of a receptor molecule expressed by a T cell. 35. A labeled protein of arrangement 34, wherein the agonist binds to CD27, CD28, CD137, or CD278. 36. The antagonist is a TGF-β receptor, PD-1, CTLA-4, Vista, steroid receptor, or A1-, A 2A -, A 2B - Or a labeled protein of arrangement 34 that binds to the A3-adenosine receptor. 37. A labeled protein of arrangement 26, wherein the loaded substance is a small molecule that regulates the activation, differentiation, proliferation, survival, or effector function of T cells. 38. The small molecule is a TGF-β receptor, PD-1, CTLA-4, VISTA, steroid receptor or A1-, A 2A -, A 2B - Or a labeled protein of arrangement 37 that inhibits either A3-adenosine receptor signaling. 39. A labeled protein of arrangement 26, wherein the loaded substance is a nucleic acid that regulates the activation, differentiation, proliferation, survival, or effector function of T cells. 40. A labeled protein of arrangement 39, wherein the nucleic acid is miRNA, shRNA, or siRNA. 41. A labeled protein of arrangement 26, wherein the loaded material is a bispecific or trispecific antibody. 42. A method for targeting and delivering one or more loadings to genetically modified cells expressing a labeled protein, To obtain a complex comprising one or more loadings and a binder that specifically binds to the labeled protein, and Bringing the genetically modified cells into contact with the complex. Methods that include... 43. The method of arrangement 42, wherein the binder is an antibody, an antibody-mimicking protein, or any other antigen-binding scaffold. 44. A method of arrangement 42, wherein one or more loadings are proteins, small molecules, nucleic acids, liposomes, or nanoparticles. 45. An antibody epitope used for detecting genetically modified cells expressing such an antibody epitope, wherein the antibody epitope is bound to a TCR chain or a chimeric antigen receptor (CAR). 46. ​​An antibody epitope of arrangement 45, wherein the antibody epitope is bound to the C-terminus or N-terminus of a TCR chain or to the C-terminus or N-terminus of a CAR. 47. An antibody epitope of arrangement 45 in which the antibody epitope is inserted into a TCR chain or CAR. 48. The gene-modified cells contain T cells in an antibody epitope of arrangement 45. 49. An antibody epitope of arrangement 48 in which T cells are modified by the introduction of a therapeutic TCR or CAR gene. 50. An antibody epitope of arrangement 49 in which T cells modified by the introduction of a therapeutic TCR or CAR gene are used to treat cancer. 51. An antibody epitope of arrangement 45, which is used to link TCRα and TCRβ chains, or to link CAR with a protein encoded by another gene. An antibody epitope of arrangement 45 comprising a 52.2A peptide sequence, an HA.11 epitope tag, a FLAG epitope tag, a Myc epitope tag, a V5 epitope tag, or a peptide containing up to the same number of amino acids as the 2A peptide. 53. An antibody epitope of arrangement 52, wherein the 2A peptide may be P2A, T2A, E2A, or F2A. An antibody epitope of arrangement 45 comprising a 54.2A peptide sequence fragment or a sequence at least 90% identical thereto, wherein the 2A peptide sequence is not a full-length 2A sequence. 55. An antibody epitope of arrangement 54, wherein the peptide sequence includes sequence number 1 (CGDVEENPG) or a sequence that is at least 75% identical thereto. 56. An antibody epitope of arrangement 54 or 55, wherein the antibody epitope can be identified by a monoclonal anti-2A peptide antibody 3H4. 57. An antibody epitope used for detecting genetically modified T cells that have been altered by the introduction of a therapeutic TCR gene, comprising the antibody epitope described in any one of arrangements 45 to 56. 58. An antibody epitope used for detecting cells modified by a novel TCR or CAR gene for cancer treatment, comprising an antibody epitope described in any one of arrangements 45 to 56. 59. A kit for the detection of genetically modified cells, comprising an antibody epitope described in any one of arrangements 45 to 56. 60. A kit of arrangement 59 in which the genetically modified cells include T cells. 61. A kit for arrangement 60, in which the T cells are modified by introducing a therapeutic TCR gene. 62. A kit for arrangement 61, in which T cells modified by the introduction of a therapeutic TCR gene are used for the treatment of cancer. A gene construct comprising a nucleotide sequence capable of expressing a 63.2A peptide sequence or a sequence at least 90% identical thereto, wherein the construct is configured to express multiple proteins from a single read frame, and the nucleotide sequence does not increase the size of the gene construct beyond 25 amino acids. 64. A gene construct of arrangement 63 in which the 2A peptide sequence comprises a 2A peptide sequence fragment or a sequence that is at least 90% identical thereto, and the 2A peptide sequence is not a full-length 2A sequence. 65. A gene construct of arrangement 64 in which the 2A peptide sequence contains the sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6, or a sequence that is at least 75% identical to SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 6 (SEQ ID NO: 2=EGRGSLLTCGDVEENPGP; SEQ ID NO: 3=ATNFSLLKQAGDVEENPGP; SEQ ID NO: 6=GDVEENPG). 66. Genetically modified cells containing a labeled protein described in any one of arrangements 1 to 15, an antibody epitope described in any one of arrangements 53 to 55, or a gene construct described in any one of arrangements 63 to 65. [Examples]

