Genetically modified rodents and rodent cells and uses thereof

Abstract

The present invention relates to transgenic rodents and rodent cells expressing components of the human immune system, and their use in the production of human T cell receptors (TCRs). The present invention relates to a genetically modified rodent comprising a TCR [alpha] locus and a TCR [beta] locus; wherein the TCR alpha gene locus comprises a human TCR alpha variable gene segment and a human TCR alpha constant region which are not rearranged, and the TCR beta gene locus comprises a human TCR beta variable gene segment and a human TCR beta constant region which are not rearranged. The present invention relates to the use of a human TCR transgenic rodent in the generation of antigen-specific CD8 + T cell response for discovering a fully human TCR sequence with therapeutic potential.

Description

Technical Field

[0001] This invention relates to genetically engineered rodents and rodent cells that express components of the human immune system. The invention also relates to methods for preparing and using these rodents to produce products with therapeutic uses, and to identifying and obtaining products through the use of these rodents. Background Technology

[0002] Currently, there is still a need for an in vivo system that can mimic some functions of the human immune system.

[0003] Existing literature involves genetically modified mice encoding chimeric (human-mouse) forms of T-cell receptors (hereinafter referred to as "TCRs"), chimeric forms of TCR co-receptors (such as CD4 and CD8), and chimeric MHC class I and MHC class II molecules. These publications specifically include EP2958937 and EP2958938B1. These chimeric TCR peptides require subsequent humanization, replacing the rodent portion to produce human therapeutics. Therefore, the resulting TCR variable region is generated in a completely different manner than that naturally occurring in mice. Chimeric loci require the use of mouse peptide portions to interact with mouse regulatory sequences and / or other mouse peptide sequences.

[0004] T cells express a variable T cell receptor (TCR) that forms a complex with the CD3 peptide. The TCR is activated by binding to a homologous peptide (i.e., the major histocompatibility complex, pMHC); pMHC is a component of the surface of most mammalian cells and can present peptide samples from within the cell via antigen processing and presentation pathways. Most mammalian cell surface MHC class I molecules can present 8-11 amino acid peptides derived from cytosol proteins, which can be recognized by CD8+ T cells—the TCR-CD3 complex and the co-receptor CD8 bind to pMHC (see [link to relevant documentation]). Figure 8 ).

[0005] A library of TCRs with distinct sequences is generated through in vivo somatic rearrangement of genomic DNA within a T cell population, with each T cell expressing a single (monoclonal) TCR containing random sequence elements. Positive and negative selection during T cell development allows T cells expressing TCRs that recognize pMHC with threshold affinity to survive, while self-reactive cells exhibiting high affinity binding are eliminated, resulting in a population of T cells that do not recognize normal "self" pMHC but can bind to unexplored pMHC. When the TCRs of CD8+ cytotoxic T lymphocytes (CTLs) recognize peptides presented by MHC class I molecules, the binding of the TCR to pMHC initiates intracellular signaling via the CD3 component of the TCR complex, activating CTLs to release cytolytic molecules and activate cytokines in immune cells. Therefore, cells expressing proteins containing novel epitopes (e.g., infected cells producing viral proteins, cells expressing mutated oncogenes) can be eliminated by the cellular adaptive immune system to maintain the body's health.

[0006] Structurally, the TCR comprises two transmembrane polypeptides (either αβ or γδ pairs) that bind to the CD3 complex. Each of the two paired TCR polypeptide chains has an N-terminal variable domain (containing three hypervariable complementarity-determining regions, CDRs), a constant domain, and a linker region connecting the transmembrane domain and the C-terminal cytoplasmic tail. Figure 9 In a structure similar to the antigen-binding fragment (Fab) binding domain of an antibody, the extracellular region of the TCR contains two paired variable domains and two paired constant domains; the pMHC binding site of the TCR is formed by a set of 6 CDRs, which include 3 CDRs from one variable domain and 3 CDRs from another variable domain.

[0007] Different TCR variable domain sequence libraries are generated from individual genomes. The sequence diversity within the variable domains arises from the combination of gene segments selected from multiple different germline gene segments within the loci encoding each TCR polypeptide chain. CDR sequences are particularly variable because they contain non-template links between rearranged gene segments. Another type of combinatorial diversity arises from the pairing of different variable domains (e.g., αβ pairing) to form TCR binding sites. Summary of the Invention

[0008] This invention relates to: A genetically modified rodent that comprises: (i) An unrearranged T-cell receptor (TCR) α variable locus comprising at least one human Vα segment and at least one human Jα segment, wherein the TCRα variable gene is operatively linked to a human TCRα constant gene sequence. (ii) An unrearranged TCRβ variable locus comprising at least one human Vβ segment, at least one human Dβ segment and at least one human Jβ segment, wherein the unrearranged TCRβ variable gene is operatively linked to a human TCRβ constant gene sequence. Among them, the aforementioned unrearranged human T cell variable region gene fragment can rearrange to form a gene encoding the variable domain of the human T cell receptor; and the unrearranged TCRα variable locus is located at the endogenous rodent TCRα locus, and the unrearranged TCRβ variable locus exists at the endogenous rodent TCRβ locus.

[0009] A genetically modified rodent cell (e.g., an ES cell) comprising: (i) An unrearranged T-cell receptor (TCR) α variable locus comprising at least one human Vα segment and at least one human Jα segment, wherein the TCRα variable gene is operatively linked to a human TCRα constant gene sequence. (ii) An unrearranged TCRβ variable locus comprising at least one human Vβ segment, at least one human Dβ segment and at least one human Jβ segment, wherein the unrearranged TCRβ variable gene is operatively linked to a human TCRβ constant gene sequence. Among them, the above-mentioned unrearranged human T cell variable region gene fragments can rearrange to form a gene encoding the variable domain of the human T cell receptor; The unrearranged TCRα variable locus is located at the endogenous rodent TCRα locus, and the unrearranged TCRβ variable locus is located at the endogenous rodent TCRβ locus.

[0010] A genetically modified rodent T cell comprising: (i) An unrearranged T-cell receptor (TCR) α variable locus comprising at least one human Vα segment and at least one human Jα segment, wherein the TCRα variable gene is operatively linked to a human TCRα constant gene sequence. (ii) An unrearranged TCRβ variable locus comprising at least one human Vβ segment, at least one human Dβ segment and at least one human Jβ segment, wherein the unrearranged TCRβ variable gene is operatively linked to a human TCRβ constant gene sequence. Among them, the above-mentioned unrearranged human T cell variable region gene fragments can rearrange to form a gene encoding the variable domain of the human T cell receptor; The unrearranged TCRα variable locus is located at the endogenous rodent TCRα locus, and the unrearranged TCRβ variable locus is located at the endogenous rodent TCRβ locus.

[0011] A genetically modified rodent antigen-presenting cell that includes any of the following: (i) The nucleic acid sequence encoding a human MHC class I polypeptide located at an endogenous rodent MHC class I locus; or (ii) The nucleic acid sequence encoding a chimeric human-rodent MHC class I polypeptide located at an endogenous rodent MHC class I locus (suitably, the nucleic acid sequence encoding the chimeric MHC class I has rodent exon 4, and exons 1-3 and 5-7 are of human origin); and (iii) Optionally, it may include a nucleic acid sequence encoding a human β2 microglobulin polypeptide; when such sequence is present, it is located at the endogenous rodent β2 microglobulin gene locus; The cells express human MHC class I or chimeric MHC class I, and optionally may also express human β2M peptide.

[0012] A method for preparing a genetically modified rodent expressing a human T-cell receptor, comprising: (i) Inserting an unrearranged human TCRα variable locus into an endogenous rodent TCRα variable locus, the unrearranged human TCRα variable locus comprising at least one human Vα fragment and at least one human Jα fragment, and operatively ligated to the human TCRα constant region. (ii) Inserting an unrearranged human TCRβ variable locus into an endogenous rodent TCRβ variable locus, the unrearranged human TCRβ variable locus comprising at least one human Vβ fragment, at least one human Dβ fragment and at least one human Jβ fragment, and operatively ligated to the human TCRβ constant region.

[0013] A method for preparing a genetically modified reproductive rodent expressing a human T-cell receptor, the method comprising deleting at least one or all of the rodent Vβ1 to Dβ1 segments from the rodent TCRβ (including deleting one or all of the serine protease genes Prss58, Prss59, Prss3b, Try4, Try5, Try10, Prss3, Prss1 and Prss2), wherein one or all of the aforementioned deleted serine protease genes Prss58, Prss59, Prss3b, Try4, Try5, Try10, Prss3, Prss1 and Prss2 are reinserted into the rodent genome.

[0014] A method for preparing a gene-knockout reproductive rodent containing an endogenous T-cell receptor β chain polypeptide, the method comprising deleting at least one or all of the rodent Vβ1 to Dβ1 fragments of the rodent TCRβ (including deletion of one or all of the serine protease genes Prss58, Prss59, Prss3b, Try4, Try5, Try10, Prss3, Prss1 and Prss2), wherein one or all of the aforementioned deleted serine protease genes Prss58, Prss59, Prss3b, Try4, Try5, Try10, Prss3, Prss1 and Prss2 are reinserted into the rodent genome.

[0015] A method for generating a human T-cell receptor targeting a target antigen, comprising: (i) Optionally, prepare rodents as disclosed herein; (ii) Immunize rodents as disclosed herein with the target antigens described above; (iii) To induce an immune response in the rodent; (iv) Determining the nucleic acid sequence of the human TCR variable region expressed by T cells from rodents responsive to the target antigen described above, optionally including the step of isolating the T cells; and (v) Expressing the human T-cell receptor or the variable region of the human T-cell receptor in cells; optionally, the expressed human T-cell receptor or the variable region of the human T-cell receptor may be further formulated with pharmaceutically acceptable excipients; or (vi) Inserting the nucleic acid encoding the human T-cell receptor or the variable region of the human T-cell receptor into cells (e.g., into human or animal cells) either in vitro or ex vivo; optionally, preparing and delivering cell lines containing the inserted nucleic acid into humans or animals; or (vii) Formulate nucleic acids (e.g., RNA or DNA) encoding the human T-cell receptor or the variable region of the human T-cell receptor using a suitable delivery carrier (e.g., lipids or liposomes) for in vivo delivery to patients who require it.

[0016] A method for treating an individual who requires treatment, the method comprising: (i) Delivering the cells as described in step (vi) above to a patient in need; or (ii) Deliver the nucleic acid (e.g., RNA, such as mRNA or DNA) that is formulated as described in step (vii) above, encoding the human T-cell receptor or the variable region of the human T-cell receptor, to the patient in need of it.

[0017] Ideally, the transgenic rodents of this invention will be able to functionally express human TCRs and provide an in vivo T-cell immune repositories highly suitable for screening candidate TCRs for medical applications (e.g., therapeutic agents for humans). The efficacy can be confirmed by validating the functional expression of the transgenic loci in rodents, for example, by evaluating data on: the use of germline gene fragments in the TCRs expressed at the transgenic TCRα and β loci; combinations of TCR gene fragments (such as v and j gene fragments); N addition; and CDR3 length.

[0018] TCRs isolated from transgenic rodents can be tested to determine their affinity for the target antigen (target peptide-MHC complex), the specificity and selectivity of target binding, and their ability to transduce signals after binding to the target pMHC when expressed in T cells (e.g., by reporter gene analysis in T cells expressing the TCR). For screening candidate therapeutics, it is particularly necessary to identify TCRs with high affinity for the target and / or efficient signal transduction capabilities, and the transgenic rodents of this invention are an abundant source of such TCRs. Advantageously, compared to isolating TCRs from human individuals, the transgenic rodents of this invention can isolate TCRs with higher affinity and selectivity for the human target pMHC complex.

[0019] Brief description of the attached figures Figure 1 shows the mouse TRA locus, the human TRA locus, and the engineered humanized mouse TRA locus. The human DNA in the third figure is derived from chromosomes 14:21570693-14:22554820 (GRCh38). This indicates that a slightly smaller than 1 Mbp human DNA cloned from the 1.1 Mbp region shown in the middle figure was inserted into the 1.95 Mbp region of the mouse DNA shown in the upper figure, resulting in the replacement of 1.8 Mbp of mouse DNA with approximately 1 Mbp of human DNA. The inserted human DNA may contain 45 functional Vα and 51 functional Jα gene fragments (…). Figure 1a Alternatively, it may contain 44 functional Vα gene fragments and 50 functional Jα gene fragments. Figure 1b ).exist Figure 1c The diagram of the human TRD loci has been revised to show human TRD gene segments (indicated by black triangles) at the plotted chromosomal regions. In human sequences, there are 3 TRDD genes, 4 TRDJ genes, and 8 TRDV genes (4 of which are shared with the TRA library).

[0020] Figure 2 shows the mouse TRB locus, the human TRB locus, and the engineered humanized mouse TRB locus (in which human DNA was inserted and rodent DNA was inverted to maintain male reproductive capacity). The human DNA in the third figure is derived from chromosomes 7:142237898-7:142813740 (GRCh38). This represents an insertion of 576 kbp of human DNA, containing the 520 kbp fragment shown in the middle figure. A 5.4 Mbp region of the mouse genomic DNA is inverted, creating a 4.8 Mbp space between the first mouse Vβ gene fragment (vβ1) and the first human Vβ gene fragment (vβ2). The inserted human DNA may contain 48 functional Vβ gene fragments and 13 functional Jβ gene fragments (…). Figure 2a Alternatively, it may contain 43 functional Vβ gene fragments and 13 functional Jβ gene fragments. Figure 2b ).

[0021] Figures 3a / b illustrate replacing the mouse CD8α-coding DNA in the mouse genome with human CD8α-coding DNA, and replacing the mouse CD8β-coding DNA in the mouse genome with human CD8β-coding DNA. This produces a chimeric CD8 polypeptide. Figure 3b illustrates in more detail one method for preparing the genome shown in Figure 3a. Figure 3a(ii) contains corrections to the genomic coordinates shown in Figure 3a(i); Figure 3b(ii) contains corrections to the genomic coordinates shown in Figure 3b(i).

[0022] Figure 4 A proposed scheme is presented: This scheme allows for the replacement of mouse CD8α-coding DNA in the mouse genome with human CD8α DNA, and mouse CD8β-coding DNA with human CD8β DNA. This would produce a fully human CD8 polypeptide.

[0023] Figures 5a / b illustrate the replacement of the mouse β2 microglobulin-encoding DNA in the mouse genome with DNA encoding human β2 microglobulin. Figure 5b illustrates in more detail one method for preparing the genome shown in Figure 5a. Figure 5a(ii) shows a modified version of the genome coordinates shown in Figure 5a(i); Figure 5b(ii) shows a modified version of the genome coordinates shown in Figure 5b(i).

[0024] Figure 6a / b shows the replacement of the mouse H2-D1 gene in the mouse genome with the human HLA_A*02:01MHC class I gene. Figure 6b The preparation is shown in more detail. Figure 6a One method of the genome shown.

[0025] Figure 7The first image shows the MHC class I region in rodents; the second image shows H2-K deletion and the human HLA-A replacing the H2-D knock-in site.

[0026] Figure 8 This diagram illustrates the immune synapse between T cells (bottom) and target cells (top). The TCR crosses the synapse and binds to the pMHC complex between adjacent cell membranes. Class I MHC consists of an α chain containing α1, α2, and α3 domains; the N-terminal α1 and α2 domains provide binding sites for peptide p; β2 microglobulin (β2m) binds to this α chain. The TCR consists of α and β chains, each containing an N-terminal variable region, a constant region, a linker region, a transmembrane region, and a short cytoplasmic C-terminal tail; the TCR forms a complex with the CD3 polypeptide. The T cell's dimerizing co-receptor CD8 binds to the α3 domain of the MHC class I molecule; CD8 is a heterodimer of the α and β chains and binds to the intracellular signaling molecule Lck. This diagram was created using BioRender.

[0027] Figure 9 This image shows the TCR in the cell membrane. The image was created using BioRender.

[0028] Figure 10 This diagram shows TRBV and TRBJ transcripts detected from batch sequencing of spleens from representative humanized TRB mice. The data shown represent the number of unique molecular identifiers (UMIs), not the total number of reads; only counts of functional TRBV and TRBJ are shown; counts of non-functional genes are typically zero (but not always).

[0029] Figure 11 The image shows TRBVUMI data obtained from bulk sequencing of spleen RNA.

[0030] Figure 12 The image shows TRBJUMI data obtained from bulk sequencing of spleen RNA.

[0031] Figure 13 This diagram shows TRAV and TRAJ transcripts detected from batch sequencing of spleens from representative humanized TRA mice. The data shown represent the number of unique molecular identifiers (UMIs), not the total number of reads; only counts of functional TRAVs and TRAJs are displayed. Counts of non-functional genes are typically zero (but not always).

[0032] Figure 14 This shows TRAVUMI data obtained from bulk sequencing of spleen RNA.

[0033] Figure 15 This shows TRAJUMI data obtained from bulk sequencing of spleen RNA.

[0034] Figure 16 This study shows a comparison of the relative usage of TRBJ1-C1 and TRBJ2-C2 in transgenic mouse transcripts with reported data in humans. The results for transgenic mice were derived from bulk sequencing of spleen RNA from OpTiMusTRB transgenic mice.

[0035] Figure 17 Compare publicly available data on TRBV expression in the spleen RNA of human TRB transgenic mice with that of human PBMCs.

[0036] Figure 18 Compare publicly available data on TRBJ expression in the spleen RNA of human TRB transgenic mice with that in human PBMCs.

[0037] Figure 19 Compare the distribution of T cell receptor CDR3 nucleotide lengths: (A) TRB in humans (Kitaura et al., 2016) (B) TRB in transgenic mice (C) TRA in humans (Kitaura et al., 2016) (D) TRA in transgenic mice.

[0038] Figure 20 Compare publicly available data on TRAV expression in the spleen RNA of human TRA transgenic mice with those in human PBMCs.

[0039] Figure 21 Compare publicly available data on TRAJ expression in the spleen of human TRA transgenic mice with that in human PBMCs.

[0040] Figure 22 shows the use of (a) TRAV, (b) TRAJ, (c) TRBV, and (d) TRBJ genes in hTCR transgenic mice. “I” indicates reported gene expression; “0” indicates reported gene loss; “~” indicates reported low expression (<0.01%); “?” indicates lack of reported data.

[0041] Figure 23 The image shows cells expressing mouse CD3 (vertical axis) and human CD8b (horizontal axis) on the surface, detected by flow cytometry (FACS) in various conjugated OpTiMusTRA / TRB transgenic mice.

[0042] Figure 24 The image shows spleen cells with surface expression of mCD3, mCD4, hCD8b, hTRBC-1, and mCD19 detected in holozygous OpTiMus mice 8. Data represent results from three mice (mice 8, 9, and 10).

[0043] Figure 25 Spleen cell populations of OpTiMus mice (mice 8, 9, and 10) that were not exposed to the antigen are shown.

[0044] Figure 26a / 26b shows a comparison of representative data from mice immunized with peptides with data from control mice that were not exposed to the antigen.

[0045] Figure 27 The immune response in hTRA+ / + (homozygous) and hTRB+ / - (heterozygous) mice after peptide immunization is shown.

[0046] Figure 28 The immune response in hTRA+ / +hTRB+ / + homozygous mice after peptide immunization is shown.

[0047] Figure 29 The yields of 124 soluble TCR-Fc fusion peptides generated by recombinant expression in Expi293T cells are shown; each dot represents one TCR.

[0048] The accompanying drawings show some preferred coordinates for mouse genome modification, but these coordinates do not constitute a limitation of the present invention.

[0049] Specifically, an insert of human DNA with the following coordinates is prepared (the inserted human DNA contains all numbered nucleotides, and the mouse genome retains the numbered nucleotides in the genome).

[0050] Tra Human DNA originates from location 14:21,570,693-14:22,554,820 (GRCh38). Human DNA was inserted into the mouse (GRCm39) at positions 14:52,664,870 or 14:52,664,818 (depending on the mouse strain) and between 14:54,463,673.

[0051] Trb Human DNA originates from location 7:142,237,898-7:142,813,740 (GRCh38). Human DNA was inserted into mouse loci between 6:36,074,375 and 6:41,535,764 (GRCm39). Mouse DNA between 6:36,070,006 and 6:36,074,375 undergoes an inversion from 6:40,868,163 to 6:41,515,110.

[0052] MHC Class I Human DNA originates from position 6:29,942,554-6:29,945,455 (GRCh38). Human DNA was inserted into the mouse loci between 17:35,482,089 and 17:35,485,847 (GRCm39).

[0053] CD8A (Figure 3a(i)) Human DNA originates from location 2:86,789,408-2:86,790,726 (GRCh38); Human DNA was inserted into mouse loci between 6:71,350,536 and 6:71,351,769 (GRCm39).

[0054] CD8A (Figure 3a(ii)) Human DNA originates from location 2:86,790,825-2:86,789,408 (GRCh38); Human DNA was inserted into mouse loci between 6:71,350,536 and 6:71,351,769 (GRCm39).

[0055] CD8B (Figure 3a(i)) Human DNA originates from location 2:86,861,751-2:86,853,004 (GRCh38). Human DNA was inserted into mouse loci between 6:71,299,840 and 6:71,306,867 (GRCm39).

[0056] CD8B (Figure 3a(ii)) Human DNA originated from location 2:86,861,865-2:86,853,004 (GRCh38); human DNA was inserted into mouse locations between 6:71,299,840 and 6:71,306,770 (GRCm39).

[0057] B2M (Figure 5a(i)) Human DNA was derived from positions 15:44,711,547–15:44,716,342 (GRCh38); human DNA was inserted into mouse positions 2:121,978,219 and 2:121,982,235 (GRCm39).

[0058] B2M (Figure 5a(ii)) Human DNA was derived from positions 15:44,711,547–15:44,716,342 (GRCh38); human DNA was inserted into mouse positions 2:121,978,218 and 2:121,982,142 (GRCm39).

[0059] The accompanying drawings are not drawn to scale, but use / / or / / / to indicate regions of chromosomes not yet included in the drawings.

[0060] In all the attached images: Gray = mouse Black = Human Enh = Enhancer The unshaded white box at the 5' end of an exon (e.g., Figure 3a) is the 5' untranslated region, which is part of the exon but not part of the coding sequence.

[0061] Implementation This invention relates to rodents and rodent cells comprising human nucleic acids encoding one or more polypeptide components of human T-cell molecular apparatuses, said human nucleic acids being inserted into an endogenous mouse locus of that component in the rodent genome. Specifically, these rodents or cells comprise human nucleic acids encoding one or more (and preferably all) of the following components: human TCRα polypeptide, human TCRβ polypeptide, human MHC class I polypeptide, human CD8α polypeptide, human CD8β polypeptide, and human β2 microglobulin polypeptide. In another aspect, these rodents or rodent cells also comprise human nucleic acids encoding a human TCRδ polypeptide. The TCRα polypeptide is encoded by the TRA locus, and the TCRβ polypeptide is encoded by the TRB locus; these loci may also be referred to herein as the TCRα and TCRβ loci.

[0062] When human TCRs (containing human TCRα and human TCRβ peptides) are expressed on the cell surface, these TCRs interact with peptides presented by MHC molecules on antigen-presenting cells. The α1 and α2 domains of MHC class I interact with the TCR. The α3 domain of MHC class I binds to CD8 molecules (which are co-receptors for the TCR). These regions of MHC class I and / or the CD8 co-receptor can be genetically engineered to achieve efficient peptide presentation to the human TCR, as described herein.

[0063] As described and asserted herein, a TCR (preferably a fully human TCR) comprising human TCRα and / or human TCRβ peptides can be combined in the genome with any of the other alleles described herein (which may be of human, chimeric, or mouse origin). The examples of other alleles described herein are merely optional examples, and other suitable alleles can be identified from common knowledge and / or earlier published literature regarding these (human / chimeric / mouse) alleles.

[0064] Other examples of alleles that can be used with TCR include the alleles described in the following publications and patent applications, which are incorporated herein by reference: Chimaeric MHC (HHD construct with human beta2m(B2m) fused to MHCcomprising human alpha1, human alpha2 and mouse alpha3, TM and cytoplasmicregions). 1997 J. Exp. Med. 185(12):2043–2051, HLA-A2.1–restricted Educationand Cytolytic Activity of CD8-T Lymphocytes from beta2-microglobulin (B2m)HLA-A2.1 Monochain Transgenic H-2Db B2m Double Knockout Mice. Steve Pascolo, Nathalie Bervas, Jan M. Ure, Austin G. Smith, François A. Lemonnier, and Béatrice Pérarnau.

[0065] The MHC class I and / or CD8 alleles described in WO02 / 059263.

[0066] The MHC class I, B2m, and / or CD8 alleles are described in US2005066375 (WO03006639A1).

[0067] The MHC class I and / or B2m alleles in ABabDII mice are described in the following literature: (i) 2010 Nature Medicine 16(9):1029 Transgenic mice with a diversehuman T cell antigen receptor repertoire, Liang-Ping Li, J Christoph Lampert,Xiaojing Chen, Catarina Leitao, Jelena Popović, Werner Müller&Thomas Blankenstein; (ii) 2015 Nature Biotechnology 33(4):402-407. Identification of humanT-cell receptors with optimal affinity to cancer antigens using antigen-negative humanized mice Matthias Obenaus, Catarina Leitão, MatthiasLeisegang, Xiaojing Chen, Ioannis Gavvovidis, Pierre van der Bruggen,Wolfgang Uckert, Dolores J Schendel&Thomas Blankenstein.

[0068] The MHC class I and CD8 alleles described in WO2014 / 130671.

[0069] MHCI in 2016 Cancer Immunology Research 4(3):204, “Identification of T-cell Receptors Targeting KRAS-Mutated Human Tumors”. Qiong J. Wang, ZhiyaYu, Kayla Griffith, Ken-ichi Hanada, Nicholas P. Restifo, and James C. Yang.

[0070] The MHC class I, B2m, and CD8 alleles described in Moore et al., SCIENCE IMMUNOLOGY “Humanization of T cell–mediated immunity in mice”, 6(66) 2021.

[0071] Suitable MHCI and B2m alleles as described in WO2021139799.

[0072] It is highly desirable to use all or most of the human V, D, and J gene fragments inserted into the expressed human TCR chain in rodents. In this way, a complete library of human TCR molecules can be potentially expressed in the rodent. It has been demonstrated that rodents expressing human TCRs, as described herein, utilize a large number of inserted human α and β gene fragments to generate TCR chains.

[0073] Furthermore, it has been demonstrated that the rodents of this invention can express TCRβDJC2 derived from the inserted fully human TrbDNA, without the need for mouse intron sequences.

[0074] Therefore, the present invention relates to a rodent that expresses at least 35, at least 40, at least 41, at least 42, at least 43, at least 44, or all 45 of the inserted human variable Vα gene fragments in its own TCRα chain library.

[0075] Therefore, the present invention relates to a rodent that expresses at least 35, at least 40, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, or all 51 of the inserted human Jα gene fragments in its own TCRα chain library.

[0076] Therefore, the present invention relates to a rodent that expresses at least 35, at least 40, at least 45, at least 46, at least 47, or all 48 of the inserted human variable Vβ gene fragments in its own TCRβ chain library.

[0077] Therefore, the present invention relates to a rodent that expresses at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, or all 13 of the inserted human Jβ gene fragments in its own TCRβ chain library.

[0078] Therefore, the present invention relates to a rodent having a library comprising a TCRβ chain expressed from both the TRBDJC1 and DJC2 gene clusters; suitably, wherein the inserted human DNA does not contain mouse intron sequences, for example, mouse intron sequences are not present between human D or J gene fragments present in the genome.

[0079] A preferred aspect of the present invention relates to a genetically modified rodent comprising: (i) An unrearranged T-cell receptor (TCR) α variable locus comprising at least one human Vα segment and at least one human Jα segment, wherein the TCRα variable gene is operatively linked to a human TCRα constant gene sequence. (ii) An unrearranged TCRβ variable locus comprising at least one human Vβ segment, at least one human Dβ segment and at least one human Jβ segment, wherein the unrearranged TCRβ variable gene is operatively linked to a human TCRβ constant gene sequence. Among them, the aforementioned unrearranged human T cell variable region gene fragments can be rearranged to form a gene encoding the variable domain of the human T cell receptor; and the unrearranged TCRα variable locus is located at the endogenous rodent TCRα locus, and the unrearranged TCRβ variable locus is located at the endogenous rodent TCRβ locus.

