Genetically modified rodents and rodent cells and their use

Abstract

Transgenic rodents and rodent cells expressing components of the human immune system, and their use in the generation of human T cell receptors (TCRs). Genetically modified rodents containing a TCRα gene locus with an unreorganized human TCRα variable gene segment and human TCRα constant region, and a TCRβ gene locus with an unreorganized human TCRβ variable gene segment and human TCRβ constant region. Antigen-specific CD8 for finding fully human TCR sequences with therapeutic potential. + Use of hTCR transgenic rodents that generate T cell responses.

Description

[Technical Field]

[0001] The present invention relates to rodents and rodent cells genetically modified to express components of the human immune system. The present invention also relates to methods for creating and using such rodents to produce therapeutically useful products, as well as products identified and obtained using the rodents. [Background technology]

[0002] There is still a need for in vivo systems that mimic the conditions of the human immune system.

[0003] Publications have been made relating to chimeric (human-mouse) forms of T cell receptors (hereinafter referred to as "TCRs"), chimeric forms of TCR coreceptors such as CD4 and CD8, and genetically modified mice encoding chimeric MHC class I and chimeric MHC class II. These publications include, in particular, Patent Documents 1 and 2. Such chimeric TCR polypeptides subsequently require humanization, i.e., replacement of the rodent portion to produce human therapeutic agents, and the resulting TCR variable regions are never provided in exactly the same way as those generated in vivo in mice. The chimeric locus is generated along with a mouse polypeptide portion that interacts with mouse regulatory sequences and / or other mouse polypeptide sequences.

[0004] T cells express a variable T cell receptor (TCR) that forms a complex with the CD3 polypeptide. The TCR is activated by binding to its cognitive peptide-major histocompatibility complex (pMHC), which is a cell surface component present on most mammalian cells that presents a portion of peptides from within the cell via antigen processing and presentation pathways. MHC class I molecules on the surface of most mammalian cells present 8-11 m-mer peptides derived from the cell's cytoplasmic proteins, and the TCR-CD3 complex and co-receptor CD8 bind to the pMHC. + It is recognized by T cells (see Figure 8).

[0005] In vivo, somatic rearrangement of genomic DNA within a T cell population generates a repertoire of TCRs with diverse sequences, where each T cell expresses a single (monoclonal) TCR containing random sequence elements. Positive and negative selection during T cell development allows T cells expressing TCRs that recognize pMHCs at threshold affinity to survive, eliminating self-reactive cells that exhibit high affinity binding. This results in a T cell population that does not recognize normal "self" pMHCs but can bind to pMHCs it has not encountered before. CD8 + When the TCR of cytotoxic T lymphocytes (CTLs) recognizes a peptide displayed on MHC class I, the TCR binds to pMHC, initiating intracellular signaling via the CD3 component of the TCR complex. This activates the CTL, releasing cytolytic molecules and immune cell-activating cytokines. Consequently, cells expressing proteins with neoepitopes (e.g., infected cells producing viral proteins and cells expressing mutated oncogenes) can be eliminated by the cellularly adaptive immune system, thus maintaining health.

[0006] Structurally, the TCR comprises two transmembrane polypeptides, which may be αβ or γδ pairs associated with 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 connecting region linked to 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 comprises two paired variable domains and two paired constant domains, and the pMHC binding site of the TCR is formed by a set of six CDRs, including three CDRs from the first variable domain and three CDRs from the second variable domain.

[0007] The repertoire of different TCR variable domain sequences is generated from individual genomes, where the sequence diversity within the variable domains arises from combining gene segments individually selected from multiple different germline gene segments within the locus encoding each TCR polypeptide chain. CDRs are particularly sequence-diverse because they contain non-template junctions between rearranged gene segments. Further combination diversity arises as different variable domains pair up (e.g., αβ pairing) to form TCR binding sites. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] European Patent No. 2958937 [Patent Document 2] European Patent No. 2958938 [Overview of the project]

[0009] The present invention (i) an unreorganized T cell receptor (TCR)α variable gene locus comprising at least one human Vα segment and at least one human Jα segment, wherein the TCRα variable gene is operably linked to the human TCRα constant gene sequence, (ii) an unreorganized TCRβ variable gene locus comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment, wherein the unreorganized TCRβ variable gene is operably linked to a human TCRβ constant gene sequence, Including, here, This relates to genetically modified rodents in which the unreorganized human T cell variable region gene segment can be rearranged to form a gene encoding the human T cell receptor variable domain, the unreorganized TCRα variable gene locus is located at the endogenous rodent TCRα gene locus, and the unreorganized TCRβ variable gene locus is located at the endogenous rodent TCRβ gene locus.

[0010] (i) an unreorganized T cell receptor (TCR)α variable gene locus comprising at least one human Vα segment and at least one human Jα segment, wherein the TCRα variable gene is operably linked to the human TCRα constant gene sequence, (ii) an unreorganized TCRβ variable gene locus comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment, wherein the unreorganized TCRβ variable gene is operably linked to a human TCRβ constant gene sequence, Including, here, Unreorganized human T cell variable region gene segments can be rearranged to form genes encoding the human T cell receptor variable domain. Genetically modified rodent cells, such as ES cells, in which the unreorganized TCRα variable gene locus is located at the endogenous rodent TCRα gene locus, and the unreorganized TCRβ variable gene locus is located at the endogenous rodent TCRβ gene locus.

[0011] (i) an unreorganized T cell receptor (TCR)α variable gene locus comprising at least one human Vα segment and at least one human Jα segment, wherein the TCRα variable gene is operably linked to the human TCRα constant gene sequence, (ii) An unrearranged TCRβ variable gene 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 operably linked to a human TCRβ constant gene sequence, and the unrearranged TCRβ variable gene locus, comprising, wherein, The unrearranged human T cell variable region gene segment can be rearranged to form a gene encoding a human T cell receptor variable domain. The unrearranged TCRα variable gene locus is present in the endogenous rodent TCRα gene locus, and the unrearranged TCRβ variable gene locus is present in the endogenous rodent TCRβ gene locus, a genetically modified rodent T cell.

[0012] A genetically modified rodent antigen-presenting cell, (i) A nucleic acid sequence encoding a human MHC class I polypeptide present in the endogenous rodent MHC class I gene locus, or (ii) A nucleic acid sequence encoding a chimeric human-rodent MHC class I polypeptide present in the endogenous rodent MHC class I gene locus, preferably, the nucleic acid sequence encoding the chimeric MHC class I has exon 4 of the rodent and exons 1-3 and 5-7 are of human origin, the nucleic acid sequence, either of which, and (iii) Optionally, if present, a nucleic acid sequence encoding a human β2 microglobulin polypeptide located in the endogenous rodent β2 microglobulin gene locus, and comprising, wherein, This cell expresses either human MHC class I or chimeric MHC class I, and optionally expresses human β2M polypeptide, a genetically modified rodent antigen-presenting cell.

[0013] A method of making a genetically modified rodent that expresses a human T cell receptor, (i) Inserting an unreorganized human TCRα variable gene locus into the endogenous rodent TCRα variable gene locus, which includes at least one human Vα segment and at least one human Jα segment operably linked to the human TCRα constant region, (ii) Inserting an unreorganized human TCRβ variable gene locus into the endogenous rodent TCRβ variable gene locus, comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment operably linked to the human TCRβ constant region, Methods that include...

[0014] A method for creating a genetically modified fertile rodent expressing a human T cell receptor, comprising deleting one or more or all of the serine protease genes Prss58, Prss59, Prss3b, Try4, Try5, Try10, Prss3, Prss1, and Prss2, and deleting at least rodent Vβ1 to Dβ1 of the rodent TCRβ, 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.

[0015] A method for producing a fertile rodent comprising a genetic knockout of an endogenous T cell receptor β chain polypeptide, comprising deleting one or more or all of the serine protease genes Prss58, Prss59, Prss3b, Try4, Try5, Try10, Prss3, Prss1, and Prss2, comprising deleting at least rodent Vβ1 to Dβ1 of the rodent TCRβ, 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.

[0016] A method for producing a human T cell receptor for a target antigen, (i) optionally, a process for producing a rodent as disclosed herein, (ii) A step of immunizing a rodent disclosed herein with a target antigen, (iii) A process to initiate an immune response in rodents, (iv) Optionally, a step of determining the nucleic acid sequence of the human TCR variable region expressed by T cells from a rodent reactive to the antigen of the target, including a step of isolating T cells, (v) A step of expressing a human T cell receptor or a human T cell receptor variable region in cells, and optionally further formulating the expressed human T cell receptor or human T cell receptor variable region together with a pharmaceutically acceptable excipient, or (vi) Inserting a nucleic acid encoding a human T cell receptor or a human T cell receptor variable region into human cells or animal cells, etc., ex vivo or in vitro, and optionally formulating the cells containing the inserted nucleic acid for delivery to humans or animals, respectively, or (vii) A process of formulating a nucleic acid (e.g., RNA or DNA) encoding the human T cell receptor or the human T cell receptor variable region together with an appropriate delivery carrier such as a lipid or liposome, and delivering it in vivo to a patient who requires delivery; Methods that include...

[0017] A method of treating individuals that require treatment, (i) Delivering the cells described in step (vi) above to a patient who requires delivery, (ii) Delivering a nucleic acid (e.g., RNA, e.g., mRNA or DNA) that has been formulated in accordance with step (vii) described above to a patient requiring delivery. Methods that include...

[0018] Preferably, the transgenic rodents according to the present invention exhibit functional expression of human TCRs and provide an in vivo T-cell immune repertoire highly suitable for selecting candidate TCRs for use in medical applications such as therapeutic use in humans. The functional expression of transgenic gene loci in rodents can be demonstrated and their effective performance confirmed by evaluating data on the use of germline gene segments in TCRs expressed from, for example, the transgenic TCRα locus and the transgenic TCRβ locus, combinations of TCR gene segments (e.g., v gene segment and j gene segment), N addition, and CDR3 length.

[0019] TCRs isolated from transgenic rodents can be tested to determine their affinity for target antigens (target peptide MHC complexes), their specificity and selectivity for binding to targets, and their ability to signal when bound to target pMHCs when expressed in T cells (e.g., measured by a reporter gene assay in T cells expressing the TCR). In particular, to select candidate therapeutic agents, it is desirable to identify TCRs that exhibit high affinity for targets and / or high efficacy in signaling, and the transgenic rodents of the present invention are an extremely rich source of such TCRs. Advantageously, the transgenic rodents according to the present invention may enable the isolation of TCRs that exhibit higher affinity and selectivity for binding to human target pMHC complexes compared to isolating TCRs from human individuals. [Brief explanation of the drawing]

[0020] [Figure 1]This figure shows the mouse TRA locus, the human TRA locus, and the manipulated human mouse TRA locus. The human DNA is located from chromosome 14:21570693 to chromosome 14:22554820 (GRCh38) in the third panel. This represents the insertion of slightly less than 1 Mbp of human DNA, cloned from the 1.1 Mbp region shown in the middle panel, into the 1.95 Mbp region of mouse DNA shown in the top panel, resulting in a replacement of 1.8 Mbp of mouse DNA with approximately 1 Mbp of human DNA. The inserted human DNA may contain 45 functional Vα gene segments and 51 functional Jα gene segments (Figure 1a). Alternatively, the inserted human DNA may contain 44 functional Vα gene segments and 50 functional Jα gene segments (Figure 1b). In Figure 1c, the illustration of the human TRD locus is modified, with the human TRD gene segments shown in the depicted chromosomal region (indicated by black triangles). In human sequences, there are 3×TRD D genes, 4×TRD J genes, and 8×TRD V genes (four of the V genes are shared with the TRA repertoire). [Figure 2] This figure shows the mouse TRB locus, the human TRB locus, and an engineered human-mouse TRB locus in which human DNA is inserted and rodent DNA is inverted to maintain male fertility. The human DNA is located on chromosomes 7:142237898 to 7:142813740 (GRCh38) in the third panel. This represents a 576 kbp insertion of human DNA encompassing the 520 kbp shown in the center panel. A 5.4 Mbp region of mouse genomic DNA is inverted, introducing a 4.8 Mbp segregation between the first mouse Vβ gene segment (vβ1) and the first human Vβ gene segment (vβ2). The inserted human DNA may contain 48 functional Vβ gene segments and 13 functional Jβ gene segments (Figure 2a). Alternatively, the inserted human DNA may contain 43 functional Vβ gene segments and 13 functional Jβ gene segments (Figure 2b). [Figure 3a-3b]This figure shows the replacement of the DNA encoding mouse CD8α in the mouse genome with the DNA encoding human CD8α, and also the replacement of the DNA encoding mouse CD8β in the mouse genome with the DNA encoding human CD8β. A chimeric CD8 polypeptide is produced. Figure 3b shows the process of creating the genome in Figure 3a in more detail. Figure 3a(ii) includes the modification of the genome coordinates shown in Figure 3a(i). Figure 3b(ii) includes the modification of the genome coordinates shown in Figure 3b(i). [Figure 4] This figure illustrates a hypothetical scheme in which the DNA encoding human CD8α can be replaced in the mouse genome with DNA encoding human CD8α, and also shows the replacement of the DNA encoding mouse CD8β with DNA encoding human CD8β. This would result in the complete generation of human CD8 polypeptide. [Figures 5a-5b] This figure shows the process of replacing the DNA encoding mouse β2-microglobulin in the mouse genome with the DNA encoding human β2-microglobulin. Figure 5b shows the process of creating the genome in Figure 5a in more detail. Figure 5a(ii) includes the modification of the genome coordinates shown in Figure 5a(i). Figure 5b(ii) includes the modification of the genome coordinates shown in Figure 5b(i). [Figures 6a-6b] This figure shows the process of replacing mouse H2-D1 with the human HLA_A*02:01 MHC class I gene in the mouse genome. Figure 6b shows the process of creating the genome in Figure 6a in more detail. [Figure 7] This diagram shows the MHC class I region of rodents, and the second panel shows the H2-K deletion and the knock-in site that replaces H2-D with human HLA-A. [Figure 8]This figure illustrates the representation of immunological synapses between T cells (bottom) and target cells (top). The TCR engages with the pMHC complex through synapses between adjacent cell membranes. Class I MHC consists of an α-chain containing domains α1, α2, and α3. The N-terminal domains α1 and α2 provide binding sites for peptide p. β2 microglobulin (β2m) associates with the α-chain. The TCR consists of an α-chain and a β-chain. Each chain includes an N-terminal variable region, a constant region, a connecting region, a transmembrane region, and a short cytoplasmic C-terminal tail. The TCR complexes with the CD3 polypeptide. The T cell dimeric coreceptor CD8 binds to the α3 domain of MHC class I molecules. CD8 is a heterodimer of α-chain and β-chain and associates with the intracellular signaling molecule Lck. This figure was created using BioRender. [Figure 9] This figure shows the TCR in the cell membrane. This figure was created using BioRender. [Figure 10] This figure shows TRBV and TRBJ transcripts detected from bulk sequencing of the spleen of a representative humanized TRB mouse. The data shown are the number of unique molecular identifiers (UMIs), not the total number of reads. Only the counts of functional TRBV and TRBJ are shown. Non-functional genes are often counted at zero, though not always. [Figure 11] This figure shows the UMI of TRBV from bulk sequencing of spleen RNA. [Figure 12] This figure shows the UMI of TRBJs from bulk sequencing of spleen RNA. [Figure 13] This figure shows TRAV and TRAJ transcripts detected from bulk sequencing of the spleen of a typical humanized TRA mouse. The data shown are the number of unique molecular identifiers (UMIs), not the total number of reads. Only the counts of functional TRAV and TRAJ are shown. Non-functional genes are often counted at zero, though not always. [Figure 14] This figure shows the UMI of TRAV from bulk sequencing of spleen RNA. [Figure 15] This figure shows the UMI of TRAJ from bulk sequencing of spleen RNA. [Figure 16] This figure compares the relative use of TRBJ1-C1 and TRBJ2-C2 in transcripts from transgenic mice with data reported in humans. The results for transgenic mice were obtained by bulk sequencing of spleen RNA from OpTiMus TRB transgenic mice. [Figure 17] This figure compares TRBV expression from spleen RNA of human TRB transgenic mice with published data from human PBMCs. [Figure 18] This figure compares TRBJ expression from spleen RNA of human TRB transgenic mice with published data from human PBMCs. [Figure 19] This figure compares the length distribution of T cell receptor CDR3 nucleotides for (A) TRB in humans (Kitaura et al, 2016), (B) TRB in transgenic mice, (C) TRA in humans (Kitaura et al, 2016), and (D) TRA in transgenic mice. [Figure 20] This figure compares TRAV expression from spleen RNA of human TRA transgenic mice with published data from human PBMCs. [Figure 21] This figure compares TRAJ expression from spleen RNA of human TRA transgenic mice with published data from human PBMCs. [Figure 22] This figure shows the use of (a) the TRAV gene, (b) the TRAJ gene, (c) the TRBV gene, and (d) the TRBJ gene in hTCR transgenic mice. "I" indicates that gene expression was reported. "0" indicates that gene expression was not reported. "~" indicates that low expression (less than 0.01%) was reported. "?" indicates that no data was reported. [Figure 23]This figure shows the detection of cells with surface expression of mouse CD3 (vertical axis) and human CD8b (horizontal axis) by FACS in OpTiMus TRA / TRB transgenic mice of various zygosities. [Figure 24] This figure shows the detection of splenocytes with surface expression of mCD3, mCD4, hCD8b, hTRBC-1, and mCD19 in fully homozygous OpTiMus mouse 8. The data represents results from three mice (mouse 8, mouse 9, and mouse 10). [Figure 25] This figure shows splenocyte populations from unsensitized OpTiMus mice (mouse 8, mouse 9, and mouse 10). [Figure 26] This figure compares representative data from peptide-immunized mice with data from unsensitized control mice. [Figure 27] This figure shows the immune response in hTRA+ / + (homozygous) and hTRB+ / - (heterozygous) mice after peptide immunization. [Figure 28] This figure shows the immune response in mice that are completely homozygous for hTRA+ / + and hTRB+ / + after peptide immunization. [Figure 29] This figure shows the yield of 124 soluble TCR-Fc fusion polypeptides produced by recombinant expression in Expi293T cells. Each dot represents one TCR. [Modes for carrying out the invention]

[0021] These figures illustrate certain preferred coordinates for genome modification in mice, but they do not limit the invention.

[0022] In particular, human DNA with the following coordinates is inserted (the inserted human DNA contains all the numbered nucleotides, and the mouse genome holds the numbered nucleotides within its genome).

[0023] Tra Human DNA from position 14:21,570,693 to position 14:22,554,820 (GRCh38) Human DNA insertion located between mouse positions 14:52,664,870 or 14:52,664,818 (depending on the mouse strain) and 14:54,463,673 (GRCm39).

[0024] Trb Human DNA from position 7:142,237,898 to position 7:142,813,740 (GRCh38) Human DNA insertion located between mouse positions 6:36,074,375 and 6:41,535,764 (GRCm39). Inversion of mouse DNA between 6:36,070,006 and 6:36,074,375, from 6:40,868,163 to 6:41,515,110.

[0025] MHC Class I Human DNA from position 6:29,942,554 to position 6:29,945,455 (GRCh38) Human DNA insertion located between mouse positions 17:35,482,089 and 17:35,485,847 (GRCm39).

[0026] CD8A (Figure 3a(i)) Human DNA from position 2:86,789,408 to position 2:86,790,726 (GRCh38) Human DNA insertion located between mouse positions 6:71,350,536 and 6:71,351,769 (GRCm39).

[0027] CD8A (Figure 3a(ii)) Human DNA from position 2:86,790,825 to position 2:86,789,408 (GRCh38) Human DNA insertion located between mouse positions 6:71,350,536 and 6:71,351,769 (GRCm39).

[0028] CD8B (Figure 3a(i)) Human DNA from position 2:86,861,751 to position 2:86,853,004 (GRCh38) Human DNA insertion located between mouse positions 6:71,299,840 and 6:71,306,867 (GRCm39).

[0029] CD8B (Figure 3a(ii)) Human DNA from position 2:86,861,865 to position 2:86,853,004 (GRCh38) Human DNA insertion located between mouse positions 6:71,299,840 and 6:71,306,770 (GRCm39).

[0030] B2M (Figure 5a(i)) Human DNA from position 15:44,711,547 to position 15:44,716,342 (GRCh38) Human DNA insertion located between mouse positions 2:121,978,219 and 2:121,982,235 (GRCm39).

[0031] B2M (Figure 5a(ii)) Human DNA from position 15:44,711,547 to position 15:44,716,342 (GRCh38) Human DNA insertion located between mouse positions 2:121,978,218 and 2:121,982,142 (GRCm39).

[0032] The diagram is not to scale, but the use of / / or / / / indicates that regions of chromosomes are not included in the diagram.

[0033] In all figures: Gray = Mouse Black = Human Enh = Enhancer The unshaded white box at the 5' end of the exon (e.g., Figure 3a) is the 5' untranslated region, which is part of the exon but not part of the coding sequence.

[0034] Detailed explanation The present invention relates to rodents and rodent cells, comprising human nucleic acids encoding one or more polypeptide components of the human T cell molecular apparatus, inserted into the rodent genome at endogenous mouse loci for those components. In particular, the rodent or cell comprises human nucleic acids encoding one or more, preferably all, of the following: human TCRα polypeptide, human TCRβ polypeptide, human MHC class I polypeptide, human CD8α polypeptide, human CD8β polypeptide, and human β2 microglobulin polypeptide. In another embodiment, the rodent or rodent cell also comprises human nucleic acids encoding human TCRδ polypeptide. The TCRα polypeptide is encoded by the TRA locus and the TCRβ polypeptide is encoded by the TRB locus, but these loci may also be referred to herein as the TCRα locus and the TCRβ locus.

[0035] If a human TCR (including human TCRα polypeptide and human TCRβ polypeptide) is expressed on a cell, that human TCR will interact with peptides presented by MHC molecules on the antigen-presenting cell. The α1 and α2 domains of MHC class I interact with the TCR. The α3 domain of MHC class I binds to the CD8 molecule, which is a co-receptor for the TCR. These regions of MHC class I and / or the CD8 co-receptor may be engineered to enable effective peptide presentation to the human TCR as described herein.

[0036] A TCR comprising human TCRα polypeptide and / or human TCRβ polypeptide, preferably a fully human TCR, as described herein and in the claims, can be combined in the genome with any of the other alleles described herein, which may be human, chimeric, or mouse. The examples of other alleles described herein are merely examples, and other suitable alleles can be identified from common general knowledge and / or previous publications of these (human / chimeric / mouse) alleles.

[0037] Other alleles that can be used with TCR are described in the following publications and patent applications which are incorporated herein by reference:

[0038] Chimeric MHC (HHD construct containing human β2m (B2m) fused to human α1, human α2, and mouse α3, TM region, and cytoplasmic region of MHC) (1997 J. Exp. Med. 185(12):2043-2051, "HLA-A2.1-restricted education and 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, Francois A. Lemonnier, and Beatrice Perarnau).

[0039] The MHC1 and / or CD8 alleles described in International Publication No. 02 / 059263.

[0040] MHC1, B2m, and / or CD8 alleles described in U.S. Patent Application Publication No. 2005 / 066375 (International Publication No. 03 / 006639).

[0041] The following MHC1 and / or B2m alleles of ABAbDII mice: (i) 2010 Nature Medicine 16(9):1029 "Transgenic mice with a diverse human T cell antigen receptor repertoire", Liang-Ping Li, J Christoph Lampert, Xiaojing Chen, Catarina Leitao, Jelena Popovic, Werner Mueller & Thomas Blankenstein, (ii) 2015 Nature Biotechnology 33(4):402-407. "Identification of human T-cell receptors with optimal affinity to cancer antigens using antigen-negative humanized mice." Matthias Obenaus, Catarina Leitao, Matthias Leisegang, Xiaojing Chen, Ioannis Gavvovidis, Pierre van der Bruggen, Wolfgang Uckert, Dolores J Schendel & Thomas Blankenstein.

[0042] The MHC1 and CD8 alleles described in International Publication No. 2014 / 130671.

[0043] 2016 Cancer Immunology Research 4(3):204, "Identification of T-cell Receptors Targeting KRAS-Mutated Human Tumors". MHCI in Qiong J. Wang, Zhiya Yu, Kayla Griffith, Ken-ichi Hanada, Nicholas P. Restifo, and James C. Yang.

[0044] The MHC1, B2m, and CD8 alleles described in Moore et al., SCIENCE IMMUNOLOGY, "Humanization of T cell-mediated immunity in mice", 6(66) 2021.

[0045] Appropriate MHCI, B2m allele as described in International Publication No. 2021 / 139799.

[0046] It is highly desirable to use all or most of the human V, human D, and human J gene segments inserted into the human TCR chain expressed in rodents. In this way, it may be possible to express the entire repertoire of human TCR molecules in rodents. It has been shown that rodents expressing the human TCRs described herein use a considerable number of inserted human α and human β gene segments to construct the TCR chain.

[0047] Furthermore, it has been shown that the rodents of the present invention express TCRβ DJC2 from entirely human Trb DNA without the need to include mouse intron sequences.

[0048] Accordingly, the present invention relates to rodents that express at least 35, at least 40, at least 41, at least 42, at least 43, at least 44, or all 45 inserted human variable Vα gene segments in the rodent TCRα chain repertoire.

[0049] Accordingly, the present invention relates to rodents that express 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 inserted human Jα gene segments in the rodent TCRα chain repertoire.

[0050] Accordingly, the present invention relates to rodents that express at least 35, at least 40, at least 45, at least 46, at least 47, or all 48 inserted human variable Vβ gene segments in the rodent TCRβ chain repertoire.

[0051] Accordingly, the present invention relates to rodents that express at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, or all thirteen inserted human Jβ gene segments in the rodent TCRβ chain repertoire.

[0052] Accordingly, the present invention relates to a rodent having a repertoire comprising TCRβ chains expressed from both the DJC1 and DJC2 gene clusters of TRB, wherein the inserted human DNA does not contain mouse intron sequences, for example, between human D gene segments or human J gene segments present in the genome.

