Mice expressing humanized T cell co-receptors

Abstract

To provide mice expressing a humanized T cell co-receptor.SOLUTION: The invention provides genetically modified non-human animals that express chimeric human / non-human T cell co-receptor polypeptides (e.g., CD4, CD8α, CD8β), as well as embryos, cells and tissues comprising the polypeptides. Also provided are constructs for making the genetically modified animals and methods of making the same. In various embodiments, provided herein is a genetically modified non-human animal, comprising a nucleotide sequence encoding a chimeric human / non-human T cell co-receptor polypeptide.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Cross-reference with related applications This application claims, under 35 U.S.C. §119(e), the benefit of U.S. Provisional Patent Application No. 61 / 890,915, filed on 15 October 2013, and U.S. Provisional Patent Application No. 61 / 766,762, filed on 20 February 2013, which are incorporated herein by reference in their entirety.

[0002] Sequence List This specification refers to the sequence listing submitted electronically on February 20, 2014, as an ascii.txt file named "2010794-0441_ST25". This .txt file was created on February 13, 2014, and has a size of 47kb.

[0003] Field of Invention This invention relates to non-human animals (e.g., rodents, e.g., mice or rats) genetically engineered to express humanized T cell co-receptors. This invention relates to non-human animals genetically engineered to express humanized CD4 or CD8 co-receptors, as well as embryos, tissues, and cells expressing said co-receptors. This invention further relates to non-human animals engineered to co-express humanized CD4 co-receptor and humanized major histocompatibility complex (MHC) II. This invention further relates to non-human animals engineered to co-express humanized CD8 co-receptor and humanized MHC I. Methods for creating genetically engineered animals that express humanized T cell co-receptors (e.g., humanized CD4 or CD8) are also provided. Methods for using genetically engineered animals expressing humanized T cell co-receptors to develop human therapeutic drugs are also provided. [Background technology]

[0004] Background of the Invention In adaptive immune responses, foreign antigens are recognized by receptor molecules on B lymphocytes (e.g., immunoglobulins) and T lymphocytes (e.g., T cell receptors, or TCRs). These foreign antigens are presented on the cell surface as peptide fragments by specialized proteins commonly referred to as major histocompatibility complex (MHC) molecules. During T cell-mediated responses, antigens presented by MHC molecules are recognized by T cell receptors. However, for an effective immune response, something beyond T cell receptor recognition of the MHC-antigen complex is necessary. It is also necessary for T cell co-receptor molecules (e.g., CD4 or CD8) to bind to the invariant portion of the MHC.

[0005] There are several types of T cells, including helper T cells and cytotoxic T cells. Helper T cells express the co-receptor CD4 and recognize antigens bound to MHC II molecules. CD4+ T cells activate other effector cells in the immune system, such as activating MHC II-expressing B cells to produce antibodies and activating MHC II-expressing macrophages to destroy pathogens. CD4 and the T cell receptor share the same MHC Binding to the foreign antigen presented in II significantly increases the T cell's sensitivity to that antigen.

[0006] In contrast, cytotoxic T cells (CTLs) express the co-receptor CD8 and recognize foreign antigens bound to MHC I molecules. CTLs are specialized to kill any cell carrying MHC I-binding peptides recognized by the TCR bound to their own membrane. When cells display peptides derived from cellular proteins that are not normally present (e.g., viruses, tumors, or other non-self-derived substances), such peptides are recognized by CTLs, thereby activating the CTLs and killing the cells displaying those peptides. Similar to CD4, the association of CD8 increases the sensitivity of CTLs to antigens presented on MHC I. 。 [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] Due to tolerance mechanisms, not all antigens induce T cell activation. However, in some diseases (e.g., cancer, autoimmune diseases), peptides derived from self-proteins target cellular components of the immune system, leading to the destruction of cells presenting such peptides. Significant progress has been made in the recognition of clinically important antigens (e.g., antigens associated with various types of cancer). However, to improve the identification and selection of peptides that elicit appropriate responses in human T cells, particularly for peptides of clinically important antigens, in vivo and in vitro systems that mimic aspects of the human immune system are still needed. Therefore, biological systems capable of displaying components of the human immune system (e.g., genetically modified non-human animals and cells) are required. [Means for solving the problem]

[0008] Non-human animals are provided, including non-human cells expressing human or humanized molecules that function in cellular immune responses. Humanized rodent loci encoding human or humanized T cell co-receptor (e.g., CD4 and / or CD8) proteins are also provided. Humanized rodent cells expressing human or humanized T cell co-receptor (e.g., CD4 and / or CD8) proteins are also provided. In vivo and in vitro systems are provided, including humanized rodent cells expressing one or more human or humanized immune system molecules.

[0009] This specification provides genetically modified non-human animals containing a nucleotide sequence encoding a human or humanized T cell co-receptor polypeptide within its genome. In various embodiments, this specification provides genetically modified non-human animals containing a nucleotide sequence encoding a chimeric human / non-human T cell co-receptor polypeptide. In one embodiment, the nucleotide sequence is located at an endogenous T cell co-receptor locus. In one embodiment, the human portion of the chimeric T cell co-receptor polypeptide comprises all or substantially all of the extracellular domain of the human T cell co-receptor, and the non-human animal expresses a functional chimeric T cell co-receptor polypeptide. In one embodiment, the non-human portion of the chimeric T cell co-receptor polypeptide comprises at least the transmembrane domain and cytoplasmic domain of the non-human T cell co-receptor, and the non-human animal expresses a functional chimeric T cell co-receptor polypeptide. In one aspect of the present invention, the chimeric T cell co-receptor polypeptide is expressed only on T cells of the non-human animal and not, for example, on B cells of the non-human animal. In one aspect, the animal does not express a functional non-human T cell co-receptor derived from its endogenous non-human T cell co-receptor locus. In one aspect of the present invention, the chimeric T cell co-receptor polypeptide is contained in the germline of a non-human animal. In one aspect, the animal contains one or two copies of the nucleotide sequence encoding the chimeric T cell co-receptor polypeptide at the endogenous T cell co-receptor locus, and therefore the animal may be heterozygous or homozygous with respect to the nucleotide sequence encoding the chimeric T cell co-receptor polypeptide.

[0010] In one embodiment, the T cell co-receptor is CD4. Accordingly, in one embodiment, the present invention provides a genetically modified non-human animal comprising a nucleotide sequence encoding a chimeric human / non-human CD4 polypeptide. In one embodiment, the nucleotide sequence is located at an endogenous CD4 locus. In one embodiment, the animal is a rodent, such as a mouse or rat. Accordingly, in one embodiment, a genetically modified mouse is provided that expresses a functional chimeric human / mouse CD4, wherein the endogenous CD4 locus comprises a nucleotide sequence encoding a chimeric human / mouse CD4 polypeptide, the human portion of the chimeric CD4 polypeptide comprises all or substantially all of the extracellular domain of the human CD4 polypeptide, and the mouse portion of the chimeric CD4 polypeptide comprises at least the transmembrane domain and cytoplasmic domain of the mouse CD4 polypeptide. In one embodiment, this specification provides a genetically modified mouse that expresses functional chimeric human / mouse CD4, wherein the endogenous CD4 locus contains a nucleotide sequence encoding a chimeric human / mouse CD4 polypeptide, the human portion of the chimeric polypeptide contains at least all or substantially all of the domains D1-D3 of the human CD4 polypeptide, and the mouse portion of the chimeric polypeptide contains at least the transmembrane domain and cytoplasmic domain of mouse CD4. In one embodiment, the mouse does not express functional endogenous mouse CD4 derived from its endogenous mouse CD4 locus. In one embodiment, the nucleotide sequence encoding the chimeric human / mouse CD4 polypeptide is operably linked to an endogenous mouse promoter sequence and a regulatory sequence. Therefore, in one embodiment, the mouse does not express the chimeric CD4 protein on B cells or CD8-lineage T cells. In one embodiment, the human portion of the chimeric CD4 protein contains the amino acid sequence described in SEQ ID NO: 57. In one embodiment, the chimeric human / mouse CD4 polypeptide is described in SEQ ID NO: 4.

[0011] In one embodiment, a genetically modified non-human animal containing the chimeric CD4 polypeptide described herein, such as a genetically modified mouse, further comprises a human or humanized MHC II protein, the MHC II protein comprising the extracellular domain of a human MHC IIα polypeptide and the extracellular domain of a human MHC IIβ polypeptide. In one embodiment, the animal comprises a humanized MHC II protein. In one embodiment, the animal is a mouse, and the mouse has at the endogenous MHC II locus (1) a nucleotide sequence encoding a chimeric human / mouse MHC IIα polypeptide, wherein the human portion of the MHC IIα polypeptide comprises a nucleotide sequence comprising the extracellular domain of human MHC IIα and the transmembrane and cytoplasmic domains of the endogenous mouse MHC IIα polypeptide, and (2) a nucleotide sequence encoding a chimeric human / mouse MHC IIβ polypeptide, the MHC The human portion of the IIβ polypeptide comprises a nucleotide sequence including the extracellular domain of human MHC IIβ and the transmembrane and cytoplasmic domains of the endogenous mouse MHC IIβ polypeptide. Genetically modified non-human animals, such as mice, containing nucleotide sequences (may be multiple) encoding chimeric human / non-human MHC II, such as human / mouse MHC II, are described in more detail in U.S. Patent Applications 13 / 661,116 and 13 / 793,935, which are incorporated herein by reference in their entirety. In one embodiment, an animal expressing humanized CD4 and / or MHC II proteins is created by replacing portions of endogenous non-human, such as the mouse CD4 gene and / or MHC II gene, at the CD4 locus and / or MHC II locus, respectively.

[0012] Therefore, a method is also provided for modifying the CD4 locus of a non-human animal, e.g., a rodent, e.g., mouse, to express a chimeric human / mouse CD4 polypeptide, the method comprising the step of replacing the nucleotide sequence encoding an endogenous non-human, e.g., mouse CD4 polypeptide at the endogenous CD4 locus with the nucleotide sequence encoding a chimeric human / mouse CD4 polypeptide. In one embodiment, the chimeric human / non-human, e.g., human / rodent, e.g., human / mouse CD4 polypeptide comprises at least all or substantially all of the domains D1-D3 of the human CD4 polypeptide and at least the transmembrane domain and cytoplasmic domain of the endogenous non-human, e.g., rodent, e.g., mouse CD4 polypeptide. In one embodiment, the chimeric human / mouse CD4 to be expressed is described in Sequence ID No. 4.

[0013] In another embodiment, the T cell co-receptor is CD8. Thus, in one embodiment, the present invention provides a genetically modified non-human animal comprising a nucleotide sequence(s) encoding a chimeric human / non-human CD8 polypeptide, such as a chimeric human / non-human CD8α and / or CD8β polypeptide. In one embodiment, the nucleotide sequence is located at the endogenous CD8 locus. In one embodiment, the animal is a rodent, such as a mouse or a rat. Accordingly, in one embodiment, a genetically modified mouse is provided that expresses a functional chimeric human / mouse CD8 protein, wherein the endogenous CD8 locus (e.g., the endogenous CD8α and / or CD8β locus) contains a first nucleotide sequence encoding a chimeric human / mouse CD8α polypeptide and a second nucleotide sequence encoding a chimeric human / mouse CD8β polypeptide, wherein the first nucleotide sequence contains a sequence encoding all or substantially all of the extracellular domain of the human CD8α polypeptide, as well as at least the transmembrane and cytoplasmic domains of the mouse CD8α polypeptide, and the second nucleotide sequence contains a sequence encoding all or substantially all of the extracellular domain of the human CD8β polypeptide, as well as at least the transmembrane and cytoplasmic domains of the mouse CD8β polypeptide. In one embodiment, the mouse does not express a functional endogenous mouse CD8 polypeptide derived from its endogenous mouse CD8 locus. In one embodiment, the first nucleotide sequence is operably linked to the endogenous mouse CD8α promoter sequence and regulatory sequence, and the second nucleotide sequence is operably linked to the endogenous mouse CD8β promoter sequence and regulatory sequence. Therefore, in one embodiment, the mouse does not express the chimeric CD8 protein on B cells or CD4-lineage T cells. In one embodiment, the human portion of the chimeric CD8α and / or CD8β polypeptide includes the immunoglobulin V-like domain of the human CD8α and / or CD8β polypeptide. In one embodiment, the human portion of the chimeric human / mouse CD8α polypeptide includes the amino acid sequence described in SEQ ID NO: 59. In one embodiment, the human portion of the chimeric human / mouse CD8β polypeptide includes the amino acid sequence described in SEQ ID NO: 58.In one embodiment, the chimeric human / mouse CD8α polypeptide is described in SEQ ID NO: 54, and the chimeric human / mouse CD8β polypeptide is described in SEQ ID NO: 53.

[0014] In one embodiment, a genetically modified non-human animal comprising the chimeric CD8α and / or CD8β polypeptide described herein, such as a genetically modified mouse, further comprises a human or humanized MHC I protein, the MHC I protein comprising the extracellular domain of the human MHC I polypeptide. In one embodiment, the animal comprises a humanized MHC I complex. Thus, the animal may comprise a humanized MHC I protein and a human or humanized β2 microglobulin polypeptide. In one embodiment, the animal is a mouse, which comprises a nucleotide sequence encoding a chimeric human / mouse MHC I polypeptide at the endogenous MHC I locus, the human portion of the MHC I polypeptide comprising the extracellular domain of the human MHC I polypeptide as well as the transmembrane and cytoplasmic domains of the endogenous mouse MHC I polypeptide, and the animal also comprises a nucleotide sequence encoding human or humanized β2 microglobulin at the endogenous β2 microglobulin locus. Chimeric human / non-human, genetically modified non-human animals, such as mice, containing nucleotide sequences(s) encoding human / mouse MHC I and β2 microglobulin, are described in more detail in U.S. Patent Applications 13 / 661,159 and 13 / 793,812, which are incorporated herein by reference in their entirety. In one embodiment, an animal expressing humanized CD8, MHC I, and / or β2 microglobulin protein(s) is created by replacing portions of endogenous non-human, such as mouse CD8, MHC I, and / or β2 microglobulin genes at the CD8 locus, MHC I locus, and / or β2 microglobulin locus, respectively.