[0080] In a preferred aspect, a T-cell receptor expressed by rodents or rodent cells is capable of interacting with peptides presented on MHC class I molecules within the rodent body, thereby appropriately activating and proliferating the T cells. In another preferred aspect, a CD8 protein expressed by the rodent or rodent cells is capable of binding to MHC class I molecules expressed by the rodent. Preferably, the regions of the interacting CD8 and MHC class I peptides originate from the same species, for example, both are human or both are rodent-derived.

[0081] Other preferred aspects of the invention include: A rodent whose genome encodes an MHC class I polypeptide, wherein the α1 and α2 domains of the MHC class I polypeptide are of human origin.

[0082] A rodent wherein the MHC class I polypeptide contains an α3 domain capable of binding to CD8 expressed in rodents.

[0083] A rodent in which the α3 domain is of human origin and CD8 contains a human MHC class I binding domain.

[0084] A rodent in which CD8 contains human CD8α.

[0085] A rodent in which CD8 comprises a human-rodent chimeric CD8α.

[0086] A rodent in which CD8 includes human CD8β.

[0087] A rodent in which CD8 comprises a human-rodent chimeric CD8β.

[0088] A rodent whose genome contains a human or human-rodent chimeric CD8 gene locus at the endogenous rodent CD8 locus.

[0089] A rodent in which an MHC class I polypeptide contains a human transmembrane domain and a cytoplasmic domain.

[0090] A rodent in which the MHC class I α3 domain is of rodent origin.

[0091] A rodent in which CD8 comprises endogenous rodent CD8.

[0092] A rodent in which DNA encoding an MHC class I polypeptide is integrated at an endogenous rodent MHC class I locus.

[0093] The present invention also relates to methods for preparing these rodents and cells, and the use of these rodents and cells in producing molecules of therapeutic use (e.g., human T-cell receptors produced in rodents as disclosed herein), including obtaining the nucleic acid sequence of a human TCR from rodents and expressing all or a variable portion or binding portion of the human TCR in cells (e.g., production cell lines, such as CHO cells) or human or animal cells. Alternatively, the nucleic acid encoding the human TCR or a variable portion or binding portion of the TCR can be appropriately formulated for delivery as a molecule of therapeutic use.

[0094] In some respects, a nucleic acid encoding a fully human polypeptide is provided in rodents or rodent cells, which is operatively linked to one or more endogenous rodent regulatory sequences, such as rodent promoters, enhancers or other regulatory sequences.

[0095] The rodents or rodent cells of the present invention preferably do not express endogenous rodent polypeptides from rodent loci modified to express equivalent human polypeptides. In this way, the inserted human sequence can form most or all of the expressed polypeptide in or from the rodent or cell. For example, in one aspect, the inserted human TCRα polypeptide is expressed from an endogenous TRA locus, but the endogenous rodent TCRα polypeptide is not expressed from that locus.

[0096] In the above preferred aspects: Preferably, the rodents or cells disclosed herein do not express functional endogenous TCRα polypeptides from endogenous TCRα variable loci; Preferably, the rodents or cells disclosed herein do not express functional endogenous TCRβ polypeptides from endogenous TCRβ variable loci; Preferably, the rodents or cells disclosed herein do not express functional endogenous rodent CD8 co-receptors from the endogenous CD8 locus; Preferably, the rodents or cells disclosed herein do not express functional endogenous rodent MHC class I polypeptides from endogenous MHC class I loci; and / or Preferably, when the rodent or rodent cell disclosed herein contains a nucleic acid sequence encoding human β2 microglobulin, the rodent or cell does not express functional endogenous rodent β2 microglobulin from the endogenous rodent β2 microglobulin locus.

[0097] The term "does not express / no expression" for endogenous peptides refers to the absence of significant / abundant expression of rodent peptides, typically not exceeding 10% of the wild-type expression level of endogenous rodent peptides. Preferably, the modified locus does not express rodent peptides and has completely blocked the expression of host peptides from the modified locus. In this way, the expression of the human equivalent peptide inserted at the rodent locus can be maximized.

[0098] In one aspect, the assessment of expression levels is considered at the level of the modified loci, and therefore expression from other unmodified alleles may be present. However, preferably, endogenous polypeptides are not expressed, which may result, for example, from homozygous modification of two rodent loci. This applies independently to each modified locus. Thus, in one aspect, one or more loci mentioned herein may be homozygous for the insertion of a human polypeptide, and no endogenous polypeptide corresponding to any allele is expressed at each locus. In one aspect, one, two, three, four, five, six, or all seven loci disclosed herein (loci encoding TCRα polypeptide, TCRβ polypeptide, MHC class I polypeptide (including those with knockout or inactivation modifications of both rodent H2K and H2D loci), human CD8α polypeptide, human CD8β polypeptide, and human β2 microglobulin polypeptide) may be homozygous. In another aspect, one or more loci disclosed herein may be heterozygous, where only one allele is modified. In one respect, the 1, 2, 3, 4, 5 or all 6 loci disclosed in this article may be heterozygous.

[0099] In some respects, the nucleic acid loci encoding rodent polypeptides that are equivalent to the human polypeptides described herein are completely or partially deleted in the rodent genome to prevent the expression of the host polypeptide; or endogenous loci are inactivated through inversion or other means.

[0100] Preferably, the inversion moves the rodent DNA (e.g., the rodent V gene fragment) away from the natural locus by at least 0.5 Mb, for example, at least 1 Mb, at least 1.5 Mb, at least 2 Mb, at least 2.5 Mb, at least 3 Mb, at least 3.5 Mb, at least 4 Mb, or at least 4.5 Mb or more.

[0101] The preferred rodents express all of the following components: human TCRα peptide, TCRβ peptide, MHC class I peptide, CD8α peptide, CD8β peptide and β2 microglobulin peptide, and do not express rodent TCRα peptide, TCRβ peptide, MHC class I peptide, CD8α peptide, CD8β peptide and β2 microglobulin peptide.

[0102] Other preferred rodent expressions: At least human TCRα peptide and TCRβ peptide to form a functional TCR; Human TCRα peptide, TCRβ peptide, MHC class I peptide, CD8α peptide and CD8β peptide.

[0103] In one aspect, the genome of rodents or rodent cells contains a fully human TCR after rearrangement of the TCR gene fragment. Therefore, this fully human TCR can be expressed in rodents or cells without further humanization, meaning any potential impact from subsequent humanization processes (e.g., subsequent replacement of mouse constant regions with human constant regions). This method minimizes the impact of structural differences between chimeric TCR molecules and fully human TCRs arising from the interaction between the human Vα / Vβ domain and the mouse Cα / Cβ domain in the chimeric molecule.

[0104] The insertion of human nucleic acids is targeted, inserting into endogenous rodent loci in the rodent genome, which makes it possible to combine naturally located endogenous regulatory sequences with inserted human DNA.

[0105] Human TCRδ gene fragments can also be inserted to allow TCRδ chain expression and the expression of TCRδγ with at least the human δ chain.

[0106] TCRα This article provides genetically modified rodents or rodent cells (such as ES cells or T cells) comprising: an unrearranged T cell receptor (TCR) α variable locus, the locus comprising at least one human Vα segment and at least one human Jα segment, wherein the TCRα variable gene is operatively linked to a human TCRα constant gene sequence; the aforementioned unrearranged human T cell variable region gene segment is capable of rearranging to form a gene encoding a human T cell receptor variable domain; and the unrearranged TCRα variable locus is located at an endogenous rodent TCRα locus.

[0107] In one aspect of the invention, the T-cell helper receptor polypeptide is expressed only on rodent T cells, for example, not on rodent B cells.

[0108] In one aspect of the invention, T-cell helper receptor peptides α and / or β are present in the reproductive system of rodents.

[0109] In one respect, rodents or rodent cells may be heterozygous for the nucleotide sequence encoding the human TCRα polypeptide.

[0110] In one respect, rodents or rodent cells may be homozygous for the nucleotide sequence encoding the human TCRα polypeptide.

[0111] In one aspect, the inserted human TCRα nucleic acid contains up to or at least 5, 10, 15, 20, 25, 30, 35, 40, or more TCRVα gene fragments, such as up to 44, 45, or at least 44, 45 Vα gene fragments. In another aspect, the inserted mouse TCRα nucleic acid contains up to or at least 5, 10, 15, 20, 25, 30, 35, 40, 44, or 45 functional TCRVα gene fragments. The inserted TCRα nucleic acid may also contain non-functional TCRVα gene fragments or pseudogenes.

[0112] In one aspect, the inserted human TCRα nucleic acid contains up to or at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or 51 TCRJα gene fragments. In another aspect, the inserted mouse TCRα nucleic acid contains up to or at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or 51 functional Jα gene fragments. The inserted TCRα nucleic acid may also contain non-functional TCRJα gene fragments or pseudogenes.

[0113] In one respect, the unrearranged TCRα variable locus contains a complete library of the human Jα fragment and a complete library of the human Vα fragment.

[0114] In one aspect, the inserted human TCRα nucleic acid contains all functional TCRVα gene segments and all functional Jα gene segments. The inserted human TCRα nucleic acid may contain 44 functional TCRVα gene segments and 50 functional Jα gene segments. In another aspect, the inserted human TCRα nucleic acid contains 45 functional TCRVα gene segments and 51 functional Jα gene segments. In one aspect, the rodent is a mouse.

[0115] The TCRVα gene fragment in the transgenic locus is optionally selected from the TRAV fragments listed in Table 1. The TCRα variable locus may contain some or all of the TRAV gene fragments listed in Table 1, preferably including all functional TRAV gene fragments. TRAV7 and / or TRAV18 may be absent; if present, they may not be expressed. Optionally, the TCRα variable locus may contain any, more, or all of the following TRAV gene fragments, which are capable of rearranging with TRAJ gene fragments to form a gene encoding the variable domain of the human T cell receptor: TRAV1-1, TRAV1-2, TRAV2, TRAV3, TRAV4, TRAV5, TRAV6, TRAV8-1, TRAV8-2, TRAV8-3, TRAV8-4, TRAV8-6, TRAV9-1, TRAV9-2, TRAV10, TRAV12-1, TRAV12-2. TRAV12-3, TRAV13-1, TRAV13-2, TRAV14 / DV4, TRAV16, TRAV17, TRAV19, TRAV20, TRAV21, TRAV22, TRAV23 / DV6, TRAV24, TRAV25 , TRAV26-1, TRAV26-2, TRAV27, TRAV29 / DV5, TRAV30, TRAV34, TRAV36 / DV7, TRAV38-1, TRAV38-2 / DV8, TRAV39, TRAV40, TRAV41.

[0116] The TCRJα gene fragment in the transgenic locus may be selected from the TRAJ fragments listed in Table 1 as needed. The inserted human TCRα nucleic acid may contain some or all of the TRAJ gene fragments listed in Table 1, preferably including all functional TRAJ gene fragments. Optionally, the TCRα variable locus may contain any, more, or all of the following TRAJ gene fragments, which are capable of rearranging with TRAV gene fragments to form a gene encoding the variable domain of the human T cell receptor: TRAJ3, TRAJ4, TRAJ5, TRAJ6, TRAJ7, TRAJ9, TRAJ10, TRAJ11, TRAJ12, TRAJ13, TRAJ14, TRAJ15, TRAJ16, TRAJ17, TRAJ18, TRAJ20, TRAJ21, TRAJ22, TRAJ23, TRAJ24. 4. TRAJ26, TRAJ27, TRAJ28, TRAJ29, TRAJ30, TRAJ31, TRAJ32, TRAJ33, TRAJ34, TRAJ35, TRAJ36, TRAJ37, TRAJ38, TRAJ39, TRAJ40 , TRAJ41, TRAJ42, TRAJ43, TRAJ44, TRAJ45, TRAJ46, TRAJ47, TRAJ48, TRAJ49, TRAJ50, TRAJ52, TRAJ53, TRAJ54, TRAJ56, TRAJ57.

[0117] In one respect, the human TCRα nucleic acid inserted into the mouse genome forms a continuous human insert fragment.

[0118] In one respect, the missing TCRαDNA includes genomic DNA from Vα1 to TCRαC (inclusive).

[0119] In one respect, the inserted human DNA includes DNA from human Vα1 to TCRαC (inclusive).

[0120] In one respect, the TCRα constant region is the human TCRα constant region.

[0121] In one aspect, the inserted human TCRα locus is operatively linked to a rodent enhancer located downstream (3' end) of the rodent TCRα constant region in the rodent genome, preferably a rodent enhancer at its native location in the genome. In another aspect, unrearranged human T cell variable region TCRαV and J gene fragments are operatively linked to an endogenous rodent TCRα enhancer downstream of the TCRα constant region.

[0122] In one aspect, the rodent or rodent cell contains or retains endogenous rodent promoters and / or other regulatory elements that regulate the expression of the human TCRα locus. In one aspect, the rodent or rodent cell provides a nucleic acid encoding a fully human polypeptide, operatively linked to one or more endogenous rodent regulatory sequences, such as rodent promoters, enhancers, or other regulatory sequences. In a preferred aspect, one or more promoters of the inserted human TCRα human V gene segment are human. In one aspect, the expression of one or more human V or J gene segments of the TCRα locus is regulated by a human promoter. In one aspect, all V promoters are human.

[0123] In one respect, there is no functional endogenous rodent TCRα variable locus, for example, caused by deletions in the rodent genome, such as (a) deletions of all endogenous Vα gene segments, (b) deletions of all endogenous Jα gene segments, or (c) a combination of the two.

[0124] In one aspect, the endogenous rodent TCRα locus is deleting the distal 3' end V (Vα1) from the rodent genome into the rodent 3' end constant region TCRαC (inclusive), thereby operatively linking the rodent enhancer downstream of TCRαC in the rodent genome to the unrearranged human T cell receptor (TCR)α variable locus.

[0125] In a preferred aspect, the rodent is a mouse, and the enhancer at the 3' end of the constant region is a mouse enhancer.

[0126] In one aspect, rodents or rodent cells retain endogenous TCRα variable loci and / or endogenous rodent TCRβ variable loci, wherein any retained endogenous rodent TCRα variable locus is a nonfunctional locus and any retained endogenous rodent TCRβ variable locus is a nonfunctional locus.

[0127] In a preferred aspect, there is no rodent TCRα constant region at the rodent TCRα locus.

[0128] In one respect, this locus exhibits appropriate spatial and temporal protein expression and supports T cell development and selection.

[0129] TCRδ In one aspect, the rodents or rodent cells (such as ES cells or T cells) disclosed herein contain a library of unrearranged human TCRδ variable region fragments at the rodent TCRα locus.

[0130] In one aspect, the inserted human TCRα locus contains an unrearranged T-cell receptor (TCR)δ variable locus, which contains at least one human Vδ fragment, at least one Jδ gene fragment, and at least one human Jα fragment, wherein these TCRδ variable gene fragments are operatively linked to a human TCRδ constant gene sequence.

[0131] In one respect, the genome of rodents or rodent cells contains a complete library of human Vδ, Dδ, and Jδ gene fragments.

[0132] In one aspect, the rodents or rodent cells disclosed herein contain a complete library of the human Vδ fragment, a complete library of the human Dδ fragment, and a complete library of the human Jδ fragment at the human TCRα variable locus.

[0133] In one respect, the rodents or rodent cells disclosed herein contain a fully human Vδ constant region, and furthermore, a rodent Vδ constant region is not present at the TCRα variable locus.

[0134] The DNA encoding TCRδ is naturally present within the TCRα locus, so insertion into the entire human TCRA locus would naturally result in the insertion of a human TCRδ gene segment (including the δ constant region). In one respect, the inserted human DNA includes DNA from human Vα-1 to TCRαC (inclusive), so that all human TCRδ will be included in the rodent genome.

[0135] Even if the entire TCRα locus is not inserted, the human TCRδ nucleic acid described herein can still be inserted into the genome. Insertion into the TCRα locus is preferred.

[0136] In a preferred aspect, the genome of a rodent or rodent cell does not contain the rodent TCRδ constant region.

[0137] In one respect, the human TCRδ locus is operatively linked to a human enhancer and / or a human promoter. In another respect, all promoters of the TCRδV gene fragment are of human origin.

[0138] In one respect, the enhancer at the 5' end of the constant region (TRDC) is human-derived.

[0139] The rodent or rodent cell preferably contains the unrearranged human TCRδ locus at the unrearranged human TCRα locus.

[0140] In one respect, as disclosed herein, in modified rodents or rodent cells, unrearranged TCRα variable loci replace all or part of the endogenous rodent TCRα locus.

[0141] In one respect, human TCRδ nucleic acids inserted into the rodent genome form continuous human insert fragments.

[0142] In one respect, this locus exhibits appropriate spatial and temporal protein expression and supports T cell development and selection.

[0143] TCRβ This article provides a genetically modified rodent or rodent cell (such as ES cells or T cells) comprising an unrearranged TCRβ variable locus containing at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment, wherein the unrearranged TCRβ variable gene is operatively linked to a human TCRβ constant gene sequence; the aforementioned unrearranged human T cell variable region gene segment is capable of rearranging to form a gene encoding a variable domain of a human T cell receptor; and the unrearranged TCRβ variable locus is located at an endogenous rodent TCRβ locus.

[0144] In one aspect, the human TCRβ locus is operatively linked to a rodent enhancer that is naturally located downstream (3' end) of the rodent TCRβ constant region in wild-type rodents. Appropriately, this enhancer is located at its natural position in the rodent genome.

[0145] In one respect, the human TCRβ locus is operatively linked to a human enhancer, which is typically located downstream of the human TCRβC2 constant region in the human locus.

[0146] In one respect, the promoter of one or more human V gene fragments of the inserted human TCRα is human. In another respect, all promoters are human.

[0147] In one respect, the rodents or rodent cells disclosed herein do not contain rodent Vβ1 gene fragments.

[0148] In one aspect, the human insert comprises human DNA from Vβ1 to Vβ30 (inclusive). In another aspect, the human insert contains human DNA from human Prss58 to Vβ30 (inclusive). In yet another aspect, the inserted DNA comprises one or more human genomic segments.

[0149] In one respect, the inserted human TCRβ nucleic acid contains at most or at least, or exactly 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 43, 45, 46, 47, 48 or more gene fragments. The inserted TCRβ nucleic acid may also contain non-functional TCRVβ gene fragments or pseudogenes.

[0150] In one respect, the inserted human TCRβ nucleic acid contains at most or at least, or exactly, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or thirteen Jβ gene fragments. The inserted TCRβ nucleic acid may also contain non-functional TCRJβ gene fragments or pseudogenes.

[0151] In one respect, the inserted human TCRβ nucleic acid contains one or two Dβ gene fragments.

[0152] In one respect, the inserted human TCRβ nucleic acid contains gene fragments from Dβ1 to Jβ1.1-1.6 and from Dβ2 to Jβ2.1-2.7.

[0153] The inserted human TCRβ nucleic acid may contain 43 functional TCRVβ gene segments and 13 functional Jβ gene segments. The rodent may be a mouse. In one aspect, the rodent is a mouse, and the inserted human TCRβ nucleic acid contains 48 functional TCRVβ gene segments and 13 functional Jβ gene segments.

[0154] The TCRVβ gene fragment in the transgenic locus may be selected from the TRBV fragments listed in Table 1 as needed. The TCRβ variable locus may contain some or all of the TRBV gene fragments listed in Table 1, preferably including all functional TRBV gene fragments. Optionally, the TCRβ variable locus may contain any, more, or all of the following TRBV gene fragments, which can be rearranged with the TRBJ gene fragment to form a gene encoding the variable domain of the human T cell receptor: TRBV2, TRBV3-1, TRBV4-1, TRBV4-2, TRBV5-1, TRBV5-4, TRBV5-5, TRBV5-6, TRBV6-1, TRBV6-2, TRBV6-4, TRBV6-5, TRBV6-6, TRBV6-8, TRBV7-2, TRBV7-3, TRBV7- 4. TRBV7-6, TRBV7-7, TRBV7-9, TRBV9, TRBV10-1, TRBV10-2, TRBV10-3, TRBV11-1, TRBV11-2, TRBV11-3, TRBV12-3, TRBV12-4, TR BV12-5, TRBV13, TRBV14, TRBV15, TRBV16, TRBV18, TRBV19, TRBV20-1, TRBV24-1, TRBV25-1, TRBV27, TRBV28, TRBV29-1, TRBV30.

[0155] The TCRJβ gene fragment in the transgenic locus may be selected from the TRBJ fragments listed in Table 1 as needed. The inserted human TCRβ nucleic acid may contain some or all of the TRBJ gene fragments listed in Table 1, preferably including all functional TRBJ gene fragments. Optionally, the TCRβ variable locus may contain any, more, or all of the following TRBJ gene fragments, which can be rearranged with TRBV gene fragments to form a gene encoding the variable domain of the human T cell receptor: TRBJ1-1, TRBJ1-2, TRBJ1-3, TRBJ1-4, TRBJ1-5, TRBJ1-6, TRBJ2-1, TRBJ2-2, TRBJ2-3, TRBJ2-4, TRBJ2-5, TRBJ2-6, and TRBJ2-7.

[0156] In one respect, the inserted human TCRβ nucleic acid contains all functional TCRVβ gene fragments and all functional Jβ gene fragments.

[0157] In one respect, rodents are mice.

[0158] In one respect, the inserted human TCRβ nucleic acid contains human TRBC1 and human TCRBC2.

[0159] In one respect, the human TCRβ nucleic acid inserted into the mouse genome forms a continuous human insert fragment.

[0160] In one respect, rodents may be heterozygous for the nucleotide sequence encoding the human TCRβ polypeptide.

[0161] In one respect, rodents may be homozygous for the nucleotide sequence encoding the human TCRβ polypeptide.

[0162] In one aspect, the rodent or rodent cell contains or retains endogenous rodent promoters, enhancers, and / or other regulatory elements that regulate the expression of the human TCRβ locus. In another aspect, the rodent or rodent cell provides a nucleic acid encoding a fully human polypeptide, which is operatively linked to one or more endogenous rodent regulatory sequences, such as rodent promoters, enhancers, or other regulatory sequences.

[0163] In one aspect, the rodent is a mouse, and the enhancers at the 3' end and Vβ315' end of the constant region (TRBC) are mouse enhancers.

[0164] In one respect, the inserted human loci exhibit appropriate spatial and temporal protein expression and support T cell development and selection.

[0165] In one respect, there is no functional endogenous rodent TCRβ variable locus, for example, caused by deletions in the rodent genome, such as (a) deletions of all endogenous Vβ gene segments, (b) deletions of all endogenous Jβ gene segments, (c) deletions of all endogenous Jδ gene segments, (d) deletions of the host rodent constant region, or (e) any combination of the above.

[0166] Preferably, both the rodent TCRα and TCRβ loci have deletions as described herein, or one or both are inactivated by inversions as described herein.

[0167] In one aspect, rodents retain endogenous TCRβ variable loci, wherein the retained endogenous rodent TCRβ variable loci are nonfunctional loci, for example, endogenous loci that have been inverted.

[0168] Reproductive capacity In one aspect, the present invention is based on the discovery that the TCRβ locus contains genes that affect mouse fertility.

[0169] We have observed (see Example 1) that mice lacking the TCRβ locus to prevent host TCRβ chain expression have lower fertility than mice that retain the inverted TCRβ variable locus (the latter also do not produce the TCRβ chain, but retain the DNA of the locus in their genome, some of which is inverted relative to the wild type).

[0170] Specifically, homozygous mice with duplicated TCRβ loci (human TRβVJ1D1C1J2J2C2 and mouse Trβv1J1D1) have better reproductive capacity than mice with deletions.

[0171] Analysis of the TCRβ locus suggests that fertility problems are likely caused by the deletion of one or all of the serine proteases Prss58, Prss59, Prss3b, Try4, Try5, Try10, Prss3, Prss1, and Prss2 located in the deletion region of the TCRβ locus.

[0172] Knocking out PRSS58 alone does not cause reproductive problems in mice (Park et al., Biology of Reproduction, 2020, 103(2):195-204), therefore it is believed that the deletion of this gene is not the cause of the observed decline in fertility, or is not the only cause.

[0173] Therefore, other aspects of the invention relate to modified rodents with knockout TCRβ chain expression at the TCRβ locus, wherein the genome of these rodents (e.g., mice) retains one or more serine protease genes from the rodent (e.g., mouse) TCRβV region at that locus. One or more serine protease genes may be retained at an endogenous location at the locus (e.g., the modification involves only a portion of the TCRβ locus without affecting the expression of the one or more serine protease genes); or may be translocated as part of a TCRβV region inversion; or may be inserted into an ectopic location in the genome after being excised from an endogenous location (e.g., as part of a TCRβV region deletion, such as a deletion at the TCRβ locus).

[0174] In one respect, no inversion occurred in the host TCRβ constant region.

[0175] Male rodents are preferred to retain reproductive capacity comparable to that of the wild type, which can be measured by the number of offspring as needed.

[0176] As described herein, one or more serine protease genes used to restore fertility can be obtained from the DNA of a host rodent strain or from other rodents containing equivalents of these serine protease genes.

[0177] Therefore, the present invention relates to a rodent or rodent cell (preferably mouse or mouse cells, such as ES cells) wherein the host TCRβ locus has been inactivated, preventing the rodent from forming the natural rodent TCRβ chain, but retaining a wild-type or near-wild-type level of fertility. This wild-type or near-wild-type fertility can be achieved by: retaining the TCRβ locus DNA in the rodent genome in a form that does not express the TCRβ polypeptide (e.g., with some or all of the variable regions inverted genomic orientation relative to any TCRβ constant region), thereby retaining the serine protease gene; or by deleting all or part of the locus region sufficient to prevent the expression of the natural rodent TCRβ chain, and reintroducing any, more, or all of the deleted Prss58, Prss59, Prss3b, Try4, Try5, Try10, Prss3, Prss1, and Prss2 genes from the rodent TCRβ locus (preferably all of these genes if the TCRβ locus is deleted).

[0178] It is evident that when the deletion or other inactivation of the TCRβ locus has led to a decline in fertility, each deleted or inactivated serine protease gene can be replaced individually or through a limited combination of genes to determine one or more genes required to restore fertility. It is not necessary to include all serine protease genes (Prss58, Prss59, Prss3b, Try4, Try5, Try10, Prss3, Prss1, and Prss2). Therefore, those skilled in the art can determine the serine protease genes (Prss58, Prss59, Prss3b, Try4, Try5, Try10, Prss3, Prss1, and Prss2) required to restore fertility by practically assessing fertility (e.g., by assessing litter size).

[0179] The host TRB locus can also be effectively inactivated by other methods that preserve the expression of fertility genes, such as by inserting DNA (such as human variable TCRβ DNA) between mouse TCRβ DNA and the constant region, thus moving the variable portion of the locus away from the constant region.

[0180] In one aspect, the rodent TCRβ locus is deleted from the genome, resulting in the deletion of the Prss58, Prss59, Prss3b, Try4, Try5, Try10, Prss3, Prss1, and Prss2 genes present at that locus. These genes are then replaced with one or more of them to restore male rodent fertility, while inserting DNA encoding at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment as disclosed herein. The host TCRβ locus is inactivated, and the human TCRβ variable region is expressed, preferably in a combination of the fully human strand and the human constant region.

[0181] The present invention also relates to a method for replacing a rodent TCRβ locus with a human TCRβ locus, the method comprising: replacing the rodent TCRβ locus with the human TCRβ locus, and then reintroducing a rodent serine protease gene (optionally, identical to a gene deleted from the rodent) (such as one or all of the genes Prss58, Prss59, Prss3b, Try4, Try5, Try10, Prss3, Prss1, and Prss2) into the rodent genome. In one aspect of the method, the deleted rodent locus includes a Vβ1 gene fragment and / or the region between Vβ1 and Vβ2. In another aspect of the method, the deleted rodent DNA includes the genomic region between Vβ29 and Dβ1. The host TCRβ locus is inactivated.

