Non-human animals having a humanized programmed cell death 1 gene

Abstract

Non-human animals, and methods and compositions for making and using the same, are provided, wherein the non-human animals comprise a humanization of a Programmed cell death 1 (Pdcd1) gene. The non-human animals may be described, in some embodiments, as having a genetic modification to an endogenous Pdcd1 gene so that the non-human animals express a PD-1 polypeptide that includes a human portion and an endogenous portion (e.g., a non-human portion).

We claim: 
     
         1 . A rodent that expresses a PD-1 polypeptide, which PD-1 polypeptide comprises a human portion and an endogenous portion. 
     
     
         2 . The rodent of  claim 1 , wherein the PD-1 polypeptide is translated in a cell of the rodent with a rodent signal peptide. 
     
     
         3 . The rodent of  claim 1  or  2 , wherein the endogenous portion comprises an intracellular portion of an endogenous PD-1 polypeptide. 
     
     
         4 . The rodent of  claim 3 , wherein the endogenous portion further comprises a transmembrane portion of an endogenous PD-1 polypeptide. 
     
     
         5 . The rodent of  claim 4 , wherein the endogenous portion has an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical to a corresponding amino acid sequence of a mouse PD-1 polypeptide that appears in  FIG. 8 . 
     
     
         6 . The rodent of  claim 4 , wherein the endogenous portion has an amino acid sequence that is identical to a corresponding amino acid sequence of a mouse PD-1 polypeptide that appears in  FIG. 8 . 
     
     
         7 . The rodent of any one of  claims 1 - 6 , wherein the human portion comprises amino acids 35-145 of a human PD-1 polypeptide. 
     
     
         8 . The rodent of any one of  claims 1 - 6 , wherein the human portion comprises amino acids 27-145 of a human PD-1 polypeptide. 
     
     
         9 . The rodent of any one of  claims 1 - 6 , wherein the human portion comprises amino acids 26-169 of a human PD-1 polypeptide. 
     
     
         10 . The rodent of any one of  claims 1 - 6 , wherein the human portion comprises an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical to a corresponding amino acid sequence of a human PD-1 polypeptide that appears in  FIG. 8 . 
     
     
         11 . The rodent of any one of  claims 1 - 6 , wherein the human portion comprises an amino acid sequence that is identical to a corresponding amino acid sequence of a human PD-1 polypeptide that appears in  FIG. 8 . 
     
     
         12 . The rodent of any one of the preceding claims, wherein the PD-1 polypeptide, which comprises a human portion and an endogenous portion, is encoded by an endogenous Pdcd1 gene. 
     
     
         13 . The rodent of  claim 12 , wherein the endogenous Pdcd1 gene comprises endogenous Pdcd1 exons 1, 4 and 5, 
     
     
         14 . The rodent of  claim 13 , wherein the endogenous Pdcd1 gene further comprises an endogenous Pdcd1 exon 3 in whole or in part. 
     
     
         15 . A rodent comprising a Pdcd1 gene that comprises an endogenous portion and a human portion, wherein the endogenous and human portions are operably linked to a rodent Pdcd1 promoter. 
     
     
         16 . The rodent of  claim 15 , wherein the rodent Pdcd1 promoter is an endogenous rodent Pdcd1 promoter. 
     
     
         17 . The rodent of  claim 15  or  16 , wherein the endogenous portion comprises endogenous Pdcd1 exons 1, 4 and 5. 
     
     
         18 . The rodent of  claim 17 , wherein the endogenous portion further comprises endogenous Pdcd1 exon 3 in whole or in part. 
     
     
         19 . The rodent of  claim 17  or  18 , wherein exons 1, 3 in whole or in part, 4 and 5 of the endogenous Pdcd1 gene are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical to the corresponding exons 1, 3 in whole or in part, 4 and 5 of an endogenous Pdcd1 gene that appears in  FIG. 8 . 
     
     
         20 . The rodent of any one of  claims 15 - 19 , wherein the human portion encodes amino acids 26-169 of a human PD-1 polypeptide. 
     
     
         21 . The rodent of any one of  claims 15 - 19 , wherein the human portion comprises exon 2 of a human Pdcd1 gene. 
     
     
         22 . The rodent of  claim 21 , wherein the human portion further comprises a human Pdcd1 exon 3 in whole or in part. 
     
     
         23 . The rodent of  claim 22 , wherein human Pdcd1 exons 2 and 3, in whole or in part, are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical to the corresponding exons 2 and 3, in whole or in part, of a human Pdcd1 gene that appears in  FIG. 8 . 
     
     
         24 . The rodent of  claim 22 , wherein the human portion comprises a sequence that is codon-optimized for expression in the rodent. 
     
     
         25 . The rodent of  claim 22 , wherein the human portion comprises SEQ ID NO: 23, 
     
     
         26 . The rodent of any one of  claims 1 - 25 , wherein the rodent is a rat or mouse. 
     