[0102] Example 1 This example demonstrates that simply incorporating 10 amino acid residues derived from the A and B strands of mouse Trbc2 and an FG loop into a human TRBC2 sequence enables TCR expression and recognition by the H57 antibody.

[0103] FACS analysis was performed on human primary T cells in which the NY-ESO-1 1G4 TCR was knocked into the endogenous TRAC locus using various repair templates. + T cells were selected, activated with anti-CD3 / CD28 beads for 2 days, and then electroporated with a repair template leading to TRAC RNP and TCR knock-in. Cells were harvested 5 days after electroporation and analyzed by co-staining with APC anti-mouse TCR Cβ chain antibody (clone H57-597, catalog number 109212, BioLegend) and PE-Cy7 anti-human TCR Vβ13.1 antibody (clone H131, catalog number 362406, BioLegend), followed by FACS analysis.

[0104] The results are shown in Figure 3: huTRBC1 represents the full-length human TRBC1 sequence; muTrbc2 represents the full-length mouse Trbc2 sequence with the polyA signal removed; muTrbc2-BGHpA represents the full-length mouse Trbc2 sequence with the polyA signal of BGH added; and huTRBC1-muFG represents the human TRBC1 sequence incorporating the FG loop of mouse Trbc2.

[0105] This data shows that when using the full-length mouse Trbc2 sequence, the poly(A) signaling pathway is useful for TCR expression, and that cells expressing the full-length mouse Trbc2 sequence can be recognized by the H57 antibody. However, even when the mouse Trbc2FG loop was incorporated into the human TRBC1 sequence, while TCR expression was possible, it was not sufficient for recognition by the H57 antibody.

[0106] Next, the human TRBC2 sequence was tested by incorporating 10 amino acid residues and an FG loop derived from the A and B strands of mouse Trbc2. Similarly, human primary CD3 +T cells were selected, activated with anti-CD3 / CD28 beads for 2 days, and then electroporated with a repair template leading to TRAC RNP and TCR knock-in. Cells were harvested 5 days after electroporation and analyzed by co-staining with APC anti-mouse TCR Cβ chain antibody (clone H57-597, catalog number 109212, BioLegend) and PE-Cy7 anti-human TCR Vβ13.1 antibody (clone H131, catalog number 362406, BioLegend), followed by FACS analysis.

[0107] The results are shown in Figure 4: huTRBC1 represents the full-length human TRBC1 sequence; muTrbc2-BGHpA represents the full-length mouse Trbc2 sequence with the poly(A) signal of BGH added; and huTRBC2-muABFG represents the human TRBC2 sequence incorporating 10 amino acid residues derived from the A and B strands of mouse Trbc2 and an FG loop.

[0108] This data shows that simply incorporating 10 amino acid residues and an FG loop derived from the A and B strands of mouse Trbc2 into the human TRBC2 sequence enables TCR expression and recognition by the H57 antibody (the amino acid mutations are K4R, F7T, Y37F, N106E, E108K, T110P, Q111E, D112G, R113S, and A114P).

[0109] Example 2 This example demonstrates that by simply incorporating six amino acid residues derived from the A strand of mouse Trbc2 and an FG loop into the human TRBC2 sequence, TCR expression and recognition by the H57 antibody become possible.

[0110] FACS analysis was performed on human primary T cells in which the NY-ESO-1 1G4 TCR was knocked into the endogenous TRAC locus using various repair templates. +T cells were selected, activated with anti-CD3 / CD28 beads for 2 days, and then electroporated with a repair template leading to TRAC RNP and TCR knock-in. Cells were harvested 5 days after electroporation and analyzed by co-staining with APC anti-mouse TCR Cβ chain antibody (clone H57-597, catalog number 109212, BioLegend) and PE-Cy7 anti-human TCR Vβ13.1 antibody (clone H131, catalog number 362406, BioLegend), followed by FACS analysis.