[0182] The present invention also relates to a genetically modified male rodent that is substantially wild-type fertile; wherein the rodent TCRβ locus has deleted at least the region from rodent Vβ1 to Dβ1, and the genome also contains one or all of the deleted rodent serine protease genes Prss58, Prss59, Prss3b, Try4, Try5, Try10, Prss3, Prss1 and Prss2; wherein the rodent genome is not a wild-type rodent genome, or does not have a wild-type genome at the TCRβ locus.

[0183] The present invention also relates to a rodent or rodent cell expressing a fully human TCRα and / or fully human TCRβ chain as disclosed herein, wherein the rodent retains an endogenous TCRα variable locus and / or an endogenous rodent TCRβ variable locus; and any retained endogenous rodent TCRα variable locus is a nonfunctional locus, and / or any retained endogenous rodent TCRβ variable locus is a nonfunctional locus; optionally, all or part of the retained endogenous rodent TCRα and / or β variable locus is inverted relative to its normal orientation in the rodent genome.

[0184] The invention also relates to a reproductive rodent or rodent cell in which a rodent TCRβ locus from at least rodent Vβ1 to Dβ1 is inverted in the rodent genome relative to its natural orientation in the rodent genome; optionally, the method further includes inserting nucleic acid (e.g., DNA) encoding all or part of TCRβ from another species different from the rodent into the rodent genome, for example, inserting human DNA encoding all or part of TCRβ.

[0185] The present invention also relates to a genetically modified rodent or rodent cell, wherein the rodent retains an endogenous rodent TCRβ variable locus, and the endogenous rodent TCRβ variable locus is a non-functional locus because all or part of the locus has been inverted in the genome.

[0186] Other aspects of the invention include: Based on any of the published information in this paper, rodents (e.g., mice) and cells in which the TRBVDJ locus is preserved in the genome and not deleted.

[0187] As disclosed herein, any method or rodent or rodent cell in which the cell does not express the host rodent TCRβ chain.

[0188] A genetically modified male rodent that is essentially wild-type fertility; wherein all or part of the variable VDJ region of the rodent's TCRβ locus is inverted in the rodent genome relative to its natural orientation in the rodent genome.

[0189] A rodent cell (such as an ES cell) capable of developing into a male rodent with essentially wild-type reproductive capacity, wherein all or part of the variable VDJ region of the TCRβ locus in the rodent genome is inverted relative to the natural orientation in the rodent genome.

[0190] Optionally, a rodent genome (such as a male rodent genome) may contain any or more TCRα, TCRβ, MHC class I, CD8α, CD8β, or β2 microglobulin alleles as described herein.

[0191] The present invention also relates to a method for preparing a breedable rodent, the method comprising the step of inverting the rodent TCRβ in the rodent genome from at least rodent Vβ1 to Dβ1; optionally, the method further comprises inserting nucleic acid (e.g., DNA) encoding all or part of the TCRβ from another species different from the rodent into the rodent genome, for example, inserting human DNA encoding all or part of the TCRβ.

[0192] The present invention also relates to a method for preparing a breedable rodent, the method comprising, in any order: (i) Deleting at least the region from rodent Vβ1 to Dβ1 from the rodent TCRβ genomic DNA, including deletion of one or all of the serine protease genes Prss58, Prss59, Prss3b, Try4, Try5, Try10, Prss3, Prss1 and Prss2. (ii) Insert one or all of the above serine protease genes Prss58, Prss59, Prss3b, Try4, Try5, Try10, Prss3, Prss1 and Prss2 into the rodent genome.

[0193] In one aspect, a genetically modified rodent or rodent cell as disclosed herein is provided, wherein at least the region from rodent Vβ1 to Dβ1 is deleted from the rodent TCRβ locus, and one or all of the deleted serine protease genes Prss58, Prss59, Prss3b, Try4, Try5, Try10, Prss3, Prss1, and Prss2 are reinserted into the rodent genome; wherein the rodent is not a wild-type rodent.

[0194] In one aspect, the modified rodent or rodent cell disclosed herein is provided, wherein an unrearranged human TCRβ variable locus replaces all or part of an endogenous rodent TCRβ locus.

[0195] Human introns / intergenic DNA In one aspect, the inserted TCRβ locus contains fully human TCRβD and J gene fragments, including introns and intergenic sequences. In this invention, the TCRβDJC2 gene cluster can be expressed without rodent DNA, which differs from the problem observed in the study by Moore et al. [Moore et al., Sci. Immunol. 6:eabj4026 (2021), December 17, 2021]. That study, under the heading “genehumanisation in mice” on page 8 of 11 pages, states: “Initial TRb humanization replaced the coding, intergenic, and intronic regions with human sequences. These mice expressed a diverse TRBV library, but only TRBDJC1 was detected, with no utilization of the DJC2 gene cluster. To address this issue, we re-mouse-ized the intergenic and intronic sequences in this region (starting from steps 5 and 6 of Figure S4), thereby achieving efficient utilization of both DJC clusters (Figure S12B).”

[0196] An example of a variable TRB locus containing human introns is shown in Figure 2: human genome TCRB variable region genome TCRβDJC2 DNA was inserted into a rodent genome, and the inserted DNA contained all human intron sequences between the D and J gene segments.

[0197] In one respect, it is not necessary to have all human TCRβDJC2D and J intron sequences; some mouse D and J intron sequences can be combined with human intron sequences.

[0198] Example 2 confirms that when inserted into the mouse genome, the human intron sequence located between the TCRβDJC2D and J gene segments is sufficient to support the expression of human V, D, and J gene segments.

[0199] Therefore, the present invention comprises rodents or rodent cells having the TCRβ locus as described herein, the TCRβ locus comprising a fully human TCRβDJC2J intron and / or a fully human TCRβDJC2D intron; in one aspect, the present invention comprises rodents having fully human TCRβDJC2D and TCRβJ gene fragments comprising a fully human TCRβJ intron and a fully human TCRβD human intron. In one aspect, the rodent TCRβDJC2J intron and / or the rodent TCRβDJC2D intron are absent from the genome.

[0200] The rodents of the present invention are preferably also capable of expressing TCR receptors derived from human gene fragments inserted into the human DJC2 gene cluster.

[0201] Therefore, the present invention further comprises rodents or rodent cells having the TCRβ locus as described herein, the TCRβ locus comprising a fully human TCRβ intergenic sequence located between one or more of the gene segments D1 and J1, J1 and C1, C1 and D2, and D2 and J2. In one aspect, there is no rodent TCRβ intergenic segment between the gene segments D1 and C2 in the genome; preferably, the insert is a contiguous human genomic insert between human D1 and human C2.

[0202] Therefore, the present invention further comprises a rodent or rodent cell (optionally, any rodent or rodent cell described herein) whose genome contains DNA encoding a fully human TCRβ chain and including human V, D, J, and C gene segments, wherein the rodent or rodent cell is capable of expressing the fully human TCRβ chain; and the rodent genome contains human intergenic and / or human intron DNA sequences located between human D1 and human C2 gene segments, preferably human intergenic and / or intron sequences naturally coexisting with human D and J gene segments; optionally, the human DNA sequence between human D1 and human C2 gene segments may be entirely genomic human DNA. Therefore, in one aspect, the rodent (e.g., a mouse, preferably the rodent and mouse of the present invention) preferably contains one or more human TRBD and J introns at the mouse TRBDJC2 cluster, and preferably only human introns at the DJC2 cluster.

[0203] Therefore, in one aspect, rodents (e.g., mice, preferably the rodents and mice of the present invention) do not contain any mouse TRBD and / or J introns at the mouse TRBDJC2 cluster.

[0204] Human CD8α and CD8β The present invention further relates to a genetically modified rodent or rodent cell (such as ES cells or T cells) comprising a nucleic acid sequence encoding a human CD8α polypeptide, wherein the nucleic acid sequence encoding the human CD8α polypeptide is located at an endogenous rodent CD8α co-receptor locus; and / or a genetically modified rodent or rodent cell (such as ES cells or T cells) comprising a nucleic acid sequence encoding a human CD8β polypeptide, wherein the nucleic acid sequence encoding the human CD8β polypeptide is located at an endogenous rodent CD8β co-receptor locus.

[0205] CD8 interacts with MHC class I molecules. CD8 can be a heterodimer of α and β chains, or a homodimer of either α or β chains. CD8 dimers on the surface of T cells interact with MHC class I molecules in adjacent cells. Figure 8 CD8 binding enhances the affinity of TCRs for MHC class I antigens. The binding of CD8 to MHC class I molecules is species-specific. Therefore, using human CD8 can be helpful in studying T cell responses to human MHC class I antigens.

[0206] CD8 expression is regulated by multiple enhancer elements located throughout the CD8 locus. A hypersensitive region of deoxyribonuclease I associated with regulatory factor binding has been identified at the CD8 locus [Hosert et al. (1997), J. Immunol. 158:4270-81]. Enhancer elements have also been identified at this CD8 locus [Kioussis et al. (2002), Nature Rev. 2:909-919 and online errata; Ellmeier et al. (1998), Immunity 9:485-96].

[0207] In some respects, the rodent or rodent cell contains or retains endogenous rodent promoters, enhancers, and / or regulatory elements that regulate the expression of human CD8α and / or CD8β. In some respects, the rodent or rodent cell provides a nucleic acid encoding a fully human CD8α or CD8β polypeptide, which is operatively linked to one or more endogenous rodent regulatory sequences, such as rodent promoters, enhancers, or other regulatory sequences.

[0208] In one respect, human CD8α and / or human CD8β are operatively linked to rodent promoters.

[0209] In one aspect, apart from cells that normally express CD8, the rodents of the present invention do not express CD8 protein on immune cells; for example, rodents do not express CD8 on B cells or CD4+SPT cells.

[0210] Rodents or rodent cells (e.g., rodent T cells) do not express functional endogenous mouse CD8α and / or CD8β peptides from their endogenous CD8 locus.

[0211] In one aspect, human proteins are displayed on the cell surface of rodents or rodent cells expressing functional CD8αβ proteins from CD8 loci as described herein. In another aspect, the rodent expresses human CD8 proteins on its cell surface in the same or substantially the same cellular distribution as observed in humans. In yet another aspect, the human CD8 proteins expressed in rodents are capable of interacting with MHC class I proteins (such as human MHC class I) expressed on a second cell surface.

[0212] In one aspect, the endogenous rodent CD8α and CD8β helper receptor loci contain an inserted human DNA encoding CD8α and CD8β polypeptides, which is operatively bound to at least one mouse enhancer (optionally, to two or three mouse enhancers) and, optionally, also operatively bound to one or more human enhancers; such that the CD8 loci (simultaneously expressing CD8α and CD8β) are under the co-regulation of human and rodent regulatory elements.

[0213] In one preferred aspect, the rodent is a mouse, and there are 4 mouse enhancers.

[0214] In one aspect, the rodent or rodent cell may be a heterozygote for the nucleotide sequence encoding the human CD8α polypeptide. In another aspect, the rodent or rodent cell may be a homozygote for the nucleotide sequence encoding the human CD8α polypeptide.

[0215] In one aspect, the rodent or rodent cell may be a heterozygote for a nucleotide sequence encoding a human CD8β polypeptide. In another aspect, the rodent or rodent cell may be a homozygote for a nucleotide sequence encoding a human CD8β polypeptide.

[0216] In one respect, rodents or rodent cells contain inserted human CD8α (from exons 1 to 4) and human CD8β (from exons 1 to 3).

[0217] In one respect, the inserted human loci exhibit appropriate spatial and temporal protein expression and support T cell development and selection.

[0218] Chimeric CD8α and CD8β In an alternative aspect, the invention further relates to a genetically modified rodent or rodent cell (such as an ES cell or a T cell) comprising a nucleic acid sequence encoding a chimeric rodent-human CD8α polypeptide (chimeric CD8α polypeptide), wherein the nucleic acid sequence encoding the chimeric CD8α polypeptide is located at an endogenous rodent CD8α co-receptor locus; and / or a genetically modified rodent or rodent cell (such as an ES cell or a T cell) comprising a nucleic acid sequence encoding a chimeric rodent-human CD8β polypeptide (chimeric CD8β polypeptide), wherein the nucleic acid sequence encoding the chimeric CD8β polypeptide is located at an endogenous rodent CD8β co-receptor locus.

[0219] The chimeric CD8α polypeptide is suitably encoded by genomic DNA comprising some or all of human exons 1, all of human exons 2, all of human exons 3, and some of human exons 4; in one aspect, human DNA from human exon 4 is partially fused with mouse exon 3. Preferably, the rodent genome also comprises human genomic DNA located between exons 1-2, exons 2-3, and exons 3-4.

[0220] In one aspect, a fusion is performed between human exon 4 and mouse exon 3, such that the expressed polypeptide is divided into human and mouse derivatives both extracellularly and intracellularly. This fusion appropriately occurs only in the linker region from the N-terminus to the transmembrane domain.

[0221] In one respect, fusion is performed at sites identical to the amino acid sequences (preferably amino acids and DNA sequences) in mice and humans to avoid introducing structural changes.

[0222] CD8A DNA fusion appropriately occurs in the following regions of the exons in both humans and mice: Human exon 4, bases 18-32; Mouse exon 3, bases 18-32.

[0223] The amino acid sequences of both species in this region are DFACD (i.e., aspartic acid, phenylalanine, alanine, cysteine, aspartic acid).

[0224] The chimeric CD8β polypeptide is suitably encoded by genomic DNA containing some or all of human exon 1, all of human exon 2 and some of human exon 3; in one aspect, human DNA from exon 3 is fused with a partial fragment of mouse exon 3; preferably, the rodent genome also contains human genomic DNA located between exons 1-2 and exons 2-3.

[0225] In one aspect, fusion was performed between human and mouse exons, such that the expressed CD8β peptide was either human-derived extracellularly or mouse-derived intracellularly.

[0226] In one respect, the genome contains human coding sequences, rather than the 5' untranslated region.

[0227] Figure 3 and Figure 4 Examples of suitable chimeric CD8 peptides are shown.

[0228] Chimeric human-rodent CD8 proteins can bind to human MHC class I proteins (e.g., fully human MHC class I) expressed on host cells, allowing T cells expressing chimeric CD8 to interact with antigen-presenting cells possessing MHC class I proteins. This enables antigen presentation to T cells and allows for T cell activation. The ability of chimeric human-rodent peptides to bind to human MHC class I proteins is achieved by preserving relevant regions of the human CD8 protein expressed on the T cell surface; these sites of interaction between human CD8 and human MHC class I proteins are known, for example, see *Fronts in Immunology*, July 22, 2013, Vol. 4, 2013, Sec. T Cell Biology. T cell activation assays are also known and can be used to measure T cell activation, thereby confirming suitable chimeric structures.

[0229] CD8 expression is regulated by multiple enhancer elements located throughout the CD8 locus. A deoxyribonuclease I hypersensitive region associated with regulatory factor binding has been identified at this CD8 locus [Hosert et al. (1997), J. Immunol. 158:4270-81]. Enhancer elements have also been identified at this CD8 locus [Kioussis et al. (2002), Nature Rev. 2:909-919 and online errata; Ellmeier et al. (1998), Immunity 9:485-96].

[0230] In some respects, the rodent or rodent cell contains or retains endogenous rodent promoters, enhancers, and / or regulatory elements that regulate the expression of chimeric CD8α and / or CD8β. In some respects, the rodent or rodent cell provides a nucleic acid encoding a chimeric CD8α or CD8β polypeptide, which is operatively linked to one or more endogenous rodent regulatory sequences, such as rodent promoters, enhancers, or other regulatory sequences.

[0231] In one respect, chimeric CD8α and / or chimeric CD8β are operatively linked to rodent promoters.

[0232] In one respect, chimeric CD8α and / or chimeric CD8β are operatively linked to human promoters.

[0233] In one aspect, apart from cells that normally express CD8, the rodents of the present invention do not express CD8 protein on immune cells; for example, rodents do not express CD8 on B cells or CD4+SPT cells.

[0234] Rodents or rodent cells (e.g., rodent T cells) do not express functional endogenous mouse CD8α and / or CD8β peptides from their endogenous CD8 locus.

[0235] In one aspect, a chimeric protein is expressed on the cell surface of a rodent or rodent cell expressing a functional CD8αβ protein from a CD8 locus as described herein. In another aspect, the rodent expresses the chimeric CD8 protein on its cell surface in the same or substantially the same cellular distribution as observed in humans. In yet another aspect, the chimeric CD8 protein expressed in the rodent is capable of interacting with MHC class I proteins (such as human MHC class I) expressed on a second cell surface.

[0236] In one aspect, the endogenous rodent CD8α and CD8β helper receptor loci contain an inserted human DNA encoding CD8α and CD8β polypeptides, which is operatively bound to at least one mouse enhancer (optionally, to two or three mouse enhancers) and, optionally, also operatively bound to one or more human enhancers; such that the CD8 loci (simultaneously expressing CD8α and CD8β) are under the co-regulation of human and rodent regulatory elements.

[0237] In one preferred aspect, the rodent is a mouse, and there are 4 mouse enhancers.

[0238] In one aspect, the rodent or rodent cell may be a heterozygote encoding a nucleotide sequence of a chimeric CD8α polypeptide. In another aspect, the rodent or rodent cell may be a homozygote encoding a nucleotide sequence of a chimeric CD8α polypeptide.

[0239] In one aspect, the rodent or rodent cell may be a heterozygote encoding a nucleotide sequence of a chimeric CD8β polypeptide. In another aspect, the rodent or rodent cell may be a homozygote encoding a nucleotide sequence of a chimeric CD8β polypeptide.

[0240] In one respect, this locus exhibits appropriate spatial and temporal protein expression and supports T cell development and selection.

[0241] EP2958938B1, derived from WO2014130671, discloses examples of chimeric MHC class I molecules and chimeric CD8 peptides applicable to the present invention.

[0242] rodent CD8α and CD8β In another aspect of the invention, host rodent CD8α and CD8β genes may be used. Preferably, these genes are under normal regulated control of the host. In this aspect, the rodent genome may contain a chimeric MHC class I polypeptide that interacts with rodent CD8 proteins through a portion of its contained rodent amino acid. The chimeric MHC class I polypeptide appropriately possesses a rodent α3 domain, and the invention also relates to nucleic acids encoding the chimeric polypeptide.

[0243] Alternatively, host rodent CD8 can combine with MHC class I containing the human α3 domain. This rodent may contain endogenous CD8α and / or β loci (both homozygous, if desired) and an MHC class I locus encoding a human MHC class I polypeptide as described herein. Species mismatch between (host) CD8 and (human) MHC class I may reduce their interactions compared to binding to species-matched polypeptides (e.g., both human or both mouse). These rodents may exhibit a weaker immune response and produce fewer antigen-specific T cells against the target antigen compared to rodents with species-matched CD8 and MHC class I α3 domains (e.g., both human or both mouse). However, antigen-specific T cells obtained from these immunized mice may bind to the target antigen with high affinity. Reduced binding affinity between the helper receptor and MHC may have an overall impact on the selection of high-affinity TCRs in rodents, as higher affinity TCR:MHC interactions are required to reach the threshold for antigen-dependent TCR signaling within T cells. This could be advantageous in scenarios where rodents are used to obtain T-cell receptors with high affinity for specific antigens (e.g., for the production of soluble TCR molecules that function as therapeutic agents by binding to pMHC in the absence of co-receptor binding).

[0244] MHC Class I The present invention further relates to a genetically modified rodent or rodent cell (such as ES cells or antigen-presenting cells) containing a nucleic acid sequence encoding a human MHC class I polypeptide, wherein the nucleotide sequence encoding the human MHC class I polypeptide is located at an endogenous rodent MHC class I locus.

[0245] This rodent allows the human CD8 protein expressed on the surface of its T cells, as revealed in this paper, to interact with human MHC class I molecules expressed on the surface of the rodent's second cells (e.g., antigen-presenting cells).

[0246] Human MHC class I peptides may be derived from human HLA class I proteins selected from the group consisting of HLA-A, HLA-B, and HLA-C, such as HLA-A2, HLA-B27, HLA-B7, HLA-Cw6, or any other HLA class I molecules present in the human population.

[0247] The rodent genome can contain more than one nucleic acid encoding a human MHC class I molecule. For example, DNA encoding a human MHC class I molecule can be inserted into the H2D and H2K loci of rodents (such as mice). The human MHC class I molecule at each locus can be different. In one respect, considering the heterozygosity of each of these loci, there may be two, three, or four different human MHC class I molecules in the rodent genome.

[0248] In some rodent strains, there are three rodent MHC class I loci. Therefore, there may be 1, 2, 3, 4, 5 or even 6 different MHC class I molecules in the rodent genome. These molecules may be human or humanized molecules, and may be HLA-A, HLA-B and HLA-C molecules.

[0249] In some respects, rodents or rodent cells contain or retain endogenous rodent promoters, enhancers, and / or regulatory elements that regulate the expression of human MHC class I molecules.

[0250] In some aspects, a nucleic acid encoding a complete human MHC class I polypeptide is introduced into a rodent or rodent cell, the nucleic acid being operatively linked to one or more endogenous rodent regulatory sequences, such as rodent promoters, enhancers, or other regulatory sequences. In one aspect, the rodent is a mouse, and the human MHC class I polypeptide is operatively linked to a mouse promoter and a mouse enhancer located at the 5' end of the complete human MHC class I polypeptide.

[0251] In one aspect, rodents may be heterozygous for a nucleotide sequence encoding a human MHC class I polypeptide. In another aspect, rodents may be homozygous for a nucleotide sequence encoding a human MHC class I polypeptide.

[0252] In one aspect, the insertion site in human DNA (such as HLA-A02 DNA) is the endogenous H2-D locus. The endogenous H2-D locus may also be wholly or partially deleted or otherwise inactivated. In another aspect, the H2-K1 gene in rodents may also be deleted or inactivated.

[0253] In one aspect, the insertion site in human DNA (such as HLA-A02 DNA) is the endogenous H2-K locus. The endogenous H2-K locus may also be entirely or partially deleted or otherwise inactivated. In another aspect, the H2-D gene in rodents may also be deleted or inactivated.

[0254] Preferably, the H2-D and H2-K genes of rodents are both deleted or otherwise inactivated to eliminate the expression of mouse class I molecules, so that only the required human or humanized MHC class I molecules are expressed in the rodent.

[0255] Preferably, all genes encoding rodent MHC class I molecules are deleted or otherwise inactivated to eliminate the expression of rodent class I molecules themselves.

[0256] In one aspect, the rodent contains a nucleic acid insertion encoding the complete coding sequence of human HLA-A02. In another aspect, the inserted human DNA comprises or consists of human DNA (nucleotide positions 6:29,942,554-29,945,450, GRCh38 version).

[0257] In one aspect, human HLA-A02 is inserted into the endogenous H2-D locus.

[0258] In one aspect, the H2-K1 gene in rodents is deleted or inactivated.

[0259] In one aspect, more than one rodent HLA locus can be inactivated. For example, human DNA described herein can be inserted into a rodent MHC class I locus, inactivating endogenous expression at that locus. Furthermore, a second rodent MHC class I locus can be inactivated (e.g., knocked out) without requiring replacement of the second locus with a human MHC class I molecule. For example, the rodent H2-D locus can be replaced with nucleic acid encoding a human HLA class I gene, while the rodent H2-K gene can be deleted without inserting any human DNA into the H2-K locus.

[0260] In one aspect, the inserted human loci showed appropriate spatial and temporal protein expression.

[0261] Chimeric MHC Class I The present invention further relates to a genetically modified rodent or rodent cell (such as ES cells or antigen-presenting cells) comprising a nucleic acid sequence encoding a chimeric (rodent-human) MHC class I polypeptide, wherein the nucleotide sequence encoding the chimeric MHC class I polypeptide is located at an endogenous rodent MHC class I locus.

[0262] Figure 6 shows a suitable example of a chimeric MHC class I peptide.

[0263] Chimeric MHC class I proteins can bind to rodent CD8 expressed on host cells, enabling T cells expressing this rodent CD8 to interact with antigen-presenting cells containing chimeric MHC class I molecules, thereby presenting antigens to T cells and activating them. The binding ability of rodent CD8 peptides to chimeric rodent-human MHC class I molecules is achieved by preserving the rodent regions of the rodent MHC class I protein that interact with CD8; these sites of interaction between rodent CD8 and rodent MHC class I molecules are known, for example, see *Front. Immunol.*, July 22, 2013, T Cell Biology, Vol. 4, 2013. Methods for detecting T cell activation are also known and can be used to determine T cell activation levels, thereby identifying suitable chimeric structures.

[0264] EP2958938B1, derived from WO2014130671, discloses examples of chimeric MHC class I molecules and chimeric CD8 peptides suitable for use in this invention.

[0265] Therefore, MHC class I loci can express chimeric (human-rodent) proteins to achieve the interaction between MHC class I molecules and human TCR, while retaining the ability to bind to rodent CD8.

[0266] Specifically, when CD8 is endogenous rodent CD8, the MHC class I molecule is preferably humanized, but may retain the rodent α3 domain—the site of interaction with CD8. Preferably, the MHC class I molecule has human α1 and human α2 domains, which interact with the human TCR domain, thus the MHC-TCR interaction interface is fully humanized.

[0267] Therefore, in one aspect, the rodent contains a nucleic acid insertion encoding the complete coding sequences of human HLA-A02-01 exons 1-3 and 5-8, wherein exon 4 is the natural rodent exon 4.

[0268] In one aspect, the inserted human DNA contains or consists of human DNA (nucleotide positions 6:29,942,554-29,945,450, GRCh38 version), but does not contain human exon 4, instead being replaced by rodent exon 4 and integrated into the genome.

[0269] Complete chimeric MHC class I molecules can be inserted into the genome, or existing rodent exon 4 in the genome can be retained, and the flanking regions of the genome can be modified to introduce the required human nucleic acids.

[0270] In one aspect, the regions between human exons 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, and 7-8 are all human-derived sequences.

[0271] This rodent allows the rodent CD8 protein expressed on the surface of its T cells, as revealed in this paper, to interact with chimeric (rodent-human) MHC class I molecules expressed on the surface of the rodent's second cells (e.g., antigen-presenting cells).

[0272] Human MHC class I peptides may be derived from human HLA class I proteins selected from the group consisting of HLA-A, HLA-B, and HLA-C, such as HLA-A2, HLA-B27, HLA-B7, HLA-Cw6, or any other HLA class I molecules present in the human population.

[0273] The rodent genome may contain more than one nucleic acid encoding a chimeric MHC class I molecule. For example, DNA encoding a chimeric MHC class I molecule can be inserted into the H2D and H2K loci of rodents (such as mice). In one respect, considering the heterozygosity of each of these loci, there may be two, three, or four different human MHC class I molecules in the rodent genome.

[0274] In some rodent strains, three rodent MHC class I loci are present. Therefore, the rodent genome may contain 1, 2, 3, 4, 5, or even 6 different MHC class I molecules. These molecules can be chimeric and can be HLA-A, HLA-B, and HLA-C molecules. In some cases, rodents or rodent cells contain or retain endogenous rodent promoters, enhancers, and / or regulatory elements that regulate the expression of human MHC class I molecules.

[0275] In some aspects, a nucleic acid encoding a complete human MHC class I polypeptide is provided to a rodent or rodent cells, the nucleic acid being operatively linked to one or more endogenous rodent regulatory sequences, such as rodent promoters, enhancers, or other regulatory sequences. In one aspect, the rodent is a mouse, and the human MHC class I polypeptide is operatively linked to a mouse promoter and a mouse enhancer located at the 5' end of the complete human MHC class I polypeptide.

[0276] In one aspect, rodents may be heterozygous for a nucleotide sequence encoding a human MHC class I polypeptide. In another aspect, rodents may be homozygous for a nucleotide sequence encoding a human MHC class I polypeptide.

[0277] In one aspect, the insertion site in human DNA (such as HLA-A02 DNA) is the endogenous H2-D locus. The endogenous H2-D locus may also be wholly or partially deleted or otherwise inactivated. In another aspect, the H2-K1 gene in rodents may also be deleted or inactivated.

[0278] In one aspect, the insertion site in human DNA (such as HLA-A02 DNA) is the endogenous H2-K locus. The endogenous H2-K locus may also be entirely or partially deleted or otherwise inactivated. In another aspect, the H2-D gene in rodents may also be deleted or inactivated.

[0279] Preferably, the H2-D and H2-K genes of rodents are both deleted or otherwise inactivated to eliminate the expression of mouse class I molecules, so that only the required human or humanized MHC class I molecules are expressed in the rodent.

