Humanized t cell mediated immune responses in non-human animals

Abstract

Disclosed herein are non-human animals (e.g., rodents, e.g., mice or rats) genetically engineered to express a humanized T cell co-receptor (e.g., humanized CD4 and/or CD8 (e.g., CD8α and/or CD8β)), a human or humanized T cell receptor (TCR) comprising a variable domain encoded by at least one human TCR variable region gene segment and/or a human or humanized major histocompatibility complex that binds the humanized T cell co-receptor (e.g., human or humanized MHC II (e.g., MHC II α and/or MHC II β chains) and/or MHC I (e.g., MHC Iα) respectively, and optionally human or humanized β2 microglobulin). Also provided are embryos, tissues, and cells expressing the same. Methods for making a genetically engineered animal that expresses at least one humanized T cell co-receptor (e.g., humanized CD4 and/or CD8), at least one humanized MHC that associates with the humanized T cell co-receptor (e.g., humanized MHC II and/or MHC I, respectively) and/or the humanized TCR are also provided. Methods for using the genetically engineered animals that mount a substantially humanized T cell immune response for developing human therapeutics are also provided.

What is claimed is: 
     
         1 . A mouse T cell expressing a TCR protein comprising a human TCR variable domain specific for an antigen, the mouse T cell comprising in its genome
 (a) a first nucleotide sequence encoding a chimeric human/mouse CD4 co-receptor that comprises D1, D2 and D3 domains of a human CD4 polypeptide and transmembrane and cytoplasmic domains of a mouse CD4 polypeptide;   (b) a second nucleotide sequence encoding a chimeric human/mouse CD8α polypeptide and a third nucleotide sequence encoding a chimeric human/mouse CD8β polypeptide,   wherein the chimeric human/mouse CD8α polypeptide comprises an IgV-like domain of a human CD8α polypeptide and transmembrane and cytoplasmic domains of a mouse CD8α polypeptide,   wherein the chimeric human/mouse CD8β polypeptide comprises an IgV-like domain of a human CD8β polypeptide and transmembrane and cytoplasmic domains of mouse CD8β polypeptide;   (c) a first nucleic acid sequence encoding a chimeric human/mouse MHC II α polypeptide and a second nucleic acid sequence encoding a chimeric human/mouse MHC II β polypeptide,   wherein the chimeric human/mouse MHC II α polypeptide comprises an extracellular domain of an HLA class II α polypeptide and transmembrane and cytoplasmic domains of a mouse MHC II α polypeptide,   wherein the chimeric human/mouse MHC IIβ polypeptide comprises an extracellular domain of an HLA class IIβ polypeptide and transmembrane and cytoplasmic domains of a mouse MHC II β polypeptide;   (d) a third nucleic acid sequence encoding a chimeric human/mouse MHC I polypeptide,   wherein the chimeric human/mouse MHC I polypeptide comprises an extracellular domain of an HLA class I polypeptide and transmembrane and cytoplasmic domains of a mouse MHC I polypeptide; and   (e) a rearranged human TCR Vα/Jα sequence operably linked to a mouse TCRα constant region sequence that encodes a humanized TCRα chain comprising a human TCRα variable domain operably linked to a mouse TCRα constant domain, and   a rearranged human TCR Vβ/Dβ/Jβ sequence operably linked to a mouse TCRβ constant region sequence that encodes a humanized TCRβ chain comprising a human TCRβ variable domain operably linked to a mouse TCRβ constant domain, and   wherein the TCR protein comprises the humanized TCRα chain and the humanized TCRβ chain.   
     
     
         2 . The mouse T cell of  claim 1 , wherein
 (a) the first nucleotide sequence is present at an endogenous CD4 locus;   (b) the second nucleotide sequence is present at an endogenous CD8α locus and the third nucleotide sequence is present at an endogenous CD8β locus;   (c) the first nucleic acid sequence is present at an endogenous MHC II α locus and the second nucleic acid sequence is present at an endogenous MHC II β locus;   (d) the third nucleic acid sequence is present at an endogenous MHC I locus;   (e) the rearranged human TCR Vα/Jα sequence is present at an endogenous TCRα variable region locus and the rearranged human TCR Vβ/Dβ/Jβ sequence is present at an endogenous TCRβ variable region locus, or   (f) any combination of (a)-(e).   
     