[0111] The results are shown in Figure 5: huTRBC2 is the full-length human TRBC2 sequence; huTRBC2-muABFG is the human TRBC2 sequence incorporating 10 amino acids derived from the A and B strands of mouse Trbc2 and an FG loop (presumably a H57-binding epitope); muABFG R4K is the human TRBC2 sequence incorporating 9 amino acid residues derived from the A and B strands of mouse Trbc2 and an FG loop, with one amino acid residue replaced by the human counterpart to position the minimum H57-binding epitope (R4K); muABFG T7F is the human TRBC2 sequence incorporating 9 amino acid residues derived from the A and B strands of mouse Trbc2 and an FG loop, with one amino acid residue replaced by the human counterpart to position the minimum H57-binding epitope (T7F); muABFG F37Y is a human TRBC2 sequence (F37Y) in which nine amino acid residues derived from the A and B strands of mouse Trbc2 and an FG loop are incorporated, and one amino acid residue is replaced with that of the human counterpart to position the minimum H57-binding epitope; muABFG. E106N is a human TRBC2 sequence (E106N) in which nine amino acid residues derived from the A and B strands of mouse Trbc2 and an FG loop are incorporated, and one amino acid residue is replaced with that of the human counterpart to position the minimum H57-binding epitope; muABFG. K108E is a human TRBC2 sequence (K108E) in which nine amino acid residues derived from the A and B strands of mouse Trbc2 and an FG loop are incorporated, and one amino acid residue is replaced with that of the human counterpart to position the minimum H57-binding epitope; muABFG. P110T is a human TRBC2 sequence (P110T) that incorporates nine amino acid residues derived from the A and B strands of mouse Trbc2 and an FG loop, with one amino acid residue replaced by that of the human counterpart to position the minimum H57-binding epitope; muABFG E111Q is a human TRBC2 sequence (E111Q) that incorporates nine amino acid residues derived from the A and B strands of mouse Trbc2 and an FG loop, with one amino acid residue replaced by that of the human counterpart to position the minimum H57-binding epitope;muABFG G112D shows the human TRBC2 sequence (G112D) incorporating nine amino acid residues and an FG loop derived from the A and B strands of mouse Trbc2, with one amino acid residue replaced by the human counterpart to position the minimum H57-binding epitope; muABFG S113R shows the human TRBC2 sequence (S113R) incorporating nine amino acid residues and an FG loop derived from the A and B strands of mouse Trbc2, with one amino acid residue replaced by the human counterpart to position the minimum H57-binding epitope; and muABFG P114A shows the human TRBC2 sequence (P114A) incorporating nine amino acid residues and an FG loop derived from the A and B strands of mouse Trbc2, with one amino acid residue replaced by the human counterpart to position the minimum H57-binding epitope. This data indicates that six amino acid residues derived from the A strand of mouse Trbc2 and an FG loop are required for recognition by the H57 antibody (the amino acids are R4, K108, P110, E111, G112, and S113).

[0112] Next, the first human CD3 + T cells were selected, activated with anti-CD3 / CD28 beads for 2 days, and then electroporated with a repair template leading to TRAC RNP and TCR knock-in. Cells were harvested 6 days after electroporation and analyzed by co-staining with APC anti-mouse TCR Cβ chain antibody (clone H57-597, catalog number 109212, BioLegend) and PE-Cy7 anti-human TCR Vβ13.1 antibody (clone H131, catalog number 362406, BioLegend), followed by FACS analysis.

[0113] The results are shown in Figure 6: huTRBC2 represents the full-length human TRBC2 sequence; huTRBC2-mu10 represents the human TRBC2 sequence incorporating 10 amino acid residues from the A and B strands of mouse Trbc2 and an FG loop (a putative H57-binding epitope); huTRBC2-mu6 represents the human TRBC2 sequence incorporating 6 amino acid residues from the A strand of mouse Trbc2 and an FG loop (the smallest identified H57-binding epitope); and huTRBC2-mu7 represents the human TRBC2 sequence incorporating 7 amino acid residues from the A strand of mouse Trbc2 and an FG loop (the smallest identified H57-binding epitope + T7). This data shows that simply incorporating six amino acid residues and an FG loop derived from the A strand of mouse Trbc2 into the human TRBC2 sequence enables TCR expression and recognition by the H57 antibody (the amino acid mutations are K4R, E108K, T110P, Q111E, D112G, and R113S).