[0015] Therefore, a method is also provided for modifying the CD8 locus of a non-human animal, e.g., a rodent, e.g., mouse, to express a chimeric human / mouse CD8 polypeptide, the method comprising the step of replacing the nucleotide sequence encoding an endogenous non-human, e.g., mouse CD8 polypeptide at the endogenous CD8 locus with the nucleotide sequence encoding the chimeric human / mouse CD8 polypeptide. In one embodiment, the CD8 polypeptide is selected from the group consisting of CD8α, CD8β, and combinations thereof. In one embodiment, the chimeric human / non-human, e.g., human / rodent, e.g., human / mouse CD8 polypeptide (CD8α and / or CD8β) comprises all or substantially all of the extracellular domain of the human CD8 polypeptide, as well as at least the transmembrane domain and cytoplasmic domain of the endogenous non-human, e.g., rodent, e.g., mouse CD8 polypeptide.

[0016] This specification also provides cells, such as T cells, derived from non-human animals (e.g., rodents, such as mice or rats) as described herein. Tissues and embryos derived from non-human animals as described herein are also provided.

[0017] The present invention provides, for example, the following items: (Item 1) It contains a nucleotide sequence encoding a chimeric human / non-human T cell co-receptor polypeptide, The non-human portion of the chimeric polypeptide comprises at least the transmembrane domain and cytoplasmic domain of a non-human T cell co-receptor in a genetically modified non-human animal, Non-human animals that express chimeric T cell co-receptor polypeptides. (Item 2) The animals described in item 1, wherein the aforementioned nucleotide sequence is located at the endogenous T cell co-receptor gene locus. (Item 3) Animals as described in item 1 that do not express a functional endogenous non-human T cell co-receptor derived from an endogenous gene locus. (Item 4) The animal according to item 1, wherein the chimeric T cell co-receptor polypeptide is not expressed on B cells of the non-human animal. (Item 5) The animal according to item 1, wherein the T cell co-receptor locus is the CD4 locus and the T cell co-receptor polypeptide is CD4. (Item 6) The animal according to item 5, wherein the chimeric CD4 polypeptide is not expressed on B cells or T cells of the CD8 lineage. (Item 7) The animal according to item 1, wherein the chimeric T cell co-receptor locus is the CD8 locus and the T cell co-receptor polypeptide is a CD8 polypeptide selected from CD8α, CD8β, or a combination thereof. (Item 8) The animal according to item 7, which expresses a chimeric human / non-human CD8 protein comprising a CD8α polypeptide and a CD8β polypeptide. (Item 9) The animal according to item 8, wherein the chimeric CD8 protein is not expressed on B cells or T cells of the CD4 lineage. (Item 10) The animal according to item 1, which is a rodent. (Item 11) The animal according to item 10, which is a mouse. (Item 12) Comprising a nucleotide sequence encoding a chimeric human / mouse CD4 polypeptide, wherein the human portion of the chimeric polypeptide comprises at least the domains of the human CD4 polypeptide described in FIG. 1, wherein the mouse portion of the chimeric polypeptide comprises at least the transmembrane domain and the cytoplasmic domain of the mouse CD4 polypeptide, a genetically modified mouse, A mouse expressing chimeric human / mouse CD4. (Item 13) The mouse according to item 12, wherein the human portion comprises the extracellular domain of the human CD4 polypeptide. (Item 14) The mouse described in item 12, wherein the aforementioned nucleotide sequence is located at the endogenous CD4 gene locus. (Item 15) A mouse as described in item 12, which does not express functional endogenous mouse CD4 derived from the endogenous mouse CD4 gene locus. (Item 16) The mouse according to item 12, wherein the nucleotide sequence is operably linked to a mouse promoter sequence and a regulatory sequence. (Item 17) The mouse described in item 12, wherein the aforementioned chimeric CD4 protein is not expressed on B cells or CD8-derived T cells. (Item 18) The mouse according to item 12, further comprising a human or humanized MHC II protein, wherein the MHC II protein comprises an extracellular domain of a human MHC IIα polypeptide and an extracellular domain of a human MHC IIβ polypeptide. (Item 19) The mouse according to item 12, wherein the nucleotide sequence is included in the germline of the mouse. (Item 20) A method for modifying the CD4 locus of a mouse to express a chimeric human / mouse CD4 polypeptide, comprising the step of replacing the nucleotide sequence encoding the endogenous mouse CD4 polypeptide with the nucleotide sequence encoding the chimeric human / mouse CD4 polypeptide at the endogenous CD4 locus of a mouse. (Item 21) The method according to item 20, wherein the human portion of the chimeric human / mouse CD4 polypeptide comprises at least the domain of the human CD4 polypeptide shown in Figure 1, and comprises at least the transmembrane domain and cytoplasmic domain of the endogenous mouse CD4 polypeptide. (Item 22) The method according to item 20, wherein the human portion comprises the extracellular domain of the human CD4 polypeptide. (Item 23) The first nucleotide sequence comprises a first nucleotide sequence encoding a chimeric human / mouse CD8α polypeptide and a second nucleotide sequence encoding a chimeric human / mouse CD8β polypeptide, wherein the first nucleotide sequence comprises a sequence encoding the extracellular domain of the human CD8α polypeptide and at least the transmembrane domain and cytoplasmic domain of the mouse CD8α polypeptide. A genetically modified mouse wherein the second nucleotide sequence comprises sequences encoding the extracellular domain of human CD8β polypeptide and at least the transmembrane domain and cytoplasmic domain of mouse CD8β polypeptide, A chimeric human / mouse mouse expressing the CD8 protein. (Item 24) The mouse described in item 23, wherein the first nucleotide sequence and the second nucleotide sequence are located at the endogenous CD8α locus and the endogenous CD8β locus, respectively. (Item 25) A mouse described in item 23 that does not express a functional endogenous CD8 protein derived from the endogenous CD8 gene locus. (Item 26) The mouse described in item 23, wherein the aforementioned chimeric human / mouse CD8 protein is not expressed on B cells or CD4-derived T cells. (Item 27) The mouse according to item 23, wherein the first nucleotide sequence is operably ligated to the mouse CD8α promoter sequence and regulatory sequence, and the second nucleotide sequence is operably ligated to the mouse CD8β promoter sequence and regulatory sequence. (Item 28) The mouse according to item 23, further comprising a human or humanized MHC I polypeptide, wherein the MHC I polypeptide comprises the extracellular domain of a human MHC I polypeptide. (Item 29) A mouse as described in item 28, further comprising human or humanized β2-microglobulin polypeptide. (Item 30) The mouse according to item 23, wherein the first nucleotide sequence and the second nucleotide sequence are included in the germline of the mouse. (Item 31) A method for modifying the CD8 locus of a mouse to express a chimeric human / mouse CD8 polypeptide, comprising the step of replacing the nucleotide sequence encoding the endogenous mouse CD8 polypeptide with the nucleotide sequence encoding the chimeric human / mouse CD8 polypeptide at the endogenous CD8 locus of a mouse. (Item 32) The method according to item 31, wherein the CD8 polypeptide is selected from CD8α, CD8β, or a combination thereof. (Item 33) The method according to item 31, wherein the chimeric human / mouse CD8 polypeptide comprises the extracellular domain of the human CD8 polypeptide and at least the transmembrane domain and cytoplasmic domain of the endogenous mouse CD8 polypeptide. (Item 34) It contains a nucleotide sequence encoding a chimeric human / mouse CD8α polypeptide, The chimeric human / mouse CD8α polypeptide is a genetically modified mouse comprising the extracellular domain of the human CD8α polypeptide and at least the transmembrane domain and cytoplasmic domain of the mouse CD8α polypeptide, A mouse expressing the chimeric human / mouse CD8α polypeptide. (Item 35) The mouse described in item 34, wherein the aforementioned nucleotide sequence is present at the endogenous mouse CD8 gene locus. (Item 36) A mouse described in item 34 that does not express a functional endogenous CD8α polypeptide derived from the endogenous CD8 gene locus. (Item 37) The mouse according to item 34, wherein the nucleotide sequence is operably linked to the mouse CD8α promoter sequence and regulatory sequence. (Item 38) It contains a nucleotide sequence encoding a chimeric human / mouse CD8β polypeptide, The chimeric human / mouse CD8β polypeptide is a genetically modified mouse comprising the extracellular domain of a human CD8β polypeptide and at least the transmembrane domain and cytoplasmic domain of a mouse CD8β polypeptide. A mouse expressing the chimeric human / mouse CD8β polypeptide. (Item 39) The mouse described in item 38, wherein the aforementioned nucleotide sequence is present at the endogenous mouse CD8 gene locus. (Item 40) A mouse described in item 38 that does not express a functional endogenous CD8β polypeptide derived from the endogenous CD8 gene locus. (Item 41) The mouse according to item 38, wherein the nucleotide sequence is operably linked to the mouse CD8β promoter sequence and regulatory sequence. Unless otherwise specified or evident from the context, any embodiment and aspect described herein can be used in conjunction with one another. Other embodiments will become apparent to those skilled in the art from a consideration of the following detailed description. The following detailed description includes illustrative representations of various embodiments of the invention, which are not intended to limit the claimed invention. The accompanying drawings constitute part of this specification and, together with the description, serve merely to illustrate embodiments and are not intended to limit the invention. [Brief explanation of the drawing]

[0018] [Figure 1] Figure 1 illustrates the method for creating a humanized CD4 locus (scale not specified). First, the sequences of mouse exons 3-6, starting immediately after the signal peptide, were replaced with the sequence of human exon 3 downstream of the signal peptide (top). Then, human exons 4-6 were inserted downstream of human exon 3 by restriction digestion / ligation.

[0019] [Figure 2]Figure 2 shows FACS analysis (A) of splenocytes derived from WT mice or mice heterozygous for human CD4 (1766HET) using anti-human CD4 antibodies and anti-mouse CD4 antibodies, and a graph showing FACS analysis of T cells derived from WT mice, T cells derived from 1766HET mice, and Jurkat human CD4 T cells.

[0020] [Figure 3] Figure 3 illustrates (scale not specified) the method for creating a humanized CD8b locus (MAID1737) by replacing mouse CD8β exons 2-3 with human CD8β exons 2-3. Mouse exon sequences are represented by black squares, and human exon sequences are represented by squares with diagonal lines.

[0021] [Figure 4] Figure 4 illustrates (scale not specified) a method for creating a humanized CD8a locus (MAID1738) by replacing parts of mouse exon 1 and exon 2 with human exons 2-3 while retaining the mouse leader sequence at the start of exon 1. Mouse exon sequences are represented by black squares, and human exon sequences are represented by squares with diagonal lines.

[0022] [Figure 5] Figure 5 illustrates (unspecified scale) a sequential targeting method for creating humanized loci containing sequences encoding the humanized CD8b and CD8a genes. Mouse exon sequences are represented by black squares, and human exon sequences are represented by diagonally lined squares.

[0023] [Figure 6] Figure 6 shows FACS analysis of splenocytes derived from either wild-type mice or mice (1739Het, 1740Het) that are heterozygous for both human CD8b and CD8a with the selection cassette removed, using either mouse CD8b antibody, mouse CD8a antibody, human CD8b antibody, or human CD8a antibody.

[0024] [Figure 7] Figure 7 is a graph showing FACS analysis of thymocytes obtained from either WT mice or 1739HET / 1740HET (mouse heterozygous for both CD8b and CD8a) mice, using either mouse CD8b, mouse CD8a, human CD8b, human CD8a, or CD4. [Modes for carrying out the invention]

[0025] definition The present invention provides genetically modified non-human animals (e.g., mice, rats, rabbits, etc.) expressing humanized T cell co-receptor polypeptides; embryos, cells, and tissues containing said polypeptides; methods for producing them; and methods for using them. Unless otherwise defined, all terms and phrases used herein have the meanings they have acquired in the art unless explicitly stated otherwise or it is evident from the context in which they are used.

[0026] The term "conservative," when used to describe a conservative amino acid substitution, includes the substitution of an amino acid residue with another amino acid residue having a side-chain R group of similar chemical properties (e.g., charge or hydrophobicity). Conservative amino acid substitutions can be achieved by modifying the nucleotide sequence so as to introduce a change in the nucleotide encoding the conservative substitution. Generally, conservative amino acid substitutions substantially alter the desired functional properties of a protein, such as the ability of CD4 or CD8 to bind to MHC II or MHC I, respectively, and increase the sensitivity of the TCR to antigens presented on the MHC. Examples of groups of amino acids having side chains with similar chemical properties include aliphatic side chains, e.g., glycine, alanine, valine, leucine, and isoleucine; aliphatic hydroxyl side chains, e.g., serine and threonine; amide-containing side chains, e.g., asparagine and glutamine; aromatic side chains, e.g., phenylalanine, tyrosine, and tryptophan; basic side chains, e.g., lysine, arginine, and histidine; acidic side chains, e.g., aspartic acid and glutamic acid; and sulfur-containing side chains, e.g., 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. In some embodiments, the conserved amino acid substitution may be the substitution of any native residue in the protein with alanine, for example, as used in alanine scanning mutagenesis. In some embodiments, conservative substitutions are made to have a positive value in the PAM250 log-likelihood matrix, as disclosed by reference herein by Gonnet et al. (Exhaustive Matching of the Entire Protein Sequence Database, Science Vol. 256: pp. 1443-45, 1992). In some embodiments, the substitutions are moderately conservative substitutions, where the substitution has a non-negative value in the PAM250 log-likelihood matrix.

[0027] Therefore, the present invention also includes genetically modified non-human animals that contain a nucleotide sequence encoding a humanized T cell co-receptor polypeptide (e.g., CD4 or CD8 polypeptide) in their genome (e.g., at an endogenous locus), and in which the polypeptide contains a conserved amino acid substitution of the amino acid sequence(s) described herein.

[0028] Those skilled in the art will understand that, in addition to the nucleic acid residues encoding the humanized T cell co-receptor polypeptide described herein, other nucleic acids may encode the polypeptide of the present invention due to degeneracy of the genetic code. Therefore, in addition to genetically modified non-human animals containing nucleotide sequences in their genomes that encode a T cell co-receptor polypeptide (e.g., CD4 or CD8 polypeptide) with conserved amino acid substitutions, non-human animals containing nucleotide sequences in their genomes that differ from those described herein due to degeneracy of the genetic code are also provided.