[0053] A preferred embodiment of the present invention is, (i) an unreorganized T cell receptor (TCR)α variable gene locus comprising at least one human Vα segment and at least one human Jα segment, wherein the TCRα variable gene is operably linked to the human TCRα constant gene sequence, (ii) an unreorganized TCRβ variable gene locus comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment, wherein the unreorganized TCRβ variable gene is operably linked to a human TCRβ constant gene sequence, Including, here, This relates to genetically modified rodents in which the unreorganized human T cell variable region gene segment can be rearranged to form a gene encoding the human T cell receptor variable domain, the unreorganized TCRα variable gene locus is located at the endogenous rodent TCRα gene locus, and the unreorganized TCRβ variable gene locus is located at the endogenous rodent TCRβ gene locus.

[0054] In a preferred embodiment, a rodent or rodent cell expresses a T cell receptor capable of interacting with peptides presented on MHC class I molecules in the rodent, and appropriately activating and proliferating T cells. In a preferred embodiment, a rodent or rodent cell expresses a CD8 protein capable of binding to MHC class I expressed by the rodent. Preferably, the interacting CD8 and MHC class I polypeptide regions are derived from the same species (e.g., both from humans or both from rodents).

[0055] Other preferred embodiments of the present invention include: A rodent containing a genome that encodes an MHC class I polypeptide in which the α1 and α2 domains are human. A rodent in which an MHC class I polypeptide contains an α3 domain that binds to CD8, which is expressed in rodents. A rodent whose α3 domain is human and whose CD8 contains a human MHC class I binding domain. CD8 is found in rodents, including human CD8α. CD8 is a rodent that includes the human-rodent chimera CD8α. CD8 is found in rodents, including human CD8β. CD8 is a rodent that contains the human-rodent chimera CD8β. A rodent containing a genome in which human CD8 or human-rodent chimeric CD8 is located at the endogenous rodent CD8 gene locus. A rodent in which MHC class I polypeptides contain both a human transmembrane domain and a human cytoplasmic domain. A rodent in which the α3 domain of MHC class I is that of a rodent. CD8 is a rodent that includes endogenous rodents CD8. A rodent in which the DNA encoding an MHC class I polypeptide is incorporated into the endogenous rodent MHC class I gene locus.

[0056] The present invention also relates to methods for producing such rodents and cells, and to the use of such rodents and cells in the production of therapeutically useful molecules such as human T cell receptors produced in the rodents disclosed herein, comprising obtaining a nucleic acid sequence of human TCR from a rodent and expressing the whole of the human TCR, its variable portion, or its binding portion in cells such as a producing cell lineage such as CHO cells, or in human or animal cells. Alternatively, the nucleic acids encoding such human TCR, or the variable portion of the TCR, or the binding portion of the TCR may be therapeutically useful molecules formulated appropriately for delivery.

[0057] In a particular embodiment, a nucleic acid that fully encodes a human polypeptide is provided operably ligated to one or more endogenous rodent regulatory sequences, such as rodent promoters, enhancers, or other regulatory sequences, in a rodent or rodent cell.

[0058] 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 forms most or all of the polypeptides expressed in the rodent or from the cell. For example, in one embodiment, the inserted human TCRα polypeptide is expressed, but the endogenous rodent TCRα polypeptide is not expressed from the endogenous TRA locus.

[0059] In a preferred embodiment of the above: Preferably, the rodents or cells disclosed herein do not express a functional endogenous TCRα polypeptide from an endogenous TCRα variable gene locus. Preferably, the rodents or cells disclosed herein do not express a functional endogenous TCRβ polypeptide from an endogenous TCRβ variable gene locus. Preferably, the rodents or cells disclosed herein do not express a functional endogenous rodent CD8 coreceptor 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, the rodents or cells disclosed herein do not express functional endogenous rodent β2-microglobulin from the endogenous rodent β2-microglobulin locus if the rodent or rodent cell contains a nucleic acid sequence encoding human β2-microglobulin.

[0060] The reference to "not expressing" or "absence of expression" of endogenous polypeptides means that there is no significant / substantial expression of rodent polypeptides, generally less than 10% of the wild-type expression of endogenous rodent polypeptides. Preferably, there is no expression of rodent polypeptides from the modified locus, and the expression of host polypeptides from the modified locus is completely blocked. In this way, the expression of equivalent human polypeptides inserted into the rodent locus can be maximized.

[0061] In one embodiment, since the evaluation of expression levels is considered at the level of the modified locus, expression from other unmodified alleles may be observed. However, it is preferable that no expression of endogenous polypeptides is observed, which may be, for example, the result of homozygous modification of both rodent loci. This applies independently to each modified locus. Therefore, in one embodiment, one or more or all of the loci referred to herein are homozygous for the inserted human polypeptide, and no expression is observed from any allele at each locus. In one embodiment, all one, two, three, four, five, six, or seven loci disclosed herein (loci encoding TCRα polypeptide, TCRβ polypeptide, MHC class I polypeptide (including both knockout or inactivation modification of both the rodent H2K and rodent H2D loci), human CD8α polypeptide, human CD8β polypeptide, and human β2 microglobulin polypeptide) may be homozygous. In another embodiment, one or more or all of the loci disclosed herein may be heterozygous, with only one allele modified. In one embodiment, all one, two, three, four, five, or six of the loci disclosed herein may be heterozygous.

[0062] In a particular embodiment, all or part of a rodent nucleic acid locus (encoding a rodent polypeptide equivalent to the human polypeptide described herein) is deleted from the rodent genome to prevent the expression of the host polypeptide, or the endogenous locus is inactivated, for example, by inversion.

[0063] Preferably, the inversion moves the rodent DNA, for example, the rodent V gene segment, at least 0.5 MB away from the native locus, for example, at least 1 Mbp, for example, at least 1.5 Mbp, at least 2 Mbp, at least 2.5 Mbp, at least 3 Mbp, at least 3.5 Mbp, at least 4 Mbp, or at least 4.5 Mbp away from the native locus.

[0064] Preferred rodents express all of the following: human TCRα polypeptide, TCRβ polypeptide, MHC class I polypeptide, CD8α polypeptide, CD8β polypeptide, and β2 microglobulin polypeptide, and do not express rodent TCRα polypeptide, TCRβ polypeptide, MHC class I polypeptide, CD8α polypeptide, CD8β polypeptide, and β2 microglobulin polypeptide.

[0065] Other preferred rodents are, By expressing at least human TCRα polypeptide and TCRβ polypeptide, a functional TCR is formed. It expresses human TCRα polypeptide, TCRβ polypeptide, MHC class I polypeptide, CD8α polypeptide, and CD8β polypeptide.

[0066] In one embodiment, the genome of a rodent or rodent cell contains a fully human TCR after rearrangement of the TCR gene segment. Therefore, a fully human TCR can be expressed in a rodent or cell without further humanization. This means that any potential influence of subsequent humanization processes (e.g., subsequent replacement of mouse constant regions with human constant regions) is eliminated. This approach minimizes the impact of structural differences between the chimeric TCR molecule and the fully human TCR, where these differences arise from the interface between the human Vα / Vβ domain and the mouse Cα / Cβ domain in the chimeric molecule.

[0067] Human nucleic acid insertions are targeted and inserted into endogenous rodent loci within the rodent genome. This creates the possibility of using them in combination with naturally occurring endogenous regulatory sequences inserted into human DNA.

[0068] By inserting a human TCRδ gene segment, it is also possible to enable the expression of the TCRδ chain and, at least, the expression of TCRδγ which has a human δ chain.

[0069] TCRα In this specification, genetically modified rodents or rodent cells, such as ES cells or T cells, A genetically modified rodent or rodent cell is provided, comprising an unreorganized T cell receptor (TCR)α variable gene locus containing at least one human Vα segment and at least one human Jα segment, wherein the TCRα variable gene is operably linked to a human TCRα constant gene sequence, the unreorganized human T cell variable region gene segment can be rearranged to form a gene encoding a human T cell receptor variable domain, and the unreorganized TCRα variable gene locus is located at the endogenous rodent TCRα gene locus.

[0070] In one embodiment of the present invention, the T cell coreceptor polypeptide is expressed only on rodent T cells, for example, the T cell coreceptor polypeptide is not expressed on rodent B cells.

[0071] In one embodiment of the present invention, T cell coreceptor polypeptide α and / or T cell coreceptor polypeptide β are included in the germ cell lineage of rodents.

[0072] In one embodiment, a rodent or rodent cell may be heterozygous for the nucleotide sequence encoding the human TCRα polypeptide.

[0073] In one embodiment, a rodent or rodent cell may be homozygous for the nucleotide sequence encoding the human TCRα polypeptide.

[0074] In one embodiment, the inserted human TCRα nucleic acid contains up to or at least 5, 10, 15, 20, 25, 30, 35, or 40 or more TCR Vα gene segments, for example, up to 44 or 45 or at least 44 or 45 Vα gene segments. In one embodiment, 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 segments. The inserted TCRα nucleic acid may contain non-functional TCR Vα gene segments or pseudogenes.

[0075] In one embodiment, the inserted human TCRα nucleic acid contains up to or at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or 51 TCR Jα gene segments. In one embodiment, 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 segments. The inserted TCRα nucleic acid may contain non-functional TCR Jα gene segments or pseudogenes.

[0076] In one embodiment, the unreorganized TCRα variable gene locus includes the complete repertoire of human Jα segments and the complete repertoire of human Vα segments.

[0077] In one embodiment, 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 one embodiment, the inserted human TCRα nucleic acid contains 45 functional TCRVα gene segments and 51 functional Jα gene segments. In one embodiment, the rodent is a mouse.

[0078] The TCRVα gene segment at the transgenic locus is arbitrarily selected from the TRAV segments listed in Table 1. The TCRα variable gene locus may include some or all of the TRAV gene segments listed in Table 1, preferably all functional TRAV gene segments. TRAV7 and / or TRAV18 may be absent or, if present, not expressed. Optionally, the TCRα variable gene locus may include one or more or all of the following TRAV gene segments that can rearrange with the TRAJ gene segment to form a gene encoding the human T cell receptor variable domain: 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, T RAV25, TRAV26-1, TRAV26-2, TRAV27, TRAV29 / DV5, TRAV30, TRAV34, TRAV36 / DV7, TRAV38-1, TRAV38-2 / DV8, TRAV39, TRAV40, TRAV41.

[0079] The TCRJα gene segment at the transgenic locus is arbitrarily selected from the TRAJ segments listed in Table 1. The inserted human TCRα nucleic acid may contain some or all of the TRAJ gene segments listed in Table 1, preferably all functional TRAJ gene segments. Optionally, the TCRα variable locus may contain any one or more or all of the following TRAJ gene segments that can be rearranged with the TRAV gene segment to form a gene encoding the human T cell receptor variable domain: TRAJ3, TRAJ4, TRAJ5, TRAJ6, TRAJ7, TRAJ9, TRAJ10, TRAJ11, TRAJ12, TRAJ13, TRAJ14, TRAJ15, TRAJ16, TRAJ17, TRAJ18, TRAJ20, TRAJ21, TRAJ22, T RAJ23, TRAJ24, 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.

[0080] In one embodiment, the inserted human TCRα nucleic acid forms a continuous human insert in the mouse genome.

[0081] In one embodiment, the deleted TCRα DNA includes genomic DNA from Vα1 to TCRαC.

[0082] In one embodiment, the inserted human DNA includes DNA from human Vα1 to TCRαC.

[0083] In one embodiment, the TCRα constant region is the human TCRα constant region.

[0084] In one embodiment, the inserted human TCRα gene locus is operably linked to a rodent enhancer, which is preferably a rodent enhancer located downstream (3') of the rodent TCRα constant region in the rodent genome, and preferably at its native location within the genome. In one embodiment, the V and J gene segments of the unreorganized human T cell variable region TCRα are operably linked to an endogenous rodent TCRα enhancer downstream of the TCRα constant region.

[0085] In one embodiment, a rodent or rodent cell contains or retains endogenous rodent promoters and / or other regulatory elements that control the expression of the human TCRα locus. In one embodiment, a nucleic acid that fully encodes a human polypeptide is provided in a rodent or rodent cell operably ligated to one or more endogenous rodent regulatory sequences, such as rodent promoters, enhancers, or other regulatory sequences. In a preferred embodiment, one or more promoters of the inserted human TCRα human V gene segment are human. In one embodiment, the expression of one or more human V gene segments or human J gene segments of the TCRα locus is under the control of human promoters. In one embodiment, all V promoters are human.

[0086] In one embodiment, a functional endogenous rodent TCRα variable locus is absent, which may result from deletions in the rodent genome that could be, for example, (a) deletion of all endogenous Vα gene segments, (b) deletion of all endogenous Jα gene segments, or (c) a combination thereof.

[0087] In one embodiment, the endogenous rodent TCRα locus is deleted from the rodent genome, including from the distal 3'V(Vα1) to the rodent 3' constant region TCRαC, leaving a rodent enhancer downstream of TCRαC in the rodent genome, which is operably linked to an unreorganized human T cell receptor (TCR)α variable gene locus.

[0088] In a preferred embodiment, the rodent is a mouse, and the enhancer located at 3' of the constant region is a mouse enhancer.

[0089] In one embodiment, rodents or rodent cells retain endogenous TCRα variable gene loci and / or endogenous rodent TCRβ variable gene loci, where the retained endogenous rodent TCRα variable gene loci are all non-functional loci, and the retained endogenous rodent TCRβ variable gene loci are all non-functional loci.

[0090] In one preferred embodiment, the rodent TCRα constant region is absent at the rodent TCRα gene locus.

[0091] In one embodiment, this gene locus exhibits appropriate spatial and temporal protein expression, aiding in T cell development and selection.

[0092] TCR δ In one embodiment, rodents or rodent cells such as ES cells or T cells disclosed herein contain a repertoire of unreorganized human TCRδ variable region segments at the rodent TCRα locus.

[0093] In one embodiment, the inserted human TCRα locus comprises an unreorganized T cell receptor (TCR)δ variable gene locus including at least one human Vδ segment, at least one Jδ gene segment, and at least one human Jα segment, wherein the TCRδ variable gene segment is operably linked to a human TCRδ constant gene sequence.

[0094] In one embodiment, a rodent or rodent cell contains a complete repertoire of human Vδ gene segments, human Dδ gene segments, and human Jδ gene segments in the rodent genome.

[0095] In one embodiment, the rodents or rodent cells disclosed herein include a complete repertoire of human Vδ segments, a complete repertoire of human Dδ segments, and a complete repertoire of human Jδ segments at the human TCRα variable locus.

[0096] In one embodiment, the rodents or rodent cells disclosed herein contain the entire human Vδ constant region, wherein the rodent Vδ constant region is absent at the TCRα variable locus.

[0097] Since the DNA encoding TCRδ is naturally present within the TCRα locus, insertion of the entire human TCRα locus naturally leads to the insertion of the human TCRδ gene segment, including the δ constant region. In one embodiment, the inserted human DNA includes DNA from human Vα-1 to TCRαC, and as a result, the rodent genome contains all of the human TCRδ.

[0098] The human TCRδ nucleic acids described herein can be inserted into the genome without the entire TCRα locus being inserted. Preferably, the human TCRδ nucleic acids are inserted into the TCRα locus.

[0099] In one preferred embodiment, the genome of a rodent or rodent cell does not contain the rodent TCRδ constant region.

[0100] In one embodiment, the human TCRδ locus is operably linked to a human enhancer and / or human promoter(s). In one embodiment, all promoters of the V gene segment of TCRδ are human.

[0101] In one embodiment, the enhancer located at 5' of the steady-state region (TRDC) is human.

[0102] The rodent or rodent cell preferably contains an unreorganized human TCRα locus and an unreorganized human TCRδ locus.

[0103] In one embodiment, a modified rodent or rodent cell is provided in which all or part of the endogenous rodent TCRα gene locus is replaced with a TCRα variable gene locus that has not been rearranged, as disclosed herein.

[0104] In one embodiment, the inserted human TCRδ nucleic acid forms a continuous human insert in the rodent genome.

[0105] In one embodiment, this gene locus exhibits appropriate spatial and temporal protein expression, aiding in T cell development and selection.

[0106] TCRβ This specification provides a genetically modified rodent or rodent cell, such as an ES cell or T cell, comprising an unreorganized TCRβ variable gene locus including at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment, wherein the unreorganized TCRβ variable gene is operably linked to a human TCRβ constant gene sequence, the unreorganized human T cell variable region gene segment can be rearranged to form a gene encoding a human T cell receptor variable domain, and the unreorganized TCRβ variable gene locus is located at an endogenous rodent TCRβ gene locus.

[0107] In one embodiment, the human TCRβ locus is operably ligated to a rodent enhancer that is naturally located downstream (3') of the rodent TCRβ constant region in wild-type rodents. Ideally, this enhancer is located in its native position within the rodent genome.

[0108] In one embodiment, the human TCRβ locus is operably linked to a human enhancer that is typically located downstream of the human TCRβC2 constant region in the human locus.

[0109] In one embodiment, one or more promoters of the inserted human TCRα human V gene segment are human. In another embodiment, all promoters are human.

[0110] In one embodiment, the rodents or rodent cells disclosed herein do not contain the rodent Vβ1 gene segment.

[0111] In one embodiment, the human insert comprises human DNA including Vβ1 to Vβ30. In one embodiment, the inserted human insert comprises human DNA including human Prss58 to Vβ30. In one embodiment, the inserted DNA comprises human genome fragments (or more).

[0112] In one embodiment, the inserted human TCRβ nucleic acid contains at most, at least, or exactly one, two, three, four, five, ten, fifteen, twenty, twenty-five, thirty, thirty, thirty-five, forty, forty, forty-three, forty-five, forty-six, forty-seven, forty-eight or more gene segments. The inserted TCRβ nucleic acid may contain non-functional TCR Vβ gene segments or pseudogenes.

[0113] In one embodiment, the inserted human TCRβ nucleic acid contains at most, at least, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 Jβ gene segments. The inserted TCRβ nucleic acid may contain Jβ gene segments or pseudogenes of non-functional TCRs.

[0114] In one embodiment, the inserted human TCRβ nucleic acid includes one or both Dβ gene segments.

[0115] In one embodiment, the inserted human TCRβ nucleic acid includes the Dβ1-Jβ1.1-1.6 gene segment and the Dβ2-Jβ2.1-2.7 gene segment.

[0116] 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 embodiment, the rodent is a mouse, and the inserted human TCRβ nucleic acid contains 48 functional TCRVβ gene segments and 13 functional Jβ gene segments.

[0117] The TCRVβ gene segment at the transgenic locus is arbitrarily selected from the TRBV segments listed in Table 1. The TCRβ variable gene locus may include some or all of the TRBV gene segments listed in Table 1, preferably all functional TRBV gene segments. Optionally, the TCRβ variable gene locus may include any one or more or all of the following TRBV gene segments that can rearrange with the TRBJ gene segment to form a gene encoding the human T cell receptor variable domain: 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, TRB V7-3, TRBV7-4, TRBV7-6, TRBV7-7, TRBV7-9, TRBV9, TRBV10-1, TRBV10-2, TRBV10-3, TRBV11-1, TRBV11-2, TRBV11-3, TRBV12-3, TRBV1 2-4, TRBV12-5, TRBV13, TRBV14, TRBV15, TRBV16, TRBV18, TRBV19, TRBV20-1, TRBV24-1, TRBV25-1, TRBV27, TRBV28, TRBV29-1, TRBV30.

[0118] The TCRJβ gene segment at the transgenic locus is arbitrarily selected from the TRBJ segments listed in Table 1. The inserted human TCRβ nucleic acid may contain some or all of the TRBJ gene segments listed in Table 1, preferably all functional TRBJ gene segments. Optionally, the TCRβ variable locus may contain any one or more or all of the following TRBJ gene segments that can be rearranged with the TRBV gene segment to form a gene encoding the human T cell receptor variable domain: 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.

[0119] In one embodiment, the inserted human TCRβ nucleic acid includes all of the functional TCRVβ gene segment and all of the functional Jβ gene segment.

[0120] In one embodiment, the rodent is the mouse.

[0121] In one embodiment, the inserted human TCRβ nucleic acid includes human TRBC1 and human TCRBC2.

[0122] In one embodiment, the inserted human TCRβ nucleic acid forms a continuous human insert in the mouse genome.

[0123] In one embodiment, rodents may be heterozygous for the nucleotide sequence encoding the human TCRβ polypeptide.

[0124] In one embodiment, rodents may be homozygous for the nucleotide sequence encoding the human TCRβ polypeptide.

[0125] In one embodiment, a rodent or rodent cell contains or retains endogenous rodent promoters, enhancers, and / or other regulatory elements that control the expression of the human TCRβ locus. In one embodiment, a nucleic acid that fully encodes a human polypeptide is provided operably ligated in a rodent or rodent cell to one or more endogenous rodent regulatory sequences, such as rodent promoters, enhancers, or other regulatory sequences.

[0126] In one embodiment, the rodent is a mouse, and the enhancer located at 3' of the constant region (TRBC) and 5' of Vb31 is a mouse enhancer.

[0127] In one embodiment, the inserted human gene locus exhibits appropriate spatial and temporal protein expression, aiding in T cell development and selection.

[0128] In one embodiment, a functional endogenous rodent TCRβ variable locus is absent, which may result from deletions in the rodent genome, such as (a) deletion of all endogenous Vβ gene segments, (b) deletion of all endogenous Jβ gene segments, (c) deletion of all endogenous Jδ gene segments, (d) deletion of host rodent constant regions (which may be multiple), and (e) combinations thereof.

[0129] Preferably, both the TCRα and TCRβ loci of a rodent have deletions as described herein, or one or both are inactivated by inversion as described herein.

[0130] In one embodiment, rodents retain an endogenous TCRβ variable gene locus, where the retained endogenous rodent TCRβ variable gene locus is a non-functional locus, for example, the endogenous locus is inverted.

[0131] fertility In a different embodiment, the present invention relates to the recognition that the TCRβ locus contains genes that affect mouse fertility.

[0132] Mice in which the TCRβ gene locus was deleted, thereby inhibiting the expression of the host TCRβ chain, were observed to have lower fertility than mice containing an inverted TCRβ variable locus, which does not produce TCRβ chains but retains the DNA of that locus in the genome, with a portion of it oriented inverted relative to the wild type (see Example 1).

[0133] In particular, homozygous mice with duplicated TCRβ loci (human TRβVJ1D1C1J2J2C2 and mouse Trβv1J1D1) were able to breed more successfully than mice with the deletion.

[0134] Analysis of the TCRβ locus suggests that fertility is likely caused by deletions of one or more 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.

[0135] Individual knockout of PRSS58 does not cause fertility problems in mice (Park et. al, 2020 Biology of Reproduction, 103(2), 195-204), therefore, deletion of this gene is not considered to be the cause or sole cause of the observed fertility decline.

[0136] Accordingly, a further aspect of the present invention relates to a rodent comprising a modification at a TCRβ locus that knocks out the expression of a TCRβ chain, wherein the rodent, for example, the mouse, carries in its genome one or more of the serine protease genes from the TCRβV region of the locus of the rodent, for example, the mouse. The serine protease genes(s) may be retained at their endogenous locations within the locus (for example, if the modification is part of a TCRβ locus that does not affect the expression of the serine protease genes(s)), or they may be moved as part of an inversion of the TCRβV region, or they may be cleaved from their endogenous locations (for example, as part of a deletion in the TCRβV region, e.g., a deletion in the TCRβ locus) and inserted at an ectopic location within the genome.

[0137] In one embodiment, the host TCRβ constant region is not inverted.

[0138] Male rodents preferably retain fertility comparable to that of the wild type, which can optionally be measured by litter size.

[0139] The serine protease genes(s) used for fertility restoration as described herein may, alternatively, be obtained or available from the DNA of a host rodent strain or from other rodents containing genetic equivalents of these serine proteases.

[0140] Accordingly, the present invention relates to a rodent or rodent cell (preferably a mouse or mouse cell, e.g., ES cell) in which the host TCRβ locus is inactivated so that the rodent does not produce native rodent TCRβ chains, but retains wild-type or substantially wild-type levels of fertility. Such wild-type or substantially wild-type fertility can be achieved by either retaining the TCRβ locus DNA in the rodent genome in a form that does not express the TCRβ polypeptide (for example, having some or all of the variable region in a genomic direction inverted relative to any TCRβ constant region), thereby retaining the serine protease gene, or by deleting all or part of the locus sufficient to prevent native rodent TCRβ chain expression, and reintroducing one or more or all of the Prss58, Prss59, Prss3b, Try4, Try5, Try10, Prss3, Prss1, and Prss2 genes (preferably all of these genes if the TRβ locus is deleted) from the deleted rodent TCRβ locus into the rodent genome.

[0141] If a decrease in fertility is associated with a deletion or other inactivation of the TCRβ locus, it will become clear that the genes (or multiple genes) necessary for restoring fertility can be determined by replacing each deleted or inactivated serine protease gene individually or in limited combinations of genes. It may not be necessary to include all serine protease genes (Prss58, Prss59, Prss3b, Try4, Try5, Try10, Prss3, Prss1, and Prss2). Therefore, a person skilled in the art can determine which serine protease genes (Prss58, Prss59, Prss3b, Try4, Try5, Try10, Prss3, Prss1, and Prss2) are necessary for restoring fertility, for example, by practical evaluation of fertility based on the number of littermates.

[0142] The host TRB locus can also be effectively inactivated in other ways that preserve the expression of fertility genes (or multiple genes). For example, by inserting DNA such as human variable TCRβ DNA between the mouse TCRβ DNA and the constant region, thereby further moving the variable portion of the locus away from the constant region.

[0143] In one embodiment, as disclosed herein, a rodent TCRβ gene locus is deleted from the genome, thereby deleting the Prss58, Prss59, Prss3b, Try4, Try5, Try10, Prss3, Prss1, and Prss2 genes present within that locus. This deletion is then combined with the insertion of DNA encoding at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment to replace one or more or all of these genes in a manner suitable for restoring fertility in male rodents. The host TCRβ locus can be inactivated, and the human TCRβ variable region can be expressed as a fully human strand, preferably in combination with the human constant region.

[0144] The present invention also relates to a method for replacing a rodent TCRβ locus with a human TCRβ locus, comprising replacing the rodent TCRβ locus with a human TCRβ locus and then reintroducing one or more or all of the rodent serine protease genes, such as the Prss58 gene, Prss59 gene, Prss3b gene, Try4 gene, Try5 gene, Try10 gene, Prss3 gene, Prss1 gene, and Prss2 gene (optionally the same genes as those deleted from the rodent), into the rodent genome. In one embodiment of this method, the deleted rodent locus is the Vβ1 gene segment and / or the region between Vβ1 and Vβ2. In one embodiment of this method, the deleted rodent DNA is the genomic region between Vβ29 and Dβ1. The host TCRβ locus is inactivated.