[0114] Example 3 This example demonstrates that human primary T cells modified to express the NY-ESO-1 1G4 TCR containing the T2A peptide sequence can be detected with an anti-2A peptide antibody.

[0115] FACS analysis was performed on human primary T cells in which the NY-ESO-1 1G4 TCR was knocked into the endogenous TRAC locus using various repair templates. + T cells were selected, activated with anti-CD3 / CD28 beads for 2 days, and then electroporated with a repair template leading to TRAC RNP and TCR knock-in. Ten days after electroporation, the cells were harvested, permeabilized, and then subjected to FACS analysis by staining with unconjugated mouse anti-2A peptide antibody (clone 3H4, catalog no. NBP2-59627, Novus Biologicals) and BV421 rat anti-mouse IgG1 antibody (clone A85-1, catalog no. 562580, BD Bioscience). BD permeabilization / wash buffer (1x) was used for all staining. FACS analysis was then performed.

[0116] The results are shown in Figure 7: Simulated electroporation represents cells that have not undergone electroporation; TRAC RNP represents cells that have been electroporated with TRAC RNP only; TRAC RNP + circular repair template represents cells that have been electroporated with both TRAC RNP and a circular repair template; and TRAC RNP + linear repair template represents cells that have been electroporated with both TRAC RNP and a linear repair template. This data demonstrates that human primary T cells modified to express the NY-ESO-1 1G4 TCR containing the T2A peptide sequence can be detected with an anti-2A peptide antibody.

[0117] Example 4 This example demonstrates that human primary T cells modified to express the NY-ESO-1 1G4 TCR containing the T2A peptide sequence can be detected with an anti-2A peptide antibody, and that there is a correlation between the staining of this 2A peptide and the staining of TCR Vβ13.1.

[0118] FACS analysis was performed on human primary T cells in which the NY-ESO-1 1G4 TCR was knocked into the endogenous TRAC locus using various repair templates. + T cells were selected, activated with anti-CD3 / CD28 beads for 2 days, and then electroporated with a repair template leading to TRAC RNP and TCR knock-in. Cells were harvested 11 days after electroporation, permeabilized, and then subjected to FACS analysis by staining with unconjugated mouse anti-2A peptide antibody (clone 3H4, catalog no. NBP2-59627, Novus Biologicals), BV421 rat anti-mouse IgG1 antibody (clone A85-1, catalog no. 562580, BD Biosciences), and PE-Cy7 anti-human TCR Vβ13.1 antibody (clone H131, catalog no. 362406, BioLegend). BD permeabilization / wash buffer (1x) was used for all staining. FACS analysis was then performed.

[0119] The results are shown in Figure 8: Simulated electroporation represents cells that have not undergone electroporation; TRAC RNP represents cells that have been electroporated with TRAC RNP only; TRAC RNP + circular repair template represents cells that have been electroporated with both TRAC RNP and a circular repair template; and TRAC RNP + linear repair template represents cells that have been electroporated with both TRAC RNP and a linear repair template. This data demonstrates that human primary T cells modified to express the NY-ESO-1 1G4 TCR containing the T2A peptide sequence can be detected with an anti-2A peptide antibody, and that there is a correlation between the staining of this 2A peptide and the staining of TCR Vβ13.1.

[0120] Example 5 This example demonstrates that human primary T cells modified to express a CD19 CAR construct containing a 2A peptide sequence can be detected by an anti-2A peptide antibody, and that a correlation can be observed between the staining of this 2A peptide and the staining of a transduction marker protein (Ly6G) contained in the same construct.

[0121] FACS analysis was performed on human primary T cells transduced via retrovirus to express the CD19 CAR construct. + T cells were selected, activated with anti-CD3 / CD28 beads for 2 days, and then infected with retrovirus as described above. Cells were harvested 14 days after transduction, permeabilized, and then subjected to FACS analysis by staining with unconjugated mouse anti-2A peptide antibody (clone 3H4, catalog number NBP2-59627, Novus Biologicals), BV421 rat anti-mouse IgG1 antibody (clone A85-1, catalog number 562580, BD Biosciences), and PE / Dazzle 594 anti-Ly6G antibody (clone 1A8, catalog number 127647, BioLegend). BD permeabilization / wash buffer (1x) was used for all staining. Subsequently, FACS analysis was performed.