[0029] When used in relation to sequences, the term "identity" encompasses identity determined by several different algorithms known in the art that can be used to measure nucleotide and / or amino acid sequence identity. In some embodiments described herein, identity is determined using ClustalW v.1.83(slow) alignment with a Gonnet similarity matrix (MacVector® 10.0.2, MacVector Inc., 2008) and employing an open gap penalty of 10.0 and an extend gap penalty of 0.1. The lengths of the sequences being compared for sequence identity depend on the specific sequence. In various embodiments, identity is determined by comparing the sequences from the N-terminus to the C-terminus of mature proteins. In various embodiments, when comparing a chimeric human / non-human sequence with a human sequence, the human portion of the chimeric human / non-human sequence is used for comparison (the non-human portion is not used) to confirm the level of identity between the human sequence and the human portion of the chimeric human / non-human sequence (for example, comparing the human external domain of a chimeric human / mouse protein with the human external domain of a human protein).

[0030] The term "homologous" or "homologous" means, with respect to sequences, such as nucleotide or amino acid sequences, two sequences that, when optimally aligned and compared, are identical in, for example, at least about 75%, at least about 80%, at least about 90-95%, or more than 97% of their nucleotides or amino acids. For optimal gene targeting, it will be understood by those skilled in the art that the targeting construct should contain an arm homologous to the endogenous DNA sequence (i.e., a "homologous arm"), thereby enabling homologous recombination between the targeting construct and the targeted endogenous sequence.

[0031] The term "operably linked" refers to a proximal relationship that enables the components described in this way to function in their intended manner. Thus, a protein-coding nucleic acid sequence can be operably linked to a regulatory sequence (e.g., a promoter, enhancer, or silencer sequence) so that appropriate transcriptional regulation is maintained. Furthermore, various parts of the chimeric or humanized protein of the present invention 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, the various domains of the chimeric or humanized protein of the present invention are operably linked to one another.

[0032] The term "replacement," in the context of gene replacement, refers to the placement of exogenous genetic material into an endogenous locus, thereby replacing all or part of the endogenous gene with an orthologous or homologous nucleic acid sequence. As demonstrated in the examples below, the nucleic acid sequences of endogenous loci encoding portions of mouse CD4 or CD8 (CD8α and / or CD8β) polypeptides were replaced with nucleotide sequences encoding portions of human CD4 or CD8 (CD8α and / or CD8β) polypeptides, respectively.

[0033] As used herein, "functional" refers, for example, with respect to functional polypeptides, to a polypeptide that retains at least one biological activity typically associated with a native protein. For example, in some embodiments of the present invention, substitution at an endogenous locus (e.g., substitution at an endogenous non-human CD4 or CD8 locus) results in a locus that cannot express a functional endogenous polypeptide.

[0034] Several aspects of the following regarding genetically modified CD4 non-human animals, such as animal species; animal strains; cell types; screening, detection and other methods; and methods of use, are applicable to genetically modified CD8 animals. Genetically modified CD4 animals

[0035] In various embodiments, the present invention generally provides genetically modified non-human animals that contain a nucleotide sequence encoding a humanized CD4 polypeptide in the genome, for example, at an endogenous CD4 locus, and therefore express a humanized CD4 polypeptide.

[0036] The human CD4 gene is located on chromosome 12 and is thought to contain 10 exons. The CD4 gene encodes a protein with an amino-terminal hydrophobic signal sequence encoded by exons 2 and 3 of the gene. This protein contains four immunoglobulin-like domains, commonly referred to as the D1-D4 domains. (Maddon et al., 1987, Structure and expression of the human and mouse T4 genes, Proc. Natl. Acad. Sci. USA, Vol. 84: pp. 9155-9159). The D1 domain is thought to be encoded by exon 3 (the downstream sequence of the signal peptide) and exon 4, while D2, D3, and D4 are thought to be encoded by separate exons—exon 5, exon 6, and exon 7, respectively. Littman (1987) The Structure of CD4 and CD8 Genes, Ann. Rev. Immunol. 5:561-84; Hanna et al. (1994) Specific Expression of the Human CD4 Gene in Mature CD4+CD8- and Immature CD4+CD8+ T cells and in Macrophages of Transgenic Mice, Mol. Cell. Biol. Vol. 14 (No. 2): pp. 1084-1094; Maddon et al., above. In regions with high protein concentration, such as the area where T cells and antigen-presenting cells come into contact, molecules tend to homodimerize through interactions between opposing D4 domains. Zamoyska (1998) CD4 and CD8: modulators of T cell receptor recognition of antigen and of Immune responses? Curr. Opin. Immunol. Vol. 10: pp. 82-87; Wu et al. (1997) Dimeric Association and segmental variability in the structure of human CD4, Nature 387: p. 527; Moldovan et al. (2002) CD4 Dimers Constitute the Functional Component Required for T Cell Activation, J. Immunol. 169: pp. 6261-6268.

[0037] The D1 domain of CD4 is similar to the immunoglobulin variable (V) domain and, along with a portion of the D2 domain, is thought to bind to MHC II. (Huang et al., 1997: Analysis of the contact sites on the CD4) Molecule with Class II MHC Molecule, J. Immunol. Vol. 158: pp. 216-2125. This time, MHC II, MHC II It interacts with the T cell co-receptor CD4 in the hydrophobic gap at the junction between the α2 domain and the MHC II β2 domain. Wang and Reinherz (2002) Structural Basis of T Cell Recognition of Peptides Bound to MHC Molecules, Molecular Immunology, vol. 38: pp. 1039-1049.

[0038] Domains D3 and D4 of the CD4 co-receptor are thought to interact with the TCR-CD3 complex because substituting these two domains inhibits CD4's ability to bind to the TCR. (Vignali et al., 1996, The Two Membrane Proximal Domains of CD4 Interact with the T Cell Receptor, J. Exp. Med., Vol. 183: pp. 2097-2107). The CD4 molecule exists as a dimer, and residues within the D4 domain of the molecule are thought to be involved in CD4 dimerization. (Moldovan et al., 2002, CD4 Dimers Constitute the Functional Components Required for T Cell Activation, J. Immunol. Vol. 169: pp. 6261-6268.

[0039] Exon 8 of the CD4 gene encodes the transmembrane domain, while the remainder of the gene encodes the cytoplasmic domain. The CD4 cytoplasmic domain has many distinct functions. For example, the cytoplasmic domain of CD4 recruits the tyrosine kinase Lck. Lck is a Src family kinase that associates with the cytoplasmic domains of CD4 and CD8, and its simultaneous binding of the co-receptor and the same MHC to the TCR increases the phosphorylation of tyrosine on the CD3 and ζ chains of the TCR complex, in turn recruiting other factors that play a role in T cell activation. Itano and collaborators proposed that the cytoplasmic tail of CD4 also promotes differentiation from CD4+CD8+ T cells to the CD4+ lineage by designing and testing the expression of a hybrid protein containing the extracellular domain of CD8 and the cytoplasmic tail of CD4 in transgenic mice. Itano et al. (1996) The Cytoplasmic Domain of CD4 Promotes the Development of CD4 Lineage T Cells, J. Exp. Med. 183: pp. 731-731. Expression of the hybrid protein led to the development of MHC I-specific CD4-lineage T cells. (Ibid.)

[0040] The CD4 co-receptor appears to be the primary receptor for the HIV virus, and CD4+ T cell depletion is an indicator of disease progression. The cytoplasmic tail of CD4 seems essential for the delivery of apoptotic signals to CD4+ T cells in HIV-induced apoptosis. In particular, the interaction between CD4 and Lck has been shown to enhance HIV-induced apoptosis in these cells. (Corbeil et al. (1996) HIV-induced Apoptosis Requires the CD4 Receptor Cytoplasmic Tail and Is Accelerated by Interaction of CD4 with p56lck, J. Exp. Med. 183: pp. 39-48.)

[0041] T cells develop in the thymus, progressing from immature CD4- / CD8- (double-negative or DN) thymocytes to CD4+ / CD8+ (double-positive or DP) thymocytes, and ultimately becoming either CD4+ or CD8+ (single-positive or SP) T cells through positive selection. DP thymocytes that receive signals through the MHC I restriction TCR differentiate into CD8+ T cells, while DP thymocytes that receive signals through the MHC II restriction TCR differentiate into CD4+ T cells. The triggers that lead DP cells to differentiate into CD4+ or CD8+ T cells have been the subject of numerous studies. Various models of CD4 / CD8 lineage selection have been proposed, and are outlined in Singer et al. (2008) Lineage fate and intense debate: myths, models and mechanisms of CD4- versus CD8- lineage choice, Nat. Rev. Immunol. 8: pp. 788-801.

[0042] The deactivation of specific T cell co-receptors as a result of positive selection is a product of transcriptional regulation. Regarding CD4, it has been shown that an enhancer located 13kb upstream of exon 1 of CD4 upregulates CD4 expression in CD4+ T cells and CD8+ T cells. (Killeen et al., 1993, Regulated expression of human CD4 rescues helper T cell development in mice lacking expression of endogenous CD4, EMBO J., Vol. 12: pp. 1547-1553). A cis-acting transcriptional silencer located within the first intron of the mouse CD4 gene functions to silence CD4 expression in cells other than CD4+ T cells. (Siu et al., 1994, A transcriptional silencer control the developmental expression of the CD4 gene, EMBO J., Vol. 13: pp. 3570-3579).

[0043] Some previously developed lines of transgenic mice expressing human CD4 lacked key transcriptional regulators (e.g., promoters, enhancers, silencers) that control CD4 lineage selection. As a result, these mice could not reproduce normal T cell lineage development and produced immune cells other than CD4+ T cells that express CD4. For example, Law et al. (1994) Human CD4 Restores Normal T Cell Development and Function in Mice Deficient in CD4, J. Exp. Med. 179: pp. 1233-1242 (CD4 expression in CD8+ T cells and B cells); Fugger et al. (1994) Expression of HLA-DR4 and human CD4 transgenes in mice determines the variable region β-chain T-cell repertoire and mediates an HLA-D-restricted immune response, Proc. Natl. Acad. Sci. USA, vol. 91:6151-55 (CD4 expressed See (on all CD3+ thymocytes and B cells). Therefore, in one embodiment, there may be advantages to developing genetically modified animals that retain the endogenous mouse promoter and other regulatory elements in order to produce T cells that can undergo normal T cell development and lineage selection.

[0044] Accordingly, in various embodiments, the present invention provides a genetically modified non-human animal comprising, for example, an endogenous T cell co-receptor locus (e.g., the CD4 locus) containing a nucleotide sequence encoding a chimeric human / non-human T cell co-receptor polypeptide. In one embodiment, the human portion of the chimeric polypeptide comprises all or substantially all of the extracellular domain of the human T cell co-receptor. In one embodiment, the non-human portion of the chimeric polypeptide comprises the transmembrane and cytoplasmic domains of the non-human T cell co-receptor. In one embodiment, the non-human animal expresses a functional chimeric T cell co-receptor polypeptide. Accordingly, in one aspect, the present invention provides a genetically modified non-human animal comprising an endogenous CD4 locus containing a nucleotide sequence encoding a chimeric human / non-human CD4 polypeptide, wherein the human portion of the chimeric polypeptide comprises all or substantially all of the extracellular domain of human CD4, and the non-human portion comprises at least the transmembrane and cytoplasmic domains of non-human CD4, and the animal expresses a functional chimeric CD4 polypeptide. In one embodiment, a non-human animal expresses only humanized CD4 polypeptides, i.e., chimeric human / non-human CD4 polypeptides, and does not express functional endogenous non-human CD4 proteins derived from its endogenous CD4 locus.

[0045] In one embodiment, the human portion of the chimeric human / non-human CD4 polypeptide comprises all or substantially all of the extracellular domains of the human CD4 polypeptide. In another embodiment, the chimeric human / non-human CD4 polypeptide comprises at least all or substantially all of the MHC II-binding domains of the human CD4 polypeptide (e.g., substantial portions of the D1 and D2 domains), in one embodiment, the human portion of the chimeric human / non-human CD4 polypeptide comprises all or substantially all of the D1, D2, and D3 domains of the human CD4 polypeptide, and in yet another embodiment, the human portion of the chimeric human / non-human CD4 polypeptide comprises all or substantially all of the immunoglobulin-like domains of CD4, e.g., domains referred to as D1, D2, D3, and D4. In yet another embodiment, the human portion of the chimeric human / non-human CD4 polypeptide comprises all or substantially all of the extracellular portion of the human CD4 sequence and / or T cell receptor involved in interaction with MHC II. In yet another embodiment, the human portion of the chimeric human / non-human CD4 polypeptide includes all or substantially all of the extracellular portion of human CD4 and / or the variable domain of the T cell receptor involved in interaction with MHC II. Thus, in one embodiment, the nucleotide sequence encoding the human portion of the chimeric CD4 polypeptide includes all or substantially all of the coding sequences of the D1-D2 domains of human CD4 (e.g., a portion of exon 3 and exons 4-5 of the human CD4 gene), and in another embodiment, the nucleotide sequence encoding the human portion of the chimeric CD4 polypeptide includes all or substantially all of the coding sequences of the D1-D3 domains of human CD4 (e.g., a portion of exon 3 and exons 4-6 of human CD4). Thus, in one embodiment, the nucleotide sequence encoding the chimeric human / non-human CD4 includes the nucleotide sequence encoding all or substantially all of the D1-D3 domains of human CD4. In another embodiment, the nucleotide sequence encoding the human portion of the chimeric CD4 polypeptide includes the coding sequences of the D1-D4 domains of the human CD4 gene.In another embodiment, the nucleotide sequence may include a nucleotide sequence encoding a mouse CD4 signal peptide, for example, a region encoded by a portion of exons 2-3 of a mouse gene. In yet another embodiment, the nucleotide sequence may include a nucleotide sequence encoding a human CD4 signal peptide. In one embodiment, the chimeric human / non-human CD4 polypeptide comprises the amino acid sequence described in SEQ ID NO: 4, wherein the human portion of the chimeric polypeptide spans approximately amino acids 27-319 of SEQ ID NO: 4 (as separately described in SEQ ID NO: 57).

[0046] In one embodiment, a non-human animal expresses a chimeric human / non-human CD4 polypeptide sequence. In one embodiment, the human portion of the chimeric CD4 sequence includes one or more conserved or non-conserved modifications.

[0047] In one embodiment, a non-human animal expressing a human CD4 sequence is provided, wherein the human CD4 sequence is at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the human CD4 sequence. In a particular embodiment, the human CD4 sequence is at least about 90%, 95%, 96%, 97%, 98%, or 99% identical to the human CD4 sequence described in the example. In one embodiment, the human CD4 sequence includes one or more conserved substitutions. In one embodiment, the human CD4 sequence includes one or more non-conserved substitutions.