[0145] The present invention also relates to a genetically modified male rodent having substantially wild-type fertility, wherein the rodent TCRβ locus is deleted from at least rodent Vβ1 to Dβ1, and the genome additionally contains one or more 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 or does not have a wild-type genome at the TCRβ locus.

[0146] The present invention also relates to a rodent or rodent cell that fully expresses human TCRα chains and / or human TCRβ chains as disclosed herein, wherein the rodent retains endogenous TCRα variable gene loci and / or endogenous rodent TCRβ variable gene loci, and the retained endogenous rodent TCRα variable gene loci are all non-functional loci, and / or the retained endogenous rodent TCRβ variable gene loci are all non-functional loci, and optionally, all or part of the retained endogenous rodent TCRα variable gene loci and / or TCRβ variable gene loci are inverted relative to their normal orientation in the rodent genome.

[0147] The present invention also relates to a fertile rodent or rodent cell in which at least the rodent TCRβ loci from rodent Vβ1 to Dβ1 are inverted within the rodent genome with respect to their native orientation within the rodent genome, and optionally further comprises inserting nucleic acids, such as DNA encoding all or part of TCRβ from a different species than the rodent, into the rodent genome, such as inserting human DNA encoding all or part of TCRβ.

[0148] The present invention also relates to a genetically modified rodent or rodent cell, wherein the rodent possesses an endogenous rodent TCRβ variable gene locus, and the endogenous rodent TCRβ variable gene locus is a non-functional locus resulting from the inversion of all or part of a gene locus in the genome.

[0149] Other embodiments of the present invention include the following: Rodents, such as mice and cells, as disclosed herein, in which the VDJ locus of TRB is retained in the genome and not deleted. Any method disclosed herein or a rodent or rodent cell in which the cell does not express the host rodent TCRβ chain. A genetically modified male rodent having substantially wild-type fertility, wherein all or part of the variable VDJ region of the rodent TCRβ locus is reversed within the rodent genome relative to its natural orientation within the rodent genome. Rodent cells, such as ES cells, that can develop into substantially wild-type fertile male rodents, wherein all or part of the variable VDJ region of the rodent genome TCRβ gene locus is reversed within the rodent genome relative to its natural orientation within the rodent genome.

[0150] Optionally, a rodent genome, such as a male rodent genome, may include any of the TCRα alleles, TCRβ alleles, MHC class I alleles, CD8α alleles, CD8β alleles, or β2 microglobulin alleles (or more) described herein.

[0151] The present invention also relates to a method for producing a fertile rodent, comprising the step of inverting rodent TCRβ in the rodent genome from at least rodent Vβ1 to Dβ1, and optionally further comprising inserting nucleic acids, such as DNA encoding all or part of TCRβ from a different species than the rodent, into the rodent genome, such as human DNA encoding all or part of TCRβ.

[0152] The present invention also relates to a method for producing fertile rodents, in any order: (i) Deleting at least rodent Vβ1 to Dβ1 of rodent TCRβ genomic DNA, including deleting one or more or all of the serine protease genes Prss58, Prss59, Prss3b, Try4, Try5, Try10, Prss3, Prss1, and Prss2, and (ii) Inserting one or more or all of the serine protease genes Prss58, Prss59, Prss3b, Try4, Try5, Try10, Prss3, Prss1, and Prss2 into the rodent genome. This includes methods.

[0153] In one embodiment, a genetically modified rodent or rodent cell is provided herein, wherein the rodent TCRβ locus is deleted from at least rodent Vβ1 to Dβ1, and 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, wherein the rodent is not a wild-type rodent.

[0154] In one embodiment, a modified rodent or rodent cell disclosed herein is provided, in which all or part of the endogenous rodent TCRβ gene locus is replaced with an unreorganized human TCRβ variable gene locus.

[0155] Human intronic DNA / intergenetic DNA In one embodiment, the inserted TCRβ locus contains the D and J gene segments of the human TCRβ, including introns and intergenic sequences. Unlike the problem observed in Moore et al. [Moore et al., Sci. Immunol. 6, eabj4026 (2021) 17 December 2021], rodent DNA was not required for the expression of the TCRβ DJC2 gene cluster in this invention. Moore et al., under the heading "gene humanisation in mice" on page 8 of 11, state that "an initial Trb humanization replaced coding, intergenic, and intronic regions with human sequences." These mice expressed a diverse TRBV repertoire, but only the use of the TRBDJC1 gene cluster was detected, and the use of the DJC2 gene cluster was not detected. To address this, we re-mouse-modified the intergenetic and intronic sequences in this region (starting from steps 5 and 6 in Figure S4), resulting in robust use of both DJC clusters (Figure S12B).

[0156] Figure 2 shows an example of a variable TRB locus containing human introns. Here, the human genome TCRB variable region genome TCRβ DJC2 DNA is inserted into the rodent genome, and the inserted DNA contains all of the human intron sequences between the D gene segment and the J gene segment.

[0157] In one embodiment, it is not necessary for all human TCRβ DJC2 D-intron and J-intron sequences to be present, and D-intron and J-intron sequences from some rodents may be used in combination with human intron sequences.

[0158] Example 2 confirms that inserting a human intron sequence located between the TCRβ DJC2 D gene segment and J gene segment into the mouse genome is sufficient for the expression of the human V gene segment, human D gene segment, and human J gene segment.

[0159] Accordingly, the present invention comprises a rodent or rodent cell having the TCRβ locus described herein, which comprises a complete human TCRβ DJC2 J intron and / or a complete human TCRβ DJC2 D intron. In one embodiment, the present invention comprises a rodent having a complete human TCRβ DJC2 D gene segment and a TCRβ J gene segment, which comprises a complete human TCRβ J intron and a complete human TCRβ D human intron. In one embodiment, the rodent TCRβ DJC2 J intron and / or rodent TCRβ DJC2 D intron are not present in the genome.

[0160] The rodents of the present invention may also preferably express a TCR receptor derived from a human gene segment located in an inserted human DJC2 gene cluster.

[0161] Accordingly, the present invention further includes a rodent or rodent cell having the TCRβ locus described herein, wherein one or more of the inter-gene sequences between the D1 gene segment and the J1 gene segment, between the J1 gene segment and the C1 gene segment, between the C1 gene segment and the D2 gene segment, and between the D2 gene segment and the J2 gene segment are entirely human TCRβ inter-gene sequences. In one embodiment, the rodent TCRβ inter-gene segment is not present between the D1 gene segment and the C2 gene segment in the genome, and preferably the insertion is a continuous human genomic insertion between human D1 and human C2.

[0162] Accordingly, the present invention further includes a rodent or rodent cell, optionally any of those described herein, comprising DNA that fully encodes the human TCRβ chain and having a genome comprising a human V gene segment, a human D gene segment, a human J gene segment, and a human C gene segment, wherein the rodent or rodent cell is capable of fully expressing the human TCRβ chain, and the rodent genome comprises a human intergene DNA sequence and / or human intron DNA sequence, preferably a human intergene DNA sequence and / or human intron DNA sequence that is naturally occurring with the human D gene segment and the human J gene segment, and optionally, all of the human DNA sequences between the human D1 gene segment and the human C2 gene segment are genomic human DNA. Therefore, in one embodiment, it is preferable that the rodent of the present invention, for example, a mouse, and the rodent and mouse contain one or more human TRB D introns and J introns in the mouse TRB DJC2 cluster, and that this DJC2 cluster contains only human introns.

[0163] Therefore, in one embodiment, it is preferable that the rodent of the present invention, for example, a mouse, and the rodent and mouse, do not contain any mouse TRB D introns and / or J introns in the mouse TRB DJC2 cluster.

[0164] Human CD8α and human CD8β The present invention further relates to a genetically modified rodent or rodent cell, for example, an ES cell or T cell, comprising a nucleic acid sequence encoding human CD8α polypeptide, wherein the nucleic acid sequence encoding human CD8α polypeptide is located at the endogenous rodent CD8α coreceptor locus, and / or a genetically modified rodent or rodent cell, for example, an ES cell or T cell, comprising a nucleic acid sequence encoding human CD8β polypeptide, wherein the nucleic acid sequence encoding human CD8β polypeptide is located at the endogenous rodent CD8β coreceptor locus.

[0165] CD8 interacts with MHC class I molecules. CD8 may be an α-chain and β-chain heterodimer, or an α-chain or β-chain homodimer. CD8 dimers on the T cell surface interact with MHC class I molecules on adjacent cells (Figure 8). CD8 binding increases the affinity of TCRs to MHC class I. The binding of CD8 to MHC class I molecules is species-specific. Therefore, the use of human CD8 is beneficial for studying T cell responses to antigens presented by human MHC class I.

[0166] CD8 expression is regulated by various enhancer elements located throughout the CD8 locus. DNase I hypersensitivity regions associated with regulatory factor binding have been identified at the CD8 locus (Hosert et al. (1997) J. Immunol. 158:4270-81). Enhancer elements have also been identified at the CD8 locus (Kioussis et al. (2002) Nature Rev. 2:909-919 and online errata, Ellmeier et al. (1998) Immunity 9:485-96).

[0167] In some embodiments, rodents or rodent cells contain or retain endogenous rodent promoters, enhancers, and / or regulatory elements that control the expression of human CD8α and / or CD8β. In some embodiments, nucleic acids encoding a whole human CD8α polypeptide or a whole human CD8β polypeptide are provided operably ligated in a rodent or rodent cell to one or more endogenous rodent regulatory sequences, such as rodent promoters, enhancers, or other regulatory sequences.

[0168] In one embodiment, human CD8α and / or human CD8β are operably linked to a rodent promoter.

[0169] In one embodiment, the rodents of the present invention do not express the CD8 protein on immune cells other than those that normally express CD8, for example, rodents express CD8 on B cells or CD4 + CD8 is not expressed on SP T cells.

[0170] Rodents or rodent cells, such as rodent T cells, do not express functional endogenous mouse CD8α polypeptide and / or mouse CD8β polypeptide from their endogenous CD8 locus.

[0171] In one embodiment, a rodent or rodent cell expressing a functional CD8αβ protein from the CD8 locus described herein displays human proteins on its cell surface. In one embodiment, the rodent expresses human CD8 protein on its cell surface in the same or substantially the same cellular distribution as observed in humans. In one embodiment, the human CD8 protein expressed in the rodent can interact with MHC class I proteins, such as human MHC class I, expressed on the surface of a second cell.

[0172] In one embodiment, the endogenous rodent CD8α coreceptor loci and CD8β coreceptor loci contain inserted human DNA encoding CD8α and CD8β nucleic acids, which are operably associated with at least one mouse enhancer, optionally two or three mouse enhancers, and optionally one or more human enhancers, so that the CD8 loci (expressing both CD8α and CD8β) are under the control of regulatory elements in both humans and rodents.

[0173] In a preferred embodiment, the rodent is a mouse, and there are four mouse enhancers.

[0174] In one embodiment, a rodent or rodent cell may be heterozygous for the nucleotide sequence encoding the human CD8α polypeptide. In another embodiment, a rodent or rodent cell may be homozygous for the nucleotide sequence encoding the human CD8α polypeptide.

[0175] In one embodiment, a rodent or rodent cell may be heterozygous for the nucleotide sequence encoding the human CD8β polypeptide. In another embodiment, a rodent or rodent cell may be homozygous for the nucleotide sequence encoding the human CD8β polypeptide.

[0176] In one embodiment, a rodent or rodent cell contains insertions of human CD8α (exons 1 to 4) and human CD8β (exons 1 to 3).

[0177] In one embodiment, the inserted human gene locus exhibits appropriate spatial and temporal protein expression, aiding in T cell development and selection.

[0178] Chimeric CD8α and Chimeric CD8β In an alternative embodiment, the present invention further relates to a genetically modified rodent or rodent cell, e.g., an ES cell or 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 the endogenous rodent CD8α coreceptor locus, and / or a genetically modified rodent or rodent cell, e.g., an ES cell or 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 the endogenous rodent CD8β coreceptor locus.

[0179] The chimeric CD8α polypeptide is appropriately encoded by genomic DNA containing part or all of human exon 1, all of human exon 2, all of human exon 3, and part of human exon 4, wherein, in one embodiment, human DNA derived from human exon 4 is fused to part of mouse exon 3. Preferably, the rodent genome further contains human genomic DNA between exon 1 and exon 2, between exon 2 and exon 3, and between exon 3 and exon 4.

[0180] In one embodiment, a fusion is performed between human exon 4 and mouse exon 3 such that the extracellular portion of the expressed polypeptide is human and the intracellular portion of the polypeptide is mouse. The fusion is appropriately performed at the connection region located precisely at the N-terminus of the transmembrane domain.

[0181] In one embodiment, to avoid introducing structural changes, fusion is performed between the mouse and the human at a site where the amino acid sequences (preferably the amino acid sequence and DNA sequence) are identical.

[0182] The CD8A DNA fusion occurs in the following regions of the exons in humans and mice: Human exon 4, bases 18-32, Mouse exon 3, bases 18-32, It will be carried out appropriately in that context.

[0183] The amino acid sequence for both species in this region is DFACD (i.e., aspartic acid, phenylalanine, alanine, cysteine, aspartic acid).

[0184] The chimeric CD8β polypeptide is appropriately encoded by genomic DNA comprising some or all of human exon 1, all of human exon 2, and part of human exon 3, wherein in one embodiment, human DNA from exon 3 is fused to part of mouse exon 3, and preferably, the rodent genome further comprises human genomic DNA between exon 1 and exon 2, and between exon 2 and exon 3.

[0185] In one embodiment, human and mouse exons are fused such that the extracellular portion of the expressed CD8β polypeptide is human, and the intracellular portion of the polypeptide is mouse.

[0186] In one embodiment, the human coding sequence is included in the genome, but the 5' untranslated region is not.

[0187] Figures 3 and 4 show examples of appropriate chimeric CD8 polypeptides.

[0188] Chimeric human-rodent CD8 proteins can bind completely to human MHC class I expressed by host cells, for example, human MHC class I, enabling T cells expressing chimeric CD8 to interact with antigen-presenting cells possessing MHC class I, thereby facilitating antigen presentation to T cells and T cell activation. The ability of chimeric human-rodent polypeptides to bind to human MHC class I may be achieved by retaining regions of human CD8 protein expressed on the T cell surface, and these interaction sites with human MHC class I are well known. See, for example, Front. Immunol., July 22, 2013, Sec. T Cell Biology, Vol. 4 - 2013. T cell activation assays are also well known and can be used to determine T cell activation, thereby confirming the appropriate chimeric structure.

[0189] CD8 expression is regulated by various enhancer elements located throughout the CD8 locus. DNase I hypersensitivity regions associated with regulatory factor binding have been identified at the CD8 locus (Hosert et al. (1997) J. Immunol. 158:4270-81). Enhancer elements have also been identified at the CD8 locus (Kioussis et al. (2002) Nature Rev. 2:909-919 and online errata, Ellmeier et al. (1998) Immunity 9:485-96).

[0190] In some embodiments, a rodent or rodent cell contains or retains endogenous rodent promoters, enhancers, and / or regulatory elements that control the expression of chimeric CD8α and / or CD8β. In some embodiments, a nucleic acid encoding a chimeric CD8α polypeptide or a chimeric CD8β polypeptide is provided operably ligated in a rodent or rodent cell to one or more endogenous rodent regulatory sequences, such as rodent promoters, enhancers, or other regulatory sequences.

[0191] In one embodiment, chimeric CD8α and / or chimeric CD8β are operably linked to a rodent promoter.

[0192] In one embodiment, chimeric CD8α and / or chimeric CD8β are operably linked to a human promoter.

[0193] In one embodiment, the rodents of the present invention do not express the CD8 protein on immune cells other than those that normally express CD8, for example, rodents express CD8 on B cells or CD4 + CD8 is not expressed on SP T cells.

[0194] Rodents or rodent cells, such as rodent T cells, do not express functional endogenous mouse CD8α polypeptide and / or CD8β polypeptide from their endogenous CD8 locus.

[0195] In one embodiment, a rodent or rodent cell expressing a functional CD8αβ protein from the CD8 locus described herein displays the chimeric protein on its cell surface. In one embodiment, 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 one embodiment, the chimeric CD8 protein expressed in the rodent can interact with MHC class I proteins, such as human MHC class I, expressed on the surface of a second cell.

[0196] In one embodiment, the endogenous rodent CD8α coreceptor loci and CD8β coreceptor loci contain inserted human DNA encoding CD8α polypeptides and CD8β polypeptides that are operably associated with at least one mouse enhancer, optionally two or three mouse enhancers, and optionally one or more human enhancers, thereby placing the CD8 locus (expressing both CD8α and CD8β) under the control of regulatory elements in both humans and rodents.

[0197] In a preferred embodiment, the rodent is a mouse, and there are four mouse enhancers.

[0198] In one embodiment, a rodent or rodent cell may be heterozygous for the nucleotide sequence encoding the chimeric CD8α polypeptide. In another embodiment, a rodent or rodent cell may be homozygous for the nucleotide sequence encoding the chimeric CD8α polypeptide.

[0199] In one embodiment, a rodent or rodent cell may be heterozygous for the nucleotide sequence encoding the chimeric CD8β polypeptide. In another embodiment, a rodent or rodent cell may be homozygous for the nucleotide sequence encoding the chimeric CD8β polypeptide.

[0200] In one embodiment, this gene locus exhibits appropriate spatial and temporal protein expression, aiding in T cell development and selection.

[0201] Patent Document 2, derived from International Publication No. 2014 / 130671, discloses examples of chimeric MHC class I molecules and chimeric CD8 polypeptides suitable for use in the present invention.

[0202] Rodent CD8α and Rodent CD8β In another embodiment of the present invention, the CD8α and CD8β genes of a host rodent can be used. Preferably, these are under the normal regulatory control of the host. In this embodiment, the rodent is used together with a chimeric MHC class I genome and can interact with the rodent CD8 protein via a rodent amino acid subdomain present in the MHC class I polypeptide. The chimeric MHC class I polypeptide appropriately has a rodent α3 domain, and the present invention also relates to the nucleic acid encoding this chimeric polypeptide.

[0203] Alternatively, CD8 from a host rodent is combined with MHC class I having a human α3 domain. Each rodent may optionally contain a homozygous endogenous CD8α locus and / or CD8β locus, 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 interaction compared to the binding of species-matched polypeptides (e.g., both human or both mouse). Such rodents may exhibit a weaker immune response and generate fewer antigen-specific T cells against the target antigen compared to rodents where species matching is observed between the α3 domains of CD8 and MHC class I (e.g., both human or both mouse). However, antigen-specific T cells obtained through such immunization can bind to the target antigen with high affinity. A decrease in binding affinity between coreceptors and MHC may have an overall effect of selecting higher affinity TCRs in rodents, as a higher affinity TCR:MHC interaction is required to meet the threshold for antigen-dependent TCR signaling within T cells. This could be advantageous in situations where rodents are used to obtain T cell receptors with high affinity for a particular antigen, for example, in producing soluble TCR molecules that act by binding to pMHC in the absence of coreceptor engagement, which could be used as therapeutic agents.

[0204] MHC Class I The present invention further relates to a genetically modified rodent or rodent cell, such as an ES cell or antigen-presenting cell, comprising 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.

[0205] Such rodents enable the human CD8 protein expressed on the surface of T cells of the rodents disclosed herein to interact with human MHC class I expressed on the surface of a second cell in the rodent, such as an antigen-presenting cell.

[0206] Human MHCI polypeptides 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 molecule present in the human population.

[0207] The genome of a rodent may contain two or more nucleic acids encoding human MHC class I. For example, DNA encoding human MHC class I can be inserted into both the H2D and H2K loci of a rodent (e.g., a mouse). Human MHC may differ at each locus. In one embodiment, considering the possibility of heterozygosity at each of these loci, the rodent genome may contain two, three, or four different human MHC class I molecules.

[0208] In some lineages of rodents, there are three rodent MHC class I loci, so a rodent genome may contain one, two, three, four, five, or even six different MHC class I molecules, which may be human molecules or humanized molecules, and may be HLA-A, HLA-B, and HLA-C.

[0209] In some embodiments, rodents or rodent cells contain or retain endogenous rodent promoters, enhancers, and / or regulatory elements that control the expression of human MHC class I.

[0210] In some embodiments, nucleic acids that fully encode human MHC class I polypeptides are provided operably ligated to one or more endogenous rodent regulatory sequences, such as rodent promoters, enhancers, or other regulatory sequences, in a rodent or rodent cell. In one embodiment, the rodent is a mouse, and the human MHC class I polypeptide is operably ligated to a mouse promoter and mouse enhancer located at the 5' position of the human MHC class I polypeptide.

[0211] In one embodiment, rodents may be heterozygous for nucleotide sequences encoding human MHC class I polypeptides. In another embodiment, rodents may be homozygous for nucleotide sequences encoding human MHC class I polypeptides.

[0212] In one embodiment, the inserted human DNA, for example, HLA-A02 DNA, is inserted into the endogenous H2-D locus. The endogenous H2-D locus may be deleted in whole or in part, or inactivated by other means. In one embodiment, the rodent H2-K1 gene is also deleted or inactivated.

[0213] In one embodiment, the inserted human DNA, for example, HLA-A02 DNA, is inserted into the endogenous H2-K locus. The endogenous H2-K locus may be deleted in whole or in part, or inactivated by other means. In one embodiment, the rodent H2-D gene is also deleted or inactivated.

[0214] Preferably, both the H2-D and H2-K genes in the rodent are deleted or inactivated by other means, resulting in the elimination of mouse class I expression, and only the desired human or humanized MHC class 1 molecule(s) are expressed in the rodent.

[0215] Preferably, all rodent genes encoding MHC class I are deleted or inactivated by other means, resulting in the elimination of rodent class I expression.

[0216] In one embodiment, the rodent includes an insertion of a nucleic acid encoding the complete coding sequence of human HLA-A02. In one embodiment, the inserted human DNA includes or comprises human DNA containing nucleotide 6:29,942,554~29,945,450 (GRCh38).

[0217] In one embodiment, human HLA-A02 is inserted into the endogenous H2-D locus.

[0218] In one embodiment, the H2-K1 gene in rodents is deleted or inactivated.

[0219] In one embodiment, two or more rodent HLA loci may be inactivated. For example, a rodent may include an insertion of human DNA as described herein at one rodent MHC class I locus (where endogenous expression is inactivated), in which case the inactivation (e.g., knockout) of a second rodent MHC class I locus may be included without replacement by human MHC class I at the second locus. For example, the rodent H2-D locus may be replaced with nucleic acid encoding a human HLA class I gene, and the rodent H2-K gene may be deleted at the H2-K locus without any human insertion.

[0220] In one embodiment, the inserted human gene locus exhibits appropriate spatial and temporal protein expression.

[0221] Chimera MHC Class I The present invention further relates to a genetically modified rodent or rodent cell, such as an ES cell or antigen-presenting cell, 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.

[0222] Figure 6 shows an example of a suitable chimeric MHC class 1 polypeptide.

[0223] Chimeric MHC class I proteins bind to rodent CD8 expressed by host cells, enabling T cells expressing rodent CD8 to interact with antigen-presenting cells containing chimeric MHC class I, thereby facilitating antigen presentation to T cells and T cell activation. The ability of rodent CD8 polypeptides to bind to chimeric rodent-human MHC class I may be achieved by retaining the rodent region of the rodent MHC class I protein that interacts with CD8, and these interaction sites between rodent CD8 and rodent MHC class I are well known. See, for example, Front. Immunol., July 22, 2013, Sec. T Cell Biology, Vol. 4 - 2013. T cell activation assays are also well known and can be used to determine T cell activation, thereby confirming the appropriate chimeric structure.

[0224] Patent Document 2, derived from International Publication No. 2014 / 130671, discloses examples of chimeric MHC class I molecules and chimeric CD8 polypeptides suitable for use in the present invention.

[0225] Therefore, the MHC class I locus may express a chimeric (human-rodent) protein, which not only allows MHC class I to interact with the human TCR but also allows it to retain its binding to rodent CD8.

[0226] In particular, when the CD8 is the endogenous rodent CD8, MHC class I is preferably of human origin, except for the presence of the rodent α3 domain, which is the interaction site with CD8. Preferably, MHCI has a human α1 domain and a human α2 domain, which interact with the human TCR domain, so that the MHC:TCR interface is entirely of human origin.

[0227] Thus, in one aspect, the rodent comprises the insertion of a nucleic acid encoding the complete coding sequences of exons 1 to 3 and exons 5 to 8 of human HLA - A02 - 01, wherein exon 4 is the native rodent exon 4.

[0228] In one aspect, the inserted human DNA comprises, or consists of, human DNA containing nucleotides 6:29,942,554 - 29,945,450 (GRCh38), with the exception of human exon 4, which is instead provided as the rodent exon 4 within the genome.

[0229] The entire chimeric MHC class I may be inserted into the genome, or the rodent exon 4 existing within the genome may be retained, and either side of the genome may be modified to introduce the desired human nucleic acid.

[0230] In one aspect, the regions between human exons 1 - exon 2, exon 2 - exon 3, exon 3 - exon 4, exon 4 - exon 5, exon 5 - exon 6, exon 6 - exon 7, and exon 7 - exon 8 are all of human origin.

[0231] In such rodents, the rodent CD8 protein expressed on the surface of the rodent T cells disclosed herein is capable of interacting with the chimeric (rodent - human) MHC class I expressed on the surface of a second cell in the rodent, such as an antigen - presenting cell.

[0232] Human MHCI polypeptides 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 molecule present in the human population.

[0233] The genome of a rodent may contain two or more nucleic acids encoding chimeric MHC class I. For example, DNA encoding chimeric MHC class I may be inserted into the H2D and H2K loci of a rodent (e.g., a mouse). In one embodiment, considering the possibility of heterozygosity at each of these loci, the rodent genome may contain two, three, or four different human MHC class I molecules.