     
         27 . A PD-1 polypeptide produced by a rodent of any one of  claims 1 - 26 . 
     
     
         28 . The PD-1 polypeptide of  claim 27 , wherein the PD-1 polypeptide comprises an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical to a humanized. PD-1 polypeptide that appears in  FIG. 8 . 
     
     
         29 . An isolated rodent cell or tissue whose genome comprises a Pdcd1 gene that encodes a PD-1 polypeptide having a human portion and an endogenous portion, which portions are operably linked to a rodent Pdcd1 promoter. 
     
     
         30 . The isolated rodent cell or tissue of  claim 29 , wherein the rodent Pdcd1 promoter is an endogenous rodent Pdcd1 promoter. 
     
     
         31 . The isolated cell or tissue of  claim 29  or  30 , wherein the human portion comprises human Pdcd1 exon 2. 
     
     
         32 . The isolated cell or tissue of  claim 31 , wherein the human portion further comprises human Pdcd1 exon 3 in whole or in part. 
     
     
         33 . The isolated rodent cell or tissue of any one of  claims 29 - 32 , wherein the rodent cell or tissue is a mouse cell or mouse tissue, or a rat cell or rat tissue. 
     
     
         34 . A rodent embryonic stem cell whose genome comprises a Pdcd1 gene that encodes a PD-1 polypeptide having a human portion and an endogenous portion, which portions are operably linked to a rodent Pdcd1 promoter. 
     
     
         35 . The rodent embryonic stem cell of  claim 34 , wherein the rodent Pdcd1 promoter is an endogenous rodent Pdcd1 promoter. 
     
     
         36 . The rodent embryonic stem cell of  claim 34  or  35 , wherein the human portion comprises human Pdcd 1 exon 2. 
     
     
         37 . The rodent embryonic stem cell of  claim 36 , wherein the human portion further comprises human Pdcd1 exon 3 in whole or in part. 
     
     
         38 . The rodent embryonic stem cell of any one of  claims 34 - 37 , wherein the rodent embryonic stem cell is a mouse embryonic stem cell and is from a 129 strain, C57BL strain, or a mixture thereof. 
     
     
         39 . The rodent embryonic stem cell of  claim 38 , wherein the rodent embryonic stem cell is a mouse embryonic stern cell and is a mixture of 129 and C57BL strains. 
     
     
         40 . A rodent embryo generated from the embryonic stem cell of any one of  claims 34 - 39 , 
     
     
         41 . A method of making a rodent that expresses a PD-1 polypeptide from an endogenous Pdcd1 gene, wherein the PD-1 polypeptide comprises a human sequence, the method comprising
 (a) inserting a genomic fragment into an endogenous Pdcd1 gene in a rodent embryonic stem cell, said genomic fragment comprising a nucleotide sequence that encodes a human PD-1 polypeptide in whole or in part;   (b) obtaining the rodent embryonic stem cell generated in (a); and,   (c) creating a rodent using the rodent embryonic stem cell of (b).   
     
     
         42 . The method of  claim 41 , wherein the human sequence comprises amino acids 35-145 of a human PD-1 polypeptide. 
     
     
         43 . The method of  claim 41 , wherein the human sequence comprises amino acids 27-145 of a human PD-1 polypeptide. 
     
     
         44 . The method of  claim 41 , wherein the human sequence comprises amino acids 26-169 of a human PD-1 polypeptide. 
     
     
         45 . The method of any one of  claims 41 - 44 , wherein the nucleotide sequence comprises human Pdcd1 exon 2. 
     
     
         46 . The method of  claim 45 , wherein the nucleotide sequence further comprises human Pdcd1 exon 3 in whole or in part. 
     
     
         47 . The method of any one of  claims 41 - 46 , wherein the nucleotide sequence comprises one or more selection markers. 
     
     
         48 . The method of any one of  claims 41 - 47 , wherein the nucleotide sequence comprises one or more site-specific recombination sites. 
     
     
         49 . A method of making a rodent whose genome comprises a Pdcd1 gene that encodes a P1)-1 polypeptide having a human portion and an endogenous portion, which portions are operably linked to a rodent Pdcd1 promoter, the method comprising
 modifying the genome of a rodent so that it comprises a Pdcd1 gene that encodes a PD-1 polypeptide having a human portion and an endogenous portion, which portions are operably linked to a rodent Pdcd1 promoter, thereby making said rodent.   
     
     
         50 . The method of  claim 49 , wherein the rodent Pdcd1 promoter is an endogenous rodent Pdcd1 promoter. 
     
     
         51 . The method of  claim 49 , wherein the human portion comprises amino acids 35-145 of a human PD-1 polypeptide. 
     
     
         52 . The method of  claim 49 , wherein the human portion comprises amino acids 27-145 of a human PD-1 polypeptide. 
     