[0048] In some embodiments, a portion of the chimeric CD4, for example, the human portion, may comprise substantially all of the sequences shown herein (e.g., substantially all of the protein domains shown herein). Substantially all sequences generally contain 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the amino acids that are considered to represent a particular portion of the protein (e.g., a particular functional domain). Those skilled in the art will understand that the boundaries of functional domains may vary slightly depending on the alignment and domain prediction method used.

[0049] In one embodiment, the non-human portion of the chimeric human / non-human CD4 polypeptide comprises at least the transmembrane domain and cytoplasmic domain of the non-human CD4 polypeptide. Due to the important functions performed by the CD4 cytoplasmic domain, the retention of the endogenous non-human (e.g., mouse) sequence within the genetically engineered animal ensures the preservation of appropriate intracellular signaling and other functions of the co-receptor. In one embodiment, the non-human animal is a mouse, and the non-human CD4 polypeptide is a mouse CD4 polypeptide. While specific mouse CD4 sequences are described in the examples, any suitable sequence derived therefrom, such as sequences containing conserved / non-conserved amino acid substitutions, is incorporated herein. In one embodiment, the non-human portion of the chimeric CD4 co-receptor comprises any sequence of unhumanized endogenous CD4.

[0050] The non-human animals described herein may contain nucleotide sequences encoding a chimeric human / non-human CD4 polypeptide at their endogenous loci. In one embodiment, this is done by replacing a portion of the endogenous CD4 gene with a nucleotide sequence encoding a portion of the human CD4 polypeptide. In one embodiment, such replacement is the replacement of an endogenous nucleotide sequence encoding all or substantially all of the extracellular domain of non-human CD4, for example, a sequence encoding all or substantially all of at least the first immunoglobulin-like domain (i.e., D1) of non-human CD4 (e.g., a sequence encoding all or substantially all of domains D1-D2 of non-human CD4, for example, a sequence encoding all or substantially all of domains D1-D3 of non-human CD4, for example, a sequence encoding all or substantially all of domains D1-D4 of non-human CD4), with a human nucleotide sequence encoding it. In one embodiment, the replacement results in a chimeric protein containing the extracellular portion of the human CD4 sequence and / or T cell receptor involved in interaction with MHC II. In yet another embodiment, the substitution results in a chimeric protein containing a human CD4 sequence and / or a variable domain of the T cell receptor involved in interaction with MHC II. In one embodiment, the substitution does not involve the substitution of a CD4 sequence encoding at least the transmembrane and cytoplasmic domains of the non-human CD4 polypeptide. Thus, in one embodiment, a non-human animal expresses a chimeric human / non-human CD4 polypeptide derived from an endogenous non-human CD4 locus. In yet another embodiment, the substitution results in a protein containing the polypeptide sequence described in Sequence ID No. 4.

[0051] In one embodiment, a nucleotide sequence of a chimeric human / non-human CD4 locus (e.g., chimeric human / rodent CD4 locus, e.g., chimeric human / mouse CD4 locus) described herein is provided. In one embodiment, the chimeric human / non-human (e.g., human / rodent, e.g., human / mouse) CD4 sequence is located at an endogenous non-human (e.g., rodent, e.g., mouse) CD4 locus and therefore retains a CD4 enhancer element located upstream of the first CD4 exon. In one embodiment, substitutions at the endogenous non-human (e.g., rodent, e.g., mouse) CD4 locus include, for example, substitutions of a portion of exon 3 encoding D1 of the CD4 polypeptide, and the remainder of D1 and exons 4-6 encoding D2-D3, and therefore, in one embodiment, the chimeric CD4 locus retains a cis-acting silencer located at intron 1 of the non-human (e.g., mouse) CD4 gene. Therefore, in one embodiment, the chimeric locus retains an endogenous non-human (e.g., rodent, e.g., mouse) CD4 promoter and regulatory elements. In another embodiment, the chimeric locus may contain human promoters and regulatory elements, insofar as they enable appropriate CD4 expression, CD4+ T cell development, CD4 lineage selection, and co-receptor function. Therefore, in some embodiments, the animals of the present invention include genetic modifications that do not alter appropriate lineage selection and development of T cells. In one embodiment, the animals of the present invention (e.g., rodents, e.g., mice) do not express the chimeric CD4 polypeptide on immune cells other than those that normally express CD4. In one embodiment, the animals do not express CD4 on B cells or CD8+SP T cells. In one embodiment, the substitution results in the retention of elements that enable appropriate spatial and temporal regulation of CD4 expression.

[0052] The genetically modified non-human animals of the present invention can be selected from the group consisting of mice, rats, rabbits, pigs, cattle (e.g., cows, bulls, water buffalo), deer, sheep, goats, chickens, cats, dogs, ferrets, and primates (e.g., marmosets, rhesus monkeys). For non-human animals for which suitable genetically modifiable ES cells are not readily available, other methods are used to produce genetically modified non-human animals. Such methods include, for example, modifying a non-ES cell genome (e.g., fibroblasts or induced pluripotent cells), transplanting the modified genome into suitable cells, such as oocytes, using nuclear transfer, and maturing the modified cells (e.g., modified oocytes) under suitable conditions in a non-human animal to form an embryo.

[0053] In one embodiment, the non-human animal is a mammal. In one embodiment, the non-human animal is, for example, a small mammal of the superfamily Dipodoidea or Muroidea. In one embodiment, the genetically modified animal is a rodent. In one embodiment, the rodent is selected from mice, rats, and hamsters. In one embodiment, the rodent is selected from the superfamily Muroidea. In one embodiment, the genetically modified animal is an animal from a family selected from Calomyscidae (e.g., mouse-like hamster), Cricetidae (e.g., hamster, New World rats and mice, field vole), Muridae (true mice and rats, gerbil, spiny mouse, maned mouse), Nesomyidae (climbing mouse, rock mouse, with-tailed rat, Malagasy rat and mouse), Platacanthomyidae (e.g., spiny dormouse), and Spalacidae (e.g., mole rate, bamboo rat, and burrowing mouse). In certain embodiments, the genetically modified rodent is selected from true mice or true rats (Muridae family), gerbils, spiny mice, and maned mice. In one embodiment, the genetically modified mouse is a member of the Muridae family. In one embodiment, the animal is a rodent. In certain embodiments, the rodent is selected from mice and rats. In one embodiment, the non-human animal is a mouse.

[0054] In certain embodiments, the non-human animal is a rodent that is a C57BL strain mouse selected from C57BL / A, C57BL / An, C57BL / GrFa, C57BL / KaLwN, C57BL / 6, C57BL / 6J, C57BL / 6ByJ, C57BL / 6NJ, C57BL / 10, C57BL / 10ScSn, C57BL / 10Cr, and C57BL / Ola. In another embodiment, the mice are 129 strains selected from a group consisting of strains 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1 / SV, 129S1 / SvIm), 129S2, 129S4, 129S5, 129S9 / SvEvH, 129S6 (129 / SvEvTac), 129S7, 129S8, 129T1, and 129T2 (see, for example, Festing et al. (1999) Revised nomenclature for strain 129 mice, Mammalian Genome Vol. 10: p. 836, Auerbach et al. (2000) Establishment and Chimera Analysis (See also of 129 / SvEv- and C57BL / 6-Derived Mouse Embryonic Stem Cell Lines). In certain embodiments, the genetically modified mouse is a mixture of the 129 strain and the C57BL / 6 strain described above. In another particular embodiment, the mouse is a mixture of the 129 strain or a mixture of the BL / 6 strain described above. In a particular embodiment, the 129 strain in the mixture is the 129S6(129 / SvEvTac) strain. In another embodiment, the mouse is a BALB strain, for example, the BALB / c strain. In yet another embodiment, the mouse is a mixture of the BALB strain and another strain described above.

[0055] In one embodiment, the non-human animal is a rat. In one embodiment, the rat is selected from Wistar rat, LEA strain, Sprague-Dolly strain, Fisher strain, F344, F6, and Dark Agouti. In one embodiment, the rat strain is a mixture of two or more strains selected from the group consisting of Wistar, LEA, Sprague-Dolly, Fisher, F344, F6, and Dark Agouti.

[0056] Accordingly, in one embodiment, the present invention provides a genetically modified mouse that expresses chimeric human / mouse CD4, wherein the nucleotide sequence encoding a chimeric human / mouse CD4 polypeptide is included in the genome, for example, at the endogenous CD4 locus, and the mouse portion of the chimeric polypeptide includes at least the transmembrane domain and the cytoplasmic domain of the mouse CD4 polypeptide. In one embodiment, the human portion of the chimeric polypeptide includes at least all or substantially all of the extracellular domain of the human CD4 polypeptide. In one embodiment, the human portion of the chimeric polypeptide includes at least all or substantially all of the D1 domain of the human CD4 protein. In one embodiment, the human portion of the chimeric polypeptide includes at least all or substantially all of the D1-D2 domains of the human CD4 protein, for example, at least all or substantially all of the D1-D3 domains of the human CD4 protein, for example, all or substantially all of the D1-D4 domains of the human CD4 protein. Therefore, in one embodiment, the mouse contains a nucleotide sequence at the endogenous CD4 locus that includes at least all or substantially all of exons 4, 5, and 6 of the human CD4 gene, for example, the sequence of exon 3 of the human CD4 gene encoding a portion of the D1 domain of human CD4 and a nucleotide sequence containing exons 4-6 of the human CD4 gene. In one embodiment, the mouse contains a chimeric human / mouse CD4 at the endogenous CD4 locus that includes a human CD4 sequence involved in interaction with MHC II and / or the extracellular portion of the T cell receptor. In another embodiment, the mouse contains a chimeric human / mouse CD4 at the endogenous CD4 locus that includes a human CD4 sequence involved in interaction with MHC II and / or the variable domain of the T cell receptor. In one embodiment, the nucleotide sequence includes a sequence encoding a mouse CD4 signal peptide. In one embodiment, the mouse contains a substitution of the nucleotide sequence encoding the mouse CD4 extracellular domain with a nucleotide sequence encoding the human CD4 extracellular domain.In another embodiment, the mouse includes substitution of a human nucleotide sequence encoding at least all or substantially all of the mouse CD4 D1 domain, for example, a nucleotide sequence encoding at least all or substantially all of the mouse CD4 D1-D2 domains, for example, a nucleotide sequence encoding at least all or substantially all of the mouse CD4 D1-D3 domains. In one embodiment, the mouse does not express functional endogenous mouse CD4 derived from its endogenous mouse CD4 locus. In one embodiment, the mouse described herein contains a chimeric human / mouse CD4 nucleotide sequence within the mouse germline. In one embodiment, the mouse retains any non-humanized endogenous sequence, for example, in an embodiment in which the mouse includes substitution of a nucleotide sequence encoding all or substantially all of the D1-D3 domains, the mouse retains an endogenous nucleotide sequence encoding the mouse CD4 D4 domain as well as nucleotide sequences encoding the transmembrane and cytoplasmic domains of mouse CD4.

[0057] In one embodiment, a mouse expressing a chimeric human / mouse CD4 protein retains a mouse CD4 promoter sequence and regulatory sequences. For example, the nucleotide sequence within the mouse encoding chimeric human / mouse CD4 is operably linked to the endogenous mouse CD4 promoter sequence and regulatory sequences. In one embodiment, these mouse regulatory sequences retained within the genetically engineered animal of the present invention include sequences that regulate the expression of the chimeric protein at appropriate stages during T cell development. Thus, in one embodiment, the mouse does not express chimeric CD4 on B cells or CD8 lineage T cells. In another embodiment, the mouse similarly does not express chimeric CD4 on any cell type that does not normally express endogenous CD4, e.g., any immune cell type.

[0058] In various embodiments, non-human animals (e.g., rodents, e.g., mice or rats) expressing a functional chimeric CD4 protein derived from the chimeric CD4 locus as described herein display the chimeric protein on the cell surface, for example, on the surface of T cells. In one embodiment, the non-human animal expresses the chimeric CD4 protein on the cell surface in the same cellular distribution as observed in humans. In one aspect, the CD4 protein of the present invention can interact with an MHC II protein expressed on the surface of a second cell, for example, an antigen-presenting cell (APC).

[0059] In one embodiment, the non-human animal of the present invention (e.g., a rodent, e.g., a mouse) further comprises a nucleotide sequence encoding a human or humanized MHC II protein, and thus a chimeric CD4 protein expressed on the surface of the animal's T cells can interact with human or humanized MHC II expressed on the surface of a second cell, e.g., an antigen-presenting cell. In one embodiment, the MHC II protein comprises the extracellular domains of a human MHC IIα polypeptide and a human MHC IIβ polypeptide. Examples of genetically modified animals expressing human or humanized MHC II polypeptides are described in U.S. Patent Application No. 13 / 661,116 filed October 26, 2012, and U.S. Patent Application No. 13 / 793,935 filed March 11, 2013, which are incorporated herein by reference in their entirety. Accordingly, in one embodiment, the animal comprising the chimeric CD4 protein described herein may further comprise a humanized MHC II protein comprising (1) a humanized MHC IIα polypeptide comprising a human MHC IIα extracellular domain and an endogenous transmembrane domain and cytoplasmic domain of, for example, mouse MHC II, wherein the human MHC IIα extracellular domain comprises the α1 and α2 domains of human MHC IIα, and (2) a humanized MHC IIβ polypeptide comprising a human MHC IIβ extracellular domain and an endogenous transmembrane domain and cytoplasmic domain of, for example, mouse MHC II, wherein the human MHC IIβ extracellular domain comprises the β1 and β2 domains of human MHC IIβ. In one embodiment, both the humanized MHC IIα polypeptide and the humanized MHC IIβ polypeptide are encoded by nucleotide sequences located at the endogenous MHC IIα locus and the endogenous MHC IIβ locus, respectively, and in one embodiment, the animal does not express functional endogenous MHC IIα polypeptide and endogenous MHC IIβ polypeptide. Therefore, the MHC II expressed by animals may be chimeric human / non-human, for example, human / rodent (e.g., human / mouse) MHC II proteins.The human portion of the chimeric MHC II protein may be derived from a human HLA class II protein selected from the group consisting of HLA-DR, HLA-DQ, and HLA-DP, such as HLA-DR4, HLA-DR2, HLA-DQ2.5, HLA-DQ8, or any other HLA class II molecule present in the human population. In embodiments where the animal is a mouse, the non-human (i.e., mouse) portion of the chimeric MHC II polypeptide may be derived from a mouse MHC II protein selected from H-2E and H-2A. In one embodiment, a non-human animal containing chimeric human / non-human CD4 and humanized MHC II as described in U.S. Patent Applications No. 13 / 661,116 and No. 13 / 793,935 can be produced by crossing an animal containing the chimeric CD4 locus described herein with an animal containing the humanized MHC II locus. Animals can also be created, for example, by introducing a nucleotide sequence encoding chimeric CD4 into animal ES cells containing a humanized MHC II locus for replacement at the endogenous CD4 locus, or by introducing a nucleotide sequence encoding humanized MHC II into animal ES cells containing a chimeric CD4 locus.