[0122] The results are shown in Figure 9: Simulated transduction was performed on untransformed cells; Ly6G-Puro was performed on cells transformed with a retrovirus containing the Ly6G-P2A-Puro construct; 1 st Gen CAR-Ly6G-Puro is 1 st Cells transformed with a retrovirus containing the Gen CD19 CAR-T2A-Ly6G-P2A-Puro construct;2 nd Gen CAR-Ly6G-Puro is 2 nd This image shows cells transformed with a retrovirus containing the Gen CD19 CAR-T2A-Ly6G-P2A-Puro construct. This data demonstrates that human primary T cells modified to express a CD19 CAR construct containing the 2A peptide sequence can be detected by an anti-2A peptide antibody, and that there is a correlation between the staining of this 2A peptide and the staining of a transduction marker protein (Ly6G) contained within the same construct.

[0123] Example 6 This example shows a labeled protein that can be used for targeted delivery of one or more payloads to genetically modified cells expressing such a labeled protein. Delivery of one or more payloads is achieved by conjugation to an H57 antibody.

[0124] The N-terminus of IL2v is fused to the C-terminus of the H57 IgG Fc domain, optionally via a (G4S)3 linker peptide. The Fc domain contains one or more amino acid substitutions, which weaken binding to the Fc receptor. The substitutions are L234A, L235A, and P329G (LALAPG based on the human IgG1 Fc domain).

[0125] FACS analysis was performed on TCR knockout CD8-high-expression Jarcut cells modified to express the human TRBC2-Mur6 1G4 TCR. FACS analysis was also performed on human primary T cells in which the huTRBC2-Mur6 1G4 TCR was knocked into the endogenous TRAC locus. huTRBC2-mur6-expressing cells were pre-incubated for 30 minutes with various concentrations of H57 antibody, or with H57-IL2 or H57-IL2v immunocytokines. Then, FACS analysis of the cells was performed using staining with APC anti-mouse TCR-Cβ antibody (clone H57-597, catalog number 553174, BD Bioscience) and FITC anti-human IL2 antibody (clone 5344.111, catalog number 340448, BD Bioscience). Staining was performed in PBS containing 2% FBS.

[0126] Figure 18 shows that in huTRBC2-Mur6 1G4 jarcut cells, the binding of huTRBC2-Mur6 to APC anti-mouse TCR-Cβ antibody is inhibited by 20 ng / mL of H57, as well as H57-IL2 and H57-IL2v. Figure 19 shows that H57, as well as H57-IL2 and H57-IL2v, inhibit the binding of huTRBC2-Mur6 to APC anti-mouse TCR-Cβ antibody in human primary huTRBC2-Mur6 1G4 TCR T cells. As seen in Figure 20, huTRBC2-Mur6 1G4 T cells incubated with H57-IL2 or H57-IL2v were strongly stained with FITC anti-human IL2 antibody, indicating the presence of IL2 on the surface of these cells. When human primary huTRBC2 1G4 T cells that do not express the Mur6 epitope were incubated with H57-IL2 and H57-IL2v, IL2 staining was observed, suggesting that immunocytokine-mediated IL2 targeting specifically to Mur6. These data together demonstrate that targeted delivery of cytokines to huTRBC2-Mur6 constant domain-expressing 1G4 TCR-modified cells by H57-IL2 antibody- and H57-IL2 antibody-cytokine fusion proteins can be mediated using the Mur6 epitope.

[0127] Example 7 In this example, the stimulating ability of the H57-IL2v complex to T cell proliferation is evaluated in vitro.

[0128] The H57-IL2v complex from Example 6 was added to CFSE-labeled primary human T cells that had been activated overnight with PHA, and the cells were then cultured for a further 4 days. An unrelated antibody-IL2v complex was used as a control. After 4 days, the stimulating effect of the H57-IL2v complex on T cell proliferation was evaluated by measuring the dilution of CFSE and by measuring the increase in CD25 expression in T cells using flow cytometry.

[0129] In experiments using CFSE substitutes, primary human T cells labeled with celltrace violet (CTV) were treated with knock-in of either huTRBC2 1G4 TCR or huTRBC2-Mur6 1G4 TCR at the endogenous TRAC locus. The H57-IL2 and H57-IL2v complexes from Example 6 were then added, and the cells were cultured for a further 4 days. Non-targeted recombinant IL-2 was used as a control. The following day, the stimulating activity of the H57-IL2 and H57-IL2v complexes was evaluated by measuring the expression of CD69 and CD25, which are T cell activation markers. FACS analysis was performed by co-staining with PE anti-human CD69 antibody (clone FN50, catalog number 557050, BD Bioscience) and BV711 anti-human CD25 antibody (clone 2A3, catalog number 563159, BD Bioscience). After culturing for 4 days, the dilution of CTV was measured, and the number of T cells was counted using flow cytometry to evaluate the stimulating effect of the H57-IL2 and H57-IL2v complexes on T cell proliferation. All staining was performed in PBS containing 2% FBS.