     
         3 . The mouse T cell of  claim 2 , wherein
 (a) the first nucleotide sequence is present at the endogenous CD4 locus and operably linked to mouse CD4 promoter and regulatory elements;   (b) the second nucleotide sequence is present at the endogenous CD8α locus and operably linked to mouse CD8α promoter and regulatory elements, and the third nucleotide sequence is present at an endogenous CD8β locus and operably linked to mouse CD8β promoter and regulatory elements;   (c) the first nucleic acid sequence is present at the endogenous MHC II α locus and operably linked to mouse MHC II α promoter and regulatory elements, and the second nucleic acid sequence is present at an endogenous MHC II β locus and operably linked to mouse MHC II β promoter and regulatory elements;   (d) the third nucleic acid sequence is present at the endogenous MHC I locus and operably linked to mouse MHC I promoter and regulatory elements; or   (e) any combination of (a)-(d).   
     
     
         4 . The mouse T cell of  claim 3 , wherein the chimeric human/mouse CD4 co-receptor comprises D1, D2 and D3 domains of the human CD4 polypeptide operably linked to D4, transmembrane, and cytoplasmic domains of the mouse CD4 polypeptide. 
     
     
         5 . The mouse T cell of  claim 1 , wherein
 (a) the HLA class II α polypeptide is selected from the group consisting of any α chain of HLA-DR, HLA-DQ, and HLA-DP;   (b) the HLA class II β polypeptide is selected from the group consisting of any β chain of HLA-DR, HLA-DQ, and HLA-DP;   (c) the HLA class I polypeptide is selected from the group consisting of HLA-A, HLA-B, and HLA-C; or   (d) any combination of (a)-(c).   
     
     
         6 . The mouse T cell of  claim 5 , wherein the HLA class II α polypeptide is the α chain of HLA-DR and the HLA class II β polypeptide is the β chain of HLA-DR, and
 wherein the HLA class I polypeptide is HLA-A. 
 
     
     
         7 . The mouse T cell of  claim 6 , wherein the chimeric human/mouse MHC II α polypeptide comprises α1 and α2 domains of the α chain of a HLA-DR2 protein, and wherein the chimeric human/mouse MHC II β polypeptide comprises β1 and β2 domains of the β chain of the HLA-DR2 protein. 
     
     
         8 . The mouse T cell of  claim 7 , wherein the chimeric human/mouse MHC I polypeptide comprises α1, α2, and α3 domains of a HLA-A2 protein. 
     
     
         9 . The mouse T cell of  claim 1 , wherein:
 (i) an endogenous TCRα variable region locus (a) lacks all or substantially all functional endogenous Vα gene segments, (b) lacks all or substantially all functional endogenous Jα gene segments, or (c) lacks all or substantially all functional endogenous Vα gene segments and lacks all or substantially all functional endogenous Jα gene segments;   (ii) an endogenous TCRβ variable region locus (a) lacks all or substantially all functional endogenous Vβ gene segments, (b) lacks all or substantially all functional endogenous Dβ gene segments, (c) lacks all or substantially all functional endogenous Jβ gene segments, or (d) lacks all or substantially all functional endogenous Vβ gene segments, lacks all or substantially all functional DR gene segments, and lacks all or substantially all functional Jβ gene segments; or   (iii) the endogenous TCRα variable region locus lacks all or substantially all functional endogenous Vα gene segments and lacks all or substantially all functional endogenous Jα gene segments, and the endogenous TCRβ variable region locus lacks all or substantially all functional endogenous Vβ gene segments, lacks all or substantially all functional Dβ gene segments, and lacks all or substantially all functional Jβ gene segments.   
     