[0234] In some lineages of rodents, there are three rodent MHC class I loci, so a rodent genome may contain one, two, three, four, five, or even six different MHC class I molecules, which may be chimeric molecules and may be HLA-A, HLA-B, and HLA-C. In some embodiments, rodents or rodent cells contain or retain endogenous rodent promoters, enhancers, and / or regulatory elements that control the expression of human MHC class I.

[0235] In some embodiments, nucleic acids that fully encode human MHC class I polypeptides are provided operably ligated to one or more endogenous rodent regulatory sequences, such as rodent promoters, enhancers, or other regulatory sequences, in a rodent or rodent cell. In one embodiment, the rodent is a mouse, and the human MHC class I polypeptide is operably ligated to a mouse promoter and mouse enhancer located at the 5' position of the human MHC class I polypeptide.

[0236] In one embodiment, rodents may be heterozygous for nucleotide sequences encoding human MHC class I polypeptides. In another embodiment, rodents may be homozygous for nucleotide sequences encoding human MHC class I polypeptides.

[0237] In one embodiment, the inserted human DNA, for example, HLA-A02 DNA, is inserted into the endogenous H2-D locus. The endogenous H2-D locus may be deleted in whole or in part, or inactivated by other means. In one embodiment, the rodent H2-K1 gene is also deleted or inactivated.

[0238] In one embodiment, the inserted human DNA, for example, HLA-A02 DNA, is inserted into the endogenous H2-K locus. The endogenous H2-K locus may be deleted in whole or in part, or inactivated by other means. In one embodiment, the rodent H2-D gene is also deleted or inactivated.

[0239] Preferably, both the H2-D and H2-K genes in the rodent are deleted or inactivated by other means, resulting in the elimination of mouse class I expression, and only the desired human or humanized MHC class 1 molecule is expressed in the rodent.

[0240] Preferably, all rodent genes encoding MHC class I are deleted or inactivated by other means, resulting in the elimination of rodent class I expression.

[0241] In one embodiment, two or more rodent HLA loci may be inactivated. For example, a rodent may include an insertion of human DNA as described herein at one rodent MHC class I locus (where endogenous expression is inactivated), in which case the inactivation (e.g., knockout) of a second rodent MHC class I locus may be included without replacement by human MHC class I at the second locus. For example, the rodent H2-D locus may be replaced with nucleic acid encoding a human HLA class I gene, and the rodent H2-K gene may be deleted at the H2-K locus without any human insertion.

[0242] In one embodiment, the inserted human gene locus exhibits appropriate spatial and temporal protein expression.

[0243] β2M The present invention relates to a genetically modified rodent or rodent cell, such as an ES cell or antigen-presenting cell, comprising a nucleic acid sequence encoding a human β2-microglobulin polypeptide, which is located at the endogenous rodent β2-microglobulin locus.

[0244] In one embodiment, all or part of the nucleic acid encoding endogenous rodent β2 microglobulin is deleted.

[0245] In one embodiment, a rodent or rodent cell contains an insertion from the ATG of exon 1 to the stop codon of exon 3. In one embodiment, the inserted human DNA contains nucleotides 15:44,711,547~44,716,342 (GRCh38). In one embodiment, the insertion is performed in the mouse genome, and 2:121,978,219~121,982,140 (GRCm39 C57BL / 6J) is deleted. In another embodiment, the insertion is performed in the mouse genome, and the region between 2:121,978,218 and 121,982,142 (GRCm39 C57BL / 6J) is deleted, but these endpoints are excluded (i.e., the deleted region starts 1 bp inside the bases on both sides and ends 1 bp inside).

[0246] In some embodiments, rodents or rodent cells contain or retain endogenous rodent promoters and / or regulatory elements that control the expression of human β2M.

[0247] In some embodiments, a nucleic acid that fully encodes human β2M is provided operably ligated to one or more endogenous rodent regulatory sequences, such as rodent promoters, enhancers, or other regulatory sequences, in a rodent or rodent cell. In one embodiment, upstream rodent enhancers and promoters are operably ligated to the nucleic acid encoding β2M at an endogenous locus. Preferably, the enhancers and promoters are promoters and enhancers that are naturally present in naturally occurring locations within the genome in a rodent or rodent cell and have not been removed or replaced.

[0248] Preferably, the rodent is a mouse, and the enhancer is a mouse enhancer located at 5' of the gene and promoter, sharing MHC class I with cis- and trans-activating factors, thereby coordinating expression.

[0249] In one embodiment, a rodent or rodent cell may be heterozygous for the nucleotide sequence encoding the human β2M polypeptide.

[0250] In one embodiment, rodents may be homozygous for the nucleotide sequence encoding the human β2M polypeptide.

[0251] In one embodiment, the inserted human gene locus exhibits appropriate spatial and temporal protein expression.

[0252] Combinations of human gene loci As used herein, rodents and cells are disclosed that contain nucleic acids encoding fully human polypeptides for one or more or all of the TCRα locus, TCRβ locus, MHC class I locus, CD8α locus, CD8β locus, and β2M locus, wherein these are inserted into the endogenous rodent genomic loci for TCRα, TCRβ, MHC class I, CD8α, CD8β, and β2M, respectively.

[0253] Preferred rodents contain insertions in a number of these rodent loci, where preferred combinations include human DNA inserted into the TCRα locus, TCRβ locus, MHC class I locus, CD8α locus, and CD8β locus, preferably including human DNA inserted into all of the TCRα locus, TCRβ locus, MHC class I locus, CD8α locus, CD8β locus, and B2M locus. In this way, in rodents, antigens for human TCR and human co-receptor CD8 can be presented by human MHC class I polypeptides.

[0254] However, it will be understood that the rodents and cells of the present invention may contain only some, but not all, of the human loci described herein, for example, only one, two, three, four, five, or six rodent loci that have been modified to express human polypeptides (which may be more). For example, a rodent may contain DNA encoding human MHC class I, which may be used in combination with rodent polypeptides expressed at other loci listed above, or in combination with chimeric rodent-human polypeptides expressed at other loci listed above, or in combination with both. Therefore, any one, two, three, four, five, six, or seven of the following loci (TCRα, TCRβ, MHC class I (H2K and / or H2D), CD8α, CD8β, and β2M) may be entirely human and may be used in combination with rodent and / or chimeric (e.g., rodent-human chimeric) forms of polypeptides from other loci disclosed herein.

[0255] Rodents and cells having at least human TCRα and human TCRβ insertions are preferred so that they can completely produce human TCR in rodents.

[0256] As a first example, a rodent may contain nucleic acids that express chimeric rodent-human TCRα and / or TCRβ in combination with human MHC class I. In other examples, a rodent may contain nucleic acids that express chimeric rodent-human TCRα and / or TCRβ in combination with human CD8α and / or human CD8β. A rodent may contain nucleic acids that express chimeric rodent-human TCRα and / or TCRβ in combination with human CD8α and / or CD8β and human β2M. A rodent may contain nucleic acids that express chimeric rodent-human MHC class I in combination with human TCRα and / or TCRβ. A rodent may contain nucleic acids that express chimeric rodent-human MHC class I in combination with human CD8α and / or CD8β. Rodents may contain nucleic acids that express chimeric rodent-human MHC class I in combination with human CD8α and / or CD8β and β2M. These are non-limiting examples, and other combinations of entirely human loci and rodent loci and / or chimeric loci are also possible.

[0257] Examples of chimeric TCRα and TCRβ loci, MHC class I loci, and CD8α and CD8β loci are shown, for example, in International Publication Nos. 2014 / 130671, 2014 / 130667, and 2016 / 085889, and specific examples of these loci and the specific polypeptides produced therefrom, by reference, form part herein as polypeptides and modified rodent loci that can be used in the present invention in combination with one or more fully human loci / polypeptides described herein.

[0258] In one embodiment, a rodent containing multiple human insertions at different loci is obtained by mating, preferably in a manner that homozygous for each of the loci containing the inserted human DNA. Thus, the rodent may be homozygous for one, two, three, four, five, six, or seven of the TCRα, TCRβ, MHC class I (H2D and H2K) loci, CD8α, CD8β, and B2M loci (depending on the number of inserted human loci).

[0259] In one embodiment, a rodent is fertile, for example, as evaluated by the litter size. The litter size is preferably at least 50%, 60%, 70%, 80%, 90%, preferably at least 95%, and preferably 100% of the litter size of a wild-type rodent.

[0260] Preferably, the genetically modified rodent or rodent cell of the present invention is (i) an unreorganized T cell receptor (TCR)α variable gene locus comprising at least one human Vα segment and at least one human Jα segment, wherein the TCRα variable gene is operably linked to the human TCRα constant gene sequence, (ii) an unreorganized TCRβ variable gene locus comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment, wherein the unreorganized TCRβ variable gene is operably linked to a human TCRβ constant gene sequence, Including, here, Unreorganized human T cell variable region gene segments can be rearranged to form genes encoding human T cell receptor variable domains, the unreorganized TCRα variable gene locus is located at the endogenous rodent TCRα gene locus, and the unreorganized TCRβ variable gene locus is located at the endogenous rodent TCRβ gene locus. (iii) A nucleic acid sequence encoding a human CD8α polypeptide located at the endogenous rodent CD8α coreceptor gene locus, (iv) A nucleic acid sequence encoding a human CD8β polypeptide, which is located at the endogenous rodent CD8β coreceptor locus, (v) 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 the endogenous rodent MHC class I gene locus, (vi) If present, a nucleic acid sequence encoding a human β2-microglobulin polypeptide located at the endogenous rodent β2-microglobulin locus, Includes.

[0261] Functional aspects of inserted human DNA In one embodiment, a rodent expresses human CD8 protein, including CD8α polypeptide and CD8β polypeptide, and human MHC class I polypeptide, and human CD8 and human MHC class I can interact within the rodent.

[0262] In one embodiment, a rodent expresses human TCR, which includes TCRα polypeptide and TCRβ polypeptide, and human MHC class I polypeptide, and the human TCR and human MHC class I can interact within the rodent.

[0263] In one embodiment, a rodent or rodent cell, such as a T cell, expresses a human CD8 protein comprising CD8α polypeptide and CD8β polypeptide, and a human TCR comprising TCRα polypeptide and TCRβ polypeptide, and both human CD8 and human TCR can bind to MHC class I in rodents.

[0264] In one embodiment, a rodent expresses a human CD8 protein containing CD8α polypeptide and CD8β polypeptide, and also expresses a human MHC class I polypeptide, and also expresses a human TCR containing TCRα polypeptide and TCRβ polypeptide, and the human TCR, human MHC class I, and human CD8 interact in the rodent, making it possible to present an antigen to the TCR via MHC class I with the help of CD8.

[0265] In one embodiment, rodents express a repertoire of TCRs. In one embodiment, rodents express a repertoire of both TCRα polypeptides and TCRβ polypeptides.

[0266] In one embodiment, the human variable region of a human TCR protein can interact with various proteins on the surface of the same cell or another cell. In one embodiment, the human variable region of the human TCR interacts with an MHC protein (e.g., MHC class I) that presents an antigen on the surface of a second cell, such as an antigen-presenting cell (APC). In some embodiments, the MHC class I protein is a non-human (e.g., rodent, e.g., mouse or rat) protein. In other embodiments, the MHC class I protein is a human protein. In one embodiment, the second cell, e.g., the APC, is an endogenous non-human cell expressing a human MHC molecule. In different embodiments, the second cell is a human cell expressing a human MHC molecule.

[0267] In one embodiment, the human variable region of a human TCR protein can interact with endogenous rodent MHC class II molecules on the surface of another cell, such as an antigen-presenting cell.

[0268] In one embodiment, the human variable region of a human TCR protein can interact with non-polymorphic MHC molecules, such as human or rodent CD1 or MR1.

[0269] In one embodiment, the human TCR receptor can interact with endogenous rodent molecules such as signaling molecules or portions thereof. In one embodiment, human TCR can interact in vivo with the rodent CD3 protein complex and CD3 zeta (ζ chain) in rodents, potentially resulting in T cell activation. Therefore, the rodent genome may be wild-type with respect to CD3 polypeptides, e.g., CD3ε, CD3γ, CD3δ, and / or CD3ζ. This specification exemplifies transgenic rodents in which the CD3 protein complex is endogenous and not humanized, demonstrating that human TCRαβ functions with rodent CD3 to successfully induce a T cell immune response to an antigen (see Example 6). Alternatively, transgenic mice in which one or more CD3 polypeptides or regions thereof are humanized can be described, and such human CD3 transgenes can be combined with the transgenic locus of the present invention. For example, International Publication No. 2016 / 085889 (Regeneron) described the targeted insertion of the CD3ε, CD3γ, and CD3δ EC domains into endogenous loci via homologous recombination in mice, generating transgenic mice that express a chimeric CD3εδγ polypeptide in which the extracellular domain is human and the cytoplasmic domain is endogenous mouse. Ueda et al. (Scientific Reports 7:45839 2017) described the generation of transgenic mice that fully express human CD3εγδ polypeptide by replacing mouse CD3ε, mouse CD3γ, and mouse CD3δ with full-length human counterparts in C57BL / 6N ESCs using cre-lox recombination. Elsewhere, individual epitopes of the CD3 complex were humanized. For example, Crespo et al. (PLoS One 2021) reported a mouse in which the mouse CD3ε exon (mouse exon 5) was replaced with a humanized CD3ε exon (human exon 6 with one substitution) that contained an epitope for the anti-CD3 antibody OKT3.The humanized exon encodes 11 additional amino acids compared to mouse exon 5, some of which form a binding epitope for OKT3. In addition to other human transgenes described herein, humanization of CD3 (in whole or in part) gives mice an immune system that is even closer to the human immune system, which is useful as an experimental model. Advantages of human CD3 or humanized CD3 in animals include the ability to target the TCR-CD3 complex in mice using antibodies against human CD3, for example, to evaluate the effectiveness of candidate therapeutic molecules. However, it is noteworthy that a functional repertoire of T cells and T cell receptors can be adequately generated in rodents with endogenous CD3 in which the CD3 complex is not humanized.

[0270] CD3 polypeptides (particularly CD3ε, CD3γ, and CD3δ), when combined with human TCR polypeptides, especially the TCRα constant domain, can signal and activate T cells with lower efficiency compared to the corresponding endogenous mouse TCR polypeptides. This may be due to the lower effectiveness of the interaction between CD3δ (endogenous rodent CD3δ, or even human CD3δ) and human TCR Cα compared to endogenous rodent TCR Cα. Consistent with this, it has been previously reported that "mouseization" of the human TCRα constant region enhances TCR expression by supporting preferential pairing with CD3 in human T cells (Sommermeyer & Uckert, J. Immunol. 184:6223-6231 2010).

[0271] In some embodiments, it is preferable that there is no functional expression of endogenous TCRα polypeptide in rodents. This avoids the expression of endogenous TRAC, otherwise endogenous TRAC may dominate binding to CD3 (endogenous CD3 or human CD3). In rodents with heterozygous human TCRα (one allele being human and the other wild-type), human and endogenous TCRα polypeptides compete for binding to CD3 (e.g., endogenous CD3), and T cells with TCRs containing endogenous TCRα may outcompete human TCRα-containing T cells due to the former's more effective TCR signaling. This is avoided by homozygosity of human TRAC.

[0272] Pairing human TCRs with CD3 may result in less efficient TCR signaling and T cell activation compared to TCRs containing the mouse TCRα constant region. This, perhaps counterintuitively, could be an advantage when using rodents as a source of high-affinity human TCRs. An individual's TCR repertoire is formed during T cell development by positive and negative selection for the individual's MHC alleles and autoantigens, where certain levels of activation signaling are required for T cell survival (positive selection) or elimination (negative selection). Slightly less efficient activation signaling may require higher affinity to reach the same level of activation required for positive and negative selection, thus leading to an upward shift in the affinity distribution of the repertoire. Therefore, rodents with fully human TCRs containing homozygous loci expressing human TCRα are indeed well-suited to generating high-affinity TCRs for therapeutic use, with or without humanized CD3. Therefore, rodents may express rodent CD3 (e.g., rodent CD3δ, optionally rodent CD3ε, CD3γ, and CD3δ, and optionally rodent CD3ζ). Optionally, the rodent CD3 locus is wild-type. Alternatively, rodents may contain human CD3 transgenes (e.g., CD3εδγ, preferably in single copy number or low copy number insertions).

[0273] In one embodiment, the rodent is a mouse having the following control region: TCRα: The enhancer located at 3' of the constant region (TRAC) is from mouse. TCRδ: The enhancer located at 5' of the constant region (TRDC) is human (e.g., from within a human BAC clone). The TCRβ mouse enhancer is located at 3' of the mouse constant region (TRBC) in the mouse genome and originates from the region at 5' of mouse Vb31, but alternatively, this can be replaced by an equivalent human enhancer. The enhancer at 5' of MHC class I:H2-D1 is from the mouse. b2 microglobulin: The enhancer and promoter at the 5' position of this gene are from a mouse. CD8a and CD8b: There are four enhancer regions (shown in Figure 3) that belong to the mouse: E8iv, E8iii, E8ii, and E8i.

[0274] How to create a rodent Various methods for producing rodents of the present invention are described herein. These include the following methods:

[0275] A method for modifying a rodent to express human TCRα polypeptide and / or TCRβ polypeptide, comprising (i) inserting a nucleotide sequence encoding human TCRα polypeptide into the endogenous TCRα gene locus of the rodent, and / or (ii) inserting a nucleotide sequence encoding human TCRβ polypeptide into the endogenous TCRβ gene locus of the rodent, wherein the rodent expresses human TCR comprising human α polypeptide (or more) and / or human β polypeptide (or more).

[0276] A method for modifying a rodent to express human CD8α polypeptide and / or human CD8β polypeptide, comprising (i) inserting a nucleotide sequence encoding human CD8α polypeptide into the endogenous CD8α locus of the rodent, and / or (ii) inserting a nucleotide sequence encoding human CD8β polypeptide into the endogenous CD8β locus of the rodent, wherein the rodent expresses human CD8α polypeptide (or more) and / or human CD8β polypeptide (or more).

[0277] A method for modifying a rodent to express a human MHC class I polypeptide, comprising (i) inserting a human MHC class I polypeptide into an endogenous rodent MHC class I gene locus, wherein the rodent expresses a human MHC class I polypeptide from the rodent MHC class I gene locus.

[0278] A method for modifying a rodent to express human β2-microglobulin polypeptide, comprising (i) inserting human β2-microglobulin polypeptide into the endogenous rodent β2-microglobulin gene locus, wherein the rodent expresses human β2-microglobulin polypeptide from the rodent β2-microglobulin gene locus.

[0279] A method for creating genetically modified rodents expressing human T cell receptor, human MHC1, and human CD8, comprising: inserting an unreorganized human TCRα variable gene locus into the endogenous rodent TCRα variable gene locus, comprising at least one human Vα gene segment and at least one human Jα gene segment operably linked to the human TCRα constant region; and inserting at least one human vβ gene segment and at least one human Dα gene segment operably linked to the human TCRβ constant region into the endogenous rodent TCRβ variable gene locus. A method comprising: inserting an unreorganized human TCRβ variable gene locus containing a β gene segment and at least one human Jβ gene segment; inserting a nucleic acid sequence encoding a human CD8α polypeptide into the endogenous rodent CD8α gene locus; inserting a nucleic acid sequence encoding a human CD8β polypeptide into the endogenous rodent CD8β gene locus; and optionally inserting a nucleic acid sequence encoding human or chimeric human-rodent β2 microglobulin into the endogenous rodent β2 microglobulin gene locus.

[0280] Human DNA insertion may or may not be accompanied by DNA deletion at the insertion site. If endogenous rodent DNA deletion occurs, this may occur simultaneously with human DNA insertion, or at an earlier or later stage. If rodent genes, the locus equivalent of the inserted human DNA, or parts thereof remain in the genome, they are inactivated or substantially inactivated, for example, by inversion of the rodent locus, destructive insertion into the locus, or destructive point mutation or substitution. If a locus is inverted, any non-TCR genes located within the inverted region preferably remain active and are expressed appropriately with respect to level (amount), tissue specificity (space), and temporal regulation.

[0281] Therefore, the method for inserting human DNA described herein may further include a step of deleting host rodent DNA at an equivalent endogenous rodent locus, preferably resulting in inactivation of the host locus and prevention of host polypeptide expression from that locus.

[0282] The method for inserting human DNA as described herein may further include a step of inactivating host rodent DNA at an equivalent endogenous rodent locus. It will also become clear that the present invention discloses rodents in which equivalent rodent genomic loci are inactivated so as not only to include insertion at the loci described herein, but also so as not to produce equivalent rodent polypeptides.

[0283] Therefore, it will be found that, as with the methods relating to human DNA insertion described herein, all methods can be extended to methods in which rodent DNA is replaced with equivalent human DNA from equivalent human loci, resulting in the deletion of rodent DNA from the associated locus (for example, deletion of rodent TCRα DNA and insertion of human TCRα DNA into the TRA locus).

[0284] Methods for large-scale replacement of rodent DNA with human DNA are well known, for example, in the context of humanization of the mouse IG locus. See Lee et al. (Lee et al., 2014, Vol. 32, No. 4, April 2014, Nature Biotechnology), Macdonald et al. 2014, and Murphy et al. 2014. Such methods involve recombineering of BACs to generate human DNA suitable for insertion using recombineering, followed by insertion of the recombineering BACs into ES cells, and then integration of the human DNA into the ES cell genome by means of, for example, cassette exchange via recombinase, or repeated homologous recombination, or a combination thereof.

[0285] If necessary, host DNA may be deleted before inserting human DNA. Such deletions can be achieved by inserting SSR sites on both sides of the locus and by deleting DNA using recombinase sites.

[0286] In one embodiment, the initial insertion event for any locus may involve a targeted insertion of an initiation cassette, establishing a unique site within the genome, followed by the insertion of the desired human DNA. Therefore, only one targeted event to the genome is required to initiate a targeted insertion of human DNA at each locus. The initiation cassette may contain one or more SSRs and / or transposase sites that allow for repeated use of the cassette in insertions. See the technique disclosed in Lee et al. above.

[0287] Cells such as ES cells can be screened using techniques well known in the field, such as Southern blotting, PCR, quantitative PCT (e.g., real-time PCR using TAQMAN®), fluorescence in situ hybridization, Northern blotting, flow cytometry, Western blotting, immunocytochemistry, and immunohistochemistry, to evaluate correct insertion events.

[0288] In one embodiment, the present invention relates to a method for producing a genetically modified fertile rodent expressing a human T cell receptor, comprising deleting serine protease genes Prss58, Prss59, Prss3b, Try4, Try5, Try10, Prss3, Prss1, and Prss2, and deleting at least rodent Vβ1 to Dβ1 of the rodent TCRβ, 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. In one embodiment, the deletion is homozygous.

[0289] The present invention also relates to a method for producing a fertile rodent comprising a genetic knockout of an endogenous T cell receptor β chain polypeptide, comprising deleting the serine protease genes Prss58, Prss59, Prss3b, Try4, Try5, Try10, Prss3, Prss1, and Prss2, and deleting at least rodent Vβ1 to Dβ1 of the rodent TCRβ, 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.

[0290] In one embodiment, the deletion is homozygous.

[0291] Use of genetically modified rodents Genetically modified rodents containing human CD8, human MHC class I, and human TCR can present peptides to T cells in a "human-like" manner because substantially all components of the complex are human. Using the genetically modified rodents of the present invention, the function of the human immune system in rodents can be studied, and antigens and antigenic epitopes that induce immune responses can be identified. Rodents can be used, for example, to identify T cell epitopes or human cancer epitopes in vaccine development, to identify TCR sequences and T cells with affinity for human pathogens or cancer antigens, to identify TCRs for use in adoptive T-cell therapy, to evaluate vaccine candidates and vaccination methods, to study human autoimmunity, and to study human infectious diseases.

[0292] Therefore, the genetically modified rodents of the present invention are particularly useful for evaluating the ability of antigens to initiate an immune response in humans, and for identifying specific antibodies that can generate diverse antigens and be used in human vaccine development.

[0293] Using a rodent containing inserted DNA encoding a human TCR as disclosed herein, the rodent can generate a human TCR / TCR polypeptide or functional fragment(s) thereof, which is a TCR directed to an immunized antigen, or generate sequence information relating to such TCR / TCR polypeptide and functional fragment(s).

[0294] T-cell therapy is currently attracting medical attention. Cytotoxic T cells, in particular, can recognize and eliminate target antigens, such as viral or bacterial antigens, or tumor antigens, or cells that present them. Adoptive cell therapy aims to isolate tumor-infiltrating lymphocytes from tumor cell masses, expand and proliferate them in vitro using T-cell growth factors, and then transfer them back to the patient. The rodents described herein may be a source of human T-cell receptors, such as high-affinity human T-cell receptors used in adoptive T-cell transfer.

[0295] The methods described below for generating human TCRs appropriately include the use of rodents containing inserted human DNA encoding human TCR polypeptides and human TCRb polypeptides.

[0296] Methods for generating or using human TCRs are similarly applicable to generating functional portions of human TCRs that adequately include at least the TCR binding site. Preferably, the binding of the TCR fragment to the antigen is the same as, or substantially the same as, that of the full-length TCR.

[0297] The methods for using or generating TCRs disclosed herein are equally applicable to the use or generation of functional fragments of TCRs.

[0298] In one embodiment, a method for generating T cell receptors for a human antigen is disclosed, comprising immunizing a rodent described herein with the antigen of interest, initiating an immune response in the rodent, isolating activated T cells having specificity for the antigen of interest from the animal, and determining the nucleic acid sequence of the T cell receptor expressed by the antigen-specific T cells. Methods for immunizing animals to generate a T cell response are known in the art. Immunization may involve administering the target peptide antigen of interest to the rodent in an immunization regimen comprising a prime and at least one boost. The immunization regimen may consist of a prime followed by one boost, or a prime followed by two or three boosts. The peptide may be administered by subcutaneous injection to perform prime immunization and / or boost immunization. The peptide may be administered in combination with an adjuvant. Examples of immunization regimens and adjuvants are shown in Example 6.

[0299] In one embodiment, antigen-specific CD8 cells can be sorted using a labeled MHC multimer (e.g., a tetramer or a higher-order multimer), and then the intracellular TCR nucleic acids can be sequenced to identify the heavy and light chains of paired TCRs.