[0280] Preferably, all genes encoding rodent MHC class I molecules are deleted or otherwise inactivated to eliminate the expression of rodent class I molecules themselves.

[0281] In one aspect, more than one rodent HLA locus can be inactivated. For example, human DNA described herein can be inserted into a rodent MHC class I locus, inactivating endogenous expression at that locus. Furthermore, a second rodent MHC class I locus can be inactivated (e.g., knocked out) without requiring replacement of the second locus with a human MHC class I molecule. For example, the rodent H2-D locus can be replaced with nucleic acid encoding a human HLA class I gene, while the rodent H2-K gene can be deleted without inserting any human DNA into the H2-K locus.

[0282] In one aspect, the inserted human locus can reveal appropriate spatial and temporal protein expression.

[0283] β2-microglobulin (β2M) This invention relates to a genetically modified rodent or rodent cell (such as ES cells or antigen-presenting cells) containing a nucleic acid sequence encoding a human β2-microglobulin polypeptide located at an endogenous rodent β2-microglobulin gene locus.

[0284] In one aspect, the nucleic acid encoding endogenous rodent β2-microglobulin is completely or partially missing.

[0285] In one aspect, the rodent or rodent cell contains an insertion fragment from the ATG of exon 1 to the stop codon of exon 3. In one aspect, the inserted human DNA contains the sequence at nucleotide position 15: 44,711,547-44,716,342 (GRCh38 version). In one aspect, the insertion is performed in the mouse genome, with a deletion at position 2: 121,978,219-121,982,140 (GRCm39C57BL / 6J version). In another aspect, the insertion is performed in the mouse genome, with a deletion of the region between positions 2: 121,978,218-121,982,142 (GRCm39C57BL / 6J version) (excluding the endpoints of this region; i.e., the deleted region begins and terminates within these flanking bases, each offset by 1 bp).

[0286] In some respects, rodents or rodent cells contain or retain endogenous rodent promoters and / or regulatory elements that regulate the expression of human β2M.

[0287] In some aspects, a nucleic acid encoding complete human β2M is introduced into rodents or rodent cells, which is operatively linked to one or more endogenous rodent regulatory sequences, such as rodent promoters, enhancers, or other regulatory sequences. In one aspect, upstream rodent enhancers and promoters are operatively linked to the nucleic acid encoding β2M at an endogenous locus. Where appropriate, these enhancers and promoters are naturally occurring promoters and enhancers in rodents or rodent cells, located at their natural positions in the genome, and have not been removed or replaced.

[0288] Preferably, the rodent is a mouse, and the enhancer is a mouse enhancer located at the 5' end of the gene. The promoter shares cis and trans-acting factors with MHC class I molecules, thereby achieving synergistic expression.

[0289] In one aspect, rodents or rodent cells may be heterozygous for a nucleotide sequence encoding a human β2M polypeptide.

[0290] In one respect, rodents may be homozygous for the nucleotide sequence encoding the human β2M polypeptide.

[0291] In one aspect, the inserted human locus enables appropriate spatial and temporal protein expression.

[0292] Combination of human loci This article reveals rodents and cells containing nucleic acids encoding complete human polypeptides of one or more (or all) genes at loci of TCRα, TCRβ, MHC class I molecules, CD8α, CD8β, and β2M, which are inserted into endogenous rodent genome loci of TCRα, TCRβ, MHC class I, CD8α, CD8β, and β2M, respectively.

[0293] Preferred rodents have insertions at the aforementioned rodent loci, with preferred combinations including human DNA insertions at the TCRα, TCRβ, MHC class I, CD8α, and CD8β loci; more preferably, human DNA is inserted at all TCRα, TCRβ, MHC class I, CD8α, CD8β, and β2M loci. In this manner, antigens in rodents can be presented to the human TCR and human co-receptor CD8 via human MHC class I peptides.

[0294] However, it should be understood that the rodents and cells of the present invention may contain only some (but not all) of the complete human loci described herein, for example, only one, two, three, four, five, or six rodent loci may be modified to express human peptides. For example, the rodent may contain DNA encoding human MHC class I molecules, which may be used in combination with rodent peptides expressed at the other loci described above, or in combination with chimeric (rodent-human) peptides expressed at the other loci described above, or both. Therefore, any one, two, three, four, five, six, or seven of the following loci can be fully humanized: TCRα, TCRβ, MHC class I (one or both of H2K and H2D), CD8α, CD8β, and β2M, and may be used in combination with rodent and / or chimeric (e.g., rodent-human chimeric) peptides at other loci disclosed herein.

[0295] Preferred rodents and cells contain at least human TCRα and TCRβ inserts to generate a complete human TCR in the rodent.

[0296] As illustrated in the first example, the rodent may contain nucleic acids expressing combinations of chimeric rodent-human TCRα and / or TCRβ with intact human MHC class I molecules. In other examples, the rodent may contain nucleic acids expressing combinations of chimeric rodent-human TCRα and / or TCRβ with intact human CD8α and / or intact human CD8β. The rodent may contain nucleic acids expressing combinations of chimeric rodent-human TCRα and / or TCRβ with intact human CD8α and / or human CD8β and human β2M. The rodent may contain nucleic acids expressing combinations of chimeric rodent-human MHC class I molecules with intact human TCRα and / or TCRβ. The rodent may contain nucleic acids expressing combinations of chimeric rodent-human MHC class I molecules with intact human CD8α and / or CD8β. The rodent may contain nucleic acids expressing combinations of chimeric rodent-human MHC class I molecules with intact human CD8α and / or β and β2M. These are all non-limiting examples; other combinations of complete human loci with rodent and / or chimeric loci are also possible.

[0297] Examples of chimeric TCRα, TCRβ, MHC class I molecules, and CD8α and CD8β loci are given, for example, in WO2014130671, WO2014130667 and WO2016085889A1. Specific examples of these loci and the specific polypeptides they produce are incorporated herein by reference and may be used in combination with one or more complete human loci / polypeptides described herein for the purposes of this invention.

[0298] In one aspect, rodents containing multiple human inserts at different loci are obtained through breeding, preferably breeding to a homozygous state at each locus containing a human DNA insert. Thus, the rodent can be homozygous at one, two, three, four, five, six, or all seven loci (depending on the number of human inserts at each locus) of the TCRα, TCRβ, MHC class I (H2D and H2K), CD8α, CD8β, and β2M loci.

[0299] In one aspect, the rodent is reproductively capable, which can be assessed by indicators such as litter size. The litter size is preferably at least 50%, 60%, 70%, 80%, or 90% of the litter size of wild-type rodents, more preferably at least 95%, and most preferably the same as the litter size of wild-type rodents (100%).

[0300] Preferably, the genetically modified rodent or rodent cell of the present invention comprises: (i) An unrearranged T-cell receptor (TCR) α variable locus comprising at least one human Vα fragment and at least one human Jα fragment, wherein the TCRα variable gene is operatively linked to a human TCRα constant gene sequence. (ii) An unrearranged TCRβ variable locus comprising at least one human Vβ segment, at least one human Dβ segment and at least one human Jβ segment, and the unrearranged TCRβ variable gene is operatively linked to a human TCRβ constant gene sequence. These unrearranged human T cell variable region gene fragments can rearrange to form a gene encoding the variable domain of the human T cell receptor, and the unrearranged TCRα variable gene locus is located at the endogenous rodent TCRα gene locus, and the unrearranged TCRβ variable gene locus is located at the endogenous rodent TCRβ gene locus. (iii) A nucleic acid sequence encoding a human CD8α polypeptide, wherein the nucleic acid sequence encoding a human CD8α polypeptide is located at an endogenous rodent CD8α co-receptor locus. (iv) The nucleic acid sequence encoding the human CD8β polypeptide, which is located at the endogenous rodent CD8β helper receptor locus. (v) The nucleic acid sequence encoding a human MHC class I polypeptide located at an endogenous rodent MHC class I locus. (vi) The nucleic acid sequence encoding the human β2-microglobulin polypeptide (if present), located at the endogenous rodent β2-microglobulin locus.

[0301] Functional aspects of inserting human DNA In one aspect, rodents express human CD8 protein containing CD8α and CD8β peptides as well as human MHC class I peptides, and these human CD8 and human MHC class I molecules can interact within the rodent.

[0302] In one aspect, rodents express human TCRs containing TCRα and TCRβ peptides, as well as human MHC class I peptides, and these human TCRs and human MHC class I molecules can interact within the rodent body.

[0303] In one aspect, rodents or rodent cells (such as T cells) express human CD8 protein containing CD8α and CD8β peptides and human TCR containing TCRα and TCRβ peptides, and both human CD8 and human TCR can bind to MHC class I molecules in rodents.

[0304] In one aspect, rodents express human CD8 protein containing CD8α and CD8β peptides, human MHC class I peptides, and human TCR containing TCRα and TCRβ peptides, and the human TCR, human MHC class I molecules, and human CD8 can interact in the rodent body to achieve antigen presentation from MHC class I molecules to TCR with the assistance of CD8.

[0305] In one aspect, a rodent expresses a TCR library. In another aspect, the rodent expresses a library of TCRα and TCRβ peptides.

[0306] In one aspect, the human variable region of a human TCR protein is capable of interacting with multiple proteins on the surface of the same cell or another cell. In one aspect, the human variable region of the human TCR interacts with an antigen-presenting MHC protein (such as a MHC class I molecule) on the surface of a second cell (such as an antigen-presenting cell, APC). In some aspects, the MHC class I protein is a non-human protein (such as a rodent protein, for example, a mouse or rat). In other embodiments, the MHC class I protein is a human protein. In one aspect, the second cell (such as an APC) is an endogenous non-human cell expressing a human MHC molecule. In yet another embodiment, the second cell is a human cell expressing a human MHC molecule.

[0307] In one aspect, the human variable region of the human TCR protein is able to interact with endogenous rodent MHC class II molecules on the surface of another cell, such as antigen-presenting cells.

[0308] In one aspect, the human variable region of the human TCR protein is able to interact with non-polymorphic MHC molecules, such as human or rodent CD1 or MR1.

[0309] In one aspect, the human TCR can interact with endogenous rodent molecules, such as signaling molecules or portions thereof. In another aspect, the human TCR can interact with the rodent CD3 protein complex and the CD3-ζ chain (ζ chain) in the rodent genome, thereby activating T cells. Therefore, the CD3 peptides in the rodent genome can be wild-type, such as CD3ε, CD3γ, CD3δ, and / or CD3ζ. This paper illustrates a transgenic rodent in which the CD3 protein complex is endogenous and non-humanized, but the human TCRαβ in this animal can co-act with rodent CD3 to successfully generate a T-cell immune response against an antigen (see Example 6). Alternatively, transgenic mice in which one or more CD3 peptides or regions thereof are humanized have been described in the literature; these human CD3 transgenes can be used in combination with the transgenic loci of this invention. For example, WO2016 / 085889 (Regeneron) describes the generation of transgenic mice expressing a chimeric CD3εδγ polypeptide (extracellular domains are human, cytoplasmic domains are endogenous mouse-derived) through homologous recombination in mice, targeting the extracellular domains of CD3ε, CD3γ, and CD3δ into an endogenous locus. Ueda et al. (Scientific Reports 7:45839, 2017) described the generation of transgenic mice expressing the complete human CD3εγδ polypeptide by replacing mouse CD3ε, CD3γ, and CD3δ with full-length human homologs using cre-lox recombination technology in C57BL / 6NES cells. Other examples of humanized individual epitopes of the CD3 complex include Crespo et al. (PLoS One 2021), who reported a mouse in which a single mouse CD3ε exon (mouse exon 5) was replaced by a humanized CD3ε exon (containing one substituted human exon 6) containing an epitope of the anti-CD3 antibody OKT3. This humanized exon encodes 11 additional amino acids compared to mouse exon 5, some of which form the OKT3 binding epitope. In addition to other human transgenes described herein, humanization (in whole or in part) of CD3 can make the mouse immune system more similar to that of humans, thus serving as a laboratory model. Advantages of expressing human or humanized CD3 in animals include the ability to target the TCR-CD3 complex in mice with antibodies against human CD3, for example, to evaluate the effects of candidate therapeutic molecules. However, it should be noted that functional T cells and T cell receptor repertoires can be suitably generated in rodents with endogenous CD3 without the need for humanization of the CD3 complex.

[0310] Compared to their corresponding endogenous mouse TCR peptides, CD3 peptides (especially CD3ε, CD3γ, and CD3δ) may exhibit lower efficiency in signal transduction and T cell activation when combined with human TCR peptides (especially the TCRα constant domain). This may be because the interaction efficiency of CD3δ (endogenous rodent CD3δ and even human CD3δ) with human TCRCα is lower than that of endogenous rodent TCRCα. Consistent with this, previous studies have reported that “mouse-derived” human TCRα constant domains can enhance TCR expression by supporting preferential pairing with CD3 in human T cells (Sommermeyer and Uckert, J. Immunol. 184:6223-6231, 2010).

[0311] In some implementations, it is preferable that the rodent does not express a functional endogenous TCRα peptide. This avoids the expression of endogenous TRAC—which would otherwise preferentially bind to CD3 (endogenous CD3 or human CD3). In rodents heterozygous for human TCRα (one allele is human-derived, and one is wild-type), the human and endogenous TCRα peptides compete for binding to CD3 (e.g., endogenous CD3), and T cells with a TCR containing endogenous TCRα may competitively suppress T cells expressing human TCRα because the former have higher TCR signaling efficiency. This situation can be avoided by ensuring the homozygosity of human TRAC.

[0312] While the pairing of human TCRs with CD3 may result in lower efficiency of TCR signaling and T cell activation compared to TCRs containing the mouse TCRα constant region, this can surprisingly become an advantage when rodents are used as a source of high-affinity human TCRs. During T cell development, an individual's TCR repertoire is formed through positive and negative selection targeting that individual's MHC alleles and self-antigens, where T cell survival (positive selection) or clearance (negative selection) requires a certain level of activation signaling. Due to the slightly lower efficiency of activation signaling, higher affinity is required to achieve the same level of activation needed for both positive and negative selection, thus shifting the overall affinity distribution of the repertoire upwards. Therefore, rodents with intact human TCRs (including homozygous loci expressing human TCRα) are actually well-suited for generating high-affinity TCRs for therapeutic use, regardless of whether CD3 is humanized. Therefore, the rodent may express rodent CD3 (e.g., rodent CD3δ, optionally rodent CD3ε, CD3γ, and CD3δ, and optionally rodent CD3ζ). Optionally, the CD3 locus of the rodent is wild-type. Alternatively, the rodent may contain human CD3 transgenes (e.g., CD3εδγ, preferably inserted as a single copy or low copy).

[0313] In one aspect, the rodent is a mouse with the following control area: The 3' enhancer of the TCRα: constant region (TRAC) is mouse-derived.

[0314] The 5' enhancer of the TCRδ: constant region (TRDC) is human (e.g., from within a human BAC clone).

[0315] TCRβ: The mouse enhancer is derived from the 3' end of the mouse constant region (TRBC) and the 315' end of the mouse Vβ in the mouse genome, and can also be replaced with the human equivalent enhancer.

[0316] The 5' enhancer of MHC class I: H2-D1 is mouse-derived.

[0317] β2-microglobulin: The enhancer and promoter at the 5' end of the gene are mouse-derived.

[0318] CD8α and CD8β: There are 4 enhancer regions (as shown in Figure 3), all of which are mouse-derived: E8iv, E8iii, E8ii and E8i.

[0319] Methods for preparing rodents This article describes various methods for preparing the rodents of the present invention, including: A method for modifying rodents to express human TCRα and / or TCRβ peptides, the method comprising: (i) inserting a nucleotide sequence encoding a human TCRα peptide at an endogenous TCRα locus in a rodent; and / or (ii) inserting a nucleotide sequence encoding a human TCRβ peptide at an endogenous TCRβ locus in a rodent, wherein the rodent expresses a human TCR containing human α and / or β peptides.

[0320] A method for modifying rodents to express human CD8α and / or β peptides, the method comprising: (i) inserting a nucleotide sequence encoding a human CD8α peptide at an endogenous CD8α locus in a rodent; and / or (ii) inserting a nucleotide sequence encoding a human CD8β peptide at an endogenous CD8β locus in a rodent, wherein the rodent expresses human CD8α and / or CD8β peptides.

[0321] A method for modifying rodents to express human MHC class I peptides, the method comprising: (i) inserting a human MHC class I peptide into an endogenous rodent MHC class I locus, wherein the rodent expresses the human MHC class I peptide from the rodent MHC class I locus.

[0322] A method for modifying rodents to express human β2-microglobulin polypeptide, the method comprising: (i) inserting human β2-microglobulin polypeptide into an endogenous rodent β2-microglobulin locus, wherein the rodent expresses human β2-microglobulin polypeptide from the rodent β2-microglobulin locus.

[0323] A method for preparing genetically modified rodents expressing human T-cell receptor, human MHC class I molecules, and human CD8, the method comprising: inserting an unrearranged human TCRα variable locus (containing at least one human Vα gene fragment and at least one human Jα gene fragment, operably linked to the human TCRα constant region) at an endogenous rodent TCRα variable locus; inserting an unrearranged human TCRβ variable locus (containing at least one human Vβ gene fragment, at least one human Dβ gene fragment, and at least one human Jβ gene fragment, operably linked to the human TCRβ constant region) at an endogenous rodent TCRβ variable locus; inserting a nucleic acid sequence encoding a human CD8α polypeptide at an endogenous rodent CD8α locus; inserting a nucleic acid sequence encoding a human CD8β polypeptide at an endogenous rodent CD8β locus; and, if necessary, inserting a nucleic acid sequence encoding human or chimeric human-rodent β2-microglobulin at an endogenous rodent β2-microglobulin locus.

[0324] Human DNA insertion may or may not be accompanied by DNA deletion at the insertion site. If endogenous rodent DNA deletion occurs, it may occur simultaneously with human DNA insertion, or at an earlier or later stage. If the rodent gene or locus homolog of the inserted human DNA, or a portion thereof, remains in the genome, it must be inactivated or substantially inactivated, for example, through inversion of the rodent locus, or through disruptive insertion, disruptive point mutation, or substitution of that locus. If the locus is inverted, any non-TCR genes located within the inverted region preferably remain active and appropriately expressed in terms of expression level (quantity), tissue specificity (spatial), and temporal regulation.

[0325] Therefore, the method for inserting the human DNA described herein may further include the step of deleting host rodent DNA at a homologous endogenous rodent locus, preferably such deletion resulting in inactivation of the host locus and preventing expression of the host polypeptide from the locus.

[0326] The method for inserting the human DNA described herein may further include the step of inactivating the host rodent DNA at a homologous endogenous rodent locus. Clearly, the present invention also discloses rodents that not only contain the insert fragment at the locus described herein, but also inactivate the homologous rodent genome locus (thus preventing the production of homologous rodent polypeptides).

[0327] In addition to the method of inserting human DNA mentioned in this article, it should be understood that all methods can be extended to replace rodent DNA with homologous human DNA at homologous human loci, such that rodent DNA is missing from the relevant locus (e.g., missing rodent TCRα DNA at the TRA locus and inserting human TCRα DNA).

[0328] Methods for large-scale replacement of rodent DNA with human DNA are known, as demonstrated in studies on the humanization of mouse immunoglobulin (IG) loci (see Lee et al., 2014, Vol. 32, No. 4, April 2014, *Nature Biotechnology*), Macdonald et al. (2014), and Murphy et al. (2014). These methods involve recombinantly engineering BACs to produce human DNA suitable for insertion, then inserting the recombinant BACs into ES cells, and finally integrating the human DNA into the ES cell genome through recombinase-mediated cassette exchange, iterative homologous recombination, or combinations thereof.

[0329] If necessary, host DNA can be deleted before the insertion of human DNA. This deletion can be achieved by inserting SSR sites on both sides of the locus and using recombinase site-specific deletion DNA.

[0330] In one aspect, the first insertion event at any locus may involve a targeted insertion initiation cassette to provide a unique site in the genome for subsequent insertion of the required human DNA. Therefore, only one targeting event is needed per locus to initiate targeted insertion of human DNA. The initiation cassette may contain one or more SSR and / or transposase sites, allowing for iterative use of the cassette structure during insertion, as illustrated by the technique revealed by Lee et al. above.

[0331] Cells (such as ES cells) can be screened using techniques known in the art (such as Southern blotting, PCR, quantitative PCR (such as real-time PCR using TAQMAN®), fluorescence in situ hybridization (FISH), Northern blotting, flow cytometry, Western blotting, immunocytochemistry, immunohistochemistry, etc.) to assess whether the insertion event is correct.

[0332] In one aspect, the present invention relates to a method for preparing a genetically modified reproductive rodent expressing a human T-cell receptor, the method comprising deleting at least the rodent Vβ1 to Dβ1 sequence (including the deletion of serine protease genes Prss58, Prss59, Prss3b, Try4, Try5, Try10, Prss3, Prss1, and Prss2) from the rodent TCRβ gene, and reinserting one or more (or all) of the deleted serine protease genes Prss58, Prss59, Prss3b, Try4, Try5, Try10, Prss3, Prss1, and Prss2 into the rodent genome. In one aspect, the deletion is a homozygous deletion.

[0333] The present invention also relates to a method for preparing a gene knockout reproductive rodent containing an endogenous T cell receptor β chain polypeptide, the method comprising deleting at least the rodent Vβ1 to Dβ1 sequence (including the deletion of serine protease genes Prss58, Prss59, Prss3b, Try4, Try5, Try10, Prss3, Prss1 and Prss2) from the rodent TCRβ, and reinserting one or more (or all) of the deleted serine protease genes Prss58, Prss59, Prss3b, Try4, Try5, Try10, Prss3, Prss1 and Prss2 into the rodent genome.

[0334] In one aspect, the deletion is a homozygous deletion.

[0335] Uses of genetically modified rodents Genetically modified rodents containing human CD8, human MHC class I molecules, and human TCRs can present peptides to T cells in a "human-like" manner because almost all elements of the complex are human-derived. The genetically modified rodents of this invention can be used to study the function of the human immune system in rodents and to identify antigens and epitopes that elicit immune responses. These rodents can be used for a variety of studies, such as identifying T cell epitopes or human cancer epitopes in vaccine development; identifying TCR sequences and T cells with affinity for human pathogens or cancer antigens for the identification of TCRs used in adoptive T-cell therapy; evaluating candidate vaccines and inoculation methods; studying human autoimmune diseases; and studying human infectious diseases.

[0336] Therefore, the genetically engineered rodents of the present invention are particularly suitable for evaluating the ability of antigens to elicit an immune response in humans, and can also be used to generate a variety of antigens and identify specific antigens that can be used for human vaccine development.

[0337] The rodents disclosed in this article, which contain insert DNA encoding a human TCR, can be used to generate human TCR / TCR peptides or functional fragments thereof, or to generate sequence information about such TCR / TCR peptides and functional fragments thereof—the TCR being targeted at an antigen used to immunize rodents.

[0338] T-cell therapy is a hot topic in current medical research. Specifically, cytotoxic T cells can recognize and eliminate target antigens (such as viral or bacterial antigens, tumor antigens, etc.) or cells that present these antigens. The approach to adoptive cell therapy involves isolating tumor-infiltrating lymphocytes from tumor cell clusters, expanding them in vitro using T-cell growth factors, and then reinfusing them into the patient. The rodents described in this article can serve as a source of human T-cell receptor repositories, for example, providing high-affinity human T-cell receptors for adoptive T-cell transfer.

[0339] The methods for generating human TCRs described below appropriately include the use of rodents containing inserted human DNA encoding human TCRα and human TCRβ peptides.

[0340] The methods used to generate or use a human TCR are also applicable to generating a functional portion of that human TCR, appropriately including at least the binding portion of the TCR. Preferably, the binding affinity of the TCR fragment to the antigen is the same as or substantially the same as that of the full-length TCR.

[0341] The methods for using or generating a TCR as disclosed in this article are also applicable to using or generating functional fragments of that TCR.

[0342] In one aspect, a method for generating a T-cell receptor against a human antigen is disclosed, the method comprising: immunizing a rodent described herein with a target antigen; inducing an immune response in the rodent; optionally isolating activated T cells specific to the target antigen from the animal; and determining the nucleic acid sequence of a T-cell receptor expressed by the antigen-specific T cells. Methods for generating a T-cell response by immunizing an animal are known in the art. Immunization may include administering the target peptide antigen to the rodent in an immunization regimen comprising a primary immunization and at least one booster immunization. The immunization regimen may be “primary immunization + single booster immunization” or “primary immunization + two or three booster immunizations”. The peptide may be administered via subcutaneous injection for primary immunization and / or booster immunization. The peptide may be administered in combination with an adjuvant. An exemplary immunization regimen and adjuvant are provided in Example 6.

[0343] In one aspect, labeled MHC multimers (such as tetramers or higher-order multimers) can be used to sort antigen-specific CD8 cells, and then the TCR nucleic acids in the cells can be sequenced to identify the pairing sequences of the TCR heavy and light chains.

[0344] In one aspect, the present invention provides a method for generating a human T-cell receptor specific to a target antigen, the method comprising: immunizing the rodent of the present invention as described herein with the target antigen; inducing an immune response in the animal; optionally isolating T cells responsive to the target antigen from the animal; determining the nucleic acid sequence of the variable region of a human TCR expressed by the antigen-specific T cells; and expressing a nucleotide construct encoding a polypeptide chain of a human T-cell receptor specific to the target antigen. The expression of the nucleotide construct may be of a membrane-bound TCR or a soluble TCR.

[0345] In one aspect, a method for identifying T cells specific to a target antigen (such as a tumor-associated antigen) is disclosed, the method comprising: immunizing the rodent of the present invention described herein with the target antigen; inducing an immune response in the animal; and isolating T cells specific to the antigen from the non-human animal.

[0346] In one aspect, a method for identifying human T cell epitopes is disclosed, the method comprising: exposing the rodent of the present invention described herein to an antigen containing a putative T cell epitope; inducing an immune response in the rodent; isolating MHC class I restricted T cells that bind the epitope from the rodent; and identifying the epitope bound by the T cells.

[0347] In one aspect, a method for determining whether a peptide will elicit a cellular immune response in a human body is disclosed, the method comprising: exposing a genetically modified rodent (the rodent of the present invention as described herein) to the peptide; inducing an immune response in the rodent; and detecting CD8+ T cells comprising human TCRs containing sequences that bind to human MHC class I molecules.

[0348] In one aspect, a method is disclosed for determining whether a putative antigen containing an epitope will elicit a class-HLAI-restricted immune response upon exposure to the human immune system, the method comprising: exposing a rodent (the rodent of the present invention as described herein) to the antigen; and measuring an antigen-specific class-HLAI-restricted immune response in mice.

[0349] The genetically engineered rodents described herein can be used in methods for identifying T-cell receptors (such as high-affinity T-cell receptors) capable of recognizing target antigens (such as tumor or other disease antigens). This method may include: exposing the rodent (the rodents of the present invention described herein) to the antigen; inducing an immune response in the rodent against the antigen; and determining the sequence of the T-cell receptor that presents the antigen by binding to human MHC class I molecules.

[0350] The genetically engineered rodents described herein can be used to identify T cell receptors (such as high-affinity T cell receptors) capable of recognizing target antigens (such as tumor or other disease antigens). The method may include: exposing the rodents of the present invention to an antigen; inducing an immune response in the rodent against the antigen; optionally isolating T cells from the rodent containing T cell receptors that bind to human MHC class I molecules to present antigens; and determining the sequence of the T cell receptors that bind to the antigen.

[0351] The target antigen mentioned herein can be any antigen known to be associated with or causing a disease or condition, such as tumor-associated antigens, viral antigens, bacterial antigens, etc. Tumor-associated antigens are known and are available in the Cancer Immunity Peptide Database (archive.cancerimmunity.org / peptidedatabase / Tcellepitopes.htm). In some embodiments of the invention, the target antigen is a human antigen, such as a human tumor-associated antigen. In some embodiments, the antigen is a cell type-specific intracellular antigen, and the T cell receptor is used to guide the killing of cells expressing the antigen.