     
         10 . The mouse T cell of  claim 1 , wherein
 (a) the first nucleotide sequence comprises a sequence encoding D1, D2 and D3 domains of the human CD4 polypeptide that, at an endogenous CD4 locus, (i) replaces a sequence encoding D1, D2 and D3 domains of a mouse CD4 polypeptide and (ii) is operably linked to mouse CD4 D4, transmembrane and cytoplasmic domains encoding sequences;   (b) the second nucleotide sequence comprises a sequence encoding the IgV-like domain of the human CD8α polypeptide that, at an endogenous CD8α locus, (i) replaces a sequence encoding the IgV-like domain of a mouse CD8α polypeptide and (ii) is operably linked to mouse CD8α transmembrane and cytoplasmic domain encoding sequences, and   the third nucleotide sequence comprises a sequence encoding the IgV-like domain of the human CD8β polypeptide that, at an endogenous CD8β locus, (i) replaces a sequence encoding the IgV-like domain of a mouse CD8β polypeptide and (ii) is operably linked to mouse CD8β transmembrane and cytoplasmic domain encoding sequences;   (c) the first nucleic acid sequence comprises a sequence encoding α1 and α2 domains of the HLA class II α polypeptide that, at an endogenous MHC II α locus, (i) replaces a sequence encoding α1 and α2 domains of a mouse MHC II α polypeptide and (ii) is operably linked to mouse MHC II α polypeptide transmembrane and cytoplasmic domain encoding sequences, and   the second nucleic acid comprises a sequence encoding β1 and β2 domains of the HLA class II β polypeptide that, at an endogenous MHC II β locus, (i) replaces a sequence encoding β1 and β2 domains of a mouse MHC II β polypeptide and (ii) is operably linked to mouse MHC II β polypeptide transmembrane and cytoplasmic domain encoding sequences;   (d) the third nucleic acid sequence comprises a sequence encoding α1, α2, and α3 domains of the HLA class I polypeptide that, at an endogenous MHC I locus, (i) replaces a sequence encoding α1, α2, and α3 domains of a mouse MHC I polypeptide and (ii) is operably linked to mouse MHC I polypeptide transmembrane and cytoplasmic domain encoding sequences;   (e) the rearranged human TCR Vα/Jα sequence replaces one or more endogenous Vα and/or Jα gene segments at an endogenous TCRα variable region locus and the rearranged human TCR Vβ/Dβ/Jβ sequence replaces one or more endogenous Vβ, Dβ and/or Jβ gene segments at an endogenous TCRβ variable region locus; or   (f) any combination of (a)-(e).   
     
     
         11 . The mouse T cell  claim 1 , wherein the mouse T cell does not express:
 (a) a functional mouse CD4 co-receptor from an endogenous CD4 locus;   (b) a functional mouse CD8 co-receptor from an endogenous CD8 locus;   (c) a mouse TCRα variable domain from an endogenous TCRα locus;   (d) a mouse TCRβ variable domain from an endogenous TCRβ locus;   (e) on a cell surface, an extracellular domain of a classical MHC class I polypeptide from an endogenous MHC I locus; or   (f) any combination of (a)-(e).   
     
     
         12 . The mouse T cell of  claim 1 , further comprising a β2 microglobulin locus comprising a sequence encoding a polypeptide comprising a human β2 microglobulin amino acid sequence, wherein the mouse T cell expresses a human or humanized β2 microglobulin polypeptide. 
     
     
         13 . The mouse T cell of  claim 12 , wherein the mouse T cell does not express a functional endogenous mouse β2 microglobulin polypeptide from an endogenous mouse β2 microglobulin locus. 
     
     
         14 . The mouse T cell of  claim 12 , wherein the sequence encoding the polypeptide comprising the human β2 microglobulin amino acid sequence is operably linked to mouse β2 microglobulin regulatory elements. 
     
     
         15 . The mouse T cell of  claim 12 , wherein the β2 microglobulin locus comprises a nucleotide sequence set forth in exon 2, exon 3, and exon 4 of a human β2 microglobulin gene. 
     
     
         16 . The mouse T cell of  claim 15 , wherein the β2 microglobulin locus further comprises a nucleotide sequence set forth in exon 1 of a mouse β2 microglobulin gene. 
     