[0130] Figure 21 is a dot plot showing the expression of T cell activation markers CD69 and CD25 after incubation for 1 day with 1 nM IL2, H57-IL2, and H57-IL2v in primary human T cells knocked in with huTRBC2 1G4 TCR or huTRBC2-Mur6 1G4 TCR. Graphs in Figures 22A and 22B show the percentage of CD69-expressing T cells (Figure 22A) and CD25-high-expressing T cells (Figure 22B) at varying concentrations of (immuno)cytokines. These results indicate that the H57-IL2 and H57-IL2v complex strongly and specifically stimulates huTRBC2-Mur6 1G4 TCR-expressing T cells.

[0131] The histogram in Figure 23 shows the dilution of CTV in primary human T cells after incubation for 4 days with 1 nM IL2, H57-IL2, and H57-IL2v in T cells knocked in with huTRBC2 1G4 TCR or huTRBC2-Mur6 1G4 TCR. Furthermore, Figure 24 shows the FACS measurement of CTV, where the average fluorescence intensity of all cells is divided by the average fluorescence intensity of undiminal cells for each (immuno)cytokine concentration, and the logarithm squared (2log of MFI) is calculated. undivided / MFI all cells) The average proliferation cycle calculated is shown. These results indicate that in huTRBC2-Mur6 1G4 TCR-expressing T cells, targeting of mur6 using H57-IL2 and H57-IL2v specifically promotes T cell proliferation. These data together indicate that the presence of the Mur6 epitope in 1G4 TCR-modified T cells enables the targeting of specific cytokines by H57-IL2 and H57-IL2v immunocytokines, thereby inducing epitope-specific T cell stimulation.

[0132] Example 8 This example demonstrates how the H57-IL2v complex influences tumor growth and T cell accumulation within tumors in vivo.

[0133] NSG mice are used here. NSG mice (NOD severe combined immunodeficiency gamma mice) are a trademark of Jackson Laboratory, which develops and sells research-use immunodeficiency mice, and the NOD strain is included in this trademark. NY-ESO-1+A375 melanoma cells are subcutaneously transplanted into NSG mice, followed by administration of NY-ESO-1 TCR-expressing primary human T cells. Subsequently, the mice are injected weekly with either the H57-IL2v complex or a solvent control. The effects on tumor growth and T cell accumulation within the tumor are then measured over time.

[0134] Example 9 This example demonstrates that TCR expression and recognition by the H57 antibody can be enabled simply by incorporating six amino acid residues derived from the A strand of mouse Trbc2 and an FG loop into a human TRBC1 or TRBC2 sequence.

[0135] FACS analysis was performed on human primary T cells in which NY-ESO-1 specific 1G4 TCRs were knocked into the endogenous TRAC locus using various repair templates. Human primary CD3+ T cells from three healthy donors (BC45, BC46, and BC48) were selected, activated for 2 days with anti-CD3 / CD28 beads (catalog number 40203D, Thermo Fisher Scientific), and then electroporated with DNA repair templates that lead to TRAC RNP and TCR knock-in, as well as TRBC RNPs that lead to endogenous TCRβ chain knockout. Five days after electroporation, 1G4 TCR-modified T cells were collected and subjected to FACS analysis by co-staining with PE anti-mouse TCR Cβ chain antibody (clone H57-597, catalog number 553172, BD Bioscience) and Brilliant Violet 421 anti-human TCR Cβ chain antibody (clone IP26, catalog number 306722, BioLegend).

[0136] The results are shown in Figure 25: muTrbc2 shows a 1G4 TCR with the full-length mouse Trbc2 constant domain sequence added; huTRBC1-Mur6 shows a 1G4 TCR with the human TRBC1 constant domain sequence incorporating six amino acid residues derived from the A strand of mouse Trbc2 and an FG loop (the smallest identified H57-binding epitope); huTRBC2-Mur6 shows a 1G4 TCR with the human TRBC2 constant domain sequence incorporating six amino acid residues derived from the A strand of mouse Trbc2 and an FG loop (the smallest identified H57-binding epitope). This data demonstrates that TCR expression and recognition by H57 antibody can be enabled simply by incorporating six amino acid residues and an FG loop derived from the A strand of mouse Trbc2 into a human TRBC1 or TRBC2 sequence (the amino acid mutations are N4R, E108K, T110P, Q111E, D112G, R113S for huTRBC1-Mur6 and K4R, E108K, T110P, Q111E, D112G, R113S for huTRBC2-Mur6).