[0060] In one embodiment, a genetically modified non-human animal (e.g., mouse) containing both chimeric human / non-human CD4 and human or humanized MHC II may contain one or two copies of the genes encoding these proteins, and therefore the animal may be heterozygous or homozygous with respect to the genes encoding chimeric CD4 and MHC II (i.e., MHC IIα and / or MHC IIβ), respectively.

[0061] In addition to genetically modified non-human animals, the Specified Publication also provides non-human embryos (e.g., rodent embryos, e.g., mouse or rat embryos) comprising donor ES cells derived from the non-human animals described herein (e.g., rodents, e.g., mouse or rat). In one embodiment, the embryo comprises ES donor cells containing a chimeric CD4 gene and host embryonic cells.

[0062] Tissues derived from non-human animals (e.g., rodents, e.g., mice or rats) that express chimeric CD4 proteins are also provided herein.

[0063] Furthermore, non-human cells isolated from non-human animals as described herein are provided. In one embodiment, the cells are ES cells. In one embodiment, the cells are T cells, for example, CD4+ T cells. In one embodiment, the cells are helper T cells (T H It is a cell. In one embodiment, T H Cells are effector T H Cells, for example, T H 1 cell or T H It is a two-celled organism.

[0064] Non-human cells containing chromosomes or fragments thereof from non-human animals described herein are also provided. In one embodiment, the non-human cell contains the nucleus of a non-human animal described herein. In one embodiment, the non-human cell contains chromosomes or fragments thereof as a result of nuclear transfer.

[0065] In one embodiment, a non-human induced pluripotent cell is provided that contains a gene encoding the chimeric CD4 polypeptide described herein. In one embodiment, the induced pluripotent cell is derived from a non-human animal described herein.

[0066] In one embodiment, a hybridoma or quadroma derived from cells of a non-human animal described herein is provided. In one embodiment, the non-human animal is a mouse or a rat.

[0067] In one embodiment, an in vitro preparation is provided comprising a T cell carrying a chimeric CD4 protein on its surface and a second cell that binds to the chimeric CD4. In one embodiment, the second cell is a cell expressing an MHC II polypeptide, such as a chimeric human / non-human MHC II protein, e.g., an APC. In one embodiment, the chimeric CD4 on the surface of the first cell interacts with the chimeric MHC II on the surface of the second cell. In one embodiment, the chimeric CD4 protein maintains interactions with endogenous cytosolic molecules, such as endogenous cytosolic signaling molecules (e.g., endogenous Lck).

[0068] Methods for producing genetically modified non-human animals (e.g., genetically modified rodents, e.g., mice or rats) as described herein are also provided. In one embodiment, the method for producing a genetically modified non-human animal results in an animal having a nucleotide sequence encoding a chimeric human / non-human CD4 polypeptide at the endogenous CD4 locus. In some embodiments, the method utilizes a targeting construct produced using VELOCIGENE® technology, introduction of the construct into ES cells, and introduction of a targeted ES cell clone into a mouse embryo using VELOCIMOUSE® technology, as described in the examples.

[0069] In one embodiment, the present invention includes a method for modifying the CD4 locus of a non-human animal to express the chimeric human / non-human CD4 polypeptide described herein. In one embodiment, the present invention provides a method for modifying the CD4 locus of a mouse to express a chimeric human / mouse CD4 polypeptide, comprising the step of replacing the nucleotide sequence encoding the endogenous mouse CD4 polypeptide at the endogenous CD4 locus of a mouse with the nucleotide sequence encoding the chimeric human / mouse CD4 polypeptide. In one aspect of the method, the chimeric human / mouse CD4 polypeptide comprises all or substantially all of the extracellular domain of the human CD4 polypeptide and at least the transmembrane domain and cytoplasmic domain of the endogenous mouse CD4 polypeptide. In another aspect of the method, the chimeric human / mouse CD4 polypeptide comprises all or substantially all of the D1-D2 domains of the human CD4 polypeptide. In yet another embodiment, the chimeric human / mouse CD4 polypeptide comprises all or substantially all of the D1-D3 domains of the human CD4 polypeptide. In yet another embodiment, the chimeric human / mouse CD4 polypeptide comprises all or substantially all of the amino acid sequence of the extracellular domain of human CD4 and / or T cell receptor involved in interaction with MHC II. In yet another embodiment, the chimeric human / mouse CD4 polypeptide comprises all or substantially all of the amino acid sequence of the variable domain of human CD4 and / or T cell receptor involved in interaction with MHC II.

[0070] Accordingly, nucleotide constructs for constructing the genetically modified animals described herein are also provided. In one embodiment, the nucleotide sequence comprises 5' and 3' homology arms, a DNA fragment containing a human CD4 gene sequence (e.g., a human CD4 extracellular domain gene sequence, e.g., all or substantially all gene sequences of domains D1-D2 of human CD4, e.g., all or substantially all gene sequences of domains D1-D3 and / or D2-D3 of human CD4, e.g., all or substantially all gene sequences of domains D1-D4 of human CD4), and a selection cassette adjacent to a recombination site. In one embodiment, the human CD4 gene sequence is a genomic sequence containing the introns and exons of human CD4. In one embodiment, the homology arms are homologous to a non-human (e.g., mouse) CD4 genomic sequence. Exemplary constructs of the present invention are shown in Figure 1, below.

[0071] A selection cassette is a nucleotide sequence inserted into a targeting construct to facilitate the selection of cells (e.g., ES cells) incorporated into the construct of interest. Several suitable selection cassettes are known in the art. Generally, selection cassettes enable positive selection in the presence of specific antibiotics (e.g., Neo, Hyg, Pur, CM, SPEC, etc.). Furthermore, selection cassettes may be adjacent to recombination sites that allow deletion of the selection cassette upon treatment with a recombinase enzyme. Commonly used recombination sites are loxP and Frt, recognized by Cre and Flop enzymes, respectively, but others are known in the art. Selection cassettes may be located anywhere outside the coding region of the construct. In one embodiment, the selection cassette is located between exons 3 and 4 of the human CD4 sequence.

[0072] Once gene targeting is complete, ES cells or genetically modified non-human animals are screened to confirm that the target exogenous nucleotide sequence has been successfully incorporated or that the exogenous polypeptide is expressed. Numerous techniques are known to those skilled in the art, including (but not limited to) Southern blotting, long PCR, quantitative PCR (e.g., real-time PCR using TAQMAN®), fluorescence in situ hybridization, Northern blotting, flow cytometry, Western blotting, immunocytochemical analysis, and immunohistochemical analysis. In one example, non-human animals (e.g., mice) carrying the target gene modification were described in Valenzuela et al. (2003) High-throughput engineering of the mouse genome coupled with high-resolution Mouse allele loss and / or human allele acquisition can be identified by screening using a modified allele assay described in expression analysis, Nature Biotech, Vol. 21 (No. 6): pp. 652-659. Other assays for identifying specific nucleotide or amino acid sequences in genetically modified animals are known to those skilled in the art.

[0073] In one embodiment, a method is provided for producing a chimeric human / non-human CD4 molecule, comprising the step of expressing a chimeric CD4 protein from a nucleotide construct described herein in a single cell. In one embodiment, the nucleotide construct is a viral vector, and in a particular embodiment, the viral vector is a lentiviral vector. In one embodiment, the cells are selected from CHO, COS, 293, HeLa, and retinal cells that express a viral nucleic acid sequence (e.g., PERC.6® cells).

[0074] In one embodiment, cells expressing a chimeric CD4 protein are provided. In one embodiment, the cells include an expression vector containing the chimeric CD4 sequence described herein. In one embodiment, the cells are selected from CHO, COS, 293, HeLa, and retinal cells expressing a viral nucleic acid sequence (e.g., PERC.6® cells).

[0075] Chimeric CD4 molecules produced by non-human animals as described herein are also provided, in one embodiment the chimeric CD4 molecule comprises all or substantially all of the extracellular domain of the human CD4 protein, as well as the amino acid sequence of at least the transmembrane and cytoplasmic domains derived from a non-human CD4 protein, e.g., mouse CD4 protein. In another embodiment the chimeric CD4 molecule produced by a non-human animal as described herein is provided, comprising at least all or substantially all of the D1 domain of human CD4, e.g., at least all or substantially all of the D1-D2 domains of human CD4, e.g., at least all or substantially all of the amino acid sequences of the D1-D3 domains of human CD4, e.g., the amino acid sequence of human CD4 involved in the binding of the extracellular domain of MHC II and / or TCR, e.g., the amino acid sequence of human CD4 involved in the binding of the variable domain of MHC II and / or TCR, and the remainder of the protein (e.g., any unhumanized portion of the transmembrane domain, cytoplasmic domain, or extracellular domain) is derived from an endogenous non-human protein sequence.

[0076] The various embodiments described above are applicable to animals expressing chimeric CD4 proteins, as well as non-human animals expressing chimeric human / non-human CD8 proteins, or other important chimeric human / non-human T cell co-receptors, in relation to cells and tissues containing such proteins.

[0077] Genetically modified CD8 animals In various embodiments, the present invention generally provides a genetically modified non-human animal that contains a nucleotide sequence encoding a humanized CD8 polypeptide in its genome, for example, at the endogenous CD8 locus, and therefore expresses a humanized CD8 polypeptide. In various embodiments, the present invention provides a non-human animal that contains a nucleotide sequence encoding a humanized CD8α polypeptide and / or a humanized CD8β polypeptide in its genome, for example, at the endogenous CD8 locus. Therefore, the genetically modified non-human animal of the present invention expresses a humanized CD8α and / or humanized CD8β polypeptide(s).

[0078] Human CD8 protein is generally expressed on the cell surface as a heterodimer of two polypeptides, CD8α and CD8β, but disulfide-linked homodimers and homomultimers have also been detected (e.g., NK cells and intestinal γδT cells expressing CD8αα). The genes encoding human CD8α and CD8β are located very close to each other on chromosome 2. Nakayama et al. (1992) Recent Duplication of the Two Human CD8 β-chain genes, J. Immunol. 148: pp. 1919-1927. The CD8α protein contains a leader peptide, an immunoglobulin V-like region, a hinge region, a transmembrane domain, and a cytoplasmic tail. Norment et al. (1989) Alternatively Spliced ​​mRNA Encodes a Secreted Form of Human CD8α. Characterization of the Human CD8α gene, J. Immunol. Vol. 142: pp. 3312-3319. The exons / introns of the CD8α gene are schematically shown in Figures 4 and 5.

[0079] The human CD8β gene is located upstream of the CD8α gene on chromosome 2. Numerous isoforms have been reported to be created by alternative splicing of the CD8β gene, and one isoform lacks a transmembrane domain and is predicted to produce a secreted protein. (Norment et al., 1988: A second subunit of CD8) is expressed in human T cells, EMBO J. Vol. 7: pp. 3433-39. The exons / introns of the CD8β gene are schematically shown in Figures 3 and 5.

[0080] The membrane-bound CD8β protein contains an N-terminal signal sequence, followed by an immunoglobulin V-like domain, a short extracellular hinge region, a transmembrane domain, and a cytoplasmic tail. See Littman (1987) The structure of CD4 and CD8 genes, Ann Rev. Immunol. Vol. 5: pp. 561-584. The hinge region is a site of extensive glycosylation where conformation is maintained and the protein is protected from protease cleavage. See Leahy (1995) A structural view of CD4 and CD8, FASEB J. Vol. 9: pp. 17-25.

[0081] The CD8 protein is generally expressed on cytotoxic T cells and interacts with MHC I molecules. This interaction is mediated by the binding of CD8 to the α3 domain of MHC I. Although the binding of CD8 to MHC class I is about 1 / 100th of the binding of TCRs to MHC class I, CD8 binding enhances the affinity for TCR binding. Wooldridge et al. (2010) MHC Class I Molecules with Superenhanced CD8 Binding Properties Bypass the Requirement for Cognate TCR Recognition and Nonspecifically Activate CTL, J. Immunol. 184: pp. 3357-3366.

[0082] The binding of CD8 to MHC class I molecules is species-specific, and the mouse homolog of CD8, Lyt-2, has H-2D binding in its α3 domain. d It was shown that it binds to the molecule but not to the HLA-A molecule. Connolly et al. (1988) The Lyt-2 Molecule Recognizes Residues in the Class I α3 Domain in Allogeneic Cytotoxic T Cell Responses, J. Exp. Med. 168: pp. 325-341. The differential binding was probably due to non-conservative CDR-like determinants (CDR1-like and CDR2-like) on CD8 between humans and mice. Sanders et al. (1991) Mutations in CD8 that Affect Interactions with HLA Class I and Monoclonal Anti-CD8 Antibodies, J. Exp. Med. 174: pp. 371-379; Vitiello et al. (1991) Analysis of the HLA-restricted Influenza-specific Cytotoxic T Lymphocyte Response in Transgenic Mice Carrying a Chimeric Human-Mouse Class I Major Histocompatibility Complex, J. Exp. Med. Vol. 173: pp. 1007 - 1015, and Gao et al. (1997) Crystal structure of the complex between human CD8αα and HLA - A2, Nature Vol. 387: pp. 630 - 634. CD8 has been reported to bind to HLA - A2 in the conserved region (positions 223 - 229) of the α3 domain. A single substitution (V245A) in HLA - A reduces the binding of CD8 to HLA - A, and at the same time, T - cell - mediated lysis is greatly reduced. Salter et al. (1989), Polymorphism in the α3 domain of HLA - A molecules affects binding to CD8, Nature Vol. 338: pp. 345 - 348. In general, polymorphisms in the α3 domain of HLA - A molecules also affect binding to CD8. Ibid. In mice, an amino - acid substitution at residue 227 of H - 2D d affects the binding of mouse Lyt - 2 to H - 2D d , and cells transfected with mutant H - 2D d were not lysed by CD8+ T cells. Potter et al. (1989) Substitution at residue 227 of H - 2 class I molecules abrogates recognition by CD8 - dependent, but not CD8 - independent, cytotoxic T lymphocytes, Nature Vol. 337: pp. 73 - 75. Therefore, the expression of human or humanized CD8 may be useful for testing T - cell responses to antigens presented by human or humanized MHC I.