[0300] In one embodiment, the present invention provides a method for producing a human T cell receptor specific to a target antigen, comprising: immunizing a rodent of the present invention as described herein with the target antigen; initiating an immune response in the animal; optionally isolating T cells reactive to the target antigen from the animal; determining the nucleic acid sequence of the human TCR variable region expressed by the antigen-specific T cells; and expressing a nucleotide construct encoding a human T cell receptor polypeptide chain specific to the target antigen. The expression of the nucleotide construct may be a cell membrane-expressed TCR or a soluble TCR.

[0301] In one embodiment, a method for identifying T cells having specificity for a target antigen, such as a tumor-associated antigen, is disclosed, comprising immunizing a rodent of the present invention as described herein with the target antigen, initiating an immune response in the animal, and isolating T cells having specificity for the antigen from a non-human animal.

[0302] In one embodiment, a method for identifying a human T cell epitope is disclosed, comprising exposing a rodent of the present invention as described herein to an antigen containing a putative T cell epitope; initiating an immune response in the rodent; isolating MHCI T cells bound to the epitope from the rodent; and identifying the epitope bound by the T cells.

[0303] In one embodiment, a method for determining whether a peptide induces a cellular immune response in humans, comprising: exposing a genetically modified rodent of the present invention as described herein to the peptide; initiating an immune response in the rodent; and a CD8 containing a human TCR that binds to the sequence of the peptide presented by human MHC class I. + A method is disclosed that includes detecting T cells.

[0304] In one embodiment, a method is disclosed for determining whether a putative antigen contains an epitope that generates an HLA class I-restricted immune response when exposed to the human immune system, the method comprising exposing a rodent of the present invention as described herein to the antigen and measuring the antigen-specific HLA class I-restricted immune response in a mouse.

[0305] The genetically modified rodents described herein may be useful in methods for identifying T cell receptors that recognize a target antigen, such as a tumor antigen or another disease antigen, such as a high-affinity T cell receptor. The method may include exposing the rodents of the present invention described herein to an antigen, initiating an immune response in the rodents to the antigen, and determining the sequence of a T cell receptor that binds to the antigen presented by human MHC I.

[0306] The genetically modified rodents described herein may be useful in identifying T cell receptors, such as high-affinity T cell receptors, that recognize a target antigen, such as a tumor antigen or another disease antigen. The method may include exposing the rodents of the present invention described herein to an antigen, initiating an immune response in the rodents to the antigen, optionally isolating T cells from the rodents that contain T cell receptors that bind to the antigen presented by human MHC class I, and determining the sequence of the T cell receptors that bind to the antigen.

[0307] The target antigens listed herein may be any antigen known to cause or be associated with a disease or pathological condition, such as tumor-associated antigens, viral antigens, or bacterial antigens. Tumor-associated antigens are known and available in the Cancer Immunity Peptide Database (archive.cancerimmunity.org / peptidedatabase / Tcellepitopes.htm). In some embodiments of the present 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 a T cell receptor is used to induce the killing of cells expressing the antigen.

[0308] In one embodiment, a method for determining T cell activation by a putative human therapeutic agent is disclosed, comprising: exposing a rodent of the present invention as described herein to a putative human therapeutic agent (or exposing human MHC class I expressing cells of such a rodent to the peptide sequence of the putative therapeutic agent); exposing cells of a genetically modified rodent that display a human MHC class I / peptide complex to T cells containing human CD8 that can bind to cells of the genetically modified animal; and measuring the T cell activation induced by peptide-displaying cells of the genetically modified animal.

[0309] The genetically modified rodents of the present 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 the present invention can be used to study various aspects of human autoimmune diseases and can be utilized as autoimmune disease models.

[0310] In one embodiment, the present invention relates to a method for producing a human T cell receptor for a target antigen, or a receptor variable region, or an antigen-binding portion of a T cell receptor, (i) optionally create genetically modified rodents as disclosed herein, (ii) Immunizing genetically modified rodents disclosed herein with the antigen of interest, (iii) Initiating an immune response in rodents, (iv) Determining the nucleic acid sequence or TCR variable region sequence of a human TCR expressed by T cells from a rodent reactive to the target antigen (optionally including the step of isolating T cells expressing such a TCR), (v) Expressing the human T cell receptor, the human T cell receptor variable region, or the antigen-binding portion of the human T cell receptor within cells, and optionally further formulating the expressed human T cell receptor, the human T cell receptor variable region, or their antigen-binding portions together with a pharmaceutically acceptable excipient, or (vi) Inserting a nucleic acid encoding the human T cell receptor, the human T cell receptor variable region, or its antigen-binding portion into human cells or animal cells, ex vivo or in vitro, and further optionally, formulating the cells containing the inserted nucleic acid for delivery to humans or animals, respectively, or (vii) optionally, formulation a nucleic acid (e.g., RNA or DNA) encoding the human T cell receptor or the human T cell receptor variable region, together with an appropriate delivery carrier such as a lipid or liposome, and delivering it in vivo to a patient in need. One of the following, This includes methods.

[0311] The present invention also relates to a method for treating an individual in need of treatment, (i) Delivering the cells described in step (vi) above to a patient who requires delivery, or (ii) optionally deliver a nucleic acid (e.g., RNA or DNA) that has been formulated in the manner described in step (vii) above to a patient requiring delivery, in accordance with the method described above. This includes methods as well.

[0312] In one embodiment, a method is provided for producing human TCRα and / or human TCRβ, or functional fragments thereof (e.g., their antigen-binding portions), comprising expressing human TCRα polypeptide(or more) and / or human TCRβ polypeptide(or more), or functional fragments thereof(or more) in a cell from nucleotide sequences encoding TCR or functional fragments thereof as described herein, obtained from or identified from the rodents described herein.

[0313] In one embodiment, the nucleic acid to be expressed is contained within a viral vector, and in a particular embodiment, the viral vector is a lentiviral vector.

[0314] In one embodiment, cells expressing nucleic acids (e.g., encoding human TCR or a functional fragment thereof) are selected from COS cells, CHO cells, 293 cells, HeLa cells, and retinal cells (e.g., PERC.6® cells) expressing viral nucleic acid sequences.

[0315] In one embodiment, cells expressing a human polypeptide having a sequence obtained from the immunization of a rodent having the human TCR described herein are provided.

[0316] The TCRα and TCRβ variable domains isolated from rodents described herein can be recombinantly produced in the context of soluble molecules containing the human TCR constant region. The TCR variable domain sequences according to the present invention, in a form in which the variable domain is linked to the human TCR constant region, are particularly well suited for the development of soluble TCR therapeutics for use in human patients, including bispecific T cell engagers. As reported herein (Example 8), 100% (124 out of 124) of the tested TCR chronotypes were recombinantly expressible in soluble form. No chronotypes that could not be expressed in soluble form containing the human constant region were encountered. The good expression levels and solubility of the TCR binding fragments observed here may be at least in part due to species matching (being entirely human), as the interaction between the variable and constant regions of the mouse TCR is known to be important for folding. Since the TCR in the rodents of this invention is entirely human, T cell selection for antigen binding occurs in vivo in the context of the human TCR variable region linked to the human TCR constant region. Therefore, when the selected TCR is converted to a soluble form in which the isolated TCR variable region is linked to the human TCR constant region, the context in which the TCR was selected in vivo is reflected. In contrast, the transgenic "VelociT" mouse described by Moore et al., Sci Immunol 6 2021 produced a TCR with both a human variable domain and a mouse constant domain. TCRs from VelociT mice require reformatting, in which the mouse constant domain is replaced with a human constant domain, to reduce immunogenicity in human patients. When a chimeric TCR is reformatted in this way and the constant region is altered, its expression may be reduced or inactivated because the human variable region was not originally selected in the context of a fully human TCR in vivo.

[0317] Cells and tissues In one embodiment, the present invention relates to cells derived from rodents described herein, such as T cells or APCs. The present invention also relates to tissues and embryos derived from rodents described herein.

[0318] In one embodiment, the cells isolated from rodents described herein are ES cells or hematopoietic stem cells, or other cells that can be the precursors of tissues and organs of non-human mammals. The cells may be iPS cells generated from cells obtained from rodents.

[0319] In one embodiment, the isolated cells are T cells. In one embodiment, the T cells express human TCRα and / or human TCRβ, and / or the T cells express human CD8α and / or human CD8β. In one embodiment, the T cells express CD8 + These are T cells.

[0320] In one embodiment, the isolated cells are antigen-presenting cells. In one embodiment, the antigen-presenting cells express human MHC class I molecules and / or human β2M molecules.

[0321] The modification of ES cells, such as mouse or rat ES cells, by manipulation is well known in the art. The present invention also extends to the modification of other cells that can be the source of hematopoietic stem cells or non-human mammalian tissues and organs, preferably those that can produce a repertoire of human TCRs.

[0322] In one embodiment, the present invention relates to pluripotent cells comprising unreorganized human TCR loci encoding human TCRα polypeptide and human TCRβ polypeptide as described herein. In one embodiment, the induced pluripotent cells are derived from a rodent as described herein.

[0323] The present invention also relates to cell lines grown from or otherwise derived from the cells described herein. In one embodiment, the cell line is an immortalized cell line.

[0324] The cell lineage may contain a VDJ gene or VJ gene that has been rearranged or has not been rearranged from the TCRα polypeptide and / or the TCRβ polypeptide.

[0325] TCR-producing cells and cell lines can be obtained by immortalizing cells through fusion with tumor cells, or they can be created through direct cell immortalization.

[0326] In one embodiment, a hybridoma or quadroma derived from the cells of a rodent described herein is provided.

[0327] Developing rodents from rodent ES cells is well known in the art and involves the steps of injecting ES cells into blastocysts, then transplanting the chimeric blastocysts into females to produce offspring, propagating them, and selecting the necessary insertions.

[0328] In one embodiment, the present invention relates to a chimeric rodent comprising ES cell-derived tissue and host embryo-derived tissue.

[0329] In one embodiment, the present invention relates to a rodent embryo comprising rodent-derived donor ES cells as described herein. In one embodiment, the embryo comprises ES donor cells containing an unreorganized human TCR locus and host embryonic cells.

[0330] In one embodiment, a tissue derived from a rodent described herein and expressing a human TCR polypeptide (e.g., TCRα and / or TCRβ) is provided.

[0331] rodent strains The preferred rodents are rats or mice. Mice are particularly preferred.

[0332] In one embodiment, the mouse may be a strain selected from the BL6 strains such as the C57BL strain, for example, 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. In another embodiment, the mice may be 129 strains selected from a group consisting of, for example, 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1 / SV, 129S1 / SvIm), 129S2, 129S4, 129S5, 129S9 / SvEvH, 129S6 (129 / SvEvTac), 129S7, 129S8, 129T1, and 129T2.

[0333] In one embodiment, the genetically modified mouse is a hybrid of the 129 strain and the C57BL / 6 strain. In another specific embodiment, the mouse is a hybrid of the 129 strain or the BL / 6 strain. In a specific embodiment, the 129 strain of the hybrid is the 129S6(129 / SvEvTac) strain.

[0334] In another embodiment, the mouse is a BALB strain, for example, a BALB / c strain. In yet another embodiment, the mouse is a hybrid of a BALB strain and the aforementioned strain.

[0335] If the rodent is a rat, the rat may be selected from Wistar rat, LEA strain, Sprague Dolly strain, Fisher strain, F344, F6, and Dark Agouti. In one embodiment, the rat strain is a hybrid of two or more strains selected from this group.

[0336] The present invention relates to any of the above-mentioned mouse or rat strains comprising any of the genetic modifications disclosed herein.

[0337] Cell development / T cell development Insertion of human DNA into endogenous rodent loci does not affect the ability of the associated rodent cells to develop normally, for example, to present MHC class I binding antigens and / or to recognize such binding antigens in the TCR.

[0338] In one embodiment, the rodents and rodent cells described herein include genetic modifications that do not alter the appropriate lineage selection and development of T cells.

[0339] In one embodiment, appropriate spatial and temporal protein expression of the inserted human DNA is observed.

[0340] In one embodiment, rodents and rodent T cells comprising human TCRα and / or human TCRβ as described herein produce T cells that, upon development in the thymus, can progress from DN1 to DN2, DN3, DN4, DP, and CD8 SP T cells. Such T cells of the rodents of the present invention express cell surface molecules (e.g., CD25, CD44, Kit, CD3, pTcc, etc.) that are typically produced by T cells during specific stages of development in the thymus. Thus, the rodents described herein express pTα that has complexed with TCRα at the DN3 stage of development in the thymus. The non-human animals described herein, upon development in the thymus, express CD8 + This represents a T cell capable of producing T cells. In one embodiment, rodents produce T cells capable of undergoing normal T cell differentiation in the periphery. In some embodiments, the non-human animals described herein can produce a normal repertoire of effector T cells, such as CTLs (cytotoxic T lymphocytes), TH1, TH2, and Treg.

[0341] In one aspect, the rodents and rodent T cells comprising the human CD8α polypeptide and / or human CD8β polypeptide described herein undergo thymic development and produce T cells that can progress from DN1 to DN2, DN3, DN4, DP, and CD8 SP T cells. Such T cells of the rodents of the present invention express cell surface molecules typically produced by T cells during specific stages of thymic development (e.g., CD25, CD44, Kit, CD3, pTcc, etc.). Thus, the rodents described herein express pTα complexed with TCRα at the DN3 stage of thymic development. The non-human animals described herein undergo thymic development to generate T cells that can produce CD8 + T cells. In one aspect, the rodents produce T cells that can undergo normal T cell differentiation in the periphery. In some embodiments, the non-human animals described herein can produce a normal repertoire of effector T cells, such as CTL (cytotoxic T lymphocytes), TH1, TH2, Treg, etc. In one aspect, the CD8 locus exhibits proper expression of CD8α and / or CD8β (proper spatial and temporal protein expression), CD8 + T cell development, CD8 lineage selection, and co-receptor function.

[0342] In one aspect, rodents comprising the human MHC class I polypeptide described herein can develop essentially normally and produce antigen-presenting cells that express MHC class I capable of presenting antigens to TCRs such as at least human TCR.

[0343] General features of the invention The general features of the invention can be combined separately with all aspects of the present disclosure.

[0344] In one aspect, one or more or all of the inserted fully human sequences are operably linked to one or more endogenous rodent control sequences such as promoters and / or enhancers.

[0345] In one embodiment, the orientation of the inserted human DNA relative to the centromere is the same as that of an equivalent 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 an equivalent endogenous rodent locus (for example, insertion of a human TRA occurs in the same orientation, at the same or substantially the same position as a rodent TRA, and at an endogenous locus).

[0346] In one embodiment, mutations in the inserted human sequence are permitted. For example, the human TCRα polypeptide, human TCRβ polypeptide, human MHC class I polypeptide, human CD8α polypeptide, and / or human CD8β polypeptide may contain one or more conserved or non-conserved modifications. Thus, a rodent is provided that expresses a human TCRα polypeptide sequence and / or a human TCRβ polypeptide sequence, a human CD8α polypeptide sequence and / or a human CD8β polypeptide sequence, a human MHC class I polypeptide, or a human β2M polypeptide sequence, wherein the human polypeptide sequence is at least approximately 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the human TCRα polypeptide sequence, human TCRβ polypeptide sequence, human CD8α polypeptide sequence, human CD8β polypeptide sequence, human MHC class I polypeptide sequence, or human β2M polypeptide sequence, respectively.

[0347] In another option, the human polypeptide sequence is at least approximately 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the human TCRα polypeptide sequence, human TCRβ polypeptide sequence, human CD8α polypeptide sequence, human CD8β polypeptide sequence, human MHC class I polypeptide sequence, or human β2M polypeptide sequence, across functional parts of the polypeptide or protein complex, such as the polypeptide or protein domain.

[0348] In one embodiment, the human polypeptide sequence contains one or more conserved substitutions. Conservative substitutions include substituting an amino acid residue with another amino acid residue having a side chain R group with similar chemical properties (e.g., charge or hydrophobicity). Conservative amino acid substitutions can be achieved by modifying the nucleotide sequence to introduce nucleotide changes that encode the conservative substitution. Generally, conservative amino acid substitutions do not substantially alter the desired functional properties of the protein, such as the ability of MHC class 1 to present the desired peptide. Examples of amino acids with side chains having similar chemical properties include aliphatic side chains such as glycine, alanine, valine, leucine, and isoleucine; aliphatic hydroxyl side chains such as serine and threonine; amide-containing side chains such as asparagine and glutamine; aromatic side chains such as phenylalanine, tyrosine, and tryptophan; basic side chains such as lysine, arginine, and histidine; acidic side chains such as aspartic acid and glutamic acid; and sulfur-containing side chains such as cysteine ​​and methionine. Examples of conserved amino acid substitutions include valine / leucine / isoleucine, phenylalanine / tyrosine, lysine / arginine, alanine / valine, glutamic acid / aspartic acid, and asparagine / glutamine. Due to the degeneracy of the genetic code, other nucleic acids, in addition to the nucleic acid residues encoding the human MHC class I polypeptide described herein, may also encode the disclosed polypeptide. Thus, this disclosure provides not only genetically modified non-human animals containing nucleotide sequences encoding MHC class I polypeptides with conserved amino acid substitutions in their genomes, but also non-human animals having genomes containing nucleotide sequences different from those described herein due to the degeneracy of the genetic code.

[0349] In one embodiment, the human DNA inserted into the rodent genome is human genomic DNA.

[0350] In another embodiment, the human DNA inserted into the rodent genome may be in the form of non-genomic DNA. For example, when a human TCR locus is inserted, the DNA may be in the form of a minigene, in which elements of human genomic DNA are deleted, resulting in a smaller insertion size compared to genomic human DNA. For example, non-coding sequences or non-functional sequences may be deleted.

[0351] When multiple DNA fragments are inserted, the inserted human DNA may be continuous or non-contiguous. Preferably, the inserted human DNA is in the form of a continuous gene or a continuous human locus within a rodent genome.

[0352] The human polypeptides described herein may be expressed under the control of rodent regulatory elements, such as endogenous non-human regulatory elements including promoters, enhancers, or silencers. These elements may be naturally occurring rodent regulatory elements in rodents that are in their native locations in the rodent, in particular, and have not been removed or rearranged from the genome (although their position relative to other rodent gene elements may change, for example, due to insertion of human DNA). Alternatively, the human polypeptides described herein may be expressed under the control of human regulatory elements, such as promoters, enhancers, or silencers.

[0353] In one embodiment, the inserted locus is regulated individually or collectively by at least one human regulatory element and at least one endogenous rodent regulatory element. For example, a human promoter(s), a host enhancer sequence, and one or more loci may be used.

[0354] In this specification, references to “rodents containing ~” or “rodent cells containing ~” mean that the genome of a rodent or rodent cell contains certain nucleic acids or DNA, unless otherwise specified.

[0355] The term "operably linked" refers to a juxtaposition of components that are in a relationship that allows them to function as intended. Thus, a protein-coding nucleic acid sequence can be operably linked to a regulatory sequence (e.g., promoter sequence, enhancer sequence, silencer sequence, etc.) to maintain proper transcriptional regulation. Furthermore, various parts of a human protein can be operably linked to maintain the proper folding, processing, targeting, expression, and other functional properties of the protein within the cell. Unless otherwise specified, various domains of a human protein are operably linked to one another.

[0356] In this specification, for example, as used with respect to a functional polypeptide, the polypeptide retains at least one biological activity typically associated with a native protein. Substitution at an endogenous locus (e.g., substitution at an endogenous non-human MHC locus) results in a locus that cannot express a functional endogenous polypeptide, such as an MHC class I polypeptide. Similarly, as used in this specification with respect to a functional portion of a protein, the extracellular domain retains its functionality, for example, in the case of a functional portion of the TCR, it retains the ability to bind to an antigen on MHC class I.

[0357] As described above, the rodents of the present invention preferably do not express functional rodent polypeptides from modified endogenous loci. For example, rodents do not express rodent TCRα polypeptides from endogenous TRA loci when DNA encoding human TCRα polypeptide is inserted into the endogenous locus. For example, an endogenous locus can be replaced before, simultaneously with, or after the insertion of human DNA into that locus. Alternatively, the endogenous locus may be inactivated, for example, by mutation or inversion, resulting in no expression of rodent polypeptides. In this specification, an endogenous locus refers to a rodent genomic locus including coding regions (which may be multiple), any intervening DNA such as introns, and promoter regions. In one embodiment, all or part of a host locus is replaced by the inserted human DNA. In one embodiment, the host locus may be retained, but shifted upstream or downstream by the insertion of human DNA at the endogenous locus. Endogenous gene expression from endogenous loci is preferably inactivated, but in one embodiment, essential genes that do not encode TCR / MHC class I, CD8, or B2M may be retained or reinserted. For example, one or more non-TCR related genes associated with fertility, such as genes encoding serine proteases located at the TCRβ locus, may be retained at the TCRβ locus.

[0358] In one embodiment, a TCR locus or TCR site as used herein refers to genomic DNA containing the TCR coding region, and this DNA includes the entire TCR coding region, which includes an unreorganized V(D)J sequence, promoter, enhancer sequence(s), constant sequence(s), and any upstream or downstream (UTR, regulatory region, etc.) or intervening DNA sequence(s) (intron, etc.). A TCR variable locus or TCR variable site refers to genomic DNA that includes the TCR variable region segment (V(D)J region) but excludes the TCR constant sequence.

[0359] It will be understood that the specific embodiments described herein are illustrative and not limit the invention. The main features of the invention can be used in various embodiments without departing from the scope of the invention. A person skilled in the art will recognize a number of procedures equivalent to the specific procedures described herein, or can verify them by ordinary study alone. Such equivalents are considered to be within the scope of the invention and are included in the claims. All publications and patent applications cited herein represent the level of skill of a person skilled in the art to which the invention pertains. All publications and patent applications are incorporated herein by reference to the same extent that each individual publication or patent application is specifically and individually indicated to be incorporated herein by reference. Where used in conjunction with the term “comprising” in the claims and / or specification, the use of the words “a” or “an” may mean “one,” but also coincides with the meanings of “one or more,” “at least one,” and “one or more than one.” The use of the term "or" in a claim is used to mean "and / or" unless it is explicitly indicated that it refers to an option alone or that the options are mutually exclusive, but this disclosure corresponds to the definitions of an option alone and "and / or". Throughout this application, the term "about" is used to indicate that a value includes variations in errors inherent in the device or method used to determine the value, or variations that exist between the subjects of study.

[0360] As used herein and in claims (which may be more), “comprising” (and any form of “comprising,” such as “comprise” and “comprises”), “having” (and any form of “having,” such as “have” and “has”), “including” (and any form of “including,” such as “includes” and “include”), or “containing” (and any form of “containing,” such as “contains” and “contain”) are comprehensive or open-ended and do not exclude any additional unlisted elements or process steps.

[0361] As used herein, the term “or combinations thereof” refers to all permutations and combinations of the listed items preceding the term. For example, “A, B, C, or combinations thereof” is intended to include at least one of A, B, C, AB, AC, BC, or ABC, and also intended to include BA, CA, CB, CBA, BCA, ACB, SAC, or CAB, where the order is important in the particular context. Continuing this example, combinations containing repetitions of one or more items or terms, such as BB, AAA, MB, BBC, AAABCCCC, CBBAAA, CABABB, etc. A person skilled in the art will understand that, unless otherwise evident from the context, there is typically no limit to the number of items or terms in any combination.

[0362] Various uses of TCRs produced in mice are disclosed in Patent Document 1, European Patent No. 2771357, and Patent Document 2, the contents of which are specifically incorporated herein by reference in the context of rodents, including all human loci disclosed herein.

[0363] The following detailed description includes illustrative representations of various embodiments of the present invention, but these are not intended to limit the invention as described in the claims. The accompanying drawings constitute part of this specification and serve only to illustrate embodiments together with the detailed description, and are not intended to limit the invention.

[0364] The following numbered sections represent embodiments of the present invention.

[0365] 1. (i) an unreorganized T cell receptor (TCR)α variable gene locus comprising at least one human Vα segment and at least one human Jα segment, wherein the TCRα variable gene is operably linked to the human TCRα constant gene sequence, (ii) an unreorganized TCRβ variable gene locus comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment, wherein the unreorganized TCRβ variable gene is operably linked to a human TCRβ constant gene sequence, Including, here, A genetically modified rodent in which the unreorganized human T cell variable region gene segment can be rearranged to form a gene encoding the human T cell receptor variable domain, the unreorganized TCRα variable gene locus is located at the endogenous rodent TCRα gene locus, and the unreorganized TCRβ variable gene locus is located at the endogenous rodent TCRβ gene locus.

[0366] 2. A rodent as described in Section 1, comprising a genome encoding an MHC class I polypeptide in which the α1 and α2 domains are human.

[0367] 3. MHC class I polypeptides are rodents as described in Section 2, which contain an α3 domain that binds to CD8 expressed in rodents.

[0368] 4. A rodent as described in Section 3, wherein the α3 domain is human and the CD8 contains a human MHC class I binding domain.

[0369] 5. CD8 refers to the rodents described in Section 4, including human CD8α.

[0370] 6. CD8 is a rodent described in Section 4, including the human-rodent chimera CD8α.

[0371] 7. CD8 refers to any rodent described in any of sections 4-6, including human CD8β.

[0372] 8. CD8 is a rodent described in any of sections 4-6, including the human-rodent chimera CD8β.

[0373] 9. A rodent as described in any of sections 5-8, whose genome contains human CD8 or human-rodent chimeric CD8 at the endogenous rodent CD8 locus.

[0374] 10. MHC class I polypeptides comprising a human transmembrane domain and a human cytoplasmic domain, as described in any of sections 2 to 9.

[0375] 11. The α3 domain of MHC class 1 is found in rodents, as described in Section 3.

[0376] 12. CD8 is a rodent described in Section 11, including the endogenous rodent CD8.

[0377] 13. The DNA encoding an MHC class I polypeptide is integrated into the endogenous rodent MHC class I gene locus in any of the rodents described in sections 2-12.

[0378] 14. The rodent genome further comprises a nucleic acid sequence encoding an MHC class I polypeptide, where MHC class I is (i) entirely human, or (ii) endogenous rodent MHC class I, or (iii) chimeric human-rodent MHC class I, for example, a chimeric MHC class I containing a rodent α3 domain capable of binding to the rodent CD8 protein, and optionally, the nucleic acid sequence encoding the MHC class I polypeptide is located at an endogenous locus, as described in Section 1 of the genetically modified rodents.

