Non-human animals expressing humanized cd3 complex

Abstract

Non-human animals, expressing humanized CD3 proteins are provided. Non-human animals, e.g., rodents, genetically modified to comprise in their genome humanized CD3 proteins are also provided. Additionally, provided are methods and compositions of making such non-human animals, as well as methods of using said non-human animals.

1 . A genetically modified non-human animal comprising an endogenous non-human CD3 locus genetically modified to encode an extracellular domain of a human CD3 protein, wherein the human CD3 protein is CD3ε, CD3δ, CD3γ, CD3ζ, or any combination thereof. 
     
     
         2 . The animal of  claim 1 , wherein the endogenous non-human CD3 locus is genetically modified to encode an extracellular domain of human CD3ε, an extracellular domain of human CD3δ, and an extracellular domain of human CD3γ. 
     
     
         3 . The animal of  claim 1 , wherein the endogenous non-human CD3 locus is genetically modified not to express functional extracellular domain(s) of non-human CD3 protein(s) corresponding to the human CD3 protein(s). 
     
     
         4 . The animal of  claim 1 , wherein the endogenous locus further encodes transmembrane and cytoplasmic domains of a CD3 protein of the endogenous non-human animal, wherein the animal expresses a chimeric CD3 protein on the surface of its T-cells comprising the extracellular domain of the human CD3 protein and the transmembrane and cytoplasmic domains of the endogenous non-human animal CD3 protein. 
     
     
         5 . The animal of  claim 2 , wherein the animal comprises:
 at an endogenous CD3ε locus a nucleic acid sequence encoding an extracellular domain of a human CD3ε operably linked to a nucleic acid sequence encoding transmembrane and cytoplasmic domains of CD3ε of the endogenous non-human animal,   at an endogenous CD3δ locus a nucleic acid sequence encoding an extracellular domain of a human CD3δ operably linked to a nucleic acid sequence encoding transmembrane and cytoplasmic domains of CD3δ protein of the endogenous non-human animal, and   at an endogenous CD3γ locus a nucleic acid sequence encoding an extracellular domain of a human CD3γ operably linked to a nucleic acid sequence encoding transmembrane and cytoplasmic domains of an CD3γ protein of an endogenous non-human animal CD3γ;   wherein the non-human animal expresses chimeric CD3ε, CD3δ, and CD3γ proteins on the surface of its T cells.   
     
     
         6 . The animal of  claim 1  wherein the animal comprises extracellular domains of human CD3 proteins which comprise the sequences of SEQ ID NO:33, SEQ ID NO:34 and SEQ ID NO:35. 
     
     
         7 . The animal of  claim 1 , wherein the animal is a mammal. 
     
     
         8 . The animal of  claim 1 , wherein the animal is a rodent. 
     
     
         9 . The animal of  claim 8 , wherein the animal is a mouse. 
     
     
         10 . The mouse of  claim 9 , wherein the mouse comprises:
 at an endogenous mouse CD3ε locus a nucleic acid sequence encoding an extracellular domain of a human CD3ε operably linked to a nucleic acid sequence encoding transmembrane and cytoplasmic domains of an endogenous mouse CD3ε,   at an endogenous mouse CD3δ locus a nucleic acid sequence encoding an extracellular domain of a human CD3δ operably linked to a nucleic acid sequence encoding transmembrane and cytoplasmic domains of an endogenous mouse CD3δ, and   at an endogenous mouse CD3γ locus a nucleic acid sequence encoding an extracellular domain of a human CD3γ operably linked to a nucleic acid sequence encoding transmembrane and cytoplasmic domains of an endogenous mouse CD3γ, and   wherein the mouse expresses chimeric CD3ε, CD3δ, and CD3γ proteins on the surface of its T cells.   
     
     
         11 . The mouse of  claim 10 , wherein the amino acid sequence of the chimeric CD3ε protein is set forth in SEQ ID NO:24, the amino acid sequence of the chimeric CD3δ protein is set forth in SEQ ID NO:25, and the amino acid sequence of the chimeric CD3γ protein is set forth in SEQ ID NO:26. 
     
     
         12 . The animal of  claim 1 , which is heterozygous for the modified endogenous non-human CD3 locus. 
     
     
         13 . The animal of  claim 1 , which is homozygous for the modified endogenous non-human CD3 locus. 
     
     
         14 . A method of making a genetically modified non-human animal as defined in  claim 1 , comprising:
 introducing a nucleic acid sequence encoding an extracellular domain of human CD3 protein, wherein the human CD3 protein is CD3ε, CD3δ, CD3γ, CD3ζ, or any combination thereof into the genome of a cell of non-human animal at an endogenous CD3 locus; and   propagating the genetically modified non-human animal from the cell.   
     
     
         15 . The method of  claim 14 , wherein the cell is a single ES cell, and the single ES cell is introduced into a mouse embryo to propagate a mouse. 
     
