Genetically modified T cell receptor mice

#Present invention relates to a genetically modified non-human animal, e.g., a rodent (e.g., a mouse or a rat), that comprises in its genome human or humanized T Cell Receptor (TCR) variable gene loci (e.g., TCRα and TCRβ variable gene loci and/or TCRδ and TCRγ variable gene loci), and expresses human or humanized TCR polypeptides (e.g., TCRα and TCRβ polypeptides and/or TCRδ and TCRγ polypeptides) from the human or humanized TCR variable gene loci. A non-human animal with human or humanized TCR variable gene loci of the invention comprises unrearranged human TCR variable region gene segments (e.g., V, D, and/or J segments) at endogenous non-human TCR gene loci. The invention also relates to embryos, tissues, and cells (e.g., T cells) that comprise human or humanized TCR variable gene loci and express human or humanized TCR polypeptides. Also provided are methods for making the genetically modified non-human animal comprising human or humanized TCR variable gene loci; and methods of using non-human animals, embryos, tissues, and cells that comprise human or humanized TCR variable gene loci and express human or humanized TCR polypeptides from those loci.

#In the adaptive immune response, foreign antigens are recognized by receptor molecules on B lymphocytes (e.g., immunoglobulins) and T lymphocytes (e.g., T cell receptors or TCRs). While pathogens in the blood and extracellular space are recognized by antibodies in the course of humoral immune response, destruction of pathogens inside cells is mediated in the course of cellular immune response by T cells.

#T cells recognize and attack antigens presented to them in the context of a Major Histocompatibility Complex (MHC) on the cell surface. The antigen recognition is mediated by TCRs expressed on the surface of the T cells. Two main classes of T cells serve this function: cytotoxic T cells, which express a cell-surface protein CD8, and helper T cells, which express a cell-surface protein CD4. Cytotoxic T cells activate signaling cascades that result in direct destruction of the cell presenting the antigen (in the context of MHC I), while helper T cells differentiate into several classes, and their activation (primed by recognition of antigen presented in the context of MHC II) results in macrophage-mediated pathogen destruction and stimulation of antibody production by B cells.

#FIG. 1 depicts interaction in a mouse between a TCR molecule and an MHC molecule: the left panel shows a mouse T cell (top) from a humanized TCR mouse comprising a T cell receptor with human variable TCR domains and mouse constant TCR domains, which recognizes an antigen (grey ball) presented through an MHC class I by an antigen presenting cell (bottom); the right panel shows the same for an MHC class II. The MHC I and MHC II complexes are shown together with their respective co-receptors, CD8 and CD4. Mouse regions are in black and human regions are in white.

#FIG. 2 depicts (not to scale) the general organization of a mouse (top panel, first locus) and human (top panel, second locus) TCRα locus. The bottom panel illustrates a strategy for replacing TCRα variable region segments in a mouse (closed symbols) with human TCRα variable region segments (open symbols) at the endogenous mouse locus on chromosome 14; a humanized TCRα locus having human Vα and Jα segments is shown with a mouse constant region and a mouse enhancer; in the embodiment shown, the TCRδ locus is deleted in the course of humanization.

#FIG. 3 depicts (not to scale) a progressive strategy for humanization of the mouse TCRα locus, wherein TCRα variable region gene segments are sequentially added upstream of an initial humanization of a deleted mouse locus (MAID1540). Mouse sequence is indicated by closed symbols; human sequence is indicated by open symbols. MAID refers to modified allele ID number. TRAV=TCR Vα segment, TRAJ=TCR Jα segment (hTRAJ=human TRAJ), TRAC=TCR Cα domain, TCRD=TCRδ.

#The present invention provides genetically modified non-human animals, e.g., rodents, e.g., mice or rats, that express humanized T cell receptors. The present invention also relates to genetically modified non-human animals that comprise in their germline unrearranged T cell receptor variable gene loci. Also provided are embryos, cells, and tissues comprising the same; methods of making the same; as well as methods of using the same. Unless defined otherwise, all terms and phrases used herein include the meanings that the terms and phrases have attained in the art, unless the contrary is clearly indicated or clearly apparent from the context in which the term or phrase is used.

#The term “conservative,” when used to describe a conservative amino acid substitution, includes substitution of an amino acid residue by another amino acid residue having a side chain R group with similar chemical properties (e.g., charge or hydrophobicity). Conservative amino acid substitutions may be achieved by modifying a nucleotide sequence so as to introduce a nucleotide change that will encode the conservative substitution. In general, a conservative amino acid substitution will not substantially change the functional properties of interest of a protein, for example, the ability of a T cell to recognize a peptide presented by an MHC molecule. Examples of groups of amino acids that have side chains with similar chemical properties include aliphatic side chains such as glycine, alanine, valine, leucine, and isoleucine; aliphatic-hydroxyl side chains such as serine and threonine; amide-containing side chains such as asparagine and glutamine; aromatic side chains such as phenylalanine, tyrosine, and tryptophan; basic side chains such as lysine, arginine, and histidine; acidic side chains such as aspartic acid and glutamic acid; and, sulfur-containing side chains such as cysteine and methionine. Conservative amino acids substitution groups include, for example, valine/leucine/isoleucine, phenylalanine/tyrosine, lysine/arginine, alanine/valine, glutamate/aspartate, and asparagine/glutamine. In some embodiments, a conservative amino acid substitution can be a substitution of any native residue in a protein with alanine, as used in, for example, alanine scanning mutagenesis. In some embodiments, a conservative substitution is made that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. ((1992) Exhaustive Matching of the Entire Protein Sequence Database, Science 256:1443-45), hereby incorporated by reference. In some embodiments, the substitution is a moderately conservative substitution wherein the substitution has a nonnegative value in the PAM250 log-likelihood matrix.

#Thus, encompassed by the invention is a genetically modified non-human animal expressing humanized TCR α and β polypeptides (and/or humanized TCRδ and TCRγ polypeptides) comprising conservative amino acid substitutions in the amino acid sequence described herein.