[0137] Example 10 This example demonstrates that incorporating six amino acid residues derived from the A strand of mouse Trbc2 and an FG loop into a human TRBC2 sequence does not interfere with the function of the TCR, which is measured as cytokine production by T cells.

[0138] FACS analysis was performed on human primary T cells in which NY-ESO-1 specific 1G4 TCRs were knocked into the endogenous TRAC locus using various repair templates. Human primary CD3+ T cells from three healthy donors (BC20, BC93, and BC97) were selected and activated with anti-CD3 / CD28 beads (catalog number 40203D, Thermo Fisher Scientific) for 2 days. Subsequently, DNA repair templates leading to TRAC RNP and TCR knock-in, as well as TRBC RNPs leading to endogenous TCRβ chain knockout, were electroporated. 12 days after electroporation, the 1G4 TCR-modified T cells were harvested and co-cultured with JY target cells loaded with various concentrations of NY-ESO-1 peptide (SLLMWITQC) ranging from 101 to 107 pg / mL. After co-culturing for 4 hours, a GolgiPlug protein transport inhibitor (catalog number 555028, BD Biosciences) was added, and the cells were co-culturised for a further 16 hours. The cells were then harvested, permeabilized, and subjected to FACS analysis by co-staining with PE anti-human IFN-γ antibody (clone 25723.11, catalog number 340452, BD Biosciences) and FITC anti-human IL-2 antibody (clone 5344.111, catalog number 340448, BD Biosciences). BD permeabilization / wash buffer (1x dilution; catalog number 555028, BD Biosciences) was used for all staining.

[0139] The results are shown in Figures 26A-26D: huTRBC2-Mur6 represents a 1G4 TCR with a human TRBC2 constant domain sequence incorporating six amino acid residues derived from the A strand of mouse Trbc2 and an FG loop (the smallest identified H57-binding epitope); huTRBC2 represents a 1G4 TCR with the full-length human TRBC2 constant domain sequence; muTrbc2 represents a 1G4 TCR with the full-length mouse Trbc2 constant domain sequence; and Unelectroporated represents unmodified T cells. Figure 26A shows IFN-γ production by T cells from a representative healthy donor (BC93) in response to different doses of peptide. The sign indicates the geometric mean of the percentage of IFN-γ-positive T cells calculated by performing three technical replicates for each dose of peptide; the error bars indicate the geometric standard deviation. Figure 26B summarizes IFN-γ production in T cells from three healthy donors, corresponding to the highest dose of peptide tested (107 pg / mL). The sign indicates the percentage of IFN-γ-positive T cells calculated from three technical replicates for each donor's T cells, and the line shows its geometric mean. Figure 26C shows IL-2 production in T cells from one representative healthy donor (BC97), corresponding to different doses of peptide. The sign indicates the geometric mean of the percentage of IL-2-positive T cells, calculated from three technical replicates for each dose of peptide; the error bars indicate the geometric standard deviation. Figure 26D summarizes IL-2 production in T cells from three healthy donors, corresponding to the highest dose of peptide tested (107 pg / mL). The sign indicates the percentage of IL-2-positive T cells, calculated from three technical replicates for each donor's T cells, and the line shows its geometric mean. These data together demonstrate that 1G4 TCR-modified T cells expressing the huTRBC2-Mur6 constant domain can mediate cytokine production equivalent to that of 1G4 TCR-modified T cells expressing the huTRBC2 or muTrbc2 constant domain. This suggests that incorporating the Mur6 epitope into human TRBC2 does not impair TCR function.

[0140] Example 11 This example demonstrates that incorporating six amino acid residues and an FG loop derived from a mouse Trbc2 A strand into a human TRBC2 sequence does not interfere with the function of TCR, which is measured as the killing of T cells' target cells.

[0141] Human primary T cells in which NY-ESO-1 specific 1G4 TCRs were knocked into the endogenous TRAC locus using various repair templates were analyzed using IncuCyte. Human primary CD3+ T cells from three healthy donors (BC20, BC93, and BC97) were selected, activated for two days with anti-CD3 / CD28 beads (catalog number 40203D, Thermo Fisher Scientific), and then electroporated with DNA repair templates that lead to TRAC RNP and TCR knock-in, as well as TRBC RNPs that lead to endogenous TCRβ chain knockout. 1G4 TCR-modified T cells were harvested 12 days after electroporation and co-cultured with NY-ESO-1+A375-GFP+ tumor cells in an incubator S3 live cell analysis system (Sartorius). Viable GFP+ tumor cells were quantified every 2 hours for a total of 72 hours.