[0352] In one aspect, a method for determining T cell activation using a hypothetical human therapeutic agent is disclosed, the method comprising: exposing the rodent of the present invention described herein to a hypothetical human therapeutic agent (or exposing cells in the rodent expressing human MHC class I molecules to a peptide sequence of the hypothetical therapeutic agent); exposing cells of the genetically modified rodent displaying human MHC class I molecule / peptide complexes to T cells containing human CD8 capable of binding to the genetically modified rodent cells; and measuring the activation of T cells induced by the peptide-displaying cells in the genetically modified rodent.

[0353] The genetically modified rodents of this invention can be used to identify autoantigens associated with human autoimmune diseases such as type 1 diabetes and multiple sclerosis. Furthermore, the genetically modified rodents of this invention can be used to study multiple aspects of human autoimmune diseases and can be used as models of autoimmune diseases.

[0354] In one aspect, the present invention relates to a method for generating a human T-cell receptor, receptor variable region, or T-cell receptor antigen-binding portion against a target antigen, the method comprising: (i) Optionally, prepare the genetically modified rodents disclosed herein; (ii) Immunize the genetically modified rodents disclosed in this paper with the target antigen; (iii) Induce an immune response in the rodent; (iv) Determining the nucleic acid sequence or variable region sequence of a human TCR expressed on rodent T cells that is responsive to the target antigen, optionally including the step of isolating T cells expressing the TCR; and any of the following steps: (v) Expressing a human T-cell receptor, a variable region of a human T-cell receptor, or an antigen-binding moiety of a human T-cell receptor in cells; optionally further formulating the expressed human T-cell receptor, a variable region of a human T-cell receptor, or an antigen-binding moiety thereof with pharmaceutically acceptable excipients; or (vi) Inserting a nucleic acid encoding the human T-cell receptor, the variable region of the human T-cell receptor, or its antigen-binding portion into cells (such as human or animal cells) in vitro or in vitro, optionally preparing and delivering cell lines containing the inserted nucleic acid into humans or animals; or (vii) Optionally, nucleic acids (such as RNA or DNA) encoding the human T-cell receptor or the variable region of the human T-cell receptor are formulated with a suitable delivery carrier (such as lipids or liposomes) for in vivo delivery to patients in need.

[0355] The present invention also includes a method for treating an individual in need, the method comprising: (i) Deliver the cells obtained according to step (vi) above to a patient in need; or (ii) Deliver the nucleic acid (such as RNA or DNA) encoding the human T-cell receptor or the variable region of the human T-cell receptor according to the above method (optionally prepared as described in step (vii) above) to the patient in need.

[0356] In one aspect, a method is provided for preparing human TCRα and / or TCRβ or functional fragments thereof (such as antigen-binding portions), the method comprising: expressing human TCRα and / or TCRβ polypeptides or functional fragments thereof in cells from a nucleotide sequence obtained from or identified from rodents described herein (the nucleotide sequence encoding the TCR or functional fragments thereof described herein).

[0357] In one aspect, the nucleic acid to be expressed is contained in a viral vector; in a particular embodiment, the viral vector is a lentiviral vector.

[0358] In one implementation, the cell line used to express nucleic acids (such as nucleic acids encoding human TCR or functional fragments thereof) is selected from COS cells, CHO cells, 293 cells, HeLa cells, and retinal cells (such as PERC.6™ cells) expressing viral nucleic acid sequences.

[0359] In one aspect, a cell expressing a human polypeptide is provided, the sequence of which is derived from a sequence obtained by immunizing rodents with the human TCR described herein.

[0360] The TCR α and β variable domains isolated from the rodents described herein can be recombined in soluble molecules containing the human TCR constant region. The TCR variable domain sequences according to the invention are particularly suitable for developing soluble TCR therapeutics for human patients, comprising bispecific T cell connectives (wherein the variable domains are linked to the human TCR constant region). As described herein (Example 8), 100% (124 / 124) of the tested TCR clontypes were recombinantly expressed in a soluble form. No clontypes were found to be unable to express in a soluble form containing the human constant region. The observed good expression levels and solubility of the TCR-binding fragments are at least partly attributable to species matching (fully human) – the interaction between the variable and constant regions of the mouse TCR is known to be crucial for folding. Because the TCRs in the rodents of the invention are fully human, antigen-binding T cells are selected in vivo in an environment where the human TCR variable region is linked to the human TCR constant region. Therefore, converting screened TCRs into a soluble form (where the isolated TCR variable region is linked to the human TCR constant region) reflects the in vivo environment in which these TCRs were screened. In contrast, the TCRs produced by transgenic “VelociT” mice, as described by Moore et al. (Sci Immunol 6, 2021), possess both a human variable domain and a mouse constant domain. TCRs from VelociT mice require modification to replace the mouse constant region with the human constant region to reduce immunogenicity in human patients. This chimeric TCR modification to alter the constant region may reduce or eliminate its expression—because the human variable region was not initially screened in vivo in a fully human TCR environment.

[0361] Cells and tissues In one aspect, the present invention relates to cells derived from the rodents described herein, such as T cells or antigen-presenting cells (APCs). The invention also relates to tissues and embryos derived from the rodents described herein.

[0362] In one aspect, the cell line isolated from the rodent described herein is an ES cell or hematopoietic stem cell, or other cells capable of differentiating into non-human mammalian tissues and organs. The cell may also be an induced pluripotent stem cell (iPS cell) induced from cells obtained from the rodent.

[0363] In one aspect, the isolated cell is a T cell. In one aspect, the T cell expresses human TCRα and / or TCRβ, and / or expresses human CD8α and / or CD8β. In one aspect, the T cell is a CD8+ T cell.

[0364] In one aspect, the isolated cells are antigen-presenting cells. In another aspect, the antigen-presenting cells express human MHC class I molecules and / or human β2M molecules.

[0365] Methods for preparing genetically modified cells by modifying ES cells (such as mouse or rat ES cells) are known in the art. This invention also extends to modifying hematopoietic stem cells or other cells capable of differentiating into non-human mammalian tissues and organs, preferably cells that can generate a human TCR library.

[0366] In one aspect, the present invention relates to a pluripotent cell comprising an unrearranged human TCR locus encoding the human TCRα and β polypeptides described herein. In one embodiment, the induced pluripotent cell is derived from the rodents described herein.

[0367] The present invention also relates to a cell line derived from or derived from the cells described herein. In one aspect, the cell line is an immortalized cell line.

[0368] This cell line may contain rearranged or unrearranged VDJ or VJ genes from TCRα and / or TCRβ peptides.

[0369] Cell immortalization can be achieved by fusing cells with tumor cells (to provide cells and cell lines that produce TCRs) or by directly immortalizing cells.

[0370] In one aspect, a hybridoma or quadroma derived from the cells of the rodents described herein is provided.

[0371] Methods for developing rodents from rodent ES cells are known in the art, comprising the following steps: injecting ES cells into a blastocyst, and then transplanting the chimeric blastocyst into a female rodent to produce fertile offspring that can be selected for the desired insert fragment.

[0372] In one aspect, the present invention relates to a chimeric rodent comprising ES cell-derived tissue and host embryo-derived tissue.

[0373] In one aspect, the present invention relates to a rodent embryo comprising donor ES cells derived from the rodent described herein. In another aspect, the embryo comprises ES donor cells containing unrearranged human TCR loci and host embryonic cells.

[0374] In one aspect, an tissue derived from the rodents described herein is provided that expresses human TCR peptides (such as TCRα and / or TCRβ).

[0375] rodent strains The preferred rodents are rats or mice, with mice being particularly preferred.

[0376] In one aspect, the mice may be BL6 strains, such as the C57BL strain, for example selected from C57BL / A, C57BL / An, C57BL / GrFa, C57BL / KaLwN, C57BL / 6, C57BL / 6J, C57BL / 6ByJ, C57BL / 6NJ, C57BL / 10, C57BL / 10ScSn, C57BL / 10Cr and C57BL / Ola strains. In another aspect, the mouse can be a 129 strain, for example selected from 129P1, 129P2, 129P3, 129X1, 129S1 (such as 129S1 / SV, 129S1 / Svlm), 129S2, 129S4, 129S5, 129S9 / SvEvH, 129S6 (129 / SvEvTac), 129S7, 129S8, 129T1, and 129T2 strains.

[0377] In one aspect, the genetically modified mouse is a hybrid offspring of the 129 strain and the C57BL / 6 strain. In another particular aspect, the mouse is a hybrid offspring of the 129 strain or the BL / 6 strain. In a particular embodiment, the 129 strain of the hybrid offspring is the 129S6 (129 / SvEvTac) strain.

[0378] In another embodiment, the mouse is a BALB strain, such as the BALB / c strain. In yet another embodiment, the mouse is a hybrid offspring of a BALB strain and the aforementioned strains.

[0379] If the rodent is a rat, it may be selected from Wistar rats, the LEA strain, the Sprague-Dawley strain, the Fischer strain, the F344 strain, the F6 strain, and the Dark-Agouti strain. In one embodiment, the rat strain is a hybrid offspring selected from two or more strains within that strain group.

[0380] This invention relates to any of the mouse or rat strains described above, and these strains contain any genetic modifications disclosed herein.

[0381] Cell development / T cell development Inserting human DNA into endogenous rodent loci can appropriately avoid affecting the normal developmental capacity of relevant rodent cells, such as not affecting the cell's participation in the presentation of MHC class I molecules bound to antigens and / or the TCR's recognition of these bound antigens.

[0382] In one respect, the genetic modifications contained in rodents and rodent cells described herein do not alter the normal lineage selection and development of T cells.

[0383] In one respect, the inserted human DNA enables appropriate spatial and temporal protein expression.

[0384] In one aspect, the rodent and rodent T cells described herein, comprising human TCRα and / or TCRβ, can generate T cells capable of undergoing thymic development—developing from the DN1 stage to the DN2, DN3, DN4, and DP stages, ultimately evolving into CD8 single-positive (SP) T cells. These rodent T cells of the present invention can express cell surface molecules (such as CD25, CD44, Kit, CD3, pTcc, etc.) that are typically produced by T cells at specific stages of thymic development. Therefore, the rodents described herein can express pTα, which binds to TCRα, at the DN3 stage of thymic development. The non-human animals described herein can generate T cells capable of undergoing thymic development and differentiating into CD8+ T cells. In one aspect, these rodents can generate T cells capable of undergoing normal T cell differentiation in the periphery. In some embodiments, the non-human animals described herein can generate a normal repertoire of effector T cells (such as cytotoxic T lymphocytes (CTLs), TH1, TH2, regulatory T cells (Tregs), etc.).

[0385] In one aspect, the rodent and rodent T cells described herein, comprising human CD8α and / or CD8β peptides, can generate T cells capable of undergoing thymic development—developing from the DN1 stage to the DN2, DN3, DN4, and DP stages, ultimately evolving into CD8 single-positive (SP) T cells. These rodent T cells of the present invention can express cell surface molecules (such as CD25, CD44, Kit, CD3, pTcc, etc.) that are typically produced by T cells at specific stages of thymic development. Therefore, the rodents described herein can express pTα, which binds to TCRα, at the DN3 stage of thymic development. The non-human animals described herein can generate T cells capable of undergoing thymic development and differentiating into CD8+ T cells. In one aspect, these rodents can generate T cells capable of undergoing normal T cell differentiation in the periphery. In some embodiments, the non-human animals described herein can generate a normal repertoire of effector T cells (such as cytotoxic T lymphocytes (CTLs), TH1, TH2, regulatory T cells (Tregs), etc.). In one aspect, the CD8 locus enables the proper expression of CD8α and / or β (appropriate spatial and temporal protein expression), CD8+ T cell development, CD8 lineage selection, and helper receptor function.

[0386] In one aspect, the rodents described herein that contain human MHC class I peptides can produce antigen-presenting cells that express MHC class I molecules—cells that are capable of substantially normal development and can present antigens to TCRs (such as human TCRs).

[0387] General features of the present invention The general features of the present invention can be combined with all aspects of the present invention.

[0388] In one aspect, one or more (or even all) complete human sequences are inserted, operatively linked to one or more endogenous rodent regulatory sequences, such as promoters and / or enhancers.

[0389] In one aspect, the orientation of the inserted human DNA relative to the centromere is the same as that of the homologous endogenous rodent locus; and the position of the inserted human DNA relative to the centromere is the same as or substantially the same as that of the homologous endogenous rodent locus (e.g., the orientation and position of the human TRA insert fragment are the same as or substantially the same as those of the rodent TRA, and it is inserted at the endogenous locus).

[0390] In one aspect, the inserted human sequence may allow for variation. For example, human TCRα, TCRβ, MHC class I molecules, CD8α and / or CD8β peptides may contain one or more conserved or non-conserved modifications. Therefore, the present invention provides a rodent expressing human TCRα and / or β peptide sequences, CD8α and / or β peptide sequences, MHC class I molecules, or β2M, wherein the human peptide sequence has at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the human TCRα, TCRβ, CD8α, CD8β, MHC class I molecules, or β2M peptide sequences, respectively.

[0391] In another option, the human polypeptide sequence has at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with human TCRα, TCRβ, CD8α, CD8β, MHC class I molecules, or β2M polypeptide sequences in the functional part (such as the domain of the polypeptide or protein) of the polypeptide or protein complex.

[0392] In one embodiment, the human polypeptide sequence contains one or more conserved substitutions. A conserved substitution is the replacement of one amino acid residue with another amino acid residue of a side chain R group that has similar chemical properties (such as charge or hydrophobicity). Conserved amino acid substitutions can be achieved by modifying the nucleotide sequence to introduce nucleotide changes that encode the conserved substitution. Generally, conserved amino acid substitutions do not substantially alter the target functional properties of a protein, such as the ability of MHC class I molecules to present the target peptide. Examples of amino acid groups with side chains of similar chemical properties include: aliphatic side chains (such as glycine, alanine, valine, leucine, isoleucine); aliphatic hydroxyl side chains (such as serine, threonine); amide-containing side chains (such as asparagine, glutamine); aromatic side chains (such as phenylalanine, tyrosine, tryptophan); basic side chains (such as lysine, arginine, histidine); acidic side chains (such as aspartic acid, glutamic acid); and sulfur-containing side chains (such as cysteine, methionine). Conserved amino acid substitution groups include, for example, valine / leucine / isoleucine, phenylalanine / tyrosine, lysine / arginine, alanine / valine, glutamic acid / aspartic acid, and asparagine / glutamine. Besides the nucleic acid residues encoding the human MHC class I polypeptides described herein, other nucleic acids can also encode the polypeptides disclosed herein due to the degeneracy of the genetic code. Therefore, in addition to genetically modified non-human animals whose genomes contain nucleotide sequences encoding MHC class I polypeptides with conserved amino acid substitutions, non-human animals whose genomes contain nucleotide sequences different from those described herein due to the degeneracy of the genetic code are also within the scope of protection of this invention.

[0393] In one aspect, human DNA inserted into the rodent genome is human genomic DNA.

[0394] In another aspect, the human DNA inserted into the rodent genome can be in the form of non-genomic DNA. For example, in the case of inserting a human TCR locus, the DNA can be in the form of a "mini-gene"—a smaller insert fragment than human genomic DNA, achieved by deleting certain elements of human genomic DNA. For example, non-coding or non-functional sequences can be deleted.

[0395] When multiple DNA fragments are inserted, the inserted human DNA can be continuous or discontinuous. Preferably, the inserted human DNA is in the form of a continuous gene or a continuous human locus in the rodent genome.

[0396] The human peptides described herein can be expressed under the regulation of endogenous non-human regulatory elements, such as rodent regulatory elements, including promoters, enhancers, or silencers. These elements can be natural rodent regulatory elements located in their native positions within the rodent genome; specifically, even if their position relative to other rodent gene elements is altered due to factors such as the insertion of human DNA, the element has not been removed or relocated from the genome. Alternatively, the human peptides described herein can also be expressed under the regulation of human regulatory elements, such as human promoters, enhancers, or silencers.

[0397] In one aspect, the inserted locus may be regulated, alone or together, by at least one human regulatory element and at least one endogenous rodent regulatory element. For example, human promoter and host enhancer sequences and one or more loci may be used.

[0398] Unless otherwise specified, the terms "rodent containing..." or "rodent cell containing..." used in this document refer to a rodent or rodent cell whose genome contains a specific nucleic acid or DNA.

[0399] The term "operable linkage" refers to the arrangement of elements that enables them to function in the intended manner. Therefore, the nucleic acid sequence encoding a protein is operably linked to regulatory sequences (such as promoters, enhancers, silencers, etc.) to achieve appropriate transcriptional regulation. Furthermore, multiple parts of human proteins are operably linked to ensure that the protein possesses appropriate folding, processing, targeting, expression, and other functional properties in the cell. Unless otherwise stated, multiple domains of human proteins are operably linked to each other.

[0400] As mentioned in this article regarding functional peptides, "functional" means that the peptide retains at least one biological activity associated with the native protein. Substitution at an endogenous locus (such as at an endogenous non-human MHC locus) can prevent that locus from expressing a functional endogenous peptide (such as an MHC class I peptide). Similarly, as mentioned in this article regarding the functional portion of a protein, "functional" means that the extracellular domain retains its function; for example, the functional portion of the TCR retains its ability to bind to MHC class I molecules and present antigens.

[0401] As mentioned above, the rodents of the present invention preferably do not express functional rodent polypeptides derived from modified endogenous loci. For example, in the case where DNA encoding a human TCRα polypeptide has been inserted into an endogenous locus, the rodent does not express the rodent TCRα polypeptide derived from the endogenous TRA locus. For example, the endogenous locus may be substituted before, simultaneously with, or after the insertion of human DNA. Alternatively, the endogenous locus may be inactivated by mutation or inversion, thereby preventing the expression of rodent polypeptides. The term "endogenous locus" as used herein refers to a locus in the rodent genome that contains a coding region, any intercalated DNA (such as introns), and a promoter region. In one aspect, the inserted human DNA replaces all or part of the host locus. In another aspect, the host locus may be retained, but substitution is achieved by inserting human DNA upstream or downstream of the endogenous locus. Preferably, the expression of endogenous genes at the endogenous locus is inactivated; however, in one aspect, essential genes that do not encode TCR / MHC class I molecules, CD8, or β2M may be retained or reinserted. For example, non-TCR-related genes associated with reproductive capacity may be retained at the TCRβ locus, such as genes encoding one or more serine proteases located at the TCRβ locus.

[0402] In one aspect, the TCR locus (TCRlocus or TCRgenelocus) in this paper refers to genomic DNA containing the TCR coding region, including the entire TCR coding region (including the unrearranged V(D)J sequence, promoter, enhancer sequence, constant sequence, and any upstream or downstream sequences (such as untranslated regions, regulatory regions, etc.) or intercalated DNA sequences (such as introns, etc.)). The TCR variable locus (TCR variable gene locus) refers to a genomic DNA region containing a TCR variable region segment (V(D)J region) but not the TCR constant sequence.

[0403] It should be understood that the specific embodiments described herein are shown by way of example and are not intended to limit the invention. The key features of the invention can be applied to a variety of embodiments without departing from the scope of the invention. Many equivalents of the specific methods described herein will be apparent to those skilled in the art, or can be determined by conventional study alone. These equivalents are considered to be within the scope of the invention and are covered by the appended claims. All publications and patent applications mentioned in this specification reflect the prior art of those skilled in the art to which this invention pertains. All publications and patent applications are incorporated herein by reference to the extent that each publication or patent application is explicitly and separately cited and incorporated herein by reference. In the appended claims and / or this specification, the term “a (or an)” used in conjunction with “comprising” may mean “one,” but is also consistent with the meaning of “one or more,” “at least one,” or “one or more.” The term “or” used in the appended claims is intended to mean “and / or” unless otherwise expressly stated to refer only to alternatives or these alternatives are mutually exclusive; however, the invention supports the definition of referring only to alternatives and “and / or.”

[0404] Throughout this manual, the term “about” is used to indicate that a value includes the inherent error variation of the apparatus or method used to measure that value, or the differences between individuals studied.

[0405] As used in this specification and the accompanying claims, the terms “comprising” (and any form thereof, such as “comprise” and “comprises”), “having” (and any form thereof, such as “have” and “has”), “including” (and any form thereof, such as “includes” and “include”) or “containing” (and any form thereof, such as “contains” and “contains”) are inclusive or open-ended expressions that do not exclude additional, unlisted elements or method steps.

[0406] As used herein, the term "or combinations thereof" refers to all permutations and combinations of the items listed preceding the term. For example, "A, B, C or combinations thereof" is intended to include at least one of the following: A, B, C, AB, AC, BC, or ABC; and if the order is important in a particular context, it may also include BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, combinations containing one or more repeated items or terms are explicitly included, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, etc. Those skilled in the art will understand that there is generally no limit to the number of items or terms in any combination unless it is clearly determined from the context.

[0407] Various uses of TCRs prepared in mice are disclosed in EP2958937B1, EP2771357B1 and EP2958938B1, which are expressly incorporated herein by reference but apply to rodent scenarios containing complete human loci as disclosed herein.

[0408] The following detailed description contains exemplary embodiments of the invention, which are not intended to limit the invention as claimed. The accompanying drawings, which form part of this specification, are used together with the specification to illustrate embodiments only and are not intended to limit the invention.

[0409] The following numbered clauses represent embodiments of the present invention.

[0410] 1. A genetically modified rodent comprising: (i) An unrearranged T-cell receptor (TCR) α variable locus comprising at least one human Vα segment and at least one human Jα segment, wherein the TCRα variable gene is operatively linked to a human TCRα constant gene sequence. (ii) An unrearranged TCRβ variable locus comprising at least one human Vβ segment, at least one human Dβ segment and at least one human Jβ segment, wherein the unrearranged TCRβ variable gene is operatively linked to a human TCRβ constant gene sequence. These unrearranged human T cell variable region gene segments can rearrange to form genes encoding the variable domain of the human T cell receptor; the unrearranged TCRα variable locus is located at the endogenous rodent TCRα locus, and the unrearranged TCRβ variable locus is located at the endogenous rodent TCRβ locus.

[0411] 2. The rodents described in Clause 1 whose genome encodes MHC class I polypeptides, wherein the α1 and α2 domains are of human origin.

[0412] 3. The rodent as described in Clause 2, wherein the MHC class I polypeptide contains an α3 domain that binds to CD8 expressed in the rodent.

[0413] 4. The rodent as described in Clause 3, wherein the α3 domain is of human origin and the CD8 contains a human MHC class I binding domain.

[0414] 5. The rodent as described in Clause 4, wherein the CD8 comprises human CD8α.

[0415] 6. The rodent as described in Clause 4, wherein the CD8 comprises a human-rodent chimeric CD8α.

[0416] 7. The rodent as described in any of Clauses 4 to 6, wherein the CD8 comprises human CD8β.

[0417] 8. The rodent as described in any of Clauses 4 to 6, wherein the CD8 comprises a human-rodent chimeric CD8β.

[0418] 9. In any of the provisions of clauses 5 to 8, the human or human-rodent chimeric CD8 gene is located at the endogenous rodent CD8 locus in the genome of the rodent.

[0419] 10. The rodent as described in any of the clauses 2 to 9, wherein the MHC class I polypeptide comprises a human transmembrane domain and a cytoplasmic domain.

[0420] 11. The rodent as described in Clause 3, wherein the MHC class I α3 domain is of rodent origin.

[0421] 12. The rodent as described in Clause 11, wherein the CD8 comprises an endogenous rodent CD8.

[0422] 13. The rodent as described in any of Clauses 2 to 12, wherein the DNA encoding the MHC class I polypeptide is integrated at an endogenous rodent MHC class I locus.

[0423] 14. A genetically modified rodent as described in Clause 1, wherein the rodent genome further comprises a nucleic acid sequence encoding an MHC class I polypeptide, the MHC class I polypeptide being: (i) a complete human MHC class I, (ii) an endogenous rodent MHC class I, or (iii) a human-rodent chimeric MHC class I (e.g., a chimeric MHC class I containing a rodent α3 domain capable of binding rodent CD8 protein); optionally, wherein the nucleic acid sequence encoding the MHC class I polypeptide is located at an endogenous locus.

[0424] 15. A genetically modified rodent as described in any of Clauses 1 or 14, wherein the rodent genome further comprises: a nucleic acid sequence encoding a CD8α polypeptide at an endogenous rodent CD8α co-receptor locus, and a nucleic acid sequence encoding a CD8β polypeptide at an endogenous rodent CD8β co-receptor locus; optionally, these nucleic acid sequences are selected from: (i) The nucleic acid sequence encoding the human CD8α polypeptide (located at the endogenous rodent CD8α co-receptor locus) and the nucleic acid sequence encoding the human CD8β polypeptide (located at the endogenous rodent CD8β co-receptor locus). (ii) The nucleic acid sequence encoding the human-rodent chimeric CD8α polypeptide (located at the endogenous rodent CD8α co-receptor locus) and the nucleic acid sequence encoding the human-rodent chimeric CD8β polypeptide (located at the endogenous rodent CD8β co-receptor locus); and (iii) Nucleic acid sequences encoding rodent CD8α polypeptide at endogenous loci and nucleic acid sequences encoding rodent CD8β polypeptide at endogenous loci.

[0425] 16. A genetically modified rodent as described in any of Clauses 1, 14 or 15, wherein the rodent genome contains a nucleic acid sequence encoding a human β2 microglobulin polypeptide.

[0426] 17. A genetically modified rodent as described in Clauses 1 and 14 to 16, wherein the rodent genome contains a nucleic acid sequence encoding a human β2 microglobulin polypeptide, optionally located at an endogenous rodent β2 microglobulin locus.

[0427] 18. A genetically modified rodent as described in Clause 1, comprising: (i) An unrearranged T-cell receptor (TCR) α variable locus comprising at least one human Vα segment and at least one human Jα segment, wherein the TCRα variable gene is operatively linked to a human TCRα constant gene sequence. (ii) An unrearranged TCRβ variable locus comprising at least one human Vβ segment, at least one human Dβ segment and at least one human Jβ segment, wherein the unrearranged TCRβ variable gene is operatively linked to a human TCRβ constant gene sequence. These unrearranged human T cell variable region gene segments can rearrange to form genes encoding the variable domain of the human T cell receptor; and the unrearranged TCRα variable locus is located at the endogenous rodent TCRα locus, and the unrearranged TCRβ variable locus is located at the endogenous rodent TCRβ locus; and The rodent genome contains any of the following: [A] The nucleic acid sequence encoding the human CD8α polypeptide (located at the endogenous rodent CD8α co-receptor locus), the nucleic acid sequence encoding the human CD8β polypeptide (located at the endogenous rodent CD8β co-receptor locus), and the nucleic acid sequence encoding the human MHC class I polypeptide located at the endogenous rodent MHC class I locus; or [B] Nucleic acid sequences encoding a human-rodent chimeric CD8α polypeptide (located at an endogenous rodent CD8α co-receptor locus), a human-rodent chimeric CD8β polypeptide (located at an endogenous rodent CD8β co-receptor locus), and a nucleic acid sequence encoding a human MHC class I polypeptide located at an endogenous rodent MHC class I locus; wherein the chimeric CD8 protein can bind to human MHC class I; or [C] Nucleic acid sequences encoding rodent CD8α polypeptide at endogenous loci, nucleic acid sequences encoding rodent CD8β polypeptide at endogenous loci, and nucleic acid sequences encoding human-rodent chimeric MHC class I polypeptide at endogenous rodent MHC class I loci; wherein the rodent CD8 protein can bind to the chimeric MHC class I. Optionally, the rodent genome contains a nucleic acid sequence encoding a human β2 microglobulin polypeptide, which, when present, is located at the endogenous rodent β2 microglobulin locus.

[0428] 19. A genetically modified rodent cell (e.g., embryonic stem cell, ES cell) comprising: (i) An unrearranged T-cell receptor (TCR) α variable locus comprising at least one human Vα segment and at least one human Jα segment, wherein the TCRα variable gene is operatively linked to a human TCRα constant gene sequence. (ii) An unrearranged TCRβ variable locus comprising at least one human Vβ segment, at least one human Dβ segment and at least one human Jβ segment, wherein the unrearranged TCRβ variable gene is operatively linked to a human TCRβ constant gene sequence. These unrearranged human T cell variable region gene segments can rearrange to form genes encoding the variable domain of the human T cell receptor. Furthermore, the unrearranged TCRα variable locus is located at the endogenous rodent TCRα locus, and the unrearranged TCRβ variable locus is located at the endogenous rodent TCRβ locus.