[0083] Similar to CD4, the cytoplasmic domain of CD8 interacts with the tyrosine kinase Lck, which in turn leads to T cell activation. While Lck appears to interact with the cytoplasmic domain of CD8α, mutations or deletions in the CD8β cytoplasmic domain reduce CD8α-related Lck activity, suggesting that this interaction is regulated by the presence of the CD8β cytoplasmic domain. (Irie et al., 1998, The cytoplasmic domain of CD8β Regulates Lck Kinase Activation and CD8 T cell Development, J. Immunol., Vol. 161: pp. 183-191). Reduced Lck activity was associated with defects in T cell development. (Ibid.)

[0084] CD8 expression in appropriate cells, such as cytotoxic T cells, is tightly regulated by various enhancer elements located throughout the CD8 locus. For example, at least four DNase I hypersensitivity regions, often associated with regulatory factor binding, have been identified within the CD8 locus. (Hosert et al., 1997) A CD8 Genomic fragment that directs subset-specific expression of CD8 in transgenic mice, J. Immunol. Vol. 158: pp. 4270-4281. Since the discovery of these DNase I-sensitive regions at the CD8 locus, at least five enhancer elements have been identified that extend across the entire CD8 locus and regulate the expression of CD8α and / or CD8β in various lineages of T cells, including DP, CD8 SP T cells, or cells expressing γδTCR. For example, Kioussis et al. (2002) Chromatin and CD4, CD8A, and CD8B gene expression during thymic differentiation, Nature Rev. Vol. 2: pp. 909-919 and Online Erratum; Ellmeier et al. (1998) Multiple Development See "Stage-Specific Enhancers Regulate CD8 Expression in Developing Thymocytes and in Thymus-Independent T Cells," Immunity Vol. 9: pp. 485-4896.

[0085] Therefore, similar to the advantages obtained from retaining endogenous CD4 promoters and regulatory elements in human or humanized CD4 genetically modified animals, in some embodiments, there may be advantages to developing genetically modified non-human animals that retain endogenous mouse promoters and regulatory elements that control the expression of human or humanized CD8. There may be specific advantages to creating genetically modified animals that include the replacement of endogenous non-human sequences encoding CD8α and / or CD8β proteins with sequences encoding human or humanized CD8α and / or CD8β proteins, as described herein.

[0086] In various embodiments, the present invention provides a genetically modified non-human animal that expresses the chimeric CD8 polypeptide (e.g., CD8α and / or CD8β polypeptide), wherein the genome contains at least one nucleotide sequence encoding a chimeric human / non-human CD8 polypeptide (e.g., CD8α and / or CD8β polypeptide), the human portion of which comprises all or substantially all of the extracellular domain of human CD8 (e.g., CD8α and / or CD8β), and the non-human portion comprises at least the transmembrane domain and cytoplasmic domain of non-human CD8 (e.g., CD8α and / or CD8β). Accordingly, in one embodiment, the present invention provides a genetically modified non-human animal that expresses a functional chimeric human / non-human CD8 protein, wherein the endogenous non-human CD8 locus contains a first nucleotide sequence encoding a chimeric human / non-human CD8α polypeptide and a second nucleotide sequence encoding a chimeric human / non-human CD8β polypeptide, the first nucleotide sequence comprising a sequence encoding all or substantially all of the extracellular domain of the human CD8α polypeptide, as well as at least the transmembrane and cytoplasmic domains of the non-human CD8α polypeptide, and the second nucleotide sequence comprising a sequence encoding all or substantially all of the extracellular domain of the human CD8β polypeptide, as well as at least the transmembrane and cytoplasmic domains of the non-human CDβ polypeptide. In one embodiment, the non-human animal expresses only humanized CD8 polypeptides (e.g., chimeric human / non-human CD8α and / or CD8β polypeptides) derived from the endogenous CD8 locus, and does not express the corresponding functional non-human CD8 polypeptide(s).

[0087] In one embodiment, the chimeric human / non-human CD8α polypeptide includes all or substantially all of the extracellular domain of the human CD8α polypeptide in the human portion. In one embodiment, the human portion of the chimeric CD8α polypeptide includes at least the MHC I-binding domain of the human CD8α polypeptide. In one embodiment, the human portion of the chimeric CD8α polypeptide includes at least all or substantially all of the sequence of the immunoglobulin V-like domain of human CD8α. In one embodiment, the nucleotide sequence encoding the human portion of the chimeric CD8α polypeptide includes at least the exon encoding the extracellular domain of the human CD8α polypeptide. In one embodiment, the nucleotide sequence includes at least the exon encoding the Ig V-like domain. In one embodiment, the extracellular domain of the human CD8α polypeptide is a region encompassing a polypeptide domain that is neither a transmembrane domain nor a cytoplasmic domain. In one embodiment, the nucleotide sequence encoding the chimeric human / non-human CD8α polypeptide includes a sequence encoding a non-human (e.g., rodent, e.g., mouse) CD8α signal peptide. Alternatively, the nucleotide sequence may include a sequence encoding a human CD8α signal sequence. In one embodiment, the chimeric human / non-human CD8α polypeptide contains the amino acid sequence described in SEQ ID NO: 54, and the human portion of the chimeric polypeptide is described in amino acids 28-179 of SEQ ID NO: 54 (also shown in SEQ ID NO: 59).

[0088] Similarly, in one embodiment, the chimeric human / non-human CD8β polypeptide includes all or substantially all of the extracellular domain of the human CD8β polypeptide in the human portion. In one embodiment, the human portion of the chimeric CD8β polypeptide includes all or substantially all of the sequence of the immunoglobulin V-like domain of CD8β. In one embodiment, the nucleotide sequence encoding the human portion of the chimeric CD8β polypeptide includes at least the exon encoding the extracellular domain of the human CD8β polypeptide. In one embodiment, the human portion of the chimeric human / non-human CD8β polypeptide includes at least the exon encoding the IgG V-like domain of CD8β. In one embodiment, the nucleotide sequence encoding the chimeric human / non-human CD8β polypeptide includes the sequence encoding a non-human (e.g., rodent, e.g., mouse) CD8β signal peptide. Alternatively, the nucleotide sequence may include the sequence encoding the human CD8β signal sequence. In one embodiment, the chimeric human / non-human CD8β polypeptide includes the amino acid sequence described in SEQ ID NO: 53, and the human portion of the chimeric polypeptide is described in amino acids 15-165 of SEQ ID NO: 53 (represented separately in SEQ ID NO: 58).

[0089] In one embodiment, a non-human animal expresses a chimeric human / non-human CD8α and / or CD8β polypeptide. In some embodiments, the human portion of the chimeric human / non-human CD8α and / or CD8β polypeptide includes one or more conserved or non-conserved modifications.

[0090] In one embodiment, a non-human animal is provided that expresses a human CD8α and / or CD8β polypeptide sequence, wherein the human CD8α and / or CD8β polypeptide sequence is at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the human CD8α and / or CD8β polypeptide sequence, respectively. In a particular embodiment, the human CD8α and / or CD8β polypeptide sequence is at least about 90%, 95%, 96%, 97%, 98%, or 99% identical to the human CD8α and / or CD8β polypeptide sequence described in the example. In one embodiment, the human CD8α and / or CD8β polypeptide sequence includes one or more conserved substitutions. In one embodiment, the human CD8α and / or CD8β polypeptide sequence includes one or more non-conserved substitutions.

[0091] In some embodiments, a portion of the chimeric CD8, for example, the human portion, may comprise substantially all of the sequences shown herein (e.g., substantially all of the protein domains shown herein). Substantially all sequences generally contain 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the amino acids that are considered to represent a particular portion of the protein (e.g., a particular functional domain). Those skilled in the art will understand that the boundaries of functional domains may vary slightly depending on the alignment and domain prediction method used.

[0092] In one embodiment, the non-human portion of the chimeric human / non-human CD8α and / or CD8β polypeptide comprises at least the transmembrane and / or cytoplasmic domains of the non-human CD8α and / or CD8β polypeptide, respectively. Due to the important functions performed by the CD8 cytoplasmic domain, the retention of endogenous non-human (e.g., mouse) sequences within the genetically engineered animal ensures the conservation of appropriate intracellular signaling and other functions of co-receptors. In one embodiment, the non-human animal is a mouse, and the non-human CD8α and / or CD8β polypeptides are mouse CD8α and / or CD8β polypeptides, respectively. While specific mouse CD8α and CD8β sequences are described in the examples, any suitable sequence derived therefrom, including sequences with conserved / non-conserved amino acid substitutions, is incorporated herein. In one embodiment, the non-human animal (e.g., a rodent, e.g., a mouse) retains any non-humanized endogenous sequence.

[0093] The non-human animals described herein may contain nucleotide sequences encoding chimeric human / non-human CD8α and / or CD8β polypeptides at their endogenous loci. In one embodiment, this is done by replacing a portion of the endogenous CD8α gene with a nucleotide sequence encoding a portion of the human CD8α polypeptide, and / or replacing a portion of the endogenous CD8β gene with a nucleotide sequence encoding a portion of the human CD8β polypeptide. In one embodiment, such replacement is the replacement of an endogenous nucleotide sequence encoding all or substantially all of the extracellular domains of non-human CD8α and / or CD8β with the human nucleotide sequence encoding them. In one embodiment, such replacement is the replacement of a sequence encoding at least all or substantially all of the immunoglobulin V-like domains of non-human CD8α and / or CD8β with the human nucleotide sequence encoding them. In one embodiment, the replacement does not involve the replacement of CD8α and / or CD8β sequences encoding the transmembrane and cytoplasmic domains of the non-human CD8α and / or CD8β polypeptides. Therefore, non-human animals express chimeric human / non-human CD8α and / or CD8β polypeptides derived from the endogenous non-human CD8 locus. In yet another embodiment, substitution results in CD8α and / or CD8β proteins containing polypeptide sequences described in SEQ ID NO: 54 and / or 53, respectively.

[0094] In one embodiment, a nucleotide sequence of a chimeric human / non-human CD8 locus (e.g., a chimeric rodent CD8 locus, e.g., a chimeric mouse CD8 locus) is provided. In one embodiment, the chimeric human / non-human (e.g., human / rodent, e.g., human / mouse) CD8α and / or CD8β sequences are located at their respective endogenous non-human (e.g., rodent, e.g., mouse) CD8α and / or CD8β loci and retain the endogenous CD8α and / or CD8β promoters and regulatory elements. In another embodiment, the chimeric locus may contain human CD8α and / or CD8β promoters and regulatory elements, insofar as they enable appropriate CD8α and / or CD8β expression (appropriate spatial and temporal protein expression), CD8+ T cell development, CD8 lineage selection, and co-receptor function. Thus, in one embodiment, the animal of the present invention includes a genetic modification in which appropriate lineage selection and development of T cells are not altered. In one embodiment, the animal of the present invention (e.g., a rodent, e.g., a mouse) does not express the chimeric CD8 protein on immune cells other than those that normally express CD8; for example, the animal does not express CD8 on B cells or CD4+SP T cells. In one embodiment, the substitution retains elements that allow for appropriate spatial and temporal regulation of CD8α and / or CD8β expression.

[0095] The genetically modified non-human animals containing human or humanized CD8 polypeptides described herein can be selected from any of the animals described in the section on humanized CD4 animals above. In one embodiment, the non-human animal may be a rodent, such as a rat or mouse.

[0096] Accordingly, in one embodiment, the present invention provides a genetically modified mouse comprising a first nucleotide sequence encoding a chimeric human / mouse CD8α polypeptide and a second nucleotide sequence encoding a chimeric human / mouse CD8β polypeptide in the genome, for example, at the endogenous CD8 locus. In one embodiment, the first nucleotide sequence comprises all or substantially all of the extracellular domain of the human CD8α polypeptide, as well as sequences encoding at least the transmembrane and cytoplasmic domains of the mouse CD8α polypeptide, and the second nucleotide sequence comprises all or substantially all of the extracellular domain of the human CD8β polypeptide, as well as sequences encoding at least the transmembrane and cytoplasmic domains of the mouse CD8β polypeptide, and the mouse expresses a functional chimeric human / mouse CD8 protein. In one embodiment, the first nucleotide sequence comprises sequences encoding at least the immunoglobulin V-like domain of the human CD8α polypeptide and sequences other than the immunoglobulin V-like domain of the mouse CD8α polypeptide, and the second nucleotide sequence comprises sequences encoding at least the immunoglobulin V-like domain of the human CD8β polypeptide and sequences other than the immunoglobulin V-like domain of the mouse CD8β polypeptide. In one embodiment, the first nucleotide sequence includes at least an MHC I-binding domain of the human CD8α polypeptide. In one embodiment, the first and second nucleotide sequences each include an exon encoding at least the extracellular domain of the human CD8α and / or CD8β polypeptide. In one embodiment, the extracellular domain of the human CD8α polypeptide and / or CD8β polypeptide is a region encompassing a domain that is neither a transmembrane domain nor a cytoplasmic domain of the human CD8α polypeptide and / or CD8β polypeptide. In one embodiment, the domain of the human CD8α polypeptide is schematically shown in Figures 4 and 5. In one embodiment, the domain of the human CD8β polypeptide is schematically shown in Figures 3 and 5.In one embodiment, the nucleotide sequences encoding the chimeric human / mouse CD8α polypeptide and / or CD8β polypeptide each include sequences encoding the mouse CD8α and / or CD8β signal peptides. Alternatively, the nucleotide sequences may include sequences encoding the human CD8α and / or CD8β signal sequences. In one embodiment, the mouse includes the replacement of the nucleotide sequences encoding all or substantially all of the mouse CD8α and / or CD8β extracellular domains with nucleotide sequences encoding all or substantially all of the human CD8α and / or CD8β extracellular domains.

[0097] In one embodiment, the mouse does not express functional endogenous mouse CD8α and / or CD8β polypeptides derived from its endogenous CD8 locus. In one embodiment, the mouse described herein contains a chimeric human / mouse CD8 sequence in its germline.