[0142] The results are shown in Figures 27A and 27B: huTRBC2-Mur6 shows a 1G4 TCR containing a human TRBC2 constant domain sequence incorporating six amino acid residues derived from the A strand of mouse Trbc2 and an FG loop (the smallest identified H57-binding epitope); huTRBC2 shows a 1G4 TCR containing the full-length human TRBC2 constant domain sequence; muTrbc2 shows a 1G4 TCR containing the full-length mouse Trbc2 constant domain sequence; and Unelectroporated indicates unmodified T cells. Figure 27A shows the dynamics of A375-GFP+ tumor cell death by T cells from one representative healthy donor (BC97). The sign represents the geometric mean of the number of viable tumor cells calculated from three technical iterations at each time point; the error bars represent the geometric standard deviation. Figure 27B is a summary of relative A375-GFP+ tumor cell death by T cells from three healthy donors at the final time point (72 hours) tested. The signs indicate the number of surviving tumor cells calculated from three technical iterations for T cells derived from each donor; the lines show their geometric mean. To calculate the relative number of tumor cells, the data were standardized against the geometric mean of unelectroporated T cells, set to 100%. These data together demonstrate that 1G4 TCR-modified T cells expressing the huTRBC2-Mur6 constant domain can mediate target cell death at a similar rate to 1G4 TCR-modified T cells expressing the huTRBC2 or muTrbc2 constant domain, suggesting that incorporating the Mur6 epitope into human TRBC2 does not impair TCR function.

Claims

1. A labeled protein, A human TCR constant domain, wherein the TCR constant domain comprises a TCR alpha or a TCR beta constant domain; and A labeled protein comprising an exogenous amino acid variant present in a TCR constant domain and comprising a detectable and identifiable sequence, said exogenous amino acid variant being of murine origin.

2. The labeled protein of claim 1, wherein the labeled protein comprises huTRBC1-mur6 or huTRBC2-mur6.

3. The labeled protein of claim 1 or 2, wherein the exogenous amino acid variants comprise mutations in a mouse TCR constant domain sequence, the mutations including 10 amino acid mutations numbered as K4R, F7T, Y37F, N106E, E108K, T110P, Q111E, D112G, R113S, and A114P according to the numbering system of SEQ ID NO:

8.

4. The labeled protein of any one of claims 1 to 3, wherein the exogenous amino acid variants comprise mutations in a mouse TCR constant domain sequence, the mutations including six amino acid mutations numbered as K4R, E108K, T110P, Q111E, D112G, and R113S according to the numbering system of SEQ ID NO:

8.

5. The labeled protein of any one of claims 1 to 4, wherein the exogenous amino acid variant can be detected and identified with anti-mouse TCR Cβ antibody H57-597.

6. A marker protein for use in the detection, isolation or reduction of genetically modified cells expressing such a marker protein, comprising the marker protein according to any one of claims 1 to 5.

7. A marker protein for use in targeted delivery of one or more payloads to genetically modified cells expressing such a marker protein, comprising the marker protein of any one of claims 1 to 5.

8. The labeled protein of claim 7, wherein the payload is a cytokine selected from the group consisting of IL-1β, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, IL-23, IL-27, IFN-α, IFN-β, IFN-γ, and TNF-α.

9. An antibody epitope for use in the detection of genetically modified cells expressing such an antibody epitope, wherein said antibody epitope is bound to a TCR chain or a chimeric antigen receptor (CAR).

10. 1. A genetic construct comprising a nucleotide sequence capable of expressing a 2A peptide sequence or a sequence at least 90% identical thereto, wherein said construct is configured to express multiple proteins from a single open reading frame, and wherein said nucleotide sequence does not increase the size of said genetic construct beyond 25 amino acids.

11. The genetic construct of claim 10, wherein the 2A peptide sequence comprises the sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:6, or a sequence at least 75% identical to SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:6 (SEQ ID NO:2=EGRGSLLTCGDVEENPGP; SEQ ID NO:3=ATNFSLLKQAGDVEENPGP; SEQ ID NO:6=GDVEENPG).

12. A genetically modified cell comprising a marker protein according to any one of claims 1 to 5 or a gene construct according to any one of claims 10 to 11.

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