[0429] 20. Genetically modified rodent cells as described in Clause 19, wherein the rodent genome further comprises a nucleic acid sequence encoding an MHC class I polypeptide located at an endogenous rodent MHC class I locus, the MHC class I polypeptide being: (i) a complete human MHC class I, (ii) an endogenous rodent MHC class I, or (iii) a human-rodent chimeric MHC class I (e.g., a chimeric MHC class I containing a rodent α3 domain capable of binding rodent CD8 protein).

[0430] 21. Genetically modified rodent cells as described in any of Clauses 19 or 20, wherein the rodent genome further comprises: nucleic acid sequences encoding a CD8α polypeptide at an endogenous rodent CD8α co-receptor locus, and nucleic acid sequences encoding a CD8β polypeptide at an endogenous rodent CD8β co-receptor locus; optionally, these nucleic acid sequences are selected from: (i) The nucleic acid sequence encoding the human CD8α polypeptide (located at the endogenous rodent CD8α co-receptor locus) and the nucleic acid sequence encoding the human CD8β polypeptide (located at the endogenous rodent CD8β co-receptor locus). (ii) The nucleic acid sequence encoding the human-rodent chimeric CD8α polypeptide (located at the endogenous rodent CD8α co-receptor locus) and the nucleic acid sequence encoding the human-rodent chimeric CD8β polypeptide (located at the endogenous rodent CD8β co-receptor locus). (iii) Nucleic acid sequences encoding rodent CD8α polypeptide at endogenous loci and nucleic acid sequences encoding rodent CD8β polypeptide at endogenous loci.

[0431] 22. Genetically modified rodent cells as described in any of Clauses 19 to 21, wherein the rodent genome contains a nucleic acid sequence encoding a human β2 microglobulin polypeptide, optionally located at an endogenous rodent β2 microglobulin locus.

[0432] 23. Genetically modified rodent cells as described in Clause 19, comprising: (i) An unrearranged T-cell receptor (TCR) α variable locus comprising at least one human Vα segment and at least one human Jα segment, wherein the TCRα variable gene is operatively linked to a human TCRα constant gene sequence. (ii) An unrearranged TCRβ variable locus comprising at least one human Vβ segment, at least one human Dβ segment and at least one human Jβ segment, wherein the unrearranged TCRβ variable gene is operatively linked to a human TCRβ constant gene sequence. These unrearranged human T cell variable region gene segments can rearrange to form genes encoding the variable domain of the human T cell receptor; and the unrearranged TCRα variable locus is located at the endogenous rodent TCRα locus, and the unrearranged TCRβ variable locus is located at the endogenous rodent TCRβ locus; and The rodent genome contains any of the following: [A] The nucleic acid sequence encoding the human CD8α polypeptide (located at the endogenous rodent CD8α co-receptor locus), the nucleic acid sequence encoding the human CD8β polypeptide (located at the endogenous rodent CD8β co-receptor locus), and the nucleic acid sequence encoding the human MHC class I polypeptide located at the endogenous rodent MHC class I locus; or [B] Nucleic acid sequences encoding a human-rodent chimeric CD8α polypeptide (located at an endogenous rodent CD8α co-receptor locus), a human-rodent chimeric CD8β polypeptide (located at an endogenous rodent CD8β co-receptor locus), and a nucleic acid sequence encoding a human MHC class I polypeptide located at an endogenous rodent MHC class I locus; wherein the chimeric CD8 protein can bind to human MHC class I; or [C] Nucleic acid sequences encoding rodent CD8α polypeptide at endogenous loci, nucleic acid sequences encoding rodent CD8β polypeptide at endogenous loci, and nucleic acid sequences encoding human-rodent chimeric MHC class I polypeptide at endogenous rodent MHC class I loci; wherein the rodent CD8 protein can bind to the chimeric MHC class I. Optionally, the rodent genome contains a nucleic acid sequence encoding a human β2 microglobulin polypeptide, which, when present, is located at the endogenous rodent β2 microglobulin locus.

[0433] 1. A genetically modified rodent T cell comprising: (i) An unrearranged T-cell receptor (TCR) α variable locus comprising at least one human Vα segment and at least one human Jα segment, wherein the TCRα variable gene is operatively linked to a human TCRα constant gene sequence. (ii) An unrearranged TCRβ variable locus comprising at least one human Vβ segment, at least one human Dβ segment and at least one human Jβ segment, wherein the unrearranged TCRβ variable gene is operatively linked to a human TCRβ constant gene sequence. These unrearranged human T cell variable region gene fragments can rearrange to form genes encoding the variable domain of the human T cell receptor; and the unrearranged TCRα variable locus is located at the endogenous rodent TCRα locus, and the unrearranged TCRβ variable locus is located at the endogenous rodent TCRβ locus.

[0434] 2. Genetically modified rodent T cells as described in Clause 24, further comprising any of the following: [A] The nucleic acid sequence encoding the human CD8α polypeptide (located at the endogenous rodent CD8α co-receptor locus), and the nucleic acid sequence encoding the human CD8β polypeptide (located at the endogenous rodent CD8β co-receptor locus); or [B] Nucleic acid sequences encoding human-rodent chimeric CD8α polypeptides (located at the endogenous rodent CD8α co-receptor locus) and nucleic acid sequences encoding human-rodent chimeric CD8β polypeptides (located at the endogenous rodent CD8β co-receptor locus); or [C] Nucleic acid sequences encoding rodent CD8α polypeptide at endogenous loci and nucleic acid sequences encoding rodent CD8β polypeptide at endogenous loci; The cell expresses any of the following: [A] Human TCR and human CD8 peptide; or [B] Human TCR and chimeric CD8 peptide; or [C] Human TCR and rodent CD8 peptide.

[0435] 26. A genetically modified rodent antigen-presenting cell comprising any of the following: (i) The nucleic acid sequence encoding a human MHC class I polypeptide located at an endogenous rodent MHC class I locus; or (ii) The nucleic acid sequence encoding a human-rodent chimeric MHC class I polypeptide located at an endogenous rodent MHC class I locus, wherein, where appropriate, the nucleic acid sequence encoding this chimeric MHC class I has rodent exon 4, and exons 1 to 3 and 5 to 7 are of human origin; and (iii) Optionally, a nucleic acid sequence encoding a human β2 microglobulin polypeptide, which, when present, is located at the endogenous rodent β2 microglobulin gene locus; The cells express human MHC class I or chimeric MHC class I, and optionally express human β2M peptide.

[0436] 27. Genetically modified rodents or rodent cells as described in any of the clauses 1 to 26, wherein the endogenous locus modified to contain DNA encoding human or chimeric polypeptides does not express an equivalent rodent polypeptide.

[0437] 28. Genetically modified rodents or rodent cells as described in any of Clauses 1 to 27, which do not express a functional endogenous TCRα polypeptide from an endogenous TCRα variable locus.

[0438] 29. Genetically modified rodents or rodent cells as described in Clause 28, which are homozygous at the TCRα locus.

[0439] 30. Genetically modified rodents or rodent cells as described in any of the clauses 1 to 29, which do not express a functional endogenous TCRβ polypeptide from an endogenous TCRβ variable locus.

[0440] 31. Genetically modified rodents or rodent cells as described in Clause 30, which are homozygous at the TCRβ locus.

[0441] 32. A genetically modified rodent or rodent cell as described in any of Clauses 1 to 31, wherein when the rodent or cell expresses a complete human MHC class I, it does not express a functional endogenous rodent CD8 co-receptor from the endogenous CD8 locus.

[0442] 33. Genetically modified rodents or rodent cells as described in any of Clauses 1 to 32, which do not express functional endogenous rodent MHC class I peptides from endogenous MHC class I loci.

[0443] 34. A genetically modified rodent or rodent cell as described in any of Clauses 1 to 33, wherein the rodent or rodent cell contains a nucleic acid sequence encoding human β2 microglobulin, and does not express functional endogenous rodent β2 microglobulin from the endogenous rodent β2 microglobulin locus.

[0444] 35. Genetically modified rodents or rodent cells as described in any of Clauses 28 to 34, wherein the deletion of the functional endogenous rodent TCRα variable locus includes deletions selected from the group consisting of: (a) deletion of all endogenous Vα gene segments, (b) deletion of all endogenous Jα gene segments, (c) a combination of the above deletions; and / or the deletion of the functional endogenous rodent TCRβ variable locus, including deletions selected from the group consisting of: (a) deletion of all endogenous Vβ gene segments, (b) deletion of all endogenous Dβ gene segments, (c) deletion of all endogenous Jβ gene segments, (d) a combination of the above deletions.

[0445] 36. A genetically modified rodent or rodent cell as described in any of Clauses 1 to 35, wherein the endogenous rodent TCRα locus is deleted from the rodent genome at a distal 3'V (Vα1) to the rodent 3' constant region TCRαC (including the constant region), such that a rodent enhancer downstream of TCRαC in the rodent genome is operatively linked to an unrearranged human T cell receptor (TCR)α variable locus.

[0446] 37. Genetically modified rodents or rodent cells as described in any of the preceding clauses, wherein the human Vα and Jα segments are rearranged to form rearranged human Vα / Jα sequences; and the human Vβ, Dβ, and Jβ segments are rearranged to form rearranged human Vβ / Dβ / Jβ sequences.

[0447] 38. A genetically modified rodent or rodent cell as described in any of the preceding clauses, wherein the rodent or rodent cell expresses a human T cell receptor containing the human TCRα and TCRβ variable domains on the surface of a T cell.

[0448] 39. Genetically modified rodents or rodent cells as described in any of the preceding clauses, wherein the unrearranged TCRα variable locus contains a complete human Jα fragment library and a complete human Vα fragment library; and / or the unrearranged TCRβ variable locus contains a complete human Jβ fragment library, a complete human Dβ fragment library, and a complete human Vβ fragment library.

[0449] 40. A genetically modified rodent or rodent cell that is any of the following: (i) a rodent or rodent cell as described in any of the preceding clauses, wherein the rodent retains an endogenous TCRα variable locus and / or an endogenous rodent TCRβ variable locus, and any retained endogenous rodent TCRα variable locus is a nonfunctional locus, and / or any retained endogenous rodent TCRβ variable locus is a nonfunctional locus; optionally, all or part of the retained endogenous rodent TCRα and / or β variable locus is inverted relative to its normal orientation in the rodent genome; or (ii) A reproductive rodent or rodent cell wherein at least a segment of the rodent TCRβ locus from rodent Vβ1 to Dβ1 is inverted in the rodent genome relative to its natural orientation; optionally, the method further includes inserting all or part of a nucleic acid (e.g., DNA) encoding TCRβ from another species different from the rodent into the rodent genome, such as inserting all or part of human DNA encoding TCRβ.

[0450] 41. A genetically modified male rodent that is any of the following: (i) A rodent as described in any of the preceding clauses, which is substantially capable of wild-type reproduction, wherein the rodent's TCRβ locus is missing at least a segment from rodent Vβ1 to Dβ1, and its genome also contains one or more, or all of the serine protease genes Prss58, Prss59, Prss3b, Try4, Try5, Try10, Prss3, Prss1 and Prss2 that are missing; (ii) A rodent that is substantially capable of wild-type reproduction, wherein at least a segment from rodent Vβ1 to Dβ1 is missing from the rodent TCRβ locus, and its genome also contains one or more, or all of the rodent serine protease genes Prss58, Prss59, Prss3b, Try4, Try5, Try10, Prss3, Prss1 and Prss2 that are missing; wherein the rodent genome is not a wild-type rodent genome, and / or does not have a wild-type genome at the TCRβ locus, and / or cannot form a rodent TCRβ chain.

[0451] 42. A genetically modified rodent or rodent cell as described in any of the preceding clauses, wherein the rodent retains an endogenous rodent TCRβ variable locus, and the endogenous rodent TCRβ variable locus is a nonfunctional locus because all or part of the locus has been inverted in the genome.

[0452] 43. Genetically modified rodents or rodent cells as described in any of the preceding clauses, wherein the unrearranged TCRα variable locus replaces all or part of the endogenous rodent TCRα locus; and / or the unrearranged TCRβ variable locus replaces all or part of the endogenous rodent TCRβ locus.

[0453] 44. A genetically modified rodent or rodent cell as described in any of the preceding clauses, wherein the rodent further comprises an unrearranged library of human TCRδ variable region fragments at the rodent TCRα locus, optionally comprising one or more human TCRδV gene fragments, one or more human TCRδD gene fragments, and one or more human TCRδJ gene fragments, which are capable of rearranging to form the human TCRD variable region.

[0454] 45. A rodent or cell as described in Clause 44, wherein the rodent contains a complete human Vδ fragment library, a complete human Dδ fragment library, and a complete human Jδ fragment library at the rodent TCRα locus.

[0455] 46. ​​The rodent or cell as described in Clause 44 or 45, wherein the rodent or cell contains a complete human Vδ constant region at the rodent TCRα locus, the constant region being operatively linked to a human TCR variable region fragment, and does not contain a rodent Vδ constant region at the rodent TCRα variable locus, thereby expressing complete human TCRδ.

[0456] 47. The rodent or cell as described in any of the preceding clauses, further comprising an unrearranged library of human TCRγ variable region fragments, such as one or more or complete human Vγ fragment libraries and one or more or complete human Jγ fragment libraries, which are operatively linked to the human TCRγ constant gene sequence.

[0457] 48. A genetically modified rodent or rodent cell as described in any of the preceding clauses, wherein the unrearranged human T cell variable region TCRαV and J gene fragments are operatively linked to an endogenous rodent TCRα enhancer downstream of the TCRα constant region.

[0458] 49. Genetically modified rodents or rodent cells as described in any of the preceding clauses, wherein the unrearranged human T cell variable region V, (D) and / or J gene fragments of the TCRα and / or TCRβ, and / or the TCRα and / or TCRβ constant region, are operatively linked to one or more endogenous rodent regulatory sequences, such as rodent promoters or enhancers.

[0459] 50. A genetically modified rodent or rodent cell as described in any of the preceding clauses, wherein the genome comprises: (i) a nucleotide sequence encoding a human CD8α polypeptide, operatively linked to one or more rodent CD8α promoters and / or another rodent regulatory sequence; and a nucleic acid sequence encoding a human CD8β polypeptide, operatively linked to one or more rodent CD8β promoters and / or other rodent regulatory sequences; optionally, the rodent regulatory sequence is an endogenous rodent promoter or regulatory sequence; or (ii) a nucleotide sequence encoding a human-rodent chimeric CD8α polypeptide, the sequence being operatively linked to one or more rodent CD8α promoters and / or another rodent regulatory sequence; and a nucleic acid sequence encoding a human-rodent chimeric CD8β polypeptide, the sequence being operatively linked to one or more rodent CD8β promoters and / or other rodent regulatory sequences; optionally, the rodent regulatory sequence is an endogenous rodent promoter or regulatory sequence.

[0460] 51. Genetically modified rodents or rodent cells as described in any of the preceding clauses, wherein: (i) The genome contains nucleic acid encoding a human MHC class I polypeptide, which is operatively linked to a rodent MHC class I promoter, enhancer, and / or other rodent regulatory sequence; optionally, the rodent promoter or regulatory element is an endogenous rodent promoter or regulatory sequence; or (ii) The genome contains a nucleic acid sequence encoding a chimeric MHC class I polypeptide, which is operatively linked to a rodent MHC class I promoter, enhancer and / or other rodent regulatory sequence; optionally, the rodent promoter or regulatory element is an endogenous rodent promoter or regulatory sequence.

[0461] 52. A genetically modified rodent or rodent cell as described in any of Clauses 49 to 51, wherein the endogenous rodent regulatory sequence is a naturally occurring rodent genome regulatory sequence located at its native genomic location.

[0462] 53. A genetically modified rodent or rodent cell as described in any of the preceding clauses, wherein the human or chimeric MHC class I polypeptide is selected from the group consisting of HLA-A, HLA-B, and HLA-C.

[0463] 54. A genetically modified rodent or rodent cell as described in any of the preceding clauses, wherein the rodent genome contains a rodent HLA locus, the locus contains an insert cassette containing a site-specific recombinase site; these sites allow the insertion of human HLA DNA into the rodent genome via recombinase-mediated cassette exchange, such that different human HLA molecules can be expressed from the same genomic location.

[0464] 55. A genetically modified rodent or rodent cell as described in any of the preceding clauses, comprising DNA encoding human or chimeric HLA-A02, wherein the rodent H2D locus is inactivated; optionally, the rodent H2D locus has been replaced by human or chimeric DNA encoding human HLA-A02; furthermore, optionally, the rodent H2-K locus has been deleted or inactivated.

[0465] 56. A genetically modified rodent or rodent cell as described in any of the preceding clauses, wherein the rodent or rodent cell expresses the human β2 microglobulin polypeptide at an endogenous non-human β2 microglobulin locus.

[0466] 57. Genetically modified rodents as described in any of the preceding clauses, wherein nucleic acids encoding human TCRα and human TCRβ are present in the rodent germline; and if present, human or human-rodent chimeric CD8α polypeptide, human or human-rodent chimeric CD8β polypeptide, human or chimeric MHC class I polypeptide and / or human β2 microglobulin are also present in the rodent germline.

[0467] 58. Genetically modified rodents as described in any of the preceding clauses, wherein: (i) The nucleic acids encoding human-rodent chimeric CD8α peptide, human-rodent chimeric CD8β peptide, human TCRα, human TCRβ, human MHC class I, and human β2 microglobulin are contained in rodent germline cells; or (ii) The nucleic acids encoding human CD8α polypeptide, human CD8β polypeptide, human TCRα, human TCRβ, human MHC class I and human β2 microglobulin are contained in rodent germ cell lines; or (iii) Nucleic acids encoding rodent CD8α polypeptide, rodent CD8β polypeptide, human TCRα, human TCRβ, human-rodent chimeric MHC class I and human β2 microglobulin are contained in rodent germ cell lines.

[0468] 59. Genetically modified rodents or rodent cells as described in any of the preceding clauses, wherein the rodent is a mouse or rat, preferably a mouse.

[0469] 60. A genetically modified rodent or rodent cell as described in any of the preceding clauses, comprising a TCRα variable locus that includes one or more, optionally all, of the following human Vα gene segments: TRAV1-1, TRAV1-2, TRAV2, TRAV3, TRAV4, TRAV5, TRAV6, TRAV8-1, TRAV8-2, TRAV8-3, TRAV8-4, TRAV8- 6. TRAV9-1, TRAV9-2, TRAV10, TRAV12-1, TRAV12-2, TRAV12-3, TRAV13-1, TRAV13-2, TRAV14 / DV4, TRA V16, TRAV17, TRAV19, TRAV20, TRAV21, TRAV22, TRAV23 / DV6, TRAV24, TRAV25, TRAV26-1, TRAV26-2, TR AV27, TRAV29 / DV5, TRAV30, TRAV34, TRAV36 / DV7, TRAV38-1, TRAV38-2 / DV8, TRAV39, TRAV40, TRAV41; and / or contains one or more of the following human Jα gene fragments, optionally all of them: TRAJ3, TRAJ4, TRAJ5, TRAJ6, TRAJ7, TRAJ9, TRAJ10, TRAJ11, TRAJ12, TRAJ13, TRAJ14, TRAJ15, TRAJ16, T RAJ17, TRAJ18, TRAJ20, TRAJ21, TRAJ22, TRAJ23, TRAJ24, TRAJ26, TRAJ27, TRAJ28, TRAJ29, TRAJ30, TRA J31, TRAJ32, TRAJ33, TRAJ34, TRAJ35, TRAJ36, TRAJ37, TRAJ38, TRAJ39, TRAJ40, TRAJ41, TRAJ42, TRAJ4 3. TRAJ44, TRAJ45, TRAJ46, TRAJ47, TRAJ48, TRAJ49, TRAJ50, TRAJ52, TRAJ53, TRAJ54, TRAJ56, TRAJ57.

[0470] 61. A genetically modified rodent or rodent cell as described in any of the preceding clauses, comprising a TCRβ variable locus that includes one or more, optionally all, of the following human Vβ gene segments: TRBV2, TRBV3-1, TRBV4-1, TRBV4-2, TRBV5-1, TRBV5-4, TRBV5-5, TRBV5-6, TRBV6-1, TRBV6-2, TRBV6-4, TRBV6-5, TRBV6-6, TRBV6-8, TRBV7-2, TRBV7-3, TRBV7-4, TRBV7-6, TRBV7-7, TRBV7-9, TRBV9, TRBV10-1 , TRBV10-2, TRBV10-3, TRBV11-1, TRBV11-2, TRBV11-3, TRBV12-3, TRBV12-4, TRBV12-5, TRBV13, TRBV14 , TRBV15, TRBV16, TRBV18, TRBV19, TRBV20-1, TRBV24-1, TRBV25-1, TRBV27, TRBV28, TRBV29-1, TRBV30; and / or contains one or more of the following human Jβ gene fragments, optionally all of them: TRBJ1-1, TRBJ1-2, TRBJ1-3, TRBJ1-4, TRBJ1-5, TRBJ1-6, TRBJ2-1, TRBJ2-2, TRBJ2-3, TRBJ2-4, TRBJ2-5, TRBJ2-6, TRBJ2-7.

[0471] 62. Genetically modified rodents or rodent cells as described in any of the preceding clauses, wherein CD3ε, CD3γ, CD3δ and / or CD3ζ in their genome are endogenous wild-type.

[0472] 63. Genetically modified rodents or rodent cells as described in Clause 62, wherein CD3ε, CD3γ, CD3δ and CD3ζ are all endogenous wild-type.

[0473] 64. A method for preparing a genetically modified rodent expressing a human T-cell receptor, the method comprising: (i) Inserting an unrearranged human TCRα variable locus into an endogenous rodent TCRα variable locus, the unrearranged human TCRα variable locus comprising at least one human Vα fragment and at least one human Jα fragment, and operatively ligated to the human TCRα constant region. (ii) Inserting an unrearranged human TCRβ variable locus into an endogenous rodent TCRβ variable locus, the unrearranged human TCRβ variable locus comprising at least one human Vβ fragment, at least one human Dβ fragment and at least one human Jβ fragment, and operatively ligated to the human TCRβ constant region.

[0474] 65. The method as described in Clause 64, further comprising one or more, or all, of the following steps: (iii) Insert the nucleic acid sequence encoding the human CD8α polypeptide into the endogenous rodent CD8α gene locus; (iv) Insert the nucleic acid sequence encoding the human CD8β polypeptide into the endogenous rodent CD8β gene locus; (v) Insert the nucleic acid sequence encoding a human MHC class I polypeptide into an endogenous MHC class I locus; (vi) Optionally, a nucleic acid sequence encoding human β2 microglobulin is inserted into an endogenous rodent β2 microglobulin locus.

[0475] 66. The method as described in Clause 64, further comprising one or more, or all, of the following steps: (iii) Insert the nucleic acid sequence encoding the human-rodent chimeric CD8α polypeptide into the endogenous rodent CD8α gene locus, or modify the host rodent CD8α gene locus by replacing rodent DNA with human DNA, to produce DNA encoding the human-rodent chimeric CD8α polypeptide. (iv) Insert the nucleic acid sequence encoding the human-rodent chimeric CD8β polypeptide into the endogenous rodent CD8β locus, or modify the host rodent CD8β locus by replacing rodent DNA with human DNA, to produce DNA encoding the human-rodent chimeric CD8β polypeptide. (v) Insert the nucleic acid sequence encoding a human MHC class I polypeptide into an endogenous MHC class I locus; (vi) Optionally, a nucleic acid sequence encoding human β2 microglobulin is inserted into an endogenous rodent β2 microglobulin locus.

[0476] 67. The method as described in Clause 64, further comprising one or more, or all, of the following steps: (iii) Insert the nucleic acid sequence encoding a human-rodent chimeric MHC class I polypeptide into an endogenous MHC class I locus; or modify the host rodent MHC class I locus by replacing rodent DNA with human DNA to produce DNA encoding a human-rodent chimeric MHC class I polypeptide. (iv) Optionally, a nucleic acid sequence encoding human β2 microglobulin is inserted into an endogenous rodent β2 microglobulin locus.

[0477] 68. The method as described in any of the clauses 64 to 67, wherein the host locus at each insertion site is inactivated or deleted such that the rodent does not express native rodent TCR, MHC class I or CD8, and optionally does not express rodent β2 microglobulin.

[0478] 69. A method for preparing a proliferable rodent or rodent cell, the method comprising the step of inverting a segment of rodent TCRβ from rodent Vβ1 to Dβ1 in the rodent genome; optionally, the method further comprising inserting all or part of a nucleic acid (e.g., DNA) encoding TCRβ from another species different from the rodent into the rodent genome, such as inserting all or part of human DNA encoding TCRβ.

[0479] 70. A method for preparing a genetically modified reproductive rodent expressing a human T-cell receptor, the method comprising deleting at least a segment of rodent Vβ1 to Dβ1 from the rodent TCRβ, including the deletion of serine protease genes Prss58, Prss59, Prss3b, Try4, Try5, Try10, Prss3, Prss1, and Prss2; wherein one or more, or all of the deleted serine protease genes Prss58, Prss59, Prss3b, Try4, Try5, Try10, Prss3, Prss1, and Prss2 are reinserted into the rodent genome.

[0480] 71. A method for preparing a reproductive rodent containing a knockout gene of an endogenous T cell receptor β chain polypeptide, the method comprising deleting at least a segment of rodent Vβ1 to Dβ1 from the rodent TCRβ gene, including deleting serine protease genes Prss58, Prss59, Prss3b, Try4, Try5, Try10, Prss3, Prss1, and Prss2; wherein one or more, or all of the deleted serine protease genes Prss58, Prss59, Prss3b, Try4, Try5, Try10, Prss3, Prss1, and Prss2 are reinserted into the rodent genome.

[0481] 72. A method for preparing a breedable rodent, the method comprising the following steps in any order: (i) Deleting at least the rodent Vβ1 to Dβ1 segments from the rodent TCRβ genomic DNA, including deletion of one or more, or all of the serine protease genes Prss58, Prss59, Prss3b, Try4, Try5, Try10, Prss3, Prss1 and Prss2. (ii) Insert one or more, or all of, the serine protease genes Prss58, Prss59, Prss3b, Try4, Try5, Try10, Prss3, Prss1 and Prss2 into the rodent genome.

[0482] 73. The method as described in any of the provisions of clauses 69 to 72, wherein the method further comprises any of the steps (i) to (vi) of clauses 59 and 60.

[0483] 74. The method described in any of the provisions 69 to 73, wherein the deletion is a homozygous deletion.

[0484] 75. A rodent or rodent cell as described in any one of Clauses 1 to 74, or a method for preparing a rodent or rodent cell, wherein the rodent genome contains DNA encoding a complete human TCRβ chain, and the DNA includes human V, D, J, and C gene segments; wherein the rodent or rodent cell is capable of expressing the complete human TCRβ chain; and the rodent genome contains human intergenic and / or human intron DNA sequences located between human D1 and human C2 gene segments, preferably human intergenic and / or intron sequences naturally coexisting with human D and J gene segments; optionally, wherein the human DNA sequences located between human D1 and human C2 gene segments are all genomic human DNA.

[0485] 76. A rodent or rodent cell whose genome contains DNA encoding a complete human TCRβ chain, and the DNA includes human V, D, J, and C gene segments; wherein the rodent or rodent cell is capable of expressing the complete human TCRβ chain; and the rodent genome contains human intergenic and / or human intron DNA sequences located between human D1 and human C2 gene segments, preferably human intergenic and / or intron sequences naturally coexisting with human D and J gene segments; optionally, wherein the human DNA sequences located between human D1 and human C2 gene segments are all genomic human DNA.

[0486] 77. The rodent, rodent cell, or method as described in Clause 75 or Clause 76, wherein the sequence contained in the TCRβ chain is encoded by rearrangement of a V gene segment at a locus with a segment from the DJC2 cluster.

[0487] 78. A method for preparing a human T-cell receptor in a rodent, the method comprising inserting DNA encoding a complete human TCRβ chain into the genome of a rodent, the DNA comprising human V, D, J, and C gene fragments; wherein the rodent is capable of expressing the complete human TCRβ chain; wherein the inserted DNA comprises a human intergenic and / or human intron DNA sequence located between human D1 and human C2 gene fragments, preferably a human intergenic and / or intron sequence naturally coexisting with human D and J gene fragments; optionally, the human DNA sequence located between human D1 and human C2 gene fragments is genomic human DNA.