[0379] 15. The rodent genome further includes a nucleic acid sequence encoding a CD8α polypeptide at the endogenous rodent CD8α coreceptor locus and a nucleic acid sequence encoding a CD8β polypeptide at the endogenous rodent CD8β coreceptor locus, where optionally, the nucleic acid sequence is (i) A nucleic acid sequence encoding a human CD8α polypeptide located at the endogenous rodent CD8α coreceptor locus, and a nucleic acid sequence encoding a human CD8β polypeptide located at the endogenous rodent CD8β coreceptor locus, (ii) A nucleic acid sequence encoding a chimeric human-rodent CD8α polypeptide located at the endogenous rodent CD8α coreceptor locus, and a nucleic acid sequence encoding a chimeric human-rodent CD8β polypeptide located at the endogenous rodent CD8β coreceptor locus, (iii) A nucleic acid sequence encoding a rodent CD8α polypeptide located at an endogenous locus, and a nucleic acid sequence encoding a rodent CD8β polypeptide located at an endogenous locus, A genetically modified rodent selected from either section 1 or 14.

[0380] 16. A genetically modified rodent described in any one of sections 1, 14, or 15, whose rodent genome contains a nucleic acid sequence encoding human β2-microglobulin polypeptide.

[0381] 17. A genetically modified rodent described in any one of sections 1 and 14-16, wherein the rodent genome contains a nucleic acid sequence encoding a human β2-microglobulin polypeptide located at the endogenous rodent β2-microglobulin locus.

[0382] 18. (i) an unreorganized T cell receptor (TCR)α variable gene locus comprising at least one human Vα segment and at least one human Jα segment, wherein the TCRα variable gene is operably linked to the human TCRα constant gene sequence, (ii) an unreorganized TCRβ variable gene locus comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment, wherein the unreorganized TCRβ variable gene is operably linked to a human TCRβ constant gene sequence, Including, here, Unreorganized human T cell variable region gene segments can rearrange to form genes encoding human T cell receptor variable domains, the unreorganized TCRα variable gene locus is located at the endogenous rodent TCRα gene locus, and the unreorganized TCRβ variable gene locus is located at the endogenous rodent TCRβ gene locus, and Rodent genomes are, [A] A nucleic acid sequence encoding a human CD8α polypeptide located at the endogenous rodent CD8α coreceptor locus, a nucleic acid sequence encoding a human CD8β polypeptide located at the endogenous rodent CD8β coreceptor locus, and a nucleic acid sequence encoding a human MHC class I polypeptide located at the endogenous rodent MHC class I locus, or [B] A nucleic acid sequence encoding a chimeric human-rodent CD8α polypeptide located at the endogenous rodent CD8α coreceptor locus, a nucleic acid sequence encoding a chimeric human-rodent CD8β polypeptide located at the endogenous rodent CD8β coreceptor locus, and a nucleic acid sequence encoding a human MHC class I polypeptide located at the endogenous rodent MHC class I locus (where the chimeric CD8 protein can bind to human MHC class I), or [C] A nucleic acid sequence encoding a rodent CD8α polypeptide at an endogenous locus, a nucleic acid sequence encoding a rodent CD8β polypeptide at an endogenous locus, and a nucleic acid sequence encoding a 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). Includes any of the following: Optionally, the genetically modified rodents described in Section 1, whose rodent genome, if present, includes a nucleic acid sequence encoding a human β2-microglobulin polypeptide located at the endogenous rodent β2-microglobulin locus.

[0383] 19. (i) an unreorganized T cell receptor (TCR)α variable gene locus comprising at least one human Vα segment and at least one human Jα segment, wherein the TCRα variable gene is operably linked to the human TCRα constant gene sequence, (ii) an unreorganized TCRβ variable gene locus comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment, wherein the unreorganized TCRβ variable gene is operably linked to a human TCRβ constant gene sequence, Including, here, Unreorganized human T cell variable region gene segments can be rearranged to form genes encoding the human T cell receptor variable domain. Genetically modified rodent cells, such as ES cells, in which the unreorganized TCRα variable gene locus is located at the endogenous rodent TCRα gene locus, and the unreorganized TCRβ variable gene locus is located at the endogenous rodent TCRβ gene locus.

[0384] 20. The rodent genome further comprises a nucleic acid sequence encoding an MHC class I polypeptide located at the endogenous rodent MHC class I locus, where MHC class I is (i) entirely human, or (ii) endogenous rodent MHC class I, or (iii) chimeric human-rodent MHC class I, for example, a chimeric MHC class I containing a rodent α3 domain capable of binding to the rodent CD8 protein, as described in Section 19 of the genetically modified rodent cells.

[0385] 21. The rodent genome further includes a nucleic acid sequence encoding a CD8α polypeptide at the endogenous rodent CD8α coreceptor locus and a nucleic acid sequence encoding a CD8β polypeptide at the endogenous rodent CD8β coreceptor locus, where optionally, the nucleic acid sequence is (i) A nucleic acid sequence encoding a human CD8α polypeptide located at the endogenous rodent CD8α coreceptor locus, and a nucleic acid sequence encoding a human CD8β polypeptide located at the endogenous rodent CD8β coreceptor locus, (ii) A nucleic acid sequence encoding a chimeric human-rodent CD8α polypeptide located at the endogenous rodent CD8α coreceptor locus, and a nucleic acid sequence encoding a chimeric human-rodent CD8β polypeptide located at the endogenous rodent CD8β coreceptor locus, (iii) A nucleic acid sequence encoding a rodent CD8α polypeptide located at an endogenous locus, and a nucleic acid sequence encoding a rodent CD8β polypeptide located at an endogenous locus, Genetically modified rodent cells as described in either section 19 or 20, selected from the above.

[0386] 22. A genetically modified rodent cell as described in any one of sections 19-21, wherein the rodent genome contains a nucleic acid sequence encoding a human β2-microglobulin polypeptide located at the endogenous rodent β2-microglobulin locus.

[0387] twenty three. (i) an unreorganized T cell receptor (TCR)α variable gene locus comprising at least one human Vα segment and at least one human Jα segment, wherein the TCRα variable gene is operably linked to the human TCRα constant gene sequence, (ii) an unreorganized TCRβ variable gene locus comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment, wherein the unreorganized TCRβ variable gene is operably linked to a human TCRβ constant gene sequence, Including, here, Unreorganized human T cell variable region gene segments can rearrange to form genes encoding human T cell receptor variable domains, the unreorganized TCRα variable gene locus is located at the endogenous rodent TCRα gene locus, and the unreorganized TCRβ variable gene locus is located at the endogenous rodent TCRβ gene locus, and Rodent genomes are, [A] A nucleic acid sequence encoding a human CD8α polypeptide located at the endogenous rodent CD8α coreceptor locus, a nucleic acid sequence encoding a human CD8β polypeptide located at the endogenous rodent CD8β coreceptor locus, and a nucleic acid sequence encoding a human MHC class I polypeptide located at the endogenous rodent MHC class I locus, or [B] A nucleic acid sequence encoding a chimeric human-rodent CD8α polypeptide located at the endogenous rodent CD8α coreceptor locus, a nucleic acid sequence encoding a chimeric human-rodent CD8β polypeptide located at the endogenous rodent CD8β coreceptor locus, and a nucleic acid sequence encoding a human MHC class I polypeptide located at the endogenous rodent MHC class I locus (where the chimeric CD8 protein can bind to human MHC class I), or [C] A nucleic acid sequence encoding a rodent CD8α polypeptide at an endogenous locus, a nucleic acid sequence encoding a rodent CD8β polypeptide at an endogenous locus, and a nucleic acid sequence encoding a 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). Includes any of the following: Optionally, genetically modified rodent cells as described in Section 19, the rodent genome, if present, containing a nucleic acid sequence encoding a human β2-microglobulin polypeptide located at the endogenous rodent β2-microglobulin locus.

[0388] twenty four. (i) an unreorganized T cell receptor (TCR)α variable gene locus comprising at least one human Vα segment and at least one human Jα segment, wherein the TCRα variable gene is operably linked to the human TCRα constant gene sequence, (ii) an unreorganized TCRβ variable gene locus comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment, wherein the unreorganized TCRβ variable gene is operably linked to a human TCRβ constant gene sequence, Including, here, Unreorganized human T cell variable region gene segments can be rearranged to form genes encoding the human T cell receptor variable domain. Genetically modified rodent T cells in which the unreorganized TCRα variable gene locus is located at the endogenous rodent TCRα gene locus, and the unreorganized TCRβ variable gene locus is located at the endogenous rodent TCRβ gene locus.

[0389] 25. In addition, [A] A nucleic acid sequence encoding a human CD8α polypeptide located at the endogenous rodent CD8α coreceptor locus, a nucleic acid sequence encoding a human CD8β polypeptide located at the endogenous rodent CD8β coreceptor locus, or [B] A nucleic acid sequence encoding a chimeric human-rodent CD8α polypeptide located at the endogenous rodent CD8α coreceptor locus, a nucleic acid sequence encoding a chimeric human-rodent CD8β polypeptide located at the endogenous rodent CD8β coreceptor locus, or [C] Nucleic acid sequence encoding rodent CD8α polypeptide at an endogenous locus, and nucleic acid sequence encoding rodent CD8β polypeptide at an endogenous locus. This includes any of the following, where the cell is [A] Human TCR polypeptide and human CD8 polypeptide, or [B] Human TCR polypeptide and chimeric CD8 polypeptide, or [C] Human TCR polypeptide and rodent CD8 polypeptide, Genetically modified rodent T cells as described in Section 24, expressing one of the following.

[0390] 26. Antigen-presenting cells of genetically modified rodents, (i) A nucleic acid sequence encoding a human MHC class I polypeptide located at the endogenous rodent MHC class I gene locus, or (ii) A nucleic acid sequence encoding a chimeric human-rodent MHC class I polypeptide located at the endogenous rodent MHC class I gene locus, wherein the nucleic acid sequence encoding the chimeric MHC class I has rodent exon 4, and exons 1 to 3 and exons 5 to 7 are human, One of the following, (iii) Optionally, if present, a nucleic acid sequence encoding a human β2-microglobulin polypeptide located at the endogenous rodent β2-microglobulin locus, Including, here, These cells are antigen-presenting cells from genetically modified rodents that express either human MHC class I or chimeric MHC class I, and optionally express human β2M polypeptide.

[0391] 27. A genetically modified rodent or rodent cell described in any one of sections 1 to 26, wherein the endogenous locus modified to contain DNA encoding a human polypeptide or chimeric polypeptide does not express an equivalent rodent polypeptide.

[0392] 28. A genetically modified rodent or rodent cell described in any one of sections 1 to 27 that does not express a functional endogenous TCRα polypeptide from an endogenous TCRα variable gene locus.

[0393] 29. A genetically modified rodent or rodent cell described in Section 28 that is homozygous at the TCRα gene locus.

[0394] 30. A genetically modified rodent or rodent cell described in any of sections 1 to 29 that does not express a functional endogenous TCRβ polypeptide from an endogenous TCRβ variable gene locus.

[0395] 31. A genetically modified rodent or rodent cell described in Section 30 that is homozygous at the TCRβ gene locus.

[0396] 32. A genetically modified rodent or rodent cell described in any one of sections 1 to 31, in which the rodent or rodent cell fully expresses human MHC class I and does not express a functional endogenous rodent CD8 coreceptor from the endogenous CD8 locus.

[0397] 33. A genetically modified rodent or rodent cell described in any one of sections 1 to 32, which does not express a functional endogenous rodent MHC class I polypeptide from an endogenous MHC class I gene locus.

[0398] 34. A genetically modified rodent or rodent cell described in any one of sections 1 to 33, which does not express functional endogenous rodent β2 microglobulin from the endogenous rodent β2 microglobulin locus if the rodent or rodent cell contains a nucleic acid sequence encoding human β2 microglobulin.

[0399] 35. A genetically modified rodent or rodent cell as described in any one of sections 28-34, wherein a deletion of a functional endogenous rodent TCRα variable locus includes (a) deletion of all endogenous Vα gene segments, (b) deletion of all endogenous Jα gene segments, and (c) a deletion selected from the group consisting of combinations thereof, and / or a deletion of a functional endogenous rodent TCRβ variable locus includes (a) deletion of all endogenous Vβ gene segments, (b) deletion of all endogenous Dβ gene segments, (c) deletion of all endogenous Jβ gene segments, and (d) a deletion selected from the group consisting of combinations thereof.

[0400] 36. A genetically modified rodent or rodent cell as described in any one of sections 1 to 35, wherein the endogenous rodent TCRα locus is deleted from the rodent genome, including from the distal 3'V(Vα1) to the rodent 3' constant region TCRαC, and a rodent enhancer remains downstream of TCRαC in the rodent genome, which is operably linked to an unreorganized human T cell receptor (TCR)α variable gene locus.

[0401] 37. Any genetically modified rodent or rodent cell as described in the preceding section, wherein the human Vα segment and human Jα segment are rearranged to form a rearranged human Vα / Jα sequence, and the human Vβ segment, human Dβ segment, and human Jβ segment are rearranged to form a rearranged human Vβ / Dβ / Jβ sequence.

[0402] 38. Any genetically modified rodent or rodent cell described in the preceding section, which expresses a human T cell receptor containing a human TCRα variable domain and a human TCRβ variable domain on the surface of the T cell.

[0403] 39. Any genetically modified rodent or rodent cell described in the above section, wherein the unreorganized TCRα variable gene locus contains the complete repertoire of human Jα segments and the complete repertoire of human Vα segments, and / or the unreorganized TCRβ variable gene locus contains the complete repertoire of human Jβ segments, the complete repertoire of human Dβ segments, and the complete repertoire of human Vβ segments.

[0404] 40. (i) Any rodent or rodent cell described in the above section, wherein the rodent possesses an endogenous TCRα variable gene locus and / or an endogenous rodent TCRβ variable gene locus, and both of the retained endogenous rodent TCRα variable gene locus are non-functional loci, and / or both of the retained endogenous rodent TCRβ variable gene locus are non-functional loci, and optionally, all or part of the retained endogenous rodent TCRα variable gene locus and / or TCRβ variable gene locus are inverted relative to their normal orientation in the rodent genome, or, (ii) Fertile rodents or rodent cells wherein at least the rodent TCRβ loci from rodent Vβ1 to Dβ1 are inverted within the rodent genome with respect to their natural orientation within the rodent genome, and optionally further comprising inserting nucleic acids such as DNA encoding all or part of TCRβ from a species other than rodents into the rodent genome, such as human DNA encoding all or part of TCRβ, A genetically modified rodent or rodent cell that is one of the following.

[0405] 41. (i) Any rodent described in the above section that is substantially wild-type fertility, wherein the rodent TCRβ locus is deleted from at least rodent Vβ1 to Dβ1, and the genome additionally contains one or more or all of the deleted serine protease genes Prss58, Prss59, Prss3b, Try4, Try5, Try10, Prss3, Prss1, and Prss2, (ii) A rodent that is substantially wild-type fertile, wherein the rodent TCRβ locus is deleted from at least rodent Vβ1 to Dβ1, and the genome additionally contains one or more 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 and / or does not have a wild-type genome at the TCRβ locus and / or cannot form a rodent TCRβ chain, a rodent that is substantially wild-type fertile. A genetically modified male rodent that is one of the following:

[0406] 42. Any genetically modified rodent or rodent cell described in the preceding section, wherein the rodent possesses an endogenous rodent TCRβ variable gene locus, and the endogenous rodent TCRβ variable gene locus is a non-functional locus resulting from the inversion of all or part of the gene locus in the genome.

[0407] 43. Any genetically modified rodent or rodent cell described in the preceding section, wherein all or part of the endogenous rodent TCRα gene locus is replaced with an unreorganized TCRα variable gene locus, and / or all or part of the endogenous rodent TCRβ gene locus is replaced with an unreorganized TCRβ variable gene locus.

[0408] 44. Any genetically modified rodent or rodent cell as described in the above section, further comprising a repertoire of human TCRδ variable region segments that have not been rearranged at the rodent TCRα locus, and optionally comprising one or more human TCRδ V gene segments, one or more human TCRδ D gene segments, and one or more human TCRδ J gene segments that can be rearranged to form a human TCRD variable region.

[0409] 45. Rodents or cells of section 44, which include the complete repertoire of human Vδ segments, human Dδ segments, and human Jδ segments at the rodent TCRα variable gene locus.

[0410] 46. ​​Rodents or cells of section 44 or 45, wherein the rodent TCRα locus contains the entire human Vδ constant region, and the rodent TCRα variable locus does not contain the rodent Vδ constant region, resulting in the complete expression of human TCRδ.

[0411] 47. Any rodent or cell of the above-mentioned section, further comprising one or more or a complete repertoire of human Vγ segments and one or more or a complete repertoire of unreorganized human TCRγ variable region segments, such as human Jγ segments, operably linked to a human TCRγ constant gene sequence.

[0412] 48. Any genetically modified rodent or rodent cell described in the preceding section, wherein the unreorganized human T cell variable region TCRα V and J gene segments are operably linked to an endogenous rodent TCRα enhancer downstream of the TCRα constant region.

[0413] 49. Any genetically modified rodent or rodent cell described in the above section, wherein the unreorganized human T cell variable regions V gene segment, (D) gene segment, and / or J gene segment, and / or TCRα constant region and / or TCRβ constant region are operably linked to one or more endogenous rodent regulatory elements, such as rodent promoters or enhancers.

[0414] 50. The genome is, (i) a nucleotide sequence encoding a human CD8α polypeptide operably ligated to a rodent CD8α promoter and / or another rodent regulatory sequence(s), and a nucleic acid sequence encoding a human CD8β polypeptide operably ligated to a rodent CD8β promoter and / or another rodent regulatory sequence(s), where optionally the rodent regulatory sequence is an endogenous rodent promoter or regulatory sequence, (ii) a nucleotide sequence encoding a chimeric human-rodent CD8α polypeptide operably ligated to a rodent CD8α promoter and / or other rodent regulatory sequences(if any), and a nucleic acid sequence encoding a chimeric human-rodent CD8β polypeptide operably ligated to a rodent CD8β promoter and / or other rodent regulatory sequences(if any) (wherein optionally, the rodent regulatory sequences are endogenous rodent promoters or regulatory sequences), Genetically modified rodents or rodent cells as described in any of the above sections, including.

[0415] 51. (i) The genome comprises nucleic acids encoding human MHC class I polypeptides operably linked to rodent MHC class I promoters, enhancers, and / or other rodent regulatory sequences (optionally, the rodent promoter or regulatory element(s) are endogenous rodent promoters or regulatory sequences), or (ii) Any genetically modified rodent or rodent cell described in the above section, the genome comprising a nucleic acid sequence encoding a chimeric MHC class I polypeptide operably linked to a rodent MHC class I promoter, enhancer, and / or other rodent regulatory sequence (optionally, the rodent promoter or regulatory element(s) are endogenous rodent promoters or regulatory sequences).

[0416] 52. An endogenous rodent regulatory sequence is a naturally occurring rodent genome regulatory sequence located in its natural genomic location, in a genetically modified rodent or rodent cell as described in any one of sections 49-51.

[0417] 53. Human MHC class I polypeptide or chimeric MHC class I polypeptide is any genetically modified rodent or rodent cell as described in the above section, selected from the group consisting of HLA-A, HLA-B, and HLA-C.

[0418] 54. A rodent genome comprising a rodent HLA locus containing an insertion cassette, wherein the cassette contains a site-specific recombinase site that enables insertion of human HLA DNA into the rodent genome by cassette exchange via recombinase, thereby allowing different human HLA molecules to be expressed from the same genomic location, in any genetically modified rodent or rodent cell as described in the preceding section.

[0419] 55. Any genetically modified rodent or rodent cell as described in the preceding section, comprising DNA encoding human HLA-A02 or chimeric HLA-A02, wherein the rodent H2D locus is inactive, optionally replaced by human DNA or chimeric DNA encoding human HLA-A02, and further optionally, the rodent H2-K locus is deleted or inactivated.

[0420] 56. A genetically modified rodent or rodent cell as described in any of the above sections, wherein the endogenous non-human β2-microglobulin locus contains a nucleotide sequence encoding a human β2-microglobulin polypeptide, wherein the rodent or rodent cell expresses the human β2-microglobulin polypeptide.

[0421] 57. Nucleic acids encoding human TCRα and human TCRβ are present in the germline of rodents, and if present, human CD8α polypeptide or chimeric human-rodent CD8α polypeptide, human CD8β polypeptide or chimeric human-rodent CD8β polypeptide, human MHC class I polypeptide or chimeric MHC class I polypeptide, and / or human β2 microglobulin are present in the germline of any genetically modified rodents as described in the above section.

[0422] 58. (i) The rodent germline contains nucleic acids encoding chimeric human-rodent CD8α polypeptide, chimeric human-rodent CD8β polypeptide, human TCRα, human TCRβ, human MHC class I, and human β2 microglobulin, or (i) The germline of rodents contains nucleic acids encoding human CD8α polypeptide, human CD8β polypeptide, human TCRα, human TCRβ, human MHC class I, and human β2 microglobulin, or (ii) Any genetically modified rodent described in the above section, wherein the nucleic acids encoding rodent CD8α polypeptide, rodent CD8β polypeptide, human TCRα, human TCRβ, chimeric human-rodent MHC class I, and human β2 microglobulin are included in the rodent germline.

[0423] 59. The rodent is a mouse or rat, preferably a mouse, a genetically modified rodent or rodent cell as described in any of the above sections.

[0424] 60. 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, One or more of, including all of them, and / or The following human Jα gene segments: 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, Any genetically modified rodent or rodent cell described in the above section, containing one or more, or any all, of the TCRα variable gene loci.

[0425] 61. 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, One or more of, including all of them, and / or 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, Any genetically modified rodent or rodent cell described in the above section, containing one or more, or any all, of the TCRβ variable gene loci.

[0426] 62. Any genetically modified rodent or rodent cell described in the above section, comprising a genome in which CD3ε, CD3γ, CD3δ, and / or CD3ζ are endogenous wild-type.

[0427] 63. All of CD3ε, CD3γ, CD3δ, and CD3ζ are endogenous wild-type in the genetically modified rodents or rodent cells described in Section 62.

[0428] 64. A method for creating a genetically modified rodent that expresses the human T cell receptor, (i) Inserting an unreorganized human TCRα variable gene locus into the endogenous rodent TCRα variable gene locus, which includes at least one human Vα segment and at least one human Jα segment operably linked to the human TCRα constant region, (ii) Inserting an unreorganized human TCRβ variable gene locus into the endogenous rodent TCRβ variable gene locus, comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment operably linked to the human TCRβ constant region, Methods that include...

[0429] 65. The following steps: (iii) A step of inserting a nucleic acid sequence encoding a human CD8α polypeptide into the endogenous rodent CD8α gene locus, (iv) A step of inserting a nucleic acid sequence encoding a human CD8β polypeptide into the endogenous rodent CD8β gene locus, (v) The step of inserting a nucleic acid sequence encoding a human MHC class I polypeptide into an endogenous MHC class I gene locus, and (vi) Optionally, inserting a nucleic acid sequence encoding human β2-microglobulin into the endogenous rodent β2-microglobulin gene locus, The method described in Section 64, further including one or more of the above.

[0430] 66. The following steps: (iii) A step of modifying the host rodent CD8α gene locus by inserting a nucleic acid sequence encoding a chimeric human-rodent human CD8α polypeptide into the endogenous rodent CD8α gene locus, or by replacing the rodent DNA with human DNA, thereby producing DNA encoding a chimeric human-rodent human CD8α polypeptide. (iv) A step of modifying the host rodent CD8β gene locus by inserting a nucleic acid sequence encoding a chimeric human-rodent CD8β polypeptide into the endogenous rodent CD8β gene locus, or by replacing the rodent DNA with human DNA, thereby producing DNA encoding a chimeric human-rodent human CD8β polypeptide. (v) The step of inserting a nucleic acid sequence encoding a human MHC class I polypeptide into an endogenous MHC class I gene locus, and (vi) Optionally, inserting a nucleic acid sequence encoding human β2-microglobulin into the endogenous rodent β2-microglobulin gene locus, The method described in Section 64, further including one or more of the above.

[0431] 67. The following steps: (iii) A step of inserting a nucleic acid sequence encoding a chimeric rodent-human MHC class I polypeptide into an endogenous MHC class I gene locus, or a step of modifying the host rodent MHC class I gene locus by replacing rodent DNA with human DNA to produce DNA encoding a chimeric human-rodent-human MHC class I polypeptide. (iv) Optionally, inserting a nucleic acid sequence encoding human β2-microglobulin into the endogenous rodent β2-microglobulin gene locus, The method described in Section 64, further including one or more of the above.

[0432] 68. The method according to any one of sections 64-67, wherein the host gene locus at each insertion site is inactivated or deleted, and as a result the rodent does not express native rodent TCR, MHC class I, or CD8, and optionally also does not express rodent β2 microglobulin.

[0433] 69. A method for producing a fertile rodent or rodent cell, comprising the step of inverting rodent TCRβ in the rodent genome from at least rodent Vβ1 to Dβ1, and optionally further comprising inserting nucleic acids such as DNA encoding all or part of TCRβ from a species other than the rodent, such as human DNA encoding all or part of TCRβ, into the rodent genome.

[0434] 70. A method for producing a genetically modified fertile rodent expressing a human T cell receptor, comprising deleting serine protease genes Prss58, Prss59, Prss3b, Try4, Try5, Try10, Prss3, Prss1, and Prss2, and deleting at least rodent Vβ1 to Dβ1 of the rodent TCRβ, 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.

[0435] 71. A method for producing a fertile rodent comprising a genetic knockout of an endogenous T cell receptor β chain polypeptide, comprising deleting serine protease genes Prss58, Prss59, Prss3b, Try4, Try5, Try10, Prss3, Prss1, and Prss2, comprising deleting at least rodent Vβ1 to Dβ1 of the rodent TCRβ, 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.

[0436] 72. A method for producing fertile rodents, comprising any of the following steps: (i) Deleting at least rodent Vβ1 to Dβ1 of rodent TCRβ genomic DNA, including deleting one or more or all of the serine protease genes Prss58, Prss59, Prss3b, Try4, Try5, Try10, Prss3, Prss1, and Prss2, (ii) Inserting one or more or all of the serine protease genes Prss58, Prss59, Prss3b, Try4, Try5, Try10, Prss3, Prss1, and Prss2 into the rodent genome, Methods that include...