[0098] In one embodiment, a mouse expressing a chimeric human / mouse CD8α and / or CD8β polypeptide retains mouse CD8α and / or CD8β promoter sequences and regulatory sequences, for example, the nucleotide sequence encoding chimeric human / mouse CD8 in the mouse is operably linked to the endogenous mouse CD8 promoter sequence and regulatory sequences. In one embodiment, these regulatory sequences retained in the mouse include sequences that regulate the expression of the CD8 protein at appropriate stages of T cell development. In one embodiment, the genetically modified mouse does not express chimeric CD8 on B cells or CD4-lineage T cells, or any cell that does not normally express endogenous CD8, such as immune cells.

[0099] In various embodiments, non-human animals (e.g., rodents, e.g., mice or rats) expressing a functional chimeric CD8 protein (e.g., CD8αβ or CD8αα) derived from the chimeric CD8 locus described herein display the chimeric protein on the cell surface. In one embodiment, the non-human animal expresses the chimeric CD8 protein on the cell surface in the same cellular distribution as observed in humans. In one aspect, the CD8 protein of the present invention can interact with an MHC I protein expressed on the surface of a second cell.

[0100] In one embodiment, the non-human animal of the present invention (e.g., a rodent, e.g., a mouse) further comprises a nucleotide sequence encoding a human or humanized MHC I protein, and thus the chimeric CD8 protein expressed on the surface of the animal's T cells can interact with human or humanized MHC I expressed on the surface of a second cell, e.g., an antigen-presenting cell. In one embodiment, the MHC I protein comprises the extracellular domain of a human MHC I polypeptide. In one embodiment, the animal further comprises a human or humanized β-2 microglobulin polypeptide. Examples of genetically modified animals expressing human or humanized MHC I polypeptide and / or β-2 microglobulin polypeptide are described in U.S. Patent Application No. 13 / 661,159 filed October 26, 2012, and U.S. Patent Application No. 13 / 793,812 filed March 11, 2013, both of which are incorporated herein by reference in their entirety. Thus, in one embodiment, the animal comprising the chimeric CD8 protein described herein may further comprise a humanized MHC I complex, and said humanized MHC The I complex includes (1) a humanized MHC I polypeptide comprising, for example, the extracellular domain of human MHC I and the transmembrane and cytoplasmic domains of endogenous (e.g., mouse) MHC I, for example, a humanized MHC I polypeptide comprising the α1, α2, and α3 domains of human MHC I polypeptide, and (2) a human or humanized β2 microglobulin polypeptide (for example, an animal has in its genome the nucleotide sequences shown in exons 2, 3, and 4 of human β2 microglobulin). In one embodiment, both humanized MHC I and human or humanized β2 microglobulin polypeptides are encoded by nucleotide sequences located at the endogenous MHC I locus and β2 microglobulin locus, respectively; in one embodiment, the animal does not express functional endogenous MHC I and β2 microglobulin polypeptides. Thus, the MHC I expressed by the animal may be a chimeric human / non-human, e.g., human / rodent (e.g., human / mouse) MHC I polypeptide. The human portion of the chimeric MHC I polypeptide may be derived from a human HLA class I protein 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. In embodiments where the animal is a mouse, the non-human (i.e., mouse) portion of the chimeric MHC I polypeptide may be derived from a mouse MHC I protein selected from H-2D, H-2K, and H-2L. In one embodiment, non-human animals containing the chimeric human / non-human CD8 described herein and humanized MHC I and / or β-2 microglobulin described in U.S. Patent Applications No. 13 / 661,159 and No. 13 / 793,812 can be produced by crossing an animal containing the chimeric CD8 locus (e.g., chimeric CD8α and / or CD8β locus) described herein with an animal containing the humanized MHC I and / or β-2 microglobulin locus.Animals can also be created, for example, by introducing nucleotide sequences encoding chimeric CD8 (e.g., chimeric CD8α and / or CD8β) into animal ES cells containing humanized MHC I and / or β-2 microglobulin loci for replacement at endogenous CD8 loci (e.g., chimeric CD8α and / or CD8β loci), or by introducing nucleotide sequences (multiple) encoding humanized MHC I and / or β-2 microglobulin into animal ES cells containing chimeric CD8 loci (e.g., chimeric CD8α and / or CD8β loci).

[0101] In addition to genetically modified non-human animals, non-human embryos (e.g., rodent embryos, e.g., mouse or rat embryos) are also provided, which include donor ES cells derived from the non-human animals (e.g., rodents, e.g., mouse or rat) described herein. In one embodiment, the embryo includes ES donor cells containing a chimeric CD8 gene and host embryonic cells.

[0102] Tissues derived from non-human animals (e.g., rodents, e.g., mice or rats) that express the chimeric CD8 protein are also provided herein.

[0103] Furthermore, non-human cells isolated from non-human animals described herein are provided. In one embodiment, the cells are ES cells. In one embodiment, the cells are T cells, for example, CD8+ T cells. In one embodiment, the cells are cytotoxic T cells.

[0104] Non-human cells containing chromosomes or fragments thereof from non-human animals described herein are also provided. In one embodiment, the non-human cell contains the nucleus of a non-human animal described herein. In one embodiment, the non-human cell contains chromosomes or fragments thereof as a result of nuclear transfer.

[0105] In one embodiment, a non-human induced pluripotent cell is provided that contains a gene encoding a chimeric CD8 polypeptide (e.g., CD8α and / or CD8β polypeptide) as described herein. In one embodiment, the induced pluripotent cell is derived from a non-human animal as described herein.

[0106] In one embodiment, a hybridoma or quadroma derived from cells of a non-human animal described herein is provided. In one embodiment, the non-human animal is a mouse or a rat.

[0107] In one embodiment, an in vitro preparation is provided comprising a T cell carrying a chimeric CD8 protein on its surface and a second cell that binds to the chimeric CD8. In one embodiment, the second cell is a cell expressing an MHC I polypeptide, such as a chimeric human / non-human MHC I protein. In one embodiment, the chimeric CD8 on the surface of the first cell interacts with the chimeric MHC I on the surface of the second cell. In one embodiment, the chimeric CD8 protein maintains interactions with endogenous cytosolic molecules, such as endogenous cytosolic signaling molecules (e.g., endogenous Lck).

[0108] This specification also provides a method for producing the genetically modified non-human animals described herein. This method results in an animal having an endogenous CD8 locus containing nucleotide sequences(s) encoding chimeric human / non-human CD8α and / or CD8β polypeptides. This method can utilize targeting constructs produced using VELOCIGENE® technology, introduction of the constructs into ES cells, and introduction of targeted ES cell clones into mouse embryos using VELOCIMOUSE® technology, as described in the examples.

[0109] In one embodiment, the present invention provides a method for modifying the CD8 locus of a non-human animal to express a chimeric human / non-human CD8 polypeptide as described herein. In one embodiment, a method is provided for modifying the CD8 locus of a mouse to express a chimeric human / mouse CD8 polypeptide, comprising the step of replacing the nucleotide sequence encoding the endogenous mouse CD8 polypeptide at the endogenous CD8 locus of a mouse with the nucleotide sequence encoding the chimeric human / mouse CD8 polypeptide. The CD8 polypeptide can be selected from CD8α, CD8β, and combinations thereof. In one embodiment, the chimeric polypeptide comprises all or substantially all of the extracellular domain of the human CD8 polypeptide, as well as at least the transmembrane domain and cytoplasmic domain of the endogenous mouse CD8 polypeptide.

[0110] Accordingly, nucleotide constructs for constructing the genetically modified animals described herein are also provided. In one embodiment, the sequence of the nucleotide construct includes 5' and 3' homology arms, a DNA fragment containing a human CD8α or CD8β sequence, and a selection cassette adjacent to the recombination site. In some embodiments, the human sequence includes introns and exons of human CD8α or CD8β, for example, exons encoding the extracellular domain of human CD8α or CD8β, respectively. In one embodiment, the homology arms are homologous to a non-human CD8α or CD8β sequence. Examples of constructs for CD8α and CD8β are shown in Figures 4 and 3, respectively.

[0111] Since the genes encoding CD8α and CD8β are localized close together within the chromosome, sequentially targeting the two genes improves the chances of successful humanization. In one embodiment, the targeting strategy includes the steps of introducing the chimeric CD8β construct described herein into ES cells, generating a mouse from the target ES cells, extracting genetically modified ES cells from the mouse, and introducing the chimeric CD8α construct described herein into the genetically modified ES cells. In another embodiment, the targeting strategy includes the steps of introducing the chimeric CD8β construct described herein into ES cells, selecting cells into which the chimeric CD8β construct has been incorporated, introducing the chimeric CD8α construct described herein into ES cells into which the chimeric CD8β construct has been incorporated and which possess it, and selecting cells into which both chimeric CD8β and CD8α have been incorporated. In one aspect of this embodiment, the selection step is carried out using different selection markers. In an alternative embodiment, humanization of CD8α can be achieved first. Once gene targeting is complete, genetically modified non-human animal ES cells can be screened to confirm that the target exogenous nucleotide sequence has been successfully incorporated or that the exogenous polypeptide has been expressed, using various methods (e.g., the method described above for CD4 humanization, e.g., Valenzuela et al., modification of the allele assay described above).

[0112] In one embodiment, a method is provided for producing a chimeric human / non-human CD8 molecule (e.g., CD8α and / or CD8β), comprising the step of expressing a chimeric CD8 polypeptide(s) from a nucleotide construct(s) described herein in a single cell. In one embodiment, the nucleotide construct(s) is a viral vector, and in a particular embodiment, the viral vector(s) is a lentiviral vector. In one embodiment, the cells are selected from CHO, COS, 293, HeLa, and retinal cells expressing a viral nucleic acid sequence (e.g., PERC.6® cells).

[0113] In one embodiment, cells expressing a chimeric CD8 protein are provided. In one embodiment, the cells include an expression vector containing the chimeric CD8 sequence(s) described herein. In one embodiment, the cells are selected from CHO, COS, 293, HeLa, and retinal cells expressing a viral nucleic acid sequence (e.g., PERC.6® cells).

[0114] This specification also provides chimeric CD8 molecules produced by non-human animals, comprising all or substantially all of the extracellular domains derived from human CD8 proteins (e.g., CD8α and / or CD8β), as well as at least a transmembrane domain and a cytoplasmic domain derived from a non-human CD8 protein, such as mouse CD8 protein. An exemplary chimeric CD8α polypeptide is described in SEQ ID NO: 54, and an exemplary chimeric CD8β protein is described in SEQ ID NO: 53.

[0115] Use of genetically modified CD4 animals and genetically modified CD8 animals Genetically modified non-human animals containing either humanized CD4 and MHC II or humanized CD8 and MHC I, such as rodents, mice, or rats, present peptides to T cells (CD4+ or CD8+ T cells, respectively) in a human-like manner because substantially all of the components of the complex are human or humanized. The genetically modified non-human animals of the present invention can be used to test the function of the human immune system in humanized animals; to identify antigens and antigen epitopes (e.g., T cell epitopes, e.g., unique human cancer epitopes) that elicit an immune response, for use in vaccine development; to identify high-affinity T cells against human pathogens or cancer antigens (i.e., T cells that bind to antigens with high binding activity in the context of human MHC I complexes), for use in adaptive T cell therapy; to evaluate vaccine candidates and other vaccine strategies; to study human autoimmunity; to study human infectious diseases; and, in other respects, to devise better therapeutic strategies based on human MHC and CD4 / CD8 expression.

[0116] Therefore, in various embodiments, the genetically modified animals of the present invention are useful, in particular, for evaluating the ability of antigens to initiate an immune response in humans, and for identifying specific antigens that can be used in human vaccine development, thereby generating antigenic diversity.

[0117] In one embodiment, a method is provided for determining whether a peptide induces a cellular immune response in humans, comprising the steps of: exposing a genetically modified non-human animal described herein to the peptide; initiating an immune response in the non-human animal; and detecting non-human animal cells (e.g., CD8+ or CD4+ T cells containing human CD8 or CD4, respectively) that bind to the sequence of the peptide presented by a chimeric human / non-human MHC I or MHC II molecule described herein. In one embodiment, the non-human animal after exposure contains MHC class I restriction CD8+ cytotoxic T lymphocytes (CTLs) that bind to the peptide. In another embodiment, the non-human animal after exposure contains MHC II restriction CD4+ T cells that bind to the peptide.

[0118] In one embodiment, a method for identifying a human T cell epitope is provided, comprising the steps of: exposing a non-human animal described herein to an antigen containing a putative T cell epitope; initiating an immune response in the non-human animal; isolating MHC class I restriction T cells or MHC class II restriction T cells bound to the epitope from the non-human animal; and identifying the epitope to which the T cells are bound.

[0119] In one embodiment, a method is provided for identifying an antigen that elicits a T cell response in humans, comprising the steps of exposing a putative antigen to a mouse described herein, inducing an immune response in the mouse, and identifying the antigen to which an HLA class I restriction molecule or a class II restriction molecule is bound.

[0120] In one embodiment, a method is provided for determining whether a putative antigen contains an epitope that, upon exposure to the human immune system, elicits an HLA class I or class II restrictive immune response, comprising the steps of exposing a mouse described herein to the putative antigen and measuring the antigen-specific HLA class I or HLA class II restrictive immune response in the mouse.

[0121] Furthermore, the genetically modified non-human animals described herein may be useful for identifying T cell receptors that recognize an antigen of interest, such as an antigen of a tumor or another disease, such as a T cell receptor with high binding activity. The method may include the steps of exposing the non-human animals described herein to an antigen, initiating an immune response in the non-human animals to the antigen, isolating non-human animal T cells containing a T cell receptor that binds to the antigen presented by human or humanized MHC I or MHC II, and determining the sequence of the T cell receptor.

[0122] In one embodiment, a method is provided for determining T cell activation by a putative human therapeutic agent, comprising the steps of: exposing a genetically modified animal described herein to a putative human therapeutic agent (or, for example, exposing human or humanized MHC II-expressing cells or MHC I-expressing cells of such an animal to the peptide sequence of the putative therapeutic agent); exposing cells of the genetically modified animal displaying human or humanized MHC / peptide complexes to T cells containing chimeric human / non-human (e.g., human / mouse) CD4 or CD8 that can bind to cells of the genetically modified animal; and measuring the T cell activation induced by the cells displaying the peptides of the genetically modified animal.

[0123] In addition to being able to identify antigens and antigenic epitopes from human pathogens or neoplasms, the genetically modified animals 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 animals of the present invention can be used to study various aspects of human autoimmune diseases and can also be used as autoimmune disease models.