[0488] 79. The method as described in Clause 78, wherein the TCR contains a TCRβ variable region whose TCRβ chain sequence is encoded by rearranging a V gene fragment from the T cell's TCRβ locus with a fragment from the DJC2 cluster.

[0489] 80. A method for generating a human T-cell receptor against a target antigen, the method comprising: (i) Optionally, a rodent as described in any of the preceding clauses is prepared; (ii) Immunize the rodents described in any of the preceding clauses with the target antigen; (iii) Induce an immune response in the rodent; (iv) Determining the nucleic acid sequence of the human TCR variable region expressed by T cells from rodents responsive to the target antigen, optionally including the step of isolating the T cells; and (v) Expressing the human T-cell receptor or the variable region of the human T-cell receptor in cells; optionally, further formulating the expressed human T-cell receptor or the variable region of the human T-cell receptor with a pharmaceutically acceptable excipient; or (vi) Inserting a nucleic acid encoding the human T-cell receptor or the variable region of the human T-cell receptor into cells (e.g., into human or animal cells) in vitro or in vitro, optionally preparing a cell line containing the inserted nucleic acid and delivering it into humans or animals; or (vii) Formulate nucleic acids (e.g., RNA or DNA) encoding the human T-cell receptor or the variable region of the human T-cell receptor using a suitable delivery carrier (e.g., lipids or liposomes) for in vivo delivery to patients who require it.

[0490] 81. The method of claim 80, wherein the TCR comprises a TCRβ variable region of a TCRβ chain, the sequence of which is encoded by rearranging a V gene fragment from the T cell's TCRβ locus with a fragment from the DJC2 cluster.

[0491] 82. A method for treating an individual in need, the method comprising: (i) Delivering a soluble TCR molecule containing the variable region of the human T cell receptor as described in step (v) of clause 80 to the individual; (ii) Deliver the cells from step (vi) of clause 80 to patients who require them; (iii) Deliver the nucleic acid (e.g., RNA, such as mRNA or DNA) formulated in step (vii) of clause 80, which encodes the human T-cell receptor or the variable region of the human T-cell receptor, to the patient in need of it.

[0492] Example Example 1 Transgenic mice have been prepared in which the TRBVDJ locus is deleted; at the same time, transgenic mice in which the TRBVDJ locus is inverted but still retained in the genome have also been prepared.

[0493] The inversion of the mouse variable region is shown in Figure 2, ranging from coordinates 6:40868163 (GRCm39) to 6:41515110 (GRCm39).

[0494] The mouse homeostatic region was missing.

[0495] When the TRBVDJ locus is present in its native or inverted position in mice, homozygous male mice produced offspring in 92% of cases (n=12 male mice). However, in male mice with a missing TRBVDJ locus, only 35% of cases resulted in offspring (n=34 male mice). Therefore, variable region inversions can preserve genomic components, and the loss of these components significantly affects reproductive capacity.

[0496] Example 2 As described in this paper, previously prepared mice expressing human TRB DNA (see Moore et al., Science Immunology, T-cell-mediated humanization of the immune system in mice, 6(66), 2021) required the use of mouse TRB D and J introns, as well as intergenic sequences in the human D1 to J2 regions containing the DJC2 gene cluster. Without re-micetization, T-cell receptors from the DJC2 gene cluster could not be detected. However, T-cell receptors have been successfully generated in transgenic mice containing human D and J introns at the mouse TRBDJC2 cluster (without requiring mouse TRBD and J introns or intergenic sequences).

[0497] This mouse contains an inversion of the TCRβ variable locus, which significantly reduces or even prevents the production of the TCRβ chain from the host mouse's TCRβ chain variable gene segment, because the variable region is located at an inversion position far from the wild-type locus.

[0498] In the genomes of these mice, approximately 520 kb of human genomic DNA from coordinates 7:142,237,898 [GRCh38] to 7:142,813,740 [GRCh38] was inserted at position 6:36,074,375, representing a complete human insert. Measured in human genome coordinates, this insert is 576 kb of human DNA. As described in Example 1, these mice retained the murine TRB variable gene region in an inverted manner.

[0499] The mouse 3' enhancer is retained downstream of TrbC2.

[0500] Example 3 The accompanying figures illustrate preferred embodiments of the genome design features. Each allele and genomic intermediate described below is an independent inventive element and can be used in combination with other alleles and intermediates described herein.

[0501] Figure 1 illustrates the mouse TRA locus, the human TRA locus, and the engineered human-mouse TRA locus. The human DNA in the third figure is derived from chromosomes 14:21,570,693 to 14:22,554,820 (GRCh38). The mouse DNA is numbered according to GRCm39. A preferred mouse locus contains human TRA (45 V and 51 J) and Trd (6 V and 2 J) DNA, replacing the mouse Tra and Trd gene segments. Another preferred mouse locus contains human TRA (44 V and 50 J) and Trd (8 V and 4 J) DNA, replacing the mouse Tra and Trd gene segments. The enhancer downstream of TraC is preferably retained.

[0502] The preparation method of the TRA locus of the present invention is as follows: Approximately 1.1 Mb (specifically 984,127 bp) of human genomic DNA from coordinates 14:21,570,693 [GRCh38] to 14:22,554,820 [GRCh38] is inserted into the mouse genome after the following coordinate positions: 14:52,664,870 or 14:52,664,818 (depending on the mouse strain). The following regions of the mouse TRA and Trd loci are deleted from the mouse genome: 14:52,664,870 to 14:54,463,673, or 14:52,664,818 to 14:54,463,673. The engineering of this locus involved: (i) a large deletion of the landing site into the mouse locus; (ii) insertion into the human BAC (bacterial artificial chromosome) using RMCE (recombinase-mediated cassette exchange); and (iii) excision using piggyBac (transposon)-mediated deletion of the selective marker in the locus. For the techniques used, see Lee et al., Nature Biotechnology, Vol. 32, No. 4, April 2014, and Boroviak et al., Genesis 54:78-85 (2016).

[0503] Figure 2 illustrates the mouse TRB locus, the human TRB locus, and an engineered human-mouse TRB locus with inserted human DNA and mouse DNA inverted to maintain male reproductive capacity. The human DNA in the third figure originates from chromosomes 7:142,237,898 to 7:142,813,740 (GRCh38). Mouse DNA is numbered according to GRCm39. Preferred mouse loci contain human TRb (47 V and 13 J). Preferred mice retain the mouse TRb locus in an inverted orientation within their genome.

[0504] The method for preparing the TRB locus of this invention is as follows: Approximately 520 kb of human genomic DNA from positions 7:142,237,898 [GRCh38] to 7:142,813,740 [GRCh38] is inserted into the mouse genome at position 6:36,074,375. Measured in human genome coordinates, this inserted fragment is 576 kb of human DNA. The inserted human DNA is ligated to the mouse genomic DNA at position 6:41,535,764 (GRCm39). Using a lox-mediated inversion method, following the principles described in Figure 1b of Lee et al., Nature Biotechnology, April 2014; 32(4):356-63, doi: 10.1038 / nbt.2825. Epub, March 16, 2014, the naturally occurring mouse TCRβ region (from 6:40,868,163 (GRCm39) to 6:41,515,110 (GRCm39)) was inverted in the genome. The mouse enhancer, naturally located downstream of mouse trbc2, is present in the mouse genome downstream of human trbc2.

[0505] Figures 3a / b illustrate the generation of chimeric CD8α and chimeric CD8β polypeptides by replacing the mouse genome DNA encoding mouse CD8α with human CD8α DNA and by replacing the mouse genome DNA encoding mouse CD8β with human CD8β DNA. For the locations of enhancers and deoxyribonuclease sites shown in Figure 3a, see “Chromatin and CD4, CD8A, and CD8B Gene Expression During Thymic Differentiation,” Kioussis D and Ellmeier W, *Nature Reviews Immunology*, December 2002; 2(12):909-19, doi: 10.1038 / nri952.

[0506] Figure 3b illustrates in more detail a method for preparing the genome shown in Figure 3a: using a dual-targeting vector approach known in the art, chimeric CD8α and CD8β loci with 5' human DNA and 3' mouse DNA are generated.

[0507] These two loci are tightly linked on mouse chromosome 6, so the best approach is to humanize them sequentially. Alternatively, they could be modified simultaneously, but targeting both at the same time is technically more challenging due to the distance between them.

[0508] To modify CD8b and CD8a sequentially, either gene can be chosen as the starting point. Example Figure 3b shows modification of CD8b first, followed by CD8a; however, it should be understood that CD8a can also be humanized first, followed by CD8b modification. Short fragments of exons 1 to 3 of mouse CD8b, along with the intron sequences they encode, are replaced by the corresponding regions of human CD8B. The first targeting vector contains homologous arms flanking the regions to be replaced, each approximately 3 to 4 kb in length. To achieve positive selection, the vector contains a puro-δ-tk (puromycin-thymidine kinase deletion) selector flanked by PiggyBac inverted repeat sequences. This selector is inserted into intron 2 of the human DNA insert. Its precise location is not critical, as it is seamlessly removed via PBase (PiggyBac transposase) in the second step; therefore, the selector could also be inserted into intron 1 or 3, for example. After transfection and selection with puroycin, the targeted clone is identified by ligation PCR on the 5' and 3' sides. After targeting is completed, the vector is removed without leaving a trace by expressing PBase, and cells that have completed this step are selected in FIAU (fluoroiododiaduridine).

[0509] Cells with successfully fully humanized CD8b can be used to humanize CD8a using a similar vector design, as shown in Figure 3b. Targeting clones are selected using puromycin, and after expression in PBase, cells with the selection marker removed are selected using FIAU. Exons 1 to 3 of mouse CD8a are replaced with exons 1 to 4 of human CD8a. Because human CD8a exon 4 is fused to mouse CD8a exon 3, the resulting chimeric locus has six exons. Second targeting can be performed in cis (same chromosome) or trans (different chromosome) as with the first targeting. Due to tight gene linkage, cloning requires cis targeting. Cis-targeted clones are identified using methods known in the art, such as pulsed-field gel electrophoresis and Southern blotting.

[0510] In an alternative approach, both CD8a and CD8b can be targeted simultaneously to generate a chimeric locus. A first targeting vector is prepared containing human CD8b exons 1 through 3 and CD8a exons 1 through 4, separated by a selection marker (e.g., PGKneo, phosphoglycerate kinase promoter-neomycin resistance gene). This is inserted into the mouse genome via homologous recombination and selection. Mouse CD8b exons 1 through 6 and CD8a exons 1 through 3 are deleted. A second targeting vector containing a second selection marker (e.g., PGKpuroδTK) inserts the desired mouse genome fragments downstream of the human CD8b at the CD8b chimeric locus. These fragments include a portion of mouse exon 3 and all of mouse exons 4, 5, and 6. This second targeting vector also replaces the human 5'UTR of human CD8a exon 1 with the mouse 5'UTR (untranslated region).

[0511] Regardless of the construction method used, the final result is the same (Figure 3a). In addition to the human coding sequences for CD8α and CD8β portions, human intercalation sequences between exons 1 and 2 and between exons 2 and 3 are inserted into the rodent genome as part of the human genome insert; and (for CD8A) human intercalation sequences between exons 3 and 4 are also inserted into the rodent genome, preferably at the coordinates shown in this paper. The rodent CD8A locus contains human exons 1 (without UTR), 2, and 3, as well as at least a portion of human exon 4 fused with a portion of mouse exon 3, complete mouse exon 4, and complete mouse exon 5. The rodent CD8B locus contains human exons 1 (without UTR), 2, and at least a portion of human exon 3.

[0512] Figure 4 A hypothetical scheme is presented: replacing the mouse genome DNA encoding mouse CD8α with human CD8α DNA, and replacing the mouse genome DNA encoding mouse CD8β with human CD8β DNA, thereby generating the complete human CD8 polypeptide. For the locations of enhancers and deoxyribonuclease sites shown in the figure, see “Chromatin and CD4, CD8A, and CD8B Gene Expression During Thymic Differentiation,” Kioussis D and Ellmeier W, *Nature Reviews Immunology*, December 2002; 2(12):909-19, doi: 10.1038 / nri952.

[0513] Figures 5a / b illustrate the replacement of the mouse β2-microglobulin-encoding DNA in the mouse genome with DNA encoding human β2-microglobulin. Figure 5b shows in more detail the method for preparing the genome shown in Figure 5a. The preferred rodent genome contains human exons 1, 2, and 3, along with intercalated human genome sequences, preferably inserted according to the coordinates shown herein.

[0514] The preparation method of the β2 microglobulin gene locus of the present invention is as follows: A targeting DNA vector is constructed, which contains sequences encoding exons 1, 2, and 3 of human β2 microglobulin and genomic interpolation sequences. This vector is then inserted into the mouse genome to target the locus by replacing the equivalent sequences of mouse exons 1 to 3 with sequences controlled by the 5' enhancer of mouse exon 1. This targeting will result in the deletion of mouse exons 1 to 3, thereby preventing the expression of mouse β2 microglobulin.

[0515] Figure 6a / b demonstrates the replacement of the mouse H2-D1 gene in the mouse genome with the human HLA_A*02MHC class I gene. Figure 6b The preparation process was shown in more detail. Figure 6a The method for the genome shown. The preferred rodent genome contains the coding regions of human exons 1 to 8, and intercalated human genome sequences between exons 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7 and 7 to 8, preferably intercalated according to the coordinates shown herein.

[0516] The method for preparing the modified MHC class I locus of this invention is as follows: A targeting DNA vector is constructed, containing sequences encoding human exons 1 to 7 and part of exon 8, as well as genomic intercalation sequences. After replacing mouse H2-D1 DNA with homologous recombination (preventing the expression of mouse MHCH2-D1 DNA), a second targeting vector is used to replace human exon 4 with mouse exon 4. All other intercalated DNA sequences are of human origin. This method generates human MHC class I, whose mouse exon 4 is regulated by a mouse enhancer upstream of mouse exon 1. If necessary, the H2K gene can be further knocked out or mutated in the mouse genome to prevent the expression of host MHC class I molecules.

[0517] Although the accompanying drawings indicate some preferred coordinates for mouse genome modification, these coordinates do not constitute a limitation on the present invention.

[0518] Example 4 Transgenic mice were prepared using the human TRA locus shown in Figure 1 and / or the human TRB locus shown in Figure 2. The hTCR (human T-cell receptor) transgenic mice prepared by the inventors may be referred to herein as OpTiMus® mice.

[0519] Table 1 shows gene fragments of the human TCR variable region that are theoretically located at the TRAV and TRBV loci of the mouse, based on genes believed to exist in the BAC (bacterial artificial chromosome) used to produce the mouse.

[0520] Table 1: Variable and linker complements of OpTiMus® hTCR transgenic mice. TRAV genes: 44 functional genes and 10 non-functional genes. TRAJ genes: 50 functional genes and 12 non-functional genes. TRBV genes: 43 functional genes and 17 non-functional genes. TRBJ genes: 13 functional genes and 1 non-functional gene. Non-functional genes are labeled as (P) pseudogenes and (ORF) open reading frames. The following genes are absent from the human BAC sequence used to prepare hTCR transgenic mice, therefore these genes cannot be expressed in mice: TRBV4-3 (related to TRBV4-2, 4-1 and TRBV1) TRBV5-8 (related to TRBV5-5, 5-3, 5-7, 5-4, etc.) TRBV6-3 (same as TRBV6-2) TRBV6-9 (related to TRBV6-5, 6-6, 6-8, 6-7, etc.) TRBV7-8 (related to TRBV7-4, 7-6, 7-7, etc.).

[0521] Gene expression was assessed by sequencing a large amount of RNA extracted from the spleen of OpTiMus® mice. All transcripts that could be assigned to any gene segment were counted, regardless of whether they were in-frame or out-of-frame expressed. To reduce PCR (polymerase chain reaction) bias, the number of UMIs (unique molecular identifiers) rather than sequence reads was counted. Two transcripts (with different UMIs) may have the same nucleotide sequence. The presence and expression of a gene segment were confirmed by detecting at least one transcript from that segment.

[0522] A) Use of VJ clips in TRBV The transgenic human BAC sequence contains 43 functional TRBV genes and 13 functional TRBJ genes (Table 1). Extensive splenic RNA sequencing of TRB genes expressed in homozygous TRB transgenic mice confirmed extensive recombination and expression of TRBV and TRBJ genes. Figure 10 , Figure 11 , Figure 12Since each TRBD is associated with both the TRBJ and the constant region, it is expected that the complete complement of the TRBD gene fragment will also be expressed.

[0523] The batch sequencing results represent all functional TRBJ and TRBV genes. This confirms that all functional human gene fragments of the TCRβ variable region can be expressed from transgenic mouse loci.

[0524] B) Use of VJ clips in TRAV The transgenic human BAC sequence contains 44 functional TRAV genes and 50 functional TRAJ genes (Table 1). Extensive splenic RNA sequencing of the TRB gene expressed in homozygous TRA transgenic mice confirmed widespread recombination and expression of the TRAV and TRAJ genes.

[0525] The batch sequencing results represent all functional TRAJ genes and all functional TRAV genes except for two genes (TRAV7 and TRAV18). Figure 13 , Figure 14 , Figure 15 This confirms that the TCRα human gene fragment can be expressed from the transgenic mouse locus.

[0526] C) Relative usage of TRBJ1-C1 and TRBJ2-C2 Batch sequencing of TRB transgenic spleen RNA showed that the ratio of TRBJ1-C1:TRBJ2-C2 used was approximately 40:60. Figure 16 ).

[0527] D) Comparison with TCR gene fragment expression in humans The formation of the peripheral TCR expression library can be attributed to two processes: (i) certain VDJ (variable region-diversity region-connecting region) fragments are readily recombined by somatic cells; and (ii) thymic positive and negative selection, which depends on the pMHC (peptide-MHC complex) encountered by the TCR during thymic development.

[0528] Results from OpTiMus® mice were compared with two publicly available TCR gene expression databases from healthy humans. Freeman et al. (Genomics Research 19(10):1817-1824, 2009) reported the expression of TRBV / TRBJ in mixed PBMC (peripheral blood mononuclear cell) RNA from 550 healthy individuals. Kitaura et al. (BMC Immunology 17:38, 2016) reported the expression of TRAV / TRAJ and TRBV / TRBJ from 20 healthy Japanese individuals.

[0529] The use of TRBV and TRBJ genes in Optimus® mice was compared with publicly available data from humans. The TRBV and TRBJ genes expressed in TRB transgenic mice and humans are broadly similar. In particular, TRBV20.1 and TRBV5.1 are the two genes with the highest expression levels in both humans and transgenic mice. Figure 11 , Figure 17 The use of TRBJ in humans and transgenic mice is also highly consistent. Figure 18 ).

[0530] The length of the CDR3 region (complementarity-determining region 3) spanning the highly variable VDJ connective region ranges from approximately 20 bp to 60 bp, peaking at 42 bp. This is consistent with the CDR3 region length found in humans (Kitaura et al., 2016). Figure 19 .

[0531] The TRBC usage ratio observed in humans is approximately 40:60 (biased towards TRBJ2-C2), which has also been replicated in transgenic mice. Figure 16 ).

[0532] Comparing the use of TRAV and TRAJ genes in OpTiMus® mice with publicly available data from humans revealed that most TRAV genes, which are well expressed in human datasets, are also expressed in mice. However, expression at distal TRAV loci was relatively lower compared to the human dataset. Figure 14 , Figure 20 In both humans and TRA transgenic mice, all functional TRAJ genes showed good expression levels. Figure 15 , Figure 21 ).

[0533] The length of the CDR3 region spanning the highly variable VJ connection ranges from approximately 24 bp to 57 bp, peaking at 42 bp, which is consistent with the CDR3 region length found in humans (Kitaura et al., 2016). Figure 19 .

[0534] Furthermore, the connectivity diversity resulting from recombination of V and J gene fragments ( Figure 10 , Figure 13 This also reflects the diversity of connections observed in humans. This is important because the connections between these gene segments form the CDR3 region of the TCR, which is the most variable part of the TCR and interacts directly with peptides.

[0535] Comparison of E)OpTiMus® mice with other humanized TCR mice Humanized TCRab mice have been reported by two companies: Regeneron and its VelociT mice, and T-knife and its MyT mice.

[0536] The Regeneron humanized mouse (“VelociT”) contains humanized gene targets TRA / TRB / CD8ab / CD4 / b2m / MHC I and MHC II (Moore et al., Sci. Immunol. 6, eabj4026 2021). TRA and TRB contain murine constant region genes. The initial version of the Regeneron mouse with complete human TRBJ1 and TRBJ2 sequences expressed only the J1-C1 cluster; converting the intergenic JC region to mice corrected this deficiency. OpTiMus® mice did not exhibit this problem and expressed both TRBJ clusters in proportions similar to those found in humans.

[0537] T-Knife mice (Li et al., Nature Medicine 16:9. 1029-1034 2010) were generated by randomly integrating YAC clones containing TRA and TRB into a Tra / Trb knockout background, and also included humanized MHC class I molecules fused to b2-microglobulin. TCR usage and expression were assessed by the simple presence or absence of amplified PCR products derived from the recombinant VJ gene or obtained via RNA RT-PCR, without involving quantitative information on the relative expression of different genes.

[0538] In the genomic regions inserted in previous transgenic mice, the expression status of each V and J gene does not appear to have been reported. Figure 22 summarizes the gene expression status in the TCR loci of previous transgenic mice based on publicly available information (Moore et al., 2021 and Li et al., 2010, cited above) and compares it with the gene expression status in the TCR loci of OpTiMus mice characterized in this paper.

[0539] Batch sequencing analysis of OpTiMus® mice showed that the expression of the inserted TCR gene fragment was complete or nearly complete. The only transcripts not detected in this analysis were TRAV7 and TRAV18. The TRBJ2-6 gene was particularly present. Figure 10 , Figure 12 , Figure 22d The deletion of this gene in Regeneron mice is due to the fact that Trbj2-6 is a pseudogene in mice. In OpTiMus® mice, TRBJ2-6 is clearly expressed, and the expression level is similar to that observed in humans.

[0540] The data used to confirm gene fragment expression in transgenic mice are shown in Figure 22 and summarized in Table 2 below.

[0541] Table 2. Confirmed gene expression in hTCR mice Based on current analysis of gene expression in OpTiMus® TRA and TRB mouse strains, OpTiMus® mice with the transgenic TCRαβ locus appear to possess the most diverse human pool to date among any humanized TCR mouse.

[0542] Example 5 OpTiMus® mice are produced according to the following genotypes, indicating whether they are homozygous (“hom”), heterozygous (“het”), or wild-type (“WT”) at a locus.

[0543] Table 3. Genotypes of OpTiMus mice used to assess naïve immune cell populations. "Conjugativity code" is an abbreviation for mouse genotype. Refer to the table for the transgene status and conjugativity of loci containing human transgene insertions. For example, mice homozygous for transgene HLA-A*02, homozygous for human B2m, heterozygous for human CD8a and CD8b, homozygous for human TRA, and heterozygous for human TRB are called conjugativity code E. Heterozygous loci have one human transgene allele and one wild-type (endogenous mouse) allele; homozygous loci have two human transgene alleles; wild-type (WT) loci have two endogenous alleles and no human transgene insertion at that locus.

[0544] The loci are explained below: H2K: Inactivated mouse MHC class I locus H2K HLA-A02: The mouse HLC1 locus H2D contains an insertion of nucleic acid encoding the fully human HLA-A02, replacing the mouse gene, such as... Figure 6a As shown hB2M: The mouse β2-microglobulin gene locus has an inserted nucleic acid encoding the complete human β2m, replacing the mouse gene, as shown in Figure 5a. hCD8a / b: Nucleic acids encoding complete human CD8a and complete human CD8b are inserted into the mouse CD8 locus, replacing the corresponding mouse genes, as shown in Figure 3a. hTRA: Nucleic acid encoding the fully human TRAV and TRAC genes was inserted into the mouse TCRα locus, replacing the corresponding mouse genes, such as... Figure 1b As shown hTRB: The mouse TCRβ locus contains an insertion of nucleic acid encoding the complete human TRBV and TRBC, with an inactivation inversion in the endogenous mouse TRBV region and a deletion of mouse TRBC, such as... Figure 2b As shown No other transgenes were introduced, therefore all other genes are considered to be WT.

[0545] The T lymphocyte population and CD8 cell surface expression in blood samples from antigen-naïve OpTiMus® mice (samples 1–7) were evaluated. Fresh blood was assessed using FACS to detect mouse CD3ε and human CD8b. Results are shown in [data missing]. Figure 23 CD3+ cells were detected in all mice. Except for mouse 2, hCD8+ cells were detected at normal levels in all mice, ranging from 1% to 5% of whole blood peripheral blood mononuclear cells (PBMCs), which is expected since mouse 2 only expresses mouse CD8 and cannot be detected by human CD8b probes. The levels of CD3+ and CD8+ cells were within the normal range, comparable to those in wild-type mice, indicating normal T lymphocyte development in the transgenic mice.

[0546] In addition, immune cells were collected from the spleens of three other homozygous mice that had not been exposed to the antigen (mice 8, 9, and 10, with the same homozygous genotype as mouse 7 in Table 3 above), and the immune cells were stained with cell surface markers mCD3, mCD4, hCD8b, hTRBC-1, and mCD19. Figure 24 Naïve mice with fully humanized, homozygous transgenes (HLA, B2M, CD8a, CD8b, TRA, and TRB) are indicated in Table 3 as conjugative K-coding mice. Their lymphocyte populations were within the normal range, with B cells comprising 57% to 65%, CD4+ T cells 10% to 15%, and CD8+ T cells 2.6% to 4.5%. Furthermore, the CD3+ T cell population contained a TRBC-1 subset of 5% to 7.5% and a TRBC-1-negative subset of 4.1% to 5.8%. These data confirm that these mice can normally generate key lymphocyte populations (including CD4 and CD8 T cells, as well as B cells).

[0547] In addition to staining for cell surface markers, a gene expression library was constructed from spleen cells of mice (numbers 8, 9, and 10) using single-cell mRNA sequencing technology. Immune cell types and T cell subset phenotypes were classified according to gene expression profiles, including B cells, T cells, natural killer (NK) cells, monocytes, and dendritic cells (cDCs). The proportions of CD4 T cells, CD8 T cells, and γ / δ T cells in the total spleen cells were within the known range for mouse spleen cell proportions. CD4 T cells comprised 11% to 14% of the entire spleen cell population, CD8 T cells comprised 5% to 9.5%, and γ / δ T cells comprised approximately 1% of the entire population. Figure 25 ).

[0548] Example 6 In OpTiMus® mice with multiple conjugative genotypes, the T-cell immune response following peptide immunization was characterized.

[0549] Mice were immunized with a combination of purified target peptide and adjuvants (incomplete Freund's adjuvant and CpGODN1826). The target peptide in this study is a cancer-associated T-cell antigen epitope known to be presented on HLA-A*02.

[0550] Materials for tissue preparation / cell staining and sorting Culture media: Tissue preparation / medium consisted of RPMI-1640 + 10% fetal bovine serum (FBS) + 2 mM glutamine + 20 mM HEPES; Cell enrichment / staining buffer consisted of phosphate-buffered saline (PBS) + 2% FBS + 20 mM HEPES + 1 mM EDTA; Cell sorting and collection buffer consisted of PBS + 20% FBS + 20 mM HEPES; Cell freezing medium consisted of FBS + 10% dimethyl sulfoxide (DMSO).

[0551] Reagents: EasySep mouse CD8+ T cell isolation kit (STEMCELL, #19853), anti-human CD8b antibody-FITC (Miltenyi, 130-11-567), anti-human CD8b-APC (Biolegend, 376705), anti-human CD8b-Viablue (Miltenyi, 130-110-515), anti-mouse CD8b-FITC (Biolegend, 126605), anti-mouse CD3ε-FI TC (Biolegend, 100306), anti-mouse CD3-BV421 (Biolegend, 100228), anti-mouse TCRβ-BV421 (Biolegend, 109230), anti-human TRBC-1-FITC (Biolegend, 383510), anti-mouse CD4-PE (Biolegend, 130310), anti-mouse CD19-BV605 (Biolegend, 115539), eFluor780 immobilizable dye (ThermoFisher, 65-0865-18), Lightening Link barcode binding kit (Abcam, ab270703 / 05 / 09), TotalSeq-C series hash-labeled antibodies (Biolegend, 155861, 155863, etc.).