     
         53 . The method of  claim 49 , wherein the human portion comprises amino acids 26-169 of a human PD-1 polypeptide. 
     
     
         54 . The method of any one of  claims 49 - 53 , wherein the Pdcd1 gene is modified to include human Pdcd1 exon 2. 
     
     
         55 . The method of  claim 54 , wherein the Pdcd1 gene is modified to include human Pdcd1 exon 2 and human Pdcd1 exon 3 in whole or in part. 
     
     
         56 . The method of any one of  claims 41 - 55 , wherein the rodent is a mouse or rat. 
     
     
         57 . A rodent obtainable from the method of any one of  claims 41 - 56 . 
     
     
         58 . The rodent of  claim 57 , wherein the rodent is a mouse or a rat. 
     
     
         59 . A method of reducing tumor growth in a rodent, the method comprising the steps of
 administering a drug targeting human PD-1 to a rodent whose genome comprises a Pdcd1 gene that encodes a PD-1 polypeptide having a human portion and an endogenous portion, which portions are operably linked to a rodent Pdcd1 promoter;   the administering being performed under conditions and for a time sufficient that tumor growth is reduced in the rodent.   
     
     
         60 . A method of killing tumor cells in a rodent, the method comprising the steps of
 administering a drug targeting human PD-1 to a rodent whose genome comprises a Pdcd1 gene that encodes a PD-1 polypeptide having a human portion and an endogenous portion, which portions are operably linked to a rodent Pdcd1 promoter;   the administering being performed under conditions and for a time sufficient that the drug mediates killing of the tumor cells.   
     
     
         61 . A method of assessing the pharmacokinetic properties of a drug targeting human PD-1, the method comprising the steps of
 administering the drug to a rodent whose genome comprises a Pdcd1 gene that encodes a PD-1 polypeptide having a human portion and an endogenous portion, which portions are operably linked to a rodent Pdcd1 promoter; and   performing an assay to determine one or more pharmacokinetic properties of the drug targeting human PD-1.   
     
     
         62 . The method of any one of  claims 59 - 61 , wherein the human portion comprises amino acids 35-145 of a human PD-1 polypeptide. 
     
     
         63 . The method of any one of  claims 59 - 61 , wherein the human portion comprises amino acids 27-145 of a human PD-1 polypeptide. 
     
     
         64 . The method of any one of  claims 59 - 61 , wherein the human portion comprises amino acids 26-169 of a human PD-1 polypeptide. 
     
     
         65 . The method of any one of  claims 59 - 64 , wherein the drug targeting human PD-1 is a PD-1 antagonist. 
     
     
         66 . The method of any one of  claims 59 - 64 , wherein the drug targeting human PD-1 is a PD-1 agonist. 
     
     
         67 . The method of any one of  claims 59 - 64 , wherein the drug targeting human PD-1 is an anti-PD-1 antibody. 
     
     
         68 . The method of any one of  claims 59 - 67 , wherein the drug targeting human PD-1 is administered to the rodent intravenously. 
     
     
         69 . The method of any one of  claims 59 - 67 , wherein the drug targeting human PD-1 is administered to the rodent intraperitoneally. 
     
     
         70 . The method of any one of  claims 59 - 67 , wherein the drug targeting human PD-1 is administered to the rodent subcutaneously. 
     
     
         71 . The method of any one of  claims 59 - 70 , wherein the rodent Pdcd1 promoter is an endogenous rodent Pdcd1 promoter. 
     
     
         72 . The method of any one of  claims 59 - 71 , wherein the rodent is a mouse or a rat. 
     
     
         73 . A rodent tumor model, which rodent expresses a PD-1 polypeptide comprising a human portion and an endogenous portion. 
     
     
         74 . A rodent tumor model, which rodent has a genome comprising a Pdcd1 gene that comprises an endogenous portion and a human portion, wherein the endogenous and human portions are operably linked to a rodent Pdcd1 promoter. 
     
     
         75 . A rodent tumor model obtained by
 a) providing a rodent whose genome comprises a Pdcd1 gene that includes an endogenous portion and a human portion, which endogenous and human portions are operatively linked to a rodent Pdcd1 promoter; and   b) implanting one or more tumor cells in the rodent of (a);   thereby providing said rodent tumor model.   
     
     
         76 . The rodent tumor model of any one of  claims 73 - 75 , wherein the rodent is a mouse or a rat. 
     
     
         77 . The rodent tumor model of  claim 76 , wherein the rodent is a mouse. 
     
     
         78 . The rodent tumor model of  claim 77 , wherein the mouse is selected from the group consisting of a 129 strain, a C57BL/6 strain, and a mixed 1129×C57BL/6 strain. 
     
     
         79 . The rodent tumor model of  claim 78 , wherein the mouse is 25% 129 and 75% C57BL/6.