     
         17 . The mouse T cell of  claim 3 , wherein the chimeric human/mouse CD8α polypeptide comprises an extracellular portion of the human CD8α polypeptide operably linked to transmembrane and cytoplasmic domains of the mouse CD8α polypeptide and the chimeric human/mouse CD8β polypeptide comprises an extracellular portion of the human CD8β polypeptide operably linked to transmembrane and cytoplasmic domains of the mouse CD8β polypeptide. 
     
     
         18 . A non-human hybridoma expressing a TCR protein comprising a human TCR variable domain specific for an antigen, wherein the non-human hybridoma is produced from the mouse T cell of  claim 1 . 
     
     
         19 . A composition for evaluating a humanized T cell immune response between mouse cells, the composition comprising a first mouse cell and second mouse cell,
 wherein the first mouse cell and the second mouse cell each comprise:   (a) a first nucleotide sequence encoding a chimeric human/mouse CD4 co-receptor that comprises D1, D2 and D3 domains of a human CD4 polypeptide and transmembrane and cytoplasmic domains of a mouse CD4 polypeptide;   (b) a second nucleotide sequence encoding a chimeric human/mouse CD8α polypeptide and a third nucleotide sequence encoding a chimeric human/mouse CD8β polypeptide,   wherein the chimeric human/mouse CD8α polypeptide comprises an IgV-like domain of a human CD8α polypeptide and transmembrane and cytoplasmic domains of a mouse CD8α polypeptide,   wherein the chimeric human/mouse CD8β polypeptide comprises an IgV-like domain of a human CD8β polypeptide and transmembrane and cytoplasmic domains of a mouse CD8β polypeptide;   (c) a first nucleic acid sequence encoding a chimeric human/mouse MHC II α polypeptide and a second nucleic acid sequence a chimeric human/mouse MHC II β polypeptide,   wherein the chimeric human/mouse MHC II α polypeptide comprises an extracellular domain of an HLA class II α polypeptide and transmembrane and cytoplasmic domains of a mouse MHC II α polypeptide,   wherein the chimeric human/mouse MHC II β polypeptide comprises an extracellular domain of an HLA class II β polypeptide and at least the transmembrane and cytoplasmic domains of a mouse MHC II β polypeptide;   (d) a third nucleic acid sequence encoding a chimeric human/mouse MHC I polypeptide,   wherein the chimeric human/mouse MHC I polypeptide comprises an extracellular domain of an HLA class I polypeptide and transmembrane and cytoplasmic domains of a mouse MHC I polypeptide; and   (e) at least one human Vα segment and at least one human Jα segment operably linked to a mouse TCRα constant region sequence, and   at least one human VR segment, at least one human DR segment, and at least one human Jβ segment operably linked to a mouse TCRβ constant region sequence   wherein the first mouse cell is a mouse T cell that expresses
 (a) the chimeric human/mouse CD4 co-receptor and/or the chimeric human/mouse CD8 co-receptor comprising the chimeric human/mouse CD8α polypeptide and the chimeric human/mouse CD8β polypeptide, and 
 (b) a T cell receptor comprising a humanized TCRα chain and a humanized TCRβ chain,
 wherein the humanized TCRα chain is encoded by a rearranged human TCR Vα/Jα sequence operably linked to the mouse TCRα constant region sequence, 
 wherein the rearranged human TCR Vα/Jα sequence is formed by rearrangement of the at least one human Vα segment and the at least one human Jα segment, and 
 wherein the humanized TCRβ chain is encoded by a rearranged human TCR Vβ/Dβ/Jβ sequence operably linked to the mouse TCRβ constant region sequence, 
 wherein the rearranged human TCR Vβ/Dβ/Jβ sequence is formed by rearrangement of the at least one human Vβ segment, the at least one Dβ segment, and the at least one human Jβ segment; and 
 
   wherein the second mouse cell expresses the chimeric human/mouse MHC I polypeptide and the human or humanized β2 microglobulin.   
     
     
         20 . The composition of  claim 19 , wherein the second cell is a mouse antigen presenting cell, and wherein the second cell further expresses the chimeric human/mouse MHC II complex comprising the chimeric human/mouse MHC II α polypeptide and the chimeric human/mouse MHC II β polypeptide.