[0552] Tetramers for detecting antigen-specific CD8T cells were purchased from MBL.

[0553] method Immunotherapy: Peptide-based immunization Immunogen: Purified peptide (purity >95%, purchased from Genscript) Adjuvants: Incomplete Freund's adjuvant (IFA, Invivogen), CpGODN1826 (Invivogen) Initial dose (per mouse): 100 μg peptide and 50 μg CpGODN dissolved in 50 μl PBS, emulsified with 50 μl IFA. Enhanced dose (per mouse): 50 μg peptide and 50 μg CpGODN dissolved in 50 μl PBS and emulsified with 50 μl IFA.

[0554] Peptide-based immunogens are administered via subcutaneous (SC) injection.

[0555] Such studies typically employ a primary immunization followed by 2 to 3 booster immunizations, with a 21-day interval between the primary and booster immunizations, and a 14-day interval between each booster immunization. Alternative primary-booster immunization regimens also use weekly booster immunizations. Tissue samples are usually collected on day 7 after each booster immunization.

[0556] Tissue preparation, CD8+ T cell enrichment and sorting staining Mesenteric lymph nodes, inguinal lymph nodes, and the entire spleen were collected from each mouse for sorting. Briefly, the lymph nodes and spleen were cut into small pieces and strained through a 40 μm cell filter to prepare a homogeneous cell suspension. The cells were further enriched into a CD8+ T cell suspension (typically 80% purity) using the Stemcell kit (19853). The enriched cells were centrifuged and stained on ice.

[0557] MBL tetramer was used for each staining of the target tetramer-PE and the negative control tetramer-APC, with a final culture volume of 0.05 to 0.1 μg / 100 μl, for a maximum of 2.5 × 10^6 enriched CD8+ mouse T cells. After adding the tetramer, the cells were placed on ice and incubated for 15 minutes. A staining mixture containing anti-hCD8b-viablue (Miltenyi), anti-hTRBC-1-FITC (Biolegend), and eFluor780 immobilizable dyes was then added. At this point, TotalSeq-C series hash-labeling antibodies were also added to each mouse sample for labeling purposes. The cells were then incubated on ice for another 30 minutes and washed twice with staining buffer.

[0558] Tetramer-specific CD8+ T cell sorting Resuspend the cells in 300 to 400 μl of staining buffer, pass them through a 35 μm cell filter, place them on ice, and then collect them using a cell sorter.

[0559] Cells were gating using the following strategy: lymphocytes > single cells > live cells > hCD8b+ > target tetramer positive and negative control tetramer negative. Cells were sorted into 1.5 ml centrifuge tubes containing approximately 300 μl of cold collection buffer.

[0560] The immune response rate was determined based on the percentage of a population of CD8+ lymphocytes specific to the target antigen (measured by antigen-specific tetramer gating in FACS) and compared to control (unimmunized) mice. Mice exhibiting an immune response had a higher percentage of antigen-specific cell populations compared to control mice. The percentage of responders was typically >0.1% of the population, while the percentage of controls was typically <0.05% of the population.

[0561] Single-cell mRNA sequencing (mRNA-Seq), library preparation, and next-generation sequencing (NGS) Freshly collected target cells were immediately subjected to mRNA extraction for single-cell RNA sequencing, and mouse TCRVDJ libraries, gene expression (GEX) libraries, and cell surface protein libraries were constructed. The final libraries underwent quality control before sequencing.

[0562] Single-cell NGS data were analyzed to determine whether homozygous reactive TCRs possessed a fully human TCR, with a focus on CD8+ cells exhibiting clonal expansion and displaying activation / exhaustion-like / proliferative phenotypes.

[0563] result In the first study, hTCR mice with multiple conjugative alleles received peptide-based immunization, spleen cells were stained to detect antigen-specific tetramer binding, and sorted to collect a target tetramer-specific CD8+ population.

[0564] Table 4. Summary of antigen-specific CD8+ immune responses in OpTiMus mice with multiple conjugations. Locus descriptions are the same as in Example 5.

[0565] Table 5. Conjugation and immune status of OpTiMus mice. Related data are shown in... Figures 23 to 28 middle.

[0566] Figure 26a Representative atlases of six mice with different conjugations (conjugations B, D, E, I, J, and K, see Table 4) are shown, which have responded from cells with fully humanized TCRs. Atlases from mouse 16 show that, for naïve control mice, the target tetramer level accounts for 0.015% of the total CD8+ population. The tetramer-specific population range for immunized mice with the six different conjugations is 0.18% to 1.74%. This suggests that not only fully humanized homozygous mice (conjugation K) but also mice with other conjugations can be used to discover fully human TCRs.

[0567] Two mice (mouse 2 and mouse 28) with murine WTCD8 (conjugative G) did not respond to the same type of immunity as mice with human CD8. Figure 26b This indicates that in this immune environment using homozygous fully human MHC class I, human CD8 can enable human TCRα / βTCR to signal transduction.

[0568] The T-cell responses of mice were specifically examined in mice that were heterozygous for TRB and homozygous for TRA (conjugative I) and mice that were homozygous for both TRB and TRA (conjugative K).

[0569] exist Figure 27 In this study, OptiMus mice with conjugative I immunization exhibited antigen-specific CD8+ T cell responses to weekly booster-based peptide immunizations. Ten OptiMus mice were immunized with the target peptide using a weekly booster regimen. Data were collected from mice of 2, 2, 3, and 3 after 1, 2, 3, and 4 booster immunizations, respectively. Mice numbered 17, 18, 19, and 20 represent data from 1, 2, 3, and 4 booster immunizations, respectively. These data indicate that OptiMus mice with homozygous TRA and heterozygous TRB (conjugative I) can produce antigen-specific T cell responses via a primary-booster immunization regimen (single or multiple booster injections). Furthermore, the range of responses varied among individuals (from 0.8% to 4.5% of the overall CD8 population).

[0570] exist Figure 28 In this study, OptiMus mice with a fully humanized allele (conjugative K) exhibited an antigen-specific CD8+ T cell response to peptide-based immunization. A total of 23 OptiMus mice were immunized with the target peptide using a two-week booster regimen. Data were collected on day 7 after the second and third booster immunizations. Mice numbered 16 and 24 served as initial controls after the second and third booster immunizations, respectively. Mice 21, 22, and 23 represent data after the second booster immunization. Figure 28 (a) Mice numbered 25, 26, and 27 represent data after three booster immunizations. Figure 28 (b) OptiMus mice with conjugative K showed a wide range of responses among individuals, and exhibited significant antigen-specific responses after two booster immunizations (0.2–1%) and three booster immunizations (0.1–4.7%). Furthermore, the overall response rate of these 23 mice was 21 / 23 (>90%), indicating a high response rate.

[0571] Comparing the immune responses of genetically modified homozygous and heterozygous alleles, it should be noted that the strongest hTCR immune response was achieved in animals lacking mouse TCRα. Although heterozygous mice at the TRA locus can still respond to the immune response and produce a complete human TCR (e.g., mice 11), animals with homozygous human TCRα are generally a superior source of human TCRs compared to heterozygous mice at the TRα locus. This may be because in heterozygotes, the endogenous mouse TCRα constant domain competes for binding to CD3, thus overriding the human TCRα constant domain and primarily producing T cells with mouse TCRα; this does not occur in homozygotes because mouse TRAC is not expressed. Mice homozygous at the TCRα locus (TRA), and homozygous or heterozygous at the TCRβ locus (TRB) produce target-specific CD8+ T cell responses after immunization, confirming the utility of these mice in discovering fully human TCR sequences with therapeutic potential.

[0572] Example 7 Seventeen male mice (with homozygous human TRB insertions at the endogenous TRB locus) were mated, and the endogenous TRBV was inverted to an ectopic position via chromosomal region inversion, as shown in Figure 2. Reproductive data are shown in Table 6.

[0573] Table 6. Reproductive capacity of male mice with homozygous hTRB insertion and mTRBV inversion All matings produced offspring, confirming that mice with the endogenous locus translocation and retained TRBV were reproductively capable. This avoided the unexpected loss of reproductive capacity observed in male mice lacking TRBV (Example 1).

[0574] Example 8 124 T-cell receptor sequences (isolated in antigen-binding form from mice immunized with target peptides as described in Example 6) were selected for expression as soluble “T-receptors”. The TCRα and β variable domains from these mice were integrated into a bivalent soluble molecule containing two TCR “Fab”-like regions dimerized by the antibody Fc region. Each TCR antigen-binding “Fab” consists of a fully human α and a fully human β polypeptide chain: the α chain contains the human TCRα variable domain from mice, with a human TCRα constant domain linked at its C-terminus, and pairs with the β chain (containing the human TCRβ variable domain from mice, with a human TCRβ constant domain linked at its C-terminus). These TCRs contain different combinations of vα and vβ gene fragments (Table 7).

[0575] Table 7. TCR germline genes of selected antigen-specific homozygous types Nucleic acid encoding soluble TCRs was transfected into Expi293T cells for small-scale (3 ml) protein expression. Five days after transfection, the protein was purified using protein A affinity purification. The concentration of soluble TCRs was measured using UV / Vis spectrophotometry. All TCR molecules were successfully expressed, and the purified protein concentrations ranged from 0.01 to 1.9 mg / ml. Figure 29 ).

Claims

1. A genetically modified rodent comprising: (i) An unrearranged T-cell receptor (TCR) α variable locus comprising at least one human Vα segment and at least one human Jα segment, wherein the TCRα variable gene is operatively linked to a human TCRα constant gene sequence. (ii) An unrearranged TCRβ variable locus comprising at least one human Vβ segment, at least one human Dβ segment and at least one human Jβ segment, wherein the unrearranged TCRβ variable gene is operatively linked to a human TCRβ constant gene sequence. in, The unrearranged human T cell variable region gene fragments mentioned above can rearrange to form a gene encoding the variable domain of the human T cell receptor; The unrearranged TCRα variable locus is located at the endogenous rodent TCRα locus, and the unrearranged TCRβ variable locus is located at the endogenous rodent TCRβ locus.

2. The genetically modified rodent of claim 1, comprising: (i) An unrearranged T-cell receptor (TCR) α variable locus comprising at least one human Vα segment and at least one human Jα segment, wherein the TCRα variable gene is operatively linked to a human TCRα constant gene sequence. (ii) An unrearranged TCRβ variable locus comprising at least one human Vβ segment, at least one human Dβ segment and at least one human Jβ segment, wherein the unrearranged TCRβ variable gene is operatively linked to a human TCRβ constant gene sequence. in, The aforementioned unrearranged human T cell variable region gene fragments can rearrange to form a gene encoding the variable domain of the human T cell receptor, wherein the unrearranged TCRα variable locus is located at the endogenous rodent TCRα locus, and the unrearranged TCRβ variable locus is located at the endogenous rodent TCRβ locus. Furthermore, the rodent genome contains any of the following: [A] A nucleic acid sequence encoding a human CD8α polypeptide, wherein the nucleic acid sequence encoding the human CD8α polypeptide is located at an endogenous rodent CD8α co-receptor locus; a nucleic acid sequence encoding a human CD8β polypeptide, wherein the nucleic acid sequence encoding the human CD8β polypeptide is located at an endogenous rodent CD8β co-receptor locus; and a nucleic acid sequence encoding a human MHC class I polypeptide located at an endogenous rodent MHC class I locus; or [B] A nucleic acid sequence encoding a chimeric human-rodent CD8α polypeptide, wherein the nucleic acid sequence encoding the chimeric human-rodent CD8α polypeptide is located at an endogenous rodent CD8α co-receptor locus; a nucleic acid sequence encoding a chimeric human-rodent CD8β polypeptide, wherein the nucleic acid sequence encoding the chimeric human-rodent CD8β polypeptide is located at an endogenous rodent CD8β co-receptor locus; and a nucleic acid sequence encoding a human MHC class I polypeptide located at an endogenous rodent MHC class I locus; wherein the chimeric CD8 protein is capable of binding to human MHC class I; or [C] The nucleic acid sequences encoding rodent CD8α polypeptide at an endogenous locus, the nucleic acid sequences encoding rodent CD8β polypeptide at an endogenous locus, and the nucleic acid sequences encoding chimeric human-rodent MHC class I polypeptide at an endogenous rodent MHC class I locus; wherein the rodent CD8 protein is capable of binding chimeric MHC class I. Optionally, the rodent genome contains a nucleic acid sequence encoding a human β2 microglobulin polypeptide; when such a sequence is present, it is located at the endogenous rodent β2 microglobulin gene locus.

3. A genetically modified rodent cell, such as an ES cell, comprising: (i) An unrearranged T-cell receptor (TCR) α variable locus comprising at least one human Vα segment and at least one human Jα segment, wherein the TCRα variable gene is operatively linked to a human TCRα constant gene sequence. (ii) An unrearranged TCRβ variable locus comprising at least one human Vβ segment, at least one human Dβ segment and at least one human Jβ segment, wherein the unrearranged TCRβ variable gene is operatively linked to a human TCRβ constant gene sequence. in, The unrearranged human T cell variable region gene fragment can rearrange to form a gene encoding the variable domain of the human T cell receptor. The unrearranged TCRα variable locus is located at the endogenous rodent TCRα locus, and the unrearranged TCRβ variable locus is located at the endogenous rodent TCRβ locus.

4. The genetically modified rodent cell of claim 3, comprising: (i) An unrearranged T-cell receptor (TCR) α variable locus comprising at least one human Vα segment and at least one human Jα segment, wherein the TCRα variable gene is operatively linked to a human TCRα constant gene sequence. (ii) An unrearranged TCRβ variable locus comprising at least one human Vβ segment, at least one human Dβ segment and at least one human Jβ segment, wherein the unrearranged TCRβ variable gene is operatively linked to a human TCRβ constant gene sequence. in, The unrearranged human T cell variable region gene fragment can rearrange to form a gene encoding the variable domain of the human T cell receptor. The unrearranged TCRα variable locus is located at the endogenous rodent TCRα locus, and the unrearranged TCRβ variable locus is located at the endogenous rodent TCRβ locus. Furthermore, the rodent genome described herein contains any of the following: [A] Nucleic acid sequences encoding human CD8α polypeptide, human CD8β polypeptide, and human MHC class I polypeptide located at an endogenous rodent MHC class I locus, wherein the nucleic acid sequence encoding human CD8α polypeptide is located at an endogenous rodent CD8α co-receptor locus, and the nucleic acid sequence encoding human CD8β polypeptide is located at an endogenous rodent CD8β co-receptor locus; or [B] Nucleic acid sequences encoding a chimeric human-rodent CD8α polypeptide, a chimeric human-rodent CD8β polypeptide, and a nucleic acid sequence encoding a human MHC class I polypeptide located at an endogenous rodent MHC class I locus, wherein the nucleic acid sequence encoding the chimeric human-rodent CD8α polypeptide is located at an endogenous rodent CD8α co-receptor locus, and the nucleic acid sequence encoding the chimeric human-rodent CD8β polypeptide is located at an endogenous rodent CD8β co-receptor locus; wherein the chimeric CD8 protein can bind to human MHC class I; or [C] Nucleic acid sequences encoding rodent CD8α polypeptide at an endogenous locus, nucleic acid sequences encoding rodent CD8β polypeptide at an endogenous locus, and nucleic acid sequences encoding chimeric human-rodent MHC class I polypeptide at an endogenous rodent MHC class I locus, wherein the rodent CD8 protein can bind to chimeric MHC class I polypeptides. Optionally, the rodent genome contains a nucleic acid sequence encoding a human β2 microglobulin polypeptide; when such a sequence is present, it is located at the endogenous rodent β2 microglobulin locus.

5. A genetically modified rodent T cell comprising: (i) An unrearranged T-cell receptor (TCR) α variable locus comprising at least one human Vα segment and at least one human Jα segment, wherein the TCRα variable gene is operatively linked to a human TCRα constant gene sequence. (ii) An unrearranged TCRβ variable locus comprising at least one human Vβ segment, at least one human Dβ segment and at least one human Jβ segment, wherein the unrearranged TCRβ variable gene is operatively linked to a human TCRβ constant gene sequence. in, The unrearranged human T cell variable region gene fragment can rearrange to form a gene encoding the variable domain of the human T cell receptor. The unrearranged TCRα variable locus is located at the endogenous rodent TCRα locus, and the unrearranged TCRβ variable locus is located at the endogenous rodent TCRβ locus.

6. The genetically modified rodent or rodent cell as described in any one of claims 1 to 5, wherein it does not express a functional endogenous TCRα polypeptide from the endogenous TCRα variable locus, and is homozygous at the TCRα locus; and It does not express functional endogenous TCRβ polypeptide from the endogenous TCRβ variable locus, and is homozygous at the TCRβ locus.

7. A genetically modified rodent or rodent cell that is any of the following: (i) A rodent or rodent cell as described in any of the preceding claims, wherein the rodent retains an endogenous TCRα variable locus and / or an endogenous rodent TCRβ variable locus; and wherein any retained endogenous rodent TCRα variable locus is a nonfunctional locus and / or wherein any retained endogenous rodent TCRβ variable locus is a nonfunctional locus; optionally, all or part of the retained endogenous rodent TCRα and / or β variable locus are inverted relative to the normal orientation in the rodent genome; or (ii) A reproductive rodent or rodent cell wherein the orientation of at least the rodent TCRβ locus from rodent Vβ1 to Dβ1 in the rodent genome is reversed relative to the natural orientation; optionally, the method may further include inserting nucleic acid such as DNA encoding all or part of TCRβ from another species different from the rodent into the rodent genome, for example, inserting human DNA encoding all or part of TCRβ.

8. A genetically modified male rodent that is any of the following: (i) The rodent as described in any of the preceding claims, wherein the rodent is substantially capable of wild-type reproduction; wherein... The rodent TCRβ locus is missing at least rodent Vβ1 to Dβ1, and its genome is also missing one or all of the serine protease genes Prss58, Prss59, Prss3b, Try4, Try5, Try10, Prss3, Prss1 and Prss2. (ii) A rodent that is essentially capable of wild-type reproduction, wherein the rodent's TCRβ locus has been deleted at least rodent Vβ1 to Dβ1, and its genome is also missing one or all of the rodent serine protease genes Prss58, Prss59, Prss3b, Try4, Try5, Try10, Prss3, Prss1 and Prss2; wherein the rodent's genome is not a wild-type rodent genome, and / or does not have a wild-type genome at the TCRβ locus, and / or cannot form a rodent TCRβ chain.

9. The genetically modified rodent or rodent cell as described in any of the preceding claims, comprising the TCRα variable locus, the locus comprising one or more, optionally all, of the following human Vα gene fragments: TRAV1-1, TRAV1-2, TRAV2, TRAV3, TRAV4, TRAV5, TRAV6, TRAV8-1, TRAV8-2, TRAV8-3, TRAV8-4, TRAV8- 6. TRAV9-1, TRAV9-2, TRAV10, TRAV12-1, TRAV12-2, TRAV12-3, TRAV13-1, TRAV13-2, TRAV14 / DV4, TRA V16, TRAV17, TRAV19, TRAV20, TRAV21, TRAV22, TRAV23 / DV6, TRAV24, TRAV25, TRAV26-1, TRAV26-2, TR AV27, TRAV29 / DV5, TRAV30, TRAV34, TRAV36 / DV7, TRAV38-1, TRAV38-2 / DV8, TRAV39, TRAV40, TRAV41; and / or contains one or more, optionally all of the following human Jα gene fragments: TRAJ3, TRAJ4, TRAJ5, TRAJ6, TRAJ7, TRAJ9, TRAJ10, TRAJ11, TRAJ12, TRAJ13, TRAJ14, TRAJ15, TRAJ16, T RAJ17, TRAJ18, TRAJ20, TRAJ21, TRAJ22, TRAJ23, TRAJ24, TRAJ26, TRAJ27, TRAJ28, TRAJ29, TRAJ30, TRA J31, TRAJ32, TRAJ33, TRAJ34, TRAJ35, TRAJ36, TRAJ37, TRAJ38, TRAJ39, TRAJ40, TRAJ41, TRAJ42, TRAJ4 3. TRAJ44, TRAJ45, TRAJ46, TRAJ47, TRAJ48, TRAJ49, TRAJ50, TRAJ52, TRAJ53, TRAJ54, TRAJ56, TRAJ57.

10. The genetically modified rodent or rodent cell as described in any of the preceding claims, comprising the TCRβ variable locus, the locus comprising one or more, optionally all, of the following human Vβ gene fragments: TRBV2, TRBV3-1, TRBV4-1, TRBV4-2, TRBV5-1, TRBV5-4, TRBV5-5, TRBV5-6, TRBV6-1, TRBV6-2, TRBV6-4, TRBV6-5, TRBV6-6, TRBV6-8, TRBV7-2, TRBV7-3, TRBV7-4, TRBV7-6, TRBV7-7, TRBV7-9, TRBV9, TRBV10-1 , TRBV10-2, TRBV10-3, TRBV11-1, TRBV11-2, TRBV11-3, TRBV12-3, TRBV12-4, TRBV12-5, TRBV13, TRBV14 , TRBV15, TRBV16, TRBV18, TRBV19, TRBV20-1, TRBV24-1, TRBV25-1, TRBV27, TRBV28, TRBV29-1, TRBV30; and / or contains one or more, optionally all of the following human Jβ gene segments: TRBJ1-1, TRBJ1-2, TRBJ1-3, TRBJ1-4, TRBJ1-5, TRBJ1-6, TRBJ2-1, TRBJ2-2, TRBJ2-3, TRBJ2-4, TRBJ2-5, TRBJ2-6, TRBJ2-7.

11. The genetically modified rodent or rodent cell as described in any of the preceding claims, wherein CD3ε, CD3γ, CD3δ and CD3ζ are all endogenous wild-type.

12. A method for preparing a genetically modified rodent expressing a human T-cell receptor, comprising: (i) Inserting an unrearranged human TCRα variable locus into an endogenous rodent TCRα variable locus, the unrearranged human TCRα variable locus comprising at least one human Vα fragment and at least one human Jα fragment, and operatively ligated to the human TCRα constant region. (ii) Inserting an unrearranged human TCRβ variable locus into an endogenous rodent TCRβ variable locus, the unrearranged human TCRβ variable locus comprising at least one human Vβ fragment, at least one human Dβ fragment and at least one human Jβ fragment, and operatively ligated to the human TCRβ constant region. (iii) Insert the nucleic acid sequence encoding the human CD8α polypeptide into the endogenous rodent CD8α gene locus; (iv) Insert the nucleic acid sequence encoding the human CD8β polypeptide into the endogenous rodent CD8β gene locus; (v) Insert the nucleic acid sequence encoding a human MHC class I polypeptide into an endogenous MHC class I locus; (vi) Insert the nucleic acid sequence encoding human β2 microglobulin into the endogenous rodent β2 microglobulin gene locus; in, The host loci at each insertion site are inactivated or deleted, so that the rodent does not express the native rodent TCR, MHC class I or CD8, and optionally, does not express rodent β2 microglobulin.

13. A method for preparing a breedable rodent or rodent cell, the method comprising the step of inverting at least rodent TCRβs from rodent Vβ1 to Dβ1 in the rodent genome; optionally, the method may further comprise inserting nucleic acids, such as DNA, encoding all or part of TCRβs from another species different from the rodent into the rodent genome, such as inserting human DNA encoding all or part of TCRβs.

14. A method for preparing a genetically modified reproductive rodent expressing a human T-cell receptor, the method comprising deleting at least rodent Vβ1 to Dβ1 from the rodent TCRβ gene, including deleting serine protease genes Prss58, Prss59, Prss3b, Try4, Try5, Try10, Prss3, Prss1, and Prss2, wherein one or all of the aforementioned deleted serine protease genes Prss58, Prss59, Prss3b, Try4, Try5, Try10, Prss3, Prss1, and Prss2 are reinserted into the rodent genome.

15. A method for preparing a reproductive rodent comprising knockout of an endogenous T cell receptor β chain polypeptide gene, the method comprising deleting at least rodent Vβ1 to Dβ1 from the rodent TCRβ gene, including deleting serine protease genes Prss58, Prss59, Prss3b, Try4, Try5, Try10, Prss3, Prss1 and Prss2, wherein one or all of the above-deleted serine protease genes Prss58, Prss59, Prss3b, Try4, Try5, Try10, Prss3, Prss1 and Prss2 are reinserted into the rodent genome.

16. A method for preparing a breedable rodent, the method comprising, in any order: (i) Deleting at least rodent Vβ1 to Dβ1 from the rodent TCRβ genomic DNA, including deletion of one or all of the serine protease genes Prss58, Prss59, Prss3b, Try4, Try5, Try10, Prss3, Prss1, and Prss2; and (ii) Insert one or all of the above serine protease genes Prss58, Prss59, Prss3b, Try4, Try5, Try10, Prss3, Prss1 and Prss2 into the rodent genome.

17. The method of any one of claims 13 to 16, wherein the deficiency is homozygous.

18. The rodent or rodent cell as claimed in any one of claims 1 to 17, or a method for preparing a rodent or rodent cell, wherein the rodent genome comprises DNA encoding the full human TCRβ chain and comprises human V, D, J and C gene fragments, wherein the rodent or rodent cell is capable of expressing the full human TCRβ chain; and wherein the rodent genome comprises a human intergenic and / or human intron DNA sequence located between human D1 and human C2 gene fragments, preferably a human intergenic and / or intron sequence naturally coexisting with human D and J gene fragments; Optionally, the human DNA sequence between the human D1 and human C2 gene fragments is entirely genomic human DNA.

19. A rodent or rodent cell whose genome contains DNA encoding the full human TCRβ chain and contains human V, D, J and C gene segments, wherein the rodent or rodent cell is capable of expressing the full human TCRβ chain; and wherein the rodent genome contains human intergenic and / or human intron DNA sequences located between human D1 and human C2 gene segments, preferably human intergenic and / or intron sequences naturally coexisting with human D and J gene segments; Optionally, the entire human DNA sequence between the human D1 and human C2 gene fragments is genomic human DNA.

20. A method for preparing a human T-cell receptor in a rodent, the method comprising inserting DNA encoding a fully human TCRβ chain and containing fragments of human V, D, J and C genes into a rodent genome, wherein the rodent is capable of expressing the fully human TCRβ chain; in, The insertion comprises a human intergenic and / or human intron DNA sequence located between human D1 and human C2 gene segments, preferably a human intergenic and / or intron sequence that naturally coexists with human D and J gene segments; Optionally, the human DNA sequence between the human D1 and human C2 gene fragments is entirely genomic human DNA.

21. A method for generating a human T-cell receptor against a target antigen, comprising: (i) Optionally, a rodent as described in any of the preceding claims is prepared; (ii) Immunizing the rodents as described in any of the preceding claims with the target antigen; (iii) Allow the rodent to produce an immune response; (iv) Determining the nucleic acid sequence of the human TCR variable region expressed by T cells from rodents responsive to the aforementioned target antigen, optionally including the step of isolating the T cells; and (v) Expressing the human T-cell receptor or the variable region of the human T-cell receptor in cells; optionally, further formulating the expressed human T-cell receptor or the variable region of the human T-cell receptor with a pharmaceutically acceptable excipient; or (vi) Inserting a nucleic acid encoding the human T-cell receptor or the variable region of the human T-cell receptor into cells, such as human or animal cells, either in vitro or in vitro; optionally, wherein the cells containing the inserted nucleic acid are formulated for delivery to humans or animals; or (vii) Using a suitable delivery carrier, such as lipids or liposomes, formulate a nucleic acid (e.g., RNA or DNA) encoding the human T-cell receptor or the variable region of the human T-cell receptor for in vivo delivery to patients who require it.

22. A method for treating an individual who requires treatment, the method comprising: (i) Delivering a soluble TCR molecule containing the human T cell receptor variable region as described in step (v) of claim 21 to the individual; (ii) Delivering the cells as described in step (vi) of claim 21 to a patient in need of them; (iii) Delivering the nucleic acid (e.g., RNA, such as mRNA or DNA) formulated as in step (vii) of claim 21 to a patient in need of it.

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