[0437] 73. The method described in any one of Sections 69 to 72, further comprising one of steps (i) to (vi) of Sections 59 and 60.

[0438] 74. The deletion is homozygous, as described in any of sections 69-73.

[0439] 75. A rodent or rodent cell as described in any one of Sections 1 to 74, or a method for producing said rodent or rodent cell, wherein the rodent genome comprises DNA that completely encodes the human TCRβ chain and includes the human V gene segment, the human D gene segment, the human J gene segment, and the human C gene segment, wherein the rodent or rodent cell is capable of fully expressing the human TCRβ chain, and the rodent genome comprises a human intergene DNA sequence and / or human intron DNA sequence which is located between the human D1 gene segment and the human C2 gene segment and is preferably a human intergene DNA sequence and / or human intron sequence that naturally occurs together with the human D gene segment and the human J gene segment. A rodent or rodent cell, or method, in which, optionally, all of the human DNA sequence between the human D1 gene segment and the human C2 gene segment is genomic human DNA.

[0440] 76. A rodent or rodent cell having a genome that fully encodes the human TCRβ chain and includes DNA comprising the human V gene segment, the human D gene segment, the human J gene segment, and the human C gene segment, wherein the rodent or rodent cell is capable of fully expressing the human TCRβ chain, and the rodent genome comprises a human intergene DNA sequence and / or human intron DNA sequence which is located between the human D1 gene segment and the human C2 gene segment, preferably a human intergene DNA sequence and / or human intron sequence that is naturally present together with the human D gene segment and the human J gene segment. A rodent or rodent cell in which, optionally, all of the human DNA sequence between the human D1 gene segment and the human C2 gene segment is genomic human DNA.

[0441] 77. A rodent, rodent cell, or method as described in Section 75 or Section 76, wherein the TCRβ chain comprises a sequence encoded by a rearrangement of the V gene segment and segments from the DJC2 cluster at the gene locus.

[0442] 78. A method for producing a human T cell receptor in a rodent, comprising inserting DNA into the genome of the rodent that completely encodes a human TCRβ chain and includes a human V gene segment, a human D gene segment, a human J gene segment, and a human C gene segment, wherein the rodent is able to fully express a human TCRβ chain, and here, The insertion comprises a human intergenetic DNA sequence and / or human intronic DNA sequence, which is preferably a human intergenetic sequence and / or human intronic sequence that is naturally present together with the human D gene segment and the human J gene segment, located between the human D1 gene segment and the human C2 gene segment. The method is such that, optionally, all of the human DNA sequence between the human D1 gene segment and the human C2 gene segment is genomic human DNA.

[0443] 79. The method according to Section 78, wherein the TCR comprises a TCRβ variable region of the TCRβ chain having a sequence encoded by a rearrangement of the V gene segment and segments from the DJC2 cluster at the TCRβ locus of the T cell.

[0444] 80. A method for producing a human T cell receptor for a target antigen, (i) optionally, the process of creating any of the rodents described in the above section, (ii) The process of immunizing any of the rodents described in the above section with the target antigen, (iii) A process to initiate an immune response in rodents, (iv) Optionally, a step of determining the nucleic acid sequence of the human TCR variable region expressed by T cells from a rodent reactive to the antigen of the target, including a step of isolating T cells, (v) A step of expressing a human T cell receptor or a human T cell receptor variable region in cells, and optionally further formulating the expressed human T cell receptor or human T cell receptor variable region together with a pharmaceutically acceptable excipient, or (vi) Inserting a nucleic acid encoding a human T cell receptor or a human T cell receptor variable region into human cells or animal cells, etc., ex vivo or in vitro, and optionally formulating the cells containing the inserted nucleic acid for delivery to humans or animals, respectively, or (vii) A process of formulating a nucleic acid (e.g., RNA or DNA) encoding the human T cell receptor or the human T cell receptor variable region together with an appropriate delivery carrier such as a lipid or liposome, and delivering it in vivo to a patient who requires delivery; Methods that include...

[0445] 81. The method according to Section 80, wherein the TCR comprises a TCRβ variable region of the TCRβ chain having a sequence encoded by a rearrangement of the V gene segment and segments from the DJC2 cluster at the TCRβ locus of the T cell.

[0446] 82. A method for treating an individual in need of treatment, (i) Delivering to an individual a soluble TCR molecule that includes the human T cell receptor variable region of step (v) of Section 80, and optionally includes the human T cell receptor constant region, (ii) Delivering the cells described in step (vi) of Section 80 to patients who require delivery, (iii) Delivering a nucleic acid (e.g., RNA, e.g., mRNA or DNA) that is formulated in accordance with step (vii) of Section 80 to a patient who requires delivery, Methods that include... [Examples]

[0447] Example 1 We generated transgenic mice in which the TRB VDJ gene locus was deleted, and also generated transgenic mice in which the TRB VDJ gene locus was inverted but retained within the genome.

[0448] The inversion of the mouse variable region is from coordinate 6:40868163 (GRCm39) to 6:41515110 (GRCm39), as shown in Figure 2.

[0449] The mouse constant region is missing.

[0450] When the mouse TRB VDJ locus was present in its natural or inverted orientation, homozygous male mice produced offspring in 92% of cases (n=12 male mice). However, in male mice with a deletion of the mouse TRB VDJ locus, offspring were produced in only 35% of cases (n=34 male mice). Therefore, inversion of the variable region preserves a genetic component that, when deleted, significantly impacts fertility.

[0451] Example 2 As described herein, mice expressing human TRB DNA that have been generated to date (see Moore et. al., Science Immunology, "Humanization of T cell-mediated immunity in mice", 6(66) 2021) require the use of mouse D and J introns of TRB, as well as intergenetic sequences in the human D1-J2 region, including the DJC2 gene cluster. Without re-mouse re-transgenication, T cell receptors from the DJC2 gene cluster were not observed. However, transgenic mice possessing human D and J introns in the mouse TRB DJC2 cluster successfully generated T cell receptors without requiring mouse TRB D and J introns or intergenetic sequences.

[0452] This mouse contains an inversion of the TCRβ variable locus, which means the variable region is located distally from the wild-type locus, potentially leading to a significant reduction and even inhibition of TCRβ chain production expressed from the host mouse TCRβ chain variable gene segment.

[0453] Such mice contain a fully human insertion of approximately 520 KB of human genomic DNA at position 6:36,074,375 in the mouse genome, from position 7:142,237,898[GRCh38] to position 7:142,813,740[GRCh38]. When measured between human genomic coordinates, this represents a 576 kbp insertion of human DNA. The mice retain the mouse TRB variable gene region in an inverted form, as described above for Example 1.

[0454] The mouse 3' enhancer downstream of TrbC2 was retained.

[0455] Example 3 The figure shows a preferred example of genome design features. Each individual allele and genomic intermediate described below is a distinct element of the present invention and can be combined with other alleles and intermediates described herein.

[0456] Figure 1 shows the mouse TRA locus, the human TRA locus, and the engineered human mouse TRA locus. The human DNA is from chromosome 14:21,570,693 to chromosome 14:22,554,820 (GRCh38) in the third panel. The mouse DNA is numbered relative to GRCm39. A preferred mouse locus contains human Tra(45 V and 51 J)DNA 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)DNA and Trd(8 V and 4 J)DNA replacing the mouse Tra and Trd gene segments. Downstream enhancers of TraC are preferably retained.

[0457] The TRA locus of the present invention was generated as follows: Approximately 1.1 Mbp (specifically 984127 bp) of human genomic DNA from position 14:21,570,693 [GRCh38] to position 14:22,554,820 [GRCh38] was inserted into the mouse genome after mouse coordinate position 14:52,664,870 or 14:52,664,818 (depending on the mouse strain). The mouse TRA locus and Trd locus were deleted from the mouse genome from position 14:52,664,870 to 14:54,463,673, or from 14:52,664,818 to 14:54,463,673. This locus manipulation includes (i) landing pad insertion with large deletion of mouse loci, (ii) insertion of human BAC using RMCE, and (iii) deletion of selectable markers at the locus using piggyBac-mediated removal, using techniques described in Lee et al., Vol. 32, No. 4, April 2014, Nature Biotechnology, and Boroviak et al., Genesis 54:78-85 (2016).

[0458] Figure 2 shows the mouse TRB locus, the human TRB locus, and an engineered human mouse TRB locus in which human DNA is inserted and mouse DNA is inverted to maintain male fertility. The human DNA is from chromosome 7:142,237,898 to chromosome 7:142,813,740 (GRCh38) in the third panel. The mouse DNA is numbered relative to GRCm39. The preferred mouse locus contains human Trb (47 Vs and 13 Js). The preferred mouse retains the mouse TRb locus in the genome in an inverted orientation.

[0459] An inverted TRB locus was generated as follows: approximately 520 KB of human genomic DNA, from position 7:142,237,898 [GRCh38] to position 7:142,813,740 [GRCh38], was inserted at position 6:36,074,375 in the mouse genome. When measured between human genomic coordinates, this represents a 576 kbp human DNA insertion. The inserted human DNA is ligated to the mouse genomic DNA at position 6:41,535,764 (GRCm39) in the mouse. The naturally occurring mouse TCRβ region from 6:40,868,163(GRCm39) to 6:41,515,110(GRCm39) was inverted within the genome using a lox-mediated inversion process based on the principle described in Figure 1b of Lee et al. 2014 Nat Biotechnol April 2014;32(4):356-63, doi: 10.1038 / nbt.2825. Epub March 16, 2014. The mouse enhancer, naturally located downstream of mouse trbc2, was found in the mouse genome downstream of human trbc2.

[0460] Figures 3a and 3b show the replacement of the DNA encoding mouse CD8α with the DNA encoding human CD8α in the mouse genome, and also the replacement of the DNA encoding mouse CD8β with the DNA encoding human CD8β in the mouse genome. Chimeric CD8α polypeptide and chimeric CD8β polypeptide are generated. The locations of the enhancer and DNase sites shown in Figure 3a are described in "Chromatin and CD4, CD8A and CD8B gene expression during thymic differentiation. Kioussis D, Ellmeier W. Nat Rev Immunol. 2002 / 2(12):909-19. doi: 10.1038 / nri952."

[0461] Figure 3b shows in more detail the process of constructing the genome shown in Figure 3a using a dual-targeting vector technique well known in the art, and generating chimeric CD8α and chimeric CD8β loci containing 5' human DNA and 3' mouse DNA.

[0462] Since the two gene loci are closely linked on mouse chromosome 6, the optimal approach is to sequentially humanize them. Alternatively, while it is theoretically possible to modify them together, targeting both simultaneously is technically more difficult given their relative distances.

[0463] To sequentially modify CD8b and CD8a, either gene can be used as a starting point. While the example in Figure 3b shows CD8b first, followed by CD8a, it should be understood that CD8a can be humanized first, followed by CD8b. A short segment from exon 1 to exon 3 of mouse CD8b, along with its encoded intron sequence, is replaced with the equivalent region of human CD8B. The first targeting vector contains homology arms on both sides of the region to be replaced, each approximately 3kb–4kb in length. To yield positive selection, the vector contains a puro-delta-tk selection marker with PiggyBac inverted repeats on both sides. This selection marker is inserted into intron 2 of the human DNA insertion. The exact location is not critical, as this selection marker is seamlessly removed using PBase in the second step, thus the marker could be inserted into, for example, intron 1 or intron 3. Following transfection and selection with puromycin, targeted clones are identified by junction PCR at the 5' and 3' ends. Subsequently, the targeting vector is completely removed by PBase expression, and cells achieving this are selected in FIAU.

[0464] Next, cells successfully humanized to CD8b can be used to humanize CD8a using a similar vector design, as shown in Figure 3b. Targeted clones are selected in puromycin, and cells with the selection marker removed are selected in FIAU following PBase expression. The mouse CD8a sequence from exons 1 to 3 is replaced with the human CD8a sequence from exons 1 to 4. The resulting chimeric locus has six exons as a result of the fusion of human CD8a exon 4 and mouse CD8a exon 3. The second targeting can be performed cis or trans relative to the first targeting. Clones must be targeted cis due to the close linkage of the genes. Clones targeted by cis are identified using methods well known in the art, such as pulsed-field gel electrophoresis and Southern blotting.

[0465] Alternatively, it is also possible to target both CD8a and CD8b together to generate a chimeric locus. A first targeting vector is generated containing exons 1-3 of human CD8b and exons 1-4 of CD8a, isolated by a selectable marker (e.g., PGK neo). This is inserted into the mouse genome using homologous recombination and selection. Exons 1-6 of mouse CD8b and exons 1-3 of CD8a are deleted. A second targeting vector, equipped with a second selectable marker (e.g., PGK puro delta TK), inserts the desired mouse gene components of the CD8b chimeric locus downstream of human CD8b. These include a portion of mouse exon 3, as well as all of mouse exons 4, 5, and 6. The second targeting vector also replaces the human 5'UTR of human CD8a exon 1 with the mouse 5'UTR.

[0466] Regardless of which construction method is followed, the final result is the same (Figure 3a). Similar to the human coding sequences for parts of CD8α and CD8β, human intercalation sequences between exon 1-exon 2 and exon 2-exon 3 are inserted into the rodent genome as part of the human genome insertion, and (additionally in the case of CD8A) a human intercalation sequence between exon 3-exon 4 is inserted into the rodent genome using the coordinates shown. The rodent CD8A locus contains at least parts of human exon 1 (excluding UTR), human exon 2, human exon 3, and human exon 4, fused to part of mouse exon 3, all of mouse exon 4, and all of mouse exon 5. The rodent CD8B locus contains at least parts of human exon 1 (excluding UTR), human exon 2, and human exon 3.

[0467] Figure 4 shows a hypothetical scheme in which the DNA encoding mouse CD8α can be replaced in the mouse genome with the DNA encoding human CD8α, and also shows the replacement of the DNA encoding mouse CD8β with the DNA encoding human CD8β in the mouse genome. This would result in the complete generation of human CD8 polypeptide. The locations of the enhancer and DNase sites shown are described in "Chromatin and CD4, CD8A and CD8B gene expression during thymic differentiation. Kioussis D, Ellmeier W. Nat Rev Immunol. 2002 / 2(12):909-19. doi: 10.1038 / nri952."

[0468] Figures 5a and 5b show the replacement of the DNA encoding mouse β2-microglobulin in the mouse genome with the DNA encoding human β2-microglobulin. Figure 5b shows in more detail the process of creating the genome in Figure 5a. A preferred rodent genome includes human exon 1, human exon 2, and human exon 3 having the coordinates shown, along with intervening human genome sequences.

[0469] The β2 microglobulin locus of the present invention was generated by constructing a targeted DNA vector encoding human β2 microglobulin exons 1, 2, and 3, as well as a genome-interrupted sequence. When this vector is targeted to the mouse genome, the equivalents of mouse exons 1 to 3 are replaced with a sequence under the control of the mouse enhancer located at 5' of exon 1. This targeting results in deletion of mouse exons 1 to 3, thereby inhibiting the expression of mouse β2 microglobulin.

[0470] Figures 6a / 6b show that mouse H2-D1 in the mouse genome is converted to human HLA_A * 02:01 indicates replacement with an MHC class I gene. Figure 6b shows in more detail the process of constructing the genome in Figure 6a. A preferred rodent genome includes the coding regions of human exons 1 to 8 and intervening human genome sequences having the preferred coordinates shown between exon 1 and exon 2, exon 2 and exon 3, exon 3 and exon 4, exon 4 and exon 5, exon 5 and exon 6, exon 6 and exon 7, and exon 7 and exon 8.

[0471] The modified MHC class 1 locus of the present invention was generated by constructing a targeted DNA vector encoding human exons 1-7 and a portion of exon 8, as well as genome-intervening sequences. Following homologous recombination to replace mouse H2-D1 DNA (preventing the expression of mouse MHC H2-D1 DNA), mouse exon 4 was replaced with human exon 4 using a second targeted vector. All other intervening DNA sequences were human. This method results in human MHC class I having mouse exon 4 under the control of a mouse enhancer upstream of human exon 1. The mouse genome may optionally have additional H2K gene knockouts or mutations to prevent the expression of host MHC class I molecules.

[0472] These figures illustrate certain preferred coordinates for genome modification in mice, but they are not limitations to the present invention.

[0473] Example 4 Transgenic mice were created that possess the human TRA gene locus shown in Figure 1 and / or the human TRB gene locus shown in Figure 2. The hTCR transgenic mice produced by the inventors may be referred to herein as OpTiMus® mice.

[0474] Table 1 shows the human TCR variable region gene segments theoretically present at the TRAV and TRBV loci in mice, based on the genes thought to be present in the BAC used to generate the mice.

[0475] [Table 1]

[0476] The following genes are not present in the human BAC sequence used to generate the hTCR transgenic mice, therefore their expression is impossible: TRBV4-3 (related to TRBV4-2, TRBV4-1, and TRBV1), TRBV5-8 (related to TRBV5-5, TRBV5-3, TRBV5-7, TRBV5-4, etc.) TRBV6-3 (same as TRBV6-2), TRBV6-9 (related to TRBV6-5, TRBV6-6, TRBV6-8, TRBV6-7, etc.) TRBV7-8 (related to TRBV7-4, TRBV7-6, TRBV7-7, etc.).

[0477] Gene segment expression was evaluated by sequencing bulk RNA extracted from the spleen of OpTiMus® mice. All transcripts that could be assigned to individual gene segments were counted, regardless of whether they were expressed in-frame or out-of-frame. To reduce PCR bias, UMIs (Unique Molecular Identifiers) were counted instead of the number of sequence reads. Two transcripts (different UMIs) may have the same nucleotide sequence. The presence and expressibility of a gene segment is confirmed by the detection of at least one transcript from that segment.

[0478] A) Use of VJ segments in TRBV The transgenic human BAC sequence contains 43 functional TRBV genes and 13 functional TRBJ genes (Table 1). Bulk spleen RNA sequencing of TRB genes expressed in homozygous TRB transgenic mice demonstrated widespread recombination and expression of TRBV and TRBJ genes (Figures 10, 11, and 12). Since each TRBD is associated with a TRBJ and a constant region, it is expected that all members of the TRBD gene segment can also be expressed.

[0479] All functional TRBJ and TRBV genes were expressed in the bulk sequencing results. This confirms that all functional human gene segments of the TCRβ variable region are expressed from the transgenic mouse locus.

[0480] B) Use of VJ segments in TRAV The transgenic human BAC sequence contains 44 functional TRAV genes and 50 functional TRAJ genes (Table 1). Bulk spleen RNA sequencing of TRB genes expressed in homozygous TRA transgenic mice demonstrated widespread recombination and expression of TRAV and TRAJ genes.

[0481] All functional TRAJ genes and all but two functional TRAV genes (TRAV7 and TRAV18) were represented in the bulk sequencing results (Figures 13, 14, and 15). This confirms the expression of the TCRα human gene segment from the transgenic mouse locus.

[0482] C) Relative use of TRBJ1-C1 and TRBJ2-C2 Bulk sequencing of TRB transgenic spleen RNA indicates a split of approximately 40:60 in the use of TRBJ1-C1:TRBJ2-C2 (Figure 16).

[0483] D) Comparison with human TCR gene segment expression The repertoire of peripheral TCR expression is thought to be shaped by two processes: (i) the tendency of certain VDJs to undergo somatic recombination, and (ii) positive and negative selection in the thymus, which depends on the pMHC encountered by the TCR during development in the thymus.

[0484] We compared two publicly available datasets of TCR gene expression in healthy humans with results from OpTiMus™ mice. Freeman et al. (Genome Res 19(10):1817-1824, 2009) reported TRBV / TRBJ expression in PBMC RNA collected from 550 healthy individuals. Kitaura et al. (BMC Immunology 17:38, 2016) reported TRAV / TRAJ and TRBV / TRBJ expression in 20 healthy Japanese individuals.

[0485] The use of TRBV and TRBJ genes in Optimus® mice was compared with publicly available data from humans. TRB transgenic mice and humans express nearly identical TRBV and TRBJ genes. In particular, TRBV20.1 and TRBV5.1 are the two most expressed genes in humans and transgenic mice (Figures 11 and 17). Very close consistency was also observed between TRBJ use in humans and transgenic mice (Figure 18).

[0486] The length of the CDR3 region spanning the highly variable VDJ ligation region ranges from approximately 20 bp to 60 bp, with a peak at 42 bp, which is indistinguishable from that observed in humans (Kitaura et al, 2016) (Figure 19).

[0487] The approximately 40:60 TRBC utilization split favorable to TRBJ2-C2 observed in humans is also reproduced in transgenic mice (Figure 16).

[0488] The use of the TRAV and TRAJ genes in OpTiMus® mice was compared with publicly available human data. Most of the TRAV gene, which is sufficiently expressed in the human dataset, is also expressed in mice. Compared to the human dataset, expression at the distal end of the TRAV locus is relatively low (Figures 14 and 20). Good expression levels are observed throughout the functional TRAJ gene in both human and TRA transgenic mice (Figures 15 and 21).

[0489] The length of the CDR3 region spanning the highly variable VJ ligation region ranges from approximately 24 bp to 57 bp, with a peak at 42 bp, which is indistinguishable from that observed in humans (Kitaura et al, 2016) (Figure 19).

[0490] Furthermore, the linkage diversity created by recombination of the v and j gene segments (Figures 10 and 13) also reflects the linkage diversity observed in humans. This is important because the linkages between gene segments form the CDR3 of the TCR (which is the most variable component and directly interacts with peptides).

[0491] E) Comparison between OpTiMus® mice and other humanized TCR mice Humanized TCRab mice have been described by two companies: VelociT mice from Regeneron and MyT mice from T-knife.

[0492] Humanized mice of Regeneron ("VelociT") contain humanized genes targeting TRA / TRB / CD8ab / CD4 / b2m / MHC I and MHC II (Moore et al, Sci. Immunol. 6, eabj4026 2021). TRA and TRB include mouse constant region genes. Early types of Regeneron mice, which possessed fully human TRBJ1 and TRBJ2 sequences, expressed only the J1-C1 cluster. This defect was corrected by converting the intergenic JC region to that of the mouse. OpTiMus™ mice did not face this problem and expressed both TRBJ clusters at rates similar to those seen in humans.

[0493] T-Knife mice (Li et al, Nature Medicine 16:9. 1029-1034 2010) were generated by randomly inserting YAC clones containing TRA and TRB into a Tra / Trb knockout background, and also included humanized MHC class I fused to b2 microglobulin. The use and expression of TCRs were evaluated simply by the presence or absence of PCR products amplified by RT PCR from recombinant VJ genes or RNA, without quantitative information on the relative expression of different genes.

[0494] In previous transgenic mice, the expression status of all V and J genes within the inserted genomic region does not appear to have been reported. Figure 22 summarizes the gene expression status at the TCR locus of previous transgenic mice from publicly available data (Moore et al., 2021 and Li et al., 2010 cited above), and compares this with the gene expression status at the TCR locus of OpTiMus mice characterized herein.

[0495] Bulk sequencing analysis of OpTiMus® mice revealed complete or near-complete expression of the inserted TCR gene segment. The only transcripts not detected in this analysis were TRAV7 and TRAV18. Notably, the TRBJ2-6 gene was found (Figures 10, 12, and 22d). The absence of this gene in Regeneron mice was attributed to the fact that Trbj2-6 in mice is a pseudogene. In OpTiMus® mice, TRBJ2-6 was clearly expressed at levels similar to those observed in humans.

[0496] The gene segment expression data identified in transgenic mice is shown in Figure 22 and summarized in Table 2 below.

[0497] [Table 2]

[0498] Based on the latest analysis of gene expression in OpTiMus®TRA mouse strains and OpTiMus®TRB mouse strains, OpTiMus® mice possessing the transgenic TCRαβ locus appear to have the most diverse human repertoire ever observed in any humanized TCR mouse.

[0499] Example 5 OpTiMus® mice were produced with the following genotypes, which are homozygous ("hom"), heterozygous ("het"), or wild-type ("WT") at the indicated gene locus.

[0500] [Table 3]

[0501] The gene loci were as follows: H2K inactivated mouse MHC class I gene locus H2K, HLA-A *02 A completely human HLA-A that replaces the mouse gene shown in Figure 6a * Mouse MHC I locus H2D, containing a nucleic acid insertion encoding 02. The β2 microglobulin locus, which contains a nucleic acid insertion that completely encodes human β2m, replaces the mouse gene shown in Figure 5a, hCD8a / b: The CD8 locus contains insertions of nucleic acids that completely encode human CD8a and completely human CD8b, replacing the corresponding mouse genes shown in Figure 3a. hTRA Figure 1b contains the insertion of nucleic acids that completely encode human TRAV and TRAC into the mouse TCRα locus, replacing the corresponding mouse genes. hTRB: The TCRβ locus, shown in Figure 2b, contains an insertion of nucleic acids that completely encode human TRBV and TRBC, along with inactivation and inversion of the endogenous mouse TRBV region and deletion of mouse TRBC.

[0502] Since no other introduced genes were introduced, it was assumed that all other genes were wild-type (WT).

[0503] T lymphocyte populations and CD8 cell surface expression were evaluated in blood samples from antigen-unsensitized OpTiMus™ mice 1-7. Fresh blood was evaluated using FACS to detect mouse CD3ε and human CD8b. The results are shown in Figure 23. CD3 in all mice + Cells were detected. In all mice except mouse 2, normal levels of hCD8 were present in 1% to 5% of total blood PBMCs. + The cells were detected. This was expected because mouse 2 only expresses mouse CD8, which is not detected even when using a probe for human CD8b. CD3 + Cells and CD8 + The cell levels were within the normal range, and were comparable to those in wild-type mice, indicating that T lymphocytes in transgenic mice develop normally.

[0504] Furthermore, immune cells were collected from the spleens of three additional antigen-unsensitized fully homozygous mice (mice 8, 9, and 10, which have the same fully homozygous genotype as mouse 7 in Table 3 above) and stained for cell surface markers mCD3, mCD4, hCD8b, hTRBC-1, and mCD19 (Figure 24). Unsensitized mice with fully humanized fully homozygous transgenes (HLA, B2M, CD8a, CD8b, TRA, and TRB), indicated as zygosity code K in Table 3, had 57%-65% B cells and CD4 + T cells 10%-15%, and CD8 + The lymphocyte population showed a normal range of 2.6% to 4.5% T cells. Furthermore, CD3 + The T cell population showed a TRBC-1 subgroup of 5%–7.5% and a TRBC-1-negative subgroup of 4.1%–5.8%. The data demonstrate that the mice exhibit normal production of major lymphocyte populations, including CD4 T cells, CD8 T cells, and B cells.