     
         16 . A mouse model for testing a CD3-based bispecific antigen-binding protein, wherein the antigen-binding protein is capable of binding both CD3 and a non-mouse antigen of interest, comprising: a genetically modified mouse as defined by  claim 9  further comprising a cell expressing or comprising the non-mouse antigen of interest. 
     
     
         17 . (canceled) 
     
     
         18 . A method of screening drug candidates that target an antigen of interest comprising:
 a. introducing into a genetically modified mouse as defined by  claim 9  the antigen of interest,   b. contacting said mouse with a drug candidate of interest, wherein the drug candidate is directed against the human CD3 and the antigen of interest, and   c. determining if the drug candidate is efficacious in preventing, reducing or eliminating cells characterized by the presence or expression of the antigen of interest.   
     
     
         19 . The method of  claim 18 , wherein the step of introducing comprises expressing in the mouse the antigen of interest. 
     
     
         20 . The method of  claim 18 , wherein the step of introducing comprises infecting the mouse with the antigen of interest. 
     
     
         21 . The method of  claim 19 , wherein the step of expressing in the mouse the antigen of interest comprises genetically modifying the mouse to express the antigen of interest. 
     
     
         22 . The method of  claim 18 , wherein the step of introducing comprises introducing into said mouse a cell expressing the antigen of interest. 
     
     
         23 . The method of  claim 16 , wherein the cell is a tumor cell. 
     
     
         24 . The method of  claim 22 , wherein the cell is a bacterial cell. 
     
     
         25 . The method of  claim 20 , wherein the infecting comprising performing viral or bacterial infection. 
     
     
         26 . The method of  claim 18 , wherein the mouse is an immunocompetent mouse. 
     
     
         27 . The method of  claim 18 , wherein the antigen of interest is a tumor associated antigen. 
     
     
         28 . The method of  claim 27 , wherein the tumor associated antigen is selected from the group consisting of ALK, BAGE proteins, BIRC5 (survivin), BIRC7, CA9, CALR, CCR5, CD19, CD20 (MS4A1), CD22, CD27, CD30, CD33, CD38, CD40, CD44, CD52, CD56, CD79, CDK4, CEACAM3, CEACAM5, CLEC12A, EGFR, EGFR variant III, ERBB2 (HER2), ERBB3, ERBB4, EPCAM, EPHA2, EPHA3, FCRL5, FLT3, FOLR1, GAGE proteins, GD2, GD3, GPNMB, GM3, GPR112, IL3RA, KIT, KRAS, LGR5, EBV-derived LMP2, L1CAM, MAGE proteins, MLANA, MSLN, MUC1, MUC2, MUC3, MUC4, MUC5, MUC16, MUM1, ANKRD30A, NY-ESO1 (CTAG1B), OX40, PAP, PAX3, PAX5, PLAC1, PRLR, PMEL, PRAME, PSMA (FOLH1), RAGE proteins, RET, RGS5, ROR1, SART1, SART3, SLAMF7, SLC39A6 (LIV1), STEAP1, STEAP2, TERT, TMPRSS2, Thompson-nouvelle antigen, TNFRSF17, TYR, UPK3A, VTCN1, and WT1. 
     
     
         29 - 30 . (canceled) 
     
     
         31 . The method of  claim 18 , wherein the antigen is a viral antigen selected from the group consisting of HIV; hepatitis A; hepatitis B; hepatitis C; herpes virus such as HSV-1, HSV-2, CMV, HAV-6, VZV, and Epstein Barr virus; adenovirus; influenza virus; flavivirus; echovirus; rhinovirus; coxsackie virus; coronavirus; respiratory syncytial virus; mumps virus; rotavirus; measles virus; rubella virus; parvovirus; vaccinia virus; HTLV; dengue virus; papillomavirus; molluscum virus; poliovirus; rabies virus; JC virus; ebola virus; and arboviral encephalitis virus antigen. 
     
     
         32 . (canceled) 
     
     
         33 . The method of  claim 18 , wherein the antigen is a bacterial antigen selected from the group consisting of chlamydia, rickettsia, mycobacteria, staphylococci, streptococci, pneumonococci, meningococci, gonococci, klebsiella, proteus, serratia, pseudomonas, legionella, diphtheria, salmonella, bacilli, cholera, tetanus, botulism, anthrax, plague, leptospira, and Lyme disease bacterial antigen. 
     
     
         34 . The method of  claim 18 , wherein the drug candidate is an antibody or an antigen-binding protein. 
     
     
         35 - 36 . (canceled) 
     
     
         37 . The method of  claim 34 , wherein the antibody or the antigen-binding protein is a bispecific antibody or a bispecific antigen-binding protein, respectively, which is capable of binding both human CD3 protein and the antigen of interest. 
     
     
         38 . The method of  claim 18 , wherein the drug candidate is capable of recognizing a monkey CD3 protein. 
     
     
         39 - 44 . (canceled)