[0124] Other uses of the genetically modified animals described herein, namely those comprising human or humanized T cell coreceptors (e.g., chimeric human / non-human CD4 or CD8), and optionally further comprising human or humanized MHC II or I proteins, will become apparent from this disclosure. [Examples]

[0125] The present invention is further illustrated by the following non-limiting examples. These examples are provided to aid in understanding the present invention and should not be interpreted as limiting its scope in any way. The examples do not include detailed descriptions of conventional methods (such as molecular cloning techniques) that would be well known to those skilled in the art. Unless otherwise specified, parts are by weight, molecular weights are average molecular weights, temperatures are given in Celsius, and pressures are atmospheric or near atmospheric pressure.

[0126] (Example 1) Construction and characterization of genetically modified CD4 mice Example 1.1: Engineering of the Chimeric CD4 Locus The mouse CD4 gene locus was extracted from human bacterial artificial chromosome (BAC) DNA and mouse bacterial artificial chromosome (BAC) DNA using VELOCIGENE® technology (e.g., U.S. Patent No. 6,586,251, and Valenzuela et al. (2003) High-throughput engineering of the mouse genome). coupled with high-resolution expression Humanization was achieved in a single step by constructing a unique targeting vector using analysis. (See Nat. Biotech. Vol. 21 (No. 6): pp. 652-659). A series of bacterial homologous recombination (BHR) and other engineering steps were performed using bacterial artificial chromosome (BAC) DNA to construct the targeting vector.

[0127] In short, four DNA fragments were joined by infusion ligation (Clonetech): (1) a fragment containing a mouse signal peptide (encoded by exons 2 and 3 of the mouse CD4 gene), (2) a fragment containing human exon 3 downstream of the mouse signal peptide, (3) a fragment containing a SPEC-resistant cassette adjacent to the Asc I and PI-SceI sites, and (4) a 160 bp fragment containing mouse CD4 intron 6 (an intron between exons 6 and 7) starting approximately 200 bp downstream of mouse CD4 exon 6. The resulting DNA fragment contained, from 5' to 3', mouse exon 2, mouse intron 2, a portion of mouse exon 3 containing the signal peptide, human exon 3 downstream of the human signal peptide, a portion of human intron 3, a SPEC cassette, and a portion of mouse intron 6. This DNA fragment was used in BHR to modify mouse BAC clone BMQ391F08 so that the mouse sequences encoding mouse CD4 Ig-like domains 1-3 were deleted and human CD4 exon 3 was introduced. The SPEC cassette was replaced with the CM cassette of BAC, thereby yielding the first BAC vector (Figure 1, top image).

[0128] Human BAC RP11-101F21 was modified using BHR so that the AscI-LoxP-PGK-neo-loxP cassette was introduced 60 bp downstream of human exon 3, and the PI-SceI restriction site and SPEC cassette were introduced approximately 100 bp downstream of exon 6, thereby yielding a second BAC vector (Figure 1, center). Following this step, digestion with AscI restriction enzymes and PI-SceI restriction enzymes was performed, and then BAC ligation of the first and second BAC vectors was carried out to construct a CD4 targeting vector (Figure 1, bottom). The upstream and downstream junctions of the mouse-human and human-mouse sequences are listed in Table 1 below, and are also listed in the sequence listing. The sequence spanning the human intron 3-lox-neo cassette junction (the 5' end of the cassette) is described in SEQ ID NO: 55, and the sequence spanning the lox-neo cassette-human intron 3 junction (the 3' end of the cassette) is described in SEQ ID NO: 56. Both sequences are also listed in Table 1. The complete nucleic acid sequence of the humanized CD4 fragment, including the pgk-neo cassette shown in Figure 1, is described in SEQ ID NO: 3. The pgk-neo cassette spans residues 307-2176 in SEQ ID NO: 3, the two lox sites are located at residues 267-300 and 2182-2215, and the human sequence spans residues 1-234 and 2222-18263. The amino acid sequence of the fully humanized CD4 protein is described in SEQ ID NO: 4, with the human sequence spanning amino acids 27-319 (described in SEQ ID NO: 57). [Table 1]

[0129] A human CD4 targeting vector was linearized using NotI and introduced into F1H4 mouse ES cells by electroporation. Target ES cells carrying the humanized CD4 locus were identified by genotyping using a modified allele assay (Valenzuela et al.), which detected the presence of a neomycin cassette, the human CD4 gene, and one copy of the mouse CD4 gene. The primers and probes used in this assay are shown in Table 2 and are also listed in the sequence listing. [Table 2]

[0130] Floxed neomycin-resistant cassettes were removed by introducing a plasmid expressing Cre recombinase into ES cells containing the humanized CD4 locus via electroporation.

[0131] Target ES cells carrying a humanized CD4 locus lacking resistance markers were identified by genotyping, which revealed the absence of neomycin cassettes and the presence of one copy of the human CD4 gene and one copy of the mouse CD4 gene.

[0132] The target ES cells described above were used as donor ES cells and introduced into 8-cell stage mouse embryos using the VELOCIMOUSE® method (see, for example, U.S. Patent No. 7,294,754, and Poueymirou et al. (2007) F0 generation mice that are essentially fully derived from the donor gene-targeted ES cells allowing immediate phenotypic analyses. Nature Biotech. Vol. 25 (No. 1): pp. 91-99). VELOCIMICE® (F0 mice completely derived from donor ES cells) independently carrying the chimeric CD4 gene were identified by genotyping using a modified allele assay (Valenzuela et al., above), thereby detecting the presence of a unique human CD4 gene sequence.

[0133] (Example 1.2) Chimeric CD4 expression in genetically modified mice Spleens from wild-type (WT) or heterozygous humanized CD4 mice ("1766HET") were perfused with collagenase D (Roche Bioscience), and erythrocytes were lysed with ACK lysis buffer. Cell surface expression of human CD4 or mouse CD4 was analyzed by FACS using either an anti-human CD4 antibody or an anti-mouse CD4 antibody, respectively. As shown in Figures 2A and 2B, human CD4 was expressed on the surface of T cells obtained from mice heterozygous for the humanized CD4 described herein.

[0134] (Example 2) Construction and characterization of genetically modified CD8 mice The CD8 protein exists either as a disulfide-linked homodimer (e.g., CD8α homodimer), a homomultimer of two subunits, or as a heterodimer of two proteins, CD8α (CD8a) and CD8β (CD8b). The CD8α and CD8β genes colocalize within the genome, for example, on mouse chromosome 6, located approximately 37kb apart from each other. Due to this close proximity, it is extremely difficult to create genetically modified mice that include humanization at both the CD8α and CD8β loci through crossbreeding. Therefore, sequential targeting is performed, in which one gene, for example CD8β, is introduced first, followed by the introduction of a second gene, for example CD8α.

[0135] (Example 2.1) Engineering of the chimeric CD8β gene locus The mouse CD8b locus was humanized in a single step by constructing a unique targeting vector from mouse bacterial artificial chromosome (BAC) DNA using VELOCIGENE® technology (see, e.g., U.S. Patent No. 6,586,251, and Valenzuela et al. (2003) High-throughput engineering of the mouse genome coupled with high-resolution expression analysis. Nat. Biotech. Vol. 21 (No. 6): pp. 652-659). A large targeting vector (LTVEC), MAID1737, was constructed by modifying DNA from BAC RP23-431M6 using BHR to contain homologous human sequence replacements in mouse exons 2-3 encoding the CD8ecto domain (from the 5' junction in intron 1 to the 3' junction in intron 3) (Figure 3). The loxp-Ub-Hyg cassette was inserted into the 3' junction in intron 3. The nucleotide sequences at various junctions of the resulting vectors are listed in Table 3 and are also listed in the sequence listing. The complete amino acid sequence of the humanized CD8β protein is described in SEQ ID NO: 53, and the human sequence spans amino acids 15-165 (described in SEQ ID NO: 58). [Table 3]

[0136] The targeting vector was introduced into F1H4 mouse ES cells by electroporation (Figure 5, left). Target ES cells carrying the humanized CD8b locus were identified by genotyping using a modified allele assay (Valenzuela et al.), thereby detecting the presence of the human CD8b gene. The primers and probes used in this assay are shown in Table 4 and are also listed in the sequence listing. [Table 4]

[0137] The target ES cells described above were used as donor ES cells and introduced into 8-cell stage mouse embryos using the VELOCIMOUSE® method (see, for example, U.S. Patent No. 7,294,754, and Poueymirou et al. (2007) F0 generation mice that are essentially fully derived from the donor gene-targeted ES cells allowing immediate phenotypic analyses. Nature Biotech. Vol. 25 (No. 1): pp. 91-99). VELOCIMICE® (F0 mice completely derived from donor ES cells) independently carrying the chimeric CD8b gene were identified by genotyping using a modified allele assay (Valenzuela et al., above), thereby detecting the presence of a unique human CD8b gene sequence.

[0138] The selective cassette can be removed by methods known to those skilled in the art. For example, to remove a "loxed" hygromycin cassette introduced by inserting a targeting construct containing the human CD8b gene sequence, an ES cell carrying a chimeric human / mouse CD8b locus can be transfected with a construct expressing Cre. The hygromycin cassette can optionally be removed by crossing with a mouse expressing Cre recombinase. Optionally, the hygromycin cassette is retained within the mouse. In one embodiment, MAID1739 is produced by deleting the cassette.

[0139] (Example 2.2) Engineering of the chimeric CD8α gene locus The mouse CD8a locus was humanized in a single step by constructing a unique targeting vector from mouse bacterial artificial chromosome (BAC) DNA using VELOCIGENE® technology (e.g., U.S. Patent No. 6,586,251 and Valenzuela et al., see above). A large targeting vector (LTVEC), MAID1738, was constructed by modifying DNA from BAC RP23-431M6 using BHR to include a replacement of the homologous human sequence (from the 5' junction in human exon 2 to the 3' junction in intron 3 (Figure 4)) in mouse exons 1-2, which encode the CD8a ecto domain (from the 5' junction at Ala codon 27 in mouse exon 1 to the 3' junction in mouse intron 2). This preserves the mouse reader sequence at the start of exon 1. The lox2372-Ub-Neo cassette was inserted into the 3' junction of the human / mouse sequence. The nucleotide sequences at various junctions of the resulting vectors are listed in Table 5 and are also listed in the sequence listing. The complete amino acid sequence of the humanized CD8α polypeptide is described in SEQ ID NO: 54, and the human sequence spans amino acids 28-179 (described in SEQ ID NO: 59). [Table 5]

[0140] The above-mentioned humanized CD8a targeting vector was introduced by electroporation into mouse ES cells containing the humanized CD8b locus to create modified ES cells containing both humanized CD8b and CD8a loci (Figure 5). Target ES cells carrying the humanized CD8a locus were identified by genotyping using a modified allele assay (Valenzuela et al.), thereby detecting the presence of the human CD8a gene. The primers and probes used in this assay are shown in Table 6 and are also listed in the sequence listing. [Table 6]

[0141] The target ES cells described above were used as donor ES cells and introduced into 8-cell stage mouse embryos using the VELOCIMOUSE® method (see, for example, U.S. Patent No. 7,294,754 and Poueymirou et al., see above). VELOCIMICE® mice (F0 mice completely derived from donor ES cells) carrying the chimeric CD8b and chimeric CD8a genes were identified by genotyping using a modified allele assay (Valenzuela et al., see above), thereby detecting the presence of unique human CD8b and CD8a gene sequences.

[0142] Alternatively, the humanized CD8a targeting vector described herein can be introduced by electroporation into mouse ES cells that do not contain the humanized CD8b locus to produce mice containing only the humanized CD8a locus.

[0143] The selective cassette within the CD8a gene locus can be removed by methods known to those skilled in the art, for example, as described in Example 2.1 above. In one embodiment, a MAID1740 mouse is produced by deleting the selective cassette.

[0144] (Example 2.3) Chimeric CD8 expression in genetically modified mice We analyzed the expression of human CD8 in mice that were heterozygous for both the humanized CD8a gene (MAID1740) and the CD8b gene (MAID1739).

[0145] Human CD8a and CD8b expression was clearly detectable on the surface of CD3+CD4-T cells derived from heterozygous spleens, but not clearly detectable in wild-type animals (Figure 6).

[0146] Human CD8a and CD8b expression was detectable on the surface of heterozygous thymocytes, but not clearly detectable in wild-type animals (Figure 7).

[0147] Equal parts Those skilled in the art can understand or confirm many equivalents of the specific embodiments of the invention described herein by means of conventional experiments. Such equivalents shall be covered by the following claims.

[0148] All non-patent literature, and the entire contents of all patent applications and patents cited throughout this application, are incorporated herein by reference in their entirety.

Claims

1. A chimeric human / mouse nucleic acid molecule, where from 5' to 3', (i) A nucleotide sequence encoding a functional chimeric human / mouse CD8β polypeptide containing the sequence described in Sequence ID No. 53, and / or (ii) Nucleotide sequences encoding a functional chimeric human / mouse CD8α polypeptide containing the sequence described in Sequence ID No. 54 Chimeric human / mouse nucleic acid molecules, including those mentioned above.

2. (i) The nucleotide sequence encoding the functional chimeric human / mouse CD8β polypeptide is operably linked to a mouse CD8β promoter sequence, and / or (ii) The nucleotide sequence encoding the functional chimeric human / mouse CD8α polypeptide is operably linked to a mouse CD8α promoter sequence. The chimeric human / mouse nucleic acid molecule according to claim 1.

3. A functional chimeric human / rodent CD8α polypeptide comprising an amino acid sequence encoded by a chimeric human / mouse nucleic acid molecule according to any one of claims 1 to 2.

4. A functional chimeric human / rodent CD8α polypeptide according to claim 3, wherein the rodent is a mouse.

5. A functional chimeric human / rodent CD8β polypeptide comprising an amino acid sequence encoded by a chimeric human / mouse nucleic acid molecule according to any one of claims 1 to 2.

6. A functional chimeric human / rodent CD8β polypeptide according to claim 5, wherein the rodent is a mouse.

7. A composition comprising mouse T cells containing a functional chimeric human / mouse nucleic acid molecule according to any one of claims 1 to 2 for generating an HLA class I restricted immune response, A composition in which the T cells express the functional chimeric human / mouse CD8β polypeptide and / or the functional chimeric human / mouse CD8α polypeptide.

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