[0505] Gene expression libraries were constructed from splenocytes of mice 8, 9, and 10 using single-cell mRNA sequencing in addition to cell surface marker staining. Immune cell types and T cell subset phenotypes were classified based on gene expression profiles including B cells, T cells, NK cells, monocytes, and dendritic cells (cDCs). The proportions of total splenocytes identified as CD4 T cells, CD8 T cells, and gamma / delta T cells were within the generally known range for mouse splenocytes. CD4 T cells accounted for 11%–14% of the total splenocyte population, while CD8 T cells accounted for 5%–9.5%. Gamma / delta T cells accounted for approximately 1% of the total population (Figure 25).

[0506] Example 6 The T-cell immune response to peptide immunization was characterized in OpTiMus® mice, which have various conjugation genotypes.

[0507] Mice were immunized with purified target peptides combined with adjuvants (incomplete Freund's adjuvant and CpG ODN 1826). The target peptides in this study are cancer-associated T cell epitopes known to be presented on HLA-A*02.

[0508] Materials for tissue preparation / cell staining and sorting Culture media: Tissue preparation / culture medium: RPMI-1640 + 10% FBS + 2 mM L-glutamine + 20 mM HEPES; Cell concentration / staining buffer: PBS + 2% FBS + 20 mM HEPES + 1 mM EDTA; Collection buffer for sorted cells: PBS + 20% FBS + 20 mM HEPES; Cell freezing medium: FBS + 10% DMSO.

[0509] Reagent: EasySep mouse CD8 + T cell isolation kit (STEMCELL, No. 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-FITC (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), eFluor 780 Fixable Viability dye (ThermoFisher, 65-0865-18), Lightening link barcode conjugation kit (Abcam, ab270703 / 05 / 09), TotalSeq-C series hashtag antibodies (Biolegend, 155861, 155863, etc.).

[0510] A tetramer obtained from MBL for the detection of antigen-specific CD8 T cells.

[0511] method Immunization method: Peptide-based immunization Immunogen: Purified peptide (over 95% purity, Genscript) Adjuvant: Incomplete Freund's adjuvant (IFA, Invivogen), CpG ODN 1826 (Invivogen) Prime dose (per mouse): 100 μg peptide emulsified with 50 μl IFA and 50 μg CpG ODN in 50 μl PBS Boost dose (per mouse): 50 μg peptide emulsified with 50 μl of IFA and 50 μg CpG ODN in 50 μl of PBS. Peptide-based immunogens were administered via subcutaneous injection.

[0512] These studies typically used a prime and 2-3 boost schedule, with a 21-day interval between primes and boosts, and a 14-day interval between each boost. Alternative prime-boost regimens also used weekly boosts. Typically, tissue was collected 7 days after each boost.

[0513] Tissue preparation, CD8 + Staining for T cell enrichment and sorting. Mesenteric lymph nodes, inguinal lymph nodes, and the entire spleen were collected from each mouse for sorting. In short, the lymph nodes and spleen were diced and passed through a 40 μM cell strainer to produce a homogeneous cell suspension. The cells were further concentrated using the Stemcell Kit (19853) to obtain CD8 + The T cells were prepared as a suspension (typically 80% purity). The concentrated cells were centrifuged and kept on ice for staining.

[0514] MBL tetramer, enriched with up to 2.5 million CD8 +For mouse T cells, both the target tetramer-PE and the negative control tetramer-APC were used at a final incubation volume of 0.05 μg / 100 μl to 0.1 μg / 100 μl for each staining stage. After adding the tetramers, the cells were kept on ice and incubated for 15 minutes. Subsequently, a staining cocktail containing anti-hCD8b-viablue (Miltenyi), anti-hTRBC-1-FITC (Biolegend), and eFluor 780 Fixable Viability dye was added. At this point, a TotalSeq-C series hashtag antibody was also added to each mouse sample for the purpose of hashtagging. After incubating the cells on ice for a further 30 minutes, they were washed twice with staining buffer.

[0515] Tetramer-specific CD8 + T cell sorting The cells were resuspended in 300 μl–400 μl of staining buffer, pushed through a 35 μM cell strainer, held on ice, and then captured with a cell sorter.

[0516] Cells were gated using the following gating strategy: lymphocytes > single cells > living cells > hCD8b + >Target Tetramer + and negative control tetramer - The cells were sorted into 1.5 ml microcentrifuge tubes containing approximately 300 μl of cold collection buffer.

[0517] The immune response rate was measured by antigen-specific tetramergating in FACS, using CD8 specific to the target antigen. + Responders were defined based on the percentage of lymphocyte population compared to control (non-immunized) mice. Mice exhibiting an immune response had a higher percentage of antigen-specific cells compared to control mice. The percentage in responders was typically at least 0.1% of the population, while the percentage in controls was typically less than 0.05% of the population.

[0518] Single-cell mRNA sequencing, library preparation, and NGS Immediately after collection, target cells were isolated, and mRNA extraction was performed for single-cell RNA sequencing to generate mouse TCR VDJ libraries, gene expression (GEX) libraries, and cell surface protein libraries. The final libraries were then sequenced after quality control.

[0519] Analysis of single-cell NGS data revealed CD8 cells exhibiting clonal expansion and proliferation, as well as activation, exhaustion, and proliferation phenotypes. + We focused on the cells and determined whether the responding TCR clone type possessed a completely human TCR.

[0520] result In the initial study, hTCR mice with various conjugation alleles were subjected to peptide-based immunization, and splenocytes were stained for antigen-specific tetramer binding and sorted to identify target tetramer-specific CD8. + They gathered the group.

[0521] [Table 4]

[0522] [Table 5]

[0523] Figure 26a shows representative plots from six mice with different zygosities (zygosities B, D, E, I, J, K - Table 4) that generated responses from cells with fully humanized TCRs. The plot from mouse 16 shows that in the case of unsensitized control mice, the level of the target tetramer was CD8 +This indicates that it represents 0.015% of the entire population. Immunized mice with six different zygosities showed a range of tetramer-specific populations from 0.18% to 1.74%. This indicates that a fully human TCR can be found not only using fully humanized homozygous mice (zygosity K) but also using other zygosities.

[0524] Two mice (mouse 2 and mouse 28) carrying mouse WT CD8 (conjugated G) did not respond to the same type of immunization as mice carrying human CD8 (Figure 26b). This indicates that human CD8 enables human TCRα / β TCR signaling capability via homozygous, fully human MHC class I in this immunization context.

[0525] The T cell response in mice that are heterozygous for TRB and homozygous for TRA (zygosity I) was specifically investigated in mice that are homozygous for both TRB and TRA (zygosity K).

[0526] In Figure 27, OptiMus mice with conjugation I showed antigen-specific CD8 in response to weekly peptide-based boost immunization. + The mice exhibited a T cell response. A total of 10 OptiMus mice were immunized with the target peptide using weekly booster regimens. Data were collected from 2 mice, 2 mice, 3 mice, and 3 mice for booster 1, booster 2, booster 3, and booster 4, respectively. Mice numbered 17, 18, 19, and 20 represent data for booster 1, booster 2, booster 3, and booster 4, respectively. The data demonstrate that homozygous for TRA and heterozygous for TRB (zygosity I) OptiMus mice can generate an antigen-specific T cell response from prime-boost regimens with single or multiple booster injections. Furthermore, they generated a wide range of responses from separate individuals (from 0.8% to 4.5% of the entire CD8 population).

[0527] In Figure 28, OptiMus mice with a fully humanized allele (conjugating K) showed antigen-specific CD8 in response to peptide-based immunization. + The mice exhibited a T cell response. A total of 23 OptiMus mice were immunized with the target peptide using bi-weekly boost regimens. Data were collected 7 days after boost 2 and boost 3. Mice numbered 16 and 24 are unsensitized controls for boost 2 and boost 3, respectively. Mice 21, 22, and 23 represent data for boost 2 (Figure 28a), while mice numbered 25, 26, and 27 represent data for boost 3 (Figure 28b). OptiMus mice with conjugation K showed a wide range of responses from individuals and demonstrated significant antigen-specific responses from both boost 2 (0.2%–1%) and boost 3 (0.1%–4.7%). Furthermore, the overall response rate of these 23 mice was 21 out of 23 (over 90%), which is a high response rate.

[0528] When comparing immune responses from homozygous and heterozygous individuals to the modified allele, it is noteworthy that the strongest hTCR immune response was achieved in animals lacking mouse TCRα. While mice heterozygous at the TRA locus were still able to fully generate human TCRs in response to immunization (e.g., mouse 11), animals with homozygous human TCRα were generally a better source of human TCRs than animals heterozygous at the TCRα locus. This may be because, in heterozygotes, the endogenous mouse TCRα constant domain competes with the human TCRα constant domain for CD3 binding, resulting in the generation of T cells primarily possessing mouse TCRα, which is unlikely to occur in homozygotes as mouse TRAC is not expressed. Immunizing mice homozygous at the TCRα locus (TRA) and mice that are either homozygous or heterozygous at the TCRβ locus (TRB) yielded target-specific CD8 +The generation of a T-cell response confirmed that these mice are useful for identifying fully human TCR sequences with therapeutic potential.

[0529] Example 7 As shown in Figure 2, we prepared for mating using 17 male mice that had a homozygous insertion of human TRB at the endogenous TRB gene locus and in which the endogenous TRBV was inverted to an ectopic position by chromosomal region inversion. The breeding data are shown in Table 6.

[0530] [Table 6]

[0531] Since littermates were produced in all matings, the fertility of mice carrying the mouse TRBV, which had been moved from the endogenous locus, was confirmed. This avoids the unwanted decrease in fertility observed in male mice lacking TRBV (Example 1).

[0532] Example 8 124 T cell receptor sequences isolated as antigen binders from mice immunized with the target peptide described in Example 6 were selected for expression in soluble "T-ceptor" form. The TCRα variable domain and TCRβ variable domain from the mouse were incorporated into a bivalent soluble molecule containing two TCR "Fab"-like regions dimerized via the Fc region of the antibody. Each TCR antigen-binding "Fab" consisted entirely of a human α polypeptide chain and a human β polypeptide chain: an α chain containing the human TCRα variable domain from the mouse, linked at the C-terminus to the human TCRα constant domain, and a β chain containing the human TCRβ variable domain from the mouse, linked at the C-terminus to the human TCRβ constant domain. The TCRs contained diverse combinations of vα and vβ gene segments (Table 7).

[0533] [Table 7] TIFF2026528791000009.tif221170TIFF2026528791000010.tif122170

[0534] Nucleic acids encoding soluble TCRs were transfected into Expi293T cells, and small-scale (3 ml) protein expression was performed. The proteins were purified using protein A affinity purification 5 days after transfection. The concentration of soluble TCRs was measured using UV / Vis spectroscopy. All TCR molecules were expressed, and the concentration of purified proteins ranged from 0.01 mg / ml to 1.9 mg / ml (Figure 29).

Claims

1. (i) an unreorganized T cell receptor (TCR) α variable gene locus comprising at least one human Vα segment and at least one human Jα segment, wherein the TCRα variable gene is operably linked to the human TCRα constant gene sequence, (ii) an unreorganized TCRβ variable gene locus comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment, wherein the unreorganized TCRβ variable gene is operably linked to a human TCRβ constant gene sequence, Includes, Unreorganized human T cell variable region gene segments can be rearranged to form genes encoding human T cell receptor variable domains, the unreorganized TCRα variable gene locus is located at the endogenous rodent TCRα gene locus, and the unreorganized TCRβ variable gene locus is located at the endogenous rodent TCRβ gene locus. Genetically modified rodents.

2. (i) an unreorganized T cell receptor (TCR) α variable gene locus comprising at least one human Vα segment and at least one human Jα segment, wherein the TCRα variable gene is operably linked to the human TCRα constant gene sequence, (ii) an unreorganized TCRβ variable gene locus comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment, wherein the unreorganized TCRβ variable gene is operably linked to a human TCRβ constant gene sequence, Includes, Unreorganized human T cell variable region gene segments can be rearranged to form genes encoding human T cell receptor variable domains, the unreorganized TCRα variable gene locus is located at the endogenous rodent TCRα gene locus, and the unreorganized TCRβ variable gene locus is located at the endogenous rodent TCRβ gene locus. The aforementioned rodent genome is, [A] A nucleic acid sequence encoding a human CD8α polypeptide located at the endogenous rodent CD8α coreceptor locus, a nucleic acid sequence encoding a human CD8β polypeptide located at the endogenous rodent CD8β coreceptor locus, and a nucleic acid sequence encoding a human MHC class I polypeptide located at the endogenous rodent MHC class I locus, or [B] A nucleic acid sequence encoding a chimeric human-rodent CD8α polypeptide located at the endogenous rodent CD8α coreceptor locus, a nucleic acid sequence encoding a chimeric human-rodent CD8β polypeptide located at the endogenous rodent CD8β coreceptor locus, and a nucleic acid sequence encoding a human MHC class I polypeptide located at the endogenous rodent MHC class I locus (wherein the chimeric CD8 protein can bind to the human MHC class I), or [C] A nucleic acid sequence encoding a rodent CD8α polypeptide located at an endogenous locus, a nucleic acid sequence encoding a rodent CD8β polypeptide located at an endogenous locus, and a nucleic acid sequence encoding a chimeric human-rodent MHC class I polypeptide located at an endogenous rodent MHC class I locus (wherein the rodent CD8 protein can bind to the chimeric MHC class I). Includes any of the following: The genetically modified rodent according to claim 1, wherein the rodent genome, if present, comprises a nucleic acid sequence encoding a human β2-microglobulin polypeptide located at the endogenous rodent β2-microglobulin locus.

3. (i) an unreorganized T cell receptor (TCR) α variable gene locus comprising at least one human Vα segment and at least one human Jα segment, wherein the TCRα variable gene is operably linked to the human TCRα constant gene sequence, (ii) an unreorganized TCRβ variable gene locus comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment, wherein the unreorganized TCRβ variable gene is operably linked to a human TCRβ constant gene sequence, Includes, Unreorganized human T cell variable region gene segments can be rearranged to form genes encoding the human T cell receptor variable domain. Genetically modified rodent cells, such as ES cells, in which the unreorganized TCRα variable gene locus is located at the endogenous rodent TCRα gene locus, and the unreorganized TCRβ variable gene locus is located at the endogenous rodent TCRβ gene locus.

4. (i) an unreorganized T cell receptor (TCR) α variable gene locus comprising at least one human Vα segment and at least one human Jα segment, wherein the TCRα variable gene is operably linked to the human TCRα constant gene sequence, (ii) an unreorganized TCRβ variable gene locus comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment, wherein the unreorganized TCRβ variable gene is operably linked to a human TCRβ constant gene sequence, Includes, Unreorganized human T cell variable region gene segments can be rearranged to form genes encoding human T cell receptor variable domains, the unreorganized TCRα variable gene locus is located at the endogenous rodent TCRα gene locus, and the unreorganized TCRβ variable gene locus is located at the endogenous rodent TCRβ gene locus. The aforementioned rodent genome is, [A] A nucleic acid sequence encoding a human CD8α polypeptide located at the endogenous rodent CD8α coreceptor locus, a nucleic acid sequence encoding a human CD8β polypeptide located at the endogenous rodent CD8β coreceptor locus, and a nucleic acid sequence encoding a human MHC class I polypeptide located at the endogenous rodent MHC class I locus, or [B] A nucleic acid sequence encoding a chimeric human-rodent CD8α polypeptide located at the endogenous rodent CD8α coreceptor locus, a nucleic acid sequence encoding a chimeric human-rodent CD8β polypeptide located at the endogenous rodent CD8β coreceptor locus, and a nucleic acid sequence encoding a human MHC class I polypeptide located at the endogenous rodent MHC class I locus (wherein the chimeric CD8 protein can bind to the human MHC class I), or [C] A nucleic acid sequence encoding a rodent CD8α polypeptide located at an endogenous locus, a nucleic acid sequence encoding a rodent CD8β polypeptide located at an endogenous locus, and a nucleic acid sequence encoding a chimeric human-rodent MHC class I polypeptide located at an endogenous rodent MHC class I locus (wherein the rodent CD8 protein can bind to the chimeric MHC class I). Includes any of the following: The genetically modified rodent cell according to claim 3, wherein the rodent genome, if present, comprises a nucleic acid sequence encoding a human β2-microglobulin polypeptide located at the endogenous rodent β2-microglobulin locus.

5. (i) an unreorganized T cell receptor (TCR) α variable gene locus comprising at least one human Vα segment and at least one human Jα segment, wherein the TCRα variable gene is operably linked to the human TCRα constant gene sequence, (ii) an unreorganized TCRβ variable gene locus comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment, wherein the unreorganized TCRβ variable gene is operably linked to a human TCRβ constant gene sequence, Includes, Unreorganized human T cell variable region gene segments can be rearranged to form genes encoding the human T cell receptor variable domain. A genetically modified rodent T cell in which the unreorganized TCRα variable gene locus is located at the endogenous rodent TCRα gene locus, and the unreorganized TCRβ variable gene locus is located at the endogenous rodent TCRβ gene locus.

6. The endogenous TCRα variable gene locus does not express a functional endogenous TCRα polypeptide, and is homozygous at the TCRα gene locus, A genetically modified rodent or rodent cell according to any one of claims 1 to 5, wherein it does not express a functional endogenous TCRβ polypeptide from an endogenous TCRβ variable gene locus and is homozygous at the TCRβ gene locus.

7. (i) A rodent or rodent cell according to any one of the preceding claims, wherein the rodent holds an endogenous TCRα variable gene locus and / or an endogenous rodent TCRβ variable gene locus, the retained endogenous rodent TCRα variable gene locus is a non-functional locus, and / or the retained endogenous rodent TCRβ variable gene locus is a non-functional locus, and optionally, all or part of the retained endogenous rodent TCRα variable gene locus and / or TCRβ variable gene locus are inverted relative to their normal orientation in the rodent genome, or (ii) A fertile rodent or rodent cell wherein at least the rodent TCRβ loci from rodent Vβ1 to Dβ1 are inverted within the rodent genome with respect to their natural orientation within the rodent genome, and optionally further comprising inserting nucleic acids, such as DNA encoding all or part of TCRβ from a different species than the rodent, into the rodent genome, such as inserting human DNA encoding all or part of TCRβ. A genetically modified rodent or rodent cell that is one of the following.

8. (i) A rodent according to any one of the preceding claims having substantially wild-type fertility, wherein the rodent TCRβ locus is deleted from at least rodent Vβ1 to Dβ1, and the genome additionally includes one or more or all of the deleted serine protease genes Press58, Press59, Press3b, Try4, Try5, Try10, Press3, Press1, and Press2, (ii) A rodent that is substantially wild-type fertile, wherein the rodent TCRβ locus is deleted from at least rodent Vβ1 to Dβ1, and the genome additionally contains one or more or all of the deleted rodent serine protease genes Press58, Press59, Press3b, Try4, Try5, Try10, Press3, Press1, and Press2, 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, a rodent that is substantially wild-type fertile. A genetically modified male rodent that is one of the following:

9. 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, One or more of, including all of them, and / or The following human Jα gene segments: 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, A genetically modified rodent or rodent cell according to any one of the preceding claims, comprising one or more, or optionally all, of the TCRα variable gene loci.

10. 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, One or more of, including all of them, and / or 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, A genetically modified rodent or rodent cell according to any one of the preceding claims, comprising one or more, or any all, of the TCRβ variable gene loci.

11. A genetically modified rodent or rodent cell according to any one of the preceding claims, wherein all of CD3ε, CD3γ, CD3δ, and CD3ζ are endogenous wild-type.

12. A method for creating genetically modified rodents that express human T cell receptors, (i) Inserting an unreorganized human TCRα variable gene locus into the endogenous rodent TCRα variable gene locus, which includes at least one human Vα segment and at least one human Jα segment operably linked to the human TCRα constant region, (ii) Inserting an unreorganized human TCRβ variable gene locus into the endogenous rodent TCRβ variable gene locus, comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment operably linked to the human TCRβ constant region, (iii) Inserting a nucleic acid sequence encoding a human CD8α polypeptide into the endogenous rodent CD8α gene locus, (iv) Inserting a nucleic acid sequence encoding a human CD8β polypeptide into the endogenous rodent CD8β gene locus, (v) Inserting a nucleic acid sequence encoding a human MHC class I polypeptide into the endogenous MHC class I gene locus, (vi) Inserting a nucleic acid sequence encoding human β2-microglobulin into the endogenous rodent β2-microglobulin gene locus, Includes, A method wherein the host gene loci at each insertion site are inactivated or deleted, and as a result, the rodent does not express native rodent TCR, MHC class I, or CD8, and optionally does not express rodent β2 microglobulin.

13. A method for producing a fertile rodent or rodent cells, comprising the step of inverting the rodent TCRβ in the rodent genome from at least rodent Vβ1 to Dβ1, and further comprising optionally inserting nucleic acids such as DNA encoding all or part of TCRβ from a different species than the rodent into the rodent genome, such as human DNA encoding all or part of TCRβ.

14. A method for creating a genetically modified fertile rodent that expresses a human T cell receptor, comprising deleting the serine protease genes Press58, Press59, Press3b, Try4, Try5, Try10, Press3, Press1, and Press2, and deleting at least the rodent TCRβ from rodent Vβ1 to Dβ1, wherein one or more or all of the deleted serine protease genes Press58, Press59, Press3b, Try4, Try5, Try10, Press3, Press1, and Press2 are reinserted into the rodent genome.

15. A method for producing a fertile rodent comprising a genetic knockout of an endogenous T cell receptor β chain polypeptide, comprising deleting the serine protease genes Press58, Press59, Press3b, Try4, Try5, Try10, Press3, Press1, and Press2, and deleting the rodent TCRβ from at least rodent Vβ1 to Dβ1, wherein one or more or all of the deleted serine protease genes Press58, Press59, Press3b, Try4, Try5, Try10, Press3, Press1, and Press2 are reinserted into the rodent genome.

16. A method for creating fertile rodents, in any order: (i) Deleting one or more or all of the serine protease genes Press58, Press59, Press3b, Try4, Try5, Try10, Press3, Press1, and Press2, thereby deleting the rodent TCRβ genomic DNA from at least rodent Vβ1 to Dβ1, and (ii) Inserting one or more or all of the serine protease genes Press58, Press59, Press3b, Try4, Try5, Try10, Press3, Press1, and Press2 into the rodent genome. Methods that include...

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

18. A rodent or rodent cell according to any one of claims 1 to 17, or a method for producing the rodent or rodent cell, wherein the rodent genome comprises DNA that completely encodes the human TCRβ chain and includes a human V gene segment, a human D gene segment, a human J gene segment, and a human C gene segment, wherein the rodent or rodent cell is capable of fully expressing the human TCRβ chain, and the rodent genome comprises a human intergene DNA sequence and / or human intron DNA sequence which is located between the human D1 gene segment and the human C2 gene segment and is preferably a human intergene DNA sequence and / or human intron sequence that is naturally present together with the human D gene segment and the human J gene segment. A method wherein, optionally, all of the human DNA sequences between the human D1 gene segment and the human C2 gene segment are genomic human DNA.

19. A rodent or rodent cell having a genome that fully encodes the human TCRβ chain and includes DNA comprising the human V gene segment, the human D gene segment, the human J gene segment, and the human C gene segment, wherein the rodent or rodent cell is capable of fully expressing the human TCRβ chain, and the rodent genome comprises a human intergene DNA sequence and / or human intron DNA sequence that is located between the human D1 gene segment and the human C2 gene segment, preferably a human intergene DNA sequence and / or human intron sequence that is naturally present together with the human D gene segment and the human J gene segment. A rodent or rodent cell in which, optionally, all of the human DNA sequence between the human D1 gene segment and the human C2 gene segment is genomic human DNA.

20. A method for producing a human T cell receptor in a rodent, comprising inserting DNA into the genome of the rodent that completely encodes a human TCRβ chain and includes a human V gene segment, a human D gene segment, a human J gene segment, and a human C gene segment, wherein the rodent is capable of fully expressing the human TCRβ chain, and here, The insertion includes a human intergene DNA sequence and / or human intron DNA sequence, which is preferably a human intergene sequence and / or human intron sequence that is naturally present together with the human D gene segment and the human J gene segment, located between the human D1 gene segment and the human C2 gene segment. A method wherein, optionally, all of the human DNA sequences between the human D1 gene segment and the human C2 gene segment are genomic human DNA.

21. A method for producing a human T cell receptor for a target antigen, (i) optionally a step of making a rodent as described in any one of the preceding claims, (ii) a step of immunizing a rodent described in any one of the preceding claims with the target antigen, (iii) A step of initiating an immune response in the rodent, (iv) optionally, a step of isolating the T cells and determining the nucleic acid sequence of the human TCR variable region expressed by the T cells from the rodent that are reactive with the antigen of the object, (v) A step of expressing the human T cell receptor or human T cell receptor variable region in cells, and optionally further formulating the expressed human T cell receptor or human T cell receptor variable region together with a pharmaceutically acceptable additive, or (vi) Inserting a nucleic acid encoding the human T cell receptor or the human T cell receptor variable region into a human cell or animal cell, etc., ex vivo or in vitro, and optionally formulating the cell containing the inserted nucleic acid for delivery to a human or animal, respectively, or (vii) A step of formulating the nucleic acid (e.g., RNA or DNA) encoding the human T cell receptor or the human T cell receptor variable region together with an appropriate delivery carrier such as a lipid or liposome, and delivering it in vivo to a patient who requires delivery; Methods that include...

22. A method of treating individuals that require treatment, (i) Delivering to the individual a soluble TCR molecule comprising the human T cell receptor variable region of step (v) of claim 21, and optionally comprising the human T cell receptor constant region, (ii) Delivering the cells described in step (vi) of claim 21 to a patient who requires delivery, (iii) Delivering a nucleic acid (e.g., RNA, e.g., mRNA or DNA) that is formulated according to step (vii) of claim 21 to a patient in need of delivery, Methods that include...

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